Aza-benzaza immunoconjugates and uses thereof

Abstract

The present invention provides immunoconjugates of Formula I, which immunoconjugates comprise an antibody linked by conjugation to one or more aza-benzaza derivatives. The present invention also provides an aza-benzaza derivative intermediate composition comprising a reactive functional group. Such intermediate compositions are suitable substrates for forming the immunoconjugates via linkers or linking moieties. The invention further provides methods of treating cancer with the immunoconjugates. Ab-[L-D] pI

Description

[0001] Cross Reference to Related Applications

[0002] This non-provisional application claims the benefit of priority of U.S. Provisional Application No. 63 / 445,390, filed February 14, 2023, which is incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present invention generally relates to immunoconjugates comprising antibodies conjugated to one or more azido-benzazido moieties. BACKGROUND

[0004] There is a need for new compositions and methods for delivering antibodies and immunoadjuvants in order to reach difficult-to-access tumors and / or to expand treatment options for cancer patients and other subjects. The present invention provides such compositions and methods. SUMMARY

[0005] The present invention generally relates to immunoconjugates comprising antibodies covalently attached via a linker to one or more azido-benzazido TLR (toll-like receptor) agonist moieties:

[0006]

[0007] wherein one or both of Z 1 , Z 2 , Z 3 , and Z 4 is N, and wherein one substituent is attached to the linker. Various substituents are defined herein.

[0008] Another aspect of the invention is a method of making an immunoconjugate by conjugating one or more azido-benzazido -linker compounds to an antibody.

[0009] Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate comprising an antibody covalently attached via a linker to one or more azido-benzazido moieties and one or more pharmaceutically acceptable diluents, vehicles, carriers, or excipients.

[0010] Another aspect of the invention is an azido-benzazido -linker compound.

[0011] 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 via a linker to one or more azido-benzazido moieties. 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 via a linker to one or more azido-benzazido moieties.

[0012] Another aspect of the application is the use of an immunoconjugate comprising an antibody covalently attached via a linker to one or more azo-Benzoazoles moieties in the treatment of disease, in particular cancer.

[0013] BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 shows the hydrolysis of the amidine group of the comparator compound CBz-3 to the lactam comparator compound CBz-5 over time in PBS buffer at 40 °C.

[0015] Figure 2A shows the hydrolysis of the amidine group of the comparator compound CBz-1 and azo-Benzoazoles

[0016] Figure 2B shows the hydrolysis of the amidine group of the comparator compound CBz-1 and azo-Benzoazoles

[0017] Figure 3A shows the hydrolysis of the amidine group of the comparator compound CBz-4 and CBz-6 and azo-Benzoazoles

[0018] Figure 3B shows the hydrolysis of the amidine group of the comparator compound CBz-4 and CBz-6 and azo-Benzoazoles

[0019] Figure 4 shows the hydrolysis of the amidine group of the comparator compound CBz-4 and CBz-6 and azo-Benzoazoles ​​​​​​​​​Plots of the hydrolysis of the amidine groups of compounds azaBa-3, azaBz-5, azaBz-6, azaBz-7, and azaBz-8 in PBS and formulation buffer, plotted against the appearance of the corresponding lactam compounds over 2 days. The amount of lactam at the start (to) of each sample was normalized for easier rate comparison.

[0020] Figure 5 Benzazepines are shown Comparison of compounds CBz-2 and CBz-7 and azo-benzazepines Plots of the hydrolysis of the amidine groups of compounds azaBa-6 and azaBz-8 in PBS, plotted against the appearance of the corresponding lactam compounds over 2 days. The amount of lactam at the start (to) of each sample was normalized for easier rate comparison. DETAILED DESCRIPTION

[0021] Reference will now be made in detail to certain embodiments of the application, examples of which are illustrated in the accompanying structures and formulas. While the application will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the application to these embodiments. On the contrary, the application is intended to cover all alternatives, modifications, and equivalents, which can be included within the scope of the application as defined by the claims.

[0022] 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 application. The present application is in no way limited to the methods and materials described.

[0023] Definitions

[0024] The term "immunoconjugate" or "immuno-stimulatory antibody conjugate" refers to an antibody construct covalently bonded to an adjuvant moiety via a linker. The term "adjuvant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant.

[0025] An "adjuvant moiety" refers to an adjuvant covalently bonded to an antibody construct, e.g., via a linker as described herein. The adjuvant moiety can elicit an immune response when bonded to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immunoconjugate to a subject.

[0026] An "adjuvant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant.

[0027] 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 serve as key signaling elements in innate immunity. They are single-pass transmembrane receptors that recognize structurally conserved molecules derived from microorganisms, often expressed on sentinel cells such as macrophages and dendritic cells. Once these microorganisms reach a physical barrier such as the skin or intestinal mucosa, they are recognized by TLRs, activating an immune cell response. TLR polypeptides share a characteristic structure comprising an extracellular domain with leucine-rich repeat sequences, a transmembrane domain, and an intracellular domain involved in TLR signaling. The terms "Toll-like receptor 7" and "TLR7" refer to a nucleic acid or polypeptide that shares at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity with a publicly available TLR7 sequence (e.g., GenBank Accession No. AAZ99026 for a human TLR7 polypeptide or GenBank Accession No. AAK62676 for a murine TLR7 polypeptide). The terms "Toll-like receptor 8" and "TLR8" refer to a nucleic acid or polypeptide that shares at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity with a publicly available TLR7 sequence (e.g., GenBank Accession No. AAZ95441 for a human TLR8 polypeptide or GenBank Accession No. AAK62677 for a murine TLR8 polypeptide).

[0028] A "TLR agonist" is a compound that directly or indirectly binds to a TLR (e.g., TLR7 and / or TLR8) to induce TLR signaling. Any detectable difference in TLR signaling can indicate that the agonist stimulates or activates the TLR. The difference in signaling can be manifested, for example, as changes in the expression of target genes, phosphorylation of signaling components, intracellular localization of downstream elements such as nuclear factor-kappa B (NF-κΒ), association of certain components (e.g., IL-1 receptor-associated kinase (IRAK)) with other proteins or intracellular structures, or biochemical activity of components such as kinases (e.g., mitogen-activated protein kinase (MAPK)).

[0029] An "antibody" refers to a polypeptide comprising an antigen binding region (including complementarity determining regions (CDRs)) from an immunoglobulin gene or fragments thereof. The term "antibody" specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they 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" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). Each chain is composed of structural domains called immunoglobulin domains. These domains are of size and function to be classified into distinct categories, e.g., variable domains or regions (V L and V H ) on the light and heavy chains, respectively, and constant domains or regions (C L and C H ) on the light and heavy chains, respectively. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is referred to as the paratope, and is primarily responsible for antigen recognition, i.e., the antigen binding domain. The light chain is classified as kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are large molecules of about 150 kDa, composed of four peptide chains. IgG antibodies contain two identical heavy chains of the gamma class of about 50 kDa and two identical light chains of about 25 kDa, and thus have a tetrameric quaternary structure. The two heavy chains are connected to each other and to the light chains via disulfide bonds. The resulting tetramer has two identical halves that together form a Y-like shape. Each end of the fork contains the same antigen binding domain. Humans have four IgG subclasses (IgG1, IgG2, IgG3, and IgG4), which are named in order of abundance in serum (i.e., IgG1 is the most abundant). Typically, the antigen binding domain of an antibody will be most critical in terms of specificity and affinity for binding to a cancer cell.

[0030] “Bispecific” antibodies (bsAbs) are antibodies that bind two different epitopes to cancer (Suurs F.V. et al. (2019) Pharmacology & Therapeutics 201 : 103-119). Bispecific antibodies can engage immune cells to destroy tumor cells, deliver a payload to a tumor, and / or block a tumor signaling pathway. Antibodies targeting specific antigens include bispecific or multispecific antibodies having at least one antigen binding region that targets a specific antigen. In some embodiments, the targeting monoclonal antibody is a bispecific antibody having at least one antigen binding region that targets a tumor cell. Such antigens include, but are not limited to: mesothelin, prostate-specific membrane antigen (PSMA), HER2, TROP2, CEA, EGFR, 5T4, Nectin4, CD19, CD20, CD22, CD30, CD70, B7H3, B7H4 (also known as 08E), protein tyrosine kinase 7 (PTK7), Glypican-3, RG1, Fucosyl-GMl, CTLA-4, and CD44 (WO 2017 / 196598).

[0031] In some embodiments, the antibody construct is an antigen-binding antibody “fragment” comprising at least an antigen-binding region of an antibody, alone or together with other components, that make up the antibody construct. Many different types of antibody “fragments” are known in the art, including, for example, (i) a Fab fragment, which is a monovalent fragment consisting of the V L , V H , C L , and CH1 domains, (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the V L , and V H domains of a single arm of an antibody, (iv) a Fab’ fragment, which is produced by breaking disulfide bridges of F(ab’)2 fragments using mild reducing conditions, (v) a disulfide stabilized Fv fragment (dsFv), and (vi) a single chain Fv (scFv), which is a monovalent molecule consisting of two domains of a Fv fragment (i.e., V L , and V H ) connected by a synthetic linker that enables the two domains to be synthesized as a single polypeptide chain. In some embodiments, the antibody construct is an antibody or fusion protein comprising (i) an antigen-binding domain and (ii) an Fc domain.

[0032] The antibody or antibody fragment can be part of a larger construct, such as a conjugate or fusion construct of the antibody fragment with additional regions. For example, in some embodiments, the antibody fragment can be fused to an Fc region as described herein. In other embodiments, the antibody fragment (e.g., Fab or scFv) can be part of a chimeric antigen receptor or chimeric T cell receptor, such as by fusion to a transmembrane domain, optionally with an intervening linker or "stalk" (e.g., a hinge region), and an optional intercellular signaling domain. For example, the antibody fragment can be fused to the gamma and / or delta chain of a T cell receptor to provide a T cell receptor-like construct that binds 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.

[0033] 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 can be a single chain variable region fragment (scFv). A single chain variable region fragment (scFv) is a truncated Fab fragment that includes an antibody heavy chain variable (V) domain linked to an antibody light chain V domain by a synthetic peptide and can be generated using conventional recombinant DNA technology. Similarly, disulfide stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology. The antibody construct or antigen binding domain can 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 a CDR1, CDR2, and CDR3.

[0034] A "cysteine mutant antibody" is an antibody in which one or more amino acid residues of the antibody are 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; US 7521541; US 7723485; US2012 / 0121615; WO 2009 / 052249). The cysteine residues provide site-specific conjugation of an adjuvant such as a TLR agonist to the antibody through the reactive cysteine thiol groups at the engineered cysteine sites, without interfering with immunoglobulin folding and assembly or altering antigen binding and effector functions. The cysteine mutant antibodies can be conjugated to TLR agonist-linker compounds, and the stoichiometry of the immunoconjugates is uniform (e.g., a maximum of two TLR agonist moieties per antibody in antibodies having a single engineered mutant cysteine site). The TLR agonist-linker compounds have a reactive electrophilic group to specifically react with the free cysteine thiol groups of the cysteine mutant antibodies.

[0035] An "epitope" means any antigenic determinant or epitope determinant of an antigen that binds to an antigen binding domain (i.e., at the paratope of the antigen binding domain). Antigenic determinants are usually groups of chemically active surface groups of molecules such as amino acids or sugar side chains, and are generally located on the surface of a molecule. An epitope typically has specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0036] The term "Fc receptor" or "FcR" means a receptor which binds to the Fc region of an antibody. There are three types of Fc receptors: (1) Fc gamma R which binds to IgG, (2) Fc alpha R which binds to IgA, and (3) Fc epsilon R which binds to IgE. The Fc gamma R family includes several members, such as Fc gamma I (CD64), Fc gamma RIIA (CD32A), Fc gamma RII B (CD32B), Fc gamma RIIIA (CD16A) and Fc gamma RIIIB (CD16B). Fc gamma receptors differ in their affinity for IgG and also have different affinities for IgG subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0037] Nucleic acid or amino acid sequence "identity" as referred to herein can be determined by comparing the nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are identical (i.e., are the same) between the best aligned sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). Alignment and calculation of percent identity of sequences can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, BLASTp, BLASTn, etc.), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also disclosed in, e.g., 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)). Percent identity (%) of sequences can also be calculated, e.g., as 100 x [(identical positions) / min(TG A ,TG B )], where TG A and TG B are the sum of residue numbers and internal gap positions in the alignment that minimizes TG A and TG B . See, e.g., Russell et al., J. Mol Biol., 244:332-350 (1994).

[0038] 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. The antibody construct can be any of a variety of types of binding agents known in the art comprising Ig heavy and light chains. For example, the binding agent can be an antibody, an antigen-binding antibody“fragment,” or a T cell receptor.

[0039] A“biosimilar” refers to an approved antibody construct that has similar activity properties to, for example, a previously approved antibody construct targeting PD-L1, such as atezolizumab (TECENTRIQ® TM , Genentech, Inc.), durvalumab (IMFINZI® TM , AstraZeneca), and avelumab (BAVENCIO® TM , EMD Serono, Pfizer); a previously approved antibody construct targeting HER2, such as trastuzumab (HERCEPTIN® TM , Genentech, Inc.), and pertuzumab (PERJETA® TM , Genentech, Inc.); or an antibody targeting CEA, such as labetuzumab (CEA-CIDE® TM , MN-14, hMN14, Immunomedics) CAS Registry Number 219649-07-7).

[0040] A“biobetter” refers to an approved antibody construct that improves upon a previously approved antibody construct, such as atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, and labetuzumab. The biobetter can have one or more modifications (e.g., altered glycan profile or unique epitope) that are superior to the previously approved antibody construct.

[0041] An“amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally occurring a-amino acids and stereoisomers thereof, as well as non-natural (non-naturally occurring) amino acids and stereoisomers thereof. A“stereoisomer” of a given amino acid refers to an isomer having the same molecular formula and intramolecular bonds but a different three-dimensional arrangement of bonds and atoms (e.g., L-amino acids and corresponding D-amino acids). An amino acid can be glycosylated (e.g., N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan, or glycosylphosphatidylinositolated) or deglycosylated. Amino acids can be represented herein by the commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0042] Naturally occurring amino acids are those encoded by the genetic code and those modified later, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Naturally occurring a-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 (lie), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring a-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.

[0043] Naturally occurring amino acids include those formed in proteins by post-translational modification, such as citrulline (Cit).

[0044] 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 act in a manner similar to naturally occurring amino acids. For example, an “amino acid analog” can be a non-natural amino acid having 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 with a modified side chain group or a modified peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. An “amino acid mimetic” refers to a chemical compound having a structure that is different from the general chemical structure of an amino acid but which acts in a manner similar to a naturally occurring amino acid.

[0045] "Linker" refers to a bifunctional or multifunctional moiety that covalently bonds two or more moieties in an immunoconjugate, such as an adjuvant moiety, to an antibody. Useful linkages for attaching linker moieties and adjuvant moieties to antibodies include, but are not limited to, amide, amine, ester, carbamate, disulfide, urea, thioether, thiocarbamate, thiocarbonate, and thiourea.

[0046] "Linker" refers to a bifunctional or multifunctional moiety that covalently bonds two or more moieties in an immunoconjugate, such as an adjuvant moiety, to an antibody. Useful linkages for attaching linker moieties and adjuvant moieties to antibodies include, but are not limited to, amide, amine, ester, carbamate, disulfide, urea, thioether, thiocarbamate, thiocarbonate, and thiourea.

[0047] "Bivalent" refers to a chemical moiety that contains two attachment points for linking two functional groups; a multivalent linker moiety can have additional attachment points for linking further functional groups. Bivalent groups can be indicated with the suffix "diyl." For example, bivalent linker moieties include bivalent polymeric moieties such as bivalent poly(ethylene glycol), bivalent cycloalkyl, bivalent heterocycloalkyl, bivalent aryl, and bivalent heteroaryl. "Bivalent cycloalkyl, heterocycloalkyl, aryl, or heteroaryl" refers to a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group having two attachment points for covalently linking two moieties or materials. 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, hydroxyl, amino, alkylamino, amido, acyl, nitro, cyano, alkoxy, and the like.

[0048] Wavy line represents the point of attachment of a particular chemical moiety. If two wavy lines are present then it is understood that the chemical moiety can be used bidirectionally, i.e., read from left to right or right to left. In some embodiments, a particular moiety with two wavy lines is considered to be used as read from left to right.

[0049] "Alkyl" refers to a straight / linear or branched saturated aliphatic group having the indicated number of carbon atoms. Alkyl groups can include any number of carbons, for example, one to twelve. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1 -propyl (n-Pr, n-butyl, -CH2CH2CH3), 2-propyl (i-Pr, isopropyl, -CH(CH3)2), 1 -butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-l -propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, tert-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-l -butyl (-CH2CH2CH(CH3)2), 2-methyl-l -butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 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(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3, 1-heptyl, 1-octyl, and the like. Alkyl groups can be substituted or unsubstituted. "Substituted alkyl" can be substituted with one or more groups selected from halo, hydroxyl, amino, oxo (=0), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0050] The term "alkyldiyl" refers to a divalent alkyl group. Examples of alkyldiyl groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and the like. Alkyldiyl groups can also be referred to as "alkylene."

[0051] "Alkenyl" refers to a straight chain (linear) or branched unsaturated aliphatic group having the indicated number of carbon atoms and at least one carbon-carbon double bond sp2. Alkenyl groups can include from two to about 12 or more carbon atoms. Alkenyl groups are groups having "cis" and "trans" orientations or alternatively "E" and "Z" orientations. Examples include, but are not limited to, ethenyl or vinyl (-CH=CH2), allyl (-CH2CH=CH2), butenyl, pentenyl, and isomers thereof. Alkenyl groups can be substituted or unsubstituted. "Substituted alkenyl" can be substituted with one or more groups selected from halo, hydroxyl, amino, oxo (=0), alkylamino, acylamino, acyl, nitro, cyano, and alkoxy.

[0052] The term "alkenylene" or "alkenyl diradical" refers to a straight chain or branched divalent hydrocarbon radical. Examples include, but are not limited to, ethenylene or vinylene (-CH=CH-), allylene (-CH2CH=CH-), and the like.

[0053] "Alkynyl" refers to a straight chain (linear) or branched unsaturated aliphatic group having the indicated number of carbon atoms and at least one carbon-carbon triple bond sp. Alkynyl groups can include from two 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 isomeric alkynyl groups. Alkynyl groups can be substituted or unsubstituted. "Substituted alkynyl" can be substituted with one or more groups selected from halo, hydroxyl, amino, oxo (=0), alkylamino, acylamino, acyl, nitro, cyano, and alkoxy.

[0054] The term "alkynylene" or "alkynyl diradical" refers to a divalent alkynyl radical.

[0055] "Heteroalkyl" or "heteroalkylene" refers to a monovalent straight chain or branched alkyl radical as defined above containing at least one heteroatom (including but not limited to Si, N, O, P, or S) within or at the end of the alkyl chain. In some embodiments, the heteroatom is within the alkyl chain. In other embodiments, the heteroatom is at the end of the alkylene group and thus serves to link the alkyl group to the remainder of the molecule. In some embodiments, the heteroalkyl group can have from 1 to 12 carbon atoms (Ci-C 12 heteroalkyl). In some embodiments, the heteroalkyl group can have from 1 to 24 carbon atoms (Ci-C 24 heteroalkyl). In some embodiments, the heteroalkyl group can have from 1 to 40 carbon atoms (Ci-C 40Heteroalkyl groups are optionally substituted unless otherwise explicitly noted in the specification. For example, a heteroalkyl group can be substituted with 1-6 fluoro (F) substituents, such as 1-6 fluoro (F) substituents on the carbon backbone (e.g., -CHF- or -CF2-) or 1-6 fluoro (F) substituents on the terminal carbon of a straight chain or branched heteroalkyl (e.g., -CHF2or -CF3). Examples of heteroalkyl groups include, but are not limited to, -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 heteroatoms can be consecutive, such as, for example, -CH2NHOCH3and -CH2OSi(CH3)3. Terminal polyethylene glycol (PEG) moieties are one type of heteroalkyl. Exemplary heteroalkyl groups also include oxiranes (e.g., polyoxiranes), oxetanes, amino acid chains (i.e., short to medium length peptides, such as containing 1-15 amino acids), and alkyl chains linked via various functional groups, such as amides, disulfides, ketones, phosphonates, phosphates, sulfates, sulfones, sulfonamides, esters, ethers, -S-, carbamates, ureas, thioureas, anhydrides, and the like (including combinations thereof). In some embodiments, a heteroalkyl includes a polyamino acid having 1-10 amino acids. In some embodiments, a heteroalkyl includes a polyamino acid having 1-5 amino acids.

[0056] Heteroalkyl groups include solubilizing units that comprise one or more polyglycines, polysarcosines, polyethylenes (PEGs), and glycosides, or combinations thereof.

[0057] "Heteroalkenyl" refers to a heteroalkyl group as defined above containing at least one carbon-carbon double bond. "Heteroalkynyl" refers to a heteroalkyl group as defined above containing at least one carbon-carbon triple bond.

[0058] "Heteroalkyldiyl" refers to the divalent form of heteroalkyl as defined above. In some embodiments, a heteroalkyldiyl can have 1 to 12 carbon atoms (Ci-Ci2). In some embodiments, a heteroalkyldiyl can have 1 to 24 carbon atoms (Ci-Ci2). In some embodiments, a heteroalkyldiyl can have 1 to 40 carbon atoms (Ci-Ci2). Examples of heteroalkyldiyl groups include, but are not limited to, -CH2CH2OCH2-, -CH2CH2OCF2-, -CH2CH2NHCH2-, -CH2OC(=0)NH-, -CH2OP(=0)(OH)OCH2-, -C(=0)NHCH2CH2NHCH2-, -C(=0)N(CH3)CH2CH2N(CH3)CH2-, -C(=0)NHCH2CH2NHC(=0)CH2CH2-, -C(=0)N(CH3)CH2CH2N(CH3)C(=0)CH2CH2-, -OCH2CH2OCH2CH2-, -OCH2CH2OCH2C(=0)-, -OCH2CH2OCH2CH2C(=0)-, -OCH2CH2NHCH2-, -OCH2CH2N(CH3)CH2-, -OCH2CH2CH2NHCH2-, -OCH2CH2CH2N(CH3)CH2-, -OCH2CH2CH2NHC(=0)CH2CH2-, -OCH2CH2CH2N(CH3)C(=0)CH2CH2-, -CH2CH2CH2NHCH2-, -CH2CH2CH2N(CH3)CH2-, -CH2CH2CH2NHC(=0)CH2CH2-, -CH2CH2CH2N(CH3)C(=0)CH2CH2-, -CH2CH2NHC(=0)-, -CH2CH2N(CH3)CH2-, -CH2CH2N 12 (CH3)2-, -NHCH2CH2(NH2)CH2-, and -NHCH2CH2(NHCH3)CH2-. A divalent polyethylene glycol (PEG) moiety having from one to about 50 -OCH2CH2- units is one type of heteroalkyldiyl. "Heteroalkenyldiyl" refers to the divalent form of heteroalkenyl. "Heteroalkynyldiyl" refers to the divalent form of heteroalkynyl. 24 (CH3)2-, -NHCH2CH2(NH2)CH2-, and -NHCH2CH2(NHCH3)CH2-. A divalent polyethylene glycol (PEG) moiety having from one to about 50 -OCH2CH2- units is one type of heteroalkyldiyl. "Heteroalkenyldiyl" refers to the divalent form of heteroalkenyl. "Heteroalkynyldiyl" refers to the divalent form of heteroalkynyl. 40 (CH3)2-, -NHCH2CH2(NH2)CH2-, and -NHCH2CH2(NHCH3)CH2-. A divalent polyethylene glycol (PEG) moiety having from one to about 50 -OCH2CH2- units is one type of heteroalkyldiyl. "Heteroalkenyldiyl" refers to the divalent form of heteroalkenyl. "Heteroalkynyldiyl" refers to the divalent form of heteroalkynyl. + (CH3)2-, -NHCH2CH2(NH2)CH2-, and -NHCH2CH2(NHCH3)CH2-. A divalent polyethylene glycol (PEG) moiety having from one to about 50 -OCH2CH2- units is one type of heteroalkyldiyl. "Heteroalkenyldiyl" refers to the divalent form of heteroalkenyl. "Heteroalkynyldiyl" refers to the divalent form of heteroalkynyl.

[0059] The terms "carbocyclic," "carbocyclyl," "carbocyclic ring," 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 carbocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocyclic rings include, for example, norbornane, [2.2.2]bicyclooctane, decalin, and adamantane. Carbocyclic groups can also be partially unsaturated, having 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.

[0060] The term "cycloalkyldiyl" refers to a divalent cycloalkyl group.

[0061] "Aryl" means a monovalent aromatic hydrocarbon group 6-20 carbon atoms (C6-C 20 ) derived by removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Aryl groups can be monocyclic, fused to form a bicyclic or tricyclic ring system, or linked by a bond to form a biaryl radical. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl groups having a methylene linker. Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl.

[0062] The term "aryldiyl" or "aryl diradical" means a divalent aromatic hydrocarbon group 6-20 carbon atoms (C6-C 20 ) derived by removal of two hydrogen atoms from two carbon atoms of a parent aromatic ring system. Some aryl diradicals are represented in exemplary structures as "Ar." Aryl diradicals include bicyclic groups comprising an aromatic ring fused to a saturated, partially unsaturated, or aromatic carbocyclic ring. Typical aryl diradicals include, but are not limited to, groups derived from benzene (phenyldiyl), substituted benzenes, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthalene, and the like. Aryl diradical groups are also referred to as "arylene," and are optionally substituted with one or more substituents described herein.

[0063] The terms “heterocycle,” “heterocyclic group,” and “heterocyclic ring” are used interchangeably herein and refer to a carbocyclic group of 3 to 20 ring atoms that is saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring), wherein at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, and the remaining ring atoms are carbon, wherein one or more ring atoms are optionally independently substituted by one or more of the following substituents. The heterocycle can 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), for example: bicyclic [4,5], [5,5], [5,6], or [6,6] systems. Heterocyclic compounds are described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (WA Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons, New York, 1950–present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. “Heterocyclic group” also includes groups in which the heterocyclic group is fused with a saturated, partially unsaturated ring or aromatic carbide ring or heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, morpholino-4-yl, piperidino-1-yl, piperazino, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidine-1-yl, thiomorpholino-4-yl, S-dioxothiomorpholino-4-yl, azacyclooctane-1-yl, azacyclobutane-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazacycloheptane-1-yl, pyrrolylyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiopheneyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiaranyl, piperidinyl, morpholino, thiomorpholino, thiaxylyl, piperazino, homopiperazino, azacyclobutane, oxacyclobutane, thiohexacyclobutane, homopiperidino, oxacycloheptane, thiohexacycloheptane, oxacyclobutane basalt, diazoxide Basic, sulfur-nitrogen The following groups are included: 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxalyl, 1,3-dioxolane, pyrazolinyl, dithiaylyl, dithiopentanyl, dihydropyranyl, dihydrothiophenyl, dihydrofuranyl, pyrazolaneylimidazolinyl, imidazolinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 3H-indolylquinazinyl, and N-pyridylurea. Spiroheterocyclic moieties are also included within the scope of this definition. Examples of spiroheterocyclic moieties include azaspiro[2.5]octyl and azaspiro[2.4]heptyl. Examples of heterocyclic groups in which two ring atoms are substituted by an oxygen (=O) moiety are pyrimidinone and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups described herein may be optionally and independently substituted by one or more substituents as described herein.

[0064] The term "heterocyclic dimethyl" refers to a divalent saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic group of 3 to 20 ring atoms, wherein at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, and the remaining ring atoms are C, wherein one or more ring atoms are optionally and independently substituted by one or more substituents as described. Examples of 5- and 6-membered heterocyclic dimethyl groups include morpholinyl dimethyl, piperidinyl dimethyl, piperazinyl dimethyl, pyrrolidinyl dimethyl, dioxinyl dimethyl, thiomorpholinyl dimethyl, and S-dioxothiomorpholinyl dimethyl.

[0065] The term "heteroaryl" refers to a monovalent aromatic group of a 5, 6, or 7-membered ring and includes a fused ring system 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), imidazole, imidazole-pyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thiophene, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrroleyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, borazolyl, indazole, inazinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purine, oxadiazolyl, thiazolyl, thiazolyl, furazolyl, benzofuranyl, benzothiophene, benzothiazolyl, benzooxazolyl, quinazolinyl, quinoxolinyl, naphthidyl, and furanylpyridinyl. The heteroaryl group may be optionally and independently substituted by one or more substituents as described herein.

[0066] The term "heteroaryldiyl" refers to a 5-, 6-, or 7-membered ring bivalent aromatic radical and includes fused ring systems of 5-20 atoms (at least one of which is aromatic) containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of 5- and 6-membered heteroaryldiyl groups include pyridyldiyl, imidazolyldiyl, pyrimidinyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furanyldiyl, thienyldiyl, isoxazolyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl.

[0067] A heterocycle or heteroaryl can be carbon (carbon-linked) or nitrogen (nitrogen-linked) bonded where possible. By way of example and not limitation, a carbon- bonded heterocycle or heteroaryl is bonded at the 2, 3, 4, 5, or 6 position of a pyridine; the 3, 4, 5, or 6 position of a pyridazine; the 2, 4, 5, or 6 position of a pyrimidine; the 2, 3, 5, or 6 position of a pyrazine; the 2, 3, 4, or 5 position of a furan, tetrahydrofuran, thiophene, pyrrole, or tetrahydropyrrole; the 2, 4, or 5 position of an oxazole, imidazole, or thiazole; the 3, 4, or 5 position of an isoxazole, pyrazole, or isothiazole; the 2 or 3 position of an aziridine; the 2, 3, or 4 position of an azetidine; the 2, 3, 4, 5, 6, 7, or 8 position of a quinoline; or the 1, 3, 4, 5, 6, 7, or 8 position of an isoquinoline.

[0068] By way of example and not limitation, a nitrogen-bonded heterocycle or heteroaryl is bonded at the 1 position of an 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; the 2 position of isoindole or isoindoline; the 4 position of morpholine; and the 9 position of carbazole or β-carboline.

[0069] The terms "halo" and "halogen" by themselves or as part of another substituent refer to a fluorine, chlorine, bromine, or iodine atom.

[0070] The term "carbonyl" by itself or as part of another substituent refers to C(=O) or -C(=O)-, that is, a carbon atom bonded to a double bond of oxygen.

[0071] As used herein, the phrase "quaternary ammonium salt" refers to a tertiary amine that has been quaternized with an alkyl substituent (e.g., a C1-C4 alkyl group, such as a methyl, ethyl, propyl, or butyl group).

[0072] The term "chiral" refers to a molecule that has the property of non-superimposability of its mirror image partner, while the term "achiral" refers to a molecule that is superimposable on its mirror image partner.

[0073] The term "stereoisomers" refers to compounds which have the same chemical constitution, but which differ in the arrangement of atoms or groups in space.

[0074] Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the application can contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the application, including but not limited to, diastereomeric, enantiomeric forms, and mixtures thereof (such as racemates), fall within the scope of the application. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate 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 1 or (+) and (-) are employed to designate the sign of the rotation of plane-polarized light by the compound, (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer can also be referred to as an enantiomer when there are one or more chiral centers in the molecule. Mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which can occur where there has been no stereocontrol in a chemical reaction or process. The terms "racemic mixture" and "racemate" mean an equimolar mixture of two enantiomeric species without optical activity.

[0075] "Diastereomer" refers to a stereoisomer of a compound that has at least two chiral centers and whose molecules are not mirror images of one another.

[0076] "Enantiomer" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0077] The term "tautomer" or "tautomeric form" refers to different energy structures that are interconvertible via a low energy barrier. For example, prototropic tautomers (also known as prototropic isomer) include interconversions that occur via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions that occur by reorganization of some of the bonding electrons.

[0078] The term "salt" refers to an acid or base salt of a compound disclosed herein. Illustrative examples of pharmaceutically acceptable salts are mineral acid (hydrochloric, hydrobromic, phosphoric, etc.) salts, organic acid (acetic, propionic, glutamic, citric, 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 ammonium salts, such as ammonium, trimethylammonium, diethylammonium, and tri(hydroxymethyl)ammonium salts. Similarly, acid addition salts, such as mineral acid, organic carboxylic, and organic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid, maleic acid, are possible, provided that the basic group, such as pyridyl, forms part of the structure. The neutral forms of the compounds can be regenerated by contacting the salts 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 otherwise the salts are equivalent to the parent form of the compound for the purposes of the present disclosure.

[0079] Any compound or formula given herein is intended to represent both the unlabeled form as well as isotopically labeled form (i.e., "isotopically enriched" or "isotopically substituted") of the compound. Isotopically labeled compounds have structures depicted by the formulas given 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 enco 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 disclosure, for example, those into which radioactive isotopes such as3 H, 13 C and 14 C) of those. Such isotopically labeled compounds can be useful in enhancing therapeutic activity, can be useful in metabolic studies, in reaction kinetic studies, in detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) (including drug or substrate tissue distribution assays), or can be used in radiotherapeutic procedures.

[0080] The present disclosure also includes "deuterated analogs" of the compounds described herein, wherein one to n hydrogens attached to a carbon atom are replaced by deuterium ( 2 H) where n is the number of hydrogens in the molecule. Such compounds exhibit enhanced anti-metabolic properties when administered to mammals, particularly humans, and are thus useful for increasing the half-life of any compound. See, e.g., Foster, "Deuterium Isotope Effects in Studies of Drug Metabolism," Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compounds are synthesized by methods well known in the art, e.g., by using starting materials in which one or more hydrogens are replaced by deuterium. Deuterium labeled or substituted therapeutic compounds of the present disclosure can have improved DMPK (drug metabolism and pharmacokinetics) properties relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and / or an improvement in therapeutic index. 18 F, 3 H or 11 C labeled compounds can be useful in PET or SPECT or other imaging studies. Isotopically labeled compounds of the present disclosure, and prodrugs thereof, can generally be prepared by carrying out the procedures described below, or by the procedures disclosed in the Examples and Preparations, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. It will be appreciated that deuterium in the present context is regarded as a substituent in the compounds described herein. The concentration of such a heavier isotope, particularly deuterium, can be defined in terms of an isotopic enrichment factor. In the compounds of the present disclosure, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of said atom. Unless otherwise indicated, when a position is designated to be "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 meant to represent deuterium.

[0081] The terms“treat,”“treatment,” and“treating” refer to any indicia of success in the treatment or amelioration of an injury, pathology, disease (e.g., cancer), or condition (e.g., cognitive impairment), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the patient more comfortable; decreasing of disease progression; frequency or duration; or in some circumstances, prevention of onset. Treatment or amelioration can be graded or assessed using any objective or subjective parameter, including, for example, results of a physical examination.

[0082] The terms“cancer,”“neoplasm,” and“tumor” are used herein to refer to cells that exhibit autonomous, unregulated growth such that the cells exhibit an abnormal growth phenotype characterized by a marked loss of control of cell proliferation. Cells of interest for detection, analysis, and / or treatment in the context of the present application include cancer cells (e.g., cancer cells from an individual having a cancer), malignant cancer cells, pre-metastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. Cancer is known for almost every tissue. The phrase“cancer burden” refers to the number of cancer cells or the volume of cancer in a subject. Reducing cancer burden accordingly refers to reducing the number of cancer cells or the volume of cancer cells in a subject. The term“cancer cell” as used herein refers to any cell that is or is derived from a cancer cell (e.g., from any cancer that an individual can be treated for, e.g., isolated from an individual having a cancer), or is a clone of a cancer cell, for example. For example, a cancer cell can be from an established cancer cell line, can be a primary cell isolated from an individual having a cancer, can be a daughter cell from a primary cell isolated from an individual having a cancer, and the like. In some embodiments, the term can also refer to a portion of a cancer cell, such as a subcellular portion of a cancer cell, a cell membrane portion, or a cell lysate. Many types of cancer are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, and circulating cancers such as leukemias.

[0083] As used herein, the term“cancer” includes any form of cancer, including but not limited to solid tumor cancers (e.g., skin cancer, lung cancer, prostate cancer, breast cancer, gastric cancer, bladder cancer, colon cancer, ovarian cancer, pancreatic cancer, renal cancer, liver cancer, glioblastoma, medulloblastoma, leiomyosarcoma, head and neck squamous cell carcinoma, melanoma, and neuroendocrine cancer) and liquid cancers (e.g., blood cancers); carcinoma; sarcoma; sarcomas; teratocarcinoma; melanoma; leukemia; lymphoma; and brain cancer, including minimal residual disease, and includes both primary tumors and metastatic tumors.

[0084] “PD-L1 expression” refers to a cell having a PD-L1 receptor on its surface. As used herein,“PD-L1 overexpression” refers to a cell having more PD-L1 receptors compared to a corresponding non-cancer cell.

[0085] “HER2” refers to the protein human epidermal growth factor receptor 2.

[0086] “HER2 expression” refers to a cell having HER2 receptors on the surface of the cell. For example, a cell can have from about 20,000 to about 50,000 HER2 receptors on the surface of the cell. As used herein, “HER2 overexpression” refers to a cell having greater than about 50,000 HER2 receptors. For example, a cell has 2-, 5-, 10-, 100-, 1,000-, 10,000-, 100,000-, or 1,000,000-fold more HER2 receptors than a corresponding non-cancerous cell (e.g., about 1 million or 2 million HER2 receptors). HER2 is estimated to be overexpressed in about 25% to about 30% of breast cancers.

[0087] The “pathology” of a cancer includes all phenomena that impair the health of a patient. This includes, but is not limited to, abnormal or uncontrolled cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immune responses, neoplasia, pre-cancer, malignancy, and invasion of surrounding or distant tissues or organs such as lymph nodes.

[0088] As used herein, the phrases “cancer recurrence” and “tumor recurrence” and grammatical variations thereof refer to further growth of tumor or cancer cells after diagnosis of cancer. In particular, recurrence can occur when further cancer cell growth occurs in the cancerous tissue. Similarly, “tumor spread” occurs when tumor cells disseminate to local or distant tissues and organs, and thus, tumor spread encompasses tumor metastasis. “Tumor invasion” occurs when tumor growth locally spreads to impair the function of the affected tissue by compression, destruction, or prevention of normal organ function.

[0089] 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 original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancer cells in an organ or body part that is not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from the original tumor site, and the migration and / or invasion of cancer cells into other parts of the body.

[0090] The phrases "effective amount" and "therapeutically effective amount" mean the dose or amount of a substance, such as an immunoconjugate, that will exert the desired therapeutic effect. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled 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 Ed. (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22nd Ed. (Pharmaceutical Press, London, 2012)). In the context of cancer, a therapeutically effective amount of an immunoconjugate can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the immunoconjugate can prevent development of or

[0091] "Recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., human) in need of diagnosis, treatment, or therapy. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain embodiments, the mammal is a human.

[0092] 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 combination elicits a synergistic effect on immunity relative to the administration of either alone. In particular, the immunoconjugates disclosed herein comprise a synergistic combination of the claimed adjuvant and antibody construct. These synergistic combinations elicit a greater immune effect upon administration, for example, relative to the administration of the antibody construct or adjuvant in the absence of the other. Further, the amount of the immunoconjugate that can be administered is reduced (as measured by the total number of antibody constructs or total number of adjuvant administered as part of the immunoconjugate) compared to the administration of the antibody construct or adjuvant alone.

[0093] The term "administering" as used herein 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 slow-release device, such as a microosmotic pump, to a subject.

[0094] The terms "about" and "approximately," as used herein to modify a value, indicate a close range around that value. Thus, if "X" is the value, "about X" or "approximately X" indicates a value of 0.9X to 1.1X, such as 0.95X to 1.05X or 0.99X to 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 written description support for claim limitations of, for example, "0.98X."

[0095] Antibody

[0096] The immunoconjugates of the present invention comprise an antibody. The scope of embodiments of the present invention includes functional variants of the antibody constructs or antigen binding domains described herein. The term "functional variant" as used herein refers to an antibody construct of an antigen binding domain having substantial or significant sequence identity or similarity to a parent antibody construct or antigen binding domain, which functional variant retains the biological activity of the antibody construct or antigen binding domain of which it is a variant. Functional variants encompass, for example, those variants of the antibody constructs or antigen binding domains described herein (parent antibody construct or antigen binding domain) that retain the ability to recognize a target cell to a similar, identical, or greater extent than the parent antibody construct or antigen binding domain.

[0097] A functional variant can comprise, for example, an amino acid sequence of a parent antibody construct or antigen binding domain having at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can comprise an amino acid sequence of a parent antibody construct or antigen binding domain having 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, such that the biological activity of the functional variant is increased compared to the parent antibody construct or antigen binding domain.

[0098] A functional variant can comprise, for example, an amino acid sequence of a parent antibody construct or antigen binding domain having at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can comprise an amino acid sequence of a parent antibody construct or antigen binding domain having 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, such that the biological activity of the functional variant is increased compared to the parent antibody construct or antigen binding domain.

[0099] Antibodies comprising the immunoconjugates of the present application include Fc engineered variants. In some embodiments, mutations in the Fc region that result in modulation of binding to one or more Fc receptors can include one or more of 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), GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R), and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at E345R, E233, G237, P238, H268, P271, L328, and A330. Additional Fc region modifications for modulating Fc receptor binding are described in, for example, US 2016 / 0145350, US 7416726, and US 5624821, which are hereby incorporated by reference in their entireties.

[0100] Antibodies comprising the immunoconjugates of the present application include glycan variants, such as afucosylation. In some embodiments, the Fc region of the binding agent is modified to have an altered Fc region glycosylation pattern compared to a naturally unmodified Fc region.

[0101] Amino acid substitutions of the antibody constructs or antigen binding domains of the application are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which an amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain substituted for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, lie, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid having a polar side chain substituted for another uncharged amino acid having a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid having a beta-branched side chain substituted for another amino acid having a beta-branched side chain (e.g., lie, Thr, and Val), an amino acid having an aromatic side chain substituted for another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0102] The antibody construct or antigen binding domain can consist essentially of one or more of the specified amino acid sequences described herein, such that other components (e.g., other amino acids) do not materially change the biological activity of the functional variant of the antibody construct or antigen binding domain.

[0103] In some embodiments, the antibody in the immunoconjugate contains a modified Fc region, wherein the modification modulates binding of the Fc region to one or more Fc receptors.

[0104] In some embodiments, the antibody in the immunoconjugate (e.g., the antibody conjugated to at least two adjuvant moieties) contains one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region that result in modulated (e.g., increased binding or decreased binding) binding to one or more Fc receptors (e.g., FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a), and / or FcyRIIIB (CD16b)) as compared to a native antibody that lacks the mutations in the Fc region. In some embodiments, the antibody in the immunoconjugate contains one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region that decrease binding of the Fc region of the antibody to FcyRIIB. In some embodiments, the antibody in the immunoconjugate contains 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 FcyRIIB while maintaining the same binding or increasing binding to FcyRI (CD64), FcyRIIA (CD32A), and / or FcyRIIIA (CD16a) as compared to a native antibody that lacks the mutations in the Fc region. In some embodiments, the antibody in the immunoconjugate contains one or more modifications in the Fc region that increase binding of the Fc region of the antibody to FcyRIIB.

[0105] In some embodiments, the modulated binding is provided by mutations in the Fc region of the antibody relative to a 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 found in Fc regions found in nature or identical to that found in the Fc region in a native antibody (e.g., cetuximab). Native sequence human Fc regions include a native sequence human IgGl Fc region, a native sequence human IgG2 Fc region, a native sequence human IgG3 Fc region, and a native sequence human IgG4 Fc region as well as naturally occurring variants thereof. Native sequence Fc includes the various allotypes of Fc (Jefferis et al., (2009) mAbs, 1(4):332-338).

[0106] In some embodiments, the Fc region of the antibody of the immunoconjugate is modified to have an altered Fc region glycosylation pattern as compared to a native unmodified Fc region.

[0107] 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- acetylglucosamine4mannose3(GlcNAc4Man3). Removal of the heptasaccharide with endoglycosidases or PNGase F is known to cause a conformational change in the Fc region of antibodies, which can significantly reduce the binding affinity to antibodies that activate FcγR and lead to reduced effector function. The core heptasaccharide is often modified with galactose, bisecting GlcNAc, fucose or sialic acid, which differentially affects Fc binding to activating and inhibitory FcγR. In addition, α2,6-sialylation has been shown to enhance anti-inflammatory activity in vivo, while afucosylation leads to improved FcγRIIIa binding and a 10-fold increase in antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. Specific glycosylation patterns can thus be used to control inflammatory effector functions.

[0108] In some embodiments, the modification that alters the glycosylation pattern is a mutation. For example, a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods of using antibodies that modulate signaling through FcγR to control immune responses are described in, for example, US 7416726, US 2007 / 0014795, and US 2008 / 0286819, which are hereby incorporated by reference in their entireties.

[0109] 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, a hybridoma can be genetically engineered to secrete an afucosylated mAb, desialylated mAb, or deglycosylated Fc with specific mutations that enable increased FcRγIIIa binding and effector function. In some embodiments, the antibody of the immunoconjugate is engineered to be afucosylated.

[0110] In some embodiments, the entire Fc region of the antibody in the immunoconjugate is exchanged for 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 an IgG2, IgG3, IgG4, or IgA Fc region, or the Fab region of nivolumab, which normally contains an IgG4 Fc region, can be conjugated to an IgG1, IgG2, IgG3, IgA1, or IgG2 Fc region. In some embodiments, the Fc-modified antibody with a non-native Fc domain further comprises one or more amino acid modifications, such as the S228P mutation within the IgG4 Fc, which modulates the stability of the Fc domain. In some embodiments, the Fc-modified antibody with a non-native Fc domain further comprises one or more of the amino acid modifications described herein that modulate Fc binding to FcR.

[0111] In some embodiments, the modification that modulates Fc region binding to an FcR does not alter the binding of the Fab region of the antibody to its antigen when compared to a naturally unmodified antibody. In other embodiments, the modification that modulates Fc region binding to an FcR also increases the binding of the Fab region of the antibody to its antigen when compared to a naturally unmodified antibody.

[0112] In some embodiments, the antibody in the immunoconjugate contains a modified Fc region, wherein the modification modulates binding of the Fc region to one or more Fc receptors.

[0113] In some embodiments, the Fc region is modified by inclusion of a transforming growth factor beta 1 (TGFβ1) receptor or fragment thereof that is capable of binding 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 fusion to the C-terminus of the TGFβRII extracellular domain (ECD), as described in US 9676863, which is incorporated herein. An “Fc linker” can be used to attach the IgG to the TGFβRII extracellular domain. The Fc linker can be a short, flexible peptide that allows the molecule to fold correctly in three dimensions 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.

[0114] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0115] In some embodiments, the antibody in the immunoconjugate is a cysteine engineered antibody, which provides site-specific conjugation of an adjuvant, label, or drug moiety to the antibody through a cysteine substitution at a site engineered for conjugation of the cysteine, without perturbing immunoglobulin folding and assembly or altering antigen binding and effector functions (Junutula et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al., (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; US2012 / 0121615; WO 2009 / 052249). The cysteine engineered antibody can be conjugated via an azido-benzazido - linker compound to an azido-benzazido Adjuvant moieties are conjugated (e.g., in antibodies having a single engineered cysteine site, up to two nitrogen- benzazepine moieties per antibody).

[0116] In some embodiments, cysteine engineered antibodies are used to make immunoconjugates. Immunoconjugates can introduce a reactive cysteine thiol residue at a site on the light chain (e.g., 149-lysine site (LC K149C)) or a site on the heavy chain (e.g., 122-serine site (HC S122C)), as numbered by Kabat numbering. In other embodiments, the cysteine engineered antibody has a cysteine residue introduced at the 118-alanine site (EU numbering) of the heavy chain (HC A118C). This site is alternatively numbered sequentially as 121, or as 114 by 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; WO 2011 / 156328; US 9000130.

[0117] In exemplary embodiments, the immunoconjugates of the application comprise an antibody construct comprising an antigen binding domain that specifically recognizes and binds PD-L1.

[0118] Programmed 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 the programmed cell death protein 1 (PD-1, PDCD1, cluster of differentiation 279, or CD279). PD-L1 can also interact with B7.1 (CD80), and this interaction is thought to inhibit T cell priming. The PD-L1 / PD-1 axis plays an important role in suppressing adaptive immune responses. More specifically, it is believed that engagement of PD-L1 with its receptor, PD-1, delivers a signal that suppresses the activation and proliferation of T cells. Agents that bind to PD-L1 and prevent the ligand from binding to the PD-1 receptor prevent this immune suppression and, thus, can enhance immune responses when needed, such as for treating cancer or infection. The PD-L1 / PD-1 pathway also contributes to preventing autoimmunity, so agonists against PD-L1 or agents that deliver immune suppressive payloads can be helpful in treating autoimmune disorders.

[0119] Several antibodies targeting PD-L1 have been developed for treating cancer, including atezolizumab (TECENTRIQ TM ), durvalumab (IMFINZI TM ), and avelumab (BAVENCIO TM ). Nonetheless, there remains a need for new PD-L1 antibody constructs, including agents that bind PD-L1 with high affinity and effectively block PD-L1 / PD-1 signaling, as well as agents that can deliver therapeutic payloads to cells expressing PD-L1. In addition, there is a need for new PD-L1 binding agents to treat autoimmune disorders and infections.

[0120] The present invention provides methods of delivering a TLR agonist payload to a cell expressing 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 covalently attached to one or more TLR agonist moieties.

[0121] Also provided are methods of enhancing or reducing or inhibiting an immune response in a mammal and methods of treating a disease, disorder, or condition in a mammal that is responsive to PD-L1 inhibition, the methods comprising administering to the mammal an immunoconjugate thereof to PD-L1.

[0122] This invention provides a PD-L1 antibody comprising a polypeptide of the variable region of the immunoglobulin heavy chain and a polypeptide of the variable region of the immunoglobulin light chain. The PD-L1 antibody specifically binds to PD-L1. This binding specificity allows targeting of cells expressing PD-L1, for example, delivering a therapeutic payload to such cells. In some embodiments, the PD-L1 antibody binds to human PD-L1. However, antibodies that bind to any PD-L1 fragment, homolog, or parahomolog are also covered.

[0123] In some embodiments, the PD-L1 antibody binds to PD-L1 without substantially inhibiting or preventing the binding of PD-L1 to its receptor PD-1. However, in other embodiments, the PD-L1 antibody may completely or partially block (inhibit or prevent) the binding of PD-L1 to its receptor PD-1, making the antibody usable for inhibiting PD-L1 / PD-1 signaling (e.g., for therapeutic purposes). The antibody or antigen-binding antibody fragment may be monospecific to PD-L1, or it may be bispecific or multispecific. For example, in bivalent or multivalent antibodies or antibody fragments, the binding domains may be different, targeting different epitopes of the same antigen or different antigens. Methods for constructing multivalent binding constructs are known in the art. Bispecific and multispecific antibodies are known in the art. Furthermore, biantibodies, triantibodies, or tetraantibodies may be provided, which are dimers, trimers, or tetramers of polypeptide chains, each polypeptide chain containing a peptide linker connected to V L Connected V H The peptide linker is too short to allow V on the same polypeptide chain to connect. H With V L Pairing between them to drive different V H -V L Pairing between complementary domains on polypeptide chains can generate multimeric molecules with two, three, or four functional antigen-binding sites. Additionally, dual scFv fragments—small scFv fragments with two different variable domains—can be generated to produce bispecific dual scFv fragments capable of binding two different epitopes. Genetic engineering methods can be used to generate Fab dimers (Fab2) and Fab trimers (Fab3) to create multispecific constructs based on Fab fragments.

[0124] PD-L1 antibodies can be or are derived from human antibodies, non-human antibodies, humanized antibodies, or chimeric antibodies or corresponding antibody fragments. A “chimeric” antibody is an antibody or fragment thereof that typically contains a human constant region and a non-human variable region. A “humanized” antibody is a monoclonal antibody that typically contains a human antibody scaffold but has non-human derived amino acids or sequences in at least one CDR (e.g., 1, 2, 3, 4, 5, or all six CDRs).

[0125] The PD-L1 antibody can be internalizing, as described in WO 2021 / 150701 and incorporated herein by reference, or the PD-L1 antibody can be non-internalizing, as described in WO 2021 / 150702 and incorporated herein by reference.

[0126] In exemplary embodiments, the immunoconjugates of the application comprise an antibody construct comprising an antigen binding domain that specifically recognizes and binds HER2.

[0127] A number of anti-HER2 monoclonal antibodies have been approved and are in clinical development (Costa, RLB, et al. (2020) Breast Cancer 6(10): 1-11.

[0128] In certain embodiments, the immunoconjugates of the application comprise an anti-HER2 antibody, such as an antibody prepared by the method of Example 201. In one embodiment of the application, the anti-HER2 antibody of the immunoconjugates of the application comprises a humanized anti-HER2 antibody, for example, huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8, as described in Table 3 of US 5821337, which patent is specifically incorporated herein by reference. These antibodies contain human framework regions and the complementarity determining regions of the murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also known as trastuzumab, which is commercially available under the trade name HERCEPTIN TM (Genentech, Inc.).

[0129] Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, Trastuzumab (HERCEPTIN®, Genentech, Inc.) is a recombinant DNA-derived IgGl kappa monoclonal antibody that is a humanized form of the murine anti-HER2 antibody (4D5) that binds selectively to the extracellular domain of HER2 with high affinity (Kd = 5 nM) in cell-based assays (US 5677171; US 5821337; US 6054297; US 6165464; US 6339142; US 6407213; US 6639055; US 6719971; US 6800738; US 7074404; Coussens et al. (1985) Science 230: 1132-9; Slamon et al. (1989) Science 244: 707-12; Slamon et al. (2001) New Engl. J. Med. 344: 783-792).

[0130] In embodiments of the application, the antibody construct or antigen binding domain comprises the CDR regions of trastuzumab. In embodiments of the application, the anti-HER2 antibody further comprises the framework regions of trastuzumab. In embodiments of the application, the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.

[0131] In another embodiment of the application, the anti-HER2 antibody of the immunoconjugate of the application comprises a humanized anti-HER2 antibody, such as humanized 2C4, as described in US 7862817. Exemplary humanized 2C4 antibodies are pertuzumab (CAS Registry Number 380610-27-5), PERJETA TM (Genentech, Inc.). Pertuzumab is a HER dimerization inhibitor (HDI) and is used to inhibit the ability of HER2 to form active heterodimers or homodimers with other HER receptors, such as 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 TM is approved for the treatment of breast cancer.

[0132] In embodiments of the present invention, the antibody construct or antigen-binding domain includes the CDR region of pertuzumab. In embodiments of the present invention, the anti-HER2 antibody further includes the framework region of pertuzumab. In one embodiment of the present invention, the anti-HER2 antibody further includes one or two variable regions of pertuzumab.

[0133] Margetucizumab (MGAH22, MARGENZA) TM Magetuximab (MacroGenics, Inc.), CAS Registry No. 1350624-75-7, is an FDA-approved anti-HER2 monoclonal antibody. The Fc region of magetuximab 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). Magetuximab has been approved by the FDA for the treatment of patients with relapsed or refractory advanced breast cancer whose tumors express HER2 at a 2+ level as determined by immunohistochemistry and who lack evidence of HER2 gene amplification as determined by FISH.

[0134] HT-19 is another anti-HER2 monoclonal antibody that binds to an epitope in human HER2 that differs from the epitopes of trastuzumab or pertuzumab. HT-19 exhibits comparable inhibition of HER2 signaling to trastuzumab and enhances HER2 degradation when combined 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).

[0135] In an exemplary embodiment, the immunoconjugate of the present invention comprises an antibody construct containing an antigen-binding domain that specifically recognizes and binds to CEA. Carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), also known as CD66e (differentiation cluster 66e), is a member of the carcinoembryonic antigen (CEA) gene family.

[0136] Elevated expression of carcinoembryonic antigens (CEA, CD66e, CEACAM5) has been implicated in various biological aspects of tumorigenesis, particularly in tumor cell adhesion, metastasis, blocking of cellular immune mechanisms, and anti-apoptotic functions. CEA is also used as a blood biomarker for many cancers. Labectotuzumab (CEA-CIDE) TMLabetuzumab (IMMU-105), also known as hMN14, is a humanized IgGl monoclonal antibody and has been investigated for the treatment of colorectal cancer (Blumenthal, R. et al. (2005) Cancer Immunology Immunotherapy 54(4):315-327). Labetuzumab conjugated to a camptothecin analogue (golvituximab labetuzumab, IMMU-130) targets carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and is being investigated in patients with relapsed 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 embodiments of the present application, the CEA-targeting antibody construct or antigen binding domain comprises the variable light chain (VLK) of hMN-14 / labetuzumab as disclosed in US 6,676,924, which is incorporated herein by reference for this purpose.

[0137] In embodiments of the present application, the heavy chain (HC) of the CEA-targeting antibody is selected from the group consisting of SEQ ID NOs: 1-5.

[0138]

[0139]

[0140] In embodiments of the present application, the light chain (LC) of the CEA-targeting antibody is selected from the group consisting of SEQ ID NOs: 6-9.

[0141]

[0142]

[0143] In exemplary embodiments, the immunoconjugates of the present application comprise an antibody construct comprising an antigen binding domain that specifically recognizes and binds to TROP2. Tumor-associated calcium signal transducer 2 (TROP-2) 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 transducer that is differentially expressed in many cancers and signals to the cell to self-renew, proliferate, invade, and survive. TROP2 is considered a stem cell marker and is expressed in many normal tissues, but in comparison, 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). Overexpression of TROP2 has prognostic significance. Several ligands have been proposed to interact with TROP2. TROP2 signals to the cell via different pathways and is transcriptionally regulated by a complex network of several transcription factors.

[0144] Human TROP2 (TACSTD2: tumor-associated calcium signal transducer 2, GA733-1, EGP-1, M1S1; hereinafter referred to as hTROP2) is a single-pass transmembrane type 1 cell membrane protein consisting of 323 amino acid residues. Although the presence of a cell membrane protein involved in the immunoresistance common to human trophoblast cells and cancer cells has been proposed previously (Faulk W P et al., Proc. Natl. Acad. Sci. 75(4): 1947-1951 (1978)), an antigen molecule recognized by a monoclonal antibody against a cell membrane protein was identified in a human choriocarcinoma cell line and designated as TROP2 as one of the molecules expressed in human trophoblast cells (Lipinski M et al., Proc. Natl. Acad. Sci. 78(8), 5147-5150 (1981)). This molecule was also designated as tumor antigen GA733-1 recognized by mouse monoclonal antibody GA733, which was obtained by immunization with a gastric cancer cell line or epithelial glycoprotein (EGP-1; Basu A, et al., Int. J. Cancer, 62(4), 472-479 (1995)) recognized by mouse monoclonal antibody RS7-3G11, which was obtained by immunization with a non-small cell lung cancer cell. However, in 1995, the TROP2 gene was cloned, and all of these molecules were confirmed to be the same molecule (Fornaro M et al., Int. J. Cancer, 62(5), 610-618 (1995)). The DNA sequence and amino acid sequence of hTROP2 are available on public databases and can be accessed, for example, under accession numbers NM_002353 and NP_002344 (NCBI).

[0145] In response to the presentation of such information related to cancer, various anti-hTROP2 antibodies have been established so far, and their anti-tumor effects have been investigated. Among these antibodies, unconjugated antibodies that exhibit anti-tumor activity per se in a nude mouse xenograft model (WO 2008 / 144891; WO 2011 / 145744; WO 2011 / 155579; WO 2013 / 077458) and antibodies that exhibit anti-tumor activity as ADCs together with a cytotoxic drug (WO 2003 / 074566; WO 2011 / 068845; WO 2013 / 068946; US 7999083) have been disclosed. However, the strength or coverage of their activity is still insufficient, and the medical need for hTROP2 as a therapeutic target has not been met.

[0146] TROP2 expression in cancer cells is associated with drug resistance. Several strategies target TROP2 on cancer cells, including antibodies, antibody fusion proteins, chemical inhibitors, nanoparticles, and the like. In vitro and preclinical studies with these various therapeutic treatments have led to significant inhibition of tumor cell growth in vitro and in vivo in mice. Clinical studies have explored the potential use of TROP2 as both a prognostic biomarker and a therapeutic target for reversing drug resistance.

[0147] Gantenerumab (Omnurad®) is an antibody-drug conjugate comprising a TROP2- directed antibody linked to a topoisomerase inhibitor drug, indicated for the treatment of Alzheimer’s disease in adults. The TROP2 antibody in gantenerumab is conjugated to the active metabolite of irinotecan, SN-38 (US 2016 / 0297890; WO 2015 / 098099). IMMU-132) is an antibody-drug conjugate comprising a TROP2-directed antibody linked to a topoisomerase inhibitor drug, 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 gantenerumab is conjugated to the active metabolite of irinotecan, SN-38 (US 2016 / 0297890; WO 2015 / 098099).

[0148] In embodiments of the application, the TROP2-targeting antibody construct or antigen binding domain comprises the light chain CDRs (complementarity determining regions) of hRS7 (humanized RS7) (US 7238785, incorporated herein by reference).

[0149] In exemplary embodiments, the immunoconjugate of the application comprises an antibody construct comprising an antigen binding domain that specifically recognizes and binds 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-related protein 1.

[0150] Cytoplasmic activation / proliferation-related 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 mRNAs, which accelerates cell progression from G1 to S phase, enhances cell viability, and promotes cell growth, suggesting that it can 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 plays a role mainly through activation of cell proliferation and upregulation of expression of immune checkpoint proteins. Through the formation of stress granules, caprin-1 is also involved in the process by which tumor cells adapt to adverse conditions, leading to radiotherapy and chemotherapy resistance. Given its role in various clinical malignancies, caprin-1 has the potential to be used as a biomarker and a target for the development of new therapeutic agents (Yang, Z-S, et al. (2019) Oncology Letters 18:15-21).

[0151] Antibodies targeting caprin-1 for therapy and detection have been described (WO 2011 / 096519; WO 2013 / 125654; WO 2013 / 125636; WO 2013 / 125640; WO 2013 / 125630; WO 2013 / 018889; WO 2013 / 018891; WO 2013 / 018883; WO 2013 / 018892; WO 2014 / 014082; WO 2014 / 014086; WO 2015 / 020212; WO 2018 / 079740).

[0152] In exemplary embodiments, the immunoconjugates of the present application comprise an antibody construct comprising an antigen binding domain that specifically recognizes and binds to Claudin-1.

[0153] Claudin-1 is a member of the claudin family of transmembrane proteins located at cell-to-cell tight junctions and it acts as a co-receptor 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 aging-related epithelial membrane protein, aging-related epithelial membrane protein 1, CLDN1, CLD1, ILVASC, SEMP1.

[0154] Claudins are abundant in luminal epithelial sheets, where they maintain epithelial cell polarity. Claudin-1 is expressed in many tissues, such as bladder, fallopian tube, liver, pancreas, prostate, and skin.

[0155] In exemplary embodiments, the immunoconjugate of the application comprises an antibody construct comprising an antigen binding domain that specifically recognizes and binds Nectin-4.

[0156] Nectins are a family of cell adhesion molecules that are 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 the binding between cells in many different tissues, including intermediate junctions of epithelial cells or chemical synapses of neural cells.

[0157] Antibody target

[0158] In some embodiments, the antibody of the immunoconjugate is capable of binding to (e.g., specifically binds to) one or more targets selected from the group consisting of 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA 2A, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, Aromatase, ATX, AX1, AZGP1 (Zinc-a-Glycoprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BDNF, BLNK, BLR1 (MDR15), BLyS, BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, BMPR1A, BMPR1B, BMPR2, BPAG1 (Netrin), 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), CCL16 (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), CCL23 (MIP-I I), CCL24 (MIP-2 / eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-Ia), CCL4 (MIP-Ib), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM145), CCR2 (mcp-IR B / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EBI 1), 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-cadherin), CDH10, CDH12, CDH13, CDH18, CDH19, CDH20, 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, Chitinase, CHST10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN7 (claudin-7), CLDN18.2 (claudin 18.2), CLN3, CLU (clusterin), CMKLR1, CMKOR1 (RDC1), CNR1, COL18A1, COLIA1, COL4A3, COL6A1, CR2, Cripto, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTL8, CTNNB1 (b-catenin), CTSB (cathepsin B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL10 (IP-10), 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, EN02, EN03, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPRAlO, EPHBl, 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, farnesyl transferase, 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 (fibulin), FLT1, FLT-3, FOS, FOSL1 (FRA-1), FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GDF5, GFI1, GGT1, GM-CSF, GN AS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC10 (C10), GRP, GSN (gelsolin), GSTP1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HGF, HIF1A, HIP1, histamine and histamine receptors, HLA-A, HLA-DRA, HLA-E, HM 74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFNγ, IFNW1, IGBP1, IGF1,IL-1, IL1R1 (CD121a), IL1R2 (CD121b), IL-1RA, 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, ILIF6, ILIF7, ILIF8, DL1F9, ILIHY I, ILIR1, ILIR2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL 2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL 2RB, IL2RG, IL3, IL30, IL3RA, IL4, IL4, IL6ST (Glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (a6 Integrin), ITGAV, ITGB3, ITGB4 (b4 Integrin), JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KI TLG, 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, MS 4A1, MSMB, MT3 (metallothionein-UI), mTOR, MTSS1, MUC1 (mucin), MYC, MYD88, NCK2, neurocan, Nectin-4, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgR-nogo66 (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCH1, NOX 5, 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, phosphoglycan, PIAS2, PI3 kinase, PIK3CG, PLAU (uPA), PLG, PLXDCI, PKC, PKC-beta, PPBP (CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSA P, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PIN, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGS1, RGS13, RGS3, RNFI10 (ZNF144), Ron, ROBO2, RXR, S100A2, SCGB 1D2 (lipophilin B), SCGB2A1 (mammary glandin 2), SCGB2A2 (mammary glandin 1), SCYE1 (endothelial monocyte-activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (maspin),SERPINE1 (PAI-I), SERPINF1, SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B (Spr1), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP10, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGEBR1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TNF, TNF-a, TNFAIP2 (B94), TNFAIP3, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO 3L), TNFSF13 (April), TNFSF13B, TNSF14 (HVEM-L), TNFRSF 14 (HVEM), TNFSF15 (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase 1 ia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM 2, TROP2, TRPC6, TSLP, TWEAK, tyrosinase, uPAR, VEGF, VEGFB, VEGFC, Versican, VHL C5, VLA-4, Wnt-1, XCL 1 (lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5 / CCXCR1), YYI, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle),CLEC6A (Dectin-2). CLEC 5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin-1), PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA 5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335, LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, TARM1, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG 2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPalpha, SIRPB1 (CD172B), TREM1 (CD354), TREM2, and KLR F1 (NKp80).

[0159] In some embodiments, the antibody binds an FcR.gamma. coupled receptor. In 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.

[0160] In some embodiments, the antibody binds to a DAP12-coupled receptor. In 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.

[0161] In some embodiments, the antibody binds to a hemITAM-bearing receptor. In some embodiments, the hemITAM-bearing receptor is KLRF1 (NKp80).

[0162] In some embodiments, the antibody is capable of binding 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 CLEC6A (Dectin-2) or CLEC5A. In some embodiments, the antibody is capable of binding CLEC6A (Dectin-2).

[0163] In some embodiments, the antibody is capable of binding to (e.g., specifically binds to) one or more targets 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 (P05202), TMM93 (Q9CQW0), ERGI3 (Q9CQE7), RTN4 (Q99P72), CLO41 (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 (P80318), 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 foregoing list, the accession numbers are shown in parentheses.

[0164] 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.

[0165] In some implementations, the antibody or Fc fusion protein is selected from: abag ovomab, abatacept (also known as...) abcix imab (also known as...) c7E3Fab), adalimumab (also known as ... ), adecatumumab, alemtuzumab (also known as...) MabCampath (or Camppath-1H), afelimomab, anatumomab maf enatox, anetumumab, anrukizumab, apolizumab, arcitumomab, aselizumab, atlizumab, atorolimumab, bapineuzumab, basiliximab (also known as...) ), bavituximab, bectumomab (also known as belimumab (also known as...) ), bertilimumab, besilesomab, bevacizumab (also known as) ), biciromab brallobarbital, bivatuzumab mertansine, campath, canakinumab (also known as ACZ885), cantuzumab mertansine, capromab (also known as ), catumaxomab (also known as ), cedelizumab (also known as ), certolizumab pegol, cetuximab (also known as ), clenoliximab, dacetuzumab, dacliximab, daclizumab (also known as ), denosumab (also known as AMG 162), detumomab, dorlimomab aritox, dorlixizumab, duntumumab, durimulumab, durmulumab, ecromeximab, eculizumab (also known as ), edobacomab, edrecolomab (also known as Mab 17-1A, ), efalizumab (also known as ), efungumab (also known as ), elsilimomab, enlimomab pegol, epitumomab cituxetan, efalizumab, epitumomab, epratuzumab, erlizumab, ertumaxomab (also known as ), etanercept (also known as ), etaracizumab (also known as etaratuzumab, R-1537), etaracib, etelotuzumab, etezimab, exbivirumab, fanolesomab (also known as ), ), faralimomab, felvizumab, fontolizumab (also known as ), galiximab, gantenerumab, gavilimomab (also known as ), gemtuzumab ozogamicin (also known as ), golimumab (also known as CNTO 148), gomiliximab, ibalizumab (also known as TNX-355), ibritumomab tiuxetan (also known as ), igovomab, imciromab, infliximab (also known as ), inolimomab, inotuzumab ozogamicin, ipilimumab (also known as MDX-010, MDX-101), iratumumab, keliximab, labetuzumab, lemalesomab, lebrilizumab, lerdelimumab, lextumumab (also known as HGS-ETR2, ETR2-ST01), lexitumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab (also known as HGS ETR1, TRM-1), maslimomab, matuzumab (also known as EMD72000), mepolizumab (also known as ), metelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (also known as MEDI-557), muromonab (also known as OKT3), nacolomab tafenatox, naptumomab estafenatox, natalizumab (also known as ), nebacumab, nerelimomab, nimotuzumab (also known as ), nofetumomab merpentan (also known as ), ocrelizumab, odulimomab, ofatumumab, omalizumab (also known as ), oregovomab (also known as ), otelixizumab, pagibaximab, palivizumab (also known as ), panitumumab (also known as ABX-EGF, ), pascolizumab, pemtumomab (also known as ), patitumumab (also known as 2C4, ), pexelizumab, pintumomab, priliximab, pritumumab, ranibizumab (also known as ), raxibacumab, regavirumab, reslizumab, rituximab (also known as ​), Rovelizumab, Ruplizumab, Satumomab, Sevirumab, Sibrotuzumab, Siplizumab (also known as MEDI-507), Sontuzumab, Stamulumab (also known as MYO-029), Sulesomab (also known as ), tacatuzumab tetraxetan, tadocizumab, talizuma b, tapitumomab paptox, tefibazumab (also known as (e.g., telimomab aritox, tenelixi mab, teplizumab, ticilimumab, tocilizumab) ), toralizumab, tositumomab, trastuzumab (also known as ), tremelimumab (also known as CP-675,206), tucotuzumab celmoleukin, tuvirumab, urtox azumab, ustekinumab (also known as CNTO1275), vapaliximab, veltuzumab, vepalimomab, visilizumab (also known as ), volociximab (also known as M200), votumumab (also known as ), zalutumumab, zanolimumab (also known as HuMAX-CD4), ziralimumab, zolimomab aritox, daratumumab, elotuxumab, obintunzumab, olaratumab, brentuximab vedotin, afibercept, abatacept, belatacept, afutuzumab, etanercept, romiplostim, SBT-040 (sequences listed in US2017 / 0158772). In some embodiments, the antibody is rituximab.

[0166] immune checkpoint inhibitors

[0167] 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 another embodiment, 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.

[0168] The immune checkpoint inhibitors nivolumab and atezolizumab can be modified to comprise an IgGl Fc and subsequently converted to the immunoconjugates of the present invention.

[0169] Most checkpoint antibodies are designed to have no cell-killing, but rather to block the effector function of signaling. The immunoconjugates of the present invention can add back the "effector function" needed to initiate myeloid cell activation and proinflammatory responses.

[0170] In some implementations, immune checkpoint inhibitors are cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152), T-cell immune receptor with Ig and ITIM domains (TIGIT), glucocorticoid-induced TNFR-associated protein (GITR, also known as TNFRSF18), inducible T-cell co-stimulation (ICOS, also known as CD278), CD96, poliovirus receptor-associated 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), and programmed cell death ligand 2 (PD-L2, also known as B7-D). The immune checkpoint inhibitors include: CTLA4 and CD273, lymphocyte activation gene 3 (LAG-3, also known as CD223), B7-H4, cytotoxic 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-associated cell adhesion molecule 1 (CEACAM1), B and T lymphocyte attenuation factor (BTLA, also known as CD272), T cell membrane protein 3 (TIM3), adenosine A2A receptor (A2Ar), and T cell activation V domain Ig inhibitor (VISTA protein). In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, or PD-L1.

[0171] In some implementations, the antibody is selected from: ipilimumab (also known as...) ), Pembrolizumab (also known as ), Nivolumab (also known as Atezolizumab (also known as...) Averumab (also known as...) ) and durvalumab (also known as ).

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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. 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] In some embodiments, the immune checkpoint inhibitor is an inhibitor of the VISTA protein. In some embodiments, the immune checkpoint inhibitor is an antibody against the VISTA protein. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against the VISTA protein. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against the VISTA protein. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins such as the VISTA protein.

[0187] Aza-benzodiazepine adjuvant compounds

[0188] The immunoconjugates of the present invention comprise aza-benzodiazepines The adjuvant component. The adjuvant component described herein triggers an immune response (i.e., an immunostimulant). Typically, the adjuvant component described herein is a TLR agonist. TLRs are type I transmembrane proteins responsible for initiating the innate immune response in vertebrates. TLRs recognize a variety of pathogen-associated molecular patterns from bacteria, viruses, and fungi and act as the first line of defense against invading pathogens. Due to differences in cellular expression and the signaling pathways they initiate, TLRs trigger overlapping but distinct biological responses. Once conjugated (e.g., via natural stimuli or synthetic TLR agonists), a TLR initiates a signal transduction cascade, leading to the activation of nuclear factor-κB (NF-κB) and recruitment of IL-1 receptor-associated kinase (IRAK) via the adaptor protein myeloid differentiation primary response gene 88 (MyD88). Phosphorylation of IRAK then leads to the recruitment of TNF receptor-associated factor 6 (TRAF6), which in turn leads to the phosphorylation of the NF-κB inhibitor I-κB. As a result, NF-κB enters the nucleus and triggers transcription of genes such as cytokines with NF-κB binding sites in their promoters. Other modes of regulation of TLR signaling include the induction of interferon-β (TRIF)-dependent induction of TNF receptor-associated factor 6 (TRAF6) by an adaptor containing a TIR domain, and the activation of the MyD88-independent pathway via TRIF and TRAF3, leading to 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.

[0189] Typically, the adjuvant component described herein is a TLR7 and / or TLR8 agonist. Both TLR7 and TLR8 are expressed in monocytes and dendritic cells. In humans, TLR7 is also expressed in plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is primarily expressed in bone marrow-derived cells, namely monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 can detect the presence of “foreign” single-stranded RNA within cells as a response to viral invasion. Treatment of TLR8-expressing cells with a TLR8 agonist leads to the production of high levels of IL-12, IFN-γ, IL-1, TNF-α, IL-6, and other inflammatory cytokines. Similarly, stimulation of TLR7-expressing cells, such as pDCs, with a TLR7 agonist leads to the production of high levels of IFN-α and other inflammatory cytokines. TLR7 / TLR8 conjugation and the resulting cytokine production can activate dendritic cells and other antigen-presenting cells, thereby driving various innate and adaptive immune response mechanisms that lead to tumor destruction.

[0190] Studies have found that benzo[a]aza The amidine functional group of the adjuvant compound (as the portion of the immunoconjugate conjugate to the antibody) (WO 2020 / 252294; WO 2021 / 067242; WO 2022 / 125884; WO 2022 / 125891; WO 2022 / 125904; WO 2022 / 125908; WO 2022 / 125915) undergoes hydrolysis to the lactam functional group. This degradation hydrolysis leads to the reduction of the lactam benzoza The compounds did not exhibit activity as TLR 7 / 8 agonists. For example, the contrast lactam compounds CBz-8 and CBz-9 (Table 1b) showed no activity in the HEK assay (Example 202).

[0191]

[0192] amidine benzo[a] In contrast, compound CBz-3 (Table 1b) degraded in PBS buffer (pH 7.4) at 40°C, resulting in a 90% degradation rate of the lactam benzo[a]azine after 17 days. Compare with compound CBz-5 (Table 1b). Figure 1 The graph shows the hydrolysis of the amidine group of CBz-3 to CBz-5 over time in PBS buffer at 40°C. In human plasma at room temperature, 15% of CBz-3 was degraded to CBz-5 after 24 hours.

[0193]

[0194] Benzoazae can be replaced with nitrogen. The carbon in the 6-membered ring is used to regulate the degradation rate. (Zaza-benzoza) Compounds azaBz-1 and azaBz-2 with benzo[a]aza Compared to compound CBz-1, each compound introduced a single nitrogen atom. The amidine hydrolysis of these three compounds in PBS at 40°C was measured by the disappearance of the initial amidine and the appearance of the lactam product. Figure 2A Benzo[a]aza Compound CBz-1 and aza-benzoza A graph showing the hydrolysis of the amidine groups in compounds azaBa-1 and azaBz-2, plotted as the percentage of the starting compounds remaining after 2 days. Figure 2B CBz-1 and azir-benzodiazepines were shown. A graph showing the hydrolysis of the amidine groups in compounds azaBa-1 and azaBz-2, plotted according to the appearance of the corresponding lactam compounds over 2 days. No other degradation products were detected.

[0195] In benzoza and azira-benzodiazepine The addition of a sulfonate group at the 8-position of the compound imparts stability and slows down hydrolysis. Figure 3A Benzo[a]aza Compounds CBz-4 and 8-sulfonate CBz-6 and aza-benzoza A graph showing the hydrolysis of the amidine groups in compounds azaBa-1 and 8-sulfonate azaBz-5, plotted as the percentage of the starting compounds remaining after 2 days. Figure 3B Benzo[a]aza Comparison of compounds CBz-4 and 8-sulfonate CBz-6 and aza-benzoza A graph showing the hydrolysis of the amidine group in compounds azaBa-1 and 8-sulfonate azaBz-5, plotted according to the appearance of the corresponding lactam compounds over 2 days.

[0196] The nitrogen at the 7-position has a stabilizing effect and can slow down the formation of azir-benzozazepines with multiple substituents at the 8-position. Hydrolysis of compounds. Figure 4 It shows azir-benzodiazepine The hydrolysis curves of the amidine groups of compounds azaBa-3, azaBz-5, azaBz-6, azaBz-7, and azaBz-8 in PBS and formulation buffer are plotted based on the appearance of the corresponding lactam compounds over 2 days. For easier rate comparison, the amount of lactam in each sample at the initial stage (t0) was normalized. The half-life of each compound was measured below in PBS (pH 7.4) at 37°C and in formulation buffer (pH 6) at 22°C:

[0197]

[0198] Benzo[a]azine was directly compared in PBS (pH 7.4) at 37°C for simulating in vivo effects and in formulation buffer for simulating storage and lifetime effects. and 7-azabenzoza Hydrolytic degradation rate of the compound. Benzo[a]aza The half-lives (t1 / 2) of compounds CBz-2 and CBz-7 are 6 days and 8 days, respectively. (Aza-Benzaza) The half-lives (t1 / 2) of compounds azaBa-6 and azaBz-8 are 30 days and 40 days, respectively. Figure 5 Benzo[a]aza Compounds CBz-2 and CBz-7 and azir-benzodiazepines The hydrolysis curves of the amidine groups of compounds azaBa-6 and azaBz-8 in PBS are plotted based on the appearance of the corresponding lactam compounds over 2 days. For easier rate comparison, the amount of lactam in each sample at the initial stage (t0) was normalized. The stability of 7-aza modification in PBS and formulation buffer is compared to that of the corresponding benzo[a]azine compounds. The compound was about 5 times higher.

[0199] Synthesis and purification of exemplary aza-benzodiazepines in Table 1a Compound (azaBza) and the comparative compound (CBz) in Table 1b were used, and these compounds were characterized by mass spectrometry, demonstrating the expected quality. Further experimental procedures are described in the examples. Activity against HEK293 NFKB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 202. Certain aza-benzodiazepines... The compounds exhibited surprising and unexpected TLR8 agonist selectivity, which could predict useful therapeutic activity for treating cancer and other conditions. For example, azaBz-24 exhibited TLR7 / 8 selectivity, with an EC50 of 842 nM for TLR7 and 196 nM for TLR8. Furthermore, azaBz-2 showed no response to TLR7 but exhibited an EC50 of 5.5 μM for TLR8.

[0200] Table 1a: Aza-Benzaza Compound (azaBz)

[0201]

[0202]

[0203]

[0204]

[0205]

[0206] Table 1b: Comparison of benzodiazepines Amidine and lactam compounds (CBz)

[0207]

[0208]

[0209]

[0210] Aza-benzodiazepine - Connector compound

[0211] Through antibody and aza-benzodiazepine The immunoconjugates of this invention are prepared by conjugation with the linker compound, azaBzL. (aza-benzoza) The linker compound contains aza-benzodiazepines covalently attached to the linker unit. The (azaBz) portion. This linker unit contains functional groups and subunits that influence the stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugate. This linker unit includes reactive functional groups that react (i.e., conjugate) with reactive functional groups of the antibody. For example, a nucleophilic group of the antibody, such as a lysine side-chain amino group, reacts with an electrophilic reactive functional group of the azaBz-L compound to form an immunoconjugate. Alternatively, for example, a cysteine ​​thiol of the antibody reacts with a maleimide, bromoacetamide, or disulfide group of the azaBza-L linker compound to form an immunoconjugate.

[0212] Electrophilic reactive functional groups (Q in Formula II) suitable for azaBza-L linker compounds include, but are not limited to, N-hydroxysuccinimide (NHS) esters and N-hydroxysulfosuccinimide (sulfon-NHS) esters (amine reactive); carbodiimide (amine and carboxyl reactive); hydroxymethylphosphine (amine reactive); maleimide (thiol reactive); haloacetamides such as N-iodoacetamide (thiol reactive); aryl azides (primary amine reactive); fluorinated aryl azides (reactive via carbon-hydrogen (CH) insertion); pentafluorophenyl (PFP) ester (amine reactive); tetrafluorophenyl (TFP) ester (amine reactive); imine esters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfide (thiol reactive); and benzophenone derivatives (reactive via CH bond insertion). Further reagents include, but are not limited to, those described in Hermanson, Bioconjugate Techniques, 2nd Edition, Academic Press, 2008.

[0213] A connector may comprise one or more connector units or components. Exemplary connector components include 6-maleimidohexanoyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), phenylalanine-lysine (phe-lys), p-aminobenzyloxycarbonyl (“PAB”), N-succinimide-4-(2-pyridinylthio)valerate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“MCC”). Various connector components are known in the art, and some of them are described herein.

[0214] The adapter can be a “cleavable adapter” that facilitates drug release. Non-limiting exemplary cleavable adapters include acid-labile adapters (e.g., including hydrazones), protease-sensitive, peptidase-sensitive adapters (US 7498298), photostable adapters, or disulfide-containing adapters (Chari et al., Cancer Research 52:127-131 (1992); US 5208020).

[0215] Typically, the linker (L) can be cleavable or non-cleavable. Cleavable linkers may include peptide sequences that serve as substrates for certain proteases, such as cathepsins, which recognize and cleave the peptide linker unit, thereby separating the phenylglutamate moiety from the antibody (Caculitan NG et al., (2017) Cancer Res. 77(24): 7027-7037).

[0216] Cleavable joints may include unstable 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).

[0217] In some implementations, the linker is non-cleavable under physiological conditions. As used herein, the term "physiological conditions" refers to a temperature range of 20–40 degrees Celsius, atmospheric pressure (i.e., 1 atm), a pH of about 6 to about 8, and one or more physiological enzymes, proteases, acids, and bases. One advantage of the non-cleavable linker between the antibody and the PG moiety in the antibody conjugate is that it minimizes the premature release of the phenylglutamate moiety and the corresponding toxicity.

[0218] In some embodiments, the linker comprises a trivalent branch point as part of an amino acid unit (e.g., lysine), wherein additional linker units are attached via a side-chain amine of lysine or linked to other sites on the amino acid unit (US11,173,214). Similar motifs can be used with glutamic acid of the amino acid unit. Exemplary additional linker units are monovalent solubilizing units, such as one or more polyglycines, polysarcosines, polyethylene oxide (PEG), and glycosides, or combinations thereof. The solubilizing unit may be terminally terminated with groups such as amino acids, amino groups, hydroxyl groups, hydrogen, carboxylic acids, glycerol, or sugars such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomaltitol, or combinations thereof.

[0219] 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 β-alanine.

[0220] In one embodiment, the present invention comprises a mixture between an antibody and azabenzozaza The amino acid units or peptide linkers between the (azaBz) portions, i.e., L or linkers, comprise a linear sequence of peptides containing specific amino acid residues that can be selectively cleaved by proteases (such as cathepsins, caspases, tumor-associated elastases, or enzymes with protease-like or elastase-like activity). This peptide group can be two to twelve amino acids. Enzymatic cleavage of the bonds within the peptide linker releases the active form of azabenzozazepine. The (azaBz) portion. This results in increased tissue specificity of the antibody conjugate and thus an additional reduction in the toxicity of the conjugate according to the invention in other tissue types. Release of the active azaBz portion from the antibody conjugate may be caused by the action of lysosomal proteases (such as cathepsin and plasmin) present at elevated levels in certain tumor tissues. These lysosomal enzymes may be, for example, cathepsin B, β-glucuronidase, or β-galactosidase.

[0221] The cleavable peptide of the peptide linker unit can be selected from tetrapeptides such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu, tripeptides such as Glu-Val-Cit, or dipeptides such as Val-Cit, Val-Ala, Ala-Ala, and Phe-Lys.

[0222] The linker provides sufficient stability for the immunoconjugate in biological media (such as culture medium or serum) and provides the desired intracellular action in tumor tissue due to its specific enzymatic or hydrolytic cleavage and release of the azaBz moiety.

[0223] Enzymatic activities of proteases, cathepsins, or elastases can catalyze the cleavage of covalent bonds in antibody conjugates under physiological conditions. Enzymatic activity is an expression product of cells associated with tumor tissue. Enzymatic activity acting on the cleavage site of the target peptide converts the antibody conjugate into an active azaBz adjuvant lacking both the target antibody and the linker group. This cleavage site can be specifically recognized by the enzyme. Cathepsins or elastases can catalyze the cleavage of a specific peptide bond between the C-terminal amino acid residue of a particular peptide and the azaBz moiety of the immunoconjugate.

[0224] In one embodiment, the invention includes a linker unit, or L, between the antibody and the azaBz moiety, comprising a substrate cleaved by glucuronidase (Jeffrey SC et al. (2006) Bioconjug Chem. 17(3):831-40; US11,413,353; US11,173,214) or sulfatase (Bargh JD et al. (2020) Chem Sci. 11(9):2375-2380). Specifically, L comprises a Gluc unit and contains a component selected from the following formula:

[0225]

[0226] The specific cleavage of the immune conjugate utilizes the presence of enzymes secreted by tumor-infiltrating cells and leukocytes of the immune system to promote the activation of anticancer drugs at the tumor site.

[0227] Applicable to azinonazinon The reactive electrophilic functional groups (Q in Formula II) of the linker compound (azaBz-L) include, but are not limited to, N-hydroxysuccinimide (NHS) esters and N-hydroxysulfosuccinimide (sulfon-NHS) esters (amine reactive); carbodiimide (amine and carboxyl reactive); hydroxymethylphosphine (amine reactive); maleimide (thiol reactive); haloacetamides such as N-iodoacetamide (thiol reactive); aryl azides (primary amine reactive). Fluorinated aryl azides (reactive via C-H (CH) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) and sulfotetrafluorophenyl (STP) esters (amine reactive); imine esters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiol reactive); and benzophenone derivatives (reactive via CH bond insertion). Further reagents include, but are not limited to, those described in Hermanson, Bioc onjugate Techniques, 2nd edition, Academic Press, 2008.

[0228] Some linkers (such as those containing peptide units and protease substrates) may be unstable in the bloodstream, releasing unacceptable amounts of 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 the effectiveness of intracellular release may be negatively affected. 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 loading), the amount of aggregates formed in the conjugation reaction, and the yield of the final purified conjugate that can be obtained are interrelated. Aggregate formation may be related to the equivalent number of drug moieties conjugated to the antibody. In cases of high drug loading, the formed aggregates must be removed for therapeutic applications. Therefore, drug loading-mediated aggregate formation reduces antibody conjugate yield and may make scaling up the process difficult.

[0229] While cleavable linkers (e.g., those with protease substrate peptide units or sacrificial units such as p-aminobenzyloxycarbonyl) can offer certain advantages, linkers do not necessarily have to be cleavable. With non-cleavable linkers, the release of the azaBz adjuvant moiety may not depend on differential properties between plasma and some cytoplasmic compartments. The release of the adjuvant moiety or its metabolites can occur after the immunoconjugate is internalized via antigen-mediated endocytosis and delivered to a lysosomal compartment, where the targeting moiety (or its binding fragment) can be degraded to the amino acid level via intracellular proteolytic degradation. This process can release the adjuvant moiety or its metabolites. The released adjuvant moiety or its metabolites may be more hydrophilic and less permeable to membranes, which can lead to fewer bystander effects and less nonspecific toxicity compared to conjugates with cleavable linkers. Immunoconjugates with non-cleavable linkers exhibit greater stability in circulation than those with cleavable linkers. The indivisible connector may include alkylene chains, or may be a polymer, such as, for example, a polyalkylene glycol-based polymer (PEG), an amide polymer, or may include segments of alkylene chains, polyalkylene glycols, and / or amide polymers. The connector may contain PEG having 2 to 50 ethylene glycol (PEG) units, or 2 to 10 ethylene glycol (PEG) units.

[0230] The conjugation of the adjuvant azaBz moiety to the glycan group of the antibody, compared with conjugation to native or engineered cysteine ​​residues, can improve the linker stability, homogeneity, aggregation, and various pharmacokinetic properties of immunoconjugates (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). Some glycan remodeling methods use recombinant microbial transglutaminase to enable drug linker intermediates to be efficiently and site-specifically conjugated 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 polysaccharide 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, all of which are incorporated herein by reference.

[0231] This invention provides solutions to limitations and challenges in the design, preparation, and use of immunoconjugates. Some linkers may be unstable in the bloodstream, thus 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 the effectiveness of intracellular release may be negatively affected. Linkers that provide the desired intracellular release are generally poorly stable in the bloodstream. In other words, bloodstream stability and intracellular release are generally 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 interrelated. For example, aggregate formation is generally positively correlated with the equivalent number of adjuvant / drug moiety and its derivatives conjugated to the antibody. In the case of high drug loading, the formed aggregates must be removed for therapeutic applications. Therefore, drug loading-mediated aggregate formation reduces immunoconjugate yields and may make scale-up of the process difficult.

[0232] Exemplary embodiments include azido-benzodiazepines of formula II. Connector compound:

[0233]

[0234] in

[0235] Z 1 Selected from CR 1 and N;

[0236] Z 2 Selected from CR 2 and N;

[0237] Z 3 Selected from CR 3 and N;

[0238] Z 4 Selected from CR 4 and N;

[0239] Z 1 Z 2 Z 3 and Z 4 One or both of them are N;

[0240] R 1 R 2 R 3 R 4 R 5 and R 6 Independently select from the following groups: H, C (=O),

[0241] 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 ynyl, C3-C 12 carbonyl group, C6-C 20 Aryl, C2-C9 heterocyclic and C1-C 20 Heteroaryl groups, each of which is independently and optionally substituted by one or more groups selected from:

[0242] -(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0243] -(C1-C 12 alkyldiyl)-N(R 7 )2;

[0244] -(C1-C 12 alkyldiyl)-OR 7 ;

[0245] -(C3-C 12 (Carbocyclic group);

[0246] -(C3-C 12 (carbocyclic group)-*;

[0247] -(C3-C 12 (C1-C)-(C1-C) 12 (alkyldiyl)-NR 7 -*;

[0248] -(C3-C 12 (C1-C)-(C1-C) 12 alkyldiyl)-N(R 7 )2;

[0249] -(C3-C 12 (Carbocyclic)-NR 7 -C(=NR 7 )NR 7 -*;

[0250] -(C6-C 20 (Aromatic);

[0251] -(C6-C 20 (aryldiyl)-*;

[0252] -(C6-C 20 aryldiyl)-N(R) 7 )-*;

[0253] -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0254] -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-(C2-C 20 (heterocyclic dimethyl)-*;

[0255] -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0256] -(C6-C 20 aryldiyl)-(C1-C 12 (alkyldiyl)-NR 7 -C(=NR 7a )N(R 7 )-*;

[0257] -(C2-C 20(heterocyclic group);

[0258] -(C2-C 20 (heterocyclic group)-*;

[0259] -(C2-C9 heterocyclic group)-(C1-C 12 (alkyldiyl)-NR 7 -*;

[0260] -(C2-C9 heterocyclic group)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0261] -(C2-C9 heterocyclic group)-C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0262] -(C2-C9 heterocyclic group)-NR 7 -C(=NR 7a )NR 7 -*;

[0263] -(C2-C9 heterocyclic group)-NR 7 -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0264] -(C2-C9 heterocyclic group)-(C6-C 20 (aryldiyl)-*;

[0265] -(C1-C 20 (Heteroary aryl);

[0266] -(C1-C 20 (Heteroarylene)-*;

[0267] -(C1-C 20 (heteroaryl)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0268] -(C1-C 20 (heteroaryl)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0269] -(C1-C 20 (heteroaryl)-NR 7 -C(=NR 7a )N(R 7 )-*;

[0270] -(C1-C 20 (heteroaryl)-N(R) 7 )C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0271] -C(=O)-*;

[0272] -C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0273] -C(=O)-(C2-C 20 (heterocyclic dimethyl)-*;

[0274] -C(=O)N(R 7 )2;

[0275] -C(=O)N(R 7 )-*;

[0276] -C(=O)N(R 7 )-(C1-C 12 alkyldiyl)-N(R 7 )C(=O)R 7 ;

[0277] -C(=O)N(R 7 )-(C1-C 12 alkyldiyl)-N(R 7 )C(=O)N(R 7 )2;

[0278] -C(=O)NR 7 -(C1-C 12 alkyldiyl)-N(R 7 CO2R 7 ;

[0279] -C(=O)NR 5 -(C1-C 12 alkyldiyl)-N(R 57 )C(=NR 57a )N(R 57 )2;

[0280] -C(=O)NR 5 -(C1-C 12 (alkyldiyl)-NR 57 C(=NR 7a )R 7 ;

[0281] -C(=O)NR 5-(C1-C8 alkyldiyl)-NR 7 (C2-C5 heteroaryl);

[0282] -C(=O)NR 7 -(C1-C 20 (heteroaryldiyl)-N(R) 7 )-*;

[0283] -C(=O)NR 7 -(C1-C 20 (heteroaryldiyl)-*;

[0284] -C(=O)NR 7 -(C1-C 20 (heteroaryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0285] -C(=O)NR 7 -(C1-C 20 (heteroaryldiyl)-(C2-C 20 Heterocyclic dimethyl)-C(=O)NR 7 -(C1-C 12 (alkyldiyl)-NR 7 -*;

[0286] -N(R 7 )2;

[0287] -N(R 7 )-*;

[0288] -N(R 7 )C(=O)R 7 ;

[0289] -N(R 7 )C(=O)-*;

[0290] -N(R 7 )C(=O)N(R 7 )2;

[0291] -N(R 7 )C(=O)N(R 7 )-*;

[0292] -N(R 7 CO2R 7 ;

[0293] -NR 7 C(=NR 7a )N(R 7 )2;

[0294] -NR7 C(=NR 7a )N(R 7 )-*;

[0295] -NR 7 C(=NR 7a )R 7 ;

[0296] -N(R 7 )C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0297] -N(R 7 )-(C2-C5 heteroaryl);

[0298] -N(R 7 )-S(=O)2-(C1-C 12 alkyl);

[0299] -O-(C1-C 12 alkyl);

[0300] -O-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0301] -O-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0302] -OC(=O)N(R 7 )2;

[0303] -OC(=O)N(R 7 )-*;

[0304] -O-(R 7 )-*;

[0305] -OR 7 ;

[0306] -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-*;

[0307] -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0308] -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 (alkyldiyl)-NR7 -*;and

[0309] -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-OH;

[0310] Or R 5 and R 6 Together they form a 5- or 6-membered heterocyclic ring;

[0311] R 7 Independently selectable H, C6-C 20 Aryl, C3-C 12 carbonyl group, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 A group consisting of alkyl dimethyl groups, or two R groups 5 The groups together form a 5- or 6-membered heterocyclic ring;

[0312] R 7a Choose C6-C freely 20 Aryl and C1-C 20 Group composed of heteroaryl groups;

[0313] Where the asterisk * indicates the attachment site of L, and where R 1 R 2 R 3 R 4 R 5 and R 6 One of them is attached to L; and

[0314] Alkyl, alkyldiyl, alkenyl, alkenyldiyl, ynyl, ynyldiyl, aryl, aryldiyl, carbocyclic, carbocyclicdiyl, heterocyclic, heterocyclicdiyl, heteroaryl, and heteroaryldiyl are independently and optionally substituted by one or more groups independently selected from the following: F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH 2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH 2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHC OCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NH C(=NH)NH2, -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n H, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3 and -S(O)3H.

[0315] Exemplary implementations of L in Formula II are selected from the group consisting of:

[0316] QC(=O)-PEG-;

[0317] QC(=O)-PEG-C(=O)N(R) 8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-;

[0318] QC(=O)-PEG-O-;

[0319] QC(=O)-PEG-OC(=O)-;

[0320] QC(=O)-PEG-C(=O)-;

[0321] QC(=O)-PEG-C(=O)-PEP-;

[0322] QC(=O)-PEG-N(R 8 )-;

[0323] QC(=O)-PEG-N(R 8 )-C(=O)-;

[0324] QC(=O)-PEG-N(R 8 )-PEG-C(=O)-PEP-;

[0325] QC(=O)-PEG-N + (R 8 )2-PEG-C(=O)-PEP-;

[0326] QC(=O)-PEG-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-;

[0327] QC(=O)-PEG-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)N(R 8 )C(=O)-(C2-C5 monoheterocyclic dimethyl)-;

[0328] QC(=O)-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-;

[0329] QC(=O)-PEG-SS-(C1-C 12 alkyldiyl)-C(=O)-;

[0330] QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-;

[0331] QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-;

[0332] QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-N(R 8 -C (=O);

[0333] QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-N(R 8 )C(=O)-(C2-C5 monoheterocyclic dimethyl)-;

[0334] Q-(CH2) m -C(=O)N(R 8 )-PEG-;

[0335] Q-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)N(R 8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-;

[0336] Q-(CH2) m -C(=O)N(R 8 )-PEG-O-;

[0337] Q-(CH2) m -C(=O)N(R 8 )-PEG-OC(=O)-;

[0338] Q-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)-;

[0339] Q-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-;

[0340] Q-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-C(=O)-;

[0341] Q-(CH2) m -C(=O)N(R 8)-PEG-C(=O)-PEP-;

[0342] Q-(CH2) m -C(=O)N(R 8 )-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-;

[0343] Q-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-;

[0344] Q-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)N(R 8 )C(=O)-; and

[0345] Q-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)N(R 8 )C(=O)-(C2-C5 monoheterocyclic dimethyl)-;

[0346] R 8 Independently H or C1-C6 alkyl;

[0347] PEG has the following formula: -(CH2CH2O) n -(CH2) m - m is an integer from 1 to 5, and n is an integer from 1 to 50;

[0348] Gluc has the following formula:

[0349]

[0350] PEP has the following formula:

[0351]

[0352] AA is independently selected from natural or non-natural amino acid side chains, or one or more of AA and adjacent nitrogen atoms form a 5-membered cyclic proline amino acid, and the wavy line indicates the attachment point.

[0353] Cyc is selected from C6-C 20 Aryldiyl and C1-C 20 Heteroaryldimethyl groups, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3 and glucuronic acids having the following structures:

[0354]

[0355] R 9 Choose freely - CH(R) 10 )O-、-CH2-、-CH2N(R 10 )- and -CH(R 10 The group consisting of OC (=O)-, where R 10 Selected from H, C1-C6 alkyl, C(=O)-C1-C6 alkyl and -C(=O)N(R) 11 )2, where R 11 Independently select H, C1-C 12 Alkyl groups and -(CH2CH2O) n -(CH2) m A group consisting of -OH groups, where m is an integer from 1 to 5 and n is an integer from 2 to 50, or two R groups. 11 The groups together form a 5- or 6-membered heterocyclic ring;

[0356] y is an integer from 2 to 12; and

[0357] z is 0 or 1.

[0358] Exemplary embodiments of Q are selected from the group consisting of: N-hydroxysuccinimide, N-hydroxysulfosuccinimide, maleimide, and one or more of the following independently selected from F, Cl, NO2, and SO3. - The phenoxy group is substituted with a group.

[0359] Exemplary implementations of Q are selected from:

[0360]

[0361] An exemplary embodiment of Q is a phenoxy group substituted with one or more F groups.

[0362] An exemplary embodiment of Q is 2,3,5,6-tetrafluorophenoxy.

[0363] Formula II aza-benzodiazepine Exemplary embodiments of the connector compounds are selected from Tables 2a and 2b. Each compound was synthesized, purified, and characterized by mass spectrometry, demonstrating the indicated quality. Further experimental procedures are described in the examples. Tables 2a and 2b contain aza-benzodiazepines. The linker compound (azaBzL) exhibited surprising and unexpected TLR8 agonist selectivity, which could predict useful therapeutic activity for treating cancer and other conditions. Tables 2a and 2b show aza-benzodiazepines. The linker intermediate (compound of formula II) was used to conjugate with antibodies by the method of Example 201 to form the immunoconjugates in Tables 3a and 3b.

[0364] Table 2a Aza-Benzaza Connector (azaBzL) compound

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371] Table 2b Aza-Benzaza Connector (azaBzL) compound

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416] The comparative adapter compounds (CL) from Table 2c have: (i) an activated ester, tetrafluorophenyl, or sulfotetrafluorophenyl that reacts with a lysine residue, or (ii) a maleimide group that reacts with a cysteine ​​residue of an antibody to form an immunoconjugate with the antibody and TLR agonist adapter moiety according to Example 201. The comparative adapter compounds CL-4,5,6,7,8 have an aza-benzo[a]aza Lactam structure.

[0417] Table 2c TLR agonist-connector comparison compounds (CL)

[0418]

[0419]

[0420]

[0421]

[0422] Aza-benzodiazepine Immunoconjugates

[0423] Immunostimulatory antibody conjugates (i.e., immunoconjugates) guide TLR7 / 8 agonists into the tumor to activate tumor-infiltrating myeloid cells and initiate a wide range of innate and adaptive antitumor immune responses (Ackerman et al., (2021) Nature Cancer 2:18-33).

[0424] Exemplary embodiments of the immunoconjugate include covalent attachment via a linker to one or more aza-benzodiazepines. Partial antibodies and having Formula I:

[0425] Ab-[LD] p I

[0426] Or its pharmaceutically acceptable salt, wherein:

[0427] Ab is an antibody;

[0428] p is an integer from 1 to 8;

[0429] L stands for connector;

[0430] D is an aza-benzozaza with the following formula part:

[0431]

[0432] Z 1 Selected from CR 1 and N;

[0433] Z 2 Selected from CR 2 and N;

[0434] Z 3 Selected from CR 3 and N;

[0435] Z 4 Selected from CR 4 and N;

[0436] Z 1 Z 2 Z 3 and Z 4 One or both of them are N;

[0437] R 1 R 2 R 3 R 4 R 5 and R 6 Independently select from the following groups: 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 ynyl, C3-C 12 carbonyl group, C6-C 20 Aryl, C2-C9 heterocyclic and C1-C 20 Heteroaryl groups, each of which is independently and optionally substituted by one or more groups selected from:

[0438] -(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0439] -(C1-C 12 alkyldiyl)-N(R 7 )2;

[0440] -(C1-C 12 alkyldiyl)-OR 7 ;

[0441] -(C3-C12 (Carbocyclic group);

[0442] -(C3-C 12 (carbocyclic group)-*;

[0443] -(C3-C 12 (C1-C)-(C1-C) 12 (alkyldiyl)-NR 7 -*;

[0444] -(C3-C 12 (C1-C)-(C1-C) 12 alkyldiyl)-N(R 7 )2;

[0445] -(C3-C 12 (Carbocyclic)-NR 7 -C(=NR 7 )NR 7 -*;

[0446] -(C6-C 20 (Aromatic);

[0447] -(C6-C 20 (aryldiyl)-*;

[0448] -(C6-C 20 aryldiyl)-N(R) 7 )-*;

[0449] -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0450] -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-(C2-C 20 (heterocyclic dimethyl)-*;

[0451] -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0452] -(C6-C 20 aryldiyl)-(C1-C 12 (alkyldiyl)-NR 7 -C(=NR 7a )N(R 7 )-*;

[0453] -(C2-C 20 (heterocyclic group);

[0454] -(C2-C 20 (heterocyclic group)-*;

[0455] -(C2-C9 heterocyclic group)-(C1-C 12 (alkyldiyl)-NR 7 -*;

[0456] -(C2-C9 heterocyclic group)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0457] -(C2-C9 heterocyclic group)-C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0458] -(C2-C9 heterocyclic group)-NR 7 -C(=NR 7a )NR 7 -*;

[0459] -(C2-C9 heterocyclic group)-NR 7 -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0460] -(C2-C9 heterocyclic group)-(C6-C 20 (aryldiyl)-*;

[0461] -(C1-C 20 (Heteroary aryl);

[0462] -(C1-C 20 (Heteroarylene)-*;

[0463] -(C1-C 20 (heteroaryl)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0464] -(C1-C 20 (heteroaryl)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0465] -(C1-C 20 (heteroaryl)-NR 7 -C(=NR 7a )N(R 7 )-*;

[0466] -(C1-C 20 (heteroaryl)-N(R) 7 )C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0467] -C(=O)-*;

[0468] -C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0469] -C(=O)-(C2-C 20 (heterocyclic dimethyl)-*;

[0470] -C(=O)N(R 7 )2;

[0471] -C(=O)N(R 7 )-*;

[0472] -C(=O)N(R 7 )-(C1-C 12 alkyldiyl)-N(R 7 )C(=O)R 7 ;

[0473] -C(=O)N(R 7 )-(C1-C 12 alkyldiyl)-N(R 7 )C(=O)N(R 7 )2;

[0474] -C(=O)NR 7 -(C1-C 12 alkyldiyl)-N(R 7 CO2R 7 ;

[0475] -C(=O)NR 5 -(C1-C 12 alkyldiyl)-N(R 57 )C(=NR 57a )N(R 57 )2;

[0476] -C(=O)NR 5 -(C1-C 12 (alkyldiyl)-NR 57 C(=NR 7a )R 7 ;

[0477] -C(=O)NR 5-(C1-C8 alkyldiyl)-NR 7 (C2-C5 heteroaryl);

[0478] -C(=O)NR 7 -(C1-C 20 (heteroaryldiyl)-N(R) 7 )-*;

[0479] -C(=O)NR 7 -(C1-C 20 (heteroaryldiyl)-*;

[0480] -C(=O)NR 7 -(C1-C 20 (heteroaryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0481] -C(=O)NR 7 -(C1-C 20 (heteroaryldiyl)-(C2-C 20 Heterocyclic dimethyl)-C(=O)NR 7 -(C1-C 12 (alkyldiyl)-NR 7 -*;

[0482] -N(R 7 )2;

[0483] -N(R 7 )-*;

[0484] -N(R 7 )C(=O)R 7 ;

[0485] -N(R 7 )C(=O)-*;

[0486] -N(R 7 )C(=O)N(R 7 )2;

[0487] -N(R 7 )C(=O)N(R 7 )-*;

[0488] -N(R 7 CO2R 7 ;

[0489] -NR 7 C(=NR 7a )N(R 7 )2;

[0490] -NR7 C(=NR 7a )N(R 7 )-*;

[0491] -NR 7 C(=NR 7a )R 7 ;

[0492] -N(R 7 )C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0493] -N(R 7 )-(C2-C5 heteroaryl);

[0494] -N(R 7 )-S(=O)2-(C1-C 12 alkyl);

[0495] -O-(C1-C 12 alkyl);

[0496] -O-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0497] -O-(C1-C 12 alkyldiyl)-N(R 7 )-*;

[0498] -OC(=O)N(R 7 )2;

[0499] -OC(=O)N(R 7 )-*;

[0500] -O-(R 7 )-*;

[0501] -OR 7 ;

[0502] -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-*;

[0503] -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-N(R 7 )2;

[0504] -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 (alkyldiyl)-NR7 -*;and

[0505] -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-OH;

[0506] Or R 5 and R 6 Together they form a 5- or 6-membered heterocyclic ring;

[0507] R 7 Independently selectable H, C6-C 20 Aryl, C3-C 12 carbonyl group, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 A group consisting of alkyl dimethyl groups, or two R groups 5 The groups together form a 5- or 6-membered heterocyclic ring;

[0508] R 7a Choose C6-C freely 20 Aryl and C1-C 20 Group composed of heteroaryl groups;

[0509] Where the asterisk * indicates the attachment site of L, and where R 1 R 2 R 3 R 4 R 5 and R 6 One of them is attached to L; and

[0510] Alkyl, alkyldiyl, alkenyl, alkenyldiyl, ynyl, ynyldiyl, aryl, aryldiyl, carbocyclic, carbocyclicdiyl, heterocyclic, heterocyclicdiyl, heteroaryl, and heteroaryldiyl are independently and optionally substituted by one or more groups independently selected from the following: F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH 2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH 2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHC OCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NH C(=NH)NH2, -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n H, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3 and -S(O)3H.

[0511] Exemplary embodiments of the immunoconjugate of Formula I include wherein L is selected from the group consisting of:

[0512] -C(=O)-PEG-;

[0513] -C(=O)-PEG-C(=O)N(R) 8 )-(C1-C 12alkyldiyl)-C(=O)-Gluc-;

[0514] -C(=O)-PEG-O-;

[0515] -C(=O)-PEG-OC(=O)-;

[0516] -C(=O)-PEG-C(=O)-;

[0517] -C(=O)-PEG-C(=O)-PEP-;

[0518] -C(=O)-PEG-N(R 8 )-;

[0519] -C(=O)-PEG-N(R 8 )-C(=O)-;

[0520] -C(=O)-PEG-N(R 8 )-PEG-C(=O)-PEP-;

[0521] -C(=O)-PEG-N + (R 8 )2-PEG-C(=O)-PEP-;

[0522] -C(=O)-PEG-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-;

[0523] -C(=O)-PEG-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)N(R 8 )C(=O)-(C2-C5 monoheterocyclic dimethyl)-;

[0524] -C(=O)-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-;

[0525] -C(=O)-PEG-SS-(C1-C 12 alkyldiyl)-C(=O)-;

[0526] -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-;

[0527] -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12alkyldiyl)-;

[0528] -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-N(R 8 -C (=O);

[0529] -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-N(R 8 )C(=O)-(C2-C5 monoheterocyclic dimethyl)-;

[0530] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-;

[0531] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)N(R 8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-;

[0532] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-O-;

[0533] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-OC(=O)-;

[0534] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)-;

[0535] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-;

[0536] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-C(=O)-;

[0537] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)-PEP-;

[0538] -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-;

[0539] -succinimide-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-;

[0540] -succinimide-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)N(R 8 )C(=O)-; and

[0541] -succinimide-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)N(R 8 )C(=O)-(C2-C5 monoheterocyclic dimethyl)-;

[0542] R 8 Independently H or C1-C6 alkyl;

[0543] PEG has the following formula: -(CH2CH2O) n -(CH2) m - m is an integer from 1 to 5, and n is an integer from 1 to 50;

[0544] Gluc has the following formula:

[0545]

[0546] PEP has the following formula:

[0547]

[0548] AA is independently selected from natural or non-natural amino acid side chains, or one or more of AA and adjacent nitrogen atoms form a 5-membered cyclic proline amino acid, and the wavy line indicates the attachment point.

[0549] Cyc is selected from C6-C 20Aryldiyl and C1-C 20 Heteroaryldimethyl groups, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3 and glucuronic acids having the following structures:

[0550]

[0551] R 9 Choose freely - CH(R) 10 )O-、-CH2-、-CH2N(R 10 )- and -CH(R 10 The group consisting of OC (=O)-, where R 10 Selected from H, C1-C6 alkyl, C(=O)-C1-C6 alkyl and -C(=O)N(R) 11 )2, where R 11 Independently select H, C1-C 12 Alkyl groups and -(CH2CH2O) n -(CH2) m A group consisting of -OH groups, where m is an integer from 1 to 5 and n is an integer from 2 to 50, or two R groups. 11 The groups together form a 5- or 6-membered heterocyclic ring;

[0552] y is an integer from 2 to 12;

[0553] z is 0 or 1.

[0554] An exemplary embodiment of the immunoconjugate of Formula I includes Z 1 It is N.

[0555] An exemplary embodiment of the immunoconjugate of Formula I includes Z 2 It is N.

[0556] An exemplary embodiment of the immunoconjugate of Formula I includes Z 3 It is N.

[0557] An exemplary embodiment of the immunoconjugate of Formula I includes Z 4 It is N.

[0558] An exemplary embodiment of the immunoconjugate of Formula I includes R 5 and R 6 Independently selected from C1-C8 alkyl groups, -O-(C1-C 12 alkyl), -(C1-C 12 alkyldiyl)-OR 5 -(C1-C8 alkyldiyl)-N(R) 5 CO2R 5 -(C1-C 12alkyl)-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.

[0559] An exemplary embodiment of the immunoconjugate of Formula I includes R 5 It is a C1-C8 alkyl group and R 6 It is -O-(C1-C 12 alkyl).

[0560] An exemplary embodiment of the immunoconjugate of Formula I includes R 5 It is -CH2CH2CH3 and R 6 Selected from -CH2CH2CH2NHCO2(t-Bu), -OCH2CH2NHCO2(cyclobutyl) and -CH2CH2CH2NHCO2(cyclobutyl).

[0561] An exemplary embodiment of the immunoconjugate of Formula I includes R 5 and R 6 Each is independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, -CH2CH2CF3, -OCH2CH2OH and -CH2CH2CH2OH.

[0562] An exemplary embodiment of the immunoconjugate of Formula I includes R 5 It is -CH2CH2CH3 and R 6 It is -OCH2CH3.

[0563] An exemplary embodiment of the immunoconjugate of Formula I includes R 6 Choose from the following groups:

[0564]

[0565] An exemplary embodiment of the immunoconjugate of Formula I includes R 1 Attach to L.

[0566] An exemplary embodiment of the immunoconjugate of Formula I includes R 2 Attach to L.

[0567] An exemplary embodiment of the immunoconjugate of Formula I includes R 3 Attach to L.

[0568] An exemplary embodiment of the immunoconjugate of Formula I includes R4 Attach to L.

[0569] An exemplary embodiment of the immunoconjugate of Formula I includes R 5 or R 6 Attach to L.

[0570] Exemplary embodiments of the immunoconjugate of Formula I include those in which L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0571] An exemplary embodiment of the immunoconjugate of Formula I includes a cysteine ​​thiol wherein L is attached to an antibody.

[0572] An exemplary embodiment of the immunoconjugate of Formula I includes a PEG in which m is 1 or 2 and n is an integer from 2 to 10, or where n is 10.

[0573] An exemplary embodiment of the immunoconjugate of Formula I includes a region where L comprises PEP, and PEP is a dipeptide having the following formula:

[0574]

[0575] Exemplary embodiments of the immunoconjugate of Formula I include wherein the AA is independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H and -CH2CH2CH2NHC(O)NH2; or the two AAs form a 5-membered cyclic proline amino acid.

[0576] An exemplary embodiment of the immunoconjugate of Formula I includes a PEP that is a dipeptide and has the following formula:

[0577]

[0578] AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids.

[0579] An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0580] An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA1 and AA2 are independently selected from GlcNAc aspartic acid, -CH2SO3H and -CH2OPO3H.

[0581] An exemplary embodiment of the immunoconjugate of Formula I includes a PEP that is a tripeptide and has the following formula:

[0582]

[0583] An exemplary embodiment of the immunoconjugate of Formula I includes a PEP that is a tetrapeptide and has the following formula:

[0584]

[0585] Exemplary embodiments of the immunoconjugate of Formula I include wherein

[0586] AA1 chooses the group consisting of Abu, Ala, and Val;

[0587] AA2 is selected from the group consisting of Nle (O-Bzl), Oic, and Pro;

[0588] AA3 is selected from the group consisting of Ala and Met(O)2; and

[0589] AA4 is selected from the group consisting of Oic, Arg(NO2), Bpa, and Nle(O-Bzl).

[0590] An exemplary embodiment of the immunoconjugate of Formula I includes L comprising PEP, and the 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.

[0591] An exemplary embodiment of the immunoconjugate of Formula I includes a region L comprising PEP, wherein the PEP is selected from the following structures:

[0592]

[0593] Exemplary embodiments of the immunoconjugate of Formula I include wherein L is selected from the following structures:

[0594]

[0595] The wavy line indicates R. 1 R 2 R 3 R 4 R 5 and R 6 The attachment of one of them.

[0596] This invention includes all reasonable combinations and arrangements of the features of the embodiment of Formula I.

[0597] In some embodiments, the immunoconjugated compounds of the present invention include those immunoconjugated compounds having immunostimulatory activity. The immunoconjugated compounds of the present invention selectively deliver an effective dose of aza-benzodiazepines to tumor tissue. (azaBz) drugs or metabolites that allow for greater selectivity (i.e., lower effective doses) while increasing the therapeutic index (“therapeutic window”) relative to unconjugated azaBz.

[0598] Each of the immunoconjugates in Tables 3a, 3b, and 3c was prepared according to the method of Example 201, purified by HPLC, and characterized by mass spectrometry.

[0599] Table 3a Aza-Benzaza Immunoconjugates (ICs)

[0600]

[0601]

[0602] N / A = EC50 not calculable, ND = not measured

[0603] Table 3b Aza-Benzaza Immunoconjugates (ICs)

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615] N / A = EC50 not calculable, ND = not measured

[0616] Table 3c compares immunoconjugates (CIC)

[0617]

[0618] The levels of secreted cytokines in the supernatant were determined using the LegendPlex Cytokine Bead Array Kit. The immunoconjugates (ICs) in Tables 3a and 3b induce the secretion of the cytokine TNFα(alpha), which is associated with the establishment of an immune response against cancer, and exhibit activation of myeloid cells upon exposure to tumor cells expressing antigens such as HER2. The aza-benzodiazepines in Tables 3a and 3b... The level of TNFα stimulated by the immunoconjugate was higher than that of the control immunoconjugate CIC-1. Notably, the levels of TNFα stimulated by the aza-benzodiazepine were higher. The payload represents a more efficient payload that provides increased activity while reducing molecular weight and hydrophobicity. The naked antibody does not induce myeloid activation, demonstrating dependence on the TLR7 / 8-activated payload.

[0619] Drug loading is expressed as p, where p is the aza-benzodiazepine content of each antibody in the immunoconjugate of formula I. The number of (azaBz) portions, and expressed as measurements (DAR) in the exemplary immunoconjugates of Table 3a. The amount of drug-loaded (azaBz) can range from 1 to about 8 drug portions (D) / antibody. Immunoconjugates of Formula I comprise a mixture or collection of antibodies conjugated to drug portions ranging from 1 to about 8. In some embodiments, the number of drug portions that can be conjugated to the antibody is limited by the number of reactive or available amino acid side chain residues such as lysine and cysteine. In some embodiments, free cysteine ​​residues are introduced into the antibody amino acid sequence by the methods described herein. In such an aspect, p can be 1, 2, 3, 4, 5, 6, 7 or 8 and ranges thereof, such as 1 to 8 or 2 to 5. In any such aspect, 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 having 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 to form intra- and inter-chain disulfide bonds (natural disulfide groups) without the need for engineering; in this case, the present free reduced cysteine ​​residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to conjugation to generate one or more free cysteine ​​residues.

[0620] For some immunoconjugates, p may be limited by the number of attachment sites on the antibody. For example, in the case where the attachment is a cysteine ​​thiol, as in some exemplary embodiments described herein, the antibody may have only one or a limited number of cysteine ​​thiol groups, or may have only one or a limited number of sufficiently reactive thiol groups that can attach to the drug. In other embodiments, one or more lysine amino groups in the antibody may be available and reactive for conjugation with the azaBz-connector compound of formula II. In some embodiments, higher drug loading (e.g., p > 5) may lead to aggregation, insolubility, toxicity, or loss of cellular permeability of some antibody-drug conjugates. In some 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 some embodiments, the antibody is subjected to denaturing conditions to exhibit reactive nucleophilic groups, such as lysine or cysteine.

[0621] The loading (drug / antibody ratio) of the immunoconjugate can be controlled in different ways, for example by: (i) limiting the molar excess of the azaBz-linker intermediate compound relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partially or limiting reductive denaturation conditions to optimize antibody reactivity.

[0622] It should be understood that when more than one nucleophilic group of an antibody reacts with a drug, the resulting product is a mixture of immunoconjugated compounds having a distribution of one or more drug moieties attached to the antibody. The drug mean for each antibody can be calculated from the mixture by a dual ELISA antibody assay that is specific for both the antibody and the drug. Individual immunoconjugate molecules in a mixture can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, for example, McDonald 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 AA CR, Vol. 45, March 2004; Alley, SC et al., “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American Association for Cancer Research). Research, 2004 Annual Meeting, March 27-31, 2004 (Proceedings of the AACR, Volume 45, March 2004). In some embodiments, homogeneous immunoconjugates with a single loading value can be separated from the conjugated mixture by electrophoresis or chromatography.

[0623] The in vitro activity of the immunoconjugate can be evaluated according to the method of Example 203.

[0624] Composition of immune conjugates

[0625] This invention provides a composition, such as a pharmaceutically or pharmacologically acceptable composition or formulation, comprising a variety of immunoconjugates as described herein and optionally a carrier therefor, such as a pharmaceutically or pharmacologically acceptable carrier. The immunoconjugates in the composition may be the same or different; that is, the composition may comprise an immunoconjugate having the same number of adjuvants linked to the same position on an antibody construct and / or having the same number of aza-benzodiazepines linked to different positions on an antibody construct. Immunoconjugates of (azaBz) adjuvant, immunoconjugates having different numbers of azaBz adjuvants linked to the same position on the antibody construct, or immunoconjugates having different numbers of azaBz adjuvants linked to different positions on the antibody construct.

[0626] In an exemplary embodiment, the composition comprising an immunoconjugation compound comprises a mixture of immunoconjugation compounds, wherein the average drug loading (aza-Bz) (DAR) of each antibody in the mixture of immunoconjugation compounds is about 2 to about 5.

[0627] The average adjuvant-to-antibody construct ratio (DAR) of the immunoconjugate compositions of the present invention can be from about 0.4 to about 10. Those skilled in the art will recognize that, in compositions comprising various immunoconjugates of the present invention, the aza-benzodiazepine conjugated to the antibody construct... The number of adjuvants may vary depending on the immunoconjugate, so the adjuvant-to-antibody construct (e.g., antibody) ratio can be measured as an average value, which may 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 method, many of which are known in the art, including conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the immunoconjugate in the composition, expressed as p, can also be determined. In some cases, homogeneous immunoconjugates can be separated, purified, and characterized by methods such as reversed-phase HPLC or electrophoresis, where p is a value derived from immunoconjugates with other drug loadings.

[0628] 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 the lumen of a body cavity or organ. Alternatively, the immunoconjugates can be injected intratumorally. Injectable compositions will typically comprise a solution of the immunoconjugate dissolved in a pharmaceutically acceptable carrier. Among acceptable media and solvents that can be used are water and isotonic solutions of one or more salts, such as sodium chloride, for example, Ringer's solution. In addition, sterile nonvolatile oils can conventionally be used as solvents or suspension media. For this purpose, any mild nonvolatile oil, including synthetic monoglycerides or diglycerides, can be used. In addition, fatty acids (such as oleic acid) can also be used in the preparation of injectable solutions. These compositions are ideally sterile and generally free of undesirable substances. These compositions can be sterilized using conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients close to physiological conditions, such as pH adjusters and buffers, toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0629] The composition may contain any suitable concentration of the immunoconjugate. The concentration of the immunoconjugate in the composition can vary widely and will be selected based primarily on fluid volume, viscosity, body weight, etc., depending on the specific administration method chosen and the patient's needs. In some embodiments, the concentration of the immunoconjugate in the injectable solution formulation will range from about 0.1% (w / w) to about 10% (w / w).

[0630] Methods of treating cancer with immunoconjugates

[0631] This invention provides a method for treating cancer. The method includes administering a therapeutically effective amount of an immunoconjugate as described herein (e.g., as a composition as described herein) to a subject in need, such as a subject suffering from cancer and requiring cancer treatment. The method includes administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 3a.

[0632] The immunoconjugates of the present invention are intended to be used to treat a variety of hyperproliferative diseases or conditions, such as those characterized by the overexpression of tumor antigens. Exemplary hyperproliferative conditions include benign or malignant solid tumors and hematologic disorders such as leukemia and lymphoid malignancies.

[0633] On the other hand, immunoconjugates are provided for use as pharmaceuticals. In some embodiments, the invention provides immunoconjugates for a method of treating an individual, the method comprising administering an effective amount of the immunoconjugate to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one other therapeutic agent to the individual, for example, as described herein.

[0634] In a further aspect, the present invention provides the use of immune conjugates in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical medicament is used to treat cancer, and the method includes administering an effective amount of the pharmaceutical medicament to an individual suffering from cancer. In one such embodiment, the method further includes administering an effective amount of at least one other therapeutic agent to the individual, such as that described herein.

[0635] Cancer is a malignant tumor originating from epithelial tissue. Epithelial cells cover the outer surface of the body, line internal cavities, and form the lining of glandular tissue. Examples of cancer include, but are not limited to, adenocarcinoma (cancer originating from glandular (secreting) cells, such as breast cancer, pancreatic cancer, lung cancer, prostate cancer, stomach cancer, gastroesophageal junction cancer, and colon cancer), 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 cancer; small cell lung cancer; non-small cell lung cancer; and so on. Cancer can be found in the prostate, pancreas, colon, brain (often as a secondary metastasis), lung, breast, and skin. In some implementations, methods of treating non-small cell lung cancer include administering an immunoconjugate containing an antibody construct capable of binding to tumor-associated antigens.

[0636] Soft tissue tumors are a highly diverse group of rare tumors originating from connective tissue. Examples of soft tissue tumors include, but are not limited to, alveolar soft tissue sarcoma; hemangioma-like fibrous histiocytoma; chondromycinous fibroma; osteochondrosarcoma; extraosseous myxoid chondrosarcoma; clear cell sarcoma; fibroproliferative small round cell tumor; dermatofibrosarcoma protuberans; endometrial stromal tumor; Ewing's sarcoma; fibromatosis (demodex sarcoma); infantile fibrosarcoma; gastrointestinal stromal tumor; giant cell tumor of bone; giant cell tumor of tenosynovium; inflammatory myofibroblastic tumor; uterine fibroma; leiomyosarcoma; lipoblastoma; and typical lipomas. Lipomas; spindle cell or pleomorphic lipomas; atypical lipomas; chondroid lipomas; well-differentiated liposarcomas; myxoid / round cell liposarcomas; pleomorphic liposarcomas; myxoid malignant fibrous histiocytomas; highly malignant fibrous histiocytomas; myxofibrosarcomas; malignant peripheral nerve sheath tumors; mesotheliomas; neuroblastomas; osteochondromas; osteosarcomas; primitive neuroectodermal tumors; alveolar rhabdomyosarcomas; embryonal rhabdomyosarcomas; benign or malignant schwannomas; synovial sarcomas; Evan's tumors. Tumors; nodular fasciitis; destenoid fibromatosis; solitary fibroma; 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; soft tissue pleomorphic adenoma; and tumors originating from fibroblasts, myofibroblasts, histiocytes, angiocytes / endothelial cells, and nerve sheath cells.

[0637] Sarcomas are rare types of cancer that originate from mesenchymal cells, such as those found in the body's bones or soft tissues, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supporting tissues. Different types of sarcomas are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examples of sarcomas include, but are not limited to, primitive neuroectodermal tumors (PNETs) of the thoracic and pulmonary regions (Askin's tumor); botryoidal sarcoma; chondrosarcoma; malignant angioendothelioma; malignant schwannoma; osteosarcoma; and soft tissue sarcoma (e.g., alveolar soft tissue sarcoma; angiosarcoma; phyllodes cystosarcoma; dermatofibrosarcoma protuberans (DFSP); desmoidoma; connective tissue proliferative small round cell tumor; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); hemangiopericytoma; hemangioendothelioma (more commonly known as "angiosarcoma"); Kaposi's sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve schwannoma (MPNST); neurofibrosarcoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma).

[0638] A teratoma is a type of germ cell tumor that can contain several different types of tissue (e.g., it can include tissues derived from any and / or all three germ layers: endoderm, mesoderm, and ectoderm), including, for example, hair, muscle, and bone. Teratomas most commonly occur in the ovaries of women, the testes of men, and the coccyx of children.

[0639] Melanoma is a form of cancer that begins in melanocytes (the cells that produce melanin). Melanoma can begin in a mole (skin melanoma), but it can also begin in other pigmented tissues, such as the eyes or intestines.

[0640] Merkel cell carcinoma is a rare type of skin cancer that typically presents as flesh-colored or bluish-red nodules on the face, head, or neck. It is also known as cutaneous neuroendocrine carcinoma. In some embodiments, treatment of Merkel cell carcinoma involves administering an immunoconjugate containing an antibody construct capable of binding, for example, CEA (e.g., labetuzumab, its biosimilars, or a biomodified version thereof). In some embodiments, the Merkel cell carcinoma has already metastasized at the time of administration.

[0641] Leukemia is a cancer that begins in tissues that form blood, such as bone marrow, and causes a large number of abnormal blood cells to be produced and enter the bloodstream. For example, leukemia can originate from bone marrow-derived cells that normally mature in the bloodstream. Leukemia is named according to how quickly the disease develops and progresses (e.g., acute vs. chronic) and the type of white blood cells affected (e.g., myeloid vs. lymphoid). Myeloid leukemia is also called myeloid or myeloblastic leukemia. Lymphoid leukemia is also called lymphoblastic or lymphocytic leukemia. Lymphoid leukemia cells can accumulate in lymph nodes, causing the lymph nodes 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).

[0642] Lymphoma is a cancer that originates in the cells of the immune system. For example, lymphoma can originate from 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), characterized by the presence of a cell type known as Reed-Stedberg cells. There are currently six recognized types of HL. Examples of Hodgkin lymphoma include tuberous sclerosis classic Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte-depleted CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0643] Another type of lymphoma is non-Hodgkin lymphoma (NHL), which comprises cancers of a large number of different immune system cells. Non-Hodgkin lymphoma can be further divided into cancers with an indolent (slow-growing) process and cancers with an aggressive (rapid-growing) process. There are currently 61 recognized types of NHL. Examples of non-Hodgkin's lymphoma include, but are not limited to, AIDS-related lymphoma, anaplastic large cell lymphoma, angioimmunoblastic lymphoma, blastic NK cell lymphoma, Burkitt lymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, enteropathy-type T-cell lymphoma, follicular lymphoma, hepatosplenic γ-δ T-cell lymphoma, T-cell leukemia, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-cell lymphoma, pediatric lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, treatment-related T-cell lymphoma, and Waldenström macroglobulinemia.

[0644] Brain cancer includes any cancer of the brain tissue. Examples of brain cancer 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).

[0645] The immunoconjugates of the present invention can be used alone or in combination with other agents in a therapy. For example, the immunoconjugates can be administered co-administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. Such combination therapies encompass both combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and single administration, in the latter case, where the administration of the immunoconjugate can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The immunoconjugates can also be used in combination with radiotherapy.

[0646] The immunoconjugates (and any other therapeutic agents) of the present invention can be administered by any suitable means, including oral, parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be made by any suitable route, such as by injection, like intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing regimens are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusion.

[0647] The immunoconjugate may be administered to a subject in need at any therapeutically effective amount using any suitable dosing regimen, such as that used for labetuzumab, its biosimilars, and their biomodifications. For example, the method may include administering the immunoconjugate to a subject at a dose of about 100 ng / kg to about 50 mg / kg. The immunoconjugate dose may range from about 5 mg / kg to about 50 mg / kg, from about 10 μg / kg to about 5 mg / kg, or from about 100 μg / kg to about 1 mg / kg. The immunoconjugate dose may be about 100, 200, 300, 400, or 500 μg / kg. The immunoconjugate dose may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The immunoconjugate dose may also be outside these ranges, depending on the specific conjugate and the type and severity of the cancer being treated. The frequency of administration may range from a single weekly dose to multiple weekly doses, or more frequently. In some embodiments, the immunoconjugate is administered from about once a month to about five times a week. In some implementations, the immunoconjugate is administered once a week.

[0648] On the other hand, the present invention provides a method for cancer prevention. The method includes administering a therapeutically effective amount of an immunoconjugate (e.g., as the composition described above) to a subject. In some embodiments, the subject is susceptible to a certain type of cancer to be prevented.

[0649] Some embodiments of the present invention provide methods for treating cancers as described above, wherein said cancer is breast cancer. Breast cancer can originate in different areas of the breast and many different types of breast cancer have been characterized. For example, the immunoconjugates of the present invention can be used to treat ductal carcinoma in situ; invasive ductal carcinoma (e.g., tubular carcinoma; medullary carcinoma; mucinous carcinoma; papillary carcinoma; or cribriform carcinoma of the breast); lobular carcinoma in situ; invasive lobular carcinoma; inflammatory breast cancer; and other forms of breast cancer, such as triple-negative (negative for estrogen receptor, progesterone receptor, and overexpressing HER2 protein tests) breast cancer. In some embodiments, the method of treating breast cancer includes administering an immunoconjugate containing an antibody construct capable of binding to tumor-associated antigens (TAAs) or overexpressing TAAs of the tumor.

[0650] In some implementations, the cancer is sensitive to pro-inflammatory responses induced by TLR7 and / or TLR8.

[0651] In some implementations, therapeutically effective doses of the immunoconjugate are administered to patients in need to treat cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, or breast cancer. Merkel cell carcinoma can be metastatic Merkel cell carcinoma. Breast cancer can be triple-negative breast cancer. Esophageal cancer can be adenocarcinoma of the gastroesophageal junction.

[0652] Example

[0653] Example 3 2-Amino-4-((2-((tert-butoxycarbonyl)amino)ethoxy)(propyl)carbamoyl)-3H-pyrido[4,3-b]aza Synthesis of 8-carboxylic acid, azaBz-3

[0654]

[0655] Preparation of (2,5-dibromopyridin-4-yl)carbamate tert-butyl ester 3b

[0656] To a solution of 2,5-dibromopyridine-4-carboxylic acid, 3a (25.0 g, 89.0 mmol, 1 equivalent) and Et3N (27.0 g, 267 mmol, 37.2 mL, 3 equivalent) in tert-butanol (200 mL), diphenylphosphohydrazide DPPA (49.0 g, 178 mmol, 38.6 mL, 2 equivalent) was added, and the mixture was stirred at 80 °C for 12 h to achieve the Kurteu rearrangement by adding tert-butanol to the intermediate isocyanate. The mixture was concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 120g Purification was performed using a silica gel rapid column at 100 mL / min with a 0–25% ethyl acetate / petroleum ether gradient elution to give 3b as a white solid (25 g, 71.02 mmol, 79.80% yield). 1 ¹H NMR (CDCl₃, 400MHz) 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 (measured).

[0657] Preparation of (2-bromo-5-formylpyridin-4-yl)carbamate tert-butyl ester 3c

[0658] n-BuLi (2.5 M, 27.3 mL, 2.4 equivalents) was added to a solution of 3b (10 g, 28.4 mmol, 1 equivalent) in THF (150 mL) at -78 °C and N2, and the mixture was stirred at this temperature for 0.5 h. Then DMF (10.4 g, 142 mmol, 10.9 mL, 5 equivalents) was added at -78 °C and stirred for 0.5 h. The mixture was quenched with an aqueous solution of NH4Cl, diluted with 50 mL of water, and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 25g Purification was performed using a silica gel rapid column at 80 mL / min with a 0–40% ethyl acetate / petroleum ether gradient elution to give 3C (4.5 g, 14.94 mmol, 52.60% yield) as a white solid. 1 H NMR (CDCl3, 400MHz) δ9.93 (s, 1H), 8.62 (s, 1H), 8.51 (s, 1H), 1.55 (s, 9H).

[0659] Preparation of ethyl 3(E)-3-(6-bromo-4-((tert-butoxycarbonyl)amino)pyridin-3-yl)-2-(cyanomethyl)acrylate, 3d

[0660] A solution of 3c (4.5 g, 14.9 mmol, 1 equivalent) and ethyl 3-cyano-2-(triphenyl-λ5-phosphonylidene)propionate (6.37 g, 16.4 mmol, 1.1 equivalent) in DCM (50 mL) was stirred at 30 °C for 2 hours. The mixture was concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 25g Purification was performed using a silica gel rapid column at 80 mL / min with a 0–50% ethyl acetate / petroleum ether gradient elution to give 3d (5 g, 12.2 mmol, 81.56% yield) as a yellow solid. 1 ¹H NMR (CDCl₃, 400MHz) δ 8.44 (s, 1H), 8.07 (s, 1H), 7.68 (s, 1H), 6.48–6.43 (m, 1H), 4.41 (q, J = 7.2Hz, 2H), 3.40 (s, 2H), 1.55 (s, 9H), 1.43 (t, J = 7.2Hz, 3H). LC / MS [M+H] 410.01 (calculated); LC / MS [M+H] 410.1 (measured).

[0661] 2-Amino-8-bromo-3H-pyrido[4,3-b]aza Preparation of ethyl 4-carboxylate 3e

[0662] To a solution of 3d (5 g, 12.2 mmol, 1 equivalent) in EtOAc (10 mL), HCl / EtOAc (4 M, 22.2 mL, 7.29 equivalents) was added, and the mixture was stirred at 25 °C for 16 hours. 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. 1¹H NMR (MeOD, 400MHz) δ 8.62 (s, 1H), 7.96 (s, 1H), 7.61 (s, 1H), 4.36 (q, J = 7.2Hz, 2H), 3.65 (s, 2H), 1.38 (t, J = 7.2Hz, 3H). LC / MS [M+H] 310.01 (calculated); LC / MS [M+H] 310.1 (measured).

[0663] 2-Amino-8-bromo-3H-pyrido[4,3-b]aza Preparation of 3f-4-carboxylic acid

[0664] Lithium hydroxide hydrate, LiOH·H₂O (484 mg, 11.5 mmol, 2 equivalents), was added to a solution of 3e (2 g, 5.77 mmol, 1 equivalent) in THF (15 mL) and water (5 mL), and the mixture was stirred at 45 °C for 1 hour. The mixture was quenched with 1 N HCl until pH 2–3 and extracted with DCM / i-prOH (10 mL x 3, 3 / 1). The organic layer was washed with brine (10 mL x 2), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give 3f as a white solid (800 mg, 2.84 mmol, 49.15% yield). LC / MS [M+H] 282.0 (calculated); LC / MS [M+H] 282.1 (found).

[0665] (2-((2-amino-8-bromo-N-propyl-3H-pyrido[4,3-b]aza) Preparation of 3g of 4-formamido)oxy)ethyl)carbamate tert-butyl ester

[0666] To a solution of 3f (800 mg, 2.84 mmol, 1 equivalent) in DCM (8 mL) and DMA (3 mL), methanesulfonic acid (273 mg, 2.84 mmol, 202 μL, 1 equivalent), N-[2-(propylaminooxy)ethyl]carbamate tert-butyl ester (743 mg, 3.40 mmol, 1.2 equivalent) and EDCI (2.17 g, 11.3 mmol, 4 equivalent) were added, and the mixture was stirred at 25 °C for 1 hour. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (15 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 20g Purification was performed using a silica gel rapid column (eluting with a 0–100% ethyl acetate / petroleum ether gradient at 45 mL / min) to give 3 g (800 mg, 1.66 mmol, 58.48% yield) of a brown oil. LC / MS [M+H] 482.1 (calculated); LC / MS [M+H] 482.2 (measured).

[0667] 2-Amino-4-[2-(tert-Butoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-pyrido[4,3-b]aza Preparation of methyl 8-carboxylate, azaBz-4

[0668] A mixture of 3 g (450 mg, 933 μmol, 1 equivalent), Pd(dppf)Cl2 (68.3 mg, 93.3 μmol, 0.1 equivalent), and Et3N (283 mg, 2.80 mmol, 390 μL, 3 equivalent) in MeOH (15 mL) was degassed and purged three times with CO, and 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 conditions column: Waters Xbridge Prep OBD C18 150*40 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 10%-40%, 8 min) to give azaBz-4 as a white solid (100 mg, 216.68 μmol, 23.23% yield). 1 ¹H NMR (MeOD, 400MHz) δ 8.57 (s, 1H), 7.80 (s, 1H), 7.36 (s, 1H), 3.96 (s, 3H), 3.91 (t, J = 5.2Hz, 2H), 3.72 (t, J = 7.2Hz, 2H), 3.23 (t, J = 5.2Hz, 2H), 3.00 (s, 2H), 1.76 (sxt, J = 7.2Hz, 2H), 1.32 (s, 9H), 0.98 (t, J = 7.2Hz, 3H). LC / MS [M+H] 462.2 (calculated); LC / MS [M+H] 462.3 (measured).

[0669] Preparation of azaBz-3

[0670] LiOH·H₂O (21.8 mg, 520 μmol, 1.2 equivalent) was added to a solution of azaBz-4 (200 mg, 433 μmol, 1 equivalent) in THF (1 mL) and water (0.3 mL), and the mixture was stirred at 25 °C for 1 hour. The mixture was filtered and purified by preparative HPLC (neutral conditions column: Waters Xbridge prep OBD C18 150*40 mm*10 μm; mobile phase: [water (NH₄HCO₃)-ACN]; B%: 5%-35%, 8 min) to give azaBz-3 (20 mg, 44.69 μmol, 10.31% yield) as a white solid. 1 ¹H NMR (MeOD, 400MHz) δ 8.57 (s, 1H), 7.80 (s, 1H), 7.37 (s, 1H), 3.90 (t, J = 5.2Hz, 2H), 3.73 (t, J = 7.2Hz, 2H), 3.23 (t, J = 5.2Hz, 2H), 3.10 (s, 1H), 1.82–1.71 (m, 2H), 1.33 (s, 9H), 0.98 (t, J = 7.2Hz, 3H). LC / MS [M+H] 448.2 (calculated); LC / MS [M+H] 448.2 (measured).

[0671] Example 6 2-Amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza Synthesis of 4-formamide, azaBz-6

[0672]

[0673] N-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-pyrido[4,3-b]aza Preparation of tert-butyl carbamate, 6a, with the following formula: [8-yl]pyrimidin-2-yl]methyl]-N-tert-butoxycarbonyl-carbamate.

[0674] 2-amino-8-bromo-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza The mixture of 4-carboxamide L-4a (200 mg, 545 μmol, 1 equivalent), N-tert-butoxycarbonyl-N-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyrimidin-2-yl]methyl]carbamate tert-butyl ester (285 mg, 654 μmol, 1.2 equivalent), K2CO3 (151 mg, 1.09 mmol, 2 equivalent), and Pd(dppf)Cl2 (19.9 mg, 27.23 μmol, 0.05 equivalent) in dioxane (6 mL) and H2O (1.2 mL) was degassed and purged three times with N2, and then stirred at 95 °C for 2 hours under N2 atmosphere. The reaction mixture was poured into H2O (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give 6a as a yellow solid (320 mg, 537 μmol, 98.6% yield). LC / MS [M+H] 596.3 (calculated); LC / MS [M+H] 596.4 (analytical).

[0675] Preparation of azaBz-6

[0676] To a solution of 6a (600 mg, 1.01 mmol, 1 equivalent) in EtOAc (12 mL), HCl / EtOAc (20 mL) was added. The mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 μm; mobile phase: [water (TFA)-ACN]; B%: 1%-20%, 8 min) to give azaBz-6 (350 mg, 885 μmol, 87.87% yield, TFA) as a yellow solid. 1 ¹H NMR (MeOD, 400MHz) 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 = 7.2Hz, 2H), 3.76 (t, J = 7.2Hz, 2H), 3.57 (s, 2H), 1.83–1.70 (m, 2H), 1.21 (t, J = 7.2Hz, 3H), 1.00 (t, J = 7.2Hz, 3H). LC / MS [M+H] 396.2 (calculated); LC / MS [M+H] 396.0 (measured).

[0677] Example L-1 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolo-1-yl)acetyl]amino]ethoxy ... Synthesis of 4-carbonyl)-propyl-amino]oxyethyl]carbamate, azaBzL-1

[0678]

[0679] Preparation of (E)-3-[3-(tert-butoxycarbonylamino)-4-pyridyl]-2-(cyanomethyl)prop-2-enoic acid ethyl ester, L-1b

[0680] Add 3-cyano-2-(triphenyl-λ) to a solution of N-(4-formyl-3-pyridyl)carbamate, L-1a (500 mg, 2.25 mmol, 1.0 equivalent) in THF (5 mL). 5 Ethyl phosphonyl propionate (871 mg, 2.25 mmol, 1.0 equivalent). The mixture was stirred at 55 °C for 1 h. The reaction mixture was quenched by adding H2O (5 mL) and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (5 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give L-1b (2.8 g, crude) as a yellow oil. LC / MS [M+H] 332.2 (calculated); LC / MS [M+H] 332.0 (found).

[0681] 2-Amino-3H-pyrido[3,4-b]aza Preparation of ethyl 4-carboxylate, L-1c

[0682] To a solution of L-1b (2.6 g, 7.85 mmol, 1.0 equivalent) in EtOAc (1 mL), HCl / EtOAc (4 M, 20 mL, 10.2 equivalent) was added. The mixture was stirred at 25 °C for 12 h, and then at 50 °C for another 2 h. The reaction mixture was filtered. The filter cake was then dried under reduced pressure to give the residue. Compound L-1c (980 mg, 4.24 mmol, 54.0% yield) was given as a yellow oil. 1¹H NMR (400MHz, DMSO-d⁶) δ 10.27 (s, 1H), 9.27 (s, 1H), 8.72 (s, 1H), 8.55 (d, J = 5.2Hz, 1H), 7.88 (s, 1H), 7.74 (d, J = 5.2Hz, 1H), 4.29 (q, J = 7.2Hz, 2H), 3.57 (s, 2H), 1.32 (t, J = 7.2Hz, 3H). LC / MS [M+H] 232.1 (calculated); LC / MS [M+H] 232.2 (measured).

[0683] 2-Amino-3H-pyrido[3,4-b]aza Preparation of 4-carboxylic acid, L-1d

[0684] LiOH (149 mg, 6.23 mmol, 3.0 equivalent) was added to a solution of L-1c (480 mg, 2.08 mmol, 1.0 equivalent) in H2O (1 mL) and EtOH (5 mL). 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 the residue, L-1d (250 mg, 1.23 mmol, 59.2% yield), obtained as a white solid. 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.68 (s, 1H), 8.56 (d, J = 5.6Hz, 1H), 7.86 (s, 1H), 7.79 (d, J = 5.6Hz, 1H). LC / MS [M+H] 204.1 (calculated); LC / MS [M+H] 204.0 (measured).

[0685] N-[2-[(2-amino-3H-pyrido[3,4-b]aza] Preparation of tert-butyl carbamate (-4-carbonyl)-propyl-amino]oxyethyl]carbamate, L-1e

[0686] MsOH (236 mg, 2.46 mmol, 1.0 equivalent) and EDCI (943 mg, 4.92 mmol, 4.0 equivalent) were added to a solution of L-1d (250 mg, 1.23 mmol, 1.0 equivalent) in DCM (7.5 mL) and DMA (1.5 mL). 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 an aqueous solution of Na2CO3 at 0 °C. Extraction was then performed with EtOAc (10 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to give L-1e as a white solid (420 mg, 1.04 mmol, 84.6% yield). 1 ¹H NMR (400MHz, MeOD) δ 8.37 (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 = 5.2Hz, 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 (measured).

[0687] 2-Amino-N-(2-aminoethoxy)-N-propyl-3H-pyrido[3,4-b]aza Preparation of 4-formamide, L-1f

[0688] To a solution of L-1e (55 mg, 136 μmol, 1.0 equivalent) in EtOAc (1 mL), HCl / EtOAc (4 M, 10 mL, 293.0 equivalent) was added, and the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue as a white solid, L-1f (51.2 mg, crude product, 2 HCl). LC / MS [M+H] 304.2 (calculated); LC / MS [M+H] 304.2 (measured).

[0689] Preparation of azaBzL-1

[0690] Add DIEA (60.8 mg, 470 μmol, 81.9 μL 5.0 equivalent) and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolo-1-yl)acetyl]amino]ethoxy ... The mixture was then filtered and purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 5%-35%, 8min) to obtain azaBzL-1 (40mg, 41.3umol, 43.9% yield) as a yellow oil. 1 H NMR(400MHz,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=4.8Hz,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 = 7.6Hz, 3H). LC / MS[M+H] 968.5 (calculated value); LC / MS[M+H] 968.5 (measured value).

[0691] Example L-3 2-amino-4-(((40-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-4,39-dioxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-3,38-diazatetrazyl)oxy)(propyl)carbamoyl)-3H-pyrido[4,3-b]aza Synthesis of 8-sulfonic acid, azaBzL-3

[0692]

[0693]

[0694] (2-((2-amino-8-(benzylthio)-N-propyl-3H-pyrido[4,3-b]aza) Preparation of tert-butyl 4-carbamate (4-carbamate)oxy)ethyl)carbamate, L-3a

[0695] N-[2-[(2-amino-8-bromo-3H-pyrido[4,3-b]aza] The mixture of (-4-carbonyl)-propyl-amino]oxyethyl]carbamate tert-butyl ester (3 g) (400 mg, 829 μmol, 1 equivalent), phenylmethanethiol (680 mg, 5.47 mmol, 642 μL, 6.60 equivalent), (1E,4E)-1,5-diphenylpentan-1,4-dien-3-one; palladium (Pd2(dba)3) (75.9 mg, 82.9 μmol, 0.1 equivalent), diisopropylethylamine (DIEA) (214 mg, 1.66 mmol, 289 μL, 2 equivalent) and (5-diphenylphosphino-9,9-dimethyl-xanthan-4-yl)-diphenyl-phosphine, Xantphos (96.0 mg, 166 μmol, 0.2 equivalent) in dioxane (5 mL) was degassed and purged three times with N2, and then stirred at 110 °C for 2 hours under N2 atmosphere. The mixture was filtered and concentrated under reduced pressure. The residue was diluted with 50 mL of H₂O at 0 °C and then extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was subjected to rapid silica gel chromatography (…). 80g Purification was performed using a silica gel rapid column at 80 mL / min with a 0–100% ethyl acetate / petroleum ether gradient elution to give L-3a (350 mg, 665.83 μmol, 80.29% yield) as a yellow solid. 1 ¹H NMR (MeOD, 400MHz) δ 8.37 (s, 1H), 7.41 (d, J = 7.2Hz, 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 = 5.2Hz, 2H), 3.71 (t, J = 7.2Hz, 2H), 3.23 (t, J = 5.2Hz, 2H), 3.00–2.93 (m, 2H), 1.75 (m, 2H), 1.33 (s, 9H), 0.97 (t, J = 7.6Hz, 3H). LC / MS [M+H] 526.2 (calculated); LC / MS [M+H] 526.3 (measured).

[0696] (2-((2-amino-8-(benzylthio)-N-propyl-3H-pyrido[4,3-b]aza) Preparation of tert-butyl 4-carbamate (L-3b)

[0697] TrtCl (318 mg, 1.14 mmol, 2 equivalents) and Et3N (231 mg, 2.28 mmol, 318 μL, 4 equivalents) were added to a solution of L-3a (300 mg, 571 μL, 1 equivalent) in DCM (10 mL) at 25 °C, and the mixture was stirred at 50 °C for 16 hours. The reaction mixture was diluted with 20 mL of water and extracted with DCM (50 mL x 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue as a yellow solid, compound L-3b (350 mg, 456 μL, 79.86% yield). LC / MS [M+H] 768.4 (calculated); LC / MS [M+H] 768.3 (found).

[0698] (2-((8-(chlorosulfonyl)-N-propyl-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza) Preparation of 4-formamido)oxy)ethyl)tert-butyl carbamate, L-3c

[0699] Sulfonyl chloride (211 mg, 1.56 mmol, 156 μL, 4 equivalents) was added to a solution of L-3b (300 mg, 391 μL, 1 equivalent) in DCM (5 mL) and water (2 mL), and the mixture was stirred at 0 °C for 1 hour. The reaction mixture was quenched by adding 10 mL of saturated NaHCO3 at 0 °C and extracted with DCM (15 mL x 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The crude product L-3c (300 mg, crude) as a yellow solid was used in the next step without further purification. LC / MS [M+H] 744.2 (calculated); LC / MS [M+H] 744.3 (found).

[0700] 4-((2-((tert-Butoxycarbonyl)amino)ethoxy)(propyl)carbamoyl)-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza Preparation of 8-sulfonic acid, L-3d

[0701] A solution of L-3c (300 mg, 403 μmol, 1 equivalent) in water (3 mL) and MeCN (5 mL) was stirred at 100 °C for 0.5 h. The mixture was concentrated under reduced pressure. The crude L-1d (270 mg, 372 μmol, 92.23% yield), a yellow solid, was used in the next step without further purification. LC / MS [M+H] 726.3 (calculated); LC / MS [M+H] 726.3 (measured).

[0702] 2-Amino-4-[2-aminoethoxy(propyl)carbamoyl]-3H-pyrido[4,3-b]aza Preparation of 8-sulfonic acid, L-3e

[0703] A solution of L-3d (200 mg, 276 μmol, 1 equivalent) in DCM (3 mL) and TFA (1 mL) was stirred at 25 °C for 4 hours. 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. The aqueous layer was concentrated to give the crude product. The crude product L-3e (100 mg, crude) as a yellow solid was used in the next step without further purification. LC / MS [M+H] 384.1 (calculated); LC / MS [M+H] 384.2 (found).

[0704] Preparation of azaBzL-3

[0705] At 0°C, 2-[ ... 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%, 8min) to obtain azaBzL-3 as a white solid (15mg, 14.31umol, 10.97% yield). 1¹H NMR (MeOD, 400MHz) 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 = 4.6Hz, 2H), 3.87 (br d, J = 4.0Hz, 2H), 3.77 (br t, J = 7.2Hz, 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 (measured).

[0706] Example L-4 2-Amino-8-(2-(38-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,37-dioxo-6,9,12,15,18,21,24,27,30,33-decaoxa-2,36-diazaoctadecyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza Synthesis of 4-formamide, azaBzL-4

[0707]

[0708] To 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza 3-[2 ... The reaction mixture was filtered and purified by preparative HPLC (water-ACN conditions) (column: Waters Xbridge BEH C18100*25mm*5um; mobile phase: [water-acetonitrile]; B%: 5%-35%, 20min) to give azaBzL-4 as a yellow solid (33.3mg, 31.89umol, 16.25% yield). 1HNMR(MeOD,400MHz)δ9.36(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 value); LC / MS[M+H] 1044.5 (measured value).

[0709] Example L-5 2-Amino-N8-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentadecane-35-yl)-N4-ethoxy-N4-propyl-3H-pyrido[4,3-b]aza Synthesis of 4,8-dicarboxamide, azaBzL-5

[0710]

[0711] (1-(4-(ethoxy(propyl)carbamoyl)-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza Preparation of tert-butyl carbamate, L-5a (-8-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azatritetradecane-3,4-yl)carbamate

[0712] N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]tert-butyl carbamate (NH2-PEG) 10 -NHBoc)(355mg, 591umol, 1.2 equivalents), 8-bromo-N-ethoxy-N-propyl-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza A mixture of 4-carboxamide (L-6b) (300 mg, 492.16 μmol, 1 equivalent), triethylamine (TEA) (249 mg, 2.46 mmol, 343 μL, 5 equivalents), and Pd(dppf)Cl2 (72.02 mg, 98.4 μmol, 0.2 equivalents) in DMF (8 mL) was degassed, purged three times with carbon monoxide (CO), and then stirred at 80 °C under a CO (50 Psi) atmosphere for 16 h. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, 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 (measured).

[0713] 2-Amino-N8-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxatridodecyl)-N4-ethoxy-N4-propyl-3H-pyrido[4,3-b]aza Preparation of 4,8-dicarboxamide, L-5b

[0714] The mixture of L-5a (50 mg, 43.2 μmol, 1 equivalent) and TFA (98.5 mg, 864 μmol, 63.9 μL, 20 equivalent) in DCM (1 mL) was degassed and purged three times with N2, then stirred at 40 °C under N2 atmosphere for 16 hours. The reaction mixture was concentrated under reduced pressure to give L-5b (40 mg, crude) as a yellow oil, which was used in the next step without further purification. LC / MS [M+H] 815.5 (calculated); LC / MS [M+H] 815.4 (measured).

[0715] Preparation of azaBzL-5

[0716] To a solution of L-5b (35 mg, 37.68 μmol, 1 equivalent, TFA) and 2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetic acid 2,5-dioxopyrrolo-1-yl ester (9.50 mg, 37.7 μmol, 1 equivalent) in DMF (0.2 mL), DIEA (19.5 mg, 151 μmol, 26.3 μL, 4 equivalents) was added, and the mixture was stirred at 25 °C for 1 h. The reaction mixture was filtered and purified by preparative HPLC (TFA conditions) (column: Phenomenex Luna 80*30 mm*3 μm; mobile phase: [water (TFA)-ACN]; B%: 10%-40%, 8 min) to give azaBzL-5 as a white solid (20 mg, 21.01 μmol, 55.76% yield). 1 H NMR(MeOD,400MHz)δ8.79(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=7.2Hz,3H),0.98(t,J=7.2Hz,3H). LC / MS[M+H] 952.5 (calculated value); LC / MS[M+H] 952.5 (measured value).

[0717] Example L-6 2-Amino-8-(N-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentadecane-35-yl)aminesulfonyl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza Synthesis of 4-formamide, azaBzL-6

[0718]

[0719]

[0720] 2-Amino-8-bromo-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza Preparation of 4-formamide, L-6a

[0721] To 2-amino-8-bromo-3H-pyrido[4,3-b]aza 4-Carboxylic acid, 3f (3 g, 10.6 mmol, 1 equivalent) was added to a solution of methanesulfonic acid (1.02 g, 10.6 mmol, 0.757 mL, 1 equivalent), N-ethoxypropyl-1-amine (1.29 g, 9.25 mmol, 0.9 equivalent, HCl), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDCI, CAS Registry No. 1892-57-5 (8.15 g, 42.5 mmol, 4 equivalent) in DCM (40 mL) and DMA (10 mL). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was diluted with water (80 mL) and extracted with DCM (50 mL x 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The crude product was ground together 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 (measured).

[0722] 8-Bromo-N-ethoxy-N-propyl-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza Preparation of 4-formamide, L-6b

[0723] Triphenylmethyl chloride (TrtCl) (1.33 g, 4.77 mmol, 2.5 equivalents) and triethylamine, Et3N (964 mg, 9.53 mmol, 1.33 mL, 5 equivalents) were added to a solution of L-6a (700 mg, 1.91 mmol, 1 equivalent) in DCM (20 mL), and the mixture was stirred at 50 °C for 16 hours. The reaction mixture was diluted with water (20 mL) and extracted with DCM (20 mL x 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 40g Purification was performed using a silica gel rapid column (eluting with a gradient of 0–60% ethyl acetate / petroleum ether at 50 mL / min) to give L-6b as a white solid (1 g, 1.64 mmol, 86.07% yield). LC / MS [M+H] 609.2 (calculated); LC / MS [M+H] 609.0 (measured).

[0724] 8-(benzylthio)-N-ethoxy-N-propyl-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza Preparation of 4-formamide, L-6c

[0725] To a solution of L-6b (600 mg, 0.986 mmol, 1 equivalent) and phenylmethanethiol, BnSH (306 mg, 2.46 mmol, 0.289 mL, 2.5 equivalent) in dioxane (30 mL), (1E,4E)-1,5-diphenylpentan-1,4-dien-3-one; palladium, Pd2(dba)3 (90.3 mg, 98.6 μmol, 0.1 equivalent), DIEA (255 mg, 1.97 mmol, 0.343 mL, 2 equivalent) and (5-diphenylphosphino-9,9-dimethyl-xanthon-4-yl)-diphenyl-phosphine, Xantphos, CAS Registry No. 161265-03-8 (114 mg, 0.197 mmol, 0.2 equivalent) were added, the mixture was stirred at 110 °C for 2 hours under N2. The reaction mixture was quenched by adding water (50 mL) at 0 °C and then extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 40g Purification was performed using a silica gel rapid column (eluting 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 (measured).

[0726] 4-(ethoxy(propyl)carbamoyl)-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza Preparation of 8-sulfonyl chloride, L-6d

[0727] Sulfonyl chloride and SO2Cl2 (393 mg, 2.91 mmol, 0.291 mL, 5 equivalents) were added to a solution of L-6c (380 mg, 0.582 mmol, 1 equivalent) in DCM (2 mL) and water (0.3 mL) at 0 °C, and then stirred at 25 °C for 1 hour. The reaction mixture was quenched by adding saturated NaHCO3 (15 mL) aqueous solution at 0 °C, and then extracted with DCM (20 mL x 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 (found).

[0728] N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[ethoxy(propyl)carbamoyl]-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza Preparation of tert-butyl carbamate, L-6e

[0729] At 0°C, N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]tert-butyl carbamate, NH2-PEG 10 -NHBoc (458 mg, 0.76 mmol, 1.2 equivalents) and Et3N (322 mg, 3.18 mmol, 0.442 mL, 5 equivalents) were added to a solution in DCM (8 mL) with L-6d (400 mg, 0.64 mmol, 1 equivalent), and then stirred at 25 °C for 0.5 h. The mixture was filtered and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 20g Purification was performed using a silica gel rapid column (eluting with a gradient of 0–100% ethyl acetate / petroleum ether at 50 mL / min) to give L-6e as a colorless oil (500 mg, 0.419 mmol, 65.90% yield). LC / MS [M+H] 1193.6 (calculated); LC / MS [M+H] 1193.3 (measured).

[0730] 2-Amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxatridodecyl)aminosulfonyl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza Preparation of 4-formamide, L-6f

[0731] TFA (191 mg, 1.68 mmol, 124 μL, 20 equivalents) was added to a solution of L-6e (100 mg, 83.8 μmol, 1 equivalent) in DCM (0.3 mL), and the mixture was stirred 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 (measured).

[0732] Preparation of azaBzL-6

[0733] Diisopropylethylamine, DIEA (53.6 mg, 414 μmol, 72.2 μL, 4 equivalents), and 2-(2,5-dioxopyrrolo-1-yl)acetic acid (2,5-dioxopyrrolo-1-yl) ester (26.13 mg, 104 μmol, 1 equivalent) were added to a solution of L-6f (100 mg, 104 μmol, 1 equivalent) in DMF (0.3 mL) at 0 °C, and the mixture was 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%-30%, 8 min) to give a colorless oily substance azaBzL-6 (53 mg, 53.6 μmol, 51.8% yield). 1 ¹H NMR (MeOD, 400MHz) 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 = 7.2Hz, 2H), 3.76 (t, J = 7.2Hz, 2H), 3.66–3.59 (m, 32H), 3.56–3.47 (m, 10H), 3.37 (td, J = 4.8, 9.6Hz, 4H), 1.78 (m, 2H), 1.20 (t, J = 7.2Hz, 3H), 1.00 (t, J = 7.6Hz, 3H). LC / MS [M+H] 988.5 (calculated); LC / MS [M+H] 988.2 (measured).

[0734] Example L-7 2-Amino-8-(N-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentadecane-35-yl)-N-methylaminesulfonyl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza Synthesis of 4-formamide, azaBzL-7

[0735]

[0736] (2-((4-(ethoxy(propyl)carbamoyl)-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza Preparation of --8-yl)sulfonyl)-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azatritetradecane-3,4-yl)carbamate, L-7a

[0737] To N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[ethoxy(propyl)carbamoyl]-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza [-8-yl]sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] tert-butyl]carbamate, L-6e (200 mg, 168 μmol, 1 equivalent) was added to a solution in MeCN (1 mL) with cesium carbonate Cs2CO3 (109 mg, 0.335 mmol, 2 equivalents) and iodomethane, MeI (28.5 mg, 201 μmol, 12.5 μL, 1.2 equivalents). The mixture was stirred at 25 °C for 1 hour. The resulting mixture was filtered and concentrated under reduced pressure to give L-7a (200 mg, crude product) as a colorless oil. 1 H NMR(MeOD,400MHz)δ8.44(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=6.8Hz,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=7.2Hz,3H),1.01(t,J=7.6Hz,3H). LC / MS[M+H] 1207.6 (calculated value); LC / MS[M+H] 1207.8 (measured value).

[0738] 2-Amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxatridodecyl)-N-methylaminosulfonyl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]aza Preparation of 4-formamide, L-7b

[0739] TFA (264 mg, 2.32 mmol, 172 μL, 20 equivalents) was added to a solution of L-7a (140 mg, 116 μmol, 1 equivalent) in DCM (0.3 mL), and the mixture was stirred at 50 °C for 1 hour. The mixture was filtered and concentrated under reduced pressure to give L-7b (100 mg, crude product) as a yellow solid. LC / MS [M+H] 865.5 (calculated); LC / MS [M+H] 865.5 (measured).

[0740] Preparation of azaBzL-7

[0741] At 0 °C, 2-(2,5-dioxopyrrolo-1-yl)acetic acid (2,5-dioxopyrrolo-1-yl) ester (25.8 mg, 102 μmol, 1 equivalent) was added to a solution of L-7b (100 mg, 102 μmol, 1 equivalent) and DIEA (52.8 mg, 409 μmol, 71.2 μL, 4 equivalent) in DMF (0.5 mL), and the mixture was stirred at 25 °C for 0.5 h. The mixture was filtered and purified by preparative HPLC (TFA column: Phenomenex Luna 80*30 mm*3 μm; mobile phase: [water (TFA)-ACN]; B%: 5%-35%, 8 min) to give azaBzL-7 (45 mg, 44.90 μmol, 43.96% yield) as a yellow oil. 1 H NMR(MeOD,400MHz)δ8.83(s,1H),7.87(s,1H),7.51(s,1H),6.89(s,2H),4.17(s,2H),3.98(q,J= 7.2Hz,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=5.2Hz,2H),3.06(s,2H),1.78(m,2H),1.20(t,J=7.2Hz,3H),1.00(t,J=7.6Hz,3H). LC / MS[M+H] 1002.5 (calculated value); LC / MS[M+H] 1002.2 (measured value).

[0742] Example L-8 2-Amino-N8-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentadecane-35-yl)-N4-ethoxy-N8-methyl-N4-propyl-3H-pyrido[4,3-b]aza Synthesis of 4,8-dicarboxamide, azaBzL-8

[0743]

[0744] N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[ethoxy(propyl)carbamoyl]-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza Preparation of tert-butyl carbamate, L-8a, with the following structure: [-8-carbonyl]-methyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethyl]carbamate]tert-butyl]urethane]

[0745] At 25°C, N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[ethoxy(propyl)carbamoyl]-2-(triphenylmethylamino)-3H-pyrido[4,3-b]aza [-8-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] tert-butyl]carbamate,"" tert-BuOK (1M, 276uL, 2 equivalents)" and"MeI (19.6mg, 138uL, 8.61uL, 1 equivalent)" were added to a solution of THF (2mL) and maintained for 2 hours. The mixture was filtered and purified by preparative HPLC (TFA conditions; column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 35%-65%, 8min) to give L-6a (80mg, 68.3uL, 49.40% yield) as a colorless oil. LC / MS[M+H] 1171.7 (calculated value); LC / MS[M+H] 1171.3 (measured value).

[0746] 2-Amino-N8-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxatridodecyl)-N4-ethoxy-N8-methyl-N4-propyl-3H-pyrido[4,3-b]aza Preparation of 4,8-dicarboxamide, L-8b

[0747] TFA (68.1 mg, 59.8 μmol, 1 equivalent) was added to a solution of L-8a (70 mg, 59.8 μmol, 1 equivalent) in DCM (0.2 mL), 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%-28%, 8 min) to give L-8b (30 mg, 31.8 μmol, 53.24% yield, TFA) as a yellow oil. 1¹H NMR (DMSO-d⁶, 400MHz) 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), 3.65–0.48 (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 (measured).

[0748] Preparation of azaBzL-8

[0749] To a solution of L-8b (20 mg, 24.1 μmol, 1 equivalent) in DMF (0.5 mL), DIEA (12.5 mg, 96.5 μmol, 16.8 μL, 4 equivalents) and 2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetic acid 2,5-dioxopyrrolidine-1-yl ester (6.08 mg, 24.1 μmol, 1 equivalent) were added, and the mixture was 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%-35%, 8 min) to give azaBzL-8 (15 mg, 15.5 μmol, 64.4% yield) as a yellow oil. 1 ¹H NMR (MeOD, 400MHz) 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 (measured).

[0750] Example L-9 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolo-1-yl)acetyl]amino]ethoxy ... Synthesis of (-8-carbonyl)-propyl-amino]oxyethyl]carbamate, azaBzL-9

[0751]

[0752]

[0753] Preparation of 4-(tert-butyl)-1-ethyl(E)-2-((3-((tert-butyloxycarbonyl)amino)pyridin-2-yl)methylene)succinate, L-9b

[0754] A solution of tert-butyl (2-formylpyridin-3-yl)carbamate, L-9a (1 g, 4.50 mmol, 1 equivalent), and O4-tert-butylO1-ethyl 2-(triphenyl-λ5-phosphine)succinate (2.50 g, 5.40 mmol, 1.2 equivalent) in DCM (5 mL) was stirred at 50 °C for 2 hours. The mixture was filtered and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 40g Purification was performed using a silica gel rapid column at 60 mL / min with a 0–30% ethyl acetate / petroleum ether gradient elution to give L-9b (350 mg, 861 μmol, 19.1% yield) as a colorless oil. 1 ¹H NMR (CDCl₃, 400MHz) δ 8.36–8.31 (m, 2H), 7.76 (s, 1H), 7.25 (dd, J = 4.8, 8.4Hz, 1H), 6.72 (s, 1H), 4.32 (q, J = 7.2Hz, 2H), 3.70 (s, 2H), 1.54 (s, 9H), 1.42 (s, 9H), 1.36 (t, J = 7.2Hz, 3H). LC / MS [M+H] 407.2 (calculated); LC / MS [M+H] 407.2 (measured).

[0755] 6-Hydroxy-7H-pyrido[3,2-b]aza Preparation of ethyl 8-carboxylate, L-9c

[0756] TFA (982 mg, 8.61 mmol, 638 μL, 10 equivalents) was added to a solution of L-9b (350 mg, 861 μmol, 1 equivalent) in DCM (5 mL) and stirred at 50 °C for 3 hours. The mixture was concentrated under reduced pressure to give L-9c (300 mg, crude product) as a yellow solid. 1 ¹H NMR (CDCl₃, 400MHz) δ 8.78 (s, 1H), 8.65 (dd, J = 1.2, 4.8Hz, 1H), 8.05 (s, 1H), 7.64–7.60 (m, 1H), 7.53 (dd, J = 4.4, 8.4Hz, 1H), 4.36 (q, J = 7.2Hz, 2H), 3.43 (s, 2H), 1.39 (t, J = 7.2Hz, 3H). LC / MS [M+H] 233.0 (calculated); LC / MS [M+H] 233.2 (measured).

[0757] 6-Amino-7H-pyrido[3,2-b]aza Preparation of ethyl 8-carboxylate, L-9d

[0758] Compound L-9c (700 mg, 3.01 mmol, 1 equivalent) was dissolved in POCl3 (6.93 g, 45.2 mmol, 4.21 mL, 15 equivalents), and the mixture was stirred at 90 °C under N2 for 16 hours. The mixture was concentrated under reduced pressure. The residue was then dissolved in MeCN (20 mL), and NH3·H2O (27.30 g, 234 mmol, 30 mL, 30% purity, 65.1 equivalents) was added to the solution, followed by stirring at 25 °C for 0.5 hours. The reaction mixture was extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over Na2SO4, 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 value); LC / MS[M+H]232.1 (measured value).

[0759] 6-Amino-7H-pyrido[3,2-b]aza Preparation of -8-carboxylic acid, L-9e

[0760] Lithium hydroxide hydrate, LiOH·H₂O (327 mg, 7.78 mmol, 3 equivalents), was added to a solution of L-9d (600 mg, 2.59 mmol, 1 equivalent) in MeOH (1 mL) and water (0.3 mL), and the mixture was stirred at 25 °C for 2 hours. The pH of the mixture was adjusted to 5–6 with 1 N HCl, the precipitate was filtered, and the solid was 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 (measured).

[0761] (2-((6-amino-N-propyl-7H-pyrido[3,2-b]aza) Preparation of tert-butyl 8-carbamate (L-9f)

[0762] To a solution of L-9e (240 mg, 1.18 mmol, 1 equivalent) and N-[2-(propylaminooxy)ethyl]carbamate tert-butyl ester (335 mg, 1.54 mmol, 1.3 equivalent) in DCM (3 mL) and dimethylacetamide, DMA (2 mL), methanesulfonic acid (170 mg, 1.77 mmol, 127 μL, 1.5 equivalent) and EDCI (906 mg, 4.72 mmol, 4 equivalent) were added, and the mixture was stirred at 25 °C for 1 hour. The reaction mixture was diluted with 10 mL of water and extracted with EtOAc (15 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 25g Purification was performed using a silica gel rapid column at 45 mL / min with a gradient elution of 0–100% ethyl acetate / petroleum ether to 50% ethyl acetate / MeOH, yielding 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 (measured).

[0763] 6-Amino-N-(2-aminoethoxy)-N-propyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-9g

[0764] HCl / dioxane (4M, 4.00 mL, 32 equivalents) was added to a solution of L-9f (200 mg, 496 μmol, 1 equivalent) in dioxane (1 mL), and the mixture was stirred at 25 °C for 0.5 h. The mixture was concentrated under reduced pressure to give crude product L-9g (200 mg, crude product, HCl) as a brown solid. LC / MS [M+H] 304.2 (calculated); LC / MS [M+H] 304.2 (measured).

[0765] Preparation of azaBzL-9

[0766] Add 2-[ ...5-dioxopyrrole-1-yl]acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eth The mixture was filtered, and the filtrate was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna C1875*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 10%-40% B, for 8.0 min) to obtain azaBzL-9 (50 mg, 51.6 μmol, 11.7% yield), which was a yellow oil. 1 H NMR (MeOD, 400MHz) δ8.66 (dd, J=1.2, 4.4Hz, 1H), 7.89 (d, J=8.0Hz, 1H), 7.58 (dd, J= 4.4,8.4Hz,1H),7.37(s,1H),6.89(s,2H),4.17(s,2H),3.99(t,J=5.2Hz,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 (measured value).

[0767] Example L-10 6-Amino-3-[2-[[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolo-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionylamino]methyl]pyrimidin-5-yl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Synthesis of 8-formamide, azaBzL-10

[0768]

[0769]

[0770] Preparation of (E)-2-(5-bromo-3-nitro-2-pyridyl)-N,N-dimethyl-ethylamine, L-10b

[0771] DMF-DMA (165 g, 1.38 mol, 184 mL, 2 equivalents) was added to a solution of 5-bromo-2-methyl-3-nitropyridine, L-10a (150 g, 691 mmol, 1 equivalent) in DMF (750 mL) at 25 °C, and then heated to 90 °C and stirred at 90 °C for 1 hour. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure to give a residue. The residue was diluted with H2O (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, 400MHz) 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).

[0772] Preparation of 5-bromo-3-nitro-pyridine-2-carboxaldehyde, L-10c

[0773] NaIO4 (156 g, 728 mmol, 40.3 mL, 2.2 equivalents) was added fractionally to a solution of L-10b (90 g, 331 mmol, 1 equivalent) in THF (750 mL) and H2O (750 mL) at 0 °C, and then the mixture was heated to 25 °C and stirred at 25 °C for 1 hour. The reaction mixture was filtered, the filtrate was diluted with H2O (500 mL), and extracted with EtOAc (500 mL x 3). The combined organic layers were washed with saturated Na2S2O3 (150 mL x 3) and brine (150 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, 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 ¹H NMR (CDCl₃, 400MHz) δ 10.22 (s, 1H), 9.04 (d, J = 1.6Hz, 1H), 8.38 (d, J = 1.6Hz, 1H). LC / MS [M+H] 230.9 (calculated); LC / MS [M+H] 231.0 (measured).

[0774] Preparation of O4-tert-butylO1-ethyl(2E)-2-[(5-bromo-3-nitro-2-pyridyl)methylene]succinate, L-10d

[0775] O4-tert-butylO1-ethyl 2-(triphenyl-phosphine-1,401,090 ... 1 ¹H NMR (CDCl₃, 400MHz) δ 8.89 (d, J = 2.0Hz, 1H), 8.48 (d, J = 2.0Hz, 1H), 8.01 (s, 1H), 4.33 (q, J = 7.2Hz, 2H), 3.75 (s, 2H), 1.42 (s, 9H), 1.36 (t, J = 7.2Hz, 3H). LC / MS [M+Na] 437.0 (calculated); LC / MS [M+Na] 436.9 (measured).

[0776] Preparation of O4-tert-butylO1-ethyl(2E)-2-[(3-amino-5-bromo-2-pyridyl)methylene]succinate, L-10e

[0777] Iron filings and Fe (20.2 g, 362 mmol, 5 equivalents) were added in portions to a solution of L-10d (30.1 g, 72.5 mmol, 1 equivalent) in AcOH (500 mL) at 25 °C under N2, and the mixture was stirred at 25 °C for 2 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (500 mL) and adjusted to pH 8-9 with saturated NaHCO3 at 0 °C. The mixture was filtered and the filtrate was extracted with EtOAc (400 mL x 3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give L-10e (27.3 g, 71 mmol, 97.9% yield) as a yellow solid. 1 H NMR (MeOD, 400MHz) δ 7.92 (d, J = 2.0Hz, 1H), 7.71 (s, 1H), 7.30 (d, J = 2.0Hz, 1H), 4.28 (q, J = 7.2Hz, 2H), 3.80 (s, 2H), 1.42 (s, 9H), 1.34 (t, J = 7.2Hz, 3H). LC / MS [M+H] 385.1 (calculated); LC / MS [M+H] 385.0 (measured).

[0778] 8-(dipropylcarbamoyl)-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of methyl 3-carboxylate, L-10f

[0779] TFA (243 g, 2.13 mol, 158 mL, 10 equivalents) was added to a solution of L-10e (82.0 g, 213 mmol, 1 equivalent) in DCM (900 mL) at 25 °C, and then heated to 50 °C and stirred for 2 hours. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure to obtain a residue. The residue was poured into ice water (1000 mL) at 0 °C and adjusted to pH 8-9 with saturated NaHCO3 at 0 °C. The aqueous phase was extracted with EtOAc (500 mL x 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give L-10f (74.0 g, crude product) as a yellow solid. 1¹H NMR (DMSO-d⁶, 400MHz) δ 10.64 (s, 1H), 8.58 (d, J = 2.0Hz, 1H), 7.79 (d, J = 2.0Hz, 1H), 7.67 (s, 1H), 4.26 (q, J = 7.2Hz, 2H), 3.24 (s, 2H), 1.29 (t, J = 7.2Hz, 3H). LC / MS [M+H] 310.9 (calculated); LC / MS [M+H] 310.9 (measured).

[0780] 6-Amino-3-bromo-7H-pyrido[3,2-b]aza Preparation of ethyl 8-carboxylate, L-10g

[0781] POCl3 (24.9 g, 162 mmol, 15.1 mL, 5 equivalents) was added to a solution of L-10f (10.1 g, 32.5 mmol, 1 equivalent) in dioxane (100 mL) at 25 °C, and then heated to 120 °C and stirred for 6 hours. The reaction mixture was cooled to 0 °C and added to NH3·H2O (275 g, 1.96 mol, 303 mL, 25% purity, 58.3 equivalents) in CH3CN (40 mL), and then stirred at 0 °C for 1 hour. The reaction mixture was filtered, and the filter cake was dried under reduced pressure as a pure product. The filtrate was extracted with (150 mL x 3). The combined organic layers were washed with brine (100 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the 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 (9.1g, 29.3mmol, 87.1% yield) as a brown solid. 1 ¹H NMR (DMSO-d⁶, 400MHz) δ 8.34 (d, J = 2.0Hz, 1H), 7.66 (s, 1H), 7.62 (d, J = 2.0Hz, 1H), 7.33 (s, 2H), 4.25 (q, J = 7.2Hz, 2H), 2.96 (s, 2H), 1.30 (t, J = 7.2Hz, 3H). LC / MS [M+H] 310.0 (calculated); LC / MS [M+H] 309.9 (measured).

[0782] 6-Amino-3-bromo-7H-pyrido[3,2-b]aza Preparation of 8-carboxylic acid, L-10h

[0783] A solution of LiOH·H₂O (11.0 g, 261 mmol, 3 equivalents) in H₂O (100 mL) was added to a solution of L⁻¹⁰ g (27.0 g, 87.1 mmol, 1 equivalent) in THF (300 mL) at 0 °C, and then the mixture was heated to 25 °C and stirred for 2 hours. The reaction mixture was cooled to 0 °C and 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 off, 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. 1 ¹H NMR (DMSO-d⁶, 400MHz) 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); LC / MS [M+H] 281.9 (measured).

[0784] 6-Amino-3-bromo-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-10i

[0785] EDCI (24.5 g, 128 mmol, 3 equivalents) was added to a solution of L-10h (12.0 g, 42.5 mmol, 1 equivalent), N-ethoxypropyl-1-amine (5.94 g, 42.5 mmol, 1 equivalent, HCl) and MsOH (4.09 g, 42.5 mmol, 3.04 mL, 1 equivalent) in DCM (120 mL) and DMA (12 mL) at 0 °C, and then the mixture was heated to 25 °C and stirred for 1 hour. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was quenched by adding H2O (50 mL) at 0 °C, and then adjusted to pH 8-9 with aqueous NaHCO3 solution at 0 °C, and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (30 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the 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. 1¹H NMR (DMSO-d⁶, 400MHz) δ 8.28 (d, J = 2.0Hz, 1H), 7.57 (d, J = 2.0Hz, 1H), 7.24 (s, 1H), 7.17 (s, 1H), 7.02 (s, 1H), 3.85 (q, J = 7.2Hz, 2H), 3.62 (t, J = 7.2Hz, 2H), 2.84 (s, 2H), 1.69–1.59 (m, 2H), 1.05 (t, J = 7.2Hz, 3H), 0.90 (t, J = 7.2Hz, 3H). LC / MS [M+H] 367.1 (calculated); LC / MS [M+H] 367.0 (measured).

[0786] N-[[5-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]aza Preparation of --3-yl]pyrimidin-2-yl]methyl]-N-tert-butoxycarbonyl-tert-butyl carbamate, L-10j

[0787] A solution of tert-butyl carbamate (180 mg, 414 μmol, 0.8 equivalents) in dioxane (2 mL) was added to a solution of L-10i (190 mg, 517 μmol, 1 equivalent) and K₂CO₃ (143 mg, 1.03 mmol, 2 equivalents) in water (0.2 mL), followed by the addition of Pd(dppf)Cl₂ (37.9 mg, 51.7 μmol, 0.1 equivalents), and the mixture was stirred at 100 °C for 1 hour under a N₂ atmosphere. The mixture was then concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; gradient: 5%-35% B, for 8 min) to obtain L-10j as a yellow solid (160 mg, 268 μmol, 51.9% yield). LC / MS [M+H] 596.3 (calculated); LC / MS [M+H] 596.3 (measured).

[0788] 6-Amino-3-(2-(aminomethyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-10k

[0789] To a solution of L-10j (160 mg, 269 μmol, 1 equivalent) in EtOAc (2 mL), HCl / EtOAc (4 M, 5 mL, 74.5 equivalents) was added, and the mixture was stirred at 25 °C for 1 hour. 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 (measured).

[0790] Preparation of azaBzL-10

[0791] N-methylmorpholine, NMM (46.8 mg, 463 μmol, 50.9 μL, 2 equivalents), and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrole-1-yl)acetyl]amino]ethoxy ... The reaction mixture was quenched with TFA until pH 5–6, and then purified by preparative HPLC (TFA conditions; column: Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 10%–40% B, for 8.0 min) to give azaBzL-10 (50 mg, 47.8 μmol, 20.6% yield) as a yellow oil. 1 H NMR (MeOD, 400MHz) δ9.17 (s, 2H), 9.06 (d, J = 2.0Hz, 1H), 8.14 (d, J = 2.0Hz, 1H), 7.49(s,1H),6.91(s,2H),4.72(s,2H),4.18(s,2H),3.81(q,J=7.2Hz,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=6.0Hz,2H),1.84-1.78(m,2H),1.24(t,J=7.2Hz,3H),1.03(t,J=7.2Hz,3H). LC / MS[M+H] 1044.5 (calculated value); LC / MS[M+H] 1044.3 (measured value).

[0792] Example L-17 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]aza Synthesis of 2,3,5,6-tetrafluorophenyl propionic acid ester, azaBzL-17

[0793]

[0794] Add 6-amino-3-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azapropionic acid (2,3,5,6-tetrafluorophenyl) ester (97.3 mg, 0.114 mmol, 2.5 equivalents) to a solution of 3-[2-[2-[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]propionic acid (2,3,5,6-tetrafluorophenyl) ester (97.3 mg, 0.114 mmol, 2.5 equivalents) in DMF (0.5 mL) at 0 °C. 8-Formamide, L-10k (18 mg, 0.0455 mmol, 1 equivalent) and DIEA (35.3 mg, 0.273 mmol, 0.476 mL, 6 equivalent) were dissolved in DMF (0.5 mL), and the mixture was stirred at 25 °C for 1 hour. The reaction mixture was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 20%-50% B, for 8.0 min) to give azaBzL-17 (12 mg, 0.111 mmol, 24.3% yield) as a colorless oil. 1H NMR (MeOD, 400MHz) δ9.16 (s, 2H), 9.03 (d, J = 2.0Hz, 1H), 8.12 (d, J = 2.0Hz, 1H), 7. 47-7.38(m,2H),4.72(d,J=5.6Hz,2H),4.02(q,J=7.2Hz,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=6.0Hz,2H),2.62( t,J=6.0Hz,2H),1.85-1.76(m,2H),1.24(t,J=7.2Hz,3H),1.01(t,J=7.2Hz,3H). LC / MS[M+H] 1084.5 (calculated value); LC / MS[M+H] 1084.7 (measured value).

[0795] Example L-20 2-Amino-N-((40-(2,5-dimethylene-2,5-dihydro-1H-pyrrolo-1-yl)-4,39-dioxo-8,11,14,17,20,23,26,29,32,35-decaoxa-3,5,38-triazatetradecyl)oxy)-N-propyl-3H-pyrido[3,4-b]aza Synthesis of 4-formamide, azaBzL-20

[0796]

[0797]

[0798] Preparation of (32-isocyano-3,6,9,12,15,18,21,24,27,30-decaoxatridodecyl)carbamate tert-butyl ester, L-20b

[0799] TEA (0.348 mL, 2.5 mmol, 10 equivalents) was added to a solution of (32-amino-3,6,9,12,15,18,21,24,27,30-decaoxatridodecyl)carbamate, L-20a (0.15 g, 0.25 mmol, 1 equivalent) in DCM, followed by the addition of phosgene (0.892 mL, as a 1.4 M solution in toluene, 0.25 mmol, 1 equivalent). The reaction mixture was monitored by LCMS, concentrated, and purified by reversed-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 (found).

[0800] 2-Amino-N-(2-aminoethoxy)-N-propyl-3H-pyrido[3,4-b]aza Preparation of 4-formamide, L-20d

[0801] (2-((2-amino-N-propyl-3H-pyrido[3,4-b]aza) 4-(formamido)oxy)ethyl)tert-butyl carbamate, L-20c (6.1 mg, 0.015 mmol, 1 equivalent) was suspended in a very small amount of TFA. After 15 minutes, 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 (found).

[0802] (39-(2-amino-3H-pyrido[3,4-b]aza) Preparation of tert-butyl carbamate, L-20e, with the following composition: (-4-carbonyl)-3,4-oxo-3,6,9,12,15,18,21,24,27,30,38-undecoxa-33,35,39-triazatetradodecyl)carbamate.

[0803] TEA (0.17 mL, 1.24 mmol, 10 equivalents) was added to a mixture of L-20d (37.8 mg, 0.124 mmol, 1 equivalent) and 2Am4CBza-L-18b (78 mg, 0.124 mmol, 1 equivalent) in DMF. The reaction mixture was stirred at room temperature, diluted with water, and purified by reversed-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 (found).

[0804] 2-Amino-N-((37-amino-4-oxo-8,11,14,17,20,23,26,29,32,35-decaoxa-3,5-diazaheptadecyl)oxy)-N-propyl-3H-pyrido[3,4-b]aza Preparation of 4-formamide, L-20f

[0805] L-20e (48 mg, 0.052 mmol, 1 equivalent) was dissolved in a very small amount of TFA. After 15 minutes, the reaction mixture was concentrated to give L-20f (0.053 g, 0.050 mmol, 96%) as a TFA salt. LC / MS [M+H] 830.49 (calculated); LC / MS [M+H] 830.76 (found).

[0806] Preparation of azaBzL-20

[0807] TEA (0.09 mL, 0.64 mmol, 1 equivalence) was added to a solution of L-20f (0.053 g, 0.050 mmol, 1 equivalence) in DMF (0.5 mL), followed by the addition of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetic acid 2,5-dioxopyrrolidine-1-yl ester (0.016 g, 0.064 mmol, 1.28 equivalence). The reaction mixture was concentrated, diluted with 1% TFA in water, and purified by reversed-phase HPLC to give azaBzL-20 (38.5 mg, 0.040 mmol, 80%). LC / MS [M+H] 967.50 (calculated); LC / MS [M+H] 967.80 (found).

[0808] Alternatively, azaBzL-20 can be synthesized as follows:

[0809]

[0810] 2-(triphenylmethylamino)-3H-pyrido[3,4-b]aza Preparation of ethyl 4-carboxylate, L-20h

[0811] To 2-amino-3H-pyrido[3,4-b]aza Ethyl 4-carboxylate, L-20 g (4 g, 17.3 mmol, 1 equivalent) was added to a solution in DCM (50 mL) along with Et3N (3.50 g, 34.5 mmol, 4.82 mL, 2 equivalents) and TrtCl (12.0 g, 43.2 mmol, 2.5 equivalents), and then stirred at 50 °C for 16 hours. The mixture was filtered, washed with EtOAc (50 mL), and the filtrate was concentrated. The residue was subjected to rapid silica gel chromatography (…). 80g Purification was performed using a silica gel rapid column chromatography at 50 mL / min with a 0–40% ethyl acetate / MeOH gradient elution, yielding 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 (measured).

[0812] 2-(triphenylmethylamino)-3H-pyrido[3,4-b]aza Preparation of 4-carboxylic acid, L-20i

[0813] A solution of LiOH·H₂O (1.20 g, 28.51 mmol, 3 equivalents) in H₂O (10 mL) was added to a solution of L-20h (4.5 g, 9.50 mmol, 1 equivalent) in THF (30 mL) and MeOH (15 mL), and the mixture was stirred at 60 °C for 2 hours. 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 ¹H NMR (MeOD, 400MHz) δ 7.84 (d, J = 5.2Hz, 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); LC / MS [M+H] 446.1 (measured).

[0814] N-[2-[propyl-[2-(triphenylmethylamino)-3H-pyrido[3,4-b]aza] Preparation of tert-butyl carbamate, L-20j

[0815] To a solution of L-20i (3.5 g, 7.86 mmol, 1 equivalent) in DMA (8 mL) and DCM (40 mL), methanesulfonic acid (755 mg, 7.86 mmol, 561 μL, 1 equivalent), N-[2-(propylaminooxy)ethyl]carbamate tert-butyl ester (1.71 g, 7.86 mmol, 1 equivalent) and EDCI (4.52 g, 23.57 mmol, 3 equivalent) were added, and the mixture was stirred at 0 °C for 0.5 h. The mixture was concentrated to remove DCM and adjusted to pH 10 with aqueous Na₂CO₃ solution, followed by extraction with DCM (50 mL x 3). The combined organic phases were washed with brine (50 mL), dried over Na₂SO₄, and concentrated to give L-20j (4.5 g, 6.97 mmol, 88.70% yield) as a pale yellow oil. 1 ¹H NMR (MeOD, 400MHz) 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); LC / MS [M+H] 646.2 (measured).

[0816] N-(2-aminoethoxy)-N-propyl-2-(triphenylmethylamino)-3H-pyrido[3,4-b]aza Preparation of 4-formamide, L-20k

[0817] To a solution of L-20j (2.7 g, 4.18 mmol, 1 equivalent) in EtOAc (20 mL), HCl / EtOAc (4 M, 52.26 mL, 50 equivalents) was added, and the mixture was stirred 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 (measured).

[0818] N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[Propyl-[2-(triphenylmethylamino)-3H-pyrido[3,4-b]aza Preparation of tert-butyl carbamate, L-20l, with the following structure: [-4-carbonyl]amino]oxyethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethyl]carbamate]tert-butyl]urethane]

[0819] To N-(2-aminoethoxy)-N-propyl-2-(triphenylmethylamino)-3H-pyrido[3,4-b]aza 4-Carboxamide (2 g, 3.44 mmol, 1 equivalent, HCl) was added to a solution in DMF (15 mL) along with DIEA (888 mg, 6.87 mmol, 1.20 mL, 2 equivalents) and N-[2-[2-[2-[2-[2-[2-[2-[2-(2-isocyanoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl, L-20b (2.05 g, 3.26 mmol, 0.95 equivalents), and then stirred at 0 °C for 1 hour. The mixture was adjusted to pH 5 at 0 °C with TFA and filtered. The filtrate was purified by preparative HPLC (column: Phenomenex luna C18 (250*70mm, 15um); mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 30%-60% B, for 20.0 min) to obtain L-20l (3.7g, 3.16mmol, 91.8% yield), which was a yellow oil. 1H NMR(MeOD,400MHz)δ8.13(d,J=6.0Hz,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=7.2Hz,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=5.2Hz,2H),1.88-1.75(m,2H),1.43(s,9H),1.03(t,J=7.2Hz,3H). LC / MS[M+H] 1172.6 (calculated value); LC / MS[M+H] 1172.9 (measured value).

[0820] N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N-propyl-2-(triphenylmethylamino)-3H-pyrido[3,4-b]aza Preparation of 4-formamide, L-20m

[0821] To a solution of L-20l (1.5 g, 1.28 mmol, 1 equivalent) in EtOAc (20 mL), HCl / EtOAc (4 M, 15.9 mL, 50 equivalents) was added, and the mixture was stirred at 25 °C for 0.5 h. The mixture was concentrated to give crude L-20m (1.5 g, crude product, HCl) as a pale yellow solid. 1 ¹H NMR (MeOD, 400MHz) δ 8.23 ​​(d, J = 6.0Hz, 1H), 7.95 (d, J = 6.0Hz, 1H), 7.90 (s, 1H), 7.49–7.20 (m, 16H), 4.05 (t, J = 5.2Hz, 2H), 3.87–3.76 (m, 4H), 3.74–3.56 (m, 38H), 3.44 (t, J = 5.2Hz, 2H), 3.38 (t, J = 5.2Hz, 2H), 3.18–3.16 (m, 4H), 1.89–1.78 (m, 2H), 1.04 (t, J = 7.2Hz, 3H). LC / MS [M+H] 1072.6 (calculated); LC / MS [M+H] 1072.4 (measured).

[0822] N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolo-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N-propyl-2-(triphenylmethylamino)-3H-pyrido[3,4-b]aza Preparation of 4-formamide, L-20n

[0823] To a solution of L-20m (1.5 g, 1.35 mmol, 1 equivalent, HCl) in CH3CN (30 mL), DIEA (874 mg, 6.76 mmol, 1.18 mL, 5 equivalents) and 2-(2,5-dioxopyrrolo-1-yl)acetic acid (2,5-dioxopyrrolo-1-yl) ester (307 mg, 1.22 mmol, 0.9 equivalents) were added, and the mixture was stirred at 0 °C for 0.5 h. The mixture was adjusted to pH 4 with TFA and then concentrated to remove CH3CN. 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 (measured).

[0824] Preparation of azaBzL-20

[0825] TFA (4.15 g, 36.3 mmol, 2.70 mL, 20 equivalents) was added to a solution of L-20n (2.2 g, 1.82 mmol, 1 equivalent) in DCM (20 mL), and the mixture was stirred 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%-95% B, for 25.0 min) to give azaBzL-20 as a light orange oil (858.6 mg, 887.84 μmol, 48.81% yield). 1H NMR(MeOD,400MHz,)δ8.66(s,1H),8.53(d,J=5.2Hz,1H),7.61(d,J=5.2Hz,1H),7.39(s,1H),6.89(s,2H),4.17(s,2H),3.95(t,J=5.2Hz,2 H),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=5.2Hz,2H),1.85-1.69(m,2H),1.00(t,J=7.2Hz,3H). LC / MS[M+H] 967.5 (calculated value); LC / MS[M+H] 967.5 (measured value).

[0826] Example L-28 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[6-amino-8-(dipropylcarbamoyl)-7H-pyrido[3,2-b]aza Synthesis of 2,3,5,6-tetrafluorophenyl propionic acid ester, azaBzL-28

[0827]

[0828] 6-Amino-3-bromo-N,N-dipropyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-28a

[0829] To 6-amino-3-bromo-7H-pyrido[3,2-b]aza -8-carboxylic acid, L-10h (0.6 g, 2.13 mmol, 1 equivalent) was added to a solution of HATU (889 mg, 2.34 mmol, 1.1 equivalent), N-propylpropyl-1-amine (430 mg, 4.25 mmol, 586 μL, 2 equivalent), and DIEA (824 mg, 6.38 mmol, 1.11 mL, 3 equivalent) in DMF (10 mL), and then stirred at 0 °C for 0.5 h. The mixture was diluted with ice water (50 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 20g Purification was performed using a silica gel rapid column at 60 mL / min with a gradient elution of 0–100% ethyl acetate / petroleum ether to EtOAc = 5 / 1, yielding L-28a (0.53 g, 1.45 mmol, 68.2% yield) as a yellow oil. 1 H NMR (MeOD, 400MHz) δ 8.31 (d, J = 2.0Hz, 1H), 7.71 (d, J = 2.0Hz, 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); LC / MS [M+H] 365.0 (measured).

[0830] N-[[5-[6-amino-8-(dipropylcarbamoyl)-7H-pyrido[3,2-b]aza] Preparation of --3-yl]pyrimidin-2-yl]methyl]-N-tert-butoxycarbonyl-tert-butyl carbamate, L-28b

[0831] Under a nitrogen atmosphere, tert-butyl carbamate (615 mg, 1.41 mmol, 1 equivalent) of L-28a (0.43 g, 1.18 mmol, 1 equivalent), N-tert-butoxycarbonyl-N-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)pyrimidin-2-yl]methyl]carbamate (615 mg, 1.41 mmol, 1.2 equivalent) in a mixture of dioxane (3 mL) and H2O (0.3 mL) was added to K2CO3 (325.41 mg, 2.35 mmol, 2 equivalent) and Pd(dppf)Cl2 (86.1 mg, 117.7 μmol, 0.1 equivalent), and then stirred at 100 °C under a nitrogen atmosphere for 2 hours. The mixture was filtered and concentrated to obtain the residue. The residue was subjected to rapid silica gel chromatography (…). 20g Purification was performed using a silica gel rapid column at 75 mL / min with a gradient elution of 0–100% ethyl acetate / petroleum ether to EtOAc / MeOH = 5 / 1, yielding L-28b (0.6 g, 1.01 mmol, 85.8% yield) as a pale yellow solid. 1¹H NMR (MeOD, 400MHz) δ 9.11 (s, 2H), 8.63 (d, J = 2.0Hz, 1H), 7.85 (d, J = 2.0Hz, 1H), 6.94 (s, 1H), 5.07 (s, 2H), 3.45 (t, J = 7.2Hz, 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); LC / MS [M+H] 594.4 (measured).

[0832] 6-Amino-3-[2-(aminomethyl)pyrimidin-5-yl]-N,N-dipropyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-28c

[0833] To a solution of L-28b (100 mg, 168 μmol, 1 equivalent) in EtOAc (2 mL), HCl / EtOAc (4 M, 2.11 mL, 50 equivalents) was added, and the mixture was stirred at 25 °C for 0.5 h. The mixture was concentrated to L-28c (70 mg, 162 μmol, 96.6% yield, HCl) as a grayish-white solid. 1 ¹H NMR (MeOD, 400MHz) δ 9.30 (s, 2H), 9.06 (d, J = 2.0Hz, 1H), 8.26 (d, J = 2.0Hz, 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); LC / MS [M+H] 394.1 (measured).

[0834] Preparation of azaBzL-28

[0835] A mixture of L-28c (50 mg, 116 μmol, 1 equivalent, HCl) and DIEA (45.0 mg, 348 μmol, 60.7 μL, 3 equivalents) in DMF (1 mL) was added to a solution of 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]propionic acid (2,3,5,6-tetrafluorophenyl) ester (119 mg, 139 μmol, 1.2 equivalents) in DMF (1 mL), and then stirred at 0 °C for 0.5 h. The mixture was adjusted to pH 5 with TFA and filtered. The filtrate was purified by preparative HPLC (column: Phenomenex Luna C1875*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 25%-45% B, for 8.0 min) to obtain azaBzL-28 (64.5 mg, 49.2 μmol, 42.3% yield, 2 TFA), which was a light yellow oil. 1 HNMR(MeOD,400MHz)δ9.17(s,2H),9.04(d,J=2.0Hz,1H),8.15(d,J=2.0Hz,1H),7.48-7.40(m,1H),7.11(s,1H),4.72(s,2H),3.88(t,J=6.0Hz,2H) ,3.82(t,J=6.0Hz,2H),3.71-3.59(m,36H),3.58-3.43(m,6H),2.99(t,J =6.0Hz,2H),2.62(t,J=6.0Hz,2H),1.82-1.67(m,4H),1.00-0.97(m,6H). LC / MS[M+H] 1082.5 (calculated value); LC / MS[M+H] 1082.4 (measured value).

[0836] Example L-32 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[3-[[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]aza Synthesis of 2,3,5,6-tetrafluorophenyl propionic acid ester, azaBzL-32, [-3-carbonyl]amino]-7,8-dihydro-5H-1,6-naphthid-6-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ester]propionic acid ester), azaBzL-32]

[0837]

[0838]

[0839] 3-Bromo-N-ethoxy-N-propyl-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza -8-Formamide and 3-bromo-6-(bis(triphenylmethylamino)-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-32a

[0840] At 25°C under N2, the 6-amino-3-bromo-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza 8-Carboxamide, L-10i (25.0 g, 68.1 mmol, 1 equivalent), and TEA (20.7 g, 204 mmol, 28.4 mL, 3 equivalents) were added to a solution in DCM (500 mL) with triphenylchloromethane and TrtCl (28.5 g, 102 mmol, 1.5 equivalents), and then heated to 50 °C and stirred for 12 hours. The reaction mixture was quenched by adding H2O (300 mL) at 0 °C and then extracted with DCM (150 mL x 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, 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-(bis(triphenylmethylamino))-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza as a yellow solid. 8-Formamide, L-32b (8.8 g, 10.3 mmol, 15.1% yield). LC / MS [M+H] 609.2 (calculated); LC / MS [M+H] 609.1 (analytical). LC / MS [M+H] 851.3 (calculated); LC / MS [M+H] 851.3 (analytical).

[0841] 8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of methyl 3-carboxylate, L-32c

[0842] A mixture of L-32a (5.60 g, 9.19 mmol, 1 equivalent), Pd(dppf)Cl2 (672 mg, 918 μmol, 0.1 equivalent), and TEA (2.79 g, 27.6 mmol, 3.84 mL, 3 equivalent) in MeOH (50 mL) was degassed and purged three times with CO, then heated to 80 °C and stirred for 16 hours under a CO atmosphere (50 psi). The reaction mixture was cooled to 25 °C and filtered, and the filter cake was dried under reduced pressure as a pure product. The filtrate was 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-32c (3.50 g, 5.95 mmol, 64.7% yield) as a brown solid. 1 ¹H NMR (CDCl₃, 400MHz) 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 (measured).

[0843] Methyl 8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of 3-carboxylic acid, L-32d

[0844] A solution of LiOH·H₂O (588 mg, 14.0 mmol, 3 equivalents) in H₂O (10 mL) was added to a solution of L-32c (2.75 g, 4.67 mmol, 1 equivalent) in THF (30 mL) at 0 °C, and then the mixture was heated to 25 °C and stirred for 2 hours. The reaction mixture was cooled to 0 °C and diluted with H₂O (30 mL), and the pH was adjusted to 5 with 2N HCl at 0 °C. The mixture was then 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 product) as a pale yellow solid. 1¹H NMR (DMSO-d⁶, 400MHz) 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 = 7.2Hz, 2H), 3.70 (t, J = 6.8Hz, 2H), 3.03 (s, 2H), 1.77–1.64 (m, 2H), 1.14 (t, J = 6.8Hz, 3H), 0.94 (t, J = 7.2Hz, 3H). LC / MS [M+H] 575.3 (calculated); LC / MS [M+H] 575.3 (measured).

[0845] 3-[[8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of tert-butyl 3-[3-carbonyl]amino]-7,8-dihydro-5H-1,6-naphthyl-6-carboxylic acid, L-32e

[0846] N-methylimidazole, NMI (1.36 g, 16.5 mmol, 1 equivalent) and tert-butyl 3-amino-7,8-dihydro-5H-1,6-naphthyl-6-carboxylic acid (618 mg, 2.48 mmol, 1.5 equivalent) were added to a solution of L-32d (0.95 g, 1.65 mmol, 1 equivalent) in acetonitrile, ACN (10 mL), followed by TCFH (1.86 g, 6.61 mmol, 4 equivalent). The mixture was stirred at 25 °C for 2 h, quenched by adding H2O (20 mL) at 0 °C, and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 20g Purification was performed using a silica gel rapid column chromatography at 80 mL / min with a 70% ethyl acetate / petroleum ether gradient elution to obtain L-32e (2.3 g, crude product) as a yellow oil. LC / MS [M+H] 806.4 (calculated value); LC / MS [M+H] 806.3 (measured value).

[0847] N8-ethoxy-N8-propyl-N3-(5,6,7,8-tetrahydro-1,6-naphthidin-3-yl)-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of 3,8-dicarboxamide, L-32f

[0848] HCl / EtOAc (4M, 10.7mL, 15 equivalents) was added to a solution of L-32e (2.30 g, 2.85 mmol, 1 equivalent) in EtOAc (20 mL), and the mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (column: Welch Xtimate C18 250*70mm*10µm; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 25%-55% B, for 20.0 min) to give L-32e (1 g, 1.42 mmol, 49.7% yield) as a yellow solid. 1 H NMR (MeOD, 400MHz) δ8.81(s,1H),8.71(d,J=2.0Hz,1H),8.23(d,J=2.0Hz,1H),7.66(d,J=2.0Hz,1H),7.45-7.22(m,16H),4.48(s,2H),4.05(q,J =7.2Hz,2H),3.81(t,J=6.8Hz,2H),3.65(t,J=6.4Hz,2H),3.23(t,J=6.4 Hz, 4H), 1.88-1.77 (m, 2H), 1.26 (t, J = 7.2Hz, 3H), 1.04 (t, J = 7.2Hz, 3H). LC / MS[M+H] 706.3 (calculated value); LC / MS[M+H] 706.4 (measured value).

[0849] 6-Amino-N8-ethoxy-N8-propyl-N3-(5,6,7,8-tetrahydro-1,6-naphthidin-3-yl)-7H-pyrido[3,2-b]aza Preparation of 3,8-dicarboxamide, L-32g

[0850] TFA (969 mg, 8.50 mmol, 631 μL, 20 equivalents) was added to a solution of L-32f (0.30 g, 425 μmol, 1 equivalent) in DCM (3 mL), and the mixture was then heated to 50 °C and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 1%-30% B, for 8.0 min) to give L-32g (0.18 g, 388 μmol, 91.4% yield) as a white solid. 1¹H NMR (MeOD, 400MHz) 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 = 7.2Hz, 2H), 3.77 (t, J = 7.2Hz, 2H), 3.66 (t, J = 6.4Hz, 2H), 3.51 (s, 2H), 3.23 (t, J = 6.4Hz, 2H), 1.84–1.74 (m, 2H), 1.21 (t, J = 7.2Hz, 3H), 1.01 (t, J = 7.2Hz, 3H). LC / MS [M+H] 464.2 (calculated); LC / MS [M+H] 464.1 (measured).

[0851] Preparation of azaBzL-32

[0852] Add dropwise a solution of DIEA (22.3 mg, 173 μmol, 30.1 μL, 1 equivalent) and L-32 g (0.08 g, 173 μmol, 1 equivalent) in DMF (2 mL) to a solution of 3-[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]propionic acid (2,3,5,6-tetrafluorophenyl) ester (295 mg, 345 μmol, 2 equivalents) in DMF (2 mL), and then stir at 0 °C for 1 hour. The reaction mixture was adjusted to pH 6 with TFA and then purified by preparative HPLC (column: Phenomenex LunaC18 75*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 15%-45%B, for 8.0 min) to obtain a colorless oily substance azaBzL-32 (0.04 g, 34.2 μmol, 19.8% yield, 98.5% purity). 1H NMR(MeOD,400MHz)δ9.20(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=6.0Hz,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 (measured value).

[0853] Example L-37 6-Amino-3-[3-[3-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrole-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionylamino]methyl]azacyclobutane-1-yl]sulfonylphenyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Synthesis of 8-formamide, azaBzL-37

[0854]

[0855]

[0856] Preparation of N-[[1-(3-bromophenyl)sulfonylazonylbutane-3-yl]methyl]tert-butyl carbamate, L-37a

[0857] Triethylamine, Et3N (9.50 g, 93.9 mmol, 13.1 mL, 2 equivalents), and 3-bromobenzenesulfonyl chloride (12.0 g, 46.9 mmol, 6.77 mL, 1 equivalent) were added to a solution of N-(azacyclobutane-3-ylmethyl)carbamate tert-butyl hydrochloride (10.5 g, 46.9 mmol, 1 equivalent) in DCM (25 mL) at 0 °C, and the mixture was stirred at 25 °C for 1 hour. The residue was poured into water (15 mL). The aqueous phase was extracted with DCM (20 mL x 3). The combined organic phases were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was ground 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 value); LC / MS [M+Na] 427.0 (measured value).

[0858] Preparation of N-[[1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl]sulfonylazonobutan-3-yl]methyl]tert-butyl carbamate, L-37b]

[0859] A mixture of L-37a (3.00 g, 7.40 mmol, 1 equivalent), KOAc (1.45 g, 14.8 mmol, 2 equivalents), Pin2B2 (2.44 g, 9.62 mmol, 1.3 equivalents), and Pd(dppf)Cl2 (270 mg, 370 μmol, 0.05 equivalents) in dioxane (30 mL) was degassed at 25 °C and purged three times with N2. The mixture was then heated to 95 °C and stirred under N2 atmosphere for 2 hours. The reaction mixture was concentrated under vacuum. The residue was analyzed by rapid silica gel chromatography (…). 40g Purification was performed using a silica gel rapid column at 80 mL / min with a 0–35% ethyl acetate / petroleum ether gradient elution to give L-37b (3.30 g, 7.29 mmol, 98.6% yield) as a yellow oil. 1¹H NMR (CDCl₃, 400MHz) δ 8.26 (s, 1H), 8.06 (d, J = 7.2Hz, 1H), 7.91 (d, J = 8.0Hz, 1H), 7.57 (t, J = 8.0Hz, 1H), 3.82 (t, J = 8.0Hz 2H), 3.55–3.46 (m, 2H), 3.16 (t, J = 6.0Hz, 2H), 2.65–2.55 (m, 1H), 1.36 (s, 9H), 1.27 (s, 12H). LC / MS [M+Na] 475.2 (calculated); LC / MS [M+Na] 475.1 (measured).

[0860] N-[[1-[3-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]aza] Preparation of --3-yl]phenyl]sulfonylazacyclobutane-3-yl]methyl]carbamate tert-butyl ester, L-37c

[0861] At 25°C, K2CO3 (147 mg, 1.06 mmol, 2 equivalents) and 6-amino-3-bromo-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azapyridine were added to a solution of L-37c (312 mg, 689 μmol, 1.3 equivalents) in dioxane (5.00 mL) and H2O (0.5 mL). -8-formamide, L-10i (195 mg, 531 μmol, 1 equivalent) and Pd(dppf)Cl2 (19.4 mg, 26.5 μmol, 0.05 equivalent). The solution was degassed and purged three times with N2, then heated to 95 °C and stirred for 2 hours under N2 atmosphere. The reaction mixture was cooled to 25 °C, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was subjected to rapid silica gel chromatography (…). 4g Purification was performed using a silica gel rapid column at 60 mL / min with a 0–40% ethyl acetate / petroleum ether gradient elution to give L-37c (300 mg, 489 μmol, 92.3% yield), which was a brown oil. 1H NMR(CDCl3,400MHz)δ8.78(s,1H),8.12(s,1H),7.90-7.97(m,3H),7.65-7.77(m,2H),3.98(q,J=7.2Hz,2H),3.92-3.84(m,2H),3.75(t,J=7.2Hz, 2H),3.62-3.55(m,2H),3.21-3.11(m,2H),3.10(s,2H),2.72-2.59(m,1H ),1.78-1.75(m,2H),1.39(s,9H),1.31-1.28(m,3H),0.99(t,J=7.6,3H). LC / MS[M+H] 613.3 (calculated value); LC / MS[M+H] 613.2 (measured value).

[0862] 6-Amino-3-[3-[3-(aminomethyl)azacyclobutane-1-yl]sulfonylphenyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-37d

[0863] TFA (465 mg, 4.08 mmol, 1 equivalent) was added to a solution of L-37c (0.25 g, 408 μmol, 1 equivalent) in DCM (1 mL), and the mixture was then heated to 50 °C and stirred for 1 hour. The reaction mixture was cooled to 25 °C and concentrated under vacuum. The residue was purified by preparative HPLC (column: Phenomenex luna C18 250*50 mm*15 μm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 5%-30% B, for 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 (measured).

[0864] Preparation of azaBzL-37

[0865] At 0 °C, DIEA (37.8 mg, 293 μmol, 50.9 μL, 3 equivalents) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrole-1-yl)acetyl]amino]ethoxy ... The filtrate was purified by preparative HPLC (column: Phenomenex luna C18 100*40mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 10%-45%B, for 8.0 min) to obtain azaBzL-37 (33.0 mg, 28.4 μmol, 29.1% yield) as a colorless oil. 1 HNMR(MeOD,400MHz)δ9.05(d,J=2.0Hz,1H),8.19-8.16(m,3H),7.98(d,J=8.0Hz,1H),7.89(t,J=8.0H z,1H),7.48(s,1H),6.89(s,2H),4.16(s,2H),4.05-3.99(m,2H),3.89(t,J=8.0Hz,2H),3.79-3.75(m, 2H), 3.65-3.59(m,38H), 3.58-3.56(m,2H), 3.55-3.53(m,4H), 3.36(s,2H), 3.15(d,J=6.4Hz,2H), 2.73-2.64(m,1H), 2.29(t,J=6.0Hz,2H), 1.84-1.75(m,2H), 1.23(t,J=7.2Hz,3H), 1.01(t,J=7.2Hz,3H). LC / MS[M+H] 1161.5 (calculated value); LC / MS[M+H] 1161.7 (measured value).

[0866] Example L-38 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[3-[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]aza Synthesis of 2,3,5,6-tetrafluorophenyl propionic acid ester, azaBzL-38

[0867]

[0868] At 0 °C, DIEA (52.9 mg, 409 μmol, 71.3 μL, 3 equivalents) and 6-amino-3-[3-[3-(aminomethyl)azacyclobutane-1-yl]sulfonylphenyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azapropionic acid (2,3,5,6-tetrafluorophenyl) ester (140 mg, 164 μmol, 1.2 equivalents) were added to a solution of 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]propionic acid (2,3,5,6-tetrafluorophenyl) ester (140 mg, 164 μmol, 1.2 equivalents) in DMF (1 mL) were added. -8-Formamide, L-37d (70 mg, 136 μmol, 1 equivalent), was prepared and stirred at 25 °C for 1 hour. The reaction mixture was acidified to pH 6 with TFA and filtered. The filtrate was purified by preparative HPLC (column: Phenomenex Luna C18 80*30 mm*3 μm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 30%-60% B, for 8.0 min) to give azaBzL-38 (30.0 mg, 24.9 μmol, 18.3% yield) as a colorless oil. 1H NMR(MeOD,400MHz)δ9.04(d,J=2.0Hz,1H),8.18-8.16(m,3H),7.98(d,J=8.0Hz,1H),7.89(t,J=8.0Hz,1H),7 .48(s,1H),7.44-7.37(m,1H),4.01(q,J=7.2Hz,2H),3.91-3.87(m,2H),3.87-3.84(m,2H),3.77(t,J=7.2Hz ,2H), 3.64-3.59(m,36H), 3.57-3.55(m,2H), 3.54-3.51(m,4H), 3.14(d,J=6.4Hz,2H), 2.97(t,J=6.00Hz,2H), 2.74-2.64(m,1H), 2.28(t,J=6.00Hz,2H), 1.82-1.75(m,2H), 1.22(t,J=7.2Hz,3H), 1.02(t,J=7.2Hz,3H). LC / MS[M+H]1201.5 (calculated value); LC / MS[M+H]1201.4 (measured value).

[0869] Example L-39 6-Amino-3-[(3S)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrole-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionylamino]piperidin-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Synthesis of 8-formamide, azaBzL-39

[0870]

[0871] N-[(3S)-1-[8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of tert-butyl carbamate [-3-carbonyl]-3-piperidinyl]carbamate, L-39a

[0872] To 8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza NMI (171 mg, 2.09 mmol, 167 μL, 4 equivalents) and N,N,N,N′,N′-tetramethylformamidinium hexafluorophosphate, TCFH, CAS Registry No. 207915-99-9 (219 mg, 783 μmol, 1.5 equivalents) were added to a mixture of 3-carboxylic acid, L-32d (300 mg, 522 μmol, 1 equivalent) and N,N,N′,N′-tetramethylformamidinium hexafluorophosphate, TCFH, CAS Registry No. 207915-99-9 in MeCN (8 mL), and then stirred at 25 °C for 1 hour. Alternatively, HATU can be used as a coupling agent. The reaction mixture was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with brine (8 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was subjected to rapid silica gel chromatography ( 12g Purification was performed using a silica gel rapid column at 80 mL / min with a 0–45% ethyl acetate / petroleum ether gradient eluent, yielding 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 (measured).

[0873] 6-Amino-3-[(3S)-3-aminopiperidin-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Preparation of 8-carboxamide, L-39b

[0874] TFA (1.58 g, 13.8 mmol, 1.03 mL, 30 equivalents) was added to a solution of L-39a (350 mg, 462 μmol, 1 equivalent) in DCM (7 mL), and the mixture was heated to 50 °C and stirred for 2 hours. The reaction mixture was concentrated under vacuum. 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 (found).

[0875] Preparation of azaBzL-39

[0876] At 0°C, DIEA (56.1 mg, 434 μmol, 75.6 μL, 3 equivalents) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrole-1-yl)acetyl]amino]ethoxy ... The pH of the reaction solution was adjusted to approximately 9 by adding TFA, and purified by preparative HPLC (column: Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 5%-45%B, for 8.0 min) to obtain azaBzL-39 (35.0 mg, 32.9 μmol, 22.7% yield) as a colorless oil. 1 HNMR(DMSO-d6,400MHz)δ9.28(s,1H),8.64(s,1H),8.21(t,J=5.2Hz,1H),7.93(d,J=7.2Hz,1H),7.79(s,1H ),7.21(s,1H),7.08(s,2H),4.01(s,2H),3.90(q,J=7.2Hz,2H),3.64(t,J=7.2Hz,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=7.2Hz,3H), 0.91(t,J=7.2Hz,3H). LC / MS[M+H] 1063.5 (calculated value); LC / MS[M+H] 1063.4 (measured value).

[0877] Example L-416-amino-3-[(3R)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrole-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionylamino]piperidin-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Synthesis of 8-formamide, azaBzL-41

[0878]

[0879] N-[(3R)-1-[8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of tert-butyl carbamate [-3-carbonyl]-3-piperidinyl]carbamate, L-41a

[0880] At 0 °C, 8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza L-32d (300 mg, 522 μmol, 1 equivalent) of 3-carboxylic acid was added to a solution in DMF (5.00 mL) with HATU (297 mg, 783 μmol, 1.5 equivalent), DIEA (134 mg, 1.04 mmol, 181 μL, 2 equivalent), and N-[(3R)-3-piperidinyl]carbamate tert-butyl ester (125 mg, 626 μmol, 1.2 equivalent). The mixture was then heated to 25 °C and stirred for 1 hour. 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 Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was subjected to rapid silica gel chromatography (…). 12g Purification was performed using a silica gel rapid column at 60 mL / min with a 0–100% ethyl acetate / petroleum ether gradient elution to obtain L-41a (443 mg, crude product) as a yellow oil. LC / MS [M+H] 757.4 (calculated value); LC / MS [M+H] 757.2 (measured value).

[0881] 6-Amino-3-[(3R)-3-aminopiperidin-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-41b

[0882] TFA (1.33 g, 11.7 mmol, 869 μL, 20 equivalents) was added to a solution of L-41a (443 mg, 585 μmol, 1 equivalent) in DCM (5 mL) at 25 °C, and then the mixture was heated to 50 °C and stirred for 12 hours. After this, the reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was ground together 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 product) as a reddish-brown solid. 1¹H NMR (MeOD, 400MHz) δ 8.7 (s, 1H), 7.90 (d, J = 1.6Hz, 1H), 7.41 (s, 1H), 3.98 (q, J = 7.2Hz, 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 = 7.2Hz, 3H), 1.01 (t, J = 7.2Hz, 3H). LC / MS [M+H] 415.2 (calculated); LC / MS [M+H] 415.2 (measured).

[0883] Preparation of azaBzL-41

[0884] At 0 °C, DIEA (35.2 mg, 272 μmol, 47.4 μL, 3 equivalents) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrole-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 C18100*40mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 5%-40%B, for 8.0 min) to obtain azaBzL-41 (20 mg, 18.8 μmol, 20.7% yield) as a colorless oil. 1H NMR(DMSO-d6,400MHz)δ8.52(s,1H),7.96-7.90(m,1H),7.68(d,J=1.0Hz,1H),7.63(d,J=7.2Hz,1H),7.19( s,1H),7.01(s,2H),4.03(s,2H),3.92(q,J=7.0Hz,2H),3.75-3.71(m,1H),3.66(t,J=7.0Hz,3H),3.62-3.5 7 (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 (measured value).

[0885] Example L-42 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolo-1-yl)acetyl]amino]ethoxy ... Synthesis of [-3-carbonyl]azacyclobutane-3-yl]methyl]carbamate, azaBzL-42

[0886]

[0887]

[0888] N-[[1-[8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of L-42a of [-3-carbonyl]azacyclobutane-3-yl]methyl]carbamate tert-butyl ester

[0889] To 8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza 3-carboxylic acid, L-32d (450 mg, 783 μmol, 1 equivalent), HATU (298 mg, 783 μmol, 1 equivalent), and DIEA (304 mg, 2.35 mmol, 409 μL, 3 equivalent) in a mixture in DMF (4 mL) were mixed with N-(azacyclobutane-3-ylmethyl)carbamate tert-butyl ester (190 mg, 1.02 mmol, 1.3 equivalent) and stirred at 25 °C for 1 hour. The reaction mixture was diluted with H2O (30 mL) and then extracted with EtOAc (10 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was subjected to rapid silica gel chromatography (…). 12g Purification was performed using a silica gel rapid column at 80 mL / min with a 0–100% ethyl acetate / petroleum ether gradient elution to give L-42a (463 mg, 623 μmol, 79.6% yield) as a pale yellow solid. 1 H NMR (MeOD, 400MHz,) δ8.35 (d, J=2.0Hz, 1H), 7.36-7.32 (m, 6H), 7.29-7.16 (m, 10H), 7.03 (d, J= 2.0Hz,1H),4.32(t,J=8.8Hz,1H),4.18(t,J=9.2Hz,1H),4.01(q,J=7.2Hz,2H),3.98-3.93(m,1 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 (measured value).

[0890] 6-Amino-3-[3-(aminomethyl)azacyclobutane-1-carbonyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-42b

[0891] TFA (1.11 g, 9.69 mmol, 720 μL, 20 equivalents) was added to a solution of L-42a (360 mg, 485 μmol, 1 equivalent) in DCM (2 mL), and the mixture was stirred 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: [H2O (0.1% TFA)-ACN]; gradient: 5%-35% B, for 8.0 min) to give L-42b (180 mg, 350 μmol, 72.2% yield, TFA) as a pale yellow solid. 1 H NMR(MeOD,400MHz,)δ8.85(d,J=1.6Hz,1H),8.07(d,J=1.6Hz,1H),7.40(s, 1H),4.62(t,J=8.8Hz,1H),4.39(t,J=9.6Hz,1H),4.31-4.24(m,1H),4.05- 3.94(m,3H),3.76(t,J=7.2Hz,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=7.2Hz,3H),1.01(t,J=7.2Hz,3H). LC / MS[M+H]401.2 (calculated value); LC / MS[M+H]401.2 (measured value).

[0892] Preparation of azaBzL-42

[0893] At 0°C under N2 下To a solution of L-42b (45 mg, 87.5 μmol, 1 equivalent, TFA) in DMF (1 mL), DIEA (33.9 mg, 262 μmol, 45.7 μL, 3 equivalents) and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrole-1-yl)acetyl]amino]ethoxy ... The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 20%-40% B, for 8.0 min) to obtain azaBzL-42 (53.0 mg, 44.9 μmol, 51.4% yield, TFA), which was a pale yellow oil. 1 H NMR(MeOD,400MHz,)δ8.87(d,J=1.6Hz,1H),8.05(s,1H),7.42(s,1H),6.89(s,2H),4. 52(t,J=8.8Hz,1H),4.28(t,J=9.6Hz,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 (measured value).

[0894] Example L-51 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[[6-amino-8-[ethoxy(propyl)carbamoyl]-7H-pyrido[3,2-b]aza Synthesis of 2,3,5,6-tetrafluorophenyl propionic acid ester, azaBzL-51

[0895]

[0896]

[0897] N3-(5-bromo-3-pyridyl)-N8-ethoxy-N8-propyl-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of 3,8-dicarboxamide, L-51a

[0898] At 0 °C, 8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza 3-carboxylic acid, L-32d (1.00 g, 1.74 mmol, 1 equivalent), 5-bromopyridin-3-amine (452 ​​mg, 2.61 mmol, 1.5 equivalent), and NMI (429 mg, 5.22 mmol, 416 μL, 3 equivalent) were added to a solution of 3-carboxylic acid, L-32d (1.00 g, 1.74 mmol, 1 equivalent) in CH3CN (10 mL), and then the mixture was heated to 20 °C and stirred at 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (30 mL) and extracted with EtOAc (30 mL x 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 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 (analytical).

[0899] N-[5-[8-[ethoxy(propyl)carbamoyl]-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of L-51b: 3-carbonyl]amino]-3-pyridyl]methyl]tert-butyl carbamate

[0900] A mixture of L-51a (1.00 g, 1.37 mmol, 1 equivalent), potassium (tert-butoxycarbonylamino)methyltrifluoroborate (357 mg, 1.51 mmol, 1.1 equivalent), [2-(2-aminophenyl)phenyl]-chloro-palladium; bis(1-adamantyl)-butylphosphine (91.6 mg, 137 μmol, 0.1 equivalent) and Cs₂CO₃ (893 mg, 2.74 mmol, 2 equivalent) in dioxane (10 mL) and H₂O (2 mL) was degassed and purged three times with N₂, and then stirred at 110 °C under N₂ 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(CDCl3,400MHz)δ8.73(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=5.6Hz,2H),3.98(q,J=7.2Hz,2H),3.77(t,J=7.2Hz,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=7.2Hz,3H). LC / MS[M+H] 780.4 (calculated value); LC / MS[M+H] 780.3 (measured value).

[0901] 6-Amino-N3-[5-(aminomethyl)-3-pyridyl]-N8-ethoxy-N8-propyl-7H-pyrido[3,2-b]aza Preparation of 3,8-dicarboxamide, L-51c

[0902] TFA (2.63 g, 23.1 mmol, 1.71 mL, 30 equivalents) was added to a solution of L-51b (0.6 g, 769 μmol, 1 equivalent) in DCM (10 mL) at 25 °C under N2, and then heated to 50 °C and stirred at 50 °C for 2 hours. The reaction mixture was cooled to 25 °C, filtered, and concentrated under reduced pressure to give a residue. The residue was ground together 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. 1¹H NMR (MeOD, 400MHz) δ 9.17 (d, J = 1.2Hz, 1H), 8.91 (d, J = 1.2Hz, 1H), 8.59 (s, 1H), 8.48 (s, 1H), 8.39 (d, J = 1.2Hz, 1H), 7.45 (s, 1H), 4.26 (s, 2H), 4.00 (q, J = 7.2Hz, 2H), 3.77 (t, J = 7.2Hz, 2H), 3.52 (s, 2H), 1.86–1.72 (m, 2H), 1.21 (t, J = 7.2Hz, 3H), 1.02 (t, J = 7.2Hz, 3H). LC / MS [M+H] 438.2 (calculated); LC / MS [M+H] 438.2 (measured).

[0903] Preparation of azaBzL-51

[0904] DIEA (66.3 mg, 513 μmol, 89.4 μL, 4 equivalents) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]eth ... The reaction mixture was adjusted to pH 5-6 with TFA at 0℃ and purified by preparative HPLC (column: Phenomenex LunaC18 75*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 20%-50%B, for 8.0 min) to obtain azaBzL-51 (70.7 mg, 47.8 μmol, 37.2% yield, 91.5% purity, 2 TFA) as a yellow oil. 1HNMR(MeOD,400MHz)δ9.21(d,J=2.0Hz,1H),9.18(d,J=2.0Hz,1H),8.51(s,1H),8.47(s,1 H),8.38(d,J=2.0Hz,1H),7.50-7.37(m,2H),4.62-4.53(m,2H),4.00(q,J=6.8Hz,2H),3.8 5 (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 (measured value).

[0905] Example L-52 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[3-[[6-amino-8-(dipropylcarbamoyl)-7H-pyrido[3,2-b]aza Synthesis of 2,3,5,6-tetrafluorophenyl propionic acid ester, azaBzL-52, [-3-carbonyl]amino]-7,8-dihydro-5H-1,6-naphthid-6-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid ester, 2,3,5,6-tetrafluorophenyl) ester, azaBzL-52

[0906]

[0907] 3-Bromo-N,N-dipropyl-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of 8-formamide, L-52a

[0908] At 25°C, 6-amino-3-bromo-N,N-dipropyl-7H-pyrido[3,2-b]aza 8-Formamide, L-28a (10.0 g, 27.4 mmol, 1 equivalent), and TEA (11.1 g, 110 mmol, 15.2 mL, 4 equivalents) were added to a solution in DCM (200 mL) with TrtCl (19.1 g, 68.4 mmol, 2.5 equivalents), and the mixture was then heated to 50 °C and stirred at 50 °C for 12 hours. The reaction mixture was quenched by adding H2O (300 mL) at 0 °C and extracted with DCM (150 mL x 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give L-52a (6.00 g, 9.88 mmol, 36.1% yield) as a yellow solid and 3-bromo-6-(bis(triphenylmethylamino))-N,N-dipropyl-7H-pyrido[3,2-b]aza -8-Formamide L-52b (2.5 g, 2.94 mmol, 10.7% yield). LC / MS [M+H] 607.2 (calculated); LC / MS [M+H] 607.2 (analytical). LC / MS [M+H] 849.3 (calculated); LC / MS [M+H] 849.3 (analytical).

[0909] 8-(dipropylcarbamoyl)-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of methyl 3-carboxylate, L-52c

[0910] A mixture of L-52a (4.60 g, 7.57 mmol, 1 equivalent), Pd(dppf)Cl2 (554 mg, 757 μmol, 0.1 equivalent), and TEA (2.30 g, 22.7 mmol, 3.16 mL, 3 equivalent) in MeOH (50 mL) was degassed and purged three times with CO, then heated to 80 °C and stirred at 80 °C under a CO atmosphere (50 psi) for 16 hours. The reaction mixture was 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-52c (3.50 g, 5.97 mmol, 78.8% yield) as a brown solid. 1¹H NMR (CDCl₃, 400MHz) δ 8.75 (d, J = 2.0Hz, 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); LC / MS [M+H] 587.3 (measured).

[0911] 8-(dipropylcarbamoyl)-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of 3-carboxylic acid, L-52d

[0912] A solution of LiOH·H₂O (1.07 g, 25.6 mmol, 6 equivalents) in H₂O (10 mL) was added to a solution of L-52c (2.50 g, 4.26 mmol, 1 equivalent) in MeOH (30 mL) at 25 °C, and then heated to 80 °C and stirred at 80 °C for 20 hours. The reaction mixture was filtered. The filtrate was cooled to 0 °C and diluted with H₂O (30 mL), adjusted to pH 5 with 2N 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 ¹H NMR (DMSO-d⁶, 400MHz) 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); LC / MS [M+H] 573.3 (measured).

[0913] 3-[[8-(dipropylcarbamoyl)-6-(triphenylmethylamino)-7H-pyrido[3,2-b]aza Preparation of tert-butyl 3-[carbonyl]amino]-7,8-dihydro-5H-1,6-naphthyl-6-carboxylic acid, L-52e

[0914] NMI (222 mg, 2.71 mmol, 215 μL, 5 equivalents) and TCFH (303 mg, 1.08 mmol, 2 equivalents) were added to a solution of L-52d (310 mg, 541 μmol, 1 equivalent) and 3-amino-7,8-dihydro-5H-1,6-naphthyl-6-carboxylic acid tert-butyl ester (148 mg, 595 μmol, 1.1 equivalents) in MeCN (5 mL), and the mixture was stirred 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 x 3). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was subjected to rapid silica gel chromatography (…). 12g Purification was performed using a silica gel rapid column chromatography at 40 mL / min with a 0–100% ethyl acetate / petroleum ether gradient elution, yielding L-52e as a yellow solid (0.38 g, 472 μmol, 87.3% yield). LC / MS [M+H] 804.4 (calculated); LC / MS [M+H] 804.5 (measured).

[0915] 6-Amino-N8,N8-dipropyl-N3-(5,6,7,8-tetrahydro-1,6-naphthid-3-yl)-7H-pyrido[3,2-b]aza Preparation of 3,8-dicarboxamide, L-52f

[0916] TFA (1.35 g, 11.8 mmol, 877 μL, 25 equivalents) was added to a solution of L-52e (0.38 g, 472 μmol, 1 equivalent) in DCM (10 mL), and the mixture was stirred at 50 °C for 15 hours. 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 (measured).

[0917] Preparation of azaBzL-52

[0918] At 0°C, a solution of DIPEA (64.3 mg, 498 μmol, 86.7 μL, 4 equivalents) and L-52f (0.1 g, 124 μmol, 1 equivalent, 3 TFA) in DMF (1 mL) was added to a solution of 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]propionic acid (2,3,5,6-tetrafluorophenyl) ester (266 mg, 311 μmol, 2.5 equivalents) in DMF (1 mL), and then stirred 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 C1875*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 20%-50%B, for 8.0 min) to obtain azaBzL-52 (35.4 mg, 25.7 μmol, 20.6% yield, 2 TFA) as a yellow oil. 1 H NMR(MeOD,400MHz)δ9.21-9.19(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 0.66-3.56 (m, 38H), 3.50-3.45 (m, 4H), 3.40-3.36 (m, 2H), 3.19 (t, J = 5.6Hz, 1H), 3.06 (t, J = 5.6Hz, 1H), 2.97 (t, J = 6.0Hz, 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 (measured value).

[0919] Example L-53 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[[6-amino-8-(dipropylcarbamoyl)-7H-pyrido[3,2-b]aza Synthesis of 2,3,5,6-tetrafluorophenyl propionic acid ester, azaBzL-53

[0920] N3-(5-bromo-3-pyridyl)-N8,N8-dipr...

Claims

1. A device comprising covalently attached to one or more aza-benzodiazepines via a connector Partial antibody-formula I immunoconjugates: Ab-[L-D] p I Or its pharmaceutically acceptable salt, wherein: Ab is an antibody; p is an integer from 1 to 8; L stands for connector; D is an aza-benzozaza with the following formula part: Z 1 Selected from CR 1 and N; Z 2 Selected from CR 2 and N; Z 3 Selected from CR 3 and N; Z 4 Selected from CR 4 and N; Z 1 Z 2 Z 3 and Z 4 One or both of them are N; R 1 R 2 R 3 R 4 ,R 5 and R 6 Independently select from the following groups: 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 ynyl, C3-C 12 carbonyl group, C6-C 20 Aryl, C2-C9 heterocyclic and C1-C 20 Heteroaryl groups, each of which is independently and optionally substituted by one or more groups selected from: -(C1-C 12 alkyldiyl)-N(R 7 )-*; -(C1-C 12 alkyldiyl)-N(R 7 )2; -(C1-C 12 alkyldiyl)-OR 7 ; -(C3-C 12 (Carbocyclic group); -(C3-C 12 (carbocyclic group)-*; -(C3-C 12 (C1-C)-(C1-C) 12 (alkyldiyl)-NR 7 -*; -(C3-C 12 (C1-C)-(C1-C) 12 alkyldiyl)-N(R 7 )2; -(C3-C 12 (Carbocyclic)-NR 7 -C(=NR 7 )NR 7 -*; -(C6-C 20 (Aromatic); -(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 20 Heterocyclic dimethyl)-*;-(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 (heterocyclic group); -(C2-C 20 (heterocyclic group)-*; -(C2-C9 heterocyclic group)-(C1-C 12 (alkyldiyl)-NR 7 -*; -(C2-C9 heterocyclic group)-(C1-C 12 alkyldiyl)-N(R 7 )2; -(C2-C9 heterocyclic group)-C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*; -(C2-C9 heterocyclic group)-NR 7 -C(=NR 7a )NR 7 -*; -(C2-C9 heterocyclic group)-NR 7 -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*; -(C2-C9 heterocyclic group)-(C6-C 20 (aryldiyl)-*; -(C1-C 20 (Heteroary aryl); -(C1-C 20 (Heteroarylene)-*; -(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 Heterocyclic dimethyl)-*; -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 Heterocyclic dimethyl)-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 )-*; -O-C(=O)N(R 7 )2; -O-C(=O)N(R 7 )-*; -O-(R 7 )-*; -OR 7 ; -S(=O)2-(C2-C 20 Heterocyclic dimethyl)-*; -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-N(R 7 )2; -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 (alkyldiyl)-NR 7 -*;and -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-OH; Or R 5 and R 6 Together they form a 5- or 6-membered heterocyclic ring; R 7 Independently selectable H, C6-C 20 Aryl, C3-C 12 carbon cyclo group, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 A group consisting of alkyl dimethyl groups, or two R groups 5 The groups together form a 5- or 6-membered heterocyclic ring; R 7a Choose C6-C freely 20 Aryl and C1-C 20 Group composed of heteroaryl groups; Where the asterisk * indicates the attachment site of L, and where R 1 R 2 R 3 R 4 R 5 and R 6 One of them is attached to L; and Alkyl, alkyldiyl, alkenyl, alkenyldiyl, ynyl, ynyldiyl, aryl, aryldiyl, carbocyclic, carbocyclicdiyl, heterocyclic, heterocyclicdiyl, heteroaryl, and heteroaryldiyl are independently and optionally substituted by one or more groups independently selected from the following: F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -C H2OH, -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, -CH2N HSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, -C ONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH 3. -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 H, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3 and -S(O)3H.

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

3. The immunoconjugate of claim 1, wherein the linker L is selected from the group consisting of: -C(=O)-PEG-; -C(=O)-PEG-C(=O)N(R) 8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-; -C(=O)-PEG-(C2-C 20 (heterocyclic dimethyl)-; -C(=O)-PEG-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-; -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 monoheterocyclic dimethyl)-; -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 12 alkyldiyl)-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 monoheterocyclic dimethyl)-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)N(R 8 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-; -succinimide-(CH2) m -C(=O)N(R 3 )-(C2-C 20 (heterocyclic dimethyl)-; -succinimide-(CH2) m -C(=O)N(R 3 )-PEG-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-O-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-OC(=O)-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-N(R 8 )-C(=O)-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-C(=O)-PEP-; -succinimide-(CH2) m -C(=O)N(R 8 )-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-; -succinimide-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)-; -succinimide-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)N(R 8 )C(=O)-; and -succinimide-(CH2) m -C(=O)-PEP-N(R 8 )-(C1-C 12 alkyldiyl)N(R 8 )C(=O)-(C2-C5 monoheterocyclic dimethyl)-; R 8 Independently H or C1-C6 alkyl; PEG has the following formula: -(CH2CH2O) n -(CH2) m - m is an integer from 1 to 5, and n is an integer from 1 to 50; Gluc has the following formula: PEP has the following formula: AA is independently selected from natural or non-natural amino acid side chains, or one or more of AA and adjacent nitrogen atoms form a 5-membered cyclic proline amino acid, and the wavy line indicates the attachment point. Cyc is selected from C6-C 20 Aryldiyl and C1-C 20 Heteroaryldimethyl groups, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3 and glucuronic acids having the following structures: R 9 Choose freely - CH(R) 10 )O-、-CH2-、-CH2N(R 10 )- and -CH(R 10 The group consisting of OC (=O)-, where R 10 Selected from H, C1-C6 alkyl, C(=O)-C1-C6 alkyl and -C(=O)N(R) 11 )2, where R 11 Independently select H, C1-C 12 Alkyl groups and -(CH2CH2O) n -(CH2) m A group consisting of -OH groups, where m is an integer from 1 to 5 and n is an integer from 2 to 50, or two R groups. 11 The groups together form a 5- or 6-membered heterocyclic ring; y is an integer from 2 to 12; and 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 antigens selected from PD-L1, HER2, CEA and TROP2.

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

7. The immunoconjugate of claim 1, wherein Z 1 Z 2 Z 3 and Z 4 One of them is N.

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

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

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

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

12. The immunoconjugate of claim 1, wherein Z 1 Z 2 Z 3 and Z 4 Both of them are N.

13. The immunoconjugate according to any one of claims 1 to 12, wherein R 5 and R 6 Independently selected from C1-C8 alkyl groups, -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.

14. The immunoconjugate of claim 13, wherein R 5 It is a C1-C8 alkyl group and R 6 It is -O-(C1-C 12 alkyl).

15. The immunoconjugate of claim 13, wherein R 5 It is -CH2CH2CH3 and R 6 Selected from -CH2CH2CH2NHCO2(t-Bu), -OCH2CH2NHCO2(cyclobutyl) and -CH2CH2CH2NHCO2(cyclobutyl).

16. The immunoconjugate of claim 13, wherein R 5 and R 6 Each is independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, -CH2CH2CF3, -OCH2CH2OH and -CH2CH2CH2OH.

17. The immunoconjugate of claim 16, wherein R 5 It is -CH2CH2CH3 and R 6 It is -OCH2CH3.

18. The immunoconjugate according to any one of claims 1 to 12, wherein R 6 Choose from the following groups:

19. The immunoconjugate according to any one of claims 1 to 12, wherein R 1 Attached to L.

20. The immunoconjugate according to any one of claims 1 to 12, wherein R 2 Attached to L.

21. The immunoconjugate according to any one of claims 1 to 12, wherein R 3 Attached to L.

22. The immunoconjugate according to any one of claims 1 to 12, wherein R 4 Attached to L.

23. The immunoconjugate according to any one of claims 1 to 12, wherein R 5 Or R 6 Attached to L.

24. The immunoconjugate according to 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 the 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. The immunoconjugate of claim 26, wherein n is 10.

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

29. The immunoconjugate of claim 28, wherein the AA is independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H and -CH2CH2CH2NHC(O)NH2; or the two AAs form a 5-membered cyclic proline amino acid.

30. The immunoconjugate of claim 28, wherein PEP is a dipeptide and has the following formula: AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids.

31. The immunoconjugate of claim 30, wherein AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

32. The immunoconjugate according to any one of claims 1 to 12, wherein L is selected from the following structures: The wavy line indicates R. 1 R 2 R 3 R 4 R 5 and R 6 The attachment of one of them.

33. A type II aza-benzodiazepine - Connector compound: in Z 1 Selected from CR 1 and N; Z 2 Selected from CR 2 and N; Z 3 Selected from CR 3 and N; Z 4 Selected from CR 4 and N; Z 1 Z 2 Z 3 and Z 4 One or both of them are N; R 1 R 2 R 3 R 4 R 5 and R 6 Independently select from the following groups: 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 ynyl, C3-C 12 carbon cyclo group, C6-C 20 Aryl, C2-C9 heterocyclic and C1-C 20 Heteroaryl groups, each of which is independently and optionally substituted by one or more groups selected from: -(C1-C 12 alkyldiyl)-N(R 7 )-*; -(C1-C 12 alkyldiyl)-N(R 7 )2; -(C1-C 12 alkyldiyl)-OR 7 ; -(C3-C 12 (Carbocyclic group); -(C3-C 12 (carbocyclic group)-*; -(C3-C 12 (C1-C)-(C1-C) 12 (alkyldiyl)-NR 7 -*; -(C3-C 12 (C1-C)-(C1-C) 12 alkyldiyl)-N(R 7 )2; -(C3-C 12 (Carbocyclic)-NR 7 -C(=NR 7 )NR 7 -*; -(C6-C 20 (Aromatic); -(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 20 Heterocyclic dimethyl)-*; -(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 Heterocyclic group); -(C2-C 20 (heterocyclic group)-*; -(C2-C9 heterocyclic group)-(C1-C 12 (alkyldiyl)-NR 7 -*; -(C2-C9 heterocyclic group)-(C1-C 12 alkyldiyl)-N(R 7 )2; -(C2-C9 heterocyclic group)-C(=O)-(C1-C 12 alkyldiyl)-N(R 7 )-*; -(C2-C9 heterocyclic group)-NR 7 -C(=NR 7a )NR 7 -*; -(C2-C9 heterocyclic group)-NR 7 -(C6-C 20 aryldiyl)-(C1-C 12 alkyldiyl)-N(R 7 )-*; -(C2-C9 heterocyclic group)-(C6-C 20 (aryldiyl)-*; -(C1-C 20 (Heteroary aryl); -(C1-C 20 (Heteroarylene)-*; -(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 Heterocyclic dimethyl)-*; -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 Heterocyclic dimethyl)-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 )-*; -O-C(=O)N(R 7 )2; -O-C(=O)N(R 7 )-*; -O-(R 7 )-*; -OR 7 ; -S(=O)2-(C2-C 20 Heterocyclic dimethyl)-*; -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-N(R 7 )2; -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 (alkyldiyl)-NR 7 -*;and -S(=O)2-(C2-C 20 (heterocyclic dimethyl)-(C1-C 12 alkyldiyl)-OH; Or R 5 and R 6 Together they form a 5- or 6-membered heterocyclic ring; R 7 Independently selectable H, C6-C 20 Aryl, C3-C 12 carbonyl group, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 A group consisting of alkyl dimethyl groups, or two R groups 5 The groups together form a 5- or 6-membered heterocyclic ring; R 7a Choose C6-C freely 20 Aryl and C1-C 20 Group composed of heteroaryl groups; Where the asterisk * indicates the attachment site of L, and where R 1 R 2 R 3 R 4 R 5 and R 6 One of them is attached to L; and Alkyl, alkyldiyl, alkenyl, alkenyldiyl, ynyl, ynyldiyl, aryl, aryldiyl, carbocyclic, carbocyclicdiyl, heterocyclic, heterocyclicdiyl, heteroaryl, and heteroaryldiyl are independently and optionally substituted by one or more groups independently selected from the following: F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -C H2OH, -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, -CH2N HSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, -C ONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH 3. -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 H, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3 and -S(O)3H.

34. The azido-benzodiazepine as described in claim 33 - A connector compound, wherein L is selected from: QC(=O)-PEG-; QC(=O)-PEG-C(=O)N(R) 8 )-(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-; Q-C(=O)-PEG-N(R 8 )-; Q-C(=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 monoheterocyclic dimethyl)-; 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 12 alkyldiyl)-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 monoheterocyclic dimethyl)-; 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-O-C(=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 monoheterocyclic dimethyl)-; R 8 Independently H or C1-C6 alkyl; PEG has the following formula: -(CH2CH2O) n -(CH2) m -; m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the following formula: PEP has the following formula: AA is independently selected from natural or non-natural amino acid side chains, or one or more of AA and adjacent nitrogen atoms form a 5-membered cyclic proline amino acid, and the wavy line indicates the attachment point. Cyc is selected from C6-C 20 Aryldiyl and C1-C 20 Heteroaryldimethyl groups, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3 and glucuronic acids having the following structures: R 9 Choose freely - CH(R) 10 )O-、-CH2-、-CH2N(R 10 )- and -CH(R 10 The group consisting of OC (=O)-, where R 10 Selected from H, C1-C6 alkyl, C(=O)-C1-C6 alkyl and -C(=O)N(R) 11 )2, where R 11 Independently select H, C1-C 12 Alkyl groups and -(CH2CH2O) n -(CH2) m A group consisting of -OH groups, where m is an integer from 1 to 5 and n is an integer from 2 to 50, or two R groups. 11 The groups together form a 5- or 6-membered heterocyclic ring; y is an integer from 2 to 12; z is 0 or 1; and Q is selected from the group consisting of: N-hydroxysuccinimide, N-hydroxysulfosuccinimide, maleimide, and one or more independently selected from F, Cl, NO2, and SO3. - The phenoxy group is substituted with a group.

35. The azido-benzodiazepine as described in claim 34 - A connector compound, wherein Q is selected from:

36. The azido-benzodiazepine as described in claim 34 - A connector compound, wherein Q is a phenoxy group substituted with one or more F groups.

37. The azido-benzodiazepine as described in claim 34 - A connector compound, wherein Q is 2,3,5,6-tetrafluorophenoxy.

38. The azido-benzodiazepine as described in claim 34 - A connector compound, wherein Q is maleimide.

39. A azinon-benzodiazepine - Connector compounds, selected from Table 2a or Table 2b.

40. An immunoconjugate, which is obtained by conjugating an antibody with an aza-benzodiazepine selected from Table 2a or Table 2b. -Prepared by conjugation of connector compounds.

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

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

43. The method of claim 42, wherein the cancer is sensitive to pro-inflammatory responses induced by TLR7 and / or TLR8 activation.

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

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

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

47. Use of the immunoconjugate according to any one of claims 1 to 32 for the treatment of 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 carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer.

48. A method for preparing the immunoconjugate of formula I according to claim 1, wherein the aza-benzodiazepine according to claim 34 is used. - The adapter compound is conjugated to the antibody.

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