JP2019043892A - Oligopeptide derivatives and pharmaceuticals using the same - Google Patents

Abstract

To provide oligopeptide derivatives characterized by having: high affinity to αβintegrin; versatility for diagnosis of primary lesion of various tumors; higher accumulation in tumors than in organs; simple and rapid production thereof; safety and utility for use in radiodiagnosis and radionuclide administration therapy as pharmaceuticals; and a radio-labeled arginine-glycine-aspartic acid sequence, and to provide pharmaceuticals using the same.SOLUTION: An oligopeptide derivative of the invention comprises a cyclic oligopeptide sequence including arginine-glycine-aspartic acid sequence and is labeled with a radioisotope. A pharmaceutical of the invention comprises the oligopeptide derivative.SELECTED DRAWING: None

Description

  TECHNICAL FIELD The present invention relates to an oligopeptide derivative used for radiological treatment by binding to a ligand of a cancer cell or a tissue promoted thereby, and a medicine using the same.

Radiopharmaceuticals having a radioisotope, a radionuclide (RI), are used in medical care such as treatment and diagnosis. For example, ⁸⁹ Sr, ⁹⁰ Y, ¹³⁵ I, ¹³¹ I, ¹²⁵ I, ¹⁵³ Sm, ^186/188 Re, ²¹¹ At, ²²³ Ra, etc. are known as such radionuclides for therapeutic use, and ¹⁸ F for in vivo diagnosis. , ^67/68 Ga, ^99m Tc, ¹¹¹ In, ¹²³ I, ¹³³ Xe, etc. are known, and ³ H, ¹²⁵ I etc. are known for in vitro diagnosis.

  Radionuclide internal therapy (Radionuclide Therapy) consists of intravenous or oral administration of a radionuclide therapeutic drug, which accumulates the radiopharmaceutical at focal sites such as primary or metastatic cancer, and releases the radiopharmaceutical from the radiopharmaceutical Radiation such as alpha rays or beta rays destroys cancer cells at the lesion site.

Sodium iodide (Nuclide: ¹³¹ I), Strontium chloride ( ⁸⁹ Sr), and radio-labeled anti-CD20 antibody ( ⁹⁰ Y) that emit β-rays as medicines for radionuclide internal medicine that are covered by insurance in Japan And radium chloride (the same ²²³ Ra) that emits alpha rays.

  For radionuclide internal therapy using these agents, for example, the thyroid uptake property of iodine in hyperthyroidism and thyroid cancer is used, and radium accumulates in the area of pathological conditions in which bone metabolism such as bone metastasis is enhanced. Taking advantage of the properties, the drug of radionuclide sodium iodide or radium chloride is administered, and alpha rays and beta rays are emitted at focal sites such as thyroid cancer and bone metastasis areas to destroy cancer cells. To kill and to exert an antitumor effect.

  Even if the radionuclide internal therapy drug that emits beta radiation accumulates in the cancer cells at the focal site, the beta radiation has a relatively long range of about 0.5 mm and reaches the cancer cells and nearby normal cells, Not only destruction of cancer cells but also normal cells are damaged. On the other hand, when the radionuclide internal therapy drug that emits alpha rays accumulates in the cancer cells at the lesion site, the alpha rays have a linear energy transfer (LET) about 400 times higher than beta rays (alpha rays: 80 keV / μm) , Β rays: 0.2 keV / μm), having strong cytotoxicity and extremely short range. Therefore, alpha rays only reach the target site of the cancer cells and destroy the cancer cells, but do not damage neighboring normal cells.

²¹¹ At is also known as a radionuclide that emits such alpha rays. ²¹¹ At is a halogen element whose detailed physical properties are still unknown because stable isotope does not exist. At has similar chemical properties and chemical reactivity to other halogens such as I and Br. Therefore, synthetic methods with known halogen derivatives can be applied to At labeling to introduce ²¹¹ At as a radionuclide for treatment. ²¹¹ At has a short half life of 7.2 hours and high safety. If such ²¹¹ At can be efficiently accumulated at target sites such as various lesion sites, a high therapeutic effect can be expected in radionuclide internal therapy.

By the way, 18 types of α subunits and 8 types of β subunits have been confirmed as integrins that are receptors that penetrate the plasma membrane and are involved in cell adhesion (Non-Patent Document 1). Among them, α _v β ₃ integrin is a marker of angiogenesis and is overexpressed in various cancer cells (Non-patent Document 2). Or expression of α _v β ₃ integrin in endothelial cells of the blood vessel at the time of the enhancement of angiogenesis, the α _v β ₃ integrin on the cancer cells of the proto-primary tumor of the tumor is or expressed by the tumor. A peptide containing an arginine-glycine-aspartic acid (RGD) sequence exhibits high affinity for α _v β ₃ integrin (Non-patent Document 3)

Patent Document 1 describes an antagonist compound of α _v β ₃ integrin showing selective affinity to α _v β ₃ integrin, which contains a cyclic RGD motif and two echistatin C-terminal moieties covalently linked by a spacer sequence. And chelating agents capable of complexing radioactive isotopes are exemplified.

Patent Document 2 discloses that at least one of α ₃ β ₁ -integrin, α ₅ β ₁ -integrin, α _v β ₃ -integrin and α _v β ₅ -integrin, and other integrins is inhibited, and Or a method of inhibiting bFGF production in an animal subject, comprising the step of administering to the subject an effective amount of an RG cysteic acid peptide or derivative thereof ing. Patent Document 2 exemplifies a method in which RG cysteic acid peptide or a derivative thereof is radioactively labeled and used to detect a tumor.

There is a need for a peptide having an arginine-glycine-aspartate sequence that exhibits high affinity to α _v β ₃ integrin and can be used for radiodiagnosis of cancer primary lesions of various tumors and internal radionuclide therapy.

Margret Schottelius et al., “Ligands for Mapping αvβ3-Integrin Expression in Vivo”, Account of Chemical Research., 42, 2009, p. 969-980 Roland Haubner et al., “Radiolabeled αvβ3 Integrin Antagonists: A New Class of Tracers for Tumor Targeting”, Journal of Nuclear Medicine, 1999, Vol. 40, p. 1061-1071. Ulrich Hersel et al., "RGD modified polymers: biomaterials for stimulated cell adhesion and beyond", Biomaterials, 2003, 24: 4385-4415.

Japanese Patent Application Publication No. 2009-512643 JP, 2017-105830, A

The present invention was made to solve the above-mentioned problems, and has high affinity to α _v β ₃ integrin, which is versatile for medical treatment of cancer primary lesions of various tumors, and is higher for tumors than organs. An oligopeptide comprising a radiolabeled arginine-glycine-aspartic acid sequence that exhibits accumulation, can be simply and rapidly manufactured, is safe, can be used for radiodiagnosis and radionuclide internal therapy, and can be used as a medicine It aims at providing a derivative and a medicine using the same.

  The oligopeptide derivative of the present invention made to achieve the above-mentioned purpose is that it is labeled with a radioactive isotope while being composed of a cyclic oligopeptide sequence and having an arginine-glycine-aspartic acid sequence.

  The oligopeptide derivative is capable of binding, coordinating and / or affinity to an integrin.

  This oligopeptide derivative is any radioactive nuclide selected from the group consisting of radioactive iodine, radioactive astatine, radioactive strontium, radioactive yttrium, radioactive radium, radioactive bismuth, radioactive actinium, radioactive lutetium, radioactive rhenium, and radioactive copper Is preferably contained.

  This oligopeptide derivative is one in which the radioactive isotope emits alpha rays.

The oligopeptide derivative is such that the radioactive isotope contains, for example, ²¹¹ At.

  In this oligopeptide derivative, the radioisotope is attached to an aromatic ring group linked directly to an aromatic ring group of an amino acid residue of the cyclic oligopeptide sequence or to the cyclic oligopeptide sequence via or without a spacer group. It is preferred that they be linked.

  In this oligopeptide derivative, it is more preferable that the cyclic oligopeptide sequence has an arginine-glycine-aspartic acid-D-amino acid sequence.

  In this oligopeptide derivative, it is even more preferable that the D-amino acid in the cyclic oligopeptide sequence is D-phenylalanine or D-tyrosine.

  This oligopeptide derivative is, for example, cyclic (arginine-glycine-aspartate-phenylalanine-lysine) oligopeptide derivative, cyclic (arginine-glycine-aspartate-tyrosine-lysine) oligopeptide derivative, cyclic (arginine-glycine-aspartic acid- Phenylalanine-valine) oligopeptide derivatives or cyclic (arginine-glycine-aspartate-tyrosine-valine) oligopeptide derivatives.

  This oligopeptide derivative is a cyclic (arginine-glycine-aspartate-D-phenylalanine-lysine) oligopeptide derivative, a cyclic (arginine-glycine-aspartic acid-D-tyrosine-lysine) oligopeptide derivative, a cyclic (arginine-glycine- More preferably, they are aspartic acid-D-phenylalanine-valine) oligopeptide derivatives or cyclic (arginine-glycine-aspartic acid-D-tyrosine-valine) oligopeptide derivatives.

  The medicament of the present invention made to achieve the above-mentioned purpose is one containing the above-mentioned oligopeptide derivative.

The oligopeptide derivative of the present invention has a simple structure and can selectively bind, coordinate, and / or have high affinity for integrins, particularly α _v β ₃ integrin, and can be a conventional oligopeptide derivative or the like Compared to radioactive isotope labeled compounds, it is superior in tumor specificity which shows higher accumulation in tumors than organs.

Since this oligopeptide derivative can be easily and rapidly produced in high purity simply and easily, it can be contained as a radioactive isotope having a relatively long half life or a relatively short isotope date of about several days, and the safety is high. Is excellent in quality. Furthermore, this oligopeptide derivative can be used as a medicine. In particular, even if ²¹¹ At, which has a short half-life of 7.2 hours, is used as a radioactive isotope, it can be prepared a short time before administration by a simple synthesis method. It can be demonstrated.

  This oligopeptide derivative is due to the high accumulation of tumor tissue, for example, cancer cells in the primary tumor of a tumor, metastatic micrometastatic tissue or metastasized metastatic tissue, and neovascularization by the tumor. It has high tumor tissue selective imaging ability by radioactive isotopes, and cancer cells and killing ability due to it.

In particular, when the radioactive isotope is ²¹¹ At that emits an alpha ray, only cancer cells are destroyed and normal cells are not damaged.

A drug containing this oligopeptide derivative is allowed to accumulate the oligopeptide derivative on integrins, particularly α _v β ₃ integrin of cancer cells and their neovascular endothelial cells by oral administration, intravenous administration or direct administration to tumor tissue. To use it for selective radiodiagnosis of tumor tissue for accurate and reliable image diagnosis and radionuclide internal therapy for accumulating active ingredients in tumor tissue for reliable and safe destruction of cancer cells and apoptosis. It can be induced.

  In addition, since the drug containing this oligopeptide derivative has high accumulation in tumor tissue, it can also be used for radiodiagnosis, and it is safe and reliable for tumor tissue site, blood vessel site enhanced by tumor tissue, micro cancer metastasis The site of cancer metastasis, the axillary lymph node site to be dissected, etc. can be identified or used to diagnose the progress of the tumor.

It is a graph which shows the result of in-body radioactivity distribution in a tumor-bearing model mouse about the oligopeptide derivative which applies this invention. It is a graph which shows the result of in-body radioactivity distribution in a tumor-bearing model mouse about another oligopeptide derivative to which this invention is applied. It is a graph which shows the result of in-body radioactivity distribution in a tumor-bearing model mouse about another oligopeptide derivative which applies this invention. It is a graph which shows the result of in-body radioactivity distribution in a tumor-bearing model mouse about another oligopeptide derivative which applies this invention. It is a graph which shows the result of the inhibition test by a non-radioactive ligand about the oligopeptide derivative which applies this invention.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, the scope of the present invention is not limited to these forms.

  The oligopeptide derivative of the present invention is one in which the amino acid sequence consists only of a cyclic oligopeptide sequence and is labeled with a radioactive isotope while having an arginine-glycine-aspartic acid sequence.

The oligopeptide derivative, having an arginine-glycine-aspartic acid sequence, recognizes, binds to, coordinates with, and / or affinity to an integrin, particularly an α _v β ₃ integrin among the family. It is possible to

The radioactive isotopes are radioactive iodine ( ¹³⁵ I, ¹³¹ I, ¹²⁵ I), radioactive astatine ( ²¹¹ At), radioactive strontium ( ⁸⁹ Sr), radioactive yttrium ( ⁹⁰ Y), radioactive radium ( ²²³ Ra), radioactive bismuth ( ^212/213 Bi), radioactive actinium ( ²²⁵ Ac), radioactive lutetium ( ¹⁷⁷ Lu), radioactive rhenium ( ^186/188 Re), and any radioactive nuclide selected from radioactive copper ( ^64/67 Cu) and their decay Or a stable isotope or radionuclide thereof.

Among them, ¹²⁵ I emitting X-rays and ²¹¹ At emitting α-rays are preferable. In particular, when the radioactive isotope is ²¹¹ At, an oligopeptide derivative used as a medicine for internal radionuclide therapy binds to α _v β ₃ integrin of cancer cells in the lesion site or vascular endothelial cells enhanced thereby. It accumulates in a short time, and alpha rays reach only tissues such as cancer cells to be killed, causing damage and inducing destruction and apoptosis but not neighboring normal cells.

  The radioactive peptide may be bound to an aromatic ring group linked directly or with or without a spacer group to a cyclic oligopeptide sequence containing an arginine-glycine-aspartic acid sequence. The aromatic ring group may be a hydrocarbon aromatic ring such as a phenyl group or a heteroaromatic ring. In the oligopeptide derivative, when there is a phenylalanine residue or a tyrosine residue in the cyclic oligopeptide sequence, the radioactive isotope may be directly bound to the aromatic ring. An aromatic ring group linked via a spacer group is, for example, a benzoyl group linked to a free ε-amino group at its end when a lysine residue is present in the cyclic oligopeptide sequence, and in which the radioactive isotope is linked. And benzyloxycarbonyl group.

  The oligopeptide derivative is composed of, for example, a cyclic pentaoligopeptide sequence, and the functional group may be protected with a non-amino acid, but another amino acid is not bound to the cyclic oligopeptide sequence. is there. This cyclic pentaoligopeptide sequence may be composed of naturally occurring L amino acids, or may be composed of one or more being substituted with non-naturally occurring D amino acids.

  In the oligopeptide derivative, the cyclic oligopeptide sequence has, for example, an arginine-glycine-aspartic acid-D-amino acid sequence. Among the oligopeptide derivatives, cyclic oligopeptide sequences are preferably cyclic (arginine-glycine-aspartic acid-any amino acid-lysine) or cyclic (arginine-glycine-aspartic acid-any amino acid-valine), cyclic (arginine-arginine) It is more preferable that -glycine-aspartate-D-amino acid-lysine or cyclic (arginine-glycine-aspartate-D-amino acid-valine) is used. Preferably, any amino acid in the cyclic pentapeptide sequence is tyrosine or phenylalanine, more preferably D-tyrosine or D-phenylalanine.

The oligopeptide derivative is a cyclic (arginine-glycine-aspartic acid-D-amino acid-lysine) oligopeptide derivative labeled with a radioactive isotope, or a cyclic (arginine-glycine-aspartic acid-D-labeled with a radioactive isotope) The amino acid-valine) has a high affinity for α _v β ₃ integrin.

（式(Ｉ)中、Ｘ^１は前記の放射性同位体を示す）で表わされるもので、フェニルアラニン残基のベンゼン環に直接放射性同位体¹²⁵Ｉである放射性ヨウ素又は²¹¹Ａｔである放射性アスタチンで直接標識した環状（アルギニン−グリシン−アスパラギン酸−フェニルアラニン−リジン）オリゴペプチド誘導体であることが好ましい。中でもＸ^１が¹²⁵Ｉ又は²¹¹Ａｔである化合物は、生体内放射能分布試験を行ったところ、腫瘍組織への高い集積を示した。 More specifically, the oligopeptide derivative has the following chemical formula (I):

(Wherein, in the formula (I), X ¹ represents the above-mentioned radioactive isotope), radioactive iodine which is radioactive isotope ¹²⁵ I directly to the benzene ring of the phenylalanine residue or radioactive astatine which is ²¹¹ At It is preferable that it is a labeled cyclic (arginine-glycine-aspartic acid-phenylalanine-lysine) oligopeptide derivative. Among them, compounds in which X ¹ is ¹²⁵ I or ²¹¹ At showed high accumulation in tumor tissue when in vivo radioactivity distribution test was performed.

The synthesis method of the oligopeptide derivative represented by the chemical formula (I) is, for example, a solid phase synthesis of a cyclic (arginine-glycine-aspartic acid-iodine substituted phenylalanine-lysine) oligopeptide having a nonradioactive iodine substituted phenylalanine residue by peptide solid phase synthesis A nonradioactive iodine-radioactive iodine substitution reaction, such as substituting the iodine group with an organotin, in the presence of an oxidizing agent such as chloramine T or NCS, for example, radioactive iodine ion ( ¹²⁵ I ⁻ ) or radioactive astatine ion ( ²¹¹ At ^-) is that substituting.

で表わされるもので、チロシン残基のベンゼン環に放射性同位体¹²⁵Ｉである放射性ヨウ素で直接標識した環状（アルギニン−グリシン−アスパラギン酸−Ｄ−チロシン−リジン）オリゴペプチド誘導体（式(II)中、Ｘ^２は前記の放射性同位体を示す）であってもよい。中でもＸ^２が¹²⁵Ｉである化合物は、生体内放射能分布試験を行ったところ、腫瘍組織への高い集積を示した。 Further, another oligopeptide derivative is specifically a cyclic (arginine-glycine-aspartic acid-tyrosine-lysine) oligopeptide derivative, in particular, a compound represented by the following chemical formula (II):

And a cyclic (arginine-glycine-aspartic acid-D-tyrosine-lysine) oligopeptide derivative directly labeled with radioactive iodine, which is a radioactive isotope ¹²⁵ I on the benzene ring of a tyrosine residue , X ² may represent the above-mentioned radioactive isotopes). Among them, compounds in which X ² is ¹²⁵ I showed high accumulation in tumor tissue when in vivo radioactivity distribution test was performed.

The synthetic method of the oligopeptide derivative represented by the chemical formula (II) is to prepare cyclic (arginine-glycine-aspartic acid-D-tyrosine-lysine) oligopeptide by peptide solid phase synthesis, and then radioiodine ion in the presence of chloramine T ( ¹²⁵ I ⁻ ) is to be introduced next to the hydroxyl group of a tyrosine residue.

（式(III)中、Ｘ^３は前記の放射性同位体を示す）で表わされるもので、放射性同位体¹²⁵Ｉである放射性ヨウ素又は²¹¹Ａｔである放射性アスタチンで標識されたベンゾアミドでリジン残基が保護されている環状（アルギニン−グリシン−アスパラギン酸−フェニルアラニン−リジン）オリゴペプチド誘導体であってもよい。中でも、Ｘ³が¹²⁵Ｉである化合物は、生体内放射能分布試験を行ったところ、腫瘍組織へ幾分低い集積性を示したが、腸への高い集積性も示した。 Furthermore, another oligopeptide derivative is specifically represented by the following chemical formula (III):

(Wherein, in the formula (III), X ³ represents the above-mentioned radioactive isotope) and the radioactive residue is a radioactive isotope that is the radioactive isotope ¹²⁵ I or a radioactive astatine that is ²¹¹ At, and a lysine residue is a benzoamide labeled It may be a protected cyclic (arginine-glycine-aspartic acid-phenylalanine-lysine) oligopeptide derivative. Among them, compounds in which X ³ is ¹²⁵ I showed somewhat lower accumulation in tumor tissue when subjected to in vivo radioactivity distribution tests, but also showed high accumulation in the intestine.

The synthesis method of the oligopeptide derivative represented by the chemical formula (III) is, for example, t- to N-succinimidyl 3- (tri-n-butyltin) benzoate (N-Succinimidyl 3- (tri-n-butylstannyl) benzoate: ATE) butyl hydroperoxide (tert-butyl hydroperoxide) radioactive iodide ions in the presence of ⁽¹²⁵ I ^-) and was reacted N- succinimidyl 3- ¹²⁵ iodobenzoate ^{(N-succinimidyl 3- 125 iodobenzoate)} , unsubstituted cyclic (arginine -Glycine-aspartate-phenylalanine-lysine) oligopeptide is reacted to obtain a cyclic (arginine-glycine-aspartate-phenylalanine-lysine) oligopeptide compound protected as radioiodine-labeled bennzamide in the presence of triethylamine It is a thing. Radioactive iodine ( ^125I ) substitution can be obtained from cyclic (arginine-glycine-aspartic acid-tyrosine-lysine) in the same manner.

  The medicament of the present invention contains the oligopeptide derivative as an active ingredient.

  This drug can be used to conduct epidermal growth factor receptor (EGFR) imaging studies.

This drug contains the oligopeptide derivative as an active ingredient that binds to, coordinates with, and / or interacts with the α _v β ₃ integrin and, if necessary, a non-toxic inactive pharmaceutically acceptable agent. It is formulated by mixing with excipients, for example, solid, semisolid or liquid diluents, dispersants, fillers and carriers. Stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, flavor preservatives, medium, saline, and other medicinal effects The medicine which it has may be included as an additive.

  The dosage form of this medicine is, for example, elixir, capsule, granule, pill, ointment, suspension, solution, enteric agent, emulsion, plaster, suppository, powder, tablet, syrup, injection, troche Agents, ointments, haptics, liniments, limonade agents, lotions. It may be dissolved or suspended in a liquid medium, or may be dispersed in a solid medium.

  Although this medicine may be administered orally, it is preferable to administer by intravenous injection / instillation from the viewpoint of absorption, distribution and metabolism, and may be directly injected in the vicinity of tumor tissue.

  In this medicine, the oligopeptide derivative is labeled with a very small amount of stoichiometry which does not show the pharmacological effect of a tracer amount, and is administered after purification.

  The dosage and dose of this medicine depend on the efficacy of the active ingredient that is the oligopeptide derivative, the form and route of administration, the stage of cancer progression, the patient's body type, body weight and age, and other therapeutic agents used in combination. It is selected appropriately according to the amount. The administration is administered intermittently at intervals of 1 to 30 days or at intervals of 1 to 6 months.

This medicine is used as follows to exert its effect. For example, a medicament containing an oligopeptide derivative having ²¹¹ At is administered to a patient whose tumor is to be treated. The oligopeptide derivative contained in the drug is flowed through blood or the like by oral administration or blood administration or is administered near the tumor site to reach the tumor site. The oligopeptide derivative is expressed in the α _v β ₃ integrin expressed in the cancer cells of the primary tumor of the tumor or in the endothelial cells of blood vessels in promoting tumor angiogenesis by the presence of the cyclic oligopeptide sequence. -Bind to, coordinate with, and / or affinity receptor sites for the glycine-aspartate sequence. As a result, this oligopeptide derivative is accumulated exclusively in cancer cells and vascular endothelial cells at the time of tumor-induced angiogenesis. At that time, the radioactive isotope ²¹¹ At of the oligopeptide derivative emits alpha rays. The alpha ray has a short range distance, and selectively destroys cancer cells and the like of the tumor tissue which has been bound or the like by radioactivity to induce apoptosis and to be killed. However, alpha rays do not damage normal tissues because they can not reach normal cells in the vicinity.

  Although the case where this medicine is used for radiation therapy has been described as an example, since it is accumulated similarly even when used for radiodiagnosis, cancer cells and vascular endothelial cells at the time of promotion of angiogenesis by tumors are also accumulated at the time of radiodiagnosis But can not damage normal tissue in the vicinity.

  The following is an example of synthesizing the oligopeptide derivative of the present invention and demonstrating its utility as a pharmaceutical.

The reagents and instruments used are as follows.
(Reagents and equipment)
Peptide solid phase is a reagent necessary for the synthesis of 2-chlorotrityl chloride resin (2-chlorotrityl chloride resin), Fmoc-Arg (Pbf) -OH, Fmoc-Asp (O t Bu) -OH, Fmoc-Gly-OH, ^{Fmoc-D-Tyr (t Bu} ) -OH, Fmoc-D-Phe-OH Watanabe chemical industry Co., Ltd. (Tokyo, Japan), Fmoc-Lys (Boc) -OH, human purified α _v β ₃ integrin, a thin layer Chromatography (TLC Silica gel 60F ₂₅₄ ) is Merck (Darmstadt, Germany), 1-hydroxybenzotriazole hydrate) 1-hydroxybenzotriazole hydrate (HOBt), 1,3-diisopropylcarbodiimide (DIPCDI) is a domestic chemical corporation (Tokyo, Japan) N, N-dimethylformamide (DMF) is purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), [ ¹²⁵ I] NaI is purchased from Perkin Elmer (Waltham, MA, USA), Other reagents were purchased from Nacalai Tesque (Kyoto, Japan), and all reagents used were special grade reagents. ²¹¹ At ^- was supplied by the national university corporation Osaka University. U87MG (human glioma cell line) was purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). About reverse phase HPLC, Comosil 5C ₁₈ -AR-II (4.6 x 150 mm), Comosil 5C ₁₈ -AR-II (10 x 150 mm) column is Nacalai Tesque, Inc. (Kyoto, Japan), liquid transfer unit LC-20 AD And LC-20AR, column ovens CTO-10A and CTO-20A, and SPD-10A and SPD-20A of absorbance detectors manufactured by Shimadzu Corporation (Kyoto, Japan) using an electron spray ionization mass spectrometer (ESI- MS) JMS-T100TD from Nippon Denshi Co., Ltd. (JEOL) (Tokyo, Japan), AccuFLEX gamma ARC-7010 for radiation measurement system autowell gamma system from Hitachi Healthcare Manufacturing Co., Ltd. (柏, Japan), and The Wallac Wizard 1470 Gamma Counter used Perkin Elmer (Waltham, MA, USA).

Example 1: Oligopeptide Derivative of the Formula (I) Type
The oligopeptide derivative represented by the above chemical formula (I) was synthesized by the method shown in the following chemical reaction formulas [1] to [4]. Moreover, the oligopeptide derivative obtained was used for the efficacy test. The details of the synthesis are as follows.

Synthesis of Fmoc-4-iodo-D-phenylalanine (Fmoc-4-iodo-D-phenylalanine) (1) According to the existing method, as shown in the chemical reaction formula [1], I ₂ and NaIO _{3 for} D-phenylalanine To give iodo-D-phenylalanine (D-Phe (4-I) -OH) and then reacting Fmoc-OSu in the presence of K ₂ CO ₃ to give Fmoc-4-iodo-D-phenylalanine ( 1) (Fmoc-D-Phe (4-I) -OH) was obtained. The physicochemical analysis result is DART-MS (m / z calcd for ([M + H] ⁺ ): 514.04 found: 514.04), which supports the structure of compound (1).

c with Compound 1 [Arg (Pbf) -Gly-Asp (O t Bu) -4-iodo-D-Phe-Lys (Boc)] (2), as shown in reaction formula [2] The resin-bound peptide and Fmoc amino acid are combined by stirring in N, N-dimethylformamide (DMF) in the presence of N, N-diisopropylcarbodiimide and HOBt according to a conventional Fmoc solid phase synthesis method, and the Fmoc group is obtained by piperidine The peptide chain is extended by repeating the procedure of deprotecting it to form a linear oligopeptide bound to the solid support resin (black circle) shown by the same formula, followed by solid phase with 30% hexafluoroisopropanol (HFIP) Cleavage from the support resin to H ₂ N-Asp (O ^t Bu) -D-Phe-Lys (Boc) -Arg (Pbf) -Gly-OH in the presence of diphenyl phosphate azide (DPPA) and NaHCO ₃ c was synthesized [Arg (Pbf) -Gly-Asp (O t Bu) -4-iodo-D-Phe-Lys (Boc)] (2). Purification by reverse phase HPLC gave the compound (2) (63.8 mg, 37.4%) as a white powder. Reverse phase HPLC using a Cosmosil 5C ₁₈ -AR-II (10 x 150 mm) column, mobile phase from water: methanol (20:80) containing 0.1% tetrafluoroacetic acid (TFA) over 10 minutes It was carried out under the conditions of UV = 220 nm and a flow rate of 4.0 mL / min by the gradient method of conversion to (10:90). The physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] ⁺ ): 1138.41 found: 1138.66), which supports the structure of compound (2).

Synthesis of Ic (Arg-Gly-Asp-D-Phe-Lys) (3) As shown in the chemical reaction formula [4], compound (2) (10 mg: 8.79 μmol) was dissolved in TFA: water: triisopropylsilane ((( It was dissolved in a mixed solution of i-Pr) ₃ SiH) (38: 1: 1, 500 μL) and stirred at room temperature for 2 hours. The solvent was distilled off with nitrogen gas, and the residue was purified by reverse phase HPLC to obtain Compound Ic (Arg-Gly-Asp-D-Phe-Lys) (3) (6.1 mg, 95.2%) as a white powder. The Reverse phase HPLC uses a Cosmosil 5C ₁₈ -AR-II (10 x 150 mm) column and the mobile phase is from 50: 50 in 10 minutes from water: methanol (70:30) containing 0.1% TFA. It carried out on the conditions of UV = 220 nm and the flow rate of 4.0 mL / min by the gradient method converted to (iii). Its physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] ⁺ ): 730.21 found: 730.18), which supports the structure of compound (3).

Sn (nBu) ₃ -c, as shown in [Arg (Pbf) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] Synthesis reaction formula (4) [3], compound (2 ) (5.6 mg 4.9 μmol) is dissolved in anhydrous 1,4-dioxane (400 μL), bis (tributyltin ([(nBu) ₃ Sn] ₂ ) (5.96 μL: 2.4 equivalents), and bis (triphenylphosphine) Add palladium (II) dichloride (PdCl ₂ [P (C ₆ H ₅ ) ₃ ] ₂ ) (517.26 μL: 0.15 equivalents), stir for 2 hours while heating to 60 ° C. under nitrogen atmosphere, then purify by reverse phase HPLC was carried out to obtain a white powder of _{Sn (nBu) 3 -c [Arg} (Pbf) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] (4) (512μg, 8.00%). Reverse phase HPLC uses a Cosmosil 5C ₁₈ -AR-II (10 x 150 mm) column and the mobile phase is from water: methanol (5:95) containing 0.1% TFA in 10 minutes (2.5: 97.5) It was performed under the condition of UV = 220 nm, flow rate 4.0 mL / min by the gradient method to be converted to ESI-MS (m / z calcd for ([M + H] ⁺ ): 1302.62 found]. : 1302.62), and the structure of compound (4) is To equity.

Synthesis of Sn (nBu) ₃ -c (Arg-Gly-Asp-D-Phe-Lys) (5) As shown in the chemical reaction formula [4], Compound (3) (2.0 mg 2.74 μmol) was dissolved in anhydrous methanol Dissolve in (300 μL), add N, N-diisopropylmethylamine (DIEA, 2.5 equivalents), and bis (tributyltin) ([(nBu) ₃ Sn] ₂ ) (5.82 μL, 4.0 equivalents), and add nitrogen gas. I sprayed it. Thereafter, tris (dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃ ) (384.6 μg, 0.15 equivalent) is added, and after stirring for 12 hours while heating to 70 ° C. under nitrogen atmosphere, reversed phase HPLC Purification was performed to obtain Sn (nBu) ₃ -c (Arg-Gly-Asp-D-Phe-Lys) (5) (839.7 μg, 34.3%) as white powder. Reversed phase HPLC uses a Cosmosil 5C ₁₈ -AR-II (10 x 150 mm) column and the mobile phase is from water: methanol (30:70) containing 0.1% TFA for 10 minutes (25:75) It carried out on the conditions of UV = 220 nm and the flow rate of 4.0 mL / min by the gradient method converted to (iii). The physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] ⁺ ): 894.42 found: 894.44), which supports the structure of compound (5).

¹²⁵ Ic as shown in [Arg (Pbf) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] Preparation chemical equation (6) [3], compound (4) (30 [mu] g, 23.0 nmol) is dissolved in ethanol (79 μL), chloramine T / ethanol solution (1 mg / mL, 15 μL), 1% acetic acid / ethanol solution (21 μL), and [ ¹²⁵ I] NaI (3.7 MBq, 3 μL) ) was added and left at room temperature for 10 ^{minutes, 125 Ic [Arg (Pbf)} -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] by purified by reverse phase HPLC (6) (Radiochemical yield: 82.9%, radiochemical purity: 96.6%) was obtained. Reversed phase HPLC uses a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column and the mobile phase is 0.1% TFA in water: methanol (20:80) over 20 minutes (0: 100) The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into

Preparation of ¹²⁵ Ic (Arg-Gly-Asp-D-Phe-Lys) (7) (using Compound (6))
As shown in the chemical reaction formula [3], the compound (6) was dissolved in a mixed solution of TFA: water: triisopropylsilane (38: 1: 1, 80 μL) and stirred at room temperature for 90 minutes. The solvent is distilled off with nitrogen gas, the residue is purified by reverse phase HPLC, and ¹²⁵ Ic (Arg-Gly-Asp-D-Phe-Lys) (7) (radiochemical yield: 75.1%, radiochemistry Target purity: 97.0% (hereinafter sometimes abbreviated as ¹²⁵ Ic (RGDfK)). Reverse phase HPLC using a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (70:30) containing 0.1% TFA (30:70) for 20 minutes The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into Reversed-phase HPLC analysis and comparison of this compound (7) with the radioactive isotope unlabeled and iodine-substituted compound (3) Ic (Arg-Gly-Asp-D-Phe-Lys) (3) As a result, each single elution peak was detected at the same retention time, and it was confirmed to be compound (7).

Preparation of ¹²⁵ Ic (Arg-Gly-Asp-D-Phe-Lys) (7) (Use Compound (5))
As shown in the chemical reaction formula [4], the compound (5) (30 μg, 33.6 nmol) is dissolved in acetonitrile (5 μL), 1% acetic acid / acetonitrile solution (10 μL), [ ¹²⁵ I] NaI (3.7 MBq , 3 μL), and N-chlorosuccinimide (NCS) / acetonitrile solution (1 mg / mL, 15 μL) and react for 15 minutes while heating to 80 ° C., and then an aqueous solution of sodium bisulfite (NaHSO ₃ ) (NaHSO ₃ ) The reaction was quenched with 1 mg / mL, 15 μL) and purified by reverse phase HPLC to obtain a compound (7) (radiochemical yield: 83.1%, radiochemical purity: 96.1%). Reverse phase HPLC using a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (70:30) containing 0.1% TFA (30:70) for 20 minutes The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into Similar to the above, when the compound (7) and the compound (3) Ic (Arg-Gly-Asp-D-Phe-Lys) are analyzed by reversed-phase HPLC, the single retention time is identical to the single retention time. One elution peak was detected and confirmed to be compound (7).

^{211 At-c [Arg (Pbf} ) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] (8) and ^{211 At-c (Arg-Gly} -Asp-D-Phe-Lys) ( Preparation of 9) As shown in the chemical reaction formula [3], compound (4) (30 μg, 23.0 nmol) is dissolved in acetonitrile (5 μL), 1% acetic acid / acetonitrile solution (10 μL), [ ²¹¹ At] After adding At ⁻ / H ₂ O (3.7 MBq, 3 μL) and N-chlorosuccinimide (NCS) / acetonitrile solution (1 mg / mL, 15 μL) and reacting for 15 minutes while heating to 80 ° C. sodium bisulfite (NaHSO ₃₎ solution (1 mg / mL, 15 μL ) by stopping the reaction at ^{211 at-c [Arg (Pbf} ) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc ] (8) (radiochemical yield: 91.2%) was obtained. After that, the solvent was distilled off with nitrogen gas, the residue was dissolved in a mixed solution of TFA: water: triisopropylsilane (38: 1: 1, 80 μL), and shaken at room temperature for 10 minutes. After distilling off the solvent with nitrogen gas, the residue is purified by reverse phase HPLC, and ²¹¹ At-c (Arg-Gly-Asp-D-Phe-Lys) (9) (radiochemical yield: 33.3%, Radiochemical purity: 97.7% (hereinafter sometimes abbreviated as ²¹¹ At-c (RGDfK)). Reversed phase HPLC uses a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column, the mobile phase is water: methanol (70:30) containing 0.1% TFA, and the flow rate is 1.0 mL / min. It went on condition. Comparing this compound (9) with the radioactive isotope unlabeled and iodine-substituted compound (3) Ic (Arg-Gly-Asp-D-Phe-Lys) by reversed-phase HPLC, A single elution peak was detected near the retention time, and it was confirmed that this was a compound (9).

Preparation of ²¹¹ At-c (Arg-Gly-Asp-D-Phe-Lys) (9) (using Compound 5)
Compound (5) (30 μg, 33.6 nmol) is dissolved in acetonitrile (5 μL) as shown in the chemical reaction formula [4], 1% acetic acid / acetonitrile solution (10 μL), [ ²¹¹ I] At ⁻ / H _After adding ₂ O (7.4 MBq, 100 μL) and N-chlorosuccinamide (NCS) / acetonitrile solution (1 mg / mL, 15 μL) and reacting for 15 minutes while heating to 80 ° C., sodium bisulfite (NaHSO 4) ₃ ) The reaction is quenched with an aqueous solution (1 mg / mL, 15 μL) and purified by reverse phase HPLC to give ²¹¹ At-c (Arg-Gly-Asp-D-Phe-Lys) (9) (radiochemistry) Target yield: 50.1%, radiochemical purity: 96.9%). Reversed phase HPLC uses a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column, the mobile phase is water: methanol (70:30) containing 0.1% TFA, and the flow rate is 1.0 mL / min. It went on condition. Similar to the above, when compared with the analysis by reversed phase HPLC of this compound (9) and Ic (Arg-Gly-Asp-D-Phe-Lys) (3) which is the above-mentioned compound (3), retention At times where the times were close, single elution peaks were detected, respectively, and it was confirmed that this was compound (9).

で示すI-c(Arg-Gly-Asp-D-Phe-Lys) (3)（以下、I-c(RGDfK)と略記することがある）は、実施例１の途中で合成したものである。その理化学分析結果は、ESI-MS (m/z calcd for ([M+H]⁺): 730.21 found: 730.18)であり、化合物(3)の構造を支持する。
なお、実施例１で合成した²¹¹Ａｔで標識した誘導体（化合物(9)）と比較例１-1における非標識のＩで置換した誘導体（化合物(3)）との逆相HPLCによる分析にはCosmosil 5C₁₈-AR-II (4.6 × 150 mm)カラムを使用し、標識した誘導体に対し放射性強度と非標識の誘導体に対し220nm紫外線とで検出し、移動相は0.1%のTFAを含有する水:メタノール(70:30)、流速1.0 mL/minの条件で行ったところ、²¹¹Ａｔで標識した誘導体の方がやや遅いリテンションタイムに、夫々単一の溶出ピークが現れ、高い純度であることが示された。 Comparative Example 1-1: Radioisotope-Unlabeled Oligopeptide Derivative of the Formula (I) Type
A radioisotopic unlabeled oligopeptide derivative for the type of formula (I) type

Ic (Arg-Gly-Asp-D-Phe-Lys) (3) (hereinafter sometimes abbreviated as Ic (RGDfK)) shown in the following is synthesized in the middle of Example 1. Its physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] ⁺ ): 730.21 found: 730.18), which supports the structure of compound (3).
For analysis of the ²¹¹ At-labeled derivative (compound (9)) synthesized in Example 1 and the unlabeled I-substituted derivative (compound (3)) in Comparative Example 1-1 by reverse-phase HPLC Using a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column, the mobile phase is detected with radioactive intensity for labeled derivatives and 220 nm UV for unlabeled derivatives, the mobile phase is water containing 0.1% TFA : When conducted under conditions of methanol (70:30) and a flow rate of 1.0 mL / min, a single elution peak appears at a slightly slower retention time for the derivative labeled with ²¹¹ At, and the purity is high Indicated.

その理化学分析結果は、ESI-MS (m/z calcd for ([M+H]⁺): 604.31 found: 604.41) であり、化合物(10)の構造を支持する。 Comparative Example 1-2: Halogen-unsubstituted Oligopeptide Derivative of the Formula (I) Type
The halogen-unsubstituted oligopeptide derivative c (Arg-Gly-Asp-D-Phe-Lys) (10) (hereinafter sometimes abbreviated as c (RGDfK)) to the chemical formula (I) type is an example It synthesize | combined as follows according to the method of Example 1 except having used iodine non-substituted D-phenylalanine as a raw material in 1.

The physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] ⁺ ): 604.31 found: 604.41) and supports the structure of compound (10).

で示す¹²⁵I-c(Arg-Gly-Asp-D-Tyr-Lys) (11) (以下、¹²⁵I-c(RGDyK)と略記することがある)は、下記の方法で合成した。c(Arg-Gly-Asp-D-Tyr-Lys) (13) (100 μg, 0.16 μmol)を0.1Mリン酸緩衝液(PH:7.4)(100 μL)に溶解し、[¹²⁵I]NaI (3.7 MBq, 1 μL)、クロラミンT/水溶液(1 mg/mL, 10 μL)加えて室温にて10分間放置したのち、亜硫酸水素ナトリウム(NaHSO₃)水溶液(1 mg/mL, 10 μL)で反応を停止し、逆相HPLCにて精製することで¹²⁵I-c(Arg-Gly-Asp-D-Tyr-Lys) (11) (放射化学的収率: 84.7%、放射化学的純度: 98.2%)を得た。逆相HPLCにはCosmosil 5C₁₈-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(80:20)から20分間で(50:50)に変換するグラジエント法にて、流速1.0 mL/minの条件で行った。この化合物(11)と、後述する放射性同位体非標識でヨウ素置換体である化合物(12)であるI-c(Arg-Gly-Asp-D-Tyr-Lys)との逆相HPLCによる分析と比較したところ、同じリテンションタイムに、夫々単一の溶出ピークが検出され、化合物(11)であることが確認された。 Example 2 Oligopeptide Derivative of the Formula (II) Type
The oligopeptide derivative represented by the above chemical formula (II) is a compound described later according to the method of Example 1 except that it is desired to use D-tyrosine instead of iodine-substituted D-phenylalanine as a raw material in Example 1 (13) is synthesized, the following chemical formula

¹²⁵ Ic (Arg-Gly-Asp-D-Tyr-Lys) (11) (hereinafter sometimes abbreviated as ¹²⁵ Ic (RGDyK)) is synthesized by the following method. c (Arg-Gly-Asp-D-Tyr-Lys) (13) (100 μg, 0.16 μmol) is dissolved in 0.1 M phosphate buffer (PH: 7.4) (100 μL), and [ ¹²⁵ I] NaI ( 3.7 MBq, 1 μL), chloramine T / water solution (1 mg / mL, 10 μL), left at room temperature for 10 minutes, reacted with sodium bisulfite (NaHSO ₃ ) aqueous solution (1 mg / mL, 10 μL) Is stopped and purified by reverse phase HPLC to give ¹²⁵ Ic (Arg-Gly-Asp-D-Tyr-Lys) (11) (radiochemical yield: 84.7%, radiochemical purity: 98.2%) Obtained. Reverse phase HPLC using a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (80: 20) containing 0.1% TFA (50: 50) for 20 minutes The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into This compound (11) was compared with the analysis by reversed phase HPLC of Ic (Arg-Gly-Asp-D-Tyr-Lys) which is compound (12) which is a radioactive isotope unlabeled and iodine-substituted form described later However, at the same retention time, each single elution peak was detected, and it was confirmed that it was compound (11).

で示すI-c(Arg-Gly-Asp-D-Tyr-Lys) (12) (以下、I-c(RGDyK)と略記することがある)は、下記の方法で合成した。ヨウ素(I₂) (254mg 1mmol)、亜硝酸ナトリウム(NaNO₂) (69mg 1mmol)を水:メタノール(1:1)溶液10mLに室温で30分以上撹拌した。この溶液68.3μLにc(RGDyK) (3.25mg 5.25μmol)を加えて室温で3時間撹拌後、逆相HPLCによる精製を行い、白色粉末のI-c(Arg-Gly-Asp-D-Tyr-Lys) (12) (1.28mg, 32.71%)を得た。逆相HPLCにはCosmosil 5C₁₈-AR-II (10 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(80:20)から、10分間で(65:35)に変換するグラジエント法にてUV = 220 nm、流速4.0 mL/minの条件で行った。その理化学分析結果は、ESI-MS (m/z calcd for ([M+H]⁺): 746.20 found: 746.25)であり、化合物(12)の構造を支持する。 Comparative Example 2-1: Nonradioactive halogen-substituted oligopeptide derivative of the formula (II) type
A nonradioactive halogen-substituted oligopeptide derivative to the formula (II) type:

Ic (Arg-Gly-Asp-D-Tyr-Lys) (12) (hereinafter sometimes abbreviated as Ic (RGDyK)) represented by is synthesized by the following method. The iodine (I ₂ ) (254 mg 1 mmol) and sodium nitrite (NaNO ₂ ) (69 mg 1 mmol) were stirred in 10 mL of a water: methanol (1: 1) solution at room temperature for 30 minutes or more. After adding c (RGDyK) (3.25 mg 5.25 μmol) to 68.3 μL of this solution and stirring at room temperature for 3 hours, purification by reverse phase HPLC is performed to obtain white powder Ic (Arg-Gly-Asp-D-Tyr-Lys). (12) (1.28 mg, 32.71%) was obtained. Reverse phase HPLC uses a Cosmosil 5C ₁₈ -AR-II (10 x 150 mm) column and the mobile phase is from water: methanol (80:20) containing 0.1% TFA for 10 minutes (65:35) It carried out on the conditions of UV = 220 nm and the flow rate of 4.0 mL / min by the gradient method converted to (iii). The physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] ⁺ ): 746.20 found: 746.25), which supports the structure of compound (12).

で示すc(Arg-Gly-Asp-D-Tyr-Lys) (13) (以下、c(RGDyK)と略記することがある)は、実施例１での原料としてＤ−チロシンを用いたいこと以外は、実施例２の方法に準じて合成した。その理化学分析結果は、ESI-MS (m/z calcd for ([M+H]⁺): 620.31 found: 620.27)であり、化合物(13)の構造を支持する。 Comparative Example 2-2: Halogen-unsubstituted Oligopeptide Derivative of Chemical Formula (II) Type
The following chemical formula, which is a non-halogenated oligopeptide derivative to the chemical formula (II) type:

C (Arg-Gly-Asp-D-Tyr-Lys) (13) (hereinafter sometimes abbreviated as c (RGDyK)) shown in the above is the case except that D-tyrosine is used as a raw material in Example 1 Was synthesized according to the method of Example 2. The physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] ⁺ ): 620.31 found: 620.27), which supports the structure of compound (13).

に示す方法で、リジン残基が放射性同位体¹²⁵Ｉである放射性ヨウ素で標識されたベンゾアミドとして保護されている環状（アルギニン−グリシン−アスパラギン酸−フェニルアラニン−リジン）オリゴペプチド化合物(14)（以下、¹²⁵IB-c(RGDfK)と略記することがある）として、以下のようにして合成した。
ATE (0.5 mg, 0.98 μmol)をエタノール(55 μL)に溶解し、クロラミンT/エタノール溶液(4 mg/mL, 15 μL)、1%酢酸/エタノール溶液(21 μL)、及び[¹²⁵I]NaI (3.7 MBq, 24 μL)を加えて室温にて10分間放置し、溶媒を、窒素ガスにより約50 μL程度に濃縮後、水(50 μL)を加え、逆相HPLCを用いて精製し、[¹²⁵I]SIBを得た。逆相HPLCにはCosmosil 5C₁₈-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(50:50)から20分間で(30:70)に変換するグラジエント法にて、流速1.0 mL/minの条件で行った。[¹²⁵I]SIBを含む溶液を、50℃に加熱しながら、窒素ガスにて溶媒を完全に留去し、DMF (50 μL)に溶解後、c(RGDfK) (50 μg, 68 nmol) に加え、TEA (0.5 μL)を加えて室温にて40分インキュベートを行った後に逆相HPLCにより精製を行い、化合物(14) (放射化学的収率: 30%、放射化学的純度: 99%)を得た。逆相HPLCにはCosmosil 5C₁₈-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(60:40)から20分間で(10:90)に変換するグラジエント法にて、流速1.0 mL/minの条件で行った。
なお、実施例３で合成した標識した誘導体(化合物(14))と後述する比較例３で合成した非標識の誘導体(化合物(15))との逆相HPLCによる分析にはCosmosil 5C₁₈-AR-II (4.6 × 150 mm)カラムを使用し、標識した誘導体に対し放射性強度と非標識の誘導体に対し220nm紫外線とで検出し、移動相は0.1%のTFAを含有する水と0.1%のTFAを含有するメタノールとの60:40〜10:90での0〜20minのグラジエントにて、流速1.0 mL/minの条件で行ったところ、同じリテンションタイムに夫々単一の溶出ピークが現れ、標識／非標識以外は同一であることが示された。 Example 3 Oligopeptide Derivative of the Formula (III) Type
The oligopeptide derivative represented by the above chemical formula (III) is a compound represented by the following chemical reaction formula [5], using the compound (10) (c (RGDfK)) obtained in Comparative Example 1-2.

Cyclic (arginine-glycine-aspartate-phenylalanine-lysine) oligopeptide compound (14) (hereinafter referred to as a cyclic arginine-glycine-aspartic acid-phenylalanine-lysine) protected as a radioactive iodine-labeled benzamide in which the lysine residue is a radioactive isotope ¹²⁵ I ^The compound was synthesized as ¹²⁵ IB-c (sometimes abbreviated as RGDfK) as follows.
ATE (0.5 mg, 0.98 μmol) is dissolved in ethanol (55 μL), chloramine T / ethanol solution (4 mg / mL, 15 μL), 1% acetic acid / ethanol solution (21 μL), and [ ¹²⁵ I] NaI Add (3.7 MBq, 24 μL) and leave at room temperature for 10 minutes. Concentrate the solvent to about 50 μL with nitrogen gas, add water (50 μL), and purify using reverse phase HPLC [ < ¹²⁵ > I] SIB was obtained. Reverse phase HPLC using a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (50:50) containing 0.1% TFA (30:70) for 20 minutes The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into The solution containing [ ¹²⁵ I] SIB was completely distilled off with nitrogen gas while heating to 50 ° C., dissolved in DMF (50 μL), and then dissolved in c (RGDfK) (50 μg, 68 nmol) In addition, TEA (0.5 μL) was added and incubation was carried out at room temperature for 40 minutes, followed by purification by reverse phase HPLC to obtain compound (14) (radiochemical yield: 30%, radiochemical purity: 99%) I got Reverse phase HPLC using a Cosmosil 5C ₁₈ -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (60:40) containing 0.1% TFA for 20 minutes (10:90) The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into
In addition, Cosmosil 5C ₁₈ -AR is used for analysis of the labeled derivative (compound (14)) synthesized in Example 3 and the unlabeled derivative (compound (15)) synthesized in Comparative Example 3 described later by reverse phase HPLC. Using a -II (4.6 x 150 mm) column and detecting radioactive intensity for labeled derivatives and 220 nm UV for unlabeled derivatives, the mobile phase is water containing 0.1% TFA and 0.1% TFA And a gradient of 0 to 20 min at 60: 40 to 10: 90 with methanol containing a water at a flow rate of 1.0 mL / min, each single elution peak appears at the same retention time, and It was shown to be identical except for unlabeled.

で示す非放射性同位体のヨウ素で置換ベンゾアミドとして保護されている環状（アルギニン−グリシン−アスパラギン酸−フェニルアラニン−リジン）オリゴペプチド化合物(15)（以下、IB-c(RGDfK)と略記することがある）は、実施例３での原料中間体としてＮ-スクシンイミジル ３-(¹²⁵ヨード)ベンゾエートに代えて、Ｎ-スクシンイミジル ３-ヨードベンゾエートを用いたこと以外は実施例３の方法に準じて合成した。その理化学分析結果は、
ESI-MS (m/z calcd for ([M+H]⁺): 834 found: 834)であり、化合物(15)の構造を支持する。 Comparative Example 3 Radioisotope-Unlabeled Oligopeptide Derivative of the Formula (III) Type
The following chemical formula is a radioisotopic unlabeled oligopeptide derivative to the chemical formula (III) type

A cyclic (arginine-glycine-aspartate-phenylalanine-lysine) oligopeptide compound (15) (hereinafter referred to as IB-c (RGDfK)) protected as a non-radioactive isotope iodine-substituted benzamide shown in ) Was synthesized according to the method of Example 3 except that N-succinimidyl 3-iodobenzoate was used in place of N-succinimidyl 3- ( ¹²⁵ iodo) benzoate as a raw material intermediate in Example 3. The physicochemical analysis result is
ESI-MS (m / z calcd for ([M + H] ⁺ ): 834 found: 834) and supports the structure of compound (15).

  Below, the method to which the oligopeptide derivative which applied this invention and the oligopeptide derivative which does not apply this invention were evaluated, and the result are shown.

(Evaluation test 1: In vitro binding assay for α _v β ₃ integrin (Binding Assay))
Evaluation of affinity for α _v β ₃ integrin has been reported in the past (Dijkgraaf I et al., “Improved targeting of the αv β3 integral by multimerisation of RGD peptides.” EJNMMI., 2007, Vol. 34, No. 2, We changed p. 267-273) slightly and carried out as follows. Coating buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl ₂ 0.5 mM MgCl ₂ , 1 mM MnCl ₂ , pH so that human purified α _v β ₃ integrin (200 μg / mL) is 600 ng / mL Then, 100 μL of each was added to a 96-well immuno lock well module plate (Thermo, Waltham, Mass., USA) and allowed to stand overnight at 4 ° C. Thereafter, the wells are washed twice with binding buffer [0.1% bovine serum albumin (BSA) / coating buffer, 200 μL], blocking buffer (1% BSA / coating buffer, 200 μL) is added, and left at room temperature for 2 hours. Then, it was washed twice with binding buffer (200 μL). Next, ¹²⁵ Ic (RGDyK) as the radioligand, Ic (RGDfK) and c (RGDfK) as the nonradioactive ligand, and ¹²⁵ Ic (RGDfK) and ²¹¹ At-c (RGDfK) as the radioligand as the nonradioactive ligand c (RGDfK), radioligand (37 kBq, 95 μL) and non-radioactive ligand (2 nM-2 mM, 5 μL) are added to the wells and shaken at 37 ° C for 1 hour, then the wells Were washed with binding buffer (200 μL, 300 μL, 350 μL, 3 times in total) to measure the radioactivity of the wells.

In addition, since a stable isotope does not exist in astatine in this evaluation test 1, it evaluates, changing a radioligand, fixing a non-radioactive ligand. The results are shown in Table 1 below.

As is clear from Table 1, when the affinity of ¹²⁵ Ic (RGDyK), ¹²⁵ Ic (RGDfK), and ²¹¹ At-c (RGDfK) to α _v β ₃ integrin was examined, the result of having high affinity was obtained. It was done.

Further, in the same manner as in this evaluation test 1, in the case of the nuclide in which a stable isotope is present, the radioligand was fixed and the cold body was compared and evaluated. The results are shown in Table 2 below.

(Evaluation test 2: Radioactivity distribution in tumor-bearing model mice)
U87MG human glioma cells are cultured in EMEM medium at 5% CO ₂ at 37 ° C., and the cell number is 5 × 10 ⁶ in nude mice [4 weeks, BALB / c Slc-nu / nu, female (15-19 g; Japan SLC)] was implanted subcutaneously on the back of the patient. Twenty-eight days later, a mixed solution (37 kBq, 74 kBq / 100 μL) of ¹²⁵ Ic (RGDfK) and ²¹¹ At-c (RGDfK) was administered to the tumor-bearing model mice via the tail vein. One and four hours after administration, the mice were sacrificed, each organ was removed, and the weight and radioactivity of the organ were measured. ²¹¹ radioactivity At performs measurements at the energy range of 70-120 keV, ¹²⁵ I is ²¹¹ At was measured with an energy range of 1-80 keV after attenuation.

The results are shown in FIG. As is clear from FIG. 1, both ¹²⁵ Ic (RGDfK) and ²¹¹ At-c (RGDfK) were highly accumulated in tumor tissue. Further, ¹²⁵ Ic (RGDfK), and ²¹¹ both compounds of At-c (RGDfK) indicate the biodistribution similar, it iodine anion, is radioactive accumulation in the stomach known as integrated organ A statin anion lower From the above, it was found that significant deiodination and deastatination did not occur in vivo.

The radioactivity distribution in the body was similarly measured except that ¹²⁵ Ic (RGDyK) and ¹²⁵ Ic (RGDfK), ¹²⁵ Ic (RGDyK), and ¹²⁵ IB-c (RGDfK) were used alone. The results are shown in FIGS. 2 to 4 respectively. As apparent from FIG. 2, three compounds of ¹²⁵ Ic (RGDyK), ¹²⁵ Ic (RGDfK) and ¹²⁵ Ic (RGDyK) were highly accumulated in the tumor tissue. ^{Further, 125 Ic (RGDyK), 125} Ic (RGDfK) and three compounds of ¹²⁵ Ic (RGDyK) indicate the biodistribution similar, that radioactive accumulation in the stomach known as integrated organ iodine anion is low From the results, it was found that significant deiodination did not occur in vivo. Also, as is clear from FIG. 4, ¹²⁵ IB-c (RGDfK) showed somewhat lower accumulation in tumor tissues as well as in tumor tissues, but high accumulation in the intestine (small intestine; 54.9% one hour after administration ID / g) is shown.

(Evaluation test 3: inhibition test by non-radioactive ligand)
Tumor-bearing model mice are prepared in the same manner as above, and a mixture of c (RGDfK) (0.2 mg / mouse), ¹²⁵ Ic (RGDfK), and ²¹¹ At-c (RGDfK) (37 kBq, 74 kBq / 100 μL) At the same time, tail vein was administered. One hour after administration, the mice were sacrificed, each organ was removed, and the weight and radioactivity of the organ were measured. ²¹¹ radioactivity At performs measurements at the energy range of 70-120 keV, ¹²⁵ I is ²¹¹ At was measured with an energy range of 1-80 keV after attenuation.

The results are shown in FIG. Figure 5 is clear from, than the accumulation in the tumor was significantly reduced by c (RGDfK) blocking experiments by simultaneous administration, ¹²⁵ Ic (RGDfK), and accumulation in tumors of ²¹¹ At-c (RGDfK) has, alpha It turned out that _v beta ₃ integrin is mediated.

From these results, it was found that Ic (RGDfK) exhibits as high binding affinity as that of c (RGDfK) for α _v β ₃ integrin. Further, in the body radioactivity distribution experiment ¹²⁵ Ic (RGDfK) it was observed distribution tendency similar to ¹²⁵ Ic (RGDyK). Furthermore, ²¹¹ At-c (RGDfK) showed similar pharmacokinetics to ¹²⁵ Ic (RGDfK). ^{In 125} Ic (RGDfK), radioactive accumulation in the kidney (6.7% ID / g one hour after administration) was observed early in administration, but it was cleared without retention, and radioactive accumulation in tumor (post-administration) 4.1% ID / g in 1 hour) is higher than the ¹²⁵ IB-c (RGDfK), biodistribution tendency similar to ¹²⁵ Ic (RGDyK) was observed.

  In addition, although the oligopeptide derivative of this invention itself was described as an example which served as a pharmaceutical, when administering to a patient, it is possible to prepare and use as a pharmaceutical which is a pharmaceutical formulation of a tablet or an injection.

  INDUSTRIAL APPLICABILITY The oligopeptide derivative of the present invention is useful as a medicine that specifically attacks and destroys cancer cells in a primary tumor or a metastatic tissue, and has industrial applicability.

Claims (11)

  An oligopeptide derivative characterized by comprising a cyclic oligopeptide sequence and labeled with a radioactive isotope while having an arginine-glycine-aspartic acid sequence.   The oligopeptide derivative according to claim 1, characterized in that it binds, coordinates and / or has an affinity to an integrin.   The radioactive isotope includes any radioactive nuclide selected from radioactive iodine, radioactive astatine, radioactive strontium, radioactive yttrium, radioactive radium, radioactive bismuth, radioactive actinium, radioactive lutetium, radioactive rhenium, and radioactive copper The oligopeptide derivative according to any one of claims 1 to 2 characterized by the above.   The oligopeptide derivative according to any one of claims 1 to 3, wherein the radioactive isotope emits an alpha ray. The oligopeptide derivative according to any one of claims 1 to 4, wherein the radioactive isotope contains ²¹¹ At.   The radioisotope is bound to an aromatic ring group linked directly to an aromatic ring group of an amino acid residue of the cyclic oligopeptide sequence or to the cyclic oligopeptide sequence via or without a spacer group. The oligopeptide derivative according to any one of claims 1 to 5 characterized by the above.   The oligopeptide derivative according to any one of claims 1 to 6, wherein the cyclic oligopeptide sequence has an arginine-glycine-aspartic acid-D-amino acid sequence.   The oligopeptide derivative according to claim 7, wherein the D-amino acid in the cyclic oligopeptide sequence is D-phenylalanine or D-tyrosine.   Cyclic (arginine-glycine-aspartate-phenylalanine-lysine) oligopeptide derivative, cyclic (arginine-glycine-aspartate-tyrosine-lysine) oligopeptide derivative, cyclic (arginine-glycine-aspartate-phenylalanine-valine) oligopeptide derivative The oligopeptide derivative according to any one of claims 1 to 8, which is or a cyclic (arginine-glycine-aspartic acid-tyrosine-valine) oligopeptide derivative.   Cyclic (arginine-glycine-aspartic acid-D-phenylalanine-lysine) oligopeptide derivative, cyclic (arginine-glycine-aspartic acid-D-tyrosine-lysine) oligopeptide derivative, cyclic (arginine-glycine-aspartic acid-D-phenylalanine The oligopeptide derivative according to any one of claims 1 to 9, which is-valine) oligopeptide derivative or cyclic (arginine-glycine-aspartic acid-D-tyrosine-valine) oligopeptide derivative.   A medicament comprising the oligopeptide derivative according to any one of claims 1 to 10.

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