KR20200097846A - New 18F-labeled dienophiles and 18F-labeling method using IeDDA reaction with tetrazines - Google Patents

Abstract

The present invention relates to a method of labeling of F-18 radioisotopes using an inverse electron demand Diels-Alder (IeDDA) reaction between a tetrazine compound and a dienophile. A ketone compound according to the present invention easily forms an enamine with a secondary amine in an aqueous solution phase to cause significantly rapid conjugation with a tetrazine compound. Particularly, as the amount of water is increased in the aqueous solution, the reaction proceeds well, so that the compound may be suitable for a bioactive compound, such as a peptide or antibody. Unlike conventional reactions producing different stereoisomers, the reaction produces a small amount of only one type of stereoisomer, besides a main product, and the product can be purified through high performance liquid chromatography, etc. Further, as compared to the conventional ^18F-labeled tetrazine or ^18F-labeled TCO synthesis with a low yield, the ^18F-labeled ketone compound represented by the following chemical formula 1 can be obtained with a high ^18F-labeling yield from a precursor thereof.

Description

New 18F-labeled dienophiles and 18F-labeling method using IeDDA reaction with tetrazines}

The present invention relates to a technique for labeling F-18 radioisotopes using an IeDDA (Inverse electron Demand Diels-Alder) reaction between a tetrazine compound and a diene body.

A lot of research is being conducted to develop compounds having new functions by chemically modifying biological compounds such as proteins, nucleotides, and sugars that make up a living body. In the case of peptide-based biochemicals, a general amide bond formation reaction occurs without selectivity at the lysine and amine ends, resulting in a variety of mixtures. In order to use biological compounds for various purposes, a selective chemical bonding method is essentially required. To this end, many researchers are putting a lot of effort into developing a bioorthogonal method in which reactions proceed only in specific combinations by introducing functional groups that do not exist in nature.

1,2,4,5-tetrazine (hereinafter referred to as tetrazine) is characterized by a lack of electrons in the heteroaromatic ring, and this is an electron-rich dienophile and easily reversed Diels-Alder. It is known to cause a reaction. (Non-Patent Literature 1, Chem. Soc. Rev., 2013, 42, 5131-5142) This reaction is the opposite of the normal Diels-Alder reaction between an electron-rich diene and an electron-deficient electrophile. It is called IeDDA (Inverse electron demand Diels-Alder).

Up to now, the IeDDA reaction between tetrazine and diene bodies of various structures has been studied, and a study applying this to a protein called Thioredoxin was reported in 2008. In this study, trans-cyclooctene (TCO) with a very large ring-strain was used as a diene body to react with tetrazine, and the secondary reaction rate constant (k ₂ ) was 2000 M ^-1 s. ^{It turned out} to be very fast with ^-1 . However, the TCO compound is very difficult to synthesize and has a disadvantage that it is not easy to handle due to an unstable structure having a large ring-strain. In addition, since it has a very lipophilic structure, it is not well soluble in an aqueous solution, and there is a disadvantage that the product obtained from the reaction with tetrazine is a mixture of at least four or more. In order to compensate for the shortcomings of the TCO compound, various diene bodies have been developed, but the stability of the compounds has not been greatly improved, the reaction rate is relatively low, and there is still a problem that several mixtures are generated.

Positron Emission Tomography (PET) is a technology for imaging the human body using disease-specific compounds labeled with positron emission isotopes, and has the feature of early diagnosis of disease. F-18 has a half-life of 110 minutes, and even if injected into the human body, radiation exposure is not large, and it can be produced at high doses through a cyclotron (particle accelerator), and it has high resolution due to its low energy, and can be used for several hours after manufacture. It is an applied positron emitting isotope.

Since biological compounds such as peptides and antibodies have higher disease selectivity than low-molecular organic synthetic compounds and have excellent pharmacokinetic properties in vivo due to their hydrophilic properties, efforts to develop them as diagnostic drugs for PET imaging by labeling F-18 have continued. have. However, since the F-18 labeling reaction, which is a nucleophilic substitution reaction, must be reacted at a high temperature under anhydrous conditions and an excessive amount of base must be used, it cannot be used directly for biological compounds such as peptides or antibodies. Therefore, a method of labeling ¹⁸ F is used by making a prosthetic group compound labeled with F-18 through a general F-18 labeling reaction, and reacting a biological compound with a functional group that can easily react with it under mild conditions. do.

The inventors of the present invention found that a compound having a ketone group easily forms an enamine in an aqueous solution with a secondary amine, and this causes an IeDDA reaction with a tetrazine compound within a short time, and then F-18 is labeled on the biological compound. It was confirmed that it can be used as a method of making and completed the present invention.

An object in one aspect of the present invention is to provide an ¹⁸ F-labeled dienophile.

Another object of the present invention is to provide an ¹⁸ F-labeling method using IeDDA (Inverse electron Demand Diels-Alder) reaction between the ¹⁸ F-labeled diene body and a tetrazine compound.

Another object of the present invention is to provide a method for preparing the ¹⁸ F-labeled diene body.

Another object of the present invention is to provide a compound prepared through the IeDDA reaction of the ¹⁸ F-labeled dienophile and a tetrazine compound.

Another object of the present invention is to provide a composition for diagnosing diseases comprising a compound prepared through IeDDA reaction between the ¹⁸ F-labeled dienophile and a tetrazine compound.

Another object of the present invention is to provide an intermediate compound formed upon IeDDA reaction between the ¹⁸ F-labeled dienophile and a tetrazine compound.

Another object of the present invention is to provide a compound prepared by reacting an RGD-tetrazine compound with the ¹⁸ F-labeled diene body.

To achieve the above object,

One aspect of the present invention provides a compound represented by the following formula (1).

[Formula 1]

(In Formula 1,

A is a bond, C _1-6 straight or branched chain alkylene, or -C(=O)-;

D is a bond, -O-, -NH-, or -C(=O)-;

E is a bond, -O-, or -NH-;

G is a bond or C _1-6 straight or branched chain alkylene, wherein the carbon of the alkylene may be substituted with oxygen;

J is a bond, a C _6-10 arylene, or a 6 to 10 ring heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S;

K is a bond, or C _1-6 straight or branched chain alkylene, wherein the carbon of the alkylene may be substituted with oxygen;

F is fluorine-18 or fluorine-19;

m and n are independently integers of 1 or 2; And

Hydrogen of -CH ₂ -in parentheses may be substituted with methyl).

Another aspect of the present invention is as shown in Scheme 1 below,

Reacting the compound represented by Formula 1 with a tetrazine compound represented by Formula 2 below; It provides a method for preparing a compound represented by the following Chemical Formulas 3 and 3', including.

[Scheme 1]

(In the above Scheme 1,

A, D, E, G, J, K, F, m, n and -CH ₂ -in parentheses are as defined in Formula 1 above;

In Formula 2, R ₁ and R ₂ are independently C _1-12 straight or branched chain alkyl, unsubstituted or substituted 5 to 10 each containing at least one heteroatom selected from the group consisting of N, O and S Ring heteroaryl, unsubstituted or substituted C _6-10 aryl, or unsubstituted or substituted C _6-10 aryl C _1-12 straight or branched chain alkyl,

The carbon of the straight chain or branched chain alkyl of C _1-12 may be substituted with oxygen, hydrogen may be substituted with halogen,

The substituted 5 to 10 ring heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl is each of 5 to 10 rings substituted with one or more additional substituents. Heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl).

Another aspect of the present invention provides a compound represented by the following formula (5).

[Formula 5]

(In Formula 5.

A, D, E, G, J, K, m, n and -CH ₂ -in parentheses are as defined in Formula 1 above;

W is -C(=O)-, or -C(OR ₅ )(OR ₆ )-,

The R ₅ and R ₆ are connected to each other to form a C _3-10 alkylene, but carbon in the alkylene may be substituted with oxygen or -NH-, or

The R ₅ and R ₆ are independently hydrogen, C _1-12 straight or branched chain alkyl, N, O and S unsubstituted or substituted 5 to 10 each containing at least one heteroatom selected from the group consisting of Ring heteroaryl, unsubstituted or substituted C _6-10 aryl, or unsubstituted or substituted C _6-10 aryl C _1-12 straight or branched chain alkyl,

The carbon of the straight chain or branched chain alkyl of C _1-12 may be substituted with oxygen, hydrogen may be substituted with halogen,

The substituted 5 to 10 ring heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl is each of 5 to 10 rings substituted with one or more additional substituents. Heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl; And

X is -OS(O ₂ )R ₇ , and R ₇ is methyl, trifluoromethyl, para-toluene, or para-nitrobenzene).

Another aspect of the present invention is as shown in Scheme 2 below,

Preparing a compound represented by Formula 1 from the compound represented by Formula 5; It provides a method of preparing the compound represented by Formula 1 including.

[Scheme 2]

(In Scheme 2,

The compound represented by Formula 1 is as defined above;

The compound represented by Formula 5 is as defined above; And

M is sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), N,N,N,N-tetraalkylammonium salt, or Cryptofix[2.2.2]-potassium salt (kryptofix[2.2) .2])-K)).

Another aspect of the present invention provides a compound represented by the following formula 3A or 3B.

[Chemical Formula 3A]

[Chemical Formula 3B]

(In Chemical Formula 3A or 3B,

A, D, E, G, J, K, F, m, n and -CH ₂ -, R ₁ and R ₂ in parentheses are as defined in Scheme 1).

Another aspect of the present invention provides a composition for diagnosing diseases comprising at least one compound selected from the group consisting of Formulas 3A and 3B.

Another aspect of the present invention provides an intermediate compound represented by the following formula A or A'.

[Formula A]

;

;

[Formula A']

;

;

(In Formula A or A',

A, D, E, G, J, K, F, m and n are as defined in Formula 1 above;

R ₃ and R ₄ are as defined in Chemical Formula 4; And

The hydrogen of -CH- in parentheses of m and -CH ₂ -in parentheses of n may be independently substituted with methyl).

Another aspect of the present invention provides a compound represented by the following formula (B).

[Formula B]

(In Formula B,

A, D, E, G, J, K and F are the same as defined in Formula 1).

The ketone compound of the present invention easily forms an enamine in an aqueous solution with a secondary amine and causes a conjugation reaction with a tetrazine compound very quickly. In particular, as the amount of water in the aqueous solution increases, the reaction proceeds well, so it is suitable for biological compounds such as peptides and antibodies. In addition, unlike conventional reactions that form several stereoisomers, only a small amount of structural isomers are generated in addition to the main product, and there is an advantage that it can be purified using high-performance liquid chromatography. Furthermore, compared to the conventional low yield of ¹⁸ F-labeled tetrazine or ¹⁸ F-labeled TCO synthesis, the ¹⁸ F-labeled ketone compound of Formula 1 of the present invention can be synthesized with a high ¹⁸ F-labeled yield from its precursor. .

FIG. 1 is a graph showing a graph of changes in UV absorbance intensity at 531 nm over time obtained in divisions 14 to 17 of Table 1 of Example 1. FIG.
2 is a graph showing a graph of changes in UV absorbance intensity at 531 nm over time obtained in Example 4. FIG.
3 is a view showing the results of high performance liquid chromatography (HPLC) of the F-18-labeled compound [ ¹⁸ F] 1a synthesized in Experimental Example 3.
4 is a diagram showing the results of high performance liquid chromatography (HPLC) after the conjugation reaction carried out in Experimental Example 4.
5 is a diagram showing the results of high performance liquid chromatography (HPLC) after the conjugation reaction performed in Experimental Example 5.
6 is a diagram showing a MicroPET image of a tumor model mouse performed in Experimental Example 6.
The graph on the left of FIG. 7 shows the change in quantitative intake (SUV) for each tissue obtained from the MicroPET image of Experimental Example 6, and the graph on the right shows the intake ratio between tumors and muscles in Experimental Example 6.

Hereinafter, the present invention will be described in detail.

On the other hand, 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. In addition, embodiments of the present invention are provided in order to more completely explain the present invention to those having average knowledge in the art. Furthermore, "including" a certain component throughout the specification means that other components may be further included, rather than excluding other components unless specifically stated to the contrary.

One aspect of the present invention provides a compound represented by the following formula (1).

[Formula 1]

(In Formula 1,

A is a bond, C _1-6 straight or branched chain alkylene, or -C(=O)-;

D is a bond, -O-, -NH-, or -C(=O)-;

E is a bond, -O-, or -NH-;

G is a bond or C _1-6 straight or branched chain alkylene, wherein the carbon of the alkylene may be substituted with oxygen;

J is a bond, a C _6-10 arylene, or a 6 to 10 ring heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S;

K is a bond, or C _1-6 straight or branched chain alkylene, wherein the carbon of the alkylene may be substituted with oxygen;

F is fluorine-18 or fluorine-19;

m and n are independently integers of 1 or 2; And

Hydrogen of -CH ₂ -in parentheses may be substituted with methyl).

The compound represented by Formula 1 is a ketone compound, and the ring size may vary depending on m and n. However, when the ring of the compound represented by Formula 1 is pentagonal, it is preferable in terms of reaction speed compared to the case of a square or hexagonal ring.

In addition, another aspect of the present invention, as shown in the following Scheme 1,

Reacting a compound represented by the following Formula 1 with a tetrazine compound represented by the following Formula 2; It provides a method for preparing a compound represented by the following Chemical Formulas 3 and 3', including.

[Scheme 1]

(In the above Scheme 1,

A, D, E, G, J, K, F, m, n and -CH ₂ -in parentheses are as defined in Formula 1 above;

In Formula 2, R ₁ and R ₂ are independently C _1-12 straight or branched chain alkyl, unsubstituted or substituted 5 to 10 each containing at least one heteroatom selected from the group consisting of N, O and S Ring heteroaryl, unsubstituted or substituted C _6-10 aryl, or unsubstituted or substituted C _6-10 aryl C _1-12 straight or branched chain alkyl,

The carbon of the straight chain or branched chain alkyl of C _1-12 may be substituted with oxygen, hydrogen may be substituted with halogen,

The substituted 5 to 10 ring heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl is each of 5 to 10 rings substituted with one or more additional substituents. Heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl).

In this case, the additional substituents are C _1-12 straight or branched chain alkyl, amine C _1-12 straight or branched chain alkyl, C _1-12 straight or branched chain alkoxy, nitro, nitrile, halogen, hydroxy , N, O and S 5 to 10 ring heteroaryl containing at least one heteroatom selected from the group consisting of, C _6-10 aryl, or C _6-10 aryl C _1-12 straight chain or branched Chain alkyl, etc.,

One or more hydrogens of the amine (H ₂ N-) may be protected with a protecting group (eg, -Boc), and one or more hydrogens present in the alkyl may be substituted with halogen.

The additional substituent may be further substituted with a second additional substituent, and the second additional substituent may be the same as or different from the definition of the additional substituent described above.

The R ₁ and R ₂ may be bound to a disease-targeting compound, and the disease-targeting compound may be a protein including a peptide or antibody that binds to a disease-specific protein, but is not limited thereto.

The compound represented by Formula 3 and/or 3'prepared above may be used as an image probe for positron emission tomography.

In addition, the reaction of the compound represented by Formula 1 and the tetrazine compound represented by Formula 2 is preferably carried out in the presence of a secondary amine represented by Formula 4.

[Formula 4]

(In Chemical Formula 4,

R ₃ and R ₄ are linked to each other to form a C _3-10 alkylene, but carbon in the alkylene may be substituted with oxygen or -NH-; or

R ₃ and R ₄ are independently C _1-12 straight or branched chain alkyl, unsubstituted or substituted 5 to 10 ring heteroaryl containing at least one heteroatom selected from the group consisting of N, O and S , Unsubstituted or substituted C _6-10 aryl, or unsubstituted or substituted C _6-10 aryl C _1-12 straight or branched chain alkyl,

The carbon of the straight chain or branched chain alkyl of C _1-12 may be substituted with oxygen, hydrogen may be substituted with halogen,

The substituted 5 to 10 ring heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl is each of 5 to 10 rings substituted with one or more additional substituents. Heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl).

Specifically, the secondary amine represented by Formula 4 may be a secondary amine represented by Formula 4-1 below.

[Formula 4-1]

(In Formula 4-1,

R ₃ ^" and R ₄ ^" are independently C _2-4 alkylene, but carbon in the alkylene may be substituted with oxygen or -NH-; And

X is a bond, methylene, oxygen, or -NH-).

More specifically, the secondary amine may be used alone or in combination with pyrrolidine, dimethylamine, diethylamine, diisopropylamine, piperidine, morpholine, proline, etc., preferably pyrrolidine, dimethyl Amine, diethylamine, diisopropylamine, and piperidine can be used alone or in combination.

When the reaction of Scheme 1 is performed in the presence of a secondary amine, the reaction of Scheme 1 may proceed through the process of Scheme 1'below.

[Scheme 1']

In Scheme 1', A, D, E, G, J, K, F, m, n, in parentheses -CH ₂ -, R ₁ , R ₂ , R ₃ and R ₄ are independently the same as defined herein Do.

The reaction of Scheme 1 and/or Scheme 1'may be carried out in various types of solvents. However, preferably, one or more solvents selected from the group consisting of DMF, DMSO, EtOH, and water may be used alone or in combination.

If a mixture of DMF and water is used as a reaction solvent, the mixing volume ratio of DMF and water may be 1:1 to 5, 1:2 to 5, 1:3 to 5, and 1:1 to It may be 4, may be 1:1 to 3, may be 1:2 to 4.5, may be 1:2.5 to 3.5, and may be 1:3.

If a mixture of DMSO and water is used as a reaction solvent, the mixing volume ratio of DMSO and water may be 1:1 to 5, 1:2 to 5, 1:3 to 5, and 1:1 to It may be 4, may be 1:1 to 3, may be 1:2 to 4.5, may be 1:2.5 to 3.5, and may be 1:3.

If a mixture of EtOH and water is used as a reaction solvent, the mixing volume ratio of EtOH and water may be 1:1 to 5, 1:2 to 5, 1:3 to 5, and 1:1 to It may be 4, may be 1:1 to 3, may be 1:2 to 4.5, may be 1:2.5 to 3.5, and may be 1:3.

Further, another aspect of the present invention provides a compound represented by the following formula (5).

[Formula 5]

(In Formula 5.

A, D, E, G, J, K, m, n and -CH ₂ -in parentheses are as defined in Formula 1 above;

W is -C(=O)-, or -C(OR ₅ )(OR ₆ )-,

The R ₅ and R ₆ are connected to each other to form a C _3-10 alkylene, but carbon in the alkylene may be substituted with oxygen or -NH-, or

The R ₅ and R ₆ are independently hydrogen, C _1-12 straight or branched chain alkyl, N, O and S unsubstituted or substituted 5 to 10 each containing at least one heteroatom selected from the group consisting of Ring heteroaryl, unsubstituted or substituted C _6-10 aryl, or unsubstituted or substituted C _6-10 aryl C _1-12 straight or branched chain alkyl,

The carbon of the straight chain or branched chain alkyl of C _1-12 may be substituted with oxygen, hydrogen may be substituted with halogen,

The substituted 5 to 10 ring heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl is each of 5 to 10 rings substituted with one or more additional substituents. Heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl; And

X is -OS(O ₂ )R ₇ , and R ₇ is methyl, trifluoromethyl, para-toluene, or para-nitrobenzene).

In addition, another aspect of the present invention is as shown in Scheme 2 below,

Preparing a compound represented by Formula 1 from the compound represented by Formula 5; It provides a method of preparing the compound represented by Formula 1 including.

[Scheme 2]

(In Scheme 2,

The compound represented by Formula 1 is as defined above;

The compound represented by Formula 5 is as defined above; And

M is sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), N,N,N,N-tetraalkylammonium salt, or Cryptofix[2.2.2]-potassium salt (kryptofix[2.2) .2])-K)).

Further, another aspect of the present invention provides a compound represented by the following formula 3A or 3B.

[Chemical Formula 3A]

[Chemical Formula 3B]

(In Chemical Formula 3A or 3B,

A, D, E, G, J, K, F, m, n and -CH ₂ -, R ₁ and R ₂ in parentheses are independently as defined in Scheme 1 above).

In addition, another aspect of the present invention provides a composition for diagnosing diseases comprising at least one compound selected from the group consisting of Formulas 3A and 3B. The disease diagnosis composition may be commercialized and used as a disease diagnosis kit.

At this time, the disease may be benign and malignant tumors, cardiovascular disease related to atherosclerotic plaque, degenerative brain diseases such as dementia and Parginson's disease.

Further, another aspect of the present invention provides an intermediate compound represented by the following formula A or A'.

[Formula A]

;

;

[Formula A']

;

;

(In Formula A or A',

A, D, E, G, J, K, F, m and n are as defined in Formula 1 above;

R ₃ and R ₄ are as defined in Chemical Formula 4; And

The hydrogen of -CH- in parentheses of m and -CH ₂ -in parentheses of n may be independently substituted with methyl).

In addition, another aspect of the present invention provides a compound represented by the following formula (B).

[Formula B]

(In Formula B,

A, D, E, G, J, K, and F are independently as defined in Formula 1 above).

In one aspect of the present invention, the compound for final use in disease diagnosis may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Do. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. Get from Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, i. Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Rate, sebacate, fumarate, maleate, butine-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, Glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. and adding an organic or inorganic acid to filter the precipitate, It can be prepared by drying, or it can be prepared by distilling a solvent and an excess of acid under reduced pressure and then drying to crystallize under an organic solvent.

In addition, a pharmaceutically acceptable metal salt can be made using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate). Further, the compound and its pharmaceutically acceptable salts, as well as solvates, optical isomers, and hydrates that may be prepared therefrom may be included.

The compound for final use in diagnosing the disease or a composition for diagnosing a disease containing the same may be administered parenterally, and the parenteral administration is by a method of injecting a subcutaneous injection, an intravenous injection, an intramuscular injection, or an intrathoracic injection.

At this time, in order to formulate a formulation for parenteral administration, the compound or a pharmaceutically acceptable salt thereof is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which can be prepared in ampoules or vials unit dosage form. . The composition may be sterilized and/or contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts and/or buffers for controlling the osmotic pressure, and other therapeutically useful substances. It can be formulated according to the method of painting or coating.

The ketone compound of the present invention easily forms an enamine in an aqueous solution with a secondary amine and causes a conjugation reaction with a tetrazine compound very quickly. In particular, as the amount of water in the aqueous solution increases, the reaction proceeds well, so it is suitable for biological compounds such as peptides and antibodies. In addition, unlike conventional reactions that form several stereoisomers, only a small amount of structural isomers are generated in addition to the main product, and there is an advantage that it can be purified using high-performance liquid chromatography. Furthermore, compared to the conventional low yield of ¹⁸ F-labeled tetrazine or ¹⁸ F-labeled TCO synthesis, the ¹⁸ F-labeled ketone compound of Formula 1 of the present invention can be synthesized with a high ¹⁸ F-labeled yield from its precursor. . This is supported by Examples and Experimental Examples described later.

Hereinafter, the present invention will be described in detail through Preparation Examples, Examples and Experimental Examples to be described later. However, the preparation examples, examples, and experimental examples to be described later are merely illustrative of the present invention, and the present invention is not limited thereto.

<Production Example 1> Preparation of compound 2a

Step 1:

After dissolving 4-(aminomethyl)benzonitrile hydrogen chloride ( 6 , 2.0 g, 11.86 mmol) in dichloromethane (100 mL), di-tert-butyl dicarbonate ((Boc) ₂ O, 4.09 g, 17.79 mmol) Was added. Triethylamine (4.13 mL, 29.65mmol) was diluted in dichloromethane (20 mL) and then slowly added at 0 °C, followed by stirring at room temperature for 12 hours. Water was added to the reaction mixture, the organic compound was extracted using dichloromethane, and moisture was removed with anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography (50% ethyl acetate/n-hexane) was performed to obtain a white solid compound 7 (2.42g, 88%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.46 (s, 9H), 4.37 (d, J = 5.2 Hz, 2H), 4.99 (br s, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H).

¹³ C NMR (100 MHz, CDCl ₃ ) δ 27.4, 28.3, 44.2, 111.0, 118.8, 127.8, 132.4, 144.7, 155.9.

MS (ESI) m/z 257 [M+Na] ⁺

Step 2:

Compound 7 (1 g, 4.31 mmol) obtained in step 1 was put in a pressure tube, dissolved in acetonitrile (30 mL), and nickel (II) trifluoromethanesulfonate (0.77 g, 2.16 mmol) was added. And then stirred. Hydrazine hydrate (10.48 mL, 215.5 mmol) was added and reacted at 70° C. for 12 hours, the temperature was gradually lowered, NaNO ₂ (aq.) was added, and a 2 N HCl solution was added dropwise at 0° C. After the pH was adjusted to 3 using litmus paper, the organic compound was extracted with ethyl acetate, and moisture was removed with anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography (2% ethyl acetate/dichloromethane) was performed to obtain a purple solid compound 2a (0.32g, 28%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.48 (s, 9H), 3.10 (s, 3H), 4.44 (d, J = 6.0 Hz, 2H), 5.02 (dr s, 1H), 7.50 (d, J = 8.4 Hz, 2H), 8.55 (d, J = 8.4 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 21.2, 28.4, 44.4, 79.8, 128.0, 128.2, 130.7, 143.9, 156.0, 163.9, 167.2

MS (ESI) m/z 324 [M+Na] ⁺ .

<Preparation Example 2> Preparation of compounds 2b and 2c

Step 1:

Compound 2a (1.26 g, 4.2 mmol) obtained in Preparation Example 1 was dissolved in 4.0 M HCl solution (dioxane dissolved, 15 mL), and then stirred at room temperature for 1 hour. The reaction solution was filtered, washed with ethyl ether, and dried to obtain a solid compound 2b (0.99 g, 98%). ¹ H NMR (400 MHz, MeOD) δ 3.06 (s, 3H), 4.26 (s, 2H), 7.72 (d, J = 8.0 Hz, 2H), 8.64 (d, J = 8.0 Hz, 2H).

Step 2:

Compound 2b (0.45 g, 2.23 mmol) obtained in step 1 was dissolved in methanol (10 mL), succinic anhydride (0.268 g, 2.68 mmol) and triethylamine (0.62 mL, 4.46 mmol) were added, followed by stirring for 18 hours. I did. The solvent was removed under reduced pressure and column chromatography was performed to obtain compound 2c (0.34 g, 50%).

¹ H NMR (400 MHz, MeOD) δ 2.55-2.63 (m, 4H), 3.04 (s, 3H), 3.66 (s, 2H), 7.55 (d, J = 8.0 Hz, 2H), 8.51 (d, J = 8.0 Hz, 2H).

<Production Example 3> Preparation of compound 11

Step 1:

2,2,6,6-tetramethylpiperidin-4-one ( 8 , 20 g, 128.8 mmol) was dissolved in acetic acid (60 mL) and dissolved by stirring with a mechanical stirrer, and then cooled to 0 °C. Br ₂ (26.48 mL, 515.0 mmol) was slowly added to acetic acid (60 mL) to dissolve it, and the solution was transferred to a dropping funnel, and then it was slowly added dropwise at 0 °C to a flask in which Compound 8 was dissolved. After 10 minutes, the mixture was raised to room temperature and stirred for 3 hours, and then ethyl ether (400 mL) was slowly added to collect the precipitate and filtered to obtain a solid compound 9 (49.7 g, 82%), followed by drying in air. ¹ H NMR (400 MHz, MeOD) δ 1.47 (s, 3H), 1.63 (s, 3H), 1.69 (s, 3H), 1.89 (s, 3H), 5.40 (s, 2H);

MS (ESI) m/z 394.0 [M+H] ⁺ .

Step 2:

Compound 9 (10 g, 25.3 mmol) obtained in step 1 was added to a mixed solution of 25% aqueous ammonia (120 mL) and 1,4-dioxane (100 mL) and stirred at 40° C. for 1 hour and a half. The solvent was removed under reduced pressure, and the concentrate was subjected to column chromatography (8% methanol/dichloromethane) to obtain compound 10 (1.1 g, 26%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.30 (s, 6H), 1.45 (s, 6H), 5.20-5.80 (br, 2H), 6.17 (s, 1H);

MS (ESI) m/z 169.2 [M+H] ⁺ .

Step 3:

Sodium hydroxide (0.713 g, 17.8 mmol) was added to a two-necked round flask, dissolved in water (6 mL), cooled to 0 ℃, and then Br ₂ (0.188 mL, 3.65 mmol) was slowly added thereto and water ( The compound (0.5 g, 2.97 mmol) obtained in step 2 dissolved in 9 mL) was added dropwise. After raising the temperature to 100 °C, the mixture was stirred for 1 hour. After making again at 0 ℃, 1 M aqueous sodium hydroxide solution (20 mL) was added to terminate the reaction, and the organic compound was extracted with ethyl ether (20 mL X 2). After removing moisture with anhydrous sodium sulfate, it was concentrated under reduced pressure to obtain compound 11 (0.1 g, 24%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.24 (s, 6H), 1.29 (s, 6H), 2.37 (s, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 27.8, 30.0, 30.1, 30.3, 30.9, 50.8;

MS (ESI) m/z 142.2 [M+H] ⁺ .

<Production Example 4> Preparation of compound 15

Step 1:

Pyrrolidinol ( 12 , 2 g, 23.0 mmol) was dissolved in methanol (50 mL), 2-bromo-2-methylpropanoyl bromide ( 13 , 4.46 mL, 34.5 mmol) was added at room temperature, and then to 60 °C. Raised. Triethylamine (9.6 mL, 69.0 mmol) was diluted in methanol (20 mL) and then slowly added dropwise at 60° C. and stirred for 12 hours. The temperature was lowered to room temperature, concentrated under reduced pressure, and then subjected to column chromatography (5% methanol/dichloromethane) to obtain a yellow liquid compound 14 (1.28 g, 30%).

Step 2:

Nitrogen was filled in the reaction vessel, and a solution of DMSO (0.52 mL, 10.98 mmol) in dichloromethane (5 mL) was added. The temperature was lowered to 0 °C, and a solution in which oxalyl chloride (0.47 mL, 5.49 mmol) was dissolved in dichloromethane (10 mL) was slowly added to the reaction vessel. Compound 14 (0.69 g, 3.66 mmol) obtained in step 1 was dissolved in dichloromethane (10 mL) and slowly added to the reaction. Triethylamine (1.53 mL, 10.98 mmol) was diluted in dichloromethane (10 mL) and slowly added to the reaction vessel, followed by stirring at room temperature for 12 hours. After adding water and extracting the organic compound with dichloromethane, water was removed with anhydrous sodium sulfate, concentrated under reduced pressure, and then subjected to column chromatography (60% ethylacetate/n-hexane) to obtain a light yellow liquid compound 15 (0.54 g). , 80%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.40 (s, 6H), 2.44 (t, J = 7.2 Hz, 2H), 3.13 (t, J = 7.0 Hz, 2H), 3.25 (s, 2H), 3.72 (s, 3H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 23.9, 38.0, 44.8, 51.6, 55.8, 60.9, 174.4, 214.3;

MS (ESI) m/z 186 [M+H] ⁺ .

< Manufacturing example 5> Preparation of compound 1a

Step 1:

4-nitrophenyl chloroformate (3.50 g, 17.36 mmol) and 3-hydroxypropyl 4-toluenesulfonate ( 16 , 4.80 g, 20.83 mmol) were sequentially dissolved in tetrahydrofuran (250 mL). After lowering the temperature to 0 °C, pyridine (4.20 mL, 52.08 mmol) diluted in tetrahydrofuran (30 mL) was slowly added dropwise. After 1 hour, water is added, the organic compound is extracted using ethyl acetate, and moisture is removed with anhydrous sodium sulfate. Concentrated under reduced pressure and subjected to column chromatography (30% ethyl acetate/n-hexane) to give a colorless viscous liquid compound 17 (5.03 g, 73%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 2.08-2.14 (m, 2H), 2.43 (s, 3H), 4.19 (t, J = 6.0 Hz, 2H), 4.34 (d, J = 6.2 Hz, 2H) , 7.34-7.37 (m, 4H), 7.80 (d, J = 8.4 Hz, 2H), 8.27 (d, J = 9.6, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 21.7, 28.2, 65.0, 66.2, 121.8, 125.3, 127.9, 130.0, 132.7, 145.1, 145.4, 152.2, 155.4;

MS (ESI) 418 m/z [M+Na] ⁺ ;

HRMS (ESI) m/z calcd. For C ₁₇ H ₁₇ NO ₈ S+Na: 418.0567, found: 418.0675.

Step 2:

Compound 17 (4.85 g, 12.27 mmol) obtained in step 1 was dissolved in tetrahydrofuran (200 mL), and then pyrrolidin-3-one HCl (1.79 g, 14.72 mmol) was added to the reaction vessel. Sodium deuterium (3.10 g, 36.81 mmol) was dissolved in water (100 mL), and an aqueous solution was slowly added to the reaction, followed by stirring at 50° C. for 1 hour. The temperature was gradually lowered to room temperature, extracted with ethyl acetate, and water was removed with anhydrous sodium sulfate. After removing the solvent under reduced pressure, column chromatography (50% EA/Hx) was performed to obtain a beige solid compound 5a (3.36 g, 80%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.99-2.04 (m, 2H), 2.45 (s, 3H), 2.59 (t, J = 7.8 Hz, 2H), 3.63 (s, 1H), 3.76-3.80 ( m, 3H), 4.11-4.14 (m, 2H), 4.19 (t, J = 6.0 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 21.6, 28.5, 36.5, 37.0, 42.5, 52.2, 52.5, 61.3, 66.8, 127.9, 129.9,132.8, 145.0, 154.5, 210.0, 210.4;

MS (ESI) m/z 364 [M+Na] ⁺ .

Mass: HRMS (ESI) m/z calcd. For C ₁₅ H ₁₉ NO ₆ S+: 342.1011, found: 342.1006.

Step 3:

Compound 5a (0.5 g, 1.65 mmol) obtained in step 2 was dissolved in toluene (10 mL), and then toluenesulfonic acid (142 mg, 0.82 mmol) and trimethyl orthoformate (0.90 mL, 8.22 mmol) were added, respectively. After adding methanol (2 mL), the mixture was stirred at 80° C. for 12 hours, and then the solvent was removed under reduced pressure, water was added, and extraction was performed using ethyl acetate. Water was removed with anhydrous sodium sulfate and the solvent was removed under reduced pressure, followed by column chromatography (50% ethylacetate/n-hexane) to obtain a yellow liquid compound 5b (0.38 g, 68%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.92-1.96 (m, 2H), 1.98 (dt, J = 1.87, 6.4 Hz, 2H), 2.41 (s, 3H), 3.21 (s, 6H), 3.26- 3.30 (m, 2H), 3.38-3.42 (m, 2H), 4.04-4.10 (m, 4H), 7.31 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 21.6, 28.6, 32.0, 32.7, 43.9, 44.2, 49.7, 49.8, 51.6, 51.8, 60.7, 60.8, 67.0, 106.9, 107.6, 127.8, 129.8, 132.9, 144.8, 154.4 , 154.5.;

MS (ESI) m/z 356[M-OMe] ⁺ _;

Mass: HRMS (ESI) m/z calcd. For C ₁₇ H ₂₅ NO ₇ S: 387.1452, found: 387.1365.

Step 4:

Compound 5b (150 mg, 0.39 mmol) obtained in step 3 was dissolved in acetonitrile (5 mL), and tetrabutylammonium fluoride (300 mg, 1.16 mmol) was added. After stirring at 80° C. for 1 hour, the solvent was removed under reduced pressure, and then column chromatography (50% ethyl acetate/n-hexane) was performed to obtain a colorless liquid compound 18 (30 mg, 33%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.97-2.02 (m, 1H), 2.03-2.10 (m, 3H), 3.26 (s, 6H), 3.43-3.50 (m, 4H), 4.22 (t, J = 6.2 Hz, 2H), 4.48 (t, J = 6.0 Hz, 1H), 4.60 (t, J = 6.0 Hz, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 30.2 ( J = 19 Hz), 32.0, 32.8, 44.0, 44.2, 49.7, 49.8, 51.8, 61.02, 61.04, 61.07, 80.8 ( J = 165 Hz), 107.0, 107.7 , 154.8;

MS (ESI) m/z 258 [M+Na] ⁺ ;

HRMS (ESI) m/z calcd. For C ₁₀ H ₁₈ FNO ₄ +Na: 258.1118, found: 258.1113.

Step 5:

Compound 18 (10 mg, 0.043 mmol) obtained in step 5 was dissolved in acetonitrile (0.5 mL), and 1 N HCl (0.1 mL) was added, followed by stirring at 60° C. for 5 minutes. Concentrated under reduced pressure at room temperature to obtain a colorless liquid compound 1a (8 mg, 0.042 mmol).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.99-2.08 (m, 5H), 3.54-3.63 (m, 3H), 4.22 (t, J = 6.2 Hz, 2H), 4.48 (t, J = 5.6 Hz, 1H), 4.60 (t, J = 5.8 Hz, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 30.1 (d, J = 20 Hz), 36.5, 37.0, 42.5, 52.3, 61.6, 80.6 (d, J = 164 Hz), 122.1, 125.18;

HRMS (ESI) m/z calcd. For C ₈ H ₁₃ FNO ₃ : 190.0879, found: 190.0874.

<Example 1> IeDDA reaction with tetrazine compounds in various solvents

Dissolve N-Boc-3-pyrrolidinone ( 19 , 5 μmol) and tetrazine compound 2a (0.5 μmol) in the organic solvent shown in Table 1 below, and in case of mixing water, add 2 mL of water separately. A reaction solution was prepared. After measuring UV with the blank solution, the reaction solution was placed in a UV cell and placed on a UV spectrometer. A pyrrolidine solution (10 μL, 5 μmol) diluted in each solution was added to a UV cell, mixed quickly, and UV was measured for 10 minutes at 15 second intervals. The UV wavelength was fixed at 519-543 nm and scanned, and the change in UV absorption intensity at 531 nm, which is the λ _max value of the tetrazine compound, was plotted in one phase exponential decay mode using the GraphPad Prism program. The velocity constant (k _obs ) value and half-life under each condition were obtained and summarized in Table 1.

division Solvent (ratio, v/v) k _obs (s ^-1 ) Half life (min) Rel. k _obs ^* One THF 0.00005679 203.43 0.000819 2 DMF 0.00009232 125.13 0.001332 3 DMF/H ₂ O (1/1) 0.002469 4.678 0.035617 4 DMF/H ₂ O (1/3) 0.02834 0.408 0.408829 5 DMSO 0.001314 8.788 0.018956 6 DMSO/H ₂ O (1/1) 0.02490 0.464 0.359204 7 DMSO/H ₂ O (1/3) 0.06932 0.167 1.00 8 MeCN 0.00003437 336.10 0.000496 9 MeCN/H ₂ O (1/1) 0.001391 8.307 0.020066 10 MeCN/H ₂ O (1/3) 0.01265 0.913 0.182487 11 MeOH 0.0009177 12.588 0.013239 12 MeOH/H ₂ O (1/1) 0.004883 2.367 0.070441 13 MeOH/H ₂ O (1/3) 0.03199 0.361 0.461483 14 EtOH 0.00001505 767.35 0.000217 15 EtOH/H ₂ O (3/1) 0.001224 9.437 0.017657 16 EtOH/H ₂ O (1/1) 0.008642 1.337 0.124668 17 EtOH/H ₂ O (1/3) 0.03184 0.363 0.459319 18 EtOH/H ₂ O pH4 (1/1) - ^** - ^** - ^** 19 EtOH/H ₂ O pH4 (1/3) 0.002009 5.750 0.028982 20 EtOH/H ₂ O pH7 (1/1) 0.001258 9.18 0.018148 21 EtOH/H ₂ O pH7 (1/3) 0.0003011 38.367 0.004344 22 EtOH/H ₂ O pH8 (1/1) 0.005613 2.058 0.080972 23 EtOH/H ₂ O pH8 (1/3) 0.005940 1.945 0.085690

* Relative velocity constant divided by the k _obs value of Category 7 in Table 1

** The reaction does not proceed

As a result of Example 1, the half-life of division 7 in Table 1 was the fastest at 0.167, and divisions 4 and 17 also showed fast reaction rates. In addition, it can be seen that the higher the water ratio, the faster the reaction rate, which is consistent with the Diels-Alder reaction proceeding well by hydrophobic interactions. It was found that the reaction rate was relatively low in various pH buffer solutions.

<Example 2> IeDDA reaction with tetrazine compounds at various concentration ratios

Dissolve N-Boc-3-pyrrolidinone ( 19 , 5-10 μmol) in ethanol (0.5 mL), transfer to a container containing tetrazine compound 2a (0.5 μmol), dissolve sufficiently, and add water (1.5 mL). And transferred to a UV cell container. After measuring UV with the blank solution, the UV cell containing the reaction solution was placed on a UV spectrometer and measured. After fixing the UV wavelength at 519 nm to 543 nm, water (10 μL) in which pyrrolidine 4a (5 to 10 μmol) was dissolved was added, and then UV was scanned at 15 second intervals for 10 minutes, and the λ _max value of the tetrazine compound was 531. The change in UV absorption intensity by time in nm was plotted in one phase exponential decay mode using the GraphPad Prism program. The velocity constant (k _obs ) value and half-life under each condition were obtained and summarized in Table 2.

division Concentration (mM) k _obs (S ^-1 ) Half life (min) Rel. k _obs * 2a 19 4a One 0.25 1.25 1.25 0.01203 0.96 0.27 2 0.25 1.875 1.875 0.03140 0.37 0.70 3** 0.25 2.5 2.5 0.04473 0.26 1.00

* Relative velocity constant divided by the k _obs value of Category 3

** Same as Category 17 in Table 1

It was confirmed that the higher the concentration of ketone compound 19, the higher the reaction rate. The secondary reaction rate constant was calculated by _plotting k _obs values measured at three different concentrations according to the concentration, and the secondary reaction rate constant (k ₂ ) was calculated as 26.16 (M ^-1 s ^{- 1} ).

<Example 3> Tetrazine conjugation reaction according to amine concentration

Except for varying the concentration of pyrrolidine ( 4a ), it was carried out in the same manner as in _Section 3 of Example 2, and the rate constant (k _obs ) value and half-life were obtained and summarized in Table 3.

division Concentration (mM) k _obs (S ^-1 ) Half life (min) Rel. k _obs * 2a 19 4a One 0.25 2.5 0.5 0.01083 1.067 0.34014 2 0.25 2.5 1.25 0.02929 0.394 0.91991 3** 0.25 2.5 2.5 0.03184 0.363 1.00

* Relative velocity constant divided by the k _obs value of Category 3

** Same as Category 17 in Table 1

As in Example 2, the reaction rate increased as the concentration of the ketone compound 19 increased, and it was found that the reaction rate increased in proportion to the concentration of the amine pyrrolidine.

<Example 4> Tetrazine conjugation reaction using various amines

Except for the use of other types of amines other than pyrrolidine ( 4a ), it was carried out in the same manner as in _Section 3 of Example 2, and the rate constant (k _obs ) value and half-life were obtained and summarized in Table 4.

division Secondary amine k _obs (S ^-1 ) Half life (min) Rel. k _obs * One** Pyrrolidine
pyrrolidine ( 4a ) 0.03184 0.363 0.844 2 Dimethylamine
dimethylamine ( 4b ) 0.03772 0.306 1.00 3 Diethylamine
diethylamine ( 4c ) 0.03025 0.382 0.802 4 Diisopropylamine
diisopropylamine ( 4d ) 0.03433 0.337 0.910 5 Piperidine
piperidine ( 4e ) 0.03425 0.337 0.908 6 Morpholine
morpholine ( 4f ) 0.001094 10.563 0.029 7 Proline
proline ( 4g ) -*** -*** -*** 8 N,N'-dimethylethylenediamine
N,N'-dimethylethylenediamine ( 4h ) 0.004465 2.587 0.118

* Relative velocity constant divided by the k _obs value of Category 2

** Same as Category 17 in Table 1

*** Reaction does not proceed

4a to In the case of 4e , there was no significant difference in the reaction rate, whereas when morpholine ( 4f ) was used, the reaction rate was significantly lowered, and when proline ( 4g ) was used, the reaction did not proceed.

<Example 5> Tetrazine conjugation reaction using several ketone compounds

Except for the use of various ketone compounds 1 , it was carried out in the same manner as in _Section 3 of Example 2, and the rate constant (k _obs ) value and half-life were obtained and summarized in Table 5.

division Ketone k _obs (S ^-1 ) Half life (min) Rel. k _obs (*) One** 1-Boc-3-pyrrolidinone
1-Boc-3-pyrrolidinone ( 19 ) 0.04473 0.26 1.00 2 1-Boc-3-azetidinone
1-Boc-3-azetidinone 0.0002645 43.7 0.0059 3 1-Boc-4-piperidone
1-Boc-4-piperidone 0.00004357 265.2 0.00097

* Relative velocity constant divided by the k _obs value of Category 1

** Same as Category 7 in Table 1

In the case of compound 19, which is a pentagonal ring, the IeDDA reaction with the tetrazine compound is very fast, whereas 1-Boc-3-azetidinone of the quadrangle ring (Table 5, Category 2) is about 170 times slower and 1 of the hexagonal ring. -Boc-4-piperidone (Table 5, Category 3) was more than 1,000 times slower.

<Example 6> Tetrazine conjugation reaction

Tetrazine compound 2a (63 mg, 0.20 mmol) and 1-Boc-3-pyrrolidinone ( 19 , 44 mg, 0.24 mmol) were dissolved in EtOH/H ₂ O (1/3 v/v) solvent and then at room temperature. Pyrrolidine ( 4a , 82 μL, 1.0 mmol) was added. The reaction solution was stirred well for 10 minutes, and then the solvent was removed under reduced pressure, followed by column chromatography (2% methanol/dichloromethane) to obtain a yellow solid compound (63 mg, 70%). After separation by high performance liquid chromatography (HPLC), 3a (58 mg, 64%) and 3b (3 mg, 3%) were obtained.

3a :

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.46 (s, 9H), 1.58 (s, 9H), 2.79 (s, 3H), 3.39 (t, J = 8.8 Hz, 2H), 4.27 (t, J = 8.8 Hz, 2H), 4.36 (s, 2H), 5.36 (br s, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 20.6, 27.5, 27.9, 28.3, 29.7, 44.0, 50.7, 80.0, 84.8, 127.9, 128.7, 135.8; 143.5, 148.5, 149.2, 150.7, 154.2, 156.3;

MS (ESI) m/z 441 [M+H] ⁺ ;

Mass: HRMS (ESI) m/z For C ₂₄ H ₃₃ N ₄ O ₄ , calcd. 441.2502, found: 441.2496.

3b :

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.47 (s, 9H), 1.52 (d, J = 8.0 Hz, 9H), 2.80 (d, J = 10.0 Hz, 3H), 4.37 (s, 2H), 4.84 (s, 1H), 5.00 (d, J = 18.4 Hz, 2H), 5.43 (br s, 1H), 7.45 (dd, J = 7.8 Hz, 16.2 Hz, 2H), 7.70 (t, J = 18.4 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 19.7, 28.4 28.5, 44.3, 50.6, 52.0, 52.2, 53.4, 79.6, 80.8, 80.9, 127.8, 128.2, 134.8, 134.9, 135.1, 135.2, 137.4, 137.8, 140.8, 140.9, 153.9, 154.1, 154.3, 155.9;

MS (ESI) m/z 441 [M+H] ⁺ ;

Mass: HRMS (ESI) m/z For C ₂₄ H ₃₃ N ₄ O ₄ , calcd. 441.2502, found: 441.2497.

<Example 7> Tetrazine conjugation reaction

Tetrazine compound 2a (5 mg, 0.017 mmol) and 2,2,5,5-tetramethylpyrrolidin-3-one ( 11 , 5 mg, 0.035 mmol) obtained in Preparation Example 3 were added to EtOH/H ₂ O (1/1 v/v) was dissolved in a solvent (1.0 mL), and then pyrrolidine (5 μL, 0.07 mmol) was added. The reaction mixture was stirred for 6 hours, but the reaction did not proceed.

<Example 8> Tetrazine conjugation reaction

Tetrazine compound 2a (30 mg, 0.10 mmol) and methyl 2-methyl-2- (3-oxopyrrolidin-1-yl) propanoate ( 15 , 22 mg, 0.12 mmol) were added to EtOH/H ₂ O ( 1/3 v/v) was dissolved in a solvent, and then pyrrolidine (8.2 μL, 0.10 mmol) was added at room temperature. The reaction solution was stirred well for 10 minutes, and then the solvent was removed under reduced pressure, followed by column chromatography (3% methanol/dichloromethane) to obtain a yellow solid compound 3d (25 mg, 57%). At this time, no other structural isomers were formed.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.48 (s, 9H), 2.10-2.22 (m, 2H), 2.84 (s, 3H), 3.40 (t, J = 8.2 Hz, 2H), 4.31 (t, J = 8.0 Hz, 2H), 4.39 (s, 2H), 4.46 (td, J = 2.0 Hz, 6.2 Hz, 2H), 4.55 (td, J = 2.1 Hz, 5.6 Hz, 1H), 4.67 (td, J = 2.1 Hz, 5.5 Hz, 1H), 5.41 (br s, 1H), 7.43 (d, J = 6.8 Hz, 2H), 7.67 (d, J = 6.8 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 19.7, 25.4, 26.7, 28.4, 44.3, 51.8, 52.1, 53.6, 60.9, 79.6, 127.6, 128.3, 135.7, 137.4, 139.8, 140.3, 153.9, 154.3, 155.9, 175.1 ;

MS (ESI) m/z 441 [M+H] ⁺ ;

HRMS(ESI) m/z For C ₂₄ H ₃₃ N ₄ O ₄ , calcd. 441.2502, found: 441.2496.

<Example 9> Tetrazine conjugation reaction

Tetrazine compound 2a (70mg, 0.23 mmol) and tert-butyl 4-oxopiperidine-1-carboxylate (55 mg, 0.28 mmol) were dissolved in EtOH/H ₂ O (1/3 v/v) solvent. At room temperature, pyrrolidine (19 μL, 0.23 mmol) was added. After the reaction solution was stirred well for 6 hours, the solvent was removed under reduced pressure, and column chromatography (5% methanol/dichloromethane) was performed to obtain a beige solid compound 3e (98 mg, 93%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.48 (s, 9H), 1.52 (s, 9H), 2.65 (s, 3H), 2.77 (t, J = 5.0, 2H), 3.59 (t, J = 5.8 , 2H), 4.39 (t, J = 5.6, 2H), 4.54 (s, 2H), 5.00 (br s, 1H), 7.40 (d, J = 8.0, 2H), 7.52 (d, J = 8.4, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 19.7, 25.4, 26.7, 28.4, 44.3, 51.8, 52.1, 53.4, 53.5, 53.6, 60.9, 79.6, 127.7, 128.3, 128.6, 135.7, 137.4, 139.8, 140.3, 153.9 , 154.3, 156.0, 175.1;

MS (ESI) m/z 455 [M+H] ⁺ ;

HRMS(ESI) m/z For C ₂₅ H ₃₅ N ₄ O ₄ . calcd.: 455.2658, found: 455.2653.

<Example 10> Tetrazine conjugation reaction

Tetrazine compound 2a (70mg, 0.23 mmol) and tert-butyl 2-oxo-7-azabicyclo[2.2.1]heptane-7-carboxylate ( 20 , 59 mg, 0.28 mmol) were added to EtOH/H ₂ O ( 1/3 v/v) was dissolved in a solvent, and then pyrrolidine (19 μL, 0.23 mmol) was added at room temperature. After the reaction solution was stirred well for 12 hours, the solvent was removed under reduced pressure, and column chromatography (2% methanol/dichloromethane) was performed to obtain a white solid compound 3f (58 mg, 63%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.31-1.35 (m, 2H), 1.37 (s, 9H), 1.48 (s, 9H), 2.27-2.29 (m, 2H), 2.00 (s, 3H), 4.41 (d, J = 5.6 Hz, 2H), 5.17 (br s, 1H), 5.26 (s, 1H), 5.42 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 19.4, 24.7, 25.2, 28.1, 28.4, 44.3, 58.8, 59.8, 79.6, 81.2, 127.8, 128.5, 135.2, 140.5, 141.3, 143.4, 151.0, 151.8, 154.8, 156.0 ;

MS (ESI) m/z 467 [M+H] ⁺ , mp: 75.0-75.5 ^o C;

HRMS(ESI) m/z For C ₂₆ H ₃₅ N ₄ O ₄ . calcd. 467.2658. found: 467.2653.

< Experimental example 1> using 1a Tetrazine Conjugation reaction

Tetrazine compound 2a (70 mg, 0.23 mmol) and 3-fluoropropyl 3-oxopyrrolidine-1-carboxylate ( 1a , 53 mg, 0.28 mmol) obtained in Preparation Example 5 were added to EtOH/H ₂ O (1 /3 v/v) was dissolved in a solvent, and then pyrrolidine (19 μL, 0.23 mmol) was added at room temperature. After the reaction solution was stirred well for 10 minutes, the solvent was removed under reduced pressure, and column chromatography (4% methanol/dichloromethane) was performed to obtain 3g (42 mg, 41%) and 3h (12 mg, 12%) of a yellow liquid compound. Got it.

3g :

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.48 (s, 9H), 2.06-2.17 (m, 2H), 2.83 (s, 1H), 4.36-4.41 (m, 4H), 4.50-4.55 (m, 1H) ), 4.62-4.67 (m, 1H), 4.91 (d, J = 8.0 Hz, 2H), 5.03 (s, 1H), 5.09 (s, 1H), 7.48 (t, J = 7.0 Hz, 2H), 7.72 -7.78 (m, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 17.9, 18.0, 28.3, 30.0 (d, J =20 Hz), 44.1, 45.4, 50.5, 51.1, 52.1, 52.9, 62.4, 62.5, 80.1, 80.6 (d, J =166 Hz), 128.0, 128.4, 128.5, 128.6, 131.9, 132.1, 139.6, 142.5, 142.7, 154.1, 154.2, 155.3, 156.1, 160.2, 160.6;

MS (ESI) m/z 445 [M+H] ⁺ ;

HRMS (ESI) m / z For C 23 H 30 FN 4 O 4. calcd .: 445.2251 found: 445.2246.

3h :

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.48 (s, 9H), 2.11-2.21 (m, 2H), 2.83 (s, 3H), 3.40 (t, J = 8.4 Hz, 2H), 4.33 (t, J = 8.4 Hz, 2H), 4.39 (br s, 2H), 4.46 (t, J = 6.2 Hz, 2H), 4.55 (t, J = 5.8 Hz, 1H), 4.66 (t, J = 5.6 Hz, 1H ), 5.18 (br s, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.66 (d, J = 6.8 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 20.5, 27.9, 28.3, 29.7 (d, J =20 Hz), 44.0, 50.3, 64.0, 64.1, 80.0, 80.6 (d, J =166 Hz), 127.8, 128.7 , 130.0, 135.2, 142.9, 147.7, 148.7, 152.1, 155.0, 156.1;

MS (ESI) m/z 445 [M+H] ⁺ ;

HRMS (ESI) m / z For C 23 H 30 FN 4 O 4. calcd .: 445.2251 found: 445.2246.

< Experimental example 2> 2e compound preparation

cRGDfK (30 mg, 0.050 mmol) was dissolved in N,N-dimethylformamide (0.5 mL), and diisopropylethylamine (DIEA, 26 μL, 0.150 mmol) was added. Compound 2c obtained in Preparation Example 2 was reacted with N-hydroxysuccinimide to dissolve the tetrazine NHS ester 2d (20 mg, 0.050 mmol) in N,N-dimethylformamide (0.5 mL), and then 30 Stir for a minute. After the reaction solution was filtered, the solvent was removed under reduced pressure, and the concentrate was separated using high performance liquid chromatography. The purified compound was lyophilized to obtain a white solid compound 2e (25 mg, 57%).

MS (ESI) m/z 887.7 [M+H] ⁺ .

< Experimental example 3> compound [ ¹⁸ F]3- Fluoropropyl 3- Oxopyrrolidine -One- Carboxylate ([ ¹⁸ Preparation of F]1a)

QMA (HCO ₃ ^-), distilled water (10 mL) to the washed ^[18 F] was passed through suyongaekreul that the fluoride is dissolved ^[18 F] After capturing the fluoride Krytofix [222] Cartridge-potassium sulfonate salt ( [ ¹⁸ F] fluoride (43.5 mCi) was eluted by flowing a QMA cartridge with an ethanol solution (1 mL) in which 10 mg, 19.6 μmol) was dissolved. Blow nitrogen gas at 100 ℃ to remove the solvent, and then 3-(tosyloxy)propyl 3,3-dimethoxypyrrolidine-1-carboxylate ( 5b , 3 mg, 7.7 μmol) dissolved in acetonitrile (0.5 mL) After putting it, it was reacted at 100° C. for 10 minutes. After the reaction, 1 N HCl aqueous solution (0.1 mL) was added, followed by stirring at 60° C. for 10 minutes. After cooling the reaction mixture to room temperature, 1 N NaOH aqueous solution (98 μL) was added, distilled water (0.300 mL) was added, filtered, and then separated by high-performance liquid chromatography. The separated solution was diluted with distilled water (15 mL), passed through a Light C-18 Sep-Pak cartridge, and nitrogen gas was blown to remove water, and the compound was eluted with ethanol (1 mL). The eluent was dried by blowing nitrogen gas at room temperature. The final compound [ ¹⁸ F] 1a (3.7 mCi) was obtained, and the synthesis time was 71 minutes.

High performance liquid chromatography (HPLC) separation conditions:

Column: YMC-Pack ODS-A (S-5 m, 12 nm); Mobile phase: 30-100% A in B 20 min, A (acetonitrile), B (0.1% TFA aqueous solution); Flow rate: 4 mL/min; Detector: UV (254 nm) and RI detector; Retention time: 14.5 minutes.

<Experimental Example 4> F-18 labeling reaction through tetrazine conjugation

In the reaction vessel containing [ ¹⁸ F]3-fluoropropyl 3-oxopyrrolidine-1-carboxylate ([ ¹⁸ F] 1a , 3.7 mCi) synthesized in Experimental Example 3, tetrazine compound 2a (1 mg, 3.3 μmol) in ethanol (50 μL) solution was added, diluted with distilled water (150 μL), pyrrolidine (1 μL, 12.2 μmol) was added, and then stirred at room temperature for 10 minutes. After the reaction, 30% acetonitrile/distilled water (1 mL) was added to the reaction vessel, and the reaction solution was filtered. The filtrate was injected into high-performance liquid chromatography to separate 3 g of the product [ ¹⁸ F]. The final 0.96 mCi was obtained, and the synthesis time was 140 minutes.

High performance liquid chromatography (HPLC) separation conditions:

Column: Xterra perp MS C18, 10 micron, 10 mmx250 mm; Mobile phase: 30% A in B 30 min, A (acetonitrile), B (0.1% TFA aqueous solution); Flow rate: 4 mL/min; Detector: UV (254 nm) and RI detector; Holding time: 24-26 minutes.

< Experimental example 5> Tetrazine Conjugation through ¹⁸ F-cover RGD Compound synthesis

RGD obtained in Experimental Example 2 in a reaction vessel containing [ ¹⁸ F]3-fluoropropyl 3-oxopyrrolidine-1-carboxylate ([ ¹⁸ F] 1a , 4.21 mCi) synthesized in Experimental Example 3 -Tetrazine 2e (1 mg, 1.1 μmol) is dissolved in ethanol/distilled water (1/3, 0.4 mL) and added. Pyrrolidine (1 μL, 12.2 μmol) was added, stirred at room temperature for 10 minutes, distilled water (0.5 mL) was added, and then filtered. The filtrate was separated using high performance liquid chromatography (HPLC) to obtain 0.61 mCi of the final compound [ ¹⁸ F] 3i , and the synthesis time was 74 minutes.

High performance liquid chromatography (HPLC) separation conditions:

Column: YMC-Pack ODS-A (S-5 m, 12 nm); Mobile phase: 0-80% A in B 30 min, A (acetonitrile), B (0.1% TFA aqueous solution); Flow rate: 3 mL/min; Detector: UV-254 nm, RI detector; Holding time: 18.5 minutes.

<Experimental Example 6> ¹⁸ Tumor PET imaging test using F-labeled RGD compound

The human glioma cell line (U87MG) was purchased from the American Type Culture Collection (ATCC) and used. Human glioma cell line was used as a culture medium in which 10% fetal bovine serum (FBS) and 1% antibiotic/antifungal agents were added to MEM medium. The experimental animal was a male nude mouse 6 weeks old (Narabio, Seoul, Korea), and was prepared by injecting 5×10 ⁶ cells subcutaneously in the right hind limb. It was used when the tumor was grown for 14 days and the tumor size reached 5.0 mm. Compound [ ¹⁸ F] 3i (4.8-7.4 MBq, 200 μL) synthesized in Experimental Example 5 was injected intravenously and then PET/CT imaged for 90 minutes after injection using small animal nanoScan PET/CT (Mediso, Budapest, Hungary) Was obtained. The obtained PET/CT image result was quantitatively analyzed using InterView ^TM FUSION software (Mediso). The results are shown in FIG. 6, and quantitative results of intake for each organ are shown in FIG. 7 and Table 6 below.

SUV value Time (minutes) Tumor Kidney Liver Muscle Tumor to muscle ratio 10 0.59 3.34 1.82 0.30 2.00 20 0.80 2.02 1.77 0.30 2.62 30 0.78 1.55 1.55 0.27 2.89 40 0.72 1.39 1.38 0.22 3.28 50 0.67 1.22 1.26 0.20 3.32 60 0.61 1.08 1.14 0.19 3.18 70 0.58 1.03 1.00 0.16 3.62 80 0.60 0.98 0.93 0.14 4.13 90 0.53 0.83 0.86 0.14 3.81

According to FIG. 6, it can be seen that ingestion in human glioma tumors (white circles in FIG. 6) starts rapidly within 10 minutes after injection of the compound, and intake in the tumor for 90 minutes is stably maintained. In addition, as the tumor/muscle intake ratio increased with time through FIG. 7 and Table 6, the target ability for glioma was confirmed.

Claims (10)

Compound represented by the following formula (1):
[Formula 1]

(In Formula 1,
A is a bond, C _1-6 straight or branched chain alkylene, or -C(=O)-;
D is a bond, -O-, -NH-, or -C(=O)-;
E is a bond, -O-, or -NH-;
G is a bond or C _1-6 straight or branched chain alkylene, wherein the carbon of the alkylene may be substituted with oxygen;
J is a bond, a C _6-10 arylene, or a 6 to 10 ring heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S;
K is a bond, or C _1-6 straight or branched chain alkylene, wherein the carbon of the alkylene may be substituted with oxygen;
F is fluorine-18 or fluorine-19;
m and n are independently integers of 1 or 2; And
Hydrogen of -CH ₂ -in parentheses may be substituted with methyl).
As shown in Scheme 1 below,
The step of reacting the compound represented by the formula (1) of claim 1 and a tetrazine compound represented by the following formula (2); A method for preparing a compound represented by the following formulas 3 and 3', including:
[Scheme 1]

(In the above Scheme 1,
A, D, E, G, J, K, F, m, n and -CH ₂ -in parentheses are as defined in Formula 1 of claim 1;

In Formula 2, R ₁ and R ₂ are independently C _1-12 straight or branched chain alkyl, unsubstituted or substituted 5 to 10 each containing at least one heteroatom selected from the group consisting of N, O and S Ring heteroaryl, unsubstituted or substituted C _6-10 aryl, or unsubstituted or substituted C _6-10 aryl C _1-12 straight or branched chain alkyl,
The carbon of the straight chain or branched chain alkyl of C _1-12 may be substituted with oxygen, hydrogen may be substituted with halogen,
The substituted 5 to 10 ring heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl is each of 5 to 10 rings substituted with one or more additional substituents. Heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl).
The method of claim 2,
The reaction of the compound represented by Formula 1 with the tetrazine compound represented by Formula 2 is carried out in the presence of a secondary amine represented by Formula 4:
[Formula 4]

(In Chemical Formula 4,
R ₃ and R ₄ are linked to each other to form a C _3-10 alkylene, but carbon in the alkylene may be substituted with oxygen or -NH-; or

R ₃ and R ₄ are independently C _1-12 straight or branched chain alkyl, unsubstituted or substituted 5 to 10 ring heteroaryl containing at least one heteroatom selected from the group consisting of N, O and S , Unsubstituted or substituted C _6-10 aryl, or unsubstituted or substituted C _6-10 aryl C _1-12 straight or branched chain alkyl,
The carbon of the straight chain or branched chain alkyl of C _1-12 may be substituted with oxygen, hydrogen may be substituted with halogen,
The substituted 5 to 10 ring heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl is each of 5 to 10 rings substituted with one or more additional substituents. Heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl).
The method of claim 3,
The secondary amine represented by Formula 4 is a secondary amine represented by Formula 4-1 below:
[Formula 4-1]

(In Formula 4-1,
R ₃ ^" and R ₄ ^" are independently C _2-4 alkylene, but carbon in the alkylene may be substituted with oxygen or -NH-; And
X is a bond, methylene, oxygen, or -NH-).
Compound represented by the following formula (5):
[Formula 5]

(In Formula 5.
A, D, E, G, J, K, m, n and -CH ₂ -in parentheses are as defined in Formula 1 of claim 1;

W is -C(=O)-, or -C(OR ₅ )(OR ₆ )-,
The R ₅ and R ₆ are connected to each other to form a C _3-10 alkylene, but carbon in the alkylene may be substituted with oxygen or -NH-, or
The R ₅ and R ₆ are independently hydrogen, C _1-12 straight or branched chain alkyl, N, O and S unsubstituted or substituted 5 to 10 each containing at least one heteroatom selected from the group consisting of Ring heteroaryl, unsubstituted or substituted C _6-10 aryl, or unsubstituted or substituted C _6-10 aryl C _1-12 straight or branched chain alkyl,
The carbon of the straight chain or branched chain alkyl of C _1-12 may be substituted with oxygen, hydrogen may be substituted with halogen,
The substituted 5 to 10 ring heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl is each of 5 to 10 rings substituted with one or more additional substituents. Heteroaryl, C _6-10 aryl, or C _6-10 aryl C _1-12 straight or branched chain alkyl; And

X is -OS(O ₂ )R ₇ , and R ₇ is methyl, trifluoromethyl, para-toluene, or para-nitrobenzene).
As shown in Scheme 2 below,
Preparing a compound represented by Formula 1 from the compound represented by Formula 5; A method for preparing a compound represented by Formula 1 of claim 1 including:
[Scheme 2]

(In Scheme 2,
The compound represented by Formula 1 is as defined in claim 1;
The compound represented by Formula 5 is as defined in claim 5; And
M is sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), N,N,N,N-tetraalkylammonium salt, or Cryptofix[2.2.2]-potassium salt (kryptofix[2.2) .2])-K)).
Compound represented by the following formula 3A or 3B:
[Chemical Formula 3A]

[Chemical Formula 3B]

(In Chemical Formula 3A or 3B,
A, D, E, G, J, K, F, m, n and -CH ₂ -, R ₁ and R ₂ in parentheses are as defined in Scheme 1 of claim 2).
A composition for diagnosing diseases comprising at least one compound selected from the group consisting of Formulas 3A and 3B of claim 7.
Intermediate compound represented by the following formula A or A':
[Formula A]

;

[Formula A']

;
(In Formula A or A',
A, D, E, G, J, K, F, m and n are as defined in Formula 1 of claim 1;
R ₃ and R ₄ are as defined in Formula 4 of claim 3; And
The hydrogen of -CH- in parentheses of m and -CH ₂ -in parentheses of n may be independently substituted with methyl).
Compound represented by the following formula B:
[Formula B]

(In Formula B,
A, D, E, G, J, K, and F are as defined in Formula 1 of claim 1).

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