Heterotandem bicyclic peptide complexes

Abstract

To provide heterotandem bicyclic peptide complexes useful for preventing, suppressing or treating cancer and uses thereof.SOLUTION: The present invention relates to a heterotandem bicyclic peptide complex which comprises a first peptide ligand, which (a) binds to a component present on an immune cell, and (b) is conjugated via a linker to a second peptide ligand, which binds to a component present on a cancer cell, where each of the peptide ligands comprises a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for treating cancer cells comprising administering a linker to a second peptide ligand that binds to a component present on the cancer cell. A first peptide ligation that binds to a component present on immune cells, the first peptide ligation being conjugated via the The present invention also relates to a heterotandem bicyclic peptide complex containing the same. The present invention relates to the use of said heterotandem bicyclic peptide complexes in the prevention or treatment of diseases. [Background technology]

[0002] Cyclic peptides can bind to proteins with high affinity and target specificity; Thus, they are an attractive class of molecules for the development of therapeutic agents. The drug may be, for example, the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine or the anticancer drug Octreotide is already being used successfully in the clinic (Driggers et al. al. (2008), Nat Rev Drug Discov 7(7), 608-2 4) The excellent binding properties are due to the relatively large interaction surface formed between the peptide and the target. This is due to the reduced structural flexibility of the ring structure as well as the reduced conformational flexibility of the ring structure. Typically, macrocycles are For example, the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 ;Wu et al (2007), Science 330, 1066-71), integrin αVb3 Cyclic peptide with Arg-Gly-Asp motif (355 Å) 2 )(Xio ng et al. (2002), Science 296(5565), 151-5) or urokinase-type plasminogen activator-binding cyclic peptide inhibitor Upai upain-1 (603Å 2 ;Zhao et al.(2007),JS truct Biol 160(1),1-10) with a few hundred square angstroms Bind to the surface.

[0003] Due to their cyclic structure, peptide macrocycles are less flexible than linear peptides and are difficult to target. This results in less entropy loss during binding to the target, leading to higher binding affinity. In addition, it provides target-specific conformational locking, increasing binding specificity compared to linear peptides. This effect is due to the potent selective inhibition of matrix metalloproteinase 8 (MMP-8). This is exemplified by the inhibitor, which upon ring opening loses its selectivity against other MMPs. (Cherney et al. (1998), J Med Chem 41(11 ), 1749-51). Favorable binding properties achieved through macrocyclization are e.g. Polypeptides with two or more peptide rings, such as coimycin, nisin, and actinomycin, This is even more pronounced in cyclic peptides.

[0004] Various research teams have previously linked polypeptides containing cysteine ​​residues to synthetic molecular structures. (Kemp and McNamara (1985), J. Org. Chem;Ti mmerman et al. (2005), ChemBioChem). Meloen and coworkers have demonstrated the synthesis of multiple peptide groups on a synthetic scaffold to structurally mimic protein surfaces. Tris(bromomethyl)benzene and related molecules have been used for rapid and quantitative cyclization of aryl groups. (Timmerman et al. (2005), ChemBioChem). Cysteine-containing polypeptides can be synthesized, for example, by the molecule tris(bromomethyl)benzene. The method for generating candidate drug compounds by linking to a scaffold is described in WO 2005 / 023363. 004 / 077062 and WO 2006 / 078161.

[0005] Creating and screening large libraries of bicyclic peptides against targets of interest A phage display-based combinatorial approach has been developed to (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). In short, the three systems A linear peptide containing two regions of Cys- The combinatorial library (Xaa)6-Cys-(Xaa)6-Cys) was The cysteine ​​side chain was attached to a small molecule (tris(bromomethyl)benzene). The compound was cyclized by covalently linking it to Summary of the Invention

[0006] According to a first aspect of the present invention, (a) a first peptide ligand that binds to a component present on an immune cell, (b) A second peptide ligand that binds to a component present on a cancer cell is attached via a linker. A first peptide ligand conjugated with A heterotandem bicyclic peptide complex comprising: Each of the peptide ligands comprises at least two small loop sequences separated by at least two loop sequences. A polypeptide containing at least three cysteine ​​residues and at least two polypeptide groups. The peptide is covalently linked to a cysteine ​​residue in a polypeptide so that the peptide is formed on a molecular scaffold. and a molecular scaffold that binds the tandem bicyclic peptide.

[0007] According to a further aspect of the invention, the present invention provides a method for the preparation of a pharmaceutical composition comprising administering to a patient a therapeutically effective amount of the composition in combination with one or more pharma- ceutically acceptable excipients. In addition, a pharmaceutical composition comprising the heterotandem bicyclic peptide complex as defined herein is provided. Provided.

[0008] According to a further aspect of the invention, there is provided a method for the prevention, inhibition or treatment of cancer comprising administering to said subject matter, There is provided a heterotandem bicyclic peptide conjugate as defined herein. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of a heterotandem bicyclic peptide complex comprising EphA2 and CD137 peptide ligands bound to both immune and cancer cells. [Diagram 2] FIG. 1 shows the structure and composition of the EphA2-CD137 heterotandem bicyclic peptide complex BCY7985. [Diagram 3] FIG. 13 shows analysis of the EphA2-CD137 heterotandem bicyclic peptide complex BCY7985 in a Promega CD137 luciferase reporter assay (CS196008) in the presence of EphA2-expressing HT1080 cells. [Figure 4] FIG. 1 shows that EphA2 / CD137 heterotandems are active in CD137 reporter assays and that the fold-induction of activation is dependent on the tumor target expression level on the cell line used in the co-culture. [Diagram 5] FIG. 1 shows that EphA2 / CD137 heterotandem induces tumor cell killing in primary human T cell and cancer cell co-culture assays. Tumor cell killing is assessed by counting viable Nuclight red positive tumor cells over time. Caspase 3 / 7 dye is used to identify apoptotic tumor cells. [Figure 6]FIG. 1 shows that Nectin-4 / CD137 heterotandems are active in CD137 reporter assays and the fold-induction of activation depends on the tumor target expression levels on the cell lines used in co-culture (HT1376: Nectin-4 high and NCI-H292: Nectin-4 medium). [Figure 7] Figure 1 shows that Nectin-4 / CD137 heterotandem induces IL-2 and IFN-γ cytokine secretion in PBMC-4T1 co-culture assays. BCY9350 and BCY9351 are non-binding controls for Nectin-4 and CD137, respectively. [Figure 8] Figure 1 shows that Nectin-4 / CD137 heterotandem induces target-dependent cytokine release in ex vivo cultures of primary patient-derived lung tumors. (A) Ex vivo patient-derived tumor cells form 3D spheroids within 4 hours in culture, 10x light microscope image. (B) Flow analysis of Nectin-4 expression in patient-derived tumor samples from three donors. (C) Table shows % CD137+T cells and % Nectin-4+ cells in samples from three donors. (D) Heatmap showing % changes in immune markers (normalized to vehicle) in response to treatment with control / test compounds. (E) % CD8+ki67+T cells in response to treatment with control / test compounds (vehicle is shown as dotted line). [Figure 9] FIG. 2 shows that PD-L1 / CD137 heterotandems are active in CD137 reporter assays in the presence of the PD-L1 expressing cell line RKO. [Figure 10] Figure 13 shows the pharmacokinetics of heterotandems in SD rats: BCY10572 and BCY10000 were administered IV at 2 mg / kg (n=3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] According to a first aspect of the present invention, (b) A second peptide ligand that binds to a component present on a cancer cell is attached via a linker. Conjugated with (a) a first peptide ligand that binds to a component present on an immune cell; A heterotandem bicyclic peptide complex comprising: Each of the peptide ligands comprises at least two loop sequences separated by at least two loop sequences. A polypeptide containing at least three cysteine ​​residues and at least two polypeptide loops. The peptide is attached to a cysteine ​​residue of a polypeptide to form a covalent bond so that the peptide is formed on a molecular scaffold. and a molecular scaffold comprising:

[0011] First Peptide Ligand Reference herein to the term "immune cell" includes any cell in the immune system. Suitable examples include lymphocytes (e.g., T lymphocytes or T cells, B cells, or natural In one embodiment, the T cells are CD8 or C4 cells. In a further embodiment, the T cells are CD8. Other examples of immune cells include , dendritic cells, follicular dendritic cells and granulocytes.

[0012] In one embodiment, the component present on an immune cell is CD137.

[0013] CD137 is a member of the tumor necrosis factor (TNF) receptor family. The name is tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-IB. CD137 is induced by B and lymphocyte activation (ILA). It can be expressed by various T cells, but is more abundantly expressed on CD8+ T cells than on CD4+ T cells. In addition, CD137 expression is expressed on dendritic cells, follicular dendritic cells, natural killer cells, and granulocytes. It is found on cells of the inflammatory bowel and in the blood vessel walls at sites of inflammation. One characterized activity of CD137 is , and its costimulatory activity on activated T cells. Crosslinking of CD137 promotes T cell proliferation, IL-2 secretion, survival and cytolytic activity are enhanced. By using this method, immune activity to eliminate tumors can be enhanced.

[0014] CD137 is a T cell costimulatory receptor induced by TCR activation (Nam et al. al.,Curr.Cancer Drug Targets,5:357-363(2 005);Waits et al.,Annu.Rev,Immunol.,23:2 3-68(2005)). In addition to its expression on activated CD4+ and CD8+ T cells, CD137 also binds to CD4+CD25+ regulatory T cells, natural killer (NK) and It is also expressed on NK-T cells, monocytes, neutrophils and dendritic cells. Its natural ligand, CD137L is expressed on antigen-presenting cells, including B cells, monocytes / macrophages, and dendritic cells. (Watts et al. Annu. Rev. Immunol, 23:2 Upon interaction with its ligand, CD137 promotes increased TC R-induced T cell proliferation, cytokine production, functional maturation and extended CD8+ T cell survival (Nam et al, Curr. Cancer Drug Targets, 5:357-363(2005),Watts et dl.,Annu.Rev.I mmunol,23:23-68(2005)).

[0015] Agonistic monoclonal antibodies (mAbs) against CD137L or CD137 Signaling through CD137 induces increased TCR-induced T cell proliferation, cytokine production, and These effects result in increased viability and functional maturation, as well as extended CD8+ T cell survival. (1) NF-κB, c-Jun NH2-terminal kinase / stress-activated protein kinase JNK / SAPK and p38 mitogen-activated protein kinase (MAP K) activation of signal transduction pathways and (2) anti-apoptotic and cell cycle-related genes This is due to the control of offspring expression.

[0016] Experiments performed in mice lacking both CD137 and CD137L showed that they were fully competent. These results further demonstrate the importance of CD137 costimulation in generating tumor T cell responses.

[0017] IL-2 and IL-15-activated NK cells express CD137 and respond to agonistic mAbs Ligation of CD137 by CD4 stimulates NK cell proliferation and IFN-γ secretion, It does not stimulate the cytolytic activity of

[0018] Furthermore, CD137-stimulated NK cells promote the expansion of activated T cells in vitro.

[0019] Through its costimulatory function, agonistic mAbs against CD137 are involved in the regulation of cardiac and cutaneous Promotes rejection of seed grafts, eradicates established tumors, and induces primary antiviral CD8+ T cell responses These studies have shown that CD4+ enhances the proliferation of CD4+ and increases the cytolytic capacity of T cells. 137 Signaling promotes T cell function that can enhance immunity against tumors and infections. This supports the idea that...

[0020] In one embodiment, the first peptide ligand is a CD137-binding bicyclic peptide ligand. include.

[0021] Suitable examples of CD137-binding bicyclic peptide ligands are listed below. No. 1712589.9 and No. This is disclosed in US Pat. No. 1802934.8.

[0022] In one embodiment, the CD137 binding bicyclic peptide ligand has the amino acid sequence: C i IEEGQYC ii FADPY[Nle]C iii (SEQ ID NO:1); C i [tBuAla]PE[D-Ala]PYC ii FADPY[Nle]C iii (Distribution column number 3); C i IEEGQYC ii F[D-Ala]DPY[Nle]C iii (SEQ ID NO:4); C i [tBuAla]PK[D-Ala]PYC ii FADPY[Nle]C iii (Distribution column number 5); C i [tBuAla]PE[D-Lys]PYC ii FADPY[Nle]C iii (Distribution Column number 6); C i [tBuAla]P[K(PYA)][D-Ala]PYC ii FADPY [Nle ]C iii (SEQ ID NO:7); C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:8); C i IEE[D-Lys(PYA)]QYC ii FADPY(Nle)C iii (Sequence number No. 9); and [dC i ][dI][dE][dE][K(PYA)][dQ][dY][dC ii ][ dF][dA][dD][dP][dY][dNle][dC iii ] (SEQ ID NO: 10) ; (In the formula, C i , C ii and C iii are the first, second and third cysteine ​​residues, respectively. Nle stands for norleucine, tBuAla stands for t-butyl-alanine, and P YA stands for 4-pentynoic acid) or a pharma- ceutically acceptable salt thereof.

[0023] In one particular embodiment that may be mentioned, the CD137 binding bicyclic peptide ligand is Amino acid sequence: C i IEEGQYC ii FADPY[Nle]C iii (SEQ ID NO:1); (In the formula, C i , C ii and C iii are the first, second and third cysteine ​​residues, respectively. and Nle stands for norleucine). or a pharma- ceutically acceptable salt thereof.

[0024] In a further embodiment, the CD137 binding bicyclic peptide ligand comprises an N-terminal modification and Contains a C-terminal modification, Ac-A-(SEQ ID NO:1)-Dap (hereafter referred to as BCY7732); Ac-A-(SEQ ID NO:1)-Dap(PYA) (hereafter referred to as BCY7741); Ac-(SEQ ID NO:3)-Dap (hereafter referred to as BCY9172); Ac-(SEQ ID NO:3)-Dap(PYA) (hereinafter referred to as BCY11014); Ac-A-(SEQ ID NO:4)-Dap (hereafter referred to as BCY8045); Ac-(SEQ ID NO:5)-A (hereafter referred to as BCY8919); Ac-(SEQ ID NO:6)-A (hereafter referred to as BCY8920); Ac-(SEQ ID NO:7)-A (hereafter referred to as BCY8927); Ac-(SEQ ID NO:8)-A (hereafter referred to as BCY8928); Ac-A-(SEQ ID NO:9)-A (hereinafter referred to as BCY7744); and Ac-[dA]-(SEQ ID NO: 10)-[dA]-NH2 (hereinafter referred to as BCY11506) ); (In the formula, Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4- (representing pentynic acid) or a pharma- ceutically acceptable salt thereof.

[0025] In a further embodiment which may be mentioned, the CD137-binding bicyclic peptide ligand is including terminal and C-terminal modifications, Ac-A-(SEQ ID NO:1)-Dap (hereafter referred to as BCY7732); (In the formula, Ac represents an acetyl group, and Dap represents diaminopropionic acid.) or a pharma- ceutically acceptable salt thereof.

[0026] Second Peptide Ligand Reference herein to the term "cancer cells" refers to cells that are known to be involved in cancer. Cancer cells are formed when genes responsible for controlling cell division are damaged. Carcinogenesis is the process by which genes that cause abnormalities in normal cells that upset the normal balance between proliferation and cell death. It is caused by mutations and epimutations of genetic material. This leads to uncontrolled cell division and the evolution of these cells through natural selection within the body. The uncontrolled, usually rapid proliferation of cells can lead to benign or malignant tumors (cancer). Benign tumors do not spread to other parts of the body or invade other tissues. Tumors can invade other organs and spread to distant locations (metastasize) and become life threatening.

[0027] In one embodiment, the cancer cells are HT1080, SC-OV-3, PC3, H1376, Selected from NCI-H292, LnCap, MC38, 4T1-D02 and RKO tumor cells It is selected.

[0028] In one embodiment, the component present on a cancer cell is EphA2.

[0029] Eph receptor tyrosine kinases (Ephs) phosphorylate proteins on tyrosine residues It belongs to a large group of receptor tyrosine kinases (RTKs), which are kinases that act on the h and its membrane-bound ephrin ligands (ephrins) regulate cell positioning and tissue organization. (Poliakov et al. (2004) Dev Cell 7, 465- 80) Functional and biochemical Eph responses occur at higher ligand oligomerization states. (Stein et al. (1998) Genes Dev 12,667-67 8).

[0030] Among other patterning functions, various Ephs and ephrins play important roles in vascular development. Knockout of EphB4 and ephrin-B2 has been shown to play a role in , the inability to remodel capillary beds into blood vessels (Poliakov et al., supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrins leads to This has also been observed in newly formed adult microvessels (Brantley-Sieders et al.(2004)Curr Pharm Des 10,3431-42;Ad ams(2003)J Anat 202,105-12).

[0031] Dysregulated re-expression of some ephrins and their receptors in adults may mediate tumor invasion , and have also been observed to contribute to metastasis and angiogenesis (Nakamoto et al. (2002)Microsc Res Tech 59,58-67;Brantle In addition, several Eph family members Brain has been shown to be overexpressed on tumor cells from a variety of human tumors ( ntley-Sieders et al., supra; Marme (2002) Ann H ematol 81 Suppl 2,S66;Booth et al.(2002) Nat Med 8,1360-1).

[0032] In humans, EPH receptor A2 (ephrin type A receptor 2) is expressed by the EPHA2 gene. The encoded protein.

[0033] Eph2 is expressed in several human cancers, e.g., usually correlated with disease progression, metastasis, and poor prognosis. For example, breast cancer (Zelinski et al. (2001) Cancer Res. 61 ,2301-2306;Zhuang et al(2010)Cancer Res. 70,299-308;Brantley-Sieders et al(2011)P LoS One 6, e24426), lung cancer (Brannan et al (2009 )Cancer Prev Res (Phila)2,1039-1049;Kinc h et al(2003)Clin Cancer Res.9,613-618;G uo et al(2013)J Thorac Oncol.8,301-308), Gastric cancer (Nakamura et al. (2005) Cancer Sci. 96, 42 -47;Yuan et al(2009)Dig Dis Sci 54,2410- 2417), pancreatic cancer (Mudali et al (2006) Clin Exp Me tastasis 23,357-365), prostate cancer (Walker-Daniel s et al (1999) Prostate 41, 275-280), liver cancer (Y ang et al(2009)Hepatol Res.39,1169-1177) and glioblastoma (Wykosky et al (2005) Mol Cancer Res .3,541-551;Li et al(2010)Tumour Biol.31, 477-488) is upregulated.

[0034] Although the full role of EphA2 in cancer progression remains to be defined, tumor cells Evidence for interactions at multiple stages of cancer progression, including growth, survival, invasion and angiogenesis There is evidence that downregulation of EphA2 expression suppresses tumor cancer cell proliferation (Binda et al(2012) Cancer Cell 22,765-780), EphA2 Blockade of VEGF-induced cell migration (Hess et al (2001) Cancer Re s.61,3250-3255), sprouting and angiogenesis (Cheng et al. (20 02)Mol Cancer Res.1,2-11;Lin et al(2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sied ers et al (2005)FASEB J.19,1884-1886) do.

[0035] Antibody-drug conjugates to EphA2 inhibit tumor growth in rat and mouse xenograft models It has been shown to significantly reduce cer Research 68, 9367-9374), and a similar approach is being tested in humans. However, treatment had to be discontinued due to treatment-related adverse events ( Annunziata et al(2013)Invest New drugs 3 1,77-84).

[0036] In one embodiment, the second peptide ligand comprises an EphA2-binding bicyclic peptide ligand. include.

[0037] Suitable examples of EphA2-binding bicyclic peptide ligands are disclosed herein. No. 1721259.8 and No. It is disclosed in 1804102.0.

[0038] In one embodiment, the EphA2 binding bicyclic peptide ligand has the amino acid sequence: C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:2) ; and C i LWDPTPC ii ANLHL[HArg]C iii (SEQ ID NO:11); (In the formula, C i , C ii and Ciii are the first, second and third cysteine ​​residues, respectively. HyP stands for hydroxyproline, dD stands for aspartic acid in the D configuration, H Arg stands for homoarginine) or a pharma- ceutically acceptable salt thereof.

[0039] In one embodiment that may be mentioned, the EphA2-binding bicyclic peptide ligand has the amino acid sequence Column: C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:2) ; (In the formula, C i , C ii and C iii are the first, second and third cysteine ​​residues, respectively. HyP stands for hydroxyproline, dD stands for aspartic acid in the D configuration, H Arg stands for homoarginine) or a pharma- ceutically acceptable salt thereof.

[0040] In a further embodiment, the EphA2-binding bicyclic peptide ligand comprises an N-terminal modification. , A-HArg-D-(SEQ ID NO:2) (hereafter referred to as BCY9594); [B-Ala]-[Sar 10 ]-A-[HArg]-D-(SEQ ID NO: 2) (hereinafter, BC Call it Y6099); [PYA]-[B-Ala]-[Sar 10 ]-A-[HArg]-D-(SEQ ID NO: 2) (hereinafter referred to as BCY6169); and [PYA]-[B-Ala]-[Sar 10 ]-VGP-(SEQ ID NO: 11) (hereinafter, BC Call it Y8941); (wherein HArg represents homoarginine, PYA represents 4-pentynoic acid, and Sar10 represents 10 sarcosine units, and B-Ala represents β-alanine). or a pharma- ceutically acceptable salt thereof.

[0041] In further embodiments that may be mentioned, the EphA2-binding bicyclic peptide ligand is Including end modifications, A-HArg-D-(SEQ ID NO:2) (hereinafter referred to as BCY9594) (wherein HArg represents homoarginine) or a pharma- ceutically acceptable salt thereof.

[0042] In an alternative embodiment, the component present on a cancer cell is PD-L1.

[0043] Programmed cell death 1 ligand 1 (PD-L1) is expressed on mouse chromosome 19 and human chromosome 19. It is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on PD-L1 expression is associated with chronic infections, such as chronic viral infections (e.g., HI, among others). HBV, HCV, and HTLV), chronic bacterial infections (e.g., Helicobacter pylori, among others), Helicobacter pylori and chronic parasites infections (e.g., Schistosoma mansoni) PD-L1 expression is involved in evading immune responses involving T cells, B cells, and macrophages. Phages, dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, muscle cells, and placenta It has been detected in several tissues and cell types, including:

[0044] PD-L1 expression is also involved in suppressing antitumor immune activity. Tumors are inhibited by host T cells. Although tumors express antigens that can be recognized by the immune system, immunological elimination of tumors is rare. This is due to immunosuppression by the tumor microenvironment. PD-L1 expression in many tumors is a key factor in this It is a component of the suppressive environment and acts in conjunction with other immunosuppressive signals. Breast, lung, colon, ovary, melanoma, bladder, liver, saliva, stomach, glioma, thyroid, supraspinatus It has been shown in situ on a wide variety of solid tumors, including those of the skin, head and neck (Brown JA et al.2003 Immunol.170:1257-66;Dong H et al.2002 Nat.Med.8:793-800;Hamanishi J,et al.2007 Proc.Natl.Acad.Sci.USA 104 :3360-65;Strome SE et al.2003 Cancer Res .63:6501-5;Inman BA et al.2007 Cancer 10 9:1499-505;Konishi J et al.2004 Clin.Can cer Res.10:5094-100;Nakanishi J et al.20 07 Cancer Immunol.Immunother.56:1173-82; Nomi T et al.2007 Clin.Cancer Res.13:215 1-57;Thompson RH et al.2004 Proc.Natl.Ac ad.Sci.USA 101:17174-79;Wu C et al.2006 Acta Histochem.108:19-24). In addition, the receptor for PD-L1, Expression of programmed cell death protein 1 (also known as PD-1 and CD279) The expression of IL-1 is upregulated in tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression ( Blank C et al.2003 Immunol.171:4574-81). Most importantly, studies linking PD-L1 expression on tumors with disease outcome have shown that PD-L1 Expression is associated with poor prognosis in kidney, ovarian, bladder, breast, gastric and pancreatic cancers. It has been shown that the effect is strongly correlated with the improvement of the quality of life (Hamanishi J et al. 200 7 Proc.Natl.Acad.Sci.USA 104:3360-65;Inm an BA et al.2007 Cancer 109:1499-505;Kon ishi J et al.2004 Clin.Cancer Res.10:509 4-100;Nakanishi J et al.2007 Cancer Immu nol.Immunother.56:1173-82;Nomi T et al.2 007 Clin.Cancer Res.13:2151-57;Thompson RH et al.2004 Proc.Natl.Acad.Sci.USA 101 :17174-79;Wu C et al.2006 Acta Histochem 108:19-24). Furthermore, these studies have shown that higher levels of PD-L1 on tumors These results suggest that expression may promote tumor stage progression and invasion into deeper tissue structures. do.

[0045] The PD-1 pathway may also play a role in hematological malignancies. is expressed on multiple myeloma cells but not on normal plasma cells (Liu J et al.2007 Blood 110:296-304). PD-L1 is It is expressed on some primary T-cell lymphomas, especially anaplastic large cell T-lymphoma (Bro wn JA et al, 2003 Immunol. 170:1257-66). PD PD-L1 is highly expressed on T cells in angioimmunoblastic lymphoma and PD-L1 is associated Expressed on the follicular dendritic cell network (Dorfman DM et al. 200 6 Am. J. Surg. Pathol. 30:802-10). Nodular lymphocyte-predominant type In Hodgkin lymphoma, T cell associated lymphocytic or histiocytic (L&H) cells PD-1 expression. Microarray using readout of genes induced by PD-1 ligation Local array analysis shows that tumor-associated T cells express PD-1 in situ in Hodgkin lymphoma This suggests that the cerebellum responds to the signal (Chemnitz JM et al. 07 Blood 110:3226-33). PD-1 and PD-L1 are involved in the expression of HTLV- SH is expressed on CD4 T cells in SH-1-mediated adult T-cell leukemia and lymphoma imauchi T et al.2007 Int.J.Cancer 121:25 85-90) These tumor cells are hyporesponsive to TCR signals.

[0046] Studies in animal models have shown that PD-L1 on tumors blocks T cell activation and tumor cell lysis have demonstrated that tumor-specific T cell death is associated with increased tumor-specific T cell death in some cases (Don g H et al.2002 Nat.Med.8:793-800;Hirano F et al.2005 Cancer Res.65:1089-96). Tumor-related APCs can also utilize the PD-1:PD-L1 pathway to regulate antitumor T cell responses. PD-L1 expression on a population of tumor-associated myeloid DCs is upregulated by tumor environmental factors. (Curiel TJ et al. 2003 Nat. Med. 9:562-6 7) Plasmacytoid dendritic cells (DCs) in tumor-draining lymph nodes of B16 melanoma tumors are regulatory Regulatory T cells express IDO, which potently activates the suppressive activity of T cells. The suppressive activity of the cells required cell contact with IDO-expressing DCs (Sharma MD et al.2007 Clin.Invest.117:2570-82).

[0047] In one embodiment, the second peptide ligand is a PD-L1 binding bicyclic peptide ligand. include.

[0048] Suitable examples of PD-L1 binding bicyclic peptide ligands are listed below. No. 1820956.9 and No. This is disclosed in US Pat. No. 1820969.2.

[0049] In one embodiment, the PD-L1 bicyclic peptide ligand is C i [HArg]DWC ii HWTFSHGHPC iii (SEQ ID NO:12); C i SAGWLTMC ii QKLHLC iii (SEQ ID NO: 13); and C i SAGWLTMC ii Q[K(PYA)]LHLC iii (SEQ ID NO:14); (In the formula, C i , C ii and C iii are the first, second and third cysteine ​​residues, respectively. where HArg stands for homoarginine and PYA stands for 4-pentynoic acid. or a pharma- ceutically acceptable salt thereof.

[0050] In a further embodiment, the PD-L1 binding bicyclic peptide ligand comprises an N-terminal modification and and / or a C-terminal modification, [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO: 12) (hereinafter, BCY893 Call it 8); [PYA]-[B-Ala]-[Sar 10 ]-SDK-(SEQ ID NO: 13) (hereinafter, BC Call it Y10043); NH2-SDK-(SEQ ID NO:13)-[Sar 10 ]-[K(PYA)] (hereinafter referred to as BCY Call it 10044); NH2-SDK-(SEQ ID NO: 14) (hereinafter referred to as BCY10045); and Ac-SDK-(SEQ ID NO:14)-PSH (hereafter referred to as BCY10861); (wherein PYA represents 4-pentynoic acid, B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units) or a pharma- ceutically acceptable salt thereof.

[0051] In an alternative embodiment, the component present on cancer cells is Nectin-4.

[0052] Nectin-4 is a surface receptor that belongs to the nectin family of proteins, which contains four members. Nectins are essential proteins for epithelial, endothelial, immune and neuronal cells during development and adulthood. Cells play important roles in various biological processes such as cell polarity, proliferation, differentiation and migration. These are cell adhesion molecules that are involved in several pathological processes in humans. is the primary receptor for poliovirus, herpes simplex virus, and measles virus. Mutations in the genes encoding pectin-1 (PVRL1) or nectin-4 (PVRL4) Mutations in Nectin-4 cause ectodermal dysplasia syndrome, which is associated with other abnormalities. It is expressed during development. In adult tissues, its expression is higher than that of other members of the family. Nectin-4 is also restricted in 50%, 49% and 60% of breast, ovarian and lung cancers, respectively. It is a tumor-associated antigen in 86% of tumors, mostly on tumors with poor prognosis. Its expression is It was not detected in the corresponding normal tissues. In breast tumors, Nectin-4 was mainly expressed in triple negative tumors. It is expressed in ERBB2+ and ERBB2+ cancers. In the serum of patients with these cancers, Detection of the soluble form of nectin-4 is associated with poor prognosis. Serum nectin-4 levels are associated with poor prognosis in patients with metastatic disease. These results suggest that Nectin-4 may be an important therapeutic tool for cancer. These results suggest that Nectin-4 may be a reliable target. Antibodies have been described in the prior art, in particular enfortumab vedotin (ASG-22ME). is an antibody-drug conjugate (ADC) targeting Nectin-4 in patients with solid tumors. It is currently under clinical investigation for the treatment of patients.

[0053] In one embodiment, the second peptide ligand is a Nectin-4 binding bicyclic peptide ligand. Includes.

[0054] Suitable examples of Nectin-4 binding bicyclic peptide ligands include those cited herein. No. 1810250.9, incorporated herein by reference in its entirety. This is disclosed in US Pat. Nos. 815684.4 and 1818499.4.

[0055] In one embodiment, the Nectin-4 binding bicyclic peptide ligand is C i P[1Nal][dD]C iiM[HArg]DWSTP[HyP]WC iii (Distribution Column number 15; hereafter referred to as BCY8116); C i P[1Nal][dD]C ii M[HArg]D[dW]STP[HyP][dW] C iii (SEQ ID NO: 16; hereafter referred to as BCY11415); and C i P[1Nal][dK](Sar 10 -(B-Ala))C ii M[HArg]DW STP[HyP]WC iii (SEQ ID NO:17); C i PFGC ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 18; , called BCY11414); (In the formula, C i , C ii and C iii are the first, second and third cysteine ​​residues, respectively. , 1Nal represents 1-naphthylalanine, HArg represents homoarginine, and H yP stands for hydroxyproline, and Sar 10 represents 10 sarcosine units, and BA la stands for β-alanine) or a pharma- ceutically acceptable salt thereof.

[0056] In a further embodiment, the Nectin-4 binding bicyclic peptide ligand is optionally Including terminal modifications, SEQ ID NO:15 (hereinafter referred to as BCY8116); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO: 15) (hereinafter, BCY884 Call it 6); SEQ ID NO:16 (hereinafter referred to as BCY11415); [PYA]-[B-Ala]-[Sar10 ]-(SEQ ID NO: 16) (hereinafter, BCY119 Call it 42); Ac-(SEQ ID NO: 17) (hereinafter referred to as BCY8831); and SEQ ID NO:18 (hereinafter referred to as BCY11414); (wherein PYA represents 4-pentynoic acid, B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units) or a pharma- ceutically acceptable salt thereof.

[0057] In an alternative embodiment, the component present on the cancer cell is prostate specific membrane antigen (PSMA). do.

[0058] Prostate-specific membrane antigen (PSMA) (glutamic acid carboxypeptidase II (GCP II), N-acetyl-L-aspartyl-L-glutamic acid peptidase I (NAAL) ADase I) and NAAG peptidase (also known as FO It is an enzyme encoded by the LH1 (folate hydrolase 1) gene. Human GCPII It contains 750 amino acids and weighs approximately 84 kDa.

[0059] Human PSMA is highly expressed in the prostate, approximately 100-fold higher than in most other tissues In some prostate cancers, PSMA is the second most commonly upregulated gene product. The expression of β-lactamase is 8-12 times higher than the levels in non-cancerous prostate cells. Therefore, PSMA is a potential biomarker for therapy and imaging of several cancers. In human prostate cancer, tumors with higher expression are more likely to progress The time to disease is rapid and is accompanied by a high proportion of patients suffering from recurrence.

[0060] In one embodiment, the second peptide ligand comprises a PSMA-binding bicyclic peptide ligand. nothing.

[0061] Suitable examples of PSMA-binding bicyclic peptide ligands are listed below, where the peptide is cited. No. 1810318.4 and No. 1810318.5, both of which are incorporated herein by reference. 0325.9 and 1820325.7.

[0062] Linker The first peptide ligand is linked to the second peptide ligand via any suitable linker. It will be appreciated that the linker can be conjugated to the The design involves two bicyclic peptides that bind either alone or simultaneously to both target receptors. such that each of the target molecules is provided with unhindered binding to its respective target. Additionally, the linker maintains the appropriate distance between target cells to produce the desired functional outcome. The linker properties should be tailored to suit the desired function while allowing simultaneous binding to both targets. The length, stiffness, or solubility of the peptide can be adjusted to increase the peptide's activity to optimize therapeutic outcomes. The linker can also be designed to allow two or more bicycles to be attached to the same target. Increasing the valency of either binding peptide can enhance the heterologous binding to target cells. This may help increase the affinity of the tandem or target receptors for one or both of the This may help induce oligomerization.

[0063] In one embodiment, the linker has the following sequence: -CH2-, -PEG5-, -PEG 10 -,-PEG 12 -,-PEG 23 -,-PEG 24 -,-PEG15 -Sar5-,- PEG 10 -Sar 10 -,-PEG5-Sar 15 -, -PEG5-Sar5-, -B -Ala-Sar 20 -,-B-Ala-Sar 10 -PEG 10 -,-B-Ala-S ar5-PEG 15 - and B-Ala-Sar5-PEG5-.

[0064] Structural representations of suitable linkers are detailed below.

[0065] [ka]

[0066] [ka]

[0067] Heterotandem complexes In one specific embodiment, the first peptide ligand is a C D137-binding bicyclic peptide ligand, wherein the second peptide ligand is a TATA scaffold. wherein the heterotandem complex comprises an EphA2-binding bicyclic peptide ligand bound to

[0068] [Table 1] is selected from.

[0069] Heterotandem bicyclic peptide conjugate BCY7985 is a PEG- 12 Via EphA 2. CD137-specific peptide linked to the N-terminal PYA group of the specific peptide BCY6169 The enzyme is BCY7859 (pictured in Figure 2).

[0070] CD137 is a homotrimeric protein, and its natural ligand, CD137L, is expressed on immune cells. It exists as a homotrimer that is expressed in the immune system or secreted. Cells are highly dependent on multimerization to induce CD137 activity. One way in which CD4+ receptors can be produced is through interactions with specific receptors present on other cells. 137-specific agonist cell crosslinking.

[0071] EphA2 is highly expressed on tumor cells and its receptor is upregulated by ephrin-A ligands. Oligomerization of tyrosine kinases drives their activation. Although not limited to the above, the present inventors have demonstrated that one CD137-specific peptide is coupled to one The EphA2-CD137 heterotandem, consisting of EphA2-specific peptides, was expressed on CD1 37. It appears that CD137 acts to bind to cells, such as tumor cells. It appears that EphA2 is involved in the localization and activation of EphA2. This would drive CD137 immune cell activation in the tumor environment (Figure 1).

[0072] This hypothesis was tested in the CD137 cell activity reporter assay described herein, The results are shown herein in FIG. 3, in which BCY7985 inhibited EphA2-expressing HT1080 Promega CD137 luciferase reporter assay (CS1 96008) showed a strong induction of CD137 cell activity.

[0073] In one alternative specific embodiment, the first peptide ligand is bound to a TATA scaffold. and a second peptide ligand, A scaffold is bound to a nectin-4 binding bicyclic peptide ligand, The complex is

[0074] [Table 2-1] [Table 2-2] is selected from.

[0075] Without being bound by theory, the inventors have determined that one CD137-specific peptide Nectin-4-CD consists of one nectin-4 specific peptide coupled to a tide 137 heterotandems express CD137, similar to those previously described for EphA2. It is believed to act as a bridge.

[0076] In one embodiment, the Nectin-4-CD137 heterotandem is Other than one or more of CY11858 and / or BCY11859 be.

[0077] In one alternative specific embodiment, the first peptide ligand is bound to a TATA scaffold. and a second peptide ligand, A scaffold is attached to a PD-L1-binding bicyclic peptide ligand, The combination,

[0078] [Table 3] is selected from.

[0079] Without being bound by theory, the inventors have determined that one CD137-specific peptide PD-L1-CD13, consisting of a single PD-L1-specific peptide coupled to a CD13 peptide 7 heterotandem crosslinks CD137, similar to that previously described for EphA2. We believe that this will have an effect on

[0080] Unless otherwise defined, all technical and scientific terms used herein refer to peptides. and those skilled in the art, such as those in the fields of biochemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. The term "common sense" has the same meaning as commonly understood by those skilled in the art. Standard techniques are used for molecular biology, genetics and biochemistry methods, which shall form part of the (Sambrook et al., Molecular Cloning: A La boratory manual,3rd ed.,2001,Cold Spring Harbor Laboratory Press,Cold Spring Har bor,NY;Ausubel et al.,Short Protocols in Molecular Biology(1999)4th ed.,John Wil (See ey&Sons, Inc.).

[0081] nomenclature Numbering When referring to amino acid residue positions within the compounds of the invention, a cysteine ​​residue (C i , C ii and C iii ) is omitted from the numbering because it is invariant, so the amino acid The residue numbering is C i -I1-E2-E3-G4-Q5-Y6-C ii -F7-A8-D9-P10 -Y1 1-[Nle] 12 -C iii (SEQ ID NO:1) It is called.

[0082] For purposes herein, all bicyclic peptides are referred to as TBMB (1,3,5-Tris( bromomethyl)benzene) or 1,1',1''-(1,3,5-triazinane-1, 1,1',1''-(1,3,5-triazinane-1,3 It is hypothesized that the cyclization with 1,5-triyl)triprop-2-en-1-one (TATA) leads to a trisubstituted structure. Cyclization with TBMB and TATA is i , C ii and C iii occurs in.

[0083] Molecular Format N- or C-terminal extensions to the bicyclic core sequence are added to the left or right side of the sequence, For example, the N-terminal βAla-Sar10-Ala tail is It is represented as βAla-Sar10-A-(SEQ ID NO:X).

[0084] Reverse peptide sequence Nair et al(2003)J Immunol 170(3),1362-1 In light of the disclosure of 373, it is understood that the peptide sequences disclosed herein are not intended to be construed as being similar to those disclosed in 373, but are instead intended to be It is anticipated that retro-inverso forms may also find utility. For example, , the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and vice versa), and The stereochemistry of is similarly reversed (i.e., the D-amino acid becomes the L-amino acid, and and vice versa).

[0085] Peptide Ligands A peptide ligand as referred to herein refers to a peptide covalently attached to a molecular scaffold. Typically, such peptides contain two or more peptides capable of forming covalent bonds with the scaffold. reactive groups (i.e., cysteine ​​residues) and when the peptide is attached to the scaffold Since a loop is formed, the sequence defined between the reactive groups is called a loop sequence. In this case, the peptide contains at least three cysteine ​​residues (referred to herein as C i , C ii and C iii ), which form at least two loops on the scaffold.

[0086] Pharmaceutically acceptable salts Salt forms are within the scope of the present invention and references to peptide ligands include salt forms of said ligands. It will be understood that this includes:

[0087] The salts of the present invention are described in Pharmaceutical Salts: Properties, Selection,and Use,P. Heinrich Stahl(Edit or), Camille G. Wermuth (Editor), ISBN:3-906 39-026-8, Hardcover, 388 pages, August 2002 The parent compound containing a basic or acidic moiety can be synthesized by conventional chemical methods, such as those described herein. Generally, such salts can be synthesized from the free acid forms of these compounds. or the free base form, in water or an organic solvent, or a mixture of the two, with an appropriate base or acid It can be prepared by reacting

[0088] Acid addition salts (monosalts or disalts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbyl acetate, and the like. ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, Benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+) camphoric acid, camphorsulfonic acid , (+)-(1S)-Camphor-10-sulfonic acid, Capric acid, Caproic acid, Caprylic acid Acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfone Acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid, glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, Licholic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), Isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobion Acid, Maleic acid, Malic acid, (-)-L-Malic acid, Malonic acid, (±)-DL-Mandelic acid Acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfone Acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, Uronic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamine Acid, Salicylic Acid, 4-Aminosalicylic Acid, Sebacic Acid, Stearic Acid, Succinic Acid, Sulfuric Acid, Tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid and valeric acid, and acylated amino acids and cation exchange resins. Examples of the salt include mono- or di-salts formed with a selected acid.

[0089] One particular group of salts is acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, Lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid , toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylic acid), ethanesulfonic acid, naphtha Thalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid and lactate It comprises salts formed from tobionic acid. One particular salt is the hydrochloride salt. Another particular salt is It is an acetate salt.

[0090] The compound is anionic or has a functional group that can become anionic (e.g., - COOH is -COO - In the case of alkoxysilanes, salts are formed with organic or inorganic bases, as appropriate. Examples of suitable inorganic cations include, but are not limited to, L i + , Na + and K. + Alkali metal ions such as Ca 2+ and Mg 2+ Al etc. Potassium earth metal cations, and Al 3+ or Zn + Other cations include Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., Wow, NH4 + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + Examples of some suitable substituted ammonium ions are: , methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine amine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine Nolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglutamine amine and tromethamine, and are derived from amino acids such as lysine and arginine. A common example of a quaternary ammonium ion is N(CH3)4 + It is.

[0091] When the compounds of the invention contain amine functions, these can be, for example, cleaved by methods well known to those skilled in the art. Thus, by reaction with an alkylating agent, a quaternary ammonium salt can be formed. Such quaternary ammonium compounds are within the scope of the present invention.

[0092] modified derivatives It is understood that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. It will be appreciated that examples of such suitable modified derivatives include N-terminal modifications and / or C-terminal modification; one or more amino acid residues are modified by one or more non-natural amino acid residues Replacement of one or more equivalent or isoelectronic amino acid residues of one or more polar amino acid residues replacement of one or more non-polar amino acid residues with other non-natural equivalents or equivalents replacement with an electrophilic amino acid); addition of a spacer group; one or more oxidation-sensitive Replacement of one or more amino acid residues with oxidation-resistant amino acid residues; replacement of one or more L-amino acid residues with alanine; replacement of one or more D-amino acid residues; one or more in a bicyclic peptide ligand N-alkylation of several amide bonds; replacement of one or more peptide bonds by surrogate bonds modification of peptide backbone length; replacement of hydrogen on the α-carbon of one or more amino acid residues Substitution by chemical groups: Cysteine, Lysine, Glutamic / Aspartic Acid, and Tyrosine and the like, suitable amines, thiols, carboxylates, etc., for functionalizing said amino acids. Orthogonal functional groups suitable for modification with acid- and phenol-reactive reagents, respectively Allows functionalization with amino acids that introduce reactive groups, e.g., azides, or alkynes one selected from an alkyne-containing amino acid, or the introduction or replacement of an azide-containing moiety; Or multiple modifications may be included.

[0093] In one embodiment, the modified derivative comprises an N-terminal modification and / or a C-terminal modification. In embodiments, the modified derivatives include N-terminal modifications using appropriate amino-reactive chemistry, and / or or C-terminal modification using appropriate carboxy-reactive chemistry. The N- or C-terminal modification may be, but is not limited to, a cytotoxic agent, a radioactive agent, These include the addition of effector groups, including photoactivators or chromophores.

[0094] In a further embodiment, the modified derivative comprises an N-terminal modification. The end modification comprises an N-terminal acetyl group. In this embodiment, an N-terminal cysteine ​​group (as defined herein) is C i The carboxyl group, called the carboxyl group, is capped with acetic anhydride or other suitable reagent during peptide synthesis. This embodiment is an aminopeptidase This offers the advantage of eliminating potential recognition points for the cyclic peptides, and reduces the possibility of degradation of the bicyclic peptides. Avoid sex.

[0095] In an alternative embodiment, the N-terminal modification is in the form of a conjugation of an effector group and a bicyclic This involves the addition of molecular spacer groups which facilitate retention of efficacy of the peptide on its target.

[0096] In a further embodiment, the modified derivative comprises a C-terminal modification. The terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine ​​group (referred to herein as C iii During peptide synthesis, the C-terminal amidated group is synthesized as an amide. This embodiment eliminates potential recognition points for carboxypeptidases. This provides the advantage of removing the carboxyl group from the bicyclic peptide, reducing the likelihood of proteolysis of the bicyclic peptide.

[0097] In one embodiment, the modified derivative comprises one or more non- In this embodiment, the amino acid residues are replaced by natural amino acid residues. Non-natural arylsulfates with equivalent / isoelectronic side chains that are not recognized by the agonist and have no adverse effects on target efficacy. amino acids may be selected.

[0098] Alternatively, proteolytic hydrolysis of nearby peptide bonds may result in conformational and and non-natural amino acids with constrained amino acid side chains are used so that they are sterically hindered. In particular, these include proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g. , aminoisobutyric acid, Aib) and cycloamino acids, aminocyclopropyl carboxylic acids. Concerning certain simple derivatives.

[0099] In one embodiment, the modified derivative comprises the addition of a spacer group. The derivative has an N-terminal cysteine ​​(C i ) and / or C-terminal cysteine ​​(C iii ) This includes the addition of a spacer group to the

[0100] In one embodiment, the modified derivative comprises one or more oxidation-sensitive amino acid residues. comprises replacement with multiple oxidation-resistant amino acid residues. The body contains replacement of tryptophan residues with naphthylalanine or alanine residues. This embodiment provides a method for determining the pharmaceutical stability profile of the resulting bicyclic peptide ligand. This provides the advantage of improving the

[0101] In one embodiment, the modified derivative comprises one or more of the charged amino acid residues. In an alternative embodiment, the modified derivative comprises the replacement of one or more replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is essential for the synthesis of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding. It affects the concentration of the free available fraction in plasma, while the charged Amino acid residues (especially arginine) affect the interaction of peptides with phospholipid membranes on cell surfaces. The two combine to affect the half-life, volume of distribution, and exposure of peptide drugs. The dose can affect the exposure and can be adjusted according to clinical endpoints. In addition, the correct combination and number of charged versus hydrophobic amino acid residues is required to ensure optimal delivery at the injection site. This may reduce irritation of the endothelial cells (when the peptide drug is administered subcutaneously).

[0102] In one embodiment, the modified derivative comprises one or more of the L-amino acid residues. with a D-amino acid residue. This embodiment is advantageous in that it avoids steric hindrance and D-amino Increased proteolytic stability due to the tendency of acids to stabilize β-turn conformations (Tugyi et al. (2005) PNAS, 102(2), 4 13-418).

[0103] In one embodiment, the modified derivative comprises the removal of any amino acid residue and replacing it with alanine. This embodiment includes the advantage of removing potential proteolytic attack site(s). Provides benefits.

[0104] Each of the above modifications may serve to deliberately improve the potency or stability of the peptide. It should be noted that further improvements in potency based on modifications include: - Utilizing the hydrophobic effect to achieve higher affinity and lower dissociation rates Incorporating hydrophobic moieties; - Utilizes long-range ionic interactions resulting in faster association rates and higher affinity Incorporating charged groups that can be used to ectrostatically assisted association of proteins(1996),Nature Struct.Biol.3,427- 31); and - For example, amino acid side chains should be oriented correctly to minimize entropy loss upon target binding. The torsion angles of the backbone are tightly constrained to minimize entropy loss upon target binding. For the same reason, additional constraints can be introduced by introducing additional cyclization into the molecule. Incorporating bundles into peptides This can be achieved through a mechanism (see Gentilucci et al. ,Curr.Pharmaceutical Design,(2010),16,31 85-203, and Nestor et al, Curr. Medicinal Ch. em(2009), 16, 4399-418).

[0105] Isotope Variants The present invention relates to an element in which one or more atoms have the same atomic number but are different from those commonly found in nature. The atomic mass or mass number of the atom is replaced by an atom having a different atomic mass or mass number. All pharma- ceutically acceptable (radio)isotope-labeled peptide ligands of the present invention, and metal chelating groups capable of carrying related (radioactive) isotopes ( The peptide ligands of the present invention (referred to as "effectors") and certain functional groups are associated with The present invention relates to a method for the preparation of a compound according to the present invention, which is covalently substituted with a (radioactive) isotope or an isotopically labeled functional group. The peptide ligands include:

[0106] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include: 2 H(D) and 3 Hydrogen isotopes such as H(T), 11 C. 13 C and 14 Carbon isotopes such as C, 36 Isotopes of chlorine such as Cl, 18 Fluorine isotopes such as F, 123 I, 125 I and 13 1 Iodine isotopes such as I 13 N and 15 Nitrogen isotopes such as N 15 O. 17 O Call 18 Oxygen isotopes such as O 32 Isotopes of phosphorus such as P 35 Sulfur isotopes such as S , 64 Copper isotopes such as Cu,67 Ga or 68 Gallium isotopes such as Ga, 90 Y Isotopes of yttrium such as 177 Ruthenium isotopes such as Lu, 213 Bi isotopes of bismuth such as Bi.

[0107] Certain isotopically labeled peptide ligands of the invention, e.g., those incorporating a radioisotope. in drug and / or substrate tissue distribution studies, as well as on diseased tissues The peptides of the present invention are useful for clinically assessing the presence and / or absence of a target. Ligands are used to bind labeled compounds to other molecules, peptides, proteins, enzymes or receptors. This is a useful diagnostic in that it can be used to detect or identify the formation of complexes Detection or identification methods include radioisotope, enzyme, fluorescent, etc. Substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, etc. Compounds labeled with labeling agents such as fluorouracil and luciferase can be used. The isotope tritium, i.e. 3 H(T) and carbon-14, i.e. 14 C is a group Given their ease of incorporation and rapid means of detection, they are particularly useful for this purpose.

[0108] Deuterium, i.e. 2 Substitution with heavier isotopes such as H(D) provides greater metabolic stability. Certain therapeutic advantages resulting from qualitative differences, such as increased in vivo half-life or reduced dosage requirements. A reduction can be achieved and may therefore be preferred in some circumstances.

[0109] 11 C. 18 F,15 O and 13 Substitution with positron emitting isotopes such as N improves target occupancy. Positron Emission Tomography (PETO) for investigating the rate of Positive echocardiography (PET) may be useful.

[0110] Isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared using conventional methods known to those skilled in the art. Depending on the technique, or by using appropriate isotope-labeled reagents in place of previously used non-labeled reagents and can be prepared by methods similar to those described in the accompanying examples.

[0111] Molecular scaffolds Molecular scaffolds are described, for example, in WO 2009 / 098450 and the compounds cited therein. References, in particular WO 2004 / 077062 and WO 2006 / 078 It is described in issue 161.

[0112] As mentioned in the above document, the molecular scaffold may be a small molecule, such as a small organic molecule.

[0113] In one embodiment, the molecular scaffold can be a polymer. In one embodiment, the molecular scaffold can be an amino They are polymers made up of acids, nucleotides, or carbohydrates.

[0114] In one embodiment, the molecular scaffold reacts with a functional group(s) of the polypeptide to form a covalent bond. The reactive group is capable of forming

[0115] The molecular scaffolds are amines, thiols, alcohols, ketones, aldehydes, nitriles, carbo Acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, Chemical groups that form bonds with peptides, such as alkyl halides and acyl halides, It may include.

[0116] In one embodiment, the molecular scaffold is a hexahydro-1,3,5-triazine, in particular a 1,3, 5-Triacryloylhexahydro-1,3,5-triazine ("TATA") or its It may comprise or consist of a derivative of

[0117] In one embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to 1,3,5-tris(bromomethyl)benzene (TBMB), but It contains three additional methyl groups attached to the benzene ring. This is because the additional methyl groups are The advantage of this is that it can form additional contacts with the peptide, thus adding additional structural constraints. Yes.

[0118] The molecular scaffolds of the invention are characterized in that the functional groups of the polypeptides of the encoded library of the invention are The chemical group comprises a chemical group that allows for the formation of a covalent bond with a molecular scaffold. amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, Alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides The functional groups are selected from a wide range of groups including acyl and acyl halides.

[0119] Scaffold reactive groups that can be used on the molecular scaffold to react with the thiol group of cysteine ​​include: Alkyl halides (also called halogenoalkanes or haloalkanes) do.

[0120] Examples include bromomethylbenzene (a scaffold reactive group exemplified by TBMB) or Examples of suitable cysteine-binding sites include iodoacetamide. Other scaffold reactive groups used for coupling are maleimides, αβ-unsaturated carbonyls, The molecular scaffolds used in the present invention are α-halomethylcarbonyl-containing compounds and α-halomethylcarbonyl-containing compounds. Examples of maleimides that can be used include tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)amine, bis-(2-maleimidoethyl)benzene, tris-(maleimido)benzene Examples of αβ-unsaturated carbonyl-containing compounds are 1,1',1''-(1,3,5-triazine Nan-1,3,5-triyl)triprop-2-en-1-one (TATA) (Ange wandte Chemie,International Edition(2014 ), 53(6), 1602-1606). An example is N,N',N''-(benzene-1,3,5-triyl)tris(2-bromoacetate). Selenocysteine ​​is also a cysteine ​​analogue, and is capable of undergoing the same reactions. Thus, unless the context suggests otherwise, Whenever cysteine ​​is mentioned, substitution with selenocysteine ​​is typically permitted. It is tolerated.

[0121] synthesis The peptides of the invention can be synthetically produced by standard techniques and subsequently purified in vitro. The molecule may be reacted with the molecular scaffold using standard chemistries to accomplish this. This allows for rapid, large-scale preparation of soluble material for further downstream experimentation or validation. Such methods include conventional methods such as those disclosed in Timmerman et al. (supra). This could be achieved using the chemistry

[0122] Thus, the present invention also relates to a method for the preparation of a selected polypeptide or conjugate as set forth herein. The present invention relates to the manufacture of a gate, the manufacture including any further steps described below. In one embodiment, these steps are carried out on a final product polypeptide prepared by chemical synthesis. This is done on the tide / conjugate.

[0123] When producing a conjugate or complex, it is sometimes necessary to select an agonist in the polypeptide of interest. The amino acid residue may be substituted.

[0124] The peptide can be extended, for example, to incorporate additional loops, thus introducing multiple specificities. It is also possible.

[0125] To extend the peptide, orthogonally protected orthogonally protected lysine (and analogues) To this end, peptides can be easily chemically extended at their N- or C-terminus or within loops. Standard (bio)conjugation techniques can be used to activate or can introduce an activatable N-terminus or C-terminus. Alternatively, for example, wson et al.1994.Synthesis of Proteins by Native Chemical Ligation.Science 266:77 6-779) by fragment condensation or native chemical ligation. Therefore, or (Chang et al. Proc Natl Acad Sci U S A.1994 Dec 20;91(26):12544-8 or Hikar i et al Bioorganic&Medicinal Chemistry L etters Volume 18, Issue 22,15 November 20 08, Pages 6000-6003 The addition can be carried out enzymatically using a ribozyme (ribozyme Gase).

[0126] Alternatively, the peptide can be conjugated via a disulfide bond. The first and second peptides can be extended or modified by the addition of a peptide to the cellular It has the added advantage of allowing them to dissociate from one another once in the environment. In this case, A molecular scaffold (e.g., TBMB) is added during the chemical synthesis of the first peptide to form the three systems. Then, additional cysteines or thiols can be reacted with the first peptide group; The peptide is then added to the N- or C-terminus of the peptide, resulting in only this cysteine ​​or thiol being the first 2 reacts with a free cysteine ​​or thiol of the peptide to produce a disulfide-linked bicyclic peptide. A peptide-peptide conjugate could be formed.

[0127] The same technique is equally applicable to the synthesis / coupling of two bicyclic and bispecific polymers In this way, tetraspecific molecules are potentially created.

[0128] Additionally, suitable coupling chemistries at the N-terminus or C-terminus or via side chains are available. The addition of other functional or effector groups can be similarly accomplished using In embodiments, the coupling is performed so as not to inhibit the activity of either entity.

[0129] Pharmaceutical Compositions According to a further aspect of the invention, the present invention provides a method for the preparation of a pharmaceutical composition comprising administering to a patient a therapeutically effective amount of the composition in combination with one or more pharma- ceutically acceptable excipients. Additionally, pharmaceutical compositions comprising the peptide ligands defined herein are provided.

[0130] Generally, the peptide ligand is combined with a pharmacologically appropriate excipient or carrier and Typically, these excipients or carriers include saline and and / or buffered media, aqueous or alcoholic / aqueous solutions, emulsions Parenteral vehicles include sodium chloride solution, Ringer's dextrose, and the like. These include sugar, dextrose, and sodium chloride and lactated Ringer's solution. If necessary to maintain the polypeptide complex in suspension, an appropriate physiologically acceptable The adjuvants are carboxymethylcellulose, polyvinylpyrrolidone, gelatin and a The thickening agent may be selected from thickening agents such as arginates.

[0131] Intravenous vehicles include those based on Ringer's dextrose that provide fluids and nutrients. Fluid replacement and electrolyte replacement. Antimicrobials, antioxidants, chelating agents and inert gases. Preservatives and other additives such as sucrose may also be present (Mack (1982) Rem. ington's Pharmaceutical Sciences,16th Ed ition).

[0132] The peptide ligands of the present invention can be administered either as a separately administered composition or in combination with other agents. These can be used in combination with antibodies, antibody fragments, and cyclosporine, methotrexate, and other anti-inflammatory drugs. Various immunotherapeutic drugs, such as Rexartan, Adriamycin or Cisplatin, and immunotherapy The pharmaceutical composition may contain a protein ligand of the invention or a toxin prior to administration. Polypeptides selected using various targeting ligands, whether pooled or not. Combinations of selected polypeptides according to the present invention having different specificities, such as The therapeutic agent may include a "cocktail" of various cytotoxic or other agents in combination with

[0133] The route of administration of the pharmaceutical composition according to the present invention may be any of those commonly known to those skilled in the art. For therapy, the peptide ligands of the invention can be administered in any form according to standard techniques. The administration can be parenteral, intravenous, intramuscular, intraperitoneal, transdermal, or pulmonary. via any suitable route, including by direct injection via a catheter, as appropriate. Preferably, the pharmaceutical composition according to the present invention is administered by inhalation. The dosage and frequency of administration should be determined based on the age, sex, and condition of the patient, concurrent administration of other drugs, and prohibited substances. It depends on the contraindications, as well as other parameters that should be taken into account by the clinician.

[0134] The peptide ligands of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and is compatible with freezing methods known in the art. Drying and reconstitution techniques can be used. Freeze-drying and reconstitution can be performed to varying degrees. This can result in loss of activity and the levels must be adjusted upward to compensate. It will be appreciated by those skilled in the art that this may be the case.

[0135] Compositions containing the peptide ligands or cocktails thereof are used for prophylactic and / or therapeutic purposes. In certain therapeutic applications, the administration of a selected population of cells At least partial inhibition, suppression, modulation, killing or some other measurable parameter An amount sufficient to achieve this is defined as a "therapeutically effective dose." The amount required to administer the drug depends on the severity of the disease and the overall state of the patient's own immune system. Typically, 0.005-5.0 mg of selected peptide ligase per kilogram of body weight is administered. The range is from 0.05 to 2.0 mg / kg / dose, with doses of 0.05 to 2.0 mg / kg / dose being more commonly used. In therapeutic applications, compositions containing the peptide ligands or cocktails thereof may also be used in the same manner. or slightly lower doses.

[0136] Compositions containing peptide ligands according to the invention are useful for the modulation of selected target cell populations in mammals. These agents can be utilized in prophylactic and therapeutic settings to aid in the modification, inactivation, killing or removal of pathogens. Additionally, the peptide ligands described herein can be used to identify target cell populations from heterogeneous populations of cells. In vitro or ex vivo selection to kill, deplete, or effectively remove a group Blood from the mammal can be purified by standard techniques to select peptide fragments. Combine it with a ribozyme in vitro, thereby killing unwanted cells, or return it to the mammal. can be removed from the blood.

[0137] therapeutic use According to a further aspect of the invention there is provided a method for preventing, inhibiting or treating cancer.

[0013] Provided is a heterotandem bicyclic peptide complex as defined herein.

[0138] Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include Tumors of epithelial origin (various types including, but not limited to, adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other carcinomas) various types of adenomas and carcinomas), e.g., bladder and urinary tract, breast, gastrointestinal tract (esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas kidney, lung (e.g., adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma and mesothelial carcinoma) tumors), head and neck (e.g., tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity and Cancer of the paranasal sinuses), ovaries, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, and uterus Membranes, thyroid (e.g., follicular thyroid carcinoma), adrenal glands, prostate, skin and adnexa (e.g., black cancers (i.e. keratoacanthoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies (i.e. leukemia, lymphoma) and hematological malignancies and associated conditions of the lymphatic system (e.g. , acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphoma, e.g. For example, diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK ] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal immunoglobulin of undetermined significance erythrocyte sedimentation, plasmacytoma, multiple myeloma and post-transplant lymphoproliferative disorder), and hematology Malignancies and myeloid lineage-related conditions (e.g., acute myeloid leukemia [AML], chronic bone marrow disease, Myelogenous leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders Proliferative disorders, e.g., polycythemia vera, essential thrombocythemia and primary myelofibrosis, myeloproliferative disorders Premalignant blood disorders and borderline disorders, including myelodysplastic syndromes and promyelocytic leukemia malignant disorders; tumors of mesenchymal origin, such as sarcomas of the soft tissue, bone or cartilage, e.g. osteosarcoma; Fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytoma, and dermatofibrosarcoma protuberans; tumors of the central and peripheral nervous system (e.g., astrocytomas, gliomas, and endocrine tumors (e.g., glioblastomas, meningiomas, ependymomas, pineal tumors, and schwannomas); Pituitary tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and thyroid medullary tumors ophthalmic and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., for example, teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and pediatric and and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumors) tumors); or congenital or other syndromes that predispose the patient to malignancies ( For example, xeroderma pigmentosum.

[0139] In further embodiments, the cancer is non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B-cell chronic lymphocytic leukemia (B-CLL), B-cell Myeloid and T-cell acute lymphoblastic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid Myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL) and chronic myeloid leukemia (CHL) The cancer is selected from hematological malignancies such as those selected from myeloid leukemia (CML).

[0140] References herein to the term "prevention" refer to administration of the protective composition prior to the induction of the disease. "Suppression" refers to administration of the composition after an inductive event but before the clinical appearance of the disease. "Treatment" involves administration of the protective composition after disease symptoms become evident.

[0141] Screening peptide ligands for efficacy in protecting against or treating disease Animal model systems are available that can be used to monitor the activity of the antibody. The polypeptides are capable of cross-reacting with human and animal targets, allowing the use of animal models. This is facilitated by the present invention which allows the development of polypeptide ligands.

[0142] The invention will now be further described with reference to the following examples. EXAMPLES

[0143] [Example 1] Synthesis of the linker COM128

[0144] [ka] Compound 1 (700.0 mg, 1.18 mmol, 1.0 equiv.), 3-azidopropane- 1-Amine (117.66 mg, 1.18 mmol, 1.0 equiv), EDCI (270. 4mg, 1.41mmol, 1.2eq), HOBt (190.6mg, 1.41mmo A mixture of 1.2 equiv. of 1H2O (1.1 eq.) was dissolved in DCM (20 mL, pre-degassed and purged 3 times with N2). After dissolution, the mixture was stirred at 20-25°C for 1 hour under N2 atmosphere. LC-MS indicates that compound 1 is completely consumed and the desired m / z (calculated MW: 677.3 3, Actual measurement m / z: 678.2 ([M+H] + )) was detected. The solvent was evaporated to give compound 2 (600 mg, crude) as a white solid.

[0145] Compound 2 (600.0 mg, 885.3 μmol, 1.0 equivalent), N-ethylethanediamine A mixture of amine (1.29 g, 15.19 mmol, 1.50 mL, 17.2 equiv.) was added to DC M (3 mL, previously degassed and purged with N2 three times) and the mixture was then placed in a N2 atmosphere. The mixture was stirred at 25-30°C for 2 hours under atmospheric pressure. LC-MS showed that compound 2 was completely consumed. The desired m / z (calculated MW: 455.51, observed m / z: 456.3 ([M +H] + One main peak having the formula 40 was detected. The solvent was evaporated to give compound 3 (40 Compound 3 (150.0 mg, 329.3 μmol) was obtained as a colorless oil. l, 1.0 eq), compound 4 (320.1 mg, 329.3 μmol, 1.0 eq), H ATU (125.2 mg, 329.3 μmol, 1.0 equiv.), DIEA (42.6 mg, A mixture of 329.3 μmol, 57.4 μL, 1.0 equiv.) in DMF (2 mL, pre-degassed The mixture was then heated at 25-30°C under a N2 atmosphere. The mixture was stirred at rt for 2 h. LC-MS showed that compound 3 was completely consumed, and the desired m / z z (calculated MW: 1408.76, measured m / z: 705.3 ([M / 2+H] + )) One major peak was detected, corresponding to the methyl group. The solvent was evaporated to give compound 5 (400 mg, crude). was obtained as an oil.

[0146] Compound 5 (400 mg, 283.77 μmol, 1.0 equiv.) was dissolved in DMF (4 mL, The mixture was degassed and purged with N2 three times) and subsequently dissolved in piperidine (862.2 mg, 1 0.13 mmol, 1 mL, 35.7 equiv.) was added and the mixture was then cooled under a N The mixture was stirred at 25-30° C. for 15 min. LC-MS showed that compound 5 was completely consumed. The desired m / z (calculated MW: 1187.37, measured m / z: 594.4 ([M / 2+H] + , 1187.4[M+H] + One main peak was detected with the solvent. Evaporation afforded COM128 (250 mg, crude) as a colorless oil.

[0147] COM129

[0148] [ka] Compound 1 (1.4 g, 1.47 mmol, 1.0 equiv.), 3-azidopropane-1-azide amine (162.1 mg, 1.62 mmol, 1.1 equiv.), EDCI (338.6 mg, 1.77mmol, 1.2eq), HOBt (238.7mg, 1.77mmol, 1. 2 equiv.) was dissolved in DCM (5 mL, previously degassed and purged with N2 three times) and then The mixture was then stirred at 20-25°C for 1 hour under N2 atmosphere. LC-MS showed that compound It was shown that 1 was completely consumed, and the desired m / z (calculated MW: 1033.14, measured m / z: 1033.2 ([M+H] + One main peak with the 2-amino acid residue was detected. The mixture was treated with a few drops of 1M HCl and the organic layer was evaporated under reduced pressure to remove the solvent. Compound 2 (1.1 g, crude) was obtained as a yellow oil.

[0149] Compound 2 (1.1 g, 1.06 mmol, 1 equiv.), N-ethylethanamine (3.8 9 g, 53.24 mmol, 5.48 mL, 50 equiv.) in DCM (5 mL, The mixture was then heated at 40° C. for 20-24 h under a N2 atmosphere. The mixture was stirred at 5° C. for 1 h. LC-MS showed that compound 2 was completely consumed. Desired m / z (calculated MW: 810.90, measured m / z: 810.9 ([M+H] + )) The reaction mixture was evaporated under reduced pressure to give compound 3 (810 mM). g, crude) as a white solid.

[0150] Compound 3 (810.0mg, 998.9μmol, 1.0eq), compound 4 (810.0mg, 998.9μmol, 1.0eq). 7mg, 1.10mmol, 1.1eq), HATU (455.8mg, 1.20mmol l, 1.2 eq.), DIEA (258.2 mg, 2.00 mmol, 348.0 μL, 2 A mixture of 1.0 eq. of 1H 2 O (1.0 eq.) was dissolved in DMF (2 mL, previously degassed and purged with N2 3 times) and The mixture was then stirred at 25-30°C under N2 atmosphere for 2 h. LC-MS showed that compound 3 This indicates that the desired m / z (calculated MW: 1530.72, measured m / z:765.5([M / 2+H] + One main peak with the 2-amino acid residue was detected. The mixture was treated with a few drops of 1M HCl, the organic layer was collected and evaporated under reduced pressure to remove the solvent. Compound 5 (1.1 g, crude) was obtained as a yellow solid.

[0151] Compound 5 (1 g, 653.29 μmol, 1 equiv.) was dissolved in DCM (10 mL, pre-degassed, purged 3 times with N2) followed by dissolving in piperidine (2.39 g, 32.66 mmHg). ol, 3.36 mL, 50 equiv.) was added, and the mixture was then heated for 25-3 The mixture was stirred at 0° C. for 2 h. LC-MS showed that compound 5 was completely consumed. Desired m / z (calculated MW: 1308.47, measured m / z: 1308.4 ([M+H] + )) One main peak having the following structure was detected. The residue was purified by preparative HPLC (TFA conditions: A phase: H2 0.075% TFA in O, B phase: MeCN, column: Luna 200 * 25mm 10 μm, C18, 110A and Gemin150 * 30mm, C18, 5μm, 110A , connected, 50 °C). COM129 (700 mg, 463.72 μmol , 70.98% yield) as a yellow solid.

[0152] COM130

[0153] [ka] Compound 1 (291 mg, 222.75 μmol, 1.0 equivalent), 3-azidopropane- 1-Amine (24.53 mg, 245.02 μmol, 1.1 equiv.), EDCI (51. 24mg, 267.30μmol, 1.2eq), HOBt (36.12mg, 267. 30 μmol, 1.2 equiv.) in DCM (3 mL, pre-degassed and purged three times with N2 The mixture was then stirred at 20-25°C for 1 hour under a N2 atmosphere. C-MS showed that compound 1 was completely consumed and the desired m / z (MW: 138 8.53, actual m / z:694.7([M / 2+H] + One main peak with The residue was purified by preparative HPLC (neutral conditions). Compound 2 (200 mg) , 144.04 μmol, 64.66% yield) was obtained as a white solid.

[0154] Compound 2 (200 mg, 144.04 μmol, 1.0 equiv.), N-ethylethaneamine A mixture of 210.7 mg, 2.88 mmol, 297 μL, 20.0 equiv. (3 mL, previously degassed and purged with N2 three times), and the mixture was then placed under a N2 atmosphere. The mixture was stirred at 20-25° C. for 1 hour under atmospheric pressure. LC-MS showed that compound 2 was completely consumed. The desired m / z (MW: 1166.29, measured m / z: 1166.3 ([M+ H]+ One major peak was detected, which had the compound 3 (1 50 mg, crude) was obtained as a yellow oil.

[0155] Compound 3 (150 mg, 128.61 μmol, 1.0 equivalent), compound 4 (75 mg, 144.91 μmol, 1.13 eq), HATU (58.7 mg, 154.34 μmol l, 1.2 equiv.) and DIEA (33.24 mg, 257.23 μmol, 44.80 A mixture of 1.0 μL, 2.0 equiv.) in DMF (5 mL, previously degassed and purged with N2 three times) After dissolution, the mixture was stirred at 20-25°C for 2 hours under N2 atmosphere. LC-MS indicates that compound 3 is completely consumed, and the desired m / z (MW: 1665.84 , Actual m / z:833.2([M / 2+H] + One main peak with The solvent was removed under reduced pressure to give compound 5 (300 mg, crude) as a yellow oil.

[0156] Crude compound 5 (300 mg, dissolved in 10 mL of DMF) was treated with piperidine (2 mL). was added and the mixture was stirred at 30° C. for 2 h. LCMS showed the desired m / z (MW: 1443 .60 Actual m / z:722.7([M / 2+H] + )) was detected. The residue was purified by preparative HPLC (neutral conditions). 0 (140 mg, 58.19 μmol, yield 32.31%, purity 60%) was obtained as a white solid. I got it.

[0157] COM131

[0158] [ka] Compound 1 (700.0 mg, 1.18 mmol, 1.0 equiv.), 3-azidopropane- 1-Amine (117.7 mg, 1.18 mmol, 1.0 equiv.), HOBt (190.6 mg, 1.41mmol, 1.2eq), EDCI (270.4mg, 1.41mmol A mixture of 1.2 equiv. (1.2 equiv.) was dissolved in DCM (20 mL, previously degassed and purged with N2 three times). The mixture was stirred at 25-30°C for 2 hours under N2 atmosphere. , indicating that compound 1 is completely consumed and shows the desired m / z (calculated MW: 677.75 , Actual m / z:678.2([M+H] + One main peak with the 1-amino acid residue was detected. The reaction mixture was treated with a few drops of 1M HCl and the organic layer was collected and evaporated under reduced pressure. Obtained 2 (600.0 mg, crude) as a white solid.

[0159] Compound 2 (600.0mg, 885.2μmol, 1.0eq) was added to DMF (3mL, pre-prepared). The mixture was degassed for 30 min and purged with N2 3 times) and then dissolved in piperidine (1.29 g, 15. 19 mmol, 1.50 mL, 17.2 equiv.) was added and the mixture was stirred for 25 The mixture was stirred at 30° C. for 2 hours. LC-MS showed that compound 2 was completely consumed. , desired m / z (calculated MW: 455.51, observed m / z: 456.3 ([M+H] + )) The reaction mixture was subjected to preparative HPLC (TFA conditions). The compound was purified by HPLC to give compound 3 (400.0 mg, 879.1 μmol) as a colorless oil. .

[0160] Compound 3 (250.0 mg, 548.83 μmol, 1.0 equivalent), compound 4 (284 .1 mg, 548.83 μmol, 1 eq), HATU (229.6 mg, 603.72 μmol, 1.1 eq.), DIEA (141.9 mg, 1.10 mmol, 191.19 1 μL, 2.0 equiv.) in DCM (20 mL, previously degassed and purged with N 3 times). The mixture was then stirred at 25-30°C for 2 hours under N2 atmosphere. Compound 3 was completely consumed, indicating that the desired m / z (calculated MW: 955.06, experimental Measured m / z:955.6([M+H] + One main peak was detected with the residue was purified by preparative HPLC (TFA conditions). Compound 5 (400.0 mg, 419. Compound 5 (400.0 mg, 418.82 μmol) was obtained as a white solid. , 1.0 equiv.) in DMF (4 mL, previously degassed and purged with N2 three times). and then piperidine (862.2 mg, 10.13 mmol, 1 mL, 24.2 equiv. ) was added and the mixture was stirred at 25-30°C for 2 hours under N2 atmosphere. LC-MS showed Compound 5 was completely consumed, indicating the desired m / z (MW: 732.83 Measured) m / z: 733.3 ([M+H] + One main peak was detected with the residue Purified by preparative HPLC (TFA conditions) and isolated as COM131 (200 mg, 272.9 μ mol) was obtained as a colorless oil.

[0161] COM470

[0162] [ka] COM122 (228mg, 149.83μmol, 1.0eq), compound 1 (51. To a solution of HATU (31 mg, 164.82 μmol, 1.1 equiv.) in DMF (6 mL), (85.40 mg, 224.75 μmol, 1.5 equiv.) and DIEA (19.37 m g, 149.83 μmol, 26.10 μL, 1.0 equiv.) was added to the mixture. The mixture was stirred at 30° C. for 2 hours. LC-MS showed that compound 1 was completely consumed. Desired m / z (MW: 1814.99, Measured m / z: 908.2 ([M / 2+H] + )) One major peak having the formula: The residue was purified by preparative HPLC (neutral conditions). Compound 2 (54 mg, 29.75 μmol, 19.86% yield) as a white solid.

[0163] To a solution of compound 2 (54 mg, 29.8 μmol, 1.0 equiv.) in DMF (2 mL), Piperidine (61 mg, 715 μmol, 71 μL, 24.0 equiv.) was added. The mixture was stirred at 25-30° C. for 2 h. LC-MS showed that compound 2 was completely consumed. The desired m / z (MW: 1592.75) was measured at 796.27 ([M / 2+ H] + One main peak having the formula ) was detected. The reaction mixture was concentrated under reduced pressure to obtain the solvent. The residue was purified by preparative HPLC (TFA conditions). OM470 (40 mg, 25.11 μmol, 84.41% yield) was obtained as a white solid. .

[0164] COM471

[0165] [ka] Compound 1 (900 mg, 1.23 mmol, 1.0 equiv.) and compound 2 (1.0 g, 3.21 mmol, 2.6 equiv.) in DCM (20 mL) and subsequently 284.0mg, 1.48mmol, 1.2eq), HOBt(200.2mg, 1.4 8 mmol, 1.20 equiv.) was added. The mixture was stirred at 25° C. for 2 h. LC-MS indicates that compound 1 has been completely consumed and the desired m / z (calculated MW: 1021. 49, actual measurement m / z: 1022.2 ([M+H] + )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (TFA conditions). ) Compound 3 (0.900 g, 880.53 μmol, yield 71.30 %) as a white solid.

[0166] Compound 3 (500.0 mg, 489.19 μmol, 1.0 equivalent), compound 4 (257 0.6 mg, 489.19 μmol, 1.0 equiv.) in DCM (5 mL) Subsequently, HOBt (132.2 mg, 978.37 μmol, 2.0 equiv.) and EDCI (187.6 mg, 978.37 μmol, 2.0 equiv.) was added to the mixture. The mixture was stirred at 0° C. for 2 h. LC-MS showed that compound 3 was completely consumed. Nominal m / z (MW: 1529.80 Measured m / z: 765.9 ([M / 2+H] + ))of The reaction mixture was concentrated under reduced pressure to remove the solvent and give The residue was purified by preparative HPLC (neutral conditions). Compound 3 (420 mg, 246.94 μmol, 50.48% yield) as a colorless oil.

[0167] Compound 5 (420 mg, 274.38 μmol, 1.0 equiv.) was dissolved in DMF (4 mL). followed by piperidine (865.2 mg, 10.16 mmol, 1 mL, 37 equiv. The mixture was stirred at 25-30°C for 2 h. LC-MS showed that compound 5 was completely The desired m / z (calculated MW: 1308.48, observed m / z: 654.8([M / 2+H] + One main peak with the methyl group was detected. Purified by preparative HPLC (TFA conditions). COM471 (386 mg, 265.50 1 μmol, yield 96.76%) was obtained as a colorless oil.

[0168] COM472

[0169] [ka] Compound 1 (0.5g, 839.43μmol, 1.0eq), compound 2 (627.0m g, 839.43 μmol, 1.0 equiv.) and DIEA (217.0 mg, 1.68 m mol, 292.4 μL, 2.0 equiv.) in DMF (2 mL) and then HATU (319.2 mg, 839.4 μmol, 1.0 equiv.) was added to the mixture. The mixture was then stirred at 25° C. for 30 min. TLC (DCM:CH3OH=10:1, R f = 0.24), compound 1 is completely consumed and one new spot is formed. The solvent was evaporated to give compound 3 (0.45 g, 339.75 μmol, yield The product was obtained as a colorless oil (40.47% yield, crude), which was used in the next step without further purification. Used for tepp.

[0170] Compound 3 (450.0 mg, 339.75 μmol, 1.0 equiv.) in DMF (8 mL) The mixture was stirred at 25° C. for 15 min. LC-MS showed that compound 3 was completely consumed, and the desired (calculated MW: 1102.27, actual m / z:552.1([M / 2+H] + )) with one main piece The residue was purified by preparative HPLC (TFA conditions). Compound 3 (3 Obtained 70.0 mg, 335.67 μmol, 98.80% yield) as a colorless oil.

[0171] COM126 (60mg, 54.45μmol, 1.0eq), compound 4 (15.5m g, 81.68 μmol, 1.5 equiv.) in DMF (5 mL) was added to a solution of HATU (31 m g, 81.68 μmol, 1.5 equiv.) and DIEA (10.5 mg, 61.68 μm ol, 15 μL, 1.5 eq.) was added. The mixture was stirred at 30° C. for 2 h. LC-M S indicates that COM126 is completely consumed and the desired single main peak is detected. The mixture was evaporated to remove the solvent and compound 5 (30 mg, crude) was obtained as a colorless oil. which was used in the next step without further purification.

[0172] Compound 5 (30 mg, 23.57 μmol, 1.0 equiv.) was dissolved in DCM (4.5 mL). Then, TFA (0.5 mL) was added, and the mixture was stirred at 25-30° C. for 2 hours. LC-MS showed that compound 5 was completely consumed, with one main peak of the desired The residue was purified by preparative HPLC (TFA conditions). 0 mg, 8.52 μmol) as a white solid.

[0173] COM473

[0174] [ka] Compound 1 (300 mg, 449.96 μmol, 1.0 equivalent), Compound 2 (138 mg , 449.96 μmol, 1.0 equivalent), HOBt (122 mg, 899.93 μmol , 2.0 equiv.), EDCI (173 mg, 899.93 μmol, 2.0 equiv.) was dissolved in DCM (10 mL, previously degassed and purged with N2 three times) and the mixture was then The mixture was stirred at 20-25° C. for 1 hour under a N2 atmosphere. LC-MS showed that compound 1 was completely consumed. The desired (MW: 955.06, measured m / z: 955.3 ([M+ H] + One main peak having the formula ) was detected. The reaction mixture was concentrated under reduced pressure to obtain the solvent. The mixture was evaporated under reduced pressure to give compound 3 (300 mg, crude) as a yellow oil. And obtained.

[0175] Compound 3 (300 mg, 314.12 μmol, 1.0 equiv.) was dissolved in DMF (4 mL). Then piperidine (1 mL) was added and the mixture was stirred at 20-25° C. for 1 hour. LC-MS showed that compound 3 was completely consumed and the desired m / z (MW: 7 32.83, actual m / z:733.2([M+H] + )) was detected. The residue was purified by preparative HPLC (neutral conditions). g, 218.33 μmol, 69.51% yield) as a colorless oil.

[0176] [Example 2] Synthesis of EphA2 / CD137-binding heterotandem bicyclic peptides BCY9173

[0177] [ka] Procedure for preparing BCY9172-PEG12-N3

[0178] [ka] BCY9172 (520 mg, 248.16 μmol, 1 equiv.) and compound 1 (37 0 mg, 499.47 μmol, 2.01 equiv.) was dissolved in DMF (5 mL) and DIEA (48.11 mg, 372.24 μmol, 64.84 μL, 1.5 eq.) was added, followed by The mixture was stirred at 30° C. for 12 h. LC-MS showed that BCY9172 was completely consumed. The desired m / z (calculated MW: 2721.12, measured m / z: 136 0.9([M / 2+H] + One main peak having the formula: The compound was purified by HPLC (TFA conditions) to obtain compound 2 (284 mg, 101.10 μmol The compound was obtained as a white solid (40.74% yield, 96.87% purity).

[0179] [ka] Procedure for preparing BCY9173 This reaction was carried out in parallel in two separate vessels. In one vessel, compound 2 ( 100 mg, 36.75 μmol, 1.0 equiv.) and BCY6169 (120 mg, 3 (6.78 μmol, 1.0 equiv.) was first dissolved in 10 mL of t-BuOH / H2O (1:1). Then, CuSO4 (0.4 M, 91.9 μL, 1.0 equiv.), VcNa (0.4 M, 183.8 μL, 2.0 equiv.) and THPTA (0.4 M, 91.9 μL, 1.0 Finally, 1M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was stirred for 40 The mixture was stirred at 5° C. for 16 h. LC-MS showed that compound 2 was completely consumed and Desired m / z (calculated MW: 5983.85, measured m / z: 997.6600 ([M / 6+H ] + ) and 1197.2300([M / 5+H] + )) shows one main peak The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain BCY9173 (218 mg, 34.97 μmol, 47.58% yield, 96% purity) as a white solid.

[0180] BCY7985

[0181] [ka] General procedure for preparing BCY7859

[0182] [ka] N3-PEG12-COOH (250 mg, 388 μmol) and HOSu (67. 0 mg, 583 μmol) in DMA (4.5 mL) and DCM (1.5 mL) Then, EDCI (89.3 mg, 466 μmol) was added and the mixture was stirred at 20° C. for 16 hours. Another mixture containing CY7732 (855 mg, 388 μmol) in 5 mL of DMA was In a 50 mL round-bottom flask, add DIEA (186 mg, 1.44 mmol, 250 μL). The initial reaction mixture was then added to the flask and stirred for 10 minutes at 20° C. The mixture was stirred for an additional 5 hours. LC-MS (ES8396-307-P1B1) showed that BCY7 One major peak with the desired mass was detected, indicating complete consumption of 732. The resulting reaction mixture was directly purified by preparative HPLC (TFA conditions) to give compound (III). BCY7859 (621 mg, 200 μmol, 51.6% yield, TFA salt) was obtained as a white solid. Obtained as.

[0183] General procedure for preparing BCY6169

[0184] [ka] A solution of BCY6099 (300 mg, 94.3 μmol) in DMA (2 mL) was added to DI EA (36.6 mg, 283 μmol, 49.3 μL) was added and stirred for 10 minutes. Then, PYA-NHS (36.8 mg, 189 μmol) was added and incubated at 20° C. for another 15 min. The mixture was stirred for an additional hour. LC-MS showed that BCY6099 was completely consumed. The reaction mixture was purified by preparative HPLC (neutral The compound BCY6169 (299 mg, 86.2 μmol, yield 91.5%) as a white solid.

[0185] General procedure for preparing BCY7985

[0186] [ka] BCY7859 (220 mg, 77.8 μmol) and BCY6169 (251 mg A solution of 1,3-dichlorophenyl ether (77.1 μmol) in DMF (5 mL) that had been purged with nitrogen for 2 h was Aqueous solvate (0.8 M, 963 μL) was added, followed by heating under a nitrogen atmosphere. Aqueous SO4 solution (0.8 M, 289 μL) was added. The mixture was then stirred at 20 °C for 2 h. LC-MS showed that BCY6169 was completely consumed and the desired mass was obtained. The reaction mixture was subjected to preparative HPLC (TFA conditions). and purified directly to give compound BCY7985 (283 mg, 43.4 μmol, yield 56.3 %, TFA) as a white solid.

[0187] BCY8942

[0188] [ka] General procedure for preparing BCY8940

[0189] [ka] N3-PEG12-COOH (120 mg, 186 μmol, 1.0 equiv.) DMA ( To a solution of HOSu (32.2 mg, 280 μmol, 1.5 eq.) was added and stirred. Then, EDCI (42.9 mg, 224 μmol, 1.2 eq.) was added to the mixture and further stirred at 20° C. for an additional 7 h. LCMS showed: This indicated complete formation of the activated ester. BCY804 in DMA (3 mL) In a separate flask containing 5 (410 mg, 186 μmol, 1.0 equiv.), DIEA (12 0 mg, 932 μmol, 162 μL, 5.0 eq.) was added, stirred, and then activated The ester was added and the mixture was stirred at 20° C. for 18 h. LC-MS showed the desired m / z. The reaction mixture was concentrated in vacuo to give DC M was removed. The resulting mixture was purified by preparative HPLC (TFA condition) to give BCY. Obtained 8940 (190 mg, 67.2 μmol, 36.1% yield) as a white solid.

[0190] General procedure for preparing BCY8942

[0191] [ka] BCY8940 (28.6 mg, 10.1 μmol, 1.1 equiv.) and BCY616 To a solution of 9 (30.0 mg, 9.19 μmol, 1.0 equiv) in DMF (2.0 mL), (2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2 H-furan-5-one (1.0 M, 92.0 μL) and CuSO4 (1.0 M, 27. 6 μL) was added and stirred at 20° C. for 2 hours under a nitrogen atmosphere. 69 was completely consumed, indicating the desired m / z (calculated MW: 6089.91). Measured m / z:1218.4([M / 5+H] + ), 1016.0([M / 6+H] + ), 8 70.7([M / 7+H] + One main peak with the following structure was detected. The compound BCY8942 (15.4 mg, 2. 46 μmol, 26.8% yield, 97.3% purity) was obtained as a white solid.

[0192] BCY8943

[0193] [ka] General procedure for preparing BCY8941

[0194] [ka] BCY6015 (a peptide identical to BCY8941 except for the absence of the PYA moiety) A solution of 100 mg of dimethyl ether (12. 8 mg, 98.7 μmol, 17.2 μL) was added and stirred for 10 minutes. ,5-dioxopyrrolidin-1-yl)pent-4-ynoate (12.8 mg, 65. 8 μmol) was added to the mixture, followed by further stirring at 20° C. for 16 hours. S indicates that compound 1 is completely consumed and the desired m / z (calculated MW: 3119 .60, actual m / z:1040.5([M / 3+H] + ) was detected. The mixture was purified by preparative HPLC (neutral conditions) to obtain compound BCY8941 ( The compound was obtained as a white solid (90.0 mg, 28.9 μmol, 87.7% yield).

[0195] General procedure for preparing BCY8943

[0196] [ka] BCY7859 (can be prepared as described in BCY7985; 40.0 mg, 1 4.2 μmol) and BCY8941 (42.0 mg, 13.5 μmol) in DMSO To a solution of (2 mL, pre-purged with nitrogen for 1 h) was added (2R)-2-[(1S) -1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (1 (0.0M, 270 μL) and CuSO4 (1.0M, 80.9 μL) were added. The mixture was purged with nitrogen three times and stirred at 15 °C for 2 h. LC-MS showed that BCY8941 was completely isolated. The desired m / z (calculated MW: 5946.77, measured m / z: 1190.2([M / 5+H] + ), 991.5([M / 6+H] + ), 849.9([ M / 7+H] + One main peak with the following structure was detected: A: 0.075% TFA in HO, B: ACN) to give compound BCY8943 (11.5 mg, 1.90 μmol, yield 14.1%, purity 98.1%) was obtained as a white solid. I got it.

[0197] BCY9647

[0198] [ka] Procedure for preparing compound 2

[0199] [ka] COM134 (300.0 mg, 57.0 μmol, 1.0 equivalent), compound 1 (17. To a solution of TEA (8.2 mg, 85.3 μmol, 1.5 equiv.) in DCM (0.5 mL) was added .65 mg, 11.9 μL, 1.5 eq.) was added. The mixture was stirred at 25° C. for 1 h. LC-MS showed that COM134 was completely consumed, and the desired mass (calculated M W: 691.72, Measured m / z: 692.3 ([M+H] + ) and 709.3 ([M+ NH4] + One main peak having the formula: The mixture was then freeze-dried to obtain crude compound 2 (30.5 mg, crude) as a white solid.

[0200] Procedure for preparing compound 3

[0201] [ka] To a solution of compound 2 (10 mg, 1.0 equiv.) in DMF (1 mL), BCY6099 (4 6mg, 1.0 equiv.) and DIEA (5.61mg, 7.55μL, 3.0 equiv.) were added. The mixture was stirred at 30° C. for 2 h. LC-MS showed that compound 2 was completely consumed. The desired m / z (calculated MW: 3735.28, measured m / z: 1245.9) was ([M / 3+H] + ) and 934.5([M / 4+H] + )) one main peak The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase chromatography. The compound was purified by HPLC (TFA condition). Compound 3 (34 mg, yield 62.96%, pure) was obtained. The compound was obtained as a white solid (100%).

[0202] Procedure for preparing BCY9647

[0203] [ka] Compound 3 (34mg, 9.10μmol, 1.0eq), BCY7741 (23mg, 10.08 μmol, 1.11 equiv.) and THPTA (0.4 M, 11.4 μL, 0. 5 equiv.) in t-BuOH / H2O (1:1, 2 mL, pre-degassed and flushed three times with N2). (0.4 M, 11.4 μL, 0.5 equiv.) and and VcNa (0.4 M, 22.8 μL, 1 eq.) were added under N2. 0.2 M NH4 The pH of this solution was adjusted by dropwise addition of HCO3 (in 1:1 t-BuOH / H2O). The reaction mixture was heated at 25-30°C under a N2 atmosphere. The mixture was stirred at rt for 12 h. LC-MS showed that compound 3 was completely consumed and the desired m / z (calculated MW: 6016.82, measured m / z: 1204.1 ([M / 5+H] + ) , 1003.5([M / 6+H] + ), 860.3([M / 7+H] + ) The reaction mixture was directly purified by preparative HPLC (TFA conditions). CY9647 (31.2 mg, 54.67% yield, 95.96% purity) as a white solid Got it.

[0204] BCY9648

[0205] [ka] Procedure for preparing compound 2

[0206] [ka] COM135 (30mg, 27.29μmol, 1.0eq), compound 1 (8.25m g, 40.94 μmol, 1.5 equiv) in DCM (0.5 mL) was treated with TEA (4. 14 mg, 40.94 μmol, 5.70 μL, 1.5 eq.) was added to the mixture. The mixture was stirred at 30° C. for 1 h. LC-MS showed that COM135 was completely consumed. Desired mass [calculated MW: 1264.41, observed m / z: 1281.4 ([M+NH 4] + ), 649.8([M / 2+H] + One main peak with the following structure was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. The residue was purified by preparative HPLC ( TFA condition) to give compound 2 (18 mg, 14.2 μmol, yield 52.1 4%).

[0207] Procedure for preparing compound 3

[0208] [ka] To a solution of compound 3 (9 mg, 7.12 μmol, 1 equiv.) in DMF (1 mL), BCY 6099 (23 mg, 7.23 μmol, 1.02 equiv.) and DIEA (2.76 mg , 21.35 μmol, 3.72 μL, 3.0 equiv.) was added. The mixture was incubated at 30° C. for 2 h. The mixture was stirred for 1 h. LC-MS showed that compound 2 was completely consumed and the desired m / z (Calculated MW: 4307.96 Measured m / z: 1436.9 ([M / 3+H] + ), 107 7.9([M / 4+H] + ), 862.5([M / 5+H] + )) with one main piece The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The compound was purified by phase HPLC (TFA conditions). Compound 3 (14.6 mg, yield 47.61 %, 100% purity) was obtained as a white solid.

[0209] Procedure for preparing BCY9648

[0210] [ka] Compound 3 (14.6 mg, 3.39 μmol, 1 equivalent), BCY7741 (8.5 mg , 3.73 μmol, 1.1 equiv) and THPTA (0.4 M, 4.3 μL, 0.5 equiv). A mixture of t-BuOH / HO (1:1, 2 mL, pre-degassed and purged three times with N (0.4M, 4.3 μL, 0.5 equiv.) and VcN a (0.4 M, 8.6 μL, 1.0 equiv.) was added under N2. 0.2 M NH4HCO The pH of this solution was adjusted to 8 by dropwise addition of 3 (in 1:1 t-BuOH / H2O). The solution turned pale yellow after adjustment. The reaction mixture was stirred at 25-30°C for 12 h under a N2 atmosphere. The mixture was stirred for 2 h. LC-MS showed that compound 3 was completely consumed and the desired m / z was obtained. z (MW: 6589.50, measured m / z: 1098.8 ([M / 6+H] + ), 942. 1([M / 7+H] + ), 824.6([M / 8+H] + )) one main peak The reaction mixture was directly purified by preparative HPLC (TFA conditions). BCY964 Obtained 8 (14.7 mg, 63.34% yield, 96.22% purity) as a white solid.

[0211] BCY9655

[0212] [ka] Procedure for preparing compound 2

[0213] [ka] COM128 (120mg, 101.06μmol, 1.0eq), compound 1 (25m g, 124.03 μmol, 1.25 equiv) in DCM (0.5 mL) was treated with TEA ( 15.34 mg, 151.59 μmol, 21.10 μL, 1.5 equivalents) was added. The mixture was stirred at 25° C. for 1 h. LC-MS showed the desired m / z (calculated MW: 1352.4 8, Actual m / z: 676.8 ([M / 2+H] + ), 1369.3 ([M+NH4] + ) The reaction mixture was concentrated under reduced pressure. The solvent was removed to give a residue, which was purified by preparative HPLC (neutral conditions). Compound 2 (14 mg, 8.99 μmol, yield 8.90%, purity 86.86%) was obtained without any Obtained as a coloured oil.

[0214] Procedure for preparing compound 3

[0215] [ka] Compound 2 (7 mg, 5.18 μmol, 1.0 equiv.) and BCY6099 (16 mg , 5.03 μmol, 1.0 equiv) in DMF (2 mL) was added DIEA (2.01 m g, 15.53 μmol, 2.70 μL) was added. The mixture was stirred at 30° C. for 2 hours. LC-MS showed that compound 2 was completely consumed and had the desired m / z (calculated MW :4396.02, actual m / z:879.8([M / 5+H] + ) and 1099.8( [M / 4+H] + One major peak having the formula: The crude product was purified by reverse phase HPLC (0.1% TFA). Compound 3 (11.8 mg, yield 48.29%, purity 93.11%) was obtained as a white solid. Obtained as.

[0216] Procedure for preparing BCY9655

[0217] [ka] Compound 3 (11.8mg, 2.69μmol, 1.0eq), BCY7741 (7.0 mg, 3.07 μmol, 1.14 equiv.) and THPTA (0.4 M, 6.8 μL, 1 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off) and then added CuSO4 (0.4 M, 6.8 μL, 1.0 equiv.) and V cNa (0.4M, 13.6 μL, 2.0 equiv.) was added under N2. 0.2M NH4 The pH of this solution was adjusted by dropwise addition of HCO3 (in 1:1 t-BuOH / H2O). The reaction mixture was heated at 25-30°C under a N2 atmosphere. The mixture was stirred at rt for 12 h. LC-MS showed that compound 3 was completely consumed and the desired m / z (calculated MW: 6677.57, measured m / z: 1113.7 ([M / 6+H] + ) , 954.7([M / 7+H] + The reaction mixture was fractionated to show one major peak with The product was directly purified by preparative HPLC (TFA conditions). BCY9655 (1.9 mg, 0.2 6 μmol, 9.65% yield, 91.15% purity) was obtained as a white solid.

[0218] BCY9656

[0219] [ka] Procedure for preparing compound 2

[0220] [ka] COM129 (30.0mg, 22.93μmol, 1.0eq), compound 1 (6.9 To a solution of TEA (3.5 mg, 34.39 μmol, 1.5 equiv.) in DCM (3 mL), mg, 34.39 μmol, 4.8 μL, 1.5 equiv.) was added. The mixture was degassed and N The mixture was then purged with 2 three times and stirred at 25° C. for 1 h under N2 atmosphere. MS showed that COM129 was completely consumed and had the desired m / z (calculated MW: 1 473.58, actual m / z:737.3([M / 2+H] + )) one main peak The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. was purified by preparative HPLC (neutral conditions) to give compound 2 (12.3 mg, 8.35 μm ol, 36.41% yield) was obtained as a white solid.

[0221] Procedure for preparing compound 3

[0222] [ka] Compound 2 (9.26 mg, 6.28 μmol, 1.0 equiv.) and BCY6099 (1 To a solution of TEA (0.70 mg, 3.14 μmol, 0.5 equiv.) in DMF (3 mL), mg, 6.93 μmol, 1 μL, 1.1 equiv.) was added. The mixture was degassed and flushed with N2 for 3 h. The mixture was then purged once and stirred at 25-30°C for 1 hour under N2 atmosphere. MS showed that compound 2 was completely consumed and had the desired m / z (calculated MW: 451 7.12, actual m / z:1129.8([M / 4+H] + ), 904.1([M / 5+H ] + ), 753.7([M / 6+H] + One main peak with the 2-aminopropyl group was detected. The mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC (TFA method). Compound 3 (12 mg, yield 72.36%, purity 85.58%) was purified by Obtained as a white solid.

[0223] Procedure for preparing BCY9656

[0224] [ka] Compound 3 (11mg, 2.44μmol, 1.0eq), BCY7741 (6.0mg , 2.63 μmol, 1.08 equiv.) and THPTA (0.4 M, 6.1 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off) and then added CuSO4 (0.4 M, 6.1 μL, 1.0 equiv.) and V cNa (0.4M, 12.2 μL, 2.0 equiv.) was added under N2. 0.2M NH4 The pH of this solution was adjusted by dropwise addition of HCO3 (in 1:1 t-BuOH / H2O). The reaction mixture was heated at 25-30°C under a N2 atmosphere. The mixture was stirred at rt for 12 h. LC-MS showed that compound 3 was completely consumed, and the desired m / z (calculated MW: 6798.66, measured m / z: 1133.8 ([M / 6+H] + ) , 971.9([M / 7+H] + ), 850.7([M / 8+H] + )) The main peak was detected. The reaction mixture was directly purified by preparative HPLC (TFA conditions). BCY9656 (6.8 mg, yield 37.36%, purity 90.97%) was obtained as a white solid. I got it.

[0225] BCY9657

[0226] [ka] Procedure for preparing compound 2

[0227] [ka] COM130 (30.0mg, 20.78μmol, 1.0eq), compound 1 (6.3 To a solution of TEA (3.2 mg, 31.17 μmol, 1.5 equiv.) in DCM (3 mL), mg, 31.17 μmol, 4.4 μL, 1.5 equiv.) was added to the mixture. The mixture was stirred at 5° C. for 1 h. LC-MS showed that COM130 was completely consumed. Desired m / z (calculated MW: 1608.7, observed m / z: 804.8 ([M / 2+H] + ) The reaction mixture was concentrated under reduced pressure and lyophilized to give Compound 2 (7.9 mg, crude) was obtained as a white solid.

[0228] Procedure for preparing compound 3

[0229] [ka] Compound 2 (7.9 mg, 4.91 μmol, 1.0 equiv.) and BCY6099 (16 mg, 5.03 μmol, 1.02 equiv) in DMF (1 mL) was treated with DIEA (1. 9 mg, 14.73 μmol, 2.6 μL, 3.0 equiv.) was added. The mixture was incubated at 30° C. After stirring for 2 h, LC-MS showed that compound 2 was completely consumed, and the desired m / z (calculated MW: 4652.25, measured m / z: 1551.3 ([M / 3+H] + ), 1 163.6([M / 4] + ), 931.1([M / 5+H] + , 776.1([M / 6+ H] + One major peak having the formula: ) was detected. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by reverse phase HPLC (TFA condition). Compound 3 (13.3 mg, 2.86 μmol, yield 53.22%, purity 91.42%) was obtained as a white solid Obtained as.

[0230] Procedure for preparing BCY9657

[0231] [ka] Compound 3 (13.3mg, 2.86μmol, 1.0eq), BCY7741 (7.0 mg, 3.07 μmol, 1.03 equiv.) and THPTA (0.4 M, 7.5 μL, 1 A mixture of 1.0 equiv.) in t-BuOH / H2O (1:1, 2 mL, pre-degassed and flushed with N2 three times) was (purged) and then added CuSO4 (0.4 M, 7.5 μL, 1.0 equiv.) and and VcNa (0.4 M, 15 μL, 2.0 equiv.) were added under N2. 0.2 M NH4 The pH of this solution was adjusted by dropwise addition of HCO3 (in 1:1 t-BuOH / H2O). The reaction mixture was heated at 25-30°C under a N2 atmosphere. The mixture was stirred at rt for 12 h. LC-MS showed that compound 3 was completely consumed, and the desired m / z [MW: 6933.78, measured m / z: 1156.7 ([M / 6+H] + ), 9 91.4([M / 7+H] + ), 867.4([M / 8+H] + )]. The reaction mixture was directly purified by preparative HPLC (TFA conditions). CY9657 (8.4 mg, 40.21% yield, 94.9% purity) was obtained as a white solid. .

[0232] BCY9658

[0233] [ka] Procedure for preparing compound 2

[0234] [ka] COM131 (167.0 mg, 227.89 μmol, 1.0 equivalent), compound 1 (5 To a solution of TEA (5.0 mg, 272.87 μmol, 1.2 equiv.) in DCM (5 mL), (36.4 mg, 359.23 μmol, 50.0 μL, 1.6 equiv.) was added. The mixture was stirred at 25-30° C. for 1 h. LC-MS showed the desired m / z (MW: 897.93 Measured value: 920.3 ([M+Na + ]) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. The residue was purified by preparative HPLC. Compound 2 (35 mg, 33.74 μmol, yield 1 4.81%, purity 86.56%) was obtained as a colorless oil.

[0235] Procedure for preparing compound 3

[0236] [ka] Compound 2 (15 mg, 16.71 μmol, 1.0 equiv.) and BCY6099 (53 To a solution of 6. The mixture was stirred for 30 The mixture was stirred at 5° C. for 2 h. LC-MS showed that compound 2 was completely consumed, and the desired m / z (MW: 3941.47 Measured m / z: 986.0 ([M / 4+H] + )) One major peak was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC (TFA condition). COM131] (5 mg, yield 50.48%, purity 94.96%) was obtained as a white solid. .

[0237] Procedure for preparing BCY9658

[0238] [ka] Compound 3 (35mg, 8.88μmol, 1.0eq), BCY7741 (21mg, 9.20 μmol, 1.03 equiv.) and THPTA (0.4 M, 22.2 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off), then CuSO4 (0.4 M, 22.2 μL, 1.0 equiv.) and VcNa (0.4 M, 44.4 μL, 2.0 equiv.) was added under N2. 0.2 M NH The p of this solution was then diluted by dropwise addition of 4HCO3 (in 1:1 t-BuOH / H2O). When H was adjusted to 8, the solution turned pale yellow. The reaction mixture was stirred for 25-30 minutes under a N2 atmosphere. The mixture was stirred at 5° C. for 12 hours. LC-MS showed that compound 3 was completely consumed. Nominal m / z [MW: 6223.01 Measured m / z: 1038.0 ([M / 6+H] + )oh and 889.8([M / 8+H] + One main peak was detected with the 2-aminopropyl group. The product was directly purified by preparative HPLC (TFA conditions). BCY9658 (13.2 mg The compound was obtained as a white solid (yield 21.54%, purity 90.16%).

[0239] BCY9659

[0240] [ka] Procedure for preparing compound 2

[0241] [ka] COM132 (20.0mg, 65.28μmol, 1.0eq), compound 1 (15. To a solution of TEA (36.8 mg, 78.34 μmol, 1.2 equiv.) in DCM (5 mL) was added The mixture was incubated at 25° C. The mixture was stirred at 4°C for 1 hour. The desired m / z (MW: 471.46, measured m / z: 489.2 ([M +NH4] + One major peak was detected by LC-MS with the compound 200 mg / ml. The reaction mixture was diluted with water and decompressed. Concentration under reduced pressure gave compound 2 (26 mg, crude) as a colorless oil.

[0242] Procedure for preparing compound 3

[0243] [ka] Compound 2 (15.0 mg, 4.71 μmol, 1.0 equiv.) and BCY6099 (3 To a solution of TEA (0.33 mg, 7.07 μmol, 1.5 equiv.) in DMF (3 mL), The mixture was incubated at 30° C. for 2 h. The mixture was stirred for 1 h. LC-MS showed that compound 2 was completely consumed and the desired m / z (MW: 3515.01, measured m / z: 1172.1 ([M / 3+H] + ) 879.5 ([M / 4+H] + One major peak with the methyl group was detected. The reaction mixture was filtered and Concentration under reduced pressure gave a residue. The crude product was purified by reverse phase HPLC (TFA conditions). Compound 3 (12.7 mg, 3.26 μmol, yield 69.23%, purity 90.3%) was obtained as a white solid.

[0244] Procedure for preparing BCY9659

[0245] [ka] Compound 3 (12.7mg, 2.89μmol, 1.0eq), BCY7741 (6.8 0 mg, 2.98 μmol, 1.03 equiv.) and THPTA (1.3 mg, 2.99 μmol, 1.03 equiv.) mol, 1.03 equiv.) in t-BuOH / HO (1:1, 2 mL, pre-degassed , purged with N2 three times) and then added CuSO4 (0.4 M, 7.3 μL, 1. 0 equiv) and VcNa (0.4 M, 14.6 μL, 2.0 equiv) were added under N2. By dropwise addition of 0.2M NH4HCO3 (in 1:1 t-BuOH / H2O) The pH of the solution was adjusted to 8, and the solution turned pale yellow. The reaction mixture was cooled under a N2 atmosphere. The mixture was stirred at 25-30° C. for 12 hours under reduced pressure. LC-MS showed that compound 3 was completely consumed. The desired m / z [MW: 5796.54] was 1159.8 ([M / 5+H] + ) 966.7([M / 6+H] + One main peak with The reaction mixture was directly purified by preparative HPLC (TFA conditions). 0.2 mg, 1.06 μmol, 36.58% yield, 98.86% purity) as a white solid Got it.

[0246] BCY9758

[0247] [ka] Procedure for preparing compound 2

[0248] [ka] Compound 1 (5.0 mg, 3.54 μmol, 1.0 equivalent), BCY6099 (11.3 To a solution of 0.9 mg, 3.54 μmol, 1.0 equiv) in DMF (3 mL), mg, 7.07 μmol, 1.2 μL, 2.0 equiv.) was added to the mixture. The mixture was then heated at 25-30°C. The mixture was stirred at RT for 20 min. LC-MS showed the desired m / z (MW: 4481.11, observed m / z :1101.3([M / 4+H] + )) was detected. The reaction mixture was filtered, concentrated under reduced pressure, and lyophilized to give compound 2 (15 mg, crude). Obtained as a coloured solid.

[0249] Procedure for preparing BCY9758

[0250] [ka] Compound 2 (15 mg, 3.35 μmol, 1.0 equiv.) and BCY7732 (14. To a solution of 1.7 mg, 6.69 μmol, 2.0 equiv.) in DMF (3 mL), DIEA (0 The mixture was stirred for 25-3 hours. The mixture was stirred at 0° C. for 2 h. LC-MS showed that compound 2 was completely consumed. Nozomi's m / z (MW: 6567.48, measured m / z: 1095.1 ([M / 6+H]), 9 38.8([M / 7+H] + One major peak having the structure ) was detected. The reaction mixture was filtered. The crude product was purified by reverse phase HPLC (TFA condition). BCY9758 (5.8 mg, yield 24.26%, purity 91.97%) was purified by white chromatography. Obtained as a coloured solid.

[0251] BCY10568

[0252] [ka] Procedure for preparing BCY8919-PEG12-N3

[0253] [ka] BCY8919 (80.0 mg, 38.47 μmol, 1.0 equiv.) and compound 1 ( 29.6 mg, 40.01 μmol, 1.04 equiv) was dissolved in DMSO (1 mL). The solution was then treated with DIPEA (7.46 mg, 55.71 μmol, 10.0 μl, 1.5 (eq.) was added, and the mixture was then stirred at 25-30° C. for 2 h. LC-MS showed that BC It shows that most of the Y8919 has been consumed, indicating that the desired m / z (calculated MW: 2705. 16, Actual measurement m / z: 1353.1 ([M / 2+H] + )) was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to give compound 2 (18.6). mg, 6.86 μmol, 17.83% yield, 99.76% purity) was obtained as a white solid .

[0254] Procedure for preparing BCY10568

[0255] [ka] Compound 2 (9.0 mg, 3.33 μmol, 1.0 equiv.) and BCY6169 (11 0.0 mg, 3.36 μmol, 1.01 equiv) first in t-BuOH / HO (1:1) Dissolve in 2 mL of 0.4 M of CuSO4 (8.3 μL, 1.0 equiv.), VcNa (1.4 mg, 7.06 μmol, 2.1 equiv.) and THPTA (1.4 mg, 3.2 2 μmol, 1.0 equiv.) was added. Finally, 0.4 M NH4HCO3 was added to The pH was adjusted to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture The mixture was stirred at 30° C. for 16 h under N2 atmosphere. LC-MS showed that compound 2 was completely consumed. and the desired m / z (calculated MW: 5967.90, observed m / z: 995. 00([M / 5+H] + ) and 1194.70([M / 6+H] + )) The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain BCY1 0568 (13.4 mg, 2.16 μmol, yield 69.44%, purity 96.3%) Obtained as a coloured solid.

[0256] BCY10570

[0257] [ka] Procedure for preparing BCY8920-PEG12-N3

[0258] [ka] BCY8920 (37 mg, 17.31 μmol, 1.0 equiv.) and compound 1 (15 To a solution of DIEA (3 mg, 20.25 μmol, 1.2 equiv.) in DMSO (2 mL), The mixture was stirred for 30 minutes. The mixture was stirred at 5° C. for 12 hours. LC-MS showed that BCY8920 was completely consumed. and the desired m / z (calculated MW: 2763.2, measured m / z: 689.07 ([M / 4-H + One main peak having the formula ]) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. Stripping gave a residue which was then purified by preparative HPLC (neutral conditions). Compound 2 (22.8 mg, 8.15 μmol, yield 47.09%, purity 98.78%) Obtained as a white solid.

[0259] Procedure for preparing BCY10570

[0260] [ka] Compound 2 (6 mg, 2.17 μmol, 1.0 equiv.) and BCY6169 (7.08 mg, 2.17 μmol, 1.0 equiv.) in 2 mL of t-BuOH / HO (1:1) Then, CuSO4 (0.4 M, 5.4 μL, 1.0 equiv.), VcNa (0. 4M, 10.8 μL, 2.0 equiv.) and THPTA (0.4M, 5.4 μL, 1.0 equiv. Finally, 0.2 M NH4HCO3 was added to adjust the pH to 8. Here, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred for 3 h under N2 atmosphere. The mixture was stirred at 0° C. for 4 hours. LC-MS showed that compound 2 was completely consumed. Desired m / z (MS: 6025.93, actual m / z: 1004.56 ([M / 6+H] + ) and 861.48([M / 7+H] + One main peak with the 2-aminopropyl group was detected. The mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA method). The product was purified by BCY10570 (7.2 mg, 1.17 μmol, yield 53. 90%, purity 97.95%) as a white solid.

[0261] BCY10574

[0262] [ka] Procedure for preparing compound 2

[0263] [ka] BCY9594 (65 mg, 27.07 μmol, 1 eq.), compound 1 (12.00 m g, 27.75 μmol, 1.02 equiv.) in DMSO (1 mL) was added to DIEA (5 The mixture was mixed with 2 The mixture was stirred at 5-30°C for 2 hours. LC-MS showed that BCY9594 was completely consumed. and the desired m / z (calculated MW: 2718.13, measured m / z: 906.04 ([M / 3+H] + ), 1359.07([M / 2+H] + )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and obtain a residue. The compound was purified by HPLC (TFA condition). Compound 2 (42.6 mg, 15.67 μmol l, 57.89% yield, 100% purity) was obtained as a white solid.

[0264] Procedure for preparing BCY10574

[0265] [ka] Compound 2 (20 mg, 7.36 μmol, 1.0 equivalent), BCY8927 (17 mg, 7.87 μmol, 1.07 equiv.) and THPTA (0.4 M, 18.4 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off), then CuSO4 (0.4 M, 18.4 μL, 1.0 equiv.) and VcNa (0.4 M, 36.8 μL, 2.0 equiv.) was added under N2. 0.2 M NH The p of this solution was then diluted by dropwise addition of 4HCO3 (in 1:1 t-BuOH / H2O). When H was adjusted to 8, the solution turned pale yellow. The reaction mixture was stirred for 25-30 minutes under a N2 atmosphere. The mixture was stirred at 5° C. for 12 hours. LC-MS showed that compound 2 was completely consumed. Desired m / z (calculated MW: 4877.68, measured m / z: 1219.42 ([M / 4+H] + ) and 975.54([M / 5+H] + )) was detected. The reaction mixture was directly purified by preparative HPLC (TFA conditions). 7.6 mg, 3.41 μmol, 46.29% yield, 94.40% purity) was obtained as a white solid. I got it.

[0266] BCY10575

[0267] [ka] Procedure for preparing compound 2

[0268] [ka] BCY9594 (65 mg, 27.07 μmol, 1 equivalent), compound 1 (12.0 mg , 27.75 μmol, 1.02 equiv) in DMSO (1 mL) was added DIEA (5. 25 mg, 40.61 μmol, 7.07 μL, 1.5 eq.) was added to the mixture. The mixture was stirred at 30° C. for 2 hours. LC-MS showed that compound 1 was completely consumed. , desired m / z [calculated MW: 2718.13 Measured m / z: 906.04 ([M / 3+H ] + ) and 1359.07([M / 2+H] + )] was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. The residue was purified by preparative HP The product was purified by LC (TFA condition). Compound 2 (42.6 mg, 15.67 μmol, Yield 57.89%, purity 100%) obtained as a white solid.

[0269] Procedure for preparing BCY10575

[0270] [ka] Compound 2 (20mg, 7.36μmol, 1.0eq), BCY8928 (17mg, 7.67 μmol, 1.04 equiv.) and THPTA (0.4 M, 18.4 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off), then CuSO4 (0.4 M, 18.4 μL, 1.0 equiv.) and VcNa (0.4 M, 36.8 μL, 2.0 equiv.) was added under N2. 0.2 M NH The p of this solution was then diluted by dropwise addition of 4HCO3 (in 1:1 t-BuOH / H2O). When H was adjusted to 8, the solution turned pale yellow. The reaction mixture was stirred for 25-30 minutes under a N2 atmosphere. The mixture was stirred at 5° C. for 12 hours. LC-MS showed that compound 2 was completely consumed. Desired m / z [calculated MW: 4935.71, measured m / z: 1234.59 ([M / 4+H] + ) and 987.71([M / 5+H] +One major peak having the structure )] was detected. The reaction mixture was directly purified by preparative HPLC (TFA conditions). 2 mg, 2.37 μmol, 32.27% yield, 97.67% purity) was obtained as a white solid. Ta.

[0271] BCY10576

[0272] [ka] Procedure for preparing compound 2

[0273] [ka] BCY9594 (30.0 mg, 12.50 μmol, 1.0 equivalent), compound 1 (5. 54 mg, 12.81 μmol, 1.02 equiv.) in DMSO (1 mL), A (2.42 mg, 18.74 μmol, 3.3 μL, 1.5 equiv) was added. Mixture The mixture was stirred at 25-30° C. for 2 hours. LC-MS showed that compound 1 was completely consumed. The desired m / z [calculated MW: 2718.13, observed m / z: 906.45 ([M / 3+H] + ) and 1359.50([M / 2+H] + One main peak with The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. The product was purified by preparative HPLC (TFA condition). Compound 2 (16 mg, 5.80 μmol, Yield 46.42%, purity 98.54%) was obtained as a white solid.

[0274] Procedure for preparing BCY10576

[0275] [ka] Compound 2 (17.0 mg, 6.25 μmol, 1.0 equivalent), BCY11014 (13 .6 mg, 6.25 μmol, 1.0 equiv.) and THPTA (0.4 M, 1.8 μL, 2.0 equiv.) in t-BuOH / HO (1:1, 2 mL, pre-degassed and purified with N for 3 min. purged twice) and then dissolved in CuSO4 (0.4 M, 15.6 μL, 1.0 equiv.) and VcNa (0.4M, 1.84 μL, 2.0 equiv.) was added under N2. The solution was diluted with NH4HCO3 (in 1:1 t-BuOH / H2O) by dropwise addition. The pH of the solution was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred for 25 The mixture was stirred at 30° C. for 12 hours. LC-MS showed that most of compound 2 had been consumed. Desired m / z [calculated MW: 4893.63, observed m / z: 1224.7 ([M / 4 +H] + ) and 980.01([M / 6+H] + )] was detected. The reaction mixture was directly purified by preparative HPLC (TFA conditions). 6 (20.5 mg, 4.13 μmol, yield 66.02%, purity 98.57%) was obtained as a white solid. Got it as a body.

[0276] BCY10577

[0277] [ka] Procedure for preparing compound 2

[0278] [ka] To a solution of compound 1 (5.0 mg, 49.5 μmol, 1.0 equiv.) in DMF (1 mL) , EDCI (8.5 mg, 54.8 μmol, 1.1 equiv.) and HOSu (5.7 mg The mixture was stirred at 25-30°C for 30 minutes. TLC showed that compound 1 was completely consumed and one new spot was formed. Then, BCY9172 (53 mg, 25.29 μmol, 0.47 equiv.) and DIEA (3.27 mg, 25.29 μmol, 4.4 μL, 0.47 equiv.) The mixture was stirred at 25-30°C for 2 hours. LC-MS showed that BCY9 The desired m / z (MW: 2178.46, measured) was confirmed, indicating that 172 had been completely consumed. m / z: 1089.5700 ([(M / 2+H + One main peak with The reaction mixture was concentrated under reduced pressure to remove the solvent and give a residue. The compound was purified by preparative HPLC (neutral conditions). Compound 2 (30 mg, 13.77 μmol The compound was obtained as a white solid (54.45% yield, 100% purity).

[0279] Procedure for preparing BCY10577

[0280] [ka] Compound 2 (20 mg, 9.18 μmol, 1.0 equiv.) and BCY6169 (32. 95 mg, 10.10 μmol, 1.1 equiv) first in t-BuOH / HO (1:1) 2 mL, then CuSO4 (0.4 M, 23 μL, 1 eq.), VcNa (0. (4M, 46 μL, 2.0 equiv.) and THPTA (0.4M, 23 μL, 1.0 equiv.) Finally, 1M NH4HCO3 was added to adjust the pH to 8. The entire solvent was degassed and purged with N2 three times. The reaction mixture was incubated at 30 °C under N2 atmosphere for 4 h. The mixture was stirred for 1 h. LC-MS showed that compound 2 was completely consumed and the desired m / z (Calculated MS: 5441.20, Actual m / z: 1361.8 ([M / 4+H] + )) One major peak was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA conditions). BCY10577(16. 2 mg, 2.98 μmol, 32.43% yield) was obtained as a white solid.

[0281] [Example 3] Synthesis of nectin-4 / CD137-binding heterotandem bicyclic peptides BCY8854

[0282] [ka] General procedure for preparing BCY8846

[0283] [ka] BCY8234 (a peptide identical to BCY8846 except for the absence of the PYA moiety) To a solution of 1.2g (300 mg, 102 μmol, 1.0 equiv.) in DMA (3 mL), Add A (52.5 mg, 406 μmol, 70.8 μL, 4.0 equiv.) and stir for 10 min. Then, (2,5-dioxopyrrolidin-1-yl)pent-4-ynoate ( 25.8 mg, 132 μmol, 1.3 equivalents) was added thereto, and the mixture was further incubated at 20° C. After stirring for an additional 16 hours, LC-MS showed that BCY8234 was completely consumed. The desired m / z (calculated MW: 3034.43, observed m / z: 1011.8 ([M / 3+H]+ ), 1517.0([M / 2+H] + One main peak with The reaction mixture was purified by preparative HPLC (neutral conditions) to give compound BCY8846 ( Obtained 290 mg, 95.6 μmol, 94.1% yield as a white solid.

[0284] General procedure for preparing BCY8854

[0285] [ka] BCY8846 (234 mg, 77.1 μmol, 1.0 equiv) in DMF (5 mL) To the solution was added BCY7859 (which can be prepared as described in BCY7985; 220 mg , 77.8 μmol, 1.0 equiv.) was added, followed by (2R)-2-[(1S)-1 ,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (0.8 0M, 963μL, 1.0 equiv.) and CuSO4 (0.80M, 289μL, 0.3 equiv. The mixture was stirred at 20° C. for 2 h. LC-MS showed that BCY8846 was completely converted. The desired m / z (calculated MW: 5861.59, measured m / z :837.9([M / 7+H] + ), 977.6([M / 6+H] + ), 1173.3( [M / 5+H] + One main peak having the structure (R = 1.0, R = 1.0) was detected. The reaction mixture was subjected to preparative HPLC. (A: 0.075% TFA in HO, B: ACN) to give compound BCY88 54 (292 mg, 46.8 μmol, yield 60.8%, purity 95.9%, TFA) was purified by HPLC. Obtained as a coloured solid.

[0286] BCY9350

[0287] [ka] General procedure for preparing BCY8782-PYA

[0288] [ka] BCY8782 (identical to BCY11942 except for the absence of the PYA moiety) To a solution of 1.0 ethyl ketone (peptide; 20.0 mg, 6.77 μmol, 1.0 equiv.) in DMA (1 mL), DIEA (4.37 mg, 33.9 μmol, 5.90 μL, 5.0 equiv.) and (2, 5-dioxopyrrolidin-1-yl)pent-4-ynoate (2.64 mg, 13.5 2.0 μmol, 2.0 equivalents) was added and stirred at 25° C. for 12 hours. LC-MS showed that BCY8 782 was completely consumed, indicating the desired m / z (calculated MW: 3034.43, Actual m / z:1012.1[M / 3+H] + One main peak with the following structure was detected. The reaction mixture was purified by preparative HPLC (neutral conditions) to give BCY11942 (20.0 ml). g, 6.00 μmol, 88.6% yield, 91.0% purity) as a white solid.

[0289] General procedure for preparing BCY9350

[0290] [ka] BCY11942 (20 mg, 6.59 μmol, 1.0 equiv.) and BCY7859 (Can be prepared as described in BCY7985; 20.5 mg, 7.25 μmol, (1.1 equiv.) in DMF (1 mL) was added to a solution of (2R)-2-[(1S)-1,2-dihydrazine [3,4-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (0.4M, 330μ Add CuSO4 (0.4M, 98.9μL, 6.0eq.) and mix. The mixture was stirred at 25° C. for 2 hours. LC-MS showed BCY8782-P The YA was completely consumed, indicating that the desired m / z (calculated MW: 5861.59, experimental Measured m / z:1173.3[M / 5+H] + ) was detected. The mixture was purified by preparative HPLC (A: 0.075% TFA in HO, B: ACN). The resultant product was BCY9350 (14.5 mg, 2.40 μmol, yield 36.5%, purity 97.2%). %) as a white solid.

[0291] BCY9351

[0292] [ka] General procedure for preparing BCY9351

[0293] [ka] BCY8940 (can be prepared as described in BCY8942; 9.4 mg, 3. 33 μmol, 1.01 equiv.) and BCY8846 (10.0 mg, 3.30 μmol To a solution of Vc (0.4 M, 165 (0.4M, 49.4μL, 6.0 equiv.) and CuSO4 (0.4M, 49.4μL, 6.0 equiv.) were added. The mixture was stirred at 25° C. for 1 h. LC-MS showed that BCY8940 was completely consumed. The desired m / z (calculated MW: 5861.59, observed m / z: 975. 4[M / 6+H] + , 1172.3[M / 5+H]+ ) was detected. The reaction mixture was purified by preparative HPLC (A: 0.075% TFA in H2O, B: ACN). The product was purified to obtain BCY9351 (5.30 mg, 0.904 μmol, yield 26.3%, The compound was obtained as a white solid (96.0% purity).

[0294] BCY9399

[0295] [ka] Procedure for preparing compound 2

[0296] [ka] COM134 (30 mg, 56.97 μmol), compound 1 (17.22 mg, 85. To a solution of TEA (8.65 mg, 85.45 μmol) in DCM (0.5 mL), The mixture was stirred at 25° C. for 1 h. LC-MS showed: This indicates that COM134 has been completely consumed, and the desired m / z (calculated MW: 691.7 2, Actual measurement m / z: 692.3 ([M+H] + ) and 709.3 ([M+NH4] + )) One major peak having the formula: The residue was purified by preparative HPLC (neutral conditions). Compound 2 (30 0.5 mg) was obtained as a colourless oil.

[0297] Procedure for preparing compound 3

[0298] [ka] Compound 2 (15 mg, 21.68 μmol) and BCY8116 (47 mg, 21. To a solution of DIEA (8.41 mg, 65.05 μmol) in DMF (1 mL), ol, 11.33 μL) was added. The mixture was stirred at 30° C. for 2 h. LC-MS showed Compound 2 was completely consumed, indicating the desired m / z (MW: 2725.1 Measured) m / z: 1362.7 ([M / 2+H] + ), 909.0([M / 3+H] + )) One major peak was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC (TFA condition). Compound 3 (20 mg, yield 33.41%, purity 98.71%) as a white solid.

[0299] Procedure for preparing BCY9399

[0300] [ka] Compound 3 (20.0 mg, 5.35 μmol, 1.0 equivalent), BCY7741 (13. 0 mg, 5.70 μmol, 1.01 equiv) and THPTA (0.4 M, 13.4 μL , 1.0 equiv.) in t-BuOH / HO (1:1, 2 mL, pre-degassed and flushed with N 3 times) and then added CuSO4 (0.4 M, 13.4 μL, 1.0 equiv. ) and VcNa (0.4 M, 26.8 μL, 2.0 equiv.) were added under N2. This was then cooled to room temperature by dropwise addition of M NH4HCO3 (in 1:1 t-BuOH / H2O). The pH of the solution was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred for 2 h under a N2 atmosphere. The mixture was stirred at 5-30° C. for 12 h. LC-MS showed that compound 3 was completely consumed. Desired m / z [MW: 5006.64 Measured m / z: 834.9 ([M / 6+H] + ), 1002.3([M / 5+H] + ), 1252.4([M / 4+H] + )] The reaction mixture was directly subjected to preparative HPLC (TFA conditions). BCY9399 (9.1 mg, yield 27.20%, purity 96.29%) was obtained as a white Obtained as a solid.

[0301] BCY9400

[0302] [ka] Procedure for preparing compound 2

[0303] [ka] COM135 (can be prepared as described in BCY9648; 30.0 mg, 27 0.29 μmol), compound 1 (8.3 mg, 40.94 μmol) in DCM (2 mL) To the solution was added TEA (4.14 mg, 40.94 μmol, 5.7 μL). The reaction mixture was stirred at 25-30° C. for 1 h. LC-MS showed that COM135 had completely disappeared. The desired m / z (calculated MW: 1264.40, measured m / z: 12 81.4([M+NH4] + One major peak with the methyl group was detected. The solvent was removed by concentration under reduced pressure to give a residue. The residue was subjected to preparative HPLC (neutral conditions). Thus, purification afforded compound 2 (18 mg) as a white solid.

[0304] Procedure for preparing compounds

[0305] [ka] Compound 2 (15.5 mg, 7.12 μmol) and BCY8116 (9 mg, 7.1 2 μmol) in DMF (2 mL), The mixture was stirred at 30° C. for 2 hours. LC-MS showed that compound 2 This indicates that the desired m / z (MW: 3297.78, actual m / z :1099.7([M / 3+H] + One main peak was detected with the reaction mixture The product was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC (TFA condition). Compound 3 (19.5 mg, 5.91 μmol, yield 33.41%, pure) was purified by The compound was obtained as a white solid (83.07%).

[0306] Procedure for preparing BCY9400

[0307] [ka] Compound 3 (19.5mg, 5.91μmol), BCY7741 (14mg, 6.14 μmol, 1.01 equiv.) and THPTA (0.4 M, 15 μL, 1 equiv.) Dissolved in t-BuOH / H2O (1:1, 2 mL, previously degassed and purged with N2 three times). , followed by addition of CuSO4 (0.4 M, 15 μL, 1 eq.) and VcNa (0.4 M, 30 0.2M NH4HCO3 (1:1 t-BuOH The pH of this solution is adjusted to 8 by dropwise addition of 1000 mM NaCl / H2O, which causes the solution to turn pale yellow. The reaction mixture was stirred under N2 atmosphere at 25-30°C for 12 hours. LC-MS indicates that compound 3 is completely consumed, and the desired m / z [MW: 5579.31 Actual m / z:930.5([M / 6+H] + ), 1116.6([M / 5+H] + )] The reaction mixture was subjected to preparative HPLC (TFA conditions). BCY9400 (13.9 mg, 2.33 μmol, yield 27.20%) , purity 93.56%) as a white solid.

[0308] BCY9401

[0309] [ka] Procedure for preparing compound 3

[0310] [ka] Compound 1 (50.0 mg, 31.39 μmol, 1 equivalent), compound 2 (6.6 mg, 3 To a solution of TEA (4.8 mg, The mixture was incubated at 25-30°C for 2 The mixture was stirred for 2 h. LC-MS showed that compound 1 was completely consumed and the desired m / z z(MW:1757.86 Measured m / z:879.10([M / 2+H] + )) One major peak was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and the residue The residue was purified by preparative HPLC (TFA conditions). Compound 3 (0.02 g , 6.56 μmol, 20.91% yield, 57.7% purity) was obtained as a white solid.

[0311] Procedure for preparing compound 4

[0312] [ka] Compound 3 (20 mg, 11.38 μmol, 1 equivalent), BCY8116 (25 mg, 1 To a solution of 1.51 μmol, 1.01 equiv) in DMF (4 mL), DIEA (2.2 mg , 17.07 μmol, 2.97 μL, 1.5 equivalents) was added. The mixture was heated at 25-30°C. The mixture was stirred at rt for 12 h. LC-MS showed that compound 3 was completely consumed, and the desired m / z (MW: 3791.23, measured m / z: 1263.2 ([M / 3+H] + ))of One main peak was detected, which was ascribed to the reaction product. The reaction product was directly purified by preparative HPLC (neutral conditions). Compound 4 (10 mg, 2.43 μmol, yield 21.33%, purity 92%) was obtained without any Obtained as a coloured oil.

[0313] Procedure for preparing BCY9401

[0314] [ka] Compound 4 (10mg, 2.43μmol, 0.9eq), BCY7741 (6.32m g, 2.77 μmol, 1.0 equiv) and THPTA (0.4 M, 6.7 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off) and then added CuSO4 (0.4 M, 6.7 μL, 1.0 equiv.) and V cNa (0.4M, 13.4 μL, 2.0 equiv.) was added under N2. 0.2M NH4 The pH of this solution was adjusted by dropwise addition of HCO3 (in 1:1 t-BuOH / H2O). The reaction mixture was heated at 25-30°C under a N2 atmosphere. The mixture was stirred at rt for 12 h. LC-MS showed that compound 4 was completely consumed, and the desired m / z [MW: 6072.77, measured m / z: 1012.00 ([M / 6+H] + One main peak having the formula (I) was detected. The reaction mixture was subjected to preparative HPLC (TFA conditions). BCY9401 (8.4 mg, 1.56 μmol, yield 59.3 1%, purity 95.52%) as a white solid.

[0315] BCY9403

[0316] [ka] Procedure for preparing compound 2

[0317] [ka] COM471 (100.0mg, 76.42μmol, 1.0eq), 4-nitrophe Nyl chloroformate (16.2 mg, 80.25 μmol, 1.05 equiv.) in DCM ( To a solution of TEA (11.6 mg, 114.64 μmol, 16.0 μL, 1 The mixture was stirred at 25-30 °C for 2 h. LC-MS showed COM The desired m / z (MW: 1473.58, measured) was confirmed, indicating that 471 had been completely consumed. m / z: 736.83 ([M / 2+H] + One main peak with the 1-amino acid residue was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. The residue was purified by preparative HPLC ( Compound 2 (62.8 mg, 42.67 μmol, yield 5 5.84%, purity 48.37%) was obtained as a white oil.

[0318] Procedure for preparing compound 3

[0319] [ka] Compound 2 (44mg, 29.46μmol, 1.0eq), BCY8116 (63mg To a solution of DIEA (5.66 mg, 43.77 μmol, 7.62 μL, 1.5 equiv.) was added. The mixture was stirred at 40° C. Stirred for 12 h. LC-MS showed that compound 2 was completely consumed, and the desired m / z (MW: 3506.95, measured m / z: 1168.58 ([M / 3+H] + ))of One main peak was detected. The residue was purified by preparative HPLC (TFA conditions). Compound 3 (20 mg, 5.42 μmol, yield 18.57%, purity 95.04%) was obtained as a white solid.

[0320] Procedure for preparing BCY9403

[0321] [ka] Compound 3 (10.0 mg, 2.71 μmol, 1.0 equivalent), BCY7741 (6.8 3 mg, 2.99 μmol, 1.1 equiv.) and THPTA (0.4 M, 7 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off) and then added CuSO4 (0.4 M, 7 μL, 1.0 equiv.) and VcN a (0.4M, 14 μL, 2.0 equiv.) was added under N2. 0.2M NH4HCO3 The pH of this solution was adjusted to 8 by dropwise addition of (1:1 t-BuOH / H2O). The reaction mixture was stirred at 25-30°C for 12 hours under a N2 atmosphere. The mixture was stirred for 1 h. LC-MS showed that compound 3 was completely consumed and the desired m / z [MW: 5788.49, Measured m / z: 1157.00 ([M / 5+H] + ) and 96 4.60([M / 6+H] + One major peak with the methyl group was detected. The product was directly purified by preparative HPLC (TFA conditions). BCY9403 (2.1 mg, 0.3 4 μmol, 11.93% yield, 93.80% purity) was obtained as a white solid.

[0322] BCY9405

[0323] [ka] Procedure for preparing compound 2

[0324] [ka] COM472 (44.7 mg, 38.1 μmol), compound 1 (9.2 mg, 45.7 To a solution of TEA (5.8 mg, 57.14 μmol, 8 μL) was added. The mixture was stirred at 25 °C for 2 h. LC-MS showed that COM472 It shows complete consumption and the desired m / z (MW: 1338.45, actual m / z: 686.23([M / 2+NH4 + One main peak with the 1,2-dichlorophenyl 1,4-dichlorophenyl 2,5-dichlorophenyl 2,6-dichlorophenyl 2,7-dichlorophenyl 2,8-dichlorophenyl 2,9-dichlorophenyl 2,5-dichlorophenyl 2,6-dichlorophenyl 2,7 ... The mixture was concentrated under reduced pressure to remove the solvent to give a residue. The residue was purified by preparative HPLC (neutral Compound 2 (20 mg, 14.94 μmol, yield 39.2%) was purified by the same procedure as above. was obtained as a colorless oil.

[0325] Procedure for preparing compound 3

[0326] [ka] Compound 2 (20 mg, 14.94 μmol) and BCY8116 (38.96 mg, To a solution of DIEA (1.9 mg, 14.94 μmol) in DMF (4 mL), The mixture was stirred at 30° C. for 2 hours. LC-MS showed that Compound 2 was completely consumed, indicating the desired m / z (MW: 3371.82, Measured m / z:1123.94([M / 3+H + One main peak with The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC (T Compound 3 (10 mg, yield 99.07%, purity 19.66%) was purified by FA. %) as a white solid.

[0327] Procedure for preparing BCY9405

[0328] [ka] Compound 3 (10.0 mg, 2.97 μmol, 1.0 equivalent), BCY7741 (7.4 mg, 3.26 μmol, 1.1 equiv) and THPTA (1.3 mg, 2.97 μmol l, 1.0 equiv.) in t-BuOH / HO (1:1, 2 mL, pre-degassed and N (purged 3 times with 500 cc) and then dissolved in CuSO4 (0.4 M, 7.5 μL, 1.0 equiv. ) and VcNa (0.4 M, 151 μL, 2.0 equiv.) were added under N2. The solution was diluted with NH4HCO3 (in 1:1 t-BuOH / H2O) by dropwise addition. The pH of the solution was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred for 25 The mixture was stirred at 30° C. for 12 hours. LC-MS showed that compound 3 was completely consumed. The desired m / z [MW: 5653.36, measured m / z: 1130.47 ([M / 5+H ] + The reaction mixture was subjected to preparative HPLC (TFA conditions). ) and purified directly by HPLC. BCY9405 (7.8 mg, yield 46.08%, purity 97. 8%) as a white solid.

[0329] BCY9406

[0330] [ka] Procedure for preparing compound 2

[0331] [ka] COM473 (130.0 mg, 177.40 μmol, 1.0 equiv.), (4-nitro phenyl) carbonochloridate (36.4 mg, 180.59 μmol, 1.02 equiv. ) in DCM (3 mL), TEA (27.0 mg, 266.09 μmol, 37 μ L, 1.5 eq.) was added. The mixture was stirred at 35° C. for 2 h. LC-MS showed that COM The desired m / z (MW: 897.93, actual m / z:897.65([M+H] + ), 914.60([M+NH4] + 1) Two main peaks were detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and the residue was The residue was purified by preparative HPLC (TFA condition). Compound 2 (90 mg, 9 5.87 μmol, 54.04% yield, 95.65% purity) was obtained as a colorless oil.

[0332] Procedure for preparing compound 3

[0333] [ka] Compound 2 (10 mg, 11.14 μmol, 1 eq), BCY8116 (25 mg, 1 To a solution of 1.51 μmol, 1.03 equiv.) in DMF (2 mL), DIEA (2.16 m g, 16.71 μmol, 2.91 μL, 1.5 equiv.) was added to the mixture. The mixture was stirred at RT for 12 h. LC-MS showed the desired m / z (MW: 2931.30, observed m / z z:977.00([M / 3+H] + )) is detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC. (FTA conditions). Compound 3 (15 mg, 5.12 μmol, yield 45. 79%, purity 99.66%) as a white solid.

[0334] Procedure for preparing BCY9406

[0335] [ka] Compound 3 (15mg, 5.12μmol, 1.0eq), BCY7741 (12mg, 5.26 μmol, 1.03 equiv.) and THPTA (0.4 M, 12.8 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off), then CuSO4 (0.4 M, 12.8 μL, 1.0 equiv.) and VcNa (0.4 M, 25.6 μL, 2.0 equiv.) was added under N2. 0.2 M NH The p of this solution was then diluted by dropwise addition of 4HCO3 (in 1:1 t-BuOH / H2O). When H was adjusted to 8, the solution turned pale yellow. The reaction mixture was stirred for 25-30 minutes under a N2 atmosphere. The mixture was stirred at 5° C. for 12 hours. LC-MS showed that compound 3 was completely consumed. Nominal m / z [MW: 5212.84 Measured m / z: 1042.74 ([M / 4+H] + ) One main peak with the following structure was detected. The reaction mixture was subjected to preparative HPLC (TFA conditions). The product was purified directly by HPLC. BCY9406 (14.4 mg, 2.57 μmol, yield 50.21 %, purity 93.01%) as a white solid.

[0336] BCY9407

[0337] [ka] Procedure for preparing compound 2

[0338] [ka] COM128 (60mg, 50.53μmol, 1.0eq), compound 1 (13mg, 64.50 μmol, 1.28 eq), DIEA (9.80 mg, 75.80 μmol, 13. A solution of 20 μL (1.5 equiv.) in DCM (5 mL) was degassed and purged with N2 three times. The mixture was then stirred at 25-30°C for 1 h under N2 atmosphere. LC-MS showed It indicates that OM128 is completely consumed and shows the desired m / z (calculated MW: 1352.4 8, Actual m / z: 676.7 ([M / 2+H] + One main peak with The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. The residue was purified by preparative HP The compound was purified by LC (TFA condition). Compound 2 (12 mg, 8.87 μmol, yield 1 7.56%) as a colorless oil.

[0339] Procedure for preparing [BCY8116]-[COM128]

[0340] [ka] Compound 2 (7 mg, 5.18 μmol, 1.0 equiv.) and BCY8116 (11 mg , 5.06 μmol, 1.0 eq), DIEA (2.01 mg, 15.53 μmol, 2 A solution of 1.70 μL, 3.0 equiv.) in DMF (3 mL) was degassed and purged with N2 three times, then The mixture was then stirred at 25-30°C for 1 h under N2 atmosphere. LC-MS showed the desired m / z (calculated MW: 3385.85, measured m / z: 1129.3 ([M / 3+H] + )) The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by reverse phase HPLC (TFA condition). Y8116]-[COM128] (15.6 mg, 4.46 μmol, yield 86.13%) , purity 96.75%) as a white solid.

[0341] Procedure for preparing BCY9407

[0342] [ka] [BCY8116]-[COM128](15.6mg, 4.61μmol, 1.0 equivalent amount), BCY7741 (11 mg, 4.82 μmol, 1.05 equiv.) and THPTA A mixture of (0.8 M, 5.8 μL, 1.0 equiv.) was dissolved in t-BuOH / HO (1:1, 2 ml L, previously degassed and purged with N2 three times), and then (0.4 M, 23.2 μL, 2.0 equiv.) and VcNa (0.4 M, 23.2 μL, 2.0 equiv.) in N 2. 0.2M NH4HCO3 (in 1:1 t-BuOH / H2O) was added dropwise. The pH of the solution was adjusted to 8 by adding 100 ml of water, which turned the solution pale yellow. The mixture was stirred at 25-30° C. for 12 hours under N2 atmosphere. LC-MS showed the desired m / z ( Calculated MW: 5667.39, Measured m / z: 945.6 ([M / 6+H] + ) and 113 4.2([M / 5+H] + )) was detected. The mixture was directly purified by preparative HPLC (TFA conditions). BCY9407 (1.3 mg , 0.23 μmol, 4.33% yield, 86.90% purity) was obtained as a white solid.

[0343] BCY9408

[0344] [ka] Procedure for preparing compound 2

[0345] [ka] COM129 (45.0mg, 34.39μmol, 1.0eq), compound 1 (15. To a solution of TEA (5.0 mg, 74.42 μmol, 2.1 equiv.) in DCM (5 mL) was added 5 mg, 53.88 μmol, 7.5 μL, 1.5 eq.) was added and the mixture was then The mixture was stirred at 25-30°C for 1 hour under a N2 atmosphere. LC-MS showed that COM129 had completely disappeared. The desired m / z (MW: 1473.58, measured m / z: 737. 3([M / 2+H] + One major peak having the formula ) was detected. The reaction mixture was diluted with water under reduced pressure. The solvent was removed by concentration to give a residue. The residue was purified by preparative HPLC (TFA conditions). Compound 2 (9 mg, 6.11 μmol, yield 17.01%, purity 95.76 %) as a white solid.

[0346] Procedure for preparing compound 3

[0347] [ka] Compound 2 (9.0 mg, 6.11 μmol, 1.0 equiv.) and BCY8116 (13 To a solution of 2.3 mg, 6.11 μmol, 1.0 equiv) in DMF (3 mL), DIEA ( 0.4 mg, 18.32 μmol, 3.2 μL, 3.0 equiv.) was added. All the solvent was removed. The mixture was then stirred under N2 atmosphere at 25-30°C for 1 hour. LC-MS showed that compound 2 was completely consumed and the desired m / z (M W: 3506.95, Measured m / z: 877.4 ([M / 4+H] + ) and m / z: 11 69.6([M / 3+H] + One major peak having the structure ) was detected. The reaction mixture was filtered. The crude product was purified by reverse phase HPLC (TFA condition). Compound 3 (7.2 mg, 2.05 μmol, yield 31.93%, purity 95%) was purified. ) as a white solid.

[0348] Procedure for preparing BCY9408

[0349] [ka] Compound 3 (7.2mg, 2.05μmol, 1.0eq), BCY7741 (5.0m g, 2.19 μmol, 1.03 equiv.) and THPTA (0.4 M, 5.1 μL, 1. A mixture of 1000 eq.) in t-BuOH / H2O (1:1, 2 mL, pre-degassed and flushed three times with N2) was (0.4 M, 5.1 μL, 1.0 equiv.) and VcNa (0.4 M, 10.2 μL, 2.0 equiv.) was added under N2. 0.2 M NH The p of this solution was then diluted by dropwise addition of 4HCO3 (in 1:1 t-BuOH / H2O). When H was adjusted to 8, the solution turned pale yellow. The reaction mixture was stirred for 25-30 minutes under a N2 atmosphere. The mixture was stirred at 5° C. for 12 hours. LC-MS showed that compound 3 was completely consumed. Nominal m / z [MW: 5788.49 Measured m / z: 968.9 ([M / 6+H] + ) and and 1158.0([M / 5+H] + One main peak was detected with the 2-aminopropyl group. The product was directly purified by preparative HPLC (TFA conditions). BCY9408 (3.1 mg, 4.97e-1μmol, 24.23% yield, 92.87% purity) was obtained as a white solid. .

[0350] BCY9409

[0351] [ka] Procedure for preparing compound 2

[0352] [ka] Compound 1 (30mg, 20.78μmol), COM130 (6.28mg, 31.1 To a solution of TEA (3.15 mg, 31.17 μmol) in DCM (3 mL), , 4.34 μL, 1.5 equivalents) was added. The mixture was stirred at 25-30°C for 1 hour. C-MS showed that compound 1 was completely consumed and the desired m / z (MW: 160 8.70 Actual m / z:804.8([M / 2+H] + ) was detected. The reaction mixture was concentrated under reduced pressure and then freeze-dried to give compound 2 (10.2 mg). (crude) was obtained as a white solid.

[0353] Procedure for preparing compound 3

[0354] [ka] Compound 2 (10.2 mg, 6.34 μmol) and BCY8116 (13.50 mg To a solution of 0.8 mg (6.22 μmol) of DIEA (0.8 mg, 6.22 μmol) in DMF (2 mL), mol, 1.1 μL, 1.0 equiv.) was added. The mixture was stirred at 30° C. for 2 h. LC -MS showed the desired m / z (MW: 3642.08, observed m / z: 1214.4 ([M / 3 +H] + The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The product was purified by reverse phase HPLC (TFA condition). Compound 3 (15.0 mg, 4.12 μmol, 62.94% yield, 95% purity) was obtained as a white solid.

[0355] Procedure for preparing BCY9409

[0356] [ka] Compound 3 (15mg, 4.12μmol, 1.0eq), BCY7741 (10mg, 4.38 μmol, 1.03 equiv.) and THPTA (0.4 M, 10.3 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off), then CuSO4 (0.4 M, 10.3 μL, 1.0 equiv.) and VcNa (0.4 M, 20.6 μL, 2.0 equiv.) was added under N2. 0.2 M NH The p of this solution was then diluted by dropwise addition of 4HCO3 (in 1:1 t-BuOH / H2O). When H was adjusted to 8, the solution turned pale yellow. The reaction mixture was stirred for 25-30 minutes under a N2 atmosphere. The mixture was stirred at 5° C. for 12 hours. LC-MS showed that compound 3 was completely consumed. Nominal m / z [MW: 5923.61, measured m / z: 988.2 ([M / 6+H] + )]of The reaction mixture was subjected to preparative HPLC (TFA conditions) to detect one main peak having the compound 1. Directly purified. BCY9409 (3.1 mg, 0.52 μmol, yield 12.62%, pure 90.89%) as a white solid.

[0357] BCY9410

[0358] [ka] Procedure for preparing compound 2

[0359] [ka] COM131 (167.0 mg, 227.89 μmol, 1.0 equivalent), compound 1 (5 To a solution of TEA (5.0 mg, 272.87 μmol, 1.2 equiv.) in DCM (5 mL), (36.4 mg, 359.23 μmol, 50.0 μL, 1.6 equiv.) was added. The mixture was stirred at 25-30° C. for 1 h. LC-MS showed the desired m / z (MW: 897.93 Measured value: 920.3 ([M+Na +]) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. The residue was purified by preparative HPLC. Compound 2 (35 mg, 33.74 μmol, yield 1 4.81%, purity 86.56%) was obtained as a colorless oil.

[0360] Procedure for preparing compound 3

[0361] [ka] Compound 2 (20 mg, 22.27 μmol, 1.0 equiv.) and BCY8116 (48 To a solution of 8. 64 mg, 66.82 μmol, 11.64 μL, 3.0 equiv.) was added to the mixture. The mixture was stirred at 0° C. for 2 h. LC-MS showed that compound 3 was completely consumed. Nominal m / z (MW: 2931.32, measured m / z: 977.7 ([M+H] + )) The residue was purified by preparative HPLC (TFA conditions). Compound 3 (40 mg, 13.08 μmol, yield 58.7%, purity 95.82%) was purified by HPLC. Obtained as a coloured solid.

[0362] Procedure for preparing BCY9410

[0363] [ka] Compound 3 (40mg, 13.08μmol, 1.0eq), BCY7741 (35mg , 15.34 μmol, 1.17 equiv) and THPTA (0.4 M, 34 μL, 1.0 A mixture of 1:1 (1 equiv.) t-BuOH / H2O (2 mL, pre-degassed and perfused three times with N2) was added. (distilled off) and then added CuSO4 (0.4 M, 34 μL, 1.0 equiv.) and Vc Na (0.4 M, 68 μL, 2.0 equiv.) was added under N2. 0.2 M NH4HCO The pH of this solution was adjusted to 8 by dropwise addition of 3 (in 1:1 t-BuOH / H2O). The solution turned pale yellow after adjustment. The reaction mixture was stirred at 25-30°C for 12 h under a N2 atmosphere. The mixture was stirred for 2 h. LC-MS showed that compound 3 was completely consumed and the desired m / z z[MW: 5212.85, measured m / z: 1043.2 ([M / 5+H] + )] One major peak was detected. The reaction mixture was directly purified by preparative HPLC (TFA conditions). BCY9410 (38.6 mg, 6.78 μmol, yield 49.71%, purity 9 1.6%) as a white solid.

[0364] BCY9411

[0365] [ka] Procedure for preparing compound 2

[0366] [ka] COM132 (5 mg, 16.32 μmol, 1 eq.), compound 1 (4 mg, 19.8 To a solution of TEA (2.8 mg, 24.5 μmol, 1.22 equiv.) in DCM (5 mL) was added 48 μmol, 3.4 μL, 1.5 eq.) was added. The mixture was stirred at 25° C. for 1 h. LC-MS showed the desired m / z (calculated MW: 471.46, observed m / z: 489.2 ([ M+NH4] + The reaction mixture was decompressed and one peak having the formula The mixture was concentrated under reduced pressure to remove the solvent and then lyophilized to give compound 2 (8 mg, crude) as a white solid. Got it as a body.

[0367] Procedure for preparing compound 3

[0368] [ka] Compound 2 (3.3 mg, 6.9 μmol, 1.5 equiv.) and BCY8116 (10. To a solution of 0.7 mg, 4.6 μmol, 1.0 equiv) in DMF (5 mL), DIEA (0.7 mg, 6.90 μmol, 1 μL, 1.5 equiv.) was added. The mixture was stirred at 30° C. for 2 h. LC-MS showed that compound 2 was completely consumed and the desired m / z (calculated Calculated MW: 2504.83, Measured m / z: 1252.3 ([M / 2+H] + 1) Two major peaks were detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The product was purified by reverse phase HPLC (TFA conditions). Compound 3 (4.2 mg, 1.5 Obtained 1 μmol, 32.78% yield, 90% purity) as a white solid.

[0369] Procedure for preparing BCY9411

[0370] [ka] Compound 3 (4.2mg, 1.68μmol, 1.0eq), BCY7741 (4.0m g, 1.75 μmol, 1.05 equiv.) and THPTA (0.04 M, 84 μL, 2. A mixture of 1000 eq.) in t-BuOH / H2O (1:1, 2 mL, pre-degassed and flushed three times with N2) was (0.04 M, 84 μL, 2.0 equiv.) and VcNa (0.04M, 168 μL, 4.0 equiv.) was added under N2. 0.2M NH The p of this solution was then diluted by dropwise addition of 4HCO3 (in 1:1 t-BuOH / H2O). When H was adjusted to 8, the solution turned pale yellow. The reaction mixture was stirred for 25-30 minutes under a N2 atmosphere. The mixture was stirred at 5° C. for 12 hours. LC-MS showed that compound 3 was completely consumed. Desired m / z [MW: 4786.37, Measured m / z: 1596.2 ([M / 3+H] + ), 1196.9([M / 4+H] + One main peak was detected with the following structure: was directly purified by preparative HPLC (TFA conditions). BCY9411 (4.1 mg, 0 0.86 μmol, 50.20% yield, 98.26% purity) was obtained as a white solid.

[0371] BCY9759

[0372] [ka] Procedure for preparing compound 2

[0373] [ka] Compound 1 (5.0mg, 3.54μmol, 1.0eq), BCY8116 (7.7m g, 3.54 μmol, 1.0 equiv) in DMF (3 mL) was added DIEA (0.9 mM g, 7.07 μmol, 1.2 μL, 2.0 equiv.) was added. The mixture was incubated at 0° C. for 20 min. The mixture was stirred. LC-MS showed that the mass (calculated MW: 3470.95, Hydrolysis MW: 3373.81, Measured m / z: 1125.0 ([M / 3 +H] + The reaction mixture was filtered, concentrated under reduced pressure, and lyophilized to give compound 2. (15 mg, crude) was obtained as a white solid.

[0374] Procedure for preparing BCY9759

[0375] [ka] Compound 2 (20 mg, 5.76 μmol, 1.0 equiv.) and BCY7732 (12. To a solution of 1.7 mg, 5.76 μmol, 1.0 equiv) in DMF (3 mL) was added DIEA (1. 5 mg, 11.52 μmol, 2.0 μL, 2.0 equiv.) was added to the mixture. The mixture was stirred at 0° C. for 2 h. LC-MS showed that compound 2 was completely consumed. Nominal m / z (MW: 5557.3, measured m / z: 927.0 ([M / 6+H] + ) and 1112.2([M / 5+H] + One main peak was detected with the reaction mixture The mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC (TFA conditions). BCY9759 (2.3 mg, yield 6.92%, purity 96.29%) was purified by Obtained as a white solid.

[0376] BCY10000

[0377] [ka] Procedure for preparing BCY9172-PEG12-N3

[0378] [ka] BCY9172 (520 mg, 248.16 μmol, 1.0 equiv.) and compound 1 ( (370 mg, 499.47 μmol, 2.01 eq.) was dissolved in DMF (5 mL) and then The mixture was added with DIEA (48.11 mg, 372.24 μmol, 64.84 μL, 1. 5 equivalents) was added and stirred at 30° C. for 12 hours. LC-MS showed that BCY9172 was completely It shows that the desired m / z (calculated MW: 2721.12, observed m / z: 1 360.9([M / 2+H] + One major peak having the structure Compound 2 (284 mg, 101.10 μm) was purified by preparative HPLC (TFA conditions). ol, 40.74% yield, 96.87% purity) was obtained as a white solid.

[0379] Procedure for preparing BCY10000

[0380] [ka] This reaction was carried out in parallel in two separate vessels. In one vessel, compound 2 ( 142 mg, 52.18 μmol, 1.0 equiv.) and BCY8846 (157 mg, 5 (1.74 μmol, 1.0 equiv.) was first dissolved in 10 mL of t-BuOH / H2O (1:1). Then, CuSO4 (0.4M, 130.5μL, 1.0 equivalent), VcNa (0. 4M, 261.0 μL, 2.0 equiv.) and THPTA (0.4M, 130.5 μL, 1 0 equiv.) was added. Finally, 1M NH4HCO3 was added to adjust the pH to 8. Here, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred under N2 atmosphere for 1 hour at 37°C for 2 hours. The mixture was stirred at 30° C. for 12 hours. LC-MS showed that compound 2 was completely consumed. , desired m / z (calculated MW: 5755.54, observed m / z: 959.60 ([M / 6+H ] + ) and 1151.55([M / 5+H] + One main peak with The reaction mixture was purified by preparative HPLC (TFA conditions) to give BCY10000 (31 4.9 mg, 51.99 μmol, 49.82% yield, 95.03% purity) as a white solid And obtained.

[0381] BCY10567

[0382] [ka] Procedure for preparing BCY8919-PEG12-N3

[0383] [ka] BCY8919 (60.0 mg, 28.85 μmol, 1.0 equiv.) and compound 1 ( 22.2 mg, 30.01 μmol, 1.04 equiv) was dissolved in DMSO (1 mL). To the solution was added DIPEA (5.6 mg, 43.28 μmol, 7.6 μl, 1.5 equiv.). The mixture was then stirred at 25-30° C. for 2 hours. LC-MS showed that BCY8919 It was shown to be completely consumed, and the desired m / z (calculated MW: 2705.16, measured m / z z:1353.15([M / 2+H] + One main peak with the 2-aminopropyl group was detected. The mixture was purified by preparative HPLC (TFA conditions) to obtain compound 2 (BCY8919-PE G12-N3, 18.5 mg, 6.77 μmol, yield 23.47%, purity 99.04% ) as a white solid.

[0384] Procedure for preparing BCY10567

[0385] [ka] NOTE: This reaction was performed twice, the first reaction is described below. Compound 2 (9.0 mg, 3.33 μmol, 1.0 equiv.) and BCY8846 (10 .1 mg, 3.33 μmol, 1.0 equiv.) was first dissolved in t-BuOH / H2O (1:1) 1 mL, then CuSO4 (0.4 M, 8.3 μL, 1.0 equiv.), VcN (1 .3 mg, 6.56 μmol, 2.0 equiv.) and THPTA (1.4 mg, 3.22 μmol, 2.0 equiv.) mol, 1.0 equiv.) was added. Finally, 0.4 M NH4HCO3 was added to adjust the pH The pH was adjusted to 8. All solvents were then degassed and purged with N2 three times. The reaction mixture was stirred at 37°C for 1 hour at 4°C for 24 hours. The mixture was stirred at 30° C. for 16 hours under a N2 atmosphere. LC-MS showed that compound 2 was completely consumed. and the desired m / z (calculated MS: 5739.58, observed m / z: 956.75 ([M / 6+H] + The reaction mixture was subjected to preparative HPLC ( TFA condition) and purified to obtain BCY10567 (6.85 mg, 1.18 μmol, yield The compound was obtained as a white solid in a yield of 35.48% and purity of 98.91%.

[0386] BCY10569

[0387] [ka] Procedure for preparing compound 3

[0388] [ka] Compound BCY8920 (40.0mg, 18.71μmol, 1.0eq), Compound 2 (16.0 mg, 21.6 μmol, 1.15 equiv.) and DIEA (5.0 μL, 28 A mixture of 1.0 μmol, 1.5 equiv.) was dissolved in DMF. LC-MS showed the desired m / z (Calculated MW: 2763.2, Measured m / z: 912.17 ([(M-28) / 2+H] + ) Stir the reaction mixture at 40 °C for 1 h until one main peak with The reaction mixture was then concentrated under reduced pressure to remove the solvent to give a residue that was subsequently The compound was purified by preparative HPLC (TFA condition). Compound 3 (23.4 mg, 8.47 μmol, 45.25% yield, 99.0% purity) was obtained as a white solid.

[0389] Procedure for preparing BCY10569

[0390] [ka] Compound 3 (5.0mg, 1.81μmol, 1.0eq), BCY8846 (5.8m g, 1.9 μmol, 1.05 equiv.) and THPTA (1.0 mg, 2.3 μmol, 1.3 equiv.) in t-BuOH / HO (1:1, 1 mL, pre-degassed and purified with N (purged twice) and then dissolved in CuSO4 (0.4 M, 5.0 μL, 1.0 equiv.) and VcNa (0.4 M, 5.0 μL, 1.0 equiv.) were added under N2. The solution was cooled to room temperature by dropwise addition of H4HCO3 (in 1:1 t-BuOH / H2O). The pH was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred at 40° C. under a N2 atmosphere. After stirring for 2 h, LC-MS showed that compound 3 was completely consumed and the desired m / z (calculated MW: 5797.62, measured m / z: 1160.7 ([M / 5+H] + ) One major peak was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA conditions) to give BCY10569 (5.7 mg, 1.18 μmol, 52.25% yield, 96.16% purity) was obtained as a white solid .

[0391] BCY10571

[0392] [ka] Procedure for preparing BCY8116-PEG5-N3

[0393] [ka] BCY8116 (60 mg, 27.62 μmol, 1.0 equiv.) and compound 1 (12 0.0 mg, 27.75 μmol, 1.0 equiv.) was first dissolved in DMSO (1 mL) and then Then, the mixture was treated with DIEA (5.4 mg, 41.43 μmol, 7.22 μL, 1.5 equiv. ) was added. The mixture was stirred at 30° C. for 12 h. LC-MS showed the desired m / z (MW :2489.82, actual m / z:1245.1700([M / 2+H] + 1) The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (TFA condition). Compound 2( 48 mg, 19.28 μmol, 69.80% yield, 100% purity) was obtained as a white solid. Ta.

[0394] Procedure for preparing BCY10571

[0395] [ka] This reaction was carried out in parallel in two separate vessels. In one vessel, compound 2 ( 10 mg, 4.02 μmol, 1.0 equiv.) and BCY8927 (9 mg, 4.17 μmol, 1.0 equiv.) mol, 1.04 equiv.) was first dissolved in 2 mL of t-BuOH / H2O (1:1), and then In, CuSO4 (0.4 M, 10.0 μL, 1.0 equiv.), VcNa (0.4 M, 20. 1 μL, 2.0 equiv.) and THPTA (0.4 M, 10.0 μL, 1 equiv.) were added. Finally, 0.4 M NH4HCO3 was added to adjust the pH to 8. The solvent was degassed and purged with N2 three times. The reaction mixture was stirred under N2 atmosphere at 30 °C for 4 h. LC-MS showed that compound 3 was completely consumed and the desired m / z (M W: 4649.36, Measured m / z: 1162.57 ([M / 4+H] + ), 1549.6 9([M / 3+H] + One main peak with the methyl group was detected. The residue was purified by preparative HPLC. (TFA condition). BCY10571 (13 mg, 2.79 μmol, yield The compound was obtained as a white solid in a yield of 34.88% and purity of 96.48%.

[0396] BCY10572

[0397] [ka] Procedure for preparing BCY8116-PEG5-N3

[0398] [ka] BCY8116 (60 mg, 27.62 μmol, 1.0 equiv.) and compound 1 (12 0.0 mg, 27.75 μmol, 1.0 equiv.) was first dissolved in DMSO (1 mL) and then Then, the mixture was treated with DIEA (5.4 mg, 41.43 μmol, 7.22 μL, 1.5 equiv. ) was added. The mixture was stirred at 30° C. for 12 h. LC-MS showed the desired m / z (MW :2489.82, actual m / z:1245.1700([M / 2+H] + 1) The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (TFA condition). Compound 2( 48 mg, 19.28 μmol, 69.80% yield, 100% purity) was obtained as a white solid. Ta.

[0399] Procedure for preparing BCY10572

[0400] [ka] This reaction was carried out in parallel in two separate vessels. In one vessel, compound 2 ( 10 mg, 4.02 μmol, 1.0 equiv.) and BCY8928 (9 mg, 4.06 μmol, 1.0 equiv.) mol, 1.01 equiv.) was first dissolved in 2 mL of t-BuOH / H2O (1:1), and then In the solution, CuSO4 (0.4 M, 10.1 μL, 1 equiv.), VcNa (0.4 M, 20.2 μ (0.4 M, 10.1 μL, 1.0 equiv.) and THPTA (0.4 M, 10.1 μL, 1.0 equiv.) were added. Finally, 0.4 M NH4HCO3 was added to adjust the pH to 8. The solvent was degassed and purged with N2 three times. The reaction mixture was stirred under N2 atmosphere at 30 °C for 4 h. LC-MS showed that compound 1 was completely consumed and the desired m / z (M W: 4707.40, Measured m / z: 1568.29 ([M / 3+H] + ) and 1176 .83([M / 4+H] + One major peak having the formula: The solvent was removed by concentration under reduced pressure to give a residue. The residue was subjected to preparative HPLC (TFA condition). The BCY10572 (21 mg, 4.46 μmol, yield 55.7%, pure The compound was obtained as a white solid (97.51%).

[0401] BCY10573

[0402] [ka] Procedure for preparing compound 2

[0403] [ka] BCY8116 (35 mg, 16.11 μmol, 1 equivalent), compound 1 (7.00 mg , 16.19 μmol, 1 equiv.) in DMSO (1 mL) was added to DIEA (3.12 m g, 24.17 μmol, 4.21 μL, 1.5 equivalents) was added to the mixture. The mixture was stirred at 5°C for 2 h. LC-MS showed that most of the BCY8116 had been consumed. The desired m / z (calculated MW: 2489.82, measured m / z: 1245.37 ([M / 2 +H] + ) and 830.25([M / 3+H] + One main peak with The reaction mixture was concentrated under reduced pressure to remove the solvent and obtain a residue. The product was purified by PLC (TFA condition). Compound 2 (26.8 mg, 10.76 μmol , 66.81% yield, 100% purity) as a white solid.

[0404] Procedure for preparing BCY10573

[0405] [ka] Compound 2 (15mg, 6.02μmol, 1.0eq), BCY11014 (13.5 0 mg, 6.21 μmol, 1.03 equiv.) and THPTA (0.4 M, 15.1 μL , 1.0 equiv.) in t-BuOH / HO (1:1, 2 mL, pre-degassed and flushed with N 3 times) and then added CuSO4 (0.4 M, 15.1 μL, 1.0 equiv. ) and VcNa (0.4 M, 30.2 μL, 2.0 equiv.) were added under N2. This was then cooled to room temperature by dropwise addition of M NH4HCO3 (in 1:1 t-BuOH / H2O). The pH of the solution was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred for 2 h under a N2 atmosphere. The mixture was stirred at 5-30° C. for 12 hours. LC-MS showed that compound 2 was completely consumed. The desired m / z [MW: 4665.32, observed m / z: 1167.50 ([M / 4+ H] + The reaction mixture was subjected to preparative HPLC (TFA conditions). The product was purified directly by HPLC using the method described above. BCY10573 (11.5 mg, 2.42 μmol, yield 40.14%, purity 98.11%) as a white solid.

[0406] BCY10578

[0407] [ka] Procedure for preparing compound 2

[0408] [ka] Compound 1 (5.0 mg, 49.5 μmol, 1.0 equiv.) was first diluted with EDCI (8.5 mM g, 54.8 μmol, 1.1 eq) and HOSu (5.7 mg, 49.5 μmol, The mixture was stirred at 25-30°C for 30 min. TLC showed that compound 1 was completely consumed and one new spot was formed. Then, compound BCY9172 (80.0 mg, 38.18 μmol, 0 .8 equiv.) and DIEA (6.3 mg, 8.5 μL, 49.5 μmol, 1.0 equiv.) was added to this mixture and LC-MS showed the desired m / z (calculated MW: 2178.46, observed m / z: 1089.44 ([M / 2+H] + ) is detected. The reaction mixture was then concentrated under reduced pressure to remove the solvent. The residue was removed and subsequently purified by preparative HPLC (TFA conditions). Product 2 (15 mg, 6.88 μmol, yield 18.66%, purity 73.3%) was obtained as a white solid. And obtained.

[0409] Procedure for preparing BCY10578

[0410] [ka] Compound 2 (9.8mg, 4.5μmol, 1.0eq), BCY8846 (14.0m g, 4.6 μmol, 1.0 equiv.) and THPTA (2.0 mg, 4.6 μmol, 1 A mixture of 1.0 equiv.) in t-BuOH / H2O (1:1, 1 mL, pre-degassed and flushed with N2 three times) was (purged), and then CuSO4 (0.4 M, 12 μL, 1.0 equiv.) and VcNa (0.4 M, 24 μL, 2.0 equiv.) was added under N2. 0.2 M NH4H The pH of this solution was adjusted by dropwise addition of CO3 (in 1:1 t-BuOH / H2O). The reaction mixture was incubated at 40° C. for 2 hours under a N2 atmosphere. LC-MS showed that compound 2 was completely consumed and the desired m / z ( Calculated MW: 5212.88, Measured m / z: 1304.2 ([M / 4+H] + )) One major peak was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA conditions) to give BCY10578 (13.78 mg, 2.64 μmol, 58.66% yield, 96.23% purity) was obtained as a white solid .

[0411] BCY10917

[0412] [ka] Procedure for preparing BCY8831-PEG12-N3

[0413] [ka] BCY8831 (40.0 mg, 13.29 μmol, 1.0 equiv.) and compound 1 ( 10.5 mg, 14.17 μmol, 1.07 equiv.) was dissolved in DMF (1 mL). DIPEA (2.6 mg, 20.09 μmol, 3.5 μl, 1.5 equivalents) was added to the solution. The mixture was then stirred at 30° C. for 16 h. LC-MS showed that BCY8831 was completely It shows that the desired m / z (calculated MW: 3635.16, measured m / z: 1 212.0([M / 3+H] + One major peak having the structure The compound was purified by preparative HPLC (TFA conditions) and obtained as compound 2 (22.0 mg, 5.83 μmol l, 43.85% yield, 96.39% purity) was obtained as a white solid.

[0414] Procedure for preparing BCY10917

[0415] [ka] Note: Two batches were made, the first one listed for the final report. Compound 2 (10.0 mg, 2.75 μmol, 1.0 equiv.) and BCY11014 ( 5.98 mg, 2.75 μmol, 1.0 equiv.) was first dissolved in t-BuOH / HO (1:1 ) in 2 mL of 0.4 M HCl, then dissolved in CuSO4 (0.4 M, 13.7 μL, 2.0 equiv.), Vc Na (1.1 mg, 5.55 μmol, 2.0 equiv.) and THPTA (1.2 mg, 2 .76 μmol, 1.0 equiv.) was added. Finally, 1M NH4HCO3 was added to The pH was adjusted to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture The mixture was stirred at 30° C. for 16 h under N2 atmosphere. LC-MS showed that compound 2 was completely consumed. and the desired m / z (calculated MW: 5810.66, measured m / z: 1163 .0([M / 5+H] + The reaction mixture showed one main peak with C (TFA condition), and BCY10917 (6.4 mg, 1.07 μmol, Yield 39.03%, purity 97.49%) obtained as a white solid.

[0416] BCY11020

[0417] [ka] Procedure for preparing BCY8831-PEG5-N3

[0418] [ka] BCY8831 (25.0 mg, 8.31 μmol, 1.0 equiv.) and compound 1 (3 0.9 mg, 9.02 μmol, 1.09 equiv.) was dissolved in DMF (1 mL). IPEA (1.6 mg, 12.46 μmol, 2.2 μl, 1.5 equiv) was added, followed by The mixture was stirred at 35° C. for 2 h. LC-MS showed that BCY8831 was completely consumed. The desired m / z (calculated MW: 3326.79, observed m / z: 1109. 66([M / 3+H] + One main peak having the formula: The compound was purified by PLC (TFA condition) to obtain compound 2 (7.3 mg, 2.09 μmol, yield 25.20%, purity 95.41%) as a white solid.

[0419] Procedure for preparing BCY11020

[0420] [ka] Compound 2 (7.3 mg, 2.19 μmol, 1.0 equiv.) and BCY11014 (4 .8 mg, 2.19 μmol, 1.0 equiv.) was first dissolved in t-BuOH / H2O (1:1) 1 mL, then CuSO4 (0.4 M, 5.5 μL, 1.0 equiv.), VcNa( 1.0 mg, 5.05 μmol, 2.3 equiv.) and THPTA (1.0 mg, 2.30 1.0 eq.) was added. Finally, 1M NH4HCO3 was added to adjust the pH The reaction mixture was adjusted to 8, where all solvents were degassed and purged with N2 three times. The mixture was stirred at 30° C. for 12 hours under 2 atmosphere. LC-MS showed that compound 2 was completely consumed. The desired m / z (calculated MW: 5502.29, measured m / z: 1101.74) was obtained. ([M / 5+H] + One main peak with the following structure was detected: C (TFA condition), and BCY11020 (3.3 mg, 0.577 μmol , 26.30% yield, 96.24% purity) as a white solid.

[0421] BCY11373

[0422] [ka] Procedure for preparing compound 2

[0423] [ka] To a solution of compound 1 (5.0 mg, 49.5 μmol, 1.0 equiv.) in DMF (1 mL) , EDCI (8.5 mg, 54.8 μmol, 1.1 equiv.) and HOSu (5.7 mg The mixture was stirred at 25-30°C for 30 minutes. TLC showed that compound 1 was completely consumed and one new spot was formed. Then, 0.3 mL of this mixture was added with BCY8116 (30.0 mg, 13.8 1 μmol, 0.28 equiv.) and DIEA (2.4 μL, 13.81 μmol, 0.2 8 equivalents) was added and stirred at 25-30°C for 2 hours. LC-MS showed that BCY8116 was completely converted. The desired m / z (calculated MW: 2255.53, measured m / z :1128.34([M / 2+H] + ) was detected. Then, The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue which was subsequently purified by preparative HPLC. (TFA condition). Compound 2 (21 mg, 8.9 μmol, yield 64.4 3%, purity 95.56%) as a white solid.

[0424] Procedure for preparing BCY11373

[0425] [ka] Compound 2 (5mg, 2.22μmol, 1.0eq), BCY8928 (4.79mg , 2.22 μmol, 1.0 equiv) and THPTA (1.0 mg, 2.30 μmol, 1.0 equiv.) in t-BuOH / HO (1:1, 1 mL, pre-degassed and purified with N (purged twice) and then dissolved in CuSO4 (0.4 M, 5.6 μL, 1.0 equiv.) and VcNa (0.4 M, 5.6 μL, 1.0 equiv.) were added under N2. The solution was cooled to room temperature by dropwise addition of H4HCO3 (in 1:1 t-BuOH / H2O). The pH was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred at 40° C. under a N2 atmosphere. After stirring for 2 h, LC-MS showed that compound 2 was completely consumed, and the desired m / z (calculated MW: 4415.07, measured m / z: 1471.5 ([M / 3+H] + and 1103.8([M / 4+H] + One major peak having the formula ##STR1## was detected. Filtration and concentration under reduced pressure gave a residue. The crude product was purified by preparative HPLC (TFA conditions). The product was purified by HPLC and purified to obtain BCY11373 (4.9 mg, 1.03 μmol, yield 46.26%, purity 1.0%). The compound was obtained as a white solid (92.4%).

[0426] BCY11374

[0427] [ka] Procedure for preparing BCY11374

[0428] [ka] Compound 2 (can be prepared as described in the procedure for preparing BCY11373; 5 mg , 2.22 μmol, 1.0 equivalent), BCY8928 (4.9 mg, 2.22 μmol, 1.0 equiv.) and THPTA (1.0 mg, 2.30 μmol, 1.0 equiv.) Dissolve in t-BuOH / H2O (1:1, 1 mL, previously degassed and purged with N2 three times). Then, CuSO4 (0.4 M, 5.6 μL, 1.0 equiv.) and VcNa (0.4 M, 5.6 μL, 1.0 equiv) was added under N2. 0.2 M NH4HCO3 (1:1 The pH of this solution is adjusted to 8 by dropwise addition of t-BuOH / H2O. The solution turned pale yellow. The reaction mixture was stirred at 40° C. for 2 hours under N2 atmosphere. LC -MS showed that compound 2 was completely consumed, and the desired m / z (calculated MW: 44 73.11, actual m / z: 1491.5 ([M / 3+H] + and 1118.5([M / 4+H] + One main peak with the formula 3 was detected. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by preparative HPLC (TFA condition) to give BCY1 1374 (4.1 mg, 1.27 μmol, yield 38.04%, purity 92.0%) was white Obtained as a solid.

[0429] BCY11375

[0430] [ka] Procedure for preparing BCY11375

[0431] [ka] Compound 2 (can be prepared as described in BCY11373; 5 mg, 2.22 μm ol, 1.0 equivalent), BCY11014 (4.8 mg, 2.22 μmol, 1.0 equivalent) and THPTA (0.5 mg, 2.30 μmol, 1.0 equiv.) were dissolved in t-BuO H / H2O (1:1, 1 mL, previously degassed and purged 3 times with N2) and then CuSO4 (0.4 M, 5.6 μL, 1.0 equiv.) and VcNa (0.4 M, 5.6 μL, 1.0 equiv.) L, 1.0 equiv.) was added under N2. 0.2M NH4HCO3 (1:1 t-BuO The pH of this solution is adjusted to 8 by dropwise addition of 1H2O (in H2O / HO), causing the solution to turn pale yellow. The reaction mixture was stirred at 40 °C under N2 atmosphere for 2 h. LC-MS showed Several desired m / z (calculated MW: 4431.03, observed m / z: 1107.59 ([M / 4+H] + and 1477.90([M / 3+H] + The reaction mixture was filtered. The crude product was purified by preparative HPLC (TFA conditions). Purified and isolated BCY11375 (6 mg, 1.31 μmol, yield 59.13%, purity 96. 8%) as a white solid.

[0432] BCY11616

[0433] [ka] Procedure for preparing compound 3

[0434] [ka] Compound BCY8116 (30.0mg, 13.81μmol, 1.0eq), Compound 2 (6.0 mg, 13.88 μmol, 1.0 equiv.) and DIEA (2.4 μL, 13. A mixture of 1, 82 μmol, 1.0 equiv. was dissolved in DMF. LC-MS confirmed that compound 1 was completely synthesized. The desired m / z (calculated MW: 2489.82, measured m / z :1245.4([M / 2+H] + The reaction mixture was stirred until one major peak with The mixture was stirred at 40° C. for 1 h. The reaction mixture was then concentrated under reduced pressure to remove the solvent. The residue was subsequently purified by preparative HPLC (TFA conditions). Compound 3 ( 27 mg, 10.29 μmol, 74.52% yield, 94.9% purity) as a white solid Got it.

[0435] Procedure for preparing BCY11616

[0436] [ka] Compound 3 (5 mg, 2.01 μmol, 1.0 equivalent), BCY7744 (5.2 mg, 2.21 μmol, 1.1 equiv) and THPTA (1.0 mg, 2.30 μmol, 1 A mixture of 1.0 equiv.) in t-BuOH / H2O (1:1, 1 mL, pre-degassed and flushed with N2 three times) was (purged), and then added CuSO4 (0.4 M, 5.0 μL, 1.0 equiv.) and and VcNa (0.4 M, 5.0 μL, 1.0 equiv.) were added under N2. The p of this solution was then diluted by dropwise addition of 4HCO3 (in 1:1 t-BuOH / H2O). The H was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred at 40°C for 2 h under a N2 atmosphere. The mixture was stirred for 2 h. LC-MS showed that compound 3 was completely consumed and the desired m / z z (calculated MW: 4827.46, measured m / z: 1207.12 ([M / 4+H]+ ) One major peak was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA conditions) to give BCY11616 (4.7 mg, 1.0 μmol, 48.48% yield, 94.7% purity) as a white solid.

[0437] BCY11617

[0438] [ka] Procedure for preparing BCY11617

[0439] [ka] Compound 3 (can be prepared as described in the procedure for preparing BCY11616; 5 mg , 2.01μmol, 1.0eq), BCY11506 (5.2mg, 2.21μmol , 1.1 equiv.) and THPTA (1.0 mg, 2.30 μmol, 1.1 equiv.) The material was dissolved in t-BuOH / H2O (1:1, 1 mL, previously degassed and purged with N2 three times). The solution was then dissolved in CuSO4 (0.4 M, 5.0 μL, 1.0 equiv.) and VcNa (0. 4M, 5.0 μL, 1.0 eq) was added under N2. 0.2M NH4HCO3 (1: The pH of this solution is adjusted to 8 by dropwise addition of 1 t-BuOH / H2O. The solution turned pale yellow. The reaction mixture was stirred at 40°C under N2 atmosphere for 2 hours. C-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 4 828.45, actual m / z: 1206.97 ([M / 4+H] + ) and 965.91( [M / 5+H] +One major peak having the formula: The crude product was purified by preparative HPLC (TFA conditions) to give a residue. BCY11617 (3.2 mg, 0.63 μmol, yield 31.37%, purity 95.05 %) as a white solid.

[0440] BCY11857

[0441] [ka] Procedure for preparing BCY11414-PEG5-N3

[0442] [ka] BCY11414 (60.0 mg, 29.06 μmol, 1.0 equiv.) and compound 1 (13.0 mg, 30.06 μmol, 1.03 equiv.) in MeCN / HO (1:1) The pH was adjusted to 8 with NaHCO3 (0.4 M), and the mixture was then diluted with 25 mL of The mixture was stirred at 30° C. for 2 h. LC-MS showed the desired m / z (calculated MW: 2381.72, Actual m / z:1191.07([M / 2+H] + One main peak with The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain compound 2 ( 38.0 mg, 15.9 μmol, 54.71% yield, 97.35% purity) as a white solid And obtained.

[0443] Procedure for preparing BCY11857

[0444] [ka] Compound 2 (10.0 mg, 4.20 μmol, 1.0 equiv.) and BCY7744 (1 1.5 mg, 4.92 μmol, 1.2 equiv.) first in t-BuOH / HO (1:1) 2 mL, then CuSO4 (0.4 M, 11.0 μL, 1.0 equiv.), VcN a (2.0 mg, 10 μmol, 2.4 equiv.) and THPTA (2.0 mg, 4.6 μmol). mol, 1.1 equiv.) was added. Finally, 0.2 M NH4HCO3 was added to bring the pH The pH was adjusted to 8. All solvents were then degassed and purged with N2 three times. The reaction mixture was stirred at 37°C for 1 hour at 4°C for 24 hours. The mixture was stirred at 30° C. for 16 hours under a N2 atmosphere. LC-MS showed that compound 2 was completely consumed. and the desired m / z (calculated MW: 4719.37, observed m / z: 1180.2 4([M / 4+H] + The reaction mixture was subjected to preparative HPLC. (TFA condition), and BCY11857 ​​(10.3 mg, 2.18 μmol, Yield 51.90%, purity 96.02%) obtained as a white solid.

[0445] BCY11858

[0446] [ka] Procedure for preparing BCY11414-PEG5-N3

[0447] [ka] BCY11414 (60.0 mg, 29.06 μmol, 1.0 equiv.) and compound 1 (13.0 mg, 30.06 μmol, 1.03 equiv.) in MeCN / HO (1:1) The pH was adjusted to 8 with NaHCO3 (0.4 M), and the mixture was then diluted with 25 mL of The mixture was stirred at 30° C. for 2 h. LC-MS showed the desired m / z (calculated MW: 2381.72, Actual m / z:1191.07([M / 2+H] + One main peak with The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain compound 2 ( 38.0 mg, 15.9 μmol, 54.71% yield, 97.35% purity) as a white solid And obtained.

[0448] Procedure for preparing BCY11858

[0449] [ka] Compound 2 (20.0 mg, 8.40 μmol, 1.0 equiv.) and BCY8928 (2 2.0 mg, 9.92 μmol, 1.1 equiv.) first in t-BuOH / HO (1:1) Dissolve in 2 mL of 0.4 M of CuSO4 (0.4 M, 21.0 μL, 1.0 equiv.), VcN a (4.0 mg, 20.19 μmol, 2.4 equiv.) and THPTA (4.0 mg, 9 Finally, 0.4M NH4HCO3 was added. The pH was adjusted to 8 by adding 100 ml of water. All solvents were then degassed and purged with N2 three times. The mixture was stirred at 30° C. under N2 atmosphere for 16 h. LC-MS showed that compound 2 was completely consumed. The desired m / z (calculated MW: 4599.30, observed m / z: 92 0.38([M / 5+H] + ), 1150.79([M / 4+H] + ), 1533.35 ([M / 3+H] + The reaction mixture was subjected to preparative HPLC ( TFA condition) and purified to obtain BCY11858 (16.9 mg, 3.67 μmol, yield The compound was obtained as a white solid in a yield of 43.43% and purity of 99.25%.

[0450] BCY11859

[0451] [ka] Procedure for preparing BCY11415-PEG5-N3

[0452] [ka] BCY11415 (30.0 mg, 13.81 μmol, 1.0 equiv.) and compound 1 (6.0 mg, 30.06 μmol, 1.0 equiv.) in 2 mL of MeCN / HO (1:1) The pH was adjusted to 8 with NaHCO3 (0.4 M), and the mixture was then diluted with 25–3 Stirred for 2 h at 0° C. LC-MS showed the desired m / z (calculated MW: 2489.82, observed m / z: 1245.18 ([M / 2+H] + One main peak with The reaction mixture was purified by preparative HPLC (TFA conditions) to give compound 2 (24 0.0 mg, 9.63 μmol, 69.7% yield, 99.28% purity) was obtained as a white solid. Ta.

[0453] Procedure for preparing BCY11859

[0454] [ka] Compound 2 (20.0 mg, 8.03 μmol, 1.0 equiv.) and BCY8928 (2 1.0 mg, 9.47 μmol, 1.1 equiv.) first in t-BuOH / HO (1:1) Dissolve in 2 mL of 0.4 M of CuSO4 (0.4 M, 21.0 μL, 1.0 equiv.), VcN a (4.0 mg, 2.5 eq.) and THPTA (4.0 mg, 1.1 eq.) were added. Finally, 1M NH4HCO3 was added to adjust the pH to 8. The mixture was degassed and purged with N2 three times. The reaction mixture was stirred under N2 atmosphere at 30 °C for 16 h. LC-MS confirmed that compound 2 was completely consumed and had the desired m / z (calculated MW: 4707.40, measured m / z: 941.7 ([M / 5+H] + ), 1176.9( [M / 4+H] + ), 1569.6([M / 3+H] + )) shows one main peak. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain BCY11859 (1 9.2 mg, 4.01 μmol, 49.87% yield, 98.22% purity) was obtained as a white solid. I got it.

[0455] [Example 4] Synthesis of PD-L1 / CD137-binding heterotandem bicyclic peptides BCY8939

[0456] [ka] General procedure for preparing BCY8939

[0457] [ka] N3-PEG12-COOH (250 mg, 388 μmol) and HOSu (67. 0 mg, 583 μmol) in DMA (4.5 mL) and DCM (1.5 mL) Then, EDCI (89.3 mg, 466 μmol) was added and the mixture was stirred at 20° C. for 16 hours. CMS showed that the desired intermediate was completely formed. .97 mg, 388.37 μmol, 1 equiv.) and DIEA (186 mg, 1.44 m mol, 250 μL) was added to the mixture and further stirred at 20° C. for another 5 h. LC -MS shows that BCY7732 is completely consumed and one with the desired mass The reaction mixture was purified by preparative HPLC (TFA conditions) to give the main peak: Compound BCY7859 (621 mg, 200.58 μmol, yield 51.65%, purity 9 5%, TFA) as a white solid. Calculated MW: 2817.16, Found m / z: 942 .7[M / 3+H] + .

[0458] General procedure for preparing BCY8939

[0459] [ka] BCY7859 (31.1 mg, 11.0 μmol) and BCY8938 (30.0 mg, 10.0 μmol) in DMF (2 mL), ,2-Dihydroxyethyl]-3,4-dihydroxyl-2H-furan-5-one (1M , 100 μL) and CuSO4 (1M, 30.0 μL) were added, and the mixture was stirred for 20 The mixture was stirred at 37 °C for 2 h. LC-MS showed that BCY7859 was completely consumed. One major peak with the desired mass was detected. The reaction mixture was purified by preparative HPLC (TFA The compound BCY8939 (16.1 mg, 2.72 μmol, yield) was obtained. The compound was obtained as a white solid in a yield of 27.1% and purity of 98.3%. Calculated MW: 5823.49, actual MW: 5823.49. Measured m / z:1165.4[M / 5+H] + , 971.0[M / 6+H] + , 832.9[ M / 7+H] +

[0460] BCY10580

[0461] [ka] Procedure for preparing BCY9172-PEG12-N3

[0462] [ka] BCY9172 (100.0 mg, 47.72 μmol, 1 eq. in DMSO (2 mL) ) and compound 1 (40.0 mg, 54.00 μmol, 1.13 equivalents) were added to DIEA ( 9.25 mg, 71.58 μmol, 12.47 μL, 1.5 eq.) was added to the mixture. The mixture was stirred at 30° C. for 12 h. LC-MS showed that BCY9172 was completely consumed. The desired m / z (MW: 2721.12, measured m / z: 1361.07 ([(M / 2+H + One main peak having the formula ]) was detected. The reaction mixture was concentrated under reduced pressure to give The solvent was removed to obtain a residue, which was then purified by preparative HPLC (neutral conditions). Compound 2 (48 mg, 17.44 μmol, yield 45.68%, purity 98.87 %) as a white solid.

[0463] Procedure for preparing BCY10580

[0464] [ka] Compound 2 (20 mg, 7.35 μmol, 1.0 equiv.) and BCY10043 (23 .1 mg, 7.35 μmol, 1.0 equiv.) was first dissolved in t-BuOH / H2O (1:1) 1 mL, then CuSO4 (0.4 M, 18.4 μL, 1.0 equiv.), VcNa (0.4M, 36.8μL, 2.0 equiv.) and THPTA (0.4M, 18.4μL, Finally, 1M NH4HCO3 was added to adjust the pH to 8. Here, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred under N2 atmosphere for 1 hour. The mixture was stirred at 30° C. for 4 hours. LC-MS showed that compound 3 was completely consumed. , desired m / z (MW: 5855.74, measured m / z: 976.40 ([M / 6+H] + ) and 1171.67([M / 5+H] + )) was detected. The residue was purified by preparative HPLC (TFA conditions). BCY10580 (29 mg, Obtained 4.85 μmol, 65.95% yield, 97.879% purity) as a white solid.

[0465] BCY10581

[0466] [ka] Procedure for preparing BCY9172-PEG12-N3

[0467] [ka] BCY9172 (100 mg, 47.72 μmol, 1 eq.) in DMSO (2 mL) and compound 1 (40.00 mg, 54.00 μmol, 1.13 equivalents) were added to DIEA (9 0.25 mg, 71.58 μmol, 12.47 μL, 1.5 equiv.) was added to the mixture. The mixture was stirred at 30° C. for 12 hours. LC-MS showed that BCY9172 was completely consumed. The desired m / z (MW: 2721.12, measured m / z: 1361.07 ([(M / 2+H +The reaction mixture was concentrated under reduced pressure to give a single main peak having the formula: The solvent was removed to give a residue, which was then purified by preparative HPLC (neutral conditions). Compound 2 (48 mg, 17.44 μmol, yield 45.68%, purity 98.87%) ) as a white solid.

[0468] Procedure for preparing BCY10581

[0469] [ka] Compound 2 (12 mg, 4.41 μmol, 1 equiv.) and BCY10044 (14.0 8 mg, 4.41 μmol, 1 equiv.) in 2 mL of t-BuOH / HO (1:1) Dissolve and then add CuSO4 (0.4 M, 11.02 μL, 1 eq.), VcNa (0.4 M, 22.05 μL, 2 equiv.) and THPTA (0.4 M, 10.04 μL, 1 equiv.) Finally, 1M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30 °C for 4 h under N2 atmosphere. The mixture was stirred for 2 h. LC-MS showed that compound 3 was completely consumed and the desired m / z z (MW: 5912.84, measured m / z: 985.90 ([M / 6+H] + ) and 11 83.28([M / 5+H] + One main peak having the formula: The product was purified by HPLC (TFA condition). BCY10581 (9.3 mg, 1.47 μ mol, 33.36% yield, 93.541% purity) as a white solid.

[0470] BCY10582

[0471] [ka] Procedure for preparing compound 2

[0472] [ka] BCY9172 (100.0 mg, 47.7 μmol, 1.0 equivalent), compound 1 (40 To a solution of 1.0 mg, 54.0 μmol, 1.13 equiv) in DMSO (2 mL), (9.2 mg, 71.6 μmol, 12.5 μL, 1.5 equiv.) was added. The mixture was mixed at 3 The mixture was stirred at 0° C. for 12 hours. LC-MS showed that BCY9172 was completely consumed. The desired m / z (calculated MW: 2721.12, observed m / z: 1361.07 ([M / 2+H] + The reaction mixture was concentrated under reduced pressure to give a single main peak having the formula: The solvent was removed to give a residue, which was purified by preparative HPLC (TFA conditions). Compound 2 (37 mg, 13.60 μmol, 28.49% yield) was obtained as a white solid. .

[0473] Procedure for preparing BCY10582

[0474] [ka] Compound 2 (16.0 mg, 5.9 μmol, 1.0 equivalent), BCY10045 (14. 0 mg, 6.0 μmol, 1.01 equiv) and THPTA (0.4 M, 14.7 μL, 1.0 equiv.) in t-BuOH / HO (1:1, 2 mL, pre-degassed and purified with N for 3 min. purged twice) and then dissolved in CuSO4 (0.4 M, 14.7 μL, 1.0 equiv.) and VcNa (0.4M, 29.4 μL, 2.0 equiv.) was added under N2. The solution was diluted with NH4HCO3 (in 1:1 t-BuOH / H2O) by dropwise addition. The pH of the solution was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred for 25 The mixture was stirred at 30° C. for 12 hours. LC-MS showed that compound 2 was completely consumed. The desired m / z [calculated MW: 5073.89, measured m / z: 1015.24 ([M / 5 +H] + ) and 1268.97([M / 4+H] + ) was detected. The reaction mixture was directly purified by preparative HPLC (TFA conditions). 2 (10 mg, 1.92 μmol, yield 32.58%, purity 97.21%) was obtained as a white solid. And obtained.

[0475] BCY11017

[0476] [ka] Procedure for preparing BCY11017

[0477] [ka] Compound 2 (can be prepared as described in the procedure for preparing BCY10567; 7.0 mg, 2.59 μmol, 1.0 equiv) and BCY10861 (7.03 mg, 2.5 9 μmol, 1.0 equiv.) was first dissolved in 2 mL of t-BuOH / H2O (1:1), then Then, CuSO4 (0.4 M, 13.0 μL, 2.0 equiv.), VcNa (1.0 mg, 5 .03 μmol, 2.0 equiv.) and THPTA (1.1 mg, 2.53 μmol, 1. 0 equiv.) was added. Finally, 1M NH4HCO3 was added to adjust the pH to 8. Here, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred for 3 h under N2 atmosphere. The mixture was stirred at 5° C. for 16 hours. LC-MS showed that compound 2 was completely consumed. Desired m / z (calculated MW: 5421.30, observed m / z: 1084.7 ([M / 5+H] + The reaction mixture was subjected to preparative HPLC (TFA conditions). The product was purified by HPLC and purified to obtain BCY11017 (6.6 mg, 1.17 μmol, yield 45.24%, pure). 96.16%) as a white solid.

[0478] BCY11018

[0479] [ka] Procedure for preparing BCY11018

[0480] [ka] Compound 2 (can be prepared as described in the procedure for preparing BCY10570; 6.0 mg, 2.17 μmol, 1.0 equiv) and BCY10861 (5.9 mg, 2.17 μmol, 1.0 equiv.) was first dissolved in 2 mL of t-BuOH / H2O (1:1), and then In the solution, CuSO4 (0.4 M, 11.0 μL, 2.0 equiv.), VcNa (1.0 mg, 2. 3 equiv.) and THPTA (1.1 mg, 1.0 equiv.). Finally, 1M NH 4HCO3 was added to adjust the pH to 8. All solvents were degassed and washed with N2 three times. The reaction mixture was stirred at 35° C. under N2 atmosphere for 16 h. LC-MS showed: Compound 2 was confirmed to be completely consumed and had the desired m / z (calculated MW: 5479.34 , Actual measurement m / z:1096.40([M / 5+H]+ )) showed one main peak. The reaction mixture was purified by preparative HPLC (TFA conditions) to give BCY11018 (2.3 mg, 0.40 μmol, 18.31% yield, 94.73% purity) was obtained as a white solid .

[0481] BCY11019

[0482] [ka] Procedure for preparing BCY11019

[0483] [ka] Compound 2 (can be prepared as described in the procedure for preparing BCY10581; 8.0 mg, 2.94 μmol, 1.0 equiv) and BCY10861 (8.0 mg, 2.95 μmol, 1.0 equiv.) was first dissolved in 2 mL of t-BuOH / H2O (1:1), and then In the solution, CuSO4 (0.4 M, 14.7 μL, 2.0 equiv.), VcNa (1.2 mg, 6. 05 μmol, 2.0 equiv) and THPTA (1.3 mg, 2.99 μmol, 1.0 Finally, 1M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was stirred for 35 min under N2 atmosphere. The mixture was stirred at 5° C. for 16 h. LC-MS showed that compound 2 was completely consumed and Desired m / z (calculated MW: 5437.26, measured m / z: 1088.09 ([M / 5+H] + ) and 1360.19([M / 4+H] + )) showed one main peak. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain BCY11019 (7.6 mg , 1.36 μmol, 46.09% yield, 96.95% purity) was obtained as a white solid.

[0484] BCY11376

[0485] [ka] Procedure for preparing compound 2

[0486] [ka] To a solution of compound 1 (5.0 mg, 49.5 μmol, 1.0 equiv.) in DMF (1 mL) , EDCI (8.5 mg, 54.8 μmol, 1.1 equiv.) and HOSu (5.7 mg The mixture was stirred at 25-30°C for 30 minutes. TLC showed that compound 1 was completely consumed and one new spot was formed. Then, 0.2 mL of this mixture was added with BCY8919 (20.0 mg, 9.62 The mixture was stirred for 25 min at 4°C for 30 min at 4°C. The mixture was stirred at 30° C. for 2 hours. LC-MS showed that BCY8919 was completely consumed. The desired m / z (calculated MW: 2162.51, observed m / z: 1081.8 ([M / 2+H] + One major peak having the formula 3 was detected. The reaction mixture was then concentrated under reduced pressure. The solvent was removed to give a residue, which was subsequently purified by preparative HPLC (TFA conditions). Compound 2 (12 mg, 5.55 μmol, yield 56.28%, purity 97.54 %) as a white solid.

[0487] Procedure for preparing BCY11376

[0488] [ka] Compound 2 (3mg, 1.39μmol, 1.0eq), BCY10861 (3.8mg , 1.40 μmol, 1.0 equiv) and THPTA (1.2 mg, 2.76 μmol, 2.0 equiv.) in t-BuOH / HO (1:1, 1 mL, pre-degassed and purified with N (purged twice) and then dissolved in CuSO4 (0.4 M, 3.5 μL, 1.0 equiv.) and VcNa (0.4 M, 3.5 μL, 1.0 equiv.) were added under N2. The solution was cooled to room temperature by dropwise addition of H4HCO3 (in 1:1 t-BuOH / H2O). The pH was adjusted to 8, and the solution turned pale yellow. The reaction mixture was stirred at 40° C. under a N2 atmosphere. Stirred for 2 h. LC-MS showed complete consumption of BCY10861. Desired m / z (calculated MW: 4878.64, observed m / z: 1220.8 ([M / 4+H] + The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA condition) to give BCY11376. (1.9 mg, 1.0 μmol, 27.01% yield, 96.2% purity) as a white solid Got it.

[0489] BCY11377

[0490] [ka] Procedure for preparing compound 2

[0491] [ka] To a solution of compound 1 (5.0 mg, 49.5 μmol, 1.0 equiv.) in DMF (1 mL) , EDCI (8.5 mg, 54.8 μmol, 1.1 equiv.) and HOSu (5.7 mg The mixture was stirred at 25-30°C for 30 minutes. TLC showed that compound 1 was completely consumed and one new spot was formed. Then, 0.2 mL of this mixture was added with BCY8920 (20.0 mg, 9.36 The mixture was stirred for 25 min at 4°C for 1 h. The mixture was stirred at 30° C. for 2 hours. LC-MS showed that BCY8920 was completely consumed. The desired m / z (calculated MW: 2220.54, observed m / z: 1110.90 ([M / 2+H] + One major peak having the formula 3 was detected. The reaction mixture was then concentrated under reduced pressure. The solvent was removed to give a residue, which was subsequently purified by preparative HPLC (TFA conditions). Compound 2 (12 mg, 5.15 μmol, yield 56.28%, purity 95.3%) ) as a white solid.

[0492] Procedure for preparing BCY11377

[0493] [ka] Compound 2 (3mg, 1.35μmol, 1.0eq), BCY10861 (3.8mg , 1.35 μmol, 1.0 equiv.) and THPTA (0.6 mg, 1.0 equiv.) The material was dissolved in t-BuOH / H2O (1:1, 1 mL, previously degassed and purged with N2 three times). Then, CuSO4 (0.4M, 3.4 μL, 1 eq.) and VcNa (0.4M , 3.4 μL, 1 eq.) was added under N2. 0.2M NH4HCO3 (1:1 t- The pH of this solution was adjusted to 8 by dropwise addition of 1,3-dihydro-2,4-tetrahydrofuran (BuOH / H2O). The reaction mixture was stirred at 40° C. for 2 h under N2 atmosphere. LC-MS is the desired m / z (calculated MW: 4936.68, observed m / z: 1234.9 ([M / 4+ H] + The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by preparative HPLC (TFA conditions) to give a residue. BCY11377 (3.5 mg, 0.66 μmol, yield 48.86%, purity 93.1%) ) as a white solid.

[0494] BCY11378

[0495] [ka] Procedure for preparing compound 2

[0496] [ka] To a solution of compound 1 (5.0 mg, 49.5 μmol, 1.0 equiv.) in DMF (1 mL) , EDCI (8.5 mg, 54.8 μmol, 1.1 equiv.) and HOSu (5.7 mg The mixture was stirred at 25-30°C for 30 minutes. TLC showed that compound 1 was completely consumed and one new spot was formed. Then, 0.2 mL of this mixture was added to BCY9172 (20.0 mg, 9.54 The mixture was added to 25 mL of DIEA (1.7 μL, 9.62 μmol). The mixture was stirred at 30° C. for 2 hours. LC-MS showed that compound 1 was completely consumed. , desired m / z (calculated MW: 2176.49, observed m / z: 1090.0 ([M / 2+H ] + The reaction mixture was then concentrated under reduced pressure to give The solvent was removed to give a residue which was subsequently purified by preparative HPLC (TFA conditions). Compound 2 (20.2 mg, 7.48 μmol, yield 78.34%, purity 80.57%) was obtained as a white solid.

[0497] Procedure for preparing BCY11378

[0498] [ka] Compound 2 (5mg, 2.30μmol, 1.0eq), BCY10861 (6.24m g, 2.30 μmol, 1.0 equiv.) and THPTA (1.0 mg, 1.0 equiv.) The mixture was dissolved in t-BuOH / H2O (1:1, 1 mL, previously degassed and purged with N2 three times). Dissolve and then add CuSO4 (0.4 M, 5.8 μL, 1.0 equiv.) and VcNa(0 0.4M, 5.8μL, 1.0 eq) was added under N2. 0.2M NH4HCO3 (1 Adjust the pH of this solution to 8 by dropwise addition of 1:1 t-BuOH / H2O. The solution turned pale yellow upon addition of the solvent, and the reaction mixture was stirred at 40° C. for 2 hours under a N2 atmosphere. LC-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 4894.61, actual measurement m / z:1224.3([M / 4+H] + ) with one main piece The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Purification by preparative HPLC (TFA conditions) yielded BCY11378 (1.2 mg, 0.34 μg). mol, 10.07% yield, 94.3% purity) as a white solid.

[0499] BCY11379

[0500] [ka] Procedure for preparing BCY8919-PEG5-N3

[0501] [ka] BCY8919 (30.0 mg, 14.43 μmol, 1.0 equiv.) and compound 1 ( 6.3 mg, 14.57 μmol, 1.01 equiv.) in MeCN (1 mL) and HO ( The solution was adjusted to pH 8 by adding 1M NaHCO3. The mixture was then stirred at 35° C. for 2 h. LC-MS showed that BCY8919 was completely consumed. The desired m / z (calculated MW: 2396.79, measured m / z: 119 8.74([M / 2+H] + ) and 799.50([M / 4+H] + )) The reaction mixture was purified by preparative HPLC (TFA conditions) and Compound 2 (20 mg, 8.07 μmol, yield 55.92%, purity 96.68%) was obtained as a white solid. Got it as a body.

[0502] Procedure for preparing BCY11379

[0503] [ka] Compound 2 (3.0 mg, 1.25 μmol, 1.0 equiv.) and BCY10861 (3 .4 mg, 1.25 μmol, 1.0 equiv.) was first dissolved in t-BuOH / H2O (1:1) 1 mL, then CuSO4 (0.4 M, 7 μL, 2.24 equiv.), VcNa (1 mg, 5.04 μmol, 4.03 equiv.) and THPTA (1 mg, 2.30 μmol Finally, 1M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was cooled to room temperature under a N2 atmosphere. The mixture was stirred at 25-30°C for 16 hours under atmospheric pressure. LC-MS showed that compound 2 was completely consumed. and the desired m / z (calculated MW: 5112.93, measured m / z: 1022.96) ([M / 5+H] + ) and 1278.74([M / 4+H] + ) with one main piece The reaction mixture was purified by preparative HPLC (TFA conditions) to give BCY1137. 9 (3.4 mg, 0.615 μmol, yield 52.00%, purity 97.88%) was obtained as a white solid. Got it as a body.

[0504] BCY11380

[0505] [ka] Procedure for preparing BCY8920-PEG5-N3

[0506] [ka] BCY8920 (30.0 mg, 14.04 μmol, 1.0 equiv.) and compound 1 ( 6.1 mg, 14.11 μmol, 1.01 equiv.) in MeCN (1 mL) and HO ( The solution was adjusted to pH 8 by adding 1M NaHCO3. The mixture was then stirred at 35° C. for 2 h. LC-MS showed that BCY8920 was completely consumed. The desired m / z (calculated MW: 2454.83, measured m / z: 122 7.63([M / 2+H] +) and 818.66([M / 3+H] + )) The reaction mixture was purified by preparative HPLC (TFA conditions) and Compound 2 (20 mg, 8.03 μmol, yield 57.21%, purity 98.56%) was obtained as a white solid. Got it as a body.

[0507] Procedure for preparing BCY11380

[0508] [ka] Compound 2 (3.5 mg, 1.43 μmol, 1.0 equiv.) and BCY10861 (3 .9 mg, 1.44 μmol, 1.0 equiv.) was first dissolved in t-BuOH / H2O (1:1) 1 mL, then CuSO4 (0.4 M, 8 μL, 2.24 equiv.), VcNa (1 mg, 5.04 μmol, 3.52 equiv.) and THPTA (1 mg, 2.30 μmol Finally, 1M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was cooled to room temperature under a N2 atmosphere. The mixture was stirred at 25-30°C for 16 hours under atmospheric pressure. LC-MS showed that most of compound 2 was consumed. and the desired m / z (calculated MW: 5170.97, observed m / z: 1034.2 8([M / 5+H] + ) and 1293.10([M / 4+H] + )) with one main The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain BCY11 380 (1.6 mg, 0.296 μmol, yield 20.77%, purity 96.77%) Obtained as a coloured solid.

[0509] BCY11381

[0510] [ka] Procedure for preparing BCY8920-PEG5-N3

[0511] [ka] BCY9172 (30.0 mg, 14.32 μmol, 1.0 equiv.) and compound 1 ( 6.2 mg, 14.34 μmol, 1.0 equiv.) in MeCN (1 mL) and HO (1 m The solution was adjusted to pH 8 by adding 1M NaHCO3, and then The mixture was stirred at 35° C. for 2 h. LC-MS showed that BCY9172 was completely consumed. The desired m / z (calculated MW: 2412.75, measured m / z: 1206) was obtained. .72([M / 2+H] + One main peak having the formula: The compound was purified by HPLC (TFA condition) to obtain compound 2 (15 mg, 6.14 μmol, yield 42.87%, purity 98.75%) as a white solid.

[0512] Procedure for preparing BCY11381

[0513] [ka] Compound 2 (3.0 mg, 1.24 μmol, 1.0 equiv.) and BCY10861 (3 .4 mg, 1.25 μmol, 1.01 equiv.) first in t-BuOH / HO (1:1) 2 mL, then CuSO4 (0.4 M, 7 μL, 2.25 equiv.), VcNa( 1 mg, 5.04 μmol, 4.06 equiv.) and THPTA (1 mg, 2.30 μmol Finally, 1M NH4HCO3 was added to bring the pH to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was cooled to room temperature under a N2 atmosphere. The mixture was stirred at 25-30° C. for 16 hours under atmospheric pressure. LC-MS showed the desired m / z (calculated MW: 5 128.89, actual m / z: 1026.05 ([M / 5+H] + ) and 1282.50 ([M / 4+H] + The reaction mixture was subjected to preparative HPLC (T FA conditions) and purified to obtain BCY11381 (1.6 mg, 0.295 μmol, yield 23.73%, purity 94.59%) as a white solid.

[0514] [Example 5] Generation of CD137 monoclonal antibody agonists The CD137 monomers used for comparison with CD137 heteromers in the experiments presented herein The sequences of the monoclonal antibody agonists are disclosed in U.S. Patent No. 7,288,638. G4 isotype antibodies were expressed on expiry following transient transfection of DNA expression constructs. CHO Expression System (Thermo Fisher Scie The antibodies were purified by Protein A affinity chromatography. The compound was purified by HPLC and formulated in phosphate buffered saline (PBS) pH 7.2. Purity analysis using SEC (column GF-250, Agilent) confirmed the presence of CD137 monoclonal antibody. The monoclonal antibody was found to be approximately 95% monomeric. CD137 monoclonal antibodies at concentrations of more than μg / ml were used to target CD137-expressing C It was shown that ToxinSensor™ Chr can bind to HO cells. omogenic LAL Endotoxin Assay Kit(Genscri Endotoxin analysis using pt) demonstrated that the CD137 monoclonal antibody preparation was It was shown to contain less than U / mg of endotoxin.

[0515] Biological Data 1.CD137 Biacore Experimental Description Biacore experiments were performed to identify heterotandem IgG1 binding to human CD137 protein. Peptide k a (M -1 s -1 ), k d (s -1 ), K D (nM) values ​​were determined. Human CD137 (R&D systems) was resuspended in PBS and incubated as per the manufacturer's suggested protocol. The protocol involves the use of EZ-Link™ Sulfo-NHS-LC-LC-Biotin Reagent Proteins were desalted and biotinylated using the reagent (Thermo Fisher). Uncoupled biotin was removed into PBS using a spin column.

[0516] To analyze peptide binding, Biacore T200 or Biacore 3 The 000 instrument was used with a XanTec CMD500D chip. Streptavidin The electrophoresis buffer was HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) Immobilize on chip using standard amine coupling chemistry at 25 °C. Briefly, the carboxymethyl dextran surface was immersed in water for 1 h at a flow rate of 10 μl / min. :1 ratio of 0.4M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide salt A 7-minute injection of 0.1M N-hydroxysuccinimide (NHS) / 0.1M EDC was performed. To capture streptavidin, the protein was activated with 10 mM acetic acid. The solution was diluted to 0.2 mg / ml in sodium chloride (pH 4.5) and 120 μl was injected. The remaining activated groups were captured on the activated chip surface using 1 M ethanolamine (pH 8.5) for 7 minutes to block, and then inject biotinylated CD137 at 270-1500 R. The buffer was changed to PBS / 0.05% Tween 20, and peptide A dilution series of peptides was prepared in this buffer with a final DMSO concentration of 0.5%. The concentration of the buffer was 500 nM and was further diluted 2-fold or 3-fold six times. SPR analysis was performed for 60 s. Binding and dissociation were performed for 900 seconds at a flow rate of 90 μl / min at 25° C. After each cycle, A regeneration step (10 μl of 10 mM glycine pH 2) was used. Data were corrected for volume effects excluding O. All data were processed using standard processing procedures. The blank injection and the reference surface were double-referenced using the Scrubber software. Data were collected using BioLogic Software, version 2.0c. Processing and kinetic fitting were performed. Mass transfer effects were calculated where applicable. The data were fitted using a simple 1:1 binding model that allows for a linear regression analysis of the binding efficiency.

[0517] Certain heterotandem peptides were tested in this assay and the results are shown in Table 1 below.

[0518] [Table 4]

[0519] 2. Nectin-4 Biacore Experimental Description Biacore experiments were performed to identify human nectin-4 protein (Charles Ri k of hetero tandem peptides bound toa (M -1 s -1 ), k d (s -1 ), K D The (nM) values ​​were determined. The gp67 signal sequence and C-terminal FLAG tag were Human nectin-4 (residues Gly32-Ser349; NCBI reference sequence: NP_1 12178.2) was cloned into pFastbac-1 and used as a standard Bac-to- Baculoviruses were cultured using the Bac™ protocol (Life Technologies). Virus was produced at 1 × 10 in Excell-420 medium (Sigma) at 27 °C. 6 pieces ml -1 Sf21 cells were infected with the P1 virus stock at an MOI of 2 and the supernatant was diluted with 72% CO. The supernatant was transferred to anti-FLAG M2 affinity agarose gel, washed with PBS. Resin (Sigma) was batch bound for 1 h at 4°C, after which the resin was transferred to a column and PB The column was washed extensively with S. Proteins were eluted with 100 μg / ml FLAG peptide. The eluted protein was concentrated to 2 ml and purified by S200 Superd in PBS at 1 ml / min. The column was loaded with 2 ml fractions. The fractions containing Chin-4 protein were concentrated to 16 mg / ml. Proteins were incubated with the EZ-Link™ Spindle according to the manufacturer's suggested protocol. Phosphatase was assayed using Lupho-NHS-LC-LC-biotin reagent (Thermo Fisher). Proteins were extensively desalted and purified using spin columns. Uncoupled biotin was removed into PBS using PBS. To analyze peptide binding, a Biacore 3000 instrument was equipped with a CM5 chip (GE Streptavidin was used in combination with HO as the running buffer. BS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) was used for 25 The antibodies were immobilized on the chip using standard amine coupling chemistry at 37 °C. The carboxymethyldextran surface was coated with a 1:1 ratio of 0.4M 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M was activated by a 7 min injection of N-hydroxysuccinimide (NHS). To capture streptavidin, the protein was diluted with 10 mM sodium acetate (pH 4. 5) Dilute the plate to 0.2 mg / ml in PBS and inject 120 μl of streptavidin. The remaining activated groups were captured on the activated chip surface using 1M ethanolamine (pH 8. 5) was injected for 7 minutes to block the cells, and biotinylated nectin-4 was injected at 1200-1800 R. The buffer was changed to PBS / 0.05% Tween 20, and peptide A dilution series of peptides was prepared in this buffer with a final DMSO concentration of 0.5%. The peptide concentration was 100 nM and was further diluted 2-fold six times. SPR analysis was performed on individual peptides. Depending on the assay, the incubation time was 60 s for binding and 400-1200 s for dissociation, at a flow rate of 50 μl / min, and at 25 °C. Data were corrected for volume effects excluding DMSO. All data were A comprehensive procedure was used to double reference and scrub the blank injection and reference surface. er software, version 2.0c (BioLogic Software) was used. Data processing and kinetic fitting were performed using the In this case, the data was fitted using a simple 1:1 binding model that allows for mass transfer effects. I did.

[0520] Certain hetero-tandem peptides of the present invention were tested in the Nectin-4 binding assay described above, The results are shown in Table 2 below.

[0521] [Table 5]

[0522] 3.EphA2 Biacore Experimental Description Biacore experiments were performed to identify heterotandem IgG1 binding to human EphA2 protein. Peptide k a (M -1 s -1 ), k d (s -1 ), K D (nM) values ​​were determined. EphA2 was incubated with 4 mM sodium acetate at 3-fold molar excess of biotin over protein. 1 hour in 100 mM NaCl, pH 5.4, using EZ-Link™ Slu Biotinylated using HO-NHS-LC-biotin. After dialysis of the reaction mixture into PBS , Fluorescence Biotin Quantification Kit( The extent of labeling was determined using a Thermo Scientific. A Biacore T200 instrument was used with a XanTec CMD500D chip. Streptavidin was incubated in HBS-N (10 mM HEPES, 0.1 The reaction was carried out using standard amine coupling chemistry with 5M NaCl, pH 7.4 at 25 °C. Briefly, the carboxymethyl dextran surface was immobilized on the chip using a 10 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)propionate in a 1:1 ratio at a flow rate of 1 μl / min. EDTA / 0.1M N-hydroxysuccinimide (NH The plate was activated by injecting 100 mM NaCl (NaCl) for 7 min. The protein was diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and The remaining activated groups were captured on the activated chip surface by injecting 1 μl of Blocking was performed by injecting ethanolamine (pH 8.5):HBS-N (1:1) for 7 min. The buffer was changed to PBS / 0.05% Tween 20, and up to 0.2 μM of Protein dilutions were used to induce biotinylated EphA2 to a level of 500–1500 RU. A dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5%. The top peptide concentration was 50 or 100 nM and was further diluted 2-fold six times. The analysis was performed at 25°C with a flow rate of 90 μl / min for 60 s binding and 900–1200 s dissociation. Data were corrected for volume effects excluding DMSO. All data were A comprehensive procedure was used to double reference and scrub the blank injection and reference surface. er software, version 2.0c (BioLogic Software) was used. Data processing and kinetic fitting were performed using the In this case, the data was fitted using a simple 1:1 binding model that allows for mass transfer effects. I did.

[0523] Certain heterogeneous tandem peptides of the invention were tested in an EphA2 binding assay, and the results are given below. As shown in Table 3 below.

[0524] [Table 6]

[0525] 4. CD137 Reporter Assay Co-culture with Tumor Cells 1% FBS in RPMI-1640 (a component of Promega kit CS196005) A culture medium called R1 medium is prepared by adding: Dilutions are prepared in sterile 96-well plates. Place in designated wells of white cell culture plates. In contrast, 25 μl of test article or R1 (as background control) per well Use: Tumor cells * Harvest and resuspend in R1 medium at a concentration of 400,000 cells / mL. Twenty-five (25) μL / well of tumor cells are used in white cell culture plates. Thaw AT cells (Promega kit CS196005, 0.5 mL) in a water bath and then Add twenty-five (25) μL / well of Jurkat cells to the 5 ml pre-warmed R1 medium. Use in white cell culture plates. Incubate cells and test materials for 6 hours at 37°C and 5% CO2. At the end of the 6 hours, 75 μl / well of Bio-Glo™ (Pr omega) was added and incubated for 10 minutes, after which the plate was read by a plate reader (Clari Luminescence was read using ostar and BMG. Fold changes relative to the agonist (cell line) were calculated and expressed as log(agonist) vs. response using GraphPad. Plotted in Prism to show EC50 (nM) and fold induction over background. (Maximum) is determined.

[0526] The tumor cell types used in the co-culture were heterotandem specific, as shown in Table 4 below. The therapeutic approach depends on the specific tumor targeting.

[0527] [Table 7]

[0528] EphA2-CD137 heterotandem BCY7985 was detected in EphA2-expressing HT108 0 cells in the presence of CD137 luciferase reporter assays. The data showing that HT1080 showed a strong induction of 137 cell activity is shown in FIG. In the absence of cells, there is no CD137 induction by the heterotandem.

[0529] EphA2 / CD137 heterotandems show strong CD137 expression in the CD137 reporter assay 137 activation was induced, and the induction fold of activation was Tumor target expression levels on SC-OV-3: EphA2 high and LNCaP: EphA2 low Data showing that the effect of β-amyloid on the β-amyloid complex is dependent on the enzyme activity are shown in Figure 4.

[0530] Nectin-4 / CD137 heterotandem shows strong C-receptor activity in CD137 reporter assay D137 activation was induced, and the induction fold of activation was compared with that of the cell line used for co-culture (HT1376 Tumor target expression levels on NCI-H292:Nectin-4 high and NCI-H292:Nectin-4 medium Data demonstrating this dependency are shown in FIG.

[0531] PD-L1 / CD137 heterotandem is associated with CD13 in the presence of PD-L1-expressing cell lines. 7 Data showing that it induces potent activation of CD137 in reporter assays are shown in Figure 9. Heterotandem assays using CD137 reporter assays in co-culture with various cell lines. The EC50 (nM) and fold induction induced by the Dem peptides are summarized in Table 5 below. Report.

[0532] [Table 8-1] [Table 8-2] [Table 8-3]

[0533] 5. Primary human T cell-A549 co-culture (tumor cell killing) PBMCs were isolated from three healthy donors and cultured at two concentrations in the presence of anti-CD3 stimulation. At two prescribed ratios, Nuclight Red-labeled tumor target cells (human lung cancer cells A549 (registered trademark) Tumor cells:PBMC co-cultures The compounds were incubated with lead bicyclics at three concentrations. To detect toxicity, all test conditions were performed in the absence of stimulated PBMCs and tumor cells. The tumor was then cultured in a 30-well plate. Viable Nucelight red-positive tumor cells were counted over time. In addition, a caspase 3 / 7 dye was used to assess tumor killing. The IncuCyte CT scan enabled real-time live cell fluorescence imaging. Cultures were analyzed using an S3 machine. Co-cultures were imaged for 72 hours. Each condition was imaged for 3 It was established in a series.

[0534] EphA2 / CD137 heterotandems enhance the uptake of primary human T cells and cancer cell co-cultures Data demonstrating that anti-CD40 induces cell killing in tumor cells is shown in Figure 5. The 137 mAb agonist is used as a control.

[0535] 6. Human PBMC-4T1 Co-culture (Cytokine Release) Assay Mouse mammary tumor cell line 4T1-1 (4T1-parent) and mouse nectin-4 overexpression T1 (4T1-D02) was incubated with 10% heat-inactivated fetal bovine serum, 100 I.U / ml penicillin and 100 I.U / streptomycin, 20 mM HEPES, 1× non-essential amino acids and cultured in RPMI 1640 supplemented with 2 mM L-glutamine (RPMI working medium). Frozen PBMCs from healthy human donors were thawed, washed once in room temperature PBS, and then For tumor cell and PBMC co-cultures, 100 100 PBMCs and 2000 tumor cells (5:1) were mixed and plated in a 384-well plate. To stimulate human PBMCs, 125 ng / ml of soluble anti-CD3 mAb (clone OKT3) was added to the cultures on day 0. Test compounds, control compounds, or or vehicle control was added to each well to a final volume of 100 μl per well. Plates were incubated at 37 °C in a cell culture incubator with 5% CO2 for up to 3 days. Supernatants were harvested 48 hours after stimulation and human IL-1 was detected using the HTRF assay. The assay was performed using Excel or Prism software. Data was analyzed, a standard curve was constructed, and protein concentrations were interpolated. Data were , represents one study using PBMCs from three different donors tested in the experiment.

[0536] Data shown in Figure 7 demonstrate that Nectin-4 / CD137 heterotandems inhibit PBMC-4T 1. Induce Robust IL-2 and IFN-γ Cytokine Secretion in Co-culture Assays BCY9350 and BCY9351 inhibited nectin-4 and C, respectively. Non-binding control for D137.

[0537] Nectin-4 selected in human PBMC-4T1 co-culture (cytokine release) assay EC50 (nM) and maximum I induced by CD137 heterotandem peptides A summary of FN-γ cytokine secretion (pg / ml) is reported in Table 6 below.

[0538] [Table 9]

[0539] 7. Ex Vivo Culture Protocol Discovery Life Sciences (DLS) primary patient-derived tumor cells Thaw the cells gently in 10 mL prewarmed wash medium freshly supplemented with Benzonase. Using the Greiner 3D Spheroid Kit (Cat. No. 655840) Maintain the cells in culture for 2 days. Briefly, tumor cells were counted using a hemocytometer. Count with lipan blue. Wash and pellet cells by centrifugation at 1500 rpm for 5 min. , 1×10 6 Resuspend the cells in 100 μL of N3D nanoshuttle per cell. To activate the cells, spun down at 1500 rpm for 5 minutes and resuspended; this process After the final centrifugation, the cells were resuspended in an appropriate amount of fresh Lung DTC medium (DLS). Suspend the cells to obtain 50,000-100,000 cells per well in 100 μL / well. Greiner cell-repellent 96-well plate Use the 655976 PBS sieve for this experiment. If fragments are present, apply them to a 70-100 μm filter before plating the samples. At least 50,000 cells were collected for the day 0 flow cytometry panel. The cells are then stained, fixed, and stored at 4°C for later flow analysis. Test compound dilutions were prepared in separate plates in 2x in Lung DTC medium and 100 μL / well. Add these 2x drug solutions to the wells as described by the plate map. The assay plate was then incubated at 37°C in a humidified chamber with 5% CO2 on a 96-well magnetic plate. Place the cells in the magnetic spheroid drive. After 24 hours, remove the magnetic spheroid drive. At 8 hours, media was collected for cytokine analysis and cells were analyzed by flow cytometry on day 2. The Luminex reader is used to collect the samples for the R&D Systems camera panel. Custom built cytokine / chemokine panel (IP-10, Granzyme B, IFNγ , IL-2, IL-6, TNFα, IL-8, MIP-1a, MIP-1b, MCP-1 , IL-10, MIG) are used to quantify cytokines. Flow panel: Day 0 = live / dead, CD45, EpCAM, Nectin4, CD3, CD4, CD8, CD137; Day 2 = Live / Dead, CD45, EpCAM, Nectin4, CD3, CD8, Ki67, and and counting beads. Flow data are analyzed with Flowjo software.

[0540] Data shown in Figure 8 demonstrate that Nectin-4 / CD137 heterotandems are demonstrated that it induces target-dependent cytokine release in ex vivo cultures of lung tumors. Treatment with BCY10572 increased several immune markers in patient samples. (normalized to vehicle) and CD8 + ki67 + Nectin-4-dependent transformation of T cells was induced.

[0541] 8. Pharmacokinetics of CD137 bispecifics in SD rats Male SD rats were administered 25 mM histidine HCl, 10% sucrose, pH 7. Each bicyclic multimer was administered at 2 mg / kg at 4 °C for 1 h. At each time point, the subjects were administered the same dose intravenously via the submandibular or saphenous vein. Subsequent bleeding (approximately 80 μL blood / time point) was performed. All blood samples were immediately diluted with 2 mL of anticoagulant. Transfer to a pre-chilled microcentrifuge tube containing 1 μL K2-EDTA (0.5 M) and place on wet ice. The blood samples were purified for plasma by centrifugation at approximately 4° C. and 3000 g. The precipitant containing the internal standard was immediately added to the plasma, mixed thoroughly, and then diluted with 12,000 The supernatant was transferred to a pre-labeled polypropylene microcentrifuge and centrifuged at 4°C for 10 min. The samples were transferred to tubes and then flash frozen on dry ice. The supernatant sample (7.5 μL) was analyzed by Orbitrap Q in positive ion mode. Using Exactive, bicyclic multimers were directly injected for LC-MS / MS analysis. The concentrations of 100 and 150 mg of 100% ethanol in the body were determined using the Phoenix WinNonlin 6.3 software program. The program was used to obtain plasma concentration data versus time using a non-compartmental approach. Analyzed: C0, Cl, Vdss, T1 / 2, AUC(0-last), AUC(0-i nf), MRT(0-last), MRT(0-inf) and plasma concentration profiles versus time A profile graph was reported.

[0542] FIG. 10 shows the effect of BCY105 on the cellular metabolism in SD rats (n=3) after IV administration of 2 mg / kg. The plasma concentration curves of 72 and BCY10000 versus time are shown. Pharmacokinetics from the experiment The parameters are as shown in Table 7.

[0543] [Table 10]

Claims

1. (a) a first peptide ligand that binds to a component present on an immune cell, said component present on said immune cell being CD137, said CD137-binding bicyclic peptide ligand comprising: C ｉ IEEGQYC ｉｉ FADPY[Nle]C ｉｉｉ (SEQ ID NO: 1); C ｉ [tBuAla]PE[D-Ala]PYC ｉｉ FADPY[Nle]C ｉｉｉ (SEQ ID NO:3); C ｉ IEEGQYC ｉｉ F[D-Ala]DPY[Nle]C ｉｉｉ (SEQ ID NO:4); C ｉ [tBuAla]PK[D-Ala]PYC ｉｉ FADPY[Nle]C ｉｉｉ (SEQ ID NO:5); C ｉ [tBuAla]PE [D-Lys] PYC ｉｉ FADPY[Nle]C ｉｉｉ (Allocation number 6); C ｉ [tBuAla]P [K (PYA)] [D-Ala] PYC ｉｉ FADPY[Nle]C ｉｉｉ (Allocation number 7); C ｉ [tBuAla]PE [D-Lys (PYA)] PYC ｉｉ FADPY[Nle]C ｉｉｉ (Allocation number 8); C ｉ IEE[D-Lys(PYA)]QYC ｉｉ FADPY(Nle)C ｉｉｉ (SEQ ID NO: 9); and [dC ｉ ][dI][dE][dE][K(PYA)][dQ][dY][dC ｉｉ ] [dF] [dA] [dD] [dP] [dY] [dNle] [dC ｉｉｉ ] (SEQ ID NO: 10); or a modified derivative thereof, (b) a second peptide ligand that binds to a component present on a cancer cell, the component present on a cancer cell being EphA2, PD-L1, or Nectin-4; (i) the EphA2-binding bicyclic peptide ligand is C ｉ [HyP]LVNPLC ｉｉ LHP[dD]W[HArg]C ｉｉｉ (SEQ ID NO:2) and C ｉ LWDPTPC ｉｉ ANLHL[HArg]C ｉｉｉ (SEQ ID NO: 11); or a modified derivative thereof, (ii) the PD-L1-binding bicyclic peptide ligand is C ｉ [HArg] DWC ｉｉ HWTFSHGHPC ｉｉｉ (SEQ ID NO: 12); C ｉ SAGWLTMC ｉｉ QKLHLC ｉｉｉ (SEQ ID NO: 13); and C ｉ SAGWLTMC ｉｉ Q[K(PYA)]LHLC ｉｉｉ (SEQ ID NO: 14); or a modified derivative thereof, and (iii) the Nectin-4 binding bicyclic peptide ligand is C ｉ P[1Nal][dD]C ｉｉ M[HArg]DWSTP[HyP]WC ｉｉｉ (SEQ ID NO: 15; hereafter referred to as BCY8116); C ｉ P[1Nal][dD]C ｉｉ M[HArg]D[dW]STP[HyP][dW]C ｉｉｉ (SEQ ID NO: 16; hereafter referred to as BCY11415); C ｉ P[1Nal][dK](Sar １０ -(B-Ala))C ｉｉ M[HArg]DWSTP[HyP]WC ｉｉｉ (SEQ ID NO: 17); and C ｉ PFGC ｉｉ M[HArg]DWSTP[HyP]WC ｉｉｉ (SEQ ID NO: 18; hereinafter referred to as BCY11414) or a modified derivative thereof, (In the formula, C ｉ , C ｉｉ and C ｉｉｉ represent the first, second and third cysteine ​​residues, respectively; 1Nal represents 1-naphthylalanine; B-Ala represents β-alanine; dD represents aspartic acid in the D configuration; HArg represents homoarginine; HyP represents hydroxyproline; Nle represents norleucine; PYA represents 4-pentynoic acid; Sar １０ represents 10 sarcosine units, and tBuAla represents t-butyl-alanine. Each of said peptide ligands comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide such that at least two of the polypeptide loops are formed on the molecular scaffold, said molecular scaffold being 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA); The modified derivative is replacement of one or more amino acid residues with one or more non-naturally occurring amino acid residues; replacement of one or more polar amino acid residues with one or more equivalent or isoelectronic amino acids; and / or independently comprising one or more replacements of one or more L-amino acid residues with one or more D-amino acid residues; A heterotandem bicyclic peptide complex, or a pharma- ceutically acceptable salt thereof, comprising: (a) a first peptide ligand, conjugated to (b) a second peptide ligand via a linker.

2. 2. The heterotandem bicyclic peptide complex of claim 1, wherein the immune cell is selected from white blood cells; lymphocytes; CD8 or CD4; CD8; dendritic cells, follicular dendritic cells, and granulocytes.

3. 3. The heterotandem bicyclic peptide conjugate of claim 2, wherein the lymphocyte is a T lymphocyte or T cell, a B cell, or a natural killer cell.

4. the CD137-binding bicyclic peptide ligand comprises an N-terminal modification and a C-terminal modification; Ac-A-(SEQ ID NO:1)-Dap (hereinafter referred to as BCY7732); Ac-A-(SEQ ID NO:1)-Dap(PYA) (hereinafter referred to as BCY7741); Ac-(SEQ ID NO:3)-Dap (hereinafter referred to as BCY9172); Ac-(SEQ ID NO:3)-Dap(PYA) (hereinafter referred to as BCY11014); Ac-A-(SEQ ID NO:4)-Dap (hereinafter referred to as BCY8045); Ac-(SEQ ID NO:5)-A (hereinafter referred to as BCY8919); Ac-(SEQ ID NO:6)-A (hereinafter referred to as BCY8920); Ac-(SEQ ID NO:7)-A (hereinafter referred to as BCY8927); Ac-(SEQ ID NO:8)-A (hereinafter referred to as BCY8928); Ac-A-(SEQ ID NO:9)-A (hereinafter referred to as BCY7744); and Ac-[dA]-(SEQ ID NO: 10)-[dA]-NH ２ (hereinafter referred to as BCY11506); (In the formula, Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentynoic acid.) 2. The heterotandem bicyclic peptide complex of claim 1, or a pharma- ceutically acceptable salt thereof, including a modified derivative thereof as defined in claim 1.

5. 5. The heterotandem bicyclic peptide complex of claim 4, wherein the CD137-binding bicyclic peptide ligand comprises Ac-(SEQ ID NO:8)-A (hereinafter referred to as BCY8928), wherein Ac represents an acetyl group.

6. 6. The heterotandem bicyclic peptide complex of any one of claims 1 to 5, wherein the cancer cells are selected from HT1080, SC-OV-3, PC3, H1376, NCI-H292, LnCap, MC38 and RKO tumor cells.

7. 7. The heterotandem bicyclic peptide complex of any one of claims 1 to 6, wherein the second peptide ligand comprises an EphA2-binding bicyclic peptide ligand, the EphA2-binding bicyclic peptide ligand comprising: C ｉ [HyP]LVNPLC ｉｉ LHP[dD]W[HArg]C ｉｉｉ (SEQ ID NO:2); and C ｉ LWDPTPC ｉｉ ANLHL[HArg]C ｉｉｉ (SEQ ID NO: 11); (In the formula, C ｉ , C ｉｉ and C ｉｉｉ represent the first, second and third cysteine ​​residues, respectively; HyP represents hydroxyproline; dD represents aspartic acid in the D configuration; and HArg represents homoarginine. or a modified derivative thereof as defined in claim 1, or a pharma- ceutically acceptable salt thereof.

8. the EphA2-binding bicyclic peptide ligand comprises an N-terminal modification; A-HArg-D-(SEQ ID NO:2) (hereinafter referred to as BCY9594); [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO:2) (hereinafter referred to as BCY6099); [PYA]-[B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO:2) (hereinafter referred to as BCY6169); and [PYA]-[B-Ala]-[Sar １０ ]-VGP-(SEQ ID NO:11) (hereinafter referred to as BCY8941); (Wherein, HArg represents homoarginine, PYA represents 4-pentynoic acid, and Sar １０ represents 10 sarcosine units, and B-Ala represents β-alanine).

8. The heterotandem bicyclic peptide complex of claim 7, or a pharma- ceutically acceptable salt thereof, comprising:

9. [PYA]-[B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169), in which PYA at the N-terminus of BCY6169 is linked to Dap at the C-terminus of BCY9172 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169), in which PYA at the N-terminus of BCY6169 is linked to Dap at the C-terminus of BCY7732 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169), in which PYA at the N-terminus of BCY6169 is linked to Dap at the C-terminus of BCY8045 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-VGP-(SEQ ID NO: 11) (BCY8941), in which PYA at the N-terminus of BCY8941 is linked to Dap at the C-terminus of BCY7732, -PEG １２ -linked via a linker; [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099), in which the N-terminus of BCY6099 is linked to the C-terminus Dap (PYA) of BCY7741 by -PEG １０ -linked via a linker; [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099), in which the N-terminus of BCY6099 is linked to the C-terminus Dap (PYA) of BCY7741 by -PEG ２３ -linked via a linker; [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099), in which the N-terminus of BCY6099 is linked to the C-terminus Dap (PYA) of BCY7741 by -PEG １５ -Sar ５ -linked via a linker; [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099), in which the N-terminus of BCY6099 is linked to the C-terminus Dap (PYA) of BCY7741 by -PEG １０ -Sar １０ -linked via a linker; [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099), in which the N-terminus of BCY6099 is linked to the C-terminus Dap (PYA) of BCY7741 by -PEG ５ -Sar １５ -linked via a linker; [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099), in which the N-terminus of BCY6099 is linked to the C-terminus Dap (PYA) of BCY7741 by -PEG ５ -Sar ５ -linked via a linker; [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099), in which the N-terminus of BCY6099 is linked to the C-terminus Dap (PYA) of BCY7741 by -PEG ５ -linked via a linker; [B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO:2) (BCY6099), in which the N-terminus of BCY6099 is linked to the C-terminus Dap of BCY7732 by -PEG ２４ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169), in which PYA at the N-terminus of BCY6169 is linked to Lys3 of BCY8919 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169), in which PYA at the N-terminus of BCY6169 is linked to dLys4 of BCY8920 by -PEG １２ -linked via a linker; A-HArg-D-(SEQ ID NO:2) (BCY9594) linked to Ac-(SEQ ID NO:7)-A (BCY8927), in which the N-terminus of BCY9594 is linked to Lys(PYA)3 of BCY8927 by -PEGylation. ５ -linked via a linker; A-HArg-D-(SEQ ID NO:2) (BCY9594) linked to Ac-(SEQ ID NO:8)-A (BCY8928), in which the N-terminus of BCY9594 is linked to dLys(PYA)4 of BCY8928 by -PEG ５ -linked via a linker; A-HArg-D-(SEQ ID NO:2) (BCY9594) linked to Ac-(SEQ ID NO:3)-Dap(PYA) (BCY11014), in which the N-terminus of BCY9594 is linked to the C-terminus of Dap(PYA) of BCY11014 by -PEG ５ -linker; and [PYA]-[B-Ala]-[Sar １０ ]-A-[HArg]-D-(SEQ ID NO:2) (BCY6169), in which the N-terminus of BCY6169 is linked to the C-terminus Dap of BCY9172 by -CH ２ -Linked via linker 9. The heterotandem bicyclic peptide complex of claim 7 or 8, which is a CD137 / EphA2 complex selected from the group consisting of:

10. the second peptide ligand comprises a PD-L1 binding bicyclic peptide ligand; The PD-L1 binding bicyclic peptide ligand is C ｉ [HArg] DWC ｉｉ HWTFSHGHPC ｉｉｉ (SEQ ID NO: 12); C ｉ SAGWLTMC ｉｉ QKLHLC ｉｉｉ (SEQ ID NO: 13); and C ｉ SAGWLTMC ｉｉ Q[K(PYA)]LHLC ｉｉｉ (SEQ ID NO: 14); (In the formula, C ｉ , C ｉｉ and C ｉｉｉ represent the first, second and third cysteine ​​residues, respectively, HArg represents homoarginine, and PYA represents 4-pentynoic acid. or a modified derivative thereof as defined in claim 1, or a pharma- ceutically acceptable salt thereof.

11. the PD-L1 binding bicyclic peptide ligand comprises an N-terminal modification and / or a C-terminal modification; [PYA]-[B-Ala]-[Sar １０ ] - (SEQ ID NO: 12) (hereinafter referred to as BCY8938); [PYA]-[B-Ala]-[Sar １０ ]-SDK-(SEQ ID NO:13) (hereinafter referred to as BCY10043); N.H. ２ -SDK- (SEQ ID NO: 13) - [Sar １０ ]-[K(PYA)] (hereafter referred to as BCY10044); N.H. ２ -SDK- (SEQ ID NO: 14) (hereinafter referred to as BCY10045); and Ac-SDK-(SEQ ID NO:14)-PSH (hereinafter referred to as BCY10861); (Wherein, PYA represents 4-pentynoic acid, B-Ala represents β-alanine, and Sar １０ represents 10 sarcosine units) 11. The heterotandem bicyclic peptide complex of claim 10, or a pharma- ceutically acceptable salt thereof, comprising:

12. [PYA]-[B-Ala]-[Sar １０ ]-(SEQ ID NO: 12) (BCY8938), in which PYA at the N-terminus of BCY8938 is linked to Dap at the C-terminus of BCY7732 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-SDK-(SEQ ID NO: 13) (BCY10043), in which PYA at the N-terminus of BCY10043 is linked to Dap at the C-terminus of BCY9172, -PEG １２ -linked via a linker; NH bound to Ac-(SEQ ID NO:3)-Dap (BCY9172) ２ -SDK- (SEQ ID NO: 13) - [Sar １０ ]-[K(PYA)](BCY10044), in which Lys(PYA) at the C-terminus of BCY10044 is linked to Dap at the C-terminus of BCY9172 by -PEG １２ -linked via a linker; NH bound to Ac-(SEQ ID NO:3)-Dap (BCY9172) ２ -SDK- (SEQ ID NO: 14) (BCY10045), in which Lys(PYA)9 of BCY10045 is linked to Dap at the C-terminus of BCY9172 by -PEG １２ -linked via a linker; Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:5)-A (BCY8919), in which Lys(PYA)9 of BCY10861 is linked to Lys3 of BCY8919 by -PEGyl １２ -linked via a linker; Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:6)-A (BCY8920), in which Lys(PYA)9 of BCY10861 is linked to dLys4 of BCY8920 by -PEG １２ -linked via a linker; Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:3)-Dap (BCY9172), in which Lys(PYA)9 of BCY10861 is linked to the C-terminal Dap of BCY9172 by -PEG １２ -linked via a linker; Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:5)-A (BCY8919), in which Lys(PYA)9 of BCY10861 is linked to Lys3 of BCY8919 by -CH ２ -linked via a linker; Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:6)-A (BCY8920), in which Lys(PYA)9 of BCY10861 is linked to dLys4 of BCY8920 by -CH ２ -linked via a linker; Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:3)-Dap (BCY9172), in which Lys(PYA)9 of BCY10861 is linked to the C-terminal Dap of BCY9172 by -CH ２ -linked via a linker; Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:5)-A (BCY8919), in which Lys(PYA)9 of BCY10861 is linked to Lys3 of BCY8919 by -PEGyl ５ -linked via a linker; Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:6)-A (BCY8920), in which Lys(PYA)9 of BCY10861 is linked to dLys4 of BCY8920 by -PEG ５ -linked via a linker; or Ac-SDK-(SEQ ID NO:14)-PSH (BCY10861) bound to Ac-(SEQ ID NO:3)-Dap (BCY9172), in which Lys(PYA)9 of BCY10861 is linked to the C-terminal Dap of BCY9172 by -PEG ５ -linked via a linker; The heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof according to claim 10 or 11, wherein the CD137 / PD-L1 complex is selected from the group consisting of:

13. the second peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand; The Nectin-4 binding bicyclic peptide ligand is C ｉ P[1Nal][dD]C ｉｉ M[HArg]DWSTP[HyP]WC ｉｉｉ (SEQ ID NO: 15; hereafter referred to as BCY8116); C ｉ P[1Nal][dD]C ｉｉ M[HArg]D[dW]STP[HyP][dW]C ｉｉｉ (SEQ ID NO: 16; hereafter referred to as BCY11415); and C ｉ P[1Nal][dK](Sar １０ -(B-Ala))C ｉｉ M[HArg]DWSTP[HyP]WC ｉｉｉ (SEQ ID NO: 17); C ｉ PFGC ｉｉ M[HArg]DWSTP[HyP]WC ｉｉｉ (SEQ ID NO: 18; hereafter referred to as BCY11414); (In the formula, C ｉ , C ｉｉ and C ｉｉｉ represent the first, second and third cysteine ​​residues, respectively; 1Nal represents 1-naphthylalanine; HArg represents homoarginine; HyP represents hydroxyproline; Sar １０ represents 10 sarcosine units, and B-Ala represents β-alanine). or a modified derivative thereof as defined in claim 1, or a pharma- ceutically acceptable salt thereof.

14. said Nectin-4 binding bicyclic peptide ligand optionally comprising an N-terminal modification; SEQ ID NO:15 (hereinafter referred to as BCY8116); [PYA]-[B-Ala]-[Sar １０ ] - (SEQ ID NO: 15) (hereinafter referred to as BCY8846); SEQ ID NO:16 (hereinafter referred to as BCY11415); [PYA]-[B-Ala]-[Sar １０ ] - (SEQ ID NO: 16) (hereinafter referred to as BCY11942); Ac-(SEQ ID NO:17) (hereinafter referred to as BCY8831); and SEQ ID NO:18 (hereinafter referred to as BCY11414); or modified derivatives thereof. (Wherein, PYA represents 4-pentynoic acid, B-Ala represents β-alanine, and Sar １０ represents 10 sarcosine units) 14. The heterotandem bicyclic peptide complex of claim 13, or a pharma- ceutically acceptable salt thereof.

15. The heterotandem bicyclic peptide complex of claim 13 or 14, wherein the Nectin-4 binding bicyclic peptide ligand comprises SEQ ID NO: 15 (hereinafter referred to as BCY8116).

16. [PYA]-[B-Ala]-[Sar １０ ]-(SEQ ID NO: 15) (BCY8846), in which PYA at the N-terminus of BCY8846 is linked to Dap at the C-terminus of BCY7732 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-(SEQ ID NO: 16) (BCY11942), in which PYA at the N-terminus of BCY11942 is linked to Dap at the C-terminus of BCY7732 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-(SEQ ID NO: 15) (BCY8846), in which PYA at the N-terminus of BCY8846 is linked to Dap at the C-terminus of BCY8045 by -PEG １２ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -PEG １０ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -PEG ２３ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -B-Ala-Sar ２０ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -B-Ala-Sar １０ -PEG １０ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -B-Ala-Sar ５ -PEG １５ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -B-Ala-Sar ５ -PEG ５ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -PEG １５ -Sar ５ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -PEG １０ -Sar １０ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -PEG ５ -Sar １５ linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -PEG ５ -Sar ５ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:1)-Dap(PYA) (BCY7741), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY7741 by -PEG ５ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:1)-Dap (BCY7732), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap of BCY7732 by -PEG ２４ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-(SEQ ID NO: 15) (BCY8846), in which PYA at the N-terminus of BCY8846 is linked to Dap at the C-terminus of BCY9172 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-(SEQ ID NO: 15) (BCY8846), in which PYA at the N-terminus of BCY8846 is linked to Lys3 of BCY8919 by -PEG １２ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-(SEQ ID NO: 15) (BCY8846), in which PYA at the N-terminus of BCY8846 is linked to dLys4 of BCY8920 by -PEG １２ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-(SEQ ID NO:7)-A (BCY8927), in which the N-terminus of BCY8116 is linked to Lys(PYA)3 of BCY8927 by -PEGylation. ５ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-(SEQ ID NO:8)-A (BCY8928), in which the N-terminus of BCY8116 is linked to dLys(PYA)4 of BCY8928 by -PEGylation. ５ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-(SEQ ID NO:3)-Dap(PYA) (BCY11014), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY11014 by -PEGylation. ５ -linked via a linker; [PYA]-[B-Ala]-[Sar １０ ]-(SEQ ID NO:15) (BCY8846), in which PYA at the N-terminus of BCY8846 is substituted with Dap at the C-terminus of BCY9172 by -CH ２ -linked via a linker; Ac-(SEQ ID NO:17) (BCY8831) bound to Ac-(SEQ ID NO:3)-Dap(PYA) (BCY11014), １０ )-(B-Ala))4 is attached to the C-terminal Dap(PYA) of BCY11014 by -PEG １２ -linked via a linker; Ac-(SEQ ID NO:17) (BCY8831) bound to Ac-(SEQ ID NO:3)-Dap(PYA) (BCY11014), １０ )-(B-Ala))4 is attached to the C-terminal Dap(PYA) of BCY11014 by -PEG ５ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-(SEQ ID NO:7)-A (BCY8927), in which the N-terminus of BCY8116 is linked to Lys(PYA)3 of BCY8927 by -CH ２ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-(SEQ ID NO:8)-A (BCY8928), in which the N-terminus of BCY8116 is linked to dLys(PYA)4 of BCY8928 by -CH ２ -linked via a linker; SEQ ID NO:15 (BCY8116) bound to Ac-(SEQ ID NO:3)-Dap(PYA) (BCY11014), in which the N-terminus of BCY8116 is linked to the C-terminus of Dap(PYA) of BCY11014 by -CH ２ -linked via a linker; SEQ ID NO:15 (BCY8116) linked to Ac-A-(SEQ ID NO:9)-A (BCY7744), in which the N-terminus of BCY8116 is linked to dLys(PYA)4 of BCY7744 by -PEG ５ -linked via a linker; Ac-[dA]-(SEQ ID NO: 10)-[dA]-NH ２（ SEQ ID NO: 15 (BCY8116) linked to Lys(PYA)4 of BCY11506, wherein the N-terminus of BCY8116 is linked to Lys(PYA)4 of BCY11506 by -PEGylation. ５ -linked via a linker; SEQ ID NO:18 (BCY11414) linked to Ac-A-(SEQ ID NO:9)-A (BCY7744), in which the N-terminus of BCY11414 is linked to dLys(PYA)4 of BCY7744 by -PEG ５ -linked via a linker; SEQ ID NO:18 (BCY11414) linked to Ac-(SEQ ID NO:8)-A (BCY8928), in which the N-terminus of BCY11414 is linked to dLys(PYA)4 of BCY8928 by -PEGylation. ５ -linked via a linker; or SEQ ID NO:16 (BCY11415) linked to Ac-(SEQ ID NO:8)-A (BCY8928), in which the N-terminus of BCY11415 is linked to dLys(PYA)4 of BCY8928 by -PEG ５ -linked via a linker; The heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof according to any one of claims 13 to 15, which is a CD137 / nectin-4 complex selected from the group consisting of:

17. The linker is -CH ２ -, -PEG ５ --PEG １０ -, -PEG １２ -, -PEG ２３ -, -PEG ２４ -, -PEG １５ -Sar ５ -, -PEG １０ -Sar １０ --PEG ５ -Sar １５ -, -PEG ５ -Sar ５ -, -B-Ala-Sar ２０ -, -B-Ala-Sar １０ -PEG １０ -, -B-Ala-Sar ５ -PEG １５ - and B-Ala-Sar ５ -PEG ５ The heterotandem bicyclic peptide complex of claim 1, wherein said heterotandem bicyclic peptide complex is selected from the group consisting of -

18. 18. The heterotandem bicyclic peptide complex of any one of claims 1 to 17, wherein the pharma- ceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salt.

19. 19. The heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 18, further comprising a cytotoxic agent, a radioactive chelator, and / or a chromophore.

20. 20. A pharmaceutical composition comprising a heterotandem bicyclic peptide complex according to any one of claims 1 to 19 in combination with one or more pharma- ceutically acceptable excipients.

21. 20. A heterotandem bicyclic peptide complex according to any one of claims 1 to 19 for use in the prevention, suppression or treatment of cancer.

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