Anti-CEA immune complexes and uses thereof

Abstract

The present invention provides immunoconjugates of Formula I comprising an anti-CEA antibody linked by conjugation to one or more 8-Het-2-aminobenzazepine derivatives. The present invention also provides 8-Het-2-aminobenzazepine derivative intermediate compositions containing reactive functional groups. Such intermediate compositions are suitable substrates for forming immunoconjugates via linkers or linking moieties. The present invention further provides methods for treating cancer using the immunoconjugates.

Description

Technical field

[0001] Cross-reference of related applications This application claims the benefit of priority from U.S. Provisional Application No. 63 / 124,328, filed December 11, 2020, which is incorporated by reference in its entirety.

[0002] Sequence list This application has been filed electronically in ASCII format and includes a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy created on December 3, 2021 is named 17019_010WO1_SL.txt and is 55,248 bytes in size.

[0003] The present invention generally relates to immunoconjugates comprising anti-carcinoembryonic antigen (CEA) antibodies conjugated to one or more 8-Het-2-aminobenzazepine molecules. [Background technology]

[0004] New compositions and methods for the delivery of antibodies and immune adjuvants are needed to reach inaccessible tumors and / or to expand treatment options for cancer patients and other subjects. . The present invention provides such compositions and methods. [Summary of the invention]

[0005] The present invention generally relates to immunoconjugates comprising an anti-CEA antibody linked by conjugation to one or more 8-Het-2-aminobenzazepine derivatives. The present invention is further directed to 8-Het-2-aminobenzazepine derivative intermediate compositions containing reactive functional groups. Such an intermediate composition is a suitable substrate for the formation of an immunoconjugate, in which the antibody, by linker L, has the formula: [ka] 8-Het-2-aminobenzazepine (HxBz) moiety, where Het is selected from heterocyclyldiyl and heteroaryldiyl, and R 1 ,R 2 ,R 3 , and R 4 One of them is connected to L. R 1-4 and X 1-4 Substituents are defined herein.

[0006] The present invention is further directed to the use of such immunoconjugates in the treatment of diseases, particularly cancer.

[0007] One aspect of the invention is an immunoconjugate comprising an anti-CEA antibody covalently attached to a linker that is covalently attached to one or more 8-Het-2-aminobenzazepine moieties.

[0008] Another embodiment of the invention is 8-Het-2-aminobenzazepine-linker compounds.

[0009] Another embodiment of the invention comprises administering a therapeutically effective amount of an immunoconjugate comprising an anti-CEA antibody linked by conjugation to one or more 8-Het-2-aminobenzazepine moieties. It is a method for treating cancer.

[0010] Another embodiment of the invention is the use of an immunoconjugate comprising an anti-CEA antibody linked by conjugation to one or more 8-Het-2-aminobenzazepine moieties to treat cancer.

[0011] Another embodiment of the invention is a method of preparing an immunoconjugate by conjugation of an anti-CEA antibody and one or more 8-Het-2-aminobenzazepine moieties. [Brief explanation of drawings]

[0012]

Figure 1

[0013] Reference will now be made in detail to certain specific embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. Although the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the claims.

[0014] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The invention is in no way limited to the methods and materials described.

[0015] definition The term "immunoconjugate" or "immunostimulatory antibody conjugate" refers to an antibody construct that is covalently attached to an adjuvant moiety via a linker. The term "adjuvant" refers to a substance that is capable of eliciting an immune response in a subject exposed to the adjuvant.

[0016] "Adjuvant moiety" refers to an adjuvant that is covalently attached to an antibody construct via a linker, eg, as described herein. The adjuvant moiety can elicit an immune response while bound to the antibody construct or after cleavage (eg, enzymatic cleavage) from the antibody construct following administration of the immune complex to a subject.

[0017] "Adjuvant" refers to a substance that is capable of eliciting an immune response in a subject exposed to the adjuvant.

[0018] The terms "Toll-like receptor" and "TLR" refer to any member of a family of highly conserved mammalian proteins that recognize pathogen-associated molecular patterns and function as important signaling elements in innate immunity. . TLR polypeptides share a characteristic structure that includes an extracellular domain with leucine-rich repeats, a transmembrane domain, and an intracellular domain involved in TLR signaling.

[0019] The terms "Toll-like receptor 7" and "TLR7" refer to publicly available TLR7 sequences, e.g., GenBank accession number AAZ99026 for human TLR7 polypeptide, or GenBank accession number AAK62676 for mouse TLR7 polypeptide. Refers to nucleic acids or polypeptides that share at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity.

[0020] The terms "Toll-like receptor 8" and "TLR8" refer to publicly available TLR7 sequences, e.g., GenBank accession number AAZ95441 for human TLR8 polypeptide, or GenBank accession number AAK62677 for mouse TLR8 polypeptide. Refers to nucleic acids or polypeptides that share at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity.

[0021] A "TLR agonist" is a substance that directly or indirectly binds to TLR (eg, TLR7 and / or TLR8) and induces TLR signal transduction. Any detectable difference in TLR signaling can indicate that the agonist stimulates or activates TLRs. Differences in signal transduction may include, for example, the expression of target genes, the phosphorylation of signaling components, the subcellular localization of downstream elements such as nuclear factor-κB (NF-κB), and the It can be manifested as an association of related kinases (such as IRAK) with other proteins or intracellular structures, or a change in the biochemical activity of components such as kinases (such as mitogen-activated protein kinases (MAPK)).

[0022] "Antibody" refers to a polypeptide that includes an antigen-binding region (including complementarity determining regions (CDRs)) from an immunoglobulin gene or fragment thereof. The term "antibody" specifically refers to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibodies that exhibit a desired biological activity. Contains fragments. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair consisting of one "light" (approximately 25 kDa) and one "heavy" chain (approximately 50 kDa) connected by disulfide bonds. ~70kDa). Each chain is made up of structural domains called immunoglobulin domains. These domains vary in size and function, e.g., variable domains or regions on the light and heavy chains (respectively, V L and V H ), and constant domains or regions on the light and heavy chains (respectively, C L and C H ) are classified into different categories. The N-terminus of each chain defines a variable region of about 100-110 or more amino acids called a paratope, which is primarily responsible for antigen recognition, ie, the antigen-binding domain. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are large molecules of approximately 150 kDa consisting of four peptide chains. IgG antibodies contain two identical class gamma heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus a tetrameric quaternary structure. The two heavy chains are linked to each other by disulfide bonds and each to a light chain. The resulting tetramer has two identical halves, which together form a Y-like shape. Each end of the fork contains the same antigen binding domain. There are four IgG subclasses in humans (IgG1, IgG2, IgG3, and IgG4), named in order of their abundance in serum (ie, IgG1 is the most abundant). Typically, the antigen-binding domain of an antibody will be most important in the specificity and affinity of binding to cancer cells.

[0023] "Antibody construct" refers to an antibody or fusion protein that includes (i) an antigen binding domain and (ii) an Fc domain.

[0024] In some embodiments, the binding agent is an antigen-binding antibody "fragment," which is a construct that includes at least the antigen-binding region of an antibody, alone or together with other components that constitute an antigen-binding construct. be. For example, (i)V L ,V H ,C L , and CH 1 (ii) F(ab’), a bivalent fragment consisting of two Fab fragments connected by a disulfide bridge in the hinge region; 2 fragment, (iii) the single arm V of the antibody L and V H (iv) F(ab’) using mild reducing conditions 2 Fab' fragments resulting from breaking the disulfide bridges of the fragments, (v) disulfide-stabilized Fv fragments (dsFv), and (vi) allowing the two domains to be synthesized as a single polypeptide chain. The two domains of the Fv fragment (i.e., V L and V H Many different types of antibody "fragments" are known in the art, including single chains (scFvs), which are monovalent molecules consisting of (scFv).

[0025] The antibody or antibody fragment may be part of a larger construct, eg, a conjugate or fusion construct of the antibody fragment to additional regions. For example, in some embodiments, antibody fragments can be fused to an Fc region as described herein. In other embodiments, the antibody fragment (e.g., Fab or scFv) includes, for example, a transmembrane domain (optionally with an intervening linker or "stalk" (e.g., hinge region)) and any cell-to-cell signaling. By being fused to a domain, it can be part of a chimeric antigen receptor or a chimeric T cell receptor.

[0026] "Epitope" means any antigenic or epitopic determinant of an antigen to which an antigen-binding domain binds (ie, in a paratope of the antigen-binding domain). Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics and specific charge characteristics.

[0027] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. There are three major classes of Fc receptors: (1) FcγRs that bind IgG, (2) FcαRs that bind IgA, and (3) FcεRs that bind IgE. The FcγR family includes several members such as FcγI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), and FcγRIIIB (CD16B). Fcγ receptors have different affinities for IgG and also have different affinities for IgG subclasses (eg, IgG1, IgG2, IgG3, and IgG4).

[0028] 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. Percent identity measures the number of nucleotides or amino acid residues that are the same (i.e., identical) between an optimally aligned sequence of interest and a reference sequence over the length of the longest sequence (i.e., the sequence of interest or the length of the reference array (whichever is longer). Sequence alignments and percent identity calculations can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g. BLAST2.1, BL2SEQ, BLASTp, BLASTn, etc.), and FASTA programs (e.g. , FASTA3x, FASTM, and SSEARCH) (for sequence alignments and sequence similarity searches). Sequence alignment algorithms are also used, 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). sea ​​bream. The percent (%) identity of a sequence can also be calculated as, for example, 100x[(identical positions) / min(TG A , T.G. B )] can be calculated as TG A and T.G. B is T.G. A and T.G. B is the sum of the number of residues and internal gap positions in peptide sequences A and B in the alignment that minimizes . See, eg, Russell et al., J. Mol Biol., 244:332-350 (1994).

[0029] The binding agent includes Ig heavy chain and light chain variable region polypeptides that together form an antigen binding site. Each heavy and light chain variable region is a polypeptide that includes three complementarity determining regions (CDR1, CDR2, and CDR3) connected by framework regions. The binding agent can be any of the various types of binding agents known in the art, including Ig heavy and light chains. For example, a binding agent can be an antibody, an antigen-binding antibody "fragment," or a T cell receptor.

[0030] A “biosimilar” has similar activity properties to a CEA-targeted antibody, such as labetuzumab (CEA-CIDE™, MN-14, hMN14, Immunomedical) CAS Reg. Refers to approved antibody constructs with

[0031] "Biobetter" refers to an approved antibody construct that is an improvement on a previously approved antibody construct, such as labetuzumab. A biobetter can have one or more modifications to a previously approved antibody construct (eg, an altered glycan profile, or a unique epitope). Biobetters are recombinant protein drugs in the same class as existing biopharmaceuticals, but they are not identical and are better than the original. BioBetter is not just a new drug, nor is it a generic version of a drug. Biosimilars and biobetters are both variants of a biological drug, with the former being a close copy of the original drug and the latter improving in terms of efficacy, safety, and tolerability or dosing regimen. has been done.

[0032] "Amino acid" refers to any monomer unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally occurring α-amino acids and their stereoisomers, as well as non-naturally occurring (non-naturally occurring) amino acids and their stereoisomers. A "stereoisomer" of a given amino acid refers to isomers that have the same molecular formula and intramolecular bonding, but different three-dimensional arrangements of bonds and atoms (eg, an L-amino acid and a corresponding D-amino acid). Amino acids can be glycosylated (eg, N-linked glycans, O-linked glycans, phosphoglycans, C-linked glycans, or glypication) or deglycosylated. Amino acids may be referred to herein by either the commonly known three letter symbol or the one letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0033] Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Naturally occurring alpha amino acids include alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), Arginine (Arg), Lysine (Lys), Leucine (Leu), Methionine (Met), Asparagine (Asn), Proline (Pro), Glutamine (Gln), Serine (Ser), Threonine (Thr), Valine (Val), Examples include, but are not limited to, tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), and 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.

[0034] Naturally occurring amino acids include those formed in proteins by post-translational modifications such as citrulline (Cit).

[0035] 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 in either the L or D configuration that function in a manner similar to naturally occurring amino acids. , and N-methyl amino acids. For example, an "amino acid analog" has the same basic chemical structure as a naturally occurring amino acid (i.e., hydrogen, carboxyl group, carbon attached to an amino group) but with modified side chain groups or It can be an unnatural amino acid with a peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. "Amino acid mimetic" refers to a compound that has a structure that differs from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

[0036] "Linker" refers to a functional group that covalently joins two or more moieties in a compound or material. For example, a linking moiety can serve to covalently attach an adjuvant moiety to an antibody construct in an immunoconjugate.

[0037] "Linking moiety" refers to a functional group that covalently bonds two or more moieties in a compound or material. For example, a linking moiety can serve to covalently attach an adjuvant moiety to an antibody in an immune complex. Useful linkages for linking linking moieties to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

[0038] "Divalent" refers to a chemical moiety that contains two points of attachment for linking two functional groups; a multivalent linking moiety can have additional points of attachment for linking additional functional groups. can. Divalent radicals may be designated with the suffix "diyl". For example, divalent linking moieties include divalent polymeric moieties such as divalent poly(ethylene glycol), divalent cycloalkyl, divalent heterocycloalkyl, divalent aryl, and divalent heteroaryl groups. "Divalent cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group" means a cycloalkyl, heterocycloalkyl, heterocycloalkyl, Refers to an aryl or heteroaryl group. A cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may be substituted or unsubstituted. A cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0039] Wavy line (“ [ka] ”) represents the point of attachment of the specified chemical moiety. It will be understood that when two squiggly lines are present in a designated chemical moiety, the chemical moiety may be used to be read on both sides, i.e., left to right or right to left.

[0040] "Alkyl" refers to a straight or branched saturated aliphatic radical having the indicated number of carbon atoms. Alkyl can contain any number of carbons, for example 1-12. Examples of alkyl groups include methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl(i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 ) 2 ), 2-butyl(s-Bu, s-butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl(t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl(-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl(-CH(CH 2 CH 3 ) 2 ), 2-methyl-2-butyl(-C(CH 3 ) 2 CH 2 CH 3 ), 3-methyl-2-butyl(-CH(CH 3 )CH(CH 3 ) 2 ), 3-methyl-1-butyl(-CH 2 CH 2 CH(CH 3 ) 2 ), 2-methyl-1-butyl(-CH 2 CH(CH 3 )CH 2 CH 3 ), 1-hexyl(-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-hexyl(-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 ), 3-hexyl(-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 )), 2-methyl-2-pentyl(-C(CH 3 ) 2 CH 2 CH 2 CH 3 ), 3-methyl-2-pentyl(-CH(CH 3 )CH(CH 3 )CH 2 CH 3 ), 4-methyl-2-pentyl(-CH(CH 3 )CH 2 CH(CH 3 ) 2 ), 3-methyl-3-pentyl(-C(CH 3 )(CH 2 CH 3 ) 2 ), 2-methyl-3-pentyl(-CH(CH 2 CH 3 )CH(CH 3 ) 2 ), 2,3-dimethyl-2-butyl(-C(CH 3 ) 2 CH(CH 3 ) 2 ), 3,3-dimethyl-2-butyl(-CH(CH 3 )C(CH 3 ) 3 , 1-heptyl, 1-octyl and the like, but are not limited to these. Alkyl groups may be substituted or unsubstituted. A "substituted alkyl" group may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0041] The term "alkyldiyl" refers to a divalent alkyl radical. Examples of alkyldiyl groups include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -), etc., but are not limited to these. Alkyldiyl groups may also be referred to as "alkylene" groups.

[0042] "Alkenyl" refers to a straight or branched, unsaturated aliphatic radical having the number of carbon atoms indicated and at least one carbon-carbon double bond, sp2. Alkenyl can contain from 2 to about 12 or more carbon atoms. Alkenyl groups are radicals with "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. Examples include ethylenyl or vinyl (-CHCH 2 ), allyl(-CH 2 CHCH 2 ), butenyl, pentenyl, and isomers thereof. Alkenyl groups may be substituted or unsubstituted. A "substituted alkenyl" group may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0043] The term "alkenylene" or "alkenyldiyl" refers to a straight or branched divalent hydrocarbon radical. Examples include ethylenylene or vinylene (-CHCH-), allyl (-CH 2 Examples include, but are not limited to, CHCH-) and the like.

[0044] "Alkynyl" refers to a straight or branched unsaturated aliphatic radical having the number of carbon atoms indicated and at least one carbon-carbon triple bond, sp. Alkynyl can contain from 2 to about 12 or more carbon atoms. For example, C 2 -C 6 Alkynyl is ethynyl (-C≡CH), propynyl (propargyl, -CH 2 C≡CH), butynyl, pentynyl, hexynyl, and isomers thereof. Alkynyl groups may be substituted or unsubstituted. A "substituted alkynyl" group may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0045] The term "alkynylene" or "alkynyldiyl" refers to a divalent alkynyl radical.

[0046] The terms "carbocycle," "carbocyclyl," "carbocyclic ring," and "cycloalkyl" refer to saturated or partially unsaturated, monocyclic rings containing from 3 to 12 ring atoms, or the number of atoms indicated. Refers to a cyclic, fused bicyclic, or bridged polycyclic ring assembly. 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, decahydronaphthalene, and adamantane. Carbocyclic groups can also be partially unsaturated, having one or more double or triple bonds within the ring. Representative carbocyclic groups that are partially unsaturated include cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctadiene (1 , 3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene.

[0047] The term "cycloalkyldiyl" refers to a divalent cycloalkyl radical.

[0048] "Aryl" is a group of 6 to 20 carbon atoms (C 6 ~C 20) refers to a monovalent aromatic hydrocarbon radical. Aryl groups may be monocyclic, fused to form bicyclic or tricyclic groups, or linked by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl having a methylene linking group. 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.

[0049] The term "arylene" or "aryldiyl" refers to a group of 6 to 20 carbon atoms (C 6 ~C 20 ) means a divalent aromatic hydrocarbon radical. Some aryldiyl groups are represented as "Ar" in exemplary structures. Aryldiyl includes bicyclic radicals containing saturated, partially unsaturated rings, or aromatic rings fused to aromatic carbocyclic rings. Typical aryldiyl groups include radicals derived from benzene (phenyldiyl), substituted benzene, naphthalene, anthracene, biphenylene, indenylene, indanilene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, etc. These include, but are not limited to: Aryldiyl groups are also referred to as "arylene" and are optionally substituted with one or more substituents as described herein.

[0050] The terms "heterocycle," "heterocyclyl," and "heterocyclic ring" are used interchangeably herein and refer to a heteroatom in which at least one ring atom is selected from nitrogen, oxygen, phosphorus, and sulfur. a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic ring of from 3 to about 20 ring atoms, with the remaining ring atoms being C Refers to a radical in which one or more ring atoms are optionally substituted independently with one or more substituents described below. Heterocycles are monocyclic rings having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S), or 7 to 10 bicyclo with ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), e.g., bicyclo[4,5], [5, 5], [5,6], or [6,6] system. Heterocyclic rings are described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7, and 9, “The Chemistry of Heterocyclic Compounds,” A series of Monographs” (John Wiley & Sons, New York, 1950 to present), especially volumes 13, 14, 16, 19, and 28, and J. Am. Chem. Soc. (1960) 82:5566. "Heterocyclyl" also includes radicals in which a heterocyclic radical is fused with a saturated, partially unsaturated ring, or an aromatic carbocyclic or heterocyclic ring. Examples of heterocyclic rings include morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thio Morpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4 ]Diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl , oxepanil, thiepanil, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, Dihydrofuranyl, pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabishico[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolidinyl, and These include, but are not limited to, N-pyridyl urea. Spiroheterocyclyl moieties are also included within this definition. Examples of spiroheterocyclyl moieties include azaspiro[2.5]octanyl and azaspiro[2.4]heptanyl. Examples of heterocyclic groups in which two ring atoms are substituted with oxo (=O) moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents as described herein.

[0051] The term "heterocyclyldiyl" refers to a dicyclic group of from 3 to about 20 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorous, and sulfur, and the remaining ring atoms are C. refers to a valent, saturated, or partially unsaturated (i.e., having one or more double and / or triple bonds in the ring) carbocyclic radical, in which one or more ring atoms are optionally independently substituted with one or more substituents, as indicated. Examples of 5- and 6-membered heterocyclyldiyl include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholyldiyl, and S-dioxothiomol. An example is horyldiyl.

[0052] The term "heteroaryl" refers to a 5-, 6-, or 7-membered monovalent aromatic radical containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Contains a fused ring system of 20 atoms (at least one is aromatic). Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, Thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, sinolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl , thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents as described herein.

[0053] The term "heteroaryldiyl" refers to a 5-, 6-, or 7-membered divalent aromatic radical containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Contains a fused ring system of 5 to 20 atoms (at least one is aromatic). Examples of 5- and 6-membered heteroaryldiyl include pyridyldiyl, imidazolyldiyl, pyrimidyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyldiyl, thiazolyldiyl, oxadiazo Included are lyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl.

[0054] A heterocycle or heteroaryl group may be carbon (carbon-bonded) or nitrogen (nitrogen-bonded) bonded, where such is possible. By way of example and not limitation, a carbon-bonded heterocycle or heteroaryl can be at the 2, 3, 4, 5, or 6 position of pyridine, at the 3, 4, 5, or 6 position of pyridazine, at the 2, 4, 5, or position 6, position 2, 3, 5, or 6 of pyrazine; position 2, 3, 4, or 5 of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole; position 2, 4, or 4 of oxazole, imidazole, or thiazole; or position 5, position 3, 4, or 5 of isoxazole, pyrazole, or isothiazole; position 2 or 3 of aziridine; position 2, 3, or 4 of azetidine; position 2, 3, 4, 5, or 6 of quinoline; 7, or 8, or the 1, 3, 4, 5, 6, 7, or 8 position of the isoquinoline.

[0055] By way of example and not limitation, nitrogen-bonded heterocycles or heteroaryls include aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2- It is attached to the 1st position of pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2nd position of isoindole or isoindoline, the 4th position of morpholine, and the 9th position of carbazole or β-carboline.

[0056] The terms "halo" and "halogen" by themselves or as part of another substituent refer to a fluorine, chlorine, bromine, or iodine atom.

[0057] The term "carbonyl" by itself or as part of another substituent refers to C(=O) or -C(=O)-, i.e., any other group within a moiety double-bonded to an oxygen and bearing a carbonyl. refers to a carbon atom bonded to two groups.

[0058] As used herein, the phrase "quaternary ammonium salt" refers to an alkyl substituent (e.g., C 1 ~C 4 Refers to a tertiary amine that has been quaternized with alkyl).

[0059] The terms "treat," "treatment," and "treating" refer to alleviation, amelioration, alleviation of symptoms, or making a symptom, injury, pathology, or disease more tolerable by a patient, or reducing the rate of progression of symptoms. an injury, pathology, disease (e.g. cancer), or condition, including any objective or subjective parameter, such as reducing, reducing the frequency or duration of a symptom or disease, or in some circumstances preventing the onset of a symptom. Refers to any sign of success in treating or ameliorating (e.g., cognitive impairment). Treatment or amelioration of symptoms may be based on any objective or subjective parameter, including, for example, the results of a physical exam.

[0060] The terms "cancer," "neoplasm," and "tumor" refer to the autonomous, uncontrolled growth of cells in which they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Used herein to refer to cells that exhibit Cells of interest for detection, analysis and / or treatment in the context of the present invention include cancer cells (e.g. cancer cells from individuals with cancer), malignant cancer cells, pre-metastatic cancer cells. , metastatic cancer cells, and non-metastatic cancer cells. Cancers of almost all tissues are known. The phrase "cancer burden" refers to the amount of cancer cells or cancer volume in a subject. Therefore, reducing cancer burden refers to reducing the number of cancer cells or cancer cell volume in a subject. As used herein, the term "cancer cell" is a cancer cell (e.g., from any cancer that an individual may be treated with, e.g., that may be isolated from an individual who has cancer) ), or any cell derived from a cancer cell, e.g., a clone of a cancer cell. For example, cancer cells may be from an established cancer cell line, may be primary cells isolated from an individual with cancer, or may be primary cells isolated from an individual with cancer. For example, it may be a progeny cell from a cell. In some embodiments, the term may also refer to a portion of a cancer cell, a subcellular portion of a cancer cell, a cell membrane portion, or a cell lysate. Many types of cancer are known to those skilled in the art, including solid tumors such as carcinoma, sarcoma, glioblastoma, melanoma, lymphoma, and myeloma, and circulating cancers such as leukemia.

[0061] As used herein, the term "cancer" refers to solid tumor cancers (e.g., skin, lung, prostate, breast, stomach, bladder, colon, ovary, pancreas, kidney, liver, glioblastoma, marrow). blastoma, leiomyosarcoma, head and neck squamous cell carcinoma, melanoma, and neuroendocrine), and liquid cancer (e.g., blood cancer), carcinoma, soft tissue tumor, sarcoma, metamorphoma, melanoma, leukemia, lymphoma, and microscopic Includes any form of cancer that includes residual disease and includes, but is not limited to, brain cancer, including both primary and metastatic tumors.

[0062] Cancer "pathology" includes all phenomena that impair patient well-being. These include abnormal or uncontrolled cell proliferation, metastasis, interference with the normal function of neighboring cells, release of cytokines or other secreted products at abnormal levels, and suppression or exacerbation of inflammatory or immunological responses. including, but not limited to, neoplasms, premalignant tumors, malignant tumors, and invasion of surrounding or distant tissues or organs, such as lymph nodes.

[0063] As used herein, the phrases "cancer recurrence" and "tumor recurrence" and grammatical variants thereof refer to the further growth of neoplastic or cancerous cells after diagnosis of cancer. . In particular, recurrence may occur when further cancerous cell growth occurs in the cancerous tissue. Similarly, "tumor spread" occurs when cells of a tumor spread to local or distant tissues and organs, and thus includes tumor metastasis. "Tumor invasion" occurs when tumor growth spreads locally and impairs the function of the involved tissues by compacting, destroying, or preventing normal organ function.

[0064] 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 undetectable amounts of cancerous 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 in the process, such as detachment of cancer cells from the original tumor site and migration and / or invasion of cancer cells to other parts of the body.

[0065] The phrases "effective amount" and "therapeutically effective amount" refer to the dose or amount of a substance, such as an immune complex, to which it is administered that produces a therapeutic effect. The exact dose depends on the purpose of treatment and can be ascertained by one skilled in the art using known techniques (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,11 th Edition (McGraw-Hill, 2006), and Remington: The Science and Practice of Pharmacy, 22 nd Edition, (Pharmaceutical Press, London, 2012). ) In the case of cancer, a therapeutically effective amount of an immune complex reduces the number of cancer cells, reduces tumor size, inhibits (i.e. slows to some extent, and preferably slows) the invasion of cancer cells into peripheral organs. may inhibit (i.e., slow, preferably halt) tumor metastasis to some extent, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with cancer. . To the extent that the immune complex can prevent the growth and / or death of existing cancer cells, it may be cytostatic and / or cytotoxic. In the case of cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and / or determining response rate (RR).

[0066] "Recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., human) for whom diagnosis, treatment, or therapy is desired. Point. "Mammal" means any animal classified as a mammal, including humans, domesticated animals, and farm animals, as well as zoo animals such as dogs, horses, cats, cows, sheep, goats, pigs, camels, or sporting animals; Refers to pet animals. In certain embodiments, the mammal is a human.

[0067] The term "synergistic adjuvant" or "synergistic combination" in the context of the present invention refers to receptor agonists, cytokines, and adjuvants that, in combination, elicit a synergistic effect on immunity compared to the administration of either alone. Contains a combination of two immunomodulatory agents such as polypeptides. In particular, the immunoconjugates disclosed herein include a synergistic combination of the claimed adjuvant and antibody construct. These synergistic combinations upon administration induce a greater effect on immunity compared to, for example, when the antibody construct or adjuvant is administered in the absence of the other moiety. Additionally, there was a reduction (as measured by the total number of antibody constructs or the total number of adjuvants administered as part of an immune complex) compared to when either the antibody construct or the adjuvant were administered alone. amount of immune complex may be administered.

[0068] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal, or subcutaneous administration, oral administration, as a suppository, etc. refers to administration, topical contact, intrathecal administration, or implantation of a sustained release device, such as a mini-osmotic pump, into the subject.

[0069] When used herein to modify numerical values, the terms "about" and "approximately" indicate a close range around the numerical value. Therefore, when "X" is a value, "about X" or "approximately References to “about X” or “approximately Be specific. Thus, "about X" and "approximately X" are intended to teach and provide written descriptive support for a claim limitation of, for example, "0.98X."

[0070] CEA antibody The immunoconjugates of the invention include antibodies that target, bind, or recognize carcinoembryonic antigens (CEA, CD66e, CEACAM5). Included within the scope of embodiments of the invention are functional variants of the antibody constructs or antigen binding domains described herein. As used herein, the term "functional variant" refers to an antibody construct that has an antigen binding domain that has substantial or significant sequence identity or similarity to a parent antibody construct or antigen binding domain; A functional variant retains the biological activity of the antibody construct or antigen binding domain of which it is a variant. A functional variant, e.g., as described herein, retains the ability to recognize target cells expressing CEA to a similar extent, to the same extent, or to a higher extent than the parent antibody construct or antigen binding domain. (parent antibody construct or antigen binding domain).

[0071] With respect to an antibody construct or antigen binding domain, a functional variant is, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90% different in amino acid sequence from the antibody construct or antigen binding domain. %, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more.

[0072] A functional variant can include, for example, a parent antibody construct or antigen binding domain amino acid sequence with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can include an amino acid sequence of a parent antibody construct or antigen binding domain that has at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of a functional variant such that the biological activity of the functional variant is increased compared to the parent antibody construct or antigen binding domain.

[0073] Antibodies comprising immunoconjugates of the invention include Fc engineered variants. In some embodiments, mutations in the Fc region that result in regulated binding to one or more Fc receptors include the following mutations: SD(S239D), SDIE(S239D / I332E), SE(S267E), SELF( S267E / L328F), SDIE(S239D / I332E), SDIEAL(S239D / I332E / A330L), GA(G236A), ALIE(A330L / I332E), GASDALIE(G236A / S239D / A330L / I332E), V9(G237D / P238D / P271G / A330R), and one or more of V11 (G237D / P238D / H268D / P271G / A330R), and / or the following amino acids: 1 in E345R, E233, G237, P238, H268, P271, L328, and A330 can contain more than one mutation. Additional Fc region modifications to modulate Fc receptor binding are described, for example, in US2016 / 0145350 and US7416726 and US5624821, which are incorporated herein by reference in their entirety.

[0074] Antibodies containing the immunoconjugate of the present invention include sugar chain variants such as afucosylation. In some embodiments, the Fc region of the binding agent is modified to have an altered glycosylation pattern of the Fc region compared to the native, unmodified Fc region.

[0075] Amino acid substitutions in antibody constructs or antigen binding domains of the invention are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art in which one amino acid with certain physical and / or chemical properties is replaced with another amino acid with the same or similar chemical or physical properties. Contains amino acid substitutions. For example, conservative amino acid substitutions include an acidic / loaded polar amino acid (e.g. Asp or Glu) substituted with another acidic / loaded polar amino acid, a nonpolar side substituted with another amino acid with a nonpolar side chain. Amino acids with chains (e.g. Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), basic / positive polarity substituted with another basic / positive polarity amino acid Amino acids (e.g. Lys, His, Arg, etc.), uncharged amino acids with a polar side chain substituted by another uncharged amino acid with a polar side chain (e.g. Asn, Gln, Ser, Thr, Tyr, etc.), beta Beta Amino acids with branched side chains substituted with another amino acid with a branched side chain (e.g., Ile, Thr, and Val), Amino acids with an aromatic side chain substituted with another amino acid with an aromatic side chain (eg, His, Phe, Trp, and Tyr).

[0076] An antibody construct or antigen binding domain can consist essentially of the specified amino acid sequence(s) described herein, such that other components, e.g. does not substantially alter the biological activity of the construct or antigen-binding domain functional variant.

[0077] In some embodiments, the antibody in the immunoconjugate contains a modified Fc region, and the modification modulates the binding of the Fc region to one or more Fc receptors.

[0078] In some embodiments, the antibody in the immunoconjugate (e.g., the antibody conjugated to at least two adjuvant moieties) has one or more Fc regions compared to a native antibody that lacks mutations in the Fc region. Modulated binding (e.g., increased binding or decreased binding) to receptors (e.g., FcγRI(CD64), FcγRIIA(CD32A), FcγRIIB(CD32B), FcγRIIIA(CD16a), and / or FcγRIIIB(CD16b)) Contains one or more modifications (eg, amino acid insertions, deletions, and / or substitutions) within the Fc region that result in. In some embodiments, the antibody in the immunoconjugate has one or more modifications within the Fc region (e.g., amino acid insertions, deletions, and / or or substitution). In some embodiments, the antibody in the immunoconjugate reduces binding of the antibody to FcγRIIB compared to a native antibody lacking mutations in the Fc region, while ), and / or one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region of the antibody that maintain or have increased binding to FcRγIIIA (CD16a). Contains. In some embodiments, the antibody in the immunoconjugate contains one or more modifications within the Fc region that increase binding of the Fc region of the antibody to FcγRIIB.

[0079] In some embodiments, modulated binding is provided by mutations within the Fc region of the antibody compared to the native Fc region of the antibody. The mutation can be in the CH2 domain, CH3 domain, or a combination thereof. "Native Fc region" is synonymous with "wild-type Fc region" and is the same as the amino acid sequence of an Fc region found in nature or the amino acid sequence of an Fc region found in a naturally occurring antibody (e.g., cetuximab). Contains identical amino acid sequences. Native sequence human Fc regions include native sequence human IgG1 Fc regions, native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Natural sequence Fc includes various allotypes of Fc (Jefferis et al., (2009) mAbs, 1(4):332-338).

[0080] In some embodiments, mutations in the Fc region that result in modulated binding to one or more Fc receptors include the following mutations: SD(S239D), SDIE(S239D / I332E), SE(S267E), SELF (S267E / L328F), SDIE(S239D / I332E), SDIEAL(S239D / I332E / A330L), GA(G236A), ALIE(A330L / I332E), GASDALIE(G236A / S239D / A330L / I332E), V9(G237D / P238D / P271G / A330R), and one or more of V11 (G237D / P238D / H268D / P271G / A330R), and / or one of the following amino acids: E233, G237, P238, H268, P271, L328, and A330 The above mutations can be included. Additional Fc region modifications to modulate Fc receptor binding are described, for example, in US2016 / 0145350 and US7416726 and US5624821, which are incorporated herein by reference in their entirety.

[0081] In some embodiments, the Fc region of the antibody of the immunoconjugate is modified to have an altered glycosylation pattern of the Fc region compared to the native, unmodified Fc region.

[0082] Human immunoglobulins are glycosylated at residue Asn297 within the Cγ2 domain of each heavy chain. This N-linked oligosaccharide is composed of the core heptasaccharide, N-acetylglucosamine 4 mannose 3 (GlcNAc4Man3). Removal of heptasaccharides by endoglycosidase or PNGase F is known to result in conformational changes in the antibody Fc region, which significantly reduces antibody binding affinity for FcγR activation and reduces effector function. Can be done. Core heptasaccharides are often decorated with galactose, bisected GlcNAc, fucose, or sialic acid, which differentially affects Fc binding to activating and inhibitory FcγRs. Furthermore, α2,6-sialylation enhances anti-inflammatory activity in vivo, while defucosylation leads to improved FcγRIIIa binding and a 10-fold increase in antibody-dependent cytotoxicity and phagocytosis. This has been proven. Thus, specific glycosylation patterns can be used to control inflammatory effector functions.

[0083] In some embodiments, the modification to alter the glycosylation pattern is a mutation. For example, substitution with Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods for controlling immune responses with antibodies that modulate FcγR-regulated signaling are described, for example, in U.S. Pat. are incorporated herein by reference in their entirety.

[0084] In some embodiments, the antibodies of the immunoconjugate are modified to contain engineered Fab regions with non-naturally occurring glycosylation patterns. For example, hybridomas can be genetically engineered to secrete afucosylated mAbs, deallylated mAbs, or deglycosylated Fc with specific mutations that allow for increased FcRγIIIa binding and effector function. In some embodiments, the antibodies of the subject immunoconjugates are engineered to be afucosylated.

[0085] In some embodiments, the entire Fc region of the antibody within the immunoconjugate is exchanged with a different Fc region, such that the Fab region of the antibody is conjugated to the non-native Fc region. For example, the Fab region of cetuximab, which typically includes an IgG1Fc region, may be conjugated to IgG2, IgG3, IgG4, or IgA, or the Fab region of nivolumab, which typically includes an IgG4Fc region, may be conjugated to IgG1, IgG2, IgG3, IgA1 or may be conjugated to IgG2. In some embodiments, Fc-modified antibodies with non-natural Fc domains also include one or more amino acid modifications, such as the S228P mutation within the IgG4Fc, that modulate the stability of the described Fc domain. In some embodiments, Fc-modified antibodies with non-natural Fc domains also include one or more amino acid modifications described herein that modulate Fc binding to FcR.

[0086] In some embodiments, a modification that modulates the binding of an Fc region to an FcR does not alter the binding of an antibody's Fab region to its antigen when compared to a native, unmodified antibody. In other embodiments, modifications that modulate the binding of the Fc region to FcR also increase the binding of the Fab region of the antibody to its antigen when compared to a native, unmodified antibody.

[0087] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct that includes an antigen binding domain that specifically recognizes and binds CEA.

[0088] Elevated expression of carcinoembryonic antigens (CEA, CD66e, CEACAM5) has been implicated in various biological aspects of tumors, especially tumor cell adhesion, metastasis, blocking cellular immune mechanisms, and having anti-apoptotic functions. There is. CEA is a cell surface antigen and is also used as a blood marker for many cancers. Labetuzumab (CEA-CIDE™, Immunomedics, CAS Reg. No. 219649-07-7), also known as MN-14 and hMN14, is a humanized IgG1 monoclonal antibody for the treatment of colorectal cancer. (Blumenthal, R. et al (2005) Cancer Immunology Immunotherapy 54(4):315-327). Labetuzumab conjugated to a camptothecin analog (rabetuzumab govitecan, IMMU-130) targets CEA and has been studied in patients with relapsed or refractory metastatic colorectal cancer (Sharkey, R. et al. (2018), Molecular Cancer Therapeutics 17(1):196-203, Dotan, E. et al (2017), Journal of Clinical Oncology 35(9):3338-3346). Also, 131 Labetuzumab conjugated to I has been evaluated in clinical trials for the treatment of colon cancer and other solid malignancies (Sharkey, R. et al (1995), Cancer Research (Suppl.) 55(23) :5935s-5945s, Liersch, T. et al(2005), Journal of Clinical Oncology 23(27):6763-6770, Sahlmann, C.-O.et al(2017), Cancer 123(4):638-649 ).

[0089] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VL kappa) of hMN-14 / Labetuzumab SEQ ID NO: 1, as disclosed in US6676924, which is used for this purpose. incorporated herein by reference. DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK Sequence number 1

[0090] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of hMN-14 / Labetuzumab SEQ ID NOs: 2-8. (US6676924).

table 1

[0091] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of hMN-14 / Labetuzumab SEQ ID NO: 9, as disclosed in US6676924, which is suitable for this purpose. is incorporated herein by reference. EVQLVESGGGVVQPGRSLRLSCSSSGFDFTTYWMSWVRQAPGKGLEWVAEIHPDSSTINYAPSLKDRFTISRDNSKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS SEQ ID NO. 9

[0092] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hMN-14 / Labetuzumab SEQ ID NOs: 10-16. (US6676924). [Table 2]

[0093] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VL kappa) of hPR1A3 SEQ ID NO: 17, as disclosed in US8642742, which for this purpose incorporated herein by. DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIK Sequence number 17

[0094] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of hPR1A3 SEQ ID NOs: 18-24 (US8642742). [Table 3]

[0095] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hPR1A3 SEQ ID NOs: 25-31 (US8642742). [Table 4]

[0096] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VL kappa) of hMFE-23 SEQ ID NO: 32, as disclosed in US7232888, which is used for this purpose. is incorporated herein by reference. ENVLTQSPSSMSASVGDRVNIACSASSSVSYMHWFQQKPGKSPKLWIYSTSNLASGVPSRFSGSGSGTDYSLTISSMQPEDAATYYCQQRSSYPLTFGGGTKLEIK Sequence number 32

[0097] In one embodiment of the invention, the CEA-targeted antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of hMFE-23 SEQ ID NOs: 33-40 (US7232888 ). This embodiment includes two variants of LFR1, SEQ ID NO: 33 and SEQ ID NO: 34. [Table 5]

[0098] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of hMFE-23 SEQ ID NO: 41 (US7232888). QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSS Sequence number 41

[0099] In one embodiment of the invention, the CEA-targeted antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hMFE-23 SEQ ID NOs: 42-49 (US7232888 ). This embodiment includes two variants of HFR1, SEQ ID NO: 42 and SEQ ID NO: 43. [Table 6]

[0100] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VL kappa) of SM3E SEQ ID NO: 50 (US7232888). ENVLTQSPSSMSVSVGDRVTIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIK Sequence number 50

[0101] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of SM3E SEQ ID NOs: 51-56 and 38-39. (US7232888). This embodiment includes two variants of LFR1, SEQ ID NO: 51 and SEQ ID NO: 52. [Table 7]

[0102] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain of NP-4 / arsitumomab SEQ ID NO: 57. QTVLSQSPAILSASPGEKVTMTCRASSSVTYIHWYQQKPGSSPKSWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQHWSSKPPTFGGGTKLEIK SEQ ID NO: 57

[0103] In one embodiment of the invention, the CEA-targeted antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of NP-4 / Arsitumomab SEQ ID NOs: 58-64. . [Table 8]

[0104] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of NP-4 / arsitumomab SEQ ID NO: 65. EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPPGKALEWLGFIGNKANGYTTEYSASVKGRFTISRDKSQSILYLQMNTLRAEDSATYYCTRDRGLRFYFDYWGQGTTLTVSS SEQ ID NO: 65.

[0105] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of NP-4 SEQ ID NOs: 66-72. [Table 9]

[0106] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VL kappa) of M5A / hT84.66 SEQ ID NO: 73, disclosed in US7776330, which is used for this purpose. incorporated herein by reference. DIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVGFLHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQGTKVEIK Sequence number 73

[0107] In one embodiment of the invention, the CEA-targeted antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of M5A / hT84.66 SEQ ID NOs: 74-80. (US7776330). [Table 10]

[0108] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of M5A / hT84.66 SEQ ID NO: 81 (US7766330). EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSS Sequence number 81

[0109] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of M5A / hT84.66 SEQ ID NOs: 82-88. (US7776330). [Table 11]

[0110] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VL kappa) of hAb2-3 SEQ ID NO: 89, as disclosed in US9617345, which is used for this purpose. is incorporated herein by reference. DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK SEQ ID NO: 89

[0111] In one embodiment of the invention, the CEA-targeted antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of hAb2-3 SEQ ID NOs: 90-96 (US9617345 ). [Table 12]

[0112] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of SEQ ID NO: 97 (US9617345). EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS SEQ ID NO: 97

[0113] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hAb2-3 SEQ ID NOs: 98-104. [Table 13]

[0114] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VL kappa) of A240VL-B9VH / AMG-211 SEQ ID NO: 105, as disclosed in US9982063, which is incorporated herein by reference for this purpose. QAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVL Sequence number 105

[0115] In one embodiment of the invention, the CEA-targeted antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of A240VL-B9VH / AMG-211 SEQ ID NOs: 106-112. (US9982063). [Table 14]

[0116] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises a variable heavy chain (VH) of B9VH SEQ ID NO: 113 (US9982063). EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS Sequence number 113

[0117] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of SEQ ID NOs: 114-121 (US9982063). This embodiment includes two variants of CDR-H2, SEQ ID NO: 117 and SEQ ID NO: 118. [Table 15]

[0118] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of E12VH SEQ ID NO: 122 (US9982063). EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFILNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS Sequence number 122

[0119] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of SEQ ID NOs: 123-129 (US9982063). [Table 16]

[0120] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises a variable heavy chain (VH) of PR1A3 VH SEQ ID NO: 130 (US8642742). QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSS Sequence number 130

[0121] In some embodiments, the antibody construct further 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). Single-chain variable region fragments (scFvs), which are truncated Fab fragments containing the variable (V) domain of an antibody heavy chain linked to the V domain of a light antibody chain via a synthetic peptide, are routinely used in recombinant DNA can be generated using technology. Similarly, disulfide stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology. The antibody construct or antigen binding domain may comprise one or more variable regions (eg, two variable regions) of the antigen binding domain of an anti-CEA antibody, each variable region comprising CDR1, CDR2, and CDR3.

[0122] In some embodiments, the antibody in the immunoconjugate contains a modified Fc region, and the modification modulates the binding of the Fc region to one or more Fc receptors.

[0123] In some embodiments, the Fc region is modified by including a transforming growth factor beta 1 (TGFβ1) receptor, or a 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 C-terminal fusion to the TGFβRII extracellular domain (ECD) as described in US9676863, incorporated herein. An "Fc linker" may be used to attach the IgG to the TGFβRII extracellular domain. Fc linkers may be short flexible peptides that allow proper three-dimensional folding of the molecule while maintaining binding specificity to the target. In some embodiments, the N-terminus of the TGFβ receptor is fused to the Fc of the antibody construct (with or without an Fc linker). In some embodiments, the C-terminus of the antibody construct heavy chain is fused to a 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.

[0124] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0125] In some embodiments, the antibody in the immunoconjugate is engineered at a site where the engineered cysteine ​​is available for conjugation but does not disrupt immunoglobulin folding and assembly or antigen binding and effector function. Cysteine-engineered antibodies that provide site-specific conjugation of adjuvants, labels, or drug moieties via cysteine ​​substitution (Junutula, et al., 2008b Nature Biotech., 26(8):925-932, Dornan et al. al. (2009) Blood 114(13):2721-2729, US7521541, US7723485, US2012 / 0121615, WO2009 / 052249). A "cysteine ​​engineered antibody" or "cysteine ​​engineered antibody variant" is an antibody in which one or more residues of the antibody are replaced with a cysteine ​​residue. The cysteine ​​engineered antibody can be conjugated to the 8-Het-2-aminobenzazepine adjuvant moiety as an 8-Het-2-aminobenzazepine-linker compound with uniform stoichiometry (e.g., a single For antibodies with one engineered cysteine ​​site, up to two 8-Het-2-aminobenzazepine moieties per antibody).

[0126] In some embodiments, the cysteine ​​engineered antibodies used to prepare the immunoconjugates of Table 3 have a cysteine ​​residue introduced at the 149-lysine site of the light chain (LC K149C). In other embodiments, the cysteine ​​engineered antibody has a cysteine ​​residue introduced at the 118-alanine site (EU numbering) (HC A118C) of the heavy chain. This site is alternatively numbered 121 by sequential numbering or 114 by Kabat numbering. In other embodiments, cysteine ​​engineered antibodies have cysteine ​​residues introduced in the light chain of Kabat numbering G64C or R142C, or in the heavy chain of Kabat numbering D101C, V184C, or T205C.

[0127] 8-HET-2-aminobenzazepine adjuvant compound The immunoconjugate of the invention includes an 8-Het-2-aminobenzazepine adjuvant moiety. An adjuvant moiety described herein is a compound that elicits an immune response (ie, an immunostimulant). Generally, the adjuvant moieties described herein are TLR agonists. TLRs are type I transmembrane proteins involved in the initiation of innate immune responses in vertebrates. TLRs recognize a variety of pathogen-associated molecular patterns from bacteria, viruses, and fungi and serve as the first line of defense against invading pathogens. TLRs elicit overlapping but distinct biological responses due to differences in cellular expression and the signaling pathways they initiate. When engaged (e.g., by natural stimuli or synthetic TLR agonists), TLRs activate nuclear factor-κB (NF-κB) and IL-1 through the adapter protein myeloid differentiation primary response gene 88 (MyD88). Initiates a signaling cascade that leads to the recruitment of receptor-associated kinase (IRAK). Phosphorylation of IRAK then leads to recruitment of TNF-receptor-associated factor 6 (TRAF6), which results in phosphorylation of the NF-κB inhibitor I-κB. As a result, NF-κB enters the cell nucleus and initiates transcription of genes whose promoters contain NF-κB binding sites, such as cytokines. Additional regulatory modes for TLR signaling include TIR domain-containing adapter-induced interferon-β (TRIF)-dependent induction of TNF receptor-associated factor 6 (TRAF6) and activation of the MyD88-independent pathway through 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.

[0128] Typically, the adjuvant moieties described herein are TLR7 and / or TLR8 agonists. Both TLR7 and TLR8 are expressed on monocytes and dendritic cells. In humans, TLR7 is also expressed on plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is primarily expressed in cells of myeloid origin, namely monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 can detect the presence of "foreign" single-stranded RNA within cells as a means of responding to viral invasion. Treatment of TLR8-expressing cells with TLR8 agonists can result in 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 TLR7 agonists can result in the production of high levels of IFN-α and other inflammatory cytokines. TLR7 / TLR8 engagement and resultant cytokine production can activate dendritic cells and other antigen-presenting cells and drive diverse innate and acquired immune response mechanisms leading to tumor destruction.

[0129] Exemplary 8-Het-2-aminobenzazepine compounds (Hx) of the invention are shown in Table 1. Each compound was synthesized, purified, characterized by mass spectrometry and shown to have the indicated mass. Additional experimental procedures can be found in the Examples. Activity against human embryonic kidney (HEK) 293NFKB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 202. The 8-Het-2-aminobenzazepine compounds of Table 1 demonstrate surprising and unexpected properties of TLR8 agonist selectivity that may predict useful therapeutic activity for treating cancer and other disorders. . [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] [Table 17-6] [Table 17-7] [Table 17-8] [Table 17-9] [Table 17-10] [Table 17-11] [Table 17-12]

[0130] 8-HET-2-aminobenzazepine-linker compound The immunoconjugates of the invention are prepared by conjugation of anti-CEA antibodies with the 8-Het-2-aminobenzazepine-linker compound, HxBzL. 8-Het-2-aminobenzazepine-linker compounds include an 8-Het-2-aminobenzazepine (HxBz) moiety covalently attached to a linker unit. The linker unit includes functional groups and subunits that affect the stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugate. The linker unit includes a reactive functional group that reacts with, or conjugates to, a reactive functional group of the antibody. For example, a nucleophilic group such as a lysine side chain amino of an antibody reacts with an electrophilic reactive functional group of a HxBzL linker compound to form the subject immunoconjugate. Also, for example, the cysteine ​​thiol of the antibody reacts with the maleimide or bromoacetamide group of the Hx-linker compound to form the subject immunoconjugate.

[0131] Suitable electrophilic reactive functional groups for HxBzL linker compounds include N-hydroxysuccinimidyl (NHS) ester and N-hydroxysulfosuccinimidyl (sulfo-NHS) ester (amine reactive), carbodiimide (amine and carboxyl reactivity), hydroxymethylphosphine (amine reactivity), maleimide (thiol reactivity), halogenated acetamides (thiol reactivity) such as N-iodoacetamide, aryl azides (primary amine reactivity), fluorinated aryl azides (reactivity due to carbon hydrogen (C-H) insertion), pentafluorophenyl (PFP) ester (amine reactivity), terafluorophenyl (TFP) ester (amine reactivity), imido ester (amine reactivity), isocyanate (hydroxyl reaction) Examples include, but are not limited to, vinyl sulfones (thiol-, amine-, and hyphoroxyl-reactive), pyridyl disulfides (thiol-reactive), and benzophenone derivatives (reactive via C-H bond insertion). Additional reagents include Hermanson, Bioconjugate Techniques 2 nd Edition, Academic Press, 2008, but is not limited to these.

[0132] The present invention provides a solution to the limitations and challenges to the design, preparation, and use of immunoconjugates. Some linkers can be unstable in the bloodstream, thereby releasing an unacceptable amount of adjuvant / drug before internalization within target cells (Khot, A. et al (2015) Bioanalysis 7(13):1633-1648). Other linkers may provide stability in the bloodstream, but intracellular release efficacy may be negatively affected. Linkers that provide the desired intracellular release typically have low stability in the bloodstream. Stated another way, blood flow stability and intracellular release are typically inversely related. Additionally, standard conjugation processes depend on 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 final purified conjugate that can be obtained. are interrelated. For example, aggregate formation generally correlates positively with the number of equivalents of adjuvant / drug moieties and derivatives thereof conjugated to the antibody. Under high drug loading, the formed aggregates must be removed for therapeutic use. As a result, drug loading-mediated aggregate formation can reduce immune complex yields and make process scale-up difficult.

[0133] An exemplary embodiment has the formula II: [ka] 8-Het-2-aminobenzazepine-linker compound, wherein Het is selected from heterocyclyldiyl and heteroaryldiyl; R 1 ,R 2 ,R 3 , and R 4 But independently, H, C 1 -C 12 Alkyl, C 2 -C 6 alkenyl, C 2 -C 6 Alkynyl, C 3 -C 12 Carbocyclyl, C 6 -C 20 Aryl, C 2 -C 9 heterocyclyl, and C 1 -C 20 selected from the group consisting of heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally -(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 1 -C 12 alkyldiyl)-OR 5 , -(C 3 -C 12 carbocyclyl), -(C 3 -C 12 carbocyclyl)-*, -(C 3 -C 12 carbocyclyl)-(C 1 -C 12 alkyldiyl)-NR 5 -*, -(C 3 -C 12 carbocyclyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 3 -C 12 carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*, -(C 6 -C 20 aryl), -(C 6 -C 20 aryldiyl)-*, -(C 6 -C 20 aryldiyl)-N(R 5 )-*, -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-*, -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-NR 5 -C(=NR 5a )N(R 5 )-*, -(C 2 -C 20 heterocyclyl), -(C 2 -C 20 heterocyclyl)-*, -(C 2 -C 9 heterocyclyl)-(C 1 -C 12 alkyldiyl)-NR 5 -*, -(C 2 -C 9 heterocyclyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 2 -C 9 heterocyclyl)-C(=O)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 2 -C 9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*, -(C 2 -C 9 heterocyclyl)-NR 5 -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 2 -C 9 heterocyclyl)-(C 6 -C 20 aryldiyl)-*, -(C 1 -C 20 heteroaryl), -(C 1 -C 20 heteroaryldiyl)-*, -(C 1 -C 20 heteroaryl)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 1 -C 20 heteroaryl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 1 -C 20 heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*, -(C 1 -C 20 heteroaryl)-N(R 5 )C(=O)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -C(=O)-*, -C(=O)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -C(=O)-(C 2 -C 20 heterocyclyldiyl)-*, -C(=O)N(R 5 ) 2 , -C(=O)N(R 5 )-*, -C(=O)N(R 5 )-(C 1 -C 12 alkyldiyl)-N(R 5 )C(=O)R 5 , -C(=O)N(R 5 )-(C 1 -C 12 alkyldiyl)-N(R 5 )C(=O)N(R 5 ) 2 , -C(=O)NR 5 -(C 1 -C 12 alkyldiyl)-N(R 5 )CO 2 R 5 , -C(=O)NR 5 -(C 1 -C 12 alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 ) 2 , -C(=O)NR 5 -(C 1 -C 12 alkyldiyl)-NR 5 C(=NR 5a )R 5 , -C(=O)NR 5 -(C 1 -C 8 alkyldiyl)-NR 5 (C 2 -C 5 heteroaryl), -C(=O)NR 5 -(C 1 -C 20 heteroaryldiyl)-N(R 5 )-*, -C(=O)NR 5 -(C 1 -C 20 heteroaryldiyl)-*, -C(=O)NR 5 -(C 1 -C 20 heteroaryldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -C(=O)NR 5 -(C 1 -C 20 heteroaryldiyl)-(C 2 -C 20 heterocyclyldiyl)-C(=O)NR 5 -(C 1 -C 12 alkyldiyl)-NR 5 -*, -N(R 5 ) 2 , -N(R 5 )-*, -N(R 5 )C(=O)R 5 , -N(R 5 )C(=O)-*, -N(R 5 )C(=O)N(R 5 ) 2 , -N(R 5 )C(=O)N(R 5 )-*, -N(R 5 )CO 2 R 5 , -NR 5 C(=NR 5a )N(R 5 ) 2 , -NR 5 C(=NR 5a )N(R 5 )-*, -NR 5 C(=NR 5a )R 5 , -N(R 5 )C(=O)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -N(R 5 )-(C 2 -C 5 heteroaryl), -N(R 5 )-S(=O) 2 -(C 1 -C 12 alkyl), -O-(C 1 -C 12 alkyl), -O-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -O-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -O-C(=O)N(R 5 ) 2 , -O-C(=O)N(R 5 )-*, -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-*, -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-(C 1 -C 12 alkyldiyl)-NR 5 -*,as well as -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-(C 1 -C 12 substituted with one or more groups selected from alkyldiyl)-OH, or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring, X 1 ,X 2 ,X 3 , and X 4 but independently, the combination, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O) 2 , and S(O) 2 N(R 5 ), R 5 But independently, H, C 6 -C 20 Aryl, C 3 -C 12 Carbocyclyl, C 6 -C 20 Aryljiyl, C 1 -C 12 alkyl, and C 1 -C 12 selected from the group consisting of alkyldiyl or two R 5 the groups together form a 5- or 6-membered heterocyclyl ring, R 5a However, C 6 -C 20 Aryl and C 1 -C 20 selected from the group consisting of heteroaryl, In the formula, the asterisk * indicates the binding site of L, and R 1 ,R 2 ,R 3 , and R 4 One of them is bonded to L, L is Q-C(=O)-PEG-, Q-C(=O)-PEG-C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-C(=O)-Gluc-, Q-C(=O)-PEG-O-, Q-C(=O)-PEG-O-C(=O)-, Q-C(=O)-PEG-C(=O)-, Q-C(=O)-PEG-C(=O)-PEP-, Q-C(=O)-PEG-N(R 6 )-, Q-C(=O)-PEG-N(R 6 )-C(=O)-, Q-C(=O)-PEG-N(R 6 )-PEG-C(=O)-PEP-, Q-C(=O)-PEG-N + (R 6 ) 2 -PEG-C(=O)-PEP-, Q-C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-, Q-C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 monoheterocyclyldiyl)-, Q-C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-, Q-C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-C(=O)-, Q-C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-, Q-C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-, Q-C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 5 )-C(=O), Q-C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 6 )C(=O)-(C 2 -C 5 monoheterocyclyldiyl)-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-C(=O)-Gluc-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-C(=O)-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-C(=O)-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-, Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-, Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-, Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-, and Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 a linker selected from the group consisting of monoheterocyclyldiyl)-, R 6 but independently, H or C 1 -C 6 is alkyl, PEG has the formula:-(CH 2 CH 2 O) n -(CH 2 ) m -, m is an integer from 1 to 5, n is an integer from 2 to 50, Gluc has the formula: [ka] has PEP is the formula: [ka] , where AA is independently selected from natural or non-natural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered proline amino acid. form, the wavy lines indicate the bonding points, Cyc is optionally F, Cl, NO 2 , -OH, -OCH 3 , and C substituted with one or more groups selected from glucuronic acid having the following structure: 6 -C 20 Aryldiyl and C 1 -C 20 selected from heteroaryldiyl, [ka] R 7 But -CH(R 8 )O-, -CH 2 -,-CH 2 N(R 8 )-, and -CH(R 8 )O-C(=O)-, where R 8 But, H, C 1 -C 6 Alkyl, C(=O)-C 1 -C 6 Alkyl, and -C(=O)N(R 9 ) 2 selected from, where R 9 But independently, H, C 1 -C 12 alkyl, and -(CH 2 CH 2 O) n -(CH 2 ) m -OH, where m is an integer from 1 to 5, n is an integer from 2 to 50, or two R 9 the groups together form a 5- or 6-membered heterocyclyl ring, y is an integer from 2 to 12, z is 0 or 1, Q is F, Cl, NO 2 , and S.O. 3 - selected from the group consisting of N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with one or more groups independently selected from Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally F, Cl, Br, I, -CN, -CH 3 ,-CH 2 CH 3 ,-CH=CH 2 , -C≡CH, -C≡CCH 3 ,-CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 ,-CH 2 CH(CH 3 ) 2 ,-CH 2 OH, -CH 2 OCH 3 ,-CH 2 CH 2 OH, -C(CH 3 ) 2 OH, -CH(OH)CH(CH 3 ) 2 , -C(CH 3 ) 2 CH 2 OH, -CH 2 CH 2 S.O. 2 CH 3 ,-CH 2 OP(O)(OH) 2 ,-CH 2 F, -CHF 2 ,-CF 3 ,-CH 2 C.F. 3 ,-CH 2 CHF 2 , -CH(CH 3 )CN, -C(CH 3 ) 2 CN, -CH 2 CN, -CH 2 N.H. 2 ,-CH 2 NHSO 2 CH 3 ,-CH 2 NHCH 3 ,-CH 2 N(CH 3 ) 2 ,-CO 2 H,-COCH 3 ,-CO 2 CH 3 ,-CO 2 C(CH 3 ) 3 , -COCH(OH)CH 3 ,-CONH 2 ,-CONHCH 3 ,-CON(CH 3 ) 2 , -C(CH 3 ) 2 CONH 2 ,-NH 2 , -NHCH 3 , -N(CH 3 ) 2 ,-NHCOCH 3 , -N(CH 3 )COCH 3 ,-NHS(O) 2 CH 3 , -N(CH 3 )C(CH 3 ) 2 CONH 2 , -N(CH 3 )CH 2 CH 2 S(O) 2 CH 3 , -NHC(=NH)H, -NHC(=NH)CH 3 , -NHC(=NH)NH 2 ,-NHC(=O)NH 2 ,-NO 2 , =O, -OH, -OCH 3 ,-OCH 2 CH 3 ,-OCH 2 CH 2 OCH 3 ,-OCH 2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 , -O(CH 2 CH 2 O) n -(CH 2 ) m C.O. 2 H, -O(CH 2 CH 2 O) n H, -OCH 2 F,-OCHF 2 ,-OCF 3 , -OP(O)(OH) 2 , -S(O) 2 N(CH 3 ) 2 ,-SCH 3 , -S(O) 2 CH 3 , and -S(O) 3 Substituted with one or more groups independently selected from H.

[0134] Exemplary embodiments of the 8-Het-2-aminobenzazepine-linker compounds of Formula II provide that Q is: [ka] including being selected from.

[0135] Exemplary embodiments of the 8-Het-2-aminobenzazepine-linker compounds of Formula II include Q is phenoxy substituted with one or more F.

[0136] An exemplary embodiment of the 8-Het-2-aminobenzazepine-linker compound of Formula II includes Q is 2,3,5,6-tetrafluorophenoxy.

[0137] Exemplary embodiments of 8-Het-2-aminobenzazepine-linker (HxBzL) compounds are selected from Table 2a and Table 2b. Each compound was synthesized, purified, characterized by mass spectrometry and shown to have the indicated mass. Additional experimental procedures can be found in the Examples. The 8-Het-2-aminobenzazepine-linker compounds of Tables 2a and 2b exhibit surprising and unexpected TLR8 agonist selectivity that may predict useful therapeutic activity for treating cancer and other disorders. Demonstrate characteristics that do not. The 8-Het-2-aminobenzazepine-linker intermediate, Formula II compound of Table 2 is used conjugated to an antibody by the method of Example 201 to form the immunoconjugates of Table 3a and Table 3b. do. [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5] [Table 18-6] [Table 18-7] [Table 18-8] [Table 18-9] [Table 18-10] [Table 18-11] [Table 18-12] [Table 18-13] [Table 18-14] [Table 18-15] [Table 18-16] [Table 18-17] [Table 18-18] [Table 18-19] [Table 18-20] [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4] [Table 19-5] [Table 19-6] [Table 19-7]

[0138] CEA immune complex Immunostimulatory antibody complexes, or immune complexes, target TLR7 / 8 agonists into tumors to activate tumor-infiltrating myeloid cells and initiate a broad range of innate and adaptive anti-tumor immune responses. (Ackerman, et al., (2021) Nature Cancer 2:18-33).

[0139] CEA (CEACAM5) is a well-validated cell surface antigen that is highly expressed on multiple solid tumors. The favorable properties of CEA, including robust cell surface expression, low internalization rate, and limited normal tissue expression, make the antigen a suitable candidate for immune complexes in a multifunctional approach to treating CEA-expressing cancers. Suggests it may be a target.

[0140] An exemplary embodiment of an immunoconjugate comprises an anti-CEA antibody covalently attached to one or more 8-Het-2-aminobenzazepine (Hx) moieties by a linker and has the formula I: Ab-[L-Hx] p I has During the ceremony, the Ab is an antibody construct having an antigen binding domain that binds CEA; p is an integer from 1 to 8, Hx is the formula: [ka] 8-Het-2-aminobenzazepine moiety with Het selected from heterocyclyldiyl and heteroaryldiyl, R 1 ,R 2 ,R 3 , and R 4 But independently, H, C 1 -C 12 Alkyl, C 2 -C 6 alkenyl, C 2 -C 6 Alkynyl, C 3 -C 12 Carbocyclyl, C 6 -C 20 Aryl, C 2 -C 9 heterocyclyl, and C 1 -C 20 selected from the group consisting of heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally -(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 1 -C 12 alkyldiyl)-OR 5 , -(C 3 -C 12 carbocyclyl), -(C 3 -C 12 carbocyclyl)-*, -(C 3 -C 12 carbocyclyl)-(C 1 -C 12 alkyldiyl)-NR 5 -*, -(C 3 -C 12 carbocyclyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 3 -C 12 carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*, -(C 6 -C 20 aryl), -(C 6 -C 20 aryldiyl)-*, -(C 6 -C 20 aryldiyl)-N(R 5 )-*, -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-*, -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl)-NR 5 -C(=NR 5a )N(R 5 )-*, -(C 2 -C 20 heterocyclyl), -(C 2 -C 20 heterocyclyl)-*, -(C 2 -C 9 heterocyclyl)-(C 1 -C 12 alkyldiyl)-NR 5 -*, -(C 2 -C 9 heterocyclyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 2 -C 9 heterocyclyl)-C(=O)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 2 -C 9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*, -(C 2 -C 9 heterocyclyl)-NR 5 -(C 6 -C20 aryldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 2 -C 9 heterocyclyl)-(C 6 -C 20 aryldiyl)-*, -(C 1 -C 20 heteroaryl), -(C 1 -C 20 heteroaryl), -(C 1 -C 20 heteroaryl)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -(C 1 -C 20 heteroaryl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -(C 1 -C 20 heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*, -(C 1 -C 20 heteroaryl)-N(R 5 )C(=O)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -C(=O)-*, -C(=O)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -C(=O)-(C 2 -C 20 heterocyclyldiyl)-*, -C(=O)N(R 5 ) 2 , -C(=O)N(R 5 )-*, -C(=O)N(R 5 )-(C 1 -C 12 alkyldiyl)-N(R 5 )C(=O)R 5 , -C(=O)N(R 5 )-(C 1 -C 12 alkyldiyl)-N(R 5 )C(=O)N(R 5 ) 2 , -C(=O)NR 5 -(C 1 -C 12 alkyldiyl)-N(R 5 )CO 2 R 5 , -C(=O)NR 5 -(C 1 -C 12 alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 ) 2 , -C(=O)NR 5 -(C 1 -C 12 alkyldiyl)-NR 5 C(=NR 5a )R 5 , -C(=O)NR 5 -(C 1 -C 8 alkyldiyl)-NR 5 (C 2 -C 5 heteroaryl), -C(=O)NR 5 -(C 1 -C 20 heteroaryldiyl)-N(R 5 )-*, -C(=O)NR 5 -(C 1 -C 20 heteroaryldiyl)-*, -C(=O)NR 5 -(C 1 -C 20 heteroaryldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -C(=O)NR 5 -(C 1 -C 20 heteroaryldiyl)-(C 2 -C 20 heterocyclyldiyl)-C(=O)NR 5 -(C 1 -C 12 alkyldiyl)-NR 5 -*, -N(R 5 ) 2 , -N(R 5 )-*, -N(R 5 )C(=O)R 5 , -N(R 5 )C(=O)-*, -N(R 5 )C(=O)N(R 5 ) 2 , -N(R 5 )C(=O)N(R 5 )-*, -N(R 5 )CO 2 R 5 , -NR 5 C(=NR 5a )N(R 5 ) 2 , -NR 5 C(=NR 5a )N(R 5 )-*, -NR 5 C(=NR 5a )R 5 , -N(R 5 )C(=O)-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -N(R 5 )-(C 2 -C 5 heteroaryl), -N(R 5 )-S(=O) 2 -(C 1 -C 12 alkyl), -O-(C 1 -C 12 alkyl), -O-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -O-(C 1 -C 12 alkyldiyl)-N(R 5 )-*, -O-C(=O)N(R 5 ) 2 , -O-C(=O)N(R 5 )-*, -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-*, -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-(C 1 -C 12 alkyldiyl)-N(R 5 ) 2 , -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-(C 1 -C 12 alkyldiyl)-NR 5 -*,as well as -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-(C 1 -C 12 substituted with one or more groups selected from alkyldiyl)-OH, or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring, X 1 ,X 2 ,X 3 , and X 4 but independently, the combination, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O) 2 , and S(O) 2 N(R 5 ), R 5 are independently H, C 6 -C 20 Aryl, C 3 -C 12 Carbocyclyl, C 6 -C 20 Aryljiyl, C 1 -C 12 alkyl, and C 1 -C 12 selected from the group consisting of alkyldiyl or two R 5 the groups together form a 5- or 6-membered heterocyclyl ring, R 5a However, C 6 -C20 Aryl and C 1 -C 20 selected from the group consisting of heteroaryl, In the formula, the asterisk * indicates the binding site of L, and R 1 ,R 2 ,R 3 , and R 4 One of them is bonded to L, L is -C(=O)-PEG-, -C(=O)-PEG-C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-C(=O)-Gluc-, -C(=O)-PEG-O-, -C(=O)-PEG-O-C(=O)-, -C(=O)-PEG-C(=O)-, -C(=O)-PEG-C(=O)-PEP-, -C(=O)-PEG-N(R 6 )-, -C(=O)-PEG-N(R 6 )-C(=O)-, -C(=O)-PEG-N(R 6 )-PEG-C(=O)-PEP-, -C(=O)-PEG-N + (R 6 ) 2 -PEG-C(=O)-PEP-, -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-, -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 monoheterocyclyldiyl)-, -C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-, -C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-C(=O)-, -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-, -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-, -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 5 )-C(=O), -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 6 )C(=O)-(C 2 -C 5 monoheterocyclyldiyl)-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-C(=O)-Gluc-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-C(=O)-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-C(=O)-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-, -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-, -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-, -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-, and -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 a linker selected from the group consisting of monoheterocyclyldiyl)-; R 6 but independently, H or C 1 -C 6 is alkyl, PEG has the formula:-(CH 2 CH 2 O) n -(CH 2 ) m -, m is an integer from 1 to 5, n is an integer from 2 to 50, Gluc has the formula: [ka] has PEP is the formula: [ka] , where AA is independently selected from natural or non-natural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered proline amino acid. form, the wavy line indicates the bonding point, Cyc is optionally F, Cl, NO 2 , -OH, -OCH 3 , and C substituted with one or more groups selected from glucuronic acid having the following structure: 6 -C 20 Aryldiyl and C 1 -C 20 selected from heteroaryldiyl, [ka] R 7 But -CH(R 8 )O-, -CH 2 -,-CH 2 N(R 8 )-, and -CH(R 8 )O-C(=O)-, where R 8 But, H, C 1 -C 6 Alkyl, C(=O)-C 1 -C 6 Alkyl, and -C(=O)N(R 9 ) 2 selected from, where R 9 But independently, H, C 1 -C 12 alkyl, and -(CH 2 CH 2 O) n -(CH 2 ) m -OH, where m is an integer from 1 to 5, n is an integer from 2 to 50, or two R 9 the groups together form a 5- or 6-membered heterocyclyl ring, y is an integer from 2 to 12, z is 0 or 1, Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally F, Cl, Br, I, -CN, -CH 3 ,-CH 2 CH3 ,-CH=CH 2 , -C≡CH, -C≡CCH 3 ,-CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 ,-CH 2 CH(CH 3 ) 2 ,-CH 2 OH, -CH 2 OCH 3 ,-CH 2 CH 2 OH, -C(CH 3 ) 2 OH, -CH(OH)CH(CH 3 ) 2 , -C(CH 3 ) 2 CH 2 OH, -CH 2 CH 2 S.O. 2 CH 3 ,-CH 2 OP(O)(OH) 2 ,-CH 2 F, -CHF 2 ,-CF 3 ,-CH 2 C.F. 3 ,-CH 2 CHF 2 , -CH(CH 3 )CN, -C(CH 3 ) 2 CN, -CH 2 CN, -CH 2 N.H. 2 ,-CH 2 NHSO 2 CH 3 ,-CH 2 NHCH 3 ,-CH 2 N(CH 3 ) 2 ,-CO 2 H,-COCH 3 ,-CO 2 CH 3 ,-CO 2 C(CH 3 ) 3 , -COCH(OH)CH 3 ,-CONH 2 ,-CONHCH 3 ,-CON(CH 3 ) 2 , -C(CH 3 ) 2 CONH 2 , -NH 2 , -NHCH 3 , -N(CH 3 ) 2 ,-NHCOCH 3 , -N(CH 3 )COCH 3 ,-NHS(O) 2 CH 3 , -N(CH 3 )C(CH 3 ) 2 CONH 2 , -N(CH 3 )CH 2 CH 2 S(O) 2 CH 3 , -NHC(=NH)H, -NHC(=NH)CH 3 , -NHC(=NH)NH 2 ,-NHC(=O)NH 2 ,-NO 2 , =O, -OH, -OCH 3 ,-OCH 2 CH 3 ,-OCH 2 CH 2 OCH 3 ,-OCH 2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 , -O(CH 2 CH 2 O) n -(CH 2 ) m C.O. 2 H, -O(CH 2 CH 2 O) n H, -OCH 2 F,-OCHF 2 ,-OCF 3 , -OP(O)(OH) 2 , -S(O) 2 N(CH 3 ) 2 ,-SCH 3 , -S(O) 2 CH 3 , and -S(O) 3 Substituted with one or more groups independently selected from H.

[0141] Exemplary embodiments of the immunoconjugate of Formula I include that the antibody is selected from labetuzumab and alsitumomab, or biosimilars or biobetters thereof.

[0142] Exemplary embodiments of the immunoconjugate of Formula I provide that the antibody construct is a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 3, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, CDR-L3 containing the amino acid sequence of SEQ ID NO: 7, CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 19, CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, CDR-L3 containing the amino acid sequence of SEQ ID NO: 23, CDR-H1 containing the amino acid sequence of SEQ ID NO: 26, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 35, CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, CDR-L3 containing the amino acid sequence of SEQ ID NO: 39, CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 53, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, CDR-L3 containing the amino acid sequence of SEQ ID NO: 39, CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; e) CDR-L1 containing the amino acid sequence of SEQ ID NO: 59, CDR-L2 containing the amino acid sequence of SEQ ID NO: 61, CDR-L3 containing the amino acid sequence of SEQ ID NO: 63, CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71; f) CDR-L1 containing the amino acid sequence of SEQ ID NO: 75, CDR-L2 containing the amino acid sequence of SEQ ID NO: 77, CDR-L3 containing the amino acid sequence of SEQ ID NO: 79, CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 85, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; g) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 91, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 93, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 95, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 99; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 103; h) CDR-L1 containing the amino acid sequence of SEQ ID NO: 107, CDR-L2 containing the amino acid sequence of SEQ ID NO: 109, CDR-L3 containing the amino acid sequence of SEQ ID NO: 111, CDR-H1 containing the amino acid sequence of SEQ ID NO: 115, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120; or i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 107, CDR-L2 containing the amino acid sequence of SEQ ID NO: 109, CDR-L3 containing the amino acid sequence of SEQ ID NO: 111, CDR-H1 containing the amino acid sequence of SEQ ID NO: 124, It includes CDR-H2, which includes the amino acid sequence of SEQ ID NO: 126, and CDR-H3, which includes the amino acid sequence of SEQ ID NO: 128.

[0143] Exemplary embodiments of the immunoconjugate of Formula I provide that the antibody construct comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89, and 105. and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122, and 130. Including.

[0144] Exemplary embodiments of the immunoconjugate of Formula I provide that the antibody construct comprises a variable light chain comprising an amino acid sequence selected from SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89, and 105; a variable heavy chain comprising an amino acid sequence selected from numbers 9, 41, 65, 81, 97, 113, 122, and 130.

[0145] An exemplary embodiment of the immunoconjugate of Formula I provides that the antibody construct comprises a variable light chain comprising an amino acid sequence from SEQ ID NO: 105 and a heavy chain CDR (complementarity determining region) comprising an amino acid sequence from SEQ ID NO: 118. CDR-H2.

[0146] An exemplary embodiment of the immunoconjugate of Formula I provides that the antibody construct comprises a variable light chain comprising the amino acid sequence from SEQ ID NO: 105 and a variable heavy chain comprising the amino acid sequence from SEQ ID NO: 113. include.

[0147] Exemplary embodiments of the immunoconjugate of Formula I include that Het is selected from the group consisting of pyridyldiyl, pyrimidyldiyl, pyrazolyldiyl, piperazinyldiyl, piperidinyldiyl, and pyrazinyldiyl.

[0148] An exemplary embodiment of the immunoconjugate of formula I is 1 is a combination and R 1 includes that is H.

[0149] An exemplary embodiment of the immunoconjugate of formula I is 2 is a combination and R 2 However, C 1 -C 8 Including being alkyl.

[0150] An exemplary embodiment of the immunoconjugate of formula I is 2 and X 3 are each a combination and R 2 and R 3 But independently, C 1 -C 8 Alkyl, -O-(C 1 -C 12 alkyl), -(C 1 -C 12 alkyldiyl)-OR 5 , -(C 1 -C 8 alkyldiyl)-N(R 5 )CO 2 R 5 , -(C 1 -C 12 alkyl)-OC(O)N(R 5 ) 2 ,-O-(C 1 -C 12 alkyl)-N(R 5 )CO 2 R 5 , and -O-(C 1 -C 12 alkyl)-OC(O)N(R 5 ) 2 including being selected from.

[0151] An exemplary embodiment of the immunoconjugate of Formula I is R 2 However, C 1 -C 8 is alkyl and R 3 But -(C 1 -C 8 alkyldiyl)-N(R 5 )CO 2 R 5 Including being.

[0152] An exemplary embodiment of the immunoconjugate of Formula I is R 2 But -CH 2 CH 2 CH 3 and R 3 But -CH 2 CH 2 CH 2 N.H.C.O. 2 (t-Bu), -OCH 2 CH 2 N.H.C.O. 2 (cyclobutyl), and -CH 2 CH 2 CH 2 N.H.C.O. 2 (cyclobutyl).

[0153] An exemplary embodiment of the immunoconjugate of Formula I is R 2 and R 3 , each independently, -CH 2 CH 2 CH 3 ,-OCH 2 CH 3 ,-OCH 2 C.F. 3 ,-CH 2 CH 2 C.F. 3 ,-OCH 2 CH 2 OH, and -CH 2 CH 2 CH 2 Including being selected from OH.

[0154] An exemplary embodiment of the immunoconjugate of Formula I is R 2 and R 3 , respectively -CH 2 CH 2 CH 3 Including being.

[0155] An exemplary embodiment of the immunoconjugate of Formula I is R 2 But -CH 2 CH 2 CH 3 and R 3 But -OCH 2 CH 3 Including being.

[0156] An exemplary embodiment of the immunoconjugate of formula I is 3 -R 3 but, [ka] including being selected from.

[0157] An exemplary embodiment of the immunoconjugate of formula I is 4 is a combination and R 4 includes that is H.

[0158] An exemplary embodiment of the immunoconjugate of Formula I is R 1 is connected to L.

[0159] An exemplary embodiment of the immunoconjugate of Formula I is R 2 or R 3 is connected to L.

[0160] An exemplary embodiment of the immunoconjugate of formula I is 3 -R 3 -L is [ka] where the wavy line indicates the point of attachment to N.

[0161] An exemplary embodiment of the immunoconjugate of Formula I is R 4 However, C 1 -C 12 Including being alkyl.

[0162] An exemplary embodiment of the immunoconjugate of Formula I is R 4 But -(C 1 -C 12 alkyldiyl)-N(R 5 )-*, where the asterisk * indicates the binding site of L.

[0163] Exemplary embodiments of the immunoconjugate of Formula I include L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0164] An exemplary embodiment of the immunoconjugate of Formula I includes L conjugated to cysteine ​​thiol of the antibody.

[0165] Exemplary embodiments of the immunoconjugate of Formula I include, for PEG, m is 1 or 2 and n is an integer from 2 to 10.

[0166] An exemplary embodiment of the immunoconjugate of formula I is where L comprises PEP, PEP is a dipeptide, and the formula: [ka] including being selected from.

[0167] An exemplary embodiment of the immunoconjugate of formula I is AA 1 and A.A. 2 but independently, H, -CH 3 , -CH(CH 3 ) 2 ,-CH 2 (C 6 H 5 ), -CH 2 CH 2 CH 2 CH 2 N.H. 2 ,-CH 2 CH 2 CH 2 NHC(NH)NH 2 ,-CHCH(CH 3 )CH 3 ,-CH 2 S.O. 3 H, and -CH 2 CH 2 CH 2 NHC(O)NH 2 Selected from or AA 1 and A.A. 2 involves forming a five-membered ring proline amino acid.

[0168] An exemplary embodiment of the immunoconjugate of formula I is AA 1 is -CH(CH 3 ) 2 and A.A. 2 But -CH 2 CH 2 CH 2 NHC(O)NH 2 Including being.

[0169] An exemplary embodiment of the immunoconjugate of formula I is AA 1 and A.A. 2 but independently, GlcNAc aspartate, -CH 2 S.O. 3 H, and -CH2 OPO 3 Including being selected from H.

[0170] Exemplary embodiments of the immunoconjugate of formula I are those in which PEP has the formula: [ka] , where AA 1 and A.A. 2 are independently selected from the side chains of naturally occurring amino acids.

[0171] An exemplary embodiment of the immunoconjugate of formula I is where L comprises PEP, PEP is a tripeptide, and the formula: [ka] including being selected from.

[0172] An exemplary embodiment of the immunoconjugate of formula I is where L comprises PEP, PEP is a tetrapeptide, and the formula: [ka] including being selected from.

[0173] An exemplary embodiment of the immunoconjugate of Formula I is: A.A. 1 is selected from the group consisting of Abu, Ala, and Val; A.A. 2 is selected from the group consisting of Nle(O-Bzl), Oic, and Pro, A.A. 3 But Ala and Met(O) 2 selected from the group consisting of A.A. 4 But Oic, Arg(NO 2 ), Bpa, and Nle(O-Bzl).

[0174] Exemplary embodiments of the immunoconjugate of Formula I include L comprising PEP, and PEP is Ala-Pro-Val, Asn-Pro-Val, Ala-Ala-Val, Ala-Ala-Pro-Ala( SEQ ID NO: 131), Ala-Ala-Pro-Val (SEQ ID NO: 132), and Ala-Ala-Pro-Nva (SEQ ID NO: 133).

[0175] An exemplary embodiment of the immunoconjugate of Formula I is where L comprises PEP, and PEP has the structure: [ka] including being selected from.

[0176] Exemplary embodiments of the immunoconjugate of Formula I have the structure: [ka] In the formula, the wavy line is R 5 including indicating a bond to.

[0177] Formula Ia: [ka] An exemplary embodiment of an immunoconjugate of formula I having:

[0178] An exemplary embodiment of the immunoconjugate of formula Ia is 4 is a combination and R 4 includes that is H.

[0179] An exemplary embodiment of the immunoconjugate of formula Ia is 2 and X 3 are each a combination and R 2 and R 3 But independently, C 1 -C 8 Alkyl, -O-(C 1 -C 12 alkyl), -(C 1 -C 12 alkyldiyl)-OR 5 , -(C 1 -C 8 alkyldiyl)-N(R 5 )CO 2 R 5 , -(C 1 -C 12 alkyl)-OC(O)N(R 5 ) 2 , -O-(C 1 -C 12 alkyl)-N(R 5 )CO 2 R 5 , and -O-(C 1 -C 12 alkyl)-OC(O)N(R 5 ) 2 including being selected from.

[0180] Formula Ib~Ii: [ka] [ka] An exemplary embodiment of an immunoconjugate of Formula Ia selected from:

[0181] An exemplary embodiment of the immunoconjugate of formula Ia is 2 and X 3 are each a bond and R 2 and R 3 But independently, C 1 -C 8 Alkyl, -O-(C 1 -C 12 alkyl), -(C 1 -C 12 alkyldiyl)-OR 5 , -(C 1 -C 8 alkyldiyl)-N(R 5 )CO 2 R 5 , and -O-(C 1 -C 12 alkyl)-N(R 5 )CO 2 R 5 including being selected from.

[0182] An exemplary embodiment of the immunoconjugate of formula Ia is 2 and X 3 are each a combination and R 2 However, C 1 -C 8 is alkyl and R 3 But -O-(C 1 -C 12 alkyl) and -O-(C 1 -C 12 alkyl)-N(R 5 )CO 2 R 5 including being selected from.

[0183] The invention includes all reasonable combinations of the embodiments of formula I and permutations of features.

[0184] In certain embodiments, immunoconjugate compounds of the invention include those that have immunostimulatory activity. The antibody-drug conjugates of the present invention selectively deliver effective doses of 8-Het-2-aminobenzazepine drug to tumor tissue, thereby making it more effective than unconjugated 8-Het-2-aminobenzazepine. Greater selectivity (ie, lower effective doses) can be achieved while increasing the therapeutic index ("therapeutic window") by comparison.

[0185] Drug loading is expressed as p, which is the number of HxBz moieties per antibody in the immunoconjugate of formula I. Drug (HxBz) loading can range from 1 to about 8 drug moieties (D) per antibody. The immunoconjugate of Formula I comprises a mixture or collection of antibodies conjugated to a range of from 1 to about 8 drug moieties. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues, such as lysine and cysteine. In some embodiments, free cysteine ​​residues are introduced into antibody amino acid sequences by the methods described herein. In such embodiments, p may be 1, 2, 3, 4, 5, 6, 7, or 8, or ranges thereof, such as 1-8 or 2-5. In any such embodiment, p and n are equal (ie, p=n=1, 2, 3, 4, 5, 6, 7, or 8, or some range therebetween). Exemplary immunoconjugates of Formula I include, but are not limited to, antibodies with 1, 2, 3, or 4 engineered cysteine ​​amino acids (Lyon, R. et al. (2012) Methods in Enzym.502:123-138). In some embodiments, one or more free cysteine ​​residues are already present in the antibody to form an intrachain disulfide bond without the use of manipulation, in which case the existing free cysteine ​​residues are used to form an intrachain disulfide bond. , the antibody may be conjugated to a drug. In some embodiments, the antibody is exposed to reducing conditions prior to conjugation of the antibody to generate one or more free cysteine ​​residues.

[0186] In some immunoconjugates, p may be limited by the number of binding sites on the antibody. For example, if the linkage is a cysteine ​​thiol, as in certain exemplary embodiments described herein, the antibody may have only one or a limited number of cysteine ​​thiol groups; Or it may have only one or a limited number of sufficiently reactive thiol groups to which the drug may be attached. In other embodiments, one or more lysine amino groups in the antibody may be available and reactive for conjugation with an Hx-linker compound of Formula II. In certain embodiments, higher drug loading, eg, p>5, can cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading of the immunoconjugate ranges from 1 to about 8, about 2 to about 6, or about 3 to about 5. In certain embodiments, antibodies are subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.

[0187] The loading of the immune complex (drug / antibody ratio) can be controlled in different ways, e.g. by (i) limiting the molar excess of Hx-linker intermediate compound over the antibody and (ii) limiting the conjugation reaction time or temperature. and (iii) partial or limited reductive denaturation conditions for optimized antibody reactivity.

[0188] It is to be understood that when two or more nucleophilic groups of an antibody react with a drug, the resulting product is a mixture of immunoconjugated compounds with a distribution of one or more drug moieties attached to the antibody. . The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay that is specific for the antibody and specific for the drug. Individual immune complex molecules can be identified in mixtures by mass spectrometry and separated by HPLC, e.g. hydrophobic interaction chromatography (e.g. McDonagh et al. (2006) Prot. Engr. Design & Selection 19 ( 7):299-307, Hamblett et al. (2004) Clin. Cancer Res.10:7063-7070, Hamblett, K.J., et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,”Abstract No.624,American Association for Cancer Research,2004 Annual Meeting,March 27-31,2004,Proceedings of the AACR,Volume 45,March 2004,Alley,S.C.,et al.,“Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogeneous immune complexes with a single loading value can be isolated from a conjugation mixture by electrophoresis or chromatography.

[0189] Exemplary embodiments of the immunoconjugates of Formula I are selected from the anti-CEA, HxBz immunoconjugates of Table 3a and Table 3b. In vitro evaluation of immune complex activity was performed according to the method of Example 203. [Table 20-1] [Table 20-2] [Table 21]

[0190] Composition of immune complexes The present invention provides compositions, e.g., pharmaceutical or pharmacologically acceptable carriers, comprising a plurality of immunoconjugates described herein and optionally a carrier thereof, e.g. Provides a physically acceptable composition or formulation. The immunoconjugates may be the same or different in composition, i.e., the composition may include immunoconjugates with the same number of adjuvants linked to the same position on the antibody construct, and / or on the antibody construct. Immune complexes with the same number of Hx adjuvants linked to different positions, different numbers of adjuvants linked to the same position on the antibody construct, or different numbers of adjuvants linked to different positions on the antibody construct can include an immune complex having a

[0191] In an exemplary embodiment, the composition comprising the subject immunoconjugate compounds comprises a mixture of immunoconjugate compounds, and the average drug (Hx) load per antibody in the mixture of immunoconjugate compounds is from about 2 to about It is 5.

[0192] The immunoconjugate compositions of the invention can have an average adjuvant to antibody construct ratio (DAR) of about 0.4 to about 10. Those skilled in the art will appreciate that the number of 8-Het-2-aminobenzazepine adjuvants conjugated to an antibody construct can vary from immunoconjugate to immunoconjugate in a composition comprising multiple immunoconjugates of the invention; It will therefore be appreciated that the adjuvant-to-antibody construct (eg, antibody) ratio can be measured as an average, which can be referred to as the drug (adjuvant) to antibody ratio (DAR). The adjuvant-to-antibody construct (eg, antibody) ratio can be assessed by any suitable means, many of which are known in the art.

[0193] The average number of adjuvant sites per antibody (DAR) in the preparation of immune complexes from conjugation reactions can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. Quantitative distribution of immune complexes in the composition in terms of p can also be determined. In some cases, the separation, purification, and characterization of homogeneous immune complexes for which p is a particular value from other drug-loaded immune complexes is accomplished by means such as reversed-phase HPLC or electrophoresis. can be achieved.

[0194] In some embodiments, the composition further comprises one or more pharmaceutically or pharmacologically acceptable excipients. For example, the immunoconjugates of the invention can be formulated for parenteral administration, such as IV administration or administration into a body cavity or lumen of an organ. Alternatively, the immune complex can be injected intratumorally. Injectable compositions generally include a solution of the immune complex dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that may be employed are water and isotonic solutions of one or more salts such as sodium chloride, eg, Ringer's solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can similarly be used in the preparation of injectables. These compositions are desirably sterile and generally free of undesirable materials. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutical additives as necessary to approximate physiological conditions, such as pH adjusting agents and buffers, toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. It may contain acceptable auxiliary substances.

[0195] The composition can contain any suitable concentration of immunoconjugate. The concentration of immunoconjugate in the composition can vary widely and is selected based primarily on fluid volume, viscosity, body weight, etc., depending on the particular mode of administration chosen and the needs of the patient. In certain embodiments, the concentration of the immunoconjugate in the injectable solution formulation ranges from about 0.1% (w / w) to about 10% (w / w).

[0196] Biological activity of immune complexes Immune complex IC-2 differentially binds surface-expressed CEA on a panel of cell lines and correlates with CEA transcript levels, as shown in the table below. [Table 22]

[0197] Human colorectal cancer arrays (n=247), non-small cell lung cancer arrays (n=69), and gastric / gastroesophageal cancer arrays (n=114) were stained with the CEA31 IHC assay (Ventana / Cell Marque). The H-score is calculated as (percent of cells with 1+ staining intensity) + (percent of cells with 2+ staining intensity 2X) + (percent of cells with 3+ staining intensity 3X). IC-2 binding sites per cell represent the number of IC-2 molecules that a given tumor cell binds and correlates with the level of antigen expression on the surface of the cell. Viable tumor cells were harvested and labeled with Alexa Flour488-labeled IC-2 or Alexa Flour488-labeled hIgG1 isotype control at 100 nM, followed by flow cytometry analysis. IC-2 binding sites were determined using QSC beads from Bangs Laboratories. Non-specific binding sites were corrected by subtracting the hIgG1 isotype control binding site from the IC-2 binding site.

[0198] Figure 1 shows a graph of an in vivo xenograft tumor model in mice. Measurement of tumor volume over time after treatment showed that immune complex IC-2, isotype immune complex (ISAC) and naked antibody CEA.9 in tumor inhibition of mice bearing CEA-rich human pancreatic HPAF-II tumors -Compared effectiveness with G1fhL2. Immunoconjugate IC-2 exhibits dose-dependent growth inhibition of CEA-rich human pancreatic HPAF-II tumors at dose levels as low as 0.5 mg / kg. Isotype ISAC is an immunoconjugate of anti-CD20 antibody (rituximab) conjugated to HxBzL-5, the same adjuvant linker as IC-2. Isotype ISAC has the same adjuvant linker (HxBzL-5) as IC-2. Isotype ISAC serves as an off-target, negative control, showing little or no tumor growth inhibition. Naked antibody CEA.9-G1fhL2 also shows little or no tumor growth inhibition in this study. These results demonstrate dose-dependent tumor recruitment of innate effector cells and induction of immunostimulatory cytokines and suggest that the immune conjugates of the invention may be effective in treating CEA-expressing cancers.

[0199] Figure 2a–e shows CEA-high gastric carcinoma MKN-45 cells and cDC enrichment by immune complexes IC-2, IC-3, IC-4, IC-6, IC-14, and naked antibody CEA.9-G1fhL2. Figure 3 shows a graph of the induction of various cytokines in co-culture with primary cell isolates. Cytokines IL-12p70 (Figure 2a), TNFα (tumor necrosis factor α) (Figure 2b), IL-6 (interleukin-6) (Figure 2c), IFNγ (interferon γ) (Figure 2d), and CCL2 (Figure 2e) ) The level of secretion by the cells into the supernatant was measured. Induction of these cytokines is associated with mounting an immune response against cancer. Various concentrations of immune complexes IC-2, IC-3, IC-4, IC-6, IC-14, and naked antibody CEA.9-G1fhL2 were added to CEA-high MKN-45 cells and cDC-enriched primary cell preparations. (E:T=10:1) for 18 hours and then the supernatant was collected. Secreted cytokine levels were determined using the LegendPlexcytokine bead array kit. The tested immune complexes differ in the levels of cytokines induced as a function of the adjuvant. The native CEA.9-G1fhL2 antibody induces little or no cytokine secretion, demonstrating dependence on TLR7 / 8 activating adjuvants.

[0200] Figures 3a-d show CEA-high HPAFII cells (Figure 3a), CEA-medium LoVo cells (Figure 3b), CEA-low LS-174T cells (Figure 3c), and CEA-negative MDA-MB-231 cells (Figure 3c). In 3d), a graph of phagocytosis by M-CSF differentiated monocyte-derived macrophages treated with various concentrations of immune complex IC-2 is shown. CTG-labeled tumor-IC-2 immune complexes were incubated with M-CSF differentiated monocyte-derived macrophages at a 2:1 effector-to-target ratio. After 4 hours, phagocytosis was measured by flow cytometry gating on effector cells positive for CTG signals. The mean + / - standard deviation from 3 donors is shown in the graph. Antibody-dependent cellular phagocytosis (ADCP) is a mechanism by which antibody-opsonized target cells activate FcγRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cells. Immune complex IC-2 induces dose-dependent phagocytosis of CEA-high HPAFII (EC50=9.2±2.3 nM) and CEA-medium LoVo (EC50=11.4±3.5 nM). Minimal ADCP is observed for CEA-low LS-174T. IC-2 does not induce ADCP of CEA-negative-MDA-MB-231. These results demonstrate that induction of ADCP by IC-2 is dependent on medium / high CEA expression by target tumor cells.

[0201] Figures 4a–f show the supernatants after incubating various concentrations of immune complex IC-2 and naked antibody CEA.9-G1fhL2 with co-culture of cancer cells and cDC-enriched primary cell isolates. Figure 2 shows graphs of secreted cytokine levels and induction of cell surface markers. Immune complex IC-2 and naked antibody CEA.9-G1fhL2 were combined with CEA-positive tumor cells (HPAF-II, LoVo, or LS174-T) and cDC-enriched primary cell preparations (E:T=10:1). After incubating for an hour, the supernatant and cells were collected. Secreted cytokine levels in the supernatants (Figures 4a-d) were determined using the LegendPlex cytokine bead array kit. Induction of cell surface markers (Figure 4e-f) was determined by flow cytometry. In co-cultures of cancer cells and cDC-enriched primary cell isolates, the CEA-targeted immune complex IC-2 was linked to cytokines TNFalpha (Figure 4a), IL-6 (Figure 4a), and IL-6 (Figure 4a), which are associated with mounting immune responses against cancer. Figure 4b), induces secretion of IL-12p70 (Figure 4c), and CXCL10 (Figure 4d). In addition, surface levels of CD40 (Figure 4e) and CD86 (Figure 4f) antigens were elevated, consistent with activation of innate immunity (myeloid cells). Levels of cytokine and surface marker induction are similar in CEA-high HPAF-II and CEA-medium LoVo cells, but are significantly reduced in CEA-low LS-174T cells. Cytokine and surface marker studies demonstrate activation of CEA-expressing tumor cells and myeloid cells when exposed to anti-CEA ISAC IC-2. Activation is observed in CEA-high HPAF-II cells and CEA-medium LoVo cells. Activation is low or undetectable in CEA-low LS-174T cells and CEA-negative MDA-MB-231 cells. Natural antibody CEA.9-G1fhL2 does not induce bone marrow activation. Results from Figures 4a-f demonstrate the dependence of IC-2 activity on CEA expression levels associated with human cancer. The native CEA.9-G1fhL2 antibody induces little or no cytokine secretion, demonstrating dependence on the TLR7 / 8 activating payload.

[0202] How to treat cancer with immune complexes The present invention provides methods for treating cancer. The method comprises administering a therapeutically effective amount of an immune complex described herein (e.g., as a composition described herein) to a subject in need thereof, e.g., having cancer and suffering from cancer. including administering to a subject in need of treatment. The method includes administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 3a and Table 3b.

[0203] It is contemplated that the immunoconjugates of the invention can be used, for example, to treat a variety of hyperproliferative diseases or disorders characterized by overexpression of tumor antigens. Exemplary hyperproliferative disorders include solid tumors, benign or malignant, and hematological disorders such as leukemias and lymphoid malignancies.

[0204] In another aspect, an immunoconjugate for use as a pharmaceutical is provided. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual that includes administering to the individual an effective amount of the immune conjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, eg, as described herein.

[0205] In a further aspect, the invention provides the use of an immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the pharmaceutical agent is for the treatment of cancer, and the method includes administering to an individual with cancer an effective amount of the pharmaceutical agent. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, eg, as described herein.

[0206] Carcinoma is a malignant tumor derived from epithelial tissue. Epithelial cells line the external surfaces of the body, line the internal cavities, and form the lining of glandular tissue. Examples of carcinoma include adenocarcinoma (cancer that develops in glandular (secretory) cells such as breast cancer, pancreatic cancer, lung cancer, prostate cancer, stomach cancer, gastroesophageal cancer, and colon cancer), adrenocortical cancer, 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, polycystic renal cell carcinoma, oat cell carcinoma, large cell lung cancer , small cell lung cancer, non-small cell lung cancer, etc., but are not limited to these. Carcinomas may be found in the prostate, pancreas, colon, brain (usually as secondary metastases), lungs, breasts, and skin. In some embodiments, the method for treating non-small cell lung cancer comprises an immunoconjugate containing an antibody construct capable of binding CEA (e.g., labetuzumab, or a biosimilar thereof, or a biobetter thereof). including administering.

[0207] Soft tissue tumors are a highly diverse group of rare tumors derived from connective tissue. Examples of soft tissue tumors include alveolar soft tissue sarcoma, angiomatoid fibrous histiocytoma, chondromuscular oxidative fibroma, skeletal chondrosarcoma, extraskeletal myxoid chondrosarcoma, clear cell sarcoma, and desmoplastic small round cell Tumor, dermatofibrosarcoma protuberance, endometrial stromal tumor, Ewing sarcoma, fibromatosis (desmoid), infantile fibrosarcoma, gastrointestinal stromal tumor, giant cell tumor of bone, tenosynovial giant cell tumor, inflammatory muscle fiber Blastic tumor, uterine leiomyoma, leiomyosarcoma, lipoblastoma, typical lipoma, spindle cell or pleomorphic lipoma, atypical lipoma, chondrolipoma, well-differentiated liposarcoma, myxoid / round cell liposarcoma, pleomorphic liposarcoma, myxoid malignant fibrous histiocytoma, high-grade malignant fibrous histiocytoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, mesothelioma, neuroblastoma, Osteochondroma, osteosarcoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, embryonic rhabdomyosarcoma, benign or malignant schwannoma, synovial sarcoma, Evan's tumor, nodular fasciitis, desmoid type Fibromatosis, solitary fibrous tumor, dermatofibrosarcoma protuberans (DFSP), angiosarcoma, epithelioid hemangioendothelioma, tenosynovial giant cell tumor (TGCT), pigmented villonodular synovitis (PVNS), fibrosis Sexual dysplasia, myxofibrosarcoma, fibrosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumors, neurofibromas, pleomorphic adenomas of soft tissues, as well as fibroblasts, myofibroblasts, histiocytes, vascular cells / endothelium cells, and neoplasms derived from nerve sheath cells.

[0208] Sarcomas are a rare type of cancer that arises in cells of mesenchymal origin, such as bones or soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supporting tissue. Different types of sarcoma are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma in fat, and rhabdomyosarcoma in muscle. Examples of sarcomas include Askin's tumor, botryoides sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma, and soft tissue sarcomas (e.g., alveolar soft tissue sarcoma, angiosarcoma, cystic sarcoma, fjord fibrous sarcoma) protuberance (DFSP), desmoid tumor, deplastic small round cell tumor, epithelial sarcoma, extraosseous chondrosarcoma, extraosseous osteosarcoma, fibrous sarcoma, peptic gastrointestinal stromal tumor (GIST), vascular cancer, angiosarcoma ( (more commonly referred to as "angiosarcoma"), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, synovial sarcoma, and undifferentiated tumor. plastic sarcoma), but are not limited to these.

[0209] A metamorphoma can contain several different types of tissue, including, for example, hair, muscle, and bone (e.g., any and / or all of the three germ layers: endoderm, mesoderm, and ectoderm). is a type of germ cell tumor that can include tissue derived from. Deformities occur most frequently on the ovaries in women, testicles in men, and the coccyx in children.

[0210] Melanoma is a form of cancer that develops in melanocytes (cells that make the pigment melanin). Melanoma can develop in moles (cutaneous melanoma) but can also develop in other pigmented tissues, such as the eyes or intestines.

[0211] Merkel cell carcinoma is a rare type of skin cancer that usually appears as flesh-colored or bluish-red nodules on the face, head, or neck. Merkel cell carcinoma is also called neuroendocrine carcinoma of the skin. In some embodiments, the method for treating Merkel cell carcinoma comprises administering an immunoconjugate containing an antibody construct capable of binding CEA (e.g., labetuzumab, a biosimilar thereof, or a biobetter thereof). including doing. In some embodiments, the Merkel cell carcinoma has metastasized at the time the administration occurs.

[0212] Leukemia is a cancer that begins in blood-forming tissues such as the bone marrow, where large numbers of abnormal blood cells are produced and enter the bloodstream. For example, leukemia can originate from bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named by the rate of onset and progression of the disease (eg, acute versus chronic) and the type of white blood cells affected (eg, bone marrow versus lymph). Myeloid leukemia is also called myeloid or myeloblastic leukemia. Lymphocytic leukemia is also called lymphoblastic or lymphocytic leukemia. Lymphocytic leukemia cells can collect in lymph nodes, which can become swollen. Examples of leukemias include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL).

[0213] Lymphoma is a cancer that affects cells of the immune system. For example, lymphomas can be derived 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), which is characterized by the presence of a type of cell called Reed-Sternberg cells. Currently, six types of HL are recognized. Examples of Hodgkin's lymphoma include nodular sclerosis classic Hodgkin's lymphoma (CHL), mixed cellular CHL, lymphopenic CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0214] Another category of lymphoma is non-Hodgkin's lymphoma (NHL), which includes a large and diverse group of cancers of immune system cells. Non-Hodgkin's lymphoma is further divided into cancers that have an indolent (slow-growing) course and cancers that have a rapid (rapid-growing) course. Currently, 61 types of NHL are recognized. Examples of non-Hodgkin's lymphoma include AIDS-related lymphoma, anaplastic large cell lymphoma, angioimmunoblastic lymphoma, blastic NK cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma), chronic Lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, enteropathic T-cell lymphoma, follicular lymphoma, hepatosplenic gamma delta T-cell lymphoma, T-cell leukemia, lymphoma Blastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-cell lymphoma, childhood lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, therapy-related T-cell lymphoma, and Waldenstrees These include, but are not limited to, mummacroglobulinemia.

[0215] Brain tumors include any cancer of brain tissue. Examples of brain tumors include gliomas (e.g., glioblastomas, astrocytomas, oligodendrogliomas, ependymomas, etc.), meningiomas, pituitary adenomas, and vestibular Schwannomas, primitive neuroectodermal tumors ( including, but not limited to, medulloblastoma).

[0216] The immunoconjugates of the invention can be used in therapy either alone or in combination with other agents. For example, the immunoconjugate may be administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. Such combination therapy includes combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, where administration of the immunoconjugate is combined with the additional therapeutic agent and / or It may occur before, simultaneously with, and / or after administration of the adjuvant. Immunoconjugates can also be used in combination with radiation therapy.

[0217] The immunoconjugates of the invention (and any additional therapeutic agents) can be administered by any suitable means, including oral, parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for topical treatment. can be administered by. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be carried out by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[0218] The immunoconjugate can be administered to a subject in need thereof in any therapeutically effective amount using any suitable dosing regimen, such as those utilized for labetuzumab, its biosimilars, and its biobetters. Ru. For example, the method can include administering the immune complex to provide a dose of about 100 ng / kg to about 50 mg / kg to the subject. Immunoconjugate doses can range from about 5 mg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The immunoconjugate dose can be about 100, 200, 300, 400, or 500 μg / kg. The immunoconjugate dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. Immunoconjugate doses can also fall outside these ranges depending on the particular conjugate and the type and severity of cancer being treated. The frequency of administration can range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about 1 time per month to about 5 times per week. In some embodiments, the immunoconjugate is administered once a week.

[0219] In another aspect, the invention provides a method for preventing cancer. The method includes administering to a subject a therapeutically effective amount of an immunoconjugate (eg, as a composition as described above). In certain embodiments, the subject is susceptible to the particular cancer that is being prevented.

[0220] Some embodiments of the invention provide methods for treating cancer as described above, where the cancer is breast cancer. Breast cancer can arise from different areas of the breast, and a number of different types of breast cancer have been characterized. For example, the immunoconjugate of the invention can be used to detect ductal carcinoma in situ, invasive ductal carcinoma (e.g., tubular, medullary, mucinous, papillary, or cribriform carcinoma), lobular carcinoma in situ, It can be used to treat other forms of breast cancer, such as invasive lobular carcinoma, inflammatory breast cancer, and triple-negative (tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer. In some embodiments, a method for treating breast cancer comprises an antibody construct capable of binding to CEA (e.g., levetuzumab, a biosimilar thereof, or a biobetter thereof) or a tumor that overexpresses CEA. and administering an immunoconjugate to the patient.

[0221] In some embodiments, the cancer is susceptible to pro-inflammatory responses induced by TLR7 and / or TLR8.

[0222] In some embodiments, a therapeutically effective amount of the immune complex is administered to cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial cancer, lung cancer. , non-small cell lung cancer, Merkel cell cancer, colon cancer, colorectal cancer, gastric cancer, or breast cancer in need of treatment. Merkel cell carcinoma can be metastatic Merkel cell carcinoma. Breast cancer can be triple negative breast cancer. Esophageal cancer may be gastroesophageal junction adenocarcinoma.

Example

[0223] Example L-2 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-Amino-4- [ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6 -Synthesis of tetrafluoro-benzenesulfonic acid, HxBzL-2 [ka] tert-Butyl3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-(4,4,5,5-tetramethyl- Preparation of 1,3,2-dioxaborolan-2-yl)pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate, HxBzL-2a PPh 3 (1.35 g, 5.15 mmol, 1 eq.) and DEAD (0.89 g, 5.15 mmol, 0.94 mL, 1 eq.) were added at 0 °C, stirred at 25 °C for 0.5 h, then tert-butyl 3-[2- [2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid Salt (3.02g, 5.15mmol, 1eq) was added and then stirred at 25°C for 16 hours. The reaction mixture was diluted with 20 mL of water and extracted with EtOAc (50 mL*3). Wash the mixed organic layer with brine (20mL*3) and remove Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate=50 / 1 to ethyl acetate:MeOH=10:1) to obtain HxBzL-2a (3.5 g, 4.59 mmol, yield 89.04%) as a yellow oil.

[0224] tert-Butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-[ethoxy(propyl)] Preparation of HxBzL-2c HxBzL-2a (625 mg, 819 umol, 2.5 eq.), 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, HxBzL-2b (120 mg, 328 umol, 1 eq.), Na in water (0.3mL) 2 C.O. 3 (69.5 mg, 655 umol, 2 eq.) and [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride, Pd(dppf)Cl in DMF (3 mL). 2 (23.9 mg, 32.8 umol, 0.1 eq.) was degassed and then N 2 The mixture was heated to 120°C for 5 hours. The mixture was filtered, concentrated under reduced pressure, and the residue was subjected to preparative HPLC (TFA conditions, column: Phenomenex luna C18 250*50mm*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 35% ~65%, 10 min) to give HxBzL-2c (300 mg, 290 umol, 88.4% yield, TFA) as a yellow solid.

[0225] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]- Preparation of 3H-1-benzazepin-8-yl]pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL-2d To a solution of HxBzL-2c (300 mg, 325 umol, 1 eq) in water (3 mL) and MeCN (0.5 mL) was added HCl (12M, 407 uL, 15 eq) and then stirred at 80 °C for 0.5 h. The mixture was concentrated under reduced pressure to give compound 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino- 4-[Ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (200 mg , 222 umol, yield 68.1%, HCl) was obtained as a colorless oil.

[0226] Preparation of HxBzL-2 HxBzL-2d (80.0 mg, 88.7 umol, 1 eq., HCl) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (119 mg, 443 umol) in DCM (1 mL) and DMA (1 mL). , 5 eq.) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDCI (84.9 mg, 443 umol, 5 eq.) and then stirred at 25° C. for 0.5 h. The mixture was filtered, concentrated under reduced pressure, and the residue was subjected to preparative HPLC (TFA conditions, column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 25 ~ 50%, 8 min) to give HxBzL-2 (30 mg, 24.8 umol, 28.01% yield, TFA) as a yellow oil. 1 H NMR(400MHz,MeOD)δ 8.20(s,1H),7.93(s,1H),7.65-7.61(m,1H),7.59(s,1H),7.55-7.52(m,1H),7.40(s ,1H),4.36(t,J=4.8Hz,2H),3.96(q,J=7.2Hz,2H),3.89-3.82(m,4H),3.74(t,J=7.2Hz,2H),3.63 -3.52(m,36H),3.42(s,2H),2.95(t,J=5.6Hz,2H),1.76(sxt,J=7.2Hz,2H),1.20(t,J=7.2Hz,3H) ,0.99(t,J=7.6Hz,3H).LC / MS[M+H]1094.4(calculated value), LC / MS[M+H]1094.3(observed value).

[0227] Example L-4 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[4-[5-[2-amino -4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]piperazin-1-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-4 [ka] 2-Amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-1-benzazepine-4-carboxamide, HxBz -Preparation of 4b 2-Amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz-4a (0.5 g, 1.37 mmol, 1 eq.) in dioxane (10 mL), 4,4, 5,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (520 mg, 2.05 mmol, 1.5 equivalent), Pd(dpppf)Cl 2 (99.9 mg, 137 umol, 0.1 eq.), KOAc (335 mg, 3.41 mmol, 2.5 eq.) in N 2 The mixture was stirred at 100° C. for 1 hour. The crude HxBz-4b was used in the next step without purification and obtained (564 mg, 1.36 mmol, 99.96% yield) as a black liquid.

[0228] Tert-Butyl 4-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin--8-yl]pyrimidin-2-yl]piperazine-1-carboxylate, HxBz-4 preparation of HxBz-4b (0.45 g, 1.09 mmol, 1 eq.) in dioxane (4 mL) and water (0.5 mL), Pd(dppf)Cl 2 (39.8mg, 54.4umol, 0.05eq), K 2 C.O. 3 (376 mg, 2.72 mmol, 2.5 eq.), tert-butyl 4-(5-bromopyrimidin-2-yl)piperazine-1-carboxylate (374 mg, 1.09 mmol, 1 eq.) in N 2 The mixture was stirred at 100° C. for 1 hour. The mixture was concentrated to remove dioxane and the residue was diluted with EtOAc (10 mL) and water (5 mL). organic layer with Na 2 S.O. 4 Dry on top and concentrate to give a residue. The residue was subjected to column chromatography (SiO 2 , petroleum ether / ethyl acetate=1 / 0~0 / 1, then EA:MeOH=1.5:1), then preparative HPLC, column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 30% to 55%, 8 min) to obtain HxBz-4 (0.35 g, 637 umol, yield 58.5%) as a brown oil. 1 H NMR(400MHz,MeOD)δ8.74(s,2H),7.72-7.63(m,2H),7.60(d,J=1.6Hz,1H),7.45(s,1H),3.99(q,J= 7.2Hz,2H),3.93-3.88(m,4H),3.77(t,J=7.2Hz,2H),3.60-3.51(m,4H),3.43(s,2H),1.80-1.75(m,2H) ),1.51(s,9H),1.22(t,J=7.2Hz,3H),1.02(t,J=7.2Hz,3H).LC / MS[M+H]550.3(calculated value),LC / MS [M+H]550.2 (observed value).

[0229] Preparation of 2-amino-N-ethoxy-8-(2-piperazin-1-ylpyrimidin-5-yl)-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz-3 To a mixture of HxBz-4 (20 mg, 36.4 umol, 1 eq) in DCM (5 mL) was added HCl / EtOAc (4M, 5 mL, 550 eq) and stirred at 25 °C for 0.5 h. The mixture was concentrated to give HxBz-3 (10.5 mg, 21.4 umol, 58.9% yield, 99.233% purity, HCl) as a white solid. 1 H NMR(400MHz,MeOD)δ8.70(s,2H),7.65-7.47(m,3H),7.32(s,1H),4.14-3.96(m,4H),3.86(q,J=7.2Hz, 2H),3.64(t,J=7.2Hz,2H),3.31(s,2H),3.25-3.21(m,4H),1.71-1.62(m,2H),1.08(t,J=7.2Hz,3H ),0.89(t,J=7.2Hz,3H).LC / MS[M+H]450.3 (calculated value), LC / MS[M+H]450.1 (observed value).

[0230] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1[3-[2-amino-4-[3-(tert -butoxycarbonylamino)propyl-ethoxy-carbamoyl]-3H-1-benzazepin-8-yl]phenyl]sulfonylazetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy] Preparation of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL-4a A mixture of HxBz-3 (110 mg, 176 umol, 1 eq.) in DMF (3 mL) was added with DIEA (63.5 mg, 491 umol, 2.8 eq.) and 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]propanoic acid (99.2 mg, 140 umol, 0.8 eq.) was added and then stirred at 25° C. for 0.5 h. The mixture was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um, mobile phase: [water (10mM NH 4 HCO 3 )-ACN], B%: 20%-55%, 8 min) to give HxBzL-4a (28 mg, 24 umol, 13.7% yield) as a yellow oil.

[0231] Preparation of HxBzL-4 HxBzL-4a (78 mg, 78.8 umol, 1 eq) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (106 mg, 394 umol, 5 EDCI (75.5 mg, 394 umol, 5 eq.) was added to the mixture of 75.5 mg, 394 umol, 5 eq.) and then stirred at 20° C. for 0.5 h. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 20%~40%, 10 minutes) to obtain HxBzL. -4 (39.8 mg, 26.4 umol, yield 33.5%, purity 95.944%, 2TFA) was obtained as a colorless oil. 1 H NMR(400MHz,MeOD)δ8.75(s,2H),7.76-7.55(m,3H),7.45(s,1H),4.02-3.73(m,16H),3.68-3.58(m,36H), 3.37(s,2H),2.99(t,J=6.0Hz,2H),2.76(t,J=6.0Hz,2H),1.85-1.74(m,2H),1.25-1.20(m,3H),1.02 (t,J=7.2Hz,3H).LC / MS[M+H]1218.5 (calculated value), LC / MS[M+H]1218.3 (observed value).

[0232] Example L-5 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[Ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy Synthesis of ]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-5 [ka] Preparation of 5-bromo-2-(bromomethyl)pyrimidine, HxBz-5b PPh 3 (499mg, 1.90mmol, 1.2eq) and CBr 4 (631mg, 1.90mmol, 1.2eq), N 2 Added all at once at 0°C. The mixture was stirred at 20°C for 10 hours. Water (10 mL) was added, the aqueous phase was extracted with ethyl acetate (10 mL*3), the combined organic phase was washed with brine (10 mL), anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 8 / 1) to obtain HxBz-5b (290 mg, 1.15 mmol, yield). 72.4%) was obtained as a white solid. 1 H NMR(400MHz, CDCl 3 )δ8.81(s,2H),4.59(s,2H).

[0233] Preparation of Tert-Butyl N-[(5-bromopyrimidin-2-yl)methyl]-N-tert-butoxycarbonyl-carbamate, HxBz-5c Cs 2 C.O. 3 (562mg, 1.73mmol, 1.5eq), N 2 was added portionwise at 20° C. and the mixture was stirred at 20° C. for 2.5 hours. Water (5 mL) was added, the aqueous phase was extracted with ethyl acetate (5 mL*3), the combined organic phase was washed with brine (5 mL), anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 5 / 1) to obtain HxBz-5c (350 mg, 901 umol, yield 78.3%) was obtained as a white solid. 1 H NMR(400MHz, CDCl 3 )δ8.74(s,2H),5.01(s,2H),1.48(s,18H).

[0234] tert-Butyl N-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzaze-pin-8-yl]pyrimidin-2-yl]methyl]-N-tert-butoxy Preparation of carbonyl-carbamate, HxBz-5d Dioxane (10mL) and H 2HxBz-5c (184 mg, 473 umol, 1.0 equiv.) and 2-amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2- Pd(dppf)Cl was added to a mixture of dioxaborolan-2-yl)-3H-1-benzazepine-4-carboxamide (195 mg, 474 umol, 1.0 eq.). 2 ·CH 2 Cl 2 (19.3 mg, 23.7 umol, 0.05 equivalent) and K 2 C.O. 3 (163mg, 1.18mmol, 2.5eq), N 2 Degas the mixture by adding in one portion under N 2 The mixture was heated to 90°C for 2 hours. Dioxane (10 mL) was removed in vacuo, water (20 mL) was added, the aqueous phase was extracted with ethyl acetate (10 mL*3), the combined organic phases were washed with brine (10 mL) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100~200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 0 / 1 ~ ethyl acetate / methanol = 10 / 1) to obtain HxBz. -5d (280 mg, 470.83 umol, yield 99.35%) was obtained as a gray solid. 1 H NMR(400MHz,MeOD)δ9.08(s,2H),7.61(s,1H),7.59(d,J=2.8Hz,2H),7.38(s,1H),5.08(s,2H),3.98 (q,J=7.2Hz,2H),3.76(t,J=7.2Hz,2H),1.83-1.75(m,2H),1.47(s,18H),1.20(t,J=7.2Hz,3H) ,1.02(t,J=7.2Hz,3H).

[0235] Preparation of 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz-5 To a solution of HxBz-5d (20.0 mg, 33.6 umol, 1.0 eq.) in EtOAc (5 mL) was added HCl / EtOAc (4M, 8.41 uL, 1.0 eq.) with N 2 was added in one portion at 20° C. and the mixture was stirred at 20° C. for 1 hour. The reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 1%~30%, 8 min) to obtain HxBz -5 (6.2 mg, 9.84 umol, yield 29.2%, purity 98.8%, 2TFA) was obtained as a white solid. 1 H NMR(400MHz,MeOD)δ9.22(s,2H),7.82(d,J=2.0Hz,1H),7.79-7.75(m,2H),7.47(s,1H),4.49(s,2H) ,4.00(q,J=7.2Hz,2H),3.78(t,J=7.2Hz,2H),3.46(s,2H),1.85-1.77(m,2H),1.22(t,J=7.2Hz, 3H),1.03(t,J=7.2Hz,3H).LC / MS[M+H]395.2 (calculated value), LC / MS[M+H]395.1 (observed value).

[0236] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl] -3H-1-Benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL- Preparation of 5a HxBz-5 (70 mg, 149 umol, 1.0 eq., 2HCl) and 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]propanoic acid (127mg, 179umol, 1.2eq) DIEA (77.4mg, 599umol, 104uL, 4.0eq) was added to a mixture of N 2 was added in one portion at 25° C. and the mixture was stirred at 25° C. for 0.5 h. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex luna C18 80*40mm*3um, mobile phase: [water (0.04%HCl)-ACN], B%: 12%-39%, 5.5 min). Purification provided HxBzL-5a (50.0 mg, 53.4 umol, 35.7% yield) as a yellow oil. 1 H NMR(400MHz,MeOD)δ9.14(s,2H),7.86-7.81(m,1H),7.78-7.74(m,2H),7.48(s,1H),4.72(s,2H),4.00( q,J=7.2Hz,2H),3.85-3.71(m,8H),3.69-3.58(m,38H),3.47(s,2H),2.62(t,J=6.0Hz,2H),2.55(t ,J=6.4Hz,2H),1.85-1.76(m,2H),1.23(t,J=7.2Hz,3H),1.03(t,J=7.2Hz,3H).

[0237] Preparation of HxBzL-5 HxBzL-5a (60 mg, 61.7 umol, 1.0 eq., HCl) and (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxysodium ( EDCI (71.0mg, 370umol, 6.0eq) was added to a mixture of N 2 was added in one portion at 25° C. and the mixture was stirred at 25° C. for 1 h. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 20%-45%, 8 min). Purification provided HxBzL-5 (38.0 mg, 30.5 umol, 49.3% yield, 93.3% purity) as a yellow oil. 1 H NMR(400MHz,MeOD)δ9.11(s,2H),7.83-7.79(m,1H),7.77(s,1H),7.76-7.71(m,1H),7.47(s,1H),4.71( s,2H),4.00(q,J=7.2Hz,2H),3.88(t,J=5.6Hz,2H),3.85-3.75(m,5H),3.70-3.57(m,38H),3.47(s ,2H),2.99(t,J=6.0Hz,2H),2.62(t,J=4Hz,2H),1.85-1.75(m,2H),1.23(t,J=7.2Hz,3H),1.02( t,J=7.2Hz,3H).LC / MS[M+H]1163.3 (calculated value), LC / MS[M+H]1163.3 (observed value).

[0238] Example L-7 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[[5-[2 -amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]-3-pyridyl]sulfonyl]azetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-7 [ka] Preparation of Tert-butyl ((1-((5-bromopyridin-3-yl)sulfonyl)azetidin-3-yl)methyl)carbamate, HxBz-7b Tert-butyl N-(azetidin-3-ylmethyl)carbamate (762 mg, 4.09 mmol, 1.05 eq) and 5-bromopyridine-3-sulfonyl chloride, HxBz-7a (1 g, 3.90 mmol, 2.26 mL) in DCM (20 mL) , 1 equivalent) in a mixture of Et 3 N (789 mg, 7.80 mmol, 1.09 mL, 2 equivalents), N 2 was added at 25° C. and then stirred at 25° C. for 1 hour. Into the mixture, H 2 O (20 mL) was added and then concentrated in vacuo to remove DCM. The desired solid precipitated from the mixture was filtered to give the desired product HxBz-7b (1.1 g, 2.71 mmol, 69.45% yield) as a white solid. 1 H NMR(DMSO-d 6 ,400MHz)δ9.09(d,J=2.0Hz,1H),8.93(d,J=2.0Hz,1H),8.40(t,J=2.0Hz,1H),6.90(t,J=6.0Hz, 1H),3.80(t,J=8.4Hz,2H),3.52(dd,J=6.0,8.0Hz,2H),2.93(t,J=6.0Hz,2H),2.56-2.52(m,1H), 1.34(s,9H).

[0239] Tert-Butyl((1-((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)sulfonyl)azetidin-3-yl)methyl ) Preparation of carbamate, HxBz-7c HxBz-7b (0.75 g, 1.85 mmol, 1 eq) 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-) in dioxane (15 mL) Dioxaborolane-2-yl)-1,3,2-dioxaborolane, Pin 2 B 2 , bis(pinacolato)diboron, CAS Reg.No.78183-34-3 (703 mg, 2.77 mmol, 1.5 eq.) KOAc (362 mg, 3.69 mmol, 2 eq.) in a mixture of Pd(dppf)Cl 2 (67.5mg, 92.3umol, 0.05eq), N 2The mixture was added at 25° C. and then stirred at 100° C. for 1 hour. The mixture was filtered and concentrated in vacuo. HxBz-7c (0.85 g, crude) was obtained as a yellow oil.

[0240] Tert-Butyl((1-((5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyridin-3-yl)sulfonyl)azetidine-3- Preparation of yl)methyl)carbamate, HxBz-7d HxBz-7c (0.85 g, 1.87 mmol, 1 eq.) and 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, HxBzL-2b ( 755mg, 2.06mmol, 1.1eq) of H 2 K in O (3 mL) 2 C.O. 3 (518mg, 3.75mmol, 2eq) and Pd(dppf)Cl 2 (68.6 mg, 93.7 umol, 0.05 equivalent), N 2 The mixture was added at 25° C. and stirred at 100° C. for 1 hour. Mixture, H 2 Pour into O (50 mL). The aqueous phase was extracted with ethyl acetate (150 mL*3). The combined organic phases were washed with brine (100 mL) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100 to 200 mesh silica gel, petroleum ether / ethyl acetate = 5 / 1, 0 / 1 to EtOAc / MeOH = 10 / 1). HxBz-7d (1 g, 1.63 mmol, yield 87.05%) was obtained as an off-white solid. 1 H NMR(DMSO-d 6 ,400MHz)δ9.18(d,J=2.0Hz,1H),8.95(d,J=2.0Hz,1H),8.42(t,J=2.0Hz,1H),7.55-7.51(m,2H), 7.49-7.45(m,1H),7.30(s,1H),3.96(q,J=7.6Hz 2H),3.90(t,J=8.0Hz,2H),3.74(t,J=7.2Hz,2H) ,3.60(dd,J=6.0,8.0Hz,2H),3.35(s,2H),3.06(d,J=6.0Hz,2H),2.69-2.58(m,1H),1.77(sxt,J=7.2 Hz,2H),1.36(s,9H),1.17(t,J=7.2Hz,3H),0.99(t,J=7.2Hz,3H).

[0241] 2-Amino-8-[5-[3-(aminomethyl)azetidin-1-yl]sulfonyl-3-pyridyl]-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz-7 preparation of CH 3 CN (10mL) and H 2 To a mixture of HxBz-7d (0.8g, 1.31mmol, 1eq) in O (10mL) was added TFA (1.49g, 13.1mmol, 967uL, 10eq) with N 2 The mixture was added at 25° C. and then stirred at 80° C. for 1 hour. The mixture was concentrated in vacuo to CH 3 CN was removed, the aqueous solution was extracted with MTBE (20*3) and discarded, and then the aqueous phase was directly lyophilized to obtain HxBz-7 (0.9 g, 1.22 mmol, yield 93.07%, 2TFA) as off-white Obtained as a solid. 1 H NMR(MeOD,400MHz)δ9.24(d,J=2.0Hz,1H),9.04(d,J=2.0Hz,1H),8.50(t,J=2.0Hz,1H),7.87-7.78(m ,2H),7.77-7.72(m,1H),7.46(s,1H),4.06-3.94(m,4H),3.79-3.70(m,4H),3.45(s,2H),3.12(d,J =7.6Hz,2H),2.83-2.73(m,1H),1.79(sxt,J=7.2Hz,2H),1.20(t,J=7.2Hz,3H),1.01(t,J=7.2Hz,3H ).LC / MS[M+H]513.2 (calculated value), LC / MS[M+H]513.2 (observed value).

[0242] 1-(1-((5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyridin-3-yl)sulfonyl)azetidin-3-yl) Preparation of -3-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-2-azahexatriacontan-36-acid, HxBzL-7a Et 3 N (161mg, 1.60mmol, 222uL, 2.5eq), N 2 was added at 0° C. and then stirred at 0° C. for 1 hour. H the mixture 2 The pH of the mixture was adjusted to ~6 with TFA at 0 °C, then extracted with MTBE (10 mL) and discarded, and the aqueous phase was further extracted with DCM / i-PrOH (20 mL*3). did. Mix organic phase with anhydrous Na 2 S.O. 4 , filtered and concentrated in vacuo to give HxBzL-7a (0.6 g, 569.68 umol, 89.25% yield) as a pale yellow oil.

[0243] Preparation of HxBzL-7 HxBzL-7a (0.6 g, 570 umol, 1 eq) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (611 mg, 2.28 EDCI (437 mg, 2.28 mmol, 4 eq) was added to a mixture of N 2 was added at 25° C. and then stirred at 25° C. for 0.5 h. The mixture was concentrated in vacuo. The residue was filtered and purified by preparative HPLC column: Phenomenex luna C18 250*50mm*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 30%~50%, 10 minutes). HxBzL-7 (370 mg, 288.76 umol, yield 50.69%) was obtained as a white solid. 1 H NMR(MeOD,400MHz)δ9.24(d,J=2.0Hz,1H),9.03(d,J=2.0Hz,1H),8.51(t,J=2.0Hz,1H),7.91-7.84(m ,2H),7.74(d,J=8.8Hz,1H),7.47(s,1H),4.03-3.91(m,4H),3.86(t,J=6.0Hz,2H),3.76(t,J= 7.2Hz,2H),3.66-3.49(m,40H),3.47(s,2H),3.21(d,J=6.4Hz,2H),3.01-2.92(m,2H),2.79-2.68(m,1H) ),2.29(t,J=6.0Hz,2H),1.78(sxt,J=7.2Hz,2H),1.21(t,J=7.2Hz,3H),1.01(t,J=7.2Hz,3H). LC / MS[M+H]1281.5 (calculated value), LC / MS[M+H]1281.6 (observed value).

[0244] Example L-12 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[2-(cyclobutoxy-carbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-12 [ka] Cyclobutyl N-[2-[[2-amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl ] Preparation of carbamate, HxBz-15b 2-Amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBz- in DCM (4 mL) and DMA (2 mL). EDCI (468 mg, 2.44 mmol, 4.0 eq.) was added to a mixture of 15a (250 mg, 611 umol, 1.0 eq.) and cyclobutyl N-[2-(propylaminooxy)ethyl]carbamate (201 mg, 794 umol, 1.3 eq., HCL). N 2 The mixture was added in one portion at 25° C. and stirred at 25° C. for 2 hours. DCM (4 mL) was removed in vacuo, water (10 mL) was added, the aqueous phase was extracted with ethyl acetate (10 mL*3), the combined organic phases were washed with brine (5 mL*2), anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100~200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 0 / 1 ~ ethyl acetate / methanol = 10 / 1) to obtain HxBz. -15b (190 mg, 313 umol, 51.2% yield) was obtained as a brown oil. 1 H NMR(400MHz,MeOD)δ9.08(s,2H),7.63(d,J=8.0Hz,1H),7.58-7.52(m,2H),7.37(s,1H),4.74-4.67(m, 2H),4.54(s,2H),3.96(t,J=4.8Hz,2H),3.76(t,J=7.2Hz,2H),3.33(s,2H),2.20(dd,J=2.8,5.2 Hz,2H),1.94-1.86(m,2H),1.82-1.75(m,2H),1.50(s,9H),1.38(d,J=1.6Hz,2H),1.01(t,J=7.2Hz ,3H).

[0245] Cyclobutyl N-[2-[[2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, HxBz-15 preparation of CF 3 COOH (535mg, 4.69mmol, 347uL, 15eq) was converted into N 2 was added in one portion at 25° C. and then stirred at 25° C. for 1.5 hours. DCM (5 mL) was removed in vacuo, the residue was diluted with water (10 mL), the aqueous phase was extracted with MTBE (5 mL*4) to remove excess TFA, and then the aqueous phase was lyophilized. , HxBz-15 (130 mg, 169 umol, yield 54.1%, purity 95.7%, 2TFA) was obtained as a brown solid. 1 H NMR(400MHz,MeOD)δ=9.21(s,2H),7.85-7.76(m,3H),7.49(s,1H),4.66(t,J=7.2Hz,1H),4.48(s,2H) ,3.96(t,J=5.2Hz,2H),3.76(t,J=7.2Hz,2H),3.43(s,2H),3.31(s,2H),2.20-2.10(m,2H),1.91- 1.83(m,2H),1.81-1.74(m,2H),1.70-1.60(m,1H),1.57-1.47(m,1H),1.00(t,J=7.2Hz,3H).LC / MS[ M+H]508.3 (calculated value), LC / MS[M+H]508.1 (observed value).

[0246] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(cyclobutoxycarbonyl amino)ethoxy-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]propanoic acid, preparation of HxBzL-12a HxBz-15 (105 mg, 181 umol, 1.0 eq., 2HCl) and Et in DMF (1.5 mL) 3 A mixture of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo- 3-(2,3,4,5,6-pentafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, PFP-PEG10-CO2H (131mg, 181umol, 1.0 equivalent), N 2 The mixture was added at 0° C. and stirred at 0° C. for 0.5 h, then at 25° C. for an additional 0.5 h. The reaction mixture was concentrated, the residue was diluted with water (5 mL), the aqueous phase was extracted with ethyl acetate (3 mL*2) and discarded, then the aqueous phase was extracted with DCM / iPrOH=3 / 1 (5 mL*3) The mixed organic layer was further extracted with anhydrous Na 2 S.O. 4 , filtered and concentrated in vacuo to give HxBzL-12a (100 mg, 95.4 umol, 52.7% yield) as a yellow oil.

[0247] Preparation of HxBzL-12 HxBzL-12a (100 mg, 95.4 umol, 1.0 eq) and (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxysodium (128 mg, 477 umol) in DCM (1 mL) and DMA (0.5 mL) , 5.0 eq.), EDCI (91.4 mg, 477 umol, 5.0 eq.) was added to a mixture of N 2 was added in one portion at 25° C. and then stirred at 25° C. for 1 hour. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 15%~35%, 8 min). Purification provided HxBzL-12 (35.1 mg, 25.6 umol, 26.9% yield, 93.3% purity) as a pale yellow oil. 1 H NMR(400MHz,MeOD)δ9.12(s,2H),7.84-7.77(m,3H),7.52(s,1H),4.75-4.67(m,3H),3.99(t,J=5.2Hz, 2H),3.88(t,J=6.0Hz,2H),3.82(t,J=6.0Hz,2H),3.78(t,J=7.2Hz,2H),3.70-3.57(m,38H),3.45( s,2H),3.01-2.97(m,2H),2.62(t,J=6.0Hz,2H),2.24-2.14(m,2H),1.96-1.86(m,2H),1.84-1.75(m, 2H),1.73-1.61(m,1H),1.59-1.49(m,1H),1.01(t,J=7.2Hz,3H).LC / MS[M+H]1276.5(calculated value),LC / MS [M+H]1276.6 (observed value).

[0248] Example L-13 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[2-(Cyclobutylcarbamoylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy Synthesis of ]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-13 [ka] Tert-Butyl((5-(2-amino-4-((2-(3-cyclobutylureido)ethoxy)(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidin-2-yl )Methyl)carbamate, preparation of HxBz-16a 2-Amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBz- in DCM (2 mL) and DMA (2 mL). 15a (250 mg, 611 umol, 1 eq.) in a solution of 1-cyclobutyl-3-[2-(propylaminooxy)ethyl]urea (231 mg, 916 umol, 1.5 eq., HCl) with EDCI (351 mg, 1.83 mmol, 3 eq.) was added and stirred at 25°C for 0.5 hour. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with water (10 mL) and extracted with EtOAc (20 mL*3). Wash the mixed organic layer with brine (20mL*2) and remove Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate=50 / 1 to ethyl acetate:MeOH=5:1) to obtain HxBz-16a (230 mg, 380 umol, yield 62.1%) as a brown solid.

[0249] 2-Amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-[2-(cyclobutylcarbamoylamino)ethoxy]-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz- Preparation of 16 To a solution of HxBz-16a (230 mg, 0.38 mmol, 1 eq) in water (2 mL) and MeCN (2 mL) was added TFA (432 mg, 3.79 mmol, 0.28 mL, 10 eq), then at 80 °C for 0.5 h. Stirred. The mixture was concentrated under reduced pressure, the residue was diluted with water (2 mL), extracted with MTBE (3 mL*3) and discarded, the aqueous phase was concentrated under reduced pressure to obtain HxBz-16 (230 mg, 371 umol, yield 97.8% TFA) was obtained as a brown solid. 1 H NMR(400MHz,MeOD)δ 9.21(s,2H),7.84-7.73(m,3H),7.47(s,1H),4.48(s,2H),4.01-3.89(m,3H),3.75(t ,J=7.2Hz,2H),3.44(s,2H),3.33(br s,2H),2.19-2.10(m,2H),1.81-1.68(m,4H),1.64-1.55(m,2H) ,1.00(t,J=7.2Hz,3H).LC / MS[M+H]507.3 (calculated value), LC / MS[M+H]507.2 (observed value).

[0250] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(cyclobutylcarbamoyl amino)ethoxy-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]propanoic acid, preparation of HxBzL-13a HxBz-16 (100mg, 136umol, 1eq, 2TFA) and 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]propanoic acid (96.2 mg, 0.14 mmol, 1 eq. ) in a solution of Et 3 N (41.3 mg, 0.41 mmol, 56.8 uL, 3 eq.) was added and then stirred at 25° C. for 0.5 h. The pH of the mixture was adjusted to ~6 with TFA at 0 °C, extracted with EtOAc (5 mL, 3 times) and discarded, and the aqueous solution was further extracted with DCM / i-PrOH (10 mL*3, 3 / 1). did. Organic layer, Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure. The crude product HxBzL-13a (120 mg, 115 umol, 84.2% yield) was obtained as a yellow oil and used in the next step without further purification.

[0251] Preparation of HxBzL-13 HxBzL-13a (70 mg, 66.9 umol, 1 eq.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (71.7 mg, 267 umol) in DMA (0.5 mL) and DCM (1.5 mL). , 4 eq.) was added EDCI (51.3 mg, 267 umol, 4 eq.) and stirred at 25° 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 Synergi C18 150*25*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 15% to 35%, 8 minutes). . The residue was then purified by preparative HPLC (TFA conditions, column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 15%~35%, 8 minutes). Purification provided HxBzL-13 (20 mg, 13.3 umol, 19.9% ​​yield, 2TFA) as a colorless oil. 1 H NMR(400MHz,MeOD)δ 9.09(s,2H),7.80-7.71(m,3H),7.47(s,1H),4.69(s,2H),3.95(br t,J=5.2Hz,2H) ,3.86(t,J=6.0Hz,2H),3.80(t,J=6.0Hz,2H),3.75(br t,J=7.2Hz,2H),3.68-3.57(m,38H),3.45(s ,2H),2.97(t,J=6.0Hz,2H),2.60(t,J=6.0Hz,2H),2.15(br d,J=7.2Hz,2H),1.83-1.68(m,4H), 1.64-1.52(m,2H),0.99(t,J=7.2Hz,3H).LC / MS[M+H]1275.5(calculated value), LC / MS[M+H]1275.2(observed value).

[0252] Example L-14 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[3-(Cyclobutoxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy Synthesis of ]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-14 [ka] Cyclobutyl N-[3-[[2-amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl ], Preparation of HxBz-14 2-amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBz-14a (0.25 g, Et 3 N (185 mg, 1.83 mmol, 255 uL, 3.0 eq.), cyclobutyl N-[3-(propylamino)propyl]carbamate (170 mg, 678 umol, 1.11 eq., HCl), and azabenzotriazole tetramethyluronium hexafluorophosphate, HATU (232 mg, 611 umol, 1.0 eq) was added in one portion at 0°C and stirred at 0°C for 0.5 h. The mixture was then diluted with water and extracted with EtOAc (20 mL x 3). Wash the organic layer with brine and remove Na 2 S.O. 4 Dry on top, filter and concentrate. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 3 / 1) to obtain HxBz-14 (0.28 g, 462 umol, yield). 75.71%) as a yellow solid. 1 H NMR(MeOD,400MHz)δ9.04(s,2H),7.52(d,J=8.4Hz,1H),7.48(d,J=1.6Hz,1H),7.45-7.40(m,1H),6.93 (s,1H),4.84-4.84(m,1H),4.64(s,4H),3.54-3.47(m,2H),3.46-3.39(m,2H),3.30(m,2H),3.22-3.07 (m,2H),2.32-2.28(m,2H),2.10-2.00(m,2H),1.88-1.79(m,3H),1.75-1.60(m,3H),1.48(s,9H),0.90 (s,3H).LC / MS[M+H]606.3 (calculated value), LC / MS[M+H]606.2 (observed value).

[0253] Cyclobutyl N-[3-[[2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl]carbamate, HxBz-13 preparation of CH 3 CN (3mL) and H 2 To a mixture of HxBz-14 (0.26 g, 429 umol, 1.0 eq.) in O (1 mL) was added TFA (489 mg, 4.29 mmol, 318 uL, 10.0 eq.) in one portion at 25 °C, then stirred at 80 °C for 0.5 h. did. The mixture was then concentrated, the residue was diluted with water (10 mL), and the mixture was extracted with MTBE (10 mL x 2) to remove excess TFA. The aqueous layer was freeze-dried to obtain HxBz-13 (0.2 g, 323 umol, yield 75.20%, TFA) as a yellow solid. 1 H NMR(MeOD,400MHz)δ9.21(s,2H),7.84-7.71(m,3H),7.12(s,1H),4.85-4.85(m,1H),4.47(s,2H),3.54( t,J=7.2Hz,2H),3.48(s,2H),3.37(s,2H),3.15(d,J=15.6Hz,2H),2.30-2.25(m,2H),2.08-2.00(m ,2H),1.89-1.66(m,6H),1.01-0.88(m,3H).LC / MS[M+H]506.3(calculated value), LC / MS[M+H]506.2(observed value).

[0254] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutoxycarbonyl amino)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]Ethoxy]propanoic acid, preparation of HxBzL-14a Et 3 N (48.9mg, 484umol, 67.4uL, 3.0eq) and 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]propanoic acid, TFP-PEG10-CO2H (114 mg, 161 umol, 1.0 equiv) , added in one portion at 0°C and then stirred at 0°C for 0.5 h. The pH of the mixture was adjusted to 5-6 with TFA at 0 °C. The mixture was then diluted with water (5 mL) and washed with MTBE (10 mL x 3). The aqueous layer was then further extracted with DCM:i-PrOH=3:1 (20 mL x 3). organic layer with Na 2 Dry over SO4, filter, and concentrate to give HxBzL-14a (0.15 g, 129 umol, 80.11% yield, TFA) as a yellow oil.

[0255] Preparation of HxBzL-14 Sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate ( 139 mg, 517 umol, 4.0 eq) and EDCI (149 mg, 776 umol, 6.0 eq) were added in one portion at 25°C and then stirred at 25°C for 0.5 h. The mixture was concentrated and filtered. The residue was then purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 15%~40%, 8 min). , HxBzL-14 (75.3 mg, 59.1 umol, yield 45.71%) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ9.09(s,2H),7.82-7.67(m,3H),7.11(s,1H),4.86-4.82(m,1H),4.69(s,2H),3.86( t,J=6.0Hz,2H),3.80(t,J=6.0Hz,2H),3.66-3.48(m,40H),3.38(s,2H),3.22-3.06(m,2H),2.97(t ,J=6.0Hz,2H),2.64-2.58(m,2H),2.32-2.25(m,2H),2.09-1.95(m,2H),1.91-1.80(m,3H),1.75-1.61(m ,3H),0.93(s,3H).LC / MS[M+H]1274.5 (calculated value), LC / MS[M+H]1274.3 (observed value).

[0256] Example L-15 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[5-[2-amino-4 -[Ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy ]-2,3,5,6-tetrafluoro-benzenesulfonic acid, synthesis of HxBzL-15 [ka] Preparation of 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz-11b N-ethoxypropan-1-amine (9.6 g, 68.8 mmol, 1.3 eq., HCl) and 2-amino-8-bromo-3H-1-benzazepine-4-carboxylic acid in DMA (150 mL) and DCM (150 mL). , EDCI (40.6 g, 211 mmol, 4.0 eq.) was added to a mixture of HxBz-11a (14.8 g, 52.9 mmol, 1.0 eq.) with N 2 The mixture was added at 25°C. The mixture was stirred at 25°C for 2 hours. Adjust the pH of the mixture to NaHCO 3 9 and concentrated under reduced pressure to remove DCM at 45°C. The aqueous phase was extracted with ethyl acetate (100 mL x 3). The combined organic layer was washed with brine (1000mLx2) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was triturated with MTBE / PE=1 / 1 at 25° C. to obtain HxBz-11b (12.5 g, 34.1 mmol, yield 64.5%) as a white solid. 1 H NMR(MeOD,400MHz)δ7.31(d,J=2.0Hz,1H),7.26-7.22(m,1H),7.18(s,1H),7.17-7.14(m,1H),3.92(q, J=6.8Hz,2H),3.71(t,J=7.2Hz,2H),3.31(s,2H),1.79-1.70(m,2H),1.15(t,J=7.2Hz,3H),0.97( t,J=7.6Hz,3H).

[0257] 2-Amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-1-benzazepine-4-carboxamide, HxBz Preparation of -11c HxBz-11b (500 mg, 1.37 mmol, 1.0 equiv.) in dioxane (10 mL), Pin 2 B 2 (416mg, 1.64mmol, 1.2eq), KOAc (335mg, 3.41mmol, 2.5eq), and Pd(dppf)Cl 2 (99.9 mg, 136 umol, 0.1 eq.) was degassed and N 2 Purge the mixture three times with N 2 Stirred at 95° C. for 1 hour under atmosphere. The mixture was concentrated in vacuo. The residue was poured into ice water (w / w=1 / 1) (10 mL) and stirred for 5 minutes. The aqueous phase was extracted with MTBE (10 mL x 1), then the aqueous phase was further extracted with DCM / i-PrOH=3 / 1 (10 mL x 3). The mixed organic layer (DCM / i-PrOH) was diluted with anhydrous Na 2 S.O. 4 , filtered and concentrated in vacuo to give HxBz-11c (490 mg, crude), which was used in the next step without further purification as a black solid.

[0258] Preparation of methyl 5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidine-2-carboxylate, HxBz-11 Dioxane (15mL) and H 2 HxBz-11c (390 mg, 944 umol, 1.0 eq.), methyl 5-bromopyrimidine-2-carboxylate (266 mg, 1.23 mmol, 1.3 eq.), Pd(dppf)Cl in O (2 mL). 2 (69.0mg, 94.3umol, 0.1eq), K 3 P.O. 4 (401 mg, 1.89 mmol, 2.0 eq.) was degassed and N 2 Purge 3 times with N 2 Stirred at 80°C for 1 hour under atmosphere. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 5%~30%, 8 min) to obtain HxBz -11 (105 mg, 161 umol, yield 17.1%, TFA) was obtained as a white solid. 1 H NMR(MeOD,400MHz)δ9.30(s,2H),7.89(dd,J=2.0,2.0Hz,1H),7.83-7.74(m,2H),7.47(s,1H),4.06(s, 3H),4.00(t,J=6.8Hz,2H),3.76(t,J=7.2Hz,2H),3.45(s,2H),1.83-1.74(m,2H),1.21(t,J=6.8 Hz,3H),1.01(t,J=7.2Hz,3H).LC / MS[M+H]424.1(calculated value), LC / MS[M+H]424.1(observed value).

[0259] Preparation of 5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidine-2-carboxylic acid, HxBzL-15a EtOH (5 mL) and H 2 A solution of HxBz-11 (330 mg, 779 umol, 1.0 eq.) in O (0.5 mL) was added with 2 O (131 mg, 3.12 mmol, 4.0 eq.) was added. The mixture was stirred at 25°C for 2 hours. The pH of the mixture was adjusted to ~6 with HCl (4M) and concentrated in vacuo to remove EtOH. The residue was diluted with water (10 mL). The aqueous phase was extracted with DCM / i-PrOH=3 / 1 (10 mL x 3). Mix organic layer with anhydrous Na 2 S.O. 4 , filtered and concentrated in vacuo to give HxBzL-15a (200 mg, 488 umol, 62.7% yield) as a yellow solid.

[0260] tert-Butyl3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[1-[[5-[2-amino-4- [3-(3,3-dimethylbutanoylamino)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]-3-pyridyl]sulfonyl]azetidin-3-yl]methyl-methyl-amino]ethoxy Preparation of ]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, HxBzL-15b HxBzL-15a (195 mg, 332 umol, 0.8 eq) in DMF (5 mL) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2- (2-Aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, tBuOOC-PEG 10 -NH 2 (390mg, 666umol, 1.0eq.) to a mixture of Et 3 N (126 mg, 1.25 mmol, 173 uL, 3.0 eq) and HATU (158 mg, 415 umol, 1.0 eq) were added at 0°C. The mixture was stirred at 0°C for 1 hour. The mixture was purified by preparative HPLC (column: Phenomenex luna C18 80*40mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 25%~50%, 7 min) to obtain HxBzL. -15b (80 mg, 66.4 umol, 16.0% yield, TFA) was obtained as a yellow oil.

[0261] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]- Preparation of 3H-1-benzazepin-8-yl]pyrimidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL-15c To a solution of HxBzL-15b (80 mg, 66.4 umol, 1.0 eq., TFA) in MeCN (2 mL) and H2O (1 mL) was added HCl (12M, 83.0 uL, 15.0 eq.) and stirred at 80 °C for 1 h. . The mixture was concentrated in vacuo to give a residue, which was lyophilized to give HxBzL-15c (60 mg, 62.7 umol, 94.4% yield, HCl) as a colorless oil.

[0262] Preparation of HxBzL-15 HxBzL-15c (60 mg, 60.4 umol, 1.0 eq., 2HCl) and (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxysodium (64.7 EDCI (46.3 mg, 241 umol, 4.0 eq.) was added to a solution of 4.0 eq. The mixture was concentrated in vacuo and filtered. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 15%~35%, 8 min) to obtain HxBzL. -15 (36 mg, 31.3 umol, 51.9% yield) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ9.27(s,2H),7.90-7.81(m,2H),7.75(d,J=8.4Hz,1H),7.46(s,1H),3.98(q,J= 6.8Hz,2H),3.85(t,J=6.0Hz,2H),3.78-3.75(m,2H),3.73-3.72(m,2H),3.70-3.56(m,36H),3.46(s,2H) ),2.96(t,J=6.0Hz,2H),1.84-1.71(m,2H),1.21(t,J=6.8Hz,3H),1.00(t,J=7.6Hz,3H).LC / MS [M+H] 1149.4 (calculated value), LC / MS [M+H] 1149.5 (observed value).

[0263] Example L-16 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[5 -[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-16 [ka] Preparation of methyl (2S)-1-(5-bromopyrimidine-2-carbonyl)pyrrolidine-2-carboxyl-ate, HxBzL-16b 5-bromopyrimidine-2-carboxylic acid, HxBzL-16a (400mg, 1.97mmol, 1.0eq) in DMF (8mL), Et 3 HATU (749 mg, 1.97 mmol, 1.0 eq.) was added to a mixture of N (598 mg, 5.91 mmol, 822 uL, 3.0 eq.) and methyl (2S)-pyrrolidine-2-carboxylate (342 mg, 2.07 mmol, 1.05 eq., HCl). ,N 2 Added in one portion at 0° C. and stirred at 0° C. for 30 min, then heated to 25° C. and stirred for an additional 0.5 h. Water (20 mL) was added, the aqueous phase was extracted with ethyl acetate (20 mL*4), the combined organic phase was washed with brine (10 mL*1), anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 4 / 1) to obtain HxBzL-16b (320 mg, 1.02 mmol, yield). 51.7%) as a yellow oil.

[0264] Preparation of (2S)-1-(5-bromopyrimidine-2-carbonyl)pyrrolidine-2-carboxylic acid, HxBzL-16c MeOH (5 mL) and H 2 A solution of HxBzL-16b (320 mg, 1.02 mmol, 1.0 equiv) in O (5 mL) was added with LiOH H 2 O (171mg, 4.07mmol, 4.0eq), N 2 The mixture was added in one portion at 25° C. and stirred at 25° C. for 2 hours. The reaction mixture was quenched with HCl (4M) until pH=7, MeOH (5 mL) was removed in vacuo, and the desired solid precipitated from the aqueous phase, filtered, dried and purified with HxBzL-16c (300 mg, crude The product was obtained as a pale yellow solid.

[0265] tert-Butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-(5-bromopyrimidine-2 -Carbonyl)pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate, preparation of HxBzL-16d HxBzL-16c (200mg, 666umol, 1.0eq) in DMF (1mL), Et 3 N (168mg, 1.67mmol, 232uL, 2.5eq) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy ) ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate (390mg, 666umol, 1.0eq) was added with N 2 Added in one portion at 0° C. and stirred at 0° C. for 30 min, then heated to 25° C. and stirred for an additional 0.5 h. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex luna C18 250*50mm*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 20% to 60%, 10 minutes). Purification provided HxBzL-16d (300 mg, 346 umol, 51.8% yield) as a colorless oil.

[0266] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-(5-bromopyrimidine-2-carbonyl) Preparation of pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL-16e MeCN (1 mL) and H 2 To a solution of HxBzL-16d (300 mg, 345 umol, 1.0 eq.) in O (3 mL) was added HCl (12 M, 864 uL, 30 eq.) with N 2 was added in one portion at 25° C. and then stirred at 80° C. for 1 hour. The reaction mixture was concentrated in vacuo to give HxBzL-16e (250 mg, 307.99 umol, 89.09% yield) as a yellow oil.

[0267] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[5-[2-amino-4- [ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy Preparation of ethoxy]propanoic acid, HxBzL-16f Dioxane (3mL) and H 2 HxBzL-16e (150 mg, 185 umol, 1.0 equiv.) in O (0.3 mL), 2-amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2- Dioxaborolan-2-yl)-3H-1-benzazepine-4-carboxamide (91.6mg, 222umol, 1.2eq), Pd(dppf)Cl 2 (13.5 mg, 18.5 umol, 0.1 equivalent) and K 2 C.O. 3 A solution of (63.8 mg, 462 umol, 2.5 eq.) was degassed and then N 2 The mixture was heated to 95°C for 2 hours under vacuum. The reaction mixture was filtered, the filtrate was concentrated in vacuo, and the residue was purified by preparative HPLC (column: Phenomenex luna C18 80*40mm*3um, mobile phase: [water (0.04%HCl)-ACN], B%: 5% ~45%, 7 min) to give HxBzL-16f (110 mg, 108 umol, 58.4% yield) as a yellow oil.

[0268] Preparation of HxBzL-16 HxBzL-16f (110 mg, 108 umol, 1.0 eq) and (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxysodium (145 mg, 540 umol, in DCM (2 mL) and DMA (0.5 mL)) EDCI (103mg, 540umol, 5.0eq) was added to a mixture of N 2 was added in one portion at 25° C. and stirred at 25° C. for 1 hour. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 10%~40%, 8 min). Purification provided HxBzL-16 (66.5 mg, 50.9 umol, 47.1% yield, 95.3% purity) as a pale yellow oil. 1 H NMR(400MHz,MeOD)δ9.28-9.24(m,2H),7.91-7.81(m,2H),7.80-7.74(m,1H),7.50-7.47(m,1H),4.00(q,J =7.2Hz,2H),3.88(dt,J=3.2,5.6Hz,4H),3.81-3.74(m,4H),3.70-3.53(m,37H),3.50-3.32(m,5H),3.02- 2.96(m,2H),2.16-1.97(m,4H),1.84-1.76(m,2H),1.23(t,7.2Hz,3H),1.03(t,7.2Hz,3H).LC / MS[M +H]1246.5 (calculated value), LC / MS[M+H]1246.7 (observed value).

[0269] Example L-21 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[2-(dimethylcarbamoylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-21 [ka] Tert-Butyl((5-(2-amino-4-((2-(3,3-dimethylureido)ethoxy)(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidine-2- Preparation of yl)methyl)carbamate, HxBz-20a 2-Amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBz- in DCM (3 mL) and DMA (1 mL). EDCI (468 mg, 2.44 mmol) was added to a mixture of 14a (250 mg, 611 umol, 1 eq.) and 1,1-dimethyl-3-[2-(propylaminooxy)ethyl]urea (165 mg, 733 umol, 1.2 eq., HCl). , 4 equivalents) and stirred at 25°C for 1 hour. The mixture was concentrated in vacuo to remove DCM, the residue was diluted with water (10 mL), and the pH of the mixture was adjusted to Na 2 C.O. 3 It was adjusted to about 8 with an aqueous solution. The aqueous phase was extracted with ethyl acetate (10 mL*4). Wash the mixed organic layer with brine (20mL*1) and add anhydrous Na2 S.O. 4 dried, filtered and concentrated in vacuo. Purify the residue by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1, ethyl acetate / methanol = 1 / 0, 3 / 1). HxBz-20a (260 mg, 447.75 umol, yield 73.33%) was obtained as a yellow solid.

[0270] Preparation of HxBz-20 To a solution of HxBz-20a (130 mg, 224 umol, 1 eq.) in EtOAc (3.00 mL) was added HCl / EtOAc (4M, 3.00 mL, 53.60 eq.) and then stirred at 25° C. for 1 h. The mixture was concentrated to give HxBz-20 (115 mg, 207.77 umol, 92.81% yield, 2HCl) as a pale red solid. 1 H NMR(MeOD,400MHz)δ9.22(s,2H),7.86-7.80(m,2H),7.80-7.74(m,1H),7.50(s,1H),4.48(s,2H),3.97( t,J=5.2Hz,2H),3.76(t,J=7.2Hz,2H),3.45(s,2H),3.38-3.34(m,2H),2.74(s,6H),1.83-1.73(m ,2H),1.00(t,J=7.6Hz,3H).LC / MS[M+H]481.3 (calculated value), LC / MS[M+H]481.1 (observed value).

[0271] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(dimethylcarbamoylamino )ethoxy-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Preparation of ethoxy]propanoic acid, HxBzL-21a A solution of HxBz-20 (65.0 mg, 117 umol, 1 eq., 2HCl) in DMF (1.00 mL) was added with Et 3 N (48.0 mg, 470 umol, 4 eq.) and 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]propanoic acid, HxBzL-21a (83.0 mg, 117 umol, 1 eq.) was added, then Stirred at 0°C for 1 hour. The mixture was diluted with water (10 mL), the pH of the mixture was adjusted to about 6 by gradual addition of TFA, extracted with MTBE (10 mL) and discarded, and the aqueous solution was diluted with DCM:i-PrOH= It was further extracted with 3:1 (20 mL x 3). organic layer with Na 2 S.O. 4 Dry on top, filter and concentrate to give HxBzL-21a (95 mg, 93.03 umol, 79.22% yield) as a pale yellow oil.

[0272] Preparation of HxBzL-21 HxBzL-21a (90.0 mg, 88.1 umol, 1 eq) and (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy sodium (95.0 EDCI (68.0 mg, 353 umol, 4 eq.) was added to a solution of EDCI (68.0 mg, 353 umol, 4 eq.) and stirred at 25° C. for 1 hour. The mixture was concentrated and filtered. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 5%~35%, 8 min) to obtain HxBzL. -21 (51 mg, 37.41 umol, 42.45% yield, TFA) was obtained as a pale yellow oil. 1 H NMR(MeOD,400MHz)δ9.10(s,2H),7.83-7.70(m,3H),7.48(s,1H),4.69(s,2H),3.97(t,J=5.2Hz,2H) ,3.86(t,J=5.6Hz,2H),3.80(t,J=6.0Hz,2H),3.78-3.74(m,2H),3.65-3.55(m,36H),3.45(s,2H), 3.37-3.34(m,2H),2.97(t,J=5.6Hz,2H),2.74(s,6H),2.60(t,J=6.0Hz,2H),1.83-1.72(m,1H),1.00 (t,J=7.2Hz,3H).LC / MS[M+H]1249.5 (calculated value), LC / MS[M+H]1249.6 (observed value).

[0273] Example L-23 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[2-Hydroxyethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-23 [ka] tert-Butyl N-[[5-[2-amino-4-[2-hydroxyethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methyl]carbamate, HxBz-22a preparation of 2-Amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBz in DCM (6 mL) and DMA (0.5 mL) EDCI (492 mg, 2.56 mmol, 3.0 eq.) was added once to a mixture of -14a (0.35 g, 855 umol, 1.0 eq.) and 2-(propylaminooxy)ethanol (200 mg, 1.28 mmol, 1.5 eq., HCl) at 25 °C. and then stirred at 25°C for 0.5 hour. The mixture was concentrated to remove DCM and the residue was purified with H 2 Diluted with O (10 mL). Adjust the pH of the mixture to NaHCO 3 It was adjusted to about 8 with an aqueous solution. The aqueous phase was then extracted with EtOAc (20 mL x 3). The organic layer was brine, Na 2 S.O. 4 Dry on top, filter and concentrate. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, ethyl acetate / MeOH=1 / 0, 10 / 1) to obtain HxBz-22a (0.37 g, 725 umol, yield 84.77%) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ9.08-9.01(m,2H),7.59(d,J=8.0Hz,1H),7.54-7.46(m,2H),7.40(s,1H),4.56-4.49( m,2H),4.02-3.95(m,2H),3.81-3.74(m,2H),3.73-3.66(m,2H),1.88-1.72(m,2H),1.48(s,9H),0.99( t,J=7.6Hz,3H).

[0274] Preparation of 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-(2-hydroxyethoxy)-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz-22 H 2 O (4 mL) and CH 3 To a mixture of HxBz-22a (0.35 g, 685 umol, 1.0 eq.) in CN (0.5 mL) was added TFA (1.17 g, 10.3 mmol, 761 uL, 15.0 eq.) in one portion at 25 °C and then at 80 °C. Stirred for 0.5 hour. The mixture was extracted with MTBE (10 mL x 2) to remove excess TFA. The aqueous layer was then freeze-dried. The residue was further purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 1%~20%, 8 min) to obtain HxBz- 22 (0.32 g, 501 umol, yield 73.11%, 2TFA) was obtained as a white solid. 1 H NMR(MeOD,400MHz)δ9.20(s,2H),7.84-7.72(m,3H),7.56(s,1H),4.47(s,2H),4.03-3.96(m,2H),3.79( t,J=7.2Hz,2H),3.74-3.66(m,2H),3.53-3.36(m,2H),1.88-1.72(m,2H),1.00(t,J=7.6Hz,3H).LC / MS[M+H]411.2 (calculated value), LC / MS[M+H]411.1 (observed value).

[0275] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[5-[2-amino-4-[2-hydroxyethoxy(propyl)] carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, Preparation of HxBzL-23a Et 3N (170mg, 1.68mmol, 234uL, 3.0eq) and 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]propanoic acid (396 mg, 560 umol, 1.0 equiv.) was added in one portion at 0 °C. , and then stirred at 0° C. for 0.5 h. The mixture was diluted with water (5ml) and the pH of the mixture was adjusted to about 6 with TFA at 0°C. The aqueous phase was extracted with EtOAc (10 mL) and discarded. The aqueous layer was further extracted with DCM:i-PrOH=3:1 (20 mL x 2). organic layer with Na 2 S.O. 4 Dry on top, filter, and concentrate to give HxBzL-23a (0.53 g, crude, TFA) as a yellow oil.

[0276] 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[[5-[2-amino-4-[2-hydroxy ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]Propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, preparation of HxBzL-23 p HxBzL-23a (0.35 g, 329 umol, 1.0 eq., TFA) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (352 mg, 1.31 EDCI (378 mg, 1.97 mmol, 6.0 eq.) was added in one portion at 25° C. and then stirred at 25° C. for 0.5 h. The mixture was concentrated and filtered. The residue was then purified by preparative HPLC (column: Phenomenex luna C18 250*50mm*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 20%~50%, 10 min). , HxBzL-23 (80.4 mg, 68.2 umol, yield 20.75%) was obtained as a pale yellow oil. 1 H NMR(MeOD,400MHz)δ9.08(s,2H),7.82-7.70(m,3H),7.56(s,1H),4.69(s,2H),4.06-3.97(m,2H),3.86( t,J=6.0Hz,2H),3.83-3.76(m,4H),3.74-3.69(m,2H),3.65-3.57(m,36H),3.46(s,2H),3.02-2.92(m, 2H),2.60(t,J=6.0Hz,2H),1.87-1.72(m,2H),1.00(t,J=7.2Hz,3H).LC / MS[M+H]1179.4(calculated value), LC / MS[M+H]1179.3 (observed value).

[0277] Example L-27 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[2-(isopropoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy Synthesis of ]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-27 [ka] Isopropyl N-[2-[[2-amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl ] Preparation of carbamate, HxBz-27a 2-Amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBz- in DCM (5 mL) and DMA (3 mL). EDCI (656 mg, 3.42 mmol, 4.0 eq.) in a mixture of 14a (350 mg, 855 umol, 1.0 eq.) and isopropyl N-[2-(propylaminooxy)ethyl]carbamate (268 mg, 1.11 mmol, 1.3 eq., HCl). was added and stirred at 25°C for 1 hour. The mixture was concentrated under reduced pressure at 30°C. The residue was poured into ice water (w / w=1 / 1) (10 mL) and stirred for 5 minutes. Adjust the pH of the mixture to NaHCO 3 It was adjusted to about 8 with an aqueous solution. The aqueous phase was extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (10 mL x 3) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. Purify the residue by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 1 / 1, ethyl acetate / methanol = 1 / 0, 10 / 1). HxBz-27a (460 mg, 772 umol, yield 90.3%) was obtained as a yellow solid. 1 H NMR(MeOD,400MHz)δ 9.04(s,2H),7.57(d,J=8.0Hz,1H),7.51-7.44(m,2H),7.32(s,1H),4.74-4.68(m,1H) ),4.52(s,2H),3.94(t,J=5.2Hz,2H),3.73(t,J=7.2Hz,2H),3.30-3.26(m,2H),1.76(sxt,J=7.2Hz ,2H),1.47(s,9H),1.12(d,J=6.0Hz,6H),0.98(t,J=7.4Hz,3H).

[0278] Isopropyl N-[2-[[2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, HxBz-27 preparation of MeCN (0.5mL) and H 2 To a solution of HxBz-27a (410 mg, 688 umol, 1.0 eq) in O (5 mL) was added TFA (1.18 g, 10.3 mmol, 764 uL, 15.0 eq) and then stirred at 80 °C for 1 h. The mixture was concentrated in vacuo to CH 3 CN was removed. The aqueous phase was extracted with MTBE (5 mL x 3) to remove excess TFA. The aqueous phase was freeze-dried to obtain HxBz-27 (400 mg, 553 umol, 80.3% yield, 2TFA) as a white solid. 1 H NMR(MeOD,400MHz)δ 9.21(s,2H),7.86-7.74(m,3H),7.51(s,1H),4.76-4.63(m,1H),4.48(s,2H),3.98(t ,J=5.2Hz,2H),3.77(t,J=7.2Hz,2H),3.43(s,2H),1.78(sxt,J=7.2Hz,2H),1.12(d,J=6.4Hz,6H ),1.00(t,J=7.2Hz,3H).LC / MS[M+H]496.2 (calculated value), LC / MS[M+H]496.1 (observed value).

[0279] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(isopropoxy carbon ylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Preparation of ethoxy]ethoxy]propanoic acid, HxBzL-27a A solution of HxBz-27 (130mg, 180umol, 1.0eq, 2TFA) in THF (2mL) was added with 3 N (54.5mg, 539umol, 75.0uL, 3.0eq) and 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]propanoic acid (127 mg, 180 umol, 1.0 eq.) was added at 0 °C; It was then stirred for 0.5 hour. The mixture was concentrated in vacuo. The residue was diluted with water (10 mL) and the pH of the mixture was adjusted to about 6 with TFA. The aqueous phase was extracted with MTBE (5 mL x 3) and discarded. The aqueous phase was further extracted with DCM / i-PrOH=3 / 1 (10 mL x 3). The organic phase was concentrated in vacuo to give HxBzL-27a (180 mg, 174 umol, 96.7% yield) as a yellow oil.

[0280] Preparation of HxBzL-27 HxBzL-27a (180 mg, 174 umol, 1.0 eq) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (186 mg, 695 umol, in DCM (2 mL) and DMA (0.5 mL)) EDCI (266 mg, 1.39 mmol, 8.0 eq.) was added to the mixture of 4.0 eq.) and then stirred at 25° C. for 0.5 h. The mixture was concentrated in vacuo and filtered. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 15%~35%, 8 min) to obtain HxBzL. -27 (91 mg, 66.0 umol, yield 38.0%, TFA) was obtained as a yellow solid. 1H NMR(MeOD,400MHz)δ 9.08(s,2H),7.82-7.73(m,3H),7.50(s,1H),4.75-4.66(m,3H),3.97(t,J=5.2Hz,2H ),3.86(t,J=6.0Hz,2H),3.80(t,J=6.0Hz,2H),3.75(br t,J=7.2Hz,2H),3.66-3.56(m,36H),3.45- 3.42(m,2H),2.96(t,J=6.0Hz,2H),2.60(t,J=6.4Hz,2H),1.84-1.70(m,2H),1.12(d,J=6.0Hz,6H ),0.99(t,J=7.6Hz,3H).LC / MS[M+H]1264.4(calculated value), LC / MS[M+H]1264.7(observed value).

[0281] Example L-32 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[Propyl-[2-(pyrrolidine-1-carbonylamino)ethoxy]carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-32 [ka] tert-Butyl N-[[5-[2-amino-4-[propyl-[2-(pyrrolidine-1-carbonylamino)ethoxy]carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl ]Methyl]carbamate, preparation of HxBzL-32a 2-Amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBz in DCM (4 mL) and DMA (0.5 mL) -14a (0.25 g, 611 umol, 1.3 eq.) was added to a mixture of N-[2-(propylaminooxy)ethyl]pyrrolidine-1-carboxamide (118 mg, 469 umol, 1.0 eq., HCl) and EDCI (270.12 mg, 1.41 mmol). , 3.0 eq.) were added in one portion at 25°C and then stirred at 25°C for 0.5 h. The mixture was then concentrated and filtered. The mixture was purified by preparative HPLC (column: Phenomenex luna C18 100*40mm*5um, mobile phase: [water (0.1%TFA)-ACN], B%:7%~38%, 8 min) to obtain HxBzL. -32a (0.1 g, 165 umol, yield 35.09%) was obtained as a yellow solid. 1 H NMR(MeOD,400MHz)δ9.08(s,2H),7.88-7.68(m,3H),7.50(s,1H),4.54(s,2H),4.02-3.89(m,2H),3.76( t,J=7.2Hz,2H),3.44(s,2H),3.36(t,J=5.6Hz,2H),3.19-3.07(m,4H),1.86-1.68(m,6H),1.47(s ,9H),1.00(t,J=7.6Hz,3H).

[0282] 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-propyl-N-[2-(pyrrolidine-1-carbonylamino)ethoxy]-3H-1-benzazepine-4-carboxamide, Preparation of HxBzL-32b H 2 O (4 mL) and CH 3 To a mixture of HxBzL-32a (0.09 g, 148 umol, 1.0 eq.) in CN (0.5 mL) was added TFA (254 mg, 2.23 mmol, 165 uL, 15.0 eq.) in one portion at 25 °C, then at 80 °C for 0.5 h. Stirred. The mixture was then extracted with MTBE (10 mL x 3) and discarded. The aqueous layer was freeze-dried to obtain HxBzL-32b (0.1 g, 136 umol, 91.76% yield, 2TFA) as a yellow solid. 1 H NMR(MeOD,400MHz)δ9.21(s,2H),7.86-7.70(m,3H),7.49(s,1H),4.48(s,2H),3.97(t,J=5.6Hz,2H) ,3.76(t,J=7.2Hz,2H),3.48-3.43(m,2H),3.37(t,J=5.2Hz,2H),3.13(s,4H),1.81-1.71(m,6H), 1.00(t,J=7.6Hz,3H).

[0283] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(pyrrolidine-1 -carbonylamino)ethoxy]carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]propanoic acid, preparation of HxBzL-32c Et 3 N (25.0mg, 247umol, 34.4uL, 3.0eq) and 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]propanoic acid (69.9 mg, 98.9 umol, 1.2 eq) at 0 °C. Added in one portion and then stirred at 0° C. for 0.5 h. The mixture was diluted with water (5 mL) and the pH was adjusted to about 6 with TFA at 0°C. The mixture was then extracted with EtOAc (10 mL) and discarded. The aqueous layer was further extracted with DCM:i-PrOH=3:1 (10 mL x 2). organic layer with Na 2 S.O. 4 Dry on top, filter, and concentrate to give HxBzL-32c (0.1 g, crude, TFA) as a yellow oil.

[0284] Preparation of HxBzL-32 Sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate was added to a mixture of HxBzL-32c (0.1 g, 86.1 umol, 1.0 eq., TFA) in DCM (2 mL) and DMA (0.5 mL). (115 mg, 431 umol, 5.0 eq) and EDCI (116 mg, 603 umol, 7.0 eq) were added in one portion at 25°C and then stirred at 25°C for 0.5 h. The mixture was concentrated. The residue was then purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10um, mobile phase: [water (0.1% TFA)-ACN], B%: 10%~35%, 8 min). , HxBzL-32 (46.4 mg, 33.4 umol, yield 38.78%, TFA) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ9.09(s,2H),7.85-7.66(m,3H),7.49(s,1H),4.70(s,2H),3.97(t,J=5.6Hz,2H) ,3.90-3.84(m,2H),3.80(t,J=6.0Hz,2H),3.66-3.58(m,38H),3.45(s,2H),3.37(t,J=5.2Hz,2H), 3.13(s,4H),3.01-2.93(m,2H),2.60(t,J=6.0Hz,2H),1.86-1.68(m,6H),1.00(t,J=7.6Hz,3H).LC / MS[M+H]1275.5 (calculated value), LC / MS[M+H]1275.6 (observed value).

[0285] Example L-33 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[[5-[2 -amino-4-[3-(cyclobutoxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]-3-pyridyl]sulfonyl]azetidin-3-yl]methylamino]-3- Synthesis of oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-33 [ka] Ethyl 2-amino-8-(5-((3-(((tert-butoxycarbonyl)amino)methyl)azetidin-1-yl)sulfonyl)pyridin-3-yl)-3H-benzo[b]azepine-4 - Preparation of carboxylate, HxBz-32b Dioxane (50mL) and H 2 Tert-Butyl N-[[1-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-pyridyl]sulfonyl] in O (5 mL) azetidin-3-yl]methyl]carbamate, HxBz-32a (5 g, 11.0 mmol, 1 eq) and ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylate (3.41 g, 11.0 mmol, 1 equivalent) in a solution of K 2 C.O. 3 (3.05g, 22.1mmol, 2 equivalents) and Pd(dppf)Cl 2 (403 mg, 551 umol, 0.05 equivalent), N 2 The mixture was added at 25° C. and then stirred at 90° C. for 2 hours. The mixture was filtered and concentrated to give a residue. The residue was diluted with water (100 mL) and extracted with EtOAc (50 mL x 3). The organic layer was washed with brine (50 mL) and 2 S.O. 4 Dry over, filter, and concentrate to obtain HxBz-32a, which was purified by CH at 25 °C for 15 min. 3 Trituration with CN gave HxBz-32b (5.5 g, 9.90 mmol, 89.75% yield) as a gray solid. 1 H NMR(DMSO-d 6,400MHz)δ9.29(s,1H),8.94(s,1H),8.32(s,1H),7.80(s,1H),7.60(d,J=8.0Hz,1H),7.50-7.41(m ,2H),7.04-6.85(m,3H),4.25(q,J=7.2Hz,2H),3.82(t,J=8.0Hz,2H),3.58-3.52(m,2H),2.99-2.85( m,4H),2.56-2.51(m,1H),1.35-1.30(m,12H).

[0286] 2-Amino-8-(5-((3-(((tert-butoxycarbonyl)amino)methyl)azetidin-1-yl)sulfonyl)pyridin-3-yl)-3H-benzo[b]azepine-4- Preparation of carboxylic acid, HxBz-32c MeOH (40mL) and H 2 A solution of HxBz-32b (3.2 g, 5.76 mmol, 1 eq.) in O (5 mL) was added with LiOH.H 2 O (725 mg, 17.3 mmol, 3 eq.) was added and then stirred at 60° C. for 4 hours. The reaction mixture was concentrated under reduced pressure to remove EtOH. The pH of the mixture was adjusted to about 5 with HCl (12M) at 0°C, then filtered and the filter cake was dried under reduced pressure to obtain the crude product. CH the crude product for 20 min at 25 °C. 3 Trituration with CN gave HxBz-32c (2.7 g, 5.12 mmol, 88.86% yield) as a gray solid. 1 H NMR(DMSO-d 6 ,400MHz)δ9.34(s,1H),9.02(s,1H),8.42(s,1H),7.98-7.92(m,2H),7.89-7.83(m,2H),3.83(t,J= 8.0Hz,2H),3.59-3.49(m,4H),2.90(d,J=6.0Hz,2H),2.56-2.54(m,1H),1.30(s,9H).

[0287] Cyclobutyl N-[3-[[2-amino-8-[5-[3-[(tert-butoxycarbonylamino)methyl]azetidin-1-yl]sulfonyl-3-pyridyl]-3H-1-benzazepine-4 -Carbonyl]-propyl-amino]propyl]carbamate, preparation of HxBz-32d A solution of HxBz-32c (400 mg, 758 umol, 1 eq.) in DMF (10.0 mL) contains HATU (317 mg, 834 umol, 1.1 eq.), DIEA (490 mg, 3.79 mmol, 660 uL, 5 eq.), and cyclobutyl N-[ 3-(Propylamino)propyl]carbamate (380 mg, 1.52 mmol, 2 eq., HCl) was added and stirred at 25° C. for 1 hour. The mixture was diluted with water (50 mL) and extracted with EtOAc (30 mL x 3). The organic layer was washed with brine (20mLx3) and 2 S.O. 4 Dry on top, filter and concentrate. The residue was purified by flash silica gel chromatography (ISCO®, 1 g SepaFlash® silica flash column, eluent of 0-30% ethyl acetate / MeOH@35 mL / min) and purified with HxBz-32d (340 mg, 469.69 umol, yield 61.95%) was obtained as a pale yellow solid. 1 H NMR(MeOD,400MHz)δ9.18(d,J=2.0Hz,1H),8.95(d,J=2.0Hz,1H),8.42(t,J=2.0Hz,1H),7.58-7.50(m ,2H),7.49-7.43(m,1H),6.93(s,1H),4.85-4.76(m,1H),3.90(t,J=8.4Hz,2H),3.64-3.56(m,2H), 3.54-3.48(m,2H),3.47-3.39(m,2H),3.32(br s,2H),3.22-3.02(m,4H),2.70-2.57(m,1H),2.35-2.01(m, 4H),1.90-1.80(m,2H),1.77-1.47(m,4H),1.37(s,9H),1.05-0.76(m,3H).

[0288] Cyclobutyl N-[3-[[2-amino-8-[5-[3-(aminomethyl)azetidin-1-yl]sulfonyl-3-pyridyl]-3H-1-benzazepine-4-carbonyl]-propyl- Preparation of amino]propyl]carbamate, HxBz-32 CH 3 CN (2.00mL) and H 2 To a solution of HxBz-32d (340 mg, 470 umol, 1 eq.) in O (1.00 mL) was added TFA (428 mg, 3.76 mmol, 278 uL, 8 eq.) and then stirred at 80° C. for 1 h. The mixture was concentrated and filtered. The residue was purified by preparative HPLC (column: Phenomenex luna C18 100*40mm*5um, mobile phase: [water (0.1%TFA)-ACN], B%: 5%~35%, 8 min) to obtain HxBz -32 (400 mg, 470 umol, yield 99.98%, 2TFA) was obtained as a pale yellow solid. 1 H NMR(MeOD,400MHz)δ9.24(d,J=1.6Hz,1H),9.04(d,J=1.6Hz,1H),8.49(s,1H),7.88-7.71(m,3H),7.13 (br s,1H),4.85-4.80(m,1H),4.03(t,J=8.4Hz,2H),3.73(dd,J=5.6,8.4Hz,2H),3.59-3.43(m,4H) ,3.38(br s,2H),3.12(br d,J=7.6Hz,4H),2.83-2.73(m,1H),2.37-2.12(m,2H),2.00-2.10(m,4H),1.78 -1.43(m,4H),1.05-0.83(m,3H).LC / MS[M+H]624.3(calculated value), LC / MS[M+H]624.2(observed value).

[0289] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[[5-[2-Amino-4-[3- (cyclobutoxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]-3-pyridyl]sulfonyl]azetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy] Preparation of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL-33a A solution of HxBz-32 (200mg, 235umol, 1eq, 2TFA) in THF (2.00mL) was added with 3 N (71.0mg, 704umol, 98.0uL, 3eq) and 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]propanoic acid (166 mg, 235 umol, 1 eq) was added and then at 0 °C The mixture was stirred for 1 hour. The mixture was concentrated, diluted with water (10 mL), the pH of the mixture was adjusted to about 6 by gradual addition of TFA, extracted with MTBE (10 mL) and discarded, and the aqueous phase was purified with DCM: It was further extracted with i-PrOH=3:1 (20 mL x 3). organic layer with Na 2 S.O. 4 Dry on top, filter and concentrate to give HxBzL-33a (210 mg, 180.36 umol, 76.81% yield) as a pale yellow oil.

[0290] Preparation of HxBzL-33 HxBzL-33a (210 mg, 180 umol, 1 eq.) and 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonic acid (178 mg, 721 umol, 4 eq.) in DCM (4.00 mL) and DMA (0.20 mL) ) was added EDCI (138 mg, 721 umol, 4 equivalents) and then stirred at 25° C. for 1 hour. The mixture was concentrated and filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.2% FA)-ACN], B%: 15%~40%, 8 min) to obtain HxBzL-33. (98 mg, 68.13 umol, yield 37.77%, FA) was obtained as a white solid. 1 H NMR(MeOD,400MHz)δ9.23(d,J=2.0Hz,1H),9.02(d,J=2.0Hz,1H),8.48(t,J=2.0Hz,1H),7.91-7.67(m ,3H),7.13(s,1H),4.85-4.80(m,1H),3.93(t,J=8.4Hz,2H),3.86(t,J=5.6Hz,2H),3.66-3.55(m, 40H),3.54-3.48(m,4H),3.40(br s,2H),3.25-3.08(m,4H),2.97(t,J=5.6Hz,2H),2.79-2.68(m,1H), 2.29(br t,J=6.0Hz,3H),1.93-1.80(m,3H),1.77-1.52(m,4H),1.01-0.88(m,3H).LC / MS[M+H]1392.5( calculated value), LC / MS[M+H]1392.3 (observed value).

[0291] Example L-34 Cyclobutyl(2-((2-amino-8-(2-(39-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,37-dioxo -6,9,12,15,18,21,24,27,30,33-Decaoxa-2,36-diazanonatriacontyl)pyrimidin-5-yl)-N-propyl-3H-benzo[b] Synthesis of azepine-4-carboxamido)oxy)ethyl)carbamate, HxBzL-34 [ka] Cyclobutyl (2-((2-amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy) in DMF (1 ml) ethyl) carbamate, HxBzL-34a (23.6 mg, 0.046 mmol, 1 eq.), and 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10 DIPEA (49 μl, 0.28 mmol, 6 (7-azenazotriazotolyloxy-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate), PyAOP, CAS Reg.No.156311-83-0 (59 mg, 0.113 mmol, 2.4 eq.) Added. The reaction was stirred at room temperature for 2 hours, then concentrated and purified by preparative HPLC to yield HxBzL-34 (4.9 mg, 0.0042 mmol, 9%). LC / MS[M+H]1170.6 (calculated value), LC / MS[M+H]1170.9 (observed value).

[0292] Example L-37 Cyclobutyl(2-((2-amino-8-(2-(38-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,37-dioxo -6,9,12,15,18,21,24,27,30,33-decaoxa-2,36-diazaoctatriacontyl)pyrimidin-5-yl)-N-propyl-3H-benzo[b ]Azepine-4-carboxamido)oxy)ethyl)carbamate, synthesis of HxBzL-37 [ka] Cyclobutyl (2-((2-amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy in DFM (0.5 ml) )ethyl) carbamate, HxBzL-37a (12.4 mg, 0.024 mmol, 1 eq.), and 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6, DIPEA (25.5 μl , 0.15 mmol, 6 eq.) followed by PyAOPY (31.0 mg, 0.059 mmol, 2.4 eq.). The reaction was stirred at room temperature, monitored by LC / MS, then concentrated and purified by preparative HPLC to yield HxBzL-37 (6.7 mg, 0.0058 mmol, 24%). LC / MS[M+H]1156.6 (calculated value), LC / MS[M+H]1156.9 (observed value).

[0293] Example L-38 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]-2-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-38 [ka] Preparation of tert-butyl N-[(5-bromo-2-pyridyl)methyl]carbamate, HxBz-36b CH 3 TFA ( 9.19g, 80.6mmol, 5.97mL, 3eq) and Et 3 SiH (31.3g, 268.8mmol, 42.9mL, 10eq) was added at 0°C and stirred at 25°C for 3 hours. The reaction mixture was diluted with Na at 0 °C. 2 C.O. 3 Quenched by adding aqueous solution (200 mL) and concentrated under reduced pressure. The residue was diluted with (200 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (50 mL) and washed with Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:0~1:1). HxBz-36b (9 g, crude) was obtained as a pale yellow solid. 1 H NMR(CDCl 3 ,400MHz)δ8.59(d,J=2.4Hz,1H),7.78(dd,J=2.4,8.4Hz,1H),7.20(d,J=8.4Hz,1H),5.50(br s,1H) ,4.58-4.29(m,2H),1.45(s,9H)

[0294] Preparation of Tert-Butyl N-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]methyl]carbamate, HxBz-36c HxBz-36b (8.00 g, 27.9 mmol, 1 eq.) in dioxane (80 mL), Pin 2 B 2 (8.49g, 33.4mmol, 1.2eq), Pd(dppf)Cl 2 A mixture of (1.02 g, 1.39 mmol, 0.05 eq.) and KOAc (5.47 g, 55.7 mmol, 2 eq.) was degassed with N 2 I purged it 3 times. Mixture, N 2 Stirred under atmosphere at 90° C. for 2 hours and then used directly in the next step without work-up to give HxBz-36c (9.4 g, crude) as a dark brown oil.

[0295] Preparation of ethyl 2-amino-8-[6-[(tert-butoxycarbonylamino)methyl]-3-pyridyl]-3H-1-benzazepine-4-carboxylate, HxBz-36d Dioxane (80mL) and H 2 HxBz-36c (9.30 g, 27.82 mmol, 2 eq.), ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylic acid (4.30 g, 13.9 mmol, 1 eq.) in O (8 mL), Pd(dppf)Cl 2 (509mg, 695umol, 0.05eq), and K 2 C.O. 3 (3.84g, 27.8mmol, 2eq) was degassed and N 2 Purge 3 times with N 2 Stirred at 90°C for 3 hours under atmosphere. The reaction mixture was filtered and concentrated under reduced pressure. H the residue 2 Diluted with O (50 mL) and extracted with EtOAc (50 mL x 3). Wash the combined organic layers with brine (30 mL x 3) and add Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:0~0:1), then (SiO 2 , EtOAc:MeOH=1:0~5:1) to obtain HxBz-36d (2.40 g, 5.50 mmol, yield 39.5%) as a pale yellow solid. 1 H NMR(MeOD,400MHz)δ8.76(s,1H),8.10(br d,J=8.0Hz,1H),7.85(s,1H),7.58-7.33(m,4H),4.40(s,2H) ),4.32(q,J=7.2Hz,2H),3.05(s,2H),1.48(s,9H),1.38(t,J=7.2Hz,3H).

[0296] Preparation of 2-amino-8-[6-[(tert-butoxycarbonylamino)methyl]-3-pyridyl]-3H-1-benzazepine-4-carboxylic acid, HxBz-36e A solution of HxBz-36d (2.40 g, 5.50 mmol, 1 eq) in EtOH (30 mL) was added with H 2 LiOH.H in O (6 mL) 2 A solution of O (923 mg, 22.0 mmol, 4 eq.) was added and then stirred at 40° C. for 2 hours. The pH of the reaction mixture was adjusted to 5-6 by adding 1M HCl at 0 °C and then concentrated under reduced pressure to remove EtOH. H the residue 2 Diluted with O (10 mL), filtered, and dried the filter cake under reduced pressure to give HxBz-36e (1.88 g, 4.60 mmol, 83.7% yield) as a gray solid. 1 H NMR(DMSO,400MHz)δ9.01(s,1H),8.50(br d,J=8.4Hz,1H),7.93(s,1H),7.83(s,2H),7.75(s,1H), 7.73-7.66(m,1H),4.41(br s,2H),3.51(s,2H),1.40(s,9H).

[0297] Preparation of tert-butyl N-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]-2-pyridyl]methyl]carbamate, HxBz-36f EDCI (493 mg , 2.57 mmol, 3 eq.) and then stirred at 25° C. for 1 hour. The reaction mixture was concentrated under reduced pressure to remove DCM. H the residue 2 Dilute with O (10 mL) and adjust the pH of the mixture to Na at 0 °C. 2 C.O. 3Adjusted to ~9 by adding aqueous solution and extracted with EtOAc (10 mL x 3). Wash the combined organic layers with brine (5 mL x 3) and add Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:0~0:1), then (SiO 2 , EtOAc:MeOH=1:0~3:1) to obtain HxBz-36f (0.33 g, 669 umol, yield 78.0%) as a pale yellow solid. 1 H NMR(MeOD,400MHz)δ8.76(d,J=2.0Hz,1H),8.11(br d,J=8.4Hz,1H),7.47(d,J=8.4Hz,2H),7.43(d, J=2.0Hz,1H),7.40-7.34(m,1H),7.29(s,1H),4.40(s,2H),3.95(q,J=7.2Hz,2H),3.73(t,J=7.2 Hz,2H),3.31(s,2H),1.82-1.70(m,2H),1.48(s,9H),1.17(t,J=7.2Hz,3H),0.99(t,J=7.2Hz,3H ).

[0298] Preparation of 2-amino-8-[6-(aminomethyl)-3-pyridyl]-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz-36 CH 3 CN (3mL) and H 2 To a solution of HxBz-36f (0.33 g, 669 umol, 1 eq) in O (3 mL) was added TFA (610 mg, 5.35 mmol, 396 uL, 8 eq) and then stirred at 80 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. H the residue 2 Diluted with O (5 mL), extracted with MTBE (5 mL x 3) and discarded. The aqueous phase was concentrated under reduced pressure to obtain HxBz-36 (0.33 g, 530.95 umol, 79.42% yield, 2TFA) as a pale yellow solid. 1 H NMR(MeOD,400MHz)δ8.99(d,J=2.0Hz,1H),8.20(dd,J=2.4,8.4Hz,1H),7.79-7.67(m,3H),7.59(d,J= 8.4Hz,1H),7.45(s,1H),4.36(s,2H),3.98(q,J=7.2Hz,2H),3.76(t,J=7.2Hz,2H),3.43(s,2H) ,1.83-1.72(m,2H),1.26-1.16(m,3H),1.01(t,J=7.2Hz,3H).LC / MS[M+H]394.2(calculated value),LC / MS[M +H]394.2 (observed value).

[0299] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]- 3H-1-Benzazepin-8-yl]-2-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL-38a preparation A solution of HxBz-36 (0.15 g, 241 umol, 1 eq., 2 TFA) in THF (3 mL) contains TEA (73.3 mg, 724 umol, 3 eq.) and 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]propanoic acid (171 mg, 241 umol, 1 eq) was added at 0°C and stirred at 20°C for 0.5 h. The pH of the reaction mixture was adjusted to 5-6 with TFA at 0 °C, then with H 2 Diluted with O (10 mL), extracted with EtOAc (5 mL x 3) and discarded. The aqueous phase was further extracted with DCM:i-PrOH=3:1 (5mLx3). Mixed organic layer with Na 2 S.O. 4 Dry on top, filter, and concentrate under reduced pressure to give HxBzL-38a (0.23 g, 219 umol, 90.9% yield, TFA) as a colorless oil. 1 H NMR(MeOD,400MHz)δ8.91(d,J=2.0Hz,1H),8.33(dd,J=2.0,8.0Hz,1H),7.83-7.77(m,1H),7.75-7.69(m, 3H),7.47(s,1H),4.64(s,2H),3.98(q,J=7.2Hz,2H),3.83-3.74(m,4H),3.71(t,J=6.4Hz,2H), 3.66-3.50(m,36H),3.45(s,2H),2.58(t,J=6.0Hz,2H),2.53(t,J=6.0Hz,2H),1.83-1.73(m,2H),1.21 (t,J=7.2Hz,3H),1.01(t,J=7.2Hz,3H)

[0300] Preparation of HxBzL-38 A solution of HxBzL-38a (0.18 g, 172 umol, 1 eq., TFA) in DCM (3 mL) and DMA (0.3 mL) was added with (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy Sodium (184 mg, 687 umol, 4 eq.) and EDCI (132 mg, 687 umol, 4 eq.) were added and stirred at 20° C. for 0.5 h. The reaction mixture was concentrated under reduced pressure to remove DCM and filtered. The residue was purified by preparative HPLC (TFA conditions, column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 10%~35%, 8 minutes), HxBzL-38 (116.7 mg, 91.4 umol, 53.2% yield, TFA) was obtained as a white solid. 1 H NMR(MeOD,400MHz)δ9.01(d,J=2.0Hz,1H),8.57(dd,J=2.0,8.4Hz,1H),7.92(d,J=8.4Hz,1H),7.84-7.79 (m,2H),7.75-7.68(m,1H),7.45(s,1H),4.72(s,2H),3.98(q,J=7.2Hz,2H),3.85(t,J=6.0Hz, 2H),3.82-3.72(m,4H),3.67-3.51(m,36H),3.45(s,2H),2.96(t,J=6.0Hz,2H),2.59(t,J=6.0Hz,2H ),1.83-1.73(m,2H),1.21(t,J=7.2Hz,3H),1.01(t,J=7.2Hz,3H).LC / MS[M+H]1162.5(calculated value),LC / MS[M+H]1162.5 (observed value).

[0301] Example L-39 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[3-(cyclobutoxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]-2-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-39 [ka] Cyclobutyl N-[3-[[2-amino-8-[6-[(tert-butoxycarbonylamino)methyl]-3-pyridyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl] Preparation of carbamate, HxBz-38b 2-Amino-8-[6-[(tert-butoxycarbonylamino)methyl]-3-pyridyl]-3H-1-benzazepine-4-carboxylic acid, HxBz-38a (0.35 g, 857 umol) in DMF (5 mL). , 1 eq.) and cyclobutyl N-[3-(propylamino)propyl]carbamate (258 mg, 1.03 mmol, 1.2 eq., HCl), HATU (326 mg, 857 umol, 1 eq.) and DIEA (332 mg, 2.57 mmol, 448 uL, 3 eq.) was added and then stirred at 20° C. for 2 hours. Add the reaction mixture at 0 °C 2 Quenched with O (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (50 mL) and washed with Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, MeOH / ethyl acetate = 1 / 5) to obtain HxBz-38b (0.45 g, 744.12 umol, yield 86.84%) as a yellow solid.

[0302] Cyclobutyl N-[3-[[2-amino-8-[6-[(tert-butoxycarbonylamino)methyl]-3-pyridyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl] Preparation of carbamate, HxBz-38 MeCN (5 mL) and H 2 To a solution of HxBz-38b (0.45 g, 744 umol, 1 eq) in O (5 mL) was added TFA (679 mg, 5.95 mmol, 441 uL, 8 eq) and stirred at 80 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to remove MeCN and then extracted with MTBE (5 mL) to remove excess TFA. The aqueous layer was concentrated to obtain a residue, and the residue was subjected to preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 10%~40%, 8 HxBz-38 (0.4 g, 646 umol, 86.89% yield, TFA) was obtained as a yellow solid. 1H NMR(MeOD,400MHz)δ 8.99(d,J=1.8Hz,1H),8.20(dd,J=2.4,8.2Hz,1H),7.80-7.66(m,3H),7.59(d,J=8.4 Hz,1H),7.10(br s,1H),4.85-4.80(m,1H),4.36(s,2H),3.54(br t,J=7.2Hz,2H),3.47(br s,2H), 3.36(br s,2H),3.13(br s,2H),2.42-1.96(m,2H),1.92-1.79(m,3H),1.77-1.59(m,3H),0.94(br s,3H) .LC / MS[M+H]505.3 (calculated value), LC / MS[M+H]505.3 (observed value).

[0303] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutoxycarbonyl [amino)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]-2-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy Preparation of ethoxy]propanoic acid, HxBzL-39a A solution of HxBzL-39 (0.15g, 204umol, 1eq, 2TFA) in THF (5mL) was added with 3 N (62.1mg, 614umol, 85.49uL, 3eq) and 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]propanoic acid (145 mg, 205 umol, 1 eq) was added and then at 0 °C The mixture was stirred for 2 hours. Add H to the reaction mixture 2 Quenched with O (5 mL) and adjusted the pH of the mixture to ~6 with TFA, then extracted with EtOAc (10 mL) and discarded, and the aqueous phase was extracted with DCM / PrOH=3 / 1 (20 mL x 3). Further extract with Na 2 S.O. 4 Dry over, filter, and concentrate under reduced pressure to give a residue, HxBzL-39a (0.2 g, 191 umol, 93.46% yield) as a yellow oil.

[0304] Preparation of HxBzL-39 HxBzL-39a (0.2 g, 191 umol, 1 eq) and sodium in DCM (2 mL) and DMA (1 mL); 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (154 mg, 574 umol, EDCI (110 mg, 574 umol, 3 eq.) was added to a solution of 3 eq.) and then stirred at 20° C. for 1 h. The reaction mixture was concentrated under reduced pressure to remove DCM and filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 20%~40%, 8 min) to obtain HxBzL-39. (0.08g, 62.83umol, yield 32.83%) was obtained as a yellow solid. 1 H NMR(MeOD,400MHz)δ 9.03(d,J=1.8Hz,1H),8.61(br d,J=8.4Hz,1H),7.95(d,J=8.4Hz,1H),7.87-7.78(m ,2H),7.73(br s,1H),7.11(s,1H),4.73(s,3H),3.85(t,J=5.6Hz,2H),3.80(t,J=5.6Hz,2H), 3.67-3.50(m,38H),3.64(br s,1H),3.38(br s,2H),3.13(br s,2H),2.95(t,J=5.6Hz,2H),2.59(t,J =5.6Hz,2H),2.35-1.96(m,2H),1.94-1.81(m,3H),1.77-1.64(m,4H),0.93(br s,3H).LC / MS[M+H] 1273.5 (calculated value), LC / MS[M+H] 1273.7 (observed value).

[0305] Example L-40 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[5 -[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-40 [ka] Preparation of tert-butyl (2S)-1-(5-bromopyridine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-40a 5-bromopyridine-2-carboxylic acid (2.00g, 9.90mmol, 1.0eq) in DMF (10mL), Et 3 HATU (3.76 g, 9.90mmol, 1.0eq) in N 2 The mixture was stirred at 0°C for 30 min, then heated to 25°C and stirred for a further 0.5 h. Water (30 mL) was added, the aqueous phase was extracted with ethyl acetate (30 mL*3), the combined organic phase was washed with brine (30 mL*1), anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 20 / 1, 2 / 1) to obtain HxBzL-40a (3.40 g, 9.57 mmol, Yield: 96.6%) was obtained as a yellow oil. 1 H NMR(400MHz, CDCl 3 )δ8.65(d,J=1.6Hz,1H),7.96-7.92(m,2H),5.03(dd,J=3.2,8.4Hz,1H),3.91-3.85(m,2H),2.33-2.28 (m,2H),2.18-2.12(m,2H),1.55-1.48(m,9H).

[0306] Tert-Butyl(2S)-1-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carbonyl]pyrrolidine-2-carboxylate, HxBzL -40b preparation HxBzL-40a (3.40 g, 9.57 mmol, 1.0 eq) in dioxane (30 mL), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2 -dioxaborolan-2-yl)-1,3,2-dioxaborolane (2.92g, 11.5mmol, 1.2eq), Pd(dppf)Cl 2 (700 mg, 957 umol, 0.1 eq.) and AcOK (2.35 g, 23.9 mmol, 2.5 eq.) were degassed and then N 2 The mixture was heated to 100°C for 3 hours. The reaction mixture was concentrated in vacuo to give HxBzL-40b (3.60 g, crude) as a black oil, which was used directly in the next step without purification.

[0307] Preparation of ethyl 2-amino-8-[6-[(2S)-2-tert-butoxycarbonylpyrrolidine-1-carbonyl]-3-pyridyl]-3H-1-benzazepine-4-carboxylate, HxBzL-40c Dioxane (45mL) and H 2 HxBzL-40b (3.60 g, 8.95 mmol, 1.0 eq.), ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylate (2.77 g, 8.95 mmol, 1.0 eq.) in O (5 mL), Pd(dppf)Cl 2 (655 mg, 895 umol, 0.1 equivalent) and K 3 P.O. 4 A solution of (3.80g, 17.9mmol, 2.0eq) was degassed and then N 2 The mixture was heated to 95°C for 2 hours under vacuum. Dioxane (45 mL) was removed, the aqueous phase was extracted with ethyl acetate (30 mL*3), the combined organic phases were washed with brine (30 mL*1), and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 0 / 1) to obtain HxBzL-40c (1.60 g, 3.17 mmol, Yield: 35.4%) was obtained as a pale yellow solid.

[0308] Preparation of 2-amino-8-[6-[(2S)-2-tert-butoxycarbonylpyrrolidine-1-carbonyl]-3-pyridyl]-3H-1-benzazepine-4-carboxylic acid, HxBzL-40d MeOH (10mL) and H 2 A solution of HxBzL-40c (1.60 g, 3.17 mmol, 1.0 equiv) in O (5 mL) was added with LiOH H 2 O (399mg, 9.51mmol, 3.0eq), N 2 was added in one portion at 25° C. and stirred at 25° C. for 10 hours. The reaction mixture was quenched with HCl (4M) until pH=7, then MeOH (10 mL) was removed and the precipitate was filtered and dried to give HxBzL-40d (1.10 g, 2.31 mmol, 72.8% yield) was obtained as a white solid. 1 H NMR(400MHz,DMSO-d 6)δ8.86(d,J=2.0Hz,1H),8.32-8.26(m,1H),8.01(d,J=8.4Hz,1H),7.95-7.65(m,5H),5.04-5.01(m ,1H),3.79-3.82(m,2H),3.52(s,2H),2.36-2.27(m,1H),2.03-1.94(m,1H),1.89-1.77(m,2H),1.45-1.23 (m,9H).

[0309] tert-butyl(2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carboxylate, Preparation of HxBzL-40e EDCI (322 mg, 1.68 mmol, 4.0 equiv), N 2 was added in one portion at 25° C. and then stirred at 25° C. for 1 hour. DCM (4 mL) was removed, water (8 mL) was added, then the pH of the aqueous phase was adjusted to saturated NaHCO 3 8 and extracted with ethyl acetate (5 mL*3), the combined organic phase was washed with brine (5 mL*1), and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100~200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 0 / 1 ~ ethyl acetate / methanol = 10 / 1) to obtain HxBzL. -40e (200 mg, 356 umol, 84.8% yield) was obtained as a brown oil.

[0310] (2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carboxylic acid, HxBzL-40f preparation of MeCN (1 mL) and H 2 To a solution of HxBzL-40e (200mg, 356umol, 1.0eq) in O (2mL) was added HCl (12M, 890uL, 30eq) with N 2 The mixture was stirred at 80° C. for 1 h and the reaction mixture was concentrated in vacuo to give HxBzL-40f (175 mg, 346 umol, 97.2% yield) as a yellow oil.

[0311] tert-Butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[5-[2-amino -4-[Ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy Preparation of ]ethoxy]ethoxy]propanate, HxBzL-40g HxBzL-40f (175 mg, 346 umol, 1.0 eq.) in DMF (2 mL), tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2- (2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (203mg, 346umol, 1.0eq), and Et 3 HATU (132mg, 346umol, 1.0eq) was added to a mixture of N (105mg, 1.04mmol, 145uL, 3.0eq), N 2 Added in one portion at 0° C. and stirred at 0° C. for 30 min, then heated to 25° C. and stirred for an additional 0.5 h. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex luna C18 250*50mm*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 20% to 50%, 10 minutes). Purification provided HxBzL-40g (150mg, 126umol, 36.5% yield, TFA) as a pale yellow oil.

[0312] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[5-[2-amino-4- [ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy Preparation of ethoxy]propanoic acid, HxBzL-40h MeCN (0.2mL) and H 2 To a solution of HxBzL-40g (150mg, 140umol, 1.0eq) in O (2mL) was added HCl (12M, 349uL, 30eq) with N 2 was added in one portion at 25° C. and then stirred at 80° C. for 1 hour. The reaction mixture was concentrated in vacuo to CH 3 CN was removed and the aqueous phase was lyophilized to give HxBzL-40h (140 mg, 137.64 umol, 98.48% yield) as a brown oil.

[0313] Preparation of HxBzL-40 HxBzL-40h (140 mg, 138 umol, 1.0 eq.) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (185 mg, 688 umol) in DCM (1.5 mL) and DMA (0.5 mL). , 5.0 eq.), EDCI (132 mg, 688 umol, 5.0 eq.) was added to a mixture of N 2 was added in one portion at 20° C. and then stirred at 20° C. for 1 hour. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 10%~40%, 8 minutes). HxBzL-40 (46.3 mg, 35.5 umol, yield 25.8%, purity 95.5%) was obtained as a pale yellow oil. 1 H NMR(400MHz,MeOD)δ8.96(d,J=2.0Hz,1H),8.28(dd,J=2.4,8.4Hz,1H),8.01(d,J=8.4Hz,1H),7.84(s ,2H),7.82(d,J=1.6Hz,1H),7.48(s,1H),5.16(dd,J=4.4,8.0Hz,1H),4.00(q,J=7.2Hz,2H),3.90 -3.84(m,3H),3.77(t,J=7.2Hz,2H),3.68-3.56(m,36H),3.53-3.43(m,6H),3.22-3.14(m,2H),3.01-2.96 (m,2H),2.42-2.35(m,1H),2.13-1.96(m,3H),1.85-1.75(m,2H),1.23(t,J=7.2Hz,3H),1.03(t,J =7.2Hz,3H).LC / MS[M+H]1245.5 (calculated value), LC / MS[M+H]1245.4 (observed value).

[0314] Example L-42 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[5 -[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-42 [ka] Preparation of (R)-tert-butyl 1-(5-bromopyrimidine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-42b A solution of 5-bromopyrimidine-2-carboxylic acid, HxBzL-42a (200 mg, 985 umol, 1 eq) in DMF (3 mL), DIEA (509 mg, 3.94 mmol, 686 uL, 4 eq), and HATU (412 mg, 1.08 mmol, 1.1 eq.) at 0 °C, then stirred for 10 min, and to the mixture was added tert-butyl(2S)-pyrrolidine-2-carboxylate (186 mg, 1.08 mmol, 1.1 eq.), further at 25 °C. Stirred for 3 hours. The reaction mixture was diluted with 20 mL of water and extracted with EtOAc (20 mL x 3). Wash the combined organic layers with brine (20mLx2) and add Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give a residue. The residue was subjected to column chromatography (SiO 2 , petroleum ether / ethyl acetate=50 / 1 to 1 / 1) to obtain HxBzL-42b (200 mg, 561 umol, yield 56.99%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 )δ 9.18-9.10(m,2H),4.70-4.41(m,1H),3.75-3.48(m,2H),2.42-1.87(m,4H),1.56-1.30(m,9H)

[0315] (R)-tert-butyl 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL -42c preparation HxBzL-42b (200 mg, 561 umol, 1 eq.) and Pin in dioxane (5 mL) 2 B 2 (214 mg, 842 umol, 1.5 eq) was added with KOAc (110 mg, 1.12 mmol, 2 eq) and Pd(dppf)Cl 2 (41.1 mg, 56.2 umol, 0.1 equiv.) with protected N 2and then stirred at 90° C. for 2 hours. The mixture was filtered and concentrated under reduced pressure. The crude product HxBzL-42c (230 mg, crude) obtained as a brown solid was used in the next step without further purification.

[0316] (R)-tert-butyl 1-(5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidine-2-carbonyl)pyrrolidine-2-carboxy Preparation of HxBzL-42d HxBzL-42c (230 mg, 570 umol, 1 eq.) and 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide (209 mg, 570 umol, 1 eq.) in dioxane (5 mL). ) in water (0.2 mL). 2 C.O. 3 (158mg, 1.14mmol, 2eq) and Pd(dppf)Cl 2 A solution of (41.7 mg, 57 umol, 0.1 eq.) was mixed with protected N 2 and then stirred at 90° C. for 16 hours. The mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate=50 / 1 to ethyl acetate:MeOH=5:1) to obtain HxBzL-42d (240 mg, 427 umol, yield 74.8%) as a yellow oil.

[0317] (R)-1-(5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidine-2-carbonyl)pyrrolidine-2-carboxylic acid, HxBzL -42e preparation H 2 To a solution of HxBzL-42d (240 mg, 427 umol, 1 eq) in O (5 mL) and MeCN (2 mL) was added HCl (12M, 355 uL, 10 eq) and then stirred at 80 °C for 1 h. The mixture was filtered and concentrated under reduced pressure to give HxBzL-42e (170 mg, 336 umol, 78.7% yield) as a yellow oil.

[0318] tert-Butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[5-[2-amino -4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy Preparation of ]ethoxy]ethoxy]propanate, HxBzL-42f Tert-Butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy] in DMF (2 mL) In a solution of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate (167 mg, 284 umol, 1.2 eq.) and HxBzL-42e (120 mg, 237 umol, 1 eq.) and DIEA (91.9 mg, 711 umol, 124 uL, 3 eq.), HATU (90.1 mg, 237 umol, 1 eq) was added at 0°C, stirred at 0°C for 2 hours, the mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 20%~45%, 8 min) to obtain HxBzL-42f. (120 mg, 112 umol, yield 47.2%) was obtained as a pale yellow oil.

[0319] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[5-[2-Amino-4- [ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy Preparation of ethoxy]propanoic acid, HxBzL-42g H 2 To a mixture of HxBzL-42f (115 mg, 107 umol, 1 eq) in O (3 mL) was added HCl (12M, 89.2 uL, 10 eq) and then stirred at 80 °C for 1 h. The mixture was filtered and concentrated under reduced pressure to give HxBzL-42g (105mg, 103umol, 96.3% yield) as a colorless oil. 1 H NMR(MeOD,400MHz)δ9.39-9.04(m,2H),7.88-7.80(m,2H),7.78-7.74(m,1H),7.48(d,J=3.0Hz,1H),4.90- 4.62(m,1H),4.03-3.95(m,2H),3.92-3.80(m,2H),3.76(t,J=7.2Hz,2H),3.72-3.67(m,2H),3.66-3.57( m,38H),3.48-3.38(m,4H),3.29-3.11(m,2H),2.47(dt,J=2.8,6.2Hz,2H),2.15-1.98(m,4H),1.84-1.73( m,2H),1.44(s,9H),1.22(t,J=7.2Hz,3H),1.01(t,J=7.4Hz,3H)

[0320] Preparation of HxBzL-42 HxBzL-42 g (105 mg, 103 umol, 1 eq.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (111 mg, 413 umol, 4 eq.) in DCM (2 mL) and DMA (0.5 mL) ) was added EDCI (79.1 mg, 413 umol, 4 equivalents) and then stirred at 20° C. for 1 hour. The mixture was filtered and concentrated under residual pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 10%~40%, 8 minutes) to obtain HxBzL-42. (60.0 mg, 44.1 umol, yield 42.8%, TFA) was obtained as a pale yellow oil. 1 H NMR(MeOD,400MHz)δ9.27-9.21(m,2H),7.89-7.81(m,2H),7.77-7.72(m,1H),7.48-7.44(m,1H),5.05-4.62(m ,1H),3.99(q,J=7.0Hz,2H),3.89-3.83(m,4H),3.76(br t,J=7.0Hz,2H),3.66-3.53(m,36H),3.50-3.42 (m,4H),3.28-3.20(m,2H),3.16-3.05(m,1H),2.99-2.94(m,2H),2.46-2.26(m,1H),2.12-1.97(m,3H) ,1.82-1.74(m,2H),1.21(dt,J=1.8,7.2Hz,3H),1.04-0.98(m,3H).LC / MS[M+H]1246.5(calculated value),LC / MS [M+H]1246.4 (observed value).

[0321] Example L-43 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[5 -[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-43 [ka]

[0322] Preparation of tert-butyl (2R)-1-(5-bromopyridine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-43b HATU (4.53 g, 11.9 mmol, 1.1 eq.), and Et 3 N (3.29 g, 32.5 mmol, 4.52 mL, 3 eq.) was added followed by tert-butyl (2R)-pyrrolidine-2-carboxylate (2.25 g, 10.8 mmol, 1 eq., HCl). The mixture was stirred at 20°C for 0.5 hour. The reaction mixture was partitioned between EtOAc (150 mL) and water (100 mL). Separate the organic phase and remove Na 2 S.O. 4 Dry on top and concentrate to give a residue. The residue was subjected to column chromatography (SiO 2 , petroleum ether / ethyl acetate = 1 / 0 to 2 / 1) to obtain HxBzL-43b (3.8 g, 10.7 mmol, yield 98.8%) as a yellow oil. 1 H NMR(400MHz,MeOD)δ8.76-8.61(m,1H),8.17-8.13(m,1H),7.91-7.74(m,1H),5.07-4.51(m,1H),3.96-3.67(m ,2H),2.43-2.27(m,1H),2.18-1.90(m,3H),1.51(s,3H),1.37(s,6H).

[0323] Tert-Butyl(2R)-1-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carbonyl]pyrrolidine-2-carboxylate, HxBzL -43c preparation Tert HxBzL-43b (3.5 g, 9.85 mmol, 1 eq.), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3, 2-dioxaborolan-2-yl)-1,3,2-dioxaborolane, Pin2B2, bis(pinacolato)diboron, CAS Reg.No.78183-34-3 (3.75g, 14.8mmol, 1.5eq), KOAc (2.42g) , 24.6 mmol, 2.5 equivalents), Pd(dppf)Cl2 (721 mg, 985 umol, 0.1 eq.) was added and then stirred at 100° C. for 2 hours. The mixture was used in the next step without work-up and purification. HxBzL-43c (3.96 g, 9.84 mmol, yield 100.00%) was obtained as a black liquid.

[0324] Preparation of ethyl 2-amino-8-[6-[(2R)-2-tert-butoxycarbonylpyrrolidine-1-carbonyl]-3-pyridyl]-3H-1-benzazepine-4-carboxylate, HxBzL-43d Dioxane (100mL) and H 2 HxBzL-43c (3.96 g, 9.84 mmol, 1 eq.), ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylate (3.04 g, 9.84 mmol, 1 eq.) in O (8 mL), Pd(dppf)Cl 2 (360 mg, 492 umol, 0.05 equivalent), and K 2 C.O. 3 A mixture of (3.40g, 24.6mmol, 2.5eq) was stirred at 100°C for 2 hours. The reaction mixture was concentrated to obtain a residue. The residue was dissolved in EtOAc (100 mL) and washed with water (50 mL). Separate the organic phase and remove Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give a residue. The crude product was subjected to column chromatography (SiO 2 , petroleum ether / ethyl acetate = 1 / 0 to 0 / 1, EA:MeOH = 5:1) to obtain HxBzL-43d (4 g, 7.93 mmol, yield 80.5%) as a yellow solid. 1 H NMR(400MHz,MeOD)δ9.07-8.72(m,1H),8.29-8.16(m,1H),8.12-7.78(m,2H),7.62-7.40(m,3H),5.17-4.47(m ,1H),4.34(q,J=7.2Hz,2H),4.04-3.75(m,2H),3.67-2.94(m,2H),2.49-2.27(m,1H),2.22-1.88(m,3H) ),1.53(s,3H),1.43-1.34(m,9H).

[0325] Preparation of 2-amino-8-[6-[(2R)-2-tert-butoxycarbonylpyrrolidine-1-carbonyl]-3-pyridyl]-3H-1-benzazepine-4-carboxylic acid, HxBzL-43e THF (20mL) and H 2 LiOH.H 2 O (582 mg, 13.9 mmol, 2 eq.) was added and then stirred at 20° C. for 3 hours. The mixture was concentrated to remove THF, then the pH of the mixture was adjusted to about 5 with HCl (4M) and a solid formed from the mixture. The mixture was filtered and the filter cake was dried in vacuo to give HxBzL-43e (3.3 g, 6.93 mmol, 99.8% yield) as a white solid.

[0326] tert-butyl (2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carboxylate, Preparation of HxBzL-43f EDCI (483 mg, 2.52 mmol, 3 eq) was added and then stirred at 20°C for 1 hour. The reaction mixture was concentrated to remove DCM and the residue was partitioned between EtOAc (20 mL) and water (20 mL). Separate the organic phase and remove Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give a residue. The residue was subjected to column chromatography (SiO 2 , petroleum ether / ethyl acetate=1 / 0~0 / 1, EA:MeOH=5:1) to obtain HxBzL-43f (0.32 g, 570 umol, yield 67.9%) as a yellow solid. 1 H NMR(400MHz,MeOD)δ9.06-8.77(m,1H),8.26-8.17(m,1H),8.07-7.87(m,1H),7.53-7.36(m,3H),7.30(s,1H) ),5.17-4.50(m,1H),4.01-3.69(m,6H),3.01-2.88(m,2H),2.45-2.30(m,1H),2.18-2.03(m,2H),2.02-1.94 (m,1H),1.82-1.73(m,2H),1.52(s,3H),1.36(s,6H),1.18(t,J=7.2Hz,3H),1.00(t,J=7.2Hz, 3H).

[0327] (2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carboxylic acid, HxBzL-43g preparation of H 2 To a mixture of HxBzL-43f (260 mg, 463 umol, 1 eq) in O (5 mL) was added HCl (12M, 579 uL, 15 eq) and then stirred at 80 °C for 1 h. The mixture was concentrated to give HxBzL-43g (0.25g, 461umol, 99.6% yield, HCl) as a yellow oil.

[0328] tert-Butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[5-[2-amino -4-[Ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]Ethoxy]ethoxy]propanate, preparation of HxBzL-43h HxBzL-43g (200mg, 369umol, 1eq, HCl) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[ To a mixture of 2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (216mg, 369umol, 1eq), HATU (154mg, 406umol, 1.1eq) and DIEA (143 mg, 1.11 mmol, 193 uL, 3 eq) was added at 0°C and stirred at 0°C for 1 hour. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 25%~51%, 8 min) to obtain HxBzL-43h. (340 mg, 286 umol, 77.6% yield, TFA) was obtained as a yellow oil.

[0329] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[5-[2-Amino-4- [ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy Preparation of ethoxy]propanoic acid, HxBzL-43i H 2 To a mixture of HxBzL-43h (340 mg, 286 umol, 1 eq., TFA) in O (20 mL) was added HCl (12 M, 358 uL, 15 eq.) and then stirred at 80 °C for 0.5 h. The mixture was concentrated to give a residue. The crude product was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 10%~40%, 8 minutes) to obtain HxBzL- 43i (220 mg, 209 umol, 72.9% yield, HCl) was obtained as a yellow oil.

[0330] Preparation of HxBzL-43 HxBzL-43i (180 mg, 171 umol, 1 eq., HCl) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (183 mg, 683 umol, EDCI (164 mg, 854 umol, 5 eq.) was added to a mixture of 4 eq.) and stirred at 15° C. for 0.5 h. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 15%~45%, 8 min) to obtain HxBzL-43. (94 mg, 72.6 umol, yield 42.5%, purity 96.2%) was obtained as a colorless oil. 1 H NMR(400MHz,MeOD)δ9.10-8.85(m,1H),8.43-8.16(m,1H),8.11-7.94(m,1H),7.91-7.71(m,3H),7.48(s,1H) ),5.18-4.65(m,1H),4.07-3.72(m,8H),3.69-3.39(m,40H),3.30-3.13(m,2H),3.00-2.97(m,2H),2.59-2.23 (m,1H),2.19-1.66(m,5H),1.25-1.21(m,3H),1.05-1.00(m,3H).LC / MS[M+H]1245.5(calculated value),LC / MS [M+H]1245.4 (observed value).

[0331] Example L-44 2-amino-8-(2-(38-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,37-dioxo-6,9,12 ,15,18,21,24,27,30,33-Decaoxa-2,36-diazaoctatriacontyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine -4-Carboxamide, synthesis of HxBzL-44 [ka] 2-Amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide, HxBz-5 (0.0283g, 0.072mmol, 1 equivalent) and 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,15,18,21,24,27,30,33- Decaoxa-3-azatriacontan-36-acid, HxBzL-44a (0.0478g, 0.072mmol, 1 eq.) was dissolved in dimethylformamide, DMF. Diisopropylethylamine, DIPEA (0.075mol, 0.43mmol, 6eq) followed by ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate), PyAOP, CAS Reg.No.156311-83-0 (0.091g, 0.18mmol, 2.4eq) was added. The reaction was stirred at room temperature, then concentrated and purified by RP-HPLC to yield HxBzL-44 (0.0346g, 0.033mmol, 46%). LC / MS[M+H] 1043.53 (calculated value), LC / MS[M+H] 1043.84 (observed value).

[0332] Example L-47 (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3- [[5-[2-amino-4-[propyl(1H-pyrazol-5-ylmethoxy)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy Synthesis of ]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate, HxBzL-47 [ka] [ka] Preparation of 5-(chloromethyl)-1H-pyrazole, HxBz-41b A solution of 1H-pyrazol-5-ylmethanol, HxBz-41a (4 g, 40.8 mmol, 1 eq.) in DCM (10 mL) was added with thionyl chloride, SOCl 2 (9.70g, 81.55mmol, 5.92mL, 2 equivalents) was added and then stirred at 0°C to 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain HxBz-41b (4.5 g, 38.6 mmol, 94.70% yield) as a white solid. LC / MS[M+H]117.0 (calculated value), LC / MS[M+H]117.0 (observed value).

[0333] Preparation of tert-butyl N-propyl-N-(1H-pyrazol-5-ylmethoxy)carbamate, HxBz-41c To a solution of HxBz-41b (3.01 g, 17.2 mmol, 1 eq.) in DMF (20 mL) at 0 °C was added NaH (1.03 g, 25.7 mmol, 60% purity, 1.5 eq.) and the mixture was heated at this temperature. Stirred for 0.5 h then added KI (285 mg, 1.72 mmol, 0.1 eq.) and 5-(chloromethyl)-1H-pyrazole (2 g, 17.16 mmol, 1 eq.). The resulting mixture was stirred at 20°C for 12 hours. Add NH to the reaction mixture at 0 °C. 4 Quenched with 20 mL of Cl and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (50 mL) and washed with Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex luna C18 (250 * 70 mm, 15 um, mobile phase: [water (0.1% TFA)-ACN], B%: 20% ~ 45%, 20 minutes), HxBz-41c (0.6 g, 2.35 mmol, yield 13.69%) was obtained as a yellow oil. LC / MS [M+H] 256.1 (calculated), LC / MS [M+H] 256.1 (observed).

[0334] Preparation of N-(1H-pyrazol-5-ylmethoxy)propan-1-amine, HxBz-41d MeCN (2 mL) and H 2 To a solution of HxBz-41c (0.5 g, 1.96 mmol, 1 eq) in O (2 mL) was added TFA (2.23 g, 19.58 mmol, 1.45 mL, 10 eq) and then stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove MeCN. The aqueous layer was extracted with 20 mL of MTBE to remove excess TFA. The aqueous layer was lyophilized to give HxBz-41d (0.25 g, crude, TFA) as a yellow oil. 1 H NMR(MeOH,400MHz)δ 7.10(d,J=2.4Hz,1H),6.47(d,J=2.4Hz,1H),5.13(s,2H),3.30-3.20(m,2H),1.78- 1.71(m,2H),1.02(t,J=7.2Hz,2H).LC / MS[M+H]156.1(calculated value), LC / MS[M+H]156.1(observed value).

[0335] tert-butyl N-[[5-[2-amino-4-[propyl(1H-pyrazol-5-ylmethoxy)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methyl]carbamate, Preparation of HxBz-41f HxBz-41d (0.2 g, 743 umol, 1 eq., TFA salt) and 2-amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidine-5- in DCM (2 mL) and DMA (1 mL) EDCI (854 mg, 4.46 mmol, 6 eq) was added to a solution of HxBz-41e (304 mg, 743 umol, 1 eq), and then stirred at 20 °C for 2 h. did. NaHCO mixture 3 quenched to pH=~8, then extracted with EtOAc (30 mL x 4). The combined organic layers were washed with brine (30 mL) and washed with Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give a residue. The residue was subjected to column chromatography (SiO 2 , MeOH / ethyl acetate=1 / 5) to obtain HxBz-41f (0.35 g, 640.30 umol, yield 86.19%) as a yellow solid. LC / MS[M+H]547.3 (calculated value), LC / MS[M+H]547.3 (observed value).

[0336] 2-Amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-propyl-N-(1H-pyrazol-5-ylmethoxy)-3H-1-benzazepine-4-carboxamide, HxBz-41 preparation MeCN (2 mL) and H 2 To a solution of HxBz-41f (0.35 g, 640 umol, 1 eq) in O (2 mL) was added TFA (584 mg, 5.12 mmol, 379 uL, 8 eq) and then stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 1%~25%, 8 min) to obtain HxBz-41. (0.25 g, 371 umol, yield 57.88%, 2TFA) was obtained as a yellow solid. 1 H NMR(MeOH,400MHz)δ 9.20(s,2H),7.82-7.78(m,1H),7.74(d,J=2.0Hz,1H),7.69(d,J=8.4Hz,1H),7.55( d,J=2.0Hz,1H),7.26(s,1H),6.31(d,J=2.0Hz,1H),4.96(s,2H),4.48(s,2H),3.80(t,J=7.4 Hz,2H),3.26(s,2H),1.88-1.73(m,2H),1.01(t,J=7.4Hz,3H).LC / MS[M+H]447.2(calculated value),LC / MS [M+H]447.2 (observed value).

[0337] tert-Butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[propyl(1H -pyrazol-5-ylmethoxy)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Preparation of ethoxy]ethoxy]propanate, HxBzL-47a Et 3 N (90.0mg, 889umol, 124uL, 3eq) and (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[ 2-[2-(3-tert-butoxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, t-Bu-COO-PEG10-COOTFP (226mg, 296 umol, 1 eq) was added and then stirred at 0°C for 2 hours. Add H to the reaction mixture 2 Quench with 5 mL of O, adjust the pH of the mixture to ~6 with TFA at 0 °C, extract the aqueous phase with EtOAc (10 ml*2) to remove by-products, and extract the aqueous phase with DCM / PrOH=10 / 1 (20mL x 3) and the combined organic phase was extracted with Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give compound HxBzL-47a as a yellow oil. LC / MS[M+H]1043.56 (calculated value), LC / MS[M+H]1043.6 (observed value).

[0338] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[propyl(1H-pyrazole- 5-ylmethoxy)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]Propanoic acid, preparation of HxBzL-47b MeCN (2 mL) and H 2 To a solution of HxBzL-47a (0.2 g, 192 umol, 1 eq) in O (2 mL) was added HCl (12M, 320 uL, 20 eq) and then stirred at 80 °C for 2 h. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 5%~35%, 8 min) to obtain HxBzL-47b. (0.13g, 132umol, yield 68.69%) was obtained as a yellow oil. LC / MS[M+H]987.5 (calculated value), LC / MS[M+H]987.6 (observed value).

[0339] Preparation of HxBzL-47 EDCI ( 77.7mg, 405umol, 4eq) was added and then stirred at 20°C for 1 hour. The reaction mixture was filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 20%~40%, 8 minutes) to obtain HxBzL-47. (0.0216g, 19.0umol, yield 18.78%) was obtained as a yellow solid. 1 H NMR(MeOH,400MHz)δ 9.10(s,2H),7.78(dd,J=1.6,8.0Hz,1H),7.71-7.66(m,2H),7.57(d,J=2.4Hz,1H), 7.48-7.37(m,1H),7.26(s,1H),7.28-7.24(m,1H),6.31(d,J=2.4Hz,1H),4.96(s,2H),4.69(s,2H) ,3.86(t,J=6.0Hz,2H),3.83-3.76(m,4H),3.68-3.55(m,36H),3.26(s,2H),3.02-2.91(m,2H),2.60(t ,J=6.0Hz,2H),1.80(t,J=7.2Hz,2H),1.01(t,J=7.2Hz,3H).LC / MS[M+H]1135.5(calculated value),LC / MS [M+H]1135.6 (observed value).

[0340] Example L-52 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[3-(Cyclobutylcarbamoyloxy)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy Synthesis of ]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-52 [ka]

[0341] tert-Butyl N-[[5-[2-amino-4-[3-(cyclobutylcarbamoyloxy)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methyl] Preparation of carbamate, HxBz-45b 2-Amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBz-45a (180 mg, 440 umol) in DMF (3 mL) , 1 eq.), HATU (167 mg, 440 umol, 1 eq.) and DIPEA (284 mg, 2.20 mmol, 383 uL, 5 eq.) were added at 0°C. After the addition, the mixture was stirred at this temperature for 5 minutes and then 3-(propylamino)propyl N-cyclobutyl carbamate (110 mg, 440 umol, 1 eq., HCl) was added at 0.degree. The resulting mixture was stirred at 20°C for 25 minutes. Heat the reaction mixture in H at 0 °C. 2 Quenched by adding O (15 mL) and then extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (5 mL x 3) and washed with Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure. The residue was purified by preparative HPLC (TFA conditions, column: Phenomenex luna C18 250*50mm*10um, mobile phase: [water (0.1%TFA)-ACN], B%: 25% to 55%, 10 minutes). HxBz-45b (0.15 g, 208 umol, yield 47.4%, TFA) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ9.07(s,2H),7.86-7.65(m,3H),7.13(s,1H),4.53(s,2H),4.09-4.06(m,3H),3.63- 3.56(m,2H),3.51-3.45(m,2H),3.36(br s,2H),2.25-2.21(m,2H),2.04-1.87(m,4H),1.78-1.61(m,4H) ,1.48(s,9H),0.98-0.94(m,3H).

[0342] 3-[[2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl N-cyclobutylcarbamate, HxBz-45 preparation To a solution of HxBz-45b (0.15 g, 208 umol, 1 eq., TFA) in EtOAc (1 mL) was added HCl / EtOAc (4M, 10 mL, 192 eq.) and then stirred at 15° C. for 0.5 h. The reaction mixture was concentrated under reduced pressure to give HxBz-45 (135 mg, crude, 2HCl) as a yellow solid. 1 H NMR(MeOD,400MHz)δ9.21(s,2H),7.88-7.71(m,3H),7.13(s,1H),4.48(s,2H),4.16-3.97(m,3H),3.62- 3.58(m,2H),3.51-3.45(m,2H),3.38(br s,2H),2.26-2.20(m,2H),2.04-1.85(m,4H),1.75-1.53(m,4H) ,1.01-0.89(m,3H).LC / MS[M+H]506.3(calculated value), LC / MS[M+H]506.3(observed value).

[0343] tert-Butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[[5-[2-amino-4-[3-( cyclobutylcarbamoyloxy)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] Preparation of ethoxy]ethoxy]ethoxy]propanate, HxBzL-52a A solution of HxBz-45 (75mg, 130umol, 1eq, 2HCl) in DMF (1mL) was added with triethylamine, Et 3 N, TEA (39.4mg, 389umol, 54.1uL, 3eq), and (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2- [2-[2-[2-(3-tert-butoxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate (98.9mg, 130umol, 1 eq.) was added at 0°C. The mixture was stirred at 15°C for 1 hour. The pH of the reaction mixture was adjusted to ˜6 with TFA at 0° C. and then concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions, column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 25% to 55%, 8 minutes), HxBzL-52a (0.13 g, 107 umol, 82.4% yield, TFA) was obtained as a yellow oil. LC / MS[M+H]1102.6 (calculated value), LC / MS[M+H]1102.6 (observed value).

[0344] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutyl carb amoyloxy)propyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]ethoxy]propanoic acid, preparation of HxBzL-52b CH 3 CN (1mL) and H 2 To a solution of HxBzL-52a (0.13 g, 107 umol, 1 eq., TFA) in O (5 mL) was added TFA (97.5 mg, 855 umol, 63.3 uL, 8 eq.) and then stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to CH 3 CN was removed. The aqueous phase was extracted with MTBE (5 mL x 3) and discarded. The aqueous phase was concentrated under reduced pressure to give HxBzL-52b (0.14 g, crude, TFA) as a pale yellow oil. 1H NMR(MeOD,400MHz)δ9.09(s,2H),7.85-7.78(m,1H),7.77-7.69(m,2H),7.13(s,1H),4.69(s,2H),4.09- 4.05(m,2H),3.80(t,J=6.0Hz,2H),3.76-3.69(m,3H),3.66-3.58(m,38H),3.50-3.45(m,2H),3.37(br s ,2H),2.60(t,J=6.0Hz,2H),2.56-2.51(m,2H),2.35-2.07(m,2H),2.06-1.81(m,4H),1.75-1.66(m,4H ),0.98-0.91(m,3H)

[0345] Preparation of HxBzL-52 To a solution of HxBzL-52b (0.13 g, 112 umol, 1 eq., TFA) in DCM (2 mL) and DMA (0.2 mL) was added Sodium (90.1 mg, 336 umol, 3 eq.) and EDCI (85.9 mg, 448 umol, 4 eq.) were added and then stirred at 15° C. for 1 hour. The reaction mixture was concentrated under reduced pressure to remove DCM and filtered. The residue was purified by preparative HPLC (TFA conditions, column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 15% to 40%, 8 minutes), HxBzL-52 (32.3 mg, 25.4 umol, 22.6% yield) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ9.08(s,2H),7.83-7.67(m,3H),7.11(s,1H),4.69(s,2H),4.09-4.05(m,2H),3.86( t,J=6.0Hz,2H),3.80(t,J=6.0Hz,2H),3.70-3.55(m,36H),3.51-3.45(m,3H),3.38(br s,2H),3.32( br s,2H),2.97(t,J=6.0Hz,2H),2.60(t,J=5.6Hz,2H),2.25-2.20(m,2H),2.07-1.84(m,4H),1.80- 1.54(m,4H),1.10-0.82(m,3H).LC / MS[M+H]1274.5(calculated value), LC / MS[M+H]1274.7(observed value).

[0346] Example L-53 (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3- [[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]-3-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy] Synthesis of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate, HxBzL-53 [ka] Preparation of tert-butyl ((5-bromopyridin-3-yl)methyl)carbamate, HxBz-39b A solution of (5-bromo-3-pyridyl)methanamine, HxBz-39a (1 g, 5.35 mmol, 1 eq.), and TEA (649 mg, 6.42 mmol, 893 uL, 1.2 eq.) in MeOH (10 mL) at 0 °C. Boc 2 O (1.40g, 6.42mmol, 1.47mL, 1.2eq) was added and then stirred at 25°C for 2 hours. The mixture was concentrated under reduced pressure. The residue was subjected to column chromatography (SiO 2 , petroleum ether / ethyl acetate=50 / 1 to 1 / 1) to obtain HxBz-39b (1.5 g, 5.22 mmol, yield 97.7%) as a white solid.

[0347] Preparation of Tert-Butyl ((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methyl)carbamate, HxBz-39c HxBz-39b (750 mg, 2.61 mmol, 1 eq.) and Pin in dioxane (10 mL) 2 B 2 (995 mg, 3.92 mmol, 1.5 eq) was added with KOAc (513 mg, 5.22 mmol, 2 eq) and Pd(dppf)Cl 2 (191 mg, 262 umol, 0.1 equivalent), N 2 and then stirred at 90° C. for 2 hours. The mixture was filtered and concentrated under reduced pressure to give HxBz-39c (800 mg, 2.39 mmol, 91.7% yield) as a brown oil, which was used in the next step without further purification.

[0348] Preparation of Tert-butyl ((5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyridin-3-yl)methyl)carbamate, HxBz-39d HxBz-39c (800 mg, 2.39 mmol, 1 eq.) and 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide (877 mg, 2.39 mmol, 1 eq.) in water (3 mL). 2 C.O. 3 (992mg, 7.18mmol, 3eq) and Pd(dppf)Cl 2 A solution of (175 mg, 239 umol, 0.1 eq.) was prepared with N 2 and then stirred at 90° C. for 16 hours. The mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate=50 / 1 to ethyl acetate:MeOH=5:1) to obtain HxBz-39d (900 mg, 1.82 mmol, yield 76.2%) as a yellow oil.

[0349] Preparation of 2-amino-8-(5-(aminomethyl)pyridin-3-yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide, HxBz-39 CH 3 CN (2mL) and H 2 To a solution of HxBz-39d (350 mg, 709 umol, 1 eq.) in O (2 mL) was added TFA (646 mg, 5.67 mmol, 420 uL, 8 eq.) and N 2 Stirred at 80°C for 2 hours under atmosphere. The mixture was filtered and concentrated under reduced pressure to give a residue with H 2 O (15 mL) was added, the aqueous phase was extracted with MTBE (20 mL x 3) and discarded, and the aqueous phase was lyophilized. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 150*30mm*5um, mobile phase: [water (0.1%TFA)-ACN], B%: 5%~35%, 9 min) to obtain HxBz -39 (201 mg, 396 umol, 55.9% yield, TFA) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ8.96(d,J=2.0Hz,1H),8.72(d,J=2.0Hz,1H),8.33-8.27(m,1H),7.80-7.72(m,3H) ,7.46(s,1H),4.31(s,2H),3.98(q,J=7.2Hz,2H),3.76(t,J=7.2z,2H),3.44(s,2H),1.82-1.74( m,2H),1.20(t,J=7.2Hz,3H),1.01(t,J=7.4Hz,3H).LC / MS[M+H]394.2(calculated value),LC / MS[M+H ]394.2 (observed value).

[0350] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]- 3H-1-Benzazepin-8-yl]-3-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL-53a preparation HxBz-39 (150 mg, 296 umol, 1 eq., TFA) and 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]propanoic acid (209 mg, 296 umol, 1 eq.) solution , Et. 3 N (89.7mg, 887umol, 123uL, 3eq) was added and stirred at 25°C for 1 hour. Adjust the pH of the mixture to 4-5 with TFA and H 2 O (5 ml) was added, extracted with EtOAc (10 mL) and discarded, the aqueous solution was further extracted with DCM / i-prOH (20 mL*3, 3 / 1), and the organic layer was 2 S.O. 4 , filtered and concentrated under reduced pressure to give HxBzL-53a (200 mg, 214 umol, 72.4% yield) as a yellow oil.

[0351] Preparation of HxBzL-53 A mixture of HxBzL-53a (0.13 g, 139 umol, 1.0 eq.) in DCM (3 mL) and DMA (0.5 mL) was added with 2,3,5,6-tetrafluorophenol (92.5 mg, 557 umol, 4.0 eq.) and EDCI (133 mg, 696 umol, 5.0 eq) was added in one portion at 25°C and then stirred at 25°C for 0.5 h. The mixture was concentrated and filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 25%~55%, 8 min) to obtain HxBzL-53. (78 mg, 65.2 umol, yield 46.85%, TFA) was obtained as a pale yellow oil. 1H NMR(MeOD,400MHz)δ8.98(d,J=2.0Hz,1H),8.72(d,J=1.6Hz,1H),8.47(s,1H),7.86-7.81(m,1H),7.79 -7.72(m,2H),7.49-7.37(m,2H),4.63(s,2H),3.98(q,J=7.2Hz,2H),3.85(t,J=6.0Hz,2H),3.81- 3.73(m,4H),3.64-3.54(m,36H),3.45(s,2H),2.96(t,J=6.0Hz,2H),2.59-2.50(m,2H),1.87-1.72(m, 2H),1.21(t,J=7.2Hz,3H),1.01(t,J=7.6Hz,3H).LC / MS[M+H]1082.5(calculated value),LC / MS[M+H]1082.6 (observed value).

[0352] Example L-61 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4 -[2-(1-ethyl-2-oxo-imidazolidin-4-yl)ethyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo Synthesis of -propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-61 [ka] [ka] Preparation of N-but-3-enyl-4-nitro-N-propyl-benzenesulfonamide, HxBzL-61b Cs 2 C.O. 3 (40.0 g, 123 mmol, 2.5 eq.), KI (8.16 g, 49.1 mmol, 1.0 eq.), and 4-bromobutyl-1-ene (19.9 g, 147 mmol, 15.0 mL, 3.0 eq.) were then added under N2. The mixture was stirred at 40°C for 12 hours. The reaction mixture was poured into ice water (w / w=1 / 1) (150 mL) and stirred for 10 minutes. The aqueous phase was extracted with ethyl acetate (100 mL x 3). The combined organic layers were washed with brine (100mLx2) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 10 / 1) to obtain HxBzL-61b (11 g, 36.9 mmol, yield 75.1%) as a yellow solid. 1 H NMR(MeOD,400MHz)δ 8.51-8.36(m,2H),8.14-7.94(m,2H),5.77-5.70(m,1H),5.10-4.96(m,2H),3.25(t,J= 7.2Hz,2H),3.15(t,J=7.2Hz,2H),2.31(q,J=7.2Hz,2H),1.67-1.44(m,2H),0.88(t,J=7.2Hz,3H) .LC / MS[M+H]299.1 (calculated value), LC / MS[M+H]299.0 (observed value).

[0353] Preparation of 4-nitro-N-[2-(oxirane-2-yl)ethyl]-N-propyl-benzenesulfonamide, HxBzL-61c A solution of HxBzL-61b (13.5 g, 45.3 mmol, 1.0 eq.) in DCM (200 mL) at 0 °C with meta-chloroperbenzoic acid, m-CPBA (18.4 g, 90.5 mmol, 85% purity, 2.0 eq.) was added and then stirred at 20°C for 12 hours. Filter the mixture and dilute the filtrate with saturated NaHSO 3 (30 mL x 1) and brine (100 mL). Organic phase anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 3 / 1) to obtain HxBzL-61c (12 g, 38.2 mmol, yield 84.4%) was obtained as a white solid. LC / MS[M+H]315.1 (calculated value), LC / MS[M+H]315.0 (observed value).

[0354] Preparation of N-[4-(ethylamino)-3-hydroxy-butyl]-4-nitro-N-propyl-benzenesulfonamide, HxBzL-61d To a solution of HxBzL-61c (7 g, 22 mmol, 1.0 eq) in THF (100 mL) at 0 °C was added ethanamine (33.5 g, 445 mmol, 48.6 mL, 60% purity, 20 eq) at 30 °C. Stir for hours. The mixture was concentrated in vacuo at 45°C. The crude product HxBzL-61d (8 g, 22.3 mmol, 99.95% yield) was used in the next step without further purification as a yellow solid. LC / MS[M+H]360.1 (calculated value), LC / MS[M+H]360.2 (observed value).

[0355] Preparation of tert-butyl N-ethyl-N-[2-hydroxy-4-[(4-nitrophenyl)sulfonyl-propyl-amino]butyl]carbamate, HxBzL-61e HxBzL-61d (7.6g, 21.1mmol, 1.0eq) and H in THF (70mL) 2 A solution of O (10 mL) was added with NaHCO 3 (3.55g, 42.3mmol, 1.64mL, 2.0eq) and Boc 2 O (9.23g, 42.3mmol, 9.71mL, 2.0eq) was added. The mixture was stirred at 25°C for 1 hour. The resulting mixture was poured into ice water (w / w=1 / 1) (50 mL) and stirred for 10 minutes. The aqueous phase was extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (50 mL x 1) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 2 / 1) to obtain HxBzL-61e (8.6 g, 18.7 mmol, Yield: 88.5%) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ 8.46-8.39(m,2H),8.13-8.04(m,2H),3.78-3.70(m,1H),3.39-3.2(m,3H),3.29-3.22(m, 2H),3.20-3.14(m,2H),3.10-3.00(m,1H),1.79-1.69(m,1H),1.65-1.53(m,3H),1.45(s,9H),1.09(t, J=7.2Hz,3H),0.90(t,J=7.2Hz,3H).

[0356] tert-Butyl N-[2-(1,3-dioxoisoindolin-2-yl)-4-[(4-nitrophenyl)sulfonyl-propyl-amino]butyl]-N-ethyl-carbamate, HxBzL-61f preparation of Triphenylphosphine, PPh 3 (4.28 g, 16.3 mmol, 1.5 eq.) and diethyl azodicarboxylate, DEAD (2.84 g, 16.3 mmol, 2.97 mL, 1.5 eq.) were added at 0° C. and stirred at 20° C. for 1 hour. The mixture was poured into ice water (w / w=1 / 1) (50 mL) and stirred for 10 minutes. The aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with brine (30 mL) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 1 / 1) to obtain HxBzL-61f (8.8 g, crude product). was obtained as a yellow solid. LC / MS[M+H]589.2 (calculated value), LC / MS[M+H]589.2 (observed value).

[0357] Preparation of tert-butyl N-[2-amino-4-[(4-nitrophenyl)sulfonyl-propyl-amino]butyl]-N-ethyl-carbamate, HxBzL-61g A solution of HxBzL-61f (4.4 g, 7.47 mmol, 1.0 eq.) in MeOH (50 mL) was added with NH at 20 °C. 2 N.H. 2 .H 2 O (2.25g, 44.9mmol, 2.18mL, 6.0eq) was added and then stirred at 80°C for 12 hours. The mixture was filtered and the filtrate was concentrated in vacuo to give HxBzL-61g (3.4g, 7.41mmol, 99.2% yield) as a yellow oil. LC / MS[M+H]459.2 (calculated value), LC / MS[M+H]459.2 (observed value).

[0358] Preparation of N-[3-amino-4-(ethylamino)butyl]-4-nitro-N-propyl-benzenesulfonamide, HxBzL-61h To a solution of HxBzL-61g (2.9 g, 6.32 mmol, 1.0 eq) in EtOAc (30 mL) was added HCl / EtOAc (4M, 29.0 mL, 18.3 eq) and then stirred at 20 °C for 1 h. The mixture was concentrated in vacuo to give HxBzL-61h (2.7 g, crude, 2HCl) as a yellow solid. 1H NMR(MeOD,400MHz)δ 8.35(d,J=8.8Hz,2H),8.09(d,J=8.8Hz,2H),3.78-3.69(m,1H),3.45-3.31(m,4H), 3.17-3.05(m,4H),2.12-1.99(m,2H),1.57-1.43(m,2H),1.32(t,J=7.2Hz,3H),0.80(t,J=7.2Hz,3H) .LC / MS[M+H]359.17 (calculated value), LC / MS[M+H]359.1 (observed value).

[0359] Preparation of N-[2-(1-ethyl-2-oxo-imidazolidin-4-yl)ethyl]-4-nitro-N-propyl-benzenesulfonamide, HxBzL-61i HxBzL-61h (2.7g, 7.53mmol, 1.0eq) and Et in THF (30mL) 3 To a mixture of N (1.91 g, 18.8 mmol, 2.62 mL, 2.5 eq) was added carbonyldiimidazole, CDI (2.44 g, 15.1 mmol, 2.0 eq) at 0°C. The mixture was stirred at 25°C for 12 hours. The resulting mixture was poured into ice water (w / w=1 / 1) (50 mL) and stirred for 10 minutes. The aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with brine (50 mL) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to obtain HxBzL-61i (300 mg, 780 umol, yield 10.4%) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ 8.39(d,J=8.8Hz,2H),8.02(d,J=8.8Hz,2H),3.99-3.95(m,1H),3.77-3.63(t,J=8.8 Hz,1H),3.48-3.38(m,1H),3.35-3.23(m,2H),3.22-3.04(m,4H),1.98-1.74(m,2H),1.66-1.45(m,2H), 1.15(t,J=7.2Hz,3H),0.87(t,J=7.2Hz,3H)

[0360] Preparation of 1-ethyl-4-[2-(propylamino)ethyl]imidazolidin-2-one, HxBzL-61j A solution of HxBzL-61i (300mg, 780umol, 1.0eq) in MeCN (10mL) was added with LiOH.H 2 O (196 mg, 4.68 mmol, 6.0 eq.) and 2-sulfanylacetate (0.45 g, 4.24 mmol, 384 uL, 5.43 eq.) were added and then stirred at 25° C. for 2 hours. The mixture was filtered and the filtrate was concentrated in vacuo. H the residue 2 diluted with O (20 mL), then the pH of the aqueous phase was adjusted to 3-4 with HCl (1M), then extracted with EtOAc (20 mL x 3) to remove by-products, and then the aqueous phase was freeze-dried to obtain HxBzL-61j (180 mg, 763 umol, 97.8% yield, HCl) as a colorless oil. 1 H NMR(MeOD,400MHz)δ 3.83-3.73(m,1H),3.65(t,J=8.8Hz,1H),3.28-3.13(m,3H),3.12-3.03(m,2H),3.02-2.93 (m,2H),2.01-1.84(m,2H),1.79-1.67(m,2H),1.11(t,J=7.2Hz,3H),1.03(t,J=7.2Hz,3H).

[0361] tert-Butyl N-[[5-[2-amino-4-[2-(1-ethyl-2-oxo-imidazolidin-4-yl)ethyl-propyl-carbamoyl]-3H-1-benzazepine-8- Preparation of yl]pyrimidin-2-yl]methyl]carbamate, HxBzL-61l 2-Amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine-4-carboxylic acid, HxBzL-61k (210 mg, 513 umol) in DMF (6 mL) , 1.0 eq.) were added HATU (205 mg, 539 umol, 1.05 eq.), DIEA (331 mg, 2.56 mmol, 447 uL, 5.0 eq.), and HxBzL-61j (145 mg, 615 umol, 1.2 eq., HCl), then Stirred at 25°C for 1 hour. The resulting mixture was poured into ice water (w / w=1 / 1) (10 mL) and stirred for 5 minutes. The aqueous phase was extracted with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine (10 mL x 2) and anhydrous Na 2 S.O. 4 dried, filtered and concentrated in vacuo. Purify the residue by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 1 / 0, ethyl acetate / methanol = 1 / 0, 3 / 1). HxBzL-61l (300 mg, 508 umol, yield 99.0%) was obtained as a yellow solid. LC / MS[M+H]591.3 (calculated value), LC / MS[M+H]591.3 (observed value).

[0362] 2-Amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-[2-(1-ethyl-2-oxo-imidazolidin-4-yl)ethyl]-N-propyl-3H- Preparation of 1-benzazepine-4-carboxamide, HxBzL-61m To a solution of HxBzL-61l (300 mg, 508 umol, 1.0 eq.) in EtOAc (5 mL) was added HCl / EtOAc (4M, 6.00 mL, 47.3 eq.) and then stirred at 25° C. for 1 h. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 1%~30%, 8 minutes) to obtain HxBzL-61m. (142 mg, 198 umol, yield 38.91%, 2TFA) was obtained as a yellow solid. 1 H NMR(MeOD,400MHz)δ 9.22(s,2H),7.88-7.71(m,3H),7.15(s,1H),4.49(s,2H),3.75-3.60(m,2H),3.57-3.50 (m,4H),3.39(s,2H),3.28-3.18(m,3H),2.02-1.97(s,1H),1.88-1.83(m,1H),1.81-1.65(m,2H),1.15 -1.10(m,3H),1.01-0.95(m,3H).LC / MS[M+H]491.28(calculated value), LC / MS[M+H]491.3(observed value).

[0363] tert-Butyl3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-( 1-Ethyl-2-oxo-imidazolidin-4-yl)ethyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy] Preparation of ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate, HxBzL-61n HxBzL-61m (90mg, 125umol, 1.0eq, 2TFA) and Et in DMF (1mL) 3 In a solution of N (38.02 mg, 376 umol, 52.3 uL, 3.0 eq.), (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2- [2-[2-[2-(3-tert-butoxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanate, t-Bu-COO-PEG10- COOTFP (95.5 mg, 125 umol, 1.0 eq) was added at 0°C and then stirred at 25°C for 1 hour. Water (10 mL) was added and then the pH of the mixture was adjusted to about 6 with TFA. The aqueous layer was extracted with MTBE (5 mL x 3) to remove byproducts. The aqueous phase was further extracted with DCM / i-PrOH=3 / 1 (10 mL x 3). The organic phase (DCM / i-PrOH) was concentrated in vacuo to give HxBzL-61n (130 mg, 120 umol, 95.5% yield) as a yellow oil.

[0364] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(1-ethyl -2-oxo-imidazolidin-4-yl)ethyl-propyl-carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy ]Ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, preparation of HxBzL-61o MeCN (0.5mL) and H 2 To a solution of HxBzL-61n (100 mg, 92.0 umol, 1.0 eq) in O (1 mL) was added TFA (83.9 mg, 735 umol, 54.5 uL, 8.0 eq) and then stirred at 80 °C for 1 h. The mixture was concentrated in vacuo to give a residue, the residue was diluted with water (10 mL), the aqueous phase was extracted with MTBE (10 mL) to remove excess TFA, the aqueous phase was lyophilized, HxBzL-61o (100 mg, 87.3 umol, 94.9% yield, TFA) was obtained as a yellow oil.

[0365] Preparation of HxBzL-61 HxBzL-61o (100 mg, 87.3 umol, 1.0 eq., TFA) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (70.2 mg, EDCI (67.0 mg, 349 umol, 4.0 eq.) was added to a solution of 262 umol, 3.0 eq.) and then stirred at 20.degree. C. for 1 hour. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 10%~35%, 8 min) to obtain HxBzL-61. (30 mg, 23.8 umol, 27.3% yield) was obtained as a yellow oil. 1 H NMR(MeOD,400MHz)δ 9.10(s,2H),7.81-7.68(m,3H),7.14(s,1H),4.71(s,2H),3.88(t,J=6.0Hz,2H), 3.80(t,J=6.0Hz,2H),3.68-3.59(m,37H),3.55-3.50(m,3H),3.40(s,2H),3.26-3.20(m,3H),2.98(t, J=6.0Hz,2H),2.62(t,J=6.0Hz,2H),2.06-1.82(m,2H),1.80-1.67(m,2H),1.15-1.10(m,3H),1.01-0.93 (m,3H).LC / MS[M+H]1259.5 (calculated value), LC / MS[M+H]1259.6 (observed value).

[0366] Example L-65 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[(1S)-1-[[ (1S)-1-[[4-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylcarbamoyloxymethyl] phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-proxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prooxy, 2 Synthesis of ,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-65 [ka] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-5-ureidopentanamide)benzyl( Preparation of (5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidin-2-yl)methyl)carbamate, HxBzL-65a 2-Amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, HxBz-5 (41.2 mg, 96umol, 1eq, HCl), and Et 3 A solution of N (29.0 mg, 287 umol, 39.9 uL, 3 eq.) was added with (9H-fluoren-9-yl)methyl ((S)-3-methyl-1-(((S)-1- ((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-uredopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate, Fmoc-Val-Cit -PNC (110 mg, 143 umol, 1.5 eq) was added at 0°C and then stirred at 25°C for 1 hour. Piperidine (24.4 mg, 287 umol, 28.3 uL, 3 eq.) was added to the mixture and stirred for an additional 1 hour at 25°C. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions, column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 5% to 30%, 8 minutes), HxBzL-65a (60 mg, 75.0 umol, 78.4% yield) was obtained as a yellow oil. LC / MS[M+H]800.4 (calculated value), LC / MS[M+H]800.6 (observed value).

[0367] tert-Butyl3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[(1S)-1-[[(1S)-1 -[[4-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylcarbamoyloxymethyl]phenyl]carbamoyl]- Preparation of 4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, HxBzL-65b A solution of HxBzL-65a (60 mg, 65.7 umol, 1 eq., TFA) in THF (2 mL) was added with Et 3 N (19.9mg, 197umol, 27.4uL, 3eq) and (2,3,5,6-tetrafluorophenyl)3-[2-[2-[2-[2-[2-[2-[2- [2-[2-(3-tert-butoxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, t-Bu-COO-PEG10-COOTFP(50.1 mg, 66 umol, 1 eq.) and then stirred at 25° C. for 1 hour. The reaction mixture was diluted with 2 mL of water, then the pH of the aqueous phase was adjusted to 5-6 with TFA, extracted with DCM / i-prOH (5 mL x 3, 3 / 1), and the combined organic phases were diluted with Na 2 S.O. 4 Dry over, filter, and concentrate under reduced pressure to give HxBzL-65b (90 mg, 64.4 umol, 98.2% yield) as a yellow oil, which was used in the next step without further purification. LC / MS[M+H]1396.8 (calculated value), LC / MS[M+H]1396.7 (observed value).

[0368] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[(1S)-1-[[(1S)-1-[[ 4-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylcarbamoyloxymethyl]phenyl]carbamoyl]-4-ureido -Butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, preparation of HxBzL-65c To a solution of HxBzL-65b (90 mg, 64 umol, 1 eq.) in water (3 mL) and MeCN (1 mL) was added TFA (73.5 mg, 644 umol, 47.7 uL, 10 eq.) and then stirred at 80 °C for 2 h. did. The reaction mixture was diluted with 2 mL of water, the pH of the aqueous phase was adjusted to 5-6 with TFA, extracted with DCM / i-prOH (5 mL x 3, 3 / 1), and the combined organic phase was diluted with Na 2 S.O. 4 Dry on top, filter and concentrate under reduced pressure to give HxBzL-65c (100 mg, crude) as a yellow oil. LC / MS[M+H]1340.7 (calculated value), LC / MS[M+H]1340.6 (observed value).

[0369] Preparation of HxBzL-65 HxBzL-65c (100 mg, 74.6 umol, 1 eq.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (80.0 mg, 298 umol, in DCM (1 mL) and DMA (0.5 mL), EDCI (57.2 mg, 298 umol, 4 eq.) was added to a solution of 4 eq.) and then stirred at 25° C. for 1 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions, column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1%TFA)-ACN], B%: 15% to 40%, 8 minutes), HxBzL-65 (20 mg, 12.75 umol, yield 17.09%) was obtained as a white solid. 1 H NMR(400MHz,MeOD)δ 9.08(s,2H),7.83-7.78(m,1H),7.77-7.71(m,2H),7.65(br d,J=7.6Hz,2H),7.47(s, 1H),7.42-7.34(m,2H),5.12(s,2H),4.62(s,2H),4.54-4.48(m,1H),4.23-4.18(m,1H),4.02-3.98(m, 2H),3.87(t,J=6.0Hz,2H),3.80-3.75(m,2H),3.65-3.60(m,36H),3.52-3.49(m,2H),3.47(s,2H),3.21 -3.14(m,2H),2.98(t,J=6.0Hz,2H),2.61-2.53(m,2H),2.20-2.10(m,1H),2.02-1.88(m,1H),1.85-1.71 (m,3H),1.70-1.52(m,2H),1.23(t,J=7.2Hz,3H),1.05-0.99(m,9H).LC / MS[M+H]1568.6(calculated value), LC / MS[M+H]1568.6 (observed value).

[0370] Example L-70 2,3,5,6-tetrafluorophenyl 1-(5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidine- HxBzL-70 [ka] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5- [2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]Ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, 2,3,5,6-tetrafluorophenol (1.77, 10.7mmol, 2.00eq) in a solution of HxBzL-5a (5.00g, 5.35mmol, 1.00eq), Propanephosphonic anhydride (PPAA, T3P), CAS Reg. No. 68957-94-8 (50 wt% solution in MeCN, 17.0 g solution, 26.8 mmol, 5.00 eq.), and N-methylimidazole, NMI ( 2.15 mL, 26.8 mmol, 5.00 eq) were added sequentially. The mixture was stirred at 20° C. for 2 hours, then diluted with 20% aqueous NaCl (50 mL). The aqueous layer was extracted with DCM (25 mL) and the combined organic layers were washed with water (25 mL), dried (Na2SO4), filtered and concentrated in vacuo to give the crude HxBzL-70 in the form of a dark brown oil. Obtained. The material was loaded onto a Biotage column (250 mL of 7.5 mM HCl in MeCN / water 2:8, v / v), followed by a gradient step (20 column volumes of MeCN / water 2:8, then 15 column volumes of MeCN / water 3). :7). The desired fractions were combined and then extracted (2 x 300 mL of DCM) and concentrated in vacuo to give pure HxBzL-70 (5.34 g, qNMR) in the form of a dark yellow oil stored at -20 °C under nitrogen. (purity of 55.6% by weight, yield 56%), and diluted with DMA to prepare a 20mM solution of HxBzL-70. LC / MS[M+H]1083.1 (calculated value), LC / MS[M+H]1083.1 (observed value).

[0371] Example 201 Preparation of immune complex (IC) To prepare lysine conjugate immune complexes, antibodies were coated on a G-25 SEPHADEX™ desalting column (Sigma-Aldrich, St. Louis, MO) or on a Zeba™ spin desalting column (Thermo Fisher Scientific). Buffer exchange using a conjugation buffer containing 100 mM boric acid, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3. Each eluate is then adjusted to a concentration of approximately 1-10 mg / ml using buffer and then sterile filtered. The antibody was pre-warmed to 20-30°C and 2-20 (e.g. 7-10) molar equivalents of tetra of formula II dissolved in dimethylsulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5-20mM. Mix rapidly with fluorophenyl (TFP) or sulfonic acid tetrafluorophenyl (sulfoTFP) ester, 8-Het-2-aminobenzazepine-linker (HxBzL) compound. Reactions were allowed to proceed for approximately 16 h at 30°C, and immune complexes (ICs) were equilibrated on two consecutive G-25 desalting columns or in phosphate-buffered saline (PBS) at pH 7.2. Separated from the reactions by running a Zeba™ spin desalting column to provide the immunoconjugates (IC) of Tables 3a and 3b. Adjuvant-to-antibody ratio (DAR) was determined using a C4 reversed phase column on an ACQUITY™ UPLC H class (Waters Corporation, Milford, MA) connected to a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation). Determined by liquid chromatography mass spectrometry used.

[0372] To prepare cysteine ​​conjugate immune complexes, antibodies were transferred using Zeba™ spin desalting columns (Thermo Fisher Scientific) to conjugation buffer containing PBS, pH 7.2 with 2 mM EDTA. Exchange the buffer. Reduce interchain disulfides using a 2-4 molar excess of tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) for 30 min to 2 h at 37 °C. Excess TCEP or DTT was removed using a Zeba™ spin desalting column pre-equilibrated with conjugation buffer. The concentration of buffer exchanged antibodies was adjusted to approximately 5-20 mg / ml using conjugation buffer and sterile filtered. Maleimide-HxBzL compounds are dissolved in either dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5-20 mM. For conjugation, the antibody is mixed with 10-20 molar equivalents of maleimide-HxBzL. In some cases, up to 20% (v / v) of additional DMA or DMSO was added to improve the solubility of maleimide-HxBzL in the conjugation buffer. The reaction is allowed to proceed at 20°C for approximately 30 minutes to 4 hours. The resulting complex is purified from unreacted maleimide-HxBzL using two consecutive Zeba™ spin desalting columns. The column is pre-equilibrated with phosphate buffered saline (PBS) at pH 7.2. Adjuvant-to-antibody ratio (DAR) was measured using a C4 reversed phase column on an ACQUITY™ UPLC H class (Waters Corporation, Milford, MA) connected to a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation). Estimated by liquid chromatography mass spectrometry used.

[0373] For conjugation, antibodies can be dissolved in aqueous buffer systems known in the art that do not adversely affect antibody stability or antigen binding specificity. Phosphate buffered saline may also be used. The HxBzL compound is dissolved in a solvent system that includes at least one polar aprotic solvent, as described elsewhere herein. In some such embodiments, HxBzL is in a pH 8 Tris buffer (e.g., 50 mM Tris) at about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM, and from about 5 mM to about 50 mM, or It is dissolved in a concentration range such as about 10mM to about 30mM. In some embodiments, the 8-Het-2-aminobenzazepine-linker intermediate is dissolved in DMSO (dimethylsulfoxide), DMA (dimethylacetamide), acetonitrile, or another suitable dipolar aprotic solvent. Ru.

[0374] Alternatively, an equivalent excess of HxBzL solution may be diluted and combined with the antibody solution in a conjugation reaction. The HxBzL solution may be suitably diluted with at least one polar aprotic solvent and at least one polar protic solvent, examples of which include water, methanol, ethanol, n-propanol, and acetic acid. The molar equivalent of 8-Het-2-aminobenzazepine-linker intermediate to antibody is about 1.5:1, about 3:1, about 5:1, about 10:1, about 15:1, or about 20:1. 1, and about 1.5:1 to about 20:1, about 1.5:1 to about 15:1, about 1.5:1 to about 10:1, about 3:1 to about 15:1, about 3:1 to about 10:1, about 5:1 to about 15:1, or about 5:1 to about 10:1. The reaction may be suitably monitored for completion by methods known in the art such as LC-MS. The conjugation reaction is typically complete in a range of about 1 hour to about 16 hours. After the reaction is complete, reagents may be added to the reaction mixture to quench the reaction. If the antibody thiol group has been reacted with a thiol-reactive group such as the maleimide of the 8-Het-2-aminobenzazepine-linker intermediate, the unreacted antibody thiol group may be reacted with a capping reagent. . An example of a suitable capping reagent is ethylmaleimide.

[0375] After conjugation, the immune complex can be purified by chromatography, such as size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, tangential flow filtration, and combinations thereof. may be purified and separated from unconjugated reactants and / or conjugate aggregates by purification methods known in the art. For example, prior to purification, the immune complex may be diluted, such as in 20 mM sodium succinate, pH 5. The diluted solution is applied to the cation exchange column, followed by washing with, for example, at least 10 column volumes of 20 mM sodium succinate, pH 5. The complex may be suitably eluted with a buffer such as PBS.

[0376] Example 202 HEK reporter assay Human embryonic kidney (HEK293) reporter cells expressing human TLR7 or human TLR8 (InvivoGen, San Diego CA) were used with vendor protocols for cell growth and experiments. Briefly, cells were incubated with 5% CO in DMEM supplemented with 10% FBS, ZEOCIN™, and Blasticidin. 2 and grown to 80-85% fusion. Then, using HEK detection medium and substrate containing immunostimulatory molecules, 4 x 10 4 Cells were seeded in 96-well plates at cells / well. Activity was measured using a plate reader at wavelengths of 620-655 nm.

[0377] Example 203 Evaluation of in vivo immune complex activity This example shows that the immunoconjugates of the invention are effective in inducing immune activation and are therefore useful in the treatment of cancer.

[0378] a) Isolation of human antigen-presenting cells: Human bone marrow antigen-presenting cells (APCs) were isolated from the ROSETTESEP™ human monocyte enrichment cocktail containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123, and HLA-DR. Negative selection was made from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation using Stem Cell Technologies, Vancouver, Canada). Immature APCs were then harvested using the EASYSEP™ Human Monocyte Enrichment Kit without CD16 Depletion (Stem Cell Technologies) containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123, and HLA-DR. , purified to >90% purity by negative selection.

[0379] b) Myeloid APC activation assay: 2x10 5 APC was incubated in Iscove's Modified Dulbecco's Medium, 10% FBS, 100 U / mL penicillin, 100 μg / mL (micrograms per milliliter) streptomycin, 2 mM L-glutamine, sodium pyruvate, IMDM (Lonza) supplemented with non-essential amino acids, and as indicated. 96-well plates (Corning, Corning, NY) containing various concentrations of unconjugated (naked) antibodies of the invention and immune complexes (prepared according to the Examples above). Cell-free supernatants are analyzed after 18 hours via ELISA to measure TNFα secretion as a readout of the pro-inflammatory response.

[0380] c) PBMC activation assay: Human peripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation. PBMCs were incubated in 96-well plates (Corning, Corning, NY) co-cultured with CEA-expressing tumor cells (eg, MKN-45, HPAF-II) at a 10:1 effector to target cell ratio. Cells were stimulated with various concentrations of unconjugated (naked) antibodies of the invention and immune complexes (prepared according to the examples above). Cell-free supernatants were analyzed by cytokine bead arrays using the LegendPlex™ kit according to the manufacturer's guidelines (BioLegend®, SanDiego, CA).

[0381] d) Isolation of human conventional dendritic cells: Human conventional dendritic cells (cDCs) were isolated from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation. selected. Briefly, T cells are first removed from the cell preparation by enriching the cells using ROSETTESEP® Human CD3 Depletion Cocktail (Stem Cell Technologies, Vancouver, Canada). cDCs are then further enriched by negative selection using the EASYSEP® Human Myeloid DC Enrichment Kit (Stem Cell Technologies).

[0382] e) cDC activation assay: 8 x 10 4 of APCs were co-cultured with tumor cells expressing ISAC target antigens at a 10:1 effector (cDC) to target (tumor cell) ratio. Cells were cultured in 96-well plates (Corning, Inc., Corning, NY). After overnight incubation for approximately 18 hours, cell-free supernatants were collected and analyzed for cytokine secretion (including TNFα) using BioLegend LEGENDPLEX cytokine bead arrays.

[0383] Activation of bone marrow cell types can be measured using a variety of screen assays in addition to the described assays in which different bone marrow populations are utilized. These were monocytes isolated from healthy donor blood, M-CSF differentiated macrophages, GM-CSF differentiated macrophages, GM-CSF+IL-4 monocyte-derived dendritic cells, isolated from healthy donor blood. It may include conventional dendritic cells (cDCs) and myeloid cells polarized to an immunosuppressive state (also referred to as myeloid-derived suppressor cells or MDSCs). Examples of MDSC-polarized cells include monocytes that have differentiated toward an immunosuppressive state, such as M2aMΦ (IL4 / IL13), M2cMΦ (IL10 / TGFb), GM-CSF / IL6 MDSC, and tumor-educating monocytes (TEM). Can be mentioned. TEM differentiation can be performed using tumor conditioned media (e.g. 786.O, MDA-MB-231, HCC1954). Primary tumor-associated bone marrow cells may also include primary cells present in dissociated tumor cell suspensions (Discovery Life Sciences).

[0384] Assessment of activation of the described populations of bone marrow cells can be performed as monocultures or as co-cultures with cells expressing the antigen of interest to which immune complexes can bind via the CDR regions of antibodies. After 18-48 hours of incubation, activation can be assessed by upregulation of cell surface costimulatory molecules using flow cytometry or by measurement of secreted proinflammatory cytokines. For cytokine measurements, cell-free supernatants are harvested and analyzed by cytokine bead arrays (eg, LegendPlex from Biolegend) using flow cytometry.

[0385] All references, including publications, patent applications, and patents, cited herein are individually and specifically indicated to be incorporated by reference and are herein incorporated by reference in their entirety. Incorporated herein by reference as if by reference.

Claims

1. 1. The method of claim 1, further comprising administering to said patient an antibody having formula I: Ab-[L-HxBz] ｐ I An immune complex having the formula: During the ceremony, Ab is an antibody construct having an antigen-binding domain that binds to CEA; p is an integer from 1 to 8; HxBz is a compound of the formula: 【Chemistry 1】 wherein Het is selected from heterocyclyldiyl and heteroaryldiyl; R １ , R ２ , R ３ , and R ４ However, independently, H, C １ -C １２ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryl, C ２ -C ９ Heterocyclyl, and C １ -C ２０ heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally selected from the group consisting of: - (C １ -C １２ alkyldiyl)-N(R ５ ) - *, - (C １ -C １２ alkyldiyl)-N(R ５ ) ２ , - (C １ -C １２ Alkyldiyl)-OR ５ , - (C ３ -C １２ Carbocyclyl), - (C ３ -C １２ carbocyclyl)-*, - (C ３ -C １２ carbocyclyl)-(C １ -C １２ Alkyldiyl)-NR ５ - *, - (C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , - (C ３ -C １２ carbocyclyl)-NR ５ -C(=NR ５ ) N.R. ５ - *, - (C ６ -C ２０ Aryl), - (C ６ -C ２０ Aryldiyl)-*, - (C ６ -C ２０ Aryldiyl)-N(R ５ ) - *, - (C ６ -C ２０ Aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) - *, - (C ６ -C ２０ Aryldiyl)-(C １ -C １２ Alkyldiyl)-(C ２ -C ２０ Heterocyclyldiyl)-*, - (C ６ -C ２０ Aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , - (C ６ -C ２０ Aryldiyl)-(C １ -C １２ Alkyldiyl)-NR ５ -C(=NR ５ａ ) N (R ５ ) - *, - (C ２ -C ２０ Heterocyclyl), - (C ２ -C ２０ Heterocyclyl)-*, - (C ２ -C ９ Heterocyclyl)-(C １ -C １２ Alkyldiyl)-NR ５ - *, - (C ２ -C ９ Heterocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , - (C ２ -C ９ Heterocyclyl)-C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ ) - *, - (C ２ -C ９ Heterocyclyl)-NR ５ -C(=NR ５ａ ) N.R. ５ - *, - (C ２ -C ９ Heterocyclyl)-NR ５ - (C ６ -C ２０ Aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) - *, - (C ２ -C ９ Heterocyclyl)-(C ６ -C ２０ Aryldiyl)-*, - (C １ -C ２０ Heteroaryl), - (C １ -C ２０ Heteroaryl)-*, - (C １ -C ２０ Heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ５ ) - *, - (C １ -C ２０ Heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , - (C １ -C ２０ Heteroaryl)-NR ５ -C(=NR ５ａ ) N (R ５ ) - *, - (C １ -C ２０ Heteroaryl)-N(R ５ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ ) - *, -C(=O)-*, -C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ ) - *, -C(=O)-(C ２ -C ２０ Heterocyclyldiyl)-*, -C(=O)N(R ５ ) ２ 、 -C(=O)N(R ５ )-*、 -C(=O)N(R ５ )-(C １ -C １２ Alkyldiyl)-*, -C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-C(=O)N(R ５ ) - *, -C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ ) C(=O)R ５ , -C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )C(=O)N(R ５ ) ２ , -C(=O)NR ５ - (C １ -C １２ alkyldiyl)-N(R ５ ) CO ２ R ５ , -C(=O)NR ５ - (C １ -C １２ alkyldiyl)-N(R ５ ) C(=NR ５ａ ) N (R ５ ) ２ , -C(=O)NR ５ - (C １ -C １２ Alkyldiyl)-NR ５ C (=NR ５ａ ) R ５ , -C(=O)NR ５ - (C １ -C ８ Alkyldiyl)-NR ５ (C ２ -C ５ Heteroaryl), -C(=O)NR ５ - (C １ -C ２０ Heteroaryldiyl)-N(R ５ ) - *, -C(=O)NR ５ - (C １ -C ２０ Heteroaryldiyl)-*, -C(=O)NR ５ - (C １ -C ２０ Heteroaryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -C(=O)NR ５ - (C １ -C ２０ Heteroaryldiyl)-(C ２ -C ２０ Heterocyclyldiyl)-C(=O)NR ５ - (C １ -C １２ Alkyldiyl)-NR ５ - *, -N(R ５ ) ２ 、 -N(R ５ )-*、 -N(R ５ )C(=O)R ５ 、 -N(R ５ )C(=O)-*、 -N(R ５ )C(=O)N(R ５ ) ２ 、 -N(R ５ )C(=O)N(R ５ )-*、 -N(R ５ )CO ２ R ５ 、 -NR ５ C(=NR) ５ａ )N(R ５ ) ２ 、 -NR ５ C(=NR) ５ａ )N(R ５ )-*、 -NR ５ C(=NR) ５ａ )R ５ 、 -N(R ５ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ ) - *, -N(R ５ )-(C ２ -C ５ Heteroaryl), -N(R ５ ) -S(=O) ２ - (C １ -C １２ alkyl), -O-(C １ -C １２ alkyl), -O-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -O-(C １ -C １２ alkyldiyl)-N(R ５ ) - *, -O-C(=O)N(R ５ ) ２ 、 -O-C(=O)N(R ５ )-*、 -S(=O) ２ - (C ２ -C ２０ Heterocyclyldiyl)-*, -S(=O) ２ - (C ２ -C ２０ Heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -S(=O) ２ - (C ２ -C ２０ Heterocyclyldiyl)-(C １ -C １２ Alkyldiyl)-NR ５ -*, and -S(=O) ２ - (C ２ -C ２０ Heterocyclyldiyl)-(C １ -C １２ or substituted with one or more groups selected from: Or R ２ and R ３ together form a 5- or 6-membered heterocyclyl ring, X １ , X ２ , X ３ , and X ４ are independently a bond, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , and S(O) ２ N (R ５ ) selected from the group consisting of R ５ However, independently, H, C ６ -C ２０ Aryl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryldiyl, C １ -C １２ Alkyl, and C １ -C １２ alkyldiyl, or two R ５ the groups taken together form a 5- or 6-membered heterocyclyl ring; R ５ａ But, C ６ -C ２０ Aryl and C １ -C ２０ Heteroaryl; In the formula, the asterisk * indicates the binding site of L, and R １ , R ２ , R ３ , and R ４ is attached to L; L, -C(=O)-PEG-, -C(=O)-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(=O)-Gluc-, -C(=O)-PEG-O-, -C(=O)-PEG-O-C(=O)-, -C(=O)-PEG-C(=O)-, -C(=O)-PEG-C(=O)-PEP-, -C(=O)-PEG-N(R ６ )-、 -C(=O)-PEG-N(R ６ )-C(=O)-、 -C(=O)-PEG-N(R ６ )-PEG-C(=O)-PEP-、 -C(=O)-PEG-N ＋ (R ６ ) ２ 2019-02-20 10:00:00 AM -C(=O)-PEG-C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)-, -C(=O)-PEG-C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)N(R ６ ) C(=O)-(C ２ -C ５ Monoheterocyclyldiyl)-, -C(=O)-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-, -C(=O)-PEG-SS-(C １ -C １２ alkyldiyl)-C(=O)-, -C(=O)-(C １ -C １２ alkyldiyl)-C(=O)-PEP-, -C(=O)-(C １ -C １２ alkyldiyl)-C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)-, -C(=O)-(C １ -C １２ alkyldiyl)-C(=O)-PEP-N(R ６ )-(C １ -C １２ alkyldiyl)-N(R ５ )-C(=O), -C(=O)-(C １ -C １２ alkyldiyl)-C(=O)-PEP-N(R ６ )-(C １ -C １２ alkyldiyl)-N(R ６ ) C(=O)-(C ２ -C ５ Monoheterocyclyldiyl)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(=O)-Gluc-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ ) -, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ )-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-PEP-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)N(R ６ ) C(═O)—, and -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)N(R ６ ) C(=O)-(C ２ -C ５ is a linker selected from the group consisting of: R ６ are independently H or C １ -C ６ is alkyl, PEG is a group represented by the formula: -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -, m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the formula: 【Chemistry 2】 having PEP has the formula: 【Chemistry 3】 where AA are independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; Cyc is optionally F, Cl, NO ２ , -OH, -OCH ３ and glucuronic acid having the structure: ６ -C ２０ Aryldiyl and C １ -C ２０ heteroaryldiyl; 【Chemistry 4】 R ７ is -CH(R ８ ) O-, -CH ２ --, --CH ２ N (R ８ )- and -CH(R ８ )O—C(═O)—, wherein R ８ But, H, C １ -C ６ Alkyl, C(=O)-C １ -C ６ Alkyl, and -C(=O)N(R ９ ) ２ wherein R ９ However, independently, H, C １ -C １２ Alkyl, and -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -OH, wherein m is an integer from 1 to 5 and n is an integer from 2 to 50, or two R ９ the groups taken together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from F, Cl, Br, I, -CN, -CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ N.H. ２ , -CH ２ N.H.S.O. ２ CH ３ , -CH ２ N.H.C.H. ３ , -CH ２ N(CH ３ ) ２ 、-CO ２ H、-COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH ３ ) ２ 、-C(CH ３ ) ２ P. ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ -NHCOCH ３ 、-N(CH ３ )COCH ３ 、-NHS(O) ２ CH ３ 、-N(CH ３ )C(CH ３ ) ２ P. ２ 、-N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHC(=NH)H, -NHC(=NH)CH ３ -NHC(=NH)NH ２ -NHC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ SO ３ 、-OCH ２ CH ２ H ２ CH ２ N(CH ３ ) ２ 、-O(CH ２ CH ２ O) ｎ -(CH ２ ) ｍ CO ２ H, -O(CH ２ CH ２ O) ｎ H, -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H, or a pharma- ceutically acceptable salt thereof.

2. The immunoconjugate of claim 1 , wherein the antibody is selected from labetuzumab and arcitumomab.

3. The antibody construct comprises: a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:3, CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, CDR-L3 comprising the amino acid sequence of SEQ ID NO:7, CDR-H1 comprising the amino acid sequence of SEQ ID NO:11, CDR-H2 comprising the amino acid sequence of SEQ ID NO:13, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:15; b) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; c) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 35, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:53, CDR-L2 comprising the amino acid sequence of SEQ ID NO:55, CDR-L3 comprising the amino acid sequence of SEQ ID NO:39, CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:48; e) CDR-L1 comprising the amino acid sequence of SEQ ID NO:59, CDR-L2 comprising the amino acid sequence of SEQ ID NO:61, CDR-L3 comprising the amino acid sequence of SEQ ID NO:63, CDR-H1 comprising the amino acid sequence of SEQ ID NO:67, CDR-H2 comprising the amino acid sequence of SEQ ID NO:69, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:71; f) CDR-L1 comprising the amino acid sequence of SEQ ID NO:75, CDR-L2 comprising the amino acid sequence of SEQ ID NO:77, CDR-L3 comprising the amino acid sequence of SEQ ID NO:79, CDR-H1 comprising the amino acid sequence of SEQ ID NO:83, CDR-H2 comprising the amino acid sequence of SEQ ID NO:85, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:87; g) CDR-L1 comprising the amino acid sequence of SEQ ID NO:91, CDR-L2 comprising the amino acid sequence of SEQ ID NO:93, CDR-L3 comprising the amino acid sequence of SEQ ID NO:95, CDR-H1 comprising the amino acid sequence of SEQ ID NO:99, CDR-H2 comprising the amino acid sequence of SEQ ID NO:101, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:103; h) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 115, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120; or i) The immune complex of claim 1, comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109; CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 124; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 126; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:

128.

4. The immune complex of claim 1, wherein the antibody construct comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89, and 105, and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122, and 130.

5. The immune complex of claim 1, wherein the antibody construct comprises a variable light chain comprising an amino acid sequence selected from SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89, and 105, and a variable heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122, and 130.

6. The immune complex of claim 5, wherein the antibody construct comprises a variable light chain comprising an amino acid sequence from SEQ ID NO: 105 and a heavy chain CDR (complementarity determining region) CDR-H2 comprising an amino acid sequence from SEQ ID NO:

118.

7. The immunoconjugate of claim 6, wherein the antibody construct comprises a variable light chain comprising an amino acid sequence from SEQ ID NO: 105 and a variable heavy chain comprising an amino acid sequence from SEQ ID NO:

113.

8. The immunoconjugate of any one of claims 1 to 7, wherein Het is selected from the group consisting of pyridyldiyl, pyrimidyldiyl, pyrazolyldiyl, piperazinyldiyl, piperidinyldiyl, and pyrazinyldiyl.

9. X １ is a bond, and R １ The immune complex according to any one of claims 1 to 7, wherein is H.

10. X ２ is a bond, and R ２ But, C １ -C ８ The immunoconjugate of any one of claims 1 to 7, which is alkyl.

11. X ２ and X ３ are each a bond, R ２ and R ３ But independently, C １ -C ８ Alkyl, —O—(C １ -C １２ alkyl), -(C １ -C １２ Alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , -(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ , -O-(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ , and —O—(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ The immune complex according to any one of claims 1 to 7, which is selected from the group consisting of

12. R ２ But, C １ -C ８ alkyl, R ３ However, (C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ The immune complex of claim 11,

13. R ２ But -CH ２ CH ２ CH ３ and R ３ But -CH ２ CH ２ CH ２ N.H.C.O. ２ (t-Bu), -OCH ２ CH ２ N.H.C.O. ２ (cyclobutyl), and -CH ２ CH ２ CH ２ N.H.C.O. ２ 13. The immune conjugate of claim 12, wherein said aryl group is selected from the group consisting of aryl, ... and aryl.

14. R ２ and R ３ each independently represents -CH ２ CH ２ CH ３ , -OCH ２ CH ３ , -OCH ２ CF ３ , -CH ２ CH ２ CF ３ , -OCH ２ CH ２ OH and -CH ２ CH ２ CH ２ 13. The immune complex of claim 12, wherein said amino acid is selected from the group consisting of OH.

15. R ２ and R ３ are each -CH ２ CH ２ CH ３ The immune complex of claim 12,

16. R ２ But -CH ２ CH ２ CH ３ and R ３ But, -OCH ２ CH ３ The immune complex of claim 12,

17. X ３ -R ３ but, 【Chemistry 5】 The immune complex according to any one of claims 1 to 7, which is selected from the group consisting of:

18. X ４ is a bond, and R ４ The immune complex according to any one of claims 1 to 7, wherein is H.

19. R １ The immune complex according to any one of claims 1 to 7, wherein:

20. R ２ Or R ３ The immune complex according to any one of claims 1 to 7, wherein:

21. X ３ -R ３ -L is, 【Chemistry 6】 21. The immunoconjugate of claim 20, wherein the wavy line indicates the point of attachment to the N.

22. The immunoconjugate of any one of claims 1 to 7, wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

23. The immune complex of any one of claims 1 to 7, wherein L is attached to a cysteine ​​thiol of the antibody.

24. The immune conjugate of any one of claims 1 to 7, wherein for the PEG, m is 1 or 2 and n is an integer from 2 to 10.

25. 25. The immune complex of claim 24, wherein n is 10.

26. L has the structure: 【Chemistry 12】 wherein the wavy line is selected from R ５ The immune complex according to any one of claims 1 to 7, which exhibits binding to

27. Formula Ia: 【Chemistry 13】 The immune complex according to any one of claims 1 to 7, comprising:

28. X ４ is a bond, and R ４ The immune complex of claim 27, wherein is H.

29. X ２ and X ３ are each a bond, R ２ and R ３ But independently, C １ -C ８ Alkyl, —O—(C １ -C １２ alkyl)-(C １ -C １２ Alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , -(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ , -O-(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ , and —O—(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ 28. The immune complex of claim 27, selected from:

30. X ２ The immune complex of claim 27, wherein is O.

31. Formulae Ib to Ii: 【Chemistry 14-1】 【Chemistry 14-2】 28. The immune complex of claim 27, selected from:

32. X ２ and X ３ are each a bond, R ２ and R ３ But independently, C １ -C ８ Alkyl, —O—(C １ -C １２ alkyl), -(C １ -C １２ Alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , and —O—(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ The immune complex of claim 31 , selected from:

33. X ２ and X ３ are each a bond, R ２ But, C １ -C ８ alkyl, R ３ But -O-(C １ -C １２ alkyl) and -O-(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ The immune complex of claim 31 , selected from:

34. An 8-Het-2-aminobenzazepine-linker compound selected from the group consisting of:

35. 35. An immunoconjugate prepared by conjugation of an anti-CEA antibody with the 8-Het-2-aminobenzazepine-linker compound of claim 34.

36. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate of any one of claims 1 to 7 and one or more pharma- ceutically acceptable diluents, vehicles, carriers, or excipients.

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

38. A method for preparing an immunoconjugate of formula I according to claim 1, wherein the 8-Het-2-amino-thienoazepine-linker compound according to claim 34 is conjugated to said anti-CEA antibody.

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