WO2013081091A1 - Drug for producing radiolabeled polypeptide reducing non-specific renal accumulation - Google Patents

Abstract

Provided is a radiolabeled drug which contains a compound expressed in the following formula (I); the compound to which polypeptide is bound which is capable of being bound to a target site, or a pharmacologically acceptable salt of the compound; and a complex which includes a complex structure which contains the compound to which the polypeptide is bound which is capable of being bound to the target site, or the pharmacologically acceptable salt thereof, and a radioisotope. (I) Herein, R₁ is a hydrogen atom, a methyl group, or a carboxymethyl group; R₂ is CH₂-CH₂-NH-CH₂-COOH, CH₂-CH₂-N(CH₂-COOH)₂, CH₂-CH₂-NH₂, CH₂-CH₂-NHR₃, the carboxymethyl group, or CH₂-CH₂-NR₃R₄; R₃ and R₄ are alkyl groups which may be different; X, Y, and Z are amino acids which may be different; and F is a functional group which is capable of being bound to the polypeptide. Using the compound according to the present invention, the radiolabeled drug is provided which is prepared through a simple operation, has high accumulation toward the target site, and reduces non-specific renal accumulation.

Description

Drug for producing radiolabeled polypeptide with reduced nonspecific renal accumulation

The present invention relates to a drug for producing a radiolabeled polypeptide with reduced nonspecific renal accumulation. More specifically, the present invention relates to an agent for producing a radioactive technetium-labeled polypeptide or a radioactive rhenium-labeled polypeptide with reduced nonspecific renal accumulation. The present invention also provides a radiolabeled polypeptide produced using the radiolabeled polypeptide-producing agent, a diagnostic or therapeutic pharmaceutical composition comprising the radiolabeled polypeptide as an active ingredient, and the radiolabel. The present invention relates to a diagnostic imaging method and a therapeutic method using a polypeptide.

A radiolabeled drug is a drug containing a compound labeled with a radioisotope (RI), and is widely used for diagnosis and treatment of diseases, for example, diagnosis and treatment of tumors. In diagnosis and treatment using radiolabeled drugs, selection of useful nuclides and drug design for accumulating the drugs in specific tissues and cells have been performed.

Low molecular weight polypeptides such as antibody fragments labeled with radioisotopes are expected to be applied as probes for molecular imaging including cancer, and as internal radiotherapy drugs using β-ray emitting nuclides. However, when an RI-labeled low molecular weight polypeptide is administered to a living body, radioactivity is observed in the kidney for a long time from the early stage of administration. Therefore, there is a risk of kidney exposure and kidney damage, and dosage adjustment is required. Thus, accumulation of radiolabeled polypeptide in the kidney is a major obstacle to its application to diagnostic imaging and treatment.

In contrast, the present inventors have devised a drug design that binds a low molecular weight polypeptide and a highly urinary excretion labeling drug through a substrate of kidney brush border membrane enzyme. And the usefulness of this drug design was clarified from the examination using radioactive iodine (nonpatent literature 1). Furthermore, in order to verify the applicability of this drug design to metal radioisotope labeling, development to organic rhenium compounds was performed and the possibility was shown (Non-patent Document 2, Patent Document 1).

JP 2005-47821 A.

In order to produce a labeled polypeptide by designing a drug that binds a low molecular weight polypeptide and a highly urinary labeling drug via a substrate of a renal brush border membrane enzyme, a plurality of steps are required. Therefore, it is actually difficult to produce such a labeled polypeptide for preclinical or clinical use.

An object of the present invention is to provide a compound that can be produced by a simple operation and that can provide a radiolabeled drug that has high accumulation at a target site and reduced nonspecific renal accumulation.

In order to solve the above problems, the inventors of the present invention have proposed a ligand structure that forms a complex with radioactive technetium- ^99m ( ^99m Tc) and radioactive rhenium-186 and 188 ( ^186/188 Re), and a renal brush border membrane enzyme. The substrate structure was examined.

As a result, radioactive iodine labeling can be achieved by simply mixing [M (CO) ₃ (OH ₂ ) ₃ ] + (M = Tc or Re) with the polypeptide to which the compound represented by the following formula (II) is bound. We were able to produce a metal-RI-labeled polypeptide that showed almost the same pharmacokinetics as drugs and metal-rhenium-labeled drugs and reduced nonspecific renal accumulation. The present invention has been achieved based on this finding.

That is, the present invention relates to the following.
(1) a compound represented by the following formula (I), or a pharmacologically acceptable salt thereof,

Here, R ₁ is a hydrogen atom, a methyl group, or a carboxymethyl group (CH ₂ —COOH), and R ₂ is CH ₂ —CH ₂ —NH—CH ₂ —COOH, CH ₂ —CH ₂ —N (CH _2- COOH) ₂ , CH ₂ -CH ₂ -NH ₂ , CH ₂ -CH ₂ -NHR ₃ , a carboxymethyl group, or CH ₂ -CH ₂ -NR ₃ R ₄ , wherein R ₃ and R ₄ are each An alkyl group which may be different,
X, Y, and Z are all amino acids that may be different from each other,
F is a functional group that can bind to a polypeptide.
(2) The functional group F capable of binding to the polypeptide is any one selected from the group consisting of carboxylic acid and its active ester, maleimide group, bromoacetyl group, iodoacetyl group, isothiocyanate group, and amino group Or a pharmacologically acceptable salt thereof.
(3) The compound of (1) or (2) above, wherein X, Y, and Z are glycine, L-phenylalanine, and lysine, or a pharmaceutically acceptable salt thereof.
(4) The compound according to any one of (1) to (3) above, wherein the functional group F capable of binding to the polypeptide is a maleimide group, or a pharmaceutically acceptable salt thereof.
(5) A compound represented by the following formula (II) or a pharmacologically acceptable salt thereof.

(6) A compound obtained by binding a polypeptide that binds to a target molecule to a compound of any one of (1) to (5) above or a pharmacologically acceptable salt thereof, or a pharmacologically acceptable salt thereof salt.
(7) The compound of (6) above, wherein the polypeptide that binds to the target molecule is an antibody Fab fragment or Fv fragment, or a pharmaceutically acceptable salt thereof.
(8) A radiolabeled polypeptide preparation drug comprising the compound according to any one of (1) to (5) above or a pharmacologically acceptable salt thereof.
(9) A complex structure formed from a metal radioisotope and the compound of any one of (1) to (5) or a pharmacologically acceptable salt bound to a polypeptide that binds to a target molecule A radiolabeled drug comprising a complex having
(10) The metal radioisotope is any one metal radioisotope selected from technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177 (9) Radiolabeled drugs.
(11) The radiolabeled drug according to (9) or (10), wherein the polypeptide that binds to the target molecule is an antibody Fab fragment or Fv fragment.
(12) A radiolabeled drug comprising a complex having a complex structure formed from technetium-99m and a compound represented by the following formula (II) to which an antibody Fab fragment is bound.

(13) A complex structure formed from a metal radioisotope and the compound of any one of (1) to (5) or a pharmacologically acceptable salt bound to a polypeptide that binds to a target molecule The diagnostic pharmaceutical composition which contains the complex which has as an active ingredient.
(14) The metal radioisotope is any one of the metal radioisotopes selected from technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177 (13) A pharmaceutical composition for diagnosis.
(15) A complex structure formed from a metal radioisotope and the compound of any one of (1) to (5) or a pharmacologically acceptable salt bound to a polypeptide that binds to a target molecule The pharmaceutical composition for treatment which contains the complex which has as an active ingredient.
(16) The metal radioisotope is any one metal radioisotope selected from technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177 (15) Pharmaceutical composition for the treatment of
(17) Use of the compound of any one of (1) to (5) or a pharmaceutically acceptable salt thereof in the production of a radiolabeled polypeptide.

According to the present invention, a novel compound or a pharmacologically acceptable salt thereof that facilitates the production of a radiolabeled polypeptide having a low renal accumulation, and the compound or a pharmacologically acceptable salt thereof and a radioisotope A radiolabeled drug containing a complex having a complex structure formed from the above can be provided. The radiolabeled polypeptide according to the present invention can be produced by a simple method without requiring a plurality of steps.

Radiolabeled drugs produced using the compounds according to the present invention or pharmacologically acceptable salts thereof have much lower non-specific renal accumulation than before. Can be applied. In addition, the radiolabeled drug according to the present invention can greatly reduce kidney damage, which is a problem in internal radiotherapy using low molecular weight polypeptides, due to the property of low nonspecific renal accumulation, Application to labeling with ^186/188 Re that emits not only ^99m Tc but also cell-killing β-rays becomes possible.

Thus, the present invention enables safe administration of a radiolabeled polypeptide having a higher radioactivity than before, and contributes to radiographic diagnosis and internal radiotherapy.

It is a figure which shows the structure of a radiolabeled Fab fragment. ^99m Tc-PGGFML-IT-Fab is a complex having a complex structure formed from PGGFML in which a Fab fragment is bound by 2-iminothiolane (IT) and ^99m Tc. [ ¹²⁵ I] HML-IT-Fab is a complex having a complex structure formed from hippur ε-N-maleoyl-L-lysine (HML) to which a Fab fragment is bound by IT and ¹²⁵ I. ^99m Tc-PG-Fab is a complex having a complex structure formed from PG and ^99m Tc bonded via the lysine residue of the Fab fragment. (Examples 1, 2, and 3) ^99m Tc-PGGFML-IT-Fab rapidly disappeared from the blood and kidney after administration to mice, while ^99m Tc-PG-Fab disappeared from the kidney although it disappeared from the blood as well. It is a figure which shows that disappearance of was late. The disappearance rate of ^99m Tc-PGGFML-IT-Fab from blood and kidney was almost the same as that of [ ¹²⁵ I] HML-IT-Fab. (Example 3)

The present invention relates to a drug for producing a radiolabeled polypeptide with reduced nonspecific renal accumulation. The present invention also provides a radiolabeled polypeptide produced using the radiolabeled polypeptide producing agent, a diagnostic or therapeutic pharmaceutical composition comprising the radiolabeled polypeptide as an active ingredient, and the radiolabel. The present invention relates to a diagnostic imaging method and a therapeutic method using a polypeptide.

The agent for producing a radiolabeled polypeptide according to the present invention contains a compound represented by the following formula (I).

Here, R ₁ is a hydrogen atom, a methyl group, or a carboxymethyl group (CH ₂ —COOH), and R ₂ is CH ₂ —CH ₂ —NH—CH ₂ —COOH, CH ₂ —CH ₂ —N (CH _2- COOH) ₂ , CH ₂ -CH ₂ -NH ₂ , CH ₂ -CH ₂ -NHR ₃ , a carboxymethyl group, or CH ₂ -CH ₂ -NR ₃ R ₄ , wherein R ₃ and R ₄ are each An alkyl group which may be different,
X, Y, and Z are all amino acids that may be different from each other,
F is a functional group that can bind to a polypeptide.

In the compound represented by the above formula (I), the functional group F is a spacer, and the polypeptide can be bonded to the compound via the functional group F. The functional group F is not particularly limited as long as it can bind to a polypeptide, but preferably a carboxylic acid and its active ester, a maleimide group, a bromoacetyl group, an iodoacetyl group, an isothiocyanate group, and an amino group Any one functional group selected from the group consisting of and more preferably a maleimide group.

In the compound represented by the above formula (1), the amino acid sequence represented by XYZ is an amino acid sequence that is cleaved by a kidney brush border membrane enzyme in the kidney, and the presence of this amino acid sequence allows the compound to be easily metabolized in the kidney. Is discharged into the urine. The amino acid sequence represented by X-Y-Z is not particularly limited as long as it is an amino acid sequence cleaved by a kidney brush border membrane enzyme. X is not particularly limited, but is preferably any amino acid residue selected from the group consisting of glycine, alanine, glutamine, glutamic acid, asparagine, aspartic acid, methionine, leucine, methionine, and isoleucine, more preferably glycine, Any amino acid residue selected from the group consisting of alanine, glutamine, glutamic acid, asparagine, aspartic acid, and methionine. Y is not particularly limited, and is any amino acid residue selected from the group consisting of glycine, alanine, phenylalanine, histidine, leucine, isoleucine, tyrosine, and β-alanine. Z is not particularly limited as long as it is an amino acid having a functional group in the side chain, and preferably any amino acid selected from the group consisting of lysine, ornithine, tyrosine, histidine, aspartic acid, glutamic acid, serine, and cysteine Residue. Specifically, examples of X, Y, and Z include glycine, L-phenylalanine, and lysine.

Preferred examples of the compound represented by the above formula (I) include a compound represented by the following formula (II).

The compound according to the present invention may be a free form or a pharmacologically acceptable salt. Examples of pharmacologically acceptable salts include acid addition salts and base addition salts. Acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, or phosphate, and citrate, oxalate, acetate, formate, Examples thereof include organic acid salts such as propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, or paratoluenesulfonate. Examples of base addition salts include inorganic base salts such as sodium, potassium, calcium, magnesium, or ammonium salts, and organic base salts such as triethylammonium, triethanolammonium, pyridinium, and diisopropylammonium salts. .

In the present invention, it is also possible to provide a compound obtained by binding a polypeptide that binds to a target molecule to the compound represented by the above formula (I).

A polypeptide that binds to a target molecule is sometimes referred to herein as a “target molecule recognition element” and refers to a polypeptide that specifically binds to a target molecule. Specific binding refers to binding to a target molecule but not to a molecule other than the target molecule or weak binding. A target molecule refers to a molecule present in a target site, for example, a tissue or a cell, preferably a molecule that is specifically expressed, to be diagnosed or treated with a radiolabeled drug. Specifically binding means that it is expressed at a target site, but not expressed at a site other than the target site, or is low in expression. In the present invention, as a target molecule recognition element, specifically, a ligand that binds to a protein that is highly expressed in tissue construction associated with inflammation or tumor cell invasion, a protein that is specifically expressed in tumor cells, an antibody, and Examples include an antigen-binding region fragment of an antibody. Examples of antibody antigen-binding region fragments include Fab fragments, F (ab ′) ₂ fragments, F (ab) ₂ fragments, and variable region (Fv) fragments. The Fab fragment means an N-terminal product generated by papain degradation of an antibody and a fragment having the same domain structure. The F (ab ′) ₂ fragment means a fragment obtained by reducing the disulfide bond in the hinge region of F (ab ′) ₂ of an antibody and a fragment having a domain structure similar to this. The F (ab) ₂ fragment means a dimer in which two molecules of Fab fragments are bonded to each other by a disulfide bond. The Fv fragment means the smallest fragment that is an antibody fragment and has an antigen-binding activity. More specifically, an antibody against a protein specifically expressed in a specific cancer cell, and a Fab fragment or Fv fragment thereof can be exemplified. In addition, a cyclic pentapeptide having an affinity for integrin, which is highly expressed in cancer new blood vessels, such as cyclo-Arg-Gly-Asp-D-Phe-Lys [SEQ ID NO: 1, c (RGDfK) Abbreviated]. In addition, receptors for bisphosphonic acid, oligoaspartic acid, oligoglutamic acid and macrophages that have an affinity for hydroxyapatite, which is abundant in osteogenic cancer (bone metastasis), and affinity for the scanning factor on the surface of macrophages Examples thereof include fMet-Leu-Phe (fMLP), a folate that binds to a folate receptor that is expressed in cancer cells, and derivatives thereof. The target molecule recognition element is not limited to these exemplified polypeptides, and any polypeptide that binds to the target molecule can be used.

Using the compound according to the present invention, it is possible to provide a radiolabeled polypeptide preparation drug containing the compound. In addition to the compound, the radiolabeled polypeptide preparation drug can contain additives such as a pH regulator such as an aqueous buffer and a stabilizer such as ascorbic acid and p-aminobenzoic acid.

In the present invention, a radiolabeled polypeptide comprising a complex having a complex structure formed from the aforementioned compound bound to a target molecule and a metal radioisotope, and a radioactivity comprising the radiolabeled polypeptide Labeling agents can be provided. The radiolabeled drug according to the present invention may contain unreacted substances and impurities in addition to the radiolabeled polypeptide, and may be a radiolabeled polypeptide purified by a purification method such as high performance liquid chromatography (HPLC) after production. It may contain a peptide.

The term “complex” means a substance in which a ligand is coordinated around an atom or ion of a metal and a metal-like element, and is also called a coordination compound. Coordination means that a ligand forms a coordinate bond with a central metal and is arranged around the central metal. The complex is formed by a coordinate bond between a ligand and a metal. Formation of a complex of a ligand and a metal may be referred to as complex formation. A coordinate bond refers to a bond in which two valence electrons participating in one bond are provided from only one atom.

The metal radioisotope is not particularly limited as long as it forms a ligand with the compound according to the present invention, but preferably a technetium isotope, rhenium isotope, indium isotope, yttrium isotope, and lutetium isotope, More preferred examples include technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177, and more preferably technetium-99m, rhenium-186, and rhenium-188. Metal radioisotopes are not limited to these specific examples, as long as they have appropriate radiation, radiation dose, and half-life for purposes such as diagnosis using radiolabeled drugs and internal radiotherapy of diseases such as cancer diseases. Either can be used. From the viewpoint of reducing the influence on normal tissues and cells in radiographic diagnosis and internal radiotherapy, short half-life metal radioisotopes are preferably used.

The complex can be produced by in vitro complexation with a metal radioactive isotope using the above compound bound to a target molecule recognition element as a ligand. Complex formation can be performed by a simple operation using a conventionally known complex formation reaction.

The radiolabeled polypeptide according to the present invention is formed by in vitro complexation with a metal radioisotope using a compound obtained by binding an antibody Fab fragment to the compound represented by the above formula (II) as a ligand. A radiolabeled polypeptide comprising a complex having a complex structure can be preferably exemplified. More preferably, the metal radioisotope is any one metal radioisotope selected from the group consisting of technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177, Preferably, a radiolabel comprising a complex having a complex structure formed using any one metal radioisotope selected from the group consisting of technetium-99m, rhenium-186, and rhenium-188 as a metal radioisotope Drugs can be exemplified.

Since the radiolabeled polypeptide produced by the present invention has the amino acid sequence XYZ that is selectively cleaved by the kidney, it is selectively enzymatically degraded after administration to a subject, and its metabolite accumulates in the kidney. And is rapidly excreted in the urine. As described above, the radiolabeled polypeptide according to the present invention having the feature of low renal accumulation can greatly reduce kidney damage, which is a problem in isotope treatment using a low-molecular-weight polypeptide, It can be applied to labeling with metal radioisotopes that emit cell-killing β-rays as well as phase metal radioisotopes. This feature also facilitates detection / diagnosis of the abdominal lesion site because there is little radioactivity that becomes the background of the abdomen in radiological image diagnosis. Furthermore, since the radiolabeled polypeptide according to the present invention has a target molecule recognition element, it can specifically bind to the target site, and therefore efficiently accumulates in the target site. Due to such properties, the radiolabeled polypeptide according to the present invention can improve the sensitivity and therapeutic effect of diagnostic imaging.

As described above, the radiolabeled drug containing the radiolabeled polypeptide according to the present invention enables safe administration of a radiolabeled polypeptide having a higher radioactivity than the conventional one. Useful for. The radiolabeled drug according to the present invention contains the above radiolabeled polypeptide as an active ingredient and, if necessary, a pharmaceutical composition containing one or two or more pharmaceutically acceptable carriers (pharmaceutical carriers). Can be prepared. As pharmaceutical carriers, aqueous buffers, pH adjusters such as acids and bases, stabilizers such as ascorbic acid and p-aminobenzoic acid, excipients such as D-mannitol, isotonic agents, and preservatives Etc. can be illustrated. In addition, compounds such as citric acid, tartaric acid, malonic acid, sodium gluconate, sodium glucoheptonate, etc., useful for improving the radiochemical purity may be added. The radiolabeled drug according to the present invention can be provided in any form of an aqueous solution, a frozen solution, and a lyophilized product.

The radiolabeled drug according to the present invention is used for various diseases such as tumors, inflammations, infectious diseases, cardiovascular diseases, brain / central diseases, and radiographic diagnosis of organs / tissues, or internal radiotherapy. Preferably, it is used for radiological image diagnosis and internal radiotherapy of cancer diseases, but the applicable disease is not particularly limited, and any disease can be applied as long as it is image diagnosis or internal radiotherapy. By selecting the target molecule recognition element according to the characteristics of the target to be diagnosed or treated, it is possible to diagnose and treat a wide variety of targets. The radiolabeled drug according to the present invention is used in the field of diagnosis and treatment. Can be widely used in.

As the administration route of the radiolabeled drug according to the present invention, parenteral administration such as intravenous administration or intraarterial administration, or oral administration can be mentioned, and intravenous administration can be preferably mentioned. The administration route is not limited to these routes, and any route can be used as long as its action can be effectively expressed after administration of the radiolabeled drug.

The radioactivity intensity of the radiolabeled drug according to the present invention is arbitrary as long as the objective can be achieved by administering the labeled drug and the subject is exposed to the lowest possible clinical dose. is there. The radioactive intensity can be determined with reference to the radioactive intensity used in a general diagnostic method or therapeutic method using a radiolabeled drug. The dose is determined in consideration of various conditions such as the patient's age, weight, appropriate radiographic imaging apparatus, and the state of the target disease, and the radioactivity and dose that can be imaged and treated are determined. In the case of human subjects, the dosage of a diagnostic agent using a technetium-99m label is 37 MBq to 111 MBq as the radioactivity of technetium-99m. In the case of a therapeutic agent using a rhenium-186 or rhenium-188 label, the radioactivity is in the range of 37 MBq to 18500 MBq, preferably 370 MBq to 7400 MBq.

In the present invention, a kit comprising the above compound and a drug containing a metal radioisotope as separate packaging units can be provided. As a kit according to the present invention, any one selected from the group consisting of the compound represented by the above formula (II) and technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177 Preferably, a kit comprising a drug containing one metal radioisotope as a separate packaging unit can be exemplified. The agent comprising any one of the metal radioisotopes selected from the group consisting of technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177 is preferably in the form of a solution. Provided. More preferably, the drug containing a metal radioisotope is a drug containing any one metal radioisotope selected from the group consisting of technetium-99m, rhenium-186, and rhenium-188. Any of the compounds and drugs contained in the kit can contain one or more pharmaceutically acceptable carriers (pharmaceutical carriers) as described above, as necessary.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples shown below.

In the compound structure represented by the following formula (I), R ₁ is a carboxymethyl group (CH ₂ -COOH), R ₂ is a hydrogen atom, X is glycine, Y is L-phenylalanine, Z is L-lysine, and F is maleimide. The compound being a group, 2- {2- [2-({2-[(carboxymethyl-amino) -methyl] -pyridine-4-carbonyl} -amino) -acetylamino] -3-phenyl-propionylamino}- 6- (2,5-Dioxo-2,5-dihydro-pyrrol-1-yl) -hexanoic acid (2- {2- [2-({2-[(Carboxymethyl-amino) -methyl] -pyridine-4 -carbonyl} -amino) -acetylamino] -3-phenyl-propionylamino} -6- (2,5-dioxo-2,5-dihydro-pyrrol-1-yl) -hexanoic acid, abbreviated herein as PGGFML ) Was synthesized as shown in Chemical Reaction Formula 1 using methyl isonicotinate (1) as a raw material.

(Chemical reaction formula 1)

1. Synthesis of Compound 2 Methyl isonicotinate (1) (9.93 g, 72.4 mmol) was dissolved in methanol (MeOH, 100 mL), and a catalytic amount of sulfuric acid (0.33 mL) was added. A saturated aqueous solution (55 mL) of ammonium persulfate (30 g, 131.5 mol) was added dropwise under reflux with heating, and the mixture was stirred at reflux temperature for 30 minutes. After cooling, sodium carbonate was added to adjust the pH to around 7, the reaction product was filtered, the filtrate MeOH was distilled off under reduced pressure, and the aqueous layer obtained was extracted with ethyl acetate (AcOEt, 150 mL × 5), organic The layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography using a solution of AcOEt: n-hexane (Hex) = 1: 1 as a mobile phase to obtain yellow crystals. The obtained crystals were dissolved in AcOEt (20 mL), and the crystals precipitated by adding Hex (40 mL) were collected by filtration, and 4.80 g (yield 39.7%) of methyl 2-hydroxymethylisonicotinate ( 2) (methyl 2-hydroxymethylisonicotinate (2)) was obtained as white needle crystals. ¹ H-NMR (CDCl ₃ ): δ8.69-8.71 [1H, d, pyridine], δ7.81 [1H, s, pyridine], δ7.74- 7.75 [1H, d, pyridine], δ4.82 [ 2H, s, CH ₂ OH], δ 3.95 [3H, s, COOCH ₃ ]. FAB-MS calculated: C ₈ H ₉ NO ₃ [M + H] ⁺ : m / z 168, measured 168.

2. Synthesis of Compound 3 Compound 2 (4.04 g, 24.0 mmol) was dissolved in dry toluene (80 mL), and thionyl chloride (20.8 mL, 288 mmol) was added dropwise under ice cooling. Stir at room temperature for 1 hour and evaporate the solvent under reduced pressure to obtain 5.19 g (97.4% yield) of methyl 2-chloromethylisonicotinate (3) as white crystals. Obtained. ¹ H-NMR (CDCl ₃ ): δ8.79-8.81 [1H, d, pyridine], δ8.26 [1H, s, pyridine], δ8.04-8.05 [1H, d, pyridine], δ4.96 [ 2H, s, CH ₂ OH], δ4.02 [3H, s, COOCH ₃ ].

3. Synthesis of Compound 4 H-Gly-OtertBu · HCl (0.59 g, 3.51 mmol) and potassium carbonate (1.13 g, 8.19 mmol) were suspended in dry acetonitrile (MeCN, 5 mL) and dried under ice-cooling with dry MeCN ( Compound 3 (0.26 g, 1.17 mmol) dissolved in 5 mL) was added dropwise. The reaction solution was filtered after stirring at room temperature overnight, and the filtrate was depressurizingly distilled. The residue was dissolved in AcOEt (10 mL) and washed with saturated aqueous sodium chloride solution (10 mL × 3). After drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography using a solution of AcOEt: Hex = 1: 2 as a mobile phase to obtain 323.4 mg (yield: 87.7%) of Compound 4 as a yellow oily substance. ¹ H-NMR (CDCl ₃ ): δ8.71- 8.70 [1H, d, pyridine], δ8.08 [1H, s, pyridine], δ7.70-7.69 [1H, d, pyridine], δ4.01 [ 2H, s, pyr-CH ₂ ], δ 3.95 [3H, s, COOCH ₃ ], δ 3.38 [2H, s, NHCH ₂ CO], δ 1.47 [9H, s, tBu].

4. Synthesis of Compound 5 Compound 4 (287.4 mg, 1.03 mmol) was dissolved in dry dichloromethane (CH ₂ Cl _2, 3 mL), and triethylamine (0.171 mL, 1.23 mmol) was added. Dicarbonate-tert-dibutyl dicarbonate (268 mg, 1.23 mmol) dissolved in dry CH ₂ Cl ₂ (2 mL) was added dropwise under ice cooling. After stirring at room temperature for 4 hours, the reaction solution was washed with 5% aqueous citric acid solution (5 mL × 3). After drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography using a solution of AcOEt: Hex = 1: 5 as a mobile phase to obtain 246.1 mg (yield 62.8%) of Compound 5 as a colorless transparent oily substance. ¹ H-NMR (CDCl ₃ ): δ8.68-8.67 [1H, d, pyridine], δ7.87 [1H, s, pyridine], δ7.73-7.72 [1H, d, pyridine], δ4.61 [ 2H, s, pyr-CH ₂ ], δ 3.95 [3H, s, COOCH ₃ ], δ 3.86 [2H, s, NHCH ₂ CO], δ 1.43 [18H, s, tBu, Boc].

5. Synthesis of Compound 6 Compound 5 (246.1 mg, 0.65 mmol) was dissolved in MeOH (1 mL), and 2N aqueous sodium hydroxide (NaOH) solution (1 mL) was added under ice cooling. After stirring at room temperature for 3 hours, 1N hydrochloric acid (HCl) aqueous solution was added to adjust the pH to around 7, and MeOH was distilled off under reduced pressure. The obtained aqueous solution was extracted with chloroform (CHCl _3, 3 mL × 3). The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain 112.0 mg (yield 47.3%) of Compound 6 as white crystals. ¹ H-NMR (CDCl ₃ ): δ8.67-8.65 [1H, d, pyridine], δ7.94 [1H, s, pyridine], δ7.89-7.88 [1H, d, pyridine], δ4.78 [ 2H, s, pyr-CH ₂ ],, δ4.12 [2H, s, NCH ₂ ], δ1.42 [18H, s, tBu, Boc].

6. Synthesis of Compound 7 Cl-Trt (2-Cl) Resin (124.8 mg, 0.196 mmol), Fmoc-Lys (Z) -OH (98.5 mg, 0.196 mmol), N, N-diisopropylethylamine (139.3 μL, 0.78 mmol) was stirred in CH ₂ Cl ₂ for 90 min at room temperature. Next, N, N-diisopropylethylamine (139.3 μL, 0.78 mmol) and MeOH (0.38 mL) were added, and the mixture was stirred at room temperature for 15 minutes. Wash the resin with dimethylformamide (DMF, 5 mL × 3), CH ₂ Cl ₂ (5 mL × 3), isopropanol (5 mL × 3), diethyl ether (Et ₂ O, 5 mL × 3), and dry under reduced pressure did. Then, 20% piperidine / DMF (v / v) (3 mL) was added and stirred for 20 minutes at room temperature. After the resin was washed with DMF (5 mL × 8), a part of the resin was collected and subjected to a Kaiser test to confirm the deprotection of the N ^α -Fmoc group.

H-Lys (Z) -Trt (2-Cl) Resin (0.196 mmol), 2.5 equivalents of protected amino derivative, Fmoc-Phe-OH (189.6mg, 0.49 mmol), N, N-diisopropylcarbodiimide (75.4 μL) , 0.49 mmol, 1-hydroxybenzotriazole (75.0 mg, 0.49 mmol) was stirred in DMF (0.5 mL) at room temperature for 2 hours. After the resin was washed with DMF (5 mL × 8), a part of the resin was collected and subjected to a Kaiser test to confirm the completion of the condensation reaction. Subsequently, 20% piperidine / DMF (v / v) (3 mL) was added, and the mixture was stirred at room temperature for 20 minutes. The resin was washed with DMF (5 mL × 8). Furthermore, the same reaction was performed using the protected amino acid Fmoc-Gly-OH (145.7 mg, 0.49 mmol) to prepare H-Gly-Phe-Lys (Z) -Trt (2-Cl) Resin.

H-Phe-Gly-Lys (Z) -Trt (2-Cl) Resin (0.196 mmol) and compound 6 (71.8 mg, 0.196 mmol), N, N-diisopropylcarbodiimide (30.3 μL, 0.196 mmol), 1-hydroxy Benzotriazole (30.0 mg, 0.196 mmol) was stirred in DMF (1 mL) at room temperature overnight. The resin was washed with DMF (5 mL × 8) and then CH ₂ Cl ₂ (5 mL × 8) and dried under reduced pressure.

7. Synthesis of Compound 8 To PG (Boc) -OtBu-Phe-Gly-Trt (2-Cl) Resin (0.196 mmol), acetic acid (AcOH): 2,2,2-trifluoroethanol: CH ₂ Cl ₂ = 3: 1: 6 (v / v / v) (5 mL) was added, and the mixture was stirred at room temperature for 2 hours. The back solution obtained by filtering the reaction solution was distilled off under reduced pressure. The residue was azeotroped with toluene (5 mL × 3), and the resulting pale yellow oily substance was dissolved in EtOH (0.2 mL). Et ₂ O (10 mL) was added to collect the precipitated white crystals by filtration. 92.8 mg (yield 56.9%) of PG (Boc) -OtBu-Gly-Phe-Lys (Z) -OH was obtained as white crystals. This compound was used in the next reaction without further purification. Note that PG is an abbreviation for 2-((carboxyamino) methyl) isonicotinic acid.

8. Synthesis of Compound 9 Compound 8 (50 mg, 0.060 mmol) was dissolved in a solution (4 mL) of MeOH: H ₂ O: AcOH = 9: 0.5: 0.5 (v / v / v) and 10% Pd / C (5.0 mg, 0.0047 mmol) was suspended and stirred at room temperature for 8 hours under H ₂ atmosphere. The reaction solution was suction filtered using a small amount of celite (the solvent of the filtrate was distilled off under reduced pressure. Et ₂ O (5 mL) was added to the residue, and the precipitated crystals were collected by filtration to give 34.6 mg ( Yield 82.6%) of PG (Boc) -OtBu-Gly-Phe-Lys-OH was obtained as gray crystals, and this compound was used in the next reaction without further purification.

9. Synthesis of Compound 10 PG (Boc) -OtBu-Gly-Phe-Lys-OH (34.6 mg, 0.050 mmol) was dissolved in a saturated aqueous solution of sodium bicarbonate (2 mL). Under ice cooling, N- (methoxycarbonyl ) Maleimide (8.5 mg, 0.055 mmol) was added. The mixture was stirred for 40 minutes under ice cooling, and then stirred at room temperature for 50 minutes. The mixture was ice-cooled again, concentrated sulfuric acid was added to adjust the pH to around 3, and the mixture was extracted with ethyl acetate (3 mL × 3). After drying the organic layer over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to obtain 16.5 mg (yield 42.4%) of PG (Boc) -OtBu-Gly-Phe-Lys (mal) -OH as white crystals. It was. ¹ H-NMR (DMSO): δ8.98-8.97 [1H, d, NH], δ8.63 [1H, s, NH], δ8.30-8.28 [1H, m, NH], δ.16-8.14 [1H, d, pyridine], δ7.68-7.65 [2H, m, pyridine], δ7.23-7.16 [5H, m, aromatic], δ6.99 [2H, s, COCHCHCO], δ4.58-4.51 [3H, m, NHCHCH ₂ C ₆ H ₅ , pyr-CH ₂ ], δ4.13-4.12 [2H, m, NHCHCOOH], δ4.00-3.76 [4H, m, NHCH ₂ , NCH ₂ ], δ3. 16 [2H, s, CH ₂ -mal], δ2.77-2.63 [2H, q, CHCH ₂ -C ₆ H ₅ ], δ1.72-1.62 [2H, m, CHCH ₂ CH ₂ ], δ1.48 -1.47 [2H, m, CH ₂ CH ₂ NH], δ1.39-1.37 [18H, m, tBu, Boc], δ1.27-1.23 [2H, m, CHCH ₂ CH ₂ ].

10. Synthesis of Compound 11 (PGGFML) PG (Boc) -OtBu-Gly-Phe-Lys (mal) -OH (16.5 mg, 0.021 mmol) was dissolved in EtOAc (0.5 mL) and 4N HCl / EtOAc (1.5 mL) ) Was added. After stirring at room temperature for 2 hours, the precipitated crystals were collected by filtration and purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) to obtain 8.6 mg (yield: 62.3%) of PGGFML hydrochloride in white Obtained as crystals. ¹ H-NMR (DMSO): δ9.02 [1H, d, NH], δ8.69 [1H, s, NH], δ8.25-8.24 [1H, d, NH], δ8.20-8.18 [1H , d, pyridine], δ7.84 [1H, s, pyridine], δ7.69-7.68 [1H, d, pyridine], δ7.24-7.17 [5H, m, aromatic], δ6.99 [2H, s , COCHCHCO], δ4.56 [1H, m, NHCHCH ₂ C ₆ H ₅ ], δ4.11 [4H, s, NHCH ₂ COOH, pyr-CH ₂ ], δ3.91-3.79 [2H, dq, NHCH ₂ ,], δ3.05-3.00 [1H, m, NHCHCOOH], δ2.80-2.74 [2H, m, CH ₂ -mal], δ2.33 [2H, m, CHCH ₂ -C ₆ H ₅ ], δ1 .75-1.72 [2H, m, CHCH ₂ CH ₂ ], δ1.48-1.46 [2H, m, CH ₂ CH ₂ NH], δ1.28-1.24 [2H, m, CHCH ₂ CH ₂ ] .ESI- MS calculated: C ₃₀ H ₃₄ N ₆ O ₉ [MH] ^- : m / z 621, measurement 621.

The compound PGGFML-IT-Fab was prepared by binding the antibody Fab fragment to PGGFML.

First, 1 μL of PGGFML (50 mg / mL) dissolved in DMF was added to a Fab solution (100 μL) thiolated with 2-iminothiolane (2-IT), and reacted at room temperature for 4 hours. Next, an iodoacetamide solution (10 mg / mL) was prepared using 0.1 M phosphate buffer (pH 6.0), and 14.8 μL was added thereto, followed by reaction at room temperature for 30 minutes to remove unreacted thiol groups. Alkylated. Thereafter, PGGFML-IT-Fab was purified by a spin column method using Sephadex® G-50® Fine equilibrated with 0.1M phosphate buffer (pH 7.0). This reaction introduced an average of 0.95 molecules of PGGFML per antibody molecule.

(1) ^99m Tc labeling of PGGFML-IT-Fab PGGFML-IT-Fab was labeled with ^99m Tc. The labeling was performed by complexing PGGFML-IT-Fab with [ ^99m Tc (CO) ₃ (OH ₂ ) ₃ ] ⁺ . As a control compound, PG-Fab prepared by binding PG carboxylic acid to an amino group derived from lysine of Fab was similarly labeled with ^99m Tc and used.

First, [ ^99m Tc (CO) ₃ (OH ₂ ) ₃ ] ⁺ was prepared. Sodium boranocarbonate (sodium boranocarbonate, 3 mg), sodium tetraborate decahydrate (1.9 mg), tartaric acid (+)-sodium dihydrate (sodium (+) tartrate dihydrate, 4.8 mg) and sodium carbonate (5.7 mg) dissolved in 1.0 ml of nitrogen-substituted MilliQ water and dispensed into a 1.5 ml Eppendorf tube, 100 μl at a time, and lyophilized to create a ^99m Tc-Aqua kit did. ^99m Tc-aqua kit ^99m TcO ₄ eluted from the generator ^- saline solution 100 [mu] l was added and reacted at 100 ° C. 20 minutes to obtain a _{^{+ [99m Tc (CO) 3}} (OH 2) 3]. The mixture was allowed to cool to room temperature, about 10 μl of 1N HCl aqueous solution was added, and the pH was adjusted to around 7 before use. The formation of [ ^99m Tc (CO) ₃ (OH ₂ ) ₃ ] ⁺ was confirmed using RP-HPLC. ^99m TcO ₄ ⁻ was observed at 2.8 minutes and [ ^99m Tc (CO) ₃ (OH ₂ ) ₃ ] ⁺ was observed at 4.9 minutes, and the radiochemical yield was 100%.

Next, a complex formation reaction was performed. Add 10 μl of [ ^99m Tc (CO) ₃ (OH ₂ ) ₃ ] ⁺ solution to 10 μl of PGGFML-IT-Fab or PG-Fab solution adjusted to 3.0 mg / ml using 0.1 M phosphate buffered water pH 7.0 And incubated at 40 ° C. for 60 minutes. Radiochemical yields were analyzed by size exclusion HPLC (SE-HPLC) and RP-TLC. The radiochemical yield was 96.2%.

^{(2) 99m Tc-PGGFML-} IT -Fab evaluation of stability in mouse plasma in ^99m Tc-PGGFML-IT-Fab solution 0.1 M phosphate buffer adjusted to a concentration of 0.5 mg / ml in (pH 7.0) Then, 20 μL of the solution was added to 230 μL of mouse plasma. After incubation at 37 ° C, a portion of the sample was analyzed by reverse phase thin layer chromatography (RP-TLC) and SE-HPLC after 1, 3 and 6 hours to calculate the percentage of radioactivity released from the Fab.

As a result, when ^99m Tc-PGGFML-IT-Fab and ^99m Tc-Fab were incubated with mouse plasma at 37 ° C., 98.3% or more existed unchanged until 6 hours later (Table 1).

(3) Pharmacokinetic experiment of RI-labeled antibody fragment ^99m Tc-PGGFML-IT-Fab was administered from the mouse tail vein, and the time course of radioactivity of each tissue was measured. Using ^99m Tc-PG-Fab and [ ¹²⁵ I] HML-IT-Fab as control compounds, the time course of radioactivity of each tissue was measured in the same manner. [ ¹²⁵ I] HML-IT-Fab is a compound obtained by binding a Fab fragment to [ ¹²⁵ I] -iodohypril N-maleoyl-L-lysine ([ ¹²⁵ I] HML) via IT. It was produced by the method described in 1. The structure of the radiolabeled compound tested is shown in FIG.

As shown in FIG. 2, all of the radiolabeled compounds tested showed approximately the same blood elimination rate. On the other hand, ^99m Tc-PG-Fab showed high radioactivity in the kidney even 6 hours after administration, whereas ^99m Tc-PGGFML-IT-Fab greatly reduced the radioactivity in the kidney from the early stage of administration. Similar to [ ¹²⁵ I] HML-IT-Fab. The ratio of renal radioactivity to blood radioactivity was calculated. ^99m Tc-PGGFML-IT-Fab and [ ¹²⁵ I] HML-IT-Fab were significantly lower than ^99m Tc-PG-Fab It has been found.

From this result, it was revealed that ^99m Tc-PGGFML-IT-Fab has reduced nonspecific renal accumulation. In this way, by using PGGFML, a low-molecular-weight polypeptide that can bind to the target substance is bound to it, and then labeled with a radioisotope, thereby producing a radiolabeled drug with reduced non-specific renal accumulation by a simple operation. can do.

SEQ ID NO: 1: Cyclic peptide derived from the 10th fibronectin type III repeat region.

Claims (17)

1. A compound represented by the following formula (I), or a pharmaceutically acceptable salt thereof:
   

   

   Here, R ₁ is a hydrogen atom, a methyl group, or a carboxymethyl group (CH ₂ —COOH), and R ₂ is CH ₂ —CH ₂ —NH—CH ₂ —COOH, CH ₂ —CH ₂ —N (CH _2- COOH) ₂ , CH ₂ -CH ₂ -NH ₂ , CH ₂ -CH ₂ -NHR ₃ , a carboxymethyl group, or CH ₂ -CH ₂ -NR ₃ R ₄ , wherein R ₃ and R ₄ are each An alkyl group which may be different,
   X, Y, and Z are all amino acids that may be different from each other,
   F is a functional group that can bind to a polypeptide.
2. The functional group F capable of binding to the polypeptide is any one functional group selected from the group consisting of carboxylic acid and its active ester, maleimide group, bromoacetyl group, iodoacetyl group, isothiocyanate group, and amino group Or a pharmaceutically acceptable salt thereof.
3. 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein X, Y, and Z are glycine, L-phenylalanine, and lysine, respectively.
4. 4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the functional group F that can bind to a polypeptide is a maleimide group.
5. A compound represented by the following formula (II) or a pharmacologically acceptable salt thereof.
   

   
6. A compound obtained by binding a polypeptide that binds to a target molecule to the compound according to any one of claims 1 to 5 or a pharmacologically acceptable salt thereof, or a pharmacologically acceptable salt thereof .
7. 7. The compound according to claim 6, or a pharmacologically acceptable salt thereof, wherein the polypeptide that binds to the target molecule is an antibody Fab fragment.
8. 6. A radiolabeled polypeptide producing agent comprising the compound according to claim 1 or a pharmacologically acceptable salt thereof.
9. A complex structure formed from a metal radioisotope and the compound according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof, to which a polypeptide that binds to a target molecule is bound. A radiolabeled drug containing a complex having the same.
10. The radioactivity according to claim 9, wherein the metal radioisotope is any one metal radioisotope selected from technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177. Labeled drug.
11. 11. The radiolabeled drug according to claim 9 or 10, wherein the polypeptide that binds to the target molecule is an antibody Fab fragment.
12. A radiolabeled drug comprising a complex having a complex structure formed from technetium-99m and a compound represented by the following formula (II) to which an antibody Fab fragment is bound.
    

    
13. A complex structure formed from a metal radioisotope and the compound according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof, to which a polypeptide that binds to a target molecule is bound. The diagnostic pharmaceutical composition which contains the complex which has as an active ingredient.
14. The diagnostic of claim 13, wherein the metal radioisotope is any one metal radioisotope selected from technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177. Pharmaceutical composition.
15. A complex structure formed from a metal radioisotope and the compound according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof, to which a polypeptide that binds to a target molecule is bound. The pharmaceutical composition for treatment which contains the complex which has as an active ingredient.
16. The treatment according to claim 15, wherein the metal radioisotope is any one metal radioisotope selected from technetium-99m, rhenium-186, rhenium-188, indium-111, yttrium-90, and lutetium-177. Pharmaceutical composition.
17. Use of the compound according to any one of claims 1 to 5 or a pharmacologically acceptable salt thereof in the production of a radiolabeled polypeptide.

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