Novel degrader conjugates

Abstract

The present disclosure provides novel degraders and novel degraders conjugated to a binding moiety. Also provided are compositions comprising the conjugates. These compounds and compositions are useful for treating a disease or disorder, e.g., cancer, in a subject in need thereof.

Description

Technical Field

[0001] This disclosure provides novel degrader conjugates, wherein the novel degrader is conjugated to a binding moiety. Compositions comprising said conjugates are also provided. These conjugates and compositions can be used to treat cancer in subjects of need. Background Technology

[0002] The effectiveness of immunomodulatory imide drugs has demonstrated that protein degradation is a therapeutic strategy. These compounds bind to cereblon (CRBN) and promote the degradation of CRL4. CRBN E3 ubiquitin ligases possess the ability to mediate the recruitment and ubiquitination of substrate proteins. It is believed that immunomodulatory imides act as "molecular glues," filling the binding interface as hydrophobic patches, and reprogramming the protein interactions between the ligase and the new substrate.

[0003] While these compounds offer exciting potential as novel treatments for cancer, their application has so far been limited to hematologic malignancies such as multiple myeloma and myelodysplastic syndromes (MDS). Expanding the library of compounds that can function by degrading other oncoproteins (many of which are considered "undruggable") is an active area of ​​drug development. Therefore, there remains a continuous need for new compounds capable of targeting these alternative oncoproteins and treating a variety of cancers. Summary of the Invention

[0004] In some respects, this disclosure provides conjugates of formula (I):

[0005]

[0006] Or its pharmaceutically acceptable salt, wherein:

[0007] a is an integer from 1 to 10;

[0008] A is a phenyl group or a C4-C4 group. 10 cycloalkyl ring;

[0009] U is selected from NH and CF2;

[0010] R 1 Independently selected from hydrogen and halogroups;

[0011] X is selected from -NR 2 -、=C(CH3)-、-Q-(CH2) n - and -Q(CH2) m Q'(CH2) n -;in

[0012] Q and Q' are each independently O, S, or N(R) 2 )v ;

[0013] v is 1 or 2;

[0014] Each R 2 Independently hydrogen or C1-C6 alkyl;

[0015] n is an integer from 1 to 6;

[0016] m is an integer from 2 to 6;

[0017] Each group is connected to L on the left and to A on the right;

[0018] This assumes that X is NH or -Q-(CH2). n - At that time, R 1 It is a halogenated group;

[0019] L represents a cuttable or non-cuttable joint; and

[0020] Bm is the binding site that can specifically bind to proteins.

[0021] In some respects, the binding part is an antibody, an antibody fragment, or an antigen-binding fragment.

[0022] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein a is an integer from 2 to 8.

[0023] In some aspects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein L is an incisorless linker. In some aspects, L is selected from the group consisting of...

[0024]

[0025] in:

[0026] p is an integer from 1 to 10;

[0027] Let X be the connection point with X; and

[0028] This refers to the connection point with the joint portion.

[0029] In some respects, L is

[0030]

[0031] In some respects, p is 5.

[0032] In some aspects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein L is a cleavable linker. In some aspects, the cleavable linker is cleaved by a protease. In some aspects, L is selected from the group consisting of...

[0033]

[0034] in:

[0035] q is an integer from 2 to 10;

[0036] Z 1 Z 2 Z 3 and Z 4 Each naturally occurring amino acid residue, either independently absent or in L- or D-configuration, is provided that Z 1 Z 2 Z 3 and Z 4 At least two of them are amino acid residues;

[0037] Let X be the connection point with X; and

[0038] This refers to the connection point with the joint portion.

[0039] In some respects, Z 1 Z 2 Z 3 and Z 4 The following groups are independently absent or selected from: 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 Z 1 Z 2 Z 3 and Z 4 At least two of them are amino acid residues.

[0040] In some respects, Z 1 It may be absent or contain glycine; Z 2 The group consisting of or not present in the group containing: L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 Choose the group consisting of: L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z 4Choose from the following groups: L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0041] In some respects, L is

[0042]

[0043] In some respects, q is 5.

[0044] In some aspects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein L is a bioreducible linker. In some aspects, L is selected from the group consisting of...

[0045]

[0046] in:

[0047] q is an integer from 2 to 10;

[0048] R, R', R” and R'” are each independently selected from hydrogen, C1-C6 alkoxy-C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or the two geminal R groups together with the carbon atoms to which they are attached can form a cyclobutyl or cyclopropyl ring;

[0049] Let X be the connection point with X; and

[0050] This refers to the connection point with the joint portion.

[0051] In some aspects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein L is an acid-cleavable linker. In some aspects, L is selected from the group consisting of...

[0052]

[0053] in:

[0054] q is an integer from 2 to 10;

[0055] Let X be the connection point with X; and

[0056] This refers to the connection point with the joint portion.

[0057] In some aspects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein L is a click-release linker. In some aspects, L is selected from...

[0058]

[0059] in:

[0060] q is an integer from 2 to 10;

[0061] Let X be the connection point with X; and

[0062] This refers to the connection point with the joint portion.

[0063] In some aspects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein L is a pyrophosphatase-cleavable linker. In some aspects, L is...

[0064]

[0065] in:

[0066] q is an integer from 2 to 10;

[0067] Let X be the connection point with X; and

[0068] This refers to the connection point with the joint portion.

[0069] In some aspects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein L is a β-glucuronidase-cleavable linker. In some embodiments, L is selected from...

[0070]

[0071] in:

[0072] q is an integer from 2 to 10;

[0073] ----Does not exist or is a key;

[0074] Let X be the connection point with X; and

[0075] This refers to the connection point with the joint portion.

[0076] In some aspects, this disclosure provides a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein Bm is an antibody or its antigen-binding moiety. In some aspects, the protein bound to the binding moiety is a surface antigen.

[0077] In some respects, surface antigens include 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, and 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, condensation factor, cKit, closure protein 3, closure protein 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1. Growth factors, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2) ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose 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-11Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including α4, α). v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxyl esterase, 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 A1, MelanA / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagoguerin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzymes, prostate cancer cells, prostate Adenosine, Pseudomonas aeruginosa, rabies virus, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, respiratory syncytial virus, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2. TIM-1, TnAg, 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.

[0078] In some respects, surface antigens include HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or combinations thereof.

[0079] In some respects, Bm is an antibody 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, and anti-CD38. A2 antibody, HuAT13 / 5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, and veltuzumab. In some cases, the antibody is rituximab, trastuzumab, pertuzumab, OR000213 (huMy9-6 IgG4 S228P), lintuzumab, or gemtruzumab.

[0080] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein:

[0081] A is a phenyl group;

[0082] U is NH;

[0083] R 1 It is a halogenated group; and

[0084] X is -N(R) 2 ) v (CH2) m O(CH2) n -;in:

[0085] v is 1;

[0086] m and n are both 2; and

[0087] R 2It is a methyl group.

[0088] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein:

[0089] A is a phenyl group;

[0090] U is NH;

[0091] R 1 It is a halogenated group; and

[0092] X is -N(R) 2 ) v (CH2) m O(CH2) n -;in:

[0093] v is 2;

[0094] m and n are both 2; and

[0095] Each R 2 It is a methyl group.

[0096] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein:

[0097] A is a phenyl group;

[0098] U is NH;

[0099] R 1 It is a halogenated group; and

[0100] X is -O(CH2) n -;in:

[0101] n is 2.

[0102] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein:

[0103] A is a phenyl group;

[0104] U is NH;

[0105] R 1 It is a halogenated group; and

[0106] X is -S(CH2) n -;in:

[0107] n is 2.

[0108] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein:

[0109] A is a phenyl group;

[0110] U is NH;

[0111] R 1 It is hydrogen; and

[0112] X is --NR 2 -;in:

[0113] R 2 It is a methyl group.

[0114] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein:

[0115] A is a phenyl group;

[0116] U is NH;

[0117] R 1 It is a halogenated group; and

[0118] X is --NR 2 -;in:

[0119] R 2 It is hydrogen.

[0120] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein:

[0121] A is a phenyl group;

[0122] U is NH;

[0123] R 1 It is hydrogen; and

[0124] X is -C(CH3) =.

[0125] In some respects, this disclosure provides conjugates of formula (I), or pharmaceutically acceptable salts thereof, wherein:

[0126] A is C4-C 10 cycloalkyl ring;

[0127] U is NH;

[0128] R 1 It is hydrogen; and

[0129] X is -N(R) 2 (CH2) m O(CH2) n -;in:

[0130] n is 1;

[0131] m is 2; and

[0132] R 2It is a methyl group.

[0133] In some respects, this disclosure provides compounds of (II):

[0134]

[0135] Or its pharmaceutically acceptable salt, wherein:

[0136] A is a phenyl group or a C4-C4 group. 10 cycloalkyl ring;

[0137] R 1 Independently selected from hydrogen and halogroups;

[0138] U is selected from NH and CF2; and

[0139] R 2 Selected from -C(O)R 3 -N(R) 4 2、-(CH2) n OH, -(CH2) n SH, -(CH2) n N(R 4 2、-(CH2) n Q'(CH2) m OH, -(CH2) n Q'(CH2) m SH and -(CH2) n Q'(CH2) m N(R 4 )2; of which

[0140] R 3 It is hydrogen or C1-C6 alkyl;

[0141] Each R 4 Independently hydrogen or C1-C6 alkyl;

[0142] Q' is O, S, or NR 4 ;

[0143] n is 1-6; and

[0144] m is 2-5;

[0145] The premise is that when R 2 For NH2, -(CH2) n NH2 or -(CH2) n When OH, then R 1 It is a halogenated group.

[0146] In some respects, this disclosure provides compounds of (III):

[0147]

[0148] Or its pharmaceutically acceptable salt.

[0149] In some respects, this disclosure provides compounds of (IV):

[0150]

[0151] Or its pharmaceutically acceptable salt.

[0152] In some respects, this disclosure provides conjugates of formula (V):

[0153]

[0154] Or a pharmaceutically acceptable salt thereof, wherein Bm is the binding moiety that specifically binds to a protein. In some respects, Bm is an antibody or its antigen-binding moiety. In some respects, the protein specifically bound to the binding moiety is a surface antigen.

[0155] In some respects, surface antigens include 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, and 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, condensation factor, cKit, closure protein 3, closure protein 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1. Growth factors, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2) ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose 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-11Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including α4, α). v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxyl esterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, podin, LewisY, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagoguerin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzymes, prostate cancer cells, prostaglandins, copper *Pseudomonas aeruginosa*, rabies virus, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, respiratory syncytial virus, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2. TIM-1, TnAg, 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.

[0156] In some respects, surface antigens include HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or combinations thereof.

[0157] In some respects, Bm refers to antibodies, including rituximab, trastuzumab, gemtuzumab, pertuzumab, obitutuzumab, oflamuzumab, olatotuzumab, antuximab, ixartuzumab, saxitotuzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, belantanumab, indextuzumab, cetuximab, dinutotuzumab, anti-CD38 A2 antibody, HuaT13 / 5 antibody, alemtuzumab, teimomab, tosimob, bevacizumab, panitumumab, trimemumab, teimomab, caputoxumab, ozovozumab, or vetotuzumab. In some cases, the antibodies are rituximab, trastuzumab, pertuzumab, OR000213, lintuzumab, or gemtuzumab.

[0158] In some aspects, this disclosure provides a pharmaceutical composition comprising a conjugate or compound of any of the foregoing aspects or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0159] In some aspects, this disclosure provides a method for treating a subject with cancer, the method comprising administering to the subject a pharmaceutically acceptable amount of any of the conjugates, compounds, or compositions of the foregoing 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.

[0160] In some aspects, the method further includes administering a pharmaceutically acceptable amount of an adjuvant to the subject before, after, or simultaneously with the conjugate or compound or a pharmaceutically acceptable salt thereof from any of the foregoing aspects. In some aspects, the adjuvant is a cytotoxic agent or an immunomodulator. In some aspects, the immunomodulator is a checkpoint inhibitor. In some aspects, checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, and / or LAG-3 inhibitors.

[0161] In some aspects, this disclosure provides a method for preparing a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, the method comprising reacting the binding moiety with a compound of formula (I-1):

[0162]

[0163] Or a pharmaceutically acceptable salt reaction, wherein:

[0164] a is an integer from 1 to 10;

[0165] A is a phenyl group or a C4-C4 group. 10 cycloalkyl ring;

[0166] R 1 Independently selected from hydrogen and halogroups;

[0167] U is selected from NH and CF2; and

[0168] X is selected from -N(R) 2 ) v -、=C(CH3)-、-Q-(CH2) n - and -Q(CH2) m Q'(CH2) n -;in

[0169] v is 1 or 2;

[0170] Q and Q' are each independently O, S, or NR. 2 ;

[0171] Each R 2 Independently hydrogen or C1-C6 alkyl;

[0172] n is an integer from 1 to 6; and

[0173] m is an integer from 2 to 6;

[0174] Each group is connected to L' on the left and to A on the right;

[0175] This assumes that X is NH or -Q-(CH2). n - At that time, R 1 It is a halogenated group; and

[0176] L' is a cuttable or non-cuttable connector precursor that mates with the joint portion.

[0177] In some respects, the method also includes reducing the bound moiety before reacting with the compound of formula (I-1).

[0178] In some respects, a is an integer from 2 to 8.

[0179] In some respects, L' is a precursor to an uncuttable joint. In some respects, L' is selected from the group consisting of the following:

[0180]

[0181] in:

[0182] p is an integer from 1 to 10; and

[0183] Let X be the connection point with X.

[0184] In some respects, L' is

[0185]

[0186] In some respects, p is 5.

[0187] In some respects, L' is a cleavable adapter precursor. In some respects, the cleavable adapter precursor can be cleaved by proteases. In some respects, L' is selected from the group consisting of the following:

[0188]

[0189] in:

[0190] q is an integer from 2 to 10;

[0191] Z 1 Z 2 Z 3 and Z 4 Each naturally occurring amino acid residue, either independently absent or in L- or D-configuration, is provided that Z 1 Z 2 Z 3 and Z 4 At least two of them are amino acid residues; and

[0192] Let X be the connection point with X.

[0193] In some respects, Z 1 Z 2 Z 3 and Z 4 The following groups are independently absent or selected from: 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 Z 1 Z 2 Z 3 and Z 4 At least two of them are amino acid residues.

[0194] In some respects, Z 1 It may be absent or contain glycine; Z 2 The group consisting of or not present in the group containing: L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 Select the group consisting of: L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; and Z 4Choose from the following groups: L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0195] In some respects, L' is

[0196]

[0197] In some respects, q is 5.

[0198] In some respects, L' is a bioreducible adaptor precursor. In other respects, L' is selected from the group consisting of the following...

[0199]

[0200] in:

[0201] q is an integer from 2 to 10;

[0202] R, R', R" and R'" are each independently selected from hydrogen, C1-C6 alkoxy-C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or the two geminal R groups together with the carbon atoms they are attached to can form a cyclobutyl or cyclopropyl ring; and

[0203] Let X be the connection point with X.

[0204] In some respects, L' is an acid-cuttable precursor. In other respects, L' is selected from the group consisting of the following...

[0205]

[0206] in:

[0207] q is an integer from 2 to 10; and

[0208] Let X be the connection point with X.

[0209] In some respects, L' is a click-release connector precursor. In other respects, L' is selected from...

[0210]

[0211] in:

[0212] q is an integer from 2 to 10; and

[0213] Let X be the connection point with X.

[0214] In some respects, L' is a pyrophosphatase-cleavable linker precursor. In some respects, L' is...

[0215]

[0216] in:

[0217] q is an integer from 2 to 10;

[0218] Let X be the connection point with X.

[0219] In some respects, L' is a β-glucuronidase-cleavable precursor of the adaptor. In some respects, L' is selected from...

[0220]

[0221] in:

[0222] q is an integer from 2 to 10;

[0223] ----Does not exist or is a key; and

[0224] Let X be the connection point with X.

[0225] In some respects, the compound of formula (I-1) reacts with a binding moiety, which includes an antibody or its antigen-binding moiety. In some respects, the antibody or its antigen-binding moiety binds to a surface antigen.

[0226] In some respects, surface antigens include 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, and 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, condensation factor, cKit, closure protein 3, closure protein 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1. Growth factors, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2) ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose 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-11Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including α4, α). v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxyl esterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, podin, LewisY, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagoguerin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzymes, prostate cancer cells, prostaglandins, copper *Pseudomonas aeruginosa*, rabies virus, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, respiratory syncytial virus, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2. TIM-1, TnAg, 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.

[0227] In some respects, surface antigens include HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or combinations thereof.

[0228] In some respects, Bm refers to antibodies, including rituximab, trastuzumab, gemtuzumab, pertuzumab, obitutuzumab, oflamuzumab, olatotuzumab, antuximab, ixartuzumab, saxitotuzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, belantanumab, indextuzumab, cetuximab, dinutotuzumab, anti-CD38 A2 antibody, HuaT13 / 5 antibody, alemtuzumab, teimomab, tosimob, bevacizumab, panitumumab, trimemumab, teimomab, caputoxumab, ozovozumab, or vetotuzumab. In some respects, the antibodies are rituximab, trastuzumab, pertuzumab, OR000213, lintuzumab, or gemtuzumab.

[0229] In some aspects, this disclosure provides a method for preparing conjugates of formula (I) from compounds of formula (I-1), wherein:

[0230] A is a phenyl group;

[0231] U is NH;

[0232] R 1 It is a halogenated group; and

[0233] X is -N(R) 2 ) v (CH2) m O(CH2) n -;in:

[0234] v is 1;

[0235] m and n are both 2; and

[0236] R 2 It is a methyl group.

[0237] In some respects:

[0238] A is a phenyl group;

[0239] U is NH;

[0240] R 1 It is a halogenated group; and

[0241] X is -N(R) 2 ) v (CH2) m O(CH2) n -;in:

[0242] v is 2;

[0243] m and n are both 2; and

[0244] Each R2 It is a methyl group.

[0245] In some respects:

[0246] A is a phenyl group;

[0247] U is NH;

[0248] R 1 It is a halogenated group; and

[0249] X is -O(CH2) n -;in:

[0250] n is 2.

[0251] In some respects:

[0252] A is a phenyl group;

[0253] U is NH;

[0254] R 1 It is a halogenated group; and

[0255] X is -S(CH2) n -;in:

[0256] n is 2.

[0257] In some respects:

[0258] A is a phenyl group;

[0259] U is NH;

[0260] R 1 It is hydrogen; and

[0261] X is --NR 2 -;in:

[0262] R 2 It is a methyl group.

[0263] In some respects:

[0264] A is a phenyl group;

[0265] U is NH;

[0266] R 1 It is a halogenated group; and

[0267] X is --NR 2 -;in:

[0268] R 2 It is hydrogen.

[0269] In some respects:

[0270] A is a phenyl group;

[0271] U is NH;

[0272] R 1 It is hydrogen; and

[0273] X is -C(CH3) =.

[0274] In some respects:

[0275] A is C4-C 10 cycloalkyl ring;

[0276] U is NH;

[0277] R 1 It is hydrogen; and

[0278] X is -N(R) 2 (CH) 2 ) m O(CH2) n -;in:

[0279] n is 1;

[0280] m is 2; and

[0281] R 2 It is a methyl group.

[0282] In some respects, the compound of formula (I-1) is:

[0283] Attached Figure Description

[0284] Figure 1 The in vitro activity of representative novel degrader conjugates against the BT-474 cell line is depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the percentage of BT-474 cell viability when treated with trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (triangles, solid lines), trastuzumab alone (triangles, dashed lines), Kadcyla (diamonds), novel degrader P1 alone (crosses), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (circles). L represents the linker.

[0285] Figure 2The in vitro activities of representative novel degradative conjugates against the BT-474 cell line are depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the viability of BT-474 cells when treated with pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (triangle, solid line), pertuzumab alone (triangle, dashed line), Kadcyla (diamond), novel degradative P1 alone (cross), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (circle).

[0286] Figure 3 The in vitro activity of representative novel degradative conjugates against the BT-474 cancer cell line is depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the viability of BT-474 cells when treated with trastuzumab-L-P4 (e.g., trastuzumab-compound (Ic)) (triangle, solid line), trastuzumab (triangle, dashed line), Kadcyla (diamond), novel degradative P4 alone (cross), and rituximab-L-P4 (e.g., rituximab-compound (Ic)) (circle).

[0287] Figure 4 The in vitro activities of representative novel degradative conjugates against the BT-474 cell line are depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the viability of BT-474 cells when treated with pertuzumab-L-P4 (e.g., pertuzumab-compound (Ic)) (triangle, solid line), pertuzumab (triangle, dashed line), Kadcyla (diamond), novel degradative P4 alone (cross), and rituximab-L-P4 (e.g., rituximab-compound (Ic)) (circle).

[0288] Figure 5 The in vitro activity of representative novel degradation agent conjugates with different drug:antibody ratios (DAR) against the BT-474 cell line is described. The X-axis represents the logarithmic antibody concentration (M). The Y-axis shows the DAR 1.6 (upward triangle, solid line) for trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)), DAR 1.5 (downward triangle, solid line) for trastuzumab-L-P3 (e.g., trastuzumab-compound (Ib)), DAR 1.6 (circle, solid line) for trastuzumab-L-P4 (e.g., trastuzumab-compound (Ic)), DAR 1.6 (square, solid line) for trastuzumab-L-P1 (e.g., trastuzumab-compound (Id)), DAR 8 (triangle, dashed line) for trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)), and DAR 8 (solid circle, dashed line) for trastuzumab-L-P4 (e.g., trastuzumab-compound (Ic)). (Rhombus, dashed line) and trastuzumab (circle, dashed line) viability of BT-474 cells after treatment.

[0289] Figure 6 The in vitro activity of representative novel degraders against the BT-474 cell line was depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the DAR 8 (upward triangle, solid line) when pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)), pertuzumab-L-P4 (e.g., pertuzumab-compound (Ic)) DAR 8 (downward triangle, solid line), and... (Rhombus, dashed line) Viability of BT-474 cells after treatment.

[0290] Figure 7 The in vitro activity of representative novel degradative conjugates against the SK-BR-3 cell line was depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the viability of SK-BR-3 cells when treated with trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia) (triangle, solid line), trastuzumab (triangle, dashed line), Kadcyla (diamond), novel degradative P1 alone (circle), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (cross).

[0291] Figure 8 The in vitro activities of representative novel degradative conjugates against the SK-BR-3 cell line are depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the viability of SK-BR-3 cells when treated with pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (triangle, solid line), pertuzumab (triangle, dashed line), Kadcyla (diamond), novel degradative P1 alone (circle), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (cross).

[0292] Figure 9 The in vitro activity of representative novel degradation agent conjugates against the HL-60 cell line was depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the activity of OR000213-L-P1 (e.g., OR000213-compound (Ia)) (triangles). (Rhombus) and trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (circle) viability of HL-60 cells when treated.

[0293] Figure 10The in vitro activities of representative novel degradation agent conjugates against the HL-60 cell line are depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the DAR8 values ​​when using huMy9-6(IgG1)-L-P1 (e.g., huMy9-6(IgG1)-compound (Ia)) DAR8 (solid triangles upward, solid lines), huMy9-6(IgG1)-L-P1 (e.g., huMy9-6(IgG1)-compound (Id)) DAR8 (solid triangles downward, solid lines), lintuzumab IgG1-L-P1 (e.g., lintuzumab IgG1-compound (Ia)) DAR8 (hollow triangles upward, dashed lines), lintuzumab IgG1-L-P1 (e.g., lintuzumab IgG1-compound (Id)) DAR8 (hollow triangles downward, dashed lines), OR000213-L-P1 (e.g., OR000213-compound (Ia)) DAR8 (solid triangles downward, dashed lines). 8 (square) and rituximab-L-P4 (e.g., rituximab-compound (Ic)) (circle, dashed line) viability of HL-60 cells.

[0294] Figure 11 The in vitro activity of conjugates of compound (Ia) against the HL60 cell line with different drug:antibody ratios (DAR) is described. The X-axis represents the logarithmic antibody concentration (M). The Y-axis shows the viability of HL-60 cells when 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, dashed line).

[0295] Figure 12The in vitro activity of conjugates of compound (Ia) against the HL60 cell line with different drug:antibody ratios (DAR) is described. The X-axis represents the logarithmic antibody concentration (M). The Y-axis shows the viability of HL-60 cells when 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, dashed line).

[0296] Figure 13 The in vitro activity of representative novel degradative conjugates against Ramos cell lines is depicted. The X-axis represents the logarithmic antibody concentration (M), and the Y-axis represents the viability of Ramos cells when treated 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, dashed line), novel degradative agent P1 alone (cross, dashed line), novel degradative agent P4 alone (star, dashed line), and trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (circle, dashed line).

[0297] Figure 14 The in vitro activity of representative novel degradation agent conjugates against the Daudi cell line was depicted. The X-axis represents the logarithmic antibody concentration (M), and the Y-axis represents the viability of Daudi cells when treated with rituximab-L-P1 (e.g., rituximab-compound (Ia)) (upward triangle, solid line), rituximab (triangle, dashed line), and trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (circle, dashed line).

[0298] Figure 15 The in vitro activities of representative novel degrader conjugates against Ramos cell lines are depicted. The X-axis represents the logarithmic antibody concentration (M), and the Y-axis represents the viability of Ramos cells when treated 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 the novel degrader P1 alone.

[0299] Figure 16 The in vitro activity of representative novel degradative conjugates against the NCI-N87 cancer cell line is depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the viability of NCI-N87 cells when treated with trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (triangle, solid line), trastuzumab (triangle, dashed line), Kadcyla (diamond), novel degradative agent P4 alone (cross), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (circle).

[0300] Figure 17 The in vitro activity of representative novel degradative conjugates against the NCI-N87 cancer cell line is depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis represents the viability of NCI-N87 cells when treated with pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (triangle, solid line), pertuzumab (triangle, dashed line), Kadcyla (diamond), novel degradative P1 alone (cross), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (circle).

[0301] Figure 18 The in vitro activity of representative novel degradation agent conjugates against the BT-474 cell line after 3 days of incubation with human serum was depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis shows the viability of BT-474 cells when 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 shape), trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (upward triangle, dashed line), pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (downward triangle, dashed line), and OR000213-L-P1 (e.g., OR000213-compound (Ia)) (circle, dashed line).

[0302] Figure 19The in vitro activity of representative novel degradation agent conjugates against the BT-474 cell line after 3 days of incubation with mouse serum was depicted. The X-axis represents the logarithmic antibody concentration (M). The Y-axis shows the viability of BT-474 cells when 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 only (star shape), trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (upward triangle, dashed line), pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (downward triangle, dashed line), and OR000213-L-P1 (e.g., OR000213-compound (Ia)) (circle, dashed line).

[0303] Figure 20 The in vivo activity of representative novel degradation conjugates against BT-474 (Her2+) tumors in mice was depicted. The X-axis shows the number of days after administration. The Y-axis shows the tumor volume (mm) after administration of the mediator (solid circles), 5 mg / kg trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (squares), 5 mg / kg rituximab-L-P1 (e.g., rituximab-compound (Ia)) (triangles), and 5 mg / kg pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (hollow circles). 3 ).

[0304] Figure 21 The in vivo activity of representative novel degradation agent conjugates against Daudi (CD20+) tumors was depicted. The X-axis shows the number of days after administration. The Y-axis shows the tumor volume (mm) after administration of the medium (solid circles), 5 mg / kg trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (squares), 1 mg / kg rituximab-L-P1 (e.g., rituximab-compound (Ia)) (triangles), and 5 mg / kg rituximab-L-P1 (e.g., rituximab-compound (Ia)) (hollow circles). 3 ).

[0305] Figure 22The in vivo activity of representative novel degradation agent conjugates against HL-60 (CD33+) tumors was depicted. The X-axis shows the number of days after administration. The Y-axis shows the tumor volume (mm) after administration of the medium (solid circles), 5 mg / kg trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (squares), 1 mg / kg OR000213-L-P1 (e.g., OR000213-compound (Ia)) (triangles), and 5 mg / kg OR000213-L-P1 (e.g., OR000213-compound (Ia)) (hollow circles). 3 ).

[0306] Figure 23 The in vitro activity of the novel degrading agent conjugate against the HCC2157 cell line was depicted. The X-axis represents the logarithmic antibody concentration (M), and the Y-axis represents the viability of HCC2157 cells when treated with sacitrus-L-P1 (e.g., sacitrus-compound (Ia)) (line 1), sacitrus-L-P1 alone (line 2), and the novel degrading agent P1 alone (line 3).

[0307] Figure 24 The in vitro activity of the novel degrading agent conjugate against the LP1 cell line was depicted. The X-axis represents the logarithmic antibody concentration (M), and the Y-axis represents the viability of LP1 cells when treated with HuAT 13 / 5-L-P1 (e.g., HuAT 13 / 5-compound (Ia)) (line 1), HuAT 13 / 5 alone (line 2), and the novel degrading agent P1 alone (line 3).

[0308] Figure 25 The in vivo activity of representative novel degradation agent conjugates against NCI-H929 (CD38+) tumors was depicted. The X-axis shows the number of days after administration. The Y-axis shows the tumor volume (mm²) after administration with the medium (circles) or 5 mg / kg HuAT13 / 5-L-P1 (e.g., HuAT13 / 5-compound (Ia)) (squares). 3 ). Detailed Implementation

[0309] This disclosure relates to conjugates of formula (I):

[0310]

[0311] Or its pharmaceutically acceptable salt, wherein:

[0312] a is an integer from 1 to 10;

[0313] A is a phenyl group or a C4-C4 group. 10 cycloalkyl ring;

[0314] R1 Independently selected from hydrogen and halogroups;

[0315] U is selected from NH and CF2;

[0316] X is selected from -N(R) 2 ) V -、=C(CH3)-、-Q-(CH2) n - and -Q(CH2) m Q'(CH2) n -;in

[0317] Q and Q' are each independently O, S, or N(R) 2 ) V ;

[0318] v is 1 or 2;

[0319] Each R 2 Independently hydrogen or C1-C6 alkyl;

[0320] n is an integer from 1 to 6;

[0321] m is an integer from 2 to 6;

[0322] Each group is connected to L on the left and to A on the right;

[0323] This assumes that X is NH or -Q-(CH2). n - At that time, R 1 It is a halogenated group;

[0324] L represents a cuttable or non-cuttable joint; and

[0325] Bm is a binding moiety capable of specifically binding to proteins. In some cases, the binding moiety is an antibody, an antibody fragment, or an antigen-binding fragment.

[0326] The present invention also provides the above-described compound fused with the binding portion, a composition comprising the compound or conjugate, or a method for using or preparing the compound or conjugate.

[0327] I. Definition.

[0328] To make this specification easier to understand, some terms are defined first. Additional definitions are set forth throughout the detailed description.

[0329] It is important to note that the terms "an" or "a" entity refer to one or more of those entities; for example, "nucleotide sequence" is understood to represent one or more nucleotide sequences. Therefore, the terms "an (or a)", "one or more", and "at least one" are used interchangeably herein. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a prior basis for using exclusive terms such as "solely", "only", etc., or for using negative limitations in relation to the recitation of the claim elements.

[0330] Furthermore, when used herein, “and / or” should be considered as each of the two specified features or components being, or not being, a specific disclosure of the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to 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 (alone); B (alone); and C (alone).

[0331] It should be understood that whenever an aspect is described in this document using the language “comprising”, other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0332] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art relating to this disclosure. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd edition, 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd edition, 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, revised edition, 2000, Oxford University Press, provide a general dictionary for those skilled in the art of the many terms used in this disclosure.

[0333] Units, prefixes, and symbols are represented in their forms acceptable according to the Systéme International de Unites (SI). Numerical ranges include the numbers that define the range. In the case of enumerated ranges of values, it should be understood that each intermediate integer value between the upper and lower limits of the enumeration of the range, each fraction thereof, and each subrange between these values ​​are also specifically disclosed. The upper and lower limits of any range may be independently included in or excluded from the range, and each range that includes any limit value, excludes any limit value, or includes both limit values ​​is also covered within this disclosure. Therefore, the ranges enumerated herein are understood to be abbreviations of all values ​​within the range, including the enumerated endpoints. For example, the range 1 to 10 is understood to include any number, combination of numbers, or subrange of numbers derived from groups consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0334] Where values ​​are explicitly listed, it should be understood that values ​​of approximately the same quantity or amount as those listed are also within the scope of this disclosure. Where combinations are disclosed, each sub-combination of the elements of that combination is also specifically disclosed and within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element disclosed is disclosed to have multiple substitutes, instances of such disclosure where each substitute is individually excluded or in any combination with other substitutes are also disclosed; more than one element may be disclosed with such exclusions, and all combinations of elements with such exclusions are thus disclosed.

[0335] As used herein, the term "DAR" refers to the drug-antibody ratio of the conjugate, which is the average number of new degrader-connector complexes linked to each antibody. In some respects, the DAR of the conjugates described herein is from 1 to 10. In other respects, the DAR of the conjugates described herein is from 1 to 8. In some respects, the DAR of the conjugates described herein are 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, and 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.

[0336] As used herein, the term "antibody" also refers to a full-length immunoglobulin molecule or the immunoactive portion of a full-length immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that immune-specifically binds to an antigen or a portion thereof that is a target of interest, including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins disclosed 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 immunoglobulin molecules. Immunoglobulins can be derived from any species. However, in one respect, immunoglobulins are of human, mouse, or rabbit origin.

[0337] The term "single-domain antibody," also known as a nanobody, refers to an antibody fragment composed of a single monomeric variable antibody domain with a molecular weight of approximately 12 kDa to approximately 15 kDa. Monomeric antibodies can be based on either a heavy chain variable domain or a light chain. Examples of single-domain antibodies include, but are not limited to, V... H H fragment and V NAR Excerpt.

[0338] An "antibody fragment" comprises a portion of a complete antibody, typically its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv fragments; dimers; linear antibodies; fragments generated from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity-determining regions); epitope-binding fragments that specifically bind to any of the aforementioned immune-specific cancer cell antigens, viral antigens, or microbial antigens; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0339] "Complete antibody" is an antibody that contains 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 can be native sequence constant domains (e.g., human native sequence constant domains) or their amino acid sequence variants.

[0340] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations, which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets 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 characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of that antibody by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by hybridoma methods or by recombinant DNA methods. "Monoclonal antibodies" can also be isolated from phage antibody libraries.

[0341] The monoclonal antibodies discussed in this paper specifically include “chimeric” antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity. Chimeric antibodies of interest in this paper include “primatoid” antibodies, which contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0342] Various methods have been employed to produce monoclonal antibodies (MAbs). Hybridoma technology refers to the production of clonal cell lines that generate a single type of antibody, using cells from various species, including mice (rodents), hamsters, rats, and humans. Another method for preparing MAbs is through genetic engineering, including recombinant DNA technology. Monoclonal antibodies produced using these techniques include chimeric antibodies and humanized antibodies. Chimeric antibodies combine DNA coding regions from more than one species. For example, the variable region of a chimeric antibody may be derived from mice, while the constant region may be derived from humans. Humanized antibodies are primarily derived from humans, although they contain non-human portions. Like chimeric antibodies, humanized antibodies may contain a fully human constant region. However, unlike chimeric antibodies, the variable region may be partially derived from humans. The non-human synthetic portion of humanized antibodies typically comes from the CDR in mouse antibodies. In any case, these regions are crucial for antibody recognition and binding to specific antigens. While mouse antibodies can be used for diagnosis and short-term treatment, they should not be administered to humans long-term without increasing the risk of harmful immunogenic responses. This response is called human anti-mouse antibody (HAMA), which occurs when the human immune system recognizes mouse antibodies as foreign substances and attacks them. HAMA response can lead to toxic shock and even death.

[0343] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portion of the administered antibody. Furthermore, chimeric and humanized antibodies can have the additional benefit of activating secondary human immune responses, such as antibody-dependent cytotoxicity.

[0344] A complete antibody may possess one or more "effective functions," which refer to those biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs).

[0345] Based on the amino acid sequence of the heavy chain constant domain of intact antibodies, they can be classified into different "classes." There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0346] As used herein, the term "about" means approximately, roughly, about, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the stated value. Generally, the term "about" is used to modify values ​​that deviate from, for example, 10% above or below (higher or lower) the stated value.

[0347] The terms "administration" and their grammatical variations refer to the introduction of a composition, such as the EV (e.g., exogenous form) of this disclosure, into a subject via a pharmaceutically acceptable route. This includes introducing the composition, such as the EV (e.g., exogenous form) of this disclosure, into a subject via any suitable route, including intratumoral, oral, intrapulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periorbital, or topical. 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 a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein in the subject.

[0348] As used herein, the term "antibody" encompasses naturally occurring or partially or wholly synthetically produced immunoglobulins and fragments thereof. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" also includes polypeptides containing a framework region of an immunoglobulin gene or a fragment thereof that specifically binds to and recognizes an antigen. The use of the term "antibody" is intended to include complete antibodies, polyclonal, monoclonal, and recombinant antibodies, fragments thereof, and also includes single-chain antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments such as scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, biantibodies, and antibody-associated polypeptides. Antibodies include bispecific and multispecific antibodies, provided they exhibit the desired biological activity or function. In some aspects of this disclosure, the biologically active molecule is an antibody or a molecule containing its antigen-binding fragment.

[0349] The terms "antibody-drug conjugate" and "ADC" are used interchangeably to refer to an antibody linked (e.g., covalently linked) to a therapeutic agent (sometimes referred to herein as an agent, drug, or active pharmaceutical ingredient). In some aspects of this disclosure, the biologically active molecule is an antibody-drug conjugate.

[0350] As used herein, the term "approximately," when applied to one or more values ​​of interest, refers to a value similar to the reference value. In some respects, the term "approximately" refers to a range of values ​​falling within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value in either direction (greater or less), unless otherwise stated or apparent from the context (unless the number would exceed 100% of the possible value).

[0351] "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conserved. In another embodiment, an amino acid string can be conservatively substituted by a string of structurally similar members of the side chain family that differ in sequence and / or composition.

[0352] As used herein, the term "conserved" refers to nucleotide or amino acid residues in polynucleotide or polypeptide sequences that have not changed at the same position in two or more sequences being compared. Relatively conserved nucleotides or amino acids are those that are conserved in sequences that are more relevant than nucleotides or amino acids appearing elsewhere in the sequence.

[0353] In some respects, two or more sequences are said to be "completely conserved" or "identical" if they are 100% identical to each other. In some respects, two or more sequences are said to be "highly conserved" if they are at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to each other. In some respects, two or more sequences are said to be "conserved" if they are at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to each other. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to its parts, regions, or features.

[0354] As used herein, the terms “connection” and “fusing” are used interchangeably, each referring to a covalent or non-covalent connection of two or more parts comprising a novel degrading agent and a binding portion. In some respects, a connection or fusing may include a joint.

[0355] The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs to some extent from that of the native polypeptide sequence. Typically, amino acid sequence variants will have at least about 70% sequence identity with at least one receptor-binding domain of a native antibody or at least one ligand-binding domain of a native receptor, and typically, they will have at least about 80%, more typically at least about 90%, sequence homology with such receptor or ligand-binding domains. Amino acid sequence variants have substitutions, deletions, and / or insertions at certain positions in the amino acid sequence of the native amino acid sequence. Amino acids are named using conventional names, one-letter codes, and three-letter codes.

[0356] "Sequence identity" is defined as the percentage of identical residues in an amino acid sequence variant after aligning the sequence and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity. Methods and computer programs used for alignment are well known in the art. One such computer program is "Align 2," written by Genentech, Inc., which was submitted with user documentation to the United States Copyright Office, Washington, DC 20559 on December 10, 1991.

[0357] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. An exemplary FcR is the naturally occurring human FcR. Furthermore, an FcR can be an FcR that binds to IgG antibodies (γ receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA (an "inhibitory receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activating motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The term "FcR" as used herein encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus.

[0358] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to cleave its target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to a molecule (e.g., an antibody) that has a complex with a homologous antigen. To assess complement activation, a CDC assay can be performed.

[0359] "Natural antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domains of the light chains are aligned with the first constant domain of the heavy chains, and the variable domains of the light chains are aligned with the variable domains of the heavy chains. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains.

[0360] The term "variability" refers to the significant sequence differences in certain portions of the variable domain between antibodies, contributing to the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, which primarily adopt a β-sheet configuration linked by three hypervariable regions. These hypervariable regions form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held together very closely by the FRs and, together with hypervariable regions from the other chain, contribute to the formation of the antibody's antigen-binding site. The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as antibody involvement in antibody-dependent cytotoxicity (ADCC).

[0361] When used herein, the term “hypervariant region” refers to the amino acid residues of the antibody responsible for antigen binding. Hypervariant regions typically comprise 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 light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., ibid.) and / or residues from the “hypervariant ring” (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain). The “frame region” or “FR” residues are those variable domain residues other than those in the hypervariant region as defined herein.

[0362] Antibody digestion with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking the antigen.

[0363] "Fv" is the smallest antibody fragment containing both complete antigen recognition and antigen binding sites. This region consists of a dimer of a tightly non-covalently associated heavy chain variable domain and a light chain variable domain. The antigen binding site on the surface of the VH-VL dimer is defined by this configuration of three interacting hypervariable regions in each variable domain. These six hypervariable regions collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three antigen-specific hypervariable regions) can recognize and bind antigens, although its affinity is lower than the entire binding site.

[0364] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. In this paper, the Fab' fragment in which the cysteine ​​residues of the constant domain have at least one free thiol group is referred to as Fab'-SH. F(ab')2 antibody fragments were initially generated as Fab' fragment pairs with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0365] Based on the amino acid sequence of their constant structural domains, the “light chains” of antibodies from any vertebrate species can be classified into one of two distinct types (called κ(.κ.) and λ(.λ.)).

[0366] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, which are contained within a single polypeptide chain. The Fv polypeptide may also contain a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0367] The term "dual antibody" refers to a small antibody fragment with two antigen-binding sites, contained within a variable heavy domain (VH) linked to a variable light domain (VL) on the same polypeptide chain (VH-VL). By using linkers that are too short to pair between the two domains on the same chain, these domains are forced to pair with complementary domains on the other chain, creating two antigen-binding sites.

[0368] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Humanization is a method of transferring mouse antigen-binding information to a non-immunogenic human antibody receptor and has yielded many therapeutically useful drugs. Humanization generally begins by transferring all six mouse complementarity-determining regions (CDRs) onto a human antibody framework. Antibodies with these CDRs typically do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. In addition to the CDRs, selected non-human antibody framework residues must be incorporated to maintain the correct CDR conformation. It has been shown that transferring key mouse framework residues to the human receptor to support the structural conformation of the transplanted CDRs can restore antigen binding and affinity. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the receptor's hypervariable region are replaced by hypervariable region residues from non-human species (donor antibodies) such as mice, rats, rabbits, or non-human primates with the desired specificity, affinity, and ability. In some cases, the framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the receptor or donor antibody. These modifications are made to further improve antibody performance. Generally, humanized antibodies will contain substantially all, and typically both, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins and all or substantially all of the FRs are those of human immunoglobulin sequences. Optionally, humanized antibodies will also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region of human immunoglobulins.

[0369] "Isolated" antibodies are antibodies that have been identified, isolated, and / or recovered from components of their native environment. Contaminating components of their native environment are substances that can interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteins or non-protein solutes. In some respects, the antibody will be purified to (1) greater than 95% by weight or greater than 99% by weight as determined by the Lowry method, (2) to the extent that at least 15 N-terminal or internal amino acid sequence residues can be obtained using a gas-phase protein sequencer, or (3) to achieve homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include recombinant intracellular in situ antibodies, as at least one component of the antibody's native environment will be absent. However, isolated antibodies are typically prepared via at least one purification step.

[0370] "Cancer" refers to a broad category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. As used in this article, "cancer" refers to primary, metastatic, and recurrent cancers.

[0371] As used herein, the term "immune response" refers to the biological response within a vertebrate to foreign factors that protect the organism against these factors and the diseases they cause. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation within the vertebrate body. Immune responses include, for example, T cells (e.g., effector T cells or Th cells, such as CD4 cells). + or CD8 + The activation or suppression of T cells, or the suppression of Treg cells. As used herein, the terms “T cell” and “T lymphocyte” are interchangeable and refer to any lymphocyte produced or processed by the thymus. In some respects, T cells are CD4+ T cells. In some respects, T cells are CD8+ T cells. In some respects, T cells are NKT cells.

[0372] "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 respects, the subject is human. The terms "subject" and "patient" are used interchangeably herein.

[0373] The term "therapeutic effective amount" or "therapeutic effective dose" refers to the amount of an agent (e.g., a novel degrading agent or novel degrading agent conjugate disclosed herein) that provides a desired biological, therapeutic, and / or preventive outcome. This outcome can be a reduction, improvement, mitigation, alleviation, delay, and / or relief of one or more signs, symptoms, or causes of disease, or any other desired alteration of a biological system. Regarding solid tumors, an effective amount includes amounts sufficient to cause tumor shrinkage and / or a decrease in the tumor growth rate (e.g., inhibition of tumor growth) or to prevent or delay other unwanted cell proliferation. In some respects, an effective amount is an amount sufficient to delay tumor development. In some respects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. Therefore, an effective amount can be administered once or multiple times. For example, an effective amount of the composition may, for example, (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) inhibit, delay, slow down and prevent the infiltration of cancer cells into surrounding organs to a certain extent; (iv) inhibit (i.e. slow down and prevent tumor metastasis to a certain extent); (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent.

[0374] In some respects, a “therapeutic effective dose” is the amount of a novel degrading agent or a novel degrading agent conjugate that has been clinically proven to significantly reduce or slow the progression (regression) of cancers such as advanced solid tumors. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0375] As used herein, the term "standard of care" refers to a treatment approach that is accepted by medical experts as appropriate for a particular type of disease and is widely used by healthcare professionals. This term may be used interchangeably with any of the following terms: "best practice," "standard medical care," and "standard therapy."

[0376] For example, "anticancer agents" promote cancer regression or prevent further tumor growth in test subjects. In some cases, therapeutically effective doses of the drug promote cancer regression to the point of elimination.

[0377] The terms "effectiveness" and "efficacy" in treatment encompass both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to a drug's ability to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (side effects) at the cellular, organ, and / or organismal level caused by drug administration.

[0378] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins regulate the activation or function of T cells. Many checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86, and PD-1 and its ligands PD-L1 and PD-L2. Pardoll, DM, Nat Rev Cancer 12(4):252-64 (2012). These proteins are responsible for co-stimulatory or inhibitory interactions in T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or antibody-derived antibodies.

[0379] The term "treatment" refers to therapeutic treatment and preventative or preventive measures aimed at preventing or mitigating undesirable physiological changes or conditions, such as the development or spread of cancer. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of the disease state (i.e., non-deterioration), delay or slowing of disease progression, improvement or mitigation of the disease state, and elimination (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean extended survival compared to expected survival without treatment. Those requiring treatment include those already suffering from a condition or disease, those susceptible to developing a condition or disease, and those seeking to prevent a condition or disease.

[0380] II. New Degrading Agents

[0381] This disclosure provides a new degrading agent of formula (II):

[0382]

[0383] Or its pharmaceutically acceptable salt, wherein:

[0384] A is a phenyl group or a C4-C4 group. 10 cycloalkyl rings;

[0385] U is selected from NH and CF2;

[0386] R 1 Independently selected from hydrogen and halogroups;

[0387] R 2Selected from -C(O)R 3 -N(R) 4 2、-(CH2) n OH, -(CH2) n SH, -(CH2) n N(R 4 2、-(CH2) n Q'(CH2) m OH, -(CH2) n Q'(CH2) m SH and -(CH2) n Q'(CH2) m N(R 4 )2; of which

[0388] R 3 It is hydrogen or C1-C6 alkyl;

[0389] Each R 4 Independently hydrogen or C1-C6 alkyl;

[0390] Q' is O, S, or NR 4 ;

[0391] n is 1-6; and

[0392] m is 2-5;

[0393] The premise is that when R 2 For NH2, -(CH2) n NH2 or -(CH2) n When OH, then R 1 It is a halogenated group.

[0394] In some respects, this disclosure provides compounds of formula (II) or pharmaceutically acceptable salts thereof, wherein:

[0395] A is a phenyl ring or a C4-C ring. 10 cycloalkyl rings;

[0396] U is NH;

[0397] R 1 Selected from hydrogen and halogroups;

[0398] R 2 Selected from -(CH2) n Q'(CH2) m N(R 4 2、-(CH2) n OH, -(CH2) n SH, -N(R) 4 )2 and -C(O)R 3 ;in:

[0399] m is 2;

[0400] n is 2;

[0401] Q' is -O-;

[0402] R 3 It is methyl; and

[0403] Each R 4 Independently selected from hydrogen and methyl;

[0404] The premise is that when R 2 It can be NH2 or -(CH2). n When OH, then R 1 It is a halogenated group.

[0405] As used herein, the term “C1-C6 alkoxy” refers to a C1-C6 alkyl group that is attached to a portion of the parent molecule via an oxygen atom.

[0406] As used herein, the term “C1-C6 alkoxy-C1-C6 alkyl” refers to a C1-C6 alkoxy group that is attached to a parent molecule moiety via a C1-C6 alkyl group.

[0407] As used herein, the term "C1-C6 alkyl" refers to a group derived from a straight-chain or branched saturated hydrocarbon containing one to six carbon atoms.

[0408] As used in this article, the term "C4-C" 10 "Cycloalkyl" refers to a saturated monocyclic hydrocarbon ring system having four to ten carbon atoms and zero heteroatoms. Representative examples of cycloalkyl include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyls containing seven to ten atoms can be monocyclic or fused, spirocyclic, or bridged bicyclic structures.

[0409] As used in this article, the term "halogenated group" refers to F, Cl, Br, or I.

[0410] In some respects, the new degrading agent of formula (II) is a compound selected from the group consisting of:

[0411]

[0412]

[0413] In some respects, the new degrading agent of formula (II) is

[0414]

[0415] In some respects, the new degrading agent of formula (II) is

[0416]

[0417] In some respects, the new degrading agent of formula (II) is

[0418]

[0419] In some respects, the new degrading agent of formula (II) is

[0420]

[0421] In some respects, the new degrading agent of formula (II) is

[0422]

[0423] In some respects, the new degrading agent of formula (II) is

[0424]

[0425] In some respects, the new degrading agent of formula (II) is

[0426]

[0427] In some respects, the new degrading agent of formula (II) is

[0428]

[0429] In some respects, this disclosure provides novel degrading agents of formula (II) or pharmaceutically acceptable salts thereof, wherein A is phenyl; U is NH; R 1 It is a halogenated group; and R 2 -(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is 0, and an R 4 It is hydrogen and the others are methyl.

[0430] In some respects, this disclosure provides a novel degrading agent of formula (II), wherein A is phenyl; U is NH; R 1 It is a halogenated group; and R 2 -(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is 0, and each R 4 It is a methyl group.

[0431] In some respects, this disclosure provides a novel degrading agent of formula (II), wherein A is phenyl; U is NH; R 1 It is a halogenated group; and R 2 -(CH2) nOH, where n is 2.

[0432] In some respects, this disclosure provides a novel degrading agent of formula (II), wherein A is phenyl; U is NH; R 1 It is a halogenated group; and R 2 -(CH2) n SH, where n is 2.

[0433] In some respects, this disclosure provides a novel degrading agent of formula (II), wherein A is phenyl; U is NH; R 1 It is hydrogen; and R 2 -N(R) 4 )2, one of which is R 4 It is hydrogen and the others are methyl.

[0434] In some respects, this disclosure provides a novel degrading agent of formula (II), wherein A is phenyl; U is NH; R 1 It is a halogenated group; and R 2 -N(R) 4 )2, where each R 4 For hydrogen. In some aspects, this disclosure provides a novel degrading agent of formula (II), wherein A is phenyl; R is hydrogen. 1 It is hydrogen; and R 2 -C(O)R 3 , where R 3 It is a methyl group.

[0435] In some respects, this disclosure provides a novel degrading agent of formula (II), wherein A is C4-C 10 cycloalkyl ring; U is NH; R 1 It is hydrogen; and R 2 -(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is 0, and an R 4 It is hydrogen and the others are methyl.

[0436] III. New Degrading Agent Conjugates

[0437] This disclosure provides conjugates of one or more novel degradative agents and binding moieties disclosed herein. These conjugates can promote the degradation of CRL4 by binding to cerebellar protein (CRBN). CRBN E3 ubiquitin ligases mediate the recruitment and ubiquitination of substrate proteins to degrade proteins. These agents act as "molecular glues," filling the binding interface as hydrophobic patches that reprogram the protein interactions between the ligase and the new substrate.

[0438] In some respects, this disclosure provides compounds of (I),

[0439]

[0440] Or its pharmaceutically acceptable salt, wherein:

[0441] a is an integer from 1 to 10;

[0442] A is a phenyl group or a C4-C4 group. 10 cycloalkyl rings;

[0443] R 1 Selected from hydrogen and halogroups;

[0444] U is selected from NH and CF2;

[0445] X is selected from -NR 2 -、=C(CH3)-、-Q-(CH2) n - and -Q(CH2) m Q'(CH2) n -;in:

[0446] Q and Q' are each independently O, S, or NR. 2 ;

[0447] R 2 It is hydrogen or C1-C6 alkyl;

[0448] n is an integer from 1 to 6;

[0449] m is an integer from 2 to 6;

[0450] Each group is connected to L on the left and to A on the right;

[0451] This assumes that X is NH or -Q-(CH2). n - At that time, R 1 It is a halogenated group;

[0452] L represents a cuttable or non-cuttable joint; and

[0453] Bm is the connecting part.

[0454] In some respects, U is NH.

[0455] In some aspects, the novel degrading agent conjugates described herein possess in vitro antiproliferative activity against tumor cell lines. In some aspects, the novel degrading agent conjugates comprising a novel degrading agent and a binding moiety exhibit in vitro antiproliferative activity that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% higher than that of the novel degrading agent alone or the binding moiety alone. In some aspects, the novel degrading agent conjugates comprising a novel degrading agent and a binding moiety exhibit in vitro antiproliferative activity that is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times higher than that of the novel degrading agent alone or the binding moiety alone.

[0456] In some aspects, the novel degradation agent conjugates described herein exhibit in vitro antiproliferative activity against the BT-474 breast cancer cell line, for example, higher antiproliferative activity against the BT-474 breast cancer cell line compared to the novel degradation agent alone or the conjugate alone. In some aspects, the novel degradation agent conjugates described herein exhibit in vitro antiproliferative activity against the SK-BR-3 breast cancer cell line, for example, higher antiproliferative activity against the SK-BR-3 breast cancer cell line compared to the novel degradation agent alone or the conjugate alone. In some aspects, the novel degradation agent conjugates described herein exhibit in vitro antiproliferative activity against the NCI-N87 gastric cancer cell line, for example, higher antiproliferative activity against the NCI-N87 gastric cancer cell line compared to the novel degradation agent alone or the conjugate alone. In some aspects, the novel degradation agent conjugates described herein exhibit in vitro antiproliferative activity against the Daudi lymphoma cell line, for example, higher antiproliferative activity against the Daudi lymphoma cell line compared to the novel degradation agent alone or the conjugate alone. In some respects, the novel degradation agent conjugates described herein exhibit in vitro antiproliferative activity against the HL-60 acute myeloid leukemia cell line, for example, higher antiproliferative activity against the HL-60 acute myeloid leukemia cell line compared to the novel degradation agent alone or the conjugate alone. In some respects, the novel degradation agent conjugates described herein exhibit in vitro antiproliferative activity against the Ramos non-Hodgkin lymphoma cell line, for example, higher antiproliferative activity against the Ramos non-Hodgkin lymphoma cell line compared to the novel degradation agent alone or the conjugate alone. In some respects, the novel degradation agent conjugates described herein are able to maintain their antiproliferative activity in the presence of human serum. The novel degradation agent conjugates described herein may be used for the treatment of cancer.

[0457] III.A. Connector

[0458] The novel degrading agent disclosed herein can be connected to the binding portion via a connector. As used herein, the term "connector" refers to any chemical part capable of connecting the binding portion (Bm) to a group X in a compound of formula (I).

[0459] In some respects, the linker may contain a heterobifunctional group. In this disclosure, the term "heterobifunctional group" refers to the chemical portion that connects the linker to the binding site, wherein the linker is part of the binding site. A heterobifunctional group is characterized by having different reactive groups at either end of the chemical portion. Connection to "Bm" can be achieved through chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves a controlled reaction of an accessible amino acid residue on the surface of the binding site with a reaction handle on the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide conjugation, cysteine ​​conjugation, and conjugation via non-natural amino acids incorporated through genetic engineering, wherein a non-natural amino acid residue having the desired reaction handle is mounted on "Bm". In enzymatic conjugation, an enzyme mediates the conjugation of the linker to an accessible amino residue on the binding site. Examples of enzymatic conjugation include, but are not limited to, transpeptides using sorting enzymes, transpeptides using microbial transglutaminase, and N-glycan engineering. Chemical and enzymatic conjugation may also be used sequentially. For example, enzymatic conjugation can also be used to mount unique reaction handles on “Bm” for subsequent chemical conjugation.

[0460] In some respects, heterodifunctional groups are selected from:

[0461]

[0462] in

[0463] For the connection point with the remaining part of the connector; and

[0464] This is the connection point with Bm.

[0465] In some respects, the connector “L” is indestructible. As used herein, the term “indestructible connector” refers to any chemical part that can stably, covalently attach the binding portion to a novel degrading agent and does not fall into the category of “cuttable connector” as defined herein. Thus, an indestructible connector is substantially resistant to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. “Substantially resistant to cleavage” means that the chemical bonds in or adjacent to the connector in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the antibody novel degrading agent conjugate group remain uncut by acids, photoinstantaneous cleaving agents, bioreductive agents, peptidases, esterases, or chemical or physiological compounds that cleave the chemical bonds (e.g., disulfide bonds) in a cuttable connector, after continuous treatment with any of the aforementioned agents for several hours to several days. In some respects, under conditions where the new degrading agent and / or binding moiety can remain active, the linker is insensitive to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, enzymatic cleavage, etc. ADC catabolites generated from cleavable linkers contain residual amino acids from the antibody. These catabolites can exhibit unique and unexpected properties in the target cells to which they are delivered.

[0466] Those skilled in the art will readily distinguish between non-cuttable joints and cuttable joints.

[0467] Examples of non-cuttable joints include, but are not limited to, SMCC (4-(N-maleimide methyl)cyclohexane-1-carboxylic acid succinimide) joints, succinimide sulfide joints, and joints such as the following:

[0468]

[0469] in:

[0470] p is an integer from 1 to 10;

[0471] Let X be the connection point with X; and

[0472] This refers to the connection point with the joint portion.

[0473] In some respects, the connector is:

[0474]

[0475] In some respects, p is 5.

[0476] In some respects, the adapter can be cleavable. In some respects, the adapter can be sensitive to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, enzymatic cleavage, etc., provided that the new degrading agent and / or binding site can remain active.

[0477] In some respects, cleavable adapters can be enzymatically cleaved. In other respects, cleavable adapters can be cleaved by proteases, peptidases, esterases, β-glucuronidases, glycosidases, phosphodiesterases, phosphatases, pyrophosphatases, or lipases.

[0478] In some respects, cleavable linkers can be cleaved by proteases. Examples of proteases include, but are not limited to, cathepsin B and VAGP tetrapeptide.

[0479] In some respects, the cleavable linker contains a peptide. In other respects, the peptide serves as the cleavage site of the linker, thereby promoting drug release upon exposure to intracellular proteases such as lysosomal enzymes. Peptides can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasminogen activator. Examples of peptides having two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-alanine (val-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides having three amino acids include, but are not limited to, gly-valine-citrulline (gly-val-cit), aspartic-valine-citrulline (asp-val-cit), alanine-alanine-asparagine (ala-ala-asn), alanine-phenylalanine-lysine (ala-phe-lys), gly-gly-phenylalanine (gly-gly-phe), and gly-gly-gly (gly-gly-gly). Examples of peptides having four amino acids include, but are not limited to, gly-gly-valine-citrulline (gly-gly-val-cit) and gly-gly-phenylalanine-gly (gly-gly-phe-gly). The above amino acid combinations can also exist in the reverse order (i.e., cit-val).

[0480] The peptides disclosed herein may comprise 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-" indicates an amino acid having a "D" (dextrorotatory) configuration, the opposite of the configuration of naturally occurring ("L-") amino acids. The amino acids described herein are commercially available (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0481] In some respects, the adapter (“L”) is a protease-cleavable adapter selected from the following

[0482]

[0483] in:

[0484] q is an integer from 2 to 10;

[0485] Z 1 Z 2 Z 3 and Z 4 Each naturally occurring amino acid residue, either independently absent or in L- or D-configuration, is provided that Z 1 Z 2 Z 3 and Z 4 At least two of them are amino acid residues;

[0486] Let X be the connection point with X; and

[0487] This refers to the connection point with the joint portion.

[0488] In some respects, Z 1 Z 2 Z 3 and Z 4 The following groups are independently absent or selected from: 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 Z 1 Z 2 Z 3 and Z 4 At least two of them are amino acid residues.

[0489] In some respects, Z 1 It may be absent or contain glycine; Z 2 It is absent or selected from L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 Selected from L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; and Z 4 It is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine and glycine.

[0490] In some respects, L is

[0491]

[0492] In some respects, q is 5.

[0493] In some respects, L is a pyrophosphatase-cleavable linker.

[0494] In some respects, L is a pyrophosphatase-cleavable linker, which is:

[0495]

[0496] in:

[0497] q is an integer from 2 to 10;

[0498] Let X be the connection point with X; and This refers to the connection point with the joint portion.

[0499] In some respects, L is a β-glucuronidase-mediated cleavage linker.

[0500] In some respects, L is a β-glucuronidase-cleavable linker selected from the following:

[0501]

[0502] in:

[0503] q is an integer from 2 to 10;

[0504] ----Does not exist or is a key;

[0505] Let X be the connection point with X; and

[0506] This refers to the connection point with the joint portion.

[0507] In some respects, the linker is bioreducible. Bioreducible linkers utilize the difference in reduction potential between intracellular compartments and plasma. Tumor cells contain 1000 times more reduced glutathione in their cytoplasm than normal cells, and tumor cells also contain enzymes that contribute to compartment reduction. The linker remains intact during systemic circulation and is selectively cleaved by high intracellular glutathione concentrations, releasing the active drug at the tumor site via a non-toxic prodrug.

[0508] In some respects, L is selected from the following bioreducible adapters:

[0509]

[0510] in:

[0511] q is an integer from 2 to 10;

[0512] R, R', R” and R'” are each independently selected from hydrogen, C1-C6 alkoxy-C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or the two geminal R groups together with the carbon atoms to which they are attached can form a cyclobutyl or cyclopropyl ring;

[0513] Let X be the connection point with X; and

[0514] This refers to the connection point with the joint portion.

[0515] In some respects, the connector is acid-cuttable. Acid-cuttable connectors are specifically designed to remain stable at the neutral pH of the bloodstream, but can hydrolyze in the acidic environment of the cellular compartment, releasing cytotoxic drugs.

[0516] In some respects, L is selected from the following acid-cuttable connectors.

[0517]

[0518] in:

[0519] q is an integer from 2 to 10;

[0520] Let X be the connection point with X; and

[0521] This refers to the connection point with the joint portion.

[0522] In some respects, where L is a click-release connector, the release of the new degrading agent is chemically triggered by tetrazine or related compounds.

[0523] In some respects, L is selected from the following click-release connectors.

[0524]

[0525] in:

[0526] q is an integer from 2 to 10;

[0527] Let X be the connection point with X; and

[0528] This refers to the connection point with the joint portion.

[0529] III.B. Combination Part

[0530] This disclosure provides novel degradative agents conjugated with binding moieties. As used herein, the term "binding moiety" refers to any molecule that recognizes and binds to cell surface markers or receptors. In some aspects, binding moieties bind proteins, not limited to polypeptide moieties. In addition to targeting specific cells, tissues, or sites, binding moieties may also have certain therapeutic effects, such as antiproliferative (cell-inhibiting and / or cytotoxic) activity against target cells or pathways. In some aspects, binding moieties may comprise or be engineered to include at least one chemically reactive group such as a carboxylic acid, amine, thiol, or chemically reactive amino acid moieties or side chains. In some aspects, binding moieties may include a targeting moiety for a given target cell population that binds to or complexes with cell surface molecules, such as cell surface receptors or antigens. Upon receptor-specific binding or complexation, cells are allowed to take up the targeting moiety or novel degradative agent conjugate and then internalize it into the cell.

[0531] In some respects, the "Bm" group can be a portion that specifically binds to molecules on the cell surface. In other respects, the "Bm" group can be a peptide or protein that binds to cell surface receptors or antigens.

[0532] In some respects, the group "Bm" can be an antibody, an antibody fragment, or an antigen-binding fragment. An antibody is a protein produced by the immune system capable of recognizing and binding to specific antigens. Target antigens typically have many binding sites, also called epitopes, recognized by CDRs on various antibodies. Each antibody that specifically binds to different epitopes has a different structure. Therefore, an antigen can have more than one corresponding antibody. The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies, single-domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. Antibodies can be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or antibodies derived from other species.

[0533] Monoclonal antibodies that can be conjugated to novel degrading agents are a homogeneous group of antibodies targeting specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against antigens of interest can be prepared using any techniques known in the art, which generate antibody molecules from continuous cell lines in culture. These techniques include, but are not limited to, hybridoma techniques, human B-cell hybridoma techniques, and EBV-hybridoma techniques. Such antibodies can be any class of immunoglobulins, including IgG, IgM, IgE, IgA, and IgD, and any subclasses thereof. Hybridomas that produce mAbs used in this disclosure can be cultured in vitro or in vivo.

[0534] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be prepared using any of a variety of techniques known in the art.

[0535] Antibodies can also be bispecific antibodies. Methods for preparing bispecific antibodies are known in the art. Conventional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities. Due to the random allocation of the immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, typically using affinity chromatography steps, is quite cumbersome and results in low product yields.

[0536] Depending on the method, an antibody variable domain (antibody-antigen binding site) with desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion may be performed with the immunoglobulin heavy chain constant domain (including at least a portion of the hinge, C.sub.H2, and C.sub.H3 regions). The first heavy chain constant region (C.sub.H1) may contain the site necessary for light chain binding and is present in at least one fusion. Nucleic acids having sequences encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the relative proportions of the three polypeptide fragments in all aspects when unequal ratios of the three polypeptide chains used for construction provide optimal yield. However, when expression of at least two polypeptide chains at equal ratios results in high yield, or when the ratios are not particularly significant, the coding sequences of two or all three polypeptide chains can be inserted into a single expression vector.

[0537] Bispecific antibodies may have a hybrid immunoglobulin heavy chain with first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing second binding specificity) in the other arm. This asymmetric structure facilitates the isolation of the desired bispecific compound from unwanted combinations of immunoglobulin chains, as the presence of the immunoglobulin light chain in only half of the bispecific molecule provides a convenient method of separation. Using such techniques, bispecific antibodies can be prepared for conjugation with novel degrading agents in the treatment or prevention of diseases as defined herein.

[0538] Hybrid or bifunctional antibodies can be biologically derived, i.e., through cell fusion techniques, or chemically derived, especially with cross-linking agents or disulfide bridging agents, and can contain complete antibodies or fragments thereof.

[0539] Antibodies can be functionally active fragments, derivatives, or analogs of antibodies that specifically bind to cancer cell antigens, viral antigens, or microbial antigens, or other antibodies that bind to tumor cells or the matrix. In this respect, "functional activity" means that the fragment, derivative, or analog can elicit an anti-anti-idiotype antibody that recognizes the same antigen as the antigen recognized by the antibody from which the fragment, derivative, or analog is derived. Specifically, in an exemplary aspect, the antigenicity of an immunoglobulin molecule's idiotype can be enhanced by deleting the C-terminal frame and the CDR sequence of a CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind to the antigen, a synthetic peptide containing the CDR sequence can be used for an antigen-binding assay using any binding assay method known in the art.

[0540] Other useful antibodies include antibody fragments, such as, but not limited to: the F(ab')2 fragment, which contains a variable region, a light chain constant region, and a heavy chain CH1 domain, which can be produced by digesting antibody molecules with pepsin; and the Fab fragment, which can be generated by reducing the disulfide bridge of the F(ab')2 fragment. Other useful antibodies are heavy and light chain dimers of antibodies, or any of their smallest fragments, such as Fvs or single-chain antibodies (SCAs), or any other molecule with the same specificity as the antibody.

[0541] Additionally, recombinant antibodies prepared using standard recombinant DNA techniques, such as chimeric and humanized monoclonal antibodies containing both human and non-human portions, are useful antibodies. Chimeric antibodies are molecules in which different portions originate from different animal species, such as those having a variable region derived from a mouse monoclonal antibody and a constant region from human immunoglobulins. Humanized antibodies are antibody molecules derived from non-human species that have one or more complementarity-determining regions (CDRs) from the non-human species and a framework region from human immunoglobulin molecules. Such chimeric and humanized monoclonal antibodies can be produced using recombinant DNA techniques known in the art.

[0542] Fully human antibodies can be produced using transgenic mice that do not express endogenous immunoglobulin heavy and light chain genes but do express human heavy and light chain genes. The transgenic mice are immunized in a normal manner with all or part of a selected antigen, such as the polypeptide of this disclosure. Monoclonal antibodies against the antigen can be obtained using conventional hybridoma techniques. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation, followed by class switching and somatic mutations. Therefore, using such techniques, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technique for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). Other antibodies are commercially available from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0543] A technique known as “guided selection” can be used to generate fully human antibodies that recognize a selected epitope. In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope. Human antibodies can also be generated using various techniques known in the art, including phage display libraries.

[0544] Antibodies can be fusion proteins of antibodies or functionally active fragments thereof, where an antibody is fused via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus to an amino acid sequence of another protein (or a portion thereof, such as at least 10, 20, or 50 amino acid segments of a protein) that is not an antibody. Antibodies or fragments thereof may be covalently linked to other proteins at the N-terminus of a constant domain.

[0545] Antibodies include modified analogues and derivatives, i.e., those covalently linked by any type of molecule, as long as such covalent linkage allows the antibody to maintain its antigen-binding immune specificity. For example, but not limited to, antibody derivatives and analogues include those that have been further modified, for example, by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage to cellular antibody units or other proteins. Any of many chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and metabolic synthesis in the presence of tunicamycin. Additionally, analogues or derivatives may contain one or more non-natural amino acids.

[0546] The antibodies in the novel degradative conjugates may include antibodies modified (e.g., substituted, deleted, or added) in amino acid residues that interact with the Fc receptor. Specifically, the antibodies include those modified in amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor. Antibodies specific to cancer cell antigens may be commercially available from, for example, Genentech (San Francisco, Calif.) or generated by any method known to those skilled in the art (such as, for example, chemical synthesis or recombinant expression techniques). The nucleotide sequences encoding antibodies specific to cancer cell antigens may be obtained, for example, from the GenBank database or similar databases, literature publications, or by routine cloning and sequencing.

[0547] In some respects, the antibody for the novel degrading agent conjugate can be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody, or a humanized antibody. In other respects, the antibody can be an antibody fragment, such as a Fab fragment.

[0548] Known antibodies for the treatment or prevention of cancer can be conjugated to the novel degrading agents described herein. Antibodies that are immune-specific against cancer cell antigens are commercially available or produced by any method known to those skilled in the art (such as, for example, recombinant expression techniques). The nucleotide sequences encoding antibodies that are immune-specific against cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by routine cloning and sequencing.Examples of antibodies that can be used to treat cancer include, but are not limited to, a humanized anti-HER2 monoclonal antibody for treating patients with metastatic breast cancer; RITUXAN.RTM. (rituximab; Genentech), a chimeric anti-CD20 monoclonal antibody for treating patients with non-Hodgkin's lymphoma; OvaRex (ozovozimab; AltaRex Corporation, MA), a murine antibody for treating ovarian cancer; Panorex (edrecolomab; Glaxo Wellcome, NC), a murine IgG sub.2a antibody for treating colorectal cancer; and Erbitux (cetuximab; Imclone Systems). The following are listed: EGFR IgG chimeric antibody (MedImmune, Inc., NY) for the treatment of epidermal growth factor-positive cancers such as head and neck cancer; Vitaxin (etaracizumab, MedImmune, Inc., MD) for the treatment of sarcoma; CamppathI / H (leukosite, MA) for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA) for the treatment of acute myeloid leukemia (AML); LymphoCide (epratuzumab, Immunomedics, Inc., NJ) for the treatment of non-Hodgkin's lymphoma; and SmartID 10 (Protein Design Labs, Inc., NY). The following are listed: Labs, Inc., CA, a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA), a radiolabeled mouse anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA), a humanized anti-CD2mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; Avastin (bevacizumab, Genentech, Inc., CA), an anti-VEGF humanized antibody for the treatment of lung and colorectal cancer; epazolizumab (Immunomedics, Inc., NJ and Amgen, CA), an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0549] Other antibodies that can be used in novel degrading agent conjugates include, but are not limited to, trastuzumab, gemtuzumab, pertuzumab, obbituzumab, oflamuzumab, daratumumab, STI-6129, lintuzumab, huMy9-6, belantanumab, indatuzumab, danutuximab, anti-CD38 A2 antibody, Huat 13 / 5H3s antibody, teimozumab, tosimoumab, panitumumab, trimemumab, teimozumab, caputuximab, and vetuzumab. In some respects, the antibody is selected from rituximab, trastuzumab, pertuzumab, OR000213, lintuzumab, and gemtuzumab.

[0550] Other antibodies that can be used in novel degradation agent conjugates include, but are not limited to, antibodies against the following antigens: CA125 (ovary), CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), Lewis Y (cancer), Lewis X (cancer), alpha-fetoprotein (cancer), and CA. 242 (colorectal), placental alkaline phosphatase (cancer), prostate-specific antigen (prostate), prostate acid phosphatase (prostate), epidermal growth factor (cancer), MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE-4 (cancer), anti-transferrin receptor (cancer), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphoma), CD20 (non-Hodgkin lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (cancer), CD38 (multiple myeloma), CD40 (lymphoma), mucin (cancer), P21 (cancer), MPG (melanoma), and Neu oncogene product (cancer). Some specific useful antibodies include, but are not limited to, BR96 mAb (Trail, PA et al., Science (1993) 261, 212-215), BR64 (Trail, PA et al., Cancer Research (1997) 57, 100-105), mAbs targeting the CD40 antigen such as S2C6 mAb (Francisco, JA et al., Cancer Res. (2000) 60: 3225-3231), mAbs targeting the CD70 antigen such as 1F6 mAb, and mAbs targeting the CD30 antigen such as AC10. Many other internalizing antibodies that bind to tumor-associated antigens can be used, and these have been reviewed.

[0551] Other antigens that the conjugates of this invention can bind to include, but are not limited to, 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, 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, and 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, condensation factor, cKit, closure protein 3, closure protein 18.2, CLDN6, CLEC12A, CL L-1, cll3, c-MET, Cripto protein, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2E) TS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose 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, integrin (including α4, αvβ3, αvβ5, αvβ6, α1β4, α4β1, α4β7, α5β1, α6β4, αIIbβ3 integrin), integrin αV, intestinal carboxyesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, podin, LewisY, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mucin hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagoguerin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzymes, prostate cancer cells, prostaglandins, copper *Pseudomonas aeruginosa*, rabies virus, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, respiratory syncytial virus, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinogen C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2, TIM-1, TnAg, 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.

[0552] 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 novel degrading agents.

[0553] Antibodies of novel degradation agent conjugates can bind to either receptors or receptor complexes expressed on activated lymphocytes. Receptors or receptor complexes may include members of the immunoglobulin gene superfamily, members of the TNF receptor superfamily, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins, or complement control proteins. Non-restricted examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152 / CTLA-4, PD-1, and ICOS. Non-restricted examples of suitable TNF receptor superfamily members are CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotein, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Suitable, non-limiting examples of integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Suitable, non-limiting examples of lectins are C-type, S-type, and I-type lectins.

[0554] In some respects, antibodies that can be used in this disclosure include, but are not limited to, 3F8, 8H9, abavomab, and abciximab. Abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab Adacumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacizumab, ALD518, alenumab alirocumab Atumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrukinzumab, apolizumab, aprutumab, and arcitumomab Ascrinvacumab, acelizumab, atidortoxumab, atlizumab, and tocilizumab Atezolizumab Atinumab, Aterolimumab, Avelumab (Bavencio), Azintuxizumab, Belantan, Bapineuzumab, Basiliximab Bavituximab, BCD-100, and bectumomab Begelomab, belantamab, and belimumab Bemarituzumab and benralizumab Bermekimab, bersanlimab, bertilimumab, and besilesomab Bevacizumab bezlotoxumab Bicitumab Bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab, briakinumab, brodalumab (SILIQ) TM ), brolucizumab ( brontictuzumab and burosumab Cabirazine (cabiralizumab) and Caplacizumab (caplacizumab) Camidanlumab, Camrelizumab, and Canakinumab Cantuzumab, capromab, carlumab, carotuximab, catumaxomab cBR96, CC49, cedelizumab, cemiplimab Cergutuzumab, certrelimab, certolizumab, and cetuximab. Cibisatamab, Cirntuzumab, Citatuzumab, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab, Codrituzumab, Cofetuzumab, Coltuximab, Conatumumab, Concizumab, Cosfroviximab, CR6261, Crenezumab, Crizanlizumab Crotedumab, Cusatuzumab, Dacetuzumab, Daclizumab dalotuzumab, dapirolizumab, daratumumab Dectrekumab, demcizumab, denintuzumab, denosumab Depatuxizumab, derlotuximab, detumomab, dezamizumab, and dinutuximab. Diridavumab, domagrozumab, dostarlimab, dorlimomab, dolixizumab, drozitumab, DS-8201, duligotuzumab, dupilumab durvalumab dosigitumab, ecromeximab, and eculizumab Edobacomab and edrecolomab efalizumab efungumab Eldelumab, elezanumab, elgemtumab, and elotuzumab. elsilimomab, emactuzumab, and emapalumab emibetuzumab and emicizumab enapotamab, enavatuzumab, enfortumab Enlimomab, Enoblituzumab, Enokizumab, Enoticumab, Enxituximab, Epitumomab, Eptinezumab epratuzumab and erenumab erlizumab and ertumaxomab etaracizumab Itigilimab, etrolizumab, evinacumab, evolocumab Exbivirumab and Fanolesomab Varalimomab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab Foralumab, Foravirumab, and Fremanezumab Fresolimumab, frovocimab, frunevetmab, fulranumab, futuximab, and galcanezumab. Galiximab, gancotamab, ganitumab, gantenerumab, gavilimomab, gedivumab, gemtuzumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab, and golimumab are all names of Chinese herbal medicines. Gomiliximab and guselkumab huMy9-6, OR000213, ianalumab, ibalizumab IBI308, ibritumomab, icrucumab, idarucizumab Ifabotuzumab, igovomab (INDIMACIS-125), idatuzumab, IMAB362, imalumab, imaprelimab, and imciromab. Imgatuzumab, inclacumab, indatuximab, indusatumab, inebilizumab, and infliximab. Intetumumab, inolimomab, inotuzumab, iomab-B, ipilimumab, iratumumab, isatuximab Icalcimab, Istiratumab, Itolizumab, Ixekizumab keliximab and labetuzumab (CEA-CIDE) TM ), lacnotuzumab, ladiratuzumab, lampalizumab, lanadelumab Landogrozumab, Lapriximab, Larcaviximab, Lebrikizumab, Lemalesomab, Lendalizumab, Lenvervimab, Lenzilumab, Lerdelimumab, Leronlimab, Lesofavumab, Letolizumab, Lexatumumab, Libivirumab, Lifastuzumab, Liligelizumab, Lilotomab, Lintozumab, Lirilumab The following are listed: lodelcizumab, lokivetmab, loncastuximab, lorvotuzumab, losatuxizumab, lucatumumab, lulizumab, lumiliximab, lumretuzumab, lupartumab, lutikizumab, mapatumumab, margetuximab, marstacimab, maslimomab, matuzumab, mavrilimumab, and mepolizumab. Metelimumab, Milatuzumab, Minretumomab, Mirikizumab, Mivituximab, Mitumomab, Modotuximab, Molalizumab, Mogamulizumab Morolimumab, mosunetuzumab, and motavizumab Moxetumomab Muromonab-CD3 (ORTHOCLONE) Nacolomab, Namilumab, Naaptumomab, Naratuximab, Namatumab, and Natalizumab are among the following: Navicixizumab, navivumab, naxitamab, nebacumab, necitumumab Nemolizumab, NEOD001, nerelimomab, nesvacumab, netakimab, nimotuzumab Nirsevimab, nivolumab, nofetumomab, and obiltoxaximab Obinutuzumab, ocaratuzumab, ocrelizumab Odulimomab and ofatumumab olaratumab Oleclumab, olendalizumab, olokizumab, omalizumab Omburtamab, OMS721, onartuzumab, ontecizumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab, oregovomab (OVAREX), orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab pamrevlumab and panitumumab Pankomab, panobacumab, parsatuzumab, pascolizumab, pastotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab Perakizumab and pertuzumab Pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, plozalizumab (Polivy), prezalumab, plozalizumab, pogalizumab, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO 140. Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranevetmab, Ranibizumab Ravigalimab and ravulizumab Raxibacumab, refanezumab, regavirumab, REGN-EB3, renatlimab, remtolumab, and reslizumab. Rituximab, rinucumab, and risankizumab Rituximab Rivarabazumab, rmab, robatumumab, roledumab, romilkimab, romosozumab rontalizumab, rosmantuzumab, rovalpituzumab, rovelizumab Rozanolixizumab, ruplizumab (ANTOVA), SA237, sacituzumab, samalizumab, samrotamab, and sarilumab. Satralizumab, satumomab pendetide, and secukinumab Selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, SGN-CD19A, SHP647, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizu (Mab), sirtratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepizumab, sontuzumab, spartalizumab, stamulumab, STI-6129, sulesomab Suttavumab, Sutimlimab, Suvizumab, Suvratoxumab, Tabalumab, Tacatuzumab Tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab, taplitumomab paptox, tarextumab, tavolimab, tefibazumab Atemomab, telisotuzumab, tesidolumab, tetraxetan, tetulomab, tenatumomab, teneliximab, teprotumumab Teplizumab, tezepelumab, TGN1412, tibulizumab, and ticilimumab. Ticarutuzumab, timigutuzumab, timolumab, tiragolumab, tiragotumab, tislelizumab, tisotumab, tiuxetan, tildrakizumab TNX-650, tocilizumab (atlizumab) Tomuzotuximab, toralizumab, tosatoxumab, and tositumomab are some of the drugs mentioned. tovetumab, tralokinumab, trastuzumab TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab Ulituximab, ulocuplumab, urelumab, utomilumab, vadastuximab, vanalimab, vandortuzumab, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vaporizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab vobarilizumab and volociximab vonlerolizumab, vopratelimab, vorsetuzumab, votumumab, vunakizumab, xentuzumab, XMAB-5574, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), zatuximab, zenocutuzumab, ziralimumab, zolbetuximab, or zolimomab.

[0555] An antibody that "binds" to a molecular target or antigen of interest is an antibody that can bind to the antigen with sufficient affinity so that the antibody can be used to target cells that express the antigen.

[0556] In this disclosure, the group "Bm" may be conjugated with more than one new degrading agent. In some aspects, "Bm" may be conjugated with 1 to 10 new degrading agents. In some aspects, "Bm" may be conjugated with 1 to 9 new degrading agents. In some aspects, "Bm" may be conjugated with 1 to 8 new degrading agents. In some aspects, "Bm" may be conjugated with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 new degrading agents. In some aspects, "Bm" may be conjugated with 7 or 8 new degrading agents. In some aspects, "Bm" may be conjugated with 5 new degrading agents. In some aspects, "Bm" may be conjugated with 6 new degrading agents. In some aspects, "Bm" may be conjugated with 7 new degrading agents. In some aspects, "Bm" may be conjugated with 8 new degrading agents. In some aspects, "Bm" may be conjugated with 9 new degrading agents.

[0557] IV. Composition and Usage

[0558] The conjugates and / or compounds described herein may be in the form of pharmaceutically acceptable salts. In some respects, such salts are derived from inorganic or organic acids or bases.

[0559] Examples of suitable acid addition salts include acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, fumarates, gluconate, glyceryl phosphates, hemisulfates, heptanates, hexanoates, hydrochlorides, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinic acid esters, oxalates, dihydroxynaphthalate, pectates, persulfates, 3-phenylpropionates, picrates, neopentanoates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, and undecanoates.

[0560] Examples of suitable base addition salts include ammonium salts, basic metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts containing organic bases (such as dicyclohexylamine salts, N-methyl-D-glucosamine), and salts containing amino acids (such as arginine, lysine, etc.).

[0561] For example, Berge lists the following FDA-approved commercially available salts: anionic acetate, besylate, benzoate, bicarbonate, hydrogen tartrate, bromide, calcium edetate (ethylenediaminetetraacetic acid), camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetic acid), ethanedisulfonate (1,2-ethanedisulfonate), etopoate (lauryl sulfate), ethanesulfonate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycolyl larsanilate (glycollamido) phenylarsonate), hexyl resorcinol ester, hebamin (N,N'-di(dehydroabi)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, hydroxyethyl sulfonate (2-hydroxyethanesulfonate), lactate, lacturonic acid, malate, maleate, mandelic acid, methanesulfonate, methyl bromide, methyl nitrate, methyl sulfate, mucilage, naphthalenesulfonate (2-naphthalenesulfonate), nitrate, bis(hydroxynaphthoate) (pamoate, embonate), pantothenate, phosphate / bisphosphate, polygalacturonic acid, salicylate, stearate, hypoacetate, succinate, sulfate, tannate, tartrate, 8-chlorotheophylline (teoclate, 8-chlorotheophyllinate), and triethyliodide; organic cationic benzylamine (N N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucosamine), and procaine; as well as the metal cations aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0562] Berge also lists the following non-FDA-approved commercially available (outside the United States) salts: anionic adipate, alginate, aminosalicylate, dehydrated methylene citrate, arecoline, aspartate, hydrogen sulfate, butyl bromide, camphorate, diglucuronide, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoric acid, hydroiodate, methylene bis(salicylate), naphthalene disulfonate (1,5-naphthalene disulfonate), oxalate, pectinate, persulfate, phenethyl barbiturate, picrate, propionate, thiocyanate, toluene sulfonate, and undecanoate; organic cations phenethylamine (N-benzylphenethylamine), cririmazole (1-p-chlorobenzyl-2-pyrrolino-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and metal cations barium and bismuth.

[0563] Pharmaceutical compositions containing the novel degrading agent conjugates described herein may also contain suitable carriers, excipients, and adjuvants, which may vary depending on the mode of administration.

[0564] In some respects, the pharmaceutical composition can be formulated into a suitable parenteral dosage form. The formulation can be prepared by various methods known in the art. The pharmaceutical composition can be administered directly into the bloodstream, muscle, or organ. Suitable routes of parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous administration. Suitable devices for parenteral administration include needle injectors, needle-free injectors, and infusion techniques.

[0565] Parenteral compositions are typically aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers. However, the composition may also be formulated into sterile, non-aqueous or dry forms for use in conjunction with suitable media such as sterile, pyrogen-free water.

[0566] Under aseptic conditions, such as by lyophilization, the preparation of parenteral compositions can be easily accomplished using standard techniques well known to those skilled in the art.

[0567] Compositions intended for parenteral administration can be formulated to provide immediate release and / or improved release. Improved release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release, and programmed release. Therefore, compositions can be formulated as solids, semi-solids, or thixotropic liquids for administration as implantable reservoirs providing improved release of the active agent.

[0568] Parenteral formulations may be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms, such as, but not limited to, preservatives.

[0569] On the other hand, the pharmaceutical composition can be formulated into suitable oral dosage forms, such as tablets, capsules, powders, pills, suspensions, solutions, emulsions, etc. Other suitable carriers may be present, such as disintegrants, diluents, chelating agents, binders, flow aids, lubricants, fillers, swelling agents, anti-adhesion agents, etc.

[0570] Oral dosage forms may also contain other suitable pharmaceutical excipients, such as sweeteners, mediators / wetting agents, colorants, flavoring agents, preservatives, thickeners, etc.

[0571] The novel degradative conjugates described herein may be used to treat various cancers. Certain conjugates disclosed herein may be superior in terms of efficacy expression, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water solubility), interactions with other drugs (e.g., inhibition of drug-metabolizing enzymes), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central nervous system toxicity), and / or stability (e.g., chemical stability, enzyme stability), and may be used as pharmaceuticals.

[0572] The novel degradation agent conjugates disclosed herein can be used as: pharmaceuticals, such as agents for the prevention or treatment of diseases, said diseases being cancers such as 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, stomach / gastric cancer (e.g., Papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small bowel cancer, breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., ovarian epithelial carcinoma, extragonadal germ cell tumors, ovarian germ cell tumors, low-potency ovarian tumors), testicular tumors, 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 carcinoma). 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), choriocarcinoma of pregnancy, brain tumors (e.g., medulloblastoma, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., basal cell carcinoma, malignant melanoma), sarcoma (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumors, bladder cancer, blood / leukemia (e.g., multiple myeloma, leukemia (e.g., acute myeloid leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorders), cancer of unknown primary origin; cancer growth inhibitors; cancer metastasis inhibitors; apoptosis promoters; agents for treating precancerous lesions (e.g., myelodysplastic syndromes); and so on.

[0573] In some respects, the novel degradation agent conjugates disclosed herein can be used as drugs for breast cancer, gastric cancer, ovarian cancer, uterine cancer, lung cancer, pancreatic cancer, liver cancer, lymphoma, or blood cancer.

[0574] Furthermore, the novel degradation agent conjugates disclosed herein can be used concurrently with non-pharmacological therapies. Specifically, the conjugates can be combined with non-pharmacological therapies such as (1) surgery, (2) chemotherapy for hypertension using angiotensin II, (3) gene therapy, (4) thermotherapy, (5) cryotherapy, (6) laser ablation, and (7) radiotherapy.

[0575] For example, by using the novel degradation agent conjugate of this disclosure before or after the aforementioned surgeries, effects such as preventing the development of resistance, prolonging disease-free survival, inhibiting cancer metastasis or recurrence, and extending lifespan can be provided.

[0576] In addition, treatment using the novel degradation agent conjugates disclosed herein may be combined with the following supportive therapies: (i) for complications of various infectious diseases, administration of antibiotics (e.g., β-lactams such as cefotiam (pansporin), macrolides such as clarithromycin), (ii) administration of high-calorie transfusions, amino acid preparations, or general vitamin preparations to improve malnutrition, (iii) administration of morphine to relieve pain, (iv) administration of agents to improve side effects such as nausea, vomiting, anorexia, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, liver disease, kidney disease, DIC, fever, etc., and (v) administration of agents for inhibiting multidrug resistance in cancer, etc.

[0577] In some aspects, the novel degrading agents or novel degrading agent conjugates of this disclosure may be used in combination with standard care therapies such as one or more therapeutic agents (e.g., anticancer agents and / or immunomodulatory agents). Therefore, in some aspects, a method of treating the tumors disclosed herein includes administering the novel degrading agents or novel degrading agent conjugates of this disclosure in combination with one or more additional therapeutic agents. In some aspects, the novel degrading agents or novel degrading agent conjugates of this disclosure may be used in combination with one or more anticancer agents, thereby targeting multiple elements of immune pathways. In some aspects, the anticancer agents include immune checkpoint inhibitors (i.e., blocking signaling through specific immune checkpoint pathways). Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the present invention include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies), or combinations thereof. A comprehensive and non-limiting list of combination therapies is disclosed in detail in the combination therapy section of this application.

[0578] In some aspects, the novel degrading agent or novel degrading agent conjugate of this disclosure is administered to a subject before or after administration of an adjunct therapeutic agent. In other aspects, the novel degrading agent or novel degrading agent conjugate of this disclosure is administered to a subject concurrently with an adjunct therapeutic agent. In some aspects, the novel degrading agent or novel degrading agent conjugate of this disclosure and the adjunct therapeutic agent may be administered concurrently as a single composition in a pharmaceutically acceptable carrier. In other aspects, the novel degrading agent or novel degrading agent conjugate of this disclosure and the adjunct therapeutic agent are administered concurrently as a separate composition.

[0579] In some respects, the subjects to be treated with the novel degrading agents or novel degrading agent conjugates disclosed herein are non-human animals, such as rats or mice. In other respects, the subjects to be treated are humans.

[0580] V. Methods for preparing novel degradative agents and compositions

[0581] This disclosure provides a method for preparing novel degradative conjugates, the method comprising subjecting the binding moiety to a compound of formula (I-1):

[0582]

[0583] Or a pharmaceutically acceptable salt reaction, wherein:

[0584] A is a phenyl group or a C4-C4 group. 10 cycloalkyl ring;

[0585] R 1 Independently selected from hydrogen and halogroups;

[0586] U is selected from NH and CF2;

[0587] X is selected from -NR 2 -、=C(CH3)-、-Q-(CH2) n - and -Q(CH2) m Q'(CH2) n- ;in

[0588] Q and Q' are each independently O, S, or NR. 2 ;

[0589] R 2 It is hydrogen or C1-C6 alkyl;

[0590] n is an integer from 1 to 6;

[0591] m is an integer from 2 to 6; and

[0592] Each group is attached to L' on the left and to A on the right; provided that X is NH or -Q-(CH2). n - At that time, R 1 It is a halogenated group;

[0593] L' is a cuttable or non-cuttable connector precursor that is attached to the said joint portion.

[0594] As described in this article, the connector precursor contains heterobifunctional groups that are connected to the junction.

[0595] In some respects, L' is a precursor to an uncuttable joint. In some respects, L' is selected from the group consisting of the following:

[0596]

[0597] in:

[0598] p is an integer from 1 to 10; and

[0599] Let X be the connection point with X.

[0600] In some respects, L' is

[0601]

[0602] In some respects, p is 5.

[0603] In some respects, L' is a precursor to a cuttable connector.

[0604] In some respects, the adaptor precursor can be cleaved by proteases. In other respects, the adaptor precursor is selected from the following groups.

[0605]

[0606] in:

[0607] q is an integer from 2 to 10;

[0608] Z 1 Z 2 Z 3 and Z 4 Each naturally occurring amino acid residue, either independently absent or in L- or D-configuration, is provided that Z 1 Z 2 Z 3 and Z 4 At least two of them are amino acid residues;

[0609] and

[0610] Let X be the connection point with X.

[0611] In some respects, Z 1 Z 2 Z 3 and Z 4 The following groups are independently absent or selected from: 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 Z 1 Z 2 Z 3 and Z 4 At least two of them are amino acid residues.

[0612] In some respects, Z 1 It may be absent or contain glycine; Z 2The group consisting of or not present in the group containing: L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 Select the group consisting of: L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; and Z 4 Choose from the following groups: L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0613] In some respects, L' is

[0614]

[0615] In some respects, q is 5.

[0616] In some respects, L' is a bioreducible adapter precursor. In some respects, the bioreducible adapter precursor is selected from the group consisting of the following:

[0617]

[0618] in:

[0619] q is an integer from 2 to 10;

[0620] R, R', R" and R'" are each independently selected from hydrogen, C1-C6 alkoxy-C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or the two geminal R groups together with the carbon atoms they are attached to can form a cyclobutyl or cyclopropyl ring; and

[0621] Let X be the connection point with X.

[0622] In some respects, L' is an acid-cuttable precursor. In other respects, L' is selected from the group consisting of the following...

[0623]

[0624] in:

[0625] q is an integer from 2 to 10; and Let X be the connection point with X.

[0626] In some respects, L' is a click-release connector precursor. In other respects, L' is selected from...

[0627]

[0628] in:

[0629] q is an integer from 2 to 10; and Let X be the connection point with X.

[0630] In some respects, L' is a pyrophosphatase-cleavable linker precursor. In some respects, L' is...

[0631]

[0632] in:

[0633] q is an integer from 2 to 10;

[0634] Let X be the connection point with X.

[0635] In some respects, L' is a β-glucuronidase-cleavable precursor of the adaptor. In other respects, L' is selected from...

[0636]

[0637] in:

[0638] q is an integer from 2 to 10;

[0639] ----Does not exist or is a key; and

[0640] Let X be the connection point with X.

[0641] In some respects, the compounds of formula (I-1) are selected from...

[0642]

[0643] In some respects, the binding moiety is pretreated before reacting with a compound of formula (I-1). In some respects, a compound of formula (I-1) is reacted with a binding moiety comprising an antibody or its antigen-binding moiety. In the respect where the binding moiety is an antibody, the antibody may be pretreated to reduce interchain disulfide bonds before reacting with a compound of formula (I-1).

[0644] Example

[0645] General synthetic methods and intermediates

[0646] The compounds disclosed herein can be prepared by those skilled in the art based on this disclosure and the knowledge of the art, and / or by referring to the schemes and synthetic examples shown below. Exemplary synthetic routes are illustrated in the following schemes and examples. It should be understood that variables (e.g., “R” groups) appearing in the following schemes and examples should be read independently of those appearing elsewhere in this application. Those skilled in the art will readily understand how the schemes and examples shown below illustrate the preparation of the compounds described herein.

[0647] The abbreviations used in this specification generally follow conventions used in the art. The chemical abbreviations used in the specification and examples are defined as follows: "THF" represents tetrahydrofuran; "DMF" represents N,N-dimethylformamide; "Me" represents methyl; "Bu" represents butyl; "FA" represents formic acid; "PE" represents petroleum ether; "MeOH" represents methanol; "EtOH" represents ethanol; "DCM" represents dichloromethane; "BOC" or "Boc" "TFA" represents trifluoroacetic acid; "DMSO" represents dimethyl sulfoxide; "EtOAc" represents ethyl acetate; "OAc" represents acetate; "dppf" represents 1,1'-bis(diphenylphosphine)ferrocene; "dba" represents dibenzylacetone; "CDI" represents 1,1'-carbonyldiimidazole; "TBAF" represents tetrabutylammonium fluoride; "TBSC1" represents tert-butyldimethylchlorosilane. “Et2O” represents diethyl ether; “ACN” represents acetonitrile; “h” represents hours; “min” represents minutes; “rt” represents room temperature or retention time (context will determine); “aq.” represents aqueous, “sat.” represents saturated; “min” represents minutes; “HOBt” represents 1-hydroxybenzotriazole hydrate; “HATU” represents 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-methylmethylammonium hexafluorophosphate N-oxide; “DIEA” and “iPrNEt2” represent diisopropylethylamine; “Et3N” and “TEA” represent triethylamine.

[0648]

[0649] BH3-Me2S (10M THF solution) (5.80 mL, 58.0 mmol, 2.50 equivalent) was added dropwise to a stirred solution of 2-chloro-4-nitrophenyl)acetic acid (compound 1, 5.00 g, 23.19 mmol, 1.00 equivalent) in THF (75.00 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 h. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc = 1:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (3 g, 64%) as a yellow solid. 1 H NMR (400MHz, CDCl3) δ 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).

[0650]

[0651] Add Bu₄NH₄ (6.74 g, 19.84 mmol, 0.80 equivalent) to a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 2, 5.00 g, 24.800 mmol, 1.00 equivalent) and tert-butyl 2-bromoacetate (29.0 mL, 148.28 mmol, 8.00 equivalent) in toluene (150.00 mL). Add NaOH (5 M H₂O solution) (500.00 mL) dropwise to the mixture at 0 °C over 40 min. Stir the resulting mixture at 25 °C for 2 h. Extract the resulting mixture with EtOAc (3 x 500 mL). Wash the combined organic layers with brine (400 mL) and dry with anhydrous Na₂SO₄. Filter and concentrate the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (8 g, 65%), which was a yellow oil. 1 H NMR(400MHz, CDCl3)δ 8.23(d,J=4.0Hz,1H),8.10-8.04(m,1H),7.60(d,J=8.0Hz,1H),4.09(s,2H),3.83-3.80(m,2H),3.17-3.14(m,2H),1.45(s,9H).

[0652]

[0653] TFA (16.00 mL) was added dropwise to a stirred solution of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]tert-butyl acetate (compound 3, 8.00 g, 16.14 mmol, 1.00 equivalent, 63.7%) in DCM (80.00 mL) at room temperature. The resulting mixture was stirred at room temperature for 1 h.

[0654] The resulting mixture was concentrated under vacuum. The mixture was diluted with water (500 mL). The mixture was extracted with EtOAc (3 x 500 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. This yielded [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (6.5 g, crude), a yellow oil. LCMS (ESI): 517 (2M-H)-

[0655]

[0656] At room temperature, CH3NH2·HCl (1.77 g, 26.21 mmol, 1.20 equivalent, 90%) and DIEA (15.20 g, 117.8 mmol, 4.00 equivalent) were added dropwise to a stirred solution of [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 4, 6.30 g, 21.84 mmol, 1.00 equivalent) and HATU (12.46 g, 32.76 mmol, 1.50 equivalent) in DMF (65.00 mL). The resulting mixture was stirred at room temperature for 2 h. The resulting mixture was diluted with water. The resulting mixture was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH = 10:1) to give 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (10 g, purity: 50%, yield: 84%), as a yellow oil. LCMS (ESI): 273.28 (M+H) +

[0657]

[0658] At room temperature under a nitrogen atmosphere, BH3-THF (1M THF solution) (12.10 mL, 12.10 mmol, 1.00 equivalent) was added dropwise to a stirred solution of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 5, 3.3 g, 12.10 mmol, 1.00 equivalent) in 35.00 mL of THF (12.10 mL, 12.10 mmol, 1.00 equivalent). The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 h. The reaction was quenched with MeOH. The residue was acidified to pH 6 with 1N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was alkalized to pH 8 with saturated NaHCO3 (saturated aqueous solution). The resulting mixture was extracted with EtOAc (3 x 100 mL), washed with brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This produces [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (2.5 g, 80%), as a yellow oil. LCMS (ESI): 259.26 (M+H) +

[0659]

[0660] TEA (1.17 g, 11.6 mmol, 1.20 equivalent) was added dropwise 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 equivalent) and Boc₂O (2.53 g, 11.6 mmol, 1.20 equivalent) in 40 mL of THF at 25 °C. The mixture was stirred at 25 °C for 2 h. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography (DCM:MeOH = 5:1) to give N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate tert-butyl ester (1.70 g, 50%) as a yellow oil. LCMS (ESI): 359.36 (M+H) +

[0661]

[0662] Fe (1.3 g, 23.7 mmol, 5.00 equivalent) was added to a stirred solution of N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 7, 1.70 g, 4.74 mmol, 1.00 equivalent) and NH4Cl (750 mg, 14.2 mmol, 3.00 equivalent) in EtOH (85 mL) and H2O (17 mL). The mixture was stirred at 80 °C for 2 h. The mixture was cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with EtOH (3 x 50 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc = 4:1) to give N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (900 mg, 58%) as a yellow oil. LCMS (ESI): 329.33 (M+H) +

[0663]

[0664] At 25°C, diphosgene (601 mg, 3.04 mmol, 2.00 equivalent) was added dropwise to a stirred solution of N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate tert-butyl ester (compound 8, 500 mg, 1.52 mmol, 1.00 equivalent) in THF (10 mL). The mixture was stirred at 25°C for 1 h. The resulting mixture was concentrated under vacuum and redissolved in DMF (5 mL). At 25°C, the solution mentioned above was added dropwise to a stirred mixture of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidin-2,6-dione (INT1, prepared as described below, 499 mg, 1.82 mmol, 1.20 equivalent) and TEA (1.56 g, 15.45 mmol, 10.00 equivalent) in DMF (20 mL). The mixture was stirred at 25°C for 1 h. The resulting mixture was diluted with 40 mL of ice water. The mixture was extracted with EtOAc (3 x 40 mL). The combined organic layers were washed with brine (5 x 40 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH = 10:1) to give (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate tert-butyl ester (670 mg, 70%), as a white solid. LCMS: (ESI): 628.63 (M+H) +

[0665]

[0666]

[0667] TFA (2.5 mL) was added dropwise to a stirred solution of N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-N-methylcarbamate tert-butyl ester (compound 9, 670 mg, 1.07 mmol, 1 equivalent) in DCM (10 mL). The mixture was stirred at 25 °C for 1 h. The resulting mixture was concentrated under vacuum. The crude product was purified by preparative HPLC under the following conditions: column, SunFire C18 OBD Prep column, 100 μm, 19 x 250 mm; mobile phase, water (0.05% TFA) and ACN (5% B phase reaching 60% within 30 min); detector, UV 220 nm. The collected fractions were lyophilized to give 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (500 mg, 89%), as a white solid. LCMS (ESI): 528.53 (M+H) + . 1 H NMR (400MHz, methanol-d4)δ 7.77(d,J=8.0Hz,1H),7.57-7.53(m,2H),7.49(d,J=8.0Hz,1H),7.21(d,J=4.0Hz,2H),5.19-5.1(m,1H),4.55-4.41(m,4H),3.75-3.67 (m,4H),3.21-3.15(m,2H),3.03-3.96(m,2H),2.96-2.84(m,1H),2.83-2.73(m,2H),2.69(s,3H),2.55-2.42(m,1H),2.21-2.12(m,1H).

[0668]

[0669] At room temperature, 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (new degrader P1, 200 mg, 0.38 mmol, 1.00 equivalent) and dimethylpyridine (81 mg, 0.76 mmol, 2.00 equivalent) were added to DMF (10 mL). HOBT (26 mg, 0.19 mmol, 0.50 equivalent) and [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexamido]-3-methylbutamido]pentamido]phenyl]methyl 4-nitrophenyl carbonate (279 mg, 0.38 mmol, 1.00 equivalent) were added in portions to the stirred mixture. The reaction mixture was stirred at 40°C under a nitrogen atmosphere for 12 hours. After cooling to room temperature, the reaction was quenched with water (30 mL). The resulting mixture was extracted with DCM (3 x 30 mL). The combined organic layers were washed with water (2 x 30 mL) and brine (30 mL) and dried over Na2SO4. After filtration, the filtrate was concentrated to dryness under vacuum. The residue was purified by reversed-phase column (C18, mobile phase A: 0.1% FA aqueous solution, mobile phase B: ACN). The collected fraction was concentrated to dryness under vacuum. The crude product (60 mg) was purified by preparative HPLC under the following conditions (column: Xselect CSH OBD column 30x150 mm 5 μm, n; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 33 B to 50 B in 7 min; 220 nm; RT1: 5.27 min). The collected fractions were freeze-dried to obtain N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-N-methylcarbamate [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopiror-1-yl)hexamido]-3-methylbutamido]pentamido]phenyl]methyl ester (23.8 mg, 5%), as a white solid. LCMS (ESI): 1126.11 (M+H) + . 1H NMR(400MHz,DMSO-d6)δ 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).

[0670]

[0671] TEA (22.4 mL, 162.2 mmol, 2.50 equivalence) was added dropwise to a stirred mixture of methyl 4-bromo-2-(bromomethyl)benzoate (compound 10, 20.0 g, 64.8 mmol, 1.00 equivalent) and 3-aminopiperidine-2,6-dione hydrochloride (10.64 g, 83.0 mmol, 1.28 equivalent) in DMF (80 mL) at 25 °C under a nitrogen atmosphere. The mixture was stirred at 25 °C for 16 h. Subsequently, H₂O (60 mL), AcOH (23 mL), and Et₂O (60 mL) were added sequentially at 25 °C. The mixture was stirred at 25 °C for 2 h. The precipitated solid was collected by filtration and washed with Et₂O (60 mL). This yields 3-(5-bromo-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione (9.0 g, 42%), a pale blue solid. LCMS (ESI): 323.32 (M+H)+

[0672]

[0673] Zn(OAc)₂ (170 mg, 0.928 mmol, 0.30 equivalent), Zn(CN)₂ (545 mg, 4.64 mmol, 1.50 equivalent), and Pd₂(dba)₃ (28 mg, 0.031 mmol, 0.01 equivalent) were added to a stirred mixture of 3-(5-bromo-1-oxo-3H-isoindol-2-yl)piperidin-2,6-dione (compound 11, 1.00 g, 3.09 mmol, 1.00 equivalent) and dppf (51 mg, 0.093 mmol, 0.03 equivalent) in DMF (8 mL) under a nitrogen atmosphere at 25 °C. The final reaction mixture was irradiated with microwave at 120 °C for 2 h. 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 subjected to rapid chromatography (silica gel, 80 g, DCM:MeOH = 10:1) to give the desired product 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindole-5-onitrile (400 mg, 47%), as a brown solid. LCMS (ESI): 270 (M+H) +

[0674]

[0675] PtO2 (1.25 g, 5.5 mmol, 0.49 equivalent) was added to a stirred mixture of 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindole-5-nitrile (compound 12, 3.0 g, 11.14 mmol, 1.00 equivalent) and HCl (12 M) (3.6 mL) in MeOH (25 mL) at 25 °C. The mixture was hydrogenated at room temperature for 16 h using a hydrogen balloon under a hydrogen atmosphere. 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 yielded 3-[5-(aminomethyl)-1-oxo-3H-isoindole-2-yl]piperidin-2,6-dione (2.5 g, 80%) as a gray solid. LCMS (ESI): 274 (M+H) + . 1H NMR(400MHz,DMSO-d6)δ 11.02(s,1H),8.15(s,1H),7.98(d,J=8.4Hz,1H),7.89(d,J=8.4Hz,1H),5.16-5.11(m,1H),4.52(d,J=17.2 Hz,1H),4.40(d,J=17.2Hz,1H),2.96-2.90(m,1H),2.60-2.54(m,1H),2.43-2.34(m,1H),2.06-1.96(m,1H)

[0676]

[0677] BH3-Me2S (10M THF solution) (5.60 mL, 56 mmol, 2.50 equivalent) was added dropwise to a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 13, 5.00 g, 22.50 mmol, 1.00 equivalent) in THF (75 mL) at 0 °C under a nitrogen atmosphere. The mixture was stirred at 70 °C for 2 h. The resulting mixture was concentrated under vacuum. The residue was loaded onto 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 HNMR(400MHz, CDCl3)δ 8.26(d,J=4.0Hz,1H),8.10-8.05(m,1H),7.50(d,J=8.0Hz,1H),3.99-3.91(m,2H),3.16-3.09(m,2H)

[0678]

[0679] TBSC1 (6.97 g, 46.25 mmol, 2.10 equivalent) was added to a stirred mixture of 2-(2-chloro-4-nitrophenyl)ethanol (compound 14, 4.44 g, 22.02 mmol, 1.00 equivalent) and imidazole (4.50 g, 66.06 mmol, 3.00 equivalent) in DMF (50.00 mL) 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) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was loaded onto 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%), which was a colorless oil. 1HNMR (400MHz, CDCl3) δ 8.24 (s, 1H), 8.06-8.04 (m, 1H), 7.46 (d, J = 8.4Hz, 1H), 3.89-3.86 (m, 2H), 3.06-0.04 (m, 2H), 0.85 (s, 9H), 0.04 (s, 6H).

[0680]

[0681] NH4Cl (9.65 g, 180.45 mmol, 10 equivalents) was added to a mixture of tert-butyl[2-(2-chloro-4-nitrophenyl)ethoxy]dimethylsilane (compound 15, 5.70 g, 18.05 mmol, 1.00 equivalents) and Fe (10.08 g, 180.45 mmol, 10.00 equivalents) in EtOH (110 mL) / water (55 mL). The mixture was stirred at 80 °C for 2 h. The mixture was cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with EtOH (3 x 50 mL). The filtrate was concentrated under reduced pressure. The residue was diluted with water (100 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate and then evaporated to dryness under vacuum to obtain 4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chloroaniline (5.2 g, crude product), a light brown oil. LCMS (ESI): 286.29 (M+H) +

[0682]

[0683] Under nitrogen atmosphere at 0°C, a solution of CDI (113 mg, 0.70 mmol, 1.00 equivalent) in DMF (1 mL) was added dropwise to a solution of 4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chloroaniline (compound 16, 200.00 mg, 0.70 mmol, 1.00 equivalent) and TEA (141 mg, 1.40 mmol, 2.00 equivalent) in DMF (3 mL). 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 equivalent) in DMF (2 mL). The same reaction was repeated twice. The resulting mixture was stirred at 25°C for 1 hour. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, and evaporated to dryness under vacuum. The residue was purified by silica gel column chromatography (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)+

[0684]

[0685] TBAF (1N THF solution, 0.58 mL, 0.58 mmol, 2.00 equivalent) was added to a solution of 1-(4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chlorophenyl)-3-[(2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindolin-5-yl)methyl]urea (compound 17, 170.00 mg, 0.29 mmol, 1.00 equivalent) in 2.00 mL of THF 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-dioxopiridin-3-yl)-1-oxoisoindolin-5-yl)methyl)urea as a white solid. LCMS (ESI): 471.47 (M+H) +

[0686]

[0687] 2-Methyl-2-thioalkylprop-1-ol (compound 18, 1.4 g, 13.2 mmol, 1.00 equivalent) and 5-nitro-2-[(5-nitropyridin-2-yl)dithioalkyl]pyridine (compound 120, 2.05 g, 6.67 mmol, 0.50 equivalent) were added to a mixture of dichloromethane (3.50 mL) and MeOH (3.50 mL) solvent. The resulting mixture was stirred at 15 °C. Then, manganese dioxide (2.29 g, 26.2 mmol, 2 equivalent) was added in portions. The resulting mixture was stirred at 15 °C for 15 min. The LCMS trace showed that the reaction was complete. The reaction was evaporated to dryness and the residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% NH4HCO3), 10% to 100% gradient over 30 min; detector, UV 254 nm. The collected fraction was concentrated to dryness under vacuum to give 2-methyl-2-[(5-nitropyridin-2-yl)dithioalkyl]prop-1-ol (2.2 g, 58%) as a yellow solid. LCMS (ESI): 261 (M+H) + .

[0688]

[0689] To a solution of 2-methyl-2-[(5-nitropyridin-2-yl)dithioalkyl]prop-1-ol (compound 20, 1.0 g, 3.84 mmol, 1.00 equivalent) in anhydrous DCM (30 mL), MeSO₂Na (1.57 g, 15.4 mmol, 4.00 equivalent) and iodine (1.95 g, 7.68 mmol, 2.00 equivalent) were added in portions. The reaction mixture was stirred at 45 °C for 24 h. The mixture was concentrated, and the residue was purified by silica gel column chromatography (TLC:PE:EA = 3:1, Rf = 0.60; 0%-35% EtOAc in petroleum ether) to give 2-(methanesulfonylthioalkyl)-2-methylprop-1-ol (80 mg, 10%) as a yellow oil. 1 H NMR (400MHz, CD3C1): δ 3.50 (s, 2H), 3.33 (s, 3H), 2.16 (br s, 1H), 1.47 (s, 6H).

[0690]

[0691] A solution of CDI (138 mg, 0.84 mmol, 2.00 equivalent) in DMF (1 mL) was added to a solution of 1-[3-chloro-4-(2-hydroxyethyl)phenyl]-3-[[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (new degrader P3, 200.00 mg, 0.42 mmol, 1.00 equivalent) and TEA (129 mg, 1.26 mmol, 3.00 equivalent) in DMF (4 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with water (20 mL x 3) and brine (20 mL), dried over sodium sulfate, and evaporated to dryness under vacuum 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]ethylimidazolium-1-carboxylate, 200 mg), a pale yellow solid. The crude product (100.00 mg, 0.18 mmol, 1.00 equivalent) and C were heated at room temperature. S2 A solution of CO3 (115 mg, 0.35 mmol, 2.00 equivalents) in DMF (8 mL) was added dropwise to a solution of 2-(methanesulfonylthioalkyl)-2-methylprop-1-ol (compound 20, 59 mg, 0.32 mmol, 1.80 equivalents) in DMF (2 mL). The reaction was stirred at 15 °C for 22 hours. The reaction mixture was diluted with EtOAc (50 mL) and ice-cold water (100 mL). The organic layer was separated. The aqueous phase was extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (30 mL x 3), dried over anhydrous sodium sulfate, and evaporated to dryness under vacuum to give a crude product (150 mg) as a yellow solid. The crude product was purified by preparative HPLC (column: Xselect CSH OBD column 30x150mm 5um; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 38B to 58B over 7 min; 220 nm; RT1: 5.12 min). The collected fractions were lyophilized to give 1-[3-chloro-4-[2-([[2-(methanesulfonylthioalkyl)-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) + . 1H NMR (400MHz, DMSO-d6)δ 10.99(s,1H),8.86(s,1H),7.70(d,J=2.4Hz,1H),7.51(s,1H),7.44(d,J= 8.0Hz,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).

[0692]

[0693] CDI (218 mg, 1.35 mmol, 1.00 equivalent) and TEA (68 mg, 1.35 mmol, 1.00 equivalent) were added dropwise to a stirred solution of (2-aminophenyl)(methyl)carbamate (compound 22, 300 mg, 1.35 mmol, 1.00 equivalent) in DMF (20 mL) at 0 °C under a nitrogen atmosphere. The mixture was stirred at 0 °C for 2 h. 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidin-2,6-dione (INT1, 368 mg, 1.35 mmol, 1.00 equivalent) was added in portions to the above mixture. The resulting mixture was stirred at 75 °C overnight. The reaction mixture was then cooled to room temperature. The resulting mixture was quenched with water (30 mL) and extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH = 10:1) to give N-[2-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate tert-butyl ester (300 mg, 42%), as a white solid. LCMS (ESI): 522 (M+H) +

[0694]

[0695] TFA (5 mL) was added to a stirred solution of N-[2-[([[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate tert-butyl ester (compound 23, 300 mg, 1.00 equivalent) in DCM (20 mL). The mixture was stirred at 0 °C for 2 h. The resulting mixture was concentrated under vacuum. The crude product was purified by reverse-phase chromatography 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 under vacuum to give 3-[[2-(2,6-dioxopiridin-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(300MHz,DMSO-d6)δ 10.99(s,1H),7.69(d,J=7.8Hz,1H),7.60(s,1H),7.53(s,1H),7.45(d,J=8.4Hz,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).

[0696]

[0697] At room temperature under a nitrogen atmosphere, [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexamido]-3-methylbutamido]pentamido]phenyl]methyl 4-nitrophenyl carbonate (394 mg, 0.53 mmol, 1.50 equivalent) was added 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 equivalent), 2,6-dimethylpyridine (76 mg, 0.71 mmol, 2.00 equivalent) and HOBT (96 mg, 0.71 mmol, 2.00 equivalent) in DMF (3.00 mL). The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column: C18 silica gel; mobile phase: A: water (0.1% FA), B: ACN; to give a crude product (60 mg) as a white solid. The crude product (60 mg) was purified by preparative HPLC under the following conditions: column: Xselect CSH OBD column 30x150 mm 5 μm, n; mobile phase: A: water (0.1% FA), B: ACN; flow rate: 60 mL / min; gradient: 24 B to 44 B over 7 min; 220 nm; RT1: 6.33; RT2: . The collected fractions were freeze-dried to obtain N-[2-[([[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopiror-1-yl)hexamido]-3-methylbutamido]pentamido]phenyl]methyl ester (18.1 mg, 5%), as a white solid. LCMS (ESI): 1020 (M+H) + . 1H NMR(400MHz,DMSO-d6)δ 10.99(s,1H),9.96(s,1H),8.19-8.06(m,3H),7.79(d,J=8.8Hz,1H),7.70(d,J=8.0H z,1H),7.53-7.41(m,5H),7.20-7.05(m,4H),7.00(s,2H),6.95-6.90(m,1H),5.95(br s,1H),5.41(s,2H),5.18-4.89(m,3H),4.44-4.20(m,5H),4.19-4.17(m,1H),3.09(s,3H),3.07-2.85( m,3H),2.22-2.02(m,2H),2.00-1.85(m,2H),1.71-1.25(m,10H),1.20-1.12(m,3H),0.84-0.80(m,6H)

[0698] Scheme 4 shows how compound (Id) is prepared from the novel degrading agent P1.

[0699]

[0700] Synthesis of compound (Id)

[0701] DIEA (20.00 mg, 0.16 mmol, 2.04 equivalents) was added dropwise to a stirred mixture of 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (P1, 40.00 mg, 0.076 mmol, 1.00 equivalent) and 2,5-dioxopiroryl-1-yl)hexanoic acid 2,5-dioxopiroryl-1-yl ester (25.00 mg, 0.081 mmol, 1.07 equivalent) in DMF (2.00 mL). The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. The resulting mixture was quenched with water (30 mL) and extracted with DCM (3 x 30 mL). The combined organic layers were washed with water (30 mL) and brine (30 mL) and dried over Na2SO4. After filtration, the filtrate was concentrated to dryness under vacuum. The residue was purified under the following conditions: column: SunFire C18 OBD Prep column, 100 μm, 19 mm x 250 mm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 25 B to 55 B 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-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethoxy)ethyl]-6-(2,5-dioxopiror-1-yl)-N-methylhexanoamide (compound (Id), 24 mg, 43%), as a white solid. LCMS: (ES, m / s): 721, 723 (M+H) + ; 1 H NMR(400MHz,DMSO-d6)δ 10.99(s,1H),8.78(s,1H),7.70-7.66(m,2H),7.51(s,1H),7.41(d,J=9.6Hz,1H),7 .18-7.16(m,2H),7.00(d,J=5.6Hz,2H),6.85-6.80(m,1H),5.12-5.05(m,1H),4.42 -4.33(m,5H),3.39-3.36(m,3H),2.91-2.76(m,7H),2.68-2.52(m,1H),2.48-2.35( m,1H),2.33-2.20(m,3H),2.05-1.95(m,1H),1.48-1.44(m,5H),1.28-1.12(m,3H).

[0702] Schemes 5A and 5B illustrate how to prepare a complex of the novel degrader P1 with an alternative tripeptide linker.

[0703]

[0704] Schemes 6A and 6B illustrate how to prepare a complex of the novel degrading agent P1 with β-glucuronide.

[0705]

[0706] Step 1. Synthesis of Compound 25

[0707] At room temperature, 3-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]-propionic acid (compound 24, 5.00 g, 16.06 mmol, 1.00 equivalent) was added to a stirred mixture in SOCl2 (25 mL). The resulting mixture was stirred at 80 °C for 16 h. The desired product (a derivative with MeOH, MS = 326) was detected by LCMS. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under vacuum to give N-(3-chloro-3-oxopropyl)carbamate 9H-fluorene-9-ylmethyl ester (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 indicated that it was the expected product (a derivative of MeOH). 1 H-NMR(300MHz,CDCl3)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 =3.0Hz, 2H), 4.24 (t, J = 6.0Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J = 3.0Hz, 2H), 2.59 (t, J = 6.0Hz, 2H).

[0708] Step 2. Synthesis of Compound 28

[0709] Compound 26 (10.00 g, 25.17 mmol, 1.00 equivalent) was added in portions to a stirred solution of 4-formyl-2-nitrophenol (compound 27, 4.21 g, 25.19 mmol, 1.00 equivalent) and Ag₂O (7.00 g, 30.20 mmol, 1.20 equivalent) in ACN (100 mL, 190.24 mmol, 75.00 equivalent) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction was indicated by LCMS to be complete. The resulting mixture was filtered, and the filter cake was washed with DCM (50 mL x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (PE:EA = 1:2) to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 28, 10.5 g, 86%), as a white solid. 1 ¹H-NMR analysis indicated it was the expected product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR(300MHz,CDCl3)δ 10.00(s,1H),8.34(s,1H),8.13-8.09(m,1H),7.52(d,J=3.0Hz,1H),5.4 7-5.29(m,4H),4.37-4.35(m,1H),3.75-3.73(m,3H),2.17-2.06(m,9H).

[0710] Step 3. Synthesis of Compound 29

[0711] NaBH4 (0.47 g, 12.42 mmol, 1.00 equivalent) was added in portions to a stirred solution of (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 equivalent) in MeOH (50 mL) under N2 atmosphere at room temperature. The resulting mixture was stirred for 2 h at room temperature under N2 atmosphere. The reaction was indicated to be complete by LCMS. The reaction was quenched with water at room temperature. The product was dried over Na2SO4. The resulting mixture was filtered, and the filter cake was washed with DCM. The resulting mixture was concentrated under vacuum to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 29, 5.5 g, 91%), as a solid. LCMS (ES, m / z): 486 [M+H]+.

[0712] Step 4. Synthesis of Compound 30

[0713] Pd / C (1.10 g, 10%) was added in portions to a stirred mixture of (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 equivalent) in EA (60 mL) at room temperature. The resulting mixture was stirred at room temperature under H2 atmosphere for 16 h. The reaction was indicated by LCMS to be complete. The resulting mixture was filtered, the filter cake was washed with DCM and MeOH, and the filtrate was concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 30, 4.0 g, 77%) as a solid. The crude product was used directly for the next step without further purification. LCMS(ES,m / z): 456[M+H] + .

[0714] Step 5. Synthesis of Compound 31

[0715] Compound 25 (0.87 g, 2.62 mmol, 1.20 equivalent) was added in portions to a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 30, 1.00 g, 2.19 mmol, 1.00 equivalent) and NaHCO3 (0.20 g, 2.40 mmol, 1.1 equivalent) in 10 mL of THF at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 0 °C under a nitrogen atmosphere for 6 h. The reaction was indicated to be complete by LCMS. 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 (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 31, 1.1 g, 66%), as a pale yellow solid. LCMS (ES, m / z): 749 [M+H] + .

[0716] Step 6. Synthesis of Compound 33

[0717] DIEA (0.52 g, 4.01 mmol, 2.00 equivalent) was added in portions to a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 31, 1.50 g, 2.00 mmol, 1.00 equivalent) and bis(4-nitrophenyl) carbonate (compound 32, 0.68 g, 2.24 mmol, 1.12 equivalent) in 15 mL of DMF at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction was indicated by LCMS to be complete. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), in a 10% to 90% gradient over 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness under vacuum to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 33, 1.4 g, 48%), as a yellow solid. LCMS (ES, m / z): 914 [M+H] + .

[0718]

[0719] Step 7. Synthesis of Compound 34

[0720] Methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 33, 1.00 g, 1.09 mmol, 1.00 equivalent) and 1-(3-chloro-4-[2-[2-(methylamino)ethyl) [2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (new degrader P1, 0.58 g, 1.09 mmol, 1.00 equivalent) was added in portions to a stirred mixture in DMF (10 mL) with HOBT (1.18 g, 8.72 mmol, 8.00 equivalent) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equivalent). The resulting mixture was stirred at room temperature under N2 atmosphere for 16 h. LCMS indicated the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), 10% to 80% gradient over 40 min; detector, UV 254 nm. The collected fraction was concentrated under vacuum to obtain (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-fluorene-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]oxane-2-carboxylic acid methyl ester (compound 34, 800 mg, 56%), as a solid. LCMS (ES, m / z): 1302 [M+H] + .

[0721] Step 8. Synthesis of Compound 35

[0722] HCl (6N, 80 mL) was added in portions to a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propamido)phenoxy]oxane-2-carboxylic acid methyl ester (compound 34, 800.00 mg, 0.61 mmol, 1.00 equivalent) in THF (80 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 50 °C for 3 h under a nitrogen atmosphere. The reaction was indicated by LCMS to be complete. The resulting mixture was concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), gradient from 0% to 80% over 40 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl]amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 35, 230 mg, 32%), as a white solid. LCMS(ES,m / z): 1162[M+H] + .

[0723] Step 9. Synthesis of Compound 36

[0724] Piperidine (0.4 mL) was added in portions 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-fluorene-9-ylmethoxy)carbonyl]amino]propamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 35, 230 mg, 0.2 mmol, 1.00 equivalent) in DMF (2 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred for 10 min at room temperature under a nitrogen atmosphere. The reaction was indicated to be complete by LCMS. The resulting mixture was directly purified 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 B to 40 B over 7 min; 220 nm; RT). 1:5.78 min), yielded (2S,3S,4S,5R,6S)-6-[2-(3-aminopropionamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 36, 35 mg, 18%), as a white solid. LCMS (ES, m / z): 940 [M+H]+.

[0725] Step 10. Synthesis of compound (Ie)

[0726] DIEA (13 mg, 0.10 mmol, 3.00 equivalent) and compound 37 (30 mg, 0.10 mmol, 3.00 equivalent) were added in portions to a stirred solution of (2S,3S,4S,5R,6S)-6-[2-(3-aminopropionamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiridine-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 equivalent) in DMF (3 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under a nitrogen atmosphere. The reaction was indicated by LCMS to be complete. The resulting mixture was purified by preparative HPLC under the following conditions (column: Xselect CSH OBD column 30x 150mm 5um, mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 21B to 36B over 10 min; 220 nm; RT1: 11.15 min). The collected fractions were freeze-dried to obtain (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiridine-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl]amino]phenyl]ethoxy)ethyl]-(methyl)carbamoyl]oxy)methyl]-2-[3-[6-(2,5-dioxopiror-1-yl)hexamido]propamido]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid] (Compound (Ie), 10.5 mg (28%), a white solid. LCMS (ES, m / z): 1133 [M+H] + . 1H-NMR(300MHz,DMSO-d6)δ 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=3,0Hz,1H),7.18-6.99(m,7H),5.76(s,1H),5.20-5.10(m,2H),4.98(br s,2H),4.76-4.74(m,1H),4.42-4.33(m,4H),3.65(br s,1H),3.58-3.54(m,5H),3.35(d,J=6Hz,2H),2.90-2.83(m,7H),2.57-2.55(m, 3H),2.45-2.30(m,1H),2.02-1.98(m,4H),1.48-1.42(m,5H),1.40-1.20(m,3H).

[0727] Scheme 7 shows how to prepare a complex of the new degrading agent P6 with a hydrazine linker.

[0728]

[0729] Step 1. Synthesis of Compound 38

[0730] At room temperature, 0.40 mL of diphosgene was added dropwise to a stirred solution of 4-aminoacetophenone (compound 37, 100 mg, 0.73 mmol, 1.00 equivalent) in 2.00 mL of THF. The resulting mixture was stirred at 0 °C for 30 min. The mixture was concentrated under vacuum. The resulting solid was redissolved in 1.50 mL of DMF. At room temperature, a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 200 mg, 0.73 mmol, 1.00 equivalent) in 3.00 mL of DMF and 0.50 mL of TEA was added dropwise to the stirred solution. The resulting mixture was stirred at 0 °C for 1 h. The reaction was indicated to be complete by LCMS. Water (5 mL) was added to the mixture and extracted with CH2Cl2 (3 x 10 mL). The organic layer was concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.05% TFA), in a 10% to 50% gradient over 35 min; detector, UV 254 nm. The collected fractions were concentrated to dryness to give 1-(4-acetylphenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 38, 80 mg, 25%), as a pale yellow solid. LCMS (ES.m / z): 435 [M+1] + .

[0731] Step 2. Synthesis of compound (If)

[0732] A mixture of 1-(4-acetylphenyl)-3-[[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 38, 80.00 mg, 0.18 mmol, 1.00 equivalent) and 6-(2,5-dioxopiror-1-yl)hexanohydrazide trifluoroacetic acid (75 mg, 1.20 equivalent) 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 solid was collected by filtration and washed with MeOH (2 x 5 mL). The crude solid was purified by reversed-phase rapid chromatography under the following conditions: C18 column; mobile phase, aqueous solution of ACN (0.1% FA), 10% to 50% gradient over 30 min; detector, UV 254 nm. The collected fractions were extracted with DCM (3 x 5 mL) and concentrated under vacuum. This produces 3-[[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-1-[4-[(1E)-1-[[6-(2,5-dioxopiror-1-yl)hexamido]imino]ethyl]phenyl]urea (compound (If), 4.4 mg, 3.7%), as a grayish-white solid. LCMS: (ES.m / z): 642 [M+1] + . 1 H NMR(400MHz,DMSO-d6)δ 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,6H)

[0733] Scheme 8 shows how to prepare a complex of the new degrading agent P2 with a quaternary ammonium linker.

[0734]

[0735] Step 1. Synthesis of Compound 40

[0736] Under N2 and at 0 °C, a solution of SOCl2 (20 mg, 0.18 mmol, 1 equivalent) in DCM (2 mL) was added dropwise to a stirred solution of N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrolo-1-yl)hexamethylenetetramine (compound 39, 100 mg, 0.18 mmol, 1.00 equivalent) in DMF (2 mL). The resulting mixture was stirred at 0 °C for 1 h. The reaction was indicated to be complete by LCMS. The reaction mixture was diluted with ice-cold water (20 mL), extracted with DCM (10 mL x 3), and the combined organic layers were washed with water (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to dryness to give the product N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(chloromethyl)phenyl]-carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrole-1-yl)hexamethylenediamide (compound 40, 80 mg, 53%), as a white solid. LCMS (ES, m / z): 591, 593 [M+H] +

[0737] Step 2. Synthesis of Compound 42

[0738] BH3-Me2S (10.00 mL, 105.4 mmol, 2.64 equivalents) was added dropwise to a stirred mixture of (2-chloro-4-nitrophenyl)acetic acid (compound 41, 8.60 g, 39.9 mmol, 1.00 equivalent) in THF (130 mL) at 0 °C. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 4 h. TLC (PE:EA = 1:2) indicated the reaction was complete. The mixture was cooled to room temperature. The resulting mixture was concentrated under vacuum. 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 (400MHz, CDCl3) δ 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).

[0739] Step 3. Synthesis of Compound 43

[0740] Bu₄NH₄ (10.37 g, 30.6 mmol, 0.80 equivalent) was added dropwise to a stirred mixture of 2-(2-chloro-4-nitrophenyl)ethanol (compound 42, 7.70 g, 38.2 mmol, 1.00 equivalent) and tert-butyl 2-bromoacetate (57.74 g, 296.0 mmol, 7.75 equivalent) in toluene (70 mL) at 0 °C. Over 30 h, a solution of NaOH (15.00 g, 375.0 mmol, 9.82 equivalent) in H₂O was added dropwise to the mixture at 0 °C. The resulting mixture was stirred for another 4 h at room temperature. TLC (PE:EA = 3:1) indicated the reaction was complete. The resulting mixture was extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted 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 (300MHz, CDCl3)δ 8.20 (d, J = 4.0Hz, 1H), 8.07-8.03 (m, 1H), 7.61 (d, J = 8.1Hz, 1H), 4.11 (s, 2H), 3.83 (t, J = 8.1Hz, 2H), 3.16 (t, J = 8.1Hz, 2H), 1.45 (s, 9H).

[0741] Step 4. Synthesis of Compound 44

[0742] TFA (20 mL) was added dropwise to a stirred mixture of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (compound 43, 12.20 g, 38.6 mmol, 1.00 equivalent) in a DCM (120 mL) at 0 °C. The resulting mixture was stirred at room temperature for 4 h. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. This yielded [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 44, 8.4 g, 83%) as a yellow solid. LCMS: (ES, m / s): 517 (2 M-H) -1 H NMR(400MHz,DMSO-d6)δ 12.64 (s, 1H), 8.20 (d, J = 4.0Hz, 1H), 8.11-8.08 (m, 1H), 7.72 (d, J = 8.0Hz, 1H), 4.06 (s, 2H), 3.74 (t, J = 8.0Hz, 2H), 3.06 (t, J = 8.0Hz, 2H).

[0743] Step 5. Synthesis of Compound 45

[0744] CH3NH2.HCl (2.69 g, 39.79 mmol, 1.23 equivalents) and DIEA (17.31 g, 133.93 mmol, 4.14 equivalents) were added to a stirred mixture of [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 44, 8.40 g, 32.35 mmol, 1.00 equivalents) and HATU (19.19 g, 50.47 mmol, 1.56 equivalents) in DMF (80 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 4 h. The reaction was indicated to be complete by LCMS. The reaction was quenched with water / ice. The resulting mixture was extracted with DCM (3 x 50 mL). The combined organic layers were washed with brine (50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with (DCM:MeOH = 10:1) to give 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 45, 7.2 g, 81%), as a yellow oil. LCMS (ES, m / s): 273, 275 (M+H) +

[0745] Step 6. Synthesis of Compound 46

[0746] At room temperature, BH3-THF (10M THF solution, 52.0 mL, 520.0 mmol, 20 equivalents) was added dropwise to a stirred mixture of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 45, 7.20 g, 26.40 mmol, 1.00 equivalents) in THF (70 mL). The resulting mixture was stirred at 70 °C for 4 h. The reaction was indicated by LCMS to be complete. The mixture was cooled to room temperature. The reaction was quenched with MeOH. The residue was acidified to pH 6 with 1N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was alkalized to pH 8 with saturated NaHCO3 (saturated aqueous solution). The resulting mixture was extracted with EtOAc (3 x 100 mL), washed with brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with (DCM:MeOH = 8:1) to give [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (compound 46, 5.4 g, 79%), as a yellow solid. LCMS: (ES, m / s): 259, 261 (M+H) + ; 1H NMR(400MHz,DMSO-d6)δ 8.26(d,J=4.0Hz,1H),8.15-8.12(m,1H),7.73(d,J=8.0Hz,1H),3.72(t,J=8.0Hz,2 H), 3.61 (t, J = 8.0Hz, 2H), 3.10 (t, J = 8.0Hz, 2H), 2.87 (t, J = 8.0Hz, 2H), 2.40 (s, 3H).

[0747] Step 7. Synthesis of Compound 47

[0748] At room temperature, a solution (20.00 mL) of NaHCO3 (4.00 g, 15.46 mmol, 1.00 equivalent) in H2O was added dropwise 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 equivalent) and Boc2O (3.80 g, 17.41 mmol, 1.13 equivalent) in THF (20.00 mL). The resulting mixture was stirred overnight at room temperature. The reaction was indicated by LCMS to be complete. The resulting mixture was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with (DCM:MeOH = 12:1) to give N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate tert-butyl ester (compound 47, 4.8 g, 77%) as a yellow solid.

[0749] LCMS:(ES,m / s):359,361(M+H) + ; 1 H NMR(400MHz,DMSO-d6)δ 8.24(d,J=4.0Hz,1H),8.13-8.10(m,1H),7.67(d,J=8.0Hz,1H),4.05-4.00(m,1H),3.69(t,J=8.0Hz ,2H),3.50(t,J=8.0Hz,2H),3.28(t,J=8.0Hz,2H),3.07(t,J=8.0Hz,2H),2.75(s,3H),1.36(s,9H).

[0750] Step 8. Synthesis of Compound 48

[0751] At room temperature, a stirred mixture of N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate tert-butyl ester (compound 47, 5.60 g, 15.6 mmol, 1.00 equivalent) in EtOH (112.00 mL) was added to a solution of NH4Cl (2.50 g, 46.74 mmol, 2.99 equivalent) in H2O (12.00 mL) and Fe (4.40 g, 78.79 mmol, 5.05 equivalent). The resulting mixture was stirred at 80 °C for 3 h. The reaction was indicated by LCMS to be complete. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine (30 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with (DCM:MeOH = 10:1) to give N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate tert-butyl ester (compound 48, 4.2 g, 81%), as a yellow oil. LCMS: (ES, m / s): 329, 331 (M+H) + ; 1 H NMR(400MHz, DMSO-d6)δ6.96(d,J=8.0Hz,1H),6.59(d,J=4.0Hz,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).

[0752] Step 9. Synthesis of Compound 49

[0753] A solution of LiAlH4 (92 mg, 2.43 mmol, 8.00 equivalent) in THF (3 mL) was added to a solution of N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate tert-butyl ester (compound 48, 100 mg, 0.30 mmol, 1.00 equivalent) in THF (3 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 hours. Five reactions were performed in parallel. LCMS indicated that the reaction was complete. The reaction was then quenched with 1N NaOH (10 mL), filtered, concentrated to dryness under vacuum, and the residue was then purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), gradient from 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 g (180 mg, 44%), as a yellow oil. LCMS (ES, m / z): 243, 245 [M+H] +

[0754] Step 10. Synthesis of Compound 50

[0755] Diphosgene (137 mg, 0.69 mmol, 1.20 equivalence) was added to a solution of 3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]aniline (compound 49, 140 mg, 0.58 mmol, 1.00 equivalence) in THF (9 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 0 °C for 1 h. The reaction solution was then concentrated to dryness under vacuum. The residue was redissolved in DMF (2 mL) and then added dropwise under a nitrogen atmosphere to a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (158 mg, 0.58 mmol, 1.00 equivalence) and TEA (117 mg, 1.15 mmol, 2.00 equivalence) in DMF (4 mL). The resulting mixture was stirred at room temperature for 16 h. The reaction was indicated by LCMS to be complete. The reaction mixture was diluted with methanol, and the resulting solution was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), gradient from 0% to 50% over 30 min; detector, UV 254 nm, yielding 100 mg of product as a colorless solid. The crude product was purified by preparative HPLC under the following conditions: column: XBridge Shield RP18 OBD column, 19 x 250 mm, 10 μm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 14% to 32% over 7 min; 220 nm; RT1: 5.25 min. The collected fractions were freeze-dried to give 1-(3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 50, 60 mg, 18%), as a colorless solid. LCMS (ES, m / z): 542, 544 [M+H]+

[0756] Step 11. Synthesis of compound (Ig)

[0757] At room temperature, in air, N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(chloromethyl)phenyl]-carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrolo-1-yl)hexamethylene (compound 40, 66 mg, 0.11 mmol, 1.00 equivalent), 1-(3-chloro-4-[2-[2-(dimethylamino)ethyl) [2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 50, 60 mg, 0.11 mmol, 1.00 equivalent) and DIEA (29 mg, 0.22 mmol, 2.00 equivalent) were added to a solution of DMF (1 mL) with TBAI (4 mg, 0.01 mmol, 0.10 equivalent). The resulting mixture was stirred at room temperature for 16 hours. LCMS traces indicated the reaction was complete. The resulting mixture was purified by reversed-phase column chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.05% TFA), 5% to 45% gradient over 40 min; detector, UV 254 nm, yielding 90 mg of crude product as a yellow oil. The crude product was then further 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: 15B to 35B over 7 min; 220 nm; RT1: 6.00 min, yielded ([4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexamido]-3-methylbutamido]pentamido]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]dimethylammonium, compound (Ig) (19 mg, 14.8%), as a white solid. LCMS (ES, m / z): 1096 [M-FA] + ,549[1 / 2(M-FA)] + ; 1H NMR(400MHz,CD3OD)δ 8.48(s,1H),7.77-7.72(m,3H),7.55-7.47(m,3H),7.37-7.35(d,J=8.4H z,2H),7.18-7.14(m,2H),6.77(s,2H),5.17-5.13(q,J=8,4Hz,1H),4.51 -4.46(m,5H),4.35(s,2H),4.12(d,J=8.0Hz,1H),3.90(s,2H),3.79(t,J=5.6Hz,2H),3.45(t,J=7. 2Hz,4H),3.22-3.15(m,1H),3.11-3.05(m,1H),3.00(t,J=6.0Hz,2H),2.92(s,6H),2.89-2.84(m,1 H),2.81-2.73(m,1H),2.54-2.43(m,1H),2.27(t,J=7.2Hz,2H),2.21-2.12(m,1H),2.10-2.02(m,1 H),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).

[0758] Schemes 9A and 9B illustrate how to prepare a complex of the novel degradative agent P13 with a peptide linker.

[0759]

[0760]

[0761] Scheme 10 illustrates the synthesis of the compound of formula (Ih).

[0762]

[0763] Step 1. Synthesis of Compound 63

[0764] SOCl2 (25 mL) was added to a stirred mixture of 3-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionic acid (compound 62, 5.00 g, 16.06 mmol, 1.00 equivalent) at room temperature. The resulting mixture was stirred at 80 °C for 16 h. The desired product (a derivative with MeOH, MS = 326) was detected by LCMS. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under vacuum to give N-(3-chloro-3-oxopropyl)carbamate 9H-fluorene-9-ylmethyl ester (compound 63, 7.5 g, crude), as a yellow oil. The crude product was used directly in the next step without further purification. 11H NMR analysis indicated that it was the expected product (a derivative of MeOH). 1 H-NMR(300MHz,CDCl3)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 =3.0Hz, 2H), 4.24 (t, J = 6.0Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J = 3.0Hz, 2H), 2.59 (t, J = 6.0Hz, 2H).

[0765] Step 2. Synthesis of Compound 66

[0766] Methyl (2S,3S,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-bromooxane-2-carboxylate (compound 64, 10.00 g, 25.17 mmol, 1.00 equivalent) was added in portions to a stirred solution of 4-formyl-2-nitrophenol (compound 65, 4.21 g, 25.19 mmol, 1.00 equivalent) and Ag₂O (7.00 g, 30.20 mmol, 1.20 equivalent) in ACN (100 mL, 190.24 mmol, 75.00 equivalent) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction was indicated by LCMS to be complete. The resulting mixture was filtered, and the filter cake was washed with DCM (50 mL x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (PE:EA = 1:2) to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 66, 10.5 g, 86%), as a white solid. 1 ¹H-NMR analysis indicated it was the expected product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR(300MHz,CDCl3)δ 10.00(s,1H),8.34(s,1H),8.13-8.09(m,1H),7.52(d,J=3.0Hz,1H),5.4 7-5.29(m,4H),4.37-4.35(m,1H),3.75-3.73(m,3H),2.17-2.06(m,9H).

[0767] Step 3. Synthesis of Compound 67

[0768] NaBH4 (0.47 g, 12.42 mmol, 1.00 equivalent) was added in portions to a stirred solution of (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 equivalent) in 50 mL of MeOH at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 h. The reaction was indicated to be complete by LCMS. The reaction was quenched with water at room temperature. The result was dried over Na2SO4. The resulting mixture was filtered, and the filter cake was washed with DCM. The resulting mixture was concentrated under vacuum to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 67, 5.5 g, 91%), as a solid. LCMS (ES, m / z): 486 [M+H] + .

[0769] Step 4. Synthesis of Compound 68

[0770] Pd / C (1.10 g, 10%) was added in portions to a stirred mixture of (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 equivalent) in EA (60 mL) at room temperature. The resulting mixture was stirred at room temperature under H2 atmosphere for 16 h. The reaction was indicated by LCMS to be complete. The resulting mixture was filtered, the filter cake was washed with DCM and MeOH, and the filtrate was concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 68, 4.0 g, 77%) as a solid. The crude product was used directly for the next step without further purification. LCMS(ES,m / z): 456[M+H] + .

[0771] Step 5. Synthesis of Compound 70

[0772] N-(3-chloro-3-oxopropyl)carbamate 9H-fluorene-9-ylmethyl ester (compound 69, 0.87 g, 2.62 mmol, 1.20 equivalent) was added in portions to a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid (compound 68, 1.00 g, 2.19 mmol, 1.00 equivalent) and NaHCO3 (0.20 g, 2.40 mmol, 1.1 equivalent) in 10 mL of THF at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 0 °C under a nitrogen atmosphere for 6 h. The reaction was indicated to be complete by LCMS. 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 (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (Compound 70, 1.1 g, 66%), as a pale yellow solid. LCMS (ES, m / z): 749 [M+H] + .

[0773] Step 6. Synthesis of Compound 72

[0774] DIEA (0.52 g, 4.01 mmol, 2.00 equivalent) was added in portions to a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 70, 1.50 g, 2.00 mmol, 1.00 equivalent) and bis(4-nitrophenyl) carbonate (compound 71, 0.68 g, 2.24 mmol, 1.12 equivalent) in 15 mL of DMF at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction was indicated by LCMS to be complete. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), in a 10% to 90% gradient over 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness under vacuum to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 72, 1.4 g, 48%), as a yellow solid. LCMS (ES, m / z): 914 [M+H] + .

[0775] Step 7. Synthesis of Compound 73

[0776] Methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 72, 1.00 g, 1.09 mmol, 1.00 equivalent) and 1-(3-chloro-4-[2-[2-(methylamino)ethyl) [2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (new degrader P1, 0.58 g, 1.09 mmol, 1.00 equivalent) was added in portions to a stirred mixture in DMF (10 mL) with HOBT (1.18 g, 8.72 mmol, 8.00 equivalent) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equivalent). The resulting mixture was stirred at room temperature under N2 atmosphere for 16 h. LCMS indicated the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), 10% to 80% gradient over 40 min; detector, UV 254 nm. The collected fraction was concentrated under vacuum to obtain (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-fluorene-9-ylmethoxy)carbonyl]amino]propamido)phenoxy]oxane-2-carboxylic acid methyl ester (Compound 73 (800 mg, 56%), as a solid. LCMS (ES, m / z): 1302 [M+H] + .

[0777] Step 8. Synthesis of Compound 74

[0778] At room temperature and under a N2 atmosphere, (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-fluorene-9-ylmethoxy)carbonyl]amino]propionamide Methyl 2-carboxylate (compound 73, 800.00 mg, 0.61 mmol, 1.00 equivalent) was added in portions to a stirred mixture in THF (80 mL) with HCl (6 N, 80 mL). The resulting mixture was stirred at 50 °C under a nitrogen atmosphere for 3 h. LC-MS indicated the reaction was complete. The resulting mixture was concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1%). FA), gradient from 0% to 80% over 40 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 74, 230 mg, 32%), as a white solid. LCMS (ES, m / z): 1162 [M+H] + .

[0779] Step 9. Synthesis of Compound 75

[0780] Piperidine (0.4 mL) was added in portions 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-fluorene-9-ylmethoxy)carbonyl]amino]propamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid, i.e., compound 74 (230 mg, 0.2 mmol, 1.00 equivalent), in DMF (2 mL). The resulting mixture was stirred in portions for 10 min at room temperature under nitrogen atmosphere. The reaction was indicated as complete by LCMS. The resulting mixture was directly subjected to preparative HPLC under the following conditions (column: XSelect CSHPrep Cl8 OBD column, 19x250mm, 5µm; mobile phase A: water (0.05%)). TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20B to 40B over 7 min; 220 nm; RT1: 5.78 min) further purified to give (2S,3S,4S,5R,6S)-6-[2-(3-aminopropionamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 75, 35 mg, 18%), as a white solid. LCMS (ES, m / z): 940 [M+H] + .

[0781] Step 10. Synthesis of compound (Ih)

[0782] DIEA (30 mg, 0.23 mmol, 2.0 equivalent) was added in portions to a stirred solution of (2S,3S,4S,5R,6S)-6-[2-(3-aminopropamido)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxoperidin-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 equivalent) and bis(2,5-dioxoperidin-1-yl)glutarate (compound 76, 46 mg, 0.14 mmol, 1.2 equivalent) in DMF (2.0 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. LC-MS indicated the reaction was complete. The reaction mixture was purified by preparative HPLC under the following conditions (column: Kinetex EVO 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% within 7 min). B, 41% B; Wavelength: 254 nm; RT1 (min): 5.8. The collected fractions were freeze-dried to give (2S,3S,4S,5R,6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiridine-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-(3-{5-[(2,5-dioxopirol-1-yl)oxy]-5-oxopentamido}propamido)phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid (compound (Ih), 48 mg, 34%, as a white solid. LCMS (ES, m / z): 1151 [M+H] + 1173[M+Na] + . 1H-NMR(300MHz,DMSO-d6):12.80(br s,1H),10.98(s,1H),9.08(s,1H),8.79(s,1H),8.18(s,1H),7.96(s,1H),7.68-7.66(m,2H),7.51(s,1H),7.44(d,J= 8.1Hz,1H),7.25-7.00(m,4H),6.82-6.80(m,1H),5.86(s,IH),5.39-5.30(m,2H),5.14-5.07(m,1H),4.97(s,2H),4. 84(d,J=7.2Hz,1H),4.47-4.27(m,4H),3.90(d,J=9.6Hz,1H),3.56-3.48(m,4H),3.45-3.36(m,6H),2.95-2.80(m,8H ),2.75-2.65(m,3H),2.62-2.55(m,2H),2.49-2.35(m,1H),2.21-2.16(m,2H),2.01-1.95(m,1H),1.85-1..80(m,2H).

[0783]

[0784] Step 1. Synthesis of Compound 76

[0785] TEA (104 mg, 1.02 mmol, 3.0 equivalent) and 4-(chlorosulfonyl)-3-nitrobenzoic acid (181 mg, 0.68 mmol, 2.00 equivalent) were added in portions to a stirred solution of 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound P1, 180 mg, 0.34 mmol, 1.00 equivalent) in DMF (8 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 0 °C under a nitrogen atmosphere for 4 h. The reaction was indicated by LCMS to be complete. The resulting mixture was used for further purification. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), in a 10% to 60% gradient over 10 min; detector, UV 254 nm. The mixture was lyophilized to give 4-[[2-(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)aminosulfonyl]-3-nitrobenzoic acid (compound 76, 70 mg, 27%), as a pale yellow solid. LCMS (ES, m / z): 757 [M+1]+ .

[0786] Step 2 Synthesis of compound (Ii)

[0787] HATU (45 mg, 0.12 mmol, 1.5 equivalent), 1-(2-aminoethyl)pyrrole-2,5-dione hydrochloride (Compound 77, 17 mg, 0.10 mmol, 1.20 equivalent), and DIEA (31 mg, 0.24 mmol, 3.0 equivalent) were added in portions to a stirred mixture of 4-[[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)aminosulfonyl]-3-nitrobenzoic acid (Compound 76, 60 mg, 0.08 mmol, 1.00 equivalent) in DMF (6 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 4 h. The reaction was indicated by LCMS to be complete. The residue was purified by preparative HPLC (column: XBridge Prep Phenyl OBD column, 19x150mm 5um 13nm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25mL / min; gradient: 25B to 43B over 10 min; 220nm; RT1: 11.97min). The collected fractions were lyophilized to give 4-[[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)aminosulfonyl]-N-[2-(2,5-dioxopiror-1-yl)ethyl]-3-nitrobenzamide (compound (Ii), 27mg, 36%), as a white solid. LCMS (ES, m / z): 879,881 [M+H]. 1H NMR(300MHz,DMSO-d6)δ11.00(s,1H),9.01(t,J=6.0Hz,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=3.0Hz,1H),7.21-7.12(m,2H),7.02(s,2H),6.8 4(t,J=6.0Hz,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).

[0788]

[0789] Step 1. Synthesis of Compound 79

[0790] Pyridine (2.59 g, 32.74 mmol, 1.16 equivalent) was added dropwise to a stirred mixture of (2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]acetamido)acetic acid (compound 78, 10.00 g, 28.22 mmol, 1.00 equivalent) and Pb(OAc)4 (15.02 g, 33.86 mmol, 1.20 equivalent) in THF (300 mL) and toluene (100 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred overnight at 80 °C under a nitrogen atmosphere. The reaction was indicated by LCMS to be complete. The mixture was cooled 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 and 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 methyl (2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]acetamido)acetate (compound 79, 6.5 g, 56%), as a white solid. LCMS (ESI, ms): 391 [M+Na] + . 1HNMR(300MHz, CDCl3)δ 7.80(d,J=7.5Hz,2H),7.62(d,J=7.5Hz,2H),7.45(t,J=7.5Hz,2H),7.36(d,J=7.5Hz,2H),7.18(br s,1H),5.48(br s, 1H), 5.28 (d, J = 7.2Hz, 2H), 4.48 (d, J = 6.6Hz, 2H), 4.26 (t, J = 6.6Hz, 1H), 3.93 (d, 5.4Hz, 2H), 2.08 (s, 3H).

[0791] Step 2. Synthesis of Compound 81

[0792] PPTS (400 mg, 1.59 mmol, 0.29 equivalents) was added to a stirred mixture of (2-[[(9H-fluorene-9-ylmethoxy)carbonyl]-amino]acetamido)acetate, compound 79 (2.00 g, 5.43 mmol, 1.00 equivalents) and 2-(2-chloro-4-nitrophenyl)ethanol (compound 3, 3.20 g, 15.85 mmol, 2.92 equivalents) in DCM (40 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred overnight at 45 °C under a nitrogen atmosphere. 40% of the desired product was detected by LCMS. The mixture was cooled to room temperature. The reaction 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) 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 (1:9) to give N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate 9H-fluorene-9-ylmethyl ester (compound 81, 1.7 g, 55%), as a white solid. LCMS (ESI, ms): 510, 512 [M+H] + . 1 HNMR(300MHz,DMSO-d6):δ 8.58(t,J=5.1Hz,1H),8.22(dd,J=12,2.4Hz,1H),7.89(d,J=7.5Hz,1H),7.71-7.54(m,4H),7.43 -7.29(m,4H),4.56(d,J=6.9Hz,2H),4.30-4.16(m,3H),3.70-3.61(m,4H),3.04(t,J=6.3Hz,2H).

[0793] Step 3. Synthesis of Compound 82

[0794] Piperidine (1.0 mL) was added in portions to a stirred mixture of N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate 9H-fluorene-9-ylmethyl ester (compound 81, 1.60 g, 3.14 mmol, 1.00 equivalent) in DMF (5.0 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. The reaction was indicated to be complete by LCMS. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.05% TFA), gradient from 0% to 50% over 40 min; detector, UV 254 nm. This yielded 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] +

[0795] Step 4. Synthesis of Compound 83

[0796] A solution of NaHCO3 (477 mg, 5.68 mmol, 2.18 equivalents) in H2O was added dropwise 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 equivalents) and Boc2O (580 mg, 2.66 mmol, 1.02 equivalents) in DMF (10.00 mL) at 0 °C. The resulting mixture was stirred at room temperature for 3 h. The reaction was indicated to be complete by LCMS. The reaction was quenched by adding water (20 mL). The resulting mixture was extracted with EtOEt (3 x 20 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (1:2) to give N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]tert-butyl carbamate (compound 83, 650 mg, 58%), as a yellow oil. LCMS (ESI, ms), 388 [M+H] + ,332[M+H-56] + . 1HNMR(400MHz, CDCl3)δ 8.21(d,J=2.4Hz,1H),8.04(d,J=8.4Hz,2H),7.46(d,J=8.4Hz,1H),7.05(br s,1H),5.25(br s, 1H), 4.73 (d, J = 7.2Hz, 2H), 3.81-3.73 (m, 4H), 3.34-3.32 (m, 2H), 3.08 (t, J = 6.8Hz, 2H), 1.42 (s, 9H).

[0797] Step 5. Synthesis of Compound 84

[0798] At room temperature, a solution (3.00 mL) of NH₄Cl (910 mg, 17.01 mmol, 10.1 equivalents) in H₂O was added dropwise to a stirred mixture of N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]-carbamoyl)methyl]carbamate (compound 83, 650 mg, 1.68 mmol, 1.00 equivalents) and Fe (260 mg, 4.66 mmol, 2.78 equivalents) in EtOH (9.00 mL). The resulting mixture was stirred at 90 °C for 4 h. The reaction was completed by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under vacuum. The resulting mixture was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (1:1) to give N-[([[2-(4-amino-2-chlorophenyl)ethoxy]methyl]-carbamoyl)methyl]tert-butyl carbamate (compound 84, 500 mg, 83%), as a yellow solid. LCMS (ESI, ms): 358 [M+H] + 380[M+Na] + . 1 HNMR(300MHz, CDCl3)δ 7.02-6.96(m,2H),6.68(d,J=2.4Hz,1H),6.52-6.49(m,1H),5.29(br s, 1H), 4.74 (d, J = 6.9Hz, 2H), 3.80-3.78 (m, 2H), 3.69-3.63 (m, 2H), 2.88 (t, J = 7.2Hz, 2H), 1.45 (s, 9H).

[0799] Step 6. Synthesis of Compound 86

[0800] TEA (300 mg, 2.96 mmol, 2.12 equivalents) was added 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 equivalents) and CDI (450 mg, 2.78 mmol, 1.99 equivalents) in 5.00 mL of DMF at 0 °C. The resulting mixture was stirred at room temperature for 2 h. N-[([[2-(4-amino-2-chlorophenyl)ethoxy]methyl]carbamoyl)methyl]tert-butyl carbamate (compound 84, 500 mg, 1.40 mmol, 1.00 equivalents) and DMAP (550 mg, 4.50 mmol, 3.22 equivalents) were added in portions to the above mixture. The resulting mixture was stirred overnight at 60 °C. The reaction was indicated by LCMS to be complete. The mixture was cooled to room temperature. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), 0% to 50% gradient over 30 min; detector, UV 254 nm. This yielded 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)tert-butyl 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] + .

[0801] Step 7. Synthesis of Compound 87

[0802] TFA (1.00 mL) was added to a stirred mixture of 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)-tert-butyl carbamate (compound 86, 530 mg, 0.80 mmol, 1.00 equivalent) in DCM (5.00 mL). The resulting mixture was stirred at 0 °C for 30 min. The reaction was indicated to be complete by LCMS. The resulting mixture was concentrated under reduced pressure. This produces 2-amino-N-[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]acetamide trifluoroacetic acid (compound 87, (510 mg, purity: 64%, yield: 60%), as a grayish-white solid. LCMS (ESI, ms): 557 [M+H-TFA] +

[0803] Step 8. Synthesis of Compound 89

[0804] A solution (40.00 mL) of Boc₂O (1.86 g, 8.52 mmol, 1.20 equivalent) in DMF was added dropwise to a stirred mixture of (2S)-2-[2-(2-aminoacetamido)acetamido]-3-phenylpropionic acid (compound 88, 2.00 g, 7.16 mmol, 1.00 equivalent) and NaHCO₃ (1.80 g, 21.41 mmol, 3.00 equivalent) in H₂O (40.00 mL). The resulting mixture was stirred overnight at room temperature. The reaction was indicated to be complete by LCMS. 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) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, Cl8 silica gel; mobile phase, aqueous solution of ACN (0.05% TFA), gradient from 5% to 60% over 30 min; detector, UV 220 nm. This yielded (2S)-2-(2-[2-[(tert-butoxycarbonyl)amino]acetamido]acetamido)-3-phenylpropionic acid (compound 89, 1.8 g, 60%), as a white semi-solid. LCMS (ESI, ms): 380 [M+H] + 324[M+H-56] + . 1HNMR: (300MHz, DMSO-d6)δ 8.17(d,J=8.1Hz,1H),7.93(t,J=5.7Hz,1H),7.31-7.20(m,5H),7.00(t,J=6.0Hz,1H),4.46-4.39( m,1H),3.78-3.67(m,2H),3.56(d,J=5.7Hz,2H),3.09-3.02(m,1H),2.92-2.73(m,1H),1.39(s,9H).

[0805] Step 9. Compound 90

[0806] HOBT (102 mg, 0.75 mmol, 0.84 equivalent) was added in portions to a stirred mixture of (2S)-2-(2-[2-[(tert-butoxycarbonyl)amino]acetamido]-acetamido)-3-phenylpropionic acid (compound 89, 340 mg, 0.90 mmol, 1.00 equivalent) and HATU (340 mg, 0.90 mmol, 1.00 equivalent) in DMF (5.00 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 30 min. 2-Amino-N-[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]acetamide trifluoroacetic acid (compound 87, 511 mg, purity: 64%, 0.48 mmol, 0.54 equivalents) and DIEA (340 mg, 2.63 mmol, 2.94 equivalents) were added to the above mixture at 0 °C. The resulting mixture was stirred at room temperature for another 2 h. The reaction was indicated to be complete by LCMS. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), 0% to 50% gradient over 30 min; detector, UV 220 nm. The collected fractions were concentrated under vacuum. This produces 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]tert-butyl carbamate (compound 90, 210 mg, 48%), as a grayish-white solid. LCMS (ESI, ms): 918 [M+H] + 818[M+H-100] + . 1HNMR:(400MHz,DMSO-d6):δ 10.97(s,1H),8.79(s,1H),8.50(t,J=6.4Hz,1H),8.31(t,J=4,4Hz,1H),8.15(d,J=9.6Hz,1H),7.910(t,J=8,0Hz ,1H),7.68-7.64(m,2H),7.49(s,1H),7.43(d,J=9.6Hz,1H),7.24-7.12(m,7H),7.00-6.95(m,1H),6.84(t,J=6.4H z,1H),5.13-5.06(m,1H),4.55-4.27(m,7H),3.72-3.60(m,6H),3.75-3.67(m,3H),3.59-3.49(m,5H),3.07-3.01( m,1H),2.94-2.73(m,4H),2.62-2.54(m,1H),2.40-2.3l(m,1H),2.01-1.94(m,1H),2.00-1.91(m,1H),1.35(s,9H)

[0807] Step 10. Synthesis of Compound 91

[0808] TFA (1.00 mL) was added dropwise to a stirred mixture of 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]tert-butyl carbamate (compound 90, 140 mg, 0.15 mmol, 1.00 equivalent) in DCM (5.00 mL). The resulting mixture was stirred at 0 °C for 30 min. The reaction was indicated to be complete by LCMS. The resulting mixture was concentrated under reduced pressure. This produces (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-phenylpropionamide trifluoroacetic acid (compound 91, 140 mg, 79%), as a grayish-white solid. LCMS (ESI, ms): 818 [M+H-TFA] + .

[0809] Step 11. Synthesis of compound (Ij)

[0810] 2,5-dioxopyrrolo-1-yl ester of 6-(2,5-dioxopyrrolo-1-yl)hexanoate (2S)-2-[2-(2-aminoacetamido)acetamido]-N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethoxy)methyl]carbamoyl]methyl)-3-phenylpropionamide trifluoroacetic acid (compound 91, 140 mg, 0.15 mmol, 1.00 equivalent) and DIEA (70 mg, 0.54 mmol, 3.61 equivalent) in a stirred mixture of DMF (2.00 mL) was added in portions. The reaction mixture was directly purified under the following conditions: column: XSelect CSH Prep C18 OBD column, 19x250 mm, 5 μm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 25 B to 50 B over 7 min; 254 nm; RT1: 6.35 min; the collected fractions were lyophilized to obtain the crude product. The crude product was further purified under the following conditions: column: Kinetex EVO C18 column, 30x150 mm, 5 μm; mobile phase A: water (0.05% FA); mobile phase B: water (0.1% FA); mobile phase C: water (0.1% FA); mobile phase D: water (0.1% FA); mobile phase E: water (0.1% FA); mobile phase B: water (0.1% FA); mobile phase C: water (0.1% FA); mobile phase D ... TFA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 20B to 40B over 7 min, 220 nm; RT1: 6.77 min; the collected fractions were lyophilized to obtain N-[[([[(1S)-1-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiridin-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-dioxopiror-1-yl)hexamethyleneamide (compound (Ik), 22.8 mg, 14%), as a grayish-white solid. LCMS (ESI, ms): 1011 [M+H] + . 1HNMR:(400MHz,DMSO-d6):δ 10.95(s,1H),8.79(s,1H),8.51(t,J=8,4Hz,1H),8.29(t,J=8.0Hz,1H),8.12-8.01(m,3H),7.70-7.66(m,2H),7 .44(s,1H),7.42(d,J=8.0Hz,1H),7.23-7.16(m,7H),6.99(s,2H),6.82(t,J=8.0Hz,1H),5.13-5.09(m,1H),4.5 5-4.28(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)

[0811]

[0812]

[0813] Step 1. Synthesis of Compound 94

[0814] BH3-Me2S (28.00 mL, 295.23 mmol, 2.65 equivalents) was added dropwise to a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 93, 24.00 g, 111.32 mmol, 1.00 equivalent) in THF (240.00 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 hours. TLC (PE:EtOAc = 3:1) indicated the completion of the reaction. After cooling to room temperature, the resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (3:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 94, 18.00 g, 80%) as a pale yellow solid. 1 H NMR (300MHz, CDCl3) δ 8.27 (s, 1H), 8.10-8.07 (m, 1H), 7.52 (d, J = 3Hz, 1H), 3.96 (t, J = 6Hz, 2H), 3.13 (t, J = 6Hz, 2H).

[0815] Step 2. Synthesis of Compound 95

[0816] NBS (6.62 g, 1.50 equivalent) and PPh3 (9.76 g, 37.21 mmol, 1.50 equivalent) were added in portions to a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 94, 5.00 g, 24.80 mmol, 1.00 equivalent) in DCM (100.00 mL) at room temperature under N2. The resulting mixture was stirred overnight at room temperature under N2. TLC (PE:EtOAc = 10:1) indicated that the reaction was complete. The reaction mixture was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (4:1), to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 95, 5.10 g, 72%) as a red oil. 1 H NMR (400MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.18 ( dd, J = 8.4, 2.4 Hz, 1H), 7.73 ( d, J = 8.4 Hz, 1H), 3.79 4 ( t, J = 6.8 Hz, 2H), 3.38 ( t, J = 6.8 Hz, 2H).

[0817] Step 3. Synthesis of Compound 96

[0818] Potassium thioacetate (2.16 g, 18.90 mmol, 1.00 equivalent) was added to a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 95, 5.00 g, 18.90 mmol, 1.00 equivalent) in DMF (50.00 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature for 2 hours. TLC (PE:EtOAc = 10:1) indicated the reaction was complete. The reaction mixture was diluted with water (600.00 mL) and extracted with EtOAc (2000 mL x 3). The combined organic layers were washed with water (200.00 mL) and brine (200.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under vacuum to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]ethenone (compound 96, 4.50 g, 85%) as a red oil. 1 H NMR (400MHz, CDCl3) δ8.24 (d, J = 2.4Hz, 1H), 8.07 (dd, J = 8.4, 2.4Hz, 1H), 7.45 (d, J = 8.4Hz, 1H), 3.20 -3.05 (m, 4H), 2.34 (s, 3H).

[0819] Step 4. Synthesis of Compound 97

[0820] MeONa (6.93 mL, 37.33 mmol, 5.00 equivalent, 30% MeOH solution) was added to a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]ethenone (compound 96, 2.00 g, 7.70 mmol, 1.00 equivalent) in MeOH (300.00 mL) at 0 °C under N2. The resulting mixture was stirred at 0 °C under N2 for 1 h. TLC (PE:EtOAc = 10:1) indicated that the reaction was complete. The reaction 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 97, 1.35 g, 72%) as a pale yellow oil. 1 H NMR (400MHz, CDCl3) δ8.26 (d, J=2.4Hz, 1H), 8.09 (dd, J=8.4, 2.4Hz, 1H), 7.45 (d, J=8 .4Hz, 1H), 3.14 (t, J = 8.0Hz, 2H), 2.85 (dt, J = 8.0, 7.2Hz, 2H), 1.43 (t, J = 7.2Hz, 1H).

[0821] Step 5. Synthesis of Compound 99

[0822] TSTU (25.18 g, 83.52 mmol, 1.30 equivalent) and DIEA (16.60 g, 128.48 mmol, 2.00 equivalent) were added to a stirred solution of (2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionic acid (compound 98, 20.00 g, 64.24 mmol, 1.00 equivalent) in DMF (200.00 mL) under air atmosphere at room temperature. The resulting mixture was stirred at room temperature for 1 h. The reaction was indicated by LCMS to be complete. The reaction mixture was diluted with water (200.00 mL) and extracted with EtOAc (100.00 mL x 3). The combined organic layers were washed with water (100.00 mL) and brine (100.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with (PE:EtOAc = 1:2) to give (2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionic acid 2,5-dioxopyrrolidine-1-yl ester (Compound 99, 25.00 g, 83%), as a white solid. LCMS (ES, m / z): 431 [M+Na] + .

[0823] Step 6. Synthesis of Compound 100

[0824] A solution of (2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionic acid 2,5-dioxopyrrolidine-1-yl ester (compound 99, 5.00 g, 12.24 mmol, 1.00 equivalent) in DMF (50.00 mL) was added to a solution of D-alanine (1.09 g, 0.012 mmol, 1.00 equivalent) in water (50.00 mL). The resulting mixture was stirred at room temperature for 2 h. The reaction was indicated to be complete by LCMS. The pH of the reaction mixture was adjusted to 2–3 with 2N HCl. The resulting mixture was extracted with EtOAc (100.00 mL x 3), and the combined organic layers were washed with brine (100.00 mL x 3), dried over anhydrous Na₂SO₄, and concentrated to dryness under vacuum to give (2R)-2-[(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]-amino]propamido]propionic acid (compound 100, 4.00 g, 71%), as a white solid. LCMS (ES, m / z: 383 [M+H]) +

[0825] Step 7. Synthesis of Compound 101

[0826] A solution of (2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionic acid 2,5-dioxopyrrolidine-1-yl ester (compound 99, 20.00 g, 48.97 mmol, 1.00 equivalent) in DMF (200.00 mL) was added to a solution of glycine (3.68 g, 48.97 mmol, 1.00 equivalent) in water (200.00 mL). The reaction was stirred at room temperature for 2 h. The reaction was indicated to be complete by LCMS. The pH of the reaction was adjusted to 2–3 with 2N HCl. The resulting mixture was extracted with EtOAc (500.00 mL x 3), and the combined organic layers were washed with brine (500.00 mL), dried over anhydrous Na₂SO₄, and concentrated to dryness under vacuum to give [(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionamido]acetic acid (compound 101, 15.00 g, 71%), as a white solid. LCMS (ES, m / z): 369 [M+H] +

[0827] Step 8. Synthesis of Compound 102

[0828] A solution of [(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propamido]-acetic acid (compound 101, 5.00 g, 13.57 mmol, 1.00 equivalent), Pb(OAc)4 (7.22 g, 16.28 mmol, 1.20 equivalent), and pyridine (1.29 g, 16.31 mmol, 1.20 equivalent) in THF (300.00 mL) / toluene (100.00 mL) was stirred at 80 °C for 16 h under N2. The reaction was indicated to be complete by LC-MS. After cooling to room temperature, the reaction mixture was filtered. The filter cake was washed with THF (100.00 mL). The combined organic layers were concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with (PE:EtOAc = 1:2) to give methyl [(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionamido]acetate (compound 102, 2.50 g, 45%), as a white solid. LCMS (ES, m / z): 405 [M + Na] + . 1 H NMR (400MHz, chloroform-d) δ7.77(t,J=7.6Hz,2H),7.58(d,J=7.6Hz,2H),7.43–7.37(m,2H),7.36–7.29(m,2H),7. 10(s,1H),5.24(d,J=7.6Hz,2H),4.51–4.35(m,2H),4.23-4.09(m,2H),2.04(s,3H),1.39(d,J=6.8Hz,3H).

[0829] Step 9. Synthesis of Compound 103

[0830] TFA (0.27 mL, 2.37 mmol, 0.62 equivalents) was added to a stirred solution of [(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]-propamido]acetate (compound 102, 2.25 g, 5.88 mmol, 1.00 equivalent) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 97, 1.28 g, 5.88 mmol, 1.00 equivalent) in 120 mL of DCM under N2 at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was indicated to be complete by LCMS. The reaction was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with (PE:EtOAc = 1:4) to give N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]ethyl]carbamate 9H-fluorene-9-ylmethyl ester (compound 103, 3.10 g, 90%), as a yellow solid. LCMS (ES, m / z): 540 [M+H] +

[0831] Step 10. Synthesis of Compound 104

[0832] Piperidine (31.00 mL) was added to a solution of N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]ethyl]carbamate 9H-fluorene-9-ylmethyl ester (compound 103, 3.10 g, 5.74 mmol, 1.00 equivalent) in DMF (155.00 mL) at 0 °C under N2. The resulting mixture was stirred at 0 °C under N2 for 0.5 h. The reaction was indicated to be complete by LCMS. 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 layers were washed with brine (200.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under vacuum to give 3.00 g of crude product. The crude product was further purified by silica gel column chromatography, eluting with (DCM:MeOH = 3:1) to give (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)acrylamide, 104 (1.50 g, 78%), as a yellow oil. LCMS (ES, m / z): 318 [M+H] + .

[0833] Step 11. Synthesis of Compound 105

[0834] At room temperature, a solution of NaHCO3 (0.59 g, 7.08 mmol, 1.50 equivalent) in H2O and Boc2O (1.03 g, 4.72 mmol, 1.00 equivalent) in DMF (75.00 mL) was added to a solution of (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)acrylamide (compound 104, 1.50 g, 4.72 mmol, 1.00 equivalent) in DMF (10.00 mL) and Boc2O (1.03 g, 4.72 mmol, 1.00 equivalent) were added. The reaction was stirred at room temperature for 1 h. The reaction was indicated to be complete by LCMS. The reaction mixture was diluted with water (500.00 mL) and extracted with EtOAc (200.00 mL x 3). The combined organic layers were washed with brine (200.00 mL x 3), dried over anhydrous Na₂SO₄, and concentrated to dryness under vacuum to give N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]ethyl]tert-butyl carbamate (compound 105, (1.82 g, 83 g),) as a red oil. LCMS (ES, m / z): 418 [M+H] + 318[M+H-100] +

[0835] Step 12. Synthesis of Compound 106

[0836] A slurry of N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]-methyl)carbamoyl]ethyl]carbamate tert-butyl ester (compound 105, 1.82 g, 4.36 mmol, 1.00 equivalent), iron powder (2.43 g, 0.04 mmol, 10.00 equivalent), and NH4Cl (2.33 g, 0.04 mmol, 10.00 equivalent) in EtOH (100.00 mL) / H2O (50.00 mL) was stirred at 70 °C for 2 h. The reaction was indicated to be complete by LCMS. The reaction mixture was filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was concentrated to dryness, and the residue was purified by silica gel column chromatography, eluting with (DCM:MeOH = 13:1) to give N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]thioalkyl]methyl)carbamoyl]ethyl]-tert-butyl carbamate (compound 106, 1.20 g, 68%), as a yellow oil. LCMS (ES, m / z): 388 [M+H] +

[0837] Step 13. Compound 107

[0838] CDI (209.00 mg, 1.29 mmol, 1 equivalent) and TEA (260 mg, 2.58 mmol, 2 equivalents) were added 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 equivalent) in DMF (5.00 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h. Then, N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]thioalkyl]methyl)-carbamoyl]ethyl]tert-butyl carbamate (compound 106, 500.00 mg, 1.29 mmol, 1.00 equivalent) and DMAP (472 mg, 3.87 mmol, 3.00 equivalent) were added. The resulting mixture was stirred at 60 °C for 24 h. LCMS indicated the reaction was complete. After cooling to room temperature, the reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), gradient from 0% to 60% over 30 min; detector, UV 254 nm, to give N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]methyl]carbamoyl)ethyl]tert-butyl carbamate (compound 107, 450.00 mg, 48%), as a yellow solid. LCMS (ES, m / z): 687 [M+H] +

[0839] Step 14. Compound 108

[0840] TFA (2.20 mL) was added to a stirred solution of N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]-methyl]carbamoyl)ethyl]carbamate (compound 107, 440.00 mg, 0.64 mmol, 1.00 equivalent) in DCM (22.00 mL). The resulting mixture was stirred at room temperature for 0.5 h. The reaction was indicated to be complete by LCMS. The reaction mixture was concentrated to dryness under vacuum to give (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]-methyl]propionamide trifluoroacetic acid (compound 108, 400.00 mg, crude), as a red oil. The residue was used in the next step without further purification. LCMS (ES, m / z): 587 [M+H-TFA] +

[0841] Step 15. Synthesis of Compound 109

[0842] A solution of (2R)-2-[(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]-amino]propamido]propionic acid (218 mg, 0.57 mmol, 1.00 equivalent), HOBT (77 mg, 0.57 mmol, 1.00 equivalent), and HATU (216 mg, 0.01 mmol, 1.00 equivalent) was stirred in air at room temperature for 1 hour, and then (2 S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]methyl]propionamide trifluoroacetic acid (compound 108, 400 mg, 0.57 mmol, 1.00 equivalent) and DIEA (663 mg, 5.14 mmol, 9.00 equivalent). The reaction was stirred at room temperature for 2 h. LCMS indicated that the reaction was complete. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.05% TFA), gradient from 0% to 50% over 30 min; detector, UV 254 nm, 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)thioalkyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamoic acid 9H-fluorene-9-yl methyl ester (compound 109, 480.00 mg, 75%), as a green solid. LCMS (ES, m / z): 951 [M+H] +

[0843] Step 16. Compound 110

[0844] Piperidine (1.00 mL) was added to a solution of N-[(1S)-1-[(1R)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethyl)thioalkyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamoic acid 9H-fluorene-9-yl methyl ester (compound 109, 110.00 mg) in DMF (5.00 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 0.5 h. The reaction was indicated to be complete by LCMS. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.05% TFA), gradient from 0% to 60% over 40 min; detector, UV 254 nm, to give (2S)-2-[(2R)-2-[(2S)-2-aminopropionamido]propionamido]-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiridin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]-phenyl]ethyl)thioalkyl]methyl]acrylamide (compound 110, 80.00 mg, 60%), as a red solid. LCMS (ES, m / z): 729 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ9.00 (br s, 1H), 8.53br (s, 1H), 8.24 (d, J = 7.6Hz, 1H), 8.10 (br s,1H),7.69–7.62(m,2H),7.49(s,1H),7.42(d,J=8.0Hz,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).

[0845] Step 17. Synthesis of compound (Ik)

[0846] At room temperature, DIEA (22.33 mg, 0.17 mmol, 2.00 equivalent) was added to a solution of (2S)-2-[(2R)-2-[(2S)-2-aminopropamido]propamido]-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]methyl]acrylamide (compound 110, 63.00 mg, 0.09 mmol, 1.00 equivalent) and 2,5-dioxoperidin-1-yl hexanoic acid 2,5-dioxoperidin-1-yl ester (26 mg, 0.09 mmol, 1.00 equivalent) in DMF (1.50 mL, 19.38 mmol, 224.36 equivalent) in air. The reaction was stirred at room temperature for 1 h. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column: Kinetex EVO C18 column, 30x150, 5 μm; mobile phase A: Xater (0.05% TFA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 23B to 43B, 254 nm over 7 min; RT1: 6.58. The collected fractions were freeze-dried 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)thioalkyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]-6-(2,5-dioxopyrrolo-1-yl)hexanoamide (compound (Ik), 16.10 mg, 20%), as a white solid. LCMS (ES, m / z): 922,924 [M+H] + . 1HNMR (400MHz, DMSO-d6) δ11.00(s,1H),8.80(s,1H),8.44-8.41(m,1H),8.15(d,J=7.2Hz,1H),8.03-8. 00(m,2H),7.7-7.65(m,2H),7.51(s,1H),7.44(d,J=8.0Hz,1H),7.22-7.14(m,2H),6.98(s,2H),6.83- 6.81(m,1H),5.13-5.08(m,1H),4.48-4.40(m,3H),4.29-4.17(m,6H),2.96-2.85(m,3H),2.75-2.70(m ,2H),2.67-2.57(m,1H),2.40-2.33(m,1H),2.09-1.98(m,3H),1.52-1.45(m,5H),1.26-1.16(m,12H).

[0847]

[0848]

[0849] Step 1. Synthesis of Compound 112

[0850] BH3-Me2S (5.50 mL, 57.99 mmol, 2.50 equivalent) was added fractionally to a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 111, 5.00 g, 23.19 mmol, 1.00 equivalent) in THF (50 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 h. TLC (PE:EtOAc = 3:1) indicated the completion of the reaction. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (2:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 112, 4.8 g, 92%) as a pale yellow solid. 1 ¹H NMR (400MHz, chloroform-d) δ 8.27 (d, J = 2.4Hz, 1H), 8.10 (dd, J = 8.4, 2.4Hz, 1H), 7.46 (s, 1H), 3.20 -3.09 (m, 4H).

[0851] Step 2. Synthesis of Compound 113

[0852] NBS (6.36 g, 35.71 mmol, 1.50 equivalent) and PPh3 (9.37 g, 35.72 mmol, 1.50 equivalent) were added in portions to a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 112, 4.80 g, 23.81 mmol, 1.00 equivalent) in 100 mL of DCM at room temperature under air. The resulting mixture was stirred overnight at room temperature under air. TLC (PE:EtOAc = 10:1) indicated that the reaction was complete. The reaction mixture was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (4:1), to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 113, 3.9 g, 57%) as a red oil. 1 ¹H NMR (400MHz, 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).

[0853] Step 3. Synthesis of Compound 114

[0854] Potassium thioacetate (1.68 g, 14.75 mmol, 1.00 equivalent) was added to a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 113, 3.90 g, 14.75 mmol, 1.00 equivalent) in DMF (39 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 hours. TLC (PE:EtOAc = 10:1) indicated the reaction was complete. The reaction mixture was diluted with water (600 mL). The resulting mixture was extracted with EA (200 mL x 3). The combined organic layers were washed with water (200 mL) and brine (200 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under vacuum to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]ethenone (compound 114, 3.7 g, 85%) as a red oil. 1 ¹H NMR (400MHz, 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).

[0855] Step 4. Synthesis of Compound 115

[0856] MeONa (14.31 mL, 77.00 mmol, 5.00 equivalence, 30%) was added to a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]ethenone (compound 114, 4.00 g, 15.40 mmol, 1.00 equivalent) in MeOH (600 mL) for 1 h under N2 conditions. The reaction mixture was stirred at 0 °C for 1 h. TLC indicated (PE:EA = 10:1) that the reaction was complete. The reaction was quenched with AcOH. The resulting mixture was concentrated to dryness under vacuum. The residue was diluted with DCM (100 mL) and filtered. The filtrate was purified by silica gel column chromatography, eluting with (PE:EtOAc = 10:1) to give 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 115, 3 g, 80%) as a yellow oil. 1 ¹H NMR (400MHz, 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).

[0857] Step 5. Synthesis of Compound 117

[0858] Pyridine (2.59 g, 32.74 mmol, 1.16 equivalent) was added dropwise to a stirred mixture of (2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]acetamido)acetic acid (compound 116, 10 g, 28.22 mmol, 1.00 equivalent) and Pb(OAc)4 (15 g, 33.86 mmol, 1.20 equivalent) in THF (300 mL) and toluene (100 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred overnight at 80 °C under a nitrogen atmosphere. The reaction was indicated by LCMS to be complete. The mixture was cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with EA (20 mL). The filtrate was concentrated under vacuum. The residue was dissolved in EA (20 mL). The resulting mixture was washed with water and brine, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / EtOAc (1:4) to give methyl acetate (2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]acetamido)acetate (compound 117, 6.5 g, 56%), as a white solid. 1HNMR (300MHz, CDCl3) δ7.80(d,J=7.5Hz,2H),7.62(d,J=7.5Hz,2H),7.45(t,d=7.5Hz,2H),7.36(d,d=7.5Hz,2H),7.18(brs,1H),5.48(br s, 1H), 5.28 (d, J = 7.2Hz, 2H), 4.48 (d, J = 6.6Hz, 2H), 4.26 (t, J = 6.6Hz, 1H), 3.93 (d, 5.4Hz, 2H), 2.08 (s, 3H). LCMS(ESI,ms): 391[M+Na] +

[0859] Step 6. Synthesis of Compound 118

[0860] TFA (0.56 g, 4.91 mmol, 0.60 equivalent) was added to a solution of methyl 2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]acetamido)acetate (compound 117, 3.00 g, 8.14 mmol, 1.00 equivalent) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 115, 1.77 g, 8.13 mmol, 1.00 equivalent) in DCM (300 mL) at room temperature. The resulting mixture was stirred at 60 °C for 16 h. The reaction was indicated to be complete by LCMS. The reaction mixture was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with (PE:EtOAc = 2:3) to give N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]-methyl]carbamate 9H-fluorene-9-ylmethyl ester (compound 118, 3.7 g, 67%), as a grayish-white solid. LCMS (ES, m / z): 526, 528 [M+H] +

[0861] Step 7. Synthesis of Compound 119

[0862] Piperidine (8 mL) was added to a solution of N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]methyl]carbamate 9H-fluorene-9-ylmethyl ester (compound 118, 3.70 g, 7.03 mmol, 1.00 equivalent) in DMF (40 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 0.5 h. The reaction was indicated by LCMS to be complete. The resulting mixture was diluted with water (400 mL) and extracted with EA (200 mL x 3). The combined organic layers were washed with water (200 mL) and brine (200 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with (DCM:MeOH = 10:1) to give 2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)acetamide (compound 119, 1.01 g, 40%), as a yellow oil. LCMS (ES, m / z): 304, 306 [M+H] +

[0863] Step 8. Synthesis of Compound 120

[0864] A solution of 2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)-acetamide (compound 119, 1.00 g, 3.29 mmol, 1.00 equivalent) in DMF (50 mL) was added to a solution of NaHCO3 (0.33 g, 3.92 mmol, 1.20 equivalent) in water (10 mL) and Boc2O (0.72 g, 3.30 mmol, 1.00 equivalent). The resulting mixture was stirred at room temperature for 1 h. The reaction was indicated to be complete by LCMS. The reaction mixture was diluted with water (500 mL) and extracted with EtOAc (200 mL x 3). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with (PE:EtOAc = 1:3) to give N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]methyl]tert-butyl carbamate (compound 120, 810 mg, 54%), as a white solid. LCMS (ES, m / z): 404, 406 [M+H] + 304, 306 [M+H-100] +

[0865] Step 9. Synthesis of Compound 121

[0866] At room temperature, a solution of iron powder (1106 mg, 19.81 mmol, 10.00 equivalent) and NH4Cl (1059 mg, 19.81 mmol, 10.00 equivalent) in water (10 mL) was added to a solution of N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]methyl]carbamate tert-butyl ester (compound 120, 800.00 mg, 1.98 mmol, 1.00 equivalent) in EtOH (40). The resulting mixture was stirred at 70 °C for 2 h. The reaction was indicated to be complete by LCMS. The reaction mixture was filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was purified by silica gel column chromatography, eluting with (DCM:MeOH = 13:1) to give N-[[([[2-(4-amino-2-chlorophenyl)ethyl]thioalkyl]methyl)-carbamoyl]methyl]tert-butyl carbamate (compound 121, 610 mg, 74%), as a yellow oil. LCMS (ES, m / z): 374, 376 [M+H] + 374,376[M+H-100] +

[0867] Step 10. Synthesis of Compound 122

[0868] CDI (130 mg, 0.80 mmol, 1.00 equivalent) and TEA (81 mg, 0.80 mmol, 1.00 equivalent) were added 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 equivalent) in DMF (10 mL) at 0 °C in air. The resulting mixture was stirred at room temperature for 2 h. Then, N-[[([[2-(4-amino-2-chlorophenyl)ethyl]thioalkyl]methyl)carbamoyl]methyl]tert-butyl carbamate (compound 121, 300 mg, 0.80 mmol, 1.00 equivalent) and DMAP (294 mg, 2.41 mmol, 3.00 equivalent) were added to air at room temperature. The resulting mixture was stirred at 60 °C for 48 h. The reaction was indicated by LCMS to be complete. The resulting mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.05% TFA), gradient from 0% to 60% over 30 min; detector, UV 254 nm, to give N-[([[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethyl)thioalkyl]methyl]carbamoyl)methyl]tert-butyl carbamate (compound 122, 270 mg, 49%), as a yellow solid. LCMS (ES, m / z): 673, 675 [M+H] + 573,575[M+H-100] +

[0869] Step 11. Synthesis of Compound-123

[0870] At 0 °C and under N2, HCl (6 mL of 1,4-dioxane solution in 4N) was added to a solution of N-[([[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]methyl]carbamoyl)methyl]-tert-butyl carbamate (compound 122, 250 mg, 0.37 mmol) in 1,4-dioxane (12 mL). The reaction was stirred at room temperature for 2 h. LCMS indicated that the reaction was complete. The reaction mixture was concentrated to dryness under vacuum 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)thioalkyl]methyl]acetamide (compound 123, 260 mg, crude), as a brown solid. LCMS (ES, m / z): 573,575 [M+H-HCl]+

[0871] Step 12. Synthesis of Compound-125

[0872] A solution of (2S)-2-[2-(2-aminoacetamido)acetamido]-3-phenylpropionic acid (compound 124, 500 mg, 1.79 mmol, 1.00 equivalent) and 2,5-dioxopyrrolidin-1-yl ester of 6-(2,5-dioxopyrrolidin-1-yl)hexanoic acid (552 mg, 1.79 mmol, 1.00 equivalent) in DMSO (5.00 mL) was stirred in air at room temperature for 16 h. The reaction was indicated to be complete by LCMS. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), gradient from 0% to 60% over 30 min; detector, UV 220 nm, to give (2S)-2-(2-[2-[6-(2,5-dioxopyrrolo-1-yl)hexamido]acetamido]acetamido)-3-phenylpropionic acid (compound 125, 760 mg, 83%), as a white solid. LCMS (ES, m / z): 473 [M+H] +

[0873] Step 13. Synthesis of compound (II)

[0874] HATU (141 mg, 0.37 mmol, 1.00 equivalent) and HOBT (50 mg, 0.37 mmol, 1.00 equivalent) were added to a solution of (2S)-2-(2-[2-[6-(2,5-dioxopyrrolo-1-yl)hexamido]acetamido]-acetamido)-3-phenylpropionic acid (compound 125, 175 mg, 0.37 mmol, 1.00 equivalent) in DMF (5.00 mL) at room temperature. 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)thioalkyl]methyl]acetamide (compound 123, 250 mg, 0.37 mmol, 1.00 equivalent, 85%) and DIEA (240 mg, 1.85 mmol, 5.00 equivalent) were added. The resulting mixture was stirred at room temperature for 1 h. The reaction was indicated to be complete by LCMS. The reaction mixture was purified under the following conditions: column: XSelect CSH Prep C18 OBD column, 19*250 mm, 5 μm; mobile phase A: water (0.05% FA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 30 B to 60 B, 254 nm over 7 min; RT1: 6.67 min, yielding 75 mg of crude product. The crude product was further purified by reversed-phase rapid chromatography under the following conditions: column: XBridge Shield RP18 OBD column, 19*250mm, 10µm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 25mL / min; gradient: 25B to 44B over 10 min; 254nm; RT1: 10.52min. The collected fractions were lyophilized to give compound (Il) (41.6mg, 10%) as a white solid. 1HNMR (400MHz, DMSO-d6) δ10.99(s,1H),8.79(s,1H),8.38(t,J=6.0Hz,1H),8.31(t,J=6.0Hz,1H),8.12(d,J=8.4Hz,1H),8.06(t,J=5.6Hz,1H), 8.01(t,J=6.0Hz,1H),7.70-7.66(m,2H),7.51(s,1H),7.44(d,J=8.0Hz ,1H),7.25-7.21(m,5H),7.19-7.14(m,2H),6.99(s,2H),6.82(t,J=6.0H z,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,4H),1.20-1.16(m,2H). LCMS(ES,m / z): 1027,1029[M+H] +

[0875]

[0876]

[0877] Step 1. Synthesis of Compound 127

[0878] BH3-Me2S (28.00 mL, 295.23 mmol, 2.65 equivalents) was added dropwise to a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 126, 24.00 g, 111.32 mmol, 1.00 equivalent) in THF (240.00 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 hours. TLC (PE:EtOAc = 3:1) indicated the completion of the reaction. After cooling to room temperature, the resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (3:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 127, 18.00 g, 80%) as a pale yellow solid. 1H NMR (300MHz, CD3C1) δ8.27 (s, 1H), 8.10-8.07 (m, 1H), 7.52 (d, J = 3Hz, 1H), 3.96 (t, J = 6Hz, 2H), 3.13 (t, J = 6Hz, 2H).

[0879] Step 2. Synthesis of Compound 128

[0880] NBS (6.62 g, 1.50 equivalent) and PPh3 (9.76 g, 37.21 mmol, 1.50 equivalent) were added in portions to a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 127, 5.00 g, 24.80 mmol, 1.00 equivalent) in DCM (100.00 mL) at room temperature under N2. The resulting mixture was stirred overnight at room temperature under N2. TLC (PE:EtOAc = 10:1) indicated that the reaction was complete. The reaction mixture was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (4:1), to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 128, 5.10 g, 72.31%) as a red oil. 1 H NMR (400MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.18 ( dd, J = 8.4, 2.4 Hz, 1H), 7.73 ( d, J = 8.4 Hz, 1H), 3.79 ( t, J = 7.2 Hz, 2H), 3.38 ( t, J = 7.2 Hz, 2H).

[0881] Step 3. Synthesis of Compound 129

[0882] Potassium thioacetate (2.16 g, 18.91 mmol, 1.00 equivalent) was added to a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 128, 5.00 g, 18.90 mmol, 1.00 equivalent) in DMF (50.00 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature for 2 hours. TLC (PE:EtOAc = 10:1) indicated 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 layers were washed with water (200.00 mL) and brine (200.00 mL * 3), dried over anhydrous Na2SO4 and concentrated to dryness under vacuum to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]ethenone (compound 129, 4.50 g, 85%) as a red oil. 1H NMR (400MHz, CDCl3) δ8.24 (d, J = 2.4Hz, 1H), 8.07 (dd, J = 8.4, 2.4Hz, 1H), 7.45 (d, J = 8.4Hz, 1H), 3.20-3.05 (m, 4H), 2.34 (s, 3H).

[0883] Step 4. Synthesis of Compound 130

[0884] MeONa (6.93 mL, 37.33 mmol, 5.00 equivalent, 30%) was added to a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]ethenone (compound 129, 2.00 g, 7.70 mmol, 1.00 equivalent) in MeOH (300.00 mL) at 0 °C under N2. The resulting mixture was stirred at 0 °C under N2 for 1 h. TLC (PE:EtOAc = 10:1) indicated that the reaction was complete. The reaction 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 pale yellow oil. 1 H NMR (400MHz, CDCl3) δ8.26 (d, J=2.4Hz, 1H), 8.09 (dd, J=8.4, 2.4Hz, 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).

[0885] Step 5. Synthesis of Compound 132

[0886] TSTU (25.18 g, 83.52 mmol, 1.30 equivalent) and DIEA (16.60 g, 128.48 mmol, 2.00 equivalent) were added to a stirred solution of (2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionic acid (compound 131, 20.00 g, 64.24 mmol, 1.00 equivalent) in DMF (200.00 mL) at room temperature under air atmosphere. The resulting mixture was stirred at room temperature for 1 h. The reaction was indicated to be complete by LCMS. 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 layers were washed with water (100.00 mL) and brine (100.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with (PE:EtOAc = 1:2) to give (2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionic acid 2,5-dioxopyrrolidine-1-yl ester (compound 132, 25.00 g, 83%), as a white solid. LCMS (ES, m / z): 431 [M+Na] +

[0887] Step 6. Synthesis of Compound 133

[0888] A solution of (2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionic acid 2,5-dioxopyrrolidine-1-yl ester (compound 132, 20.00 g, 48.97 mmol, 1.00 equivalent) in DMF (200.00 mL) was added to a solution of glycine (3.68 g, 48.97 mmol, 1.00 equivalent) in water (200.00 mL). The reaction was stirred at room temperature for 2 h. The reaction was indicated to be complete by LCMS. The pH of the reaction was adjusted to 2–3 with 2N HCl. The resulting mixture was extracted with EtOAc (500.00 mL * 3). The combined organic layers were washed with brine (500.00 mL), dried over anhydrous Na₂SO₄, and concentrated to dryness under vacuum to give [(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionamido]acetic acid (compound 133, 15.00 g, 71%), as a white solid. LCMS (ES, m / z): 369 [M+H] +

[0889] Step 7. Synthesis of Compound 134

[0890] A solution of [(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]-propamido]acetic acid (compound 133, 5.00 g, 13.57 mmol, 1.00 equivalent), Pb(OAc)4 (7.22 g, 16.28 mmol, 1.20 equivalent), and pyridine (1.29 g, 16.31 mmol, 1.20 equivalent) in THF (300.00 mL) / toluene (100.00 mL) was stirred at 80 °C under N2 for 16 h. The reaction was indicated by LCMS to be complete. After cooling to room temperature, the reaction mixture was filtered. The filter cake was washed with THF (100.00 mL). The combined organic layers were concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with PE:ETOAC = 1:2, to give methyl [(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionamido]acetate (compound 134, 2.50 g, 45%), as a white solid. LCMS (ES, m / z): 405 [M+Na] + . 1 H NMR (400MHz, chloroform-d) δ7.77-7.73(m,2H),7.58(d,J=7.6Hz,2H),7.43–7.37(m,2H),7.36–7.29(m,2H),7.10( s,1H),5.24(d,J=7.6Hz,2H),4.51–4.35(m,2H),4.22(t,J=6.8Hz,2H),2.04(s,3H),1.39(d,J=6.8Hz,3H).

[0891] Step 8. Synthesis of Compound 135

[0892] TFA (0.27 mL, 2.376 mmol, 0.62 equivalents) was added to a stirred solution of [(2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]-propamido]acetate (compound 134, 2.25 g, 5.88 mmol, 1.00 equivalent) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 500, 1.28 g, 5.88 mmol, 1.00 equivalent) in 120 mL of DCM under N2 at room temperature. The resulting mixture was stirred at 40 °C for 16 hours. The reaction was indicated by LCMS to be complete. The reaction was concentrated to dryness under vacuum to give N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]ethyl]carbamate 9H-fluorene-9-ylmethyl ester (compound 135, 3.10 g, 90%), as a yellow solid. LCMS (ES, m / z): 540, 542 [M+H] + .

[0893] Step 9. Synthesis of Compound 136

[0894] Piperidine (31.00 mL) was added to a solution of N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)carbamoyl]ethyl]carbamate 9H-fluorene-9-ylmethyl ester (compound 135, 3.10 g, 5.74 mmol, 1.00 equivalent) in DMF (155.00 mL) at 0 °C under N2. The resulting mixture was stirred at 0 °C under N2 for 0.5 h. The reaction was indicated to be complete by LCMS. 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 layers were washed with brine (200.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under vacuum to give 3.00 g of crude product. The crude product was further purified by silica gel column chromatography, eluting with (DCM:MeOH = 3:1) to give (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)acrylamide (compound 136, 1.50 g, 78%), as a yellow oil. LCMS (ES, m / z): 318, 320 [M+H] + .

[0895] Step 10. Synthesis of Compound 137

[0896] At room temperature, a solution of (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]-methyl)acrylamide (compound 136, 1.50 g, 4.72 mmol, 1.00 equivalent) in DMF (75.00 mL) was mixed with 10.00 mL of NaHCO3 (0.59 g, 7.08 mmol, 1.50 equivalent) in H2O and Boc2O (1.03 g, 4.72 mmol, 1.00 equivalent). The reaction 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) and extracted with EtOAc (200.00 mL x 3). The combined organic layers were washed with brine (200.00 mL * 3), dried over anhydrous Na₂SO₄, and concentrated to dryness under vacuum to give N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]thioalkyl]methyl)-carbamoyl]ethyl]tert-butyl carbamate (compound 137, 1.82 g, 83%), as a red oil. LCMS (ES, m / z): 418, 420 [M+H] + 318,320[M+H-100] +

[0897] Step 11. Synthesis of Compound 138

[0898] A slurry of N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]-thioalkyl]methyl)carbamoyl]ethyl]carbamate tert-butyl ester (compound 137, 1.82 g, 4.36 mmol, 1.00 equivalent), iron powder (2.43 g, 0.04 mmol, 10.00 equivalent), and NH4Cl (2.33 g, 0.04 mmol, 10.00 equivalent) in EtOH (100.00 mL) / H2O (50.00 mL) was stirred at 70 °C for 2 h. The reaction was indicated to be complete by LCMS. The reaction mixture was filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was purified by silica gel column chromatography, eluting with (DCM:MeOH = 13:1) to give N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]thioalkyl]methyl)carbamoyl]ethyl]tert-butyl carbamate (compound 138, 1.20 g, 68%), as a yellow oil. LCMS (ES, m / z): 388, 390 [M+H] + 288,290 [M+H-100] +

[0899] Step 12. Synthesis of Compound 139

[0900] CDI (209.00 mg, 1.29 mmol, 1 equivalent) and TEA (260 mg, 2.58 mmol, 2 equivalents) were added 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 equivalent) in DMF (5.00 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h. Then, N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]thioalkyl]-methyl)carbamoyl]-ethyl]tert-butyl carbamate (compound 138, 500.00 mg, 1.29 mmol, 1.00 equivalent) and DMAP (472 mg, 3.87 mmol, 3.00 equivalent) were added. The resulting mixture was stirred at 60 °C for 24 h. LCMS indicated the reaction was complete. After cooling to room temperature, the reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), gradient from 0% to 60% over 30 min; detector, UV 254 nm, to give N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]methyl]carbamoyl)ethyl]tert-butyl carbamate (compound 139, 450.00 mg, 48%), as a yellow solid. LCMS (ES, m / z): 687, 689 [M+H] + 587,589[M+H-100] +

[0901] Step 13. Synthesis of Compound 140

[0902] TFA (2.20 mL) was added to a stirred solution of N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]-methyl]carbamoyl)ethyl]carbamate (compound 139, 440.00 mg, 0.64 mmol, 1.00 equivalent) in DCM (22.00 mL). The resulting mixture was stirred at room temperature for 0.5 h. The reaction was indicated to be complete by LCMS. The reaction mixture was concentrated to dryness under vacuum to give (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]methyl]propionamide trifluoroacetic acid (compound 140, 400.00 mg), as a red oil. LCMS (ES, m / z): 578, 589 [M+H-TFA] +

[0903] Step 14. Synthesis of Compound 142

[0904] To a slurry of L-valine (compound 141, 0.50 g, 4.27 mmol, 1.00 equivalent) in DMSO (10 mL), 2,5-dioxopyrrolidine-1-yl ester of 6-(2,5-dioxopyrrol-1-yl)hexanoate (1.32 g, 4.28 mmol, 1.00 equivalent) and DIEA (1103 mg, 8.54 mmol, 2.00 equivalent) were added. The resulting mixture was stirred at room temperature for 4 h. The reaction was indicated to be complete by LCMS. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% FA), gradient from 0% to 60% over 30 min; detector, UV 220 nm, to give (2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexamido]-3-methylbutyric acid (compound 142, 1.2 g, 72%), as a brown solid. LCMS (ES, m / z): 311 [M+H] +

[0905] Step 15. Synthesis of compound (Im)

[0906] A solution of (2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexamido]-3-methylbutyric acid (compound 142, 59 mg, 0.19 mmol, 1.00 equivalent), HOBT (26 mg, 0.19 mmol, 1.00 equivalent), and HATU (72 mg, 0.19 mmol, 1.00 equivalent) in DMF (2 mL) was stirred in air at room temperature for 1 hour. Then, (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxoperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)thioalkyl]methyl]propionamide trifluoroacetic acid (compound 140, 200 mg, 0.19 mmol, 1.00 equivalent, 66.70%) and DIEA (197 mg, 1.52 mmol, 8.00 equivalent) were added at room temperature. The reaction mixture was stirred at room temperature for 2 h. The reaction was indicated to be complete by LCMS. The resulting mixture was purified by reversed-phase rapid 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 B to 45 B, 254 nm over 10 min; RT1: 9.67 min. The collected fractions were freeze-dried to give N-[(1S)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopyridine-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]-phenyl]ethyl)thioalkyl]methyl]carbamoyl)ethyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrolo-1-yl)hexanoamide (compound (Im), 27.8 mg, 16%), as a white solid. LCMS (ES, m / z): 879,881 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ10.99(s,1H),8.80(s,1H),8.47(t,J=6.0Hz,1H),8.03(d,J=7.2Hz,1H),7.78(d,J=8.8Hz,1H),7.70-7.66(m,2H),7. 51(s,1H),7.44(d,J=8.0Hz,1H),7.21-7.14(m,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=7.2Hz,1H),4.14(t,J=6.8Hz,1H),3.38-3.36(m,1H),2.97-2.90(m,1H),2.86(t,J=7.6Hz,2H),2.73-2.67(m,2H),2.62-2.57 (m,1H),2.40-2.35(m,1H),2.20-2.05(m,2H),2.02-1.96(m,1H),1.95 -1.88(m,1H),1.48-1.46(m,4H),1.23-1.16(m,6H),0.83-0.78(m,6H).

[0907] Example 4: General procedure for the preparation and characterization of novel degradation agent conjugates

[0908] The antibody solution was treated with 30 equivalents of tri-(2-carboxyethyl)phosphine (TCEP) and incubated at 37°C for 1 hour to reduce interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 buffer using an illustra NAP column (GE Healthcare).

[0909] The conjugation was achieved as follows: a solution of 2–5 mg / mL reducing antibody in 50 mM EPPS, 5 mM EDTA pH 7.0 was treated with 12 equivalents of a new linker-degrader added as a stock solution in N,N-dimethylacetamide (DMA) to achieve a final DMA concentration of 15% (v / v). The resulting reaction mixture was incubated overnight at 4°C. The resulting new degrader conjugate was purified using an illustra NAP column (GE Healthcare) to 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 and concentrated using an Amicon ultracentrifuge (Millipore) with a molecular weight cutoff of 50 kD.

[0910] Concentrations and monomers were determined by size exclusion chromatography using a 7.8 x 300 mM TSKGel3000SWXL column (Tosoh Bioscience) with 5 μm particles, eluted isocratically at 0.5 mg / mL for 30 min with a mobile phase of 400 mM sodium perchlorate, 50 mM sodium phosphate, and 5% (v / v) isopropanol. The novel degrading agent conjugates were quantified based on an antibody standard curve detected at 214 nm.

[0911] The drug-to-antibody ratio (DAR) was determined by hydrophobic interaction chromatography using a 4.6 x 35 mm TSKgel butyl-NPR column with 2.5 μm particles. Mobile phase A consisted of 1.5 M ammonium sulfate and 25 mM sodium phosphate at pH 7.0. Mobile phase B consisted of 25 mM sodium phosphate at pH 7.0 and 25% (v / v) isopropanol. The analyte was eluted with a linear gradient of 0%–100% B over 12 min at a flow rate of 0.6 mL / min and detected at 214 nm.

[0912] The free linker loading was determined by mixed-mode chromatography using a 4.6 x 250 mm HISEP column (Supelco) with 2.5 μm particles. Mobile phase A was 100 mM ammonium acetate. Mobile phase B was 100% acetonitrile. The analyte was eluted with a gradient of 25%–40% B over 25 min, followed by a gradient of 40%–100% B over 2 min, at a flow rate of 0.7 mL / min. The column temperature was 35 °C. The free linker loading was quantified using an external standard curve detected at 254 nm.

[0913] Example 5: General procedure for in vitro antiproliferative assay of novel degrading agents and novel degrading agent conjugates 1

[0914] The ability of the novel degradation agent conjugate to inhibit cell growth was measured using an in vitro antiproliferative assay. Target cells were seeded at 1,500–5,000 cells per well in 100 μL of complete cell growth medium (RPMI 1640, 10% fetal bovine serum, and 1% penicillin-streptomycin for most cell lines; Hybri-care medium, 1.5 g / L sodium bicarbonate, 10% fetal bovine serum, and 1% penicillin-streptomycin for BT-474; RPMI 1640, 20% fetal bovine serum, and 1% penicillin-streptomycin for HL-60). The conjugate was diluted in complete cell growth medium using a 4-fold serial dilution, with 100 μL added to each well. The final concentration was typically 1 x 10⁻⁶. -8 M to 1.53 x 10 -13 M or 1x 10 -7 M to 1.53 x 10 -12Cells were incubated for 5 days in a humidified 5% CO2 incubator at 37°C. The viability of remaining cells was determined by colorimetric WST-8 assay (Dojindo Molecular Technologies, Inc., Rockville, MD, US). WST-8 was added to 10% of the final volume, and the plate was incubated for 2–4 hours in a humidified 5% CO2 incubator at 37°C. The plate was analyzed by measuring the absorbance (A450) at 450 nm in a multi-well plate reader. The background A450 absorbance of wells containing only culture medium and WST-8 was subtracted from all values. The viability percentage was calculated by dividing each treated sample value by the average of the wells containing untreated cells. For each treatment, the viability percentage values ​​were plotted against the test sample concentration in a semi-logarithmic graph. IC50 values ​​were calculated automatically.

[0915] The trastuzumab and pertuzumab conjugates of compounds (Ia) and (Ic) showed antiproliferative activity against the BT-474 breast cancer cell line. Figures 1-4 In the study (drug:antibody ratio = 8 for each new degradation agent conjugate), targeting BT-474 cells, the antibody-drug conjugate Kadcyla and the unconjugated antibodies trastuzumab and pertuzumab showed >100-fold lower activity than the new antibody degradation agent conjugates. In contrast, the non-cell-binding control new degradation agent conjugate rituximab-compound (Ia) and the released new degradation agents P1 and P4 showed >1000-fold lower activity.

[0916] The trastuzumab and pertuzumab conjugates of compounds (Ia) and (Ic) showed antiproliferative activity against the BT-474 breast cancer cell line. Figures 5-6 (Designated drug:antibody ratio). Antibody-drug conjugates were discovered. The activity of the unconjugated antibody trastuzumab against BT-474 cells was lower than that of the antibody new degradation agent conjugate.

[0917] The trastuzumab and pertuzumab conjugate of compound (Ia) showed antiproliferative activity against the SK-BR-3 breast cancer cell line. Figure 7 and Figure 8 In each novel degrader conjugate (drug:antibody ratio = 8), the conjugated novel degraders showed similar activity to the antibody-drug conjugate Kadcyla, while the unconjugated antibodies trastuzumab and pertuzumab showed significantly lower activity than the novel degrader conjugates. The non-cell-binding control novel degrader conjugate rituximab-compound (Ia) and the released novel degraders P1 and P4 showed significantly lower activity against SK-BR-3 cells.

[0918] The antiproliferative activity of compounds (Ia) and (Id) containing OR000213, huMy9-6, and lintozumab IgG1 conjugates against the HL-60 (acute myeloid leukemia) cell line was demonstrated. Figures 9-12 (Drug:antibody ratio = 8 when not specified). The new degradation agent conjugate showed similar activity against cell lines as the approved agent. Similar activity was observed, but the activity of the non-cell-binding control novel degradation conjugates trastuzumab-compound (Ia) and rituximab-compound (Id) was significantly lower.

[0919] The rituximab conjugates of compounds (Ia) and (Ic) showed antiproliferative activity against the Ramos (non-Hodgkin lymphoma) cell line. Figure 13 In the mean (drug:antibody ratio = 8 when not specified), unconjugated rituximab, the non-cell-binding control novel degrader conjugate trastuzumab-compound (Ia), and the released novel degraders P1 and P4 showed lower activity against this cell line than the novel degrader conjugate.

[0920] The rituximab conjugates of compounds (Ia) and (Ic) showed antiproliferative activity against the Daudi lymphoma cell line. Figure 14 and Figure 15 In the mean (drug:antibody ratio = 8 when not specified), unconjugated rituximab, the non-cell-binding control new degrader conjugate trastuzumab-compound (Ia), and the released new degrader P1 showed lower activity against this cell line than the new degrader conjugate.

[0921] The trastuzumab and pertuzumab conjugate of compound (Ia) showed antiproliferative activity against the NCI-N87 gastric cancer cell line. Figure 16 and Figure 17 In each novel degrader conjugate (drug:antibody ratio = 8), the conjugated novel degraders showed similar activity to the antibody-drug conjugate Kadcyla, while the unconjugated antibodies trastuzumab and pertuzumab showed significantly lower activity than the novel degrader conjugates. The non-cell-binding control novel degrader conjugate rituximab-compound (Ia) and the released novel degrader P1 showed significantly lower activity against NCI-N87 cells.

[0922] Figure 18 The antiproliferative activity of the trastuzumab and pertuzumab conjugates of compound (Ia) in BT-464 breast cancer cells after three days of incubation with human serum is shown compared to the activity of the conjugates in the absence of serum. As shown in the figure, the activity of the novel degrading conjugates was similar in the presence and absence of serum, indicating that human serum does not affect the activity. The non-cell-binding control novel degrading conjugate OR000213-compound (Ia) showed >1000-fold lower activity against this cell line.

[0923] Figure 19 The antiproliferative activity of the trastuzumab and pertuzumab conjugates of compound (Ia) in BT-464 breast cancer cells after three days of incubation with mouse serum is shown compared to the activity of the conjugates in the absence of serum. As shown in the figure, the activity of the novel degrading conjugates was similar in the presence and absence of serum, indicating that mouse serum did not affect the activity. The non-cell-binding control novel degrading conjugate OR000213-compound (Ia) showed >1000-fold lower activity against this cell line.

[0924] Tables 1 and 2 show the IC50 values ​​of trastuzumab conjugates (Ia), (Ib), (Ic), and (Id) and pertuzumab conjugates (Ia) and (Ic) against various Her2 cell lines. As shown in Table 1, the novel degradation agent conjugates exhibited improved activity in the BT-474 cell line compared to the unconjugated antibody, and also showed improved activity relative to the released payload and antibody-drug conjugates Kadcyla and Improved activity was also observed. Compared to the unconjugated antibody, the novel degradation agent conjugate also showed better activity against the SK-BR-3 breast cancer cell line and the NCI-N87 gastric cell line. As shown in Table 2, the novel antibody degradation agent conjugate also exhibited improved activity against the SNU-182 hepatitis cell line compared to the unconjugated antibody or Kadcyla.

[0925] Table 1: IC50 of novel anti-Her2 degradation agent conjugates

[0926]

[0927]

[0928] Table 2: IC50 of anti-Her2 neodegrading agent conjugates

[0929]

[0930] Table 3 shows the activity of the novel antibody degradation conjugates in the anti-CD20 cell line. Compared with the unconjugated antibody, the non-cell-binding control novel degradation conjugate trastuzumab-compound I(a), and the released payload, the novel antibody degradation conjugates exhibit superior activity against the Daudi and Ramos lymphocyte lines.

[0931] Table 3: IC50 of novel CD20 degradation agent conjugates

[0932]

[0933] Table 4 shows the IC50 values ​​of the huMy9-6 and OR000213 conjugates of compounds (Ia) and (Id), and the lintozumab IgG1 conjugates of compounds (Ia) and (Id) against the AML HL-60 cell line. As shown in Table 4, the novel degrading conjugates exhibited similar activity to IL-60 cells in the HL-60 cell line. Compared to comparable activity and improved activity compared to the non-bound conjugate rituximab-compound (Ic).

[0934] Table 4: IC50 of novel HL-60 degradation agent conjugates

[0935]

[0936] Table 5 shows the antiproliferative activity of trastuzumab and pertuzumab compound (Ia) conjugates against the BT-474 breast cancer cell line after incubation with human or mouse serum, compared to the non-cell-binding control conjugate OR000213- (Ia). As shown in the table, the activity of the new degraders in human or mouse serum was consistent with that without the introduction of serum.

[0937] Table 5: IC50 of serum stability test

[0938]

[0939] Example 6: General procedure for in vitro antiproliferative assay of novel degrading agents and novel degrading agent conjugates 2

[0940] Cell culture: Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or the German Collection of Microbial and Cell Cultures (DSMZ, Braunschweig, Germany) and maintained according to the culture conditions specified by ATCC or DSMZ. Cells were thawed and maintained in culture medium for at least two passages before experimental conditions were applied.

[0941] Cytotoxicity assays: For adherent cell lines, cells were dissociated using cell dissociation buffer based on enzyme-free PBS (Gibco, USA) and plated at appropriate cell densities according to cell doubling time on tissue culture-treated 96-well flat-bottomed polystyrene plates (Costar, Corning, USA). Eighteen hours after plating, cells were treated with the test product at an appropriate concentration starting at 100 nM and serially diluted 4-fold. For suspension cell lines, cells were seeded on the same day as treatment and treated as described above. Adherent cells were treated for 5 days and suspension cells for 3 days. Cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan), and measurements were obtained 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 in triplicate and validated using three biological replicates.

[0942] Table 6 shows the activities of the new degrading agents P1, P3, and P4 against various cancer cell lines. As shown in the table, the new degrading agents are active against each cell line.

[0943] Table 6: IC50 of representative new degradation agents in various cancer cell lines

[0944]

[0945] Table 7 shows pertuzumab-compound (Ia) conjugates and known antibody drug conjugates. Activity against various breast cancer cell lines. As shown in the table, the pertuzumab-compound (Ia) conjugate exhibited higher activity in all reported cell lines.

[0946] Table 7: IC50 of pertuzumab-compound (Ia) conjugate in various breast cancer cell lines

[0947]

[0948] Table 8 shows the activities of the pertuzumab-compound (Ia) conjugate and the known antibody drug conjugate ENHERTU against three gastric cancer cell lines. As shown in the table, the pertuzumab-compound (Ia) conjugate exhibited higher activity in all reported cell lines.

[0949] Table 8: IC50 of pertuzumab-compound I(a) conjugate in various gastric cancer cell lines

[0950]

[0951] Table 9 shows the activities of the OR000213-compound (Ia) conjugate and the known antibody drug conjugate MYLOTARG against various acute myeloid leukemia cell lines. As shown in the table, the OR000213-compound (Ia) conjugate exhibits better activity in several cell lines.

[0952] Table 9: IC50 of OR000213-compound I(a) conjugate in various acute myeloid leukemia cell lines

[0953]

[0954]

[0955] Table 10 shows the activity of three novel anti-CD38 degrader conjugates against various multiple myeloma cell lines. As shown in the table, the conjugates exhibited good activity in all cell lines.

[0956] Table 10: IC50 of novel anti-CD38 degradation conjugates in multiple myeloma cell lines

[0957]

[0958] Table 11 shows the activity of the novel anti-CD138 degrader conjugates against various multiple myeloma cell lines. As shown in the table, the conjugates exhibit good activity in all cell lines.

[0959] Table 11: IC50 of the new anti-CD38 degradation conjugate in multiple myeloma cell lines

[0960]

[0961] Table 12 shows the activity of the novel anti-BCMA degrader conjugates against various multiple myeloma cell lines. As shown in the table, the conjugates exhibit good activity in all cell lines.

[0962] Table 12: IC50 of novel anti-BCMA degradation conjugates in multiple myeloma cell lines

[0963]

[0964] Table 13 shows the activity of the novel anti-Trop-2 degrader conjugates against various cancer cell lines. As shown in the table, the conjugates exhibit good activity in all cell lines.

[0965] Table 13: IC50 of novel anti-Trop-2 degradation conjugates in various cancer cell lines

[0966]

[0967] Table 14 shows the activity of the novel anti-FGFR4 degrader conjugate against two cancer cell lines. As shown in the table, the conjugate exhibits good activity in both cell lines and better activity than the US-1784 antibody alone and the unconjugated novel degrader.

[0968] Table 14: IC50 of novel anti-FGFR4 degradation conjugates in various cancer cell lines

[0969]

[0970] Table 15 shows the activity of the novel anti-EGFR degrader conjugate against two synovial sarcoma cell lines. As shown in the table, the conjugate exhibits good activity in both cell lines and better activity than cetuximab alone and the unconjugated novel degrader.

[0971] Table 15: IC50 of anti-EGFR neodegrading conjugates in synovial sarcoma cell lines

[0972]

[0973] Table 16 shows the activity of the novel anti-PDGF-Rα degradative conjugates against two cancer cell lines. As shown in the table, the conjugates exhibited good activity in both cell lines.

[0974] Table 16: IC50 of novel anti-PDGF-Rα degradation conjugates in cancer cell lines

[0975]

[0976] Table 17 shows the activity of the novel TEM1 / CD248 degrader conjugate against rhabdomyosarcoma cell lines. As shown in the table, this conjugate exhibits good activity against this cell line. Table 17: IC50 of the novel TEM1 / CD248 degrader conjugate in rhabdomyosarcoma cell lines. ...

Claims

1. A conjugate of formula (I): (I)， Or its pharmaceutically acceptable salt, wherein: a is an integer from 1 to 10; A is a phenyl group or a C4-C4 group. 10 cycloalkyl rings; U is NH; R 1 Independently selected from hydrogen and halogroups; X is selected from -NR 2 -、=C(CH3)-、-Q-(CH2) n - and -Q(CH2) m Q'(CH2) n -;in Q and Q' are each independently O, S, or N(R) 2 ) V ; v is 1 or 2; Each R 2 Independently hydrogen or C1-C6 alkyl; n is an integer from 1 to 6; and m is an integer from 2 to 6; Each group is connected to L on the left and to A on the right; This assumes that X is NH or -Q-(CH2). n - At that time, R 1 It is a halogenated group; L is selected from the following connectors. 、 、 、 、 、 、 、 、 、 、 、 and ; in: Let X be the connection point with X; and For the connection point with the joint part, p is an integer from 1 to 10; q is an integer from 2 to 10; Z 1 It may be absent or contain glycine; Z 2 The following groups are absent or selected: L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 Select from the following groups: L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; and Z 4 Select the group consisting of the following: L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine; R, R', R" and R'" are each independently selected from hydrogen, C1-C6 alkoxy-C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or, the two geminal R groups together with the carbon atoms they are attached to can form a cyclobutyl or cyclopropyl ring, and ---Does not exist or is a key; and Bm is a binding moiety, which is an antibody or antigen-binding fragment, wherein the binding moiety specifically binds to a surface antigen, wherein the surface antigen is not CD33.

2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting of... in: p is an integer from 1 to 10; Let X be the connection point with X; and This refers to the connection point with the aforementioned connecting portion.

3. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting of... ; ; ; ; ; ;and ; in: q is an integer from 2 to 10; Z 1 It may be absent or contain glycine; Z 2 The following groups are absent or selected: L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 Select from the following groups: L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; and Z 4 Choose from the following groups: L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

4. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L is... 。 5. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting of... in: q is an integer from 2 to 10; R, R', R” and R'” are each independently selected from hydrogen, C1-C6 alkoxy-C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or the two geminal R groups together with the carbon atoms to which they are attached can form a cyclobutyl or cyclopropyl ring; Let X be the connection point with X; and This refers to the connection point with the aforementioned connecting portion.

6. The conjugate as claimed in claim 1, wherein L is ； in: q is an integer from 2 to 10; Let X be the connection point with X; and This refers to the connection point with the aforementioned connecting portion.

7. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L is selected from... in: q is an integer from 2 to 10; ---Does not exist or is a key; Let X be the connection point with X; and This refers to the connection point with the aforementioned connecting portion.

8. The conjugate of claim 1, wherein the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, 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, 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, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, condensation factor, cKit, closure protein 3, closure protein 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1. Growth factors, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ErbB3, ERG, ETBR, ETV6-AML, FAP FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.

24. HMWMAA, HPVE6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-11Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrin, integrin αV, intestinal carboxyl esterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, podocyte oleate, LewisY, LFA-1, L-selectin, LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagogue 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostaglandins Prostaglandins, survivins and telomerases, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2. TIM-1, TnAg, 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.

9. The conjugate of claim 8, wherein the surface antigen comprises HER2, CD20, CD38, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or combinations thereof.

10. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of: rituximab, trastuzumab, pertuzumab, obitutuzumab, oflamutuzumab, olatuzumab, entuximab, ixartuximab, saxituzumab, U3-1784, daratumumab, STI-6129, belantanumab, indextuzumab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuaT13 / 5 antibody, alemtuzumab, teimomab, tosimob, bevacizumab, panitumumab, trimemumab, teimomab, caputoxumab, ozovozumab, and vetuzumab.

11. The conjugate of claim 10, or a pharmaceutically acceptable salt thereof, wherein the antibody is rituximab, trastuzumab, or pertuzumab.

12. The conjugate of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein: (a) A is a phenyl group; R 1 It is a halogenated group; and X is -N(R) 2 ) v (CH2) m O(CH2) n -;in: v is 1; m and n are both 2; and R 2 It is methyl; (b) A is a phenyl group; R 1 It is a halogenated group; and X is -N(R) 2 ) v (CH2) m O(CH2) n -;in: v is 2; m and n are both 2; and Each R 2 It is methyl; (c) A is a phenyl group; R 1 It is a halogenated group; and X is -O(CH2) n -;in: n is 2; (d) A is a phenyl group; R 1 It is a halogenated group; and X is -S(CH2) n -;in: n is 2; (e) A is a phenyl group; R 1 It is hydrogen; and X is -NR 2 -;in: R 2 It is methyl; (f) A is a phenyl group; R 1 It is a halogenated group; and X is -NR 2 -;in: R 2 It is hydrogen; (g) A is a phenyl group; R 1 It is hydrogen; and X is -C(CH3)=; or (h) A is C4-C 10 cycloalkyl ring; R 1 It is hydrogen; and X is -N(R) 2 (CH2) m O(CH2) n -;in: n is 1; m is 2; and R 2 It is a methyl group.

13. A compound of formula (II): (II); Or its pharmaceutically acceptable salt, wherein: A is a phenyl group or a C4-C4 group. 10 cycloalkyl ring; R 1 Independently selected from hydrogen and halogroups; U is NH; and R 2 Selected from -C(O)R 3 -N(R) 4 2、-(CH2) n OH, -(CH2) n SH, -(CH2) n N(R 4 2、-(CH2) n Q'(CH2) m OH, -(CH2) n Q'(CH2) m SH and -(CH2) n Q'(CH2) m N(R 4 )2; of which R 3 It is hydrogen or C1-C6 alkyl; Each R 4 Independently hydrogen or C1-C6 alkyl; Q' is O, S, or NR 4 ; n is 1-6; and m is 2-5; The premise is that when R 2 For NH2, –(CH2) n NH2 or –(CH2) n When OH, then R 1 It is a halogenated group.

14. A compound of formula (III): Or its pharmaceutically acceptable salt.

15. A compound of formula (IV): (IV); Or its pharmaceutically acceptable salt.

16. A conjugate of formula (V): (V)； Or a pharmaceutically acceptable salt thereof, wherein Bm is the binding moiety, which is an antibody or an antigen-binding fragment thereof that specifically binds to a surface antigen, wherein the surface antigen is not CD33.

17. The conjugate of claim 16, or a pharmaceutically acceptable salt thereof, wherein the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, 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, CD 20. 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, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, condensation factor, cKit, closure protein 3, closure protein 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1. Growth factors, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ErbB3, ERG, ETBR, ETV6-AML, FAP FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose 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-11Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrin, integrin αV, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, podocyte oleate, LewisY, LFA-1, L-selectin, LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagogue 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostaglandins Prostaglandins, survivins and telomerases, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2. TIM-1, TnAg, 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.

18. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein the surface antigen comprises HER2, CD20, CD38, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or combinations thereof.

19. The conjugate of claim 16, or a pharmaceutically acceptable salt thereof, wherein the antibody comprises rituximab, trastuzumab, pertuzumab, obitutuzumab, oflamutumab, olatuzumab, entuximab, ixartuximab, saxituzumab, U3-1784, daratumumab, STI-6129, belantanumab, indextuzumab, cetuximab, denutuximab, anti-CD38 A2 antibody, HuaT13 / 5 antibody, alemtuzumab, teimomab, tosimob, bevacizumab, panitumumab, trimemumab, teimomab, caputoxumab, ovovabumab, or vetozumab.

20. A pharmaceutical composition for treating cancer, comprising a therapeutically effective amount of any one of claims 1 to 12 or 16 to 19, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

21. Use of the conjugate of any one of claims 1 to 12 or 16 to 19, or a pharmaceutically acceptable salt thereof, or the composition of claim 20, in the preparation of a medicament for treating cancer in a subject of need.

22. The use as described in claim 21, 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.

23. The use as claimed in claim 21, further comprising administering a pharmaceutically acceptable amount of the adjuvant to the subject before, after, or simultaneously with the said conjugate or its pharmaceutically acceptable salt or composition.

24. The use as described in claim 23, wherein the adjuvant is a cytotoxic agent or an immune response modulator.

25. The use as described in claim 24, wherein the immune response modulator is a checkpoint inhibitor.

26. The use as claimed in claim 25, 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.

27. A method for preparing the conjugate of claim 1, or a pharmaceutically acceptable salt thereof, the method comprising reacting the conjugating moiety with a compound of formula (I-1): Or a pharmaceutically acceptable salt reaction thereof, wherein the binding moiety is an antibody or an antigen-binding fragment, wherein the binding moiety specifically binds to a surface antigen, wherein the surface antigen is not CD33, wherein: a is an integer from 1 to 10; A is a phenyl group or a C4-C4 group. 10 cycloalkyl ring; R 1 Independently selected from hydrogen and halogroups; U is NH; X is selected from -N(R) 2 ) V -、=C(CH3)-、-Q-(CH2) n - and -Q(CH2) m Q'(CH2) n -;in v is 1 or 2; Q and Q' are each independently O, S, or NR. 2 ; Each R 2 Independently hydrogen or C1-C6 alkyl; n is an integer from 1 to 6; and m is an integer from 2 to 6; Each group is connected to L' on the left and to A on the right; This assumes that X is NH or -Q-(CH2). n - At that time, R 1 It is a halogenated group; L' is selected from 、 、 、 、 、 、 、 、 、 、 、 , and ; in: p is an integer from 1 to 10; q is an integer from 2 to 10; Z 1 It may be absent or contain glycine; Z 2 The following groups are absent or selected: L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 Choose from the following groups: L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z 4 Select the group consisting of the following: L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine; R, R', R” and R'” are each independently selected from hydrogen, C1-C6 alkoxy-C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or the two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring.

28. The method of claim 27, further comprising reducing the binding moiety prior to reacting with the compound of formula (I-1).

29. The method of claim 27 or 28, wherein a is an integer from 2 to 8.

30. The method of any one of claims 27 to 28, wherein the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, 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, CD 20. 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, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, condensation factor, cKit, closure protein 3, closure protein 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPC AM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ErbB3, ERG, ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose 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-11Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrin, integrin αV, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, podocyte oleate, LewisY, LFA-1, L-selectin, LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagogue 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostaglandins Prostaglandins, survivins and telomerases, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2. TIM-1, TnAg, 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.

31. The method of claim 30, wherein the surface antigen comprises HER2, CD20, CD38, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, Trop-2, or combinations thereof.

32. The method of any one of claims 27 to 28, wherein the antibody comprises rituximab, trastuzumab, pertuzumab, obitutuzumab, ofratumumab, olatuzumab, entuximab, ixartuzumab, saxituzumab, U3-1784, daratumumab, STI-6129, belantanumab, indextuzumab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuaT13 / 5 antibody, alemtuzumab, teimomab, tosimob, bevacizumab, panitumumab, trimemumab, teimomab, caputoxumab, ozovumab, or vetuzumab.

33. The method of any one of claims 27 to 28, wherein: A is a phenyl group; R 1 It is a halogenated group; and X is -N(R) 2 ) v (CH2) m O(CH2) n -;in: v is 1; m and n are both 2; and R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -N(R) 2 ) v (CH2) m O(CH2) n -;in: v is 2; m and n are both 2; and Each R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -O(CH2) n -;in: n is 2; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -S(CH2) n -;in: n is 2; or in: A is a phenyl group; R 1 It is hydrogen; and X is -NR 2 -;in: R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -NR 2 -;in: R 2 It is hydrogen; or in: A is a phenyl group; R 1 It is hydrogen; and X is -C(CH3)=; or in: A is C4-C 10 cycloalkyl ring; R 1 It is hydrogen; and X is -N(R) 2 (CH2) m O(CH2) n -;in: n is 1; m is 2; and R 2 It is a methyl group.

34. The method of claim 30, wherein: A is a phenyl group; R 1 It is a halogenated group; and X is -N(R) 2 ) v (CH2) m O(CH2) n -;in: v is 1; m and n are both 2; and R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -N(R) 2 ) v (CH2) m O(CH2) n -;in: v is 2; m and n are both 2; and Each R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -O(CH2) n -;in: n is 2; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -S(CH2) n -;in: n is 2; or in: A is a phenyl group; R 1 It is hydrogen; and X is -NR 2 -;in: R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -NR 2 -;in: R 2 It is hydrogen; or in: A is a phenyl group; R 1 It is hydrogen; and X is -C(CH3)=; or in: A is C4-C 10 cycloalkyl ring; R 1 It is hydrogen; and X is -N(R) 2 (CH2) m O(CH2) n -;in: n is 1; m is 2; and R 2 It is a methyl group.

35. The method of claim 31, wherein: A is a phenyl group; R 1 It is a halogenated group; and X is -N(R) 2 ) v (CH2) m O(CH2) n -;in: v is 1; m and n are both 2; and R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -N(R) 2 ) v (CH2) m O(CH2) n -;in: v is 2; m and n are both 2; and Each R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -O(CH2) n -;in: n is 2; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -S(CH2) n -;in: n is 2; or in: A is a phenyl group; R 1 It is hydrogen; and X is -NR 2 -;in: R 2 Methyl; or in: A is a phenyl group; R 1 It is a halogenated group; and X is -NR 2 -;in: R 2 It is hydrogen; or in: A is a phenyl group; R 1 It is hydrogen; and X is -C(CH3)=; or in: A is C4-C 10 cycloalkyl ring; R 1 It is hydrogen; and X is -N(R) 2 (CH2) m O(CH2) n -;in: n is 1; m is 2; and R 2 It is methyl.

36. The method of claim 27, wherein the compound of formula (I-1) is: 。

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