CN114805109B - Efficient preparation method of fluoro [18F ] sand fenamide and PET imaging agent application - Google Patents

Abstract

The invention discloses an efficient preparation method of fluoro[18F]safinamide and a PET imaging agent application. The method comprises the following steps: firstly designing and preparing a precursor of fluoro[18F]safinamide, and then directly radioactively fluorinating an aromatic ring in the precursor through a nucleophilic substitution reaction to obtain fluoro[18F]safinamide; the precursor is Mel-SF, SPIAd-SF, SPI5-SF, Me ₃ Sn-SF, or Bpin-SF; the prepared fluoro[18F]safinamide can be used as a PET imaging agent for imaging a cell line capable of secreting monoamine oxidase B, and can also be used for imaging of living animals, can penetrate the blood-brain barrier and realize brain region imaging, can reversibly bind to multiple targets in vivo, and can also be used for animal ex vivo tissue imaging, and has potential application prospects in the medical fields of early medical diagnosis, auxiliary treatment, pathogenesis mechanism research, and the like.

Description

High-efficiency preparation method of fluoro[18F]safinamide and its application as PET imaging agent

Technical Field

The present invention relates to the development of radioactive drugs and belongs to the field of nuclear medicine technology. In particular, it relates to an efficient preparation method of fluoro[18F]safinamide and its application as a PET probe with the ability to target monoamine oxidase B. The present invention has potential application prospects in the medical fields such as early medical diagnosis, auxiliary treatment, and pathogenesis mechanism research.

Background technique

Safinamide is an adjuvant drug currently approved for the treatment of Parkinson's disease. It can reduce the degradation of levodopa in the body and exogenously injected levodopa by inhibiting the activity of monoamine oxidase B (MAO B), thereby maintaining levodopa in the body at a high level and achieving the effect of treating Parkinson's disease. However, as the research progresses, safinamide also exhibits effects such as potassium and sodium ion channel inhibition and glutamate receptor secretion inhibition. These effects make safinamide have good therapeutic effects or show therapeutic potential in other neurological diseases besides Parkinson's disease, such as depression, Alzheimer's disease, Partington's disease, ischemic cerebral infarction, etc. Unfortunately, it took more than ten years for the application research of safinamide in the treatment of Parkinson's disease from the first clinical trial to being approved for widespread use. The main reason for limiting this progress is that the current research methods for safinamide are mainly focused on anatomical plasma methods or traumatic in vivo experiments. These experiments have long cycles, high consumption, and it is difficult to know the true metabolism, distribution, and effects of safinamide in the body. This means that even if safinamide shows a wider application prospect and greater application potential, it is difficult to be recognized and applied in other disease areas in the short term. Therefore, it is very important to conduct non-invasive and real-time in vivo research on safinamide, and then quickly obtain its therapeutic efficacy, explore the mechanism of different diseases, and shorten the research cycle.

Positron Emission Tomography (PET) is currently the only new imaging technology that can display biological molecular metabolism, receptor and neurotransmitter activities in vivo. It is now widely used in the diagnosis and differential diagnosis of various diseases, efficacy evaluation, organ function research and new drug development. PET imaging technology has high sensitivity, high specificity and strong penetration, and it can image the whole body. A one-time whole-body imaging examination can obtain images of all areas of the body. PET is an imaging that reflects molecular metabolism. When the disease is in the early stage of molecular level changes, the morphological structure of the lesion area has not yet shown abnormalities, and MRI and CT examinations cannot make a clear diagnosis, PET examinations can find the location of the lesion, obtain three-dimensional images, and perform quantitative analysis to achieve early diagnosis, which is unmatched by other imaging examinations.

PET probes, also known as PET contrast agents, refer to radioactive drugs that can be introduced into the body to perform organ, tissue or molecular imaging. After the radioactive drug is introduced into the body, it can be concentrated in the target organ or tissue. The radiation emitted by it is detected by the imaging instrument to obtain an image of the drug's distribution in the body, which is used to diagnose a variety of diseases. Among PET probes, glucose is mainly used in clinical research, such as 18-fluoro-deoxyglucose ([ ¹⁸ F]FDG), which is the most common PET contrast agent and has been studied in depth in tumor diagnosis and treatment.

Therefore, the use of 18F-labeled safinamide (fluorine [18F] safinamide) as a PET imaging agent can provide a non-invasive, rapid, sensitive, and real-time in vivo research method for safinamide research. Since the half-life of the radioactive nuclides used in PET imaging agents is relatively short (the half-life of 18F nuclides is 109.8 minutes), in order to meet the experimental needs, the radioactive synthesis yield of PET imaging agents is generally required to reach more than 10%, providing sufficient radioactive doses on the one hand, and providing sufficiently high radioactive concentrations on the other hand, so as to extend the use time range of PET imaging agents and the number of experiments that can be arranged.

By adopting the radiolabeling strategy of direct radioactive nucleophilic substitution of the aromatic ring, the present invention has determined five efficient synthetic routes for ¹⁸ F-labeled safinamide, all of which can achieve a radioactive synthesis yield of more than 10%. And through biological experiments at the cell level, living animal level, and anatomical level, the high affinity of the probe for monoamine oxidase B, the distribution of the probe in the whole body of living animals, especially in the brain, and the uptake kinetics of the probe were explored, indicating the application potential of the probe in neurological diseases such as Alzheimer's disease, Partington's disease, depression, ischemic cerebral infarction, etc.

Summary of the invention

The object of the present invention is to provide a method for preparing 18F-fluoro[18F]safinamide and its application as a PET developer in view of the deficiencies of the prior art.

The technical solution adopted by the present invention is as follows:

Fluoro[18F]safinamide has the structure shown below:

The preparation method is as follows: firstly, a precursor of fluoro[18F]safinamide is designed and prepared, and then the aromatic ring in the precursor is directly radioactively fluorinated by a nucleophilic substitution reaction to obtain fluoro[18F]safinamide. The precursor is Mel-SF, SPIAd-SF, SPI5-SF, Me ₃ Sn-SF, or Bpin-SF; its general structural formula is:

The corresponding R are:

The preparation method of the precursor is as follows:

1) For the three types of precursors, Mel-SF, SPIAd-SF and SPI5-SF, they were prepared by the following method:

Compound 4A [tert-butyl (S)-(1-((tert-butyloxycarbonyl)amino)-1-oxopropane-2-yl)(4-((3-iodophenyl)oxy)benzyl)carbamate]] (1 equivalent) and m-chloroperbenzoic acid (1 to 2 equivalents) are stirred and dissolved in an anhydrous organic solvent (such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, etc.), and stirred at 40°C to 80°C for 1 to 6 hours. The reaction mixture is cooled to room temperature, potassium hydroxide or sodium hydroxide (5 to 10 equivalents) and 1 to 2 equivalents of McBride's acid, spiro[decane-2,2'-[1,3]dioxane]-4',6'-dione (SPI5), or spiro[adamantane-2,2'-[1,3]dioxane]-4',6'-dione (SPIAd) are added, and the reaction is stirred for 30 minutes to 12 hours. Then, the reactant is diluted with a high-polarity organic solution (such as dichloromethane, methanol, ethyl acetate, etc.), filtered and concentrated until solid precipitation occurs, at which time 4 to 20 volumes of a low-polarity organic solution (such as n-hexane, petroleum ether, etc.) are added to promote solid precipitation, and the mixture is allowed to stand at -30°C to 0°C for 2 to 24 hours until all solids are precipitated. The solid is obtained by filtration and drying, and the product is further purified by column chromatography. The synthetic route is (where KOH can also be NaOH):

2) The synthesis method of Me ₃ Sn-SF precursor is:

Compound 4B [tert-butyl (S)-(1-((tert-butyloxycarbonyl)amino)-1-oxopropane-2-yl)(4-((3-bromophenyl)oxy)benzyl)carbamate]] (1 equivalent) is dissolved in an anhydrous organic solvent (tetrahydrofuran, 1,4-dioxane, etc.), and inert gas (such as nitrogen, argon) is replaced, and hexamethylditin (1 to 8 equivalents) is added under the protection of inert gas and stirred, and then triphenylphosphine palladium dichloride (0.01 to 0.5 equivalents) is added for catalysis, and heated under reflux and stirred for 2 to 48 hours to fully react. After the reaction is completed, the mixture is cooled and dried to remove the solvent, and the crude product is purified by column chromatography.

3) The synthesis method of Bpin-SF precursor is:

Compound 4B (1 equivalent) is dissolved in an anhydrous organic solvent (tetrahydrofuran, 1,4-dioxane, etc.), replaced with an inert gas, and potassium acetate (1 to 5 equivalents) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (0.01 to 0.5 equivalents) are added in sequence under an inert gas atmosphere, mixed evenly, and 4,4,5,5-tetramethyl-1,3,2-dioxaborane (1 to 10 equivalents) are added, and refluxed and stirred for 0.5 to 72 hours. After the reaction is completed, the mixture is cooled and extracted, and the extracted organic phase is dried by adding a desiccant (such as anhydrous magnesium sulfate, etc.), filtered, concentrated, and finally purified by column chromatography.

The synthesis routes of Bpin-SF precursor and Me ₃ Sn-SF precursor are as follows:

In the above technical scheme, the preparation methods of compound 4A, i.e. [tert-butyl (S)-(1-((tert-butoxycarbonyl)amino)-1-oxopropane-2-yl)(4-((3-iodophenyl)oxy)benzyl)carbamate]] and compound 4B, i.e. [tert-butyl (S)-(1-((tert-butoxycarbonyl)amino)-1-oxopropane-2-yl)(4-((3-bromophenyl)oxy)benzyl)carbamate]], are as follows:

Dissolve compound 3A or 3B (1 equivalent) in an anhydrous organic solution (such as tetrahydrofuran, dichloromethane, etc.), slowly add sodium cyanide substance (2 to 4 equivalents) at low temperature, stir evenly, and then raise the temperature of the mixture to 20 to 30 degrees Celsius. Then slowly add di-tert-butyl dicarbonate (2 to 20 equivalents), and continue to stir the mixture to react. After the reaction is completed, cool to room temperature, extract and wash the organic solution and the aqueous solution, and transfer the crude product to the organic solution. Add a desiccant to the collected organic solution to remove water and filter and concentrate. Finally, purify the crude product by column chromatography. The structural formulas of compounds 3A and 3B are as follows:

The method for radioactive fluorine labeling of the precursor is as follows:

(1) The radioactive nuclide 18F is obtained by bombarding H ₂ ¹⁸ O with a cyclotron, and then purified by an ion exchange method in the presence of an alkali and a phase transfer catalyst, or directly transferred to a reaction bottle containing an alkali and a phase transfer catalyst, and water is removed at 80°C to 120°C (anhydrous acetonitrile may be added to remove water by azeotropic method).

(2) Then, the precursor is dissolved in an anhydrous polar aprotic solution (such as dimethyl sulfoxide, acetonitrile, etc.) (the precursor concentration is 0.5 mmol/L to 20 mmol/L) and added to the reactant obtained in (1), and reacted at 80°C-160°C for 5 minutes to 40 minutes. After the reaction is completed, it is diluted and purified by a semi-preparative high performance liquid chromatography system. For Me ₃ Sn-SF and Bpin precursors, copper catalysts (such as copper trifluoromethanesulfonate, Cu(OTf) ₂ (py) ₄ ) and pyridine are also required to be added for catalytic reaction.

The fluoro[18F]safinamide prepared by the invention can be used as a PET imaging agent for imaging a cell line capable of secreting monoamine oxidase B.

Fluoro[18F]safinamide can be used as a PET imaging agent for in vivo animal imaging. Furthermore, when performing in vivo animal imaging, the PET imaging agent can penetrate the blood-brain barrier and achieve brain imaging, and can bind to multiple targets in vivo, including monoamine oxidase B, potassium and sodium ion channel receptors, and glutamate receptors, and the binding is reversible. By adding 19-fluoro-safinamide, monoamine oxidase B inhibitors, and potassium and sodium ion channel inhibitors, the fluoro[18F]safinamide that has been bound to the target can be separated from the target under competitive action.

Fluoro[18F]safinamide can also be used as a PET imaging agent for in vitro tissue imaging of animals via radioautography.

Beneficial effects of the present invention:

① The present invention provides a route for the radioactive preparation of fluoro[18F]safinamide and corresponding reaction steps and parameters, which can obtain a radioactive yield of more than 10% to provide sufficient radioactive dose and radioactive concentration to meet various experimental requirements.

② The present invention provides five precursors and three synthetic routes for radioactive preparation of fluoro[18F]safinamide, which is helpful for the demanders of fluoro[18F]safinamide to select appropriate precursors and synthetic routes according to actual production conditions.

③ The present invention discloses for the first time the biological experimental results of flu[18F]safinamide at the cellular level, the living animal level, and the animal ex vivo tissue level, which can serve as an imaging research method for users to obtain multi-level information.

④ The fluoro[18F]safinamide disclosed in the present invention has been experimentally verified to retain many characteristics of 19-fluoro-safinamide, and can therefore be used as an imaging research method for 19-fluoro-safinamide.

⑤ The biological experimental results of the flu[18F]safinamide disclosed in the present invention show that it has the properties of binding to monoamine oxidase B, potassium and sodium ion channel receptors, penetrating the blood-brain barrier, and reversible binding in vivo. Combined with the current research status of some diseases, especially neurological diseases, it is pointed out that flu[18F]safinamide can be used as a PET imaging agent in drug screening, efficacy monitoring, early disease diagnosis, and pathogenesis mechanism research for diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, depression, and ischemic cerebral infarction.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings.

Figure 1 Radiolabeled precursor of fluoro[18F]safinamide

Figure 2 Synthesis process of radiolabeled precursors of fluoro[18F]safinamide (A) Mel-SF, SPIAd-SF, SPI5-SF; (B) Me ₃ Sn-SF&Bpin-SF.

Figure 3 1H-NMR spectra of each molecule in the synthesis process of radiolabeled precursor of fluoro[18F]safinamide (A) 4A, (B) 4B, (C) Mel-SF, (D) SPIAd-SF, (E) SPI5-SF, (F) Me ₃ Sn-SF, (G) Bpin-SF

Figure 4 Radiosynthesis process of fluoro[18F]safinamide based on different precursors (A) Mel-SF, SPIAd-SF, SPI5-SF; (B) Me ₃ Sn-SF; (C) Bpin-SF

Figure 5 Experimental condition optimization of radiolabeling process of fluoro[18F]safinamide based on Mel-SF, SPIAd-SF, and SPI5-SF (A) Base dosage and type screening, (B) Precursor type and dosage screening, (C) Reaction time and temperature screening, (D) Reaction solvent screening

Figure 6 Radioactive quality control of fluoro[18F]safinamide (A) Radiochemical purity and identity test (B) Injection stability test

Figure 7 Overview of the uptake of fluoro[18F]safinamide in astrocytomas U87 cells, breast cancer MCF-7 cells, and astrocytomas cells pretreated with safinamide

Figure 8 Systemic distribution of fluoro[18F]safinamide in Sprague Dawley rats (PET dynamic scan) Figure 9 (A) Distribution of fluoro[18F]safinamide in the brain of Sprague Dawley rats (PET dynamic scan), (B) and the corresponding activity/time curve

Figure 10 Distribution of fluoro[18F]safinamide in the brain of Sprague Dawley rats (A) (PET dynamic scan), and (B) the corresponding activity/time curves under displacement experiment

Figure 11 Distribution of 18F-fluorosafinamide in isolated tissues of C57 mice (radioautographic imaging)

Detailed ways

The technical solution of the present invention is further described in detail below through the accompanying drawings and specific embodiments. Unless otherwise specified, all reagents can be purchased.

The structure of fluoro[18F]safinamide is as follows:

The present invention designs and determines an efficient preparation method thereof, wherein the structure of the labeling precursor involved is specifically shown in FIG1 ;

The synthesis routes of various precursors are shown in FIG2 . In order to better illustrate the synthesis of various precursors, the present invention is further described below in conjunction with some specific examples.

① Preparation method of molecule 4A [tert-butyl (S)-(1-((tert-butyloxycarbonyl)amino)-1-oxopropane-2-yl)(4-((3-iodophenyl)oxy)benzyl)carbamate]] and molecule 4B [tert-butyl (S)-(1-((tert-butyloxycarbonyl)amino)-1-oxopropane-2-yl)(4-((3-bromophenyl)oxy)benzyl)carbamate]]:

The molecule 3A or 3B (1 equivalent) is dissolved in an anhydrous organic solution (such as tetrahydrofuran, dichloromethane, etc.), and sodium cyanide (2 to 4 equivalents) is slowly added at low temperature. After stirring, the mixture temperature is raised to 20 to 30 degrees Celsius. Then, di-tert-butyl dicarbonate (2 to 20 equivalents) is slowly added, and the mixture is continued to be stirred and reacted. After the reaction is completed, it is cooled to room temperature, and the crude product is transferred to the organic solution by extraction and washing of the organic solution and the aqueous solution. A desiccant is added to the collected organic solution to remove water and filter and concentrate. Finally, the crude product is purified by column chromatography.

Embodiment 1:

3A (615 mg, 1.5 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and then sodium hydride (60% dispersion in mineral oil, 132 mg, 3.3 mmol) was added dropwise to the solution at 0 ° C and stirred for 1 h. The reaction mixture was heated to 25 ° C, di-tert-butyl dicarbonate (1.63 g, 24 mmol) was slowly added, and the mixture was stirred for 2 hours. Finally, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic solution was then washed with water, 1M HCl and brine, and the organic portion was collected. The final organic solution was dried over Na2SO4, filtered and concentrated. The product was finally purified by silica gel column chromatography (5-50% ethyl acetate/petroleum ether) to obtain a colorless solid (4A, 170 mg, 0.28 mmol, 68% yield). ¹ H NMR (400 MHz, Chloroform-d) δ 7.67 (d, J = 1.8 Hz, 1H), 7.54 (dt, J = 7.8, 1.5 Hz, 1H), 7.26 (dd, J = 7.8, 1.5 Hz, 1H), 7.13-6.96 (m, 3H), 6.80 (dd, J = 8.3, 4.6 Hz, 2H), 4.86 (s, 2H), 4.69-3.70 (m, 3H), 1.33 (d, J = 4.1 Hz, 18H), 1.21 (d, J = 7.0 Hz, 3H) ppm.

Embodiment 2:

Similarly, the preparation of 4B only requires replacing the molar amount of 3A in Example 1 with 3B, and the other operations are the same, and finally a colorless oily substance 4B can be obtained. ¹ H NMR (400MHz, Chloroform-d) δ7.59 (t, J = 1.8 Hz, 1H), 7.46 (dt, J = 7.9, 1.6 Hz, 1H), 7.34 (dt, J = 7.8, 1.3 Hz, 1H), 7.25 (p, J = 4.8 Hz, 3H), 6.99-6.88 (m, 2H), 5.01 (s, 2H), 4.90-3.89 (m, 3H), 1.71 (d, J = 2.8 Hz, 2H), 1.46 (d, J = 4.3 Hz, 18H), 1.34 (d, J = 7.0 Hz, 3H), 1.31-1.22 (m, 1H).

The preparation of 3A/3B can be obtained based on the existing technology. One preparation method thereof is shown in FIG2 , and the starting compound is 1A/1B.

②Synthesis of precursors Mel-SF, SPIAd-SF, and SPI5-SF

Compound 4A (1 equivalent) and m-chloroperbenzoic acid (1 to 2 equivalents) are stirred and dissolved in an anhydrous organic solvent (such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, etc.), and stirred at 40°C to 80°C for 1 to 6 hours. The reaction mixture is cooled to room temperature, potassium hydroxide or sodium hydroxide (5 to 10 equivalents) and 1 to 2 equivalents of McLaughlin's acid/spiro[decane-2,2'-[1,3]dioxane]-4',6'-dione (SPI5)/spiro[adamantane-2,2'-[1,3]dioxane]-4',6'-dione (SPIAd) are added, and the reaction is stirred for 30 minutes to 12 hours. Then, the reactant is diluted with a high-polarity organic solution (such as dichloromethane, methanol, ethyl acetate, etc.), filtered, and the filtrate is concentrated until solid precipitation occurs. At this time, 4 to 20 volumes of a low-polarity organic solution (such as n-hexane, petroleum ether, etc.) are added to promote solid precipitation, and the mixture is allowed to stand at a low temperature of -30 to 0°C for 2 to 24 hours until all solids are precipitated. The solid is obtained by filtration and drying, and the product is further purified by column chromatography.

Embodiment 3:

In a closed reaction bottle, compound 4A (1 eq.) was stirred with 85% m-chloroperbenzoic acid (1.1 eq.) in dichloromethane (DCM) at 40 °C for 80 minutes. After cooling to room temperature, KOH (7 eq.) and Mel's acid (1.3 eq.) were added and further stirred for 45 minutes. The reaction mixture was then diluted with DCM and filtered through filter paper. The solvent of the collected organic phase was removed under reduced pressure at 35 °C until the first solid precipitated. Then, hexane was slowly added and the mixture was left to stand at -20 °C for 12 hours to complete the precipitation. The solid was collected by filtration, washed with n-hexane, and dried in air and vacuum. The crude product was finally purified by silica gel column chromatography (SiO2, 40-100% ethyl acetate/petroleum ether) to obtain Mel-SF precursor.

Mel-SF (41% yield, white solid): ¹ H NMR (400 MHz, Chloroform-d) δ 7.86–7.76 (m, 1H), 7.69–7.57 (m, 1H), 7.47–7.36 (m, 1H), 7.25–7.05 (m, 2H), 6.92 (t, J = 7.6 Hz, 2H), 5.03 (d, J = 37.9 Hz, 2H), 4.90–3.80 (m, 3H), 1.72 (s, 6H), 1.49–1.41 (m, 18H), 1.33 (dd, J = 7.1, 3.0 Hz, 3H) ppm.

The same method can be used to prepare SPIAd-SF precursor and SPI5-SF precursor

SPIAd-SF (33% yield, slightly yellow solid): ¹ H NMR (400 MHz, Chloroform-d) δ 7.83 (s, 1H), 7.75–7.69 (m, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.18–6.95 (m, 3H), 6.82 (d, J = 8.6 Hz, 2H), 5.00 (s, 2H), 4.79–3.92 (m, 3H), 2.36 (s, 2H), 2.09 (d, J = 12.6 Hz, 4H), 1.78 (s, 2H), 1.64 (s, 4H), 1.39 (d, J = 12.6 Hz, 18H), 1.26 (d, J = 7.0 Hz, 3H) ppm.

SPI5-SF (36% yield, yellow solid): ¹ H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 1.7 Hz, 1H), 7.68 (dt, J = 7.9, 1.5 Hz, 1H), 7.40 (dt, J = 7.8, 1.3 Hz, 1H), 7.14 (t, J = 7.8 Hz, 3H), 6.94 (d, J = 6.6 Hz, 2H), 5.01 (s, 2H), 4.95–3.78 (m, 3H), 2.40–2.21 (m, 2H), 2.18 (d, J = 7.5 Hz, 2H), 1.98 (td, J = 4.5, 2.2 Hz, 2H), 1.95–1.88 (m, 2H), 1.48 (d, J = 4.3 Hz, 18H), 1.35 (d, J = 7.1 Hz, 3H).

③ Synthesis of precursor Me ₃ Sn-SF

Compound 4B (1 equivalent) was dissolved in an anhydrous organic solvent (tetrahydrofuran, 1,4-dioxane, etc.), and hexamethyltin (1 to 8 equivalents) was added under the protection of an inert gas (such as nitrogen and argon) and stirred, and then triphenylphosphine palladium dichloride (0.01 to 0.5 equivalents) was added for catalysis, and heated under reflux and stirred for 2 to 48 hours to fully react. After the reaction was completed, the solvent was cooled and dried to remove, and the crude product was purified by column chromatography.

Embodiment 4:

Compound 4B (282 mg, 0.500 mmol) was added to a two-necked reaction flask, and 5 mL of 1,4-dioxane was added to dissolve the mixture, and the mixture was degassed and filled with nitrogen. Hexamethyltin (193 μL, 0.930 mmol) and triphenylphosphine palladium dichloride (3.9 mg) were added. The mixture was heated to 65°C and stirred for 12 hours. The mixture was rotary evaporated to dryness and purified by silica gel column chromatography (0-80% ethyl acetate/n-hexane, containing 1% volume of triethylamine) to obtain a yellow oily product (Me ₃ Sn-SF, 21% yield). ¹ H NMR (400 MHz, Chloroform-d) δ8.80 (s, 1H), 7.35–7.18 (m, 6H), 6.88–6.72 (m, 2H), 5.14–5.02 (m, 2H), 4.68 (q, J = 6.8 Hz, 1H), 4.35 (dt, J = 12.5, 1.0 Hz, 1H), 4.28 (dt, J = 12.4, 0.9 Hz, 1H), 1.46 (d, J = 2.8 Hz, 18H), 1.38 (d, J = 6.8 Hz, 3H), 0.78 (s, 9H).

④Synthesis of precursor Bpin-SF

Compound 4B (1 equivalent) is dissolved in an anhydrous organic solvent (tetrahydrofuran, 1,4-dioxane, etc.), replaced with an inert gas, and potassium acetate (1 to 5 equivalents) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (0.01 to 0.5 equivalents) are added in sequence under an inert gas atmosphere, mixed evenly, and 4,4,5,5-tetramethyl-1,3,2-dioxaborane (1 to 10 equivalents) are added, and refluxed and stirred for 0.5 to 72 hours. After the reaction is completed, the mixture is cooled and extracted, and the extracted organic phase is dried by adding a desiccant (such as anhydrous magnesium sulfate, etc.), filtered, concentrated, and finally purified by column chromatography.

Embodiment 5:

Under nitrogen atmosphere, compound 4B (4.8 g, 8.7 mmol), potassium acetate (2.55 g, 26 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (0.63 g, 0.87 mmol) and 4,4,5,5-tetramethyl-1,3,2-dioxaborane (4.39 g) were dissolved in 1,4-dioxane (40 ml), stirred at 80 ° C under nitrogen for 2 h, and then cooled to room temperature. Water (30 ml) was added, and the mixture was extracted with ethyl acetate (3×20 ml). The combined organic phases were concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (0-40% ethyl acetate/n-hexane) to obtain a transparent colloidal substance (62% yield). ¹ H NMR (300 MHz, Chloroform-d) δ7.59–7.47 (m, 2H), 7.40–7.21 (m, 4H), 6.89–6.80 (m, 2H), 5.19–5.03 (m, 2H), 4.68 (q, J = 6.8 Hz, 1H), 4.41–4.30 (m, 1H), 4.30–4.22 (m, 1H), 1.46 (d, J = 2.2 Hz, 18H), 1.41 (d, J = 6.8 Hz, 3H), 1.24 (d, J = 15.1 Hz, 12H).

⑤ Nuclear magnetic resonance hydrogen spectrum: The 1H-NMR spectrum of each molecule mentioned above is shown in Figure 3. Figure 3 1H-NMR spectrum of each molecule in the synthesis process of fluoro[18F]safinamide radiolabeled precursor (A) 4A, (B) 4B, (C) Mel-SF, (D) SPIAd-SF, (E) SPI5-SF, (F) Me ₃ Sn-SF, (G) Bpin-SF

According to the different precursors, different radioactive synthesis processes of fluoro[18F]safinamide were designed, as shown in Figure 4. The method is: cyclotron bombardment of H ₂ ¹⁸ O to obtain radioactive nuclide 18F, purification by ion exchange method (requiring the use of eluent containing alkali and phase transfer catalyst), or directly transferred to a reaction bottle containing alkali and phase transfer catalyst; at 80℃ to 120℃, remove water (anhydrous acetonitrile can be added during the volatilization process to remove water by azeotropic method). Then, anhydrous polar aprotic solution (such as dimethyl sulfoxide, acetonitrile, DMF, DMA, etc.) is used to dissolve the precursor (0.5mmol/L to 20mmol/L) and add it to the reaction bottle. For Me ₃ Sn-SF and Bpin precursors, copper catalysts (such as copper trifluoromethanesulfonate, Cu(OTf) ₂ (py) ₄ ) and pyridine are also needed to be added for catalytic reaction, and the reaction is carried out at 80℃-160℃ for 5 minutes to 40 minutes. After the reaction is completed, dilution is performed and purification is carried out by semi-preparative high performance liquid chromatography system.

Embodiment 6:

The radiosynthesis process based on Mel-SF, SPIAd-SF, and SPI5-SF includes:

The oxygen-rich water H ₂ ¹⁸ O was bombarded by a cyclotron to obtain ^18F ions, which were adsorbed by an anion exchange column (QMA). The ^18F ions were eluted from the QMA column with 1mL of an eluent containing 0.8mL acetonitrile, 0.2mL H ₂ O, and 10mg TEAB, and transferred to a reaction bottle. 1mL of anhydrous acetonitrile was used for azeotropic dehydration. The azeotropic step was repeated three times until the product was completely dried. 3mg of the labeled precursor compound Mel-SF, SPIAd-SF or SPI5-SF prepared in Example 1 was added, dissolved in 1mL of anhydrous acetonitrile, and heated at 120°C for 15min. 1mL of 6M hydrochloric acid aqueous solution was added to the obtained crude product solution, and the target compound fluoro[18F]safinamide was hydrolyzed at 110°C for 10min to obtain a crude product solution.

Embodiment 7:

The radiosynthesis process based on Me ₃ Sn-SF includes:

The oxygen-rich water H ₂ ¹⁸ O was bombarded by a cyclotron to obtain 18F ions, which were adsorbed by an anion exchange column (Chromafix 30-PS-HCO3). The column was rinsed and activated with ⁵ mL of ethanol, 5 mL of 90 mg/mL potassium trifluoromethanesulfonate solution, and 5 mL of deionized water before use. Then, 1 mL of eluent (0.45 mL of 10 mg/mL potassium trifluoromethanesulfonate, 0.05 mL of 1 mg/mL potassium carbonate aqueous solution, and 0.5 mL of anhydrous acetonitrile) was used to elute the adsorbed fluoride ions into the reaction bottle. 1 mL of anhydrous acetonitrile was used for azeotropic dehydration, and the azeotropic step was repeated three times until the drying was complete. 3 mg of the labeled precursor compound Me ₃ Sn-SF prepared in Example 1, 0.4 mL of anhydrous dimethylacetamide (DMA), 0.1 mL of a 1 M pyridine DMA solution, and 0.067 mL of a 0.2 M copper trifluoromethanesulfonate DMA solution were added, mixed thoroughly, and heated at 110° C. for 20 min. The mixture was then cooled to room temperature, and 1 mL of a 6 M hydrochloric acid aqueous solution was added. The mixture was hydrolyzed at 110° C. for 10 min to obtain a crude product solution of the target compound fluoro[18F]safinamide.

Embodiment 8:

The radiosynthesis process based on Bpin-SF includes:

The oxygen-rich water H ₂ ¹⁸ O was bombarded by a cyclotron to obtain ^18F ions, which were adsorbed by an anion exchange column (QMA). The ^18F ions were eluted from the QMA column with 1mL of an eluent containing 0.8mL acetonitrile, 0.2mL H ₂ O, and 10mg TEAB, and transferred to a reaction bottle. 1mL of anhydrous acetonitrile was used for azeotropic dehydration. The azeotropic step was repeated three times until it was completely dried. 1-3mg of the labeled precursor compound Bpin-SF prepared in Example 1, 0.15mL 1M Cu(OTf) ₂ (py) ₄ , and 1mL of anhydrous DMA solution were added, and the reaction was heated at 140°C for 20min. 1mL of 6M hydrochloric acid aqueous solution was added to the obtained crude product solution, and the target compound fluoro[18F]safinamide was hydrolyzed at 110°C for 10min to obtain a crude product solution.

The obtained crude product solution was subjected to semi-preparative liquid chromatography purification process:

2.5 mL of solution (30% acetonitrile, 70% water) was added to the crude product reaction solution obtained in Examples 6, 7, and 8 respectively for dilution, and then purified using a semi-preparative high performance liquid chromatography system (chromatographic column C18, 250 mm*10 mm, 4.6 um particle size), the chromatographic conditions were (5 mL/min, 30%-80% acetonitrile/0.1% TFA aqueous solution), the purified product was transferred to a sterile vial, diluted with 40 mL of deionized water, and adsorbed by a Waters Sep-Pak C18 chromatographic column. The product was then eluted from the reagent loading module using 0.5 mL of ethanol and 4.5 mL of saline. The final solution of fluoro[18F]safinamide was filtered through a sterile membrane filter (Millex LG, 0.20 um) and stored in another sterile vial as an injection for subsequent experiments.

The radiochemical yield (RCY) of the above-mentioned synthesis process can basically reach 10%; in addition, we have made a lot of optimizations on the synthesis conditions of the above-mentioned various radiochemical synthesis processes to maximize the radiochemical yield to better meet the dosage requirements of biological experiments. In particular, the radiochemical yield of the radiolabeling process of fluoro[18F]safinamide based on SPIAd-SF can be optimized to reach more than 25%, and its specific synthesis conditions are:

10 mg TEAB was used in the eluent; 15 mg SPIAd-SF was used as the precursor, DMSO was used as the polar aprotic solution, and the radioactive reaction conditions were 120°C for 15 min; the rest was the same as in Example 6. Under these conditions, the highest radioactive yield was 25.6±4.2% (decay correction) and the molar activity was 286.2±31.5 GBq/umol. All operations including radiosynthesis and purification took a total of 88±4 minutes. Correspondingly, when 7 mg TEAB was used as the eluent, 2 mg SPIAd-SF was used as the precursor, N,N-dimethylacetamide was used as the polar aprotic solution, and the radioactive reaction conditions were 120°C for 10 min, the radioactive yield was only 4%-9%.

When the precursor is Me ₃ Sn-SF, the optimized radiosynthesis process is as follows: 3 to 5 mg potassium trifluoromethanesulfonate and 0.05 mg potassium carbonate are used in the eluent, the amount of the precursor is 3 to 8 mg, the polar aprotic solution is DMA solution, the radioreaction temperature is 110 to 125°C, 10 to 30 min, and the other conditions are the same as those in Example 7. The radioactive yield can exceed 10%, and the molar activity is 166 GBq/umol. All operations including radiosynthesis and purification take 94 to 106 minutes.

The precursor was Bpin-SF, and the optimized radiosynthesis process was as follows: 6 mg TEAB, 10 mg precursor, 0.25 mL Cu(OTf) ₂ (py) ₄ solution were used as the eluent, the polar aprotic solution was DMA solution, the radioactive reaction was carried out at 155°C for 10 min, and the highest radioactive yield was 11-17% (n=5, decay correction), and the molar activity was 182 GBq/umol. All operations including radiosynthesis and purification took 101 minutes.

To ensure that the quality of the prepared flu[18F]safinamide injection met the experimental requirements, a high performance liquid chromatography system was used for quality control. The results are shown in FIG6 .

① By mixing the injection with non-radioactive safinamide (red peak line) and then co-injecting into HPLC analysis, it was confirmed that the retention time difference between the product fluoro[18F]safinamide (black peak line in A in Figure 6) and 19-fluoro-safinamide (red line in 6A) was within 0.1 minutes, and fluoro[18F]safinamide. At the same time, the radiochemical purity of fluoro[18F]safinamide purified in radio-HPLC exceeded 99%. It proves that fluoro[18F]safinamide was successfully prepared and the purity meets the requirements

② The newly prepared fluoro[18F]safinamide injection was stirred (550 rpm) at 37°C for 0, 30, 90, and 180 minutes. At each time point, 1/10 volume of the solution was taken out and diluted with 10% ethanol in saline solution. The radiochemical purity was analyzed on a high performance liquid chromatography system equipped with a radioactive NaI detector. It was finally demonstrated that fluoro[18F]safinamide injection had extremely high stability (radiochemical purity greater than 98%) throughout 180 minutes.

Embodiment 9:

Through cell experiments, it was explored that the prepared fluoro[18F]safinamide can image the cell lines that secrete monoamine oxidase B. In the experiment, astrocytoma cells U87 that can produce monoamine oxidase B and breast cancer cells MCF-7 that cannot produce monoamine oxidase B were used as comparisons. The experimental results are shown in Figure 7.

The uptake of fluoro[18F]safinamide by U87 astroglioma cells (uptake rate 2.01%), which can produce monoamine oxidase B, was approximately four times that of MCF-7 breast cancer cells (uptake rate 0.53%), which cannot produce monoamine oxidase B. This result indicates that fluoro[18F]safinamide has a high affinity for monoamine oxidase B, which is consistent with the characteristics of safinamide. At the same time, compared with U87 cells, the uptake rate of the radioisotope probe in U87 cells pretreated with 100ug/mL safinamide (uptake rate 0.59%) was significantly reduced, and showed a level similar to that of MCF-7 cells. This indicates that the binding site of fluoro[18F]safinamide is consistent with that of safinamide, and the binding capacity has an upper limit.

Embodiment 10:

We performed the first in vivo experiment of flu[18F]safinamide to obtain information on its distribution, aggregation, migration, metabolism, etc. in living organisms. The uptake of major organs is shown in Figure 8.

The first in vivo PET study of fluoro[18F]safinamide was conducted on Sprague-Dawley (SD) rats to examine the biodistribution of fluoro[18F]safinamide and to further understand the in vivo mechanism and metabolism information of fluoro[18F]safinamide. SD rats were injected with fluoro[18F]safinamide via the tail vein, followed by a 3-minute CT scan to position the rats, and then a 90-minute dynamic PET scan. The PET images obtained were fused with the matched CT images and are shown in Figure 8. We delineated the region of interest (ROI) and quantified these PET imaging data by standardized uptake value (SUV = radioactivity concentration in ROI/whole body radioactivity concentration). It is particularly worth mentioning that the bone uptake detected during the PET scan was negligible, and this region also showed the lowest SUV in the whole body of 0.0573, confirming that fluoro[18F]safinamide had no or little defluorination in vivo. We then used the SUV of the bone as a baseline and calculated the SUV ratio of the ROI and the bone (labeled as SUVROI/bone).

The highest uptake of flu[18F]safinamide was observed in the bladder (SUVROI/bone=11.99), which is consistent with the metabolic pathway of safinamide, as the elimination of safinamide is mainly carried out through bladder metabolism. Similarly, some flu[18F]safinamide can also be found in the small intestine area (SUVROI/bone=1.69)33. In addition, high uptake of flu[18F]safinamide was observed in the liver, kidney and pancreas, with corresponding SUVROI/bone values of 6.56, 6.43 and 5.00, respectively. A certain amount of flu[18F]safinamide can also be found in the lung (SUVROI/bone=2.35). The heart showed an uptake level similar to that of the bone (SUVROI/bone=1.15). According to literature reports, a large amount of monoamine oxidase B was found in the liver and pancreas in the peripheral tissues of rats; the renal cortex and ureters of the kidney and lung also produce some monoamine oxidase B, and small or negligible monoamine oxidase B can be observed in the heart, spleen and muscle. Therefore, these uptakes indicate that the distribution and abundance of flu[18F]safinamide in rat peripheral tissues are closely related to MAO B, suggesting that flu[18F]safinamide and MAO B have a high affinity in these areas.

Embodiment 11:

The information of monoamine oxidase B in the brain is the most difficult to obtain by conventional means, and it is also the area that can best highlight the advantages of PET in vivo detection. In addition, changes in monoamine oxidase B in the brain are closely related to many important neurological diseases. We explored the distribution of tracers in the brain and measured the time-activity curve (TAC) to disclose its kinetic characteristics in the brain.

As shown in Figure 9B, the level of radioactivity in the rat brain was relatively high in the early stage of the PET scan (before 10 minutes) and was then effectively cleared. The SUV then slowly decreased and remained stable after 30 minutes. This TAC means that fluoro[18F]safinamide entered the rat brain, but most of the fluoro[18F]safinamide was not well accumulated and was quickly cleared from the brain with metabolism; the remaining tracer accumulated in the brain, which is considered to reflect the actual binding site of fluoro[18F]safinamide in the rat brain.

Several representative slices of dynamic PET images are shown in Figure 9, A. According to the SUV of the rat brain ROI, the cerebellum gray matter had the lowest radioactivity. For a more convenient comparison of SUV, the SUV ratio between the ROI and the cerebellum was calculated, labeled as SUV _{ROI/cerebellum} . The results showed that high accumulation of fluoro[18F]safinamide (SUV _{ROI/cerebellum} exceeded 1.5) was found in the hippocampus, septum, thalamus, striatum, cingulate cortex, accumbens, amygdala, and medial prefrontal cortex. There was also some radiotracer in the midbrain, pons, hypothalamus, medulla, pituitary, and parietal cortex, peripheral frontal cortex, auditory cortex, insular cortex, and somatosensory cortex (SUV _{ROI/cerebellum} between 1.3 and 1.5).

In addition, the distribution and abundance pattern of flu[18F]safinamide in the rat brain is not single-targeted, and it has binding ability to multiple targets in the brain. On the one hand, a large amount of flu[18F]safinamide can be seen in areas rich in monoamine oxidase B, such as the hippocampus, amygdala, striatum, septum, accumbens, medial prefrontal cortex and thalamus. On the other hand, radioactive tracers were also observed in areas such as the cingulate cortex, orbitofrontal cortex, and auditory cortex. Monoamine oxidase B is relatively scarce in these areas, where a large number of potassium/sodium channel receptors and glutamate are distributed. The above distribution pattern of flu[18F]safinamide is consistent with the multi-target action mode of safinamide reported in recent studies. This is also the first time that this has been verified through non-invasive real-time monitoring in vivo.

Embodiment 12:

In the field of PET imaging agents, whether the binding of the tracer to the target is reversible is related to whether the tracer has a high signal-to-noise ratio, excellent image accuracy and a fast kinetic curve. To this end, we disclosed the reversible binding experiment (i.e., displacement experiment) of fluoro[18F]safinamide in in vivo experiments. The results are shown in Figure 10.

To further verify the reversibility of fluoro[18F]safinamide binding and the consistency of fluoro[18F]safinamide and safinamide associated with the binding site, a displacement experiment was performed in which SD rats were injected with a safinamide methanesulfonate solution as a competitor during PET scanning (in order to improve the solubility of safinamide). As shown in Figure 10B, after the injection of safinamide methanesulfonate, the whole brain SUV decreased by more than 50% and maintained a slightly decreasing trend in subsequent PET scans. This result indicates that the binding of fluoro[18F]safinamide in the rat brain is reversible and that the binding site of fluoro[18F]safinamide is highly consistent with the binding site of safinamide. This can be more intuitively demonstrated by Figure 10A, where the degree of probe aggregation in the high uptake area was significantly reduced and showed a continuous downward trend after the injection of safinamide methanesulfonate.

Embodiment 13:

In order to verify the accuracy of 18F-fluoro[18F]safinamide in in vivo PET detection, some experimental mice were subjected to in vitro dissection and autoradiographic scanning. The accuracy was demonstrated by comparing the PET experimental results with the autoradiographic results. The autoradiographic image is shown in Figure 11.

The results showed that the in vitro systemic distribution of flu[18F]safinamide in animal body weight was basically consistent with that obtained by PET, indicating that it is feasible to use flu[18F]safinamide to replace some in vitro experiments or other traditional laboratories, which is conducive to significantly reducing the consumption of manpower and material resources and accelerating the promotion of safinamide as a drug for the treatment of other diseases. It also proves the application value of flu[18F]safinamide itself in the fields of disease diagnosis, pathological research, and efficacy evaluation.

Claims (2)

1. The preparation method of the fluoro [18F ] salfenamide is characterized in that a precursor of the fluoro [18F ] salfenamide is firstly designed and prepared, and then the aromatic ring in the precursor is directly and radially fluorinated and labeled through nucleophilic substitution reaction to obtain the fluoro [18F ] salfenamide; the structure of the fluoro [18F ] sand-fenamide is as follows:

The precursor is Mel-SF, or SPIAd-SF; the structural general formula is as follows:

The corresponding R is respectively:

the preparation method comprises the following steps:

1 equivalent of compound 4A [ tert-butyl (S) - (1- ((tert-butoxycarbonyl) amino) -1-oxypropane-2-yl) (4- ((3-iodophenyl) oxy) benzyl) carbamate ] ] and 1 to 2 equivalents of m-chloroperoxybenzoic acid are stirred and dissolved by using an anhydrous organic solution, and stirred for 1 to 6 hours at a temperature of between 40 and 80 ℃; the reaction mixture is cooled to room temperature, 5 to 10 equivalents of potassium hydroxide or sodium hydroxide and 1 to 2 equivalents of McOtstool acid or spiro [ adamantane-2, 2' - [1,3] dioxane ] -4',6' -diketone (SPIAd) are added, and stirred for reaction for 30 minutes to 12 hours; then diluting the reactant by using a high-polarity organic solution, filtering, concentrating the filtrate until solid precipitation occurs, adding a low-polarity organic solution with the volume of 4-20 times to promote the solid precipitation, standing the mixture at the temperature of minus 30 ℃ to 0 ℃ for 2-24 hours until all the solid is precipitated, obtaining the solid after filtering and drying, and finally further purifying the product by column chromatography;

The anhydrous organic solution is tetrahydrofuran, dichloromethane or N, N-dimethylformamide; the high-polarity organic solution is dichloromethane, methanol or ethyl acetate; the low-polarity organic solution is n-hexane or petroleum ether;

the preparation method of the compound 4A comprises the following steps:

Dissolving 1 equivalent of compound 3A in anhydrous organic solution, adding 2 to 4 equivalents of sodium hydride substance at 0 ℃, uniformly stirring, heating the mixture to 20 to 30 ℃, then adding 2 to 20 equivalents of di-tert-butyl dicarbonate, continuously stirring the mixture for reaction, cooling the reaction to room temperature, extracting and cleaning the organic solution and aqueous phase solution, transferring the crude product into the organic solution, adding a drying agent into the collected organic solution for removing water, filtering and concentrating, and finally purifying the crude product by column chromatography; the structural formula of compound 3A is as follows:

The method for radiolabeling the precursor comprises:

(1) Bombarding H ₂ ¹⁸ O by a cyclotron to obtain radionuclide 18F, purifying by an ion exchange method under the condition of leacheate containing alkali and a phase transfer catalyst TEAB, or directly transferring the radionuclide 18F into a reaction bottle containing the alkali and the phase transfer catalyst; then removing water at 80-120 ℃;

(2) Then using anhydrous polar aprotic solution to dissolve the precursor, wherein the concentration of the precursor is 0.5mmol/L to 20mmol/L, adding the precursor into the reactant obtained in the step (1), reacting for 5 minutes to 40 minutes at the temperature of 80 ℃ to 160 ℃, ending the reaction, diluting and purifying by a semi-preparation high performance liquid chromatography system.

2. The method for preparing fluoro [18F ] sand fenamide according to claim 1, wherein when the precursor is SPIAd-SF, the method for radiolabelling comprises: bombarding oxygen-enriched water H ₂ ¹⁸ O by a cyclotron to obtain 18F ions, adsorbing ¹⁸ F ions by an anion exchange column, eluting ¹⁸ F ions from a QMA column by using 1mL of eluent containing 0.8mL of acetonitrile, 0.2mL of H ₂ O and 10mg of TEAB, transferring the eluent into a reaction bottle, performing azeotropic dehydration by using 1mL of anhydrous acetonitrile for three times, repeating the azeotropic step until the azeotropic step is dried completely, adding 15mg of a marked precursor compound SPIAd-SF, dissolving by using a 1mL of a MSO solution, heating and reacting at 120 ℃ for 15min, adding 1mL of a 6M hydrochloric acid aqueous solution into the obtained crude product solution, and performing hydrolysis reaction at 110 ℃ for 10min to obtain the crude product solution of the target compound fluorine [18F ] sand-fenamide.