Antibody-conjugated chemical degradants and methods thereof

Abstract

The subject matter described herein is directed to molecules referred to herein as chemical degradative inducers (CIDEs) and antibody-conjugated CIDEs (Ab-CIDEs), where the Ab-CIDEs comprise an antibody covalently attached to a CIDE through a linker, which CIDEs can be further covalently attached to a phosphate moiety, and the use of such molecules in the treatment of diseases and conditions in which targeted protein degradation is beneficial.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 303,446, filed January 26, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The subject matter described herein relates generally to degrader conjugates comprising antibody-proteolytic targeting chimeric molecules useful for promoting intracellular degradation of target proteins. [Background technology]

[0003] Cellular maintenance and normal function require the controlled degradation of cellular proteins. For example, the degradation of regulatory proteins triggers events in the cell cycle such as DNA replication, chromosome segregation, etc. Thus, the degradation of such proteins has effects on cell proliferation, differentiation, and death.

[0004] Protein inhibitors can block or reduce protein activity in cells, and protein degradation in cells can also reduce activity or completely remove target proteins. Thus, harnessing cellular protein degradation pathways can provide a means to reduce or remove protein activity. One of the major degradation pathways in cells is known as the ubiquitin-proteasome system. In this system, proteins are marked for degradation by the proteasome by ubiquitinating them. Protein ubiquitination is achieved by E3 ubiquitin ligases, which bind to proteins and add ubiquitin molecules to the proteins. E3 ubiquitin ligases are part of a pathway that includes E1 and E2 ubiquitin ligases, which make ubiquitin available to the E3 ubiquitin ligase to add to the protein.

[0005] To exploit this degradation pathway, molecular constructs known as chemical degraders (CIDEs) bring E3 ubiquitin ligases together with proteins targeted for degradation. To promote proteins for degradation by the proteasome, CIDEs are composed of a group that binds to the E3 ubiquitin ligase and a group that binds to the protein target for degradation. These groups are usually connected with a linker. The CIDEs can bring the E3 ubiquitin ligase into close proximity with the protein so that it is ubiquitinated and marked for degradation. However, the relatively large size of CIDEs can be problematic for targeted delivery as well as contribute to undesirable properties such as rapid metabolism / clearance, short half-life, and low bioavailability.

[0006] There is a continuing need in the art for improvements to CIDE, including enhancing the targeted delivery of CIDE to cells containing protein targets. The subject matter described herein addresses these and other deficiencies in the art. Summary of the Invention

[0007] The present disclosure is directed to CIDE conjugated or covalently linked to an antibody by a linker. In some embodiments, CIDE is phosphorylated. In some embodiments, the linker that covalently binds the antibody to CIDE is a peptidomimetic linker. In another aspect, the phosphate moiety acts as a prodrug of the conjugate.

[0008] In some embodiments, the CIDE conjugated or covalently linked to the antibody by a peptidomimetic linker has the chemical structure: Ab-(L1-D) j where Ab is an antibody; L1 is a linker; D is CIDE; and j is an integer between 1 and 16. In such aspects, at least one phosphate moiety is covalently attached to D. In some embodiments, L1 is a peptidomimetic linker.

[0009] In some embodiments, D is a CIDE having the structure: E3LB-L2-PB, where E3LB is an E3 ligase binding ligand covalently attached to L2; L2 is a linker covalently attached to E3LB and PB; and PB is a protein binding group covalently attached to L2. In one aspect of these embodiments, a phosphate moiety is covalently attached to D at the E3LB and / or PB of D. In one aspect of these embodiments, a single phosphate moiety is covalently attached to D at the E3LB or PB of D.

[0010] In some embodiments, the E3 ligase is the von Hippel-Lindau (VHL) tumor suppressor protein.

[0011] In some embodiments, the phosphate moiety is covalently attached to E3LB of D.

[0012] In some embodiments, the phosphate moiety is covalently attached to PB of D.

[0013] In some embodiments, L1 is covalently linked to the PB of D and the phosphate moiety is covalently linked to the E3LB of D.

[0014] In some embodiments, L1 is covalently linked to the PB of D and the phosphate moiety is covalently linked to the PB of D.

[0015] In some embodiments, E3LB comprises a hydroxyproline residue and the phosphate moiety is covalently attached to the hydroxyproline residue.

[0016] In some embodiments, PB comprises a hydroxyphenyl moiety and the phosphate moiety is covalently attached to the hydroxyphenyl moiety.

[0017] In some embodiments, the PB is a BRM protein.

[0018] In some embodiments, the subject matter described herein is directed to a pharmaceutical composition comprising the Ab-L1-CIDE described herein and one or more pharma- ceutically acceptable excipients.

[0019] In some embodiments, the subject matter described herein is directed to the use of Ab-L1-CIDE described herein in methods for treating conditions and diseases by administering a pharmaceutical composition comprising Ab-L1-CIDE to a subject.

[0020] In some embodiments, the subject matter described herein is a method of making Ab-L1-CIDE.

[0021] In some embodiments, the subject matter described herein is directed to an article of manufacture comprising a pharmaceutical composition comprising Ab-L1-CIDE, a container, and a package insert or label indicating that the pharmaceutical composition can be used to treat a disease or condition.

[0022] Further embodiments are also described in detail herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Disclosed herein are antibody-conjugated chemical degraders ("antibody-conjugated CIDEs," "Ab-L1-CIDEs," or "Ab-CIDEs") that include a linker, e.g., a peptidomimetic linker, covalently attached to the CIDE and the antibody, and are useful for targeted protein degradation. In particular, the disclosure is directed to antibody-conjugated CIDEs that include a ligand that binds to von Hippel-Lindau E3 ubiquitin ligase and a moiety that binds to a target protein (e.g., BRM), such that the target protein is placed in proximity to the ubiquitin ligase to trigger degradation, thereby regulating the amount of the target protein. In some embodiments, a phosphate moiety is covalently attached to the CIDE.

[0024] The subject matter described herein utilizes antibody targeting to direct CIDE to target cells or tissues. As described herein, it has been shown that CIDE is delivered to target cells or tissues by connecting an antibody to CIDE to form Ab-CIDE. As shown herein, for example in the Examples, cells expressing an antigen can be targeted by antigen-specific Ab-CIDE, whereby the CIDE portion of Ab-CIDE is delivered intracellularly to the target cell. Furthermore, CIDEs that include antibodies directed to antigens not found on cells do not result in significant intracellular delivery of CIDE to cells.

[0025] The disclosed Ab-CIDE comprises an antibody covalently linked to a linker 1 (L1), e.g., a peptidomimetic linker, covalently linked at any point available for binding to the CIDE, the CIDE comprising an E3 ubiquitin ligase binding (E3LB) moiety, which recognizes the E3 ubiquitin ligase protein, VHL, and a linker 2 (L2) covalently connects the E3LB moiety to a protein binding moiety (PB), which is a moiety that recognizes a target protein. In some embodiments, a phosphate moiety is covalently attached to the CIDE. Thus, the subject matter described herein is useful for degrading proteins, thereby regulating protein activity and treating diseases and conditions associated with protein activity. In embodiments, phosphorylated Ab-CIDE unexpectedly enhances target degradation in vivo.

[0026] The subject matter disclosed herein is described in more detail below. However, many modifications and other embodiments of the subject matter disclosed herein will occur to those skilled in the art with the benefit of the teachings set forth in the foregoing description, which relate to the subject matter disclosed herein. It is therefore to be understood that the subject matter disclosed herein is not limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the claims. In other words, the subject matter described herein covers all variations, modifications, and equivalents. In the event that one or more of the references, patents, and similar materials differ or conflict with this application, including, but not limited to, the definitions, usage, or techniques described herein, this application shall control. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0027] I. Definition The term "CIDE" generally refers to a chemical degradant that is a proteolytic targeting chimeric molecule having three components, an E3 ubiquitin ligase binding group (E3LB), a linker L2, and a protein binding group (PB). In some embodiments, CIDE is phosphorylated.

[0028] The term "phosphorylated" as used herein refers to a CIDE that is covalently attached to a phosphate moiety to form a phosphorylated CIDE.

[0029] The term "phosphate moiety" as used herein refers to a chemical group that includes a single phosphate and its salts. Examples of phosphate moieties include -(P=O)(OH)2, -CHO(P=O)(OH)2, -(P=O)(OH)O(P=O)(OH)2, and -CH-O(P=O)(OH)O(P=O)(OH)2, and salts thereof.

[0030] The terms "residue," "moiety," "moiety," or "group" refer to a moiety that is covalently bound or linked to another moiety. The term "moiety" is also used herein to describe such residues, moieties, moieties, or groups. As an example, a residue of a compound has one or more atoms of the compound, such as hydrogen or hydroxy, replaced with a covalent bond, thereby linking the residue to another moiety of CIDE, L1-CIDE, or Ab-L1-CIDE. For example, a "residue of CIDE" refers to CIDE that is covalently linked to one or more groups, such as a linker L2, which may itself be further linked to an antibody.

[0031] The terms "covalent bond" or "covalently linked" refer to a chemical bond formed by the sharing of one or more pairs of electrons.

[0032] The term "protein binding group" or "PB" refers to a residue of a small molecule or other compound that can bind to a target protein or other polypeptide target of interest. In the conjugate molecules described herein, the PB binds to the target, thereby placing the target in proximity to a ubiquitin ligase so that degradation of the protein or polypeptide by the ubiquitin ligase can occur. Thus, the conjugates described herein can include any PB as long as it is covalently attached to L2 and interacts with or binds to a target of interest. Non-limiting examples of small molecule target protein binding moieties include compounds that bind to BRM (BRAHMA), BRG1, AKT, HPK1, IRE1, Tau and androgen receptor (AR), Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting human BET bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors such as KDM5, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting the aryl hydrocarbon receptor (AHR), among many others. In some embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of PB. In some embodiments, the CIDEs and conjugated CIDEs described herein are not limited to the type of PB, the PB is covalently linked to L2; the CIDE or conjugated CIDE comprises a phosphate moiety and an L1 peptidomimetic linker.

[0033] The term "comprising a hydroxyphenyl moiety" and the like refers to a PB compound that includes as part of its structure a hydroxyl group covalently bonded to a phenyl group, or a residue of such a moiety. In some embodiments, a compound that includes a hydroxyphenyl (phenol) group or a residue thereof is attached to a BRM.

[0034] The term "E3 ligase binding (E3LB) ligand" refers to a molecule capable of binding to von Hippel-Lindau (VHL) E3 ubiquitin ligase. The terms "VCB E3 ubiquitin ligase", "von Hippel-Lindau (or VHL) E3 ubiquitin ligase", "VHL" and "ubiquitin ligase" all refer to a target enzyme binding site for the E3LB portion of a conjugate generally described herein. VCB E3 is a protein that, in combination with an E2 ubiquitin conjugating enzyme, causes the attachment of ubiquitin to a lysine on a target protein; E3 ubiquitin ligase targets specific protein substrates for degradation by the proteasome. Thus, E3 ubiquitin ligase, alone or in complex with an E2 ubiquitin conjugating enzyme, is responsible for the transfer of ubiquitin to a target protein. In some embodiments, the CIDEs and conjugated CIDEs described herein are not limited to the type of E3LB. In some embodiments, the CIDE and conjugated CIDE described herein are not limited to the type of E3LB, and the E3LB is covalently linked to L2; the CIDE or conjugated CIDE contains a phosphate moiety and is further covalently linked to a peptidomimetic L1 linker.

[0035] The term "comprising a hydroxyproline residue" and similar expressions refer to a compound having the structure: It refers to an E3LB compound containing a group or a residue of a group having TIFF2025508328000001.tif39170. Such compounds that are E3 ligase binding (E3LB) ligands are known in the art.

[0036] The terms "linker," "linker unit," or "linkage" as used herein refer to a chemical moiety comprising a chain of one or more atoms that covalently attaches a CIDE moiety to an antibody or a residue, moiety, portion, group, or component of CIDE to another residue, moiety, portion, group, or component of CIDE. In various embodiments, the linker is a divalent group identified as Linker1, Linker2, L1, or L2.

[0037] The term "peptidomimetic" or PM as used herein refers to a non-peptide chemical moiety. A peptide is a short chain of amino acid monomers linked by peptide (amide) bonds, which are covalent chemical bonds formed when the carboxyl group of an amino acid reacts with the amino group of another amino acid. The shortest peptide is a dipeptide, which consists of two amino acids linked by a single peptide bond, followed by tripeptides, tetrapeptides, etc. A peptidomimetic chemical moiety includes a non-amino acid chemical moiety. A peptidomimetic chemical moiety may also include one or more amino acids separated by one or more non-amino acid chemical units. A peptidomimetic chemical moiety may not include two or more adjacent amino acids linked by peptide bonds in any part of its chemical structure. A "peptidomimetic linker" is a portion of a molecule that is attached to a CIDE and an antibody. Useful peptidomimetic linkers are known in the art and others are disclosed herein.

[0038] The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen usually has multiple binding sites, also called epitopes, recognized by CDRs (complementarity determining regions), on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes 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 of a target of interest or a portion thereof, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may 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 may be obtained from any species. However, in one embodiment, the immunoglobulins are of human, murine, or rabbit origin.

[0039] The term "antibody fragment" as used herein includes a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al (2004) Protein Eng. Design & Sel. 17(4):315323), fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0040] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Moreover, in contrast to polyclonal antibody preparations that contain 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 the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used by the subject matter described herein may be produced by the hybridoma method first described by Kohler et al (1975) Nature, 256:495, or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. Nos. 4,816,567; 5,807,715). Monoclonal antibodies may be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597.

[0041] Monoclonal antibodies herein include, inter alia, "chimeric" antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies obtained from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains are identical or homologous to corresponding sequences in antibodies obtained from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies which contain variable domain antigen-binding sequences obtained from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0042] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species and the remainder of the heavy and / or light chain is derived from a different source or species.

[0043] The "class" of an antibody refers to the type of constant domain or region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0044] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to a specific binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can usually be expressed by a dissociation constant (kd). Affinity can be measured by common methods known in the art, including those described herein. Certain exemplary embodiments for measuring binding affinity are described below. In some embodiments, the antibodies described herein have a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦5 nm, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 It has a dissociation constant (Kd) of 1 M.

[0045] The term "free cysteine ​​amino acid" as used herein refers to a cysteine ​​amino acid residue that has been engineered into a parent antibody, has a thiol functional group (-SH) and is not paired as an intramolecular or intermolecular disulfide bridge. The term "amino acid" as used herein means glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine or citrulline.

[0046] A "patient" or "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the patient, individual, or subject is human. In some embodiments, the patient may be a "cancer patient," i.e., one who is suffering from or at risk for one or more symptoms of cancer.

[0047] "Patient population" refers to a group of cancer patients. Populations such as these can be used to demonstrate statistically significant efficacy and / or safety of a drug.

[0048] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. A "tumor" contains one or more cancerous cells. Examples of cancer are provided elsewhere herein.

[0049] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the condition, and remission or improving prognosis. In some embodiments, the subject antibodies described herein are used to delay the onset of disease or to slow the progression of the disease.

[0050] An "effective amount" of a drug, e.g., a pharmaceutical formulation, refers to an amount effective at the dosage and for the period of time necessary to achieve a desired therapeutic or prophylactic result. For example, an effective amount of a drug for treating cancer can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that the drug can prevent the growth of existing cancer cells and / or kill existing cancer cells, the drug can be cytostatic and / or cytotoxic. An effective amount can extend progression-free survival (e.g., as measured by change in Response Evaluation Criteria in Solid Tumors (RECIST) or CA-125), produce an objective response (including partial response (PR) or complete response (CR)), extend overall survival, and / or improve one or more symptoms of the cancer (e.g., as assessed by FOSI).

[0051] The term "therapeutically effective amount" as used herein means any amount that results in the treatment of a disease, disorder, or side effect, or a slowing of the progression of a disease or disorder, compared to a corresponding subject not administered such amount. The term also includes within its scope an amount effective to enhance normal physiological function. For use in therapy, a therapeutically effective amount of Ab-CIDE and its salts can be administered as a raw chemical. In addition, the active ingredient can be presented as a pharmaceutical composition.

[0052] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredients contained therein is in a form such that it is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0053] "Pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, carriers, stabilizers, or preservatives.

[0054] The phrase "pharmaceutical acceptable salt" as used herein refers to an organic or inorganic salt of a molecule that is pharmaceutical acceptable. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, acid tartrate, ascorbate, succinate, maleate, gentianate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salt. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counter ion. A counter ion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.

[0055] Other salts, which are not pharma-ceutically acceptable, may be useful in the preparation of the compounds described herein and should be considered to form a further aspect of the subject matter. These salts, such as oxalic acid or trifluoroacetate, are not themselves pharma-ceutically acceptable but may be useful in the preparation of salts that are useful as intermediates in obtaining the compounds described herein and their pharma-ceutically acceptable salts.

[0056] The term "alkyl" as used herein refers to a saturated, straight or branched chain monovalent hydrocarbon radical of any one to six carbon atoms (C1-C6) in length, which may be independently substituted with one or more substituents as described below. In another embodiment, the alkyl radical is 1, 2, 3, 4, or 5 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl ( Examples include t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), and 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3).

[0057] "Aryl" refers to an aromatic carbocyclic group having a single ring (e.g., monocyclic) or multiple rings (e.g., bicyclic or tricyclic), including fused systems. As used herein, aryl refers to an aromatic carbocyclic group having 6 to 20 ring carbon atoms (i.e., C6-C 20 aryl), 6 to 12 carbon ring atoms (i.e., C6-C 12 aryl), or 6 to 10 carbon ring atoms (i.e., C6-C 10 Examples of the aryl group include, for example, phenyl, naphthyl, fluorenyl, and anthryl.

[0058] "Heteroaryl" refers to an aromatic group having a single ring, multiple rings, or multiple fused rings that contains one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl refers to an aromatic group having from 1 to 20 ring carbon atoms (i.e., C1-C 20 Heteroaryl), 3 to 12 ring carbon atoms (i.e., C3-C 12Heteroaryl), or 3 to 8 carbon ring atoms (i.e., C3-C8 heteroaryl), and 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur. In some cases, heteroaryl includes 9-10 membered ring systems, 6-10 membered ring systems, 5-10 membered ring systems, 5-7 membered ring systems, or 5-6 membered ring systems, each independently having 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include, for example, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzofuranyl, benzothiazolyl, benzothiadiazolyl, benzonaphthofuranyl, benzoxazolyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindoline, and the like. Examples of aryl include aryl, isoquinolyl, isoxazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, phenazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl and triazinyl. Examples of fused heteroaryl rings include, but are not limited to, benzo[d]thiazolyl, quinolinyl, isoquinolinyl, benzo[b]thiophenyl, indazolyl, benzo[d]imidazolyl, pyrazolo[1,5-a]pyridinyl, and imidazo[1,5-a]pyridinyl, where the heteroaryl may be attached via any ring of the fused system. Any aromatic ring having single or multiple fused rings and containing at least one heteroatom is considered heteroaryl, regardless of attachment to the rest of the molecule (i.e., through any one of the fused rings).Heteroaryl does not encompass or overlap with aryl, as defined above.

[0059] As used herein, the term "alkylene" refers to any alkylene group having a length of 1 to 12 carbon atoms (C-C 12 (-CH2CH2-), where the alkylene radical is optionally independently substituted with one or more substituents described below. In another embodiment, the alkylene radical is from 1 to 8 carbon atoms (C1-C8), or from 1 to 6 carbon atoms (C1-C6). Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), and propylene (-CH2CH2CH2-).

[0060] The terms "carbocycle", "carbocyclyl", "carbocyclic ring" and "cycloalkyl" refer to a monovalent non-aromatic saturated or partially unsaturated ring having from three to five carbon atoms (C3-C5) as a monocyclic ring. Examples of monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl. Carbocyclyl groups may be independently substituted with one or more alkyl groups.

[0061] "Heterocycle", "heterocyclic", "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially unsaturated group having a single ring or multiple condensed rings, including fused, bridged, or spiro ring systems, and having from 3 to 20 ring atoms, including from 1 to 10 heteroatoms. The ring atoms are selected from the group consisting of carbon, nitrogen, sulfur, or oxygen, and in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In some embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group can be oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. Examples of heterocycles include, but are not limited to, azetidine, dihydroindole, indazole, quinolizine, imidazolidine, imidazoline, piperidine, piperazine, indoline, 1,2,3,4-tetrahydroisoquinoline, thiazolidine, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, and tetrahydrofuranyl, etc. Heterocyclyl groups can be substituted as described in WO 2014 / 100762.

[0062] The term "chiral" refers to a molecule that has the property of not being superimposable on its mirror image partner, whereas the term "achiral" refers to a molecule that can be superimposed on its mirror image partner. The term "stereoisomers" refers to compounds that have identical chemical structure but differ with respect to the arrangement of atoms or groups in space. "Diastereomers" refers to stereoisomers that have two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectroscopic properties, and reactivities. Mixtures of diastereomers can be separated under high-resolution analytical procedures such as electrophoresis and chromatography. "Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another. Stereochemical definitions and conventions used herein generally follow S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they can rotate the plane of plane polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about one or more of its chiral centers. The prefixes d and l, or (+) and (-), are used to denote the sign of rotation of plane polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. Compounds with the prefix (+) or D are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0063] Additional definitions and abbreviations may be set out elsewhere herein.

[0064] II. Chemical decomposition inducers The present disclosure is directed to a CIDE conjugated or covalently linked to an antibody by a linker. The present disclosure is directed to a CIDE conjugated or covalently linked to an antibody by a peptidomimetic linker. In some embodiments, the CIDE is phosphorylated. In another aspect, the phosphate moiety acts as a prodrug of the conjugate.

[0065] In embodiments, CIDE has the structure: E3LB-L2-PB, where E3LB is an E3 ligase binding ligand covalently attached to L2; L2 is a linker covalently attached to E3LB and PB; and PB is a protein binding group covalently attached to L2. In some embodiments, the phosphate moiety is covalently attached to E3LB and / or PB of CIDE, D.

[0066] In some embodiments, the E3 ligase is the von Hippel-Lindau (VHL) tumor suppressor protein.

[0067] In some embodiments, the phosphate moiety is covalently attached to E3LB of D.

[0068] In some embodiments, the phosphate moiety is covalently attached to PB of D.

[0069] In some embodiments, L1 is covalently linked to the PB of D and the phosphate moiety is covalently linked to the E3LB of D.

[0070] In some embodiments, L1 is covalently linked to the PB of D and the phosphate moiety is covalently linked to the PB of D.

[0071] In some embodiments, E3LB comprises a hydroxyproline residue and the phosphate moiety is covalently attached to the hydroxyproline residue.

[0072] In some embodiments, PB comprises a hydroxyphenyl moiety and the phosphate moiety is covalently attached to the hydroxyphenyl moiety.

[0073] In some embodiments, the PB is a BRM protein.

[0074] In some embodiments, the CIDE conjugated or covalently linked to the antibody by a peptidomimetic linker L1 has the formula: Ab-(L1-D) j where Ab is an antibody; L1 is a linker; D is CIDE; and j is an integer between 1 and 16. In such embodiments, at least one phosphate moiety is covalently attached to D.

[0075] The following sections describe in more detail the above components that comprise the Ab-CIDE. To obtain an Ab-CIDE having one or more properties, including potent efficacy, desirable therapeutic index, the following components are provided:

[0076] 1. Antibody (Ab) As described herein, antibodies, e.g., monoclonal antibodies (mAbs), are used to deliver CIDE to target cells, e.g., cells expressing a specific protein targeted by the antibody. The antibody portion of the Ab-CIDE can target cells expressing the antigen, whereby the antigen-specific Ab-CIDE is delivered intracellularly to the target cell, typically through endocytosis. In some cases, normal cellular uptake of Ab-CIDE can occur inside antigen-expressing or non-expressing cells by pinocytosis or smaller non-specific routes of uptake. The Ab-CIDEs and methods of use described herein advantageously utilize antibody recognition of cell surfaces and / or endocytosis of Ab-CIDE to deliver the CIDE portion into cells. Antibodies are described in WO2020 / 086858, which is incorporated herein by reference in its entirety.

[0077] In certain embodiments, antibodies may be mutated to reduce effector function. Examples of mutations that modulate Fc effector function include the LALAPG and NG2LH mutations.

[0078] In certain embodiments, the antibody is THIOMAB, previously described in WO 2016 / 04856. TM Additionally, combinations are contemplated, where the antibody targeting is THIOMAB with or without any Fc effector modulation, including LALAPG or NG2LH mutations. TM Any suitable combination of mutations may be combined.

[0079] Human antibodies are, for example, human antibodies as reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). The antibody may be a library-derived antibody. Various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding properties. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0080] The antibody may be a chimeric or humanized antibody. Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "reshuffling"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0081] The antibody may be a multispecific antibody, e.g., a bispecific antibody. The term "multispecific antibody" as used herein encompasses antibodies that contain an antigen-binding domain that has multiple epitopic specificity (i.e., can bind to two or more different epitopes on one biological molecule or can bind to epitopes on two or more different molecules). The term "bispecific antibody" as used herein refers to a multispecific antibody that contains an antigen-binding domain that can bind to two different epitopes on one molecule or can bind to epitopes on two different molecules. Bispecific antibodies are also referred to herein as having "dual specificity" or being "bispecific". Exemplary bispecific antibodies can bind to both proteins and any other antigens. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983); WO 93 / 08829; and Traunecker et al., EMBO J. 10:3655 (1991)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168; WO 2009 / 089004; U.S. Patent Application Publication Nos. 2009 / 0182127; 2011 / 0287009; Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658; and Kontermann (2005) Acta Pharmacol. Sin., 26:1-9).Multispecific antibodies can be produced by a number of techniques, including the engineering of electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); the use of "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 1997, 111:111-112). al., J. Immunol., 152:5368 (1994); and by preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991). Engineered antibodies or "dual variable domain immunoglobulins" (DVDs) with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication No. 2006 / 0025576, and Wu et al. Nature Biotechnology (2007)). The antibodies or fragments herein also include "dual acting FAbs" or "DAFs" that contain antigen binding sites that bind to the target protein as well as another distinct antigen (see, for example, U.S. Patent Application Publication No. 2008 / 0069820).

[0082] The antibody may be an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of some antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458. See U.S. Patent No. 5,869,046 for a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have extended in vivo half-life. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). Single domain antibodies are antibody fragments that contain all or a portion of an antibody heavy chain variable domain or all or a portion of an antibody light chain variable domain. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells.

[0083] The antibody may be an antibody variant. In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, e.g., antigen binding.

[0084] Antibodies can be produced using recombinant methods and compositions, for example as described in U.S. Patent No. 4,816,567. With respect to antibody affinity herein, in embodiments, the antibody binds to one or more tumor-associated antigens or cell surface receptors:

[0085] In some embodiments, the tumor-associated antigen or cell surface receptor is selected from CD71, Trop2, MSLN, NaPi2b, Ly6E, EpCAM, STEAP1, HER2, CD33, and CD22. Thus, in some embodiments, the Ab-CIDE may comprise an antibody selected from an anti-anti-Ly6E antibody, an anti-NaPi2b antibody, an anti-CD22 antibody, an anti-CD71 antibody, an anti-Trop2 antibody, an anti-MSLN antibody, an anti-EpCAM antibody, an anti-Steap1 antibody, an anti-CD33 antibody, an anti-CLL1 antibody, an anti-CD123 antibody, and an anti-HER2 antibody.

[0086] Specific antibodies include, but are not limited to, the following:

[0087] i. Anti-Ly6E antibody In certain embodiments, Ab-CIDE can comprise an anti-Ly6E antibody: Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen, A Get al (2003) Int. J. Cancer 103(6), 768-774; Zammit, D J et al (2002) Mol. Cell. Biol. 22(3):946-952; WO 2013 / 17705.

[0088] Ly6E is a GPI-linked, 131 amino acid long, ~8.4 kDa protein of unknown function and no known binding partners. Ly6E was first identified as a transcript expressed in immature mouse thymocytes, medullary thymic epithelial cells (Mao, et al. (1996) Proc. Natl. Acad. Sci. USA 93:5910-5914). In some embodiments, the subject matter described herein provides an Ab-CIDE comprising the anti-Ly6E antibody described in WO 2013 / 177055.

[0089] ii. Anti-NaPi2b antibody In some embodiments, the Ab-CIDE comprises the following anti-NaPi2b antibodies: Napi2b (Napi3b, NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424) J. Biol. Chem. 277(22):19665-19672(2002), Genomics 62(2):281-284(1999), Feild, JA, et al (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO 2004022778 (claim 2); EP 1394274 (example 11); WO 2002102235 (claim 13; page 326); EP 875569 (claim 1; pages 17-19); WO 200157188 ​​(claim 20; page 329); WO 2004032842 (example IV); WO 200175177 (claim 24; pages 139-140); cross references: MIM:604217; NP_006415.1; NM_006424_1.

[0090] iii. Anti-CD22 antibody In some embodiments, Ab-CIDE can include the following anti-CD22 antibodies: CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilson et al (1991) J. Exp. Med. 173:137-146; WO 2003072036 (Claim 1; Figure 1); Cross References: MIM:107266; ​​NP_001762.1; NM_001771_1.

[0091] iv. Anti-CD71 antibody In some embodiments, Ab-CIDE can include anti-CD71 antibody. CD71 (transferrin receptor) is an integral membrane glycoprotein that plays an important role in cellular uptake of iron. It is well known as a marker of cell proliferation and activation. All proliferating cells in the hematopoietic system express CD71, but CD71 has been considered a useful erythrocyte-associated antigen. In one embodiment, anti-CD71 antibody is described in WO2016081643, which is incorporated by reference in its entirety.

[0092] v. Anti-Trop2 antibody In some embodiments, Ab-CIDE can include an anti-Trop2 antibody. Trop2 (trophoblast antigen 2) is a transmembrane glycoprotein that is an intracellular calcium signaler that is differentially expressed in many cancers. Trop2 signals cells to self-renew, grow, invade, and survive. Trop2 is also known as cell surface glycoprotein Trop-2 / Trop2, gastrointestinal tumor associated antigen GA7331, pancreatic cancer marker protein GA733-1 / GA733, membrane component chromosome 1 surface marker 1 (M1S1), epithelial glycoprotein-1, EGP-1, CAA1, gelatinous droplet-like corneal dystrophy GDLD, and TTD2. In any of the above embodiments, the anti-Trop2 antibody of Ab-CIDE is humanized. In one embodiment, the anti-Trop2 antibody is described in U.S. Patent Application Publication Nos. 2014 / 0377287 and 2015 / 0366988, each of which is incorporated by reference in its entirety.

[0093] vi. Anti-MSLN antibody In some embodiments, Ab-CIDE can include an anti-MSLN antibody. MSLN (mesothelin) is a glycosylphosphatidylinositol-anchored cell surface protein that can function as a cell adhesion protein. MSLN is also known as CAK1 and MPF. This protein is overexpressed in epithelial mesothelioma, ovarian cancer, and certain squamous cell carcinomas. In any of the above embodiments, the anti-MSLN antibody of Ab-CIDE is humanized. In one embodiment, the anti-MSLN antibody is h7D9.v3, described in Scales, SJ et al., Mol. Cancer Ther. 2014, 13(11), 2630-2640, which is incorporated by reference in its entirety.

[0094] vii. Anti-EpCAM antibody In some embodiments, Ab-CIDE can include an anti-EpCAM antibody. In one aspect, the antibody of Ab-CIDE can be an antibody against a protein found on multiple cell or tissue types. Examples of such antibodies include EpCAM. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that mediates Ca2+-independent homotypic cell-cell adhesion in epithelia (Litvinov, S. et al. (1994) Journal of Cell Biology 125(2):437-46). EpCAM, also known as DIAR5, EGP-2, EGP314, EGP40, ESA, HNPCC8, KS1 / 4, KSA, M4S1, MIC18, MK-1, TACSTD1, TROP1, is also involved in cell signaling (Maetzel, D. et al. (2009) Nature Cell Biology 11(2):162-71), migration (Osta, WA; et al. (2004) Cancer Res. 64(16):5818-24), proliferation, and differentiation (Litvinov, S. et al. (1996) Am J Pathol. 148(3):865-75). In addition, EpCAM has oncogenic potential through its ability to upregulate c-myc, e-fabp, and cyclin A & E (Munz, M. et al. (2004) Oncogene 23(34):5748-58). EpCAM is exclusively expressed in epithelial and epithelial derived neoplasms and can therefore be used as a diagnostic marker for various cancers. In other words, Ab-CIDE can be used to deliver CIDE to many cells or tissues, rather than to a specific cell or tissue type as when using targeted antibodies.

[0095] viii. Anti-Steap1 antibody In some embodiments, Ab-CIDE comprises an anti-STEAP1 antibody. STEAP1 (six transmembrane epithelial antigen of the prostate, Genbank accession no. NM_0 12449) Cancer Res. 6 1(15), 5857-5860 (2001), Hubert, RS, et al (1999) Proc. Natl. Acad. Sci. USA 96(25): 14523-14528; WO 2004065577 (claim 6); WO 2004027049 (Figure 1L); EP1394274 (Example 11); WO 2004016225 (claim 2); WO 2003042661 (claim 12); US 2003 157089 (Example 5); US 2003 185830 (Example 5); U.S. Patent Publication No. 2003064397 (Figure 2); WO 200289747 (Example 5; pages 618-619); WO 2003022995 (Example 9; Figure 13A, Example 53; page 173, Example 2; Figure 2A);

[0096] viii. Anti-Steap2 antibody In some embodiments, the Ab-CIDE comprises an anti-STEAP2 antibody. STEAP2 (HGNC 8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138) Lab. Invest. 82(11)) 1573 1582 (2002)); WO 2003087306; U.S. Patent Application Publication No. 2003064397 (Claim 1; Figure 1); WO 200272596 (Claim 13; pages 54-55); WO 200172962 (Claim 1; Figure 4B); WO 2003 104270 (Claim 11); WO 2003 104270 (claim 16); U.S. Patent Application Publication No. 2004005598 (claim 22); WO 2003042661 (claim 12); U.S. Patent Application Publication No. 2003060612 (claim 12; figure 10); WO 200226822 (claim 23; figure 2); WO 200216429 (claim 12; figure 10); Cross References: GF22655488; AAN04080.1; AF455l38_I

[0097] ix. Anti-Her2 antibody The Ab-CIDE comprises an anti-HER2 antibody. In one embodiment, the anti-HER2 antibody of the Ab-CIDE comprises a humanized anti-HER2 antibody. In some embodiments, the Ab-CIDE comprises a humanized HER2 antibody, also called trastuzumab, which is commercially available under the trade name HERCEPTIN®. In another embodiment, the anti-HER2 antibody of the Ab-CIDE comprises a humanized anti-HER2 antibody described in U.S. Pat. No. 7,862,817, such as humanized 2C4. An exemplary humanized 2C4 antibody is pertuzumab, commercially available under the trade name PERJETA®.

[0098] x.Anti-CD33 antibody In some embodiments, Ab-CIDE comprises an anti-CD33 antibody. CD33 is a member of the immunoglobulin-like lectin family that binds sialic acid and is a 67-kDa glycosylated transmembrane protein. CD33 is expressed on most myeloid and monocytic leukemia cells, as well as committed myelomonocytic and erythroid progenitors. CD33 is not found on early pluripotent stem cells, mature granulocytes, lymphocytes, or non-hematopoietic cells (Sabbath et al., (1985). Clin. Invest. 75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33 contains two tyrosine residues in its cytoplasmic tail, each of which is followed by a hydrophobic residue similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) found in many inhibitory receptors.

[0099] 2. Linker (L1) As described herein, the linker (L1) is a bifunctional or multifunctional moiety that can be used to link one or more CIDE moieties (D) to an antibody (Ab) to form an Ab-CIDE. In some embodiments, the Ab-CIDE can be prepared using a linker (L1) that has a reactive functionality for covalently binding the CIDE to the antibody. For example, in some embodiments, a cysteine ​​thiol of the antibody (Ab) can form a bond with a reactive functional group of the linker or with a linker L1-CIDE group to create the Ab-CIDE.

[0100] i. Peptidomimetic Linkers In some embodiments, the linker (L1) of Ab-CIDE is a non-peptidic peptidomimetic linker that can be cleaved by lysosomal enzymes. In such embodiments, the non-peptidic linker acts like a peptide and can be effectively cleaved by lysosomal proteases. For example, the amide bond in the middle of a dipeptide (e.g., Val-Cit) can be replaced with an amide mimic, and / or the entire amino acid (e.g., the valine amino acid in the Val-Cit dipeptide) can be replaced with a non-amino acid moiety (e.g., a cycloalkyldicarbonyl structure (e.g., a cycloalkyl with a ring size of 4 or 5)).

[0101] In some embodiments, L1 is a peptidomimetic linker having the formula: -Str-(PM)-Sp- [In formula: Str is the Stretcher unit covalently attached to Ab; Sp is a bond or a spacer unit covalently attached to the CIDE moiety; PM is a non-peptide chemical moiety selected from the group consisting of: TIFF2025508328000002.tif105170(in formula W is -NH-heterocycloalkyl- or heterocycloalkyl; Y is heteroaryl, aryl, -C(O)C1-C6 alkylene, C1-C6 alkylene-NH2, C1-C6 alkylene-NH-CH3, C1-C6 alkylene-N-(CH3)2, alkenyl, or alkylenyl; Each R 1 are independent, C1-C 10 Alkyl, C1-C 10 Alkenyl, (C1-C 10 alkyl)NHC(NH)NH2 or (C1-C 10 alkyl)NHC(O)NH2-CH2CH2CH2CH2NH2; -CH2CH2CH2CH2NH-CH3; and -CH2CH2CH2CH2N-(CH3)2; R 3 and R 2are each independently H, C1-C 10 Alkyl, C1-C 10 alkenyl, arylalkyl, or heteroarylalkyl; or R 3 and R 2 can together form a cycloalkyl; R 4 and R 5 are each independently 10 Alkyl, C1-C 10 Alkenyl, arylalkyl, heteroarylalkyl, (C1-C 10 alkyl)OCH2- or R 4 and R 5 can form a C3-C7 cycloalkyl ring).

[0102] L1 can be connected to CIDE through either the E3LB, L2 or PB group.

[0103] In some embodiments, Y is heteroaryl; R 4 and R 5 together form a cyclobutyl ring.

[0104] In some embodiments, Y is a moiety selected from the group consisting of: TIFF2025508328000003.tif21170

[0105] In some embodiments, Str is a chemical moiety represented by the formula: TIFF2025508328000004.tif43170[where, R 6 is C1-C 10 Alkylene, C1-C 10 Alkenyl, C3-C8 cycloalkyl, (C1-C8 alkylene)O-, and C1-C 10 Alkylene-C(O)N(R a)-C2-C6 alkylene, each alkylene being optionally substituted by 1 to 5 substituents selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C3-C8 cycloalkyl, C4-C7 heterocycloalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl; and each R a is independently H or C1-C6 alkyl; Sp is -Ar-R b where Ar is aryl or heteroaryl and R b (C1-C 10 Conjugation to antibodies can occur when the maleimide reacts via Michael addition with exposed Cys residues on the antibody. Exposed Cys residues can be artificially introduced by molecular engineering and / or generated by reduction of interchain disulfide bonds.

[0106] In some embodiments, Str has the formula: TIFF2025508328000005.tif18170[where, R 7 is C1-C 10 Alkylene, C1-C 10 Alkenyl, (C1-C 10 alkylene)O-, N(R c )-(C2-C6 alkylene)-N(R c ) and N(R c )-(C2-C6 alkylene); each R c is independently H or C1-C6 alkyl; Sp is -Ar-R b where Ar is aryl or heteroaryl and R b (C1-C 10 Sp-C1-C6 alkylene-C(O)NH- or Sp-C1-C6 alkylene-C(O)NH-.

[0107] In an embodiment, L1 is a non-peptide chemical moiety represented by the formula: TIFF2025508328000006.tif26170[where, R 1 is C1-C6 alkyl, C1-C6 alkenyl, (C1-C6 alkyl)NHC(NH)NH2 or (C1-C6 alkyl)NHC(O)NH2; R 3 and R 2 are each independently H or C 10 alkyl].

[0108] In an embodiment, L1 is a non-peptide chemical moiety represented by the formula: TIFF2025508328000007.tif22170[where, R 1 is C1-C6 alkyl, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2; R 4 and R 5 together form a C3-C7 cycloalkyl ring.

[0109] In an embodiment, L1 is a non-peptide chemical moiety represented by the formula: TIFF2025508328000008.tif26170[where, R 1 is C1-C6 alkyl, C1-C6 alkyl)NHC(NH)NH2 or (C1-C6 alkyl)NHC(O)NH2, and W is as defined above.

[0110] In some embodiments, the linker may be a peptidomimetic linker, such as those described in WO 2015 / 095227, WO 2015 / 095124, or WO 2015 / 095223, each of which is incorporated by reference in its entirety.

[0111] In some embodiments, L1 is selected from the group consisting of: i) TIFF2025508328000009.tif66170Z is -(CH2) p- or -CH2-(CH2-O-CH2) p -CH2-, and p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 Alkyl, or -(CH2) v -aryl, where v is 0 or 1; for example phenyl or benzyl; Q: a) TIFF2025508328000010.tif18170b) TIFF2025508328000011.tif32170Q 1 teeth, TIFF2025508328000012.tif20170R 2 is hydrogen, halo(C 1-6 ) alkyl or C 1-6 (It is alkyl) selected from the group consisting of; L 1-A teeth: TIFF2025508328000013.tif71170ii) TIFF2025508328000014.tif68170Z2 is C 1-12 Alkylene or -[CH2] g -O-[CH2] h -, g and h are each independently 0, 1 or 2; w is 0, 1, 2, 3, 4 or 5; J is for C 1-5 Alkyl, -N(R x )(R y ), -C(O)NH2, -NH-C(O)-NH2, and -NHC(=NH)NH2; R x and R y each independently represents hydrogen and C 1-3 alkyl; K is C 1-3 Alkylene, -CH(R)-, -C(O)-, -C(O)-O-CH(R)-, -CH2-OC(O)-, -CH2-OC(O)-NH-CH2-, and -CH2-OC(O)-R-[CH2] q-O-, R is hydrogen, C 1-3 Alkyl, N(R x )(R y ), -ON(R x )(R y ) or C(O)-N(R x )(R y ) where q is 0, 1, 2, or 3; R x and R y each independently represents hydrogen and C 1-3 alkyl or R x and R y each taken together with the nitrogen to which it is attached forms an optionally substituted 5- to 7-membered heterocyclyl; R a , R b , R C and R D each independently represents hydrogen and C 1-3 alkyl or R a and R b Or R C and R D Each, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R7 and R8 are each independently hydrogen, halo, C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl.

[0112] L 1-A In some embodiments, when w is 0 and J is methyl, L 1-A teeth: TIFF2025508328000015.tif49170

[0113] In some embodiments, K of L1 is selected from the group consisting of: TIFF2025508328000016.tif133170

[0114] In some embodiments, L1 is selected from the group consisting of: TIFF2025508328000017.tif114170

[0115] In some embodiments, R7 and R8 are each hydrogen.

[0116] In some embodiments, Z is -(CH) p -It is.

[0117] In some embodiments, w is 2 or 3 and J is -N(CH3)2 or -NH(CO)NH.

[0118] In some embodiments, Q is -CH2-CH2-.

[0119] In some embodiments, L1 is selected from the group consisting of: TIFF2025508328000018.tif180170[In the formula, R5 and R6 are independently hydrogen or C 1-5 alkyl; or R5 and R6, together with the nitrogen to which they are each attached, form an optionally substituted 5- to 7-membered heterocyclyl.

[0120] In some embodiments, L1 is a peptidomimetic linker selected from the group consisting of: TIFF2025508328000019.tif116170

[0121] In some embodiments, J is -NH-C(O)-NH2 or -N(CH3)2.

[0122] In some embodiments, w is 2 and J is -NH-C(O)-NH; or w is 3 and J is -N(CH).

[0123] ii. Other Linkers In some embodiments, L1 can be a peptide linker or other linker. Peptide linkers such as valine-citrulline (Val-Cit), which can be hydrolyzed by lysosomal enzymes (such as cathepsin B), have been used to connect drugs to antibodies (US Pat. No. 6,214,345). These have been particularly useful, in part, because they are relatively stable in the general circulation and can efficiently release drugs into tumors.

[0124] In one embodiment, the carbonyl group of the linker is connected to an amine group in CIDE. Note also that the sulfur atom connected to Ab is the sulfur group from a cysteine ​​in the antibody. In another embodiment, L1 has a functionality that can react with a free cysteine ​​present on the antibody to form a covalent bond. Non-limiting examples of such reactive functionality include maleimides, haloacetamides, a-haloacetyls, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, for example, Klussman, et al (2004), Bioconjugate Chemistry l5(4):765-773, page 766 for conjugation methods, and examples therein.

[0125] In some embodiments, a linker has a functionality that can react with an electrophilic group present on an antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of a linker can react with an electrophilic group on an antibody to form a covalent bond to an antibody unit. Non-limiting examples of such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0126] The linker may include one or more linker moieties. Exemplary linker moieties include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane 1 carboxylate ("MCC"). A variety of linker moieties are known in the art, some of which are described below.

[0127] The linker can be a "cleavable linker" that facilitates release of the CIDE. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0128] In some embodiments, the linker has the formula: TIFF2025508328000020.tif8170 [wherein A is a "stretcher unit" and a is an integer from 0 to 1; W is an "amino acid unit" and w is an integer from 0 to 12; Y is a "spacer unit" and y is 0, 1, or 2. Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298.

[0129] In some embodiments, a linker component comprises a "stretcher unit" that links an antibody to another linker component or to a CIDE moiety. Non-limiting exemplary stretcher units are shown below (the wavy line indicates the site of covalent attachment to an antibody, CIDE, or additional linker component): TIFF2025508328000021.tif101170

[0130] In some embodiments, the linker is: TIFF2025508328000022.tif40170

[0131] In some embodiments, L1 has the structure: TIFF2025508328000023.tif242170TIFF2025508328000024.tif87170

[0132] 3. CIDE ("D") Useful CIDEs have the general formula above. In some embodiments, CIDEs or CIDEs conjugated to Abs exhibit desirable properties such as cell targeting and protein targeting and degradation. In some embodiments, Ab-L1-CIDEs exhibit DC50 (μg / mL) of 0.0001 to less than about 2.0, or less than about 1.0, or less than about 0.8, or less than about 0.7, or less than about 0.6, or less than about 0.5, or less than about 0.4, or less than about 0.3, or less than about 0.2. In some embodiments, Ab-L1-CIDEs exhibit DCmax of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99. In some embodiments, degradation of target protein is significantly higher than comparator.

[0133] CIDEs include those that have the following components:

[0134] a. E3 ubiquitin ligase binding group (E3LB) E3 ubiquitin ligases (of which over 600 are known in humans) confer substrate specificity for ubiquitination. There are known ligands that bind to these ligases. The E3 ubiquitin ligase binding groups described herein are peptides or small molecules that can bind to the von Hippel-Lindau (VHL) E3 ubiquitin ligase.

[0135] A particular E3 ubiquitin ligase is the von Hippel-Lindau (VHL) tumor suppressor, which is the substrate recognition subunit of the E3 ligase complex VCB, which consists of elongins B and C, Cul2 and Rbxl. The main substrate of VHL is hypoxia-inducible factor la (HIF-la), a transcription factor that upregulates genes such as the proangiogenic growth factor VEGF and the erythroid-inducing cytokine erythropoietin in response to low oxygen levels.

[0136] In some embodiments, the subject matter herein is directed to an E3LB moiety that includes a hydroxyproline residue and is of the formula: TIFF2025508328000025.tif40170

[0137] In some embodiments, the subject matter herein is directed to E3LB moieties that include a phosphate moiety.

[0138] In some embodiments, the E3LB comprises: TIFF2025508328000026.tif67170

[0139] In some embodiments, the E3LB comprises: TIFF2025508328000027.tif64170

[0140] In some embodiments, the E3LB comprises: TIFF2025508328000028.tif55170 [wherein A is a group covalently bonded to group L2].

[0141] In some embodiments, the E3LB comprises: TIFF2025508328000029.tif90170R A1 , R A2 and R A3 are each independently hydrogen or C 1-5 alkyl; or R A1 , R A2 and R A3Two of them, together with the carbon to which they are attached, form C 1-5 forming a cycloalkyl].

[0142] In some embodiments, the E3LB comprises: TIFF2025508328000030.tif66170[In the formula, R 2 is hydrogen or C 1~5 is alkyl; Y 1 and Y 2 each is -CH or Y 1 and Y 2 one of is -CH and the other is N; R 3 teeth, [TIFF2025508328000031.tif29170]

[0143] In some embodiments, the E3LB comprises: TIFF2025508328000032.tif66170

[0144] In some embodiments, the E3LB comprises: TIFF2025508328000033.tif66170

[0145] In some embodiments, E3LB has the structure where R3 is cyano.

[0146] In some embodiments, E3LB is The structure is TIFF2025508328000034.tif33170.

[0147] In some embodiments, E3LB is The structure is TIFF2025508328000035.tif33170.

[0148] In some embodiments, E3LB is The structure is TIFF2025508328000036.tif29170.

[0149] In some embodiments, E3LB has a structure where R2 is hydrogen, methyl, ethyl, or propyl.

[0150] In some embodiments, E3LB has the structure where R2 is methyl.

[0151] In some embodiments, E3LB is The structure is TIFF2025508328000037.tif20170.

[0152] In some embodiments, the hydroxyproline moiety of E3LB has the following structure: TIFF2025508328000038.tif53170

[0153] The E3LB moiety has at least one end that contains a moiety that is or can be covalently linked to the L2 moiety and at least one end that contains a moiety that is or can be covalently linked to the L1 moiety, e.g., the E3LB moiety terminates in an -NHCOOH moiety that can be covalently linked to the L2 moiety through an amide bond.

[0154] In any of the aspects or embodiments described herein, the E3LB described herein may be a pharma- ceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof.

[0155] b. Protein binding group (PB) "Protein binding group" or "PB" refers to a residue of a small molecule or other compound that can bind to a target protein or other polypeptide target of interest. The PB binds to or otherwise interacts with the target, thereby placing the target in proximity to a ubiquitin ligase so that degradation of the protein or polypeptide can occur by the ubiquitin ligase. The PB can be any molecule that is covalently attached to L2 and interacts with or binds to the target of interest. Non-limiting examples of small molecule target protein binding moieties include compounds that bind to BRM (BRAHMA), Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds that target human BET bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds that target the aryl hydrocarbon receptor (AHR), among many others. In some embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of PB. In some embodiments, the CIDEs and conjugated CIDEs described herein are not limited to the type of PB, and the PB is covalently linked to L2; the CIDE or conjugated CIDE comprises a phosphate moiety and an L1 peptidomimetic linker.

[0156] In some embodiments, the PB portion of the CIDE is a small molecule that includes a hydroxyphenyl residue covalently attached to a phosphate moiety through the oxygen of the hydroxyphenyl residue. In this aspect, the PB comprises the formula: TIFF2025508328000039.tif33170, where PM is a phosphate moiety. An example of a hydroxyphenyl residue covalently attached to a phosphate moiety is: TIFF2025508328000040.tif36170

[0157] In some embodiments, PB is attached at the ortho position of: TIFF2025508328000041.tif50170An example of this type of structure is PB, which contains the following formula: TIFF2025508328000042.tif40170

[0158] In some embodiments, the PB portion of the CIDE is a small molecule moiety that binds to BRM, including all variants, mutants, splice variants, indels, and fusions of BRM. BRM is also known as subfamily A, member 2, SMARCA2, and BRAHMA. Such small molecule target protein binding moieties also include pharma- ceutically acceptable salts, enantiomers, solvates, and polymorphs of these compositions, as well as other small molecules that may target a protein of interest. The CIDEs described herein can include any residue of a known BRM binding compound, including those disclosed in WO 2019 / 195201, which is incorporated herein by reference in its entirety.

[0159] In some embodiments, the BRM binding compound has formula I: JPEG2025508328000043.jpg58170[In the formula, TIFF2025508328000044.tif218170, where for (a)-(e), * indicates the point of attachment to [X], or if [X] is absent, * indicates the point of attachment to [Y], and ** indicates the point of attachment to the phenyl ring; (i) [X] is a 3- to 15-membered heterocyclyl or a 5- to 20-membered heteroaryl; however, Instead of TIFF2025508328000045.tif45170, [Y] does not exist, [Z] is not present; or (ii) [X] is a 3- to 15-membered heterocyclyl or a 5- to 20-membered heteroaryl, and the 3- to 15-membered heterocyclyl of [X] is one or more -OH or C 1-6 may be substituted with alkyl; [Y] does not exist, [Z] is a 3- to 15-membered heterocyclyl or a 5- to 20-membered heteroaryl; however, TIFF2025508328000046.tif29170&& indicates the attachment point to [Z]), [Z] is TIFF2025508328000047.tif21170, where # indicates the point of attachment to [X] and ## indicates the point of attachment to L2; or (iii) [X] is a 3- to 15-membered heterocyclyl or a 5- to 20-membered heteroaryl; [Y] is methylene, and the methylene of [Y] may be substituted with one or more methyl groups; [Z] is a 3- to 15-membered heterocyclyl; or (iv) [X] does not exist; [Y] is ethenylene, and the ethenylene in [Y] is optionally substituted with one or more halo; [Z] is a 5-20 membered heteroaryl; however, TIFF2025508328000048.tif16170(a), (b), (d), or (e); or (v) [X] does not exist; [Y] is ethynylene; [Z] is a 5-20 membered heteroaryl; however, TIFF2025508328000049.tif16170(a), (b), (d), or (e); or (vi) [X] does not exist; [Y] is cyclopropyl or cyclobutyl; [Z] is a 5-20 membered heteroaryl; however, TIFF2025508328000050.tif16170(a), (b), (d), or (e)] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0160] In some embodiments, the BRM binding compound has the formula (IA): TIFF2025508328000051.tif61170, wherein [X], [Y] and [Z] are as defined for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0161] In some embodiments, the BRM binding compound has the formula (IB): TIFF2025508328000052.tif50170, wherein [X], [Y] and [Z] are as defined for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0162] In some embodiments, the BRM binding compound has the formula (IC): TIFF2025508328000053.tif52170, wherein [X], [Y] and [Z] are as defined for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0163] In some embodiments, the BRM binding compound has the formula (ID): TIFF2025508328000054.tif59170, wherein [X], [Y] and [Z] are as defined for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0164] In some embodiments, the BRM binding compound has the formula (IE): TIFF2025508328000055.tif58170, wherein [X], [Y] and [Z] are as defined for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0165] In some embodiments, the PB (BRM) portion of CIDE has the following structure: TIFF2025508328000056.tif153170, where This is the covalent attachment point to TIFF2025508328000057.tif12170L2.

[0166] c. Linker L2 The E3LB and PB portions of the CIDE described herein can be connected with a linker (L2, linker L2, linker 2). In some embodiments, linker L2 is covalently linked to the E3LB portion and covalently linked to the PB portion, thus constituting a CIDE.

[0167] In some embodiments, the L2 moiety can be selected from the linkers disclosed in WO 2019 / 195201, which is incorporated by reference in its entirety.

[0168] While the E3LB and PB groups may be covalently linked to the linker group through any group appropriate and stable to the linker chemistry, in some embodiments, L2 is independently covalently linked to the E3LB and PB groups through an amide, ester, thioester, keto group, carbamate (urethane) or ether, each of which may be inserted at any position on the E3LB and PB groups to allow binding of the E3LB group to a ubiquitin ligase and binding of the PB group to a target protein such as a BRM to be degraded. In other words, as shown herein, linkers can be designed and attached to the E3LB and PB groups to regulate binding of the E3LB and PB groups to their respective binding partners.

[0169] In some embodiments, L2 is a linker covalently connecting E3LB and PB, and L2 has the formula: TIFF2025508328000058.tif44170[In the formula, R4 is hydrogen or methyl; TIFF2025508328000059.tif74170[In the formula, z is 1 or 0; G is TIFF2025508328000060.tif49170For example, directly attached to PB, which is the attachment point to CIDE].

[0170] In some embodiments of L2a, R4 is hydrogen.

[0171] In some embodiments of L2a, R4 is methyl.

[0172] In some embodiments of L2a, R4 is methyl and the methyl is oriented relative to the piperazine to which it is attached, as follows: TIFF2025508328000061.tif43170

[0173] In some embodiments of L2c, z is 0.

[0174] In some embodiments of L2c, z is 1.

[0175] Referring now to Ab-CIDEs, Ab-CIDEs can include a single antibody, and a single antibody can have more than one CIDE, with each CIDE covalently linked to an antibody through a linker L1. "CIDE loading" is the average number of CIDE moieties per antibody. CIDE loading can range from 1 to 16 CIDEs (D) per antibody (Ab). That is, the Ab-CIDE formula, Ab-(L1-D) j wherein j has a value of about 1 to about 16, about 1 to about 8, about 1 to about 5, about 1 to about 4, or about 1 to about 3. Each CIDE covalently linked to the antibody through a linker L1 can be the same or a different CIDE and can have the same or a different type of linker as any other L1 covalently linked to the antibody. In some embodiments, Ab is a cysteine ​​engineered antibody and j is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0176] The average number of CIDEs per antibody in a preparation of Ab-CIDE from a conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, electrophoresis, and HPLC. The quantitative distribution of Ab-CIDEs in terms of j can also be determined. The average value of a particular preparation of Ab-CIDEs can be determined by ELISA (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p values ​​cannot be distinguished by the detection limits of antibody-antigen binding and ELISA. Also, the ELISA assay for detection of Ab-CIDE does not determine where on the antibody the CIDE moiety is attached (e.g., to the heavy or light chain fragment, or to a specific amino acid residue). In some cases, separation, purification, and characterization of homogenous Ab-CIDEs for a particular value of j from Ab-CIDEs with other CIDE loadings can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0177] For some Ab-CIDEs, j may be limited by the number of binding sites on the antibody. For example, the antibody may have only one or more cysteine ​​thiol groups through which a linker may be attached, or may have only one or more sufficiently reactive thiol groups. Another reactive site on the Ab for attaching L1-D is the amine functionality of a lysine residue. Values ​​of j include values ​​of about 1 to about 16, about 1 to about 8, about 1 to about 5, about 1 to about 4, about 1 to about 3, where j is equal to 2. In some embodiments, the subject matter described herein is directed to any Ab-CIDE, where j is about 1, 2, 3, 4, 5, 6, 7, or 8.

[0178] Generally, less than the theoretical maximum CIDE moiety is conjugated to the antibody during the conjugation reaction. The antibody may contain many lysine residues that do not react with, for example, the linker L1-CIDE group (L1-D) or the linker reagent. Only the most reactive lysine groups can react with amine-reactive linker reagents. Also, only the most reactive cysteine ​​thiol groups can react with thiol-reactive linker reagents or the linker-L1-CIDE group. Generally, the antibody does not contain many free and reactive cysteine ​​thiol groups, if any, that can be linked to the CIDE moiety. Most cysteine ​​thiol residues in the antibody of the compound exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partial or complete reducing conditions. However, the CIDE loading of the DAR (CIDE / antibody ratio, "DAR") can be controlled in several different ways, including: (i) limiting the molar excess of linker L1-CIDE groups or linker reagent compared to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing conditions for cysteine ​​thiol modification.

[0179] III. L1-CIDE Compounds The CIDEs described herein can be covalently linked to a linker L1 to prepare the L1-CIDE group. These compounds have the following general formula: L1-D where D is a CIDE having the structure E3LB-L2-PB; E3LB is an E3 ligase binding group covalently attached to L2; L2 is a linker covalently attached to E3LB and PB; PB is a target protein binding group covalently attached to L2; and L1 is a peptidomimetic linker covalently attached to D. Useful groups for each of these components are described above. In some embodiments, CIDE is phosphorylated.

[0180] In some embodiments, L1 is as described elsewhere herein and includes a peptidomimetic linker. In these embodiments, L1-CIDE has the formula: TIFF2025508328000062.tif22170[in the formula, Str is the stretcher unit; Sp is a bond or D, a spacer unit covalently attached to the CIDE moiety; R 1 is C1-C 10 Alkyl, (C1-C 10 alkyl)NHC(NH)NH2, or (C1-C 10 alkyl)NHC(O)NH2; R 4 and R 5 are each independently 10 Alkyl, arylalkyl, heteroarylalkyl, (C1-C 10 alkyl)OCH2- or R 4 and R 5 forms a C3-C7 cycloalkyl ring; D is the CIDE portion].

[0181] The L1-CIDE compound can be represented by the following formula: TIFF2025508328000063.tif28170 [wherein R6 is C1-C 10 alkylene; R 4 and R 5 together form a C3-C7 cycloalkyl ring, and D is a CIDE moiety.

[0182] The L1-CIDE compound can be prepared according to the following formula: TIFF2025508328000064.tif27170[where, R 1 , R 4 and R 5 is as described elsewhere herein and D is a CIDE moiety].

[0183] The L1-CIDE compound can be represented by the following formula: TIFF2025508328000065.tif25170[in the formula, Str is the stretcher unit; Sp is an optional spacer unit covalently attached to the D, CIDE moiety; Y is heteroaryl, aryl, -C(O)C1-C6 alkylene, C1-C6 alkylene-NH2, C1-C6 alkylene-NH-CH3, C1-C6 alkylene-N-(CH3)2, C1-C6 alkenyl, or C1-C6 alkylenyl; R 1 is C1-C 10 Alkyl, (C1-C 10 alkyl)NHC(NH)NH2, or (C1-C 10 alkyl)NHC(O)NH2; R 3 and R 2 are each independently H, C1-C 10 alkyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 may together form a C3-C7 cycloalkyl; D is the CIDE portion].

[0184] The L1-CIDE compound can be represented by the following formula: TIFF2025508328000066.tif29170[where, R 6 is C1-C 10 alkylene, R 1 , R 2 and R 3 is as described elsewhere herein and D is a CIDE moiety.

[0185] The L1-CIDE compound can be represented by the following formula: TIFF2025508328000067.tif30170[where, R 1 , R 2 and R 3 is as described elsewhere herein and D is a CIDE moiety].

[0186] In any of the above L1-CIDE compounds, Str can have the formula: TIFF2025508328000068.tif41170[where, R 6 is C1-C 10 Alkylene, C3-C8 cycloalkyl, O-(C1-C8 alkylene), and C1-C 10 Alkylene-C(O)N(R a )-C2-C6 alkylene, each alkylene can be substituted by 1 to 5 substituents selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C3-C8 cycloalkyl, C4-C7 heterocycloalkylaryl, arylalkyl, heteroarylalkyl, and heteroaryl; each R a is independently H or C1-C6 alkyl; Sp is -Ar-R b -(Ar is aryl or heteroaryl, R b is (C1-C 10 alkylene)O-).

[0187] In some L1-CIDE compounds, R 6 is C1-C 10 alkylene, and Sp is -Ar-R b -(Ar is aryl and R b is (C1-C6 alkylene)O-; or R6 is -(CH2) q is between 1 and 10;

[0188] In any of the above L1-CIDE compounds, Str can have the formula: TIFF2025508328000069.tif21170[In the formula, TIFF2025508328000070.tif11170 indicates binding to a moiety capable of being conjugated to an antibody, R 7 is C1-C 10 Alkylene, C1-C10 Alkylene-O,N(R c )-(C2-C6 alkylene)-N(R c ) and N(R c )-(C2-C6 alkylene); each R c is independently H or C1-C6 alkyl; Sp is -Ar-R b -(Ar is aryl or heteroaryl, R b is (C1-C 10 alkylene)O-; or R 6 is C1-C 10 alkylene, and Sp is -Ar-R b - (Ar is aryl and R b is (C1-C6 alkylene)O-).

[0189] The L1-CIDE can have the following formula, where D is the CIDE moiety: TIFF2025508328000071.tif184170

[0190] Ab-CIDE can include any combination of PB, E3LB, Ab, L1, and L2; one skilled in the art will appreciate that when L1 is present, the attachment points of L1 and L2 can vary, so long as the CIDE, L1-CIDE, or Ab-L1-CIDE includes a phosphate group covalently attached to PB or E3LB and a peptidomimetic linker. Furthermore, portions of the linker, such as -Str-(PM)-Sp-, can be interchangeable. Furthermore, portions of the linker, L1, can be interchangeable.

[0191] Now referring to the Ab-CIDE and L1-CIDE compounds described herein, they can exist in solid or liquid form. In the solid state, they can exist in crystalline or non-crystalline form, or as a mixture thereof. Those skilled in the art will understand that pharma- ceutically acceptable solvates can be formed for crystalline or non-crystalline compounds. In crystalline solvates, solvent molecules are incorporated into the crystal lattice during crystallization. Solvates may include non-aqueous solvents, such as but not limited to ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or may include water as the solvent incorporated into the crystal lattice. Solvates in which water is the solvent incorporated into the crystal lattice are generally referred to as "hydrates". Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The subject matter described herein includes all such solvates.

[0192] Those skilled in the art will further appreciate that certain compounds and Ab-CIDEs described herein that exist in crystalline forms, including various solvates thereof, may exhibit polymorphism (i.e., the ability to occur in different crystal structures). These different crystalline forms are typically known as "polymorphs". The subject matter disclosed herein includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometric configuration, and other descriptive properties of the crystalline solid state. Thus, polymorphs may have different physical properties, such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which can be used for identification. Those skilled in the art will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents used to make the compound. For example, changes in temperature, pressure, or solvents may result in polymorphism. In addition, one polymorph may spontaneously transform into another polymorph under certain conditions.

[0193] The compounds and Ab-CIDEs or salts thereof described herein may exist in stereoisomeric forms (e.g., they contain one or more asymmetric carbon atoms). Individual stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of the subject matter disclosed herein. Similarly, compounds or salts of formula (I) may exist in tautomeric forms other than those shown in the formula, which are also understood to be included within the scope of the subject matter disclosed herein. The subject matter disclosed herein is understood to include all combinations and subsets of the specific groups described herein. The scope of the subject matter disclosed herein includes mixtures of stereoisomers, as well as purified enantiomers or enantiomerically / diastereomerically enriched mixtures. The subject matter disclosed herein is understood to include all combinations and subsets of the specific groups defined herein above.

[0194] The subject matter disclosed herein also includes isotopically labeled forms of the compounds described herein, where in fact one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be incorporated into the compounds described herein and their pharma- ceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, e.g., 2 H, 3 H, 11 C. 13 C. 14 C. 15 N, 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 Contains I.

[0195] Compounds disclosed herein and Ab-CIDEs, and pharma- ceutically acceptable salts thereof, that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the presently disclosed subject matter. Isotopically labeled compounds, e.g. 3 H, 14 Disclosed herein are those in which radioactive isotopes such as C are incorporated and are useful for drug and / or substrate tissue distribution assays. Tritium isotopes, i.e., 3 H and carbon-14 isotopes, i.e. 14 C is commonly used due to its ease of preparation and detectability. 11 C and 18 F isotopes are useful in PET (positron emission tomography), 125 I isotopes are useful in SPECT (single photon emission computed tomography), all of which are useful in brain imaging. In addition, deuterium, i.e. 2 Substitution with heavier isotopes such as H can provide certain therapeutic benefits due to increased metabolic stability, e.g., increased half-life in vivo or reduced dosing requirements, and therefore may be preferred in some circumstances. Isotopically labeled compounds can be prepared by carrying out the procedures disclosed in the following schemes and / or examples, typically by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents.

[0196] In some embodiments, the subject matter described herein includes the following L1-CIDE: TIFF2025508328000072.tif220170TIFF2025508328000073.tif218170

[0197] In embodiments, at least one of L1Hy-PE-CIDE-1, L1-PE-CIDE-2, L1-PE-CIDE-3, L1Hy-PE-CIDE-4, L1-PE-CIDE-5, L1Hy-PE-CIDE-6, L1-PP-CIDE-1 and L1-PP-CIDE-2 is conjugated to an antibody. In embodiments, at least one L1Hy-PE-CIDE-1 is conjugated to an antibody. In embodiments, at least one L1-PE-CIDE-2 is conjugated to an antibody. In embodiments, at least one L1-PE-CIDE-3 is conjugated to an antibody. In embodiments, at least one L1Hy-PE-CIDE-4 is conjugated to an antibody. In embodiments, at least one L1-PE-CIDE-5 is conjugated to an antibody. In an embodiment, at least one L1Hy-PE-CIDE-6 is conjugated to an antibody. In an embodiment, at least one L1-PP-CIDE-1 is conjugated to an antibody. In an embodiment, at least one L1-PP-CIDE-2 is conjugated to an antibody.

[0198] The subject matter disclosed herein includes the following non-limiting embodiments: Embodiment 1. Chemical structure: Ab-(L1-D) j [In the formula, D has the structure: E3LB-L2-PB (In the formula, E3LB is the E3 ligase-binding (E3LB) ligand covalently attached to L2, and the E3 ligase is the von Hippel-Lindau (VHL) tumor suppressor protein; L2 is a linker covalently linking E3LB and PB; PB is a protein-binding group covalently attached to L1 and L2) wherein at least one phosphate moiety is covalently attached to D; Ab is antibody covalently bound to L1; L1 is a linker covalently linking Ab and D; j has a value of about 1 to about 16. A conjugate having the formula: Embodiment 2. The conjugate of embodiment 1, wherein one phosphate moiety is covalently attached to D at E3LB or PB. Embodiment 3. 3. The conjugate of embodiment 1 or 2, wherein L1 is a peptidomimetic linker covalently linking Ab and the PB of D. Embodiment 4. 3. The conjugate of embodiment 1 or 2, wherein L1 is a peptidomimetic linker covalently linking Ab to E3LB of D. Embodiment 5. L1: i) TIFF2025508328000074.tif65170Z is -(CH2) p - or -CH2-(CH2-O-CH2) p -CH2-, and p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 Alkyl, or -(CH2) v -aryl, where v is 0 or 1 (e.g., phenyl or benzyl); Q: a) TIFF2025508328000075.tif18170b) TIFF2025508328000076.tif32170Q 1 teeth, TIFF2025508328000077.tif20170R 2 is hydrogen, halo(C 1-6 ) alkyl or C 1-6 (It is alkyl) selected from the group consisting of; L 1-A teeth: TIFF2025508328000078.tif69170ii) TIFF2025508328000079.tif67170Z2 is C 1-12 Alkylene or -[CH2] g -O-[CH2] h -, g and h are each independently 0, 1 or 2; w is 0, 1, 2, 3, 4 or 5; J is for C 1-5 Alkyl, -N(R x )(R y ), -C(O)NH2, -NH-C(O)-NH2, and -NHC(=NH)NH2; R x and R y each independently represents hydrogen and C 1-3 alkyl; K is C 1-3 Alkylene, -CH(R)-, -C(O)-, -C(O)-O-CH(R)-, -CH2-OC(O)-, -CH2-OC(O)-NH-CH2-, and -CH2-OC(O)-R-[CH2] q -O-, R is hydrogen, C 1-3 Alkyl, N(R x )(R y ), -ON(R x )(R y ) or C(O)-N(R x )(R y ) where q is 0, 1, 2, or 3; R x and R y each independently represents hydrogen and C 1-3 alkyl or R x and R y each taken together with the nitrogen to which it is attached forms an optionally substituted 5- to 7-membered heterocyclyl; R a , R b , R C and R D each independently represents hydrogen and C 1-3 alkyl or R a and R b Each, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R7 and R8 are each independently hydrogen, halo, C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl. The conjugate of embodiment 1, 2, 3 or 4, selected from the group consisting of: Embodiment 6. K in L1 is: The conjugate of embodiment 5, selected from the group consisting of: TIFF2025508328000080.tif110170. Embodiment 7. L1: The conjugate of embodiment 1, 2, 3, 4 or 5, selected from the group consisting of: TIFF2025508328000081.tif112170. Embodiment 8. The conjugate of embodiment 7, wherein R7 and R8 are each hydrogen. EMBODIMENT 9. Z is -(CH2) p The conjugate of embodiment 8, wherein Embodiment 10. The conjugate of embodiment 9, wherein w is 2 or 3 and J is -N(CH3)2 or -NH(CO)NH. Embodiment 11. The conjugate of embodiment 10, wherein Q is -CH2-CH2-. Embodiment 12. L1: TIFF2025508328000082.tif172170[In the formula, R5 and R6 are independently hydrogen or C 1-5 alkyl; or R5 and R6, together with the nitrogen to which they are each attached, form an optionally substituted 5- to 7-membered heterocyclyl. The conjugate of embodiment 1, 2, 3, 4 or 5, selected from the group consisting of: Embodiment 13. L1: TIFF2025508328000083.tif108170. Embodiment 14. The conjugate of embodiment 13, wherein J is -NH-C(O)-NH2 or -N(CH3)2. EMBODIMENT 15. The conjugate of embodiment 14, wherein w is 2 and J is -NH-C(O)-NH2; or w is 3 and J is -N(CH3)2. Embodiment 16. L1 has the structure: The conjugate of embodiment 15 having TIFF2025508328000084.tif129170. EMBODIMENT 17. 17. The conjugate of any one of embodiments 1-16, wherein the phosphate moiety is selected from the group consisting of: -(P=O)(OH)2, -CHO(P=O)(OH)2, -(P=O)(OH)O(P=O)(OH)2 and -CH-O(P=O)(OH)O(P=O)(OH)2. Embodiment 18. The conjugate of any one of embodiments 1-17, wherein E3LB comprises a hydroxyproline residue. EMBODIMENT 19. 19. The conjugate of embodiment 18, wherein the phosphate moiety is covalently attached to E3LB through the oxygen of the hydroxyproline residue. EMBODIMENT 20. E3LB: The conjugate of embodiment 19, comprising TIFF2025508328000085.tif67170. EMBODIMENT 21. E3LB: The conjugate of embodiment 20, comprising TIFF2025508328000086.tif63170. EMBODIMENT 22. The antibody conjugated to L1-D is: 22. The conjugate of embodiment 21, selected from the group consisting of: TIFF2025508328000087.tif225170TIFF2025508328000088.tif49170. EMBODIMENT 23. The conjugate of any one of embodiments 1-22, wherein PB comprises a hydroxyphenyl moiety. EMBODIMENT 24. The conjugate of embodiment 23, wherein the phosphate moiety is covalently attached to PB through an oxygen of the hydroxyphenyl moiety. EMBODIMENT 25. 25. The conjugate of embodiment 24, wherein PB is a BRM ligand. 26. The antibody conjugated to L1-D is: TIFF2025508328000089.tif90170. EMBODIMENT 27. A pharmaceutical composition comprising the conjugate of any one of embodiments 1 to 26 and one or more pharma- ceutically acceptable excipients. 28. A method of treating a disease in a human, comprising administering to said human in need of such treatment an effective amount of the conjugate of any one of embodiments 1 to 26 or the composition of embodiment 27; or the conjugate of any one of embodiments 1 to 26 (or the composition thereof) or the composition of embodiment 27 for use in treating a disease in a human in need of such treatment; or the use of the conjugate of any one of embodiments 1 to 26 (or the composition thereof) or the composition of embodiment 27 for the manufacture of a medicament for treating a disease in a human in need of such treatment. 29. 29. The method of embodiment 28, wherein the disease is cancer. EMBODIMENT 30. 30. The method of embodiment 29, wherein the cancer is BRM dependent. EMBODIMENT 31. 31. The method of embodiment 30, wherein the cancer is non-small cell lung cancer. EMBODIMENT 32. A method for reducing the level of a target protein in a subject, comprising administering to said subject a conjugate of any one of embodiments 1 to 26 or a composition of embodiment 27, wherein said PB moiety binds to said target protein, and a ubiquitin ligase leads to degradation of the bound target protein, thereby reducing the level of said target protein.

[0199] In embodiments, the disclosed compounds have the chemical structure: L1-D [In the formula, L1 is a peptidomimetic linker covalently attached to D; D has the structure: E3LB-L2-PB (In the formula, E3LB is E3 ligase binding (E3LB) covalently linked to ligand L2, the E3 ligase being the von Hippel-Lindau (VHL) tumor suppressor protein, the E3LB having a hydroxyproline residue with a phosphate moiety covalently linked to the E3LB through the oxygen of the hydroxyproline residue; PB is a protein-binding group covalently attached to L1 and L2; L2 is a linker covalently linking E3LB and PB. CIDE has has.

[0200] In embodiments, the disclosed compounds have the chemical structure: L1-D [In the formula, L1 is a peptidomimetic linker covalently attached to D; D has the structure: E3LB-L2-PB (In the formula, E3LB is an E3 ligase-binding (E3LB) ligand covalently attached to L2, and the E3 ligase is the von Hippel-Lindau (VHL) tumor suppressor protein; PB is a protein-binding group covalently bonded to L1 and L2, PB comprises a hydroxyphenyl moiety having a phosphate moiety covalently bonded to PB through an oxygen of the hydroxyphenyl moiety; L2 is a linker covalently linking E3LB and PB. CIDE has has.

[0201] In embodiments, the disclosed compounds have the chemical structure: L1-D [In the formula, L1 is a peptidomimetic linker covalently attached to D; D has the structure: E3LB-L2-PB where E3LB is an E3 ligase-binding (E3LB) ligand covalently attached to L2, the E3 ligase is the von Hippel-Lindau (VHL) tumor suppressor protein, and E3LB contains a hydroxyproline residue; PB is a protein binding group covalently attached to L1 and L2, PB comprising a hydroxyphenyl moiety; L2 is a linker covalently connecting E3LB and PB; The phosphate moiety is covalently attached through the oxygen of a hydroxyphenyl moiety of PB, or the phosphate moiety is covalently attached through the oxygen of a hydroxyproline residue of E3LB. have] has.

[0202] In embodiments, the disclosed conjugates have the chemical structure: Ab-(L1-D) j [In the formula, D has the structure: E3LB-L2-PB (In the formula, E3LB is an E3 ligase-binding (E3LB) ligand covalently attached to L2, the E3 ligase being the von Hippel-Lindau (VHL) tumor suppressor protein, and E3LB contains a hydroxyproline residue with a phosphate moiety covalently attached through the oxygen of the hydroxyproline residue; PB is a protein-binding group covalently attached to L1 and L2; L2 is a linker covalently linking E3LB and PB. A CIDE having: Ab is antibody covalently bound to L1; L1 is a peptidomimetic linker covalently linking Ab and D; j has a value of about 1 to about 16. has.

[0203] In embodiments, the disclosed conjugates have the chemical structure: Ab-(L1-D) j [In the formula, D has the structure: E3LB-L2-PB (In the formula, E3LB is an E3 ligase-binding (E3LB) ligand covalently attached to L2, and the E3 ligase is the von Hippel-Lindau (VHL) tumor suppressor protein; PB is a protein-binding group covalently attached to L1 and L2, PB comprising a hydroxyphenyl moiety having a phosphate moiety covalently attached through an oxygen of the hydroxyphenyl moiety; L2 is a linker covalently linking E3LB and PB. A CIDE having: Ab is antibody covalently bound to L1; L1 is a peptidomimetic linker covalently attached to Ab and D; j has a value of about 1 to about 16. has.

[0204] In embodiments, the disclosed conjugates have the chemical structure: Ab-(L1-D)j [In the formula, D has the structure: E3LB-L2-PB where E3LB is an E3 ligase-binding (E3LB) ligand covalently attached to L2, the E3 ligase is the von Hippel-Lindau (VHL) tumor suppressor protein, and E3LB contains a hydroxyproline residue; L2 is a linker covalently connecting E3LB and PB; PB is a protein-binding group covalently attached to L1 and L2, and PB contains a hydroxyphenyl moiety. A CIDE having: Ab is antibody covalently bound to L1; L1 is a peptidomimetic linker covalently linking Ab and D; j has a value of about 1 to about 16. having The phosphate moiety is covalently attached through an oxygen of a hydroxyphenyl moiety of PB, or the phosphate moiety is covalently attached through an oxygen of a hydroxyproline residue of E3LB.

[0205] IV. Formulation The pharmaceutical formulations of the therapeutic Ab-L1-CIDE described herein can be prepared in unit dosage injectable form using a pharma- ceutical acceptable parenteral vehicle for parenteral administration, e.g., bolus, intravenous, intratumoral injection. The Ab-CIDE having the desired purity is optionally mixed with one or more pharma- ceutical acceptable additives in the form of a lyophilized formulation or an aqueous solution for reconstitution (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

[0206] Ab-L1-CIDE can be formulated as a pharmaceutical composition according to standard pharmaceutical practice. According to this embodiment, a pharmaceutical composition is provided that includes Ab-L1-CIDE in combination with one or more pharma- ceutically acceptable excipients.

[0207] A typical formulation is prepared by mixing Ab-L1-CIDE with additives such as carriers and / or diluents. Suitable carriers, diluents, and other additives are well known to those skilled in the art and include materials such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, and water. The particular carrier, diluent, or other additive used will depend on the means and purpose for which Ab-L1-CIDE is applied. Solvents are generally selected based on solvents recognized by those skilled in the art as safe (GRAS) for administration to mammals.

[0208] In general, safe solvents are non-toxic aqueous solvents, such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400, PEG300), and the like, and mixtures thereof. Acceptable diluents, carriers, excipients, and stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-chloride. resoles; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or TWEEN. TM , PLURONICS TMor non-ionic surfactants such as polyethylene glycol (PEG).

[0209] The formulations may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, flavorings, flavoring agents, and other known additives to present Ab-L1-CIDE in a presentable manner or to aid in the manufacture of pharmaceutical products. The formulations may be prepared using conventional dissolution and mixing procedures.

[0210] Formulation may be carried out by mixing at room temperature, at an appropriate pH, and at the desired purity with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to recipients at the dosages and concentrations employed. The pH of the formulation depends primarily on the particular application and the concentration of the compound, but may range from about 3 to about 8. Formulation in acetate buffer at pH 5 is a suitable embodiment.

[0211] The Ab-L1-CIDE formulation can be sterile. In particular, formulations to be used for in vivo administration must be sterile. Such sterilization is readily accomplished by filtration through sterile filtration membranes.

[0212] Ab-L1-CIDE can generally be stored as a solid composition, a lyophilized formulation or an aqueous solution.

[0213] Pharmaceutical compositions containing Ab-L1-CIDE can be formulated, dosed, and administered in a form, i.e., amount, concentration, schedule, course, vehicle, and route of administration that is in accordance with medical practice standards. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the drug, the method of administration, the administration schedule, and other factors known to medical practitioners. The "therapeutically effective amount" of the compound administered is determined by such considerations and is the minimum amount necessary to prevent, ameliorate, or treat a disorder mediated by a coagulation factor. Such an amount is preferably below an amount that is toxic to the host or that renders the host significantly more susceptible to bleeding.

[0214] Ab-L1-CIDE can be formulated into a pharmaceutical dosage form to provide an easily controllable dosage of the drug and to allow the patient to comply with the prescribed regimen. The pharmaceutical composition (or formulation) for application can be packaged in a variety of ways depending on the method used to administer the drug. Generally, the article for distribution includes a container in which the pharmaceutical formulation is disposed in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, and metal cylinders. The container may include a tamper-evident assembly to prevent inadvertent access to the contents of the package. In addition, the container has a label disposed thereon that describes the contents of the container. The label may also include appropriate warnings.

[0215] The pharmaceutical composition may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. The suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as 1,3-butanediol. The sterile injectable preparation may be prepared as a lyophilized powder. Acceptable vehicles and solvents that may be utilized are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils may be commonly used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables as well.

[0216] The amount of Ab-L1-CIDE that can be combined with carrier materials to make a single dosage form will vary depending on the host being treated and the particular mode of administration. For example, a sustained release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material mixed with an appropriate and convenient amount of carrier material, which may vary from about 5 to about 95% (weight:weight) of the total composition. Pharmaceutical compositions can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain about 3 to 500 μg of active ingredient per milliliter of solution, such that infusion of a suitable volume can occur at a rate of about 30 mL / hour.

[0217] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0218] The formulations may be packaged in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier for injection, for example, water, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or daily unit sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.

[0219] The present subject matter further provides veterinary compositions comprising at least one active ingredient as defined above together with a veterinary carrier therefor. The veterinary carrier is a material useful for the purpose of administering the composition and may be a solid, liquid, or gaseous material that is otherwise inert or acceptable in the veterinary art and compatible with the active ingredient. These veterinary compositions may be administered parenterally or by any other desired route.

[0220] V. Indications and Methods of Treatment It is contemplated that the Ab-L1-CIDE disclosed herein can be used to treat a variety of diseases or disorders. Further provided herein is a composition comprising Ab-L1-CIDE or Ab-L1-CIDE for use in therapy. In some embodiments, provided herein is a composition comprising Ab-L1-CIDE or Ab-L1-CIDE for the treatment or prevention of diseases and disorders disclosed herein. Further provided herein is the use of Ab-L1-CIDE or a composition comprising Ab-L1-CIDE in therapy. In some embodiments, provided herein is the use of Ab-L1-CIDE for the treatment or prevention of diseases and disorders disclosed herein. Further provided herein is the use of Ab-L1-CIDE or a composition comprising Ab-L1-CIDE in the manufacture of a medicament for the treatment or prevention of diseases and disorders disclosed herein. Further provided herein is the use of Ab-L1-CIDE or a composition comprising Ab-L1-CIDE in the manufacture of a medicament for the treatment or prevention of diseases and disorders disclosed herein.

[0221] Generally, the disease or disorder to be treated is a disease or disorder that depends on target protein, for example, a hyperproliferative disease such as cancer.The example of the cancer to be treated herein includes BRM-dependent cancer.In some embodiments, the cancer is non-small cell lung cancer.

[0222] In some embodiments, the subject matter described herein is directed to a method for reducing the level of a target protein in a subject, the method comprising administering to the subject Ab-L1-CIDE described herein or a composition comprising Ab-L1-CIDE described herein, wherein the PB portion binds to the target protein and the ubiquitin ligase causes degradation of the bound target protein, thereby reducing the level of the target protein.

[0223] Ab-L1-CIDE may be administered by any route appropriate to the condition being treated. Ab-L1-CIDE will typically be administered parenterally, i.e., by infusion, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally, and epidurally.

[0224] Ab-L1-CIDE can be used alone or in combination with other agents in therapy. For example, Ab-L1-CIDE can be co-administered with at least one additional therapeutic agent. Such combination therapy as described above includes co-administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration, where administration of Ab-L1-CIDE can occur prior to, simultaneously with, and / or subsequent to administration of the additional therapeutic agent and / or adjuvant. Ab-L1-CIDE can also be used in combination with radiation therapy.

[0225] Ab-L1-CIDE (and any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injections, such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time periods, bolus administration, and pulse infusion.

[0226] For the prevention or treatment of disease, the appropriate dosage of Ab-L1-CIDE (when used alone or in combination with one or more other additional therapeutic agents) will be determined depending on the type of disease being treated, the type of Ab-L1-CIDE, the severity and course of the disease, whether Ab-L1-CIDE is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to Ab-L1-CIDE, and the discretion of the attending physician. Ab-L1-CIDE is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of Ab-L1-CIDE can be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion, for example. Typical daily dosages can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is generally continued until the desired suppression of disease symptoms occurs. One exemplary dosage of Ab-L1-CIDE would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives about 2 to about 20 doses, or, for example, about 6 doses). A higher initial loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0227] The methods described herein include a method of degrading a target protein. In some embodiments, the method includes administering Ab-L1-CIDE to a subject, and the target protein is degraded. The protein degradation level can be about 1% to about 5%; or about 1% to about 10%; or about 1% to about 15%; or about 1% to about 20%; or about 1% to about 30%; or about 1% to about 40%; or about 1% to about 50%; or about 10% to about 20%; or about 10% to about 30%; or about 10% to about 40%; or about 10% to about 50%; or at least about 1%; or at least about 10%; or at least about 20%; or at least about 30%; or at least about 40%; or at least about 50%; or at least about 60%; or at least about 70%; or at least about 80%; or at least about 90%; or at least about 95%; or at least about 99%.

[0228] The methods described herein include methods of reducing the proliferation of neoplastic tissue, such as non-small cell lung cancer. In some embodiments, the methods include administering Ab-L1-CIDE to a subject, whereby the proliferation of neoplastic tissue is reduced. The level of reduction can be about 1% to about 5%; or about 1% to about 10%; or about 1% to about 15%; or about 1% to about 20%; or about 1% to about 30%; or about 1% to about 40%; or about 1% to about 50%; or about 10% to about 20%; or about 10% to about 30%; or about 10% to about 40%; or about 10% to about 50%; or at least about 1%; or at least about 10%; or at least about 20%; or at least about 30%; or at least about 40%; or at least about 50%; or at least about 60%; or at least about 70%; or at least about 80%; or at least about 90%; or at least about 95%; or at least about 99%.

[0229] VI. Manufactured products In another aspect, described herein are articles of manufacture, e.g., "kits" containing materials useful for treating the above diseases and disorders. The kits include a container containing Ab-L1-CIDE. The kits may further include a label or package insert on or associated with the container. The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products, including information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products.

[0230] Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. A "vial" is a container suitable for holding a liquid or lyophilized preparation. In one embodiment, the vial is a single-use vial, e.g., a 20 cc single-use vial with a stopper. The container can be formed from a variety of materials, such as glass or plastic. The container can hold Ab-L1-CIDE or a formulation thereof effective for treating a condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle).

[0231] At least one active agent in the composition is Ab-L1-CIDE. The label or package insert indicates that the composition is used to treat a selected condition, such as cancer. In addition, the label or package insert can indicate that the patient to be treated is a patient with a disorder, such as a hyperproliferative disorder, neurodegeneration, cardiac hypertrophy, pain, migraine, or neurotraumatic disease or event. In one embodiment, the label or package insert can indicate that the composition comprising Ab-L1-CIDE can be used to treat a disorder resulting from abnormal cell proliferation. The label or package insert can also indicate that the composition can be used to treat other disorders. Alternatively, or additionally, the article of manufacture can further comprise a second container comprising a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article of manufacture can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0232] The kit can further include instructions for administration of Ab-L1-CIDE and, if present, a second pharmaceutical formulation. For example, when the kit includes a first composition comprising Ab-L1-CIDE and a second pharmaceutical formulation, the kit can further include instructions for administering the first and second pharmaceutical compositions simultaneously, sequentially, or separately to a patient in need thereof.

[0233] In another embodiment, the kit is suitable for delivery of a solid oral form of AAb-L1-CIDE, such as a tablet or capsule. Such a kit preferably includes a plurality of unit dosages. Such a kit may include a card with the dosages arranged in the order of their intended use. One example of such a kit is a "blister pack". Blister packs are well known in the packaging industry and are widely used even to package pharmaceutical unit dosage forms. If desired, a memory aid can be provided, for example in the form of numbers, letters, or other markings, or with a calendar insert, indicating the days in the treatment schedule on which the dosages can be administered.

[0234] According to one embodiment, the kit can include (a) a first container having Ab-L1-CIDE contained therein; and optionally (b) a second container having a second pharmaceutical formulation contained therein, the second pharmaceutical formulation comprising a second compound having anti-hyperproliferative activity. Alternatively, or additionally, the kit may further comprise a third container comprising a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0235] In some other embodiments in which the kit comprises Ab-L1-CIDE and a second therapeutic agent, the kit may comprise a container for housing the separate compositions, such as a divided bottle or a divided foil packet; however, the separate compositions may be housed in a single, undivided container. Typically, the kit comprises instructions for administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), when administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

[0236] VII. Methods for making conjugates Synthetic Route The subject matter described herein is also directed to methods of preparing CIDE, L1-CIDE, and Ab-L1-CIDE from L1-CIDE. Generally, the methods include contacting an antibody, or variants, mutants, splice variants, indels, and fusions thereof, with L1-CIDE under conditions in which the antibody covalently binds to any available binding site on L1-CIDE, and Ab-L1-CIDE is prepared. The subject matter described herein is also directed to methods of preparing Ab-L1-CIDE from an Ab-L1 moiety, i.e., an antibody, or variants, mutants, splice variants, indels, and fusions thereof, covalently bound to L1, the methods including contacting CIDE with Ab-L1 under conditions in which CIDE covalently binds to any available binding site on Ab-L1, and Ab-L1-CIDE is prepared. The methods can further include routine isolation and purification of Ab-L1-CIDE.

[0237] CIDEs, L1-CIDEs and Ab-L1-CIDEs, as well as other compounds described herein, can be synthesized by synthetic routes that include processes similar to those well known in the chemical arts, particularly in light of the description contained herein, and processes for other heterocycles described in: Comprehensive Heterocyclic Chemistry II, Editors Katritzky and Rees, Elsevier, 1997, e.g. Volume 3; Liebigs Annalen der Chemie, (9): 1910-16, (1985); Helvetica Chimica Acta, 41: 1052-60, (1958); Arzneimittel-Forschung, 40(12): 1328-31, (1990). The starting materials are typically available from commercial sources such as Aldrich Chemicals (Milwaukee, WI) or are readily prepared using methods well known to those of skill in the art (e.g., prepared by the methods outlined in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 123, Wiley, NY (1967-2006 ed.), or in Beilstein's Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including appendices (also available via the Beilstein online database)).

[0238] Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in the synthesis of CIDEs, L1-CIDEs and Ab-L1-CIDEs, as well as other compounds described herein, and the necessary reagents and intermediates, are known in the art and are described, for example, in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3 rdEd., John Wiley and Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions. In the preparation of CIDEs, L1-CIDEs and Ab-CIDEs and other compounds, protection of remote functionality (e.g., primary or secondary amines) of intermediates may be necessary. The need for such protection will vary depending on the nature of the remote functionality and the conditions of the preparation method. Suitable amino protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz or CBZ), and 9-fluorenylmethylenoxycarbonyl (Fmoc). The need for such protection is readily determined by one skilled in the art. For a general review of protecting groups and their uses, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0239] The general procedures and examples provide exemplary methods for preparing CIDE, L1-CIDE, and Ab-L1-CIDE, as well as other compounds described herein. Those skilled in the art will appreciate that other synthetic routes may be used to prepare Ab-L1-CIDE and compounds. Although specific starting materials and reagents are shown and described in the schemes, general procedures, and examples, they can be readily substituted with other starting materials and reagents to provide a variety of derivatives and / or reaction conditions. In addition, many of the exemplary compounds prepared by the methods described can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

[0240] Generally, Ab-L1-CIDE can be prepared by coupling CIDE with an L1 linker reagent according to the procedures of WO 2013 / 055987; WO 2015 / 023355; WO 2010 / 009124; WO 2015 / 095227 to prepare L1-CIDE, and conjugating L1-CIDE with any of the antibodies or variants, mutants, splice variants, indels, and fusions thereof described herein, including cysteine ​​engineered antibodies. Alternatively, Ab-CIDE can be prepared by first coupling an antibody or variants, mutants, splice variants, indels, and fusions thereof described herein, including cysteine ​​engineered antibodies, with an L1 linker reagent and conjugating it with any CIDE.

[0241] The following synthetic routes describe exemplary methods for preparing CIDE, L1-CIDE, and Ab-L1-CIDE, as well as other compounds and components thereof. Other synthetic routes for CIDE, L1-CIDE, and Ab-L1-CIDE, as well as other compounds and components thereof, are disclosed elsewhere herein.

[0242] 1. Cysteine ​​Engineered Antibodies Cysteine ​​engineered antibodies (THIOMABs) TM ) can be recombinantly expressed and purified using standard methods and typically prepared for conjugation by reduction and reoxidation as follows:

[0243] Full-length, recombinantly expressed cysteine ​​engineered monoclonal antibody (THIOMAB TM THIOMAB (antibody) either has a cysteine ​​adduct (cystine) or is glutathionylated on an engineered cysteine ​​due to cell culture conditions. TMAntibodies cannot be conjugated to the Cys-reactive linker L1-CIDE intermediate. Cysteine ​​engineered antibodies can be made reactive for conjugation with the L1-PCIDE intermediate described herein by treatment with a reducing agent such as DTT (Cleland's Reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal. Biochem. Vol 273:7380; Soltec Ventures, Beverly, MA), followed by reformation of the interchain disulfide bonds (reoxidation) with a weak oxidant such as dehydroascorbic acid. A full-length cysteine ​​engineered monoclonal antibody (ThioMab) expressed in CHO cells was TM Antibodies) (Gomez et al (2010) Biotechnology and Bioeng. 105(4):748-760; Gomez et al (2010) Biotechnol. Prog. 26:1438-1445) were reduced, for example, with approximately 50-fold excess of DTT in 50 mM Tris, pH 8.0, containing 2 mM EDTA, at room temperature overnight to remove Cys and glutathione adducts and reduce interchain disulfide bonds in the antibodies. Removal of adducts was monitored by reversed-phase LCMS using a PLRP-S column.

[0244] After removal of Cys and glutathione adducts, THIOMAB TM The antibodies can be purified by methods commonly known in the art, including cation exchange chromatography as produced herein. TM can be diluted and acidified by adding to at least four volumes of 10 mM succinate (pH 5) until the pH is approximately 5 and / or by titration with 10% acetic acid. TMThe antibody can then be loaded onto a HiTrap S cation exchange column, washed with multiple column volumes of 10 mM sodium acetate, pH 5, and eluted with 50 mM Tris, pH 8.0, 150 mM sodium chloride. Reoxidation can be performed to re-establish disulfide bonds between cysteine ​​residues present in the parent Mab. The eluted reduced THIOMAB as described above. TM The antibody can be treated with 15X dehydroascorbic acid (DHAA) for about 3 hours or with 200 nM to 2 mM aqueous copper sulfate (CuSO4) overnight at room temperature. Other oxidants (i.e., oxidizing agents) and oxidation conditions known in the art can be used. Ambient air oxidation can also be effective. This gentle partial reoxidation step efficiently forms intrachain disulfides with high fidelity. Reoxidation can be monitored by reversed-phase LCMS using a PLRP-S column. The reoxidized THIOMAB is then TM The antibody can be diluted with succinate buffer to reach a pH of approximately 5 as described above and then purified on an S column as described above, except that elution was performed with a gradient of 10 mM succinate (pH 5), 300 mM sodium chloride (buffer B) and 10 mM succinate (pH 5) (buffer A). TM EDTA can be added to the antibody to a final concentration of 2 mM. TM The resulting THIOMAB can be concentrated, if necessary, to reach a final concentration of greater than 5 mg / mL. TMThe antibodies are ready for conjugation and can be stored at -20°C or -80°C. Liquid chromatography / mass spectrometry can be performed on a 6200 series TOF or QTOF Agilent LC / MS. Samples are chromatographed on a PRLP-S®, 1000A, microbore column (50mm x 2.1mm, Polymer Laboratories, Shropshire, UK) heated to 80°C. Using a linear gradient of 30-40% B (solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile), the eluent can be directly ionized using an electrospray source. Data was collected and deconvoluted by MassHunter software (Agilent). Prior to LC / MS analysis, antibodies (50 micrograms) can be treated with PNGase F (2 units / ml; PROzyme, San Leandro, Calif.) for 2 hours at 37° C. to remove N-linked carbohydrates.

[0245] Alternatively, antibodies can be partially digested with LysC (0.25 μg per 50 μg (micrograms) of antibody) for 15 minutes at 37° C. to obtain Fab and Fc fragments for analysis by LCMS.

[0246] 2. Conjugation of the Linker L1-CIDE Group to the Antibody In one method of conjugating the linker L1-PCIDE compound to an antibody, after the reduction and reoxidation procedure described above, a cysteine ​​engineered antibody (THIOMAB TMThe antibody) is pH adjusted to pH 7.5-8.5 with 1 M Tris in 10 mM succinate (pH 5), 150 mM NaCl, 2 mM EDTA. An excess of about 3 molar to 20 equivalents of a linker-PCIDE intermediate containing a thiol reactive group (e.g., maleimide or 4-nitropyridyl disulfide, or methanethiosulfonyl (MTS) disulfide), with or without propylene glycol, is dissolved in DMF or DMA and then added to the reduced, reoxidized, pH adjusted antibody. The reaction is incubated at room temperature or 37°C and monitored until completion (1 to about 24 hours) as determined by LCMS analysis of the reaction mixture. Once the reaction is complete, the conjugate is purified by one or any combination of several methods with the goal of removing remaining unreacted L1-PCIDE intermediate and aggregated protein (if present at significant levels). For example, the conjugate can be diluted with 10 mM histidine-acetate (pH 5.5) until the final pH is approximately 5.5 and purified by S cation exchange chromatography using a HiTrap S column (GE Healthcare) or an S Maxi spin column (Pierce) connected to an Akta purification system. Alternatively, the conjugate can be purified by gel filtration chromatography using an S200 column or a Zeba spin column connected to an Akta purification system. Alternatively, dialysis can be used to remove unreacted or excess linker drug. THIOMAB TM The antibody-PCIDE conjugate can be formulated in 20 mM His / acetate, pH 5, with 240 mM sucrose using gel filtration or dialysis. The purified conjugate can be concentrated by centrifugal ultrafiltration and / or filtered through a 0.2-μm filter under sterile conditions and frozen for storage. Ab-L1-PCIDE was characterized by BCA assay to determine protein concentration, analytical SEC (size-exclusion chromatography) for aggregation analysis, and LC-MS after treatment with Lysine C endopeptidase (LysC) or reduction using standard procedures, and the DAR was calculated.

[0247] Size exclusion chromatography was performed on the conjugates using a Shodex KW802.5 column in 0.2 M potassium phosphate (pH 6.2) with 0.25 mM potassium chloride and 15% IPA at a flow rate of 0.75 ml / min. The aggregation state of the conjugates was determined by integration of the elution peak area absorbance at 280 nm.

[0248] LC-MS analysis can be performed on Ab-L1-PCIDE using an Agilent QTOF 6520 ESI instrument. As an example, Ab-L1-PCIDE is treated with endoprotease Lys C (Promega) at 1:500 w / w in Tris (pH 7.5) for 30 min at 37° C. The resulting fragments are loaded onto a 1000 Å (angstrom), 8 μm (micron) PLRP-S (highly cross-linked polystyrene) column heated to 80° C. and eluted with a gradient from 30% B to 40% B in 5 min. Mobile phase A was H2O with 0.05% TFA and mobile phase B was acetonitrile with 0.04% TFA. The flow rate was 0.5 ml / min. Protein elution was monitored by UV absorbance detection at 280 nm prior to electrospray ionization and MS analysis. Chromatographic resolution of unconjugated Fc fragments, residual unconjugated Fab, and drug-labeled Fab was routinely achieved. The resulting m / z spectra were analyzed using Mass Hunter. TM Software (Agilent Technologies) was used to deconvolute and calculate the mass of the antibody fragments. Peaks in the deconvoluted LCMS spectrum can be assigned and quantified. The PCIDE-to-antibody ratio (DAR) is calculated by calculating the ratio of the intensity of one or more peaks corresponding to the antibody conjugated to PCIDE to all peaks observed.

[0249] 3. General synthetic coupling of L2 to E3LB to prepare the E3LB-L2 intermediate In some embodiments, first contact L2 with a first suitable solvent, a first base, and a first coupling reagent to prepare a first solution. In some embodiments, contacting L2 with a first suitable solvent, a first base, and a first coupling reagent proceeds at room temperature (about 25° C.) for about 15 minutes. Then, E3LB is contacted with the first solution.

[0250] In some embodiments, contacting E3LB with the first solution is allowed to proceed for about 1 hour at room temperature (about 25° C.). The solution is then concentrated and optionally purified.

[0251] In some embodiments, the molar ratio of L2 to the first base pair first coupling reagent is about 1:4:1.19. In some embodiments, the molar ratio of L2 to the first base pair first coupling reagent is about 1:2:0.5, about 1:3:1, about 1:4:2, about 1:5:3, or about 1:6:4.

[0252] In some embodiments, the molar ratio of L2 to E3LB is about 1: 1. In some embodiments, the molar ratio of L2 to E3LB is about 1:0.5, about 1:0.75, about 1:2, or about 0.5:1.

[0253] 4. General synthetic method for coupling of E3LB-L2 intermediate to PB In some embodiments, the E3LB-L2 intermediate is coupled to PB to prepare CIDE. In some embodiments, the PB is first contacted with a second suitable solvent, a second base, and a second coupling reagent. In some embodiments, the contacting proceeds for about 10 minutes at room temperature (about 25° C.). The solution is then contacted with the E3LB-L2 intermediate. In some embodiments, the contacting of the second solution with the E3LB-L2 intermediate proceeds for about 1 hour at room temperature (about 25° C.). The solution is then concentrated and optionally purified to prepare CIDE.

[0254] In some embodiments, the molar ratio of PB to the second base pair second coupling reagent is about 1:4:1.2. In some embodiments, the molar ratio of PB to the second base pair second coupling reagent is about 1:3:0.75, about 1:5:1, about 1:3:2, or about 1:5:3.

[0255] In some embodiments, the molar ratio of PB to E3LB-L2 intermediate is about 1: 1. In some embodiments, the molar ratio of PB to E3LB-L2 intermediate is about 1:0.5, about 1:0.75, about 1:2, or about 0.5:1.

[0256] 5. General synthetic method for coupling CIDE to L1 to prepare L1-CIDE In some embodiments, the contacting is allowed to proceed for about 2 hours at about (about 25° C.). The solution can then be optionally purified to prepare L1-CIDE.

[0257] In some embodiments, the molar ratio of CIDE to L1 is about 1:4. In some embodiments, the molar ratio of CIDE to L1 is about 1:1, 1:2, 1:3, 1:5, 1:6, 1:7, or about 1:8.

[0258] 6. General synthetic method for coupling L1-CIDE to antibodies In some embodiments, L1-CIDE is contacted with a thiol and a fourth suitable solvent to form a fourth solution. This solution is then contacted with an antibody to prepare the conjugate. In certain embodiments,

[0259] In some embodiments, the thiol is maleimide or 4-nitropyridindisulfide, hi some embodiments, the suitable solvent is selected from the group consisting of dimethylformamide, dimethylacetamide, and propylene glycol.

[0260] In some embodiments, the molar ratio of L1-CIDE to thiol reactive groups is from about 3:1 to about 20:1.

[0261] In some embodiments, contacting the antibody with a solution comprising L1-CIDE, a thiol reactive group, and a suitable solvent is allowed to proceed for about 1 to about 24 hours. In some embodiments, contacting the antibody with a solution comprising L1-CIDE, a thiol reactive group, and a suitable solvent is allowed to proceed at about room temperature (about 25° C.) to about 37° C.

[0262] In some embodiments of the above general method, the suitable solvent is a polar aprotic solvent selected from the group consisting of dimethylformamide, tetrahydrofuran, ethyl acetate, acetone, acetonitrile, dimethylsulfoxide, and propylene carbonate.

[0263] In some embodiments of the general method, the base is selected from the group consisting of N,N-diisopropylethylamine (DIEA), triethylamine, and 2,2,2,6,6-tetramethylpyridine. In some embodiments, the coupling reagent is selected from the group consisting of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU ... O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), O-(5-norbornene-2,3-dicarboximide)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TNTU), O-(1,2-dihydro-2-oxo-1-pyridyl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), and carbonyldiimidazole (CDI).

[0264] In some embodiments of the above general method, the contacting proceeds for about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 20 hours, 40 hours, 60 hours, or 72 hours.

[0265] In some embodiments of the above general method, the contacting proceeds at about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 60°C, 70°C, 80°C, 90°C, or 100°C.

[0266] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0267] Example 1 Synthesis of L1Hy-PE-CIDE-1 L1Hy-PE-CIDE-1: (3R,5S)-1-((2R)-2-(3-(2-(4-((1r,3R)-3-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-((2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)carbamoyl)cyclobutane-1-carboxamide)hexanamide )benzyl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000090.tif102170

[0268] Step 1: Di-tert-butyl ((3R,5S)-5-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)pyrrolidin-3-yl)phosphate TIFF2025508328000091.tif42170To a solution of (2S,4R)-N-((S)-1-(4-cyanophenyl)ethyl)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (500 mg, 0.921 mmol) and 1H-tetrazole (6.1 mL, 0.45 M in ACN) in THF (15 mL) under nitrogen was added di-tert-butyl diisopropylphosphoramidite (511 mg, 1.84 mmol) at 0° C. The resulting solution was stirred at room temperature overnight. Then t-BuOOH (5M in decane) (0.74 mL, 3.69 mmol) was added at -30°C and stirred at room temperature for 5 h. The reaction was quenched with NaHSO3. The resulting solution was extracted with EtOAc and the organic layers were combined. The organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (gradient: 0% to 87% ethyl acetate / petroleum ether) to give 420 g (62% yield) of the title compound as a colorless oil. LC-MS: (ESI, m / z): [M+H] + =735.

[0269] Step 2: (3R,5S)-5-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)pyrrolidin-3-yl dihydrophosphate TIFF2025508328000092.tif49170 Under nitrogen, a solution of di-tert-butyl((3R,5S)-5-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)pyrrolidin-3-yl)phosphate (70.0 mg, 0.100 mmol) in HCOOH (0.5 mL) and water (0.5 mL) was stirred at 60° C. for 1 h. The solution was concentrated under vacuum to give 50 mg (crude) of the title compound as a solid. LC-MS: (ESI, m / z): [M+H] + =549.

[0270] Step 3: 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025508328000093.tif49170 (3R,5S)-5-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)pyrrolidin-3-yl dihydrophosphate (62.8 mg, 0.115 mmol), 1-(((2S)-1-((4-((2-(6-amino- A solution of 5-(8-(2-((1r,3r)-3-(piperidin-4-yloxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (56.0 mg, 0.0602 mmol), CH3COONa (14.9 mg, 0.182 mmol) was stirred at room temperature for 1 h. Then NaBH3CN (5.6 mg, 0.0891 mmol) was added and stirred at room temperature for 3 h. The reaction was quenched with water. The reaction mixture was concentrated under vacuum. The residue was purified by pre-packed C18 column (solvent gradient: 0-100% ACN in water (0.05% NH4HCO3)) to give 60 mg (65% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] +=1464.

[0271] Step 4: (3R,5S)-1-((2R)-2-(3-(2-(4-((1r,3R)-3-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-((2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl) Oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000094.tif42170 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2 To a solution of 1-(2-((2-aminoethyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (10.5 mg, crude) and DIPEA (54.2 mg, 0.420 mmol) was added HATU (18.7 mg, 0.0492 mmol) at room temperature. The reaction was stirred at room temperature for 10 minutes. The crude was purified by Prep-HPLC (Column: Xselect CSH C18 OBD column 30*150mm 5μm, n; Mobile phase A: water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 14% B to 23% B in 12 min, 23% B in 12 min; Wavelength: 254 / 220nm; R T1 (min: 11.75 min) to give 20.3 mg (30% yield) of L1Hy-PE-CIDE-1 as a white solid. LC-MS: (ESI, m / z): [M+H] + =1643. 1H NMR(300MHz,DMSO-d6)δ 10.24(s,1H), 9.67(s,1H), 8.56(d,J=7.3Hz,1H), 8.12(s,1H), 7.88-7.73(m,4H), 7.68-7.49(m,4H), 7.49-7.25 (m,6H), 7.20-6.80(m,4H), 6.61(s,1H), 6.21-5.95(m,2H), 5.30-5.00(m,3H), 4.90(t,J=6.9Hz,1H), 4.76(s,1H) , 4.52-4.27(m,7H), 3.76-3.67(m,6H), 3.55-3.40(m,9H), 3.30-3.20(m,5H), 3.15-2.95(m,5H), 2.81(s,3H), 2.7 3(s,6H), 2.45-2.30(m,9H), 2.01-1.54(m,15H), 1.49-1.20(m,5H), 0.94(d,J=6.4Hz,3H), 0.79(d,J=7.0Hz,3H).

[0272] Example 2 Synthesis of L1-PE-CIDE-2 L1-PE-CIDE-2: (3R,5S)-1-((2R)-2-(3-(2-((3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl)oxy)phenyl)pyridazine- 4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000095.tif125170

[0273] Step 1: Ethyl (S)-1-((1-((4-(chloromethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025508328000096.tif30170To a solution of ethyl (S)-1-((1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carboxylate (1.00 g, 2.30 mmol) in THF (10 mL) was added SOCl2 (0.830 g, 6.97 mmol) at 0 °C. The reaction was stirred at room temperature for 1 h. MeOH was added at 0 °C to quench the reaction. The solvent was evaporated and the crude was purified by flash chromatography on silica gel (solvent gradient: 0-10% MeOH in DCM) to give 910 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =453.

[0274] Step 2: tert-Butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate To a solution of tert-butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (1.15 g, 1.86 mmol) and CsCO (1.22 g, 3.74 mmol) in DMF (10 mL) under nitrogen was added ethyl (S)-1-((1-((4-(chloromethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carboxylate (850 mg, 1.87 mmol) at 0 °C. The reaction was stirred at 0° C. for 15 min. EtOAc was added and washed with water. The organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum. The crude was purified by flash chromatography on silica gel (solvent gradient: 0-15% MeOH in DCM) to give 690 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =1034.

[0275] Step 3: 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-4-(tert-butoxycarbonyl)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid A solution of tert-butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (690 mg, 0.670 mmol) and LiOH.HO (84.2 mg, 2.00 mmol) in THF (10 mL) and water (10 mL) was stirred at room temperature for 1 h. The crude was purified by pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH4HCO3)) to give 600 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =1006.

[0276] Step 4: 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (2,2,2-trifluoroacetate) TIFF2025508328000099.tif35170A solution of 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-4-(tert-butoxycarbonyl)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (300 mg, 0.300 mmol) in 5% TFA in HFIP (9 mL) was stirred at room temperature for 1 h. The solvent was evaporated and the crude product was used directly in the next step. LCMS (ESI) [M+H] + =906.

[0277] Step 5: 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025508328000100.tif421701-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carbo A solution of 2,2,2-trifluoroacetate (crude from the last step), (3R,5S)-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (309 mg, 0.498 mmol) and NaOAc (60.0 mg, 0.731 mmol) was stirred at room temperature for 1 h. NaBH3CN (32.0 mg, 0.510 mmol) was then added and stirred at room temperature for 1 h. The crude was purified by pre-packed C18 column (0-100% MeOH in water (0.05% NH4HCO3)) to give 324 mg of the title compound as a white solid. LCMS(ESI)[M+H] + =1510.

[0278] Step 6: (3R,5S)-1-((2R)-2-(3-(2-((3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl)oxy)phenyl)pyridazin-4-yl) -3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000101.tif47170 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl in DMF A solution of 1-(5-aminopentyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (90.9 mg, crude), HATU (106 mg, 0.280 mmol) and DIPEA (277 mg, 2.15 mmol) was stirred at room temperature for 15 minutes. The crude was purified under the following conditions: Column: Xselect CSH C18 OBD column 30*150mm 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 19% B to 32% B in 10 min, 32% B; 254 / 220nm; R T(Min.): 10 grams of L1-PE-CIDE-2 is a white solid and 173.7 mg is purified by Prep HPLC. LCMS(ESI)[M+H] + =1674. 1 H NMR (300 MHz, DMSO-d6, ppm)δ 10.18(s,1H), 9.00(d,J=1.8Hz,1H), 8.47(d,J=7.7Hz,1H), 7.91(d,J=6.4Hz,1H ), 7.82(q,J=8.0,6.7Hz,2H), 7.67(d,J=8.3Hz,2H), 7.58(d,J=7.8Hz,2H), 7.53 -7.25(m,8H), 7.16(t,J=7.5Hz,1H), 6.98(s,3H), 6.71(s,1H), 6.35(s,1H), 6.1 2(s,1H), 6.01(s,1H), 5.44(s,1H), 5.10(s,2H), 4.92(t,J=7.2Hz,1H), 4.83-4.7 0(m,2H), 4.58(s,2H), 4.47(s,2H), 4.45-4.30(m,3H), 3.78-3.67(m,11H), 3.40 -3.35(m,4H), 3.25-3.17(m,3H), 3.13-3.05(m,5H), 3.00-2.92(m,2H), 2.85-2. 75(m,1H), 2.50-2.28(m,9H), 2.10-1.85(m,4H), 1.79-1.70(m,3H), 1.69(s,1H) , 1.53-1.30(m,9H), 1.27-1.10(m,5H), 0.98(d,J=6.5Hz,3H), 0.85-0.75(m,3H).

[0279] Example 3 Synthesis of L1-PE-CIDE-3 L1-PE-CIDE-3: 4-((S)-6-(dimethylamino)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl(6-(2-hydroxyphenyl)-4-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(( (S)-1-(4-(4-Methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate)

[0280] Step 1: 1-(((2S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-4-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidine- 1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025508328000102.tif43170 1-(((2S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-4-(8-(2-(2-((R)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (50.0 mg, 0.0527 mmol, same as described in Example 3a) in MeOH (3 mL) and DCM (1 ml) under nitrogen. A solution of NT, (3R,5S)-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (26.0 mg, 0.0419 mmol), HOAc (9.5 mg, 0.157 mmol) was stirred at 30° C. for 1 h. NaBH3CN (6.6 mg, 0.105 mmol) was then added and stirred at 30° C. for 0.5 h. The reaction solution was concentrated under vacuum. The residue was purified by elution with a 1:1 hexane-hexane dihydrochloride column (solvent gradient: 0-100% CH3OH in water (0.05% NH4HCO3)) to give 23.0 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =1553.

[0281] Step 2: 4-((S)-6-(dimethylamino)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl(6-(2-hydroxyphenyl)-4-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1 -(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025508328000103.tif43170

[0282] In DMF (1 mL), 1-(((2S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-4-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)- A solution of 3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (23.0 mg, 0.0148 mmol), 1-(5-aminopentyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (5.5 mg, crude), HATU (7.3 mg, 0.0192 mmol) and DIPEA (19.8 mg, 0.153 mmol) was stirred at room temperature for 10 minutes. The crude was purified under the following conditions: Column: Xselect CSH C18 OBD column 30*150mm 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 18% B to 30% B, 30% B in 12 min; Wavelength: 254 / 220nm; R T (min): Purification by Prep-HPLC using 11 gave 8.7 mg of L1-PE-CIDE-3 as a white solid. LCMS (ESI) [M+H] + =1717. 1H NMR(300MHz,DMSO-d6,ppm)δ 10.15(s,1H), 10.07(s,1H), 9.37(br,1H), 8.95(s,1H), 8.48(d,J=7.6Hz,1H), 8.00-7.80(m,4H), 7.66-7.57(m,3H), 7.49-7.28( m,6H), 7.02-6.89(m,4H), 6.78(d,J=7.1Hz,1H), 6.44(s,1H), 6.10(s,1H), 5.10(s,2H), 4.89(t,J=6.9Hz,1H), 4.80(s,1H), 4.73 (s,2H), 4.50-4.30(m,6H), 3.85-3.63(m,9H), 3.52-3.45(m,3H), 3.35(t,J=7.0Hz,3H), 3.20-2.98(m,8H), 2.90-2.70(m,8H), 2. 48-2.34(m,9H), 1.93(s,4H), 1.83-1.55(m,6H), 1.49-1.32(m,8H), 1.28-1.13(m,6H), 0.94(d,J=6.4Hz,3H), 0.85-0.74(m,3H).

[0283] Example 3a 4-((S)-6-(dimethylamino)-2-(1-((5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl(4-(8-(2-(2-((R)-4-(2-((5-((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthio) Azol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025508328000104.tif127170

[0284] Step 1: tert-Butyl (3R)-4-(2-((4-(3-(3-((((4-((S)-6-(((allyloxy)carbonyl)amino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-(((allyloxy)carbonyl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate TIFF2025508328000105.tif52170 tert-Butyl (3R)-4-(2-((4-(3-(6-(2-(((allyloxy)carbonyl)oxy)phenyl)-3-aminopyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (500 mg, 0.01 ml) in DCM (15 mL) under nitrogen. To a solution of ethyl (S)-1-((6-(((allyloxy)carbonyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (1.05 g, 2.15 mmol) and DIPEA (554 mg, 4.29 mmol) was added a solution of triphosgene (109 mg, 0.367 mmol) in DCM (1 mL) at 0° C. The reaction was stirred at room temperature for 2 h. The solvent was concentrated under vacuum. The residue was purified by pre-packed C18 column (solvent gradient: 0-100% CH3CN in water (0.05% NH4HCO3)) to give 480 mg (55% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =1216.

[0285] Step 2: tert-Butyl (3R)-4-(2-((4-(3-(3-((((4-((S)-6-amino-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate TIFF2025508328000106.tif51170Under nitrogen, tert-butyl(3R)-4-(2-((4-(3-(3-((((4-((S)-6-(((allyloxy)carbonyl)amino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-(((allyloxy)carbonyl)amino) To a solution of (3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (400 mg, 0.329 mmol), pyrrolidine (234 mg, 3.29 mmol) was added Pd(PPh3)4 (76.0 mg, 0.0658 mmol) at room temperature. The reaction was stirred at room temperature for 1 h. The reaction mixture was diluted with DCM (100 mL) and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to give 390 mg (crude) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =1048.

[0286] Step 3: tert-Butyl (3R)-4-(2-((4-(3-(3-((((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate A solution of tert-butyl (3R)-4-(2-((4-(3-(3-((((4-((S)-6-amino-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (465 mg, 0.440 mmol) and 37% aqueous CHO (465 mg, 5.73 mmol) in CHOH (5 mL) was stirred at room temperature for 1 h. NaBH3CN (140 mg, 2.22 mmol) was then added and stirred at room temperature for 1 h. The reaction was quenched with water (0.2 mL). The residue was purified by pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH4HCO3)) to give 260 mg (54% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M] + =1077.

[0287] Step 4: 1-(((2S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-4-(8-(2-(2-((R)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid A solution of tert-butyl (3R)-4-(2-((4-(3-(3-((((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (75.0 mg, 0.0697 mmol) and LiOH.HO (8.4 mg, 0.210 mmol) in THF (5 mL) and water (5 mL) was stirred at room temperature for 1 h. The solvent was concentrated in vacuo. 5% TFA in HFIP (3 mL) was then added and the solution was stirred at room temperature for 1 hour. The solvent was concentrated under vacuum. The residue was purified by pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH4HCO3)) to give 55.0 mg (83 percent yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M] + =949.

[0288] Step 5: 1-(((2S)-6-(dimethylamino)-1-((4-(((4-(8-(2-(2-((R)-4-(2-((5-((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxa 2-(2-hydroxyphenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025508328000109.tif54170Under nitrogen, 1-(((2S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-4-(8-(2-(2-((R)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexane- A solution of (2-yl)carbamoyl)cyclobutane-1-carboxylic acid (20.0 g, 0.0211 mmol), (2S,4R)-4-hydroxy-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (19.4 mg, 0.0359 mmol) and NaOAc (4.0 mg, 0.0488 mmol) was stirred at room temperature for 1 hour. NaBH3CN (5.2 mg, 0.0827 mol) was then added and stirred at room temperature for 3 hours. The reaction solution was concentrated under vacuum. The residue was purified by pre-packed C18 column (solvent gradient: 0-100% CH3OH in water (0.05% NH4HCO3)) to give 18 mg (58 percent yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =1473.

[0289] Step 6: 4-((S)-6-(dimethylamino)-2-(1-((5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl (4-(8-(2-(2-((R)-4-(2-((5-((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methyl ruthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025508328000110.tif411701-(((2S)-6-(dimethylamino)-1-((4-(((4-(8-(2-(2-((R)-4-(2-((5-((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[ To a solution of 3.2.1]octan-3-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (40.0 mg, 0.0272 mmol), 1-(2-((5-aminopentyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (9.70 mg, crude) and DIPEA (35.1 mg, 0.272 mmol) was added HATU (13.5 mg, 0.0355 mmol) at room temperature. The reaction was stirred at room temperature for 10 minutes. The crude was purified under the following conditions: Column: XSelect CSH Fluorophenyl, 30 x 150 mm, 5 μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 15% B to 20% B in 10 min; Wavelength: 254 / 220 nm; R T (min): 10.5, purified by Prep-HPLC to give 13.0 mg (28.3% yield) of the target compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =1808. 1H NMR(300MHz,DMSO-d6,ppm)δ 10.09(s,1H), 9.97(s,1H), 9.50(s,2H), 8.88(d,J=1.3Hz,1H), 8.30(d,J=7.7Hz,1 H), 8.00-7.80(m,4H), 7.58-7.50(m,3H), 7.39-7.16(m,7H), 6.96(s,2H), 6.84(dd d,J=11.0, 7.0, 2.3Hz,2H), 6.65(d,J=6.6Hz,1H), 6.29(s,1H), 6.00(s,1H), 5.01( s,2H), 4.95-4.80(m,1H), 4.59(s,2H), 4.37(s,2H), 4.35-4.21(m,4H), 4.21-4.15 (m,2H), 3.59-3.48(m,6H), 3.40-3.30(m,4H), 3.27-2.97(m,14H), 2.89(s,4H), 2. 70-2.60(m,9H), 2.45-2.34(m,4H), 2.30-2.19(m,2H), 2.17-2.03(m,2H), 2.05-1. 83(m,5H), 1.83-1.66(m,5H), 1.66-1.49(m,4H), 1.34(d,J=7.1Hz,3H), 1.25(d,J= 7.0Hz,3H), 1.20-1.10(m,6H), 0.85(d,J=6.6Hz,3H), 0.69(dd,J=10.7, 6.6Hz,3H).

[0290] Example 4 Synthesis of L1-PP-CIDE-1 L1-PP-CIDE-1: 6-(8-chloro-4-(2-(dimethylamino)ethyl)-2-((tetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6-dihydro-4H-[1,4]oxazepino[5,6,7-de]quinazolin-9-yl)-4-methyl-5-(trifluoromethyl)pyridin-2-amine (2,2,2-trifluoroacetate) TIFF2025508328000111.tif99170

[0291] Step 1: tert-Butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate A solution of tert-butyl (3R)-4-(2-((4-(3-(3-amino-6-chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (3.00 g, 5.37 mmol, provided by Genentech), 2-hydroxyphenylboronic acid (0.890 g, 6.46 mmol), Pd(PPh3)4 (1.24 g, 1.08 mmol) and K2CO3 (2.23 g, 16.1 mmol) in dioxane (37.5 mL) and HO (7.5 mL) under nitrogen was stirred at 100 °C for 1 h. The reaction was diluted with water and extracted with DCM. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting crude product was purified by flash chromatography on silica gel (solvent gradient: 0-10% MeOH / DCM) to give 2.26 g (68% yield) of the title compound as a yellow solid. LCMS (ESI, m / z): [M+H] + =617.

[0292] Step 2: tert-Butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-((di-tert-butoxyphosphoryl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate TIFF2025508328000113.tif37170 To a solution of tert-butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (500 mg, 0.811 mmol) and 1H-tetrazole (5.4 mL, 0.45 M in ACN) in THF (10 mL) under nitrogen was added di-tert-butyl diisopropylphosphoramidite (1.35 g, 4.86 mmol) dropwise at 0° C. The reaction was stirred at 0° C. for 1 h. Then t-BuOOH (5M in decane) (0.64 mL, 3.20 mmol) was added at -30°C and stirred at -30°C for 30 min. The mixture was then stirred at room temperature overnight. The reaction mixture was poured into aqueous NaHSO3 and extracted with EtOAc. The combined organic layers were washed with water, brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting crude product was purified by packed C18 column (solvent gradient: 0-100% ACN in water (0.05% NH4HCO3)) to give 140 mg (21% yield) of the title compound as a yellow oil. LCMS (ESI, m / z): [M+H] + =809.

[0293] Step 3: tert-Butyl (3R)-4-(2-((4-(3-(6-(2-((di-tert-butoxyphosphoryl)oxy)phenyl)-3-((((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate TIFF2025508328000114.tif59170 tert-Butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-((di-tert-butoxyphosphoryl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate in THF (5 mL) under nitrogen. To a solution of sylate (120 mg, 0.148 mmol), ethyl (S)-1-((6-(dimethylamino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (225 mg, 0.518 mmol) and DIPEA (303 mg, 2.34 mmol) was added triphosgene (79.8 mg, 0.269 mmol) at 0° C. The reaction was stirred at room temperature for 30 min. The reaction mixture was then purified by pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH4HCO3)) to give 35 mg of the title compound as a yellow solid. LCMS (ESI, m / z): [M+H] + =1269.

[0294] Step 4: 1-(((2S)-6-(dimethylamino)-1-((4-((((4-(8-(2-(2-((R)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025508328000115.tif65170 tert-Butyl (3R)-4-(2-((4-(3-(6-(2-((di-tert-butoxyphosphoryl)oxy)phenyl)-3-((((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamide)hexaneamine in THF (0.3 mL) under nitrogen. To a solution of 3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate (35.0 mg, 0.0280 mmol) was added a solution of LiOH.H2O (3.5 mg, 0.0850 mmol) in H2O (0.3 mL) at room temperature. The reaction was stirred at room temperature for 1 h. The resulting mixture was concentrated under vacuum. Then a solution of concentrated HCl (0.33 mL) in H2O (0.67 mL) was added and stirred at room temperature for 10 min. The resulting mixture was basified with saturated NaHCO3 solution at 0°C to pH = and purified by packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH4HCO3)) to give 13 mg of the title compound as a white solid. LCMS (ESI, m / z): [M+H] + =1029.

[0295] Step 5: 1-(((2S)-6-(dimethylamino)-1-((4-(((4-(8-(2-(2-((R)-4-(2-((5-((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)o 2-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (2,2,2-trifluoroacetate) TIFF2025508328000116.tif53170 1-(((2S)-6-(dimethylamino)-1-((4-((((4-(8-(2-(2-((R)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1- A solution of (oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (18.0 mg, 0.0180 mmol), (2S,4R)-4-hydroxy-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (9.5 mg, 0.0180 mmol) and HOAc (2.1 mg, 0.0360 mmol) was stirred at room temperature for 1 hour. NaBH3CN (1.7 mg, 0.0260 mmol) was then added and stirred at room temperature for 0.5 hours. The crude product was purified using the following conditions: Column: Xselect CSH C18 OBD column 30*150mm 5μm, n; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 11% B to 23% B in 12 min, 23% B; Wavelength: 254 / 220nm; R T Purification by Prep-HPLC at (min): 12 gave 6 mg of the title compound as a white solid. LCMS (ESI, m / z): [M+H] + =1553.

[0296] Step 6: 4-((S)-6-(dimethylamino)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl (4-(8-(2-(2-((R)-4-(2-((5-((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl )phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025508328000117.tif47170Under nitrogen, 1-(((2S)-6-(dimethylamino)-1-((4-(((4-(8-(2-(2-((R)-4-(2-((5-((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazotrimethylenediamine (DMF) (0.8 mL) was dissolved in 1-(((2S)-6-(dimethylamino)-1-((4-(((4-(8-(2-(2-((R)-4-(2-((5-((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazotrimethylenediamine (DMF) (0.8 mL). To a solution of zabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (2,2,2-trifluoroacetate) (6.0 mg, 0.00400 mmol), 1-(5-aminopentyl)pyrrole-2,5-dione (2,2,2-trifluoroacetate) (0.9 mg, crude) and DIPEA (5.0 mg, 0.0400 mmol) was added HATU (1.9 mg, 0.00500 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The resulting mixture was purified under the following conditions: Column: XBridge Prep C18 OBD column, 30*100mm, 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 15% B to 33% B in 11 min, 33% B; Wavelength: 254 / 220nm; R T (min): 11 and purified by Prep-HPLC to give 2.7 mg of L1-PP-CIDE-1 as a white solid. LCMS(ESI,m / z):[M+H] + =1717. 1H NMR(300MHz,DMSO-d6,ppm)δ 10.14(d,J=11.3Hz,2H), 8.97(s,1H), 8.40(d,J=7.7Hz,1H), 8.00-7.85(m,2 H), 7.82-7.75(s,1H), 7.70-7.60(m,3H), 7.60-7.57(m,1H), 7.57-7.42(m,6H) ), 7.35-7.30(m,1H), 6.93(s,2H), 6.66(d,J=6.6Hz,1H), 6.31(s,1H), 6.10(s ,1H), 5.10(s,2H), 4.94-4.83(m,1H), 4.58(s,2H), 4.46(s,2H), 4.43-4.32(m ,4H), 4.30-4.25(m,2H), 3.70-3.59(m,6H), 3.50-3.40(m,3H), 3.40-3.30(m, 4H), 3.31-2.92(m,12H), 2.79-2.65(m,6H), 2.50-2.35(m,7H), 2.37-2.05(m, 1H), 2.10-2.00(m,1H), 1.90(s,3H), 1.86-1.56(m,6H), 1.50-1.40(m,4H), 1. 35(d,J=7.0Hz,4H), 1.25-1.14(m,6H), 1.05-0.90(m,3H), 0.90-0.75(m,3H).

[0297] Example 5 Synthesis of L1-PP-CIDE-2 L1-PP-CIDE-2: 4-((S)-6-(dimethylamino)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl(4-(8-(2-((1R,3r)-3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyanophenyl)ethyl) carbamoyl)-4-hydroxypyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025508328000118.tif151170

[0298] Step 1: tert-Butyl 4-((1r,3r)-3-((4-(3-(6-(2-((allyloxy)carbonyl)oxy)phenyl)-3-aminopyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025508328000119.tif34170To a solution of tert-butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (535 mg, 0.832 mmol) and DIPEA (967 mg, 7.48 mmol) in DCM (6 mL) under nitrogen was added allyl carbonochloridate (350 mg, 2.91 mmol) at 0° C. The reaction was stirred at room temperature for 30 min. The crude was purified by flash chromatography on silica gel (solvent gradient: 0-10% MeOH in DCM) to give 410 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =728.

[0299] Step 2: tert-Butyl 4-((1r,3r)-3-((4-(3-(3-((((4-((S)-6-(((allyloxy)carbonyl)amino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-(((allyloxy)carbonyl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025508328000120.tif46170Under nitrogen, tert-butyl To a solution of 4-((1r,3r)-3-((4-(3-(6-(2-(((allyloxy)carbonyl)oxy)phenyl)-3-aminopyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (440 mg, 0.605 mmol) and ethyl (S)-1-((6-(((allyloxy)carbonyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (921 mg, 1.81 mmol) and DIPEA (468 mg, 3.63 mmol) was added triphosgene (89.8 mg, 0.302 mmol) at 0° C. The reaction was stirred at room temperature for 0.5 h. Then additional triphosgene (36.0 mg, 0.121 mmol) was added at 0° C. and stirred at room temperature for 0.5 h. Aqueous NH4Cl was added at 0° C. to quench the reaction. The solvent was concentrated under vacuum. The crude was purified by pre-packed C18 column (solvent gradient: 0-100% ACN in water (0.05% NH4HCO3)) to give 280 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =1244.

[0300] Step 3: tert-Butyl 4-((1r,3r)-3-((4-(3-(3-((((4-((S)-6-amino-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate To a solution of tert-butyl 4-((1r,3r)-3-((4-(3-(3-((((4-((S)-6-amino-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (260 mg, 0.209 mmol) and pyrrolidine (148 mg, 2.09 mmol) in DCM (5 mL) under nitrogen was added Pd(PPh3)4 (24.1 mg, 0.0208 mmol) at room temperature. The reaction was stirred at room temperature for 1 h. Water was added and extracted three times with DCM. The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo to give 310 mg (crude) of the title compound, which was used directly in the next step. LCMS (ESI) [M+H] + =1076.

[0301] Step 4: tert-Butyl 4-((1r,3r)-3-((4-(3-(3-((((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate A solution of tert-butyl 4-((1r,3r)-3-((4-(3-(3-((((4-((S)-6-amino-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (310 mg, crude) and aqueous CHO (37%) (259 mg, 3.19 mmol) in MeOH (3 mL) was stirred at room temperature for 1 h. NaBHCN (75.9 mg, 1.21 mmol) was then added and stirred at room temperature for 1 h. The crude was purified by pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH4HCO3)) to give 120 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =1104.

[0302] Step 5: 1-(((2S)-1-((4-((((4-(8-(2-((1r,3r)-3-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid To a solution of tert-butyl 4-((1r,3r)-3-((4-(3-(3-((((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (120 mg, 0.108 mmol) in THF (3 mL) under nitrogen was added a solution of TMSOK in THF (1 M, 2 mL) at -60 °C. POCl3 (165 mg, 1.08 mmol) was then added at -60 °C. The reaction was stirred at -60 °C for 20 min. NaOH (80.0 mg, 2.00 mmol) in H2O (1.6 mL) was then added at -60 °C and stirred at room temperature for 4 h. The crude was purified by packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH4HCO3)) to give 65.0 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =1156.

[0303] Step 6: 1-(((2S)-6-(dimethylamino)-1-oxo-1-((4-((((6-(2-(phosphonooxy)phenyl)-4-(8-(2-((1r,3r)-3-(piperidin-4-yloxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)hexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid A solution of 1-(((2S)-1-((4-((((4-(8-(2-((1r,3r)-3-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (65.0 mg, 0.056 mmo) in a solution of 5% TFA in HFIP (6.1 mL) was stirred at room temperature for 15 min. The solvent was evaporated and the crude was purified by pre-packed C18 column (0-100% ACN in water (0.05% NH4HCO3)) to give 58.0 mg of the title compound as a yellow oil. LCMS (ESI) [M+H] + =1055.

[0304] Step 7: 1-(((2S)-1-((4-(((4-(8-(2-((1R,3r)-3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-4-hydroxypyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025508328000125.tif451701-(((2S)-6-(dimethylamino)-1-oxo-1-((4-((((6-(2-(phosphonooxy)phenyl)-4-(8-(2-((1r,3r)-3-(piperidin-4-yloxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl) in MeOH (1 mL) and DCM (3 mL). A solution of (2S,4R)-N-((S)-1-(4-cyanophenyl)ethyl)-4-hydroxy-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide (73.5 mg, 0.157 mmol) and HOAc (6.6 mg, 0.11 mmol) was stirred at room temperature for 1 hour. Then NaBH3CN (5.1 mg, 0.083 mmol) was added and stirred at room temperature for 0.5 hours. The crude was purified by pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH4HCO3)) to give 44.0 mg of the title compound as a white solid. LCMS (ESI) [M+H] + =1508.

[0305] Step 8: 4-((S)-6-(dimethylamino)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl(4-(8-(2-((1R,3r)-3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyanophenyl)ethyl)carba moyl)-4-hydroxypyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(phosphonooxy)phenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025508328000126.tif45170

[0306] In DMF (0.8 mL), 1-(((2S)-1-((4-(((4-(8-(2-((1R,3r)-3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-4-hydroxypyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-(ho A solution of (sulfonoxy)phenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (44.0 mg, 0.0292 mmol) and 1-(5-aminopentyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetic acid) (19.9 mg, crude), HATU (14.4 mg, 0.0378 mmol) and DIPEA (37.7 mg, 0.290 mmol) was stirred at room temperature for 10 minutes. The crude was purified under the following conditions: Column: XBridge Shield RP18 OBD column, 30*150mm, 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 15% B to 25% B, 25% B in 20min; Wavelength: 254 / 220nm; R T (min): 20, purified by Prep HPLC to give 13.6 mg of L1-PP-CIDE-2 as a white solid. LCMS (ESI) [M+H] + =1672. 1H NMR (400 MHz, DMSO-d6, ppm)δ 10.19(s,2H), 9.45(s,1H), 8.50(d,J=7.3Hz,1H), 7.95-7.85(m,3H), 7.83- 7.76(m,3H), 7.72-7.64(m,2H), 7.62-7.49(m,2H), 7.48-7.30(m,5H), 7.24- 6.98(m,3H), 6.72(s,1H), 6.20(s,1H), 6.15(s,1H), 5.24-5.09(m,3H), 4.91 (q,J=7.2Hz,1H), 4.69(s,2H), 4.53(d,J=5.1Hz,2H), 4.47-4.24(m,5H), 3.7 0-3.60(m,7H), 3.45-3.40(m,2H), 3.38-3.34(m,2H), 3.19-3.06(m,6H), 3.0 5-2.98(m,3H), 2.75(d,J=3.8Hz,6H), 2.46-2.35(m,6H), 2.30-2.20(m,1H), 2.09-1.99(m,2H), 1.99-1.85(m,5H), 1.84-1.56(m,8H), 1.54-1.41(m,5H), 1.40-1.27(m,5H), 1.26-1.13(m,3H), 1.01-0.93(m,3H), 0.87-0.72(m,3H).

[0307] Example 6 Synthesis of L1Hy-PE-CIDE-4 L1Hy-PE-CIDE-4: (3R,5S)-1-((2R)-2-(3-(2-((3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-((5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy )phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate(2,2,2-trifluoroacetate)

[0308] Step 1: Di-tert-butyl ((3R,5S)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl)phosphate. To a solution of (2S,4R)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (500 mg, 0.81 mmol) and 1H-tetrazole (5.4 mL, 0.45 M in ACN) in THF (5 mL) under nitrogen was added di-tert-butyl diisopropyl phosphoramidite (451 mg, 1.63 mmol) dropwise at 0° C. The reaction was stirred at room temperature overnight. tBuOOH (0.4 mL, 5 M in decane) was then added at −30° C. and stirred for 30 min at −30° C. The mixture was then stirred at room temperature for 4 h. The reaction was quenched with aqueous NaHSO3 and extracted with EtOAc. The combined organic layers were washed with water and brine. The organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by packed C18 column (solvent gradient: 0-70% ACN in water (0.05% NH4HCO3)) to give 340 mg (51%) of the title compound as a yellow oil. LC-MS: (ESI,m / z): [M+H]+=807.

[0309] Step 2: (3R,5S)-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate A solution of di-tert-butyl((3R,5S)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl)phosphate (150 mg, 0.185 mmol) in HCOOH (0.5 mL) and water (0.5 mL) under nitrogen was stirred at 60° C. for 1 h. The solution was concentrated under vacuum to give 121 mg (crude) of the title compound as a solid. LC-MS: (ESI, m / z): [M+H]+=695.

[0310] Step 3: 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025508328000127.tif86170

[0311] Step 1: (3R,5S)-1-((2R)-2-(3-(2-((3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-((5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)phenyl) Pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000128.tif461701-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabiphenyl in DMF (0.8 mL). A solution of cyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (40.0 mg, 0.0265 mmol), 1-(2-((5-aminopentyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (10.7 mg, crude), HATU (12.1 mg, 0.0318 mmol) and DIPEA (34.2 mg, 0.265 mmol) was stirred at room temperature for 10 minutes. The product was purified using the following conditions: Column: XSelect CSH Fluorophenyl, 30×150 mm, 5 μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 6% B to 36% B in 7 min; Wavelength: 254 nm; R T (min): 6.5, purified by Prep-HPLC to give 7.0 mg of L1Hy-PE-CIDE-4 as a white solid. LCMS (ESI) [M+H] + =1730. 1H NMR(400MHz,DMSO-d6,ppm)δ 10.23(s,1H), 9.00(d,J=1.8Hz,1H), 8.48(d,J=7.7Hz,1H), 7.93-7.83(m,3H), 7.67(d,J=8.3Hz,2H), 7.59(d,J=7.9Hz,1H), 7.56(s,1H) ), 7.49-7.31(m,7H), 7.18-7.07(m,3H), 6.90(br,1H), 6.64(s,1H), 6.26(s,1H), 6.11(s,1H), 5.10(s,2H), 4.92(t,J=7.1Hz,1H), 4.82- 4.70(m,2H), 4.48(s,5H), 4.41-4.33(m,3H), 3.80-3.65(m,6H), 3.15-2.96(m,18H), 2.79(s,4H), 2.73(s,6H), 2.50-2.35(m,9H), 2.05 -1.85(m,6H), 1.78-1.65(m,3H), 1.65-1.40(m,8H), 1.39(d,J=6.9Hz,3H), 1.35-1.18(m,7H), 0.98(d,J=6.5Hz,3H), 0.90-0.75(m,3H).

[0312] Example 7 Synthesis of L1-PE-CIDE-5 L1-PE-CIDE-5: (3R,5S)-1-((2R)-2-(3-(2-((3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)phenyl)pyridazinyl (4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000129.tif77170

[0313] Step 1: (3R,5S)-1-((2R)-2-(3-(2-((3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)phenyl)pyridazine-4- yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000130.tif461701-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabiphenyl in DMF (3 mL). A solution of cyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (180 mg, 0.119 mmol, synthesis described in L1-PE-CIDE-4) 1-(5-aminopentyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate salt) (45.0 mg, crude), HATU (60.6 mg, 0.159 mmol) and DIPEA (158 mg, 1.22 mmol) was stirred at room temperature for 5 min. The reaction solution was eluted under the following conditions: Column: XSelect CSH Prep C18 OBD column, 19*250mm, 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: MeOH; Flow rate: 25mL / min; Gradient: 46% B to 57% B in 11 min, 57% B; Wavelength: 254 / 220nm; R T (min): 8.75, purified by Prep-HPLC to give 66.6 mg of L1-PE-CIDE-5 as a white solid. LCMS (ESI) [M+H] + =1673. 1H NMR (300MHz, DMSO-d6, ppm) δ 10.21(s,1H), 9.50(s,1H), 9.00(d,J=1.7Hz,1H), 8.49(d,J=7.6Hz,1H), 8.01-7.84(m,3H), 7.67(d,J=8.3Hz,2H), 7.59(dd,J=8.2, 6.1Hz,2H ), 7.52-7.25(m,7H), 7.21-7.08(m,2H), 6.99(s,2H), 6.73(d,J=6.7Hz,1H), 6.37(s,1H), 6.12(s,1H), 5.10(s,2H), 4.92(t,J=7.3Hz,1H), 4. 85-4.73(m,2H), 4.60(s,3H), 4.51-4.33(m,6H), 3.85-3.70(m,7H), 3. 55-3.45(m,2H), 3.40-3.30(m,4H), 3.25-2.95(m,9H), 2.90-2.70(m,7 H), 2.47-2.30(m,9H), 2.10-1.85(m,5H), 1.85-1.54(m,6H), 1.53-1.2 9(m,8H), 1.27-1.10(m,5H), 0.98(d,J=6.5Hz,3H), 0.90-0.73(m,3H).

[0314] Example 8 Synthesis of L1Hy-PE-PCIDE-6 (3R,5S)-1-((2R)-2-(3-(2-((3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-((2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)phenyl)pyri dazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000131.tif107170

[0315] Step 1: Di-tert-butyl ((3R,5S)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl)phosphate TIFF2025508328000132.tif37170 Under nitrogen, to a solution of (2S,4R)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (500 mg, 0.81 mmol) and 1H-tetrazole (5.4 mL, 0.45 M in ACN) in THF (5 mL) was added di-tert-butyl diisopropylphosphoramidite (451 mg, 1.63 mmol) dropwise at 0° C. The reaction was stirred at room temperature overnight. tBuOOH (0.4 mL, 5M in decane) was then added at -30°C and stirred at -30°C for 30 min. The mixture was then stirred at room temperature for 4 h. The reaction was quenched with aqueous NaHSO3 and extracted with EtOAc. The combined organic layers were washed with water and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by packed C18 column (solvent gradient: 0-70% ACN in water (0.05% NH4HCO3)) to give 340 mg (51%) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =807.

[0316] Step 2: (3R,5S)-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate TIFF2025508328000133.tif44170 A solution of di-tert-butyl((3R,5S)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl)phosphate (150 mg, 0.185 mmol) in HCOOH (0.5 mL) and water (0.5 mL) under nitrogen was stirred at 60° C. for 1 h. The solution was concentrated under vacuum to give 121 mg (crude) of the title compound as a solid. LC-MS: (ESI, m / z): [M+H] + =695.

[0317] Step 3: 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025508328000134.tif59170 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methylpiperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohex-2-yl in methyl alcohol (1.2 mL) and dichloromethane (0.4 mL). A solution of (3R,5S)-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (121 mg, 0.190 mmol) and NaOAc (13.6 mg, 0.170 mmol) was stirred at room temperature for 1 h. NaBH3CN (34.7 mg, 0.550 mmol) was then added and stirred at room temperature for 0.5 h. Water was added to quench the reaction. The solvent was concentrated in vacuo. The product was purified by Prep-HPLC (Column: YMC-Actus Triart C18 ExRS, 30*150mm, 5μm; Mobile phase A: Water (10mmol / L NH4HCO3), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 36% B to 60% B in 9min, 60% B; Wavelength: 254 / 220nm; RT1(min): 7.63) to give 30.0mg (18% yield) of the title compound as a white solid. LCMS(ESI)[M+H] + =1509.

[0318] Step 4: (3R,5S)-1-((2R)-2-(3-(2-((3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-((2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)carbamoyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)phenyl) Pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazin-1-yl)ethoxy)isoxazol-5-yl)-3-methylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl dihydrophosphate (2,2,2-trifluoroacetate) TIFF2025508328000135.tif51170 1-(((2S)-1-((4-((2-(6-amino-5-(8-(2-(2-((R)-2-methyl-4-(2-((5-((R)-3-methyl-1-((2S,4R)-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-4-(phosphonooxy)pyrrolidin-1-yl)-1-oxobutan-2-yl)isoxazol-3-yl)oxy)ethyl)piperazin-1-yl)ethoxy)pyridin-4-yl)-3,8-diaza in DMF (1 mL). To a solution of bicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (30.0 mg, 0.0200 mmol), 1-(2-((2-aminoethyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (12.3 mg, crude) and DIPEA (25.6 mg, 0.200 mmol) was added HATU (9.07 mg, 0.0200 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The crude was purified by Prep-HPLC (column: Xselect CSH C18 OBD column 30*150mm 5μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60mL / min; gradient: 12% B to 24% B in 10 min, 24% B; wavelength: 254 / 220nm; RT1 (min): 10) to obtain 8.70mg (24% yield) of L1Hy-PE-CIDE-6 as a white solid. LCMS (ESI) [M+H] + =1688. 1H NMR(300MHz,DMSO-d6)δ 10.26(s,1H), 8.99(s,1H), 8.50(d,J=7.5Hz,1H), 8.15(t,1H), 7.87(d,J=6.5Hz,2H), 7.65(d,J=8.3Hz,2H), 7.61- 7.51(m,2H), 7.50-7.30(m,8H), 7.20-6.90(m,5H), 6.62(s,1H), 6.14(s,1H), 5.99(s,1H), 5.09(s,2H), 4.92(t,J= 7.2Hz,1H), 4.79(s,1H), 4.55-4.25(m,8H), 3.80-3.65(m,8H), 3.25-2.95(m,17H), 2.70(s,3H), 2.62(s,6H), 2.48 -2.38(m,9H), 2.20-1.55(m,13H), 1.52-1.30(m,5H), 1.30-1.15(m,4H), 0.85(d,J=6.4Hz,3H), 0.85-0.70(m,3H).

[0319] Example 9 Conjugation of L1-CIDE to antibodies Cysteine ​​engineered antibodies (THIOMAB TM ) was pH adjusted to pH 7.5-8.5 with 1 M Tris. 3-16 equivalents of L1-CIDE (containing a thiol-reactive maleimide group) was dissolved in DMF or DMA (concentration = 10 mM) and added to the reduced, reoxidized, pH adjusted antibody. The reaction was incubated at room temperature or 37°C and monitored until completion (1 to approximately 24 hours) as determined by LC-MS analysis of the reaction mixture. Once the reaction was complete, Ab-CIDE was purified by one or any combination of several methods with the goal of removing any remaining unreacted linker-drug intermediates and aggregated proteins (if present at significant levels).

[0320] In one example, Ab-CIDE was diluted with 10 mM histidine-acetate (pH 5.5) to a final pH of approximately 5.5 and purified by S cation exchange chromatography using a HiTrap S column or an S Maxi spin column (Pierce) connected to an Akta purification system (GE Healthcare). Alternatively, Ab-CIDE was purified by gel filtration chromatography using an S200 column or a Zeba spin column connected to an Akta purification system. Dialysis was used to purify the conjugate.

[0321] THIOMAB TM Ab-CIDE was formulated in 20 mM His / acetate, pH 5, with 240 mM sucrose using gel filtration or dialysis. Purified Ab-CIDE was concentrated by centrifugal ultrafiltration, filtered through a 0.2 μm filter under sterile conditions, and frozen at −20° C. for storage.

[0322] Example 10 Cellular assay for determining DC50 and Dmax HCC515 and H1944 cells were seeded in 384-well plates at 4000 and 2500 cells / well, respectively. The next day, Ab-CIDE was added. After 24 hours of drug treatment, cells were fixed with 4% formaldehyde for 15 minutes. Plates were washed three times with PBS. Cells were incubated with IF blocking solution (10% FCS, 1% BSA, 0.1% Triton, 0.01% azide, X-100 in PBS). After 1.5 hours, a 2x solution of primary antibody diluted in IF blocking buffer: BRM (Cell signaling catalogue no. 11966, 1:2000) was added. Plates were incubated overnight at 4°C. The next morning, cells were washed three times with PBS. Cells were then incubated with secondary antibody (rabbit-Alexa 488A21206 (1:2000)) for 1 hour at room temperature in the dark. Hoechst H3570 was added to the wells at 1:5000 and the plate was incubated for an additional 30 minutes. The plate was washed 3x with PBS and incubated with OperaPhenix TMImaging was performed using a High Content Screening System. Nuclear BRM mean signal intensity was quantified using nuclear staining as a mask.

[0323] The data are shown in Table 1 below. The data demonstrate the success of the antibody targeting strategy disclosed herein. Negative control: anti-TROP2 does not interact with NCI-H1944 cells, whereas anti-TfR2 does. The data show that multiple Ab-L1-CIDEs have both desirable low DC50 values ​​and desirable high DCmax values.

[0324] Table 1. TIFF2025508328000136.tif243170TIFF2025508328000137.tif246170

[0325] Example 11 In vivo assays for PLAU and KRT80 inhibition PLAU inhibition is an indication of the suppression of the mRNA transcript associated with the PLAU gene in response to BRM-degrader treatment. BRM activity controls the production of this transcript. KRT80 is a downstream marker of BRM degradation. Therefore, both PLAU and KRT80 assays are indicators of BRM degradation in vivo.

[0326] PK / PD HCC515 Tumor Assay: The PK / PD effects of anti-CD71-BRM Ab-CIDE were evaluated in a mouse xenograft model of HCC515 human non-small cell lung cancer. HCC515 cells were obtained from Genentech's cell line repository. The cell line was authenticated by short tandem repeat (STR) profiling using the Promega PowerPlex 16 System and compared with external STR profiles of the cell line to confirm the ancestry of the cell line.

[0327] To establish this model, tumor cells (5 million in 0.1 mL Hank's Balanced Salt Solution) were inoculated subcutaneously into the flank of female CB-17 SCID mice (Charles River Laboratories). Once tumors reached the desired volume (200-300 mm3), mice were randomized into groups of n=5, each with a similar distribution of tumor sizes, and received a single intravenous injection of vehicle (histidine buffer) or test article through the tail vein. All anti-CD71-BRM Ab-CIDE and unconjugated antibodies were formulated in histidine buffer (20 mM histidine acetate (pH 5.5), 240 mM sucrose, 0.02% Tween 20). Unconjugated BRM CIDE was formulated in 10% hydroxypropyl-beta-cyclodextrin, 50 mM sodium acetate (pH 4).

[0328] Four days after dosing, mice were euthanized and tumors and whole blood were collected. Tumors were excised and divided into two aliquots prior to flash freezing in liquid nitrogen. One aliquot was used to measure the levels of released BRM CIDE, and the other was used to evaluate modulation of downstream PD markers. Whole blood was collected under surgical anesthesia by terminal cardiac puncture into tubes containing lithium heparin. Blood was placed on wet ice (within 15 minutes of collection) until centrifugation. Samples were centrifuged at 10,000 rpm for 5 minutes at 4°C, and plasma was collected, placed on dry ice, and stored at -70°C until analysis of linker stability and total antibody pharmacokinetics.

[0329] KRT80 and PLAU qRT-PCR assay: For qRT-PCR, 15-30 mg of frozen tissue was homogenized in 300 ul of lysis buffer by agitation in a Bullet Blender (Next Advance). RNA was prepared with MagMax mirVana Total RNA Isolation Kit (Applied Biosystems). Gene expression levels were detected with FAM probes (ThermoFisher Scientific) using Taqman One-Step RT-PCR Master Mix Reagent kit (ThermoFisher Scientific). Analysis was performed using a 7900HT SDS (ThermoFisher Scientific). Expression levels are presented relative to the housekeeping gene, GusB (2-ΔCt). TIFF2025508328000138.tif31170

[0330] The data are shown in Table 2 TIFF2025508328000139.tif188170

[0331] Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0332] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the subject matter described herein, and the present disclosure is not limited to only the methods and materials described.

[0333] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs.

[0334] Throughout this specification and the claims, the words "comprise" and "comprises" are used in their open-ended sense, unless the context is otherwise contradictory. The embodiments described herein are understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0335] As used herein, the term "about" when referring to a value is meant to encompass variations from the particular amount, in some embodiments, ±50%, in some embodiments, ±20%, in some embodiments, ±10%, in some embodiments, ±5%, in some embodiments, ±1%, in some embodiments, ±0.5%, and in some embodiments, ±0.1%, as appropriate for carrying out the disclosed methods or using the disclosed compositions.

[0336] Where a range of values ​​is provided, unless the context dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of the range and any other stated or intervening value within that range is included. The upper and lower limits of these smaller ranges, which may be independently included in smaller ranges, are also included, subject to any specifically excluded limit within the stated range. Where the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included.

[0337] Many modifications and other embodiments of the subject matter set forth herein will come to mind to one skilled in the art to which this subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the accompanying drawings. It is to be understood, therefore, that the subject matter is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. Chemical structure: Ab―(L1―D) ｊ [In the formula, D has the structure: E3LB-L2-PB (In the formula, E3LB is the E3 ligase-binding (E3LB) ligand covalently attached to L2, and the E3 ligase is the von Hippel-Lindau (VHL) tumor suppressor protein; L2 is a linker covalently linking E3LB and PB; PB is a protein binding group covalently attached to L1 and L2. CIDE having the formula: Ab is an antibody covalently bound to L1; L1 is a linker covalently connecting Ab and D; j has a value of from about 1 to about 16. A conjugate having the formula:

2. 2. The conjugate of claim 1, wherein one phosphate moiety is covalently attached to D at E3LB or PB.

3. 3. The conjugate of claim 1 or 2, wherein L1 is a peptidomimetic linker covalently linking Ab to the PB of D.

4. 3. The conjugate of claim 1 or 2, wherein L1 is a peptidomimetic linker covalently linking Ab to E3LB of D.

5. L1 is the following: i) Z is -(CH ２ ) ｐ -or-CH ２ - (CH ２ -O-CH ２ ) ｐ -CH ２ -, where p is 1, 2, 3, 4, 5 or 6; R Ａ is hydrogen, C １－６ Alkyl, or -(CH ２ ) ｖ -aryl, where v is 0 or 1; Q: a) b) Q １ teeth, Here, R ２ is hydrogen, halo (C １－６ ) alkyl or C １－６ is alkyl) selected from the group consisting of: L １－Ａ teeth: ii) Z ２ is C １－１２ Alkylene or -[CH ２ ] ｇ -O-[CH ２ ] ｈ -, g and h are each independently 0, 1 or 2; w is 0, 1, 2, 3, 4 or 5; J is C １－５ Alkyl, -N(R ｘ ) (R ｙ ), -C(O)NH ２ , -NH-C(O)-NH ２ , and -NHC(=NH)NH ２ R ｘ and R ｙ are each independently hydrogen and C １－３ alkyl; K is C １－３ Alkylene, —CH(R)—, —C(O)—, —C(O)—O—CH(R)—, —CH ２ -OC(O)-, -CH ２ -OC(O)-NH-CH ２ - and -CH ２ -O-C(O)-R-[CH ２ ] ｑ -O-, R is hydrogen, C １－３ Alkyl, N(R ｘ ) (R ｙ ), -O-N(R ｘ ) (R ｙ ) or C(O)-N(R ｘ ) (R ｙ ) where q is 0, 1, 2, or 3; R ｘ and R ｙ are each independently hydrogen and C １－３ alkyl, or R ｘ and R ｙ together with the nitrogen to which each is attached form an optionally substituted 5- to 7-membered heterocyclyl; R ａ , R ｂ , R Ｃ and R Ｄ are each independently hydrogen and C １－３ alkyl, or R ａ and R ｂ Each of them, together with the carbon to which it is attached, represents an optionally substituted C ３－６ Forming a cycloalkyl; R ７ and R ８ each independently represents hydrogen, halo, C １－５ Alkyl, C １－５ alkoxy or hydroxyl. The conjugate according to any one of claims 1 to 4, selected from the group consisting of:

6. K of L1 is: The conjugate of claim 5, selected from the group consisting of:

7. L1 is: The conjugate according to any one of claims 1 to 5, selected from the group consisting of:

8. R ７ and R ８ The conjugate of claim 5 , wherein each of is hydrogen.

9. Z is -(CH ２ ) ｐ The conjugate according to any one of claims 5 to 8, wherein

10. w is 2 or 3 and J is -N(CH ３ ) ２ or -NH(CO)NH.

11. Q is -CH ２ -CH ２ The conjugate according to any one of claims 5 to 10, wherein

12. L1 is: [In the formula, R ５ and R ６ are independently hydrogen or C １－５ alkyl; or R ５ and R ６ each taken together with the nitrogen to which it is attached forms an optionally substituted 5- to 7-membered heterocyclyl.

13. L1 is: The conjugate of claim 12, selected from the group consisting of:

14. J is -NH-C(O)-NH ２ or -N(CH ３ ) ２ The conjugate of claim 12 or 13,

15. w is 2 and J is -NH-C(O)-NH ２ or w is 3 and J is -N(CH ３ ) ２ 15. The conjugate according to any one of claims 12 to 14, wherein

16. L1 has the structure:

16. The conjugate of any one of claims 12 to 15, having the formula:

17. The phosphate moiety is: -(P=O)(OH) ２ ,-CH ２ O(P=O)(OH) ２ ,-(P=O)(OH)O(P=O)(OH) ２ and -CH ２ -O(P=O)(OH)O(P=O)(OH) ２ 17. The conjugate of any one of claims 1 to 16, selected from the group consisting of:

18. 18. The conjugate of any one of claims 1 to 17, wherein E3LB comprises a hydroxyproline residue.

19. 20. The conjugate of claim 18, wherein the phosphate moiety is covalently attached to E3LB through the oxygen of a hydroxyproline residue.

20. E3LB:

20. The conjugate of claim 19, comprising:

21. E3LB:

21. The conjugate of claim 20.

22. The antibody conjugated L1-D is:

22. The conjugate of claim 1, wherein the conjugate is a residue of a compound selected from the group consisting of:

23. 23. The conjugate of claim 1, wherein PB comprises a hydroxyphenyl moiety.

24. 24. The conjugate of claim 23, wherein the phosphate moiety is covalently attached to PB through an oxygen of the hydroxyphenyl moiety.

25. 25. The conjugate of claim 24, wherein PB is a BRM ligand.

26. The antibody conjugated L1-D is:

26. The conjugate of any one of claims 1 to 25, wherein the conjugate is a residue of a compound selected from the group consisting of:

27. 27. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 26 and one or more pharma- ceutically acceptable excipients.

28. 28. A method of treating a disease in a human in need of such treatment, comprising administering to said human an effective amount of a conjugate described in any one of claims 1 to 26 or a pharmaceutical composition described in claim 27.

29. 29. The method of claim 28, wherein the disease is cancer.

30. 30. The method of claim 29, wherein the cancer is BRM dependent.

31. 31. The method of claim 29 or 30, wherein the cancer is non-small cell lung cancer.

32. 28. A method for reducing the level of a target protein in a subject, comprising administering to the subject a conjugate of any one of claims 1 to 26 or a pharmaceutical composition of claim 27, wherein the PB moiety binds to the target protein and ubiquitin ligase causes degradation of the bound target protein, thereby reducing the level of the target protein.

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