Neodegrader-anti-CD33 antibody conjugate

Abstract

The present invention provides a neodegrader conjugated to an anti-CD33 antibody. Also provided is a composition comprising the conjugate. The compounds and compositions are useful for treating a disease or condition, such as cancer, in a subject in need of such treatment.

Description

[Technical field]

[0001] Reference to Electronically Provided Sequence Listings The contents of the Sequence Listing, provided electronically with this application in an ASCII text file (Name: 4547_017PC02_Seqlisting_ST25; Size: 42,724 bytes; and Created Date: May 31, 2022), are hereby incorporated by reference in their entirety.

[0002] Field The present invention provides a neodegrader conjugate, in which the neodegrader is conjugated to an anti-CD33 antibody or an antigen-binding portion thereof. Also provided is a composition comprising the conjugate. The conjugate and composition are useful for treating cancer in a subject in need of treatment. [Background technology]

[0003] background Proteolysis has been demonstrated as a therapeutic strategy by the efficacy of immunomodulatory imide drugs. These compounds bind to cereblon (CRBN) and inhibit CRL4. CRBN They have the ability to promote the recruitment and ubiquitination of substrate proteins mediated by E3 ubiquitin ligases. Immunomodulatory imides are thought to act as "molecular glue" and fill the binding interface as a hydrophobic patch that reprograms the protein interaction between the ligase and the neosubstrate.

[0004] Despite the excitement these compounds have generated as novel cancer treatments, to date their use has been limited to hematological malignancies such as multiple myeloma and myelodysplastic syndromes (MDS). The expanding library of compounds that can function by degrading other cancer proteins, many of which are considered "undruggable," is an active area of ​​drug development. Thus, there is a continuing need for novel compounds that can target these alternative cancer proteins and treat a wide range of cancers. Summary of the Invention

[0005] overview Treatment of acute myeloid leukemia (AML) patients with small molecule GSPT1 degraders has been shown to drive clinical responses but is associated with serious adverse events (AEs). Patients with AML often have high levels of CD33 on their cancer cells - supported by the clinical approval of the CD33-targeting ADC, Mylotarg, in the treatment of AML. The present invention is based on the discovery that conjugation of a GSPT1 degrading payload molecule with a CD33-targeting antibody can improve both the clinical efficacy and tolerability of GSPT1 degraders.

[0006] In a first aspect, the present invention provides a compound of formula (I): [ka] [During the ceremony, a is an integer from 1 to 10; A is phenyl or C4-C 10 is a cycloalkyl ring; U is selected from NH and CF2; R 1 is independently selected from hydrogen and halo; X is -NR 2 -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from; where Q and Q' are each independently O, S or N(R 2 ) v and; v is 1 or 2; Each R 2 are independently hydrogen or C1-C6 alkyl; n is an integer from 1 to 6; and m is an integer from 2 to 6; where the left side of each group is bonded to L and the right side is bonded to A; provided that X is NH or -Q-(CH2) n -When R 1 is a halo; L is a cleavable or non-cleavable linker; and Bm is an anti-CD33 antibody or an antigen-binding portion thereof. or a pharma- ceutically acceptable salt thereof.

[0007] In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1 (VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain variable region (VL) CDR1 (VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 5, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a constant region, the constant region differing from gemtuzumab by at least one amino acid. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof is an IgG1 antibody or antigen-binding portion thereof. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises an alanine at amino acid 297, which corresponds to the constant region. In one embodiment, the anti-CD33 antibody comprises a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:10.

[0008] In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a VH-CDR20 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 27 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof is an IgG1 antibody or antigen-binding portion thereof. In some embodiments, the anti-CD33 antibody comprises a heavy chain set forth in SEQ ID NO: 25 and a light chain set forth in SEQ ID NO: 26.

[0009] In some embodiments, a is an integer from 2 to 8.

[0010] In some embodiments, L is a non-cleavable linker. [ka] [During the ceremony, p is an integer from 1 to 10; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is selected from the group consisting of:

[0011] In some embodiments, L is [ka] It is.

[0012] In some embodiments, p is 5.

[0013] In some embodiments, L is a cleavable linker. In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, L is [ka] [During the ceremony, q is an integer from 2 to 10; Z 1 , Z 2 , Z 3 and Z 4 are each independently absent or a naturally occurring amino acid residue of the L or D configuration, with the proviso that Z 1 , Z 2 , Z 3 and Z 4 at least two of are amino acid residues; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is selected from the group consisting of:

[0014] In one embodiment, Z 1 , Z 2 , Z 3 and Z 4 is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine and glycine; with the proviso that Z is 1 , Z 2 , Z 3 and Z 4 At least two of the residues are amino acid residues.

[0015] In one embodiment, Z1 is absent or is glycine; Z 2 is absent or is selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine and glycine; Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine and glycine; and Z 4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine and glycine.

[0016] In some embodiments, L is [ka] It is.

[0017] In some embodiments, q is 5.

[0018] In some embodiments, L is a bioreducible linker. [ka] [During the ceremony, q is an integer from 2 to 10; R, R', R" and R"' are each independently selected from hydrogen, C1-C6 alkoxyC1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is selected from the group consisting of:

[0019] In some embodiments, L is an acid cleavable linker. [ka] [During the ceremony, q is an integer from 2 to 10; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is selected from the group consisting of:

[0020] In some embodiments, L is a click release linker. [ka] [During the ceremony, q is an integer from 2 to 10; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is selected from.

[0021] In some embodiments, L is a pyrophosphatase cleavable linker. [ka] [During the ceremony, q is an integer from 2 to 10; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. It is.

[0022] In some embodiments, L is a beta-glucuronidase cleavable linker. [ka] [During the ceremony, q is an integer from 2 to 10; ---- is absent or a bond; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is selected from.

[0023] In one embodiment, the present invention relates to a conjugate of formula (I), A is phenyl; U is NH; R 1 is a halo; and X is -N(R 2 ) v (CH2) m O(CH2) n - and v is 1; m and n are 2; and R 2 is methyl, A conjugate is provided.

[0024] In one embodiment, the present invention relates to a conjugate of formula (I), A is phenyl; U is NH; R 1 is a halo; and X is -N(R 2 ) v (CH2) m O(CH2) n - and v is 2; m and n are 2; and Each R 2 is methyl, A conjugate is provided.

[0025] In one embodiment, the present invention relates to a conjugate of formula (I), A is phenyl; U is NH; R 1 is a halo; and X is -O(CH2) n - and n is 2, A conjugate is provided.

[0026] In one embodiment, the present invention relates to a conjugate of formula (I), A is phenyl; U is NH; R 1 is a halo; and X is -S(CH2) n - and n is 2, A conjugate is provided.

[0027] In one embodiment, the present invention relates to a conjugate of formula (I), A is phenyl; U is NH; R 1 is hydrogen; and X is -NR 2 - and R 2 is methyl, A conjugate is provided.

[0028] In one embodiment, the present invention relates to a conjugate of formula (I), A is phenyl; U is NH; R 1 is a halo; and X is -NR 2 - and R 2 is hydrogen, A conjugate is provided.

[0029] In one embodiment, the present invention relates to a conjugate of formula (I), A is phenyl; U is NH; R 1 is hydrogen; and X is -C(CH3)= A conjugate is provided.

[0030] In one embodiment, the present invention relates to a conjugate of formula (I), A is C4-C 10 is a cycloalkyl ring; U is NH; R 1 is hydrogen; and X is -N(R 2 )(CH2) m O(CH2) n - and n is 1; m is 2; and R 2 is methyl, A conjugate is provided.

[0031] In one embodiment, the present invention provides a method for producing a compound according to formula (V): [ka] [wherein Bm is an anti-CD33 antibody or an antigen-binding portion thereof.] The anti-CD33 antibody or antigen-binding portion thereof may, for example, be a conjugate of (i) a VH-CDR1 having an amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 having an amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 having an amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 having an amino acid sequence shown in SEQ ID NO: 5, a VL-CDR2 having an amino acid sequence shown in SEQ ID NO: 6, and a VL-CDR3 having an amino acid sequence shown in SEQ ID NO: 7, (ii) a VH having an amino acid sequence shown in SEQ ID NO: 4 and a VL having an amino acid sequence shown in SEQ ID NO: 8, (iii) a heavy chain having a heavy chain shown in SEQ ID NO: 9 and a VL-CDR3 having an amino acid sequence shown in SEQ ID NO: 10, or a pharma- ceutical acceptable salt thereof. (iv) a VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 19, a VH-CDR20 having the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 21, a VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 22, a VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 23, and a VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 24, (v) a VH having the amino acid sequence shown in SEQ ID NO: 27 and a VL having the amino acid sequence shown in SEQ ID NO: 28, or (vi) a heavy chain shown in SEQ ID NO: 25 and a light chain shown in SEQ ID NO: 26.

[0032] In one embodiment, the present invention provides a compound of formula (VI): [ka] [wherein Bm is anti-CD33 or an antigen-binding portion thereof.] The anti-CD33 antibody or antigen-binding portion thereof may, for example, be a conjugate of (i) a VH-CDR1 having an amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 having an amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 having an amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 having an amino acid sequence shown in SEQ ID NO: 5, a VL-CDR2 having an amino acid sequence shown in SEQ ID NO: 6, and a VL-CDR3 having an amino acid sequence shown in SEQ ID NO: 7, (ii) a VH having an amino acid sequence shown in SEQ ID NO: 4 and a VL having an amino acid sequence shown in SEQ ID NO: 8, (iii) a heavy chain having a heavy chain shown in SEQ ID NO: 9 and a VL-CDR3 having an amino acid sequence shown in SEQ ID NO: 10, or a pharma- ceutical acceptable salt thereof. (iv) a VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 19, a VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 20, a VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 21, a VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 22, a VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 23, and a VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 24, (v) a VH having the amino acid sequence shown in SEQ ID NO: 27 and a VL having the amino acid sequence shown in SEQ ID NO: 28, or (vi) a heavy chain shown in SEQ ID NO: 25 and a light chain shown in SEQ ID NO: 26.

[0033] In one embodiment, the present invention provides a compound of formula (VI): [ka] [wherein Bm is an anti-CD33 antibody or an antigen-binding portion thereof.] The anti-CD33 antibody or antigen-binding portion thereof may, for example, be a conjugate of (i) a VH-CDR1 having an amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 having an amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 having an amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 having an amino acid sequence shown in SEQ ID NO: 5, a VL-CDR2 having an amino acid sequence shown in SEQ ID NO: 6, and a VL-CDR3 having an amino acid sequence shown in SEQ ID NO: 7, (ii) a VH having an amino acid sequence shown in SEQ ID NO: 4 and a VL having an amino acid sequence shown in SEQ ID NO: 8, (iii) a heavy chain having a heavy chain shown in SEQ ID NO: 9 and a VL-CDR3 having an amino acid sequence shown in SEQ ID NO: 10, or a pharma- ceutical acceptable salt thereof. (iv) a VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 19, a VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 20, a VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 21, a VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 22, a VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 23, and a VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 24, (v) a VH having the amino acid sequence shown in SEQ ID NO: 27 and a VL having the amino acid sequence shown in SEQ ID NO: 28, or (vi) a heavy chain shown in SEQ ID NO: 25 and a light chain shown in SEQ ID NO: 26.

[0034] In certain aspects, the present invention provides pharmaceutical compositions comprising a conjugate or compound provided herein, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers.

[0035] In some embodiments, the present invention provides a method for treating cancer or myelodysplastic syndrome in a subject in need of treatment, comprising administering to the subject a pharma- ceutically acceptable amount of the conjugate or composition or a pharma- ceutically acceptable salt thereof. In some embodiments, the cancer is a hematological / blood cancer. In some embodiments, the cancer is multiple myeloma, leukemia, malignant lymphoma, Hodgkin's disease or chronic myeloproliferative disorder. In some embodiments, the cancer is acute myelogenous leukemia or lymphoma. In some embodiments, the cancer is acute myelogenous leukemia. In some embodiments, the cancer is resistant or refractory to Mylotarg.

[0036] In some embodiments, the method further comprises administering to the subject a pharma- ceutically acceptable amount of an additional agent before, after, or simultaneously with the conjugate or a pharma- ceutically acceptable salt thereof. In some embodiments, the additional agent is a cytotoxic agent or an immune response modifier. In some embodiments, the immune response modifier is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, and / or a LAG-3 inhibitor.

[0037] In one aspect, the present invention provides a method for preparing a conjugate of formula (I) or a pharma- ceutically acceptable salt thereof, comprising combining an anti-CD33 antibody or an antigen-binding portion thereof with a conjugate of formula (I-1): [ka] [During the ceremony, a is an integer from 1 to 10; A is phenyl or C4-C 10 is a cycloalkyl ring; R 1 is independently selected from hydrogen and halo; U is selected from NH and CF2; X is -N(R 2 ) v -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from; where v is 1 or 2; Q and Q' are each independently O, S or NR 2 and; Each R 2 are independently hydrogen or C1-C6 alkyl; n is an integer from 1 to 6; and m is an integer from 2 to 6; where the left side of each group is attached to L' and the right side is attached to A; However, X is NH or -Q-(CH2) n -When R 1 is a halo; L' is a cleavable or non-cleavable linker precursor conjugated to an anti-CD33 antibody or antigen-binding portion thereof. or a pharma- ceutically acceptable salt thereof. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 1. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a constant region, the constant region differing from gemtuzumab by at least one amino acid. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof is an IgG1 antibody or antigen-binding portion thereof. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises an alanine at amino acid 297, which corresponds to the constant region. In some embodiments, the anti-CD33 antibody comprises a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR20 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:24. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:27 and a VL comprising the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof is an IgG1 antibody or antigen-binding portion thereof. In some embodiments, the anti-CD33 antibody comprises a heavy chain set forth in SEQ ID NO:25 and a light chain set forth in SEQ ID NO:26.

[0038] In certain embodiments, the method further comprises reducing the anti-CD33 antibody, or antigen-binding portion thereof, prior to reaction with the compound of Formula (I-1).

[0039] In some embodiments, a is an integer from 2 to 8. In some embodiments, L' is a non-cleavable linker precursor, a cleavable linker precursor, a bioreducible linker precursor, an acid cleavable linker precursor, a click release linker precursor, a pyrophosphatase cleavable linker precursor, a beta-glucuronidase cleavable linker precursor, or any combination thereof. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows the in vivo activity of representative neodegrader conjugates against MV411 (CD33+) tumors. The X-axis shows days after dosing. The Y-axis shows tumor volume (mm3) after dosing with vehicle, 3 mg / kg CD33AB-compound (Ia), 2.83 mg / kg CD33AB-compound (Ie), 2.99 mg / kg CD33AB-compound (1h), 0.1 mg / kg Mylotarg, 50 mg / kg venetoclax, and 5 mg / kg CC-90009.

[0041] [Diagram 2] FIG. 2 shows the in vitro activity of huMy9-6(AB1)-Compound (Ia) in CD33-positive and CD33-negative malignancies.

[0042] [Diagram 3] FIG. 3 shows the in vivo activity of AB1-based conjugates against MV4-11 (CD33+) tumors. The X-axis shows the number of days after dosing. The Y-axis shows the tumor volume (mm3) after dosing with vehicle, AB1-compound (Ia), AB1-compound (Ii), AB1-compound (Id), AB1-compound (Ij), AB1-compound (Ie), AB1-compound (Ik), Mylotarg, Gemtuzumab-compound I(a) and CC-90009.

[0043] [Figure 4]FIG. 4 shows the stability of gemtuzumab and CD33AB conjugates.

[0044] [Diagram 5] FIG. 5 shows the in vivo activity of gemtuzumab-based conjugates against MV4-11 (CD33+) tumors. The X-axis shows the number of days after dosing. The Y-axis shows the tumor volume (mm3) after dosing with vehicle, 3 mg / kg gemtuzumab-compound (Ia), 5 mg / kg gemtuzumab-compound (Ia), 3 mg / kg CD33AB-compound (Ia), 5 mg / kg CD33AB-compound (Ia), 3 mg / kg gemtuzumab IgG1 LALA-compound (Ia), 5 mg / kg gemtuzumab IgG1 LALA-compound (Ia), 5 mg / kg gemtuzumab-compound (Ie), 25 mg / kg venetoclax, and 50 mg / kg venetoclax.

[0045] [Figure 6] Figures 6A and 6B show the in vitro activity of AB1-Compound (Ia) conjugate against Mylotarg-insensitive AML cells (AML-193 (Figure 6A) and Kasumi-6 (Figure 6B)). The X-axis shows the concentration and the Y-axis shows the percent viability of the cell line after treatment.

[0046] Detailed Description The present invention relates to a compound of formula (I): [ka] [During the ceremony, a is an integer from 1 to 10; A is phenyl or C4-C 10 is a cycloalkyl ring; R 1 is independently selected from hydrogen and halo; U is selected from NH and CF2; X is -N(R 2 ) v - 、 =C(CH3)-, -Q-(CH2) n - and -Q(CH2) mQ'(CH2) n - selected from; where Q and Q' are each independently O, S or N(R 2 ) v and; v is 1 or 2; Each R 2 are independently hydrogen or C1-C6 alkyl; n is an integer from 1 to 6; m is an integer from 2 to 6; where the left side of each group is attached to L and the right side is attached to A; However, X is NH or -Q-(CH2) n -When R 1 is a halo; L is a cleavable or non-cleavable linker; and Bm is an anti-CD33 antibody or antigen-binding portion thereof, e.g., an anti-CD33 antibody or antigen-binding portion thereof, a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:3, a light chain variable region (VL) CDR1 comprising the amino acid sequence shown in SEQ ID NO:5, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:7; an anti-CD33 antibody or antigen-binding portion thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO:4 and a VL comprising the amino acid sequence shown in SEQ ID NO:8; or an anti-CD33 antibody comprising a heavy chain shown in SEQ ID NO:9 and a light chain shown in SEQ ID NO:10. or a pharma- ceutically acceptable salt thereof.

[0047] The present invention also provides the above compounds fused to an anti-CD33 antibody or antigen-binding portion thereof, compositions comprising said compounds or said conjugates or methods of using or making said compounds or said conjugates.

[0048] VI. Definitions In order that this description may be more readily understood, certain terms are first defined. Further definitions are given throughout the detailed description.

[0049] It is understood that a singular reference to an item refers to one or more of that item; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. That is, the terms "a," "one or more," and "at least one" may be used interchangeably herein. It is further noted that the claims may be drafted to exclude any optional element. That is, this description is intended to serve as a predicate for the use of exclusive terms such as "solely," "only," and the like, or the use of a negative limitation in connection with the limitation of a claim element.

[0050] Furthermore, "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used herein in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" used in a phrase such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0051] When embodiments are described herein using the term "comprising," it is understood that other similar embodiments described using "consisting of" and / or "consisting essentially of" are also provided.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000 provide those skilled in the art with a general dictionary of many of the terms used herein.

[0053] Units, prefixes, and symbols are shown in the form recognized by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. When a range of values ​​is described, it is understood that each integer value and each fraction between the stated upper and lower limits of the range is also specifically disclosed, along with each subrange of such values. The upper and lower limits of any range can be independently included or excluded within the range, and each range in which either, neither, or both of the limits are included is also included in the invention. Thus, ranges described herein are understood to include all omissions of values ​​within the range, including the stated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subranges in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0054] When values ​​are specified, it is understood that values ​​that are approximately the same quality or quantity as the stated value are also within the scope of the present invention. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the present invention. Conversely, when different elements or groups of elements are individually disclosed, their combinations are also disclosed. When any element of the present invention is disclosed as having multiple options, the disclosed examples in which each option is excluded, either alone or in any combination with other options, are also disclosed herein; more than one element of the present invention may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0055] As used herein, the term "DAR" refers to the drug-antibody ratio of a conjugate, which is the average number of neodegrader-linker complexes linked to each antibody. In some embodiments, the DAR of the conjugates described herein is 1-10. In some embodiments, the DAR of the conjugates described herein is 1-8. In some embodiments, the DAR of the conjugates described herein is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 1 .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.

[0056] As used herein, the term "antibody" refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulins can be from any species. However, in certain embodiments, the immunoglobulins are of human, murine, or rabbit origin.

[0057] The term "single domain antibody", also known as nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain with a molecular weight of about 12 kDa to about 15 kDa. Single antibodies can be based on heavy chain variable domains or on light chains. An example of a single domain antibody is the V H H fragment and V NAR Including, but not limited to, fragments.

[0058] An "antibody fragment" comprises a portion of an intact antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab').sub.2 and Fv fragments; bispecific antibodies; linear antibodies; fragments produced by a Fab expression library which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions) and epitope-binding fragments of any of the above, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0059] An "intact antibody" is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0060] The term "monoclonal antibody" as used herein refers to a substantially homogeneous antibody, i.e., an antibody obtained from a population in which the individual antibodies constituting the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to polyclonal antibody preparations which include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not intended to require production of the antibody by any particular method. For example, monoclonal antibodies for use in the present invention may be produced by hybridoma methods or by recombinant DNA methods. "Monoclonal antibodies" may also be isolated from phage antibody libraries.

[0061] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belong to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belong to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include "primatized" antibodies which contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0062] Various methods are used to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses cells from various species, including mice (murine), hamsters, rats, and humans. Other methods used to produce MAbs use genetic engineering, including recombinant DNA technology. Monoclonal antibodies produced by these techniques include chimeric and humanized antibodies, among others. Chimeric antibodies combine DNA coding regions from more than one type of species. For example, chimeric antibodies can be derived from variable regions from mouse and constant regions from human. Humanized antibodies are primarily human, even if they contain non-human portions. Like chimeric antibodies, humanized antibodies can contain fully human constant regions. However, unlike chimeric antibodies, the variable regions can be partially human. The non-human, synthetic portions of humanized antibodies are often derived from the CDRs of mouse antibodies. Either way, these regions are important for the antibody to be able to recognize and bind to a specific antigen. Although useful for diagnosis and short-term therapy, mouse antibodies cannot be administered long-term to humans without increasing the risk of adverse immunogenic responses. This response, called human anti-mouse antibody (HAMA), occurs when the human immune system recognizes the mouse antibody as foreign and attacks it. The HAMA response can lead to toxic shock or even death.

[0063] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portion of the administered antibody, and may have the added benefit of activating a secondary human immune response, such as antibody-dependent cellular cytotoxicity.

[0064] An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like.

[0065] Depending on the amino acid sequence of the constant domain of the heavy chain, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0066] The term "about" is used herein to mean approximately, roughly, in the region or area. When the term "about" is used in conjunction with a numerical value, it modifies the range by extending the boundaries above and below the numerical value set forth. In general, the term "about" can modify the numerical value by, for example, a variance of 10 percent above or below (higher or lower) the stated value.

[0067] The terms "administration", "administer" and grammatical variants thereof refer to the introduction of a composition, such as the EVs (e.g., exosomes) of the present invention, into a subject via a pharma- ceutically acceptable route. The introduction of a composition, such as the EVs (e.g., exosomes) of the present invention, into a subject can be by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal or subcutaneous), rectal, intralymphatic, intrathecal, periocular or topical. Administration includes self-administration and administration by another. 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 the subject's vein.

[0068] The term "antibody" as used herein includes immunoglobulins and fragments thereof, whether natural or partially or wholly synthetically produced. The term also encompasses any protein having a binding domain that is homologous to an immunoglobulin binding domain. "Antibody" further includes polypeptides comprising framework regions from immunoglobulin genes or fragments thereof that specifically bind and recognize an antigen. The use of the term antibody is meant to include complete antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as, for example, scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, bispecific antibodies and antibody-related polypeptides. Antibodies include bispecific and multispecific antibodies, so long as they exhibit the desired biological activity or function. In certain embodiments of the present invention, the biologically active molecule is a molecule comprising an antibody or an antigen-binding fragment thereof.

[0069] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to an antibody attached, e.g., covalently, to a therapeutic agent (sometimes referred to herein as a drug, agent or active pharmaceutical ingredient) or agent. In certain embodiments of the invention, the biologically active molecule is an antibody-drug conjugate.

[0070] As used herein, the term "approximately" when applied to one or more values ​​of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" refers to a range of values ​​that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise specified or clear from the context (except when such value exceeds 100% of possible values).

[0071] "Conservative amino acid substitution" refers to an amino acid residue that is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when an amino acid in a polypeptide is replaced with another amino acid of the same side chain family, the substitution is considered conservative. In other embodiments, strings of amino acids can be conservatively replaced with structurally similar strings that differ in the order and / or composition of side chain family members.

[0072] As used herein, the term "conserved" refers to nucleotides or amino acid residues, respectively, of a polynucleotide or polypeptide sequence that do not vary at the same position in two or more sequences being compared. A relatively conserved nucleotide or amino acid is one that is more conserved in related sequences than a nucleotide or amino acid that appears elsewhere in the sequence.

[0073] In some embodiments, two or more sequences are considered "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are considered "highly conserved" if they are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. In some embodiments, two or more sequences are considered "conserved" if they are at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to portions, parts, regions, or features thereof.

[0074] As used herein, the terms "linked" and "conjugated" are used interchangeably and refer to the covalent or non-covalent attachment of two or more moieties, including a neodegrader and an anti-CD33 antibody or antigen-binding portion thereof, respectively. In some embodiments, the linked or conjugated may include a linker.

[0075] The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Usually, an amino acid sequence variant has 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 has at least about 80% and more typically at least about 90% sequence identity with such receptor or ligand binding domain. Amino acid sequence variants have substitutions, deletions and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by trivial names, one-letter and three-letter codes.

[0076] "Sequence identity" is defined as the percentage of residues in amino acid sequence variants that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignment are well known in the art. One computer program is "Align 2" by Genentech, Inc., filed with the United States Copyright Office, Washington, DC 20559, on December 10, 1991, together with the manual.

[0077] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that is complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed.

[0078] The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. 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 domains are called framework regions (FRs). Each of the variable domains of naturally occurring heavy and light chains contains four FRs that adopt a predominantly beta-sheet configuration, connected by three hypervariable regions that connect, and in some cases form part of, a beta-sheet structure. The hypervariable regions of each chain are held in close proximity to each other by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0079] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al supra) and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0080] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0081] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions contribute to the antigen binding specificity of the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, but with a lower affinity than the complete binding site.

[0082] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0083] The "light chains" of antibodies from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0084] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0085] The term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites, which comprises a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen-binding sites.

[0086] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody is purified (1) to the extent of greater than 95% or greater than 99% by weight of the antibody as determined by the Lowry method, (2) to the extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a gas phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions by use of Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0087] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Immature cell division and proliferation leads to the formation of malignant tumors that can invade nearby tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary, metastatic and recurrent cancers.

[0088] As used herein, the term "immune response" refers to a biological response of a vertebrate to foreign agents, which response protects the organism against these agents and the diseases caused by them. Immune responses are 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, which result in the selective targeting, binding, damaging, destroying, and / or eliminating from the vertebrate body, invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses can be mediated, for example, by T cells, e.g., effector T cells or CD4 T cells, or by the action of soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which result in the selective targeting, binding, damaging, destroying, and / or eliminating from the vertebrate body, invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. + or CD8 +The term "T cell" and "T lymphocyte" as used herein are interchangeable and refer to any lymphocyte produced or processed by the thymus. In one embodiment, a T cell is a CD4 + In one embodiment, the T cells are CD8 + In some embodiments, the T cell is a NKT cell.

[0089] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0090] The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of an agent (e.g., a neodegrader or neodegrader conjugate disclosed herein) that provides a desired biological, therapeutic and / or prophylactic result. The result may be a decrease, amelioration, alleviation, reduction, delay and / or alleviation of one or more of the signs, symptoms or causes of a disease or any other desired alteration of a biological system. For solid tumors, an effective amount includes an amount that shrinks the tumor and / or reduces the tumor growth rate (e.g., to tumor growth inhibition) or prevents or delays other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered one or more times. An effective amount of the composition can, for example, (i) reduce the number of cancer cells; (ii) reduce the size of a tumor; (iii) inhibit, delay, slow or stop to some extent cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent) or stop tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0091] In some embodiments, a "therapeutically effective amount" is an amount of a neodegrader or neodegrader conjugate that has a clinically proven effect in significantly reducing cancer or slowing the progression (regression) of cancer, 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 of skill in the art, such as assaying the activity of the agent in human subjects in clinical trials, animal model systems predictive of human efficacy, or in vitro assays.

[0092] As used herein, the term "standard of care" refers to a procedure that is accepted by medical professionals as the appropriate treatment for a certain type of illness and is widely used by medical practitioners. This term may be used interchangeably with any of the following terms: "best practice," "standard of medical care," and "standard of care."

[0093] As an example, an "anti-cancer drug" promotes cancer regression or prevents further tumor growth in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer.

[0094] The terms "effective" and "effectiveness" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cell, organ and / or organism level resulting from drug administration.

[0095] The term "immune checkpoint inhibitors" as used herein refers to molecules that fully or partially reduce, inhibit, prevent or modulate one or more checkpoint proteins. Checkpoint proteins control T cell activation or function. A number of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2. Pardoll, DM, Nat Rev Cancer 12 (4): 252-64 (2012). These proteins are responsible for costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins control and maintain self-tolerance as well as the duration and strength of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies.

[0096] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent or slow down (reduce) undesired physiological changes or disorders, such as the progression or spread of cancer. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., non-worsening) of the disease state, delay or slow down of disease progression, improvement or palliative and remission (whether partial or complete) of the disease state, whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to life expectancy without treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed to the condition or disorder or those in whom the condition or disorder is to be prevented.

[0097] VII. "Neo Degrader" The present invention relates to a compound of formula (II): [ka] [During the ceremony, A is phenyl or C4-C 10 is a cycloalkyl ring; U is selected from NH and CF2; R 1 is independently selected from hydrogen and halo; R 2 HA-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; where R 3 is hydrogen or C1-C6 alkyl; Each R 4 are independently hydrogen or C1-C6 alkyl; Q' is O, S or NR 4 and; n is 1 to 6; and m is 2 to 5; However, R 2 NH2, -(CH2) n NH2 or -(CH2) n When OH, R 1 is a halo. or a pharma- ceutically acceptable salt thereof.

[0098] In one embodiment, the present invention provides a compound of formula (II) or a pharma- ceutically acceptable salt thereof, wherein: A is a phenyl ring or C4-C 10 is a cycloalkyl ring; U is NH; R 1 is selected from hydrogen and halo; R 2 Ha-(CH2) n Q'(CH2) m N(R 4 )2, -(CH2) n OH, -(CH2) n SH, -N(R 4 )2 and -C(O)R3 is selected from; where m is 2; n is 2; Q' is -O-; R 3 is methyl; and Each R 4 is independently selected from hydrogen and methyl; However, R 2 is NH2 or -(CH2) n When OH, R 1 is a halo.

[0099] The term "C1-C6 alkoxy," as used herein, refers to a C1-C6 alkyl group attached to the parent molecular moiety through an oxygen atom.

[0100] The term "C1-C6 alkoxy C1-C6 alkyl," as used herein, refers to a C1-C6 alkoxy group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0101] The term "C1-C6 alkyl" as used herein refers to a group derived from a straight or branched chain saturated hydrocarbon containing from 1 to 6 carbon atoms.

[0102] The term "C4-C 10 "Cycloalkyl" refers to a saturated monocyclic, hydrocarbon ring system having from 4 to 10 carbon atoms and 0 heteroatoms. Representative examples of cycloalkyl groups include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups containing from 7 to 10 atoms can be monocyclic or fused, spirocyclic, or bridged bicyclic structures.

[0103] The term "halo" as used herein refers to F, Cl, Br or I.

[0104] In some embodiments, the neodegrader of formula (II) is: [ka] The compound is selected from the group consisting of:

[0105] In one embodiment, the neodegrader of formula (II) is [ka] It is.

[0106] In one embodiment, the neodegrader of formula (II) is [ka] It is.

[0107] In one embodiment, the neodegrader of formula (II) is [ka] It is.

[0108] In one embodiment, the neodegrader of formula (II) is [ka] It is.

[0109] In one embodiment, the neodegrader of formula (II) is [ka] It is.

[0110] In one embodiment, the neodegrader of formula (II) is [ka] It is.

[0111] In one embodiment, the neodegrader of formula (II) is [ka] It is.

[0112] In one embodiment, the neodegrader of formula (II) is [ka] It is.

[0113] In some embodiments, the present invention provides a neodegrader of formula (II) or a pharma- ceutically acceptable salt thereof, wherein A is phenyl; U is NH; and R 1 is a halo; and R 2 Ha-(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is O, and one R 4 is hydrogen and the other is methyl.

[0114] In one embodiment, the present invention provides a neodegrader of formula (II), wherein AA is phenyl; U is NH; R 1 is a halo; and R 2 Ha-(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is O, and each R 4 is methyl.

[0115] In one embodiment, the present invention provides a neodegrader of formula (II), where AA is phenyl; U is NH; R 1 is a halo; and R 2 Ha-(CH2) n OH, where n is 2.

[0116] In one embodiment, the present invention provides a neodegrader of formula (II), where A is phenyl; U is NH; R 1 is a halo; and R 2 Ha-(CH2) n SH, where n is 2.

[0117] In one embodiment, the present invention provides a neodegrader of formula (II), where A is phenyl; U is NH; R 1 is hydrogen; and R 2 -N(R 4 )2, where one R 4 is hydrogen and the other is methyl.

[0118] In one embodiment, the present invention provides a neodegrader of formula (II), where A is phenyl; U is NH; R 1 is a halo; and R 2 -N(R 4 )2, where each R 4 is hydrogen. In one embodiment, the present invention provides a neodegrader of formula (II), wherein A is phenyl; R 1 is hydrogen; and R 2 HA-C(O)R 3 where R 3 is methyl.

[0119] In one embodiment, the present invention provides a neodegrader of formula (II), wherein AA is C4-C 10 is a cycloalkyl ring; U is NH; R 1 is hydrogen; and R 2 Ha-(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is O, and one R 4 is hydrogen and the other is methyl.

[0120] In certain embodiments, a neodegrader is a molecule that forms a ternary complex with an E3 ubiquitin ligase that can target proteins for degradation.

[0121] VIII. Neodegrader Conjugates The present invention provides conjugates of one or more of the neodegraders disclosed herein and the anti-CD33 antibodies or antigen-binding portions thereof disclosed herein. These conjugates bind to cereblon (CRBN) and inhibit CRL4. CRBN Proteins can be degraded by promoting the recruitment and ubiquitination of substrate proteins mediated by E3 ubiquitin ligases. These agents act as "molecular glue" and fill the binding interface as a hydrophobic patch that reprograms the ligase and neo-substrate protein interactions.

[0122] In one embodiment, the present invention relates to a compound represented by formula (I): [ka] [During the ceremony, a is an integer from 1 to 10; A is phenyl or C4-C 10 is a cycloalkyl ring; R 1 is selected from hydrogen and halo; U is selected from NH and CF2; X is -NR 2 -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from; where Q and Q' are each independently O, S or NR 2 and; R 2 is hydrogen or C1-C6 alkyl; n is an integer from 1 to 6; m is an integer from 2 to 6; where the left side of each group is attached to L and the right side is attached to A; However, X is NH or -Q-(CH2) n -When R 1 is a halo; L is a cleavable or non-cleavable linker; and Bm is an anti-CD33 antibody or antigen-binding portion thereof disclosed herein. or a pharma- ceutically acceptable salt thereof.

[0123] In some embodiments, U is NH.

[0124] In some embodiments, the neodegrader conjugates described herein have in vitro anti-proliferative activity against tumor cell lines. In some embodiments, the neodegrader conjugates comprising a neodegrader and an anti-CD33 antibody or antigen-binding portion thereof disclosed herein have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% greater in vitro anti-proliferative activity than the neodegrader alone or the anti-CD33 antibody or antigen-binding portion thereof disclosed herein alone. In some embodiments, the neodegrader conjugates comprising a neodegrader and an anti-CD33 antibody or antigen-binding portion thereof disclosed herein have at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater in vitro anti-proliferative activity than the neodegrader alone or the anti-CD33 antibody or antigen-binding portion thereof disclosed herein alone.

[0125] In some embodiments, the neodegrader conjugates described herein have in vitro anti-proliferative activity against Daudi lymphoma cell lines, e.g., anti-proliferative activity against Daudi lymphoma cell lines, as compared to the neodegrader alone or to an anti-CD33 antibody or antigen-binding portion thereof alone. In some embodiments, the neodegrader conjugates described herein have in vitro anti-proliferative activity against HL-60 acute myeloid leukemia cell lines, e.g., anti-proliferative activity against HL-60 acute myeloid leukemia cell lines, as compared to the neodegrader alone or to an anti-CD33 antibody or antigen-binding portion thereof alone. In some embodiments, the neodegrader conjugates described herein have in vitro anti-proliferative activity against Ramos non-Hodgkin lymphoma cell lines, e.g., anti-proliferative activity against Ramos non-Hodgkin lymphoma cell lines, as compared to the neodegrader alone or to an anti-CD33 antibody or antigen-binding portion thereof alone. In some embodiments, the neodegrader conjugates described herein can maintain anti-proliferative activity in the presence of human serum. The neodegrader conjugates described herein can be used to treat cancer.

[0126] In one embodiment, an antibody-neodegrader conjugate (AnDC) is a conjugate of one or more neodegraders disclosed herein and an anti-CD33 antibody, or antigen-binding portion thereof, disclosed herein.

[0127] III.A. Linkers The neodegraders of the present invention can be linked to an anti-CD33 antibody or antigen-binding portion thereof via a linker. As used herein, the term "linker" refers to any chemical moiety capable of connecting an anti-CD33 antibody or antigen-binding portion thereof (Bm) to the group X in the compound of formula (I).

[0128] In some embodiments, the linker may comprise a heterobifunctional group. In the present invention, the term "heterobifunctional group" refers to a chemical moiety that connects the linker of which it is a part to the anti-CD33 antibody or antigen-binding portion thereof. Heterobifunctional groups are characterized as having different reactive groups at either end of the chemical moiety. Conjugation to "Bm" can be achieved via chemical or enzymatic conjugation or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the anti-CD33 antibody or antigen-binding portion thereof with the reactive handle of the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via genetically engineered non-natural amino acids (wherein a non-natural amino acid with a desired reactive handle is inserted on "Bm"). In enzymatic conjugation, an enzyme mediates the coupling of the linker to accessible amino acid residues on the anti-CD33 antibody or antigen-binding portion thereof. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation can also be used sequentially. For example, enzymatic conjugation can be used to introduce a unique reactive handle onto "Bm" for use in subsequent chemical conjugation.

[0129] In some embodiments, the heterobifunctional group is: [ka] [During the ceremony, [ka] is the point of attachment to the remainder of the linker; and [ka] is the point of attachment to Bm. is selected from.

[0130] In some embodiments, the linker "L" is non-cleavable. As used herein, the term "non-cleavable linker" refers to any chemical moiety that can link an anti-CD33 antibody, or antigen-binding portion thereof, to a neodegrader in a stable, covalent manner and does not fall within the category defined herein as "cleavable linkers." Thus, a non-cleavable linker is substantially resistant to acid-induced cleavage, photoinduced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. "Substantially resistant to cleavage" means that 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 linker or adjacent linker chemical bonds of the antibody neodegrader conjugate population remain uncleaved by acids, photolabile cleavage agents, bioreductive agents, peptidases, esterases, or chemical or physiological compounds that cleave the chemical bonds (e.g., disulfide bonds) in the cleavable linker within hours to days of treatment with any of the above agents. In some embodiments, the linker is not susceptible to acid-induced cleavage, photoinduced cleavage, bioreductive cleavage, enzymatic cleavage, etc. under conditions under which the neodegrader and / or anti-CD33 antibody or antigen-binding portion thereof may remain active. ADC catabolic products generated from non-cleavable linkers include residual amino acids from the antibody. These catabolic products may exert unique and unexpected properties on the target cells from which they are derived.

[0131] One of skill in the art can easily distinguish between non-cleavable and cleavable linkers.

[0132] Examples of non-cleavable linkers are SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) linker, succinimide thioether linker and: [ka] [During the ceremony, p is an integer from 1 to 10; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. Linkers include, but are not limited to,

[0133] In some embodiments, the linker is: [ka] In some embodiments, p is 5.

[0134] In some embodiments, the linker may be cleavable. In some embodiments, the linker may be susceptible to acid-induced cleavage, photoinduced cleavage, bioreductive cleavage, enzymatic cleavage, and the like, under conditions where the neodegrader and / or anti-CD33 antibody or antigen-binding portion thereof may remain active.

[0135] In some embodiments, the cleavable linker can be cleaved by an enzyme, hi some embodiments, the cleavable linker can be cleaved by a protease, peptidase, esterase, beta-glucuronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, or lipase.

[0136] In some embodiments, the cleavable linker can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like.

[0137] In some embodiments, the cleavable linker comprises a peptide. In some embodiments, the peptide is the cleavage site of the linker, thereby facilitating the release of the drug by exposure to intracellular proteases, such as lysosomal enzymes. The peptide can be designed and optimized for enzymatic cleavage by a specific enzyme, such as tumor-associated proteases, cathepsin B, C and D, or plasmin proteases. Examples of peptides with 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 with three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit), aspartic acid-valine-citrulline (asp-val-cit), alanine-alanine-asparagine (ala-ala-asn), alanine-phenylalanine-lysine (ala-phe-lys), glycine-glycine-phenylalanine (gly-gly-phe) and glycine-glycine-glycine (gly-gly-gly). Examples of peptides with four amino acids include, but are not limited to, glycine-glycine-valine-citrulline (gly-gly-val-cit) and glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly). The above amino acid combinations can also occur in the reverse order (i.e., cit-val).

[0138] The peptides of the present invention may contain amino acid residues in their L- or D-isomer form. 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-" designates an amino acid having a "D" (dextrorotatory) configuration, which is the opposite of the configuration of the naturally occurring ("L-") amino acid. The amino acids described herein can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0139] In some embodiments, the linker ("L") is [ka] [During the ceremony, q is an integer from 2 to 10; Z 1 , Z 2 , Z 3 and Z 4 are each independently absent or a naturally occurring amino acid residue of the L or D configuration, with the proviso that Z 1 , Z 2 , Z 3 and Z 4 at least two of are amino acid residues; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is a protease-cleavable linker selected from

[0140] In one embodiment, Z 1 , Z 2 , Z 3 and Z 4is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine and glycine; with the proviso that Z is 1 , Z 2 , Z 3 and Z 4 At least two of the residues are amino acid residues.

[0141] In one embodiment, Z 1 is absent or is glycine; Z 2 is absent or selected from L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine and glycine; Z 3 is selected from L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine and glycine; and Z 4 is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine and glycine.

[0142] In some embodiments, L is [ka] It is.

[0143] In some embodiments, q is 5.

[0144] In certain embodiments, L is a pyrophosphatase cleavable linker.

[0145] In some embodiments, L is: [ka] [During the ceremony, q is an integer from 2 to 10; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is a pyrophosphatase cleavable linker.

[0146] In certain embodiments, L is a beta-glucuronidase cleavable linker.

[0147] In some embodiments, L is: [ka] [During the ceremony, q is an integer from 2 to 10; ---- is absent or a bond; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is a beta-glucuronidase cleavable linker selected from:

[0148] In some embodiments, the linker is bioreducible. Bioreducible linkers take advantage of the reductive potential in intracellular compartments that are different from plasma. The reduced glutathione present in the cytoplasm of tumor cells is up to 1000 times higher than that present in the cytoplasm of normal cells, and tumor cells also contain enzymes that can contribute to reduction in cellular compartments. The linker keeps the conjugate intact in the systemic circulation and is selectively cleaved by the high intracellular concentration of glutathione, releasing the active drug from the non-toxic prodrug at the tumor site.

[0149] In some embodiments, L is: [ka] [During the ceremony, q is an integer from 2 to 10; R, R', R" and R"' are each independently selected from hydrogen, C1-C6 alkoxyC1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is a bioreducible linker selected from the group consisting of

[0150] In some embodiments, the linker is acid cleavable. Acid cleavable linkers are specifically designed to remain stable at the neutral pH of the blood circulation, but to be hydrolyzed in the acidic environment of the cellular compartment, releasing the cytotoxic drug.

[0151] In some embodiments, L is [ka] [During the ceremony, q is an integer from 2 to 10; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. is an acid cleavable linker selected from

[0152] In certain embodiments, L is a click release linker in which release of the neodegrader is chemically triggered by a tetrazine or related compound.

[0153] In some embodiments, L is [ka] [During the ceremony, q is an integer from 2 to 10; [ka] is the point of attachment to X; and [ka] is the point of attachment for the anti-CD33 antibody or antigen-binding portion thereof. The click release linker is selected from the group consisting of:

[0154] III.B. Anti-CD33 antibody The present invention provides a neodegrader conjugated to an anti-CD33 antibody, or antigen-binding portion thereof.

[0155] CD33 is a transmembrane receptor expressed on both myeloid and lymphoid cells. It binds sialic acid and is therefore a member of the sialic acid-binding immunoglobulin-type lectin (SIGLEC) family. CD33 plays a role in mediating cell interactions and maintaining the quiescent state of immune cells. Upon binding, CD33's immunoreceptor tyrosine-based inhibitory motifs (ITIMs), present in the cytoplasmic portion of the protein, are phosphorylated and act as docking sites for Src homology 2 (SH2) domain-containing proteins such as SHP phosphatases. This can result in a cascade that inhibits phagocytosis of cells. Structurally, the extracellular portion of CD33 contains two immunoglobulin domains and the intracellular portion contains the ITIMs. Synonyms for CD33 include, but are not limited to, sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, and p67.

[0156] The canonical amino acid sequence and known isoforms of human CD33 are shown in Table 1 (UniProtKB-P20138; SEQ ID NOs: 13 to 18). [Table 1]

[0157] CD33 is expressed in approximately 90% of acute myeloid leukemia (AML) cases, demonstrating its usefulness as a target for therapeutic antibodies. High CD33 expression on AML blasts was reported approximately 30 years ago. CD33 was detected on blasts in 85-90% of patients presenting with AML as well as on normal myeloid progenitor cells and myelocytes. CD33 is restricted to hematopoietic cells, but not normal hematopoietic stem cells, making it an ideal therapeutic for the treatment of AML.

[0158] The anti-CD33 antibodies for the conjugates of the present invention can specifically bind to CD33. In some embodiments, the anti-CD33 antibodies described herein have high affinity, e.g., 10 -6 Below, 10 -7 Below, 10 -8 Below, 10 -9 Below, 10 -10 Below, 10-11 Below, 10 -12 Below, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M or 10 -9 M~10 -7 K of M D and binds to human CD33.

[0159] In some embodiments, the anti-CD33 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) and the light chain comprises a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1 (VH-CDR1), VH-CDR2 and VH-CDR3, and the VL comprises a VL-CDR1, VL-CDR2 and VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the anti-CD33 antibody comprises a VH-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the anti-CD33 antibody comprises a VH-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD33 antibody comprises a VL-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-CD33 antibody comprises a VL-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD33 antibody comprises a VL-CDR3 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the CDR comprises a sequence as shown in Table 2 below. [Table 2]

[0160] In certain embodiments, the anti-CD33 antibody heavy chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the anti-CD33 antibody light chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0161] In certain embodiments, the anti-CD33 antibody comprises a heavy chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:4, and a light chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0162] In certain embodiments, the anti-CD33 antibody heavy chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9 or SEQ ID NO:11. In certain embodiments, the anti-CD33 antibody comprises a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:10 or SEQ ID NO:12. [Table 3]

[0163] In certain embodiments, the anti-CD33 antibody comprises a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9, and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:10.

[0164] In certain embodiments, the anti-CD33 antibody comprises a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:11, and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:12.

[0165] In certain embodiments, the anti-CD33 antibody is disclosed in US Pat. No. 5,585,089, US Pat. No. 5,693,762, each of which is expressly incorporated herein by reference.

[0166] In some embodiments, the anti-CD33 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) and the light chain comprises a light chain variable region (VL); wherein the VH comprises a VH complementarity Determining Region (CDR) 1 (VH-CDR1), VH-CDR2 and VH-CDR3, and the VL comprises a VL-CDR1, VL-CDR2 and VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-CD33 antibody comprises a VH-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the anti-CD33 antibody comprises a VH-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the anti-CD33 antibody comprises a VL-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the anti-CD33 antibody comprises a VL-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, the anti-CD33 antibody comprises a VL-CDR3 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDR comprises a sequence as shown in Table 4 below. [Table 4]

[0167] In certain embodiments, the anti-CD33 antibody heavy chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the anti-CD33 antibody light chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:28.

[0168] In certain embodiments, the anti-CD33 antibody comprises a heavy chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:27, and a light chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:28.

[0169] In certain embodiments, the anti-CD33 antibody heavy chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 25. In certain embodiments, the anti-CD33 antibody comprises a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 26. [Table 5]

[0170] In certain embodiments, the anti-CD33 antibody comprises a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 25, and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 26. The term "CD33AB" includes the heavy chain set forth in SEQ ID NO: 25 and the light chain set forth in SEQ ID NO:26.

[0171] Anti-CD33 antibodies include modifications, i.e., analogs and derivatives, to which any type of molecule is covalently attached, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. By way of example, but not by way of limitation, antibody derivatives and analogs include those that are further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular antibody units or other proteins, and the like. Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like. Additionally, analogs or derivatives may contain one or more unnatural amino acids.

[0172] The anti-CD33 antibody in the neodegrader conjugate may include an antibody with modifications (e.g., substitutions, deletions, or additions) at amino acid residues that interact with Fc receptors. In particular, the antibody includes an antibody with modifications at amino acid residues that have been identified as involved in the interaction between the anti-Fc domain and the FcRn receptor. Antibodies immunospecific for cancer cell antigens can be obtained commercially, for example, from Genentech (San Francisco, Calif.), or produced by any method known to those skilled in the art, for example, by chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, publications, or by routine cloning and sequencing.

[0173] In some embodiments, the antibody of the neodegrader conjugate can be a monoclonal antibody, such as a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the antibody can be an antibody fragment, such as a Fab fragment.

[0174] IX. Compositions and Methods of Use The conjugates and / or compounds described herein may be in the form of a pharma- ceutically or pharma-ceutically acceptable salt. In some embodiments, such salts are derived from inorganic or organic salts.

[0175] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, cinnamate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate salts.

[0176] Examples of suitable base addition salts include ammonium salts; alkali metal salts, such as sodium salts and potassium salts; alkaline earth metal salts, such as calcium salts and magnesium salts; salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts with amino acids, such as arginine, lysine, and the like.

[0177] For example, Berge lists the following FDA-approved commercially available salts of the anions acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycolyl arsanilate (glycolamide phenylarsonate), hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-hydroxyphenyl)-2-propanediol ... the organic cations benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and the metallic cations aluminium, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0178] Berge further lists the following non-FDA approved commercially available (outside the United States) salts: anionic adipate, alginate, aminosalicylate, anhydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, cinnamate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylene bis(salicylate), napadisilate (1,5-naphthalenedisulfonate), and oxalate. salts, pectinates, persulfates, phenylethylbarbiturates, picrates, propionates, thiocyanates, tosylates and undecanoates; the organic cations benethamine (N-benzylphenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), diethylamine, piperazine and tromethamine (tris(hydroxymethyl)aminomethane); and the metal cations barium and bismuth.

[0179] Pharmaceutical compositions containing the neodegrader conjugates described herein may also contain suitable carriers, additives and adjuvants, which may vary depending on the method of administration.

[0180] In some embodiments, the pharmaceutical composition may be formulated into a suitable parenteral dosage form. The formulation may be prepared by a variety of methods known in the art. The pharmaceutical composition may be administered directly into the bloodstream, into muscle, or directly into an organ. Suitable means of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle syringes, needleless syringes, and infusion techniques.

[0181] Parenteral compositions are typically aqueous solutions which may contain additives such as salts, carbohydrates, and buffers. However, the compositions can also be formulated as sterile non-aqueous solutions or a dry form for use with a suitable vehicle such as sterile pyrogen-free water.

[0182] The preparation of parenteral compositions under sterile conditions, such as by lyophilization, may be readily accomplished using standard techniques well known to those skilled in the art.

[0183] Compositions for parenteral administration can be formulated for immediate and / or modified release.Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.Thus, the composition can be formulated as a solid, semi-solid or thixotropic liquid for administration as an implanted depot that provides modified release of active agent.

[0184] The parenteral formulation may be mixed with other suitable pharma- ceutically acceptable additives used in parenteral dosage forms, such as, but not limited to, preservatives.

[0185] In other embodiments, the pharmaceutical composition may be formulated as a suitable oral dosage form such as a tablet, capsule, powder, pellet, suspension, solution, emulsion, etc. Other suitable carriers may be present such as disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, bulking agents, anti-adherents, etc.

[0186] The oral administration formulations may also contain other suitable pharmaceutical additives such as sweeteners, vehicles / wetting agents, colorants, flavoring agents, preservatives, thickening / thickening agents, and the like.

[0187] Neodegrader or the neodegrader conjugates described herein may be used to treat various cancers. Some conjugates of the present invention may be useful as medicines because they may be superior in terms of efficacy, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., aqueous solubility), interactions with other drugs (e.g., drug-metabolizing enzyme inhibition), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central nervous toxicity) and / or stability (e.g., chemical stability, stability against enzymes).

[0188] The neodegraders or neodegrader conjugates of the invention can be used to treat diseases, such as cancers, such as colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumors), pharyngeal cancer, laryngeal cancer, esophageal cancer, stomach / gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestine cancer, breast cancer, Cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., ovarian epithelial carcinoma, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, non-hormone-dependent prostate cancer, castration-resistant prostate cancer), liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic bile duct cancer), thyroid cancer (e.g., medullary thyroid carcinoma), kidney cancer (e.g., For example, renal cell carcinoma (e.g., clear cell renal cell carcinoma), renal pelvis and ureter transitional cell carcinoma), uterine cancer (e.g., cervical cancer, uterine carcinoma, uterine sarcoma), gestational choriocarcinoma, brain tumors (e.g., medulloblastoma, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., basal cell carcinoma, malignant melanoma), sarcomas (e.g., rhabdomyosarcoma, smooth muscle cell carcinoma, gli ... The compounds may be used as pharmaceuticals for the prevention or treatment of cancers including myosinoma, soft tissue sarcoma, spindle cell sarcoma, malignant bone tumors, bladder cancer, hematological / blood cancers (e.g., multiple myeloma, leukemia (e.g., acute myeloid leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorders), and cancers of unknown primary origin; cancer growth inhibitors; cancer metastasis inhibitors; apoptosis promoters; and drugs for treating precancerous lesions (e.g., myelodysplastic syndromes).

[0189] In some embodiments, the neodegrader or neodegrader conjugate of the present invention can be used as a medicament for breast cancer, gastric cancer, ovarian cancer, uterine cancer, lung cancer, pancreatic cancer, liver cancer, lymphoma or hematological cancer.

[0190] Furthermore, the neodegraders or neodegrader conjugates of the present invention may be used simultaneously with non-pharmacological therapies, specifically, the conjugates may be combined with non-pharmacological therapies such as (1) surgery, (2) hypertensive therapy, such as with angiotensin II, (3) gene therapy, (4) hyperthermia, (5) cryotherapy, (6) laser ablation, and (7) radiation therapy.

[0191] For example, the use of the neodegrader or neodegrader conjugate of the present invention before or after the above-mentioned surgery, etc., can provide effects such as preventing the development of resistance, extending progression-free survival, extending disease-free survival, suppressing cancer metastasis or recurrence, and extending lifespan.

[0192] Furthermore, treatment with the neodegrader or neodegrader conjugate of the present invention can be combined with supportive therapies such as (i) administration of antibiotics (e.g., β-lactams such as pansporin, macrolides such as clarithromycin) for various concurrent infectious diseases, (ii) administration of high-calorie infusions, amino acid preparations or general vitamin preparations to improve nutritional disorders, (iii) administration of morphine to relieve pain, (iv) administration of drugs to reduce side effects such as nausea, vomiting, eating disorders, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, liver damage, kidney damage, DIC, fever, and (v) administration of drugs to suppress multidrug resistance of cancer.

[0193] In some embodiments, the neodegrader or neodegrader conjugate of the present invention may be used in combination with standard therapy, e.g., one or more therapeutic agents (e.g., anti-cancer agents and / or immunomodulatory agents). Thus, in some embodiments, the method of treating tumors disclosed herein comprises administering a combination of the neodegrader or neodegrader conjugate of the present invention and one or more additional therapeutic agents. In some embodiments, the neodegrader or neodegrader conjugate of the present invention may be used in combination with one or more anti-cancer agents, e.g., so that multiple elements of the immune pathway can be targeted. In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocking signaling through a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that may be used in the present methods 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 treatments is disclosed in detail in the Combination Treatments section herein.

[0194] In some embodiments, the neodegrader or neodegrader conjugate of the present invention is administered to the subject before or after administration of the additional therapeutic agent. In other embodiments, the neodegrader or neodegrader conjugate of the present invention is administered to the subject simultaneously with the additional therapeutic agent. In some embodiments, the neodegrader or neodegrader conjugate of the present invention and the additional therapeutic agent can be administered simultaneously as one composition in a pharma- ceutically acceptable carrier. In other embodiments, the neodegrader or neodegrader conjugate of the present invention and the additional therapeutic agent are administered simultaneously as separate compositions.

[0195] In some embodiments, the subject that can be treated with the neodegrader or neodegrader conjugate of the present invention is a non-human animal, such as a rat or a mouse. In some embodiments, the subject that can be treated is a human.

[0196] X. Methods of Making Neodegraders and Compositions The present invention relates to a method for producing a neodegrader conjugate, comprising combining an anti-CD33 antibody or an antigen-binding portion thereof with a compound of formula (I-1): [ka] [During the ceremony, A is phenyl or C4-C 10 is a cycloalkyl ring; R 1 is independently selected from hydrogen and halo; U is selected from NH and CF2; X is -NR 2 -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from; where Q and Q' are each independently O, S or NR 2 and; R 2 is hydrogen or C1-C6 alkyl; n is an integer from 1 to 6; m is an integer from 2 to 6; and where the left side of each group is attached to L' and the right side is attached to A; However, X is NH or -Q-(CH2) n -When R 1 is a halo; L' is a cleavable or non-cleavable linker precursor conjugated to an anti-CD33 antibody or antigen-binding portion thereof. or a pharma- ceutically acceptable salt thereof.

[0197] As described herein, the linker precursor comprises a heterobifunctional group that is attached to an anti-CD33 antibody, or antigen-binding portion thereof.

[0198] In some embodiments, L' is a non-cleavable linker precursor. [ka] [During the ceremony, p is an integer from 1 to 10; and [ka] is the point of attachment to X. is selected from the group consisting of:

[0199] In some embodiments, L' is [ka] It is.

[0200] In some embodiments, p is 5.

[0201] In some embodiments, L' is a cleavable linker precursor.

[0202] In some embodiments, the linker precursor is cleavable by a protease. [ka] [During the ceremony, q is an integer from 2 to 10; Z 1 , Z 2 , Z 3 and Z 4 are each independently absent or a naturally occurring amino acid residue of the L or D configuration, with the proviso that Z 1 , Z 2 , Z 3 and Z 4 at least two of are amino acid residues; and [ka] is the point of attachment to X. is selected from the group consisting of:

[0203] In one embodiment, Z 1 , Z 2 , Z3 and Z 4 is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine and glycine, with the proviso that Z 1 , Z 2 , Z 3 and Z 4 At least two of the residues are amino acid residues.

[0204] In one embodiment, Z 1 is absent or is glycine; Z 2 is absent or is selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine and glycine; Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine and glycine; and Z 4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine and glycine.

[0205] In some embodiments, L' is [ka] It is.

[0206] In some embodiments, q is 5.

[0207] In some embodiments, L' is a bioreducible linker precursor. In some embodiments, the bioreducible linker precursor is [ka] [During the ceremony, q is an integer from 2 to 10; R, R', R" and R"' are each independently selected from hydrogen, C1-C6 alkoxyC1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring; and [ka] is the point of attachment to X. is selected from the group consisting of:

[0208] In some embodiments, L' is an acid cleavable linker precursor. [ka] [During the ceremony, q is an integer from 2 to 10; and [ka] is the point of attachment to X. is selected from the group consisting of:

[0209] In some embodiments, L' is a click release linker precursor. [ka] [During the ceremony, q is an integer from 2 to 10; and [ka] is the point of attachment to X. is selected from.

[0210] In some embodiments, L' is a pyrophosphatase cleavable linker precursor. [ka] [During the ceremony, q is an integer from 2 to 10; [ka] is the point of attachment to X. It is.

[0211] In some embodiments, L' is a beta-glucuronidase cleavable linker precursor. [ka] [During the ceremony, q is an integer from 2 to 10; ---- is absent or a bond; and [ka] is the point of attachment to X. is selected from.

[0212] In some embodiments, the compound of formula (I-1) [ka] is selected from.

[0213] In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof is pretreated prior to reaction with the compound of formula (I-1). In some embodiments, the compound of formula (I-1) is reacted with the anti-CD33 antibody or antigen-binding portion thereof. In some embodiments, the anti-CD33 antibody or antigen-binding portion thereof may be pretreated to reduce interchain disulfides prior to reaction with the compound of formula (I-1). EXAMPLES

[0214] General Synthetic Methods and Intermediates The compounds of the present invention can be prepared by one of ordinary skill in the art in light of this disclosure and knowledge in the art and / or by reference to the following schemes and synthetic examples. Example synthetic routes are shown in the following schemes and examples. It should be understood that variables (e.g., "R" groups) used in the following schemes and examples are to be interpreted independently from those shown elsewhere in this specification. One of ordinary skill in the art will readily understand how the following schemes and examples illustrate the preparation of the compounds described herein.

[0215] Abbreviations used in the schemes follow conventions commonly used in the art. Chemical abbreviations used in the present specification and examples are defined as follows: "THF" is tetrahydrofuran; "DMF" is N,N-dimethylformamide; "Me" is methyl; "Bu" is butyl; "FA" is formic acid; "PE" is petroleum ether; "MeOH" is methanol; "EtOH" is ethanol; "DCM" is dichloromethane; "BOC" or "Boc" "TFA" is trifluoroacetic acid; "DMSO" is dimethylsulfoxide; "EtOAc" is ethyl acetate; "OAc" is acetate; "dppf" is 1,1'-bis(diphenylphosphino)ferrocene; "dba" is dibenzylideneacetone; "CDI" is 1,1'-carbonyldiimidazole; "TBAF" is tetrabutylammonium fluoride; "TBSCl" is tert-butyldimethylammonium chloride; silyl chloride; "Et2O" is diethyl ether; "ACN" is acetonitrile; "h" is hour; "min" is minute; "rt" is room temperature or retention time (determined from the context); "aq" is aqueous, "sat" is saturated; "min" is minute; "HOBt" is 1-hydroxybenzotriazole hydrate; "HATU" is 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate or N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; "DIEA" and "iPrNEt2" are diisopropylethylamine; "Et3N" and "TEA" are triethylamine.

[0216] [ka] Scheme 1: Preparation of compound (Ia)

[0217] [ka] Example 1: Synthesis of compound (Ia) [ka] Step 1: Synthesis of Compound 2 To a stirred solution of 2-chloro-4-nitrophenyl)acetic acid (compound 1, 5.00 g, 23.19 mmol, 1.00 equiv.) in THF (75.00 mL) was added BH3-Me2S (10 M in THF) (5.80 mL, 58.0 mmol, 2.50 equiv.) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at 70° C. for 2 h under nitrogen atmosphere. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=1:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (3 g, 64%) as a yellow solid. 1 H NMR (400 MHz, 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)

[0218] [ka] Step 2: Synthesis of Compound 3 To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 2, 5.00 g, 24.800 mmol, 1.00 equiv.) and tert-butyl 2-bromoacetate (29.0 mL, 148.28 mmol, 8.00 equiv.) in toluene (150.00 mL) was added Bu4NHSO4 (6.74 g, 19.84 mmol, 0.80 equiv.). To the above mixture was added NaOH (5M in H2O) (500.00 mL) dropwise for 40 min at 0° C. The resulting mixture was stirred for another 2 h at 25° C. The resulting mixture was extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (400 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=4:1) to give tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (8 g, 65%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 8.23 ​​(d, J = 4.0 Hz, 1H), 8.10-8.04 (m, 1H), 7.60 (d, J = 8.0 Hz, 1H), 4.09 (s, 2H), 3.83-3.80 (m, 2H), 3.17-3.14 (m, 2H), 1.45 (s, 9H)

[0219] [ka] Step 3: Synthesis of Compound 4 To a stirred solution of tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (compound 3, 8.00 g, 16.14 mmol, 1.00 equiv, 63.7%) in DCM (80.00 mL) was added dropwise at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (500 mL). The mixture was extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (6.5 g, crude) as a yellow oil. LCMS (ESI): 517 (2M-H)-

[0220] [ka] Step 4: Synthesis of Compound 5 To a stirred solution of [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 4, 6.30 g, 21.84 mmol, 1.00 equiv, 90%) and HATU (12.46 g, 32.76 mmol, 1.50 equiv) in DMF (65.00 mL) was added CH3NH2.HCl (1.77 g, 26.21 mmol, 1.20 equiv) and DIEA (15.20 g, 117.8 mmol, 4.00 equiv) dropwise at room temperature. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was diluted with water. The resulting mixture was extracted with EtOAc (2×100 mL). The combined organic layers were washed with brine (50 mL) 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) +

[0221] [ka] Step 5: Synthesis of Compound 6 To a stirred solution of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 5, 3.3 g, 12.10 mmol, 1.00 equiv.) in THF (35.00 mL) was added BH3-THF (1 M in THF) (12.10 mL, 12.10 mmol, 1.00 equiv.) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 70° C. for 2 h under nitrogen atmosphere. The reaction was quenched with MeOH. The residue was acidified to pH 6 with 1N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was basified to pH 8 with saturated NaHCO3 (sat., aq.). The resulting mixture was extracted with EtOAc (3×100 mL), washed with brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (2.5 g, 80%) as a yellow oil. LCMS (ESI): 259.26 (M+H). +

[0222] [ka] Step 6: Synthesis of Compound 7 To a stirred solution of [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (compound 6, 2.50 g, 9.69 mmol, 1.00 equiv.) and Boc2O (2.53 g, 11.6 mmol, 1.20 equiv.) in THF (40 mL) was added TEA (1.17 g, 11.6 mmol, 1.20 equiv.) dropwise at 25° C. The mixture was stirred at 25° C. for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=5:1) to give tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (1.70 g, 50%) as a yellow oil. LCMS (ESI): 359.36 (M+H) +

[0223] [ka] Step 7: Synthesis of Compound 8 To a stirred solution of tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 7, 1.70 g, 4.74 mmol, 1.00 equiv.) and NH4Cl (750 mg, 14.2 mmol, 3.00 equiv.) in EtOH (85 mL) and HO (17 mL) at 25° C. was added Fe (1.3 g, 23.7 mmol, 5.00 equiv.). The mixture was stirred at 80° C. for 2 h. The mixture was cooled to room temperature. The resulting mixture was filtered and the filter cake was washed with EtOH (3×50 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=4:1) to give tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (900 mg, 58%) as a yellow oil. LCMS (ESI): 329.33 (M+H) +

[0224] [ka] Step 8: Synthesis of Compound 9 To a stirred solution of tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 8, 500 mg, 1.52 mmol, 1.00 equiv.) in THF (10 mL) was added diphosgene (601 mg, 3.04 mmol, 2.00 equiv.) dropwise at 25° C. The mixture was stirred at 25° C. for 1 h. The resulting mixture was concentrated under reduced pressure and redissolved in DMF (5 mL). To a stirring mixture of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, prepared as below, 499 mg, 1.82 mmol, 1.20 equiv.) and TEA (1.56 g, 15.45 mmol, 10.00 equiv.) in DMF (20 mL) at 25° C., the above solution was added dropwise. The mixture was stirred at 25° C. for 1 h. The resulting mixture was diluted with 40 mL of ice water. The resulting mixture was extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (5×40 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=10:1) to give tert-butyl (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenetoxy)ethyl)(methyl)carbamate (670 mg, 70%) as a white solid. LCMS: (ESI): 628.63 (M+H) +

[0225] [ka] Step 9: Synthesis of Neodegrader P1 To a stirring solution of tert-butyl N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-N-methylcarbamate (compound 9, 670 mg, 1.07 mmol, 1 equiv.) in DCM (10 mL) was added TFA (2.5 mL) dropwise at 0° C. The mixture was stirred at 25° C. for 1 h. The resulting mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC using the following conditions: column, SunFire C18 OBD Prep column, 100 μm, 19×250 mm; mobile phase, water (0.05% TFA) and ACN (5% phase B to 60% 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 (400 MHz, methanol-d4) δ 7.77 (d, J = 8.0 Hz, 1H), 7.57-7.53 (m, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 4.0 Hz, 2H), 5.19-5.1 (m, 1H), 4.55-4.41 (m, 4H), 3.75-3.67 (m, 4H), 3.21-3.15 (m,2H), 3.03-3.96 (m, 2H), 2.96-2.84 (m, 1H), 2.83-2.73 (m, 2H), 2.69 (s, 3H), 2.55-2.42 (m, 1H), 2.21-2.12 (m, 1H)

[0226] [ka] Step 10: Synthesis of compound (Ia) A stirring mixture of 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (Neodegrader P1, 200 mg, 0.38 mmol, 1.00 equiv.) and lutidine (81 mg, 0.76 mmol, 2.00 equiv.) in DMF (10 mL). To this was added HOBT (26 mg, 0.19 mmol, 0.50 equiv) and [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl 4-nitrophenyl carbonate (279 mg, 0.38 mmol, 1.00 equiv) in portions at room temperature. The reaction mixture was stirred at 40° C. for 12 h under nitrogen atmosphere. After cooling the reaction to room temperature, the reaction was quenched with water (30 mL). The resulting mixture was extracted with DCM (3×30 mL). The combined organic layers were washed with water (2×30 mL), brine (30 mL) and dried over Na2SO4. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified on a reversed phase column (C18, mobile phase A: 0.1% FA in water, B: ACN). The collected fractions were concentrated to dryness under reduced pressure. The crude product (60 mg) was purified by preparative HPLC using the following conditions (Column: Xselect CSH OBD column 30 × 150 mm 5 μm, n; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 33B to 50B in 7 min; 220 nm; RT1: 5.27 min). The collected fractions were lyophilized to give [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-N-methylcarbamate (23.8 mg, 5%) as a white solid. LCMS (ESI): 1126.11 (M+H) + . 1H NMR (400 MHz, DMSO-d6) 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)

[0227]

change

[0228]

change

[0229] Example 2: Synthesis of compound (Ib)

change

[0230] [ka] Step 2: Synthesis of compound 12 To a stirring mixture of 3-(5-bromo-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione (compound 11, 1.00 g, 3.09 mmol, 1.00 equiv.) and dppf (51 mg, 0.093 mmol, 0.03 equiv.) in DMF (8 mL) was added Zn(OAc)2 (170 mg, 0.928 mmol, 0.30 equiv.), Zn(CN)2 (545 mg, 4.64 mmol, 1.50 equiv.) and Pd2(dba)3 (28 mg, 0.031 mmol, 0.01 equiv.) under nitrogen atmosphere at 25° C. The final reaction mixture was irradiated in a microwave oven at 120° C. for 2 h. The mixture was cooled to room temperature and filtered. The filter cake was washed with MeOH (3×30 mL). The filtrate was concentrated under reduced pressure. The residue was subjected to flash chromatography (silica gel, 80 g, DCM:MeOH=10:1) to give the desired product 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindole-5-carbonitrile (400 mg, 47%) as a brown solid. LCMS (ESI): 270 (M+H) +

[0231] [ka] Step 3: Synthesis of INT1 To a stirring mixture of 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindole-5-carbonitrile (compound 12, 3.0 g, 11.14 mmol, 1.00 equiv.) and HCl (12 M) (3.6 mL) in MeOH (25 mL) was added PtO2 (1.25 g, 5.5 mmol, 0.49 equiv.) at 25° C. The mixture was hydrogenated at room temperature under hydrogen atmosphere using a hydrogen balloon for 16 h. The resulting mixture was filtered and the filter cake was washed with MeOH (2×30 mL). The filtrate was concentrated under reduced pressure. The resulting solid was washed with DCM:MeOH (3:1) (3×30 mL) and dried. This gave 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (2.5 g, 80%) as a grey solid. LCMS (ESI): 274 (M+H). + . 1 H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 8.15 (s, 1H), 7.98 (d, J=8.4 Hz, 1H), 7.89 (d, J=8.4 Hz, 1H),5.16-5.11 (m, 1H), 4.52 (d, J=17.2Hz, 1H), 4.40 (d, J=17.2Hz, 1H), 2.96-2.90 (m, 1H), 2.60-2.54 (m, 1H), 2.43-2.34 (m, 1H), 2.06-1.96 (m, 1H)

[0232] [ka] Step 4: Synthesis of compound 14 To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 13, 5.00 g, 22.50 mmol, 1.00 equiv) in THF (75 mL) was added BH3-Me2S (10 M in THF) (5.60 mL, 56 mmol, 2.50 equiv) dropwise at 0° C. under nitrogen atmosphere. The mixture was stirred at 70° C. for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was applied to a silica gel column and eluted with PE / EtOAc (5:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (4.44 g, 88%) as a yellow solid. 1 H NMR (400 MHz, CDCl3) 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) [ka]

[0233] Step 5: Synthesis of Compound 15 To a stirring mixture of 2-(2-chloro-4-nitrophenyl)ethanol (compound 14, 4.44 g, 22.02 mmol, 1.00 equiv.) and imidazole (4.50 g, 66.06 mmol, 3.00 equiv.) in DMF (50.00 mL) at 25° C. was added TBSCl (6.97 g, 46.25 mmol, 2.10 equiv.). 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 (3×100 mL). The combined organic layers were washed with brine (3×100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied to a silica gel column and eluted with PE / EtOAc (10:1) to give tert-butyl[2-(2-chloro-4-nitrophenyl)ethoxy]dimethylsilane (6.6 g, 90%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 8.06-8.04 (m, 1H), 7.46 (d, J = 8.4 Hz, 1H), 3.89-3.86 (m, 2H), 3.06-0.04 (m, 2H), 0.85 (s, 9H), 0.04 (s, 6H)

[0234] [ka] Step 6: Synthesis of Compound 16 To a mixture of tert-butyl[2-(2-chloro-4-nitrophenyl)ethoxy]dimethylsilane (compound 15, 5.70 g, 18.05 mmol, 1.00 equiv.) and Fe (10.08 g, 180.45 mmol, 10.00 equiv.) in EtOH (110 mL) / water (55 mL) was added NH4Cl (9.65 g, 180.45 mmol, 10 equiv.). The mixture was stirred at 80° C. for 2 h. The mixture was cooled to room temperature. The resulting mixture was filtered and the filter cake was washed with EtOH (3×50 mL). The filtrate was concentrated under reduced pressure. The residue was diluted with water (100 mL) and extracted with EtOAc (50 mL×3). The combined organic layers were dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give 4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chloroaniline (5.2 g, crude) as a light brown oil. LCMS (ESI): 286.29 (M+H). +

[0235] [ka] Step 7: Synthesis of Compound 17 A solution of 4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chloroaniline (compound 16, 200.00 mg, 0.70 mmol, 1.00 equiv.) and TEA (141 mg, 1.40 mmol, 2.00 equiv.) in DMF (3 mL) was added dropwise under nitrogen at 0° C. to a solution of CDI (113 mg, 0.70 mmol, 1.00 equiv.) in DMF (1 mL). The resulting mixture was stirred at 25° C. for 1 h. The above solution and TEA (141 mg, 1.40 mmol) were then added dropwise to a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 192 mg, 0.70 mmol, 1.00 equiv.) in DMF (2 mL). The same reaction was repeated twice. The resulting mixture was stirred at 25° C. for 1 h. The reaction was diluted with water (20 mL) and extracted with EtOAc (20 mL×3). The combined organic layers were washed with water, brine, dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (DCM:MeOH=10:1) to give 1-(4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chlorophenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (170 mg, 21%) as a white solid. LCMS (ESI): 585.59 (M+H) +

[0236] [ka] Step 8: Synthesis of Neodegrader P3 To a solution of 1-(4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chlorophenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 17, 170.00 mg, 0.29 mmol, 1.00 equiv.) in THF (2.00 mL) was added TBAF (1N in THF, 0.58 mL, 0.58 mmol, 2.00 equiv.) at 0° C. The resulting mixture was stirred at 25° C. for 8 h. The reaction was purified by preparative TLC (DCM:MeOH=10:1) to give 147 mg of crude 1-(3-chloro-4-(2-hydroxyethyl)phenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)urea as a white solid. LCMS (ESI): 471.47 (M+H) +

[0237] [ka] Step 9: Synthesis of Compound 19 2-Methyl-2-sulfanylpropan-1-ol (compound 18, 1.4 g, 13.2 mmol, 1.00 equiv.) and 5-nitro-2-[(5-nitropyridin-2-yl)disulfanyl]pyridine (compound 120, 2.05 g, 6.67 mmol, 0.50 equiv.) were added to a solvent mixture of dichloromethane (3.50 mL) and MeOH (3.50 mL). The resulting mixture was stirred at 15° C. Then manganese dioxide (2.29 g, 26.2 mmol, 2 equiv.) was added in portions. The resulting mixture was stirred at 15° C. for 15 min. LCMS trace showed the reaction was complete. The reaction was evaporated to dryness and the residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% NH4HCO3), 10% to 100% gradient in 30 min; detector, UV 254 nm. The collected fractions were concentrated to dryness under reduced pressure to give 2-methyl-2-[(5-nitropyridin-2-yl)disulfanyl]propan-1-ol (2.2 g, 58%) as a yellow solid. LCMS (ESI): 261 (M+H). +

[0238] [ka] Step 10: Synthesis of Compound 20 To a solution of 2-methyl-2-[(5-nitropyridin-2-yl)disulfanyl]propan-1-ol (compound 20, 1.0 g, 3.84 mmol, 1.00 equiv.) in anhydrous DCM (30 mL), MeSO2Na (1.57 g, 15.4 mmol, 4.00 equiv.) and iodine (1.95 g, 7.68 mmol, 2.00 equiv.) were added portionwise. The reaction mixture was stirred at 45° C. for 24 h. The mixture was concentrated and the residue was purified by column silica gel chromatography (TLC: PE:EA=3:1, Rf=0.60; 0-35% EtOAc in petroleum ether) to give 2-(methanesulfonylsulfanyl)-2-methylpropan-1-ol (80 mg, 10%) as a yellow oil. 1 H NMR (400 MHz, CD3Cl): δ 3.50 (s, 2H), 3.33 (s, 3H), 2.16 (br s, 1H), 1.47 (s, 6H)

[0239] [ka] Step 11: Synthesis of compound (Ib) To a solution of 1-[3-chloro-4-(2-hydroxyethyl)phenyl]-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (Neodegrader P3, 200.00 mg, 0.42 mmol, 1.00 equiv.) and TEA (129 mg, 1.26 mmol, 3.00 equiv.) in DMF (4 mL) was added a solution of CDI (138 mg, 0.84 mmol, 2.00 equiv.) in DMF (1 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction was diluted with water (50 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with water (20 mL×3), brine (20 mL), dried over sodium sulfate, and evaporated to dryness under reduced pressure to give the crude product (2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl imidazole-1-carboxylate, 200 mg) as a light yellow solid. To a solution of the crude product (100.00 mg, 0.18 mmol, 1.00 equiv) and Cs2CO3 (115 mg, 0.35 mmol, 2.00 equiv) in DMF (8 mL) was added dropwise a solution of 2-(methanesulfonylsulfanyl)-2-methylpropan-1-ol (compound 20, 59 mg, 0.32 mmol, 1.80 equiv) in DMF (2 mL) at room temperature. The reaction was stirred at 15° C. for 22 h. The reaction was diluted with EtOAc (50 ml) and ice-cold water (100 mL). The organic layer was filtered off. The aqueous phase was extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine (30 mL×3), dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give the crude product (150 mg) as a yellow solid. The crude product was purified by preparative HPLC (Column: Xselect CSH OBD column 30×150 mm 5 μm; Mobile phase A: Water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 38B to 58B in 7 min; 220 nm; RT1: 5.12 min).The collected fractions were lyophilized to give 1-[3-chloro-4-[2-([[2-(methanesulfonylsulfanyl)-2-methylpropoxy]carbonyl]-oxy)ethyl]phenyl]-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (15.7 mg, 11%) as a white solid. LCMS (ESI): 681.68 (M+H). + . 1 H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.86 (s, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.51 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.24-7.17 (m, 1H), 6.87-6.84 (m, 1H), 5.76 (s, 2H), 5.13-5.11 (m, 1H), 4.42-4.40 (m, 2H), 4.32-4.28 (m, 4H), 3.54 (s, 3H), 3.00-2.87 (m, 3H), 2.62-2.58 (m, 1H), 2.44-2.34 (m, 1H), 2.01-1.95 (m, 1H), 1.45 (s, 6H)

[0240] [ka] Scheme 3: Preparation of compound (Ic)

[0241] [ka]

[0242] Example 3: Synthesis of compound (Ic) [ka] Step 1: Synthesis of compound 23 To a stirred solution of tert-butyl(2-aminophenyl)(methyl)carbamate (compound 22, 300 mg, 1.35 mmol, 1.00 equiv.) in DMF (20 mL) was added CDI (218 mg, 1.35 mmol, 1.00 equiv.) and TEA (68 mg, 1.35 mmol, 1.00 equiv.) dropwise at 0° C. under nitrogen atmosphere. The mixture was stirred at 0° C. for 2 h. To the above mixture was added 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 368 mg, 1.35 mmol, 1.00 equiv.) in portions. The resulting mixture was stirred at 75° C. overnight. The reaction mixture was then cooled to room temperature. The resulting mixture was quenched with water (30 mL) and extracted with DCM (3×30 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 tert-butyl N-[2-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate (300 mg, 42%) as a white solid. LCMS (ESI): 522 (M+H) +

[0243] [ka] Step 2: Synthesis of Neodegrader P4 To a stirring solution of tert-butyl N-[2-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate (compound 23, 300 mg, 1.00 equiv.) in DCM (20 mL) was added TFA (5 mL) at 0° C. The mixture was stirred at 0° C. for 2 h. The resulting mixture was concentrated under reduced pressure. The crude product was purified by reverse phase using the following conditions (C18, mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min). The collected fractions were concentrated under reduced pressure to give 3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-1-[2-(methylamino)phenyl]urea (210 mg, 87%) as a white solid. LCMS (ESI): 422 (M+H). + . 1 H NMR (300 MHz, DMSO-d6) δ 10.99 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.60 (s, 1H), 7.53 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.26-7.24 (m, 1H), 6.99-6.93 (m, 1H), 6.76-6.72 (m, 1H), 6.60-6.55 (m, 2H), 5.14-5.08 (m, 1H), 5.00-4.85 (br s, 1H), 4.48-4.28 (m, 4H), 2.92-2.82 (m, 1H), 2.70 (s, 3H), 2.62-2.57 (m, 1H), 2.49-2.41 (m, 1H), 2.02-1.95 (m, 1H)

[0244] [ka] Step 3: Synthesis of compound (Ic) A mixture of 3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-1-[2-(methylamino)phenyl]urea (P4, 150.00 mg, 0.36 mmol, 1.00 equiv.), 2,6-lutidine (76 mg, 0.71 mmol, 2.00 equiv.) and HOBT (96 mg, 0.71 mmol, 2.00 equiv.) was stirred at 37°C for 10 min. To a mixture of 1,2-dioxopyrrol-1-yl)hexanamido-3-methylbutanamido]pentanamido]phenyl]methyl 4-nitrophenyl carbonate (394 mg, 0.53 mmol, 1.50 equiv.) in DMF (3.00 mL) was added [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl 4-nitrophenyl carbonate (394 mg, 0.53 mmol, 1.50 equiv.) at room temperature under nitrogen atmosphere. The reaction mixture was purified by reversed-phase flash chromatography (column, C18 silica gel; mobile phase, mobile phase A: water (0.1% FA), mobile phase B: ACN) using the following conditions to give the crude product (60 mg) as a white solid. The crude product (60 mg) was purified by preparative HPLC using the following conditions (Column: Xselect CSH OBD column 30 × 150 mm 5 μm, n; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 24B to 44B in 7 min; 220 nm; RT1: 6.33; RT2:). The collected fractions were lyophilized to give [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl N-[2-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate (18.1 mg, 5%) as a white solid. LCMS (ESI): 1020 (M+H) + . 1H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 9.96 (s, 1H), 8.19-8.06 (m, 3H), 7.79 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.53-7.41 (m, 5H), 7.20-7.05 (m, 4H), 7.00 (s, 2H), 6.95-6.90 (m, 1H), 5.95 (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)

[0245] Scheme 4 shows how compound (Id) was prepared from Neodegrader P1. [ka] Scheme 4: Preparation of compound (Id)

[0246] Synthesis of compound (Id) To a stirring mixture of 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (P1, 40.00 mg, 0.076 mmol, 1.00 equiv.) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (25.00 mg, 0.081 mmol, 1.07 equiv.) in DMF (2.00 mL) was added dropwise DIEA (20.00 mg, 0.16 mmol, 2.04 equiv.) at room temperature. The resulting mixture was stirred at room temperature for 3 h under nitrogen atmosphere. The resulting mixture was quenched with water (30 mL) and extracted with DCM (3×30 mL). The combined organic layers were washed with water (30 mL), brine (30 mL) and dried over Na2SO4. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by the following conditions: Column: SunFire C18 OBD Prep column, 100 μm, 19 mm × 250 mm; Mobile phase A: water (0.05% TFA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 25B to 55B in 8.5 min; 220 nm; RT1: 8 min; Collected fractions were lyophilized to give N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethoxy)ethyl]-6-(2,5-dioxopyrrol-1-yl)-N-methylhexanamide (Compound (Id), 24 mg, 43%) as a white solid. LCMS: (ES, m / s): 721,723 (M+H) + ; 1H NMR (400 MHz, 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)

[0247] Schemes 5A and 5B show how to prepare conjugates of neodegrader P1 with alternative tripeptide linkers. [ka] Scheme 5A: Synthesis of Neodegrader P1-Tripeptide Linker Conjugate

[0248] [ka] Scheme 5B: Synthesis of Neodegrader P1-Tripeptide Linker Conjugate (cont.)

[0249] Schemes 6A and 6B show how to prepare the conjugate of Neodegrader P1 with a β-glucuronide linker. [ka] Scheme 6A: Synthesis of Neodegrader P1-β-glucuronide linker conjugate

[0250] Step 1: Synthesis of Compound 25 To a stirring mixture of 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanoic acid (compound 24, 5.00 g, 16.06 mmol, 1.00 equiv.) was added SOCl2 (25 mL) at room temperature. The resulting mixture was stirred for 16 h at 80° C. The desired product was detected by LCMS (derivative with MeOH MS=326). LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure to give 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 25, 7.5 g, crude) as a yellow oil. The crude product was used directly in the next step without further purification. 1 H-NMR analysis showed it to be the desired product (derivative with MeOH). 1 H-NMR (300 MHz, 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=3.0 Hz, 2H), 4.24 (t, J=6.0 Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J=3.0 Hz, 2H), 2.59 (t, J=6.0 Hz, 2H)

[0251] Step 2: Synthesis of compound 28 To a stirred solution of 4-formyl-2-nitrophenol (compound 27, 4.21 g, 25.19 mmol, 1.00 equiv.) and Ag2O (7.00 g, 30.20 mmol, 1.20 equiv.) in ACN (100 mL, 190.24 mmol, 75.00 equiv.) was added compound 26 (10.00 g, 25.17 mmol, 1.00 equiv.) in portions at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature overnight under N2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (50 ml x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (PE:EA=1:2) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 28, 10.5 g, 86%) as a white solid. 1 H-NMR analysis showed it to be the desired product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR (300 MHz, CDCl3) δ 10.00 (s, 1H), 8.34 (s, 1H), 8.13-8.09 (m, 1H), 7.52 (d, J=3.0 Hz, 1H), 5.47-5.29 (m, 4H), 4.37-4.35 (m, 1H), 3.75-3.73 (m, 3H), 2.17-2.06 (m, 9H)

[0252] Step 3: Synthesis of compound 29 To a stirring solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 28, 6.00 g, 12.41 mmol, 1.00 equiv.) in MeOH (50 mL) was added NaBH4 (0.47 g, 12.42 mmol, 1.00 equiv.) in portions at RT under N2 atmosphere. The resulting mixture was stirred at RT for 2 h under N2 atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at RT. The resulting material was dried over Na2SO4. The resulting mixture was filtered and the filter cake was washed with DCM. The resulting mixture was concentrated under reduced pressure 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] +

[0253] Step 4: Synthesis of compound 30 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 29, 5.50 g, 11.33 mmol, 1.00 equiv) in EA (60 mL) was added Pd / C (1.10 g, 10%) portionwise at room temperature. The resulting mixture was stirred at room temperature for 16 h under H2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered, the filter cake was washed with DCM and MeOH, and the filtrate was concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 30, 4.0 g, 77%) as a solid. The crude product was used directly in the next step without further purification. LCMS (ES, m / z): 456 [M+H] +

[0254] Step 5: Synthesis of Compound 31 To a stirred solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 30, 1.00 g, 2.19 mmol, 1.00 equiv.) and NaHCO3 (0.20 g, 2.40 mmol, 1.1 equiv.) in THF (10 mL) was added compound 25 (0.87 g, 2.62 mmol, 1.20 equiv.) in portions at 0° C. under N2 atmosphere. The resulting mixture was stirred at 0° C. for 6 h under N2 atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The resulting mixture was extracted with DCM. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (EA=100%) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 31, 1.1 g, 66%) as a light yellow solid. LCMS (ES, m / z): 749 [M+H] +

[0255] Step 6: Synthesis of compound 33 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 31, 1.50 g, 2.00 mmol, 1.00 equiv.) and bis(4-nitrophenyl)carbonate (compound 32, 0.68 g, 2.24 mmol, 1.12 equiv.) in DMF (15 mL) was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv.) in portions at 0° C. under a N2 atmosphere. The resulting mixture was stirred at room temperature overnight under a nitrogen atmosphere. LCMS indicated the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 90% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 33, 1.4 g, 48%) as a yellow solid. LCMS (ES, m / z): 914 [M+H] +

[0256] [ka] Scheme 6B: Synthesis of Neodegrader P1-β-glucuronide linker conjugate

[0257] Step 7: Synthesis of compound 34 A mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 33, 1.00 g, 1.09 mmol, 1.00 equiv.) and 1-(3-chloro-4-[2-[2-(methylamino)ethoxy To a mixture of 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (Neodegrader P1, 0.58 g, 1.09 mmol, 1.00 equiv.) in DMF (10 mL) under N2 atmosphere, HOBT (1.18 g, 8.72 mmol, 8.00 equiv.) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equiv.) were added portionwise at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature for 16 h under N2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]oxane-2-carboxylate (compound 34, 800 mg, 56%) as a solid. LCMS (ES, m / z): 1302 [M+H] +

[0258] Step 8: Synthesis of Compound 35 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]oxane-2-carboxylate (compound 34, 800.00 mg, 0.61 mmol, 1.00 equiv) in THF (80 mL) was added HCl (6N, 80 mL) in portions at room temperature under N2 atmosphere. The resulting mixture was stirred at 50° C. for 3 h under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 35, 230 mg, 32%) as a white solid. LCMS (ES, m / z): 1162 [M+H] +

[0259] Step 9: Synthesis of Compound 36 To a stirred solution of (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 35, 230 mg, 0.2 mmol, 1.00 equiv) in DMF (2 mL) was added piperidine (0.4 mL) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 10 min. LCMS indicated completion of the reaction. The resulting mixture was analyzed under the following conditions (column: XSelect CSH Prep C18 OBD column, 19 × 250 mm, 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20B to 40B in 7 min; 220 nm; RT This was used directly for further purification by preparative HPLC with 1:5.78 min) to give (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 36, 35 mg, 18%) as a white solid. LCMS (ES, m / z): 940 [M+H] +

[0260] Step 10: Synthesis of compound (Ie) To a stirring solution of (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 36, 30 mg, 0.03 mmol, 1.00 equiv.) in DMF (3 mL), DIEA (13 mg, 0.10 mmol, 3.00 equiv.) and compound 37 (30 mg, 0.10 mmol, 3.00 equiv.) were added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 h. LCMS showed the reaction was complete. The resulting mixture was purified by preparative HPLC using the following conditions: (Column: Xselect CSH OBD column 30×150 mm 5 μm, Mobile phase A: Water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 21B to 36B in 10 min; 220 nm; RT 1:11.15 min). The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-(methyl)carbamoyl]oxy)methyl]-2-[3-[6-(2,5-dioxopyrrol-1-yl)hexanamido]propanamido]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid ( Compound (Ie), 10.5 mg, 28%) was obtained as a white solid. LCMS (ES, m / z): 1133 [M+H] + . 1H-NMR (300 MHz, 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=3.0 Hz, 1H), 7.18-6.99 (m, 7H), 5.76 (s, 1H), 5.20-5.10 (m, 2H), 4.98 (br s, 2H), 4.76-4.74 (m, 1H), 4.42-4.33 (m, 4H), 3.65 (br s, 1H), 3.58-3.54 (m, 5H), 3.35 (d, J=6 Hz, 2H), 2.90-2.83 (m, 7H), 2.57-2.55 (m, 3H), 2.45-2.30 (m, 1H), 2.02-1.98 (m, 4H), 1.48-1.42 (m, 5H), 1.40-1.20 (m, 3H)

[0261] Scheme 7 shows how to prepare the conjugate of Neodegrader P6 with a hydrazine linker. [ka] Scheme 7: Synthesis of neodegrader P6-hydrazone linker conjugates

[0262] Step 1: Synthesis of compound 38 To a stirring solution of 4-aminoacetophenone (compound 37, 100 mg, 0.73 mmol, 1.00 equiv) in THF (2.00 mL) was added dropwise at room temperature. The resulting mixture was stirred for 30 min at 0 °C. The resulting mixture was concentrated under reduced pressure. The resulting solid was redissolved in DMF (1.50 mL). To the stirring solution was added a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 200 mg, 0.73 mmol, 1.00 equiv) in DMF (3.00 mL) and TEA (0.50 mL) dropwise at room temperature. The resulting mixture was stirred for 1 h at 0 °C. LCMS indicated the reaction was complete. The mixture was added water (5 mL) and extracted with CH2Cl2 (3 x 10 mL). The organic layer was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), 10% to 50% gradient in 35 min; detector, UV 254 nm. The collected fractions were concentrated to dryness to give 1-(4-acetylphenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 38, 80 mg, 25%) as a light yellow solid. LCMS: (ES.m / z): 435 [M+1] +

[0263] Step 2: Synthesis of compound (If) A mixture of 1-(4-acetylphenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 38, 80.00 mg, 0.18 mmol, 1.00 equiv.) and 6-(2,5-dioxopyrrol-1-yl)hexanehydrazide; trifluoroacetic acid (75 mg, 1.20 equiv.) in methanol (5.00 mL) was stirred at 50° C. overnight. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The precipitated solid was collected by filtration and washed with MeOH (2×5 mL). The crude solid was purified by reverse-phase flash chromatography using the following conditions: C18 column; mobile phase, ACN in water (0.1% FA), 10% to 50% gradient in 30 min; detector, UV 254 nm. The collected fractions were extracted with DCM (3×5 mL) and concentrated under reduced pressure to give 3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-1-[4-[(1E)-1-[[6-(2,5-dioxopyrrol-1-yl)hexanamide]iso]ethyl]phenyl]urea (compound (If), 4.4 mg, 3.7%) as an off-white solid. LCMS: (ES.m / z): 642 [M+1] + . 1 H NMR (400 MHz, DMSO-d6) 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)

[0264] Scheme 8 shows how to prepare the conjugate of Neodegrader P2 with a quaternary amine linker. [ka] Scheme 8: Synthesis of Neodegrader P2-Quaternary Amine Linker Conjugates

[0265] Step 1: Synthesis of compound 40 To a stirring solution of N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound 39, 100 mg, 0.18 mmol, 1.00 equiv) in DMF (2 mL) was added a solution of SOCl2 (20 mg, 0.18 mmol, 1 equiv) in DCM (2 mL) dropwise under N2 at 0 °C. The resulting mixture was stirred at 0 °C for 1 h. LCMS indicated the reaction was complete. The reaction mixture was diluted with ice-cold water (20 mL) and extracted with DCM (10 mL×3), and the combined organic layers were washed with water (10 mL), brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to give the product N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(chloromethyl)phenyl]-carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound 40, 80 mg, 53%) as a white solid. LCMS (ES, m / z): 591,593 [M+H] +

[0266] Step 2: Synthesis of compound 42 To a stirring mixture of (2-chloro-4-nitrophenyl)acetic acid (compound 41, 8.60 g, 39.9 mmol, 1.00 equiv.) in THF (130 mL) was added BH3-Me2S (10.00 mL, 105.4 mmol, 2.64 equiv.) dropwise at 0° C. The resulting mixture was stirred at 70° C. for 4 h under nitrogen atmosphere. TLC (PE:EA=1:2) indicated completion of the reaction. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 42, 7.7 g, 96%) as a yellow solid. 1H NMR (400 MHz, CDCl3) 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)

[0267] Step 3: Synthesis of compound 43 To a stirring mixture of 2-(2-chloro-4-nitrophenyl)ethanol, (compound 42, 7.70 g, 38.2 mmol, 1.00 equiv.) and tert-butyl 2-bromoacetate (57.74 g, 296.0 mmol, 7.75 equiv.) in toluene (70 mL) was added Bu4NHSO4 (10.37 g, 30.6 mmol, 0.80 equiv.) portionwise at 0 °C. To the above mixture was added a solution of NaOH (15.00 g, 375.0 mmol, 9.82 equiv.) in H2O (90 mL) dropwise for 30 h at 0 °C. The resulting mixture was stirred at room temperature for another 4 h. TLC (PE:EA=3:1) showed the completion of the reaction. 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 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 (300 MHz, CDCl3) δ 8.20 (d, J = 4.0 Hz, 1H), 8.07-8.03 (m, 1H), 7.61 (d, J = 8.1 Hz, 1H), 4.11 (s, 2H), 3.83 (t, J = 8.1 Hz, 2H), 3.16 (t, J = 8.1 Hz, 2H), 1.45 (s, 9H)

[0268] Step 4: Synthesis of compound 44 To a stirring mixture of tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (compound 43, 12.20 g, 38.6 mmol, 1.00 equiv) in DCM (120 mL) was added TFA (20 mL) dropwise at 0° C. The resulting mixture was stirred for 4 h at room temperature. LCMS indicated completion of the reaction. The resulting mixture was concentrated under reduced pressure. This afforded [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 44, 8.4 g, 83%) as a yellow solid. LCMS: (ES, m / s): 517 (2M-H) - 1 H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H), 8.20 (d, J = 4.0 Hz, 1H), 8.11-8.08 (m, 1H), 7.72 (d, J = 8.0 Hz, 1H), 4.06 (s, 2H), 3.74 (t, J = 8.0 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H)

[0269] Step 5: Synthesis of compound 45 To a stirring mixture of [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid, (compound 44, 8.40 g, 32.35 mmol, 1.00 equiv.) and HATU (19.19 g, 50.47 mmol, 1.56 equiv.) in DMF (80 mL) was added CH3NH2.HCl (2.69 g, 39.79 mmol, 1.23 equiv.) and DIEA (17.31 g, 133.93 mmol, 4.14 equiv.) under nitrogen atmosphere at 0° C. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 4 h. LCMS showed the reaction was complete. The reaction was quenched with water / ice. The resulting mixture was extracted with DCM (3×50 mL). The combined organic layers were washed with brine (50 mL) and dried over anhydrous 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)+

[0270] Step 6: Synthesis of compound 46 To a stirring mixture of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 45, 7.20 g, 26.40 mmol, 1.00 equiv.) in THF (70 mL) was added BH3-THF (10 M in THF, 52.0 mL, 520.0 mmol, 20 equiv.) dropwise at room temperature. The resulting mixture was stirred at 70° C. for 4 h. LCMS showed the reaction was 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 basified to pH 8 with saturated NaHCO3 (sat., aq.). The resulting mixture was extracted with EtOAc (3×100 mL), washed with brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with (DCM:MeOH=8:1) to give [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (compound 46, 5.4 g, 79%) as a yellow solid. LCMS: (ES, m / s): 259,261 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 4.0 Hz, 1H), 8.15-8.12 (m, 1H), 7.73 (d, J = 8.0 Hz, 1H), 3.72 (t, J = 8.0 Hz, 2H), 3.61 (t, J = 8.0 Hz, 2H), 3.10 (t, J = 8.0 Hz, 2H), 2.87 (t, J = 8.0 Hz, 2H), 2.40 (s, 3H)

[0271] Step 7: Synthesis of compound 47 To a stirring mixture of [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (compound 46, 4.00 g, 15.46 mmol, 1.00 equiv.) and Boc2O (3.80 g, 17.41 mmol, 1.13 equiv.) in THF (20.00 mL) was added dropwise a solution of NaHCO3 (4.00 g, 47.61 mmol, 3.08 equiv.) in H2O (20.00 mL) at room temperature. The resulting mixture was stirred at room temperature overnight. LCMS showed the reaction was complete. The resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with (DCM:MeOH=12:1) to give tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 47, 4.8 g, 77%) as a yellow solid. LCMS: (ES, m / s): 359,361 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 4.0 Hz, 1H), 8.13-8.10 (m, 1H), 7.67 (d, J = 8.0 Hz, 1H), 4.05-4.00 (m, 1H), 3.69 (t, J = 8.0 Hz, 2H), 3.50 (t, J = 8.0 Hz, 2H), 3.28 (t, J = 8.0 Hz, 2H), 3.07 (t, J = 8.0 Hz, 2H), 2.75 (s, 3H), 1.36 (s, 9H)

[0272] Step 8: Synthesis of compound 48 To a stirring mixture of tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate, (compound 47, 5.60 g, 15.6 mmol, 1.00 equiv.) in EtOH (112.00 mL) was added a solution of NH4Cl (2.50 g, 46.74 mmol, 2.99 equiv.) in H2O (12.00 mL) and Fe (4.40 g, 78.79 mmol, 5.05 equiv.) at room temperature. The resulting mixture was stirred at 80° C. for 3 h. LCMS showed the reaction was complete. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with DCM (3×30 mL). The combined organic layers were washed with brine (30 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with (DCM:MeOH=10:1) to give tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 48, 4.2 g, 81%) as a yellow oil. LCMS: (ES, m / s): 329,331 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 6.96 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 4.0 Hz, 1H), 6.46-6.43 (m, 1H), 5.18 (br s, 2H), 3.50-3.45 (m, 4H), 3.29-3.26 (m, 2H), 2.75-2.71 (m, 5H), 1.38 (s, 9H)

[0273] Step 9: Synthesis of compound 49 To a solution of tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 48, 100 mg, 0.30 mmol, 1.00 equiv.) in THF (3 mL) was added a solution of LiAlH4 (92 mg, 2.43 mmol, 8.00 equiv.) in THF (2 mL) at 0 °C under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 h. 5 reactions were run at equilibrium. LCMS showed the reaction was complete. The reaction was then quenched with 1N NaOH (10 mL), filtered, and concentrated to dryness under reduced pressure, and the residue was then purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 60% gradient in 30 min; detector, UV 254 nm. The collected fractions were concentrated to dryness to give 3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]aniline, 49 (180 mg, 44%) as a yellow oil. LCMS (ES, m / z): 243,245 [M+H] +

[0274] Step 10: Synthesis of Compound 50 To a solution of 3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]aniline (compound 49, 140 mg, 0.58 mmol, 1.00 equiv.) in THF (9 mL) was added diphosgene (137 mg, 0.69 mmol, 1.20 equiv.) under nitrogen atmosphere at 0° C. The resulting mixture was stirred at 0° C. for 1 h. The reaction solution was then concentrated to dryness under reduced pressure. The residue was redissolved in DMF (2 mL) and then added dropwise to a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (158 mg, 0.58 mmol, 1.00 equiv.) and TEA (117 mg, 1.15 mmol, 2.00 equiv.) in DMF (4 mL) under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 h. LCMS indicated the reaction was complete. The reaction mixture was diluted with methanol and the resulting solution was purified by reversed-phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 50% gradient in 30 min; detector, UV 254 nm) to give 100 mg of the product as a colorless solid. The crude product was purified by preparative HPLC using the following conditions: Column: XBridge Shield RP18 OBD column, 19 x 250 mm, 10 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 14% to 32% in 7 min; 220 nm; RT1: 5.25 min. The collected fractions were lyophilized to give 1-(3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 50, 60 mg, 18%) as a colorless solid. LCMS (ES, m / z): 542,544 [M+H] +

[0275] Step 11: Synthesis of compound (Ig) N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(chloromethyl)phenyl]-carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide, (compound 40, 66 mg, 0.11 mmol, 1.00 equiv.), 1-(3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl To a solution of 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 50, 60 mg, 0.11 mmol, 1.00 equiv.) and DIEA (29 mg, 0.22 mmol, 2.00 equiv.) in DMF (1 mL), TBAI (4 mg, 0.01 mmol, 0.10 equiv.) was added at room temperature in air. The resulting mixture was stirred at room temperature for 16 h. LCMS trace showed the completion of the reaction. The resulting mixture was purified by reverse phase column chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), 5% to 45% gradient in 40 min; detector, UV 254 nm) to give 90 mg of crude product as a yellow oil. The crude product was then purified under the following conditions (column: Xselect CSH OBD column 30×150 mm 5 μm, n; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 15B to 35B in 7 min; 220 nm; RT1: 6.00 min) to give ([4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl)[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]dimethylazanium compound (Ig) (19 mg, 14.8%) as a white solid. LCMS (ES, m / z): 1096 [M-FA] + , 549 [1 / 2 (M-FA)] + ; 1H NMR (400 MHz, CD3OD) δ 8.48 (s, 1H), 7.77-7.72 (m, 3H), 7.55 - 7.47 (m, 3H), 7.37-7.35 (d, J = 8.4 Hz, 2H), 7.18 - 7.14 (m, 2H), 6.77 (s, 2H), 5.17-5.13 (q, J = 8, 4Hz, 1H), 4.51 - 4.46 (m, 5H), 4.35 (s, 2H), 4.12 (d, J = 8.0 Hz, 1H), 3.90 (s, 2H), 3.79 (t, J = 5.6 Hz, 2H), 3.45 (t, J = 7.2 Hz, 4H), 3.22-3.15 (m, 1H), 3.11-3.05 (m, 1H), 3.00 (t, J = 6.0 Hz, 2H), 2.92 (s, 6H), 2.89 - 2.84 (m, 1H), 2.81 - 2.73 (m, 1H), 2.54-2.43 (m, 1H), 2.27 (t, J = 7.2 Hz, 2H), 2.21 - 2.12 (m, 1H), 2.10 - 2.02 (m, 1H), 1.95 - 1.82 (m, 1H), 1.78-1.69 (m, 1H), 1.64-1.59 (m, 7H), 1.32-1.25 (m, 2H), 0.98-0.96 (m, 6H)

[0276] Schemes 9A and 9B show how to prepare conjugates of neodegrader P13 and peptide-containing linkers. [ka] Scheme 9A: Synthesis of Neodegrader P13-Peptide Linker Conjugate

[0277] [ka] Scheme 9B: Synthesis of Neodegrader P13-Peptide Linker Conjugate

[0278] Scheme 10 illustrates the synthesis of compounds of formula (Ih). [ka] Scheme 10: Synthesis of Neodegrader P1-β-glucuronide linker conjugate (compound (Ih)) Step 1: Synthesis of compound 63

[0279] To a stirring mixture of 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoic acid (compound 62, 5.00 g, 16.06 mmol, 1.00 equiv.) was added SOCl2 (25 mL) at room temperature. The resulting mixture was stirred for 16 h at 80° C. The desired product was detected by LCMS (derivative with MeOH MS=326). LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure to give 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 63, 7.5 g, crude) as a yellow oil. The crude product was used directly in the next step without further purification. 1 1 H NMR analysis showed it to be the desired product (derivative with MeOH). 1 H-NMR (300 MHz, 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=3.0 Hz, 2H), 4.24 (t, J=6.0 Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J=3.0 Hz, 2H), 2.59 (t, J=6.0 Hz, 2H)

[0280] Step 2: Synthesis of compound 66 To a stirred solution of 4-formyl-2-nitrophenol (compound 65, 4.21 g, 25.19 mmol, 1.00 equiv.) and Ag2O (7.00 g, 30.20 mmol, 1.20 equiv.) in ACN (100 mL, 190.24 mmol, 75.00 equiv.), methyl (2S,3S,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-bromooxane-2-carboxylate (compound 64, 10.00 g, 25.17 mmol, 1.00 equiv.) was added portionwise at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature overnight under N2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (50 mL x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (PE:EA=1:2) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 66, 10.5 g, 86%) as a white solid. 1 H-NMR analysis showed it to be the desired product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR (300 MHz, CDCl3) δ 10.00 (s, 1H), 8.34 (s, 1H), 8.13-8.09 (m, 1H), 7.52 (d, J=3.0 Hz, 1H), 5.47-5.29 (m, 4H), 4.37-4.35 (m, 1H), 3.75-3.73 (m, 3H), 2.17-2.06 (m, 9H)

[0281] Step 3: Synthesis of compound 67 To a stirring solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 66, 6.00 g, 12.41 mmol, 1.00 equiv.) in MeOH (50 mL) was added NaBH4 (0.47 g, 12.42 mmol, 1.00 equiv.) at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature for 2 h under N2 atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The resulting material was dried over Na2SO4. The resulting mixture was filtered and the filter cake was washed with DCM. The resulting mixture was concentrated under reduced pressure 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] +

[0282] Step 4: Synthesis of compound 68 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 67, 5.50 g, 11.33 mmol, 1.00 equiv) in EA (60 mL) was added Pd / C (1.10 g, 10%) portionwise at room temperature. The resulting mixture was stirred at room temperature for 16 h under H2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered, the filter cake was washed with DCM and MeOH, and the filtrate was concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 68, 4.0 g, 77%) as a solid. The crude product was used directly in the next step without further purification. LCMS (ES, m / z): 456 [M+H] +

[0283] Step 5: Synthesis of Compound 70 To a stirred solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 68, 1.00 g, 2.19 mmol, 1.00 equiv.) and NaHCO3 (0.20 g, 2.40 mmol, 1.1 equiv.) in THF (10 mL) was added 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 69, 0.87 g, 2.62 mmol, 1.20 equiv.) in portions at 0° C. under N2 atmosphere. The resulting mixture was stirred at 0° C. for 6 h under N2 atmosphere. LCMS showed the reaction was complete. At room temperature, the reaction was quenched with water. The resulting mixture was extracted with DCM. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (EA=100%) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 70, 1.1 g, 66%) as a light yellow solid. LCMS (ES, m / z): 749 [M+H] +

[0284] Step 6: Synthesis of compound 72 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 70, 1.50 g, 2.00 mmol, 1.00 equiv.) and bis(4-nitrophenyl)carbonate (compound 71, 0.68 g, 2.24 mmol, 1.12 equiv.) in DMF (15 mL) was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv.) in portions at 0° C. under a N2 atmosphere. The resulting mixture was stirred at room temperature overnight under a nitrogen atmosphere. LCMS indicated the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 90% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 72, 1.4 g, 48%) as a yellow solid. LCMS (ES, m / z): 914 [M+H] +

[0285] Step 7: Synthesis of compound 73 A mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (Compound 72, 1.00 g, 1.09 mmol, 1.00 equiv.) and 1-(3-chloro-4-[2-[2-(methylamino)ethoxy To a mixture of 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (Neodegrader P1, 0.58 g, 1.09 mmol, 1.00 equiv.) in DMF (10 mL) under N2 atmosphere, HOBT (1.18 g, 8.72 mmol, 8.00 equiv.) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equiv.) were added portionwise at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature for 16 h under N2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]oxane-2-carboxylate (compound 73 (800 mg, 56%) as a solid. LCMS (ES, m / z): 1302 [M+H] +

[0286] Step 8: Synthesis of compound 74 Methyl(2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]oxane-2-carboxylate under stirring To a mixture of 73 (800.00 mg, 0.61 mmol, 1.00 equiv) in THF (80 mL) under N2 atmosphere, HCl (6N, 80 mL) was added portionwise at room temperature. The resulting mixture was stirred at 50 °C under nitrogen atmosphere for 3 h. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 74, 230 mg, 32%) as a white solid. LCMS (ES, m / z): 1162 [M+H] +

[0287] Step 9: Synthesis of Compound 75 To a stirred solution of (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid, 74 (230 mg, 0.2 mmol, 1.00 equiv) in DMF (2 mL) was added piperidine (0.4 mL) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 10 min. LCMS indicated completion of the reaction. The resulting mixture was analyzed under the following conditions (column: XSelect CSH Prep C18 Further purification directly by preparative HPLC using an OBD column, 19×250 mm, 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20B to 40B in 7 min; 220 nm; RT1: 5.78 min) afforded (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 75, 35 mg, 18%) as a white solid. LCMS (ES, m / z): 940 [M+H] +

[0288] Step 10: Synthesis of compound (Ih) (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy]-3,4 To a solution of ,5-trihydroxyoxane-2-carboxylic acid (compound 75, 110 mg, 0.12 mmol, 1.00 equiv.) and bis(2,5-dioxopyrrolidin-1-yl)pentanedioate (compound 76, 46 mg, 0.14 mmol, 1.2 equiv.) in DMF (2.0 mL), DIEA (30 mg, 0.23 mmol, 2.0 equiv.) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was purified by preparative HPLC using 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% B in 7 min, 41% B; Wavelength: 254 nm; RT1 (min): 5.8. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3 H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-(3-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanamido}propanamido)phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid (compound (Ih), 48 mg, 34%) was obtained as a white solid. LCMS (ES, m / z): 1151 [M+H] + , 1173 [M+Na] + . 1H-NMR (300MHz, DMSO-d6): 12.80 (br s, 1H), 10.98 (s, 1H), 9.08 (s, 1H), 8.79 (s, 1H), 8.18 (s, 1H), 7.96 (s, 1H), 7.68-7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J=8.1 Hz,1H), 7.25-7.00 (m, 4H), 6.82-6.80 (m, 1H), 5.86 (s, 1H), 5.39-5.30 (m, 2H), 5.14-5.07 (m, 1H), 4.97 (s, 2H), 4.84 (d, J=7.2 Hz,1H), 4.47-4.27 (m, 4H), 3.90 (d, J=9.6 Hz, 1H), 3.56-3.48 (m, 4H), 3.45-3.36 (m, 6H), 2.95-2.80 (m, 8H), 2.75-2.65 (m, 3H), 2.62-2.55 (m, 2H), 2.49-2.35 (m, 1H), 2.21-2.16 (m, 2H), 2.01-1.95 (m, 1H), 1.85-1..80 (m, 2H)

[0289]

change

[0290] Project 1: Synthesis of Compound 76 To a stirring solution of 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound P1, 180 mg, 0.34 mmol, 1.00 equiv.) in DMF (8 mL), TEA (104 mg, 1.02 mmol, 3.0 equiv.) and 4-(chlorosulfonyl)-3-nitrobenzoic acid (181 mg, 0.68 mmol, 2.00 equiv.) were added portionwise under nitrogen atmosphere at 0° C. The resulting mixture was stirred under nitrogen atmosphere for 4 h at 0° C. LCMS showed the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 60% gradient in 10 min; detector, UV 254 nm. The mixture was lyophilized to give 4-[[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)sulfamoyl]-3-nitrobenzoic acid, (compound 76, 70 mg, 27%) as a light yellow solid. LCMS (ES, m / z): 757 [M+1] +

[0291] Step 2: Synthesis of compound (Ii) To a stirring mixture of 4-[[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)sulfamoyl]-3-nitrobenzoic acid, (compound 76, 60 mg, 0.08 mmol, 1.00 equiv) in DMF (6 mL) was added portionwise at room temperature under nitrogen atmosphere HATU (45 mg, 0.12 mmol, 1.5 equiv), 1-(2-aminoethyl)pyrrole-2,5-dione hydrochloride (compound 77, 17 mg, 0.10 mmol, 1.20 equiv) and DIEA (31 mg, 0.24 mmol, 3.0 equiv). The resulting mixture was stirred at room temperature under nitrogen atmosphere for 4 h. LCMS showed the reaction was complete. The residue was purified by preparative HPLC (column: XBridge Prep Phenyl OBD column, 19×150 mm 5 μm 13 nm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 25B to 43B in 10 min; 220 nm; RT1: 11.97 min). The collected fractions were lyophilized to give 4-[[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)sulfamoyl]-N-[2-(2,5-dioxopyrrol-1-yl)ethyl]-3-nitrobenzamide (compound (Ii), 27 mg, 36%) as a white solid. LCMS (ES, m / z): 879,881 [M+H]. 1H NMR (300 MHz, DMSO-d6) δ 11.00 (s, 1H), 9.01 (t, J=6.0 Hz, 1H), 8.82 (s, 1H), 8.20 (s, 1H), 8.11 (s, 2H), 7.71-7.67 (m, 2H), 7.52 (s, 1H), 7.44 (d, J=3.0 Hz, 1H), 7.21-7.12 (m, 2H), 7.02 (s, 2H), 6.84 (t, J=6.0 Hz, 1H), 5.14-5.08 (m, 1H), 4.48-4.28 (m, 4H), 3.62-3.50 (m, 6H), 3.40-3.28 (m, 2H), 2.95-2.85 (m, 4H), 2.80-2.73 (m, 2H), 2.65-2.60 (s, 1H), 2.41-2.27 (m, 1H), 2.05-1.95 (m, 1H)

[0292]

change

[0293] Project 1: Synthesis of Compound 79 To a stirred mixture of (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-acetamido)acetic acid (compound 78, 10.00 g, 28.22 mmol, 1.00 equiv.) and Pb(OAc)4 (15.02 g, 33.86 mmol, 1.20 equiv.) in THF (300 mL) and toluene (100 mL) was added pyridine (2.59 g, 32.74 mmol, 1.16 equiv.) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 80° C. overnight under nitrogen atmosphere. LCMS showed the reaction was 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, brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:4) to give (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamide)methyl acetate (compound 79, 6.5 g, 56%) as a white solid. LCMS (ESI, ms): 391 [M+Na] + . 1 H NMR (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)

[0294] Step 2: Synthesis of Compound 81 To a stirred mixture of (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]acetamide)methyl acetate, 79 (2.00 g, 5.43 mmol, 1.00 equiv.) and 2-(2-chloro-4-nitrophenyl)ethanol (compound 3, 3.20 g, 15.85 mmol, 2.92 equiv.) in DCM (40 mL) was added PPTS (400 mg, 1.59 mmol, 0.29 equiv.) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at 45° C. overnight under nitrogen atmosphere. 40% 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 (3×20 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 9H-fluoren-9-ylmethyl N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate (compound 81, 1.7 g, 55%) as a white solid. LCMS (ESI, ms): 510,512 [M+H] + . 1 H NMR (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)

[0295] Step 3: Synthesis of Compound 82 To a stirring mixture of 9H-fluoren-9-ylmethyl N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate (compound 81, 1.60 g, 3.14 mmol, 1.00 equiv) in DMF (5.0 mL) was added piperidine (1.0 mL) portionwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 h. LCMS showed the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), 0% to 50% gradient in 40 min; detector, UV 254 nm. This afforded 2-amino-N-[[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]acetamide (compound 82, 750 mg, 76%) as a yellow oil. LCMS (ESI, ms) 288 [M+H] + ,329 [M+H+ACN] +

[0296] Step 4: Synthesis of compound 83 To a stirring mixture of 2-amino-N-[[2-(2-chloro-4-nitrophenyl)ethoxy]-methyl]acetamide (compound 82, 750 mg, 2.61 mmol, 1.00 equiv.) and Boc2O (580 mg, 2.66 mmol, 1.02 equiv.) in DMF (10.00 mL) was added dropwise a solution of NaHCO3 (477 mg, 5.68 mmol, 2.18 equiv.) in H2O (10.00 mL) at 0 °C. The resulting mixture was stirred for 3 h at room temperature. LCMS showed the reaction was complete. The reaction was quenched by the addition of water (20 mL). The resulting mixture was extracted with EtOEt (3 x 20 mL). The combined organic layers were washed with brine (20 mL) 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 tert-butyl N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate (compound 83, 650 mg, 58%) as a yellow oil. LCMS (ESI, ms), 388 [M+H] +, 332 [M+H-56] + . 1 H NMR (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)

[0297] Step 5: Synthesis of Compound 84 To a stirring mixture of tert-butyl N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]-carbamoyl)methyl]carbamate (compound 83, 650 mg, 1.68 mmol, 1.00 equiv.) and Fe (260 mg, 4.66 mmol, 2.78 equiv.) in EtOH (9.00 mL) was added dropwise a solution of NH4Cl (910 mg, 17.01 mmol, 10.1 equiv.) in H2O (3.00 mL) at room temperature. The resulting mixture was stirred for 4 h at 90 °C. LCMS showed the reaction was complete. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. 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 tert-butyl N-[([[2-(4-amino-2-chlorophenyl)ethoxy]methyl]-carbamoyl)methyl]carbamate (compound 84, 500 mg, 83%) as a yellow solid. LCMS (ESI, ms): 358 [M+H] + , 380 [M+Na] + . 1H NMR (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)

[0298] Step 6: Synthesis of Compound 86 To a stirring mixture of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione hydrochloride (compound 85, 398 mg, 1.28 mmol, 0.92 equiv.) and CDI (450 mg, 2.78 mmol, 1.99 equiv.) in DMF (5.00 mL) was added TEA (300 mg, 2.96 mmol, 2.12 equiv.) at 0° C. The resulting mixture was stirred at room temperature for 2 hours. To the above mixture was added tert-butyl N-[([[2-(4-amino-2-chlorophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate (compound 84, 500 mg, 1.40 mmol, 1.00 equiv.) and DMAP (550 mg, 4.50 mmol, 3.22 equiv.) in portions. The resulting mixture was further stirred at 60° C. overnight. LCMS indicated the reaction was complete. The mixture was cooled to room temperature. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 50% gradient in 30 min; detector, UV 254 nm. This gave tert-butyl N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethoxy)methyl]carbamoyl]methyl)carbamate (compound 86, 550 mg, 60%) as a light brown solid. LCMS (ESI, ms): 657 [M+H] + , 601 [M+H-56] + ,557 [M+H-100] +

[0299] Step 7: Synthesis of Compound 87 To a stirring mixture of tert-butyl N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]carbamoyl]methyl)-carbamate (compound 86, 530 mg, 0.80 mmol, 1.00 equiv) in DCM (5.00 mL) was added TFA (1.00 mL) at 0° C. The resulting mixture was stirred for 30 min at 0° C. LCMS indicated the reaction was complete. The resulting mixture was concentrated under reduced pressure. This gave 2-amino-N-[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]acetamide;trifluoroacetic acid (compound 87, (510 mg, purity: 64%, yield: 60%) as an off-white solid. LCMS (ESI, ms): 557 [M+H-TFA] +

[0300] Step 8: Synthesis of Compound 89 To a stirring mixture of (2S)-2-[2-(2-aminoacetamido)acetamido]-3-phenylpropanoic acid (compound 88, 2.00 g, 7.16 mmol, 1.00 equiv.) and NaHCO3 (1.80 g, 21.41 mmol, 3.00 equiv.) in H2O (40.00 mL) was added dropwise a solution of Boc2O (1.86 g, 8.52 mmol, 1.20 equiv.) in DMF (40.00 mL) at 0 °C. The resulting mixture was stirred overnight at room temperature. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The resulting mixture was extracted with EtOEt (3 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 flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN water (0.05% TFA), 5% to 60% gradient in 30 min; detector, UV 220 nm. This gave (2S)-2-(2-[2-[(tert-butoxycarbonyl)amino]acetamido]acetamido)-3-phenylpropanoic acid (compound 89, 1.8 g, 60%) as a white semi-solid. LCMS (ESI, ms): 380 [M+H] + ,324 [M+H-56] + . 1 H NMR: (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)

[0301] Step 9: Compound 90 To a stirring mixture of (2S)-2-(2-[2-[(tert-butoxycarbonyl)amino]acetamido]-acetamido)-3-phenylpropanoic acid (compound 89, 340 mg, 0.90 mmol, 1.00 equiv.) and HATU (340 mg, 0.90 mmol, 1.00 equiv.) in DMF (5.00 mL) was added HOBT (102 mg, 0.75 mmol, 0.84 equiv.) in portions at 0° C. The resulting mixture was stirred for 30 min at 0° C. To the above mixture, 2-amino-N-[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]acetamide; trifluoroacetic acid (compound 87, 511 mg, purity: 64%, 0.48 mmol, 0.54 equiv.) and DIEA (340 mg, 2.63 mmol, 2.94 equiv.) were added at 0° C. The resulting mixture was stirred for another 2 h at room temperature. LCMS showed the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 50% gradient in 30 min; detector, UV 220 nm. The collected fractions were concentrated under reduced pressure. This gave tert-butyl N-[[([[(1S)-1-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]carbamoyl]methyl)carbamoyl]-2-phenylethyl]carbamoyl]methyl)carbamoyl]methyl]carbamate (compound 90, 210 mg, 48%) as an off-white solid. LCMS (ESI, ms): 918 [M+H] + , 818 [M+H-100] + . 1H NMR: (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 =6.4Hz, 1H), 5.13-5.06 (m, 1H), 4.55-4.27 (m, 7H), 3.72-3.60 (m, 6H), 3.75-3.67 (m, 3H), 3.59-3.49 (m, 5H), 3.07-3.01 (m, 1H), 2.94-2.73 (m, 4H), 2.62-2.54 (m, 1H), 2.40-2.31 (m, 1H), 2.01-1.94 (m, 1H), 2.00-1.91 (m, 1H), 1.35 (s, 9H)

[0302] Process 10: Synthesis of Compound 91 To a stirring mixture of tert-butyl N-[[([[(1S)-1-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)-methyl]carbamoyl]methyl)carbamoyl]-2-phenylethyl]carbamoyl]methyl)carbamoyl]-methyl]carbamate (Compound 90, 140 mg, 0.15 mmol, 1.00 equiv) in DCM (5.00 mL) was added TFA (1.00 mL) dropwise at 0° C. The resulting mixture was stirred for 30 min at 0° C. LCMS indicated the reaction was complete. The resulting mixture was concentrated under reduced pressure. This gave (2S)-2-[2-(2-aminoacetamido)acetamido]-N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]-carbamoyl]methyl)-3-phenylpropanamide; trifluoroacetic acid (compound 91, 140 mg, 79%) as an off-white solid. LCMS (ESI, ms): 818 [M+H-TFA] +

[0303] Step 11: Synthesis of compound (Ij) To a stirring mixture of (2S)-2-[2-(2-aminoacetamido)acetamido]-N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethoxy)methyl]carbamoyl]methyl)-3-phenylpropanamide; trifluoroacetic acid (compound 91, 140 mg, 0.15 mmol, 1.00 equiv.) and DIEA (70 mg, 0.54 mmol, 3.61 equiv.) in DMF (2.00 mL) was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (compound 92, 70 mg, 0.23 mmol, 1.50 equiv.) in portions at 0° C. The resulting mixture was stirred for 2 hours at room temperature. LCMS showed the reaction was complete. The reaction mixture was directly purified by the following conditions: Column: XSelect CSH Prep C18 OBD column, 19×250 mm, 5 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 25B to 50B in 7 min; 254 nm; RT1: 6.35 min; The collected fractions were lyophilized to give the crude product. The crude product was repurified by the following conditions: Column: Kinetex EVO C18 column, 30 × 150, 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 20B to 40B in 7 min, 220 nm; RT1: 6.77 min; collected fractions were lyophilized to give N-[[([[(1S)-1-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]carbamoyl]methyl)carbamoyl]-2-phenylethyl]carbamoyl]methyl)carbamoyl]methyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound (Ik), 22.8 mg, 14%) as an off-white solid. LCMS (ESI, ms): 1011 [M+H] + . 1H NMR: (400MHz, DMSO-d6): δ 10.95 (s, 1H), 8.79 (s, 1H), 8.51 (t, J=8.4Hz, 1H), 8.29 (t, J =8.0Hz, 1H), 8.12-8.01 (m, 3H), 7.70-7.66 (m, 2H), 7.44 (s, 1H), 7.42 (d, J =8.0Hz, 1H), 7.23-7.16 (m, 7H), 6.99 (s, 2H), 6.82 (t, J=8.0Hz, 1H), 5.13-5.09 (m, 1H), 4.55-4.28 (m, 7H), 3.72-3.60 (m, 6H), 3.55-3.51 (m, 2H), 3.36-3.34 (m, 2H), 3.05-3.00 (m, 1H), 2.94-2.72 (m, 4H), 2.62-2.54 (m, 1H), 2.40-2.32 (m, 1H), 2.12-2.05 (m, 2H), 2.00-1.91 (m, 1H), 1.50-1.38 (m, 4H), 1.19-1.10 (m, 2H)

[0304]

change

change

[0305] Process 1: Synthesis of Compound 94 To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 93, 24.00 g, 111.32 mmol, 1.00 equiv.) in THF (240.00 mL) was added BH3-Me2S (28.00 mL, 295.23 mmol, 2.65 equiv.) dropwise under nitrogen atmosphere. The resulting mixture was stirred at 70° C. for 2 h under nitrogen atmosphere. TLC (PE:EtOAc=3:1) indicated the completion of the reaction. After cooling to room temperature, the resulting mixture was concentrated under reduced pressure. 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 light yellow solid. 1 H NMR (300 MHz, CDCl3) δ 8.27 (s, 1H), 8.10-8.07 (m, 1 H), 7.52 (d, J = 3 Hz, 1H), 3.96 (t, J = 6 Hz, 2H), 3.13 (t, J = 6 Hz, 2H)

[0306] Step 2: Synthesis of Compound 95 To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 94, 5.00 g, 24.80 mmol, 1.00 equiv) in DCM (100.00 mL) was added NBS (6.62 g, 1.50 equiv) and PPh3 (9.76 g, 37.21 mmol, 1.50 equiv) in portions at room temperature under N2. The resulting mixture was stirred overnight at room temperature under N2. TLC (PE:EtOAc=10:1) showed the reaction was complete. The reaction was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (4:1) to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 95, 5.10 g, 72%) as a red oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 8.4, 2.4 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 3.79 4 (t, J = 6.8 Hz, 2H), 3.38 (t, J = 6.8 Hz, 2H)

[0307] Step 3: Synthesis of Compound 96 To a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 95, 5.00 g, 18.90 mmol, 1.00 equiv.) in DMF (50.00 mL) was added potassium thioacetate (2.16 g, 18.90 mmol, 1.00 equiv.) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 h. TLC (PE:EtOAc=10:1) showed the reaction was complete. The reaction was diluted with water (600.00 mL) and extracted with EtOAc (2000 mL×3). The combined organic layer was washed with water (200.00 mL), brine (200.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (compound 96, 4.50 g, 85%) as a red oil. 1 H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.4 Hz, 1H), 8.07 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 3.20 - 3.05 (m, 4H), 2.34 (s, 3H)

[0308] Step 4: Synthesis of compound 97 To a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (compound 96, 2.00 g, 7.70 mmol, 1.00 equiv) in MeOH (300.00 mL) was added MeONa (6.93 mL, 37.33 mmol, 5.00 equiv, 30% in MeOH) at 0 °C under N2. The resulting mixture was stirred at 0 °C under N2 for 1 h. TLC (PE:EtOAc = 10:1) showed the completion of the reaction. The reaction was quenched with AcOH to a pH value of 3-4. The resulting mixture was concentrated to dryness under reduced pressure. The residue was diluted with DCM (50.00 mL) and filtered. The filtrate was purified by preparative TLC (PE:EtOAc=10:1) to give 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 97, 1.35 g, 72%) as a light yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.4 Hz, 1H), 8.09 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 3.14 (t, J = 8.0Hz, 2H), 2.85 (dt, J = 8.0, 7.2 Hz, 2H), 1.43 (t, J = 7.2 Hz, 1H)

[0309] Step 5: Synthesis of Compound 99 To a stirred solution of (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoic acid (compound 98, 20.00 g, 64.24 mmol, 1.00 equiv.) in DMF (200.00 mL) was added TSTU (25.18 g, 83.52 mmol, 1.30 equiv.) and DIEA (16.60 g, 128.48 mmol, 2.00 equiv.) at room temperature under air atmosphere. The resulting mixture was stirred for 1 h at room temperature. LCMS showed the reaction was complete. The reaction was diluted with water (200.00 mL) and extracted with EtOAc (100.00 mL×3). The combined organic layer was washed with water (100.00 mL), brine (100.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with (PE:EtOAc=1:2) to give 2,5-dioxopyrrolidin-1-yl(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (compound 99, 25.00 g, 83%) as a white solid. LCMS (ES, m / z): 431 [M+Na] +

[0310] Step 6: Synthesis of Compound 100 To a solution of D-alanine (1.09 g, 0.012 mmol, 1.00 equiv.) and NaHCO3 (3.09 g, 0.04 mmol, 3.00 equiv.) in water (50.00 mL) was added a solution of 2,5-dioxopyrrolidin-1-yl(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (compound 99, 5.00 g, 12.24 mmol, 1.00 equiv.) in DMF (50.00 mL). The resulting mixture was stirred at room temperature for 2 h. LCMS showed the completion of the reaction. The reaction was adjusted to a pH value of 2-3 with 2N HCl. The resulting mixture was extracted with EtOAc (100.00 mL×3) and the combined organic layers were washed with brine (100.00 mL×3), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to give (2R)-2-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propanamide]propanoic acid (compound 100, 4.00 g, 71%) as a white solid. LCMS (ES, m / z): 383 [M+H] +

[0311] Step 7: Synthesis of Compound 101 To a solution of glycine (3.68 g, 48.97 mmol, 1.00 equiv) and NaHCO3 (12.34 g, 146.89 mmol, 3.00 equiv) in water (200.00 mL) was added a solution of 2,5-dioxopyrrolidin-1-yl(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (compound 99, 20.00 g, 48.97 mmol, 1.00 equiv) in DMF (200.00 mL). The reaction was stirred at room temperature for 2 h. LCMS showed the reaction was complete. The reaction was adjusted to a pH value of 2-3 with 2N HCl. The resulting mixture was extracted with EtOAc (500.00 mL×3) and the combined organic layers were washed with brine (500.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to give [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]acetic acid (compound 101, 15.00 g, 71%) as a white solid. LCMS (ES, m / z): 369 [M+H] +

[0312] Step 8: Synthesis of Compound 102 A solution of [(2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]propanamido]-acetic acid (compound 101, 5.00 g, 13.57 mmol, 1.00 equiv.), Pb(OAc)4 (7.22 g, 16.28 mmol, 1.20 equiv.) and pyridine (1.29 g, 16.31 mmol, 1.20 equiv.) in THF (300.00 mL) / toluene (100.00 mL) was stirred at 80° C. under N2 for 16 h. LCMS showed the reaction was complete. After cooling to room temperature, the reaction was filtered. The filter cake was washed with THF (100.00 mL). The combined organic layers were concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with (PE:EtOAc=1:2) to give [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamide]methyl acetate (compound 102, 2.50 g, 45%) as a white solid. LCMS (ES, m / z): 405 [M+Na] + . 1 H NMR (400 MHz, chloroform-d) δ 7.77 (t, J = 7.6 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.43 - 7.37 (m, 2H), 7.36 - 7.29 (m, 2H), 7.10 (s, 1H), 5.24 (d, J = 7.6 Hz, 2H), 4.51 - 4.35 (m, 2H), 4.23-4.09 (m, 2H), 2.04 (s, 3H), 1.39 (d, J = 6.8 Hz, 3H)

[0313] Step 9: Synthesis of Compound 103 To a stirring solution of [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanamido]methyl acetate (compound 102, 2.25 g, 5.88 mmol, 1.00 equiv.) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 97, 1.28 g, 5.88 mmol, 1.00 equiv.) in DCM (120 mL) under N2 at room temperature was added TFA (0.27 mL, 2.37 mmol, 0.62 equiv.). The resulting mixture was stirred at room temperature for 16 h. LCMS indicated the reaction was complete. The reaction was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with (PE:EtOAc=1:4) to give 9H-fluoren-9-ylmethyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 103, 3.10 g, 90%) as a yellow solid. LCMS (ES, m / z): 540 [M+H] +

[0314] Step 10: Synthesis of Compound 104 To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 103, 3.10 g, 5.74 mmol, 1.00 equiv.) in DMF (155.00 mL) was added piperidine (31.00 mL) at 0° C. under N2. The resulting mixture was stirred at 0° C. for 0.5 h under N2. LCMS showed the reaction was complete. The reaction was diluted with water (600.00 ml). The resulting mixture was extracted with EtOAc (200.00 mL×3). The combined organic layer was washed with brine (200.00 ml), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to give 3.00 g of crude product. The crude product was repurified by silica gel column chromatography eluting with (DCM:MeOH=3:1) to give (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)propenamide, 104 (1.50 g, 78%) as a yellow oil. LCMS (ES, m / z): 318 [M+H] +

[0315] Step 11: Synthesis of Compound 105 To a solution of (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)-propenamide (compound 104, 1.50 g, 4.72 mmol, 1.00 equiv.) in DMF (75.00 mL) was added a solution of NaHCO3 (0.59 g, 7.08 mmol, 1.50 equiv.) in H2O (10.00 mL) and Boc2O (1.03 g, 4.72 mmol, 1.00 equiv.) at room temperature. The reaction was stirred at room temperature for 1 h. LCMS showed the reaction was complete. The reaction 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 reduced pressure to give tert-butyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 105, (1.82 g, 83)) as a red oil. LCMS (ES, m / z): 418 [M+H] + , 318 [M+H-100] +

[0316] Step 12: Synthesis of Compound 106 A slurry of tert-butyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)carbamoyl]ethyl]carbamate (compound 105, 1.82 g, 4.36 mmol, 1.00 equiv), iron powder (2.43 g, 0.04 mmol, 10.00 equiv) and NH4Cl (2.33 g, 0.04 mmol, 10.00 equiv) in EtOH (100.00 mL) / H2O (50.00 mL) was stirred at 70 °C for 2 h. LCMS showed the reaction was complete. The reaction was filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was concentrated to dryness, and the residue was purified by silica gel column chromatography eluting with (DCM:MeOH=13:1) to give tert-butyl N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]-carbamate (compound 106, 1.20 g, 68%) as a yellow oil. LCMS (ES, m / z): 388 [M+H] +

[0317] Step 13: Compound 107 To a stirring solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 352 mg, 1.29 mmol, 1.00 equiv.) in DMF (5.00 mL) was added CDI (209.00 mg, 1.29 mmol, 1 equiv.) and TEA (260 mg, 2.58 mmol, 2 equiv.) at 0° C. The resulting mixture was stirred at 0° C. for 2 h. Then tert-butyl N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]-methyl)-carbamoyl]ethyl]carbamate (compound 106, 500.00 mg, 1.29 mmol, 1.00 equiv.) and DMAP (472 mg, 3.87 mmol, 3.00 equiv.) were added. The resulting mixture was stirred at 60° C. for 24 hours. LCMS indicated completion of the reaction. After cooling to room temperature, the reaction mixture was purified by reverse-phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 60% gradient in 30 minutes; detector, UV 254 nm) to give tert-butyl N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamate (compound 107, 450.00 mg, 48%) as a yellow solid. LCMS (ES, m / z): 687 [M+H] +

[0318] Step 14: Compound 108 To a stirring solution of tert-butyl N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]-methyl]carbamoyl)ethyl]carbamate (compound 107, 440.00 mg, 0.64 mmol, 1.00 equiv) in DCM (22.00 mL) was added TFA (2.20 mL) at room temperature. The resulting mixture was stirred at room temperature for 0.5 h. LCMS showed the reaction was complete. The reaction was concentrated to dryness under reduced pressure to give (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]-methyl]propanamide; trifluoroacetic acid (compound 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] +

[0319] Step 15: Synthesis of Compound 109 A solution of(2R)-2-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propanamido]propanoic acid (218 mg, 0.57 mmol, 1.00 equiv.), HOBT (77 mg, 0.57 mmol, 1.00 equiv.) and HATU (216 mg, 0.01 mmol, 1.00 equiv.) was stirred at room temperature in air for 1 h, followed by addition of (2S)-2-amino-N-[[(2- [2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]propanamide; trifluoroacetic acid (compound 108, 400 mg, 0.57 mmol, 1.00 equiv.) and DIEA (663 mg, 5.14 mmol, 9.00 equiv.) were added at room temperature. The reaction was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), 0% to 50% gradient in 30 min; detector, UV 254 nm) to give 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1R)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamate (compound 109, 480.00 mg, 75%) as a green solid. LCMS (ES, m / z): 951 [M+H] +

[0320] Step 16: Compound 110 To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1R)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamate (compound 109, 110.00 mg) in DMF (5.00 mL) was added piperidine (1.00 mL) at 0° C. The resulting mixture was stirred at 0° C. for 0.5 h. LCMS indicated completion of the reaction. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), 0% to 60% gradient in 40 min; detector, UV 254n) to give (2S)-2-[(2R)-2-[(2S)-2-aminopropanamido]propanamido]-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]-phenyl]ethyl)sulfanyl]methyl]propenamide (compound 110, 80.00 mg, 60%) as a red solid. LCMS (ES, m / z): 729 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.00 (br s, 1H), 8.53 br (s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.10 (br s, 1H), 7.69 - 7.62 (m, 2H), 7.49 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.26-7.13 (m, 3H), 7.00 (br s, 1H), 5.11-5.06 (m, 1H), 4.45 - 4.36 (m, 3H), 4.35 - 4.13 (m, 6H), 2.90-2.83 (m, 3H), 2.73-2.71 (m, 2H), 2.05-1.90 (m, 1H), 1.70-1.53 ​​(m, 4H), 1.22-1.17 (m, 6H), 1.14 - 1.05 (m, 3H)

[0321] Step 17: Synthesis of compound (Ik) (2S)-2-[(2R)-2-[(2S)-2-aminopropanamido]propanamido]-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]propenamide (Compound 110, 63.00 mg To a solution of 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (26 mg, 0.09 mmol, 1.00 equiv.) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (26 mg, 0.09 mmol, 1.00 equiv.) in DMF (1.50 mL, 19.38 mmol, 224.36 equiv.) was added DIEA (22.33 mg, 0.17 mmol, 2.00 equiv.) at room temperature in air. The reaction was stirred at room temperature for 1 h. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions: Column: Kinetex EVO C18 column, 30 x 150, 5 μm; Mobile phase A: xater (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 23B to 43B in 7 min, 254 nm; RT1: 6.58). The collected fractions were lyophilized to give N-[(1S)-1-[[(1R)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound (Ik), 16.10 mg, 20%) as a white solid. LCMS (ES, m / z): 922,924 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 8.80 (s, 1H), 8.44-8.41 (m, 1H), 8.15 (d, J=7.2Hz, 1H), 8.03-8.00 (m, 2H), 7.7-7.65 (m, 2H), 7.51 (s, 1H), 7.44 (d, J =8.0Hz,1H), 7.22-7.14 (m, 2H), 6.98 (s, 2H), 6.83-6.81 (m, 1H), 5.13-5.08 (m, 1H), 4.48-4.40 (m, 3H), 4.29-4.17 (m, 6H), 2.96-2.85 (m, 3H), 2.75-2.70 (m, 2H), 2.67-2.57 (m, 1H), 2.40-2.33 (m, 1H), 2.09-1.98 (m, 3H), 1.52-1.45 (m, 5H), 1.26-1.16 (m, 12H)

[0322] [ka] [ka] Scheme 14: Synthesis of the neodegrader P14-GGFG linker conjugate (compound (Il))

[0323] Step 1: Synthesis of Compound 112 To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 111, 5.00 g, 23.19 mmol, 1.00 equiv.) in THF (50 mL) was added BH3-Me2S (5.50 mL, 57.99 mmol, 2.50 equiv.) portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 70° C. for 2 h under nitrogen atmosphere. TLC (PE:EtOAc=3:1) showed the reaction was complete. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (2:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 112, 4.8 g, 92%) as a light yellow solid. 1H NMR (400 MHz, chloroform-d) δ 8.27 (d, J = 2.4 Hz, 1H), 8.10 (dd, J = 8.4, 2.4 Hz, 1H), 7.46 (s, 1H), 3.20 -3.09 (m, 4H)

[0324] Step 2: Synthesis of Compound 113 To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 112, 4.80 g, 23.81 mmol, 1.00 equiv.) in DCM (100 mL), NBS (6.36 g, 35.71 mmol, 1.50 equiv.) and PPh3 (9.37 g, 35.72 mmol, 1.50 equiv.) were added portionwise at room temperature under air atmosphere. The resulting mixture was stirred at room temperature overnight under air atmosphere. TLC (PE:EtOAc=10:1) showed the reaction was complete. The reaction was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (4:1) to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 113, 3.9 g, 57%) as a red oil. 1 H NMR (400 MHz, chloroform-d) δ 8.29 (d, J = 2.4 Hz, 1H), 8.13 (dd, J = 8.4, 2.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 3.67 (t, J = 7.2 Hz, 2H), 3.42 (t, J = 7.2 Hz, 2H)

[0325] Step 3: Synthesis of compound 114 To a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 113, 3.90 g, 14.75 mmol, 1.00 equiv.) in DMF (39 mL) was added potassium thioacetate (1.68 g, 14.75 mmol, 1.00 equiv.) at room temperature. The resulting mixture was stirred at room temperature for 2 h. TLC ((PE:EtOAc=10:1) showed the reaction was complete. The reaction was diluted with water (600 mL). The resulting mixture was extracted with EA (200 mL×3). The combined organic layers were washed with water (200 mL), brine (200 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (compound 114, 3.7 g, 85%) as a red oil. 1 H NMR (400 MHz, chloroform-d) δ 8.27 (d, J = 2.4 Hz, 1H), 8.10 (dd, J = 8.4, 2.4 Hz, 1H), 7.46 (s, 1H), 3.21 -3.02 (m, 4H), 2.37 (s, 3H)

[0326] Step 4: Synthesis of Compound 115 To a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (compound 114, 4.00 g, 15.40 mmol, 1.00 equiv) in MeOH (600 mL) was added MeONa (14.31 mL, 77.00 mmol, 5.00 equiv, 30%) under N2 at 0 °C for 1 h. The reaction mixture was stirred at 0 °C for 1 h. TLC (PE:EA = 10:1) showed the reaction was complete. The reaction was quenched with AcOH. The resulting mixture was concentrated to dryness under reduced pressure. 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. 1H NMR (400 MHz, chloroform-d) δ 8.28 (d, J = 2.4 Hz, 1H), 8.11 (dd, J = 8.4, 2.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 3.17 (t, J = 7.2 Hz, 2H), 2.87 (dt, J = 8.0, 7.2 Hz, 2H), 1.46 (t, J = 8.0Hz, 1H)

[0327] Step 5: Synthesis of Compound 117 To a stirred mixture of (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-acetamido)acetic acid (compound 116, 10 g, 28.22 mmol, 1.00 equiv.) and Pb(OAc)4 (15 g, 33.86 mmol, 1.20 equiv.) in THF (300 mL) and toluene (100 mL) was added pyridine (2.59 g, 32.74 mmol, 1.16 equiv.) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 80° C. overnight under nitrogen atmosphere. LCMS showed the reaction was 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 reduced pressure. The residue was dissolved in EA (20 mL). The resulting mixture was washed with water, brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:4) to give (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamide)methyl acetate (compound 117, 6.5 g, 56%) as a white solid. 1H NMR (300MHz, CDCl3) δ7.80 (d, J=7.5Hz, 2H), 7.62 (d, J=7.5Hz, 2H), 7.45 (t, d=7.5Hz, 2H), 7.36 (d, d=7.5Hz, 2H), 7.18 (br s, 1H), 5.48 (br s, 1H), 5.28 (d, J=7.2Hz, 2H), 4.48 (d, J=6.6Hz, 2H), 4.26 (t, J=6.6Hz, 1H), 3.93 (d, 5.4Hz, 2H), 2.08 (s, 3H). LCMS (ESI, ms): 391 [M+Na] +

[0328] Step 6: Synthesis of Compound 118 To a solution of (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamido)methyl acetate (compound 117, 3.00 g, 8.14 mmol, 1.00 equiv.) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 115, 1.77 g, 8.13 mmol, 1.00 equiv.)) in DCM (300 mL) was added TFA (0.56 g, 4.91 mmol, 0.60 equiv.) at room temperature. The resulting mixture was stirred at 60° C. for 16 h. LCMS showed the reaction was complete. The reaction was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with (PE:EtOAc=2:3) to give 9H-fluoren-9-ylmethyl N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]-methyl]carbamate (compound 118, 3.7 g, 67%) as an off-white solid. LCMS (ES, m / z): 526,528 [M+H] +

[0329] Step 7: Synthesis of Compound 119 To a solution of 9H-fluoren-9-ylmethyl N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]methyl]carbamate (compound 118, 3.70 g, 7.03 mmol, 1.00 equiv) in DMF (40 mL) was added piperidine (8 mL) at 0° C. The resulting mixture was stirred at 0° C. for 0.5 h. LCMS showed the reaction was complete. The resulting mixture was diluted with water (400 mL) and extracted with EA (200 mL×3). The combined organic layers were washed with water (200 mL), brine (200 mL), dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with (DCM:MeOH=10:1) to give 2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)acetamide (compound 119, 1.01 g, 40%) as a yellow oil. LCMS (ES, m / z): 304,306 [M+H] +

[0330] Step 8: Synthesis of Compound 120 To a solution of 2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)-acetamide (compound 119, 1.00 g, 3.29 mmol, 1.00 equiv.) in DMF (50 mL) was added a solution of NaHCO3 (0.33 g, 3.92 mmol, 1.20 equiv.) in water (10 mL), Boc2O (0.72 g, 3.30 mmol, 1.00 equiv.) at room temperature. The resulting mixture was stirred at room temperature for 1 h. LCMS showed the reaction was complete. The reaction was diluted with water (500 mL) and extracted with EtOAc (200 mL×3). The combined organic layer was washed with brine (200 mL), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with (PE:EtOAc=1:3) to give tert-butyl N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]methyl]carbamate (compound 120, 810 mg, 54%) as a white solid. LCMS (ES, m / z): 404,406 [M+H] + , 304,306 [M+H-100]+

[0331] Step 9: Synthesis of Compound 121 To a solution of tert-butyl N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)carbamoyl]methyl]carbamate (compound 120, 800.00 mg, 1.98 mmol, 1.00 equiv) in EtOH (40) was added a solution of iron powder (1106 mg, 19.81 mmol, 10.00 equiv) and NH4Cl (1059 mg, 19.81 mmol, 10.00 equiv) in water (10 mL) at room temperature. The resulting mixture was stirred at 70 °C for 2 h. LCMS showed the reaction was complete. The reaction was filtered. The filtrate was concentrated to dryness under reduced pressure. 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 tert-butyl N-[[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]methyl)-carbamoyl]methyl]carbamate (compound 121, 610 mg, 74%) as a yellow oil. LCMS (ES, m / z): 374,376 [M+H] + , 374,376 [M+H-100] +

[0332] Step 10: Synthesis of Compound 122 To a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 219 mg, 0.80 mmol, 1.00 equiv.) in DMF (10 mL), CDI (130 mg, 0.80 mmol, 1.00 equiv.) and TEA (81 mg, 0.80 mmol, 1.00 equiv.) were added at 0° C. in air. The resulting mixture was stirred at room temperature for 2 hours. Then tert-butyl N-[[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]-methyl)carbamoyl]methyl]carbamate (compound 121, 300 mg, 0.80 mmol, 1.00 equiv.) and DMAP (294 mg, 2.41 mmol, 3.00 equiv.) were added at room temperature in air. The resulting mixture was stirred at 60° C. for 48 hours. LCMS indicated completion of the reaction. The resulting mixture was purified by reverse-phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), 0% to 60% gradient in 30 minutes; detector, UV 254 nm) to give tert-butyl N-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)methyl]carbamate (compound 122, 270 mg, 49%) as a yellow solid. LCMS (ES, m / z): 673,675 [M+H] + , 573,575 [M+H-100] +

[0333] Step 11: Synthesis of Compound 123 To a solution of tert-butyl N-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)methyl]-carbamate (compound 122, 250 mg, 0.37 mmol) in 1,4-dioxane (12 mL) was added HCl (4N in 1,4-dioxane, 6 mL) at 0° C. under N2. The reaction was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction mixture was concentrated to dryness under reduced pressure to give 2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]acetamide (compound 123, 260 mg, crude) as a brown solid. LCMS (ES, m / z): 573,575 [M+H-HCl] +

[0334] Step 12: Synthesis of Compound 125 A solution of (2S)-2-[2-(2-aminoacetamido)acetamido]-3-phenylpropanoic acid (compound 124, 500 mg, 1.79 mmol, 1.00 equiv.) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (552 mg, 1.79 mmol, 1.00 equiv.) in DMSO (5.00 mL) was stirred at room temperature under air for 16 hours. LCMS showed the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 60% gradient in 30 min; detector, UV 220 nm) to give (2S)-2-(2-[2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]acetamido]acetamido)-3-phenylpropanoic acid (compound 125, 760 mg, 83%) as a white solid. LCMS (ES, m / z): 473 [M+H] +

[0335] Step 13: Synthesis of compound (Il) To a solution of (2S)-2-(2-[2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]acetamido]-acetamido)-3-phenylpropanoic acid (compound 125, 175 mg, 0.37 mmol, 1.00 equiv.) in DMF (5.00 mL), HATU (141 mg, 0.37 mmol, 1.00 equiv.) and HOBT (50 mg, 0.37 mmol, 1.00 equiv.) were added at room temperature in air. The resulting mixture was stirred at room temperature for 1 h. Then 2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]acetamide (compound 123, 250 mg, 0.37 mmol, 1.00 equiv, 85%) and DIEA (240 mg, 1.85 mmol, 5.00 equiv) were added. The resulting mixture was stirred at room temperature for 1 h. LCMS showed the reaction was complete. The reaction mixture was purified under the following conditions (Column: XSelect CSH Prep C18 OBD column, 19×250 mm, 5 μm; Mobile phase A: water (0.05% FA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 30B to 60B in 7 min, 254 nm; RT1: 6.67 min) to give 75 mg of crude product. The crude product was repurified by reversed-phase flash chromatography using the following conditions: Column: XBridge Shield RP18 OBD column, 19×250 mm, 10 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 25B to 44B in 10 min, 254 nm; RT1: 10.52 min. The collected fractions were lyophilized to give compound (Il) (41.6 mg, 10%) as a white solid. 1H NMR (400MHz, DMSO-d6) δ10.99 (s, 1H), 8.79 (s, 1H), 8.38 (t, J=6.0Hz, 1H), 8.31 (t, J=6.0Hz, 1H), 8.12 (d, J=8.4Hz, 1H), 8.06 (t, J=5.6Hz, 1H), 8.01 (t, J=6.0Hz, 1H), 7.70-7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J=8.0Hz, 1H), 7.25-7.21 (m, 5H), 7.19-7.14 (m, 2H), 6.99 (s, 2H), 6.82 (t, J=6.0Hz,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] +

[0336]

change

change

[0337] Process 1: Synthesis of Compound 127 To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 126, 24.00 g, 111.32 mmol, 1.00 equiv.) in THF (240.00 mL) was added BH3-Me2S (28.00 mL, 295.23 mmol, 2.65 equiv.) dropwise under nitrogen atmosphere. The resulting mixture was stirred at 70° C. for 2 h under nitrogen atmosphere. TLC (PE:EtOAc=3:1) indicated the completion of the reaction. After cooling to room temperature, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (3:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 127, 18.00 g, 80%) as a light yellow solid. 1 H NMR (300 MHz, CD3Cl) δ8.27 (s, 1H), 8.10-8.07 (m, 1 H), 7.52 (d, J = 3 Hz, 1H), 3.96 (t, J = 6 Hz, 2H), 3.13 (t, J = 6 Hz, 2H)

[0338] Step 2: Synthesis of Compound 128 To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 127, 5.00 g, 24.80 mmol, 1.00 equiv) in DCM (100.00 mL) was added NBS (6.62 g, 1.50 equiv) and PPh3 (9.76 g, 37.21 mmol, 1.50 equiv) in portions at room temperature under N2. The resulting mixture was stirred overnight at room temperature under N2. TLC (PE:EtOAc=10:1) showed the reaction was complete. The reaction was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (4:1) to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 128, 5.10 g, 72.31%) as a red oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 8.4, 2.4 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 3.79 (t, J = 7.2 Hz, 2H), 3.38 (t, J = 7.2 Hz, 2H)

[0339] Step 3: Synthesis of Compound 129 To a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 128, 5.00 g, 18.90 mmol, 1.00 equiv.) in DMF (50.00 mL) was added potassium thioacetate (2.16 g, 18.91 mmol, 1.00 equiv.) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 h. TLC (PE:EtOAc=10:1) showed the reaction was complete. The reaction was diluted with water (600.00 mL). The resulting mixture was extracted with EtOAc (200.00 mL×3). The combined organic layers were washed with water (200.00 mL), brine (200.00 mL×3), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (compound 129, 4.50 g, 85%) as a red oil. 1 H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.4 Hz, 1H), 8.07 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 3.20 - 3.05 (m, 4H), 2.34 (s, 3H)

[0340] Step 4: Synthesis of Compound 130 To a stirring solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (compound 129, 2.00 g, 7.70 mmol, 1.00 equiv) in MeOH (300.00 mL) was added MeONa (6.93 mL, 37.33 mmol, 5.00 equiv, 30%) at 0 °C under N2. The resulting mixture was stirred at 0 °C under N2 for 1 h. TLC (PE:EtOAc = 10:1) showed the completion of the reaction. The reaction was quenched with AcOH to a pH value of 3-4. The resulting mixture was concentrated to dryness under reduced pressure. The residue was diluted with DCM (50.00 mL) and filtered. The filtrate was purified by preparative TLC (PE:EtOAc = 10:1) to give 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 130, 1.35 g, 72%) as a light yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.4 Hz, 1H), 8.09 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 3.14 (dd, J = 8.0, 6.8 Hz, 2H), 2.85 (dt, J = 8.0, 7.2 Hz, 2H), 1.43 (t, J= 8.0 Hz, 1H)

[0341] Step 5: Synthesis of Compound 132 To a stirred solution of (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoic acid (compound 131, 20.00 g, 64.24 mmol, 1.00 equiv.) in DMF (200.00 mL) was added TSTU (25.18 g, 83.52 mmol, 1.30 equiv.) and DIEA (16.60 g, 128.48 mmol, 2.00 equiv.) at room temperature under air atmosphere. The resulting mixture was stirred for 1 h at room temperature. LCMS showed the reaction was complete. The reaction was diluted with water (200.00 mL) and the resulting mixture was extracted with ETOAC (100.00 mL×3). The combined organic layer was washed with water (100.00 mL), brine (100.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with (PE:EtOAc=1:2) to give 2,5-dioxopyrrolidin-1-yl(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (compound 132, 25.00 g, 83%) as a white solid. LCMS (ES, m / z): 431 [M+Na] +

[0342] Step 6: Synthesis of Compound 133 To a solution of glycine (3.68 g, 48.97 mmol, 1.00 equiv) and NaHCO3 (12.34 g, 146.89 mmol, 3.00 equiv) in water (200.00 mL) was added a solution of 2,5-dioxopyrrolidin-1-yl(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (compound 132, 20.00 g, 48.97 mmol, 1.00 equiv) in DMF (200.00 mL). The reaction was stirred at room temperature for 2 h. LCMS showed the reaction was complete. The reaction was adjusted to a pH value of 2-3 with 2N HCl. The resulting mixture was extracted with EtOAc (500.00 mL×3) and the combined organic layers were washed with brine (500.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to give [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]acetic acid (compound 133, 15.00 g, 71%) as a white solid. LCMS (ES, m / z): 369 [M+H] +

[0343] Step 7: Synthesis of Compound 134 A solution of [(2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-propanamido]acetic acid (compound 133, 5.00 g, 13.57 mmol, 1.00 equiv.), Pb(OAc)4 (7.22 g, 16.28 mmol, 1.20 equiv.) and pyridine (1.29 g, 16.31 mmol, 1.20 equiv.) in THF (300.00 mL) / toluene (100.00 mL) was stirred at 80° C. under N2 for 16 h. LCMS showed the reaction was complete. After cooling to room temperature, the reaction was filtered. The filter cake was washed with THF (100.00 mL). The combined organic layers were concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with (PE:ETOAC=1:2) to give [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamide]methyl acetate (compound 134, 2.50 g, 45%) as a white solid. LCMS (ES, m / z): 405 [M+Na] + . 1H NMR (400 MHz, chloroform-d) δ 7.77-7.73 (m, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.43 - 7.37 (m, 2H), 7.36 - 7.29 (m, 2H), 7.10 (s, 1H), 5.24 (d, J = 7.6 Hz, 2H), 4.51 - 4.35 (m, 2H), 4.22 (t, J = 6.8 Hz, 2H), 2.04 (s, 3H), 1.39 (d, J = 6.8 Hz, 3H)

[0344] Step 8: Synthesis of Compound 135 To a stirred solution of [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanamido]methyl acetate (compound 134, 2.25 g, 5.88 mmol, 1.00 equiv.) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 500, 1.28 g, 5.88 mmol, 1.00 equiv.) in DCM (120 mL) was added TFA (0.27 mL, 2.376 mmol, 0.62 equiv.) at room temperature under N2. The resulting mixture was stirred at 40° C. for 16 h. LCMS indicated the reaction was complete. The reaction was concentrated to dryness under reduced pressure to give 9H-fluoren-9-ylmethyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 135, 3.10 g, 90%) as a yellow solid. LCMS (ES, m / z): 540,542 [M+H] +

[0345] Process 9 : Synthesis of compound 136 To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 135, 3.10 g, 5.74 mmol, 1.00 equiv) in DMF (155.00 mL) was added piperidine (31.00 mL) at 0° C. under N. The resulting mixture was stirred at 0° C. for 0.5 h under N. LCMS showed the reaction was complete. The reaction was diluted with water (600.00 ml). The resulting mixture was Extraction with EA (200.00 mL×3) was performed. The combined organic layers were washed with brine (200.00 ml), dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to give 3.00 g of crude product. The crude product was re-purified by silica gel column chromatography eluting with (DCM:MeOH=3:1) to give (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)propenamide (compound 136, 1.50 g, 78%) as a yellow oil. LCMS (ES, m / z): 318,320 [M+H] +

[0346] Step 10 : Synthesis of compound 137 To a solution of (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)propenamide (compound 136, 1.50 g, 4.72 mmol, 1.00 equiv.) in DMF (75.00 mL) was added a solution of NaHCO3 (0.59 g, 7.08 mmol, 1.50 equiv.) in H2O (10.00 mL) and Boc2O (1.03 g, 4.72 mmol, 1.00 equiv.) at room temperature under air. The reaction was stirred at room temperature for 1 h. LCMS showed the reaction was complete. The reaction 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 reduced pressure to give tert-butyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)-carbamoyl]ethyl]carbamate (compound 137, 1.82 g, 83%) as a red oil. LCMS (ES, m / z): 418,420 [M+H] + , 318,320 [M+H-100] +

[0347] Step 11: Synthesis of Compound 138 A slurry of tert-butyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]-sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 137, 1.82 g, 4.36 mmol, 1.00 equiv), iron powder (2.43 g, 0.04 mmol, 10.00 equiv) and NH4Cl (2.33 g, 0.04 mmol, 10.00 equiv) in EtOH (100.00 mL) / H2O (50.00 mL) was stirred at 70 °C for 2 h. LCMS showed the reaction was complete. The reaction was filtered. The filtrate was concentrated to dryness under reduced pressure. 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 tert-butyl N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 138, 1.20 g, 68%) as a yellow oil. LCMS (ES, m / z): 388,390 [M+H] + , 288,290 [M+H-100] +

[0348] Step 12: Synthesis of Compound 139 To a stirring solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 352 mg, 1.29 mmol, 1.00 equiv.) in DMF (5.00 mL) was added CDI (209.00 mg, 1.29 mmol, 1 equiv.) and TEA (260 mg, 2.58 mmol, 2 equiv.) at 0° C. The resulting mixture was stirred at 0° C. for 2 h. Then tert-butyl N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]-methyl)carbamoyl]-ethyl]carbamate (compound 138, 500.00 mg, 1.29 mmol, 1.00 equiv.) and DMAP (472 mg, 3.87 mmol, 3.00 equiv.) were added. The resulting mixture was stirred at 60° C. for 24 hours. LCMS indicated completion of the reaction. After cooling to room temperature, the reaction mixture was purified by reverse-phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 60% gradient in 30 minutes; detector, UV 254 nm) to give tert-butyl N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamate (compound 139, 450.00 mg, 48%) as a yellow solid. LCMS (ES, m / z): 687, 689 [M+H] + , 587,589 [M+H-100] +

[0349] Step 13: Synthesis of Compound 140 To a stirring solution of tert-butyl N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]-methyl]carbamoyl)ethyl]carbamate (compound 139, 440.00 mg, 0.64 mmol, 1.00 equiv) in DCM (22.00 mL) was added TFA (2.20 mL) at room temperature. The resulting mixture was stirred at room temperature for 0.5 h. LCMS showed the reaction was complete. The reaction was concentrated to dryness under reduced pressure to give (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]propanamide; trifluoroacetic acid (compound 140, 400.00 mg) as a red oil. LCMS (ES, m / z): 578, 589 [M+H-TFA] +

[0350] Step 14: Synthesis of Compound 142 To a slurry of L-valine (compound 141, 0.50 g, 4.27 mmol, 1.00 equiv) in DMSO (10 mL) was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (1.32 g, 4.28 mmol, 1.00 equiv) and DIEA (1103 mg, 8.54 mmol, 2.00 equiv). The resulting mixture was stirred at room temperature for 4 h. LCMS showed the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 60% gradient in 30 min; detector, UV 220 nm) to give (2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanoic acid (compound 142, 1.2 g, 72%) as a brown solid. LCMS (ES, m / z): 311 [M+H] +

[0351] Step 15: Synthesis of compound (Im) A solution of (2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamide]-3-methylbutanoic acid (compound 142, 59 mg, 0.19 mmol, 1.00 equiv.), HOBT (26 mg, 0.19 mmol, 1.00 equiv.) and HATU (72 mg, 0.19 mmol, 1.00 equiv.) in DMF (2 mL) was stirred at room temperature in air for 1 h. Then (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]propanamide trifluoroacetic acid (compound 140, 200 mg, 0.19 mmol, 1.00 equiv, 66.70%) and DIEA (197 mg, 1.52 mmol, 8.00 equiv) were added at room temperature. The reaction mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The resulting mixture was purified by reversed-phase flash chromatography using the following conditions: Column: YMC-Actus Triart C18, 30 mm × 150 mm, 5 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 28B to 45B in 10 min, 254 nm; RT1: 9.67 min. The collected fractions were lyophilized to give N-[(1S)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]-phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound (Im), 27.8 mg, 16%) as a white solid. LCMS (ES, m / z): 879,881 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ10.99 (s, 1H), 8.80 (s, 1H), 8.47 (t, J=6.0Hz, 1H), 8.03 (d, J=7.2Hz, 1H), 7.78 (d, J=8.8Hz,1H), 7.70-7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J=8.0Hz, 1H), 7.21-7.14 (m, 2 H), 6.99 (s, 2H), 6.82 (t, J=6.0Hz, 1H), 5.13-5.10 (m,1 H), 4.47-4.40 (m, 3H), 4.33-4.29 (m, 3H), 4.24 (t, J=7.2 Hz, 1H), 4.14 (t, J=6.8Hz, 1H), 3.38-3.36 (m, 1H), 2.97-2.90 (m, 1H), 2.86 (t, J=7.6Hz, 2H), 2.73-2.67 (m, 2H), 2.62-2.57 (m, 1H), 2.40-2.35 (m, 1H), 2.20-2.05 (m, 2H), 2.02-1.96 (m, 1H), 1.95-1.88 (m, 1H), 1.48-1.46 (m, 4H), 1.23-1.16 (m, 6H), 0.83-0.78 (m, 6H)

[0352] Example 4: Preparation and characterization of neodegrader conjugates The antibody solution was treated with 30 equivalents of tris-(2-carboxyethyl)phosphine (TCEP) and incubated for 1 hour at 37° C. to reduce interchain disulfides. The reduced antibody was purified using an illustra NAP column (GE Healthcare) into 50 mM EPPS, 5 mM EDTA pH 7.0 buffer.

[0353] Conjugation was performed by treating a reduced anti-CD33 antibody ("CD33AB") comprising a heavy chain having SEQ ID NO:9 and a light chain having SEQ ID NO:10 at 2-5 mg / mL in 50 mM EPPS, 5 mM EDTA pH 7.0 with 12 equivalents of linker-neodegrader added as a stock solution in N,N-dimethylacetamide (DMA) such that the final concentration of DMA was 15% (v / v). The resulting reaction mixture was allowed to stand overnight at 4°C. The resulting neodegrader conjugate was purified into 20 mM succinic acid, 8% sucrose, 0.01% Tween-20 pH 5.5 using illustra NAP columns (GE Healthcare) and concentrated using Amicon Ultra centrifugal concentrators with a 50 kD molecular weight cutoff (Millipore).

[0354] Concentrations and monomers were determined by size-exclusion chromatography using a 7.8 x 300 mm TSKGel 3000SWXL column (Tosoh Bioscience) with 5 μm particles, run at 0.5 mg / mL for 30 min, and eluted isocratically with a 400 mM sodium perchlorate, 50 mM sodium phosphate, 5% (v / v) isopropanol mobile phase. Neodegrader conjugates were quantified from an antibody standard curve detected at 214 nm.

[0355] Drug-to-antibody ratios (DAR) were determined by hydrophobic interaction chromatography using a 4.6 x 35 mm TSKgel Butyl-NPR column with 2.5 μm particles. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate pH 7.0. Mobile phase B was 25 mM sodium phosphate pH 7.0, 25% (v / v) isopropanol. Analytes were eluted with a linear gradient of 0-100% B in 12 min at a flow rate of 0.6 mL / min. Detection was at 214 nm.

[0356] Free linker-payload was determined by mixed-mode chromatography using a 4.6 x 250 mm HISEP column (Supelco) with 2.5 μm particles. Mobile phase A was 100 mM ammonium acetate. Mobile phase B was 100% acetonitrile. Analytes were eluted with a gradient of 25-40% B for 25 min, then 40-100% B in 2 min at a flow rate of 0.7 mL / min. Column temperature was 35°C. Free linker-payload was quantified using an external standard curve with detection at 254 nm.

[0357] [ka] Scheme 16: Preparation of anti-CD33 antibody-compound (Ie) conjugate

[0358] [ka] Scheme 17: Preparation of anti-CD33 antibody-compound (Ih) conjugate

[0359] Other conjugates can be prepared in an analogous manner using the appropriate antibodies.

[0360] Example 5: Treatment of Acute Myeloid Leukemia (AML) with Anti-CD33 Antibody-Neodegrader Conjugates Anti-CD33 antibody (CD33AB)-neodegrader compound was administered to athymic nude mice (Crl:NU(NCr)-Foxn1 nu , Charles River). 7 MV411 human acute monocytic leukemia cells (ATCC (登録商標) CRL-5991 TM ) was injected subcutaneously into the flank of the mice (0.1 mL / mouse). 3 Once the average size of the tumors had been reached, mice were administered anti-CD33 antibody-neodegrader conjugate, non-targeting neodegrader conjugate, and vehicle control.

[0361] Stock solutions of CD33AB-Compound (Ia), CD33AB-Compound (Ie) and CD33AB-Compound (Ih) were diluted with vehicle to obtain 0.3, 0.283 and 0.299 mg / mL dosing solutions at 3, 2.83 and 2.99 mg / kg, adjusted by each animal's weight, resulting in a dosing volume of 10 mL / kg (0.2 mL / 20 g mouse). This dosing strategy ensured delivery of the same amount of payload in each test group. Mylotarg was diluted to 0.01 mg / mL with 0.9% sodium chloride solution, resulting in a dosing volume of 10 mL / kg (0.2 mL / 20 g mouse) at 3 mg / kg. Venetoclax was formulated in a vehicle consisting of 60% PG, 30% PEG400, 10% ethanol by sonication to obtain a 5 mg / mL dosing suspension, which delivered 50 mg / kg when administered at a volume of 10 mL / kg. CC-90009 was centrifuged to collect the powder at the bottom; then N-methyl-2-pyrrolidinone (NMP), PEG400 and saline were added and mixed well one by one to give a 0.5 mg / mL dosing solution in 5% NMP, 45% PEG400 and 50% saline, which delivered 5 mg / kg when administered in a volume of 10 mL / kg.

[0362] Mice were divided into 7 treatment groups (N=9 / group) as follows: 1) vehicle; 2) CD33AB-compound (Ia) (3 mg / kg, iv, qd x 1); 3) CD33AB-compound (Ie) (2.83 mg / kg, iv, qd x 1); 4) CD33AB-compound (Ih) (2.99 mg / kg, iv, qd x 1); 5) Mylotarg (0.1 mg / kg, iv, qd x 1); 6) Venetoclax (50 mg / kg, po, qd x 21); 7) CC-90009 (5 mg / kg, ip, bid x 10). Test articles in groups 1-5 were administered intravenously (iv) in a single dose (qd x 1) with a volume adjusted for body weight (0.200 mL / 20 g mouse). Venetoclax was administered orally (po) while CC-90009 was administered intraperitoneally (ip) in a dose volume of 10 mL / kg (0.2 mL / 20 g mouse) adjusted for the BW of each animal.

[0363] Tumors were measured twice weekly using calipers and each animal was cultured until the tumor reached the endpoint volume (2,000 mm 3 ) or on the final day of the study (day 45), whichever occurred first. MTV(n) was defined as the median tumor volume on the final day of the study among the remaining number of animals (n) whose tumors had not been reduced to the endpoint volume.

[0364] As shown in FIG. 1, all neodegrader conjugates resulted in slower tumor growth over time compared to vehicle.

[0365] Example 6: Activity of AB1-Compound (Ia) conjugates against human leukemia models In vitro cytotoxicity was measured using a panel of CD33 positive acute myeloid leukemia cell lines and a panel of non-AML CD33 negative cells. Cells were seeded into 96-well plates at predetermined concentrations and after overnight incubation at 37°C / 5CO2, serial dilutions of each test article (TA) were added to the cells. Cells were incubated with the test articles for 72 hours and viability was detected with CellTiter-Glo® reagent (Promega). Luminescence values ​​were normalized for each cell line and IC 50 was calculated using Prizm software.

[0366] Results showed that the huMy9-6 derived antibody, AB1, conjugated to Compound Ia ("AB--Compound (Ia)") exhibited overall in vitro efficacy comparable to that of CC-885 (a known GSPT1 degrader) or Mylotarg in CD33-positive AML cells - and in some cases, superior efficacy (see Figure 2). Additionally, consistent with the hypothesis of targeted CD33-mediated delivery of the GSPT1 degrader payload, AB1-Compound (Ia) was inactive in CD33-negative cell models.

[0367] Example 7: Treatment of Acute Myeloid Leukemia (AML) with Antibody-Neodegrader Conjugates Subcutaneous tumor model MV4-11 human acute myeloid leukemia cells (1 × 10 in 0.1 mL) 6The mice were inoculated subcutaneously into the right flank of female athymic nude mice until the tumor size reached 150 mm. 3 The mice were treated with test articles either by intravenous injection into the lateral tail vein, intraperitoneal injection, oral gavage or a combination thereof starting when the tumor reached 0.01 mm H. Tumor size and mouse body weight were measured twice weekly.

[0368] Consistent with the in vitro observations, in vivo treatment of a CD33-positive AML model tumor (MV4-11) with several AB1-based conjugates that release neodegrader P1 led to tumor regression, with the most robust effects seen for conjugates containing a beta-glucuronide release trigger and native cysteine ​​conjugation (see Figure 3).

[0369] Example 8: Stability of CD33AB-Compound (Ia) and Gemtuzumab-Compound (Ia) Conjugates Stability assays of anti-CD33-compound (Ia) conjugates (approximately 8 DAR) were performed using gemtuzumab, IgG1, CD33AB and IgG1 L234A / L235A "LALA" Ab formats at 2.5 mg / mL in 20 mM succinic acid, 8% sucrose, 0.01% Tween-20 pH 5.5 over 40 days and assayed by size exclusion chromatography (SEC). At 4°C, no significant changes in concentration or monomer were observed for any of the conjugates (Figure 4). In contrast, at 37°C, native gemtuzumab-compound (Ia) and compound (Ia) conjugates using IgG1 and IgG1 LALA backbones showed significant aggregation over 39 days (rising from 15-28% over 20-39 days). However, CD33AB-compound (Ia) (using an IgG1 N297A backbone) maintained a monomeric state of over 88% over 39 days at 37° C. (FIG. 4). Maintenance of high monomericity, such as in CD33AB-compound (Ia) over several days, is generally a desirable property of ADCs, since aggregation in the blood circulation can lead to rapid clearance and increased toxicity, narrowing the therapeutic index. Furthermore, native SEC-MS analysis showed that native gemtuzumab-compound (Ia) showed much higher levels of unconjugated antibody than CD33AB-derived conjugates made with the same molar equivalent of payload-linker. In general, low levels of unconjugated antibody in ADCs, such as in CD33AB-compound (Ia), are a desirable quality attribute. Also, more TCEP was required for reduction of gemtuzumab than CD33AB (4.5 vs. 2.5 molar equivalents). The use of less reducing agent for the production of ADCs, such as in CD33AB-compound (Ia), is desirable to lower the cost of goods and simplify purification methods.

[0370] Example 9: Activity of anti-CD33 neodegrader conjugates against human leukemia models Subcutaneous tumor model MV4-11 human acute myeloid leukemia cells (1 × 10 in 0.1 mL) 6 The mice were inoculated subcutaneously into the right flank of female athymic nude mice. 3The mice were treated with test articles either by intravenous injection into the lateral tail vein, intraperitoneal injection, oral gavage or a combination thereof starting when the tumor size reached 100 μg / kg. Tumor size and mouse weights were measured twice weekly.

[0371] Consistent with the in vitro observations, in vivo treatment of a CD33-positive AML model tumor (MV4-11) with various CD33-based conjugates releasing neodegrader P1 led to tumor regression, with the most stable conjugate (CD33AB-compound (Ia)) showing robust antitumor efficacy (Figure 5).

[0372] Example 10: Activity of anti-CD33 neodegrader conjugates against Mylotarg-insensitive cell lines The cytotoxicity of the test articles (TA) was measured using a panel of CD33-positive acute myeloid leukemia cell lines known to be Mylotarg-insensitive (AML-193 and Kasumi-6). Cells were seeded into 96-well plates at the designated concentrations and after overnight incubation at 37°C / 5CO2, serial dilutions of each test article (TA) were added to the cells. Cells were incubated with the test articles for 72 hours and viability was detected with CellTiter-Glo® reagent (Promega). Luminescence values ​​were normalized for each cell line and IC 50 was calculated using Prizm software.

[0373] As shown in Figures 6A and 6B, the anti-CD33 neodegrader conjugate had good activity against both cell lines.

[0374] It is recognized that the Detailed Description section, and not the Summary and Abstract sections, are intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more, but not all, example aspects of the invention as contemplated by the inventors, and thus are not intended to limit the scope of the invention and the appended claims in any manner.

[0375] The present invention has been described above with the aid of functional components that illustrate the performance of certain functions and their interrelationships. The boundaries of these functional components have been arbitrarily defined herein for convenience of description. Other boundaries may be defined so long as the specified functions and their relationships are appropriately performed.

[0376] The above description of the specific embodiments fully reveals the generality of the invention so that others may easily modify and / or adapt such specific embodiments to various applications by application of knowledge within the skill of the art without undue experimentation without departing from the general concept of the invention. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments based on the teachings and guidance set forth herein. It should be understood that the expressions or terms herein are intended to be illustrative, not limiting, as the terms or terms herein should be interpreted by those skilled in the art in light of the teachings and guidance.

[0377] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. An antibody comprising a heavy chain shown in SEQ ID NO: 9 and a light chain shown in SEQ ID NO:

10.

2. A pharmaceutical composition comprising the antibody of Claim 1 and a pharmaceutically acceptable carrier.

3. Formula (I): 【Chemical 1】 [wherein, a is an integer from 1 to 10; A is phenyl or C ４ -C １０ is a cycloalkyl ring; U is NH or CF ２ and; R １ is independently selected from hydrogen and halo; X is -NR ２ -, =C(CH ３ )-, -Q-(CH ２ ) ｎ -, or -Q(CH ２ ) ｍ Q’(CH ２ ) ｎ -; where Q and Q' are each independently O, S, or N(R ２ ) ｖ and; v is 1 or 2; Each R ２ is independently hydrogen or C １ -C ６ alkyl; n is an integer from 1 to 6; and m is an integer from 2 to 6; here, the left side of each group is linked to L and the right side is linked to A; However, when X is NH or -Q-(CH ２ ) ｎ -, R １ is halo; L is a cleavable linker or a non-cleavable linker; and Bm is an anti-CD33 antibody or an antigen-binding portion thereof comprising a heavy chain variable region (VH) complementarity-determining region (CDR) 1 (VH-CDR1) containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, light chain variable region (VL) CDR1 (VL-CDR1) containing the amino acid sequence shown in SEQ ID NO: 5, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 6, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 7] a conjugate thereof or a pharmaceutically acceptable salt thereof.

4. The conjugate of Claim 3 or a pharmaceutically acceptable salt thereof, wherein the anti-CD33 antibody or an antigen-binding portion thereof comprises a VH containing the amino acid sequence shown in SEQ ID NO: 4 and a VL containing the amino acid sequence shown in SEQ ID NO:

8.

5. The conjugate of Claim 3 or a pharmaceutically acceptable salt thereof, wherein the anti-CD33 antibody comprises a heavy chain shown in SEQ ID NO: 9 and a light chain shown in SEQ ID NO:

10.

6. The conjugate of any one of Claims 3 to 5 or a pharmaceutically acceptable salt thereof, wherein a is an integer from 2 to 8.

7. The conjugate of any one of Claims 3 to 5 or a pharmaceutically acceptable salt thereof, wherein L is a non-cleavable linker.

8. The non-cleavable linker is 【Chemical 2】 [wherein, p is an integer from 1 to 10; [Chemical Formula 3] is the binding point to X; and 【Chemical 4】 is the binding point to the anti-CD33 antibody or an antigen-binding portion thereof] The conjugate of Claim 7 or a pharmaceutically acceptable salt thereof.

9. The conjugate of any one of Claims 3 to 5, wherein L is a cleavable linker.

10. The cleavable linker is (a) a linker cleavable by a protease, (b) an in vivo reducible linker, (c) an acid-cleavable linker, (d) a click-release linker, or (e) a pyrophosphatase-cleavable linker The conjugate of Claim 9 or a pharmaceutically acceptable salt thereof.

11. (a) a cleavable linker cleavable by protease is [Chemical Formula 5] [wherein, q is an integer from 2 to 10; Z １ 、Z ２ 、Z ３ and Z ４ each do not exist independently or are naturally occurring amino acid residues in the L or D configuration, provided that at least two of Z １ 、Z ２ 、Z ３ and Z ４ are amino acid residues; 【Chemical Formula 6】 is the binding point to X ； and 【Chemical Formula 7】 is the binding point to an anti-CD33 antibody or its antigen-binding portion]; is; (b) a bioreducible linker is 【Chemical 8】 [wherein, q is an integer from 2 to 10; R, R', R" and R''' are each independently hydrogen, C １ -C ６ alkoxy C １ -C ６ alkyl, (C １ -C ６ ) ２ NC １ -C ６ alkyl and C １ -C ６ alkyl, or two geminal R groups can combine with the carbon atom to which they are attached to form a cyclobutyl or cyclopropyl ring; 【Chemical Formula 9】 is the binding point to X; and 【Chemical 10】 is the binding point to an anti-CD33 antibody or its antigen-binding portion]; selected from the group consisting of; (c) an acid-cleavable linker is 【Chemical 11】 [wherein, q is an integer from 2 to 10; 【Chemical Formula 12】 is the binding point to X; and 【Chemical Formula 13】 is the binding point to an anti-CD33 antibody or its antigen-binding portion]; selected from the group consisting of; (d) a click-release linker is 【Chemical Formula 14】 [wherein, q is an integer from 2 to 10; 【Chemical Formula 15】 is the binding point to X; and 【Chemical 16】 is the binding point to an anti-CD33 antibody or its antigen-binding portion]; selected from; or (e) a pyrophosphatase-cleavable linker is 【Chemical 17】 [wherein, q is an integer from 2 to 10; 【Chemical Formula 18】 is the binding point to X; and 【Chemical Formula 19】 is the binding point to an anti-CD33 antibody or its antigen-binding portion]; is the conjugate of claim 10 or a pharmaceutically acceptable salt thereof.

12. The conjugate of claim 9 or a pharmaceutically acceptable salt thereof, wherein the cleavable linker is a beta-glucuronidase-cleavable linker.

13. The beta-glucuronidase-cleavable linker is 【Chemical 20】 [wherein, q is an integer from 2 to 10; ---- does not exist or is a bond; 【Chemical 21】 is the binding point to X; and 【Chemical 22】 is the binding point to an anti-CD33 antibody or its antigen-binding portion]; is the conjugate of claim 12 or a pharmaceutically acceptable salt thereof.

14. (i) A is phenyl; U is NH; R １ is halo; and X is -N(R ２ ) ｖ (CH ２ ) ｍ O(CH ２ ) ｎ -; where v is 1; m and n are 2; and R ２ is methyl, (ii) A is phenyl; U is NH; R １ is halo; and X is -N(R ２ ) ｖ (CH ２ ) ｍ O(CH ２ ) ｎ -, where v is 2; m and n are 2; and each R ２ is methyl, (iii) A is phenyl; U is NH; R １ is halo; and X is -O(CH ２ ) ｎ -; where n is 2, (iv) A is phenyl; U is NH; R １ is halo; and X is -S(CH ２ ) ｎ -; where n is 2, (v) A is phenyl; U is NH; R １ is hydrogen; and X is -NR ２ -; where R ２ is methyl, (vi) A is phenyl; U is NH; R １ is halo; and X is -NR ２ -; where R ２ is hydrogen, (vii) A is phenyl; U is NH; R １ is hydrogen; and X is -C(CH ３ )=, or (viii) AA is C ４ - C １０ is a cycloalkyl ring; U is NH; R １ is hydrogen; and X is -N(R ２ )(CH ２ ) ｍ O(CH ２ ) ｎ - where n is 1; m is 2; and R ２ is methyl, The conjugate of any one of claims 3 to 5 or a pharmaceutically acceptable salt thereof.

15. The conjugate of formula (V): 【Chemical 23】 The conjugate of formula (VI): or the conjugate of formula (VII): 【Chemical 24】 [In each formula, Bm is an anti-CD33 antibody or its antigen-binding portion comprising VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 5, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 6, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 7]] is the conjugate or a pharmaceutically acceptable salt thereof. 【Chemical 25】

16. ​ ​ ​ The conjugate of claim 15 or a pharmaceutically acceptable salt thereof, wherein the anti-CD33 antibody or antigen-binding portion thereof comprises (i) a VH comprising the amino acid sequence shown in SEQ ID NO: 4 and a VL comprising the amino acid sequence shown in SEQ ID NO: 8, or (ii) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

10.

17. A pharmaceutical composition comprising a conjugate of any one of claims 3 to 5 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

18. A pharmaceutical composition for treating cancer or myelodysplastic syndrome, comprising a conjugate of any one of claims 3 to 5 or a pharmaceutically acceptable salt thereof.

19. The pharmaceutical composition of claim 18, wherein the cancer is multiple myeloma, leukemia, malignant lymphoma, Hodgkin's disease or chronic myeloproliferative disorder.

20. A method for producing a conjugate of any one of claims 3 to 5 or a pharmaceutically acceptable salt thereof, comprising reacting an anti-CD33 antibody or antigen-binding portion thereof with a compound of formula (I-1): 【Chemical 26】 [Wherein, a is an integer from 1 to 10; A is phenyl or C ４ -C １０ is a cycloalkyl ring; R １ is independently hydrogen or halo; U is NH or CF ２ and; X is -N(R ２ ) ｖ -, =C(CH ３ )-, -Q-(CH ２ ) ｎ -, or -Q(CH ２ ) ｍ Q’(CH ２ ) ｎ -; where v is 1 or 2; Q and Q' are each independently O, S or NR ２ ; Each R ２ is independently hydrogen or C １ -C ６ alkyl; n is an integer from 1 to 6; and m is an integer from 2 to 6; wherein the left side of each group is bonded to L' and the right side is bonded to A; However, when X is NH or -Q-(CH ２ ) ｎ -, R １ is halo; L' is a cleavable or non-cleavable linker precursor conjugated to the anti-CD33 antibody or antigen-binding portion thereof, wherein the anti-CD33 antibody or antigen-binding portion thereof comprises VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, light chain variable region (VL) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, and VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 7] or a pharmaceutically acceptable salt thereof.

Images