Antibody-conjugated chemical decomposition inducers having hydrolyzable maleimide linkers and methods thereof

Abstract

The subject matter described herein relates generally to hydrolyzable maleimide-containing molecules useful as linkers to covalently attach chemical degradation inducers to antibodies, conjugates produced therefrom and their uses to treat conditions, and the use of the conjugates in the treatment of diseases and conditions in which targeted protein degradation is beneficial.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 303,447, filed January 26, 2022, the disclosure of which is incorporated by reference in its entirety into this specification.

[0002] Technical Field The subject matter described herein relates generally to hydrolyzable maleimide-containing molecules useful as linkers for covalently attaching chemical degradation inducers to antibodies, and conjugates produced therefrom. [Background technology]

[0003]

[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 is linked to cell proliferation, differentiation, and death.

[0004]

[0004] Protein inhibitors can block or reduce protein activity in cells, but intracellular proteolysis can also reduce activity or completely eliminate target proteins. Thus, harnessing cellular proteolytic pathways can provide a means to reduce or eliminate protein activity. One of the major cellular degradative pathways is known as the ubiquitin-proteasome system. In this system, proteins are targeted 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 E3 ubiquitin ligases to add to proteins.

[0005] To exploit this degradation pathway, molecular constructs known as chemically degraded inducers (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 by a linker. This CIDE can bring the E3 ubiquitin ligase into close proximity with the protein, so that the E3 ubiquitin ligase is ubiquitinated and marked for degradation. However, the relatively large size of CIDEs can contribute to undesirable properties such as rapid metabolism / clearance, short half-life, and low bioavailability, as well as problems with targeted delivery.

[0006]

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

[0007]

[0007] In certain embodiments, the present disclosure relates to a hydrolyzable maleimide-containing linker useful for covalently attaching CIDE to an antibody in the Ab conjugate CIDE to form Ab-L1-CIDE.

[0008] In certain embodiments, the present disclosure relates to a CIDE covalently attached to a hydrolyzable maleimide-containing linker.

[0009]

[0009] In certain embodiments, the present disclosure relates to Ab-L1-CIDE, where L1 is a hydrolyzable maleimide-containing linker covalently attached to the Ab and the CIDE.

[0010] In certain embodiments, the hydrolyzable maleimide-containing linker has the structure of Formula I, IA, IB, IC, or IG:

[0011]

[0011] In certain embodiments, the subject matter described herein relates to a pharmaceutical composition comprising the Ab-L1-CIDE described herein and one or more pharma- ceutically acceptable excipients.

[0012]

[0012] In certain embodiments, the subject matter described herein relates to the use of Ab-L1-CIDE described herein in a method for treating conditions and diseases by administering a pharmaceutical composition containing Ab-L1-CIDE to a subject.

[0013]

[0013] In certain embodiments, the subject matter described herein is a method of making Ab-L1-CIDE.

[0014]

[0014] In certain embodiments, the subject matter described herein relates to an article of manufacture comprising a pharmaceutical composition containing 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.

[0015] Further embodiments are also fully described herein. [Brief description of the drawings]

[0016] [Figure 1]

[0016] Mechanistic diagram of possible pathways for conjugation, hydrolysis and cleavage of Ab-L1-CIDE. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] As used herein, a hydrolyzable maleimide-containing linker (in embodiments, L 1 , L1, L X Or L ZDisclosed are linkers that are useful for covalently attaching degradation agents, also called PROTACs or CIDEs ("Chemical Inducers of Degradation"), to antibodies. Hydrolyzable maleimide-containing linkers are components of Ab-conjugate CIDEs ("antibody-conjugate CIDEs", "Ab-L1-CIDEs" or "Ab-CIDEs"), in which the hydrolyzable maleimide-containing linker is covalently attached to the CIDE and to the antibody. These conjugates are useful for the degradation of target proteins.

[0018]

[0018] The subject matter described herein utilizes a hydrolyzable maleimide-containing linker that can be stably covalently attached to increase the number of linkers attached to the antibody. Thus, the number of CIDEs per antibody can be stably increased. It is advantageous to have a relatively large number of stably attached linkers on the antibody, which can then be used to conjugate with CIDE. A higher DAR allows for a less potent targeted payload than traditional highly cytotoxic payloads. The use of such less potent payloads widens the therapeutic window. The high and low DAR conjugates can also allow for a reduced dosage.

[0019]

[0019] The subject matter described herein utilizes antibodies to direct targets or CIDEs to target cells or tissues. As described herein, delivery of CIDEs to target cells or tissues is improved by attaching an antibody to a CIDE to form an Ab-CIDE. As shown herein, e.g., in the Examples, a cell expressing an antigen can be targeted by an antigen-specific antibody of Ab-CIDE, whereby Ab-CIDE is delivered to a target cell expressing such antigen, and the CIDE portion of Ab-CIDE is delivered into the target cell.

[0020]

[0020] Thus, the subject matter described herein relates to an Ab-CIDE composition that results in ubiquitination of a target protein and subsequent degradation of the protein. The composition comprises an antibody covalently attached to a hydrolyzable linker 1 (L1), which is covalently attached at any available attachment point to CIDE. CIDE comprises an E3 ubiquitin ligase binding (E3LB) moiety that recognizes the E3 ubiquitin ligase protein, VHL, and linker 2 (L2) covalently attaches the E3LB moiety to a protein binding moiety (PB), which is a moiety that recognizes the target protein. The subject matter described herein is useful for degrading and thus regulating protein activity and treating diseases and conditions associated with protein activity.

[0021]

[0021] The subject matter disclosed herein is described in more detail below. However, modifications and other embodiments of the subject matter disclosed herein described herein will occur to those skilled in the art to which the subject matter disclosed herein pertains having the benefit of the teachings presented herein. Therefore, the subject matter disclosed herein should not be limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein encompasses all alternatives, modifications, and equivalents. In the event that one or more of the references, patents, and similar materials differ from or conflict with this application, including but not limited to defined terms, term usage, described techniques, etc., this application shall control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0022] I. Definition

[0022] The term "hydrolyzable" refers to a linker that contains a site capable of inducing hydrolysis, under physiological conditions, of a thio-substituted succinimide that is formed when the maleimide is conjugated to an antibody via a thioether bond.

[0023]

[0023] The term "CIDE" refers to Chemical Inducers of Degradation, which are protein degradation-targeting chimeric molecules that typically have three components: an E3 ubiquitin ligase binding group (E3LB), a linker 2 (L2), and a protein binding group (PB).

[0024]

[0024] The terms "residue," "moiety," "part," 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 a residue, moiety, part, or group. By way of example, a residue of a compound has an atom or atoms of the compound, such as hydrogen or hydroxy, replaced with a covalent bond, thereby linking the residue to another moiety of a CIDE, L1-CIDE, or Ab-CIDE. For example, a "residue of CIDE" refers to a CIDE that is covalently bound to one or more groups, such as a linker, L2, which itself may optionally be further linked to an antibody.

[0025]

[0025] The terms "covalently bound" or "covalently linked" refer to a chemical bond formed by the sharing of one or more electron pairs.

[0026]

[0026] 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 and brings the target into proximity with the 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 or binds to the target of interest. Non-limiting examples of small molecule target protein binding moieties include numerous compounds such as BRM (BRAHMA), Hsp90 inhibitors, kinase inhibitors such as tau and androgen receptor (AR), BRG1, AKT, HPK1 and IRE1, 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). In certain embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of PB. In certain embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of PB, the PB is covalently linked to L2; and the conjugated CIDE comprises a hydrolyzable linker.

[0027]

[0027] The term "E3 ligase binding (E3LB) ligand" refers to a molecule capable of binding 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 generally refer to the target enzyme binding site of the E3LB portion of the conjugates described herein. VCB E3 is a protein that, in combination with an E2 ubiquitin conjugating enzyme, causes the attachment of ubiquitin to lysines on target proteins. 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 target proteins. In certain embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of E3LB. In certain embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of E3LB, where the E3LB is covalently attached to L2; and the conjugated CIDE is further covalently attached to a hydrolyzable linker.

[0028]

[0028] As used herein, the term "linker", "linker unit" or "linkage" refers 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, part, group or component of CIDE to another residue, moiety, part, group or component of CIDE. In various embodiments, the linker is any of the following: Linker1, Linker2, L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L25, L26, L27, L28, L29, L30, L31, L32, L33, L41, L42, L43, L44, L45, L46, L47, L48, L49, L50, L51, L52, L53, L54, L55, L56, L67, L68, L69, L70, L71, L72, L73, L74, L75, L76, L77, L78, L79, L81, L82, L83, L84, L85, L86, L87, L88, L99, L91, L92, L93, L94, L95, L96, L97, L98, L99, L109, L1109, L1209, L131, L140, L150, L161, L172, L183, L194, L195, L109, L109, L109, L109, L109, L1109, L 1 , L2, or L 2 and the like.

[0029]

[0029] The term "peptidomimetic" 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 one 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 does not contain more than two adjacent amino acids linked by peptide bonds in any part of its chemical structure. A "peptidomimetic linker" is a portion of a molecule that connects CIDE to an antibody. Useful peptidomimetic linkers are known in the art, and others are disclosed herein. The peptidomimetic linker may be a 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.

[0030]

[0030] The term "antibody" in this specification 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, that are recognized by the CDRs (complementarity determining regions) of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have multiple corresponding antibodies. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds 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 from any species. However, in one embodiment, the immunoglobulins are of human, murine, or rabbit origin.

[0031]

[0031] As used herein, the term "antibody fragment" 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') and Fab'. 2and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al (2004) Protein Eng. Design & Sel. 17(4):315-323), fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to a cancer cell antigen, a viral antigen, or a microbial antigen, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0032]

[0032] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the subject matter described herein may be made by the hybridoma method first described by Kohler et al (1975) Nature, 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,807,715). The "monoclonal antibodies" can also 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.

[0033]

[0033] As used herein, monoclonal antibodies specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0034]

[0034] 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.

[0035]

[0035] 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 have subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0036]

[0036] "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 indicated, as used herein, "binding affinity" refers to specific binding affinity reflecting 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 specific and exemplary embodiments for measuring binding affinity are described below. In certain 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.

[0037]

[0037] As used herein, the term "free cysteine ​​amino acid" 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. As used herein, the term "amino acid" refers to glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine or citrulline.

[0038]

[0038] 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 certain 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 of suffering from one or more symptoms of cancer.

[0039]

[0039] "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.

[0040]

[0040] 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.

[0041]

[0041] As used herein, "treatment" (and grammatical variations thereof, e.g., "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, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and achieving remission or improved prognosis. In some embodiments, the subject antibodies described herein are used to delay the onset of disease or to slow the progression of disease.

[0042]

[0042] An "effective amount" of a drug, e.g., a pharmaceutical formulation, refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic or preventive 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., delay to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., delay 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 them, it can be cytostatic and / or cytotoxic. An effective amount can extend progression-free survival (e.g., as measured by Response Evaluation Criteria in Solid Tumors, RECIST, or CA-125 change), produce an objective response (including partial response, PR, or complete response, CR), increase overall survival, and / or improve one or more symptoms of cancer (e.g., as assessed by FOSI).

[0043]

[0043] As used herein, the term "therapeutically effective amount" refers to any amount that results in the treatment of a disease, disorder, or side effect, or a slowing of the rate of progression of a disease or disorder, as compared to a corresponding subject not receiving such amount. The term also includes within its scope an amount effective to enhance normal physiological function. For therapeutic use, 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.

[0044]

[0044] The term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0045]

[0045] "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.

[0046]

[0046] As used herein, the phrase "pharmaceutical acceptable salt" refers to a pharmaceutical acceptable organic or inorganic salt of a molecule. Representative salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may include 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 multiple charged atoms in its structure. An example in which multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counter ions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.

[0047]

[0047] Other salts, which are not pharma- ceutically acceptable, may be useful in the preparation of the compounds described herein and these should be considered to form a further aspect of the subject matter. These salts, such as oxalates or trifluoroacetates, are not themselves pharma- cetically acceptable but may be useful in the preparation of salts which are useful as intermediates in obtaining the compounds described herein and their pharma- cetically acceptable salts.

[0048] As used herein, the term "alkyl" refers to an alkyl group having 1 to 5 carbon atoms (C 1 -C 5(-CH)), where the alkyl group may be independently substituted with one or more substituents. In another embodiment, the alkyl group is 1, 2, 3, 4, or 5 carbon atoms. Examples of alkyl groups include methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-Butyl (n-Bu, n-Butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 ) 2 ), 2-Butyl (s-Bu, s-Butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl-2-butyl (-C(CH 3 ) 2 CH 2 CH 3 ), 3-methyl-2-butyl (-CH(CH 3 )CH(CH 3 ) 2 ), 3-methyl-1-butyl (-CH 2 CH 2 CH(CH3 ) 2 ), 2-methyl-1-butyl (-CH 2 CH(CH 3 )CH 2 CH 3 It is understood that alkyl chains covalently bonded at each end can form an alkylene chain as described below.

[0049] "Halogen" or "halo" refers to atoms occupying Group VIIA of the periodic table, eg, fluoro, chloro, bromo, or iodo.

[0050]

[0050] "Haloalkyl" refers to an unbranched or branched alkyl group, as defined above, in which one or more (e.g., 1-6, or 1-3) hydrogen atoms are replaced with halogen. For example, if a residue is substituted with multiple halogens, it can be referred to using a prefix corresponding to the number of halogen moieties attached. Dihaloalkyl and trihaloalkyl refer to an alkyl substituted with two ("di") or three ("tri") halogen groups, which may, but need not, be the same halogen. Examples of haloalkyl include, for example, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. Additionally, "halo-C 1 -C 3 "Alkyl" refers to an alkyl group of 1 to 3 carbons in which at least one hydrogen atom is replaced by a halogen. 1 -C 6 "Alkyl" refers to an alkyl group of 1 to 6 carbons in which at least one hydrogen atom is replaced with a halogen.

[0051] As used herein, the term "alkylene" refers to an alkylene group having 1 to 12 carbon atoms (C 1 -C 12(C) refers to a saturated, linear or branched, divalent hydrocarbon group having any length from 1 to 8 carbon atoms (C 1 -C 8 ) or 1 to 6 carbon atoms (C 1 -C 6 Examples of alkylene groups include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -) but are not limited to these.

[0052] The terms "carbocycle", "carbocyclyl", "carbocyclic ring", and "cycloalkyl" refer to rings having 3 to 5 carbon atoms (C 3 -C 5 ) 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, and the like. Carbocyclyl groups may be independently substituted with one or more alkyl groups.

[0053] "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., C 6 -C 20 aryl), 6 to 12 carbon ring atoms (i.e., C 6 -C 12 aryl), or 6 to 10 carbon ring atoms (i.e., C 6 -C 10 Examples of aryl groups include, for example, phenyl, naphthyl, fluorenyl, and anthryl.

[0054]

[0054] "Heteroaryl" refers to an aromatic group having a single ring, multiple rings, or multiple fused rings, with one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl refers to an aromatic group having 1 to 20 ring carbon atoms (i.e., C 1 -C 20 Heteroaryl), 3 to 12 ring carbon atoms (i.e., C 3 -C 12 heteroaryl), or 3 to 8 carbon ring atoms (i.e., C 3 -C 8Heteroaryl) and 1-5 ring heteroatoms, 1-4 ring heteroatoms, 1-3 ring heteroatoms, 1-2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, heteroaryl includes a 9-10 ring system, a 6-10 membered ring system, a 5-10 membered ring system, a 5-7 membered ring system, or a 5-6 membered ring system, each independently containing 1-4 ring heteroatoms, 1-3 ring heteroatoms, 1-2 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, and the heteroaryl may be bonded through any ring of the fused system. Aromatic rings with single or multiple fused rings containing at least one heteroatom are considered heteroaryl regardless of the bond 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.

[0055]

[0055] "Heterocycle", "heterocyclic", "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially unsaturated group having a single ring or multiple fused rings, including fused, bridged or spiro ring systems, and having 3 to 20 ring atoms, including 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 certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to N-oxide, -S(O)- or -SO 2 - 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, tetrahydrofuranyl, and the like. Heterocyclyl groups can be substituted as described in WO 2014 / 100762.

[0056]

[0056] The term "chiral" refers to molecules that have the property of not being superimposable on their mirror image partners, while the term "achiral" refers to molecules that are superimposable on their mirror image partners.

[0057]

[0057] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0058]

[0058] "Diastereomer" refers to a stereoisomer with 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, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.

[0059]

[0059] "Enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0060]

[0060] Stereochemical definitions and conventions used herein generally follow those of 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 are capable of rotating 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 its chiral center. 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.

[0061]

[0061] Other terms, definitions and abbreviations used herein include: wild-type ("WT"); cysteine ​​engineered variant antibody ("Thio"); light chain ("LC"); heavy chain ("HC"); 6-maleimidocaproyl ("MC"); maleimidopropanoyl ("MP"); valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyl ("PAB") and p-aminobenzyloxycarbonyl ("PABC"); A118C (EU numbering) = A121C (sequential numbering) = A114C (Kabat numbering) of heavy chain K149C (Kabat numbering) of light chain.

[0062]

[0062] Further definitions and abbreviations are provided elsewhere herein.

[0063] II. Hydrolyzable Linkers In an embodiment, the subject matter described herein relates to a peptidomimetic linker comprising a hydrolyzable moiety, the linker being covalently attached to a CIDE or an antibody and a CIDE. In an embodiment, the hydrolyzable moiety of the maleimide portion of the peptidomimetic linker is represented by Formula I: TIFF2025506357000001.tif46170[In the formula, Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 -wherein p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 alkyl, phenyl or benzyl; Q: i) TIFF2025506357000002.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000003.tif32170, where t is 0, 1, 2, 3, or 4; Q 1teeth, TIFF2025506357000004.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 is the remaining portion of the peptidomimetic linker. The structure includes:

[0064] In certain embodiments, the subject matter described herein has the structure: L 1 -D [In the formula, D is CIDE; L 1 is covalently attached to D and has formula I: TIFF2025506357000005.tif42170 (in the formula, Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 -wherein p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 Alkyl, phenyl, benzyl; Q: i) TIFF2025506357000006.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000007.tif32170 (in the formula, t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000008.tif20170, 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: TIFF2025506357000009.tif46170, During the ceremony, Shows the join point to TIFF2025506357000010.tif12170D; w is 0, 1, 2, 3, 4 or 5; J is for C 1-5 Alkyl, -N(R x )(R y ), -C(O)NH 2 , -NH-C(O)-NH 2 and -NH-C(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 -OC(O)-R-[CH 2 ] u -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 u 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 C and R D are each independently hydrogen and C 1-3alkyl or R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are each independently hydrogen, halo, or C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl is a linker having the structure The present invention relates to a compound having the formula:

[0065] In certain embodiments, L 1 -D is selected from the group consisting of: TIFF2025506357000011.tif221170TIFF2025506357000012.tif217170

[0066] In certain embodiments, the subject matter described herein has the structure: Ab-(L 1 -D) j [In the formula, Ab is antibody; j ranges from 1 to 16; D is CIDE; L 1 is covalently linked to Ab and D and has the formula IA: TIFF2025506357000013.tif41170 (in the formula, Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 -wherein p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 Alkyl, phenyl, benzyl; Q: i) TIFF2025506357000014.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000015.tif32170 (in the formula, t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000016.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: TIFF2025506357000017.tif47170, During the ceremony, Shows the join point to TIFF2025506357000018.tif12170D; w is 0, 1, 2, 3, 4 or 5; J is for C 1-5 Alkyl, -N(R x )(R y ), -C(O)NH 2 , -NH-C(O)-NH 2 and -NH-C(=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 -OC(O)-R-[CH 2 ] u -O-, R is hydrogen, C 1-3 Alkyl, N(R x )(R y ), -ON(R x )(Ry ) or C(O)-N(R x )(R y ) where u 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 C and R D are each independently hydrogen and C 1-3 alkyl or R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are each independently hydrogen, halo, or C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl is a linker having the structure The present invention relates to an antibody-CIDE conjugate having the formula:

[0067] In certain embodiments, the subject matter described herein has the structure: Ab-((L X )-D) j [In the formula, Ab is antibody; j ranges from 1 to 16; D is CIDE; L X L 1 and L 1h and L 1h L X At least one example of: L 1 is covalently linked to Ab and D and has the formula IA: Linker 1 having the structure of TIFF2025506357000019.tif42170, L 1h is covalently bonded to D and has the formula IB: having the structure TIFF2025506357000020.tif44170, where # indicates the point of attachment at position a or b; L 1 and L 1h In TIFF2025506357000021.tif15170 indicates the binding site for the antibody; Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 -wherein p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 Alkyl, phenyl, benzyl; Q: i) TIFF2025506357000022.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000023.tif32170 (in the formula, t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000024.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: TIFF2025506357000025.tif46170, During the ceremony, Shows the attachment point to TIFF2025506357000026.tif12170D; w is 0, 1, 2, 3, 4 or 5; J is for C 1-5 Alkyl, -N(R x )(R y ), -C(O)NH 2 , -NH-C(O)-NH 2 and -NH-C(=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 -OC(O)-R-[CH 2 ] u -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 u 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 C and R D are each independently hydrogen and C 1-3 alkyl or R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are each independently hydrogen, halo, or C1-5 Alkyl, C 1-5 alkoxy or hydroxyl is a linker having the structure The present invention relates to an antibody-CIDE conjugate having the formula:

[0068] In certain embodiments, Ab-((L X )-D) j is an Ab-CIDE, described above, that covalently bonds to an antibody and includes at least one linker having the structure of formula IB.

[0069] In certain embodiments, Ab-((L X )-D) j The conjugate is Ab-(L 1 -D) j It is the product of complete or partial hydrolysis of

[0070] In certain embodiments, the subject matter described herein has the structure: Ab-(L Z ) j [In the formula, Ab is antibody; j ranges from 1 to 16; L Z L X1 and Lx-D; X1 L Z At least one example of: L X1 is covalently attached to Ab and has the formula IG: TIFF2025506357000027.tif43170 structure, And, Lx-D (where L X L 1 and L 1h Selected from the group consisting of: L 1 is covalently linked to Ab and D and has formula IA or formula IC: Linker 1 having the structure of TIFF2025506357000028.tif92170, L 1h is covalently linked to Ab and D and has the formula IB: Linker 1 having the structure: TIFF2025506357000029.tif45170, where # indicates the point of attachment at position a or b; TIFF2025506357000030.tif15170 indicates the binding site for the antibody; Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 -wherein p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 Alkyl, phenyl, benzyl; Q: i) TIFF2025506357000031.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000032.tif32170 (in the formula, t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000033.tif20170R 2 is hydrogen, haloC 1-6 Alkyl or C 1-6 (It is alkyl) selected from the group consisting of; L 1-A teeth: TIFF2025506357000034.tif46170, During the ceremony, Shows the attachment point to TIFF2025506357000035.tif12170D; w is 0, 1, 2, 3, 4 or 5; J is for C 1-5Alkyl, -N(R x )(R y ), -C(O)NH 2 , -NH-C(O)-NH 2 and -NH-C(=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 -OC(O)-R-[CH 2 ] u -O-, R is hydrogen, C 1-3 Alkyl, N(R x )(R y ) or C(O)-N(R x )(R y ) where u 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 C and R D are each independently hydrogen and C 1-3 alkyl or R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are each independently hydrogen, halo, or C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl). The present invention relates to an antibody-linker conjugate having the formula:

[0071] In certain embodiments, Ab-((LD) j is an Ab-CIDE, described above, that covalently bonds to an antibody and includes at least one linker having the structure of formula IG.

[0072] In certain embodiments, Ab-((L z ) j The conjugate is Ab-(L 1 -D) j The product is a product of complete or partial hydrolysis and / or partial or complete cleavage of

[0073] In certain embodiments of formulas I, IA, IB, IC, and IG, Q is: TIFF2025506357000036.tif18170, wherein q is 1, 2, 3, or 4. In certain embodiments, q is 1, 2, or 3. In certain embodiments, q is 2.

[0074] In certain embodiments of formulas I, IA, IB, IC, and IG, Q is: TIFF2025506357000037.tif33170, where t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000038.tif20170 where R 2 is hydrogen, haloC 1-6 Alkyl or C 1-6 It is an alkyl.

[0075] In certain embodiments of formulas I, IA, IB, IC and IG, R 2 is hydrogen or C 1-6 In certain embodiments, R 2 is methyl, ethyl, propyl or butyl. In certain embodiments, R 2 is methyl.

[0076] In certain embodiments of formulas I, IA, IB, IC and IG, Q 1 teeth: In certain aspects of these embodiments, R 2 is hydrogen or methyl.

[0077] In certain embodiments of formulas I, IA, IB, IC, and IG, Q 1 teeth: TIFF2025506357000040.tif22170

[0078] In certain embodiments of formulas I, IA, IB, IC, and IG, t is 0.

[0079] In certain embodiments of formulas I, IA, IB, IC, and IG, Z is -(CH 2 ) p - and p is 1, 2, 3, 4, 5 or 6. In aspects of these embodiments, p is 4, 5 or 6. In aspects of these embodiments, p is 5.

[0080] In certain embodiments of formulas I, IA, IB, IC, and IG, Z is -CH 2 -(CH 2 -O-CH 2 ) p -CH 2 - and p is 1, 2, 3, 4, 5 or 6. In aspects of these embodiments, p is 1, 2 or 3. In aspects of these embodiments, p is 1.

[0081] In certain embodiments of formulas I, IA, IB, IC, and IG, R A is hydrogen or C 1-6 In an aspect of these embodiments, R A is hydrogen or methyl.

[0082] In certain embodiments of formulas I, IA, IB, IC, and IG, R A Ha-(CH 2 ) v In certain embodiments, Ar is C 6-10 In certain embodiments, Ar is C 6 In certain embodiments, v is 0 or 1. In certain embodiments, aryl is hydroxy or C 1-3 In certain embodiments, R A is phenyl or benzyl.

[0083]

[0083] L 1-A In certain embodiments of the present invention, Indicates the binding point of TIFF2025506357000041.tif12170D to PB.

[0084] In certain embodiments of formulas I, IA, IB, IC and IG, R 7 and R 8 are hydrogen.

[0085] In certain embodiments of formulas I, IA, IB, IC, and IG, R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 In an aspect of these embodiments, C 3-6 The cycloalkyl is cyclobutane.

[0086] In certain embodiments when w is 0, J is C 1-5 alkyl, e.g., methyl; L 1 , L1, L X Or L Z teeth: TIFF2025506357000042.tif33170

[0087] In certain embodiments, K is -CH(R)-, where R is hydrogen, C 1-3 Alkylene, -CH 2 O(CO)-, N(R x )(R y ), -ON(R x )(R y ) or C(O)-N(R x )(R y ) and R x and R y are each independently hydrogen and C 1-3 alkyl or R x and R y taken together with the nitrogen to which each is attached, form an optionally substituted 5- to 7-membered heterocyclyl. In aspects of these embodiments, R is hydrogen or C 1-3 In aspects of these embodiments, R is hydrogen. In certain embodiments, K is TIFF2025506357000043.tif118170.

[0088] In one embodiment, Z is -(CH 2 ) p - and p is 2 or 5; R A is hydrogen or C 1-6 is alkyl; Q is TIFF2025506357000044.tif18170q is 2; w is 2 or 3; J is for C 1-5 Alkyl, -N(R x )(R y )(R x and R y are C 1-3 alkyl), and -NH-C(O)-NH 2 selected from the group consisting of; K is C 1-3 Alkylene or -CH 2 -OC(O)-; R Cand R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are hydrogen.

[0089] In one embodiment, Z is -(CH 2 ) p - and p is 2 or 5; R A is hydrogen or C 1-6 is alkyl; Q is TIFF2025506357000045.tif33170t is 0, Q 1 teeth TIFF2025506357000046.tif20170R 2 is hydrogen or C 1-6 is alkyl; w is 3; J is for C 1-5 Alkyl, -N(R x )(R y ), R x and R y are C 1-3 is alkyl; K is C 1-3 Alkylene or -CH 2 -OC(O)-; R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are hydrogen.

[0090] In certain embodiments, the subject matter described herein has the structure: TIFF2025506357000047.tif42170[in the formula, Z is -(CH 2 ) p-or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 and p is an integer from 1 to 24; R A is hydrogen, C 1-6 Alkyl, or -(CH 2 ) v -aryl and v is 0 or 1; Q is, i) TIFF2025506357000048.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000049.tif32170, where t is 0, 1, 2, 3, or 4; Q 1 teeth, TIFF2025506357000050.tif20170R 2 is hydrogen, haloC 1-6 Alkyl or C 1-6 (It is alkyl) selected from the group consisting of The present invention relates to a compound of formula II having the formula:

[0091] In certain embodiments of formula II, Q is wherein q is 1, 2, 3, or 4. In certain embodiments of Formula II, q is 2.

[0092] In certain embodiments of formula II, Q is, TIFF2025506357000052.tif33170, where t is 0, 1, 2, 3, or 4; Q 1 teeth, TIFF2025506357000053.tif20170R 2 is hydrogen, haloC 1-6 Alkyl or C 1-6 In certain embodiments of formula II, R2 is hydrogen or C 1-6 27. The compound of claim 26, wherein C is an alkyl group. In certain aspects of these embodiments, 1-6 The alkyl is methyl.

[0093] In certain embodiments of formula II, Q 1 teeth, In certain embodiments of formula II, R 2 is hydrogen or methyl.

[0094] In certain embodiments of formula II, Q 1 teeth, TIFF2025506357000055.tif22170

[0095] In certain embodiments of Formula II, t is 0.

[0096] In certain embodiments of formula II, Z is -(CH 2 ) p - and p is 1, 2, 3, 4, 5 or 6. In certain aspects of these embodiments, p is 4, 5 or 6. In certain aspects of these embodiments, p is 5.

[0097] In certain embodiments of formula II, Z is -CH 2 -(CH 2 -O-CH 2 ) p -CH 2 - and p is 1, 2, 3, 4, 5 or 6. In certain aspects of these embodiments, p is 1, 2 or 3;

[0098] In certain embodiments of formula II, R A is hydrogen or C 1-6 In certain of these embodiments, R A is hydrogen or methyl.

[0099] In certain embodiments of formula II, R A is phenyl or benzyl.

[0100] In certain embodiments, formula II is a compound in which Q is -CH 2 CH 2 - and;R A is hydrogen and Z is -CH 2 CH 2 -; and Q is -(CH 2 ) 3 - and;R A is hydrogen and Z is -(CH 2 ) 3 -; and Q is -CH 2 CH 2 - and;R A is methyl and Z is -CH 2 CH 2 - and does not include compounds which are

[0101] III. CIDE Covalently Attached to a Hydrolyzable Linker

[0101] A chemical decomposition inducer (CIDE) molecule is covalently attached to a hydrolyzable maleimide-containing linker described herein. In certain embodiments, the CIDE comprises a degrading agent that is a bifunctional molecule having a ubiquitin-binding moiety linked to a protein targeting moiety, such as those described in WO 2017 / 201449; WO 2020 / 086858; U.S. Patent No. 7,208,157; ​​U.S. Patent Application Publication No. 2014 / 0356322; WO 2015 / 0291562; WO 2017 / 030814; U.S. Patent Application Publication No. 2017 / 0008904; U.S. Patent No. 9,938,264; U.S. Patent Application Publication Nos. 2019 / 300521, 2020 / 0038378, WO 2021 / 067606; and WO 2021 / 207291. Given the subject matter disclosed herein, one of skill in the art will understand that the point of attachment of the hydrolyzable linker to the CIDE can vary and can be any available attachment point on the CIDE.

[0102] In certain embodiments, CIDEs include those having the following components: a, E3 ubiquitin ligase binding group (E3LB).

[0103] 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 the E3 ubiquitin ligase von Hippel-Lindau (VHL).

[0103]

[0104] A specific 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 erythropoietin-inducing cytokine in response to low oxygen levels.

[0104]

[0105] In certain embodiments, the E3LB portion of CIDE can be any known E3LB ligand. In certain embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of E3LB, and the CIDE is covalently linked to a hydrolyzable linker described herein.

[0105]

[0106] The E3LB moiety has at least one terminus bearing a moiety that is or can be covalently attached to the L2 moiety and at least one terminus bearing a moiety that is or can be covalently attached to the L1 moiety, for example, the E3LB moiety terminates in an -NHCOOH moiety that can be covalently attached to the L2 moiety via an amide bond.

[0106]

[0107] 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.In addition, in any of the aspects or embodiments described herein, the E3LB described herein may be directly linked to the PB via a bond or by a chemical linker.

[0107]

[0108] In certain embodiments, E3LB comprises a compound containing a hydroxyproline moiety as described in WO 2020 / 086858, WO 2013 / 106643, and WO 2013 / 106646, each of which is incorporated by reference in its entirety.

[0108]

[0109] In certain embodiments, the subject matter described herein relates to an E3LB portion that includes a hydroxyproline residue: TIFF2025506357000056.tif36170 wherein in all embodiments and substructures the hydroxyl may be replaced with another group such as a phosphate moiety.

[0109]

[0110] In one particular embodiment, the E3LB: Contains TIFF2025506357000057.tif33170.

[0110]

[0111] In one particular embodiment, the E3LB: Contains TIFF2025506357000058.tif38170, wherein A is a group covalently bonded to L2.

[0111]

[0112] In one particular embodiment, the E3LB: Contains TIFF2025506357000059.tif43170, During the ceremony, This is the connection point to TIFF2025506357000060.tif16170L2. R A1 , R A2and R A3 are each independently hydrogen or C 1-5 alkyl; or R A1 , R A2 and R A3 Two of them, together with the carbon to which they are attached, are C 1-5 Forms a cycloalkyl.

[0112]

[0113] In one particular embodiment, the E3LB: Contains TIFF2025506357000061.tif45170, During the ceremony, R 2 is hydrogen or C 1-5 is alkyl; Y 1 and Y 2 are each -CH or Y 1 and Y 2 one of which is -CH and the other is N; R 3 teeth, The file is TIFF2025506357000062.tif35170.

[0113]

[0114] In one particular embodiment, the E3LB: Contains TIFF2025506357000063.tif46170.

[0114]

[0115] In one particular embodiment, the E3LB: Contains TIFF2025506357000064.tif45170.

[0115]

[0116] In certain embodiments, E3LB is R 3 is cyano.

[0116]

[0117] In certain embodiments, E3LB is R 3 but The structure is TIFF2025506357000065.tif30170.

[0117]

[0118] In certain embodiments, E3LB is R 3 but The structure is TIFF2025506357000066.tif30170.

[0118]

[0119] In certain embodiments, E3LB is R 3 but The structure is TIFF2025506357000067.tif27170.

[0119]

[0120] In certain embodiments, E3LB is R 2 is hydrogen, methyl, ethyl, or propyl.

[0120]

[0121] In certain embodiments, E3LB is R 2 is methyl.

[0121]

[0122] In certain embodiments, E3LB is R 2 but The structure is TIFF2025506357000068.tif15170.

[0122]

[0123] In certain embodiments, the hydroxyproline moiety of E3LB has the structure: TIFF2025506357000069.tif47170.

[0123]

[0124] The E3LB moiety has at least one terminus bearing a moiety that is or can be covalently attached to the L2 moiety and at least one terminus bearing a moiety that is or can be covalently attached to the L1 moiety, for example, the E3LB moiety terminates in an -NHCOOH moiety that can be covalently attached to the L2 moiety via an amide bond.

[0124]

[0125] 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.

[0125] b. Protein binding group (PB)

[0126] "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 interacts with the target, bringing the target into proximity with the ubiquitin ligase so that degradation of the protein or polypeptide by the ubiquitin ligase can occur. The PB can include any molecule as long as it is covalently attached to L2 and interacts or binds to the target of interest. Non-limiting examples of small molecule target protein binding moieties include numerous compounds, such as BRM (BRAHMA), Hsp90 inhibitors, kinase inhibitors such as tau and androgen receptor (AR), BRG1, AKT, HPK1 and IRE1, 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). In certain embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of PB. In certain embodiments, the CIDES and conjugated CIDEs described herein are not limited to the type of PB, and the CIDE is covalently attached to a hydrolyzable linker described herein.

[0126]

[0127] In certain embodiments, the PB portion of the CIDE is a small molecule moiety that binds to BRM, including all variants, mutations, 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 can target proteins of interest. The CIDECs described herein may include any residue of a known BRM binding compound, a binding compound, including those disclosed in WO 2019 / 195201, which is incorporated herein by reference in its entirety.

[0127]

[0128] In certain embodiments, the BRM binding compound has formula I: TIFF2025506357000070.tif58170, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein: TIFF2025506357000071.tif217170; wherein for (a) through (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, When TIFF2025506357000072.tif15170(a), [X] is TIFF2025506357000073.tif21170 (where # is TIFF2025506357000074.tif14170 indicates the connection point, and ## indicates the connection point to L2), [Y] does not exist, [Z] is absent; 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, TIFF2025506357000075.tif15170(a) and [X] is TIFF2025506357000076.tif20170 (where & is TIFF2025506357000077.tif14170, && indicates the attachment point to [Z]), [Z] is is not TIFF2025506357000078.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, TIFF2025506357000079.tif15170(a), (b), (d), or (e); or (v) [X] does not exist; [Y] is ethynylene; [Z] is a 5-20 membered heteroaryl; however, TIFF2025506357000080.tif15170(a), (b), (d), or (e); or (vi) [X] does not exist; [Y] is cyclopropyl or cyclobutyl; [Z] is a 5-20 membered heteroaryl; however, TIFF2025506357000081.tif15170(a), (b), (d), or (e).

[0128]

[0129] In certain embodiments, the BRM binding compound has the formula (IA): TIFF2025506357000082.tif61170, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein [X], [Y] and [Z] are as defined above for compounds of formula (I).

[0129]

[0130] In certain embodiments, the BRM binding compound has the formula (IB): TIFF2025506357000083.tif49170, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein [X], [Y] and [Z] are as defined above for compounds of formula (I).

[0130]

[0131] In certain embodiments, the BRM binding compound has the formula (IC): TIFF2025506357000084.tif52170, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein [X], [Y] and [Z] are as defined above for compounds of formula (I).

[0131]

[0132] In certain embodiments, the BRM binding compound has the formula (IM): TIFF2025506357000085.tif59170, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein [X], [Y] and [Z] are as defined above for compounds of formula (I).

[0132]

[0133] In certain embodiments, the BRM binding compound has the formula (IE): TIFF2025506357000086.tif58170, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein [X], [Y] and [Z] are as defined above for compounds of formula (I).

[0133]

[0134] In certain embodiments, the PB (BRM) portion of CIDE has the following structure: TIFF2025506357000087.tif154170In the formula, This is the covalent attachment point to TIFF2025506357000088.tif14170L2.

[0134] c. Linker L2 The E3LB and PB portions of the CIDE described herein can be linked with a linker (L2, linker L2, linker-2). In certain embodiments, linker L2 is covalently linked to the E3LB portion and to the PB portion, thus constituting a CIDE.

[0135] In certain embodiments, the L2 moiety may be selected from the linkers disclosed in WO 2019 / 195201, the entirety of which is incorporated by reference herein.

[0136] The E3LB and PB groups may be covalently attached to the linker group via any group that is appropriate and stable to the linker chemistry, but in certain embodiments, L2 is independently covalently attached to the E3LB and PB groups via an amide, ester, thioester, keto group, carbamate (urethane) or ether, each of which may be inserted anywhere in the E3LB and PB groups to allow binding of the E3LB group to a ubiquitin ligase and binding of the PB group to a BRM target protein for degradation. In other words, as shown herein, a linker may be designed and connected to the E3LB and PB to regulate binding of the E3LB and PB to their respective binding partners.

[0137] In certain embodiments, L2 is a linker covalently attached to E3LB and PB, and L2 has the following formula: TIFF2025506357000089.tif48170 (in the formula, R 4 is hydrogen or methyl) TIFF2025506357000090.tif68170 (in the formula, z is 1 or 0; G is TIFF2025506357000091.tif25170 or -C(O)NH-; (This is the junction point to TIFF2025506357000092.tif15170PB) has.

[0138] In certain embodiments of L2a, R 4 is hydrogen.

[0139] In certain embodiments of L2a, R 4 is methyl.

[0140] In certain embodiments of L2a, R 4 is methyl, and the methyl is oriented relative to the piperazine to which it is attached, as follows: TIFF2025506357000093.tif45170

[0141] In certain embodiments of L2c, z is 0.

[0142] In certain embodiments of L2c, z is 1.

[0143] IV. Antibodies Covalently Linked to Hydrolyzable Linkers

[0135] Referring now to antibody-hydrolyzable linker conjugates and antibody-hydrolyzable linker-CIDE conjugates, such conjugates include a single antibody, and a single antibody may have multiple hydrolyzable linkers and / or hydrolyzable linker-CIDEs. The antibody may be linked to a hydrolyzable linker (L 1 , L X Or L Z The conjugate is selected from the following formulas, as described elsewhere herein: Ab-(L 1 -D) j ; Ab-((L x )-D) j and Ab-(L z ) j .

[0144]

[0136] 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 by endocytosis. In some cases, the Ab-CIDE may be internalized intracellularly, either in antigen-expressing or non-expressing cells, by pinocytosis or similar non-specific uptake pathways. The Ab-CIDEs and methods of their use described herein advantageously utilize antibody recognition of cell surfaces and / or endocytosis of Ab-CIDE to deliver the CIDE portion intracellularly. Antibodies are described in WO2020 / 086858, which is incorporated herein by reference in its entirety.

[0145]

[0137] In certain embodiments, the antibody may be mutated to reduce effector function. Examples of mutations that modulate Fc effector function include the LALAPG and NG2LH mutations.

[0146] In certain embodiments, the antibody is THIOMAB, as previously described in WO 2016 / 04856. TM In addition, any antibody target can be targeted with THIOMAB with or without any Fc effector modulation, including LALAPG or NG2LH mutations. TM Combinations are contemplated, as can any suitable combination of mutations.

[0147] The antibody may be a human antibody, e.g., as generally described 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 with desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, e.g., 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, 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).

[0148]

[0140] The antibody may be a chimeric antibody or a humanized antibody. Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are 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 "resurfacing"); 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).

[0149]

[0141] The antibody may be a multispecific antibody, e.g., a bispecific antibody. As used herein, the term "multispecific antibody" refers to an antibody that contains an antigen-binding domain that has multiple epitopic specificity (i.e., can bind to two or more different epitopes on one molecule or can bind to epitopes on two or more different molecules). As used herein, the term "bispecific antibody" 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 a protein and any other antigen. 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 "knobs-into-holes" 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 also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 1999, 20:111-112 (1999)). al., J. Immunol., 152:5368 (1994); and by preparing trispecific antibodies, e.g., as described in Tutt et al. J. Immunol. 147:60 (1991). Engineered antibodies having three or more functional antigen binding sites, including "Octopus antibodies" or "Dual Variable Domain Immunoglobulins" (DVDs), are also included herein (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576 A1, 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 the target protein and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0150]

[0142] The antibody may be an antibody fragment. Antibody fragments include Fab, Fab', Fab'-SH, F(ab') 2Antibody fragments that can be used include, but are not limited to, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), and see WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. Fab and F(ab') fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives 2 For a description of fragments, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; 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 the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain 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 B1). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells.

[0151]

[0143] The antibody may be an antibody variant. In certain 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 can 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.

[0152]

[0144] Antibodies can be produced using recombinant methods and compositions, such as those described in, for example, 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.

[0153] In certain embodiments, the tumor-associated antigen or cell surface receptor is selected from CD71, Trop2, MSLN, NaPi2b, Ly6E, EpCAM, and CD22. Thus, in certain embodiments, the Ab-CIDE may comprise an antibody or fragment selected from an 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, and an anti-HER2 antibody.

[0154] Specific antibodies include, but are not limited to, the following: i. Anti-Ly6E antibody

[0147] In certain embodiments, Ab-CIDE is an anti-Ly6E antibody: Ly6E (may include lymphocyte antigen 6 complex: locus E; Ly67, IG-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.

[0155]

[0148] Ly6E is a GPI-linked, 131 amino acid long, approximately 8.4 kDa protein with unknown function and unknown binding partners. It was initially 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 PCT Publication WO 2013 / 177055.

[0156] ii. Anti-NaPi2b antibody In certain embodiments, the Ab-CIDE comprises an anti-Napi2b antibody: Napi2b (Napi3b, NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM_006424) J. Biol. Chem. 277(22):19665-19672 (2002); Genomics 62(2):281-284 (1999); Feild, JA, et al. al (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO 2004022778 (claim 2); EP 1394274 (example 11); WO 2004 (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.

[0157] iii. Anti-CD22 antibody

[0150] In certain embodiments, Ab-CIDE may comprise an anti-CD22 antibody: 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.

[0158] iv. Anti-CD71 antibody In certain embodiments, the Ab-CIDE may comprise an 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, which has been considered a useful erythrocyte-associated antigen. In one embodiment, the anti-CD71 antibody is described in WO2016081643, which is incorporated by reference in its entirety.

[0159] v. Anti-Trop2 antibody In certain embodiments, Ab-CIDE may comprise an anti-Trop2 antibody. Trop2 (trophoblast antigen 2) is a transmembrane glycoprotein that is an intracellular calcium signaling agent that is differentially expressed in many cancers. It signals cells for self-renewal, proliferation, invasion and survival. Trop2 is also known as cell surface glycoprotein Trop-2 / Trop2, gastrointestinal tumor associated antigen GA7331, pancreatic carcinoma marker protein GA733-1 / GA733, membrane component chromosome 1 surface marker 1M1S1, epithelial glycoprotein-1, EGP-1, CAA1, gelatinous droplet-like corneal dystrophy GDLD and TTD2. In any of the above-mentioned embodiments, the anti-Trop2 antibody of Ab-CIDE is humanized. In one embodiment, the anti-Trop2 antibodies are described in U.S. Patent Application Publication Nos. 2014 / 0377287 and 2015 / 0366988, each of which is incorporated by reference in its entirety.

[0160] vi. Anti-MSLN antibody In certain embodiments, Ab-CIDE may comprise an anti-MSLN antibody. MSLN (mesothelin) is a glycosylphosphatidylinositol-anchored cell surface protein that may 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.

[0161] vii. Anti-EpCAM antibody

[0154] In certain embodiments, Ab-CIDE may comprise an anti-EpCAM antibody. In one aspect, the antibody of Ab-CIDE may be an antibody against a protein found on multiple cell or tissue types. An example of such an antibody is 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). Furthermore, EpCAM has oncogenic potential due to its ability to upregulate c-myc, e-fabp, and cyclins A and E (Munz, M. et al. (2004) Oncogene 23(34):5748-58). Because EpCAM is exclusively expressed in epithelial and epithelial-derived neoplasms, EpCAM can 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 in the case of using a targeting antibody.

[0162] viii. Anti-Steap1 antibody In certain embodiments, Ab-CIDE comprises an anti-STEAP1 antibody. STEAP1 (six transmembrane epithelial antigen of the prostate, Genbank accession number NM_0 12449) Cancer Res. 61(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); EP 1394274 (Example 11); WO 2004016225 (claim 2); WO 2003042661 (claim 12) ; U.S. Patent Application Publication No. 2003157089 (Example 5); U.S. Patent Application Publication No. 2003185830 (Example 5); U.S. Patent Application 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).

[0163] viii. Anti-Steap2 antibody In certain 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 prostate transmembrane epithelial antigen 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 2003104270 (Claim 11); WO 2003104270 (Claim 16); U.S. Patent Application Publication No. US Patent Publication No. 2004005598 (Claim 22); WO 2003042661 (Claim 12); US Patent Publication No. 2003060612 (Claim 12; Figure 10); WO 200226822 (Claim; Figure 2); WO 200216429 (Claim 12; Figure 10); Cross References: GF22655488; AAN04080.1; AF455l38_I.

[0164] x.Anti-Her2 antibody

[0157] In certain embodiments, Ab-CIDE comprises an anti-HER2 antibody. In one embodiment, the anti-HER2 antibody of Ab-CIDE comprises a humanized anti-HER2 antibody. In some embodiments, 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 Ab-CIDE comprises a humanized anti-HER2 antibody, such as humanized 2C4, as described in U.S. Pat. No. 7,862,817. An exemplary humanized 2C4 antibody is pertuzumab, commercially available under the trade name PERJETA®.

[0165] x.Anti-CD33 antibody In certain 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 glycosylated transmembrane protein of 67 kDa. CD33 is expressed on most myeloid and monocytic leukemia cells, as well as myelomonocytic and erythroid progenitor cells. It is not found on the earliest pluripotent stem cells, mature granulocytes, lymphocytic cells, 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.

[0166]

[0159] With specific reference to antibody-hydrolyzable linker-CIDE(D) conjugates, the "CIDE loading" (CIDE / antibody ratio, "DAR" or j above) is the average number of CIDE moieties per antibody. CIDE loading can range from 1 to 20 CIDE(D) per antibody (Ab). That is, the formula for Ab-CIDE, Ab-(L1-D) j In the formula (I), j represents the number of CIDEs linked to the antibody and has a value of about to about 20, about 1 to about 16, about 1 to about 10, about 2 to about 8, or about 4 to about 7. In an embodiment, j is about 6. In an embodiment, j is about 13 or 14. Each CIDE covalently attached to the antibody via a linker L1 can be the same or a different CIDE and can have the same type or a different type of linker as any other L1 covalently attached to the antibody. In certain embodiments, the Ab is a cysteine ​​engineered antibody.

[0167]

[0160] The average number of CIDEs per antibody in a preparation of Ab-CIDEs by conjugation reaction is characterized by common means such as mass spectrometry, ELISA assay, electrophoresis, and HPLC. The quantitative distribution of Ab-CIDEs with respect to j can also be determined. By ELISA, the average value of j in a particular preparation of Ab-CIDEs can be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of j values ​​cannot be distinguished due to the detection limit of antibody-antigen binding and ELISA. Also, the ELISA assay for detection of Ab-CIDE does not determine where the CIDE moiety is attached on the antibody (such as the heavy or light chain fragment, or a specific amino acid residue). In some instances, separation, purification and characterization of homogeneous Ab-CIDEs, where j is a particular value, from Ab-CIDEs having other CIDE loadings can be accomplished by means such as reverse-phase HPLC or electrophoresis.

[0168]

[0161] 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, or only one or more sufficiently reactive thiol groups, by which a linker can be attached. Another reactive site on the Ab for connecting L1-D is the amine functionality of a lysine residue.

[0169]

[0162] In general, the CIDE moiety conjugated to the antibody during the conjugation reaction is less than the theoretical maximum. 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. In general, 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 compound's antibody 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, CIDE loading may be controlled in several different ways: (i) by limiting the molar excess of L1-CIDE groups or linker reagent relative to antibody, (ii) by limiting the conjugation reaction time or temperature, and (iii) by partial or limited reducing conditions for cysteine ​​thiol modification.

[0170]

[0163] Now referring to the Ab-CIDE and L1-CIDE compounds as described herein, they may exist in solid or liquid form. In the solid state, they may exist in crystalline or non-crystalline form, or as a mixture thereof. Those skilled in the art will appreciate that pharma- ceutical acceptable solvates may be formed for crystalline or non-crystalline compounds. In crystalline solvates, the solvent molecules are incorporated into the crystal lattice during crystallization. The 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 usually 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.

[0171]

[0164] 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 arrangement, 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 prepare the compound. For example, changes in temperature, pressure, or solvents may result in polymorphism. Furthermore, one polymorph may spontaneously transform into another polymorph under certain conditions.

[0172] The compounds and Ab-CIDEs or salts thereof described herein may exist in stereoisomeric forms (e.g., containing one or more asymmetric carbon atoms). The individual stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of the subject matter disclosed herein. Similarly, it is understood that the compounds or salts of formula (I) can exist in tautomeric forms other than those shown in the formula, which are also included within the scope of the subject matter disclosed herein. The subject matter disclosed herein should be understood to include all combinations and subsets of 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 should be understood to include all combinations and subsets of specific groups defined herein above.

[0173]

[0166] The subject matter disclosed herein also includes isotopically labeled forms of the compounds described herein, except for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can 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 I can be mentioned.

[0174] Compounds as 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 subject matter disclosed herein. Isotopically labeled compounds, e.g. 3 H, 14 Disclosed herein are those incorporating radioactive isotopes such as C, which are useful for drug and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) Isotopes are commonly used because of their 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. 2Substitution with heavier isotopes such as H may be preferred in some circumstances as it may confer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds can generally be prepared by following the procedures disclosed in the following schemes and / or examples by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents.

[0175]

[0168] The subject matter disclosed herein includes the following non-limiting embodiments: 1. Structure: L 1 -D [In the formula, D is CIDE; L 1 is covalently attached to D and has formula I: TIFF2025506357000094.tif42170 (in the formula, Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 and p is an integer from 1 to 24; R A is hydrogen, C 1-6 Alkyl, or -(CH 2 ) v -aryl and v is 0 or 1; Q is, i) TIFF2025506357000095.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000096.tif32170 (in the formula, t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000097.tif20170R 2 is hydrogen, haloC 1-6Alkyl or C 1-6 (It is alkyl) selected from the group consisting of; TIFF2025506357000098.tif61170 formula, Shows the attachment point to TIFF2025506357000099.tif12170D; 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 , -NH-C(=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 -OC(O)-R-[CH 2 ] u -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 u 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 C and R D are each independently hydrogen and C 1-3alkyl or R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are each independently hydrogen, halo, or C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl and linker 1 having the structure A compound having the formula: 2. Q is, TIFF2025506357000100.tif17170, and q is 1, 2, 3 or 4. 3. The compound according to embodiment 1 or 2, wherein q is 2. 4. Q is, TIFF2025506357000101.tif32170, where t is 0, 1, 2, 3, or 4; Q 1 but, TIFF2025506357000102.tif20170R 2 But hydrogen, halo C 1-6 Alkyl or C 1-6 The compound according to embodiment 1, wherein said compound is alkyl. 5.R 2 is hydrogen or C 1-6 The compound according to embodiment 4, wherein said compound is alkyl. 6.C 1-6 The compound according to embodiment 5, wherein the alkyl is methyl. 7.Q 1 but, TIFF2025506357000103.tif26170 Compound according to embodiment 4. 8.R 2 is hydrogen or methyl. 9.Q 1 but, TIFF2025506357000104.tif22170 Compound according to embodiment 8. 10. A compound according to any one of embodiments 4 to 9, wherein t is 0. 11.Z is -(CH 2 ) p - and p is 1, 2, 3, 4, 5 or 6. 12. The compound according to embodiment 11, wherein p is 4, 5 or 6. 13. The compound according to embodiment 11, wherein p is 5. 14.Z is -CH 2 -(CH 2 -O-CH 2 ) p -CH 2 - and p is 1, 2, 3, 4, 5 or 6. 15. The compound according to embodiment 14, wherein p is 1, 2 or 3. 16. The compound according to embodiment 14, wherein p is 1. 17.R A is hydrogen or C 1-6 The compound according to any one of embodiments 1 to 16, wherein R is alkyl. 18.R A is hydrogen or methyl. 19.R A 17. The compound according to any one of embodiments 1 to 16, wherein is phenyl or benzyl. 20.R 7 and R 8

[0036] Embodiment 20. The compound of any one of embodiments 1 to 19, wherein each is hydrogen. 21.R C and R D each of which, together with the carbon to which it is attached, is optionally substituted C 3-6 The compound according to any one of the preceding embodiments, which forms a cycloalkyl. 22.C 3-6 The compound according to embodiment 21, wherein the cycloalkyl is cyclobutane. 23. K is -CH(R)-, R is hydrogen, C 1-3 Alkyl, N(R x )(R y) or C(O)-N(R x )(R y ) and R x and R y are each independently hydrogen and C 1-3 alkyl or R x and R y and R are taken together with the nitrogen to which they are attached to form an optionally substituted 5- to 7-membered heterocyclyl. 24. R is hydrogen or C 1-3 The compound according to embodiment 23, wherein said compound is alkyl. 25. The compound according to embodiment 24, wherein R is hydrogen. 26. Structure: Ab-(L 1 -D) j [In the formula, Ab is antibody; j ranges from 1 to 16; D is CIDE; L 1 is covalently linked to Ab and D and has the formula IA: TIFF2025506357000105.tif42170 (in the formula, Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 and p is an integer from 1 to 24; R A is hydrogen, C 1-6 Alkyl, or -(CH 2 ) v -aryl and v is 0 or 1; Q is i) TIFF2025506357000106.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000107.tif32170 (in the formula, t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000108.tif20170R 2 is hydrogen, haloC 1-6 Alkyl or C 1-6 (It is alkyl) selected from the group consisting of; L 1-A teeth: TIFF2025506357000109.tif42170, (In the formula, Shows the join point to TIFF2025506357000110.tif12170D; w is 0, 1, 2, 3, 4 or 5; J is -N(R x )(Ry), -C(O)NH 2 , -NH-C(O)-NH 2 and -NH-C(=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 -OC(O)-R-[CH 2 ] u -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 u 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 ytogether with the nitrogen to which each is attached form an optionally substituted 5- to 7-membered heterocyclyl; R C and R D are each independently hydrogen and C 1-3 alkyl or R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are each independently hydrogen, halo, or C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl is a linker having the structure The antibody-CIDE conjugate having 27. Q is, 27. The antibody-CIDE conjugate of embodiment 26, wherein TIFF2025506357000111.tif17170 and q is 1, 2, 3 or 4. 28. An antibody-CIDE conjugate according to embodiment 26 or 27, wherein q is 2. 29. Q is, TIFF2025506357000112.tif32170, where t is 0, 1, 2, 3, or 4; Q 1 but, TIFF2025506357000113.tif20170R 2 But hydrogen, halo C 1-6 Alkyl or C 1-6 The antibody-CIDE conjugate of embodiment 26, which is alkyl. 30.R 2 is hydrogen or C 1-6 The antibody-CIDE conjugate of embodiment 29, which is alkyl. 31.C 1-6 The antibody-CIDE conjugate of embodiment 30, wherein the alkyl is methyl. 32.Q 1 but, TIFF2025506357000114.tif26170 An antibody-CIDE conjugate described in embodiment 29. 33.R 2 The antibody-CIDE conjugate of embodiment 29, wherein is hydrogen or methyl. 34.Q 1 but, TIFF2025506357000115.tif22170An antibody-CIDE conjugate described in embodiment 33. 35. An antibody-CIDE conjugate according to any one of embodiments 29 to 34, wherein t is 0. 36. Z is -(CH 2 ) p - and p is 1, 2, 3, 4, 5 or 6. 37. An antibody-CIDE conjugate according to embodiment 36, wherein p is 4, 5 or 6. 38. An antibody-CIDE conjugate according to embodiment 36, wherein p is 5. 39.Z is -CH 2 -(CH 2 -O-CH 2 ) p -CH 2 - and p is 1, 2, 3, 4, 5 or 6. 40. An antibody-CIDE conjugate according to embodiment 39, wherein p is 1, 2 or 3. 41. An antibody-CIDE conjugate according to embodiment 39, wherein p is 1. 42.R A is hydrogen or C 1-6 An antibody-CIDE conjugate described in any one of embodiments 26 to 41, which is alkyl. 43.R A The antibody-CIDE conjugate of embodiment 42, wherein is hydrogen or methyl. 44.R A An antibody-CIDE conjugate described in any one of embodiments 26 to 41, wherein is phenyl or benzyl. 45.R 7 and R 8 An antibody-CIDE conjugate described in any one of embodiments 26 to 44, wherein each of is hydrogen. 46.R C and R D each of which, together with the carbon to which it is attached, is optionally substituted C 3-6 An antibody-CIDE conjugate described in any one of embodiments 26 to 45, which forms a cycloalkyl. 47.C 3-6 The antibody-CIDE conjugate of embodiment 46, wherein cycloalkyl is cyclobutane. 48.K is -CH(R)-, R is hydrogen, C 1-3 Alkyl, N(R x )(R y ) or C(O)-N(R x )(R y ) and R x and R y are each independently hydrogen and C 1-3 alkyl or R x and R y An antibody-CIDE conjugate described in any one of embodiments 26 to 47, wherein, together with the nitrogen to which each is attached, form an optionally substituted 5- to 7-membered heterocyclyl. 49. R is hydrogen or C 1-3 The antibody-CIDE conjugate of embodiment 48, which is alkyl. 50. The antibody-CIDE conjugate according to embodiment 49, wherein R is hydrogen. 51. A pharmaceutical composition comprising a conjugate according to any one of embodiments 26 to 50 and one or more pharma- ceutically acceptable excipients. 52. A method for treating a disease in a human being in need of such treatment, comprising administering to said human an effective amount of a conjugate (or composition thereof) according to any one of embodiments 26 to 50 or 57 to 108 or a composition according to embodiment 51; or a conjugate (or composition thereof) according to any one of embodiments 26 to 50 or 57 to 108 or a composition according to embodiment 51 for use in the treatment of a disease in a human being in need of such treatment; or a use of a conjugate (or composition thereof) according to any one of embodiments 26 to 50 or 57 to 108 or a composition according to embodiment 51 for the manufacture of a medicament for the treatment of a disease in a human being in need of such treatment. 53. The method of embodiment 52, wherein the disease is cancer. 54. The method of embodiment 53, wherein the cancer is BRM dependent. 55. The method of embodiment 54, wherein the cancer is non-small cell lung cancer. 56. A method for reducing the level of a target protein in a subject, comprising administering to the subject a conjugate according to any one of embodiments 26 to 50 or 58 to 108 (or a composition thereof) or a composition according to embodiment 51, wherein D binds to the target protein and a ubiquitin ligase affects degradation of the bound target protein, thereby reducing the level of the target protein. 58. Structure: Ab-((L X )-D) j [In the formula, Ab is antibody; j ranges from 1 to 16; D is CIDE; L X L 1 and L 1h and L 1h L X At least one example of: L 1 is covalently linked to Ab and D and has the formula IA: Linker 1 having the structure of TIFF2025506357000116.tif42170, L 1h is covalently bonded to D and has the formula IB: having the structure TIFF2025506357000117.tif45170, where # indicates the point of attachment at position a or b; L 1 and L 1h In TIFF2025506357000118.tif15170 indicates the binding site for the antibody; Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 -wherein p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 Alkyl, or -(CH 2 ) v -aryl and v is 0 or 1; Q: i) TIFF2025506357000119.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000120.tif32170 (in the formula, t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000121.tif20170R 2 is hydrogen, haloC 1-6 Alkyl or C 1-6 (It is alkyl) selected from the group consisting of; L 1-A teeth: TIFF2025506357000122.tif46170, During the ceremony, Shows the joining point to TIFF2025506357000123.tif12170D; 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 , -NH-C(=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 -OC(O)-R-[CH 2 ] u -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 u 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 C and R D are each independently hydrogen and C 1-3 alkyl or R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R7 and R 8 are each independently hydrogen, halo, or C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl and linker 1 having the structure The antibody-CIDE conjugate having 59. Q is, The antibody-CIDE conjugate of embodiment 58, wherein TIFF2025506357000124.tif17170 and q is 1, 2, 3 or 4. 60. An antibody-CIDE conjugate according to embodiment 58 or 59, wherein q is 2. 61. Q is, TIFF2025506357000125.tif32170, where t is 0, 1, 2, 3, or 4; Q 1 but, TIFF2025506357000126.tif20170R 2 But hydrogen, halo C 1-6 Alkyl or C 1-6 The antibody-CIDE conjugate of embodiment 58, which is alkyl. 62.R 2 is hydrogen or C 1-6 The antibody-CIDE conjugate of embodiment 61, which is alkyl. 63.C 1-6 The antibody-CIDE conjugate of embodiment 62, wherein the alkyl is methyl. 64.Q 1 but, TIFF2025506357000127.tif26170 An antibody-CIDE conjugate described in embodiment 61. 65.R 2 The antibody-CIDE conjugate of embodiment 61, wherein is hydrogen or methyl. 66.Q 1 but, TIFF2025506357000128.tif22170 An antibody-CIDE conjugate described in embodiment 65. 67. An antibody-CIDE conjugate described in any one of claims 61 to 66, wherein t is 0. 68.Z is -(CH 2 ) p - and p is 1, 2, 3, 4, 5 or 6. An antibody-CIDE conjugate according to any one of embodiments 58 to 67, 69. An antibody-CIDE conjugate according to embodiment 68, wherein p is 4, 5 or 6. 70. An antibody-CIDE conjugate according to embodiment 68, wherein p is 5. 71.Z is -CH 2 -(CH 2 -O-CH 2 ) p -CH 2 - and p is 1, 2, 3, 4, 5 or 6. An antibody-CIDE conjugate according to any one of embodiments 58 to 67, 72. The antibody-CIDE conjugate according to embodiment 71, wherein p is 1, 2 or 3. 73. The antibody-CIDE conjugate according to embodiment 71, wherein p is 1. 74.R A is hydrogen or C 1-6 The antibody-CIDE conjugate of any one of embodiments 58 to 73, which is alkyl. 75.R A The antibody-CIDE conjugate of embodiment 74, wherein is hydrogen or methyl. 76.R A The antibody-CIDE conjugate described in any one of embodiments 58 to 73, wherein is phenyl or benzyl. 77.R 7 and R 8 An antibody-CIDE conjugate described in any one of embodiments 58 to 76, wherein each of is hydrogen. 78.R C and R D each of which, together with the carbon to which it is attached, is optionally substituted C 3-6 An antibody-CIDE conjugate described in any one of embodiments 58 to 77, which forms a cycloalkyl. 79.C 3-6 The antibody-CIDE conjugate of embodiment 78, wherein the cycloalkyl is cyclobutane. 80.K is -CH(R)-, R is hydrogen, C 1-3 Alkyl, N(R x )(R y ) or C(O)-N(R x )(R y ) and R x and R y are each independently hydrogen and C 1-3 alkyl or R x and R y An antibody-CIDE conjugate described in any one of embodiments 58 to 79, wherein, together with the nitrogen to which each is attached, form an optionally substituted 5- to 7-membered heterocyclyl. 81. R is hydrogen or C 1-3 The antibody-CIDE conjugate of embodiment 80, which is alkyl. 82. The antibody-CIDE conjugate according to embodiment 81, wherein R is hydrogen. 83. Structure: Ab-(L Z ) j [In the formula, Ab is antibody; j ranges from 1 to 16; L Z L X1 and Lx-D; X1 L Z At least one example of: L X1 is covalently attached to Ab and has the formula IG: TIFF2025506357000129.tif45170 structure, And, Lx-D (where L X L 1 and L 1h Selected from the group consisting of: L 1 is covalently linked to Ab and D and has formula IA or formula IC: Linker 1 having the structure of TIFF2025506357000130.tif92170, L 1h is covalently bonded to D and has the formula IB: Linker 1 having the structure: TIFF2025506357000131.tif45170, where # indicates the point of attachment at position a or b; TIFF2025506357000132.tif15170 indicates the binding site for the antibody; Z is -(CH 2 ) p -or-CH 2 -(CH 2 -O-CH 2 ) p -CH 2 -wherein p is 1, 2, 3, 4, 5 or 6; R A is hydrogen, C 1-6 Alkyl, or -(CH 2 ) v -aryl and v is 0 or 1; Q: i) TIFF2025506357000133.tif17170, where q is 1, 2, 3, or 4; and ii) TIFF2025506357000134.tif32170 (in the formula, t is 0, 1, 2, 3 or 4; Q 1 teeth, TIFF2025506357000135.tif20170R 2 is hydrogen, haloC 1-6 Alkyl or C 1-6 (It is alkyl) selected from the group consisting of; TIFF2025506357000136.tif61170 formula, Shows the attachment point to TIFF2025506357000137.tif12170D; w is 0, 1, 2, 3, 4 or 5; J is -C 1-5 Alkyl, -N(Rx )(R y ), -C(O)NH 2 , -NH-C(O)-NH 2 , -NH-C(=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 -OC(O)-R-[CH 2 ] u -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 u 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 C and R D are each independently hydrogen and C 1-3 alkyl or R C and R D Each of these, together with the carbon to which it is attached, is an optionally substituted C 3-6 Forming a cycloalkyl; R 7 and R 8 are each independently hydrogen, halo, or C 1-5 Alkyl, C 1-5 alkoxy or hydroxyl). 85. Q, TIFF2025506357000138.tif17170, and q is 1, 2, 3 or 4. 86. An antibody-conjugate according to embodiment 84 or 85, wherein q is 2. 87. Q, TIFF2025506357000139.tif32170, where t is 0, 1, 2, 3, or 4; Q 1 but, TIFF2025506357000140.tif20170R 2 But hydrogen, halo C 1-6 Alkyl or C 1-6 The antibody-conjugate according to embodiment 84, which is alkyl. 88.R 2 is hydrogen or C 1-6 The antibody-conjugate according to embodiment 87, which is alkyl. 89.C 1-6 The antibody-conjugate of embodiment 88, wherein the alkyl is methyl. 90.Q 1 but, TIFF2025506357000141.tif26170 An antibody conjugate as described in embodiment 87. 91.R 2 The antibody-conjugate of embodiment 87, wherein is hydrogen or methyl. 92.Q 1 but, TIFF2025506357000142.tif22170 An antibody conjugate as described in embodiment 91. 93. An antibody conjugate according to any one of embodiments 87 to 92, wherein t is 0. 94.Z is -(CH 2 ) p - and p is 1, 2, 3, 4, 5 or 6. 95. The antibody-conjugate according to embodiment 94, wherein p is 4, 5 or 6. 96. The antibody-conjugate according to embodiment 94, wherein p is 5. 97.Z is -CH 2 -(CH 2 -O-CH 2 ) p -CH 2 - and p is 1, 2, 3, 4, 5 or 6. 98. The antibody-conjugate according to embodiment 87, wherein p is 1, 2 or 3. 99. An antibody-conjugate according to embodiment 87, wherein p is 1. 100.R A is hydrogen or C 1-6 100. The antibody-conjugate according to any one of embodiments 84 to 99, which is alkyl. 101.R A The antibody-conjugate of embodiment 100, wherein is hydrogen or methyl. 102.R A The antibody-conjugate according to any one of embodiments 84 to 99, wherein is phenyl or benzyl. 103.R 7 and R 8 An antibody-conjugate according to any one of embodiments 84 to 102, wherein each of 104.R C and R D each of which, together with the carbon to which it is attached, is optionally substituted C 3-6 The antibody-conjugate according to any one of embodiments 84 to 103, which forms a cycloalkyl. 105.C 3-6 The antibody-conjugate of claim 104, wherein the cycloalkyl is cyclobutane. 106.K is -CH(R)-, R is hydrogen, C 1-3 Alkyl, N(R x )(R y ) or C(O)-N(R x )(R y ) and R x and R y are each independently hydrogen and C 1-3alkyl or R x and R y and R 1 and R 2 are together with the nitrogen to which they are attached to form an optionally substituted 5- to 7-membered heterocyclyl. 107. R is hydrogen or C 1-3 The antibody-conjugate according to embodiment 106, which is alkyl. 108. The antibody-conjugate according to embodiment 107, wherein R is hydrogen. 109. A method for making an Ab conjugate comprising at least six DARs, comprising contacting a compound of formula IA, formula IC or formula II with an antibody to prepare an Ab conjugate comprising at least six DARs. 110. The method of embodiment 109, wherein the DAR is from about 6 to about 16, or from about 8 to about 14, or 6, 7, 8, 9, 10, 11, 12 13, 14, 15, or 16.

[0176]

[0169] Structure L 1 Non-limiting exemplary compounds having -D are The file is TIFF2025506357000143.tif29170.

[0177]

[0170] Structure Ab-((L X )-D) j Non-limiting exemplary compounds having the formula: The file is TIFF2025506357000144.tif34170.

[0178]

[0171] Structure Ab-(L Z ) j Non-limiting exemplary compounds having the formula: The file is TIFF2025506357000145.tif35170.

[0179] A non-limiting example of a compound of formula II is: The file is TIFF2025506357000146.tif35170.

[0180]

[0173] In certain embodiments, the subject matter described herein includes the following L1-CIDES, residues of which may be included in certain Ab-Hy-B-CIDEs: JPEG2025506357000147.jpg222170JPEG2025506357000148.jpg255170JPEG2025506357000149.jpg53170

[0181] V. Preparations

[0174] Pharmaceutical formulations of therapeutic Ab-CIDEs described herein can be prepared in unit dose injectable form using a pharma- ceutically acceptable parenteral vehicle for parenteral administration, e.g., bolus, intravenous, intratumoral injection. Ab-CIDEs having the desired purity are optionally mixed with one or more pharma- ceutically acceptable excipients (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.) in the form of a lyophilized formulation or an aqueous solution for reconstitution.

[0182]

[0175] Ab-CIDE may be formulated according to standard pharmaceutical practice as a pharmaceutical composition. According to this embodiment, there is provided a pharmaceutical composition comprising Ab-CIDE in association with one or more pharma- ceutically acceptable excipients.

[0183]

[0176] A typical formulation is prepared by mixing the Ab-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 the Ab-CIDE is to be applied. Solvents are generally selected based on solvents recognized by those skilled in the art as safe (GRAS) for administration to mammals.

[0184]

[0177] Generally, 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., PEG 400, PEG 300), and the like, and mixtures thereof. Acceptable diluents, carriers, additives, and stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium 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-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; 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 non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0185] The formulations may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, aromas, flavoring agents, and other known additives to provide proper presentation of the Ab-CIDE or to aid in the manufacture of pharmaceutical preparations. The formulations may be prepared using conventional dissolution and mixing procedures.

[0186]

[0179] Formulation may be carried out at ambient temperature, at an appropriate pH, and at the desired purity, by mixing 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 will depend primarily on the particular application and the concentration of compound, but may range from about 3 to about 8. Formulation in acetate buffer at pH 5 is a suitable embodiment.

[0187]

[0180] Ab-CIDE formulations 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.

[0188]

[0181] Ab-CIDE may typically be stored as a solid composition, a lyophilized formulation, or as an aqueous solution.

[0189] Pharmaceutical compositions containing Ab-CIDE may be formulated, prescribed, and administered in a manner consistent with medical practice standards, i.e., amounts, concentrations, schedules, courses, vehicles, and routes of administration. Factors to be considered in this regard include the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to medical practitioners. The "therapeutically effective amount" of the compound to be administered is governed by such considerations and is the minimum amount necessary to prevent, reduce, 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 patient significantly more susceptible to bleeding.

[0190]

[0183] The Ab-CIDEs may be formulated into pharmaceutical dosage forms to provide easily controllable drug dosages and allow patient compliance with a prescribed regimen. The pharmaceutical compositions (or formulations) for application may be packaged in a variety of ways depending on the method used to administer the drug. Generally, the article for distribution includes a container having disposed therein the pharmaceutical formulation in a suitable 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, metal cylinders, and the like. The container may include a tamper-evident assembly to prevent inadvertent access to the contents of the package. In addition, the container has disposed thereon a label describing the contents of the container. The label may also include appropriate warnings.

[0191]

[0184] The pharmaceutical composition may be in the form of a sterile injectable preparation, such as a sterile aqueous or oleaginous injectable suspension. This suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also 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 also be prepared as a lyophilized powder. Acceptable additives and solvents that may be utilized include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils may be conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil may be used, including synthetic monoglycerides or synthetic diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables as well.

[0192] The amount of Ab-CIDE that can be combined with the carrier material to produce a single dosage form can vary depending on the host treated and the particular mode of administration. For example, a sustained release formulation intended for oral administration to humans can contain approximately 1-1000 mg of active material, compounded with an appropriate and convenient amount of carrier material, which can 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 can contain about 3-500 μg of active ingredient per milliliter of solution, such that infusion of a suitable volume at a rate of about 30 mL / hour can occur.

[0193]

[0186] 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 suspension solutions which may contain suspending agents and thickening agents.

[0194] 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 the 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 unit daily sub-dose of the active ingredient, as herein above recited, or an appropriate fraction thereof.

[0195]

[0188] The subject matter further provides veterinary compositions comprising at least one active ingredient as defined above together with a veterinary carrier for the active ingredient. The veterinary carrier is a material useful for the purposes of administration of 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.

[0196] VI. Indications and Treatment Methods It is contemplated that the Ab-CIDEs disclosed herein may be used to treat a variety of diseases or disorders associated with or involving a target protein, such as BRM. Also provided herein is Ab-CIDE or a composition comprising Ab-CIDE for use in therapy. In some embodiments, provided herein is Ab-CIDE or a composition comprising Ab-CIDE for treating or preventing diseases and disorders as disclosed herein. Also provided herein is the use of Ab-CIDE or a composition comprising Ab-CIDE in therapy. In some embodiments, provided herein is the use of Ab-CIDE for treating or preventing diseases and disorders as disclosed herein. Also provided herein is the use of Ab-CIDE or a composition comprising Ab-CIDE in the manufacture of a medicament for treating or preventing diseases and disorders as disclosed herein. Also provided herein is the use of Ab-CIDE or a composition comprising Ab-CIDE in the manufacture of a medicament for treating or preventing diseases and disorders as disclosed herein.

[0197]

[0190] Generally, the disease or disorder to be treated is a target protein dependent disease or disorder, such as a BRM dependent disease or disorder. For example, the target protein dependent disease or disorder is a hyperproliferative disease, such as cancer. Examples of cancers to be treated herein include BRM dependent cancers. In certain embodiments, the cancer is non-small cell lung cancer.

[0198]

[0191] In certain embodiments, the subject matter described herein relates to a method for reducing the level of a target protein in a subject comprising administering to the subject an Ab-CIDE or a composition comprising an Ab-CIDE described herein, wherein the PB moiety binds to the target protein and a ubiquitin ligase affects degradation of the bound target protein, thereby reducing the level of the target protein.

[0199]

[0192] In certain embodiments, Ab-CIDEs comprising antibodies as described above are used in methods of treating cancers, such as solid tumors.

[0200]

[0193] Ab-CIDE may be administered by any route appropriate to the condition being treated. Ab-CIDE is typically administered parenterally, i.e., by injection, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally, and epidurally.

[0201]

[0194] Ab-CIDE can be used alone or in combination with other agents in therapy. For example, Ab-CIDE can be co-administered with at least one additional therapeutic agent. Such combination therapy as described above includes combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration, where administration of Ab-CIDE can occur prior to, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. Ab-CIDE can also be used in combination with radiation therapy.

[0202]

[0195] Ab-CIDE (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing depends in part on whether the administration is brief or chronic, and may be by any suitable route, for example, injections, such as intravenous or subcutaneous injections. A variety of dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time periods, bolus administration, and pulse infusion.

[0203]

[0196] For the prevention or treatment of a disease, the appropriate dosage of Ab-CIDE (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of Ab-CIDE, the severity and course of the disease, whether Ab-CIDE is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to Ab-CIDE, and the discretion of the attending physician. Ab-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-CIDE may be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dosage may 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 more, depending on the condition, treatment should generally be continued until a desired suppression of disease symptoms occurs. One exemplary dosage of Ab-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 from about 2 to about 20, or for example about 6, doses). An initial high loading dose may be followed by one or more lower doses. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0204]

[0197] The methods described herein include methods of degrading a target protein. In certain embodiments, the methods include administering Ab-CIDE to a subject, whereby the target protein is degraded. The level of protein degradation 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%.

[0205]

[0198] The methods described herein include methods of reducing the proliferation of neoplastic tissue, such as non-small cell lung cancer. In certain embodiments, the methods include administering Ab-CIDE to a subject, whereby the proliferation of the neoplastic tissue is reduced. The level of reduction can be from about 1% to about 5%; or from about 1% to about 10%; or from about 1% to about 15%; or from about 1% to about 20%; or from about 1% to about 30%; or from about 1% to about 40%; or from about 1% to about 50%; or from about 10% to about 20%; or from about 10% to about 30%; or from about 10% to about 40%; or from 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%.

[0206] VI. Manufactured products

[0199] In another aspect, described herein are articles of manufacture, e.g., "kits" containing materials useful for treating the diseases and disorders described above. The kits include a container containing Ab-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 packaging of a therapeutic agent, including information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic agent.

[0207]

[0200] 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, for example, 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 may hold Ab-CIDE or a formulation thereof effective to treat a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle).

[0208] At least one active agent in the composition is Ab-CIDE. The label or package insert indicates that the composition is used to treat a selected condition, such as cancer. Additionally, the label or package insert may 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 indicates that the composition comprising Ab-CIDE can be used to treat a disorder resulting from abnormal cell proliferation. The label or package insert may also indicate that the composition can be used to treat other disorders. Alternatively, or in addition, the article of manufacture may 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 may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0209]

[0202] The kit may further include instructions for administration of Ab-CIDE and, if present, a second pharmaceutical formulation. For example, where the kit includes a first composition comprising Ab-CIDE and a second pharmaceutical formulation, the kit may further include instructions for the simultaneous, sequential, or separate administration of the first and second pharmaceutical compositions to a patient in need of the kit.

[0210] In another embodiment, the kit is suitable for delivery of a solid oral form (e.g., tablet or capsule) of Ab-CIDE. Such a kit preferably includes several unit doses. Such a kit may include a card having the doses arranged in the order of their intended use. An example of such a kit is a "blister pack." Blister packs are known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If necessary, 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 of the treatment schedule on which the dosages can be administered.

[0211] In one embodiment, the kit may comprise (a) a first container comprising Ab-CIDE, and optionally (b) a second container containing a second pharmaceutical formulation, the second pharmaceutical formulation comprising a second compound having anti-hyperproliferative activity. Additionally 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. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0212] In certain other embodiments in which the kit comprises an Ab-CIDE and a second therapeutic agent, the kit may include containers for containing the separate compositions, such as separate bottles or separate foil packets, although the separate compositions may also be contained in a single, undivided container. Typically, the kit includes 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), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

[0213] VII. Methods for making conjugates General synthesis route

[0206] The subject matter described herein also relates to a method for preparing CIDE, L1-CIDE and Ab-L1-CIDE from L1-CIDE. In general, the method includes contacting an antibody or its variants, mutations, splice variants, indels and fusions with L1-CIDE under conditions in which the antibody is covalently bound to any available binding site on L1-CIDE, and Ab-L1-CIDE is prepared. The subject matter described herein also relates to a method for preparing Ab-L1-CIDE from an Ab-L1 moiety, i.e., an antibody or its variants, mutations, splice variants, indels and fusions, covalently bound to L1, comprising contacting CIDE with Ab-L1 under conditions in which CIDE is covalently bound to any available binding site on Ab-L1, and Ab-L1-CIDE is prepared. The method may further include routine isolation and purification of Ab-L1-CIDE.

[0214]

[0207] The CIDEs, L1-CIDEs and Ab-L1-CIDEs and other compounds described herein, particularly in light of the description contained herein, can be synthesized by synthetic routes including processes similar to those well known in the chemical arts and other heterocyclic processes 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). Starting materials are generally available from commercial sources, such as Aldrich Chemicals (Milwaukee, Wis.), 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. 1-23, Wiley, NY (1967-2006 ed.) or Beilstein's Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including supplements (also available via the Beilstein online database)).

[0215]

[0208] Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful for synthesizing the CIDEs, L1-CIDEs and Ab-L1-CIDEs and other compounds described herein, as well as the necessary reagents and intermediates, are well known in the art and can be found in, for example, 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 thereof. 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 with 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 description of protecting groups and their uses, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0216] The general procedures and examples provide exemplary methods for preparing CIDE, L1-CIDE and Ab-CIDE as well as other compounds described herein. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize Ab-L1-CIDE and compounds. Although specific starting materials and reagents are shown and discussed in the schemes, general procedures and examples, other starting materials and reagents can be readily substituted 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.

[0217]

[0210] Generally, Ab-L1-CIDE can be prepared by linking CIDE with L1 linker reagent to prepare L1-CIDE, and conjugating L1-CIDE with any of the antibodies or variants, mutations, splice variants, indels and fusions thereof, including cysteine ​​engineered antibodies, described herein, according to the procedures of WO 2013 / 055987; WO 2015 / 023355; WO 2010 / 009124; WO 2015 / 095227. Alternatively, Ab-CIDE can be prepared by first combining an antibody or variants, mutations, splice variants, indels and fusions thereof, including cysteine ​​engineered antibodies, described herein, with L1 linker reagent and conjugating it with any CIDE.

[0218] 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 preparing CIDE, L1-CIDE and Ab-L1-CIDE, as well as other compounds and components thereof, are disclosed elsewhere herein.

[0219]

[0212] A non-limiting general synthetic route for preparing Ab-CIDE is shown in Scheme 1. TIFF2025506357000150.tif88170 Scheme 1

[0220] 1. Cysteine ​​Engineered Antibodies

[0213] Cysteine ​​engineered antibodies (THIOMAB TM ) can be recombinantly expressed and purified using standard methods and typically prepared for conjugation by reduction and reoxidation as follows.

[0221]

[0214] Recombinantly expressed full-length cysteine ​​engineered monoclonal antibody (THIOMAB TMThe THIOMAB antibody (antibody) has a cysteine ​​adduct (cystine) or is glutathionylated on an engineered cysteine ​​due to cell culture conditions. Thus, THIOMAB purified from mammalian cell lines TM Antibodies cannot be conjugated to the Cys-reactive linker L1-CIDE intermediate. Cysteine ​​engineered antibodies can be made reactive for conjugation with the L1-CIDE 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 273:73-80; Soltec Ventures, Beverly, Mass.), followed by reformation (reoxidation) of the interchain disulfide bonds with a mild oxidizing agent 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) are reduced with, for example, approximately 50-fold excess of DTT overnight in 50 mM Tris, pH 8.0, containing 2 mM EDTA at room temperature, which removes Cys and glutathione adducts and reduces the interchain disulfide bonds of the antibodies. Removal of adducts was monitored by reversed-phase LCMS using a PLRP-S column.

[0222] After removing Cys and glutathione adducts, THIOMAB TM The antibodies can be purified by methods commonly known in the art, including cation exchange chromatography, as described in detail herein. TM was diluted and acidified by adding at least 4 volumes of 10 mM succinic acid (pH 5) and / or titrating with 10% acetic acid until the pH was approximately 5. TMThe antibody was then loaded onto a HiTrap S cation exchange column, washed with several 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 and reduced THIOMAB as described above. TM Antibodies were incubated for approximately 3 h in 15× dehydroascorbic acid (DHAA) or in 200 nM to 2 mM aqueous copper sulfate (CuSO 4 ) at room temperature overnight. Other oxidants (i.e., oxidizing agents) and oxidation conditions known in the art may be used. Ambient air oxidation may also be effective. This mild partial reoxidation step efficiently forms intrachain disulfides with high fidelity. Reoxidation may be monitored by reversed-phase LCMS using a PLRP-S column. The reoxidized THIOMAB TM The antibody can be purified on an S column as described above, except that it is diluted with the succinate buffer described above to reach a pH of about 5, followed by elution 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 may be added to the antibody to a final concentration of 2 mM. TM The resulting THIOMAB is ready for conjugation. TMAntibodies may be stored at -20°C or -80°C. Liquid chromatography / mass spectrometry may be performed on a 6200 series TOF or QTOF Agilent LC / MS. Samples were chromatographed on a PRLP-S® (1000A, microbore column (50 mm x 2.1 mm, Polymer Laboratories, Shropshire, UK), heated to 80°C). A linear gradient of 30-40% B (solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile) was used and the eluent was directly ionized using an electrospray source. Data were 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 glycans (carbohydrates).

[0223]

[0216] Alternatively, antibodies can be partially digested with LysC (0.25 μg / 50 μg (micrograms) antibody) at 37° C. for 15 minutes to obtain Fab and Fc fragments for analysis by LCMS.

[0224] 2. Conjugation of the Linker L1-CIDE Group to the Antibody

[0217] In one method of conjugating the linker L1-CIDE 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. Approximately 3 molar to 20 equivalents of excess linker-CIDE intermediate bearing a thiol reactive group (e.g., maleimide or 4-nitropyridin disulfide, or methanethiosulfonyl (MTS) disulfide) is dissolved in DMF or DMA with or without propylene glycol and then added to the reduced, reoxidized and 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 aimed at removing remaining unreacted L1-CIDE intermediate and aggregated protein (if present at significant levels). For example, the conjugate may be diluted with 10 mM histidine-acetate (pH 5.5) until the final pH is about 5.5 and purified by S cation exchange column chromatography using either a HiTrap S column (GE Healthcare) or an S Maxi spin column (Pierce) connected to an Akta purification system. Alternatively, the conjugate may be purified by gel filtration chromatography using an S200 column or a Zeba spin column connected to an Akta purification system. Alternatively, dialysis may be used to remove unreacted or excess linker drug. THIOMAB can be purified using either gel filtration or dialysis. TM The antibody-drug conjugates can be formulated in 20 mM His / Acetate (pH 5) with 240 mM sucrose. The purified conjugates can be concentrated by centrifugation ultrafiltration and / or filtered through a 0.2 μm filter under sterile conditions and frozen for storage. Ab-L1-CIDE was characterized by BCA assay to determine protein concentration, analytical SEC (size exclusion chromatography) for aggregation analysis, and LC-MS after treatment with LysC endopeptidase (LysC) and reduction using standard procedures to calculate the DAR.

[0225]

[0218] 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.

[0226] LC-MS analysis can be performed on Ab-L1-CIDE using an Agilent QTOF 6520 ESI instrument. As an example, Ab-L1-CIDE is treated with endoproteinase Lys C (Promega) at 1:500 by weight in Tris (pH 7.5) at 37° C. for 30 min. The resulting cleaved 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 is H2SO4 with 0.05% TFA. 2 The LCMS protocol was run at 100°C for 10 min, 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 deconvoluted using Mass Hunter™ software (Agilent Technologies) to calculate the masses of antibody fragments. Peaks in the deconvoluted LCMS spectra can be assigned and quantified. The CIDE-to-antibody ratio (DAR) is calculated by calculating the ratio of the intensity of one or more peaks corresponding to CIDE-conjugated antibodies to all peaks observed.

[0227] 3. Preparation of E3LB-L2 intermediate by general synthetic coupling of L2 with E3LB In certain embodiments, L2 is first contacted with a first suitable solvent, a first base, and a first coupling reagent to prepare a first solution. In certain embodiments, contacting L2 with the first suitable solvent, the first base, and the first coupling reagent proceeds at room temperature (about 25° C.) for about 15 minutes. E3LB is then contacted with the first solution.

[0228]

[0221] In certain embodiments, contacting E3LB with the first solution proceeds for about 1 hour at room temperature (about 25° C.) The solution is then concentrated and optionally purified.

[0229] In certain embodiments, the molar ratio of L2 to the first base pair first coupling reagent is about 1:4:1.19. In certain 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.

[0230] In certain embodiments, the molar ratio of L2 to E3LB is about 1: 1. In certain 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.

[0231] 4. General synthetic method for coupling E3LB-L2 intermediate with PB

[0224] In certain embodiments, the E3LB-L2 intermediate is coupled to PB to prepare CIDE. In certain embodiments, the PB is first contacted with a second suitable solvent, a second base, and a second coupling reagent. In certain embodiments, the contacting proceeds at room temperature (about 25°C) for about 10 minutes. The solution is then contacted with the E3LB-L2 intermediate. In certain embodiments, the contacting of the second solution with the E3LB-L2 intermediate proceeds at room temperature (about 25°C) for about 1 hour. The solution is then concentrated and optionally purified to prepare CIDE.

[0232] In certain embodiments, the molar ratio of PB to the second base pair second coupling reagent is about 1:4:1.2. In certain 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.

[0233]

[0226] In certain embodiments, the molar ratio of PB to E3LB-L2 intermediate is about 1: 1. In certain 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.

[0234] 5. General synthetic method for coupling CIDE with L1 to prepare L1-CIDE In certain embodiments, CIDE is contacted with L1 and a third base in a third suitable solvent to prepare a solution. In certain embodiments, the contacting proceeds at about (about 25° C.) for about 2 hours. The solution can then be optionally purified to prepare L1-CIDE.

[0235] In certain embodiments, the molar ratio of CIDE to L1 is about 1:4. In certain 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.

[0236] 6. General synthetic method for coupling L1-CIDE to antibodies In certain 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, In certain embodiments, the thiol is maleimide or 4-nitropyridindisulfide. In certain embodiments, the suitable solvent is selected from the group consisting of dimethylformamide, dimethylacetamide, and propylene glycol.

[0237]

[0231] In certain embodiments, the molar ratio of L1-CIDE to thiol reactive groups is from about 3:1 to about 20:1.

[0238] In certain embodiments, contacting of the antibody with the solution comprising L1-CIDE, a thiol reactive group, and a suitable solvent proceeds for about 1 to about 24 hours. In certain embodiments, contacting of the antibody with the solution comprising L1-CIDE, a thiol reactive group, and a suitable solvent proceeds at about room temperature (about 25° C.) to about 37° C.

[0239] In certain 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.

[0240]

[0234] In certain embodiments of the above general method, the base is selected from the group consisting of N,N-diisopropylethylamine (DIEA), triethylamine, and 2,2,2,6,6-tetramethylpiperidine. In certain embodiments, the coupling reagent is 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'-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).

[0241] In certain 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.

[0242] In certain 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.

[0243]

[0237] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0244] Example 1 Synthesis of hydrolyzable linker fragment and PB Intermediate 1: Ethyl (S)-1-((6-(dimethylamino)-1-oxo-1-((4-((2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methyl)phenyl)amino)hexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000151.tif91170

[0245]

[0239] Process 1:N 2 -(tert-butoxycarbonyl)-N 6 ,N 6 -Dimethyl-L-lysine TIFF2025506357000152.tif31170Under hydrogen (3 atm), (tert-butoxycarbonyl)-L-lysine (20.0 g, 81.2 mmol), 37% aqueous CH 2 A solution of 2.00 g (12.2 g, 150 mmol) and 10% Pd / C (2.00 g) in methyl alcohol (100 mL) was stirred at room temperature for 4 h. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in Et 2 The solid was collected by filtration to give 20.6 g (92% yield) of the title compound as a white solid. LCMS (ESI) [M+H] + =275.

[0246] Step 2: 1-Bromo-2-((4-nitrobenzyl)oxy)benzene TIFF2025506357000153.tif411702-Bromophenol (52.6 g, 304 mmol), 1-(bromomethyl)-4-nitrobenzene (65.7 g, 304 mmol) and K 2 CO 3 A solution of (83.9 g, 608 mmol) in DMF (700 mL) was stirred at room temperature for 1 h. EtOAc was added and the organic layer was washed three times with water. The organic layer was washed with anhydrous Na 2 SO 4 Drying at rt and concentration in vacuo afforded 73.8 g (78% yield) of the title compound as a yellow solid. 1 H NMR (300MHz, DMSO-d 6 ,ppm)δ 8.34-8.24(m,2H),7.81-7.70(m,2H),7.62(dd,J=7.9,1.6Hz,1H),7.36(ddd,J=8.3,7 .3,1.6Hz,1H),7.20(dd,J=8.3,1.5Hz,1H),6.94(td,J=7.6,1.4Hz,1H),5.39(s,2H).

[0247] Step 3: 4-((2-bromophenoxy)methyl)aniline TIFF2025506357000154.tif41170Under nitrogen, 1-bromo-2-((4-nitrobenzyl)oxy)benzene (43.0 g, 139.5 mmol) and K 2 CO 3 (115 g, 837 mmol) in acetonitrile (800 mL) and water (400 mL) was added to a solution of Na 2 S 2 O 4 (242 g, 1395 mmol) was added in small portions at 0° C. The mixture was stirred at room temperature for 6 h and the product was extracted once with EtOAc. The organic layer was washed with anhydrous Na 2 SO 4 Drying at 40° C. and concentration in vacuo afforded 35 g (crude) of the title compound as a yellow solid. LCMS (ESI) [M+H]+ =278.

[0248] Step 4: tert-Butyl (S)-(1-((4-((2-bromophenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamate TIFF2025506357000155.tif38170 Nitrogen, N 2 -(tert-butoxycarbonyl)-N 6 ,N 6 To a solution of 13.3 g, 48.4 mmol) and NMM (10.3 g, 96.9 mmol) in tetrahydrofuran (200 mL) was added iso-butyl chloroformate (7.91 g, 58.1 mmol) dropwise at -25°C. The reaction was stirred at -25°C for 0.5 h. A solution of 4-((2-bromophenoxy)methyl)aniline (16.1 g, crude) in tetrahydrofuran (120 mL) was added at -25°C. The reaction was stirred at room temperature for 4 h. The solvent was concentrated under vacuum. DCM was added and washed with water. The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C. and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-9% MeOH / DCM) to give 6.70 g (25% yield) of the title compound as a white solid. LCMS (ESI) [M+H] + =534.

[0249] Step 5: (S)-2-Amino-N-(4-((2-bromophenoxy)methyl)phenyl)-6-(dimethylamino)hexanamide (2,2,2-trifluoroacetate) TIFF2025506357000156.tif37170 A solution of tert-butyl (S)-(1-((4-((2-bromophenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamate (4.00 g, 7.48 mmol) in 5% TFA / HFIP (50 mL) was stirred at room temperature for 3 h. The solvent was concentrated in vacuo and used directly in the next step. LCMS (ESI) [M+H] +=434.

[0250] Step 6: Ethyl (S)-1-((1-((4-((2-bromophenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000157.tif30170To a solution of (S)-2-amino-N-(4-((2-bromophenoxy)methyl)phenyl)-6-(dimethylamino)hexanamide (2,2,2-trifluoroacetate) (crude from step 5), 1-(ethoxycarbonyl)cyclobutane-1-carboxylic acid (1.55 g, 8.98 mmol) and DIPEA (9.65 g, 74.8 mmol) in DMF (20 mL) was added HATU (3.41 g, 8.98 mmol) at 0 °C. The mixture was stirred at room temperature for 0.5 h. The crude was purified by column chromatography on a pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH 4 HCO 3 )) to give 2.70 g (61% yield) of the title compound as a red solid. LCMS (ESI) [M+H] + =588. 1 H NMR (300MHz, DMSO-d 6 ,ppm)δ 9.87(s,1H),7.72(d,J=7.9Hz,1H),7.54-7.42(m,3H),7.30(d,J=8.6Hz,2H),7.21(dd d,J=8.8,7.3,1.6Hz,1H),7.07(dd,J=8.4,1.5Hz,1H),6.77(td,J=7.6,1.4Hz,1H),5. 02(s,2H),4.30(q,J=8.0Hz,1H),4.00(q,J=7.1Hz,2H),2.35-2.21(m,2H),2.03(t,J= 6.8Hz,2H),1.96(s,6H),1.80-1.41(m,4H),1.30-1.14(m,6H),1.06(t,J=7.1Hz,3H).

[0251] Step 7: Ethyl (S)-1-((6-(dimethylamino)-1-oxo-1-((4-((2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methyl)phenyl)amino)hexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000158.tif28170Under nitrogen, ethyl (S)-1-((1-((4-((2-bromophenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (500 mg, 0.852 mmol), B 2 Pin 2 (649mg, 2.55mmol), Pd(dppf)Cl 2 A solution of (124 mg, 0.170 mmol) and KOAc (250 mg, 2.55 mmol) in 1,4-dioxane (5 mL) was stirred at 80 °C for 2 h. The reaction was diluted with DCM and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (gradient: 0% to 20% MeOH / DCM (0.3% 7M NH 3 / MeOH) to give 390 mg (72% yield) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =636.

[0252] Example 2 Partial synthesis of E3LB Intermediate 2: (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 TIFF2025506357000159.tif89170

[0253] Step 1: tert-Butyl (S)-(1-(4-cyanophenyl)ethyl)carbamate TIFF2025506357000160.tif21170Under nitrogen, tert-butyl (S)-(1-(4-bromophenyl)ethyl)carbamate (5.00 g, 16.7 mmol), zinc cyanide (2.32 g, 20.1 mmol) and Pd(PPh 3 ) 4 A solution of (184 mg, 0.159 mmol) in DMF (50 mL) was stirred at 80° C. for 30 min. The reaction mixture was then poured into water and the solid was collected by filtration and washed with water. The solid was purified by flash chromatography on silica gel (solvent gradient: 0% to 30% EtOAc / petroleum ether) to give 2.80 g (68% yield) of the title compound as a colorless oil. LCMS (ESI): [M+H] + =247.

[0254] Step 2: (S)-4-(1-aminoethyl)benzonitrile (hydrogen chloride salt) TIFF2025506357000161.tif22170Under nitrogen, tert-butyl (S)-(1-(4-cyanophenyl)ethyl)carbamate (2.80 g, 11.4 mmol) was dissolved in CH 2 Cl 2 (10 mL) and 4M HCl / dioxane (20 mL) was stirred at room temperature for 1 h. The resulting mixture was concentrated in vacuo to give 2.8 g (crude) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =147.

[0255] Step 3: tert-Butyl (2S,4R)-2-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylate TIFF2025506357000162.tif37170A solution of (S)-4-(1-aminoethyl)benzonitrile (2.35 g, 16.1 mmol), (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (3.71 g, 16.1 mmol), HATU (7.34 g, 19.3 mmol) and DIPEA (20.7 g, 161 mmol) in DMF (20 mL) was stirred at room temperature under nitrogen for 30 min. The reaction was purified by column chromatography (solvent gradient: 0-60% ACN in water (0.05% NH 4 HCO 3 )) to give 4.60 g (50% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =360.

[0256] Step 4: (2S,4R)-N-((S)-1-(4-cyanophenyl)ethyl)-4-hydroxypyrrolidine-2-carboxamide (hydrogen chloride salt) TIFF2025506357000163.tif34170Under nitrogen, tert-butyl (2S,4R)-2-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylate (3.50 g, 9.7 mmol) was dissolved in CH 2 Cl 2 (35 mL) and 4M HCl / dioxane (70 mL) was stirred at room temperature for 30 min. The resulting mixture was concentrated in vacuo to give 3.1 g (crude) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =260.

[0257] Step 5: Methyl 2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoate TIFF2025506357000164.tif31170Methyl 2-(3-hydroxyisoxazol-5-yl)-3-methylbutanoate (10.0 g, 50.2 mmol), 2-bromo-1,1-diethoxyethane (11.3 mL, 75.1 mmol) and K2 CO 3 A solution of (13.9 g, 101 mmol) in DMF (150 mL) was stirred at 70 °C overnight. The reaction mixture was diluted with ethyl acetate and then washed with water. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on a pre-packed C18 column (solvent gradient: 0-100% ACN in water (0.05% NH 4 HCO 3 )) to give 12.5 g (79% yield) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =316.

[0258] Step 6: 2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoic acid TIFF2025506357000165.tif31170 A solution of methyl 2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoate (12.5 g, 39.6 mmol) and LiOH (4.75 g, 198 mmol) in tetrahydrofuran (100 mL) and water (100 mL) was stirred at room temperature for 4 h. The pH was adjusted to 4-5 with 1 M hydrogen chloride and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum to give the title compound 13 g (crude) as a white solid. LC-MS: (ESI, m / z): [M+Na] + =324.

[0259] Step 7: (2S,4R)-N-((S)-1-(4-cyanophenyl)ethyl)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide TIFF2025506357000166.tif36170A solution of (2S,4R)-N-((S)-1-(4-cyanophenyl)ethyl)-4-hydroxypyrrolidine-2-carboxamide (hydrochloride salt) (3.00 g, crude from step 4), 2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoic acid (3.00 g, 9.96 mmol), HATU (4.56 g, 12.0 mmol) and DIPEA (3.85 g, 29.8 mmol) in DMF (30 mL) was stirred at room temperature for 30 min. The crude was purified by column chromatography on a prepacked C18 column (solvent gradient: 0-100% ACN in water (0.05% NH 4 HCO 3 )) to give 4.3 g of a mixture of two isomers as a yellow solid. The mixture was separated by Prep Chiral SFC under the following conditions: Column: CHIRALPAK IG-3, 3.0*50mm, 3μm; Mobile phase B: IPA (0.1%DEA); Flow rate: 2mL / min; Gradient: homogeneous 10% B; Wavelength: 220nm to give 2.14 g (faster peak, undesired isomer) and 2.6 g (slower peak, desired isomer) as a yellow solid.

[0260] For the faster peak (undesired isomer): LC-MS: (ESI, m / z): [M+Na] + =565. 1 H NMR (300MHz, DMSO-d 6,ppm)δ 8.35(8.95)(d,J=7.6Hz,1H),7.85-7.70(m,2H),7.52-7.30(m,2H),6.08(6.16)(s,1H),5.10(d,J =3.6Hz,1H),5.06-4.83(m,1H),4.79(td,J=5.2,1.7Hz,1H),4.55-4.29(m,1H),4.27-4.19(m,1H) ,4.12-3.99(m,2H),3.73(d,J=8.6Hz,1H),3.67-3.38(m,6H),2.34-2.15(m,1H),2.09-1.81(m,1H ),1.78-1.60(m,1H),1.40-1.25(m,3H),1.19-1.05(m,6H),1.05-0.90(m,3H),0.85-0.70(m,3H).

[0261] For the slower peak (desired isomer), LC-MS: (ESI, m / z): [M+Na] + =565. 1 H NMR (300MHz, DMSO-d 6 ,ppm)δ 8.46(8.85)(d,J=7.4Hz,1H),7.83-7.72(m,2H),7.45(d,J=8.5Hz,2H),6.13(5.92)(m,1H),5.0 9(d,J=3.3Hz,1H),4.98-4.82(m,1H),4.79(td,J=5.2,2.0Hz,1H),4.39-4.28(m,1H),4.24(s,1 H),4.07(d,J=5.2Hz,2H),3.80-3.59(m,4H),3.56-3.39(m,3H),2.29-2.13(m,1H),2.05-1.65( m,2H),1.45-1.25(m,3H),1.20-1.00(m,6H),0.93(t,J=6.9Hz,3H),0.79(dd,J=9.4,6.7Hz,3H).

[0262] Step 8: (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 TIFF2025506357000167.tif34170(2S,4R)-N-((S)-1-(4-cyanophenyl)ethyl)-1-((R)-2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (300 mg, 0.550 mmol) was dissolved in tetrahydrofuran (3 mL) and 1 M H 2 SO 4 The solution in (3 mL) was stirred at 50° C. for 2 h. The solution was cooled and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The product was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo to give 250 mg (crude) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =469.

[0263] Example 3 Partial synthesis of E3LB Intermediate 3: (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 TIFF2025506357000168.tif69170

[0264] Step 1: tert-Butyl (2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylate TIFF2025506357000169.tif26170A solution of (S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethan-1-amine (hydrochloride) (10.0 g, 39.3 mmol), (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (9.07 g, 39.2 mmol), HATU (17.9 g, 47.0 mmol) and DIPEA (25.3 g, 195 mmol) in DMF (100 mL) was stirred at room temperature for 1 h. The reaction was diluted with EtOAc and washed with water. The organic layer was washed with anhydrous Na 2 SO 4 The residue was purified by column chromatography (solvent gradient: 0-100% ACN in water (0.05% NH 4 HCO 3 )) to give 14.2 g (83% yield) of the title compound as an off-white solid. LC-MS: (ESI, m / z): [M+H] + =432. Step 2: (2S,4R)-4-Hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (hydrochloride) TIFF2025506357000170.tif26170 A solution of tert-butyl (2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylate (6.89 g, 13.5 mmol) in 4M HCl / dioxane (50 mL) and dichloromethane (100 mL) was stirred at room temperature for 1 h. The solvent was concentrated in vacuo to give 5.8 g (crude) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =332.

[0265] Step 3: (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 TIFF2025506357000171.tif41170 A solution of 2-(3-(2,2-diethoxyethoxy)isoxazol-5-yl)-3-methylbutanoic acid (4.80 g, 15.9 mmol), (2S,4R)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (5.78 g, 15.7 mmol), HATU (7.29 g, 19.2 mmol) and DIPEA (6.19 g, 48.0 mmol) in DMF (60 mL) was stirred at room temperature for 30 min. The crude was purified by column chromatography (solvent gradient: 0-100% ACN in water (0.05% NH 4 HCO 3 )) to give 7.8 g of a mixture of two isomers as a yellow solid. The mixture was purified under the following conditions: Column: CHIRAL ART Amylose-SA, 5*25cm, 5μm; Mobile phase A: CO 2 , Mobile phase B: MeOH; Flow rate: 200 mL / min; Gradient: isocratic 40% B; Column temperature (°C): 35; Back pressure (bar): 100; Wavelength: 220 nm; RT1 (min): 2.61; RT2 (min): 3.63; Sample solvent: MeOH (0.1% 2M NH 3 --MeOH) by prep chiral SFC to give 2.57 g of the faster peak (undesired isomer) and 3.00 g of the slower peak (desired isomer) as yellow solids.

[0266] For the faster peak (undesired isomer): LC-MS: (ESI, m / z): [M+H] + =615; 1 H NMR (300MHz, DMSO-d 6,ppm)δ 9.02(s,1H),8.31(d,J=7.8Hz,1H),7.58-7.29(m,4H),6.16(6.23)(s,1 H),5.09(d,J=3.6Hz,1H),5.05-4.71(m,2H),4.52-4.38(m,1H),4.25(s ,1H),4.19-4.04(m,2H),3.83-3.39(m,7H),2.44(d,J=3.4Hz,3H),2.32 -1.74(m,3H),1.54-1.30(m,3H),1.23-1.09(m,6H),1.08-0.73(m,6H).

[0267] For the slower peak (desired isomer): LC-MS: (ESI, m / z): [M+H] + =615; 1 H NMR (300MHz, DMSO-d 6 ,ppm) δ 8.97(d,J=2.6Hz,1H),8.40(8.80)(d,J=7.7Hz,1H),7.50-7.26(m,4H),6.12(5.94)(s,1H), 5.08(d,J=3.3Hz,1H),4.95-4.80(m,1H),4.79(t,J=5.2Hz,1H),4.36(t,J=7.8Hz,1H),4.26( s,1H),4.08(d,J=5.2Hz,2H),3.73-3.36(m,7H),2.44(d,J=1.5Hz,3H),2.29-1.96(m,2H),1. 80-1.65(m,1H),1.50-1.30(m,3H),1.20-1.05(m,6H),1.05-0.90(m,3H),0.85-0.70(m,3H).

[0268] Step 4: (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 TIFF2025506357000172.tif41170(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 (300 mg, 0.490 mmol) was dissolved in tetrahydrofuran (3 mL) and 1 M H 2 SO 4 The solution in (3 mL) was stirred at 50° C. for 2 h. The solution was cooled and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The product was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under vacuum to give 260 mg (crude) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =541.

[0269] Example 4 Synthesis of part of hydrolyzable linker Intermediate 4: 1-(5-aminopentyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetic acid) TIFF2025506357000173.tif28170

[0270] Step 1: tert-Butyl (5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamate TIFF2025506357000174.tif18170Under nitrogen, tert-butyl (5-aminopentyl)carbamate (2.60 g, 12.8 mmol) in dioxane (20 mL) and saturated NaHCO 3To a solution in 100 ml of ethyl acetate (64 mL) was added methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (2.00 g, 12.9 mmol). The solution was stirred at 0° C. for 40 min. The reaction mixture was then stirred at room temperature for 1 h. The reaction was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (gradient: 0% to 30% EtOAc / petroleum ether) to give 2.00 g (54% yield) of the title compound as a brown oil. LC-MS: (ESI, m / z): [M+H] + =283.

[0271] Step 2: 1-(5-aminopentyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetic acid) TIFF2025506357000175.tif20170 Under nitrogen, a solution of tert-butyl (5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamate (200 mg, 0.709 mmol) in TFA (0.5 mL) and dichloromethane (2 mL) was stirred at room temperature for 30 min. The resulting mixture was concentrated under vacuum to give 270 mg (crude) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =183.

[0272] Example 5 Synthesis of part of hydrolyzable linker Intermediate 5: Ethyl (S)-1-((6-(((allyloxy)carbonyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000176.tif31170

[0273]

[0268] Process 1:N 6 -((allyloxy)carbonyl)-N 2 -(1-(ethoxycarbonyl)cyclobutane-1-carbonyl)-L-lysine TIFF2025506357000177.tif32170N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 A solution of -((allyloxy)carbonyl)-L-lysine (900 mg, 1.98 mmol) and DIPEA (1 g, 7.75 mmol) in DCM (10 mL) and DMF (10 mL) was added to 2-chlorotrityl chloride resin (2 g). The mixture was agitated at room temperature for 1 h. 5 mL of MeOH was added and agitated at room temperature for 0.5 h. The resin was washed three times with DMF. A solution of piperidine (4 mL) and DMF (16 mL) was added and agitated at room temperature for 0.5 h. It was then washed four times with DMF. Then, HATU (1.1 g, 2.89 mmol) and HOBt (454 mg, 2.98 mmol) were added to a solution of 1-ethoxycarbonylcyclobutanecarboxylic acid (514 mg, 2.98 mmol) and DIPEA (771 mg, 5.97 mmol) in DMF (15 mL) at 0° C. and stirred at room temperature for 0.5 h. The solution was added to the above resin and stirred at room temperature for 1 h. The resin was washed three times using DMF, followed by DCM. The resin was agitated in a solution of HFIP (4 mL) and DCM (16 mL) for 1 h. The resin was filtered and the filtrate was concentrated to give 900 mg (crude) of the title compound as a yellow oil. LCMS (ESI, m / z) [M+H] + =385.

[0274] Step 2: Ethyl (S)-1-((6-(((allyloxy)carbonyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000178.tif33170N 6 -((allyloxy)carbonyl)-N 2A solution of -(1-(ethoxycarbonyl)cyclobutane-1-carbonyl)-L-lysine (4.2 g, 10.96 mmol), (4-aminophenyl)methanol (1.48 g, 11.9 mmol), EEDQ (5.41 g, 21.8 mmol) in DCM (40 mL) and MeOH (20 mL) was stirred at 30° C. for 1 h. The solvent was evaporated and the crude was purified by flash chromatography on silica gel (solvent gradient: 0-10% MeOH in DCM) to give 2.6 g (49% yield) of the title compound as a yellow oil. LCMS (ESI, m / z) [M+H] + =490.

[0275] Example 6 Synthesis of part of linker L2 Intermediate 6: tert-Butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000179.tif74170 Step 1: (1s,3s)-3-(benzyloxy)cyclobutan-1-ol TIFF2025506357000180.tif13170Under nitrogen, a solution of 3-(benzyloxy)cyclobutan-1-one (10.0 g, 56.8 mmol) in methanol (100 mL) was diluted with NaBH 4 (2.15 g, 56.9 mmol) was added at 0° C. The resulting solution was stirred at 0° C. for 1 h. The reaction was quenched with water and extracted with EtOAc. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-50% ethyl acetate / petroleum ether) to give 9.85 g (97.4% yield) of the title compound as a colorless oil. LC-MS: (ESI, m / z): [M+H] + =179.

[0276] Step 2: 4-((1r,3r)-3-(benzyloxy)cyclobutoxy)pyridine TIFF2025506357000181.tif13170Under nitrogen, (1s,3s)-3-(benzyloxy)cyclobutan-1-ol (11.0 g, 61.7 mmol), pyridin-4-ol (8.80 g, 92.5 mmol) and PPh 3 To a solution of (19.4 g, 74.2 mmol) in tetrahydrofuran (110 mL) was added DIAD (14.9 g, 74.1 mmol) at 0° C. The reaction was stirred at 50 for 2 h. The organic layer was concentrated under vacuum. The resulting solution was diluted with water and extracted with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-50% ethyl acetate / petroleum ether) to give 15.0 g (crude) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =256.

[0277] Step 3: 1-benzyl-4-((1r,3r)-3-(benzyloxy)cyclobutoxy)pyridin-1-ium TIFF2025506357000182.tif17170 Under nitrogen, a solution of 4-((1r,3r)-3-(benzyloxy)cyclobutoxy)pyridine (7.00 g, crude) and BnBr (4.69 g, 27.4 mmol) in toluene (70 mL) was stirred at 80° C. overnight. The solution was concentrated under vacuum. Petroleum ether was added and the solution was stirred until a white solid precipitated. After filtration, the solid was collected to give 7.86 g of the title compound as a white solid. LC-MS: (ESI, m / z): [M] + =346.

[0278] Step 4: 1-Benzyl-4-((1r,3r)-3-(benzyloxy)cyclobutoxy)-1,2,3,6-tetrahydropyridine TIFF2025506357000183.tif14170Under nitrogen, a solution of 1-benzyl-4-((1r,3r)-3-(benzyloxy)cyclobutoxy)pyridin-1-ium (7.50 g, 21.7 mmol) in ethanol (75 mL) was diluted with NaBH 4 (4.90 g, 129 mmol) was added at 0° C. The resulting solution was stirred at room temperature for 1 h. The resulting solution was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (gradient: 0-100% ethyl acetate / petroleum ether) to give 4.13 g (55.1% yield) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =350.

[0279] Step 5: tert-Butyl 4-((1r,3r)-3-hydroxycyclobutoxy)piperidine-1-carboxylate TIFF2025506357000184.tif14170 Under hydrogen (3 atm), 1-benzyl-4-((1r,3r)-3-(benzyloxy)cyclobutoxy)-1,2,3,6-tetrahydropyridine (4.00 g, 11.4 mmol), Boc 2 O(7.49g, 34.3mmol), 20% Pb(OH) 2 A solution of Pd / C (800 mg) and 10% Pd / C (800 mg) in methanol (400 mL) was stirred at room temperature for 48 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-100% ethyl acetate / petroleum ether) to give 900 mg (28.9% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =272.

[0280] Step 6: tert-Butyl 4-((1r,3r)-3-((4-bromopyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000185.tif21170Under nitrogen, tert-butyl 4-((1r,3r)-3-hydroxycyclobutoxy)piperidine-1-carboxylate (3.27 g, 12.0 mmol), 4-bromo-2-fluoro-pyridine (3.92 g, 22.4 mmol) and Cs 2 CO 3 A solution of (7.81 g, 24.0 mmol) in ACN (150 mL) was stirred at 90 for 12 h. The solid was filtered and the filtrate was concentrated under vacuum. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-30% ethyl acetate / petroleum ether) to give 4.50 g (87% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =427.

[0281] Step 7: Benzyl 8-(2-((1r,3r)-3-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate TIFF2025506357000186.tif21170Under nitrogen, tert-butyl 4-((1r,3r)-3-((4-bromopyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (4.50 g, 10.5 mmol), benzyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (3.23 g, 13.1 mmol), C S2 CO 3 (16.7 g, 51.6 mmol) and Ruphos Pd G2 (1.73 g, 2.22 mmol) in toluene (300 mL) was stirred at 90° C. for 12 h. The solution was concentrated under vacuum. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-30% ethyl acetate / petroleum ether) to give 4.70 g (75.2% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =593.

[0282] Step 8: tert-Butyl 4-((1r,3r)-3-((4-(3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000187.tif21170 Under hydrogen (3 atm), benzyl 8-(2-((1r,3r)-3-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (260 mg, 0.438 mmol), 20% Pb(OH) 2 A solution of / C (61.6 mg) in ethanol (40 mL) and THF (40 mL) was stirred at 60° C. for 3 h. After filtration, the filtrate was collected and concentrated under vacuum to give 220 mg (crude) of the title compound as a black solid. LC-MS: (ESI, m / z): [M+H] + =459.

[0283] Step 9: tert-Butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000188.tif34170Under nitrogen, a solution of 4-bromo-6-chloro-pyridazin-3-amine (0.60 g, 2.98 mmol), tert-butyl 4-((1r,3r)-3-((4-(3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (1.10 g, 2.40 mmol) and DIPEA (3.10 g, 24.0 mmol) in DMSO (15 mL) was stirred at 130 °C for 18 h. The reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by column chromatography on a pre-packed C18 column (solvent gradient: 0-100% ACN in water (0.05% NH 3. H 2Purification by HPLC) afforded 1.10 g (78% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =586. 1 H NMR (300MHz, DMSO-d 6 ,ppm)δ 7.76(d,J=6.0Hz,1H),6.90(s,1H),6.50(dd,J=6.1,2.1Hz,1H),6.12(d,J=2.0Hz,1H),5.85 (s,2H),5.26-5.12(m,1H),4.45(s,2H),4.31(p,J=6.0Hz,1H),3.66(dt,J=13.4,4.7Hz,2H), 3.50-3.37(m,1H),3.18(d,J=11.5Hz,2H),2.92(dd,J=35.6,11.5Hz,4H),2.40-2.21(m,4H), 2.13(d,J=7.2Hz,2H),1.97-1.88(m,2H),1.80-1.70(m,2H),1.39(s,9H),1.33-1.20(m,2H).

[0284] Example 7 Synthesis of PB and part of linker L2 Intermediate 7: tert-Butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-(methoxymethoxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate TIFF2025506357000189.tif39170

[0285] Step 1: tert-Butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-(methoxymethoxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)ethyl)-3-methylpiperazine-1-carboxylate TIFF2025506357000190.tif36170Under nitrogen, 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 (1.00 g, 1.79 mmol, provided by Genentech), (2-(methoxymethoxy)phenyl)boronic acid (391 mg, 2.15 mmol), Pd(PPh 3 ) 4 (413 mg, 0.358 mmol) and K 2 CO 3 A solution of (741 mg, 5.37 mmol) in dioxane (10 mL) and water (2 mL) was stirred at 100° C. for 1 h. The reaction was diluted with water and extracted with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (solvent gradient: 0% to 10% MeOH / DCM) to give 670 mg (57% yield) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =661. 1 H NMR (300MHz, DMSO-d 6 ,ppm)δ 7.76(d,J=5.9Hz,1H),7.58(dd,J=7.6,1.8Hz,1H),7.34(ddd,J=9.0,7.3,1.8Hz,1H),7.18-7.01(m,3 H),6.51(dd,J=6.1,2.0Hz,1H),6.12(d,J=2.0Hz,1H),5.72(s,2H),5.14(s,2H),4.47(s,2H),4.25(t ,J=6.1Hz,2H),3.53(d,J=12.8Hz,2H),3.22(s,3H),3.14-2.67(m,8H),2.64-2.56(m,1H),2.46-2.36 (m,1H),2.32-2.22(m,1H),2.22-2.13(m,2H),2.00-1.90(m,2H),1.38(s,9H),0.96(d,J=6.2Hz,3H).

[0286] Example 8 Synthesis of hydrolyzable linker, PB, and part of linker L2 Intermediate 8: 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-oxy sobutan-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 TIFF2025506357000191.tif129170

[0287] Step 1: tert-Butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)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-methylpiperazine-1-carboxylate TIFF2025506357000192.tif39170Under nitrogen, ethyl (S)-1-((6-(dimethylamino)-1-oxo-1-((4-((2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methyl)phenyl)amino)hexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (316 mg, 0.570 mmol), 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 (538 mg, 0.850 mmol), K 3 PO4 (240 mg, 1.13 mmol) and Ad 2 nBuPPdG 2 A solution of (37.8 mg, 0.0600 mmol) in 1,4-dioxane (4 mL) and water (1 mL) was stirred at 95 °C for 3 h. Water was added and extracted three times using EtOAc. The organic solvents were combined and concentrated under vacuum. The residue was purified by column chromatography (solvent gradient: 0-100% ACN in water (0.05% NH 4 HCO 3 )) to give 230 mg (39% yield) of the title compound as a red solid. LCMS (ESI) [M+H] + =1032.

[0288] Step 2: Lithium 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)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000193.tif43170 tert-Butyl (3R)-4-(2-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)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-methylpiperazine-1-carboxylate (210 mg, 0.200 mmol) and LiOH.H 2 A solution of O (25.6 mg, 0.610 mmol) in tetrahydrofuran (3 mL) and water (1 mL) was stirred at room temperature for 1 h. The solvent was concentrated in vacuo to give 254 mg (crude) of the title compound as a yellow solid.

[0289] Step 3: 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-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid A solution of lithium 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)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (254 mg, 0.250 mmol) in 5% TFA / HFIP (20 mL) was stirred at room temperature for 3 h. The solvent was concentrated in vacuo. The residue was purified using a pre-packed C18 column (solvent gradient: 0–100% MeOH in water (0.05% NH 4 HCO 3 )) to give 102 mg (44% yield) of the title compound as a red solid. LCMS (ESI, m / z) [M+H] + =905.

[0290] Step 4: 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 TIFF2025506357000195.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. Then, tBuOOH (0.4 mL, 5 M in decane) was 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 cooled to 30° C. with NaHSO 3 The mixture was quenched with aqueous solution 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 column chromatography on a pre-packed C18 column (solvent gradient: 0-70% ACN in water (0.05% NH 4 HCO 3 )) to give 340 mg (51%) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =807.

[0291] Step 5: (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 dihydrogen phosphate TIFF2025506357000196.tif44170 Under nitrogen, 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) 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.

[0292] Step 6: 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-oxo butan-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 TIFF2025506357000197.tif591701-(((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-(dimethyl 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 dihydrogen phosphate (121 mg, 0.190 mmol) and NaOAc (13.6 mg, 0.170 mmol) in methyl alcohol (1.2 mL) and dichloromethane (0.4 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (34.7 mg, 0.550 mmol) was added and stirred at room temperature for 0.5 h. Water was added to quench the reaction. The solvent was concentrated under vacuum. The product was purified by preparative HPLC (column: YMC-Actus Triart C18 ExRS, 30*150 mm, 5 μm; mobile phase A: water (10 mmol / L NH 4 HCO 3 ), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 36% B to 60% B in 9 min, 60% B; Wavelength: 254 / 220 nm; R T (min: 7.63) to give 30.0 mg (18% yield) of the title compound as a white solid. LCMS (ESI, m / z): [M+H] + =1509.

[0293] Example 9 Synthesis of part of hydrolyzable linker Intermediate 9: Ethyl (S)-1-((6-(((allyloxy)carbonyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000198.tif30170

[0294] Step 1: (S)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)-5-ureidopentanoic acid A solution of Fmoc-CIT-OH (4.00 g, 10.0 mmol) and DIPEA (5.20 g, 40.3 mmol) in DCM (40 mL) and DMF (40 mL) was added to 2-chlorotrityl chloride resin (10.0 g). The mixture was agitated for 1 h. 8 mL of MeOH was added and the resulting solution was agitated at room temperature for 0.5 h. The resin was washed three times with DMF. A solution of piperidine (15 mL) and DMF (60 mL) was added and agitated at room temperature for 0.5 h. It was then washed four times with DMF. Then, to a solution of 1-ethoxycarbonylcyclobutanecarboxylic acid (2.60 g, 15.1 mmol) and DIPEA (3.90 g, 30.2 mmol) in DMF (60 mL) was added HATU (5.70 g, 15.0 mmol) and HOBt (2.30 g, 15.1 mmol) at 0° C. and the reaction was stirred at room temperature for 0.5 h. The resulting solution was added to the above resin and stirred at room temperature for 1 h. The resin was washed three times using DMF followed by DCM. The resin was agitated in a solution of HFIP (15 mL) and DCM (60 mL) for 1 h. The resin was filtered and the filtrate was concentrated to give 3.7 g (crude) of the title compound as a yellow oil. LCMS (ESI, m / z) [M+H] + =329.

[0295] Step 2: Ethyl (S)-1-((1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000199.tif36170 A solution of (S)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)-5-ureidopentanoic acid (3.20 g, 9.73 mmol), (4-aminophenyl)methanol (1.32 g, 10.6 mmol), EEDQ (4.81 g, 19.4 mmol) in DCM (20 mL) and MeOH (10 mL) was stirred at 30° C. for 2 h. The solvent was evaporated and the crude was purified by flash chromatography on silica gel (solvent gradient: 0-20% MeOH / DCM) to give 1.75 g of the title compound as a brown oil. LCMS (ESI) [M+H] + =434.

[0296] Example 10 Synthesis of part of hydrolyzable linker Intermediate 10: 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000200.tif29170

[0297] Step 1: 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000201.tif441701-(((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-oxohexane (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 (61.0 mg, 0.110 mmol) and CH 3 A solution of COOH (13.6 mg, 0.230 mmol) in methyl alcohol (1.2 mL) and dichloromethane (0.4 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (21.3 mg, 0.340 mmol) was added and stirred at room temperature for 0.5 h. The reaction was quenched by adding water. The solvent was concentrated under vacuum. The residue was purified by column chromatography (solvent gradient: 0-100% MeOH in water (0.05% NH 4 HCO 3)) to give 80.0 mg (49% yield) of the title compound as a yellow solid. LCMS (ESI, m / z) [M+H] + =1429.

[0298] Example 11 Synthesis of part of hydrolyzable linker Intermediate 11: Ethyl (S)-1-((6-(dimethylamino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000202.tif64170

[0299] Step 1: tert-Butyl (S)-(6-(dimethylamino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamate TIFF2025506357000203.tif31170 Nitrogen, N 2 -(tert-butoxycarbonyl)-N 6 ,N 6 A solution of 2-dimethyl-L-lysine (5.00 g, 18.2 mmol), (4-aminophenyl)methanol (2.46 g, 20.1 mmol) and ethyl 2-ethoxyquinoline-1(2H)-carboxylate (9.01 g, 36.5 mmol) in MeOH (50 mL) and DCM (20 mL) was stirred at 30° C. overnight. The solvent was concentrated under vacuum. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-20% EtOAc / petroleum ether) to give 2.32 g (33% yield) of the title compound as a white solid. LCMS (ESI): [M+H] + =380.

[0300] Step 2: (S)-2-Amino-6-(dimethylamino)-N-(4-(hydroxymethyl)phenyl)hexanamide (2,2,2-trifluoroacetate) TIFF2025506357000204.tif28170CH of tert-butyl (S)-(6-(dimethylamino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamate (2.20 g, 5.78 mmol) 2 Cl 2 (15 mL) and TFA (5 mL) was stirred at room temperature for 1 h. The solvent was concentrated in vacuo and the crude was used directly in the next step. LC-MS: (ESI, m / z): [M+H] + =280.

[0301] Step 3: 1-(2,5-dioxopyrrolidin-1-yl)1-ethyl cyclobutane-1,1-dicarboxylate TIFF2025506357000205.tif14170 A solution of 1-(ethoxycarbonyl)cyclobutane-1-carboxylic acid (3.00 g, 17.4 mmol), 1-hydroxypyrrolidine-2,5-dione (2.21 g, 19.2 mmol) and DCC (3.95 g, 19.2 mmol) in THF (50 mL) was stirred at room temperature for 5 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give 5.19 g (crude) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =270.

[0302] Step 4: Ethyl (S)-1-((6-(dimethylamino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000206.tif29170(S)-2-Amino-6-(dimethylamino)-N-(4-(hydroxymethyl)phenyl)hexanamide (2.9 g, 10.4 mmol) and NaHCO 3 (2.27g, 83.2mmol) was 2A solution of 1-(2,5-dioxopyrrolidin-1-yl)1-ethyl cyclobutane-1,1-dicarboxylate (1.51 g, 15.5 mmol) in DME (30 mL) was then added and stirred at room temperature overnight. The reaction mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by column chromatography (solvent gradient: 0-100% MeOH in water (0.05% NH 4 HCO 3 )) to give 1.26 g (49% yield) of the title compound as a colorless oil. LC-MS: (ESI, m / z): [M+H] += 434. 1 H NMR (300MHz, DMSO-d 6 ,ppm)δ 9.77(s,1H),7.73(d,J=7.9Hz,1H),7.42(d,J=8.2Hz,2H),7.12(d,J=8.2Hz,2H),4.99(s,1H),4.36-4.22(m,3H),4.05-3.97 (m,2H),2.34-2.19(m,4H),2.07(t,J=6.9Hz,2H),1.99(s,6H),1.83-1.51(m,4H),1.37-1.16(m,4H),1.07(d,J=7.0Hz,3H).

[0303] Example 12 Synthesis of part of hydrolyzable linker Intermediate 12: tert-Butyl (S)-(5-(3-(tert-butoxy)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentyl)carbamate TIFF2025506357000207.tif56170

[0304] Step 1: Benzyl (S)-(2,2,16,16-tetramethyl-4,12-dioxo-3,15-dioxa-5,11-diazaheptadecan-13-yl)carbamate TIFF2025506357000208.tif30170A solution of N-((benzyloxy)carbonyl)-O-(tert-butyl)-L-serine (1.00 g, 3.39 mmol), tert-butyl(5-aminopentyl)carbamate (684 mg, 3.39 mmol), HATU (1.67 g, 4.40 mmol) and DIPEA (1.31 g, 10.1 mmol) in DMF (10 mL) was stirred at room temperature under nitrogen for 30 min. The reaction was purified by column chromatography (solvent gradient: 0-60% ACN in water (0.05% NH 4 HCO 3 )) to give 980 mg (60% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =480.

[0305] Step 2: tert-Butyl (S)-(5-(2-amino-3-(tert-butoxy)propanamido)pentyl)carbamate TIFF2025506357000209.tif27170 Under hydrogen (3 atm), a solution of benzyl (S)-(2,2,16,16-tetramethyl-4,12-dioxo-3,15-dioxa-5,11-diazaheptadecan-13-yl)carbamate (980 mg, 2.046 mmol) and 10% Pd / C (196 mg) in MeOH (200 mL) was stirred at room temperature for 1 h. The solid was filtered through a Celite pad and the filtrate was concentrated under reduced pressure to give 750 mg (crude) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =346.

[0306] Step 3: tert-Butyl (S)-(5-(3-(tert-butoxy)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentyl)carbamate TIFF2025506357000210.tif27170Under nitrogen, tert-butyl (S)-(5-(2-amino-3-(tert-butoxy)propanamido)pentyl)carbamate (740 mg, 2.14 mmol) and NaHCO3 (540 mg, 6.43 mmol) in dioxane (5 mL) and H 2 To a solution in O (16 mL) was added methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (332 mg, 2.14 mmol) at 0° C. The solution was stirred at 0° C. for 40 min. The reaction mixture was then stirred at room temperature for 0.5 h. Extraction was performed three times using ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (gradient: 0% to 60% EtOAc / petroleum ether) to give 540 mg (59% yield) of the title compound as a colorless oil. LC-MS: (ESI, m / z): [M+H] + =426.

[0307] Example 13 Synthesis of Hy-B-CIDE-6

[0302] N-((2S)-1-((4-((2-(6-amino-5-(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-methylpiperazine-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)-N-(5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000211.tif116170

[0308] Step 1: tert-Butyl (5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)pentyl)carbamate TIFF2025506357000212.tif18170 A solution of tert-butyl (5-aminopentyl)carbamate (500 mg, 2.47 mmol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (1.25 g, 4.94 mmol) and NMM (476 mg, 4.45 mmol) in DMF (3 mL) was stirred at room temperature for 1 h. The residue was purified by pre-packed C18 column (solvent gradient: 0-100% ACN in water (0.05% TFA)) and then purified again by flash chromatography on silica gel (gradient: 0%-10% MeOH / DCM) to give 343 mg (41% yield) of the title compound as a red oil. LCMS (ESI) [M+H] + =340.

[0309] Step 2: N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide (2,2,2-trifluoroacetate) TIFF2025506357000213.tif28170 A solution of tert-butyl (5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)pentyl)carbamate (30.0 mg, 0.0900 mmol) in dichloromethane (2 mL) and 2,2,2-trifluoroacetic acid (0.5 mL) was stirred at room temperature for 1 h. The solvent was concentrated in vacuo to give 32.0 mg (crude) of the title compound as a yellow oil. LCMS (ESI) [M+H] + =240.

[0310] Step 3: N-((2S)-1-((4-((2-(6-amino-5-(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)ethyl (2,2,2-trifluoroacetate)-N-(5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000214.tif411701-(((2S)-1-((4-((2-(6-amino-5-(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]octane-3- To a solution of N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide (2,2,2-trifluoroacetate) (30.0 mg, crude) and DIPEA (54.2 mg, 0.420 mmol) in DMF (0.5 mL) was added HATU (19.2 mg, 0.0500 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The crude was purified by preparative HPLC (Column: Xselect CSH F-Phenyl OBD column, 19*250mm, 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 25mL / min; Gradient: 20% B to 27% B, 27% B in 11 min; Wavelength: 254nm; RT1(min): 10.4) to give 31.5mg (45% yield) of the title compound as a yellow solid. LCMS(ESI)[M+H] + =1650. 1H NMR(300MHz,DMSO-d6)δ 10.20(s,1H),9.51(s,1H),8.99(s,1H),8.38(d,J=7.6Hz,1H),8.11(t,J=5.4Hz,1H),7.95-7.80(m,3H),7.67(d,J=8.3Hz,2H),7.58( td,J=5.1,2.4Hz,2H),7.51-7.25(m,8H),7.23-7.03(m,5H),6.69(d,J=6.5Hz,1H),6.32(s,1H),6.11(s,1H),5.10(s,2H),4.91(t,J= 7.3Hz,1H),4.55(s,2H),4.47(s,2H),4.43-4.20(m,5H),3.99(s,2H),3.50-3.30(m,7H),3.30-3.15(m,4H),3.15-3.05(m,6H),3.05- 2.95(m,5H),2.75(d,J=4.4Hz,6H),2.50-2.35(m,8H),2.23-1.60(m,13H),1.53-1.04(m,15H),1.05-0.90(m,3H),0.85-0.70(m,3H).

[0311] Example 14 Synthesis of Hy-B-CIDE-1 N-((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)-N-(5-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamido)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000215.tif121170

[0312] Step 1: Benzyl (S)-(2,2,16,16-tetramethyl-4,12-dioxo-3,15-dioxa-5,11-diazaheptadecan-13-yl)carbamate TIFF2025506357000216.tif22170 Under nitrogen, a solution of N-((benzyloxy)carbonyl)-O-(tert-butyl)-L-serine (1.0 g, 3.390 mmol), tert-butyl(5-aminopentyl)carbamate (684 mg, 3.39 mmol), HATU (1.67 g, 4.40 mmol) and DIPEA (1.31 g, 10.1 mmol) in DMF (10 mL) was stirred at room temperature for 30 min. The reaction was purified by column chromatography (solvent gradient: 0-60% ACN in water (0.05% NH 4 HCO 3 )) to give 980 mg (60% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =480.

[0313] Step 2: tert-Butyl (S)-(5-(2-amino-3-(tert-butoxy)propanamido)pentyl)carbamate TIFF2025506357000217.tif21170 Under hydrogen (1 atm), a solution of benzyl (S)-(2,2,16,16-tetramethyl-4,12-dioxo-3,15-dioxa-5,11-diazaheptadecan-13-yl)carbamate (980 mg, 2.046 mmol) and Pd / C (196 mg) in MeOH (200 mL) was stirred at room temperature for 1 h. The solid was filtered through a Celite pad and the filtrate was concentrated under reduced pressure to give 750 mg (crude) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =346.

[0314] Step 3: tert-Butyl (S)-(5-(3-(tert-butoxy)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentyl)carbamate TIFF2025506357000218.tif24170Under nitrogen, tert-butyl (S)-(5-(2-amino-3-(tert-butoxy)propanamido)pentyl)carbamate (740 mg, 2.145 mmol) and NaHCO 3 (540 mg, 6.43 mmol) in dioxane (5 mL) and H 2 To a solution in O (16 mL) was added methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (332 mg, 2.14 mmol) at 0° C. The solution was stirred at 0° C. for 40 min. The reaction mixture was then stirred at room temperature for 0.5 h. The reaction was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (gradient: 0% to 60% EtOAc / petroleum ether) to give 540 mg (59% yield) of the title compound as a colorless oil. LC-MS: (ESI, m / z): [M+H] + =426

[0315] Step 4: (S)-N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamide (2,2,2-trifluoroacetate) TIFF2025506357000219.tif23170 A solution of tert-butyl (S)-(5-(3-(tert-butoxy)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentyl)carbamate (15.0 mg, 0.035 mmol) in TFA (0.5 mL) was stirred at room temperature under nitrogen for 1 h. The resulting mixture was concentrated in vacuo to give 20.0 mg (crude) of the title compound as a colorless oil. LC-MS: (ESI, m / z): [M+H] + =270.

[0316] Step 5: N-((2S)-1-((4-((2-(6-amino-5-(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 oxy)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)-N-(5-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamido)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000220.tif31170Under nitrogen, 1-(((2S)-1-((4-((2-(6-amino-5-(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) To a solution of pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (30.0 mg, 0.021 mmol), (S)-N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamide (2,2,2-trifluoroacetate) (20.0 mg, crude), DIPEA (27.1 mg, 0.210 mmol) in DMF (0.9 mL) was added HATU (10.4 mg, 0.027 mmol) at room temperature. The resulting solution was stirred at room temperature for 10 min. The resulting solution was purified by preparative HPLC (XBridge Shield RP18 OBD column, 19 × 250 mm 10 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20% B to 35% B in 10 min; 254 / 220 nm; R T1 : 8.26) to give 22.6 mg (64% yield) of HY-B-CIDE-1 as a white solid. LC-MS: (ESI, m / z): [M+H] + =1680. 1 H NMR (300MHz, DMSO-d 6)δ 10.21(s,1H),9.45(br,1H),9.00(d,J=1.7Hz,1H),8.40(d,J=7.8Hz,1H), 7.98-7.84(m,4H),7.67(d,J=8.3Hz,2H),7.58(d,J=7.4Hz,2H),7.52-7.28 (m,8H),7.19-6.91(m,5H),6.69(s,1H),6.31(s,1H),6.13(s,1H),5.11(s, 2H),4.95-4.80(m,1H),4.62-4.52(m,2H),4.52-4.45(m,3H),4.42-4.32(m ,4H),4.32-4.25(m,2H),3.95-3.86(m,9H),3.50-3.40(m,3H),3.40-3.28 (m,4H),3.15-3.05(m,5H),3.05-2.95(m,5H),2.75(d,J=4.7Hz,6H),2.45- 2.35(m,8H),2.25-2.10(m,1H),2.05-1.86(m,5H),1.84-1.52(m,7H),1.50 -1.30(m,8H),1.30-1.10(m,6H),0.97(d,J=6.4Hz,3H),0.88-0.76(m,3H).

[0317] Example 15 Synthesis of Hy-B-CIDE-2 N-((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)-N-(5-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methoxypropanamido)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000221.tif128170

[0318] Step 1: (S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methoxypropanoic acid TIFF2025506357000222.tif25170Methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (500 mg, 3.22 mmol) and NaHCO 3 To a solution of (810 mg, 9.64 mmol) in water (10 mL) was added methyl O-methyl-L-serine (384 mg, 3.22 mmol) at room temperature. The reaction was stirred at room temperature for 2 h. KHSO 4 The pH was adjusted to 5 using hexanes. The product was extracted with dichloromethane four times. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solvent was concentrated under vacuum to give 600 mg (93% yield) of the title compound as a white oil. LC-MS: (ESI, m / z): M - =198.

[0319] Step 2: tert-Butyl (S)-(5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methoxypropanamido)pentyl)carbamate TIFF2025506357000223.tif26170 A solution of tert-butyl (5-aminopentyl)carbamate (0.62 mL, 2.97 mmol), (S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methoxypropanoic acid (600 mg, 3.01 mmol), HATU (1.50 g, 3.94 mmol) and DIPEA (1.20 g, 9.3 mmol) in DMF (10 mL) was stirred at room temperature for 1 h. The reaction was diluted with EtOAc and washed with water. The organic layers were combined, dried under vacuum and concentrated under vacuum. The crude was purified by flash chromatography on silica gel (gradient: 0% to 100% ethyl acetate / petroleum ether) to give 150 mg (13% yield) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H]+ =384.

[0320] Step 3: (S)-N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methoxypropanamide (2,2,2-trifluoroacetate) TIFF2025506357000224.tif30170 A solution of tert-butyl (S)-(5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methoxypropanamido)pentyl)carbamate (25.0 mg, 0.0700 mmol) in dichloromethane (3 mL) and 2,2,2-trifluoroacetic acid (1 mL) was stirred at room temperature for 1 h. The solvent was concentrated in vacuo to give 18 mg (crude) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =284.

[0321] Step 4: N-((2S)-1-((4-((2-(6-amino-5-(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 oxy)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)-N-(5-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methoxypropanamido)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000225.tif411701-(((2S)-1-((4-((2-(6-amino-5-(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)pyridine To a solution of (S)-N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methoxypropanamide (2,2,2-trifluoroacetate) (18.0 mg, crude) and DIPEA (54.2 mg, 0.420 mmol) in DMF (2 mL) was added HATU (19.2 mg, 0.0500 mmol) at room temperature. The reaction was stirred at room temperature for 10 minutes. The residue was purified by preparative HPLC (XBridge Shield RP18 OBD column, 30×150 mm 5 um; mobile phase A: water (10 mmol / L NH 4 HCO 3 ), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 6% B to 21% B in 12 min; 254 nm; R T1 : 14.45) to give 34 mg (44% yield) of Hy-B-CIDE-2 as a white solid. LC-MS: (ESI, m / z): [M+H] + =1694. 11H NMR (300 MHz, DMSO-d6, ppm) δ 10.22 (s, 1H), 9.52 (s, 1H), 8.99 (d, J = 1.5 Hz, 1H), 8.40 (d, J = 7.7 Hz, 1H), 8.03 (t, J = 5.7 Hz, 1H), 7.89 (d, J = 6.6 Hz, 3H), 7.68 (d, J = 8.3 Hz, 2H), 7.57 (t, J = 7.4 Hz, 2H), 7.50 - 7.28 (m, 8H), 7.18 - 7.10 (m, 2H), 7.10 - 6.90 (m, 3H), 6.66 (s, 1H), 6.28 (s, 1H), 6.12 (s, 1H), 5.10 (s, 3H), 4.91 (t, J = 7.2 Hz, 1H), 4.71 - 4.66 (m, 1H), 4.60 - 4.45 (m, 4H), 4.40 - 4.27 (m, 5H), 3.89 - 3.79 (m, 5H), 3.70 - 3.65 (m, 4H), 3.50 - 3.40 (m, 3H), 3.17 (s, 4H), 3.13 - 3.08 (m, 4H), 3.06 - 2.96 (m, 6H), 2.75 (d, J = 4.5 Hz, 8H), 2.47 - 2.43 (m, 5H), 2.42 - 2.35 (m, 4H), 2.25 - 2.15 (m, 1H), 2.10 - 1.55 (m, 10H), 1.52 - 1.27 (m, 9H), 1.27 - 1.10 (m, 6H), 0.97 (dd, J = 6.5, 3.7 Hz, 3H), 0.88 - 0.75 (m, 3H).

[0322] Example 16 Synthesis of Hy - B - CIDE - 5 N-((2S)-1-((4-((2-(6-amino-5-(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)ethyl (2,2,2-trifluoroacetate) TIFF2025506357000226.tif81170

[0323] Step 1: N-((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)-N-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)cyclobutane-1,1-dicarboxamide(2,2,2-trifluoroacetate) TIFF2025506357000227.tif421701-(((2S)-1-((4-((2-(6-amino-5-(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]o To a solution of (2-(2-(2-aminoethyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (12.4 mg, crude) and DIPEA (54.2 mg, 0.420 mmol) in DMF (1 mL) was added HATU (19.2 mg, 0.0505 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The reaction solution was purified by preparative 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: 5% B to 25% B, 25% B in 12 min; wavelength: 254 / 220nm; R T1 (min: 12 min) to give 34.3 mg (51% yield) of Hy-B-CIDE-5 as a white solid. LC-MS: (ESI, m / z): [M+H] + =1608. 1 H NMR (300MHz, DMSO-d 6)δ 10.21(s,1H),9.59(br,1H),8.99(d,J=1.6Hz,1H),8.38(d,J=7.6Hz,1H),8.11(t,J=5.9Hz,1H),7.86(dd,J=18.6,7.0Hz,2H),7 .68-7.51(m,4H),7.49-7.26(m,9H),7.14(t,J=7.5Hz,1H),7.07(s,3H),6.67(d,J=6.5Hz,1H),6.29(s,1H),6.11(s,1H),5.10( s,2H),4.91(t,J=7.3Hz,1H),4.60-4.40(m,5H),4.38-4.31(m,3H),4.29(s,1H),3.50-3.05(m,25H),2.85(s,3H),2.75(s,6H), 2.46(s,9H),2.05-1.87(m,5H),1.86-1.55(m,8H),1.50-1.30(m,5H),1.25-1.15(m,4H),0.97(d,J=6.6,3H),0.89-0.70(m,3H).

[0324] Example 17 Synthesis of Hy-B-CIDE-8 N-((2S)-1-((4-((2-(6-amino-5-(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)ethyl (2,2,2-trifluoroacetate) TIFF2025506357000228.tif91170 Step 1: tert-Butyl (5-((2-(((benzyloxy)carbonyl)amino)ethyl)(methyl)amino)pentyl)carbamate TIFF2025506357000229.tif10170 Under nitrogen, a solution of tert-butyl (5-(methylamino)pentyl)carbamate (1.00 g, 4.62 mmol), benzyl N-(2-bromoethyl)carbamate (1.80 g, 6.97 mmol) and DIPEA (1.80 g, 13.9 mmol) in acetonitrile (10 mL) was stirred at 50° C. for 1 h. The solvent was concentrated in vacuo and water was added. The resulting solution was extracted with DCM and the organic layers were combined. The organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-5% MeOH / DCM) to give 1.30 g (71% yield) of the title compound as a colorless oil. LCMS (ESI, m / z): [M+H] + =394.

[0325] Step 2: tert-Butyl (5-((2-aminoethyl)(methyl)amino)pentyl)carbamate TIFF2025506357000230.tif10170 A solution of methylamine (2M in THF) (7.5 mL, 15.2 mmol) and 10% Pd / C (240 mg) in MeOH (50 mL) was stirred at room temperature for 30 min under hydrogen (3 atm) (to remove traces of formaldehyde in MeOH). Then, tert-butyl (5-((2-(((benzyloxy)carbonyl)amino)ethyl)(methyl)amino)pentyl)carbamate (1.20 g, 3.05 mmol) was added and the reaction was stirred at room temperature for 2 h. After filtration, the filtrate was concentrated under reduced pressure to give 810 mg (crude) of the title compound as a yellow oil. LCMS (ESI, m / z): [M+H] + =260.

[0326] Step 3: tert-Butyl (5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)pentyl)carbamate TIFF2025506357000231.tif20170Under nitrogen, tert-butyl (5-((2-aminoethyl)(methyl)amino)pentyl)carbamate (800 mg, 3.08 mmol) and maleic anhydride (300 mg, 3.05 mmol) were dissolved in CH 3 The solution in COOH (10 mL) was stirred at room temperature for 1 h. The solution was concentrated under vacuum and the residue was triturated three times with DCM / hexanes (1:4) and toluene (10 mL) was added, followed by triethylamine (1.56 g, 15.4 mmol) and 4A molecular sieves (2.86 g). The reaction was stirred at 110° C. for 1 h. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (solvent gradient: 0-100% EtOAc / petroleum ether) to give 825 mg of the title compound as a yellow oil. LCMS (ESI, m / z): [M+H] + =340.

[0327] Step 4: 1-(2-((5-aminopentyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetic acid) TIFF2025506357000232.tif28170 Under nitrogen, a solution of tert-butyl (5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)pentyl)carbamate (13.0 mg, 0.0400 mmol) in DCM (1 mL) and 2,2,2-trifluoroacetic acid (0.3 mL) was stirred at room temperature for 30 min. The solvent was evaporated to give 30 mg (crude) of the title compound as a yellow oil. LCMS (ESI, m / z): [M+H] + =240.

[0328] Step 5: N-((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)-N-(5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)pentyl)cyclobutane-1,1-dicarboxamide(2,2,2-trifluoroacetate) TIFF2025506357000233.tif33170Under nitrogen, 1-(2-((5-aminopentyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (8.0 mg, crude), 1-(((2S)-1-((4-((2-(6-amino-5-(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)isoxazo To a solution of (40.0 mg, 0.0300 mmol, intermediate 10) and DIPEA (38.7 mg, 0.300 mmol) in N,N-dimethylformamide (1 mL) was added HATU (14.0 mg, 0.0400 mmol) at room temperature. The reaction was stirred at room temperature for 10 minutes. The crude was purified by preparative HPLC under the following conditions: Column: XselectCSH C18 OBD column 30*150mm 5μm,n; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 19% B to 34% B, 34% B in 11 min; Wavelength: 254 / 220nm; RT1(min):10 to give 7.0mg of Hy-B-CIDE-8 as a white solid.

[0329] LCMS(ESI,m / z):[M+H] + =1649. 1 H NMR (300MHz, DMSO-d 6,ppm)δ 10.13(s,1H),9.57(s,1H),8.99(s,1H),8.41(d,J=7.7Hz,1H),8.00-7.8 0(m,3H),7.55(d,J=8.3Hz,2H),7.50-7.39(m,2H),7.39-7.11(m,8H),7. 08-6.97(m,2H),6.96(s,2H),6.56(d,J=6.5Hz,1H),6.18(s,1H),6.00(s ,1H),4.98(s,2H),4.79(t,J=7.2Hz,1H),4.50-4.12(m,10H),3.60-3.51 (m,5H),3.37(q,J=11.1,10.2Hz,10H),3.06-2.83(m,11H),2.72-2.57(m ,9H),2.45-2.35(m,7H),2.31-2.10(m,2H),1.99-1.85(m,3H),1.79(s,2 H),1.72-1.39(m,9H),1.34(d,J=7.0Hz,3H),1.25(d,J=7.0Hz,3H),1.20-0.90(m,7H),0.85(dd,J=6.6,3.7Hz,3H),0.69(dd,J=10.5,6.6Hz,3H).

[0330] Example 18 Synthesis of Hy-B-PCIDE-2 (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 dihydrogen phosphate (2,2,2-trifluoroacetate) TIFF2025506357000234.tif97170

[0331] 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 TIFF2025506357000235.tif41170 Under nitrogen, to 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) 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 cooled to 0 °C with NaHSO 3 The mixture was quenched with 50 ml of ethyl acetate. 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 mg (62% yield) of the title compound as a colorless oil. LC-MS: (ESI, m / z): [M+H] + =735.

[0332] 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 dihydrogen phosphate TIFF2025506357000236.tif48170 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.

[0333] 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 TIFF2025506357000237.tif51170Under nitrogen, (3R,5S)-5-(((S)-1-(4-cyanophenyl)ethyl)carbamoyl)-1-((R)-3-methyl-2-(3-(2-oxoethoxy)isoxazol-5-yl)butanoyl)pyrrolidin-3-yl Dihydrogen phosphate (62.8 mg, 0.115 mmol), 1-(((2S)-1-((4-((2-(6-amino-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), CH 3 A solution of COONa (14.9 mg, 0.182 mmol) in methanol (1.5 mL) and dichloromethane (0.5 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (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 column chromatography on a pre-packed C18 column (solvent gradient: 0-100% ACN in water (0.05% NH 4 HCO 3 )) to give 60 mg (65% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =1464.

[0334] 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-carboxamide amido)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 dihydrogen phosphate (2,2,2-trifluoroacetate) TIFF2025506357000238.tif42170

[0335] 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 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) in DMF (1 mL) was added HATU (18.7 mg, 0.0492 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The crude product was purified by preparative 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; wavelength: 254 / 220nm; R T1 (min: 11.75 min) to afford 20.3 mg (30% yield) of Hy-B-PCIDE-2 as a white solid.

[0336]

[0330] LC-MS:(ESI,m / z):[M+H] + =1643. 1 H NMR (300MHz, DMSO-d 6)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).

[0337] Example 19 Synthesis of Hy-B-CIDE-7 N-((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)-N-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000239.tif74170

[0338] Step 1: Ethyl (S)-1-((6-(dimethylamino)-1-oxo-1-((4-((2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methyl)phenyl)amino)hexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000240.tif28170Under nitrogen, ethyl (S)-1-((1-((4-((2-bromophenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (500 mg, 0.852 mmol), B 2 Pin 2 (649mg, 2.55mmol), Pd(dppf)Cl 2 A solution of (124 mg, 0.170 mmol) and KOAc (250 mg, 2.55 mmol) in 1,4-dioxane (5 mL) was stirred at 80 °C for 2 h. The reaction was diluted with DCM and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (gradient: 0% to 20% MeOH / DCM (0.3% 7M NH 3 / MeOH) to give 390 mg (72% yield) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =636.

[0339] Step 2: tert-Butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000241.tif27170Under nitrogen, ethyl (S)-1-((6-(dimethylamino)-1-oxo-1-((4-((2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methyl)phenyl)amino)hexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (390 mg, 0.614 mmol), tert-butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (395 mg, 0.676 mmol), Ad 2 nBuPPdG2 (41.0 mg, 0.061 mmol) and K 2 CO 3 (260 mg, 1.22 mmol) in dioxane (5.0 mL) and H 2 The solution in 2H2O (1.2 mL) was stirred at 95 °C for 2 h. The resulting solution was diluted with water and extracted with EtOAc. The organic layer was concentrated under vacuum. The residue was purified by column chromatography (solvent gradient: 0-100% MeOH in water (0.1% NH 4 HCO 3 )) to give 310 mg (47% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =1060.

[0340] Step 3: Lithium 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate TIFF2025506357000242.tif27170tert-Butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-(2-((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (270 mg, 0.255 mmol) and LiOH.H 2 HO (32.1 mg, 0.765 mmol) in THF (2 mL) and H 2 The solution in O (2 mL) was stirred at room temperature for 1 h. The resulting mixture was concentrated in vacuo. The crude was used directly in the next step. LC-MS: (ESI, m / z): [M+H] + =1032.

[0341] Step 4: 1-(((2S)-1-((4-((2-(6-amino-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 A solution of lithium 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (crude from the last step) in TFA (0.75 mL) and HFIP (14 mL) was stirred at room temperature for 30 min. The resulting mixture was concentrated under vacuum. The resulting crude product was purified by HPLC using a pre-packed C18 column (solvent gradient: 0–100% MeOH in water (0.1% NH 4 HCO 3 )) to give 170 mg (71% yield) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =932.

[0342] Step 5: 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000244.tif31170Under nitrogen, 1-(((2S)-1-((4-((2-(6-amino-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 A solution of 1-carboxylate (170.0 mg, 0.183 mmol), (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 (111 mg, 0.237 mmol), HOAc (21.9 mg, 0.365 mmol) in DCM (1.5 mL) and MeOH (0.5 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (17.3 mg, 0.274 mmol) was added at 0° C. and stirred at room temperature for 30 min. The reaction was quenched with water. The resulting solution was concentrated under vacuum. The resulting crude product was purified by column chromatography on a pre-packed C18 column (gradient: 0-100% MeOH in water (0.05% NH 4 HCO 3 )) to give 180 mg (71% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =1384.

[0343] Step 5: N-((2S)-1-((4-((2-(6-amino-5-(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)cyclobutene oxy)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)-N-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000245.tif311701-(((2S)-1-((4-((2-(6-amino-5-(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]octane To a solution of 1-(2-((2-aminoethyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (12.8 mg, 0.065 mmol), DIPEA (56.0 mg, 0.434 mmol) in DMF (1 mL) was added HATU (19.8 mg, 0.052 mmol) at room temperature. The reaction was stirred at room temperature for 30 min. The resulting solution was purified by preparative HPLC (Xselect CSH C18 OBD column, 30 × 150 mm 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 8% B to 32% B in 8 min; 254 / 220 nm; R T1 : 8 min) to give 29.5 mg (43% yield) of Hy-B-CIDE-7 as a white solid. LC-MS: (ESI, m / z): [M+H] + =1563. 1 H NMR (300MHz, DMSO-d 6)δ 10.22(s,1H),9.69(br,2H),8.46(d,J=7.3Hz,1H),8.12(s,1H),7.90-7.70(m,4H),7.70-7.50(m,4H),7.51-7.27(m,6H) ),7.19-6.96(m,5H),6.65(s,1H),6.17-5.98(m,2H),5.22(s,1H),5.10(s,2H),4.90(d,J=7.2Hz,1H),4.51(s,4H),4.45 -4.19(m,5H),3.76-3.65(m,7H),3.43(s,5H),3.30-3.20(m,5H),3.14-2.97(m,6H),2.84(s,3H),2.75(s,7H),2.42-2. 30(m,7H),2.27-2.24(m,1H),2.07-1.52(m,17H),1.50-1.20(m,5H),0.96(t,J=6.3Hz,3H),0.82(dd,J=9.1,6.6Hz,3H).

[0344] Example 20 Synthesis of Hy-B-CIDE-9 6-(8-chloro-4-(2-(dimethylamino)ethyl)-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) TIFF2025506357000246.tif101170

[0345] Step 1: tert-Butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-(2-(methoxymethoxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000247.tif38170Under nitrogen, tert-butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (1.50 g, 2.56 mmol), 2-(methoxymethoxy)phenylboronic acid (559 mg, 3.07 mmol), Pd(PPh 3 ) 4 (591 mg, 0.512 mmol) and K 2 CO 3 (1.06 g, 7.68 mmol) in dioxane (12.5 mL) and H 2 The solution in 2.5 mL of O was stirred at 100° C. for 1 h. The reaction mixture was diluted with water and extracted with DCM. The combined organic layers were washed with anhydrous Na 2 SO 4 The crude product was purified by flash chromatography on a silica gel column (solvent gradient: 0-10% MeOH / DCM) to give 1.33 g (75% yield) of the title compound as a yellow solid. LCMS (ESI, m / z): [M+H] + =688.

[0346] Step 2: 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-(methoxymethoxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000248.tif56170Under nitrogen, tert-butyl To a solution of 4-((1r,3r)-3-((4-(3-(3-amino-6-(2-(methoxymethoxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (400 mg, 0.582 mmol), ethyl (S)-1-((6-(dimethylamino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (882 mg, 2.04 mmol) and DIPEA (1.19 g, 9.20 mmol) in THF (10 mL) was added triphosgene (313 mg, 1.05 mmol) at 0° C. The reaction was stirred at room temperature for 0.5 h. Evaporate the solvent and purify the residue on a pre-packed C18 column (solvent gradient: 0–100% MeOH in water (0.05% NH 4 HCO 3 )) to give 160 mg (23%) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =1148.

[0347] Step 3: 1-(((2S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-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)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000249.tif54170 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-(methoxymethoxy)phenyl)pyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (60.0 mg, 0.0520 mmol) and LiOH.H 2 A solution of 2H2O (6.58 mg, 0.156 mmol) in THF (0.5 mL) and H 2 The solution in 0.25 mL was stirred at room temperature for 1 h. The resulting mixture was concentrated in vacuo. It was then washed with concentrated HCl (0.33 mL) and H 2 The reaction was stirred at room temperature for 20 min. The resulting mixture was dissolved in a solution of saturated NaHCO 3 The solution was basified to pH = 8 at 0 °C and purified using a pre-packed C18 column (solvent gradient: 0–100% MeOH in water (0.05% NH 4 HCO 3 )) to give 35 mg of the title compound as a yellow solid. LCMS (ESI, m / z): [M+H] + =976.

[0348] Step 4: 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 ethyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000250.tif51170Under nitrogen, 1-(((2S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-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)-1-oxohexan-2-yl)carbamoyl)cyclo A solution of 2-cyclobutane-1-carboxylic acid (30.0 mg, 0.0310 mmol), (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 (28.8 mg, 0.0620 mmol) and HOAc (3.69 mg, 0.0620 mmol) in DCM (0.75 mL) and MeOH (0.25 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (2.90 mg, 0.0460 mmol) was added and stirred at room temperature for 0.5 h. The reaction was quenched with water at 0 °C. The resulting mixture was concentrated under vacuum and purified by column chromatography (solvent gradient: 0-100% MeOH in water (0.05% NH 4 HCO 3 )) to give 27 mg of the title compound as a white solid. LCMS (ESI, m / z): [M+H] += 1428.

[0349] Step 5: 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-((1R,3r)-3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyano) nophenyl)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-hydroxyphenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025506357000251.tif461701-(((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-hydro To a solution of ((5-aminopentyl)(methyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (22.0 mg, 0.0150 mmol), 1-(2-((5-aminopentyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (5.5 mg, crude) and DIPEA (19.9 mg, 0.154 mmol) in DMF (1 mL) was added HATU (7.62 mg, 0.0200 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 Shield RP18 OBD column, 30*150mm, 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 2% B to 32% B, 32% B in 10 min; Wavelength: 220 / 254nm; R T (min): 11.93 and purified by preparative HPLC to give 13.8 mg of Hy-B-CIDE-9 as a white solid.

[0350]

[0344] LCMS(ESI,m / z):[M+H] += 1649. 1 H NMR (300MHz, DMSO-d 6, ppm) δ 10.17 (s, 1H), 10.03 (s, 1H), 9.70 (br, 1H), 9.47 (br, 2H), 8.47 (d, J = 7.3 Hz, 1H), 7.99 - 7.83 (m, 4H), 7.78 (m, J = 9.1, 7.3 Hz, 2H), 7.64 (d, J = 8.2 Hz, 3H), 7.45 (m, J = 8.5, 2.4 Hz, 2H), 7.37 - 7.25 (m, 3H), 7.08 (d, J = 3.9 Hz, 2H), 6.98 - 6.90 (m, 2H), 6.72 (s, 1H), 6.22 (s, 1H), 6.14 (s, 1H), 5.20 (s, 1H), 5.11 (s, 2H), 4.89 (t, J = 7.0 Hz, 1H), 4.69 (s, 2H), 4.50 (s, 2H), 4.44 - 4.38 (m, 1H), 4.31 (t, J = 11.3 Hz, 3H), 3.75 - 3.69 (m, 7H), 3.42 - 3.39 (m, 2H), 3.38 - 3.33 (m, 2H), 3.33 - 3.27 (m, 3H), 3.27 - 3.12 (m, 7H), 3.12 - 3.00 (m, 4H), 3.00 (s, 3H), 2.74 (s, 6H), 2.46 - 2.38 (m, 7H), 2.37 - 2.09 (m, 1H), 2.05 - 2.00 (m, 2H), 1.95 - 1.85 (m, 5H), 1.85 - 1.75 (m, 5H), 1.75 - 1.59 (m, 5H), 1.43 (t, J = 6.6 Hz, 3H), 1.33 (d, J = 7.0 Hz, 4H), 1.30 - 1.20 (m, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.78 (d, J = 6.8 Hz, 3H).

[0351] Example 21 Synthesis of Hy - B - CIDE - 10

[0345] 4-((S)-6-(dimethylamino)-2-(1-((5-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamido)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-hydroxyphenyl)pyridazin-3-yl)carbamate TIFF2025506357000252.tif80170

[0352] Step 1: (S)-N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamide (2,2,2-trifluoroacetic acid) TIFF2025506357000253.tif28170 A solution of tert-butyl (S)-(5-(3-(tert-butoxy)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentyl)carbamate (8.8 mg, 0.0094 mmol) in TFA (0.3 mL) was stirred at room temperature under nitrogen for 1.5 h. The solvent was concentrated in vacuo to give 10 mg (crude) of the title compound as a colorless oil. LCMS (ESI, m / z): [M+H] + =270.

[0353] Step 2: 4-((S)-6-(dimethylamino)-2-(1-((5-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamido)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-hydroxyphenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025506357000254.tif441701-(((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-hydroxyphenyl)pyridin To a solution of (S)-N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamide (2,2,2-trifluoroacetic acid) (8.8 mg, crude) and DIPEA (24.4 mg, 0.189 mmol) in DMF (0.9 mL) was added HATU (9.3 mg, 0.025 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The crude material was purified under 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: 15% B to 27% B, 27% B in 12 min; Wavelength: 254 / 220nm; R T Purification by preparative HPLC at (min):12 gave 9.9 mg of Hy-B-CIDE-10 as a white solid.

[0354] LCMS (ESI, m / z): [M+H] += 1679. 1 H NMR (300MHz, DMSO-d 6,ppm)δ 10.16(s,1H),10.04(s,1H),9.60(br,1H),9.35(br,1H),8.47(d,J=7.4Hz, 1H),8.06-7.84(m,4H),7.79(t,J=8.3Hz,2H),7.69-7.58(m,3H),7.45(d,J =8.4,2H),7.40-7.34(m,3H),7.04(s,2H),7.00-6.88(m,2H),6.72(s,1H), 6.21(s,1H),6.14(s,1H),5.19(s,1H),5.11(s,2H),4.89(t,J=7.1Hz,1H), 4.68(d,J=7.1Hz,2H),4.55-4.23(m,7H),3.97-3.82(m,2H),3.67(d,J=9.5 Hz,3H),3.62-3.52(m,8H),3.17-2.95(m,10H),2.73(d,J=4.1Hz,6H),2.45 -2.30(m,8H),2.19-1.97(m,4H),1.95-1.85(m,5H),1.82-1.53(m,8H),1.4 5-1.30(m,8H),1.25-1.15(m,4H),0.95(t,J=6.4Hz,3H),0.90-0.70(m,3H).

[0355] Example 22 Synthesis of Hy-B-CIDE-11 N-((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)-N-(5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000255.tif74170

[0356] Step 1: 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000256.tif26170Under nitrogen, tert-butyl 4-((1r,3r)-3-((4-(3-(3-amino-6-chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (550 mg, 0.900 mmol), ethyl (S)-1-((6-(dimethylamino)-1-oxo-1-((4-((2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methyl)phenyl)amino)hexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (600 mg, 0.900 mmol), Pd(dppf)Cl 2 (138 mg, 0.200 mmol) and Na 2 CO 3 A solution of (300 mg, 2.800 mmol) in 1,4-dioxane (10 mL) and water (2 mL) was stirred at 110° C. for 1 h. The reaction was cooled to room temperature and added LiOH.H 2 HO (160 mg, 3.800 mmol) was added. The reaction was stirred at room temperature for 1 h. The resulting mixture was then loaded onto a pre-packed C18 column (solvent gradient: 0-70% ACN in water (0.5% NH 4 HCO 3 )) to give 383 mg (39% yield) of the title compound as a brown solid. LCMS (ESI, m / z): [M+H] += 1032.

[0357] Step 2: 1-(((2S)-1-((4-((2-(6-amino-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 (2,2,2-trifluoroacetate) A solution of 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (383.0 mg, 0.3700 mmol) in DCM (1 mL) and 2,2,2-trifluoroacetic acid (1 mL) was stirred at room temperature for 30 min. The solvent was evaporated under vacuum and the residue was purified by pre-packed C18 column (solvent gradient: 0-100% ACN in water (0.05% TFA)) to give 290 mg of the title compound as a white solid. LCMS (ESI, m / z): [M+H] + =931.

[0358] Step 3: 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000258.tif221701-(((2S)-1-((4-((2-(6-amino-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-carvone A solution of the acid (2,2,2-trifluoroacetate salt) (260 mg, 0.248 mmol), (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 (195 mg, 0.420 mmol) and NaOAc (228 mg, 2.79 mmol) in MeOH (1 mL) and DCM (3 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (52.0 mg, 0.830 mmol) was added and stirred at room temperature for 1 h. The crude was purified by column chromatography (solvent gradient: 0-100% MeOH in water (0.05% NH 4 HCO 3 )) to give 210 mg of the title compound as a yellow solid. LCMS (ESI, m / z): [M+H] + =1383.

[0359] Step 4: N-((2S)-1-((4-((2-(6-amino-5-(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 (ii) 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)-N-(5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl) (methyl) amino) pentyl) cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000259.tif271701-(((2S)-1-((4-((2-(6-amino-5-(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]octane-3 To a solution of 1-(2-((5-aminopentyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (30.0 mg, crude) and DIPEA (46.6 mg, 0.360 mmol) in DMF (1 mL) was added HATU (18.0 mg, 0.0500 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The crude was purified under 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: 8% B to 26% B, 26% B in 12 min; Wavelength: 254 / 220nm; R T Purification by preparative HPLC at (min): 12 gave 16.7 mg of Hy-B-CIDE-11 as a white solid. LCMS (ESI, m / z): [M+H] + =1604. 1 H NMR (300MHz, DMSO-d 6, ppm) δ 10.12 (s, 1H), 9.53 (s, 2H), 8.36 (d, J = 7.4 Hz, 1H), 8.00 - 7.77 (m, 3H), 7.74 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.51 - 7.39 (m, 2H), 7.45 - 7.29 (m, 5H), 7.21 (d, J = 8.4 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.96 (s, 2H), 6.87 (s, 1H), 6.51 (s, 1H), 6.04 (d, J = 9.9 Hz, 2H), 5.09 (s, 1H), 4.97 (s, 2H), 4.80 (t, J = 7.0 Hz, 1H), 4.69 - 4.49 (m, 1H), 4.39 (s, 4H), 4.37 - 4.30 (m, 1H), 4.27 - 4.03 (m, 3H), 3.70 - 3.52 (m, 6H), 3.40 - 3.30 (m, 5H), 3.03 - 2.78 (m, 11H), 2.82 - 2.70 (m, 9H), 2.45 - 2.30 (m, 8H), 2.22 - 2.15 (m, 1H), 1.97 - 1.84 (m, 4H), 1.77 (s, 4H), 1.69 - 1.54 (m, 5H), 1.54 - 1.40 (m, 5H), 1.39 - 1.28 (m, 3H), 1.25 - 1.14 (m, 8H), 0.84 (t, J = 6.3 Hz, 3H), 0.69 (dd, J = 9.2, 6.6 Hz, 3H).

[0360] Example 23 Synthesis of Hy - B - CIDE - 12 N-((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)-N-(5-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamido)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) TIFF2025506357000260.tif74170

[0361]

[0355] Step 1: N-((2S)-1-((4-((2-(6-amino-5-(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)pi Lysin-4-yl)-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenoxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)-N-(5-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamido)pentyl)cyclobutane-1,1-dicarboxamide (2,2,2-trifluoroacetate) Under nitrogen, 1-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl To a solution of (S)-N-(5-aminopentyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropanamide (2,2,2-trifluoroacetate) (30.0 mg, crude) and DIPEA (37.0 mg, 0.290 mmol) in N,N-dimethylformamide (1 mL) was added HATU (14.0 mg, 0.0400 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The product was purified under 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: 12% B to 27% B, 27% B in 12 min; Wavelength: 254 / 220nm; R T Purification by preparative HPLC at (min):12 gave 7.2 mg of Hy-B-CIDE-12 as a white solid. LCMS(ESI,m / z):[M+H] + =1634. 1 H NMR (300MHz, DMSO-d 6,ppm)δ 10.23(s,1H),9.59(d,J=47.0Hz,2H),8.49(d,J=7.3Hz,1H),8.00-7.75(m,6H),7.68(d,J=8.2Hz,2H),7.57(t,J=8.6Hz,2H) ,7.52-7.28(m,6H),7.14(t,J=7.6Hz,1H),7.05(s,2H),6.98(s,1H),6.64(s,1H),6.17(d,J=8.2Hz,2H),5.25-5.05(m,4H), 4.92(t,J=6.8Hz,2H),4.58-4.25(m,9H),4.01-3.85(m,3H),3.74-3.66(m,3H),3.17-2.96(m,11H),2.80-2.70(m,7H),2.45 -2.30(m,8H),2.29-2.18(m,2H),2.16-1.52(m,18H),1.49-1.13(m,12H),0.97(d,J=6.4Hz,3H),0.82(dd,J=9.3,6.6Hz,3H).

[0362] Example 24 Synthesis of Hy-B-CIDE-13 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-(5-(2-((1R,3r)-3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyanophenoxy) 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)-2-oxa-5,8-diazaspiro[3.5]nonan-8-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025506357000261.tif78170

[0363] Step 1: tert-Butyl 4-((1r,3r)-3-((4-(8-(6-(2-((allyloxy)carbonyl)oxy)phenyl)-3-aminopyridazin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000262.tif39170To a solution of tert-butyl 4-((1r,3r)-3-((4-(8-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (480 mg, 7.28 mmol) and DPIEA (847 mg, 65.6 mmol) in DCM (6 mL) under nitrogen, AllocCl (306 mg, 25.5 mmol) was added at 0° C. The resulting solution was stirred at room temperature for 30 min. The crude was purified by flash chromatography on silica gel (solvent gradient: 0-5% MeOH / DCM) to afford 505 mg (86% yield) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =744.

[0364] Step 2: tert-Butyl 4-((1r,3r)-3-((4-(8-(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)-2-oxa-5,8-diazaspiro[3.5]nonan-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000263.tif55170Under nitrogen, tert-butyl To a solution of 4-((1r,3r)-3-((4-(8-(6-(2-(((allyloxy)carbonyl)oxy)phenyl)-3-aminopyridazin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (460 mg, 0.619 mmol), ethyl (S)-1-((6-(((allyloxy)carbonyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylate (908 mg, 1.86 mmol) and DIPEA (480 mg, 3.72 mmol) in THF (6 mL) was added triphosgene (208 mg, 0.433 mmol) at 0 °C. The reaction was stirred at room temperature for 0.5 h. The solvent was evaporated and the residue was purified by column chromatography (solvent gradient: 0-100% ACN in water (0.05% NH 4 HCO 3 )) to give 230 mg (29%) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =1260.

[0365] Step 3: tert-Butyl 4-((1r,3r)-3-((4-(8-(3-((((4-((S)-6-amino-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000264.tif57170Under nitrogen, tert-butyl 4-((1r,3r)-3-((4-(8-(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)-2-oxa-5,8-diazaspiro[3.5]non-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (230 mg, 0.183 mmol), pyrrolidine (131 mg, 1.83 mmol) and Pd(PPh 3 ) 4 A solution of (22.0 mg, 0.01830 mmol) in DCM (4 mL) was stirred at room temperature for 1 h. The solvent was evaporated and the crude was used directly in the next step.

[0366] Step 4: tert-Butyl 4-((1r,3r)-3-((4-(8-(3-((((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate TIFF2025506357000265.tif52170Under nitrogen, tert-butyl 4-((1r,3r)-3-((4-(8-(3-((((4-((S)-6-amino-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (230 mg, crude) and 37% aqueous CH 2A solution of 230 mg of O in 4 mL of MeOH was stirred at room temperature for 1 h. Then, NaBH 3 CN (63.5 mg, 1.06 mmol) was added and the solution was stirred at room temperature for 15 min. The reaction was quenched with water and the resulting residue was purified by column chromatography (solvent gradient: 0-90% MeOH in water (0.05% NH 4 HCO 3 )) to give 140 mg (55% yield) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =1120.

[0367] Step 5: 1-(((S)-1-((4-((((4-(5-(2-((1r,3r)-3-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-8-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000266.tif55170Under nitrogen, tert-butyl 4-((1r,3r)-3-((4-(8-(3-((((4-((S)-6-(dimethylamino)-2-(1-(ethoxycarbonyl)cyclobutane-1-carboxamido)hexanamido)benzyl)oxy)carbonyl)amino)-6-(2-hydroxyphenyl)pyridazin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-5-yl)pyridin-2-yl)oxy)cyclobutoxy)piperidine-1-carboxylate (140 mg, 0.125 mmol) and LiOH.H 2 HO (15.9 mg, 0.375 mmol) in THF (2 mL) and H 2 The solution in O (2 mL) was stirred at room temperature for 1 h. The resulting mixture was concentrated in vacuo to give 160 mg (crude) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =1092.

[0368] Step 6: 1-(((S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-4-(5-(2-((1r,3r)-3-(piperidin-4-yloxy)cyclobutoxy)pyridin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-8-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid A solution of 1-(((S)-1-((4-((((4-(5-(2-((1r,3r)-3-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)cyclobutoxy)pyridin-4-yl)-2-oxa-5,8-diazaspiro[3.5]nonan-8-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (160 mg, crude) in HFIP (4.8 mL) and 2,2,2-trifluoroacetic acid (0.25 mL) was stirred at room temperature under nitrogen for 15 min. The solvent was evaporated under vacuum and the column was purified by centrifugation using a pre-packed C18 column (solvent gradient: 0–70% ACN in water (0.05% NH 4 HCO 3 )) to give 74.0 mg (51% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =992.

[0369] Step 7: 1-(((S)-1-((4-(((4-(5-(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)-2-oxa-5,8-diazaspiro[3.5]nonan-8-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000268.tif49170Under nitrogen, 1-(((S)-6-(dimethylamino)-1-((4-((((6-(2-hydroxyphenyl)-4-(5-(2-((1r,3r)-3-(piperidin-4-yloxy)cyclobutoxy)pyridin-4-yl)-2-oxa-5,8-diazaspiro[3.5]non-8-yl)pyridazin-3-yl)carbamoyl)oxy)methyl)furan (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 (28.5 mg, 0.061 mmol) and CH 3 A solution of COOH (3.60 mg, 0.0610 mmol) in MeOH (0.25 mL) and DCM (0.75 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (3.00 mg, 0.0450 mmol) was added and stirred at room temperature for 0.5 h. The crude was purified by column chromatography (solvent gradient: 0-80% MeOH in water (0.05% NH 4 HCO 3 )) to give 18.0 mg (41% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H]+ =1444.

[0370] Step 8: 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-carboxamide)hexanamide)benzyl (4-(5-(2-((1R,3r)-3-((1-(2-((5-((R)-1-((2S,4R)-2-(((S)-1-(4-cyano Phenyl)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)-2-oxa-5,8-diazaspiro[3.5]nonan-8-yl)-6-(2-hydroxyphenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025506357000269.tif43170Under nitrogen, 1-(((S)-1-((4-((((4-(5-(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)-2-oxa-5,8-diazaspiro[3.5]non-8-yl)-6-(2-hydroxypyrrolidin-1-yl) To a solution of (hydroxyphenyl)pyridazin-3-yl)carbamoyl)oxy)methyl)phenyl)amino)-6-(dimethylamino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (18.0 mg, 0.0125 mmol), 1-(2-((5-aminopentyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione (2,2,2-trifluoroacetate) (13.0 mg, crude) and DIPEA (25.8 mg, 0.200 mmol) in DMF (1 mL) was added HATU (9.50 mg, 0.025 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The crude material was purified under 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: 14% B to 28% B, 28% B in 10 min; Wavelength: 254 / 220nm; R T (min): 10 and purified by preparative HPLC to give 14.9 mg (71.7% yield) of Hy-B-CIDE-13 as a white solid. LC-MS: (ESI, m / z): [M+H] += 1665. 1 H NMR (300MHz, DMSO-d 6, ppm) δ 10.17 (d, J = 17.2 Hz, 2H), 9.55 (br, 1H), 9.40 (br, 1H), 8.49 (d, J = 7.3 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.95 - 7.82 (m, 3H), 7.79 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.47 (m, J = 8.4, 2.7 Hz, 2H), 7.38 (t, J = 8.5 Hz, 3H), 7.10 (s, 2H), 7.02 - 6.92 (m, 2H), 6.42 - 6.34 (m, 1H), 6.16 (s, 1H), 5.75 (s, 1H), 5.20 (s, 1H), 5.14 (s, 2H), 4.91 (t, J = 7.2 Hz, 1H), 4.63 (d, J = 4.4 Hz, 3H), 4.51 (s, 2H), 4.42 (s, 1H), 4.33 (t, J = 8.2 Hz, 2H), 3.81 - 3.66 (m, 10H), 3.21 - 2.94 (m, 13H), 2.82 - 2.74 (m, 9H), 2.45 - 2.20 (m, 12H), 2.18 - 2.00 (m, 3H), 1.92 - 1.70 (m, 7H), 1.70 - 1.50 (m, 5H), 1.47 (q, J = 7.1 Hz, 3H), 1.39 - 1.21 (m, 8H), 0.96 (d, J = 6.5 Hz, 3H), 0.80 (d, J = 6.8 Hz, 3H).

[0371] Example 25 Synthesis of Hy - B - CIDE - 14 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) ethylthiazol-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) TIFF2025506357000270.tif126170

[0372] 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 TIFF2025506357000271.tif53170Under nitrogen, 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, 0.714 mmol), ethyl (S To a solution of 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) in DCM (15 mL) 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 column chromatography on a pre-packed C18 column (solvent gradient: 0-100% CH in water). 3 CN(0.05% NH 4 HCO 3 )) to give 480 mg (55% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =1216.

[0373] 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 TIFF2025506357000272.tif51170Under nitrogen, a solution of 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 (400 mg, 0.329 mmol), pyrrolidine (234 mg, 3.29 mmol) in DCM (5 mL) was added to Pd(PPh 3 ) 4 (76.0 mg, 0.0658 mmol) was added 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 in vacuo to give 390 mg (crude) of the title compound as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =1048.

[0374] 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 TIFF2025506357000273.tif51170 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 CH 2 O (465 mg, 5.73 mmol) was dissolved in CH 3 The solution in OH (5 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (140 mg, 2.22 mmol) was added and stirred at room temperature for 1 h. The reaction was quenched with water (0.2 mL). The residue was purified by column chromatography on a packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH 4 HCO 3 )) to give 260 mg (54% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M] + =1077.

[0375] 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 TIFF2025506357000274.tif50170 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.H 2 A solution of 2H2O (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 under vacuum. Then, 5% TFA in HFIP (3 mL) was added and the solution was stirred at room temperature for 1 h. The solvent was concentrated under vacuum. The residue was purified by column chromatography on a pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH 4 HCO 3 )) to give 55.0 mg (83% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M] + =949.

[0376] 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)isopropyl) Soxazol-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)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)carbamoyl)cyclobutane-1-carboxylic acid TIFF2025506357000275.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-oxohexan-2-yl)carbamoyl)cyclobutane A solution of 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) in methyl alcohol (1.5 mL) was stirred at room temperature for 1 h. 3 CN (5.2 mg, 0.0827 mol) was added and stirred at room temperature for 3 h. The reaction solution was concentrated under vacuum. The residue was purified by column chromatography (solvent gradient: 0-100% CH in water). 3 OH(0.05% NH 4 HCO 3 )) to give 18 mg (58% yield) of the title compound as a white solid. LC-MS: (ESI, m / z): [M+H] + =1473.

[0377] 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-carboxamide)hexanamide)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-hydroxyphenyl)pyridazin-3-yl)carbamate (2,2,2-trifluoroacetate) TIFF2025506357000276.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[3.2.1]octane To a solution of 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) in DMF (1 mL) was added HATU (13.5 mg, 0.0355 mmol) at room temperature. The reaction was stirred at room temperature for 10 min. The crude material 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 and purified by preparative HPLC to give 13.0 mg (28.3% yield) of Hy-B-CIDE-14 as a yellow solid. LC-MS: (ESI, m / z): [M+H] + =1808. 1 H NMR (300MHz, DMSO-d 6,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).

[0378] Example 26 Synthesis of Hy-B-CIDE-3 TIFF2025506357000277.tif63170

[0379] Step 1: tert-Butyl (2-((2-(1,3-dioxoisoindolin-2-yl)ethyl)(methyl)amino)ethyl)carbamate TIFF2025506357000278.tif20170A mixture of potassium carbonate (10.9 g, 78.7 mmol), 2-(2-bromoethyl)isoindoline-1,3-dione (10.0 g, 39.4 mmol) and tert-butyl (2-(methylamino)ethyl)carbamate (8.23 g, 47.2 mmol) in N,N-dimethylformamide (200 mL) was cooled to 37°C for 10 min at 4°C. 2The mixture was stirred at 90° C. under ambient atmosphere for 16 hours. The reaction was cooled to 25° C., diluted with water (30 mL) and extracted with ethyl acetate (50 mL×3). The organics were washed with brine (20 mL×2), dried over sodium sulfate, filtered and concentrated to dryness. The residue was purified by flash chromatography (silica gel, 100-200 mesh, 0-15% methanol in dichloromethane) to give the title compound (4.5 g, 32.9% yield) as a white solid.

[0380]

[0373] 1 H NMR (400 MHz, CDCl 3 ):δ(ppm)7.92-7.82(m,2H),7.73(m,2H),4.32-4.28(m,1H),4.01-3.98(m,1H),3.84-3.74(m,1H),3.50(s,1H),3 .37-3.09(m,3H),2.94-2.78(m,1H),2.69-2.65(m,1H),2.55-2.50(m,1H),2.31-2.35(m,1H),1.49-1.32(m,10H).

[0381] Step 2: tert-Butyl (2-((2-aminoethyl)(methyl)amino)ethyl)carbamate TIFF2025506357000279.tif11170 To a stirred solution of tert-butyl (2-((2-(1,3-dioxoisoindolin-2-yl)ethyl)(methyl)amino)ethyl)carbamate (4.00 g, 11.5 mmol) in acetonitrile (60 mL) was added hydrazine monohydrate (22.4 mL, 230 mmol) dropwise. The resulting mixture was cooled to room temperature with 500° C. for 1 hour at 4° C. for 2 hours. 2 The mixture was stirred at 70° C. under atmospheric pressure for 16 hours. The reaction mixture was poured into ice water (15.0 mL) and extracted with ethyl acetate (20.0 mL×3). The combined organic layers were washed with brine (20.0 mL×2), dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was purified by preparative TLC (5% methanol in dichloromethane) to give the title compound (1.50 g, 60% yield) as a colorless oil.

[0382]

[0375] 1 H NMR (400 MHz, CDCl 3 )δ(ppm)4.13-4.10(m,2H),3.43-3.33(m,2H),3.30-3.25(m,2H),2.96-2.92(m,5H),1.41(s,9H).

[0383] Step 3: tert-Butyl (2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)carbamate TIFF2025506357000280.tif18170 To a solution of tert-butyl (2-((2-aminoethyl)(methyl)amino)ethyl)carbamate (100 mg, 0.46 mmol) in tetrahydrofuran (2.00 mL) was added methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (107 mg, 0.69 mmol) and a solution of sodium bicarbonate (116 mg, 1.38 mmol) in water (1.00 mL). The reaction mixture was stirred at 0° C. for 2 h. The reaction mixture was filtered and the organic layer was concentrated in vacuo to dryness. To the resulting solution was added water (20.0 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to dryness. The residue was purified (silica gel, 100-200 mesh, 0-10% methanol in dichloromethane) to give the title compound (73 mg, 53.4% ​​yield) as a colorless oil.

[0384]

[0377] 1 H NMR (400 MHz, CDCl 3 )δ(ppm)6.71(s,2H),3.65-3.62(m,2H),3.16-3.13(m,2H),2.62-2.41(m,4H),2.25(s,3H),1.45(s,10H).

[0385] Step 4: 1-(2-((2-aminoethyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate TIFF2025506357000281.tif26170 To a solution of tert-butyl (2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)carbamate (73.0 mg, 0.25 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (2.31 mL, 29.95 mmol). The reaction mixture was stirred at 20° C. for 5 h. The reaction was concentrated to give the title compound (40 mg, 82.6%) as a yellow oil.

[0386] Step 5: N-((2S)-1-((4-((2-(6-amino-5-(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-methyl rupiperazin-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)-N-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)ethyl)cyclobutane-1,1-dicarboxamide TIFF2025506357000282.tif401701-(((2S)-1-((4-((2-(6-amino-5-(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)pyridazin-3-yl)phenoxy)methyl)phenyl) To a solution of 1-(2-((2-aminoethyl)(methyl)amino)ethyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (10.5 mg, 0.03 mmol) in N,N-dimethylformamide (4.00 mL) was added N,N-diisopropylethylamine (0.01 mL, 0.08 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.8 mg, 0.03 mmol). The reaction mixture was stirred at 25° C. for 3 h. The mixture was concentrated by oil pump. The residue was purified by preparative HPLC (Boston Green ODS 150*30mm*5um, water (0.075% trifluoroacetic acid)-acetonitrile, 20%-50%) to give the title compound (12.0mg, yield 26.7%) as a white solid.

[0387]

[0380] 1 H NMR (400MHz, DMSO-d 6)δ 10.19(s,1H),9.02-8.86(m,1H),8.39(d,J=8.0Hz,1H),7.94(d,J=6.8Hz,1H),7.87-7.78(m,2H),7.65(d,J=8.0Hz,2H),7.51-7.41(m,5H) ,7.36(d,J=6.8Hz,5H),7.29-7.09(m,1H),6.97(s,2H),6.76(s,1H), 6.42(s,1H),6.15-5.96(m,2H),5.07(s,2H),4.93-4.86(m,1H),4.71- 4.60(m,2H),4.52-4.24(m,9H),3.66(s,4H),3.33(d,J=7.2Hz,5H),3.09-2.96(m,8H),2.45(s,3H),2.42-2.34(m,4H),2.29-2.14(m,2H), 2.04(d,J=11.2Hz,3H),1.91(s,2H),1.83-1.55(m,5H),1.51-1.30(m,9H),1.19(d,J=5.8Hz,5H),0.96(d,J=6.4Hz,3H),0.86-0.75(m,3H).

[0388]

[0381] LCMS(ESI)m / z:1609.8[M+H] + .

[0389] Example 27 Synthesis of Hy-B-PCIDE-1 (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-carboxamide)hexanamide) 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 dihydrogen phosphate (2,2,2-trifluoroacetate) TIFF2025506357000283.tif102170

[0390] 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 TIFF2025506357000284.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. Then, tBuOOH (0.4 mL, 5 M in decane) was 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 cooled to 30° C. with NaHSO 3The mixture was quenched with aqueous solution 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 column chromatography on a pre-packed C18 column (solvent gradient: 0-70% ACN in water (0.05% NH 4 HCO 3 )) to give 340 mg (51%) of the title compound as a yellow oil. LC-MS: (ESI, m / z): [M+H] + =807.

[0391] 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 dihydrogen phosphate TIFF2025506357000285.tif44170 Under nitrogen, 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) 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.

[0392] 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-oxo butan-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 TIFF2025506357000286.tif591701-(((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-(dimethyl 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 dihydrogen phosphate (121 mg, 0.190 mmol) and NaOAc (13.6 mg, 0.170 mmol) in methyl alcohol (1.2 mL) and dichloromethane (0.4 mL) was stirred at room temperature for 1 h. Then, NaBH 3 CN (34.7 mg, 0.550 mmol) was added and stirred at room temperature for 0.5 h. Water was added to quench the reaction. The solvent was concentrated under vacuum. The product was purified by preparative HPLC (column: YMC-Actus Triart C18 ExRS, 30*150 mm, 5 μm; mobile phase A: water (10 mmol / L NH 4 HCO 3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 36% B to 60% B in 9 min, 60% B; wavelength: 254 / 220 nm; RT1 (min): 7.63) to give 30.0 mg (18% yield) of the title compound as a white solid. LCMS (ESI) [M+H] + =1509.

[0393] 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-carboxamide)hexaneamine 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 dihydrogen phosphate (2,2,2-trifluoroacetate) TIFF2025506357000287.tif521701-(((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] To a solution of 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) in DMF (1 mL) 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 preparative 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 10min, 24% B; Wavelength: 254 / 220nm; RT1(min): 10min) to give 8.70mg (24% yield) of Hy-B-PCIDE-1 as a white solid. LCMS(ESI)[M+H] + =1688. 11H NMR (300 MHz, DMSO-d6) δ 10.26 (s, 1H), 8.99 (s, 1H), 8.50 (d, J = 7.5 Hz, 1H), 8.15 (t, 1H), 7.87 (d, J = 6.5 Hz, 2H), 7.65 (d, J = 8.3 Hz, 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 = 7.2 Hz, 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 = 6.4 Hz, 3H), 0.85 - 0.70 (m, 3H).

[0394] Example 28 Cell 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 catalog number 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 488 A21206 (1:2000)) for 1 hour at room temperature in the dark. Hoechst H3570 was added to the wells at 1:5000 and the plates were incubated for an additional 30 minutes. The plates were washed 3x with PBS and imaged on the OperaPhenix™ High Content Screening System. The nuclear stain was used as a mask to quantify the nuclear BRM mean signal intensity.

[0395] The data are shown in the following table, which demonstrates that the antibody targeting strategy disclosed herein is successful and that Ab-CIDEs containing hydrolyzable linkers can degrade target proteins at similar levels as Ab-CIDEs containing non-hydrolyzable linkers. TIFF2025506357000288.tif148170

[0396] Example 29 Achieving high DAR by conjugating hydrolyzable linkers to antibodies

[0389] Hydrolyzable maleimide-containing linkers have been shown to provide high DARs (greater than 6). At this level of DAR, certain non-hydrolyzable linkers become unstable, but antibody-conjugated hydrolyzable linkers have been prepared, isolated, and characterized. Thus, hydrolyzable linkers are advantageous in obtaining high DARs, which results in a greater number of CIDEs that can be attached to a single antibody.

[0397] Data showing levels of DAR obtained with CIDEs containing hydrolyzable linkers: TIFF2025506357000289.tif81170

[0398]

[0391] The DAR levels of antibodies conjugated with hydrolyzable linkers containing CIDE are advantageously higher compared to non-hydrolyzable linkers containing CIDE: TIFF2025506357000290.tif117170

[0399]

[0392] Efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.) but some experimental error and deviation should be accounted for.

[0400]

[0393] 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. The present disclosure is in no way limited to only the methods and materials described.

[0401]

[0394] 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 the subject matter belongs and are consistent with Singleton et al (1994) Dictionary of Microbiology and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York, NY; and Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York.

[0402]

[0395] Throughout this specification and the claims, the words "comprise" (comprises) and "comprising" are used in their open-ended sense unless the context indicates otherwise. The embodiments described in this specification include embodiments "consisting of" and / or "consisting essentially of."

[0403]

[0396] As used herein, the term "about" when referring to a value is meant to encompass variations from the particular amount of, 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%, thereby appropriate for practicing the disclosed methods or using the disclosed compositions.

[0404]

[0397] Where a range of values ​​is provided, it is understood that the upper and lower limits of the range include each intervening value, to one-tenth of the unit of the lower limit, between the range and any other value or intervening value stated within the range, unless the context indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included subject to any specifically excluded limit within the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of the included limits are also included.

[0405]

[0398] Many modifications and other embodiments of the invention described 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 associated drawings. It is understood, therefore, that the invention 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. structure: L １ -D [In the formula, D is CIDE; L １ is covalently attached to D and has formula I: (In the formula, Z is -(CH ２ ) ｐ -or-CH ２ - (CH ２ -O-CH ２ ) ｐ -CH ２ - and p is an integer from 1 to 24; R Ａ is hydrogen, C １－６ Alkyl, or -(CH ２ ) ｖ -aryl, where v is 0 or 1; Q is, i) wherein q is 1, 2, 3, or 4; and ii) wherein t is 0, 1, 2, 3, or 4; Q １ teeth, R ２ is hydrogen, haloC １－６ Alkyl or C １－６ is alkyl) selected from the group consisting of: L １－Ａ teeth: and During the ceremony, indicates the point of attachment to D; 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 -NH-C(NH)-NH ２ R is selected from the group consisting of 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 ] ｕ R is selected from the group consisting of hydrogen, C １－３ Alkyl, N(R ｘ ) (R ｙ ), -O-N(R ｘ ) (R ｙ ) or C(O)-N(R ｘ ) (R ｙ ) u 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 they are attached form an optionally substituted 5- to 7-membered heterocyclyl; R Ｃ and R Ｄ are each independently hydrogen and C １－３ alkyl, or R Ｃ and R Ｄ 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 are each independently hydrogen, halo, C １－５ Alkyl, C １－５ alkoxy or hydroxyl The linker 1 has the structure A compound having the formula:

2. Q, and q is 1, 2, 3 or 4.

3. The compound of claim 2 , wherein q is 2.

4. Q, where t is 0, 1, 2, 3 or 4; Q １ but R ２ is hydrogen, haloC １－６ Alkyl or C １－６ The compound of claim 1 , wherein the aryl group is an alkyl group.

5. R ２ is hydrogen or C １－６ The compound of claim 4, which is alkyl.

6. C １－６ The compound of claim 5, wherein the alkyl is methyl.

7. Q １ but, The compound according to claim 4.

8. R ２ The compound of claim 4, wherein is hydrogen or methyl.

9. Q １ but, The compound according to claim 8.

10. The compound of claim 4, wherein t is 0.

11. Z is -(CH ２ ) ｐ - and p is 1, 2, 3, 4, 5 or 6.

12. The compound of claim 11 , wherein p is 4, 5 or 6.

13. The compound of claim 11 , wherein p is 5.

14. Z is -CH ２ - (CH ２ -O-CH ２ ) ｐ -CH ２ - and p is 1, 2, 3, 4, 5 or 6.

15. 15. The compound of claim 14, wherein p is 1, 2 or 3.

16. 15. The compound of claim 14, wherein p is 1.

17. R Ａ is hydrogen or C １－６ 17. The compound of any one of claims 1 to 16, wherein said compound is alkyl.

18. R Ａ 18. The compound of claim 17, wherein is hydrogen or methyl.

19. R Ａ 17. The compound according to any one of claims 1 to 16, wherein is phenyl or benzyl.

20. R ７ and R ８ 20. The compound of claim 1, wherein each is hydrogen.

21. R Ｃ and R Ｄ each of which, together with the carbon to which it is attached, is an optionally substituted C ３－６ 21. A compound according to any one of claims 1 to 20 which forms a cycloalkyl.

22. C ３－６ 22. The compound of claim 21 , wherein the cycloalkyl is cyclobutane.

23. K is -CH(R)-, R is hydrogen, C １－３ Alkyl, N(R ｘ ) (R ｙ ) or C(O)-N(R ｘ ) (R ｙ ) and R ｘ and R ｙ are each independently hydrogen and C １－３ alkyl, or R ｘ and R ｙ and R 1 and R 2 are taken together with the nitrogen to which they are attached to form an optionally substituted 5- to 7-membered heterocyclyl.

24. R is hydrogen or C １－３ 24. The compound of claim 23, which is alkyl.

25. 25. The compound of claim 24, wherein R is hydrogen.

26. structure: A-(L) １ (-D) ｊ [In the formula, Ab is an antibody; j is 1 to 16; D is CIDE; L １ is covalently linked to Ab and D and has the formula IA: (In the formula, Z is -(CH ２ ) ｐ -or-CH ２ - (CH ２ -O-CH ２ ) ｐ -CH ２ -, where p is an integer from 1 to 24; R Ａ is hydrogen, C １－６ Alkyl, or -(CH ２ ) ｖ -aryl, where v is 0 or 1; Q is, i) wherein q is 1, 2, 3, or 4; and ii) wherein t is 0, 1, 2, 3, or 4; Q １ teeth, R ２ is hydrogen, halo (C １－６ ) alkyl or C １－６ is alkyl) selected from the group consisting of: During the ceremony, indicates the point of attachment to D; w is 0, 1, 2, 3, 4 or 5; J is -N(R ｘ )(Ry), -C(O)NH ２ , -NH-C(O)-NH ２ , and -NH-C(=NH)-NH ２ R is selected from the group consisting of 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 ] ｕ R is selected from the group consisting of hydrogen, C １－３ Alkyl, N(R ｘ ) (R ｙ ), -O-N(R ｘ ) (R ｙ ) or C(O)-N(R ｘ ) (R ｙ ) u 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 they are attached form an optionally substituted 5- to 7-membered heterocyclyl; R Ｃ and R Ｄ are each independently hydrogen and C １－３ alkyl, or R Ｃ and R Ｄ 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 are each independently hydrogen, halo, C １－５ Alkyl, C １－５ alkoxy or hydroxyl is a linker having the structure An antibody-CIDE conjugate having the formula:

27. Q, and q is 1, 2, 3 or 4.

28. The antibody-CIDE conjugate of claim 26 or 27, wherein q is 2.

29. Q, where t is 0, 1, 2, 3 or 4; Q １ but, R ２ is hydrogen, haloC １－６ Alkyl or C １－６ The antibody-CIDE conjugate of claim 26, which is alkyl.

30. R ２ is hydrogen or C １－６ The antibody-CIDE conjugate of claim 29, which is alkyl.

31. C １－６ The antibody-CIDE conjugate of claim 30, wherein the alkyl is methyl.

32. Q １ but, The antibody-CIDE conjugate of claim 29.

33. R ２ The antibody-CIDE conjugate of claim 29, wherein is hydrogen or methyl.

34. Q １ but, The antibody-CIDE conjugate of claim 33.

35. The antibody-CIDE conjugate of any one of claims 29 to 34, wherein t is 0.

36. Z is -(CH ２ ) ｐ - and p is 1, 2, 3, 4, 5 or 6.

37. The antibody-CIDE conjugate of claim 36, wherein p is 4, 5 or 6.

38. The antibody-CIDE conjugate of claim 36, wherein p is 5.

39. Z is -CH ２ - (CH ２ -O-CH ２ ) ｐ -CH ２ - and p is 1, 2, 3, 4, 5 or 6.

40. The antibody-CIDE conjugate of claim 39, wherein p is 1, 2 or 3.

41. The antibody-CIDE conjugate of claim 39, wherein p is 1.

42. R Ａ is hydrogen or C １－６ The antibody-CIDE conjugate of any one of claims 26 to 41, which is alkyl.

43. R Ａ The antibody-CIDE conjugate of claim 42, wherein is hydrogen or methyl.

44. R Ａ The antibody-CIDE conjugate of any one of claims 26 to 41, wherein is phenyl or benzyl.

45. R ７ and R ８ The antibody-CIDE conjugate of any one of claims 26 to 44, wherein each is hydrogen.

46. R Ｃ and R Ｄ each of which, together with the carbon to which it is attached, is an optionally substituted C ３－６ The antibody-CIDE conjugate of any one of claims 26 to 45, which forms a cycloalkyl.

47. C ３－６ The antibody-CIDE conjugate of claim 46, wherein the cycloalkyl is cyclobutane.

48. K is -CH(R)-, R is hydrogen, C １－３ Alkyl, N(R ｘ ) (R ｙ ) or C(O)-N(R ｘ ) (R ｙ ) and R ｘ and R ｙ are each independently hydrogen and C １－３ alkyl, or R ｘ and R ｙ The antibody-CIDE conjugate of any one of claims 26 to 47, wherein, together with the nitrogen to which each is attached, form an optionally substituted 5- to 7-membered heterocyclyl.

49. R is hydrogen or C １－３ The antibody-CIDE conjugate of claim 48, which is alkyl.

50. 50. The antibody-CIDE conjugate of claim 49, wherein R is hydrogen.

51. A pharmaceutical composition comprising an antibody-CIDE conjugate according to any one of claims 26 to 50 and one or more pharma- ceutically acceptable excipients.

52. A method of treating a disease in a human in need thereof, comprising administering to said human an effective amount of an antibody-CIDE conjugate of any one of claims 26 to 50 and 57, an antibody-conjugate of claim 58, or a pharmaceutical composition of claim 51.

53. 53. The method of claim 52, wherein the disease is cancer.

54. 54. The method of claim 53, wherein the cancer is BRM dependent.

55. 55. The method of claim 53 or 54, wherein the cancer is non-small cell lung cancer.

56. A method for reducing the level of a target protein in a subject, comprising administering to the subject an antibody-CIDE conjugate of any one of claims 26 to 50 and 57, an antibody-conjugate of claim 58, or a pharmaceutical composition of claim 51, wherein the PB moiety binds to the target protein and a ubiquitin ligase affects degradation of the bound target protein, thereby reducing the level of the target protein.

57. structure: A-((L Ｘ )-D) ｊ [In the formula, Ab is an antibody; j is 1 to 16; D is CIDE; L Ｘ L １ and L １ｈ and L is selected from the group consisting of １ｈ L Ｘ In at least one instance of: L １ is covalently linked to Ab and D and has the formula IA: The linker 1 has the structure L １ｈ is covalently bonded to D and has the formula I-B: wherein # indicates the point of attachment at position a or b; L １ and L １ｈ In indicating the attachment points to the antibody; 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 is as follows: i) wherein q is 1, 2, 3, or 4; and ii) wherein t is 0, 1, 2, 3, or 4; Q １ teeth, R ２ is hydrogen, haloC １－６ Alkyl or C １－６ is alkyl) selected from the group consisting of: L １－Ａ teeth: and During the ceremony, indicates the point of attachment to D; 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 , -NH-C(=NH)-NH ２ R is selected from the group consisting of 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 ] ｕ R is selected from the group consisting of hydrogen, C １－３ Alkyl, N(R ｘ ) (R ｙ ), -O-N(R ｘ ) (R ｙ ) or C(O)-N(R ｘ ) (R ｙ ) u 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 they are attached form an optionally substituted 5- to 7-membered heterocyclyl; R Ｃ and R Ｄ are each independently hydrogen and C １－３ alkyl, or R Ｃ and R Ｄ 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 are each independently hydrogen, halo, C １－５ Alkyl, C １－５ alkoxy or hydroxyl The linker 1 has the structure An antibody-CIDE conjugate having the formula:

58. structure: A-(L) Ｚ ) ｊ [In the formula, Ab is an antibody; j is 1 to 16; L Ｚ L Ｘ１ and Lx-D; Ｘ１ L Ｚ In at least one instance of: L Ｘ１ is covalently attached to Ab and has the formula IG: The structure is And, Lx-D (where L Ｘ L １ and L １ｈ Selected from the group consisting of: L １ is covalently linked to Ab and D and has formula IA or formula IC: L is a linker 1 having the structure １ｈ is covalently bonded to D and has the formula I-B: where # indicates the point of attachment at position a or b; indicating the attachment points to the antibody; 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 is as follows: i) wherein q is 1, 2, 3, or 4; and ii) (Wherein, t is 0, 1, 2, 3 or 4; Q １ teeth, R ２ is hydrogen, haloC １－６ Alkyl or C １－６ is alkyl) selected from the group consisting of: L １－Ａ teeth: and During the ceremony, indicates the point of attachment to D; 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 , -NH-C(=NH)-NH ２ R is selected from the group consisting of 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 ] ｕ R is selected from the group consisting of hydrogen, C １－３ Alkyl, N(R ｘ ) (R ｙ ), -O-N(R ｘ ) (R ｙ ) or C(O)-N(R ｘ ) (R ｙ ) u 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 they are attached form an optionally substituted 5- to 7-membered heterocyclyl; R Ｃ and R Ｄ are each independently hydrogen and C １－３ alkyl, or R Ｃ and R Ｄ 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 are each independently hydrogen, halo, C １－５ Alkyl, C １－５ alkoxy or hydroxyl ］ An antibody-conjugate having the formula:

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