KR102150377B1 - Preparation method of [18F]-substituted pyrazole derivatives Radiopharmaceuticals - Google Patents

Abstract

The present invention relates to a method for manufacturing fluorine-18-substituted pyrazole derivative radiopharmaceuticals which comprises: a first step of obtaining [^18F] fluorine without moisture by adding [^18F] fluorine to a reaction vessel together with a phase change additive and a base additive under a first solvent, and blowing nitrogen at a temperature of 80 to 100°C to remove moisture; a second step of adding the moisture-removed [^18F] fluorine into the reaction vessel in which a pyrazole precursor, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) or a copper-based catalyst are dissolved in a second solvent, followed by heating the same at a temperature of 100 to 140°C to manufacture a pyrazole derivative solution; and a third step of cooling the pyrazole derivative solution to room temperature, and performing a high-performance liquid chromatography (HPLC) to separate and purify the pyrazole derivative. According to the present invention, there is an effect that alpha-synuclein, which is a major biomarker of brain disease, can be selectively tracked and explored by imaging alpha-synuclein accumulated in the brain with advanced dementia and FAF1 and alpha-synuclein conjugates present in the brain of a person with dementia.

Description

Preparation method of [18F]-substituted pyrazole derivatives Radiopharmaceuticals}

The present invention is capable of selectively tracking and searching for alpha-synuclein, a major biomarker of brain disease, by imaging alpha-synuclein accumulated in the brain with dementia disease and FAF1 and alpha-synuclein conjugates present in the brain of a person with dementia. It relates to a method for producing a pyrazole derivative radiopharmaceutical substituted with fluorine-18.

The present invention is derived from a study conducted as part of the disease recovery technology development project of the Ministry of Health and Welfare and the National Institute of Health Industry Promotion [Task management number: HI16C0947, task name: Neuroinflammation and alpha for initial diagnosis of dementia and evaluation of drug treatment effects -Synuclein target PET imaging technology development and clinical application].

Due to the recent rapid entry into an aging society, many problems have emerged in all areas such as the economy, society, and health of our society. In particular, dementia, a representative degenerative brain disease of the elderly, is threatening the future society as a health problem.

According to the report on dementia epidemiology of the central dementia center in Korea, the prevalence of degenerative brain diseases such as Alzheimer's disease is progressing faster than expected, and the estimated number of dementia patients in 2050 is expected to exceed 3 million. Since this figure is a figure that is required to care for one dementia patient per seven citizens, the economic problems surrounding it are expected to increase rapidly. Therefore, preparation of countermeasures, early diagnosis of dementia, and development of technologies and treatments to prevent it are required.

Currently, the underlying cause of dementia is not accurately identified, so it is difficult to estimate the proper treatment method and treatment time. The theory that the accumulation of beta-amyloid plaques is related to dementia has emerged worldwide, and various new drugs have been developed, but all of them have failed clinical trials, and in recent years, there is a possibility that amyloid may be a biomarker of dementia, but for early diagnosis and treatment. In terms of the cause of dementia, opinions are gathering that a new strategy must be found.

As a biomarker of interest as another cause of dementia incidence of dementia, it is the alpha-synuclein protein that has recently attracted the attention of many researchers. Alpha-synuclein is a protein composed of 140 amino acids and is mostly present in the central nervous system. Although the function of alpha-synuclein has not been clearly identified, it has been reported that it exists at the end of systemic ganglion cells and is associated with formation of vesicles and release of neurotransmitters. According to recent research results, it has been reported that abnormal accumulation and aggregation of alpha-synuclein in the central nervous system due to specific causes causes inflammatory reactions and cytotoxicity by increasing caspase-3 activity along with dysfunction and weakening of mitochondria. .

Therefore, research is being conducted to develop a new drug that can selectively target the normal accumulation and aggregation of alpha-synuclein worldwide. However, despite these efforts, a target substance capable of tracking alpha-synuclein has not yet been developed, and candidate substances being studied in the non-clinical trial stage, which are being studied recently, have high binding ability to alpha-synuclein, but patients with dementia. The selectivity between beta-amyloid plaques and tau aggregates, which are other biomarkers in the brain, is remarkably low, and there is a demand for the development of a new high-selectivity alpha-synuclein target drug.

Fas-associated factor 1 (FAF1) is known as a protein that promotes cell death. According to recent research results, abnormal expression of FAF1 was observed in the brain of patients with degenerative brain disease, and coexistence in neurons like alpha-synuclein. In addition, FAF1 promotes the aggregation and secretion of alpha-synuclein, as well as research results showing that FAF1 and alpha-cycline form a conjugate in dementia brain.

Meanwhile, positron emission tomography (PET) is a medical device capable of non-invasively qualifying and quantifying the function of vital organs including the human brain and the distribution of cancer. In addition, in order to obtain such a PET image, a drug that emits a positron from a radioisotope contained by binding to various biomarkers related to organs and diseases of interest in the body is essential, and this is called a radiopharmaceutical.

In the case of the carbon-11 labeling technique, which is currently an easy way to synthesize radiopharmaceuticals, it is possible to label hydroxy- or amine groups with a simple organic chemical method, but it is developed as a radiopharmaceutical due to the short half-life of 20 minutes and low specific activity value. While fluorine-18, which can be produced from cyclotron, has a long enough half-life (t _1/2 = 109.8 min) to image the brain using PET, it is possible to see many patients at once. In some cases, delivery is possible at other hospitals for diagnostic purposes, and it is a very suitable isotope for tracking specific biomarkers in the brain, such as degenerative brain disease, because it can have very high specific activity values depending on the labeling technique. However, since the direct aromatic fluorination reaction, which is a technique for directly labeling fluorine-18 at the aromatic position, has low reactivity, various precursors and reaction techniques to increase the reactivity have been actively studied.

An object of the present invention is to selectively track and search for alpha-synuclein, a major biomarker of brain disease, by imaging alpha-synuclein accumulated in the brain with dementia disease and FAF1 and alpha-synuclein conjugates present in the brain of a person with dementia. It is to provide a method for preparing radiopharmaceuticals of pyrazole derivatives substituted with fluorine-18.

It is to provide an imaging agent for diagnosing dementia diseases related to alpha-synuclein overexpression prepared by the method of manufacturing a pyrazole derivative radiopharmaceutical substituted with fluorine-18.

It is to provide a method for diagnosing dementia diseases related to alpha-synuclein overexpression using a pyrazole derivative radiopharmaceutical prepared by the fluorine-18-substituted pyrazole derivative radiopharmaceutical manufacturing method.

In order to achieve the above object, the method for preparing radiopharmaceuticals of pyrazole derivatives substituted with fluorine-18 according to the present invention is 80℃ by adding [ ¹⁸ F] fluorine to a reaction vessel together with a phase transfer additive and a base additive under a first solvent. A first step of obtaining [ ¹⁸ F] fluorine from which moisture is removed by blowing nitrogen at a temperature of about 100° C. to remove moisture; A reaction vessel in which the moisture-removed [ ¹⁸ F] fluorine is dissolved in a pyrazole precursor, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) or a copper-based catalyst in a second solvent A second step of preparing a pyrazole derivative solution by heating at a temperature of 100° C. to 140° C. after being added to; And a third step of cooling the pyrazole derivative solution to room temperature and performing liquid chromatography (HPLC) to separate and purify the pyrazole derivative.

The phase transfer catalyst may be Kryptofix 2.2.2 or 18-Crown-6.

The base additive may be selected from the group consisting of CsHCO ₃ , Cs ₂ CO ₃ , KHCO ₃ , K ₂ CO ₃ and tetrabutylammonium salt.

The copper-based catalyst is Cu(OTf) ₂ , Cu(py) ₄ (OTf) ₂ , Cu(NCCH ₃ ) ₄ , Cu(MeCN) ₄ BF ₄ , Cu(NCCH ₃ ) ₄ ·CF ₃ SO ₃ , [( CH ₃ CN) ₄ Cu]PF ₆ , and (CF ₃ SO ₃ Cu) ₂ ·C ₆ H ₆ It may be selected from the group consisting of.

The first solvent may be methyl alcohol or acetonitrile, and the second solvent may be acetonitrile or dimethylformaldehyde.

The pyrazole precursor may be a pyrazole precursor represented by Formula 2 below.

[Formula 2]

Here, R ₁ and R ₂ are each H or iodo phenyl tosylate, iodo toluene tosylate, iodo 4-anisole tosylate, iodo 2-thiopine tosylate, iodo phenyl bromide, iodo toluene Bromide, iodo 4-anisole bromide, iodo 2-thiopine bromide, iodo phenyl triflate, iodo toluene triflate, iodo 4-anisole triflate, iodo 2-thiopine triflate and pinacol It may be a compound selected from the group consisting of boronic esters; Either of R ₁ and R ₂ is necessarily hydrogen.

The pyrazole derivative may be a pyrazole derivative represented by Formula 1 below.

[Formula 1]

Here, X is ¹⁸ F or H, wherein X may be substituted at any one position of 4, 3, and 2 of the thiobenzene ring; Each of Y is H or ¹⁸ F, wherein Y may be substituted on any one of 4, 3 and 2 of the corresponding ethyl benzene ring; When X is H, Y is ¹⁸ F, and when X is ¹⁸ F, Y is H; Z is hydrogen, methyl, ethyl or propyl.

The present invention also provides an imaging agent for diagnosing dementia diseases related to alpha-synuclein overexpression prepared by the method of manufacturing a pyrazole derivative radiopharmaceutical in which the fluorine-18 is substituted in order to achieve the above object.

The present invention also provides a method for diagnosing dementia disease related to alpha-synuclein overexpression using a pyrazole derivative radiopharmaceutical prepared by the fluorine-18-substituted pyrazole derivative radiopharmaceutical manufacturing method to achieve the above object.

According to the present invention, a pyrazole derivative introduced with fluorine-18 having a high production efficiency and a high specific radioactivity value required as a brain diagnostic imaging agent is prepared by overcoming the disadvantages of previously known electrophilic and nucleophilic fluorination reactions. can do.

In addition, by imaging alpha-synuclein accumulated in the brain with dementia disease and FAF1 and alpha-synuclein conjugates present in the brain of a person with dementia, it is possible to selectively track and search for alpha-synuclein, a major biomarker of brain disease. There is.

In addition, there is an effect that can be usefully used for early diagnosis of degenerative diseases by using a pyrazole derivative into which fluorine-18 prepared as described above is introduced.

1 is a reaction scheme showing a synthesis route of a precursor required to synthesize a pyrazole derivative into which fluorine-18 is introduced in a method for preparing a pyrazole derivative radiopharmaceutical in which fluorine-18 is substituted according to an embodiment of the present invention. .
2 is a reaction scheme showing a process of synthesizing a pyrazole derivative into which fluorine-18 is introduced using the final precursor of FIG. 1.
FIG. 3 is a view showing [ ¹⁸ F]SNUBH-NM-026 and [ ¹⁸ F]SNUBH-NM-027, two representative pyrazole derivatives into which fluorine-18 of the present invention was introduced using the final precursor of FIG. 2 to be.
4 is a graph showing high performance liquid chromatography for separating pure fluorine-18 labeled pyrazole derivatives from a pyrazole derivative solution in a method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. .
5 is a fluorine-18-substituted pyrazole derivative production method according to an embodiment of the present invention, after separating the pyrazole derivative into which fluorine-18 is introduced through high-performance liquid chromatography, stable fluorine-19 is added. This is a graph showing the results of comparing the retention times of each of the introduced pyrazole derivatives and high performance liquid chromatography simultaneously.
Figure 6 shows the binding effectiveness of the alpha-synuclein protein using a pyrazole derivative substituted with fluorine-18 prepared by the method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. This is a graph showing the experimental results.
7 is a PET image data confirming the distribution in the body in a normal mouse using a pyrazole derivative substituted with fluorine-18 prepared through a method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. to be.
8 is a PET image data confirming the distribution in the body in a normal mouse using a pyrazole derivative substituted with fluorine-18 prepared through a method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. This is the distribution of fluorine-18 in the body by measuring the radioactivity of each major organ.
9 is a normal view of [ ¹⁸ F]SNUBH-NM-026 among the pyrazole derivatives substituted with fluorine-18 prepared through the method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. Brain ingestion PET imaging data in mice and alpha-synuclein-modified mice.
FIG. 10 is a normal view of [ ¹⁸ F]SNUBH-NM-026, among pyrazole derivatives substituted with fluorine-18 prepared through the method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. This is a graph comparing brain intake by brain region obtained based on brain intake PET images in mice and alpha-synuclein-modified mice.

The terms or words used in the present specification and claims correspond to the technical idea of the present invention based on the principle that the inventor can appropriately define the concept of the term in order to describe his or her invention in the best way. And should be interpreted as a concept.

Hereinafter, the present invention will be described in detail. However, the present invention is not limited to one embodiment disclosed below, but will be implemented in a variety of different forms, only the present one embodiment makes the disclosure of the present invention complete, and the scope of the invention to those of ordinary skill in the art. It is provided to fully inform you.

1 is a reaction scheme showing the synthesis route of a precursor required to synthesize a pyrazole derivative into which fluorine-18 is introduced in a method for preparing a pyrazole derivative radiopharmaceutical in which fluorine-18 is substituted according to an embodiment of the present invention. 2 is a reaction scheme showing a process of synthesizing a pyrazole derivative into which fluorine-18 is introduced using the final precursor of FIG. 1, and FIG. 3 shows a reaction formula using the final precursor of FIG. A drawing showing [ ¹⁸ F]SNUBH-NM-026 and [ ¹⁸ F]SNUBH-NM-027, which are two representative pyrazole derivatives into which fluorine-18 of the present invention is introduced, is disclosed.

4 is a graph showing high performance liquid chromatography for separating pure fluorine-18 labeled pyrazole derivatives from a pyrazole derivative solution in a method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. 5 shows a stable fluorine derivative after separating a pyrazole derivative into which fluorine-18 is introduced through high performance liquid chromatography in a method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. A graph showing the results of comparing the retention times of each of the pyrazole derivatives introduced with -19 and high performance liquid chromatography simultaneously is disclosed, and in FIG. 6, fluorine-18 is substituted according to an embodiment of the present invention. A graph showing the experimental results showing the binding effectiveness of the alpha-synuclein protein using a pyrazole derivative substituted with fluorine-18 prepared through a method for preparing a pyrazole derivative is disclosed.

7 shows PET image data confirming the distribution in the body in a normal mouse using a pyrazole derivative substituted with fluorine-18 prepared through a method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. It is disclosed, and Figure 8 shows the distribution in the body in a normal mouse using a pyrazole derivative substituted with fluorine-18 prepared through a method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. Through the confirmed PET image data, the body distribution data showing by measuring the radioactivity for each major organ of fluorine-18 is disclosed, and FIG. 9 shows a method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. Among the pyrazole derivatives substituted with fluorine-18 prepared through, [ ¹⁸ F]SNUBH-NM-026, brain ingestion PET imaging data in normal mice and alpha-synuclein-modified mice are disclosed.

FIG. 10 shows the normality of [ ¹⁸ F]SNUBH-NM-026 among the pyrazole derivatives substituted with fluorine-18 prepared through the method for preparing a pyrazole derivative substituted with fluorine-18 according to an embodiment of the present invention. A graph comparing intake by brain regions obtained based on PET images of brain intake in mice and alpha-synuclein-modified mice is disclosed.

Referring to these drawings, in the method for preparing radiopharmaceuticals of pyrazole derivatives substituted with fluorine-18 according to the present invention, [ ¹⁸ F] fluorine is added to a reaction vessel together with a phase transfer additive and a base additive under a first solvent to by to about 100 ℃ also remove water blowing nitrogen at a temperature, moisture pyrazol the removal of ^[18 F] a first step of obtaining the fluorine, the water is removed, ^[18 F] fluorine precursor, 2,2, 6,6-tetramethylpiperidine-N-oxyl (TEMPO) or copper-based catalyst was added to the reaction vessel dissolved in the second solvent, and then heated at a temperature of 100°C to 140°C to prepare a pyrazole derivative solution. And a third step of cooling the pyrazole derivative solution to room temperature and performing liquid chromatography (HPLC) to separate and purify the pyrazole derivative.

That is, the method for preparing radiopharmaceuticals of pyrazole derivatives substituted with fluorine-18 according to the present invention obtained high-purity fluorine-18 from which moisture was removed by using a phase transfer additive and a base additive having excellent elution efficiency for fluorine-18. Thereafter, a pyrazole precursor having excellent reactivity and a copper-based catalyst can be used to prepare a high-purity pyrazole precursor in high yield.

In addition, radiopharmaceuticals of pyrazole derivatives substituted with fluorine-18 prepared by the above manufacturing method overcome the disadvantages of previously known electrophilic and nucleophilic fluorination reactions, and thus must be equipped with high production efficiency and as a brain diagnostic imaging agent. It has the advantage of having a high specific activity value, and by imaging alpha-synuclein accumulated in the brain with dementia disease and FAF1 and alpha-synuclein conjugates present in the brain of dementia patients, alpha-synuclein, a major biomarker of brain disease, is selectively selected. It has the advantage of being able to track and navigate with.

The phase transfer catalyst may be preferably Kryptofix 2.2.2 or 18-Crown-6, and the base additive is CsHCO ₃ , Cs ₂ CO ₃ , KHCO ₃ , K ₂ CO _{3 In} the group consisting of tetrabutylammonium salt You can use the selected one. These phase transfer catalysts and base additives remove moisture from the aqueous solution in which fluorine-18 produced in cyclotron is dissolved in the direct aromatic fluorination technique to obtain fluorine-18, and the reactivity of the fluorination reaction with water There is an advantage of not only allowing fluorine-18 to be extracted with high yield by preventing deterioration, but also allowing fluorine-18 to be selectively introduced at a desired position by controlling the electron density of the aromatic ring.

The copper-based catalyst is a catalyst having excellent reactivity in the synthesis process of a pyrazole derivative. For example, Cu(OTf) ₂ , Cu(py) ₄ (OTf) ₂ , Cu(NCCH ₃ ) ₄ , Cu(MeCN) ₄ BF ₄ , Cu(NCCH ₃ ) ₄ ·CF ₃ SO ₃ , [(CH ₃ CN) ₄ Cu]PF ₆ , and (CF ₃ SO ₃ Cu) ₂ ·C ₆ H ₆ It may be selected from the group consisting of.

The base additive and the phase transfer catalyst are used in different amounts depending on the type of catalyst used in the second step, thereby controlling the elution efficiency of fluorine-18 and the reactivity and catalyst efficiency of the copper catalyst in the pyrazole derivative solution. I can.

That is, when using 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) as a catalyst in the second step, 20 to 30 parts by weight of the TEMPO based on 100 parts by weight of the pyrazole precursor , By using 20 to 30 parts by weight of the base additive and 120 to 130 parts by weight of the phase transfer catalyst, the elution efficiency of fluorine 18 can be optimized, and when a copper-based catalyst is used as a catalyst in the second step, the By using 6.7 to 13.3 parts by weight of a copper-based catalyst, 2.5 to 3.5 parts by weight of the base additive, and 6 to 7 parts by weight of the phase transfer catalyst based on 100 parts by weight of the pyrazole precursor, it is possible to optimize the elution efficiency of fluorine 18. have.

In addition, the first solvent may be methyl alcohol or acetonitrile, and the second solvent may be acetonitrile or dimethylformaldehyde, respectively.

The pyrazole precursor may be a pyrazole precursor represented by Formula 2 below.

[Formula 2]

Here, R ₁ and R ₂ are each H or iodo phenyl tosylate, iodo toluene tosylate, iodo 4-anisole tosylate, iodo 2-thiopine tosylate, iodo phenyl bromide, iodo toluene Bromide, iodo 4-anisole bromide, iodo 2-thiopine bromide, iodo phenyl triflate, iodo toluene triflate, iodo 4-anisole triflate, iodo 2-thiopine triflate and pinacol It may be a compound selected from the group consisting of boronic esters; Either of R ₁ and R ₂ is necessarily hydrogen.

R ₁ and R ₂ i.e. iodo phenyl tosylate, iodo toluene tosylate, iodo 4-anisole tosylate, iodo 2-thiopine tosylate, iodo phenyl bromide, iodo toluene bromide, iodo 4-anisole bromide, iodo 2-thiopine bromide, iodo phenyl triflate, iodo toluene triflate, iodo 4-anisole triflate, iodo 2-thiopine triflate and pinacol boronic ester The leaving group selected from the group consisting of is substituted on any one of the benzene rings of the pyrazole derivative, so that fluorine can be directly substituted on the benzene ring of the pyrazole derivative with an iodine or boron as the center, thereby reducing the electron density to control the reactivity. While playing a role, it is possible to increase the yield and selectivity.

In addition, the pyrazole precursor may be synthesized through the same process as in Scheme 1 below.

[Scheme 1]

Specifically describing the synthesis process of the pyrazole precursor, first, the step of preparing compound 2 by esterifying compound 1 and acyl chloride in a methanol solvent (precursor synthesis step 1); Reacting compound 2 with a 2-phenyl ethyl bromide pyrazole derivative with a base in a dimethylformamide solvent to prepare compound 3 (precursor synthesis step 2); Preparing compound 4 through a reduction reaction of compound 3 using hydrogen and a palladium catalyst in dichloromethane or methanol solvent (precursor synthesis step 3); Reacting compound 4 by adding triethyl amine and bromoacetyl bromide in a tetrahydrofuran solvent to prepare compound 5 (precursor synthesis step 4); Reacting compound 5 by adding a thiophenol pyrazole derivative and triethyl amine in a tetrahydrofuran solvent to prepare compound 6 (precursor synthesis step 5); Compound 6 is subjected to a metal transfer reaction using hexamethylditine and a palladium catalyst in a dioxane solvent, and then a diacetoxyiodo arene pyrazole derivative and 4-tosyl acid are added to react or compound 6 is used as a palladium catalyst. And reacting with bis(pinacolato)diborone to prepare compound 7 (precursor synthesis step 6).

In the above scheme, the compound of Formula 7 is a kind of precursor pyrazole derivative for the preparation of a pyrazole derivative into which [ ¹⁸ F] fluorine of Formula 1 is introduced, wherein A is a halogen element or H, where A is the corresponding ethyl It may be substituted at any one of 4, 3 and 2 of the benzene ring, wherein B is a halogen element or H, where B may be substituted at any one position of 4, 3, and 2 of the thiobenzene ring. There is; When A is H, B is a halogen element, when B is H, Y is a halogen element, and the halogen element is one of Cl, Br or I.

In addition, a specific example of the pyrazole precursor is 1) (4-((2-((3-(methoxycarbonyl)-1-phenethyl-1 H -pyrazol-4-yl)amino)-2- Oxoethyl)thio)phenyl(p-tolyl)iodonium tosylate; 2) (3-((2-((3-(methoxycarbonyl)-1-phenethyl- 1H -pyrazole-4) -Yl)amino)-2-oxoethyl)thio)phenyl (p-tolyl)iodonium tosylate; 3) (2-((2-((3-(methoxycarbonyl)-1-phenethyl)) -1 H -pyrazol-4-yl)amino)-2-oxoethyl)thio)phenyl(p-tolyl)iodonium tosylate; 4) (4-(2-(3-(methoxycarbonyl) )-4-(2-phenylthio)acetamido)-1 H -pyrazol-1-yl)ethyl)phenyl)(p-tolyl)iodonium tosylate; 5) (3-(2-(3-(methoxycarbonyl)-4-(2-phenylthio)acetamido)-1 H -pyrazol-1-yl)ethyl)phenyl)(p-tolyl ) Iodonium tosylate; 6) (2-(2-(3-(methoxycarbonyl)-4-(2-phenylthio)acetamido)-1 H -pyrazol-1-yl)ethyl)phenyl)(p-tolyl ) Iodonium tosylate; 7) 1-phenethyl-4-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)phenyl)thio)acetami Figure)-1 H -pyrazole-3-carboxylate methyl ester; 8) 1-phenethyl-4-(2-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)phenyl)thio)acetami Figure)-1 H -pyrazole-3-carboxylate methyl ester; 9) 1-phenethyl-4-(2-((2-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)phenyl)thio)acetami Figure)-1 H -pyrazole-3-carboxylate methyl ester; 10) 4-(2-(phenylthio)acetamido)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phen Ethyl)-1 H -pyrazole-3-carboxylate methyl ester; 11) 4-(2-(phenylthio)acetamido)-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phen Ethyl)-1 H -pyrazole-3-carboxylate methyl ester; 12) 4-(2-(phenylthio)acetamido)-1-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phen Ethyl)-1 H -pyrazole-3-carboxylate may be selected from the group consisting of methyl ester.

Meanwhile, the pyrazole derivative may be a pyrazole derivative represented by Formula 1 below.

[Formula 1]

Here, X is ¹⁸ F or H, wherein X may be substituted at any one position of 4, 3, and 2 of the thiobenzene ring;

Each of Y is H or ¹⁸ F, wherein Y may be substituted on any one of 4, 3 and 2 of the corresponding ethyl benzene ring;

When X is H, Y is ¹⁸ F, and when X is ¹⁸ F, Y is H;

Z is hydrogen, methyl, ethyl or propyl.

In addition, the pyrazole derivative is synthesized through the synthesis process as shown in Scheme 2 below, that is, by reacting with Formula 2 using [ ¹⁸ F] fluorine and a phase transfer catalyst and a base additive to introduce fluorine-18 into the pyrazole derivative. Can be.

[Scheme 2]

Here, a is a kind of pyrazole precursor of Formula 2;

X and Y are as defined above; R ₁ and R ₂ are each H or iodo phenyl tosylate, iodo toluene tosylate, iodo 4-anisole tosylate, iodo 2-thiopine tosylate, iodo phenyl bromide, iodo toluene bromide, iodo Figure 4-anisole bromide, iodo 2-thiopine bromide, iodo phenyl triflate, iodo toluene triflate, iodo 4-anisole triflate, iodo 2-thiopine triflate and pinacol boronic ester It may be a compound selected from the group consisting of; Either of R ₁ and R ₂ is necessarily hydrogen.

In addition, the pyrazole derivative of Formula 1 may be synthesized as a pyrazole derivative of another embodiment through a synthesis process as shown in Scheme 3 below. That is, compound c (pyrazole derivative) is hydrolyzed at 25°C to 60°C for 5 to 15 minutes in a 2N sodium hydroxide aqueous solution to prepare compound d (pyrazole derivatives in other examples). Can be.

[Scheme 3]

Here, X and Y are as defined above.

In addition, specific examples of the pyrazole derivatives include 1) methyl 4-(2-((4-[ ¹⁸ F]fluorophenyl)thio)acetamido)-1-phenylethyl-1 H -pyrazole-3 -Carboxylate; 2) 4-(2-((4-[ ¹⁸ F]fluorophenyl)thio)acetamido)-1-phenylethyl-1 H -pyrazole-3-carboxylic acid; 3) methyl 4-(2-((3-[ ¹⁸ F]fluorophenyl)thio)acetamido)-1-phenylethyl-1 H -pyrazole-3-carboxylate; 4) 4-(2-((3-[ ¹⁸ F]fluorophenyl)thio)acetamido)-1-phenylethyl-1 H -pyrazole-3-carboxylic acid; 5) methyl 4-(2-((2-[ ¹⁸ F]fluorophenyl)thio)acetamido)-1-phenylethyl-1 H -pyrazole-3-carboxylate; 6) 4-(2-((2-[ ¹⁸ F]fluorophenyl)thio)acetamido)-1-phenylethyl-1 H -pyrazole-3-carboxylic acid; 7) Methyl 1-(4-[ ¹⁸ F]fluorophenylethyl)-4-(2-(phenylthio)acetamido-1 H -pyrazole-3-carboxylate; 8) 1-(4- [ ¹⁸ F]fluorophenylethyl)-4-(2-(phenylthio)acetamido-1 H -pyrazole-3-carboxylic acid; 9) methyl 1-(3-[ ¹⁸ F]fluoro Phenylethyl)-4-(2-(phenylthio)acetamido-1 H -pyrazole-3-carboxylate; 10) 1-(3-[ ¹⁸ F]fluorophenylethyl)-4-(2 -(Phenylthio)acetamido-1 H -pyrazole-3-carboxylic acid; 11) methyl 1-(2-[ ¹⁸ F]fluorophenylethyl)-4-(2-(phenylthio) Acetamido-1 H -pyrazole-3-carboxylate; 12) 1-(2-[ ¹⁸ F]fluorophenylethyl)-4-(2-(phenylthio)acetamido-1 H -pyra It may be selected from the group consisting of sol-3-carboxylic acid.

The present invention also provides an imaging agent for diagnosing dementia diseases related to alpha-synuclein overexpression prepared by the method for preparing radiopharmaceuticals of pyrazole derivatives substituted with fluorine-18. Since these imaging agents for diagnosing dementia diseases related to overexpression of alpha-synuclein form a bond with alpha-synuclein in the body and act as a diagnostic radiopharmaceutical necessary for proton tomography (PET), positrons released after binding to alpha-synuclein oligomers are alpha-synuclein. By detecting the two gamma-ray energies generated by encountering and dissipating adjacent electrons in the dense region with a detector of a proton tomography machine, it is possible to directly image whether alpha-synuclein oligomers are generated.

In addition, the imaging agent for diagnosing dementia disease related to alpha-synuclein overexpression can be variously modified through known organic chemistry techniques for the substituent of Z of a pyrazole derivative substituted with [ ¹⁸ F] fluorine, and if necessary-a pyrazole derivative Brain intake and excretion and binding capacity can be controlled.

The present invention also provides a method for diagnosing dementia diseases related to alpha-synuclein overexpression using a pyrazole derivative radiopharmaceutical prepared by the fluorine-18-substituted pyrazole derivative radiopharmaceutical manufacturing method. That is, the pyrazole derivatives substituted with fluorine-18 may be very useful for early diagnosis of the presence or absence of dementia by administering to a mammal, more preferably a human.

Hereinafter, a method for producing a pyrazole derivative radiopharmaceutical substituted with fluorine-18 according to the present invention will be described in detail through examples.

Preparation Example: Preparation of a pyrazole derivative of Formula 2

<Preparation Example 1> (4-((2-((3-(methoxycarbonyl)-1-phenethyl-1H-pyrazol-4-yl)amino)-2-oxoethyl)thio)phenyl( Preparation of p-tolyl)iodonium tosylate

(Step 1) Preparation of methyl 1-phenethyl-4-(2-((4-(trimethylstenyl)phenyl)thio)acetamido)-1H-pyrazole-3-carboxylate

Methyl 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylate (0.948 g, 2.00 mmol) was added to dioxane (dioxane, 15 ml), hexamethylditin (0.86 mL, 4.16 mmol) and tetrakis (triphenylphosphine) palladium (0.34 g, 0.30 mmol) were added and stirred at 120 °C for 4 hours in an argon atmosphere in a pressure tube. Was done. The reaction product was cooled to room temperature, filtered through Celite, and extracted by adding water and ethyl acetate. The extracted organic solvent was removed by negative pressure after treatment with sodium sulfate. After removing the solvent, the remaining residue was purified by column chromatography to obtain the target compound.

¹ H NMR (400 MHz, CDCl ₃ ) δ 0.26 (s, 9H), 3.14-3.20 (m, 2H), 3.76 (s, 2H), 3.98 (s, 3H), 4.30-4.39 (m, 2H), 7.13 (d, J = 6.8 Hz, 2H, Ar), 7.20-7.31 ( m, 3H, Ar), 7.32-7.45 (m, 4H, Ar), 8.12 (s, 1H), 10.26 (s, 1H)

(Step 2) (4-((2-((3-(methoxycarbonyl)-1-phenethyl-1 H -pyrazol-4-yl)amino)-2-oxoethyl)thio)phenyl( Preparation of p-tolyl)iodonium tosylate

After dissolving 4-(diacetoxy)iodotoluene (470.0 mg, 1.4 mmol) in acetonitrile (8 mL) and chloroform (30 mL), p-toluene sulfonic acid (266.4 mg, 1.4 mmol) was added. It was stirred at room temperature for 5 minutes. Next, the product of step 1 (781 mg, 1.4 mmol) dissolved in chloroform (5 mL) was added to the reaction solution, followed by stirring at 50° C. for 18 hours. After cooling the reaction product to room temperature, the organic solvent was removed under negative pressure, the resultant was dissolved in a small amount of dichloromethane, and then added to a conical tube containing cold diethyl ether (10 mL) to separate the resulting solid to obtain the resultant product. .

¹ H NMR (400 MHz, CDCl ₃ ) δ 2.30 (s, 3H), 2.33 (s, 3H), 3.13-3.29 (m, 2H), 3.74 (s, 2H), 3.93 (s, 3H), 4.29-4.38 (m, 2H), 7.00 (d, J = 8.0 Hz, 2H, Ar), 7.08-7.15 ( m, 4H, Ar), 7.20-7.31 (m, 5H, Ar), 7.45 (d, J = 8.4 Hz, 2H, Ar), 7.75-7.81 (m, 4H, Ar), 8.08 (s, 1H), 10.23 (s, 1H)

<Production Example 2> 1-phenethyl-4-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)phenyl)thio ) Preparation of acetamido)-1 H -pyrazole-3-carboxylate methyl ester

Dimethyl containing methyl 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylate (237 mg, 0.5 mmol) [1,1'-bis(diphenylphosphino)ferro-cene]palladium(II) dichloride (82 mg, 0.1 mmol) and potassium acetate (141 mg, 1.4 mmol) in formamide (DMF, 10 mL), After adding bis(pinacolato)diboron (134 mg, 0.53 mmol), the mixture was stirred at 80 °C for 4 hours. After cooling to room temperature, a Celite filter was performed, and water and dichloromethane were added for extraction. As for the extracted organic solvent, sodium sulfate was added, the solid was removed, and the remaining solvent was removed under negative pressure. The residue was purified by column chromatography to obtain the title compound.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.31 (s, 12H), 3.13-3.20 (m, 2H), 3.80 (s, 2H), 3.95 (s, 3H), 4.30-4.39 (m, 2H), 7.13 (d, J = 6.7 Hz, 2H, Ar), 7.20-7.38 ( m, 6H, Ar), 7.71 (d, J = 6.8 Hz, 2H, Ar), 8.10 (s, 1H), 10.19 (s, 1H)

<Example 1> Preparation of methyl 4-(2-((4-[ ¹⁸ F]fluorophenyl)thio)acetamido)-1-phenylethyl-1 H -pyrazole-3-carboxylate 1

50-100 μL of water in which fluorine-18 produced in cyclotron is dissolved is added to acetonitrile in which CsHCO ₃ (1.0 mg) and 18-Crown-6 (5.0 mg) are dissolved, and then 90 Evaporate the solvent at °C. Pyrazole precursor (4 mg, (4-((2-((3-(methoxycarbonyl)-1-phenethyl-1 H -pyrazol-4-yl)amino)-2-oxoethyl)thio ) Phenyl (p-tolyl) iodonium tosylate) and 1 mg of 2,2,6,6-tetramethyl-1-piperidinyloxy were dissolved in 0.3 ml of acetonitrile, and the solution was fluorine- After adding to the reaction vessel containing 18, proceed for 10 minutes at 140° C. After the reaction, 10 mL of water was added, adsorbed to the tC18 Sep-Pak cartridge, washed with 10 mL of water, and eluted with 0.5 mL of ethanol. The resulting solution was subjected to an HPLC system including a UV detector of 254 nm and a radioactive isotope gamma ray detector (Waters, Xterra Semi-preparative C18 column, 10 x 250 mm, 10 μm; 50% acetonitrile-water, 254 nm, flow rate: 3.0 mL min) purified methyl 4-(2-((4-[ ¹⁸ F]fluorophenyl)thio)acetamido with a radiochemical yield of about 10-20% in 21 minutes )-1-phenylethyl-1 H -pyrazole-3-carboxylate can be obtained.

<Example 2> Preparation 2 of methyl 4-(2-((4-[ ¹⁸ F]fluorophenyl)thio)acetamido)-1-phenylethyl-1 H -pyrazole-3-carboxylate

CsHCO ₃ (0.1 mg) and 18-Crown-6 (0.2 mg) or K ₂ CO ₃ (0.1 mg) and K ₂₂₂ (0.2 mg) with 50-100 μL of water in which fluorine-18 produced in cyclotron is dissolved. ) Is added to the dissolved acetonitrile. Heat to 90℃ with nitrogen and evaporate all of the solvent, and then the pyrazole derivative precursor (3 mg, 1-phenethyl-4-(2-((4-(4,4,5,5-tetramethyl-1)) , 3,2- dioxa borane-2-yl) phenyl) between o) acetamido) -1 H - pyrazole-3-carboxylate methyl ester) and cooper (II) methanesulfonate trifluoroacetate ( Cu(OTf) ₂ ) (0.2 mg) or tetrakis (pyridine) Cooper (II) triflate (Cu(py) ₄ (OTf) ₂ ) (0.4 mg) was added to the reaction vessel with 0.5 ml of dimethylformamide The reaction proceeds at 110° C. for 10 minutes. After the reaction, 10 mL of water was added, adsorbed to the tC18 Sep-Pak cartridge, washed with 10 mL of water, and eluted with 0.5 mL of ethanol. The eluted solution was treated with a 254 nm UV detector and a radioactive isotope gamma ray detector in an HPLC system (Waters, Xterra Semi-preparative C18 column, 10 x 250 mm, 10 μm; 50% acetonitrile-water, 254 nm, flow rate: 3.0 mL min) and fluorine-18-labeled methyl 4-(2-((4-[ ¹⁸ F]fluorophenyl)thio) having a radiochemical yield of about 10-15-30% in 21 minutes Acetamido)-1-phenylethyl-1 H -pyrazole-3-carboxylate can be obtained.

In the case of Examples 1 and 2, as a compound labeled with fluorine-18, the retention time and purity in high-performance liquid chromatography were confirmed as shown in FIG. 3, and the fluorine-18 labeled compound obtained during the implementation of FIG. 3 was As a result of comparing the retention times in high-performance liquid chromatography with the stable isotope fluorine-19 compound as shown in FIG. 4, the retention times were accurately matched, so that fluorine-18 was successfully used in Examples 1 and 2 It proved to be substituted.

<Experimental Example 1> Protein binding ability test

A. Recombinant Human FAF1 protein (H00011124-Q01, Novus biologicals) or B. Recombinant Human alpha-synuclein protein (ab51189, abcam) or C. Human FAF1 and alpha-synuclein protein conjugate protein [ ¹⁸ F]SNUBH-NM-026 (Example 1), 20 μL of physiological saline solution containing 5% ethanol in which -027 (Example 2) was dissolved was added, followed by reaction at room temperature with continuous shaking (1500 rpm) for 1 hour. The mixture obtained after the reaction was transferred to a centrifugal filter unit, centrifuged at 13,000 rpm, and the filtered mixture was measured with a gamma-counter, and shown in FIG. 6.

<Experimental Example 2> Evaluation of distribution in normal mice

[ ¹⁸ F]SNUBH-NM-026, -027 obtained in the present invention were evaluated for intake and excretion of major organs in mice over time using 6-week-old ICR mice. The obtained [ ¹⁸ F]SNUBH-NM-026 (Example 1), -027 (Example 2) was injected into the tail vein of 200 μL (300 μCi) of physiological saline containing 5% ethanol dissolved therein. I did. After the injection, a 90-minute dynamic PET image was acquired using Animal PET/CT equipment, and the obtained radioactivity level for each major organ was expressed as %ID/g.

<Experimental Example 3> Alpha-synuclein modified gene insertion mouse brain distribution evaluation

C57BL/6 A53T transgenic mice were anesthetized with oxygen containing 2% isoflurane, and then obtained in the present invention after CT images [ ¹⁸ F]SNUBH-NM-026 (Example 1), -027 (Example 2) ) Was injected into the tail vein with 200 μL (300 μCi) of physiological saline containing 5% ethanol dissolved in each animal, and a dynamic PET image for 90 minutes was obtained through PET equipment.

As described above, although the present invention has been described by limited embodiments and drawings, the present invention is not limited to the above embodiments, and various modifications and variations from these descriptions for those of ordinary skill in the field to which the present invention pertains. This is possible.

Therefore, the scope of the present invention is limited to the described embodiments and should not be defined, but should be defined by the claims to be described later as well as equivalents to the claims.

Claims (9)

First by blowing to remove water with nitrogen in the ^[18 F] phase change to fluorine catalyst and a base additive temperature 80 ℃ to 100 ℃ be added to the reaction vessel together with a solvent, the ^[18 F] fluorine from the water is removed The first step of obtaining;
A reaction vessel in which the moisture-removed [ ¹⁸ F] fluorine is dissolved in a pyrazole precursor, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) or a copper-based catalyst in a second solvent A second step of preparing a pyrazole derivative solution by heating at a temperature of 100° C. to 140° C. after being added to; And
A third step of cooling the pyrazole derivative solution to room temperature and performing liquid chromatography (HPLC) to separate and purify the pyrazole derivative;
Including,
The pyrazole precursor is 1) (4-((2-((3-(methoxycarbonyl)-1-phenethyl-1H-pyrazol-4-yl)amino)-2-oxoethyl)thio) Phenyl(p-tolyl)iodonium tosylate; 2) (3-((2-((3-(methoxycarbonyl)-1-phenethyl-1H-pyrazol-4-yl)amino)-2) -Oxoethyl)thio)phenyl(p-tolyl)iodonium tosylate; 3) (2-((2-((3-(methoxycarbonyl)-1-phenethyl-1H-pyrazole-4)) -Yl)amino)-2-oxoethyl)thio)phenyl (p-tolyl)iodonium tosylate; 4) (4-(2-(3-(methoxycarbonyl)-4-(2-phenyl) Thio)acetamido)-1H-pyrazol-1-yl)ethyl)phenyl)(p-tolyl)iodonium tosylate; 5) (3-(2-(3-(methoxycarbonyl)-4-(2-phenylthio)acetamido)-1H-pyrazol-1-yl)ethyl)phenyl)(p-tolyl) Iodonium tosylate; 6) (2-(2-(3-(methoxycarbonyl)-4-(2-phenylthio)acetamido)-1H-pyrazol-1-yl)ethyl)phenyl)(p-tolyl) Iodonium tosylate; 7) 1-phenethyl-4-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)phenyl)thio)acetami Figure)-1H-pyrazole-3-carboxylate methyl ester; 8) 1-phenethyl-4-(2-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)phenyl)thio)acetami Figure)-1H-pyrazole-3-carboxylate methyl ester; 9) 1-phenethyl-4-(2-((2-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)phenyl)thio)acetami Figure)-1H-pyrazole-3-carboxylate methyl ester; 10) 4-(2-(phenylthio)acetamido)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phen Ethyl)-1H-pyrazole-3-carboxylate methyl ester; 11) 4-(2-(phenylthio)acetamido)-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phen Ethyl)-1H-pyrazole-3-carboxylate methyl ester; 12) 4-(2-(phenylthio)acetamido)-1-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phen Ethyl)-1H-pyrazole-3-carboxylate methyl ester is selected from the group consisting of,
The pyrazole derivatives include 1) methyl 4-(2-((4-[18F]fluorophenyl)thio)acetamido)-1-phenylethyl-1H-pyrazole-3-carboxylate; 2) 4-(2-((4-[18F]fluorophenyl)thio)acetamido)-1-phenylethyl-1H-pyrazole-3-carboxylic acid; 3) Methyl 4-(2-((3-[18F]fluorophenyl)thio)acetamido)-1-phenylethyl-1H-pyrazole-3-carboxylate; 4) 4-(2-((3-[18F]fluorophenyl)thio)acetamido)-1-phenylethyl-1H-pyrazole-3-carboxylic acid; 5) Methyl 4-(2-((2-[18F]fluorophenyl)thio)acetamido)-1-phenylethyl-1H-pyrazole-3-carboxylate; 6) 4-(2-((2-[18F]fluorophenyl)thio)acetamido)-1-phenylethyl-1H-pyrazole-3-carboxylic acid; 7) Methyl 1-(4-[18F]fluorophenylethyl)-4-(2-(phenylthio)acetamido-1H-pyrazole-3-carboxylate; 8) 1-(4-[18F ]Fluorophenylethyl)-4-(2-(phenylthio)acetamido-1H-pyrazole-3-carboxylic acid; 9) methyl 1-(3-[18F]fluorophenylethyl)-4 -(2-(phenylthio)acetamido-1H-pyrazole-3-carboxylate; 10) 1-(3-[18F]fluorophenylethyl)-4-(2-(phenylthio)acetate Amido-1H-pyrazole-3-carboxylic acid; 11) methyl 1-(2-[18F]fluorophenylethyl)-4-(2-(phenylthio)acetamido-1H-pyrazole- 3-carboxylate; 12) 1-(2-[18F]fluorophenylethyl)-4-(2-(phenylthio)acetamido-1H-pyrazole-3-carboxylic acid selected from the group consisting of Fluorine-18 substituted pyrazole derivative radiopharmaceutical manufacturing method.
The method of claim 1,
The phase transfer catalyst is Kryptofix 2.2.2 or 18-Crown-6 fluorine-18 substituted pyrazole derivative radiopharmaceutical manufacturing method.
The method of claim 1,
The base additive is selected from the group consisting of CsHCO ₃ , Cs ₂ CO ₃ , KHCO ₃ , K ₂ CO ₃ and tetrabutylammonium salts. Fluorine-18-substituted pyrazole derivatives radiopharmaceutical manufacturing method.
The method of claim 1,
The copper-based catalyst is Cu(OTf) ₂ , Cu(py) ₄ (OTf) ₂ , Cu(NCCH ₃ ) ₄ , Cu(MeCN) ₄ BF ₄ , Cu(NCCH ₃ ) ₄ ·CF ₃ SO ₃ , [( CH ₃ CN) ₄ Cu]PF ₆ , and (CF ₃ SO ₃ Cu) ₂ ·C ₆ H _{6 A} method for producing a pyrazole derivative radiopharmaceutical substituted with fluorine-18 that is selected from the group consisting of.
The method of claim 1,
The first solvent is methyl alcohol or acetonitrile, the second solvent is acetonitrile or dimethylformaldehyde fluorine-18 substituted pyrazole derivative radiopharmaceutical manufacturing method.
delete delete An imaging agent for diagnosing dementia diseases related to overexpression of alpha-synuclein prepared by the method of manufacturing a pyrazole derivative radiopharmaceutical in which any one of claims 1 to 5 is substituted.
A method for diagnosing dementia disease related to alpha-synuclein overexpression using a pyrazole derivative radiopharmaceutical prepared by the method of manufacturing a pyrazole derivative radiopharmaceutical having fluorine-18 substituted according to any one of claims 1 to 5.

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