Immunoconjugates for treatment of cancer comprising anti-CEA antibodies linked by conjugation to one or more 8-Het-2-aminobenzoaza derivatives

Abstract

The present invention provides an immunoconjugate of formula I, which comprises an anti-CEA antibody linked by conjugation to one or more 8-Het-2-aminobenzoaza # imgabs0 # derivative (s). The invention also provides an intermediate composition of the 8-Het-2-aminobenzoaza # imgabs 1 # derivative, which comprises a reactive functional group. Such intermediate compositions are suitable substrates for the formation of immunoconjugates via linkers or linking moieties. The invention further provides methods of treating cancer with the immunoconjugates.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This non-provisional application claims the benefit of priority to U.S. Provisional Application No. 63 / 124,328, filed on December 11, 2020, which is incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on December 3, 2021, is named 17019_010WO1_SL.txt, and is 55,248 bytes in size. Technical Field

[0005] The present invention generally relates to a method comprising combining with one or more 8-Het-2-aminobenzazepines Immunoconjugates of anti-carcinoembryonic antigen (CEA) antibodies conjugated to the molecule. Background Art

[0006] New compositions and methods for delivering antibodies and immunoadjuvants are needed to reach hard-to-reach tumors and / or expand treatment options for cancer patients and other subjects. The present invention provides such compositions and methods. Summary of the invention

[0007] The present invention generally relates to a method comprising reacting one or more 8-Het-2-aminobenzazepines with The present invention further relates to an immunoconjugate of an anti-CEA antibody conjugated to a derivative. Derivative intermediate compositions. Such intermediate compositions are suitable substrates for forming immunoconjugates, wherein the antibody can be linked to 8-Het-2-aminobenzazepine having the formula (HxBz) part covalently bound:

[0008]

[0009] wherein 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.

[0010] The invention further relates to the use of such immunoconjugates in the treatment of diseases, in particular cancer.

[0011] One aspect of the invention is an immunoconjugate comprising an anti-CEA antibody covalently linked to a linker that is covalently linked to one or more 8-Het-2-aminobenzazepines. Partially covalently linked.

[0012] Another aspect of the present invention is 8-Het-2-aminobenzazepine -Joint compounds.

[0013] Another aspect of the invention is a method of treating cancer comprising administering a therapeutically effective amount of an immunoconjugate comprising a molecule of the invention which is conjugated to one or more 8-Het-2-aminobenzazepines. Partially conjugated anti-CEA antibody.

[0014] Another aspect of the present invention is the use of an immunoconjugate for treating cancer, the immunoconjugate comprising a molecule of the invention which is conjugated to one or more 8-Het-2-aminobenzazepines. Partially conjugated anti-CEA antibody.

[0015] Another aspect of the present invention is to prepare a 2-aminobenzazepine comprising one or more 8-Het-2-aminobenzazepines. A method for preparing an immunoconjugate by conjugating a moiety to an anti-CEA antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of an in vivo xenograft tumor model in mice is shown. Tumor volume was measured over time after treatment to compare the efficacy of immunoconjugate IC-2 with isotype immunoconjugates (ISACs) and naked antibody CEA.9-G1fhL2 in tumor inhibition in mice bearing CEA-high human pancreatic HPAF-II tumors.

[0017] Figure 2a Graph showing the induction of the cytokine IL-12p70 by immunoconjugates IC-2, IC-3, IC-4, IC-6, IC-14 (Table 3a) and naked antibody CEA.9-G1fhL2 in co-culture of CEA-high MKN-45 cells with human conventional dendritic cell (cDC)-enriched primary cell isolates.

[0018] Figure 2b Graphic representation showing the induction of the cytokine TNFα (tumor necrosis factor α) by immunoconjugates IC-2, IC-3, IC-4, IC-6, IC-14 and naked antibody CEA.9-G1fhL2 in co-culture of CEA-high MKN-45 cells with cDC-enriched primary cell isolates.

[0019] Figure 2cGraph showing IL-6 (interleukin-6) induction by immunoconjugates IC-2, IC-3, IC-4, IC-6, IC-14 and naked antibody CEA.9-G1fhL2 in co-culture of CEA-high MKN-45 cells with cDC-enriched primary cell isolates.

[0020] Figure 2d Graph showing the induction of the cytokine IFNγ (interferon gamma) by immunoconjugates IC-2, IC-3, IC-4, IC-6, IC-14 and naked antibody CEA.9-G1fhL2 in co-culture of CEA-high MKN-45 cells with cDC-enriched primary cell isolates.

[0021] Figure 2e Graph showing cytokine CCL2 induction by immunoconjugates IC-2, IC-3, IC-4, IC-6, IC-14 and naked antibody CEA.9-G1fhL2 in co-culture of CEA-high MKN-45 cells with cDC-enriched primary cell isolates.

[0022] Figure 3a Graph showing phagocytosis of M-CSF differentiated monocyte-derived macrophages treated with various concentrations of immunoconjugate IC-2 in CEA-high HPAF II cells. 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 signal. Mean values ​​+ / - standard deviations from three donors are shown in the graph.

[0023] Figure 3b Graph showing phagocytosis of M-CSF differentiated monocyte-derived macrophages treated with various concentrations of immunoconjugate IC-2 in CEA medium LoVo cells. 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 signal. Mean values ​​+ / - standard deviations from three donors are shown in the graph.

[0024] Figure 3cGraph showing phagocytosis of M-CSF differentiated monocyte-derived macrophages treated with various concentrations of immunoconjugate IC-2 in CEA low LS-174T cells. 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 signal. Mean values ​​+ / - standard deviations from three donors are shown in the graph.

[0025] Figure 3d Graph showing phagocytosis of M-CSF differentiated monocyte-derived macrophages treated with various concentrations of immunoconjugate IC-2 in CEA-negative MDA-MB-231 cells. CTG-labeled tumor-IC-2 immune complexes were incubated with M-CSF differentiated monocyte-derived macrophages at an effector to target ratio of 2:1. After 4 hours, phagocytosis was measured by flow cytometry gating effector cells positive for CTG signal. Mean values ​​+ / - standard deviations from three donors are shown in the graph.

[0026] Figure 4a A graphical representation showing the levels of secreted TNFα (tumor necrosis factor α) cytokine after incubation of different concentrations of the immunoconjugate IC-2 and naked antibody CEA.9-G1fhL2 with co-cultures of cancer cells and cDC-enriched primary cell isolates.

[0027] Figure 4b A graphical representation showing the levels of IL-6 (interleukin-6) cytokine secreted after incubation of different concentrations of the immunoconjugate IC-2 and naked antibody CEA.9-G1fhL2 with co-cultures of cancer cells and cDC-enriched primary cell isolates.

[0028] Figure 4c A graphical representation showing the levels of secreted CXCL10 cytokine after incubation of different concentrations of the immunoconjugate IC-2 and naked antibody CEA.9-G1fhL2 with co-cultures of cancer cells and cDC-enriched primary cell isolates.

[0029] Figure 4d A graphical representation showing the levels of secreted TNFα (tumor necrosis factor α) cytokine after incubation of different concentrations of the immunoconjugate IC-2 and naked antibody CEA.9-G1fhL2 with co-cultures of cancer cells and cDC-enriched primary cell isolates.

[0030] Figure 4eA graphical representation showing the level of induction of secreted CD40 surface marker following incubation of different concentrations of the immunoconjugate IC-2 and naked antibody CEA.9-G1fhL2 with co-cultures of cancer cells and cDC-enriched primary cell isolates.

[0031] Figure 4f A graphical representation showing the level of induction of secreted CD86 surface marker following incubation of different concentrations of the immunoconjugate IC-2 and naked antibody CEA.9-G1fhL2 with co-cultures of cancer cells and cDC-enriched primary cell isolates. DETAILED DESCRIPTION

[0032] Now will be referred to in detail certain embodiments of the present invention, and its embodiment is illustrated in the appended structure and chemical formula.Although the present invention will be described in conjunction with the embodiment of enumeration, it will be appreciated that they are not intended to limit the present invention to these embodiments.On the contrary, the present invention is intended to contain all substitutions, modifications and equivalents, which can be included in the scope of the present invention as defined in the claims.

[0033] One skilled in the art will recognize that many methods and materials similar or equivalent to those described herein, which can be used to practice the present invention. The present invention is in no way limited to the methods and materials described.

[0034] definition

[0035] The term "immunoconjugate" or "immunostimulatory antibody conjugate" refers to an antibody construct covalently bonded to an adjuvant moiety via a linker. The term "adjuvant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant.

[0036] An "adjuvant moiety" refers to an adjuvant covalently bonded to an antibody construct, e.g., via a linker as described herein. The adjuvant moiety can elicit an immune response upon bonding to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct following administration of the immunoconjugate to a subject.

[0037] "Adjuvant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant.

[0038] The terms "Toll-like receptor" and "TLR" refer to any member of a highly conserved mammalian protein family that recognizes pathogen-associated molecular patterns and serves as a key signaling element 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.

[0039] The terms "Toll-like receptor 7" and "TLR7" refer 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 with a publicly available TLR7 sequence (e.g., GenBank Accession No. AAZ99026 for human TLR7 polypeptide or GenBank Accession No. AAK62676 for mouse TLR7 polypeptide).

[0040] The terms "Toll-like receptor 8" and "TLR8" refer 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 with a publicly available TLR7 sequence (e.g., GenBank Accession No. AAZ95441 for human TLR8 polypeptide or GenBank Accession No. AAK62677 for mouse TLR8 polypeptide).

[0041] "TLR agonist" is a substance that directly or indirectly binds to a TLR (e.g., TLR7 and / or TLR8) to induce TLR signaling. Any detectable difference in TLR signaling may indicate that the agonist stimulates or activates the TLR. The signaling difference may, for example, be manifested as changes in the expression of target genes, phosphorylation of signal transduction components, intracellular localization of downstream elements such as nuclear factor-κB (NF-κB), association of certain components (such as IL-1 receptor-associated kinase (IRAK)) with other proteins or intracellular structures, or biochemical activity of components such as kinases (such as mitogen-activated protein kinases (MAPK)).

[0042] "Antibody" refers to a polypeptide comprising an antigen binding region (including complementary determining regions (CDRs)) from an immunoglobulin gene or a fragment thereof. The term "antibody" specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer consists of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa) connected by disulfide bonds. Each chain consists of domains called immunoglobulin domains. These domains are divided into different categories by size and function, such as variable domains or regions on light and heavy chains (V, V, and V, respectively). L and V H ) and the constant domains or regions on the light and heavy chains (C L and C H). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is called a paratope and is primarily responsible for antigen recognition, i.e., the antigen binding domain. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are macromolecules of about 150 kDa consisting of four peptide chains. IgG antibodies contain two identical γ-class heavy chains of about 50 kDa and two identical light chains of about 25 kDa, and therefore have a tetrameric quaternary structure. The two heavy chains are connected to each other and are each connected to the light chain by a disulfide bond. The resulting tetramer has two identical hemisomes that together form a Y-like shape. Each end of the fork contains the same antigen binding domain. Humans have four IgG subclasses (IgG1, IgG2, IgG3, and IgG4), which are named in order of abundance in serum (i.e., IgG1 is the most abundant). Typically, the antigen binding domain of the antibody will be most critical in terms of specificity and affinity of binding to cancer cells.

[0043] "Antibody construct" refers to an antibody or fusion protein comprising (i) an antigen binding domain and (ii) an Fc domain.

[0044] In some embodiments, the binding agent is an antigen-binding antibody "fragment", which is a construct comprising at least the antigen-binding region of an antibody, alone or together with other components that together constitute the antigen-binding construct. Many different types of antibody "fragments" are known in the art, including, for example, (i) Fab fragments, which are composed of V L 、V H , C L and CH 1 Monovalent fragment composed of domains, (ii) F(ab') 2 A fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region, (iii) a V fragment of a single arm of the antibody L and V H (iv) Fab' fragment, which is formed by destroying F(ab') under mild reducing conditions. 2 (i) disulfide bridges of the Fv fragment, (v) disulfide-stabilized Fv fragment (dsFv), and (vi) single-chain Fv (scFv), which is two domains of the Fv fragment (i.e., V L and V H ), the synthetic linker enabling the two domains to be synthesized as a single polypeptide chain.

[0045] The antibody or antibody fragment can be part of a larger construct, such as a conjugate or fusion construct of an antibody fragment with another region. For example, in some embodiments, the antibody fragment can be fused to an Fc region as described herein. In other embodiments, the antibody fragment (e.g., Fab or scFv) can be part of a chimeric antigen receptor or a chimeric T cell receptor, for example by fusion with a transmembrane domain (optionally with an intermediate linker or "stem" (e.g., hinge region)) and an optional intercellular signaling domain.

[0046] "Epitope" means any antigenic determinant or epitopic determinant of an antigen to which an antigen binding domain binds (i.e., at the paratope of the antigen binding domain). Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0047] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. There are three main types of Fc receptors: (1) FcγR that binds to IgG, (2) FcαR that binds to IgA, and (3) FcεR that binds to 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 (e.g., IgG1, IgG2, IgG3, and IgG4).

[0048] As mentioned herein, nucleic acid or amino acid sequence "identity" can be determined by comparing the nucleic acid or amino acid sequence of interest with a reference nucleic acid or amino acid sequence. Percent identity is the number of nucleotides or amino acid residues that are identical (i.e., identical) to the sequence of the best comparison of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). Available software programs can be used to compare sequences and calculate percent identity. Examples of such programs include CLUSTAL-W, T-Coffee and ALIGN (for comparison of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, BLASTp, BLASTn, etc.) and FASTA programs (e.g., FASTA3x, FASTM and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms are also disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3):403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009); Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7):951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). The percent identity (%) of a sequence can also be calculated, for example, as 100×[(identical positions) / min(TG A ,TG B )], where TG A and TG B Is to use TG A and TG B The sum of the number of residues and internal gap positions in the peptide sequences A and B in the minimized alignment. See, e.g., Russell et al., J. Mol Biol., 244:332-350 (1994).

[0049] The binding agent comprises an Ig heavy chain and a light chain variable region polypeptide that together form an antigen binding site. Each of the heavy chain and light chain variable regions comprises three complementary determining regions (CDR1, CDR2 and CDR3) connected by a framework region. The binding agent can be any of the various types of binding agents comprising an Ig heavy chain and a light chain known in the art. For example, the binding agent can be an antibody, an antigen-binding antibody "fragment" or a T cell receptor.

[0050] A “biosimilar” is an approved antibody construct with an activity profile similar to that of, for example, a CEA-targeting antibody such as labetuzumab (CEA-CIDE TM , MN-14, hMN14, Immunomedics) CAS registration number 219649-07-7).

[0051] A “biobetter” is an approved antibody construct that improves a previously approved antibody construct, such as labetuzumab. A biobetter may have one or more modifications that are superior to a previously approved antibody construct (e.g., an altered glycan profile or a unique epitope). A biobetter is a recombinant protein drug that is in the same class as an existing biologic drug, but is not identical; and is superior to the original. A biobetter is not just a new drug, nor is it a copy of a drug. Both biosimilars and biobetters are variants of biologics; the former are close copies of the original drug, while the latter have improvements in efficacy, safety and tolerability, or dosing regimen.

[0052] "Amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide or protein. Amino acids include naturally occurring α-amino acids and stereoisomers thereof and non-natural (non-naturally occurring) amino acids and stereoisomers thereof. "Stereoisomers" of a given amino acid refer to isomers with the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., L-amino acids and corresponding D-amino acids). Amino acids can be glycosylated (e.g., N-linked polysaccharides, O-linked polysaccharides, phosphate polysaccharides, C-linked polysaccharides or glycosylphosphatidylinositolization) or deglycosylated. Amino acids can be represented herein by the commonly known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0053] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamate (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0054] Naturally occurring amino acids include those formed in proteins by post-translational modifications, such as citrulline (Cit).

[0055] Unnatural (non-naturally occurring) amino acids include, but are not limited to, amino acid analogs in the L- or D-configuration, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids that function in a manner similar to the naturally occurring amino acids. For example, an "amino acid analog" can be an unnatural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., carbon, carboxyl, amino group bonded to hydrogen) but has modified side chain groups or a modified peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to the naturally occurring amino acids.

[0056] "Linker" refers to a functional group that covalently bonds two or more moieties in a compound or material. For example, a linker moiety can be used to covalently bond an adjuvant moiety to an antibody construct in an immunoconjugate.

[0057] "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 be used to covalently bond an adjuvant moiety to an antibody in an immunoconjugate. Useful bonds for connecting a linking moiety to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

[0058] "Divalent" refers to a chemical moiety containing two connection points for connecting two functional groups; a multivalent connecting moiety may have additional connection points for connecting further functional groups. A divalent group can be represented by the suffix "diradical". For example, a divalent connecting moiety includes a divalent polymer moiety such as a divalent poly (ethylene glycol), a divalent cycloalkyl, a divalent heterocycloalkyl, a divalent aryl, and a divalent heteroaryl. "Divalent cycloalkyl, heterocycloalkyl, aryl or heteroaryl" refers to a cycloalkyl, heterocycloalkyl, aryl or heteroaryl having two connection points for covalently connecting two parts in a molecule or material. Cycloalkyl, heterocycloalkyl, aryl or heteroaryl can be substituted or unsubstituted. Cycloalkyl, heterocycloalkyl, aryl or heteroaryl can be substituted by one or more groups selected from halo, hydroxy, amino, alkylamino, acylamino, acyl, nitro, cyano and alkoxy.

[0059] Wavy Lines Represents the point of attachment of a designated chemical moiety. If two wavy lines are present for a designated chemical moiety, it will be understood that the chemical moiety can be used bidirectionally, ie, read from left to right or from right to left.

[0060] "Alkyl" refers to a straight chain (linear) or branched chain saturated aliphatic group having the indicated number of carbon atoms. An alkyl group may include any number of carbons, such as one to twelve. Examples of alkyl groups include, but are not limited to, 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, isopropyl, -CH (CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, isobutyl, -CH 2 CH(CH 3 ) 2 ), 2-butyl (s-Bu, sec-butyl, -CH (CH 3 )CH 2 CH3 ), 2-methyl-2-propyl (t-Bu, tert-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 CH3 ), 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, etc. The alkyl group may be substituted or unsubstituted. The "substituted alkyl group" may be substituted by one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, acylamino, acyl, nitro, cyano and alkoxy.

[0061] The term "alkyldiyl" refers to a divalent alkyl group. Examples of alkyldiyl groups include, but are not limited to, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -), etc. An alkyldiyl group may also be referred to as an "alkylene group".

[0062] "Alkenyl" refers to a straight chain (linear) or branched unsaturated aliphatic group having the indicated number of carbon atoms and at least one carbon-carbon double bond, sp2. An alkenyl group may include two to about 12 or more carbon atoms. An alkenyl group is a group having "cis" and "trans" orientations or, alternatively, "E" and "Z" orientations. Examples include, but are not limited to, vinyl (ethylenyl / vinyl, -CH=CH 2 ), allyl (-CH 2 CH=CH 2 ), butenyl, pentenyl and isomers thereof. Alkenyl may be substituted or unsubstituted. "Substituted alkenyl" may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, acylamino, acyl, nitro, cyano and alkoxy.

[0063] The term "alkenylene" or "alkenyldiyl" refers to a straight or branched divalent hydrocarbon radical. Examples include, but are not limited to, ethylenylene (ethylenylene / vinylene, -CH=CH-), allyl (-CH 2 CH=CH-) etc.

[0064] "Alkynyl" refers to a straight-chain (linear) or branched unsaturated aliphatic group having the indicated number of carbon atoms and at least one carbon-carbon triple bond, sp. Alkynyl groups may include two to about 12 or more carbon atoms. For example, C 2 -C 6 Alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propynyl (propargyl, -CH 2 C≡CH), butynyl, pentynyl, hexynyl and isomers thereof. Alkynyl may be substituted or unsubstituted. "Substituted alkynyl" may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, acylamino, acyl, nitro, cyano and alkoxy.

[0065] The term "alkynylene" or "alkynyldiyl" refers to a divalent alkynyl radical.

[0066] The terms "carbocycle", "carbocyclyl", "carbocyclic ring", and "cycloalkyl" refer to saturated or partially unsaturated, monocyclic, fused bicyclic, or bridged polycyclic ring combinations containing 3 to 12 ring atoms or the indicated number of atoms. Saturated monocyclic carbocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocyclic rings include, for example, norbornane, [2.2.2]bicyclooctane, decalin, and adamantane. Carbocyclic groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative partially unsaturated carbocyclic groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4-, and 1,5-isomers), norbornene, and norbornadiene.

[0067] The term "cycloalkyldiyl" refers to a divalent cycloalkyl group.

[0068] "Aryl" refers to a 6-20 carbon atom (C 2 - 40) radical derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. 6 -C 20 ) is a monovalent aromatic hydrocarbon radical having a ring structure of 1, 2, 3, 4, 5 or 6. The aryl group may be monocyclic, fused to form a bicyclic or tricyclic group, or linked by bonds to form a biaryl group. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl with a methylene linker. Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl.

[0069] The term "arylene" or "aryldiyl" refers to a 6-20 carbon atom (C 2 ) radical derived from a parent aromatic ring system by removing two hydrogen atoms from two carbon atoms. 6 -C 20 ) is a divalent aromatic hydrocarbon radical of 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, etc. Some aryl diradicals are represented as "Ar" in the exemplary structures. Aryl diradicals include bicyclic groups containing aromatic rings fused to saturated, partially unsaturated rings or aromatic carbocyclic rings. Typical aryl diradicals include, but are not limited to, groups derived from benzene (phenyl diradicals), substituted benzenes, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, etc. Aryl diradicals are also referred to as "arylene" and are optionally substituted with one or more substituents described herein.

[0070] The terms "heterocycle", "heterocyclyl" and "heterocyclic ring" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double bonds and / or triple bonds within the ring) carbocyclic group of 3 to about 20 ring atoms, wherein at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, and the remaining ring atoms are C, wherein one or more ring atoms are optionally independently substituted with one or more of the following substituents. The heterocycle can be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P and S), for example: a bicyclo [4,5], [5,5], [5,6] or [6,6] system. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82: 5566. "Heterocyclyl" also includes groups in which a heterocyclic group is fused to a saturated, partially unsaturated ring or an aromatic carbocyclic or heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepanyl, Base, diazepine Thiazepine 1,3-dioxolanyl, pyrazolyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothiophenyl, dihydrofuranyl, pyrazolidinyl imidazolinyl, imidazolidinyl, 3-azabicyclo [3.1.0] hexanyl, 3-azabicyclo [4.1.0] heptanyl, azabicyclo [2.2.2] hexanyl, 3H-indolyl quinolizinyl and N-pyridyl urea. Spiro heterocyclic moieties are also included within the scope of this definition. Examples of spiro heterocyclic moieties include azaspiro [2.5] octanyl and azaspiro [2.4] heptanyl. Examples of heterocyclic groups in which 2 ring atoms are substituted with oxo (=O) moieties include pyrimidinone and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.

[0071] The term "heterocyclyl diyl" refers to a divalent saturated or partially unsaturated (i.e., having one or more double bonds and / or triple bonds in the ring) carbocyclic group of 3 to about 20 ring atoms, wherein at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, and the remaining ring atoms are C, wherein one or more ring atoms are optionally independently substituted with one or more substituents as described. Examples of 5- and 6-membered heterocyclyl diyls include morpholinyl diyl, piperidinyl diyl, piperazinyl diyl, pyrrolidinyl diyl, dioxanyl diyl, thiomorpholinyl diyl and S-dioxothiomorpholinyl diyl.

[0072] The term "heteroaryl" refers to a monovalent aromatic group of 5, 6 or 7 ring members, and includes fused ring systems of 5-20 atoms (at least one of which is aromatic), containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. Examples of heteroaryl groups include pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolyl, isoquinolyl, tetrahydroisoquinolyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents as described herein.

[0073] The term "heteroaryldiyl" refers to a divalent aromatic group of 5, 6 or 7 ring members, and includes a fused ring system of 5-20 atoms (at least one of which is aromatic), containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. Examples of 5- and 6-membered heteroaryldiyls include pyridyldiyl, imidazolyldiyl, pyrimidinyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furanyldiyl, thienyldiyl, isoxazolyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl and pyrrolyldiyl.

[0074] The heterocyclic or heteroaryl groups may be carbon (carbon-linked) or nitrogen (nitrogen-linked) bonded where possible. By way of example and not limitation, the carbon-bonded heterocyclic or heteroaryl groups are bonded at the 2, 3, 4, 5, or 6 position of pyridine; the 3, 4, 5, or 6 position of pyridazine; the 2, 4, 5, or 6 position of pyrimidine; the 2, 3, 5, or 6 position of pyrazine; the 2, 3, 4, or 5 position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole; the 2, 4, or 5 position of oxazole, imidazole, or thiazole; the 3, 4, or 5 position of isoxazole, pyrazole, or isothiazole; the 2 or 3 position of aziridine; the 2, 3, or 4 position of azetidine; the 2, 3, 4, 5, 6, 7, or 8 position of quinoline; or the 1, 3, 4, 5, 6, 7, or 8 position of isoquinoline.

[0075] By way of example and not limitation, the nitrogen-bonded heterocyclic or heteroaryl group is bonded to the 1 position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole; the 2 position of isoindole or isoindoleline; the 4 position of morpholine; and the 9 position of carbazole or β-carboline.

[0076] The terms "halo" and "halogen," by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0077] The term "carbonyl" by itself or as part of another substituent refers to C(=O) or -C(=O)-, ie, a carbon atom double-bonded to oxygen and to two other groups in the carbonyl-bearing moiety.

[0078] As used herein, the phrase "quaternary ammonium salt" refers to a quaternary ammonium salt that has been substituted with an alkyl group (e.g., 1 -C 4 alkyl groups such as methyl, ethyl, propyl or butyl) quaternized tertiary amines.

[0079] The term "treat / treatment / treating" refers to any sign of successful treatment or improvement of an injury, pathology, disorder (e.g., cancer) or symptom (e.g., cognitive impairment), including any objective or subjective parameter, such as relief; remission; reducing symptoms or making symptoms, injury, pathology or disorder more tolerable for the patient; reducing the rate at which symptoms progress; reducing the frequency or duration of symptoms or disorder; or in some cases preventing the onset of symptoms. Treatment or improvement of symptoms can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0080] The terms "cancer", "neoplasm" and "tumor" are used herein to refer to cells that exhibit autonomous, unregulated growth, such that the cells exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. 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 every tissue are known. The phrase "cancer burden" refers to the number of cancer cells or the volume of cancer in a subject. Reducing the cancer burden accordingly refers to reducing the number of cancer cells or the volume of cancer cells in a subject. As used herein, the term "cancer cell" refers to any cell that is a cancer cell (e.g., from any cancer that can be treated by an individual, such as isolated from an individual with cancer) or derived from a cancer cell (e.g., a clone of a cancer cell). For example, a cancer cell may be from an established cancer cell line, may be a primary cell isolated from an individual with cancer, may be a progeny cell from a primary cell isolated from an individual with cancer, etc. In some embodiments, the term may also refer to a portion of a cancer cell, such as a subcellular portion, a cell membrane portion or a cell lysate of a cancer cell. Many types of cancer are known to those skilled in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, as well as circulating cancers such as leukemias.

[0081] As used herein, the term "cancer" includes any form of cancer, including, but not limited to, solid tumor cancers (e.g., skin cancer, lung cancer, prostate cancer, breast cancer, stomach cancer, bladder cancer, colon cancer, ovarian cancer, pancreatic cancer, kidney cancer, liver cancer, glioblastoma, medulloblastoma, leiomyosarcoma, head and neck squamous cell carcinoma, melanoma, and neuroendocrine cancer) and liquid cancers (e.g., hematological cancers); carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas; leukemias; lymphomas; and brain cancers, including minimal residual disease, and including both primary tumors and metastatic tumors.

[0082] The "pathology" of cancer includes all phenomena that impair the patient's health. This includes, but is not limited to, abnormal or uncontrolled cell growth, metastasis, interference with the normal function of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immune responses, neoplasia, precancer, malignancy, and invasion of surrounding or distant tissues or organs such as lymph nodes.

[0083] As used herein, the phrases "cancer recurrence" and "tumor recurrence" and grammatical variations thereof refer to further growth of a tumor or cancer cell after a cancer diagnosis. In particular, a recurrence may occur when further cancer cell growth occurs in a cancerous tissue. Similarly, "tumor spread" occurs when tumor cells spread to local or distant tissues and organs, and thus, tumor spread encompasses tumor metastasis. "Tumor invasion" occurs when tumor growth spreads outward locally to impair the function of the tissue involved by compressing, destroying, or preventing normal organ function.

[0084] As used herein, the term "metastasis" refers to the growth of a cancerous tumor in an organ or body part that is not directly related to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of undetectable amounts of cancer cells in an organ or body part that is not directly related to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from the original tumor site, and the migration and / or invasion of cancer cells into other parts of the body.

[0085] The phrases "effective amount" and "therapeutically effective amount" refer to the dosage or amount of a substance such as an immunoconjugate that produces the therapeutic effect of administration. The exact dosage will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22nd Edition, (Pharmaceutical Press, London, 2012)). In the case of cancer, a therapeutically effective amount of an immunoconjugate can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and preferably stop to some extent) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. To the extent that an immunoconjugate can prevent the growth of existing cancer cells and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0086] "Recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., a human) in need of diagnosis, treatment, or therapy. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, racetrack, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain embodiments, the mammal is a human.

[0087] The phrase "synergistic adjuvant" or "synergistic combination" in the context of the present invention includes a combination of two immunomodulators, such as a receptor agonist, a cytokine, and an adjuvant polypeptide, which combination induces a synergistic effect of immunity relative to the single administration of either one. In particular, the immunoconjugates disclosed herein comprise a synergistic combination of the claimed adjuvant and antibody construct. These synergistic combinations induce a greater immune effect upon administration, for example, relative to the administration of the antibody construct or adjuvant in the absence of the other part. Further, the amount of immunoconjugate that can be administered is reduced (as measured by the total amount of antibody constructs or the total amount of adjuvant administered as part of the immunoconjugate) compared to when the antibody construct or adjuvant is administered alone.

[0088] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal or subcutaneous administration to a subject, oral administration, administration as a suppository, topical contact, intrathecal administration, or implantation of a sustained release device, such as a mini-osmotic pump.

[0089] As used herein to modify a numerical value, the terms "about" and "approximately" represent a close range around the numerical value. Thus, if "X" is the value, "about X" or "approximately X" represents a value of 0.9X to 1.1X, such as 0.95X to 1.05X or 0.99X to 1.01X. Reference to "about X" or "approximately X" specifically represents at least values ​​of X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Therefore, "about X" and "approximately X" are intended to teach and provide written description support for claim limitations (e.g., "0.98X").

[0090] CEA antibody

[0091] The immunoconjugates of the present invention include antibodies that target, bind or recognize carcinoembryonic antigens (CEA, CD66e, CEACAM5). Functional variants of antibody constructs or antigen-binding domains described herein are included within the scope of embodiments of the present invention. The term "functional variant" as used herein refers to an antibody construct having an antigen-binding domain having substantial or significant sequence identity or similarity with a parent antibody construct or antigen-binding domain, and the functional variant retains the biological activity of the antibody construct or antigen-binding domain of which it is a variant. Functional variants encompass those variants of, for example, antibody constructs or antigen-binding domains (parent antibody constructs or antigen-binding domains) described herein, which retain the ability to recognize target cells expressing CEA to a degree similar to, the same degree or a higher degree than, the parent antibody construct or antigen-binding domain.

[0092] With respect to antibody constructs or antigen binding domains, a functional variant may, for example, be at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the antibody construct or antigen binding domain.

[0093] Functional variants may, for example, comprise an amino acid sequence of a parent antibody construct or antigen-binding domain having at least one conservative amino acid substitution. Alternatively or additionally, functional variants may comprise an amino acid sequence of a parent antibody construct or antigen-binding domain having at least one non-conservative amino acid substitution. In this case, it is preferred that 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 the functional variant, so that the biological activity of the functional variant is increased compared to the parent antibody construct or antigen-binding domain.

[0094] Antibodies comprising the immunoconjugates of the invention include Fc engineered variants. In some embodiments, mutations in the Fc region that result in modulated binding to one or more Fc receptors may include one or more of the following mutations: SD (S239D), SDIE (S239D / I332E), SE (S267E), SELF (S267E / L328F), SDIE (S239D / I332E), SDIEAL (S239D / I332E / A330L), GA (G236A), ALIE (A336A), 30L / I332E), GASDALE1 (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R) and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E345R, E233, G237, P238, H268, P271, L328 and A330. Additional Fc region modifications for modulating Fc receptor binding are described, for example, in US 2016 / 0145350 and US7416726 and US 5624821, which are hereby incorporated by reference in their entirety.

[0095] Antibodies comprising the immunoconjugates of the invention include glycan variants, such as afucosylation.In some embodiments, the Fc region of the binding agent is modified to have an altered Fc region glycosylation pattern compared to a native, unmodified Fc region.

[0096] The amino acid substitutions of the antibody constructs or antigen binding domains of the present invention are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which an amino acid with certain physical and / or chemical properties is exchanged for another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., Ile, Thr and Val), an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr), etc.

[0097] The antibody construct or antigen binding domain may consist essentially of one or more specified amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the antibody construct or antigen binding domain functional variant.

[0098] In some embodiments, the antibody in the immunoconjugate contains a modified Fc region, wherein the modification modulates binding of the Fc region to one or more Fc receptors.

[0099] In some embodiments, the antibody in the immunoconjugate (e.g., an antibody conjugated to at least two adjuvant moieties) contains one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region that result in modulated (e.g., increased binding or decreased binding) to one or more Fc receptors (e.g., FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), and / or FcγRIIIB (CD16b)) compared to a native antibody lacking a mutation in the Fc region. In some embodiments, the antibody in the immunoconjugate contains one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region that reduce binding of the Fc region of the antibody to FcγRIIB. In some embodiments, the antibody in the immunoconjugate contains one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region of the antibody that reduce binding of the antibody to FcγRIIB while maintaining the same binding to or increasing binding to FcγRI (CD64), FcγRIIA (CD32A), and / or FcRγIIIA (CD16a) compared to a native antibody lacking mutations in the Fc region. In some embodiments, the antibody in the immunoconjugate contains one or more modifications in the Fc region that increase binding of the Fc region of the antibody to FcγRIIB.

[0100] In some embodiments, regulated binding is provided by mutations in the Fc region of the antibody relative to the native Fc region of the antibody. The mutation may be in the CH2 domain, the CH3 domain, or a combination thereof. "Native Fc region" is synonymous with "wild-type Fc region", and comprises an amino acid sequence identical to the amino acid sequence of the Fc region found in nature or identical to the amino acid sequence of the Fc region found in a natural antibody (e.g., cetuximab). Native sequence human Fc regions include native sequence human IgG1 Fc regions, native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions and naturally occurring variants thereof. Native sequence Fc includes various allotypes of Fc (Jefferis et al., (2009) mAbs, 1(4): 332-338).

[0101] In some embodiments, mutations in the Fc region that result in modulated binding to one or more Fc receptors may include one or more of the following mutations: SD (S239D), SDIE (S239D / I332E), SE (S267E), SELF (S267E / L328F), SDIE (S239D / I332E), SDIEAL (S239D / I332E / A330L), GA (G236A), ALIE (A330L / I332E), GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R), and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E233, G237, P238, H268, P271, L328 and A330. Additional Fc region modifications for modulating Fc receptor binding are described in, for example, US2016 / 0145350 and US 7416726 and US 5624821, which are hereby incorporated by reference in their entirety.

[0102] In some embodiments, the Fc region of the antibody of the immunoconjugate is modified to have an altered Fc region glycosylation pattern compared to a native, unmodified Fc region.

[0103] Human immunoglobulins are glycosylated at residue Asn297 in the Cγ2 domain of each heavy chain. This N-linked oligosaccharide consists of the core heptasaccharide N-acetylglucosamine 4 mannose 3 (GlcNAc4Man3). Removal of the heptasaccharide with endoglycosidase or PNGase F is known to result in conformational changes in the Fc region of antibodies, which can significantly reduce antibody binding affinity to activating FcγRs and lead to reduced effector function. The core heptasaccharide is often modified with galactose, bisecting GlcNAc, fucose, or sialic acid, which differentially affects Fc binding to activating and inhibitory FcγRs. In addition, α2,6-sialylation has been shown to enhance anti-inflammatory activity in vivo, while defucosylation results in improved FcγRIIIa binding and a 10-fold increase in antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. Specific glycosylation patterns can therefore be used to control inflammatory effector functions.

[0104] In some embodiments, the modification that changes the glycosylation pattern is a mutation. For example, a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods for controlling immune responses with antibodies that modulate FcγR-regulated signaling are described in, for example, U.S. Pat. No. 7,416,726 and U.S. Patent Application Publications Nos. 2007 / 0014795 and 2008 / 0286819, which are hereby incorporated by reference in their entirety.

[0105] In some embodiments, the antibody of the immunoconjugate is modified to contain an engineered Fab region with a non-naturally occurring glycosylation pattern. For example, a hybridoma can be genetically engineered to secrete afucosylated mAb, a desialylated mAb, or a deglycosylated Fc with a specific mutation that enables increased FcRγIIIa binding and effector function. In some embodiments, the antibody of the immunoconjugate is engineered to be afucosylated.

[0106] In some embodiments, the entire Fc region of the antibody in the immunoconjugate is exchanged with a different Fc region, so that the Fab region of the antibody is conjugated to a non-natural Fc region. For example, the Fab region of cetuximab generally comprises an IgG1 Fc region, which can be conjugated to IgG2, IgG3, IgG4 or IgA, or the Fab region of nivolumab generally comprises an IgG4 Fc region, which can be conjugated to IgG1, IgG2, IgG3, IgA1 or IgG2. In some embodiments, the Fc-modified antibody with a non-natural Fc domain further comprises one or more amino acid modifications, such as an S228P mutation within an IgG4 Fc, which regulates the stability of the Fc domain. In some embodiments, the Fc-modified antibody with a non-natural Fc domain further comprises one or more amino acid modifications that regulate the binding of Fc to FcR as described herein.

[0107] In some embodiments, the modification that modulates the binding of the Fc region to the FcR does not alter the binding of the Fab region of the antibody to its antigen when compared to the naturally unmodified antibody. In other embodiments, the modification that modulates the binding of the Fc region to the FcR also increases the binding of the Fab region of the antibody to its antigen when compared to the naturally unmodified antibody.

[0108] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen binding domain that specifically recognizes and binds CEA.

[0109] Elevated expression of carcinoembryonic antigens (CEA, CD66e, CEACAM5) has been implicated in various biological aspects of neoplasia, particularly tumor cell adhesion, metastasis, blockade of cellular immune mechanisms, and anti-apoptotic functions. CEA is a cell surface antigen and is also used as a blood marker for many cancers. Labetuzumab (CEA-CIDE TM , Immunomedics, CAS Reg. No. 219649-07-7) also known as MN-14 and hMN14, is a humanized IgG1 monoclonal antibody and has been studied for the treatment of colorectal cancer (Blumenthal, R. et al., (2005) Cancer Immunology Immunotherapy 54(4):315-327). Labetuzumab conjugated to a camptothecin analog (govitecant labetuzumab, IMMU-130) targets CEA and is being studied in patients with recurrent 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). In addition, the combination of gliomastatin and gliomastatin has been evaluated in clinical trials. 131 I-conjugated labetuzumab is used to treat 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).

[0110] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of hMN-14 / labetuzumab SEQ ID NO. 1 as disclosed in US6676924, which is incorporated herein by reference for this purpose.

[0111]

[0112] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of hMN-14 / labetuzumab SEQ ID NO. 2-8 (US 6676924).

[0113]

[0114] In an embodiment of the present 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 incorporated herein by reference for this purpose.

[0115]

[0116] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of hMN-14 / labetuzumab SEQ ID NOs. 10-16 (US 6676924).

[0117]

[0118] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of hPR1A3 SEQ ID NO. 17 as disclosed in US8642742, which is incorporated herein by reference for this purpose.

[0119]

[0120] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of hPR1A3 SEQ ID NO. 18-24 (US 8642742).

[0121]

[0122] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of hPR1A3 SEQ ID NO. 25-31 (US 8642742).

[0123]

[0124]

[0125] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of hMFE-23 SEQ ID NO. 32 as disclosed in US7232888, which is incorporated herein by reference for this purpose.

[0126]

[0127] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of hMFE-23 SEQ ID NO.33-40 (US 7232888). This embodiment includes two variants of LFR1, SEQ ID NO.:33 and SEQ ID NO.:34.

[0128]

[0129] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of hMFE-23 SEQ ID NO. 41 ( US 7232888 ).

[0130]

[0131] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of hMFE-23 SEQ ID NO. 42-49 (US 7232888). This embodiment includes two variants of HFR1, SEQ ID NO.: 42 and SEQ ID NO.: 43.

[0132]

[0133] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of SM3E SEQ ID NO. 50 (US 7232888).

[0134]

[0135] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of SM3E SEQ ID NO.51-56 and 38-39 (US 7232888). This embodiment includes two variants of LFR1, SEQ ID NO.: 51 and SEQ ID NO.: 52.

[0136]

[0137] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain of NP-4 / Acitumomab SEQ ID NO.57

[0138]

[0139] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of NP-4 / Acitumomab SEQ ID NOs. 58-64.

[0140]

[0141] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of NP-4 / Acitumomab SEQ ID NO.65.

[0142]

[0143] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of NP-4 SEQ ID NOs. 66-72.

[0144]

[0145] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of M5A / hT84.66 SEQ ID NO. 73 as disclosed in US7776330, which is incorporated herein by reference for this purpose.

[0146]

[0147] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of M5A / hT84.66 SEQ ID NO. 74-80 ( US 7776330 ).

[0148]

[0149] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of M5A / hT84.66 SEQ ID NO. 81 ( US 7776330 ).

[0150]

[0151] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of M5A / hT84.66 SEQ ID NOs. 82-88 ( US 7776330 ).

[0152]

[0153]

[0154] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of hAb2-3 SEQ ID NO. 89 as disclosed in US9617345, which is incorporated herein by reference for this purpose.

[0155]

[0156] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of hAb2-3 SEQ ID NOs. 90-96 (US 9617345).

[0157]

[0158] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of SEQ ID NO. 97 (US 9617345).

[0159]

[0160] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of hAb2-3 SEQ ID NOs. 98-104.

[0161]

[0162]

[0163] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of A240VL-B9VH / AMG-211 SEQ ID NO. 105 as disclosed in US9982063, which is incorporated herein by reference for this purpose.

[0164]

[0165] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of A240VL-B9VH / AMG-211 SEQ ID NOs. 106-112 ( US9982063 ).

[0166]

[0167] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of B9VH SEQ ID NO. 113 (US 9982063).

[0168]

[0169] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises a heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of SEQ ID NO. 114-121 (US 9982063). This embodiment includes two variants of CDR-H2, SEQ ID NO.: 117 and SEQ ID NO.: 118.

[0170]

[0171] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of E12VH SEQ ID NO. 122 (US 9982063).

[0172]

[0173] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of SEQ ID NOs. 123-129 (US 9982063).

[0174]

[0175] In an embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of PR1A3 VH SEQ ID NO. 130 ( US 8642742 ).

[0176]

[0177] In some embodiments, the antibody construct also includes 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). The single-chain variable region fragment (scFv) is a truncated Fab fragment, including the variable (V) domain of the antibody heavy chain connected via a synthetic peptide to the V domain of a light antibody chain, which can be produced using conventional recombinant DNA techniques. Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA techniques. The antibody construct or antigen binding domain may include one or more variable regions (e.g., two variable regions) of the antigen binding domain of an anti-CEA antibody, each variable region including CDR1, CDR2, and CDR3.

[0178] In some embodiments, the antibody in the immunoconjugate contains a modified Fc region, wherein the modification modulates binding of the Fc region to one or more Fc receptors.

[0179] In some embodiments, the Fc region is modified by including a transforming growth factor β1 (TGFβ1) receptor or a fragment thereof that can bind to TGFβ1. For example, the receptor can be a TGFβ receptor II (TGFβRII). In some embodiments, the TGFβ receptor is a human TGFβ receptor. In some embodiments, IgG has a C-terminal fusion with the TGFβRII extracellular domain (ECD), as described in US 9676863, which is incorporated herein. "Fc linker" can be used to connect IgG to the TGFβRII extracellular domain. The Fc linker can be a short flexible peptide that allows the molecule to fold correctly in three dimensions while maintaining binding specificity to the target. In some embodiments, the N-terminus of the TGFβ receptor is fused to the Fc of the antibody construct (with or without an Fc linker). In some embodiments, the C-terminus of the heavy chain of the antibody construct is fused to the TGFβ receptor (with or without an Fc linker). In some embodiments, the C-terminal lysine residue of the heavy chain of the antibody construct is mutated to alanine.

[0180] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0181] In some embodiments, the antibody in the immunoconjugate is a cysteine ​​engineered antibody that provides site-specific conjugation of an adjuvant, label or drug moiety to the antibody by substitution of a cysteine ​​at a site where the engineered cysteine ​​is available for conjugation, but does not perturb immunoglobulin folding and assembly or alter antigen binding and effector function (Junutula et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al., (2009) Blood 114(13):2721-2729; US 7521541; US ​​7723485; US 2012 / 0121615; WO 2009 / 052249). A "cysteine ​​engineered antibody" or "cysteine ​​engineered antibody variant" is an antibody in which one or more residues of the antibody are substituted with a cysteine ​​residue. Cysteine ​​engineered antibodies can be used as 8-Het-2-aminobenzazepines with uniform stoichiometry. -Linker compounds with 8-Het-2-aminobenzazepine Adjuvant moiety conjugation (e.g., up to two 8-Het-2-aminobenzazepines per antibody in an antibody with a single engineered cysteine ​​site) part).

[0182] 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 (LC K149C) of the light chain. In other embodiments, the cysteine ​​engineered antibodies have a cysteine ​​residue introduced at the 118-alanine site (EU numbering) (HC A118C) of the heavy chain. This site is alternatively numbered 121 according to the sequential numbering method, or 114 according to the Kabat numbering method. In other embodiments, the cysteine ​​engineered antibodies have a cysteine ​​residue introduced at G64C or R142C in the light chain according to the Kabat numbering method, or at D101C, V184C or T205C in the heavy chain according to the Kabat numbering method.

[0183] 8-HET-2-aminobenzazepine Adjuvant compounds

[0184] The immunoconjugates of the present invention comprise 8-Het-2-aminobenzazepine Adjuvant part. The adjuvant part described herein is a compound (i.e., immunostimulant) that triggers an immune response. Generally, the adjuvant part described herein is a TLR agonist. TLR is a type I transmembrane protein responsible for initiating an innate immune response in vertebrates. TLR recognizes a variety of pathogen-related molecular patterns from bacteria, viruses and fungi, and serves as the first line of defense against invading pathogens. Due to the differences in cell expression and the signal transduction pathways they initiate, TLR triggers overlapping but different biological responses. Once engaged (e.g., by natural stimulants or synthetic TLR agonists), TLR initiates a signal transduction cascade, resulting in activation of nuclear factor-κB (NF-κB) and recruitment of IL-1 receptor-associated kinases (IRAK) via adapter protein myeloid differentiation primary response gene 88 (MyD88). Then the phosphorylation of IRAK leads to the recruitment of TNF receptor-associated factor 6 (TRAF6), which leads to the phosphorylation of NF-κB inhibitor I-κB. As a result, NF-κB enters the nucleus and triggers the transcription of genes such as cytokines whose promoters contain NF-κB binding sites. Additional modes of regulating TLR signaling include TIR domain-containing adaptor-induced interferon-β (TRIF)-dependent induction of TNF-receptor-associated factor 6 (TRAF6) and activation of MyD88-independent pathways via TRIF and TRAF3, leading to phosphorylation of interferon response factor three (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.

[0185] Typically, the adjuvant moiety described herein is a TLR7 and / or TLR8 agonist. Both TLR7 and TLR8 are expressed in monocytes and dendritic cells. In humans, TLR7 is also expressed in plasmacytoid dendritic cells (pDC) and B cells. TLR8 is mainly expressed in cells of bone marrow origin, i.e., monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 can detect the presence of "external" single-stranded RNA in cells as a means of responding to viral invasion. Cells expressing TLR8 treated 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, cells expressing TLR7 such as pDC stimulated with TLR7 agonists can result in the production of high levels of IFN-α and other inflammatory cytokines. TLR7 / TLR8 engagement and the resulting cytokine production can activate dendritic cells and other antigen presenting cells, thereby driving a variety of innate and acquired immune response mechanisms, leading to tumor destruction.

[0186] Exemplary 8-Het-2-aminobenzazepines of the present invention Compounds (Hx) are shown in Table 1. Each compound was synthesized, purified and characterized by mass spectrometry and is shown with the indicated mass. Additional experimental procedures are found in the Examples. Activity against human embryonic kidney (HEK) 293 NFKB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 202. 8-Het-2-aminobenzazepines of Table 1 The compounds demonstrate the unexpected and surprising property of TLR8 agonist selectivity, which may predict useful therapeutic activity in the treatment of cancer and other disorders.

[0187] Table 1: 8-Het-2-aminobenzazepines Compound (HxBz)

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] 8-HET-2-aminobenzazepine -Joint compounds

[0199] 8-Het-2-aminobenzazepine by anti-CEA antibody -linker compound HxBzL to prepare the immunoconjugate of the present invention. - Linker compounds comprising 8-Het-2-aminobenzazepine covalently linked to a Linker unit (HxBz) moiety. The linker unit comprises functional groups and subunits that affect the stability, permeability, solubility and other pharmacokinetic, safety and efficacy characteristics of the immunoconjugate. The linker unit includes a reactive functional group that reacts with the reactive functional group of the antibody, i.e., conjugates. For example, the nucleophilic group of the antibody, such as the lysine side chain amino group, reacts with the electrophilic reactive functional group of the HxBzL linker compound to form an immunoconjugate. Also, for example, the cysteine ​​thiol of the antibody reacts with the maleimide or bromoacetamide group of the Hx-linker compound to form an immunoconjugate.

[0200] Suitable electrophilic reactive functional groups for HxBzL linker compounds include, but are not limited to, N-hydroxysuccinimide (NHS) esters and N-hydroxysulfosuccinimide (sulfo-NHS) esters (amine reactivity); carbodiimides (amine and carboxyl reactivity); hydroxymethylphosphines (amine reactivity); maleimides (thiol reactivity); haloacetamides such as N-iodoacetamide (thiol reactivity); aryl azides (primary amine reactivity); fluorinated aryl azides (reactivity via carbon-hydrogen (CH) insertion); pentafluorophenyl (PFP) esters (amine reactivity); tetrafluorophenyl (TFP) esters (amine reactivity); imidates (amine reactivity); isocyanates (hydroxyl reactivity); vinyl sulfones (thiol, amine, and hydroxyl reactivity); pyridyl disulfides (thiol reactivity); and benzophenone derivatives (reactivity via C-H bond insertion). Further reagents include, but are not limited to, those described in Hermanson, Bioconjugate Techniques 2nd ed., Academic Press, 2008.

[0201] The present invention provides solutions to the limitations and challenges of the design, preparation and use of immunoconjugates. Some joints may be unstable in the bloodstream, thereby releasing unacceptable amounts of adjuvant / drugs before internalization in target cells (Khot, A. et al., (2015) Bioanalysis 7 (13): 1633-1648). Other joints can provide stability in the bloodstream, but the effectiveness of intracellular release may be negatively affected. Joints that provide the required intracellular release are generally poorly stable in the bloodstream. In other words, bloodstream stability and intracellular release are generally inversely correlated. In addition, in the standard conjugation process, the amount of adjuvant / drug moiety loaded on the antibody (i.e., drug loading), the amount of aggregates formed in the conjugation reaction, and the yield of the final purified conjugate that can be obtained are interrelated. For example, aggregate formation is generally positively correlated with the equivalent number of adjuvant / drug moieties and their derivatives conjugated to the same antibody. In the case of high drug loading, the aggregates formed must be removed for therapeutic applications. As a result, drug loading-mediated aggregate formation reduces the yield of immunoconjugates and may make process amplification difficult.

[0202] Exemplary embodiments include 8-Het-2-aminobenzazepine of Formula II -Joint Compound:

[0203]

[0204] wherein Het is selected from heterocyclyldiyl and heteroaryldiyl;

[0205] R 1 , R 2 , R 3 and R 4 Independently selected from H, C 1 -C 12 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 12 Carbocyclic group, C 6 -C 20 Aryl, C 2 -C 9 Heterocyclic and C 1 -C 20 The group consisting of heteroaryl, wherein alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl are independently and optionally substituted by one or more groups selected from the following:

[0206] -(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0207] -(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0208] -(C 1 -C 12 Alkyldiyl)-OR 5 ;

[0209] -(C 3 -C 12 Carbocyclic group);

[0210] -(C 3 -C 12 Carbocyclyl)-*;

[0211] -(C 3 -C 12 Carbocyclic)-(C 1 -C 12 Alkyldiyl)-NR 5 -*;

[0212] -(C 3 -C 12 Carbocyclic)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0213] -(C 3 -C 12 Carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*;

[0214] -(C 6 -C 20 Aryl);

[0215] -(C 6 -C 20 Aryldiyl)-*;

[0216] -(C 6 -C 20 Aryldiyl)-N(R 5 )-*;

[0217] -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0218] -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-*;

[0219] -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0220] -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-NR 5 -C(=NR 5a )N(R 5 )-*;

[0221] -(C 2 -C 20 heterocyclic group);

[0222] -(C 2 -C 20 Heterocyclyl)-*;

[0223] -(C 2 -C 9 Heterocyclyl)-(C 1 -C 12 Alkyldiyl)-NR 5 -*;

[0224] -(C 2 -C 9 Heterocyclyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0225] -(C 2 -C 9 heterocyclyl)-C(=O)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0226] -(C 2 -C 9 Heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*;

[0227] -(C 2 -C 9 Heterocyclyl)-NR 5 -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0228] -(C 2 -C 9 Heterocyclyl)-(C 6 -C 20 Aryldiyl)-*;

[0229] -(C 1 -C 20 heteroaryl);

[0230] -(C 1 -C 20Heteroaryldiyl)-*;

[0231] -(C 1 -C 20 Heteroaryl)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0232] -(C 1 -C 20 Heteroaryl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0233] -(C 1 -C 20 Heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*;

[0234] -(C 1 -C 20 Heteroaryl)-N(R 5 )C(=O)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0235] -C(=O)-*;

[0236] -C(=O)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0237] -C(=O)-(C 2 -C 20 Heterocyclyldiyl)-*;

[0238] -C(=O)N(R 5 ) 2 ;

[0239] -C(=O)N(R 5 )-*;

[0240] -C(=O)N(R 5 )-(C 1 -C 12 Alkyldiyl)-N(R 5 )C(=O)R 5 ;

[0241] -C(=O)N(R 5 )-(C1 -C 12 Alkyldiyl)-N(R 5 )C(=O)N(R 5 ) 2 ;

[0242] -C(=O)NR 5 -(C 1 -C 12 Alkyldiyl)-N(R 5 )CO 2 R 5 ;

[0243] -C(=O)NR 5 -(C 1 -C 12 Alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 ) 2 ;

[0244] -C(=O)NR 5 -(C 1 -C 12 Alkyldiyl)-NR 5 C(=NR 5a )R 5 ;

[0245] -C(=O)NR 5 -(C 1 -C 8 Alkyldiyl)-NR 5 (C 2 -C 5 heteroaryl);

[0246] -C(=O)NR 5 -(C 1 -C 20 Heteroaryldiyl)-N(R 5 )-*;

[0247] -C(=O)NR 5 -(C 1 -C 20 Heteroaryldiyl)-*;

[0248] -C(=O)NR 5 -(C 1 -C 20 Heteroaryldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0249] -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 -*;

[0250] -N(R 5 ) 2 ;

[0251] -N(R 5 )-*;

[0252] -N(R 5 )C(=O)R 5 ;

[0253] -N(R 5 )C(=O)-*;

[0254] -N(R 5 )C(=O)N(R 5 ) 2 ;

[0255] -N(R 5 )C(=O)N(R 5 )-*;

[0256] -N(R 5 )CO 2 R 5 ;

[0257] -NR 5 C(=NR 5a )N(R 5 ) 2 ;

[0258] -NR 5 C(=NR 5a )N(R 5 )-*;

[0259] -NR 5 C(=NR 5a )R 5 ;

[0260] -N(R 5 )C(=O)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0261] -N(R 5 )-(C 2 -C 5 heteroaryl);

[0262] -N(R 5 )-S(=O) 2 -(C 1 -C 12 alkyl);

[0263] -O-(C 1 -C 12 alkyl);

[0264] -O-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0265] -O-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0266] -OC(=O)N(R 5 ) 2 ;

[0267] -OC(=O)N(R 5 )-*;

[0268] -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-*;

[0269] -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0270] -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-(C 1 -C 12 Alkyldiyl)-NR 5 -*;and

[0271] -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-(C 1 -C12 Alkyldiyl)-OH;

[0272] or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring;

[0273] X 1 , X 2 , X 3 and X 4 are independently selected from a free bond, C(=O), C(=O)N(R 5 )、O、N(R 5 )、S、S(O) 2 and S(O) 2 N(R 5 )

[0274] R 5 Independently selected from H, C 6 -C 20 Aryl, C 3 -C 12 Carbocyclic group, C 6 -C 20 Aryldiyl, C 1 -C 12 Alkyl and C 1 -C 12 A group consisting of an alkyl diyl group, or two R 5 The groups together form a 5- or 6-membered heterocyclyl ring;

[0275] R 5a Choose from C 6 -C 20 Aryl and C 1 -C 20 A group consisting of heteroaryl groups;

[0276] wherein the asterisk * indicates the attachment site of L, and wherein R 1 , R 2 , R 3 and R 4 One of them is connected to L;

[0277] L is a linker selected from the group consisting of:

[0278] QC(=O)-PEG-;

[0279] QC(=O)-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-Gluc-;

[0280] QC(=O)-PEG-O-;

[0281] QC(=O)-PEG-OC(=O)-;

[0282] QC(=O)-PEG-C(=O)-;

[0283] QC(=O)-PEG-C(=O)-PEP-;

[0284] QC(=O)-PEG-N(R 6 )-;

[0285] QC(=O)-PEG-N(R 6 )-C(=O)-;

[0286] QC(=O)-PEG-N(R 6 )-PEG-C(=O)-PEP-;

[0287] QC(=O)-PEG-N + (R 6 ) 2 -PEG-C(=O)-PEP-;

[0288] QC(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-;

[0289] QC(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 Monoheterocyclyldiyl)-;

[0290] QC(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-;

[0291] QC(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-C(═O)-;

[0292] QC(=O)-(C 1 -C 12 alkyldiyl)-C(═O)-PEP-;

[0293] QC(=O)-(C 1 -C12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl-;

[0294] QC(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-N(R 5 )-C(=O);

[0295] QC(=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)-;

[0296] Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-;

[0297] Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-Gluc-;

[0298] Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-;

[0299] Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-OC(=O)-;

[0300] Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-;

[0301] Q-(CH 2 )m -C(=O)N(R 6 )-PEG-N(R 5 )-;

[0302] Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-C(=O)-;

[0303] Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-;

[0304] Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-;

[0305] Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-;

[0306] Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-; and

[0307] Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 Monoheterocyclyldiyl)-;

[0308] R 6 independently H or C 1 -C 6 alkyl;

[0309] PEG has the formula: -(CH 2 CH 2 O)n -(CH 2 ) m -; m is an integer from 1 to 5, and n is an integer from 2 to 50;

[0310] Gluc has the following formula:

[0311]

[0312] PEP has the following formula:

[0313]

[0314] wherein AA is independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a five-membered ring of proline amino acids, and the wavy line indicates the point of attachment;

[0315] Cyc is selected from C 6 -C 20 Aryldiyl and C 1 -C 20 Heteroaryldiyl, optionally substituted with one or more selected from F, Cl, NO 2 、-OH、-OCH 3 and a glucuronic acid radical having the following structure:

[0316]

[0317] R 7 Selected from -CH(R 8 )O-、-CH 2 -、-CH 2 N(R 8 )- and -CH(R 8 )OC(=O)-, where R 8 Selected from H, C 1 -C 6 Alkyl, C(=O)-C 1 -C 6 Alkyl and -C(=O)N(R 9 ) 2 The group consisting of R 9 Independently selected from 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 together form a 5- or 6-membered heterocyclyl ring;

[0318] y is an integer from 2 to 12;

[0319] z is 0 or 1;

[0320] Q is selected from the group consisting of: N-hydroxysuccinimide, N-hydroxysulfosuccinimide, maleimide, and one or more independently selected from F, Cl, NO 2 and SO 3 - A phenoxy group substituted with a group; and

[0321] Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl and heteroaryldiyl are independently and optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, -CN, -CH 3 、-CH 2 CH 3 、-CH=CH 2 、-C≡CH、-C≡CCH 3 、-CH 2 CH 2 CH 3 、-CH(CH 3 ) 2 、-CH 2 CH(CH 3 ) 2 、-CH 2 OH, -CH 2 OCH 3 、-CH 2 CH 2 OH, -C(CH 3 ) 2 OH, -CH(OH)CH(CH 3 ) 2 、-C(CH 3 ) 2 CH 2 OH, -CH 2 CH 2 SO 2 CH 3 、-CH 2 OP(O)(OH) 2 、-CH 2 F, -CHF 2 , -CF 3 、-CH 2 CF 3 、-CH 2 CHF 2 、-CH(CH 3 )CN、-C(CH 3) 2 CN、-CH 2 CN、-CH 2 NH 2 、-CH 2 NHSO 2 CH 3 、-CH 2 NHCH 3 、-CH 2 N(CH 3 ) 2 、-CO 2 H、-COCH 3 、-CO 2 CH 3 、-CO 2 C(CH 3 ) 3 、-COCH(OH)CH 3 、-CONH 2 、-CONHCH 3 、-CON(CH 3 ) 2 、-C(CH 3 ) 2 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 、-OCH2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 、-O(CH 2 CH 2 O) n -(CH 2 ) m CO 2 H、-O(CH 2 CH 2 O) n H、-OCH 2 F, -OCHF 2 、-OCF 3 、-OP(O)(OH) 2 、-S(O) 2 N(CH 3 ) 2 、-SCH 3 、-S(O) 2 CH 3 and -S(O) 3 H.

[0322] 8-Het-2-aminobenzazepine of formula II - Exemplary embodiments of linker compounds include those wherein Q is selected from:

[0323]

[0324] 8-Het-2-aminobenzazepine of formula II Exemplary embodiments of the -linker compounds include those wherein Q is phenoxy substituted with one or more Fs.

[0325] 8-Het-2-aminobenzazepine of formula II Exemplary embodiments of the -linker compound include where Q is 2,3,5,6-tetrafluorophenoxy.

[0326] 8-Het-2-aminobenzazepine -Exemplary embodiments of linker (HxBzL) compounds are selected from Tables 2a and 2b. Each compound was synthesized, purified, and characterized by mass spectrometry and shown to have the indicated mass. Additional experimental procedures are found in the Examples. 8-Het-2-aminobenzazepines of Tables 2a and 2b -Linker compounds demonstrate unexpected and surprising properties of TLR8 agonist selectivity, which may predict useful therapeutic activity in the treatment of cancer and other disorders. 8-Het-2-aminobenzazepines of Table 2 -Linker intermediates The compound of formula II was used to conjugate with antibodies by the method of Example 201 to form the immunoconjugates of Tables 3a and 3b.

[0327] Table 2a: 8-Het-2-aminobenzazepines -Linker intermediate formula II compound (HxBzL)

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346] Table 2b: 8-Het-2-aminobenzazepines -Linker intermediate formula II compound (HxBzL)

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354] CEA immunoconjugate

[0355] Immunostimulatory antibody conjugates (i.e., immunoconjugates) direct TLR7 / 8 agonists into tumors to activate tumor-infiltrating myeloid cells and initiate broad innate and adaptive anti-tumor immune responses (Ackerman et al., (2021) Nature Cancer 2:18-33).

[0356] CEA (CEACAM5) is a well-validated cell surface antigen that is highly expressed in a variety of solid tumors. The favorable properties of CEA include robust cell surface expression, low internalization rate, and limited normal tissue expression, suggesting that this antigen may be a suitable target for immunoconjugates in a versatile approach to treating CEA-expressing cancers.

[0357] An exemplary embodiment of the immunoconjugate comprises a molecule covalently linked to one or more 8-Het-2-aminobenzazepines via a linker. (Hx) portion of an anti-CEA antibody having Formula I:

[0358] Ab-[L-Hx] p I

[0359] or a pharmaceutically acceptable salt thereof,

[0360] in:

[0361] Ab is an antibody construct with an antigen binding domain that binds CEA;

[0362] p is an integer from 1 to 8;

[0363] Hx is 8-Het-2-aminobenzazepine having the formula part:

[0364]

[0365] Het is selected from heterocyclyldiyl and heteroaryldiyl;

[0366] R 1 , R 2 , R3 and R 4 Independently selected from H, C 1 -C 12 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 12 Carbocyclic group, C 6 -C 20 Aryl, C 2 -C 9 Heterocyclic and C 1 -C 20 The group consisting of heteroaryl, wherein alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl are independently and optionally substituted by one or more groups selected from the following:

[0367] -(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0368] -(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0369] -(C 1 -C 12 Alkyldiyl)-OR 5 ;

[0370] -(C 3 -C 12 Carbocyclic group);

[0371] -(C 3 -C 12 Carbocyclyl)-*;

[0372] -(C 3 -C 12 Carbocyclic)-(C 1 -C 12 Alkyldiyl)-NR 5 -*;

[0373] -(C 3 -C 12 Carbocyclic)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0374] -(C 3 -C 12Carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*;

[0375] -(C 6 -C 20 Aryl);

[0376] -(C 6 -C 20 Aryldiyl)-*;

[0377] -(C 6 -C 20 Aryldiyl)-N(R 5 )-*;

[0378] -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0379] -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-*;

[0380] -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0381] -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-NR 5 -C(=NR 5a )N(R 5 )-*;

[0382] -(C 2 -C 20 heterocyclic group);

[0383] -(C 2 -C 20 Heterocyclyl)-*;

[0384] -(C 2 -C 9 Heterocyclyl)-(C1 -C 12 Alkyldiyl)-NR 5 -*;

[0385] -(C 2 -C 9 Heterocyclyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0386] -(C 2 -C 9 heterocyclyl)-C(=O)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0387] -(C 2 -C 9 Heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*;

[0388] -(C 2 -C 9 Heterocyclyl)-NR 5 -(C 6 -C 20 Aryldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0389] -(C 2 -C 9 Heterocyclyl)-(C 6 -C 20 Aryldiyl)-*;

[0390] -(C 1 -C 20 heteroaryl);

[0391] -(C 1 -C 20 Heteroaryl)-*;

[0392] -(C 1 -C 20 Heteroaryl)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0393] -(C 1 -C 20 Heteroaryl)-(C1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0394] -(C 1 -C 20 Heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*;

[0395] -(C 1 -C 20 Heteroaryl)-N(R 5 )C(=O)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0396] -C(=O)-*;

[0397] -C(=O)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0398] -C(=O)-(C 2 -C 20 Heterocyclyldiyl)-*;

[0399] -C(=O)N(R 5 ) 2 ;

[0400] -C(=O)N(R 5 )-*;

[0401] -C(=O)N(R 5 )-(C 1 -C 12 Alkyldiyl)-N(R 5 )C(=O)R 5 ;

[0402] -C(=O)N(R 5 )-(C 1 -C 12 Alkyldiyl)-N(R 5 )C(=O)N(R 5 ) 2 ;

[0403] -C(=O)NR 5 -(C 1 -C 12 Alkyldiyl)-N(R 5 )CO2 R 5 ;

[0404] -C(=O)NR 5 -(C 1 -C 12 Alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 ) 2 ;

[0405] -C(=O)NR 5 -(C 1 -C 12 Alkyldiyl)-NR 5 C(=NR 5a )R 5 ;

[0406] -C(=O)NR 5 -(C 1 -C 8 Alkyldiyl)-NR 5 (C 2 -C 5 heteroaryl);

[0407] -C(=O)NR 5 -(C 1 -C 20 Heteroaryldiyl)-N(R 5 )-*;

[0408] -C(=O)NR 5 -(C 1 -C 20 Heteroaryldiyl)-*;

[0409] -C(=O)NR 5 -(C 1 -C 20 Heteroaryldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0410] -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 -*;

[0411] -N(R 5 ) 2 ;

[0412] -N(R 5 )-*;

[0413] -N(R 5 )C(=O)R 5 ;

[0414] -N(R 5 )C(=O)-*;

[0415] -N(R 5 )C(=O)N(R 5 ) 2 ;

[0416] -N(R 5 )C(=O)N(R 5 )-*;

[0417] -N(R 5 )CO 2 R 5 ;

[0418] -NR 5 C(=NR 5a )N(R 5 ) 2 ;

[0419] -NR 5 C(=NR 5a )N(R 5 )-*;

[0420] -NR 5 C(=NR 5a )R 5 ;

[0421] -N(R 5 )C(=O)-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0422] -N(R 5 )-(C 2 -C 5 heteroaryl);

[0423] -N(R 5 )-S(=O) 2 -(C 1 -C 12 alkyl);

[0424] -O-(C1 -C 12 alkyl);

[0425] -O-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0426] -O-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*;

[0427] -OC(=O)N(R 5 ) 2 ;

[0428] -OC(=O)N(R 5 )-*;

[0429] -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-*;

[0430] -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 5 ) 2 ;

[0431] -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-(C 1 -C 12 Alkyldiyl)-NR 5 -*;and

[0432] -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-(C 1 -C 12 Alkyldiyl)-OH;

[0433] or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring;

[0434] X 1 , X 2 , X 3 and X 4are independently selected from a free bond, C(=O), C(=O)N(R 5 )、O、N(R 5 )、S、S(O) 2 and S(O) 2 N(R 5 )

[0435] R 5 Independently selected from H, C 6 -C 20 Aryl, C 3 -C 12 Carbocyclic group, C 6 -C 20 Aryldiyl, C 1 -C 12 Alkyl and C 1 -C 12 A group consisting of an alkyl diyl group, or two R 5 The groups together form a 5- or 6-membered heterocyclyl ring;

[0436] R 5a Choose from C 6 -C 20 Aryl and C 1 -C 20 A group consisting of heteroaryl groups;

[0437] wherein the asterisk * indicates the attachment site of L, and wherein R 1 , R 2 , R 3 and R 4 One of them is connected to L;

[0438] L is a linker selected from the group consisting of:

[0439] -C(=O)-PEG-;

[0440] -C(=O)-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-Gluc-;

[0441] -C(=O)-PEG-O-;

[0442] -C(=O)-PEG-OC(=O)-;

[0443] -C(=O)-PEG-C(=O)-;

[0444] -C(=O)-PEG-C(=O)-PEP-;

[0445] -C(=O)-PEG-N(R6 )-;

[0446] -C(=O)-PEG-N(R 6 )-C(=O)-;

[0447] -C(=O)-PEG-N(R 6 )-PEG-C(=O)-PEP-;

[0448] -C(=O)-PEG-N + (R 6 ) 2 -PEG-C(=O)-PEP-;

[0449] -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-;

[0450] -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 Monoheterocyclyldiyl)-;

[0451] -C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-;

[0452] -C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-C(═O)-;

[0453] -C(=O)-(C 1 -C 12 alkyldiyl)-C(═O)-PEP-;

[0454] -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-;

[0455] -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C12 Alkyldiyl)-N(R 5 )-C(=O);

[0456] -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)-;

[0457] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-;

[0458] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-Gluc-;

[0459] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-;

[0460] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-OC(=O)-;

[0461] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-;

[0462] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-;

[0463] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R5 )-C(=O)-;

[0464] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-;

[0465] -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-;

[0466] -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-;

[0467] -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-; and

[0468] -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 Monoheterocyclyldiyl)-;

[0469] R 6 independently H or C 1 -C 6 alkyl;

[0470] PEG has the formula: -(CH 2 CH 2 O) n -(CH 2 ) m -; m is an integer from 1 to 5, and n is an integer from 2 to 50;

[0471] Gluc has the following formula:

[0472]

[0473] PEP has the following formula:

[0474]

[0475] wherein AA is independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a five-membered ring of proline amino acids, and the wavy line indicates the point of attachment;

[0476] Cyc is selected from C 6 -C 20 Aryldiyl and C 1 -C 20 Heteroaryldiyl, optionally substituted with one or more selected from F, Cl, NO 2 、-OH、-OCH 3 and a glucuronic acid radical having the following structure:

[0477]

[0478] R 7 Selected from -CH(R 8 )O-、-CH 2 -、-CH 2 N(R 8 )- and -CH(R 8 )OC(=O)-, wherein R 8 Selected from H, C 1 -C 6 Alkyl, C(=O)-C 1 -C 6 Alkyl and -C(=O)N(R 9 ) 2 , where R 9 Independently selected from 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 together form a 5- or 6-membered heterocyclyl ring;

[0479] y is an integer from 2 to 12;

[0480] z is 0 or 1; and

[0481] Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl and heteroaryldiyl are independently and optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, -CN, -CH 3 、-CH 2 CH 3 、-CH=CH 2 、-C≡CH、-C≡CCH 3 、-CH 2 CH 2 CH 3 、-CH(CH 3 ) 2 、-CH 2 CH(CH 3 ) 2 、-CH 2 OH, -CH 2 OCH 3 、-CH 2 CH 2 OH, -C(CH 3 ) 2 OH, -CH(OH)CH(CH 3 ) 2 、-C(CH 3 ) 2 CH 2 OH, -CH 2 CH 2 SO 2 CH 3 、-CH 2 OP(O)(OH) 2 、-CH 2 F, -CHF 2 , -CF 3 、-CH 2 CF 3 、-CH 2 CHF 2 、-CH(CH 3 )CN、-C(CH 3 ) 2 CN、-CH 2 CN、-CH 2 NH 2 、-CH 2 NHSO 2 CH 3 、-CH 2 NHCH 3 、-CH 2 N(CH 3 ) 2 , -CO2 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 CO 2 H、-O(CH 2 CH2 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.

[0482] Exemplary embodiments of the immunoconjugate of Formula I include wherein the antibody is selected from labetuzumab and acitumomab, or a biosimilar or biobetter thereof.

[0483] Exemplary embodiments of the immunoconjugates of Formula I include wherein the antibody construct comprises:

[0484] a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15;

[0485] b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30;

[0486] c) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:35, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:37, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:48;

[0487] d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:53, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:55, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:48;

[0488] e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 59, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 63, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71;

[0489] f) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 75, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 79, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 83, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 85, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87;

[0490] g) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:91, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:93, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:95, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:99, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:101, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:103;

[0491] h) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 115, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120; or

[0492] i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 124, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 126, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 128.

[0493] Exemplary embodiments of the immunoconjugate of Formula I include wherein the antibody construct comprises a variable light chain and a variable heavy chain, wherein the variable light chain comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89 and 105; and wherein the variable heavy chain comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122 and 130.

[0494] Exemplary embodiments of the immunoconjugate of Formula I include wherein the antibody construct comprises a variable light chain and a variable heavy chain, wherein the variable light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89 and 105; and wherein the variable heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122 and 130.

[0495] Exemplary embodiments of the immunoconjugate of Formula I include 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.

[0496] Exemplary embodiments of the immunoconjugate of Formula I include 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.

[0497] Exemplary embodiments of the immunoconjugate of Formula I include those wherein Het is selected from the group consisting of pyridinyldiyl, pyrimidinyldiyl, pyrazolyldiyl, piperazinyldiyl, piperidinyldiyl, and pyrazinyldiyl.

[0498] Exemplary embodiments of the immunoconjugates of Formula I include those wherein X 1 is a key, and R 1 It's H.

[0499] Exemplary embodiments of the immunoconjugates of Formula I include those wherein X 2 is a key, and R 2 It is C 1 -C 8 alkyl.

[0500] Exemplary embodiments of the immunoconjugates of Formula I include those wherein X 2 and X 3 Each is a bond, and R 2 and R 3 Independently selected from 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 .

[0501] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 2 It is C 1 -C 8 Alkyl, and R 3 Yes-(C 1 -C 8 Alkyldiyl)-N(R 5 )CO 2 R 5 .

[0502] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 2 Yes-CH 2 CH 2 CH 3 , and R 3 Selected from -CH 2 CH 2 CH2 NHCO 2 (t-Bu), -OCH 2 CH 2 NHCO 2 (cyclobutyl) and -CH 2 CH 2 CH 2 NHCO 2 (cyclobutyl).

[0503] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 2 and R 3 Each independently selected from -CH 2 CH 2 CH 3 、-OCH 2 CH 3 、-OCH 2 CF 3 、-CH 2 CH 2 CF 3 、-OCH 2 CH 2 OH and -CH 2 CH 2 CH 2 OH.

[0504] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 2 and R 3 Each is -CH 2 CH 2 CH 3 .

[0505] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 2 Yes-CH 2 CH 2 CH 3 , and R 3 Yes-OCH 2 CH 3 .

[0506] Exemplary embodiments of the immunoconjugates of Formula I include those wherein X 3 -R 3 Selected from the group consisting of:

[0507]

[0508] Exemplary embodiments of the immunoconjugates of Formula I include those wherein X 4 is a key, and R 4 It's H.

[0509] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 1 Connect to L.

[0510] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 2 or R 3 Connect to L.

[0511] Exemplary embodiments of the immunoconjugates of Formula I include those wherein X 3 -R 3 -L Select from the group consisting of:

[0512]

[0513] The wavy line indicates the connection point with N.

[0514] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 4 It is C 1 -C 12 alkyl.

[0515] Exemplary embodiments of the immunoconjugates of Formula I include those wherein R 4 Yes-(C 1 -C 12 Alkyldiyl)-N(R 5 )-*; wherein the asterisk * indicates the attachment site of L.

[0516] Exemplary embodiments of the immunoconjugate of Formula I include those wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0517] Exemplary embodiments of the immunoconjugates of Formula I include those wherein L is attached to a cysteine ​​thiol of the antibody.

[0518] Exemplary embodiments of the immunoconjugates of Formula I include those wherein, for PEG, m is 1 or 2, and n is an integer from 2 to 10.

[0519] Exemplary embodiments of the immunoconjugates of Formula I include those wherein L comprises PEP, and PEP is a dipeptide and has the formula:

[0520]

[0521] Exemplary embodiments of the immunoconjugates of Formula I include those wherein AA 1 and AA 2 are independently selected from H, -CH 3 、-CH(CH 3 ) 2 、-CH 2 (C 6 H 5),-CH 2 CH 2 CH 2 CH 2 NH 2 、-CH 2 CH 2 CH 2 NHC(NH)NH 2 、-CHCH(CH 3 )CH 3 、-CH 2 SO 3 H and -CH 2 CH 2 CH 2 NHC(O)NH 2 ; or AA 1 and AA 2 A 5-membered ring of proline amino acid is formed.

[0522] Exemplary embodiments of the immunoconjugates of Formula I include those wherein AA 1 It is -CH(CH 3 ) 2 , and AA 2 Yes-CH 2 CH 2 CH 2 NHC(O)NH 2 .

[0523] Exemplary embodiments of the immunoconjugates of Formula I include those wherein AA 1 and AA 2 are independently selected from GlcNAc, aspartic acid, -CH 2 SO 3 H and -CH 2 OPO 3 H.

[0524] Exemplary embodiments of the immunoconjugates of Formula I include those wherein the PEP has the formula:

[0525]

[0526] Among them, AA 1 and AA 2 Independently selected from the side chains of naturally occurring amino acids.

[0527] Exemplary embodiments of the immunoconjugates of Formula I include those wherein L comprises PEP, and PEP is a tripeptide and has the formula:

[0528]

[0529] Exemplary embodiments of the immunoconjugates of Formula I include those wherein L comprises PEP, and PEP is a tetrapeptide and has the formula:

[0530]

[0531] Exemplary embodiments of the immunoconjugates of Formula I include those wherein:

[0532] AA 1 Selected from the group consisting of Abu, Ala and Val;

[0533] AA 2 Selected from the group consisting of Nle(O-Bzl), Oic and Pro;

[0534] AA 3 Selected from Ala and Met(O) 2 a group consisting of; and

[0535] AA 4 Selected from Oic, Arg(NO 2 ), Bpa and Nle(O-Bzl).

[0536] Exemplary embodiments of the immunoconjugate of Formula I include those wherein L comprises PEP, and the PEP is selected from the group consisting of 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).

[0537] Exemplary embodiments of the immunoconjugates of Formula I include those wherein L comprises PEP, and PEP is selected from the following structures:

[0538]

[0539] Exemplary embodiments of the immunoconjugates of Formula I include those wherein L is selected from the following structures:

[0540]

[0541] The wavy line indicates the 5 connection.

[0542] Exemplary embodiments of immunoconjugates of Formula I having Formula Ia:

[0543]

[0544] Exemplary embodiments of the immunoconjugates of Formula Ia include those wherein X4 is a key, and R 4 It's H.

[0545] Exemplary embodiments of the immunoconjugates of Formula Ia include those wherein X 2 and X 3 Each is a bond, and R 2 and R 3 Independently selected from 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 .

[0546] Exemplary embodiments of the immunoconjugates of Formula Ia selected from Formulas Ib-Ii:

[0547]

[0548]

[0549] Exemplary embodiments of the immunoconjugates of Formula Ia include those wherein X 2 and X 3 Each is a bond, and R 2 and R 3 Independently selected from C 1 -C 8 Alkyl, -O-(C 1 -C 12 Alkyl), -(C 1 -C 12 Alkyldiyl)-OR 5 、-(C 1 -C 8 Alkyldiyl)-N(R5 )CO 2 R 5 and -O-(C 1 -C 12 Alkyl)-N(R 5 )CO 2 R 5 .

[0550] Exemplary embodiments of the immunoconjugates of Formula Ia include those wherein X 2 and X 3 Each is a key, R 2 It is C 1 -C 8 Alkyl, and R 3 Selected from -O-(C 1 -C 12 Alkyl) and -O-(C 1 -C 12 Alkyl)-N(R 5 )CO 2 R 5 .

[0551] The present invention includes all reasonable combinations and permutations of features of the formula I embodiments.

[0552] In certain embodiments, the immunoconjugate compounds of the present invention include those having immunostimulatory activity. The antibody-drug conjugates of the present invention selectively deliver an effective dose of 8-Het-2-aminobenzazepine to tumor tissue. drug, thereby achieving greater selectivity (i.e., lower effective doses) while maintaining a relatively low level of selectivity relative to unconjugated 8-Het-2-aminobenzazepine Added therapeutic index ("therapeutic window").

[0553] Drug loading is represented by p, i.e. the number of HxBz parts of each antibody in the immunoconjugate of formula I. The range of drug loading (HxBz) amount can be 1 to about 8 drug moieties (D) per antibody. The immunoconjugate of formula I includes a mixture or collection of antibodies conjugated with a range of 1 to about 8 drug moieties. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues such as lysine and cysteine. In some embodiments, free cysteine ​​residues are introduced into the antibody amino acid sequence by the methods described herein. In these aspects, p can be 1, 2, 3, 4, 5, 6, 7 or 8 and ranges thereof, such as 1 to 8 or 2 to 5. In any such aspect, p and n are equal (i.e., p=n=1, 2, 3, 4, 5, 6, 7 or 8, or a 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 intrachain disulfide bonds without the need for engineering, in which case the free cysteine ​​residues present can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to conjugation of the antibody to produce one or more free cysteine ​​residues.

[0554] For some immunoconjugates, p may be limited to the number of attachment sites on the antibody. For example, in the case where the connection 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 may have only one or a limited number of sufficiently reactive thiol groups that can be connected to the drug. In other embodiments, one or more lysine amino groups in the antibody may be available and reactive for conjugation with the Hx-linker compound of formula II. In certain embodiments, a higher drug loading (e.g., p>5) may result in the loss of aggregation, insolubility, toxicity, or cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading range of the immunoconjugate is 1 to about 8; about 2 to about 6; or about 3 to about 5. In certain embodiments, the antibody is subjected to denaturation conditions to display reactive nucleophilic groups, such as lysine or cysteine.

[0555] The loading (drug / antibody ratio) of the immunoconjugate can be controlled in different ways, for example by: (i) limiting the molar excess of the Hx-linker intermediate compound relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing denaturation conditions to optimize antibody reactivity.

[0556] It is understood that in the case where more than one nucleophilic group of an antibody reacts with a drug, the resulting product is a mixture of immunoconjugate compounds distributed with one or more drug moieties attached to the antibody. The average amount of drug per antibody can be calculated from the mixture by a dual ELISA antibody assay, which is specific for the antibody and specific for the drug. Individual immunoconjugate molecules in a mixture can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, e.g., McDonagh et al., 2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al., (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, KJ et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004; Alley, SC et al., "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004). In certain embodiments, a homogeneous immunoconjugate having a single loading value can be separated from a conjugated mixture by electrophoresis or chromatography.

[0557] Exemplary embodiments of the immunoconjugates of Formula I are selected from the anti-CEA HxBz immunoconjugates of Tables 3a and 3b. Evaluation of the in vitro activity of the immunoconjugates was performed according to the method of Example 203.

[0558] Table 3a Anti-CEA HxBz immunoconjugate (IC)

[0559]

[0560]

[0561] Table 3b Anti-CEA HxBz immunoconjugate (IC)

[0562]

[0563] Compositions of immunoconjugates

[0564] The present invention provides compositions, such as pharmaceutically or pharmacologically acceptable compositions or formulations, comprising a plurality of immunoconjugates as described herein and optionally a carrier therefor, such as a pharmaceutically or pharmacologically acceptable carrier. The immunoconjugates in the composition may be the same or different, i.e., the composition may comprise immunoconjugates having the same number of adjuvants attached to the same position on the antibody construct and / or immunoconjugates having the same number of Hx adjuvants attached to different positions on the antibody construct, immunoconjugates having different numbers of adjuvants attached to the same position on the antibody construct, or immunoconjugates having different numbers of adjuvants attached to different positions on the antibody construct.

[0565] In an exemplary embodiment, the composition comprising an immunoconjugate compound comprises a mixture of immunoconjugate compounds, wherein the average drug loading (Hx) per antibody in the mixture of immunoconjugate compounds is from about 2 to about 5.

[0566] Compositions of the immunoconjugates of the invention may have an average adjuvant to antibody construct ratio (DAR) of about 0.4 to about 10. The skilled artisan will recognize that in compositions comprising a variety of immunoconjugates of the invention, the 8-Het-2-aminobenzazepine conjugated to the antibody construct The number of adjuvants may vary depending on the immunoconjugate, so the adjuvant to antibody construct (e.g., antibody) ratio can be measured as an average value, which can be referred to as the drug (adjuvant) to antibody ratio (DAR). The adjuvant to antibody construct (e.g., antibody) ratio can be assessed by any suitable method, many of which are known in the art.

[0567] The average number of adjuvant fractions per antibody (DAR) in the preparation of the immunoconjugate from the conjugation reaction can be characterized by conventional methods, such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the immunoconjugate in the composition, represented by p, can also be determined. In some cases, separation, purification, and characterization of homogeneous immunoconjugates can be achieved by methods such as reverse phase HPLC or electrophoresis, where p is a certain value from immunoconjugates with other drug loadings.

[0568] In some embodiments, the composition further comprises one or more pharmaceutically or pharmacologically acceptable excipients. For example, the immunoconjugates of the present invention can be formulated for parenteral administration, such as IV administration or administration into the lumen of a body cavity or organ. Alternatively, the immunoconjugates can be injected intratumorally. The composition for injection will generally comprise a solution of the immunoconjugate dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be used are isotonic solutions of water and one or more salts such as sodium chloride, such as Ringer's solution. In addition, sterile fixed oils can be routinely used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used to prepare injections. These compositions are ideally sterile and generally free of undesirable substances. These compositions can be sterilized by conventional well-known sterilization techniques. The composition can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH adjustment and buffering agents, toxicity regulators, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0569] The composition may contain any suitable concentration of the immunoconjugate. The concentration of the immunoconjugate in the composition may vary widely and will be selected based primarily on fluid volume, viscosity, body weight, etc., depending on the specific mode of administration selected and the needs of the patient. In certain embodiments, the concentration of the immunoconjugate in the solution formulation for injection will range from about 0.1% (w / w) to about 10% (w / w).

[0570] Biological activity of immunoconjugates

[0571] Immunoconjugate IC-2 binds differentially to surface expressed CEA on a panel of cell lines and correlates with CEA transcript levels as shown in the table below.

[0572]

[0573] 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 was calculated as (percentage of cells with a staining intensity of 1+) + (percentage of cells with a staining intensity of 2+ at 2X) + (percentage of cells with a staining intensity of 3+ at 3X). The IC-2 binding sites per cell represent the number of IC-2 molecules that a given tumor cell will bind to and are associated with the level of antigen expression on the cell surface. Live tumor cells were harvested and labeled with Alexa Flour 488-labeled IC-2 or Alexa Flour 488-labeled hIgG1 isotype control at 100 nM, followed by flow cytometry analysis. IC-2 binding sites were determined using QSC beads from Bangs Laboratories. Nonspecific binding sites were corrected by subtracting the hIgG1 isotype control binding sites from the IC-2 binding sites.

[0574] Figure 1 A graphic representation of an in vivo xenograft tumor model in mice is shown. Tumor volume over time after treatment was measured to compare the efficacy of immunoconjugate IC-2 with isotype immunoconjugates (ISACs) and naked antibody CEA.9-G1fhL2 in tumor inhibition in mice bearing CEA-high human pancreatic HPAF-II tumors. Immunoconjugate IC-2 demonstrated dose-dependent growth inhibition of CEA-high human pancreatic HPAF-II tumors at dose levels as low as 0.5 mg / kg. Isotype ISACs are anti-CD20 antibodies (rituximab) conjugated to HxBzL-5, and the adjuvant-linker is the same as IC-2. Isotype ISACs have the same adjuvant linker (HxBzL-5) as IC-2. Isotype ISACs served as off-target negative controls, showing little or no tumor growth inhibition. Naked antibody CEA.9-G1fhL2 also showed 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 immunoconjugates of the invention are effective in treating CEA-expressing cancers.

[0575] Figure 2a -e shows a graphic representation of the induction of various cytokines by immunoconjugates IC-2, IC-3, IC-4, IC-6, IC-14 and naked antibody CEA.9-G1fhL2 in co-culture of CEA-high gastric cancer MKN-45 cells with cDC-enriched primary cell isolates. Cellular induction of the cytokine 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 ) levels secreted in the supernatant of cells cultured with CEA. The induction of these cytokines is associated with enhanced immune responses to cancer. Various concentrations of immunoconjugates IC-2, IC-3, IC-4, IC-6, IC-14 and naked antibody CEA.9-G1fhL2 were incubated with CEA-high MKN-45 cells and cDC-enriched primary cell preparations (E:T=10:1) for 18 hours, and the supernatant was then recovered. The levels of secreted cytokines were determined using the LegendPlexcytokine Bead Array Kit. The immunoconjugates tested differed in the levels of cytokines induced as a function of adjuvants. The natural CEA.9-G1fhL2 antibody induced little or no cytokine secretion, indicating dependence on TLR7 / 8 activating adjuvants.

[0576] Figure 3a -d shows HPAF II cells with high CEA ( Figure 3a ), CEA-medium LoVo cells ( Figure 3b ), LS-174T cells with low CEA ( Figure 3c ) and CEA-negative MDA-MB-231 cells ( Figure 3d ) Graph of phagocytosis of M-CSF differentiated monocyte-derived macrophages treated with various concentrations of immunoconjugate IC-2. CTG-labeled tumor-IC-2 immune complexes were incubated with M-CSF differentiated monocyte-derived macrophages at an effector to target ratio of 2:1. After 4 hours, phagocytosis was measured by flow cytometry gating effector cells that were positive for CTG signal. Mean values ​​+ / - standard deviations from three donors are 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 leading to internalization and degradation of target cells. Immunoconjugate IC-2 induced dose-dependent phagocytosis of HPAF II with high CEA (EC50 = 9.2 ± 2.3 nM) and LoVo with medium CEA (EC50 = 11.4 ± 3.5 nM). Minimal ADCP was observed for LS-174T with low CEA. IC-2 did not induce ADCP in CEA-negative MDA-MB-231. These results suggest that the induction of ADCP by IC-2 is dependent on moderate / high CEA expression in target tumor cells.

[0577] Figure 4a-f shows a graphical representation of the levels of secreted cytokines and induction of cell surface markers in the supernatant after incubation of different concentrations of immunoconjugate IC-2 and naked antibody CEA.9-G1fhL2 with co-cultures of cancer cells and cDC-enriched primary cell isolates. Immunoconjugate IC-2 and naked antibody CEA.9-G1fhL2 were incubated with CEA-positive tumor cells (HPAF-II, LoVo or LS174-T) and cDC-enriched primary cell preparations (E:T=10:1) for 18 hours, and then the supernatant and cells were recovered. The levels of secreted cytokines in the supernatant were determined using the LegendPlex Cytokine Bead Array Kit ( Figure 4a -d). Induction of cell surface markers was determined by flow cytometry ( Figure 4e -f). In co-cultures of cancer cells with cDC-enriched primary cell isolates, the CEA-targeted immunoconjugate IC-2 induced the cytokine TNFα, which is associated with enhanced immune responses to cancer ( Figure 4a )、IL-6( Figure 4b )、IL-12p70( Figure 4c ) and CXCL10( Figure 4d ) secretion. In addition, CD40 ( Figure 4e ) and CD86( Figure 4f ) antigens, consistent with activation of innate immunity (myeloid cells). Cytokine and surface marker induction levels were similar for HPAF-II with high CEA and LoVo cells with medium CEA, but significantly reduced for LS-174T cells with low CEA. Cytokine and surface marker studies demonstrated that myeloid cells were activated when exposed to tumor cells expressing CEA and the anti-CEA ISAC IC-2. Activation was observed with HPAF-II cells with high CEA and LoVo cells with medium CEA. Activation was low or undetectable with LS-174T cells with low CEA and CEA-negative MDA-MB-231 cells. The natural antibody CEA.9-G1fhL2 did not induce myeloid activation. Figure 4a -f results demonstrate the dependence of IC-2 activity on CEA expression levels associated with human cancers. Native CEA.9-G1fhL2 antibody induced little or no cytokine secretion, indicating a dependency on TLR7 / 8 activating payload.

[0578] Methods of treating cancer using immunoconjugates

[0579] The present invention provides a method for treating cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate as described herein (e.g., as a composition as described herein) to a subject in need thereof, such as a subject suffering from cancer and in need of treatment for cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate (IC) selected from Tables 3a and 3b.

[0580] It is expected that the immunoconjugates of the invention can be used to treat various hyperproliferative diseases or disorders, such as those characterized by overexpression of tumor antigens. Exemplary hyperproliferative disorders include benign or malignant solid tumors and hematological disorders, such as leukemias and lymphoid malignancies.

[0581] In another aspect, an immunoconjugate for use as a medicament is provided. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual, the method comprising administering an effective amount of the immunoconjugate to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, e.g., as described herein.

[0582] In a further aspect, the invention provides for the use of an immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat cancer, and the method comprises administering an effective amount of the medicament to an individual suffering from cancer. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, e.g., as described herein.

[0583] Cancer is a malignant tumor originating from epithelial tissue. Epithelial cells cover the outer surface of the body, line the lumen, and form the lining of glandular tissue. Examples of cancer include, but are not limited to, adenocarcinoma (cancer originating from glandular (secretory) cells, such as breast cancer, pancreatic cancer, lung cancer, prostate cancer, gastric cancer, gastroesophageal junction cancer, and colon cancer) adrenocortical carcinoma; hepatocellular carcinoma; renal cell carcinoma; ovarian cancer; carcinoma in situ; ductal carcinoma; breast cancer; basal cell carcinoma; squamous cell carcinoma; transitional cell carcinoma; colon cancer; nasopharyngeal carcinoma; multilocular cystic renal cell carcinoma; oat cell carcinoma; large cell lung cancer; small cell lung cancer; non-small cell lung cancer; etc. Cancer can be found in the prostate, pancreas, colon, brain (usually as secondary metastasis), lung, breast, and skin. In some embodiments, the method for treating non-small cell lung cancer includes administering an immunoconjugate containing an antibody construct capable of binding CEA (e.g., labetuzumab or its biosimilars or its bioimprovement medicine).

[0584] Soft tissue tumors are a highly diverse group of rare tumors that arise from connective tissue. Examples of soft tissue tumors include, but are not limited to, alveolar soft tissue sarcoma; angiomatoid fibrous histiocytoma; chondromyxoid fibroma; chondrosarcoma of the skeleton; extraosseous myxoid chondrosarcoma; clear cell sarcoma; desmoplastic small round cell tumor; dermatofibrosarcoma protuberans; endometrial stromal tumor; Ewing's sarcoma; fibromatosis (desmoid tumor); infantile fibrosarcoma; gastrointestinal stromal tumor; giant cell tumor of bone; giant cell tumor of the tendon sheath; inflammatory myofibroblastic tumor; uterine fibroids; leiomyosarcoma; lipoblastoma; typical lipoma Lipomas; spindle cell or pleomorphic lipomas; atypical lipomas; chondroid lipomas; 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; embryonal rhabdomyosarcoma; benign or malignant nerve sheath tumor; synovial sarcoma; Evan's tumor tumor); nodular fasciitis; desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcoma protuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma; tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis (PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovial sarcoma; malignant peripheral nerve sheath tumor; neurofibroma; pleomorphic adenoma of soft tissue; and neoplasia arising from fibroblasts, myofibroblasts, histiocytes, vascular cells / endothelial cells, and nerve sheath cells.

[0585] Sarcomas are rare types of cancer that originate from 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 tissues. Different types of sarcomas are based on where the cancer forms. For example, osteosarcomas form in bones, liposarcoma forms in fat, and rhabdomyosarcomas form in muscle. Examples of sarcomas include, but are not limited to, Askin tumor; botryoid sarcoma; chondrosarcoma; Ewing's sarcoma; malignant hemangioendothelioma; malignant schwannoma; osteosarcoma; and soft tissue sarcomas (e.g., alveolar soft tissue sarcoma; angiosarcoma; cystosarcoma phyllodes; dermatofibrosarcoma protuberans (DFSP); desmoid tumor; desmoplastic small round cell tumor; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); hemangiopericytoma; hemangioendothelioma (more commonly known as "angiosarcoma"); Kaposi sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma).

[0586] A teratoma is a type of germ cell tumor that can contain several different types of tissue (e.g., can include tissue derived from any and / or all three germ layers: endoderm, mesoderm, and ectoderm), including, for example, hair, muscle, and bone. Teratomas most commonly occur in the ovaries of women, the testicles of men, and the coccyx of children.

[0587] Melanoma is a form of cancer that begins in melanocytes (cells that produce the pigment melanin). Melanoma can begin in moles (skin melanoma), but can also begin in other pigmented tissues, such as the eyes or intestines.

[0588] 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 includes administering an immunoconjugate containing an antibody construct capable of binding CEA (e.g., labetuzumab, its biosimilars, or its bioimproved medicine). In some embodiments, at the time of administration, Merkel cell carcinoma has metastasized.

[0589] Leukemia is a cancer that begins in the tissue that forms blood, such as the bone marrow, and causes the production of a large number of abnormal blood cells and enters the bloodstream. For example, leukemia can originate from bone marrow-derived cells that are usually mature in the bloodstream. Leukemia is named according to how fast (for example, acute and chronic) the disease occurs and progresses and the affected white blood cell types (for example, myeloid and lymphoid). Myeloid leukemia is also referred to as myeloid or myeloblastic leukemia. Lymphoid leukemia is also referred to as lymphoblast or lymphocytic leukemia. Lymphoid leukemia cells can gather in lymph nodes, and the lymph nodes can swell. Examples of leukemia include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL).

[0590] Lymphoma is a cancer that begins in the cells of the immune system. For example, lymphoma can originate from bone marrow-derived cells that normally mature in the lymphatic system. There are two basic categories of lymphoma. One category of lymphoma is Hodgkin lymphoma (HL), which is characterized by the presence of a cell type called Reed-Sternberg cells. There are currently 6 recognized types of HL. Examples of Hodgkin lymphoma include nodular sclerosis classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte-depleted CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0591] Another category of lymphoma is non-Hodgkin lymphoma (NHL), which includes cancers of a large number of different immune system cells. Non-Hodgkin lymphoma can be further divided into cancers with an indolent (slow-growing) course and cancers with an aggressive (fast-growing) course. There are currently 61 recognized types of NHL. Examples of non-Hodgkin lymphoma include, but are not limited to, AIDS-related lymphoma, anaplastic large cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, enteropathy-type T-cell lymphoma, follicular lymphoma, hepatosplenic gamma-delta T-cell lymphoma, T-cell leukemia, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-cell lymphoma, pediatric lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, therapy-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0592] Brain cancer includes any cancer of brain tissue. Examples of brain cancer include, but are not limited to, gliomas (e.g., glioblastomas, astrocytomas, oligodendrogliomas, ependymomas, etc.), meningiomas, pituitary adenomas and vestibular schwannomas, primitive neuroectodermal tumors (medulloblastomas).

[0593] The immunoconjugates of the present invention can be used alone or in combination with other agents in therapy. For example, the immunoconjugates can be co-administered with at least one additional therapeutic agent such as a chemotherapeutic agent. Such combination therapies encompass combined administration (wherein two or more therapeutic agents are included in the same or separate preparations) and single administration, in which case the administration of the immunoconjugates can be performed before, simultaneously with, and / or after the administration of additional therapeutic agents and / or adjuvants. The immunoconjugates can also be used in combination with radiotherapy.

[0594] The immunoconjugates of the present invention (and any additional therapeutic agents) can be administered in any suitable manner, including oral, parenteral, intrapulmonary and intranasal administration, and if local treatment is required, intralesional administration is performed. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, which depends in part on whether administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations, push administrations and pulse infusions at various time points.

[0595] The immunoconjugate is administered to a subject in need in any therapeutically effective amount using any suitable dosing regimen, such as that used for labetuzumab, its biosimilars and its bioimproved drugs. For example, the method may include administering an immunoconjugate to provide a dose of about 100 ng / kg to about 50 mg / kg to the subject. The range of the immunoconjugate dose may be 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 may be about 100, 200, 300, 400 or 500 μg / kg. The immunoconjugate dose may be about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. The immunoconjugate dose may also be outside these ranges, depending on the specific conjugate and the type and severity of the cancer being treated. The frequency of administration may range from a single dose per week to multiple doses, or more frequently. In some embodiments, the immunoconjugate is administered about once a month to about five times a week. In some embodiments, the immunoconjugate is administered once a week.

[0596] In another aspect, the present invention provides a method for preventing cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate (eg, as a composition as described above) to a subject. In certain embodiments, the subject is susceptible to a cancer to be prevented.

[0597] Some embodiments of the present invention provide methods for treating cancer as described above, wherein the cancer is breast cancer. Breast cancer can originate from different regions of the breast, and many different types of breast cancer have been characterized. For example, the immunoconjugates of the present invention can be used to treat ductal carcinoma in situ; invasive ductal carcinoma (e.g., ductal carcinoma; medullary carcinoma; mucinous carcinoma; papillary carcinoma; or cribriform breast carcinoma); lobular carcinoma in situ; invasive lobular carcinoma; inflammatory breast cancer; and other forms of breast cancer, such as triple negative (negative for estrogen receptor, progesterone receptor and excessive HER2 protein test) breast cancer. In some embodiments, the method for treating breast cancer includes administering an immunoconjugate containing an antibody construct capable of binding CEA or overexpressing CEA of a tumor (e.g., labetuzumab, its biosimilars or bioimproved drugs).

[0598] In some embodiments, the cancer is sensitive to a proinflammatory response induced by TLR7 and / or TLR8.

[0599] In some embodiments, a therapeutically effective amount of an immunoconjugate is administered to a patient in need thereof to treat cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial cancer, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, or breast cancer. Merkel cell carcinoma can be metastatic Merkel cell carcinoma. Breast cancer can be triple negative breast cancer. Esophageal cancer can be gastroesophageal junction adenocarcinoma.

[0600] Example

[0601] Example L-2 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-2

[0602]

[0603] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, HxBzL-2a

[0604] To a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1 g, 5.15 mmol, 1 eq) in THF (15 mL) was added PPh 3 (1.35 g, 5.15 mmol, 1 eq) and DEAD (0.89 g, 5.15 mmol, 0.94 mL, 1 eq) and stirred at 25 ° C for 0.5 hours, then tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (3.02 g, 5.15 mmol, 1 eq) was added, and then stirred at 25 ° C for 16 hours. The reaction mixture was diluted with water 20 mL and extracted with EtOAc (50 mL * 3). The combined organic layer was washed with brine (20 mL * 3) and purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a residue.2 , petroleum ether / ethyl acetate = 50 / 1 to ethyl acetate:MeOH = 10:1) to give HxBzL-2a (3.5 g, 4.59 mmol, 89.04% yield) as a yellow oil.

[0605] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, HxBzL-2c

[0606] HxBzL-2a (625 mg, 819 umol, 2.5 equivalents), 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine -4-formamide, HxBzL-2b (120 mg, 328 umol, 1 equivalent), Na 2 CO 3 (69.5 mg, 655 umol, 2 equivalents) in water (0.3 mL) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II), Pd(dppf)Cl 2 A mixture of 2-nitropropene (23.9 mg, 32.8 umol, 0.1 eq.) in DMF (3 mL) was degassed and then heated under N 2 The mixture was heated to 120°C for 5 hours. The mixture was filtered and concentrated under reduced pressure, and the residue was purified by preparative HPLC (TFA conditions; column: Phenomenex luna C18 250*50mm*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 35%-65%, 10 minutes) to give HxBzL-2c (300 mg, 290umol, 88.4% yield, TFA) as a yellow solid.

[0607] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of ethoxy-8-yl]pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-2d

[0608] To a solution of HxBzL-2c (300 mg, 325 umol, 1 eq.) in water (3 mL) and MeCN (0.5 mL) was added HCl (12 M, 407 uL, 15 eq.), followed by stirring 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-benzazepine

[0137]

[0138]

[0139] 4-[8-yl]pyrazol-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (200 mg, 222 umol, 68.1% yield, HCl) as a colorless oil.

[0609] Preparation of HxBzL-2

[0610] To a solution of 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, 5 eq.) in DCM (1 mL) and DMA (1 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDCI (84.9 mg, 443 umol, 5 eq.), followed by stirring at 25 °C for 0.5 h. The mixture was filtered and concentrated under reduced pressure, and the residue was purified by preparative HPLC (TFA conditions; column: Phenomenex Synergi C18 150*25*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-50%, 8 minutes) to give HxBzL-2 (30 mg, 24.8umol, 28.01% yield, TFA) as a yellow oil. 1 H NMR(400MHz,MeOD)δ8.20(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); LC / MS [M+H] 1094.3 (found).

[0611] 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-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]piperazin-1-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-4

[0612]

[0613] 2-Amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-1-benzazepine Preparation of -4-formamide, HxBz-4b

[0614] 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine -4-formamide, HxBz-4a (0.5 g, 1.37 mmol, 1 eq), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1,3,2-dioxaborolane (520 mg, 2.05 mmol, 1.5 eq), Pd(dppf)Cl 2 A mixture of KOAc (99.9 mg, 137 umol, 0.1 eq.) and KOAc (335 mg, 3.41 mmol, 2.5 eq.) in dioxane (10 mL) was heated at 100 °C under N 2 The crude HxBz-4b was used in the next step without purification to give a black liquid (564 mg, 1.36 mmol, 99.96% yield).

[0615] 4-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl 1-(8-yl)pyrimidin-2-yl)piperazine-1-carboxylate, HxBz-4

[0616] HxBz-4b (0.45 g, 1.09 mmol, 1 equivalent), Pd(dppf)Cl 2 (39.8 mg, 54.4 umol, 0.05 equivalent), K 2 CO 3A mixture of tert-butyl 4-(5-bromopyrimidin-2-yl)piperazine-1-carboxylate (376 mg, 2.72 mmol, 2.5 eq.) and tert-butyl 4-(5-bromopyrimidin-2-yl)piperazine-1-carboxylate (374 mg, 1.09 mmol, 1 eq.) in dioxane (4 mL) and water (0.5 mL) was heated at 100 °C and N 2 The mixture was concentrated to remove dioxane and the residue was diluted with EtOAc (10 mL) and water (5 mL). The organic layer was washed with Na 2 SO 4 Drying and concentration gave a residue. The product was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate=1 / 0 to 0 / 1, then EA:MeOH=1.5:1) and then further purified by preparative HPLC, column: Phenomenex Synergi C18 150*25*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-55%, 8 minutes) to give HxBz-4 (0.35 g, 637umol, 58.5% yield) as a brown oil. 1 H NMR (400 MHz, MeOD) δ 8.74 (s, 2H), 7.72-7.63 (m, 2H), 7.60 (d, J = 1.6 Hz, 1H), 7.45 (s, 1H), 3.99 (q, J = 7.2 Hz, 2H), 3.93-3.88 (m, 4H), 3.77 (t, J = 7.2 Hz, 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 = 7.2 Hz, 3H), 1.02 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 550.3 (calculated); LC / MS [M+H] 550.2 (found).

[0617] 2-amino-N-ethoxy-8-(2-piperazin-1-ylpyrimidin-5-yl)-N-propyl-3H-1-benzazepine Preparation of -4-formamide, HxBz-3

[0618] To a mixture of HxBz-4 (20 mg, 36.4 umol, 1 eq.) in DCM (5 mL) was added HCl / EtOAc (4 M, 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. 1H NMR (400 MHz, MeOD) δ 8.70 (s, 2H), 7.65-7.47 (m, 3H), 7.32 (s, 1H), 4.14-3.96 (m, 4H), 3.86 (q, J = 7.2 Hz, 2H), 3.64 (t, J = 7.2 Hz, 2H), 3.31 (s, 2H), 3.25-3.21 (m, 4H), 1.71-1.62 (m, 2H), 1.08 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 450.3 (calculated); LC / MS [M+H] 450.1 (found).

[0619] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[3-[2-amino-4-[3-(tert-butoxycarbonylamino)propyl-ethoxy-carbamoyl]-3H-1-benzazepine Preparation of 3-(4-(2-(4-(2-(4-(2-(4-nitrophenyl)phenyl)sulfonylazetidin-3-yl)methylamino)-3-oxo-propoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)propanoic acid, HxBzL-4a

[0620] To a mixture of HxBz-3 (110 mg, 176 umol, 1 eq.) in DMF (3 mL) was added 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.), followed by stirring at 25° C. for 0.5 h. The mixture was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 um; mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B%: 20%-55%, 8 min) to afford HxBzL-4a (28 mg, 24 umol, 13.7% yield) as a yellow oil.

[0621] Preparation of HxBzL-4

[0622] To a mixture of HxBzL-4a (78 mg, 78.8 umol, 1 eq.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (106 mg, 394 umol, 5 eq.) in DCM (3 mL) and DMA (0.3 mL) was added EDCI (75.5 mg, 394 umol, 5 eq.), which was 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*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-40%, 10 min) to give HxBzL-4 (39.8 mg, 26.4 umol, 33.5% yield, 95.944% purity, 2TFA) as a colorless oil. 1 H NMR (400 MHz, MeOD) δ 8.75 (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 = 6.0 Hz, 2H), 2.76 (t, J = 6.0 Hz, 2H), 1.85-1.74 (m, 2H), 1.25-1.20 (m, 3H), 1.02 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1218.5 (calculated); LC / MS [M+H] 1218.3 (found).

[0623] Example L-5 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-5

[0624]

[0625] Preparation of 5-bromo-2-(bromomethyl)pyrimidine, HxBz-5b

[0626] At 0°C and N 2 To a solution of (5-bromopyrimidin-2-yl)methanol, HxBz-5a (300 mg, 1.59 mmol, 1.0 equiv) in THF (10 mL) was added PPh 3 (499 mg, 1.90 mmol, 1.2 eq) and CBr4 (631 mg, 1.90 mmol, 1.2 eq.). The mixture was stirred at 20°C for 10 hours. Water (10 mL) was added, and the aqueous phase was extracted with ethyl acetate (10 mL*3). The combined organic phases were washed with brine (10 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate 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, 8 / 1) to give HxBz-5b (290 mg, 1.15 mmol, 72.4% yield) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ8.81(s,2H),4.59(s,2H).

[0627] Preparation of N-[(5-bromopyrimidin-2-yl)methyl]-N-tert-butyloxycarbonyl-carbamic acid tert-butyl ester, HxBz-5c

[0628] At 20 °C and N 2 To a mixture of HxBz-5b (290 mg, 1.15 mmol, 1.0 equiv) and tert-butyl N-tert-butyloxycarbonylcarbamate (250 mg, 1.15 mmol, 1.0 equiv) in DMF (3 mL) was added Cs 2 CO 3 (562 mg, 1.73 mmol, 1.5 eq), the mixture was stirred at 20°C for 2.5 hours. Water (5 mL) was added, and the aqueous phase was extracted with ethyl acetate (5 mL*3), the combined organic phases were washed with brine (5 mL), and the mixture was dried with anhydrous Na 2 SO 4 Dry, filter and concentrate 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, 5 / 1) to give HxBz-5c (350 mg, 901 umol, 78.3% yield) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ8.74(s,2H),5.01(s,2H),1.48(s,18H).

[0629] N-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methyl]-N-tert-butyloxycarbonyl-carbamate, HxBz-5d

[0630] In N2 The mixture was stirred for 2 h at room temperature and then to HxBz-5c (184 mg, 473 umol, 1.0 equiv) and 2-amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-1-benzazepine. -4-formamide (195 mg, 474 umol, 1.0 equiv.) in dioxane (10 mL) and H 2 Pd(dppf)Cl was added to the mixture in 2 mL of O in one portion. 2 ·CH 2 Cl 2 (19.3 mg, 23.7 umol, 0.05 eq) and K 2 CO 3 (163 mg, 1.18 mmol, 2.5 eq), the mixture was degassed and heated under N 2 The mixture was heated to 90°C for 2 hours. Dioxane (10 mL) was removed in vacuo and water (20 mL) was added, and the aqueous phase was extracted with ethyl acetate (10 mL*3), and the combined organic phases were washed with brine (10 mL) and washed with anhydrous Na 2 SO 4 The residue was 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 ethyl acetate / methanol = 10 / 1) to afford HxBz-5d (280 mg, 470.83 umol, 99.35% yield) 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=7.2 Hz, 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).

[0631] 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-3H-1-benzazepine Preparation of -4-formamide, HxBz-5

[0632] At 20 °C and N 2To a solution of HxBz-5d (20.0 mg, 33.6 umol, 1.0 equiv) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 8.41 uL, 1.0 equiv) at once, 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*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 1%-30%, 8 minutes) to give HxBz-5 (6.2 mg, 9.84 umol, 29.2% yield, 98.8% purity, 2TFA) as a white solid. 1 H NMR (400 MHz, MeOD) δ 9.22 (s, 2H), 7.82 (d, J = 2.0 Hz, 1H), 7.79-7.75 (m, 2H), 7.47 (s, 1H), 4.49 (s, 2H), 4.00 (q, J = 7.2 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 3.46 (s, 2H), 1.85-1.77 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 395.2 (calculated); LC / MS [M+H] 395.1 (found).

[0633] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of HxBzL-5a

[0634] At 25°C and N 2To a mixture of HxBz-5 (70 mg, 149 umol, 1.0 equiv, 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]propanoic acid (127 mg, 179 umol, 1.2 equiv) in DMF (0.5 mL) was added DIEA (77.4 mg, 599 umol, 104 uL, 4.0 equiv) in one portion 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 minutes) to give 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).

[0635] Preparation of HxBzL-5

[0636] At 25°C and N 2 To a mixture of HxBzL-5a (60 mg, 61.7 umol, 1.0 equiv., HCl) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (99.3 mg, 370 umol, 6.0 equiv.) in DCM (2 mL) and DMA (0.5 mL) was added EDCI (71.0 mg, 370 umol, 6.0 equiv.) in one portion, and the mixture was stirred at 25 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex Synergi C18150*25*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-45%, 8 minutes) to give 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); LC / MS [M+H] 1163.3 (found).

[0637] 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-benzazepine Synthesis of [-8-yl]-3-pyridyl]sulfonyl]azetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-7

[0638]

[0639] Preparation of tert-butyl ((1-((5-bromopyridin-3-yl)sulfonyl)azetidin-3-yl)methyl)carbamate, HxBz-7b

[0640] At 25°C and N 2 To a mixture of 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, 1 eq.) in DCM (20 mL) was added Et 3 N (789 mg, 7.80 mmol, 1.09 mL, 2 eq) was added and then stirred at 25 °C for 1 hour. H 2 O (20 mL), then concentrated in vacuo to remove DCM. The desired solid precipitated from the mixture and was filtered to give the desired product HxBz-7b (1.1 g, 2.71 mmol, 69.45% yield) as a white solid. 1H 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).

[0641] Preparation of tert-butyl ((1-((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)sulfonyl)azetidin-3-yl)methyl)carbamate, HxBz-7c

[0642] At 25°C and N 2 Downward HxBz-7b (0.75 g, 1.85 mmol, 1 equivalent), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1,3,2-dioxaborolane, Pin 2 B 2 To a mixture of bis(pinacolato)diboron, CAS Registry No. 78183-34-3 (703 mg, 2.77 mmol, 1.5 eq.), KOAc (362 mg, 3.69 mmol, 2 eq.) in dioxane (15 mL) was added Pd(dppf)Cl 2 (67.5 mg, 92.3 umol, 0.05 equiv) and then stirred at 100°C for 1 hour. The mixture was filtered and concentrated in vacuo to afford HxBz-7c (0.85 g, crude) as a yellow oil.

[0643] ((1-((5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of tert-butyl (-8-yl)pyridin-3-yl)sulfonyl)azetidin-3-yl)methyl)carbamate, HxBz-7d

[0644] At 25°C and N 2 HxBz-7c (0.85 g, 1.87 mmol, 1 eq.) and 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine were added. -4-formamide, HxBzL-2b (755 mg, 2.06 mmol, 1.1 eq.) in dioxane (15 mL) was added in H 2 K in O (3 mL) 2 CO3 (518 mg, 3.75 mmol, 2 equiv) and Pd(dppf)Cl 2 (68.6 mg, 93.7 umol, 0.05 eq.) and stirred it at 100 °C for 1 hour. The mixture was poured into H 2 O (50 mL). The aqueous phase was extracted with ethyl acetate (150 mL * 3). The combined organic phase was washed with brine (100 mL) and washed with anhydrous Na 2 SO 4 The residue was 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 = 5 / 1, 0 / 1 to EtOAc / MeOH = 10 / 1). HxBz-7d (1 g, 1.63 mmol, 87.05% yield) 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=7.6Hz2H),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=7.2Hz,2H),1.36(s,9H),1.17(t,J=7.2Hz,3H),0.99(t,J=7.2Hz,3H).

[0645] 2-amino-8-[5-[3-(aminomethyl)azetidin-1-yl]sulfonyl-3-pyridinyl]-N-ethoxy-N-propyl-3H-1-benzazepine Preparation of -4-formamide, HxBz-7

[0646] At 25°C and N 2 HxBz-7d (0.8 g, 1.31 mmol, 1 eq.) was added to CH 3 CN (10 mL) and H 2 To the mixture in 4% CO (10 mL), TFA (1.49 g, 13.1 mmol, 967 uL, 10 equiv) was added, followed by stirring at 80 °C for 1 hour. The mixture was concentrated in vacuo to remove CH 3CN, the aqueous phase was extracted with MTBE (20*3), discarded, and then the aqueous phase was directly freeze-dried to obtain HxBz-7 (0.9 g, 1.22 mmol, 93.07% yield, 2TFA) as an off-white 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); LC / MS [M+H] 513.2 (found).

[0647] 1-(1-((5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of 6,9,12,15,18,21,24,27,30,33-decaoxa-2-azahexatricarbox-36-alkanoic acid, HxBzL-7a

[0648] At 0°C and N 2 To a mixture of HxBz-7 (451 mg, 638 umol, 1 eq.) in THF (10 mL) was added Et 3 N (161 mg, 1.60 mmol, 222 uL, 2.5 equiv) was added and then stirred at 0 °C for 1 hour. The mixture was poured into H 2 O (5 mL), and the pH of the mixture was adjusted to ~6 with TFA at 0°C, then extracted with MTBE (10 mL), discarded, and the aqueous phase was further extracted with DCM / i-PrOH (20 mL*3). The combined organic phases were washed with anhydrous Na 2 SO 4 Drying, filtration and concentration in vacuo afforded HxBzL-7a (0.6 g, 569.68 umol, 89.25% yield) as a light yellow oil.

[0649] Preparation of HxBzL-7

[0650] At 25°C and N 2To a mixture of HxBzL-7a (0.6 g, 570 umol, 1 eq.) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (611 mg, 2.28 mmol, 4 eq.) in DCM (10 mL) and DMA (1.5 mL) was added EDCI (437 mg, 2.28 mmol, 4 eq.), 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 to give HxBzL-7 (370 mg, 288.76 umol, 50.69% yield) as a white solid. 1 HNMR(MeOD,400MHz)δ9.24(d,J=2.0Hz,1H),9.03(d,J=2.0Hz,1H),8.51(t,J=2.0Hz,1H),7.91-7.8 4(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 .2 Hz, 2H), 3.66-3.49 (m, 40H), 3.47 (s, 2H), 3.21 (d, J = 6.4 Hz, 2H), 3.01-2.92 (m, 2H), 2.79-2.68 (m, 1H), 2.29 (t, J = 6.0 Hz, 2H), 1.78 (sxt, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1281.5 (calculated); LC / MS [M+H] 1281.6 (found).

[0651] Example L-12 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(cyclobutyloxy-carbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-12

[0652]

[0653] N-[2-[[2-amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine Preparation of cyclobutyl-4-carbonyl]-propyl-amino]oxyethyl]carbamate, HxBz-15b

[0654] At 25°C and N 2 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine -4-carboxylic acid, HxBz-15a (250 mg, 611 umol, 1.0 equiv) and N-[2-(propylamino-oxy)ethyl]carbamic acid cyclobutyl ester (201 mg, 794 umol, 1.3 equiv, HCl) in DCM (4 mL) and DMA (2 mL) were added EDCI (468 mg, 2.44 mmol, 4.0 equiv) in one portion and stirred at 25 ° C for 2 hours. DCM (4 mL) was removed in vacuo, water (10 mL) was added, and the aqueous phase was extracted with ethyl acetate (10 mL * 3), and the combined organic phase was washed with brine (5 mL * 2) and washed with anhydrous Na 2 SO 4 The residue was 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 ethyl acetate / methanol = 10 / 1) to afford HxBz-15b (190 mg, 313 umol, 51.2% yield) 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=2.8,5.2Hz,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).

[0655] N-[2-[[2-Amino-8-[2-(aminomethyl)pyrimidin-5-yl]-3H-1-benzazepine Preparation of cyclobutyl-4-carbonyl]-propyl-amino]oxyethyl]carbamate, HxBz-15

[0656] At 25°C and N 2To a solution of HxBz-15b (190 mg, 313 umol, 1.0 equiv) in DCM (5 mL) was added CF 3 COOH (535 mg, 4.69 mmol, 347 uL, 15 equiv), then stirred at 25 ° C for 1.5 hours. DCM (5 mL) was removed in vacuo, and the residue was diluted with water (10 mL), and the aqueous phase was extracted with MTBE (5 mL * 4) to remove excess TFA, and then the aqueous phase was freeze-dried to give HxBz-15 (130 mg, 169 umol, 54.1% yield, 95.7% purity, 2TFA) 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); LC / MS [M+H] 508.1 (found).

[0657] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(cyclobutyloxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-12a

[0658] At 0°C and N 2 HxBz-15 (105 mg, 181 umol, 1.0 eq., 2 HCl) and Et 3To a mixture of 3-[2-[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]propanoic acid, PFP-PEG10-CO2H (131 mg, 181 umol, 1.0 equiv) was added, and stirred at 0°C for 0.5 hours, and then heated at 25°C for another 0.5 hours. The reaction mixture was concentrated, the residue was diluted with water (5 mL), and the aqueous phase was extracted with ethyl acetate (3 mL*2), discarded, and then the aqueous phase was further extracted with DCM / iPrOH=3 / 1 (5 mL*3), and the combined organic phases were washed with anhydrous Na 2 SO 4 Drying, filtration and concentration in vacuo afforded HxBzL-12a (100 mg, 95.4 umol, 52.7% yield) as a yellow oil.

[0659] Preparation of HxBzL-12

[0660] At 25°C and N 2 To a mixture of HxBzL-12a (100 mg, 95.4 umol, 1.0 equiv) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (128 mg, 477 umol, 5.0 equiv) in DCM (1 mL) and DMA (0.5 mL) was added EDCI (91.4 mg, 477 umol, 5.0 equiv) in one portion, followed by stirring 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*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-35%, 8 minutes) to give HxBzL-12 (35.1 mg, 25.6 umol, 26.9% yield, 93.3% purity) as a light yellow oil. 1H 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 = 6.0 Hz, 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 = 7.2 Hz, 3H). LC / MS [M+H] 1276.5 (calculated value); LC / MS [M+H] 1276.6 (found value).

[0661] 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-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-13

[0662]

[0663] ((5-(2-amino-4-((2-(3-cyclobutylureido)ethoxy)(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of tert-butyl (8-yl)pyrimidin-2-yl)methyl)carbamate, HxBz-16a

[0664] 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine -4-carboxylic acid, HxBz-15a (250 mg, 611 umol, 1 eq.), 1-cyclobutyl-3-[2-(propylaminooxy)ethyl]urea (231 mg, 916 umol, 1.5 eq., HCl) in a solution of DCM (2 mL) and DMA (2 mL) were added EDCI (351 mg, 1.83 mmol, 3 eq.) and stirred at 25 ° C for 0.5 hours. 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). The combined organic layer was washed with brine (20 mL * 2) and washed with Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a residue. 2 , petroleum ether / ethyl acetate = 50 / 1 to ethyl acetate:MeOH = 5:1) to give HxBz-16a (230 mg, 380 umol, 62.1% yield) as a brown solid.

[0665] 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-[2-(cyclobutylcarbamoylamino)ethoxy]-N-propyl-3H-1-benzazepine Preparation of -4-formamide, HxBz-16

[0666] 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.), followed by stirring at 80° C. for 0.5 h. The mixture was concentrated under reduced pressure, the residue was diluted with water (2 mL), extracted with MTBE (3 mL*3), discarded, and the aqueous phase was concentrated under reduced pressure to give HxBz-16 (230 mg, 371 umol, 97.8% yield, TFA) as a brown solid. 1 HNMR (400 MHz, 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 = 7.2 Hz, 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 = 7.2 Hz, 3H). LC / MS [M+H] 507.3 (calculated); LC / MS [M+H] 507.2 (found).

[0667] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(cyclobutylcarbamoylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-13a

[0668] To a solution of HxBz-16 (100 mg, 136 umol, 1 eq., 2TFA) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (96.2 mg, 0.14 mmol, 1 eq.) in THF (1 mL) was added Et 3 N (41.3 mg, 0.41 mmol, 56.8 uL, 3 equivalents), and then stirred at 25 ° C for 0.5 hours. The pH of the mixture was adjusted to about 6 with TFA at 0 ° C, extracted with EtOAc (5 mL, three times), discarded, and the aqueous phase was further extracted with DCM / i-PrOH (10 mL*3, 3 / 1). The organic layer was washed with Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain the crude product HxBzL-13a (120 mg, 115 umol, 84.2% yield) as a yellow oil and used in the next step without further purification.

[0669] Preparation of HxBzL-13

[0670] To a solution of HxBzL-13a (70 mg, 66.9 umol, 1 eq.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (71.7 mg, 267 umol, 4 eq.) in DMA (0.5 mL) and DCM (1.5 mL) 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*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-35%, 8 min. The residue was then purified by preparative HPLC (TFA conditions; column: Phenomenex Synergi C18150*25*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-35%, 8 minutes) to give HxBzL-13 (20 mg, 13.3umol, 19.9% ​​yield, 2TFA) as a colorless oil. 1 H NMR(400MHz,MeOD)δ9.09(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 = 7.2 Hz, 2H), 1.83-1.68 (m, 4H), 1.64-1.52 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1275.5 (calculated value); LC / MS [M+H] 1275.2 (found value).

[0671] Example L-14 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutyloxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-14

[0672]

[0673] Cyclobutyl N-[3-[[2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine Preparation of HxBz-14

[0674] 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine was added at 0°C. -4-carboxylic acid, HxBz-14a (0.25 g, 611 umol, 1.0 equiv) in DMF (4 mL) was added Et 3 N (185 mg, 1.83 mmol, 255 uL, 3.0 equiv), N-[3-(propylamino)propyl]carbamic acid cyclobutyl ester (170 mg, 678 umol, 1.11 equiv, HCl) and tetramethyluronium hexafluorophosphate azabenzotriazole, HATU (232 mg, 611 umol, 1.0 equiv) and stirred at 0 ° C for 0.5 h. The mixture was then diluted with water and extracted with EtOAc (20 mL x 3). The organic layer was washed with brine and purified by Na 2 SO 4 Dry, 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 give HxBz-14 (0.28 g, 462 umol, 75.71% yield) 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),2.1 0-2.00(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); LC / MS [M+H] 606.2 (found).

[0675] N-[3-[[2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-3H-1-benzazepine Preparation of cyclobutyl-4-carbonyl]-propyl-amino]propyl]carbamate, HxBz-13

[0676] HxBz-14 (0.26 g, 429 umol, 1.0 equiv.) was reacted with CH 3 CN (3 mL) and H 2 In the mixture of 4-nitropropene (0.2g, 3.2mmol, 1.2mmol), TFA (489mg, 4.29mmol, 318uL, 10.0 equivalents) was added once, then stirred at 80°C for 0.5h. The mixture was then concentrated, and the residue was diluted with water (10mL), and the mixture was extracted with MTBE (10mL x 2) to remove excess TFA. The aqueous layer was freeze-dried to obtain HxBz-13 (0.2g, 323umol, 75.20% yield, TFA) as a yellow solid. 1 HNMR (MeOD, 400 MHz) δ 9.21 (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 = 7.2 Hz, 2H), 3.48 (s, 2H), 3.37 (s, 2H), 3.15 (d, J = 15.6 Hz, 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); LC / MS [M+H] 506.2 (found).

[0677] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutyloxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-14a

[0678] To a mixture of HxBz-13 (0.1 g, 161 umol, 1.0 equiv., TFA) in THF (3 mL) was added Et in one portion at 0 °C. 3N (48.9 mg, 484 umol, 67.4 uL, 3.0 equiv) 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, TFP-PEG10-CO2H (114 mg, 161 umol, 1.0 equiv) were 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). The organic layer was purified by Na 2 Drying with SO4, filtration and concentration gave HxBzL-14a (0.15 g, 129 umol, 80.11% yield, TFA) as a yellow oil.

[0679] Preparation of HxBzL-14

[0680] To a mixture of HxBzL-14a (0.15 g, 129 umol, 1.0 equiv., TFA) in DCM (3 mL) and DMA (0.5 mL) was added sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (139 mg, 517 umol, 4.0 equiv.) and EDCI (149 mg, 776 umol, 6.0 equiv.) 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*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-40%, 8 minutes) to give HxBzL-14 (75.3 mg, 59.1 umol, 45.71% yield) as a yellow oil. 1H NMR(MeOD,400MHz)δ9.09(s,2H),7.82-7.67(m,3H),7.11(s,1H),4.86-4.82(m,1 H),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 = 6.0 Hz, 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 (found value).

[0681] Example L-15 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyrimidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-15

[0682]

[0683] 2-Amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine Preparation of -4-formamide, HxBz-11b

[0684] At 25°C and N 2 N-ethoxypropan-1-amine (9.6 g, 68.8 mmol, 1.3 eq., HCl) and 2-amino-8-bromo-3H-1-benzazepine were added. -4-carboxylic acid, HxBz-11a (14.8 g, 52.9 mmol, 1.0 eq.) was added to a mixture of DMA (150 mL) and DCM (150 mL) with EDCI (40.6 g, 211 mmol, 4.0 eq.). The mixture was stirred at 25 °C for 2 h. NaHCO 3 The pH of the mixture was adjusted to ~9 and concentrated at 45 °C under reduced pressure to remove DCM. The aqueous phase was extracted with ethyl acetate (100 mL x 3). The combined organic phases were washed with brine (1000 mL x 2) and washed with anhydrous Na 2 SO 4Dry, filter and concentrate in vacuo.The residue was triturated with MTBE / PE=1 / 1 at 25°C to give HxBz-11b (12.5 g, 34.1 mmol, 64.5% yield) 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=6.8 Hz, 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).

[0685] 2-Amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-1-benzazepine Preparation of -4-formamide, HxBz-11c

[0686] HxBz-11b (500 mg, 1.37 mmol, 1.0 equivalent), Pin 2 B 2 (416 mg, 1.64 mmol, 1.2 eq), KOAc (335 mg, 3.41 mmol, 2.5 eq) and Pd(dppf)Cl 2 A mixture of 1,4-dioxane (99.9 mg, 136 umol, 0.1 eq.) and dioxane (10 mL) was degassed and washed with N 2 The mixture was then purged 3 times at 95 °C and N 2 Stir 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), and then the aqueous phase was further extracted with DCM / i-PrOH=3 / 1 (10 mL x 3). The combined organic phase (DCM / i-PrOH) was washed with anhydrous Na 2 SO 4 Drying, filtration and concentration in vacuo afforded HxBz-11c (490 mg, crude), which was used in the next step without further purification as a black solid.

[0687] 5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of methyl pyrimidine-2-carboxylate, HxBz-11

[0688] HxBz-11c (390 mg, 944 umol, 1.0 equivalent), methyl 5-bromopyrimidine-2-carboxylate (266 mg, 1.23 mmol, 1.3 equivalent), Pd(dppf)Cl 2 (69.0 mg, 94.3 umol, 0.1 equivalent), K 3 PO 4 (401 mg, 1.89 mmol, 2.0 equiv) in dioxane (15 mL) and H 2 The mixture was degassed with N 2 Purge 3 times, then at 80 °C and N 2 Stir 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 C18150*25*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%-30%, 8 minutes) to give HxBz-11 (105 mg, 161 umol, 17.1% yield, TFA) as a white solid. 1 H NMR (MeOD, 400 MHz) δ 9.30 (s, 2H), 7.89 (dd, J = 2.0, 2.0 Hz, 1H), 7.83-7.74 (m, 2H), 7.47 (s, 1H), 4.06 (s, 3H), 4.00 (t, J = 6.8 Hz, 2H), 3.76 (t, J = 7.2 Hz, 2H), 3.45 (s, 2H), 1.83-1.74 (m, 2H), 1.21 (t, J = 6.8 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 424.1 (calculated); LC / MS [M+H] 424.1 (found).

[0689] 5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of HxBzL-15a

[0690] HxBz-11 (330 mg, 779 umol, 1.0 eq.) was dissolved in EtOH (5 mL) and H 2 O (0.5 mL) was added with LiOH. 2 O (131 mg, 3.12 mmol, 4.0 equiv). 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). The combined organic phases were washed with anhydrous Na2 SO 4 Drying, filtration and concentration in vacuo afforded HxBzL-15a (200 mg, 488 umol, 62.7% yield) as a yellow solid.

[0691] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[1-[[5-[2-amino-4-[3-(3,3-dimethylbutyrylamino)propyl-propyl-carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]-3-pyridyl]sulfonyl]azetidin-3-yl]methyl-methyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, HxBzL-15b

[0692] HxBzL-15a (195 mg, 332 umol, 0.8 equiv.) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, tBuOOC-PEG 10 -NH 2 (390 mg, 666 umol, 1.0 eq.) In a mixture in DMF (5 mL) was added Et 3 N (126 mg, 1.25 mmol, 173 uL, 3.0 equiv) and HATU (158 mg, 415 umol, 1.0 equiv). 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 minutes) to give HxBzL-15b (80 mg, 66.4 umol, 16.0% yield, TFA) as a yellow oil.

[0693] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine -8-yl]pyrimidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, preparation of HxBzL-15c

[0694] To a solution of HxBzL-15b (80 mg, 66.4 umol, 1.0 equiv, TFA) in MeCN (2 mL) and H2O (1 mL) was added HCl (12 M, 83.0 uL, 15.0 equiv) and stirred at 80° C. for 1 hour. The mixture was concentrated in vacuo to give a residue, which was freeze-dried to give HxBzL-15c (60 mg, 62.7 umol, 94.4% yield, HCl) as a colorless oil.

[0695] Preparation of HxBzL-15

[0696] To a solution of HxBzL-15c (60 mg, 60.4 umol, 1.0 equiv., 2HCl) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (64.7 mg, 241 umol, 4.0 equiv.) in DCM (2 mL) and DMA (0.5 mL) was added EDCI (46.3 mg, 241 umol, 4.0 equiv.), followed by stirring at 25 °C for 1 hour. The mixture was concentrated in vacuo and filtered. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-35%, 8 minutes) to give HxBzL-15 (36 mg, 31.3 umol, 51.9% yield) 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); LC / MS [M+H] 1149.5 (found).

[0697] Example L-16 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-16

[0698]

[0699]

[0700] Preparation of methyl (2S)-1-(5-bromopyrimidine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-16b

[0701] At 0°C and N 2 Downward to 5-bromopyrimidine-2-carboxylic acid, HxBzL-16a (400 mg, 1.97 mmol, 1.0 equiv), Et 3 To a mixture of N (598 mg, 5.91 mmol, 822 uL, 3.0 equiv) and (2S)-pyrrolidine-2-carboxylic acid methyl ester (342 mg, 2.07 mmol, 1.05 equiv, HCl) in DMF (8 mL) was added HATU (749 mg, 1.97 mmol, 1.0 equiv) in one portion, then stirred at 0°C for 30 minutes, then heated to 25°C and stirred for another 0.5 hours. Water (20 mL) was added, and the aqueous phase was extracted with ethyl acetate (20 mL*4), and the combined organic phases were washed with brine (10 mL*1) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate 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 = 10 / 1, 4 / 1) to give HxBzL-16b (320 mg, 1.02 mmol, 51.7% yield) as a yellow oil.

[0702] Preparation of (2S)-1-(5-bromopyrimidine-2-carbonyl)pyrrolidine-2-carboxylic acid, HxBzL-16c

[0703] At 25°C and N 2 HxBzL-16b (320 mg, 1.02 mmol, 1.0 equiv) was dissolved in MeOH (5 mL) and H 2 LiOH·H O (5 mL) was added to the solution in one portion. 2O (171 mg, 4.07 mmol, 4.0 equiv) and stirred it 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 and dried to give HxBzL-16c (300 mg, crude) as a light yellow solid.

[0704] Preparation of tert-butyl 3-[2-[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]propanoate, HxBzL-16d

[0705] At 0°C and N 2 Downward HxBzL-16c (200mg, 666umol, 1.0 equivalent), Et 3 To a mixture of N (168 mg, 1.67 mmol, 232 uL, 2.5 eq) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (390 mg, 666 umol, 1.0 eq) in DMF (1 mL) was added HATU (253 mg, 666 umol, 1.0 eq) in one portion and stirred at 0 °C for 30 min, then heated to 25 °C and stirred for another 0.5 h. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex luna C18250*50mm*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-60%, 10 minutes) to give HxBzL-16d (300 mg, 346umol, 51.8% yield) as a colorless oil.

[0706] Preparation of 3-[2-[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]propanoic acid, HxBzL-16e

[0707] At 25°C and N 2 HxBzL-16d (300 mg, 345 umol, 1.0 equiv) was dissolved in MeCN (1 mL) and H 2To a solution in 2% O (3 mL) was added HCl (12 M, 864 uL, 30 equiv) in one portion and then stirred at 80° C. for 1 hour. The reaction mixture was concentrated in vacuo to afford HxBzL-16e (250 mg, 307.99 umol, 89.09% yield) as a yellow oil.

[0708] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine -8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, preparation of HxBzL-16f

[0709] HxBzL-16e (150 mg, 185 umol, 1.0 equivalent), 2-amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-1-benzazepine -4-formamide (91.6 mg, 222 umol, 1.2 equivalents), Pd(dppf)Cl 2 (13.5 mg, 18.5 umol, 0.1 equivalent) and K 2 CO 3 (63.8 mg, 462 umol, 2.5 eq.) in dioxane (3 mL) and H 2 The solution in O (0.3 mL) was degassed and then stirred under N 2 The mixture was heated to 95°C for 2 hours. The reaction mixture was filtered, and 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 minutes) to give HxBzL-16f (110 mg, 108umol, 58.4% yield) as a yellow oil.

[0710] Preparation of HxBzL-16

[0711] At 25°C and N 2To a mixture of HxBzL-16f (110 mg, 108 umol, 1.0 equiv) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (145 mg, 540 umol, 5.0 equiv) in DCM (2 mL) and DMA (0.5 mL) was added EDCI (103 mg, 540 umol, 5.0 equiv) in one portion 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*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-40%, 8 minutes) to give HxBzL-16 (66.5 mg, 50.9 umol, 47.1% yield, 95.3% purity) as a light 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),3.8 1-3.74(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); LC / MS [M+H] 1246.7 (found).

[0712] 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-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-21

[0713]

[0714] ((5-(2-amino-4-((2-(3,3-dimethylureido)ethoxy)(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of tert-butyl (8-yl)pyrimidin-2-yl)methyl)carbamate, HxBz-20a

[0715] 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine -4-carboxylic acid, HxBz-14a (250 mg, 611 umol, 1 eq) and 1,1-dimethyl-3-[2-(propylaminooxy)ethyl]urea (165 mg, 733 umol, 1.2 eq, HCl) in a mixture of DCM (3 mL) and DMA (1 mL) was added EDCI (468 mg, 2.44 mmol, 4 eq) 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 Na 2 CO 3 The pH of the mixture was adjusted to ~8 with aqueous solution. The aqueous phase was extracted with ethyl acetate (10 mL*4). The combined organic phases were washed with brine (20 mL*1) and washed with anhydrous Na 2 SO 4 The residue was 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, ethyl acetate / methanol = 1 / 0, 3 / 1) to give HxBz-20a (260 mg, 447.75 umol, 73.33% yield) as a yellow solid.

[0716] Preparation of HxBz-20

[0717] To a solution of HxBz-20a (130 mg, 224 umol, 1 eq.) in EtOAc (3.00 mL) was added HCl / EtOAc (4 M, 3.00 mL, 53.60 eq.), followed by stirring at 25° C. for 1 h. The mixture was concentrated to afford HxBz-20 (115 mg, 207.77 umol, 92.81% yield, 2HCl) as a light red solid. 1 H NMR (MeOD, 400 MHz) δ 9.22 (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 = 5.2 Hz, 2H), 3.76 (t, J = 7.2 Hz, 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 = 7.6 Hz, 3H). LC / MS [M+H] 481.3 (calculated); LC / MS [M+H] 481.1 (found).

[0718] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(dimethylcarbamoylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-21a

[0719] To a solution of HxBz-20 (65.0 mg, 117 umol, 1 eq., 2 HCl) in DMF (1.00 mL) was added Et 3 N (48.0 mg, 470 umol, 4 equivalents) 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, HxBzL-21a (83.0 mg, 117 umol, 1 equivalents), and then stirred at 0 ° C for 1 hour. The mixture was diluted with water (10 mL), and the pH of the mixture was adjusted to about 6 by gradually adding TFA, and extracted with MTBE (10 mL), discarded, and the aqueous phase was further extracted with DCM: i-PrOH = 3: 1 (20 mL x 3). The organic layer was purified by Na 2 SO 4 Drying, filtration and concentration gave HxBzL-21a (95 mg, 93.03 umol, 79.22% yield) as a light yellow oil.

[0720] Preparation of HxBzL-21

[0721] To a solution of HxBzL-21a (90.0 mg, 88.1 umol, 1 eq.) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (95.0 mg, 353 umol, 4 eq.) in DCM (2.00 mL) and DMA (0.10 mL) was added EDCI (68.0 mg, 353 umol, 4 eq.) and stirred at 25 °C for 1 h. The mixture was concentrated and filtered. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%-35%, 8 minutes) to give HxBzL-21 (51 mg, 37.41 umol, 42.45% yield, TFA) as a light yellow oil. 1H 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); LC / MS [M+H] 1249.6 (found).

[0722] 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-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-23

[0723]

[0724]

[0725] N-[[5-[2-amino-4-[2-hydroxyethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methyl]carbamate, HxBz-22a

[0726] 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine was added at 25°C. To a mixture of -4-carboxylic acid, HxBz-14a (0.35 g, 855 umol, 1.0 eq.) and 2-(propylaminooxy)ethanol (200 mg, 1.28 mmol, 1.5 eq., HCl) in DCM (6 mL) and DMA (0.5 mL) was added EDCI (492 mg, 2.56 mmol, 3.0 eq.) in one portion, followed by stirring at 25 °C for 0.5 h. The mixture was concentrated to remove DCM and washed with H 2 O (10 mL).3 The pH of the mixture was adjusted to about 8 with aqueous solution. The aqueous phase was then extracted with EtOAc (20 mL x 3). The organic layer was brine and washed with Na 2 SO 4 Dry, filter and concentrate. Purify the residue by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, ethyl acetate / MeOH=1 / 0, 10 / 1) to give HxBz-22a (0.37 g, 725 umol, 84.77% yield) as a yellow oil. 1 HNMR(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).

[0727] 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-(2-hydroxyethoxy)-N-propyl-3H-1-benzazepine Preparation of -4-formamide, HxBz-22

[0728] HxBz-22a (0.35 g, 685 umol, 1.0 equiv) was reacted with H at 25 °C. 2 O (4 mL) and CH 3 TFA (1.17 g, 10.3 mmol, 761 uL, 15.0 equivalents) was added to the mixture in CN (0.5 mL) at one time, and then stirred at 80 ° C for 0.5 h. 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 Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 1%-20%, 8 minutes) to obtain HxBz-22 (0.32 g, 501 umol, 73.11% yield, 2TFA) as a white solid. 1H NMR (MeOD, 400 MHz) δ 9.20 (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 = 7.2 Hz, 2H), 3.74-3.66 (m, 2H), 3.53-3.36 (m, 2H), 1.88-1.72 (m, 2H), 1.00 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 411.2 (calculated); LC / MS [M+H] 411.1 (found).

[0729] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-hydroxyethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-23a

[0730] To a mixture of HxBz-22 (0.23 g, 560 umol, 1.0 eq., 2TFA) in THF (6 mL) at 0 °C was added Et 3 N (170mg, 1.68mmol, 234uL, 3.0 equiv) 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 (396mg, 560umol, 1.0 equiv) were then stirred at 0°C for 0.5h. The mixture was diluted with water (5ml) and the pH of the mixture was adjusted to ~6 with TFA at 0°C. The aqueous phase was extracted with EtOAc (10mL) and discarded. The aqueous layer was further extracted with DCM:i-PrOH=3:1 (20mL x 2). The organic layer was purified by Na 2 SO 4 Drying, filtration and concentration afforded HxBzL-23a (0.53 g, crude, TFA) as a yellow oil.

[0731] 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-hydroxyethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-23

[0732] To a mixture of HxBzL-23a (0.35 g, 329 umol, 1.0 equiv., TFA) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (352 mg, 1.31 mmol, 4.0 equiv.) in DCM (4 mL) and DMA (0.5 mL) was added EDCI (378 mg, 1.97 mmol, 6.0 equiv.) at 25 °C, followed by stirring 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 minutes) to give HxBzL-23 (80.4 mg, 68.2 umol, 20.75% yield) as a light 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); LC / MS [M+H] 1179.3 (found).

[0733] 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-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-27

[0734]

[0735]

[0736] N-[2-[[2-amino-8-[2-[(tert-butoxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine Preparation of isopropyl-4-carbonyl]-propyl-amino]oxyethyl]carbamate, HxBz-27a

[0737] 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine -4-formic acid, HxBz-14a (350 mg, 855 umol, 1.0 eq.) and N-[2-(propylaminooxy)ethyl]carbamic acid isopropyl ester (268 mg, 1.11 mmol, 1.3 eq., HCl) were added to a mixture of DCM (5 mL) and DMA (3 mL) with EDCI (656 mg, 3.42 mmol, 4.0 eq.) and stirred at 25 ° C for 1 hour. The mixture was concentrated at 30 ° C under reduced pressure. The residue was poured into ice-water (w / w=1 / 1) (10 mL) and stirred for 5 minutes. The mixture was dried over medium-pressure. The residue was concentrated to 40 ° C under reduced pressure. The residue was poured into ice-water (w / w=1 / 1) (10 mL) and stirred for 5 minutes. The residue was dried over medium-pressure. The residue was dried over medium-pressure. The residue was dried over medium-pressure. The residue was concentrated to 40 ° C under reduced pressure ... 3 The pH of the mixture was adjusted to ~8 with aqueous solution. The aqueous phase was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with brine (10 mL x 3) and washed with anhydrous Na 2 SO 4 The residue was 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, ethyl acetate / methanol = 1 / 0, 10 / 1) to give HxBz-27a (460 mg, 772 umol, 90.3% yield) as a yellow solid. 1 H NMR (MeOD, 400MHz) δ9.04 (s, 2H), 7.57 (d, J = 8.0 Hz, 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).

[0738] N-[2-[[2-Amino-8-[2-(aminomethyl)pyrimidin-5-yl]-3H-1-benzazepine Preparation of isopropyl-4-carbonyl]-propyl-amino]oxyethyl]carbamate, HxBz-27

[0739] HxBz-27a (410 mg, 688 umol, 1.0 equiv) was dissolved in MeCN (0.5 mL) and H 2 To the solution in 2% 2-nitropropene (5 mL) was added TFA (1.18 g, 10.3 mmol, 764 uL, 15.0 equiv), followed by stirring at 80 °C for 1 hour. The mixture was concentrated in vacuo to remove CH 3 CN, the aqueous phase was extracted with MTBE (5 mL x 3) to remove excess TFA. The aqueous phase was freeze-dried to give HxBz-27 (400 mg, 553 umol, 80.3% yield, 2TFA) as a white solid. 1 H NMR (MeOD, 400 MHz) 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 = 5.2 Hz, 2H), 3.77 (t, J = 7.2 Hz, 2H), 3.43 (s, 2H), 1.78 (sxt, J = 7.2 Hz, 2H), 1.12 (d, J = 6.4 Hz, 6H), 1.00 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 496.2 (calculated); LC / MS [M+H] 496.1 (found).

[0740] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(isopropoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-27a

[0741] To a solution of HxBz-27 (130 mg, 180 umol, 1.0 eq., 2TFA) in THF (2 mL) was added Et 3N (54.5 mg, 539 umol, 75.0 uL, 3.0 equiv) 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 equiv) were then stirred at 0°C for 0.5 hours. The mixture was concentrated in vacuo. The residue was diluted with water (10 mL) and the pH of the mixture was adjusted to ~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 afford HxBzL-27a (180 mg, 174 umol, 96.7% yield) as a yellow oil.

[0742] Preparation of HxBzL-27

[0743] To a mixture of HxBzL-27a (180 mg, 174 umol, 1.0 equiv) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (186 mg, 695 umol, 4.0 equiv) in DCM (2 mL) and DMA (0.5 mL) was added EDCI (266 mg, 1.39 mmol, 8.0 equiv), followed by stirring 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*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-35%, 8 min) to give HxBzL-27 (91 mg, 66.0 umol, 38.0% yield, TFA) as a yellow solid. 1 H NMR(MeOD,400MHz)δ9.08(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 = 7.2 Hz, 2H), 3.66-3.56 (m, 36H), 3.45-3.42 (m, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 6.4 Hz, 2H), 1.84-1.70 (m, 2H), 1.12 (d, J = 6.0 Hz, 6H), 0.99 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 1264.4 (calculated); LC / MS [M+H] 1264.7 (found).

[0744] 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-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-32

[0745]

[0746] N-[[5-[2-amino-4-[propyl-[2-(pyrrolidine-1-carbonylamino)ethoxy]carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methyl]carbamate, HxBzL-32a

[0747] 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine was added at 25°C. -4-carboxylic acid, HxBz-14a (0.25 g, 611 umol, 1.3 equiv) in a mixture of DCM (4 mL) and DMA (0.5 mL) was added N-[2-(propylaminooxy)ethyl]pyrrolidine-1-carboxamide (118 mg, 469 umol, 1.0 equiv, HCl) and EDCI (270.12 mg, 1.41 mmol, 3.0 equiv) in one portion, 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 minutes) to give HxBzL-32a (0.1 g, 165 umol, 35.09% yield) 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).

[0748] 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-propyl-N-[2-(pyrrolidine-1-carbonylamino)ethoxy]-3H-1-benzazepine Preparation of -4-formamide, HxBzL-32b

[0749] HxBzL-32a (0.09 g, 148 umol, 1.0 equiv) was reacted with H at 25 °C. 2 O (4 mL) and CH 3 TFA (254 mg, 2.23 mmol, 165 uL, 15.0 equiv) was added to the mixture in CN (0.5 mL) at once and then stirred at 80 ° C for 0.5 h. The mixture was then extracted with MTBE (10 mL x 3) and discarded. The aqueous layer was freeze-dried to give 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).

[0750] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[propyl-[2-(pyrrolidine-1-carbonylamino)ethoxy]carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-32c

[0751] To a mixture of HxBzL-32b (70 mg, 82.5 umol, 1.0 eq., 3 TFA) in THF (2 mL) was added Et in one portion at 0 °C. 3N (25.0 mg, 247 umol, 34.4 uL, 3.0 equiv) 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 (69.9 mg, 98.9 umol, 1.2 equiv) were then stirred at 0°C for 0.5 h. The mixture was diluted with water (5 mL) and the pH was adjusted to ~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). The organic layer was purified by Na 2 SO 4 Drying, filtration and concentration afforded HxBzL-32c (0.1 g, crude, TFA) as a yellow oil.

[0752] Preparation of HxBzL-32

[0753] To a mixture of HxBzL-32c (0.1 g, 86.1 umol, 1.0 equiv., TFA) in DCM (2 mL) and DMA (0.5 mL) was added sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (115 mg, 431 umol, 5.0 equiv.) and EDCI (116 mg, 603 umol, 7.0 equiv.) 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*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-35%, 8 minutes) to give HxBzL-32 (46.4 mg, 33.4 umol, 38.78% yield, TFA) 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),3.01-2.9 3(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); LC / MS [M+H] 1275.6 (found).

[0754] Example L-33 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[[5-[2-amino-4-[3-(cyclobutyloxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]-3-pyridyl]sulfonyl]azetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-33

[0755]

[0756] 2-amino-8-(5-((3-(((tert-butyloxycarbonyl)amino)methyl)azetidin-1-yl)sulfonyl)pyridin-3-yl)-3H-benzo[b]azepine Preparation of ethyl 4-formate, HxBz-32b

[0757] At 25°C and N 2 To tert-butyl N-[[1-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-pyridinyl]sulfonyl]azetidin-3-yl]methyl]carbamate, HxBz-32a (5 g, 11.0 mmol, 1 equiv) and 2-amino-8-bromo-3H-1-benzazepine -4-Ethyl carboxylate (3.41 g, 11.0 mmol, 1 eq.) was dissolved in dioxane (50 mL) and H 2 O (5 mL) was added with K 2 CO 3 (3.05 g, 22.1 mmol, 2 equiv) and Pd(dppf)Cl 2 (403 mg, 551 umol, 0.05 equiv) 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 purified by Na 2 SO 4 Drying, filtration and concentration afforded HxBz-32a, which was reacted with CH 3 Trituration with CN at 25 °C for 15 min afforded 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).

[0758] 2-amino-8-(5-((3-(((tert-butyloxycarbonyl)amino)methyl)azetidin-1-yl)sulfonyl)pyridin-3-yl)-3H-benzo[b]azepine Preparation of -4-carboxylic acid, HxBz-32c

[0759] HxBz-32b (3.2 g, 5.76 mmol, 1 eq.) was dissolved in MeOH (40 mL) and H 2 O (5 mL) was added with LiOH. 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 (12 M) at 0 °C and then filtered, and the filter cake was dried under reduced pressure to obtain a crude product. The crude product was mixed with CH 3 CN were triturated at 25 °C for 20 min to afford 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),3.8 3(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).

[0760] N-[3-[[2-amino-8-[5-[3-[(tert-butoxycarbonylamino)methyl]azetidin-1-yl]sulfonyl-3-pyridinyl]-3H-1-benzazepine Preparation of cyclobutyl-4-carbonyl]-propyl-amino]propyl]carbamate, HxBz-32d

[0761] To a solution of HxBz-32c (400 mg, 758 umol, 1 eq.) in DMF (10.0 mL) was added HATU (317 mg, 834 umol, 1.1 eq.), DIEA (490 mg, 3.79 mmol, 660 uL, 5 eq.) and N-[3-(propylamino)propyl]carbamic acid cyclobutyl ester (380 mg, 1.52 mmol, 2 eq., HCl) and stirred at 25 °C for 1 h. The mixture was diluted with water (50 mL) and extracted with EtOAc (30 mL x 3). The organic layer was washed with brine (20 mL x 3) and purified by Na 2 SO 4 Dry, filter and concentrate. 1g The residue was purified by silica flash column, eluting with 0-30% ethyl acetate / MeOH @ 35 mL / min) to afford HxBz-32d (340 mg, 469.69 umol, 61.95% yield) as a light 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,1 H),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).

[0762] N-[3-[[2-amino-8-[5-[3-(aminomethyl)azetidin-1-yl]sulfonyl-3-pyridinyl]-3H-1-benzazepine Preparation of cyclobutyl-4-carbonyl]-propyl-amino]propyl]carbamate, HxBz-32

[0763] HxBz-32d (340 mg, 470 umol, 1 eq.) was added to CH 3 CN (2.00 mL) and H 2To the solution in 4% paraformaldehyde (0.1% TFA, 0.1% tFA) (1.00 mL) was added TFA (428 mg, 3.76 mmol, 278 uL, 8 equiv.), 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 minutes) to give HxBz-32 (400 mg, 470 umol, 99.98% yield, 2TFA) as a light 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 = 7.6 Hz, 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 (found value).

[0764] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[[5-[2-amino-4-[3-(cyclobutyloxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]-3-pyridyl]sulfonyl]azetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-33a

[0765] To a solution of HxBz-32 (200 mg, 235 umol, 1 eq., 2TFA) in THF (2.00 mL) was added Et 3N (71.0 mg, 704 umol, 98.0 uL, 3 equiv) 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 (166 mg, 235 umol, 1 equiv) were then stirred at 0°C for 1 h. The mixture was concentrated and diluted with water (10 mL), and the pH of the mixture was adjusted to ~6 by gradually adding TFA, extracted with MTBE (10 mL), discarded, and the aqueous phase was further extracted with DCM:i-PrOH=3:1 (20 mL x3). The organic layer was purified by Na 2 SO 4 Drying, filtration and concentration gave HxBzL-33a (210 mg, 180.36 umol, 76.81% yield) as a light yellow oil.

[0766] Preparation of HxBzL-33

[0767] To a solution of 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 eq.), followed by stirring at 25 °C for 1 h. The mixture was concentrated and filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.2% FA)-ACN]; B%: 15%-40%, 8 minutes) to give HxBzL-33 (98 mg, 68.13 umol, 37.77% yield, FA) as a white solid. 1H 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 = 6.0 Hz, 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 (found value).

[0768] Example L-34 (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-diazatriacontadecyl)pyrimidin-5-yl)-N-propyl-3H-benzo[b]azepine Synthesis of cyclobutyl-4-carboxamido)oxy)ethyl)carbamate, HxBzL-34

[0769]

[0770] To (2-((2-amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-propyl-3H-benzo[b]azepine To a solution of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28,31,34-decaoxa-4-azatriacontahedral-37-anoic acid (31.7 mg, 0.046 mmol, 1 eq.) in DMF (1 ml) was added DIPEA (49 μl, 0.28 mmol, 6 eq.) followed by ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate), PyAOP, CAS Reg. No. 156311-83-0 (59 mg, 0.113 mmol, 2.4 eq.). The reaction was stirred at room temperature for 2 hours, then concentrated and purified by preparative HPLC to give HxBzL-34 (4.9 mg, 0.0042 mmol, 9%). LC / MS [M+H] 1170.6 (calculated); LC / MS [M+H] 1170.9 (found).

[0771] Example L-37 (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-diazatriadecyl)pyrimidin-5-yl)-N-propyl-3H-benzo[b]azepine Synthesis of cyclobutyl (4-formylamino)oxy)ethyl)carbamate, HxBzL-37

[0772]

[0773] To (2-((2-amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-propyl-3H-benzo[b]azepine To a stirred solution of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azahexatriacont-36-alkanoic acid (16.3 mg, 0.024 mmol, 1 eq.) in DMF (0.5 ml) was added DIPEA (25.5 μl, 0.15 mmol, 6 eq.) followed by PyAOP (31.0 mg, 0.059 mmol, 2.4 eq.). The reaction was stirred at room temperature and monitored by LC / MS, then concentrated and purified by preparative HPLC to afford HxBzL-37 (6.7 mg, 0.0058 mmol, 24%). LC / MS [M+H] 1156.6 (calculated); LC / MS [M+H] 1156.9 (found).

[0774] Example L-38 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]-2-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-38

[0775]

[0776]

[0777] Preparation of tert-butyl N-[(5-bromo-2-pyridyl)methyl]carbamate, HxBz-36b

[0778] To 5-bromopyridine-2-carbaldehyde, HxBz-36a (5.00 g, 26.9 mmol, 1 eq.) and tert-butyl carbamate (6.30 g, 53.8 mmol, 2 eq.) was added in CH 3 To the solution in CN (250 mL) were added TFA (9.19 g, 80.6 mmol, 5.97 mL, 3 eq) and Et 3 SiH (31.3 g, 268.8 mmol, 42.9 mL, 10 equiv) was added and stirred at 25 °C for 3 h. Na 2 CO 3The reaction mixture was quenched with 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 purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure. 2 The residue was purified by HPLC-MS / MS (1:0 to 1:1) to obtain HxBz-36b (9 g, crude) as a light 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)

[0779] Preparation of tert-butyl N-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]methyl]carbamate, HxBz-36c

[0780] HxBz-36b (8.00 g, 27.9 mmol, 1 equivalent), Pin 2 B 2 (8.49 g, 33.4 mmol, 1.2 eq), Pd(dppf)Cl 2 A mixture of 2-nitropropene (1.02 g, 1.39 mmol, 0.05 eq.) and KOAc (5.47 g, 55.7 mmol, 2 eq.) in dioxane (80 mL) was degassed and washed with N 2 The mixture was heated at 90 °C and N 2 The mixture was stirred for 2 h under atmospheric pressure and then used in the next step without work-up to afford HxBz-36c (9.4 g, crude) as a dark brown oil.

[0781] 2-amino-8-[6-[(tert-butyloxycarbonylamino)methyl]-3-pyridinyl]-3H-1-benzazepine Preparation of ethyl 4-formate, HxBz-36d

[0782] HxBz-36c (9.30 g, 27.82 mmol, 2 equivalents), 2-amino-8-bromo-3H-1-benzazepine -4-Ethyl carboxylate (4.30 g, 13.9 mmol, 1 eq), Pd(dppf)Cl 2 (509 mg, 695 umol, 0.05 eq) and K 2 CO3 (3.84 g, 27.8 mmol, 2 eq.) in dioxane (80 mL) and H 2 The mixture was degassed in O (8 mL) and washed with N 2 Purge 3 times, then heat at 90 °C and N 2 The reaction mixture was filtered and concentrated under reduced pressure. The residue was washed with H 2 O (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (30 mL x 3) and purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure. 2 , petroleum ether:ethyl acetate = 1:0 to 0:1 and then SiO 2 The residue was purified by evaporation with 5% paraformaldehyde (EtOAc:MeOH=1:0 to 5:1) to afford HxBz-36d (2.40 g, 5.50 mmol, 39.5% yield) as a light 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).

[0783] 2-amino-8-[6-[(tert-butyloxycarbonylamino)methyl]-3-pyridinyl]-3H-1-benzazepine Preparation of -4-carboxylic acid, HxBz-36e

[0784] To a solution of HxBz-36d (2.40 g, 5.50 mmol, 1 eq.) in EtOH (30 mL) was added LiOH. 2 O (923 mg, 22.0 mmol, 4 equiv) in H 2 O (6 mL), which was then stirred at 40 °C for 2 h. The pH of the reaction mixture was adjusted to 5-6 by adding 1 M HCl at 0 °C, and then concentrated under reduced pressure to remove EtOH. The residue was washed with H 2 O (10 mL) and filtered, and the filter cake was dried under reduced pressure to afford HxBz-36e (1.88 g, 4.60 mmol, 83.7% yield) as a gray solid. 1H 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).

[0785] N-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]-2-pyridinyl]methyl]carbamate, HxBz-36f

[0786] To a solution of HxBz-36e (0.35 g, 857 umol, 1 eq.) and N-ethoxypropan-1-amine (144 mg, 1.03 mmol, 1.2 eq., HCl) in DCM (3 mL) and DMA (3 mL) was added EDCI (493 mg, 2.57 mmol, 3 eq.), which was then stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was washed with H 2 The residue was diluted with 10 mL of HO and the mixture was stirred for 2 h at 0 °C. 2 CO 3 The pH of the mixture was adjusted to ~9 with aqueous solution and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (5 mL x 3) and purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure. 2 , petroleum ether:ethyl acetate = 1:0 to 0:1 and then SiO 2 The residue was purified by evaporation with 5% paraformaldehyde (EtOAc:MeOH=1:0 to 3:1) to give HxBz-36f (0.33 g, 669 umol, 78.0% yield) as a light 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=7.2 Hz,2H),3.73(t,J=7.2Hz,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).

[0787] 2-amino-8-[6-(aminomethyl)-3-pyridinyl]-N-ethoxy-N-propyl-3H-1-benzazepine Preparation of -4-formamide, HxBz-36

[0788] HxBz-36f (0.33 g, 669 umol, 1 eq.) was reacted with CH 3 CN (3 mL) and H 2 To a solution in 4% 4% dHO (3 mL) was added TFA (610 mg, 5.35 mmol, 396 uL, 8 equiv), followed by stirring at 80 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by HPLC. 2 The mixture was diluted with 1% HCl (5 mL), extracted with MTBE (5 mL x 3) and discarded. The aqueous phase was concentrated under reduced pressure to give HxBz-36 (0.33 g, 530.95 umol, 79.42% yield, 2TFA) as a light yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.99 (d, J = 2.0 Hz, 1H), 8.20 (dd, J = 2.4, 8.4 Hz, 1H), 7.79-7.67 (m, 3H), 7.59 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 4.36 (s, 2H), 3.98 (q, J = 7.2 Hz, 2H), 3.76 (t, J = 7.2 Hz, 2H), 3.43 (s, 2H), 1.83-1.72 (m, 2H), 1.26-1.16 (m, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 394.2 (calculated); LC / MS [M+H] 394.2 (found).

[0789] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]-2-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-38a

[0790] To a solution of HxBz-36 (0.15 g, 241 umol, 1 eq., 2TFA) in THF (3 mL) was added 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]propanoic acid (171 mg, 241 umol, 1 eq.) 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 and then heated with H 2 O (10 mL), extracted with EtOAc (5 mL x 3) and discarded. The aqueous phase was further extracted with DCM: i-PrOH = 3: 1 (5 mL x 3). The combined organic layers were purified by Na 2 SO 4 Drying, filtration and concentration under reduced pressure afforded 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)

[0791] Preparation of HxBzL-38

[0792] To a solution of HxBzL-38a (0.18 g, 172 umol, 1 eq., TFA) in DCM (3 mL) and DMA (0.3 mL) was added sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (184 mg, 687 umol, 4 eq.) and EDCI (132 mg, 687 umol, 4 eq.), 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 Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-35%, 8 minutes) to give HxBzL-38 (116.7 mg, 91.4 umol, 53.2% yield, TFA) 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,1 H),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 = 6.0 Hz, 2H), 3.82-3.72 (m, 4H), 3.67-3.51 (m, 36H), 3.45 (s, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.59 (t, J = 6.0 Hz, 2H), 1.83-1.73 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1162.5 (calculated value); LC / MS [M+H] 1162.5 (found value).

[0793] Example L-39 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutyloxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]-2-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-39

[0794]

[0795] N-[3-[[2-amino-8-[6-[(tert-butoxycarbonylamino)methyl]-3-pyridinyl]-3H-1-benzazepine Preparation of cyclobutyl-4-carbonyl]-propyl-amino]propyl]carbamate, HxBz-38b

[0796] 2-amino-8-[6-[(tert-butyloxycarbonylamino)methyl]-3-pyridinyl]-3H-1-benzazepine To a solution of -4-carboxylic acid, HxBz-38a (0.35 g, 857 umol, 1 eq.) and cyclobutyl N-[3-(propylamino)propyl]carbamate (258 mg, 1.03 mmol, 1.2 eq., HCl) in DMF (5 mL) were added HATU (326 mg, 857 umol, 1 eq.) and DIEA (332 mg, 2.57 mmol, 448 uL, 3 eq.), followed by stirring at 20 °C for 2 hours. The mixture was heated to 40 °C by adding HCl at 0 °C. 2 The reaction mixture was quenched with 1% O (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (50 mL) and purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, MeOH / ethyl acetate = 1 / 5) to give HxBz-38b (0.45 g, 744.12 umol, 86.84% yield) as a yellow solid.

[0797] N-[3-[[2-amino-8-[6-[(tert-butoxycarbonylamino)methyl]-3-pyridinyl]-3H-1-benzazepine Preparation of cyclobutyl-4-carbonyl]-propyl-amino]propyl]carbamate, HxBz-38

[0798] HxBz-38b (0.45 g, 744 umol, 1 eq.) was dissolved in MeCN (5 mL) and H 2 O (5mL) in a solution of TFA (679mg, 5.95mmol, 441uL, 8 equivalents), and stirred at 80 ° C for 0.5 hours. The reaction mixture was concentrated under reduced pressure to remove MeCN, and then extracted with MTBE (5mL) to remove excess TFA. The aqueous layer was concentrated to give a residue, which was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-40%, 8 minutes) to give HxBz-38 (0.4g, 646umol, 86.89% yield, TFA) as a yellow solid. 1HNMR(MeOD,400MHz)δ8.99(d,J=1.8Hz,1H),8.20(dd,J=2.4,8.2Hz,1H),7.80-7.66(m,3H),7.59(d,J=8.4Hz,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); LC / MS [M+H] 505.3 (found).

[0799] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutyloxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]-2-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-39a

[0800] To a solution of HxBzL-39 (0.15 g, 204 umol, 1 eq., 2 TFA) in THF (5 mL) was added Et 3 N (62.1 mg, 614 umol, 85.49 uL, 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]propanoic acid (145 mg, 205 umol, 1 eq) were added, and then stirred at 0°C for 2 hours. By adding H 2 O (5 mL), and the pH of the mixture was adjusted to about 6 with TFA, then extracted with EtOAc (10 ml) and discarded. The aqueous phase was further extracted with DCM / PrOH=3 / 1 (20 mL x 3), and then dried over Na 2 SO 4 Drying, filtration and concentration under reduced pressure gave the residue HxBzL-39a (0.2 g, 191 umol, 93.46% yield) as a yellow oil.

[0801] Preparation of HxBzL-39

[0802] To a solution of HxBzL-39a (0.2 g, 191 umol, 1 eq.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (154 mg, 574 umol, 3 eq.) in DCM (2 mL) and DMA (1 mL) was added EDCI (110 mg, 574 umol, 3 eq.), followed by stirring at 20 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure to remove DCM and filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-40%, 8 minutes) to give HxBzL-39 (0.08 g, 62.83 umol, 32.83% yield) as a yellow solid. 1 H NMR (MeOD, 400MHz) δ9.03(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 = 5.6 Hz, 2H), 2.59 (t, J = 5.6 Hz, 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 (found value).

[0803] Example L-40 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-40

[0804]

[0805] Preparation of tert-butyl (2S)-1-(5-bromopyridine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-40a

[0806] At 0°C and N 2 To 5-bromopyridine-2-carboxylic acid (2.00 g, 9.90 mmol, 1.0 equiv), Et 3 To a mixture of N (2.50 g, 24.7 mmol, 3.45 mL, 2.5 eq.) and (2S)-pyrrolidine-2-carboxylic acid tert-butyl ester (2.06 g, 9.90 mmol, 1.0 eq., HCl) in DMF (10 mL) was added HATU (3.76 g, 9.90 mmol, 1.0 eq.) in one portion, the mixture was stirred at 0°C for 30 minutes, then heated to 25°C and stirred for another 0.5 hours. Water (30 mL) was added, and the aqueous phase was extracted with ethyl acetate (30 mL*3), the combined organic phases were washed with brine (30 mL*1), and the mixture was dried over anhydrous Na 2 SO 4 Dry, filter and concentrate 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 = 20 / 1, 2 / 1) to give HxBzL-40a (3.40 g, 9.57 mmol, 96.6% yield) as a yellow oil. 1 H NMR (400 MHz, 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).

[0807] Preparation of (2S)-1-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carbonyl]pyrrolidine-2-carboxylic acid tert-butyl ester, HxBzL-40b

[0808] HxBzL-40a (3.40 g, 9.57 mmol, 1.0 equiv), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolan (2.92 g, 11.5 mmol, 1.2 equiv), Pd(dppf)Cl 2 A solution of 1,2-dioxane (30 mL) (700 mg, 957 umol, 0.1 eq.) and AcOK (2.35 g, 23.9 mmol, 2.5 eq.) was degassed and then heated under N 2The mixture was heated to 100 °C for 3 h. The reaction mixture was concentrated in vacuo to afford HxBzL-40b (3.60 g, crude) as a black oil, which was used in the next step without purification.

[0809] 2-Amino-8-[6-[(2S)-2-tert-butyloxycarbonylpyrrolidine-1-carbonyl]-3-pyridinyl]-3H-1-benzazepine Preparation of ethyl 4-formate, HxBzL-40c

[0810] HxBzL-40b (3.60 g, 8.95 mmol, 1.0 equiv), 2-amino-8-bromo-3H-1-benzazepine -4-Ethyl carboxylate (2.77 g, 8.95 mmol, 1.0 equiv), Pd(dppf)Cl 2 (655 mg, 895 umol, 0.1 equivalent) and K 3 PO 4 (3.80 g, 17.9 mmol, 2.0 equiv) in dioxane (45 mL) and H 2 The solution in O (5 mL) was degassed and then stirred under N 2 The mixture was heated to 95°C for 2 hours. Dioxane (45 mL) was removed, and the aqueous phase was extracted with ethyl acetate (30 mL*3), and the combined organic phases were washed with brine (30 mL*1) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate 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 = 10 / 1, 0 / 1) to give HxBzL-40c (1.60 g, 3.17 mmol, 35.4% yield) as a light yellow solid.

[0811] 2-Amino-8-[6-[(2S)-2-tert-butyloxycarbonylpyrrolidine-1-carbonyl]-3-pyridinyl]-3H-1-benzazepine Preparation of -4-carboxylic acid, HxBzL-40d

[0812] At 25°C and N 2 HxBzL-40c (1.60 g, 3.17 mmol, 1.0 equiv) was dissolved in MeOH (10 mL) and H 2 LiOH·H O (5 mL) was added to the solution in one portion. 2O (399 mg, 9.51 mmol, 3.0 equiv) was added 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) as a white solid. 1 H NMR (400 MHz, 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).

[0813] (2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl pyridine-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carboxylate, HxBzL-40e

[0814] At 25°C and N 2 To a mixture of HxBzL-40d (200 mg, 420 umol, 1.0 eq.) and N-ethoxypropan-1-amine (64.5 mg, 462 umol, 1.1 eq., HCl) in DCM (4 mL) and DMA (2 mL) was added EDCI (322 mg, 1.68 mmol, 4.0 eq.) in one portion and then stirred at 25 °C for 1 hour. DCM (4 mL) was removed and water (8 mL) was added, followed by saturated NaHCO 3 The pH of the aqueous phase was adjusted to ~8, extracted with ethyl acetate (5 mL*3), the combined organic phases were washed with brine (5 mL*1), and washed with anhydrous Na 2 SO 4 The residue was 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 ethyl acetate / methanol = 10 / 1) to afford HxBzL-40e (200 mg, 356 umol, 84.8% yield) as a brown oil.

[0815] (2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of [8-yl]pyridine-2-carbonyl]pyrrolidine-2-carboxylic acid, HxBzL-40f

[0816] At 25°C and N 2 HxBzL-40e (200 mg, 356 umol, 1.0 equiv) was dissolved in MeCN (1 mL) and H 2 To a solution in 4% CO (2 mL) was added HCl (12 M, 890 uL, 30 equiv) in one portion and the mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated in vacuo to afford HxBzL-40f (175 mg, 346 umol, 97.2% yield) as a yellow oil.

[0817] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl propionate, HxBzL-40g

[0818] At 0°C and N 2 The mixture was stirred for 2 h at room temperature for 3 hours. The mixture was stirred for 2 h at room temperature for 3 hours. The mixture was stirred for 2 hours. HxBzL-40f (175 mg, 346 umol, 1.0 equiv), tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (203 mg, 346 umol, 1.0 equiv) and Et 3 To a mixture of N (105 mg, 1.04 mmol, 145 uL, 3.0 equiv) in DMF (2 mL) was added HATU (132 mg, 346 umol, 1.0 equiv) in one portion and stirred at 0 ° C for 30 minutes, then heated to 25 ° C and stirred for another 0.5 hours. 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%-50%, 10 minutes) to give HxBzL-40g (150 mg, 126 umol, 36.5% yield, TFA) as a light yellow oil.

[0819] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2S)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, HxBzL-40h

[0820] At 25°C and N 2 HxBzL-40g (150 mg, 140 umol, 1.0 equiv) was dissolved in MeCN (0.2 mL) and H 2 To a solution of 4% HCl (12 M, 349 uL, 30 equiv.) in 2% O (2 mL) was added HCl (12 M, 349 uL, 30 equiv.) in one portion and then stirred at 80 °C for 1 hour. The reaction mixture was concentrated in vacuo to remove CH 3 CN, and the aqueous phase was freeze-dried to afford HxBzL-40h (140 mg, 137.64 umol, 98.48% yield) as a brown oil.

[0821] Preparation of HxBzL-40

[0822] At 20 °C and N 2 To a mixture of HxBzL-40h (140 mg, 138 umol, 1.0 equiv) and sodium (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (185 mg, 688 umol, 5.0 equiv) in DCM (1.5 mL) and DMA (0.5 mL) was added EDCI (132 mg, 688 umol, 5.0 equiv) in one portion, followed by stirring at 20°C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-40%, 8 minutes) to give HxBzL-40 (46.3 mg, 35.5 umol, 25.8% yield, 95.5% purity) as a light yellow oil. 1H 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,2 H),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 = 7.2 Hz, 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 = 7.2 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1245.5 (calculated); LC / MS [M+H] 1245.4 (found).

[0823] Example L-42 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-42

[0824]

[0825] Preparation of (R)-tert-butyl 1-(5-bromopyrimidine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-42b

[0826] To a solution of 5-bromopyrimidine-2-carboxylic acid, HxBzL-42a (200 mg, 985 umol, 1 eq.) in DMF (3 mL) was added 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 minutes, (2S)-tert-butyl pyrrolidine-2-carboxylate (186 mg, 1.08 mmol, 1.1 eq.) was added to the mixture, and it was stirred for another 3 h at 25 °C. The reaction mixture was diluted with water 20 mL and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 2) and purified by Na2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a residue. 2 , petroleum ether / ethyl acetate = 50 / 1 to 1 / 1) to give HxBzL-42b (200 mg, 561 umol, 56.99% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d 6 )δ9.18-9.10(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)

[0827] Preparation of (R)-tert-butyl 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-42c

[0828] In N 2 Under protection, HxBzL-42b (200 mg, 561 umol, 1 equivalent) and Pin 2 B 2 To a solution of KOAc (110 mg, 1.12 mmol, 2 eq.) (214 mg, 842 umol, 1.5 eq.) and Pd(dppf)Cl were added. 2 (41.1 mg, 56.2 umol, 0.1 eq.) and then stirred at 90 °C for 2 h. The mixture was filtered and concentrated under reduced pressure. The obtained brown solid crude product HxBzL-42c (230 mg, crude) was used in the next step without further purification.

[0829] (R)-1-(5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of tert-butyl (8-yl)pyrimidine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-42d

[0830] In N 2 HxBzL-42c (230 mg, 570 umol, 1 equivalent) and 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine were reacted under protection. To a solution of -4-formamide (209 mg, 570 umol, 1 eq.) in dioxane (5 mL) was added K 2 CO 3 (158 mg, 1.14 mmol, 2 equiv) in water (0.2 mL) and Pd(dppf)Cl 2(41.7 mg, 57 umol, 0.1 eq.) and then stirred at 90 °C for 16 h. 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 give HxBzL-42d (240 mg, 427 umol, 74.8% yield) as a yellow oil.

[0831] (R)-1-(5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of HxBzL-42e

[0832] HxBzL-42d (240 mg, 427 umol, 1 eq.) was added to the mixture in H 2 To a solution of 4-(4-(4-hydroxy-1-oxo-2-nitropropene)-4-nitropropene (5 mL) and MeCN (2 mL) was added HCl (12 M, 355 uL, 10 equiv), followed by stirring at 80 ° C for 1 h. The mixture was filtered and concentrated under reduced pressure to afford HxBzL-42e (170 mg, 336 umol, 78.7% yield) as a yellow oil.

[0833] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl propionate, HxBzL-42f

[0834] To a solution of 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 (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) in DMF (2 mL) was added HATU (90.1 mg, 237 umol, 1 eq) at 0° C. and stirred for 2 h at 0° C. The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-45%, 8 minutes) to give HxBzL-42f (120 mg, 112umol, 47.2% yield) as a light yellow oil.

[0835] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine -8-yl]pyrimidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, preparation of HxBzL-42g

[0836] HxBzL-42f (115 mg, 107 umol, 1 equivalent) was added in H 2 To the mixture in 4% CO (3 mL), HCl (12 M, 89.2 uL, 10 equiv) was added, followed by stirring at 80° C. for 1 h. The mixture was filtered and concentrated under reduced pressure to afford HxBzL-42g (105 mg, 103 umol, 96.3% yield) as a colorless oil. 1H 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)

[0837] Preparation of HxBzL-42

[0838] To a solution of HxBzL-42g (105mg, 103umol, 1 eq.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (111mg, 413umol, 4 eq.) in DCM (2mL) and DMA (0.5mL), EDCI (79.1mg, 413umol, 4 eq.) was added, followed by stirring at 20°C for 1h. The mixture was filtered and concentrated under residual pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-40%, 8 minutes) to give HxBzL-42 (60.0mg, 44.1umol, 42.8% yield, TFA) as a light yellow oil. 1H 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 = 7.0 Hz, 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, 7.2 Hz, 3H), 1.04-0.98 (m, 3H). LC / MS [M+H] 1246.5 (calculated); LC / MS [M+H] 1246.4 (found).

[0839] Example L-43 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-43

[0840]

[0841] Preparation of tert-butyl (2R)-1-(5-bromopyridine-2-carbonyl)pyrrolidine-2-carboxylate, HxBzL-43b

[0842] To a mixture of 5-bromopyridine-2-carboxylic acid, HxBzL-43a (2.19 g, 10.8 mmol, 1 eq.) in DMF (50 mL) were added 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 the addition of (2R)-tert-butyl pyrrolidine-2-carboxylate (2.25 g, 10.8 mmol, 1 eq., HCl). The mixture was stirred at 20 °C for 0.5 h. The reaction mixture was partitioned between EtOAc (150 mL) and water (100 mL). The organic phase was separated and purified by Na 2 SO 4Drying and concentration gave a residue. The product was purified by column chromatography (SiO 2 The residue was purified by HPLC-MS / MS (5% CO, 4% CO, 0.1% ethanol, 1.0% ethanol, 0.2% ethanol, 0.5% ethanol, 1.0% ethanol, 0.2% ethanol, 0.3% ethanol, 0.4% ethanol, 0.5% ethanol, 1.0 ...2% ethanol, 0 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).

[0843] Preparation of (2R)-1-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carbonyl]pyrrolidine-2-carboxylic acid tert-butyl ester, HxBzL-43c

[0844] To a mixture of 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-dioxaborolan, Pin2B2, bis(pinacolato)diboron, CAS Reg. No. 78183-34-3 (3.75 g, 14.8 mmol, 1.5 eq), KOAc (2.42 g, 24.6 mmol, 2.5 eq) in dioxane (80 mL) was added Pd(dppf)Cl 2 (721 mg, 985 umol, 0.1 eq.) and then stirred at 100°C for 2 hours. The mixture was used in the next step without post-treatment and purification to obtain HxBzL-43c (3.96 g, 9.84 mmol, 100.00% yield) as a black liquid.

[0845] 2-Amino-8-[6-[(2R)-2-tert-butyloxycarbonylpyrrolidine-1-carbonyl]-3-pyridinyl]-3H-1-benzazepine Preparation of ethyl 4-formate, HxBzL-43d

[0846] HxBzL-43c (3.96 g, 9.84 mmol, 1 equivalent), 2-amino-8-bromo-3H-1-benzazepine -4-Ethyl carboxylate (3.04 g, 9.84 mmol, 1 eq), Pd(dppf)Cl 2 (360 mg, 492 umol, 0.05 eq) and K 2 CO 3(3.40 g, 24.6 mmol, 2.5 equiv) in dioxane (100 mL) and H 2 The mixture in 2% O (8 mL) was stirred at 100 ° C for 2 hours. The reaction mixture was concentrated to give a residue. The residue was dissolved in EtOAc (100 mL) and washed with water (50 mL). The organic phase was separated and purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a residue. 2 The crude material was purified by HPLC-MS / MS (50% yield, 4% by weight, 1% by weight, 0.1% by weight, 0.2% by weight, 0.4% by weight, 0.5% by weight, 1% by weight, 0.6% by weight, 0.8% by weight, 1.2% by weight, 0.9% by weight, 2.0% by weight, 0.1% by weight, 0.2% by weight, 0.4% by weight, 0.5% by weight, 1.2% by weight, 0.4% by weight, 0.5% by weight, 2.0% by weight, 0.6% by weight, 1.2% by weight, 0.8% by weight, 2.0% by weight, 0.9% by weight, 1.2% by weight, 0.4% by weight, 2.0% by weight, 0.4% by weight, 0.4% by weight, 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).

[0847] 2-Amino-8-[6-[(2R)-2-tert-butyloxycarbonylpyrrolidine-1-carbonyl]-3-pyridinyl]-3H-1-benzazepine Preparation of -4-carboxylic acid, HxBzL-43e

[0848] HxBzL-43d (3.5 g, 6.94 mmol, 1 eq.) was dissolved in THF (20 mL) and H 2 O (40 mL) was added to the mixture 2 O (582 mg, 13.9 mmol, 2 eq) was then stirred at 20 ° C for 3 hours. The mixture was concentrated to remove THF, and then the pH of the mixture was adjusted to ~5 with HCl (4M), and the resulting solid formed a mixture. The mixture was filtered and the filter cake was dried under vacuum to give HxBzL-43e (3.3 g, 6.93 mmol, 99.8% yield) as a white solid.

[0849] (2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl pyridine-8-yl]pyridine-2-carbonyl]pyrrolidine-2-carboxylate, HxBzL-43f

[0850] To a mixture of HxBzL-43e (0.4 g, 839 umol, 1 eq.) and N-ethoxypropan-1-amine (117 mg, 839 umol, 1 eq., HCl) in DCM (5 mL) and DMA (5 mL) was added EDCI (483 mg, 2.52 mmol, 3 eq.) 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). The organic phase was separated and purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a residue. 2 The residue was purified by HPLC-MS / MS (with 5% CO, 0.1% ethanol, 0.2% ethanol, 1.0% ethanol, 0.5% ethanol, 4.5% ethanol, 0.6% ethanol, 1.0 ... 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),3.01-2.8 8(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).

[0851] (2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of pyridine-2-carbonyl-8-yl]pyrrolidine-2-carboxylic acid, HxBzL-43g

[0852] HxBzL-43f (260 mg, 463 umol, 1 eq.) was added to the mixture in H 2 To the mixture in 2% CO (5 mL) was added HCl (12 M, 579 uL, 15 equiv), followed by stirring at 80° C. for 1 hour. The mixture was concentrated to afford HxBzL-43g (0.25 g, 461 umol, 99.6% yield, HCl) as a yellow oil.

[0853] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl propionate, HxBzL-43h

[0854] To a mixture of HxBzL-43g (200 mg, 369 umol, 1 eq, HCl) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (216 mg, 369 umol, 1 eq) in DMF (5 mL) was added HATU (154 mg, 406 umol, 1.1 eq) and DIEA (143 mg, 1.11 mmol, 193 uL, 3 eq) at 0° C. and stirred for 1 hour at 0° C. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-51%, 8 minutes) to give HxBzL-43h (340 mg, 286umol, 77.6% yield, TFA) as a yellow oil.

[0855] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2R)-1-[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine -8-yl]pyridine-2-carbonyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, preparation of HxBzL-43i

[0856] HxBzL-43h (340 mg, 286 umol, 1 eq., TFA) was added in H 2 HCl (12M, 358uL, 15 equiv) was added to the mixture in 4% HCl (20mL), and then stirred at 80°C for 0.5 hours. The mixture was concentrated to a residue. The crude material was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-40%, 8 minutes) to give HxBzL-43i (220mg, 209umol, 72.9% yield, HCl) as a yellow oil.

[0857] Preparation of HxBzL-43

[0858] To a mixture of HxBzL-43i (180 mg, 171 umol, 1 eq., HCl) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (183 mg, 683 umol, 4 eq.) in DMA (0.3 mL) and DCM (3 mL) was added EDCI (164 mg, 854 umol, 5 eq.) and stirred at 15 °C for 0.5 h. The mixture was concentrated to a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 8 minutes) to give HxBzL-43 (94 mg, 72.6 umol, 42.5% yield, 96.2% purity) as a colorless oil. 1 HNMR(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); LC / MS [M+H] 1245.4 (found).

[0859] 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-diazatriadecyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine -4-Formamide, Synthesis of HxBzL-44

[0860]

[0861] 2-amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine -4-formamide, HxBz-5 (0.0283 g, 0.072 mmol, 1 eq.) and 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azahexatriacont-36-alkanoic acid, HxBzL-44a (0.0478 g, 0.072 mmol, 1 eq.) were dissolved in dimethylformamide, DMF. Diisopropylethylamine, DIPEA (0.075 mol, 0.43 mmol, 6 equiv) was added followed by ((7-azabenzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate), PyAOP, CAS Reg. No. 156311-83-0 (0.091 g, 0.18 mmol, 2.4 equiv). The reaction was stirred at room temperature, then concentrated and purified by RP-HPLC to give HxBzL-44 (0.0346 g, 0.033 mmol, 46%). LC / MS [M+H] 1043.53 (calculated); LC / MS [M+H] 1043.84 (found).

[0862] Example L-47 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[propyl(1H-pyrazol-5-ylmethoxy)carbamoyl]-3H-1-benzazepine Synthesis of [(2,3,5,6-tetrafluorophenyl)-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid (2,3,5,6-tetrafluorophenyl) ester, HxBzL-47

[0863]

[0864]

[0865] Preparation of 5-(chloromethyl)-1H-pyrazole, HxBz-41b

[0866] To a solution of 1H-pyrazol-5-ylmethanol, HxBz-41a (4 g, 40.8 mmol, 1 eq.) in DCM (10 mL) was added sulfuryl chloride, SOCl 2 (9.70 g, 81.55 mmol, 5.92 mL, 2 equiv.), then stirred at 0°C to 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to give HxBz-41b (4.5 g, 38.6 mmol, 94.70% yield) as a white solid. LC / MS [M+H] 117.0 (calculated); LC / MS [M+H] 117.0 (found).

[0867] Preparation of tert-butyl N-propyl-N-(1H-pyrazol-5-ylmethoxy)carbamate, HxBz-41c

[0868] To a solution of HxBz-41b (3.01 g, 17.2 mmol, 1 eq.) in DMF (20 mL) was added NaH (1.03 g, 25.7 mmol, 60% purity, 1.5 eq.) at 0 °C, the mixture was stirred at this temperature for 0.5 h, then KI (285 mg, 1.72 mmol, 0.1 eq.) and 5-(chloromethyl)-1H-pyrazole (2 g, 17.16 mmol, 1 eq.) were added. The resulting mixture was stirred at 20 °C for 12 h. The reaction mixture was heated to 40 °C by adding NH 4 The reaction mixture was 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 purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (column: Phenomenex luna C18 (250*70mm, 15um); mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-45%, 20 minutes) to obtain HxBz-41c (0.6 g, 2.35 mmol, 13.69% yield) as a yellow oil. LC / MS [M+H] 256.1 (calculated value); LC / MS [M+H] 256.1 (found value).

[0869] Preparation of N-(1H-pyrazol-5-ylmethoxy)propan-1-amine, HxBz-41d

[0870] HxBz-41c (0.5 g, 1.96 mmol, 1 eq.) was dissolved in MeCN (2 mL) and H 2 To the solution in 4% paraformaldehyde (0.14g, 0.27mmol, 1.2mmol) was added TFA (2.23g, 19.58mmol, 1.45mL, 10 equivalents) and then stirred at 80°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove MeCN. The aqueous phase was extracted with MTBE 20mL to remove excess TFA. The aqueous layer was freeze-dried to give HxBz-41d (0.25g, thick, TFA) as a yellow oil. 1 H NMR (MeOH, 400 MHz) δ 7.10 (d, J = 2.4 Hz, 1H), 6.47 (d, J = 2.4 Hz, 1H), 5.13 (s, 2H), 3.30-3.20 (m, 2H), 1.78-1.71 (m, 2H), 1.02 (t, J = 7.2 Hz, 2H). LC / MS [M+H] 156.1 (calculated value); LC / MS [M+H] 156.1 (found value).

[0871] N-[[5-[2-amino-4-[propyl(1H-pyrazol-5-ylmethoxy)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methyl]carbamate, HxBz-41f

[0872] HxBz-41d (0.2 g, 743 umol, 1 eq., TFA salt) and 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine were added. -4-carboxylic acid, HxBz-41e (304 mg, 743 umol, 1 eq.) in DCM (2 mL) and DMA (1 mL) was added with EDCI (854 mg, 4.46 mmol, 6 eq.), followed by stirring at 20 °C for 2 h. 3 The mixture was quenched to adjust pH = ~8 and then extracted with EtOAc (30 mL x 4). The combined organic layers were washed with brine (30 mL) and purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a residue. 2 The residue was purified by HPLC (MeOH / ethyl acetate = 1 / 5) to give HxBz-41f (0.35 g, 640.30 umol, 86.19% yield) as a yellow solid. LC / MS [M+H] 547.3 (calculated value); LC / MS [M+H] 547.3 (found value).

[0873] 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-propyl-N-(1H-pyrazol-5-ylmethoxy)-3H-1-benzazepine Preparation of -4-formamide, HxBz-41

[0874] HxBz-41f (0.35 g, 640 umol, 1 eq.) was dissolved in MeCN (2 mL) and H 2 To the solution in 4% 4-nitropropene (2mL), TFA (584mg, 5.12mmol, 379uL, 8 equivalents) was added, and then stirred at 80°C for 1 hour. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 1%-25%, 8 minutes) to give HxBz-41 (0.25g, 371umol, 57.88% yield, 2TFA) as a yellow solid. 1H NMR (MeOH, 400 MHz) δ 9.20 (s, 2H), 7.82-7.78 (m, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.26 (s, 1H), 6.31 (d, J = 2.0 Hz, 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 = 7.4 Hz, 3H). LC / MS [M+H] 447.2 (calculated); LC / MS [M+H] 447.2 (found).

[0875] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[propyl(1H-pyrazol-5-ylmethoxy)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, HxBzL-47a

[0876] To a solution of HxBz-41 (0.2 g, 296 umol, 1 eq., 2TFA) in THF (10 mL) was added Et 3 N (90.0 mg, 889 umol, 124 uL, 3 equiv.) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(3-tert-butoxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (2,3,5,6-tetrafluorophenyl) ester, t-Bu-COO-PEG10-COOTFP (226 mg, 296 umol, 1 equiv.) were added, and then stirred at 0°C for 2 hours. By adding H 2 O 5mL quench the reaction mixture, and adjust the pH of the mixture to ~6 with TFA at 0°C, extract the aqueous phase with EtOAc (10ml*2) to remove the by-product, and further extract the aqueous phase with DCM / PrOH=10 / 1 (20mL x 3), and the combined organic phases are purified by Na 2 SO 4 Dry, filter and concentrate under reduced pressure to give compound HxBzL-47a as a yellow oil. LC / MS [M+H] 1043.56 (calculated); LC / MS [M+H] 1043.6 (found).

[0877] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[propyl(1H-pyrazol-5-ylmethoxy)carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-47b

[0878] HxBzL-47a (0.2 g, 192 umol, 1 eq.) was dissolved in MeCN (2 mL) and H 2 O (2mL) was added with HCl (12M, 320uL, 20 equivalents), and then stirred at 80°C for 2 hours. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%-35%, 8 minutes) to give HxBzL-47b (0.13g, 132umol, 68.69% yield) as a yellow oil. LC / MS [M+H] 987.5 (calculated); LC / MS [M+H] 987.6 (measured).

[0879] Preparation of HxBzL-47

[0880] To a solution of HxBzL-47b (0.1 g, 101 umol, 1 eq.) and 2,3,5,6-tetrafluorophenol (67.3 mg, 405 umol, 4 eq.) in DCM (1 mL) and DMA (1 mL) was added EDCI (77.7 mg, 405 umol, 4 eq.), followed by stirring at 20 °C for 1 hour. The reaction mixture was filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-40%, 8 minutes) to give HxBzL-47 (0.0216 g, 19.0 umol, 18.78% yield) as a yellow solid. 1H NMR(MeOH,400MHz)δ9.10(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 = 6.0 Hz, 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 = 6.0 Hz, 2H), 1.80 (t, J = 7.2 Hz, 2H), 1.01 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1135.5 (calculated value); LC / MS [M+H] 1135.6 (found value).

[0881] 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-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-52

[0882]

[0883]

[0884] N-[[5-[2-amino-4-[3-(cyclobutylcarbamoyloxy)propyl-propyl-carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methyl]carbamate, HxBz-45b

[0885] 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine was added at 0°C. -4-carboxylic acid, HxBz-45a (180 mg, 440 umol, 1 eq.) in DMF (3 mL) was added with HATU (167 mg, 440 umol, 1 eq.) and DIPEA (284 mg, 2.20 mmol, 383 uL, 5 eq.). After addition, the mixture was stirred at this temperature for 5 minutes, and then 3-(propylamino)propyl N-cyclobutylcarbamate (110 mg, 440 umol, 1 eq., HCl) was added at 0 °C. The resulting mixture was stirred at 20 °C for 25 minutes. The reaction mixture was stirred for 25 minutes by adding HCl at 0 °C. 2 O (15 mL) to quench the reaction mixture, and then extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (5 mL x 3) and purified by Na 2 SO 4 The mixture was dried, filtered and concentrated 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%-55%, 10 minutes) to give HxBz-45b (0.15 g, 208umol, 47.4% yield, TFA) 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).

[0886] N-cyclobutylcarbamic acid 3-[[2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-3H-1-benzazepine Preparation of [4-carbonyl]-propyl-amino]propyl ester, HxBz-45

[0887] To a solution of HxBz-45b (0.15 g, 208 umol, 1 eq., TFA) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 10 mL, 192 eq.), followed by stirring for 0.5 h at 15° C. The reaction mixture was concentrated under reduced pressure to afford HxBz-45 (135 mg, crude, 2HCl) as a yellow solid. 1H NMR (MeOD, 400 MHz) δ 9.21 (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 (brs, 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); LC / MS [M+H] 506.3 (found).

[0888] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutylcarbamoyloxy)propyl-propyl-carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-52a

[0889] To a solution of HxBz-45 (75 mg, 130 umol, 1 eq., 2 HCl) in DMF (1 mL) was added triethylamine, Et 3 N,TEA (39.4 mg, 389 umol, 54.1 uL, 3 equiv) and 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]propanoic acid (2,3,5,6-tetrafluorophenyl) ester (98.9 mg, 130 umol, 1 equiv). The mixture was stirred at 15 ° C for 1 h. 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 Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-55%, 8 minutes) to give HxBzL-52a (0.13 g, 107umol, 82.4% yield, TFA) as a light yellow oil. LC / MS [M+H] 1102.6 (calculated); LC / MS [M+H] 1102.6 (found).

[0890] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[3-(cyclobutylcarbamoyloxy)propyl-propyl-carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-52b

[0891] HxBzL-52a (0.13 g, 107 umol, 1 eq., TFA) was added to CH 3 CN (1 mL) and H 2 TFA (97.5 mg, 855 umol, 63.3 uL, 8 equiv.) was added to a solution of 1% ethanol in 4% ethanol (5 mL), and then stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove CH 3 CN. The aqueous phase was extracted with MTBE (5 mL x 3) and discarded. The aqueous phase was concentrated under reduced pressure to afford HxBzL-52b (0.14 g, crude, TFA) as a light yellow oil. 1 H 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)

[0892] Preparation of HxBzL-52

[0893] 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 (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxy (90.1 mg, 336 umol, 3 eq.) and EDCI (85.9 mg, 448 umol, 4 eq.), followed by stirring at 15 °C for 1 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 Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-40%, 8 minutes) to give HxBzL-52 (32.3 mg, 25.4 umol, 22.6% yield) as a light 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 = 6.0 Hz, 2H), 2.60 (t, J = 5.6 Hz, 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); LC / MS [M+H] 1274.7 (found).

[0894] Example L-53 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Synthesis of [2,3,5,6-tetrafluorophenyl]-8-yl]-3-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid (2,3,5,6-tetrafluorophenyl) ester, HxBzL-53

[0895]

[0896] Preparation of tert-butyl ((5-bromopyridin-3-yl)methyl)carbamate, HxBz-39b

[0897] To a solution of (5-bromo-3-pyridinyl)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) was added Boc 2 O (1.40 g, 6.42 mmol, 1.47 mL, 1.2 eq) and then stirred at 25 °C for 2 hours. The mixture was concentrated under reduced pressure. The product was purified by column chromatography (SiO 2 The residue was purified by evaporation of 4% paraffin wax (5% ethanol, petroleum ether / ethyl acetate = 50 / 1 to 1 / 1) to give HxBz-39b (1.5 g, 5.22 mmol, 97.7% yield) as a white solid.

[0898] Preparation of tert-butyl ((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methyl)carbamate, HxBz-39c

[0899] In N 2 HxBz-39b (750 mg, 2.61 mmol, 1 eq.) and Pin were added. 2 B 2 To a solution of KOAc (513 mg, 5.22 mmol, 2 eq.) (995 mg, 3.92 mmol, 1.5 eq.) and Pd(dppf)Cl were added 2 (191 mg, 262 umol, 0.1 eq.) and then stirred at 90 °C for 2 h. 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.

[0900] ((5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of tert-butyl (-8-yl)pyridin-3-yl)methyl)carbamate, HxBz-39d

[0901] In N 2 HxBz-39c (800 mg, 2.39 mmol, 1 eq.) and 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine were reacted under protection. To a solution of -4-formamide (877 mg, 2.39 mmol, 1 eq.) in dioxane (3 mL) was added K 2 CO 3 (992 mg, 7.18 mmol, 3 equiv) in water (3 mL) and Pd(dppf)Cl 2(175 mg, 239 umol, 0.1 eq.) 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 give HxBz-39d (900 mg, 1.82 mmol, 76.2% yield) as a yellow oil.

[0902] 2-amino-8-(5-(aminomethyl)pyridin-3-yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine Preparation of -4-formamide, HxBz-39

[0903] HxBz-39d (350 mg, 709 umol, 1 eq.) was reacted with CH 3 CN (2 mL) and H 2 To the solution in 2% CO (2 mL) was added TFA (646 mg, 5.67 mmol, 420 uL, 8 eq.), and the mixture was heated at 80 °C and N 2 The mixture was filtered and concentrated under reduced pressure to give a residue, and H 2 O (15mL), the aqueous phase was extracted with MTBE (20mL x 3), discarded, and the aqueous phase was freeze-dried. 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 minutes) to obtain HxBz-39 (201mg, 396umol, 55.9% yield, TFA) as a light yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.96 (d, J = 2.0 Hz, 1H), 8.72 (d, J = 2.0 Hz, 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 = 7.2 Hz, 2H), 3.76 (t, J = 7.2z, 2H), 3.44 (s, 2H), 1.82-1.74 (m, 2H), 1.20 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.4 Hz, 3H). LC / MS [M+H] 394.2 (calculated); LC / MS [M+H] 394.2 (found).

[0904] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of [-8-yl]-3-pyridyl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-53a

[0905] To a solution of 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]propanoic acid (209 mg, 296 umol, 1 eq.) in THF (5 mL) was added Et 3 N (89.7 mg, 887 umol, 123 uL, 3 equiv.) was added and stirred at 25°C for 1 hour. The pH of the mixture was adjusted to 4-5 with TFA at 0°C, and H 2 O (5 ml), and extracted with EtOAc (10 mL), discarded, the aqueous phase was further extracted with DCM / i-prOH (20 mL*3, 3 / 1), and the organic layer was purified by Na 2 SO 4 Drying, filtering and concentrating under reduced pressure afforded HxBzL-53a (200 mg, 214 umol, 72.4% yield) as a yellow oil.

[0906] Preparation of HxBzL-53

[0907] To a mixture of HxBzL-53a (0.13 g, 139 umol, 1.0 equiv) in DCM (3 mL) and DMA (0.5 mL) was added 2,3,5,6-tetrafluorophenol (92.5 mg, 557 umol, 4.0 equiv) and EDCI (133 mg, 696 umol, 5.0 equiv) 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 Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-55%, 8 minutes) to give HxBzL-53 (78 mg, 65.2 umol, 46.85% yield, TFA) as a light yellow oil. 1H NMR(MeOD,400MHz)δ8.98(d,J=2.0Hz,1H),8.72(d,J=1.6Hz,1H),8.47(s,1H),7.86-7.8 1(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 = 6.0 Hz, 2H), 3.81-3.73 (m, 4H), 3.64-3.54 (m, 36H), 3.45 (s, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.59-2.50 (m, 2H), 1.87-1.72 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 1082.5 (calculated value); LC / MS [M+H] 1082.6 (found value).

[0908] 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-benzazepine Synthesis of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-61

[0909]

[0910]

[0911]

[0912] Preparation of N-but-3-enyl-4-nitro-N-propyl-benzenesulfonamide, HxBzL-61b

[0913] To a solution of 4-nitro-N-propyl-benzenesulfonamide, HxBzL-61a (12 g, 49.1 mmol, 1.0 equiv) in DMF (150 mL) was added Cs 2 CO 3(40.0 g, 123 mmol, 2.5 eq.), KI (8.16 g, 49.1 mmol, 1.0 eq.) and 4-bromobut-1-ene (19.9 g, 147 mmol, 15.0 mL, 3.0 eq.) were then stirred at 40 °C and N2 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 phases were washed with brine (100 mL x 2) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate 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, 10 / 1) to give HxBzL-61b (11 g, 36.9 mmol, 75.1% yield) as a yellow solid. 1 H NMR (MeOD, 400 MHz) 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 = 7.2 Hz, 2H), 3.15 (t, J = 7.2 Hz, 2H), 2.31 (q, J = 7.2 Hz, 2H), 1.67-1.44 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 299.1 (calculated); LC / MS [M+H] 299.0 (found).

[0914] Preparation of 4-nitro-N-[2-(oxiran-2-yl)ethyl]-N-propyl-benzenesulfonamide, HxBzL-61c

[0915] To a solution of HxBzL-61b (13.5 g, 45.3 mmol, 1.0 eq.) in DCM (200 mL) was added m-chloroperbenzoic acid, m-CPBA (18.4 g, 90.5 mmol, 85% purity, 2.0 eq.) at 0 °C and then stirred at 20 °C for 12 h. The mixture was filtered and washed with saturated NaHSO 3 The filtrate was washed with 1% ethyl acetate (30 mL x 1) and brine (100 mL). The organic phase was washed with anhydrous Na 2 SO 4The residue was 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 afford HxBzL-61c (12 g, 38.2 mmol, 84.4% yield) as a white solid. LC / MS [M+H] 315.1 (calculated); LC / MS [M+H] 315.0 (found).

[0916] Preparation of N-[4-(ethylamino)-3-hydroxy-butyl]-4-nitro-N-propyl-benzenesulfonamide, HxBzL-61d

[0917] To a solution of HxBzL-61c (7 g, 22 mmol, 1.0 equiv) in THF (100 mL) was added ethylamine (33.5 g, 445 mmol, 48.6 mL, 60% purity, 20 equiv) at 0 °C, followed by stirring at 30 °C for 2 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); LC / MS [M+H] 360.2 (found).

[0918] Preparation of tert-butyl N-ethyl-N-[2-hydroxy-4-[(4-nitrophenyl)sulfonyl-propyl-amino]butyl]carbamate, HxBzL-61e

[0919] HxBzL-61d (7.6 g, 21.1 mmol, 1.0 equiv) was dissolved in THF (70 mL) and H 2 O (10 mL) was added with NaHCO 3 (3.55 g, 42.3 mmol, 1.64 mL, 2.0 equiv) and Boc 2 O (9.23 g, 42.3 mmol, 9.71 mL, 2.0 equiv). 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 phase was washed with brine (50 mL x 1) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate 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, 2 / 1) to give HxBzL-61e (8.6 g, 18.7 mmol, 88.5% yield) as a yellow oil. 1H NMR(MeOD,400MHz)δ8.46-8.39(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),3.20-3.1 4(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).

[0920] Preparation of N-[2-(1,3-dioxoisoindolin-2-yl)-4-[(4-nitrophenyl)sulfonyl-propyl-amino]butyl]-N-ethyl-carbamic acid tert-butyl ester, HxBzL-61f

[0921] To a mixture of HxBzL-61e (5 g, 10.9 mmol, 1.0 eq.) and isoindoline-1,3-dione (1.76 g, 12.0 mmol, 1.1 eq.) in THF (50 mL) at 0 °C were added triphenylphosphine, PPh 3 (4.28 g, 16.3 mmol, 1.5 equiv) and diethyl azodicarboxylate, DEAD (2.84 g, 16.3 mmol, 2.97 mL, 1.5 equiv) were added 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 washed with anhydrous Na 2 SO 4 The residue was 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 give HxBzL-61f (8.8 g, crude) as a yellow solid. LC / MS [M+H] 589.2 (calculated); LC / MS [M+H] 589.2 (found).

[0922] Preparation of N-[2-amino-4-[(4-nitrophenyl)sulfonyl-propyl-amino]butyl]-N-ethyl-carbamic acid tert-butyl ester, HxBzL-61g

[0923] To a solution of HxBzL-61f (4.4 g, 7.47 mmol, 1.0 equiv) in MeOH (50 mL) was added NH 2 NH 2 .H 2O (2.25 g, 44.9 mmol, 2.18 mL, 6.0 equiv) was then stirred at 80 °C for 12 h. The mixture was filtered and the filtrate was concentrated in vacuo to afford HxBzL-61g (3.4 g, 7.41 mmol, 99.2% yield) as a yellow oil. LC / MS [M+H] 459.2 (calculated); LC / MS [M+H] 459.2 (found).

[0924] Preparation of N-[3-amino-4-(ethylamino)butyl]-4-nitro-N-propyl-benzenesulfonamide, HxBzL-61h

[0925] To a solution of HxBzL-61g (2.9 g, 6.32 mmol, 1.0 eq) in EtOAc (30 mL) was added HCl / EtOAc (4 M, 29.0 mL, 18.3 eq) and then stirred for 1 h at 20° C. The mixture was concentrated in vacuo to afford HxBzL-61h (2.7 g, crude, 2HCl) as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.35 (d, J = 8.8 Hz, 2H), 8.09 (d, J = 8.8 Hz, 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 = 7.2 Hz, 3H), 0.80 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 359.17 (calculated); LC / MS [M+H] 359.1 (found).

[0926] Preparation of N-[2-(1-ethyl-2-oxo-imidazolidin-4-yl)ethyl]-4-nitro-N-propyl-benzenesulfonamide, HxBzL-61i

[0927] HxBzL-61h (2.7 g, 7.53 mmol, 1.0 equiv) and Et 3 To a mixture of N (1.91 g, 18.8 mmol, 2.62 mL, 2.5 equiv.) in THF (30 mL) was added carbonyl diimidazole, CDI (2.44 g, 15.1 mmol, 2.0 equiv.). 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 washed with anhydrous Na 2 SO 4Dry, filter and concentrate 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 give HxBzL-61i (300 mg, 780 umol, 10.4% yield) as a yellow oil. 1 H NMR (MeOD, 400MHz) δ8.39(d,J=8.8Hz,2H),8.02(d,J=8.8Hz,2H),3.99-3.95(m,1H),3.77-3.63(t,J=8.8Hz,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)

[0928] Preparation of 1-ethyl-4-[2-(propylamino)ethyl]imidazolidin-2-one, HxBzL-61j

[0929] To a solution of HxBzL-61i (300 mg, 780 umol, 1.0 eq.) in MeCN (10 mL) was added LiOH. 2 O (196 mg, 4.68 mmol, 6.0 equiv) and methyl 2-sulfanyl acetate (0.45 g, 4.24 mmol, 384 uL, 5.43 equiv) were added and then stirred at 25 °C for 2 hours. The mixture was filtered and the filtrate was concentrated in vacuo. 2 O (20 mL), and then the pH of the aqueous phase was adjusted to 3-4 with HCl (1 M), and then extracted with EtOAc (20 mL x 3) to remove the by-products, and then the aqueous phase was freeze-dried to give HxBzL-61j (180 mg, 763 umol, 97.8% yield, HCl) as a colorless oil. 1 H NMR(MeOD,400MHz)δ3.83-3.73(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).

[0930] N-[[5-[2-amino-4-[2-(1-ethyl-2-oxo-imidazolidin-4-yl)ethyl-propyl-carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methyl]carbamate, HxBzL-611

[0931] 2-amino-8-[2-[(tert-butyloxycarbonylamino)methyl]pyrimidin-5-yl]-3H-1-benzazepine -4-carboxylic acid, HxBzL-61k (210 mg, 513 umol, 1.0 equiv) in DMF (6 mL) was added with HATU (205 mg, 539 umol, 1.05 equiv), DIEA (331 mg, 2.56 mmol, 447 uL, 5.0 equiv) and HxBzL-61j (145 mg, 615 umol, 1.2 equiv, HCl), and 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 phase was washed with brine (10 mL x 2) and washed with anhydrous Na 2 SO 4 The residue was 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 / 0, ethyl acetate / methanol = 1 / 0, 3 / 1) to give HxBzL-611 (300 mg, 508 umol, 99.0% yield) as a yellow solid. LC / MS [M+H] 591.3 (calculated); LC / MS [M+H] 591.3 (found).

[0932] 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-[2-(1-ethyl-2-oxo-imidazolidin-4-yl)ethyl]-N-propyl-3H-1-benzazepine Preparation of -4-formamide, HxBzL-61m

[0933] To a solution of HxBzL-611 (300 mg, 508 umol, 1.0 equiv.) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 6.00 mL, 47.3 equiv.), followed by stirring at 25 °C for 1 hour. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 1%-30%, 8 minutes) to give HxBzL-61m (142 mg, 198 umol, 38.91% yield, 2TFA) as a yellow solid. 1H NMR (MeOD, 400 MHz) 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); LC / MS [M+H] 491.3 (found).

[0934] 3-[2-[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-benzazepine Preparation of tert-butyl]-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-61n

[0935] HxBzL-61m (90 mg, 125 umol, 1.0 eq., 2TFA) and Et 3 To a solution of N (38.02 mg, 376 umol, 52.3 uL, 3.0 equiv) in DMF (1 mL) was added 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]propanoic acid (2,3,5,6-tetrafluorophenyl) ester, t-Bu-COO-PEG10-COOTFP (95.5 mg, 125 umol, 1.0 equiv), and then stirred at 25° C. for 1 hour. Water (10 mL) was added, and the pH of the mixture was adjusted to about 6 with TFA. The aqueous phase was extracted with MTBE (5 mL x 3) to remove by-products. 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 afford HxBzL-61n (130 mg, 120 umol, 95.5% yield) as a yellow oil.

[0936] 3-[2-[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-benzazepine Preparation of [-8-yl]pyrimidin-2-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid, HxBzL-61o

[0937] HxBzL-61n (100 mg, 92.0 umol, 1.0 equiv) was dissolved in MeCN (0.5 mL) and H 2 To a solution in 4% paraformaldehyde (1% paraformaldehyde) in 4% paraformaldehyde (1% paraformaldehyde) (10% paraformaldehyde) was added TFA (83.9 mg, 735 umol, 54.5 uL, 8.0 equiv.), then stirred at 80° C. for 1 hour. The mixture was concentrated in vacuo to give a residue, which was diluted with water (10 mL), and the aqueous phase was extracted with MTBE (10 mL) to remove excess TFA, and the aqueous phase was lyophilized to HxBzL-61o (100 mg, 87.3 umol, 94.9% yield, TFA) as a yellow oil.

[0938] Preparation of HxBzL-61

[0939] To a solution of HxBzL-61o (100 mg, 87.3 umol, 1.0 equiv., TFA) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (70.2 mg, 262 umol, 3.0 equiv.) in DCM (1 mL) and DMA (0.5 mL) was added EDCI (67.0 mg, 349 umol, 4.0 equiv.), followed by stirring at 20 °C for 1 hour. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-35%, 8 minutes) to give HxBzL-61 (30 mg, 23.8 umol, 27.3% yield) as a yellow oil. 1H NMR(MeOD,400MHz)δ9.10(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,2 H),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); LC / MS [M+H] 1259.6 (found).

[0940] 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-benzazepine Synthesis of [-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]propionyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, HxBzL-65

[0941]

[0942]

[0943] ((5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl ester, HxBzL-65a

[0944] 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-3H-1-benzazepine was added at 0°C. -4-formamide, HxBz-5 (41.2 mg, 96 umol, 1 eq., HCl) and Et 3To a solution of ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester, Fmoc-Val-Cit-PNC (110 mg, 143 umol, 1.5 equiv) was added to N (29.0 mg, 287 umol, 39.9 uL, 3 equiv) in DMF (0.5 mL), and then stirred at 25° C. for 1 hour. Piperidine (24.4 mg, 287 umol, 28.3 uL, 3 equiv) was added to the mixture and stirred at 25° C. for another 1 hour. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%-30%, 8 minutes) to give HxBzL-65a (60 mg, 75.0 umol, 78.4% yield) as a yellow oil. LC / MS [M+H] 800.4 (calculated); LC / MS [M+H] 800.6 (found).

[0945] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[(1S)-1-[[(1S)-1-[[4-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine Preparation of tert-butyl]-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]propionic acid, HxBzL-65b

[0946] To a solution of HxBzL-65a (60 mg, 65.7 umol, 1 eq., TFA) in THF (2 mL) was added Et 3N (19.9 mg, 197 umol, 27.4 uL, 3 equivalents) and 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]propionic acid (2,3,5,6-tetrafluorophenyl) ester, t-Bu-COO-PEG10-COOTFP (50.1 mg, 66 umol, 1 equivalent), and then stirred at 25 ° C for 1 hour. The reaction mixture was diluted with 2 mL of water, and then the pH of the aqueous phase was adjusted to 5-6 with TFA, and extracted with DCM / i-prOH (5 mL x 3, 3 / 1), and the combined organic phase was purified by Na 2 SO 4 Drying, filtration and concentration under reduced pressure gave 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); LC / MS [M+H] 1396.7 (found).

[0947] 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[(1S)-1-[[(1S)-1-[[4-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine -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]propanoic acid, preparation of HxBzL-65c

[0948] 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.), followed by stirring at 80 °C for 2 hours. The reaction mixture was diluted with 2 mL of water, and the pH of the aqueous phase was adjusted to 5-6 with TFA, and extracted with DCM / i-prOH (5 mL x 3, 3 / 1), and the combined organic phases were purified by Na 2 SO 4 Drying, filtration and concentration under reduced pressure afforded HxBzL-65c (100 mg, crude) as a yellow oil. LC / MS [M+H] 1340.7 (calculated); LC / MS [M+H] 1340.6 (found).

[0949] Preparation of HxBzL-65

[0950] To a solution of HxBzL-65c (100 mg, 74.6 umol, 1 eq.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (80.0 mg, 298 umol, 4 eq.) in DCM (1 mL) and DMA (0.5 mL) was added EDCI (57.2 mg, 298 umol, 4 eq.), followed by stirring at 25 °C for 1 hour. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-40%, 8 minutes) to give HxBzL-65 (20 mg, 12.75 umol, 17.09% yield) as a white solid. 1 H NMR(400MHz,MeOD)δ9.08(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 = 6.0 Hz, 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 = 7.2 Hz, 3H), 1.05-0.99 (m, 9H). LC / MS [M+H] 1568.6 (calculated value); LC / MS [M+H] 1568.6 (found value).

[0951] Example L-70 1-(5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine -8-yl)pyrimidin-2-yl)-3-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-2-azahexatricarbox-36-alkanoic acid 2,3,5,6-tetrafluorophenyl ester, HxBzL-70

[0952]

[0953] According to the procedure of Example L-5, 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine To a solution of 2,3,5,6-tetrafluorophenol (1.77 g, 10.7 mmol, 2.00 equiv), propanephosphonic anhydride (PPAA, T3P), CAS registry number 68957-94-8 (50 wt % solution in MeCN, 17.0 g solution, 26.8 mmol, 5.00 equiv) and N-methylimidazole, NMI (2.15 mL, 26.8 mmol, 5.00 equiv) were added successively. The mixture was stirred at 20 °C for 2 h and then diluted with 20% aqueous NaCl solution (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 afford crude HxBzL-70 in the form of a dark brown oil. The material was loaded onto a Biotage column (250 mL 7.5 mM HCl in MeCN / water 2:8, v / v) and purified using a gradient step (20 column volumes MeCN / water 2:8, then 15 column volumes MeCN / water 3:7). The desired fractions were combined, then extracted (2 x 300 mL DCM) and concentrated in vacuo to afford pure HxBzL-70 in the form of a dark yellow oil (5.34 g, 55.6% purity by qNMR, 56% yield), which was stored at -20°C under nitrogen and then diluted with DMA to make a 20 mM solution of HxBzL-70. LC / MS [M+H] 1083.1 (calculated); LC / MS [M+H] 1083.1 (found).

[0954] Example 201 Preparation of Immunoconjugate (IC)

[0955] To prepare lysine-conjugated immunoconjugates, use G-25 SEPHADEX TM Desalting columns (Sigma-Aldrich, St. Louis, MO) or Zeba TMThe antibody was buffer exchanged into a conjugation buffer containing 100 mM boric acid, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3 using a spin desalting column (Thermo Fisher Scientific). The eluents were then adjusted to a concentration of about 1-10 mg / ml using a buffer and then sterile filtered. The antibody was pre-warmed to 20-30°C and incubated with 2-20 (e.g., 7-10) molar equivalents of tetrafluorophenyl (TFP) or sulfonic acid tetrafluorophenyl (sulfoTFP) ester, 8-Het-2-aminobenzazepine of Formula II dissolved in dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA). -Linker (HxBzL) compound was quickly mixed to a concentration of 5 to 20 mM. The reaction was allowed to proceed at 30°C for about 16 hours and passed through two consecutive G-25 desalting columns or Zeba TM The immunoconjugates (IC) were separated from the reaction products by running on a spin desalting column to obtain the immunoconjugates (IC) in Tables 3a and 3b. TM G2-XS TOF mass spectrometer (Waters Corporation) connected to H-class ACQUITY TM The adjuvant-antibody ratio (DAR) was determined by liquid chromatography-mass spectrometry analysis on a C4 reverse phase column on a UPLC (Waters Corporation, Milford, MA).

[0956] To prepare cysteine-conjugated immunoconjugates, Zeba TM The antibody was buffer exchanged into conjugation buffer containing PBS, pH 7.2 and 2 mM EDTA using a spin desalting column (Thermo Fisher Scientific). Interchain disulfides were reduced using 2-4 molar excess of tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) at 37°C for 30 min-2 h. The antibody was then eluted using a Zeba Pre-equilibrated with conjugation buffer. TM The desalting column was rotated to remove excess TCEP or DTT. The concentration of the buffer exchanged antibody was adjusted to approximately 5 to 20 mg / ml using conjugation buffer and sterile filtered. The maleimide-HxBzL compound was dissolved in dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5 to 20 mM. For conjugation, the antibody was mixed with 10 to 20 molar equivalents of maleimide-HxBzL. In some cases, up to 20% (v / v) of additional DMA or DMSO was added to increase the solubility of maleimide-HxBzL in the conjugation buffer. The reaction was allowed to proceed at 20°C for approximately 30 minutes to 4 hours. Two consecutive Zeba TMThe resulting conjugate was purified from unreacted maleimide-HxBzL by spinning a desalting column. The column was pre-equilibrated with phosphate buffered saline (PBS) at pH 7.2. TM G2-XS TOF mass spectrometer (Waters Corporation) connected to H-class ACQUITY TM The adjuvant to antibody ratio (DAR) was estimated by liquid chromatography-mass spectrometry analysis using a C4 reverse phase column on a UPLC (Waters Corporation, Milford, MA).

[0957] For conjugation, the antibody can be dissolved in an aqueous buffer system known in the art that will not adversely affect the stability or antigen binding specificity of the antibody. Phosphate buffered saline can be used. The HxBzL compou...

Claims

1. An immunoconjugate comprising a molecule of the invention linked to one or more 8-Het-2-aminobenzazepines via a linker. The antibody is covalently linked to the moiety and has Formula I: Ab-[L-HxBz] p I or a pharmaceutically acceptable salt thereof, in: Ab is an antibody construct with an antigen binding domain that binds CEA; p is an integer from 1 to 8; HxBz is 8-Het-2-aminobenzazepine having the formula part: Het is selected from heterocyclyldiyl and heteroaryldiyl; R 1 , R 2 , R 3 and R 4 Independently selected from H, C1-C 12 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 12 Carbocyclic group, C6-C 20 Aryl, C2-C9 heterocyclic and C1-C 20 The group consisting of heteroaryl, wherein alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl are independently and optionally substituted by one or more groups selected from the following: -(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C1-C 12 Alkyldiyl)-N(R 5 )2; -(C1-C 12 Alkyldiyl)-OR 5 ; -(C3-C 12 Carbocyclic group); -(C3-C 12 Carbocyclyl)-*; -(C3-C 12 Carbocyclic)-(C1-C 12 Alkyldiyl)-NR 5 -*; -(C3-C 12 Carbocyclic)-(C1-C 12 Alkyldiyl)-N(R 5 )2; -(C3-C 12 Carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*; -(C6-C 20 Aryl); -(C6-C 20 Aryldiyl)-*; -(C6-C 20 Aryldiyl)-N(R 5 )-*; -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-(C2-C 20 Heterocyclyldiyl)-*; -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2; -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -C(=NR 5a )N(R 5 )-*; -(C2-C 20 Heterocyclic group); -(C2-C 20 Heterocyclyl)-*; -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-NR 5 -*; -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2; -(C2-C9 heterocyclyl)-C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C2-C9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*; -(C2-C9 heterocyclyl)-NR 5 -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C2-C9 heterocyclyl)-(C6-C 20 Aryldiyl)-*; -(C1-C 20 heteroaryl); -(C1-C 20 Heteroaryl)-*; -(C1-C 20 Heteroaryl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C1-C 20 Heteroaryl)-(C1-C 12 Alkyldiyl)-N(R 5 )2; -(C1-C 20 Heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*; -(C1-C 20 Heteroaryl)-N(R 5 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -C(=O)-*; -C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -C(=O)-(C2-C 20 Heterocyclyldiyl)-*; -C(=O)N(R 5 )2; -C(=O)N(R 5 )-*; -C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-*; -C(=O)N(R 5 )-(C1-C 12 alkyldiyl)-C(=O)N(R 5 )-*; -C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-N(R 5 )C(=O)R 5 ; -C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-N(R 5 )C(=O)N(R 5 )2; -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-N(R 5 )CO2R 5 ; -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 )2; -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 C(=NR 5a )R 5 ; -C(=O)NR 5 -(C1-C8 alkyldiyl)-NR 5 (C2-C5 heteroaryl); -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-N(R 5 )-*; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-*; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 5 -(C1-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 )CO2R 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)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -N(R 5 )-(C2-C5 heteroaryl); -N(R 5 )-S(=O)2-(C1-C 12 alkyl); -O-(C1-C 12 alkyl); -O-(C1-C 12 Alkyldiyl)-N(R 5 )2; -O-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -O-C(=O)N(R 5 )2; -O-C(=O)N(R 5 )-*; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-*; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -*;and -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 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 are independently selected from a free bond, C(=O), C(=O)N(R 5 )、O、N(R 5 ), S, S(O)2 and S(O)2N(R 5 ) R 5 Independently selected from H, C6-C 20 Aryl, C3-C 12 Carbocyclic group, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 A group consisting of an alkyl diyl group, or two R 5 The groups together form a 5- or 6-membered heterocyclyl ring; R 5a Choose from C6-C 20 Aryl and C1-C 20 A group consisting of heteroaryl groups; wherein the asterisk * indicates the attachment site of L, and wherein R 1 , R 2 , R 3 and R 4 One of them is connected to L; L is a linker selected from the group consisting of: -C(=O)-PEG-; -C(=O)-PEG-C(=O)N(R 6 )-(C1-C 12 Alkyldiyl)-C(=O)-Gluc-; -C(=O)-PEG-O-; -C(=O)-PEG-OC(=O)-; -C(=O)-PEG-C(=O)-; -C(=O)-PEG-C(=O)-PEP-; -C(=O)-PEG-N(R 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 )-(C1-C 12 Alkyldiyl)-; -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; -C(=O)-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-; -C(=O)-PEG-SS-(C1-C 12 alkyldiyl)-C(═O)-; -C(=O)-(C1-C 12 alkyldiyl)-C(═O)-PEP-; -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-; -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-N(R 5 )-C(=O); -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C1-C 12 Alkyldiyl)-C(=O)-Gluc-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-O-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-OC(=O)-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-N(R 5 )-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-N(R 5 )-C(=O)-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-; -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-; -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-; -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-; and -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; R 6 are independently H or C1-C6 alkyl; PEG has the formula: -(CH2CH2O) n -(CH2) m -; m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the following formula: PEP has the following formula: wherein AA is independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a five-membered ring of proline amino acids, and the wavy line indicates the point of attachment; Cyc is selected from C6-C 20 Aryldiyl and C1-C 20 Heteroaryldiyl, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3 and glucuronic acid having the following structure: R 7 Selected from -CH(R 8 )O-、-CH2-、-CH2N(R 8 )- and -CH(R 8 )OC(=O)-, wherein R 8 is selected from H, C1-C6 alkyl, C(=O)-C1-C6 alkyl and -C(=O)N(R 9 )2, where R 9 Independently selected from H, C1-C 12 Alkyl and -(CH2CH2O) n -(CH2) 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 together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; and Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl and heteroaryldiyl are independently and optionally substituted with one or more groups independently selected from the following: F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -C H2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP (O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2N HSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, -C ONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH 3. -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2, -NHC (=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n H, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3 and -S(O)3H.

2. The immunoconjugate of claim 1, wherein the antibody is selected from labetuzumab and acitumomab or a biosimilar or bioimproved version thereof.

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

128.

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

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

6. The immunoconjugate of claim 5, wherein the antibody construct comprises a variable light chain and a heavy chain CDR (complementarity determining region) CDR-H2, the variable light chain comprising an amino acid sequence from SEQ ID NO: 105; and the 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 pyridinyldiyl, pyrimidinyldiyl, pyrazolyldiyl, piperazinyldiyl, piperidinyldiyl and pyrazinyldiyl.

9. The immunoconjugate of any one of claims 1 to 7, wherein X 1 is a key, and R 1 It's H.

10. The immunoconjugate of any one of claims 1 to 7, wherein X 2 is a key, and R 2 It is a C1-C8 alkyl group.

11. The immunoconjugate of any one of claims 1 to 7, wherein X 2 and X 3 Each is a bond, and R 2 and R 3 independently selected from C1-C8 alkyl, -O-(C1-C 12 Alkyl), -(C1-C 12 Alkyldiyl)-OR 5 、-(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 、-(C1-C 12 alkyl)-OC(O)N(R 5 )2. -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 and -O-(C1-C 12 alkyl)-OC(O)N(R 5 )2.

12. The immunoconjugate of claim 11, wherein R 2 is a C1-C8 alkyl group, and R 3 is -(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 .

13. The immunoconjugate of claim 12, wherein R 2 is -CH2CH2CH3, and R 3 Selected from -CH2CH2CH2NHCO2(t-Bu), -OCH2CH2NHCO2(cyclobutyl) and -CH2CH2CH2NHCO2(cyclobutyl).

14. The immunoconjugate of claim 12, wherein R 2 and R 3 Each is independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, -CH2CH2CF3, -OCH2CH2OH and -CH2CH2CH2OH.

15. The immunoconjugate of claim 12, wherein R 2 and R 3 Each is -CH2CH2CH3.

16. The immunoconjugate of claim 12, wherein R 2 is -CH2CH2CH3, and R 3 It is -OCH2CH3.

17. The immunoconjugate of any one of claims 1 to 7, wherein X 3 -R 3 Selected from the group consisting of:

18. The immunoconjugate of any one of claims 1 to 7, wherein X 4 is a key, and R 4 It's H.

19. The immunoconjugate of any one of claims 1 to 7, wherein R 1 Connect to L.

20. The immunoconjugate of any one of claims 1 to 7, wherein R 2 or R 3 Connect to L.

21. The immunoconjugate of claim 20, wherein X 3 -R 3 -L Select from the group consisting of: The wavy line indicates the connection point with N.

22. The immunoconjugate of any one of claims 1 to 7, wherein R 4 It is C1-C 12 alkyl.

23. The immunoconjugate of any one of claims 1 to 7, wherein R 4 Yes - (C1-C 12 Alkyldiyl)-N(R 5 )-*; wherein the asterisk * indicates the attachment site of L.

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

25. The immunoconjugate of any one of claims 1 to 7, wherein L is linked to a cysteine ​​thiol of the antibody.

26. The immunoconjugate 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.

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

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

29. The immunoconjugate of claim 28, wherein AA1 and AA2 are independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2; or AA1 and AA2 form a 5-membered ring proline amino acid.

30. The immunoconjugate of claim 28, wherein AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

31. The immunoconjugate of claim 28, wherein AA1 and AA2 are independently selected from GlcNAc aspartic acid, -CH2SO3H, and -CH2OPO3H.

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

33. The immunoconjugate of any one of claims 1 to 7, wherein L comprises PEP, and PEP is a tripeptide and has the formula:

34. The immunoconjugate of any one of claims 1 to 7, wherein L comprises PEP, and PEP is a tetrapeptide and has the formula:

35. The immunoconjugate of claim 34, wherein AA1 is selected from the group consisting of Abu, Ala and Val; AA2 is selected from the group consisting of Nle(O-Bzl), Oic and Pro; AA3 is selected from the group consisting of Ala and Met(O)2; and AA4 is selected from the group consisting of Oic, Arg(NO2), Bpa and Nle(O-Bzl).

36. The immunoconjugate of any one of claims 1 to 7, wherein L comprises PEP, and PEP is selected from the group consisting of 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).

37. The immunoconjugate of any one of claims 1 to 7, wherein L comprises PEP, and PEP is selected from the following structures:

38. The immunoconjugate of any one of claims 1 to 7, wherein L is selected from the following structures: The wavy line indicates the 5 connection.

39. The immunoconjugate of any one of claims 1 to 7, which has Formula Ia:

40. The immunoconjugate of claim 39, wherein X 4 is a key, and R 4 It's H.

41. The immunoconjugate of claim 39, wherein X 2 and X 3 Each is a bond, and R 2 and R 3 independently selected from C1-C8 alkyl, -O-(C1-C 12 Alkyl), -(C1-C 12 Alkyldiyl)-OR 5 、-(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 、-(C1-C 12 alkyl)-OC(O)N(R 5 )2. -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 and -O-(C1-C 12 alkyl)-OC(O)N(R 5 )2.

42. The immunoconjugate of claim 39, wherein X 2 It's O.

43. The immunoconjugate of claim 39, which is selected from Formula Ib-Ii:

44. The immunoconjugate of claim 43, wherein X 2 and X 3 Each is a bond, and R 2 and R 3 independently selected from C1-C8 alkyl, -O-(C1-C 12 Alkyl), -(C1-C 12 Alkyldiyl)-OR 5 、-(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 and -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 .

45. The immunoconjugate of claim 43, wherein X 2 and X 3 Each is a key, R 2 is a C1-C8 alkyl group, and R 3 Selected from -O-(C1-C 12 Alkyl) and -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 .

46. ​​8-Het-2-aminobenzazepine - a linker compound selected from Tables 2a and 2b.

47. An immunoconjugate comprising an anti-CEA antibody and an 8-Het-2-aminobenzazepine selected from Tables 2a and 2b. -linker compound conjugation.

48. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate according to any one of claims 1 to 7 and one or more pharmaceutically acceptable diluents, vehicles, carriers or excipients.

49. A method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of an immunoconjugate according to any one of claims 1 to 7, wherein the cancer is selected from cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer and breast cancer.

50. The method of claim 49, wherein the cancer is sensitive to a pro-inflammatory response induced by TLR7 and / or TLR8 agonism.

51. The method of claim 49, wherein the cancer is a CEA-expressing cancer.

52. The method of claim 49, wherein the breast cancer is triple-negative breast cancer.

53. The method of claim 49, wherein the Merkel cell carcinoma cancer is metastatic Merkel cell carcinoma.

54. The method of claim 49, wherein the cancer is gastroesophageal junction adenocarcinoma.

55. The method of claim 49, wherein the immunoconjugate is administered to the patient intravenously, intratumorally, or subcutaneously.

56. The method of claim 49, wherein the immunoconjugate is administered to the patient at a dose of about 0.01 to 20 mg / kg body weight.

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

58. A method for preparing an immunoconjugate of formula I as claimed in claim 1, wherein the 8-Het-2-amino-thienoazepine as claimed in claim 46 - A linker compound is conjugated to the anti-CEA antibody.

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