KR20200008942A - Method for labeling radioisotope, radiolabeled compounds and kit comprising the same for labeling radioisotope - Google Patents

Abstract

The present invention relates to a radioactive element labeling method, a radiolabeled compound, and a radioactive element labeling kit comprising the same. More specifically, the present invention relates to: a radioactive element labeling method comprising a step of providing a diaminophenyl compound represented by chemical formula (I), to which a biomolecule, a fluorescent dye or a nanoparticle compound is bound and a step of conducting a reaction of an aldehyde compound represented by chemical formula (II) to which a radioactive element is labeled at room temperature; and related technology.

Description

Method for labeling radioisotope, radiolabeled compounds and kit comprising the same for labeling radioisotope

The present invention relates to a method for labeling a radioactive element, a radiolabeling compound and a radioactive element labeling kit including the same, and more particularly, a method for labeling a radioactive element in which accumulation in the thyroid gland is significantly reduced, a radiolabeling compound and the same The radioactive element labeling kit and the like using the same technology can be applied in the field of medical diagnosis and treatment.

In order to use bioactive substances such as peptides, proteins, antibodies, etc. for molecular imaging or disease diagnosis / treatment, radiolabeling technology of biomolecules has been developed. The prior art mainly used a method of conjugating a chelator to a bioactive material. This method chemically introduces a separate chelator into the bioactive material, which reduces the reaction rate at the conjugation of the chelator or that is not suitable for the bioactive material, such as high temperature reactions, acidic or basic reactions, and toxicity. There was a fundamental limitation to improve the reaction solvent and the like.

Representative radioactive iodine has been used for decades for radiolabeling biomolecules for diagnostic and therapeutic purposes. Specifically, ¹²⁴ I is a radioactive iodine mainly used for positron emission tomography (PET), ¹²³ I and ¹²⁵ I are for single photon emission computed tomography (SPECT), and ¹³¹ I It is used for the diagnosis and treatment of diseases such as thyroid cancer.

As described above, as the radioactive iodine is widely used in medicine, various labeling methods have been developed for labeling radioactive iodine on biomolecules and small-molecule molecules, and electrophilic aromatic substitution reactions are performed directly on radioisotopes. It shows high efficiency as a way to label. However, most of the labeling compounds synthesized through the labeling method are unstable in the animal body, and thus often do not obtain desired image results, and a strong oxidant used for labeling has been shown to reduce the biological activity of the biological material. A method of indirectly labeling radioactive iodine, which can solve the above problems, has been studied, and several prosthetic groups have been developed. However, the method of indirectly labeling and synthesizing radioactive iodine developed up to now does not have a chemical selectivity for an active group, so that a random labeling compound is often produced, and the reaction rate is slow, and an excess substrate is required for high yield. have.

Therefore, there is a need for the development of a new radioactive iodine labeling method that is stable in living cells and animals and can be applied as an imaging diagnostic and therapeutic material with reduced accumulation in the body, such as the thyroid gland. In the related art, Korean Patent Publication No. 2012-0101073 relates to iodine-labeled homoglutamic acid and glutamic acid derivatives, and discloses a technique related to ¹²⁵ I-labeled homoglutamic acid and glutamic acid derivatives. However, the prior art has a problem that uptake in blood is not sufficient.

Thus, radioactive labeling compounds and biomolecules, fluorescent dyes or nanoparticle compounds can be used to label radioactive elements in biomolecules and the like with rapid reaction rates and high radiochemical yields. If a radiolabeled compound having improved uptake is provided, it is expected to be useful for radioactive labeling and medical diagnosis of biomolecules.

Accordingly, an aspect of the present invention is to provide a method for labeling radioactive elements using an aldehyde-diamine condensation reaction.

Another aspect of the present invention is to provide a radiolabeled compound for labeling molecules having an amine moiety.

Another aspect of the present invention is to provide a radioactive element labeling kit using an aldehyde-diamine condensation reaction.

Another aspect of the present invention is to provide a radiolabeled compound for labeling molecules having a thiol moiety.

Another aspect of the invention is to provide a biomolecule, fluorescent dye or nanoparticle compound labeled with a radioactive element.

Another aspect of the present invention is to provide a medical diagnostic composition comprising a biomolecule, fluorescent dye or nanoparticle compound labeled with the radioactive element of the present invention.

According to an aspect of the present invention, a step of providing a diaminophenyl compound represented by the following formula (I), the biomolecule, fluorescent dye or nanoparticle compound is bound; And it is represented by the following formula (II) and the diaminophenyl compound, a radioactive element labeled aldehyde compound comprising the step of reacting at room temperature, there is provided a method of labeling a radioactive element.

[화학식 I]

[Formula I]

(In Formula I,

이며; Z는 생체 분자, 형광 염료 또는 나노입자 화합물이다.)A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; Y is CH ₂ or

Is; Z is a biomolecule, fluorescent dye or nanoparticle compound.)

[화학식 II]

[Formula II]

(In Formula II,

b is 0 or an integer from 1 to 10; L is CH ₂ or -CONH-;

,

,

또는

이며, Q is

,

,

or

Is,

Where M, M 'and M "are radioactive elements.)

According to another aspect of the present invention, there is provided a radiolabeled compound for labeling molecules having an amine moiety, represented by the formula (II), comprising an aldehyde compound labeled with a radioactive element.

According to another aspect of the present invention, a diaminophenyl compound represented by Chemical Formula (I), to which a biomolecule, a fluorescent dye, or a nanoparticle compound is bound; And an aldehyde compound labeled with a radioactive element, represented by the formula (II), is provided.

According to still another aspect of the present invention, there is provided a radiolabeled compound for labeling molecules having a thiol moiety for labeling molecules having a thiol moiety represented by the following formula (III).

[화학식III]

[Formula III]

(In Formula III, A, a, X, b, L, Q, and Q in M, M 'and M "are as defined above.)

According to still another aspect of the present invention, there is provided a biomolecule, fluorescent dye or nanoparticle compound labeled with a radioactive element represented by the following general formula (IV).

[화학식IV]

[Formula IV]

(In Formula IV, A, a, X, b, L, Q, and Q in M, M 'and M "are as defined above, Z' is a biomolecule containing a thiol group or substituted with a thiol group , Fluorescent dye or nanoparticle compound except the thiol group structure.)

According to still another aspect of the present invention, there is provided a medical diagnostic composition comprising a biomolecule, a fluorescent dye, or a nanoparticle compound labeled with a radioactive element represented by Formula IV as an active ingredient.

The method for labeling radioactive elements and related technologies according to the present invention is based on radioisotope labeling using an aldehyde-diamine condensation reaction, and is a biomolecule, fluorescent dye or nanoparticle compound labeled with a radioactive element obtained by the present invention. Is commercialized as a medical diagnostic composition and is highly likely to be commercialized for the purpose of molecular imaging, diagnosis, treatment, etc., and is expected to contribute greatly to the health and welfare of the people.

1 is a Radio or UV / VIS-HPLC chromatogram: (a) Radio-HPLC chromatogram for crude product [ ¹²⁵ I] 5, (b) Radio- for purified [ ¹²⁵ I] 5 HPLC chromatogram, (c) UV / VIS-HPLC chromatogram for compound 5.
FIG. 2 shows the in vitro stability of the [ ¹²⁵ I] 5 compound in various media at 37 ° C. FIG.
Figure 3 shows the results of the evaluation of the biodistribution (biodistribution) of the normal ICR male mouse (n = 5) of the [ ¹²⁵ I] 9 compound.

Hereinafter, with reference to the accompanying drawings will be described a preferred embodiment of the present invention. However, 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.

According to the present invention there is provided a radioactive element labeled aldehyde compound with markedly improved accumulation on the thyroid gland, such a compound may also be referred to as a "tracer". The tracer of the present invention can label radioactive elements at high reaction rates and high radiochemical yields through condensation reactions with biomolecules, fluorescent dyes or nanoparticles containing diaminophenyl moiety.

More specifically, the labeling method of the radioactive element of the present invention uses a condensation reaction of aldehyde-diamine, which is represented by the following general formula (I), and provides a diaminophenyl compound in which a biomolecule, a fluorescent dye, or a nanoparticle compound is bound. Making; And reacting the diaminophenyl compound with an aldehyde compound labeled with a radioactive element at room temperature.

[화학식 I]

[Formula I]

이며; Z는 생체 분자, 형광 염료 또는 나노입자 화합물이다.In formula (I), A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; Y is CH ₂ or

Is; Z is a biomolecule, fluorescent dye or nanoparticle compound.

[화학식 II]

[Formula II]

In Formula II, b is 0 or an integer of 1 to 10; L is CH ₂ or -CONH-;

,

,

또는

이며, Q is

,

,

or

Is,

M, M 'and M "are radioactive elements.

That is, the present invention can be obtained by the above-mentioned biomolecule, fluorescent dye or nanoparticle compound by reacting the aldehyde compound at room temperature, high temperature reaction, acidic or basic conditions, the use of toxic reaction solvent, etc. are not required. There is no advantage. At this time, the room temperature may be 10 to 40 ℃, for example 25 to 35 ℃.

The pH conditions for obtaining the compound are not particularly limited to pH 1 to 14, but are preferably pH close to neutral, for example pH 6 to 8.

The solvent that can be used in the reaction of the present invention is not particularly limited, but is preferably a water-soluble solvent, and may be, for example, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), or the like. have.

Furthermore, the condensation reaction product of the aldehyde-diamine obtained by the labeling method of the radioactive element according to the present invention showed stable properties in vivo, and the bound diaminophenyl compound and the radioactive element were labeled.

It is preferable to use a catalyst when performing the step of reacting the diaminophenyl compound of the present invention with an aldehyde compound labeled with a radioactive element at room temperature. For example, a catalyst such as copper sulfate (CuSO ₄ ) may be used as a Cu ⁺² catalyst. Can be used, but is not limited thereto.

Process of the labeling method of the radioactive element of the present invention can be represented schematically as shown in the following formula (I), such a reaction can be obtained in a yield of about 67 to 99%.

On the other hand, the step of providing a diaminophenyl compound may include providing a compound of formula (Ia) comprising a thiol moiety (Thiol moiety); And reacting a compound of formula (Ia) with a compound of formula (Ib) to obtain a compound of formula (I).

Formula Ia

Z'-SH

In the above formula (Ia), Z 'is the same as the structure excluding the -SH portion in Z when the biomolecule, fluorescent dye or nanoparticle compound represented by Z in formula I includes a thiol portion; When the biomolecule, fluorescent dye or nanoparticle compound represented by Z does not contain a thiol moiety, Z 'is the same as Z.

That is, in the present invention, the biomolecule, fluorescent dye or nanoparticle compound containing a thiol group or substituted with a thiol group has a structure as shown in Formula (Ia).

[화학식 Ib]

Formula Ib

In formula (Ib), A, a and X are as defined in formula (I).

At this time, the reaction of the compound of formula (Ia) and the compound of formula (Ib) is preferably carried out at room temperature and atmospheric conditions under a Cu ^{+ 2} catalyst.

That is, the compound of formula (I) of the present invention reacts the structure of formula (Ib) with a biomolecule, fluorescent dye or nanoparticle compound comprising a thiol moiety, and thus contains various desired biomolecule, fluorescent dye or nanoparticle compounds. It can be designed to easily obtain a diaminophenyl compound.

Thus, providing the compound of formula (Ia) comprising the thiol moiety may comprise, for example, cysteine (C) when the biomolecule, fluorescent dye or nanoparticle compound comprises a thiol moiety. In the case of a biological molecule, a fluorescent dye, or a nanoparticle compound that does not contain a thiol moiety, the peptide may be directly applied to the peptide and the protein, and the thiol moiety is substituted with Z ′ to provide the compound of Formula (Ia). It may further comprise a step.

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 nanoparticle may be any one selected from the group consisting of gold (Au), platinum (Pt), palladium (Pd), silver (Ag), and copper (Cu); Or any one selected from the group consisting of cobalt (Co), manganese (Mn), iron (Fe), nickel (Ni), gadolinium (Gd), molybdenum (Mo), zinc (Zn), and chromium (Cr). Phosphorus metal oxide. On the other hand, the fluorescent dye may be at least one selected from cyanine, fluorescein (fluorescein) and rhodamine (rhodamine).

At this time, the peptide is a cancer targeting peptide (tumor targeting peptide), RGD peptide, CGNSNPKSC peptide, VHSPNKK peptide, CTTHWGFTLC peptide, SGKGPRQITAL peptide, SGRSA peptide, FSRYLWS peptide and the like, preferably, RGD peptide.

In addition, the affibody is not limited, Aβ peptide (peptides), Apolipo protein A1, CD25, CD28, c-Jun, EGFR, Factor VIII, Fibrinogen, Gp120, HER2, IgA, Within 7 KDa, which can bind to proteins such as IgE, IgM, IL-8, Insulin, RSV G protein, Taq polymerase, TNF-α, Transferrin, Transthyretin, etc. It may be a small molecule.

Furthermore, the antibody is not limited to, but is not limited to, Anti-VEGFR, Anti-ERBB2, Anti-CD20, Anti-CD19, Anti-CD22, Anti-CD33, Anti-CD25, Anti-HLA-DR 10β, Anti-tenascin, Anti -CEA, Anti-MUC1, Anti-TAG 72 and the like.

In addition, the oligonucleotide is not limited, but may be DNA, RNA, siRNA, antisense oligonucleotide, and the like.

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

In addition, the synthetic polymer nanoparticles are not limited, polyethylene glycol (poly (ethylene glycol)), polyvinyl alcohol (poly (vinyl alcohol)), poly acrylic acid (poly (acrylic acid)), polyhydroxy Poly (hydroxyester), polycaprolactone (poly (ε-caprolactone)), polyorthoester (poly (orthoester)), polymer anhydride (polanhydride), polyphosphagene, polypropylene fumarate (poly (propylenefumarate)), poly (glycolic acid), poly (lactic acid), poly (lactic-co-glycolic acid), polyhydric Poly (hydroxybutyate), poly (hydroxybutyrate-co-valerate), polyurethane (polyurethane), polymethylmethacrylate (poly (hydroxybutyate)) methyl methacrylate)).

Further, the biopolymer nanoparticles are not limited, but chitin, chitosan, polylysine, hyaluronic acid, alginic acid, dextran, cellulose And the like.

In addition, the nanoparticles are 1 nm to 1000 nm in size, preferably 10 m to 500 nm in size, more preferably 50 nm to 200 nm in size. When the size of the nanoparticles is less than 1 nm, there is a problem in that it is difficult to manufacture the radioactive iodine-labeled nanoparticles according to the present invention.

On the other hand, the fluorescent dye may be at least one selected from cyanine, fluorescein (fluorescein) and rhodamine (rhodamine) system.

The radioactive element that can be applied to the present invention may be selected from the group consisting of I, F, Tc, Re, Ga, In, Zr, Y, Ho, Sm and Lu, for example, I- which is a diagnostic radioisotope. 125, F-18, Sc-44, Ga-67, Ga-68, Zr-89, Tc-99m, In-111 and other therapeutic radioisotopes Sc-47, Y-90, Sm-153, Ho- 166, Lu-177, Re-188, Pb-212, Bi-213, Th-232 and the like. However, the radioactive elements that can be applied to the present invention are not limited thereto, and alpha-emitting nuclides and beta-emitting nuclides, which are therapeutic radioisotopes used for the treatment of cancer, and positron emission, which are diagnostic radioisotopes for diagnosis, are used. It includes, but is not limited to, nuclides and gamma-emitting nuclides.

In this case, the radioactive element M is I or F, M 'is an element selected from the group consisting of Tc and Re, M "may be an element selected from the group consisting of Ga, In, Y and Zr.

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

According to another aspect of the present invention, a radiolabeled compound containing an aldehyde moiety and represented by the following general formula (II), for labeling a molecule having an amine moiety, is provided.

[화학식 II]

[Formula II]

In Formula II, b is 0 or an integer of 1 to 10; L is CH ₂ or -CONH-;

,

,

또는

이며, Q is

,

,

or

Is,

In Q, M, M 'and M "are radioactive elements.

The compound of formula (II) may preferably have the same structure as in formula (IIa), wherein M may be a radioactive iodine.

[화학식 IIa]

[Formula IIa]

The aldehyde compound labeled with a radioactive element used in the present invention as described above may be applied as a radiolabeled compound for molecular labeling having an amine moiety, for example, a diamine moiety, preferably a diaminophenyl moiety.

According to another aspect of the present invention, a diaminophenyl compound represented by the following general formula (I), to which a biomolecule, fluorescent dye, or nanoparticle compound is bound; And a aldehyde compound labeled with a radioactive element, represented by the following formula (II), is provided. The radioactive element labeling kit of the present invention is based on the radioactive element labeling method of the present invention described above, and all of the above-mentioned contents apply equally.

[화학식 I]

[Formula I]

이며; Z는 생체 분자, 형광 염료 또는 나노입자 화합물이다.In formula (I), A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; Y is CH ₂ or

Is; Z is a biomolecule, fluorescent dye or nanoparticle compound.

[화학식 II]

[Formula II]

In Formula II, b is 0 or an integer of 1 to 10; L is CH ₂ or -CONH-;

,

,

또는

이며, Q is

,

,

or

Is,

In Q, M, M 'and M "are radioactive elements.

That is, the biomolecule is at least one selected from the group consisting of peptides, affibodies, antibodies and oligonucleotides; The nanoparticles are at least one selected from the group consisting of metal nanoparticles, synthetic polymer nanoparticles, and biopolymer nanoparticles; The metal nanoparticle may be any one selected from the group consisting of gold (Au), platinum (Pt), palladium (Pd), silver (Ag), and copper (Cu); Or any one selected from the group consisting of cobalt (Co), manganese (Mn), iron (Fe), nickel (Ni), gadolinium (Gd), molybdenum (Mo), zinc (Zn), and chromium (Cr). Phosphorus metal oxides; The fluorescent dye may be at least one selected from cyanine, fluorescein, and rhodamine.

The radioactive element is also not particularly limited and may be selected from the group consisting of I, F, Tc, Re, Ga, In, Zr, Y, Ho, Sm and Lu as mentioned in the labeling method of the radioactive element of the present invention. For example, the diagnostic radioisotopes I-125, F-18, Sc-44, Ga-67, Ga-68, Zr-89, Tc-99m, In-111, etc. 47, Y-90, Sm-153, Ho-166, Lu-177, Re-188, Pb-212, Bi-213, Th-232 and the like. However, radioactive elements that can be applied to the present invention are not limited thereto, and alpha-emitting nuclides and beta-emitting nuclides, which are therapeutic radioisotopes used for the treatment of cancer, and positron emission, which are diagnostic radioisotopes for diagnosis, are used. It includes, but is not limited to, nuclides and gamma-emitting nuclides.

In this case, the radioactive element M is I or F, M 'is an element selected from the group consisting of Tc and Re, M "may be an element selected from the group consisting of Ga, In, Y and Zr.

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

According to another aspect of the present invention, there is provided a radiolabeled compound for labeling molecules having a thiol moiety represented by the following general formula (III).

[화학식III]

[Formula III]

In formula (III), A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; b is 0 or an integer from 1 to 10; L is CH ₂ or -CONH-;

,

,

또는

이며, 상기 Q에서 M, M' 및 M"는 방사성 원소이다.Q is

,

,

or

In the above Q, M, M 'and M "are radioactive elements.

The radiolabeled compound represented by Chemical Formula III may be used as a radiolabeled compound for labeling molecules having a thiol moiety, and the radiolabeled compound represented by Chemical Formula III includes maleimide moieties, and thus is specific for Thiol moieties. Biocompatible molecules, fluorescent dyes or nanoparticles, for example, wherein one of the components of the amino acid sequence can be selectively conjugated to peptides and proteins bearing cysteine (C) In the case of a compound, the radiolabeled compound of the present invention may be used by substituting a thiol moiety.

According to another aspect of the present invention, there is provided a biomolecule, fluorescent dye or nanoparticle compound labeled with a radioactive element represented by the following general formula (IV).

[화학식IV]

[Formula IV]

In formula (IV), A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; Z 'is a structure excluding a thiol group in a biomolecule, fluorescent dye or nanoparticle compound containing or substituted with a thiol group; b is 0 or an integer from 1 to 10; L is CH ₂ or -CONH-;

,

,

또는

이며, Q is

,

,

or

Is,

In Q, M, M 'and M "are radioactive elements.

According to still another aspect of the present invention, there is provided a medical diagnostic and therapeutic composition comprising a biomolecule, fluorescent dye or nanoparticle compound labeled with a radioactive element represented by the above formula (IV) as an active ingredient.

The medical diagnosis is not particularly limited, but targets of single photon emission tomography (SPECT), positron emission tomography (PET), micro-PET, computed tomography (CT), magnetic resonance imaging (MRI), or radiographs The diagnosis may be by imaging.

Radioactive iodine labeling method according to the present invention, by using a compound represented by the formula (I) and / or II, it is possible to radioactive iodine labeling biomolecules, fluorescent dyes, nanoparticle compounds or combinations thereof, within 30 to 60 minutes As the reaction proceeds at a high speed and shows a high radiochemical yield value of 67% or more, it can be usefully used as a radioactive iodine labeling method.

Therefore, according to the present invention, it is expected that the resultant labeled with radioactive iodine can be effectively obtained for various target compounds, for example, peptides. In addition, this result is expected to lead to the development of radiopharmaceuticals that can be used in clinical as well as future studies.

Hereinafter, the present invention will be described in more detail with reference to specific examples. The following examples are merely examples to help understanding of the present invention, but the scope of the present invention is not limited thereto.

Example

One. ¹²⁵ I Synthesis of Labeled Compounds

(1) ¹²⁵ I-labeled aldehyde synthesis

^For the synthesis of ¹²⁵ I-labeled aldehydes [ ¹²⁵ I] 5, the non-radioactive analog 4-iodo-N- (4 is obtained by coupling 4-aminobutyraldehydediethylacetal and 4-iodo benzoic acid under basic conditions. Oxobutyl) benzamide (5) was synthesized. The next step was carried out (Scheme 1). Radioiodination of 6 was achieved using [ ¹²⁵ I] NaI and chloramines-T as oxidants (Scheme 3). The radioiodine reaction was terminated by adding an aqueous solution of sodium metabisulfite (sodium metabisulfite). [ ¹²⁵ I] 5 was obtained in high radiochemical yield (72 ± 6%, n = 5) after HPLC purification of the crude product. The specific activity was 45 GBq / μmol and the chemical purity was over 99%. HPLC chromatograms of the crude mixture clearly showed the main product [ ¹²⁵ I] 5 at 22.6 minutes (FIG. 1). Radio-labelling reactions were performed using varying amounts of radioactivity (100 μCi to 1 mCi) but radiochemical results were consistent. The radioactive iodine compound [ ¹²⁵ I] 5 was found to be stable for at least 6 months in a refrigerator (4 ° C.) and was not hydrolyzed unlike the conventionally used active ester-based prosthetic groups (Bolton-Hunter-reagent). Compound [ ¹²⁵ I] 5 was shown to be stable for 24 hours at 37 ° C. in various media including PBS, saline and mouse serum, which was confirmed using radio-HPLC (FIG. 2).

Scheme 1: Synthesis of Compound 5 and Compound 7

Reagents and conditions: (i) HBTU, DIPEA, 4-aminobutyraldehyde diethyl acetal, room temperature, 2h (ii) acid hydrolysis (iii) Pd (Ph ₃ P) ₄ , bis (tributyltin), 1, 4-dioxane, reflux, (iv) Cu ⁺² , pH 7.5, room temperature, 2h, air

(2) Synthesis of Maleimide Aryldiamine Linker

The aryl diamine compound (4) disclosed in the above (1) was synthesized by the following scheme 2.

Scheme 2: Synthesis of Compound 4

Reagents and conditions: (i) Zn / HCOOH, MeOH, 1 h (ii) (Boc) ₂ O, H ₂ O, room temperature, 2d (iii) methyl 5-bromovalerate, K ₂ CO ₃ , DMF, room temperature, 24h (iv) LiOH, Dioxane / H ₂ O, room temperature, 2h (v) HBTU, DIPEA, room temperature, N- (2-aminoethyl) -maleimidetrifluoromethylacetate (vi) HCl, Et 2 O, 2h

(3) Radiosynthesis of Radioactively Labeled Compound 7

The aldehyde diamine coupling reaction was carried out using a radioiodine compound [ ¹²⁵ I] 5 and a diamine group containing compound 4. An aryldiamine ring to which an electron withdrawing group (-OCH _2- ) was added was selected to promote the coupling reaction. Non-radioactive analogue 7 was synthesized for HPLC identification and characterization of [ ¹²⁵ I] 7 prior to ^{radiosynthesis of the} desired compound [ ¹²⁵ I] 7. Compound 7 was synthesized by stirring Compounds 4 and 5 at room temperature and in the presence of a catalytic amount of copper sulfate. Radiolabeled reactions were carried out at various concentrations of 5- (3,4-diamino phenoxy) -N- (2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl) Pentanamide 4 (5, 25 and 50 μM) and 100 μCi [ ¹²⁵ I] 5 were performed by stirring at room temperature. The reaction was monitored at different time points using radio-HPLC and showed the radiochemical yields summarized in Table 1. More than 99% and 91% of [ ¹²⁵ I] 7 were obtained within 30 minutes at room temperature using 4 of 50 μM and 25 μM, respectively (entries 2 and 3). In addition, at least 80% of [ ¹²⁵ I] 5 was converted to [ ¹²⁵ I] 7 within 30 minutes using 5 μM substrate 4 (entry 4). The reaction at 5 minutes was slow when using 50 μM of precursor 4 (entry 1), but high radiochemical yield was observed 30 minutes after incubation. The radiochemical yield and reaction kinetics have been found to be comparable to many of the bioconjugate reactions given in the literature.

Scheme 3: radiosynthesis of linker [ ¹²⁵ I] 5 compound and radiolabeled compound 7

Reagents and conditions: (i) [ ¹²⁵ I] NaI, chloramine-T, DMSO, room temperature, 10 minutes (ii) Cu ⁺² , pH 7.5, room temperature, air

Radiolabeling results for [ ¹²⁵ I] 7 and [ ¹²⁵ I] 8 Entry ^a Concentration ^b (μmol) temperament Time (min) product % RCY ^c One 50 4 5 [ ¹²⁵ I] 7 26 2 50 4 30 [ ¹²⁵ I] 7 > 99 3 25 4 30 [ ¹²⁵ I] 7 91 4 5 4 30 [ ¹²⁵ I] 7 80 5 50 8 30 [ ¹²⁵ I] 8 > 99 6 25 8 30 [ ¹²⁵ I] 8 93 7 5 8 30 [ ¹²⁵ I] 8 69

^a solvent: DMSO / PBS, Cu ⁺² (0.1 eq), 25 ° C. ^b Final concentration ^{c in the} reaction mixture Radiochemical yield determined by radio-HPLC

2. ¹²⁵ I marker cRGD Preparation of Peptides [[ ¹²⁵ I] 8 )

Scheme 4 below uses the radiolabeled compound [ ¹²⁵ I] 5 of the present invention synthesized in 1 to process radiolabeled cyclic RGD peptides with radioactive iodine as cancer targeting peptides. It is shown . Radioactively labeled cRGD peptides have high binding affinity and selectivity for the α _v β ₃ receptor and are widely used to detect metastatic disease and rapidly growing cancer in preclinical studies. Many radiolabeled cRGD peptides have been developed for SPECT or PET based studies of tumor models. We also selected cRGD as a model peptide to test the effectiveness of the radioactive element labeling strategy. The radiosynthesis of the target compound [ ¹²⁵ I] 8 is shown in Scheme 4 below. Radiolabeling reactions were carried out in the presence of a catalyst with different concentrations of aryl diamine with cRGD 8 (5, 25 and 50 μM) at 100 μCi [ ¹²⁵ I] 5 at room temperature. Radioiodination reaction was monitored using radio-HPLC and the observed radiochemical yields are summarized in Table 1 above. More than 99% of [ ¹²⁵ I] 8 was obtained using 50 μM 8 within 30 minutes and at room temperature (entry 5). In addition, 93% and 69% of [ ¹²⁵ I] 8 were obtained at 6 of 25 μM and 5 μM, respectively. There were no side reactions in the radiolabeling reaction and only one radioiodinated product was observed in the radioactive HPLC chromatogram.

Scheme 4. Radiosynthesis of Compound [ ¹²⁵ I] 8 ^a

^a Reagents and conditions: (i) [ ¹²⁵ I] 5, Cu ^{+ 2} , room temperature, air

3. Radioactive Element Mark HSA  Manufacture

(1) Preparation of ¹²⁵ I labeled HSA ([ ¹²⁵ I] 9)

To determine the radioisotope labeling efficiency of the aryldiamine alkylaldehyde coupling reaction, [ ¹²⁵ I] 5 was treated with aryl diamine containing human serum albumin protein 9. Human serum albumin protein can not only be used as a drug carrier but also improves blood circulation time by increasing the time the drug stays in the blood. In this experiment maleimide-cysteine-34 conjugation was applied.

Scheme 5: Radioactive iodide of aryl diamine functionalized human serum albumin [ ¹²⁵ I] 9 ^a

^a Reagents and conditions: (i) PBS, room temperature, pH 7.5, 10 h (ii) [ ¹²⁵ I] 5, Cu ⁺² , air, room temperature

For chemical substitution of the aryldiamine group in HSA, 4 molar excess of maleimide containing aryldiamine 4 was incubated with HSA in phosphate buffered saline at pH 7.5 for 10 hours at 25 ° C. At the end of the reaction, the mixture was passed through a PD-10 (except size) column to obtain a chemically substituted aryldiamine HSA. MALDI-Tof analysis confirmed the purity and properties of the desired product. The results showed that at room temperature 1.3 aryl diamine groups were conjugated with one HSA protein. To determine the efficiency of the alkylaldehydearyldiamine condensation reaction, various concentrations of aryl diamine were placed in HSA and incubated with ¹²⁵ μL of ¹²⁵ I-labeled alkyl aldehyde [ ¹²⁵ I] 5 in the presence of a catalytic amount of CuSO ₄ . Radiochemical yields were determined using radio-TLC and summarized in Table 2 below. The conversion yield is concentration dependent and a radiochemical yield of at least 94% was obtained for 50 μM of aryldiamine modified HSA 9 within 2 hours. Under the same reaction conditions, 89% and 67% radiochemical yields were obtained for 25% and 5 μM of aryldiamine modified HSA, respectively (entries 4 and 5). In control experiments [ ¹²⁵ I] 5 was incubated with unmodified pure HSA under the same conditions but no nonspecific interaction was observed. Preparing ^[125 I] 9 for the biodistribution study with an aldehyde ^[125 I] 5 1.0mCi incubated with 50μM of the aryl diamine The modified HSA 9 - Next, 2 hours ¹²⁵ I- labeled alkyl in the presence of a catalyst did. The crude mixture was purified using PD-10 desalting column to obtain [ ¹²⁵ I] 9 with 90% isolated radiochemical yield and at least 99% radiochemical purity. To compare in vivo behavior, HSA was radioiodinated through the tyrosine ring using [ ¹²⁵ I] NaI and chloramine-T as oxidants. ¹²⁵ I-HSA ([ ¹²⁵ I] 10) was synthesized with 83% radiochemical yield and at least 99% radiochemical purity after purification through PD-10 column.

[ ¹²⁵ I] In Vitro Radiolabeling Results of Aryldiamine Conjugated HSAs When 5 is Applied Entry ^a Concentration of 9 ^b (μmol) Time (h) % RCY ^c One 50 0.5 54 2 50 One 63 3 50 2 94 4 25 2 89 5 5 2 67

^a solvent: PBS, Cu ⁺² (0.1 eq), 25 ° C. ^b Final concentration ^{c in the} reaction mixture Radiochemical yield determined by radio-HPLC

(2) Preparation of ^99m Tc Labeled HSA

A ^99m Tc labeled HSA was prepared as in Scheme 6 below by a process similar to (1) above.

^99m Tc-labeled alkylaldehyde in the presence of catalytic amount of CuSO ₄ was purified after reaction with HSA 9 at room temperature for 2 hours and obtained with radiochemical purity of 99% or more.

스킴 6

Scheme 6

(3) Preparation of ⁶⁸ Ga, ¹¹¹ In and ⁸⁹ Zr Labeled HSAs

⁶⁸ Ga, ¹¹¹ In and ⁸⁹ Zr labeled HSA were prepared by the same procedure as in (1) above as in Scheme 7 below.

⁶⁸ Ga-, ¹¹¹ In- and ⁸⁹ Zr-labeled alkylaldehydes were purified after reaction with HSA 9 at room temperature for 2 hours in the presence of catalytic amounts of CuSO ₄ and obtained with radiochemical purity of 99% or higher.

스킴 7

Scheme 7

4. ¹²⁵ I cover Of HSA  Distribution in vivo in animals ( Biodistribution ) evaluation

Evaluation of the in vivo organ distribution and body behavior of the ¹²⁵ I labeled HSA ([125I] 9) of the present invention synthesized in (1) was performed using ICR male mice. Each mouse was injected intravenously with 1 μCi of radiolabeled product and body distribution data was collected at 0.5, 3, 6, 24 and 36 hours post injection. Initial radioactivity concentrations (24.07 ± 1.28% ID / g) were found in the blood pool. Activity levels were also significantly higher at 24 hours (8.32 ± 0.72% ID / g) and 36 hours (6.73 ± 0.48% ID / g) (Figure 3) after injection. This observation suggests that [ ¹²⁵ I] 9 has a long blood circulation time and can be used to increase the blood half-life of fast elimination drugs after binding with [ ¹²⁵ I] 9. Referring to FIG. 3, the uptake of other organs such as the spleen, liver, small intestine, large intestine, lung, heart, and gastrointestinal tract was significantly higher at an early time but decreased over time. Slightly high renal uptake suggests removal of the labeled compound through urine. Radioactivity accumulation in the thyroid gland usually indicates in vivo deiodation, at 5.20 ± 1.61% ID / g and 4.86 ± 1.01% ID / g at 24 and 36 hours after IV injection.

On the other hand, biodistribution studies and data of direct radioiodinated HSA ([ ¹²⁵ I] 10) are summarized in Table 3 below, as shown in Scheme 8, which shows the radioiodination reaction of the tyrosine ring of HSA.

Scheme 8

The radiation accumulation of [ ¹²⁵ I] 10 in the thyroid gland was normal at the first point but increased over time. Thyroid uptake was found to be 255.1 ± 66.21% ID / g and 395.2 ± 59.98% ID / g at 24 hours and 36 hours after IV injection, respectively. These results suggest a high instability of radioactive iodine to tyrosine rings in vivo. In addition, the structure of [ ¹²⁵ I] 5 has been demonstrated to be completely different from iodine tyrosine, which can easily undergo deiodation in vivo.

[ ¹²⁵ I] Biodistribution of 10 Agency blood liver spleen top Intestine Leader kidneys Heart lungs thyroid 0.5h 30.1 ± 1.28 5.1 ± 1.68 3.6 ± 2.32 1.6 ± 1.64 1.6 ± 0.86 0.88 ± 0.12 6.1 ± 0.28 5.9 ± 0.98 8.1 ± 2.31 2.1 ± 2.11 3h 25.3 ± 2.21 3.0 ± 2.03 3.2 ± 1.06 1.8 ± 0.98 2.2 ± 1.56 0.95 ± 0.17 5.3 ± 0.78 4.4 ± 1.32 7.7 ± 1.84 15.3 ± 5.61 6h 14.5 ± 6.01 2.8 ± 0.99 2.8 ± 1.55 5.8 ± 2.57 1.9 ± 1.22 1.1 ± 0.36 5.5 ± 0.31 3.5 ± 1.50 7.1 ± 0.98 75.3 ± 12.63 24h 8.3 ± 2.25 1.9 ± 1.68 1.6 ± 0.68 12.1 ± 2.21 1.7 ± 1.54 1.2 ± 0.54 4.2 ± 0.54 2.8 ± 0.87 5.2 ± 1.66 255.1 ± 66.21 36h 6.1 ± 3.25 1.8 ± 0.56 1.5 ± 1.02 14.2 ± 2.97 1.5 ± 0.91 1.0 ± 0.22 3.8 ± 0.66 2.1 ± 0.69 4.9 ± 1.59 395.2 ± 59.98

N = 5 mice per group, data shows% ID / g tissue The alkylaldehyde aryldiamine condensation reaction of the present invention has excellent radiochemical yield, and the resultant in vivo stability is obtained using harsh oxidizing agents such as chloramine-T. It is much better compared to the direct radioiodination for tyrosine. In the latter case, the protein is damaged in the radioactive element labeling process and the bioactivity of the radiolabeled protein can be reduced, while in the present invention, radioactivity of various small and large bioactive molecules based on the condensation reaction of aryldiamine with alkylaldehyde It can be used for iodide.

In addition, the in vitro and in vivo stability of the aldehyde compounds labeled with the radioactive elements of the present invention is quite high and does not react to all functional groups of biologically active proteins.

Based on these results, it can be determined that the radiolabeling method of the present invention and the compound used therein have excellent characteristics that can be used for in vivo targeting imaging.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations can be made without departing from the technical spirit of the present invention described in the claims. It will be obvious to those of ordinary skill in the field.

Claims (18)

Providing a diaminophenyl compound represented by Formula (I) below, to which a biomolecule, fluorescent dye, or nanoparticle compound is bound; And
A method for labeling radioactive elements, comprising reacting the diaminophenyl compound with an aldehyde compound labeled with a radioactive element at room temperature.

[Formula I]
(In Formula I,
A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; Y is CH ₂ or

Is; Z is a biomolecule, fluorescent dye or nanoparticle compound.)

[Formula II]
(In Formula II, b is 0 or an integer from 1 to 10; L is CH ₂ or -CONH-;
Q is

,

,

or

Is,
In Q, M, M 'and M "are radioactive elements.)
The method of claim 1, wherein providing the diaminophenyl compound comprises: providing a compound of formula (Ia) comprising a thiol moiety; And
A method of labeling a radioactive element, comprising reacting a compound of formula (Ia) with a compound of formula (Ib) to obtain a compound of formula (I).

Formula Ia
Z'-SH

(In the above formula (Ia), Z 'is the same as the structure except Z -SH portion when the biomolecule, fluorescent dye or nanoparticle compound represented by Z of the formula (I) comprises a thiol portion; If the molecule, fluorescent dye or nanoparticle compound does not contain a thiol moiety, Z 'is the same as Z.)

Formula Ib
(In the formula (Ib), wherein A, a and X are as defined in formula (I).)
The method of labeling a radioactive element according to claim 2, wherein the reaction between the compound of formula (Ia) and the compound of formula (Ib) is carried out at room temperature and atmospheric conditions under a Cu ⁺² catalyst.
The method of claim 2, wherein the providing of the compound of formula (Ia) comprising the thiol moiety is performed on a biomolecule, a fluorescent dye, or a nanoparticle compound Z ′ which does not include a thiol moiety. Substituting the thiol portion further to provide the compound of formula (Ia), the labeling method of the radioactive element.
The method of claim 1, wherein the biomolecule is at least one selected from the group consisting of peptides, affibodies, antibodies, and oligonucleotides.
The method of claim 1, wherein the nanoparticles are at least one selected from the group consisting of metal nanoparticles, synthetic polymer nanoparticles, and biopolymer nanoparticles.
The metal nanoparticle of claim 1, wherein the metal nanoparticle is any one selected from the group consisting of gold (Au), platinum (Pt), palladium (Pd), silver (Ag), and copper (Cu); Or any one selected from the group consisting of cobalt (Co), manganese (Mn), iron (Fe), nickel (Ni), gadolinium (Gd), molybdenum (Mo), zinc (Zn), and chromium (Cr). The labeling method of a radioactive element which is a metal oxide nanoparticle of.
The method of claim 1, wherein the fluorescent dye is at least one selected from cyanine, fluorescein, and rhodamine.
The method of claim 1, wherein the radioactive element is selected from the group consisting of I, F, Tc, Re, Ga, In, Zr, Y, Ho, Sm, and Lu.
The method of labeling a radioactive element according to claim 9, wherein the radioactive element M is I or F, M 'is Tc or Re, and M "is an element selected from the group consisting of Ga, In, Y and Zr.
A radiolabeled compound comprising an aldehyde moiety, wherein the radiolabeled compound represented by the following general formula (II) is used to label a molecule having an amine moiety.

[Formula II]
(In Formula II, b is 0 or an integer from 1 to 10; L is CH ₂ or -CONH-;
Q is

,

,

or

Is,
In Q, M, M 'and M "are radioactive elements.)
A diaminophenyl compound represented by the following formula (I), to which a biomolecule, fluorescent dye, or nanoparticle compound is bound; And
A radioactive element labeling kit, represented by the following formula (II), comprising an aldehyde compound labeled with a radioactive element.

[Formula I]
(In Formula I,
A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; Y is CH ₂ or

Is; Z is a biomolecule, fluorescent dye or nanoparticle compound.)

[Formula II]
(In Formula II, b is 0 or an integer from 1 to 10; L is CH ₂ or -CONH-;
Q is

,

,

or

Is,
In Q, M, M 'and M "are radioactive elements.)
The method of claim 12, wherein the biomolecule is at least one selected from the group consisting of peptides, affibodies, antibodies, and oligonucleotides; The nanoparticles are at least one selected from the group consisting of metal nanoparticles, synthetic polymer nanoparticles, and biopolymer nanoparticles; The metal nanoparticle may be any one selected from the group consisting of gold (Au), platinum (Pt), palladium (Pd), silver (Ag), and copper (Cu); Or cobalt (Co), manganese (Mn), iron (Fe), nickel (Ni), gadolinium (Gd), molybdenum (Mo), zinc (Zn) and chromium (Cr) Is a metal oxide of; And the fluorescent dye is at least one selected from cyanine, fluorescein, and rhodamine.
The radioactive element labeling kit of claim 12, wherein the radioactive element is selected from the group consisting of I, F, Tc, Re, Ga, In, Zr, Y, Ho, Sm, and Lu.
A radiolabeled compound for labeling molecules having a thiol moiety, represented by the following formula (III), for labeling molecules having a thiol moiety.

[Formula III]
(In Formula III,
A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; b is 0 or an integer from 1 to 10; L is CH ₂ or -CONH-;
Q is

,

,

or

Is,
In Q, M, M 'and M "are radioactive elements.)
A biomolecule, fluorescent dye or nanoparticle compound labeled with a radioactive element represented by Formula IV:

[Formula IV]
(In Formula IV,
A is CH ₂ or O; a is 0 or an integer from 1 to 10; X is CH ₂ or -CONH-; Z 'is a structure excluding a thiol group in a biomolecule, fluorescent dye or nanoparticle compound containing or substituted with a thiol group; b is 0 or an integer from 1 to 10; L is CH ₂ or -CONH-;
Q is

,

,

or

Is,
In Q, M, M 'and M "are radioactive elements.)
17. A medical diagnostic composition comprising a biomolecule, fluorescent dye or nanoparticle compound labeled with a radioactive element represented by Formula IV of claim 16 as an active ingredient.
18. The method of claim 17, wherein the medical diagnosis is single photon emission tomography (SPECT), positron emission tomography (PET), micro-PET, computed tomography (CT), magnetic resonance imaging (MRI), or a target of a radiograph. Medical diagnostic composition, which is diagnosed by imaging.

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