Fluorine-18 labeled dopamine receptor 2 targeting probe as well as preparation method and application thereof

Abstract

The invention discloses a fluorine-18 labeled dopamine receptor 2 targeting probe as well as a preparation method and application thereof. The probe takes an antagonist iminopyrone structure of a dopamine receptor 2 as a skeleton, comprises 1-10 polyethylene glycol chains, and is prepared by labeling radioactive signal groups in one step through nucleophilic substitution reaction. The tracer has good uptake in dopamine receptor 2 positive tumors, can distinguish dopamine receptor expression positive and dopamine receptor expression negative tumors in vivo, has strong specificity, meets the conditions of being used as a pancreatic cancer dopamine receptor 2 imaging agent, and has a structural general formula of # imgabs0 #.

Description

Technical Field

[0001] The invention belongs to the technical field of medical imaging, and in particular relates to a fluorine-18 labeled dopamine receptor 2 targeted radioactive tracer, a preparation method and an application thereof. Background Art

[0002] Dopamine receptors are common nerve-related receptors, which are divided into five subtypes according to their pharmacological properties. Among them, dopamine receptor 2 (DRD2) has received special attention in various physiological and pathological processes. DRD2 is closely related to dopaminergic neural signaling. With the further development of tumor neuroscience, the regulation of tumors by nerves has received attention. DRD2 has been found to be closely related to various properties of tumors, such as tumor proliferation, metastasis, and drug resistance. In addition, dopamine receptor 2 antagonists used as traditional antipsychotic drugs have been found to effectively inhibit tumor proliferation and metastasis. In addition, ONC201, a new anti-tumor drug entering Phase III clinical trials, has been verified to target DRD2. It can be seen that targeting dopamine receptor 2 has potential clinical application value. However, since antipsychotic drugs have certain side effects, it is necessary to screen the population for which they are suitable. At present, there is a lack of effective strategies for tumor DRD2 imaging, and it is difficult to effectively predict the sensitivity of tumor patients to drugs targeting DRD2. It is necessary to find relevant explorations to achieve DRD2 visualization. Positron emission tomography (PET) is a functional imaging technique that provides images of biochemical processes by detecting the distribution of radioactive tracers in the body. Compared with other imaging techniques, PET can provide a functional view of biological processes in vivo and can be accurately quantified. This ability makes PET particularly effective in the early diagnosis of diseases, and therefore more suitable for in vivo targeted imaging studies of DRD2. The present invention uses the DRD2-targeted small molecule drug ONC212 as a targeting molecule, modifies it with p-toluenesulfonyloxy (OTs), and then labels it with fluorine-18. The synthetic raw materials of the whole process are easy to obtain and the operation is simple. More importantly, in a pancreatic cancer-bearing mouse model, the tumor has high specific uptake and low background uptake of the DRD2 probe, and the tumor's uptake of compound C10 can be effectively inhibited by ONC212, proving the specificity of the DRD2 targeting probe. The present invention lays a foundation for the detection and application of DRD2 in tumor diseases and its prediction of the therapeutic effect of DRD2 antagonists. Summary of the invention

[0003] The primary purpose of the present invention is to provide a fluorine-18 labeled dopamine receptor 2 targeting small molecule probe, which is named compound C10. Compared with other existing probes targeting DRD2, this molecule uses the anti-tumor drug ONC212 targeting DRD2 as a substrate, can better accumulate in tumors, and effectively predict the effect of mipridones in tumors. Furthermore, one of the technical solutions adopted by the present invention to achieve the technical problem is: the tracer comprises a dopamine receptor 2 targeting group iminopyrrolidone structure in its molecule, comprising 1 to 10 polyethylene glycol chains and 18 F radioactive signal group. The specific structural formula of the tracer is as follows: In a preferred embodiment of the present invention, the reaction scheme is as follows: Here, TsO represents a p-toluenesulfonyloxy group. The second technical solution adopted by the present invention to solve the technical problem is: a method for preparing a dopamine receptor targeting radioactive tracer, which specifically comprises the following steps: 1) Compound C1, potassium carbonate and (2-bromoethoxy)(tert-butyl)dimethylsilane are dissolved in a dimethylformamide (DMF) solution at a molar ratio of (1-3):(2-4):(1-3), and reacted at 60-100° C. for 2-12 h under a nitrogen atmosphere. After the reaction, the compound C2 is purified to obtain compound C2. 2) Compound C2, ethylenediamine monohydrate, and sodium borohydride (NaBH4) were mixed in a methanol (MeOH) solution at a molar ratio of (0.5-1.5): (2-10): (0.5-1.5), and reacted at room temperature for 1-3 hours. After the reaction, the mixture was purified to obtain compound C3; 3) Compound C3 and hydrogen bromide were dissolved in ethanol (EtOH) at a molar ratio of (0.5-1.5): (0.5-5), reacted at room temperature for 1-6 hours, and purified to obtain compound C4; 4) Compound C5, compound C6, and potassium carbonate in a molar ratio of (1-3): (1-6): (1-5) Mix in acetonitrile (MeCN), react at 50-90°C for 1-12h, and purify to obtain compound C7; 5) Compound C4 and Compound C7 are dissolved in a methanol solution containing 20% ​​to 35% sodium methoxide in a molar ratio of (1 to 1.5): (1 to 2), and refluxed at 40 to 70° C. for 4 to 24 hours. After the reaction, the compound C8 is purified to obtain the compound; 6) Compound C8, triethylamine (Et3N), 4-dimethylaminopyridine (DMAP) and p-toluenesulfonyl chloride (TsCl) in a molar ratio of (1-2): (1.1-2.5): (0.04-0.08): (0.4-0.9) The compound was dissolved in dichloromethane and reacted at 0-25°C for 2-24 hours to obtain compound C9, which is the probe-labeled precursor compound of compound C10. Compound C8, triethylamine (Et3N), 4-dimethylaminopyridine (DMAP) and p-toluenesulfonyl chloride (TsCl) are dissolved in dichloromethane at a molar ratio of (1-2): (1.1-2.5): (0.04-0.08): (0.4-0.9), and reacted at 0-25°C for 2-24h to obtain compound C9, i.e., compound C10 probe-labeled precursor compound. In a preferred embodiment of the present invention, in step 6), C9 is dissolved in organic solvent A, and K is added. 222 , K2CO3 and fluoride ion, heated at 100-110°C for 10-30 min, to obtain compound C10 through nucleophilic substitution reaction. Organic solvent A is at least one of ethanol, acetonitrile and dimethyl sulfoxide. The reaction route is as follows: Fluorine-18 labeled DRD2 targeting probe compound C10 is a PET imaging agent used to target DRD2 receptors inside tumors and to evaluate the efficacy of corresponding drugs. The beneficial effects of the present invention are: (1) A fluorine-18 labeled DRD2 targeting probe in the present invention is a novel DRD2 targeting PET imaging agent. The preparation method of compound C10 has a high radiochemical yield and radiochemical purity, good stability in PBS, serum and mouse blood, and can be used for in vivo PET imaging of DRD2. (2) A fluorine-18 labeled DRD2 targeting probe compound C10 in the present invention has excellent biological properties. In a tumor-bearing mouse model with positive expression of dopamine receptor 2, compound C10 has high uptake and low background uptake at the tumor site, significant imaging effect at the tumor site, and specific uptake, meeting the conditions for use as a DRD2 targeting PET probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 The synthetic routes of the compound C10 probe-labeled precursor compound C9 and the standard compound C14 of the compound C10 of the present invention; Figure 2 The synthetic route diagram of compound C10 of the present invention; Figure 3 The radio-conversion rate of compound C10 was high in Radio-TLC analysis, and the peak time of compound C10 in high performance liquid chromatography (HPLC) analysis was the same as that of the corresponding standard substance C14, which basically confirmed that compound C10 was successfully labeled. Figure 4 HPLC analysis of the in vitro stability of DRD2-targeted PET probe compound C10; Figure 5Radio-TLC was used to analyze the in vivo stability of DRD2-targeted PET probe compound C10; Figure 6 PET imaging of DRD2-targeted PET probe compound C10 in mice bearing DRD2-positive Miapaca-2 tumors at 1h and 2h after injection of compound C10 alone or co-injection of compound C10 with standard C14, with arrows indicating tumors; and comparison of compound C10 uptake in tumors with uptake of co-injected compound C10 and standard C14; ratio of tumor to muscle uptake. DETAILED DESCRIPTION

[0005] The present invention is further illustrated and described below in conjunction with the embodiments and accompanying drawings. like Figure 1 and Figure 2 As shown, the preparation steps of the fluorine-18 labeled DRD2 targeting probe compound C10 and its standard substance C14 based on the OTs-modified DRD2 targeting small molecule ONC212 are as follows: (1) Synthesis of Compound C9, a Labeling Precursor of Compound C10 Probe ( Figure 1 ) Synthesis of 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzaldehyde (Compound C2) :To a solution of salicylaldehyde (compound C1, 2.12g, 17.4mmol) in dimethylformamide (DMF, 8.5mL), K2CO3 (7.19g, 52.0mmol) was added, and (2-bromoethoxy) (tert-butyl) dimethylsilane (4.98g, 20.8mmol) was added under N2. The reaction mixture was stirred at 80°C for 16h, and poured into water (30mL) after cooling. The resulting mixture was extracted with ethyl acetate (EtOAc, 10mL×3). The combined organic layer was washed with brine (10mL×4), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to obtain a residue, which was purified by column chromatography (silica gel, EtOAc / n-hexane=1 / 20 (v / v)) to obtain compound C1-1. Synthesis of N1-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)phenylmethyl)ethane-1,2-diamine (compound C3) : At 0°C, a solution of compound C2 (1.50 g, 5.35 mmol) in methanol (7.5 mL) was added dropwise to a stirred solution of ethylenediamine monohydrate (2.09 g, 26.8 mmol) in methanol (MeOH, 7.5 mL). The reaction mixture was poured into water (30 mL) and the resulting mixture was extracted with EtOAc (20 mL × 2). The combined organic layers were washed with brine (30 mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give a colorless oil. Synthesis of 1-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzyl)tetrahydropyrimidine-2(1H)-hydrobromide Salt (Compound C4): To a stirred solution of crude compound C3 (1.50 g, 4.62 mmol) in ethanol (EtOH, 10 mL) cooled in an ice-water bath under N2, hydrogen bromide (BrCN, 0.54 g, 5.10 mmol) was slowly added. The reaction mixture was stirred at room temperature overnight. The solvent was removed on a rotary evaporator to obtain a colorless oil. Synthesis of methyl 4-oxo-1-(4-(trifluoromethyl)benzyl)piperidine-3-carboxylate (Compound C7) :To a stirred solution of 4-oxo-1-(4-trifluoromethyl)piperidine-3-carboxylic acid methyl ester hydrochloride (compound C6, 5.00g, 25.8mmol) in DMF (75mL), K2CO3 (10.70g, 77.4mmol) and 4-(trifluoromethyl)benzyl bromide (compound C5, 7.40g, 31.0mmol) were added successively under N2. The reaction mixture was stirred at 70°C for 16h. After cooling to room temperature, the mixture was poured into water (120mL) and the resulting mixture was extracted with EtOAc (100mL×2). The combined organic layers were washed with brine (100mL×4), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to obtain a residue, which was purified by column chromatography (silica gel, EtOAc / n-hexane=1 / 20 to 1 / 10 (v / v)) to obtain a colorless oil, which was directly used in the next step. Synthesis of 3-(2-(3-hydroxypropyl)benzyl)-7-(4-(trifluoromethyl)benzyl)-2,3,6,7,8,9-hexahydroimidazole O-[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one (Compound C8) :Under N2, sodium methylate (NaOMe, 0.43g, 7.96mmol) was added to a stirred solution of crude compound C4 (1.00g, 2.01mmol) and crude compound C7 (0.71g, 2.25mmol) in MeOH (10mL). After cooling to room temperature, the mixture was poured into water (15mL) and extracted with dichloromethane (CH2Cl2, 20mL×2). The combined organic layer was washed with salt water (20mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to obtain a residue, which was purified by column chromatography (silica gel, MeOH / CH2Cl2=1 / 20 (v / v)) to obtain compound C8. Synthesis of 3-(2-((5-oxo-7-(4-(trifluoromethyl)benzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a] Pyrido[3,4-e]pyridin-3(5H)-yl)methyl)phenyl)propyl 4-methylbenzenesulfonate (Compound C9): Under N2, triethylamine (Et3N, 0.13g, 1.28mmol) and DMAP (6mg, 0.049mmol) were added to a stirred solution of compound C8 (0.21g, 0.42mmol) in CH2Cl2 (2mL) successively. Then, p-toluenesulfonyl chloride (TsCl, 0.08g, 0.42mmol) was slowly added to the reaction mixture at 0°C. The reaction mixture was stirred at room temperature for 1.5h and then poured into water (10mL). The resulting mixture was extracted with CH2Cl2 (10mL×2). The combined organic layers were washed with brine (10 mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give a residue, which was purified by column chromatography (silica gel, EtOAc / n-hexane = 1 / 1 to 1 / 0 (v / v)) to give 3-(2-((5-oxo-7-(4-(trifluoromethyl)benzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyridin-3(5H)-yl)methyl)phenyl)propyl 4-methylbenzenesulfonate, i.e., compound C10 probe labeled precursor compound. (2) Preparation of the final product compound C10 ( Figure 2 ): Take 1mL of Kryptofix222 / K2CO3 mixed solution to elute the QMA column and collect the eluent; heat the nitrogen blower to 110℃ to blow dry the eluent, then add 0.50mL of anhydrous acetonitrile 4 times continuously after drying, remove the remaining water by azeotropic distillation, cover and cool to room temperature. Transfer 3-(2-((5-oxo-7-(4-(trifluoromethyl)benzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyridin-3(5H)-yl)methyl)phenyl)propyl 4-methylbenzenesulfonate (2.00mg) dissolved in 200μL of anhydrous acetonitrile to the dried 18 The F complex was removed and reacted at 110°C for 20 min. After cooling to ambient temperature, the product was purified using a HPLC semi-preparative column, and the collected product was enriched on a Sep-Pak Plus C18 column, and the radiolabeled product was eluted with 1-2 mL of ethanol. (3) Preparation of Standard C14 ( Figure 1 ) 2-(2-Fluoroethoxy)benzaldehyde(C11):To a stirred solution of salicylaldehyde (5.00 g, 40.9 mmol) in DMF (60 mL), K2CO3 (16.98 g, 122.9 mmol) and 1-fluoro-2-iodoethane (10.68 g, 61.4 mmol) were added under N2. The reaction mixture was stirred at 50 ° C and poured into water (200 mL) after cooling to room temperature. The resulting mixture was extracted with EtOAc (100 mL × 2). The combined organic layer was washed with brine (100 mL × 4), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to obtain a residue, which was purified by column chromatography (silica gel, EtOAc / n-hexane=1 / 20 (v / v)) to obtain compound C11. N1-(2-(2-Fluoroethoxy)benzyl)ethane-1,2-diamine (C12) :To a stirred solution of ethylenediamine monohydrate (2.32 g, 29.7 mmol) in MeOH (5 mL), a solution of compound C11 (1.00 g, 5.95 mmol) in MeOH (5 mL) was slowly added at 0°C, N2. Then NaBH4 (0.22 g, 5.82 mmol) was slowly added to the reaction mixture. The mixture was poured into water (20 mL) and the resulting mixture was extracted with EtOAc (20 mL×2). The combined organic layers were washed with brine (20 mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give compound C12. 1-(2-(2-Fluoroethoxy)benzyl)tetrahydropyrimidine-2(1H)-imine hydrobromide (C13) : In an ice-water bath under N2, BrCN (0.49 g, 4.63 mmol) was slowly added to a stirred solution of compound C12 (0.90 g, 4.24 mmol) in EtOH (10 mL). The reaction mixture was stirred at room temperature overnight. The solvent was removed by rotary evaporator to obtain compound C13. 3-(2-(2-fluoroethoxy)benzyl)-7-(4-(trifluoromethyl)benzyl)-2,3,6,7,8,9-hexahydroimidazo[1, 2-a]Pyrido[3,4-e]pyrimidin-5(1H)-one(C14) : Under N2, NaOMe (0.41 g, 7.59 mmol) was added to a stirred solution of compound C·13 (0.80 g, 2.41 mmol) and crude C7 (0.99 g, 3.14 mmol) in MeOH (10 mL). The reaction mixture was stirred at 70°C for 5 h, cooled to room temperature and poured into water (25 mL). The resulting mixture was extracted with CH2Cl2 (20 mL×2). The combined organic layer was washed with brine (20 mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to obtain a residue, which was purified by column chromatography (silica gel, EtOAc / n-hexane=1 / 1 to 1 / 0 (volume fraction)) to obtain compound C14. like Figure 3 As shown, compound C10 was analyzed by TLC, and the radioactive conversion rate was high; and by HPLC analysis, the peak time of compound C10 was the same as that of standard substance C14, which basically confirmed that compound C10 was successfully labeled. Taking the above compound C10 developer as an example, the experimental results show that its basic properties are as follows: (1) Stability of compound C10 in vitro and in vivo in mice Compound C10 was added to PBS and serum, mixed, and allowed to stand for 0.5, 1, and 2 hours, and then the radiochemical purity of compound C10 was detected by TLC to determine whether it was stable. Figure 4 As shown, compound C10 remained stable in the blood for at least 2 hours after injection. Four-week-old C57BL / 6 mice were selected to study the in vivo stability of compound C10. To monitor the stability of radioactive metabolites in the blood of C57BL / 6 mice at different time points, approximately 0.3mCi / 100μL of compound C10 injection solution was injected into each mouse (n=3) through the tail vein, and then the radiochemical purity of compound C10 was monitored by TLC to determine whether it was stable. Figure 5 The compound C10 remained stable in the blood of mice for at least 2 hours after injection. (2) PET imaging of compound C10 in pancreatic cancer-bearing mice Four-week-old C57BL / 6 mice were selected and Miapaca-2 human pancreatic cancer cells were implanted in the right upper limb. PET imaging was performed when the tumor grew to a diameter of 0.6-0.8 cm. Each mouse (n=3) was injected with approximately 0.3 mCi / 100 μL of compound C10 injection solution through the tail vein, with or without injection of standard C14, and inhalation anesthesia was performed with isoflurane 1 hour and 2 hours after injection. Static PET scanning was performed after prone fixation. The above experiments were used to observe the in vivo PET imaging of compound C10 in pancreatic cancer-bearing mice. The results are shown in Figure 6 As shown in the figure, the imaging results show that compound C10 is significantly enriched in the tumor site in pancreatic cancer tumors within 1 hour. In addition, co-injection of standard C14 and compound C10 significantly blocked the enrichment of compound C10 probe in vivo, which can be further explained by the %ID / g value of the tumor site and the tumor / muscle signal ratio. The above results fully demonstrate that compound C10 has high targeting to tumors expressing DRD2. In contrast, the results of co-injection of compound C10 and standard substance C14 showed that standard substance C14 could effectively inhibit the uptake of compound C10, which fully demonstrated that compound C10 has high specificity for tumors expressing DRD2. The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of implementation of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A fluorine-18 labeled dopamine receptor 2 targeted radiotracer, characterized in that: Its molecule contains the dopamine receptor 2 targeting group iminopyrone structure, which contains 1 to 10 polyethylene glycol chains and 18 F Radioactive signal group; the general structural formula of the tracer is as follows:

2. The method for preparing a dopamine receptor 2 targeted radioactive tracer precursor according to claim 1, characterized in that: The reaction route is as follows:

3. The method for preparing a dopamine receptor 2 targeted radioactive tracer precursor according to claim 2, characterized in that: The specific steps include: 1) Compound C1, potassium carbonate and (2-bromoethoxy)(tert-butyl)dimethylsilane are dissolved in a dimethylformamide (DMF) solution at a molar ratio of (1-3):(2-4):(1-3), and reacted at 60-100° C. for 2-12 h under a nitrogen atmosphere. After the reaction, the compound C2 is purified to obtain compound C2. 2) Compound C2, ethylenediamine monohydrate, and sodium borohydride (NaBH4) in a molar ratio (0.5-1.5): (2-10): (0.5-1.5) was mixed in methanol (MeOH) solution, reacted at room temperature for 1-3 hours, and purified to obtain compound C3 after the reaction; 3) Compound C3 and hydrogen bromide were dissolved in ethanol (EtOH) at a molar ratio of (0.5-1.5): (0.5-5), reacted at room temperature for 1-6 hours, and purified to obtain compound C4; 4) Compound C5, compound C6, and potassium carbonate are mixed in acetonitrile (MeCN) in a molar ratio of (1-3): (1-6): (1-5), reacted at 50-90° C. for 1-12 h, and purified to obtain compound C7; 5) Compound C4 and Compound C7 are dissolved in a methanol solution containing 20% ​​to 35% sodium methoxide in a molar ratio of (1 to 1.5): (1 to 2), and refluxed at 40 to 70° C. for 4 to 24 hours. After the reaction, the compound C8 is purified to obtain the compound; 6) Compound C8, triethylamine (Et3N), 4-dimethylaminopyridine (DMAP) and p-toluenesulfonyl chloride (TsCl) are dissolved in dichloromethane in a molar ratio of (1-2): (1.1-2.5): (0.04-0.08): (0.4-0.9), and reacted at 0-25°C for 2-24h to obtain compound C9, i.e., the probe-labeled precursor compound.

4. A method for preparing a dopamine receptor 2 targeted radioactive tracer, characterized in that: The reaction route is as follows:

5. The method for preparing a dopamine receptor 2 targeted radioactive tracer according to claim 4, characterized in that: Specifically, the steps include: dissolving compound C9 in an organic solvent A, adding 4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacosane (K 222 ), potassium carbonate and fluoride ion, heated at 100-120° C. for 10-30 min, to obtain compound C10 through nucleophilic substitution reaction. Organic solvent A is at least one of ethanol, acetonitrile and dimethyl sulfoxide.

6. The use of a fluorine-18 labeled dopamine receptor 2 targeting probe as claimed in claim 1, characterized in that: Positron emission tomography (PET) imaging agent for labeling 18F.

7. The use of a fluorine-18 labeled dopamine receptor 2 targeting probe as claimed in claim 1, characterized in that: Imaging agent for dopamine receptor positive cells labeled with 18F.

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