Heterotandem bicyclic peptide complexes

Abstract

The present invention relates to a hetero-tandem bicyclic peptide complex comprising a first peptide ligand that binds to a component present on a cancer cell, the first peptide ligand being coupled via a linker to a second peptide ligand that binds to a component present on an immune cell. The invention also relates to application of the different tandem bicyclic peptide compound in prevention, inhibition or treatment of cancers.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202080069454.8, application date October 5, 2020, and invention name “Hetero-tandem bicyclic peptide complex”. Technical Field

[0002] The present invention relates to a heterotandem bicyclic peptide complex, which comprises a first peptide ligand that binds to a component present on cancer cells, wherein the first peptide ligand is coupled to a second peptide ligand that binds to a component present on immune cells via a linker. The present invention also relates to the use of the heterotandem bicyclic peptide complex in preventing, inhibiting or treating cancer. Background Art

[0003] Cyclic peptides can bind to protein targets with high affinity and target specificity, and are therefore an attractive class of molecules for therapeutic agent development. In fact, several cyclic peptides have been successfully used clinically, such as the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). The good binding properties are due to the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to surfaces of hundreds of square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 ( Wu et al. (2007), Science 330, 1066-71), has a binding affinity to integrin αVb3 The cyclic peptides with Arg-Gly-Asp motifs (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 ( Zhao et al. (2007), J Struct Biol 160(1), 1-10).

[0004] Due to its cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss after binding to the target and resulting in higher binding affinity. Compared with linear peptides, the reduced flexibility also leads to locking of target-specific conformations, increasing binding specificity. This effect has been exemplified by an effective and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses selectivity relative to other MMPs when the ring is opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties obtained by macrocyclization are more pronounced in polycyclic peptides with more than one peptide ring, such as in vancomycin, nisin and actinomycin.

[0005] Various research groups have previously tethered peptides with cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and coworkers have used tri(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide rings to synthetic scaffolds to structurally mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). WO 2004 / 077062 and WO 2006 / 078161 disclose methods for generating candidate drug compounds, wherein the compounds are generated by attaching cysteine-containing peptides to molecular scaffolds, such as tri(bromomethyl)benzene.

[0006] Combinatorial methods based on phage display have been developed to generate and screen large libraries of bicyclic peptides against target targets (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, a region containing three cysteine ​​residues and two six random amino acids (Cys-(Xaa) 6 -Cys-(Xaa) 6 -Cys) and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the invention

[0007] According to a first aspect of the present invention, there is provided an iso-tandem bicyclic peptide complex comprising:

[0008] (a) a first peptide ligand that binds to a component present on cancer cells; the first peptide ligand is coupled to

[0009] (b) a second peptide ligand that binds to a component present on an immune cell;

[0010] Each of the peptide ligands comprises a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three reactive groups separated by at least two ring sequences, and the molecular scaffold forms a covalent bond with the reactive groups of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold, characterized in that the hetero-tandem bicyclic peptide complex comprises the following first and second peptide ligands:

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017] Wherein 1Nal represents 1-naphthylalanine, HArg represents homoarginine, HyP represents hydroxyproline, B-Ala represents β-alanine, PYA represents 4-pentenoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cba represents β-cyclobutylalanine, hGlu represents homoglutamic acid, Nle represents norleucine, NMeAla represents N-methyl-alanine, tBuAla represents tert-butyl-alanine, Aad represents α-L-aminoadipic acid, Ac represents acetyl, Dap represents diaminopropionic acid, or a pharmaceutically acceptable salt thereof.

[0018] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising the hetero-tandem bicyclic peptide complex as defined herein, in combination with one or more pharmaceutically acceptable excipients.

[0019] According to a further aspect of the present invention, there is provided a hetero-tandem bicyclic peptide complex as defined herein for use in preventing, inhibiting or treating cancer. DETAILED DESCRIPTION

[0020] The present invention provides the following embodiments:

[0021] Embodiment 1. A hetero-tandem bicyclic peptide complex comprising:

[0022] (a) a first peptide ligand that binds to a component present on cancer cells; the first peptide ligand is coupled to

[0023] (b) a second peptide ligand that binds to a component present on an immune cell;

[0024] Each of the peptide ligands comprises a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three reactive groups separated by at least two ring sequences, and the molecular scaffold forms a covalent bond with the reactive groups of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold, characterized in that the hetero-tandem bicyclic peptide complex comprises the following first and second peptide ligands:

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] Wherein 1Nal represents 1-naphthylalanine, HArg represents homoarginine, HyP represents hydroxyproline, B-Ala represents β-alanine, PYA represents 4-pentenoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cba represents β-cyclobutylalanine, hGlu represents homoglutamic acid, Nle represents norleucine, NMeAla represents N-methyl-alanine, tBuAla represents tert-butyl-alanine, Aad represents α-L-aminoadipic acid, Ac represents acetyl, Dap represents diaminopropionic acid, or a pharmaceutically acceptable salt thereof.

[0032] Embodiment 2. A hetero-tandem bicyclic peptide complex as defined in embodiment 1, wherein the immune cell is selected from: leukocytes; lymphocytes (e.g., T lymphocytes or T cells, B cells or natural killer cells); CD8 or CD4; CD8; dendritic cells, follicular dendritic cells and granulocytes.

[0033] Embodiment 3. A hetero-tandem bicyclic peptide complex as defined in embodiment 1 or 2, wherein the second peptide ligand comprises a bicyclic peptide ligand that binds to CD137.

[0034] Embodiment 4. The hetero-tandem bicyclic peptide complex as defined in Embodiment 3, wherein the bicyclic peptide binding to CD137 is selected from any one of the peptides in SEQ ID NO: 67 to 84.

[0035] Embodiment 5. A hetero-tandem bicyclic peptide complex as defined in any one of embodiments 1 to 4, wherein the first peptide ligand comprises a bicyclic peptide ligand that binds to adhesion protein-4.

[0036] Embodiment 6. The hetero-tandem bicyclic peptide complex as defined in embodiment 5, wherein the bicyclic peptide that binds to adhesion protein-4 is selected from any one of the peptides in SEQ ID NO: 52 to 66.

[0037] Embodiment 7. The hetero-tandem bicyclic peptide complex as defined in Embodiment 5 or 6, which is selected from any one of the complexes listed in Table C, such as BCY11468, BCY11618, BCY11776, BCY11860, BCY12020, BCY12661 and BCY12969.

[0038] Embodiment 8. A heterotandem bicyclic peptide complex as defined in any one of embodiments 1 to 4, wherein the first peptide ligand comprises a bicyclic peptide ligand that binds to EphA2.

[0039] Embodiment 9. The hetero-tandem bicyclic peptide complex as defined in embodiment 8, wherein the bicyclic peptide binding to EphA2 is selected from any one of the peptides in SEQ ID NOs: 10 to 51.

[0040] Embodiment 10. The hetero-tandem bicyclic peptide complex as defined in Embodiment 8 or 9, which is selected from any one of the complexes listed in Table B, such as BCY13035, BCY13040, BCY13253, BCY13254, BCY13340 and BCY13342.

[0041] Embodiment 11. A heterotandem bicyclic peptide complex as defined in any one of Embodiments 1 to 4, wherein the first peptide ligand comprises a bicyclic peptide ligand that binds to PD-L1.

[0042] Embodiment 12. The hetero-tandem bicyclic peptide complex as defined in Embodiment 11, wherein the bicyclic peptide binding to PD-L1 is selected from any one of the peptides in SEQ ID NO: 1 to 9.

[0043] Embodiment 13. The hetero-tandem bicyclic peptide complex as defined in Embodiment 11 or 12, which is selected from any one of the complexes listed in Table A, such as BCY12375 and BCY12021.

[0044] Embodiment 14. An iso-tandem bicyclic peptide complex as defined in any one of embodiments 1 to 13, wherein the molecular scaffold is selected from 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

[0045] Embodiment 15. The hetero-tandem bicyclic peptide complex as defined in any one of embodiments 1 to 14, wherein the pharmaceutically acceptable salt is selected from free acid or sodium, potassium, calcium, ammonium salts.

[0046] Embodiment 16. A pharmaceutical composition comprising the hetero-tandem bicyclic peptide complex of any one of embodiments 1 to 15, in combination with one or more pharmaceutically acceptable excipients.

[0047] Embodiment 17. The hetero-tandem bicyclic peptide complex as defined in any one of embodiments 1 to 15, for use in preventing, inhibiting or treating cancer.

[0048] First peptide ligand

[0049] The term "cancer cell" mentioned herein includes any cell known to be involved in cancer. When the gene responsible for regulating cell division is damaged, cancer cells are produced. Carcinogenesis is caused by mutations and epimutations of normal cell genetic material, which disrupt the normal balance between proliferation and cell death. This leads to uncontrolled cell division and the evolution of these cells by natural selection in vivo. Cell proliferation is uncontrolled and usually rapid, which can lead to benign or malignant tumors (cancer). Benign tumors do not spread to other parts of the body or invade other tissues. Malignant tumors can invade other organs, spread to distant places (metastasis) and become life-threatening.

[0050] In one embodiment, the cancer cells are selected from HT1080, A549, SC-OV-3, PC3, H1376, NCI-H292, LnCap, MC38, 4T1-D02 and RKO tumor cells.

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

[0052] Adhesin-4 is a surface molecule that belongs to the adhesin family of proteins, which consists of 4 members. Adhesins are cell adhesion molecules that play key roles in various biological processes such as polarity, proliferation, differentiation and migration in epithelial, endothelial, immune and neuronal cells during development and adulthood. They are involved in a variety of pathological processes in humans. They are the main receptors for poliovirus, herpes simplex virus and measles virus. Mutations in the genes encoding adhesin-1 (PVRL1) or adhesin-4 (PVRL4) lead to ectodermal dysplasia syndrome associated with other abnormalities. Adhesin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other family members. Adhesin-4 is a tumor-associated antigen present in 50%, 49% and 86% of breast, ovarian and lung cancers, respectively, mainly in tumors with poor prognosis. Its expression is not detected in the corresponding normal tissues. In breast tumors, adhesin-4 is mainly expressed in triple-negative and ERBB2+ carcinomas. In the serum of patients with these cancers, soluble forms of nectin-4 were detected and were associated with a poor prognosis. Serum nectin-4 levels increased during metastatic progression and decreased after treatment. These results suggest that nectin-4 may be a reliable target for the treatment of cancer. Therefore, several anti-nectin-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting nectin-4 and is currently being studied clinically for the treatment of patients with solid tumors.

[0053] In one embodiment, the first peptide ligand comprises a bicyclic peptide ligand that binds to fibronectin-4.

[0054] Suitable examples of bicyclic peptide ligands that bind to adhesion protein-4 are disclosed in PCT patent application number PCT / GB2019 / 051740, the peptides of which are incorporated herein by reference.

[0055] In one embodiment, the bicyclic peptide that binds to fibronectin-4 is selected from any one of the peptides of SEQ ID NOs: 52 to 66 described herein.

[0056] In an alternative embodiment, the moiety present on cancer cells is EphA2.

[0057] Eph receptor tyrosine kinases (Eph) belong to a large class of receptor tyrosine kinases (RTKs), i.e., kinases that phosphorylate tyrosine residues of proteins. Eph and its membrane-bound ephrin ligands (ephrins) control cell localization and tissue architecture (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur in a state of higher ligand oligomerization (Stein et al. (1998) Genes Dev 12, 667-678).

[0058] Among other patterning functions, various Ephs and ephrins have been shown to play a role in vascular development. Knockout of EphB4 and ephrin-B2 results in a lack of ability to remodel capillary beds into blood vessels (Poliakov et al., supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrins has also been observed in newly formed adult microvessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

[0059] It has also been observed that the deregulation of some ephrins and their receptors in adults contributes to tumor invasion, metastasis and neoangiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., supra). In addition, some Eph family members have been found to be overexpressed on tumor cells from various human tumors (Brantley-Sieders et al., supra; Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

[0060] EPH receptor A2 (ephrin receptor type A 2) is a protein which in humans is encoded by the EPHA2 gene.

[0061] EphA2 is upregulated in a variety of human cancers and is often associated with disease progression, metastasis and poor prognosis, such as 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) PLoS One 6, e24426), lung cancer (Brannan et al. (2009) Cancer Prev Res (Phila) 2, 1039-1049; Kinch et al. (2003) Clin Cancer Res. 9, 613-618; Guo 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 Metastasis 23, 357-365), prostate cancer (Walker-Daniels et al. (1999) Prostate 41, 275-280), liver cancer (Yang 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).

[0062] Although there is evidence of interactions at multiple stages of cancer progression, including tumor cell growth, survival, invasion, and angiogenesis, the full role of EphA2 in cancer progression remains undetermined. Downregulation of EphA2 expression inhibits tumor cancer cell proliferation (Binda et al. (2012) Cancer Cell 22, 765-780), while blocking EphA2 inhibits VEGF-induced cell migration (Hess et al. (2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng et al. (2002) Mol Cancer Res. 1, 2-11; Lin et al. (2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al. (2005) FASEB J. 19, 1884-1886).

[0063] Antibody drugs conjugated to EphA2 have been shown to significantly reduce tumor growth in rat and mouse xenograft models (Jackson et al. (2008) Cancer Research 68, 9367-9374), and a similar approach has been attempted in humans, although treatment had to be stopped due to treatment-related adverse events (Annunziata et al. (2013) Invest New Drugs 31, 77-84).

[0064] In one embodiment, the first peptide ligand comprises a bicyclic peptide ligand that binds EphA2.

[0065] Suitable examples of bicyclic peptide ligands that bind to EphA2 are disclosed in WO 2019 / 122860, WO 2019 / 122861 and WO 2019 / 122863, the peptides of which are incorporated herein by reference.

[0066] In one embodiment, the bicyclic peptide that binds EphA2 is selected from any one of the peptides of SEQ ID NOs: 10 to 51 described herein.

[0067] In an alternative embodiment, the component present on cancer cells is PD-L1.

[0068] Programmed cell death 1 ligand 1 (PD-L1) is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. The expression of PD-L1 is involved in the escape of immune responses involved in chronic infections, such as chronic viral infections (especially including, for example, HIV, HBV, HCV, and HTLV, etc.), chronic bacterial infections (especially including, for example, Helicobacter pylori), and chronic parasitic infections (including, for example, Schistosoma mansoni). The expression of PD-L1 has been detected in many tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells (including endothelial cells, hepatocytes, muscle cells, and placenta).

[0069] Expression of PD-L1 is also involved in the suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immune clearance of the tumor is rare. This failure is due in part to the suppression of immunity by the tumor microenvironment. PD-L1 expression on many tumors is a component of this suppressive environment and works in concert with other immunosuppressive signals. Expression of PD-L1 has been shown in situ on a variety of solid tumors, including breast, lung, colon, ovary, melanoma, bladder, liver, salivary gland, stomach, glial, thyroid, thymic epithelial, and head and neck tumors (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 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol.Immunother.56:1173-82; Nomi T et al. 2007 Clin.Cancer Res.13:2151-57; Thompson RH et al. 2004 Proc.Natl.Acad.Sci.USA101:17174-79; Wu C et al. 2006 Acta Histochem.108:19-24). In addition, the expression of the receptor for PD-L1, programmed cell death protein 1 (also known as PD-1 and CD279), is upregulated in tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C et al. 2003 Immunol.171:4574-81).Most importantly, studies on PD-L1 expression on tumors and disease outcome have shown that PD-L1 expression is closely associated with poor prognosis in renal cancer, ovarian cancer, bladder cancer, breast cancer, gastric cancer and pancreatic cancer (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. (2007), Clin. Cancer Res. 13:2151-57; Thompson RH et al. (2004), Proc. Natl. Acad. Sci. USA 101:17174-79; Wu et al. 2005). C et al. (2006), Acta Histochem. 108: 19-24). In addition, these studies showed that higher levels of PD-L1 expression on tumors may promote tumor stage progression and invasion into deeper tissue structures.

[0070] The PD-1 pathway can also play a role in hematological malignancies. PD-L1 is expressed in multiple myeloma cells, but not in normal plasma cells (Liu J et al. 2007 Blood 110: 296-304). PD-L1 is expressed in some primary T-cell lymphomas, especially anaplastic large cell T lymphomas (Brown Ja et al., 2003 Immunol. 170: 1257-66). PD-1 is highly expressed in T cells of angioimmunoblastic lymphoma, while PD-L1 is expressed in the associated follicular dendritic cell network (Dorfman DM et al. 2006 Am. J. Surg. Pathol. 30: 802-10). In Hodgkin's lymphoma, which is mainly composed of nodular lymphocytes, T cells associated with lymphocytes or tissue cells (L&H) express PD-1. Microarray analysis using readouts of genes induced by PD-1 ligation showed that in Hodgkin lymphoma, tumor-associated T cells responded in situ to PD-1 signals (Chemnitz JM et al. (2007), Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T cell leukemia and lymphoma (Shimauchi T et al. 2007 Int. J. Cancer 121:2585-90). These tumor cells are hyporesponsive to TCR signals.

[0071] Animal model studies have shown that PD-L1 on tumors inhibits T cell activation and tumor cell lysis, and in some cases leads to an increase in tumor-specific T cell death (Dong H et al. (2002), Nat. Med. 8: 793-800; Hirano F et al. (2005), Cancer Res. 65: 1089-96). Tumor-associated APCs can also use the PD-1: PD-L1 pathway to control anti-tumor T cell responses. Tumor environmental factors upregulate PD-L1 expression on tumor-associated myeloid DC populations (Curiel TJ et al. (2003), Nat. Med. 9: 562-67). Plasmacytoid dendritic cells (DCs) in tumor-draining lymph nodes of B16 melanoma express IDO, which strongly activates the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells requires cell contact with IDO-expressing DCs (Sharma MD et al. 2007 Clin. Invest. 117: 2570-82).

[0072] In one embodiment, the first peptide ligand comprises a bicyclic peptide ligand that binds PD-L1.

[0073] Suitable examples of bicyclic peptide ligands that bind to PD-L1 are disclosed in UK Patent Application Nos. 1905631.6 and 1904622.6, the peptides of which are incorporated herein by reference.

[0074] In one embodiment, the bicyclic peptide that binds to PD-L1 is selected from any one of the peptides of SEQ ID NOs: 1 to 9 described herein.

[0075] In alternative embodiments, the component present on the cancer cells is prostate specific membrane antigen (PSMA).

[0076] Prostate-specific membrane antigen (PSMA) (also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) and NAAG peptidase) is an enzyme encoded by the FOLH1 (folate hydrolase 1) gene in humans. Human GCPII contains 750 amino acids and weighs approximately 84 kDa.

[0077] Human PSMA is highly expressed in the prostate, approximately one hundred times more than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, with an 8- to 12-fold increase over levels in noncancerous prostate cells. Due to this high expression, PSMA is being developed as a potential biomarker for some cancer treatments and imaging. In human prostate cancer, tumors with higher expression are associated with faster time to progression and a higher proportion of patients with recurrence.

[0078] In one embodiment, the first peptide ligand comprises a bicyclic peptide ligand that binds PSMA.

[0079] Suitable examples of bicyclic peptide ligands that bind PSMA are disclosed in UK Patent Application Nos. 1820325.7 and 1912723.2 and PCT Patent Application No. PCT / EP2019 / 066273, the peptides of which are incorporated herein by reference.

[0080] Second peptide ligand

[0081] The term "immune cell" as used herein includes any cell within the immune system. Suitable examples include leukocytes, such as lymphocytes (e.g., T lymphocytes or T cells, B cells, or natural killer cells). In one embodiment, the T cell is CD8 or CD4. In a further embodiment, the T cell is CD8. Other examples of immune cells include dendritic cells, follicular dendritic cells, and granulocytes.

[0082] In one embodiment, the component present on the immune cell is CD137.

[0083] CD137 is a member of the tumor necrosis factor (TNF) receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-IBB, and induced by lymphocyte activation (ILA). CD137 can be expressed by activated T cells, but the expression is greater on CD8+ than on CD4+ T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. One of the characteristic activities of CD137 is its co-stimulatory activity on activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. In addition, it can enhance immune activity to eliminate tumors in mice.

[0084] CD137 is a T cell co-stimulatory receptor induced by TCR activation (Nam et al., Curr. Cancer Drug Targets, 5: 357-363 (2005); Waits et al., Annu. Rev, Immunol., 23: 23-68 (2005)). In addition to being expressed on activated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+ regulatory T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils and dendritic cells. Its natural ligand CD137L has been described on antigen presenting cells (including B cells, monocytes / macrophages and dendritic cells) (Watts et al. Annu. Rev. Immunol, 23: 23-68 (2005)). Upon interaction with its ligand, CD137 leads to increased TCR-induced T cell proliferation, cytokine production, functional maturation and prolonged survival of CD8+ T cells (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005), Watts et al., Annu. Rev. Immunol, 23:23-68 (2005)).

[0085] Signaling through CD137 via CD137L or agonistic monoclonal antibodies (mAbs) against CD137 results in increased TCR-induced T cell proliferation, cytokine production, functional maturation, and prolonged survival of CD8+ T cells. These effects are due to: (1) activation of NF-κB, c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK / SAPK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways, and (2) control of the expression of anti-apoptotic and cell cycle-related genes.

[0086] Experiments performed in CD137- and CD137L-deficient mice additionally demonstrated the importance of CD137 costimulation in generating fully competent T-cell responses.

[0087] IL-2 and IL-15 activated NK cells express CD137, and ligation of agonistic mAb to CD137 stimulates NK cell proliferation and IFN-γ secretion, but not its cytolytic activity.

[0088] Furthermore, CD137-stimulated NK cells promoted the expansion of activated T cells in vitro.

[0089] Based on its co-stimulatory function, agonist mAbs directed against CD137 have been shown to promote rejection of cardiac and skin allografts, eradicate established tumors, amplify primary antiviral CD8+ T-cell responses, and increase the cytolytic potential of T cells. These studies support the notion that CD137 signaling promotes T-cell function, which may enhance immunity to tumors and infections.

[0090] In one embodiment, the second peptide ligand comprises a bicyclic peptide ligand that binds CD137.

[0091] Suitable examples of bicyclic peptide ligands that bind CD137 are disclosed in WO 2019 / 025811, the peptides of which are incorporated herein by reference.

[0092] In one embodiment, the bicyclic peptide that binds CD137 is selected from any one of the peptides of SEQ ID NOs: 67 to 84 described herein.

[0093] Connectors

[0094] It will be understood that the first peptide ligand can be coupled to the second peptide ligand by any suitable linker. Generally, the design of the linker will make two bicyclic peptides present in such a way that they can be unhindered to bind to their respective targets individually, or to bind to two target receptors simultaneously. In addition, the linker should allow simultaneous binding of two targets while maintaining the appropriate distance between the target cells, which will lead to the desired functional results. The characteristics of the linker can be adjusted to increase the length, rigidity or solubility to optimize the desired functional results. The linker can also be designed to allow more than one bicyclic to be connected to the same target. Increasing the valence of any binding peptide can be used to increase the affinity of heterotandems to target cells, or can contribute to the oligomerization of one or both of the inducing target receptors.

[0095] In one embodiment, the linker is selected from the following sequences: -PEG5- and TCA-[PEG 10 ] 3 .

[0096] The structural representations of these joints are detailed below:

[0097]

[0098] Heterotandem complex

[0099] In a specific embodiment, the first peptide ligand comprises a bicyclic peptide ligand that binds PD-L1 attached to a TATA scaffold, the second peptide ligand comprises a bicyclic peptide ligand that binds CD137 attached to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table A:

[0100] Table A (PD-L1:CD137; 1:1)

[0101] Compound No. PD-L1 BCY number Connect the dots Connectors CD137 BCY Number Connect the dots BCY12229 BCY11865 Lys9 Peg5 BCY8928 dLys(PYA)4 BCY12230 BCY11866 Lys2 Peg5 BCY8928 dLys(PYA)4 BCY12231 BCY11867 Lys7 Peg5 BCY8928 dLys(PYA)4 BCY12232 BCY11868 Lys8 Peg5 BCY8928 dLys(PYA)4 BCY12242 BCY11869 Lys11 Peg5 BCY8928 dLys(PYA)4 BCY12375 BCY10861 Lys(PYA)9 Peg5 BCY12023 dLys4 BCY12663 BCY12479 C-terminal Lys Peg5 BCY8928 dLys(PYA)4 BCY12796 BCY12477 C-terminal Lys Peg5 BCY8928 dLys(PYA)4 BCY12021 BCY10861 Lys(PYA)9 Peg5 BCY11144 dLys4

[0102] In one embodiment, the heterotandem bicyclic peptide complex is selected from: BCY12375 and BCY12021.

[0103] In a specific embodiment, the first peptide ligand comprises a bicyclic peptide ligand that binds EphA2 attached to a TATA scaffold, the second peptide ligand comprises a bicyclic peptide ligand that binds CD137 attached to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table B:

[0104] Table B (EphA2:CD137; 1:1)

[0105]

[0106]

[0107]

[0108] In one embodiment, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY13035, BCY13040, BCY13253, BCY13254, BCY13340 and BCY13342.

[0109] In a specific embodiment, the first peptide ligand comprises a bicyclic peptide ligand that binds to fibronectin-4 attached to a TATA scaffold, the second peptide ligand comprises a bicyclic peptide ligand that binds to CD137 attached to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table C:

[0110] Table C (fibronectin-4:CD137; 1:1)

[0111]

[0112]

[0113] In one embodiment, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY11468, BCY11618, BCY11776, BCY11860, BCY12020, BCY12661 and BCY12969.

[0114] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999), 4th edition, John Wiley & Sons, Inc.), which are incorporated herein by reference.

[0115] the term

[0116] Molecular form

[0117] The N- or C-terminal extension of the bicyclic core sequence is added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail would be represented as:

[0118] βAla-Sar10-A-(SEQ ID NO:X).

[0119] Reverse peptide sequence

[0120] According to the disclosure in Nair et al. (2003) J Immunol 170 (3), 1362-1373, it is envisioned that the peptide sequences disclosed herein will also be used in their reverse-inverse form. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and vice versa), and its stereochemistry is also reversed (i.e., the D-amino acid becomes the L-amino acid, and vice versa). For the avoidance of doubt, unless otherwise indicated, the reference to an amino acid by its full name or by its amino acid single letter or three letter code is intended to represent an L-amino acid. If such an amino acid is intended to represent a D-amino acid, the amino acid will begin with a lowercase d in square brackets, such as [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

[0121] Peptide ligand

[0122] As referred to herein, a peptide ligand refers to a peptide covalently bound to a molecular scaffold. Typically, such a peptide comprises two or more reactive groups (i.e., cysteine ​​residues) capable of forming a covalent bond with the scaffold, and a sequence present opposite to the reactive groups, the sequence being referred to as a loop sequence because a loop is formed when the peptide is bound to the scaffold. In the present case, the peptide comprises at least three reactive groups selected from cysteine, 3-mercaptopropionic acid and / or cysteamine, and forms at least two loops on the scaffold.

[0123] Reactive Group

[0124] The molecular scaffold of the present invention can be combined with the polypeptide through the functional group or reactive group on the polypeptide. It is usually formed by the side chain of the specific amino acid present in the polypeptide polymer. Such reactive groups can be cysteine ​​side chains, lysine side chains or N-terminal amine groups or any other suitable reactive groups, such as penicillamine. Specific information on suitable reactive groups can be found in WO 2009 / 098450.

[0125] Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartic acid or glutamic acid, the guanidinium group of arginine, the phenolic group of tyrosine, or the hydroxyl group of serine. Unnatural amino acids can provide a wide range of reactive groups, including azide, ketocarbonyl, alkyne, vinyl, or aryl halide groups. Amino and carboxyl groups at the ends of polypeptides can also be used as reactive groups to form covalent bonds with the molecular scaffold / molecular core.

[0126] The polypeptide of the present invention comprises at least three reactive groups. The polypeptide may also comprise four or more reactive groups. The more reactive groups used, the more rings can be formed in the molecular scaffold.

[0127] In a preferred embodiment, a polypeptide with three reactive groups is generated. The reaction of the polypeptide with a molecular scaffold / molecular core having a three-fold rotational symmetry generates a single product isomer. The generation of a single product isomer is advantageous for a variety of reasons. The nucleic acid of the compound library encodes only the primary sequence of the polypeptide, and does not encode the isomeric state of the molecule formed after the polypeptide reacts with the molecular core. If only one product isomer can be formed, the nucleic acid arrangement of the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acid cannot provide information about the properties of the product isomer isolated during the screening or selection process. If a specific member of the library of the present invention is synthesized, the formation of a single product isomer is also advantageous. In this case, the chemical reaction of the polypeptide with the molecular scaffold produces a single product isomer rather than a mixture of isomers.

[0128] In another embodiment, a polypeptide having four reactive groups is generated. The reaction of the polypeptide with a molecular scaffold / molecular core having tetrahedral symmetry generates two product isomers. Although the two different product isomers are encoded by the same nucleic acid, the isomeric properties of the isolated isomers can also be determined by chemically synthesizing the two isomers, separating the two isomers, and testing the binding of the two isomers to the target ligand.

[0129] In one embodiment of the invention, at least one of the reactive groups of the polypeptide is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the orthogonal reactive groups to be directed to a specific site in the molecular core. Connection strategies involving orthogonal reactive groups can be used to limit the number of product isomers formed. In other words, by selecting reactive groups for one or more of the at least three bonds, the reactive groups are unique or different relative to those selected for the remainder of the at least three bonds, and a specific order can be effectively achieved, with which the specific reactive groups of the polypeptide are bonded or directed to a specific position on the molecular scaffold.

[0130] In another embodiment, the reactive group of the polypeptide of the invention is reacted with a molecular linker, wherein the linker is capable of reacting with a molecular scaffold, such that the linker will be inserted between the molecular scaffold and the polypeptide in a final bonded state.

[0131] In some embodiments, the amino acid of the member of polypeptide library or group can be replaced by any natural or non-natural amino acid.Excluded from these exchangeable amino acids are those amino acids with functional groups for making polypeptide cross-linked to the molecular core, so that only the loop sequence is exchangeable.Described exchangeable polypeptide sequence has random sequence, constant sequence or has random and constant amino acid sequence.Amino acid with reactive group is located at the determined position in polypeptide, because these amino acid whose positions determine the size of ring.

[0132] In one embodiment, the polypeptide having three reactive groups has the sequence (X) l Y(X) m Y(X) n Y(X) o , wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 3 and 6, which define the length of the middle polypeptide segment, which may be the same or different, and l and o are numbers between 0 and 20, which define the length of the flanking polypeptide segments.

[0133] The method of replacing thiol-mediated coupling can be used to connect the molecular scaffold to the peptide by covalent interaction. Alternatively, these techniques can be used to modify or connect further parts (such as small molecules of interest different from the molecular scaffold) to the polypeptide after selection or separation according to the present invention-in the present embodiment, then it is obvious that the connection does not have to be covalent and can include non-covalent connection. These methods can replace the thiol-mediated method (or combine with it), by producing phages that display proteins and peptides with non-natural amino acids with necessary chemical reactive groups, and use them in combination with small molecules with complementary reactive groups, or when preparing molecules after the selection / separation stage, by merging the non-natural amino acids into chemical or recombinantly synthesized polypeptides. Further specific information can be found in WO 2009 / 098450 or Heinis et al., Nat Chem Biol 2009, 5 (7), 502-7.

[0134] In one embodiment, the reactive groups are selected from cysteine, 3-mercaptopropionic acid and / or cysteamine residues.

[0135] Pharmaceutically acceptable salts

[0136] It will be appreciated that salt forms are within the scope of the present invention and reference to a peptide ligand includes salt forms of the ligand.

[0137] The salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a suitable base or acid in water or in an organic solvent, or in a mixture of the two.

[0138] Acid addition salts (mono or di) can be formed with a wide variety of inorganic and organic acids. Examples of acid addition salts include mono or di salts formed with an acid selected from acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphor, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid , hippuric acid, hydrohalic acid (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), hydroxyethanesulfonic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic 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, as well as acylated amino acids and cation exchange resins.

[0139] A particular group of salts consists of salts formed from 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 (mesylate), ethanesulfonic acid, naphthylsulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. A particular salt is the hydrochloride. Another particular salt is the acetate.

[0140] If the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be -COO - ), then it can form a salt with an organic or inorganic base to generate a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li + 、Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH 4 + ) and substituted ammonium ions (e.g. NH 3 R+ NH 2 R 2 + 、NHR 3 + and NR 4 + Some examples of suitable substituted ammonium ions are those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .

[0141] When the compound of the present invention contains an amine functional group, it can be reacted with an alkylating agent to form a quaternary ammonium salt, for example, according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the present invention.

[0142] Modified derivatives

[0143] It will be appreciated that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from the following: N-terminal and / or C-terminal modification; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; replacement of one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues; replacement of one or more amino acid residues with alanine, and replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacement of one or more peptide bonds with alternative bonds; modification of the length of the peptide backbone; replacement of hydrogen on the α-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate / aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids, and introduction or substitution of amino acids suitable for functionalization with orthogonal reactivity, such as amino acids with azide or alkynyl groups, which allow functionalization with moieties with alkynyl or azide groups, respectively.

[0144] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry and / or a C-terminal modification using suitable carboxyl-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator, or a chromophore.

[0145] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, during peptide synthesis, the N-terminal cysteine ​​group (referred to herein as C i The group) is blocked by acetic anhydride or other suitable reagents, resulting in the molecule being acetylated at the N-terminus. This embodiment provides the advantage of removing potential recognition points of aminopeptidases and avoids the possibility of degradation of the bicyclic peptide.

[0146] In an alternative embodiment, the N-terminal modification involves the addition of a molecular spacer group that facilitates coupling of the effector group and maintains the potency of the bicyclic peptide to its target.

[0147] In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, during peptide synthesis, the C-terminal cysteine ​​group (referred to herein as C iii The group) is synthesized as an amide, resulting in a C-terminal amidation of the molecule. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidases and reduces the likelihood of proteolytic degradation of the bicyclic peptide.

[0148] In one embodiment, the modified derivative comprises replacing one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, the non-natural amino acid with an isosteric / isoelectronic side chain can be selected, which is neither recognized by the degradation protease nor has any adverse effect on the target efficacy.

[0149] Alternatively, unnatural amino acids with constrained amino acid side chains can be used so that proteolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, this involves proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid (Aib)), and cyclic amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

[0150] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine ​​(C i ) and / or C-terminal cysteine ​​(Ciii ) to add a spacer group.

[0151] In one embodiment, the modified derivative comprises replacing one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues. In a further embodiment, the modified derivative comprises replacing a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the drug stability characteristics of the resulting bicyclic peptide ligand.

[0152] In one embodiment, the modified derivative comprises replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an optional embodiment, the modified derivative comprises replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged and hydrophobic amino acid residues is an important feature of the bicyclic peptide ligand. For example, the hydrophobic amino acid residue affects the degree of plasma protein binding, thereby affecting the concentration of the free available part in the blood plasma, and the charged amino acid residue (particularly arginine) can affect the interaction of the peptide with the cell surface phospholipid membrane. The two are combined to affect the half-life, distribution volume and exposure of the peptide drug, and can be adjusted according to the clinical endpoint. In addition, the correct combination and quantity of charged and hydrophobic amino acid residues can reduce the stimulation at the injection site (if the peptide drug has been subcutaneously administered).

[0153] In one embodiment, the modified derivative comprises replacing one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by the tendency of D-amino acids to stabilize the β-turn conformation (Tugyi et al. (2005) PNAS, 102 (2), 413-418).

[0154] In one embodiment, the modified derivative comprises removing any amino acid residue and replacing it with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

[0155] It should be noted that each of the above modifications is used to intentionally improve the potency or stability of the peptide. Through modification, the potency can be further improved by the following mechanisms:

[0156] - Incorporation of hydrophobic moieties that exploit hydrophobic interactions and result in lower off-rates, allowing higher affinity to be achieved;

[0157] - incorporation of charged groups that exploit long-range ionic interactions, resulting in faster on-rates and higher affinities (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0158] - Incorporating additional constraints into the peptide, for example by properly constraining the amino acid side chains to minimize entropy loss upon target binding, by restricting the torsion angles of the backbone to minimize entropy loss upon target binding, and introducing additional cyclization into the molecule for the same reason.

[0159] (For review, see Gentilucci et al., Curr. Pharmaceutical Design (2010), 16, 3185-203 and Nestor et al. (2009), Curr. Medicinal Chem 16, 4399-418).

[0160] Isotopic variants

[0161] The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the present invention, in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number usually occurring in nature, and peptide ligands of the present invention, in which a metal chelating group (referred to as "effector") is attached, which is capable of holding the relevant (radio)isotope, and peptide ligands of the present invention, in which certain functional groups are covalently substituted by relevant (radio)isotopes or isotopically labeled functional groups.

[0162] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include hydrogen isotopes such as 2 H(D) and 3 H(T), carbon isotopes such as 11 C. 13 C and 14 C, chlorine isotopes such as 36 Cl, fluorine isotopes such as 18 F, iodine isotopes such as 123 I. 125 I and 131 I, nitrogen isotopes such as 13 N and 15 N, oxygen isotopes such as 15 O. 17 O and 18 O, phosphorus isotopes such as 32 P, sulfur isotopes such as 35 S, copper isotopes such as 64Cu, gallium isotopes such as 67 Ga or 68 Ga, yttrium isotopes such as 90 Y, and lutetium isotopes such as 177 Lu, and bismuth isotopes such as 213 Bi.

[0163] Certain isotope-labeled peptide ligands of the present invention, such as those incorporating radioactive isotopes, can be used for tissue distribution studies of drugs and / or substrates, and for clinically evaluating the presence and / or absence of the adhesion protein-4 target on diseased tissues. The peptide ligands of the present invention can further have valuable diagnostic properties, which can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods can use compounds labeled with labeling agents, such as radioactive isotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, fluorescein, aequorin and luciferase), etc. The radioactive isotope tritium, i.e. 3 H(T) and carbon-14 14 C, is particularly useful for this purpose due to its ease of incorporation and readily available detection methods.

[0164] Using heavier isotopes such as deuterium 2 H(D) substitution may offer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0165] Positron emitting isotopes such as 11 C. 18 F. 15 O and 13 N substitution can be used in positron emission tomography (PET) studies to examine target occupancy.

[0166] Isotopically-labeled compounds of the peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0167] Molecular scaffold

[0168] Molecular scaffolds are described, for example, in WO 2009 / 098450 and in the references cited therein, in particular WO 2004 / 077062 and WO 2006 / 078161.

[0169] As mentioned in the above document, the molecular scaffold may be a small molecule, such as an organic small molecule.

[0170] In one embodiment, the molecular scaffold may be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

[0171] In one embodiment, the molecular scaffold comprises reactive groups capable of reacting with functional groups of the polypeptide to form covalent bonds.

[0172] The molecular scaffold may comprise chemical groups that form linkages with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, olefins, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0173] In one embodiment, the molecular scaffold may comprise or consist of hexahydro-1,3,5-triazine, in particular 1,3,5-triacryloylhexahydro-1,3,5-triazine ("TATA") or a derivative thereof.

[0174] The molecular scaffold of the present invention comprises chemical groups that allow functional groups of polypeptides of the coding library of the present invention to form covalent bonds with the molecular scaffold. The chemical groups are selected from a wide range of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, olefins, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.

[0175] Scaffold reactive groups that can be used on the molecular scaffold to react with the thiol group of cysteine ​​are alkyl halides (or also known as halogenoalkane or haloalkane).

[0176] Examples include bromomethylbenzene or iodoacetamide. Other scaffold reactive groups used to selectively couple compounds to cysteine ​​in proteins are maleimide, compounds containing αβ unsaturated carbonyl groups, and compounds containing α-halomethyl carbonyl groups. Examples of maleimides that can be used as molecular scaffolds of the present invention include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of a compound containing an αβ unsaturated carbonyl group is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). An example of a compound containing an α-halomethyl carbonyl group is N,N',N"-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine ​​is also a natural amino acid that has similar reactivity to cysteine ​​and can be used in the same reactions. Therefore, wherever cysteine ​​is mentioned, substitution of selenocysteine ​​is generally acceptable unless the context indicates otherwise.

[0177] synthesis

[0178] The peptides of the present invention can be synthesized by standard techniques and then reacted in vitro with the molecular scaffold. When doing this, standard chemical methods can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or verification. Such methods can be accomplished using conventional chemical methods as disclosed in Timmerman et al. (supra).

[0179] Therefore, the present invention also relates to the manufacture of a polypeptide or conjugate selected as described herein, wherein said manufacture comprises optional further steps as described below. In one embodiment, these steps are performed on a final product polypeptide / conjugate prepared by chemical synthesis.

[0180] When making a conjugate or complex, amino acid residues in the target polypeptide may be optionally substituted.

[0181] The peptide may also be extended to incorporate, for example, another loop and thereby introduce multiple specificities.

[0182] To extend the peptide, conventional solid phase or solution phase chemistry can be used, using orthogonally protected lysine (and analogs) to simply chemically extend at its N-terminus or C-terminus or within a ring. Standard (bio) coupling techniques can be used to introduce activated or activatable N- or C-termini. Alternatively, addition can be made by fragment condensation or native chemical connection, such as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266: 776-779), or by enzyme addition, such as using subtiligase, such as (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20; 91 (26): 12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, 6000-6003 pages).

[0183] Alternatively, the peptide may be extended or modified by further coupling of disulfide bonds. This has the additional advantage of allowing the first and second peptides to dissociate from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA) may be added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups; further cysteine ​​or thiol may then be attached to the N- or C-terminus of the first peptide so that the cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0184] Similar techniques are also used for the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially generating tetraspecific molecules.

[0185] Furthermore, other functional groups or effector groups can be added in the same manner, at the N- or C-terminus or via side chain coupling using appropriate chemistry. In one embodiment, the coupling is performed in a way that does not block the activity of either entity.

[0186] Pharmaceutical composition

[0187] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand as defined herein, in combination with one or more pharmaceutically acceptable excipients.

[0188] Generally, the peptide ligands of the present invention will be used in purified form with a pharmacologically suitable excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / water solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride and lactated Ringer's solution. If it is necessary to keep the polypeptide complex suspended, a suitable physiologically acceptable adjuvant can be selected from thickening agents such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin and alginate.

[0189] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982), Remington's Pharmaceutical Sciences, 16th edition).

[0190] The peptide ligands of the invention can be used as a composition for administration alone or in combination with other agents. They can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, doxorubicin or cisplatin and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in combination with the protein ligands of the invention, or even in combination with polypeptides selected according to the invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are combined before administration.

[0191] The route of administration of the pharmaceutical composition according to the present invention can be any route commonly known to those of ordinary skill in the art. For treatment, the peptide ligand of the present invention can be applied to any patient according to standard techniques. The application can be carried out in any suitable manner, including parenteral, intravenous, intramuscular, intraperitoneal, percutaneous, via pulmonary approach or suitably by direct infusion with a catheter. Preferably, the pharmaceutical composition according to the present invention will be applied by inhalation. The dosage and frequency of application will depend on the patient's age, sex and condition, the simultaneous application of other drugs, contraindications and other parameters that the clinician will consider.

[0192] The peptide ligands of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and lyophilization and reconstitution techniques known in the art may be employed. Those skilled in the art will recognize that lyophilization and reconstitution may result in varying degrees of loss of activity and that levels may have to be adjusted upwards to compensate.

[0193] Compositions comprising the peptide ligands of the present invention or mixtures thereof can be administered for preventive and / or therapeutic treatment. In certain therapeutic applications, an amount sufficient to complete at least partial inhibition (inhibition), inhibition (suppression), regulation, killing or some other measurable parameters of a selected cell population is defined as a "therapeutically effective dose". The amount required to reach the dose will depend on the severity of the disease and the general state of the patient's own immune system, but is generally 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, and a more commonly used dose is 0.05 to 2.0 mg / kg / agent. For preventive applications, compositions comprising the peptide ligands of the present invention or mixtures thereof can also be administered in similar or slightly lower doses.

[0194] Compositions comprising peptide ligands according to the present invention can be used in preventive and therapeutic settings to assist in the alteration, inactivation, killing or removal of selected target cell populations in mammals. In addition, peptide ligands as described herein can be selectively used to kill, consume or otherwise effectively remove target cell populations from heterogeneous cell collections in vitro or in vitro. Blood from mammals can be combined in vitro with selected peptide ligands, thereby killing or otherwise removing undesirable cells from the blood for return to mammals according to standard techniques.

[0195] Therapeutic Uses

[0196] According to a further aspect of the present invention, there is provided a hetero-tandem bicyclic peptide complex as defined herein for use in preventing, inhibiting or treating cancer.

[0197] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and various types of carcinomas, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and others) such as bladder and urinary tract cancer, breast cancer, gastrointestinal cancer (including cancers of the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver cancer (hepatocellular carcinoma), gallbladder and biliary system cancer, exocrine pancreatic cancer, kidney cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck cancer (e.g., tongue cancer, buccal cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulvar cancer, penis cancer, cervical cancer, uterine cancer, Myometrial cancer, endometrial cancer, thyroid cancer (e.g., follicular thyroid cancer), adrenal cancer, prostate cancer, skin and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, and hyperplastic nevus); hematologic malignancies (i.e., leukemias and lymphomas) and hematologic precancerous conditions and marginal malignancies, including hematologic malignancies and related conditions of the lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, unexplained monoclonal immunoglobulinemia, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematologic malignancies and related conditions of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilia, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, such as soft tissue, bone, or chondrosarcomas such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma , Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); tumors of the eye and adnexa (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor);or congenital or other syndromes that predispose the patient to malignancy (e.g. xeroderma pigmentosum). ;

[0198] In a further embodiment, the cancer is selected from hematopoietic malignancies such as selected from: non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), chronic B lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL) and chronic myeloid leukemia (CML).

[0199] The term "prevention" as referred to herein involves administration of a protective composition prior to induction of a disease. "Suppression" refers to administration of a composition after an induction event but prior to clinical manifestation of the disease. "Treatment" involves administration of a protective composition after disease symptoms become apparent.

[0200] Animal model systems exist that can be used to screen peptide ligands for effectiveness in preventing or treating disease. The present invention facilitates the use of animal model systems, which allow the development of polypeptide ligands that can cross-react with both human and animal targets, thereby allowing the use of animal models.

[0201] The present invention is further described below with reference to the following examples.

[0202] Example

[0203] Generally, the hetero-tandem bicyclic peptide complex of the present invention can be prepared according to the following general method:

[0204]

[0205] A mixture of bicyclic 1 (1.0 eq.) and NHS-PEG5-N3 (1.6 eq.) was dissolved in MeCN / H 2 O (1:1), by dropping NaHCO 3 (0.1 M) and the pH of the solution was adjusted to 8. The reaction mixture was stirred at 30°C for 2 hours and then concentrated under reduced pressure to remove the solvent. The residue was then purified by preparative HPLC to give Intermediate 2.

[0206] A mixture of intermediate 2 (1.0 eq) and bicyclic 2 (1.0 eq) was dissolved in t-BuOH / H 2 O (1:1), then add CuSO 4 (1.0 eq), VcNa (2.3 eq) and THPTA (1.0 eq). Finally, 0.2 M NH 4 HCO 3 To adjust the pH to 8.2 The reaction mixture was stirred at 40° C. for 16 h under atmospheric pressure. The reaction mixture was directly purified by preparative HPLC.

[0207] The following provides more detailed experiments of the hetero-tandem bicyclic peptide complex of the present invention:

[0208] Example 1: Synthesis of BCY12375

[0209]

[0210] Preparation of Palmitic Acid--PEG10-N 3 The program

[0211]

[0212] A mixture of palmitic acid (100.0 mg, 282.89 μmol, 1.0 eq.), compound 2 (150.0 mg, 284.84 μmol, 1.0 eq.) and DIEA (74.5 mg, 574.11 μmol, 100.0 μL, 2.0 eq.) was dissolved in DMF (2 mL). The reaction mixture was stirred at 30 ° C for 2 hours. LC-MS showed that compound 1 was completely consumed, and a main peak with the desired m / z (MW: 765.03, observed m / z: 765.22) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). Palmitic acid-PEG10-N was obtained as a white solid. 3 (79.0 mg, 99.41 μmol, 35.14% yield, 96.27% purity).

[0213] Procedure for preparation of palmitic acid-PEG10-BCY12023

[0214]

[0215] A mixture of compound 3 (50.0 mg, 22.07 μmol, 1.0 eq.), compound 2 (17.0 mg, 22.22 μmol, 1.0 eq.) and THPTA (10.0 mg, 23.02 μmol, 1.0 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 56.0 μL, 1.0 eq.) and VcNa (10.0 mg, 50.48 μmol, 2.3 eq.). 0.2 M NH 4 HCO 3(at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 2 hours under a 4% CO atmosphere. LC-MS showed that palmitic acid-PEG10-N 3 The product was completely consumed and one product with the desired m / z (calculated MW: 3030.60, observed m / z: 1010.35 ([M / 3+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain palmitic acid-PEG10-BCY12023 (43.0 mg, 13.97 μmol, 63.30% yield, 98.46% purity) as a white solid.

[0216] Preparation of palmitic acid--PEG10-BCY12023-PEG5-N 3 The program

[0217]

[0218] A mixture of compound 5 (43.0 mg, 14.19 μmol, 1.0 eq.) and compound 6 (10.0 mg, 23.13 μmol, 1.6 eq.) was dissolved in MeCN / H 2 O (1:1, 1 mL), and then dropwise add NaHCO 3 (0.1 M) and the pH of the solution was adjusted to 8. The reaction mixture was stirred at 30°C for 2 hours. LC-MS showed that compound 5 was completely consumed and a desired m / z (MW: 3347.94, observed m / z: 1673.7 ([(M / 2+H + ]),1115.9([(M / 3+H + ])). The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). Palmitic acid-PEG10-BCY12023-PEG5-N3 (16.0 mg, 4.43 μmol, 31.25% yield, 92.78% purity) was obtained as a white solid.

[0219] Procedure for preparation of BCY12375

[0220]

[0221] A mixture of compound 7 (8.0 mg, 2.39 μmol, 1.0 eq.), compound 8 (6.5 mg, 2.39 μmol, 1.0 eq.) and THPTA (1.1 mg, 2.53 μmol, 1.0 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 6.0 μL, 1.0 eq.) and VcNa (1.0 mg, 5.05 μmol, 2.1 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 16 hours under a 4% CO atmosphere. LC-MS showed that compound 7 was completely consumed and one with the desired m / z (calculated MW: 6064.08, observed m / z: 1516.4 ([M / 4+H] + ),1212.8([M / 5+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY12375 (6.2 mg, 0.99 μmol, 41.62% yield, 97.27% purity) as a white solid.

[0222] Example 2: Synthesis of BCY12021

[0223]

[0224] Procedure for the preparation of palmitic acid-PEG10-BCY11144

[0225]

[0226] A mixture of compound 3 (160.0 mg, 69.45 μmol, 1.0 eq.), compound 4 (56.0 mg, 72.20 μmol, 1.0 eq.) and THPTA (35.0 mg, 80.55 μmol, 1.1 eq.) was dissolved in t-BuOH / H 2 O (1:1, 2 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4(0.4 M, 56.0 μL, 1.0 eq.) and VcNa (30.0 mg, 151.43 μmol, 2.2 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 16 hours under a 4% CO atmosphere. LC-MS showed the detection of a desired m / z (calculated MW: 3068.70, observed m / z: 1533.81 ([M / 2+H] + ),1023.43([M / 3+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain palmitic acid-PEG10-BCY11144 (150.0 mg, 46.83 μmol, 67.42% yield, 95.80% purity) as a white solid.

[0227] Preparation of palmitic acid--PEG10-BCY11144-PEG5-N 3 The program

[0228]

[0229] A mixture of compound 5 (47.0 mg, 15.32 μmol, 1.0 eq.), compound 6 (7.0 mg, 16.19 μmol, 1.0 eq.) and DIEA (3.0 mg, 22.97 μmol, 4.0 μL, 1.5 eq.) was dissolved in DMF (1 mL). The reaction mixture was stirred at 30 °C for 2 hours. LC-MS showed that compound 5 was completely consumed, and a desired m / z (MW: 3386.03, observed m / z: 1693.21 ([M / 2+H] + ),1129.13([M / 3+H] + )) The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). Palmitic acid--PEG10-BCY11144-PEG5-N was obtained as a white solid. 3 (20.0 mg, 5.72 μmol, 37.33% yield, 96.79% purity).

[0230] Procedure for preparing BCY12021

[0231]

[0232] A mixture of compound 7 (10.0 mg, 2.95 μmol, 1.0 eq.), compound 8 (8.2 mg, 3.02 μmol, 1.0 eq.) and THPTA (1.5 mg, 3.45 μmol, 1.1 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 8.0 μL, 1.0 eq.) and VcNa (1.5 mg, 7.57 μmol, 2.5 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 16 hours under a 4% CO atmosphere. LC-MS showed the desired m / z (calculated MW: 6102.17, observed m / z: 1525.17 ([M / 4+H] + ),1221.3([M / 5+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY12021 (6.6 mg, 1.02 μmol, 34.62% yield, 94.54% purity) as a white solid.

[0233] Example 3: Synthesis of BCY11468

[0234]

[0235] Procedure for preparing COM113

[0236]

[0237] A mixture of compound 1 (50.0 mg, 124.4 μmol, 1.0 eq), EDCI (95.4 mg, 497.7 μmol, 4.0 eq), HOBt (55.5 mg, 410.6 μmol, 3.3 eq) and DMAP (15.2 mg, 124.4 μmol, 1.0 eq) was dissolved in 2 mL of DMF, and then DIEA (134.9 mg, 1.04 mmol, 181.8 μL, 8.4 eq) was added to form a homogeneous solution. Compound 2 (200.0 mg, 379.8 μmol, 3.05 eq) dissolved in DMF (2 mL) was then added dropwise to the solution. The reaction mixture was stirred at 30 °C for 16 hours. LC-MS showed that compound 1 was completely consumed, and a desired m / z (MW: 1891.19, observed m / z: 945.8600 ([M / 2+H + ]) and 612.4400([(M-3H 2 O) / 3+H + The reaction mixture was directly purified by preparative HPLC (TFA conditions) to give COM113 (161 mg, 85.67 μmol, 68% yield) as a yellow oil after freeze-drying.

[0238] Procedure for preparing COM113-BCY8928

[0239]

[0240] First, COM113 (50.0 mg, 26.44 μmol, 1.0 eq) and BCY8928 (53.0 mg, 23.9 μmol, 0.9 eq) were dissolved in 2 mL t-BuOH / H 2 O (1:1), then add CuSO 4 (0.4M, 66.1μL, 1.0eq), VcNa (10.5mg, 53.0μmol, 2.0eq) and THPTA (23.0mg, 52.93μmol, 2.0eq). Finally, 1M NH 4 HCO 3 The pH was adjusted to 8. All solvents were degassed and heated with N 2 Purge 3 times. 2 The reaction mixture was stirred at 30°C for 16 hours under a 4% CO atmosphere. LC-MS showed a desired m / z (calculated MW: 4108.77 observed m / z: 1369.97 ([M / 3+H] +The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain compound 2 (14.0 mg, 3.21 μmol, 12.14% yield, 94.16% purity) as a white solid.

[0241] Procedure for the preparation of palmitic acid NHS ester

[0242]

[0243] EDCI (747.60 mg, 3.90 mmol, 2.0 eq) was added to a solution of palmitic acid (500 mg, 1.95 mmol, 586.85 μL, 1.0 eq) and 1-hydroxypyrrolidine-2,5-dione (250 mg, 2.17 mmol, 1.11 eq) in DCM (5 mL). The mixture was stirred at 30 ° C for 16 hours. TLC showed that reactant 1 was completely consumed and a new spot was formed. According to TLC, the reaction was thorough (clean). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, DCM: MeOH = 0 to 100: 1). The desired product was dried to obtain palmitic acid NHS ester (0.68 g, 1.92 mmol, 98.65% yield) as a white solid.

[0244] Procedure for the preparation of palmitic acid-propargylalanine

[0245]

[0246] DIEA (131.62 mg, 1.02 mmol, 177.39 μL, 3.0 eq) and DMAP (41.47 mg, 339.47 μmol, 1.0 eq) were added to a DMF (6 mL) solution of compound 3 (120 mg, 339.47 μmol, 1.0 eq) and compound 4 (57.60 mg, 509.20 μmol, 1.5 eq). The mixture was stirred at 40 ° C for 16 hours. LC-MS showed that reactant 3 was completely consumed and a main peak with the desired m / z or the desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (TFA conditions). Palmitic acid-propargyl alanine (90 mg, 256.03 μmol, 75.42% yield) was obtained as a white solid.

[0247] Procedure for the preparation of COM113-BCY8928-palmitic acid

[0248]

[0249] First, compound 2 (14.0 mg, 3.41 μmol, 1.0 eq) and compound 3 (1.1 mg, 3.13 μmol, 0.9 eq) were dissolved in 2 mL t-BuOH / H 2 O (1:1), then add CuSO 4 (0.4M, 10.0μL, 1.0eq), VcNa (2.0mg, 10.1μmol, 2.9eq) and THPTA (2.0mg, 4.6μmol, 1.3eq). Finally, 0.2M NH 4 HCO 3 To adjust the pH to 8. All solvents were degassed and heated with N 2 Purge 3 times. 2 The reaction mixture was stirred at 35°C for 16 hours under a 4% CO atmosphere. LC-MS showed a desired m / z (calculated MW: 4460.29, observed m / z: 1486.92 ([M / 3+H] + ),1115.58([M / 4+H] + ),895.83([M / 5+H] + The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain compound 4 (5.9 mg, 1.28 μmol, 37.66% yield, 97.0% purity) as a white solid.

[0250] Procedure for preparation of BCY11468

[0251]

[0252] First, compound 4 (5.9 mg, 1.32 μmol, 1.0 eq) and BCY11016 (3.0 mg, 1.29 μmol, 1 eq) were dissolved in 2 mL t-BuOH / H 2 O (1:1), then add CuSO 4 (0.4M, 8.0μL, 2.4eq), VcNa (2.0mg, 7.6eq) and THPTA (2.0mg, 3.5eq). Finally, 1M NH 4 HCO 3 The pH was adjusted to 8. All solvents were degassed and heated with N 2 Purge 3 times. 2 The reaction mixture was stirred at 30°C for 16 hours under a 4% CO atmosphere. LC-MS showed a desired m / z (calculated MW: 6783.93, observed m / z: 1131.7 ([M / 6+H] +The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain BCY11468 (2.2 mg, 0.312 μmol, 23.57% yield, 96.16% purity) as a white solid.

[0253]

[0254] Preparation of BCY8920-PEG5-N 3 The program

[0255]

[0256] BCY8920 (50.0 mg, 23.39 μmol, 1.0 eq.), compound 2 (10.2 mg, 23.51 μmol, 1.01 eq.) and NaHCO 3 A mixture of (2.0 mg, 24.8 μmol, 1.0 eq.) was dissolved in MeCN / H 2 O (1:1, 2 mL). The reaction mixture was stirred at 40 °C for 2 h until LC-MS showed complete consumption of BCY8920 and a desired m / z (calculated MW: 2454.83, observed m / z: 1227.67 ([M / 2+H] + ) and 818.74([M / 3+H] + )). The reaction mixture was then concentrated under reduced pressure to remove the solvent and produce a residue, which was then purified by preparative HPLC (TFA conditions). BCY8920-PEG5-N3 (25 mg, 9.70 μmol, 41.47% yield, 95.26% purity) was obtained as a white solid.

[0257] Procedure for the preparation of BCY11143-dK (palmitic acid)

[0258]

[0259] A mixture of BCY11143 (30.0 mg, 12.84 μmol, 1.0 eq.), compound 5 (5.0 mg, 14.12 μmol, 1.1 eq.), DIEA (1.7 mg, 12.84 μmol, 2.2 μL, 1.0 eq.) and DMAP (1.6 mg, 12.84 μmol, 1.0 eq.) was dissolved in DMF. 2 The reaction mixture was stirred at 40°C for 2 hours under a 4% CO atmosphere. LC-MS showed the detection of a desired m / z (calculated MW: 2575.14, observed m / z: 1287.68 ([M / 2+H +])). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue, which was then purified by preparative HPLC (TFA conditions). BCY11143-dK (palmitic acid) (18.3 mg, 6.95 μmol, 54.17% yield, 97.86% purity) was obtained as a white solid.

[0260] Procedure for preparation of BCY11618

[0261]

[0262] A mixture of compound 3 (5 mg, 2.04 μmol, 1.0 eq.), compound 6 (5.8 mg, 2.3 μmol, 1.1 eq.) and THPTA (0.9 mg, 2.07 μmol, 1.0 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 5.1 μL, 1.0 eq.) and VcNa (0.4 M, 5.1 μL, 1.0 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 6 hours under a 4% CO atmosphere. LC-MS showed that compound 3 was completely consumed and one with the desired m / z (calculated MW: 5029.97, observed m / z: 1257.8 ([M / 4+H] + ) and 1006.6([M / 5+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY11618 (5.3 mg, 1.0 μmol, 49.15% yield, 95% purity) as a white solid.

[0263]

[0264] Preparation of BCY8116-Peg5-N 3 The program

[0265]

[0266] BCY8116 (50.0 mg, 23.39 μmol, 1.0 eq.), compound 2 (10.2 mg, 23.51 μmol, 1.01 eq.) and NaHCO3 A mixture of (2.0 mg, 24.8 μmol, 1.0 eq.) was dissolved in MeCN / H 2 O (1:1, 2 mL). The reaction mixture was stirred at 25 °C for 1 hour until LC-MS showed complete consumption of BCY8116 and a desired m / z (calculated MW: 2454.83, observed m / z: 1227.67 ([M / 2+H + ]),818.74([M / 3+H + ])). The reaction mixture was then concentrated under reduced pressure to remove the solvent and produce a residue, which was then purified by preparative HPLC (TFA conditions). Compound 3 (25.0 mg, 9.70 μmol, 41.47% yield, 95.26% purity) was obtained as a white solid.

[0267] Procedure for the preparation of compound BCY11144-dK (palmitic acid)

[0268]

[0269] A mixture of BCY11144 (50.0 mg, 21.7 μmol, 1.0 eq.), compound 5 (8.5 mg, 23.87 μmol, 1.1 eq.), DIEA (2.81 mg, 21.7 μmol, 4.0 μL, 1.0 eq.) and DMAP (2.7 mg, 21.7 μmol, 1.0 eq.) was dissolved in DMF. 2 The reaction mixture was stirred at 25°C for 2 hours under a 4% CO atmosphere. LC-MS showed that compound 3 was completely consumed and one with the desired m / z (calculated MW: 2542.08, observed m / z: 1271.7 ([M / 2+H + ])). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue, which was then purified by preparative HPLC (TFA conditions). Compound 6 (18.3 mg, 6.95 μmol, 54.17% yield, 96.68% purity) was obtained as a white solid.

[0270] Procedure for preparation of BCY11776

[0271]

[0272] A mixture of compound 3 (10 mg, 4.0 μmol, 1.0 eq.), compound 6 (11.2 mg, 4.4 μmol, 1.1 eq.) and THPTA (1.8 mg, 1.0 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2Purge 3 times) and then in N 2 Add CuSO 4 (0.4M, 5.1μL, 1eq.) and VcNa (0.4M, 5.1μL, 1eq.). 0.2M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 6 hours under a 4% CO atmosphere. LC-MS showed that compound 3 was completely consumed and a desired m / z (calculated MW: 5031.9, observed m / z: 1258.52 ([M / 4+H + ]),1006.7([M / 5+H + ])). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY11776 (12.5 mg, 2.4 μmol, 60.11% yield, 96.6% purity) as a white solid.

[0273]

[0274] Procedure for preparation of BCY8920-Peg5-BCY11143

[0275]

[0276] BCY8920-PEG5-N 3 A mixture of 1,2-dihydro-1,4 ... 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 21.0 μL, 1.0 eq.) and VcNa (0.4 M, 21.0 μL, 1.0 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2The reaction mixture was stirred at 40°C for 4 hours under a 4% CO atmosphere. LC-MS showed that compound 1 was completely consumed and a desired m / z (calculated MW: 4791.56, observed m / z: 1597.28 ([M / 3+H] + ),1198.18([M / 4+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY8920-Peg5-BCY11143 (22.5 mg, 4.25 μmol, 52.13% yield, 90.44% purity) as a white solid.

[0277] Procedure for preparation of BCY11860

[0278]

[0279] Compound 3 (5.0 mg, 1.04 μmol, 1.0 eq.), compound 4 (1.08 mg, 1.15 μmol, 1.1 eq.), DIEA (0.4 M, 1.04 μmol, 3.0 μL, 1.0 eq.) and DMAP (0.2 mg, 1.04 μmol, 1.0 eq.) were dissolved in DMF (1.0 mL). The reaction mixture was stirred at 30 °C for 2 hours. LC-MS showed that compound 3 was completely consumed, and a desired m / z (MW: 5617.56, observed m / z: 1404.56 ([(M / 4+H + ])). The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). BCY11860 (2.9 mg, 0.48 μmol, 45.86% yield, 92.70% purity) was obtained as a white solid.

[0280]

[0281] Preparation of Palmitic Acid-PEG10-N 3 The program

[0282]

[0283] A mixture of palmitic acid-NHS (100.0 mg, 282.89 μmol, 1.0 eq.), compound 2 (150.0 mg, 284.84 μmol, 1.0 eq.) and DIEA (74.5 mg, 574.11 μmol, 100.0 μL, 2.0 eq.) was dissolved in DMF (2 mL). The reaction mixture was stirred at 30 ° C for 2 hours. LC-MS showed that compound 1 was completely consumed, and a main peak with the desired m / z (MW: 765.03, observed m / z: 765.22) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). Palmitic acid-PEG10-N was obtained as a white solid. 3 (79.0 mg, 99.41 μmol, 35.14% yield, 96.27% purity).

[0284] Procedure for the preparation of palmitic acid-PEG10-BCY11144

[0285]

[0286] A mixture of compound 3 (160.0 mg, 69.45 μmol, 1.0 eq.), compound 2 (56.0 mg, 72.20 μmol, 1.0 eq.) and THPTA (35.0 mg, 80.55 μmol, 1.1 eq.) was dissolved in t-BuOH / H 2 O (1:1, 2 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 56.0 μL, 1.0 eq.) and VcNa (30.0 mg, 151.43 μmol, 2.2 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 16 hours under a 4% CO atmosphere. LC-MS showed a desired m / z (calculated MW: 3068.70, observed m / z: 1533.81 ([M / 2+H] + ),1023.43([M / 3+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain palmitic acid-PEG10-BCY11144 (150.0 mg, 46.83 μmol, 67.42% yield, 95.80% purity) as a white solid.

[0287] Preparation of palmitic acid-PEG10-BCY11144-PEG5-N 3 The program

[0288]

[0289] A mixture of compound 5 (47.0 mg, 15.32 μmol, 1.0 eq.), compound 6 (7.0 mg, 16.19 μmol, 1.1 eq.) and DIEA (3.0 mg, 22.97 μmol, 4.0 μL, 1.5 eq.) was dissolved in DMF (1 mL). The reaction mixture was stirred at 30 °C for 2 hours. LC-MS showed that compound 5 was completely consumed, and a desired m / z (MW: 3386.03, observed m / z: 1693.21 ([M / 2+H] + ),1129.13([M / 3+H] + )) The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). Palmitic acid-PEG10-BCY11144-PEG5-N was obtained as a white solid. 3 (20.0 mg, 5.72 μmol, 37.33% yield, 96.79% purity).

[0290] Procedure for preparing BCY12020

[0291]

[0292] A mixture of compound 7 (50.0 mg, 14.77 μmol, 1.0 eq.), compound 8 (35.0 mg, 15.06 μmol, 1.0 eq.) and THPTA (10.0 mg, 23.02 μmol, 1.5 eq.) was dissolved in t-BuOH / H 2 O (1:1, 2 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 38.0 μL, 1.0 eq.) and VcNa (6.5 mg, 32.81 μmol, 2.2 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2The reaction mixture was stirred at 40°C for 16 hours under a 4% CO atmosphere. LC-MS showed a desired m / z (calculated MW: 5709.68, observed m / z: 1902.80 ([M / 3+H] + ),1427.56([M / 4+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY12020 (54.8 mg, 9.49 μmol, 64.24% yield, 98.83% purity) as a white solid.

[0293]

[0294] Procedure for the preparation of compound 2

[0295]

[0296] The peptide was synthesized using standard Fmoc chemistry. DCM was added to a mixture containing chlorotrityl resin (1 mmol, 0.91 g, 1.10 mmol / g) and Fmoc-Lys(N3)-OH (1 eq, 395.4 mg, 1 mmol) and N 2 The resin was then drained and washed 5 times with DMF. Fmoc deprotection was performed by adding 20% ​​piperidine / DMF and mixing for 30 minutes. The resin was drained and washed 5 times with DMF. For chain extension, the Fmoc-amino acid solution was first added and mixed for 30 seconds, then the activation buffer (HBTU and DIEA in DMF) was added and the mixture was washed 5 times with continuous N. 2 Stir under bubbling for 1 hour. Repeat deprotection and coupling until the peptide is complete.

[0297] # Material Coupling reagents 1 Fmoc-Lys(N3)-OH(1eq) DIEA (4.0eq) 2 Fmoc-γGlu(OtBu)-OH(3eq) HBTU (2.85 eq) and DIEA (6.0 eq) 3 Palmitic acid (3eq) HBTU (2.85 eq) and DIEA (6.0 eq)

[0298] After the last amino acid coupling, the resin was washed 3 times with MeOH and then dried in vacuo. 10 ml of cleavage mixture (95% TFA / 2.5% TIS / 2.5% H 2 O) was added to the flask containing the side chain protected peptide and stirred for 1 hour. The resin was filtered and the filtrate was concentrated to remove the solvent. The crude peptide was lyophilized to obtain the final product, compound 2 (palmitic acid azide) (200 mg, 97.78% purity, 37.06% yield). Calculated MW: 539.72, observed m / z: 540.4 ([M+H] + ).

[0299]

[0300] A mixture of compound 3 (35.0 mg, 12.48 μmol, 1.0 eq.) and compound 4 (5.4 mg, 12.48 μmol, 1.0 eq.) was dissolved in MeCN / H 2 O (1:1, 1 mL), and then dropwise add NaHCO 3 (0.1 M) and the pH of the solution was adjusted to 8. The reaction mixture was stirred at 30°C for 2 hours. LC-MS showed that compound 3 was completely consumed and a desired m / z (MW: 3121.63, observed m / z: 1561.2 ([(M / 2+H + ])). The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). BCY12023-palmitic acid-PEG5-N3 (11.4 mg, 3.46 μmol, 27.71% yield, 94.70% purity) was obtained as a white solid.

[0301] Procedure for preparation of BCY12661

[0302]

[0303] A mixture of compound 5 (11.4 mg, 3.65 μmol, 1.0 eq.), compound 6 (8.3 mg, 3.65 μmol, 1.0 eq.) and THPTA (1.6 mg, 3.65 μmol, 1.0 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 10.0 μL, 1.1 eq.) and VcNa (1.5 mg, 7.30 μmol, 2.0 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 4 hours under a 4% CO atmosphere. LC-MS showed that compound 3 was completely consumed and one with the desired m / z (calculated MW: 5374.21, observed m / z: 1344.5 ([M / 4+H] +)). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY12661 (9.8 mg, 19.63 μmol, 48.73% yield, 97.60% purity) as a white solid.

[0304]

[0305] General procedure for the preparation of compound 1

[0306]

[0307] The peptide was synthesized using standard Fmoc chemistry. DCM was added to a reaction vessel containing chlorotrityl resin (1 mmol, 0.91 g, 1.1 mmol / g) and Fmoc-γGlu(OtBu)-OH (0.425 mg, 1 mmol, 1 eq) and heated with N 2 The mixture was stirred with bubbling. DIEA (4.0 eq) was added dropwise and the mixture was stirred for 2 hours. MeOH (4.6 mL) was then added and mixed for 30 minutes. The resin was drained and washed 5 times with DMF. 20% piperidine / DMF was added to the resin and mixed for 30 minutes. The resin was drained and washed 5 times with DMF. The Fmoc-amino acid solution was added to the resin and mixed for 30 seconds, then the activator and DIPEA were added and the mixture was N 2 Bubble for 1 hour. Repeat the deprotection and coupling steps with the following reagents:

[0308] Note:

[0309] # Material Coupling reagents 1 Fmoc-Glu-OtBu(1eq) DIEA (4.0eq) 2 Fmoc-Glu-OtBu(3eq) HBTU (2.85 eq) and DIEA (6.0 eq) 3 Palmitic acid (3eq) HBTU (2.85 eq) and DIEA (6.0 eq)

[0310] After palmitic acid coupling, the resin was washed 3 times with MeOH and then dried under vacuum. The peptide was cleaved from the resin by adding 20% ​​HFIP / 80% DCM at room temperature and the mixture was stirred for 1 hour. The procedure was repeated once more, and then the resin was filtered and the filtrate was concentrated to remove the solvent. The crude peptide was lyophilized to give the final product (280 mg, 84.80% purity, 44.67% yield). Calculated MW: 626.8, observed m / z: 627.4 ([M+H] + )

[0311]

[0312]

[0313]

[0314] Preparation of BCY12860-PEG5-N 3 The program

[0315]

[0316] A mixture of BCY12860 (40.0 mg, 19.40 μmol, 1.0 eq.) and compound 2 (10.0 mg, 21.34 μmol, 1.1 eq.) was dissolved in MeCN / H 2 O (1:1, 1 mL), and then dropwise add NaHCO 3 (0.1 M) and the pH of the solution was adjusted to 8. The reaction mixture was stirred at 25°C for 1 hour. LC-MS showed a peak with the desired m / z. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). BCY12860-PEG5-N was obtained as a white solid. 3 (39.7 mg, 15.02 μmol, 77.41% yield, 90.0% purity). MW: 2378.78, observed m / z: 1190.1 ([(M / 2+H + ]),793.5([(M / 3+H + ]).

[0317] Procedure for preparation of BCY13035

[0318]

[0319] A mixture of compound 3 (39.7 mg, 16.69 μmol, 1.0 eq.), BCY8928 (41.0 mg, 18.36 μmol, 1.1 eq.) and THPTA (0.4 M, 55 μL, 1.3 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4M, 55μL, 1.3eq.) and VcNa (0.4M, 109μL, 2.6eq.). By adding 0.2M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2The reaction mixture was stirred at 40 °C for 2 hours under atmosphere. LC-MS showed that compound 3 was completely consumed and a major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY13035 (42.0 mg, 8.85 μmol, 53.04% yield, 96.41% purity) as a white solid. Calculated MW: 4596.37, observed m / z: 1532.9 ([M / 3+H] + ),1149.9([M / 4+H] + ).

[0320] Example 11: Synthesis of BCY13040

[0321]

[0322] Preparation of BCY12865-PEG5-N 3 The program

[0323]

[0324] BCY12865 (30.0 mg, 13.99 μmol, 1.0 eq) and compound 1 (6.1 mg, 14.11 μmol, 1.01 eq) were dissolved in 1 mL MeCN / H 2 O (1:1), and then add 1M NaHCO 3 The pH was adjusted to 8. The mixture was stirred at 25 °C for 2 hours. LC-MS showed that BCY12865 was completely consumed and a major peak with the desired m / z was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain compound 2 as a white solid (15.6 mg, 6.32 μmol, 45.19% yield, 99.76% purity). Calculated MW: 2461.87, observed m / z: 1231.5 ([M / 2+H] + ) and 821.3 ([M / 3+H]+).

[0325] Procedure for preparing BCY13040

[0326]

[0327] First, compound 2 (15.6 mg, 6.34 μmol, 1.0 eq) and BCY8928 (14.5 mg, 6.54 μmol, 1.03 eq) were dissolved in 2 mL t-BuOH / H 2 O (1:1), then add CuSO 4(0.4M, 16μL, 1.01eq), VcNa (3.0mg, 15.14μmol, 2.39eq) and THPTA (3mg, 6.90μmol, 1.09eq). Finally, 1M NH 4 HCO 3 The pH was adjusted to 8. All solvents were degassed and heated with N 2 Purge 3 times. 2 The reaction mixture was stirred at 40 °C for 16 hours under atmosphere. LC-MS showed that compound 2 was completely consumed and a major peak with the desired m / z was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain BCY13040 (15.8 mg, 3.31 μmol, 52.27% yield, 98.1% purity) as a white solid. Calculated MW: 4679.45, observed m / z: 1560.8 ([M / 3+H] + ),1170.9([M / 4+H] + ),936.6([M / 5+H] + ).

[0328]

[0329] Procedure for preparation of BCY13253

[0330]

[0331] A mixture of compound 3 (25.7 mg, 10.92 μmol, 1.0 eq.), compound 2 (26.6 mg, 12.02 μmol, 1.1 eq.) and THPTA (5.7 mg, 13.11 μmol, 1.2 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 33.0 μL, 1.2 eq.) and VcNa (5.2 mg, 26.21 μmol, 2.4 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 25°C for 2 hours under a 4% CO atmosphere. LC-MS showed that compound 3 was completely consumed and one with the desired m / z (calculated MW: 4570.32, observed m / z: 1143.4 ([M / 4+H] + ),914.9([M / 5+H]+ )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY13253 (17.5 mg, 3.67 μmol, 33.58% yield, 95.8% purity) as a white solid.

[0332]

[0333] Preparation of BCY13120-PEG5-N 3 The program

[0334]

[0335] A mixture of BCY13120 (40.0 mg, 17.92 μmol, 1.0 eq.) and compound 2 (8.5 mg, 19.72 μmol, 1.1 eq.) was dissolved in MeCN / H 2 O (1:1, 1 mL), and then dropwise add NaHCO 3 (0.1 M) and the pH of the solution was adjusted to 8. The reaction mixture was stirred at 25°C for 1 hour. LC-MS showed complete consumption of BCY13120 and the detection of a desired m / z (calculated MW: 2548.99, observed m / z: 1275.3 ([(M / 2+H + The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). BCY13120-PEG5-N was obtained as a white solid. 3 (27.3 mg, 10.46 μmol, 58.38% yield, 97.7% purity).

[0336] Procedure for preparing BCY13254

[0337]

[0338] A mixture of compound 3 (27.3 mg, 10.71 μmol, 1.0 eq.), compound 2 (26.1 mg, 11.78 μmol, 1.1 eq.) and THPTA (5.6 mg, 12.85 μmol, 1.2 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times). 2 Add CuSO 4 (0.4 M, 33.0 μL, 1.2 eq.) and VcNa (5.2 mg, 26.24 μmol, 2.4 eq.). 0.2 M NH 4HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 25°C for 2 hours under a 4% CO atmosphere. LC-MS showed that compound 3 was completely consumed and one with the desired m / z (calculated MW: 4766.58, observed m / z: 1192.5 ([M / 4+H] + ),954.1([M / 5+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY13254 (36.5 mg, 7.49 μmol, 69.92% yield, 97.8% purity) as a white solid.

[0339]

[0340] Preparation of BCY12865-PEG5-N 3 The program

[0341]

[0342] BCY12865 (50 mg, 23.32 μmol, 1.0 eq) and compound 1 (10.5 mg, 24.28 μmol, 1.04 eq) were dissolved in 2 mL MeCN / H 2 O (1:1), add 1M NaHCO 3 The pH was adjusted to 8. The mixture was then stirred at 25°C for 2 hours. LC-MS showed that BCY12865 was completely consumed and one compound with the desired m / z (calculated MW: 2461.87, observed m / z: 1231.6 ([M / 2+H] + ) and 821.4([M / 3+H] + The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain compound 2 (31.5 mg, 12.62 μmol, 54.14% yield, 98.66% purity) as a white solid.

[0343] Procedure for preparation of BCY13340

[0344]

[0345] A mixture of compound 2 (31.5 mg, 12.80 μmol, 1.0 eq.), BCY12353 (27 mg, 12.92 μmol, 1.0 eq.) and THPTA (5.7 mg, 13.12 μmol, 1.0 eq.) was dissolved in t-BuOH / H 2 O (1:1, 2 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4M, 32μL, 1.0eq) and VcNa (5.1mg, 25.74μmol, 2.0eq). 0.2M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 40°C for 1 hour under a 4% CO atmosphere. LC-MS showed that compound 2 was completely consumed and one with the desired m / z (calculated MW: 4551.32, observed m / z: 1517.7 ([M / 3+H] + ) and 1138.6([M / 4+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY13340 (34.7 mg, 7.62 μmol, 59.59% yield, 89.59% purity) as a white solid.

[0346]

[0347] Preparation of BCY12860-PEG5-N 3 The program

[0348]

[0349] A mixture of BCY12860 (28.0 mg, 13.58 μmol, 1.0 eq.) and compound 2 (6.5 mg, 14.94 μmol, 1.1 eq.) was dissolved in MeCN / H 2 O (1:1, 1 mL), and then dropwise add NaHCO 3 (0.1 M) and the pH of the solution was adjusted to 8. The reaction mixture was stirred at 25°C for 1 hour. LC-MS showed complete consumption of BCY12860 and the detection of a desired m / z (calculated MW: 2378.78, observed m / z: 1190.2 ([(M / 2+H +The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC (neutral conditions). BCY12860-PEG5-N was obtained as a white solid. 3 (20.7 mg, 8.41 μmol, 61.95% yield, 96.7% purity).

[0350] Procedure for preparation of BCY13342

[0351]

[0352] A mixture of compound 3 (20.7 mg, 8.70 μmol, 1.0 eq.), compound 4 (19.0 mg, 9.14 μmol, 1.05 eq.) and THPTA (5.0 mg, 11.31 μmol, 1.3 eq.) was dissolved in t-BuOH / H 2 O (1:1, 1 mL, pre-degassed and N 2 Purge 3 times) and then in N 2 Add CuSO 4 (0.4 M, 28.3 μL, 1.3 eq.) and VcNa (4.5 mg, 22.62 μmol, 2.6 eq.). 0.2 M NH 4 HCO 3 (at 1:1t-BuOH / H 2 The pH of the solution was adjusted to 8 in N O, and the solution turned light yellow. 2 The reaction mixture was stirred at 25°C for 2 hours under a 4% CO atmosphere. LC-MS showed that compound 3 was completely consumed and one with the desired m / z (calculated MW: 4468.24, observed m / z: 1118.6 ([M / 4+H] + )). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions) to obtain BCY13342 (21.7 mg, 4.60 μmol, 52.85% yield, 94.7% purity) as a white solid.

[0353] Analyze the data

[0354] The following hetero-tandem bicyclic peptide complexes of the present invention were analyzed using mass spectrometry and HPLC. The HPLC settings are as follows:

[0355] Mobile phase: A: 0.1% TFA in H 2 O Solution B: 0.1% TFA in ACN

[0356] Flow rate: 1.0ml / min

[0357] Column: Gemini-NX C18 5um 110A 150*4.6mm

[0358] Instrument: Agilent 1200HPLC-BE(1-614)

[0359] The gradient used is described in the table below:

[0360]

[0361]

[0362] The data is generated as follows:

[0363]

[0364]

[0365]

[0366]

[0367] Biological data

[0368] 1. CD137 reporter gene assay in co-culture with tumor cells

[0369] Prepare medium, called R1 medium, by adding 1% FBS to RPMI-1640 (component of Promega kit CS196005). Prepare serial dilutions of the test article in R1 in a sterile 96-well plate. Add 25 μL of the test article or R1 (as background control) to designated wells of a white cell culture plate. Harvest tumor cells * , and resuspended in R1 medium at a concentration of 400,000 cells / mL. Add 25 μL / well of tumor cells to the white cell culture plate. Thaw Jurkat cells (Promega kit CS196005, 0.5 mL) in a water bath and add 5 ml of pre-warmed R1 medium. Then add 25 μL / well of Jurkat cells to the white cell culture plate. Incubate at 37°C, 5% CO 2 Incubate cells and test articles for 6 h. At the end of 6 h, add 75 μL / well of Bio-Glo TM Reagent (Promega) and incubate for 10 min, then read the luminescence value in a plate reader (Clariostar, BMG). Calculate the fold change relative to cells only (Jurkat cells + cell lines used in co-culture) and plot as a log (agonist) vs. response curve in GraphPad Prism to determine EC50 (nM) and induction fold relative to background (maximum).

[0370] The tumor cell type used in the co-culture was NCI-H292, which has been shown to express fibronectin-4. For EphA2, the tumor cell type used in the co-culture was PC-3. For PD-L1, the tumor cell type used in the co-culture was RKO.

[0371] Table 1 below shows a summary of the fold induction induced by the fibronectin-4 / CD137 hetero-tandem peptides in a CD137 reporter co-culture assay with NCI-H292 cells. All compounds were compared to the plate control BCY10000, which had an average EC50 of 1.1 ± 0.5 nM and an Emax of 28 ± 11 fold relative to background.

[0372] Table 1: Fold induction of fibronectin-4 / CD137 heterotandem bicyclic peptide complexes in CD137 reporter gene assay

[0373]

[0374]

[0375] A summary of the fold induction induced by EphA2 / CD137 heterotandem peptides in a CD137 reporter co-culture assay with PC3 cells is shown in Table 2. All compounds were compared to the plate control BCY9173, which had a mean EC50 of 0.54 nM and an Emax of 42-fold relative to background.

[0376] Table 2: Fold induction of EphA2 / CD137 heterotandem bicyclic peptide complexes in CD137 reporter gene assay

[0377]

[0378] Table 3 shows a summary of the fold induction induced by the PD-L1 / CD137 hetero-tandem peptides in a CD137 reporter co-culture assay with RKO cells.

[0379] Table 3: Fold induction of PD-L1 / CD137 heterotandem bicyclic peptide complexes in CD137 reporter gene assay

[0380] Dual Ring ID EC50(nM) Fold induction relative to background BCY12229 18 8 BCY12230 46 10 BCY12242 20 13 BCY12375 22 3

[0381] 2. Pharmacokinetics of CD137 hetero-tandem bicyclic peptide complex in SD rats

[0382] Male SD rats were administered 2 mg / kg of each hetero-tandem bicyclic peptide complex formulated in 25 mM histidine HCl, 10% sucrose pH 7. At each time point, serial blood sampling was performed through the submandibular vein or the great saphenous vein (approximately 80 μL of blood / time point). All blood samples were immediately transferred to pre-cooled microcentrifuge tubes containing 2 μL of K2-EDTA (0.5 M) as an anticoagulant and placed on wet ice. The blood samples were immediately processed into 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 centrifuged at 12,000 rpm and 4°C for 10 minutes. The supernatant was transferred to a pre-labeled polypropylene microcentrifuge tube and then quickly frozen on dry ice. The samples were stored at 70°C or below until analysis as needed. 7.5 μL of supernatant samples were directly injected in positive ion mode using Orbitrap Q Exactive for LC-MS / MS analysis to determine the concentration of the bicyclic. The plasma concentration versus time data were analyzed by noncompartmental methods using Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T 1 / 2 , AUC (0-end point), AUC (0-infinity), MRT (0-end point), MRT (0-infinity) and plasma concentration versus time. The pharmacokinetic parameters of the experiment are shown in Table 4:

[0383] Table 4: Pharmacokinetic parameters in SD rats

[0384] Compound Route of administration Dosage (mg / kg) T1 / 2(h) Vdss(L / kg) Clp(ml / min / kg) BCY12234 IV infusion 2.0 0.42 0.95 26 BCY13035 IV infusion 3.0 1.5 0.63 11 BCY13040 IV infusion 3.0 1.6 0.63 10

Claims

1. A peptide ligand that binds to a component present on a cancer cell, the peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two loop sequences, and the molecular scaffold forms a covalent bond with the reactive groups of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold, or a pharmaceutically acceptable salt thereof; Features The polypeptide is selected from or a modified derivative thereof, wherein one or more cysteine ​​residues are replaced with a reactive group selected from 3-mercaptopropionic acid, cysteamine and penicillamine.

2. The peptide ligand according to claim 1, wherein the peptide ligand is selected from a modified derivative of one of the following peptides, One or more cysteine ​​residues are replaced with a reactive group selected from 3-mercaptopropionic acid, cysteamine and penicillamine.

3. The peptide ligand according to claim 1, which is a peptide ligand that binds to adhesion protein-4; wherein the polypeptide is selected from or a modified derivative thereof, wherein one or more cysteine ​​residues are replaced with a reactive group selected from 3-mercaptopropionic acid, cysteamine and penicillamine. The peptide ligand according to claim 1 , wherein the polypeptide is C-terminally amidated. The peptide ligand of claim 1 , wherein the polypeptide comprises an N-terminal spacer.

6. The peptide ligand of claim 1, wherein the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

7. The peptide ligand according to claim 1, wherein the pharmaceutically acceptable salt is selected from free acid or sodium, potassium, calcium, ammonium salt.

8. A peptide complex comprising a) a first peptide ligand that binds to a component present on cancer cells; said first peptide ligand being coupled to b) a second peptide; wherein the first peptide comprises a polypeptide and a molecular scaffold, the polypeptide comprises at least three reactive groups separated by at least two loop sequences, and the molecular scaffold forms a covalent bond with the reactive groups of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold, or a pharmaceutically acceptable salt thereof; Features The first peptide ligand comprises a polypeptide selected from the group consisting of: or a modified derivative thereof, wherein one or more cysteine ​​residues are replaced with a reactive group selected from 3-mercaptopropionic acid, cysteamine and penicillamine.

9. The peptide complex according to claim 8, wherein the first peptide ligand comprises a polypeptide selected from a modified derivative of one of the following peptides, One or more cysteine ​​residues are replaced with a reactive group selected from 3-mercaptopropionic acid, cysteamine and penicillamine.

10. The peptide complex according to claim 8, wherein the first peptide ligand is a peptide ligand that binds to adhesion protein-4; Wherein the first peptide ligand comprises a polypeptide selected from the group consisting of or a modified derivative thereof, wherein one or more cysteine ​​residues are replaced with a reactive group selected from 3-mercaptopropionic acid, cysteamine and penicillamine. The peptide complex according to claim 8 , wherein the first peptide ligand is C-terminally amidated.

12. The peptide complex of claim 8, wherein the first peptide ligand comprises an N-terminal spacer.

13. The peptide complex according to claim 8, wherein the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

14. The peptide complex according to claim 8, wherein the pharmaceutically acceptable salt is selected from free acid or sodium, potassium, calcium, ammonium salt.

15. Use of the peptide complex according to any one of claims 1 to 14 for the preparation of a medicament for preventing, inhibiting or treating cancer in a subject in need thereof.

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