Peptides targeting DLL3 and constructs thereof

Abstract

The present disclosure relates to targeting moieties, such as peptides and antibodies, that can bind to DLL3. The disclosure also provides targeting constructs, which may include a targeting moiety attached via an optional linker to a chelating agent for associating a load. Methods of making these constructs and formulations thereof are also provided. Also described are methods of treating a subject, e.g., treating or preventing cancer, using these constructs and / or formulations thereof.

Description

Related Applications

[0001] This application is related to U.S. Provisional Application No. 63 / 508,202, filed June 14, 2023, and U.S. Provisional Application No. 63 / 557,140, filed February 23, 2024, the entire contents of which are incorporated by reference in their entirety. The contents of each application are hereby incorporated by reference in their entirety. BACKGROUND

[0002] Radiation therapy, or radiotherapy, is a cancer treatment method that uses high doses of radiation to kill cancer cells and shrink tumors. In recent years, targeted radionuclide therapy for cancer using radiolabeled peptides has been developed as an alternative to external beam therapy. These peptides typically bind to receptors that are overexpressed by cancer cells. Despite these advances, new targeted radionuclide therapies are needed.

[0003] Delta-like ligand 3 (DLL3), a member of the Notch signaling system, is a potential target for radionuclide therapy. This evolutionarily conserved system regulates cell fate via cell-cell interactions. During embryonic development, DLL3 is highly expressed and transported to the cell membrane. Once development is complete, DLL3 expression is downregulated and restricted to the interior of the cell, typically the Golgi apparatus. However, DLL3 has been found to be highly expressed and localized to the cell membrane in multiple forms of cancer (Xiu et al., Onco. Targets Ther. (2020), 13:3881-3901).

[0004] In addition to being a biomarker, DLL3 plays a role in the modulation of cancer behavior. A study of small cell lung cancer (SCLC) showed that upregulation of DLL3 expression decreased tumor sensitivity to chemotherapy. Additionally, proliferation and migration of SCLC cells were inhibited and epithelial-mesenchymal transition (EMT) was reversed by blocking DLL3 (Huang et al., Biochem. Biophys. Res. Commun. (2019) 514(3):853-860). Carcinogenic behavior of DLL3 has also been demonstrated in pancreatic cancer, melanoma, and gastric cancer (Mullendore et al., Clin. Cancer. Res. (2009) 15(7):2291-301; Ding et al., Life Sci. (2019) 226:149-155; Hu et al., South China University of Hong Kong Journal (2018) 38(1):14-19).

[0005] Taken together, these findings suggest that DLL3 plays a key role in the regulation of oncogenic pathways and is specifically upregulated in cancer cells; therefore, the development of therapeutic approaches targeting DLL3 is useful in the clinical treatment of cancer. SUMMARY

[0006] The present disclosure relates to targeting moieties, such as peptides, proteins, and antibodies, that can bind to Delta-like canonical Notch ligand 3 (DLL3). The present disclosure also provides targeting constructs that can include a targeting moiety attached via an optional linker to a chelating agent for associating a payload. The chelating agent can be associated with a payload, such as, for example, a radionuclide or a cytotoxic agent. In particular aspects, the targeting construct comprises a targeting moiety that is a cyclic peptide targeting DLL3 attached via an optional linker to a chelating agent for associating a radioisotope or a radionuclide.

[0007] Accordingly, provided herein are cyclic peptides targeting DLL3. Thus, these peptides are useful for treating a variety of indications, including cancer.

[0008] In an aspect, provided herein is a cyclic peptide comprising an amino acid sequence having Formula A:

[0009]

[0010] (A),

[0011] or a pharmaceutically acceptable salt thereof, wherein the cyclic peptide is cyclized between Y1and Y2via a linker, wherein the remaining variables are as defined herein, wherein a chelator is optionally linked to the cyclic peptide via a linking group; and wherein the cyclic peptide binds to DLL3. The chelator can be labeled with a radionuclide.

[0012] In another aspect, provided herein is a cyclic peptide having Formula I:

[0013]

[0014] (I),

[0015] or a pharmaceutically acceptable salt thereof, wherein the variables are as defined herein.

[0016] In embodiments, the cyclic peptide having Formula I is attached via an optional linker to a chelating agent for associating a radionuclide.

[0017] In another embodiment, the cyclic peptide having Formula I is selected from the cyclic peptides in Table A. In yet another embodiment, the cyclic peptide having Formula I is selected from the cyclic peptides in Table B.

[0018] In another embodiment, the cyclic peptide of Formula I is selected from the cyclic peptides in Table A, or a pharmaceutically acceptable salt and / or solvate thereof. In yet another embodiment, the cyclic peptide of Formula I is selected from the cyclic peptides in Table B, or a pharmaceutically acceptable salt and / or solvate thereof.

[0019] In yet another aspect, provided herein is a cyclic peptide of Formula B:

[0020]

[0021] (B),

[0022] or a pharmaceutically acceptable salt thereof, wherein the variables are as defined herein.

[0023] In embodiments, the cyclic peptide of Formula B is attached via an optional linker to a chelating agent for associating a radioisotope.

[0024] In another embodiment, the cyclic peptide of Formula B is selected from the cyclic peptides in Table A. In yet another embodiment, the cyclic peptide of Formula B is selected from the cyclic peptides in Table B.

[0025] In another embodiment, the cyclic peptide of Formula B is selected from the cyclic peptides in Table A, or a pharmaceutically acceptable salt and / or solvate thereof. In yet another embodiment, the cyclic peptide of Formula B is selected from the cyclic peptides in Table B, or a pharmaceutically acceptable salt and / or solvate thereof.

[0026] In still another embodiment, the chelating agent is selected from the chelating agents in Table C. In some embodiments, the chelating agent is labeled with a radionuclide. In some embodiments, the radionuclide is selected from the group consisting of: 111 In、 99m Tc、 94m Tc、 66 Ga、 67 Ga、 68 Ga、 52 Fe、 169 Er、 72 As、 97 Ru、 203 Pb、 61 Cu、 62 Cu、 64 Cu、 67 Cu、 89 Sr、 186 Re、 188 Re、 86 Y、 90 Y、 89 Zr、 51 Cr、 52 Mn、 51 Mn、 177 Lu、169 Yb, 175 Yb, 105 Rh, 166 Dy, 166 Dy, 166 Ho, 153 Sm, 149 Pm, 151 Pm, 172 Tm, 121 Sn, 117m Sn, 212 Bi, 213 Bi, 142 Pr, 143 Pr, 198 Au, 199 Au, 123 I, 124 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, 80 Br, 82 Br, 18 F, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 211 At, 223 Ra, 227 Th, 226 Th, 82 Rb, 32 P, 76 As, 89 Zr, 111 Ag, 165 Er, 225 Ac, and 227 Ac. In some embodiments, the radionuclide is 111 In, 99m Tc, 67 Ga, 68 Ga, 203 Pb, 64 Cu, 86 Y, 89 Zr, 123 I, 124 I, 125 I, 18 F, 76 Br, 77 Br,152 Tb, 155 Tb, 44 Sc, 43 Sc, 67 Cu, 188 Re, 90 Y, 177 Lu, 213 Bi, 131 I, 47 Sc, 225 Ac, 212 Pb, 211 At or 227 Th.

[0027] In embodiments, the radioisotope is selected from the radioisotopes in Table 3.

[0028] In another aspect, provided herein is a pharmaceutical composition comprising a cyclic peptide described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0029] In yet another aspect, provided herein is a method of targeting DLL3 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein.

[0030] In still another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein. The cancer can comprise at least one cell comprising DLL3. The cancer can be urothelial carcinoma, melanoma, or squamous cell carcinoma. Additionally, the cancer can be a neuroendocrine tumor, melanoma, or a primary brain cancer. In some embodiments, the neuroendocrine tumor is selected from small cell lung cancer (SCLC), medullary thyroid cancer (MTC), large cell neuroendocrine carcinoma (LCNEC), gastroenteropancreatic neuroendocrine carcinoma (GEP NEC), neuroendocrine prostate cancer (NEPC), small cell prostate cancer (SCPC), Merkel cell carcinoma (MCC), cervical neuroendocrine carcinoma, G3 grade neuroendocrine tumor (NET), and cervical extrapulmonary neuroendocrine carcinoma (NEC). In some embodiments, the cancer can be a solid tumor with DLL3 positivity (e.g., > 1% DLL3 positive cells) as measured by immunohistochemistry (IHC).

[0031] In one aspect, provided herein is a peptide having binding specificity for DLL3, wherein the peptide binds to one or more of amino acids A81, L83, G106, A85, and R61 of the DLL3 amino acid sequence of SEQ ID NO: 1. In embodiments, the peptide binds to amino acids A81, L83, G106, A85, and R61 of the DLL3 amino acid sequence of SEQ ID NO: 1. In another embodiment, the peptide binds to backbone atoms of amino acids A81, L83, G106, and A85 of the DLL3 amino acid sequence of SEQ ID NO: 1. In yet another embodiment, the peptide binds to a side chain atom of amino acid R61 of the DLL3 amino acid sequence of SEQ ID NO: 1. In still another embodiment, the peptide binds to backbone atoms of amino acids A81, L83, G106, and A85 of the DLL3 amino acid sequence of SEQ ID NO: 1 and to a side chain atom of amino acid R61 of the DLL3 amino acid sequence of SEQ ID NO: 1. In embodiments, the peptide comprises the amino acid sequence of WTACANAKDCWP or a derivative thereof comprising one or more non-natural amino acids. In another embodiment, amino acids W1, A3, A7, and W11 bind to DLL3. In yet another embodiment, the peptide is cyclic. In some embodiments, the disclosure provides a construct or a pharmaceutically acceptable salt thereof, the construct comprising a targeting moiety attached via an optional linker to at least one chelating agent for associating a payload, wherein the targeting moiety binds to a cellular antigen, wherein the cellular antigen comprises DLL3. The payload can be a payload, such as, for example, a radionuclide or a cytotoxic agent.

[0032] In some embodiments, the disclosure provides a construct or pharmaceutically acceptable salt thereof, the construct comprising a targeting moiety attached via an optional linker to at least one chelating agent for associating a payload, wherein the targeting moiety binds to DLL3. The chelating agent can comprise a polyaminocarboxylate agent. The chelating agent can comprise ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"' -tetraacetic acid (DOTA), DOTAGA, or a derivative thereof. The chelating agent can comprise EDTA, DTPA, DOTA, DOTAGA, or a derivative thereof. The chelating agent can comprise a macrocyclic agent. The chelating agent can comprise 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"' -tetraacetic acid (TETA), 1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N""- pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N",N"',N"",N""'- hexaacetic acid (HEHA), or a derivative thereof. The chelating agent can also comprise deferoxamine (DFO), 5,11,16,22-tetraazadocosanediamide (DFO ) or N,N'-1,4-butanediylbis[N-[3-[[(1,6-dihydro-1-hydroxy-6-oxo-2-pyridinyl)carbonyl]amino]propyl]-1,6-dihydro-1-hydroxy-6-oxo-2-pyridinecarboxamide] (HOPO) or a derivative thereof.

[0033] The payload can comprise a radiopharmaceutical. The radiopharmaceutical can comprise a radioisotope. Accordingly, in some embodiments, the constructs or compounds disclosed herein optionally comprise a radioisotope. In some embodiments, the constructs or compounds disclosed herein comprise a radioisotope. The radioisotope can be a radionuclide. The radioisotope can be any of those listed in Table 3. In some embodiments, the radionuclide is selected from the group consisting of: 111 In、 99m Tc、 94m Tc、 66 Ga、 67 Ga、 68 Ga、 52 Fe、 169 Er、 72 As、 97 Ru、 203 Pb、 61 Cu、 62 Cu、 64 Cu、 67 Cu、89 Sr, 186 Re, 188 Re, 86 Y, 90 Y, 89 Zr, 51 Cr, 52 Mn, 51 Mn, 177 Lu, 169 Yb, 175 Yb, 105 Rh, 166 Dy, 166 Dy, 166 Ho, 153 Sm, 149 Pm, 151 Pm, 172 Tm, 121 Sn, 117m Sn, 212 Bi, 213 Bi, 142 Pr, 143 Pr, 198 Au, 199 Au, 123 I, 124 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, 80 Br, 82 Br, 18 F, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 43 Sc, 44 Sc, 47 Sc, 212 Pb, 211 At, 223 Ra, 227 Th, 226 Th, 82 Rb, 32 P, 76 As, 89 Zr, 111 Ag, 165 Er, 225 Ac and 227 Ac. In some embodiments, the radionuclide is 111 In, 99m Tc, 67 Ga, 68 Ga,203 Pb, 64 Cu, 86 Y, 89 Zr, 123 I, 124 I, 125 I, 18 F, 76 Br, 77 Br, 152 Tb, 155 Tb, 44 Sc, 43 Sc, 67 Cu, 188 Re, 90 Y, 177 Lu, 213 Bi, 131 I, 47 Sc, 225 Ac, 212 Pb, 211 At or 227 Th. The optional linker can comprise a cleavable linker. The optional linker can comprise a non-cleavable linker. The optional linker can comprise at least one amino acid. In some embodiments, the present disclosure provides a pharmaceutical composition comprising the construct and a pharmaceutically acceptable excipient.

[0034] In some embodiments, the present disclosure provides a method of delivering a payload to a cell, the method comprising contacting the cell or a subject comprising the cell with the construct or a pharmaceutical composition thereof. In embodiments, the payload can be a radiopharmaceutical, e.g., a radionuclide and / or a radioisotope. In other embodiments, the payload is a cytotoxic agent.

[0035] In some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, comprising administering a construct or a pharmaceutical composition thereof. In some embodiments, the disclosure provides a method of treating a subject, comprising administering a construct or a pharmaceutical composition thereof. In some embodiments, the disease or disorder is cancer (i.e., the subject has cancer). The cancer can comprise at least one cell comprising DLL3. In embodiments, the cancer expresses DLL3. The cancer can be urothelial carcinoma, melanoma, or squamous cell carcinoma. Additionally, the cancer can be a neuroendocrine tumor, melanoma, or a primary brain cancer. In some embodiments, the neuroendocrine tumor is selected from small cell lung cancer (SCLC), medullary thyroid cancer (MTC), large cell neuroendocrine carcinoma (LCNEC), gastroenteropancreatic neuroendocrine carcinoma (GEP NEC), neuroendocrine prostate cancer (NEPC), small cell prostate cancer (SCPC), Merkel cell carcinoma (MCC), cervical neuroendocrine carcinoma, G3 grade neuroendocrine tumor (NET), and cervical extrapulmonary neuroendocrine carcinoma (NEC). In some embodiments, the cancer can be a solid tumor with DLL3 positivity (by immunohistochemistry (IHC), e.g., > 1% DLL3 positive cells). DETAILED DESCRIPTION

[0036] DLL3 binds to the highly conserved notch receptor family members to regulate embryonic development. In contrast to the canonical notch ligands, DLL3 inhibits Notch signaling through interaction with the Golgi apparatus. Reflecting this function, DLL3 is normally restricted to the cytoplasm (Geffers, I. et al. J. Cell Biol. (2007) 178(3), 465-76; Zhou, B. et al. Signal Transduct. Target Ther. (2022) 7(1), 95). DLL3 is detected in the cytoplasm of healthy fetal tissues and its absence leads to severe vertebral defects in the form of autosomal recessive spondylorthoepiphyseal dysplasia (Serth, K. et al. PLoS One (2015) 10(4), e0123776; Dunwoodie, S. L. et al. Development (2002) 129(7), 1795-806). Low levels of DLL3 as an RNA transcript are detectable in the adult brain, pituitary, and testis (Sharma, S. K. et al. Cancer Res. (2017) 77(14), 3931-41). DLL3 mRNA is also detectable in the cytoplasm of the adult brain, pituitary, basophils, and pancreas (Giffin, M. J. et al. Clin. Cancer Res (2021) 27(5), 1526-37). However, significant overexpression of DLL3 leads to its aberrant localization to the cell surface (Zhou, B. et al. Signal Transduct. Target Ther. (2022) 7(1), 95; Geffers, I. et al. (2007)). Upregulated DLL3 is also observed to aberrantly localize to the cell surface in cancer (Giffin, M. J. et al. (2021); Saunders, L. R. et al. Sci. Transl. Med. (2015) 7(302), 302ra136; Sharma, S. K. et al. (2017)).

[0037] Aberrant DLL3 expression, including cell surface expression, is observed in a variety of human tumors (Saunders, L. R. et al. (2015)). Neuroendocrine neoplasms (NENs), including well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs), frequently express DLL3 on the cell surface and share common histological and transcriptomic markers of neuroendocrine lineage and transformation (Puca, L., et al. Sci. Transl. Med. (2019) 11(484); Yao, J. et al. Oncologist (2022) 27(11), 940-51). Although gain-of-function and loss-of-function experiments suggest that DLL3 can affect cell proliferation, migration, and tumor growth in vitro and in vivo, the pathophysiological role of DLL3 mislocalization on tumor cell function is poorly understood (Furuta, M. et al. Cancer Sci. (2019) 110(5), 1599-608; Huang, J. et al. (2019)). DLL3 is detectable on the tumor cell surface by immunohistochemistry (IHC), flow cytometry, and is accessible to exogenous DLL3-targeting antibodies in vivo (Dylla, S.J. Mol. Cell Oncol. (2016) 3(2), e1101515; Saunders, L. R. et al. (2015)).

[0038] DLL3 has been quantified to be in low single-digit thousands of copies per cell in representative small cell lung cancer (SCLC) and neuroendocrine prostate cancer (NEPC) cell lines (Giffin, M. J. et al. (2021); Zhang, Y. et al. Clin. Cancer Res. (2023) 29(5), 971-85). Despite the low copy number levels on a per cell basis, the feasibility of targeting DLL3 has been preclinically demonstrated in SCLC as well as neuroendocrine cancer xenograft models with a 89Zr / 177Lu radioconjugate targeting DLL3, a bispecific antibody targeting DLL3, and an antibody drug conjugate targeting DLL3 (Chou, J. et al. Cancer Res. (2023) 83(2), 301-15; Giffin, M. J. et al. (2021); Korsen, J. A. et al. Proc. Natl. Acad. Sci. USA, (2022) 119(27), e2203820119; Saunders, L. R. et al. (2015)). Furthermore, despite the low abundance of the tumor associated antigen, it was demonstrated that tumors expressing DLL3 could be visualized by 89Zr immunoPET imaging in several SCLC mouse models (Sharma, S. K. et al. (2017)).

[0039] Therapies targeting DLL3, including the antibody drug conjugate RovaT targeting DLL3, have demonstrated clinical efficacy as a target (Morgensztern, D. et al. Clin Cancer Res, (2019) 25(23), 6958-66; Rudin, C. M. et al. Nat Rev Dis Primers (2021) 7(1), 3), the potential of which was limited by payload related toxicities. Recently, the bispecific antibody targeting DLL3, tarlatamab (AMG757), produced durable responses up to 12 months in a quarter of treated patients (Paz-Ares, L. et al. J. Clin. Oncol. (2023) 41(16), 2893-903). Given the well-studied nature of DLL3 and its selective expression on the surface of cancer cells in both primary and metastatic solid tumors, it is an extremely attractive target for new therapies in NEN and other DLL3 expressing solid tumors.

[0040] Provided herein are compounds targeting DLL3. In particular, provided herein are targeting constructs (also referred to herein as "compounds") comprising a targeting moiety that is a cyclic peptide targeting DLL3, attached via an optional linker to a chelating agent for association of a radioisotope. Accordingly, these compounds, and pharmaceutical compositions comprising these compounds, are useful for treating a variety of indications, including cancer.

[0041] I. Compounds and compositions

[0042] In some embodiments, the present disclosure relates to targeting moieties, such as peptides, proteins, and antibodies, that can bind to a target. In some embodiments, the present disclosure provides constructs capable of localizing to and / or associating with a target. Such constructs, comprising any combination of a targeting moiety and a payload, are referred to herein as "targeting constructs." As used herein, the term "targeting moiety" refers to a component of a targeting construct or a combination of components that participate in the localization of a targeting construct to a target or the association of a targeting construct with a target. The payload component of a targeting construct can include any of a variety of compounds, including but not limited to chemical compounds, biological molecules, metals, polymeric molecules, therapeutic agents, cytotoxic agents, and radiopharmaceutical agents. Chelators can associate with a payload, such as a radionuclide or a cytotoxic agent.

[0043] In particular, provided herein are targeting constructs (also referred to herein as "compounds") comprising a targeting moiety that is a cyclic peptide targeting DLL3, attached via an optional linker to a chelating agent for association of a radioisotope.

[0044] Target

[0045] Targeting constructs can be directed to a variety of targets. In particular embodiments, a targeting construct comprises a peptide directed to DLL3 and further comprises a radioisotope. In some embodiments, a targeting construct can target a cell. Such targeting constructs can include a targeting moiety that can target a cell antigen, including those associated with the surface of a target cell. In this case, the cell antigen is the target of the targeting moiety and the targeting construct. An "antigen" as referred to herein is any entity that induces an immune response of an organism, or can simply refer to an antibody binding partner. An immune response is a reaction of cells, tissues, and / or organs of an organism to a foreign entity. An immune response typically results in the production of one or more antibodies by an organism against the foreign entity. As used herein, the term "target antigen" refers to an entity, protein, or epitope to which an antibody binds, or to which an antibody is intended, designed, or developed to have affinity. Such target antigens can include cancer cell antigens, such as those expressed on the surface of a cancer cell.

[0046] In some embodiments, the target antigen disclosed herein includes DLL3 or a portion thereof. A DLL3 antigen may include a DLL3 extracellular domain. A DLL3 antigen may include a DLL3 fusion protein or other entity containing a portion of DLL3.

[0047] DLL3

[0048] Delta-like classical Notch ligand 3 (also known as Drosophila delta-like protein 3, or DLL3 for short) is a member of the delta protein ligand family. It is encoded by the DLL3 gene. DLL3 inhibits primary neurogenesis and is involved in guiding neuronal differentiation along specific pathways. It also plays a role in somnolence formation during paraaxial mesodermal segmentation. Mutations in the DLL3 gene result in the autosomal recessive genetic disorder Yaho-Levin syndrome. The DLL3 gene is expressed in neuroendocrine tumors. DLL3 may be a potential target for the treatment of tumors such as lung cancer.

[0049] In some embodiments, the targeting construct includes a targeting portion that is specific to one or more DLL3 domains. In some embodiments, the targeting construct includes a targeting portion that is specific to one or more DLL3 target amino acid sequences or fragments or variants thereof listed in Table 1.

[0050] Table 1. DLL3 protein target sequences

[0051]

[0052] Targeting moieties

[0053] In some embodiments, the targeting portion localizes the targeting construct to such a target by binding to a target or its associated components. The targeting portion may bind to cells or biomolecules or other cell-associated structures. For example, in some embodiments, the targeting portion binds to cellular antigens. Such cellular antigens may be expressed specifically by, on, or otherwise associated with a particular cell type. A particular cell type can be characterized by one or more of cell size, stage, shape, location, tissue origin, organ origin, function, activity, genotype, phenotype, or association with dysfunction or disease. The targeting portion may bind to cancer cell antigens. In some embodiments, the targeting portion binds to DLL3. In some embodiments, the targeting portion binds to human DLL3.

[0054] The targeting portion may include, or be composed of, proteins, peptides, antibodies, nucleic acids, nucleic acid analogs, aptamers, lipids, carbohydrates, glycoproteins, or small molecules. In some embodiments, the targeting portion includes, or is composed of, polypeptides or peptides, antibodies, fragments or variants thereof. In a particular embodiment, the targeting portion is a cyclic peptide.

[0055] In some embodiments, the targeting portions of this disclosure (e.g., peptides and antibodies) have an affinity for human DLL3. In some embodiments, the targeting portions of this disclosure have an affinity for human DLL3 within a defined range as measured in conventional assays. "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or peptide-binding compound) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the total interaction between members of the binding pair (e.g., an antibody or peptide-binding compound and an antigen). The affinity of molecule X for its partner Y can generally be expressed using the dissociation constant (K0). D The equilibrium dissociation constant (K) is represented by [the symbol]. D ) is calculated as ratio k off / k on .

[0056] Low-affinity targeting moieties typically bind antigens slowly and tend to dissociate easily, while high-affinity targeting moieties typically bind antigens more quickly and tend to maintain the binding for a longer period. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure.

[0057] In some embodiments, the target portion disclosed herein may have the following equilibrium dissociation constant (K). D The target protein can bind to amounts ranging from about 0.001 nM to about 0.01 nM, from about 0.005 nM to about 0.05 nM, from about 0.01 nM to about 0.1 nM, from about 0.05 nM to about 0.5 nM, from about 0.1 nM to about 1.0 nM, from about 0.5 nM to about 5.0 nM, from about 2 nM to about 10 nM, from about 8 nM to about 20 nM, from about 15 nM to about 45 nM, from about 30 nM to about 60 nM, from about 40 nM to about 80 nM, from about 50 nM to about 100 nM, from about 75 nM to about 150 nM, from about 100 nM to about 500 nM, from about 200 nM to about 800 nM, from about 400 nM to about 1,000 nM, or at least 1,000 nM. In some embodiments, the target protein is DLL3.

[0058] In some embodiments, the target portion disclosed herein may have the following equilibrium dissociation constant (K). D The target protein can be bound to: about 1 nM or less, about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 300 nM. In some embodiments, the target protein is DLL3.

[0059] In some embodiments, K D The determination is made by surface plasmon resonance (SPR). Example 3 provides an exemplary SPR scheme.

[0060] Polypeptides and peptides

[0061] In some embodiments, the target portion of this disclosure is a polypeptide. According to this disclosure, any amino acid-based molecule (natural or non-natural) may be called a "polypeptide," and this term includes "peptide," "peptide mimic," and "protein." A "peptide" is generally considered to be in the size range of about 4 to about 50 amino acids. Peptides with more than about 50 amino acids are generally referred to as "proteins."

[0062] The peptides disclosed herein can be linear or cyclic. In particular, this document provides cyclic peptides targeting DLL3. Cyclic peptides include any peptide having one or more cyclic features (e.g., rings and / or internal bonds) in its structural moiety. In some embodiments, cyclic peptides are formed when a molecule acts as a bridging portion connecting two or more regions of a peptide.

[0063] As used herein, the term "bridging portion" refers to one or more components of a bridge formed between two adjacent or non-adjacent amino acids, non-natural amino acids, or non-amino acids in a peptide. A bridging portion can be of any size or composition. In some embodiments, a bridging portion may comprise one or more chemical bonds between two adjacent or non-adjacent amino acids, non-natural amino acids, non-amino acid residues, or combinations thereof. In some embodiments, such chemical bonds may be between one or more functional groups on adjacent or non-adjacent amino acids, non-natural amino acids, non-amino acid residues, or combinations thereof. A bridging portion may comprise one or more of amide bonds (lactams), disulfide bonds, thioether bonds, aromatic rings, triazole rings, and hydrocarbon chains. In some embodiments, a bridging portion comprises an amide bond between an amine functional group and a carboxylate functional group, respectively, present in the side chain of an amino acid, non-natural amino acid, or non-amino acid residue. In some embodiments, the amine or carboxylate functional group is part of a non-amino acid residue or a non-natural amino acid residue.

[0064] In some embodiments, this disclosure provides peptides that bind to human DLL3. Antibodies disclosed herein can bind to the extracellular domain of human DLL3. Antibodies disclosed herein can bind to DLL3 associated with cells (e.g., cell surfaces). Such cells may include, but are not limited to, cancer cells, such as, but not limited to, lung cancer cells, breast cancer cells, bladder cancer cells, colon cancer cells, urothelial carcinoma cells, melanoma cells, or squamous cell carcinoma cells.

[0065] DLL3 epitopes

[0066] The peptide disclosed herein binds to specific amino acids in DLL3, namely DLL3 epitopes. As used herein, the term "epitaph" refers to one or more specific portions of a target (e.g., DLL3) that interact (e.g., bind) with a binding entity (e.g., the peptide disclosed herein).

[0067] In some embodiments, the peptide has binding specificity to DLL3, wherein the peptide binds to one or more amino acids of A81, L83, G106, A85 and R61 of the DLL3 amino acid sequence of SEQ ID NO: 1.

[0068] In some embodiments, the peptide binds to amino acids A81, L83, G106, A85, and R61 of the DLL3 amino acid sequence of SEQ ID NO: 1.

[0069] In some embodiments, the peptide is bound to the backbone atoms of amino acids A81, L83, G106, and A85 of the DLL3 amino acid sequence of SEQ ID NO: 1. As used herein, the term "backbone atom" refers to an atom in the peptide backbone, which consists of a central carbon atom (i.e., α-carbon), a hydrogen atom bonded to the central carbon atom, an amino group (NH2), and a carboxyl group (COOH).

[0070] In some embodiments, the peptide is bound to the side chain atom of amino acid R61 of the DLL3 amino acid sequence of SEQ ID NO: 1. As used herein, the term "side chain atom" refers to an atom on the side chain of the peptide, and is also commonly referred to as the R group.

[0071] In some embodiments, the peptide binds to the main chain atoms of amino acids A81, L83, G106, and A85 of the DLL3 amino acid sequence of SEQ ID NO: 1, and to the side chain atoms of amino acid R61 of the DLL3 amino acid sequence of SEQ ID NO: 1.

[0072] In another embodiment, the peptide is able to [distribute at approximately 1 x 10] -8 M to approximately 1 x 10 -12 M's EC 50 The value binds to DLL3. In another embodiment, the peptide is able to bind at approximately 1 x 103 -8 M to approximately 1 x 10 -10 M's EC 50 The value is associated with DLL3.

[0073] In some embodiments, EC 50 The values ​​are determined in an enzyme-linked immunosorbent assay (ELISA). In some embodiments, a DLL3 protein concentration of about 1 µg / mL to about 5 µg / mL is used in the ELISA.

[0074] In some embodiments, the peptide comprises the amino acid sequence WTACANAKDCWP or a derivative thereof comprising one or more non-natural amino acids. In another embodiment, amino acids W1, A3, A7, and W11 are bound to DLL3. In yet another embodiment, the peptide is cyclic. In still another embodiment, the peptide has any of the formulas described herein or any of the types described herein.

[0075] Cyclic peptides

[0076] In some embodiments, the peptides disclosed herein may comprise cyclic peptides having one or more bridging moieties (e.g., cyclic structures, staples, bridges, etc.). Peptide stapling / bridging is a macrocyclic strategy that covalently modifies a peptide by forming chemical bonds (e.g., staples, bridging moieties, etc.) between the side chains of two amino acids. More specifically, a macrocyclic structure is formed by forming covalent bonds between atoms present in the linear peptide and atoms of the bridging moieties. Stapling / bridging can be used to restrict peptides to a preferred bioactive conformation (reducing conformational flexibility and rotational degrees of freedom), thereby improving affinity for specific receptor targets and improving overall pharmacokinetics. The linked residues are typically located on the same side of the peptide helix and spaced one, two, or three helical turns apart (e.g., the first amino acid at position (z) is linked to a second amino acid at position z+4, z+7, or z+11). In some embodiments, the bridging moieties may comprise one or more chemical bonds between two adjacent or non-adjacent amino acids, non-natural amino acids, non-amino acid residues, or combinations thereof. In some embodiments, such chemical bonds may be between one or more functional groups on adjacent or non-adjacent amino acids, non-natural amino acids, non-amino acid residues, or combinations thereof.

[0077] Accordingly, in one respect, this paper provides a cyclic peptide comprising an amino acid sequence having formula A:

[0078]

[0079] (A)

[0080] Or its pharmaceutically acceptable salt.

[0081] in:

[0082] X0 is any natural or non-natural amino acid, or X0 is not present.

[0083] X1 is selected from Trp, 7aza-Trp, 1Me-Trp, 5OH-Trp, 5OMe-Trp, 7OMe-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, α-Me-Trp and NMe-Trp;

[0084] X2 and X3 are each, independently, any natural or non-natural amino acid;

[0085] Y1 is Cys;

[0086] X4, X5, X6, X7, and X8 are each, independently, any natural or non-natural amino acid;

[0087] Y2 is Cys;

[0088] X9 is selected from Trp, 7aza-Trp, 1Me-Trp, 5OH-Trp, 5OMe-Trp, 7OMe-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, α-Me-Trp and NMe-Trp;

[0089] X 10 Selected from Pro, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, 5,5-diMe-Pro, trans-4NH2-Pro and cis-4NH2-Pro;

[0090] P 2’ Selected from -L 2 - Chelating agents or

[0091] ;

[0092] D 2 It is OH or NH2;

[0093] L 2 Not found or selected from:

[0094] , , ,

[0095] , , , and ;

[0096] Where L 2 The amino group is attached to P 2 Or the carbonyl group of the chelating agent to form an amide bond;

[0097] P 3 Selected from -L3 - Chelating agents,

[0098] , , , and ;

[0099] L 3 Not present or independently selected

[0100] , , , ,and ;

[0101] Where L 3 The carbonyl group is attached to P 2 The amine groups form amide bonds;

[0102] D 3 Independently, it is a -NR"- chelating agent or

[0103] ;

[0104] Each R' is independently selected from H, C(O)OH, (CH2)OH, and NHAc;

[0105] Each "R" is independently selected from H and CH3;

[0106] X is H or a halogen;

[0107] Each n is an independent integer from 0 to 16;

[0108] Each p is an independent integer from 0 to 24;

[0109] Each s is an independent integer from 0 to 16;

[0110] Each t is independently 1, 2, 3, 4, 5, or 6; and

[0111] w is selected from 1, 2, or 3;

[0112] The cyclic peptide is cyclized via a linker between Y1 and Y2; and

[0113] The cyclic peptide binds to DLL3.

[0114] In the embodiment, X2 is Thr and X8 is Asp.

[0115] In another embodiment, the cyclic peptide having formula A is a cyclic peptide comprising an amino acid sequence having formula Ai:

[0116]

[0117] (Ai)

[0118] Or its pharmaceutically acceptable salt.

[0119] In yet another embodiment, the cyclic peptide having formula A is a cyclic peptide comprising an amino acid sequence having formula Aii:

[0120]

[0121] (Aii)

[0122] Or its pharmaceutically acceptable salt.

[0123] In yet another embodiment, the cyclic peptide having formula A is a cyclic peptide comprising an amino acid sequence having formula Aiii:

[0124]

[0125] (Aiii)

[0126] Or its pharmaceutically acceptable salt.

[0127] In another embodiment, the N-terminus of the peptide is capped. In yet another embodiment, the N-terminus of the peptide adjacent to X0 is p-capped. 1 Add a cap, where P 1 Selected from H,

[0128] , , and ;

[0129] D 1 Selected from H, CH3 and C(O)OH;

[0130] L 1 Not present or selected

[0131] , ,

[0132] , and ;

[0133] Where L 1 The amino group is attached to P 1 The carbonyl group forms an amide bond;

[0134] n and q are each independent integers from 0 to 16;

[0135] Each p is an independent integer from 0 to 24;

[0136] Each s is an independent integer from 0 to 16;

[0137] Each t is independently 0, 1, 2, 3, 4, 5, or 6;

[0138] Each R' is independently selected from H, C(O)OH, (CH2)OH, and NHAc; and

[0139] Each "R" is independently selected from H and CH3.

[0140] In another embodiment of the above aspects, X0 is absent or X0 is selected from Gly, Met, D-Ala, Ala, Nle, and Nva. In yet another embodiment, X0 is absent.

[0141] In the embodiments described above, X1 is selected from Trp or 7Me-Trp. In another embodiment, X1 is Trp. If X0 does not exist, X1 can be selected from P. 1 The N-terminal group is substituted.

[0142] In the embodiments described above, X2 is selected from Thr, D-Ala, Ala, α-Me-Thr, Lys, and NMe-Thr. In another embodiment, X2 is selected from the group consisting of Thr, α-Me-Thr, and NMe-Thr. In yet another embodiment, X2 is Thr.

[0143] In another embodiment of the above aspects, X3 is selected from Ile, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, 1Nal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe-Nle, α-tert-pentyl Gly, Allo-Ile, Lys(C12), Lys(C14), Lys(C16), α-Me-Ile, and NMe-tBuAla. In an embodiment, X3 is selected from the group consisting of Ile, Allo-Ile, α-Me-Ile, and NMe-tBuAla. In another embodiment, X3 is selected from the group consisting of D-Ala, Ala, t-Bu-Ala, and NMe-tBuAla. In yet another embodiment, X3 is Ile or NMe-tBuAla. In yet another embodiment, X3 is Ile. In the embodiment, X3 is NMe-tBuAla.

[0144] In another embodiment of the above aspects, the connector between Y1 and Y2 is selected from a key, C 1-6 Alkylene and

[0145] .

[0146] In another embodiment of the above aspects, X4 is selected from Asn, D-Ala, Ala, DAB-4-NHCOC5H 11 DAB-4-NHCOC7H 15 Asp, Ser, Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, 3Pya, 4Pya, Glu, and NMe-Asn. In another embodiment of the above aspects, X4 is selected from Asn, 3-(4-piperidinyl)-Ala, and Pip(CH2CO2H)Ala. In an embodiment, X4 is 3-(4-piperidinyl)-Ala or Pip(CH2CO2H)Ala. In another embodiment, X4 is Pip(CH2CO2X)Ala, wherein X is a pharmaceutically acceptable cation (e.g., to form a pharmaceutically acceptable salt).

[0147] In another embodiment of the above aspects, X5 is selected from Asn, Ala, D-Ala, Trp, Asp, Lys, 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser. In another embodiment of the above aspects, X5 is Asn or NMe-Asn. In yet another embodiment, X5 is Asn. In still another embodiment, X5 is NMe-Asn.

[0148] In the embodiments described above, X6 is selected from Trp, 4CF3-Phe, 1Me-Trp, 7aza-Trp, BIP, 2Nal, 1Nal, aMe-Trp, D-Ala, Ala, 4F-Phe, 5F-Trp, 5Ome-Trp, Asn, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp. In another embodiment, X6 is selected from the group consisting of Trp, 1Me-Trp, 7aza-Trp, 2Nal, 1Nal, α-Me-Trp, 5F-Trp, 5MeO-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp. In yet another embodiment described above, X6 is selected from Trp, 2Nal, and 1Nal. In still another embodiment, X6 is Trp. In this embodiment, X6 is 2Nal.

[0149] In another embodiment of the above aspects, X7 is selected from 3Pya, 4Pya, Lys(Me)3, His, Ala, D-Ala, Gln, Lys, Glu, Arg, Orn, NMe-His, and Ser. In yet another embodiment, X7 is His or Lys. In still another embodiment, X7 is His. In this embodiment, X7 is Lys.

[0150] In another embodiment of the above aspects, X8 is selected from Asp, Asn, NMe-Asp, and α-Me-Asp. In yet another embodiment, X8 is Asp.

[0151] In yet another embodiment of the above aspects, X9 is Trp.

[0152] In the embodiments described above, X 10 It's Pro.

[0153] In another embodiment, P 2 Yes -L 2 - Chelating agent.

[0154] In yet another embodiment, P 2 yes

[0155] .

[0156] In yet another embodiment, P 3 Yes -L 3 - Chelating agent.

[0157] In one embodiment, P 3 yes

[0158] .

[0159] In another embodiment, the chelating agent is selected from the chelating agents in Table C.

[0160] On the other hand, this paper provides a cyclic peptide having Formula I:

[0161]

[0162] (I)

[0163] Or its pharmaceutically acceptable salt.

[0164] in:

[0165] P 1 Selected from: H,

[0166] , ,

[0167] and ;

[0168] D 1 Selected from H, CH3 and C(O)OH;

[0169] L 1 Not present or selected

[0170] , , , and

[0171] ;

[0172] Where L 1 The amino group is attached to P 1 The carbonyl group forms an amide bond;

[0173] P 2 Yes -L 2 - Chelating agents or

[0174] ;

[0175] D 2 It is OH or NH2;

[0176] L 2 Not found or selected from:

[0177] , , ,

[0178] , , , and ;

[0179] Where L 2 The amino group is attached to P 2 Or the carbonyl group of the chelating agent to form an amide bond;

[0180] P 3 Selected from -L 3 - Chelating agents,

[0181] , , ,

[0182] and ;

[0183] L 3 Not present or independently selected

[0184] , , , ,and ;

[0185] Where L 3 The carbonyl group is attached to P 2 The amine groups form amide bonds;

[0186] D 3 It is a NR-chelating agent;

[0187] R 0 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0188] R 1 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0189] R 2 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0190] R 3 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0191] B 1 It is C 1-6 Alkylene;

[0192] C 1 It is C 1-6 Alkylene;

[0193] A 1 Selected from:

[0194] , , , , , , , , and ;

[0195] Where w is selected from 1, 2, or 3;

[0196] R 4 It is the amino acid side chain of a natural amino acid or the amino acid side chain of a non-natural amino acid, both of which are optionally coated with CH2C(O)OH or C(O)(CH2CH2O). p (CH2)2N(CH3)3 + replace;

[0197] R 5 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0198] R 6 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0199] R 7 Selected from:

[0200] (i) The amino acid side chains of natural amino acids,

[0201] (ii) Amino acid side chains of non-natural amino acids, or

[0202] (iii) Choose from the following groups:

[0203] and ;

[0204] R 8 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0205] R 9 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0206] R 10 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0207] m is 0 or 1;

[0208] n, q, and u are each an independent integer from 0 to 16;

[0209] Each p is an independent integer from 0 to 24;

[0210] Each s is an independent integer from 0 to 16;

[0211] Each t is independently 1, 2, 3, 4, 5, or 6;

[0212] Each R' is independently selected from H, C(O)OH, (CH2)OH, and NHAc; and

[0213] Each "R" is independently selected from H and CH3.

[0214] In the embodiments, the nitrogen atom and α-carbon atom on the peptide backbone are optionally replaced by methyl groups.

[0215] Where the variable group R 0 R 1 R 2 R 3 R4 R 5 R 6 R 7 R 8 R 9 Or R 10 When an amino acid side chain is defined as a cyclic amino acid, the corresponding amino acid nitrogen in the peptide backbone of Formula I forms part of the cyclic group; and

[0216] Furthermore, the cyclic peptide optionally contains a radionuclide.

[0217] In the embodiments, the cyclic peptide having Formula I is a cyclic peptide having Formula I':

[0218]

[0219] (I'),

[0220] Or its pharmaceutically acceptable salt.

[0221] In another aspect, this paper provides a cyclic peptide having formula B:

[0222]

[0223] (B)

[0224] Or its pharmaceutically acceptable salt.

[0225] in

[0226] P 1 Selected from: H,

[0227] , , and ;

[0228] D 1 Selected from H, CH3 and C(O)OH;

[0229] L 1 Not present or selected

[0230] , , , and ;

[0231] Where L 1 The amino group is attached to P 1 The carbonyl group forms an amide bond;

[0232] P 2 Yes -L 2 - Chelating agents or

[0233] ;

[0234] D 2 It is OH or NH2;

[0235] L 2 Not found or selected from:

[0236] , , ,

[0237] , , , and ;

[0238] Where L 2 The amino group is attached to P 2 Or the carbonyl group of the chelating agent to form an amide bond;

[0239] P 3 Selected from -L 3 - Chelating agents,

[0240] , , , and ;

[0241] L 3 Not present or independently selected

[0242] , , , ,and ;

[0243] Where L 3 The carbonyl group is attached to P 2 The amine groups form amide bonds;

[0244] D 3 It is a NR-chelating agent;

[0245] R 0 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0246] R 1 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0247] R 2 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0248] R 3 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0249] B 1 It is C 1-6 Alkylene;

[0250] C 1 It is C 1-6 Alkylene;

[0251] A 1 Selected from:

[0252] , , , , , , , , and ;

[0253] Where w is selected from 1, 2, or 3;

[0254] R 4 It is the amino acid side chain of a natural amino acid or the amino acid side chain of a non-natural amino acid, both of which are optionally coated with CH2C(O)OH or C(O)(CH2CH2O). p (CH2)2N(CH3)3 + replace;

[0255] R 5 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0256] R 6 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0257] R 7 Selected from:

[0258] (i) The amino acid side chains of natural amino acids,

[0259] (ii) Amino acid side chains of non-natural amino acids, or

[0260] (iii) Choose from the following groups:

[0261] and ;

[0262] R 8 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0263] R 9 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0264] R 10 It is the amino acid side chain of natural amino acids or the amino acid side chain of non-natural amino acids.

[0265] m is 0 or 1;

[0266] n, q, and u are each an independent integer from 0 to 16;

[0267] Each p is an independent integer from 0 to 24;

[0268] Each s is an independent integer from 0 to 16;

[0269] Each t is independently 1, 2, 3, 4, 5, or 6;

[0270] Each R' is independently selected from H, C(O)OH, (CH2)OH, and NHAc; and

[0271] Each "R" is independently selected from H and CH3;

[0272] Where the variable group R 0 R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 Or R 10 When an amino acid side chain is defined as a cyclic amino acid, the corresponding amino acid nitrogen of the peptide backbone of formula B forms part of the cyclic group; and

[0273] Furthermore, the cyclic peptide optionally contains a radionuclide.

[0274] In the embodiments, the cyclic peptide having formula B is a cyclic peptide having formula Bi:

[0275]

[0276] (Bi)

[0277] Or its pharmaceutically acceptable salt.

[0278] In another embodiment, the cyclic peptide having formula B is a cyclic peptide having formula Bii:

[0279]

[0280] (Bii)

[0281] Or its pharmaceutically acceptable salt.

[0282] In another embodiment, the cyclic peptide having formula B is a cyclic peptide having formula Biii:

[0283]

[0284] (Biii)

[0285] Or its pharmaceutically acceptable salt.

[0286] In the embodiments, the cyclic peptide having formula B is a cyclic peptide having formula Ia:

[0287]

[0288] (Ia)

[0289] Or its pharmaceutically acceptable salt.

[0290] In another embodiment, the cyclic peptide having formula B is a cyclic peptide having formula Ib:

[0291]

[0292] (Ib)

[0293] Or its pharmaceutically acceptable salt.

[0294] In yet another embodiment, P 1 Selected from Ac,

[0295] and ;

[0296] in:

[0297] D 1 It is CH3 or C(O)OH;

[0298] L 1 Does not exist or

[0299] ;

[0300] n and s are independent integers from 2 to 15; and

[0301] p is 8, 9, 10, 11 or 12.

[0302] In yet another embodiment, P 1 It is Ac or CH3(OCH2CH2) 8-16 C(O).

[0303] In the embodiment, P 1Selected from H, Ac,

[0304] and ;

[0305] in:

[0306] n and s are independently 9, 10, 11, 12, or 13; and

[0307] D 1 It is CH3 or C(O)OH.

[0308] In another embodiment, P 2 Yes -L 2 - Chelating agent. In yet another embodiment, P 2 yes

[0309] ;

[0310] P 3 Selected from -L 3 - Chelating agents,

[0311]

[0312] L 3 Not present or independently selected

[0313] , , and .

[0314] In yet another embodiment, P 2 yes

[0315] ;

[0316] P 3 Selected from chelating agents,

[0317]

[0318] and

[0319] p is 8, 12, or 24.

[0320] In yet another embodiment, P 2 yes

[0321] ;

[0322] P 3 Selected from DOTA

[0323] , , , , , , , , , , , and ;and

[0324] p is 8, 12, or 24.

[0325] In yet another embodiment, P 3 Yes -L 3 - Chelating agent.

[0326] In one embodiment, P 3 yes

[0327] .

[0328] In another embodiment, D 3 It is a NR-chelating agent.

[0329] On the other hand, this paper provides a cyclic peptide having formula I, wherein D 3 yes

[0330] ,

[0331] Where w is selected from 1, 2, or 3; and the other variables are defined in this paper.

[0332] In the embodiment, P 3 Is it Ac or

[0333] .

[0334] In yet another embodiment, the cyclic peptide having formula B is a cyclic peptide having formula II:

[0335]

[0336] (II)

[0337] Or its pharmaceutically acceptable salt.

[0338] In the embodiments, the cyclic peptide having formula B is a cyclic peptide having formula III:

[0339]

[0340] (III)

[0341] Or its pharmaceutically acceptable salt.

[0342] In yet another embodiment,

[0343] m is 0;

[0344] P 1 Selected from H, Ac,

[0345] and ;

[0346] in:

[0347] n and s are each independently 9, 10, 11, 12 or 13;

[0348] P 2 yes ;

[0349] P 3 Selected from DOTA

[0350] , , , , , , , , , , , and ;

[0351] p is 8, 12, or 24;

[0352] D 1 It is CH3 or C(O)OH;

[0353] R 1 Select amino acid side chains from the following groups: Trp, 2Nal, 1Nal, 4CF3-Phe, 7aza-Trp, 1Me-Trp, 5OH-Trp, BIP, 5Ome-Trp, 4F-Phe, 3Pya, 4Pya, PAF, MAF, OAF, 5Qui, 7MeO-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, D-Ala, Ala, α-Me-Trp, and NMe-Trp;

[0354] R 2 Select amino acid side chains from the following groups: Thr, D-Ala, Ala, α-Me-Thr, Lys, and NMe-Thr;

[0355] R 3Select amino acid side chains from the following groups: Ile, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, 1Nal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe-Nle, α-tert-pentyl Gly, Allo-Ile, Lys(C12), Lys(C14), Lys(C16), α-Me-Ile, and NMe-tBuAla;

[0356] A 1 Choose from the following groups:

[0357] , , , , , , and ;

[0358] R 4 Select from the following groups: Asn, D-Ala, Ala, DAB-4-NHCOC5H 11 DAB-4-NHCOC7H 15 Amino acid side chains of Asp, Ser, Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, 3Pya, 4Pya, Glu, NMe-Asn, Pip(CH2CO2H)Ala and Pip(PegNMe3)Ala;

[0359] R 5 Select amino acid side chains from the following groups: Asn, Ala, D-Ala, Trp, Asp, Lys, 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser;

[0360] R 6 The amino acid side chains of the following groups were selected: Trp, 4CF3-Phe, 1Me-Trp, 7aza-Trp, BIP, 2Nal, 1Nal, α-Me-Trp, D-Ala, Ala, 4F-Phe, 5F-Trp, 5Ome-Trp, Asn, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp.

[0361] R 7 :

[0362] (i) Select the amino acid side chains of the group consisting of: 3Pya, 4Pya, Lys(Me)3, His, Ala, D-Ala, Gln, Lys, Glu, Arg, Orn, NMe-His, and Ser; or

[0363] (ii) Choose from the following groups:

[0364] , and ;

[0365] Each s is independently 3, 5, 10, 12 or 14;

[0366] R 8 Select amino acid side chains from the following groups: Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and α-Me-Asp;

[0367] R 9 The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, D-Ala, Ala, 4F-Phe, 1Nal, 2Nal, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and

[0368] R 10 The amino acid side chains of the following groups were selected: Pro, D-Ala, Ala, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, Pip, 5,5-diMe-Pro, NMe-Ser, trans-4NH2-Pro, cis-4NH2-Pro, Sar, Aze, NMe-Ala, NMe-Leu, R-3Me-Aze, α-Me-Aze, ACI, and 3Me2-Aze.

[0369] In another embodiment,

[0370] m is 1;

[0371] P 1 Selected from Ac,

[0372] and ;

[0373] in:

[0374] n and s are each independently 9, 10, 11, 12 or 13;

[0375] P2 yes

[0376] ;

[0377] P 3 Selected from DOTA

[0378] , , , , , , , , , , , and ,

[0379] in

[0380] p is 8, 12, or 24;

[0381] D 1 It is CH3 or C(O)OH;

[0382] R 0 Select amino acid side chains from the following groups: Gly, Met, D-Ala, Ala, Nle, and Nva;

[0383] R 1 Select amino acid side chains from the following groups: Trp, 2Nal, 1Nal, 4CF3-Phe, 7aza-Trp, 1Me-Trp, 5OH-Trp, BIP, 5Ome-Trp, 4F-Phe, 3Pya, 4Pya, PAF, MAF, OAF, 5Qui, 7MeO-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, D-Ala, Ala, α-Me-Trp, and NMe-Trp;

[0384] R 2 Select amino acid side chains from the following groups: Thr, D-Ala, Ala, α-Me-Thr, Lys, and NMe-Thr;

[0385] R 3Select amino acid side chains from the following groups: Ile, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, 1Nal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe-Nle, α-tert-pentyl Gly, Allo-Ile, Lys(C12), Lys(C14), Lys(C16), α-Me-Ile, and NMe-tBuAla;

[0386] A 1 Choose from the following groups:

[0387] , , , , , , and ;

[0388] R 4 Select from the following groups: Asn, D-Ala, Ala, DAB-4-NHCOC5H 11 DAB-4-NHCOC7H 15 Amino acid side chains of Asp, Ser, Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, 3Pya, 4Pya, Glu and NMe-Asn;

[0389] R 5 Select amino acid side chains from the following groups: Asn, Ala, D-Ala, Trp, Asp, Lys, 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser;

[0390] R 6 The amino acid side chains of the following groups were selected: Trp, 4CF3-Phe, 1Me-Trp, 7aza-Trp, BIP, 2Nal, 1Nal, α-Me-Trp, D-Ala, Ala, 4F-Phe, 5F-Trp, 5Ome-Trp, Asn, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp.

[0391] R 7 Select amino acid side chains from the following groups: 3Pya, 4Pya, Lys(Me)3, His, Ala, D-Ala, Gln, Lys, Glu, Arg, Orn, NMe-His, and Ser; or

[0392] R 7 Choose from the following groups:

[0393] , and ;

[0394] Each s is independently 3, 5, 10, 12 or 14;

[0395] R 8 Select amino acid side chains from the following groups: Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and α-Me-Asp;

[0396] R 9 The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, D-Ala, Ala, 4F-Phe, 1Nal, 2Nal, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and

[0397] R 10 Amino acid side chains from the following groups: Pro, D-Ala, Ala, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, Pip, 5,5-diMe-Pro, NMe-Ser, trans-4NH2-Pro, cis-4NH2-Pro, Sar, Aze, NMe-Ala, NMe-Leu, R-3Me-Aze, α-Me-Aze, ACI, and 3Me2-Aze.

[0398] In the embodiments, m is 0 or 1;

[0399] P 1 Choose from the following groups: H, Ac,

[0400] and ;

[0401] n and s are each independently 9, 10, 11, 12 or 13;

[0402] D 1 It is CH3 or C(O)OH;

[0403] P 2 yes

[0404] ;

[0405] D 2 It is OH or NH2;

[0406] P 3 Choose from the following groups: chelating agents,

[0407]

[0408] p is an integer selected from 6 to 24;

[0409] X is a halogen;

[0410] R 0 Select amino acid side chains from the following groups: Gly, Met, D-Ala, Ala, Nle, and Nva;

[0411] R 1 Select amino acid side chains from the following groups: Trp, 2Nal, 1Nal, 4CF3-Phe, 7aza-Trp, 1Me-Trp, 5OH-Trp, BIP, 5Ome-Trp, 4F-Phe, 3Pya, 4Pya, PAF, MAF, OAF, 5Qui, 7Ome-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, D-Ala, Ala, α-Me-Trp, and NMe-Trp;

[0412] R 2 Select amino acid side chains from the following groups: Thr, D-Ala, Ala, α-Me-Thr, Lys, and NMe-Thr;

[0413] R 3 Select amino acid side chains from the following groups: Ile, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, 1Nal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe-Nle, α-tert-pentyl Gly, Allo-Ile, Lys(C12), Lys(C14), Lys(C16), α-Me-Ile, and NMe-tBuAla;

[0414] A 1 Choose from the following groups:

[0415] , , , , , , and ;

[0416] R 4 Select from the following groups: Asn, D-Ala, Ala, DAB-4-NHCOC5H 11 DAB-4-NHCOC7H 15 Amino acid side chains of Asp, Ser, Lys(DOTA), Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, 3Pya, 4Pya, Glu, NMe-Asn, Pip(CH2CO2H)Ala, Pip(PegNMe3)Ala and Pip(GAE-DOTA)Ala;

[0417] R 5 Select amino acid side chains from the following groups: Asn, Ala, D-Ala, Trp, Asp, Lys, Lys(DOTA), 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser;

[0418] R 6 The amino acid side chains of the following groups were selected: Trp, 4CF3-Phe, 1Me-Trp, 7aza-Trp, BIP, 2Nal, 1Nal, aMe-Trp, D-Ala, Ala, 4F-Phe, 5F-Trp, 5Ome-Trp, Asn, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp.

[0419] R 7

[0420] (i) Select the amino acid side chains of the group consisting of: 3Pya, 4Pya, Lys(Me)3, His, Ala, Phe, D-Ala, Gln, Lys, Glu, Arg, Orn, NMe-His, Trp, and Ser; or

[0421] (ii) Choose from the following groups:

[0422] , and ;

[0423] s is 3, 5, 10, 12, or 14;

[0424] R 8 Select amino acid side chains from the following groups: Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and α-Me-Asp;

[0425] R9 The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, D-Ala, Ala, 4F-Phe, 1Nal, 2Nal, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and

[0426] R 10 The amino acid side chains of the following groups were selected: Pro, D-Ala, Ala, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, Pip, 5,5-diMe-Pro, NMe-Ser, trans-4NH2-Pro, cis-4NH2-Pro, Sar, Aze, NMe-Ala, NMe-Leu, R-3Me-Aze, α-Me-Aze, ACI, and 3Me2-Aze.

[0427] In another embodiment, A 1 Choose from the following groups:

[0428] , and .

[0429] In yet another embodiment, A 1 yes:

[0430] .

[0431] In yet another embodiment, m is 0.

[0432] In the embodiments, m is 0 or 1;

[0433] P 2 yes

[0434] ;

[0435] D 2 It is OH or NH2;

[0436] P 3 Choose from the following groups: DOTA,

[0437] , , , , , , , , , , , and ,and

[0438] p is 8, 12, or 24.

[0439] In the embodiments, m is 0 or 1;

[0440] P 1 Choose from the following groups: Ac,

[0441] and ;

[0442] n and s are each independently 9, 10, 11, 12 or 13;

[0443] D 1 It is CH3 or C(O)OH;

[0444] P 2 yes

[0445] ;

[0446] D 2 It is OH or NH2;

[0447] P 3 Choose from the following groups: chelating agents,

[0448]

[0449] p is an integer selected from 8 to 24;

[0450] R 0 Select amino acid side chains from the following groups: Gly, Met, D-Ala, Ala, Nle, and Nva;

[0451] R 1 The amino acid side chains of the following groups were selected: Trp, 2Nal, 1Nal, 7-aza-Trp, 1Me-Trp, 5OH-Trp, BIP, 5Ome-Trp, 3Pya, 4Pya, 5Qui, 7Ome-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, α-Me-Trp, and NMe-Trp.

[0452] R 2 Select amino acid side chains from the following groups: Thr, D-Ala, Ala, α-Me-Thr, Lys, and NMe-Thr;

[0453] R3 The amino acid side chains of the following groups were selected: Ile, Env, Nle, D-Ala, Ala, Leu, t-Bu-Ala, NMe-Nle, α-tert-pentyl Gly, Allo-Ile, Lys(C12), Lys(C14), Lys(C16), α-Me-Ile, and NMe-tBuAla;

[0454] A 1 Choose from the following groups:

[0455] , , , , , , and ;

[0456] R 4 Select from the following groups: Asn, D-Ala, Ala, DAB-4-NHCOC5H 11 DAB-4-NHCOC7H 15 Amino acid side chains of Asp, Ser, Lys (DOTA), Lys, 3-(4-piperidinyl)-Ala, PipA (acetyl), 3-(1-morpholinyl)-Ala, 3Pya, Glu and NMe-Asn;

[0457] R 5 Select amino acid side chains from the following groups: Asn, Ala, D-Ala, Asp, Lys, Glu, NMe-Asn, and Ser;

[0458] R 6 The amino acid side chains of the following groups were selected: Trp, 4CF3-Phe, 1Me-Trp, 7aza-Trp, BIP, 2Nal, 1Nal, aMe-Trp, 4F-Phe, 5F-Trp, 5Ome-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp.

[0459] R 7 Select amino acid side chains from the following groups: 3Pya, 4Pya, Lys(Me)3, His, Ala, Phe, D-Ala, Gln, Lys, Glu, Arg, Orn, NMe-His, Trp, and Ser; or

[0460] R 8Select amino acid side chains from the following groups: Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and α-Me-Asp;

[0461] R 9 The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, 1Nal, 2Nal, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and

[0462] R 10 The amino acid side chains of the following groups were selected: Pro, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, 5,5-diMe-Pro, trans-4NH2-Pro, cis-4NH2-Pro, Aze, ACI, and 3Me2-Aze.

[0463] In an embodiment,

[0464] P 1 yes:

[0465] ;

[0466] n is an integer selected from 0 to 16;

[0467] D 1 Choose from the following groups: H, CH3, and C(O)OH;

[0468] P 2 yes

[0469] ;

[0470] D 2 It is OH or NH2;

[0471] P 3 Choose from the following groups: chelating agents,

[0472]

[0473] p is 8, 12, or 24;

[0474] R 0 Select amino acid side chains from the following groups: Gly, Met, D-Ala, Ala, Nle, and Nva;

[0475] R 1The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, 5OH-Trp, 5Ome-Trp, 7Ome-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, α-Me-Trp and NMe-Trp;

[0476] R 2 Select amino acid side chains from the following groups: Thr, α-Me-Thr, Lys, and NMe-Thr;

[0477] R 3 The amino acid side chains of the following groups were selected: D-Ala, Ala, t-Bu-Ala, and NMe-tBuAla;

[0478] A 1 Choose from the following groups:

[0479] , , , , , , and ;

[0480] R 4 The amino acid side chains of the following groups were selected: 3-(4-piperidinyl)-Ala, PipA (acetyl) and 3-(1-morpholinyl)-Ala;

[0481] R 5 Select amino acid side chains from the following groups: Asn, Asp, and NMe-Asn;

[0482] R 6 It is an amino acid side chain of 2Nal or 1Nal;

[0483] R 7 Select amino acid side chains from the following groups: 3Pya, 4Pya, Lys(Me)3, His, Ala, Phe, D-Ala, Gln, Lys, Glu, Arg, Orn, NMe-His, Trp, and Ser.

[0484] R 8 Select amino acid side chains from the following groups: Asp, Asn, NMe-Asp, and α-Me-Asp;

[0485] R 9The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and

[0486] R 10 The amino acid side chains of the following groups were selected: Pro, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, 5,5-diMe-Pro, trans-4NH2-Pro and cis-4NH2-Pro.

[0487] In another embodiment,

[0488] R 1 It is the amino side chain of Trp;

[0489] R 2 It is the amino side chain of Thr;

[0490] R 3 It is the amino side chain of Ile or NMe-tBuAla;

[0491] R 4 The amino side chains are selected from Lys, 3-(4-piperidinyl)-Ala and PipA (acetyl);

[0492] R 5 It is an amino side chain of Asn or NMe-Asn;

[0493] R 6 It is the amino side chain of Trp or 2NaI;

[0494] R 7 Amino side chains selected from His or Lys;

[0495] R 8 It is the amino side chain of Asp;

[0496] R 9 It is the amino side chain of Trp; and

[0497] R 10 It is the amino side chain of Pro.

[0498] In another embodiment,

[0499] R 1 It is the amino side chain of Trp;

[0500] R 2 It is the amino side chain of Thr;

[0501] R3 It is the amino side chain of Ile;

[0502] R 4 The amino side chains are selected from Lys, 3-(4-piperidinyl)-Ala and PipA (acetyl);

[0503] R 5 It is an amino side chain of Asn or NMe-Asn;

[0504] R 6 It is the amino side chain of 2NaI or Trp;

[0505] R 7 The amino side chain is selected from His;

[0506] R 8 It is the amino side chain of Asp;

[0507] R 9 It is the amino side chain of Trp; and

[0508] R 10 It is the amino side chain of Pro.

[0509] In another embodiment,

[0510] R 1 It is the amino acid side chain of Trp;

[0511] R 2 It is the amino acid side chain of Thr;

[0512] R 3 It is the amino acid side chain of NMe-tBuAla;

[0513] R 4 It is an amino acid side chain of 3-(4-piperidinyl)-Ala or PipA (acetyl);

[0514] R 5 It is the amino acid side chain of Asn;

[0515] R 6 It is the amino acid side chain of 2NaI;

[0516] R 7 It is the amino acid side chain of Lys;

[0517] R 8 It is the amino acid side chain of Asp;

[0518] R 9 It is the amino acid side chain of Trp; and

[0519] R 10 It is the amino acid side chain of Pro.

[0520] In an embodiment,

[0521] P 1 yes:

[0522] ;

[0523] n is an integer from 0 to 8;

[0524] P 2 yes

[0525] ;

[0526] D 2 It is OH or NH2;

[0527] P 3 Choose from the following groups: chelating agents,

[0528]

[0529] R 1 The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, 5OH-Trp, 5Ome-Trp, 7Ome-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, α-Me-Trp and NMe-Trp;

[0530] R 2 It is the amino acid side chain of Thr;

[0531] R 3 The amino acid side chains of the following groups were selected: D-Ala, Ala, t-Bu-Ala, and NMe-tBuAla;

[0532] A 1 yes

[0533] ;

[0534] B 1 It is CH2;

[0535] C 1 It is CH2;

[0536] R 4 The amino acid side chains of the following groups were selected: 3-(4-piperidinyl)-Ala, PipA (acetyl) and 3-(1-morpholinyl)-Ala;

[0537] R 5 Select amino acid side chains from the following groups: Asn, Asp, and NMe-Asn;

[0538] R6 It is an amino acid side chain of 2Nal or 1Nal;

[0539] R 7 Select from the following groups: amino acid side chains of His, Lys, and Trp;

[0540] R 8 Select amino acid side chains from the following groups: Asp, NMe-Asp, and α-Me-Asp;

[0541] R 9 The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and

[0542] R 10 The amino acid side chains of the following groups were selected: Pro, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, 5,5-diMe-Pro, trans-4NH2-Pro and cis-4NH2-Pro.

[0543] In the embodiment, P 1 yes:

[0544] ;

[0545] P 2 yes

[0546] ;

[0547] P 3 Choose from the following groups: chelating agents,

[0548]

[0549] R 1 The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, 5OH-Trp, 5Ome-Trp, 7Ome-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, α-Me-Trp and NMe-Trp;

[0550] R 2 Select amino acid side chains from the following groups: Thr, α-Me-Thr, and NMe-Thr;

[0551] R3 The amino acid side chains of the following groups were selected: D-Ala, Ala, t-Bu-Ala, and NMe-tBuAla;

[0552] A 1 yes

[0553] ;

[0554] B 1 It is CH2;

[0555] C 1 It is CH2;

[0556] R 4 The amino acid side chains of the following groups were selected: 3-(4-piperidinyl)-Ala, PipA (acetyl) and 3-(1-morpholinyl)-Ala;

[0557] R 5 It is an amino acid side chain of Asn or NMe-Asn;

[0558] R 6 It is an amino acid side chain of 2Nal or 1Nal;

[0559] R 7 Select from the following groups: amino acid side chains of His, Lys, and Trp;

[0560] R 8 Select amino acid side chains from the following groups: Asp, NMe-Asp, and α-Me-Asp;

[0561] R 9 The amino acid side chains of the following groups were selected: Trp, 7-aza-Trp, 1Me-Trp, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and

[0562] R 10 The amino acid side chains of the following groups were selected: Pro, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, 5,5-diMe-Pro, trans-4NH2-Pro and cis-4NH2-Pro.

[0563] In an embodiment,

[0564] P 1 yes:

[0565] ;

[0566] P 2 yes

[0567] ;

[0568] P 3 Choose from the following groups: chelating agents,

[0569]

[0570] R 1 It is the amino acid side chain of Trp;

[0571] R 2 It is the amino acid side chain of Thr;

[0572] R 3 It is an amino acid side chain of Ile or NMe-tBuAla;

[0573] A 1 yes

[0574] ;

[0575] B 1 It is CH2;

[0576] C 1 It is CH2;

[0577] R 4 It is an amino acid side chain of Lys, 3-(4-piperidinyl)-Ala, or PipA (acetyl);

[0578] R 5 It is an amino acid side chain of Asn or NMe-Asn;

[0579] R 6 It is an amino acid side chain of Trp or 2NaI;

[0580] R 7 It is an amino acid side chain of His or Lys;

[0581] R 8 It is the amino acid side chain of Asp;

[0582] R 9 It is the amino acid side chain of Trp; and

[0583] R 10 It is the amino acid side chain of Pro.

[0584] In an embodiment,

[0585] P 1 yes:

[0586] ;

[0587] P 2 yes

[0588] ;

[0589] P 3 Choose from the following groups: chelating agents,

[0590]

[0591] R 1 It is the amino acid side chain of Trp;

[0592] R 2 It is the amino acid side chain of Thr;

[0593] R 3 It is the amino acid side chain of Ile;

[0594] A 1 yes

[0595] ;

[0596] B 1 It is CH2;

[0597] C 1 It is CH2;

[0598] R 4 It is an amino acid side chain of 3-(4-piperidinyl)-Ala or PipA (acetyl);

[0599] R 5 It is the amino acid side chain of NMe-Asn;

[0600] R 6 It is the amino acid side chain of 2Nal;

[0601] R 7 It is the amino acid side chain of His;

[0602] R 8 It is the amino acid side chain of Asp;

[0603] R 9 It is the amino acid side chain of Trp; and

[0604] R 10 It is the amino acid side chain of Pro.

[0605] In an embodiment,

[0606] P 1 yes:

[0607] ;

[0608] P 2 yes

[0609] ;

[0610] P 3 Choose from the following groups: DOTA,

[0611] , , , ,

[0612] ,

[0613] and

[0614] ;

[0615] R 1 It is the amino acid side chain of Trp;

[0616] R 2 It is the amino acid side chain of Thr;

[0617] R 3 It is the amino acid side chain of NMe-tBuAla;

[0618] A 1 yes

[0619] ;

[0620] B 1 It is CH2;

[0621] C 1 It is CH2;

[0622] R 4 It is an amino acid side chain of 3-(4-piperidinyl)-Ala or PipA (acetyl);

[0623] R 5 It is the amino acid side chain of Asn;

[0624] R 6 It is the amino acid side chain of 2Nal;

[0625] R 7 It is the amino acid side chain of Lys;

[0626] R 8 It is the amino acid side chain of Asp;

[0627] R 9 It is the amino acid side chain of Trp; and

[0628] R 10 It is the amino acid side chain of Pro.

[0629] In another embodiment, R 0 Select amino acid side chains from the following groups: Gly, Met, D-Ala, Ala, Nle, and Nva.

[0630] In yet another embodiment, R 1 The amino acid side chains of Trp, 2Nal, 1Nal, 4CF3-Phe, 7aza-Trp, 1Me-Trp, 5OH-Trp, BIP, 5Ome-Trp, 4F-Phe, 3Pya, 4Pya, PAF, MAF, OAF, 5Qui, 7MeO-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, D-Ala, Ala, α-Me-Trp, and NMe-Trp are selected. In yet another embodiment, R 1 The amino acid side chains of Trp, 7-aza-Trp, 1Me-Trp, 5OH-Trp, 5Ome-Trp, 7Ome-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, α-Me-Trp, and NMe-Trp are selected. In the examples, R 1 It is an amino side chain of Trp or 7Me-Trp. In another embodiment, R 1 It is the amino side chain of Trp.

[0631] In yet another embodiment, R 2 The amino acid side chains are selected from the group consisting of: Thr, D-Ala, Ala, α-Me-Thr, Lys, and NMe-Thr. In yet another embodiment, R 2 The amino acid side chains are selected from the group consisting of Thr, α-Me-Thr, and NMe-Thr. In the examples, R 2 It is the amino side chain of Thr.

[0632] In the embodiment, R 3The amino acid side chains selected are from the group consisting of: Ile, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, 1Nal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe-Nle, α-tert-pentyl Gly, Allo-Ile, Lys(C12), Lys(C14), Lys(C16), α-Me-Ile, and NMe-tBuAla. In another embodiment, R 3 The amino acid side chains are selected from the group consisting of: Ile, Allo-Ile, α-Me-Ile, and NMe-tBuAla. In yet another embodiment, R 3 The amino acid side chains are selected from the group consisting of D-Ala, Ala, t-Bu-Ala, and NMe-tBuAla. In yet another embodiment, R 3 It is an amino side chain of Ile or NMe-tBuAla. In the examples, R 3 It is the amino side chain of Ile. In another embodiment, R 3 It is the amino side chain of NMe-tBuAla.

[0633] In yet another embodiment, R 4 Select from the following groups: Asn, D-Ala, Ala, DAB-4-NHCOC5H 11 DAB-4-NHCOC7H 15 Amino acid side chains of Asp, Ser, Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, 3Pya, 4Pya, Glu, NMe-Asn, Pip(CH2CO2H)Ala, and Pip(PegNMe3)Ala. In yet another embodiment, R 4 The amino acid side chains are selected from the group consisting of 3-(4-piperidinyl)-Ala, PipA (acetyl), and 3-(1-morpholinyl)-Ala. In another embodiment, R 4 It is an amino side chain of 3-(4-piperidinyl)-Ala or PipA (acetyl).

[0634] In yet another embodiment, R 5 The amino acid side chains are selected from the group consisting of: Asn, Ala, D-Ala, Trp, Asp, Lys, 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser. In yet another embodiment, R 5 The amino acid side chains of Asn and NMe-Asn are selected from the group consisting of: Asn and NMe-Asn. In the examples, R5 It is the amino side chain of NMe-Asn. In another embodiment, R 5 It is the amino side chain of Asn.

[0635] In yet another embodiment, R 6 The amino acid side chains of Trp, 4CF3-Phe, 1Me-Trp, 7aza-Trp, BIP, 2Nal, 1Nal, α-Me-Trp, D-Ala, Ala, 4F-Phe, 5F-Trp, 5MeO-Trp, Asn, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp are selected. In yet another embodiment, R 6 The amino acid side chains of Trp, 1Me-Trp, 7-aza-Trp, 2Nal, 1Nal, α-Me-Trp, 5F-Trp, 5MeO-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp are selected. In the examples, R 6 The amino acid side chains are selected from the group consisting of Trp, 2Nal, and 1Nal. In another embodiment, R 6 It is the amino acid side chain of Trp. In yet another embodiment, R 6 It is the amino side chain of 2NaI.

[0636] In yet another embodiment, R 7 The amino acid side chains of the following groups were selected: 3Pya, 4Pya, Lys(Me)3, His, Ala, D-Ala, Gln, Lys, Glu, Arg, Orn, NMe-His, and Ser. In the examples, R 7 It is an amino acid side chain of His or Lys. In the examples, R 7 It is the amino acid side chain of His. In the examples, R 7 It is the amino acid side chain of Lys.

[0637] In yet another embodiment, R 8 The amino acid side chains selected are from the group consisting of: Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and α-Me-Asp. In the examples, R 8 The amino acid side chains are selected from the group consisting of Asp, Asn, NMe-Asp, and α-Me-Asp. In another embodiment, R 8 The amino acid side chains are selected from the group consisting of Asp, NMe-Asp, and α-Me-Asp. In yet another embodiment, R 8 It is the amino side chain of Asp.

[0638] In the embodiment, R 9 The amino acid side chains of Trp, 7-aza-Trp, 1Me-Trp, D-Ala, Ala, 4F-Phe, 1Nal, 2Nal, 5F-Trp, 5MeO-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp are selected. In the examples, R 9 The amino acid side chains of Trp, 7-aza-Trp, 1Me-Trp, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp are selected from the group consisting of: Trp, 7aza-Trp, 1Me-Trp, 5F-Trp, 5Ome-Trp, α-Me-Trp, 7Cl-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp. In the examples, R 9 It is the amino side chain of Trp.

[0639] In the embodiment, R 10 The amino acid side chains selected are from the group consisting of: Pro, D-Ala, Ala, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, Pip, 5,5-diMe-Pro, NMe-Ser, trans-4NH2-Pro, cis-4NH2-Pro, Sar, Aze, NMe-Ala, NMe-Leu, R-3Me-Aze, α-Me-Aze, ACI, and 3Me2-Aze. In the examples, R... 10 Select from the following groups: amino acid side chains, R 10 The amino acid side chains of Pro, α-Me-Pro, trans-4-fluoro-Pro, cis-4-fluoro-Pro, trans-4OH-Pro, cis-4OH-Pro, 5,5-diMe-Pro, trans-4NH2-Pro, and cis-4NH2-Pro are selected from the group consisting of: 10 It is the amino side chain of Pro.

[0640] In another embodiment, P 2 yes

[0641] ;and

[0642] D 2 It is OH or NH2.

[0643] In another embodiment, P 2 yes

[0644] ;and

[0645] D2 It is OH or NH2.

[0646] In yet another embodiment,

[0647] B 1 It is CH2 or C(CH3)2; and

[0648] C 1 It is CH2 or C(CH3)2.

[0649] In yet another embodiment,

[0650] B 1 It is CH2; and

[0651] C 1 It is CH2.

[0652] In yet another embodiment, the chelating agent is selected from the chelating agents in Table C.

[0653] In the embodiments, the chelating agent was independently selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′′′-tetraacetic acid (DOTA), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isothiocyanopyridinecarboxylic acid (Macropa), Macrodipa, 2,2′,2″,2′′′-(1,10-diaza-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid (crown ether), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-(2-Carboxyethyl) (DOTAGA), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraacetic acid (TETA), 1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′′′,N′′′′-pentaacetic acid (PEPA), and 1,4,7,10,13,16-hexaazacyclohexadecane-N,N′,N″,N′′′,N′′′′,N″′′′-hexaacetic acid (HEHA). In another embodiment, the chelating agent is selected from deferoxamine (DFO) and 5,11,16,22-tetraazahexacosanediamide (DFO). ) and N,N′-1,4-butanediylbis[N-[3-[[(1,6-dihydro-1-hydroxy-6-oxo-2-pyridyl)carbonyl]amino]propyl]-1,6-dihydro-1-hydroxy-6-oxo-2-pyridinecarboxamide] (HOPO).

[0654] In another embodiment, the chelating agent is DOTA. In yet another embodiment, the chelating agent is DOTAGA. In still another embodiment, the chelating agent is Macrodipa. In this embodiment, the chelating agent is macropa.

[0655] In another embodiment, formula B is replaced by at least one chelating agent. In yet another embodiment, formula B is replaced by one chelating agent. In still another embodiment, formula B is replaced by two chelating agents.

[0656] In the formulas provided in this article, variables (e.g., R) 0 R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 Or R 10 A cyclic group can be defined as a side chain of a cyclic amino acid (e.g., proline). In this case, the corresponding amino acid nitrogen of the peptide backbone having the general formula provided herein forms part of the cyclic group. For example, “R 10 The amino acid side chains of the selected groups, consisting of: Pro, α-Me-Pro, trans-tetrafluoro-Pro, and cis-tetrafluoro-Pro, are defined as follows:

[0657]

[0658] In this diagram, each R is independently either hydrogen or a substituent.

[0659] In another embodiment, the compound having formula B is selected from the group consisting of compounds from Table A.

[0660] Table A.

[0661]

[0662] In yet another embodiment, the cyclic peptide having formula B is selected from the peptides in Table B.

[0663] Table B.

[0664]

[0665]

[0666]

[0667]

[0668]

[0669] Or its pharmaceutically acceptable salt.

[0670] Note: In Table B and the above sequences below, C(3) and / or Pen(3) represent two cysteine ​​residues involved in the formation of a disulfide ring bond.

[0671] Note: In Table B and below, C(1) and / or D-Cys(1) represent two cysteine ​​residues involved in the formation of a cyclic bond in a thioacetal bridge (S-CH2-S or methylene crosslinker).

[0672] On the other hand, this article provides a pharmaceutical composition comprising the peptide described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0673] The compounds disclosed herein can exist as tautomers and optical isomers (e.g., enantiomers, diastereomers, mixtures of diastereomers, racemic mixtures, etc.). Absolute stereochemistry is assigned according to the Cahn-Ingold-Prelog RS system. Chiral centers of known absolute configurations are marked with prefixes R and S, which are assigned according to standard sequence rules and preceded by the appropriate position where necessary (Pure & Appl. Chem. 45, 1976, 11-30). Some instances contain (R...) ) or (S The chemical structure of (R). ) or (S When used in the name of a compound or in a chemical diagram of a compound, it is intended to indicate that the compound is a pure, single isomer at that chiral center; however, the absolute configuration of that chiral center has not been determined. Therefore, it is designated as (R A compound is defined as a single, pure isomer at the chiral center, with an absolute configuration of I or (S), and is designated as (S). A compound is a single isomer that is pure at the chiral center and has an absolute configuration of (R) or (S).

[0674] The compounds provided herein may also include all isotopes of the atoms present in the intermediates or the final compound. Isotopes include those atoms with the same number of atoms but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the present invention may be substituted or replaced by atomic isotopes of natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. For example, one or more hydrogen atoms in the compounds disclosed herein may be substituted or replaced by deuterium. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deuterium atoms. Synthetic methods for introducing isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry, Alan F. Thomas, Appleton-Century-Crofts, NY, 1971; The Renaissance of H / D Exchange, Jens Atzrodt, Volker Derdau, Thorsten Fey, and Jochen Zimmermann, Angew. Chem. Int. Ed., 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling, James R. Hanson, Royal Society of Chemistry, 2011). Isotope-labeled compounds can be used in a variety of studies, such as NMR spectroscopy, metabolic experiments, and / or assays.

[0675] In the compounds provided herein, any atom not explicitly designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise stated, when a position is specifically designated as “H” or “hydrogen”, that position is understood to have hydrogen in its native abundance isotope composition. Furthermore, unless otherwise stated, when a position is specifically designated as “D” or “deuterium”, that position is understood to have at least 3000 times the abundance of deuterium (which is 0.015%) (i.e., at least 45% deuterium doping).

[0676] In the embodiments, the compounds provided herein have an isotopic enrichment factor for each specified deuterium atom of at least 3500 (52.5% deuterium doping at each specified deuterium atom), at least 4000 (60% deuterium doping), at least 4500 (67.5% deuterium doping), at least 5000 (75% deuterium), at least 5500 (82.5% deuterium doping), at least 6000 (90% deuterium doping), at least 6333.3 (95% deuterium doping), at least 6466.7 (97% deuterium doping), at least 6600 (99% deuterium doping), or at least 6633.3 (99.5% deuterium doping).

[0677] Examples of bridging moieties / peptide stapling structures used for the compounds disclosed herein include, but are not limited to: amide-based (e.g., lactam) bridges; aromatic ring-based bridges; hydrocarbon chains; alkene-based hydrocarbon bridges (e.g., using Fmoc-'-2-(2'-pentenyl)alanine); triazole-based click chemistry bridges, such as copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reactions between side-chain azido and alkynyl moieties (e.g., Fmoc-L-Nle(εN3) and Fmoc-D-Pra) (see S. Kawamoto et al., J. Med. Chem. [Journal of Medicinal Chemistry] 2012, 55(3), 1137-1146); diynyl stapling structures for stapling linear azido-peptides (e.g., 1,4-dieethynylbenzene, dieethynylpentane, dieethynylamine); disulfide-linked disulfide, thioether, and disulfide bridges; perfluorobenzene bridges; or combinations thereof.

[0678] In some embodiments, the bridging portion comprises an amide bond between an amine functional group and a carboxylate functional group, respectively, present in the side chains of an amino acid, a non-natural amino acid, or a non-amino acid residue. In some embodiments, the amine or carboxylate functional group is part of a non-amino acid residue or a non-natural amino acid residue. In some embodiments, the bridging portion comprises an amide bond generated by a side chain reaction of the following amino acid pairs: lysine and glutamic acid; lysine and aspartic acid; ornithine and glutamic acid; ornithine and aspartic acid; high-lysine and glutamic acid; high-lysine and aspartic acid; and other combinations of amino acids, non-natural amino acids, or non-amino acid residues, including primary amines and carboxylic acids. In some embodiments, the bridging portion is formed by a cyclization reaction using olefin metathesis.

[0679] In some embodiments, the bridging portion comprises a disulfide bond formed between two thiol-containing residues. In some embodiments, the bridging portion comprises one or more thioether bonds. Such thioether bonds may include those found in cyclothioalkyl compounds. These bonds may be formed in a chemical cyclization reaction between an N-terminal chloroacetic acid modification group and a cysteine ​​residue. In some embodiments, the bridging portion comprises one or more triazole rings.

[0680] In some embodiments, the bridging portion comprises one or more hydrocarbon chains (straight-chain or branched) and / or hydrocarbon rings (cyclic, heterocyclic, aromatic, or heteroaromatic). In some embodiments, the hydrocarbon bridging portion can be introduced by reacting with a reagent containing a plurality of reactive halides, including but not limited to poly(bromomethyl)benzene, poly(bromomethyl)pyridine, poly(bromomethyl)alkylbenzene, or (E)-1,4-dibromobut-2-ene. Examples of poly(bromomethyl)benzene molecules disclosed herein may include 1,2-bis(bromomethyl)benzene; 1,3-bis(bromomethyl)benzene; and 1,4-bis(bromomethyl)benzene.

[0681] In some embodiments, the thiol group of a cysteine ​​residue crosslinks with another cysteine ​​residue to form a disulfide bond. In some embodiments, the thiol group of a cysteine ​​residue reacts with the bromomethyl group of a poly(bromomethyl)benzene molecule to form a stable bond (see, for example, Timmerman et al., ChemBioChem [Chemical Biochemistry] (2005) 6:821-824, the contents of which are incorporated herein by reference in their entirety).

[0682] In some embodiments, bis-, tri-, and tetra(bromomethyl)benzene molecules can be used to generate bridging moieties to produce peptides having one, two, or three rings, respectively. The bromomethyl groups of the poly(bromomethyl)benzene molecule can be arranged on adjacent ring carbons of the benzene ring (ortho or o-), with two groups separated by a ring carbon (meta or m-), or on opposite ring carbons (para or p-). In some embodiments, m-bis(bromomethyl)benzene (i.e., m-dibromoxylene), o-bis(bromomethyl)benzene (i.e., o-dibromoxylene), and / or p-bis(bromomethyl)benzene (i.e., p-dibromoxylene) are used to form cyclic peptides. In some embodiments, the thiol group of a cysteine ​​residue reacts with other reagents containing one or more bromine functional groups to form a stable bond. Such reagents may include, but are not limited to, poly(bromomethyl)pyridine (e.g., 2,6-bis(bromomethyl)pyridine), poly(bromomethyl)alkylbenzene (e.g., 1,2-bis(bromomethyl)-4-alkylbenzene), and / or (E)-1,4-dibromobut-2-ene.

[0683] In some embodiments, the side-chain amino groups and terminal amino groups are crosslinked with bis(succinimide) glutarate (see, for example, Millward et al., J. Am. Chem. Soc. [Journal of the American Chemical Society] (2005) 127:14142-14143). In some embodiments, an enzymatic method is used to generate the thioether bond, which relies on a reaction catalyzed by lanohydinosin synthase between (1) cysteine ​​and (2) dehydroalanine or dehydrobutyryline groups (see, for example, Levengood et al., Bioorg. and Med. Chem. Lett. [Bioorganic and Medicinal Chemistry Letters] (2008) 18:3025-3028). The dehydrogenated functional group can also be chemically generated by oxidative translation of a selenium-containing amino acid side chain (see, for example, Seebeck et al., J. Am. Chem. Soc. [Journal of the American Chemical Society] 2006).

[0684] In some embodiments, the bridging portion comprises a 6-membered aromatic ring (e.g., benzene). In some embodiments, the bridging portion comprises a 6-membered heterocycle including one nitrogen atom (e.g., pyridine). In some embodiments, the bridging portion comprises a 6-membered heterocycle including two nitrogen atoms (e.g., pyridazine, pyrimidine, pyrazine). In some embodiments, the bridging portion comprises a 6-membered heterocycle including three nitrogen atoms (e.g., triazine). In some embodiments, the bridging portion comprises a 5-membered heterocycle including one nitrogen atom (e.g., pyrrole). In some embodiments, the bridging portion comprises a 5-membered heterocycle including two nitrogen atoms (e.g., imidazole, pyrazole). In some embodiments, the bridging portion comprises a 5-membered heterocycle including three nitrogen atoms (e.g., triazole).

[0685] The peptides disclosed herein may be cyclized via a carboxyl terminus, an amino terminus, or via any other convenient attachment site, such as via the sulfur of a cysteine ​​residue (e.g., by forming a disulfide bond between two cysteine ​​residues in the sequence) or any side chain of an amino acid residue. Other bonds forming a cyclic loop may include, but are not limited to, maleimide bonds, amide bonds, ester bonds, ether bonds, thioether bonds, hydrazone bonds, or acetamide bonds.

[0686] In some embodiments, the peptides disclosed herein are formed using a lactam moiety. For example, such cyclic peptides can be formed by synthesis on a solid support, Wang resin, using standard Fmoc chemistry. In some cases, Fmoc-ASP(allyl)-OH and Fmoc-LYS(allyloxycarbonyl)-OH are incorporated into the peptide as precursor monomers for lactam bridge formation.

[0687] In some embodiments, the peptide disclosed herein is a linear peptide. In some embodiments, the peptide disclosed herein is a cyclic peptide. In some embodiments, the cyclic peptide contains a disulfide bond. In some embodiments, the peptide disclosed herein is a linear peptide prior to the cyclization step. In some embodiments, the peptide disclosed herein is a linear peptide prior to the formation of the disulfide bond.

[0688] Typically, the formation of disulfide bonds involves a reaction between the thiol (SH) side chains of two cysteine ​​residues. Proper disulfide bonds provide stability to proteins, reducing further entropic selection by restricting unfolded or misfolded conformations, and promoting protein folding towards its native state.

[0689] End modification and lacing

[0690] One method of protecting peptides from proteolytic degradation involves chemically modifying or “capping” the amino and / or carboxyl terms of the peptide. As used herein, the terms “chemical modification” or “capping” are used interchangeably to refer to the introduction of blocking groups at one or both ends of a compound through covalent modification. Suitable blocking groups are used to block the ends of the peptide without reducing its biological activity. Any residue located at the amino or carboxyl terminus of the compound, or both, can be chemically modified. In some embodiments, the peptides disclosed herein contain N-terminal and / or C-terminal modifications.

[0691] In one embodiment, the amino terminus of the compound is chemically modified by acetylation to produce an N-acetylated peptide (represented by "Ac-" in the structures or formulas disclosed herein). In another embodiment, the carboxyl terminus of the peptide is chemically modified by amidation to give a primary carboxamide (represented as "amide" in the peptide sequences, structures, or claims disclosed herein) at the C-terminus. In some embodiments, both the amino terminus and the carboxyl terminus are chemically modified by acetylation and amidation, respectively. However, other capping groups may be used. For example, the amino terminus may be capped by acylation with groups such as acetyl groups, benzoyl groups, or natural or non-natural amino acids (e.g., β-alanine capped by an acetyl group); or by alkylation with groups such as benzyl groups or butyl groups; or by sulfonation to produce a sulfonamide. Similarly, the carboxyl terminus may be esterified or converted to secondary amides and acylsulfonamides, etc.

[0692] In some embodiments, the N-terminal capping function is due to the bond formed with the terminal amino group and can be selected from the group consisting of: formyl; alkanoyl groups having 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl; enoyl groups having 1 to 10 carbon atoms, such as hex-3-enoyl; ynyl groups having 1 to 10 carbon atoms, such as hex-5-ynyl; aromatic acyl groups, such as benzoyl or 1-naphthyl; heteroaromatic acyl groups, such as 3-pyrroyl or 4-quinoloyl. l) alkylsulfonyl groups, such as methanesulfonyl; arylsulfonyl groups, such as benzenesulfonyl or sulfanyl; heteroarylsulfonyl groups, such as pyridine-4-sulfonyl; substituted alkanoyl groups having 1 to 10 carbon atoms, such as 4-aminobutyryl; substituted enoyl groups having 1 to 10 carbon atoms, such as 6-hydroxy-hex-3-enoyl; substituted alkynyl groups having 1 to 10 carbon atoms, such as 3-hydroxy-hex-5-alkynyl; substituted aromatic acyl groups, such as 4-chlorobenzoyl or 8-hydroxy-naphthalene-2-acyl; and substituted alkanoyl groups. Substituted heteroaryl groups, such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazoline-6-acyl; substituted alkylsulfonyl groups, such as 2-aminoethanesulfonyl; substituted arylsulfonyl groups, such as 5-dimethylamino-1-naphthalenesulfonyl; substituted heteroarylsulfonyl groups, such as 1-methoxy-6-isoquinolinesulfonyl; carbamoyl or thiocarbamoyl; substituted carbamoyl (R′—NH—CO) or substituted thiocarbamoyl (R′—NH—CS), wherein R′ is an alkyl group. Alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl or substituted heteroaryl; substituted carbamoyl (R′—NH—CO) and substituted thiocarbamoyl (R′—NH—CS), wherein R′ is alkanoyl, enoyl, alkynyl, aromatic acyl, heteroaryl, substituted alkanoyl, substituted enoyl, substituted alkynyl, substituted aromatic acyl or substituted heteroaryl, all as defined above; Lys-(Gly)n, where n = 1-8; or Tyr-(Gly)n, where n = 1-8.

[0693] In some embodiments, the C-terminal capping function may be due to an amide bond formed with the terminal carboxyl group, or to an ester bond formed with the terminal carboxyl group. The capping function providing the amide bond is designated as NR1R2, wherein R1 and R2 can each be independently selected from the group consisting of: hydrogen; alkyl groups having 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl; preferably alkenyl groups having 1 to 10 carbon atoms, such as prop-2-alkenyl; preferably alkynyl groups having 1 to 10 carbon atoms, such as prop-2-alynyl; substituted alkyl groups having 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxylalkyl, carbamoylalkyl; substituted alkenyl groups having 1 to 10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxylalkenyl, carbamoylalkenyl. Alkyne group; substituted alkynyl group having 1 to 10 carbon atoms, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, haloalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkylylalkynyl, carboxyalkynyl, carbamoylalkynyl; arylylalkyl group having up to 10 carbon atoms, such as benzoylmethyl or 2-benzoylethyl; aryl group, such as phenyl or 1-naphthyl; heteroaryl group, such as 4-quinoline. Linoyl; alkanoyl group having 1 to 10 carbon atoms, such as acetyl or butyryl; aromatic acyl group, such as benzoyl; heteroaromatic acyl group, such as 3-quinoline acyl; OR′ or NR′R″, wherein R′ and R″ are each independently hydrogen, alkyl, aryl, heteroaromatic, acyl, aromatic acyl, sulfonyl, sulfinyl, SO2—R′″ or SO—R′″, wherein R′″ is a substituted or unsubstituted alkyl, aryl, heteroaromatic, alkenyl or alkynyl.

[0694] In some embodiments, the capping function of the ester bond is provided as OR, where R can be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaryloxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted heteroaryloxy.

[0695] In some embodiments, the peptide disclosed herein may comprise a modification of the C-terminus of the peptide sequence with one or more of the following portions: NH2, NH-CH3; NH-CH2-CH3; NH-CH-(CH3)2, NH-CH2-CH2-CH3, NH-CH2-CH-(CH3)2, N(CH3)2, N(CH2-CH3)2 or OH.

[0696] In some embodiments, the peptide disclosed herein may comprise a modification of the N-terminus of the peptide sequence with one or more peptide-based moieties. In some embodiments, the peptide disclosed herein may comprise a modification of the C-terminus of the peptide sequence with one or more peptide-based moieties. In some embodiments, the peptide disclosed herein may comprise modifications of both the N-terminus and the C-terminus of the peptide sequence with one or more peptide-based moieties.

[0697] In some embodiments, the peptide disclosed herein may comprise a modification of the N-terminus of the peptide sequence with one or more non-peptide-based portions. In some embodiments, the peptide disclosed herein may comprise a modification of the C-terminus of the peptide sequence with one or more non-peptide-based portions. In some embodiments, the peptide disclosed herein may comprise modifications of both the N-terminus and the C-terminus of the peptide sequence with one or more non-peptide-based portions.

[0698] In some embodiments, the peptide disclosed herein may include an N-terminal modification comprising a string of 5 or 6 Glu amino acids. In some embodiments, the peptide disclosed herein may include an N-terminal modification comprising a string of 5 or 6 Lys amino acids. In some embodiments, the peptide disclosed herein may include an N-terminal modification comprising a string of 5 or 6 amino acids, each amino acid being independently selected from Glu or Lys.

[0699] In some embodiments, the peptide disclosed herein comprises an N-terminal peptide consisting of a chain of about 15 to about 400 identical amino acids. In some embodiments, the N-terminal peptide comprises about 25 to about 300 identical amino acids, about 50 to about 200 identical amino acids, about 75 to about 150 identical amino acids, about 90 to about 120 identical amino acids, or about 100 or 110 identical amino acids. In some embodiments, the N-terminal peptide comprises: poly(glutamate) polypeptide (PGa), poly(aspartic acid) polypeptide (PAs), poly(lysine) polypeptide (PLy), poly(arginine) polypeptide (PAr), poly(histidine) polypeptide (PHi), poly(ornithine) polypeptide (POr), or combinations thereof.

[0700] In some embodiments, the peptides disclosed herein comprise an N-terminal modification. In other embodiments, the N-terminal modification comprises an N-terminal acetyl group (denoted as Ac). For example, in SEQ ID NOs: 24-44 and 67-89 of Table 2, the N-terminal methionine group is capped with acetic anhydride or other suitable reagent during peptide synthesis to produce an N-terminal acetylated molecule. In some embodiments, the peptides disclosed herein comprise a C-terminal modification. In other embodiments, the C-terminal modification comprises an amide group (denoted as amide or CONH2). For example, in SEQ ID NOs: 24-44 and 67-89 of Table 2, the C-terminal serine group is synthesized as an amide during peptide synthesis to produce a C-terminal amidated molecule.

[0701] In some embodiments, the targeting portion includes one or more peptide sequences or fragments or variants thereof that bind to DLL3, as listed in Table 2. In some embodiments, the targeting construct includes targeting portions that include one or more peptide sequences or fragments or variants thereof that bind to DLL3, as listed in Table 2.

[0702] For example, the amino acid sequence having SEQ ID NO: 3-24 is an unmodified linear peptide without a capping group before disulfide bond formation. The amino acid sequence having SEQ ID NO: 24-44 is a linear peptide having an acetyl group at the N-terminus and an amide group at the C-terminus before disulfide bond formation. The amino acid sequence having SEQ ID NO: 45-66 is an unmodified cyclic peptide without a capping group after disulfide bond formation. The amino acid sequence having SEQ ID NO: 67-89 is a cyclic peptide having an acetyl group at the N-terminus and an amide group at the C-terminus after disulfide bond formation.

[0703] Table 2. Peptide sequences that bind to DLL3

[0704]

[0705]

[0706] The polypeptides disclosed herein may be peptide mimics. A “peptide mimic” or “polypeptide mimic” is a polypeptide containing structural elements in its molecule that are not found in natural polypeptides (i.e., polypeptides composed of only 20 protein amino acids). In some embodiments, peptide mimics are capable of reproducing or mimicking one or more biological effects of natural peptides. Peptide mimics can differ from natural polypeptides in many ways, for example, through changes in skeletal structure or through the presence of amino acids not found in nature. In some cases, peptide mimics may include amino acids having side chains not found in the known 20 protein amino acids; non-peptide-based bridging moieties for creating cyclization between terminal or internal portions of the molecule; substitution of the hydrogen moiety of an amide bond by a methyl group (N-methylation) or other alkyl group; substitution of the α-hydrogen moiety of an amino acid by a methyl group (α-methylation) or other alkyl group; substitution of the peptide bond by a chemical group or bond resistant to chemical or enzymatic treatment; N-terminal and C-terminal modifications; and / or conjugation with non-peptide extensions (e.g., polyethylene glycol, lipids, carbohydrates, nucleosides, nucleotides, nucleoside bases, various small molecules, or phosphate or sulfate groups).

[0707] As used herein, the term "amino acid" includes both naturally occurring amino acids and residues of non-natural amino acids. Twenty naturally occurring protein amino acids are identified and referred to herein by the following single-letter or three-letter names: aspartic acid (Asp:D), isoleucine (Ile:I), threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine ​​(Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q), methionine (Met:M), and asparagine (Asn:N). Naturally occurring amino acids exist in their levorotatory (L) stereoisomer form. Unless otherwise specified, the amino acid referred to herein is the L-stereoisomer. The term "amino acid" also includes amino acids with conventional amino protecting groups (such as acetyl or benzyloxycarbonyl), as well as natural and non-natural amino acids protected at a carboxyl terminus (e.g., as (C1-C6) alkyl, phenyl, or benzyl esters or amides; or as α-methylbenzylamides). Other suitable amino and carboxyl protecting groups are known to those skilled in the art (see, for example, Greene, TW; Wutz, PGM, Protecting Groups In Organic Synthesis; 2nd edition, 1991, New York, John Wiley & Sons, Inc., and the references cited therein, the contents of which are incorporated herein by reference in their entirety). The peptides and / or peptide compositions disclosed herein may also include modified amino acids.

[0708] “Non-natural” amino acids have side chains or other features not present in the 20 naturally occurring amino acids listed above, and include, but are not limited to: N-methyl amino acids, N-alkyl amino acids, α,α-substituted amino acids, β-amino acids, α-hydroxy amino acids, D-amino acids, and other non-natural amino acids known in the art (see, for example, Josephson et al., (2005) J. Am. Chem. Soc. [Journal of the American Chemical Society] 127: 11727-11735; Forster, AC et al. (2003) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 100: 6353-6357; Subtelny et al., (2008) J. Am. Chem. Soc. [Journal of the American Chemical Society] 130: 6131-6136; Hartman, MCT et al. (2007) PLoS ONE [PLOS ONE] 2:e972; and Hartman et al., (2006) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 103:4356-4361). Other non-natural amino acids that may be used to optimize the peptides and / or peptide compositions disclosed herein include, but are not limited to, 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, 1-amino-2,3-hydro-1H-indene-1-carboxylic acid, high lysine, high arginine, high serine, 2-aminohexanoic acid, 3-aminohexanoic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 5-aminovaleric acid, 5-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, desmosin, 2,3-Diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesin, alloleucine, N-methylpentylglycine, naphthylalanine, ornithine, pentylglycine, thioproline, valine, tert-butylglycine (also known as tert-leucine), phenylglycine, azatryptophan, 5-azatryptophan, 7-azatryptophan, 4-fluorophenylalanine, penicillamine, sarcosine, homocysteine, 1-aminocyclopropionic acid, 1-aminocyclobutylcarboxylic acid, 1-aminocyclopentylcarboxylic acid, 1-aminocyclohexylcarboxylic acid, 4-aminotetrahydro-2H-pyran-4-carboxylic acid, (S)-2-amino 3-(1H-tetrazol-5-yl)propionic acid, cyclopentylglycine, cyclohexylglycine, cyclopropylglycine, n-ω-methyl-arginine, 4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine, 5-fluorotryptophan, 5-chlorotryptophan, citrulline, 4-chloro-homophenylalanine, homophenylalanine, 4-aminomethyl-phenylalanine, 3-aminomethyl-phenylalanine, octylglycine, leucine, tranexamic acid, 2-aminovaleric acid, 2-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, 2-aminodecanoic acid, 2-aminoundecanoic acid, 2-aminododecanoic acid, aminovaleric acid, and 2-(2-aminoethoxy)acetic acid, piperidine acid. (acid), 2-carboxylated aziridine, hexafluoroleucine, 3-fluorovaline, 2-amino-4,4-difluoro-3-methylbutyric acid, 3-fluoro-isoleucine, 4-fluoro-isoleucine, 5-fluoro-isoleucine, 4-methyl-phenylglycine, 4-ethyl-phenylglycine, 4-isopropyl-phenylglycine, (S)-2-amino-5-azidopentanoic acid (also referred to as "X02" in this article), (S)-2-amino-heptane-6-enoic acid (also referred to as "X30" in this article), (S)-2-aminopentane-4-alkynic acid (also referred to as "X31" in this article), (S)-2-aminopentane-4-enoic acid (also referred to as "X12" in this article), (S)-2-amino-5-(3-methylguanidino)pentanoic acid, (S)-2-amino -3-(4-(aminomethyl)phenyl)propionic acid, (S)-2-amino-3-(3-(aminomethyl)phenyl)propionic acid, (S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl)butyric acid, (S)-leucine, (S)-valine, (S)-tert-leucine, (R)-3-methylbut-2-amine, (S)-2-methyl-1-phenylprop-1-amine, and (S)-N,2-dimethyl-1-(pyridin-2-yl)prop-1-amine, (S)-2-amino-3-(oxazol-2-yl)propionic acid, (S)-2-amino-3-(oxazol-5-yl)propionic acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl)propionic acid, (S)-2-amino-3-(1,2,4-oxadiazol-2-yl)propionic acid, (S)-2-amino-3-(1,2,4-Oxadiazol-3-yl)propionic acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propionic acid and (S)-2-amino-3-(1H-indazol-3-yl)propionic acid, (S)-2-amino-3-(oxazol-2-yl)butyric acid, (S)-2-amino-3-(oxazol-5-yl)butyric acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl) Butyric acid, (S)-2-amino-3-(1,2,4-oxadiazol-3-yl)butyric acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)butyric acid, and (S)-2-amino-3-(1H-indazol-3-yl)butyric acid, 2-(2'MeOphenyl)-2-aminoacetic acid, tetrahydro-3-isoquinolinecarboxylic acid and their stereoisomers (including but not limited to D and L isomers).

[0709] Additional non-natural amino acids that may be used to optimize the peptides or peptide compositions disclosed herein include, but are not limited to, halogenated amino acids, wherein one or more carbon-bonded hydrogen atoms are replaced by one or more halogen atoms. The number of halogen atoms included may range from 1 to include all hydrogen atoms.

[0710] In some embodiments, the non-natural amino acids that can be used to optimize the peptides or peptide compositions disclosed herein include, but are not limited to, fluoroamino acids, wherein one or more carbon-bonded hydrogen atoms are replaced by one or more fluorine atoms. The number of fluorine atoms included may range from 1 to a maximum and includes all hydrogen atoms. Examples of such amino acids include, but are not limited to, 3-fluoroproline, 3,3-difluoroproline, 4-fluoroproline, 4,4-difluoroproline, 3,4-difluoroproline, 3,3,4,4-tetrafluoroproline, 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, 7-fluorotryptophan, and their stereoisomers.

[0711] In some embodiments, the non-natural amino acids that can be used to optimize the peptides or peptide compositions disclosed herein include, but are not limited to, chlorinated amino acids, wherein one or more carbon-bonded hydrogen atoms are replaced by one or more chlorine atoms. The number of chlorine atoms included may range from 1 to a maximum and includes all hydrogen atoms.

[0712] Other non-natural amino acids that can be used to optimize the peptides disclosed herein include, but are not limited to, those that are disubstituted at the α-carbon. These include amino acids with the same two substituents at the α-carbon (e.g., α-aminoisobutyric acid and 2-amino-2-ethylbutyric acid) and those with different substituents (e.g., α-methylphenylglycine and α-methylproline). Furthermore, the substituents at the α-carbon can together form a ring, such as 1-aminocyclopentanoic acid, 1-aminocyclobutanoic acid, 1-aminocyclohexanecarboxylic acid, 3-aminotetrahydrofuran-3-carboxylic acid, 3-aminotetrahydropyran-3-carboxylic acid, 4-aminotetrahydropyran-4-carboxylic acid, 3-aminopyrrolidine-3-carboxylic acid, 3-aminopiperidine-3-carboxylic acid, 4-aminopiperidine-4-carboxylic acid, and their stereoisomers.

[0713] Additional non-natural amino acids that can be used to optimize the peptides or peptide compositions disclosed herein include, but are not limited to, analogs of tryptophan, wherein the indole ring system is replaced by another 9- or 10-membered bicyclic ring system comprising 0, 1, 2, 3, or 4 heteroatoms independently selected from N, O, or S. Each ring system may be saturated, partially unsaturated, or fully unsaturated. The ring system may be substituted at any substituted atom by 0, 1, 2, 3, or 4 substituents. Each substituent may be independently selected from H, F, Cl, Br, CN, COOR, CONRR', oxo, OR, NRR'. R and R' may each be independently selected from H, C1-C20 alkyl, or C1-C20 alkyl-O-C1-20 alkyl.

[0714] In some embodiments, tryptophan analogs (also referred to herein as "tryptophan analogs") may be used to optimize the peptides or peptide compositions disclosed herein. Tryptophan analogs may include, but are not limited to, 5-fluorotryptophan [(5-F)W], 5-methyl-O-tryptophan [(5-MeO)W], 1-methyltryptophan [(1-Me-W) or (1-Me)W], D-tryptophan (D-Trp), azatryptophan (including but not limited to 4-azatryptophan, 7-azatryptophan, and 5-azatryptophan), 5-chlorotryptophan, 4-fluorotryptophan, 6-fluorotryptophan, 7-fluorotryptophan, and their stereoisomers. Unless otherwise specified, the term "azatryptophan" and its abbreviation "azaTrp" as used herein refer to 7-azatryptophan.

[0715] Modified amino acid residues that can be used to optimize the peptides and / or peptide compositions disclosed herein include, but are not limited to, those that are chemically blocked (reversibly or irreversibly); those that are chemically modified on their N-terminal amino groups or their side chain groups; those that are chemically modified in the amide backbone, such as N-methylation, D (non-natural amino acid) and L (natural amino acid) stereoisomers; or residues in which the side chain functional group is chemically modified into another functional group. In some embodiments, the modified amino acids include, but are not limited to, methionine sulfoxide; methionine sulfone; aspartic acid-(β-methyl ester) (a modified amino acid of aspartic acid); N-ethylglycine (a modified amino acid of glycine); alanine formamide; and / or a modified amino acid of alanine. Non-natural amino acids are available from Sigma-Aldrich (St. Louis, Missouri), Bachem (Torrance, California), or other suppliers. Non-natural amino acids may further include any of those listed in Table 2 of U.S. Patent Publication US 2011 / 0172126, the contents of which are incorporated herein by reference in their entirety.

[0716] In some embodiments, the amino acids used in this disclosure are modified with organic protein or non-protein derivatizing agents. In some embodiments, the amino acids used in this disclosure are modified with post-translational modifications. In some embodiments, modification is introduced by reacting the target amino acid residues of the peptide with an organic derivatizing agent capable of reacting with selected side chain or terminal residues. In some embodiments, modification is introduced by utilizing a post-translational modification mechanism that functions in a selected recombinant host cell. Some post-translational modifications are the result of the action of the recombinant host cell on the expressed peptide. For example, glutamine acyl and asparagine acyl residues are typically deamidated post-translationally to their respective glutamine acyl and asparagine acyl residues under certain post-translational conditions (e.g., under weakly acidic conditions). Other post-translational modifications include: hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of tyrosine, serine, or threonine residues; and methylation of the α-amino groups of the lysine, arginine, and histidine side chains (see, for example, Creighton et al., Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, 1983, pp. 79-86).

[0717] In some embodiments, amino acid modification includes the bonding of a non-protein polymer to the peptide disclosed herein. Examples of non-protein polymers include hydrophilic synthetic polymers (i.e., non-natural polymers), such as hydrophilic polyethylene polymers (e.g., polyvinyl alcohol and polyvinylpyrrolidone). Examples of non-protein polymers also include polyethylene glycol, polypropylene glycol, and polyoxyethylene. In some embodiments, amino acid modification includes the bonding of a non-protein polymer to the peptide disclosed herein, as described in US4640835, US 4496689, US 4301144, US 4670417, US 4791192, and US 4179337; each of these, where applicable to amino acid modifications used in this disclosure, is incorporated herein by reference in its entirety.

[0718] Peptide synthesis

[0719] This disclosure provides methods for synthesizing the peptides and compounds disclosed herein. In some embodiments, the peptides of this disclosure can be obtained by inducing the formation of a covalent bond between an amino group (if provided) at the N-terminus of the peptide and a carboxyl group (if provided) of a reactive amino acid side chain portion. In some embodiments, the peptides and compounds of this disclosure can be synthesized using any known conventional procedure for forming peptide bonds between amino acids. Such conventional procedures include, for example, any solution-phase procedure that allows condensation between a free α-amino group of an amino acid or a residue thereof (whose carboxyl group or other reactive group is protected) and a free major carboxyl group of another amino acid or a residue thereof (whose amino group or other reactive group is protected). In some embodiments, the peptides of this disclosure can be synthesized by solid-phase synthesis and purified according to methods known in the art. The peptides of this disclosure can be prepared using any of a number of well-known procedures, utilizing a variety of resins and reagents.

[0720] In some embodiments, the method for synthesizing peptides can be performed by a procedure of sequentially adding each amino acid in the desired sequence to another amino acid or residue thereof, one at a time. In some embodiments, the method for synthesizing peptides can be performed by a procedure of first synthesizing multiple peptide fragments having portions of the desired amino acid sequence, and then condensing them to provide the desired peptide sequence.

[0721] In some embodiments, the method for synthesizing peptides can be performed using solid-phase peptide synthesis, which includes methods well-known and practiced in the art (e.g., the Symphony Multiplex Peptide Synthesizer (Rainin Instrument Company) Automated Peptide Synthesizer). In some embodiments, the method for synthesizing peptides can be performed using standard Fmoc methods on an automated synthesizer (e.g., Advanced ChemTech 440M05, Louisville, Kentucky). In some embodiments, the method for synthesizing peptides can be performed using coupling reagents (e.g., 2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) and / or 1-hydroxybenzotriazole (HOBt)).

[0722] Solid-phase peptide synthesis can be performed by sequentially incorporating the desired amino acid residues one at a time into the growing side chain, based on the general principles of solid-phase methods. These methods are disclosed in numerous references, including Merrifield et al., Solid phase synthesis (Nobel lecture), Angew. Chem. (1985) 24:799-810; Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2; Gross et al., ed., Academic Press, 1-284 (1980), the contents of which, where applicable to methods and schemes for peptide synthesis, are incorporated herein by reference in their entirety.

[0723] Solid-phase synthesis of peptides typically begins from the C-terminus of the peptide by coupling a protected α-amino acid to a suitable resin. Examples of known methods for the preparation of substituted amide derivatives on the solid phase have been described in the art (see, for example, Barn DR et al., Tetrahedron Letters (1996), 37:3213-3216; DeGrado et al., J. Org. Chem. (1982) 47:3258-3261; the contents relating to methods and systems for solid-phase peptide synthesis are each incorporated herein by reference in their entirety). For example, starting materials can be prepared by attaching an α-amino-protected amino acid to a p-benzyloxybenzyl alcohol (Wang) resin or an oxime resin via ester bonds using well-known methods. The peptide chain is grown according to the desired amino acid sequence, and then the peptide resin is treated with a solution of a suitable amine (e.g., methylamine, dimethylamine, ethylamine, etc.). Peptides using p-benzyloxybenzyl alcohol (Wang) resin can be cleaved from the resin by aluminum chloride in the DCM, while peptides using oxime resin can be cleaved by the DCM.

[0724] In some embodiments, the reactive side chain groups of various amino acid residues are protected by suitable protecting groups to prevent chemical reactions at the site until the protecting groups are removed. In some embodiments, the α-amino group of an amino acid residue or fragment is protected when the entity of the amino acid residue or fragment reacts at the carboxyl group, and then the α-amino protecting group is selectively removed to allow subsequent reactions to occur at the site. Examples of protecting groups used in this disclosure have been disclosed and are known in solid-phase synthesis methods and solution-phase synthesis methods.

[0725] In some embodiments, the α-amino group may be protected by a suitable protecting group, which includes: urethane-type protecting groups, such as benzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups, such as tert-butoxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl and allyloxycarbonyl.

[0726] In some embodiments, the guanidino amino group (e.g., those found in arginine) may be protected by a suitable protecting group (e.g., nitro, p-toluenesulfonyl (Tos), Z, pentamethylsomethanesulfonyl (Pmc), adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), and Boc).

[0727] As a non-limiting example, solid-phase synthesis of peptides can begin from the C-terminus of the peptide by coupling a protected α-amino acid to a suitable resin. Starting materials can be prepared by attaching the α-amino-protected amino acid to a p-benzyloxybenzyl alcohol (Wang) resin, a 2-chlorotriphenylmethyl chloride resin, or an oxime resin via an ester bond, an amide bond between an Fmoc-linker (e.g., p-[(R,S)-α-[1-(9H-fluorene-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyacetic acid (Rink linker)) and a diphenylmethylamine (BHA) resin, or by other methods well known in the art. Fmoc-linker-BHA resin supports are commercially available and commonly used (where feasible). If necessary, the resin is repeatedly added in cycles to sequentially add amino acids. The α-amino Fmoc protecting group is then removed under alkaline conditions (e.g., piperidine, piperazine, diethylamine, or morpholine in N,N-dimethylformamide (DMF) (20%–40% v / v)). Following removal of the α-amino protecting group, the protected amino acids are then sequentially coupled in the desired order to obtain the intermediate, the protected peptide resin. Activators used for amino acid coupling in the solid-phase synthesis of peptides are well known in the art. After peptide synthesis, orthogonally protected side-chain protecting groups can be removed using methods well known in the art to further derivatize the peptide, if desired.

[0728] Reactive groups in peptides can be selectively modified during solid-phase synthesis or after removal from the resin. For example, when on a resin, a peptide can be modified to obtain an N-terminal modification (e.g., acetylation), or it can be modified after removal from the resin using a cleavage reagent. Similarly, methods for modifying amino acid side chains are well known to those skilled in the art of peptide synthesis. The choice of modification to the reactive groups present on the peptide will be determined in part by the desired characteristics of the peptide.

[0729] In some embodiments, the N-terminal group is modified by introducing an N-acetyl group. As a non-limiting example, peptide synthesis may include the step of reacting a resin-bound peptide with acetic anhydride in dichloromethane in the presence of an organic base such as diisopropylethylamine after removing the protecting group at the N-terminus. Other methods of N-terminal acetylation are known in the art, including solution-phase acetylation.

[0730] In some embodiments, the peptides disclosed herein may comprise cyclic peptides having one or more bridging portions (e.g., cyclic structures, staple structures, bridges, etc.).

[0731] In some embodiments, peptides can be synthesized using solid-phase peptide synthesis, followed by cyclization before cleavage from a peptide resin. If the peptide is partially cyclized via a reactive side chain, the desired side chain is first deprotected in a suitable solvent under specific deprotection conditions, followed by the addition of a cyclic coupling agent. Suitable solvents include, but are not limited to, DMF, dichloromethane (DCM), and 1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling agents include, but are not limited to: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tris(pyrrolidinyl)phosphonium hexafluorophosphate (PyBOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TATU), 2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethylurea tetrafluoroborate (TPTU), and N,N′-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole (DCCI / HOBt). In some embodiments, coupling of the cyclic moiety to the peptide chain is initiated by using a suitable base such as N,N-diisopropylethylamine (DIPEA), symmetric chlorhexidine, or N-methylmorpholine (NMM).

[0732] The cyclized peptide can then be cleaved from the solid phase using any suitable reagent (e.g., ethylamine in DCM). The resulting crude peptide is dried and the remaining amino acid side-chain protecting groups (if any) are cleaved using a suitable reagent (e.g., trifluoroacetic acid (TFA) and 1,2-ethylenedithiol (EDT) in the presence of water). The final product is precipitated by adding cold diethyl ether and collected by filtration. Final purification can be performed by reversed-phase high-performance liquid chromatography (RP-HPLC) using a suitable column, such as a C18 column. Other separation or purification methods can also be used, such as methods based on peptide size or charge. Once purified, the peptide can be characterized using many methods, such as high-performance liquid chromatography (HPLC), amino acid analysis, mass spectrometry, etc.

[0733] In some embodiments, the peptides disclosed herein may comprise one or more modifications (e.g., substitution, addition, or deletion) to one or more ends of the peptide sequence (e.g., N-terminus, C-terminus, or both). In some embodiments, the terminally modified peptides may be synthesized using solid-phase peptide synthesis and then modified prior to cleavage from a peptide resin.

[0734] This disclosure considers variants and derivatives of the peptides provided herein. These include substitution, insertion, deletion, and covalent variants and derivatives. As used herein, the term “derivative” is used synonymously with the term “variant” and refers to a molecule that is modified or altered in any way relative to a reference molecule or an initial molecule.

[0735] In one embodiment, the peptide described herein comprises one or more D-amino acid residues that replace one or more L-amino acid residues. This embodiment is believed to increase proteolytic stability through steric hindrance and the tendency of D-amino acids to stabilize the β-turn conformation (Tugyi et al. (2005) PNAS [Proceedings of the National Academy of Sciences], 102(2), 413-418).

[0736] In some embodiments, the peptides disclosed herein may be in salt form. Salts of peptides can be synthesized from parent compounds containing a basic or acidic moiety using 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. Typically, such salts can be prepared by reacting the free acidic or basic form of these compounds with a suitable base or acid in water, in an organic solvent, or in a mixture of both.

[0737] Acid addition salts (monosal or diosal) can be formed from a variety of acids (both inorganic and organic). Examples of acid addition salts include monosal or diosal salts formed from acids selected from the group consisting of: 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-acetaminobenzoic acid, butyric acid, (+)camphoric acid, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaricaid, gentian acid, glucoheponic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxo- Glutamic acid, glycolic acid, hippuric acid, hydrohalic acids (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, dihydroxynaphthoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanate, p-toluenesulfonic acid, undecenoic acid and valeric acid, as well as acylated amino acids and cation exchange resins.

[0738] In some embodiments, the salt disclosed herein may be a salt formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, hydroxyethanesulfonic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (methanesulfonate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid, and lactobionic acid. In some embodiments, the salt may be a hydrochloride salt. In some embodiments, the salt may be an acetate salt.

[0739] If a compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO-), it can form a salt with an organic or inorganic base to produce a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions (e.g., Li-). + Na + and K + ), alkaline earth metal cations (e.g., Ca 2+ and Mg 2+ ) and other cations. Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4+). + ) and substituted ammonium ions (e.g., NH3R) + NH2R2+ NHR 3+ NR 4+ Some suitable examples of substituted ammonium ions are derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids (such as lysine and arginine). A common example of a quaternary ammonium ion is N(CH3). 4+ .

[0740] In the case of peptides containing amine functional groups, these can form quaternary ammonium salts, for example, by reacting with an alkylating agent according to methods known to those skilled in the art.

[0741] The peptides disclosed in this article can be dissociated by the following equilibrium dissociation constant (K). D Binding to target receptors: from about 0.001 nM to about 0.01 nM, from about 0.005 nM to about 0.05 nM, from about 0.01 nM to about 0.1 nM, from about 0.05 nM to about 0.5 nM, from about 0.1 nM to about 1.0 nM, from about 0.5 nM to about 5.0 nM, from about 2 nM to about 10 nM, from about 8 nM to about 20 nM, from about 15 nM to about 45 nM, from about 30 nM to about 60 nM, from about 40 nM to about 80 nM, from about 50 nM to about 100 nM, from about 75 nM to about 150 nM, from about 100 nM to about 500 nM, from about 200 nM to about 800 nM, from about 400 nM to about 1,000 nM or at least 1,000 nM.

[0742] In some embodiments, the peptides disclosed herein can have the following equilibrium dissociation constant (K). D Combined with DLL3: from about 0.001 nM to about 0.01 nM, from about 0.005 nM to about 0.05 nM, from about 0.01 nM to about 0.1 nM, from about 0.05 nM to about 0.5 nM, from about 0.1 nM to about 1.0 nM, from about 0.5 nM to about 5.0 nM, from about 2 nM to about 10 nM, from about 8 nM to about 20 nM, from about 15 nM to about 45 nM, from about 30 nM to about 60 nM, from about 40 nM to about 80 nM, from about 50 nM to about 100 nM, from about 75 nM to about 150 nM, from about 100 nM to about 500 nM, from about 200 nM to about 800 nM, from about 400 nM to about 1,000 nM or at least 1,000 nM.

[0743] Chelating agents

[0744] Chelating agents (CA), also referred to herein as "chelating agents," can include metal chelating agents that associate with a metal support (e.g., a metal nuclide support). Chelating agents can include macromolecular compounds. In some embodiments, chelating agents include acyclic or macrocyclic compounds.

[0745] Exemplary chelating agents (also known as "chelating agents") are shown in Table C below.

[0746] Table C

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754] In some embodiments, the chelating agent comprises an acyclic or macrocyclic compound. Non-limiting examples of chelating agents include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); DOTA derivatives: DO3A; diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA); DTPA derivatives: 2-(p-SCN-Bz)-6-methyl-DTPA, CHX-A''-DTPA, and cyclic anhydrides of DTPA (CA-DTPA); 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA); and NOA derivatives (e.g., BCNOTA, p-NCS-Bz-NOTA, BCN...). OT); 6-Hydroxynicotinamide (HYNIC); Ethylenediaminetetraacetic acid (EDTA); N,N′-Ethylene-di-L-cysteine; N,N′-Bis(2,2-dimethyl-2-mercaptoethyl)ethylenediamine-N,N′-diacetic acid (6SS); 1-(4-Carboxymethoxybenzyl)-N-N'-Bis[(2-mercapto-2,2-dimethyl)ethyl]-1,2-ethylenediamine-N,N'-diacetic acid (B6SS); Deferroamine (DFO); 1,1,1-Tris(aminomethyl)ethane (TAME); Tris(aminomethyl)ethane-N,N,N',N',N'',N''-hexaacetic acid (TAME) Hex); o-hydroxybenzyliminodiacetic acid; 1,4,7-triazacyclononane (TACN); 1,4,7,10-tetraazacyclododecane (Rhodotin); 1,4,7-triazacyclononane-1-succinic-4,7-diacetic acid (NODASA); 1-(1-carboxy-3-carboxypropyl)-4,7-bis-(carboxymethyl)-1,4,7-triazacyclononane (NODAGA); 1,4,7-tris(2-mercaptoethyl)-1,4,7-triazacyclononane (triazacyclononane−TM); 1,4,7-triazacyclononane-N,N′,N″-tris(methylenephosphine) ) acid (NOTP); 1,4,8,11-tetraazacyclotetradecane-N,N′',N″,N′′′-tetraacetic acid (TETA); 1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′′′,N′′′′-pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N'',N''',N'''',N''''-hexaacetic acid (HEHA); 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (TCMC); and their derivatives or analogues.

[0755] In the embodiments, the chelating agent was independently selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′′′-tetraacetic acid (DOTA), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isothiocyanopyridinecarboxylic acid (Macropa), Macrodipa, 2,2',2'',2'''-(1,10-diaza-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid (crown ether), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-(2-Carboxyethyl) (DOTAGA), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraacetic acid (TETA), 1,4,7,10,13-pentaazacyclopentadecane-N,N',N″,N′′′,N′′′′-pentaacetic acid (PEPA), and 1,4,7,10,13,16-hexaazacyclohexadecane-N,N′,N″,N′′′,N′′′′,N′′′″-hexaacetic acid (HEHA). In another embodiment, the chelating agent is selected from deferoxamine (DFO) and 5,11,16,22-tetraazahexacosanediamide (DFO). ) and N,N′-1,4-butanediylbis[N-[3-[[(1,6-dihydro-1-hydroxy-6-oxo-2-pyridyl)carbonyl]amino]propyl]-1,6-dihydro-1-hydroxy-6-oxo-2-pyridinecarboxamide] (HOPO).

[0756] In some embodiments, the chelating agents disclosed herein include DOTA, DOTAGA, or any derivatives / analytes thereof. Any chelating agent disclosed in Eisenwiener et al., Bioorg. Med. Chem. Lett., Vol. 10(18): 2133 (2000), the contents of which are incorporated herein by reference in their entirety.

[0757] Chelating agents (e.g., DOTA) can be attached to any position on the cyclic peptide without negatively affecting the binding of the cyclic peptide to its target (i.e., those skilled in the art will be able to discern how the placement of the chelating agent affects binding by conducting the studies described herein). In some embodiments, the chelating agent (e.g., DOTA) can be attached directly to the N-terminal amine or to a short linker attached to the same residue. Alternatively, the chelating agent (e.g., DOTA) can be attached via a short linker to the C-terminus or to a side chain that can tolerate its presence. In some embodiments, a crosslinking agent (e.g., dibromoxylene) pre-pre-functionalized with the chelating agent can be attached to the cyclic peptide.

[0758] Linkers

[0759] The targeting construct may include an optional linker connecting the chelating agent and the targeting moiety. The targeting construct linker may connect one or more chelating agents and one or more targeting moieties. The linker may include one or more of the following: ester, disulfide, amide, acylhydrazone, ether, carbamate, carbonate, sulfonamide, alkyl, aryl, heteroaryl, thioether, and urea.

[0760] In some embodiments, the linker comprises a cleavable linker. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises an amino acid.

[0761] As used herein, the term "linker" refers to the chemical moiety by which a chelating agent is linked to the peptide disclosed herein. In light of the content of this disclosure, any suitable linker known to those skilled in the art may be used herein.

[0762] Linkers can act as electrophiles and bond to the nucleophilic portion of the chelating agent. Alternatively, linkers can act as nucleophiles and bond to the electrophilic portion of the chelating agent. It should be understood that linkers can attach to the chelating agent via its carbon skeleton, thereby allowing all the "binding arms" of the chelating agent molecule to interact with the metal. Alternatively, one of these arms can attach to the linker.

[0763] For example, if the chelating agent binds to the carbonyl group of the chelating agent via the amine group of the cyclic peptide or optional linker, an amide bond is formed between the chelating agent and the cyclic peptide or optional linker.

[0764] In another example, when the chelating agent is DOTA and the linker is PEG, the resulting structure could be:

[0765] or .

[0766] In yet another example, when the chelating agent is DOTA and the amino acid is Lys, the resulting amino acid side chain can be:

[0767] .

[0768] Payloads

[0769] The targeted construct may include a variety of loads. In some embodiments, association between the load and the targeted construct is facilitated by a chelating agent. The load may include a radiopharmaceutical. A radiopharmaceutical load associated with the targeted construct via a chelating agent may include a radionuclide and / or a radioisotope. The chelating agent used for association between the targeted construct and such a load may include a metal chelating group. In embodiments, the chelating agent of the compounds provided herein further comprises a radioactive metal ion bound to the chelating agent via coordination bonding, thereby forming a radiometal complex.

[0770] Many radionuclides possess emission properties, including alpha, beta, gamma, and Auger emission. These can be used with targeted constructs for therapeutic and / or diagnostic purposes. For example, active agent Z may include radioisotopes such as Y-90, Y-86, I-131, Re-186, Re-188, Y-90, Bi-212, At-211, Zr-89, Sr-89, Ho-166, Sm-153, Cu-67, Cu-64, Lu-177, Ac-225, Pb-203, Bi-213, Th-227, Pb-212, Ra-223, P-32, Sc-47, Br-77, Rh-105, Pd-103, Ag-111, Pr-142, Pm-149, Gd-159, Ir-194, and / or Pt-199.

[0771] In some embodiments, the targeting construct used in imaging applications may include a radioactive isotope load that can be used as an imaging probe. Non-limiting examples of such radioactive isotopes include, but are not limited to, I-124, I-131, In-111, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-60, Cu-67, Cu-64, Lu-177, Ac-225, Bi-213, Th-227, Pb-212, Ra-223, P-32, and Sc-47. , Br-76, Br-77, Rh-105, Pd-103, Ag-111, Pr-142, Pm-149, Gd-159, In-111, Ir-194, Pt-199, Tc-99m , Co-57, Ga-66, Ga-67, Ga-68, Kr-81m, Rb-82, Sr-92, Tl-201, Y-86, Zr-89, C-11, N-13, O-15 and F-18.

[0772] In some embodiments, the target construct load includes any radioisotope (including the parent radionuclide and its daughter) listed in Table 3.

[0773] Table 3. Radioactive Isotopes

[0774]

[0775] In some embodiments, a radioactive isotope is referred to as a radionuclide. In some embodiments, a radionuclide is a therapeutically active radionuclide. Suitable therapeutically active radionuclides include, but are not limited to, those mentioned above. 32 P, 67 Cu、 186 Re、 188 Re、 89 Sr、 90 Y、 143 Ce、 177 Lu、 161 Tb, 166 Ho、 169 Er、 183 Ta、 153 Sm、 213 Bi、 131 I, 149 Tb, 47 Sc、 225 Ac、 212 Pb, 211 At、 223 Ra、 227 Th and 226 Th. In some embodiments, the radionuclide is selected from... 67 Cu、 188 Re、 90 Y、 177 Lu、 213 Bi、 131 I, 47 Sc、 225 Ac、 212 Pb, 211 At and 227 Therapeutic radionuclides of Th. In a particular embodiment, the radionuclide is selected from... 90 Y、 177 Lu、 131 I, 225 Ac、 211 At and 227 Therapeutic radionuclides of Th. In a specific embodiment, the therapeutically active radionuclide is... 177 Lu.

[0776] Alternatively, in some embodiments, the radionuclide is a diagnostically active radionuclide. Suitable diagnostically active radionuclides include, but are not limited to, those listed below. 111 In、 99m Tc, 94m Tc, 67 Ga、 68 Ga、 203 Pb, 64 Cu、 86 Y、 89 Zr、 51 Mn, 52 Mn, 123 I, 124 I, 125 I, 18 F, 76 Br 、77 Br、 152 Tb, 155 Tb, 44 Sc、 43 Sc and 201 Tl. In some embodiments, the radionuclide is selected from... 111 In、 99m Tc, 67 Ga、 68 Ga、 203 Pb, 64 Cu、 86 Y、 89 Zr、 123 I, 124 I, 125 I, 18 F, 76 Br 、77 Br、 152 Tb, 155 Tb, 44 Sc and 43 Sc is a diagnostically active radionuclide.

[0777] In a particular embodiment, the radionuclide is selected from... 111 In、 99m Tc, 68 Ga、 64 Cu、 89 Zr、 123 I, 124 I and 18 F is a radionuclide with diagnostic activity. In a specific embodiment, the radionuclide with diagnostic activity is... 68 Ga. In another specific embodiment, the diagnostically active radionuclide is 18 F.

[0778] In another specific embodiment, the diagnostically active radionuclide is64 Cu. In some embodiments, the radionuclide is selected from the group consisting of: 111 In、 99m Tc, 94m Tc, 66 Ga、 67 Ga、 68 Ga、 52 Fe、 169 Er、 72 As、 97 Ru、 203 Pb, 61 Cu、 62 Cu、 64 Cu、 67 Cu、 89 Sr、 186 Re、 188 Re、 86 Y、 90 Y、 89 Zr、 51 Cr 52 Mn, 51 Mn, 177 Lu、 169 Yb、 175 Yb、 105 Rh、 166 Dy、 166 Dy、 166 Ho、 153 Sm、 149 Pm, 151 Pm, 172 Tm、 121 Sn、 117m Sn、 212 Bi、 213 Bi、 142 Pr、 143 Pr、 198 Au、 199 Au、 123 I, 124 I, 125 I, 131 I, 75 Br、 76 Br、 77 Br、 80 Br、 82 Br、 18 F, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 43 Sc、 44 Sc、 47 Sc、 212Pb, 211 At、 223 Ra、 227 Th、 226 Th、 82 Rb、 32 P, 76 As、 89 Zr、 111 Ag、 165 Er、 225 Ac and 227 Ac.

[0779] In some embodiments, a radionuclide is 111 In、 99m Tc, 67 Ga、 68 Ga、 203 Pb, 64 Cu、 86 Y、 89 Zr、 123 I, 124 I, 125 I, 18 F, 76 Br、 77 Br、 152 Tb, 155 Tb, 44 Sc、 43 Sc、 67 Cu、 188 Re、 90 Y、 177 Lu、 213 Bi、 131 I, 47 Sc、 225 Ac、 212 Pb, 211 At or 227 Th.

[0780] In some embodiments, a radionuclide is 66 Ga、 67 Ga、 68 Ga、 64 Cu、 177 Lu or 225 Ac. In some embodiments, the radionuclide is 111 In、 99m Tc, 68 Ga、 64 Cu、 89 Zr、 123 I, 124 I, 18 F, 90 Y、 177 Lu、131 I, 225 Ac、 211 At or 227 Th.

[0781] In some embodiments, the radionuclide is 177 Lu. In some embodiments, the radionuclide is 225 Ac. In some embodiments, the radionuclide is 68 Ga. In some embodiments, the radionuclide is 18 F.

[0782] In some embodiments, a radionuclide is 177 Lu、 161 Tb, 90 Y、 67 Cu、 131 I, 225 Ac、 212 Pb, 211 At or 227 Th.

[0783] In some embodiments, the radionuclide is a radioactive halogen, for example... 18 F, 75 Br、 76 Br、 77 Br、 80 Br、 80m Br、 82 Br、 123 I, 124 I, 125 I, 131 I or 211 At. When the radionuclide is a radioactive halogen, the term radioactive halogen includes complexes that adapt the radioactive halogen to covalent attachment to linkers or cyclic peptides or to chelate with chelating agents or form complexes. Such complexes considered under the term radioactive halogen include Si- 18 F, B- 18 F and Al- 18 F.

[0784] In some embodiments, a radioactive halogen is directly linked to a cyclic peptide or linker. For example... 131 I and 18 F (or any other radioactive halogen) may be substituted at any position on the linker or cyclic peptide suitable for substitution by a halogenated group. In some embodiments, the radioactive halogen is 18 F. In some embodiments, a chelating agent is not present when a radioactive halogen is directly linked to a cyclic peptide or linker.

[0785] In some embodiments, the targeting construct disclosed herein can be radiolabeled with a radionuclide at any site on the peptide targeting DLL3. For example, in some embodiments, the peptide targeting DLL3 is directly conjugated to a radionuclide. In one embodiment, a radionuclide is covalently attached to the peptide targeting DLL3. In another embodiment, the radionuclide can form a peptide radionuclide salt based on ion interactions.

[0786] In some embodiments, the peptide targeting DLL3 can be conjugated to a chelating agent. In one embodiment, the peptide targeting DLL3 can be radiolabeled via chelation of a radionuclide with a chelating agent. The chelation of the radionuclide with the chelating agent can be depicted using solid single bonds, dashed single bonds, or a combination thereof. For example, the chelation of a radionuclide with DOTA can be depicted as follows using solid or dashed single bonds. In some embodiments, charge can also be indicated. For example, when a radionuclide is chelated to a chelating agent, each group of the chelating radionuclide can have a negative charge, and the chelated radionuclide can have an opposite positive charge. 68 In the case of Ga, such bonds and charges can be described in this paper as follows:

[0787] .

[0788] exist 225 In the case of Ac, such bonds and charges can be described as follows (non-limiting):

[0789] .

[0790] Radionuclides can be therapeutic, diagnostic, or both. Suitable radionuclides include, but are not limited to, Auger electron-emitting radionuclides, beta-emitting (β+ or β-emitting) radionuclides, and alpha-emitting radionuclides. The choice of radionuclide type may depend on the intended use of the peptide targeting DLL3. As those skilled in the art will understand, several factors can be considered when selecting a radionuclide for a peptide targeting DLL3, such as half-life, linear energy transfer, imaging capability, and emission range in tissue. For example, beta-emitting radionuclides typically have a long emission range in tissue (e.g., 0.1–10 mm) and emit photons in an energy range readily imaged; therefore, they can be selected for use in compounds targeting DLL3 for therapeutic, diagnostic, or therapeutic diagnostic purposes. On the other hand, alpha-emitting radionuclides have a relatively short emission range in tissues (e.g., 50-100 micrometers) and are highly efficient due to the amount of energy deposited per path length (i.e., linear energy transfer), approximately 400 times more efficient than electrons (β-particles) or positrons (β+particles). Therefore, alpha-emitting radionuclides can be selected for therapeutic applications where the high efficiency of the radionuclide is desired.

[0791] Therefore, in some embodiments, the radionuclide is an alpha-emitting radionuclide. In other embodiments, the radionuclide is a beta-emitting radionuclide. In still other embodiments, the radionuclide is an Auger electron-emitting radionuclide.

[0792] Targeting constructs

[0793] In some embodiments, this disclosure provides constructs capable of targeting and / or associating with a target. Such constructs, including any combination of a target portion and a load, are referred to herein as “target constructs.” The target constructs provided herein may be targeted at DLL3.

[0794] As used herein, the term "targeting portion" refers to a component of a targeting construct or a combination of components involved in the targeting construct's localization to or association with a target. The loading components of a targeting construct may include any of a variety of compounds, including but not limited to chemical compounds, biomolecules, metals, polymer molecules, therapeutic agents, cytotoxic agents, and radiopharmaceuticals. In a particular embodiment, the targeting construct includes a targeting portion, which is a cyclic peptide targeting DLL3, attached via an optional linker to a chelating agent for associating with a radioisotope.

[0795] The targeted constructs disclosed herein may include chelating agents. As used herein, the term "chelating agent" refers to any compound capable of forming two or more bonds with a metal atom. Chelating agents can facilitate association between the targeted construct and a load comprising a metal atom. In certain embodiments, the targeted construct comprises a chelating agent for associating a radioisotope.

[0796] As used herein, the terms “chelate to” and “complex with” are intended to indicate that two separate components are linked together, for example, by one or more non-covalent bonds, such as coordinate bonds.

[0797] As used herein, the terms "radiolabeled" or "labeled" refer to the labeling of a non-radioactive compound with a radioactive isotope. Radiolabeling can be achieved, for example, via chelation or complexation of a chelating agent with a suitable radionuclide. Radiolabeling can also refer to the chemical substitution of a group on a compound with a radionuclide, for example, by forming a covalent bond, such as in... 18 In the case of F.

[0798] The targeted construct components can be associated via one or more linkers. For example, the targeted portion can be associated with a chelating agent or a support via a linker. In some embodiments, the linker includes a chelating agent (e.g., in the case where the targeted construct support includes metal atoms).

[0799] In some embodiments, the targeted construct disclosed herein includes a targeting portion optionally attached to a load or a chelating agent for associating the load via a linker. The targeted construct may include a single targeting portion and a single chelating agent, i.e., having the structure TM-L-CA, where “TM” is the targeting portion, “L” is an optional linker, and “CA” is the chelating agent. Alternatively, the targeted construct may include a single targeting portion and more than one chelating agent, such as a construct having the structure TM-L-(CA)n, where n is an integer representing the number of chelating agents. In some embodiments, n is an integer between 1 and 50 (e.g., between 2 and 20 or between 1 and 5). The targeted construct may have the structure CA-L-TM-L-CA, where each L and each CA may be the same or different.

[0800] Targeted constructs that are optionally associated with a radioactive load may be referred to herein as the corresponding analogues, i.e., “radioactive analogues” or “non-radioactive analogues” of a given targeted construct.

[0801] In some embodiments, the targeting construct may include a detectable marker. The detectable marker can be used to detect antibody binding. Examples of detectable markers include, but are not limited to, radioisotopes, fluorophores, chromophores, chemiluminescent compounds, enzymes, enzyme cofactors, dyes, metal ions, ligands, biotin, avidin, streptavidin, haptens, quantum dots, or any other detectable marker known in the art or described herein.

[0802] Formulations

[0803] In some embodiments, the composition is administered to a human, a human patient, or a subject. For the purposes of this disclosure, the phrase "active ingredient" generally refers to the construct as described herein.

[0804] Although the description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as non-human animals, or non-human mammals. It is well known that pharmaceutical compositions suitable for administration to humans are modified to make them suitable for administration to a variety of animals, and ordinary veterinary pharmacists can design and / or perform such modifications simply through ordinary (if any) experiments. Subjects considered for administration of the pharmaceutical compositions include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0805] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or subsequently developed in the field of pharmacology. Typically, such preparation methods involve the steps of associating the active ingredient with excipients and / or one or more other auxiliary ingredients, and then, if desired and / or expected, dispensing, shaping, and / or packaging the product into desired single-dose or multi-dose units.

[0806] The pharmaceutical compositions disclosed herein may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a suitable portion of such a dose, such as one-half or one-third of such a dose.

[0807] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any other ingredients in the pharmaceutical compositions disclosed herein may vary depending on the identity, body type, and / or condition of the subject being treated, and further on the route of administration of the composition. For example, the composition may contain between 0.1% and 100%, such as between 0.5% and 50%, 1%-30%, 5%-80%, or at least 80% (w / w) of the active ingredient.

[0808] The constructs disclosed herein can be formulated using one or more excipients to: (1) increase stability; (2) allow sustained or delayed release; (3) alter biodistribution; and (4) alter the in vivo release profile of the compound. Non-limiting examples of excipients include any and all solvents, dispersion media, diluents or other liquid media, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, and preservatives. Excipients disclosed herein may also include, but are not limited to, lipids, liposomes, lipid nanoparticles, polymers, cationic lipid complexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics, and combinations thereof. Accordingly, formulations disclosed herein may include one or more excipients, the amount of each excipient collectively increasing the stability of the compound.

[0809] Excipients

[0810] When suitable for a particular desired dosage form, pharmaceutical formulations may additionally contain pharmaceutically acceptable excipients, which, as used herein, include any and all solvents, dispersion media, diluents or other liquid media, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient medium is incompatible with a substance or its derivatives, for example by producing any undesirable biological effects or otherwise interacting in a harmful manner with one or more other components of the pharmaceutical composition, its use should be considered within the scope of this disclosure.

[0811] In some embodiments, the pharmaceutically acceptable excipient has a purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipient has been approved for human and veterinary use. In some embodiments, the excipient has been approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient conforms to the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0812] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Such excipients may optionally be included in the pharmaceutical composition.

[0813] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0814] Exemplary granulating agents and / or dispersants include, but are not limited to, potato starch, corn starch, cassava starch, sodium starch glycolate, clay, alginate, guar gum, citrus pomace, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crosspovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium dodecyl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0815] Exemplary surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, astragalus gum, carrageenan, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, and high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetylglycerol, monostearate). Esters, ethylene glycol distearate, glyceryl monostearate and propylene monostearate, polyvinyl alcohol), carbomer (e.g., carboxylated polymethyl methacrylate, polyacrylic acid, acrylic polymers and carboxylated vinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolauric acid). Esters [TWEEN®20], polyoxyethylene sorbitan monooleate [TWEEN®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80], polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ®45], polyoxyethylene Hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and Kolliphor® (SOLUTOL®)), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium dodecyl sulfate, PLUORINC® F 68, POLOXAMER® 188, cetrimonium bromide, cetrimonium chloride, benzalkonium chloride, sodium docusate, and / or combinations thereof.

[0816] Exemplary adhesives include, but are not limited to, starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, molasses, lactose, lactitol, and mannitol); natural and synthetic gums (e.g., gum arabic, sodium alginate, Irish moss extract, panwar gum, ghatti gum, psyllium husk mucilage, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabinogalactan); alginates; polyoxyethylene; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohols; and combinations thereof.

[0817] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Exemplary antioxidants include, but are not limited to, α-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzyl alcohol, bromonitrile glycol, bromophthalium trimethylammonium, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, xylene, cresol, ethanol, glycerol, hexadiazine, imidazoline, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcoholic preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, and / or phenethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deferoxamine mesylate, trimethylammonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®.

[0818] Exemplary buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginate, pyrogen-free raw water, isotonic saline, Ringer's solution, ethanol, and / or combinations thereof.

[0819] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicates, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium dodecyl sulfate, sodium dodecyl sulfate, and combinations thereof.

[0820] Exemplary oils include, but are not limited to, almond oil, avocado oil, babassu oil, bergamot oil, blackcurrant seed oil, borage oil, juniper oil, chamomile oil, mustard oil, caraway oil, palm wax oil, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, flaxseed oil, geraniol oil, gourd oil, grapeseed oil, hazelnut oil, sedge oil, isopropyl myristate oil, simondyl oil, macadamia nut oil, mixed lavender oil, and more. Lavender oil, lemon oil, litsea cubeba oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange salmon oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, camellia oil, vanilla (savory) oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, ailanthus oil, vetiver oil, walnut oil, and wheat germ oil. Exemplary oils include, but are not limited to, butyl stearate, caprylic / capric triglyceride, caprylic / capric triglyceride, cyclomethyl silicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecyl alcohol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0821] Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavoring agents, and / or aromas may be present in the composition, depending on the formulator's judgment.

[0822] Administration

[0823] The construct disclosed herein can be administered via any means that produces therapeutically effective results. These routes include, but are not limited to: enteral administration, gastrointestinal administration, epidural administration, oral administration, transdermal administration, epidural (spinal) administration, intracerebral administration (administered to the brain), intraventricular administration (administered to the ventricles), epidermal administration (applied to the skin), intradermal administration (administered within the skin itself), subcutaneous administration (administered under the skin), nasal administration (administered via the nose), intravenous administration (administered to the veins), intraarterial administration (administered to the arteries), intramuscular administration (administered to the muscles), intracardiac administration (administered to the heart), intraosseous infusion (administered to the bone marrow), intrathecal administration (administered to the spinal canal), intraperitoneal administration (infused or injected into the peritoneum), intravesical infusion, intravitreal administration (administered via the eye), intracavernosal injection (administered to the base of the penis), intravaginal administration, intrauterine administration, extraamniotic administration, transdermal administration (diffusion through intact skin for systemic distribution), transmucosal administration (diffusion through the mucous membranes), inhalation (nasal inhalation), sublingual administration, sublipal administration, enema administration, eye drops (administered to the conjunctiva), or ear drops. In particular embodiments, the compositions may be administered in a manner that allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barriers.

[0824] The formulations described herein contain an effective amount of the construct in a drug delivery system suitable for administration to an individual in need. These formulations can be administered parenterally (e.g., by injection or infusion). These formulations, or variations thereof, can be administered in any manner, including enteral administration, topical administration (e.g., to the eye), or via the lungs. In some embodiments, the formulation is administered topically.

[0825] Dosing

[0826] This disclosure provides methods for administering constructs as described herein to subjects in need. Constructs as described herein can be administered to subjects in any amount and via any route of administration that is effective in preventing or treating diseases, disorders, and / or conditions (e.g., diseases, disorders, and / or conditions associated with working memory deficits) or imaging such diseases, disorders, and / or conditions. The exact amount required will vary depending on the subject's species, age, general condition, severity of the disease, the specific composition, the method of administration, activity patterns, etc.

[0827] For ease of administration and dosage uniformity, the compositions disclosed herein are typically formulated in unit dosage forms. However, it will be understood that the total daily dosage of the compositions disclosed herein may be determined by the attending physician within reasonable medical judgment. The specific therapeutically effective dose level, prophylactically effective dose level, or appropriate imaging dose level for any particular patient will depend on a variety of factors, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and factors well-known in the medical field.

[0828] In some embodiments, the dosage may be sufficient to deliver doses ranging from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, from about 25 mg / kg to about 50 mg / kg, from about 50 mg / kg to about 100 mg / kg, from about 100 mg / kg to about 125 mg / kg, or from about 125 mg / kg to about 125 mg / kg per day. The compositions according to this disclosure are administered at dose levels ranging from about 150 mg / kg to about 175 mg / kg, from about 175 mg / kg to about 200 mg / kg, and from about 200 mg / kg to about 250 mg / kg of the subject's body weight, once or more daily, to achieve the desired therapeutic, diagnostic, preventative, or imaging effect. The desired dose may be delivered three times daily, twice daily, once daily, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In some embodiments, the desired dose may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When using multiple administrations, fractionated dosing regimens, as described herein, may be used.

[0829] The concentration of the construct in the pharmaceutical composition may be from about 0.01 mg / mL to about 50 mg / mL, from about 0.1 mg / mL to about 25 mg / mL, from about 0.5 mg / mL to about 10 mg / mL, or from about 1 mg / mL to about 5 mg / mL.

[0830] As used herein, “fragmented dosing” means dividing a single unit dose or total daily dose into two or more doses, for example, administering a single unit dose twice or more. As used herein, “single unit dose” is the dose of any therapeutic agent administered in one dose / once / through a single route / at a single point of contact, i.e., a single administration event. As used herein, “total daily dose” is the amount given or inoculated over a 24-hour period. It can be administered as a single unit dose. In some embodiments, a β-emitter (e.g., like...) is included. 177 The total dose (during the treatment regimen) of the construct targeting DLL3 (Lu) is from about 1 GBq to about 200 GBq. In some embodiments, the construct targeting DLL3 containing a β-emitter is administered with a total dose delivering 40 to 100 GBq of radiation. In some embodiments, the construct targeting DLL3 containing a β-emitter is administered as a single dose delivering about 1 to about 20 GBq of radiation (administered once over a 24-hour period). In some embodiments, the construct targeting DLL3 containing a β-emitter is administered as a single dose delivering about 3 to about 15 GBq of radiation (administered once over a 24-hour period). In some embodiments, the construct targeting DLL3 containing a β-emitter is administered as a single dose delivering about 5 to about 10 GBq of radiation (administered once over a 24-hour period).

[0831] In some embodiments, an α-emitter (e.g., 225The total dose (during the treatment regimen) of the construct targeting DLL3 of Ac) is from about 1 MBq to about 100 MBq, for example, about 4 MBq to about 80 MBq, for example, about 5 MBq to about 77 MBq, for example, about 5 MBq, about 6 MBq, about 8 MBq, about 10 MBq, about 13 MBq, and about 76 MBq. In some embodiments, the construct targeting DLL3 containing an α-emitter is administered with a total dose of about 20 to about 80 MBq of radiation. In some embodiments, the construct targeting DLL3 containing an α-emitter is administered as a single dose delivering about 1 to about 40 MBq of radiation (administered once over a 24-hour period). In some embodiments, the construct targeting DLL3 containing an α-emitter is administered as a single dose delivering about 5 to about 40 MBq of radiation (administered once over a 24-hour period). In some embodiments, the construct targeting DLL3 containing an α-emitter is administered as a single dose delivering about 5 to about 25 MBq of radiation (administered once over a 24-hour period).

[0832] In some embodiments, an α-emitter will be included (e.g., 225 The total dose of the construct targeting DLL3 (Ac) is administered to the subject every 4 to 10 weeks during the treatment regimen. In another embodiment, the construct is administered to the subject approximately every 6 to 8 weeks. In yet another embodiment, the construct is administered to the subject approximately every 6 weeks. In still another embodiment, the construct is administered to the subject approximately every 6 weeks for 4 to 6 cycles.

[0833] Dosage forms

[0834] The pharmaceutical compositions described herein can be formulated into the dosage forms described herein, such as topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and subcutaneous) dosage forms.

[0835] II. Methods

[0836] In some embodiments, this disclosure provides methods for preparing, using, and evaluating the compounds (e.g., targeted constructs) and compositions disclosed herein.

[0837] Therapeutic applications

[0838] In some embodiments, the methods disclosed herein include methods of treating a therapeutic indication using the compounds and / or compositions disclosed herein. As used herein, the term "therapeutic indication" means any symptom, condition, disorder, or disease that can be relieved, stabilized, improved, cured, or otherwise resolved by some form of treatment or other therapeutic intervention. In some embodiments, the methods disclosed herein include treating a therapeutic indication using the targeted constructs disclosed herein.

[0839] In the context of disease markers or symptoms, “reduction” or “decrease” refers to a significant drop in levels that is typically statistically significant. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably a drop to a level acceptable within the normal range for individuals without such a disorder.

[0840] In the context of disease markers or symptoms, “increase” or “elevation” refers to a significant increase in such a level, which is usually statistically significant. This increase may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably an increase to a level acceptable within the normal range for individuals without such a disorder.

[0841] The efficacy of treatment or disease improvement can be assessed, for example, by measuring the level of disease progression, disease remission, symptom severity, pain reduction, quality of life, the dosage of medication required to maintain treatment effectiveness, disease biomarkers, or any other measurable parameter applicable to the given disease being treated or targeted for prevention. Monitoring the efficacy of treatment or prevention by measuring any one or any combination of such parameters is within the capabilities of those skilled in the art. Regarding the administration of the compounds or compositions described herein, "effective" for a disease or disorder means that, when administered in a clinically appropriate manner, it results in a beneficial effect for at least a subset of patients, such as symptom improvement, cure, reduction of disease burden, increased lifespan, improved quality of life, or other effects generally considered positive by physicians familiar with treating that particular type of disease or disorder.

[0842] A therapeutic or preventative effect is evident when one or more parameters of a disease symptom are significantly improved (typically statistically significant), or when the symptom fails to worsen or develop as would be expected under other circumstances. For example, a favorable change of at least 10%, and preferably at least 20%, 30%, 40%, 50%, or more, in a measurable parameter of the disease can indicate effective treatment. The efficacy of a given compound or composition can also be determined using experimental animal models of a given disease, as known in the art. When using experimental animal models, therapeutic efficacy is evident when a statistically significant marker or symptom modulation is observed.

[0843] In some embodiments, the methods disclosed herein include administering the targeted constructs described herein to treat hyperproliferative diseases, metabolic diseases, infectious diseases, and / or cancer. The targeted construct formulations can be administered via a variety of routes, including but not limited to injection, oral administration, or topical administration. In some embodiments, administration is made to mucosal surfaces (lungs, nose, mouth, buccal mucosa, sublingual region, vagina, rectum) or the eyes (intraocular or transocular).

[0844] Cancer

[0845] In one respect, this article provides a method for targeting DLL3 in a subject in need, which involves administering a therapeutically effective amount of the compound disclosed herein to the individual.

[0846] On the other hand, this article provides a method for treating cancer in a subject in need, the method comprising administering to the individual a therapeutically effective amount of the compound disclosed herein.

[0847] In one embodiment, the cancer is DLL3-mediated cancer. In another embodiment, the cancer is cancer expressing DLL3. In yet another embodiment, the cancer is small cell lung cancer, urothelial carcinoma, melanoma, or squamous cell carcinoma.

[0848] In another embodiment, the neuroendocrine tumor is selected from the group consisting of: gastrointestinal and pancreatic neuroendocrine tumors, carcinoid tumors, pheochromocytomas, paragangliomas, medullary thyroid carcinomas, pulmonary neuroendocrine tumors, thymic neuroendocrine tumors, carcinoid tumors or pancreatic neuroendocrine tumors, pituitary adenomas, adrenal tumors, Merkel cell carcinoma (MCC), breast cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, head and neck tumors, urothelial carcinoma (bladder), renal cell carcinoma, hepatocellular carcinoma, GIST, Neuroblastoma, bile duct tumor, cervical tumor, Ewing sarcoma, osteosarcoma, small cell lung cancer (SCLC), prostate cancer, melanoma, meningioma, glioma, medulloblastoma, hemangioblastoma, supratentorial primitive neuroectodermal tumor, olfactory neuroblastoma, functional carcinoid tumor, insulinoma, gastrinoma, vasoactive intestinal peptide (VIP) tumor, glucagonoma, serotoninoma, histaminema, ACTH tumor, pheochromocytoma, and somatostatinoma. In another embodiment, the cancer is a neuroendocrine tumor, melanoma, or primary brain cancer. In another embodiment, the neuroendocrine tumor is selected from small cell lung cancer (SCLC, including, for example, extensive-stage (ES)-SCLC or limited-stage (LS)-SCLC), medullary thyroid carcinoma (MTC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroendocrine prostate cancer (NEPC) (e.g., NEPC occurring during treatment), small cell prostate cancer (SCPC), Merkel cell carcinoma (MCC), cervical neuroendocrine carcinoma, and G3 grade neuroendocrine tumor (NET). In some embodiments, extrapulmonary neuroendocrine carcinoma of the cervix (NEC). In some embodiments, small cell lung cancer may be extensive-stage (ES)-SCLC or limited-stage (LS)-SCLC. In some embodiments, neuroendocrine prostate cancer (NEPC) may be NEPC occurring during treatment. In some embodiments, the cancer is a solid tumor having DLL3 positivity (e.g., ≥ 1% DLL3-positive cells) as measured by immunohistochemistry (IHC).

[0849] In yet another embodiment, the cancer is selected from the group consisting of: acoustic neuroma, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (monocytic, medulloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia. Diseases, chronic myeloid leukemia (CML), colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt lymphoma, dysplastic changes (developmental abnormalities and metaplasia), embryonal carcinoma, endometrial cancer, endothelial sarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor-positive breast cancer, essential thrombocytosis, Ewing tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glial cell carcinoma. Tumors, heavy chain disease, hemangioblastoma, liver cancer, hepatocellular carcinoma, hormone-insensitive prostate cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangiosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin's and non-Hodgkin's lymphoma), malignant tumors and hyperproliferative disorders of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin and uterus, malignant tumors of the lymphatic system of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma Tumors, melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, myeloma, myxosarcoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pineal tumor, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung carcinoma, solid tumors (carcinoma and sarcoma), small cell lung cancer, stomach cancer, squamous cell carcinoma, synovial sarcoma, sweat gland carcinoma, thyroid cancer, Waldenström macroglobulinemia, testicular tumors, uterine cancer, and Wilms' tumor.

[0850] In another embodiment, the cancer is selected from the group consisting of: primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gallbladder cancer, bile duct cancer, small intestine cancer, urethral cancer, and kidney cancer. Cancer, urothelial carcinoma, female reproductive tract cancer, uterine cancer, gestational trophoblastic disease, male reproductive tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer, pituitary cancer, hemangioma, sarcoma originating from bone and soft tissue, Kaposi's sarcoma, neurocarcinoma, eye cancer, meningeal cancer, glioblastoma, neuroma, neuroblastoma, Schwannoma, solid tumors originating from hematopoietic malignancies (e.g., leukemia), metastatic melanoma, recurrent or persistent ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumors, colorectal cancer, gastric cancer, melanoma, glioblastoma multiforme, non-squamous non-small cell lung cancer, malignant glioma, epithelial ovarian cancer, primary peritoneal serous carcinoma, metastatic liver cancer, neuroendocrine carcinoma, refractory malignant tumors, triple-negative breast cancer, HER2-amplified breast cancer, nasopharyngeal carcinoma, oral cancer, biliary duct cancer. Hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN), non-thyroid medullary carcinoma, recurrent glioblastoma multiforme, neurofibromatosis type 1, CNS carcinoma, liposarcoma, leiomyosarcoma, salivary gland carcinoma, mucosal melanoma, acral / lentigines melanoma, paraganglioma, pheochromocytoma, advanced metastatic carcinoma, solid tumors, triple-negative breast cancer, colorectal cancer, sarcoma, melanoma, renal carcinoma, endometrial cancer, thyroid cancer, rhabdomyosarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumor, mantle cell lymphoma, and refractory malignancies.

[0851] In this embodiment, cancer is selected from the group consisting of: breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, urogenital tract cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, gastric cancer, skin cancer, keratoacanthoma, lung cancer, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma, bone cancer, colon cancer, colorectal cancer, adenoma, pancreatic cancer, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver and biliary tract cancer, kidney cancer, medullary disorders, lymphatic system disorders, Hodgkin's lymphoma, pilosarcoma, oral and laryngeal cancer (oral cancer), lip cancer, tongue cancer, oral cancer, laryngeal cancer, small bowel cancer, colon cancer, rectal cancer, colorectal cancer, rectal cancer, brain and central nervous system cancer, chronic myeloid leukemia (CML), and leukemia.

[0852] In another embodiment, the cancer is selected from the group consisting of: myeloma, lymphoma, or cancer selected from gastric cancer, kidney cancer, head and neck cancer, oropharyngeal cancer, non-small cell lung cancer (NSCLC), endometrial cancer, liver cancer, non-Hodgkin lymphoma, and lung cancer.

[0853] In this embodiment, the cancer is selected from the group consisting of: prostate cancer, colon cancer, lung cancer, head and neck squamous cell carcinoma, esophageal cancer, hepatocellular carcinoma, melanoma, sarcoma, gastric cancer, pancreatic cancer, ovarian cancer, and breast cancer.

[0854] In this embodiment, cancer is selected from the group consisting of: tumor, growth, carcinoma, sarcoma, leukemia, lymphoma, etc. For example, cancer includes, but is not limited to, mesothelioma, leukemia, and lymphoma, such as cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, lymphoma associated with human T-cell lymphotropic virus (HTLV) (such as adult T-cell leukemia / lymphoma (ATLL)), B-cell lymphoma, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, lymphoma and multiple myeloma, non-Hodgkin lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodysplastic syndromes, childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft tissue sarcomas, common adult solid tumors such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal, and esophageal cancers), genitourinary cancers (e.g., prostate, bladder, kidney, uterine, ovarian, and testicular cancers), lung cancers (e.g., small cell and non-small cell carcinomas), breast cancer, pancreatic cancer, melanoma and other skin cancers, gastric cancer, brain tumors, Göring syndrome-related tumors (e.g., medulloblastoma, meningioma, etc.), and liver cancer. Other exemplary forms of cancers that can be treated with the subject compound include, but are not limited to, cancers of skeletal or smooth muscle, gastric cancer, small bowel cancer, rectal cancer, salivary gland cancer, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

[0855] Other cancers that can be treated with the compounds described herein include, for example, colon cancer, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, or melanoma. Furthermore, cancers include, but are not limited to, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), kidney cancer, renal parenchymal carcinoma, cervical cancer, uterine cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary tract cancer, melanoma, brain tumors (such as glioblastoma, astrocytoma, meningioma, medulloblastoma, and peripheral neuroectodermal tumors), gallbladder cancer, bronchial cancer, multiple myeloma, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma.

[0856] On the other hand, this disclosure provides for the use of the compounds disclosed herein or pharmaceutically acceptable salts thereof in the manufacture of medicaments for the treatment of a disease in which DLL3 plays a role.

[0857] One aspect of this disclosure provides compounds that can be used to treat diseases, disorders, and conditions characterized by excessive or abnormal cell proliferation. Such diseases include, but are not limited to, proliferative or hyperproliferative disorders and neurodegenerative diseases. Examples of proliferative and hyperproliferative disorders include, but are not limited to, cancer.

[0858] On the other hand, this document provides for the use of one or more of the compounds disclosed herein in the manufacture of a medicament for treating cancer, including but not limited to the various types of cancer disclosed herein.

[0859] In some embodiments, therapeutic indications include cancer-related indications. The term “cancer” refers to a collection of diseases characterized by disordered cell growth and division, which in some cases spread between areas of the body. As used herein, the term “cancer-related indication” refers to any disease, disorder, or condition associated with cancer, cancer treatment, or a precancerous condition. Cancer-related indications include, but are not limited to, pathological conditions characterized by malignant growths, tumors, and / or hematologic malignancies. In some embodiments, the methods disclosed herein include treating cancer-related indications using the targeted constructs disclosed herein.

[0860] In various embodiments, methods for treating cancer are provided, wherein the method includes administering a therapeutically effective amount of the construct, or its salt form, as described herein, to a subject who has cancer, is suspected of having cancer, or is predisposed to cancer. According to this disclosure, cancer includes any disease or condition characterized by the uncontrolled proliferation (e.g., excessive proliferation) of cells. Cancer can be characterized by tumors, such as solid tumors or any growths.

[0861] In some embodiments, the subject may conversely lack an indication for treatment with these constructs. In some embodiments, these methods involve the use of cancer cells, including but not limited to mammalian cancer cells. In some cases, the mammalian cancer cells are human cancer cells.

[0862] In some embodiments, the constructs according to this disclosure inhibit cancer and / or tumor growth. They may also reduce one or more of cell proliferation, invasion, and metastasis, thereby making them suitable for cancer treatment.

[0863] In some embodiments, the constructs of this teaching content can be used to prevent the growth of tumors or cancer, and / or to prevent the metastasis of tumors or cancer. In some embodiments, the compositions of this teaching content can be used to shrink or cure cancer.

[0864] In some embodiments, the constructs provided herein can be used to inhibit the proliferation of cancer cells. In some embodiments, the constructs provided herein can be used to inhibit cell proliferation, such as inhibiting cell proliferation rate, preventing cell proliferation, and / or inducing cell death. Generally, the constructs described herein can inhibit the proliferation of cancer cells, or simultaneously inhibit the proliferation of cancer cells and / or induce cell death in cancer cells. In some embodiments, compared with untreated cells, treatment with the constructs disclosed herein reduces cell proliferation by at least about 25%, about 50%, about 75%, or about 90%. In some embodiments, compared with untreated cells, treatment with the constructs disclosed herein increases the cell cycle arrest marker phosphorylated histone H3 (PH3 or PHH3) by at least about 50%, about 75%, about 100%, about 200%, about 400%, or about 600%. In some embodiments, compared with untreated cells, treatment with the constructs disclosed herein increases the apoptosis marker cleaved caspase-3 (CC3) by at least 50%, about 75%, about 100%, about 200%, about 400%, or about 600%.

[0865] Furthermore, in some embodiments, the disclosed constructs effectively inhibit tumor growth in multiple types of tumors, whether measured by net size values ​​(weight, surface area, or volume) or by rate over time.

[0866] In some embodiments, treatment with the construct disclosed herein reduces the size of the tumor by about 60% or more. In some embodiments, the tumor size is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, measured by weight and / or area and / or volume.

[0867] Cancers treatable using the methods taught herein commonly occur in mammals. Mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs, horses, pigs, sheep, goats, and cattle. In various embodiments, cancers include, but are not limited to, acoustic neuroma, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (monocytic, medulloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic, and promyelocytic), acute T-cell leukemia, basal cell carcinoma, cholangiocarcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, and chronic... Myeloid leukemia (granulocytic leukemia), chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt lymphoma, dysplastic changes (developmental abnormalities and metaplasia), embryonal carcinoma, endometrial cancer, endothelial sarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor-positive breast cancer, essential thrombocytosis, Ewing tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma. Heavy chain disease, hemangioblastoma, liver cancer, hepatocellular carcinoma, hormone-insensitive prostate cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangiosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin's and non-Hodgkin's lymphoma), malignant tumors and hyperproliferative disorders of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin and uterus, T-cell or B-cell origin malignant tumors of the lymphatic system, leukemia, lymphoma, medullary carcinoma, medulloblastoma Melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, myeloma, myxosarcoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pineal tumor, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung carcinoma, solid tumors (carcinoma and sarcoma), small cell lung cancer, stomach cancer, squamous cell carcinoma, synovial malformation, sweat gland carcinoma, thyroid cancer, Waldenström macroglobulinemia, testicular tumors, uterine cancer, and Wilms' tumor.Other cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gallbladder cancer, bile duct cancer, small intestine cancer, urethral cancer, and kidney cancer. Cancer, urothelial carcinoma, female reproductive tract cancer, uterine cancer, gestational trophoblastic disease, male reproductive tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer, pituitary cancer, hemangioma, sarcoma originating from bone and soft tissue, Kaposi's sarcoma, neurocarcinoma, eye cancer, meningeal cancer, glioblastoma, neuroma, neuroblastoma, Schwannoma, solid tumors originating from hematopoietic malignancies (e.g., leukemia), metastatic melanoma, recurrent or persistent ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumors, colorectal cancer, gastric cancer, melanoma, glioblastoma multiforme, non-squamous non-small cell lung cancer, malignant glioma, epithelial ovarian cancer, primary peritoneal serous carcinoma, metastatic liver cancer, neuroendocrine carcinoma, refractory malignant tumors, triple-negative breast cancer, HER2-amplified breast cancer, nasopharyngeal carcinoma, oral cancer, biliary duct cancer. Hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN), non-thyroid medullary carcinoma, recurrent glioblastoma multiforme, neurofibromatosis type 1, CNS carcinoma, liposarcoma, leiomyosarcoma, salivary gland carcinoma, mucosal melanoma, acral / lentigines melanoma, paraganglioma, pheochromocytoma, advanced metastatic carcinoma, solid tumors, triple-negative breast cancer, colorectal cancer, sarcoma, melanoma, renal carcinoma, endometrial cancer, thyroid cancer, rhabdomyosarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumor, mantle cell lymphoma, and refractory malignancies.

[0868] In some embodiments, the targeted constructs disclosed herein are used to target cancer cells expressing DLL3. In some embodiments, the targeted constructs disclosed herein are used to treat lung cancer, breast cancer, bladder cancer, colon cancer, urothelial carcinoma, melanoma, or squamous cell carcinoma.

[0869] Theranostics

[0870] "Therapeutic diagnostics" is a term derived from the combination of therapeutics and diagnostics, and is an emerging medical field in which specific disease-targeting agents, such as radiopharmaceuticals, can be used to simultaneously or sequentially diagnose and treat medical conditions. Therapeutic diagnostics has become an important area of ​​research and development in medical physics, where altering the isotope of a radionuclide present in a given disease-targeting agent (e.g., radioligand therapy) can transform the disease-targeting agent from an imaging probe (by, for example, using β+ or γ emission isotopes to promote positron emission tomography (PET) or single-photon emission computed tomography (CT) imaging, respectively) into a therapeutic probe (by, for example, using α or β- particles or Auger electron emission isotopes to promote targeted radiotherapy).

[0871] Molecular imaging is a well-known and useful in vivo diagnostic technique. It can be used in a wide variety of methods involving the three-dimensional mapping of molecular processes such as gene expression, blood flow, physiological changes (pH, etc.), immune responses, and cell transport. It can be used to detect and diagnose diseases, select optimal treatments, and monitor the efficacy of treatments for early readouts of effectiveness.

[0872] In principle, many different techniques, including PET, single-photon emission computed tomography (SPET), optical magnetic resonance imaging (MRI), CT, and Cerenkov luminescence imaging (CLI), can be used for molecular imaging. Combinations of these modalities, such as PET / CT and SPET / CT (“multimodal imaging”), are emerging to provide improved clinical applications.

[0873] Radionuclide imaging using PET and SPET offers advantages such as extremely high sensitivity and small contrast doses (e.g., picomolars in vivo), without disrupting molecular processes. Furthermore, the targeting principles used in radionuclide imaging can also be applied to the targeted delivery of radionuclide therapy. Typically, isotopes used as radionuclides in molecular imaging or therapy are incorporated into molecules to produce pharmaceutically acceptable radiotracers for the subject.

[0874] Therefore, the construct disclosed herein can be used in radiotherapy and medical imaging for diagnostics.

[0875] Combination therapy

[0876] In some embodiments, the construct disclosed herein is combined with at least one additional active agent. The active agent can be any suitable pharmaceutical agent. The active agent can be selected from the group consisting of: hormonal therapeutic agents, antitumor drugs, chemotherapy agents, immunotherapeutic agents, immunomodulators, radiation sensitizers, DNA damage repair inhibitors, PARP (poly-ADP-ribose polymerase) inhibitors, and combinations thereof. The construct and at least one additional active agent can be administered simultaneously, sequentially, or in any order. These constructs and at least one additional active agent can be administered at different doses, different dosing frequencies, or via different routes as needed.

[0877] In some embodiments, additional active agents affect the biodistribution (i.e., tissue distribution) of the construct disclosed herein. For example, radiopharmaceuticals may accumulate in the kidneys and may pose potential radiotoxicity problems to the kidneys and surrounding organs. Additional active agents can reduce kidney accumulation or retention time. Preferably, renal turnover of the construct is reduced, while tumor uptake of the construct is unaffected. The kidneys and surrounding organs are protected without diminishing the efficacy of the construct. In a non-limiting example, the construct disclosed herein may be administered in combination with at least one amino acid or one or more analogues thereof. The amino acid or one or more analogues thereof may be a positively charged basic amino acid, such as lysine (L-lysine or D-lysine) or arginine or a combination thereof.

[0878] The additional active agent may be selected from any of the active agents described herein, such as drugs for treating cancer. It may also be a drug for relieving cancer symptoms. Non-limiting examples of drugs for relieving symptoms include: octreotide or lanreotide; interferon, cyproheptadine, or any other antihistamine. In some embodiments, there is no drug-pharmaceutical interaction between the constructed organism disclosed herein and the additional active agent. The additional active agent may be administered concurrently with the constructed organism disclosed herein.

[0879] In some embodiments, a non-radioactive analog of the construct disclosed herein may be combined with a radioactive analog of the construct. For example, the non-radioactive construct may be administered prior to the administration of the radioactive analog. In another instance, a subject may receive a mixture of a non-radioactive construct and its radioactive analog. In yet another instance, a subject may receive treatment with the non-radioactive construct first, followed by treatment with a mixture of the non-radioactive construct and its radioactive analog.

[0880] In some embodiments, a construct of this disclosure comprising a radiolabel can be combined with at least one other construct of this disclosure comprising one or more different radiolabels. For example, a construct comprising an imaging radiolabel can be combined with a construct comprising a non-imaging radiolabel. In one embodiment, a construct associated with lutetium (Lu) can be combined with a construct associated with gallium (Ga).

[0881] The constructs described herein, or formulations containing such constructs, can be used to selectively deliver therapeutic, prophylactic, or diagnostic agents to tissues of individuals or patients in need. For example, the constructs disclosed herein are used to deliver radiopharmaceutical agents to selective tissues. These tissues may be tumor tissue. Dosing regimens can be adjusted to provide the best desired response (e.g., therapeutic or prophylactic response). For example, a single bolus injection can be administered, several split doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by an emergency situation in the treatment. As used herein, a unit dosage form refers to a physically discrete unit suitable for use as a single dose in a mammalian subject to be treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect.

[0882] Diagnostic applications

[0883] In some embodiments, this disclosure provides diagnostic methods involving the use of targeted portions and / or targeted constructs. Such methods may include detecting DLL3 using any of the targeted portions and / or targeted constructs described herein. Such methods may include contacting a subject or test sample with the targeted portions and / or targeted constructs described herein. Peptides and / or targeted constructs may bind to DLL3. Targeted portions and / or targeted constructs used in detection methods may include detectable markers. Detection methods may include using a detection reagent to detect bound antibodies or peptides. As used herein, the term "detection reagent" refers to any compound or substance used to visualize or otherwise observe an object (e.g., a bound antibody or detectable marker) or event. Detection reagents may include secondary antibodies or other high-affinity compounds (e.g., biotin or avidin) that bind to the antibody or associated conjugate to be detected. Detection reagents may be or include substrates for detecting enzyme-detectable markers (e.g., associated with primary or secondary antibodies).

[0884] The diagnostic applications disclosed herein may include detecting DLL3 in a test sample comprising cells. In some embodiments, cell-associated DLL3 may be detected. Cell-associated DLL3 in a test sample can be detected by fluorescence-associated cell sorting (FACS) analysis. In some embodiments, DLL3 in a test sample can be detected by immunohistochemistry. Such methods may include using a colorimetric system or an immunofluorescence-based system for DLL3 detection.

[0885] In some embodiments, this disclosure provides a method for stratifying subjects based on the detection of DLL3 in a subject or test sample. Such a method may include detecting DLL3 in a subject or test sample according to any of the methods described herein (e.g., using a peptide or a targeting construct containing a peptide) and categorizing subjects based on the detected DLL3 levels. In a particular embodiment, the targeting construct includes a targeting portion, which is a cyclic peptide targeting DLL3.

[0886] In some embodiments, subjects may be categorized based on the presence and / or level of DLL3 in the subject or test sample. Subjects may be further categorized based on the presence and / or level of a specific DLL3 extracellular subdomain in the subject or test sample. Categorization used in subject stratification may include, but is not limited to, categorization by disease type, disease prognosis or severity, treatment suitability, and the type of treatment most likely to be successful or appropriate.

[0887] III. Kits and devices

[0888] This disclosure provides various kits and apparatuses for conveniently and / or effectively implementing the methods disclosed herein. Typically, the kits will contain sufficient quantities and / or numbers of components to allow the user to perform multiple treatments and / or multiple experiments on one or more subjects.

[0889] In one embodiment, this disclosure provides a kit for inhibiting cancer cell growth in vitro or in vivo, the kit comprising a construct of this disclosure or a combination of constructs of this disclosure, optionally in combination with any other active agent.

[0890] The kit may further include packaging and instructions and / or delivery agents to form a formulation composition. The delivery agent may comprise saline, buffer solutions, or any delivery agent disclosed herein. The amount of each component can be varied to achieve consistent, reproducible higher concentrations of saline or simple buffer formulations. Components can also be modified to increase the stability of the construct in buffer solutions over time and / or under various conditions.

[0891] This disclosure provides devices that can include the constructs disclosed herein. These devices contain stable formulations that can be readily delivered to subjects in need, such as human patients. In some embodiments, the subject has cancer.

[0892] Non-limiting examples of these devices include pumps, catheters, needles, transdermal patches, pressurized olfactory delivery devices, iontophoresis devices, and multilayer microfluidic devices. These devices can be used to deliver the constructs disclosed herein according to single, multiple, or fractionated dosing regimens. These devices can be used to deliver the constructs disclosed herein through biological tissues, intradermally, subcutaneously, or intramuscularly.

[0893] IV. Definitions

[0894] The following are definitions of various terms used to describe the compounds and compositions disclosed herein. Unless otherwise specified individually or as part of a larger group, these definitions apply to all terms used throughout this specification and the claims.

[0895] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Typically, the nomenclature used herein, as well as laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry, are those well-known and commonly used in the art.

[0896] As used herein, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical objects of that article. For example, “an element” means one or more elements. Furthermore, the use of the term “including” and other forms (such as “include,” “includes,” and “included”) is not restrictive.

[0897] As used herein, the term “about” will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. As used herein, when referring to measurable values ​​such as quantity, time interval, etc., the term “about” means covering a variation of ±20% or ±10% from the specified value, including ±5%, ±1%, and ±0.1%, because such variation is suitable for performing the disclosed methods.

[0898] As used herein, the term "administration" or similar terms refer to the provision of a therapeutic agent to a subject. Various techniques for administering therapeutic agents exist in the art, including but not limited to intravenous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration, and topical administration.

[0899] The term "alkylene" used alone or in combination with other terms refers to a divalent alkyl linking group. An alkylene group formally corresponds to an alkane, where the two CH bonds are replaced by the alkylene group at the attachment point to the rest of the compound. The term "C..." n-m"alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, methylene, ethyl-1,2-diyl, ethyl-1,1-diyl, propyl-1,3-diyl, propyl-1,2-diyl, propyl-1,1-diyl, butyl-1,4-diyl, butyl-1,3-diyl, butyl-1,2-diyl, 2-methyl-propyl-1,3-diyl, etc.

[0900] As used herein, the terms “association,” “attachment,” “joining,” “attachment,” and “tethering,” when used with respect to two or more entities, mean that these entities are physically associated or joined directly or via one or more parts acting as a linker to another to form a structure sufficiently stable to maintain physical association, such that the entities remain physically associated, for example, under working conditions, such as physiological conditions. “Association” is not necessarily through covalent chemical bonding and may include other forms of association or bonding, provided that they are sufficiently stable to maintain physical association of the “associated” entities, such as ionic bonding, hydrogen bonding, or hybridization-based connections.

[0901] In some embodiments, this disclosure provides a method for stratifying subjects based on the detection of DLL3 in a subject or test sample. Such a method may include detecting DLL3 in a subject or test sample according to any of the methods described herein (e.g., using a peptide or a targeting construct containing a peptide) and categorizing subjects based on the detected DLL3 levels. In a particular embodiment, the targeting construct includes a targeting portion, which is a cyclic peptide targeting DLL3.

[0902] As used in this article, the term "cancer" refers to a disease characterized by abnormal cell growth and division.

[0903] As used in this article, the term "cancer cell" refers to cells that grow and divide in an abnormal and uncontrolled manner.

[0904] As used herein, the term "compound" refers to a standalone chemical entity. An builder, a targeted builder, a targeted moiety, a load, a chelating agent or other builder component, as well as any fragment or variant thereof, may be referred to individually or collectively as a compound.

[0905] Compounds may exist in one or more isomers or isotopic forms (including, but not limited to, stereoisomers, geometric isomers, tautomers, and isotopes). Compounds may be provided or used in a single form or as a mixture of two or more forms (including, but not limited to, racemic mixtures of stereoisomers). Some compounds may exist in different forms that may exhibit different properties and / or activities (including, but not limited to, biological activities). For example, compounds containing asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms. As used herein, the following structures indicate the presence of a double bond, wherein the substituents may be configured as E or Z isomers:

[0906] .

[0907] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers (e.g., enantiomers and diastereomers) are expected. Compounds of this disclosure containing asymmetrically substituted carbon atoms can be isolated in either optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, for example, by resolving racemic mixtures or by stereoselective synthesis. Many geometric isomers of alkenes, C=N double bonds, etc., may also be present in the compounds described herein, and all such stable isomers are considered in this disclosure. Cis and trans geometric isomers of the compounds of this disclosure can be isolated as mixtures of isomers or as separate isomers.

[0908] Tautomers arise from the exchange of single bonds with adjacent double bonds and the accompanying proton transfer. Tautomers include proton-transfer tautomers, which are isomeric protonated states having the same empirical formula and total charge. Examples of proton-transfer tautomers include keto-enol pairs, amide-imino pairs, lactam-lactamimide pairs, amide-imino pairs, enamine-imide pairs, and cyclic forms in which protons can occupy two or more positions in the heterocyclic system, such as 1H- and 3H-imidazolium, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindole, and 1H- and 2H-pyrazoles. Tautomers can be in equilibrium or spatially locked into one form through appropriate substitution.

[0909] The compounds described herein may be provided in the form of different isotopes comprising the atoms of the compound. An "isotope" is an atom having the same atomic number but different mass numbers due to differences in the number of neutrons in its nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0910] The compounds described herein can be provided in the form of salts and can be prepared by conventional methods by combining with solvents or water molecules to form solvates and hydrates.

[0911] As used herein, the term "hydrate" refers to a complex formed by combining a compound having formula I, formula C, or any formula disclosed herein with water.

[0912] The term "solvent" refers to a complex formed by combining a compound having formula I, formula C, or any other formula disclosed herein with a solvent or a crystalline solid containing a certain amount of solvent incorporated into the crystal structure. As used herein, the term "solvent" includes hydrates.

[0913] As used herein, the term "construction" refers to an artificially manipulated molecule. Some constructs may include nucleic acids and / or peptides, which may be products of recombinant technologies and may be artificially synthesized or expressed from recombinant nucleic acid sequences. Constructs may be combinations of nucleic acids, peptides, and / or other compounds.

[0914] As used herein, the term "cyclic" refers to the presence of an unbroken ring. Cyclic molecules do not need to be circular; they simply connect to form an unbroken chain of subunits. Cyclic peptides can include "cyclic structures" formed when two amino acids are linked by a bridging portion. The cyclic structure contains all the amino acids present on the peptide between the bridging amino acids. Cyclic structures can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0915] As used herein, the terms “effective amount,” “pharmaceutical effective amount,” and “therapeutic effective amount” refer to an amount of a drug that is non-toxic but sufficient to provide the desired biological outcome. The outcome may be a reduction or relief of signs and symptoms, or any other desired alteration of a disease or biological system. In any individual case, the appropriate therapeutic amount can be determined by a person skilled in the art using standard experimental methods.

[0916] As used herein, an epitope refers to a surface or region on one or more entities capable of interacting with antibodies or other binding biomolecules. For example, a protein epitope may contain one or more amino acids and / or post-translational modifications (e.g., phosphorylated residues) that interact with antibodies. In some embodiments, an epitope may be a "conformational epitope," which refers to an epitope relating to a specific three-dimensional arrangement of an entity having or forming the epitope. For example, a conformational epitope of a protein may include a combination of amino acids and / or post-translational modifications from folded, nonlinear amino acid chain extensions.

[0917] As used in this article, the term "equilibrium dissociation constant" or "K" is used. D"K" refers to the value indicating the tendency for two or more agents (e.g., two proteins) to separate reversibly. In some cases, K... D Indicates the concentration of the primary agent at which half the total level of the secondary agent associates with the primary agent.

[0918] As used herein, “expression” of a nucleic acid sequence means one or more of the following events: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing RNA transcripts (e.g., by splicing, editing, 5′ cap formation and / or 3′ end processing); (3) translating RNA into a peptide or protein; and (4) post-translational modifications of the peptide or protein.

[0919] As used in this article, the term "half-life" or "t" refers to the period of time in which the time elapsed. 1 / 2 "Terminal half-life" refers to the time required for a given method or compound concentration to reach half of its final value. 1 / 2 "This refers to the time required for the plasma concentration of a factor to decrease by half after the factor concentration has reached a pseudo-equilibrium.

[0920] Unless otherwise stated, the term “halogenated” or “halogen” as used herein, alone or as part of another substituent, refers to a fluorine, chlorine, bromine or iodine atom, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine.

[0921] As used herein, when referring to peptides or nucleic acids, the term "identity" refers to the comparative relationship between sequences. This term describes the degree of sequence correlation between aggregated sequences and may include the percentage of matching monomeric components with vacancy alignments (if any) resolved by a specific mathematical model or computer program (i.e., an "algorithm"). The identity of related peptides can be readily calculated using known methods. Such methods include, but are not limited to, those previously described by others (Lesk, AM, ed., *Computational Molecular Biology*, Oxford University Press, New York, 1988; Smith, DW, ed., *Biocomputing: Informatics and Genome Projects*, Academic Press, New York, 1993; Griffin, AM et al., ed., *Computer Analysis of Sequence Data, Part 1*, Humana Press, New Jersey, 1994; von Heinje, G., *Sequence Analysis in Molecular Biology*, Academic Press, 1987; Gribskov, M. et al., ed., *Sequence Analysis Primer*, M. Stockton Press, New York, 1991; and Carillo et al., *Applied Math*, SIAM J. [Journal of Applied Mathematics of the Society for Industrial and Applied Mathematics, 1988, 48, 1073].

[0922] As used herein, the term "lactam bridge" refers to an amide bond that forms a bridge between chemical groups in a molecule. In some cases, lactam bridges are formed between amino acids in a peptide.

[0923] As used herein, a "linker" refers to any chemical structure that connects two or more entities or domains. A linker may include one or more chemical bonds, atoms, groups of atoms, and / or chemical groups. Examples of chemical groups that may be introduced into a linker include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclic chemical groups, each of which may optionally be substituted as described herein. A linker may include one or more unsaturated alkanes, polyethylene glycols (e.g., ethylene glycol or propylene glycol monomer units, such as diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers. A linker may include amino acids, peptides, and / or proteins.

[0924] Linkers may include carbon chains. The length of the linker carbon chain can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more atoms. Linker carbon chains may contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.).

[0925] Entities or domains linked by linkers can include, but are not limited to, atoms, chemical groups, nucleosides, nucleotides, nucleobases, sugars, nucleic acids, amino acids, peptides, proteins, protein complexes, loadings, therapeutic agents, and detectable labels. Linkers can be used for a variety of purposes, including but not limited to forming polymers or conjugates. For example, compounds considered in this disclosure include those containing more than one target agent and / or more than one loading agent. For example, the cyclic peptides disclosed herein may contain more than one chelating agent and therefore more than one radionuclide. As another example, the constructs disclosed herein may contain more than one targeting cyclic peptide as disclosed herein.

[0926] Linkers may include cleavable elements, such as disulfide bonds (-SS-) or azo bonds (-N=N-), which can be cleaved using a reducing agent or photolysis. Selectively cleavable bonds may include amide bonds, which can be cleaved, for example, by photolysis or by using tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents. Selectively cleavable bonds may include ester bonds that can be cleaved, for example, by acidic or alkaline hydrolysis.

[0927] Linkers may include, but are not limited to, pH-sensitive linkers, protease-cleavable peptide linkers, nuclease-sensitive nucleic acid linkers, lipase-sensitive lipid linkers, glycosidase-sensitive carbohydrate linkers, hypoxia-sensitive linkers, light-cleavable linkers, thermally unstable linkers, enzyme-cleavable linkers (e.g., esterase-cleavable linkers), ultrasound-sensitive linkers, and X-ray-cleavable linkers.

[0928] As used herein, the term “modulation” refers to an upregulation (i.e., activation or stimulation) or downregulation (i.e., inhibition or repression) of a response, or a combination of both or alone. Modulation is typically compared to a baseline or reference value, which can be internal or external to the entity being treated.

[0929] As used herein, the term "peptide backbone" consists of repeating units of an amino group, an α-carbon, and a carbonyl group (e.g., -NH2-CH-C(O-)). As used herein, the term "patient" refers to a subject who seeks treatment for a particular disease, disorder, or condition, needs treatment, requests treatment, receives treatment, expects treatment, or is under the care of a trained (e.g., licensed) professional. Patients can include any organism. Patient treatment can include, but is not limited to, experimental, diagnostic, preventative, and / or therapeutic treatments. Typical patients include, but are not limited to, animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans).

[0930] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one active ingredient in a form and amount that allows for effective treatment. Pharmaceutical compositions facilitate the administration of compounds to a patient or subject. Various techniques for administering compounds exist in the art, including but not limited to intravenous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration, and topical administration.

[0931] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, which relates to the delivery or transport of compounds that may be used in this disclosure within a patient or into a patient to enable them to perform their intended function. Typically, such constructs deliver or transport compounds from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense that it is compatible with other components of the formulation, including compounds that may be used in this disclosure, and is harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered astragalus gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and compatible substances used in pharmaceutical formulations.

[0932] As used herein, "pharmaceuticalally acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delay agents that are compatible with the activity of the compounds disclosed herein and are physiologically acceptable to a patient. Additional active compounds may also be incorporated into the composition. "Pharmaceuticalally acceptable carrier" may further include pharmaceutically acceptable salts of the compounds disclosed herein. Other additional ingredients that may be included in the pharmaceutical composition are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (edited by Genaro, Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0933] As used herein, the term “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of reasonable medical judgment, and is commensurate with a reasonable benefit / risk ratio (e.g., in accordance with guidelines from government agencies or other regulatory bodies such as the U.S. Food and Drug Administration).

[0934] As used herein, the phrase “pharmaceuticalally acceptable excipient” means any component present in a pharmaceutical composition and having substantially nontoxic and noninflammatory properties in a patient (except for active agents (e.g., active agents as described herein)).

[0935] As used herein, the term "pharmaceutically acceptable salt" refers to a disclosed derivative of a cyclic peptide in which the parent compound is modified by converting an existing acid or base moiety into its salt form. In some embodiments, the side-chain amino acid groups of the cyclic peptide (e.g., R...) may be modified. 0 R 1 R 2 R 3 R 4 Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and so on. Pharmaceutically acceptable salts disclosed herein include conventionally non-toxic salts of parent compounds formed from, for example, non-toxic inorganic or organic acids. Pharmaceutically acceptable salts disclosed herein can be synthesized from parent compounds containing a basic or acidic moiety using conventional chemical methods. Typically, such salts are prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, in an organic solvent, or in a mixture of both; typically, non-aqueous media (such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile) are preferred. The phrase “pharmaceutically acceptable salt” is not limited to a single salt or a 1:1 salt. For example, “pharmaceutically acceptable salt” also includes disalts, such as dihydrochlorides. A list of suitable salts can be found in Remington's Pharmaceuticals. ca The full texts of the following articles are incorporated herein by reference: *Pharmaceutical Sciences of Remington*, 17th edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418, and *Journal of Pharmaceutical Science*, 66, 2 (1977).

[0936] As used herein, the term "sample" or "biological sample" refers to a subset of its tissues, cells, or components (e.g., bodily fluids, including but not limited to blood, serum, plasma, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, and urine). A sample may further include homogenates, lysates, or extracts prepared from a subset of the whole organism or its tissues, cells, or components, or its fractions or portions (including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, external skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs). A sample may further refer to a medium, such as a nutrient broth or gel, which may contain cellular components or other biological materials, such as proteins (e.g., antibodies) or nucleic acid molecules.

[0937] As used herein, the term "subject" refers to any entity involved in or subjected to a particular process or activity. Subjects may include any living organism. Typical subjects include, but are not limited to, animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0938] As used herein, the term "target" refers to an object or entity affected by an action, or to an activity associated with an agent targeting an object or entity (e.g., an agent that "targets" an object or entity). In some embodiments, a target refers to an antigen, epitope, or other structure that an antibody or other compound binds to or selects and / or uses in the design, development, or isolation of an antigen-specific antibody or other compound. Targets may include molecular structures, including but not limited to fragments or complexes of nucleic acids, peptides, proteins, haptens, receptors, carbohydrates, glycans, enzymes, lipids, cells, and any of the foregoing.

[0939] When used to refer to the activity of a drug against an object or entity, the term "target" can be used to describe the binding activity of a drug (e.g., an antibody or related structure) to such an object or entity (e.g., an antigen or epitope). For example, an antibody that binds to a specific antigen can be described as "targeting" or "against" a specific antigen. Similarly, a compound (e.g., a targeting construct) that exhibits activity against specific cells or tissues (e.g., therapeutic or cytotoxic activity) can be said to "target" cells or tissues.

[0940] Targets may include cells (referred to herein as “target cells”). Target cells may be in vivo or in vitro. Target cells may include, for example, blood cells, lymphocytes, cells distributed along the digestive tract (e.g., oral and pharyngeal mucosa), cells forming intestinal villi, cells distributed along the large intestine, cells distributed along the animal respiratory system (nasal cavity / lungs), dermal / epidermal cells, cells of the vagina and rectum, cells of internal organs, cells of the placenta, and cells of the blood-brain barrier. In some embodiments, target cells may be cancer cells, including but not limited to those found in leukemia or tumors (e.g., tumors of the brain, lungs (small cell and non-small cell), ovaries, prostate, breast, and colon, as well as other cancers and sarcomas). In other embodiments, target cells may be part of a tissue. Tissue having target cells or other target structures is referred to herein as target tissue. Target tissue may include, but is not limited to, neuronal tissue, intestinal tissue, pancreatic tissue, liver tissue, kidney tissue, prostate tissue, ovarian tissue, lung tissue, bone marrow tissue, and breast tissue.

[0941] As used herein, the term “therapeutic effective amount” means an amount of a delivered agent that, when administered to a subject who has or is susceptible to a disease, disorder, and / or condition, is sufficient to treat, improve the symptoms of the disease, disorder, and / or condition, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition.

[0942] As used herein, the terms “treat” or “treatment” mean any action taken to reduce or alleviate a pathological process. When referring to any therapeutic indication described herein, the terms “treat” or “treatment” mean the reduction or relief of at least one symptom associated with such indication, or the slowing or reversal of the progression or anticipated progression of such indication.

[0943] As used herein, the term "prevent" means the absence of impairment or disease progression if no condition or disease occurs, or the absence of further impairment or disease progression if a condition or disease has already developed. The ability to prevent some or all of the symptoms associated with the impairment or disease is also considered.

[0944] As used herein, the term "contact" means bringing together the indicative portions of an in vitro or in vivo system. For example, "contacting" a cell with a compound includes administering the compound of the present invention to an individual, subject, or patient (e.g., a human), and, for example, introducing the compound into a sample containing a purified formulation (containing the cell).

[0945] As used herein, the term "cell" means a cell in vitro, outside the body, or in vivo. In some embodiments, an in vitro cell may be a portion of a tissue sample excised from an organism (e.g., a mammal). In some embodiments, an in vitro cell may be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism (e.g., a mammal).

[0946] As used in this article, the term "tumor" refers to a population of cells that forms in a solid tissue due to abnormal cell growth and division. Benign or "non-cancerous" tumors remain isolated, while malignant or "cancerous" tumors include cells capable of proliferating into surrounding tissues.

[0947] As used in this article, the term "tumor cell" refers to a cell that is associated with or derived from a tumor. Benign or "non-cancerous" tumor cells remain attached to the tumor, while malignant or "cancerous" tumor cells are able to proliferate into surrounding tissues.

[0948] As used in this article, the following abbreviations are defined by the structure in Table D.

[0949]

[0950]

[0951]

[0952]

[0953]

[0954]

[0955]

[0956]

[0957]

[0958]

[0959]

[0960]

[0961]

[0962]

[0963]

[0964] V. Equivalents and scope

[0965] While various disclosed embodiments have been specifically shown and described in this disclosure, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the embodiments disclosed herein and set forth in the appended claims.

[0966] Those skilled in the art will recognize or be able to determine many equivalent forms of the specific embodiments described herein using only conventional experiments. The scope of this disclosure is not intended to be limited to the foregoing description, but rather as set forth in the appended claims.

[0967] In the claims, articles such as “a,” “an,” and “the” may mean one or more, unless otherwise indicated or otherwise apparent from the context. A claim or description including “or” among one or more members of the group is considered satisfactory if one, more than one, or all members of the group are present, used in, or otherwise related to the given product or method, unless otherwise indicated or otherwise apparent from the context. This disclosure includes embodiments in which exactly one member of the group is present, used in, or otherwise related to the given product or method. This disclosure includes embodiments in which more than one or all members of the group are present, used in, or otherwise related to the given product or method.

[0968] It should also be noted that the term “comprising” is intended to be open-ended and allows, but does not require, the inclusion of additional elements or steps. Therefore, when the term “comprising” is used herein, the terms “consisting of” and “or including” are also covered and disclosed.

[0969] Where a range is given, the endpoints are included. Furthermore, it should be understood that, unless otherwise specified or otherwise clearly apparent from the context and to those skilled in the art, numerical values ​​represented as ranges in different embodiments of this disclosure may take any specific value or subrange within the stated range up to one-tenth of the lower limit unit of that range, unless the context otherwise expressly specifies.

[0970] Furthermore, it should be understood that any particular embodiment of the present disclosure within the prior art may be expressly excluded from any one or more claims. Because such embodiments are considered to be known to those skilled in the art, they may be excluded even if such exclusion is not expressly stated herein. Any particular embodiment of the compositions disclosed herein may be excluded from any one or more claims for any reason, whether or not related to the existence of prior art.

[0971] All sources cited herein, such as references, publications, databases, database entries, and techniques cited herein, are incorporated herein by reference, even if not explicitly stated in the citations. In the event of a contradiction between the cited sources and this application, the statements in this application shall prevail.

[0972] Chapter and table headings are not intended to be restrictive. Example

[0973] The compounds and methods disclosed herein are further illustrated by the following examples, which should not be construed as further limiting. Unless otherwise indicated, the practices disclosed herein will employ conventional techniques of organic synthesis, cell biology, cell culture, and molecular biology, which are within the scope of the art.

[0974] Reagent abbreviations

[0975] ACN (or MeCN): Acetonitrile

[0976] Ac2O: Acetic anhydride

[0977] AcOH: Acetic acid

[0978] Boc: tert-butoxycarbonyl

[0979] DCM: Dichloromethane

[0980] DIC: N,N′-Diisopropylcarbodiimide

[0981] DIPEA: N,N-Diisopropylethylamine

[0982] DMF: N,N-dimethylformamide

[0983] DMSO: Dimethyl sulfoxide

[0984] Et2O: Diethyl ether

[0985] Fmoc: fluorenyl methoxy carbonyl

[0986] HATU: 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisourea

[0987] HOBt: 1-Hydroxybenzotriazole

[0988] MeOH: Methanol

[0989] Mpe: 3-Methylpentan-3-yl

[0990] MTBE: Methyl tert-butyl ether

[0991] NMP: N-methyl-2-pyrrolidone

[0992] Oxyma (or OxymaPure): Ethyl cyanohydroxyiminoethyl

[0993] Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexyl)ethyl

[0994] TFA: Trifluoroacetic acid

[0995] TIPS: Triisopropylsilane

[0996] Cp RuCl(PPh3)2: Chloro(pentamethylcyclopentadienyl)bis(triphenylphosphine)ruthenium(II)

[0997] Grubbs2:(1,3-Dimethyltrimethylimidazolidine-2-yl))(tricyclohexylphosphine)benzyl ruthenium dichloride

[0998] Amino acids and building blocks

[0999] •Na-(((9H-fluorene-9-yl)methoxy)carbonyl)-1-(tert-butoxycarbonyl)-L-tryptophan (W)

[1000] •(((9H-fluorene-9-yl)methoxy)carbonyl)-L-threonine (T)

[1001] •(((9H-fluorene-9-yl)methoxy)carbonyl)-L-isoleucine (I)

[1002] •N-(((9H-fluorene-9-yl)methoxy)carbonyl)-S-triphenylmethyl-L-cysteine ​​(C)

[1003] •(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(triphenylmethylamino)propionic acid (N)

[1004] •Na-(((9H-fluorene-9-yl)methoxy)carbonyl)-Np-(tert-butoxycarbonyl)-L-histidine(H)

[1005] •(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-((3-methylpentan-3-yl)oxy)-4-oxobutyric acid (D)

[1006] •(((9H-fluorene-9-yl)methoxy)carbonyl)-L-proline (P)

[1007] •N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(1-(4,4-dimethyl-2,6-dioxocyclohexyl)ethyl)-L-lysine (K(Dde))

[1008] •N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(1-(4,4-dimethyl-2,6-dioxocyclohexyl)ethyl)-D-lysine (D-Lys(Dde))

[1009] •N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-D-lysine (D-Lys(Boc))

[1010] •(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(naphth-2-yl)propionic acid (2Nal)

[1011] •2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid (DOTA(tBu)3)

[1012] •2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-NHS)

[1013] •(S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6,6-dimethyl-5-oxoheptanic acid (gE)

[1014] • 3-((tert-butoxycarbonyl)amino)propionic acid (Boc-βAla)

[1015] •(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)pent-4-enoic acid (allyl G)

[1016] •(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-azidopropionic acid (DapN3)

[1017] •(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)pentan-4-yneic acid (Pra)

[1018] Example 1. Preparation of Targeted Constructs

[1019] The targeted construct is prepared by binding a targeting moiety to a loading substrate. The targeting moiety incorporates a peptide sequence specific to cancer cell antigens selected from DLL3. The loading substrate includes a radiopharmaceutical comprising a radioisotope. The targeting moiety and the loading substrate are bound together using a linker.

[1020] In some embodiments, the DOTA chelator can be attached to the cyclic peptide targeting portion according to the following general method.

[1021] Universal peptide synthesis of SEQ ID NO: 3 - 89 (0.1 mmol scale):

[1022] Peptides were synthesized using a CEM Liberty Blue microwave peptide synthesizer. Standard Fmoc chemistry and coupling were used, along with ProTide Rink amide resin (0.19 g / mmol).

[1023] Step 1: Deprotection: Add 20% piperidine (3 mL, 75 equivalents) from DMF to the resin and heat the mixture to 90°C for 1 minute. Then wash the resin 4 times with 4 mL of DMF.

[1024] Step 2: Dual Coupling: Add Fmoc-protected amino acids (5 equivalents, 0.2 M) from DMF, DIC (10 equivalents, 1 M) from DMF, and OxymaPure (5 equivalents, 1 M) from DMF to the microwave reaction vessel. Then heat the microwave reaction vessel to 90°C for 4 minutes, and then drain.

[1025] Step 3: Repeat steps 1 and 2 for the remaining amino acids according to Table 4 (coupling numbers 2-12).

[1026] Step 4: DOTA Single Coupling (Coupling No. 13): Remove the Fmoc group on AA No. 12 using the normal deprotection protocol (Step 1). Add 2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid (5 equivalents, 0.2 M), DIC (10 equivalents, 1 M), and OxymaPure (5 equivalents, 1 M) from DMF to the microwave reaction vessel. Then heat the microwave reaction vessel to 50°C for 10 minutes, and then drain.

[1027] Step 5: Capping (Coupling No. 14; if no DOTA chelating agent is attached): Remove the Fmoc group on AA No. 12 using the normal deprotection protocol (Step 1). After washing 4 times with DMF, add 2.5 mL of 10% Ac2O in DMF to the microwave reaction vessel and heat to 65°C for 2 minutes. Then wash the resin 4 times with 4 mL of DMF.

[1028] Table 4.

[1029]

[1030] Peptide cleavage, cyclization, and purification:

[1031] Using the CEM Razor peptide lysis system, peptide resin was transferred to porous lysis tubes, and 8 mL of lysis solution (TFA / EDT / H2O / TIS, 94 / 2.5 / 2.5 / 1, v / v / v / v) was added to each tube. The peptide resin in the lysis solution was heated to 40°C for 40 minutes.

[1032] Filter each tube and collect the filtrate in centrifuge tubes.

[1033] The peptide was then precipitated with cold diethyl ether (45 mL). The precipitated peptide was then placed on a centrifuge and allowed to rotate for at least 10 minutes. The ether in each tube was then decanted, leaving the peptide at the bottom. This ether precipitation process was then repeated.

[1034] The peptide was then dissolved in 15 mL of MeCN / H₂O (1 / 1, v / v), and 0.1 M I₂ in MeOH was added dropwise until the yellow color remained stable. The mixture was stirred at 20°C for 10 minutes. The reaction was quenched by adding 0.1 M Na₂S₂O₃ dropwise until the yellow color disappeared. The reaction solution was then concentrated under reduced pressure. The crude cyclized peptide was then dissolved in 2 mL of DMSO for purification.

[1035] The crude peptide in DMSO was directly purified by preparative HPLC (acidic conditions, TFA) and then lyophilized to obtain the product.

[1036] Purification conditions:

[1037]

[1038] Example 2. Synthesis Scheme

[1039] Program D

[1040] Example: Compound number 75

[1041]

[1042] Step A. Synthesis of intermediate A of compound

[1043] Peptides were synthesized using Fmoc / t-Bu chemistry via standard solid-phase peptide synthesis (SPPS). Assembly was performed on Rink-amide MBHA resin (250 μmol, 100-200 mesh; loading 0.42 mmol / g) on ​​a CEM LibertyBlue microwave peptide synthesizer (CEM). During solid-phase peptide assembly, the side chain protecting groups were: tert-butyl for T; triphenylmethyl for C and N; Boc for W and H; Mpe for D; and Dde for D-Lys.

[1044] All amino acids were dissolved in DMF at a concentration of 0.2 M. The acylation reaction was carried out at 90°C under MW radiation for 2 minutes, wherein the activated amino acid was in 4-fold excess relative to the free amino groups in the resin. The amino acids were activated with equimolar amounts of 1 M DIC solution and 1 M Oxyma solution in DMF.

[1045] For T 2 H 8 N 6 and W 11 A double acylation reaction is carried out.

[1046] N-terminal acetylation was performed at 65°C for 2 minutes using a 10% v / v Ac2O solution in DMF under MW irradiation.

[1047] At the end of assembly, a 3% hydrazine monohydrate solution (50 mL) in DMF was percolated onto the resin for 15 minutes to selectively remove the Dde protecting group from D-Lys.

[1048] DOTA was manually incorporated into an equimolar solution of DOTA(tBu)3, DIC, and HOBt (3 Eq, 1:1:1) in NMP at room temperature, and complete acylation was monitored by an ninhydrin test.

[1049] At the end of assembly, the resin was washed with DMF, DCM, and Et2O. The peptide was lysed from the solid support at room temperature using 50 mL of TFA solution (v / v: 87.5% TFA, 5% H2O, 2.5% TIPS, 5% phenol) for approximately 4 hours. The resin was then filtered and concentrated to approximately 10 mL and precipitated in cold MTBE (80 mL). After centrifugation, the peptide precipitate was washed with cold, fresh Et2O to remove organic scavenging agents. This process was repeated twice. The final precipitate was dried, resuspended in H2O and ACN (1:1), and stirred overnight. It was then lyophilized to give crude intermediate A (376 mg, yield: 70.2%). LCMS analysis was performed for C... 98 H 133 N 25 O 26Calculated value of S2: 2141.4; Measured value: 715.6 (M+3) 3+

[1050] Step B. Synthesis of compound number 75

[1051] Intermediate compound A (intermediate A) was dissolved in H2O / ACN (1 mg / mL). Iodine (saturated solution in AcOH) was added until the yellow color remained stable. The mixture was stirred at room temperature for 5 min, then quenched with ascorbic acid and lyophilized. The crude peptide was purified by two rounds of reversed-phase HPLC (using a preparative Waters XBridge C18 column (150 x 50 mm, 130 Å, 5 µm)). Mobile phase A: H2O + 0.1% TFA, mobile phase B: ACN + 0.1% TFA. The following gradient of elution buffer B was used: 20% B to 20% B after 5 min; to 35% B after 25 min, flow rate: 80 mL / min, wavelength: 214 nm. The collected fractions were lyophilized to give compound number 75 (58 mg, yield: 15.4%).

[1052] Compound 262 was prepared using the methods described herein and the general procedure for compound 75, with 2-chlorotriphenylmethyl chloride resin instead of Rink amide resin.

[1053] Program F

[1054] Example: Compound number 158

[1055]

[1056] Step A. Synthesis of compound intermediate B

[1057] Peptides were synthesized using Fmoc / t-Bu chemistry via standard solid-phase peptide synthesis (SPPS). Assembly was performed on Rink-amide MBHA resin (100 μmol, 100-200 mesh; loading 0.42 mmol / g) on ​​a CEM LibertyBlue microwave peptide synthesizer (CEM). During solid-phase peptide assembly, the side chain protecting groups were: tert-butyl for T; triphenylmethyl for C and N; Boc for W and D-Lys; Mpe for D; and Dde for K.

[1058] All amino acids were dissolved in DMF at a concentration of 0.2 M. The acylation reaction was carried out at 90°C under MW radiation for 2 minutes, wherein the activated amino acid was in 4-fold excess relative to the free amino groups in the resin. The amino acids were activated with equimolar amounts of 1 M DIC solution and 1 M Oxyma solution in DMF.

[1059] For T2 K 8 N 6 and W 11 A double acylation reaction is carried out.

[1060] N-terminal acetylation was performed at 65°C for 2 minutes using a 10% v / v Ac2O solution in DMF under MW irradiation.

[1061] At the end of assembly, a 3% hydrazine monohydrate solution (50 mL) in DMF was percolated onto the resin for 15 minutes to selectively remove the Dde protecting group from Lys.

[1062] C12 (dodecanoic acid) was manually incorporated into an equimolar solution of C12, DIC, and HOBt (3 Eq, 1:1:1) in NMP at room temperature, and complete acylation was monitored by a ninhydrin test.

[1063] At the end of assembly, the resin was washed with DMF, DCM, and Et2O. The peptides were lysed from the solid support at room temperature using 15 mL of TFA solution (v / v: 87.5% TFA, 5% H2O, 2.5% TIPS, 5% phenol) for approximately 1.5 hours. The resin was then filtered and concentrated to approximately 4 mL and precipitated in cold MTBE (40 mL). After centrifugation, the peptide precipitate was washed with cold, fresh Et2O to remove organic scavenging agents. This process was repeated twice. The final precipitate was dried, and the crude material was used directly for the next step.

[1064] The crude peptide was dissolved in H2O / ACN (1 mg / mL). Iodine (saturated solution in AcOH) was added until the yellow color remained stable. The mixture was stirred at room temperature for 5 min, then quenched with ascorbic acid and lyophilized. Half of the material was purified by reversed-phase HPLC (using a preparative Waters Deltapak C4 column (200 x 25 mm, 300 Å, 15 µm)). Mobile phase A: H2O + 0.1% TFA, mobile phase B: ACN + 0.1% TFA. The following gradient of elution buffer B was used: 30% B to 30% B over 5 min; to 45% B over 25 min, flow rate: 50 mL / min, wavelength: 214 nm. The collected fractions were lyophilized to give compound intermediate B (8.8 mg, yield: 9.7%). LCMS analysis yielded a calculated value of 1915.28 for C92H131N21O20S2; the measured value was 959.0 (M+2). 2+

[1065] Step B. Synthesis of compound number 158

[1066] Intermediate compound B (intermediate B) was dissolved in DMSO (20 mg / mL). DIPEA (10 equivalents) was added, followed by DOTA-NHS (3 equivalents). After 4 h, the reaction was complete (monitored by UPLC-MS), and the mixture was quenched with TFA. The crude peptide was purified by reverse-phase HPLC (using a preparative Waters XBridge C4 column (150 x 19 mm, 300 Å, 5 µm)). Mobile phase A: H2O + 0.1% TFA, mobile phase B: ACN + 0.1% TFA. The following gradient of elution buffer B was used: 35% B to 35% B over 5 min; to 55% B over 25 min, flow rate: 25 mL / min, wavelength: 214 nm. The collected fractions were lyophilized to give compound number 158 (7.8 mg, yield: 74%).

[1067] Program G

[1068] Example: Compound number 252

[1069]

[1070] Solid-phase peptide synthesis:

[1071] Peptides were synthesized using Fmoc / t-Bu chemistry via standard solid-phase peptide synthesis (SPPS). Assembly was performed on ProTide Rink amide resin (0.1 mmol scale, 0.19 g / mmol) on a CEM LibertyBlue microwave peptide synthesizer.

[1072] Fmoc deprotection was performed by adding 20% ​​(v / v) piperidine (3 mL) in DMF to the resin, and the mixture was heated to 90°C for 1 minute. The resin was then washed four times with DMF.

[1073] Amide coupling: Add Fmoc-protected amino acids (5 equivalents), DIC in DMF (10 equivalents, 1 M), and OxymaPure in DMF (5 equivalents, 1 M) to a microwave reaction vessel. Then heat the microwave reaction vessel to 90°C for 4 minutes, and then drain. For dual coupling, repeat this process twice.

[1074] Except for Fmoc-D-Lys(Dde)-OH (single coupling) at position 13, all positions undergo double acylation.

[1075] After final Fmoc deprotection, N-terminal acetylation was performed at 65°C under MW irradiation for 2 minutes using 2.5 mL of 10% v / v Ac2O solution in DMF. The resin was then washed four times with 4 mL of DMF.

[1076] Orthogonal deprotection of Dde from D-Lys: Add 4 mL of 2% hydrazine monohydrate solution in DMF to a microwave reaction vessel and mix for 30 minutes at room temperature. Then wash the resin 6 times with 4 mL of DMF.

[1077] The remaining residues are assembled as follows: Fmoc-PEG8-OH (double coupling), Dde-D-Lys(Fmoc)-OH (double coupling), DOTA(tBu)3 (tri-coupling), orthogonal protection to remove the Dde group (2% hydrazine in DMF, 30 min), Fmoc-PEG12-OH (double coupling), and finally, 4-bromophenylacetic acid (double coupling).

[1078] At the end of assembly, the resin is washed with DMF and DCM.

[1079] Peptide cleavage, cyclization, and purification:

[1080] Using a CEM Razor peptide lysis system, peptides were lysed from the resin for 30 minutes at 40°C using 8 mL of lysis solution (TFA / DODT / H2O / TIS, 92.5 / 2.5 / 2.5 / 2.5, v / v / v / v). The resin was then filtered, and the peptides were precipitated with cold diethyl ether (45 mL). The precipitated peptides were then placed on a centrifuge and allowed to rotate for at least 5 minutes. The ether was decanted, leaving the peptides at the bottom of the centrifuge tube. This ether precipitation process was then repeated.

[1081] The peptide was then dissolved in 15 mL of MeCN / H₂O (1 / 1, v / v), and 0.1 M I₂ in MeOH was added dropwise until the yellow color remained stable. The mixture was shaken at 20°C for 10 minutes. The reaction was quenched by adding 0.1 M Na₂S₂O₃ dropwise until the yellow color disappeared. The reaction solution was then concentrated under reduced pressure. The crude cyclized peptide was then dissolved in 2 mL of DMSO for purification.

[1082] The crude peptide was purified using a preparative HPLC column (using a preparative Waters XBridge C18 column (100x19 mm, 5 µm OBD)). Mobile phase A: H₂O + 0.1% TFA, Mobile phase B: ACN + 0.1% TFA. The following elution gradient of elution buffer B was used: 0–0.5 min, 20% B; 0.5–14 min, 20%–40% B; 14–15 min, 40%–95% B; 15–17 min, hold at 95% B; 17–18 min, 20% B. Column temperature: Ambient temperature. Flow rate: 24 mL / min. The collected fractions were lyophilized to obtain the desired product.

[1083] General Program H

[1084] Example: Compound number 354

[1085]

[1086] Step A. Synthesis of compound intermediate C

[1087] Peptides were synthesized using Fmoc / t-Bu chemistry via standard solid-phase peptide synthesis (SPPS). Assembly was performed on Rink-amide MBHA resin (100 μmol, 100-200 mesh; loading 0.42 mmol / g) on ​​a CEM LibertyBlue microwave peptide synthesizer (CEM). During solid-phase peptide assembly, the side chain protecting groups were: tert-butyl for T and gE; triphenylmethyl for C and N; Boc for W and H; Mpe for D; and Dde for D-Lys.

[1088] All amino acids were dissolved in DMF at a concentration of 0.2 M. The acylation reaction was carried out at 90°C under MW radiation for 2 minutes, wherein the activated amino acid was in 4-fold excess relative to the free amino groups in the resin. The amino acids were activated with equimolar amounts of 1 M DIC solution and 1 M Oxyma solution in DMF.

[1089] For T 2 H 8 N 6 and W 11 A double acylation reaction is carried out.

[1090] N-terminal acetylation was performed at 65°C for 2 minutes using a 10% v / v Ac2O solution in DMF under MW irradiation.

[1091] At the end of assembly, a 3% hydrazine monohydrate solution (50 mL) in DMF was percolated onto the resin for 15 minutes to selectively remove the Dde protecting group from Lys.

[1092] D-Lys side chain derivatization was performed manually using Fmoc-Glu-OtBu(gE) and DOTA(tBu)3. Acylation was carried out at room temperature using an equimolar solution of acid, DIC, and HOBt (3 Eq, 1:1:1) in NMP, and complete acylation was monitored by a ninhydrin test. Fmoc deprotection was performed at room temperature using a 20% piperidine solution in DMF (2 x 5 min).

[1093] At the end of assembly, the resin was washed with DMF, DCM, and Et2O. The peptide was lysed from the solid support at room temperature using 20 mL of TFA solution (v / v: 87.5% TFA, 5% H2O, 2.5% TIPS, 5% phenol) for approximately 4 hours. The resin was then filtered and concentrated to approximately 4 mL and precipitated in cold MTBE (40 mL). After centrifugation, the peptide precipitate was washed with cold, fresh Et2O to remove organic scavenging agents. This process was repeated twice. The final precipitate was dried, resuspended in H2O and ACN (1:1), and stirred overnight. It was then lyophilized to give crude intermediate C (186 mg, yield: 82%). LCMS analysis was performed for C. 103 H 140 N 26 O 29 Calculated value of S2: 2270.50; Measured value: 758.5 ​​(M+3) 3+

[1094] Step B. Synthesis of compound number 354

[1095] Intermediate compound A was dissolved in H₂O / ACN (1 mg / mL). Iodine (saturated solution in AcOH) was added until the yellow color remained stable. The mixture was stirred at room temperature for 5 min, then quenched with ascorbic acid and lyophilized. The crude peptide was purified by reversed-phase HPLC (using a preparative Waters XBridge C18 column (250 x 50 mm, 130 Å, 5 µm)). Mobile phase: Al₂O + 0.1% TFA; Mobile phase B: ACN + 0.1% TFA. The following gradient of elution buffer B was used: 20% B to 20% B after 5 min; to 35% B after 25 min, flow rate: 80 mL / min, wavelength: 214 nm. The collected fractions were lyophilized to give 23 mg of compound number 354 (purity less than 90%).

[1096] The peptide was then repurified by reversed-phase HPLC (using a preparative Phenomenex Luna C18 column (250 x 30 mm, 100 Å, 5 µm)). Mobile phase A: H₂O + 0.1% TFA, mobile phase B: ACN + 0.1% TFA. The following gradient was used with elution buffer B: 20% B to 20% B over 5 min; to 35% B over 25 min, flow rate: 30 mL / min, wavelength: 214 nm. The collected fractions were lyophilized to give compound number 354 (7 mg, yield: 30.4%).

[1097] Program L

[1098] Example: Compound number 329

[1099]

[1100] Step A. Synthesis of intermediate H of the compound

[1101] Peptides were synthesized using Fmoc / t-Bu chemistry via standard solid-phase peptide synthesis (SPPS). Assembly was performed on Rink-amide MBHA resin (250 μmol, 100-200 mesh; loading 0.42 mmol / g) using a Cem LibertyBlue microwave peptide synthesizer (CEM). During solid-phase peptide assembly, the side chain protecting groups were: tert-butyl for T; triphenylmethyl for C and N; Boc for W and H; Mpe for D; and Dde for L-Lys.

[1102] All amino acids were dissolved in DMF at a concentration of 0.2 M. The acylation reaction was carried out at 90°C under MW radiation for 2 minutes, wherein the activated amino acid was in 4-fold excess relative to the free amino groups in the resin. The amino acids were activated with equimolar amounts of 1 M DIC solution and 1 M Oxyma solution in DMF.

[1103] For T 2 H 8 and W 11 A double acylation reaction is carried out.

[1104] N-terminal acetylation was performed at 65°C for 2 minutes using a 10% v / v Ac2O solution in DMF under MW irradiation.

[1105] At the end of assembly, a 4% hydrazine monohydrate solution (20 mL) in DMF was percolated onto the resin for 15 minutes to selectively remove the Dde protecting group from Lys.

[1106] DOTA was manually incorporated into an equimolar solution of DOTA(tBu)3, DIC, and HOBt (3 Eq, 1:1:1) in NMP at room temperature, and complete acylation was monitored by an ninhydrin test.

[1107] At the end of assembly, the resin was washed with DMF, DCM, and Et2O. The peptide was lysed from the solid support at room temperature using 20 mL of TFA solution (v / v: 87.5% TFA, 5% H2O, 2.5% TIPS, 5% phenol) for approximately 4 hours. The resin was then filtered and concentrated to approximately 5 mL and precipitated in cold MTBE (45 mL). After centrifugation, the peptide precipitate was washed with cold, fresh Et2O to remove organic scavenging agents. This process was repeated twice. The final precipitate was dried, resuspended in H2O and ACN (1:1), and stirred overnight. It was then lyophilized to give the crude compound intermediate H. LCMS analysis was performed for C... 98 H 133 N 25 O 26 Calculated value of S2: 2141.39; Measured value: 715.1 (M+3) 3+ .

[1108] Step B. Synthesis of compound number 329

[1109] Intermediate compound A (210 mg) was dissolved in H₂O / ACN (1 mg / mL). TCEP HCl (3 equivalents) was added, followed by diiodomethane (50 equivalents) and DIPEA (0.5% v / v). The mixture was stirred at room temperature. After 3 h, the reaction was complete (monitored by UPLC-MS). The reaction was quenched with TFA and lyophilized.

[1110] The crude peptide was purified by two rounds of reversed-phase HPLC (using a preparative Waters XBridge C18 column (150x50 mm, 130 Å, 5 µm)). Mobile phase A: H₂O + 0.1% TFA, mobile phase B: ACN + 0.1% TFA. The following gradient of elution buffer B was used: 20% B to 20% B after 5 min; to 35% B after 25 min, flow rate: 80 mL / min, wavelength: 214 nm. The collected fractions were lyophilized to obtain the desired TFA salts of the peptides.

[1111] The purified peptide was dissolved in H2O / ACN (1 mg / mL) and 50 mM HCl solution was added (10 equivalents relative to each amino group). The resulting solution was lyophilized, then redissolved in H2O / ACN (1 mg / mL) and lyophilized to obtain compound number 329.

[1112] General Program N

[1113] Peptides were synthesized using a CEM Liberty Blue microwave peptide synthesizer. Standard Fmoc chemistry and coupling were used, along with Wang resin (0.75 mmol / g).

[1114] Manually load Wang resin using the following procedure. Transfer Wang resin to a multi-well syringe and swell it with DMF. Add 4 eq Fmoc-protected amino acids, 4 eq HOBT, 4 eq DIC, and 1 eq DMAP to the multi-well syringe in a total of 5 mL DMF. Allow the reaction mixture to shake for 4 h. Filter the reaction mixture and wash with DMF (4 x 5 mL, 30 s each) and DCM (4 x 5 mL, 30 s each). Then dry the resin under vacuum.

[1115] Step 1. Deprotection: Add 20% piperidine (3 mL, 75 equivalents) from DMF to the resin and heat the mixture to 90°C for 1 minute. Then wash the resin with DMF (4 x 4 mL).

[1116] Step 2. Dual Coupling: Add Fmoc-protected amino acids (5 equivalents), DIC (10 equivalents, 1 M) in DMF, and OxymaPure (5 equivalents, 1 M) in DMF to the microwave reaction vessel. Then heat the microwave reaction vessel to 90°C for 4 minutes, and then drain.

[1117] Step 3. Repeat steps 1 and 2 for the remaining amino acids.

[1118] Step 4. Capping: Remove the Fmoc group on the last amino acid using the normal deprotection protocol (Step 1). After washing with DMF (4 x 4 mL), add 2.5 mL of 10% Ac2O in DMF to the microwave reaction vessel and heat to 65°C for 2 minutes. Then wash the resin with DMF (4 x 4 mL).

[1119] Step 5. Orthogonal deprotection: Wash the resin with DMF (2 x 4 mL). Add 4 mL of 2% hydrazine in DMF to the microwave reaction vessel and mix for 30 minutes at room temperature. Then wash the resin with DMF (5 x 4 mL).

[1120] Step 6. DOTA Coupling: Add the final DOTA coupling under triple coupling conditions. Add DOTA(tBu)3 (5 equivalents), DIC (10 equivalents, 1 M) in DMF, and OxymaPure (5 equivalents, 1 M) in DMF to the microwave reaction vessel. Then heat the microwave reaction vessel to 90°C for 4 minutes, and then drain. Repeat this step twice more.

[1121] At the end of assembly, the resin is washed with DMF and DCM.

[1122] Using a CEM Razor peptide lysis system, peptides were lysed from the resin for 30 minutes at 40°C using 8 mL of lysis solution (TFA / DODT / H2O / TIS, 92.5 / 2.5 / 2.5 / 2.5, v / v / v / v). The resin was then filtered, and the peptides were precipitated with cold ether (45 mL). The precipitated peptides were then placed on a centrifuge and allowed to rotate for at least 5 minutes. The ether was decanted, leaving the peptides at the bottom of the centrifuge tube. This ether precipitation process was then repeated. The peptides were then dissolved in 10 mL of MeCN / H2O (1 / 1, v / v) and lyophilized to dryness.

[1123] The crude linear peptide was dissolved in 25 mL of ACN / H₂O (1:1). DODT (2 equivalents, 32 μL) was added to the reaction mixture, followed by DIPEA (250 μL–400 μL) to adjust the pH of the reaction solution to 8–10. Finally, diiodomethane (20 equivalents, 150 μL) was added to the reaction solution, and the mixture was shaken at room temperature for 2 hours. The reaction was monitored by HPLC, and once complete, it was quenched with 100 μL of TFA. The reaction solvent was removed by lyophilization, and the sample was purified by HPLC.

[1124] The crude peptide was purified by preparative HPLC using a preparative Waters XSelect peptide CSH C18 column (1.9 cm inner diameter × 25 cm (5 µm / 130 Å)). Mobile phase A: 20 mM NH4HCO3 in water; Mobile phase B: ACN. Gradient: 0–1 min, 20% B; 1–35 min, 20%–50% B; 35–36 min, 50%–90% B; 36–36.5 min, 90%–95% B; 36.5–40 min, 95% B; 40–41 min, 95%–20% B; 41–45 min, 20% B. Column temperature: Ambient temperature. Flow rate: 24 mL / min. The collected fractions were lyophilized to obtain the desired product.

[1125] Program Q

[1126] Example: Compound number 256

[1127]

[1128] Solid-phase peptide synthesis:

[1129] Peptides were synthesized using Fmoc / t-Bu chemistry via standard solid-phase peptide synthesis (SPPS). Assembly was performed on ProTide Rink amide resin (0.1 mmol scale, 0.19 g / mmol) on a CEM LibertyBlue microwave peptide synthesizer.

[1130] Fmoc deprotection was performed by adding 20% ​​(v / v) piperidine (3 mL) in DMF to the resin, and the mixture was heated to 90°C for 1 minute. The resin was then washed four times with DMF.

[1131] Amide coupling: Add Fmoc-protected amino acids (5 equivalents), DIC in DMF (10 equivalents, 1 M), and OxymaPure in DMF (5 equivalents, 1 M) to a microwave reaction vessel. Then heat the microwave reaction vessel to 90°C for 4 minutes, and then drain. For dual coupling, repeat this process twice.

[1132] For all positions except DOTA(tBu)3, perform Fmoc unprotection and double coupling loop.

[1133] N-terminal acetylation was performed at 65°C for 2 minutes using 2.5 mL of 10% v / v Ac₂O solution in DMF under MW irradiation. The resin was then washed four times with 4 mL of DMF.

[1134] Orthogonal deprotection of Dde from D-Lys: At the end of assembly, 4 mL of a 2% hydrazine monohydrate solution in DMF was added to the microwave reaction vessel and mixed for 30 minutes at room temperature. The resin was then washed 6 times with 4 mL of DMF.

[1135] To incorporate DOTA using a triple coupling condition: Add DOTA(tBu)3 (5 equivalents), DIC (10 equivalents, 1 M) in DMF, and OxymaPure (5 equivalents, 1 M) in DMF to a microwave reactor. Repeat this process three times.

[1136] At the end of assembly, the resin is washed with DMF and DCM.

[1137] Peptide cleavage, cyclization, and purification:

[1138] Using a CEM Razor peptide lysis system, peptides were lysed from the resin for 30 minutes at 40°C using 8 mL of lysis solution (TFA / DODT / H2O / TIS, 92.5 / 2.5 / 2.5 / 2.5, v / v / v / v). The resin was then filtered, and the peptides were precipitated with cold diethyl ether (45 mL). The precipitated peptides were then placed on a centrifuge and allowed to rotate for at least 5 minutes. The ether was decanted, leaving the peptides at the bottom of the centrifuge tube. This ether precipitation process was then repeated.

[1139] The peptide was then dissolved in 10 mL MeCN / H2O (1 / 1, v / v) and lyophilized to dryness. The resulting peptide was then dissolved in 60 mL MeCN / H2O (1 / 1, v / v), and 100 µL of DIPEA was added to the mixture, along with 30 mg of para-DBX in 0.5 mL of ACN. The mixture was stirred at RT for 2 h. The reaction was quenched by adding 50 µL of TFA. The reaction solution was then concentrated under reduced pressure. The crude cyclized peptide was then purified by dissolving it in 2 mL DMSO.

[1140] The crude peptide was purified by preparative HPLC using a preparative Waters XBridge C18 column (100x19mI 5µm OBD). Mobile phase A: H2O + 0.1% TFA, mobile phase B: ACN + 0.1% TFA. The following eluent gradients were used with elution buffer B: 0–0.5 min, 20% B; 0.5–14 min, 20%–40% B; 14–15 min, 40%–95% B; 15–17 min, hold at 95% B; 17–18 min, 20% B. Column temperature: ambient temperature. Flow rate: 24 mL / min. The collected fractions were lyophilized to obtain the desired product, compound number 256.

[1141] Table 4 below shows the synthetic procedures used to synthesize the compounds described herein.

[1142]

[1143]

[1144] Table 5 below shows the LCMS analysis of the compounds described in this paper.

[1145] Table 5

[1146]

[1147]

[1148]

[1149]

[1150]

[1151] Example 3. Binding affinity of the targeted partial peptide to DLL3

[1152] Affinity determination by surface plasmon resonance (SPR)

[1153] The following protocol was used to conduct SPR studies on the compounds disclosed in this paper.

[1154] Procedure: Binding affinity and binding kinetics at 25°C were determined using a Biacore S200 or Biacore 8K instrument. The biotinylated target ligand, human DLL3, was immobilized on a streptavidin sensor chip. A reference surface was established for nonspecific binding and refractive index changes. To analyze the interaction kinetics, samples of different concentrations were injected at a flow rate of 100 μL / min using a running buffer containing 20 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, 0.05% surfactant P20, and 2% DMSO. Sensing curves were fitted using a 1:1 binding model, and affinity and kinetic parameters were obtained using BiacoreInsight evaluation 4.0 software.

[1155] Table 6 below shows the K values ​​of a selected group of compounds obtained by SPR determination. D Value. In this table, "A" represents K. D ≤ 1.0 nM; “B” indicates 1.0 nM < K D ≤ 10 nM; “C” indicates 10 nM < K D ≤ 100 nM; “D” means 100 nM < K D ≤ 300 nM.

[1156]

[1157]

[1158] Example 4. Cell binding, internalization, and radiolabeling of targeted peptides

[1159] use[ 177 A general procedure for radiolabeling the chelating agent-peptide RPT with LuCl3.

[1160] [ 177 LuCl3 was obtained from the supplier as an HCl solution. 1 mCi was added to each reaction vial (1.5 mL Eppendorf vial).177 Lu]LuCl3, ammonium acetate buffer (0.2 M, pH 4.9, 100 µL, containing 1% w / v ascorbic acid and 6% v / v ethanol) and peptide conjugate (1 nmol). The peptide conjugate disclosed herein is also referred to herein as "chelating agent-peptide". The pH of the solution was determined to be approximately 5 using pH test paper. The reaction flask was incubated at 80°C, 700 RPM for 17–20 min. Samples from the reaction were analyzed by RP-HPLC using an Agilent Infinity II 1260 HPLC to determine the completion of the reaction and [ 177 Radiochemical purity of Lu-Lu chelating agent-peptide. HPLC conditions: Waters XBridge BEH C18 column, 130 Å, 3.5 μm, 4.6 mm x 250 mm; mobile phase: solvent A = water (containing 0.1% formic acid), solvent B = acetonitrile (containing 0.1% formic acid). Gradient: 25%-45% B over 10 min; 45%-65% B over 12 min; 65%-90% B over 6 min; flow rate: 0.5 mL / min. The required amount of this product was prepared in 1% PBSA for cell studies.

[1161] use[ 225 Ac]Ac General Procedure for Radiolabeling of Chelating Agent-Peptides

[1162] The peptide conjugates disclosed herein (also referred to herein as "chelating agent-peptides") are used in reactions comprising acetate or equivalent buffer, excipients, and ethanol. 225 Ac]Ac isotopes are radiolabeled. The reaction mixture is heated to achieve radiolabeling, for example, by heating the reaction mixture at 90°C for 15 minutes. The radiolabeled [Ac] is then placed in a preparation buffer containing additional excipients. 225 Ac]Ac-chelating agent-peptide is further diluted to the desired radioactive concentration. [The radiolabeled [] 225 DTPA was incorporated into the Ac-chelating agent-peptide product sample, and the fraction was analyzed by RP-HPLC, with fractions collected every 12 seconds. 225 After Ac[Ac]Ac and its daughter isotopes reached long-term equilibrium (>6 hours after collection), the fractions were analyzed using gamma-ray spectroscopy, and the resulting CPM was plotted as a function of time. Radiochemical purity (% RCP) data were obtained from this reconstructed chromatography.

[1163] Reagents and materials:

[1164]

[1165] instrument:

[1166]

[1167] method:

[1168] Cell preparation

[1169] This study used SHP-77, CT26.WT, and CT26.DLL3 cells (Table 7).

[1170] Adherent cell studies: Cells were cultured in appropriate medium (20 mL) in a tissue culture-treated T150 flask at 37°C and 5% CO2. Adherent cells were isolated at 37°C (60%-70% confluence) for 3 minutes using 5 mL of virginium. After confirming cell viability and count using trypan blue (with a Countess counter), the cells were centrifuged at 4°C (1,000 rpm) for 5 minutes. The resulting cell pellet was washed once with PBS and resuspended in 1% PBSA to obtain the desired cell concentration (5-25 million cells / mL).

[1171] Suspension cell studies: Cells were cultured in appropriate medium (20 mL) in a tissue culture-treated T150 flask at 37°C and 5% CO2. After confirming cell viability and count using trypan blue, the cells were centrifuged at 4°C (1,000 rpm) for 5 minutes. The resulting cell pellet was washed once with PBS and resuspended in 1% PBSA to obtain the desired cell concentration (5-25 million cells / mL).

[1172] The number of cells used for the assay under each condition ranged from 5 million to 25 million cells.

[1173] Table 7: Cell line information

[1174]

[1175] Research Implementation

[1176] Total uptake: Cells were incubated with 300 μL (0.8 µCi) of incubation buffer (containing radiolabeled peptides). 177 The Lu-test peptide was incubated at 37°C for 1 hour (Table 8). After incubation, the cells were pelleted and washed twice with cold PBS + 1% BSA. The supernatant and wash buffer were combined and counted together in a gamma counter as the "unbound fraction". The cells were then resuspended in 300 μL of 1% PBS + 1% BSA and counted in a gamma counter as the "bound fraction".

[1177] Internalization: Cells resuspended in 300 µl of 1% PBSA and counted as the "bound fraction" using a gamma counter were collected in 1.5 mL Eppendorf tubes and precipitated. The precipitated cells were incubated with 300 µl of 50 mM glycine (pH = 2.5) for 3 min, then precipitated again, and the supernatant was discarded. This process was repeated once more. The precipitated cells were washed with 300 µl of cold 1% PBSA and then resuspended in 300 µl of 0.3 M NaOH. This suspension was counted as the "internalized fraction" using a gamma counter.

[1178] Table 8: Summary of Experimental Conditions

[1179]

[1180] SHP-77 cells are human-derived, non-engineered SCLC cells.

[1181] CT26.WT cells do not express DLL3 and were used as a negative control.

[1182] CT26.DLL3 is a cell engineered to express DLL3.

[1183] Tables 9 and 10 below show cytometric data for a selected set of compounds. In these tables, "A" indicates % ≤ 25%; "B" indicates 25 < % ≤ 50%; and "C" indicates % > 50%.

[1184] Table 9: Cell data (CT26 DLL3)

[1185]

[1186] Table 10: Cell data (SHP77)

[1187]

[1188] Example 5. Biological Distribution

[1189] General protocol for in vitro biological distribution studies

[1190] For xenograft mice, when the tumor volume is 200-400 mm... 3 Within the specified range, for tumor-free mice, when the mice were 5-7 weeks old, a bolus dose of the radiolabeled peptide was administered intravenously (via the lateral tail vein) for in vitro biodistribution studies. To assess the amount of radionuclide injected into each mouse, [the following was performed after injection]. 177 The syringe was calibrated using a dose calibrator before and after the Lu-peptide procedure.

[1191] Mice were euthanized at predetermined time points, and selected tissues were excised and collected in pre-weighed test tubes. The test tubes were weighed again after tissue excision; the difference between the two weighings was the weight of each tissue. Radioactivity in each tissue was measured using a gamma counter. The counts were decay-corrected to the injection time, and for each tissue, the percentage of injected activity per gram (%IA / g) was calculated based on the injected activity of each individual mouse. Based on the sensitivity of the gamma counter, the injected activity was converted to counts. The counts in the tissue were then converted to the percentage of injected activity and divided by the tissue mass to obtain %IA / g.

[1192] Table 11: Radioactive concentrations (%IA / g) of the compound tested in different organs of nude mice (n = 3) carrying SHP-77 tumors 24 hours after injection. “A” indicates in the tumor, 0 < %IA / g ≤ 5; “B” indicates in the tumor, %IA / g > 5; “C” indicates in the kidney, 0 < %IA / g ≤ 10; “D” indicates in the kidney, %IA / g > 10.

[1193]

[1194] The subject matter disclosed is not limited in scope to the specific embodiments and examples described herein. In fact, various modifications to this disclosure will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those described herein. These modifications are intended to fall within the scope of the appended claims.

[1195] All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein by reference in their entirety, and for all purposes, to the extent that each individual reference (e.g., publications, patents, or patent applications) is specifically and individually indicated to be incorporated herein by reference in its entirety for all purposes. Further embodiments are within the scope of the following claims.

Claims

1. A cyclic peptide of Formula B: Formula B or a pharmaceutically acceptable salt thereof, (B)、 wherein: wherein w is selected from 1, 2, or 3; P 1 selected from: H, , , and ; D 1 selected from H, CH3, and C(O)OH; L 1 absent or is selected from 、 、 , and ; wherein L 1 is an amino group attached to P 1 is a carbonyl group to form an amide bond; P 2 is -L 2 - chelators or ； D 2 is OH or NH2; L 2 absent or is selected from: , , , , , , and ; wherein L 2 the amino group of the amino acid is attached to P 2 or the carbonyl group of the chelator to form an amide bond; P 3 selected from -L 3 - chelating agents, 、 、 , and ； L 3 is absent or is independently selected from , , , , and ; wherein L 3 is a carbonyl group attached to the P 2 amine group to form an amide bond; D 3 is -NR"-chelator; R 0 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; R 1 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; R 2 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; R 3 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; B 1 is C 1-6 alkylene; C 1 is C 1-6 alkylene; A 1 selected from the group consisting of 、 、 、 、 、 、 、 、 and ; (i) an amino acid side chain of a natural amino acid, R 4 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid, both of which are optionally substituted with CH2C(O)OH or C(O)(CH2CH2O) p (CH2)2N(CH3)3 + ; R 5 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; R 6 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; R 7 selected from the group consisting of: (ii) an amino acid side chain of an unnatural amino acid, or (iii) is selected from the group consisting of: m is 0 or 1; and ; R 8 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; R 9 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; R 10 is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; n, q, and u are each independently an integer from 0 to 16; each p is independently an integer from 0 to 24; each s is independently an integer from 0 to 16; each t is independently 1, 2, 3, 4, 5, or 6; each R’ is independently selected from H, C(O)OH, (CH2)OH, and NHAc; and each R” is independently selected from H and CH3; and each alpha carbon atom on the peptide backbone is optionally substituted with a methyl group; wherein the cyclic peptide optionally comprises a radionuclide. wherein when the variable group R 0 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 or R 10 is defined as an amino acid side chain of a cyclic amino acid, the corresponding amino acid nitrogen of the peptide backbone of Formula B forms part of the cyclic group; and 2. The cyclic peptide of claim 1, wherein the cyclic peptide of Formula B is a cyclic peptide of Formula la: (Ia), or a pharmaceutically acceptable salt thereof.

3. The cyclic peptide of claim 1, wherein the cyclic peptide of Formula B is a cyclic peptide of Formula lb: (Ib), or a pharmaceutically acceptable salt thereof.

4. The cyclic peptide of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein wherein: P 1 selected from Ac, and ; n and s are independently an integer from 2 to 15; and D 1 is CH3or C(O)OH; L 1 absent or ; p is 8, 9, 10, 11, or 12. wherein:

5. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 3, wherein P 1 is Ac or CH3(OCH2CH2) 8-16 C(O).

6. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 3, wherein P 1 is selected from H, Ac, and ; n and s are independently 9, 10, 11, 12, or 13; and p is 8, 12, or 24. D 1 is CH3or C(O)OH.

7. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 3, wherein P 2 is -L 2 - a chelator.

8. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 3, wherein P 2 is 。 9. The cyclic peptide, or pharmaceutically acceptable salt thereof, of claim 8, wherein P 3 is selected from DOTA, 、 、 、 、 、 、 、 、 、 、 、 and ; and 10. The cyclic peptide of claim 1, wherein the cyclic peptide of Formula B is a cyclic peptide of Formula II: (II), or a pharmaceutically acceptable salt thereof.

11. The cyclic peptide of claim 1, wherein the cyclic peptide of Formula B is a cyclic peptide of Formula III: (III), or a pharmaceutically acceptable salt thereof.

12. The cyclic peptide of claim 1, wherein m is 0; n and s are each independently 9, 10, 11, 12, or 13; P 1 selected from H, Ac, and ; p is 8, 12, or 24; P 2 is ； P 3 selected from DOTA, 、 、 、 、 、 、 、 、 、 、 、 and ; (i) is an amino acid side chain selected from the group consisting of 3Pya, 4Pya, Lys(Me)3, His, Ala, D-Ala, Gin, Lys, Glu, Arg, Orn, NMe-His, and Ser; or D 1 is CH3or C(O)OH; R 1 an amino acid side chain selected from the group consisting of Trp, 2Nal, INal, 4CF3-Phe, 7aza-Trp, IMe-Trp, 5OH-Trp, BIP, 5OMe-Trp, 4F-Phe, 3Pya, 4Pya, PAF, MAF, OAF, 5Qui, 7MeO-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, D-Ala, Ala, a-Me-Trp, and NMe-Trp; R 2 an amino acid side chain selected from the group consisting of Thr, D-Ala, Ala, a-Me-Thr, Lys, and NMe-Thr; R 3 is an amino acid side chain selected from the group consisting of lie, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, INal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe-Nle, a-tert- AmylGly, Allo-lle, Lys(C12), Lys(C14), Lys(C16), a-Me-lle, and NMe-tBuAla; A 1 selected from the group consisting of: , , , , , , and ; R 4 is selected from the group consisting of Asn, D-Ala, Ala, DAB-4-NHCOC5H 11 , DAB-4-NHCOC7H 15 , Asp, Ser, Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, 3Pya, 4Pya, Glu, NMe-Asn, Pip(CH2CO2H)Ala, and Pip(PegNMe3)Ala amino acid side chains; R 5 is selected from the group consisting of: Asn, Ala, D-Ala, Trp, Asp, Lys, 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser; R 6 an amino acid side chain selected from the group consisting of Trp, 4CF3-Phe, 1Me-Trp, 7aza-Trp, BIP, 2Nal, 1Nal, a-Me-Trp, D-Ala, Ala, 4F-Phe, 5F-Trp, 50me-Trp, Asn, 50H-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp; R 7 ： (ii) is selected from the group consisting of: each s is independently 3, 5, 10, 12, or 14; , and ; 13. The cyclic peptide of claim 1 or 3, or a pharmaceutically acceptable salt thereof, wherein R 8 an amino acid side chain selected from the group consisting of Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and a-Me-Asp; R 9 is selected from the group consisting of: Trp, 7aza-Trp, 1 Me-Trp, D-Ala, Ala, 4F-Phe, 1 Nal, 2Nal, 5F-Trp, 50me-Trp, a-Me-Trp, 7CI-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and R 10 an amino acid side chain selected from the group consisting of Pro, D-Ala, Ala, a-Me-Pro, trans 4 fluoro-Pro, cis 4 fluoro-Pro, trans 4 OH-Pro, cis 4 OH-Pro, Pip, 5,5-diMe-Pro, NMe-Ser, trans 4 NH2-Pro, cis 4 NH2-Pro, Sar, Aze, NMe-Ala, NMe-Leu, R-3Me-Aze, a-Me-Aze, ACI, and 3Me2-Aze. m is 1; n and s are each independently 9, 10, 11, 12, or 13; P 1 selected from Ac, and ; p is 8, 12, or 24; P 2 is ； P 3 selected from DOTA, 、 、 、 、 、 、 、 、 、 、 、 and ; each s is independently 3, 5, 10, 12, or 14; D 1 is CH3or C(O)OH; R 0 an amino acid side chain selected from the group consisting of Gly, Met, D-Ala, Ala, Nle, and Nva; R 1 is selected from the group consisting of Trp, 2Nal, INal, 4CF3-Phe, 7aza-Trp, IMe-Trp, 5OH-Trp, BIP, 50me-Trp, 4F-Phe, 3Pya, 4Pya, PAF, MAF, OAF, 5Qui, 7MeO-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, D-Ala, Ala, a-Me-Trp, and NMe-Trp; R 2 an amino acid side chain selected from the group consisting of Thr, D-Ala, Ala, a-Me-Thr, Lys, and NMe-Thr; R 3 is selected from the group consisting of the amino acid side chains of Ile, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, INal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe-Nle, a-tert- AmylGly, Allo-Ile, Lys(C12), Lys(C14), Lys(C16), a-Me-Ile, and NMe-tBuAla; A 1 selected from the group consisting of: , , , , , , and ; R 4 is selected from the group consisting of Asn, D-Ala, Ala, DAB-4-NHCOC5H 11 , DAB-4-NHCOC7H 15 , Asp, Ser, Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, 3Pya, 4Pya, Glu, and the amino acid side chain of NMe-Asn; R 5 an amino acid side chain selected from the group consisting of Asn, Ala, D-Ala, Trp, Asp, Lys, 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser; R 6 an amino acid side chain selected from the group consisting of Trp, 4CF3-Phe, 1Me-Trp, 7aza-Trp, BIP, 2Nal, 1Nal, a-Me-Trp, D-Ala, Ala, 4F-Phe, 5F-Trp, 50Me-Trp, Asn, 50H-Trp, 7Me-Trp, 7MeO-Trp, 7Cl-Trp, and NMe-Trp; R 7 an amino acid side chain selected from the group consisting of 3Pya, 4Pya, Lys(Me)3, His, Ala, D-Ala, Gin, Lys, Glu, Arg, Orn, NMe-His, and Ser; or R 7 selected from the group consisting of: , and ； 14. The cyclic peptide of any one of claims 1 to 9, 12, and 13, or a pharmaceutically acceptable salt thereof, wherein R 8 an amino acid side chain selected from the group consisting of Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and a-Me-Asp; R 9 is selected from the group consisting of: Trp, 7aza-Trp, 1 Me-Trp, D-Ala, Ala, 4F-Phe, 1 Nal, 2Nal, 5F-Trp, 50me-Trp, a-Me-Trp, 7CI-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; and R 10 an amino acid side chain selected from the group consisting of Pro, D-Ala, Ala, a-Me-Pro, trans 4 fluoro-Pro, cis 4 fluoro-Pro, trans 4 OH-Pro, cis 4 OH-Pro, Pip, 5,5-diMe-Pro, NMe-Ser, trans 4 NH2-Pro, cis 4 NH2-Pro, Sar, Aze, NMe-Ala, NMe-Leu, R-3Me-Aze, a-Me-Aze, ACI, and 3Me2-Aze.

15. The cyclic peptide of any one of claims 1 to 9, 12, and 13, or a pharmaceutically acceptable salt thereof, wherein B 1 is CH2or C(CH3)2; and C 1 is CH2or C(CH3)2. ​ B 1 is CH2; and C 1 is CH2.

16. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 15, wherein the chelator is independently selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetra-azacyclododecane-N,N',N",N"' -tetraacetic acid (DOTA), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4- isothiocyanatopicolinic acid (Macropa), Macrodipa, 2,2',2",2"'-(1,10-dioxa-4,7,13,16- tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid) (crown ether), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, alpha-(2-carboxyethyl) (DOTAGA), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"' -tetraacetic acid (TETA), 1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N'''-pentaacetic acid (PEPA), and 1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N",N"',N'''',N''' '-hexaacetic acid (HEHA).

17. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 16, wherein the cyclic peptide of Formula B is substituted with at least one chelator.

18. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 17, wherein the cyclic peptide of Formula B is selected from the cyclic peptides in Table A.

19. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 17, wherein the cyclic peptide of Formula B is selected from the cyclic peptides in Table B.

20. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 19, wherein the cyclic peptide further comprises a radioisotope.

21. The cyclic peptide, or a pharmaceutically acceptable salt thereof, of claim 20, wherein the radioisotope is selected from the radioisotopes in Table 3.

22. The cyclic peptide, or pharmaceutically acceptable salt thereof, of claim 20, wherein the radioisotope is selected from the group consisting of: 111 In、 99m Tc、 94m Tc、 66 Ga、 67 Ga、 68 Ga、 52 Fe、 169 Er、 72 As、 97 Ru、 203 Pb、 61 Cu、 62 Cu、 64 Cu、 67 Cu、 89 Sr、 186 Re、 188 Re、 86 Y、 90 Y、 89 Zr、 51 Cr、 52 Mn、 51 Mn、 177 Lu、 169 Yb、 175 Yb、 105 Rh、 166 Dy、 166 Dy、 166 Ho、 153 Sm、 149 Pm、 151 Pm、 172 Tm、 121 Sn、 117m Sn、 212 Bi、 213 Bi、 142 Pr、 143 Pr、 198 Au、 199 Au、 123 I、 124 I、 125 I、 131 I、 75 Br、 76 Br、 77 Br、 80 Br、 82 Br、 18 F、 149 Tb、 152 Tb、 155 Tb、 161 Tb、 43 Sc、 44 Sc、 47 Sc, 212 Pb, 211 At, 223 Ra, 227 Th, 226 Th, 82 Rb, 32 P, 76 As, 89 Zr, 111 Ag, 165 Er, 225 Ac and 227 Ac.

23. A pharmaceutical composition comprising the cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 22, and a pharmaceutically acceptable carrier.

24. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the cyclic peptide, or a pharmaceutically acceptable salt thereof, of any one of claims 1 to 24, or the pharmaceutical composition of claim 23, to the subject.

25. The method of claim 24, wherein the cancer is a DLL3-mediated cancer.

26. The method of claim 24 or 25, wherein the cancer is a neuroendocrine tumor, melanoma, or a primary brain cancer.

27. The method of claim 26, wherein the neuroendocrine tumor is selected from small cell lung cancer (SCLC), medullary thyroid cancer (MTC), large cell neuroendocrine carcinoma (LCNEC), gastroenteropancreatic neuroendocrine carcinoma (GEP NEC), neuroendocrine prostate cancer (NEPC), small cell prostate cancer (SCPC), Merkel cell carcinoma (MCC), cervical neuroendocrine carcinoma, and G3 grade neuroendocrine tumor (NET).

28. A peptide having binding specificity for DLL3, wherein the peptide binds to one or more of amino acids A81, L83, G106, A85, and R61 of the DLL3 amino acid sequence of SEQ ID NO: 1, wherein the peptide comprises an amino acid sequence of Formula A: Formula A or a pharmaceutically acceptable salt thereof, (A)、 wherein: X0is any natural or unnatural amino acid or X0is absent; X1is selected from Trp, 7aza-Trp, 1Me-Trp, 5OH-Trp, 5OMe-Trp, 7OMe-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, a-Me-Trp, and NMe-Trp; X2and X3are each independently any natural or unnatural amino acid; Y1is Cys; X4, X5, X6, X7, and X8are each independently any natural or unnatural amino acid; Y2is Cys; X9is selected from Trp, 7aza-Trp, 1Me-Trp, 5OH-Trp, 5OMe-Trp, 7OMe-Trp, 7Me-Trp, 5F-Trp, 7Cl-Trp, a-Me-Trp, and NMe-Trp; each R’ is independently selected from H, C(O)OH, (CH2)OH, and NHAc; X 10 selected from Pro, a-Me-Pro, trans 4 fluoro-Pro, cis 4 fluoro-Pro, trans 4 OH-Pro, cis 4 OH-Pro, 5,5-diMe-Pro, trans 4NH2-Pro, and cis 4NH2-Pro; P 2’ selected from -L 2 - chelating agents or ； D 2 is OH or NH2; L 2 absent or is selected from: 、 、 、 , , , and ; wherein L 2 the amino group of the amino acid is attached to P 2 or the carbonyl group of the chelator to form an amide bond; P 3 selected from -L 3 - chelating agents, , , , and ; L 3 is absent or is independently selected from , , , , and ; wherein L 3 is a carbonyl group attached to the P 2 amine group to form an amide bond; D 3 independently -NR"-chelator or ； each R” is independently selected from H and CH3; X is H or halogen; each n is independently an integer from 0 to 16; each p is independently an integer from 0 to 24; each s is independently an integer from 0 to 16; each t is independently 1, 2, 3, 4, 5, or 6; and w is selected from 1, 2, or 3; wherein the cyclic peptide is cyclized via a linker between Y1and Y2; and wherein the cyclic peptide binds to DLL3.

29. The peptide of claim 27, which binds to amino acids A81, L83, G106, A85, and R61 of the DLL3 amino acid sequence of SEQ ID NO:

1.

30. The peptide of claim 27 or 28, which binds to backbone atoms of amino acids A81, L83, G106, and A85 of the DLL3 amino acid sequence of SEQ ID NO:

1.

31. The peptide of any one of claims 27 to 29, which binds to a side chain atom of amino acid R61 of the DLL3 amino acid sequence of SEQ ID NO:

1. ​ 32. The peptide of any one of claims 27-30, which binds to the main chain atoms of amino acids A81, L83, G106, and A85 of the DLL3 amino acid sequence of SEQ ID NO: 1 and to the side chain atom of amino acid R61 of the DLL3 amino acid sequence of SEQ ID NO:

1.

33. The peptide of any one of claims 27 to 31, which is capable of binding to DLL3 with a K -8 D of about 1 x 10 -10 M to about 1 x 10 D M.

34. The peptide of any one of claims 27-32, which comprises the amino acid sequence of WTACANAKDCWP or a derivative thereof comprising one or more non-natural amino acids.

35. The peptide of claim 33, wherein amino acids W1, A3, A7, and W11 bind to DLL3.

36. A pharmaceutical composition comprising the peptide of any one of claims 27-34, and a pharmaceutically acceptable carrier.

37. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the peptide of any one of claims 27-34, or the pharmaceutical composition of claim 35, to the subject.

38. The method of claim 36, wherein the cancer is a DLL3-mediated cancer.

39. The method of claim 36 or 37, wherein the cancer is a neuroendocrine tumor, a melanoma, or a primary brain cancer.

40. The method of claim 38, wherein the neuroendocrine tumor is selected from small cell lung cancer (SCLC), medullary thyroid cancer (MTC), large cell neuroendocrine carcinoma (LCNEC), gastroenteropancreatic neuroendocrine carcinoma (GEP NEC), neuroendocrine prostate cancer (NEPC), small cell prostate cancer (SCPC), Merkel cell carcinoma (MCC), cervical neuroendocrine carcinoma, and G3 grade neuroendocrine tumor (NET).

41. The method of claim 26 or 39, wherein the neuroendocrine tumor is cervical extrapulmonary neuroendocrine carcinoma (NEC).

42. The cyclic peptide of claim 21, wherein the cyclic peptide and pharmaceutically acceptable salts and solvates thereof are radiolabeled with F-18, Ga-68, In-111, Lu-177, or Ac-225.

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