Cyclic peptides, methods for preparing same, complexes containing same and uses thereof

Abstract

The array is cyclo(X 1 X 2 X 3 X 4 X 5 X 6 ) wherein X 1 is asparagine, and said X 2 is glycine or sarcosine, and X 3 is arginine, and X 4 is selected from the group consisting of threonine, tyrosine and phenylalanine, 5 is lysine, and X 6 is selected from the group consisting of tyrosine, valine, and glutamic acid. A method for preparing a cyclic peptide is provided. A conjugate is provided comprising the cyclic peptide, a linker, and a chelating agent. Use of the conjugate as a targeting molecule for radionuclide labeling is provided. A method for labeling with a radionuclide is provided, comprising contacting a conjugate chelated with a radionuclide with a target to be labeled with the radionuclide.

Description

[Technical Field]

[0001] The present invention relates to the pharmaceutical field, in particular to cyclic peptides, methods for their preparation, conjugates containing them and uses thereof. [Background technology]

[0002] For a long time, therapeutic diagnostics has been the main development direction in the field of nuclear medicine, and at the same time, radiotherapy diagnostics is the most mature and widely applied clinical field in the field of therapeutic diagnostics. One of the notable advantages of radiotherapy diagnostics is that the treatment site is the diagnostic image of the patient's lesion, and imaging and therapeutic intervention are closely related. Therefore, the development of specific imaging tracers with high affinity and specificity, low nonspecific uptake, sufficient retention and effective penetration has become a key research direction in the field of nuclear medicine.

[0003] Aminopeptidase N Aminopeptidase N (APN, also known as CD13) is a Zn2 + CD13 is a membrane-dependent metalloprotein hydrolase that can cleave neutral amino acids at the N-terminus of proteins or polypeptides. CD13 was first purified in 1963 and has since been found to be overexpressed in tumors, tumor angiogenesis, and cardiovascular angiogenesis.

[0004] CD13 is expressed in a range of different human cells, including macrophages, stromal cells, smooth muscle cells, and fibroblasts. It is involved in peptide cleavage, viral infection, endocytosis, and cell signaling. Abnormally high levels of CD13 expression occur in various cancers, including breast cancer, ovarian cancer, thyroid cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer. In gastric cancer, CD13 and TGF-β1 expression levels are associated with tumor size, lymph node metastasis, and tumor differentiation. Similarly, in pancreatic cancer, serum CD13 levels are associated with tumor size, lymph node metastasis, and metastatic stage. It is a biomarker for early pancreatic cancer diagnosis and prognosis, and can predict the mortality and overall survival of pancreatic cancer patients. In colorectal cancer, higher plasma CD13 activity is associated with lower overall survival. Studies have also shown that CD13 expression is correlated with osteosarcoma; immunohistochemistry showed positive CD13 expression in 77% of osteosarcoma patients, and high CD13 expression correlated with poor overall survival. In summary, CD13 can be used as a cancer biomarker for the clinical detection and evaluation of tumors.

[0005] NGR Ligand Studies have shown that CD13 is not expressed on the surface of normal blood vessels, but is typically highly expressed on angiogenic vessels, such as tumor vessels. Furthermore, certain peptide structures can bind to the CD13 active site without being cleaved. The most well-known peptide sequence is a polypeptide containing asparagine-glycine-arginine (NGR), which can target tumor vascular tissues through its interaction with CD13. NGR polypeptides were isolated in 1998 by screening a phage-display peptide library. Experiments have shown that polypeptides containing the NGR structure can bind to CD13 receptor-positive blood vessels in tumor tissues but cannot bind to other tissues rich in CD13 receptors. These results further demonstrate the feasibility of using NGR-containing polypeptides as potential tumor-targeting diagnostic and therapeutic agents.

[0006] Currently, there are many studies on polypeptides containing NGR fragment sequences as carriers for delivering chemotherapy drugs, nanoparticles, and radioisotopes to tumors. Preclinical and clinical studies have shown that radiolabeled NGR peptides have great potential in tumor vascular diagnostic imaging and targeted radionuclide or ion therapy.

[0007] Cyclic peptides Cyclic peptide compounds are cyclic compounds with unique structures, broad biological activities, and unique mechanisms of action. The conformation of peptide molecules is stable and uniform, with high selective affinity to receptors and strong metabolic stability. As drug molecules, cyclic peptide compounds have a wide range of biological activities, including anticancer, antiviral, antibacterial, antifungal, and enzyme inhibitory. Therefore, research into cyclic peptide drugs has attracted increasing attention.

[0008] Due to the limited conformational change caused by the cyclic structure, cyclic peptide compounds generally have a large surface area, resulting in high affinity and recognition specificity with target proteins. The limited conformational flexibility of the macrocyclic structure also reduces the entropy value of the binding between the drug and the target, improving the binding stability. Secondly, due to the characteristics of their amino acid composition, cyclic peptide compounds generally have very low or no cytotoxicity. Furthermore, cyclic peptide compounds are easy to produce using automated chemical synthesis processes, and are convenient for various modifications, processing, and monitoring, all of which are very advantageous in the drug development process.

[0009] Currently, the most commonly used cyclic peptide in the study of tumor radioactive tracers targeting NGR is the cyclo(CNGRC) cyclic peptide, and initial studies have used this cyclic peptide. 99m Tc, 68 Ga, 64It has been labeled with radionuclides such as Cu and used for molecular imaging of tumor neovascularization. However, the disulfide bond in the cyclo(CNGRC) cyclic peptide structure is susceptible to biodegradation or chemical modification, so its biological stability and in vivo retention time need to be further optimized, which to some extent limits its use. Summary of the Invention

[0010] In some embodiments, a cyclic peptide is provided, the sequence of which is cyclo(X 1 X 2 X 3 X 4 X 5 X 6 ) X 1 is asparagine, particularly L-asparagine or D-asparagine, especially L-asparagine. X 2 is glycine or sarcosine. X 3 is arginine, in particular L-arginine or D-arginine, in particular L-arginine. X 4 is selected from the group consisting of threonine, tyrosine and phenylalanine, in particular selected from the group consisting of L-threonine, D-threonine, L-tyrosine, D-tyrosine, L-phenylalanine and D-phenylalanine, in particular threonine, more in particular L-threonine or D-threonine, more in particular L-threonine. X 5 is lysine, in particular L-lysine or D-lysine, in particular L-lysine. X 6 is selected from the group consisting of tyrosine, valine and glutamic acid, in particular selected from the group consisting of L-tyrosine, D-tyrosine, L-valine, D-valine, L-glutamic acid and D-glutamic acid, in particular tyrosine, more in particular L-tyrosine or D-tyrosine, more in particular L-tyrosine.

[0011] In some embodiments, the X 5 The C-terminus of 6 and binds to the N-terminus of said X 4 The C-terminus of 5 and binds to the N-terminus of said X 3 The C-terminus of 4 and binds to the N-terminus of said X 2 The C-terminus of 3 and binds to the N-terminus of said X 1 The C-terminus of 2 and binds to the N-terminus of said X 6 The C-terminus of 1 The present invention provides a method for preparing a cyclic peptide, comprising linking to the N-terminus of

[0012] In some embodiments, the method comprises: 5 and the C-terminus of X, the C-terminus of which is protected or bound to a solid phase material. 6 In some embodiments, the method comprises condensing the N-terminal protected X 4 and the C-terminus of X, the C-terminus of which is protected or bound to a solid phase material. 5 In some embodiments, the method comprises condensing the N-terminal protected X 3 and the C-terminus of X, the C-terminus of which is protected or bound to a solid phase material. 4 In some embodiments, the method comprises condensing the N-terminal protected X 2 and the C-terminus of X, the C-terminus of which is protected or bound to a solid phase material. 3 In some embodiments, the method comprises condensing the N-terminal protected X 1 and the C-terminus of X, the C-terminus of which is protected or bound to a solid phase material. 2 In some embodiments, the method comprises condensing the N-terminal protected X 6 and the C-terminus of X, the C-terminus of which is protected or bound to a solid phase material. 1 and the N-terminus of

[0013] In some embodiments, a conjugate is provided comprising the cyclic peptide, a linker, and a chelator. In some embodiments, a radionuclide formulation is provided comprising the conjugate and a radionuclide chelated to the chelator of the conjugate.

[0014] In some embodiments, there is provided a use of a cyclic peptide or conjugate of the present invention in radionuclide labeling. In some embodiments, there is provided a use of a cyclic peptide or conjugate of the present invention in preparing a targeting molecule for radionuclide labeling. In some embodiments, there is provided a use of a cyclic peptide or conjugate of the present invention in preparing a radionuclide labeling reagent. In some embodiments, there is provided a use of a cyclic peptide or conjugate of the present invention in preparing a drug carrier. In some embodiments, there is provided a use of a cyclic peptide or conjugate of the present invention as a drug carrier. In some embodiments, there is provided a use of a cyclic peptide, conjugate, or radionuclide formulation of the present invention in preparing a drug for use in cancer detection, cancer diagnosis, cancer progression monitoring, cancer therapy monitoring, or cancer treatment. In some embodiments, there is provided a use of a cyclic peptide, conjugate, or radionuclide formulation of the present invention in cancer detection, cancer diagnosis, cancer progression monitoring, cancer therapy monitoring, or cancer treatment.

[0015] In some embodiments, cyclic peptides or conjugates of the present invention are provided for use in radionuclide labeling. In some embodiments, radionuclide-labeled targeting molecules comprising or consisting of the cyclic peptides or conjugates of the present invention are provided. In some embodiments, radionuclide-labeled reagents are provided for use in or consisting of the cyclic peptides or conjugates of the present invention. In some embodiments, drug carriers are provided for use in or consisting of the cyclic peptides or conjugates of the present invention. In some embodiments, formulations are provided for use in cancer detection, cancer diagnosis, cancer progression monitoring, cancer therapy monitoring, or cancer treatment, comprising or consisting of the cyclic peptides, conjugates, or radionuclide formulations of the present invention. In some embodiments, cyclic peptides, conjugates, or radionuclide formulations of the present invention are provided for use in cancer detection, cancer diagnosis, cancer progression monitoring, cancer therapy monitoring, or cancer treatment.

[0016] In some embodiments, a method for labeling with a radionuclide is provided, comprising contacting the radionuclide-chelated conjugate or radionuclide formulation with a radionuclide-labeled target. In some embodiments, a method for use in cancer detection, cancer diagnosis, cancer progression monitoring, or cancer treatment monitoring is provided. The method includes administering the radionuclide-chelated conjugate or radionuclide formulation to a subject in need of cancer detection, cancer diagnosis, cancer progression monitoring, or cancer treatment monitoring; detecting the radionuclide to determine the level and location of the radionuclide in the subject's body; and comparing the level and location with the level and location of the radionuclide in an otherwise identical location in an unaffected subject or in an unaffected area of ​​the subject. wherein a higher level or different location of the radionuclide in the body of the test / treated subject compared to the level and location of the radionuclide in an unaffected subject or in the sample from an unaffected area from the test / treated subject indicates that the test / treated subject is afflicted with cancer, thereby detecting cancer, diagnosing cancer, monitoring cancer progression, or monitoring cancer treatment. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is an electrospray mass spectrum of APN21-Bn-SCN-NOTA in some examples of the present invention. [Figure 2] FIG. 1 shows the emission HPLC profile of 68Ga-APN21-Bn-SCN-NOTA in some examples of the present invention. [Figure 3A] These are static PET / CT images taken 0.5, 1, and 2 hours after injection of HT1080 tumor-bearing mice injected with only Comparative Example 68Ga-CG6-Bn-SCN-NOTA (CG6 group), Comparative Example 68Ga-KE5-Bn-SCN-NOTA (KE5 group), Example 68Ga-APN21-Bn-SCN-NOTA (APN21 group), and Example 68Ga-APN21-Bn-SCN-NOTA co-injected with unlabeled NGR peptide (APN21 blockade group). [Figure 3B] Figure 1 shows the tumor uptake calculated based on the 68Ga-APN21-Bn-SCN-NOTA signal intensity 0.5, 1, and 2 hours after injection in HT1080 tumor-bearing mice injected with only Comparative Example 68Ga-CG6-Bn-SCN-NOTA (CG6 group), Comparative Example 68Ga-KE5-Bn-SCN-NOTA (KE5 group), Example 68Ga-APN21-Bn-SCN-NOTA (APN21 group), or Example 68Ga-APN21-Bn-SCN-NOTA co-injected with unlabeled NGR peptide (APN21 blockade group). DETAILED DESCRIPTION OF THE INVENTION

[0018] As used herein, the terms "a," "an," and "an element" refer to one or more elements.

[0019] As stated above, the term "about" refers to an approximation of a value that is roughly or nearby in range. When the term "about" is used in conjunction with a numerical range, it refers to modifying that range by extending the limits above or below the stated numerical values. Generally, the use of the term "about" herein varies a numerical value by plus or minus 10% from the stated value. Conversely, the term "about" refers to adding or subtracting 20% ​​from the modified numerical value. For example, "about 50%" refers to a range of 45%-55%. Numerical ranges expressed herein by endpoints include all integers and fractions within this range (e.g., "1 to 5" includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all integers and fractions thereof are considered to be modified by the term "about."

[0020] "Comprise" or "comprising" means that a combination (e.g., an apparatus, composition, or method) includes listed elements (e.g., each unit of an apparatus, each component of a composition, or substantial step of a method), but does not exclude other elements. When defining compositions and methods, "consisting essentially of" means excluding other elements that are significant to the intended combination. Thus, a combination of elements essentially defined herein does not exclude other elements that do not materially affect the substantial and novel characteristics of the claimed invention. "Consisting of" means excluding other combinations of elements, unit components, and substantial method steps. Embodiments defined by each of these transition terms are within the scope of the invention.

[0021] The term "amino acid" can be used interchangeably with "amino acid residue" and refers to free amino acids and peptide amino acid residues. The context will indicate whether the term refers to a free amino acid or a peptide residue. As used herein, "amino acid" includes naturally occurring and synthetic amino acids and is intended to include both D- and L-amino acids. A "standard amino acid" typically refers to any of the 20 standard L-amino acids (including glycine) found in naturally occurring peptides. The terms "D-" and "L-" amino acids used herein are not intended to exclude non-chiral amino acids, such as glycine, unless otherwise specified. A "non-standard amino acid residue" refers to an amino acid other than a standard amino acid, whether synthetic or naturally occurring. As used herein, "synthetic amino acid" further includes chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (e.g., amides), and substitutions. Amino acids contained in the peptides of the present invention, particularly those located at the C- or N-terminus, may be modified by methylation, amidation, acetylation, or substitution with other chemical groups that alter the circulating half-life of the peptide without adversely affecting its activity. Additionally, disulfide bonds may or may not be present in the peptides of the present invention.

[0022] As used herein, the term "pharmaceutical composition" refers to a composition containing at least one active ingredient, which is acceptable for studying a specific effective outcome in a mammal (including, for example, a human). As needed by the artisan, those skilled in the art will understand and comprehend techniques suitable for determining whether an active ingredient has a desired effective effect.

[0023] As used herein, the term "pharmaceutically acceptable carrier" refers to a chemical composition that can be combined with a suitable compound or derivative and, after combination, can be applied to administer the suitable compound to a subject.

[0024] As used herein, the term "physiologically acceptable" ester or salt refers to an ester or salt form of an active ingredient that is compatible with any other ingredients of the pharmaceutical composition, and such form is not harmful to the subject to which the composition is administered.

[0025] As used herein, "pharmaceutically acceptable" refers to physiologically acceptable for human or veterinary use.

[0026] As used herein, a "pharmaceutical composition" refers to a formulation for use in human or veterinary medicine.

[0027] As used herein, "plurality" refers to at least two.

[0028] As used herein, a "polynucleotide" refers to a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide can be a single-stranded or double-stranded nucleic acid.

[0029] As used herein, "polypeptide" refers to a polymer of amino acid residues joined by peptide bonds, and related naturally occurring structural variants and synthetic non-naturally occurring analogues thereof.

[0030] As used herein, a "synthetic peptide or polypeptide" refers to a non-naturally formed peptide or polypeptide. For example, a synthetic peptide or polypeptide can be synthesized using an automated peptide synthesizer.

[0031] As used herein, "N-terminal protection" refers to binding of the terminal amino group of a peptide to any one of a variety of N-terminal protecting groups commonly used in peptide synthesis. As used herein, "C-terminal protection" refers to binding of the terminal amino group of a peptide to any one of a variety of C-terminal protecting groups commonly used in peptide synthesis.

[0032] As used herein, the term "high expression" indicates that the expression level in a subject is higher than that in a normal individual. In particular, the value or level of a particular substance, e.g., a particular biomarker or protein, in a biological sample from a subject is higher than the value or level of that particular substance detected in a biological sample obtained from a healthy or wild-type (normal) individual. As used herein, the term "low expression" indicates that the expression level in a subject is lower than that in a normal individual. In particular, the value or level of a particular substance, e.g., a particular biomarker or protein, in a biological sample from a subject is lower than the value or level of that particular substance detected in a biological sample obtained from a healthy or wild-type (normal) individual. Compared to the "normal" expression level or value of a biomarker, the terms "high expression" and "low expression" can refer to "differential levels," "differential values," or "differential expression," and can include quantitative and qualitative differences in expression levels.

[0033] In some embodiments, the X 1 In some embodiments, X is L-asparagine. In some embodiments, X is D-asparagine. 3 is L-arginine. 3 is D-arginine. 5 is L-lysine. 5 is D-lysine. 4 is L-threonine. 6 is L-tyrosine. 4 is L-threonine, and X 6 is L-tyrosine.

[0034] In some embodiments, the cyclic peptide is a compound of Formula (I) or a derivative thereof. In some embodiments, the cyclic peptide is a compound of Formula (I). [ka]

[0035] In some embodiments, the method for preparing a cyclic peptide further comprises providing an N-terminally protected X 1 Deprotecting the N-terminus of X 1 and obtaining the N-terminally protected X 2 Deprotecting the N-terminus of X 2 and obtaining the N-terminally protected X 3 Deprotecting the N-terminus of X 3 and obtaining the N-terminally protected X 4 Deprotecting the N-terminus of X 4 and obtaining the N-terminally protected X 5 Deprotecting the N-terminus of X 5 and / or the N-terminally protected X 6 Deprotecting the N-terminus of X 6 This includes obtaining

[0036] In some embodiments, the method for preparing a cyclic peptide further comprises providing the X 1 The N-terminus of X is deprotected to obtain a compound having a protected C-terminus. 1 and obtaining the N-terminally and C-terminally protected X 2 The N-terminus of X is deprotected to obtain a compound having a protected C-terminus. 2 and obtaining the N-terminally and C-terminally protected X 3 The N-terminus of X is deprotected to obtain a compound having a protected C-terminus. 3 and obtaining the N-terminally and C-terminally protected X 4 The N-terminus of X is deprotected to obtain a compound having a protected C-terminus. 4 and obtaining the N-terminally and C-terminally protected X 5 The N-terminus of X is deprotected to obtain a compound having a protected C-terminus. 5 and / or the N-terminal and C-terminal protected X 6 The N-terminus of X is deprotected to obtain a compound having a protected C-terminus. 6 This includes obtaining

[0037] In some embodiments, the method for preparing the cyclic peptide comprises providing the X 1 The C-terminus of X is deprotected to obtain a compound having an N-terminus protected therein. 1 and obtaining the N-terminally and C-terminally protected X 2 The C-terminus of X is deprotected to obtain a compound having an N-terminus protected therein. 2 and obtaining the N-terminally and C-terminally protected X 3 The C-terminus of X is deprotected to obtain a compound having an N-terminus protected therein. 3 and obtaining the N-terminally and C-terminally protected X 4 The C-terminus of X is deprotected to obtain a compound having an N-terminus protected therein. 4 and obtaining the N-terminally and C-terminally protected X 5 The C-terminus of X is deprotected to obtain a compound having an N-terminus protected therein. 5 and / or the N-terminal and C-terminal protected X 6 The C-terminus of X is deprotected to obtain a compound having an N-terminus protected therein. 6 This includes obtaining

[0038] In some embodiments, the method for preparing a cyclic peptide further comprises providing a C-terminally protected X 1 Deprotecting the C-terminus of X 1 and obtaining the C-terminally protected X 2 Deprotecting the C-terminus of X 2 and obtaining the C-terminally protected X 3 Deprotecting the C-terminus of X 3 and obtaining the C-terminally protected X 4 Deprotecting the C-terminus of X 4 and obtaining the C-terminally protected X 5 Deprotecting the C-terminus of X 5 and / or obtaining the C-terminally protected X 6 The C-terminus of X is deprotected to form 6 This includes obtaining

[0039] In some embodiments, the method for preparing a cyclic peptide further comprises: 1The C-terminus of X is separated from the solid phase material. 1 and obtaining the X having a solid phase material bound to its C-terminus. 2 The C-terminus of X is separated from the solid phase material. 2 and obtaining the X having a solid phase material bound to its C-terminus. 3 The C-terminus of X is separated from the solid phase material. 3 and obtaining the X having a solid phase material bound to its C-terminus. 4 The C-terminus of X is separated from the solid phase material. 4 and obtaining the X having a solid phase material bound to its C-terminus. 5 The C-terminus of X is separated from the solid phase material. 5 or the X having a solid phase material bound to its C-terminus. 6 The C-terminus of X is separated from the solid phase material. 6 This includes obtaining

[0040] In some embodiments, the X 1 and the C-terminus of X 2 The condensation reaction with the N-terminus of X 1 , the X 2 In some embodiments, the X 2 and the C-terminus of X 3 The condensation reaction with the N-terminus of X 2 , the X 3 In some embodiments, the X 3 and the C-terminus of X 4 The condensation reaction with the N-terminus of X 3 , the X 4 In some embodiments, the X 4 and the C-terminus of X 5 The condensation reaction with the N-terminus of X 4 , the X 5 In some embodiments, the X 5 and the C-terminus of X 6The condensation reaction with the N-terminus of X 5 , the X 6 In some embodiments, the X 6 and the C-terminus of X 1 The condensation reaction with the N-terminus of X 6 , the X 1 , HBTU and DIEA.

[0041] In some embodiments, the N-terminus is protected with Fmoc. In some embodiments, the solid phase material is a resin.

[0042] In some embodiments, a radionuclide is chelated to the complex. 44 Sc, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 99 mTc, 110m In, 111 In, 113 m In, 114m In, 177 Lu, 203 Pb, 212 Pb, 212 Bi, 213 Bi, and 225 In some embodiments, the radionuclide comprises or consists of at least one selected from the group consisting of: Ac 44 Sc, 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 90 Y, 99m Tc, 111 In, 177 Lu, 212 Pb, 213Bi, and 225 In some embodiments, the radionuclide comprises or consists of at least one selected from the group consisting of: Ac 99m Tc, 68 Ga, and 64 In some embodiments, the linker comprises or consists of at least one selected from the group consisting of: Cu. In some embodiments, the linker comprises or consists of polyethylene glycol (PEG). In some embodiments, the radionuclide is 68In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (DTPA), or 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (DTPA). acid, TETA), 2,2'-((6-amino-1-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)hexan-2-yl)azadiacyl)diacetic acid (2,2'-((6-amino-1-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)hexan-2-yl)azanediyl)diacetic acid (NETA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid acid, DO3A), ethylenebis(o-hydroxyphenyl)glycine (EHPG), N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED), 1,4,7,10-tetraazacyclododecane-α,α',α'',α'''-tetramethyl-N,N',N'',N'''-tetraacetic acid,DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid), TETMA, ethylenediamine tetraacetic acid (EDTA), 1,3-propylenediaminetetraacetic acid (PDTA), triethylenetetraaminehexaacetic acid The compound may comprise or consist of at least one selected from the group consisting of 1,5,10-N,N',N''-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM), 1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM), 6-hydrazinonicotinic acid (HYNIC), and ions formed therefrom by losing one or more hydrogen ions.

[0043] In some embodiments, the cyclic peptide is linked to a linker. In some embodiments, X in the cyclic peptide 5 In some embodiments, a lysine in the cyclic peptide, particularly X 5 Lysine, especially X 5 The ε-amino of the lysine is attached to a linker. In some embodiments, the linker is attached to a chelator. In some embodiments, the linker is attached to a cyclic peptide and a chelator, particularly where a first end of the linker is attached to the cyclic peptide and a second end of the linker, different from the first end, is attached to the chelator.

[0044] In some embodiments, the cancer is a tumor with high CD13 expression. In some embodiments, the cancer is selected from the group consisting of head and neck cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, prostate cancer, adrenal cancer, lymphoma, salivary gland cancer, bone cancer, brain tumor, cerebellar cancer, colon cancer, rectal cancer, colorectal cancer, oropharyngeal cancer, kidney cancer, bladder cancer, skin cancer, melanoma, basal cell carcinoma, cancer of the hard palate, squamous cell carcinoma of the tongue, meningioma, pleomorphic adenoma, astrocytoma, soft tissue sarcoma, chondrosarcoma, cortical adenoma, mesothelioma, squamous cell carcinoma, and adenocarcinoma. In some embodiments, the cancer is selected from the group consisting of breast cancer, ovarian cancer, thyroid cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, and osteosarcoma. In some embodiments, the cancer is selected from the group consisting of esophageal cancer, pancreatic cancer, and gastric cancer.

[0045] We have developed an NGR cyclic peptide with a new structure that has higher in vivo stability, higher affinity for the CD13 receptor, and higher targeting ability. After being labeled with a radionuclide, it can accurately identify the location of the CD13 receptor in the body, image tumor molecules using PET to achieve diagnostic purposes, and after being labeled with a therapeutic radionuclide, achieve targeted tumor treatment purposes.

[0046] Provided are NGR cyclic peptides with novel structures and the use of these polypeptides in the preparation of radiopharmaceuticals. In some embodiments, the radiopharmaceuticals and their labeling techniques of the present invention can be applied to molecular imaging and treatment of malignant tumors by targeting the CD13 receptor in neovasculature. In some embodiments, the receptors of the present invention have good targeting properties, high stability, and ease of labeling, making them highly applicable.

[0047] In order to further explain the technical means and effects used by the present invention to achieve the desired objectives, the specific embodiments, structures, features and effects of the present invention will be described in detail below with reference to the drawings and preferred examples.

[0048] Synthesis of cyclic peptides Polypeptides are synthesized from the C-terminus to the N-terminus of the sequence using a solid-phase continuous polypeptide synthesizer.

[0049] 1n equivalents of the first amino acid (C-terminal amino acid) CTC resin bearing the protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) was placed in a reactor and swollen with dichloromethane (DCM) for half an hour. The DCM was removed by suction filtration, and a 2% piperidine N,N'-dimethylformamide (DMF) solution was added to remove the N-terminal Fmoc protecting group of the first amino acid on the resin. In some embodiments, the sequence is cyclo(X 1 X 2 X 3 X 4 X 5 X 6 ) in the cyclic peptide of X 1 , X 2 , X 3 , X 4 , X 5 and X 6 Any one of the amino acids may be the first amino acid.

[0050] 3n equivalents of the next amino acid, 3n equivalents of HBTU (O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), and 10n equivalents of DIEA (N,N-diisopropylethylamine) were added to the reactor, and the condensation reaction was carried out for 5 minutes under continuous reflux. The resin in the reactor was then washed four times with DMF. A 2% piperidine DMF solution was added to remove the N-terminal Fmoc protecting group of the next amino acid, and the resin was washed four times with DMF. In this way, the polypeptide sequence was synthesized.

[0051] After drying the resin with nitrogen blowing, it was removed from the reaction column and placed in a flask. Next, a certain amount (approximately 10 mL of cleavage solution / 1 g of resin) of cleavage solution (composed of 30% trifluoroethanol and 70% dichloromethane) was added to the flask, shaken, and the resin was filtered off.

[0052] After obtaining the filtrate, a large amount of ethyl ether was added to the filtrate to precipitate the crude product, which was then centrifuged and washed to obtain a crude linear polypeptide sequence product with a protecting group.

[0053] The protected peptide segment was dissolved in DCM, and 2n equivalents of PyBop and 10n equivalents of DIEA were added. The mixture was refluxed at 45°C overnight, and the solvent was removed using a rotary evaporator to obtain a cyclic peptide with a protected group.

[0054] A protecting group cleavage solution (consisting of 95% trifluoroacetic acid (TFA), 2% ethanedithiol, 2% triisopropylsilane, and 1% water) was prepared and added to the container containing the cyclic peptide from the previous step, followed by reaction for 120 minutes with shaking.

[0055] After the filtrate was obtained, a large amount of ethyl ether was added to the filtrate to precipitate the crude product, which was then centrifuged and washed to obtain a crude product of the desired cyclic peptide sequence.

[0056] The crude polypeptide sequence product was purified using high performance liquid chromatography (HPLC), lyophilized, and the molecular weight of the desired product was confirmed using liquid chromatography-mass spectrometry (LC-MS).

[0057] Affinity evaluation of cyclic peptides and CD13 protein The binding affinity of the polypeptides of the Examples and Comparative Examples in the HT-1080 cell line was determined by cellular uptake studies.

[0058] Culture of the human fibrosarcoma cell line HT-1080 The human fibrosarcoma cell line HT-1080 was cultured in Gibco's Dulbecco's Modified Eagle's Medium (DMEM) containing 10% sterile-filtered fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. Cell cultures were maintained in a controlled environment at 37°C and 5% CO2, with subcultures performed once every 3–4 days. 24 hours prior to the experiment, cells were seeded into 96-well plates (5,000 cells / well) and cultured overnight until cells adhered to the wells. Before the experiment, cells were washed twice with 1 mL of PBS balanced salt solution to remove the growth medium.

[0059] Preparation of Cy3 fluorescent peptides The cyclic peptides of Examples 1-28 (the sequences of which are shown in SEQ ID NOs: 1-28, respectively, hereinafter referred to as APN1-APN28, and collectively referred to as APN) or the cyclic peptide of Comparative Example 1 (the sequence of which is shown in SEQ ID NO: 29, hereinafter referred to as KE5) were subjected to a condensation reaction with Cy3-NHS fluorescent dye in a DMF solution, and then prepared and purified by HPLC to obtain the APN-Cy3 fluorescent peptides of Examples 1-28 or the KE5-Cy3 fluorescent peptide of Comparative Example 1.

[0060] Treatment of HT-1080 cells with fluorescent peptides In addition to the blank control group, six groups treated with the KE5-Cy3 fluorescent peptide of Comparative Example 1 were prepared at a concentration gradient (3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, and 100 μmol / L (6 wells / group) respectively).

[0061] Comparative Example - HT-1080 Cell Treatment with Cy3 Fluorescent Peptide

[0062] After incubation at 37°C for 30 min, the cells were washed twice with PBS and read using a fluorescent microplate reader (emission wavelength 488 nm, excitation wavelength 520 nm) to detect changes in intracellular fluorescence intensity and evaluate the uptake of the KE5-Cy3 fluorescent peptide into HT-1080 cells.

[0063] 10 μL of CCK-8 was added to the cells of each group, and the cells were incubated for 4 hours. After that, the fluorescence intensity was read using a microplate reader (wavelength 490 nm). Errors in the fluorescence intensity of each group due to differences in cell number were corrected using CCK-8.

[0064] Each concentration group was repeated three times. The optimal concentration for uptake of KE5-Cy3 fluorescent peptide by HT-1080 cells was determined.

[0065] HT-1080 cells were treated with the APN-Cy3 fluorescent peptide of each example, at a concentration that was determined based on the optimal concentration for uptake of the KE5-Cy3 fluorescent peptide of the comparative example by HT-1080 cells.

[0066] After incubation at 37°C for 30 min, the cells were washed twice with PBS and read using a fluorescent microplate reader (emission wavelength 488 nm, excitation wavelength 520 nm) to measure changes in intracellular fluorescence intensity, thereby assessing the uptake of each APN-Cy3 fluorescent peptide by HT-1080 cells.

[0067] 10 μL of CCK-8 was added to the cells of each group, and the cells were incubated for 4 hours. The fluorescence intensity was then read using a microplate reader (wavelength 490 nm). Errors in the fluorescence intensity of each group due to differences in cell number were corrected using CCK-8.

[0068] The relative cell uptake (RCU) between each APN-Cy3 fluorescent peptide and KE5-Cy3 fluorescent peptide was calculated using the following formula.

[0069]

number

[0070] Table 1: Specific sequences of cyclic peptides and relative cellular uptake rates of fluorescent peptides [Table 1]

[0071] In Table 1, Orn represents ornithine, Dab represents 2,4-diaminobutyric acid, Sar represents sarcosine, Nme represents N-methyl-asparagine, and each lowercase letter represents a D-amino acid.

[0072] As can be seen from Table 1, in Examples 2, 3, 4, 5, 6, 9, 15, 16, 18, 19, 21, and 22, the relative cellular uptake of the APN-Cy3 fluorescent peptide was at least twice that of the KE5-Cy3 fluorescent peptide of the comparative example. Among these, in Examples 9, 15, 19, 21, and 22, the relative cellular uptake of the APN-Cy3 fluorescent peptide was at least four times that of the KE5-Cy3 fluorescent peptide. The relative cellular uptake of the APN21-Cy3 fluorescent peptide of Example 9 was approximately 5.03 times that of the KE5-Cy3 fluorescent peptide, and the relative cellular uptake of the APN21-Cy3 fluorescent peptide of Example 21 was approximately 5.68 times that of the KE5-Cy3 fluorescent peptide. This indicates that these cyclic peptides can be particularly effectively taken up by cells.

[0073] From Table 1, the sequence cyclo(X 1 X 2 X 3 X 4 X5 X 6 ) the following can be seen for the cyclic peptide: -X 1 may be asparagine, and X 1 Compared with the case where N-methyl-asparagine is used, the Cy3 fluorescent peptide can achieve a relatively high relative cellular uptake rate. -X 2 X may be glycine or sarcosine, which can provide a higher relative cellular uptake rate. 2 In particular, sarcosine may be used, and its Cy3 fluorescent peptide can achieve a higher relative cellular uptake rate. -X 4 may be selected from the group consisting of threonine, tyrosine and phenylalanine, which can provide a relatively high relative cellular uptake rate. 4 may be particularly threonine, more particularly L-threonine, and the Cy3 fluorescent peptide may have a higher relative cellular uptake rate. -X 5 may be lysine, and X 5 The Cy3 fluorescent peptide can achieve a higher relative cellular uptake rate than when ornithine, 2,4-diaminobutyric acid, or cysteine ​​is used. -X 6 may be selected from the group consisting of tyrosine, valine and glutamic acid, which can result in a higher relative cellular uptake rate. 6 may be, in particular, tyrosine, more particularly L-tyrosine, and the Cy3 fluorescent peptide may have a higher relative cellular uptake rate.

[0074] stability studies In this experiment, the stability of the cyclic peptide APN21 (Example 21) and the cyclic peptide KE5 (Comparative Example 1) (1.0 μg / mL) in mouse plasma or PBS was investigated using an in vitro incubation method at constant temperature (37°C). These two cyclic peptides were dissolved in 1 mL of mouse serum and incubated in vitro for 0 h, 0.5 h, 1 h, 4 h, 24 h, and 48 h, after which the percentage of drug remaining was measured. PBS was used as a negative control. The peak areas of the drug and internal standard were measured using LC-MS / MS, and the ratio was used to calculate the drug concentration.

[0075] The results are shown in Table 2.

[0076] Table 2: Percentage of drug remaining after in vitro incubation in mouse plasma or PBS for the cyclic peptides of Example 21 and the comparative example [Table 2]

[0077] As can be seen from Table 2, compared with the cyclic peptide of Comparative Example 1, the cyclic peptide of Example 21 has a significantly higher drug residual percentage after in vitro incubation at constant temperature in mouse plasma or PBS, indicating its significantly higher stability.

[0078] Animal experiments 1. Synthesis of APN21-Bn-SCN-NOTA 1) 3.8 mg (5.18 μmol) of the cyclic peptide (APN21) of Example 21 was weighed and dissolved in 50 μL of DMF to obtain a DMF solution of APN21. 6.5 mg of N,N-diisopropylethylamine (DIEA) (52 μmol) was added to the above APN21 solution. 2) 2.9 mg (5.18 μmol) of NOTA-Bn-SCN ester trihydrochloride was weighed and dissolved in 50 μL of DMF. 3) The NOTA-Bn-SCN solution was slowly added dropwise to the APN21 solution under shaking, and the reaction mixture was stirred at room temperature for 4 hours. 4) The products were separated by high-performance liquid chromatography (HPLC). The stationary phase was a semi-preparative C18 column. The mobile phase was gradient elution, with a flow rate of 4 mL / min, changing from 5% acetonitrile to 50% acetonitrile within 18 min. 5) The product was analyzed and identified by high performance liquid chromatography and mass spectrometry. Measured by electrospray ionization mass spectrometry (ESI-MS), the m / z [M+H] + =1184.87(Chemical formula:C 53 H 82 N 12 O 16 The molecular weight was 1183.54). The mass spectrum is shown in Figure 1. 6) The fraction containing the desired product was freeze-dried overnight.

[0079] 2.APN21-PEG4-DOTA 68 Ga labeling process 1) 0.75 μmol of APN21-Bn-SCN-NOTA was dissolved in 1.5 mL of 0.25 M sodium acetate solution. 2) Elute the gallium germanium generator with 4 mL of 0.05 M hydrochloric acid solution and rinse. 68 GaCl3 was prepared. 3) Add 1 m of Ci radioactive rinse solution to the APN21-Bn-SCN-NOTA solution and place it in a 60°C heater for 10 min to react. 68 The Ga-APN21-Bn-SCN-NOTA conjugate was obtained. The resulting mixture was monitored by radio-high performance liquid chromatography to quantify the label (purity >97%). The radio-chromatogram is shown in Figure 2.

[0080] In addition, the 68Ga-APN21-Bn-SCN-NOTA conjugate of Comparative Example 1 was prepared by the method for preparing the 68Ga-APN21-Bn-SCN-NOTA conjugate of the Example. 68 Ga-KE5-Bn-SCN-NOTA conjugate and Comparative Example 2 68 A Ga-CG6-Bn-SCN-NOTA conjugate was prepared (where "CG6" refers to a cyclic peptide with the sequence cyclo(CNGRC), set forth in SEQ ID NO: 30).

[0081] 3. Construction of a Human Fibrosarcoma Xenograft Tumor Model HT-1080 cells were collected from normal NCr nude mice (18-25 g, 4-6 weeks old, n=3) and placed in 200 μL of a mixture of phosphate buffer and matrix gel (v / v, 1 / 1). 6 Mice were inoculated subcutaneously into the right shoulder of each mouse. After an average of 1.5 weeks, tumors were approximately 10 mm in diameter, which was sufficient for biodistribution and PET imaging studies.

[0082] 4. Small Animal Positron Emission Computed Tomography (PET / CT) PET / CT and image analysis were performed using a small animal NovelMedcal PET / CT scanner (Beijing Yongxin), with a maximum tangential and radial half-width at the center of the field of view of 1.5 mm and a maximum tangential and radial half-width at the edge of the field of view of 1.8 mm.

[0083] For the CG6 group, after anesthesia with isoflurane, approximately 3.7 MBq (100 μCi) of Comparative Example 2 was administered. 68 The Ga-CG6-Bn-SCN-NOTA compound was injected into HT1080 tumor-bearing mice via the tail vein. 15-minute static PET / CT images were acquired 0.5, 1, and 2 hours after intravenous injection.

[0084] For the KE5 group, after anesthesia with isoflurane, approximately 3.7 MBq (100 μCi) of Comparative Example 1 was administered. 68 The Ga-KE5-Bn-SCN-NOTA compound was injected into HT1080 tumor-bearing mice via the tail vein. Static PET / CT images were acquired for 15 minutes at 0.5, 1, and 2 hours after intravenous injection.

[0085] The APN21 group was anesthetized with isoflurane and then administered approximately 3.7 MBq (100 μCi) of the compound of Example 21. 68 The Ga-APN21-Bn-SCN-NOTA compound was injected into HT1080 tumor-bearing mice via the tail vein. Static PET / CT images were acquired for 15 minutes at 0.5, 1, and 2 hours after intravenous injection.

[0086] For the APN21 blockade group, approximately 3.7 MBq (100 μCi) of Example 21 68 Ga-APN21-Bn-SCN-NOTA and unlabeled NGR peptide (15 mg / kg each) were co-injected into HT1080 tumor-bearing mice (n = 3 / group), and 15-minute static PET / CT images were obtained 0.5, 1, and 2 hours later.

[0087] 3A and 3B are 68 Ga-CG6-Bn-SCN-NOTA only injection (CG6 group), 68 Ga-KE5-Bn-SCN-NOTA only injection (KE5 group), 68 Ga-APN21-Bn-SCN-NOTA injection only (APN21 group), and 68 Static PET / CT images of HT1080 tumor-bearing mice co-injected with Ga-APN21-Bn-SCN-NOTA and unlabeled NGR peptide (APN21 blockade group) at 0.5, 1, and 2 hours after injection, and 68 3A and 3B show the tumor uptake calculated based on the Ga-APN21-Bn-SCN-NOTA signal intensity. 68 The signal intensity of Ga-APN21-Bn-SCN-NOTA was significantly weaker than that of the unblocked APN21 group and was close to that of the KE5 group, suggesting that blocking the CD13 receptor with the NGR peptide 68 It has been shown that the target of action of Ga-APN21-Bn-SCN-NOTA is blocked, i.e. 68 The targeting of Ga-APN21-Bn-SCN-NOTA to the CD13 receptor has been demonstrated.

[0088] PET and CT images were acquired by NMSoft workstation software (Beijing Yongxin), and data were given as a percentage of the injected dose per gram of tissue or organ (ID / g), determined by decay correction for each sample (normalized to a known weight representing the injected dose).

[0089] The above embodiments are merely preferred embodiments of the present invention and do not limit the scope of protection of the present invention. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention will fall within the scope of protection of the present invention.

Claims

1. The sequence is cyclo(X １ X ２ X ３ X ４ X ５ X ６ ) a cyclic peptide having the formula The X １ is asparagine, The X ２ is glycine or sarcosine, The X ３ is arginine, The X ４ is selected from the group consisting of threonine, tyrosine, and phenylalanine; The X ５ is lysine, The X ６ is selected from the group consisting of tyrosine, valine, and glutamic acid.

2. The X ４ is L-threonine or D-threonine, and / or The X ６ The cyclic peptide according to claim 1, wherein is L-tyrosine or D-tyrosine.

3. The X １ is L-asparagine, The X ３ is L-arginine, The X ４ is L-threonine, The X ５ is L-lysine, and / or The X ６ is L-tyrosine, Optionally, the X １ is L-asparagine, and X ２ is sarcosine, and said X ３ is L-arginine, and X ４ is L-threonine, and X ５ is L-lysine, and X ６ is L-tyrosine, Optionally, the X １ is L-asparagine, and X ２ is glycine, and said X ３ is L-arginine, and X ４ is L-threonine, and X ５ is L-lysine, and X ６ is L-tyrosine, or Optionally, the X １ is L-asparagine, and X ２ is sarcosine, and said X ３ is L-arginine, and X ４ is L-threonine, and X ５ is L-lysine, and X ６ The cyclic peptide according to claim 1 or 2, characterized in that is L-tyrosine.

4. A cyclic peptide according to any one of claims 1 to 3; a linker attached to the cyclic peptide; a chelator attached to the linker; A complex comprising:

5. the linker is polyethylene glycol, and / or The chelating agent may be 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid, 1,4,7-triazacyclononane-N,N',N''-triacetic acid, diethylenetriamine-N,N,N',N'',N''-pentaacetic acid, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, 2,2'-((6-amino-1-(4,7-bis(carboxymethyl)-1,4,7-triazan-1-yl)hexan-2-yl)azadiacyl)diacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, ethylenebis(o-hydroxyphenyl)glycine, N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid, comprising or consisting of at least one selected from the group consisting of 1,4,7,10-tetraazacyclododecane-α,α',α'',α'''-tetramethyl-N,N',N'',N'''-tetraacetic acid, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyltetraacetic acid), ethylenediaminetetraacetic acid, 1,3-propylenediaminetetraacetic acid, triethylenetetraminehexaacetic acid, 1,5,10-N,N',N''-tri(2,3-dihydroxybenzoyl)-tricatechol, 1,3,5-N,N',N''-tri(2,3-dihydroxybenzoyl)aminomethylbenzene, 6-hydrazinonicotinic acid, and ions formed therefrom by losing one or more hydrogen ions; Optionally, the complex according to claim 4, wherein the chelating agent comprises 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid.

6. Use of a cyclic peptide according to any one of claims 1 to 3 or a conjugate according to claim 4 or 5 in radionuclide labelling, preparation of a radionuclide labelling reagent, or preparation of a drug carrier.

7. The composite of claim 4 or 5, a radionuclide chelated with a chelating agent of said complex; A radionuclide formulation comprising:

8. The radionuclide is ４４ Sc, ４７ Sc, ６４ Cu, ６７ Cu, ６７ Ga, ６８ Ga, ９９ｍ Tc, ９０ Y. １１１ In, １７７ Lu, ２１２ Pb, ２１３ Bi, Japanese ２２５ 8. The radionuclide preparation according to claim 7, characterized in that it is at least one selected from the group consisting of Ac.

9. 10. Use of a cyclic peptide according to any one of claims 1 to 4, a conjugate according to claim 5 or 6, or a radionuclide formulation according to claim 7 or 8 in the preparation of a medicament for use in the detection of cancer, the diagnosis of cancer, the monitoring of cancer progression, the monitoring of cancer therapy, or the treatment of cancer, Optionally, the cancer is selected from the group consisting of head and neck cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, prostate cancer, adrenal cancer, lymphoma, salivary gland cancer, bone cancer, brain tumor, cerebellar cancer, colon cancer, rectal cancer, colorectal cancer, oropharyngeal cancer, kidney cancer, bladder cancer, skin cancer, melanoma, basal cell carcinoma, cancer of the hard palate, squamous cell carcinoma of the tongue, meningioma, pleomorphic adenoma, astrocytoma, soft tissue sarcoma, chondrosarcoma, cortical adenoma, mesothelioma, squamous cell carcinoma, and adenocarcinoma.

10. the cancer is a CD13-high expressing cancer, and / or 10. The use according to claim 9, characterized in that the cancer is selected from the group consisting of breast cancer, ovarian cancer, thyroid cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer and osteosarcoma.

Images