Azabenzazepine immunoconjugates and uses thereof

Abstract

The present invention provides immunoconjugates of Formula I comprising an antibody linked by conjugation to one or more azabenzazepine derivatives. The present invention also provides azabenzazepine derivative intermediate compositions containing reactive functional groups. Such intermediate compositions are suitable substrates for forming the immunoconjugates via a linker or linking moiety. The present invention further provides methods for treating cancer using the immunoconjugates. [Formula 1] TIFF2026506037000158.tif17165

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This non-provisional application claims the benefit of priority to U.S. Provisional Application No. 63 / 445,390, filed February 14, 2023, which is incorporated by reference in its entirety.

[0002] The present invention relates generally to immunoconjugates comprising an antibody conjugated to one or more azabenzazepine molecules. [Background technology]

[0003] New compositions and methods for delivering antibodies and immune adjuvants are needed to reach inaccessible tumors and / or expand treatment options for cancer patients and other subjects. The present invention provides such compositions and methods. Summary of the Invention

[0004] The present invention generally relates to a linker having the formula: [ka] and wherein the immunoconjugate comprises an antibody covalently linked to one or more azabenzazepine TLR (toll-like receptor) agonist moieties having the formula: In the formula, Z 1 , Z 2 , Z 3 , and Z 4 wherein one or two of the substituents are N and one of the substituents is attached to a linker. The various substituents are defined herein.

[0005] Another aspect of the invention is a method for preparing an immunoconjugate by conjugation of one or more azabenzazepine-linker compounds with an antibody.

[0006] Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate comprising an antibody covalently attached by a linker to one or more azabenzazepine moieties, and one or more pharmaceutically acceptable diluents, vehicles, carriers, or excipients.

[0007] Another aspect of the present invention is an azabenzazepine-linker compound.

[0008] Another aspect of the invention is a method for treating cancer comprising administering a therapeutically effective amount of an immunoconjugate comprising an antibody covalently attached by a linker to one or more azabenzazepine moieties.

[0009] Another aspect of the invention is the use of an immunoconjugate comprising an antibody covalently attached by a linker to one or more azabenzazepine moieties in the treatment of disease, particularly cancer. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows a time course plot of the hydrolysis of the amidine group of benzazepine comparative compound CBz-3 to form lactam comparative compound CBz-5 in PBS buffer at 40° C. [Figure 2A] 1 shows a plot of the hydrolysis of the amidine group of the benzazepine comparison compound CBz-1 and the azabenzazepine compounds azaBa-1 and azaBz-2, as a percentage of the starting compound remaining over two days. [Figure 2B] 1 shows a plot of the hydrolysis of the amidine group of the benzazepine comparison compound CBz-1 and the azabenzazepine compounds azaBa-1 and azaBz-2 with the appearance of the corresponding lactam compound over a two day period. [Figure 3A] 1 shows a plot of the hydrolysis of the amidine group of benzazepine comparison compounds CBz-4 and CBz-6 and azabenzazepine compounds azaBa-1 and azaBz-5, as a percentage of the starting compound remaining over two days. [Figure 3B]1 shows a plot of the hydrolysis of the amidine group of benzazepine comparison compounds CBz-4 and CBz-6 and azabenzazepine compounds azaBa-1 and azaBz-5 with the appearance of the corresponding lactam compounds over a two day period. [Figure 4] Figure 1 shows a plot of the hydrolysis of the amidine group of azabenzazepine compounds azaBa-3, azaBz-5, azaBz-6, azaBz-7, and azaBz-8 in PBS and formulation buffer, with the appearance of the corresponding lactam compound over a 2-day period. To facilitate rate comparisons, the amount of lactam is normalized to the starting time (t0) for each sample. [Figure 5] Figure 1 shows a plot of the hydrolysis of the amidine group of benzazepine comparison compounds CBz-2 and CBz-7 and azabenzazepine compounds azaBa-6 and azaBz-8 in PBS, showing the appearance of the corresponding lactam compound over a two-day period. To facilitate rate comparisons, the amount of lactam is normalized to the starting time (t0) for each sample. DETAILED DESCRIPTION OF THE INVENTION

[0011] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0012] 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 present invention, and the present invention is in no way limited to the methods and materials described.

[0013] definition The term "immunoconjugate" or "immunostimulatory antibody conjugate" refers to an antibody construct covalently attached via a linker to an adjuvant moiety. The term "adjuvant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant.

[0014] "Adjuvant moiety" refers to an adjuvant that is covalently attached to an antibody construct, e.g., via a linker, as described herein. The adjuvant moiety is capable of eliciting an immune response while attached to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immunoconjugate to a subject.

[0015] An "adjuvant" refers to a substance that can elicit an immune response in a subject exposed to the adjuvant.

[0016] The terms "Toll-like receptor" and "TLR" refer to any member of a family of highly conserved mammalian proteins that recognize pathogen-associated molecular patterns and function as key signaling elements in innate immunity. They are single-pass transmembrane receptors typically expressed in sentinel cells, such as macrophages and dendritic cells, that recognize structurally conserved molecules derived from microorganisms. When these microorganisms reach physical barriers, such as the skin or intestinal mucosa, they are recognized by TLRs, activating immune cell responses. TLR polypeptides share a characteristic structure, including an extracellular domain containing leucine-rich repeats, a transmembrane domain, and an intracellular domain involved in TLR signaling. The terms "Toll-like receptor 7" and "TLR7" refer to nucleic acids or polypeptides that share at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with a publicly available TLR7 sequence (e.g., GenBank Accession No. AAZ99026 for human TLR7 polypeptide or GenBank Accession No. AAK62676 for mouse TLR7 polypeptide). The terms "Toll-like receptor 8" and "TLR8" refer to nucleic acids or polypeptides that share at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with a publicly available TLR7 sequence (e.g., GenBank Accession No. AAZ95441 for human TLR8 polypeptide or GenBank Accession No. AAK62677 for mouse TLR8 polypeptide).

[0017] A "TLR agonist" is a compound that directly or indirectly binds to a TLR (e.g., TLR7 and / or TLR8) and induces TLR signaling. A detectable difference in TLR signaling can indicate that the agonist stimulates or activates the TLR. Differences in signaling can be manifested, for example, as changes in target gene expression, changes in phosphorylation of signaling components, changes in the subcellular localization of downstream elements such as nuclear factor kappa B (NF-κB), changes in the association of certain components (e.g., IL-1 receptor-associated kinase (IRAK)) with other proteins or subcellular structures, or changes in the biochemical activity of components such as kinases (e.g., mitogen-activated protein kinases (MAPKs)).

[0018] "Antibody" refers to a polypeptide comprising an antigen-binding region (including complementarity-determining regions (CDRs)) derived from an immunoglobulin gene or a fragment thereof. The term "antibody" specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa) connected by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains include, for example, the variable domains or regions of the light and heavy chains (V and V, respectively). L and V H ), light and heavy chain constant domains or regions (C L and C H) are classified into various categories based on size and function. The N-terminus of each chain defines a variable region of approximately 100–110 amino acids, or antigen-binding domain, called the paratope, which is primarily responsible for antigen recognition. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and these heavy chains define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are large molecules of approximately 150 kDa composed of four peptide chains. IgG antibodies contain two identical class gamma heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thus forming a tetrameric quaternary structure. The two heavy chains are linked to each other and to each light chain by disulfide bonds. The resulting tetramer has two identical halves that together form a Y-shape. Both ends of the branch contain identical antigen-binding domains. Humans have four IgG subclasses (IgG1, IgG2, IgG3, and IgG4), named in order of their abundance in serum (i.e., IgG1 is the most abundant). The antigen-binding domain of an antibody is usually most important for the specificity and affinity of binding to cancer cells.

[0019] A "bispecific" antibody (bsAb) is an antibody that binds two different epitopes to cancer (Suurs FV et al (2019) Pharmacology & Therapeutics 201:103-119). Bispecific antibodies can engage immune cells to destroy tumor cells, deliver payloads to tumors, and / or block tumor signaling pathways. Antibodies that target specific antigens include bispecific or multispecific antibodies with at least one antigen-binding region that targets a specific antigen. In some embodiments, the targeting monoclonal antibody is a bispecific antibody with at least one antigen-binding region that targets tumor cells. Such antigens include, but are not limited to, mesothelin, prostate-specific membrane antigen (PSMA), HER2, TROP2, CEA, EGFR, 5T4, nectin-4, CD19, CD20, CD22, CD30, CD70, B7H3, B7H4 (also known as 08E), protein tyrosine kinase 7 (PTK7), glypican-3, RG1, fucosyl-GM1, CTLA-4, and CD44 (WO2017 / 196598).

[0020] In some embodiments, the antibody construct is an antigen-binding antibody "fragment," which comprises at least the antigen-binding region of an antibody, either alone or together with other components that together comprise the antibody construct. Many different types of antibody "fragments" are known in the art, including, for example, (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region, and (iii) a V fragment of a single arm of an antibody. L and V H(iv) Fab' fragments, which are obtained by cleavage of the disulfide bridges of the F(ab')2 fragment under mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) two domains of the Fv fragment joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (i.e., V L and V H In some embodiments, an antibody construct refers to an antibody or fusion protein comprising (i) an antigen-binding domain and (ii) an Fc domain.

[0021] Antibodies or antibody fragments can be part of larger constructs, such as conjugates or fusion constructs of antibody fragments with additional regions. For example, in some embodiments, antibody fragments can be fused to an Fc region as described herein. In other embodiments, antibody fragments (e.g., Fab or scFv) can be part of a chimeric antigen receptor or chimeric T cell receptor, e.g., by fusion to a transmembrane domain (optionally with an intervening linker or "stalk" (e.g., hinge region)) and optional intercellular signaling domains. For example, antibody fragments can be fused to the gamma and / or delta chains of a T cell receptor to provide a T cell receptor-like construct that binds to PD-L1. In yet another embodiment, the antibody fragment is part of a bispecific T cell engager (BiTE) comprising a CD1- or CD3-binding domain and a linker.

[0022] In some embodiments, the antibody construct comprises an Fc domain. In certain embodiments, the antibody construct is an antibody. In certain embodiments, the antibody construct is a fusion protein. The antigen-binding domain may be a single-chain variable fragment (scFv). A single-chain variable fragment (scFv) is a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to the V domain of an antibody light chain via a synthetic peptide and can be produced using conventional recombinant DNA technology techniques. Similarly, a disulfide-stabilized variable fragment (dsFv) can be prepared by recombinant DNA technology. The antibody construct or antigen-binding domain may comprise one or more variable regions (e.g., two variable regions) of the antigen-binding domain of an anti-CEA antibody, each variable region comprising CDR1, CDR2, and CDR3.

[0023] A "cysteine ​​mutant antibody" is an antibody in which one or more amino acid residues of the antibody have been replaced with cysteine ​​residues. Cysteine ​​mutant antibodies can be prepared from a parent antibody by antibody engineering methods (Junutula, et al., (2008b) Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US7521541; US7723485; US2012 / 0121615; WO2009 / 052249). The cysteine ​​residues provide site-specific conjugation of adjuvants, such as TLR agonists, to the antibody via reactive cysteine ​​thiol groups at the engineered cysteine ​​sites without interfering with immunoglobulin folding and assembly or altering antigen binding and effector function. Cysteine ​​mutated antibodies can be conjugated to TLR agonist-linker compounds with uniform stoichiometry of the immunoconjugate (e.g., up to two TLR agonist moieties per antibody in an antibody with a single engineered mutated cysteine ​​site). The TLR agonist-linker compounds have reactive electrophilic groups that react specifically with the free cysteine ​​thiol groups of the cysteine ​​mutated antibodies.

[0024] "Epitope" refers to any antigenic or epitopic determinant of an antigen to which an antigen-binding domain binds (i.e., at the paratope of the antigen-binding domain). Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0025] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. There are three major classes of Fc receptors: (1) FcγR, which binds IgG; (2) FcαR, which binds IgA; and (3) FcεR, which binds IgE. The FcγR family includes several members, including FcγI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), and FcγRIIIB (CD16B). Fcγ receptors have different affinities for IgG and for IgG subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0026] The "identity" of a nucleic acid or amino acid sequence referred to herein can be determined by comparing a subject nucleic acid or amino acid sequence with a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., identical) between optimally aligned subject and reference sequences, divided by the length of the longest sequence (i.e., the length of either the subject or reference sequence, whichever is longer). Sequence alignment and percent identity calculations can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffe, and ALIGN (for nucleic acid and amino acid sequence alignment), BLAST programs (e.g., BLAST2.1, BL2SEQ, BLASTp, BLASTn, etc.), and FASTA programs (e.g., FASTA3x, FASTM, and SRESEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms are also described, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. Usa, 106(10):3770-3775 (2009); Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7):951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997). The percent sequence identity (%) is calculated, for example, as 100 × [(identical positions) / min(TG A ,TG B)], where TG A and T.G. B , T.G. A and T.G. B is the sum of the number of residues and internal gap positions of peptide sequences A and B in the alignment that minimizes (see, for example, Russell et al., J. Mol. Biol., 244:332-350 (1994)).

[0027] An "antibody construct" or "binding agent" comprises Ig heavy and light chain variable region polypeptides that together form an antigen-binding site. Each of the heavy and light chain variable regions is a polypeptide comprising three complementarity-determining regions (CDR1, CDR2, and CDR3) connected by framework regions. An antibody construct can be any of the various types of binding agents known in the art that comprise Ig heavy and light chains. For example, the binding agent can be an antibody, an antigen-binding antibody "fragment," or a T-cell receptor.

[0028] "Biosimilar" refers to an approved antibody construct that has similar activity profiles to previously approved PD-L1-targeting antibody constructs, such as atezolizumab (TECENTRIQ™, Genentech, Inc.), durvalumab (IMFINZI™, AstraZeneca), and avelumab (BAVENCIO™, EMDSerono, Pfizer); previously approved HER2-targeting antibody constructs, such as trastuzumab (HERCEPTIN™, Genentech, Inc.) and pertuzumab (PERJETA™, Genentech, Inc.); or a CEA-targeting antibody, such as labetuzumab (CEA-CIDE™, MN-14, hMN14, Immunomedics) CAS Registry Number 219649-07-7).

[0029] "Biobetter" refers to an approved antibody construct that is an improvement over a previously approved antibody construct, such as atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, and labetuzumab. A biobetter can have one or more modifications (e.g., an altered glycan profile, or a unique epitope) relative to the previously approved antibody construct.

[0030] "Amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally occurring α-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers. A "stereoisomer" of a given amino acid refers to an isomer that has the same molecular formula and intramolecular bond(s) but differs in the three-dimensional arrangement of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid). Amino acids can be glycosylated (e.g., N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan, or glypication) or deglycosylated. Amino acids may be represented herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0031] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0032] Naturally occurring amino acids include those formed in proteins by post-translational modifications, such as citrulline (Cit).

[0033] Non-natural (non-naturally occurring) amino acids include, but are not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in the L- or D-configuration that function similarly to naturally occurring amino acids. For example, an "amino acid analog" can be a non-natural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., a carbon bonded to a hydrogen, a carboxyl group, and an amino group) but has a modified side group or peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0034] "Linker" refers to a bifunctional or polyfunctional moiety that covalently joins two or more moieties, such as an adjuvant moiety and an antibody of an immunoconjugate. Useful linkages for connecting the linking moiety, the adjuvant moiety, to the antibody include, but are not limited to, amide, amine, ester, carbamate, disulfide, urea, thioether, thiocarbamate, thiocarbonate, and thiourea.

[0035] A "linking moiety" refers to a functional group that covalently bonds two or more moieties in a compound or material. For example, a linking moiety can serve to covalently attach an adjuvant moiety to an antibody of an immunoconjugate. Useful bonds for connecting linking moieties to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

[0036] "Divalent" refers to a chemical moiety that contains two points of attachment for linking two functional groups. A polyvalent linking moiety can have additional points of attachment for linking additional functional groups. A divalent radical can be indicated by the suffix "diyl." For example, divalent linking moieties include divalent polymer moieties such as divalent poly(ethylene glycol), divalent cycloalkyl, divalent heterocycloalkyl, divalent aryl, and divalent heteroaryl groups. A "divalent cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group" refers to a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group that has two points of attachment for covalently linking two moieties in a molecule or material. The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be substituted or unsubstituted. The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, alkoxy, etc.

[0037] Wavy line [ka] represents the point of attachment of a particular chemical moiety. [ka] When present, it is understood that the chemical moiety can be used in both ways, that is, reading from left to right or right to left. In some embodiments, when two wavy lines are present, [ka] are considered to be used as read from left to right.

[0038] "Alkyl" refers to a straight-chain (linear) or branched saturated aliphatic radical having the number of carbon atoms indicated. Alkyl can contain any number of carbons, for example, from 1 to 12. Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), and 2-methyl-3-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3). Butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2C H2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2 Examples of alkyl groups include, but are not limited to, 2,3-dimethyl-2-butyl (-C(CH)CH(CH)), 2,3-dimethyl-2-butyl (-C(CH)CH(CH)), 3,3-dimethyl-2-butyl (-CH(CH)C(CH), 1-heptyl, 1-octyl, and the like. An alkyl group can be substituted or unsubstituted. A "substituted alkyl group" can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0039] The term "alkyldiyl" refers to a divalent alkyl radical. Examples of alkyldiyl groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and the like. An alkyldiyl group may also be referred to as an "alkylene" group.

[0040] "Alkenyl" refers to a straight-chain (linear) or branched unsaturated aliphatic radical having the indicated number of carbon atoms and at least one carbon-carbon double bond, sp2. Alkenyl can contain from 2 to about 12 or more carbon atoms. Alkenyl groups are radicals having "cis" and "trans" orientations, alternatively "E" and "Z" orientations. Examples include, but are not limited to, ethylenyl or vinyl (-CH=CH), allyl (-CHCH=CH), butenyl, pentenyl, and isomers thereof. Alkenyl groups can be unsubstituted or substituted. "Substituted alkenyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0041] The terms "alkenylene" or "alkenyldiyl" refer to a straight- or branched-chain divalent hydrocarbon radical. Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (-CHCH=CH-), and the like.

[0042] "Alkynyl" refers to a straight-chain (linear) or branched unsaturated aliphatic radical having the indicated number of carbon atoms and at least one carbon-carbon triple bond, sp. Alkynyl can contain from 2 to about 12 or more carbon atoms. For example, C2-C6 alkynyl includes, but is not limited to, ethynyl (-C≡CH), propynyl (propargyl, -CH2C≡CH), butynyl, pentynyl, hexynyl, and isomers thereof. Alkynyl groups can be substituted or unsubstituted. "Substituted alkynyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0043] The terms "alkynylene" or "alkynyldiyl" refer to a divalent alkynyl radical.

[0044] "Heteroalkyl" or "heteroalkylene" refers to a monovalent straight or branched chain alkyl group, as defined above, that includes at least one heteroatom, including, but not limited to, Si, N, O, P, or S, within the alkyl chain or at the terminus of the alkyl chain. In some embodiments, the heteroatom is within the alkyl chain. In other embodiments, the heteroatom is at the terminus of the alkylene, thereby serving to attach the alkyl to the remainder of the molecule. In some embodiments, the heteroalkyl group can have 1 to 12 carbon atoms (C1-C6). 12 In some embodiments, a heteroalkyl group can have 1 to 24 carbon atoms (C-C 24 In some embodiments, a heteroalkyl group can have 1 to 40 carbon atoms (C-C 40Heteroalkyl). Unless otherwise specified herein, heteroalkyl groups are optionally substituted. For example, heteroalkyl groups can be substituted with 1 to 6 fluoro (F) substituents. For example, they can be substituted on the carbon backbone of a linear or branched heteroalkyl (as -CHF- or -CF2-) or on a terminal carbon of a linear or branched heteroalkyl (e.g., -CHF2 or -CF3). Examples of heteroalkyl groups include -CH2CH2OCH3, -CH2CH2NHCH3, -CH2CH2N(CH3)2, -C(=O)NHCH2CH2NHCH3, -C(=O)N(CH3)CH2CH2N(CH3)2, -C(=O)NHCH2CH2NHC(=O)CH2CH3, -C(=O)N(CH3)CH2CH2N(CH3)C(=O)CH2CH3, -OCH2CH2CH2NH(CH3), -OCH2CH2CH2N(CH3)2, -OCH2CH2CH2NHC(=O)CH2CH3 , -OCH2CH2CH2N(CH3)C(=O)CH2CH3, -CH2CH2CH2NH(CH3), -OCH2CH2CH2N(CH3)2, -CH2CH2CH2NHC(=O)CH2CH3, -CH2CH2CH2N(CH3)C(=O)CH2CH3, -CH2SCH2CH3, -CH2CH2S(O)CH3, -NHCH2CH2NHC(=O)CH2CH3, -CH2CH2S(O)2CH3, -CH2CH2OCF3, and -Si(CH3)3. Up to two consecutive heteroatoms may be present, for example, -CH2NHOCH3 and -CH2OSi(CH3)3. A terminal polyethylene glycol (PEG) moiety is a type of heteroalkyl group. Exemplary heteroalkyl groups include ethylene oxide (e.g., polyethylene oxide), propylene oxide, amino acid chains (i.e., short to medium length peptides, such as those containing 1 to 15 amino acids), and alkyl chains connected through various functional groups, such as amide, disulfide, ketone, phosphonate, phosphate, sulfate, sulfone, sulfonamide, ester, ether, -S-, carbamate, urea, thiourea, anhydride, and the like, including combinations thereof.In some embodiments, the heteroalkyl group comprises a polyamino acid having 1 to 10 amino acids. In some embodiments, the heteroalkyl group comprises a polyamino acid having 1 to 5 amino acids.

[0045] Heteroalkyl groups include solubilizing units that include one or more groups of polyglycine, polysarcosine, polyethyleneoxy (PEG), and glycosides, or combinations thereof.

[0046] "Heteroalkenyl" refers to a heteroalkyl group, as defined above, that contains at least one carbon-carbon double bond. "Heteroalkynyl" refers to a heteroalkyl group, as defined above, that contains at least one carbon-carbon triple bond.

[0047] "Heteroalkyldiyl" refers to a divalent form of a heteroalkyl group, as defined above. In some embodiments, a heteroalkyldiyl group can have 1 to 12 carbon atoms (C1-C6). 12 In some embodiments, a heteroalkyldiyl group can have 1 to 24 carbon atoms (C-C 24 In some embodiments, a heteroalkyldiyl group can have 1 to 40 carbon atoms (C-C 40Heteroalkyldiyl). Examples of heteroalkyldiyl groups include -CH2CH2OCH2-, -CH2CH2OCF2-, -CH2CH2NHCH2-, -CH2OC(=O)NH-, -CH2OP(=O)(OH)OCH2-, -C(=O)NHCH2CH2NHCH2-, -C(=O)N(CH3)CH2CH2N(CH3)CH2-, -C(=O)NHCH2CH2NHC(=O)CH2CH2-, -C(=O)N(CH3)CH2CH2N(CH3)C(=O)CH2CH2-, -OCH2CH2OCH2CH2-, -OCH2CH2OCH2C(=O)-, and -OCH2CH2OCH2CH2C(=O)- , -OCH2CH2NHCH2-, -OCH2CH2N(CH3)CH2-, -OCH2CH2CH2NHCH2-, -OCH2CH2CH2N(CH3)CH2-, -OCH2CH2CH2NHC(=O)CH2CH2-, -OCH2CH2CH2N(CH3)C(=O)CH2CH2-, - CH2CH2CH2NHCH2-, -CH2CH2CH2N(CH3)CH2-, -CH2CH2CH2NHC(=O)CH2CH2-, -CH2CH2CH2N(CH3)C(=O)CH2CH2-, -CH2CH2NHC(=O)-, -CH2CH2N(CH3)CH2-, -CH2CH2N + Examples include, but are not limited to, -(CH3)2-, -NHCH2CH2(NH2)CH2-, and -NHCH2CH2(NHCH3)CH2-. A divalent polyethylene glycol (PEG) moiety having 1 to about 50 -OCH2CH2- units is a type of heteroalkyldiyl group. "Heteroalkenyldiyl" refers to the divalent form of a heteroalkenyl group. "Heteroalkynyldiyl" refers to the divalent form of a heteroalkynyl group.

[0048] The terms "carbocycle," "carbocyclyl," "carbocycle," and "cycloalkyl" refer to saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring assemblies containing 3 to 12 ring atoms, or the number of atoms indicated. Saturated monocyclic carbocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocycles include, for example, norbornane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. Carbocyclic groups are partially unsaturated and may contain one or more double or triple bonds in the ring. Representative partially unsaturated carbocyclic groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene.

[0049] The term "cycloalkyldiyl" refers to a divalent cycloalkyl radical.

[0050] "Aryl" means an aromatic ring system of 6 to 20 carbon atoms (C6-C8) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. 20 (aryl) refers to a monovalent aromatic hydrocarbon radical. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl, which has a methylene linking group. Some aryl groups, such as phenyl, naphthalene, or biphenyl, have 6 to 12 ring members. Other aryl groups, such as phenyl or naphthyl, have 6 to 10 ring members.

[0051] The term "arylene" or "aryldiyl" refers to an aryl group of 6 to 20 carbon atoms (C6-C7) derived by removing two hydrogen atoms from two carbon atoms of a parent aromatic ring system.20 ) divalent aromatic hydrocarbon radical. Some aryldiyl groups are represented by "Ar" in the exemplary structures. Aryldiyl includes bicyclic radicals containing an aromatic ring fused to a saturated ring, a partially unsaturated ring, or an aromatic carbocyclic ring. Typical aryldiyl groups include, but are not limited to, radicals derived from benzene (phenyldiyl), substituted benzene, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. Aryldiyl groups, also known as "arylenes," are optionally substituted with one or more substituents described herein.

[0052] The terms "heterocycle," "heterocyclyl," and "heterocyclic ring" are used interchangeably herein and refer to saturated or partially unsaturated (i.e., having one or more double and / or triple bonds in the ring) carbocyclic radicals of 3 to about 20 ring atoms, where at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms are C, and one or more ring atoms are optionally substituted independently with one or more substituents described below. The heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), e.g., a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950-present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. "Heterocyclyl" also includes radicals in which the heterocycle radical is fused with a saturated, partially unsaturated, or aromatic carbocyclic or heterocyclic ring.Examples of heterocyclic rings include morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidin ... Examples of heterocyclic ... Examples of spiroheterocyclyl moieties include azaspiro[2.5]octanyl and azaspiro[2.4]heptanyl. Examples of heterocyclic groups in which two ring atoms are substituted with oxo (=O) moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.

[0053] The term "heterocyclyldiyl" refers to a divalent saturated or partially unsaturated (i.e., having one or more double and / or triple bonds in the ring) carbocyclic radical of 3 to about 20 ring atoms, where at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, and the remaining ring atoms are C, and one or more ring atoms are optionally substituted independently with one or more substituents as described. Examples of 5- and 6-membered heterocyclyldiyls include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholinyldiyl, and S-dioxothiomorpholinyldiyl.

[0054] The term "heteroaryl" refers to a 5-, 6-, or 7-membered monovalent aromatic radical, including fused ring systems of 5 to 20 atoms, at least one of which is aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

[0055] The term "heteroaryldiyl" refers to a 5-, 6-, or 7-membered divalent aromatic radical, including fused ring systems of 5 to 20 atoms containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, at least one of which is aromatic. Examples of 5- and 6-membered heteroaryldiyls include pyridyldiyl, imidazolyldiyl, pyrimidinyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyldiyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl.

[0056] The heterocycle or heteroaryl group may be carbon (carbon-linked) or nitrogen (nitrogen-linked) linked, where possible. By way of example and not limitation, a carbon-linked heterocycle or heteroaryl is bonded at the 2-, 3-, 4-, 5-, or 6-position of pyridine, the 3-, 4-, 5-, or 6-position of pyridazine, the 2-, 4-, 5-, or 6-position of pyrimidine, the 2-, 3-, 5-, or 6-position of pyrazine, the 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2-, 4-, or 5-position of oxazole, imidazole, or thiazole, the 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole, the 2-, or 3-position of aziridine, the 2-, 3-, or 4-position of azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline.

[0057] By way of example, and without limitation, a nitrogen-linked heterocycle or heteroaryl is bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or β-carboline.

[0058] The terms "halo" and "halogen," by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0059] The term "carbonyl" by itself or as part of another substituent refers to C(=O) or -C(=O)-, i.e., a carbon atom double-bonded to oxygen and to two other groups in the carbonyl-containing moiety.

[0060] As used herein, the phrase "quaternary ammonium salt" refers to a tertiary amine that is quaternized with an alkyl substituent (e.g., C1-C4 alkyl such as methyl, ethyl, propyl, or butyl).

[0061] The term "chiral" refers to a molecule that has the property of not being superimposable on its mirror image partner, while the term "achiral" refers to a molecule that is superimposable on its mirror image partner.

[0062] 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.

[0063] 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," John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in various stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, and atropisomers, as well as mixtures thereof (e.g., racemic mixtures), are intended to form part of the present invention. Many organic compounds exist in optically active forms, i.e., are capable of rotating the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to indicate the sign of rotation of plane-polarized light by a compound; (-) or l means the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. 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 can occur when there is 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.

[0064] "Diastereomer" refers to a stereoisomer with two or more chiral centers 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 can separate under high-resolution analytical procedures such as electrophoresis and chromatography.

[0065] "Enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0066] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0067] The term "salt" refers to an acid or base salt of a compound disclosed herein. Illustrative examples of pharmaceutically acceptable salts are inorganic acid (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid, etc.) salts, and quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. It is understood that pharmaceutically acceptable salts are non-toxic. Pharmaceutically acceptable salts of acidic compounds disclosed herein are salts formed with bases, i.e., cationic salts such as alkali and alkaline earth metal salts such as sodium, lithium, potassium, calcium, magnesium, and the like, and ammonium salts such as ammonium, trimethylammonium, diethylammonium, and tris(hydroxymethyl)methylammonium salts. Similarly, where a basic group, such as pyridyl, forms part of the structure, acid addition salts with inorganic acids, organic carboxylic acids, and organic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid, maleic acid, and the like, are also possible. Neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to the parent form of the compound for the purposes of this disclosure.

[0068] Any compound or formula provided herein is intended to represent unlabeled forms and isotopically labeled forms (i.e., "isotopic analogs") of the compound. Isotopically labeled compounds have a structure represented by the formula provided herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, e.g., 2 H 、3 H 、11 C 、13 C 、14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P,35 S, 18 F, 36 Cl, 123 I, and 125 I. Various isotopically labeled compounds of the present invention, for example, 3 H, 13 C, and 14 Compounds incorporating a radioactive isotope, such as C. Such isotopically labeled compounds may be useful in enhancing therapeutic activity, metabolic studies, reaction kinetic studies, detection or imaging techniques (e.g., positron emission tomography (PET) or single photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays), or radiation treatment of patients.

[0069] The present disclosure also includes "deuterated analogs" of the compounds described herein, which are analogs in which 1 to n hydrogens bonded to a carbon atom are replaced with deuterium ( 2 H), where n is the number of hydrogen atoms in the molecule. Such compounds are highly resistant to metabolism and are therefore useful for extending the half-life of any compound when administered to mammals, particularly humans. See, for example, Foster, "Deuterium Isotope Effects in Studies of Drug Metabolism," Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example, by using starting materials in which one or more hydrogen atoms have been replaced by deuterium. Therapeutic compounds of the present invention labeled or substituted with deuterium may have improved DMPK (drug metabolism and pharmacokinetic) properties in terms of distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes, such as deuterium, may confer certain therapeutic advantages resulting from higher metabolic stability, such as increased in vivo half-life, reduced dose requirements, and / or improved therapeutic index. 18 F, 3 H, or 11C-labeled compounds may be useful for PET or SPECT or other imaging studies. Isotopically labeled compounds of the present invention and their prodrugs can generally be prepared by carrying out the procedures disclosed in the following schemes or examples and preparations by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents. It is understood that deuterium in this context is considered a substituent in the compounds described herein. The concentration of such heavier isotopes, specifically deuterium, can be defined by an isotopic enrichment factor. In the compounds of the present disclosure, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise specified, when a position is specifically designated as "H" or "hydrogen," the position is understood to have hydrogen at its natural abundance isotopic composition. Thus, in the compounds of the present disclosure, any atom specifically designated as deuterium (D) is intended to represent deuterium.

[0070] The terms "treat," "treatment," and "treating" refer to any indication of success in treating or ameliorating an injury, condition, state (e.g., cancer) or symptom (e.g., cognitive impairment), and include any objective or subjective parameter, such as remission, remission, reduction of symptoms, or making the symptom, injury, condition, or symptom more tolerable to the patient, reducing the rate of progression of the symptom, reducing the frequency or duration of the symptom or condition, or, in some circumstances, preventing the onset of the symptom. Treatment or amelioration of the symptom can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0071] The terms "cancer," "neoplasm," and "tumor" are used herein to refer to cells exhibiting autonomous, uncontrolled growth, such as those exhibiting an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Cells subject to detection, analysis, and / or treatment in the context of the present invention include cancer cells (e.g., cancer cells from an individual with cancer), malignant cancer cells, premetastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. Cancers of virtually all tissues are known. The phrase "cancer burden" refers to the amount or volume of cancer cells in a subject. Thus, reducing cancer burden refers to reducing the number or volume of cancer cells in a subject. As used herein, the term "cancer cell" refers to any cell that is a cancer cell (e.g., derived from any cancer for which an individual may be treated, e.g., isolated from an individual with cancer) or any cell derived from a cancer cell (e.g., a clone of a cancer cell). For example, a cancer cell may be from an established cancer cell line, a primary cell isolated from an individual with cancer, a progeny cell from a primary cell isolated from an individual with cancer, etc. In some embodiments, the term can also refer to a portion of a cancer cell, such as an intracellular portion of the cancer cell, a cell membrane portion, or a cell lysate. Many types of cancer are known to those skilled in the art, including solid tumors such as carcinoma, sarcoma, glioblastoma, melanoma, lymphoma, and myeloma, as well as circulating cancers such as leukemia.

[0072] As used herein, the term "cancer" includes any form of cancer, including, but not limited to, solid tumor cancers (e.g., skin, lung, prostate, breast, stomach, bladder, colon, ovary, pancreas, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head and neck squamous cell carcinoma, melanoma, and neuroendocrine), including minimal residual disease, and including both primary and metastatic tumors; and liquid cancers (e.g., blood cancers); carcinoma; soft tissue tumor; sarcoma; teratoma; melanoma; leukemia; lymphoma; and brain tumor.

[0073] "PD-L1 expression" refers to cells that have PD-L1 receptors on their surface. As used herein, "PD-L1 overexpression" refers to cells that have more PD-L1 receptors than corresponding non-cancerous cells.

[0074] "HER2" refers to the protein human epidermal growth factor receptor 2.

[0075] "HER2 expression" refers to a cell that has HER2 receptors on its surface. For example, a cell may have about 20,000 to about 50,000 HER2 receptors on its surface. As used herein, "HER2 overexpression" refers to a cell that has more than about 50,000 HER2 receptors. For example, the cell has 2, 5, 10, 100, 1,000, 10,000, 100,000, or 1,000,000 times the number of HER2 receptors compared to a corresponding non-cancerous cell (e.g., about 1 or 2 million HER2 receptors). HER2 is estimated to be overexpressed in about 25% to about 30% of breast cancers.

[0076] The "pathology" of cancer includes all phenomena that compromise the patient's well-being, including, but not limited to, abnormal or uncontrolled cell proliferation, metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretions, suppressed or exacerbated inflammatory or immune responses, neoplasia, premalignant tumors, malignant tumors, and invasion of surrounding or distant tissues or organs, such as lymph nodes.

[0077] As used herein, the phrases "cancer recurrence" and "tumor recurrence," as well as grammatical variations thereof, refer to the further growth of tumors or cancer cells after a cancer diagnosis. In particular, recurrence can occur when further cancer cell proliferation occurs in cancerous tissue. Similarly, "tumor spread" occurs when tumor cells disseminate to local or distant tissues or organs, and thus includes tumor metastasis. "Tumor invasion" occurs when tumor growth spreads locally and impairs the function of the involved tissue by compressing, destroying, or preventing normal organ function.

[0078] As used herein, the term "metastasis" refers to the growth of a cancerous tumor in an organ or body part that is not directly connected to the organ of the primary cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of undetectable amounts of cancer cells in an organ or body part that is not directly connected to the organ of the primary cancerous tumor. Metastasis can also be defined as a several-step process, including the separation of cancer cells from the primary tumor site and the migration and / or infiltration of cancer cells to other parts of the body.

[0079] The phrases "effective amount" and "therapeutically effective amount" refer to the dosage or amount of a substance, such as an immunoconjugate, that produces the therapeutic effect for which it is administered. The actual dosage will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press, London, 2012)). In the case of cancer, a therapeutically effective amount of immunoconjugate may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent and 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 immunoconjugate may inhibit the growth of and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. With respect to cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0080] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., a human) for whom diagnosis, treatment, or therapy is desired. For purposes of treatment, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain embodiments, the mammal is a human.

[0081] The phrase "synergistic adjuvant" or "synergistic combination" in the context of the present invention includes a combination of two immunomodulatory agents, such as a receptor agonist, a cytokine, and an adjuvant polypeptide, which, when combined, induce a synergistic effect on immunity compared to the administration of either alone. In particular, the immunoconjugates disclosed herein comprise synergistic combinations of the claimed adjuvants and antibody constructs. These synergistic combinations, when administered, induce a greater effect on immunity compared to, for example, when the antibody construct or adjuvant is administered in the absence of the other moiety. Furthermore, reduced amounts of the immunoconjugate can be administered (as measured by the total number of antibody constructs or the total number of adjuvants administered as part of the immunoconjugate) compared to when either the antibody construct or the adjuvant is administered alone.

[0082] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal, or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or implantation of a sustained-release device, e.g., a mini-osmotic pump, into a subject.

[0083] The terms "about" and "approximately" used herein to modify a numerical value indicate a close range surrounding that numerical value. Thus, where "X" is a value, "about X" or "approximately X" indicates a value between 0.9X and 1.1X, e.g., between 0.95X and 1.05X, or between 0.99X and 1.01X. Reference to "about X" or "approximately X" specifically indicates at least the values ​​X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" and "approximately X" are intended to teach and provide support herein for a claim limitation, e.g., "0.98X."

[0084] antibody The immunoconjugates of the present invention comprise antibodies. Included within the scope of the present embodiments are functional variants of the antibody constructs or antigen-binding domains described herein. As used herein, the term "functional variant" refers to an antibody construct having an antigen-binding domain that has substantial or significant sequence identity or similarity with the parent antibody construct or antigen-binding domain, and this functional variant retains the biological activity of the antibody construct or antigen-binding domain from which it is a variant. Functional variants include, for example, variants of the antibody constructs or antigen-binding domains described herein (parent antibody constructs or antigen-binding domains) that retain a similar, equal, or greater ability to recognize target cells than the parent antibody construct or antigen-binding domain.

[0085] With respect to an antibody construct or antigen-binding domain, a functional variant can be, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the antibody construct or antigen-binding domain.

[0086] A functional variant can, for example, comprise the amino acid sequence of a parent antibody construct or antigen-binding domain with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can comprise the amino acid sequence of a parent antibody construct or antigen-binding domain with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, resulting in an increased biological activity compared to the parent antibody construct or antigen-binding domain.

[0087] Antibodies, including immunoconjugates of the invention, comprise Fc-engineered variants. In some embodiments, the Fc region mutations that result in modulated binding to one or more Fc receptors include the following mutations: SD(S239D), SDIE(S239D / I332E), SE(S267E), SELF(S267E / L328F), SDIE(S239D / I332E), SDIEAL(S239D / I332E / A330L), GA(G236A), ALIE(A330L / I332E), GA(G236B), GA(G236C), GA(G236D), GA(G236E), GA(G236F ... ), GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R), and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E345R, E233, G237, P238, H268, P271, L328, and A330. Further Fc region modifications to modulate Fc receptor binding are described, for example, in US2016 / 0145350, US7416726, and US5624821, which are incorporated by reference in their entireties.

[0088] Antibodies comprising immunoconjugates of the invention include glycan variants, such as defucosylated, hi some embodiments, the Fc region of the binding agent is modified to have an altered glycosylation pattern of the Fc region compared to a native, unmodified Fc region.

[0089] Amino acid substitutions in the antibody construct or antigen-binding domain of the present invention are preferably conservative amino acid substitutions, which are known in the art and involve replacing one amino acid with particular physical and / or chemical properties with another amino acid with the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid (e.g., Asp or Glu) substituted for another acidic / negatively charged polar amino acid; an amino acid having a nonpolar side chain substituted for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.); a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) substituted for another basic / positively charged polar amino acid; an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.); an amino acid with a beta-branched side chain substituted for another amino acid with a beta-branched side chain (e.g., Ile, Thr, and Val); an amino acid with an aromatic side chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0090] An antibody construct or antigen-binding domain may consist essentially of the specific amino acid sequence(s) described herein, such that other components, e.g., other amino acids, do not substantially alter the biological activity of the antibody construct or antigen-binding domain functional variant.

[0091] In some embodiments, the antibody in the immunoconjugate comprises a modified Fc region, wherein the modification modulates binding of the Fc region to one or more Fc receptors.

[0092] In some embodiments, an antibody in an immunoconjugate (e.g., an antibody conjugated to at least two adjuvant moieties) comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in an Fc region that result in modulated binding (e.g., increased or decreased binding) to one or more Fc receptors (e.g., FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), and / or FcγRIIIB (CD16b)) compared to a native antibody lacking the Fc region mutations. In some embodiments, an antibody in an immunoconjugate comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in an Fc region that decrease binding of the Fc region of the antibody to FcγRIIB. In some embodiments, the antibody in the immunoconjugate comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region of the antibody that decrease binding of the antibody to FcγRIIB while maintaining the same or increasing binding to FcγRI (CD64), FcγRIIA (CD32A), and / or FcγRIIIA (CD16a) compared to a native antibody lacking the Fc region mutations. In some embodiments, the antibody in the immunoconjugate comprises one or more modifications in the Fc region that increase binding of the antibody Fc region to FcγRIIB.

[0093] In some embodiments, the modulated binding is provided by mutations in the Fc region of the antibody compared to the native Fc region of the antibody. The mutations can be in the CH2 domain, the CH3 domain, or a combination thereof. A "native Fc region" is synonymous with a "wild-type Fc region" and comprises an amino acid sequence identical to that of an Fc region found in nature or identical to that of an Fc region found in a native antibody (e.g., cetuximab). Native-sequence human Fc regions include native-sequence human IgG1 Fc regions, native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native-sequence Fc includes various Fc allotypes (Jefferis et al., (2009) mAbs, 1(4):332-338).

[0094] In some embodiments, the Fc region of the antibody of the immunoconjugate is modified to have an altered glycosylation pattern of the Fc region compared to the native, unmodified Fc region.

[0095] Human immunoglobulins are glycosylated at the Asn297 residue in the Cγ2 domain of each heavy chain. This N-linked oligosaccharide consists of a core heptasaccharide, N-acetylglucosamine 4 mannose 3 (GlcNAc4Man3). Removal of the heptasaccharide by endoglycosidases or PNGaseF is known to cause conformational changes in the antibody Fc region, potentially significantly reducing antibody binding affinity to activating FcγRs and reducing effector function. The core heptasaccharide is often modified with galactose, bisecting GlcNAc, fucose, or sialic acid, which differentially affect Fc binding to activating and inhibitory FcγRs. Furthermore, α2,6-sialylation has been shown to enhance anti-inflammatory activity in vivo, while defucosylation improves FcγRIIIa binding and leads to a 10-fold increase in antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. Thus, specific glycosylation patterns can be used to control inflammatory effector functions.

[0096] In some embodiments, the modification to alter the glycosylation pattern is a mutation, e.g., a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods of regulating immune responses with antibodies that modulate FcγR regulatory signaling are described, for example, in US7416726, US2007 / 0014795, and US2008 / 0286819, which are incorporated by reference in their entireties.

[0097] In some embodiments, the antibody of the immunoconjugate is modified to contain an engineered Fab region with a non-naturally occurring glycosylation pattern. For example, hybridomas can be engineered to secrete a defucosylated mAb, a desialylated mAb, or a deglycosylated Fc with specific mutations that allow for increased FcRγIIIa binding and effector function. In some embodiments, the antibody of the immunoconjugate is engineered to be defucosylated.

[0098] In some embodiments, the entire Fc region of an antibody in an immunoconjugate is replaced with a different Fc region, such that the Fab region of the antibody is conjugated to a non-native Fc region. For example, the Fab region of cetuximab, which normally contains an IgG1 Fc region, can be conjugated to IgG2, IgG3, IgG4, or IgA, or the Fab region of nivolumab, which normally contains an IgG4 Fc region, can be conjugated to IgG1, IgG2, IgG3, IgA1, or IgG2. In some embodiments, an Fc-modified antibody having a non-native Fc domain also contains one or more amino acid modifications, such as the S228P mutation in the IgG4 Fc, that modulate the stability of the described Fc domain. In some embodiments, an Fc-modified antibody having a non-native Fc domain also contains one or more amino acid modifications, described herein, that modulate Fc binding to FcR.

[0099] In some embodiments, modifications that modulate binding of the Fc region to an FcR do not alter binding of the Fab region of the antibody to its antigen compared to the native, unmodified antibody, hi other embodiments, modifications that modulate binding of the Fc region to an FcR also increase binding of the Fab region of the antibody to its antigen compared to the native, unmodified antibody.

[0100] In some embodiments, the antibody in the immunoconjugate comprises a modified Fc region, wherein the modification modulates binding of the Fc region to one or more Fc receptors.

[0101] In some embodiments, the Fc region is modified by including a TGFβ1 receptor or a fragment thereof capable of binding to transforming growth factor β1 (TGFβ1). For example, the receptor can be TGFβ receptor II (TGFβRII). In some embodiments, the TGFβ receptor is a human TGFβ receptor. In some embodiments, the IgG has a C-terminal fusion to the TGFβRII extracellular domain (ECD), as described in US9676863, which is incorporated herein. An "Fc linker" may be used to connect the IgG to the TGFβRII extracellular domain. The Fc linker may be a short, flexible peptide that allows proper three-dimensional folding of the molecule while maintaining binding specificity to the target. In some embodiments, the N-terminus of the TGFβ receptor is fused to the Fc of the antibody construct (with or without an Fc linker). In some embodiments, the C-terminus of the antibody construct heavy chain is fused to the TGFβ receptor (with or without an Fc linker). In some embodiments, the C-terminal lysine residue of the antibody construct heavy chain is mutated to alanine.

[0102] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0103] In some embodiments, the antibody in the immunoconjugate is a cysteine ​​engineered antibody, which provides for site-specific conjugation of adjuvants, labels, or drug moieties to the antibody via cysteine ​​substitution at sites where the engineered cysteine ​​is available for conjugation but does not disrupt immunoglobulin folding and assembly or alter antigen binding and effector function (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US7521541; US7723485; US2012 / 0121615; WO2009 / 052249). Cysteine ​​engineered antibodies can be conjugated to azabenzazepine adjuvant moieties via azabenzazepine-linker compounds with uniform stoichiometry (e.g., up to two azabenzazepine moieties per antibody in an antibody with a single engineered cysteine ​​site).

[0104] In some embodiments, cysteine ​​engineered antibodies are used to prepare immunoconjugates. The immunoconjugates may have a reactive cysteine ​​thiol residue introduced into a site on the light chain, e.g., the 149-lysine site (LC K149C), or a site on the heavy chain, e.g., the 122-serine site (HC S122C), as numbered by Kabat numbering. In other embodiments, the cysteine ​​engineered antibody has a cysteine ​​residue introduced into the 118-alanine site (EU numbering) of the heavy chain (HC A118C). This site is numbered 121 in SEQ ID NO: 1 and 114 in Kabat numbering. In other embodiments, the cysteine ​​engineered antibody has a cysteine ​​residue introduced at a site described in Bhakta, S. et al. (2013) "Engineering THIOMABs for Site-Specific Conjugation of Thiol-Reactive Linkers", Laurent Ducry (ed.), Antibody-Drug Conjugates, Methods in Molecular Biology, vol. 1045, pp. 189-203; WO2011 / 156328; US9000130.

[0105] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to PD-L1.

[0106] Programmed cell death ligand 1 (PD-L1, cluster of differentiation 274, CD274, B7-homolog 1, or B7-H1) belongs to the B7 protein superfamily and is a ligand for programmed cell death protein 1 (PD-1, PDCD1, cluster of differentiation 279, or CD279). PD-L1 can also interact with B7.1 (CD80), and such interaction is thought to inhibit T cell priming. The PD-L1 / PD-1 axis plays a major role in suppressing adaptive immune responses. More specifically, binding of PD-L1 to its receptor, PD-1, is thought to result in a signal that inhibits T cell activation and proliferation. Agents that bind to PD-L1 and prevent its ligand from binding to the PD-1 receptor can prevent this immune suppression and thus enhance immune responses as needed, for example, to treat cancer or infectious diseases. The PD-L1 / PD-1 pathway also contributes to the prevention of autoimmunity, and therefore agonistic agents against PD-L1, or agents that deliver an immunoinhibitory payload, may be useful in the treatment of autoimmune disorders.

[0107] Several antibodies targeting PD-L1, such as atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™), and avelumab (BAVENCIO™), are being developed for the treatment of cancer. Nevertheless, there remains a need for new PD-L1 antibody constructs, including agents that bind to PD-L1 with high affinity and effectively inhibit PD-L1 / PD-1 signaling, and agents that can deliver therapeutic payloads to PD-L1-expressing cells. Additionally, there is a need for novel PD-L1-binding agents for the treatment of autoimmune and infectious diseases.

[0108] Provided are methods for delivering a TLR agonist payload to a cell that expresses PD-L1, comprising administering to the cell or a mammal comprising the cell an immunoconjugate comprising an anti-PD-L1 antibody covalently attached to a linker that is covalently attached to one or more TLR agonist moieties.

[0109] Also provided are methods for enhancing, reducing or inhibiting an immune response in a mammal, and methods for treating a disease, disorder, or condition in a mammal that responds to PD-L1 inhibition, comprising administering to the mammal the PD-L1 immunoconjugate.

[0110] The present invention provides PD-L1 antibodies comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide. The PD-L1 antibodies specifically bind to PD-L1. The binding specificity of the antibodies allows for targeting of cells that express PD-L1, for example, delivery of a therapeutic payload to such cells. In some embodiments, the PD-L1 antibodies bind to human PD-L1. However, antibodies that bind to any PD-L1 fragment, homolog, or paralog are also encompassed.

[0111] In some embodiments, the PD-L1 antibody binds to PD-L1 without substantially inhibiting or preventing PD-L1 from binding to its receptor, PD-1. However, in other embodiments, the PD-L1 antibody can completely or partially block (inhibit or prevent) the binding of PD-L1 to its receptor, PD-1, and the antibody can be used to inhibit PD-L1 / PD-1 signaling (e.g., for therapeutic purposes). The antibody or antigen-binding antibody fragment can be monospecific for PD-L1, or can be bispecific or multispecific. For example, in a bivalent or multivalent antibody or antibody fragment, the binding domains can be different, targeting different epitopes of the same antigen or targeting different antigens. Methods for constructing multivalent binding constructs are known in the art. Bispecific and multispecific antibodies are known in the art. Additionally, diabodies, triabodies, or tetrabodies, which are dimers, trimers, or tetramers of polypeptide chains, can be provided, where each polypeptide chain is composed of a V H and V L V due to a peptide linker that is too short to allow pairing between LV connected to H and thereby different V H -V L Pairing between complementary domains on the polypeptide chains is driven to generate multimeric molecules with two, three, or four functional antigen-binding sites. Bis-scFv fragments, small scFv fragments with two different variable domains, can also be generated to produce bispecific bis-scFv fragments capable of binding to two different epitopes. Fab dimers (Fab2) and Fab trimers (Fab3) can be generated using genetic engineering methods to create multispecific constructs based on Fab fragments.

[0112] PD-L1 antibodies can be, or can be derived from, human, non-human, humanized, or chimeric antibodies, or corresponding antibody fragments. A "chimeric" antibody is typically an antibody or fragment thereof that comprises a human constant region and a non-human variable region. A "humanized" antibody is typically a monoclonal antibody that comprises a human antibody scaffold but contains non-human-derived amino acids or sequences in at least one CDR (e.g., one, two, three, four, five, or all six CDRs).

[0113] The PD-L1 antibody may be internalizing as described in WO2021 / 150701, which is incorporated herein by reference, or the PD-L1 antibody may be non-internalizing as described in WO2021 / 150702, which is incorporated herein by reference.

[0114] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to HER2.

[0115] Several anti-HER2 monoclonal antibodies have been approved and are in clinical development (Costa, RLB et al (2020) Breast Cancer 6 (10): 1-11).

[0116] In certain embodiments, the immunoconjugates of the invention comprise an anti-HER2 antibody, e.g., one prepared by the method of Example 201. In one embodiment of the invention, the anti-HER2 antibody of the immunoconjugates of the invention comprises a humanized anti-HER2 antibody, e.g., huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8, as described in Table 3 of US Pat. No. 5,821,337, which is specifically incorporated herein by reference. These antibodies comprise human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also known as trastuzumab and is commercially available under the trade name HERCEPTIN™ (Genentech, Inc.).

[0117] Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, HERCEPTIN®, Genentech, Inc.) is a recombinant DNA-derived IgG1κ monoclonal antibody, a humanized version of the murine anti-HER2 antibody (4D5) that selectively binds with high affinity (Kd = 5 nM) to the extracellular domain of HER2 in cell-based assays (US5677171; US5821337; US6054297; US6165464; US6339142; US6407213; US6639055; US6719971; US6800738; US7074404; Coussens et al (1985) Science, 230:1132-9; Slamon et al. (1985) Science, 230:1132-9). al(1989)Science,244:707-12, Slamon et al(2001)New Engl.J.Med.344:783-792).

[0118] In one embodiment of the invention, the antibody construct or antigen-binding domain comprises the CDR regions of trastuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises the framework regions of trastuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.

[0119] In another embodiment of the present invention, the anti-HER2 antibody of the immunoconjugate of the present invention comprises a humanized anti-HER2 antibody, e.g., humanized 2C4, as described in U.S. Patent No. 7,862,817. An exemplary humanized 2C4 antibody is pertuzumab (CAS Registry Number 380610-27-5), PERJETA™ (Genentech, Inc.). Pertuzumab is a HER dimerization inhibitor (HDI) that functions by inhibiting the ability of HER2 to form active heterodimers or homodimers with other HER receptors (e.g., EGFR / HER1, HER2, HER3, and HER4). See, e.g., Harari and Yarden, Oncogene 19:6102-14 (2000); Yarden and Sliwkowski, Nat Rev Mol Cell Biol 2:127-37 (2001); Sliwkowski Nat Struct Biol 10:158-9 (2003); Cho et al. Nature 421:756-60 (2003); and Malik et al. Pro Am Soc Cancer Res 44:176-7 (2003). PERJETA™ is approved for the treatment of breast cancer.

[0120] In one embodiment of the invention, the antibody construct or antigen-binding domain comprises the CDR regions of pertuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises the framework regions of pertuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises one or both variable regions of pertuzumab.

[0121] Margetuximab (MGAH22, MARGENZA™, MacroGenics, Inc.), CAS Registry Number 1350624-75-7, is an FDA-approved anti-HER2 monoclonal antibody. The Fc region of margetuximab has been optimized to increase binding to activating FcγRs but decrease binding to inhibitory FcγRs on immune effector cells (Nordstrom, JL, et al. (2011) Breast Cancer Res. 13(6):R123; Rugo, HS, et al. (2021) JAMA Oncol.; 7(4):573-584; Markham, A. (2021) Drugs 81:599-604). Margetuximab is approved by the FDA for the treatment of patients with recurrent or refractory advanced breast cancer whose tumors express HER2 at a 2+ level by immunohistochemistry and lack HER2 gene amplification by FISH.

[0122] HT-19 is another anti-HER2 monoclonal antibody that binds to a different epitope on human HER2 than that of trastuzumab or pertuzumab. HT-19 has been shown to inhibit HER2 signaling as effectively as trastuzumab and enhance HER2 degradation in combination with trastuzumab and pertuzumab. XMT-1522 is an antibody-drug conjugate containing the HT-19 antibody (Bergstrom DA et al., (2015) Cancer Res.; 75:LB-231).

[0123] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to CEA. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), also known as CD66e (cluster of differentiation 66e), is a member of the carcinoembryonic antigen (CEA) gene family.

[0124] Increased expression of carcinoembryonic antigens (CEA, CD66e, CEACAM5) is associated with various biological aspects of tumors, particularly tumor cell adhesion, metastasis, blockade of cellular immune mechanisms, and anti-apoptotic functions. CEA is also used as a blood marker for many carcinomas. Labetuzumab (CEA-CIDE™, Immunomedics, CAS Registry Number 219649-07-7), also known as MN-14 and hMN14, is a humanized IgG1 monoclonal antibody that is being investigated for the treatment of colorectal cancer (Blumenthal, R. et al. (2005) Cancer Immunology Immunotherapy 54(4):315-327). Labetuzumab conjugated to a camptothecin analog (labetuzumab govitecan, IMMU-130) targets carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and has been studied in patients with recurrent or refractory metastatic colorectal cancer (Sharkey, R. et al. (2018) Molecular Cancer Therapeutics 17(1):196-203; Cardillo, T. et al. (2018) Molecular Cancer Therapeutics 17(1):150-160). In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hMN-14 / labetuzumab, as disclosed in US6676924 (hereby incorporated by reference for this purpose).

[0125] In one embodiment of the present invention, the heavy chain (HC) of the CEA-targeting antibody is selected from SEQ ID NOs: 1-5. [Table 1]

[0126] In one embodiment of the present invention, the light chain (LC) of the CEA-targeting antibody is selected from SEQ ID NOs: 6-9. [Table 2]

[0127] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to TROP2. Tumor-associated calcium signal transduction pathway 2 (TROP2) is a transmembrane glycoprotein encoded by the TACSTD2 gene (Linnenbach AJ, et al (1993) Mol Cell Biol. 13(3):1507-15; Calabrese G, et al (2001) Cytogenet Cell Genet. 92(1-2):164-5). TROP2 is an intracellular calcium signal transduction pathway that is differentially expressed in many cancers and signals cells for self-renewal, proliferation, invasion, and survival. TROP2 is considered a stem cell marker and is expressed in many normal tissues. However, in contrast, it is overexpressed in many cancers (Ohmachi T, et al., (2006) Clin. Cancer Res., 12(10), 3057-3063; Muhlmann G, et al., (2009) J. Clin. Pathol., 62(2), 152-158; Fong D, et al., (2008) Br. J. Cancer, 99(8), 1290-1295; Fong D, et al., (2008) Mod. Pathol., 21(2), 186-191; Ning S, et al., (2013) Neurol. Sci., 34(10), 1745-1750). TROP2 overexpression is important for prognosis. Several ligands that interact with TROP2 have been proposed. TROP2 signals to cells through different pathways, which are transcriptionally regulated by a complex network of several transcription factors.

[0128] Human TROP2 (TACSTD2: tumor-associated calcium signaling substrate 2, GA733-1, EGP-1, M1S1; hereafter referred to as hTROP2) is a 323-amino acid single-spanning type 1 plasma membrane protein. The existence of a plasma membrane protein common to human trophoblasts and cancer cells has previously been suggested to be involved in immune resistance (Faulk WP, et al., Proc. Natl. Acad. Sci. 75(4):1947-1951(1978)). Furthermore, an antigenic molecule recognized by a monoclonal antibody against the plasma membrane protein in a human choriocarcinoma cell line was identified and named TROP2 as one of the molecules expressed in human trophoblasts (Lipinski M, et al., Proc. Natl. Acad. Sci. 78(8):5147-5150(1981)). This molecule was also named tumor antigen GA733-1, recognized by the mouse monoclonal antibody GA733 (Linnenbach AJ, et al., Proc. Natl. Acad. Sci. 86(1), 27-31 (1989)), obtained by immunization with gastric cancer cell lines, and epithelial glycoprotein (EGP-1; Basu A, et al., Int. J. Cancer, 62(4), 472-479 (1995)), recognized by the mouse monoclonal antibody RS7-3G11 obtained by immunization with non-small cell lung cancer cells. However, in 1995, the cloning of the TROP2 gene confirmed that these molecules were all identical (Fornaro M, et al., Int. J. Cancer, 62(5), 610-618 (1995)). The DNA and amino acid sequences of hTROP2 are available in public databases and can be found, for example, under accession numbers NM_002353 and NP_002344 (NCBI).

[0129] Based on this information suggesting a link with cancer, several anti-hTROP2 antibodies have been established and their antitumor effects have been investigated. Among these antibodies, for example, unconjugated antibodies that exhibit antitumor activity by themselves in nude mouse xenograft models (WO2008 / 144891; WO2011 / 145744; WO2011 / 155579; WO2013 / 077458) and antibodies that exhibit antitumor activity as ADCs with cytotoxic drugs (WO2003 / 074566; WO2011 / 068845; WO2013 / 068946; US7999083) have been disclosed. However, the potency or scope of their activity remains insufficient, and hTROP2 remains a therapeutic target with unmet medical needs.

[0130] TROP2 expression in cancer cells correlates with drug resistance. Several strategies have been developed to target TROP2 on cancer cells, including antibodies, antibody fusion proteins, chemical inhibitors, and nanoparticles. In vitro and preclinical studies using these various therapeutic treatments have significantly suppressed tumor cell growth both in vitro and in vivo in mice. Clinical studies are exploring the potential application of TROP2 both as a prognostic biomarker and as a therapeutic target for reversing resistance.

[0131] Sacituzumab govitecan (TRODELVY®, Immunomedics, IMMU-132), an antibody-drug conjugate comprising a TROP2-directed antibody linked to a topoisomerase inhibitor, is indicated for the treatment of metastatic triple-negative breast cancer (mTNBC) in adult patients who have received at least two prior therapies. The TROP2 antibody in sacituzumab govitecan is conjugated to SN-38, an active metabolite of irinotecan (US 2016 / 0297890; WO 2015 / 098099).

[0132] In one embodiment of the present invention, the TROP2-targeting antibody construct or antigen-binding domain comprises the light chain CDRs (complementarity-determining regions) of hRS7 (humanized RS7) (US7238785, incorporated herein by reference).

[0133] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to Caprin-1 (Ellis JA, Luzio JP (1995) J Biol Chem. 270(35):20717-23; Wang B, et al (2005) J Immunol. 175(7):4274-82; Solomon S, et al (2007) Mol Cell Biol. 27(6):2324-42). Caprin-1 is also known as GPIAP1, GPIP137, GRIP137, M11S1, RNG105, p137GPI, and cell cycle-associated protein 1.

[0134] Cytoplasmic activation / proliferation-associated protein-1 (Caprin-1) is an RNA-binding protein involved in the regulation of cell cycle control-related genes. Caprin-1 selectively binds to c-Myc and cyclin D2 mRNA, accelerating cell progression from G1 to S phase, increasing cell survival, and promoting cell proliferation. This suggests that Caprin-1 may play an important role in tumorigenesis (Wang B, et al. (2005) J. Immunol. 175:4274-4282). Caprin-1 acts alone or in combination with other RNA-binding proteins, such as RasGAP SH3 domain-binding protein 1 and fragile X mental retardation protein. During tumorigenesis, Caprin-1 primarily functions by activating cell proliferation and upregulating the expression of immune checkpoint proteins. Through the formation of stress granules, Caprin-1 is also involved in the adaptation of tumor cells to adverse conditions, which contributes to radiation and chemotherapy resistance. Given its role in various clinical malignancies, caprin-1 may be used as a biomarker and a target for the development of novel therapeutic agents (Yang, ZS, et al (2019) Oncology Letters 18:15-21).

[0135] Antibodies targeting Caprin-1 for treatment and detection have been described (WO2011 / 096519; WO2013 / 125654; ​​WO2013 / 125636; WO2013 / 125640; WO2013 / 125630; WO2013 / 018889; WO2013 / 018891; WO2013 / 018883; WO2013 / 018892; WO2014 / 014082; WO2014 / 014086; WO2015 / 020212; WO2018 / 079740).

[0136] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to claudin-1.

[0137] Claudin 1 is a member of the claudin family of transmembrane proteins located in tight junctions between cells and acts as a coreceptor for HCV entry into hepatocytes (Kniesel U, et al (2000). Cell. Mol. Neurobiol. 20(1):57-76; Furuse M, et al (1998). J. Cell Biol. 141(7):1539-50; Swisshelm K, et al (2005) Adv. Drug Deliv. Rev. 57(6):919-28). Claudin 1 is also known as senescence-associated epithelial membrane protein, senescence-associated epithelial membrane protein 1, CLDN1, CLD1, ILVASC, and SEMP1.

[0138] Claudins are abundant in luminal epithelial sheets, where they maintain epithelial cell polarity. Claudin 1 is expressed in most tissues, including the bladder, fallopian tube, liver, pancreas, prostate, and skin.

[0139] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to Nectin-4.

[0140] Nectins are a family of cell adhesion molecules involved in calcium-dependent cell adhesion (Takai Y. et al. (2003) Cancer Science 94(8):655-67; ​​Fuchs, A. et al. (2006) Seminars in Cancer Biology 16(5):359-366; Miyoshi J. et al. (2007) American Journal of Nephrology 27(6):590-604). Nectins play an important role in cell-cell junctions in many different tissues, including intermediate junctions in epithelial cells and chemical synapses in neurons.

[0141] antibody target In some embodiments, the antibody of the immunoconjugate is 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, aromatase, ATX, AX1, AZGP1 (zinc-α-glycoprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAG1, BAI1, B CR, BCL2, BCL6, BDNF, BLNK, BLR1 (MDR15), BIyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMPRTA, BMPR1B, BMPR2, BPAG1 (plectin), BRCA1, C19orflO (IL27w), C3, C4A, C5, C5R1, CANT1, CAPRIN-1, CASP1, CASP4, CAV1, CCBP2(D6 / JAB61), CCLI(1-309), CCLI1(eotaxin), CCL13(MCP-4), CCL15(MIP-Id), C CL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC, Exodus-2, CCL22 (MDC / STC-1), CC L23(MPIF-I), CCL24(MPIF-2 / eotaxin-2), CCL25(TECK), CCL26(eotaxin-3), CCL27(CTACK / ILC), CCL28, CCL3(MIP-Ia), CCL4(MIPIb), CCL5(RANTES), C CL7(MCP-3), CCL8(mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1(CKR1 / HM145), CCR2(mcp-IRB / RA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / Che mR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBI1), CCR8(CMKBR8 / TERI / CKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1), CCRL2(L-CCR), CD164,CD19、CDIC、CD2、CD20、CD21、CD200、CD-22、CD24、CD27、CD28、CD3、CD33、CD35、CD37、CD38、CD3E、CD3G、CD3Z、CD4、CD38、CD40、CD40L、CD44、CD45RB、CD47、CD52、CD69、CD72、CD74、CD79A、CD79B、CD8、CD80、CD81、CD83、CD86、CD137、CD152、CD274、CDH1(Eカドヘリン)、CDH1O、CDH12、CDH13、CDH18、CDH19、CDH2O、CDH5、CDH7、CDH8、CDH9、CDK2、CDK3、CDK4、CDK5、CDK6、CDK7、CDK9、CDKN1A(p21Wap1 / Cip1)、CDKN1B(p27Kip1)、CDKN1C、CDKN2A(p16INK4a)、CDKN2B、CDKN2C、CDKN3、CEBPB、CERI、CHGA、CHGB、キチナーゼ、CHST1O、CKLFSF2、CKLFSF3、CKLFSF4、CKLFSF5、CKLFSF6、CKLFSF7、CKLFSF8、CLDN3、CLDN7(クローディン-7)、CLDN18.2(クローディン18.2)、CLN3、CLU(クラステリン)、CMKLR1、CMKOR1(RDC1)、CNR1、COL18A1、COLIA1、COL4A3、COL6A1、CR2、Cripto、CRP、CSF1(M-CSF)、CSF2(GM-CSF)、CSF3(GCSF)、CTL8、CTNNB1(b-カテニン)、CTSB(カテプシンB)、CX3CL1(SCYD1)、CX3CR1(V28)、CXCL1(GRO1)、CXCL1O(IP-IO)、CXCLI1(1-TAC / IP-9)、CXCL12(SDF1)、CXCL13、CXCL14、CXCL16、CXCL2(GRO2)、CXCL3(GRO3)、CXCL5(ENA-78 / LIX)、CXCL6(GCP-2)、CXCL9(MIG)、CXCR3(GPR9 / CKR-L2)、CXCR4、CXCR6(TYMSTR / STRL33 / Bonzo)、CYB5、CYC1、CYSLTR1、DAB2IP、DES、DKFZp451J0118、DNCL1、DPP4、E2F1、Engel、Edge、Fennel、EFNA3、EFNB2、EGF、EGFR、ELAC2、ENG, Enola, ENO2, ENO3, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPRA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN- A1, EPHRIN-A2, EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERBB2(Her-2), EREG, ERK8, estrogen receptor, Earl, ESR2, F3(TF), FADD, farnesyltransferase, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1(aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF20, FGF21, FGF22, FGF23, FGF3(int-2), FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR3, FIGF( VEGFD), FILI (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (fibronectin), FLT1, FLT-3, FOS, FOSL1 (FRA-1), FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GDF5, GFI1, GGT1, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (Gelsoli) ), GSTP1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, hedgehog, HGF, HIF1A, HIP1, histamine and histamine receptor, HLA-A, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-α, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFNγ, IFNW1, IGBP1, IGF1, IGFIR, IGF2, IGFBP2, IGFBP3, IGFBP6,DL-1, ILIO, ILIORA, ILIORB, IL-1, IL1R1 (CD121a), IL1R2 (CD121b), IL-IRA, IL-2, IL2RA (CD25), IL2RB (CD122), IL2RG (CD132), IL-4, IL-4R (CD123), IL-5, IL5RA (CD125), IL3RB (CD131), IL-6, IL6RA, (CD126), IR6RB (CD130), IL-7, IL7RA (CD127), IL-8, CXCR1 (IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R (CD129), IL-10, IL10RA (CD210), IL10RB (CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHYI, ILIR1, ILIR2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, IL4, IL6ST (Glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α6 integrin), ITGAV, ITGB3, ITGB4 (β4 integrin), JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (Keratin 19), KRT2A, KRTHB6 (Hair-specific type II keratin), LAMA5, LEP (Leptin), Lingo-p75,Lingo-Troy, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​MACMARCKS, MAG or OMgp, MAP2K7 (c-Jun), MCP-1, MDK, MIB1, midkine, MIF, MISRII, MJP-2, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionein-UI), mTOR, MTSS1, MUC1 (mucin), MYC, MYD88, NCK2, neurocan, nectin-4, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCH1, NOX5, NPPB, NROB1, NROB2, NRID1, NR1D2, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZI, OPRDI, P2RX7, PAP, PART1, PATE, PAWR, PCA3, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, PEG-asparaginase , PF4(CXCL4), PGF, PGR, phosphacan, PIAS2, PI3 kinase, PIK3CG, PLAU(uPA), PLG, PLXDCI, PKC, PKC-beta, PPBP(CXCL7), PPID, P R1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2(COX-2), PIN, RAC2(P21Rac2), RANK, RANK ligand, RARB, RGS1, RGS13, RGS3, RNFI1O (ZNF144), Ron, ROBO2, RXR, S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial monocyte-activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (maspin), SERPINEI (PAI-I), SERPINFI, SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLC2A2, SLC33A1, SLC43A1, SL IT2, SPP1, SPRR1B(Spr1), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP1O, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGEBR 1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, T LR10, TLR11, TNF, TNF-a, TNFAIP2(B94), TNFAIP3, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6(Fas), TNFRSF7, TNFRSF8, TNFRSF9, T NFSF1O(TRAIL), TNFSF11(TRANCE), TNFSF12(APO3L), TNFSF13(April), TNFSF13B, TNSF14(HVEM-L), TNFRSF14(HVEM), TNFSF15(VEGI), TNFSF18, TNFS F4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase 1ia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, tyrosinase, uPAR, VEGF, VEGFB,VEGFC, versican, VHLC5, VLA-4, Wnt-1, XCL1 (lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5 / CCXCR1), YYI, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL- 1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin-1), PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD8 5I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335 , LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, TARM1, CD300C, CD300E, CD300LB (CD 300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (C The antibody can bind to (e.g., specifically bind to) one or more targets selected from) the following: SIGLEC1 (CD169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPalpha, SIRPB1 (CD172B), TREM1 (CD354), TREM2, and KLRF1 (NKp80).

[0142] In some embodiments, the antibody binds to an FcR gamma coupled receptor, hi some embodiments, the FcR gamma coupled receptor is selected from the group consisting of GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335, LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, and TARM1.

[0143] In some embodiments, the antibody binds to a DAP12 coupled receptor, hi some embodiments, the DAP12 coupled receptor is selected from the group consisting of CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPB1 (CD172B), TREM1 (CD354), and TREM2.

[0144] In some embodiments, the antibody binds to a hemITAM-containing receptor. In some embodiments, the hemITAM-containing receptor is KLRF1 (NKp80).

[0145] In some embodiments, the antibody is capable of binding to one or more targets selected from CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), and CLEC7A (Dectin-1). In some embodiments, the antibody is capable of binding to CLEC6A (Dectin-2) or CLEC5A. In some embodiments, the antibody is capable of binding to CLEC6A (Dectin-2).

[0146] In some embodiments, the antibody is selected from the group consisting of ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (O88967), LPPRC (Q6PB66), LONM (Q8CGK3), ACON (Q99KI0), ODO1 (Q60597), IDHP (P54071), ALDH2 (P47738), ATPB (P56480), AATM (P052 02), TMM93(Q9CQW0), ERGI3(Q9CQE7), RTN4(Q99P72), CL041(Q8BQR4), ERLN2(Q8BFZ9), TERA(Q01853), DAD1(P61804), CALX(P35564), CALU(O35887), VAPA(Q9WV55), MOGS(Q80UM7), GANAB(Q8BHN3), ERO1A(Q8R180), UGGG1(Q6P5E4), P4HA1(Q60715), HYEP(Q9D379), CALR (P14211), AT2A2(O55143), PDIA4(P08003), PDIA1(P09103), PDIA3(P27773), PDIA6(Q922R8), CLH(Q68FD5), PPIB(P24369), TCPG(P80 318), MOT4(P57787), NICA(P57716), BASI(P18572), VAPA(Q9WV55), ENV2(P11370), VAT1(Q62465), 4F2(P10852), ENOA(P17182), ILK( O55222), GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH, (Q68FD5), DSG1A (Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), and CH10 (Q64433). In the preceding list, accession numbers are given in parentheses.

[0147] In some embodiments, the antibody binds to an antigen selected from CDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, EpCAM, MUC1, MUC16, EGFR, Her2, SLAMF7, and gp75. In some embodiments, the antigen is selected from CD19, CD20, CD47, EpCAM, MUC1, MUC16, EGFR, and Her2. In some embodiments, the antibody binds to an antigen selected from Tn antigen and Thomsen-Friedenreich antigen.

[0148] In some embodiments, the antibody or Fc fusion protein is selected from the group consisting of abagovomab, abatacept (also known as ORENCIA®), abciximab (also known as REOPRO®), c7E3Fab), adalimumab (also known as HUMIRA®), adecatumumab, alentuzumab (also known as CAMPATH®), MabCampath or Campath-1H), altumomab, afelimomab, anatumomab mafenatox, anetumumab, anrukizumab , apolizumab, arcitumomab, acelizumab, atlizumab, atolizumab, bapineuzumab, basiliximab (also known as SIMULECT®), bavituximab, bectumomab (also known as LYMPHSCAN®), belimumab (also known as LYMPHO-STAT-B®), bertilimumab, becilisomab, bevacizumab (also known as AVASTIN®), biciromab, bralobarbital, bivatuzumab mertansine, Campath, canakinumab (ACZ88 5), cantuzumab mertansine, capromab (also known as PROSTASCINT®), catumaxomab (also known as REMOVAB®), cedelizumab (also known as CIMZIA®), certolizumab pegol, cetuximab (also known as ERBITUX®), clenoliximab, decatuzumab, dacliximab, daclizumab (also known as ZENAPAX®), denosumab (also known as AMG162), detu Momab, dorlimomab alitox, dorlixizumab, dantumumab, durimumab, durumulumab, ecloneximab, eculizumab (also known as SOLIRIS®), edovacomab, edrecolomab (Mab17-1A, also known as PANOREX®), efalizumab (also known as RAPTIVA®), efungumab (also known as MYCOGRAB®), elsilimomab, enlimomab pegol, epitumomab situxetan, efalizumab, epitumomab,Epratuzumab, erlizumab, ertumaxomab (also known as REXOMUN®), etanercept (also known as ENBREL®), etaracizumab (also known as etaratuzumab, VITAXIN®, ABEGRIN®), exbivirumab, fanolesomab (also known as NEUTROSPEC®), faralimomab, felvizumab, fontolizumab (also known as HUZAF®), gallinirab, ximab, gantenerumab, gavilimomab (also known as ABXCBL®), gemtuzumab ozogamicin (also known as MYLOTARG®), golimumab (also known as CNTO148), golimumab, ibalizumab (also known as TNX-355), ibritumomab tiuxetan (also known as ZEVALIN®), igovomab, imciromab, infliximab (also known as REMICADE®), ino Rimomab, inotuzumab ozogamicin, ipilimumab (also known as MDX-010, MDX-101), iratumumab, keliximab, labetuzumab, remaresomab, revrilizumab, lerdelimumab, lexatumumab (also known as HGS-ETR2, ETR2-ST01), lexitumumab, ribivirumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab (also known as HGSETR1, TRM-1), maslimomab, matuzumab (also known as EMD72000) (also known as BOSATRIA®), mepolizumab (also known as BOSATRIA®), metelimu- mab, miratumumab, minletumomab, mitumomab, morolimu- mab, motavizumab (also known as NUMAX®), muromonab (also known as OKT3), nacolomab butafenatox, naptumomab estafenatox, natalizumab (also known as TYSABRI®, ANTEGREN®), nebacumab, nerelimomab, nimotuzumab (also known as THERACIM hR3®, THERA-CIM-hR3®, THERA-LIM-hR3®, THERA-LIM-hR3®),Nofetumomab merpentane (also known as VERLUMA®), ocrelizumab, odulimomab, ofatumumab, omalizumab (also known as XOLAIR®), oregovomab (also known as OVAREX®), otelixizumab, pagibaximab, palivizumab (also known as SYNAGIS®), panitumumab (also known as ABX-EGF, VECTIBIX®), pascolizumab, pemtumomab (also known as THERAGYN®) (also known as OMNITARG®), pertuzumab (2C4), pexelizumab, pintumomab, priliximab, pritumumab, ranibizumab (also known as LUCENTIS®), raxibacumab, regavirumab, reslizumab, rituximab (also known as RITUXAN®, MabTHERA®), lovelizumab, ruplizumab, satumomab, cevirumab, sibrotuzumab, siplizumab (also known as MEDI-507), sontuzumab, stamumab (also known as MYO-029), sulesomab (also known as LEUKOSCAN®), tacatuzumab tetraxetan, tadocizumab, talizumab, taplitumomab paptox, tefibazumab (also known as AUREXIS®), terimomab alitox, teneliximab, teplizumab, ticilimumab, tocilizumab (also known as ACTEMRA®), toralizumab, tositumomab, trastuzumab (also known as HERCEPTIN®), tremelimumab (also known as CP-675,206), tucotuzumab, celmoleukin, tuvilumab, urtoxazumab, ustekinumab (also known as CNTO1275), bapaliximab, veltuzumab, bepalimomab, visilizumab (also known as NUVION®), volociximab (also known as M200), votumumab (also known as HUMASPECT®), zalutumumab, zanolimumab (also known as HuMAX-CD4), zoralimumab, zolimomab-allitoxin, daratumumab,Selected from elotuxumab, obintunzumab, olaratumab, brentuximab vedotin, afibercept, abatacept, belatacept, afibercept, etanercept, romiplostim, SBT-040 (sequences listed in US2017 / 0158772). In some embodiments, the antibody is rituximab.

[0149] Immune checkpoint inhibitors In some embodiments, the antibody of the immunoconjugate is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins. In other embodiments, the immune checkpoint inhibitor reduces the interaction between one or more immune checkpoint proteins and their ligands. Inhibitory nucleic acids that reduce the expression and / or activity of immune checkpoint molecules can also be used in the methods disclosed herein.

[0150] The immune checkpoint inhibitors nivolumab and atezolizumab can be modified to contain an IgG1 Fc and subsequently converted into the immunoconjugates of the invention.

[0151] Most checkpoint antibodies are designed to block signaling rather than have cell-killing effector function. The immunoconjugates of the present invention can restore the "effector functionality" necessary to induce myeloid cell activation and a pro-inflammatory response.

[0152] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-inducible TNFR-related protein (GITR, also known as TNFRSF18), inducible T-cell costimulatory (ICOS, also known as CD278), CD96, poliovirus receptor-related 2 (PVRL2, also known as CD112R), programmed cell death protein 1 (PD-1, also known as CD279), programmed cell death 1 ligand 1 (PD-L1, also known as B7-H3 and CD274), programmed cell death ligand 2 (PD-L2, also known as B7-H3 and CD273), and the like. (also known as CD40), lymphocyte-activation gene 3 (LAG-3, also known as CD223), B7-H4, killer immunoglobulin receptor (KIR), tumor necrosis factor receptor superfamily member 4 (TNFRST4, also known as OX40 and CD134) and its ligand OX40L (CD252), indoleamine 2,3-dioxygenase 1 (IDO-1), indoleamine 2,3-dioxygenase 2 (IDO-2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), B- and T-lymphocyte attenuator factor (BTLA, also known as CD272), T-cell membrane protein 3 (TIM3), adenosine A2A receptor (A2Ar), and V-domain Ig suppressor of T-cell activation (VISTA protein). In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, or PD-L1.

[0153] In some embodiments, the antibody is selected from ipilimumab (also known as Yervoy®), pembrolizumab (also known as Keytruda®), nivolumab (also known as Opdivo®), atezolizumab (also known as Tecentriq®), avelumab (also known as Bavencio®), and durvalumab (also known as Imfinzi®).

[0154] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4. In some embodiments, the immune checkpoint inhibitor is an antibody against CTLA4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against CTLA4. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against CTLA4. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as CTLA4.

[0155] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-1. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against PD-1. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-1.

[0156] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-L1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-L1. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against PD-L1. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-L1. In some embodiments, the immune checkpoint inhibitor reduces the interaction between PD-1 and PD-L1.

[0157] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L2. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-L2. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-L2. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against PD-L2. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-L2. In some embodiments, the immune checkpoint inhibitor reduces the interaction between PD-1 and PD-L2.

[0158] In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG-3. In some embodiments, the immune checkpoint inhibitor is an antibody against LAG-3. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against LAG-3. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against LAG-3. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as LAG-3.

[0159] In some embodiments, the immune checkpoint inhibitor is an inhibitor of B7-H4. In some embodiments, the immune checkpoint inhibitor is an antibody against B7-H4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against B7-H4. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against B7-H4. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as B7-H4.

[0160] In some embodiments, the immune checkpoint inhibitor is an inhibitor of KIR. In some embodiments, the immune checkpoint inhibitor is an antibody against KIR. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against KIR. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against KIR. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as KIR.

[0161] In some embodiments, the immune checkpoint inhibitor is an inhibitor of TNFRSF4. In some embodiments, the immune checkpoint inhibitor is an antibody against TNFRSF4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against TNFRSF4. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against TNFRSF4. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as TNFRSF4.

[0162] In some embodiments, the immune checkpoint inhibitor is an inhibitor of OX40L. In some embodiments, the immune checkpoint inhibitor is an antibody against OX40L. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against OX40L. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against OX40L. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as OX40L. In some embodiments, the immune checkpoint inhibitor reduces the interaction between TNFRSF4 and OX40L. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO-1. In some embodiments, the immune checkpoint inhibitor is an antibody against IDO-1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against IDO-1, and in some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against IDO-1. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as IDO-1.

[0163] In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO-2. In some embodiments, the immune checkpoint inhibitor is an antibody against IDO-2. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against IDO-2. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against IDO-2. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as IDO-2.

[0164] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CEACAM1. In some embodiments, the immune checkpoint inhibitor is an antibody against CEACAM1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against CEACAM1. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against CEACAM1. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as CEACAM1.

[0165] In some embodiments, the immune checkpoint inhibitor is an inhibitor of BTLA. In some embodiments, the immune checkpoint inhibitor is an antibody against BTLA. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against BTLA. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against BMA. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as BTLA.

[0166] In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3. In some embodiments, the immune checkpoint inhibitor is an antibody against TIM3. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against TIM3. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against TIM3. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as TIM3.

[0167] In some embodiments, the immune checkpoint inhibitor is an inhibitor of A2Ar. In some embodiments, the immune checkpoint inhibitor is an antibody against A2Ar. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against A2Ar. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against A2Ar. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as A2Ar.

[0168] In some embodiments, the immune checkpoint inhibitor is an inhibitor of VISTA protein. In some embodiments, the immune checkpoint inhibitor is an antibody against VISTA protein. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against VISTA protein. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against VISTA protein. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as VISTA protein.

[0169] Azabenzazepine adjuvant compounds The immunoconjugates of the present invention comprise an azabenzazepine adjuvant moiety. The adjuvant moieties described herein induce an immune response (i.e., are immunostimulants). Generally, the adjuvant moieties described herein are TLR agonists. TLRs are type I transmembrane proteins involved in the initiation of innate immune responses in vertebrates. TLRs recognize various pathogen-associated molecular patterns from bacteria, viruses, and fungi and serve as a first line of defense against invading pathogens. TLRs elicit overlapping yet distinct biological responses due to differences in cellular expression and the signaling pathways they initiate. Once engaged (e.g., by natural stimulation or synthetic TLR agonists), TLRs initiate a signaling cascade that leads to the activation of nuclear factor-κB (NF-κB) via the recruitment of the adaptor proteins myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor-associated kinase (IRAK). Subsequent phosphorylation of IRAK leads to the recruitment of TNF receptor-associated factor 6 (TRAF6), which phosphorylates the NF-κB inhibitor I-κB. As a result, NF-κB enters the cell nucleus and initiates the transcription of genes whose promoters contain NF-κB binding sites, such as cytokines. Additional regulatory modes of TLR signaling include the TIR domain-containing adaptor-inducing interferon-β (TRIF)-dependent induction of TNF receptor-associated factor 6 (TRAF6) and the activation of a MyD88-independent pathway via TRIF and TRAF3, which leads to the phosphorylation of interferon response factor 3 (IRF3). Similarly, the MyD88-dependent pathway also activates several IRF family members, including IRF5 and IRF7, while the TRIF-dependent pathway also activates the NF-κB pathway.

[0170] Typically, the adjuvant moieties described herein are TLR7 and / or TLR8 agonists. Both TLR7 and TLR8 are expressed on monocytes and dendritic cells. In humans, TLR7 is also expressed on plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is primarily expressed on cells of myeloid origin, i.e., monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 can detect the presence of "foreign" single-stranded RNA within cells as a means of responding to viral invasion. Treatment of TLR8-expressing cells with a TLR8 agonist can result in the production of high levels of IL-12, IFN-γ, IL-1, TNF-α, IL-6, and other proinflammatory cytokines. Similarly, stimulation of TLR7-expressing cells, such as pDCs, with a TLR7 agonist can result in the production of high levels of IFN-α and other proinflammatory cytokines. TLR7 / TLR8 engagement and resulting cytokine production activates dendritic cells and other antigen-presenting cells, promoting a variety of innate and adaptive immune response mechanisms that lead to tumor destruction.

[0171] The amidine functional group of benzazepine adjuvant compounds, moieties conjugated to antibodies as immunoconjugates (WO2020 / 252294; WO2021 / 067242; WO2022 / 125884; WO2022 / 125891; WO2022 / 125904; WO2022 / 125908; WO2022 / 125915), was found to undergo hydrolysis to a lactam functional group. This degradative hydrolysis renders the lactam benzazepine compounds inactive as TLR7 / 8 agonists. For example, the comparative lactam compounds CBz-8 and CBz-9 (Table 1b) were inactive in the HEK assay (Example 202). [ka]

[0172] The amidine benzazepine comparative compound CBz-3 (Table 1b) decomposes in PBS buffer (pH 7.4) at 40° C. to form the lactam benzazepine comparative compound CBz-5 (Table 1b) by 90% over 17 days. Figure 1 shows a time course plot of the hydrolysis of the amidine group of CBz-3 to form CBz-5 in PBS buffer at 40° C. After 24 hours in room temperature human plasma, 15% of CBz-3 decomposes to CBz-5. [ka]

[0173] The decomposition rate can be controlled by nitrogen substitution of carbons in the six-membered benzazepine ring. Compared to benzazepine compound CBz-1, azabenzazepine compounds azaBz-1 and azaBz-2 each incorporate a single nitrogen. Amidine hydrolysis of the three compounds in PBS at 40°C was measured by the disappearance of the starting amidine and the appearance of lactam products. Figure 2A shows a plot of the hydrolysis of the amidine group in benzazepine compound CBz-1 and azabenzazepine compounds azaBa-1 and azaBz-2, as a percentage of the starting compound remaining over two days. Figure 2B shows a plot of the hydrolysis of the amidine group in CBz-1 and azabenzazepine compounds azaBa-1 and azaBz-2, as a percentage of the starting compound remaining over two days. No other decomposition products were detected.

[0174] The addition of a sulfonic acid group at the 8-position of both benzazepine and azabenzazepine compounds confers stability and retards hydrolysis. Figure 3A shows plots of the hydrolysis of the amidine group in benzazepine compounds CBz-4 and 8-sulfonic acid CBz-6 and azabenzazepine compounds azaBa-1 and 8-sulfonic acid azaBz-5, as a percentage of the starting compound remaining over two days. Figure 3B shows plots of the hydrolysis of the amidine group in benzazepine comparison compounds CBz-4 and 8-sulfonic acid CBz-6 and azabenzazepine compounds azaBa-1 and 8-sulfonic acid azaBz-5, as a percentage of the starting compound remaining over two days, as a percentage of the starting compound remaining over two days.

[0175] In azabenzazepine compounds with various substituents at the 8-position, the nitrogen at the 7-position stabilizes and retards hydrolysis. Figure 4 shows plots of the hydrolysis of the amidine group of azabenzazepine compounds azaBa-3, azaBz-5, azaBz-6, azaBz-7, and azaBz-8 in PBS and formulation buffer, showing the appearance of the corresponding lactam compound over a 2-day period. To facilitate rate comparisons, the amount of lactam for each sample is normalized to the starting time (t0). The half-life of each compound was measured at 37°C in PBS (pH 7.4) and at 22°C in formulation buffer (pH 6) as follows: [Table 3]

[0176] The hydrolytic degradation rates of benzazepine and 7-azabenzazepine compounds were directly compared at 37°C in PBS (pH 7.4) to mimic in vivo effects, and in formulation buffer to simulate storage and shelf-life effects. The half-lives (t) of benzazepine compounds CBz-2 and CBz-7 were 6 and 8 days, respectively. The half-lives (t) of azabenzazepine compounds azaBa-6 and azaBz-8 were 30 and 40 days, respectively. Figure 5 shows plots of the hydrolysis of the amidine group of benzazepine compounds CBz-2 and CBz-7 and azabenzazepine compounds azaBa-6 and azaBz-8 in PBS, showing the appearance of the corresponding lactam compounds over a 2-day period. To facilitate rate comparisons, the amount of lactam for each sample is normalized to the starting time (t). The 7-aza modification provides approximately 5-fold stabilization in PBS and formulation buffers compared to the corresponding benzazepine compound.

[0177] The exemplary azabenzazepine compound (azaBza) in Table 1a and the comparative compound (CBz) in Table 1b were synthesized, purified, characterized by mass spectrometry, and shown to have the expected masses. Additional experimental procedures are found in the Examples. Activity against HEK293 NFKB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 202. Certain azabenzazepine compounds exhibit the surprising and unexpected property of TLR8 agonist selectivity, which may predict useful therapeutic activity for treating cancer and other disorders. For example, azaBz-24 exhibited TLR7 / 8 selectivity with an EC50 of 842 nM for TLR7 and 196 nM for TLR8. Additionally, azaBz-2 showed no response to TLR7 and an EC50 of 5.5 micromolar (uM) for TLR8. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 5-1] [Table 5-2] [Table 5-3]

[0178] Azabenzazepine-Linker Compounds The immunoconjugates of the present invention are prepared by conjugation of an antibody with an azabenzazepine linker compound, azaBzL. The azabenzazepine linker compound comprises an azabenzazepine (azaBz) moiety covalently attached to a linker unit. The linker unit contains functional groups and subunits that affect the stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugate. The linker unit contains a reactive functional group that reacts with, i.e., conjugates with, a reactive functional group on an antibody. For example, a nucleophilic group, such as a lysine side chain amino group on an antibody, can react with an electrophilic reactive functional group on an azaBz-L compound to form an immunoconjugate. Alternatively, for example, a cysteine ​​thiol on an antibody can react with a maleimide group, bromoacetamide group, or disulfide group on an azaBza-L linker compound to form an immunoconjugate.

[0179] Suitable reactive electrophilic functional groups (Q in Formula II) for azaBza-L linker compounds include, but are not limited to, N-hydroxysuccinimidyl (NHS) esters and N-hydroxysulfosuccinimidyl (sulfo-NHS) esters (amine reactive); carbodiimides (amine and carboxyl reactive); hydroxymethylphosphines (amine reactive); maleimides (thiol reactive); halogenated acetamides such as N-iodoacetamide (thiol reactive); aryl azides (primary amine reactive); fluorinated aryl azides (reactive via carbon-hydrogen (CH) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) esters (amine reactive); imide esters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiol reactive); and benzophenone derivatives (reactive via C-H bond insertion). Additional reagents include, but are not limited to, those described in Hermanson, Bioconjugate Techniques 2nd Edition, Academic Press, 2008.

[0180] A linker may comprise one or more linker units or moieties. Exemplary linker moieties include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), phenylalanine-lysine (phe-lys), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("MCC"). A variety of linker moieties are known in the art, some of which are described herein.

[0181] The linker may be a "cleavable linker" that facilitates release of the drug. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive, peptidase-substrate linkers (US 7,498,298), photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); US 5,208,020).

[0182] Generally, the linker (L) may be cleavable or non-cleavable. A cleavable linker may contain a peptide sequence that is a substrate for a specific protease, such as a cathepsin, that recognizes and cleaves the peptide linker unit, separating the phenylglutarimide moiety from the antibody (Caculitan NG, et al. (2017) Cancer Res. 77(24):7027-7037).

[0183] Cleavable linkers may contain labile functional groups such as acid-sensitive disulfide groups (Kellogg, BA et al (2011) Bioconjugate Chem. 22, 717-727; Ricart, AD et al (2011) Clin. Cancer Res. 17, 6417-6427; Pillow, T., et al (2017) Chem. Sci. 8:366-370; Zhang D, et al (2016) ACS Med Chem Lett. 7(11):988-993).

[0184] In some embodiments, the linker is not cleavable under physiological conditions. As used herein, the term "physiological conditions" refers to a temperature range of 20 to 40 degrees Celsius, atmospheric pressure (i.e., 1 atmosphere), a pH of about 6 to about 8, and one or more physiological enzymes, proteases, acids, and bases. One advantage of a non-cleavable linker between the antibody and the PG moiety in an antibody conjugate is that it minimizes premature release of the phenylglutarimide moiety and the corresponding toxicity.

[0185] In some embodiments, the linker includes a trivalent branch point as part of the amino acid unit (e.g., lysine), in which case additional linker units are attached via the side chain amine of the lysine or linked to other portions of the amino acid unit (US Pat. No. 1,173,214). A similar motif can be utilized with glutamic acid in the amino acid unit. Exemplary additional linker units are monovalent solubilizing units such as one or more units of polyglycine, polysarcosine, polyethyleneoxy (PEG), and glycosides, or combinations thereof. The solubilizing units may terminate with groups such as amino acids, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or sugars (e.g., pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, etc.), or combinations thereof.

[0186] In some embodiments, the amino acid unit or peptide unit comprises one or more amino acids selected from the group consisting of glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, sarcosine, and beta-alanine.

[0187] In one embodiment, the present invention includes an amino acid or peptide linking unit, i.e., L, or linker, between the antibody and the azabenzazepine (azaBz) moiety, comprising a peptide containing a linear sequence of specific amino acid residues that can be selectively cleaved by a protease, such as a cathepsin, a caspase, a tumor-associated elastase enzyme, or an enzyme with protease-like or elastase-like activity. The peptide radical may be from 2 to about 12 amino acids. Enzymatic cleavage of the bond within the peptide linker releases the active form of the azabenzazepine (azaBz) moiety. This results in increased tissue specificity of the antibody conjugate, further reducing the toxicity of the conjugates of the present invention in other tissue types. Release of the active azaBz moiety from the antibody conjugate may occur through the action of lysosomal proteases, such as cathepsin and plasmin, which may be present at elevated levels in certain tumor tissues. The lysosomal enzyme may be, for example, cathepsin B, β-glucuronidase, or β-galactosidase.

[0188] The cleavable peptide of the peptide linker unit may be selected from tetrapeptides such as Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, tripeptides such as Glu-Val-Cit, or dipeptides such as Val-Cit, Val-Ala, Ala-Ala, and Phe-Lys.

[0189] The linker provides sufficient stability of the immunoconjugate in biological media, such as culture medium or serum, while providing the desired intracellular effect within tumor tissue as a result of its specific enzymatic or hydrolytic cleavability with release of the azaBz moiety.

[0190] The enzymatic activity of proteases, cathepsins, or elastases can catalyze the cleavage of the covalent bond of the antibody conjugate under physiological conditions. The enzymatic activity is an expression product of cells associated with tumor tissue. Enzymatic activity at the cleavage site of the targeting peptide converts the antibody conjugate into an activated azaBz adjuvant that does not contain the targeting antibody or the linking group. The cleavage site may be specifically recognized by the enzyme. Cathepsins or elastases may catalyze the cleavage of a specific peptide bond between the C-terminal amino acid residue of a specific peptide and the azaBz portion of the immunoconjugate.

[0191] In one embodiment, the invention includes a linking unit, i.e., L or linker, between the antibody and the azaBz moiety that comprises a substrate for cleavage by glucuronidase (Jeffrey SC, et al (2006) Bioconjug Chem. 17(3):831-40; US 11,413,353; US 11,173,214) or sulfatase (Bargh JD, et al (2020) Chem Sci. 11(9):2375-2380). In particular, L includes a Glu unit and has a formula selected from the following: [ka]

[0192] Specific cleavage of the immunoconjugate takes advantage of the presence of tumor-infiltrating cells of the immune system and leukocyte-secreted enzymes to facilitate activation of anticancer drugs at the tumor site.

[0193] Suitable electrophilic reactive functional groups (Q in Formula II) for azabenzazepine linker compounds (azaBz-L) include, but are not limited to, N-hydroxysuccinimidyl (NHS) esters and N-hydroxysulfosuccinimidyl (sulfo-NHS) esters (amine reactive); carbodiimides (amine and carboxyl reactive); hydroxymethylphosphines (amine reactive); maleimides (thiol reactive); halogenated acetamides such as N-iodoacetamide (thiol reactive); aryl azides (primary amine reactive); fluorinated aryl azides (reactive via carbon-hydrogen (CH) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) and sulfotetrafluorophenyl (STP) esters (amine reactive); imide esters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiol reactive); and benzophenone derivatives (reactive via C-H bond insertion). Additional reagents include, but are not limited to, those described in Hermanson, Bioconjugate Techniques 2nd Edition, Academic Press, 2008.

[0194] Some linkers, such as those containing peptide units and substrates for proteases, may be unstable in the bloodstream, thereby releasing unacceptable amounts of adjuvant / drug before internalization in target cells (Khot, A. et al. (2015) Bioanalysis 7(13):1633-1648). Other linkers may provide stability in the bloodstream but may adversely affect the efficacy of intracellular release. Linkers that provide the desired intracellular release may have poor stability in the bloodstream. Furthermore, in standard conjugation processes, the amount of adjuvant / drug moiety loaded onto the antibody (i.e., drug load), the amount of aggregates formed in the conjugation reaction, and the yield of the final purified conjugate that can be obtained are correlated. Aggregate formation may correlate with the number of equivalents of drug moieties conjugated to the antibody. Under high drug loads, the formed aggregates must be removed for therapeutic use. As a result, drug-loading-mediated aggregate formation can reduce the yield of antibody conjugates and make process scale-up difficult.

[0195] Although cleavable linkers (e.g., those with a protease substrate peptide unit or an immolative unit such as para-aminobenzyloxycarbonyl) may offer certain advantages, the linker need not be cleavable. With non-cleavable linkers, release of the azaBz adjuvant moiety may not depend on the differences in properties between plasma and some cytoplasmic compartments. Release of the adjuvant moiety or its metabolites may occur after internalization of the immunoconjugate via antigen-mediated endocytosis and delivery to the lysosomal compartment. At this time, the targeting moiety (or its binding fragments) may be degraded to the amino acid level by intracellular protease degradation. This process may release the adjuvant moiety or its metabolites. The released adjuvant moiety or its metabolites may be more hydrophilic and less membrane-permeable, resulting in reduced bystander effects and nonspecific toxicity compared to conjugates with cleavable linkers. Immunoconjugates with non-cleavable linkers may be more stable in circulation than immunoconjugates with cleavable linkers. Non-cleavable linkers may include those containing alkylene chains, or polymeric ones based on, for example, polyalkylene glycol polymers (PEGs), amide polymers, or those containing alkylene chain, polyalkylene glycol, and / or amide polymer segments. Linkers may contain 2 to 50 ethylene glycol (PEG) units, or PEGs with 2 to 10 ethylene glycol (PEG) units.

[0196] Conjugation of adjuvant azaBz moieties to glycan groups of antibodies can improve the linkage stability, homogeneity, aggregation, and various pharmacokinetic properties of immunoconjugates compared to conjugation to natural or engineered cysteine ​​residues (Zhou, Q., et al (2014) Bioconjugate Chem. 25(3), 510-520; Okeley, NM, et al (2013) Bioconjugate Chem. 24(10):1650-1655; US10,072,096; WO2015057063; WO2021248048). Several glycan remodeling methods use recombinant microbial transglutaminase to enable efficient site-specific conjugation of drug-linker intermediates to the HC-Q295 position of native, fully glycosylated IgG antibodies (Dickgeisser, S., et al. (2020) Bioconjugate Chemistry 31(4), 1070-1076). Natural and modified glycan groups and conjugation methods can be those taught in Qasba, PK (2015) Bioconjugate Chem. 26:2170-2175; Jaramillo, ML et al. (2023) MABS, VOL. 15, NO. 1:1-15; Zhang, X., et al. (2021) ACS Chem. Biol. 16:2502-2514 (each of which is incorporated herein by reference in its entirety).

[0197] The present invention provides solutions to limitations and challenges in the design, preparation, and use of immunoconjugates. Some linkers may be unstable in the bloodstream, thereby releasing unacceptable amounts of adjuvant / drug before internalization in target cells (Khot, A. et al. (2015) Bioanalysis 7(13):1633-1648). Other linkers may provide stability in the bloodstream but may adversely affect the efficacy of intracellular release. Linkers that provide desired intracellular release typically exhibit poor stability in the bloodstream. In other words, bloodstream stability and intracellular release are typically inversely related. Furthermore, in standard conjugation processes, the amount of adjuvant / drug moiety loaded onto the antibody (i.e., drug loading), the amount of aggregates formed in the conjugation reaction, and the yield of the final purified conjugate that can be obtained are correlated. For example, aggregate formation generally correlates directly with the number of equivalents of adjuvant / drug moiety and its derivatives conjugated to the antibody. Under high drug loading, the aggregates formed must be removed for therapeutic use. As a result, drug-loading-mediated aggregate formation can reduce the yield of immunoconjugates and make process scale-up difficult.

[0198] An exemplary embodiment is an azabenzazepine linker compound of formula II: [ka] wherein Z 1 is CR 1 and N, Z 2 is CR 2 and N, Z 3 is CR 3 and N, Z 4 is CR 4 and N, Z 1 , Z 2 , Z 3 , and Z 4one or two of are N, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently H, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O)2, S(O)2N(R 5 ), C1-C 12 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 12 Carbocyclyl, C6-C 20 Aryl, C2-C9 heterocyclyl, and C1-C 20 heteroaryl, each of which is independently and optionally selected from the group consisting of: -(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C1-C 12 alkyldiyl)-N(R 7 )2, -(C1-C 12 Alkyldiyl)-OR 7 , -(C3-C 12 carbocyclyl), -(C3-C 12 carbocyclyl)-*, -(C3-C 12 Carbocyclyl)-(C1-C 12 Alkyldiyl)-NR 7 -*, -(C3-C 12 Carbocyclyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -(C3-C 12 Carbocyclyl)-NR 7 -C(=NR 7 )NR 7 -*, -(C6-C 20 aryl), -(C6-C 20 Aryldiyl)-*, -(C6-C20 Aryldiyl)-N(R 7 )-*, -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-(C2-C 20 Heterocyclyldiyl)-*, -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-NR 7 -C(=NR 7a )N(R 7 )-*, -(C2-C 20 heterocyclyl), -(C2-C 20 heterocyclyl)-*, -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-NR 7 -*, -(C2-C9 heterocyclyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -(C2-C9 heterocyclyl)-C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C2-C9 heterocyclyl)-NR 7 -C(=NR 7a )NR 7 -*, -(C2-C9 heterocyclyl)-NR 7 -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C2-C9 heterocyclyl)-(C6-C 20Aryldiyl)-*, -(C1-C 20 heteroaryl), -(C1-C 20 Heteroaryl)-*, -(C1-C 20 Heteroaryl)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C1-C 20 Heteroaryl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -(C1-C 20 Heteroaryl)-NR 7 -C(=NR 7a )N(R 7 )-*, -(C1-C 20 heteroaryl)-N(R 7 )C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -C(=O)-*, -C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -C(=O)-(C2-C 20 Heterocyclyldiyl)-*, -C(=O)N(R 7 )2, -C(=O)N(R 7 )-*, -C(=O)N(R 7 )-(C1-C 12 alkyldiyl)-N(R 7 )C(=O)R 7 , -C(=O)N(R 7 )-(C1-C 12 alkyldiyl)-N(R 7 )C(=O)N(R 7 )2, -C(=O)NR 7 -(C1-C 12 alkyldiyl)-N(R 7 )CO2R 7 , -C(=O)NR 5 -(C1-C 12 alkyldiyl)-N(R 57 )C(=NR 57a )N(R 57 )2, -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 57 C(=NR 7a )R 7 , -C(=O)NR 5 -(C1-C8 alkyldiyl)-NR 7 (C2-C5 heteroaryl), -C(=O)NR 7 -(C1-C 20 heteroaryldiyl)-N(R 7 )-*, -C(=O)NR 7 -(C1-C 20 Heteroaryldiyl)-*, -C(=O)NR 7 -(C1-C 20 Heteroaryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -C(=O)NR 7 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 7 -(C1-C 12 Alkyldiyl)-NR 7 -*, -N(R 7 )2, -N(R 7 )-*, -N(R 7 )C(=O)R 7 , -N(R 7 )C(=O)-*, -N(R 7 )C(=O)N(R 7 )2, -N(R 7 )C(=O)N(R 7 )-*, -N(R 7 )CO2R 7 , -NR 7 C(=NR 7a )N(R 7 )2, -NR 7 C(=NR 7a )N(R 7 )-*, -NR 7 C(=NR 7a )R 7 , -N(R 7 )C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -N(R 7 )-(C2-C5 heteroaryl), -N(R 7 )-S(=O)2-(C1-C 12 alkyl), -O-(C1-C 12 alkyl), -O-(C1-C 12 alkyldiyl)-N(R 7 )2, -O-(C1-C 12 alkyldiyl)-N(R 7 )-*, -OC(=O)N(R 7 )2, -OC(=O)N(R 7 )-*, -O-(R 7 )-*, -OR 7 , -S(=O)2-(C2-C 20 Heterocyclyldiyl)-*, -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-NR 7 -*, and -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 or substituted with one or more groups selected from: or R 5 and R 6 together form a 5- or 6-membered heterocyclyl ring, R 7 are independently H, C6-C 20 Aryl, C3-C 12 Carbocyclyl, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 alkyldiyl, or two R 5 the groups together form a 5- or 6-membered heterocyclyl ring; R 7a is C6-C 20 Aryl and C1-C 20 heteroaryl; where the asterisk * indicates the binding site of L and R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 One of them is bound to L, Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from F, Cl, Br, I, —CN, —CH3, —CH2CH3, —CH=CH2, —C≡CH, —C≡CCH3, —CH2 CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3) 2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN , -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH( OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2 , -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n Substituted with one or more groups independently selected from H, —OCH2F, —OCHF2, —OCF3, —OP(O)(OH)2, —S(O)2N(CH3)2, —SCH3, —S(O)2CH3, and —S(O)3H.

[0199] Exemplary embodiments of L in formula II are: QC(=O)-PEG-, QC(=O)-PEG-C(=O)N(R8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-, QC(=O)-PEG-O-, QC(=O)-PEG-OC(=O)-, QC(=O)-PEG-C(=O)-, QC(=O)-PEG-C(=O)-PEP-, QC(=O)-PEG-N(R 8 )-, QC(=O)-PEG-N(R 8 )-C(=O)-, QC(=O)-PEG-N(R 8 )-PEG-C(═O)-PEP-, QC(=O)-PEG-N + (R 8 )2-PEG-C(=O)-PEP-, QC(=O)-PEG-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-, QC(=O)-PEG-C(=O)-PEP-N(R 8 )-(C1-C 12 Alkyldiyl)N(R 8 )C(=O)-(C2-C5 monoheterocyclyldiyl)-, QC(=O)-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-, QC(=O)-PEG-SS-(C1-C 12 alkyldiyl)-C(=O)-, QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-, QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-, QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12alkyldiyl)-N(R 8 )-C(=O), QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-N(R 8 )C(=O)-(C2-C5 monoheterocyclyldiyl)-, Q-(CH2) m -C(=O)N(R 8 )-PEG-, Q-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)N(R 8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-, Q-(CH2) m -C(=O)N(R 8 )-PEG-O-, Q-(CH2) m -C(=O)N(R 8 )-PEG-OC(=O)-, Q-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)-, Q-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-, Q-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-C(=O)-, Q-(CH2) m -C(=O)N(R 8 )-PEG-C(═O)-PEP-, Q-(CH2) m -C(=O)N(R 8 )-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-, Q-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-, Q-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 Alkyldiyl)N(R 8 )C(=O)-, and Q-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 Alkyldiyl)N(R 8 )C(═O)—(C2-C5 monoheterocyclyldiyl)-; R 8 are independently H or C1-C6 alkyl; PEG has the formula: -(CH2CH2O) n -(CH2) m wherein m is an integer from 1 to 5 and n is an integer from 1 to 50; Gluc has the formula: [ka] and PEP has the formula: [ka] wherein AA are independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; Cyc is a C-C alkyl group optionally substituted with one or more groups selected from F, Cl, NO, —OH, —OCH, and glucuronic acid having the structure: 20 Aryldiyl and C1-C 20 heteroaryldiyl; [ka] R 9 is -CH(R 10 )O-, -CH2-, -CH2N(R 10 )-, and -CH(R 10 )OC(=O)-, wherein R 10is H, C1-C6 alkyl, C(=O)-C1-C6 alkyl, and -C(=O)N(R 11 )2, where R 11 are independently H, C1-C 12 Alkyl, and -(CH2CH2O) n -(CH2) m -OH (wherein m is an integer of 1 to 5 and n is an integer of 2 to 50), or two R 11 the groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1.

[0200] Exemplary embodiments of Q include F, Cl, NO and SO. - and N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, each substituted with one or more groups independently selected from:

[0201] An exemplary embodiment of Q is [ka] is selected from.

[0202] An exemplary embodiment of Q is phenoxy substituted with one or more F.

[0203] An exemplary embodiment of Q is 2,3,5,6-tetrafluorophenoxy.

[0204] Exemplary embodiments of azabenzazepine linker compounds of Formula II are selected from Tables 2a and 2b. Each compound was synthesized, purified, characterized by mass spectrometry, and shown to have the indicated mass. Additional experimental procedures are found in the Examples. The azabenzazepine linker compounds (azaBzL) of Tables 2a and 2b exhibit the surprising and unexpected property of TLR8 agonist selectivity, which may predict useful therapeutic activity for treating cancer and other disorders. The azabenzazepine linker intermediates of Formula II compounds of Tables 2a and 2b are used in conjugation with antibodies by the method of Example 201 to form the immunoconjugates of Tables 3a and 3b. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] Table 7-6 Table 7-7 Table 7-8 Table 7-9 Table 7-10 Table 7-11 Table 7-12 Table 7-13 Table 7-14 Table 7-15 Table 7-16 Table 7-17 Table 7-18 Table 7-19 Table 7-20 Table 7-21 Table 7-22 Table 7-23 Table 7-24 Table 7-25 Table 7-26 Table 7-27 Table 7-28 Table 7-29 Table 7-30 Table 7-31 Table 7-32 Table 7-33 Table 7-34 Table 7-35 Table 7-36 Table 7-37 Table 7-38 Table 7-39 [Table 7-40] [Table 7-41] [Table 7-42] [Table 7-43] [Table 7-44] [Table 7-45] [Table 7-46] [Table 7-47]

[0205] Comparative linker compounds (CL) in Table 2c have (i) an active ester, i.e., a tetrafluorophenyl group or a sulfotetrafluorophenyl group, that reacts with a lysine residue of an antibody, or (ii) a maleimide group that reacts with a cysteine ​​residue of an antibody to form an immunoconjugate having an antibody and a TLR-agonist-linker moiety according to Example 201. Comparative linker compounds CL-4, 5, 6, 7, and 8 have an azabenzazepine, lactam structure. [Table 8-1] [Table 8-2] [Table 8-3]

[0206] Azabenzazepine immunoconjugates Immunostimulatory antibody conjugates, or immunoconjugates, target TLR7 / 8 agonists into tumors to activate tumor-infiltrating myeloid cells and initiate broad innate and adaptive antitumor immune responses (Ackerman, et al., (2021) Nature Cancer 2:18-33).

[0207] An exemplary embodiment of an immunoconjugate comprises an antibody covalently attached by a linker to one or more azabenzazepine moieties and has Formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein: Ab is antibody, p is an integer from 1 to 8; L is a linker, D is an azabenzazepine moiety having the formula: [ka] Z 1 is CR 1 and N, Z 2 is CR 2 and N, Z 3 is CR 3 and N, Z 4 is CR 4 and N, Z 1 , Z 2 , Z 3 , and Z 4 one or two of are N, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently H, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O)2, S(O)2N(R 5 ), C1-C12 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 12 Carbocyclyl, C6-C 20 Aryl, C2-C9 heterocyclyl, and C1-C 20 heteroaryl, each of which is independently and optionally selected from the group consisting of: -(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C1-C 12 alkyldiyl)-N(R 7 )2, -(C1-C 12 Alkyldiyl)-OR 7 , -(C3-C 12 carbocyclyl), -(C3-C 12 carbocyclyl)-*, -(C3-C 12 Carbocyclyl)-(C1-C 12 Alkyldiyl)-NR 7 -*, -(C3-C 12 Carbocyclyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -(C3-C 12 Carbocyclyl)-NR 7 -C(=NR 7 )NR 7 -*, -(C6-C 20 aryl), -(C6-C 20 Aryldiyl)-*, -(C6-C 20 Aryldiyl)-N(R 7 )-*, -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-(C2-C 20Heterocyclyldiyl)-*, -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-NR 7 -C(=NR 7a )N(R 7 )-*, -(C2-C 20 heterocyclyl), -(C2-C 20 heterocyclyl)-*, -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-NR 7 -*, -(C2-C9 heterocyclyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -(C2-C9 heterocyclyl)-C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C2-C9 heterocyclyl)-NR 7 -C(=NR 7a )NR 7 -*, -(C2-C9 heterocyclyl)-NR 7 -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C2-C9 heterocyclyl)-(C6-C 20 Aryldiyl)-*, -(C1-C 20 heteroaryl), -(C1-C 20 Heteroaryl)-*, -(C1-C 20 Heteroaryl)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -(C1-C 20Heteroaryl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -(C1-C 20 Heteroaryl)-NR 7 -C(=NR 7a )N(R 7 )-*, -(C1-C 20 heteroaryl)-N(R 7 )C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -C(=O)-*, -C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -C(=O)-(C2-C 20 Heterocyclyldiyl)-*, -C(=O)N(R 7 )2, -C(=O)N(R 7 )-*, -C(=O)N(R 7 )-(C1-C 12 alkyldiyl)-N(R 7 )C(=O)R 7 , -C(=O)N(R 7 )-(C1-C 12 alkyldiyl)-N(R 7 )C(=O)N(R 7 )2, -C(=O)NR 7 -(C1-C 12 alkyldiyl)-N(R 7 )CO2R 7 , -C(=O)NR 5 -(C1-C 12 alkyldiyl)-N(R 57 )C(=NR 57a )N(R 57 )2, -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 57 C(=NR 7a )R7 , -C(=O)NR 5 -(C1-C8 alkyldiyl)-NR 7 (C2-C5 heteroaryl), -C(=O)NR 7 -(C1-C 20 heteroaryldiyl)-N(R 7 )-*, -C(=O)NR 7 -(C1-C 20 Heteroaryldiyl)-*, -C(=O)NR 7 -(C1-C 20 Heteroaryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -C(=O)NR 7 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 7 -(C1-C 12 Alkyldiyl)-NR 7 -*, -N(R 7 )2, -N(R 7 )-*, -N(R 7 )C(=O)R 7 , -N(R 7 )C(=O)-*, -N(R 7 )C(=O)N(R 7 )2, -N(R 7 )C(=O)N(R 7 )-*, -N(R 7 )CO2R 7 , -NR 7 C(=NR 7a )N(R 7 )2, -NR 7 C(=NR 7a )N(R 7 )-*, -NR 7C(=NR 7a )R 7 , -N(R 7 )C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*, -N(R 7 )-(C2-C5 heteroaryl), -N(R 7 )-S(=O)2-(C1-C 12 alkyl), -O-(C1-C 12 alkyl), -O-(C1-C 12 alkyldiyl)-N(R 7 )2, -O-(C1-C 12 alkyldiyl)-N(R 7 )-*, -OC(=O)N(R 7 )2, -OC(=O)N(R 7 )-*, -O-(R 7 )-*, -OR 7 , -S(=O)2-(C2-C 20 Heterocyclyldiyl)-*, -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2, -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-NR 7 -*, and -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 or substituted with one or more groups selected from: or R 5 and R 6 together form a 5- or 6-membered heterocyclyl ring, R 7 are independently H, C6-C20 Aryl, C3-C 12 Carbocyclyl, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 alkyldiyl, or two R 5 the groups together form a 5- or 6-membered heterocyclyl ring; R 7a is C6-C 20 Aryl and C1-C 20 heteroaryl; where the asterisk * indicates the binding site of L and R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 One of them is bound to L, Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from F, Cl, Br, I, —CN, —CH3, —CH2CH3, —CH=CH2, —C≡CH, —C≡CCH3, —CH2 CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3) 2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN , -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH( OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2 , -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n Substituted with one or more groups independently selected from H, —OCH2F, —OCHF2, —OCF3, —OP(O)(OH)2, —S(O)2N(CH3)2, —SCH3, —S(O)2CH3, and —S(O)3H.

[0208] Exemplary embodiments of the immunoconjugate of Formula I include those in which L is —C(═O)-PEG-, -C(=O)-PEG-C(=O)N(R 8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-, —C(═O)-PEG-O—, —C(═O)-PEG-OC(═O)-, —C(═O)-PEG-C(═O)—, -C(=O)-PEG-C(=O)-PEP-, -C(=O)-PEG-N(R 8 )-, -C(=O)-PEG-N(R 8 )-C(=O)-, -C(=O)-PEG-N(R 8 )-PEG-C(═O)-PEP-, -C(=O)-PEG-N + (R 8 )2-PEG-C(=O)-PEP-, -C(=O)-PEG-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-, -C(=O)-PEG-C(=O)-PEP-N(R 8 )-(C1-C 12 Alkyldiyl)N(R 8 )C(=O)-(C2-C5 monoheterocyclyldiyl)-, -C(=O)-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-, -C(=O)-PEG-SS-(C1-C 12 alkyldiyl)-C(=O)-, -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-, -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-, -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12alkyldiyl)-N(R 8 )-C(=O), -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-N(R 8 )C(=O)-(C2-C5 monoheterocyclyldiyl)-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)N(R 8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-O-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-OC(=O)-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-C(=O)-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-C(═O)-PEP-, -Succinimidyl-(CH2) m -C(=O)N(R 8 )-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-, -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-, -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 Alkyldiyl)N(R 8 )C(=O)-, and -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 Alkyldiyl)N(R 8 )C(═O)—(C2-C5 monoheterocyclyldiyl)-; R 8 are independently H or C1-C6 alkyl; PEG has the formula: -(CH2CH2O) n -(CH2) m wherein m is an integer from 1 to 5 and n is an integer from 1 to 50; Gluc has the formula: [ka] and PEP has the formula: [ka] wherein AA are independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; Cyc is a C-C alkyl group optionally substituted with one or more groups selected from F, Cl, NO, —OH, —OCH, and glucuronic acid having the structure: 20 Aryldiyl and C1-C 20 heteroaryldiyl; [ka] R 9 is -CH(R10 )O-, -CH2-, -CH2N(R 10 )-, and -CH(R 10 )OC(=O)-, wherein R 10 is H, C1-C6 alkyl, C(=O)-C1-C6 alkyl, and -C(=O)N(R 11 )2, where R 11 are independently H, C1-C 12 Alkyl, and -(CH2CH2O) n -(CH2) m -OH (wherein m is an integer of 1 to 5 and n is an integer of 2 to 50), or two R 11 the groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z may be 0 or 1.

[0209] Exemplary embodiments of the immunoconjugate of Formula I include 1 is N.

[0210] Exemplary embodiments of the immunoconjugate of Formula I include 2 is N.

[0211] Exemplary embodiments of the immunoconjugate of Formula I include 3 is N.

[0212] Exemplary embodiments of the immunoconjugate of Formula I include 4 is N.

[0213] Exemplary embodiments of the immunoconjugate of Formula I include R 5 and R 6 are independently C1-C8 alkyl, -O-(C1-C 12 alkyl), -(C1-C 12 Alkyldiyl)-OR 5 , -(C1-C8 alkyldiyl)-N(R 5 )CO2R 5, -(C1-C 12 alkyl)-OC(O)N(R 5 )2, -O-(C1-C 12 alkyl)-N(R 5 )CO2R 5 , and -O-(C1-C 12 alkyl)-OC(O)N(R 5 )2.

[0214] Exemplary embodiments of the immunoconjugate of Formula I include R 5 is C1-C8 alkyl, and R 6 -O-(C1-C 12 alkyl).

[0215] Exemplary embodiments of the immunoconjugate of Formula I include R 5 is -CH2CH2CH3 and R 6 is selected from -CH2CH2CH2NHCO2(t-Bu), -OCH2CH2NHCO2(cyclobutyl), and -CH2CH2CH2NHCO2(cyclobutyl).

[0216] Exemplary embodiments of the immunoconjugate of Formula I include R 5 and R 6 are each independently selected from —CH 2 CH 2 CH 3 , —OCH 2 CH 3 , —OCH 2 CF 3 , —CH 2 CH 2 CF 3 , —OCH 2 CH 2 OH, and —CH 2 CH 2 CH 2 OH.

[0217] Exemplary embodiments of the immunoconjugate of Formula I include R 5 is -CH2CH2CH3 and R 6 is -OCH2CH3.

[0218] Exemplary embodiments of the immunoconjugate of Formula I include R 6 but, [ka] and wherein the hydroxyl group is selected from the group consisting of:

[0219] Exemplary embodiments of the immunoconjugate of Formula I include R 1 is attached to L.

[0220] Exemplary embodiments of the immunoconjugate of Formula I include R 2 is attached to L.

[0221] Exemplary embodiments of the immunoconjugate of Formula I include R 3 is attached to L.

[0222] Exemplary embodiments of the immunoconjugate of Formula I include R 4 is attached to L.

[0223] Exemplary embodiments of the immunoconjugate of Formula I include R 5 or R 6 is attached to L.

[0224] Exemplary embodiments of the immunoconjugate of Formula I include where L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0225] An exemplary embodiment of the immunoconjugate of Formula I includes where L is attached to a cysteine ​​thiol of the antibody.

[0226] Exemplary embodiments of the immunoconjugate of Formula I include those in which m is 1 or 2 and n is an integer from 2 to 10, or n is 10, for PEG.

[0227] Exemplary embodiments of the immunoconjugate of Formula I include wherein L comprises PEP, wherein PEP is a dipeptide and has the formula: [ka] This includes having:

[0228] Exemplary embodiments of the immunoconjugate of Formula I include where AA are independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2, or where two AAs form a five-membered ring proline amino acid.

[0229] Exemplary embodiments of the immunoconjugate of Formula I include wherein PEP is a dipeptide and has the formula: [ka] and wherein AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids.

[0230] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0231] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 and AA2 are independently selected from GlcNAc aspartic acid, -CH2SO3H, and -CH2OPO3H.

[0232] Exemplary embodiments of the immunoconjugate of Formula I include wherein PEP is a tripeptide and has the formula: [ka] This includes having:

[0233] Exemplary embodiments of the immunoconjugate of Formula I include wherein PEP is a tetrapeptide and has the formula: [ka] This includes having:

[0234] Exemplary embodiments of the immunoconjugate of Formula I include: AA1 is selected from the group consisting of Abu, Ala, and Val; AA2 is selected from the group consisting of Nle(O-Bzl), Oic, and Pro; AA3 is selected from the group consisting of Ala and Met(O)2; AA4 is selected from the group consisting of Oic, Arg(NO2), Bpa, and Nle(O-Bzl).

[0235] Exemplary embodiments of the immunoconjugate of Formula I include those in which L comprises PEP, wherein PEP is selected from the group consisting of Ala-Pro-Val, Asn-Pro-Val, Ala-Ala-Val, Ala-Ala-Pro-Ala, Ala-Ala-Pro-Val, and Ala-Ala-Pro-Nva.

[0236] Exemplary embodiments of the immunoconjugate of Formula I include those in which L comprises a PEP, and the PEP is selected from the following structures: [ka]

[0237] Exemplary embodiments of the immunoconjugate of Formula I include those in which L has the structure: [ka] is selected from The wavy line is R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 It includes showing binding to one of:

[0238] The present invention includes all rational combinations and permutations of features of the embodiments of Formula I.

[0239] In certain embodiments, immunoconjugate compounds of the invention include those with immunostimulatory activity. The immunoconjugates of the invention selectively deliver effective doses of azabenzazepine (azaBz) drugs or metabolites to tumor tissue, thereby achieving greater selectivity (i.e., lower effective doses) while increasing the therapeutic index ("therapeutic window") compared to unconjugated azaBz.

[0240] Each immunoconjugate in Tables 3a, 3b, and 3c was prepared according to the method of Example 201, purified by HPLC, and characterized by mass spectrometry. [Table 9] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 11]

[0241] Secreted cytokine levels in the supernatants were determined using a LegendPlex cytokine bead array kit. The immunoconjugates (IC) in Tables 3a and 3b induced the secretion of TNFα (alpha), a cytokine associated with the initiation of immune responses against cancer, indicating activation of myeloid cells upon exposure to antigen-expressing tumor cells such as HER2. The azabenzazepine immunoconjugates in Tables 3a and 3b stimulated higher levels of TNFα than the comparative immunoconjugate CIC-1. Notably, the azabenzazepine payload is a more efficient payload, decreasing molecular weight and hydrophobicity while increasing activity. Naked antibodies did not induce myeloid activation, indicating dependence on the TLR7 / 8-activating payload.

[0242] Drug loading is represented by p, which is the number of azabenzazepine (azaBz) moieties per antibody in the immunoconjugate of Formula I, measured as (DAR) for the exemplary immunoconjugates of Table 3a. Drug (azaBz) loading can range from 1 to about 8 drug moieties (D) per antibody. The immunoconjugates of Formula I comprise a mixture or population of antibodies conjugated to a range of 1 to about 8 drug moieties. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues, such as lysine and cysteine. In some embodiments, a free cysteine ​​residue is introduced into the antibody amino acid sequence by the methods described herein. In such aspects, p can be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges therein, e.g., 1 to 8 or 2 to 5. In any such embodiment, p and n are equal (i.e., p = n = 1, 2, 3, 4, 5, 6, 7, or 8, or some range therebetween). Exemplary immunoconjugates of Formula I include, but are not limited to, antibodies with 1, 2, 3, or 4 engineered cysteine ​​amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine ​​residues are already present in the antibody that form intra- and inter-chain disulfide bonds (natural disulfide groups) without the use of genetic engineering, in which case the existing free, reduced cysteine ​​residue may be used to conjugate the antibody to a drug. In some embodiments, the antibody is exposed to reducing conditions to generate one or more free cysteine ​​residues prior to conjugation of the antibody.

[0243] For some immunoconjugates, p may be limited by the number of binding sites on the antibody. For example, if the linkage is a cysteine ​​thiol, as in certain exemplary embodiments described herein, the antibody may have only one or a limited number of cysteine ​​thiol groups, or only one or a limited number of reactive and sufficient thiol groups to which a drug may be attached. In other embodiments, one or more lysine amino groups on the antibody may be available and reactive for conjugation with an azaBz-linker compound of Formula II. In certain embodiments, high drug loading, e.g., p greater than 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading of the immunoconjugate ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0244] The loading (drug / antibody ratio) of the immunoconjugate may be controlled in different ways, as well as by, for example, (i) limiting the molar excess of the azaBz-linker intermediate compound compared to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reductive denaturation conditions for optimized antibody reactivity.

[0245] It should be understood that if more than one nucleophilic group on an antibody reacts with a drug, the resulting product will be a mixture of immunoconjugate compounds with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a double ELISA antibody assay, specific for the antibody and specific for the drug. Individual immunoconjugate molecules may be identified in the mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography (see, e.g., McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, KJ, et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, SC, et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract (See, e.g., American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004, No. 627.) In certain embodiments, homogeneous immunoconjugates having a single loading value may be isolated from a complex mixture by electrophoresis or chromatography.

[0246] Assessment of immunoconjugate activity in vitro can be carried out according to the method of Example 203.

[0247] Composition of Immunoconjugates The present invention provides compositions, e.g., pharmaceutically or pharmacologically acceptable compositions or formulations, comprising a plurality of immunoconjugates described herein and, optionally, a carrier therefor, e.g., a pharmaceutically or pharmacologically acceptable carrier. The immunoconjugates may be the same or different in the composition; i.e., the composition can include immunoconjugates having the same number of adjuvants linked to the same position on the antibody construct, and / or immunoconjugates having the same number of azabenzazepine (azaBz) adjuvants linked to different positions on the antibody construct, different numbers of azaBz adjuvants linked to the same position on the antibody construct, or different numbers of azaBz adjuvants linked to different positions on the antibody construct.

[0248] In an exemplary embodiment, the composition comprising immunoconjugate compounds comprises a mixture of immunoconjugate compounds, wherein the average drug (aza-Bz) loading per antibody in the mixture of immunoconjugate compounds is about 2 to about 5.

[0249] Immunoconjugate compositions of the invention can have an average adjuvant-to-antibody construct ratio (DAR) of about 0.4 to about 10. Those skilled in the art will recognize that in compositions comprising multiple immunoconjugates of the invention, the number of azabenzazepine adjuvants conjugated to the antibody constructs can vary from one immunoconjugate to another, and therefore the adjuvant-to-antibody construct (e.g., antibody) ratio can be measured as an average value, which can be referred to as the drug-to-antibody ratio (DAR). The adjuvant-to-antibody construct (e.g., antibody) ratio can be assessed by any suitable means, many known in the art, including conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of immunoconjugates in a composition can also be determined in terms of p. In some cases, separation, purification, and characterization of homogeneous immunoconjugates with a particular value of p from immunoconjugates with other drug loads can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0250] In some embodiments, the composition further comprises one or more pharmaceutically or pharmacologically acceptable excipients. For example, the immunoconjugates of the present invention can be formulated for parenteral administration, such as IV administration or administration into a body cavity or organ lumen. Alternatively, the immunoconjugates can be injected intratumorally. An injectable composition will generally comprise a solution of the immunoconjugate dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be used include water and isotonic solutions of one or more salts, such as sodium chloride, such as Ringer's solution. Additionally, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Additionally, fatty acids, such as oleic acid, may also be used in the preparation of injectables. These compositions are desirably sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, etc., required to approximate physiological conditions, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0251] The composition can contain any suitable concentration of immunoconjugate. The concentration of immunoconjugate in the composition can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., in accordance with the particular mode of administration selected and the patient's needs. In certain embodiments, the concentration of immunoconjugate in an injectable solution formulation ranges from about 0.1% (w / w) to about 10% (w / w).

[0252] Immunoconjugates for cancer therapy The present invention provides methods for treating cancer. The methods include administering a therapeutically effective amount of an immunoconjugate described herein (e.g., a composition described herein) to a subject in need thereof, e.g., a subject having cancer and in need of cancer treatment. The methods include administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 3a.

[0253] It is contemplated that the immunoconjugates of the invention can be used to treat a variety of hyperproliferative diseases or disorders, such as those characterized by overexpression of tumor antigens. Exemplary hyperproliferative disorders include benign or malignant solid tumors, and hematological disorders such as leukemia and lymphoid malignancies.

[0254] In another aspect, immunoconjugates are provided for use as pharmaceuticals. In certain embodiments, the invention provides immunoconjugates for use in methods of treating an individual, comprising administering to the individual an effective amount of the immunoconjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0255] In a further aspect, the present invention provides use of the immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer, and the method comprises administering an effective amount of the medicament to an individual having cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0256] Carcinomas are malignant tumors that arise from epithelial tissue. Epithelial cells line the exterior surfaces of the body, interior cavities, and form the lining of glandular tissue. Examples of carcinomas include, but are not limited to, adenocarcinoma (cancer that begins in glandular (secretory) cells, e.g., breast, pancreas, lung, prostate, stomach, gastroesophageal junction, and colon); adrenocortical carcinoma; hepatocellular carcinoma; renal cell carcinoma; ovarian cancer; carcinoma in situ; ductal carcinoma; breast cancer; basal cell carcinoma; squamous cell carcinoma; transitional cell carcinoma; colon cancer; nasopharyngeal carcinoma; multilocular cystic renal cell carcinoma; oat cell carcinoma; large cell lung carcinoma; small cell lung carcinoma; non-small cell lung carcinoma, etc. Carcinomas can be found in the prostate, pancreas, colon, brain (usually as secondary metastasis), lung, breast, and skin. In some embodiments, a method for treating non-small cell lung cancer comprises administering an immunoconjugate containing an antibody construct capable of binding to a tumor-associated antigen.

[0257] Soft tissue tumors are a highly diverse group of rare tumors derived from connective tissue. Examples of soft tissue tumors include alveolar soft part sarcoma, hemangiomatoid fibrous histiocytoma, chondromyxoid fibroma, skeletal chondrosarcoma, extraskeletal myxoid chondrosarcoma, clear cell sarcoma, desmoplastic small round cell tumor, dermatofibrosarcoma protuberans, endometrial stromal tumor, Ewing's sarcoma, fibromatosis (desmoid), infantile fibrosarcoma, gastrointestinal stromal tumor, giant cell tumor of bone, giant cell tumor of tendon sheath, inflammatory myofibroblastic tumor, uterine fibroid, leiomyosarcoma, lipoblastoma, typical lipoma, spindle cell or pleomorphic lipoma, atypical lipoma, chondroid lipoma, well-differentiated liposarcoma, myxoid / round cell liposarcoma, pleomorphic liposarcoma, myxoid malignant fibrous histiocytoma, high-grade malignant fibrous histiocytoma, myxofibrosarcoma, and malignant peripheral nerve tumor. These include, but are not limited to, sheath tumor, mesothelioma, neuroblastoma, osteochondroma, osteosarcoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, benign or malignant nerve sheath tumor, synovial sarcoma, Evans' tumor, nodular fasciitis, desmoid fibromatosis, solitary fibrous tumor, dermatofibrosarcoma protuberans (DFSP), angiosarcoma, epithelioid hemangioendothelioma, giant cell tumor of tendon sheath (TGCT), pigmented villonodular synovitis (PVNS), fibrous dysplasia, myxofibrosarcoma, fibrosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, neurofibroma, pleomorphic adenoma of soft tissue, and tumors derived from fibroblasts, myofibroblasts, histiocytes, vascular cells / endothelial cells, and nerve sheath cells.

[0258] Sarcoma is a rare type of cancer that arises in cells of mesenchymal origin, such as bone, or in the soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissues. Different types of sarcoma are based on where the cancer forms. For example, osteosarcoma arises in bone, liposarcoma arises in fat, and rhabdomyosarcoma arises in muscle. Examples of sarcomas include, but are not limited to, primitive neuroectodermal tumor (PNET) of the thoracic and pulmonary region (Askin tumor), botryoid sarcoma, chondrosarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcomas (e.g., alveolar soft part sarcoma, angiosarcoma, cystosarcoma lobularis, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrous sarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma (more commonly referred to as "angiosarcoma"), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, synovial sarcoma, and undifferentiated pleomorphic sarcoma).

[0259] Teratomas are a type of germ cell tumor that can contain several different types of tissue (e.g., tissue derived from any and / or all of the three germ layers: endoderm, mesoderm, and ectoderm), including, for example, hair, muscle, and bone. Teratomas most commonly occur in the ovaries in women, the testes in men, and the coccyx in children.

[0260] Melanoma is a form of cancer that begins in melanocytes (cells that make the pigment melanin). Melanoma can start in a mole (skin melanoma) but can also start in other pigmented tissues, such as in the eye or in the intestine.

[0261] Merkel cell carcinoma is a rare type of skin cancer that usually appears as flesh-colored or bluish-red nodules on the face, head, or neck. Merkel cell carcinoma is also called cutaneous neuroendocrine carcinoma. In some embodiments, a method for treating Merkel cell carcinoma includes administering, for example, an immunoconjugate containing an antibody construct capable of binding to CEA (e.g., labetuzumab, its biosimilar, or its biobetter). In some embodiments, the Merkel cell carcinoma has metastasized at the time of administration.

[0262] Leukemia is a cancer that originates in blood-forming tissues, such as bone marrow, and causes the production and entry of large numbers of abnormal blood cells into the bloodstream. For example, leukemia can arise in bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named for the rate at which the disease develops and progresses (e.g., acute vs. chronic) and the type of white blood cell affected (e.g., myeloid vs. lymphoid). Myeloid leukemias are also called myelogenous leukemias or myeloblastic leukemia. Lymphoid leukemias are also called lymphoblastic leukemia or lymphocytic leukemia. Lymphoid leukemia cells may collect in lymph nodes, causing them to swell. Examples of leukemia include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL).

[0263] Lymphoma is a cancer that begins in cells of the immune system. For example, lymphoma can arise in bone marrow-derived cells that normally mature in the lymphatic system. There are two basic categories of lymphoma. One category of lymphoma is Hodgkin lymphoma (HL), which is characterized by the presence of a type of cell called Reed-Sternberg cells. There are currently six recognized types of HL. Examples of Hodgkin lymphoma include nodular sclerosing classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte-reduced CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0264] Another category of lymphoma is non-Hodgkin's lymphoma (NHL), which includes a large and diverse group of cancers of immune system cells. Non-Hodgkin's lymphoma can be further divided into indolent (slow-growing) and aggressive (fast-growing) cancers. There are currently 61 recognized types of NHL. Examples of non-Hodgkin's lymphomas include, but are not limited to, AIDS-related lymphoma, anaplastic large cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small non-cleavable cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, enteropathic T-cell lymphoma, follicular lymphoma, hepatosplenic gamma-delta T-cell lymphoma, T-cell leukemia, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-cell lymphoma, childhood lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, treatment-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0265] Brain tumors include cancers of brain tissue. Examples of brain tumors include, but are not limited to, gliomas (e.g., glioblastoma, astrocytoma, oligodendroglioma, ependymoma, etc.), meningiomas, pituitary adenomas, and vestibular schwannomas, and primitive neuroectodermal tumors (medulloblastomas).

[0266] The immunoconjugates of the present invention can be used in therapy either alone or in combination with other agents. For example, the immunoconjugates can be co-administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. Such combination therapy includes combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of the immunoconjugate can occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The immunoconjugates can also be used in combination with radiation therapy.

[0267] The immunoconjugates of the invention (and any additional therapeutic agents) can be administered by any suitable means, including oral, parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized therapy. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0268] The immunoconjugate is administered to a subject in need thereof in any therapeutically effective amount using any suitable dosing regimen, such as those utilized for labetuzumab, its biosimilars, and its biobetters. For example, the method can include administering the immunoconjugate to provide the subject with a dose of about 100 ng / kg to about 50 mg / kg. The dose of the immunoconjugate can range from about 5 mg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The dose of the immunoconjugate can be about 100, 200, 300, 400, or 500 μg / kg. The dose of the immunoconjugate can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The dosage of the immunoconjugate may fall outside these ranges, depending on the particular conjugate and the type and severity of the cancer being treated. The frequency of administration may range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week.

[0269] In another aspect, the present invention provides a method for preventing cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate (e.g., as a composition as described above) to a subject. In certain embodiments, the subject is susceptible to the particular cancer to be prevented.

[0270] Some embodiments of the present invention provide methods for treating cancers such as those described above, wherein the cancer is breast cancer. Breast cancer can arise from various regions of the breast, and various types of breast cancer have been characterized. For example, the immunoconjugates of the present invention can be used to treat other forms of breast cancer, such as ductal carcinoma in situ; invasive ductal carcinoma (e.g., tubular, medullary, mucinous, papillary, or cribriform carcinoma of the breast); lobular carcinoma in situ; invasive lobular carcinoma; inflammatory breast cancer; and triple-negative (tests negative for estrogen receptor, progesterone receptor, and excess HER2 protein) breast cancer. In some embodiments, the method for treating breast cancer comprises administering an immunoconjugate containing an antibody construct capable of binding to a tumor-associated antigen (TAA) or a tumor overexpressing a TAA.

[0271] In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8.

[0272] In some embodiments, a therapeutically effective amount of the immunoconjugate is administered to a patient in need of treatment for cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial cancer, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, or breast cancer. The Merkel cell carcinoma can be metastatic Merkel cell carcinoma. The breast cancer can be triple-negative breast cancer. The esophageal cancer can be gastroesophageal junction adenocarcinoma. [Example]

[0273] Example 3: Synthesis of 2-amino-4-((2-((tert-butoxycarbonyl)amino)ethoxy)(propyl)carbamoyl)-3H-pyrido[4,3-b]azepine-8-carboxylic acid, azaBz-3 [ka] Preparation of tert-butyl (2,5-dibromopyridin-4-yl)carbamate, 3b To a solution of 2,5-dibromopyridine-4-carboxylic acid 3a (25.0 g, 89.0 mmol, 1 equiv.) and EtN (27.0 g, 267 mmol, 37.2 mL, 3 equiv.) in tert-butanol (200 mL), diphenylphosphoryl azide, DPPA (49.0 g, 178 mmol, 38.6 mL, 2 equiv.), was added and stirred at 80 °C for 12 h to induce a Curtius rearrangement via the addition of tert-butanol to the intermediate isocyanate. The mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® silica flash column, elution with a gradient of 0–25% ethyl acetate / petroleum ether at 100 mL / min) to give 3b (25 g, 71.02 mmol, 79.80% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 8.49 (s, 1H), 8.43 (s, 1H), 1.65 (s, 9H). LC / MS [M+H] 350.9 (calculated); LC / MS [M+H] 350.9 (observed).

[0274] Preparation of tert-butyl (2-bromo-5-formylpyridin-4-yl)carbamate, 3c To a solution of 3b (10 g, 28.4 mmol, 1 equiv.) in THF (150 mL) was added n-BuLi (2.5 M, 27.3 mL, 2.4 equiv.) at −78° C. under N2, and the mixture was stirred at this temperature for 0.5 h. Then, DMF (10.4 g, 142 mmol, 10.9 mL, 5 equiv.) was added at −78° C. and stirred for 0.5 h. The mixture was quenched with aqueous NH4Cl, diluted with 50 mL of water, and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 25 g SepaFlash® silica flash column, elution with a gradient of 0–40% ethyl acetate / petroleum ether at 80 mL / min) to give 3c (4.5 g, 14.94 mmol, 52.60% yield) as a white solid. 1H NMR (CDCl3, 400 MHz) δ 9.93 (s, 1H), 8.62 (s, 1H), 8.51 (s, 1H), 1.55 (s, 9H).

[0275] 3: Preparation of ethyl (E)-3-(6-bromo-4-((tert-butoxycarbonyl)amino)pyridin-3-yl)-2-(cyanomethyl)acrylate, 3d A solution of 3c (4.5 g, 14.9 mmol, 1 equiv.) and ethyl 3-cyano-2-(triphenyl-λ5-phosphanylidene)propanoate (6.37 g, 16.4 mmol, 1.1 equiv.) in DCM (50 mL) was stirred at 30 °C for 2 h. The mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 25 g SepaFlash® silica flash column, elution with a gradient of 0–50% ethyl acetate / petroleum ether at 80 mL / min) to give 3d (5 g, 12.2 mmol, 81.56% yield) as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ 8.44 (s, 1H), 8.07 (s, 1H), 7.68 (s, 1H), 6.48-6.43 (m, 1H), 4.41 (q, J=7.2 Hz, 2H), 3.40 (s, 2H), 1.55 (s, 9H), 1.43 (t, J=7.2 Hz, 3H). LC / MS [M+H] 410.01 (calculated value); LC / MS [M+H] 410.1 (observed value).

[0276] Preparation of ethyl 2-amino-8-bromo-3H-pyrido[4,3-b]azepine-4-carboxylate, 3e To a solution of 3d (5 g, 12.2 mmol, 1 equiv) in EtOAc (10 mL) was added HCl / EtOAc (4 M, 22.2 mL, 7.29 equiv), followed by stirring for 16 h at 25° C. The mixture was filtered and concentrated under reduced pressure to give 3e (4 g, 11.54 mmol, 94.69% yield, HCl) as a yellow solid. 1H NMR (MeOD, 400 MHz) δ 8.62 (s, 1H), 7.96 (s, 1H), 7.61 (s, 1H), 4.36 (q, J=7.2 Hz, 2H), 3.65 (s, 2H), 1.38 (t, J=7.2 Hz, 3H). LC / MS [M+H] 310.01 (calculated value); LC / MS [M+H] 310.1 (observed value).

[0277] Preparation of 2-amino-8-bromo-3H-pyrido[4,3-b]azepine-4-carboxylic acid, 3f To a solution of 3e (2 g, 5.77 mmol, 1 equiv., HCl) in THF (15 mL) and water (5 mL), lithium hydroxide monohydrate, LiOH.HO (484 mg, 11.5 mmol, 2 equiv.), was added and stirred at 45 °C for 1 h. The mixture was quenched with 1 N HCl until pH = 2-3 and extracted with DCM / i-prOH (10 mL × 3, 3 / 1). The organic layer was washed with brine (10 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give 3f (800 mg, 2.84 mmol, 49.15% yield) as a white solid. LC / MS [M+H] 282.0 (calculated); LC / MS [M+H] 282.1 (observed).

[0278] Preparation of tert-butyl (2-((2-amino-8-bromo-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamido)oxy)ethyl)carbamate, 3 g To a solution of 3f (800 mg, 2.84 mmol, 1 equiv) in DCM (8 mL) and DMA (3 mL), methanesulfonic acid (273 mg, 2.84 mmol, 202 μL, 1 equiv), tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (743 mg, 3.40 mmol, 1.2 equiv), and EDCI (2.17 g, 11.3 mmol, 4 equiv) were added, and the mixture was stirred at 25 °C for 1 h. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (15 mL × 2), dried over Na SO , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® silica flash column, elution with a gradient of 0-100% ethyl acetate / petroleum ether at 45 mL / min) to give 3g (800 mg, 1.66 mmol, 58.48% yield) as a brown oil. LC / MS [M+H] 482.1 (calculated); LC / MS [M+H] 482.2 (observed).

[0279] Preparation of methyl 2-amino-4-[2-(tert-butoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-pyrido[4,3-b]azepine-8-carboxylate, azaBz-4 A mixture of 3g (450 mg, 933 μmol, 1 equiv.), Pd(dppf)Cl (68.3 mg, 93.3 μmol, 0.1 equiv.), and EtN (283 mg, 2.80 mmol, 390 μL, 3 equiv.) in MeOH (15 mL) was degassed and purged with CO three times, then stirred at 80 °C for 16 h (50 PSI). The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (neutral column: Waters Xbridge Prep OBD C18 150*40 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 10% to 40%, 8 min) to give azaBz-4 (100 mg, 216.68 μmol, 23.23% yield) as a white solid. 1H NMR (MeOD, 400 MHz) δ 8.57 (s, 1H), 7.80 (s, 1H), 7.36 (s, 1H), 3.96 (s, 3H), 3.91 (t, J=5.2 Hz, 2H), 3.72 (t, J=7.2 Hz, 2H), 3.23 (t, J=5.2 Hz, 2H), 3.00 (s, 2H), 1.76 (sxt, J=7.2 Hz, 2H), 1.32 (s, 9H), 0.98 (t, J=7.2 Hz, 3H). LC / MS [M+H] 462.2 (calculated value); LC / MS [M+H] 462.3 (observed value).

[0280] Preparation of azaBz-3 To a solution of azaBz-4 (200 mg, 433 μmol, 1 equiv) in THF (1 mL) and water (0.3 mL) was added LiOH.HO (21.8 mg, 520 μmol, 1.2 equiv), followed by stirring at 25° C. for 1 h. The mixture was filtered and purified by preparative HPLC (neutral column: Waters Xbridge Prep OBD C18 150*40 mm*10 μm; mobile phase: [water(NHHCO)-ACN]; B%: 5%–35%, 8 min) to give azaBz-3 (20 mg, 44.69 μmol, 10.31% yield) as a white solid. 1 H NMR (MeOD, 400 MHz) δ 8.57 (s, 1H), 7.80 (s, 1H), 7.37 (s, 1H), 3.90 (t, J=5.2 Hz, 2H), 3.73 (t, J=7.2 Hz, 2H), 3.23 (t, J=5.2 Hz, 2H), 3.10 (s, 1H), 1.82-1.71 (m, 2H), 1.33 (s, 9H), 0.98 (t, J=7.2 Hz, 3H). LC / MS [M+H] 448.2 (calculated value); LC / MS [M+H] 448.2 (observed value).

[0281] Example 6: Synthesis of 2-amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, azaBz-6 [ka] Preparation of tert-butyl N-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-pyrido[4,3-b]azepin-8-yl]pyrimidin-2-yl]methyl]-N-tert-butoxycarbonylcarbamate, 6a 2-Amino-8-bromo-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, L-4a (200 mg, 545 umol, 1 equiv.), tert-butyl N-tert-butoxycarbonyl-N-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxabo]azepine-4-carboxamide], L-4b (200 mg, 545 umol, 1 equiv.), tert-butyl N-tert-butoxycarbonyl-N-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxabo]azepine-4-carboxamide], L-4c (200 mg, 545 umol, 1 equiv.), tert-butyl N-tert-butoxycarbonyl-N-[[5-(4,4,5,5-tetramethyl A mixture of [(loran-2-yl)pyrimidin-2-yl]methyl]carbamate (285 mg, 654 μmol, 1.2 equiv.), K2CO3 (151 mg, 1.09 mmol, 2 equiv.), and Pd(dppf)Cl2 (19.9 mg, 27.23 μmol, 0.05 equiv.) was degassed and purged with N2 three times. The mixture was then stirred under N2 atmosphere at 95 °C for 2 h. The reaction mixture was poured into HO (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give 6a (320 mg, 537 μmol, 98.6% yield) as a yellow solid. LC / MS [M+H] 596.3 (calculated); LC / MS [M+H] 596.4 (observed).

[0282] Preparation of azaBz-6 To a solution of 6a (600 mg, 1.01 mmol, 1 equiv) in EtOAc (12 mL) was added HCl / EtOAc (20 mL). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by preparative HPLC column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (TFA)-ACN]; B%: 1% to 20%, 8 min to give azaBz-6 (350 mg, 885 umol, yield 87.87% TFA) as a yellow solid.1 H NMR (MeOD, 400 MHz) δ 9.51 (s, 2H), 8.96 (s, 1H), 8.10 (s, 1H), 7.52 (s, 1H), 4.50 (s, 2H), 3.97 (q, J=7.2 Hz, 2H), 3.76 (t, J=7.2 Hz, 2H), 3.57 (s, 2H), 1.83-1.70 (m, 2H), 1.21 (t, J=7.2 Hz. 3H), 1.00 (t, J=7.2 Hz, 3H). LC / MS [M+H] 396.2 (calculated value); LC / MS [M+H] 396.0 (observed value).

[0283] Example L-1: Synthesis of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl N-[2-[(2-amino-3H-pyrido[3,4-b]azepine-4-carbonyl)-propylamino]oxyethyl]carbamate, azaBzL-1 [ka] Preparation of ethyl (E)-3-[3-(tert-butoxycarbonylamino)-4-pyridyl]-2-(cyanomethyl)prop-2-enoate, L-1b To a solution of tert-butyl N-(4-formyl-3-pyridyl)carbamate, L-1a (500 mg, 2.25 mmol, 1.0 equiv.) in THF (5 mL) was added ethyl 3-cyano-2-(triphenyl-λ 5To the resulting mixture was added 1.2-phosphanylidene)propanoate (871 mg, 2.25 mmol, 1.0 equiv). The mixture was stirred at 55° C. for 1 h. The reaction mixture was quenched by adding HO (5 mL) and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (5 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give L-lb (2.8 g, crude) as a yellow oil. LC / MS [M+H] 332.2 (calculated); LC / MS [M+H] 332.0 (observed).

[0284] Preparation of ethyl 2-amino-3H-pyrido[3,4-b]azepine-4-carboxylate, L-1c To a solution of L-1b (2.6 g, 7.85 mmol, 1.0 equiv.) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 20 mL, 10.2 equiv.). The mixture was stirred at 25° C. for 12 hours, then at 50° C. for an additional 2 hours. The reaction mixture was filtered. The filter cake was then dried under reduced pressure to obtain a residue. Compound L-1c (980 mg, 4.24 mmol, 54.0% yield) was obtained as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ10.27 (s, 1H), 9.27 (s, 1H), 8.72 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 7.88 (s, 1H), 7.74 (d, J = 5.2 Hz, 1H), 4.29 (q, J = 7.2 Hz, 2H), 3.57 (s, 2H), 1.32 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 232.1 (calculated value); LC / MS [M+H] 232.2 (observed value).

[0285] Preparation of 2-amino-3H-pyrido[3,4-b]azepine-4-carboxylic acid, L-1d To a solution of L-1c (480 mg, 2.08 mmol, 1.0 equiv) in HO (1 mL) and EtOH (5 mL) was added LiOH (149 mg, 6.23 mmol, 3.0 equiv). The mixture was stirred at 50 °C for 2 h. The reaction mixture was quenched with 2.5 N HCl at 0 °C until pH = 5-6, and the resulting mixture was concentrated under reduced pressure to remove EtOH. The solid was filtered, and the filter cake was dried under reduced pressure to give residue L-1d (250 mg, 1.23 mmol, 59.2% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.56 (d, J = 5.6 Hz, 1H), 7.86 (s, 1H), 7.79 (d, J = 5.6 Hz, 1H). LC / MS [M+H] 204.1 (calculated); LC / MS [M+H] 204.0 (observed).

[0286] Preparation of tert-butyl N-[2-[(2-amino-3H-pyrido[3,4-b]azepine-4-carbonyl)-propylamino]oxyethyl]carbamate, L-1e To a solution of L-1d (250 mg, 1.23 mmol, 1.0 equiv) and tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (537 mg, 2.46 mmol, 2.0 equiv) in DCM (7.5 mL) and DMA (1.5 mL) was added MsOH (236 mg, 2.46 mmol, 175 μL, 2.0 equiv) and EDCI (943 mg, 4.92 mmol, 4.0 equiv). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated to remove DCM, and the residue was diluted with water (30 mL). The pH of the aqueous phase was then adjusted to 8-9 with aqueous NaCO at 0 °C. It was then extracted with EtOAc (10 mL × 3). The organic layer was washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to give L-1e (420 mg, 1.04 mmol, yield 84.6%) as a white solid. 1H NMR (400 MHz, MeOD) δ8.37 (s, 1H), 8.08 (d, J = 5.2Hz, 1H), 7.37 (d, J = 5.4Hz, 1H), 7.20 (s, 1H), 4.61 (s, 1H), 3.91 (t, J = 5.2Hz, 2H), 3.72 (t, J = 7.2Hz, 2H), 3.23 (t, J = 5.2 Hz, 2H), 2.96 (s, 2H), 1.76 (sxt, J = 7.6Hz, 2H), 1.34 (s, 9H), 0.98 (t, J = 7.6Hz, 3H). LC / MS [M+H] 404.2 (calculated); LC / MS [M+H] 404.2 (observed).

[0287] Preparation of 2-amino-N-(2-aminoethoxy)-N-propyl-3H-pyrido[3,4-b]azepine-4-carboxamide, L-1f To a solution of L-1e (55 mg, 136 μmol, 1.0 equiv) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 10 mL, 293.0 equiv), followed by stirring at 25° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give residue L-1f (51.2 mg, crude, 2HCl) as a white solid. LC / MS [M+H] 304.2 (calculated); LC / MS [M+H] 304.2 (observed).

[0288] Preparation of azaBzL-1 To a solution of L-1f (50 mg, 94.1 μmol, 1.0 equiv, 2 TFA) in DMF (1 mL) was added DIEA (60.8 mg, 470 μmol, 81.9 μL 5.0 equiv) and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl)carbonate (75.6 mg, 94.1 μmol, 1.0 equiv), followed by stirring at 25° C. for 1 h. The mixture was quenched with TFA until the pH reached approximately 6. The mixture was then filtered and purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 5%~35%, 8 min) to give azaBzL-1 (40 mg, 41.3 umol, 43.9% yield) as a yellow oil. 1 H NMR (400 MHz, MeOD -d4) δ8.68 (s, 1H), 8.54 (d, J = 5.6Hz, 1H), 7.63 (d, J = 5.2Hz, 1H), 7.38 (s, 1H), 6.89 (s, 2H), 4.17 (s, 2H), 3.97 (t, J = 4.8 Hz, 2H), 3.89-3.81 (m, 2H), 3.75 (t, J = 7.2Hz, 2H), 3.71-3.57 (m, 38H), 3.54 (t, J = 5.6Hz, 2H), 3.52-3.48 (m, 2H), 3.45 (s, 2H), 3.38 (q, J = 5.2Hz, 2H), 1.85-1.73 (m, 2H), 1.00 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 968.5 (calculated value); LC / MS [M+H] 968.5 (observed value).

[0289] Example L-3: Synthesis of 2-amino-4-(((40-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,39-dioxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-3,38-diazatetracontyl)oxy)(propyl)carbamoyl)-3H-pyrido[4,3-b]azepine-8-sulfonic acid, azaBzL-3 [ka] Preparation of tert-butyl (2-((2-amino-8-(benzylthio)-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamido)oxy)ethyl)carbamate, L-3a tert-Butyl N-[2-[(2-amino-8-bromo-3H-pyrido[4,3-b]azepine-4-carbonyl)-propyl-amino]oxyethyl]carbamate 3g (400mg, 829umol, 1eq), phenylmethanethiol (680mg, 5.47mmol, 642uL, 6.60eq), (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one in dioxane (5mL); palladium, Pd A mixture of 2(dba)3 (75.9 mg, 82.9 μmol, 0.1 equiv), diisopropylethylamine, DIEA (214 mg, 1.66 mmol, 289 μL, 2 equiv), and (5-diphenylphosphanyl-9,9-dimethyl-xanthen-4-yl)diphenylphosphane, XantPhos (96.0 mg, 166 μmol, 0.2 equiv) was degassed and purged with N three times and then stirred at 110 °C under a N atmosphere for 2 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with 50 mL of HO at 0 °C and then extracted with EtOAc (60 mL × 3). The combined organic layers were washed with brine (50 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® silica flash column, elution with a gradient of 0–100% ethyl acetate / petroleum ether at 80 mL / min) to afford L-3a (350 mg, 665.83 μmol, 80.29% yield) as a yellow solid.1 H NMR (MeOD, 400 MHz) δ 8.37 (s, 1H), 7.41 (d, J = 7.2 Hz, 2H), 7.32-7.25 (m, 3H), 7.24-7.19 (m, 1H), 6.92 (s, 1H), 4.37 (s, 2H), 3.89 (t, J = 5.2 Hz, 2H), 3.71 (t, J = 7.2 Hz, 2H), 3.23 (t, J = 5.2 Hz, 2H), 3.00-2.93 (m, 2H), 1.75 (m, 2H), 1.33 (s, 9H), 0.97 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 526.2 (calculated); LC / MS [M+H] 526.3 (observed).

[0290] Preparation of tert-butyl (2-((2-amino-8-(benzylthio)-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamido)oxy)ethyl)carbamate, L-3b To a solution of L-3a (300 mg, 571 umol, 1 equiv) in DCM (10 mL) was added TrtCl (318 mg, 1.14 mmol, 2 equiv) and EtN (231 mg, 2.28 mmol, 318 μL, 4 equiv) at 25 °C, followed by stirring at 50 °C for 16 h. The reaction mixture was diluted with 20 mL of water and extracted with DCM (50 mL × 3). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue, affording compound L-3b (350 mg, 456 umol, 79.86% yield) as a yellow solid. LC / MS [M+H] 768.4 (calculated); LC / MS [M+H] 768.3 (observed).

[0291] Preparation of tert-butyl (2-((8-(chlorosulfonyl)-N-propyl-2-(tritylamino)-3H-pyrido[4,3-b]azepine-4-carboxamido)oxy)ethyl)carbamate, L-3c To a solution of L-3b (300 mg, 391 umol, 1 equiv) in DCM (5 mL) and water (2 mL) was added sulfuryl chloride (211 mg, 1.56 mmol, 156 uL, 4 equiv), followed by stirring at 0 °C for 1 h. The reaction mixture was quenched by adding 10 mL of saturated NaHCO at 0 °C and extracted with DCM (15 mL × 3). The combined organic layers were washed with brine (10 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The crude product L-3c (300 mg, crude) was used in the next step without further purification as a yellow solid. LC / MS [M+H] 744.2 (calculated); LC / MS [M+H] 744.3 (observed).

[0292] Preparation of 4-((2-((tert-butoxycarbonyl)amino)ethoxy)(propyl)carbamoyl)-2-(tritylamino)-3H-pyrido[4,3-b]azepine-8-sulfonic acid, L-3d A solution of L-3c (300 mg, 403 μmol, 1 equiv.) in water (3 mL) and MeCN (5 mL) was stirred at 100° C. for 0.5 h. The mixture was concentrated under reduced pressure. Crude L-1d (270 mg, 372 μmol, 92.23% yield) was obtained as a yellow solid and used in the next step without further purification. LC / MS [M+H] 726.3 (calculated); LC / MS [M+H] 726.3 (observed).

[0293] Preparation of 2-amino-4-[2-aminoethoxy(propyl)carbamoyl]-3H-pyrido[4,3-b]azepine-8-sulfonic acid, L-3e A solution of L-3d (200 mg, 276 μmol, 1 equiv) in DCM (3 mL) and TFA (1 mL) was stirred at 25° C. for 4 h. The mixture was concentrated under reduced pressure. The residue was diluted with water (10 mL) and extracted with MTBE (5 mL) to remove excess TFA, and the aqueous layer was concentrated to give the crude product. The crude product L-3e (100 mg, crude) was used in the next step without further purification as a yellow solid. LC / MS [M+H] 384.1 (calculated); LC / MS [M+H] 384.2 (observed).

[0294] Preparation of azaBzL-3 To a solution of L-3e (50 mg, 130 μmol, 1 equivalent of TFA) and DIEA (67.4 mg, 522 μmol, 90.9 μL, 4 equivalents) in DMF (0.5 mL) was added 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl)carbonate (105 mg, 130 μmol, 1 equivalent) at 0°C, followed by stirring at 0°C for 0.5 hours. The mixture was filtered and purified by preparative HPLC (TFA conditions; column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 5%~25%, 8 min) to give azaBzL-3 (15 mg, 14.31 umol, 10.97% yield) as a white solid. 1 H NMR (MeOD, 400 MHz) δ 8.79 (s, 1H), 7.90 (s, 1H), 7.51 (s, 1H), 6.91 (s, 2H), 4.19 (s, 2H), 3.99 (br t, J=4.6 Hz, 2H), 3.87 (br d, J=4.0 Hz, 2H), 3.77 (br t, J=7.2 Hz, 2H), 3.67-3.61 (m, 38H), 3.58-3.48 (m, 6H), 3.42-3.38 (m, 2H), 1.83-1.75 (m, 2H), 1.05-1.00 (m, 3H). LC / MS [M+H] 1048.4 (calculated); LC / MS [M+H] 1048.5 (observed).

[0295] Example L-4: Synthesis of 2-amino-8-(2-(38-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,37-dioxo-6,9,12,15,18,21,24,27,30,33-decaoxa-2,36-diazaoctatriacontyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, azaBzL-4 [ka] To a mixture of 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, azaBz-6 (100 mg, 196 umol, 1 equiv., TFA) and DIEA (101 mg, 785 umol, 137 uL, 4 equiv.) in DMF (1 mL) was added 2,3,5,6-tetrafluorophenyl AzaBzL-4 (33.3 mg, 31.89 μmol, 16.25% yield) was obtained as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ 9.36 (s, 2H), 8.59 (s, 1H), 7.56 (s, 1H), 7.32 (s, 1H), 6.87 (s, 1H), 4.67 (s, 2H), 4.15 (s, 2H), 3.95-3.85 (m, 2H), 3.75-3.65 (m, 4H), 3.64-3.49 (m, 38H), 3.35-3.32 (m, 2H), 3.02 (s, 2H), 2.62-2.55 (m, 2H), 1.77-1.71 (m, 2H), 1.21-1.12 (m, 3H), 1.02-0.95 (m, 3H). LC / MS [M+H] 1044.3 (calculated); LC / MS [M+H] 1044.5 (observed).

[0296] Example L-5: Synthesis of 2-amino-N8-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl)-N4-ethoxy-N4-propyl-3H-pyrido[4,3-b]azepine-4,8-dicarboxamide, azaBzL-5 [ka] Preparation of tert-butyl (1-(4-(ethoxy(propyl)carbamoyl)-2-(tritylamino)-3H-pyrido[4,3-b]azepin-8-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azatetratriacontan-34-yl)carbamate, L-5a tert-Butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate, NH2-PEG in DMF (8 mL) 10A mixture of N-NHBoc (355 mg, 591 μmol, 1.2 equiv.), 8-bromo-N-ethoxy-N-propyl-2-(tritylamino)-3H-pyrido[4,3-b]azepine-4-carboxamide, L-6b (300 mg, 492.16 μmol, 1 equiv.), triethylamine, TEA (249 mg, 2.46 mmol, 343 μL, 5 equiv.), and Pd(dppf)Cl (72.02 mg, 98.4 μmol, 0.2 equiv.) was degassed and purged with carbon monoxide (CO) three times and then stirred at 80 °C under a CO atmosphere (50 Psi) for 16 h. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (100 mL × 3). The combined organic layer was washed with brine (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 0 / 1 to 1 / 1) to give L-5a (530 mg, 458 μmol, 93.04% yield) as a yellow oil. LC / MS [M+H] 1157.6 (calculated); LC / MS [M+H] 1157.6 (observed).

[0297] Preparation of 2-amino-N8-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-N4-ethoxy-N4-propyl-3H-pyrido[4,3-b]azepine-4,8-dicarboxamide, L-5b A mixture of L-5a (50 mg, 43.2 μmol, 1 equiv) and TFA (98.5 mg, 864 μmol, 63.9 μL, 20 equiv) in DCM (1 mL) was degassed and purged with N three times, then stirred under N atmosphere at 40° C. for 16 h. The reaction mixture was concentrated under reduced pressure to give L-5b (40 mg, crude), which was used in the next step as a yellow oil without further purification. LC / MS [M+H] 815.5 (calculated); LC / MS [M+H] 815.4 (observed).

[0298] Preparation of azaBzL-5 To a solution of L-5b (35 mg, 37.68 μmol, 1 equiv., TFA) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (9.50 mg, 37.7 μmol, 1 equiv.) in DMF (0.2 mL) was added DIEA (19.5 mg, 151 μmol, 26.3 μL, 4 equiv.), followed by stirring at 25 °C for 1 h. The reaction mixture was filtered and purified by preparative HPLC (TFA conditions) using a Phenomenex Luna 80*30 mm*3 μm column; mobile phase: [water (TFA)-ACN]; B%: 10% to 40% over 8 min to give azaBzL-5 (20 mg, 21.01 μmol, 55.76% yield) as a white solid. 1 H NMR (MeOD, 400 MHz) δ 8.79 (s, 1H), 8.01 (s, 1H), 7.50 (s, 1H), 6.87 (s, 2H) 4.14 (s, 2H), 3.92-3.98 (m, 2H), 3.77-3.72 (m, 2H), 3.7-3.65 (m, 2H), 3.63-3.57 (m, 38H), 3.55-3.48 (m, 2H), 3.47-3.44 (m, 2H), 3.41-3.32 (m, 2H), 1.78-1.72 (m, 2H), 1.18 (t, J=7.2 Hz, 3H), 0.98 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 952.5 (calculated); LC / MS [M+H] 952.5 (observed).

[0299] Example L-6: Synthesis of 2-amino-8-(N-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl)sulfamoyl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, azaBzL-6 [ka] Preparation of 2-amino-8-bromo-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, L-6a To a solution of 2-amino-8-bromo-3H-pyrido[4,3-b]azepine-4-carboxylic acid, 3f (3 g, 10.6 mmol, 1 equiv.) in DCM (40 mL) and DMA (10 mL) was added methanesulfonic acid (1.02 g, 10.6 mmol, 0.757 mL, 1 equiv.), N-ethoxypropan-1-amine (1.29 g, 9.25 mmol, 0.9 equiv., HCl), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDCI, CAS Registry Number 1892-57-5 (8.15 g, 42.5 mmol, 4 equiv.), and the mixture was stirred at 25° C. for 1 h. The reaction mixture was diluted with water (80 mL) and extracted with DCM (50 mL × 3). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The crude product was triturated with MTBE (10 mL) at 25 °C for 10 min to give L-6a (3.2 g, 8.71 mmol, 81.9% yield) as a white solid. LC / MS [M+H] 367.1 (calculated); LC / MS [M+H] 367.0 (observed).

[0300] Preparation of 8-bromo-N-ethoxy-N-propyl-2-(tritylamino)-3H-pyrido[4,3-b]azepine-4-carboxamide, L-6b To a solution of L-6a (700 mg, 1.91 mmol, 1 equiv.) in DCM (20 mL) was added triphenylmethyl chloride, trityl chloride, TrtCl (1.33 g, 4.77 mmol, 2.5 equiv.), and triethylamine, EtN (964 mg, 9.53 mmol, 1.33 mL, 5 equiv.), followed by stirring at 50 °C for 16 h. The reaction mixture was diluted with water (20 mL) and extracted with DCM (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, elution with a gradient of 0–60% ethyl acetate / petroleum ether at 50 mL / min) to give L-6b (1 g, 1.64 mmol, 86.07% yield) as a white solid. LC / MS [M+H] 609.2 (calculated); LC / MS [M+H] 609.0 (observed).

[0301] Preparation of 8-(benzylthio)-N-ethoxy-N-propyl-2-(tritylamino)-3H-pyrido[4,3-b]azepine-4-carboxamide, L-6c To a solution of L-6b (600 mg, 0.986 mmol, 1 equiv.) and phenylmethanethiol, BnSH (306 mg, 2.46 mmol, 0.289 mL, 2.5 equiv.) in dioxane (30 mL) was added (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one; palladium, Pd(dba) (90.3 mg, 98.6 mmol, 0.1 equiv.), DIEA (255 mg, 1.97 mmol, 0.343 mL, 2 equiv.), and (5-diphenylphosphanyl-9,9-dimethyl-xanthen-4-yl)-diphenyl-phosphane, Xantphos, CAS Registry Number 161265-03-8 (114 mg, 0.197 mmol, 0.2 equiv.), and the mixture was stirred at 110 °C under N for 2 h. The reaction mixture was quenched at 0°C by adding water (50 mL) and then extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (30 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, elution with a gradient of 0-80% ethyl acetate / petroleum ether at 70 mL / min) to give L-6c (500 mg, 0.767 mmol, 77.8% yield) as a yellow solid. LC / MS [M+H] 653.3 (calculated); LC / MS [M+H] 653.0 (observed).

[0302] Preparation of 4-(ethoxy(propyl)carbamoyl)-2-(tritylamino)-3H-pyrido[4,3-b]azepine-8-sulfonyl chloride, L-6d To a solution of L-6c (380 mg, 0.582 mmol, 1 equiv) in DCM (2 mL) and water (0.3 mL) was added sulfuryl chloride, SO2Cl2 (393 mg, 2.91 mmol, 0.291 mL, 5 equiv) at 0 °C, followed by stirring at 25 °C for 1 h. The reaction mixture was quenched by adding saturated aqueous NaHCO3 (15 mL) at 0 °C and then extracted with DCM (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude L-6d (400 mg, crude) as a yellow oil, which was used in the next step without further purification. LC / MS [M+H] 629.2 (calculated); LC / MS [M+H] 629.0 (observed).

[0303] Preparation of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[ethoxy(propyl)carbamoyl]-2-(tritylamino)-3H-pyrido[4,3-b]azepin-8-yl]sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate, L-6e tert-Butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate, NH2-PEG 10To a solution of -NHBoc (458 mg, 0.76 mmol, 1.2 equiv) and EtN (322 mg, 3.18 mmol, 0.442 mL, 5 equiv) in DCM (8 mL) was added L-6d (400 mg, 0.64 mmol, 1 equiv) at 0 °C, followed by stirring at 25 °C for 0.5 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® silica flash column, elution with a gradient of 0–100% ethyl acetate / petroleum ether at 50 mL / min) to give L-6e (500 mg, 0.419 mmol, 65.90% yield) as a colorless oil. LC / MS [M+H] 1193.6 (calculated); LC / MS [M+H] 1193.3 (observed).

[0304] Preparation of 2-amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)sulfamoyl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, L-6f To a solution of L-6e (100 mg, 83.8 μmol, 1 equiv) in DCM (0.3 mL) was added TFA (191 mg, 1.68 mmol, 124 μL, 20 equiv), followed by stirring at 50° C. for 0.5 h. The mixture was filtered and concentrated under reduced pressure to give L-6f (100 mg, crude) as a yellow oil, which was used in the next step without further purification. LC / MS [M+H] 851.4 (calculated); LC / MS [M+H] 851.2 (observed).

[0305] Preparation of azaBzL-6 To a solution of L-6f (100 mg, 104 μmol, 1 equiv., TFA) in DMF (0.3 mL), diisopropylethylamine, DIEA (53.6 mg, 414 μmol, 72.2 μL, 4 equiv.), and (2,5-dioxopyrrolidin-1-yl)2-(2,5-dioxopyrrol-1-yl)acetate (26.13 mg, 104 μmol, 1 equiv.) were added at 0°C and stirred at 25°C for 0.5 h. The mixture was filtered and purified by preparative HPLC (TFA conditions; column: Phenomenex Luna 80*30 mm*3 μm; mobile phase: [water (TFA)-ACN]; B%: 1% to 30%, 8 min) to give azaBzL-6 (53 mg, 53.6 μmol, 51.8% yield) as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ 8.86 (s, 1H), 7.94 (s, 1H), 7.52 (s, 1H), 6.89 (s, 2H), 4.17 (s, 2H), 3.98 (q, J=7.2 Hz, 2H), 3.76 (t, J=7.2 Hz, 2H), 3.66-3.59 (m, 32H), 3.56-3.47 (m, 10H), 3.37 (td, J=4.8, 9.6 Hz, 4H), 1.78 (m, 2H), 1.20 (t, J=7.2 Hz, 3H), 1.00 (t, J=7.6 Hz, 3H). LC / MS [M+H] 988.5 (calculated); LC / MS [M+H] 988.2 (observed).

[0306] Example L-7: Synthesis of 2-amino-8-(N-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl)-N-methylsulfamoyl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, azaBzL-7 [ka] Preparation of tert-butyl (2-((4-(ethoxy(propyl)carbamoyl)-2-(tritylamino)-3H-pyrido[4,3-b]azepin-8-yl)sulfonyl)-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azatetratriacontan-34-yl)carbamate, L-7a To a solution of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[ethoxy(propyl)carbamoyl]-2-(tritylamino)-3H-pyrido[4,3-b]azepin-8-yl]sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate, L-6e (200 mg, 168 μmol, 1 equiv) in MeCN (1 mL) was added cesium carbonate, CsCO (109 mg, 0.335 mmol, 2 equiv) and methyl iodide, MeI (28.5 mg, 201 μmol, 12.5 μL, 1.2 equiv), and the mixture was stirred at 25 °C for 1 h. The resulting mixture was filtered and concentrated under reduced pressure to afford L-7a (200 mg, crude) as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ 8.44 (s, 1H), 7.35-7.31 (m, 7H), 7.25-7.16 (m, 9H), 6.91 (s, 1H), 4.61 (s, 1H), 4.05-3.99 (m, 2H), 3.79 (t, J=6.8 Hz, 2H), 3.64-3.48 (m, 42H), 3.23-3.19 (m, 2H), 3.04 (s, 2H), 2.89 (s, 3H), 1.83-1.77 (m, 2H), 1.43 (s, 9H), 1.24 (t, J=7.2 Hz, 3H), 1.01 (t, J=7.6 Hz, 3H). LC / MS [M+H] 1207.6 (calculated); LC / MS [M+H] 1207.8 (observed).

[0307] Preparation of 2-amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-N-methylsulfamoyl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4-carboxamide, L-7b To a solution of L-7a (140 mg, 116 μmol, 1 equiv) in DCM (0.3 mL) was added TFA (264 mg, 2.32 mmol, 172 μL, 20 equiv), followed by stirring at 50° C. for 1 h. The mixture was filtered and concentrated under reduced pressure to give L-7b (100 mg, crude) as a yellow solid. LC / MS [M+H] 865.5 (calculated); LC / MS [M+H] 865.5 (observed).

[0308] Preparation of azaBzL-7 To a solution of L-7b (100 mg, 102 μmol, 1 equiv., TFA) and DIEA (52.8 mg, 409 μmol, 71.2 μL, 4 equiv.) in DMF (0.5 mL) was added (2,5-dioxopyrrolidin-1-yl)2-(2,5-dioxopyrrol-1-yl)acetate (25.8 mg, 102 μmol, 1 equiv.) at 0° C. and stirred at 25° C. for 0.5 h. The mixture was filtered and purified by preparative HPLC (TFA condition: Phenomenex Luna 80*30 mm*3 μm; mobile phase: [water (TFA)-ACN]; B%: 5% to 35%, 8 min) to give azaBzL-7 (45 mg, 44.90 μmol, 43.96% yield) as a yellow oil. 1H NMR (MeOD, 400 MHz) δ 8.83 (s, 1H), 7.87 (s, 1H), 7.51 (s, 1H), 6.89 (s, 2H), 4.17 (s, 2H), 3.98 (q, J=7.2 Hz, 2H), 3.78-3.73 (m, 2H), 3.66-3.58 (m, 34H), 3.56-3.51 (m, 4H), 3.51-3.46 (m, 7H), 3.37 (br t, J=5.2 Hz, 2H), 3.06 (s, 2H), 1.78 (m, 2H), 1.20 (t, J = 7.2 Hz, 3H), 1.00 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 1002.5 (calculated); LC / MS [M+H] 1002.2 (observed).

[0309] Example L-8: Synthesis of 2-amino-N8-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl)-N4-ethoxy-N8-methyl-N4-propyl-3H-pyrido[4,3-b]azepine-4,8-dicarboxamide, azaBzL-8 [ka] Preparation of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[ethoxy(propyl)carbamoyl]-2-(tritylamino)-3H-pyrido[4,3-b]azepine-8-carbonyl]-methyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate, L-8a To a solution of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[ethoxy(propyl)carbamoyl]-2-(tritylamino)-3H-pyrido[4,3-b]azepine-8-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate, L-5a (160 mg, 138 μmol, 1 equiv.) in THF (2 mL) was added t-BuOK (1 M, 276 μL, 2 equiv.) and MeI (19.6 mg, 138 μmol, 8.61 μL, 1 equiv.) at 25° C. for 2 hours. The mixture was filtered and purified by preparative HPLC (TFA condition; column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 35%-65%, 8 min) to give L-6a (80 mg, 68.3 umol, 49.40% yield) as a colorless oil. LC / MS [M+H] 1171.7 (calculated); LC / MS [M+H] 1171.3 (observed).

[0310] Preparation of 2-amino-N8-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-N4-ethoxy-N8-methyl-N4-propyl-3H-pyrido[4,3-b]azepine-4,8-dicarboxamide, L-8b To a solution of L-8a (70 mg, 59.8 μmol, 1 equiv.) in DCM (0.2 mL) was added TFA (68.1 mg, 598 μmol, 44.2 μL, 10 equiv.), and the mixture was stirred at 50°C for 0.5 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna 80*30 mm*3 μm; mobile phase: [water (TFA)-ACN]; B%: 1% to 28%, 8 min) to give L-8b (30 mg, 31.8 μmol, 53.24% yield, TFA) as a yellow oil. 1H NMR (DMSO-d6, 400 MHz,) δ 8.52 (s, 1H), 8.51-8.46 (m, 1H), 7.72 (s, 1H), 7.68-7.49 (m, 2H), 7.21 (s, 1H), 3.86 (q, J = 6.8 Hz, 2H), 3.65-.48 (m, 44H), 3.04-2.97 (m, 4H), 2.90 (s, 3H), 1.72-1.63 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H), 0.96-0.91 (m, 3H). LC / MS [M+H] 829.5 (calculated); LC / MS [M+H] 829.5 (observed).

[0311] Preparation of azaBzL-8 A solution of L-8b (20 mg, 24.1 μmol, 1 equiv) in DMF (0.5 mL) was added with DIEA (12.5 mg, 96.5 μmol, 16.8 μL, 4 equiv) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (6.08 mg, 24.1 μmol, 1 equiv) and stirred at 25 °C for 0.5 h. The mixture was filtered and purified by preparative HPLC (TFA conditions, column: Phenomenex Luna 80*30 mm*3 μm; mobile phase: [water (TFA)-ACN]; B%: 5% to 35%, 8 min) to give azaBzL-8 (15 mg, 15.5 μmol, 64.4% yield) as a yellow oil. 1 H NMR (MeOD, 400 MHz) δ 8.80 (s, 1H), 8.16 (s, 1H), 7.52 (s, 1H), 6.91 (s, 2H), 4.19 (s, 2H), 4.02-3.94 (m, 2H), 3.77 (t, J = 7.2 Hz, 2H), 3.72 (m, 2H), 3.69-3.61 (m, 36H), 3.56 (m, 2H), 3.44-3.36 (m, 6H), 3.17 (s, 3H), 1.79 (m, 2H), 1.23-1.17 (m, 3H), 1.05-0.99 (m, 3H). LC / MS [M+H] 966.5 (calculated); LC / MS [M+H] 966.2 (observed).

[0312] Example L-9: Synthesis of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl N-[2-[(6-amino-7H-pyrido[3,2-b]azepine-8-carbonyl)-propyl-amino]oxyethyl]carbamate, azaBzL-9 [ka] Preparation of 4-(tert-butyl) 1-ethyl (E)-2-((3-((tert-butoxycarbonyl)amino)pyridin-2-yl)methylene)succinate, L-9b A solution of tert-butyl (2-formylpyridin-3-yl)carbamate, L-9a (1 g, 4.50 mmol, 1 equiv.) and O4-tert-butyl O1-ethyl 2-(triphenyl-λ5-phosphanylidene)butanedioate (2.50 g, 5.40 mmol, 1.2 equiv.) in DCM (5 mL) was stirred at 50 °C for 2 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, elution with a gradient of 0–30% ethyl acetate / petroleum ether at 60 mL / min) to afford L-9b (350 mg, 861 μmol, 19.1% yield) as a colorless oil. 1 H NMR (CDCl3, 400 MHz) δ 8.36-8.31 (m, 2H), 7.76 (s, 1H), 7.25 (dd, J = 4.8, 8.4 Hz, 1H), 6.72 (s, 1H), 4.32 (q, J = 7.2 Hz, 2H), 3.70 (s, 2H), 1.54 (s, 9H), 1.42 (s, 9H), 1.36 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 407.2 (calculated value); LC / MS [M+H] 407.2 (observed value).

[0313] Preparation of ethyl 6-hydroxy-7H-pyrido[3,2-b]azepine-8-carboxylate, L-9c To a solution of L-9b (350 mg, 861 umol, 1 equiv) in DCM (5 mL) was added TFA (982 mg, 8.61 mmol, 638 uL, 10 equiv) and stirred at 50° C. for 3 h. The mixture was concentrated under reduced pressure to give L-9c (300 mg, crude) as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ 8.78 (s, 1H), 8.65 (dd, J = 1.2, 4.8 Hz, 1H), 8.05 (s, 1H), 7.64-7.60 (m, 1H), 7.53 (dd, J = 4.4, 8.4 Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H), 3.43 (s, 2H), 1.39 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 233.0 (calculated value); LC / MS [M+H] 233.2 (observed value).

[0314] Preparation of ethyl 6-amino-7H-pyrido[3,2-b]azepine-8-carboxylate, L-9d Compound L-9c (700 mg, 3.01 mmol, 1 equiv) was dissolved in POCl (6.93 g, 45.2 mmol, 4.21 mL, 15 equiv), and the mixture was stirred at 90 °C for 16 h under N. The mixture was concentrated under reduced pressure. The residue was then dissolved in MeCN (20 mL), and NH.HO (27.30 g, 234 mmol, 30 mL, 30% purity, 65.1 equiv) was added to the solution, followed by stirring at 25 °C for 0.5 h. The reaction mixture was extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give L-9d (600 mg, 2.59 mmol, 72.2% yield) as a brown solid. LC / MS [M+H] 232.1 (calculated); LC / MS [M+H] 232.1 (observed).

[0315] Preparation of 6-amino-7H-pyrido[3,2-b]azepine-8-carboxylic acid, L-9e To a solution of L-9d (600 mg, 2.59 mmol, 1 equiv.) in MeOH (1 mL) and water (0.3 mL) was added lithium hydroxide monohydrate, LiOH.HO (327 mg, 7.78 mmol, 3 equiv.), followed by stirring at 25 °C for 2 h. The pH of the mixture was adjusted to 5-6 with 1 N HCl, and the precipitate was then filtered and the solid concentrated under reduced pressure to give L-9e (300 mg, 1.48 mmol, 56.9% yield) as a brown solid. LC / MS [M+H] 204.1 (calculated); LC / MS [M+H] 204.2 (observed).

[0316] Preparation of tert-butyl (2-((6-amino-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamido)oxy)ethyl)carbamate, L-9f To a solution of L-9e (240 mg, 1.18 mmol, 1 equiv.) and tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (335 mg, 1.54 mmol, 1.3 equiv.) in DCM (3 mL) and dimethylacetamide, DMA (2 mL), methanesulfonic acid (170 mg, 1.77 mmol, 127 μL, 1.5 equiv.) and EDCI (906 mg, 4.72 mmol, 4 equiv.) were added and then stirred at 25° C. for 1 h. The reaction mixture was diluted with 10 mL of water and extracted with EtOAc (15 mL × 3). The combined organic layers were washed with brine (10 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 25 g SepaFlash® silica flash column, elution with a gradient of 0-100% ethyl acetate / petroleum ether to 50% ethyl acetate / MeOH, 45 mL / min) to afford L-9f (300 mg, 743 μmol, 62.9% yield) as a brown oil. LC / MS [M+H] 404.2 (calculated); LC / MS [M+H] 404.3 (observed).

[0317] Preparation of 6-amino-N-(2-aminoethoxy)-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-9g To a solution of L-9f (200 mg, 496 μmol, 1 equiv.) in dioxane (1 mL) was added HCl / dioxane (4 M, 4.00 mL, 32 equiv.), followed by stirring at 25° C. for 0.5 h. The mixture was concentrated under reduced pressure to give the crude product L-9g (200 mg, crude, HCl) as a brown solid. LC / MS [M+H] 304.2 (calculated); LC / MS [M+H] 304.2 (observed).

[0318] Preparation of azaBzL-9 To a solution of L-9g (150 mg, 441 μmol, 1 equiv., HCl) and diisopropylethylamine, DIEA (285 mg, 2.21 mmol, 384 μL, 5 equiv.) in DMF (1 mL) was added 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl(4-nitrophenyl)carbonate (248 mg, 309 μmol, 0.7 equiv.) and then stirred at 25° C. for 1 h. The mixture was filtered, and the filtrate was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 10% to 40% of B over 8.0 min) to give azaBzL-9 (50 mg, 51.6 μmol, 11.7% yield) as a yellow oil. 1H NMR (MeOD, 400 MHz) δ 8.66 (dd, J = 1.2, 4.4 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.58 (dd, J = 4.4, 8.4 Hz, 1H), 7.37 (s, 1H), 6.89 (s, 2H), 4.17 (s, 2H), 3.99 (t, J = 5.2 Hz, 2H), 3.94-3.88 (m, 2H), 3.77 (t, J = 7.2 Hz, 2H), 3.66-3.59 (m, 38H), 3.56-3.52 (m, 2H), 3.52-3.49 (m, 2H), 3.47 (s, 2H), 3.41-3.35 (m, 2H), 1.85-1.75 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 968.5 (calculated value); LC / MS [M+H] 968.3 (observed value).

[0319] Example L-10: Synthesis of 6-amino-3-[2-[[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]methyl]pyrimidin-5-yl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, azaBzL-10 [ka] [ka] Preparation of (E)-2-(5-bromo-3-nitro-2-pyridyl)-N,N-dimethyl-ethenamine, L-10b To a solution of 5-bromo-2-methyl-3-nitro-pyridine, L-10a (150 g, 691 mmol, 1 equiv.) in DMF (750 mL) was added DMF-DMA (165 g, 1.38 mol, 184 mL, 2 equiv.) at 25 °C, then heated to 90 °C and stirred at 90 °C for 1 h. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure to give a residue. The residue was diluted with HO (1000 mL) at 0 °C, the solid was filtered, and the filter cake was dried under reduced pressure to give L-10b (181 g, 665 mmol, 96.2% yield) as a red solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.44 (d, J = 2.4 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.14 (d, J = 12.4 Hz, 1H), 6.07 (d, J = 12.4 Hz, 1H), 3.04 (s, 6H).

[0320] Preparation of 5-bromo-3-nitro-pyridine-2-carbaldehyde, L-10c To a solution of L-10b (90 g, 331 mmol, 1 equiv.) in THF (750 mL) and HO (750 mL), NaIO (156 g, 728 mmol, 40.3 mL, 2.2 equiv.) was added portionwise at 0 °C, then warmed to 25 °C and stirred at 25 °C for 1 h. The reaction mixture was filtered, and the filtrate was diluted with HO (500 mL) and extracted with EtOAc (500 mL × 3). The combined organic layers were washed with saturated NaSO (150 mL × 3), brine (150 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give L-10c (54.3 g, 235 mmol, 71.1% yield) as a brown solid. 1 H NMR (CDCl3, 400 MHz) δ 10.22 (s, 1H), 9.04 (d, J = 1.6 Hz, 1H), 8.38 (d, J = 1.6 Hz, 1H). LC / MS [M+H] 230.9 (calculated); LC / MS [M+H] 231.0 (observed).

[0321] Preparation of O4-tert-butyl O1-ethyl (2E)-2-[(5-bromo-3-nitro-2-pyridyl)methylene]butanedioate, L-10d To a solution of L-10c (103 g, 446 mmol, 1 equiv.) in DCM (1500 mL), O4-tert-butyl O1-ethyl 2-(triphenylphosphanylidene)butanedioate (186 g, 401 mmol, 0.9 equiv.) was added portionwise under N2 at 25 °C, then heated to 50 °C and stirred for 4 h. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give L-10d (150.8 g, 363 mmol, 81.5% yield) as a pale yellow solid. 1 H NMR (CDCl3, 400 MHz) δ 8.89 (d, J = 2.0 Hz, 1H), 8.48 (d, J = 2.0 Hz, 1H), 8.01 (s, 1H), 4.33 (q, J = 7.2 Hz, 2H), 3.75 (s, 2H), 1.42 (s, 9H), 1.36 (t, J = 7.2 Hz, 3H). LC / MS [M+Na] 437.0 (calculated value); LC / MS [M+Na] 436.9 (observed value).

[0322] Preparation of O4-tert-butyl O1-ethyl (2E)-2-[(3-amino-5-bromo-2-pyridyl)methylene]butanedioate, L-10e To a solution of L-10d (30.1 g, 72.5 mmol, 1 equiv.) in AcOH (500 mL), iron powder, Fe (20.2 g, 362 mmol, 5 equiv.) was added portionwise under N at 25 °C, followed by stirring at 25 °C for 2 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was diluted with HO (500 mL) and adjusted to pH 8–9 with saturated NaHCO at 0 °C. The mixture was filtered, and the filtrate was extracted with EtOAc (400 mL × 3). The combined organic layers were washed with brine (200 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give L-10e (27.3 g, 71 mmol, 97.9% yield) as a yellow solid. 1H NMR (MeOD, 400 MHz) δ 7.92 (d, J = 2.0 Hz, 1H), 7.71 (s, 1H), 7.30 (d, J = 2.0 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 3.80 (s, 2H), 1.42 (s, 9H), 1.34 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 385.1 (calculated value); LC / MS [M+H] 385.0 (observed value).

[0323] Preparation of methyl 8-(dipropylcarbamoyl)-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylate, L-10f To a solution of L-10e (82.0 g, 213 mmol, 1 equiv) in DCM (900 mL) was added TFA (243 g, 2.13 mol, 158 mL, 10 equiv) at 25 °C, then heated to 50 °C and stirred for 2 h. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure to give a residue. The residue was poured into ice water (1000 mL) at 0 °C and adjusted to pH = 8-9 with saturated NaHCO at 0 °C. The aqueous phase was extracted with EtOAc (500 mL × 3). The combined organic layers were washed with brine (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give L-10f (74.0 g, crude) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 10.64 (s, 1H), 8.58 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.67 (s, 1H), 4.26 (q, J = 7.2 Hz, 2H), 3.24 (s, 2H), 1.29 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 310.9 (calculated value); LC / MS [M+H] 310.9 (observed value).

[0324] Preparation of ethyl 6-amino-3-bromo-7H-pyrido[3,2-b]azepine-8-carboxylate, L-10g To a solution of L-10f (10.1 g, 32.5 mmol, 1 equiv) in dioxane (100 mL) was added POCl (24.9 g, 162 mmol, 15.1 mL, 5 equiv) at 25 °C, then heated to 120 °C and stirred for 6 h. The reaction mixture was cooled to 0 °C and added to NH.HO (275 g, 1.96 mol, 303 mL, 25% purity, 58.3 equiv) in CHCN (40 mL), then stirred at 0 °C for 1 h. The reaction mixture was filtered, and the filter cake was dried under reduced pressure to obtain the pure product. The filtrate was extracted with EtOAc (150 mL × 3). The combined organic layers were washed with brine (100 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to ethyl acetate / methanol = 10:1) to give L-10g (9.1 g, 29.3 mmol, 87.1% yield) as a brown solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.34 (d, J = 2.0 Hz, 1H), 7.66 (s, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.33 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 2.96 (s, 2H), 1.30 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 310.0 (calculated value); LC / MS [M+H] 309.9 (observed value).

[0325] Preparation of 6-amino-3-bromo-7H-pyrido[3,2-b]azepine-8-carboxylic acid, L-10h A solution of LiOH.HO (11.0 g, 261 mmol, 3 equiv.) in HO (100 mL) was added to a solution of L-10g (27.0 g, 87.1 mmol, 1 equiv.) in THF (300 mL) at 0 °C, then warmed to 25 °C and stirred for 2 h. The reaction mixture was cooled to 0 °C, adjusted to pH 5-6 with 1 N HCl at 0 °C, and then concentrated under reduced pressure to remove THF. The solid precipitate was filtered, and the filter cake was dried under reduced pressure to give L-10h (24.1 g, 85.5 mmol, 98.1% yield) as a brown solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.32 (d, J = 2.0 Hz, 1H), 7.61 (s, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.28 (s, 2H), 2.94 (s, 2H). LC / MS [M+H] 282.0 (calculated value); LC / MS [M+H] 281.9 (observed value).

[0326] Preparation of 6-amino-3-bromo-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-10i To a solution of L-10h (12.0 g, 42.5 mmol, 1 equiv.), N-ethoxypropan-1-amine (5.94 g, 42.5 mmol, 1 equiv., HCl), and MsOH (4.09 g, 42.5 mmol, 3.04 mL, 1 equiv.) in DCM (120 mL) and DMA (12 mL) was added EDCI (24.5 g, 128 mmol, 3 equiv.) at 0 °C, then warmed to 25 °C and stirred for 1 h. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was quenched by adding HO (50 mL) at 0 °C, then adjusted to pH = 8-9 with aqueous NaHCO at 0 °C, and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to ethyl acetate / methanol = 10:1) to give L-10i (15 g, 41.0 mmol, 96.3% yield) as a brown solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.28 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.24 (s, 1H), 7.17 (s, 1H), 7.02 (s, 1H), 3.85 (q, J = 7.2 Hz, 2H), 3.62 (t, J = 7.2 Hz, 2H), 2.84 (s, 2H), 1.69-1.59 (m, 2H), 1.05 (t, J = 7.2 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 367.1 (calculated); LC / MS [M+H] 367.0 (observed).

[0327] Preparation of tert-butyl N-[[5-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]azepin-3-yl]pyrimidin-2-yl]methyl]-N-tert-butoxycarbonyl-carbamate, L-10j To a solution of tert-butyl N-tert-butoxycarbonyl-N-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl]methyl]carbamate (180 mg, 414 μmol, 0.8 equiv.) in dioxane (2 mL), L-10i (190 mg, 517 μmol, 1 equiv.) and KCO (143 mg, 1.03 mmol, 2 equiv.) in water (0.2 mL) were added, followed by the addition of Pd(dppf)Cl (37.9 mg, 51.7 μmol, 0.1 equiv.) and stirring at 100° C. under N for 1 h. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (TFA)-ACN]; gradient: 5% to 35% of B in 8 min) to give L-10j (160 mg, 268 μmol, 51.9% yield) as a yellow solid. LC / MS [M+H] 596.3 (calculated); LC / MS [M+H] 596.3 (observed).

[0328] Preparation of 6-amino-3-(2-(aminomethyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-10k To a solution of L-10j (160 mg, 269 μmol, 1 equiv) in EtOAc (2 mL) was added HCl / EtOAc (4 M, 5 mL, 74.5 equiv), followed by stirring at 25 °C for 1 h. The mixture was concentrated under reduced pressure to give L-10k (100 mg, 231 μmol, 86.2% yield, HCl) as a brown solid. LC / MS [M+H] 396.2 (calculated); LC / MS [M+H] 396.2 (observed).

[0329] Preparation of azaBzL-10 To a solution of L-10k (100 mg, 232 μmol, 1 equiv., HCl) in DMF (1 mL) was added N-methylmorpholine, NMM (46.8 mg, 463 μmol, 50.9 μL, 2 equiv.) and (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (245 mg, 301 μmol, 1.3 equiv.) at 0° C., followed by stirring at 0° C. for 0.5 h. The reaction mixture was quenched with TFA to pH = 5–6 and then purified by preparative HPLC (TFA conditions; column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 10% to 40% of B over 8.0 min) to give azaBzL-10 (50 mg, 47.8 μmol, 20.6% yield) as a yellow oil. 1H NMR (MeOD, 400 MHz) δ 9.17 (s, 2H), 9.06 (d, J = 2.0 Hz, 1H), 8.14 (d, J = 2.0 Hz, 1H), 7.49 (s, 1H), 6.91 (s, 2H), 4.72 (s, 2H), 4.18 (s, 2H), 3.81 (q, J = 7.2 Hz, 2H), 3.83-3.75 (m, 4H), 3.65-3.60 (m, 36H), 3.54-3.52 (m, 4H), 3.38-3.36 (m, 2H), 2.62 (t, J = 6.0 Hz, 2H), 1.84-1.78 (m, 2H), 1.24 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1044.5 (calculated value); LC / MS [M+H] 1044.3 (observed value).

[0330] Example L-17: Synthesis of (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]azepin-3-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, azaBzL-17 [ka] A solution of (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (97.3 mg, 0.114 mmol, 2.5 equiv) in DMF (0.5 mL) To the mixture was added a solution of 6-amino-3-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-10k (18 mg, 0.0455 mmol, 1 equiv.) and DIEA (35.3 mg, 0.273 mmol, 0.476 mL, 6 equiv.) in DMF (0.5 mL) at 0° C., and the mixture was then stirred at 25° C. for 1 h. The reaction mixture was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 20% to 50% of B over 8.0 min) to give azaBzL-17 (12 mg, 0.111 mmol, 24.3% yield) as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ 9.16 (s, 2H), 9.03 (d, J = 2.0 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 7.47-7.38 (m, 2H), 4.72 (d, J = 5.6 Hz, 2H), 4.02 (q, J = 7.2 Hz, 2H), 3.90-3.86 (m, 2H), 3.83-3.77 (m, 4H), 3.68-3.60 (m, 36H), 3.54 (s, 2H), 2.99 (t, J = 6.0 Hz, 2H), 2.62 (t, J = 6.0 Hz, 2H), 1.85-1.76 (m, 2H), 1.24 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1084.5 (calculated value); LC / MS [M+H] 1084.7 (observed value).

[0331] Example L-20: Synthesis of 2-amino-N-((40-(2,5-dimethylene-2,5-dihydro-1H-pyrrol-1-yl)-4,39-dioxo-8,11,14,17,20,23,26,29,32,35-decaoxa-3,5,38-triazatetracontyl)oxy)-N-propyl-3H-pyrido[3,4-b]azepine-4-carboxamide, azaBzL-20 [ka] [ka] Preparation of tert-butyl (32-isocyanato-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate, L-20b To a solution of tert-butyl (32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate, L-20a (0.15 g, 0.25 mmol, 1 equiv.) in DCM was added TEA (0.348 mL, 2.5 mmol, 10 equiv.), followed by phosgene (0.892 mL as a 1.4 M solution in toluene, 0.25 mmol, 1 equiv.). The reaction mixture was monitored by LCMS, concentrated, and purified by reverse-phase HPLC to give L-20b (78 mg, 0.125 mmol, 50%). LC / MS [M+H] 627.37 (calculated); LC / MS [M+H] 627.64 (observed).

[0332] Preparation of 2-amino-N-(2-aminoethoxy)-N-propyl-3H-pyrido[3,4-b]azepine-4-carboxamide, L-20d tert-Butyl (2-((2-amino-N-propyl-3H-pyrido[3,4-b]azepine-4-carboxamido)oxy)ethyl)carbamate, L-20c (6.1 mg, 0.015 mmol, 1 equiv.) was suspended in minimal TFA. After 15 min, the reaction mixture was concentrated to give crude L-20d (12.7 mg, 0.031 mmol, 100%). LC / MS [M+H] 304.18 (calculated); LC / MS [M+H] 304.28 (observed).

[0333] Preparation of tert-butyl (39-(2-amino-3H-pyrido[3,4-b]azepine-4-carbonyl)-34-oxo-3,6,9,12,15,18,21,24,27,30,38-undecaoxa-33,35,39-triazadtetracontyl)carbamate, L-20e To a mixture of L-20d (37.8 mg, 0.124 mmol, 1 equiv.) and 2Am4CBza-L-18b (78 mg, 0.124 mmol, 1 equiv.) in DMF was added TEA (0.17 mL, 1.24 mmol, 10 equiv.). The reaction was stirred at room temperature, then diluted with water and purified by reverse-phase HPLC to give L-20e (48 mg, 0.052 mmol, 41%). LC / MS [M+H] 930.54 (calculated); LC / MS [M+H] 930.54 (observed).

[0334] Preparation of 2-amino-N-((37-amino-4-oxo-8,11,14,17,20,23,26,29,32,35-decaoxa-3,5-diazaheptatriacontyl)oxy)-N-propyl-3H-pyrido[3,4-b]azepine-4-carboxamide, L-20f L-20e (48 mg, 0.052 mmol, 1 equiv.) was dissolved in a minimum amount of TFA. After 15 min, the reaction mixture was concentrated to give L-20f as a TFA salt (0.053 g, 0.050 mmol, 96%). LC / MS [M+H] 830.49 (calculated); LC / MS [M+H] 830.76 (observed).

[0335] Preparation of azaBzL-20 To a solution of L-20f (0.053 g, 0.050 mmol, 1 equiv.) in DMF (0.5 mL) was added TEA (0.09 mL, 0.64 mmol, 12.8 equiv.), followed by 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (0.016 g, 0.064 mmol, 1.28 equiv.). The reaction mixture was concentrated, diluted with 1% TFA in water, and purified by reverse-phase HPLC to give azaBzL-20 (38.5 mg, 0.040 mmol, 80%). LC / MS [M+H] calculated: 967.50; LC / MS [M+H] observed: 967.80.

[0336] Alternatively, azaBzL-20 may be synthesized as follows. [ka] Preparation of ethyl 2-(tritylamino)-3H-pyrido[3,4-b]azepine-4-carboxylate, L-20h To a solution of ethyl 2-amino-3H-pyrido[3,4-b]azepine-4-carboxylate, L-20g (4 g, 17.3 mmol, 1 equiv.) in DCM (50 mL) was added EtN (3.50 g, 34.5 mmol, 4.82 mL, 2 equiv.) and TrtCl (12.0 g, 43.2 mmol, 2.5 equiv.), followed by stirring at 50 °C for 16 h. The mixture was filtered, washed with EtOAc (50 mL), and the filtrate was concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® silica flash column, elution with a gradient of 0–40% ethyl acetate / MeOH at 50 mL / min) to afford L-20h (5.2 g, 10.9 mmol, 63.4% yield) as a pale yellow solid. LC / MS [M+H] 474.2 (calculated); LC / MS [M+H] 474.1 (observed).

[0337] Preparation of 2-(tritylamino)-3H-pyrido[3,4-b]azepine-4-carboxylic acid, L-20i To a solution of L-20h (4.5 g, 9.50 mmol, 1 equiv) in THF (30 mL) and MeOH (15 mL) was added a solution of LiOH.HO (1.20 g, 28.51 mmol, 3 equiv) in HO (10 mL), followed by stirring at 60° C. for 2 h. The mixture was concentrated to remove THF and MeOH, the suspension was filtered, and the filter cake was concentrated to give L-20i (3.5 g, 7.86 mmol, 82.68% yield) as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ 7.84 (d, J = 5.2 Hz, 1H), 7.64 (s, 1H), 7.43 (s, 1H), 7.35-7.12 (m, 16H), 2.93 (s, 2H). LC / MS [M+H] 446.2 (calculated value); LC / MS [M+H] 446.1 (observed value).

[0338] Preparation of tert-butyl N-[2-[propyl-[2-(tritylamino)-3H-pyrido[3,4-b]azepine-4-carbonyl]amino]oxyethyl]carbamate, L-20j To a solution of L-20i (3.5 g, 7.86 mmol, 1 equiv.) in DMA (8 mL) and DCM (40 mL), methanesulfonic acid (755 mg, 7.86 mmol, 561 μL, 1 equiv.), tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (1.71 g, 7.86 mmol, 1 equiv.), and EDCI (4.52 g, 23.57 mmol, 3 equiv.) were added and then stirred at 0 °C for 0.5 h. The mixture was concentrated to remove DCM, adjusted to pH = 10 with aqueous NaCO solution, and extracted with DCM (50 mL × 3). The combined organic phase was washed with brine (50 mL), dried over NaSO, and concentrated to give L-20j (4.5 g, 6.97 mmol, 88.70% yield) as a pale yellow oil. 1H NMR (MeOD, 400 MHz) δ 7.92-7.82 (m, 1H), 7.67 (s, 1H), 7.41-7.32 (m, 6H), 7.29-7.13 (m, 11H), 3.97 (t, J = 5.2 Hz, 2H), 3.77 (t, J = 6.8 Hz, 2H), 3.28-3.26 (m, 2H), 3.06 (s, 2H), 1.89-1.73 (m, 2H), 1.32 (s, 9H), 1.00 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 646.3 (calculated value); LC / MS [M+H] 646.2 (observed value).

[0339] Preparation of N-(2-aminoethoxy)-N-propyl-2-(tritylamino)-3H-pyrido[3,4-b]azepine-4-carboxamide, L-20k To a solution of L-20j (2.7 g, 4.18 mmol, 1 equiv) in EtOAc (20 mL) was added HCl / EtOAc (4 M, 52.26 mL, 50 equiv), followed by stirring at 25 °C for 0.5 h. The mixture was concentrated to give crude L-20k (2.5 g, crude, HCl) as a pale yellow solid. LC / MS [M+H] 546.3 (calculated); LC / MS [M+H] 546.2 (observed).

[0340] Preparation of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[propyl-[2-(tritylamino)-3H-pyrido[3,4-b]azepine-4-carbonyl]amino]oxyethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate, L-20l To a solution of N-(2-aminoethoxy)-N-propyl-2-(tritylamino)-3H-pyrido[3,4-b]azepine-4-carboxamide (2 g, 3.44 mmol, 1 equiv., HCl) in DMF (15 mL) was added DIEA (888 mg, 6.87 mmol, 1.20 mL, 2 equiv.) and tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-isocyanatoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate, L-20b (2.05 g, 3.26 mmol, 0.95 equiv.), followed by stirring at 0° C. for 1 h. The mixture was adjusted to pH 5 with TFA at 0°C and filtered. The filtrate was purified by preparative HPLC (column: Phenomenex luna C18 (250*70mm, 15um); mobile phase [HO (0.1% TFA)-ACN]; gradient: 30% to 60% of B in 20.0 min) to give L-20l (3.7g, 3.16mmol, 91.8% yield) as a yellow oil. 1 H NMR (MeOD, 400 MHz) δ 8.13 (d, J = 6.0 Hz, 1H), 7.87-7.81 (m, 2H), 7.36-7.33 (m, 7H), 7.30-7.15 (m, 9H), 4.02-4.01 (m, 2H), 3.80 (t, J = 7.2 Hz, 2H), 3.72-3.52 (m, 39H), 3.50-3.44 (m, 2H), 3.41-3.33 (m, 4H), 3.25-3.17 (m, 4H), 3.05 (t, J = 5.2 Hz, 2H), 1.88-1.75 (m, 2H), 1.43 (s, 9H), 1.03 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1172.6 (calculated value); LC / MS [M+H] 1172.9 (observed value).

[0341] Preparation of N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoylamino]ethoxy]-N-propyl-2-(tritylamino)-3H-pyrido[3,4-b]azepine-4-carboxamide, L-20m To a solution of L-20l (1.5 g, 1.28 mmol, 1 equiv) in EtOAc (20 mL) was added HCl / EtOAc (4 M, 15.9 mL, 50 equiv), followed by stirring for 0.5 h at 25° C. The mixture was concentrated to give crude L-20m (1.5 g, crude, HCl) as a pale yellow solid. 1 H NMR (MeOD,400 MHz,) δ 8.23 ​​(d, J = 6.0 Hz, 1H), 7.95 (d, J = 6.0 Hz, 1H), 7.90 (s, 1H), 7.49-7.20 (m, 16H), 4.05 (t, J = 5.2 Hz, 2H), 3.87-3.76 (m, 4H), 3.74-3.56 (m, 38H), 3.44 (t, J = 5.2 Hz, 2H), 3.38 (t, J = 5.2 Hz, 2H), 3.18-3.16 (m, 4H), 1.89-1.78 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1072.6 (calculated); LC / MS [M+H] 1072.4 (observed).

[0342] Preparation of N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoylamino]ethoxy]-N-propyl-2-(tritylamino)-3H-pyrido[3,4-b]azepine-4-carboxamide, L-20n To a solution of L-20m (1.5 g, 1.35 mmol, 1 equiv., HCl) in CHCN (30 mL) was added DIEA (874 mg, 6.76 mmol, 1.18 mL, 5 equiv.) and (2,5-dioxopyrrolidin-1-yl)2-(2,5-dioxopyrrol-1-yl)acetate (307 mg, 1.22 mmol, 0.9 equiv.), followed by stirring at 0 °C for 0.5 h. The mixture was adjusted to pH = 4 with TFA and then concentrated to remove CHCN. Crude L-20n (1.6 g, crude) was obtained as a yellow oil. LC / MS [M+H] 1209.6 (calculated); LC / MS [M+H] 1209.9 (observed).

[0343] Preparation of azaBzL-20 To a solution of L-20n (2.2 g, 1.82 mmol, 1 equiv) in DCM (20 mL) was added TFA (4.15 g, 36.3 mmol, 2.70 mL, 20 equiv), followed by stirring at 50 °C for 16 h. The mixture was concentrated to remove DCM and TFA. The residue was purified by preparative HPLC (column: Welch Ultimate XB-Diol 250*50*10 μm; mobile phase: [heptane-EtOH]; gradient: 5% to 95% B over 25.0 min) to afford azaBzL-20 (858.6 mg, 887.84 μmol, 48.81% yield) as a pale orange oil. 1 H NMR (MeOD, 400 MHz,) δ 8.66 (s, 1H), 8.53 (d, J = 5.2 Hz, 1H), 7.61 (d, J = 5.2 Hz, 1H), 7.39 (s, 1H), 6.89 (s, 2H), 4.17 (s, 2H), 3.95 (t, J = 5.2 Hz, 2H), 3.82-3.73 (m, 2H), 3.70-3.52 (m, 38H), 3.46 (s, 2H), 3.40-3.38 (m, 6H), 3.11 (t, J = 5.2 Hz, 2H), 1.85-1.69 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 967.5 (calculated); LC / MS [M+H] 967.5 (observed).

[0344] Example L-28: Synthesis of (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[6-amino-8-(dipropylcarbamoyl)-7H-pyrido[3,2-b]azepin-3-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, azaBzL-28 [ka] Preparation of 6-amino-3-bromo-N,N-dipropyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-28a To a solution of 6-amino-3-bromo-7H-pyrido[3,2-b]azepine-8-carboxylic acid, L-10h (0.6 g, 2.13 mmol, 1 equiv.) in DMF (10 mL) was added HATU (889 mg, 2.34 mmol, 1.1 equiv.), N-propylpropan-1-amine (430 mg, 4.25 mmol, 586 μL, 2 equiv.), and DIEA (824 mg, 6.38 mmol, 1.11 mL, 3 equiv.), followed by stirring at 0° C. for 0.5 h. The mixture was diluted with ice-water (50 mL) and extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® silica flash column, elution with a gradient of 0–100% ethyl acetate / petroleum ether to 5 / 1 EtOAc at 60 mL / min) to afford L-28a (0.53 g, 1.45 mmol, 68.2% yield) as a yellow oil. 1H NMR (MeOD, 400 MHz) δ 8.31 (d, J = 2.0 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 6.84 (s, 1H), 3.51-3.38 (m, 4H), 3.30 (s, 2H), 1.78-1.56 (m, 4H), 1.08-0.77 (m, 6H). LC / MS [M+H] 365.1 (calculated value); LC / MS [M+H] 365.0 (observed value).

[0345] Preparation of tert-butyl N-[[5-[6-amino-8-(dipropylcarbamoyl)-7H-pyrido[3,2-b]azepin-3-yl]pyrimidin-2-yl]methyl]-N-tert-butoxycarbonyl-carbamate, L-28b To a mixture of L-28a (0.43 g, 1.18 mmol, 1 equiv.), tert-butyl N-tert-butoxycarbonyl-N-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl]methyl]carbamate (615 mg, 1.41 mmol, 1.2 equiv.) in dioxane (3 mL) and HO (0.3 mL) was added KCO (325.41 mg, 2.35 mmol, 2 equiv.) and Pd(dppf)Cl (86.1 mg, 117.7 μmol, 0.1 equiv.) under N under N, followed by stirring at 100 °C for 2 h under N. The mixture was filtered and concentrated to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® silica flash column, elution with a gradient of 0–100% ethyl acetate / petroleum ether to 5 / 1 EtOAc / MeOH, 75 mL / min) to afford L-28b (0.6 g, 1.01 mmol, 85.8% yield) as a pale yellow solid. 1H NMR (MeOD, 400 MHz) δ 9.11 (s, 2H), 8.63 (d, J = 2.0 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H), 6.94 (s, 1H), 5.07 (s, 2H), 3.45 (t, J = 7.2 Hz, 4H), 3.32 (s, 2H), 1.79-1.61 (m, 4H), 1.45 (s, 18H), 1.09-0.78 (m, 6H). LC / MS [M+H] 594.3 (calculated value); LC / MS [M+H] 594.4 (observed value).

[0346] Preparation of 6-amino-3-[2-(aminomethyl)pyrimidin-5-yl]-N,N-dipropyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-28c To a solution of L-28b (100 mg, 168 μmol, 1 equiv) in EtOAc (2 mL) was added HCl / EtOAc (4 M, 2.11 mL, 50 equiv), followed by stirring for 0.5 h at 25° C. The mixture was concentrated to give L-28c (70 mg, 162 μmol, 96.6% yield, HCl) as an off-white solid. 1 H NMR (MeOD, 400 MHz) δ 9.30 (s, 2H), 9.06 (d, J = 2.0 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 7.12 (s, 1H), 4.52 (s, 2H), 3.61-3.42 (m, 6H), 1.84-1.67 (m, 4H), 1.11-0.87 (m, 6H). LC / MS [M+H] 394.2 (calculated value); LC / MS [M+H] 394.1 (observed value).

[0347] Preparation of azaBzL-28 A mixture of L-28c (50 mg, 116 μmol, 1 equiv., HCl) and DIEA (45.0 mg, 348 μmol, 60.7 μL, 3 equiv.) in DMF (1 mL) was added to a solution of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]eth ... The filtrate was purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 25% to 45% of B in 8.0 min) to give azaBzL-28 (64.5 mg, 49.2 μmol, 42.3% yield, 2% TFA) as a pale yellow oil. 1 H NMR (MeOD, 400 MHz) δ 9.17 (s, 2H), 9.04 (d, J = 2.0 Hz, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.48-7.40 (m, 1H), 7.11 (s, 1H), 4.72 (s, 2H), 3.88 (t, J = 6.0 Hz, 2H), 3.82 (t, J = 6.0 Hz, 2H), 3.71-3.59 (m, 36H), 3.58-3.43 (m, 6H), 2.99 (t, J = 6.0 Hz, 2H), 2.62 (t, J = 6.0 Hz, 2H), 1.82-1.67 (m, 4H), 1.00-0.97 (m, 6H). LC / MS [M+H] 1082.5 (calculated); LC / MS [M+H] 1082.4 (observed).

[0348] Example L-32: Synthesis of (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]azepine-3-carbonyl]amino]-7,8-dihydro-5H-1,6-naphthyridin-6-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, azaBzL-32 [ka] [ka] Preparation of 3-bromo-N-ethoxy-N-propyl-6-(tritylamino)-7H-pyrido[3,2-b]azepine-8-carboxamide and 3-bromo-6-(ditritylamino)-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-32a To a solution of 6-amino-3-bromo-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-10i (25.0 g, 68.1 mmol, 1 equiv.) and TEA (20.7 g, 204 mmol, 28.4 mL, 3 equiv.) in DCM (500 mL) was added trityl chloride, TrtCl (28.5 g, 102 mmol, 1.5 equiv.) at 25 °C under N, then heated to 50 °C and stirred for 12 h. The reaction mixture was quenched at 0 °C by the addition of HO (300 mL) and then extracted with DCM (150 mL × 3). The combined organic layers were washed with brine (50 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give L-32a (11.3 g, 18.5 mmol, 27.2% yield) as a yellow solid and 3-bromo-6-(ditritylamino)-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-32b (8.8 g, 10.3 mmol, 15.1% yield) as a yellow solid. LC / MS [M+H] 609.2 (calculated); LC / MS [M+H] 609.1 (observed). LC / MS [M+H] 851.3 (calculated); LC / MS [M+H] 851.3 (observed).

[0349] Preparation of methyl 8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylate, L-32c A mixture of L-32a (5.60 g, 9.19 mmol, 1 equiv.), Pd(dppf)Cl (672 mg, 918 μmol, 0.1 equiv.), and TEA (2.79 g, 27.6 mmol, 3.84 mL, 3 equiv.) in MeOH (50 mL) was degassed and purged with CO three times, then heated to 80 °C and stirred under CO atmosphere (50 psi) for 16 h. The reaction mixture was cooled to 25 °C, filtered, and the filter cake was dried under reduced pressure to give the pure product. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give L-32c (3.50 g, 5.95 mmol, 64.7% yield) as a brown solid. 1 H NMR (CDCl3, 400 MHz) δ 8.78 (s, 1H), 7.50 (s, 2H), 7.35-7.28 (m, 6H), 7.26-7.15 (m, 9H), 6.33 (s, 1H), 3.98-3.96 (m, 2H), 3.90 (s, 3H), 3.76 (t, J = 6.8 Hz, 2H), 2.83 (s, 2H), 1.78-1.75 (m, 2H), 1.28 (t, J = 6.8 Hz,3H), 0.98 (t, J = 6.8 Hz, 3H). LC / MS [M+H] 589.3 (calculated); LC / MS [M+H] 589.2 (observed).

[0350] Preparation of methyl 8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylic acid, L-32d To a solution of L-32c (2.75 g, 4.67 mmol, 1 equiv) in THF (30 mL) was added a solution of LiOH.HO (588 mg, 14.0 mmol, 3 equiv) in HO (10 mL) at 0 °C, then warmed to 25 °C and stirred for 2 h. The reaction mixture was cooled to 0 °C, diluted with HO (30 mL), adjusted to pH = 5 with 2 N HCl at 0 °C, and concentrated under reduced pressure to remove THF. The mixture was filtered, and the filter cake was dried under reduced pressure to give L-32d (2.80 g, crude) as a pale yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.57 (s, 1H), 8.39 (s, 1H), 7.33-7.20 (m, 12H), 7.19-7.14 (m, 4H), 7.12 (s, 1H), 3.96 (q, J = 7.2 Hz, 2H), 3.70 (t, J = 6.8 Hz, 2H), 3.03 (s, 2H), 1.77-1.64 (m, 2H), 1.14 (t, J = 6.8 Hz, 3H), 0.94 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 575.3 (calculated value); LC / MS [M+H] 575.3 (observed value).

[0351] Preparation of tert-butyl 3-[[8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carbonyl]amino]-7,8-dihydro-5H-1,6-naphthyridine-6-carboxylate, L-32e To a solution of L-32d (0.95 g, 1.65 mmol, 1 equiv.) in acetonitrile and ACN (10 mL) was added N-methylimidazole, NMI (1.36 g, 16.5 mmol, 1.32 mL, 10 equiv.), and tert-butyl 3-amino-7,8-dihydro-5H-1,6-naphthyridine-6-carboxylate (618 mg, 2.48 mmol, 1.5 equiv.), followed by TCFH (1.86 g, 6.61 mmol, 4 equiv.). The mixture was stirred at 25 °C for 2 h, quenched at 0 °C by the addition of HO (20 mL), and then extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® silica flash column, elution with a gradient of 70% ethyl acetate / petroleum ether at 80 mL / min) to give L-32e (2.3 g, crude) as a yellow oil. LC / MS [M+H] 806.4 (calculated); LC / MS [M+H] 806.3 (observed).

[0352] Preparation of N8-ethoxy-N8-propyl-N3-(5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3,8-dicarboxamide, L-32f To a solution of L-32e (2.30 g, 2.85 mmol, 1 equiv) in EtOAc (20 mL) was added HCl / EtOAc (4 M, 10.7 mL, 15 equiv), followed by stirring at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Welch Xtimate C18 250*70 mm*10 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 25% to 55% of B over 20 min) to give L-32e (1 g, 1.42 mmol, 49.7% yield) as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.81 (s, 1H), 8.71 (d, J = 2.0 Hz, 1H), 8.23 ​​(d, J = 2.0 Hz, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.45-7.22 (m, 16H), 4.48 (s, 2H), 4.05 (q, J = 7.2 Hz, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 3.23 (t, J = 6.4 Hz, 4H), 1.88-1.77 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H), 1.04 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 706.3 (calculated value); LC / MS [M+H] 706.4 (observed value).

[0353] Preparation of 6-amino-N8-ethoxy-N8-propyl-N3-(5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-7H-pyrido[3,2-b]azepine-3,8-dicarboxamide, L-32g To a solution of L-32f (0.30 g, 425 μmol, 1 equiv.) in DCM (3 mL) was added TFA (969 mg, 8.50 mmol, 631 μL, 20 equiv.), which was then heated to 50 °C and stirred for 16 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 1% to 30% B over 8.0 min) to give L-32g (0.18 g, 388 μmol, 91.4% yield) as a white solid. 1 H NMR (MeOD, 400 MHz) δ 9.15 (s, 1H), 8.80 (s, 1H), 8.35 (s, 1H), 8.28 (s, 1H), 7.45 (s, 1H), 4.49 (s, 2H), 4.10 (q, J = 7.2 Hz, 2H), 3.77 (t, J = 7.2 Hz, 2H), 3.66 (t, J = 6.4 Hz, 2H), 3.51 (s, 2H), 3.23 (t, J = 6.4 Hz, 2H), 1.84-1.74 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 464.2 (calculated); LC / MS [M+H] 464.1 (observed).

[0354] Preparation of azaBzL-32 To a solution of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (295 mg, 345 μmol, 2 equiv.) in DMF (2 mL) was added dropwise a solution of DIEA (22.3 mg, 173 μmol, 30.1 μL, 1 equiv.) and L-32g (0.08 g, 173 μmol, 1 equiv.) in DMF (2 mL), followed by stirring at 0° C. for 1 hour. The reaction mixture was adjusted to pH = 6 with TFA and then purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 15% to 45% of B in 8.0 min) to give azaBzL-32 (0.04 g, 34.2 μmol, 19.8% yield, 98.5% purity) as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ 9.20 (s, 1H), 8.95 (s, 1H), 8.41-8.22 (m, 2H), 7.50-7.36 (m, 2H), 4.03-3.95 (m, 4H), 3.85 (t, J = 6.0 Hz, 2H), 3.82-3.74 (m, 4H), 3.64-3.56 (m, 34H), 3.53-3.51 (m, 5H), 3.39-3.37 (m, 1H), 3.18 (t, J = 6.0 Hz, 1H), 3.08-3.01 (m, 1H), 2.96 (t, J = 6.0 Hz, 2H), 2.83-2.77 (m, 2H), 1.83-1.74 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7 Hz, 3H). LC / MS [M+H] 1152.5 (calculated value); LC / MS [M+H] 1152.6 (observed value).

[0355] Example L-37: Synthesis of 6-amino-3-[3-[3-[[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]methyl]azetidin-1-yl]sulfonylphenyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, azaBzL-37 [ka] Preparation of tert-butyl N-[[1-(3-bromophenyl)sulfonylazetidin-3-yl]methyl]carbamate, L-37a To a solution of tert-butyl N-(azetidin-3-ylmethyl)carbamate hydrochloride (10.5 g, 46.9 mmol, 1 equiv.) in DCM (25 mL) were added triethylamine, EtN (9.50 g, 93.9 mmol, 13.1 mL, 2 equiv.), and 3-bromobenzenesulfonyl chloride (12.0 g, 46.9 mmol, 6.77 mL, 1 equiv.) at 0 °C, followed by stirring at 25 °C for 1 h. The residue was poured into water (15 mL). The aqueous phase was extracted with DCM (20 mL × 3). The combined organic phase was washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was triturated with MTBE (30 mL) at 0 °C for 15 min to give L-37a (14.0 g, 34.5 mmol, 73.6% yield) as a white solid: LC / MS [M+Na] 427.0 (calculated); LC / MS [M+Na] 427.0 (observed).

[0356] Preparation of tert-butyl N-[[1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methyl]carbamate, L-37b A mixture of L-37a (3.00 g, 7.40 mmol, 1 equiv.), KOAc (1.45 g, 14.8 mmol, 2 equiv.), Pin2B2 (2.44 g, 9.62 mmol, 1.3 equiv.), and Pd(dppf)Cl2 (270 mg, 370 μmol, 0.05 equiv.) in dioxane (30 mL) was degassed and purged with N2 three times at 25 °C, heated to 95 °C, and stirred under N2 atmosphere for 2 h. The reaction mixture was concentrated in vacuo. The residue was purified by flash silica gel chromatography (biotage®; 40 g SepaFlash® silica flash column, elution with a gradient of 0–35% ethyl acetate / petroleum ether at 80 mL / min) to afford L-37b (3.30 g, 7.29 mmol, 98.6% yield) as a yellow oil. 1 H NMR (CDCl3, 400 MHz) δ 8.26 (s, 1 H), 8.06 (d, J=7.2 Hz, 1 H), 7.91 (d, J = 8.0 Hz, 1 H), 7.57 (t, J = 8.0 Hz, 1 H), 3.82 (t, J = 8.0 Hz 2 H), 3.55-3.46 (m, 2H), 3.16 (t, J = 6.0 Hz, 2H),2.65-2.55(m, 1H), 1.36 (s, 9H), 1.27 (s, 12H). LC / MS [M+Na] 475.2 (calculated value); LC / MS [M+Na] 475.1 (observed value).

[0357] Preparation of tert-butyl N-[[1-[3-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]azepin-3-yl]phenyl]sulfonylazetidin-3-yl]methyl]carbamate, L-37c To a solution of L-37c (312 mg, 689 μmol, 1.3 equiv) in dioxane (5.00 mL) and HO (0.5 mL) was added KCO (147 mg, 1.06 mmol, 2 equiv), 6-amino-3-bromo-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-10i (195 mg, 531 μmol, 1 equiv), and Pd(dppf)Cl (19.4 mg, 26.5 μmol, 0.05 equiv) at 25 °C. The solution was degassed and purged with N three times, then heated to 95 °C and stirred under a N atmosphere for 2 h. The reaction mixture was cooled to 25 °C, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (biotage®; 4 g SepaFlash® silica flash column, elution with a gradient of 0–40% ethyl acetate / petroleum ether at 60 mL / min) to afford L-37c (300 mg, 489 μmol, 92.3% yield) as a brown oil. 1 H NMR (CDCl3, 400 MHz) δ 8.78 (s, 1 H), 8.12 (s, 1 H) , 7.90-7.97 (m, 3 H), 7.65-7.77 (m, 2 H), 3.98 (q, J = 7.2 Hz, 2 H), 3.92-3.84 (m, 2 H), 3.75 (t, J = 7.2 Hz, 2 H), 3.62-3.55 (m, 2 H), 3.21-3.11 (m, 2 H), 3.10 (s, 2 H), 2.72-2.59 (m, 1 H), 1.78-1.75 (m, 2 H), 1.39 (s, 9 H), 1.31-1.28 (m, 3 H), 0.99 (t, J = 7.6, 3 H). LC / MS [M+H] 613.3 (calculated); LC / MS [M+H] 613.2 (observed).

[0358] Preparation of 6-amino-3-[3-[3-(aminomethyl)azetidin-1-yl]sulfonylphenyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-37d To a solution of L-37c (0.25 g, 408 μmol, 1 equiv.) in DCM (1 mL) was added TFA (465 mg, 4.08 mmol, 303 μL, 10 equiv.), which was then heated to 50 °C and stirred for 1 h. The reaction mixture was cooled to 25 °C and concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex luna C18 250*50 mm*15 μm; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 5% to 30% B over 10.0 min) to give L-37d (100 mg, 195 μmol, 47.8% yield) as a white solid. LC / MS [M+H] 513.2 (calculated); LC / MS [M+H] 513.2 (observed).

[0359] Preparation of azaBzL-37 To a solution of L-37d (50.0 mg, 97.5 μmol, 1 equiv.) in DMF (0.5 mL), DIEA (37.8 mg, 293 μmol, 50.9 μL, 3 equiv.) and (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy ... The filtrate was purified by preparative HPLC (column: Phenomenex luna C18 100*40mm*3um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 10% to 45% of B over 8.0 min) to give azaBzL-37 (33.0 mg, 28.4 μmol, 29.1% yield) as a colorless oil. 1H NMR (MeOD, 400 MHz) δ 9.05 (d, J = 2.0 Hz, 1 H), 8.19-8.16 (m, 3 H), 7.98 (d, J = 8.0 Hz, 1 H), 7.89 (t, J = 8.0 Hz, 1 H), 7.48 (s, 1 H), 6.89 (s, 2 H), 4.16 (s, 2 H), 4.05-3.99 (m, 2 H), 3.89 (t, J = 8.0 Hz, 2 H), 3.79-3.75 (m, 2 H), 3.65-3.59 (m, 38 H), 3.58-3.56 (m, 2 H), 3.55-3.53 (m, 4 H), 3.36 (s, 2 H), 3.15 (d, J = 6.4 Hz, 2 H), 2.73-2.64 (m, 1 H), 2.29 (t, J = 6.0 Hz, 2 H), 1.84-1.75 (m, 2 H), 1.23 (t, J = 7.2 Hz, 3 H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1161.5 (calculated value); LC / MS [M+H] 1161.7 (observed value).

[0360] Example L-38: Synthesis of (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[3-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]azepin-3-yl]phenyl]sulfonylazetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, azaBzL-38 [ka] (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (140 mg, 164 μmol, 1.2 equiv.) in DMF (1 mL To the solution of 1,2-dimethyl-3,4-dichloro-2 ... The filtrate was purified by preparative HPLC (column: Phenomenex Luna C18 80*30mm*3um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 30% to 60% of B over 8.0 min) to give azaBzL-38 (30.0 mg, 24.9 μmol, 18.3% yield) as a colorless oil. 1H NMR (MeOD, 400 MHz) δ 9.04 (d, J = 2.0 Hz, 1 H), 8.18-8.16 (m, 3 H), 7.98 (d, J = 8.0 Hz, 1 H), 7.89 (t, J = 8.0 Hz, 1 H), 7.48 (s, 1 H), 7.44-7.37 (m, 1 H), 4.01 (q, J = 7.2 Hz, 2 H), 3.91-3.87 (m, 2 H), 3.87-3.84 (m, 2 H), 3.77 (t, J = 7.2 Hz, 2 H), 3.64-3.59 (m, 36 H), 3.57-3.55 (m, 2H), 3.54-3.51 (m, 4 H), 3.14 (d, J = 6.4 Hz, 2 H), 2.97 (t, J = 6.00 Hz, 2 H), 2.74-2.64 (m, 1 H), 2.28 (t, J = 6.00 Hz, 2 H), 1.82-1.75 (m, 2 H), 1.22 (t, J = 7.2 Hz, 3 H), 1.02 (t, J = 7.2 Hz, 3 H). LC / MS [M+H] 1201.5 (calculated value); LC / MS [M+H] 1201.4 (observed value).

[0361] Example L-39: Synthesis of 6-amino-3-[(3S)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]piperidine-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, azaBzL-39 [ka] Preparation of tert-butyl N-[(3S)-1-[8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carbonyl]-3-piperidyl]carbamate, L-39a To a mixture of 8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylic acid, L-32d (300 mg, 522 μmol, 1 equiv.) and tert-butyl N-[(3S)-3-piperidyl]carbamate (125 mg, 627 μmol, 1.2 equiv.) in MeCN (8 mL), NMI (171 mg, 2.09 mmol, 167 μL, 4 equiv.) and chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate, TCFH, CAS Registry Number 207915-99-9 (219 mg, 783 μmol, 1.5 equiv.) were added and stirred at 25 °C for 1 h. Alternatively, HATU may be used as the coupling reagent. The reaction mixture was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with brine (8 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (biotage®; 12 g SepaFlash® silica flash column, elution with a gradient of 0–45% ethyl acetate / petroleum ether at 80 mL / min) to afford L-39a (350 mg, 462 μmol, 88.6% yield) as a yellow oil. LC / MS [M+H] 757.4 (calculated); LC / MS [M+H] 757.4 (observed).

[0362] Preparation of 6-amino-3-[(3S)-3-aminopiperidine-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-39b To a solution of L-39a (350 mg, 462 μmol, 1 equiv) in DCM (7 mL) was added TFA (1.58 g, 13.8 mmol, 1.03 mL, 30 equiv), which was then heated to 50° C. and stirred for 2 h. The reaction mixture was concentrated in vacuo. The crude product was purified by recrystallization from MTBE (15 mL) at 0° C. to give L-39b (160 mg, 386 μmol, 83.5% yield) as a yellow solid. LC / MS [M+H] 415.2 (calculated); LC / MS [M+H] 415.3 (observed).

[0363] Preparation of azaBzL-39 To a mixture of L-39b (60.0 mg, 145 μmol, 1 equiv.) in DMF (2 mL) was added DIEA (56.1 mg, 434 μmol, 75.6 μL, 3 equiv.) and (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (118 mg, 145 μmol, 1 equiv.) at 0° C., followed by stirring at 25° C. for 0.5 h. The pH of the reaction solution was adjusted to approximately 9 by adding TFA, and the resulting mixture was purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 5% to 45% of B over 8.0 min) to give azaBzL-39 (35.0 mg, 32.9 μmol, 22.7% yield) as a colorless oil. 1 H NMR (DMSO-d6, 400 MHz) δ 9.28 (s, 1H), 8.64 (s, 1H), 8.21 (t, J = 5.2 Hz, 1H), 7.93 (d, J = 7.2 Hz, 1H), 7.79 (s, 1H), 7.21 (s, 1H), 7.08 (s, 2H), 4.01 (s, 2H), 3.90 (q, J = 7.2 Hz, 2H), 3.64 (t, J = 7.2 Hz, 2H), 3.62-3.58 (m, 1H), 3.52-3.47 (m, 38H), 3.45-3.43 (m, 4H), 3.43-3.42 (m, 2H), 3.41-3.40 (m, 2H), 3.21-3.17 (m, 2H), 2.36-2.29 (m, 2H), 1.89-1.77 (m, 2H), 1.68-1.63 (m, 2H), 1.57-1.45 (m, 2H), 1.09 (t, J = 7.2 Hz, 3H), 0.91 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1063.5 (calculated value); LC / MS [M+H] 1063.4 (observed value).

[0364] Example L-41: Synthesis of 6-amino-3-[(3R)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]piperidine-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, azaBzL-41 [ka] Preparation of tert-butyl N-[(3R)-1-[8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carbonyl]-3-piperidyl]carbamate, L-41a To a solution of 8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylic acid, L-32d (300 mg, 522 μmol, 1 equiv.) in DMF (5.00 mL), HATU (297 mg, 783 μmol, 1.5 equiv.), DIEA (134 mg, 1.04 mmol, 181 μL, 2 equiv.), and tert-butyl N-[(3R)-3-piperidyl]carbamate (125 mg, 626 μmol, 1.2 equiv.) were added at 0° C., warmed to 25° C., and stirred for 1 h. The reaction mixture was diluted with 5 mL of ice water and extracted with 15 mL of ethyl acetate (5 mL × 3). The organic layer was washed with water (5 mL) and brine (5 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica flash column, elution with a gradient of 0-100% ethyl acetate / petroleum ether at 60 mL / min) to give L-41a (443 mg, crude) as a yellow oil. LC / MS [M+H] 757.4 (calculated); LC / MS [M+H] 757.2 (observed).

[0365] Preparation of 6-amino-3-[(3R)-3-aminopiperidine-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-41b To a solution of L-41a (443 mg, 585 μmol, 1 equiv.) in DCM (5 mL) was added TFA (1.33 g, 11.7 mmol, 869 μL, 20 equiv.) at 25° C., followed by heating to 50° C. and stirring for 12 h. The reaction mixture was then concentrated under reduced pressure to give a residue. The residue was triturated with MTBE (5 mL) at 25° C. for 20 min, filtered, and the filter cake was concentrated under reduced pressure to give L-41b (285 mg, crude) as a reddish-brown solid. 1 H NMR (MeOD, 400 MHz,) δ 8.7 (s, 1H), 7.90 (d, J = 1.6 Hz, 1H), 7.41 (s, 1H), 3.98 (q, J = 7.2 Hz, 2H), 3.80-3.67 (m, 4H), 3.50 (s, 2H), 3.45-3.36 (m, 2H), 2.23-2.14 (m, 1H), 1.88-1.86 (m, 2H), 1.83-1.64 (m, 4H), 1.20 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 415.2 (calculated); LC / MS [M+H] 415.2 (observed).

[0366] Preparation of azaBzL-41 To a solution of L-41b (48 mg, 90.8 μmol, 1 equivalent, TFA salt) in DMF (1.5 mL) were added DIEA (35.2 mg, 272 μmol, 47.4 μL, 3 equivalents) and (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy ... The reaction mixture was adjusted to pH = 6 with TFA at 0 °C and then purified by preparative HPLC (column: Phenomenex luna C18 100*40 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 5% to 40% of B in 8.0 min) to give azaBzL-41 (20 mg, 18.8 μmol, 20.7% yield) as a colorless oil. 1 H NMR (DMSO-d6, 400 MHz) δ 8.52 (s, 1H), 7.96-7.90 (m, 1H), 7.68 (d, J = 1.0 Hz, 1H), 7.63 (d, J = 7.2 Hz, 1H), 7.19 (s, 1H), 7.01 (s, 2H), 4.03 (s, 2H), 3.92 (q, J = 7.0 Hz, 2H), 3.75-3.71 (m, 1H), 3.66 (t, J = 7.0 Hz, 3H), 3.62-3.57 (m, 2H), 3.55-3.49 (m, 36H), 3.44 (t, J = 5.9 Hz, 2H), 3.31 (d, J = 1.1 Hz, 2H), 3.26-3.20 (m, 6H), 2.35-2.31 (m, 2H), 1.92-1.74 (m, 2H), 1.73-1.66 (m, 2H), 1.50-1.49 (m, 2H), 1.12 (t, J = 7.0 Hz, 3H), 0.94 (t, J = 7.5 Hz, 3H). LC / MS [M+H] 1063.5 (calculated value); LC / MS [M+H] 1063.4 (observed value).

[0367] Example L-42: Synthesis of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl N-[[1-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]azepine-3-carbonyl]azetidin-3-yl]methyl]carbamate, azaBzL-42 [ka] [ka] Preparation of tert-butyl N-[[1-[8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carbonyl]azetidin-3-yl]methyl]carbamate, L-42a To a mixture of 8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylic acid, L-32d (450 mg, 783 μmol, 1 equiv.), HATU (298 mg, 783 μmol, 1 equiv.), and DIEA (304 mg, 2.35 mmol, 409 μL, 3 equiv.) in DMF (4 mL) was added tert-butyl N-(azetidin-3-ylmethyl)carbamate (190 mg, 1.02 mmol, 1.3 equiv.), followed by stirring at 25 °C for 1 h. The reaction mixture was diluted with HO (30 mL) and then extracted with EtOAc (10 mL × 3). The organic layer was washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica flash column, elution with a gradient of 0–100% ethyl acetate / petroleum ether at 80 mL / min) to afford L-42a (463 mg, 623 μmol, 79.6% yield) as a pale yellow solid. 1H NMR (MeOD, 400 MHz,) δ 8.35 (d, J = 2.0 Hz, 1H), 7.36-7.32 (m, 6H), 7.29-7.16 (m, 10H), 7.03 (d, J = 2.0 Hz, 1H), 4.32 (t, J = 8.8 Hz, 1H), 4.18 (t, J = 9.2 Hz, 1H), 4.01 (q, J = 7.2 Hz, 2H), 3.98-3.93 (m, 1H), 3.86 (dd, J = 5.2, 10.4 Hz, 1H), 3.80 (t, J = 6.8 Hz, 2H), 3.28 (d, J = 6.8 Hz, 2H), 3.01-2.98 (m, 2H), 2.88-2.85 (m, 1H), 1.85-1.76 (m, 2H), 1.41 (s, 9H), 1.24-1.20 (m, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 743.4 (calculated value); LC / MS [M+H] 743.3 (observed value).

[0368] Preparation of 6-amino-3-[3-(aminomethyl)azetidine-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-42b To a solution of L-42a (360 mg, 485 μmol, 1 equiv.) in DCM (2 mL) was added TFA (1.11 g, 9.69 mmol, 720 μL, 20 equiv.), followed by stirring at 50 °C for 12 h. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 5% to 35% B over 8.0 min) to give L-42b (180 mg, 350 μmol, 72.2% yield, TFA) as a pale yellow solid. 1H NMR (MeOD, 400 MHz,) δ 8.85 (d, J = 1.6 Hz, 1H), 8.07 (d, J = 1.6 Hz, 1H), 7.40 (s, 1H), 4.62 (t, J = 8.8 Hz, 1H), 4.39 (t, J = 9.6 Hz, 1H), 4.31-4.24 (m, 1H), 4.05-3.94 (m, 3H), 3.76 (t, J = 7.2 Hz, 2H), 3.48 (s, 2H), 3.31-3.29 (m, 2H), 3.12-3.02 (m, 1H), 1.82-1.73 (m, 2H), 1.20 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 401.2 (calculated value); LC / MS [M+H] 401.2 (observed value).

[0369] Preparation of azaBzL-42 To a solution of L-42b (45 mg, 87.5 μmol, 1 equiv., TFA) in DMF (1 mL), DIEA (33.9 mg, 262 μmol, 45.7 μL, 3 equiv.) and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl)carbonate (70.3 mg, 87.5 μmol, 1 equiv.) was added under N at 0° C., followed by warming to 25° C. and stirring for 1 h. The pH of the mixture was adjusted to 6 with TFA, and the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 20% to 40% of B in 8.0 min) to give azaBzL-42 (53.0 mg, 44.9 μmol, 51.4% yield, TFA) as a pale yellow oil. 1H NMR (MeOD, 400 MHz,) δ 8.87 (d, J = 1.6 Hz, 1H), 8.05 (s, 1H), 7.42 (s, 1H), 6.89 (s, 2H), 4.52 (t, J = 8.8 Hz, 1H), 4.28 (t, J = 9.6 Hz, 1H), 4.21-4.13 (m, 5H), 4.02-3.94 (m, 3H), 3.76 (t, J = 7.2 Hz, 2H), 3.68-3.61 (m, 38H), 3.56-3.53 (m, 2H), 3.50 (s, 2H), 3.42-3.36 (m, 4H), 2.97-2.88 (m, 1H), 1.83-1.73 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.00 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1065.5 (calculated value); LC / MS [M+H] 1065.4 (observed value).

[0370] Example L-51: Synthesis of (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]azepine-3-carbonyl]amino]-3-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, azaBzL-51 [ka] [ka] Preparation of N3-(5-bromo-3-pyridyl)-N8-ethoxy-N8-propyl-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3,8-dicarboxamide, L-51a To a solution of 8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylic acid, L-32d (1.00 g, 1.74 mmol, 1 equiv.), 5-bromopyridin-3-amine (452 ​​mg, 2.61 mmol, 1.5 equiv.), and NMI (429 mg, 5.22 mmol, 416 μL, 3 equiv.) in CH3CN (10 mL) was added TCFH (732 mg, 2.61 mmol, 1.5 equiv.) at 0 °C, followed by warming to 20 °C and stirring at 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with HO (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give L-51a (1.00 g, 1.34 mmol, 77.2% yield) as a yellow solid. LC / MS [M+H] 729.2 (calculated); LC / MS [M+H] 729.2 (observed).

[0371] Preparation of tert-butyl N-[[5-[[8-[ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carbonyl]amino]-3-pyridyl]methyl]carbamate, L-51b A mixture of L-51a (1.00 g, 1.37 mmol, 1 equiv.), potassium; (tert-butoxycarbonylamino)methyl-trifluoroboranide (357 mg, 1.51 mmol, 1.1 equiv.), [2-(2-aminophenyl)phenyl]-chloropalladium; bis(1-adamantyl)-butyl-phosphane (91.6 mg, 137 μmol, 0.1 equiv.), and CsCO (893 mg, 2.74 mmol, 2 equiv.) in dioxane (10 mL) and HO (2 mL) was degassed and purged with N three times and then stirred at 110 °C under a N atmosphere for 1 h. The reaction mixture was cooled to 20 °C, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give L-51b (1.00 g, 1.28 mmol, 93.6% yield) as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ 8.73 (s, 1H), 8.61 (s, 1H), 8.34 (s, 1H), 8.20 (s, 1H), 7.98 (s, 1H), 7.54 (s, 1H), 7.35-7.28 (m, 6H), 7.26-7.16 (m, 9H), 6.42 (s, 1H), 5.02 (s, 1H), 4.36 (d, J = 5.6 Hz, 2H), 3.98 (q, J = 7.2 Hz, 2H), 3.77 (t, J = 7.2 Hz, 2H), 2.86 (s, 2H), 1.83-1.76 (m, 2H), 1.47 (s, 9H), 1.32-1.26 (m, 3H), 0.99 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 780.4 (calculated value); LC / MS [M+H] 780.3 (observed value).

[0372] Preparation of 6-amino-N3-[5-(aminomethyl)-3-pyridyl]-N8-ethoxy-N8-propyl-7H-pyrido[3,2-b]azepine-3,8-dicarboxamide, L-51c To a solution of L-51b (0.6 g, 769 μmol, 1 equiv) in DCM (10 mL) was added TFA (2.63 g, 23.1 mmol, 1.71 mL, 30 equiv) under N at 25 °C, then heated to 50 °C and stirred at 50 °C for 2 h. The reaction mixture was cooled to 25 °C, filtered, and concentrated under reduced pressure to give a residue. The residue was triturated with MTBE (10 mL) at 25 °C for 10 min, the mixture was filtered, and the filter cake was dried under reduced pressure to give L-51c (0.5 g, 611 μmol, 79.4% yield, 95.2% purity, 3% TFA) as a pale yellow solid. 1 H NMR (MeOD, 400 MHz) δ 9.17 (d, J = 1.2 Hz, 1H), 8.91 (d, J = 1.2 Hz, 1H), 8.59 (s, 1H), 8.48 (s, 1H), 8.39 (d, J = 1.2 Hz, 1H), 7.45 (s, 1H), 4.26 (s, 2H), 4.00 (q, J = 7.2 Hz, 2H), 3.77 (t, J = 7.2 Hz, 2H), 3.52 (s, 2H), 1.86-1.72 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.02 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 438.2 (calculated); LC / MS [M+H] 438.2 (observed).

[0373] Preparation of azaBzL-51 To a solution of L-51c (0.1 g, 128 μmol, 1 equivalent, 3TFA) in DMF (1 mL), DIEA (66.3 mg, 513 μmol, 89.4 μL, 4 equivalents) and (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (274 mg, 321 μmol, 2.5 equivalents) were added at 0° C., followed by stirring at 0° C. for 0.5 hours. The reaction mixture was adjusted to pH 5–6 with TFA at 0°C and purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 20%–50% B over 8.0 min) to give azaBzL-51 (70.7 mg, 47.8 μmol, 37.2% yield, 91.5% purity, 2% TFA) as a yellow oil. 1 H NMR (MeOD, 400 MHz) δ 9.21 (d, J = 2.0 Hz, 1H), 9.18 (d, J = 2.0 Hz, 1H), 8.51 (s, 1H), 8.47 (s, 1H), 8.38 (d, J = 2.0 Hz, 1H), 7.50-7.37 (m, 2H), 4.62-4.53 (m, 2H), 4.00 (q, J = 6.8 Hz, 2H), 3.85 (t, J = 6.0 Hz, 2H), 3.82-3.73 (m, 4H), 3.67-3.55 (m, 36H), 3.52 (s, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.54 (t, J = 6.0 Hz, 2H), 1.83-1.74 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1126.5 (calculated value); LC / MS [M+H] 1126.5 (observed value).

[0374] Example L-52: Synthesis of (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[6-amino-8-(dipropylcarbamoyl)-7H-pyrido[3,2-b]azepine-3-carbonyl]amino]-7,8-dihydro-5H-1,6-naphthyridin-6-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, azaBzL-52 [ka] Preparation of 3-bromo-N,N-dipropyl-6-(tritylamino)-7H-pyrido[3,2-b]azepine-8-carboxamide, L-52a To a solution of 6-amino-3-bromo-N,N-dipropyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-28a (10.0 g, 27.4 mmol, 1 equiv.) and TEA (11.1 g, 110 mmol, 15.2 mL, 4 equiv.) in DCM (200 mL) was added TrtCl (19.1 g, 68.4 mmol, 2.5 equiv.) at 25 °C, then heated to 50 °C and stirred at 50 °C for 12 h. The reaction mixture was quenched at 0 °C by the addition of HO (300 mL) and extracted with DCM (150 mL × 3). The combined organic layers were washed with brine (50 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give L-52a (6.00 g, 9.88 mmol, 36.1% yield) and 3-bromo-6-(ditritylamino)-N,N-dipropyl-7H-pyrido[3,2-b]azepine-8-carboxamide, L-52b (2.5 g, 2.94 mmol, 10.7% yield) as a yellow solid. LC / MS [M+H] 607.2 (calculated); LC / MS [M+H] 607.2 (observed). LC / MS [M+H] 849.3 (calculated); LC / MS [M+H] 849.3 (observed).

[0375] Preparation of methyl 8-(dipropylcarbamoyl)-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylate, L-52c A mixture of L-52a (4.60 g, 7.57 mmol, 1 equiv.), Pd(dppf)Cl (554 mg, 757 μmol, 0.1 equiv.), and TEA (2.30 g, 22.7 mmol, 3.16 mL, 3 equiv.) in MeOH (50 mL) was degassed and purged with CO three times, then heated to 80 °C and stirred under CO atmosphere (50 psi) at 80 °C for 16 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give L-52c (3.50 g, 5.97 mmol, 78.8% yield) as a brown solid. 1 H NMR (CDCl3, 400 MHz) δ 8.75 (d, J = 2.0 Hz, 1H), 7.49 (s, 1H), 7.37-7.31 (m, 5H), 7.26-7.15 (m, 10H), 6.91 (s, 1H), 6.30 (s, 1H), 3.90 (s, 3H), 3.69-3.36 (m, 4H), 2.77 (s, 2H), 1.74-1.62 (m, 4H), 1.00-0.85 (m, 6H). LC / MS [M+H] 587.3 (calculated value); LC / MS [M+H] 587.3 (observed value).

[0376] Preparation of 8-(dipropylcarbamoyl)-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylic acid, L-52d To a solution of L-52c (2.50 g, 4.26 mmol, 1 equiv) in MeOH (30 mL) was added a solution of LiOH.HO (1.07 g, 25.6 mmol, 6 equiv) in HO (10 mL) at 25 °C, followed by heating to 80 °C and stirring at 80 °C for 20 h. The reaction mixture was filtered. The filtrate was cooled to 0 °C, diluted with HO (30 mL), adjusted to pH = 5 with 2 N HCl at 0 °C, and concentrated under reduced pressure to remove MeOH. The mixture was filtered, and the filter cake was dried under reduced pressure to give L-52d (671.5 mg, 1.17 mmol, 27.5% yield) as a pale yellow solid.1 H NMR (DMSO-d6, 400 MHz) δ 8.56 (d, J = 2.0 Hz, 1H), 8.40 (s, 1H), 7.33-7.28 (m, 6H), 7.27-7.20 (m, 7H), 7.19-7.13 (m, 3H), 6.77 (s, 1H), 3.45-3.37 (m, 4H), 2.99 (s, 2H), 1.69-1.56 (m, 4H), 1.04-0.75 (m, 6H). LC / MS [M+H] 573.3 (calculated value); LC / MS [M+H] 573.3 (observed value).

[0377] Preparation of tert-butyl 3-[[8-(dipropylcarbamoyl)-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carbonyl]amino]-7,8-dihydro-5H-1,6-naphthyridine-6-carboxylate, L-52e To a solution of L-52d (310 mg, 541 μmol, 1 equiv.) and tert-butyl 3-amino-7,8-dihydro-5H-1,6-naphthyridine-6-carboxylate (148 mg, 595 μmol, 1.1 equiv.) in MeCN (5 mL) was added NMI (222 mg, 2.71 mmol, 215 μL, 5 equiv.) and TCFH (303 mg, 1.08 mmol, 2 equiv.), followed by stirring at 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with ice-water (w / w = 1 / 1) (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL × 3). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica flash column, elution with a gradient of 0-100% ethyl acetate / petroleum ether at 40 mL / min) to give L-52e (0.38 g, 472 μmol, 87.3% yield) as a yellow solid. LC / MS [M+H] 804.4 (calculated); LC / MS [M+H] 804.5 (observed).

[0378] Preparation of 6-amino-N8,N8-dipropyl-N3-(5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-7H-pyrido[3,2-b]azepine-3,8-dicarboxamide, L-52f To a solution of L-52e (0.38 g, 472 μmol, 1 equiv) in DCM (10 mL) was added TFA (1.35 g, 11.8 mmol, 877 μL, 25 equiv), followed by stirring at 50° C. for 15 h. The mixture was concentrated under reduced pressure to give L-52f (0.25 g, 434 μmol, 91.9% yield, TFA) as a yellow oil. LC / MS [M+H] 462.3 (calculated); LC / MS [M+H] 462.3 (observed).

[0379] Preparation of azaBzL-52 To a solution of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (266 mg, 311 μmol, 2.5 equiv) in DMF (1 mL) was added a solution of DIPEA (64.3 mg, 498 μmol, 86.7 μL, 4 equiv) and L-52f (0.1 g, 124 μmol, 1 equiv, 3TFA) in DMF (1 mL) at 0° C., followed by stirring at 0° C. for 0.5 h. The reaction mixture was adjusted to pH 5–6 with TFA at 0°C and purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 20%–50% of B in 8.0 min) to give azaBzL-52 (35.4 mg, 25.7 μmol, 20.6% yield, 2% TFA) as a yellow oil. 1H NMR (MeOD, 400 MHz) δ 9.21-9.19 (m, 1H), 8.98 (s, 1H), 8.40-8.36 (m, 1H), 8.35-8.27 (m, 1H), 7.49-7.37 (m, 1H), 7.10 (s, 1H), 4.05-3.95 (m, 2H), 3.90-3.83 (m, 2H), 3.80-3.78 (m, 3H), 3.66-3.56 (m, 38H), 3.50-3.45 (m, 4H), 3.40-3.36 (m, 2H), 3.19 (t, J = 5.6 Hz, 1H), 3.06 (t, J = 5.6 Hz, 1H), 2.97 (t, J = 6.0 Hz, 2H), 2.85-2.74 (m, 2H), 1.76-1.67 (m, 4H), 1.05-0.85 (m, 6H). LC / MS [M+H] 1150.5 (calculated value); LC / MS [M+H] 1150.7 (observed value).

[0380] Example L-53: Synthesis of (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[[6-amino-8-(dipropylcarbamoyl)-7H-pyrido[3,2-b]azepine-3-carbonyl]amino]-3-pyridyl]methylamino]-3-oxopropoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, azaBzL-53 [ka] [ka] Preparation of N3-(5-bromo-3-pyridyl)-N8,N8-dipropyl-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3,8-dicarboxamide, L-53a To a solution of 8-(dipropylcarbamoyl)-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carboxylic acid, L-52d (450 mg, 785 μmol, 1 equiv.) and 5-bromopyridin-3-amine (149 mg, 864 μmol, 1.1 equiv.) in MeCN (5 mL) was added NMI (322 mg, 3.93 mmol, 313 μL, 5 equiv.) and TCFH (440 mg, 1.57 mmol, 2 equiv.), followed by stirring at 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was diluted with ice water (w / w = 1 / 1) (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL × 3). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica flash column, elution with a gradient of 0–100% ethyl acetate / petroleum ether at 40 mL / min) to afford L-53a (410 mg, 563 μmol, 71.7% yield) as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.66 (d, J = 2.0 Hz, 1H), 8.48 (d, J = 2.0 Hz, 1H), 8.41 (t, J = 2.0 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 2.0 Hz, 1H), 7.30-7.28 (m, 6H), 7.15-7.10 (m, 6H), 7.10-7.05 (m, 3H), 6.79 (s, 1H), 3.43-3.38 (m, 4H), 2.88 (s, 2H), 1.68-1.58 (m, 4H), 0.94-0.82 (m, 6H). LC / MS [M+H] 727.2 (calculated); LC / MS [M+H] 727.4 (observed).

[0381] Preparation of tert-butyl N-[[5-[[8-(dipropylcarbamoyl)-6-(tritylamino)-7H-pyrido[3,2-b]azepine-3-carbonyl]amino]-3-pyridyl]methyl]carbamate, L-53b A mixture of L-53a (360 mg, 494 μmol, 1 equiv.), potassium; (tert-butoxycarbonylamino)methyl-trifluoroboranide (129 mg, 544 μmol, 1.1 equiv.), [2-(2-aminophenyl)phenyl]chloropalladium; bis(1-adamantyl)-butyl-phosphane (33.1 mg, 49.4 μmol, 0.1 equiv.), and CsCO (322 mg, 989 μmol, 2 equiv.) in dioxane (5 mL) and HO (1 mL) was degassed and purged with N three times, then stirred at 110 °C under N for 15 h. The mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica flash column, elution with a gradient of 0–100% ethyl acetate / petroleum ether at 40 mL / min) to afford L-53b (0.35 g, 445 μmol, 90.9% yield) as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.75 (s, 1H), 8.60 (d, J = 2.0 Hz, 1H), 8.26 (s, 1H), 8.16 (s, 1H), 7.46 (s, 1H), 7.45-7.40 (m, 6H), 7.27-7.18 (m, 9H), 6.91 (s, 1H), 4.33 (s, 2H), 3.55-3.50 (m, 4H), 2.99 (s, 2H), 1.78-1.72 (m, 4H), 1.49 (s, 9H), 1.02-0.95 (m, 6H). LC / MS [M+H] 778.4 (calculated); LC / MS [M+H] 778.3 (observed).

[0382] Preparation of 6-amino-N3-[5-(aminomethyl)-3-pyridyl]-N8,N8-dipropyl-7H-pyrido[3,2-b]azepine-3,8-dicarboxamide, L-53c To a mixture of L-53b (0.35 g, 450 μmol, 1 equiv) in DCM (1 mL) was added TFA (1.54 g, 13.5 mmol, 1.00 mL, 30 equiv) at 20 °C, then warmed to 50 °C and stirred for 1 h. The mixture was concentrated. The crude product was purified by recrystallization from MTBE (10 mL) at 20 °C to give L-53c (0.3 g, crude, TFA) as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ 9.17 (d, J = 2.0 Hz, 1H), 8.90 (d, J = 2.0 Hz, 1H), 8.59 (s, 1H), 8.47 (s, 1H), 8.38 (d, J = 2.0 Hz, 1H), 7.10 (s, 1H), 4.26 (s, 2H), 3.57-3.42 (m, 4H), 1.79-1.64 (m, 4H), 1.05-0.86 (m, 6H). LC / MS [M+H] 436.2 (calculated value); LC / MS [M+H] 436.3 (observed value).

[0383] Preparation of azaBzL-53 To a mixture of L-53c (0.1 g, 182 μmol, 1 equiv., TFA) in DMF (1 mL) was added DIEA (70.6 mg, 546 μmol, 95.1 μL, 3 equiv.) and (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (389 mg, 455 μmol, 2.5 equiv.) in one portion at 0° C. and stirred at 0° C. for 0.5 h. The mixture was adjusted to pH 6 with TFA and purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [HO (0.1% TFA)-ACN]; gradient: 20% to 50% of B over 8.0 min) to give azaBzL-53 (63.8 mg, 54.30 μmol, 29.8% yield, 95.6% purity) as a yellow oil. 1H NMR (MeOD, 400 MHz) δ 9.24-9.20 (m, 1H), 8.53-8.52 (m, 1H), 8.39 (d, J = 1.6 Hz, 1H), 7.47-7.40 (m, 1H), 7.10 (s, 1H), 4.59 (s, 2H), 3.85 (t, J = 6.0 Hz, 2H), 3.78 (t, J = 6.0 Hz, 2H), 3.63-3.58 (m, 38H), 3.47-3.31 (m, 4H), 2.96 (t, J = 6.0 Hz, 2H), 2.55 (t, J = 6.0 Hz, 2H), 1.76-1.67 (m, 4H), 0.98-0....

Claims

1. An immunoconjugate of formula I comprising an antibody covalently attached by a linker to one or more azabenzazepine moieties. 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody, p is an integer from 1 to 8; L is a linker, D is an azabenzazepine moiety having the formula: 【Chemistry 2】 Z １ is CR １ and N; Z ２ is CR ２ and N; Z ３ is CR ３ and N; Z ４ is CR ４ and N; Z １ , Z ２ , Z ３ , and Z ４ one or two of are N; R １ , R ２ , R ３ , R ４ , R ５ , and R ６ are independently H, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , S(O) ２ N (R ５ ), C １ -C １２ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryl, C ２ -C ９ Heterocyclyl, and C １ -C ２０ heteroaryl, each of which is independently and optionally selected from the group consisting of: -(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C １ -C １２ alkyldiyl)-OR ７ , -(C ３ -C １２ carbocyclyl), -(C ３ -C １２ carbocyclyl)-*, -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-NR ７ - *, -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C ３ -C １２ carbocyclyl)-NR ７ -C(=NR ７ ) NR ７ - *, -(C ６ -C ２０ aryl), -(C ６ -C ２０ Aryldiyl)-*, -(C ６ -C ２０ aryldiyl)-N(R ７ )-*, -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-(C ２ -C ２０ heterocyclyldiyl)-*, -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-NR ７ -C(=NR ７ａ ) N (R ７ )-*, -(C ２ -C ２０ heterocyclyl), -(C ２ -C ２０ heterocyclyl)-*, -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-NR ７ - *, -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C ２ -C ９ heterocyclyl)-C(═O)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C ２ -C ９ Heterocyclyl)-NR ７ -C(=NR ７ａ ) NR ７ - *, -(C ２ -C ９ Heterocyclyl)-NR ７ -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C ２ -C ９ heterocyclyl)-(C ６ -C ２０ Aryldiyl)-*, -(C １ -C ２０ heteroaryl), -(C １ -C ２０ heteroaryl)-*, -(C １ -C ２０ heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C １ -C ２０ heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C １ -C ２０ Heteroaryl)-NR ７ -C(=NR ７ａ ) N (R ７ )-*, -(C １ -C ２０ heteroaryl)-N(R ７ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -C(=O)-*, -C(=O)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -C(=O)-(C ２ -C ２０ heterocyclyldiyl)-*, -C(=O)N(R ７ ) ２ 、 -C(=O)N(R ７ )-*、 -C(=O)N(R ７ )-(C １ -C １２ alkyldiyl)-N(R ７ ) C(=O)R ７ , -C(=O)N(R ７ )-(C １ -C １２ alkyldiyl)-N(R ７ )C(=O)N(R ７ ) ２ , -C(=O)NR ７ -(C １ -C １２ alkyldiyl)-N(R ７ ) CO ２ R ７ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-N(R ５７ ) C(=NR ５７ａ ) N (R ５７ ) ２ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-NR ５７ C (=NR ７ａ ) R ７ , -C(=O)NR ５ -(C １ -C ８ alkyldiyl)-NR ７ (C ２ -C ５ heteroaryl), -C(=O)NR ７ -(C １ -C ２０ heteroaryldiyl)-N(R ７ )-*, -C(=O)NR ７ -(C １ -C ２０ heteroaryldiyl)-*, -C(=O)NR ７ -(C １ -C ２０ heteroaryldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -C(=O)NR ７ -(C １ -C ２０ heteroaryldiyl)-(C ２ -C ２０ heterocyclyldiyl)-C(=O)NR ７ -(C １ -C １２ alkyldiyl)-NR ７ - *, -N(R ７ ) ２ 、 -N(R ７ )-*、 -N(R ７ )C(=O)R ７ 、 -N(R ７ )C(=O)-*、 -N(R ７ )C(=O)N(R ７ ) ２ 、 -N(R ７ )C(=O)N(R ７ )-*、 -N(R ７ )CO ２ R ７ 、 -NR ７ C(=NR) ７ａ )N(R ７ ) ２ 、 -NR ７ C(=NR) ７ａ )N(R ７ )-*、 -NR ７ C(=NR) ７ａ )R ７ 、 -N(R ７ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -N(R ７ )-(C ２ -C ５ heteroaryl), -N(R ７ ) -S(=O) ２ -(C １ -C １２ alkyl), -O-(C １ -C １２ alkyl), -O-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -O-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -O-C(=O)N(R ７ ) ２ 、 -O-C(=O)N(R ７ )-*、 -O-(R ７ )-*、 -OR ７ 、 -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-*, -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-NR ７ -*, and -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ or substituted with one or more groups selected from: or R ５ and R ６ together form a 5- or 6-membered heterocyclyl ring, R ７ are independently H, C ６ -C ２０ Aryl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryldiyl, C １ -C １２ Alkyl, and C １ -C １２ alkyldiyl, or two R ５ the groups together form a 5- or 6-membered heterocyclyl ring; R ７ａ is C ６ -C ２０ Aryl and C １ -C ２０ heteroaryl; where the asterisk * indicates the binding site of L, and R １ , R ２ , R ３ , R ４ , R ５ , and R ６ one of which is attached to L; Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N(CH) ３ ) ２ 、-CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-C(CH ３ ) ２ CONG ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ 、-NHCOCH ３ 、-N(CH ３ COCH ３ 、-NHS(O) ２ CH ３ 、-N(CH ３ )C(CH ３ ) ２ CONG ２ 、-N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-O(CH ２ CH ２ O) ｎ -(CH) ２ ) ｍ CO ２ H, —O(CH ２ CH ２ O) ｎ H, -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H, or a pharmaceutically acceptable salt thereof.

2. The immunoconjugate of claim 1 , wherein the linker L is a bivalent or branched trivalent linker.

3. The linker L is -C(=O)-PEG-, -C(=O)-PEG-C(=O)N(R ８ )-(C １ -C １２ alkyldiyl)-C(═O)-Gluc-, -C(=O)-PEG-(C ２ -C ２０ heterocyclyldiyl)-, -C(=O)-PEG-(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-, -C(=O)-PEG-O-, -C(=O)-PEG-O-C(=O)-, -C(=O)-PEG-C(=O)-, -C(=O)-PEG-C(=O)-PEP-, - C ( = O ) - PEG - N ( R ) ８ )-、 - C ( = O ) - PEG - N ( R ) ８ )-C(=O)-、 - C ( = O ) - PEG - N ( R ) ８ )-PEG-C(=O)-PEP-、 - C ( = O ) - PEG - N ＋ (R) ８ ) ２ -PEG-C(=O)-PEP- -C(=O)-PEG-C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-, -C(=O)-PEG-C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)N(R ８ )C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-, -C(=O)-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-, -C(=O)-PEG-SS-(C １ -C １２ alkyldiyl)-C(=O)-, -C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-PEP-, -C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-, -C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-N(R ８ )-C(=O), -C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-N(R ８ )C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-C(=O)N(R ８ )-(C １ -C １２ alkyldiyl)-C(═O)-Gluc-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ３ )-(C ２ -C ２０ heterocyclyldiyl)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ３ )-PEG-(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-O-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-O-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-N(R ８ ) -, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-N(R ８ )-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-C(=O)-PEP-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)N(R ８ )C(═O)—, and -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)N(R ８ )C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-, R ８ are independently H or C １ -C ６ is alkyl, PEG has the formula: -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -, m is an integer from 1 to 5, and n is an integer from 1 to 50; Gluc has the formula: 【Transformation 3】 and PEP has the formula: 【Chemistry 4】 wherein AA are independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; Cyc is F, Cl, NO ２ , —OH, —OCH ３ and glucuronic acid having the structure: ６ -C ２０ Aryldiyl and C １ -C ２０ heteroaryldiyl; 【Transformation 5】 R ９ is -CH(R １０ ) O—, —CH ２ -, -CH ２ N (R １０ )-, and -CH(R １０ )O—C(═O)—, where R １０ is H, C １ -C ６ Alkyl, C(=O)-C １ -C ６ Alkyl, and —C(═O)N(R １１ ) ２ where R １１ are independently H, C １ -C １２ Alkyl, and -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -OH (wherein m is an integer from 1 to 5 and n is an integer from 2 to 50), or two R １１ the groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; The immunoconjugate of claim 1 , wherein z is 0 or 1.

4. The immunoconjugate of claim 1 , wherein the antibody is an immune checkpoint inhibitor.

5. The immunoconjugate of claim 1 , wherein the antibody is an antibody construct having an antigen-binding domain that binds to an antigen selected from PD-L1, HER2, CEA, and TROP2.

6. 6. The immunoconjugate of claim 5, wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, margetuximab, HT-19, labetuzumab, and sacituzumab.

7. Z １ , Z ２ , Z ３ , and Z ４ The immunoconjugate of claim 1 , wherein one of

8. Z １ The immunoconjugate of claim 7 , wherein is N.

9. Z ２ The immunoconjugate of claim 7 , wherein is N.

10. Z ３ The immunoconjugate of claim 7 , wherein is N.

11. Z ４ The immunoconjugate of claim 7 , wherein is N.

12. Z １ , Z ２ , Z ３ , and Z ４ The immunoconjugate of claim 1 , wherein two of

13. R ５ and R ６ became independent and C １ -C ８ Alkyl, —O—(C １ -C １２ alkyl), -(C １ -C １２ alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , -(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ , —O—(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ , and —O—(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ The immunoconjugate of any one of claims 1 to 12, selected from:

14. R ５ is C １ -C ８ alkyl, and R ６ -O-(C １ -C １２ The immunoconjugate of claim 13, wherein the aryl group is aryl, ...

15. R ５ Ga-CH ２ CH ２ CH ３ and R ６ Ga-CH ２ CH ２ CH ２ NHCO ２ (t-Bu), -OCH ２ CH ２ NHCO ２ (cyclobutyl), and —CH ２ CH ２ CH ２ NHCO ２ 14. The immunoconjugate of claim 13, wherein the aryl group is selected from the group consisting of aryl, ...

16. R ５ and R ６ are each independently —CH ２ CH ２ CH ３ , -OCH ２ CH ３ , -OCH ２ CF ３ , -CH ２ CH ２ CF ３ , -OCH ２ CH ２ OH, and -CH ２ CH ２ CH ２ 14. The immunoconjugate of claim 13, wherein the aryl group is selected from the group consisting of aryl, aryl- ...

17. R ５ Ga-CH ２ CH ２ CH ３ and R ６ Ga-OCH ２ CH ３ The immunoconjugate of claim 16, wherein:

18. R ６ but, 【Transformation 6】 The immunoconjugate of any one of claims 1 to 12, selected from the group consisting of:

19. R １ The immunoconjugate of any one of claims 1 to 12, wherein: is attached to L.

20. R ２ The immunoconjugate of any one of claims 1 to 12, wherein: is attached to L.

21. R ３ The immunoconjugate of any one of claims 1 to 12, wherein: is attached to L.

22. R ４ The immunoconjugate of any one of claims 1 to 12, wherein: is attached to L.

23. R ５ or R ６ The immunoconjugate of any one of claims 1 to 12, wherein: is attached to L.

24. The immunoconjugate of any one of claims 1 to 12, wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

25. The immunoconjugate of any one of claims 1 to 12, wherein L is attached to a cysteine ​​thiol of the antibody.

26. The immunoconjugate of any one of claims 1 to 12, wherein for the PEG, m is 1 or 2 and n is an integer from 2 to 10.

27. 27. The immunoconjugate of claim 26, wherein n is 10.

28. L comprises PEP, wherein PEP is a dipeptide and has the formula: 【Transformation 7】 The immunoconjugate of any one of claims 1 to 12, having the formula:

29. AA independently represents H, —CH ３ , -CH(CH ３ ) ２ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ NH ２ , -CH ２ CH ２ CH ２ NHC (NH) NH ２ , -CHCH(CH ３ ) CH ３ , -CH ２ SO ３ H, and -CH ２ CH ２ CH ２ NHC(O)NH ２ or two AA form a five-membered ring proline amino acid.

30. PEP is a dipeptide of the formula: 【Transformation 8】 and In the formula, AA １ and A.A. ２ 29. The immunoconjugate of claim 28, wherein: are independently selected from the side chains of naturally occurring amino acids.

31. A.A. １ -CH(CH ３ ) ２ and AA ２ Ga-CH ２ CH ２ CH ２ NHC(O)NH ２ 31. The immunoconjugate of claim 30, wherein:

32. L has the structure: 【Chemistry 9】 is selected from The wavy line is R １ , R ２ , R ３ , R ４ , R ５ , and R ６ The immunoconjugate of any one of claims 1 to 12, which exhibits binding to one of:

33. An azabenzazepine-linker compound of formula II, 【Chemistry 10】 During the ceremony, Z １ is CR １ and N; Z ２ is CR ２ and N; Z ３ is CR ３ and N; Z ４ is CR ４ and N; Z １ , Z ２ , Z ３ , and Z ４ one or two of are N; R １ , R ２ , R ３ , R ４ , R ５ , and R ６ are independently H, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , S(O) ２ N (R ５ ), C １ -C １２ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryl, C ２ -C ９ Heterocyclyl, and C １ -C ２０ heteroaryl, each of which is independently and optionally selected from the group consisting of: -(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C １ -C １２ alkyldiyl)-OR ７ , -(C ３ -C １２ carbocyclyl), -(C ３ -C １２ carbocyclyl)-*, -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-NR ７ - *, -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C ３ -C １２ carbocyclyl)-NR ７ -C(=NR ７ ) NR ７ - *, -(C ６ -C ２０ aryl), -(C ６ -C ２０ Aryldiyl)-*, -(C ６ -C ２０ aryldiyl)-N(R ７ )-*, -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-(C ２ -C ２０ heterocyclyldiyl)-*, -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-NR ７ -C(=NR ７ａ ) N (R ７ )-*, -(C ２ -C ２０ heterocyclyl), -(C ２ -C ２０ heterocyclyl)-*, -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-NR ７ - *, -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C ２ -C ９ heterocyclyl)-C(═O)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C ２ -C ９ Heterocyclyl)-NR ７ -C(=NR ７ａ ) NR ７ - *, -(C ２ -C ９ Heterocyclyl)-NR ７ -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C ２ -C ９ heterocyclyl)-(C ６ -C ２０ Aryldiyl)-*, -(C １ -C ２０ heteroaryl), -(C １ -C ２０ heteroaryl)-*, -(C １ -C ２０ heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -(C １ -C ２０ heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -(C １ -C ２０ Heteroaryl)-NR ７ -C(=NR ７ａ ) N (R ７ )-*, -(C １ -C ２０ heteroaryl)-N(R ７ )C(=O)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -C(=O)-*, -C(=O)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -C(=O)-(C ２ -C ２０ heterocyclyldiyl)-*, -C(=O)N(R ７ ) ２ 、 -C(=O)N(R ７ )-*、 -C(=O)N(R ７ )-(C １ -C １２ alkyldiyl)-N(R ７ ) C(=O)R ７ , -C(=O)N(R ７ )-(C １ -C １２ alkyldiyl)-N(R ７ )C(=O)N(R ７ ) ２ , -C(=O)NR ７ -(C １ -C １２ alkyldiyl)-N(R ７ ) CO ２ R ７ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-N(R ５７ ) C(=NR ５７ａ ) N (R ５７ ) ２ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-NR ５７ C (=NR ７ａ ) R ７ , -C(=O)NR ５ -(C １ -C ８ alkyldiyl)-NR ７ (C ２ -C ５ heteroaryl), -C(=O)NR ７ -(C １ -C ２０ heteroaryldiyl)-N(R ７ )-*, -C(=O)NR ７ -(C １ -C ２０ heteroaryldiyl)-*, -C(=O)NR ７ -(C １ -C ２０ heteroaryldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -C(=O)NR ７ -(C １ -C ２０ heteroaryldiyl)-(C ２ -C ２０ heterocyclyldiyl)-C(=O)NR ７ -(C １ -C １２ alkyldiyl)-NR ７ - *, -N(R ７ ) ２ 、 -N(R ７ )-*、 -N(R ７ )C(=O)R ７ 、 -N(R ７ )C(=O)-*、 -N(R ７ )C(=O)N(R ７ ) ２ 、 -N(R ７ )C(=O)N(R ７ )-*、 -N(R ７ )CO ２ R ７ 、 -NR ７ C(=NR) ７ａ )N(R ７ ) ２ 、 -NR ７ C(=NR) ７ａ )N(R ７ )-*、 -NR ７ C(=NR) ７ａ )R ７ 、 -N(R ７ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -N(R ７ )-(C ２ -C ５ heteroaryl), -N(R ７ ) -S(=O) ２ -(C １ -C １２ alkyl), -O-(C １ -C １２ alkyl), -O-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -O-(C １ -C １２ alkyldiyl)-N(R ７ )-*, -O-C(=O)N(R ７ ) ２ 、 -O-C(=O)N(R ７ )-*、 -O-(R ７ )-*、 -OR ７ 、 -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-*, -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ７ ) ２ , -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-NR ７ -*, and -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ or substituted with one or more groups selected from: or R ５ and R ６ together form a 5- or 6-membered heterocyclyl ring, R ７ are independently H, C ６ -C ２０ Aryl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryldiyl, C １ -C １２ Alkyl, and C １ -C １２ alkyldiyl, or two R ５ the groups together form a 5- or 6-membered heterocyclyl ring; R ７ａ is C ６ -C ２０ Aryl and C １ -C ２０ heteroaryl; where the asterisk * indicates the binding site of L, and R １ , R ２ , R ３ , R ４ , R ５ , and R ６ one of which is attached to L; Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N(CH) ３ ) ２ 、-CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-C(CH ３ ) ２ CONG ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ 、-NHCOCH ３ 、-N(CH ３ COCH ３ 、-NHS(O) ２ CH ３ 、-N(CH ３ )C(CH ３ ) ２ CONG ２ 、-N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-O(CH ２ CH ２ O) ｎ -(CH) ２ ) ｍ CO ２ H, —O(CH ２ CH ２ O) ｎ H, -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ The azabenzazepine-linker compound is substituted with one or more groups independently selected from H.

34. L, Q-C(=O)-PEG-, Q-C(=O)-PEG-C(=O)N(R ８ )-(C １ -C １２ alkyldiyl)-C(═O)-Gluc-, Q-C(=O)-PEG-O-, Q-C(=O)-PEG-O-C(=O)-, Q-C(=O)-PEG-C(=O)-, Q-C(=O)-PEG-C(=O)-PEP-, Q-C(=O)-PEG-N(R ８ )-、 Q-C(=O)-PEG-N(R ８ )-C(=O)-、 Q-C(=O)-PEG-N(R ８ )-PEG-C(=O)-PEP-、 Q-C(=O)-PEG-N ＋ (R) ８ ) ２ -PEG-C(=O)-PEP- Q-C(=O)-PEG-C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-, Q-C(=O)-PEG-C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)N(R ８ ) C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-, Q-C(=O)-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-, Q-C(=O)-PEG-SS-(C １ -C １２ alkyldiyl)-C(=O)-, Q-C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-PEP-, Q-C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-, Q-C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-N(R ８ )-C(=O), Q-C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-N(R ８ ) C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-, Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-、 Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-C(=O)N(R ８ )-(C １ -C １２ alkyldiyl)-C(═O)-Gluc-, Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-O-、 Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-O-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-N(R ８ )-、 Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-N(R ８ )-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-C(=O)-PEP-、 Q-(CH ２ ) ｍ -C(=O)N(R ８ )-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-, Q-(CH ２ ) ｍ -C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)-, Q-(CH ２ ) ｍ -C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)N(R ８ )C(═O)—, and Q-(CH ２ ) ｍ -C(=O)-PEP-N(R ８ )-(C １ -C １２ alkyldiyl)N(R ８ ) C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-; R ８ are independently H or C １ -C ６ is alkyl, PEG has the formula: -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -, m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the formula: 【Chemistry 11】 and PEP has the formula: 【Chemistry 12】 wherein AA are independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; Cyc is F, Cl, NO ２ , —OH, —OCH ３ and glucuronic acid having the structure: ６ -C ２０ Aryldiyl and C １ -C ２０ heteroaryldiyl; 【Chemistry 13】 R ９ is -CH(R １０ ) O—, —CH ２ -, -CH ２ N (R １０ )-, and -CH(R １０ )O—C(═O)—, where R １０ is H, C １ -C ６ Alkyl, C(=O)-C １ -C ６ Alkyl, and —C(═O)N(R １１ ) ２ where R １１ are independently H, C １ -C １２ Alkyl, and -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -OH (wherein m is an integer from 1 to 5 and n is an integer from 2 to 50), or two R １１ the groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; Q is F, Cl, NO ２ and SO ３ － 34. The azabenzazepine-linker compound of claim 33, wherein the linker is selected from the group consisting of N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, substituted with one or more groups independently selected from:

35. Q is, 【Chemistry 14】 35. The azabenzazepine-linker compound of claim 34, selected from:

36. 35. The azabenzazepine-linker compound of claim 34, wherein Q is phenoxy substituted with one or more F.

37. 35. The azabenzazepine-linker compound of claim 34, wherein Q is 2,3,5,6-tetrafluorophenoxy.

38. 35. The azabenzazepine-linker compound of claim 34, wherein Q is maleimide.

39. An azabenzazepine-linker compound selected from Table 2a or Table 2b.

40. An immunoconjugate prepared by conjugation of an antibody with an azabenzazepine-linker compound selected from Table 2a or Table 2b.

41. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate of any one of claims 1 to 32 and one or more pharmaceutically acceptable diluents, vehicles, carriers, or excipients.

42. 34. A method for treating cancer, comprising administering a therapeutically effective amount of the immunoconjugate of any one of claims 1 to 32 to a patient in need thereof, wherein the cancer is selected from cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial cancer, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer.

43. 43. The method of claim 42, wherein the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8 agonism.

44. 43. The method of claim 42, wherein the cancer is selected from triple-negative breast cancer, metastatic Merkel cell carcinoma, and gastroesophageal junction adenocarcinoma.

45. 43. The method of claim 42, wherein the immunoconjugate is administered to the patient intravenously, intratumorally, or subcutaneously.

46. 43. The method of claim 42, wherein the immunoconjugate is administered to the patient at a dose of about 0.01 to 20 mg per kg of body weight.

47. 33. Use of the immunoconjugate of any one of claims 1 to 32 for treating cancer, wherein the cancer is selected from cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial cancer, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer.

48. 35. A method for preparing an immunoconjugate of Formula I according to claim 1, wherein the azabenzazepine-linker compound of claim 34 is conjugated to said antibody.

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