Peptide of targeted somatostatin receptor, coupling medicine and application of peptide and coupling medicine

Abstract

The invention provides a peptide targeting at SSTR2, a peptide coupling medicine targeting at SSTR2 and application of the peptide coupling medicine. The peptide / peptide coupling medicine can be used for diagnosing, preventing, inhibiting or treating cancers, especially solid tumors derived from neuroendocrine cells, such as neuroendocrine tumors of digestive systems of stomach, intestine, pancreas and the like and small cell lung cancer.

Description

A peptide targeting somatostatin receptor, a conjugated drug and its application Technical Field

[0001] The present invention relates to a novel polypeptide targeting SSTR2, and in particular to a peptide-drug conjugate (PDC) comprising a peptide coupled to one or more effectors and / or functional groups, a pharmaceutical composition comprising the peptide ligand and the drug conjugate, and uses of the peptide ligand or drug conjugate in preventing, inhibiting or treating diseases with high SSTR2 expression or in related pharmaceutical preparations. Background Art

[0002] Somatostatin receptors (SSTRs) are G protein-coupled receptors (GPCRs) that specifically bind to somatostatin on the cell membrane and belong to the GPCR family. SSTRs have five distinct molecular subtypes, SSTR1-SSTR5. SSTR2 has two distinct isoforms (subunits), SSTR2A and SSTR2B. Based on the degree of amino acid sequence homology and the similarity in selectivity and functional responses to their ligands, the SSTR family is divided into two major subfamilies: SSTR1 and SSTR4, and SSTR2, SSTR3, and SSTR5.

[0003] At the 19th European Society of Neuroendocrine Tumors Annual Meeting in 2022, research on the somatostatin receptor (SSTR) became a hot topic. The high heterogeneity and rarity of NETs pose challenges to precision cancer treatment. In recent years, the SSTR family of targets has been recognized as the most important targets in NET treatment and diagnosis.

[0004] The most widely studied subtype of the somatostatin receptor (SSTR) family is the somatostatin receptor subtype SSTR2. Clinical reports indicate that SSTR expression is found in over 70% of both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCL), with SSTR2 being the predominant expression in SCLC and squamous cell carcinoma. Extensive data demonstrate that SSTR2 is specifically expressed in neuroendocrine tumors.

[0005] Reverse transcription-PCR was used to detect the expression of somatostatin receptor subtypes SSTR2 and SSTR3 mRNA in the cancerous and adjacent tissues of 27 patients with primary liver cancer. Results: The positive rates of SSTR2 mRNA expression in the cancerous and adjacent tissues of the 27 patients with primary liver cancer were 81.5% (22 / 27) and 96.3% (26 / 27), respectively. The positive rates of SSTR3 mRNA expression in the cancerous tissues were 66.7% (18 / 27) and 51.9% (14 / 27) in the adjacent tissues. Most primary liver cancer tissues express more than one somatostatin receptor subtype gene.

[0006] The expression of SSTRs in the human nasopharyngeal carcinoma cell line CNE2 was detected by combined RT-PCR and SP immunohistochemistry. The PCR mRNA products were sequenced for identification. Results: RT-PCR indicated that the human nasopharyngeal carcinoma cell line CNE2 expressed the somatostatin receptor subtypes SSTR1, SSTR2, and SSTR4. Immunohistochemistry revealed that the human nasopharyngeal carcinoma cell line CNE2 was strongly positive for SSTR1 and SSTR2A (60%), weakly positive for SSTR4, intermediate for SSTR2, and absent for SSTR3 and SSTR5. Conclusion: Our study confirmed that the human nasopharyngeal carcinoma cell line CNE2 expresses multiple somatostatin receptor subtypes, with SSTR1 and SSTR2 being the most abundant.

[0007] This suggests that SSTR2 plays a key targeting role in NET treatment, providing a better strategy for NET treatment. Therefore, research on drugs targeting SSTR2 is crucial for the diagnosis, prevention, inhibition, or treatment of diseases with high SSTR2 expression.

[0008] Summary of the Invention

[0009] The present application provides a peptide targeting SSTR2, or a peptide specific for SSTR2; the present application also provides a drug conjugate of the peptide and applications thereof.

[0010] In a first aspect, the present application provides a peptide targeting SSTR2, which has the following structure: Cys-Xaa-Trp-Lys-Thr-Cys, wherein a disulfide bond is formed between two Cys groups.

[0011] In a preferred embodiment, Xaa is selected from one or more of Tyr and Phe.

[0012] In a preferred embodiment, Trp is D-Trp.

[0013] In a preferred embodiment, the peptide targeting SSTR2 has the following sequence: Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH; wherein the two Cys groups within the square brackets [] form a disulfide bond; X1 and X2 can be any amino acid residues; and X3 can be any amino acid residue or not exist.

[0014] Among them, X1 is preferably Arg or Leu, X2 is preferably Trp or Asp, and X3 is preferably Asp.

[0015] In a preferred embodiment, the peptide targeting SSTR2 has the following sequence: X0-Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH; wherein a disulfide bond is formed between the two Cys groups in the square brackets [].

[0016] Wherein, X0 can be an amino acid residue with a group capable of reacting with an amino group, or other carboxyl-containing groups. The group capable of reacting with an amino group is preferably a carboxyl group. Examples of X0 include Arg or Gly.

[0017] A second aspect of the present application provides a peptide ligand targeting SSTR2, or a drug targeting SSTR2 containing the peptide ligand, comprising the peptide and a spacer connected to the peptide, wherein the spacer has the following structure: X4-X6-X5, wherein X4 may be absent or a single bond, an acetyl group, or a polyether structure connecting the peptide; X5 is a polyether structure, such as a polyethylene glycol structure having 2-12 repeating units; X6 may be selected from amino acid residues containing active groups, such as carboxyl, hydroxyl, amino, thiol, or aldehyde groups; preferably, X6 may be selected from cysteine, lysine, ornithine, glutamic acid, aspartic acid, 6-azido-L-norleucine, 2-amino-5-hydroxyvaleric acid, 2-amino-5-ureidopentanoic acid, or 2-amino-5-cyanobenzoic acid. For example, the spacer is X4-Cys-X5.

[0018] The spacer may further contain other amino acid residues. For example, the spacer may have the following structure: X4'-X6-{Lys(X4-X6'-X5)} x -AA y -X5'. AA is a random sequence of amino acid residues, y is an integer of 0-4, and x is an integer of 0-3, wherein X4' and X4 may be the same or different and may be absent or independently represent a single bond, an acetyl group, or a polyether structure connecting the peptide, such as a polyethylene glycol structure having 2-12 repeating units; X5' and X5 may be the same or different and may be independently represent a polyether structure, such as a polyethylene glycol structure having 2-12 repeating units; X6 and X6' may be the same or different and may be independently selected from amino acid residues containing active groups, such as carboxyl, hydroxyl, amino, thiol, aldehyde, etc. Preferably, X6 and X6' may be independently selected from cysteine ​​residues, lysine residues, ornithine residues, glutamic acid residues, aspartic acid residues, 6-azido-L-norleucine, 2-amino-5-hydroxyvaleric acid residues, 2-amino-5-ureidopentanoic acid residues, 2-amino-5-cyanobenzoic acid residues, etc.

[0019] The peptide ligand or drug described in the present application further comprises a linker for connecting to an active drug, wherein the linker is connected to X6 of the spacer and / or to X6'.

[0020] As a preferred example, the Cys-Xaa-Trp-Lys-Thr-Cys is selected from: Cys-Tyr-Trp-Lys-Thr-Cys or Cys-Phe-Trp-Lys-Thr-Cys.

[0021] As a preferred example, the peptide targeting SSTR2 is selected from the following sequences:

[0022] SEQ ID NO.1: H-Arg-Arg-Leu-Phe-[Cys-Tyr-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0023] SEQ ID NO.2: H-Arg-Arg-Leu-Phe-[Cys-Phe-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0024] SEQ ID NO.3: H-Gly-Arg-Arg-Phe-[Cys-Tyr-Trp-Lys-Thr-Cys]-Thr-Asp-Arg-NH2

[0025] SEQ ID NO.4: H-Arg-Arg-Leu-Phe-[Cys-Tyr-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-OH

[0026] As a preferred example, the spacer is selected from: X6-(AEEA) r PEG z -X6-PEG z 、Ac-X6-PEG z 、Ac-X6-(AEEA) r 、Ac-X6-PEG z -{Lys(Ac-X6'-PEG6)} x -PEG6, X6-Glu-Glu-Glu-AEEA, wherein r is an integer, such as an integer of 0-6, more preferably an integer of 1-5, such as 2, 3, 4, etc.

[0027] As a preferred example, the linker can be MC-Val-Ala-PABC, MC-Val-Cit-PABC, MC-Gly-Gly-Phe-Gly-NH-CH2, MCC, MC-betaglucuronide, MC-Glu-Val-Ala-PABC, MC-Glu-Val-Cit-PABC, etc., wherein,

[0028] MC is Maleimidocaproyl,

[0029] PABC is p-aminobenzyl alcohol,

[0030] Cit is citrulline,

[0031] MCC is 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic acid

[0032] The peptide-conjugated drug targeting SSTR2 described in the present application also includes an active drug (also called an effector), which is connected to a linker.

[0033] The drug can be connected to the linker by condensation, esterification, etherification, amidation, etc.

[0034] As a preferred example, the active drug can be a cytotoxic agent selected from: microtubule inhibitors (calendula, maytansine derivatives, tubulysin, cryptocolistin, etc.), DNA damaging drugs (pyrrole benzazepines and indolechlorobenzazepines, dukamycin, camptothecin, calicheamicin, etc.), amatoxin, apoptosis inducers, camptothecins, etc.

[0035] As a more preferred example, the cytotoxic agent is selected from MMAF, MMAE, PBD, DM1, DM4, Dxd, SN38, paclitaxel, docetaxel, exotecan, etc.

[0036] However, it should be noted that the peptides described above in this application may also be in the reverse-inverse form of the aforementioned sequences, such as by swapping the N-terminal and C-terminal positions and the corresponding amino acid sequences. Similarly, reverse-inverse forms of the amino acid stereochemical structures are also included, such as by swapping D-amino acids with L-amino acids. According to the disclosure of Nair et al. (J Immunol, 2003 170(3), pp. 1362-1373), the aforementioned reverse-inverse forms have the same or similar technical effects and are therefore encompassed within the scope of the peptides described herein.

[0037] However, it should be noted that the peptides or peptide ligands mentioned above in this application may also be peptides or peptide ligands labeled with any pharmaceutically acceptable (radioactive) isotope or labeling agent, or the active drug is an isotope or labeling agent. For example, one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number usually present in nature, or the active drug is a metal complex group capable of carrying a (radioactive) isotope or labeling agent, or certain functional groups in the peptide or peptide ligand are covalently replaced by functional groups labeled with a related (radioactive) isotope or labeling agent.

[0038] Examples of isotopes suitable for inclusion in the present invention include hydrogen isotopes such as 2 H and 3 H, carbon isotopes such as 11 C. 13 C and 14 C, chlorine isotopes such as 36 Cl, fluorine isotopes such as 18 F, iodine isotopes such as 123 I. 125 I and 131 I, nitrogen isotopes such as 13 N and 15 N, oxygen isotopes such as 15 O. 17 O and 18 O, phosphorus isotopes such as 32 P, sulfur isotopes such as 35 S, copper isotopes such as 64 Cu, gallium isotopes such as 67 Ga or 68 Ga, yttrium isotopes such as 90 Y, and lutetium isotopes such as 177 Lu, and bismuth isotopes such as 213 Bi.

[0039] The labeling agent suitable for inclusion in the present invention may be, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance (eg, luminol, luminol derivatives, luciferin, aequorin, and luciferase), and the like.

[0040] As a preferred example, the peptide-coupled drug targeting SSTR2 is selected from:

[0041] Drug 1: Cys(MC-Val-Cit-PABC-MMAE)-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0042] Drug 2: PEG6-Cys(MC-Val-Cit-PABC-MMAE)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0043] Drug 3: Ac-Cys(MC-Val-Cit-PABC-MMAE)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0044] Drug 4: Ac-Cys(MC-betaglucuronide-MMAE)-AEEA-AEEA-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0045] Drug 5: PEG6-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0046] Drug 6: Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0047] Drug 7: Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Lys(Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6)-Lys(Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0048] Drug 8: Ac-Cys(MC-Val-Cit-PABC-MMAE)-AEEA-AEEA-AEEA-Arg-Arg-Leu-Phe-[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0049] Drug 9: Cys(MC-Val-Cit-PABC-MMAE)-Glu-Glu-Glu-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0050] Drug 10: Ac-Cys(MC-Val-Cit-PABC-MMAE)-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0051] The third aspect of the present application is to provide a use of the above-mentioned peptide or peptide ligand or drug targeting SSTR2 for preventing, inhibiting or treating diseases with high SSTR2 expression; or for preparing a drug for treating, inhibiting or preventing diseases with high SSTR2 expression.

[0052] The peptide or peptide ligand or drug is used for diagnosing, preventing, inhibiting or treating cancer, or for preparing a drug for diagnosing, preventing, inhibiting or treating cancer.

[0053] Among them, the cancer is preferably a solid tumor originating from neuroendocrine cells, such as neuroendocrine tumors of the digestive system such as the stomach, intestine, and pancreas, and small cell lung cancer.

[0054] The peptides, peptide ligands, and related drugs provided by the present invention target SSTR2 and can be used to diagnose, prevent, inhibit, or treat cancer, particularly solid tumors originating from neuroendocrine cells, such as gastric, intestinal, and pancreatic neuroendocrine tumors of the digestive system and small cell lung cancer. DETAILED DESCRIPTION

[0055] The present application provides a peptide and a peptide ligand targeting SSTR2, and further provides a peptide-conjugated drug targeting SSTR2, which is used for diagnosing, preventing, inhibiting or treating diseases with high expression of SSTR2.

[0056] In one embodiment, the peptide contains the following sequence: Cys-Tyr / Phe-Trp-Lys-Thr-Cys, wherein preferably, a disulfide bond is formed between the two Cys.

[0057] In a further preferred embodiment, the peptide comprises the following sequence: Arg-X1-Phe-[Cys-Tyr / Phe-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH

[0058] Alternatively, the peptide has the following sequence: X0-Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH

[0059] Among them, a disulfide bond is formed between the two Cys groups in the square brackets [].

[0060] Wherein, X1 and X2 are any amino acid residues, X1 is such as Arg or Leu, X2 is such as Trp or Asp; X3 is any amino acid residue or none, such as Asp;

[0061] Wherein, X0 can be an amino acid residue with a group capable of reacting with an amino group, or other carboxyl-containing groups. The group capable of reacting with an amino group is preferably a carboxyl group. Examples of X0 include Arg or Gly.

[0062] Specifically, the peptides described in this application are selected from:

[0063] SEQ ID NO.1: H-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0064] SEQ ID NO.2: H-Arg-Arg-Leu-Phe-[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0065] SEQ ID NO.3: H-Gly-Arg-Arg-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Asp-Arg-NH2

[0066] SEQ ID NO.4: H-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-OH

[0067] In one embodiment, the peptide ligand further comprises a spacer and may further comprise a linker.

[0068] spacer

[0069] The functionalized spacer of the present invention is located at the N-terminus of the peptide and added to the left side of the peptide sequence. In one embodiment, the functionalized spacer has the general formula X4-X6-X5, where X4 can be polyethylene glycol or an acetyl group, preferably with 2-12 repeating units, or none; X5 can be polyethylene glycol, preferably with 2-12 repeating units. X6 is an amino acid residue containing an active group, which can be a hydroxyl group, a carboxyl group, an amino group, a sulfhydryl group, or the like. For example, X6 can be selected from cysteine ​​residues, lysine residues, ornithine residues, glutamic acid residues, aspartic acid residues, 6-azido-L-norleucine, 2-amino-5-hydroxyvaleric acid residues, 2-amino-5-ureidopentanoic acid residues, 2-amino-5-cyanobenzoic acid residues, and the like.

[0070] Alternatively, the spacer can be X4'-X6-{Lys(X4-X6'-X5)} x -AA y -X5', AA is a random sequence of amino acid residues, y is an integer from 0 to 4, and x is an integer from 0 to 3, wherein X4' and X4 may be the same or different and may each independently be absent or represent a single bond, an acetyl group, or a polyether structure connecting the peptide, such as a polyethylene glycol structure having 2 to 12 repeating units; X5' and X5 may be the same or different and may each independently represent a polyether structure, such as a polyethylene glycol structure having 2 to 12 repeating units; X6 and X6' are each an amino acid residue containing an active group, such as a hydroxyl group, a carboxyl group, an amino group, a thiol group, or the like; for example, X6 and X6' may each independently be selected from a cysteine ​​residue, a lysine residue, an ornithine residue, a glutamic acid residue, an aspartic acid residue, a 6-azido-L-norleucine residue, a 2-amino-5-hydroxyvaleric acid residue, a 2-amino-5-ureidopentanoic acid residue, a 2-amino-5-cyanobenzoic acid residue, or the like.

[0071] For example, the spacer may be preferably selected from: Ac-Cys-(AEEA) r PEG z -Cys-PEG z , Ac-Cys-PEG z 、Ac-Cys-(AEEA) r , Ac-Cys-PEG6-{Lys(Ac-Cys-PEG6)} x -PEG6, Ac-Cys-Glu-Glu-Glu-AEEA, Ac-Lys-(AEEA) r PEG z -Lys-PEG z 、Ac-Lys-PEG z 、Ac-Lys-(AEEA) r, Ac-Lys-PEG6-{Lys(Ac-Cys-PEG6)} x -PEG6, Ac-Cys-PEG6-{Lys(Ac-Lys-PEG6)} x -PEG6, Ac-Lys-Glu-Glu-Glu-AEEA, Ac-Clu-(AEEA) r PEG z -Glu-PEG z 、Ac-Clu-PEG z 、Ac-Clu-(AEEA) r , Ac-Clu-PEG6-{Lys(Ac-Org-PEG6)} x -PEG6, Ac-Asn-Glu-Glu-Glu-AEEA, Ac-Org-PEG z PEG z -Asn-PEG z .

[0072] Linker

[0073] One end of the linker is connected to a functional spacer (connected to X6 and / or X6' of the spacer) via a maleimidopropionic acid linker. In one embodiment, the linker is MC-Val-Ala-PABC, MC-Val-Cit-PABC, MC-Gly-Gly-Phe-Gly-NH-CH2, MCC, MC-betaglucuronide, MC-Glu-Val-Ala-PABC, MC-Glu-Val-Cit-PABC, etc.

[0074] In one embodiment, the peptide-conjugated drug further comprises an active drug (also called an effector) based on the peptide ligand, and the other end of the linker is connected to the effector.

[0075] effector

[0076] The effector is coupled to the linker through amide condensation, esterification reaction, etc. In one embodiment, the effector is a cytotoxic agent, such as a microtubule inhibitor (calendula, maytansine derivatives, tubulysin, cryptocolistin, etc.), a DNA damaging drug (pyrrole benzazepines and indolechlorobenzazepines, dukamycin, camptothecin, calicheamicin, etc.), amatoxin, apoptosis inducers, camptothecins, etc.

[0077] In a further embodiment of the present invention, the cytotoxic agent used is MMAF, MMAE, PBD, DM1, DM4, Dxd, SN38, paclitaxel, docetaxel, exitecan, etc.

[0078] In one embodiment, the peptide, peptide ligand or peptide-conjugated drug of the present application may also contain a pharmaceutically acceptable (radioactive) isotope label or labeling agent.

[0079] mark

[0080] One or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, or certain functional groups are covalently substituted by related (radioactive) isotopes or isotope-labeled functional groups, or the active drug is a metal conjugate group capable of carrying a related (radioactive) isotope, or the peptide / peptide ligand is linked to a metal conjugate group capable of carrying a related (radioactive) isotope.

[0081] Examples of suitable isotopes for inclusion in the peptide ligands of the present invention include: hydrogen isotopes such as 2 H and 3 H, carbon isotopes such as ll C. 13 C and 14 C, chlorine isotopes such as 36 Cl, fluorine isotopes such as 18 F, iodine isotopes such as 123 I. 125 I and 131 I, nitrogen isotopes such as 13 N and 15 N, oxygen isotopes such as 15 O. 17 O and 18 O, phosphorus isotopes such as 32 P, sulfur isotopes such as 35 S, copper isotopes such as 64 Cu, gallium isotopes such as 67 Ga or 68 Ga, yttrium isotopes such as 90 Y, and lutetium isotopes such as 177 Lu, and bismuth isotopes such as 213 Bi.

[0082] Certain isotope-labeled peptides / peptide ligands / peptide-coupled drugs can be used for tissue distribution studies of drugs and / or substrates, as well as for clinically evaluating the presence and / or absence of SSTR2 targets in diseased tissues. The present application can further be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes or receptors. The detection or identification method can use compounds labeled with labeling agents, such as radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, fluorescein, aequorin and luciferase), etc. The radioisotope tritium (i.e. 3 H) and carbon-14 (i.e. 14C), are particularly useful for this detection and identification purpose.

[0083] If heavier isotopes such as deuterium (i.e. 2 Deuterium substitution may be preferred in some circumstances because it may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0084] If a positron-emitting isotope such as ll C. 18 F. 15 O and 13 N substitution can be used in positron emission tomography (PET) studies to examine target occupancy.

[0085] The labeling of the peptides / peptide ligands / peptide-conjugated drugs of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the accompanying examples, using appropriate isotope-labeled reagents or labeling agents instead of the non-labeled reagents previously used.

[0086] Example 1

[0087] Synthesis of Cys(MC-Val-Cit-PABC-MMAE)-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0088] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling agents were DIC and HOBt, and deprotection was achieved using 20% ​​piperidine in DMF.

[0089] Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent. Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were sequentially condensed using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent.

[0090] 5 times the amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction was completed, VDMF :V ACN :V H2O =90:5:5, and then washed with DMF three times.

[0091] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, sequentially condense Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, AEEA, and Fmoc-Cys(Trt)-OH. Finally, acetylate with 5-20 equivalents of acetic anhydride / N-methylmorpholine for 30-60 minutes. To start, dissolve the amino acid and HOBt in DMF and add DIC dropwise for activation. The coupling reaction typically takes 45 minutes, and the deprotection time is 20 minutes. After peptide chain synthesis, wash the resin with DCM and dry it.

[0092] Using V TFA :V TIS :V H2O =90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C, centrifuged, the supernatant discarded, and the solid washed more than twice with cold methyl tert-butyl ether. A small amount of sample was taken to analyze the molecular weight of the crude product by mass spectrometry, and then the peptide was dissolved in 10-20 mL of acetonitrile-water and lyophilized. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 28-48% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and lyophilized. The lyophilized peptide powder was called naked peptide.

[0093] Naked peptide is dissolved into the solution that concentration is about 1mg / mL with acetonitrile and PBS (pH7.0), takes a small amount of liquid and carries out HPLC analysis, determines elution time, then naked peptide solution is added drop-wise in the VcMMAE (1.1 times of naked peptide amount) dissolved with acetonitrile and PBS (pH7.0), stirring reaction 30 minutes, takes a small amount of reacted liquid and carries out HPLC analysis, contrasts the elution time of naked peptide, if time appears to shift after, and mass spectrum confirms and theoretical molecular weight error within ± 1.0, then determines that reaction is successful. Reaction solution is loaded on C8 preparative HPLC post after adding pure water dilution once, flow velocity is 13mL / min, 0.1%TFA / acetonitrile mobile phase, and gradient is 30min 40-60% acetonitrile. Merge PDC with purity more than 90%, freeze-drying, the powder after freeze-drying is placed in-20 ℃ of preservations.

[0094] Example 2

[0095] Synthesis of PEG6-Cys(MC-Val-Cit-PABC-MMAE)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0096] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0097] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF :V ACN :V H2O =90:5:5, and then washed with DMF three times.

[0098] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, the following reactions were performed: Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-NH-PEG6-CH2CH2COOH, Fmoc-Cys(Trt)-OH, and Fmoc-NH-PEG6-CH2CH2COOH. To start the process, the amino acid and HOBt were dissolved in DMF and activated with DIC dropwise. The coupling reaction typically took 45 minutes, and the deprotection time was 20 minutes. After peptide chain synthesis, the resin was washed with DCM and dried.

[0099] Using V TFA :V TIS :V H2O=90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C, centrifuged, the supernatant discarded, and the solid washed more than twice with cold methyl tert-butyl ether. A small amount of sample was taken to analyze the molecular weight of the crude product by mass spectrometry, and then the peptide was dissolved in 10-20 mL of acetonitrile-water and lyophilized. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 20-40% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and lyophilized. The lyophilized peptide powder was called naked peptide.

[0100] The naked peptide was dissolved in acetonitrile and PBS (pH 7.0) to a concentration of approximately 1 mg / mL. A small amount of the liquid was taken for HPLC analysis to determine the peak time. The naked peptide solution was then added dropwise to VcMMAE (1.1 times the amount of naked peptide) dissolved in acetonitrile and PBS (pH 7.0). The reaction was stirred for 30 minutes. A small amount of the reaction liquid was taken for HPLC analysis to compare the peak time of the naked peptide. If the time was shifted and the mass spectrometry confirmed that the error with the theoretical molecular weight was within ±1.0, the reaction was determined to be successful. The reaction solution was diluted once with pure water and loaded onto a C8 preparative HPLC column at a flow rate of 13 mL / min. The system was flushed for 20 minutes with 95% 20 mmol / L ammonium acetate and 5% acetonitrile. The mobile phase was 0.1% acetic acid / acetonitrile with a gradient of 30-50% acetonitrile over 30 minutes. PDC with a purity of more than 90% was combined and freeze-dried. The freeze-dried powder was stored at -20°C.

[0101] Example 3

[0102] Synthesis of Ac-Cys(MC-Val-Cit-PABC-MMAE)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0103] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0104] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF V ACN V H2O =90:5:5, and then washed with DMF three times.

[0105] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, the reaction was sequentially condensed with Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-NH-PEG6-CH2CH2COOH, and Fmoc-Cys(Trt)-OH, followed by acetylation. To start the process, the amino acid and HOBt were dissolved in DMF and activated with DIC dropwise. The coupling reaction typically took 45 minutes, and the deprotection time was 20 minutes. After peptide chain synthesis, the resin was washed with DCM and dried.

[0106] Using V TFA :V TIS :V H2O=90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C. Centrifuge, discard the supernatant, and wash the solid more than twice with cold methyl tert-butyl ether. Take a small amount of sample to analyze the molecular weight of the crude product by mass spectrometry, and then dissolve the peptide in 10-20 mL of acetonitrile-water and freeze-dry. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 27-47% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and freeze-dried. The freeze-dried peptide powder was called naked peptide. The naked peptide was dissolved in acetonitrile and PBS (pH 7.0) to a concentration of about 1 mg / mL. A small amount of liquid was taken for HPLC analysis to determine the elution time. The naked peptide solution was then added dropwise to VcMMAE (1.1 times the amount of naked peptide) dissolved in acetonitrile and PBS (pH 7.0). The reaction was stirred for 30 minutes. A small amount of the reacted liquid was taken for HPLC analysis to compare the elution time of the naked peptide. If the time shifted and the mass spectrometry confirmed that the error with the theoretical molecular weight was within ± 1.0, the reaction was determined to be successful. The reaction solution was diluted to half with pure water and loaded onto a C8 preparative HPLC column with a flow rate of 13 mL / min. The system was flushed for 20 min with 95% 20 mmol / L ammonium acetate and 5% acetonitrile. The mobile phase was 0.1% acetic acid / acetonitrile with a gradient of 30-50% acetonitrile over 30 min. PDC with a purity of more than 90% was combined and freeze-dried. The freeze-dried powder was stored at -20°C.

[0107] Example 4

[0108] Synthesis of Ac-Cys(MC-betaglucuronide-MMAE)-AEEA-AEEA-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0109] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0110] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF :V ACN :V H2O =90:5:5, and then washed with DMF three times.

[0111] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, the reaction was sequentially condensed with Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, AEEA, AEEA, AEEA, Fmoc-Cys(Trt)-OH, and acetylated. To start, the amino acid and HOBt were dissolved in DMF and activated with DIC dropwise. The coupling reaction typically took 45 minutes, and the deprotection time was 20 minutes. After peptide chain synthesis, the resin was washed with DCM and dried.

[0112] Using V TFA :V TIS :V H2O =90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C. Centrifuge, discard the supernatant, and wash the solid more than twice with cold methyl tert-butyl ether. Take a small amount of sample to analyze the molecular weight of the crude product by mass spectrometry, and then dissolve the peptide in 10-20 mL of acetonitrile-water and freeze-dry. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 25-45% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and freeze-dried. The freeze-dried peptide powder was called naked peptide.

[0113] The naked peptide was dissolved in acetonitrile and PBS (pH 7.0) to a concentration of about 1 mg / mL. A small amount of liquid was taken for HPLC analysis to determine the peak time. The naked peptide solution was then added dropwise to MC-betaglucuronide-MMAE-1 (1.1 times the amount of naked peptide) dissolved in acetonitrile and PBS (pH 7.0). The reaction was stirred for 30 minutes. A small amount of the reaction liquid was taken for HPLC analysis to compare the peak time of the naked peptide. If the time was shifted and the mass spectrometry confirmed that the error with the theoretical molecular weight was within ±1.0, the reaction was determined to be successful. The reaction solution was diluted once with pure water and loaded onto a C8 preparative HPLC column at a flow rate of 13 mL / min. The system was flushed for 20 min with 95% 20 mmol / L ammonium acetate and 5% acetonitrile. The mobile phase was 0.1% acetic acid / acetonitrile with a gradient of 28-48% acetonitrile over 30 min. PDC with a purity of more than 90% was combined and freeze-dried. The freeze-dried powder was stored at -20°C.

[0114] Example 5

[0115] Synthesis of PEG6-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0116] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0117] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF V ACN V H2O =90:5:5, and then washed with DMF three times.

[0118] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, the following reactions were performed: Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-NH-PEG6-CH2CH2COOH, Fmoc-Cys(Trt)-OH, and Fmoc-NH-PEG6-CH2CH2COOH. To start the process, the amino acid and HOBt were dissolved in DMF and activated with DIC dropwise. The coupling reaction typically took 45 minutes, and the deprotection time was 20 minutes. After peptide chain synthesis, the resin was washed with DCM and dried.

[0119] Using V TFA :V TIS :V H2O =90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C, centrifuged, the supernatant discarded, and the solid washed more than twice with cold methyl tert-butyl ether. A small amount of sample was taken to analyze the molecular weight of the crude product by mass spectrometry, and then the peptide was dissolved in 10-20 mL of acetonitrile-water and lyophilized. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 20-40% acetonitrile over 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and lyophilized. The lyophilized peptide powder was called naked peptide.

[0120] The naked peptide was dissolved in acetonitrile and PBS (pH 7.0) to a concentration of about 1 mg / mL. A small amount of liquid was taken for HPLC analysis to determine the peak time. The naked peptide solution was then added dropwise to MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd (1.1 times the amount of naked peptide) dissolved in acetonitrile and PBS (pH 7.0). The reaction was stirred for 30 minutes. A small amount of the reacted liquid was taken for HPLC analysis to compare the peak time of the naked peptide. If the time shifted and the mass spectrometry confirmed that the error with the theoretical molecular weight was within ±1.0, the reaction was determined to be successful. The reaction solution was diluted to half with pure water and loaded onto a C8 preparative HPLC column with a flow rate of 13 mL / min. The system was flushed for 20 min with 95% 20 mmol / L ammonium acetate and 5% acetonitrile. The mobile phase was 0.1% acetic acid / acetonitrile with a gradient of 21-41% acetonitrile over 30 min. PDC with a purity of more than 90% was combined and freeze-dried. The freeze-dried powder was stored at -20°C.

[0121] Example 6

[0122] Synthesis of Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0123] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0124] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF :V ACN :V H2O =90:5:5, and then washed with DMF three times.

[0125] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, the reaction was sequentially condensed with Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-NH-PEG6-CH2CH2COOH, and Fmoc-Cys(Trt)-OH, followed by acetylation. To start the process, the amino acid and HOBt were dissolved in DMF and activated with DIC dropwise. The coupling reaction typically took 45 minutes, and the deprotection time was 20 minutes. After peptide chain synthesis, the resin was washed with DCM and dried.

[0126] Using V TFA :V TIS :V H2O=90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C, centrifuged, the supernatant discarded, and the solid washed more than twice with cold methyl tert-butyl ether. A small amount of sample was taken to analyze the molecular weight of the crude product by mass spectrometry, and then the peptide was dissolved in 10-20 mL of acetonitrile-water and lyophilized. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 27-47% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and lyophilized. The lyophilized peptide powder was called naked peptide.

[0127] The naked peptide was dissolved in acetonitrile and PBS (pH 7.0) to a concentration of about 1 mg / mL. A small amount of liquid was taken for HPLC analysis to determine the peak time. The naked peptide solution was then added dropwise to MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd (1.1 times the amount of naked peptide) dissolved in acetonitrile and PBS (pH 7.0). The reaction was stirred for 30 minutes. A small amount of the reacted liquid was taken for HPLC analysis to compare the peak time of the naked peptide. If the time shifted and the mass spectrometry confirmed that the error with the theoretical molecular weight was within ±1.0, the reaction was determined to be successful. The reaction solution was diluted once with pure water and loaded onto a C8 preparative HPLC column with a flow rate of 13 mL / min. The system was flushed for 20 min with 95% 20 mmol / L ammonium acetate and 5% acetonitrile. The mobile phase was 0.1% acetic acid / acetonitrile with a gradient of 24-44% acetonitrile over 30 min. PDC with a purity of more than 90% was combined and freeze-dried. The freeze-dried powder was stored at -20°C.

[0128] Example 7

[0129] Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Lys(Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6)-Lys(Ac-Cys(MC- Synthesis of Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0130] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0131] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF :V ACN : V H2O =90:5:5, and then washed with DMF three times.

[0132] Then, a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent were used to sequentially condense Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-NH-PEG6-CH2CH2COOH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Dde)-OH, Fmoc-NH-PEG6-CH2CH2COOH, and acetylate. When adding the materials, the amino acid and HOBt were dissolved in DMF and activated by adding DIC dropwise. The coupling reaction generally took 45 minutes, and the deprotection time was 20 minutes.

[0133] The Dde protecting group was removed using 15% hydrazine hydrate / DMF. Then, a 6-fold excess of starting material and a 6-fold excess of coupling reagent was used to sequentially condense Fmoc-NH-PEG6-CH2CH2COOH, followed by Fmoc-Cys(Trt)-OH, and then acetylation. After peptide chain synthesis, the resin was washed with DCM and dried.

[0134] Using V TFA :V TIS :V H2O=90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C. Centrifuge, discard the supernatant, and wash the solid more than twice with cold methyl tert-butyl ether. Take a small amount of sample to analyze the molecular weight of the crude product by mass spectrometry, and then dissolve the peptide in 10-20 mL of acetonitrile-water and freeze-dry. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 20-40% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and freeze-dried. The freeze-dried peptide powder was called naked peptide.

[0135] The naked peptide was dissolved in acetonitrile and PBS (pH 7.0) to a concentration of about 1 mg / mL. A small amount of liquid was taken for HPLC analysis to determine the peak time. The naked peptide solution was then added dropwise to MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd (3.3 times the amount of naked peptide) dissolved in acetonitrile and PBS (pH 7.0). The reaction was stirred for 30 minutes. A small amount of the reacted liquid was taken for HPLC analysis to compare the peak time of the naked peptide. If the time was shifted and the mass spectrometry confirmed that the error with the theoretical molecular weight was within ±1.0, the reaction was determined to be successful. The reaction solution was diluted once with pure water and loaded onto a C8 preparative HPLC column with a flow rate of 13 mL / min. The system was flushed for 20 min with 95% 20 mmol / L ammonium acetate and 5% acetonitrile. The mobile phase was 0.1% acetic acid / acetonitrile with a gradient of 30-50% acetonitrile over 30 min. PDC with a purity of more than 90% was combined and freeze-dried. The freeze-dried powder was stored at -20°C.

[0136] Example 8

[0137] Synthesis of Ac-Cys(MC-Val-Cit-PABC-MMAE)-AEEA-AEEA-AEEA-Arg-Arg-Leu-Phe-[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0138] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0139] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF :V ACN :V H2O =90:5:5, and then washed with DMF three times.

[0140] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, the reaction was sequentially condensed with Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, AEEA, AEEA, AEEA, Fmoc-Cys(Trt)-OH, and acetylated. To start, the amino acid and HOBt were dissolved in DMF and activated with DIC dropwise. The coupling reaction typically took 45 minutes, and the deprotection time was 20 minutes. After peptide chain synthesis, the resin was washed with DCM and dried.

[0141] Using V TFA :V TIS :V H2O =90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C, centrifuged, the supernatant discarded, and the solid washed more than twice with cold methyl tert-butyl ether. A small amount of sample was taken to analyze the molecular weight of the crude product by mass spectrometry, and then the peptide was dissolved in 10-20 mL of acetonitrile-water and lyophilized. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 27-47% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and lyophilized. The lyophilized peptide powder was called naked peptide.

[0142] The naked peptide was dissolved in acetonitrile and PBS (pH 7.0) to a concentration of about 1 mg / mL. A small amount of liquid was taken for HPLC analysis to determine the elution time. The naked peptide solution was then added dropwise to VcMMAE (1.1 times the amount of naked peptide) dissolved in acetonitrile and PBS (pH 7.0). The reaction was stirred for 30 minutes. A small amount of the reacted liquid was taken for HPLC analysis to compare the elution time of the naked peptide. If the time shifted and the mass spectrometry confirmed that the error with the theoretical molecular weight was within ± 1.0, the reaction was determined to be successful. The reaction solution was diluted to half with pure water and loaded onto a C8 preparative HPLC column with a flow rate of 13 mL / min. The system was flushed for 20 min with 95% 20 mmol / L ammonium acetate and 5% acetonitrile. The mobile phase was 0.1% acetic acid / acetonitrile with a gradient of 33-53% acetonitrile over 30 min. PDC with a purity of more than 90% was combined and freeze-dried. The freeze-dried powder was stored at -20°C.

[0143] Example 9

[0144] Synthesis of Cys(MC-Val-Cit-PABC-MMAE)-Glu-Glu-Glu-AEEA-Arg-Arg-Leu-Phe-[Cys- Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0145] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0146] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF :V ACN :V H2O =90:5:5, and then washed with DMF three times.

[0147] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, the following reactions were performed: Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, AEEA, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Cys(Trt)-OH. To add the starting materials, the amino acids and HOBt were dissolved in DMF and activated with DIC dropwise. The coupling reaction typically took 45 minutes, and the deprotection time was 20 minutes. After peptide chain synthesis, the resin was washed with DCM and dried.

[0148] Using V TFA :V TIS :V H2O =90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C, centrifuged, the supernatant discarded, and the solid washed more than twice with cold methyl tert-butyl ether. A small amount of sample was taken to analyze the molecular weight of the crude product by mass spectrometry, and then the peptide was dissolved in 10-20 mL of acetonitrile-water and lyophilized. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 28-48% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and lyophilized. The lyophilized peptide powder was called naked peptide.

[0149] Naked peptide is dissolved into the solution that concentration is about 1mg / mL with acetonitrile and PBS (pH7.0), takes a small amount of liquid and carries out HPLC analysis, determines elution time, then naked peptide solution is added drop-wise in the VcMMAE (1.1 times of naked peptide amount) dissolved with acetonitrile and PBS (pH7.0), stirring reaction 30 minutes, takes a small amount of reacted liquid and carries out HPLC analysis, contrasts the elution time of naked peptide, if time appears to shift after, and mass spectrum confirms and theoretical molecular weight error within ± 1.0, then determines that reaction is successful. Reaction solution is loaded on C8 preparative HPLC post after adding pure water dilution once, flow velocity is 13mL / min, 0.1%TFA / acetonitrile mobile phase, and gradient is 30min 41-61% acetonitrile. Merge PDC with purity more than 90%, freeze-drying, the freeze-dried powder is placed in-20 ℃ of preservations.

[0150] Example 10

[0151] Synthesis of Ac-Cys(MC-Val-Cit-PABC-MMAE)-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2

[0152] Peptide synthesis was based on Fmoc chemistry using solid-phase synthesis using 0.2 mmol / g Rink Amide-AM Resin and standard Fmoc-amino acids. Coupling reagents were DIC and HOBt, and deprotection was achieved with 20% piperidine in DMF. Resin modification was performed using a 1.5-fold excess of Fmoc-Asp(OtBu)-OH and a 1.5-fold excess of coupling reagent.

[0153] Using 3 times excess Fmoc-amino acid and 3 times excess coupling reagent, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH were condensed in sequence. A 5-fold amount of iodine was dissolved in DMF for solid phase cyclization for 45 minutes. After the reaction, V was used. DMF :V ACN :V H2O =90:5:5, and then washed with DMF three times.

[0154] Then, using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, sequentially condense Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, AEEA, and Fmoc-Cys(Trt)-OH. Finally, acetylate with 5-20 equivalents of acetic anhydride / N-methylmorpholine for 30-60 minutes. To start, dissolve the amino acid and HOBt in DMF and add DIC dropwise for activation. The coupling reaction typically takes 45 minutes, and the deprotection time is 20 minutes. After peptide chain synthesis, wash the resin with DCM and dry it.

[0155] Using V TFA :V TIS :V H2O=90:5:5 for cleavage for 1 hour. After cleavage, the spent resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20°C, centrifuged, the supernatant discarded, and the solid washed more than twice with cold methyl tert-butyl ether. A small amount of sample was taken to analyze the molecular weight of the crude product by mass spectrometry, and then the peptide was dissolved in 10-20 mL of acetonitrile-water and lyophilized. The crude peptide was dissolved in about 15% acetonitrile-water and loaded on a C8 preparative HPLC column with a flow rate of 13 mL / min, 0.1% TFA / acetonitrile mobile phase, and a gradient of 28-48% acetonitrile in 30 min. Peptides with a purity of more than 85% (confirmed by mass spectrometry and HPLC analysis) were combined and lyophilized. The lyophilized peptide powder was called naked peptide.

[0156] The naked peptide was dissolved in acetonitrile and PBS (pH 7.0) to a solution having a concentration of about 1 mg / mL. A small amount of liquid was taken for HPLC analysis to determine the elution time. The naked peptide solution was then added dropwise to VcMMAE (1.1 times the amount of naked peptide) dissolved in acetonitrile and PBS (pH 7.0). The reaction was stirred for 30 minutes. A small amount of the reacted liquid was taken for HPLC analysis to compare the elution time of the naked peptide. If the time shifted, and the mass spectrometry confirmed that the error with the theoretical molecular weight was within ± 1.0, the reaction was determined to be successful. The reaction solution was diluted with pure water and then loaded onto a C8 preparative HPLC column. The flow rate was 13 mL / min, with a 0.1% TFA / acetonitrile mobile phase and a gradient of 40-60% acetonitrile over 30 min. The PDC with a purity of more than 90% was combined and freeze-dried. The freeze-dried powder was placed at -20°C for preservation.

[0157] In the above examples and contents, the sources or meanings of the compounds used are as follows:

[0158] Table 1. Sources of compounds used in Examples 1-10

[0159] Effect Example 1: Target affinity test of PDC

[0160] SSTR2 cAMP experiment:

[0161] 1) Compound preparation: dilute the test compound 4-fold with DMSO to 10 different concentrations. Transfer 100 nL of the diluted compound to a cell reaction plate and centrifuge for later use.

[0162] 2) Cell preparation: Cultured SSTR2 / CHO cells were collected into a sterile centrifuge tube, centrifuged and the supernatant was discarded.

[0163] 3) Resuspend the cells with detection buffer, count them using a Vi-cell counter, and dilute the cells with detection buffer.

[0164] 4) Transfer 100 nL of 100 μM Forskolin to the cell reaction plate using an Echo, centrifuge, and then add 10 μL of assay buffer containing cells and incubate.

[0165] 5) Add 10 μL of cAMP detection reagent solution to the corresponding wells of the cell reaction plate and incubate at room temperature in the dark.

[0166] 6) Place the plate in a microplate reader and read the result. The final value is the ratio of OD665nm to OD615nm.

[0167] 7) Calculate the cAMP value for each well: Activity (%) = (sample well signal value - average low-signal control well signal value) / (average high-signal control well signal value - average low-signal control well signal value). The EC50 of each sample was calculated using the "Nonlinear regression (curve fit) - log (agonist) vs. response - Variable slope" model in GraphPad Prism 5.0, with the logarithmic value of compound concentration as the abscissa and the cAMP activation rate as the ordinate.

[0168] Table 2, SSTR2 cAMP assay results:

[0169] It can be seen that the polypeptide sequence of the present application has good affinity for SSTR2.

[0170] Effect Example 2: Cell Killing Ability Test of PDC

[0171] CGT assay to detect cell killing of NCI-H69 cells:

[0172] a) Cell line: NCI-H69

[0173] b) Detection steps

[0174] (1) Centrifuge NCI-H69 suspension cells, resuspend in fresh growth medium, and count using a cell counter;

[0175] (2) Dilute the cells to 3.125 w / mL with growth medium;

[0176] (3) The cell suspension was inoculated into a 96-well plate at a volume of 95 μL per well;

[0177] (4) diluting the peptide-coupled drug of the present application with growth medium containing DMSO, adding 5 μL of the peptide-coupled drug to the corresponding well plate position, and comparing with a DMSO control group without the peptide-coupled drug of the present application, as well as PTX, MMAE, and Dxd, wherein the final concentration of DMSO in each well is 0.2%;

[0178] (5) Place the cells in a 37°C, 5% CO2 incubator and culture for 96 h.

[0179] (6) Return the cell culture plate to room temperature and add 50uL to each well. Reagent;

[0180] (7) Place the plate in a shaker for 2 minutes to ensure complete cell lysis;

[0181] (8) After mixing, place the plate at room temperature for 10 minutes until the signal reaches stability and read the fluorescence value (RLU).

[0182] c) Data analysis

[0183] (1) The inhibition rate (Inh%) relative to the DMSO control group was calculated as follows: Inhibition rate (Inh%) = 100 - (RLU compound - RLU DMSO control group) / (RLU DMSO control group - RLU blank) * 100%

[0184] (2) Graphpad 5.0 software was used to analyze the data, fit the 4-parameter equation, generate the concentration response curve, and obtain the half-inhibitory concentration IC 50 .

[0185] Table 3. Results of NCI-H69 cell killing experiments

[0186] Detection of HL-60 cell killing by CCK8 assay:

[0187] a) Cell line: HL-60

[0188] b) Detection steps

[0189] (1) Take the HL-60 cell suspension in the logarithmic growth phase of normal culture, centrifuge at 800 rpm for 5 min, discard the supernatant, add 3 mL of normal culture medium, gently pipette to suspend the cell pellet, count the cells, and inoculate the cells on a 96-well plate.

[0190] (2) Prepare sample solutions with different concentration gradients, take 10 μL and add them to a 96-well plate, with 3 to 6 replicate wells for each concentration, and incubate at 37°C, 5% CO2 for an appropriate time of 48 h.

[0191] (3) Return CCK-8 to room temperature before use and add 10 μL of CCK-8 solution to each well.

[0192] (4) Incubate the plate in a dark incubator for 0.5 to 4 hours. The length of time depends on the experimental conditions such as cell type and cell density.

[0193] (5) Measure the absorbance at 450 nm using an enzyme reader.

[0194] c) Data analysis Cell survival rate = [(A s -A b ) / (A c -A b )]×100% Inhibition rate=[(A c -A s ) / (A c -A b )]×100%

[0195] A s : Absorbance of experimental wells (containing cells + culture medium + CCK-8 solution + drug solution);

[0196] A c : Absorbance of control well (containing cells + culture medium + CCK-8 solution);

[0197] A b : absorbance of blank well (culture medium + CCK-8 solution);

[0198] The results were processed and analyzed using Excel and Graphpad Prism to obtain the half-inhibitory concentration (IC) 50 .

[0199] Table 4, HL-60 48h cell killing test results

[0200] Effect Example 3: In vivo efficacy test of CDX

[0201] EX3.1: In vivo efficacy of the test article on NCI-H69 cells in a subcutaneous tumor model of Balb / c nude mice. Animals:

[0202] Species: Mouse

[0203] Strain: Balb / c nude mice

[0204] Age: 6-8 weeks

[0205] Gender: Female

[0206] Animal source: Shanghai Jihui Experimental Animal Breeding Co., Ltd.

[0207] Experimental methods and steps

[0208] a) Cell culture

[0209] NCI-H69 cells were cultured in RPMI1640 medium supplemented with 20% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 5% CO2 incubator at 37°C, with passages occurring 2-3 times per week. When cells reached the logarithmic growth phase, they were harvested and washed twice with serum-free medium. Finally, the cells were resuspended in serum-free medium, counted, and harvested for plating.

[0210] b) Animal grouping and drug administration

[0211] Each mouse was subcutaneously inoculated with 0.1 mL of NCI-H69 tumor cell solution (5.00 × 10 6 / mouse, and the day of inoculation was designated as day 0. When the tumor volume reached about 100 mm 3 Mice were selected and randomly divided into groups according to tumor size and body weight using Excel (solvent control, positive control, and drug treatment according to Example 4, 6 mice in each group). They were intravenously injected with 5% DMSO + 95% saline, Etoposide (10 mg / kg), and the drug of Example 4 of the present application (22.8 mg / kg), twice a week.

[0212] c) Tumor measurement

[0213] The weight of mice was measured twice a week using an electronic balance. The tumor diameter was measured three times a week using a vernier caliper. The tumor volume was calculated using the formula: V = 0.5 × a × b 2 , where a and b are the length and width of the tumor, respectively.

[0214] Relative tumor growth rate: T / C (%) = T RTV / C RTV ×100%(T RTV :RTV, C RTV : RTV of solvent control group, RTV=Vt / V0).

[0215] Tumor growth inhibition rate: TGI (%) = (1-T / C) × 100%.

[0216] Table 5. Tumor volume of mice in each group (mm 3 , mean ± SEM)

[0217] Experimental results:

[0218] This study evaluated the anti-tumor effect of the test article in the NCI-H69 subcutaneous model of BALB / c nude mice. As of day 32, both the positive control group (Etoposide, 10 mg / kg) and the Example 4 group (22.8 mg / kg) had significant tumor inhibitory effects compared with the solvent control group (p<0.001). Among them, in the experimental group treated with Example 4 of the present application, 2 / 6 of the mouse tumors achieved PR (partial response, tumor reduction of more than 50%), and 1 / 6 of the mouse tumors did not change (tumor reduction was less than 50% or increase was less than 25%). The TGI of this application is 87.90%.

[0219] EX3.2: In vivo efficacy of the test article on NCI-H69 cells in a subcutaneous tumor model of Balb / c nude mice. Animals:

[0220] Balb / c nude mice, 6-8 weeks old, female, were purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd.

[0221] a) Cell culture

[0222] NCI-H69 cells were cultured in RPMI1640 medium supplemented with 20% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 5% CO2 incubator at 37°C, with passages occurring 2-3 times per week. When cells reached the logarithmic growth phase, they were harvested and washed twice with serum-free medium. Finally, the cells were resuspended in serum-free medium, counted, and harvested for plating.

[0223] b) Animal grouping and drug administration

[0224] Each mouse was subcutaneously inoculated with 0.1 mL of NCI-H69 tumor cell solution (5.00 × 10 6 / mouse, and the day of inoculation was designated as day 0. When the tumor volume reached about 100 mm 3 Mice were selected and randomly divided into groups according to tumor size and body weight using Excel (solvent control group and Example 4 drug group, 6 mice in each group), and were intravenously injected with 5% DMSO + 95% saline and the drug of Example 4 (53.7 mg / kg), respectively. The injection was continuous for 4 days / rest for 3 days as one cycle.

[0225] c) Tumor measurement

[0226] The weight of mice was measured twice a week using an electronic balance. The tumor diameter was measured three times a week using a vernier caliper. The tumor volume was calculated using the formula: V = 0.5 × a × b 2 , where a and b are the length and width of the tumor, respectively.

[0227] Relative tumor growth rate: T / C (%) = T RTV / C RTV ×100% (RTV=Vt / V0).

[0228] Tumor growth inhibition rate: TGI (%) = (1-T / C) × 100%.

[0229] The results of the in vivo efficacy test of the test article on NCI-H69 cells in the Balb / c nude mouse subcutaneous tumor model are shown in Table 5 below.

[0230] Table 5. Tumor volume of mice in each group (mm 3 ,average value)

[0231] Experimental results:

[0232] This study evaluated the anti-tumor effect of the test article in the NCI-H69 subcutaneous model of BALB / c nude mice. As of day 34, the application had a significant tumor inhibition effect compared with the solvent control group (p<0.001), with a TGI of 93.9%.

[0233] EX3.3: In vivo pharmacodynamic study of the test drug in a CB17 SCID mouse model bearing subcutaneous human multiple myeloma MM.1S cells

[0234] a) Cell culture:

[0235] Human multiple myeloma MM.1S cells were cultured in suspension in 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Suspension cells were harvested by centrifugation twice a week, and adherent cells were digested with trypsin-EDTA to prepare cell suspensions for subculture. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0236] b) Animals:

[0237] CB17 SCID mice, female, 6-8 weeks old, weighing 18-22 g, were provided by Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd.

[0238] c) Model building:

[0239] 5 × 106 MM.1S cells in 100 μL PBS were mixed with 100 μL Matrigel (final volume was 200 μL) and inoculated subcutaneously into the right forelimb axilla of each mouse. On the 20th day after cell inoculation, the average tumor volume reached 109 mm 3Dosing began at 4:00 pm (solvent control, ixazomib citrate group, and drug group treated with Example 1 of the present application, 6 rats per group). Ixazomib citrate was orally administered (4 mg / kg) twice weekly via gavage; the solvent control group (5% DMSO + 95% saline) and drug group treated with Example 1 of the present application (1.75 mg / kg) were administered via slow intravenous injection, with each injection lasting approximately 30 seconds, twice weekly.

[0240] d) Experimental indicators:

[0241] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice a week with a vernier caliper. The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0242] The anti-tumor efficacy of a drug is evaluated using the TGI (%) or relative tumor growth rate (T / C) (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) is calculated as follows: TGI (%) = [(1 - (mean tumor volume at the end of treatment in a given treatment group - mean tumor volume at the start of treatment in that treatment group)) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%.

[0243] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C (%) = T RTV / C RTV × 100%. The relative tumor volume (RTV) was calculated based on the tumor measurement results. The calculation formula is RTV = V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., d0), V t is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0244] After the experiment, the tumor weight will be measured and T / C will be calculated. weight Percentage, T weight and C weight Represent the tumor weights of the drug-treated group and the vehicle control group, respectively.

[0245] e) Data analysis:

[0246] Statistical analysis, including the mean and standard error (SEM) of tumor volume at each time point in each group (see Table 5-1 in Section 5.2 for specific data), was performed based on the data of the treatment group on day 18 after administration at the end of the experiment to evaluate the differences between the groups. T-Test was used for analysis between two groups, and one-way ANOVA was used for analysis between three or more groups. If there was a significant difference in the F value, the Games-Howell method was used for testing. If there was no significant difference in the F value, the Dunnet (2-sided) method was used for analysis. All data analyses were performed using SPSS 17.0. p<0.05 was considered to be significantly different.

[0247] f) Tumor volume:

[0248] Tumor volume (mm) of female CB17 SCID mice with MM.1S cell subcutaneous xenograft tumor after treatment with the test drugs 3 )The changes in average values ​​are shown in the following table.

[0249] Table 6, Tumor volume of mice in each group (mm 3 , mean ± SEM)

[0250] Experimental results:

[0251] In this study, we evaluated the in vivo efficacy of the test drug in a MM.1S cell subcutaneous xenograft tumor model. On day 18 after the start of drug administration, the tumor volume of the tumor-bearing mice in the solvent control group reached 2487 mm 3 Compared with the solvent control group, Ixazomib Citrate and the drug in Example 1 of the present application both had significant tumor inhibition effects, with tumor volumes of 781 mm and 136 mm, respectively. 3 (T / C=38.38%, TGI=71.76%, p=0.040), and 23 mm 3 (T / C=0.94%, TGI=103.59%, p=0.010) All 6 animals treated with the drug in Example 1 of the present application achieved complete remission of tumors. During the experiment, all drug-treated groups showed no significant weight loss and no morbidity.

[0252] In summary, in this experiment, the test drug of this application was well tolerated by the animals. The drug of Example 1 showed significant anti-tumor effects at a dose of 1.75 mg / kg in a subcutaneous xenograft model of human multiple myeloma MM.1S cells, and the tumor inhibition effect was significant, with a complete tumor remission rate of 100%.

[0253] The specific embodiments of the present application have been described in detail above, but these are merely examples, and the present application is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present application are also within the scope of the present application. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present application should be included within the scope of the present application.

[0254] Sequence Listing Information:

[0255] DTD version: V1_3

[0256] File name: PCT23347-A371-231.xml

[0257] Software Name:WIPO Sequence

[0258] Software version: 2.3.0

[0259] Date of creation: 2023-08-30

[0260] Basic Information:

[0261] Current application / applicant file name: PCT23347-A371-231

[0262] Name of applicant: Mainstream Biotechnology (Shanghai) Co., Ltd.

[0263] Applicant's name / language:zh

[0264] Applicant's name or title / Latin name: Mainline Biosciences (Shanghai) Co., Ltd.

[0265] Inventor Name: Zhang Junge

[0266] Inventor Name / Language:zh

[0267] Inventor Name / Latin Name:Zhang Junge

[0268] Invention Title: A peptide targeting somatostatin receptor, a conjugated drug, and its application (zh)

[0269] Total number of sequences: 6

[0270] sequence:

Claims

1. A peptide targeting SSTR2, characterized in that: With the following sequence: Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH; wherein a disulfide bond is formed between the two Cys in [ ]; Xaa is selected from one or more of Tyr and Phe; X1 and X2 are selected from any amino acid residues; and X3 is selected from any amino acid residue or does not exist.

2. The peptide targeting SSTR2 according to claim 1, characterized in that X1 is Arg or Leu, X2 is Trp or Asp, X3 is Asp; Trp is D-Trp.

3. The peptide targeting SSTR2 according to claim 1, characterized in that The sequence is: X0-Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH; wherein X0 is selected from an amino acid residue capable of reacting with an amino group, or other carboxyl-containing groups, and X0 is more preferably Arg or Gly.

4. The peptide targeting SSTR2 according to claim 3, characterized in that The peptide targeting SSTR2 is selected from the following sequences: SEQ ID NO.1: H-Arg-Arg-Leu-Phe-[Cys-Tyr-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 SEQ ID NO.2: H-Arg-Arg-Leu-Phe-[Cys-Phe-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 SEQ ID NO.3: H-Gly-Arg-Arg-Phe-[Cys-Tyr-Trp-Lys-Thr-Cys]-Thr-Asp-Arg-NH2 SEQ ID NO.4: H-Arg-Arg-Leu-Phe-[Cys-Tyr-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-OH.

5. A peptide-coupled drug targeting SSTR2, characterized in that: The invention comprises a peptide targeting SSTR2, and a spacer connected to the peptide, wherein: The peptide targeting SSTR2 has the following sequence: Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH; wherein the two Cys in [] form a disulfide bond; Xaa is selected from one or more of Tyr and Phe; X1 and X2 are selected from any amino acid residue; X3 is selected from any amino acid residue or does not exist; The spacer has the following structure X4-X6-X5, wherein X4 may not exist or is a single bond, acetyl group, or polyether structure connecting the peptide; X5 is a polyether structure; X6 contains an amino acid residue of an active group, and the active group is preferably selected from carboxyl, hydroxyl, amino, thiol, and aldehyde.

6. The peptide-coupled drug targeting SSTR2 according to claim 5, characterized in that: The polyether is preferably a polyethylene glycol structure having 2 to 12 repeating units.

7. The peptide-coupled drug targeting SSTR2 according to claim 5, characterized in that: The amino acid residue containing the active group is selected from cysteine ​​residues, lysine residues, ornithine residues, glutamic acid residues, aspartic acid residues, 6-azido-L-norleucine, 2-amino-5-hydroxypentanoic acid residues, 2-amino-5-ureidopentanoic acid residues, and 2-amino-5-cyanobenzoic acid residues.

8. The peptide-coupled drug targeting SSTR2 according to claim 5, characterized in that: The sequence is: X0-Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH; wherein X0 is selected from an amino acid residue capable of reacting with an amino group, or other carboxyl-containing groups, and X0 is more preferably Arg or Gly.

9. The peptide-coupled drug targeting SSTR2 according to claim 5, characterized in that: The spacer may also contain other amino acid residues. For example, the spacer may have the following structure: X4'-X6-{Lys(X4-X6'-X5)} x -AA y -X5', wherein AA is a random sequence of amino acid residues, y is an integer of 0-4, and x is an integer of 0-3, wherein X4' and X4 may be the same or different, and may be absent or independently be a single bond, an acetyl group, or a polyether structure connecting the peptide; X5' and X5 may be the same or different, and may be independently a polyether structure; X6 and X6' may be the same or different and are independently amino acid residues containing active groups.

10. The peptide-coupled drug targeting SSTR2 according to claim 9, characterized in that: The spacer is selected from: X6-(AEEA) r PEG z -X6-PEG z 、Ac-X6-PEG z 、Ac-X6-(AEEA) r ,Ac-X6-PEG6-{Lys(Ac-X6'-PEG6)} x -PEG6, X6-Glu-Glu-Glu-AEEA, r is an integer, preferably an integer of 0-6, more preferably an integer of 1-5, such as r=2, 3 or 4.

11. The peptide-coupled drug targeting SSTR2 according to claim 9, characterized in that: It also includes a linker for connecting an active drug, which is connected to the X6 and / or X6' residues of the spacer; preferably, the linker is selected from MC-Val-Ala-PABC, MC-Val-Cit-PABC, MC-Gly-Gly-Phe-Gly-NH-CH2, MCC, MC-betaglucuronide, MC-Glu-Val-Ala-PABC, and MC-Glu-Val-Cit-PABC.

12. The peptide-coupled drug targeting SSTR2 according to claim 7, characterized in that: It also includes an active drug, which is connected to the linker; preferably, the active drug is connected to the linker by one or more of condensation, esterification, etherification, and amidation.

13. The peptide-conjugated drug according to claim 12, characterized in that: The active drug is selected from cytotoxic agents, preferably, selected from: microtubule inhibitors (such as calendula, maytansine derivatives, tubulysin, cryptocolistin, etc.), DNA damaging drugs (such as pyrrolebenzazepines and indolechlorobenzazepines, dukamycin, camptothecin, calicheamicin, etc.), amatoxin, apoptosis inducers, camptothecins; more preferably, the cytotoxic agent is selected from MMAF, MMAE, PBD, DM1, DM4, Dxd, SN38, paclitaxel, docetaxel, exotecan.

14. The peptide-coupled drug targeting SSTR2 according to claim 12, characterized in that: The peptide-coupled drug is selected from Drug 1: Cys(MC-Val-Cit-PABC-MMAE)-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-T hr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 2: PEG6-Cys(MC-Val-Cit-PABC-MMAE)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp- Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 3: Ac-Cys(MC-Val-Cit-PABC-MMAE)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys -Thr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 4: Ac-Cys(MC-betaglucuronide-MMAE)-AEEA-AEEA-AEEA-Arg-Arg-Leu-Phe-[C ys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 5: PEG6-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Arg-Arg-Leu-Phe-[Cys-T yr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 6: Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr- D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 7: Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6-Lys(Ac-Cys(MC-Gly-Gly-Ph e-Gly-NH-CH2-Dxd)-PEG6)-Lys(Ac-Cys(MC-Gly-Gly-Phe-Gly-NH-CH2-Dxd)-PEG6)-PEG6-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 8: Ac-Cys(MC-Val-Cit-PABC-MMAE)-AEEA-AEEA-AEEA-Arg-Arg-Leu-Phe-[Cys- Phe-D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 9: Cys(MC-Val-Cit-PABC-MMAE)-Glu-Glu-Glu-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr D-Trp-Lys-Thr-Cys]-Thr-Trp-Arg-Asp-NH2 Drug 10: Ac-Cys(MC-Val-Cit-PABC-MMAE)-AEEA-Arg-Arg-Leu-Phe-[Cys-Tyr-D-Trp-Ly s-Thr-Cys]-Thr-Trp-Arg-Asp-NH2。 15. The peptide-conjugated drug according to claim 12, characterized in that: The peptide-coupled drug contains a pharmaceutically acceptable (radioactive) isotope label or labeling agent label, or the active drug is an isotope or labeling agent, for example, one or more atoms of the peptide-coupled drug are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number usually present in nature, or the active drug is a metal complex group capable of carrying a radioactive isotope or labeling agent, or certain functional groups in the peptide-coupled drug are covalently replaced by functional groups labeled with relevant radioactive isotopes or labeling agents.

16. The peptide-conjugated drug according to claim 15, characterized in that: Examples of such isotopes include: hydrogen isotopes such as 2 H and 3 H, carbon isotopes such as 11 C. 13 C and 14 C, chlorine isotopes such as 36 Cl, fluorine isotopes such as 18 F, iodine isotopes such as 123 I. 125 I and 131 I, nitrogen isotopes such as 13 N and 15 N, oxygen isotopes such as 15 O. 17 O and 18 O, phosphorus isotopes such as 32 P, sulfur isotopes such as 35 S, copper isotopes such as 64 Cu, gallium isotopes such as 67 Ga or 68 Ga, yttrium isotopes such as 90 Y, and lutetium isotopes such as 177 Lu, and bismuth isotopes such as 213 Bi.

17. A use of a peptide targeting SSTR2 or a peptide-coupled drug targeting SSTR2, characterized in that: The peptide-coupled drug targeting SSTR2 contains a peptide targeting SSTR2 and a spacer connected to the peptide, and may further contain a linker connected to the spacer, or an active drug connected to the linker; Wherein, the peptide of SSTR2 or the peptide-coupled drug targeting SSTR2 is used to prevent, inhibit or treat diseases with high expression of SSTR2; or is used to prepare drugs for treating, inhibiting or preventing diseases with high expression of SSTR2; Among them, the peptide targeting SSTR2 has the following sequence: Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH; wherein the two Cys in [] form a disulfide bond; Xaa is selected from one or more of Tyr and Phe; X1 and X2 are selected from any amino acid residue; X3 is selected from any amino acid residue or does not exist; The spacer has the following structure X4-X6-X5, wherein X4 may not exist or be a spacer for connecting the peptide. single bond, acetyl group, polyether structure; X5 is a polyether structure; X6 contains an amino acid residue of an active group, and the active group is preferably selected from carboxyl, hydroxyl, amino, thiol, and aldehyde.

18. The use according to claim 17, characterized in that The disease is cancer, and in particular preferably solid tumors originating from neuroendocrine cells, such as neuroendocrine tumors of the digestive system such as the stomach, intestine, pancreas, and small cell lung cancer.

19. The use according to claim 17, characterized in that: The sequence is: X0-Arg-X1-Phe-[Cys-Xaa-Trp-Lys-Thr-Cys]-Thr-X2-Arg-X3-NH2 / OH; wherein X0 is selected from an amino acid residue capable of reacting with an amino group, or other carboxyl-containing groups, and X0 is more preferably Arg or Gly.

20. The use according to claim 17, characterized in that The spacer may have the following structure: X4'-X6-{Lys(X4-X6'-X5)} x -AA y -X5', wherein AA is a random sequence of amino acid residues, y is an integer of 0-4, and x is an integer of 0-3, wherein X4' and X4 may be the same or different, and may be absent or independently be a single bond, an acetyl group, or a polyether structure connecting the peptide; X5' and X5 may be the same or different, and may be independently a polyether structure; X6 and X6' may be the same or different and are independently amino acid residues containing active groups; the linker is connected to the X6 and / or X6' residues of the spacer.