Peptides and conjugates comprising the same

Abstract

This invention relates to peptides, conjugates comprising the peptides, and compositions comprising the like. The peptides of this invention comprise the following amino acid sequence: da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-S-3Py6NH2-HseMe-dk-Y; or have substitution, addition, deletion, or insertion of 1-10 amino acid residues selected from the 1st, 2nd, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, and 11th positions of the above amino acid sequence, and the conjugates comprise the above peptides.

Description

Technical Field

[0001] This invention relates to peptides, conjugates comprising the peptide and a payload (sometimes also referred to as "peptide conjugates", "peptide complexes" or simply "conjugates"), and the use of the peptide or conjugate. Background Technology

[0002] CA9 (Carbonic anhydrase IX) is one of 15 carbonic anhydrases, a transmembrane protein that exists locally as a homodimer on the cell membrane (Non-Patent Literature 1). Furthermore, CA9 possesses three domains: a catalytic domain, a proteoglycan-like domain, a membrane-type transmembrane domain, and a cytoplasmic tail. One of its functions is to exhibit zinc-demanding activity in the catalytic domain, converting water molecules and carbon dioxide into hydrogen ions and bicarbonate ions, and to adjust the intracellular and extracellular pH to a state suitable for cell proliferation (Non-Patent Literature 2). In fact, the slower proliferation of CA9-knockout tumor cells compared to wild-type cells has been confirmed in in vitro and tumor-bearing mouse studies, experimentally demonstrating CA9's involvement in tumor cell proliferation (Non-Patent Literature 3). Furthermore, in various cancer patients, CA9 expression levels were found to be significantly correlated with overall survival, disease-free survival, local control, disease-specific survival, metastasis-free survival, and progression-free survival. This indicates that patients with tumors exhibiting high CA9 expression, regardless of tumor type or location, have a higher risk of local recurrence, disease progression, and metastasis (Non-Patent Literature 4). Based on these reports, CA9 shows promise as a target for detecting tumors with poor prognosis.

[0003] As a mechanism for inducing CA9 expression, activation of the hypoxia-inducible factor (HIF) 1α pathway has been reported (Non-Patent Literature 5), particularly in clear cell renal cell carcinoma and colorectal cancer, where a large number of patients have high CA9 expression levels (Non-Patent Literature 6) (Non-Patent Literature 7). In colorectal cancer, due to activation of the HIF1α pathway associated with hypoxia, approximately 70% of patients have high CA9 expression in their tumor tissues (Non-Patent Literature 7). On the other hand, in clear cell renal cell carcinoma patients, more than half have somatic mutations or hypermethylation of the promoter region of the VHL (Von Hippel-Lindau) gene, a tumor suppressor gene (Non-Patent Literature 8), and the HIF1α pathway is chronically activated even under normoxic conditions (Non-Patent Literature 5), with approximately 90% of patients having high CA9 expression in their tumor tissues (Non-Patent Literature 6). Metastatic colorectal cancer and metastatic renal cell carcinoma have a 5-year survival rate of less than 20%, making them diseases with very poor prognosis. Therefore, establishing methods for their diagnosis and treatment is of paramount importance.

[0004] To date, cancer therapies targeting CA9 have been developed using antibodies such as girentuximab, but have failed to demonstrate efficacy (Non-Patent Literature 9). Therefore, an antibody-RI complex was developed that conjugates a chelating agent to girentuximab and a therapeutic radionuclide for PET imaging. While tumors can be detected with high sensitivity in antibody-RI complex-based PET imaging, the long blood half-life of the antibody necessitates the use of a high-energy radionuclide, and tumors can only be detected 5 days after administration, thus requiring considerable time to detect the tumor (Non-Patent Literature 10). Furthermore, the long blood half-life during treatment also leads to prolonged thrombocytopenia caused by RI, making repeated dosing difficult. In fact, in a Phase II clinical trial, three repeated doses could not be performed due to the failure to recover from thrombocytopenia (Non-Patent Literature 11).

[0005] Therefore, there is still a need to develop new cancer treatments and diagnostic drugs that target CA9 and contain non-antibody compounds as active ingredients, such as imaging drugs for PET.

[0006] Existing technical documents

[0007] Non-patent literature

[0008] Non-patent literature 1: Mayank Aggarwal, et al. J. Enzyme Inhib. Med. Chem. 2013Apr;28(2): 267-77.

[0009] Non-patent literature 2: Pawel Swietach, et al. J. Biol Chem. 2009 Jul 24; 284(30): 20299-310.

[0010] Non-patent literature 3: Scott K Parks, et al. Oncotarget 2017 Feb 7; 8(6):10225-10237

[0011] Non-patent literature 4: Simon JA van Kuijk, et al. Front Oncol. 2016 Mar 29:6:69.

[0012] Non-patent literature 5: CC Wykoff, NJ Beasley et al. Cancer Res. 2000 Dec 15;60(24): 7075-83.

[0013] Non-patent literature 6: Matthew HT Bui, et al. Clin. Cancer Res. 2003 Feb; 9(2): 802-11.

[0014] Non-patent literature 7: Emese Sarolta Badon, et al. Int. J. Mol. Sci. 2023 Jan30; 24(3): 2581.

[0015] Non-patent literature 8: Yusuke Sato, et al. Nat. Genet. 2013 Aug; 45(8): 860-7.

[0016] Non-patent literature 9: Karim Chamie, et al. JAMA Oncol. 2017 Jul 1; 3(7): 913-920.

[0017] Non-patent document 10: Brian M. Shuch, et, al. Results from phase 3 study of89Zr-DFO-girentuximab for PET / CT imaging of clear cell renal cell carcinoma(ZIRCON). 2023 ASCO Genitourinary Cancers Symposium Meeting Abstract

[0018] Non-patent literature 11: Constantijn HJ Muselaers, et al. Eur. Urol. 2016 May;69(5): 767-70. Summary of the Invention

[0019] Problems solved by the invention

[0020] The inventors conducted in-depth research with the aim of developing compounds with CA9 binding activity, which led to the development of this invention. Furthermore, the inventors also discovered conjugates of compounds with CA9 binding activity and payloads such as radioactive substances and fluorescent substances. This invention relates to novel peptides with CA9 binding activity, conjugates of these peptides and payloads, compositions containing these peptides or conjugates (pharmaceutical compositions, diagnostic compositions, research compositions), and uses of these peptides or conjugates.

[0021] Problem Solving Methods

[0022] In a non-limiting sense, this application includes the following inventions. [1]

[0024] A peptide comprising the following amino acid sequence:

[0025] da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-S-3Py6NH2-HseMe-dk-Y (SEQ ID NO. 1); or

[0026] The amino acid sequence described above has 1-10 amino acid residues selected from the 1st, 2nd, 3rd, 4th, 5th, 7th, 8th, 9th, 10th and 11th positions, which have substitution, addition, deletion or insertion of amino acid residues. [2]

[0028] According to the peptide described in [1], wherein,

[0029] (1) In the amino acid sequence of SEQ ID NO. 1, MeC, C, or MeCt is added as the 13th amino acid residue; or

[0030] (2) In the amino acid sequence of SEQ ID NO. 1, MeA, MeS, MeN, Hpr, G or P are added as the 13th amino acid residue, and MeG, G, da or Meda are added as the 14th amino acid residue. [3]

[0032] According to the peptide described in [1] or [2], it comprises:

[0033] The amino acid sequence of SEQ ID NO. 1 has 1-7 amino acid residues selected from the 1st, 4th, 7th, 8th, 9th, 10th and 11th positions, which have substitution, addition, deletion or insertion of amino acid residues. [4]

[0035] The peptide according to any one of [1] to [3] has one or more of the following features:

[0036] The amino acid residue at position 1 is da, dq, de, dhgl, or dk;

[0037] The second amino acid residue is I or Tbg;

[0038] The third amino acid residue is 3Py6NH2 or 3Py6CON;

[0039] The fourth amino acid residue is S, Hgl, Hgl(PEG8Me), alT, Hgn, SMe, KCOpipzaa, or DabAc;

[0040] The fifth amino acid residue is PeG, MeG, Meda, or MedkCOpipzaa;

[0041] The amino acid residue at position 7 is PeG, pMeOPeG, MsMeapG, 3OMePeG, pHPeG, PpG, pFPeG, mCPeG, pCPeG, 4HPpG, 4OMePpG, 4PypG, 4COOPeG, 3COOPeG, or 3FPeG.

[0042] The amino acid residue at position 8 is H, S, P, D, KCOpipzaa, Q (PEG8Me), P4Sh, Hse, SMe, Qmm, or Qdm;

[0043] The amino acid residue at position 9 is 3Py6NH2, 5Inda, Y, or F4OMe;

[0044] The 10th amino acid residue is Ahp, HseMe, or E; and

[0045] The 11th amino acid residue is dk, G, da, dq, dkCOpipzaa, dk(t4amCh), de, Acb, or dkAc. [5]

[0047] The peptide according to any one of [1] to [4] has one or more of the following features:

[0048] The second amino acid residue is Tbg;

[0049] The third amino acid residue is 3Py6NH2;

[0050] The fifth amino acid residue is Meda;

[0051] The amino acid residue at position 7 is either PeG or pHPeg;

[0052] The 9th amino acid residue is 3Py6NH2 or 5Inda; and

[0053] The 10th amino acid residue is HseMe. [6]

[0055] The peptide according to any one of [1] to [4], wherein,

[0056] The second amino acid residue is Tbg.

[0057] The third amino acid residue is 3Py6NH2.

[0058] The fifth amino acid residue is Meda.

[0059] The amino acid residue at position 7 is either PeG or pHPeg.

[0060] The 9th amino acid residue is 3Py6NH2 or 5Inda, and

[0061] The 10th amino acid residue is HseMe. [7]

[0063] The peptide according to any one of [1] to [6] comprises the amino acid sequence described in any one of SEQ ID NO. 2-54, or is composed of the amino acid sequence described in any one of SEQ ID NO. 2-54. [8]

[0065] The peptide according to any one of [1] to [7] further comprises added amino acid residues. [9]

[0067] The peptide according to any one of [1] to [8], wherein,

[0068] The above-mentioned peptides are cyclic peptides.

[10]

[0070] The peptide according to any one of [1] to [9] has the following characteristics:

[0071] The cyclic structure is formed by the combination of the first amino acid residue of the chloroacetylated amino acid sequence of SEQ ID NO. 1 with the cysteine ​​residue contained in the peptide.

[11]

[0073] The peptide according to any one of [1] to [9] has the following characteristics:

[0074] The peptide contains a cyclic structure formed by the amino group of the first amino acid residue of SEQ ID NO. 1 and the carboxyl group of the 14th amino acid residue.

[12]

[0076] The peptide according to any one of [1] to

[11] , wherein,

[0077] (a) The C-terminus of the above peptide can bind a linker and / or a payload; or

[0078] (b) The amino acid residues at positions 1, 4, 8, 11 or 13 of the amino acid sequence of SEQ ID NO. 1 are amino acid residues capable of binding linkers and / or payloads.

[13]

[0080] A conjugate comprising any one of [1] to

[12] a peptide and a payload, wherein,

[0081] (i) The amino acid residue at position 13 of SEQ ID NO. 1 is bound to any payload, with or without a linker; or

[0082] (ii) As shown in formula (I) below, the amino acid residue at position 1, 4, 8, or 11 of SEQ ID NO. 1 is bound with a payload.

[0083] [Chemical Formula 1]

[0084]

[0085] In formula (I), R1 is H or C1-3 alkyl;

[0086] R2 is C1-6 alkyl-NH-, C1-6 alkyl-C6 aryl-O-C1-3 alkyl-NH-, C1-6 alkyl-NH(=O)-CH(C1-6 alkylphenyl)-NH-, C1-6 alkyl-NH(=O)-CH(C1-6 alkyl)-NH-, or C1-6 alkyl-NH(=O)-C3-8 cycloalkyl-C1-3 alkyl-NH-;

[0087] X is any payload.

[14]

[0089] According to the conjugate described in

[13] , wherein,

[0090] R2 is a C1-6 alkyl-NH-.

[15]

[0092] According to the conjugate described in

[13] or

[14] , wherein,

[0093] X is a chelating agent.

[16]

[0095] According to the conjugate described in

[15] , wherein,

[0096] The aforementioned chelating agents bind to radioactive substances.

[17]

[0098] According to the conjugate described in

[15] or

[16] , wherein,

[0099] The chelating agents mentioned above are DOTA, DOTAGA, or NODAGA.

[18]

[0101] A pharmaceutical composition comprising any one of the peptides described in [1] to

[12] .

[19]

[0103] A pharmaceutical composition for the prevention or treatment of CA9-related diseases, comprising any one of the peptides described in [1] to

[12] .

[20]

[0105] A pharmaceutical composition comprising any one of the conjugates described in

[13] to

[17] . [twenty one]

[0107] A pharmaceutical composition for the prevention or treatment of CA9-related diseases, comprising any one of the conjugates described in

[13] to

[17] . [twenty two]

[0109] A diagnostic or research composition comprising any one of the peptides described in [1] to

[12] . [twenty three]

[0111] A composition for diagnosis or research purposes for diagnosing CA9-related diseases, comprising any one of the peptides described in [1] to

[12] . [twenty four]

[0113] A diagnostic or research composition comprising any one of the conjugates described in

[13] to

[17] .

[25]

[0115] A composition for diagnosis or research purposes for diagnosing CA9-related diseases, comprising any one of the conjugates described in

[13] to

[17] .

[26]

[0117] An imaging agent comprising any one of the conjugates described in

[13] to

[17] .

[27]

[0119] An imaging agent for tumor diagnosis, comprising any one of the conjugates described in

[13] to

[17] .

[28]

[0121] A radioactive ligand imaging agent for positron emission tomography, comprising any one of the conjugates described in

[13] to

[17] .

[29]

[0123] A method for testing peptides, which is applicable to peptide testing for at least one of the following a) to d):

[0124] a) Solubility in solvents

[0125] b) CA9 binding activity,

[0126] c) Toxicity to cells and / or tissues, or

[0127] d) Toxicity to laboratory animals,

[0128] The peptides mentioned above are any one of [1] to

[12] .

[30]

[0130] A method for testing conjugates, wherein the conjugates are tested for at least one of the following a) to d):

[0131] a) Solubility in solvents

[0132] b) CA9 binding activity,

[0133] c) Toxicity to cells and / or tissues, or

[0134] d) Toxicity to laboratory animals,

[0135] The above-mentioned conjugates are any one of

[13] to

[17] .

[31]

[0137] The peptide according to any one of [1] to [3] has one or more of the following features:

[0138] The amino acid residue at position 1 is de(PEG8Me), dk, dyae, or dkCOpipzaa;

[0139] The second amino acid residue is I or Tbg;

[0140] The third amino acid residue is 3Py6NH2 or 3Py6CON;

[0141] The fourth amino acid residue is K, KCOpipzaa, Hgn(Qglucamine-NH2), Hgn(KCOpipzaa-NH2), or Qglucamine;

[0142] The fifth amino acid residue is PeG, MeG, Meda, or MedkCOpipzaa;

[0143] The amino acid residue at position 7 is PeG, pMeOPeG, MsMeapG, 3OMePeG, pHPeG, PpG, pFPeG, mCPeG, pCPeG, 4HPpG, 4OMePpG, 4PypG, 4COOPeG, 3COOPeG, or 3FPeG.

[0144] The 8th amino acid residue is K;

[0145] The amino acid residue at position 9 is 3Py6NH2, 5Inda, Y, or F4OMe;

[0146] The 10th amino acid residue is Ahp, HseMe, or E; and

[0147] The amino acid residue at position 11 is either dk(F) or dk(PEG8c).

[32]

[0149] The peptide according to any one of [1] to [6] comprises or is composed of the amino acid sequence described in any one of SEQ ID NO. 55-103.

[33]

[0151] A conjugate comprising the peptide described in

[32] and a payload, wherein,

[0152] The amino acid residues at positions 1, 4, 8, 11, or 13 may be linked with an arbitrary payload, with or without a linker.

[34]

[0154] A conjugate, which is any of the conjugate numbers 1-81 listed in Table 8 or Table 18.

[0155] The effects of the invention

[0156] The peptides and conjugates of the present invention have CA9 binding activity, and are therefore useful as pharmaceutical compositions, diagnostic compositions, and research compositions for the prevention or treatment of CA9-related symptoms. Attached Figure Description

[0157] [ Figure 1 ] Figure 1 This demonstrates that the various peptides of the present invention possess DOTA- 67 This figure shows the in vivo distribution of various Ga conjugates as payloads in female BALB / cSlc-nu / nu tumor transplantation model mice 4 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0158] [ Figure 2 ] Figure 2 This demonstrates that the various peptides of the present invention possess DOTA- 67 This figure shows the in vivo distribution of various Ga conjugates as payloads in female BALB / cSlc-nu / nu tumor transplantation model mice 24 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0159] [ Figure 3 ] Figure 3 This demonstrates that the various peptides of the present invention possess DOTA- 67 This figure shows the in vivo distribution of various Ga conjugates as payloads in female BALB / cSlc-nu / nu tumor transplantation model mice 72 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0160] [ Figure 4 ] Figure 4 This demonstrates that the various peptides of the present invention possess DOTA- 64 The figure shows the in vivo distribution of Cu conjugates as payloads in female BALB / cSlc-nu / nu tumor transplantation model mice 4 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0161] [ Figure 5 ] Figure 5 This demonstrates that the various peptides of the present invention possess DOTA- 64 This figure shows the in vivo distribution of various conjugates containing Cu as the payload in female BALB / cSlc-nu / nu tumor transplantation model mice 24 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0162] [ Figure 6 ] Figure 6 This demonstrates that the various peptides of the present invention possess DOTA- 64 Cu、DOTAGA- 64 Cu or NODAGA- 64 The figure shows the in vivo distribution of Cu conjugates as payloads in female BALB / cSlc-nu / nu tumor transplantation model mice 4 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0163] [ Figure 7 ] Figure 7This demonstrates that the various peptides of the present invention possess DOTA- 64 Cu、DOTAGA- 64 Cu or NODAGA- 64 This figure shows the in vivo distribution of various conjugates containing Cu as the payload in female BALB / cSlc-nu / nu tumor transplantation model mice 24 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0164] [ Figure 8 ] Figure 8 The peptide of this invention contains DOTA- 64 Cu as an effective load conjugate Conjugate No. 64- 64 Representative PET / CT images of female BALB / cSlc-nu / nu tumor transplantation model mice at 1, 4, and 23.5 hours after Cu administration. White arrows in the images indicate the local presence of transplanted tumors.

[0165] [ Figure 9 ] Figure 9 The peptide of this invention contains DOTA- 64 Cu as an effective load conjugate Conjugate No. 62- 64 Representative PET / CT images of female BALB / cSlc-nu / nu tumor transplantation model mice at 1, 4, and 23.5 hours after Cu administration. White arrows in the images indicate the local presence of transplanted tumors.

[0166] [ Figure 10 ] Figure 10 The peptide of this invention contains DOTA- 64 Cu as an effective load conjugate No. 2- 64 Representative PET / CT images of female BALB / cSlc-nu / nu tumor transplantation model mice at 1, 4, and 23.5 hours after Cu administration. White arrows in the images indicate the local presence of transplanted tumors.

[0167] [ Figure 11 ] Figure 11 The peptide of this invention contains rDOTAGA- 64 Cu as an effective load conjugate Conjugate No. 56- 64 Representative PET / CT images of female BALB / cSlc-nu / nu tumor transplantation model mice at 1, 4, and 23.5 hours after Cu administration. White arrows in the images indicate the local presence of transplanted tumors.

[0168] [ Figure 12 ] Figure 12 The peptide of this invention contains rNODAGA- 64 Cu as an effective load conjugate Conjugate No. 58- 64 Representative PET / CT images of female BALB / cSlc-nu / nu tumor transplantation model mice at 1, 4, and 23.5 hours after Cu administration. White arrows in the images indicate the local presence of transplanted tumors.

[0169] [ Figure 13 ] Figure 13 This demonstrates that DOTA- is given in various peptides of the present invention. 177 A graph showing the shift in mean tumor volume in female BALB / cSlc-nu / nu tumor transplantation model mice using Lu as the conjugate, saline, or cabozantinib. Note that the horizontal axis represents days after administration, and the vertical axis represents tumor volume (mm). 3 Additionally, the short black dotted circle represents saline solution, the solid black triangular line represents cabozantinib, and the long black dotted circle represents Conjugate No. 64. 177 Lu (3 doses), solid white circular line indicates Conjugate No. 62- 177 Lu (3 doses), solid X indicates Conjugate No. 64- 177 Lu (single dose), the solid black square line indicates Conjugate No. 62- 177 Lu (single dose), the solid black circle indicates Conjugate No. 65- 177 Lu (60 MBq, administered once).

[0170] [ Figure 14 ] Figure 14 This demonstrates that DOTA- is given in various peptides of the present invention. 177 A graph showing the mean rate of change in body weight in female BALB / cSlc-nu / nu tumor transplantation model mice with Lu as the effective payload, saline, or cabozantinib. It should be noted that the horizontal axis represents days after administration, and the vertical axis represents the rate of change in body weight (%). Additionally, short black dashed circles represent saline, solid black triangles represent cabozantinib, and long black dashed circles represent Conjugate No. 64- 177 Lu (3 doses), solid white circular line indicates Conjugate No. 62- 177 Lu (3 doses), solid X indicates Conjugate No. 64- 177Lu (single dose), the solid black square line indicates Conjugate No. 62- 177 Lu (single dose), the solid black circle indicates Conjugate No. 65- 177 Lu (60 MBq, administered once).

[0171] [ Figure 15 ] Figure 15 This illustrates that the peptide of the present invention contains DOTA- 64 Cu as the payload Conjugate No. 62- 64 The graph shows the in vivo distribution of Cu in VMRC-RCW tumor-bearing mice at 4, 24, and 48 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0172] [ Figure 16 ] Figure 16 This illustrates that the peptide of the present invention contains DOTA- 177 Lu as the payload Conjugate No. 62- 177 The graph shows the in vivo distribution of Lu in VMRC-RCW tumor-bearing mice at 1, 24, and 48 hours after drug administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0173] [ Figure 17 ] Figure 17 This illustrates that the peptide of the present invention contains DOTA- 64 Cu as the payload Conjugate No. 62- 64 Representative PET / CT images of VMRC-RCW tumor-bearing mice at 1, 4, 23.5, and 47.5 hours after Cu administration.

[0174] [ Figure 18 ] Figure 18 This demonstrates that the peptide of the present invention contains DOTA- 177 Lu as the payload Conjugate No. 62- 177 A graph showing the mean tumor volume in VMRC-RCW tumor-bearing mice treated with Lu, saline, or cabozantinib. Note that the horizontal axis represents days after administration, and the vertical axis represents tumor volume (mm). 3 Additionally, the black circle represents Group 1: saline, the X represents Group 2: cabozantinib, and the checkerboard triangle represents Group 3: Conjugate No. 62- 177 Lu (30 MB q once), vertical stripes and triangles indicate 4 groups: Conjugate No. 62-177 Lu (30 MBq, administered twice daily), horizontal stripes and triangles indicate 5 groups: Conjugate No. 62- 177 Lu (30 MBq, 3 doses), black triangles indicate group 6: Conjugate No. 62- 177 Lu (30 MBq, 4 doses), checkerboard pattern squares represent 7 groups: Conjugate No. 62- 177 Lu (60 MBq, single dose), vertical stripes and squares indicate 8 groups: Conjugate No. 62- 177 Lu (60 MBq, administered twice).

[0175] [ Figure 19 ] Figure 19 This demonstrates that the peptide of the present invention contains DOTA- 177 Lu as the payload Conjugate No. 62- 177 A graph showing the mean change in body weight in VMRC-RCW tumor-bearing mice treated with Lu, saline, or cabozantinib. Note that the horizontal axis represents days after administration, and the vertical axis represents the rate of change in body weight (%). Additionally, black circles represent group 1: saline, X represents group 2: cabozantinib, and checkerboard triangles represent group 3: Conjugate No. 62- 177 Lu (30 MB q once), vertical stripes and triangles indicate 4 groups: Conjugate No. 62- 177 Lu (30 MBq, administered twice daily), horizontal stripes and triangles indicate 5 groups: Conjugate No. 62- 177 Lu (30 MBq, 3 doses), black triangles indicate group 6: Conjugate No. 62- 177 Lu (30 MBq, 4 doses), checkerboard pattern squares represent 7 groups: Conjugate No. 62- 177 Lu (60 MBq, single dose), vertical stripes and squares indicate 8 groups: Conjugate No. 62- 177 Lu (60 MBq, administered twice).

[0176] [ Figure 20 ] Figure 20 This illustrates that the peptide of the present invention contains DOTA- 225 Ac as the payload Conjugate No. 62- 225 The graph shows the distribution of Ac in HT-29 tumor-bearing mice at 4, 24, and 48 hours after administration. It should be noted that the horizontal axis represents individual tissues, and the vertical axis represents the radioactivity concentration (%ID / g) in each tissue.

[0177] [ Figure 21 ] Figure 21 This demonstrates that the peptide of the present invention contains DOTA- 225 Ac as the payload Conjugate No. 62- 225 A graph showing the average tumor volume in HT-29 tumor-bearing mice treated with Ac and saline. It should be noted that the horizontal axis represents the number of days after drug administration, and the vertical axis represents the tumor volume (mm). 3 Additionally, black squares represent Group 1: saline solution, and black triangles represent Group 2: Conjugate No. 62- 225 Ac (30 MBq dosing), white triangles indicate 3 groups: Conjugate No. 62- 225 Ac (60 MBq dosing), X indicates 4 groups: Conjugate No. 62- 225 Ac (90 MBq dosing), black circles indicate 5 groups: Conjugate No. 62- 225 Ac (120 MBq dosing), white circles indicate 6 groups: Conjugate No. 62- 225 Ac (150 MBq administration).

[0178] [ Figure 22 ] Figure 22 This demonstrates that the peptide of the present invention contains DOTA- 225 Ac as the payload Conjugate No. 62- 225 A graph showing the change in mean body weight in HT-29 tumor-bearing mice treated with saline and acetic acid. It should be noted that the horizontal axis represents the number of days post-transplantation, and the vertical axis represents the mean body weight (g). Additionally, black squares represent Group 1: saline, and black triangles represent Group 2: Conjugate No. 62- 225 Ac (30 MBq dosing), white triangles indicate 3 groups: Conjugate No. 62- 225 Ac (60 MBq dosing), X indicates 4 groups: Conjugate No. 62- 225 Ac (90 MBq dosing), black circles indicate 5 groups: Conjugate No. 62- 225 Ac (120 MBq dosing), white circles indicate 6 groups: Conjugate No. 62- 225 Ac (150 MBq administration). Detailed Implementation

[0179] This invention includes, without limitation, the following embodiments. Unless otherwise stated, the technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art. The substances, materials, and examples disclosed in this specification are merely illustrative and not intended to be limiting. In this specification, the reference to "in one embodiment" is not intended to limit the scope of the embodiment, and is therefore non-limiting.

[0180] 1. Abbreviation

[0181] In this specification, unless otherwise specified, the following abbreviations are used with the following meanings.

[0182] Abbreviations (general)

[0183] Å: Angstrom (unit)

[0184] ClAc: Chloroacetyl group;

[0185] Cy5SAlk: Sulfo-Cy5-alkyne;

[0186] DCM: Dichloromethane;

[0187] TIPS: Triisopropylsilyl;

[0188] tert: Uncle;

[0189] DTT: Dithiothreitol;

[0190] DMSO: Dimethyl sulfoxide;

[0191] Trt: Triphenylmethyl;

[0192] Boc: tert-butyloxycarbonyl;

[0193] DMF: N,N-dimethylformamide;

[0194] DIEA or DIPEA: N,N-diisopropylethylamine;

[0195] DIPCI or DIC: N,N'-diisopropylcarbodiimide;

[0196] Oxyma pure: Ethyl cyano(hydroxyimino)acetate;

[0197] DODT: 3,6-dioxa-1,8-octanedithiol;

[0198] DOTA-NHS ester: 2,2',2''-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0199] DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;

[0200] Fmoc: 9-fluorenylmethoxycarbonyl;

[0201] g: gram (unit);

[0202] H2O: water;

[0203] HCl: Hydrogen chloride;

[0204] HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate;

[0205] HOSu: N-hydroxysuccinimide;

[0206] HPLC: High Performance Liquid Chromatography;

[0207] LC-MS or LC / MS: Liquid Chromatography-Mass Spectrometry;

[0208] MeOH: Methanol;

[0209] mL: milliliters (units);

[0210] M: Moles per liter (unit);

[0211] μL: microliter (unit);

[0212] mM: millimoles per liter (unit);

[0213] μM: micromoles per liter (unit);

[0214] mmol: millimole (unit);

[0215] mg: milligram (unit);

[0216] MeCN or CH3CN: Acetonitrile;

[0217] min: minutes (unit):

[0218] mm: millimeter (unit);

[0219] μm: micrometer (unit);

[0220] nm: nanometer (unit);

[0221] nM: nanomoles per liter (unit);

[0222] NMP: N-methyl-2-pyrrolidone

[0223] OSu: Succinimide;

[0224] PEG: Polyethylene glycol;

[0225] rpm: revolutions per minute (unit);

[0226] tBu: tert-butyl;

[0227] TFA: Trifluoroacetic acid;

[0228] TIS: Triisopropylsilane;

[0229] Trt or Tr: Triphenylmethyl;

[0230] H-PEG4Me: 2,5,8,11-tetraoxatridecane-13-amine;

[0231] H-PEG8Me: 2,5,8,11,14,17,20,23-octaoxaconicosode-25-amine;

[0232] AA: Amino acid;

[0233] PyAOP: 7-((azabenzotriazol-1-yloxy)tripyrroleyl hexafluorophosphate ;

[0234] CSA: 10-Camphorsulfonic acid;

[0235] Fmoc-OSu: N-(9-fluorenylmethoxycarbonyloxy)succinimide;

[0236] THF: Tetrahydrofuran;

[0237] ClAcOSu: N-(chloroacetoxy)succinimide;

[0238] Pd2(dba)3·CHCl3: tris(dibenzylacetone)dipalladium(O)-chloroform complex;

[0239] SPhos: 2-Bicyclohexylphosphine-2',6'-dimethoxybiphenyl;

[0240] EDCI·HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0241] Pd(PPh3)4: tetrakis(triphenylphosphine)palladium(0);

[0242] ClAcOH: Chloroacetic acid;

[0243] conc: concentration;

[0244] HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol;

[0245] Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;

[0246] Alloc: allyloxycarbonyl;

[0247] Allyl: allyl group;

[0248] Fmoc-Qglucamine-NH2: (9H-fluorene-9-yl)methyl((S)-1-amino-1,5-dioxo-5-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)pentane-2-yl)carbamate;

[0249] H-Qglucamine-NH2:(S)-4-amino-N1-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)glutaramide;

[0250] H-KCOpipzaa(Mpe)-NH2: 3-Methylpentane-3-yl(S)-2-(4-((5,6-diamino-6-oxohexyl)carbamoyl)piperazin-1-yl);

[0251] TEA: Triethylamine.

[0252] Abbreviation (non-natural amino acids)

[0253] The abbreviations associated with the following non-natural amino acids also include substances in which the amino group of the main chain is protected by conventional protecting groups such as the Boc group and the Fmoc group.

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] 2. Peptides

[0263] This invention relates to peptides, or pharmaceutically acceptable salts thereof. In this specification, reference to "peptide" includes, unless otherwise stated, references to its pharmaceutically acceptable salts, isomers, or solvates.

[0264] In one embodiment, the peptide of the present invention comprises the following amino acid sequence:

[0265] da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-S-3Py6NH2-HseMe-dk-Y (SEQ ID NO. 1); or

[0266] The amino acid sequence described above has 1-10 amino acid residues selected from the 1st, 2nd, 3rd, 4th, 5th, 7th, 8th, 9th, 10th and 11th positions, which have substitution, addition, deletion or insertion of amino acid residues.

[0267] The peptide of the present invention may be a peptide comprising an amino acid sequence having substitutions, additions, deletions or insertions in 1 to 9 amino acid residues selected from the 1st, 2nd, 3rd, 4th, 5th, 7th, 8th, 9th and 11th amino acid residues of the amino acid sequence of SEQ ID NO. 1.

[0268] The amino acid sequence of SEQ ID NO. 1 may have substitutions, additions, deletions, or insertions in 1-10 amino acid residues selected from positions 1, 2, 3, 4, 5, 7, 8, 9, 10, and 11. The number of amino acids substituted, deleted, added, and / or inserted may be more than one and less than ten, with a lower limit of one. The upper limits are 10, 9, 8, 7, 6, 5, 4, 3, and 2, with a minimum of one.

[0269] Preferably, the amino acid sequence may have substitutions, additions, deletions, or insertions in 1-7 amino acid residues selected from positions 1, 4, 7, 8, 9, 10, and 11 of the amino acid sequence in SEQ ID NO. 1. The number of amino acids that are substituted, deleted, added, and / or inserted may be more than one and less than seven, with a lower limit of one, an upper limit of seven, six, five, four, three, or two, and a minimum of one. Alternatively, the amino acid sequence may have substitutions, additions, deletions, or insertions in 1-6 amino acid residues selected from positions 1, 4, 7, 8, 9, and 11 of the amino acid sequence in SEQ ID NO. 1.

[0270] In one approach, the preferred method is the "substitution" of amino acids. Non-limitingly, such amino acid substitution is preferably a conservative amino acid substitution.

[0271] "Conservative amino acid substitution" refers to the substitution of an amino acid with a functionally equivalent or similar amino acid. Conservative amino acid substitutions in peptides result in a static change in the peptide's amino acid sequence. For example, if one or more amino acids of the same polarity function equivalently, it results in a static change in the amino acid sequence of such a peptide. Generally, substitutions within a group can be considered conserved in terms of both structure and function. However, it will be apparent to those skilled in the art that the role of a particular amino acid residue can be determined based on the significance of the molecule containing that amino acid in its three-dimensional structure. For example, a cysteine ​​residue can take the form of an oxidized form (disulfide) with lower polarity compared to its reduced (thiol) form. The long aliphatic portion of the arginine side chain can constitute an important structural and functional feature. Additionally, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic interactions or cation-pi interactions. In such cases, even if these amino acids with side chains are replaced with amino acids belonging to the acidic or nonpolar group, the structure and function can still be conserved. Residues such as proline, glycine, and cysteine ​​(in disulfide form) can directly affect the three-dimensional structure of the main chain and often cannot be substituted without structural distortion.

[0272] As shown below, conserved amino acid substitutions include specific substitutions based on side chain similarity (e.g., substitutions described in Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions. Furthermore, for conserved amino acid substitutions, for example, in groups of naturally occurring amino acids classified based on the properties of their common side chains, it is preferable to substitute an amino acid from the same group as the group to which the particular amino acid belongs.

[0273] Hydrophobic (also known as nonpolar) amino acids: Amino acids that exhibit hydrophobic (nonpolar) properties, including alanine (“Ala” or also abbreviated as “A”), glycine (“Gly” or also abbreviated as “G”), valine (“Val” or also abbreviated as “V”), leucine (“Leu” or also abbreviated as “L”), isoleucine (“Ile” or also abbreviated as “I”), proline (“Pro” or also abbreviated as “P”), phenylalanine (“Phe” or also abbreviated as “F”), tryptophan (“Trp” or also abbreviated as “W”), tyrosine (“Tyr” or also abbreviated as “Y”), and methionine (“Met” or also abbreviated as “M”).

[0274] It should be noted that hydrophobic amino acids can be further divided into the following groups.

[0275] Aliphatic amino acids: Amino acids with fatty acid or hydrogen in their side chain, including Ala, Gly, Val, Ile, and Leu.

[0276] Aliphatic / branched-chain amino acids: Amino acids with branched fatty acids in their side chains, including Val, Ile, and Leu.

[0277] Aromatic amino acids: Amino acids with an aromatic ring in their side chain, including Trp, Tyr, and Phe.

[0278] Hydrophilic (also known as polar) amino acids: Amino acids that exhibit hydrophilicity (polarity) include serine (“Ser” or also abbreviated as “S”), threonine (“Thr” or also abbreviated as “T”), cysteine ​​(“Cys” or also abbreviated as “C”), asparagine (“Asn” or also abbreviated as “N”), glutamine (“Gln” or also abbreviated as “Q”), aspartic acid (“Asp” or also abbreviated as “D”), glutamic acid (“Glu” or also abbreviated as “E”), lysine (“Lys” or also abbreviated as “K”), arginine (“Arg” or also abbreviated as “R”), and histidine (“His” or also abbreviated as “H”).

[0279] It should be noted that hydrophilic amino acids can be further divided into the following groups.

[0280] Acidic amino acids: Amino acids whose side chains show acidity, including Asp and Glu.

[0281] Basic amino acids: Amino acids whose side chains show basicity, including Lys, Arg, and His.

[0282] Neutral amino acids: Amino acids whose side chains show neutrality, including Ser, Thr, Asn, Gln, and Cys.

[0283] Additionally, Gly and Pro can also be classified as "amino acids that affect the orientation of the main chain," and amino acids containing sulfur molecules in their side chains, Cys, and Met can also be classified as "sulfur-containing amino acids."

[0284] In this specification, "amino acid" includes not only naturally occurring amino acids but also non-natural amino acids. Non-natural amino acids include, for example, naturally occurring amino acids described above that have been N-alkylated, such as N-alkyl amino acids, and amino acids in which the nitrogen atoms forming peptide bonds are modified with branched or unbranched lower-order alkyl groups (e.g., C1-C5, preferably C1-C3, more preferably C1). Among N-alkyl amino acids, N-ethyl amino acids, N-butyl amino acids, or N-methyl amino acids are preferred, with N-methyl amino acids being more preferred. Furthermore, non-natural amino acids include chemically modified amino acids such as D-type amino acids (also called D-amino acids), β-amino acids, γ-amino acids, amino acid mutants, and amino acid derivatives, as well as amino acids such as ortholeucine and ornithine that do not form protein building blocks in living organisms. In addition, it also includes: amino acids formed by further adding functional groups to the side chains of natural amino acids or by replacing them with other functional groups (e.g., amino acids with substitutions or additions in the arylene, alkylene, or other parts of the side chain; amino acids with increased C numbers in the arylene, alkylene, or alkyl groups of the side chain; amino acids with substitutions in the aromatic ring of the side chain; amino acids that have undergone heterocyclization or fused cyclization).

[0285] It should be noted that by adding or substituting functional groups into the side chains of natural amino acids, different properties can be imparted to them. For example, Dap is an amino acid with an amino group on the side chain of alanine. By adding this amino group, unlike alanine which belongs to the nonpolar amino acid group, it exhibits the properties of a basic polar amino acid. That is, in the above-mentioned group that classifies natural amino acids based on their common side chain properties, non-natural amino acids with the same side chain properties can be included. For example, N-methylarginine (MeR), an amino acid formed by methylating the nitrogen atom of the main chain of arginine, which belongs to the basic amino acid group, is a non-natural amino acid. Because it exhibits basicity, it can be classified as a basic amino acid. Thus, non-natural amino acids exhibiting the same side chain properties as a certain amino acid can also be included as objects of conserved amino acid substitution. It should be noted that D-amino acids such as de (D-glutamic acid) can be classified as D-amino acids, or they can be classified according to the properties of their side chains; N-methyl amino acids can also be classified as N-alkyl amino acids, or they can be classified according to the properties of the original amino acid side chain that has not undergone N-methylation.

[0286] Non-natural amino acids include, without limitation, N-methyl amino acids, D-amino acids, etc., and more specifically, da, de, dhgl, dq, dk, dyae, df, dkCOpipzaa, Tbg, 3Py6CON, 3Py6NH2, Hgl, alT, Hgn, SMe, DabAc, KCOpipzaa, Meda, PeG, MeG, MedkCOpipzaa, Ahp, pHPeG, pMeOPeG, MsMeapG, 3OMePeG, PpG, pFPeG, and mCPe. Amino acids including G, pCPeG, 4HPpG, 4OMePpG, 4PypG, 4COOPeG, 3COOPeG, 3FPeG, P4Sh, Hse, Qmm, Qdm, QPEG8Me, 5Inda, F4OMe, HseMe, Acb, dkAc, dnle, t4amCh, Nle, MeC, MeA, MeS, MeN, Hpr, Hgn(Qglucamine-NH2), Hgn(KCOpipzaa-NH2), Qglucamine, and MeCt.

[0287] "Pharmaceutically acceptable salts" refers to any salt of a peptide. Examples of pharmaceutically acceptable salts include salts formed with inorganic acids such as sulfuric acid, hydrochloric acid, and phosphoric acid; salts formed with organic acids such as acetic acid, oxalic acid, lactic acid, tartaric acid, fumaric acid, maleic acid, methanesulfonic acid, and benzenesulfonic acid; salts formed with amines such as trimethylamine and methylamine; or salts formed with metal ions such as sodium, potassium, and calcium ions. Compounds that contain water over time are also included in pharmaceutically acceptable salts.

[0288] In addition, the aforementioned peptides can be isomers, such as stereoisomers. Peptides sometimes have one or more stereocenters, which can exist independently in either (R) or (S) configurations. In this specification, the aforementioned peptides can be optically active or racemic; "can be isomers" means including racemic, optically active, positional, and stereoisomers, or combinations thereof. Furthermore, in one embodiment, it can be a mixture of one or more isomers. It should be noted that "stereoisomer of peptide" refers to the stereoisomer of the aforementioned peptide. In this specification, the term "peptide" includes its isomers unless otherwise specified.

[0289] Furthermore, the aforementioned peptides can be solvates. Solvation refers to the phenomenon where ions generated by the ionization of solute molecules (peptides or conjugates) combine with and surround solvent molecules through electrostatic forces, hydrogen bonds, etc., thereby causing the solute to diffuse into the solvent. The type of solvent is not particularly limited. When the solvent is water, it is specifically called a "hydrate."

[0290] The following peptides are examples of peptides that have been confirmed to have CA9 binding activity in the embodiments of this specification.

[0291] da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-S-3Py6NH2-HseMe-dk-Y (SEQ ID NO. 1);

[0292] A peptide in which MeC, C, or MeCt is added as the 13th amino acid residue in the amino acid sequence of SEQ ID NO. 1; and

[0293] A peptide in which MeA, MeS, MeN, Hpr, G or P are added as the 13th amino acid residue and MeG, G, da or Meda are added as the 14th amino acid residue in the amino acid sequence of SEQ ID NO. 1.

[0294] In one embodiment, the peptide of the present invention comprises:

[0295] (1) In the amino acid sequence of SEQ ID NO. 1, MeC, C, or MeCt is added as the 13th amino acid residue; or

[0296] (2) In the amino acid sequence of SEQ ID NO. 1, MeA, MeS, MeN, Hpr, G or P are added as the 13th amino acid residue, and MeG, G, da or Meda are added as the 14th amino acid residue.

[0297] The amino acid residue at position 13 in (1) and (2) above are different. In one embodiment, when the peptide of the present invention is a cyclic peptide, the number of amino acid residues contained in the cyclic structure is not limited to 13 or 14, and may be more than 14 if there are further added amino acids at the end.

[0298] In one embodiment, the peptide of the present invention comprises one or more of the following elements:

[0299] The amino acid residue at position 1 of (SEQ ID NO. 1) is da, dq, de, dhgl, or dk;

[0300] The second amino acid residue is I or Tbg;

[0301] The third amino acid residue is 3Py6NH2 or 3Py6CON;

[0302] The fourth amino acid residue is S, Hgl, Hgl(PEG8Me), alT, Hgn, SMe, KCOpipzaa, or DabAc;

[0303] The fifth amino acid residue is PeG, MeG, Meda, or MedkCOpipzaa;

[0304] The amino acid residue at position 7 is PeG, pMeOPeG, MsMeapG, 3OMePeG, pHPeG, PpG, pFPeG, mCPeG, pCPeG, 4HPpG, 4OMePpG, 4PypG, 4COOPeG, 3COOPeG, or 3FPeG.

[0305] The amino acid residue at position 8 is H, S, P, D, KCOpipzaa, Q (PEG8Me), P4Sh, Hse, SMe, Qmm, or Qdm;

[0306] The amino acid residue at position 9 is 3Py6NH2, 5Inda, Y, or F4OMe;

[0307] The 10th amino acid residue is Ahp, HseMe, or E; and

[0308] The 11th amino acid residue is dk, G, da, dq, dkCOpipzaa, dk(t4amCh), de, Acb, or dkAc.

[0309] The above-described option for the amino acid residues at positions 1 through 12 can be selected in any combination. It is not limited to having one, two, three, four, five, six, seven, eight, nine, ten, or eleven of the above-described requirements. Preferably, all twelve of the above-described requirements are included.

[0310] In one embodiment, the peptide of the present invention comprises one or more of the following elements:

[0311] The amino acid residue at position 1 of (SEQ ID NO. 1) is da, dq, de, dhgl, or dk;

[0312] The second amino acid residue is I or Tbg;

[0313] The third amino acid residue is 3Py6NH2 or 3Py6CON;

[0314] The fourth amino acid residue is S, Hgl, Hgl(PEG8Me), alT, Hgn, SMe, KCOpipzaa, or DabAc;

[0315] The fifth amino acid residue is PeG, MeG, Meda, or MedkCOpipzaa;

[0316] The amino acid residue at position 7 is PeG, pMeOPeG, MsMeapG, 3OMePeG, pHPeG, PpG, pFPeG, mCPeG, pCPeG, 4HPpG, 4OMePpG, 4PypG, 4COOPeG, 3COOPeG, or 3FPeG.

[0317] The amino acid residue at position 8 is H, S, P, D, KCOpipzaa, Q (PEG8Me), P4Sh, Hse, SMe, Qmm, or Qdm;

[0318] The amino acid residue at position 9 is 3Py6NH2, 5Inda, Y, or F4OMe;

[0319] The 10th amino acid residue is Ahp, HseMe, or E; and

[0320] The 11th amino acid residue is dk, G, da, dq, dkCOpipzaa, dk(t4amCh), de, Acb, or dkAc.

[0321] and,

[0322] (1) In the amino acid sequence of SEQ ID NO. 1, MeC, C or MeCt is added as the 13th amino acid residue;

[0323] or

[0324] (2) In the amino acid sequence of SEQ ID NO. 1, MeA, MeS, MeN, Hpr, G or P are added as the 13th amino acid residue, and MeG, G, da or Meda are added as the 14th amino acid residue.

[0325] The above-described option for the amino acid residues at positions 1 through 12 can be selected in any combination. It is not limited to having one, two, three, four, five, six, seven, eight, nine, ten, or eleven of the above-described requirements. Preferably, all twelve of the above-described requirements are included.

[0326] In one embodiment, the peptide of the present invention comprises one or more of the following elements:

[0327] The second amino acid residue of (SEQ ID NO. 1) is Tbg;

[0328] The third amino acid residue is 3Py6NH2;

[0329] The fifth amino acid residue is Meda;

[0330] The amino acid residue at position 7 is either PeG or pHPeg;

[0331] The 9th amino acid residue is 3Py6NH2 or 5Inda; and

[0332] The 10th amino acid residue is HseMe.

[0333] The above-described option for the amino acid residues at positions 1 through 10 can be selected in any combination. It is not limited to having one, two, three, four, or five of the above requirements. Preferably, all six requirements are present.

[0334] In one embodiment, the peptide of the present invention comprises one or more of the following elements:

[0335] The second amino acid residue of (SEQ ID NO. 1) is Tbg;

[0336] The third amino acid residue is 3Py6NH2;

[0337] The fifth amino acid residue is Meda;

[0338] The amino acid residue at position 7 is either PeG or pHPeg;

[0339] The 9th amino acid residue is 3Py6NH2 or 5Inda; and

[0340] The 10th amino acid residue is HseMe.

[0341] and

[0342] (1) In the amino acid sequence of SEQ ID NO. 1, MeC, C or MeCt is added as the 13th amino acid residue;

[0343] or

[0344] (2) In the amino acid sequence of SEQ ID NO. 1, MeA, MeS, MeN, Hpr, G or P are added as the 13th amino acid residue, and MeG, G, da or Meda are added as the 14th amino acid residue.

[0345] The above-described option for the amino acid residues at positions 1 through 10 can be selected in any combination. It is not limited to having one, two, three, four, or five of the above requirements. Preferably, all six requirements are present.

[0346] In one embodiment, the peptide of the present invention comprises the following requirements:

[0347] The second amino acid residue of (SEQ ID NO. 1) is Tbg;

[0348] The third amino acid residue is 3Py6NH2;

[0349] The fifth amino acid residue is Meda;

[0350] The amino acid residue at position 7 is either PeG or pHPeg;

[0351] The 9th amino acid residue is 3Py6NH2 or 5Inda; and

[0352] The 10th amino acid residue is HseMe.

[0353] In one embodiment, the peptide of the present invention,

[0354] The second amino acid residue of (SEQ ID NO. 1) is Tbg;

[0355] The third amino acid residue is 3Py6NH2;

[0356] The fifth amino acid residue is Meda;

[0357] The amino acid residue at position 7 is either PeG or pHPeg;

[0358] The 9th amino acid residue is 3Py6NH2 or 5Inda; and

[0359] The 10th amino acid residue is HseMe.

[0360] and

[0361] (1) In the amino acid sequence of SEQ ID NO. 1, MeC, C, or MeCt is added as the 13th amino acid residue; or

[0362] (2) In the amino acid sequence of SEQ ID NO. 1, MeA, MeS, MeN, Hpr, G or P are added as the 13th amino acid residue, and MeG, G, da or Meda are added as the 14th amino acid residue.

[0363] In one embodiment, the peptide of the present invention comprises one or more of the following elements:

[0364] The amino acid residue at position 1 of (SEQ ID NO. 1) is de(PEG8Me), dk, dyae, or dkCOpipzaa;

[0365] The second amino acid residue is I or Tbg;

[0366] The third amino acid residue is 3Py6NH2 or 3Py6CON;

[0367] The fourth amino acid residue is K, KCOpipzaa, Hgn(Qglucamine-NH2), Hgn(KCOpipzaa-NH2), or Qglucamine;

[0368] The fifth amino acid residue is PeG, MeG, Meda, or MedkCOpipzaa;

[0369] The amino acid residue at position 7 is PeG, pMeOPeG, MsMeapG, 3OMePeG, pHPeG, PpG, pFPeG, mCPeG, pCPeG, 4HPpG, 4OMePpG, 4PypG, 4COOPeG, 3COOPeG, or 3FPeG.

[0370] The 8th amino acid residue is K;

[0371] The amino acid residue at position 9 is 3Py6NH2, 5Inda, Y, or F4OMe;

[0372] The 10th amino acid residue is Ahp, HseMe, or E; and

[0373] The amino acid residue at position 11 is either dk(F) or dk(PEG8c).

[0374] The above-described option for the amino acid residues at positions 1 through 12 can be selected in any combination. It is not limited to having one, two, three, four, five, six, seven, eight, nine, ten, or eleven of the above-described requirements. Preferably, all twelve of the above-described requirements are included.

[0375] Furthermore, in one embodiment of the peptide of the present invention, the contained amino acids may be further modified. Modification refers to, for example, attaching other compounds to the side chain ends of the amino acids. For example, low / medium molecular weight compounds such as PEG, sugar chains, or other amino acids may be attached to the side chain ends of lysine, aspartic acid, glutamic acid, arginine, glutamine, or tyrosine.

[0376] In one embodiment, the aforementioned peptide is a cyclic peptide. A "cyclic peptide" refers to a peptide in which two amino acids are linked together, and which is wholly or partially cyclic. The aforementioned peptide also includes: peptides in which the amino acids form a cross-linked structure; peptides that form a cyclic structure through lactam ring formation or macrocyclization; and peptides with a lasso peptide-like structure. That is, the aforementioned cyclic peptide only needs to have a portion forming a cyclic structure; it may have a linear portion.

[0377] Peptides often suffer from poor metabolic stability in vivo and are difficult to pass through cell membranes due to their large size. To address these issues, peptide cyclization has been employed. It has been shown that peptide cyclization improves protease tolerance and metabolic stability, and because conformational changes are restricted, rigidity is enhanced, resulting in improved membrane permeability and affinity for target proteins.

[0378] In one embodiment, the peptide has a cyclic structure formed by the combination of a chloroacetylated amino acid (preferably the N-terminal amino acid (the amino acid residue at position 1)) and a cysteine ​​residue (including a cysteine ​​substitute such as N-methylcysteine) contained in the peptide. In another embodiment, the peptide has a cyclic structure formed by the combination of an N-terminal amino acid (the amino acid residue at position 1) and a cysteine ​​residue (or a compound containing an optional substituted cysteine ​​residue or an internally -SH group) contained in the peptide. In yet another embodiment, the peptide has a cyclic structure formed by the combination of an N-terminal amino acid (the amino acid residue at position 1) and a cysteine ​​residue at position 13 contained in the peptide (including a cysteine ​​substitute such as N-methylcysteine). In yet another embodiment, the peptide has a cyclic structure formed by the combination of a chloroacetylated N-terminal amino acid (the amino acid residue at position 1) and a cysteine ​​residue at position 13 contained in the peptide or a -SH group contained in a compound (base) having a -SH group. The compound (base) having a -SH group includes, for example, MeCt. "Chloroacetylation" can also be "haloacetylation" based on other halogens. Additionally, "acetylation" can also be "acylation" based on acyl groups other than acetyl groups. In one embodiment, the peptide has a cyclic structure obtained by combining the amino acid residue at position 1 of the chloroacetylated amino acid sequence of SEQ ID NO. 1 with a cysteine ​​residue contained in the peptide. In another embodiment, the peptide can be a structure formed by combining the amino acid sequence of any of SEQ ID NO. 1-103 with a cysteine ​​residue or its substitution contained in the peptide via an acetyl group. That is, in this specification, for example, "a cyclic peptide composed of SEQ ID NO. XX" also includes: a cyclic structure formed by combining the amino acid sequence shown in SEQ ID NO. XX, the acetyl group contained in the amino acid sequence bound to the first amino acid, the cysteine ​​residue or its substitution, and the S (sulfur atom) contained in a compound having a -SH group.

[0379] In this specification, sometimes a portion of the amino acids is modified for peptide cyclization. Such partially modified amino acids are also included. For example, as described above, sometimes a chloroacetyl group (ClAc group) is added to the N-terminal amino acid, which cyclizes by binding to a cysteine ​​residue or an amino acid residue with a -SH group in the peptide. Various (natural / non-natural) amino acids with added chloroacetyl groups are also included in the amino acids of this application.

[0380] In one embodiment, the peptide has a cyclic structure formed by the combination of an N-terminal amino acid (the amino acid residue at position 1) and a C-terminal amino acid (the amino acid located at the C-terminus when an amino acid residue at position 13 or 14, etc., is added to the C-terminus of the amino acid sequence of SEQ ID NO. 1). In another embodiment, the peptide has a cyclic structure formed by the combination of the amino group of the N-terminal amino acid (the amino acid residue at position 1) and the carboxyl group of the amino acid at position 13 contained in the peptide. In yet another embodiment, it may have a cyclic structure formed by the combination of an added amino acid at position 13 and an added amino acid at position 14 or 15, for example, the combination of the N-terminal amino acid (the amino acid residue at position 1) and the amino acid at position 13. In one embodiment, the peptide has a cyclic structure formed by the combination of the amino group of the amino acid residue at position 1 of the amino acid sequence of SEQ ID NO. 1 contained in the peptide and the carboxyl group of the amino acid residue at position 14. In another embodiment, the peptide has a cyclic structure formed by combining the amino group of the N-terminal amino acid (the amino acid residue at position 1) with a functional group present at the end of the side chain of the C-terminal amino acid contained in the peptide.

[0381] In one embodiment, the peptide comprises or is composed of the amino acid sequence described in any one of SEQ ID NO. 2-54. In another embodiment, the peptide is a cyclic peptide comprising the amino acid sequence described in any one of SEQ ID NO. 2-54, or a cyclic peptide composed of the amino acid sequence described in any one of SEQ ID NO. 2-54. The peptide is a peptide composed of the amino acid sequence described in any one of SEQ ID NO. 2-54, or a peptide composed of an amino acid sequence consisting of 1-13 or 14 (preferably 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2, or 1) amino acid residues substituted, deleted, inserted, or added therefrom. It should be noted that, regarding the sequence with added amino acid residues, if the added portion is a linker, multiple amino acid residues (e.g., 1-14 residues) can be further added. Furthermore, peptides composed of amino acid sequences in which these amino acid residues have been substituted, deleted, inserted, or added are preferably, for example, peptides with CA9 binding activity.

[0382] In another embodiment, the peptide comprises or is composed of the amino acid sequences described in any of SEQ ID NO. 55-103. In another embodiment, the peptide is a cyclic peptide comprising the amino acid sequences described in any of SEQ ID NO. 55-103, or a cyclic peptide composed of the amino acid sequences described in any of SEQ ID NO. 55-103. The peptide is either a peptide composed of the amino acid sequences described in any of SEQ ID NO. 55-103, or a peptide composed of amino acid sequences from which 1-13 or 14 (preferably 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2, or 1) amino acid residues have been replaced, deleted, inserted, or added. It should be noted that, regarding sequences with added amino acid residues, if the added portion is a linker, multiple amino acid residue sequences (e.g., 1-14 residues) can be further added. Furthermore, peptides composed of amino acid sequences with substitutions, deletions, insertions, or additions of these amino acid residues are preferably, for example, peptides with CA9 binding activity. In one embodiment, the peptides of the present invention do not include: peptides comprising SEQ ID NO. 103 or peptides composed of SEQ ID NO. 103.

[0383] Furthermore, in one embodiment, the peptide comprises or is composed of the amino acid sequence described in any of SEQ ID NO. 55-102. In another embodiment, the peptide is a cyclic peptide comprising or composed of the amino acid sequence described in any of SEQ ID NO. 55-103. The peptide may be a peptide composed of the amino acid sequence described in any of SEQ ID NO. 55-102, or a peptide composed of an amino acid sequence from which 1-13 or 14 (preferably 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2, or 1) amino acid residues have been substituted, deleted, inserted, or added.

[0384] In addition, in one embodiment, the peptide may be a peptide consisting of the amino acid sequence described in SEQ ID NO. 2 or 21, or a peptide consisting of an amino acid sequence in which 1-13 or 14 (preferably 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2 or 1) amino acid residues have been replaced, deleted, inserted or added.

[0385] The peptide described above may contain additional amino acid residues in addition to the amino acid sequence of SEQ ID NO. 1. Non-limitingly, the peptide described above may contain additional amino acid residues in addition to the amino acid sequences described in SEQ ID NO. 2-54, 55-102, or 55-103.

[0386] The "added amino acid residues" can be contained within the peptide forming the cyclic structure, or can be added further based on the cyclic peptide in the form of a linker. In one manner, they can be added to the side chain of the amino acid residues contained in the cyclic peptide. There is no particular limitation on the number (number / length of amino acids) of amide bonds in the peptide or peptide site.

[0387] Furthermore, linkers can be added based on cyclic peptides. Examples of linkers include, for instance, amino acid linkers (peptide linkers), chemical linkers, fatty acid linkers, nucleic acid linkers, and glycan linkers, as described above. Complexes of chemical linkers and peptide linkers are also possible. For example, a PEG linker composed of 1-24 ethylene glycol units can be used as a chemical linker. Alternatively, the linker can be a fatty acid linker containing a divalent chemical moiety derived from a fatty acid. Amino acid (peptide) linkers are linkers containing at least one amino acid, and can be glycine-rich peptides such as those with the sequence [Gly-Gly-Gly-Gly-Ser]n (where n is 1, 2, 3, 4, 5, or 6) as described in U.S. Patent No. 7,271,149, or serine-rich peptide linkers as described in U.S. Patent No. 5,525,491. Additionally, peptide linkers consisting of amino acids such as K, dk, F, G, df, Nle, dnle, and t4amCh, or two or more amino acids linked together, can be used without limitation. It should be noted that in peptide linkers, amino acid-amino acid or amino acid-chemical linker bonding can also occur via the side chains of amino acids. Without limitation, the addition of linkers can sometimes alter the physical properties of the peptide (e.g., solubility).

[0388] The linker can be added at any position. For example, it can bind to an amino acid residue located at the C-terminus or to an amino acid residue contained in a cyclic peptide. In one embodiment, the peptide contains a linker at the C-terminus. Preferably, it binds to the side chain of an amino acid residue contained in a cyclic peptide, or to a Cys, a compound having a -SH group, or any amino acid residue located at the C-terminus, more preferably to the side chain of an amino acid residue at position 1, 4, 8, or 11.

[0389] In one embodiment, the peptide is a peptide capable of binding a linker and / or payload at its C-terminus.

[0390] In one embodiment, the peptide is an amino acid residue at position 1, 4, 8, or 11, or an amino acid residue located at the C-terminus, or an amino acid residue with added amino acids that can bind linkers and / or payloads.

[0391] An amino acid residue capable of binding a linker and / or payload refers to an amino acid residue having a functional group capable of binding a linker and / or payload at the end of its side chain, or, in the case of an amino acid residue located at the C-terminus or an added amino acid residue, having a functional group capable of binding a linker and / or payload at either the end of its side chain or the C-terminus. It should be noted that the payload will be explained later.

[0392] In one manner, the amino acid residues capable of binding linkers and / or payloads refer to structures having the structure shown in formula (II) below.

[0393] [Chemical Formula 2]

[0394]

[0395] In formula (II),

[0396] R1 is H or a C1-3 alkyl group;

[0397] R2 can be one of the following: C1-6 alkyl-NH-R3, C1-6 alkyl-C6 aryl-O-C1-3 alkyl-NH-R3, C1-6 alkyl-NH(=O)-CH(C1-6 alkylphenyl)-NH-R3, C1-6 alkyl-NH(=O)-CH(C1-6 alkyl)-NH-R3, or C1-6 alkyl-NH(=O)-C3-8 cycloalkyl-C1-3 alkyl-NH-R3.

[0398] R3 can be H or any functional group.

[0399] R3 is H or any functional group, including functional groups protected by any protecting group. Preferably, it is a structure capable of binding a linker and / or a payload in R3. Structures capable of binding linkers and / or payloads can be, for example, functional groups such as amide groups, or well-known functional groups and compounds used in click chemistry, such as alkynes or azides, when binding compounds. The term "protecting group" is not particularly limited and includes, for example, Fmoc (9-fluorenylmethoxycarbonyl), tertBu (tert-butyl), Alloc (allyloxycarbonyl), Boc (tert-butyloxycarbonyl), etc.

[0400] Furthermore, the aforementioned peptides can form polymers via linkers, etc. The number of peptides contained in the polymer is not limited. In one embodiment, the polymer is a dimer, trimer, tetramer, pentamer, hexamer, octamer, or more. The polymer can contain multiple identical peptides or multiple different peptides.

[0401] In one embodiment, the peptide is preferably CA9-binding activity.

[0402] CA9 is one of 15 carbonic anhydrases known as CA9 (Carbonic anhydrase IX). It is a transmembrane protein that exists locally on the cell membrane as a homodimer and plays an important role in maintaining intracellular pH. CA9 is known to be expressed under hypoxic conditions, particularly in specific cancers of the tumor, cervix, uterine body, ovary, kidney, esophagus, lung, breast, brain, digestive tract, liver, pancreas, head and neck, salivary glands, body cavities, and skin. Therefore, CA9 is considered a general marker of hypoxia in various solid tumors.

[0403] In addition, CA9 is known to be used not only as a marker for evaluating the presence and malignancy of tumors, but also as a marker for poor prognosis of cervical tumors, rectal tumors, breast tumors, lung tumors, and brain tumors.

[0404] Unless otherwise specified, the term "CA9" or "CA-9" in this specification refers to carbonic anhydrase-9, which is found in mammals, preferably rodents such as mice, and primates such as humans. More preferably, it refers to human CA9. It should be noted that "human CA9" in this specification refers to the naturally occurring CA9 found in humans (e.g., Gene ID: 768). It should also be noted that the amino acid sequences of CA9 in humans, mice, rats, etc., are known to be similar.

[0405] In this specification, CA9 binding activity refers to specific binding with CA9, specifically specific binding with human CA9. Unless otherwise stated, CA9 binding activity in this specification refers to binding activity with human CA9.

[0406] In a non-limiting sense, the binding state of the peptides of the present invention to CA9 can be expressed by indicators such as affinity constant KA, dissociation constant KD, binding rate constant kon, and dissociation rate constant koff.

[0407] The affinity constant KA and dissociation constant KD are indicators of the binding affinity, or the strength of the bond, between two molecules in equilibrium. The dissociation constant KD is the reciprocal of the affinity constant KA. Furthermore, a smaller value of the dissociation constant KD indicates a stronger bond. On the other hand, the rate of the binding / dissociation reaction between two molecules in equilibrium can be represented by the binding rate constant kon and the dissociation rate constant koff, obtained from reaction kinetic analysis, where KD = koff / kon. Therefore, even with the same dissociation constant KD, there can be cases of slow association but slow dissociation (both kon and koff values ​​are small), and cases of rapid association and rapid dissociation (both kon and koff values ​​are large), with completely different bond states.

[0408] The affinity constants KA, dissociation constant KD, binding rate constant kon, and dissociation rate constant koff, which represent the binding state of the above peptides to CA9, can be determined using any intermolecular interaction determination method known to those skilled in the art.

[0409] The binding state of the aforementioned peptide to CA9 can be determined using known methods. For example, it can be determined by surface plasmon resonance spectroscopy (SPR). Non-limitingly, SPR can be performed using, for example, the BIACORE system (Cytiva) as a biosensor (a device for analyzing biomolecular interactions).

[0410] One indicator of the CA9 binding activity of the aforementioned peptides is the KD dissociation constant. The lower the dissociation constant KD, the higher the binding activity (affinity). Based on surface plasmon resonance spectroscopy analysis, the dissociation constant KD associated with the binding of the aforementioned peptides to, for example, human CA9 is non-limitingly 50 nM or less, 30 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 8 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, and 1 nM or less. There is no particular limitation on the lower limit of the dissociation constant KD for the binding of the aforementioned peptides to human CA9. Non-limitingly, the dissociation constant KD of the aforementioned peptides is 0.01 nM or more, 0.05 nM or more, 0.1 nM or more, 0.2 nM or more, 0.3 nM or more, 0.4 nM or more, and 0.5 nM or more. Non-limiting, the dissociation constant KD of the above peptide is 0.01-50 nM, preferably 0.05-30 nM, more preferably 0.2-15 nM, particularly preferably 0.3-10 nM, and most preferably 0.4-5 nM.

[0411] The binding of the aforementioned peptides to CA9 can also be studied using other known methods for measuring binding ability, such as ELISA (enzyme-linked immunosorbent assay). ELISA can be performed, for example, using CA9 beads and peptides with an HA tag sequence added to the C-terminus to study binding activity. Alternatively, FACS (fluorescence-activated cell sorting) can also be used, for example.

[0412] Non-limiting, the binding of the above peptide to CA9 is preferably non-covalent.

[0413] In one embodiment, the peptide described above has a cyclic structure as shown by the peptide portion contained in the chemical formulas of Examples 1-1 to 1-16 and Examples 11-1 to 5.

[0414] Peptides with CA9 binding activity are effective in the evaluation, diagnosis, and treatment of various diseases involving CA9.

[0415] In one embodiment, the present invention relates to the above-mentioned peptide having CA9 binding activity.

[0416] In one embodiment, the present invention relates to the use of the aforementioned peptide for binding with CA9.

[0417] In one embodiment, the present invention relates to the aforementioned peptide for binding with CA9.

[0418] In this invention, the peptide of this invention binds to CA9 in vitro or in vivo.

[0419] Unless otherwise specified, the information described in other items also applies to this item.

[0420] 3. Peptide Manufacturing

[0421] The peptides of the present invention can be manufactured, for example, by any known method for manufacturing peptides.

[0422] Unless otherwise specified, the matters described in other items also apply to this item.

[0423] Chemical synthesis methods include liquid-phase methods, solid-phase methods, and hybrid methods combining liquid-phase and solid-phase methods; gene recombination methods, etc.

[0424] Solid-phase methods, for example, involve esterifying the hydroxyl groups of a resin with hydroxyl groups with the carboxyl group of the first amino acid (usually the C-terminal amino acid of the target peptide) protected by a protecting group. Known dehydrating condensing agents such as 1-mesinesulfone-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC) can be used as esterification catalysts.

[0425] Next, the protecting group of the α-amino group of the first amino acid is removed, and simultaneously, a second amino acid with all functional groups protected except for the carboxyl group of the main chain is added, activating the carboxyl group and causing the first and second amino acids to bind. Further, the α-amino group of the second amino acid is deprotected, and a third amino acid with all functional groups protected except for the carboxyl group of the main chain is added, activating the carboxyl group and causing the second and third amino acids to bind. This process is repeated until a peptide of the target length is synthesized, at which point all functional groups are deprotected.

[0426] Examples of resins synthesized in the solid phase include Merrifield resin, MBHA resin, Cl-Trtresin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), and HMPA-PEGA resin (Merck). These resins can be used after being cleaned with solvents such as dimethylformamide (DMF), 2-propanol, and dichloromethane.

[0427] Examples of protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, 9-fluorenylmethoxycarbonyl (Fmoc) group, benzyl, allyl, and allyloxycarbonyl (Alloc) group. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment based on piperidine or pyrrolidine.

[0428] For example, methyl ester, ethyl ester, allyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc. can be used to protect the α-carboxyl group.

[0429] The activation of the carboxyl group can be achieved using condensing agents. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), and (1H-benzotriazol-1-yloxy)tris(dimethylamino) Hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazole -3-oxide hexafluorophosphate (HBTU), etc.

[0430] Peptide chains can be cleaved from resin by treatment with acids such as TFA or hydrogen fluoride (HF).

[0431] The manufacture of peptides based on gene recombination (translation synthesis system) can be carried out using nucleic acids encoding the aforementioned peptides. The nucleic acids encoding these peptides can be DNA or RNA.

[0432] The nucleic acid encoding the aforementioned peptide can be prepared by known methods or methods based on known methods. For example, it can be synthesized using an automated synthesis apparatus. To insert the obtained DNA into a vector, a restriction endonuclease recognition site can be added. Alternatively, a base sequence encoding the amino acid sequence for cleavage by enzymes or the like can be introduced.

[0433] To inhibit degradation caused by host-derived proteases, chimeric protein expression, in which the target peptide is expressed as a chimeric peptide with other peptides, can also be used. In such cases, the nucleic acid used can be a nucleic acid encoding both the target peptide and the peptide bound to it.

[0434] Next, an expression vector is prepared using the nucleic acid encoding the peptide. The nucleic acid can be inserted downstream of the promoter of the expression vector directly, or by digestion with restriction endonucleases, or by adding a linker. Examples of vectors include E. coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retroviruses, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), and granules.

[0435] The promoter can be appropriately selected based on the type of host. When the host is an animal cell, promoters derived from SV40 (Simian virus 40) or CMV (cytomegalovirus) can be used. When the host is E. coli, promoters such as trp, T7, and lac can be used.

[0436] Nucleic acids such as those encoding DNA replication origin (ori), selection markers (antibiotic resistance, auxotrophic traits, etc.), enhancers, splicing signals, polyA addition signals, and tags (FLAG, HA, GST, GFP, etc.) can also be integrated into expression vectors.

[0437] Next, appropriate host cells are transformed using the aforementioned expression vector. The host can be selected appropriately based on its relationship to the vector. Suitable hosts include, for example, *Escherichia coli*, *Bacillus subtilis*, *Bacillus* spp., yeast, insects or insect cells, and animal cells. Suitable animal cells include, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells. Transformation can be performed using known methods such as liposome transfection, calcium phosphate transfection, electroporation, microinjection, and gene gun transfection, depending on the host species. The target peptide can be expressed by culturing the transformants using conventional methods.

[0438] For purifying peptides from transformant cultures, the cultured cells are recovered, suspended in an appropriate buffer, and then disrupted using methods such as sonication, freeze-thaw, etc. The crude extract is obtained by centrifugation or filtration. If the peptide is secreted into the culture medium, the supernatant is recovered.

[0439] Purification from crude extracts or culture supernatants can also be performed by known methods or methods based on known methods (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reversed-phase high-performance liquid chromatography, etc.).

[0440] The obtained peptides can be converted from free bodies to salts, or from salts to free bodies, by known methods or methods based on known methods.

[0441] In one approach, the translation synthesis system can be a cell-free translation system. With a cell-free translation system, the expression product can typically be obtained in high purity without purification. A cell-free translation system contains, for example, ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF), elongation factor (EF), termination factor (RF), and ribosome regeneration factor (RRF), as well as other factors required for translation. To improve expression efficiency, *E. coli* extract or wheat germ extract can be added. Additionally, rabbit erythrocyte extract or insect cell extract can also be added.

[0442] By continuously supplying energy to these systems via dialysis, proteins in the range of several hundred μg to several mg / mL can be produced without limitation. Systems containing RNA polymerase can also be used to facilitate transcription from genetic DNA. Commercially available cell-free translation systems, such as those derived from *E. coli*, include Roche Diagnostics' RTS-100 (registered trademark), GENE FRONTIER's PURESYSTEM, and NEW ENGLAND Biolabs' PURExpressIn Vitro Protein Synthesis Kit. Systems using wheat germ extract, such as those from ZOEGENE and CellFree Sciences, can also be used.

[0443] In cellular translation systems, artificial aminoacyl-tRNAs obtained by linking (acylizing) desired amino acids or hydroxy acids to tRNA can be used instead of aminoacyl-tRNAs synthesized by natural aminoacyl-tRNA synthetases. Such aminoacyl-tRNAs can be synthesized using artificial ribozymes.

[0444] Examples of such ribozymes include Flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; and WO2007 / 066627, etc.). Flexizyme is also known by the names of the original Flexizyme (Fx), as well as modified forms such as dinitrobenzyl Flexizyme (dFx), enhanced Flexizyme (eFx), and aminoFlexizyme (aFx).

[0445] By using tRNA generated by Flexizyme and linked to a desired amino acid or hydroxy acid, a desired codon can be associated with the desired amino acid or hydroxy acid for translation. Specific amino acids can also be used as the desired amino acid. For example, non-natural amino acids required for the aforementioned cyclization can also be introduced into the binding peptide using this method.

[0446] The chemical synthesis of this peptide can be carried out by various methods commonly used in the art, including stepwise solid-phase synthesis, semi-synthesis of peptide fragments via conformational-assisted relinking, and chemical linking. The synthesis of the aforementioned peptide is a chemical synthesis using various solid-phase techniques described, for example, KJ Jensen, PT Shelton, SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013, etc. As a preferred strategy, it is based on a combination of a Fmoc group that temporarily protects the α-amino group and can be selectively removed by a base, and a protecting group that temporarily protects the side chain functional groups and is stable under Fmoc removal conditions. Such general selection of peptide side chains is described in Peptide Synthesis and Applications, 2nd Edition, GB Fields, RL Noble, Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids, Int. J. Peptide Protein Res. 35, 1990. Among 161-214, preferred peptide side chain protecting groups include, for example: benzyl, tert-butyl, and triphenylmethyl (Trt) groups for the hydroxyl groups of serine and threonine; 2-bromobenzyloxycarbonyl and tert-butyl groups for the hydroxyl groups of tyrosine; Boc, methyltetrazole thiol (Mtt), Alloc, and ivDde groups for the amino groups of the lysine side chain; Tlt and Boc groups for the imidazole groups of histidine; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) groups for the guanidinium groups of arginine; tert-butyl, allyl, and 3-methylpentane (Mpe) groups for the carboxyl groups represented by glutamic acid and aspartic acid; Tlt groups for the formamide groups of glutamine and asparagine; and Tlt groups and monomethoxytriphenylmethyl (Mmt) groups for the thiol groups of cysteine.

[0447] The aforementioned peptides can be synthesized stepwise on the aforementioned solid-phase resin. For the C-terminal amino acid used and for all amino acids or peptides used in the synthesis, the α-amino protecting group must be selectively removed during the synthesis process. Preferably, the aforementioned solid-phase resin is used, and the C-terminal carboxyl group of the peptide with an N-terminus appropriately protected by an Fmoc group, or the C-terminal carboxyl group of an amino acid protected by an Fmoc group, is prepared into an active ester using appropriate reagents and then added to the amino group on the solid-phase resin, thus initiating the process. Subsequent peptide chain elongation can be achieved by sequentially repeating the removal of the N-terminal protecting group (Fmoc group) and then the condensation of the protected amino acid derivative, according to the amino acid sequence of the target peptide. It should be noted that this can liberate the target peptide in the final stage. For example, as a release condition, TFA solutions containing water / hydrosyl / thiol as trapping agents can be used, as listed in Teixeira, WE Benckhuijsen, PE de Koning, ARPM Valentijn, JW Drijfhout, Protein Pept. Lett., 2002, 9, 379-385. A typical example is TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5).

[0448] The synthesis of the peptides described in this specification can be performed using a single- or multi-channel peptide synthesizer, such as the Liberty Blue synthesizer from CEM or the Syro I synthesizer from Biotage, or their successors.

[0449] The activation of the carboxyl group can be achieved using condensing agents. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), and (1H-benzotriazol-1-yloxy)tris(dimethylamino) Hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazole -3-oxide hexafluorophosphate (HBTU), etc.

[0450] Cyclation of peptides can be performed using known methods. Non-limitingly, for example, by designing the peptide to contain two or more cysteine ​​residues, a cyclic structure can be formed post-translationally via disulfide bonds. Alternatively, cyclization can be performed by synthesizing peptides with an N-terminal chloroacetyl group using genetic code reprogramming techniques, as described by Goto et al. (Y. Goto, et al. ACS Chem. Biol. 3 120-129 (2008)). Cyclation is then performed by pre-configuring cysteine ​​residues containing sulfur molecules within the peptide. Post-translational, the thiol group spontaneously nucleophilically attacks the chloroacetyl group, and the peptide cyclizes via a thioether bond. Cyclation can also be achieved by incorporating combinations of other amino acids that would otherwise form a cyclic structure into the peptide using genetic code reprogramming. Alternatively, cyclization can be achieved by incorporating L−2-aminoadipic acid residues into the peptide, allowing them to bind to the N-terminal main chain amino group. Alternatively, cyclization can be achieved by linking the amide group of an N-terminal amino acid residue to the carboxyl group of a C-terminal amino acid residue via an amide bond. Alternatively, cyclization can be achieved by binding the amino group of the N-terminal amino acid (the amino acid residue at position 1) to the functional group at the end of the side chain of the C-terminal amino acid contained in the aforementioned peptide. Thus, any known cyclization method can be used without particular restriction.

[0451] Unless otherwise specified, the information described in other items also applies to this item.

[0452] 4. Conjugates (complexes)

[0453] This invention relates to complexes comprising peptides and payloads (also known as "peptide conjugates," "peptide complexes," or simply "conjugates") or pharmaceutically acceptable salts thereof. The peptides in conjugates, as described in section "2. Peptides," include all forms (including salts, isomers, solvates, etc.). In this specification, references to "peptide conjugates," "conjugates," or "peptide complexes" also include, unless contradictory, references to their pharmaceutically acceptable salts, isomers, or solvates.

[0454] In one embodiment, the conjugate of the present invention represents a complex in which the peptide and payload of the present invention are bound via a linker or not.

[0455] In one embodiment, the conjugate of the present invention has a payload attached to the side chain of the amino acid residues contained in the peptide of the present invention, with or without a linker.

[0456] In one embodiment, the conjugate of the present invention is bound to any payload via or without a linker at the amino acid residue at position 13 of SEQ ID NO. 1.

[0457] In one embodiment, the conjugate of the present invention is as shown in the following formula (I), wherein an amino acid residue at position 1, 4, 8 or 11 of SEQ ID NO. 1 is bound with a payload.

[0458] [Chemical Formula 3]

[0459]

[0460] In formula (I),

[0461] R1 is H or a C1-3 alkyl group;

[0462] R2 is C1-6 alkyl-NH-, C1-6 alkyl-C6 aryl-O-C1-3 alkyl-NH-, C1-6 alkyl-NH(=O)-CH(C1-6 alkylphenyl)-NH-, C1-6 alkyl-NH(=O)-CH(C1-6 alkyl)-NH-, or C1-6 alkyl-NH(=O)-C3-8 cycloalkyl-C1-3 alkyl-NH-;

[0463] X is any payload.

[0464] It should be noted that the C1-6 alkyl-NH- of R2 refers to residues of K and residues in the alkyl chain extension modification of the side chain of K; the C1-6 alkyl-C6 aryl-O-C1-3 alkyl-NH- refers to residues of dyae and residues in the alkyl chain extension modification of the side chain of dyae; the C1-6 alkyl-NH(=O)-CH(C1-6 alkylphenyl)-NH- refers to residues of dk-df and residues in the compound formed by combining the alkyl chain extension modification of the side chain of dk with the alkyl chain extension modification of the side chain of df; and the C1-6 alkyl... The residues in compounds formed by combining alkyl-NH(=O)-CH(C1-6 alkyl)-NH- are residues of dk-dnle and alkyl chain elongation modifiers in the side chain of dk with alkyl chain elongation or shortening modifiers in the side chain of dnle, and C1-6 alkyl-NH(=O)-C3-8 cycloalkyl-C1-3 alkyl-NH- are residues of dk-t4amCh and alkyl chain elongation modifiers in the side chain of dk with modifiers that increase or decrease the number of carbons in the cycloalkyl structure in the side chain of t4amCh.

[0465] It should be noted that the curve shown in the following chemical formula 4 in compound (I) represents the combination of its carboxyl or amide group with the amide or carboxyl group of adjacent amino acid residues in the amino acid sequence.

[0466] [Chemical Formula 4]

[0467]

[0468] In this specification, C1-3 alkyl refers to a monovalent group derived from a straight-chain or branched saturated aliphatic hydrocarbon having 1-3 carbon atoms by removing any one hydrogen atom. Specific examples include methyl, ethyl, n-propyl, isopropyl, etc.

[0469] In this specification, C1-6 alkyl refers to a monovalent group derived from a straight-chain or branched saturated aliphatic hydrocarbon having 1-6 carbon atoms by removing any one hydrogen atom. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 1-methylpropyl, n-pentyl, isopentyl, 2-methylbutyl, 1,1-dimethylpropyl, 1-ethylpropyl, hexyl, 4-methylpentyl, etc.

[0470] In this specification, C1-6 alkylphenyl refers to the structure obtained by combining a phenyl group with a monovalent group derived from a straight-chain or branched saturated aliphatic hydrocarbon having 1-6 carbon atoms by removing any one hydrogen atom.

[0471] In this specification, C3-8 cycloalkyl refers to a monovalent group derived from a cyclic saturated aliphatic hydrocarbon having 3-8 carbon atoms by removing any one hydrogen atom.

[0472] It should be noted that, without limitation, in this invention, the conjugates include the conjugates shown in Conjugate No. 1-81, such as the conjugates shown in Conjugate No. 1-69 and 74-81, and also include conjugates in which the payload contained in these conjugates is replaced with different payloads. The conjugates that are replaced with different payloads can be, for example, the payloads in Conjugate No. 1-81 (in one embodiment, Conjugate No. 1-69 and 74-81) shown in Table 8 or Table 18, DOTA, DOTALu, SulfoCy5, DOTAZr, DOTALu-df, DOTALu-dnle, DOTALu-t4amCh, DOTALu-F, DOTALu-Nle, SulfoCy5-PEG8c, DOTACu, DOTAGA, dDOTAGA, rNODAGAa, DOTA-Ga (representing a substance formed by combining Ga (gallium) with DOTA), and DOTALa (representing a substance formed by combining La (lanthanum) with DOTA) that are replaced with different payloads.

[0473] For example, in the conjugate described in Conjugate No. 1, the sulfoCy5 that serves as the payload, bound to the linker G-PEG10c-K, can be a conjugate formed by further binding a chelating agent such as DOTA to a radioactive element, or it can be a conjugate formed by binding sulfoCy5 to a non-radioactive isotope such as an antibody as the payload. Additionally, for example, in the conjugate described in Conjugate No. 2, it can be a conjugate formed by further binding a radioactive isotope to the chelating agent DOTA bound via linker K. For example, in the conjugate described in Conjugate No. 4, it can be a conjugate formed by binding Lu (non-radioactive lutetium) to the chelating agent DOTA bound via linker K as the payload, or a radioactive isotope. 177 A conjugate formed by replacing non-radioactive lutetium with Lu.

[0474] Payload

[0475] In this invention, the payload refers to a known functional molecule. There is no particular limitation on the functional molecule; it refers to a molecule that exhibits a desired function by binding to or interacting with a target. Examples of desired functions include pharmacological activity, labeling, and delivery to the target site. It should be noted that the term "payload" in this specification includes pharmacologically active compounds, labeled compounds, fluorescent substances, peptides, proteins, nucleic acids, and molecules used in drug delivery systems. These molecules can be low-molecular-weight compounds, medium-molecular-weight compounds, or high-molecular-weight compounds.

[0476] In this specification, a pharmacologically active compound refers to a compound that has pharmacological activity. Preferred pharmacologically active compounds include, for example, antibodies or proteins that are high molecular weight compounds, nucleic acids, peptides that are medium molecular weight compounds, and low molecular weight compounds. Alternatively, compounds formed by encapsulating low molecular weight compounds with pharmacological activity in the form of liposomes or micelles may also be used.

[0477] In addition, the term "payload" in this specification includes any cell inhibitor that is toxic to cells, particularly killing them. Examples, without limitation, include paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxanthraquinone, scintillans, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologs. Additionally, the “payload” in this specification includes therapeutic agents with anticancer effects, such as metabolic antagonists (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepa, chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiammineplatin(II) (DDP) cisplatin), and anthracyclines (e.g., daunorubicin). The treatment agents include, but are not limited to, doxorubicin (formerly known as "daunomycin"), antibiotics (e.g., actinomycin D (formerly known as "daunomycin"), bleomycin, scintillans, and atrazodine (AMC), and antimitotic agents (e.g., vincristine and vinblastine). As a preferred embodiment, the treatment agent is a cytotoxic agent. In another embodiment, the treatment agent is an immunosuppressant. In yet another embodiment, the treatment agent is GM-CSF. As a preferred embodiment, the treatment agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A.

[0478] In this specification, a labeled compound refers to a compound that has been labeled with pigments, fluorescent substances, tags, or radioactive isotopes. Specifically, a compound labeled with a radioactive isotope refers to a compound obtained by labeling low-molecular-weight or medium-molecular-weight compounds, antibodies, etc., with a radioactive isotope. Furthermore, the labeled compound can be the pigment, fluorescent substance, tag, or the radioactive isotope itself. The radioactive isotope can coordinate with chelating agents such as DOTA.

[0479] In this specification, the radioactive isotope may be a known radioactive isotope. In one embodiment, the radioactive isotope includes actinium-225, actinium-227 (225Ac, 227Ac), astatine-211 (211At), bismuth-212 and bismuth-213 (212Bi, 213Bi), copper-61, copper-62, copper-64 and copper-67 (61Cu, 62Cu, 64Cu, 67Cu), gallium-64, gallium-67 and gallium-68 (64Ga, 67Ga and 68Ga), indium-111 (111In), iodine-123, iodine-124, iodine-125 or iodine-131 (123I, 124I, 125I, 131I) (123I), lead-203, lead-212 ( 203Pb, 212Pb), Ruthenium-97 (97Ru), Lutetium-177 (177Lu), Radium-223 (223Ra), Samarium-153 (153Sm), Scandium-44 and Scandium-47 (44Sc, 47Sc), Iron-52 (52Fe), Arsenic-72, Arsenic-76 (72As, 76As), Erbium-169 (169Er), Strontium-89, Strontium-90 (89Sr, 90Sr), Technetium-99 (99mTc), Technetium-94 (94mTc), Yttrium-86 and Yttrium-90 (86Y, 90Y), Chromium-51 (51Cr), Rhenium-186, Rhenium-188 (186Re, 188R) e), Zirconium-89 (89Zr), Manganese-52, Manganese-51 (52Mn, 51Mn), Terbium-149, Terbium-152, Terbium-155, Terbium-161 (149Tb, 152Tb, 155Tb, 161Tb), Ytterbium-169, Ytterbium-175 (169Yb, 175Yb), Rhodium-105 (105Rh), Dysprosium-166 (166Dy), Holmium-166 (166Ho), Samarium-153 (153Sm), Promethium-149, Promethium-151 (149Pm, 151Pm), Thulium-172 (172Tm), Tin-121 (121Sn), Praseodymium-142, Praseodymium-143 (14 2Pr, 143Pr), gold-198, gold-199 (198Au, 199Au), bromine-75, bromine-76, bromine-77, bromine-80, bromine-82 (75Br, 76Br, 77Br, 80Br, 82Br), fluorine-18 (18F), scandium-43, scandium-44, scandium-47 (43Sc, 44Sc, 47Sc), astatine-211 (211At), thorium-227, thorium-226 (227Th, 226Th), lanthanum-140 (140La), rubidium-82 (82Rb), phosphorus-32 (32P), silver-111 (111Ag), erbium-165 (165Er), etc.

[0480] In this specification, a chelating agent is an organic compound used to stably bind radioactive isotopes, particularly metallic radioactive isotopes, to metal ions to form a cyclic complex. In one embodiment, the chelating agent is EDTA (ethylenediaminetetraacetic acid), DPTA (diethylenetriaminepentaacetic acid), 1,4,8,11-tetraazatetradecane, 1,4,8,11-tetraazatetradecane-1,4,8,11-tetraacetic acid, 1-oxa-4,7,12,15-tetraazaheptadecane-4,7,12,15-tetraacetic acid, DOTAGA (α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (including the racemic rDOTAGA), TMT (6,6''-bis[N,N'',N... [Tetra(carboxymethyl)aminomethyl]-4'-(3-amino-4-methoxyphenyl)-2,2':6',2''-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N''(N'''-tetraacetic acid), TCMC (tetra-primary amide of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), NOTA (1,4,7-triazacyclononane-triacetic acid), Diamsar (3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosano-1,8-diamine), DTPA (pentacycline or diethylenetriaminepentaacetic acid), CHX-A''-DTPA ([(R)-2-amino-3-(4-isothiocyanate phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diaminepentaacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), Te2A (4,11-di... (Carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), HBED, DFO (deferroamine), DFOsq (DFO-squamamide), HOPO (3,4,3-(LI-1,2-HOPO)), NODAGA (1,4,7-triazacyclononane-1-1-glutamic acid-4,7-acetic acid) (including the racemic rNODAGA), etc., and also include their modifications. DOTA, DOTAGA, or NODAGA are preferred. It should be noted that, where optical isomers exist for each chelating agent, these optical isomers are included.

[0481] In one embodiment, the payload is preferably a radioactive isotope bound to a chelating agent. Examples, non-limitingly, include structures formed by binding radioactive isotopes of lutetium to DOTA, structures formed by binding radioactive isotopes of zirconium to DOTA, structures formed by binding radioactive isotopes of copper to DOTA, and structures formed by binding radioactive isotopes of gallium to DOTA.

[0482] In another approach, the payload may also be a radioactive isotope not bound to a chelating agent. This can be a compound used in evaluation methods or imaging employing radioactive isotopes; for example, non-limitingly, it can be a known compound containing a radioactive isotope used as a radioligand imaging agent in positron emission tomography methods such as PET and SPECT described later. For example, non-limitingly, it can be 18F-fluorodeoxyglucose (18F-FDG), 15O-H2O, 11C-methionine, 11C-acetic acid, 11C-choline, 11C-ralopride, 11C-flumazenil, 13N-ammonia, 18F-sodium fluoride, 82Rn+ (82RbCl), 18F-flurpiridazine, 123I-IMP, 99mTc-ECD, 99Tc-HMPAO, 123I-iodomazenil, 123I-iodoflupane, 11... 1In-DTPA, 99mTcO4-(Na99mTcO4), 99mTcMIBI, 201TI+(201TICI), 99mcTc-tetrophosphine, 99mcTc-human serum albumin, 131O-adosterol, 99mTc-phytic acid, 90Y-zevalin, 67Ga+(67GaC6H5O7), 111In-teimomab, 111In-pentetreotide, etc.

[0483] In this specification, "protein" refers to any protein that exists in a living organism and performs a useful function in the body. It can be any protein, such as proteins with pharmacological effects or proteins with molecular recognition functions. Specifically, examples include fibronectin, avidin, antibodies, proteins with Fc sites, immune checkpoint proteins, and enzymes such as proteolytic enzymes.

[0484] In this specification, nucleic acids are defined as polymers of nucleotides and can be any type of nucleic acid. Specifically, examples include DNA and RNA. Nucleic acids can be modified, for example, by 2'-methoxyethyl (MOE) modification, the use of 2'-deoxynucleotides in RNA, nucleoside binding based on phosphate thioesters, nucleoside binding based on phosphodiester, and the conversion of cytosine to 5-methylcytosine.

[0485] In this specification, a drug delivery system refers to a system that delivers an active ingredient into target cells. Examples of carriers include water-soluble polymers, nanoparticles (nanospheres), liposomes, and micelles. Drug delivery system (DDS) molecules may further contain low-molecular-weight compounds, proteins, peptides, nucleic acids, vaccines, and other drugs.

[0486] In one embodiment, the present invention relates to the above-mentioned conjugates having CA9 binding activity.

[0487] In one embodiment, the present invention relates to the use of the aforementioned conjugate in combination with CA9.

[0488] In one embodiment, the present invention relates to the aforementioned conjugates for use in combination with CA9.

[0489] In this invention, the binding of the conjugate to CA9 is in vitro or in vivo.

[0490] Unless otherwise specified, the information described in other items also applies to this item.

[0491] 5. Compositions, etc.

[0492] The present invention also relates to compositions comprising the peptides, conjugates, or pharmaceutically acceptable salts thereof of the present invention. These compositions are not limited to pharmaceutical compositions (medical compositions), diagnostic compositions, or research compositions. In one embodiment, the pharmaceutical composition is a composition for use in radiotherapy (one embodiment of a therapeutic composition).

[0493] The present invention also relates to imaging agents comprising the conjugates of the present invention. The imaging agents can also be used as diagnostic compositions. In one embodiment, the diagnostic composition is an imaging agent.

[0494] Unless otherwise specified, the information described in other items also applies to this item.

[0495] Pharmaceutical Composition

[0496] In one embodiment, the present invention relates to a pharmaceutical composition comprising the above-described peptide, conjugate, or a pharmaceutically acceptable salt thereof.

[0497] In one embodiment, the above-mentioned pharmaceutical composition has CA9 binding activity.

[0498] In one embodiment, the above-mentioned pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of CA9-related diseases or symptoms.

[0499] "CA9-related diseases or symptoms" includes well-known CA9-related diseases or symptoms. For example, it includes tumors, especially malignant tumors, and solid tumors.

[0500] In one approach, examples of CA9-related diseases include tumors originating from the cervix and uterine body, breast, ovary, digestive tract (including esophagus, stomach, small intestine, colon, and rectum), kidney, liver, gallbladder and biliary system, pancreas (including pancreatic duct and exocrine pancreas), lung, head and neck, salivary glands, bladder and urinary tract, body cavities, and epithelial tissues. Other examples include cancers of the head and neck, ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid, adrenal glands, prostate, skin, and appendages; hematologic malignancies; borderline malignancies including premalignant hematologic disorders and lymphatic hematologic malignancies and related diseases; bone marrow hematologic malignancies and related diseases; mesenchymal tumors; tumors of the central or peripheral nervous system; endocrine tumors; tumors of the eyeball and its appendages; germ cell and trophoblastic tumors; and pediatric and fetal tumors. Non-limiting, renal cell carcinoma, head and neck cancer, urothelial carcinoma, pancreatic duct cancer, colorectal cancer, etc. are preferred.

[0501] In one embodiment, the aforementioned pharmaceutical composition is a composition for radiotherapy (one aspect of a therapeutic composition). In this case, the components included in the pharmaceutical composition contain a radioactive isotope. Preferably, it is a conjugate containing a radioactive isotope as an effective payload. It should be noted that radiotherapy refers to a treatment method that damages the intracellular DNA of tumors, especially cancer cells, by irradiating them with radiation or by delivering substances that emit radiation to the affected area, thereby causing the cells to die.

[0502] The pharmaceutical compositions described above may contain the peptide or conjugate itself, or pharmaceutically acceptable salts, isomers, or solvates of the peptide. In this specification, unless otherwise specified, "peptide" and "conjugate" may contain pharmaceutically acceptable salts, isomers, or solvates thereof. Preferably, the pharmaceutical compositions contain an effective amount of the peptide or conjugate as the active ingredient.

[0503] In this specification, there is no particular limitation on the form of administration of the pharmaceutical composition; it may be administered orally or not orally. Examples of non-oral administration include injection (such as intramuscular injection, intravenous injection, subcutaneous injection, etc.), transdermal administration, and administration via mucous membranes (nasal, oral, ocular, pulmonary, vaginal, or rectal).

[0504] Given the readily metabolizable and excreted nature of peptides, various modifications can be made to the aforementioned pharmaceutical compositions. For example, polyethylene glycol (PEG) and sugar chains can be added to the peptides to prolong their retention time in the blood and reduce their antigenicity. Additionally, biodegradable polymers such as polylactic-co-glycolic acid (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared from unsaturated fatty acids, nanoparticles, and nanospheres can be used as sustained-release agents to encapsulate the peptides. In the case of transdermal administration, a weak electric current can be passed through the skin surface to allow it to penetrate the stratum corneum (iontophoresis).

[0505] The aforementioned pharmaceutical compositions can use the active ingredient directly, or they can be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquid preparations (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, granules, capsules, syrups, lozenges, inhalers, ointments, eye drops, nasal drops, ear drops, poultices, etc. Formulation can be carried out using, for example, appropriate excipients, binders, disintegrants, lubricants, solubilizers, dissolving agents, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc., through conventional methods.

[0506] Examples of ingredients used in formulations include, but are not limited to: purified water, physiological saline, phosphate buffer, glucose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicate, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carbomer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth gum, casein, agar, polyethylene glycol, diglycerides, glycerol, propylene glycol, petrolatum, paraffin, octyl dodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, etc.

[0507] Since peptides are generally difficult to absorb through mucous membranes, the above-mentioned pharmaceutical compositions may contain absorption enhancers that improve the absorption of poorly absorbed drugs. Such absorption enhancers may include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylates; fatty acids such as hexanoic acid, decanoic acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0508] When the above-mentioned pharmaceutical composition is in the form of pills or tablets, it can be coated with sugar coating, gastric-soluble, or enteric-soluble substances.

[0509] When the above-mentioned pharmaceutical composition is an injectable preparation, it may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. In addition, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solvents, preservatives, etc., may also be added.

[0510] Furthermore, the target of the above-mentioned pharmaceutical composition can be not only humans, but also non-human mammals or birds. Examples of non-human mammals include primates other than humans (monkeys, chimpanzees, gorillas, etc.), livestock (pigs, cattle, horses, sheep, etc.), or dogs, cats, rats, mice, guinea pigs, rabbits, etc.

[0511] Especially when administered to humans, the dosage varies depending on symptoms, patient age, sex, weight, sensitivity differences, method of administration, dosing interval, type of active ingredient, and type of formulation. Non-limitingly, for example, 30 μg to 100 g, 100 μg to 500 mg, or 100 μg to 100 mg can be administered in a single or multiple doses. In cases of injection administration, the dosage can be administered in a single or multiple doses of 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg, depending on the patient's weight.

[0512] In one embodiment, the present invention relates to a method for preventing or treating CA9-related diseases or symptoms by administering the peptides or conjugates of the present invention (comprising pharmaceutically acceptable salts, isomers, or solvates thereof). Non-limitingly, the treatment methods of the present invention include the step of administering the peptides or conjugates of the present invention to a subject. The subject and method of administration are the same as described in the "Pharmaceutical Compositions" section. Additionally, non-limitingly, the treatment methods of the present invention, aimed at delivering the active ingredient of the treatment method to any part (affected area) of the subject using the peptides or conjugates of the present invention, include the step of administering the peptides or conjugates of the present invention to the subject.

[0513] In one embodiment, the present invention relates to the use of the peptides, conjugates or pharmaceutically acceptable salts thereof for the prevention or treatment of CA9-related diseases or symptoms.

[0514] In one embodiment, the present invention relates to the use of the peptides, conjugates, or pharmaceutically acceptable salts thereof of the present invention in the manufacture of pharmaceutical compositions for the prevention or treatment of CA9-related diseases or symptoms.

[0515] In one embodiment, the present invention relates to the use of the peptides, conjugates, or pharmaceutically acceptable salts thereof as pharmaceutical compositions for the prevention or treatment of CA9-related diseases or symptoms.

[0516] In one embodiment, the present invention relates to peptides, conjugates, or pharmaceutically acceptable salts thereof used in methods for the prevention or treatment of CA9-related diseases or symptoms.

[0517] In one embodiment, the present invention relates to peptides, conjugates, or pharmaceutically acceptable salts thereof for use in pharmaceutical compositions for the prevention or treatment of CA9-related diseases or symptoms.

[0518] Diagnostic compositions / imaging agents

[0519] The present invention also relates to diagnostic compositions for diagnosing CA9-related diseases or symptoms, comprising the peptides, conjugates or pharmaceutically acceptable salts thereof of the present invention.

[0520] The peptides and conjugates of the present invention can also be used as diagnostic compositions for CA9-related diseases or symptoms. As diagnostic agents, they can be used as test agents for diagnosing whether one has CA9-related diseases or symptoms, for diagnosing the severity of symptoms of such diseases, or for diagnosing poor prognosis of such diseases. When used as test agents, the aforementioned peptides or conjugates can be detectably labeled, and can be conjugates containing a detectable payload or conjugates containing a known imaging agent as a payload.

[0521] In one embodiment, the present invention comprises an imaging agent containing a conjugate. The imaging agent can be used as a diagnostic composition. In one embodiment, the diagnostic composition is an imaging agent. In this specification, "imaging agent" refers to a compound capable of performing diagnostic imaging, having one or more properties that enable direct or indirect detection of its presence and / or location. Examples of such imaging agents include proteins, peptides, medium-molecular-weight compounds, small-molecular-weight compounds, and radioisotopes themselves, incorporating a detectable labeled portion.

[0522] The labeling components that can be detected can include enzymes such as peroxidase and alkaline phosphatase; radioactive isotopes (which can be bound to chelating agents or compounds such as glucose); fluorescein isothiocyanate (FITC); rhodamine; dansyl chloride; phycoerythrin; tetramethylrhodamine isothiocyanate; fluorescein phosphorus amide (FAM); eosin; carboxyfluorescein; erythrosine; carboxytetramethylrhodamine (TAMRA); tetramethylrhodamine (TMR); and sulforhodamine or near-infrared fluorescent materials, as well as luminescent substances such as luciferase, luciferin, and jellyfish luminescent proteins. Antibodies labeled with these luminescent substances can also be used. In addition, antibodies labeled with nanoparticles such as colloidal gold and quantum dots can also be detected.

[0523] For example, by creating a complex from the aforementioned antibody and peptide, a complex labeled with the antibody or peptide can be prepared, and then administered and tested to determine the severity of CA9-related diseases or symptoms. Alternatively, in immunoassays, the aforementioned peptide can be biotin-labeled, allowing for the binding of enzyme-labeled avidin or streptavidin for detection.

[0524] In immunoassays, enzyme-labeled ELISA is preferred because it allows for simple and rapid antigen detection. For example, after immobilizing an antibody on a solid support and adding a sample to react, a labeled peptide is added and reacted again. After washing, the sample reacts with an enzyme substrate to develop color, and the severity of CA9-related diseases or symptoms can be detected by measuring the absorbance. Alternatively, after reacting the antibody immobilized on the solid support with the sample, an unlabeled peptide can be added, followed by enzyme labeling of the antibody against the peptide and further addition. The antibody can be immobilized on the surface of the solid support or inside it.

[0525] For enzyme substrates, when the enzyme is a peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used; when the enzyme is an alkaline phosphatase, p-nitrophenyl phosphate (pNPP), etc. can be used.

[0526] In this specification, "solid support" is not specifically limited to any carrier capable of immobilizing antibodies. Examples include microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, etc., made of glass, metal, or resin. The target substance can be immobilized on these solid supports using known methods.

[0527] Furthermore, in this specification, diagnostic imaging includes imaging based on immunohistochemistry, immunofluorescence staining, etc.; optical imaging such as positron emission tomography (PET, including PET-CT) and single-photon emission computed tomography (SPECT); and magnetic resonance imaging (MRI), as well as non-invasive (molecular) diagnostic imaging containing iron oxide nanoparticles and carbon-coated iron-cobalt nanoparticles. The imaging agents of the present invention can be used for any diagnostic imaging by appropriately selecting detectable labeled portions. Preferably, they are imaging agents for PET, PET-CT, or SPECT as optical imaging. In one embodiment, the present invention relates to a radioligand imaging agent for positron emission tomography comprising the conjugate of the present invention.

[0528] This invention relates to diagnostic kits comprising the peptides or conjugates of the present invention. The diagnostic kits comprise the reagents and apparatus required for the aforementioned detections (non-limitingly including any or all of the peptides or conjugates of the present invention, antibodies, solid-phase carriers, buffer solutions, enzyme reaction termination solutions, ELISA readers, etc.).

[0529] This invention relates to a method for diagnosing CA9-related diseases or symptoms, which is performed using the peptide or conjugate of the present invention. "Diagnostic method" includes in vivo or in vitro diagnostic methods. Non-limitingly, the method of the present invention includes the step of administering the peptide or conjugate of the present invention to a subject or a sample obtained from the subject. Non-limitingly, the method of the present invention includes the steps of administering the peptide or conjugate of the present invention to a subject or a sample obtained from the subject, and detecting the binding of the peptide or conjugate of the present invention to CA9. The subject and method of administration are the same as described in the "Pharmaceutical Composition" section. "Sample obtained from the subject" includes, for example, blood, urine, feces, tears, nasal discharge, tissue sections, etc.

[0530] This invention also relates to a method for detecting diseases, which is performed using the peptides or conjugates of this invention. For example, disease detection can be performed in research institutions (including educational institutions such as universities), enterprises, etc., by examination technicians other than doctors, researchers, etc. In one embodiment, the disease detection method does not involve medical procedures. Preferably, diagnostic methods using PET, PET-CT, or SPECT are employed.

[0531] This invention relates to the use of the peptides or conjugates of this invention in the diagnosis of CA9-related diseases or symptoms.

[0532] This invention relates to the use of the peptides or conjugates of the present invention in the manufacture of diagnostic compositions for the diagnosis of CA9-related diseases or symptoms.

[0533] This invention relates to the use of the peptides or conjugates of the present invention as diagnostic compositions for the diagnosis of CA9-related diseases or symptoms.

[0534] This invention relates to peptides or conjugates of the present invention for use in methods for diagnosing CA9-related diseases or symptoms.

[0535] This invention relates to peptides or conjugates of the present invention for use as diagnostic compositions for diagnosing CA9-related diseases or symptoms.

[0536] This invention relates to test reagents comprising the peptides or conjugates of the present invention. This invention also relates to diagnostic or detection test reagents comprising the peptides or conjugates of the present invention.

[0537] Research Composition

[0538] This invention also relates to research compositions comprising the peptides or conjugates of this invention. "Research compositions" include compositions used by researchers, technicians, students, doctors, etc., in research institutions (including educational institutions such as universities), businesses, hospitals, etc.

[0539] The aforementioned research compositions can be used, for example, for the detection of CA9, and for the detection of CA9-related diseases or symptoms.

[0540] In this specification, the carrier used to immobilize peptides or conjugates is not particularly limited, and examples include microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, etc., made of glass, metal, or resin.

[0541] This invention includes a method for detecting CA9 using the peptide or conjugate of the present invention. "Detection method" includes in vivo or in vitro detection methods. Non-limitingly, the method of the present invention includes the step of administering the peptide or conjugate of the present invention to a subject or a sample obtained from the subject. Non-limitingly, the method of the present invention includes the step of administering the peptide or conjugate of the present invention to a subject or a sample obtained from the subject, and detecting the binding of the peptide or conjugate of the present invention to CA9. The subject and method of administration are the same as described in the "Pharmaceutical Composition" section. "Sample obtained from the subject" includes, for example, blood, urine, feces, tears, nasal discharge, tissue sections, etc.

[0542] This invention also includes the use of the peptides or conjugates of this invention in the detection of CA9.

[0543] This invention relates to diagnostic or detection kits comprising the peptides or conjugates of the present invention. The aforementioned diagnostic or detection kits comprise the reagents and apparatus required for the aforementioned detections (non-limitingly including any one or all of the peptides, antibodies, solid-phase carriers, buffer solutions, enzyme reaction termination solutions, ELISA readers, etc. of the present invention).

[0544] In one embodiment, the present invention includes a method for delivering any payload to a subject using the peptide or conjugate of the present invention. Non-limitingly, the method of the present invention includes the steps of: manufacturing a conjugate comprising the peptide of the present invention and any payload, and administering the peptide to a subject. The subject and method of administration are the same as described in the "Pharmaceutical Compositions" section.

[0545] In one embodiment, the present invention relates to a method of using the peptide or conjugate of the present invention to deliver any payload to any part (affected area) of a subject. Non-limitingly, the method of delivery to any part of a subject in the present invention includes the steps of: manufacturing a conjugate comprising the peptide of the present invention and any payload, and administering the peptide to the subject. The subject and method of administration are the same as described in the "Pharmaceutical Compositions" section.

[0546] Unless otherwise specified, the information described in other items also applies to this item.

[0547] 7. Testing Methods

[0548] The present invention also relates to a method for testing peptides or pharmaceutically acceptable salts thereof, the method for testing peptides or pharmaceutically acceptable salts thereof for at least one of the following a) to d): a) solubility in a solvent, b) CA9 binding activity, c) toxicity to cells and / or tissues, or d) toxicity to experimental animals, wherein the aforementioned peptides or pharmaceutically acceptable salts thereof are the peptides or pharmaceutically acceptable salts thereof of the present invention.

[0549] The present invention also relates to a method for testing conjugates, wherein the method tests at least one of the following a) to d): a) solubility in a solvent, b) CA9 binding activity, c) toxicity to cells and / or tissues, or d) toxicity to experimental animals, wherein the conjugates are conjugates of the present invention.

[0550] "Solubility in a solvent" can be determined using known methods. There are no limitations on the solvent used for solubility determination; it can be freely chosen depending on the purpose. Furthermore, the method for determining solubility can be any known method appropriately selected depending on the type of solvent. Non-limitingly, it can be the solubility of the peptide or conjugate in known solvents such as water, glycerol, PBS, and DMSO.

[0551] "CA9 binding activity" can be measured, for example, as described in "2. Peptide" and other descriptions.

[0552] "Toxicity to cells and / or tissues" can be determined using known methods. For example, toxicity tests to cells and / or tissues can be known toxicity evaluation tests that use cells and / or tissues, and can be performed in vitro. Cells and tissues can be any cells and / or tissues that are typically used in toxicity evaluation tests for pharmaceuticals, without limitation.

[0553] "Toxicity to laboratory animals" can be determined using known methods. For example, there are no particular limitations on laboratory animals; any commonly used animal can be used, such as mice, rats, guinea pigs, gerbils, hamsters, ferrets, rabbits, dogs, cats, pigs, goats, horses, cattle, birds (e.g., chickens, quails, etc.), monkeys, and primates other than humans (e.g., cynomolgus monkeys, marmosets, rhesus monkeys, etc.).

[0554] Furthermore, there are no limitations on the toxicity evaluation tests mentioned above. These can be safety-related tests that are typically conducted in non-clinical trials of drugs. Examples include: general toxicity tests (single-dose toxicity tests / repeated-dose toxicity tests), genotoxicity tests (Ames test / chromosomal aberration test / in vitro micronucleus test), carcinogenicity tests, reproductive and developmental toxicity tests (ICH-I, II, III), local irritation tests (eye irritation test, skin irritation test, etc.), other toxicity tests (skin sensitization test, phototoxicity test, antigenicity test), chemical analysis / biological analysis (TK / PK), etc.

[0555] Unless otherwise specified, the information described in other items also applies to this item.

[0556] 8. Combination of uses, combination

[0557] The peptides or conjugates of the present invention can be used in combination with other pharmaceuticals for the prevention or treatment of CA9-related diseases or symptoms. In one embodiment, the present invention relates to the combination of the aforementioned peptides or conjugates with other pharmaceuticals for the prevention or treatment of CA9-related diseases or symptoms.

[0558] The aforementioned peptides or conjugates can be administered simultaneously or sequentially with other drugs used to prevent or treat CA9-related diseases or symptoms. Preferably, administration is performed in a manner that achieves an additive effect between the aforementioned peptides or conjugates and other drugs used to prevent or treat CA9-related diseases or symptoms, and more preferably in a manner that achieves a synergistic effect. In the case of sequential administration, the order of administration is not limited. In the case of sequential administration, it is non-limitingly preferred that both be ingested within 2 hours, 1 hour, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes.

[0559] Unless otherwise specified, the matters described in other items also apply to this item.

[0560] Example

[0561] The present invention will be described in more detail below through examples, but the invention is not limited thereto. Those skilled in the art can easily modify / change the invention based on the description herein, and all such modifications / changes are included within the technical scope of the invention. It should be noted that the compound names shown in the following reference examples and embodiments do not necessarily follow the IUPAC nomenclature. It should also be noted that abbreviations are sometimes used for simplicity, as described above.

[0562] The raw materials, building blocks, reagents, acids, bases, solid resins, and solvents used in the chemical synthesis of the compounds were commercially available, or, where otherwise described, synthesized using organic chemical methods. It should be noted that amino acids containing protecting groups were commercially available.

[0563] As a method of counting peptide residues, the amino acid residue to be ClAc-treated is counted as the first residue, and then the residues are counted towards the resin as the second, third, and so on. The commonly used amino acids are listed below, with side chain protecting groups shown in parentheses.

[0564] Fmoc-Ile-OH;

[0565] Fmoc-Ser(Trt)-OH;

[0566] Fmoc-Ser(tBu)-OH;

[0567] Fmoc-His(Boc)-OH;

[0568] Fmoc-Pro-OH;

[0569] Fmoc-Asp(OMpe)-OH;

[0570] Fmoc-Glu(tBu)-OH;

[0571] Fmoc-Y(tBu)-OH;

[0572] Fmoc-Gly-OH;

[0573] Fmoc-Cys(Trt)-OH;

[0574] Boc-Phe-OH;

[0575] Fmoc-Lys(Boc)-OH.

[0576] In addition, regarding non-natural amino acids, the following substances and substances listed in abbreviations were used.

[0577] Fmoc-da-OH;

[0578] Fmoc-de(tBu)-OH;

[0579] Fmoc-dgln(Trt)-OH;

[0580] Fmoc-dhgl(tBu)-OH;

[0581] Fmoc-dkCOpipzaa(tBu)-OH;

[0582] Fmoc-Tbg-OH;

[0583] Fmoc-3Py6CON-OH;

[0584] Fmoc-3Py6NH2(Boc)-OH;

[0585] Fmoc-Hgl(tBu)-OH;

[0586] Fmoc-alT(tBu)-OH;

[0587] Fmoc-Hgn(Trt);

[0588] Fmoc-SMe-OH;

[0589] Fmoc-DacAc-OH;

[0590] Fmoc-KCOpipzaa(tBu)-OH;

[0591] Fmoc-N-Me-da-OH;

[0592] Fmoc-PeG-OH;

[0593] Fmoc-MeG-OH;

[0594] Fmoc-MedkCOpipzaa(tBu)-OH;

[0595] Fmoc-Ahp-OH;

[0596] Fmoc-pHPeG(tBu)-OH;

[0597] Fmoc-pMeOPeG-OH;

[0598] Fmoc-MsMeapG-OH;

[0599] Fmoc-3OMePeG-OH;

[0600] Fmoc-PpG-OH;

[0601] Fmoc-pFPeG-OH;

[0602] Fmoc-mCPeG-OH;

[0603] Fmoc-pCPeG-OH;

[0604] Fmoc-4HPpG-OH;

[0605] Fmoc-4OMePpG-OH;

[0606] Fmoc-4PypG-OH;

[0607] Fmoc-4COOPeG(tBu)-OH;

[0608] Fmoc-3COOPeG(tBu)-OH;

[0609] Fmoc-3FPeG-OH;

[0610] Fmoc-Hse(Trt)-OH;

[0611] Fmoc-P4Sh(tBu)-OH;

[0612] Fmoc-Qmm-OH;

[0613] Fmoc-Qdm-OH;

[0614] Fmoc-QPEG8Me-OH;

[0615] Fmoc-5Inda-OH;

[0616] Fmoc-F4OMe-OH;

[0617] Fmoc-HseMe-OH;

[0618] Fmoc-Acb-OH;

[0619] Fmoc-dkAc-OH;

[0620] Boc-df-OH;

[0621] Boc-dnle-OH;

[0622] Fmoc-t4amCh-OH;

[0623] Boc-Nle-OH;

[0624] Fmoc-N-Me-Cys(Trt)-OH;

[0625] Fmoc-N-Me-Ala-OH;

[0626] Fmoc-N-Me-Ser(Trt)-OH;

[0627] Fmoc-N-Me-Asn(Trt)-OH;

[0628] Fmoc-Hpr-OH;

[0629] Boc-PEG8c-OH;

[0630] Fmoc-PEG10c-OH;

[0631] Fmoc-dk(Boc)-OH;

[0632] Fmoc-dp-OH;

[0633] Fmoc-dyae(Boc)-OH:

[0634] Alloc-dk(Fmoc)-OH;

[0635] Boc-PEG8c-OH;

[0636] Fmoc-de(Allyl)-OH;

[0637] Fmoc-Hgl(Allyl)-OH;

[0638] Fmoc-dk(Alloc)-OH;

[0639] Fmoc-Qglucamine(acetonide)-OH.

[0640] In the structural determination of chemically synthesized peptides, the molecular weight, calculated considering the amino acids used according to the target sequence and the building blocks used as required, was confirmed by ESI-MS(+) mass spectrometry. It should be noted that "ESI-MS(+)" indicates electrospray ionization mass spectrometry performed in positive ion mode. The detected mass is reported in m / z units. It should also be noted that compounds with molecular weights greater than approximately 1000 are often detected as divalent or trivalent ions.

[0641] Example 1: Synthesis of cyclic peptides and conjugates

[0642] The identification of chemically synthesized peptides is performed by the retention time obtained by any of the following analytical methods (detection: UV wavelength 225nm, Shimadzu Corporation "SPD-M20A").

[0643] Analysis condition A

[0644] Column: Kinetex (registered trademark) EVO C18 2.6μm, 2.1 ID x 150mm, 100Å

[0645] Mobile phase: A = 0.025% TFA in water; B = 0.025% TFA in CH3CN.

[0646] Temperature: 60℃

[0647] Flow rate: 0.5 mL / min

[0648] Gradient: 20-60 / 7.15 minutes, 60-95 / 0.3 minutes.

[0649] Analysis condition B

[0650] Column: Kinetex (registered trademark) EVO C18 2.6μm, 2.1 ID x 150mm, 100Å

[0651] Mobile phase: A = 0.025% TFA in water; B = 0.025% TFA in CH3CN.

[0652] Temperature: 60℃

[0653] Flow rate: 0.5 mL / min

[0654] Gradient: 5-45 / 7.15 minutes, 45-95 / 0.3 minutes.

[0655] For peptide chain elongation in solid-phase resin, commercially available resin was used as the starting material via the Fmoc method. Specifically, using Sieber amide resin (Watanabe Chemical Industry Co., Ltd.), the target peptide was synthesized by repeatedly introducing and deprotecting Fmoc groups, starting with the removal of Fmoc groups. Unless otherwise specified, peptide chain elongation was performed automatically using a CEM Liberty Blue or Liberty Blue HT solid-phase synthesizer according to the manufacturer's instructions. For the introduction of each residue, double coupling was performed, repeating the peptide coupling reaction twice as needed. On the other hand, for the introduction of some amino acids, the resin was removed from the automated synthesizer, and the reaction was performed using reagents prepared immediately before use (manual coupling).

[0656] For example, for deFmoc-modified peptide chains supported on solid resin, approximately 4 equivalents of Fmoc amino acids, approximately 8 equivalents of DIC, and approximately 4 equivalents of Oxyma pure were added, and the reaction was carried out under any of the reaction conditions shown in the table below, followed by 1-2 oscillations, and then the resin was washed with DMF.

[0657]

[0658] The removal of the Fmoc group is performed, for example, by the following method. For peptide chains loaded on a solid resin with introduced Fmoc amino acids, the resin is washed with DMF after 1-2 oscillations under any of the conditions shown in the table below.

[0659]

[0660] Following the method described above, the introduction of each Fmoc amino acid and the deprotection of the Fmoc group were repeated until the target peptide chain was extended, followed by the introduction of chloroacetyl groups. The introduction method can be any of the methods listed in the table below. For solid-phase resins containing the Fmoc-protected peptides obtained in the previous step, after removing the Fmoc groups, a solution of the following solvent containing approximately 5-10 equivalents of chloroacetic acid, approximately 5-10 equivalents of a condensing agent, and approximately 5-10 equivalents of an additive was added to the solid-phase resin, and the mixture was shaken at room temperature for 30-60 minutes.

[0661]

[0662] For the deprotection of side chains and the removal from solid resin, the peptide bound to the resin after the introduction of chloroacetyl groups is added to any of the reaction mixtures listed in the table below (a mixture of TFA / H2O / TIS / DODT), and shaken at room temperature for 30-120 minutes.

[0663]

[0664] The reaction solution was then recovered by filtration through a filter plate. When the filtrate was added to an excess of diisopropyl ether or a mixture of diisopropyl ether and hexane or diethyl ether and hexane (e.g., 1 / 1, v / v) cooled to 0°C, a white, turbid precipitate was formed. To obtain this precipitate as a solid, centrifugation was performed, and the supernatant was decanted. The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C and then dried under reduced pressure.

[0665] The solid obtained above was used for peptide cyclization. In the peptide cyclization reaction, the peptide was dissolved in DMSO or a mixed solvent of DMSO / H2O, MeCN / H2O, or DMSO / MeCN / H2O at a final concentration of 4-5 mM based on the molar amount of the solid resin used. Then, 5-35 equivalents of triethylamine were added, and the mixture was shaken at room temperature for 2 hours to overnight. The resulting reaction solution was concentrated under reduced pressure and then purified.

[0666] The resulting residues were purified by reverse-phase preparative HPLC according to any of the methods listed in the table below.

[0667]

[0668] The following shows the specific structures of the synthesized peptides and conjugates.

[0669] Examples 1-1 to 1-3 involve the synthesis of peptides.

[0670] Example 1-1 Synthesis of peptide SEQ ID NO. 45.

[0671] [Chemical Formula 5]

[0672]

[0673] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.60 mmol / g, 1.67 g). The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer, following the manufacturer's instructions.

[0674] To remove Fmoc from the solid resin, a reaction was carried out at 75°C for 3 minutes at a time using 10% pyrrolidine (in DMF).

[0675] To introduce each residue, 0.21M Fmoc-AA (in DMF) / 1.0M DIC (in DMF) / 0.5M Oxymapure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents) were used relative to 1 resin equivalent, and the reaction was carried out once every 10 minutes at 75°C. Specifically, residues 3 and 9 were treated with 0.21M Fmoc-AA (in NMP) / 1.0M DIC (in DMF) / 0.5M Oxymapure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents). Residues 4 and 6 were treated twice at 75°C for 20 minutes. Residue 12 was treated twice at 75°C for 30 minutes. Residue 13 was treated once at 50°C for 30 minutes.

[0676] After each residue was introduced, to remove the Fmoc group, for the solid-phase resin containing the peptide with Fmoc protection, a reaction was performed at 75°C with 4% pyrrolidine + 83 mM Oxyma pure (in DMF) every 3 minutes. For the 4th residue, a 10% pyrrolidine DMF solution was used, and the reaction was performed at room temperature every 5 minutes twice. For the 6th and 12th residues, a 10% pyrrolidine DMF solution was used, and the reaction was performed at room temperature every 5 minutes and 10 minutes respectively.

[0677] For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, the α-amino Fmoc group was removed by reacting it with a 10% pyrrolidine DMF solution at 75°C for 3 minutes at a time. Further shaking with 1.0 MClAcNHS (in DMF, 10 equivalents) at room temperature for 60 minutes introduced the chloroacetyl group.

[0678] To deprotect the side chains and remove them from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed once with DMF, dichloromethane, and diethyl ether, and then dried under reduced pressure. Next, the solid resin was roughly divided into half and placed in a reaction vessel. A reaction mixture (10 mL of a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to each half, and the mixture was shaken at room temperature for 40 minutes. The reaction solution was filtered through a filter plate and recovered. The remaining solid resin in the reaction vessel was shaken again with a cleavage mixture (cocktail for cleavage), and the solution components were recovered through a filter plate and mixed with the filtrate. When the filtrate was divided into four equal portions and 40 mL of a 1 / 1 mixture of diethyl ether and hexane was added to each, precipitation occurred.

[0679] The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with diethyl ether and dried under reduced pressure. The resulting solids (peptides) were combined for the subsequent cyclization reaction. The peptides were dissolved in water / acetonitrile (1 / 1) to a final concentration of 5 mM based on the molar amount of solid resin, and then 10 equivalents of triethylamine were added. The mixture was stirred at room temperature for 60 minutes to carry out the peptide cyclization reaction. The reaction was stopped by adding acetic acid to the reaction solution, and the reaction solution was concentrated under reduced pressure using Genevac EZ-II elite.

[0680] The crude product was purified under the following conditions (column: Waters Xbridge C18 5μm 50x250mm; mobile phase: A = 0.1% TFA (water), B = 0.1% TFA (MeCN); temperature: 50℃; gradient (%B): 0.1 min 4.2–0.0%, 4.9 min 0.0–0.0%, 2 min 0.0–4.2%, 3 min 4.2–28.6%, 15 min 28.6–33.7%, 3 min 33.7–60%; flow rate: 0.1 min 118–18 mL / min, 4.9 min 18–18 mL / min, 2 min 18–118 mL / min, followed by 118 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0681] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 96.7%.

[0682] Analysis condition A: Retention time = 2.86 minutes

[0683] ESI-MS(+) observation m / z = 862.37 (M+2H) 2+

[0684] Examples 1-2: Synthesis of peptide SEQ ID NO. 46

[0685] [Chemical Formula 6]

[0686]

[0687] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.60 mmol / g, 1.67 g). The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer, following the manufacturer's instructions.

[0688] To remove Fmoc from the solid resin, a reaction was carried out at 75°C for 3 minutes at a time using 10% pyrrolidine (in DMF).

[0689] To introduce each residue, a reaction was performed at 75°C for 10 minutes at a time using 0.21M Fmoc-AA (in DMF) / 0.5M HATU (in DMF) / 1.0M DIPEA (in DMF) (4.2 equivalents / 4 equivalents / 8 equivalents) relative to 1 resin equivalent. Specifically, residues 3 and 9 were treated with 0.21M Fmoc-AA (in NMP) / 0.5M HATU (in DMF) / 1.0M DIPEA (in DMF) (4.2 equivalents / 4 equivalents / 8 equivalents). Residues 4, 6, and 12 were treated twice at 75°C for 10 minutes. Residue 13 was treated once at 40°C for 30 minutes.

[0690] After each residue was introduced, to remove the Fmoc group, for the solid-phase resin containing the peptide with Fmoc protection, a reaction was performed every 3 minutes at 75°C using 4% pyrrolidine + 83 mM Oxyma pure (in DMF). For residues 4, 6, and 12, a DMF solution of 10% pyrrolidine was used, and the reaction was performed twice at room temperature for 5 minutes.

[0691] For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, the α-amino Fmoc group was removed by reacting it with a 10% pyrrolidine DMF solution at 75°C for 3 minutes at a time. Further shaking with 0.25 MClAcNHS (in DMF, 5 equivalents) at room temperature for 60 minutes introduced chloroacetyl groups.

[0692] To deprotect the side chains and remove them from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed once with DMF, dichloromethane, and diethyl ether, and then dried under reduced pressure. Next, the solid resin was roughly divided into half and placed in a reaction vessel. A reaction mixture (20 mL of a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to each half, and the mixture was shaken at room temperature for 30 minutes. The reaction solution was filtered through a filter plate and recovered. The remaining solid resin in the reaction vessel was shaken again with a cleavage mixture (cocktail for cleavage), and the solution components were recovered through a filter plate and mixed with the filtrate. When the filtrate was divided into eight equal portions and 40 mL of a 1 / 1 mixture of diisopropyl ether and hexane was added, a precipitate formed.

[0693] The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with diethyl ether and dried under reduced pressure. The resulting solids (peptides) were combined for the subsequent cyclization reaction. The peptides were dissolved in water / acetonitrile (1 / 1) to a final concentration of 2.5 mM based on the molar amount of solid resin, and then 10 equivalents of triethylamine were added. The mixture was stirred at room temperature for 16 hours to carry out the peptide cyclization reaction. The reaction was stopped by adding acetic acid to the reaction solution, and the reaction solution was concentrated under reduced pressure using Genevac HT-12.

[0694] The crude product was purified under the following conditions (column: Waters XSelect C18 5μm 50x150mm; mobile phase: A = 0.1% TFA (water), B = 0.1% TFA (MeCN); temperature: 40℃; gradient (%B): 2 min 5.0-5.0%, 1 min 5.0-25.0%, 8 min 25.0-30.0%, 1 min 30.0-60.0%; flow rate: 1 min 20-20 mL / min, 1 min 20-120 mL / min, followed by 120 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0695] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 96.3%.

[0696] Analysis condition A: Retention time = 3.30 minutes

[0697] ESI-MS(+) observation m / z = 882.36 (M+2H) 2+

[0698] Examples 1-3 Synthesis of peptide SEQ ID NO. 47

[0699] [Chemical Formula 7]

[0700]

[0701] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.57 mmol / g, 0.53 g). The synthesis was performed using a CEM Liberty Prime solid-phase synthesizer, following the manufacturer's instructions.

[0702] To remove Fmoc from the solid resin, a reaction was carried out at 110°C for 1 minute at a time using 10% pyrrolidine (in DMF).

[0703] To introduce each residue, 0.21M Fmoc-AA (in DMF) / 2.0M DIC (in DMF) / 0.25M Oxymapure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents) were used relative to 1 resin equivalent, and the reaction was carried out at 105°C for 2 minutes at a time. Specifically, residues 3 and 9 were treated with 0.21M Fmoc-AA (in NMP) / 2.0M DIC (in DMF) / 0.25M Oxymapure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents), and residue 4 was treated at 105°C for 3 minutes at a time, twice. Additionally, residues 6 and 12 were treated at 90°C for 10 minutes at a time, twice, and residue 13 was treated at 40°C for 30 minutes at a time, once.

[0704] After each residue was introduced, to remove the Fmoc group, for the solid-phase resin containing the peptide with Fmoc protection, a reaction was performed every 3 minutes at 110°C using 4% pyrrolidine + 83 mM Oxyma pure (in DMF). For residues 4, 6, and 12, a reaction was performed every 1 minute at room temperature using a DMF solution of 10% pyrrolidine.

[0705] For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, the α-amino Fmoc group was removed by reacting it once per minute at 110°C with a 4% pyrrolidine / 83mM / Oxyma Pure DMF solution. Further, the mixture was shaken at 25°C for 30 minutes with 0.1M ClAcOH (in DMF) / 0.1M HATU (in DMF) / 0.2M DIEA (in DMF) (5 equivalents / 5 equivalents / 10 equivalents) to introduce a chloroacetyl group.

[0706] To deprotect the side chains and remove them from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed once with DMF, dichloromethane, and diethyl ether, and then dried under reduced pressure. Next, a reaction mixture (14 mL of a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid resin, and the mixture was shaken for 60 minutes at room temperature. The reaction solution was filtered through a filter plate and recovered. The remaining solid resin in the reaction vessel was shaken again with a cleavage mixture (cocktail for cleavage), and the solution components were recovered through a filter plate and mixed with the filtrate. When the filtrate was divided into three equal portions and 40 mL of a 1 / 1 mixture of diisopropyl ether and hexane was added to each portion, precipitation occurred.

[0707] The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with diethyl ether and dried under reduced pressure. The resulting solids (peptides) were combined for the subsequent cyclization reaction. The peptides were dissolved in water / DMSO (1 / 9) to a final concentration of 5 mM based on the molar amount of solid resin, and then 10 equivalents of triethylamine were added. The mixture was stirred overnight at room temperature to carry out the peptide cyclization reaction. The reaction was stopped by adding acetic acid to the reaction solution, and the reaction solution was concentrated under reduced pressure using a Genevac HT-12.

[0708] The crude product was purified under the following conditions (column: Waters Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 40℃; gradient (%B): 2 min 5.0-5.0%, 1 min 5.0-27%, 8 min 27-32%, 1 min 32-60%; flow rate: 1 min 20-20 mL / min, 1 min 20-120 mL / min, followed by 120 mL / min). The fraction containing the target substance was collected and lyophilized, and then purified again (column: Waters XSelect (registered trademark) C18 5μm 30x150mm; mobile phase: A = 1.0% AcOH (water), B = 1.0% AcOH (MeCN); temperature: 40℃; gradient (%B): 3 min 2.0-19%, 8 min 19-24%, 1 min 24-60%; flow rate: 45 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0709] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 99.6%.

[0710] Analysis condition B: Retention time = 5.71 minutes

[0711] ESI-MS(+) observation m / z = 855.30 (M+2H) 2+

[0712] Examples 1-4 to 1-16 involve the synthesis of conjugates.

[0713] Examples 1-4: Synthesis of Conjugate No. 64

[0714] [Chemical Formula 8]

[0715]

[0716] The peptide SEQ ID NO. 45 (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-S-3Py6NH2-HseMe-dk-Y-MeC) synthesized in Example 1-1 (200 mg) and DIEA (225 mg) were dissolved in DMF (4.84 mL), and then DOTA-NHS ester / hexafluorophosphate trifluoroacetate (147 mg) was added under ice-cold conditions. The reaction mixture was stirred at room temperature for 90 minutes.

[0717] The resulting reaction mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C185μm 30x150mm; mobile phase: A = 0.1% TFA (water), B = 0.1% TFA (MeCN); temperature: 50℃; gradient (%B): 0.1 min 4.0–0.0%, 4.9 min 0.0–0.0%, 1 min 0.0–4.0%, 3 min 4.0–28.5%, 9 min 28.5–33.6%, 1 min 33.6–60%; flow rate: 0.1 min 44–9 mL / min, 4.9 min 9–9 mL / min, 1 min 9–44 mL / min, followed by 44 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0718] The purity of the target substance was calculated based on the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analytical conditions. The purity of the target substance was 97.3%.

[0719] Analysis condition B: Retention time = 5.74 minutes

[0720] ESI-MS(+) observation m / z = 704.13 (M+H) 3+

[0721] Examples 1-5: Synthesis of Conjugate No. 62

[0722] [Chemical Formula 9]

[0723]

[0724] The peptide SEQ ID NO. 46 (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk-Y-MeC) synthesized in Examples 1-2 (100 mg) and DIEA (117 mg) were dissolved in DMF (2.51 mL), and then DOTA-NHS ester / hexafluorophosphate trifluoroacetate (76 mg) was added under ice-cold conditions. The reaction mixture was stirred at room temperature for 120 minutes.

[0725] The resulting reaction mixture was purified under the following conditions (column: Waters XBridge (registered trademark) C185μm 50x250mm; mobile phase: A = 0.1% TFA (water), B = 0.1% TFA (MeCN); temperature: 50℃; gradient (%B): 0.1 min 4.2–0.0%, 4.9 min 0.0–0.0%, 2 min 0.0–4.2%, 3 min 4.2–25.6%, 15 min 25.6–30.7%, 3 min 30.7–60%; flow rate: 0.1 min 118–18 mL / min, 4.9 min 18–18 mL / min, 2 min 18–118 mL / min, followed by 118 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0726] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 96.6%.

[0727] Analysis condition A: Retention time = 3.27 minutes

[0728] ESI-MS(+) observation m / z = 717.38 (M+H) 3+

[0729] Examples 1-6: Synthesis of Conjugate No. 65

[0730] [Chemical Formula 10]

[0731]

[0732] DOTA (0.42 g, CAS: 60239-18-1), DIEA (0.14 g), and N-hydroxysuccinimide (0.08 g, CAS: 6066-82-6) were dissolved in water (3.3 mL). Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.13 g, CAS: 25952-53-8) dissolved in water (1.9 mL) was added under ice-cold conditions. The reaction mixture was stirred under ice-cold conditions for 30 minutes.

[0733] The peptide SEQ ID NO. 47 (sequence: ClAc-da-Tbg-3Py6CON-Hgl-MeG-Ahp-PeG-S-3Py6NH2-HseMe-dk-Y-MeC) synthesized in Examples 1-3 (23 mg) and DIEA (13.0 mg) were dissolved in DMF (561 μL), and the solution prepared above (561 μL) was added under ice-cold conditions. The reaction mixture was stirred at room temperature for 1 hour. DIEA (13.0 mg) was added to the reaction solution and stirred for 1 hour to quench the reaction.

[0734] The resulting reaction mixture was purified under the following conditions (column: Waters Xbridge (registered trademark) C185μm 19x150mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 50℃; gradient (%B): 3 min 5.0-28%, 8 min 28-33%, 1 min 33-60%; flow rate: 17 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0735] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 97.6%.

[0736] Analysis condition A: Retention time = 3.14 minutes

[0737] ESI-MS(+) observation m / z = 699.33 (M+H) 3+

[0738] Examples 1-7 Synthesis of Conjugate No. 26

[0739] [Chemical Formula 11]

[0740]

[0741] DOTA (0.79 g, CAS: 60239-18-1), DIEA (0.34 mL), and N-hydroxysuccinimide (0.15 g, CAS: 6066-82-6) were dissolved in water (7.2 mL). Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.25 g, CAS: 25952-53-8) dissolved in water (4.17 mL) was added under ice-cold conditions. The reaction mixture was stirred under ice-cold conditions for 60 minutes.

[0742] The peptide SEQ ID NO. 46 (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk-Y-MeC) synthesized in Examples 1-2 (40 mg) and DIEA (31.6 μL) were dissolved in DMF (1.00 mL), and the solution prepared above (1004 μL) was added under ice-cold conditions. The reaction mixture was stirred at room temperature for 60 minutes. DIEA (31.6 μL) was added to the reaction mixture, and the mixture was stirred at room temperature for 60 minutes. Lutetium(III) hexahydrate (86.0 mg, CAS: 15230-79-2) and ammonium acetate (17 mg, CAS: 631-61-8) dissolved in water (4420 μL) were added to the reaction mixture. The reaction mixture was stirred at 90 °C for 1 hour.

[0743] The resulting reaction mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C185μm 30x150mm; mobile phase: A = 1.0% AcOH (water), B = 1.0% AcOH (MeCN); temperature: 40℃; gradient (%B): 3 min 5.0-27%, 8 min 27-32%, 1 min 32-60%; flow rate: 45 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0744] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 99.4%.

[0745] Analysis condition A: Retention time = 3.48 minutes

[0746] ESI-MS(+) observation m / z = 774.79 (M+H) 3+

[0747] Examples 1-8 Synthesis of Conjugate No. 51

[0748] [Chemical Formula 12]

[0749]

[0750] The target peptide (sequence: ClAc-de(PEG8Me)-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk-Y-MeC) (SEQ ID NO. 55) was synthesized using Sieber amide resin (Watanabe Chemicals, 0.60 mmol / g, 0.584 g). The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer according to the manufacturer's instructions.

[0751] To remove Fmoc from the solid resin, a reaction was carried out at 90°C for 1 minute at a time using 10% pyrrolidine (in DMF).

[0752] To introduce each residue, 0.21M Fmoc-AA (in DMF) / 1.0M DIC (in DMF) / 0.5M Oxyma pure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents) were used relative to 1 resin equivalent, and the reaction was carried out once every 3 minutes at 90°C. Specifically, the third residue was treated with 0.21M Fmoc-AA (in NMP) / 1.0M DIC (in DMF) / 0.5M Oxyma pure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents). For the fourth, sixth, and twelfth residues, the reaction was carried out twice every 10 minutes at 90°C. For the thirteenth residue, the reaction was carried out once every 30 minutes at 40°C. The first residue was treated with Fmoc-de(Allyl)-OH. The obtained solid resin was shaken with 0.19 M Fmoc-OSu (in DMF, 5 equivalents) at room temperature for 1 hour, followed by washing with DMF. For the deprotection of the first residue side chain, tetrakis(triphenylphosphine)palladium(0) (CAS: 14221-01-3) / phenylsilane (CAS: 694-53-1) (0.2 equivalents / 10 equivalents) was used relative to 1 equivalent of resin in DCM (15 mL) at room temperature, reacting once every 60 minutes. Regarding the side chain extension reaction, after deprotection with Allyl, followed by washing with DCM and DMF, H-PEG8Me / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used relative to 1 equivalent of resin in DMF (13 mL) at 75°C, reacting once every 30 minutes.

[0753] After each residue was introduced, to remove the Fmoc group, for the solid-phase resin containing the peptide with Fmoc protection, a reaction was performed once per minute at 90°C using 4% pyrrolidine + 83 mM Oxyma pure (in DMF). For residues 4, 6, and 12, a reaction was performed twice per minute at room temperature using a DMF solution of 10% pyrrolidine.

[0754] For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, the α-amino Fmoc group was removed by reacting twice for 1 minute at room temperature with a 10% pyrrolidine DMF solution. Further shaking with 0.19 MClAcNHS (in DMF, 5 equivalents) at room temperature for 60 minutes introduced the chloroacetyl group.

[0755] To deprotect the side chains and remove them from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed once with DMF, dichloromethane, and diethyl ether, and then dried under reduced pressure. Next, a reaction mixture (16 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel and shaken for 30 minutes at room temperature. The reaction solution was filtered through a filter plate and recovered. The remaining solid resin in the reaction vessel was shaken again with a cleavage mixture (cocktail for cleavage), and the solution components were recovered through a filter plate and mixed with the filtrate. When the filtrate was divided into three equal portions and 40 mL of a 1 / 1 mixture of diethyl ether and hexane was added to each portion, precipitation occurred.

[0756] The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with diethyl ether and dried under reduced pressure. The resulting solids (peptides) were combined for the subsequent cyclization reaction. The peptides were dissolved in water / acetonitrile (1 / 1) to a final concentration of 3.0 mM based on the molar amount of solid resin, and then 10 equivalents of triethylamine were added. The mixture was stirred at room temperature for 15 hours to carry out the peptide cyclization reaction. The reaction was stopped by adding acetic acid to the reaction solution, and the reaction solution was concentrated under reduced pressure using Genevac EZ-II elite.

[0757] The crude product was purified under the following conditions (column: Waters XSelect C18 5μm 50x250mm; mobile phase: A = 1.0% AcOH (water), B = 1.0% AcOH (MeCN); temperature: 50℃; gradient (%B): 5.1 min 0-0%, 1.9 min 0-4.2%, 3 min 4.2-25.6%, 15.5 min 25.6-30.7%, 1.5 min 30.7-60%, 4 min 60-90%; flow rate: 8 min 18-18 mL / min, 2 min 18-118 mL / min, followed by 118 mL / min). The fraction containing the target substance was collected and lyophilized to obtain cyclic peptides.

[0758] Using the obtained cyclic peptide as a raw material, the target peptide was obtained under the same reaction and purification conditions as in Examples 1-7.

[0759] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 98.5%.

[0760] Analysis condition A: Retention time = 4.12 minutes

[0761] ESI-MS(+) observation m / z = 915.83 (M+H) 3+

[0762] Examples 1-9: Synthesis of Conjugate No. 35

[0763] [Chemical Formula 13]

[0764]

[0765] The target peptide (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-D-3Py6NH2-HseMe-dk(F)-Y-MeC) (SEQ ID NO. 56) was synthesized using Sieber amide resin (Watanabe Chemicals, 0.54 mmol / g, 1.39 g). The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer according to the manufacturer's instructions.

[0766] To remove Fmoc from the solid resin, a reaction was carried out at 75°C for 3 minutes at a time using 10% pyrrolidine (in DMF).

[0767] To introduce each residue, a reaction was performed at 75°C for 10 minutes at a time, using 0.21M Fmoc-AA (in DMF) / 1.0M DIC (in DMF) / 0.5M Oxyma pure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents) relative to 1 resin equivalent. Specifically, residue 3 was treated with 0.21M Fmoc-AA (in NMP) / 1.0M DIC (in DMF) / 0.5M Oxyma pure (in DMF), and residue 9 was treated with 0.21M Fmoc-AA (in NMP) / 0.5M HATU (in DMF) / 1.0M DIEA (in DMF) (4.2 equivalents / 4 equivalents / 8 equivalents). For residue 3, the reaction was performed at 75°C for 30 minutes at a time. For residues 4, 6, and 12, the reaction was performed twice at 75°C for 10 minutes at a time. For residue 13, the reaction was carried out at 50 °C every 15 minutes. For residue 11, Fmoc-dk(Alloc)-OH was used. The resulting solid resin was shaken with 0.38 M Fmoc-OSu (in DMF, 5 equivalents) at room temperature for 1 hour, followed by washing with DMF. For deprotection of the side chain of residue 11, tetrakis(triphenylphosphine)palladium(0) (CAS: 14221-01-3) / phenylsilane (CAS: 694-53-1) (0.2 equivalents / 10 equivalents) was used relative to 1 equivalent of resin in DCM / HFIP (30 mL / 0.3 mL) every 60 minutes at room temperature. Regarding the side chain extension reaction, after deprotection of Allyl and washing with DCM and DMF, Boc-F-OH / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) were used in DMF (10 mL) at 75°C for 30 minutes per reaction relative to the obtained 1 equivalent of resin.

[0768] After each residue was introduced, to remove the Fmoc group, for the solid-phase resin containing the peptide with Fmoc protection, two reactions were performed at room temperature for 5 minutes each using 10% pyrrolidine (in DMF). For residues 9, 10, 11, 12, and 13, reactions were performed at 75°C for 3 minutes each using 4% pyrrolidine + 83 mM / Oxyma pure (in DMF).

[0769] For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, the α-amino Fmoc group was removed by reacting it once per minute at room temperature with a 10% piperidine DMF solution. Further shaking with 0.25 MClAcNHS (in DMF, 10 equivalents) at room temperature for 60 minutes introduced the chloroacetyl group.

[0770] To deprotect the side chains and remove them from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed once with DMF, dichloromethane, and diethyl ether, and then dried under reduced pressure. Next, the solid resin was divided into three equal portions and placed in a reaction vessel. A reaction mixture (12 mL of a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to each portion, and the mixture was shaken at room temperature for 40 minutes. The reaction solution was filtered through a filter plate and recovered. The remaining solid resin in the reaction vessel was shaken again with a cleavage mixture (cocktail for cleavage), and the solution components were recovered through a filter plate and mixed with the filtrate. When the filtrate was divided into two equal portions and 30 mL of a 1 / 1 mixture of diisopropyl ether and hexane was added, a precipitate formed.

[0771] The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with diethyl ether and dried under reduced pressure. The resulting solids (peptides) were combined for the subsequent cyclization reaction. The peptides were dissolved in DMSO / water / acetonitrile (1 / 2 / 2) to a final concentration of 5.0 mM based on the molar amount of solid resin, and then 10 equivalents of triethylamine were added. The mixture was stirred at room temperature for 1 hour to carry out the peptide cyclization reaction. The reaction was stopped by adding acetic acid to the reaction solution, and the reaction solution was concentrated under reduced pressure using Genevac EZ-II elite.

[0772] The crude product was purified under the following conditions (column: Waters XBridge (registered trademark) C18 5μm 50x250mm; mobile phase: A = 0.1% TFA (water), B = 0.1% TFA (MeCN); temperature: 50℃; gradient (%B): 0.1 min 5.3-1.1%, 4.9 min 1.1-1.1%, 2 min 1.1-5.3%, 3 min 5.3-30.7%, 15 min 30.7-35.8%, 3 min 35.8-60%; flow rate: 0.1 min 118-18 mL / min, 4.9 min 18-18 mL / min, 2 min 18-118 mL / min, followed by 118 mL / min). The fraction containing the target substance was collected and lyophilized to obtain cyclic peptides.

[0773] Using the obtained cyclic peptide as a raw material, the target peptide was obtained under the same reaction and purification conditions as in Examples 1-7.

[0774] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 98.7%.

[0775] Analysis condition A: Retention time = 3.79 minutes

[0776] ESI-MS(+) observation m / z = 819.85 (M+H) 3+

[0777] Examples 1-10: Synthesis of Conjugate No. 38

[0778] [Chemical Formula 14]

[0779]

[0780] The target peptide (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk(PEG8c)-Y-MeC) (SEQ ID NO. 57) was synthesized using Sieber amide resin (Watanabe Chemicals, 0.60 mmol / g, 0.21 g). The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer according to the manufacturer's instructions.

[0781] To remove Fmoc from the solid resin, a reaction was carried out at 90°C for 1 minute at a time using 10% pyrrolidine (in DMF).

[0782] To introduce each residue, 0.21M Fmoc-AA (in DMF) / 1.0M DIC (in DMF) / 0.5M Oxyma pure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents) were used relative to 1 equivalent of resin, and the reaction was carried out at 90°C every 3 minutes. Alloc-dk(Fmoc)-OH was used for residue 11. For extension from residue 13 to residue 11, after removing the Fmoc group from the side chain, Boc-PEG8c-OH was used for the extension reaction from the side chain. For deprotection of the main chain Alloc group, tetrakis(triphenylphosphine)palladium(0) (CAS: 14221-01-3) / dimethylamineborane (CAS: 74-94-2) (0.2 equivalents / 10 equivalents) was used relative to 1 equivalent of resin, and the reaction was carried out at room temperature in DCM / HFIP (5 mL / 0.1 mL) every 60 minutes. The resulting solid resin was subjected to subsequent extension reactions. For residue 9, a mixture of 0.21M Fmoc-AA (in NMP) / 1.0M DIC (in DMF) / 0.5M Oxyma pure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents) was used. For residues 4, 6, 7, and 12, the reaction was carried out twice at 90°C for 10 minutes. For residue 13, the reaction was carried out once at 40°C for 20 minutes.

[0783] After each residue was introduced, in order to remove the Fmoc group, for the solid-phase resin containing the peptide that retained Fmoc protection, a reaction was performed twice per minute at room temperature using 10% pyrrolidine (in DMF). For the side chains of residues 10 and 11, a reaction was performed once per minute at 90°C using 4% pyrrolidine + 83 mM / Oxyma pure (in DMF).

[0784] For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, the α-amino Fmoc group was removed by reacting twice for 1 minute at room temperature with a 10% piperidine DMF solution. Further shaking with 0.16MClAcNHS (DMF / DCM = 1 / 1, 5 equivalents) at room temperature for 60 minutes introduced the chloroacetyl group.

[0785] To deprotect the side chains and remove them from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed once with DMF, dichloromethane, and diethyl ether, and then dried under reduced pressure. Next, a reaction mixture (3 mL of a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 90 / 2.5 / 2.5 / 5.0) was added to the reaction vessel and shaken for 120 minutes at room temperature. The reaction solution was filtered through a filter plate and recovered. The remaining solid resin in the reaction vessel was shaken again with a cleavage mixture (cocktail for cleavage), and the solution components were recovered through a filter plate and mixed with the filtrate. When this filtrate was added to 35 mL of a 1 / 1 mixture of diethyl ether and hexane, a precipitate formed.

[0786] The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with diethyl ether and dried under reduced pressure. The resulting solid (peptide) was used for the subsequent cyclization reaction. The peptide was dissolved in water / acetonitrile (1 / 1) to a final concentration of 5.0 mM based on the molar amount of solid resin, and then 10 equivalents of triethylamine were added. The mixture was stirred at room temperature for 1 hour to carry out the peptide cyclization reaction. The reaction was stopped by adding acetic acid to the reaction solution, and the reaction solution was concentrated under reduced pressure using Genevac EZ-IIelite.

[0787] The resulting residue was dissolved in DMSO / water (9 / 1) to a final concentration of 25 mM based on the molar amount of the solid resin. Then, DIEA / SulfoCy5-NHS ester (5 equivalents / 1.2 equivalents) was added, and the mixture was stirred at room temperature for 1 hour. The reaction was stopped by adding acetic acid to the reaction solution.

[0788] The resulting reaction mixture was purified under the following conditions (column: Waters XSelect, C185 μm, 19 x 150 mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 60 °C; gradient (%B): 3 min 12-37%, 8 min 37-42%, 1 min 42-60%; flow rate: 17 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0789] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 96.0%.

[0790] Analysis condition A: Retention time = 5.12 minutes

[0791] ESI-MS(+) observation m / z = 942.68 (M+H) 3+

[0792] Examples 1-11 Synthesis of Conjugate No. 67

[0793] [Chemical Formula 15]

[0794]

[0795] The target peptide (sequence: da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk-Y-MeA-MeG) (SEQ ID NO. 58) was synthesized using H-Ser(tBu)-Trt(2-Cl) resin (Watanabe Chemicals, 0.78 mmol / g, 1.28 g). The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer according to the manufacturer's instructions.

[0796] To introduce each residue, a reaction was performed at room temperature for 30 minutes at a time using 0.21M Fmoc-AA (in DMF) / 0.5M HATU (in DMF) / 1.0M DIPEA (in DMF) (4.2 equivalents / 4 equivalents / 8 equivalents) relative to 1 resin equivalent. Specifically, residue 3 was treated with 0.21M Fmoc-AA (in NMP) / 0.5M HATU (in DMF) / 1.0M DIPEA (in DMF) (4.2 equivalents / 4 equivalents / 8 equivalents), residue 9 with 0.21M Fmoc-AA (in NMP) / 1.0M DIC (in DMF) / 0.5M Oxyma pure (in DMF) (4.2 equivalents / 8 equivalents / 4 equivalents), and residues 4, 6, 7, and 12 were treated twice at room temperature for 30 minutes each. Residue 9 was treated once at room temperature for 60 minutes each. For residue 13, the reaction was carried out twice at room temperature for 60 minutes.

[0797] After each residue was introduced, in order to remove the Fmoc group, for the solid-phase resin containing the peptide with Fmoc protection, two reactions were performed at room temperature for 5 minutes each using 10% pyrrolidine (in DMF). For the first residue, two reactions were performed at room temperature for 1 minute each using 10% pyrrolidine (in DMF).

[0798] To remove the solid resin, a reaction mixture of HFIP / DCM (9 mL, 1 / 4) was added and shaken for 60 minutes at room temperature. The reaction mixture was concentrated under reduced pressure using Genevac EZ-II elite. The residue was solidified by adding diisopropyl ether, centrifuged, and the solution was decanted. The resulting solid was washed again with diethyl ether and dried under reduced pressure. The resulting residue was dissolved in DMF to a final concentration of 5 mM based on the molar amount of the solid resin, and then DIEA / HATU (5 equivalents / 1.2 equivalents) was added and stirred for 1 hour at room temperature. The reaction was stopped by adding acetic acid to the reaction solution. The reaction mixture was concentrated under reduced pressure using Genevac HT−12. A reaction mixture (a mixture of TFA / H2O / TIS at a volume ratio of 95 / 2.5 / 2.5) was added to the resulting residue and shaken for 40 minutes at room temperature. A precipitate formed upon the addition of 40 mL of diisopropyl ether to the reaction mixture.

[0799] The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with diethyl ether and then dried under reduced pressure.

[0800] The resulting residue was purified under the following conditions (column: Waters XSelect C18 5μm 50x150mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 40℃; gradient (%B): 2 min 5-5%, 1 min 5-27%, 8 min 27-32%, 1 min 32-60%; flow rate: 1 min 20-20 mL / min, 1 min 20-120 mL / min, followed by 120 mL / min). Fractions containing the target substance were collected and lyophilized.

[0801] Using the obtained cyclic peptide as a raw material, the target peptide was obtained under the same reaction and purification conditions as in Examples 1-7.

[0802] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 99.7%.

[0803] Analysis condition A: Retention time = 4.27 minutes

[0804] ESI-MS(+) observation m / z = 768.69 (M+H) 3+

[0805] Examples 1-12 Synthesis of Conjugate No. 56

[0806] [Chemical Formula 16]

[0807]

[0808] The peptide SEQ ID NO. 46 (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk-Y-MeC) synthesized in Examples 1-2 (20 mg) and DIEA (26.3 μL) were dissolved in DMSO (0.50 mL), and then 2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (8.1 mg, CAS: 1375475-53-8) was added at room temperature. The reaction mixture was stirred at room temperature for 5 hours.

[0809] The resulting reaction mixture was purified under the following conditions (column: Waters XBridge (registered trademark) C185μm 19x150mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 50℃; gradient (%B): 3 min 5.0-27%, 8 min 27-32%, 1 min 32-60%; flow rate: 17 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0810] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 97.8%.

[0811] Analysis condition A: Retention time = 3.40 minutes

[0812] ESI-MS(+) observation m / z = 741.40 (M+H) 3+

[0813] Examples 1-13 Synthesis of Conjugate No. 58

[0814] [Chemical Formula 17]

[0815]

[0816] The peptide SEQ ID NO. 46 (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk-Y-MeC) synthesized in Examples 1-2 (20 mg) and DIEA (26.3 μL) were dissolved in DMSO (0.50 mL), and then 2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidine-1-yl)oxy)-4-oxobutyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid (14 mg, CAS: 1407166-70-4) was added at room temperature. The reaction mixture was stirred at room temperature for 7 hours.

[0817] The resulting reaction mixture was purified under the following conditions (column: Waters XBridge (registered trademark) C185μm 19x150mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 50℃; gradient (%B): 3 min 5.0-28%, 8 min 28-33%, 1 min 33-60%; flow rate: 17 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0818] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 96.4%.

[0819] Analysis condition A: Retention time = 3.70 minutes

[0820] ESI-MS(+) observation m / z = 1060.97 (M+2H) 2+

[0821] Examples 1-14 Synthesis of Conjugate No. 21

[0822] [Chemical Formula 18]

[0823]

[0824] The conjugate No. 2 (10 mg) obtained by the same synthesis method as in Examples 1-4 was dissolved in DMF (0.40 mL), and copper(II) chloride dihydrate (0.90 mg, CAS: 10125-13-0) and 100 mM ammonium acetate aqueous solution (0.20 mL) were dissolved. The reaction mixture was stirred at 40 °C for 60 minutes.

[0825] The resulting reaction mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C185μm 30x150mm; mobile phase: A = 1.0% AcOH (water), B = 1.0% AcOH (MeCN); temperature: 40℃; gradient (%B): 3 min 6.0-31%, 8 min 31-36%, 1 min 36-60%; flow rate: 45 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0826] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 97.7%.

[0827] Analysis condition A: Retention time = 4.32 minutes

[0828] ESI-MS(+) observation m / z = 768.95 (M+H) 3+

[0829] Examples 1-15 Synthesis of Conjugate No. 22

[0830] [Chemical Formula 19]

[0831]

[0832] The cyclic peptide (sequence: ClAc-da-Tbg-3Py6CON-Hgl-PeG-Ahp-PeG-S-3Py6NH2-HseMe-GY-MeC-K) (SEQ ID NO. 59) (15 mg) obtained by the same synthesis method as in Examples 1-1 was dissolved in DMF (0.40 mL). DOTA-NHS ester / hexafluorophosphate trifluoroacetate (7.8 mg) and DIPEA (0.018 mL) were added under ice-cold conditions. The reaction mixture was stirred at room temperature for 120 minutes. Zirconium acetylacetonate (IV) (5.1 mg, CAS: 17501-44-9) was added to the reaction mixture, and after stirring at 50 °C for 90 minutes, DOTA (10 mg) was added.

[0833] The resulting reaction mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C185μm 50x250mm; mobile phase: A = 1.0% AcOH (water), B = 1.0% AcOH (MeCN); temperature: 50℃; gradient (%B): 5.1 min 0-0%, 1.9 min 0-4.2%, 3 min 4.2-25.6%, 15.5 min 25.6-30.7%, 1.5 min 30.7-60%, 4 min 60-90%; flow rate: 5.1 min 18-18 mL / min, 1.9 min 18-118 mL / min, followed by 118 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[0834] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 99.7%.

[0835] Analysis condition A: Retention time = 3.66 minutes

[0836] ESI-MS(+) observation m / z = 1165.62 (M+H) 2+

[0837] Examples 1-16 Synthesis of Conjugate No. 45

[0838] [Chemical Formula 20]

[0839]

[0840] Using Cl-Trt(2-Cl) resin (Watanabe Chemical) and Fmoc-MeCt (CAS: 2932449-54-0) as raw materials, the target peptide (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk-Y-MeCt) (SEQ ID NO. 60) was synthesized using a solid-phase synthesizer (1.007 mmol / g, 0.500 g) supported in the same manner as described in Tetrahedron Letters, 43(2002) 3419-3421. The synthesis was performed using a Liberty Blue solid-phase synthesizer from CEM, following the manufacturer's instructions. The synthesis of the cyclic peptide was the same as in Examples 1-2, and the introduction of the chelating agent and metal was carried out under the same reaction and purification conditions as in Examples 1-7, yielding the target peptide.

[0841] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 99.5%.

[0842] Analysis condition A: Retention time = 3.85 minutes

[0843] ESI-MS(+) observation m / z = 760.33 (M+H) 3+

[0844] Example 2: Synthesis of cyclic peptides and conjugates

[0845] The target peptides and their conjugates with the payload listed below were synthesized according to the general method described in Example 1.

[0846]

[0847]

[0848] Table 7 records the peptides of the target substance, analytical conditions and retention times, and the observed values ​​in ESI-MS (+); Table 8 records the conjugates of the target substance, analytical conditions and retention times, and the observed values ​​in ESI-MS (+).

[0849] In Table 7, "terminus" indicates the functional group at the C-terminus (in the table, "-OH" represents COOH, and "-NH2" represents CO(NH2)). If not listed, it indicates the absence of a functional group at the C-terminus. Regarding "Cyclization" in the table, "ClAc" indicates cyclization by combining the amino acid at position 1, which has a chloroacetyl group introduced, with a C-terminal C or MeC group present at the C-terminus. "Free" indicates that the N-terminal amino group of the amino acid at position 1 is combined with the C-terminal carboxyl group of the amino acid at position 13 or 14. Additionally, "Additional / Linker" in the table indicates a linker or added amino acid not included in the cyclic structure. For example, in Ca9_No1 (SEQ ID NO. 3), it indicates that the amino acid sequence X1-X13 forms a cyclic structure, with glycine added as a linker at the C-terminus X13.

[0850] Table 8 uses the same format as Table 7. Furthermore, in Table 8, substances enclosed in parentheses () represent payloads. For example, Conjugate No. 1 indicates that X1 and the amino acid residue C of X13 form a cyclic structure, further including a linker structure such as G-PEG10c-K starting from the C of X13, and with SulfoCy5 attached to the side chain of the terminal K as the payload. Similarly, Conjugate No. 6 indicates that X1 and the amino acid residue MeC of X13 form a cyclic structure, and the side chain of X1's dk is attached to Lu-bound DOTA as the payload. Likewise, Conjugate No. 19 in Table 8 also indicates that X1 and the amino acid residue MeC of X13 form a cyclic structure, and the side chain of X1's dk is attached to Lu-bound DOTA as the payload via df. Additionally, NT in Table 8 indicates not tested.

[0851] Example 3: Intermolecular interactions between human CA9 and peptides or conjugates based on surface plasmon resonance (SPR) Evaluation test

[0852] For the various peptides or conjugates synthesized in Examples 1 and 2, surface plasmon resonance (SPR)-based intermolecular interaction experiments of the peptides with human CA9 protein were conducted using the methods shown below. The specific experimental methods are as follows.

[0853] SPR measurement

[0854] The Protein A sensor chip (Cytiva) was inserted into the Biacore T200 (Cytiva) and pretreated with running buffer: 1X HBS-P+ (Cytiva), 1.0% DMSO (Fujifilm and Koichi Chemicals Co., Ltd.), and 50 μM zinc acetate dihydrate (Hampton Research), and equilibrated at a flow rate of 30 μL / min. 50 nM Human CA9-Fc (SinoBiological) diluted with running buffer was reacted at a flow rate of 5 μL / min for 60 seconds and captured into a flow cell.

[0855] The peptides or conjugates synthesized in Example 1 or 2 were diluted with running buffer to prepare 1 mM solutions in DMSO solution to achieve a final concentration of 100 nM, resulting in 25 nM and 5 nM peptide or conjugate solutions. Additionally, based on the affinity of the peptides or conjugates, they were diluted with running buffer to achieve a final concentration of 40 nM, resulting in 10 nM and 2 nM peptide or conjugate solutions.

[0856] Using the above samples, the kinetics of the peptides or conjugates for human CA9 were obtained by SPR assay. The kinetic evaluation model was set as Single Cycle Kinetics, and curve fitting was performed using Biacore T200 Evaluation Software Version 3.0 (Cytiva). The obtained sensor spectra were subjected to least squares-based curve fitting to determine their KD values, thereby evaluating the binding of the peptides or conjugates to human CA9.

[0857] The results are shown in Tables 7 and 8. As shown in Tables 7 and 8, the peptides and conjugates of the present invention exhibit binding activity against human CA9.

[0858] Example: Synthesis of non-natural amino acids

[0859] This reference example illustrates the synthesis of various non-natural amino acids.

[0860] Unless otherwise explicitly stated, the following devices, abbreviations, and analysis conditions are used in the reference examples.

[0861] Unless otherwise specified, the proton nuclear magnetic resonance (¹H-NMR) measurements of the following synthetic examples were performed using a JNM-ECP300 manufactured by JEOL Ltd., or a JNM-ECX300 manufactured by JEOL Ltd., or an Ascend™500 manufactured by Bruker Ltd., in deuterated chloroform or deuterated dimethyl sulfoxide solvent. The chemical shift is expressed as δ value (ppm) with tetramethylsilane as the internal standard (0.0 ppm).

[0862] In NMR spectroscopy, "s" indicates a singlet, "d" indicates a doublet, "t" indicates a triplet, "q" indicates a quartet, "dd" indicates a double doublet, "dt" indicates a double triplet, "m" indicates a multiplet, "br" indicates a broad peak, "J" indicates the coupling constant, "Hz" indicates Hertz, "CDCl3" indicates deuterated chloroform, and "DMSO-d6" indicates deuterated dimethyl sulfoxide.

[0863] Unless otherwise specified, high-speed liquid chromatography / mass spectrometry analysis was performed using any one of the following: Waters ACQUITY UPLC H-Class / QDa, Waters ACQUITY UPLC H-Class / SQD2, or Shimadzu LC-20AD / Triple Tof5600.

[0864] In the documentation of high-performance liquid chromatography / mass spectrometry (HPLC / MS), ESI+ represents the positive mode of electrospray ionization, (M+H)+ represents the proton addition ion, and (M-C4H8+H)+ represents the ion generated by proton addition and tert-butyl desorption.

[0865] In the documentation of high-performance liquid chromatography / mass spectrometry, ESI- represents the negative mode of electrospray ionization, and MH represents ions produced by proton desorption.

[0866] Analysis condition C

[0867] Column: ACQUITY (registered trademark) 1.7μm BEH C18, 2.1x100mm, Waters

[0868] Mobile phase A: 0.025% TFA in water

[0869] Mobile phase B: 0.025% TFA in CH3CN

[0870] Column temperature: 60℃

[0871] Gradient (%B): 5.56 min 5-95%, followed by 5.56 min~7.22 min 95%, flow rate: 0.6 mL / min

[0872] Detection: UV 254nm.

[0873] Refer to Example 1 for the synthesis of Fmoc-4COOPeG(tBu)-OH

[0874] [Chemical Formula 21]

[0875]

[0876] 10 g of methyl 2-aminoacetate hydrochloride (CAS: 5680-79-5) was dissolved in 200 mL of MeOH (CAS: 67-56-1) at room temperature. Triethylamine (16.12 g, CAS: 121-44-8) and tert-butyl acrylate (20.42 g, CAS: 1663-39-4) were added. The reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The resulting residue (15 g) was dissolved in 150 mL of DCM (CAS: 75-09-2), and DIPEA (35.69 g, CAS: 7087-68-5) and N-[(9H-fluorene-9-ylmethoxy)carbonyloxy]succinimide (22.12 g, CAS: 82911-69-1) were added under ice-cold conditions. The reaction was stopped by adding 1 M hydrochloric acid (300 mL, CAS: 7647-01-0) after stirring at room temperature for 2 hours. Extracted with DCM, the resulting organic layer was washed with saturated brine and dried with sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1), and the fraction containing the target substance was concentrated under reduced pressure.

[0877] The product (31 g) obtained from the above reaction was dissolved in DCM (300 mL), and a 1,4-dioxane solution (300 mL) of 4 M HCl was added under ice-cold conditions. The reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The resulting residue (25 g) was dissolved in DCM (150 mL) at room temperature, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.75 g, CAS: 25952-53-8) and N-hydroxyphthalimide (11.70 g, CAS: 524-38-9) were added. The reaction mixture was stirred at room temperature for 2 hours, and then the reaction was stopped by adding saturated sodium bicarbonate aqueous solution (200 mL). The resulting mixture was washed with saturated brine and dried with sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate = 1 / 1), and the fraction containing the target substance was concentrated under reduced pressure.

[0878] Under a nitrogen atmosphere, nickel(II) bromide trihydrate (4.64 g, CAS: 7789-49-3) and 4,4'-di-tert-butyl-2,2'-bipyridine (4.57 g, CAS: 72914-19-3) were added to N,N-dimethylacetamide (300 mL, CAS: 127-19-5), and the mixture was stirred at room temperature for 15 minutes. Tert-butyl 4-bromobenzoate (29.19 g, CAS: 59247-47-1) and the product obtained from the previous step (30 g) were added to the resulting mixture, followed by zinc (18.56 g, CAS: 7440-66-6) over 10 minutes at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. Water was added to the mixture to stop the reaction, and the insoluble matter was filtered off and washed with ethyl acetate. After extracting the filtrate with ethyl acetate, the resulting organic layer was washed with saturated brine and dried with sodium sulfate. After filtration and vacuum concentration, the residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate = 1 / 1), and the fraction containing the target substance was concentrated under vacuum.

[0879] The obtained product (20 g) was dissolved in isopropanol (600 mL, CAS: 67-63-0), and under ice-cold conditions, an aqueous solution (150 mL) of calcium chloride (68.88 g, CAS: 10043-52-4) and an aqueous solution (50 mL) of lithium hydroxide (3.72 g, CAS: 1310-65-2) were added. The reaction mixture was stirred at room temperature for 16 hours. The pH was adjusted to 5 by adding a saturated aqueous solution of sodium dihydrogen phosphate (CAS: 7558-80-7), and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine and dried with sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by reversed-phase silica gel rapid column chromatography (column: C18 silica gel, gradient: 10 mM ammonium bicarbonate aqueous solution / acetonitrile = 95 / 5-0 / 100). The fraction containing the target substance was concentrated under reduced pressure to give the title compound (10.1 g).

[0880] 1H NMR(500MHz, DMSO-d6) δ12.68(br, 1H), 7.92-7.86(m, 2H), 7.86-7.55(m, 4H), 7.47-7.00(m, 6H), 4.52-4.45(m, 1H), 4.28-4.14(m, 2H), 4.00-3.69(m,2H), 3.56-3.07(m, 2H), 2.91-2.41(m, 2H), 1.55-1.51(m, 9H).

[0881] Analysis conditions C: Retention time = 5.44 minutes; ESI-MS(+) observation m / z = 446(M-C4H8+H) + The theoretical value is m / z = 501.

[0882] Refer to Example 2 for the synthesis of Fmoc-3COOPeG(tBu)-OH

[0883] [Chemical Formula 22]

[0884]

[0885] Fmoc-3COOPeG(tBu)-OH was synthesized in the same manner as in Reference Example 1, Fmoc-4COOPeG(tBu)-OH. In this synthesis, tert-butyl 3-bromobenzoate (CAS: 69038-74-0) was used instead of tert-butyl 4-bromobenzoate as the starting material.

[0886] 1H NMR(500MHz, DMSO-d6) δ12.70(br, 1H), 7.92-7.82(m, 2H), 7.81-7.55(m, 4H), 7.52-7.10(m, 6H), 4.51-4.32(m, 1H), 4.32-4.09(m, 2H), 4.09-3.74(m,2H), 3.53-3.12(m, 2H), 2.92-2.46(m, 2H), 1.55-1.47(m, 9H).

[0887] Analysis conditions C: Retention time = 5.19 minutes; ESI-MS (+) observation m / z = 446(M-C4H8+H) + The theoretical value is m / z = 501.

[0888] Example 4 67 In vivo distribution of Ga-labeled conjugates

[0889] For peptides 67 Ga mark

[0890] The following conjugates were synthesized using the same methods as in Examples 1 and 2.

[0891] Conjugate No. 65

[0892] Conjugate No. 64

[0893] Conjugate No. 70 (the payload portion of Conjugate No. 18 is only DOTA and not bound to a lutetium conjugate (sequence: ClAc-de-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-D-3Py6NH2-HseMe-dk(DOTA)-Y-MeC) (DOTA is bound to the side chain of dk at position 11 of the amino acid sequence described in SEQ ID NO. 73)).

[0894] Conjugate No. 71 (the payload portion of Conjugate No. 29 is only DOTA and not bound to lutetium (sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-D-5Inda-HseMe-dk(DOTA)-Y-MeC) (DOTA is bound to the side chain of dk at position 11 of the amino acid sequence described in SEQ ID NO. 77).

[0895] Conjugate No. 72 (a conjugate of Conjugate No. 15 whose payload is only DOTA and not bound to lutetium (sequence: ClAc-dk(DOTA)-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-D-3Py6NH2-HseMe-dq-Y-MeC) (DOTA is bound to the side chain of dk at position 1 of the amino acid sequence described in SEQ ID NO. 70).

[0896] Conjugate No. 62

[0897] as well as

[0898] Conjugate No. 73 (The payload portion of Conjugate No. 31 is only DOTA-t4amCh and not bound with lutetium (Sequence: ClAc-da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-D-5Inda-HseMe-dk(DOTA-t4amCh)-Y-MeC) (DOTA is bound to the side chain of dk at position 11 of the amino acid sequence described in SEQ ID NO. 77 via t4amCh))

[0899] The labeled reaction solutions containing each conjugate (Table 9) were heated at 95°C for 10 minutes. 67 Ga marker.

[0900]

[0901] Radioactive HPLC

[0902] The synthesized as described above 67 The Ga-labeled conjugate (hereinafter also referred to as the test substance) was mixed with acetonitrile, and the radiochemical purity before and after administration was determined using a TSK gel (registered trademark) ODS-80Ts QA, 4.6mm ID×250mm, 5μm (Tosoh Corporation). The results showed a purity of 95.61-91.27%.

[0903] Radioactive TLC

[0904] The test substance was spotted onto TLC silica gel 60 RP-18 F. 254 The radioactivity of the sample (Merck, 115389) was determined by radiochromatography using a 1:1 mixture of 2 mol / L ammonium acetate and acetone, followed by a 10-minute collimator (energy range 60-350 keV, collimator 1.0 cm, V-shaped BGO crystal). The results showed a purity of 96.67-94.27%.

[0905] Preparation of evaluation animals (HT-29 tumor-bearing mice)

[0906] Five-week-old female BALB / cSlc-nu / nu mice were purchased from SLC (Shizuoka, Japan) for animal evaluation. After acclimatization, 0.1 mL of a suspension containing 5 × 10⁻⁶ mice was used. 7 A 1:2 mixture of PBS pH 7.4 (1×) (Thermo Fisher Scientific, 10010-023) and VitroGel (The WellBioscience, VHM01) containing CA9-positive human colon cancer-derived HT-29 cells / mL was transplanted subcutaneously into the right abdomen of nude mice (6 weeks old) using a disposable syringe with a needle.

[0907] The evaluation animals consisted of 3 animals per group, for a total of 12 groups. At the time of evaluation, the animals weighed 18.2g-20.7g and had tumor diameters ranging from 216-908mm. 3 .

[0908] Distribution in the body

[0909] Each test substance was administered intravenously as a single dose to HT-29 tumor-bearing mice. Dissections were performed at 4, 24, and 72 hours post-administration to determine the radioactivity concentration (%ID / g) in each tissue.

[0910] result

[0911] Will 67The results of the in vivo distribution of Ga-labeled compounds are shown in Figure 1 , Figure 2 , Figure 3 It should be noted that "Conjugate No. XX" in the figure indicates that the payload portion of the conjugate recorded in that Conjugate No. is DOTA- 67 Various Ga conjugates.

[0912] Figure 1 Tissue distribution %ID / g 4 hours after drug administration (mean ± SD, n = 3)

[0913] Figure 2 Tissue distribution %ID / g 24 hours after drug administration (mean ± SD, n = 3)

[0914] Figure 3 Tissue distribution %ID / g 72 hours after drug administration (mean ± SD, n = 3)

[0915] Example 5 64 In vivo distribution and PET imaging of Cu-labeled conjugates

[0916] For peptide conjugates 64 Cu marking

[0917] The labeled reaction solution (Table 10) containing each peptide conjugate (Conjugate No. 64, Conjugate No. 62, Conjugate No. 2, Conjugate No. 56, Conjugate No. 58) synthesized in Examples 1 or 2 was heated at 95°C for 10 minutes. 64 Cu labelling.

[0918]

[0919] Radioactive HPLC

[0920] The synthesized as described above 64 The Cu-labeled conjugate (hereinafter also referred to as the test substance) was mixed with acetonitrile, and the radiochemical purity before and after administration was determined using a TSK gel (registered trademark) ODS-80Ts QA, 4.6mm ID×250mm, 5μm (Tosoh Corporation). The results showed a purity of 94.14-100%.

[0921] Radioactive TLC

[0922] The test substance was spotted onto TLC silica gel 60 RP-18 F. 254The radioactivity of the sample (Merck, 115389) was determined by radiochromatography using a 2 mol / L ammonium acetate / acetone mixture (1:1), followed by collimation (60-350 keV, 1.0 cm collimator, V-shaped BGO crystal, 10 min assay). The results showed a purity of 97.60-98.79%.

[0923] Preparation of evaluation animals (HT-29 tumor-bearing mice)

[0924] Five-week-old female BALB / cSlc-nu / nu mice were purchased from SLC (Shizuoka, Japan) for animal evaluation. After acclimatization, 0.1 mL of a suspension containing 5 × 10⁻⁶ mice was used. 7 A 1:2 mixture of PBS pH 7.4 (1×) (Thermo Fisher Scientific, 10010-023) and VitroGel (The WellBioscience, VHM01) containing CA9-positive human colon cancer-derived HT-29 cells / mL was transplanted subcutaneously into the right abdomen of nude mice (6 weeks old) using a disposable syringe with a needle.

[0925] The evaluation animals consisted of 3 animals per group, for a total of 12 groups. At the time of evaluation, the animals weighed 17.7g-21.5g and had tumor diameters ranging from 165-523mm. 3 .

[0926] In vivo distribution and PET imaging

[0927] Each test substance was administered intravenously in HT-29 tumor-bearing mice at a single dose of 80 μL / head. In vivo distribution was evaluated by dissection at 4 and 24 hours post-administration, with the radioactivity concentration (%ID / g) measured in each tissue. Additionally, PET / CT images were acquired using a Si78 (Bruker BioSpin MRI GmbH) under isoflurane gas anesthesia at 1, 4, and 23.5 hours post-administration.

[0928] result

[0929] Will 64 The results of the in vivo distribution of the Cu-labeled compound are shown in Figures 4-7 .

[0930] Figure 4 Tissue distribution %ID / g 4 hours after drug administration (mean ± SD, n = 3)

[0931] Figure 5 Tissue distribution %ID / g 24 hours after drug administration (mean ± SD, n = 3)

[0932] Figure 6 Tissue distribution %ID / g 4 hours after drug administration (mean ± SD, n = 3)

[0933] Figure 7 Tissue distribution %ID / g 24 hours after drug administration (mean ± SD, n = 3)

[0934] PET / CT imaging

[0935] exist 64 PET / CT images were collected at 1, 4, and 23.5 hours after administration of the Cu-labeled compound (4.62–4.72 MBq / head) under isoflurane gas anesthesia, as shown below. Figures 8-13 Aggregation towards tumors was confirmed in each compound. It should be noted that each column represents the same individual; for example, the left column shows results at 1 hour, 4 hours, and 23.5 hours after administration to the same individual. Additionally, white arrows indicate transplanted tumors.

[0936] Figure 8 : 64 Cu-labeled conjugate No. 64- 64 Cu

[0937] Figure 9 : 64 Cu-labeled conjugate No. 62- 64 Cu

[0938] Figure 10 : 64 Cu-labeled conjugate No. 2- 64 Cu

[0939] Figure 11 : 64 Cu-labeled conjugate No. 56- 64 Cu

[0940] Figure 12 : 64 Cu-labeled conjugate No. 58- 64 Cu

[0941] Example 6 177 Pharmacodynamic testing of Lu-labeled conjugates

[0942] For peptides 177 Lu mark

[0943] The labeled reaction solution (Table 11) containing each peptide of the conjugates Conjugate No. 64, Conjugate No. 62, and Conjugate No. 65 synthesized in Example 1 or 2 was heated at 95°C for 10 minutes. 177 Lu tag.

[0944]

[0945] Radioactive HPLC

[0946] The synthesized as described above 177 The Lu-labeled conjugate (hereinafter also referred to as the test substance) was mixed with acetonitrile, and the radiochemical purity before and after administration was determined using a TSK gel (registered trademark) ODS-80Ts QA, 4.6mm ID×250mm, 5μm (Tosoh Corporation). The results showed a purity of 90.31-100%.

[0947] Radioactive TLC

[0948] The test substance was spotted onto TLC silica gel 60 RP-18 F. 254 The radioactivity of the sample (Merck, 115389) was determined by radiochromatography using a 1:1 mixture of 2 mol / L ammonium acetate and acetone. The analysis was performed using a radiochromatogram (energy range 60-350 keV, collimator 1.0 cm, V-shaped BGO crystal, measurement time 10 minutes). The results showed a purity of 97.25-98.23%.

[0949] Preparation of cabozantinib

[0950] As a comparative example, cabozantinib was used. It is a well-known treatment for renal cell carcinoma and hepatocellular carcinoma, and its efficacy in colorectal cancer is also known. Cabozantinib (Selleckchem; No. S1119) was dissolved in DMSO to achieve a concentration of 100 mg / mL, aliquoted, and frozen. This was used as a cabozantinib DMSO solution. On the day of administration, the cabozantinib DMSO solution was thawed and added to water for injection to achieve a concentration of 30 mg / mL. This was used as a cabozantinib solution.

[0951] Evaluation of animal preparation

[0952] Five-week-old female BALB / cSlc-nu / nu mice were purchased from SLC (Shizuoka, Japan) for animal evaluation. After acclimatization, 0.1 mL of a suspension containing 5 × 10⁻⁶ mice was used. 7Human colon cancer-derived HT-29 cells / mL in PBS pH 7.4 (1×) (Thermo Fisher Scientific, 10010-023) and VitroGel (The Well Bioscience, VHM01) was transplanted subcutaneously into the right abdomen of nude mice (6 weeks old) using a disposable syringe with a needle.

[0953] Nine evaluation animals were used, with six animals per group. At the time of evaluation, the animals weighed between 16.28g and 20.05g, and had tumor diameters between 88 and 229mm. 3 .

[0954] Drug efficacy evaluation

[0955] On Days 0, 11, and 18, HT-29 tumor-bearing mice were intravenously administered saline (Saline) at 30 MBq / head or 60 MBq / head. 177 Lu-labeled peptide. Additionally, for the cabozantinib group, the dosage was calculated based on body weight data on the dosing day to reach 30 mg / kg, and this dosage was administered orally daily from day 0 to day 27. Body weight and tumor volume were measured twice weekly until day 49. On the final day of measurement, all surviving individuals were euthanized.

[0956] Tumor diameter results

[0957] The changes in tumor diameter in each group are shown in the figure. Figure 13 Conjugate No. 64- 177 Lu (30 MBq / head, 3 doses), Conjugate No. 62- 177 Lu (30 MBq / head, 3 doses), Conjugate No. 65- 177 Lu (60 MBq / head, single dose) showed excellent antitumor effects.

[0958] Weight results

[0959] The weight changes of each group are shown in the figure. Figure 14 Conjugate No. 64, which exhibited excellent anti-tumor effects, 177 Lu (30 MBq / head, 3 doses), Conjugate No. 62- 177 Lu (30 MBq / head, 3 doses) and Conjugate No. 65- 177No significant weight loss was observed in Lu (60 MBq / head, single dose).

[0960] Example 7 64 Cu or 177 In vivo distribution of Lu-labeled conjugates (VMRC-RCW xenograft model)

[0961] For peptides 64 Cu or 177 Lu's mark

[0962] Will contain Conjugate No. 62 64 Cu-labeled reaction solution (Table 12) or 177 The Lu-labeled reaction solution (Table 13) was heated at 95°C for 10 minutes to label peptides.

[0963] The composition of the labeled reaction solution (Conjugate No. 62-) 64 Cu)

[0964]

[0965] The composition of the labeled reaction solution (Conjugate No. 62-) 177 Lu)

[0966]

[0967] Radioactive HPLC

[0968] Conjugate No. 62 synthesized as described above 64 Cu or Conjugate No.62- 177 After Lu was mixed with acetonitrile, the radiochemical purity before and after administration was determined using a TSK gel (registered trademark) ODS-80Ts QA, 4.6mm ID×250mm, 5μm (Tosoh). The result was 100% purity.

[0969] Radioactive TLC

[0970] Each test substance (Conjugate No. 62-) was spotted. 64 Cu or Conjugate No.62- 177 Lu) TLC silicone 60 RP-18 F 254 The radioactivity of s (Merck, 115389) was determined by radiochromatography (10 minutes) after development with a 2 mol / L ammonium acetate / acetone mixture. The results showed a purity of 98.26–99.27%.

[0971] Preparation of evaluation animals (VMRC-RCW tumor-bearing mice)

[0972] Five-week-old female BALB / c-nu mice were purchased from Jackson Laboratory Japan (Kanagawa, Japan) for animal evaluation. After acclimatization, 0.1 mL of a suspension containing 5 × 10⁻⁶ mice was used. 7 EMEM (Fujifilm and Hikari Pure Chemicals, Osaka, Japan) of human renal cell carcinoma-derived VMRC-RCW cells per mL were transplanted subcutaneously into the right ventral region of nude mice (6 weeks old) using a disposable syringe with a needle.

[0973] The animals evaluated during use had a body weight of 18.2g–21g and a tumor diameter of 80–386mm. 3 .

[0974] Distribution in the body

[0975] Each test substance was administered intravenously as a single dose to VMRC-RCW tumor-bearing mice. Dissections were performed 4, 24, and 48 hours after administration, and the radioactivity concentration (%ID / g) of each tissue was measured.

[0976] result

[0977] Conjugate No.62- 64 The results of Cu's bulk distribution are shown in Figure 15 , Conjugate No.62- 177 The results of Lu's in vivo distribution are shown in Figure 16 .

[0978] PET / CT imaging

[0979] In Conjugate No.62- 64 PET / CT images were collected under isoflurane gas anesthesia at 1, 4, 23.5, and 47.5 hours after Cu administration, confirming the detection of tumors. Images at each time point are shown below. Figure 17 It should be noted that each column represents the same individual. For example, the left column represents the results of the same individual at 1 hour, 4 hours, 23.5 hours and 47.5 hours after drug administration.

[0980] Example 8 177 Pharmacodynamic assay of Lu-labeled peptides (VMRC-RCW xenograft model)

[0981] For peptides 177 Lu's mark

[0982] Preparation containing Conjugate No. 62 177 Lu-labeled reaction solution was administered at 30 MBq / head or 60 MBq / head (Table 14) and labeled by heating at 95°C for 10 minutes.

[0983] Conjugate No. 62- (labeled reaction solution) 177 Composition of Lu

[0984]

[0985] Radioactive HPLC

[0986] Conjugate No. 62 synthesized as described above 177 After Lu was mixed with acetonitrile, the radiochemical purity before and after administration was determined using a TSK gel (registered trademark) ODS-80Ts QA, 4.6mm ID×250mm, 5μm (Tosoh Corporation). The results showed a purity of 99.6%–100%.

[0987] Radioactive TLC

[0988] Conjugate No. 62 was sampled. 177 Lu's TLC Silicone 60 RP-18 F 254 The radioactivity of s (Merck, 115389) was determined by radiochromatography using a 1:1 mixture of 2 mol / L ammonium acetate and acetone, followed by a 13-250 keV energy range, 1.0 cm collimator, V-shaped BGO crystal, and a 10-minute measurement time. The results showed a purity of 98.1-98.6%.

[0989] Preparation of cabozantinib

[0990] As a comparative example, cabozantinib (Selleckchem; No. S1119) was dissolved in DMSO to achieve a concentration of 100 mg / mL, aliquoted, and frozen. This was used as a cabozantinib DMSO solution. On the day of administration, the cabozantinib DMSO solution was thawed and added to water for injection to achieve a dosing concentration of 12.3 mg / mL. This was used as a cabozantinib solution.

[0991] Preparation of evaluation animals (VMRC-RCW tumor-bearing mice)

[0992] Five-week-old female BALB / c-nu mice were purchased from Jackson Laboratory Japan (Kanagawa, Japan) for animal evaluation. After acclimatization, 0.1 mL of a suspension containing 5 × 10⁻⁶ mice was used. 7 Human renal cell carcinoma-derived VMRC-RCW cells per mL were transplanted subcutaneously into the right ventral region of nude mice (6 weeks old) using EMEM (Eagle Minimum Essential Culture Medium, Fujifilm and Hikari Pure Chemicals, Osaka, Japan) via a disposable syringe with a needle.

[0993] The animals evaluated during use weighed 19.3g to 21.9g, and the tumor diameter ranged from 216 to 445mm. 3 .

[0994] Drug efficacy evaluation

[0995] Physiological saline solution (SAline) and Conjugate No. 62 prepared as described above. 177 Lu, mice in each group (8 weeks old at Day 0) were administered a single dose (80 μL / head) via the tail vein according to Table 15 below.

[0996] Relationship between the drug solutions and administration dates for each group

[0997]

[0998] The dosage for the cabozantinib group was calculated based on body weight data on the dosing day to reach 12.3 mg / kg, and was administered orally daily from Day 0 to Day 27 at this dosage.

[0999] Tumor diameter results

[1000] The changes in tumor diameter in each group are shown in the figure. Figure 18 Conjugate No. 62- 177 Lu showed excellent anti-tumor effects.

[1001] Weight results

[1002] The average rate of change in body weight for each group is shown in the figure. Figure 19 After administering Conjugate No. 62- 177 No significant weight loss was identified in any of Lu's groups.

[1003] Example 9 225 Pharmacodynamics / In vivo distribution of Ac-labeled peptides

[1004] For peptides 225 Ac mark

[1005] The labeled reaction solution containing Conjugate No. 62 (Table 16) was heated at 95°C for 20 minutes for labeling, yielding... 225 Ac-labeled compounds.

[1006] Composition of the labeling reaction solution (50 μL)

[1007]

[1008] Radioactive HPLC

[1009] The test substance was mixed with acetonitrile, and its radiochemical purity before administration was determined using a TSK gel (registered trademark) ODS-80Ts QA, 4.6 mm ID × 150 mm, 5 μm (Tosoh Corporation). The result was a purity of 97.9%.

[1010] Radioactive TLC

[1011] The TLC silica gel 60 RP-18 F254s (Merck, 115389) sampled with the test substance was developed with a 2 mol / L ammonium acetate / acetone mixture (1:1), and the radioactivity was determined using a radiochromatogram (energy range 180–500 keV, collimator 1.0 cm, V-shaped BGO crystal, measurement time 10 min). The result showed a purity of 97.7%.

[1012] Preparation of evaluation animals (HT-29 tumor-bearing mice)

[1013] Six-week-old female BALB / cSlc-nu / nu mice were purchased from SLC (Shizuoka, Japan) for animal evaluation. After domestication, 0.1 mL of a suspension containing 5 × 10⁻⁶ mice was used. 7 Human colon cancer-derived HT-29 cells / mL in PBS pH 7.4 (1×) (Thermo Fisher Scientific, 10010-023) and VitroGel (The Well Bioscience, VHM01) was transplanted subcutaneously into the right abdomen of nude mice (7 weeks old) using a disposable syringe with a needle.

[1014] Three animals were used in each evaluation group, for a total of three groups. At the time of evaluation, the animals weighed between 17.8g and 19.63g, and the tumor diameter ranged from 151 to 275 mm. 3 .

[1015] Distribution in the body

[1016] A single intravenous administration was administered to HT-29 tumor-bearing mice (9 weeks old). 225 Ac-labeled compounds were used. Dissections were performed at 4, 24, and 48 hours after administration, and the radioactivity concentration (%ID / g) of each tissue was measured.

[1017] result

[1018] Will 225 The results of the in vivo distribution of the Ac-labeled compound are shown in Figure 20 .

[1019] Example 10 225 Pharmacodynamic testing of Ac-labeled peptides

[1020] For peptides 225 Ac mark

[1021] The labeled reaction solution containing Conjugate No. 62 (Table 17) was heated at 95°C for 20 minutes to label the test substance.

[1022] Composition of the test substance

[1023]

[1024] Radioactive HPLC

[1025] For the test substance, the radiochemical purity before administration was determined using a Jupiter (registered trademark) C18 5μm 4.6x150 mm (Phenomenex) instrument. The result was a purity of 97.25%.

[1026] Radioactive TLC

[1027] The test substance was spotted onto a TLC-SG paper (Agilent, SGI0001), developed with 25 mM EDTA and 0.1 mol / L ammonium acetate solution, and its radioactivity was determined using a Bioscan AR-2000 (Eckert & Ziegler). The result showed a purity of over 99%.

[1028] Preparation of evaluation animals (HT-29 tumor-bearing mice)

[1029] Five-week-old female BALB / cSlcnude mice were purchased from Charles River Laboratories (Wilmington, USA) for animal evaluation. After acclimatization, 0.1 mL of a suspension containing 5 × 10⁻⁶ mice was used. 7 Human colon cancer-derived HT-29 cells / mL in a 1:2 mixture of PBS pH 7.4 (1×) (Thermo Fisher Scientific, 10010-023) and VitroGel (The Well Bioscience, VHM01) was transplanted subcutaneously into the right abdomen of nude mice (7 weeks old) using a disposable syringe with a needle.

[1030] The animals were evaluated using 10 cases per group, for a total of 6 groups. 225 The animals evaluated the day before administration of the Ac-labeled peptide weighed 16.95 g to 17.83 g and had tumor diameters of 189.08 to 201.72 mm. 3 .

[1031] Drug efficacy evaluation

[1032] Ten days after HT-29 cell transplantation, PBS was administered intravenously to HT-29 tumor-bearing mice at doses of 30 kBq / head, 60 MBq / head, 90 MBq / head, 120 MBq / head, or 150 MBq / head. 225 Ac-labeled peptides. Body weight and tumor volume were measured twice a week until Day 70, at which point all surviving individuals were euthanized.

[1033] Tumor diameter results

[1034] Tumor diameter changes in each group are shown in Figure 21 . 225 Ac-labeled Conjugate No. 62 exhibited excellent radiodependent antitumor effects.

[1035] Weight results

[1036] The weight changes of each group are shown in the figure. Figure 22 Unconfirmed source 225 The administration of Ac-labeled Conjugate No. 62 resulted in significant weight loss.

[1037] Example 11 Synthesis of cyclic peptides and conjugates

[1038] Example 11-1 Synthesis of Conjugate No. 74

[1039] [Chemical Formula 23]

[1040]

[1041] The peptide conjugate Conjugate No. 62 (10 mg) obtained in Examples 1-5 was dissolved in 100 mM sodium acetate aqueous solution (0.465 mL), and lanthanum(III) chloride heptahydrate (25.9 mg, CAS: 10025-84-0) was dissolved. The reaction mixture was stirred at 45 °C for 60 minutes.

[1042] The resulting reaction mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C185μm 50x250mm; mobile phase: A = 0.1% TFA (water), B = 0.1% TFA (MeCN); temperature: 50℃; gradient (%B): 5 min 0-0%, 2 min 0-4.2%, 3 min 4.2-24.6%, 15.5 min 24.6-29.7%, 1.5 min 29.7-60%, 4 min 60-90%; flow rate: 5.0 min 18-18 mL / min, 2 min 18-118 mL / min, followed by 118 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[1043] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 96.5%.

[1044] Analysis condition A: Retention time = 3.54 minutes

[1045] ESI-MS(+) observation m / z = 1143.45 (M+2H) 2+

[1046] Example 11-2 Synthesis of Conjugate No. 75

[1047] [Chemical Formula 24]

[1048]

[1049] The peptide SEQ ID NO. 46 (10 mg) and DIEA (15.7 μL), DOTA-NHS ester hexafluorophosphate trifluoroacetate (5.04 mg) obtained in Examples 1-2 were dissolved in DMF (0.50 mL) at 0 °C. After stirring the reaction mixture at room temperature for 60 minutes, gallium(III) chloride (3.52 mg, CAS: 13450-90-3) and 50 mM sodium acetate aqueous solution (0.40 mL) were added to the reaction mixture. The reaction mixture was stirred at 90 °C for 1 hour.

[1050] The resulting reaction mixture was purified under the following conditions (column: Waters XBridge (registered trademark) C185μm 30x150mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 50℃; gradient (%B): 3 min 5.0-27%, 8 min 27-32%, 1 min 32-60%; flow rate: 45 mL / min). The fraction containing the target substance was collected and lyophilized to obtain the target peptide.

[1051] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 99.5%.

[1052] Analysis condition A: Retention time = 3.24 minutes

[1053] ESI-MS(+) observation m / z = 1108.94 (M+2H) 2+

[1054] Example 11-3 Synthesis of Conjugate No. 78

[1055] [Chemical Formula 25]

[1056]

[1057] The cyclic peptide (sequence: da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk(Alloc)-Y-MeA-MeG (SEQ ID NO. 103)) was synthesized using the same method as Conjugate No. 67. Fmoc-dk(Alloc)-OH was used instead of Fmoc-dk(Boc)-OH.

[1058] Purification was performed under the following conditions (column: Waters XBridge (registered trademark) C18 5μm 50x150mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 40℃; gradient (%B): 2 min 8-8%, 1 min 8-33%, 8 min 33-38%, 1 min 38-60%; flow rate: 1 min 20-20 mL / min, 1 min 20-120 mL / min, followed by 120 mL / min). The fraction containing the target substance was collected and lyophilized.

[1059] The cyclic peptide (38 mg) obtained by the above method was dissolved in DMF (0.65 mL), and a DMF solution of 2,4,6-trimethylpyridine (25.8 μL, CAS: 108-75-8) and 0.5 M HATU (58.7 μL) was added and stirred at 25 °C for 5 minutes. Fmoc-Qglucamine-NH2 (50 mg) was dissolved in DMSO (0.94 mL), and TEA (0.20 mL) was added. After stirring at 60 °C for 1 hour, the solution was concentrated under reduced pressure using a Biotage V-10 pump. Trituration was performed with methyl tert-butyl ether. A DMSO solution (0.50 mL) of the synthesized H-Qglucamine-NH2 (7.9 mg) was added to the reaction solution of the cyclic peptide, and the mixture was stirred for 2 hours. After adding water (50 μL), the solution was concentrated under reduced pressure using a Biotage V-10 pump. The resulting mixture was dissolved in DMF (0.67 mL), and phenylsilane (25 μL, CAS: 694-53-1) and Pd(PPh3)4 (4.6 mg, CAS: 14221-01-3) were added. The mixture was stirred at room temperature for 50 minutes. DIPEA (68 μL) and DOTA-NHS TFA salt (36 mg, CAS: 2832911-57-4) were added to the resulting mixture, and the mixture was stirred overnight at 25 °C. After adding water (100 μL), half of the resulting mixture was diluted in DMSO (1 mL) and water (1 mL), and then stirred with a 0.5 M lutetium(III) ammonium acetate aqueous solution (2.4 mL, pH 5.0) at 90 °C for 1 hour. The resulting suspension was concentrated under reduced pressure using a Biotage V-10 solution and then dissolved in DMSO / water (9 / 1, 4 mL).

[1060] The resulting aqueous solution was purified under the following conditions (column: Waters XSelect C18 5μm 50x250mm; mobile phase: A = 0.1% TFA (water), B = 0.1% TFA (MeCN); temperature: 50℃; gradient (%B): 5 min 0-0%, 2 min 0-4.2%, 3 min 4.2-19.5%, 15.5 min 19.5-24.6%, 1.5 min 24.6-60%; flow rate: 5 min 18-18 mL / min, 2 min 18-118 mL / min, followed by 118 mL / min). Fractions containing the target substance were collected and lyophilized.

[1061] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 97.7%.

[1062] Analysis condition B: Retention time = 5.72 minutes

[1063] ESI-MS(+) observation m / z = 865.85 (M+H) 3+

[1064] Examples 11-4 Synthesis of Conjugate No. 79

[1065] [Chemical Formula 26]

[1066]

[1067] The cyclic peptide (sequence: da-Tbg-3Py6CON-Hgl-Meda-Ahp-pHPeG-S-5Inda-HseMe-dk(Alloc)-Y-MeA-MeG (SEQ ID NO. 103)) was synthesized using the same method as Conjugate No. 67. Fmoc-dk(Alloc)-OH was used instead of Fmoc-dk(Boc)-OH.

[1068] Purification was performed under the following conditions (column: Waters XBridge (registered trademark) C18 5μm 50x150mm; mobile phase: A = 0.1% TFA (in water), B = 0.1% TFA (in MeCN); temperature: 40℃; gradient (%B): 2 min 8-8%, 1 min 8-33%, 8 min 33-38%, 1 min 38-60%; flow rate: 1 min 20-20 mL / min, 1 min 20-120 mL / min, followed by 120 mL / min). The fraction containing the target substance was collected and lyophilized.

[1069] The cyclic peptide (38 mg) obtained by the above method was dissolved in DMF (0.65 mL), and a DMF solution of 2,4,6-trimethylpyridine (25.8 μL, CAS: 108-75-8) and 0.5 M HATU (58.7 μL) was added and stirred at 25 °C for 5 minutes. A DMSO solution of H-KCOpipzaa(Mpe)-NH2 (10.2 mg) (0.50 mL) was added to the reaction solution of the cyclic peptide, and the mixture was stirred for 1.5 hours. After adding water (50 μL), the mixture was concentrated under reduced pressure using a Biotage V-10 pump. The resulting mixture was dissolved in TFA / TIS / H2O (95 / 2.5 / 2.5, 0.5 mL) and stirred for 30 minutes. The solid produced by adding diisopropyl ether / heptane (1 / 1, 5 mL) to the resulting mixture was centrifuged. The supernatant was removed, and the mixture was washed with diisopropyl ether / heptane (1 / 1, 5 mL). After drying under reduced pressure, the solution was dissolved in DMF (0.67 mL), and phenylsilane (25 μL, CAS: 694-53-1) and Pd(PPh3)4 (4.6 mg, CAS: 14221-01-3) were added. The mixture was stirred at room temperature for 50 minutes. DIPEA (68 μL) and DOTA-NHS TFA salt (36 mg, CAS: 2832911-57-4) were added to the resulting mixture, and the mixture was stirred overnight at 25 °C. After adding water (100 μL), half of the resulting mixture was diluted with DMSO (1 mL) and water (1 mL), and then stirred with 0.5 M lutetium(III) in ammonium acetate aqueous solution (2.4 mL, pH 5.0) at 90 °C for 1 hour. The obtained suspension was concentrated under reduced pressure using Biotage V-10 and then dissolved in DMSO / water (9 / 1, 4 mL).

[1070] The resulting aqueous solution was purified under the following conditions (column: Waters XSelect C18 5μm 50x250mm; mobile phase: A = 0.1% TFA (water), B = 0.1% TFA (MeCN); temperature: 50℃; gradient (%B): 5 min 0-0%, 2 min 0-4.2%, 3 min 4.2-19.5%, 15.5 min 19.5-24.6%, 1.5 min 24.6-60%; flow rate: 5 min 18-18 mL / min, 2 min 18-118 mL / min, followed by 118 mL / min). Fractions containing the target substance were collected and lyophilized.

[1071] The purity of the target substance was calculated based on the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under the analytical conditions. The purity of the target substance was 96.6%.

[1072] Analysis condition B: Retention time = 5.70 minutes

[1073] ESI-MS(+) observation m / z = 867.91 (M+H) 3+

[1074] Examples 11-5 Synthesis of Conjugates

[1075] The target peptides and their conjugates with payloads listed below were synthesized according to the general methods described in Examples 1 and 11-1, 2.

[1076]

[1077] Table 18 records the conjugates of the target substance, analytical conditions and retention times, and ESI-MS+ observations.

[1078] In Table 18, "terminus" indicates the C-terminal functional group (in the table, "-OH" represents COOH, and "-NH2" represents CO(NH2)), and the absence of a terminus indicates the absence of a C-terminal functional group. Regarding "Cyclization" in the table, "ClAc" indicates cyclization by binding the amino acid at position 1, which has a chloroacetyl group introduced, to a C-terminal C or MeC group; "Free" indicates that the N-terminal amino group of the amino acid at position 1 binds to the C-terminal carboxyl group of the amino acid at position 13 or 14. Furthermore, for example, the conjugate No. 75 indicates that the amino acid residues C of X1 and X13 form a cyclic structure, and the side chain of dk of X11 is further bound to Ga-bound DOTA as the payload. Similarly, the conjugate No. 76 described in Table 8 indicates that the amino acid residue MeG of X1 and X14 form a cyclic structure, and the side chain of dk of X11 is bound to Lu-bound DOTA as the payload.

[1079]

[1080]

[1081]

[1082]

[1083] Table 19 shows only the amino acid sequences of the peptides and peptide moieties contained in the peptides synthesized in Examples 2 and 11-3. Table 20 shows a comparison of the SEQ ID NO. of the amino acid sequences contained in the cyclic structure of the conjugates synthesized / evaluated in this example. It should be noted that in conjugates No. 2-5, 21, 22, 35, 38, and 49, they are respectively recorded as 4 (59), 4 (62), 76 (55), 46 (57), and 88 (86). This indicates that, for example, in conjugate No. 2, the amino acid sequence contained in the cyclic structure is the amino acid sequence recorded in SEQ ID No. 4, and the peptide-linker complex formed by binding the payload DOTA to its C-terminus (K, lysine) as a linker is the amino acid sequence shown in SEQ ID No. 59. Furthermore, in Conjugate No. 35, the amino acid sequence contained in the cyclic structure is the amino acid sequence described in SEQ ID No. 76, and the peptide-linker complex formed by binding the payload DOTALu and its associated linker F (phenylalanine) to the side chain of the 11th dk position of the amino acid sequence contained in the cyclic structure is the amino acid sequence shown in SEQ ID No. 56. Similarly, in Conjugate No. 38, the amino acid sequence contained in the cyclic structure is the amino acid sequence described in SEQ ID No. 46, and the peptide-linker complex formed by binding the payload SulfoCy5 and its associated linker PEG8c to the side chain of the 11th dk position of the amino acid sequence contained in the cyclic structure is the amino acid sequence shown in SEQ ID No. 57. That is, the numbers shown in parentheses in Table 20 indicate the amino acid sequence SEQ ID NO. when a linker is attached or an added amino acid is added.

[1084] For example, Conjugate No. 1 is a complex obtained by having the amino acid sequence shown in SEQ ID NO. 2 as a cyclic structural part and by further adding G, PEG10c, and K linkers at the C-terminus to bind the payload SulfoCy5. Here, whether it is only the cyclic amino acid sequence part as shown in Table 7, or the conjugate formed with a certain payload as shown in Table 8, all have the ability to bind CA9. In addition, there are several conjugates (Conjugate No. 14, 37, 55, 57, 64) with the amino acid shown in SEQ ID No. 45 as the cyclic structural part and only different payloads. Whether it is SEQ ID No. 45 without a payload, or various chelating agents such as DOTA, structures with metal ions bound to the chelating agent, or low molecular weight substances such as SulfoCy5, all have the ability to bind CA9. That is, it can be clearly stated that if a certain conjugate has the ability to bind CA9, a cyclic peptide having the amino acid sequence of the cyclic structure portion contained in the conjugate but not containing a payload, or a conjugate with a different payload, also has the ability to bind CA9.

[1085] Thus, the peptides of the present invention have CA9 binding ability regardless of the presence or type of payload.

[1086] Reference Example 3: Synthesis of Fmoc-Qglucamine-NH2

[1087] [Chemical Formula 27]

[1088]

[1089] A solution (3.3 mL) of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N5-((2S)-2-hydroxy-2-((4'R,5S)-2,2,2',2'-tetramethyl-[4,4'-bi(1,3-dioxocyclo)]-5-yl)ethyl)-L-glutamine (200 mg, CAS: 1227510-37-3) and ammonium chloride (34.9 mg, CAS: 12125-02-9) in DMF was mixed with DIPEA (0.14 mL) and HATU (0.19 mg) and stirred at 0 °C for 1 hour. Purification was performed using silica gel rapid column chromatography (DCM / MeOH = 100 / 0-80 / 20), and the fraction containing the target substance was recovered. A fraction (111 mg) of the obtained compound was dissolved in DCM (2.7 mL) and ethanol (1.8 mL), and stirred with TFA (1 mL) at 25 °C for one week. The resulting suspension was diluted with methyl tert-butyl ether and centrifuged. After removing the supernatant, the mixture was slurried with methyl tert-butyl ether. The title compound was obtained.

[1090] ESI-MS(+) observation m / z = 532.2 (M+H) +

[1091] Reference Example 4: Synthesis of H-KCOpipzaa(Mpe)-NH2

[1092] [Chemical Formula 28]

[1093]

[1094] A solution (3.2 mL) of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(4-(2-(((3-methylpentan-3-yl)oxy)-2-oxoethyl)piperazine-1-carbonyl)-L-lysine (200 mg, CAS: 2973753-38-5) and ammonium chloride (34.4 mg, CAS: 12125-02-9) in DMF was added with DIPEA (0.14 mL) and HATU (0.18 mg), and the mixture was stirred at 25 °C for 2 hours. Purification was performed by silica gel rapid column chromatography (DCM / MeOH = 100 / 0-90 / 10), and the fraction containing the target substance was recovered. A portion (100 mg) of the obtained compound was dissolved in DMSO (1.6 mL), and TEA (0.34 mL) was added. The mixture was stirred at 60 °C for 1 hour. The compound was concentrated under reduced pressure using a Biotage V-10 pump and then pulped with methyl tert-butyl ether. The title compound was obtained.

[1095] ESI-MS(+) observation m / z = 400.3 (M+H) +

[1096] Industrial applicability

[1097] The peptides and conjugates of the present invention have CA9 binding activity and can be used as pharmaceutical compositions, diagnostic compositions, and research compositions for the prevention or treatment of CA9-related symptoms.

Claims

1. A peptide comprising the following amino acid sequence: da-Tbg-3Py6CON-Hgl-Meda-Ahp-PeG-S-3Py6NH2-HseMe-dk-Y (SEQ ID NO. 1); or The amino acid sequence having substitutions, additions, deletions, or insertions in 1-10 amino acid residues selected from the 1st, 2nd, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, and 11th amino acid residues.

2. The peptide according to claim 1, wherein, (1) In the amino acid sequence of SEQ ID NO. 1, MeC, C, or MeCt is added as the 13th amino acid residue; or (2) In the amino acid sequence of SEQ ID NO. 1, MeA, MeS, MeN, Hpr, G or P are added as the 13th amino acid residue, and MeG, G, da or Meda are added as the 14th amino acid residue.

3. The peptide according to claim 1 or 2, comprising: The amino acid sequence of SEQ ID NO. 1 has 1-7 amino acid residues selected from the 1st, 4th, 7th, 8th, 9th, 10th and 11th positions, which have substitution, addition, deletion or insertion of amino acid residues.

4. The peptide according to any one of claims 1 to 3, wherein it comprises one or more of the following elements: The amino acid residue at position 1 is da, dq, de, dhgl, or dk; The second amino acid residue is I or Tbg; The third amino acid residue is 3Py6NH2 or 3Py6CON; The fourth amino acid residue is S, Hgl, Hgl(PEG8Me), alT, Hgn, SMe, KCOpipzaa, or DabAc; The fifth amino acid residue is PeG, MeG, Meda, or MedkCOpipzaa; The amino acid residue at position 7 is PeG, pMeOPeG, MsMeapG, 3OMePeG, pHPeG, PpG, pFPeG, mCPeG, pCPeG, 4HPpG, 4OMePpG, 4PypG, 4COOPeG, 3COOPeG, or 3FPeG. The amino acid residue at position 8 is H, S, P, D, KCOpipzaa, Q (PEG8Me), P4Sh, Hse, SMe, Qmm, or Qdm; The amino acid residue at position 9 is 3Py6NH2, 5Inda, Y, or F4OMe; The 10th amino acid residue is Ahp, HseMe, or E; and The 11th amino acid residue is dk, G, da, dq, dkCOpipzaa, dk(t4amCh), de, Acb, or dkAc.

5. The peptide according to any one of claims 1 to 4, wherein it comprises one or more of the following elements: The second amino acid residue is Tbg; The third amino acid residue is 3Py6NH2; The fifth amino acid residue is Meda; The amino acid residue at position 7 is either PeG or pHPeg; The 9th amino acid residue is 3Py6NH2 or 5Inda; and The 10th amino acid residue is HseMe.

6. The peptide according to any one of claims 1 to 4, wherein, The second amino acid residue is Tbg. The third amino acid residue is 3Py6NH2. The fifth amino acid residue is Meda. The amino acid residue at position 7 is either PeG or pHPeg. The 9th amino acid residue is 3Py6NH2 or 5Inda, and The 10th amino acid residue is HseMe.

7. The peptide according to any one of claims 1 to 6, comprising or consisting of the amino acid sequence described in any one of SEQ ID NO. 2-54.

8. The peptide according to any one of claims 1 to 7, further comprising added amino acid residues.

9. The peptide according to any one of claims 1 to 8, wherein, The peptide is a cyclic peptide.

10. The peptide according to any one of claims 1 to 9, comprising: A cyclic structure formed by the first amino acid residue of the chloroacetylated amino acid sequence of SEQ ID NO. 1 and the cysteine ​​residue contained in the peptide.

11. The peptide according to any one of claims 1 to 9, comprising: The peptide contains a cyclic structure formed by the amino group of the first amino acid residue of SEQ ID NO. 1 and the carboxyl group of the 14th amino acid residue.

12. The peptide according to any one of claims 1 to 11, wherein, (a) The peptide is capable of binding a linker and / or payload at its C-terminus; or (b) The amino acid residues at positions 1, 4, 8, 11 or 13 of the amino acid sequence of SEQ ID NO. 1 are amino acid residues capable of binding linkers and / or payloads.

13. A conjugate comprising the peptide and payload as described in any one of claims 1 to 12, wherein, (i) The amino acid residue at position 13 of SEQ ID NO. 1 is bound to any payload, with or without a linker; or (ii) As shown in formula (I) below, the amino acid residue at position 1, 4, 8, or 11 of SEQ ID NO. 1 is bound with a payload. [Chemical Formula 1] In formula (I), R1 is H or C1-3 alkyl; R2 is C1-6 alkyl-NH-, C1-6 alkyl-C6 aryl-O-C1-3 alkyl-NH-, C1-6 alkyl-NH(=O)-CH(C1-6 alkylphenyl)-NH-, C1-6 alkyl-NH(=O)-CH(C1-6 alkyl)-NH-, or C1-6 alkyl-NH(=O)-C3-8 cycloalkyl-C1-3 alkyl-NH-; X is any payload.

14. The conjugate according to claim 13, wherein, R2 is a C1-6 alkyl-NH-.

15. The conjugate according to claim 13 or 14, wherein, X is a chelating agent.

16. The conjugate according to claim 15, wherein, The chelating agent binds to the radioactive material.

17. The conjugate according to claim 15 or 16, wherein, The chelating agent is DOTA, DOTAGA, or NODAGA.

18. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 12.

19. A pharmaceutical composition for the prevention or treatment of CA9-related diseases, comprising any one of claims 1 to 12.

20. A pharmaceutical composition comprising the conjugate according to any one of claims 13 to 17.

21. A pharmaceutical composition for the prevention or treatment of CA9-related diseases, comprising the conjugate according to any one of claims 13 to 17.

22. A composition for diagnostic or research use, comprising the peptide according to any one of claims 1 to 12.

23. A composition for diagnosis or research of CA9-related diseases, comprising the peptide according to any one of claims 1 to 12.

24. A composition for diagnostic or research use, comprising the conjugate according to any one of claims 13 to 17.

25. A composition for diagnosis or research of CA9-related diseases, comprising the conjugate according to any one of claims 13 to 17.

26. An imaging agent comprising the conjugate according to any one of claims 13 to 17.

27. An imaging agent for tumor diagnosis, comprising the conjugate according to any one of claims 13 to 17.

28. A radioactive ligand imaging agent for positron emission tomography, comprising any one of claims 13 to 17.

29. A method for testing peptides, wherein at least one of the following (a) to (d) is used for peptide testing: a) Solubility in solvents b) CA9 binding activity, c) Toxicity to cells and / or tissues, or d) Toxicity to laboratory animals, The peptide is any one of claims 1 to 12.

30. A method for testing conjugates, wherein the conjugates are tested for at least one of the following a) to d): a) Solubility in solvents b) CA9 binding activity, c) Toxicity to cells and / or tissues, or d) Toxicity to laboratory animals, The conjugate is any one of claims 13 to 17.

31. The peptide according to any one of claims 1 to 3, wherein it comprises one or more of the following elements: The amino acid residue at position 1 is de(PEG8Me), dk, dyae, or dkCOpipzaa; The second amino acid residue is I or Tbg; The third amino acid residue is 3Py6NH2 or 3Py6CON; The fourth amino acid residue is K, KCOpipzaa, Hgn(Qglucamine-NH2), Hgn(KCOpipzaa-NH2), or Qglucamine; The fifth amino acid residue is PeG, MeG, Meda, or MedkCOpipzaa; The amino acid residue at position 7 is PeG, pMeOPeG, MsMeapG, 3OMePeG, pHPeG, PpG, pFPeG, mCPeG, pCPeG, 4HPpG, 4OMePpG, 4PypG, 4COOPeG, 3COOPeG, or 3FPeG. The 8th amino acid residue is K; The amino acid residue at position 9 is 3Py6NH2, 5Inda, Y, or F4OMe; The 10th amino acid residue is Ahp, HseMe, or E; and The amino acid residue at position 11 is either dk(F) or dk(PEG8c).

32. The peptide according to any one of claims 1 to 6, comprising or consisting of the amino acid sequence described in any one of SEQ ID NO. 55-103.

33. A conjugate comprising the peptide of claim 32 and a payload, wherein, The amino acid residues at positions 1, 4, 8, 11, or 13 may be linked with an arbitrary payload, with or without a linker.

34. A conjugate, which is any of the conjugate numbers 1-81 listed in Table 8 or Table 18.

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