CN118978517A

CN118978517A - Preparation method and application of a type of radioactive probe targeting monoamine oxidase B

Info

Publication number: CN118978517A
Application number: CN202411061261.4A
Authority: CN
Inventors: 杨文江; 张格�; 张智雄; 刘宇
Original assignee: Jinan Zhongke Nuclear Technology Research Institute; Institute of High Energy Physics of CAS
Current assignee: Jinan Zhongke Nuclear Technology Research Institute; Institute of High Energy Physics of CAS
Priority date: 2024-08-05
Filing date: 2024-08-05
Publication date: 2024-11-19

Abstract

The invention discloses a preparation method and application of a radioactive probe targeting monoamine oxidase B. The invention first prepares a 4-methylcoumarin precursor compound containing a chlorine substituent or a boric acid pinacol ester substituent and a corresponding ¹⁹ F standard compound, and then uses the prepared 4-methylcoumarin precursor compound containing a Cl or Bpin substituent to carry out a nucleophilic substitution reaction on the aromatic ring or a copper-catalyzed Chan-Evans-Lam coupling reaction with [ 18 F]F to achieve ¹⁸ F radioactive labeling on the benzene ring, and prepares two ¹⁸ F-labeled 4-methylcoumarin radioactive probes, namely [ ¹⁸ F ^] FCOB02 and [ ¹⁸ F]FCOB04. The obtained radioactive labeled products [ ¹⁸ F]FCOB02 and [ ¹⁸ F]FCOB04 are compared ^with their respective ¹⁹ F standards to determine that the radioactive labeled products are correctly synthesized.

Description

Preparation method and application of radioactive probe targeting monoamine oxidase B

Technical Field

The invention belongs to the field of radiopharmaceuticals, and relates to a preparation method and application of a radioactive probe targeting monoamine oxidase B.

Background

Monoamine oxidase B (MAO B) is a flavin enzyme on the outer mitochondrial membrane, mainly present in astrocytes, 5-hydroxytryptamine neurons and histamine neurons, responsible for catalyzing oxidative deamination of biogenic amines. In the human brain and rodent brain, MAO B degrades dopamine into 3, 4-dihydroxyphenylacetic acid and homovanillic acid, which are important for maintaining the balance of dopamine in the brain. The MAO B irreversible inhibitor L-desmenyl has been widely used as a therapeutic aid for Parkinson's Disease (PD), which increases the striatal dopaminergic availability in dopamine deficient states mainly by inhibiting dopamine decomposition. With the age, MAO B activity in the brain also appears to increase physiologically, which is markedly pronounced in neurodegenerative diseases such as Alzheimer's Disease (AD), PD, etc. These neurodegenerative diseases have typical detrimental characteristics of astrocyte proliferation secondary to neuronal loss and pathological protein aggregation. In PD conditions, astrocyte activation causes up-regulation of MAO B, which in turn leads to increased dopamine loss, and in addition, free radicals (e.g., H ₂O₂) generated during dopamine metabolism by MAO B further induce cell damage, which in turn further exacerbates PD disease progression. Many studies have shown that there is a correlation between the number of MAO B and PD, and that visualizing and quantifying MAO B by Positron Emission Tomography (PET) will help to understand the molecular mechanisms of the underlying pathophysiology of MAO B in neurological diseases and to aid in the discovery of therapeutic drugs.

Coumarin is a natural compound which exists in a large number in nature and has a plurality of medicinal activities such as antioxidation, antibiosis, anticancer and the like. In recent years, coumarin has been found to have MAO B inhibitory activity and its structure is easily modified. Although MAO B inhibition activity of natural coumarin is generally poor, potential MAO B selective inhibitors can be obtained through structural modification.

The radionuclide ¹⁸ F has lower positron energy (average 0.25 MeV), is easy to obtain a high-resolution image, is the radionuclide most commonly used in the PET imaging of a nervous system, and has a clinical application prospect by carrying out chemical modification on the structure of a coumarin MAO B inhibitor and carrying out ¹⁸ F radiolabeling to obtain a selective MAO B radioactive probe.

Currently, there are three major categories of PET radioactive probes targeting MAO B: (1) an irreversible probe; (2) metabolizing the capture class probe; (3) reversible probes. Irreversible probes include clinically applied L- [ ¹¹ C ] decrynyl and its isotopologue L- [ ¹¹ C ] decrynyl-D2, which are clinically applied to human neuropsychiatric related MAO B assessment. Although they have been the "gold standard" for PET imaging of human brain MAO B, inhibitors of this irreversible binding present a great challenge in pharmacokinetic analysis, firstly, this irreversible suicide-type binding mode (the formation of a strong covalent bond between the probe and the flavin cofactor of MAO B) cleared very slowly from the brain, and thus the time activity profile (TAC) of this probe is susceptible to blood flow, complicating quantification of the binding parameters and making pharmacokinetic simulation difficult. Second, the radioactive metabolite (R) -methamphetamine produced by their metabolism in vivo is able to cross the blood brain barrier and bind off-target with monoamine transporter, causing signal noise interference and not accurate quantification. Metabolic capture class probes include [ ¹¹ C ] PHXY and [ ¹¹ C ] COU, which are metabolized by MAO B in vivo, resulting in highly polar radioactive enzyme catalytic activity products that are retained in the target tissue, are not easily excluded from the brain, and have similar irreversible time activity profiles, are difficult to perform pharmacokinetic analysis, but can be used to assess MAO B enzyme activity in vivo, yet their rapid capture rate makes it difficult to distinguish between high and low activity regions of MAO B enzyme. at present, more attention is paid to the study of MAO B PET probes, which have a TAC profile that shows rapid brain uptake and subsequent faster brain clearance during scanning, which is generally insensitive to blood flow changes, easy to perform pharmacokinetic analysis, giving quantitative information on various binding parameters. Reversible radiotracers are suitable for measuring an index related to MAO density. Representative reversible probes are characterized by [ ¹¹C]SL25.1188、[¹⁸F]FSL25.1188.[¹¹ C ] SL25.1188 having high reversible specific binding and selectivity for MAO-B, high brain uptake and no brain penetrating radiometabolites, while [ ¹¹ C ] SL25.1188 has been modeled in humans, the short half-life of ¹¹ C (t1/2=20.4 minutes) and its very complex synthetic preparation process have limited its widespread use, particularly in research environments where there are no radioligand production conditions. Then again a ¹⁸ F labelled SL25.1188 analogue [ ¹⁸ F ] FSL25.1188 appears, which has good MAO B specific binding in the cynomolgus brain but is prone to defluorination in vivo, the radioactivity of the skull increases over time, which means that the radioactivity of the brain area in the vicinity of the skull may be subject to errors.

At present, there is no reversible MAO B PET radioactive probe for clinical use. Accordingly, the present invention aims to develop a novel ¹⁸ F-labeled high MAO B affinity and reversible radioactive probe with excellent metabolic properties to complement the short plates of clinical demand for reversible MAOB PET probes.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to provide a preparation method and application of a radioactive probe targeting monoamine oxidase B. The invention provides a ¹⁸ F marked 4-methylcoumarin compound which has very simple synthesis process, high MAO B affinity, gao Biaoji rate and high specific activity, and is applied to the field of nuclear medicine MAO B PET imaging, and a preparation method thereof.

Coumarin compounds have been widely studied in recent years due to their strong MAO B inhibitory activity, and 4-methylcoumarin compound 3 has high MAO B affinity (IC ₅₀ =7 nM) and subtype selectivity (IC _50,MAO _A/IC_50,MAO _B =743), and further modified compound NW-1772 has good oral bioavailability although the MAO B affinity (IC ₅₀ =13 nM) is slightly inferior to subtype selectivity (IC _50,MAO _A/IC_50,MAO _B =457), and is expected to be a promising clinical candidate for the treatment of neurodegenerative diseases. In addition, the inclusion of 4-methylcoumarin in the structure of [ ¹¹ C ] COU makes it a distinct feature in a number of metabolism-capture MAO B probes of the 1-methyl-4-aryloxy-1, 2,3, 6-tetrahydropyridine structure. The molecular structures based on compound 3 and NW-1772 are as follows:

In view of the superior properties of the 4-methylcoumarin MAO B ligands, such as Compound 3, compound NW-1772 and [ ¹¹ C ] COU, the present invention is directed to the development of reversible MAO B radioactive probes of 4-methylcoumarin type by structural modification. Studies have shown that the introduction of a large steric substituent, particularly benzyloxy, at the C7 position of the coumarin scaffold is important for improving MAO B affinity and selectivity. The structure-activity relationship research shows that coumarin skeleton occupies the substrate cavity of MAO B protein, the benzyloxy group at C7 position is located in the inlet cavity surrounded by the hydrophobic residue of MAO B protein, forms an 'open' conformation with Ile199 residue, enhances the selectivity to MAO-B, the methylene in benzyloxy group is located between the inlet and the substrate cavity, the space length of the methylene is important for MAO B inhibition activity, and the activity is impaired by increasing the methylene number (such as phenylpropyloxy group and the like) or removing the methylene (such as phenoxy group). Replacement of halogen atoms (e.g., F, cl) to the meta or para positions of benzyloxy groups, ligands establish hydrophobic interactions in the inlet chamber of the protein to enhance activity with MAO-B. In addition, the benzene ring of the benzyloxy group is replaced by a pyridine ring, and the nucleophilic reaction is easy to carry out at the ortho position because the carbon electron cloud density at the ortho position of the N atom on the pyridine ring is lower, so that the radioactive fluorination marking can be realized very simply, the lipophilicity of the compound can be reduced after the replacement, and the method has certain help in reducing nonspecific uptake in the brain and improving the clarity rate. The introduction of a small hydrophobic substituent at the C4 position of the coumarin scaffold does not adversely affect the structure, and the introduced methyl group is capable of forming Pi-alkyl interactions with Phe343 residues, further increasing binding with MAO B. Based on the design, the invention introduces fluoropyridine methoxy or fluorobenzyloxy at the C7 position of the 4-methylcoumarin skeleton, synthesizes two 4-methylcoumarin MAO B PET probes, namely [ ¹⁸ F ] FCOB02 and [ ¹⁸ F ] FCOB04, and evaluates the potential of the probes as MAO B PET probes.

The invention designs and synthesizes two 4-methylcoumarin MAO B radioactive probes (the C7 position is [ ¹⁸ F ] FCOB02 of fluoropyridine methoxy group, the C7 position is [ ¹⁸ F ] FCOB04 of fluorobenzyloxy group), wherein the molecular structural formula of the MAO B-targeting radioactive probe [ ¹⁸ F ] FCOB02 is shown as follows:

the molecular structural formula of [ ¹⁸ F ] FCOB04 is shown below:

Firstly, preparing a precursor compound of 4-methylcoumarin containing chlorine (Cl) substituent or pinacol borate (Bpin) substituent and a corresponding ¹⁹ F standard compound, and then carrying out nucleophilic substitution reaction on an aromatic ring or copper-catalyzed Chan-Evans-Lam coupling reaction on the precursor compound of the 4-methylcoumarin containing Cl or Bpin substituent and [ ¹⁸F]F^- ] to realize ¹⁸ F radiolabelling on a benzene ring, so as to prepare two ¹⁸ F-labeled 4-methylcoumarin radioactive probes, namely [ ¹⁸ F ] FCOB02 with pyridine methoxy at C7 and [ ¹⁸ F ] FCOB04 with benzyloxy at C7. The resulting radiolabeled products [ ¹⁸ F ] FCOB02 and [ ¹⁸ F ] FCOB04 were compared to their respective ¹⁹ F standards to confirm that the radiolabeled products were synthesized correctly.

The general formula of the synthetic route of the precursor compounds of the 4-methylcoumarin containing Cl or Bpin substituent and the respective ¹⁹ F standard compound is shown in figure 1. The synthesis method is that 4-methylumbelliferone and bromo-or chloro-derivatives are used as raw materials, DMF or ethanol is used as solvent under the catalysis of potassium carbonate, and Williamson ether synthesis reaction is carried out at room temperature or under the condition of reflux. The method is characterized by simple and easily obtained raw materials, simple synthesis, and the synthesis process of the precursor and the standard substance can be completed by one step.

The preparation methods of the precursor and the standard substance are as follows:

(1) The synthesis method of the Cl substituent precursor (2 a) of [ ¹⁸ F ] FCOB02 with the C7 position being pyridylmethoxy is as follows:

2-chloro-5-chloromethyl pyridine (1-1.1 mol equivalent) is dissolved in ethanol (100-200 mol equivalent), 4-methylumbelliferone (1 mol equivalent) and potassium carbonate (2-3 mol equivalent) are added, and the mixture is stirred under reflux for 8-15 hours to fully react. After the reaction, insoluble matters are removed by filtration, the solvent is dried by spin-drying, the residue is washed by adding water, and the crude product is purified by flash silica gel purification column chromatography to obtain the precursor compound of 4-methylcoumarin containing chlorine (Cl) substituent.

(2) The synthesis method of the [ ¹⁸ F ] FCOB02 with the C7 position being pyridylmethoxy and the ¹⁹ F standard (2 b) comprises the following steps:

5-chloromethyl-2-fluoropyridine (1-1.1 mol equivalent) was dissolved in ethanol (100-200 mol equivalent), 4-methylumbelliferone (1 mol equivalent) and potassium carbonate (2-3 mol equivalent) were added, and the mixture was stirred under reflux for 8-15 hours to effect a sufficient reaction. At the end of the reaction, insoluble materials were removed by filtration, the solvent was dried by spin-drying, the residue was washed with water, filtered, and the crude product was purified by flash silica gel purification column chromatography to give its ¹⁹ F standard.

(3) The synthesis method of the [ ¹⁸ F ] FCOB04 with benzyloxy at the C7 position and the Bpin substituent precursor (2C) comprises the following steps:

3-bromomethylphenylboronic acid farnesol ester (1-1.1 mol equivalent) was dissolved in anhydrous DMF (30-50 mol equivalent), 4-methylumbelliferone (1 mol equivalent) and potassium carbonate (2-3 mol equivalent) were added and stirred at room temperature for 8-15 hours to effect a complete reaction. At the end of the reaction, insoluble materials are removed by filtration, the solvent is dried by spin-drying, and the crude product is transferred to the organic solution by extraction and washing of the organic solution and aqueous solution. The collected organic solution is added with a drying agent to remove water, filtered and concentrated, and then the crude product is purified by flash silica gel purification column chromatography to obtain the 4-methylcoumarin precursor compound containing the pinacol borate (Bpin) substituent.

(4) The synthesis method of the [ ¹⁸ F ] FCOB04 with benzyloxy at the C7 position and the ¹⁹ F standard (2 d) comprises the following steps:

3-fluorobenzyl bromide (1-1.1 molar equivalent) was dissolved in anhydrous DMF (30-50 molar equivalent), 4-methylumbelliferone (1 molar equivalent) and potassium carbonate (2-3 molar equivalent) were added and stirred at room temperature for 8-15 hours to effect a complete reaction. At the end of the reaction, insoluble materials are removed by filtration, the solvent is dried by spin-drying, and the crude product is transferred to the organic solution by extraction and washing of the organic solution and aqueous solution. The collected organic solution was added with a desiccant to remove water and filtered to concentrate, and then the crude product was purified by flash silica gel purification column chromatography to give its ¹⁹ F standard.

The method for radiolabelling the precursor compounds is as follows:

(1) Bombarding H ₂ ¹⁸ O with a cyclotron to obtain a radionuclide ¹⁸ F, namely [ ¹⁸F]F^-; leaching [ ¹⁸F]F^- ] into a reaction bottle by an ion exchange method under the condition of leaching solution containing alkali and a phase transfer catalyst, and removing water (adding three times of anhydrous acetonitrile, 0.5mL each time, and removing water by an azeotropic mode) at 100-120 ℃.

(2) Precursor compound 2a containing Cl substituents was radiolabelled by nucleophilic substitution reaction on aromatic ring at ¹⁸ F and precursor compound 2c containing Bpin substituents was radiolabelled by copper-catalyzed Chan-Evans-Lam coupling reaction at ¹⁸ F. The precursor compound (precursor compound concentration of 4mg-10m in 0.5-1mL solvent) is dissolved using an anhydrous polar aprotic solvent (such as dimethyl sulfoxide, N-dimethylacetamide) and added to the reaction flask of step (1) and reacted at 120-150 ℃ for 10 min to 30min. And (3) diluting after the reaction is finished, removing fluorine ions through a C18 column, and purifying through a semi-preparative high performance liquid chromatography system. For Bpin precursor (2 c), a copper catalyst (Cu (OTf) ₂(py)₄, 1-2 times the precursor equivalent) and t-butanol (0.5 times the volume of the anhydrous polar aprotic solvent) were also added for catalytic reaction.

The invention aims at providing an application for detecting the quantity of monoamine oxidase B in a living body and evaluating monoamine oxidase B inhibitor medicines.

The invention has the following advantages:

1. The invention provides two MAO B PET probes containing a 4-methylcoumarin structure, which realize ¹⁸ F radiolabelling on an aromatic ring by using positron nuclide ¹⁸ F to obtain [ ¹⁸ F ] FCOB02 and [ ¹⁸ F ] FCOB04.

2. The [ ¹⁸ F ] FCOB02 and [ ¹⁸ F ] FCOB04 prepared by the invention show high affinity to MAO B in an in-vitro receptor ligand competitive binding experiment, and particularly, the [ ¹⁸ F ] FCOB04 shows excellent sub-nanomolar MAO B affinity, so that the specificity of the probe is improved.

3. The invention provides a preparation method of a labeling precursor and ¹⁹ F standard substance, wherein the labeling precursor has the advantages of easily available raw materials, simple preparation and one-step preparation, is beneficial to reducing the cost and reducing the introduction of impurities, and the yield of the purified product is more than 80 percent.

4. The invention realizes the efficient radiolabelling of [ ¹⁸ F ] FCOB02 and [ ¹⁸ F ] FCOB04 by utilizing the nucleophilic substitution reaction of [ ¹⁸F]F^- and halogen substituent on pyridine ring and the Chan-Evans-Lam coupling reaction of boric acid ester substituent on benzene ring, provides the route for preparing [ ¹⁸ F ] FCOB02 and [ ¹⁸ F ] FCOB04 by radioactivity and the corresponding reaction steps and parameters, the labeling rate can reach 90%, and can provide enough radioactive dose and radioactive concentration to meet various experimental requirements.

5. The invention carries out module automatic synthesis on [ ¹⁸ F ] FCOB04 to obtain 366 GBq/. Mu.mol high-specific activity radiolabeled product, which is superior to other similar probes, and effectively avoids saturation of MAO B binding sites in brain.

6. The [ ¹⁸ F ] FCOB04 prepared by the invention shows excellent brain uptake capacity and faster clearance rate, and has reversible combination in vivo. It has good stability in brain and is not easy to defluorinate in vivo. Significant brain uptake differences can be observed in PET images before and after inhibition of the MAO B inhibitor L-Deprenyl, and after inhibition, the total brain uptake is reduced by 30%, which is significantly superior to the existing probes, and has important value for realizing specific MAOB imaging and quantitative analysis in vivo.

Drawings

FIG. 1 is a general formula of a synthesis route of a precursor and a standard; wherein R ₁ is represented as H or N, R ₂ is represented as H or F or Cl, R ₃ is represented as H or F or Bpin, and R ₄ is represented as Cl or Br.

FIG. 2 is a synthetic route diagram of [ ¹⁸ F ] FCOB02 precursor (2 a) and ¹⁹ F standard (2 b).

FIG. 3 is a synthetic route diagram of [ ¹⁸ F ] FCOB04 precursor (2 c) and ¹⁹ F standard (2 d).

FIG. 4 is a radiolabeled roadmap of [ ¹⁸ F ] FCOB 02.

FIG. 5 is a radiolabeled roadmap of [ ¹⁸ F ] FCOB 04.

FIG. 6 shows the radioactive HPLC peak (red) and the standard UV peak (black) of the probe;

(a) A radioactive HPLC peak (red) for [ ¹⁸ F ] FCOB02 with a standard UV peak (black);

(b) The radioactive HPLC peak (red) for [ ¹⁸ F ] FCOB04 and the standard UV peak (black).

FIG. 7 shows uptake of the MAO B inhibitor L-deprenyl in rat brain before and after [ ¹⁸ F ] FCOB04 inhibition;

(a) A TAC curve of [ ¹⁸ F ] FCOB04 on rat whole brain and a TAC curve after L-deprenyl inhibition, labeled for module automation;

(b) Normal SD rat cross-section, sagittal and coronal PET images 0-10min,10-20min,20-30min,30-40min after injection of [ ¹⁸ F ] FCOB 04;

(c) Is a PET image after L-deprenyl (1 mg/kg) inhibition.

FIG. 8 shows uptake of the MAO B inhibitor L-deprenyl in rat liver, [ ¹⁸ F ] FCOB04 before and after inhibition;

(a) TAC curve for SD rat liver uptake at baseline conditions and under L-deprenyl (1 mg/kg) inhibition conditions for [ ¹⁸ F ] FCOB 04;

(b) Is MIP image of normal SD rat 0-10,10-20,20-30,30-40min under baseline condition.

FIG. 9 shows the results of radioactive HPLC analysis of blood, brain, urine of mice after 2 and 30 minutes of [ ¹⁸ F ] FCOB04 injection.

Fig. 10 is a inhibition curve corresponding to R ² =0.95.

Fig. 11 is a suppression curve corresponding to R ² =0.86.

Detailed Description

The invention will now be described in further detail with reference to the accompanying drawings, which are given by way of illustration only and are not intended to limit the scope of the invention.

The technical scheme of the invention, including the preparation method of the precursor compound and ¹⁹ F standard substance and the radiolabelling method, and the module automatic labeling process are detailed below by the accompanying drawings and examples.

The synthetic routes of the precursor compound of [ ¹⁸F]FCOB02、[¹⁸ F ] FCOB04 and the standard compound are shown in FIGS. 2 and 3, and the chemical synthesis of the precursor compound of the present invention and the standard is described in detail by way of specific examples.

Example 1: synthesis of [ ¹⁸ F ] FCOB02 precursor Compound (2 a)

2-Chloro-5-chloromethylpyridine (1 mmol,162 mg) was dissolved in 10ml of ethanol, and 4-methylumbelliferone (1 mmol,176 mg) and K ₂CO₃ (1.1 mmol,152 mg) were added. The mixture was stirred under reflux with heating overnight. After the reaction was completed, the solvent was removed by rotary evaporation, 20ml of distilled water was added, cooled to room temperature, and then filtered, and the residue was purified by silica gel flash purification column chromatography (dichloromethane/methanol=1/0→100/1), to obtain a product 2a. Yield 81%, off-white powder was [ ¹⁸ F ] FCOB02 precursor compound (i.e. precursor compound of 4-methylcoumarin class containing chlorine substituents) with the molecular structure shown below:

The nuclear magnetic hydrogen spectrum of the [ ¹⁸ F ] FCOB02 precursor compound is characterized as ：¹H NMR(500MHz,CDCl₃)δ8.48(d,J＝2.5Hz,1H),7.76(dd,J＝8.2,2.5Hz,1H),7.53(d,J＝8.8Hz,1H),7.39(d,J＝8.2Hz,1H),6.92(dd,J＝8.8,2.5Hz,1H),6.88(d,J＝2.5Hz,1H),6.17(t,J＝1.4Hz,1H),5.12(s,2H),2.41(d,J＝1.3Hz,3H).

Example 2: synthesis of [ ¹⁸ F ] FCOB02 Standard Compound (2 b)

5- (Chloromethyl) -3-fluoropyridine (1 mmol,145 mg) was dissolved in 10ml ethanol, and 4-methylumbelliferone (1 mmol,176 mg) and K ₂CO₃ (1.1 mmol,152 mg) were added. The mixture was stirred under reflux with heating overnight. After the reaction was completed, the solvent was removed by rotary evaporation, 20ml of distilled water was added, cooled to room temperature, and then filtered, and the residue was purified by silica gel flash purification column chromatography (dichloromethane/methanol=1/0→100/1), to obtain product 2b. Yield 86%, off-white powder was a standard compound corresponding to [ ¹⁸ F ] FCOB02, whose molecular structure is shown below:

[ ¹⁸ F ] the nuclear magnetic hydrogen spectrum of the FCOB02 standard compound is characterized as ：¹H NMR(500MHz,Chloroform-d)δ8.48(d,J＝2.5Hz,1H),7.76(dd,J＝8.2,2.5Hz,1H),7.53(d,J＝8.8Hz,1H),7.39(d,J＝8.2Hz,1H),6.92(dd,J＝8.8,2.5Hz,1H),6.88(d,J＝2.5Hz,1H),6.17(t,J＝1.4Hz,1H),5.12(s,2H),2.41(d,J＝1.3Hz,3H).

Example 3: synthesis of [ ¹⁸ F ] FCOB04 precursor Compound (2 c)

Pinacol (3-bromomethylphenyl) borate (327 mg,1.1 mmol) was dissolved in 3mL of DMF, 4-methylumbelliferone (176 mg,1 mmol) and potassium carbonate (414 mg,3 mmol) were added thereto, and the reaction was stirred at room temperature overnight. At the end of the reaction, insoluble matter was removed by filtration, washed with ultra-dry acetonitrile, concentrated solution was distilled off with 30mL of water, extracted twice with an equal amount of ethyl acetate, the organic phase was mixed, washed once with 10mL of saturated brine, dried over anhydrous sodium sulfate, filtered, solvent was removed by distillation with water, and the residue was purified by silica gel Flash purification column chromatography (n-hexane/ethyl acetate=1/0→2/1) to give product 2c. Yield 84%, white powder is [ ¹⁸ F ] FCOB04 precursor compound (i.e. 4-methylcoumarin precursor compound containing pinacol borate substituent), its molecular structure is shown below:

The nuclear magnetic hydrogen spectrum of the [ ¹⁸ F ] FCOB04 precursor compound is characterized as ：¹H NMR(500MHz,CDCl₃)δ7.93-7.76(m,2H),7.56-7.47(m,2H),7.41(t,J＝7.5Hz,1H),6.97-6.85(m,2H),6.14(d,J＝1.0Hz,1H),5.12(s,2H),2.40(d,J＝1.0Hz,3H),1.36(s,12H).

Nuclear magnetic hydrogen spectrum characterization and mass spectrum characterization prove that the [ ¹⁸ F ] FCOB04 precursor compound is synthesized; i.e. LC-MS(ESI):m/zcalcd for C₂₃H₂₅BO₅[M+Na]⁺:415.17;found:415.28;[2M+Na]⁺:807.35;found:807.37.

Example 4: synthesis of [ ¹⁸ F ] FCOB04 Standard Compound (2 d)

3-Fluorobromobenzyl (208 mg,1.1 mmol) was dissolved in 3mL of DMF, 4-methylumbelliferone (176 mg,1 mmol) and potassium carbonate (414 mg,3 mmol) were added thereto, and the reaction was stirred at room temperature overnight. At the end of the reaction, insoluble matter was removed by filtration, washed with ultra-dry acetonitrile, concentrated solution was distilled off with 30mL of water, extracted twice with an equal amount of ethyl acetate, the organic phase was mixed, washed once with 10mL of saturated brine, dried over anhydrous sodium sulfate, filtered, solvent was removed by distillation with water, and the residue was purified by silica gel Flash purification column chromatography (n-hexane/ethyl acetate=1/0→2/1) to give product 2d. The yield was 88%, the pale pink powder was a standard compound corresponding to [ ¹⁸ F ] FCOB04, and the molecular structure was as follows:

[ ¹⁸ F ] the nuclear magnetic hydrogen spectrum of the FCOB04 standard compound is characterized as ：¹H NMR(500MHz,CDCl₃)δ7.52(dd,J＝8.8,2.2Hz,1H),7.42-7.34(m,1H),7.23-7.18(m,1H),7.16(dd,J＝9.6,5.7Hz,1H),7.04(q,J＝8.4Hz,1H),6.97-6.91(m,1H),6.87(t,J＝2.3Hz,1H),6.15(d,J＝3.8Hz,1H),5.21-5.04(m,1H),2.40(d,J＝3.0Hz,1H).

Mass spectrometry characterization of [ ¹⁸ F ] FCOB04 standard compound was as follows LC-MS(ESI):m/z calcd for C₁₇H₁₃FO₃[M+Na]⁺:307.07;found:307.15;[2M+Na]⁺:591.15;found:591.21.

According to the difference of precursor leaving groups (Cl and Bpin), the corresponding radioactive synthesis processes of [ ¹⁸ F ] FCOB02 and [ ¹⁸ F ] FCOB04 are designed, and the synthesis process comprises the steps of fluoride ion leaching, water removal, heating reaction, adsorption, HPLC separation and purification, ethanol/physiological saline preparation and the like. The radiolabeled routes of [ ¹⁸ F ] FCOB02 and [ ¹⁸ F ] FCOB04 are shown in FIGS. 4 and 5.

Example 5: radioactive synthesis process of [ ¹⁸ F ] FCOB02

The accelerator produced [ ¹⁸ F ] F-solution was captured on a QMA column (pre-activated with 10m 0.5M sodium bicarbonate solution and 10mI water), after which ¹⁸F^- was rinsed from the QMA column into a 10mL penicillin bottle with a rinse solution (11.1 mg K _2,2,2 and 6mg K ₂CO₃ in water/acetonitrile (1/2)), the solution was dried by heating with nitrogen at 105℃on a nitrogen blower, water was removed, and 0.5m acetonitrile was added to azeotropically dry three times, and the water was removed sufficiently. After sufficient drying, 4mg of the labeled precursor dissolved in 1ml of ultra-dry DMSO was added to a penicillin bottle and the reaction was sealed with a gland at 120℃for 20 minutes. After the reaction was cooled slightly, 10mL of water was added to dilute the reaction, the unreacted ¹⁸F^- was removed by passing through a C18 column, 10mL of water was used to wash the C18 column once, the labeled product was rinsed to penicillin vials with 2mL of acetonitrile, heated and concentrated to about 200% C on a nitrogen blower, then subjected to HPLC separation and purification, HPLC mobile phase conditions (55% mecn/45% h ₂ O), the fraction of the position of the radiolabeled product [18F ] FCOB02 (which was previously confirmed by the HPLC position of its ¹⁹ F standard) was collected, 3 volumes of water was added to dilute the reaction product, the C18 column was washed once with 10mL of water, the labeled product was rinsed to penicillin vials with 2m of ethanol, heated and concentrated to a volume at 70 ℃ on a nitrogen blower, and then subjected to formulation with physiological saline (requiring ethanol content < 9%) to give [ ¹⁸ F ] FCOB02 for subsequent experiments.

As shown in FIG. 6 (a), the radiochemical purity of [ ¹⁸ F ] FCOB02 was greater than 99% as determined by HPLC analysis. The retention time in HPLC was 8.0min. The HPLC conditions for analysis of the final radioactive product are high performance liquid chromatograph WATERSALLIANCE HPLC; krmaisl C18 column 250x4.6 mm,5um100A; mobile phase conditions (55% mecn/45% h ₂ O) flow rate 1mL/min.

Example 6: radioactive synthesis process of [ ¹⁸ F ] FCOB04

The accelerator produced [ ¹⁸F]F^- solution was captured on a QMA column (pre-activated with 10mL of 0.5M sodium bicarbonate solution and 10mL of water), after which ¹⁸F^- was back-rinsed from the QMA column into a 10mL penicillin bottle with a rinse solution (2.3 mg tetrabutylammonium bicarbonate and 0.4mg potassium carbonate in water/acetonitrile (1/4)), the solution was dried by heating with nitrogen at 105℃on a nitrogen blower, water was removed, and 0.5mL acetonitrile was added for three azeotropic drying to fully remove water. After sufficient drying, 10mg [ Cu (py) ₄(OTf)₂ ],0.3mL tert-BuOH and 4mg of the labeled precursor (1 c) in 1mL of ultra-dry DMA were dissolved in 0.36mL of ultra-dry DMA, added to penicillin bottles, and the reaction was sealed with a gland at 120℃for 15 minutes. After the reaction was cooled slightly, 10mL of water was added to dilute the reaction solution, the unreacted ¹⁸F^- was removed by passing through a C18 column, 10mL of water was used to wash the C18 column once, the labeled product was rinsed to penicillin vials with 2mL of acetonitrile, heated and concentrated to about 200 μl on a nitrogen blower, then subjected to HPLC separation and purification, HPLC mobile phase conditions (60% mecn/40% h ₂ O), and fractions of the position of the radiolabeled product [ ¹⁸ F ] FCOB04 (which was previously confirmed by the HPLC position of its ¹⁹ F standard 2 d) were collected, 3 volumes of water were added to dilute the reaction solution, the C18 column was washed once with 10mL of water, the labeled product was rinsed to penicillin vials with 2mL of ethanol, heated and concentrated to a volume at 70 ℃ on a nitrogen blower, and formulated with physiological saline (requiring ethanol content < 9%) to give [ ¹⁸ F ] FCOB04 for subsequent experiments.

As shown in FIG. 6, the radiochemical purity of [ ¹⁸ F ] FCOB04 was greater than 99% as determined by HPLC analysis. The retention time in HPLC was 11.2min. The HPLC conditions for analysis of the final radioactive product were: high performance liquid chromatograph WATERSALLIANCE HPLC; kromaisl C18 column 250X 4.6mm,5 μmMobile phase conditions (60% mecn/40% h ₂ O), flow rate 1mL/min.

Example 7: module automatic radiosynthesis process of [ ¹⁸ F ] FCOB04

1. Automated synthesis preparation: the He valve and vent valve of the mobile phase solvent bottle were opened for venting, the vent valve was closed after three minutes, the HPLC pump was turned on, and the preparation column was equilibrated with mobile phase (63% acetonitrile and 37% water) at a flow rate of 10mL/min (to which the flow rate was gradually controlled manually). Packing work was performed during equilibration of the column: the rinse solution (2.3 mg tetrabutylammonium bicarbonate and 0.4mg potassium carbonate in water/acetonitrile (1/4)) was added to bottle 1; bottle No.2 was added with the reaction solution (4 mg of the labeled precursor 1c,10mg of [ Cu (py) ₄(OTf)₂ ] dissolved in 0.27mL of ultra-dry DMA with 0.13 mLtert-BuOH); bottle 4 was charged with 3.3mL HPLC mobile phase; 20mL of physiological saline is added into a No. 12 bottle, and 2mL of ethanol is added into a No. 13 bottle; bottle No. 14 was filled with 10mL of water; adding 30mL of water into a round bottom big flask; light QMA column (46 mg) is loaded between interface 10 and interface 11; a C18 column is fitted between interface 17 and interface 15.

2. Automated tag synthesis: the automatic synthesis program is imported into a computer operated by a module, the automatic synthesis is started by a starting program, and the running program is as follows:

1. the [ ¹⁸F]F^- ] produced by the accelerator is transmitted to the synthesizer module through the underground pipeline, and after the transmission is finished, the synthesis is started by clicking an OK button.

2. The pump in the module pumps [ ¹⁸F]F^- ] to capture on QMA and TBAHCO ₃/K₂CO₃ eluent is pumped to rinse [ ¹⁸F]F^- ] on QMA column to reaction tube 1.

3. The reaction tube 1 was blown dry with He gas at 90℃and cooled to 40℃and a mixed solution of the precursor and [ Cu (py) ₄(OTf)₂ ] was added.

4. After the reaction flask was evacuated by pumping for 5 seconds, air was sucked in by the negative pressure of the reaction tube 1.

5. The reaction tube was sealed and heated to 120℃for 15min.

6. The reaction was cooled to 40℃at the end of the reaction, and 3.3mL of HPLC mobile phase was added for dilution.

7. The mixture was transferred from the reaction tube 1 to the reaction tube 2, and was separated and purified by blowing He gas into the HPLC system (mobile phase condition: 63% acetonitrile/37% water, 10 mL/min).

8. The collected components are connected into a round-bottom large flask, diluted by water in the flask, then pass through a C18 column, a No. 12 bottle is used for washing, a No. 13 bottle is used for leaching the product into a collecting bottle, the saline of the No. 14 bottle is added into the collecting bottle for preparation, and finally the obtained product is transmitted into a split charging hot chamber for split charging.

The radiochemical yield was 52±6% (n=2, decay correction) and the radiochemical purity was greater than 99%. The molar activity was 366.+ -.15 GBq/. Mu.mol (n=2, decay correction).

The invention takes coumarin compound containing Cl or Bpin substituent as precursor, and carries out nucleophilic substitution reaction or copper catalyzed Chan-Evans-Lam coupling reaction with [ ¹⁸F]F^- to prepare two ¹⁸ F labeled coumarin radioactive probes. All probes have good chemical stability, no visible decomposition exists when the probes are placed for 4 hours at room temperature, the radiochemical purity of HPLC analysis is more than 97%, and the radiochemical yield of [ ¹⁸ F ] FCOB04 is up to 52%. In vitro binding experiments showed that the probes had nanomolar to subnanomolar high MAO B affinities as shown in table 1 below.

Table 1 shows in vitro receptor binding data for 2B, 2d and MAO B

Brain probes are susceptible to saturation of the receptor binding sites, leading to failure of PET imaging, and therefore high specific activity is important for probes to have good brain imaging effects. The invention obtains the [ ¹⁸ F ] FCOB04 with high specific activity of 366 GBq/. Mu.mol through module automation, and the high specific activity can not cause saturation of MAO B binding site basically. And the product produced by the module reaches more than 200mCi, so that sufficient radioactive dose is provided, subsequent experiments are conveniently arranged, and the service time and distance of the PET imaging agent can be prolonged.

The invention carries out rat PET/CT imaging on [ ¹⁸ F ] FCOB04, and the whole brain TAC curve shows that the uptake of [ ¹⁸ F ] FCOB04 reaches a peak value (SUV-1.43) in about 1 minute, has better clearance rate in brain, and the clearance rate of the imaging agent is 83% after 40 minutes of administration. After inhibition of the MAO B inhibitor L-deprenyl, [ ¹⁸ F ] FCOB04 was significantly inhibited in the whole brain (shown in FIG. 7), with a 30% inhibition rate.

According to the existing research report, the rat autoradiography study of [ ³ H ] Ro19-6327 shows that MAO B is highly expressed in the peripheral organs of rats by the liver, and almost no MAO B is contained in pancreas and cardiac muscle. As shown in FIG. 8, the study also observed a high uptake of [ ¹⁸ F ] FCOB04 in the liver region (SUV _max:2.68), and uptake in the liver was inhibited by L-deprenyl by 42% in 40 minutes. The uptake of the heart and pancreas was not apparent from the PET images. No significant bone uptake was seen throughout the MIP map, indicating that no significant defluorination occurred in the rat. The MIP image shows that the gallbladder uptake increases with time, indicating that the imaging agent is cleared through liver and gall metabolic pathways.

HPLC analysis of radioactivity in mouse plasma as shown in FIG. 9 shows that [ ¹⁸ F ] FCOB04 is rapidly metabolized in plasma, is relatively stable at 2min, and radioactivity is mainly derived from the parent compound, whereas by 30min only about 8% of the parent compound remains, radioactivity is mainly derived from the radioactive metabolite that is more hydrophilic than the parent compound. The radioactivity in the brain is all from the parent compound at 2min, and the [ ¹⁸ F ] FCOB04 still maintains more than 95% of integrity in the brain at 30min, which indicates that the [ ¹⁸ F ] FCOB04 is metabolically stable in the brain and the metabolites in the blood are not easy to enter the brain. The radioactive components in urine at 30min are consistent with the radioactive metabolites in plasma.

Although specific embodiments of the invention have been disclosed for illustrative purposes, it will be appreciated by those skilled in the art that the invention may be implemented with the help of a variety of examples: various alternatives, variations and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will have the scope indicated by the scope of the appended claims.

Claims

1. A method for preparing a radioactive probe targeting monoamine oxidase B, comprising the steps of:

1) preparing a precursor compound of 4-methylcoumarin containing a chlorine substituent;

2) The precursor compound is subjected to a nucleophilic substitution reaction on the aromatic ring with [ ¹⁸ F] ^F- to achieve ¹⁸ F radioactive labeling on the benzene ring, and a radioactive labeled product [ ¹⁸ F]FCOB02 is obtained as a radioactive probe targeting monoamine oxidase B; the radioactive labeled product [ ¹⁸ F]FCOB02 is [ ¹⁸ F]FCOB02 with a pyridinemethoxy group at the C7 position, and its molecular structure is as follows:

2. The method according to claim 1, characterized in that a ¹⁹ F standard compound corresponding to [ ¹⁸ F]FCOB02 in which the C7 position is a pyridinemethoxy group is prepared; the radioactive labeled product [ ¹⁸ F]FCOB02 is compared with the ¹⁹ F standard compound to determine whether the synthesis of the radioactive labeled product [ ¹⁸ F]FCOB02 is correct.

3. The method according to claim 2, characterized in that the method for preparing the ¹⁹ F standard compound is: dissolving 1-1.1 molar equivalents of 5-chloromethyl-2-fluoropyridine in 100-200 molar equivalents of ethanol, then adding 1 molar equivalent of 4-methylumbelliferone and 2-3 molar equivalents of potassium carbonate, after reflux stirring reaction, filtering to remove insoluble matter, spin-drying the solvent, adding water to wash the residue, filtering to obtain a first crude product; then purifying the first crude product by flash silica gel purification column chromatography to obtain the ¹⁹ F standard compound.

4. The method according to claim 1, 2 or 3, characterized in that the method for obtaining the precursor compound of the chlorine-containing 4-methylcoumarin is as follows: 1-1.1 molar equivalents of 2-chloro-5-chloromethylpyridine are dissolved in 100-200 molar equivalents of ethanol, and then 1 molar equivalent of 4-methylumbelliferone and 2-3 molar equivalents of potassium carbonate are added. After the reaction is refluxed and stirred, the insoluble matter is filtered out, the solvent is spin-dried, the residue is washed with water, and filtered to obtain a second crude product; and then the second crude product is purified by flash silica gel purification column chromatography to obtain the precursor compound of the chlorine-containing 4-methylcoumarin.

5. The method according to claim 1, 2 or 3, characterized in that the method for obtaining the radioactive labeled product [ ^18F ]FCOB02 is:

21) bombarding H ₂ ¹⁸ O with a cyclotron to obtain the radioactive nuclide ¹⁸ F, namely [ ¹⁸ F]F ^- ; then eluting [ ¹⁸ F]F ^- into a reaction flask by an ion exchange method under the condition of an eluent containing a base and a phase transfer catalyst, and then removing the water in the reaction flask;

22) dissolving the precursor compound of the chlorine-containing 4-methylcoumarin in an anhydrous polar aprotic solvent;

23) The solution obtained in step 22) is added to the reaction bottle in step 21), and the reaction is carried out at 120° C.-150° C. for 10 to 30 minutes to carry out a nucleophilic substitution reaction on the aromatic ring to achieve radioactive labeling of ¹⁸ F; after the reaction is completed, the solution is diluted and passed through a C18 column to remove fluoride ions, and then purified by a semi-preparative high performance liquid chromatography system to obtain the radioactive labeled product [ ¹⁸ F]FCOB02.

6. A method for preparing a radioactive probe targeting monoamine oxidase B, comprising the steps of:

1) preparing a precursor compound of 4-methylcoumarin containing a boronic acid pinacol ester substituent;

2) The precursor compound is subjected to a copper-catalyzed Chan-Evans-Lam coupling reaction with [ ¹⁸ F] ^F- to achieve ¹⁸ F radioactive labeling on the benzene ring, and a radioactive labeled product [ ¹⁸ F]FCOB04 is obtained as a radioactive probe targeting monoamine oxidase B; the radioactive labeled product [ ¹⁸ F]FCOB04 is [ ¹⁸ F]FCOB04 with a benzyloxy group at the C7 position, and its molecular structure is as follows:

7. The method according to claim 6, characterized in that a ¹⁹ F standard compound corresponding to the [ ¹⁸ F]FCOB04 in which the C7 position is a benzyloxy group is prepared; and the radioactive labeled product [ ¹⁸ F]FCOB04 is compared with the ¹⁹ F standard compound to determine whether the synthesis of the radioactive labeled product [ ¹⁸ F]FCOB04 is correct.

8. The method according to claim 7, characterized in that the method for preparing the ¹⁹ F standard compound is:

a) dissolving 1-1.1 molar equivalents of 3-fluorobenzyl bromide in 30-50 molar equivalents of anhydrous DMF, then adding 1 molar equivalent of 4-methylumbelliferone and 2-3 molar equivalents of potassium carbonate, stirring at room temperature after the reaction is completed, filtering to remove insoluble matter, and spinning the solvent to dryness;

b) extracting and washing the product obtained in step a) with an organic solution and an aqueous solution, and transferring the washed product into an organic solution;

c) removing water from the organic solution obtained in step b), filtering and concentrating the solution to obtain a third crude product, and then purifying the third crude product by flash silica gel purification column chromatography to obtain the ¹⁹ F standard compound.

9. The method according to claim 6, 7 or 8, characterized in that the method for obtaining the precursor compound of the 4-methylcoumarin containing the boronic acid pinacol ester substituent is:

11) Dissolve 1-1.1 molar equivalents of 3-bromomethylphenylboronic acid pinesol ester in 30-50 molar equivalents of anhydrous DMF, then add 1 molar equivalent of 4-methylumbelliferone and 2-3 molar equivalents of potassium carbonate, and stir at room temperature until the reaction is complete.

Filter to remove insoluble matter and spin dry the solvent;

12) extracting and washing the product obtained in step 11) with an organic solution and an aqueous solution, and transferring the washed product into an organic solution;

13) removing water from the organic solution obtained in step 12) and filtering and concentrating the solution to obtain a fourth crude product, and then purifying the fourth crude product by flash silica gel purification column chromatography to obtain the precursor compound of the 4-methylcoumarin containing the boronic acid pinacol ester substituent.

10. The method according to claim 6, 7 or 8, characterized in that the method for obtaining the radioactive labeled product [ ^18F ]FCOB04 is:

21) using a cyclotron to bombard H ₂ ¹⁸ O to obtain a radioactive nuclide ¹⁸ F, namely [ ¹⁸ F]F ^- ; then eluting [ ¹⁸ F]F ^- into a reaction flask by an ion exchange method under the conditions of an eluent containing a base, a copper catalyst, tert-butyl alcohol and a phase transfer catalyst, and then removing the water in the reaction flask; the copper catalyst is Cu(OTf) ₂ (py) ₄ , and the amount used is 1-2 times the amount of the precursor compound; the amount of the tert-butyl alcohol used is 0.5 times the volume of the anhydrous polar aprotic solvent;

22) radiolabeling the precursor compound of 4-methylcoumarin containing boronic acid pinacol ester substituent with ¹⁸ F through a copper-catalyzed Chan-Evans-Lam coupling reaction, and then dissolving it in an anhydrous polar aprotic solvent;

23) The solution obtained in step 22) is added to the reaction bottle in step 21) and reacted at 120°C-150°C for 10 to 30 minutes; after the reaction, the solution is diluted and passed through a C18 column to remove fluoride ions and then purified by a semi-preparative HPLC system to obtain the radiolabeled product [ ¹⁸ F]FCOB04.

11. Use of a radioactive probe prepared by the method of claim 1 or 6 in detecting the amount of monoamine oxidase B in vivo and evaluating monoamine oxidase B inhibitor drugs.