Radioactive element labeling method using quinone compound, radioactive labeling compound and radioactive element labeling kit

Abstract

The present invention relates to a radioactive isotope labeling method using a quinone compound, a radioactive labeled compound, and a radioactive element labeling kit containing the same. The radioactive isotope labeling method comprises: providing a cyclooctyne compound represented by chemical formula (I) and bonded to a biomolecule, a fluorescent dye or a nanoparticle compound; the cyclooctyne compound reacts with a quinone compound represented by chemical formula (II) and labeled with a radioactive isotope at room temperature. The labeled compound is represented by chemical formula (II) and is used to label a molecule having a cyclooctyne group. The radioactive isotope labeling kit comprises: a cyclooctyne compound represented by chemical formula (I) and bonded to a biomolecule, a fluorescent dye or a nanoparticle compound, and a quinone compound represented by chemical formula (II) and labeled with a radioactive isotope, #imgabs0#

Description

Technical Field

[0001] The present invention relates to a radioactive element labeling method using a quinone compound, a radioactive labeling compound, and a radioactive element labeling kit containing the same, and more specifically, to a radioactive element labeling method using a quinone compound that does not use an organic solvent and has a fast reaction speed, a radioactive labeling compound, and a radioactive element labeling kit containing the same, which can be applied to the fields of medical diagnosis and treatment. Background Art

[0002] In order to use physiologically active substances such as peptides, proteins, antibodies for purposes such as molecular imaging, disease diagnosis or treatment, radioactive labeling technology of biomolecules has been developed. The prior art mainly adopts the method of combining physiologically active substances with chelating agents. The above method is a method of introducing chelating agents into physiologically active substances by chemical means. However, when the chelating agent is combined, there is a fundamental limitation that the reaction conditions that are not suitable for physiologically active substances, such as high temperature reaction, acidic or alkaline reaction, or toxic reaction solvents, need to be improved.

[0003] Typically, radioactive iodine has been used in the past decades for radioactive labeling of biomolecules for diagnostic and therapeutic purposes. 124 I is radioactive iodine, which is mainly used in positron emission tomography (PET). 123 I and 125 I is radioactive iodine mainly used in single photon emission computed tomography (SPECT). 131 I is used to diagnose and treat diseases such as thyroid cancer.

[0004] As mentioned above, with the widespread use of radioactive iodine in medicine, various labeling methods for labeling radioactive iodine on biomolecules and relatively small molecules have been developed, wherein, electrophilic aromatic substitution reaction as a method capable of directly labeling radioisotopes shows high efficiency. However, since most of the labeled compounds synthesized by the labeling method are not stable in animals, it is impossible to obtain expected imaging results in most cases, and the strong oxidants used for labeling show the results of reducing the physiological activity of biological substances. In order to solve the above problems, several prosthetic groups have been developed by studying the method of indirectly labeling and synthesizing radioactive iodine. However, there are some problems in the indirect labeling and synthesizing radioactive iodine methods developed so far, namely, due to the lack of chemical selectivity for the active group of the compound, the situation of randomly obtaining the labeled compound often occurs, and due to the slow reaction rate, it is necessary to use excessive substrate to ensure high yield.

[0005] Therefore, it is necessary to develop a new labeling method of radioactive iodine that can maintain stability in living cells and animals. The related prior art WO2006-050262 discloses a reaction using a cyclooctyne compound and an azide compound, but in the process of this reaction, an organic solvent such as toluene or DMSO is required, which limits the use of living cells and animals in some aspects.

[0006] To this end, by providing a radioactive labeled compound that can be used with a biomolecule, a fluorescent dye or a nanoparticle compound to label a radioactive element on a biomolecule, etc. with a rapid reaction rate and a high radiochemical yield, and without the need for the use of an organic solvent, it is expected that the compound can be effectively used for radioactive element labeling and medical diagnosis of biomolecules, etc. Summary of the invention

[0007] Technical issues

[0008] One aspect of the present invention provides a method for labeling a radioactive element which does not use an organic solvent and has a fast reaction rate.

[0009] Another aspect of the present invention provides a radioactive element-labeled compound which does not use an organic solvent and has a fast reaction rate.

[0010] Another aspect of the present invention provides a kit for labeling radioactive elements which does not use an organic solvent and has a fast reaction speed.

[0011] Another aspect of the present invention provides a biomolecule, fluorescent dye or nanoparticle compound labeled with a radioactive element.

[0012] Another aspect of the present invention provides a composition for medical diagnosis comprising the same.

[0013] Another aspect of the present invention provides a composition for treating cancer comprising the same.

[0014] Problem Solving Methods

[0015] According to one aspect of the present invention, a method for labeling a radioactive element is provided, wherein the method for labeling a radioactive element comprises the steps of providing a cyclooctyne compound represented by the following chemical formula I and bonded to a biomolecule, a fluorescent dye or a nanoparticle compound; and

[0016] A reaction step of reacting the cyclooctyne compound with a quinone compound represented by the following chemical formula II and labeled with a radioactive element.

[0017]

[0018] In the chemical formula I, Z is a biomolecule, a fluorescent dye or a nanoparticle compound.

[0019]

[0020] In the chemical formula II, b is 0 or an integer from 1 to 10; L is CH2, -COO- or -CONH-; and M is a radioactive element.

[0021] According to another aspect of the present invention, a radioactive labeled compound is provided, wherein the radioactive labeled compound is represented by the following chemical formula II or chemical formula IIa and is used to label a molecule having a cyclooctyne moiety.

[0022]

[0023] In the chemical formula II and chemical formula IIa, b is 0 or an integer from 1 to 10; L is CH2, -COO- or -CONH-; and M is a radioactive element.

[0024] According to another aspect of the present invention, a kit for labeling radioactive elements is provided, wherein the kit for labeling radioactive elements comprises: a cyclooctyne compound represented by the following chemical formula I and bonded to a biological molecule, a fluorescent dye or a nanoparticle compound; and a quinone compound represented by the following chemical formula II or chemical formula IIa and labeled with a radioactive element.

[0025]

[0026] In the chemical formula I, Z is a biomolecule, a fluorescent dye or a nanoparticle compound.

[0027]

[0028] In the chemical formula II and chemical formula IIa, b is 0 or an integer from 1 to 10; L is CH2, -COO- or -CONH-; and M is a radioactive element.

[0029] According to another aspect of the present invention, there is provided a biomolecule, fluorescent dye or nanoparticle compound represented by chemical formula III and labeled with a radioactive element.

[0030]

[0031] In the chemical formula III, Z is a biomolecule, a fluorescent dye or a nanoparticle compound; b is 0 or an integer from 1 to 10; L is CH2, -COO- or -CONH-;

[0032] M is a radioactive element.

[0033] According to another aspect of the present invention, a composition for medical diagnosis is provided, wherein the composition for medical diagnosis comprises as an active ingredient a biomolecule, a fluorescent dye or a nanoparticle compound represented by chemical formula III and labeled with a radioactive element.

[0034] According to another aspect of the present invention, a composition for cancer treatment is provided, wherein the composition for cancer treatment comprises as an active ingredient a biomolecule, a fluorescent dye or a nanoparticle compound represented by chemical formula III and labeled with a radioactive element.

[0035] Effects of the Invention

[0036] When radioactive elements are labeled with the quinone compound labeled with radioactive elements according to the present invention, the deposition of iodine-125 in the thyroid gland can be significantly reduced due to its higher in vivo stability compared to the prior art. Furthermore, since the reaction rate with the cyclooctyne compound bonded with a biological molecule, a fluorescent dye or a nanoparticle compound is fast and it has excellent in vivo stability, it can be applied to compositions used for medical diagnosis and treatment, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 To show that radioiodinated HSA [ 125 I] Graph of biodistribution results of 7 with n=5 male mice per group.

[0038] Figure 2 The SPECT / CT images of normal mice at different time periods after intravenous injection of radioiodinated HSA are shown in (B) using the (([ 125I]7) to synthesize purified 150 μCi radioiodinated HSA, (A) shows the direct use of the radioiodinated ([ 125 I]8) was used to synthesize purified 150 μCi radioiodinated HSA.

[0039] Figure 3 To show the effect of radioiodinated HSA [ 125 I]7, 8 and 9 free 125 Diagram of the extent of I absorption within the thyroid gland. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0041] According to the present invention, a radioactive element labeled compound is provided, and more specifically, a radioactive element labeled quinone compound is provided, which can also be called a "tracer". The tracer of the present invention can be used to label radioactive elements with a fast reaction rate and high radiochemical yield by cyclizing with a biomolecule, fluorescent dye or nanoparticle containing a cyclooctyne moiety.

[0042] More specifically, according to the present invention, a method for labeling a radioactive element is provided, wherein the method for labeling a radioactive element comprises: a step of providing a cyclooctyne compound represented by the following chemical formula I and bonded to a biomolecule, a fluorescent dye or a nanoparticle compound; and a reaction step of reacting the cyclooctyne compound with a quinone compound represented by the following chemical formula II and labeled with a radioactive element.

[0043]

[0044] In the chemical formula I, Z is a biomolecule, a fluorescent dye or a nanoparticle compound.

[0045]

[0046] In the chemical formula II, b is 0 or an integer from 1 to 10; L is CH2, -COO- or -CONH-; and M is a radioactive element.

[0047] The reaction steps can be carried out at room temperature and under atmospheric conditions, and therefore have the advantages of not requiring the use of high temperature reactions, acidic or alkaline conditions, toxic reaction solvents, organic solvents, and the like.

[0048] At this time, the normal temperature may be 10-40° C., for example, 25-35° C. On the other hand, the pH atmosphere for obtaining the compound includes but is not limited to pH 1 to 14, but is preferably close to neutral pH, for example, pH 6 to 8.

[0049] The solvent that can be used in the reaction in the present invention is not particularly limited, but water is preferably used, that is, an aqueous solution is preferably used, and there is no need to use an organic solvent such as DMSO. Therefore, the reaction step can be carried out in water, that is, in an aqueous solution.

[0050] The biomolecule may be at least one selected from the group consisting of peptides, affibodies, antibodies, and oligonucleotides; the nanoparticles may be at least one selected from the group consisting of metal nanoparticles, synthetic polymer nanoparticles, and biopolymer nanoparticles; in addition, the metal nanoparticles may be any metal selected from the group consisting of gold (Au), platinum (Pt), palladium (Pd), silver (Ag), and copper (Cu); or any metal oxide selected from the group consisting of cobalt (Co), manganese (Mn), iron (Fe), nickel (Ni), gadolinium (Gd), molybdenum (Mo), zinc (Zn), and chromium (Cr). On the other hand, the fluorescent dye may be at least one selected from cyanine, fluorescein, and rhodamine dyes.

[0051] In this case, the peptide is a tumor targeting peptide such as RGD peptide, CGNSNPKSC peptide, VHSPNKK peptide, CTTHWGFTLC peptide, SGKGPRQITAL peptide, SGRSA peptide, or FSRYLWS peptide, among which RGD peptide is preferred.

[0052] In addition, the affinity body is not particularly limited and can be a small molecule of 7 KDa or less that can bind to proteins such as Aβ peptides, apolipoprotein A1, CD25, CD28, c-Jun, EGFR, Factor VIII, fibrinogen, Gp120, HER2, IgA, IgE, IgM, IL-8, insulin, RSV G protein, Taq polymerase, TNF-α, transferrin or transthyretin.

[0053] Furthermore, the antibody is not particularly limited, but may be Anti-VEGFR, Anti-ERBB2, Anti-CD20, Anti-CD19, Anti-CD22, Anti-CD33, Anti-CD25, Anti-HLA-DR10β, Anti-tenascin, Anti-CEA, Anti-MUC1, or Anti-TAG72, etc.

[0054] Furthermore, the oligonucleotide is not particularly limited, but may be DNA, RNA, siRNA, or antisense oligonucleotide, etc.

[0055] The metal nanoparticles are not particularly limited, but can be metal nanoparticles such as gold (Au), platinum (Pt), palladium (Pd), silver (Ag) or copper (Cu); or can be metal oxides of metals such as cobalt (Co), manganese (Mn), iron (Fe), nickel (Ni), gadolinium (Gd), molybdenum (Mo), zinc (Zn) or chromium (Cr), etc., and are preferably gold nanoparticles or iron nanoparticles.

[0056] In addition, the synthetic polymer nanoparticles are not particularly limited, but can be polyethylene glycol, polyvinyl alcohol, polyacrylic acid, polyhydroxyester, poly(ε-caprolactone), polyorthoester, polyanhydride, polyphosphagene, poly(propylenefumarate), poly(glycolic acid), polylactic acid, poly(lactic-co-glycolic acid), poly(hydroxybutyrate), poly(hydroxybutyrate-co-valerate), polyurethane, or poly(methylmethacrylate).

[0057] Furthermore, the biopolymer nanoparticles are not particularly limited, and may be chitin, chitosan, polylysine, hyaluronic acid, alginic acid, dextran, or cellulose.

[0058] In addition, the particle size of the nanoparticles is 1 to 1000 nm, preferably 10 to 500 nm, and more preferably 50 to 200 nm. When the particle size of the nanoparticles is less than 1 nm, it is difficult to prepare the nanoparticles labeled with radioactive iodine according to the present invention; when the particle size exceeds 1000 nm, the inherent properties of the nanoparticles may be lost.

[0059] On the other hand, the fluorescent dye may be at least one selected from cyanine, fluorescein, and rhodamine-based dyes.

[0060] The radioactive elements applicable to the present invention can be selected from the group consisting of I, F, Tc, Re, Ga, In, Zr, Y, Ho, Sm and Lu, for example, they can include diagnostic radioisotopes I-125, F-18, Sc-44, Ga-67, Ga-68, Zr-89, Tc-99m or In-111, etc., or therapeutic radioisotopes Sc-47, Y-90, Sm-153, Ho-166, Lu-177, Re-188, Pb-212, Bi-213 or Th-232, etc. However, the radioactive elements applicable to the present invention are not limited thereto, but can include, without limitation, α-ray radionuclides and β-ray radionuclides as therapeutic radioisotopes for treating cancer, etc., and positron-emitting radionuclides and γ-ray radionuclides as diagnostic radioisotopes for diagnosis. The radioactive element M is preferably I or F.

[0061] The radioactive iodine may be obtained from, for example 122 I. 123 I. 124 I. 125 I. 127 I. 131 I and 132 At least one radioactive iodine selected from the group consisting of I, preferably 125 I.

[0062] According to another aspect of the present invention, a radioactive labeled compound is provided, wherein the radioactive labeled compound is represented by the following chemical formula II or chemical formula IIa and is used to label a molecule having a cyclooctyne moiety.

[0063]

[0064] In the chemical formula II and chemical formula IIa, b is 0 or an integer from 1 to 10; L is CH2, -COO- or -CONH-; and M is a radioactive element.

[0065] At this time, when the radioactive labeled compound represented by the chemical formula IIa is used, the compound of the chemical formula II can be obtained by additionally performing a step of oxidizing the -OH group of the dihydroxyphenyl part to a =O group. At this time, the method for performing the oxidation step is not particularly limited, for example, oxidation can be performed by using sodium periodate (NaIO4) or the like.

[0066] As described above, the quinone compound labeled with a radioactive element used in the present invention can be suitably used as a radioactive labeled compound for labeling a molecule having a cyclooctyne moiety.

[0067] According to another aspect of the present invention, a kit for labeling radioactive elements is provided, wherein the kit for labeling radioactive elements comprises: a cyclooctyne compound represented by the following chemical formula I and bonded to a biomolecule, a fluorescent dye or a nanoparticle compound; and a quinone compound represented by the following chemical formula II or chemical formula IIa and labeled with a radioactive element. The kit for labeling radioactive elements of the present invention is based on the labeling method of radioactive elements of the present invention, and therefore is also applicable to the contents mentioned here.

[0068]

[0069] In the chemical formula I, Z is a biomolecule, a fluorescent dye or a nanoparticle compound.

[0070]

[0071] In the chemical formulae II and IIa, b is 0 or an integer from 1 to 10; L is CH2, -COO- or -CONH-; and M is a radioactive element.

[0072] At this time, when the radioactive labeled compound represented by the chemical formula IIa is used, the compound of the chemical formula II can be obtained by additionally performing a step of oxidizing the -OH group of the dihydroxyphenyl part to a =O group. At this time, the method for performing the oxidation step is not particularly limited, for example, oxidation can be performed by using sodium periodate (NaIO4) or the like.

[0073] That is, the biomolecule may be at least one selected from the group consisting of peptides, affibodies, antibodies and oligonucleotides; the nanoparticles may be at least one selected from the group consisting of metal nanoparticles, synthetic polymer nanoparticles and biopolymer nanoparticles; the metal nanoparticles may be any metal selected from the group consisting of gold (Au), platinum (Pt), palladium (Pd), silver (Ag) and copper (Cu), or any metal oxide selected from the group consisting of cobalt (Co), manganese (Mn), iron (Fe), nickel (Ni), gadolinium (Gd), molybdenum (Mo), zinc (Zn) and chromium (Cr); in addition, the fluorescent dye may be at least one selected from cyanine, fluorescein and rhodamine-based dyes.

[0074] According to another aspect of the present invention, there is provided a biomolecule, fluorescent dye or nanoparticle compound represented by chemical formula III and labeled with a radioactive element.

[0075]

[0076] In the chemical formula III,

[0077] Z is a biomolecule, a fluorescent dye or a nanoparticle compound; b is 0 or an integer from 1 to 10; L is CH2, -COO- or -CONH-;

[0078] M is a radioactive element.

[0079] According to another aspect of the present invention, a composition for medical diagnosis and treatment is provided, wherein the composition for medical diagnosis and treatment comprises a biological molecule, fluorescent dye or nanoparticle compound represented by chemical formula III and labeled with a radioactive element as an active ingredient.

[0080] The medical diagnosis is not particularly limited, but can be a diagnosis performed by single photon emission tomography (SPECT), positron emission tomography (PET), micro-positron tomography, computed tomography (CT), magnetic resonance imaging (MRI) or target imaging of radiation diagnostic equipment.

[0081] Furthermore, according to another aspect of the present invention, a composition for treating cancer is provided, wherein the composition for treating cancer comprises as an active ingredient a biomolecule, fluorescent dye or nanoparticle compound represented by the chemical formula III and labeled with a radioactive element.

[0082] The radioactive element may be 131I, and the cancer can be, for example, thyroid cancer.

[0083] The radioactive iodine labeling method according to the present invention can label biological molecules, fluorescent dyes, nanoparticle compounds or a combination thereof at room temperature without the need for additional organic solvents by using compounds represented by chemical formula I and / or II, and can be effectively used as a radioactive iodine labeling method due to its fast reaction speed and high radiochemical yield.

[0084] Therefore, according to the present invention, it is expected that the results of effectively labeling various target compounds such as peptides with radioactive iodine can be obtained. In addition, it is expected that this result will not only be used for future research, but also lead to the development of radiopharmaceuticals that can be used clinically.

[0085] The present invention will be described in more detail below with reference to specific examples. The following examples are only used as examples to help understand the present invention, and the scope of the present invention is not limited thereto.

[0086] Example

[0087] 1. Materials and Equipment

[0088] All chemicals, reagents, solvents and materials including human serum albumin (HSA) and human transferrin (THF) were purchased from Sigma Aldrich and were not purified before use. Peptide c[RGDyK] (FC-2202) was purchased from FUTURECHEM Co., South Korea. All newly synthesized intermediate precursors were purified by silica gel column chromatography using high-purity silica gel with an average particle size of 60 μm. The progress of the reaction was monitored using glass or aluminum-based silica gel plates (60F254 pure silica gel). The compound spots on the TLC were observed by using ultraviolet light with a wavelength of 254 nm, or by using a color developer or heating. The reaction was carried out at low temperature (4°C) or room temperature (25°C). NMR ( 1 H and 13C) Analysis was performed by using a JEOL NMR spectrometer (spectrometer) (500MHz, KAERI, Jingyu City, South Korea). NMR spectra were obtained by using chloroform-d (CDCl3-d1), dimethyl sulfoxide-d6 (DMSO-d6) or methanol-d4 (CD3OD) as solvents. High-resolution mass spectrometry was performed on low molecular weight precursors using a Bruker Q-TOF high-resolution mass spectrometry system. In order to perform MALDI-TOF analysis of proteins and peptides, an AB SCIEX TOF / TOF 5800 MALDI-TOF system (Korea Institute of Radiation Medicine) was used. 3,5-dimethoxy-4-hydroxycinnamic acid (Sinapinic Acid) or 2,5-dihydroxybenzoic acid (DHB) was used as a matrix for MALDI-TOF analysis. For biodistribution studies, the radioactivity deposited in each organ was detected using an automatic gamma counter (PerkinElmer, USA). All SPECT / CT images were acquired using an Inveon (Siemens) small animal multimodality SPECT / CT system.

[0089] The analytical HPLC system supplied by Agilent Technologies was used for analysis. An Eclipse XDB-C18 column (4.6x 250mm, 5 μm) and a gamma detector were installed in the analytical HPLC system (1290infinite). For purification HPLC, an Agilent Technologies separation and purification (preparative) HPLC system (1260infinite) equipped with a gamma detector and an Eclipse XDB-C18 column (7 μm, 21.2x150mm) was used. All HPLC analyses were performed using solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). For HPLC purification, HPLC method A for separation and purification (flow rate: 10 mL / min, eluent gradient: 0-3 min: 95% A / 5% B; 3-15 min: linear gradient from 35% A / 65% B to 95% A / 5% B; 15-35 min: linear gradient from 35% A / 65% B to 5% A / 95% B; 35-45 min: 5% A / 95% B) or method B (flow rate: 10 mL / min, eluent gradient: 0-5 min: 95% A / 5% B; 5-20 min: linear gradient from 95% A / 5% B to 50% A / 50% B; 20-35 min: linear gradient from 50% A / 50% B to 5% A / 95% B; 35-45 min: linear gradient from 5% A / 95% B to 3% A / 97% B) was applied. The radioactive product was trapped in a Sep Pak C18 column conditioned with 5 mL of ethanol and 10 mL of water. 125 I] NaI aqueous solution (0.1 M in NaOH) was purchased from PerkinElmer (Korea).

[0090] 2. Synthesis of quinone compounds

[0091] (1) Synthesis of the intermediate compound N-(3,4-dihydroxyphenethyl)-4-iodobenzamide (1)

[0092] 4-Iodobenzoic acid (1 g, 4 mmol) and HBTU coupling agent (1.5 g, 3.9 mol) were dissolved in dimethylformamide DMF (10 mL). 4-(2-aminoethyl)benzene-1,2-diol hydrochloride (0.76 g, 4 mmol) and N,N-diisopropylethylamine (DIPEA) base (1 g, 7.7 mmol) were added under a nitrogen atmosphere. The reaction mixture was stirred for 2.5 hours, and 1N HCL was added to terminate the reaction. The crude product was dissolved in ethyl acetate, then washed with a saturated NaCl solution, and dried using anhydrous Na2SO4. The crude product was purified by silica gel chromatography and ethyl acetate / n-hexane (1:1) to obtain the intermediate compound N-(3,4-dihydroxyphenylethyl)-4-iodobenzamide (1) (0.9 g, 2.3 mmol, 58%). In the case of using method B, the HPLC retention time of product 1 was 22.0 minutes.

[0093] 1 H NMR (500MHz, DMSO-d6) δ8.722 (s, 1H), 8.609 (s, 1H), 8.550 (t, J = 6.5Hz, 1H), 7.797 (d, J = 8.5Hz, 2 H), 7.556 (d, J=8.5Hz, 2H), 6.406-6.596 (m, 3H, Ph), 3.319 (q, J=6.5Hz, 2H), 2.593 (t, J=8.5, 2H); 13 C NMR (125MHz, DMSO-d6)165.862, 145.593, 144.058, 137.658, 133.825, 130.71 3. 129.655, 119.744, 116.511, 116.005, 99.161, 41.854, 35.073; HRMS([M+H] + )calculated: 384.1854; found: 384.1855.

[0094] (2) Synthesis of the intermediate compound N-(3,4-dihydroxyphenylethyl)-4-(tributylstannyl)benzamide (2)

[0095] The reagent bis(tributyltin) (1.5 g, 2.6 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) (0.15 g, 0.13) and intermediate compound 1 (0.5 g, 1.3 mmol) were dissolved in 25 mL of 1,4-dioxane. The reaction mixture was refluxed for 16 hours. The crude product was cooled at 25°C and filtered using Whatman cellulose filter paper to remove undissolved impurities. The crude product was dissolved in ethyl acetate, then washed with saturated NaCl solution and dried using anhydrous Na2SO4. The crude product was purified by silica gel chromatography and ethyl acetate / n-hexane (3:7) to obtain the intermediate compound N-(3,4-dihydroxyphenethyl)-4-(tributyltinalkyl)benzamide (2) (0.9 g, 2.3 mmol, 70%).

[0096] 1 H NMR (500MHz, DMSO-d6) δ8.732 (s, 1H), 8.616 (s, 1H), 8.442 (t, J = 5.5Hz, 1H), 7.695 (d, J = 7.5Hz, 2H), 7.470 (d, J = 7.5Hz, 2H), 6.415-6.602 (m, 3 H, Ph), 3.343 (q, J=7.5Hz, 2H), 2.597 (t, J=7.5Hz, 2H), 1.401-1.559 (m, 6H), 1.211-1.284 (m, 6H), 0.956-1.136 (m, 6H), 0.806 (t, J=8.5Hz, 9H); 13 C NMR (125MHz, DMSO-d6) 166.845, 146.089, 145.593, 144.038, 136.551, 134.910, 130.799, 126. 869, 119.744, 116.511, 116.005, 41.807, 35.206, 29.111, 27.204, 14.088, 9.720; HRMS([M+H] + )calculated: 548.3390; found: 548.3395.

[0097] 3. Synthesis of compounds bonded with cyclooctyne

[0098] (1) Synthesis of cRGD peptide 5a bonded to BCN

[0099] Peptide (cRGDyK) (20 mg, 0.032 mmol) was added to a solution of (1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN-succinimidyl ester) (9.3 mg, 0.31 mmol) in DMSO (100 μL). The pH of the solution was adjusted to 8.2-8.5 using (DIPEA) (17 μL). After 2.5 hours of continuous reaction, the reaction mixture was purified by HPLC (method A) to obtain 5a (19 mg, 0.23 mmol, 74%, retention time: 15.9 min). MALDI-TOF ([M+H] + )Actual measured value: 796.4450.

[0100] (2) Synthesis of human transferrin 6a bound to BCN

[0101] TF (Transferrin human protein) (500 μM, pH 8.2, 100 μL, 1xPBS) was cultured with BCN-succinimidyl ester (20 mM, 10 μL in DMSO) for 4 hours at 4 ° C. For 6a purification, the crude product was diluted with saline (2.5 mL) and passed through a PD-10 desalting column. The extracted product was centrifuged (4500 rpm, 12 min, 4 ° C) using an Amicon filter (30 kDa). BCN-modified protein 6a was found in the residue, analyzed using a MALDI-TOF mass spectrometry system, and the concentration (mg / mL) was determined using a protein quantification system.

[0102] (3) Synthesis of human serum albumin 7a bonded to BCN

[0103] Human serum albumin bonded to BCN was synthesized using the same protocol as that employed for human transferrin.

[0104] 4. Synthesis of biomolecules labeled with radioactive elements

[0105] Embodiment 1: 125 I-cRGD peptide ([ 125 I]5) radiosynthesis

[0106] (1) Compound [ 125 Synthesis of 5

[0107] According to Radiosynthesis of 123I-labeled hesperetin for biodistributions study of orally administered hesperetin (Journal of Radioanalytical and Nuclear Chemistry, 306 (2), pp. 437-443, 1. Jeon, J. et al.) was synthesized by slightly modifying the procedure disclosed in [ 125 I]1. Briefly, the intermediate precursor 2 (1 mg) obtained by the above 2 was dissolved in 1 mL DMSO. 0.5 M phosphoric acid (250 μL) and 0.5 M peracetic acid (500 μL) were added. To start the reaction, 0.1 M NaOH was added [ 125 I]NaI (2mCi, 7μL), and reacted at room temperature for 10 minutes. The reaction was quenched by adding an excess of sodium metabisulfite aqueous solution (0.5M). The crude product was diluted with distilled water to a final volume of 5mL and purified by separation and purification using an HPLC system (Method B). The radioiodinated product [ 125 I]1 was placed in a pre-treated Sep Pac C18 column and eluted with methanol (100 μl), thereby obtaining [ 125 I] (retention time by analytical HPLC: 22.5 min).

[0108]

[0109] Scheme 1: N-(3,4-dihydroxyphenethyl)-4-iodobenzamide 1, N-(3,4-dihydroxyphenethyl)-4-(tributylstannyl)benzamide 2 and [ 125 I] Synthesis of 1-compound

[0110] (2) 125 I-cRGD peptide ([ 125 I]5) Synthesis

[0111] The purified compound [ 125I]1 (200 μCi, 10 μL in methanol solution) was treated for 10 minutes. Compound 5a obtained by 3. (1) (40 μL in methanol solution) was added to the solution. The total reaction volume was 100 μL, and the final concentration of BCN bonded to the cRGD peptide was 25 μM, 10 μM or 5 μM. The radiochemical yield of the reaction process was monitored at different reaction times using a radio-HPLC system (method A). The radiochemical yield results are summarized in Table 1 below.

[0112] Embodiment 2 and 3: 125 I-TF([ 125 I]6) and 125 I-HSA([ 125 I]7) Synthesis

[0113] The purified intermediate precursor [ 125 I]1 (200μCi, 10μL in methanol solution) was treated for 10 minutes. The reaction mixture was diluted with 40mL of distilled water and captured in a Sep Pac C18 column pretreated with radioactivity to remove unreacted NaIO4. The oxidized radiotracer was eluted with 100μL of methanol and the solvent volume was reduced to 10μL under reduced pressure. The radioactive product was then incubated together with 6a or 7a (100uL, 1×PBS) obtained by 3.(2) and 3.(3), respectively, to a final concentration of 6a or 7a of 25μM, 10μM or 5μM. The progress of the iodination reaction of the antibody according to the reaction time and concentration was monitored using a radio-TLC system at different time points, and the results are summarized in Table 1 below.

[0114]

Table 1

[0115] For use with [ 125 I] BCN-containing substrates and conditions for the reaction

[0116] <![CDATA[Project a > Substrate installed with BCN Concentration (μmol) Time (min) product <![CDATA[%RCY * ]]> 1 c(RGDyK) / 5a 25 5 <![CDATA[[ 125 I]5]]> <![CDATA[83 b,c ]]> 2 c(RGDyK) / 5a 25 10 <![CDATA[[ 125 I]5]]> <![CDATA[>99 b,c ]]> 3 c(RGDyK) / 5a 10 10 <![CDATA[[ 125 I]5]]> <![CDATA[95 b,c ]]> 4 <![CDATA[THP protein ** / 6a]]> 25 15 <![CDATA[[ 125 I]6]]> <![CDATA[97 b,d ]]> 5 <![CDATA[THP protein ** / 6a]]> 10 15 <![CDATA[[ 125 I]6]]> <![CDATA[90 b,d ]]> 6 <![CDATA[THP protein ** / 6a]]> 5 15 <![CDATA[[ 125 I]6]]> <![CDATA[79 b,d ]]> 7 <![CDATA[HSA protein *** / 7a]]> 25 5 <![CDATA[[ 125 I]7]]> <![CDATA[77 b,d ]]> 8 <![CDATA[HSA protein *** / 7a]]> 25 15 <![CDATA[[ 125 I]7]]> <![CDATA[>99 b,d <!-- 10 -->]]> 9 <![CDATA[HSA protein *** / 7a]]> 10 15 <![CDATA[[ 125 I]7]]> <![CDATA[89 b,d ]]>

[0117] *RCY: Radiochemical yield **THP protein: Transferrin human protein

[0118] ***HSA protein: Human serum albumin protein

[0119] aSolvent system and reaction conditions; Items 1-3: methanol / water (1:1), (pH 7.5), room temperature; Items 4-9: 10% methanol in 1x PBS solvent, (pH 7.5), room temperature.

[0120] b After treatment with sodium periodate (80 μg), the purified 125 I]1 to react.

[0121] c Radiochemical yields determined by radio-HPLC.

[0122] d Radiochemical yields determined by radio-TLC.

[0123]

[0124] R=c(RGDyK)peptide 5THF protein 6HSA protein 7

[0125] Solution 2: 125 I]5 to[ 125 Synthesis of 7

[0126] Comparative Example 1: Synthesis of iodinated cRGD peptide 5b

[0127] Intermediate compound 1 (5 mg, 0.013 mmol) dissolved in 1 mL MeOH was treated with sodium periodate (NaIO4) (1 equivalent, 1 mL H2O) for 10 minutes. Intermediate compound 5a (10 mg, 0.012 mmol) was added to the reaction mixture. After 30 minutes, the crude product was purified by HPLC (method A) to obtain iodinated cRGD peptide (5b) (12.5 mg, 0.010 mmol, retention time: 19.0 minutes). MALDI-TOF ([M+H] + )Actual measured value: 1177.3569.

[0128]

[0129] Scheme 3: Synthesis of iodinated cRGD peptide 5b

[0130] Comparative Example 2: 125 Synthesis of I-HSA (8) (direct radioiodination)

[0131] Use at 4℃ 125HSA (25 μM, 100 μL saline) was treated with aqueous NaI (1 mCi, 3.5 μL) and chloramine-T (100 μg, 10 μL) for 10 minutes. The reaction was terminated with 10 μL of sodium metabisulfite aqueous solution (1 mg, 20 μl). The crude product was purified using a PD-10 desalting column to obtain 8 ( ) with a radiochemical yield of 93% and a radiochemical purity of more than 99% as determined by a radio-TLC system. 125 I-HSA).

[0132] Comparative Example 3: 125 Synthesis of I-HSA (9)

[0133] The reagent 3-(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester was purchased from Sigma Aldrich, South Korea. The radioiodination of HSA protein was based on the preparation of the iodine-124 derivative of the Bolton-Hunter reagent ([ 124 I]I-SHPP) and its use for labelling a VEGF antibody as a PETtracer (Journal of Labelled Compounds and Radiopharmaceuticals. 2002 Oct 30; 45(12): 1077-90, Glaser M et al.) was used to perform the procedure published in the literature. The crude product was purified using a PD-10 desalting column to obtain 9[I]I-SHPP with a radiochemical yield of 85% and a radiochemical purity of more than 99%. 125 I-HSA].

[0134] 5. 125 Biodistribution study of I-HSA

[0135] All animal experiments followed the guidelines of the institutional animal ethics committee. Twenty-five ICR male mice were purchased from Orientbio Co., Ltd (Korea) for detailed tissue distribution studies. The animals were randomly divided into five groups, each containing five mice. Each mouse was injected with the 125 I-HSA([ 125I]7) (1 μCi, 100 μL saline). Animals were sacrificed and key organs and blood were collected at the time points given in Table 2 below. The collected organs and blood were weighted and the accumulated radioactivity was measured using a gamma counter. The final tissue distribution data are shown as the percentage of injected dose per gram of blood or mouse organ (injected dose per gram, %ID / g). The results are shown in Figure 1 .

[0136] The tissue distribution of radioiodinated HSA proteins 8 and 9 obtained in Comparative Examples 2 and 3, respectively, was also performed in the same manner. The results are shown in Tables 2 and 3.

[0137]

Table 2

[0138] Radioiodinated HSA[ 125 Biodistribution results of 8

[0139]

[0140]

[0141] a n = 5, 5 animals per group; percentage of radioactive injection dose / gram of organ

[0142]

Table 3

[0143] Radioiodinated HSA[ 125 I]9 biodistribution results

[0144]

[0145] a n = 5, 5 animals per group; percentage of radioactive injection dose / gram of organ

[0146] 6. Competitive reaction studies

[0147] In order to determine the oxidized 125 I]1Two groups of experiments were conducted to investigate the response preference of the BCN group.

[0148] In experiment (I), oxidized [ 125 I] 1 (200 μCi, 20 μL in methanol), cRGDP peptide bonded to BCN (25 μM) and a mixture of non-denatured HSA protein (25 μM) containing. The reaction was performed for 30 minutes and centrifuged using a 50 kDa Amicon filter (4500 RPM, 12 min, 4 ° C). The radioactivity associated with cRGD peptide (filtrate) or HSA protein (residue) was measured using a gamma counter system.

[0149] In experiment (II), under conditions similar to those in experiment (1), oxidized [ 125 I]1 (200 μCi, 20 μL in methanol), HSA protein conjugated to BCN (25 μM), and native cRGD peptide (25 μM).

[0150] Each experiment was repeated three times, and the product identity was confirmed by radio-HPLC or radio-TLC system. The final radiolabeling results are summarized in Table 4.

[0151]

Table 4

[0152] Radiolabeling results for competitive reaction studies

[0153] experiment molecular %RCY <![CDATA[ 125 I-Molecule]]> II c(RGDyK)25μmol+HSA-BCN 25μmol 93-97 <![CDATA[ 125 I-HSA]]> I c(RGDyK)-BCN 25μmol+HSA 25μmol 90-94 <![CDATA[ 125 I-c(RGDyK)]]>

[0154] *RCY: Radiochemical yield

[0155] 7. 125 SPECT / CT imaging of I-HSA

[0156] ICR mice were injected intravenously 125 I-HSA([ 125 I]7) (150 μCi, 100 μL saline). SPECT / CT images were acquired at given times (0.25 hours, 1 hour, 4 hours, 16 hours, and 24 hours). The results are shown in Figure 2 (B).

[0157] Use the same process to obtain information about [ 125 The SPECT / CT images of 18 samples were used for comparison. The results are shown in Figure 2 (A).

[0158] 8. From [ 125 Comparison of thyroid uptake of 7, 8, and 9 free radioactive iodine

[0159] Deiodination in vivo, i.e. from radioiodinated HSA[ 125 I]7, 8 and 9 free 125 The results of the absorption degree of I in the thyroid gland showed that compared with the other two radiotherapy methods, the absorption degree of I in the thyroid gland obtained by Example 3 was significantly higher than that of the other two radiotherapy methods. 125 I-HSA([ 125 I]7) has remarkable stability. The results are shown in Figure 3 .

[0160] Although the embodiments of the present invention have been described in detail above, the scope of rights of the present invention is not limited thereto. A technician with common knowledge in the technical field to which the present invention belongs can obviously make various modifications and variations to the present invention without departing from the scope of the technical idea of ​​the present invention recorded in the claims.

Claims

1. A method for labeling a radioactive element, comprising: A step of providing a cyclooctyne compound represented by the following chemical formula I and bonded to a biomolecule; as well as A step of reacting the cyclooctyne compound with a quinone compound represented by the following chemical formula II and labeled with a radioactive element to obtain a reaction of chemical formula III, wherein the reaction step is carried out in an aqueous solution at room temperature and atmospheric conditions; In the chemical formula I, Z is a biomolecule, and the biomolecule is at least one selected from the group consisting of a peptide, an affibody, an antibody, and an oligonucleotide; In the chemical formula II, b is 0 or an integer from 1 to 10; L is -CONH-; M is a radioactive element, and the radioactive element is at least one of an α-ray radionuclide, a β-ray radionuclide, a positron-emitting radionuclide, and a γ-ray radionuclide; In chemical formula III, Z is a biological molecule, which is at least one selected from the group consisting of peptides, affibodies, antibodies and oligonucleotides; b is 0 or an integer from 1 to 10; L is -CONH-; and M is a radioactive element, which is at least one of α-ray radionuclides, β-ray radionuclides, positron-emitting radionuclides and γ-ray radionuclides.

2. The method for labeling a radioactive element according to claim 1, wherein: The radioactive element M is selected from the group consisting of I, F, Tc, Re, Ga, In, Zr, Y, Ho, Sm and Lu.

3. A radiolabeled compound represented by the following chemical formula III, wherein: The radiolabeled compound is obtained by reacting the following chemical formula II with a molecule having a cyclooctyne moiety, In the chemical formula II, b is 0 or an integer from 1 to 10; L is -CONH-; M is a radioactive element, and the radioactive element is at least one of an α-ray radionuclide, a β-ray radionuclide, a positron-emitting radionuclide, and a γ-ray radionuclide; In chemical formula III, Z is a biological molecule, which is at least one selected from the group consisting of peptides, affibodies, antibodies and oligonucleotides; b is 0 or an integer from 1 to 10; L is -CONH-; and M is a radioactive element, which is at least one of α-ray radionuclides, β-ray radionuclides, positron-emitting radionuclides and γ-ray radionuclides.

4. A kit for labeling radioactive elements, comprising: A cyclooctyne compound represented by the following chemical formula I and bonded to a biomolecule, a fluorescent dye or a nanoparticle compound; as well as A quinone compound represented by the following chemical formula II or chemical formula IIa and labeled with a radioactive element, In the chemical formula I, Z is a biomolecule, and the biomolecule is at least one selected from the group consisting of a peptide, an affibody, an antibody, and an oligonucleotide; In the chemical formula II and the chemical formula IIa, b is 0 or an integer from 1 to 10; L is -CONH-; M is a radioactive element, and the radioactive element is at least one of an α-ray radionuclide, a β-ray radionuclide, a positron-emitting radionuclide, and a γ-ray radionuclide; Wherein, chemical formula I reacts with chemical formula II or chemical formula IIa to obtain chemical formula III, In chemical formula III, Z is a biological molecule, which is at least one selected from the group consisting of peptides, affibodies, antibodies and oligonucleotides; b is 0 or an integer from 1 to 10; L is -CONH-; and M is a radioactive element, which is at least one of α-ray radionuclides, β-ray radionuclides, positron-emitting radionuclides and γ-ray radionuclides.

5. A biomolecule represented by chemical formula III and labeled with a radioactive element, In the chemical formula III, Z is a biomolecule, and the biomolecule is at least one selected from the group consisting of a peptide, an affibody, an antibody, and an oligonucleotide; b is 0 or an integer from 1 to 10; L is -CONH-; M is a radioactive element, and the radioactive element is at least one of α-ray radionuclides, β-ray radionuclides, positron-emitting radionuclides, and γ-ray radionuclides.

6. A composition for medical diagnosis, comprising the biomolecule represented by chemical formula III according to claim 5 and labeled with a radioactive element as an effective ingredient.

7. A composition for treating cancer, comprising the biomolecule represented by chemical formula III and labeled with a radioactive element according to claim 5 as an effective ingredient.

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