Preparation method of alpha-type 2-amino-dihydrotetrabenazine

Abstract

The invention provides a preparation method of alpha-type 2-amino-dihydrotetrabenazine, which comprises the following steps: 1) reacting raw materials containing tetrabenazine, ammonium acetate and sodium cyanoborohydride, and post-treating a reaction mixed solution to obtain a crude product; (2) carrying out column chromatography on the crude product in the step (1), and separating to obtain alpha-type 2-amino-dihydrotetrabenazine; a stationary phase used by the column chromatography is silica gel; the diameter-height ratio of the stationary phase is 1: (6-8), a mixed solution of dichloromethane and methanol is adopted as an elution system, and the volume ratio of dichloromethane to methanol is (9-11): 1; the elution flow rate is 4 to 6 mL / min. Radioactive labeling of the alpha-type 2-amino-dihydrotetrabenazine is realized through a fluorine [< 18 > F] labeling technology, a key technical support is provided for development of a novel positron emission tomography (PET) specific tracing probe based on a molecular skeleton, and an important technical foundation is laid for development of a novel VMAT2 targeted tracing probe based on a compound skeleton.

Description

Technical Field

[0001] This invention relates to the field of medical technology, specifically to a method for preparing α-type 2-amino-dihydrobutanazine. Background Technology

[0002] Parkinson's disease (PD) is a neurodegenerative disease characterized primarily by motor dysfunction, with its core pathological mechanism being the progressive degeneration of dopaminergic neurons in the substantia nigra of the midbrain. Currently, PD diagnosis largely relies on typical clinical manifestations, but early-stage PD patients often present with atypical symptoms. Therefore, developing imaging probes capable of early detection of dopaminergic neuronal degeneration is of significant clinical importance. Currently, imaging probes targeting PD primarily target key sites in the dopamine metabolic pathway, including aromatic amino acid decarboxylases (AADCs), dopamine transporters (DATs), and vesicular monoamine transporter 2 (VMAT2). Among these, VMAT2 has become a research hotspot in recent years due to its central role in vesicle storage of monoamine neurotransmitters. VMAT2 is a transport protein located on the synaptic vesicle membrane, responsible for transporting monoamine neurotransmitters (such as dopamine, norepinephrine, and serotonin) from the cytoplasm to vesicles for storage. In Parkinson's disease (PD), the expression level of VMAT2 is closely related to the survival status of dopaminergic neurons, and its reduction directly reflects the degree of neuronal degeneration. Therefore, the development of VMAT2 imaging probes not only helps in the early diagnosis of PD but can also be used for disease progression monitoring and treatment efficacy evaluation.

[0003] Dihydrotetrabenazine (DTBZ) is a high-affinity inhibitor of VMAT2 and has been successfully used in the development of radiographic probes. For example, [ 11 C]DTBZ ] As a positron emission tomography (PET) probe, it can specifically bind to VMAT2, but its short half-life (T0) 1 / 2 The limited clinical application of the 20-minute half-life (T20) model was initially limited. Subsequently, research focus gradually shifted to longer half-lives (T20). 1 / 2 =110min) 18 Development of F-labeled DTBZ derivatives, such as [ 18F]FP-DTBZ has been proven to be a potential VMAT2 imaging probe with high safety, high initial brain uptake, rapid brain clearance, and a high target / non-target ratio. To advance the development of novel VMAT2 imaging probes, the synthesis and structure-activity optimization of dihydrobutylbenazine 2-position amino derivatives have become important research directions in this field. Due to the existence of multiple isomers with significant differences in biological activity, isomer separation and purification techniques have become essential research content in this field. However, the diastereomeric separation technique for 2-amino-dihydrobutylbenazine (2-NH2-DTBZ) remains unsolved, and the products obtained by literature preparation methods are mixed diastereomeric forms. Summary of the Invention

[0004] To achieve efficient separation of diastereomers of 2-amino-dihydrobenzonazine (2-NH2-DTBZ) and prepare VMAT2-targeted radioimaging probes with superior performance, this invention proposes a method for preparing α-type 2-amino-dihydrobenzonazine. The technical solution adopted in this invention is as follows:

[0005] A method for preparing α-type 2-amino-dihydrobutanazine includes the following steps:

[0006] Step 1) React the raw materials containing butylbenazine, ammonium acetate and sodium cyanoborohydride to obtain a reaction mixture; perform post-treatment on the reaction mixture to obtain the crude product;

[0007] Step 2) The crude product from Step 1 is subjected to column chromatography to separate α-type 2-amino-dihydrobenzenarazine; the stationary phase used in the column chromatography is silica gel; the stationary phase diameter-to-height ratio is 1:6-8, and a mixture of dichloromethane and methanol is used as the elution system, with a volume ratio of dichloromethane to methanol of 9-11:1; the elution flow rate is 4-6 mL / min.

[0008] Optionally, in step one), the molar ratio of bubenazine: ammonium acetate: sodium cyanoborohydride is 1-2:9-11:1-2;

[0009] Preferably, the raw materials in step one further include a solvent, wherein the solvent comprises at least one of methanol, ethanol, and propanol;

[0010] Preferably, in the raw materials of step one), the mass fraction of butylbenazine is 2-6%.

[0011] Optionally, the post-treatment method for the reaction mixture includes: diluting and filtering the reaction product with a solvent; removing the solvent from the filtrate and dissolving the residue in water to obtain a reaction product solution; adjusting the pH of the reaction product solution to <4 and then extracting it with ether; adjusting the pH of the raffinate to >10 and then extracting it with ether; combining the ether extracts, washing and drying to obtain the crude product.

[0012] Optionally, in step two), the separation product is monitored by thin-layer chromatography during column chromatography to separate α-type 2-amino-dihydrobutanazine.

[0013] Optional steps include the following:

[0014] α-type 2-amino-dihydrobutanazine was prepared using the above-described method.

[0015] The α-type 2-amino-dihydrobutanazine is labeled with a radioactive isotope, and a group containing the radioactive isotope is attached to the amino group of the α-type 2-amino-dihydrobutanazine to obtain the radioactively labeled α-type 2-amino-dihydrobutanazine.

[0016] Optionally, the radioactive isotope is 18 F.

[0017] Optionally, the labeling reaction of α-type 2-amino-dihydrobenazine with a radioactive isotope includes: 18 F - The mixture was reacted with 1,3-bis(toluenesulfonyloxy) at a constant temperature of 80–100 °C for 10–30 minutes, and then reacted with the α-type 2-amino-dihydrobutanazine at 110–150 °C for 15–35 minutes to obtain radioisotope-labeled α-type 2-amino-dihydrobutanazine.

[0018] The present invention also proposes radioisotope-labeled α-amino-dihydrobutanazine prepared by the above method.

[0019] The present invention also proposes the application of the above-mentioned radioisotope-labeled α-amino-dihydrobutenazine in the preparation of products for diagnosing diseases related to type 2 vesicular monoamine transporters.

[0020] Optionally, the disease associated with the type 2 vesicular monoamine transporter is Parkinson's disease.

[0021] The beneficial effects of this invention are:

[0022] This invention successfully achieved the efficient separation of the diastereomers of 2-amino-dihydrobutanazine using column chromatography, and confirmed the chemical structures of the two pairs of diastereomers through systematic structural characterization. Furthermore, this invention also utilizes fluorine... 18 The F-labeling technology enabled the radioactive labeling of α-type 2-amino-dihydrobenazine, providing key technical support for the development of novel positron emission tomography (PET) specific tracer probes based on this molecular framework, and laying an important technical foundation for the development of novel VMAT2 targeted tracer probes based on this compound framework. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is the synthetic route for 2-amino-dihydrobutenazine;

[0025] Figure 2 It is the fluorine of 2-amino-dihydrobutenazine. 18 Synthetic routes for F-labeled and non-radioactive control compounds; where (a) is fluorine [ 18 F] indicates the synthetic route, and (b) is the synthetic route of the non-radioactive control compound;

[0026] Figure 3 Here are the chemical structure diagrams of the two pairs of diastereomers of 2-amino-dihydrobutanazine;

[0027] Figure 4 The results are column chromatography and related mass spectrometry characterization of crude 2-amino-dihydrobutanazine, where (a) is the TLC monitoring result, (b) is the mass spectrometry characterization of fraction I, (c) is the mass spectrometry characterization of fraction II, and (d) is the mass spectrometry characterization of fraction III.

[0028] Figure 5 The results are the 1H NMR characterization of the diastereomers of 2-amino-dihydrobutanazine, where (a) is the 1H NMR spectrum of α-type 2-amino-dihydrobutanazine and (b) is the 1H NMR spectrum of β-type 2-amino-dihydrobutanazine.

[0029] Figure 6 The results are the carbon NMR spectra of the diastereomers of 2-amino-dihydrobutanazine, where (a) is the carbon NMR spectrum of α-type 2-amino-dihydrobutanazine and (b) is the carbon NMR spectrum of β-type 2-amino-dihydrobutanazine.

[0030] Figure 7 It is 2-amino-dihydrobutanazine fluoride. 18 F] indicates the chemical structure verification results, where (a) is [ 18 (a) Chromatographic comparison of F5 and non-radioactive compound 5, and (b) Mass spectrometric characterization of non-radioactive compound 5.

[0031] Figure 8The column chromatography and related mass spectrometry characterization of crude 2-amino-dihydrobutanazine in Comparative Example 1 are shown in (a) column chromatography TLC monitoring results, (b) mass spectrometry characterization of fraction I, and (c) mass spectrometry characterization of fraction II.

[0032] Figure 9 The column chromatography and related mass spectrometry characterization of crude 2-amino-dihydrobutanazine in Comparative Example 2 are shown in (a) column chromatography TLC monitoring results, (b) mass spectrometry characterization of fraction I, and (c) mass spectrometry characterization of fraction II.

[0033] Figure 10 The column chromatography and related mass spectrometry characterization of crude 2-amino-dihydrobutanazine in Comparative Example 3 are shown in (a) column chromatography TLC monitoring results, (b) mass spectrometry characterization of fraction I, and (c) mass spectrometry characterization of fraction II. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0035] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Example 1

[0039] Synthesis and purification of 2-amino-dihydrobutenazine: The synthetic route of 2-amino-dihydrobutenazine is as follows... Figure 1 As shown, butylbenazine (TBZ, compound 1) is converted to 2-amino-dihydrobutylbenazine (2-NH2-DTBZ, compound 2) by Borch reduction ammoniation in the presence of ammonium acetate and sodium cyanoborohydride.

[0040] (1) Chemical synthesis: Bubenazine (2g, 6.3mmol), ammonium acetate (4.86g, 63mmol) and sodium cyanoborohydride (0.4g, 6.3mmol) were dissolved in 50mL of anhydrous methanol, and 3g of molecular sieve was added. The mixture was stirred continuously at room temperature for 24 hours. After the reaction, the reaction solution was diluted with 100 mL of methanol and filtered. The methanol solvent was removed by concentration under reduced pressure, and the residue was dissolved in 50 mL of water. The aqueous phase was adjusted to pH < 4 with concentrated hydrochloric acid, followed by extraction with diethyl ether (100 mL × 2). After discarding the organic phase, the aqueous phase was adjusted to pH > 10 with 5M sodium hydroxide solution and extracted again with diethyl ether (100 mL × 2). The ether extracts were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain the crude product.

[0041] (2) Separation and Purification: 300-400 mesh dry silica gel was wet-packed into a 4 cm diameter glass chromatography column. During packing, a rubber mallet was used to gently tap the column wall to remove air bubbles, ensuring dense packing and forming a stable silica gel column system with a diameter-to-height ratio of approximately 1:7. Approximately 2 g of the crude 2-NH2-DTBZ obtained from the previous synthesis was completely dissolved in 2 mL of anhydrous methanol and slowly loaded onto the silica gel surface along the column wall. A layer of defatted cotton was then placed over the silica gel surface to effectively prevent eluent erosion and column surface disturbance. A dichloromethane-methanol (10:1, v / v) mixture was used as the elution system, with an elution flow rate of approximately 5 mL / min. Eluent was collected in conical flasks at approximately 30 mL / s, and the separation process was monitored in real-time using thin-layer chromatography (TLC). Finally, the target components were differentially collected based on the TLC detection results, achieving effective separation of the diastereomers of 2-NH2-DTBZ.

[0042] Example 2

[0043] Fluorine of 2-amino-dihydrobutenazine 18 F] Label: Fluorine of 2-amino-dihydrobutanazine [ 18 F] Marking process such as Figure 2 As shown in Figure a, a two-step one-pot method was used for radioactive fluorine labeling of this compound. The synthetic route for the non-radioactive control compound (5) is as follows. Figure 2 As shown in b, this control is used as a key reference for the structural confirmation of the radiolabeled product.

[0044] (1) Preparation of non-radioactive control compound: Compound 2 (5 mg, 12.8 μmol) was placed in a nitrogen-protected three-necked flask, dissolved in anhydrous DMF (1.0 mL), and then Cs₂CO₃ (20.85 mg, 64 μmol) and 1-bromo-3-fluoropropane (1.5 μL, 15.96 μmol) were added sequentially. The mixture was heated and stirred in an oil bath at 90 °C for 12 h until the reaction solution turned brown. After the reaction was completed, the mixture was quenched with 10 mL of water. The aqueous phase was extracted with ethyl acetate (3 × 25 mL), and the combined organic phases were dried over anhydrous sodium sulfate. The crude product 5 was concentrated and purified by semi-preparative HPLC. Chromatographic column: Phenemenex Gemini, 5 μm, C18, 10 × 250 mm; column temperature: room temperature; mobile phase: acetonitrile, water and trifluoroacetic acid (20:80:0.1); flow rate: 3.0 mL / min; detection wavelength: 280 nm.

[0045] (2) Radioactive fluorine [ 18 F] Marking: Fluorine [ 18 F] labeled compounds ([ 18 The preparation of F]5) was carried out using a two-step, one-pot manual radiosynthesis process. ① Fluorine [ 18 F] ion preparation: via 18 O(p,n) 18 The F nuclear reaction produces fluorine in the cyclotron. 18 F] ions, after being captured by a QMA column, were treated with 1 mL of Stock solution (150 mg K) 222 Elution was performed using a solution of 50 mg K₂CO₃ and 1 mL water dissolved in 8 mL acetonitrile. The collected [ 18 F]F - The eluent (initial activity 11.1 GBq) was evaporated and concentrated under a nitrogen stream at 95°C for 3 minutes, followed by the addition of 2 mL of acetonitrile and further drying for 2 minutes. ② Radionuclide labeling reaction: […] 18 F]F - 0.5 mL of acetonitrile solution containing 8 mg of 1,3-bis(toluenesulfonyloxy)propane was added to the system, and the mixture was stirred at 95 °C for 15 minutes. After removing the solvent with nitrogen, 0.3 mL of DMF solution containing 8 mg of compound 2 was injected, and the reaction was continued at 135 °C for 25 minutes. ③ Product separation and purification: After the reaction was terminated, purification was performed using a semi-preparative HPLC system (Waters XBridge C18 column, 10×250 mm, 5 μm). The mobile phase was acetonitrile / water / triethylamine (35:65:0.1, v / v / v), and the flow rate was 3.0 mL / min. The collected radioactive products [ 18 The F5 fraction was diluted with 50 mL of deionized water and enriched using a Sep-Pak C18 column. The product was then eluted with 2 mL of ethanol. 18F]5. ④ Product analysis and verification: HPLC identification was performed using an analytical Phenomenex Gemini C18 column (4.6×150mm, 5μm). The mobile phase was methanol / water / trifluoroacetic acid (35:65:0.1, v / v / v), and the flow rate was 1.0 mL / min.

[0046] Experimental example:

[0047] This invention systematically studies the isolation of diastereomers of 2-amino-dihydrobutenazine. The molecular structure of this compound contains three chiral centers, theoretically allowing for eight stereoisomers. However, its synthetic starting material (butenazine) contains only two enantiomers: (R,R) and (S,S). Based on this stereochemical characteristic, the newly generated chiral center during the introduction of the amino group theoretically limits the number of stereoisomers of the target product, 2-amino-dihydrobutenazine, to four, including two pairs of diastereomers: α-configuration [(R,R,R) / (S,S,S)] and β-configuration [(R,R,S) / (S,S,R)]. Figure 3 Given the potentially significant differences in biological activity between α and β configurations, establishing an effective method for separating diastereomers is a crucial prerequisite for conducting stereoselective pharmacodynamic studies. However, current technologies have not yet achieved this key separation, severely hindering further research.

[0048] This invention successfully solves this technical problem through systematic optimization of column chromatography conditions. For example... Figure 4 As shown in Figure a, column chromatography (TLC) monitoring of the crude 2-amino-dihydrobutanazine revealed three characteristic fractions (I, II, and III). Mass spectrometry characterization indicated that fraction I was an impurity from the chemical synthesis reaction. Figure 4 b) Fractions II and III exhibited the same quasi-molecular ion peaks, confirming them to be two isomers of the target compound 2-amino-dihydrobutanazine. Figure 4 cd; ESI-MS:m / z[M+H] + calcd forC 19 H 31 N2O2 + (319.46, found 319.49). It is worth noting that some cross-mixing occurs during the current column chromatography fraction collection process. Subsequent repeated column chromatography experiments can further improve separation efficiency by reducing the amount of eluent collected per bottle.

[0049] This invention successfully achieved the efficient separation of two pairs of diastereomers of 2-amino-dihydrobutanazine, providing key technical support for subsequent chiral resolution and structure-activity relationship studies of single isomers. Since the α-configuration is slightly less polar than the β-configuration, based on this characteristic and the elution order of column chromatography in this invention (fraction II is eluted before fraction III), fraction II is α-type 2-amino-dihydrobutanazine, and fraction III is β-type 2-amino-dihydrobutanazine.

[0050] Furthermore, we characterized the structures of two pairs of diastereomers of 2-amino-dihydrobutenazine using nuclear magnetic resonance (NMR) technology. Figure 5 , Figure 6 ).

[0051] α-type 2-amino-dihydrobutenazine: 1 H NMR(400MHz,Chloroform-d)δ6.69(s,1H),6.58(s,1H),3.85(s,6H),3.41(d,J=11.5Hz,1H),3.25(q,J =3.4Hz,1H),3.12(ddd,J=16.5,11.9,6.0Hz,1H),2.96(ddd,J=11.4,6.2,1.8Hz,1H),2.64(td,J=12.1 ,4.2Hz,2H),2.54(td,J=11.5,4.1Hz,1H),2.42-2.25(m,2H),2.03(dtt,J=11.4,7.3,3.8Hz,1H),1.80 -1.54(m,4H),1.21(dt,J=13.9,7.1Hz,1H),1.11(dt,J=13.8,7.4Hz,1H),0.93(dd,J=8.2,6.5Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ147.37,147.10,130.10,126.93,111.53,108.07,56.45 ,56.13,55.99,55.83,52.61,47.91,39.64,39.45,37.27,29.16,24.84,23.13,22.89.

[0052] β-type 2-amino-dihydrobutenazine: 1H NMR(400MHz,Chloroform-d)δ6.70(s,1H),6.57(s,1H),3.84(s,6H),3.16-2.9 4(m,4H),2.68-2.58(m,1H),2.47(dddt,J=15.5,11.6,7.6,3.4Hz,3H),1.97(t ,J=11.2Hz,1H),1.81(s,2H),1.67(d,J=3.9Hz,1H),1.57-1.44(m,2H),1.44-1 .23(m,1H),1.04(ddd,J=14.1,10.5,4.2Hz,1H),0.93(dd,J=11.4,6.5Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ147.41,147.19,129.62,126.41,111.45,107.99,61.73 ,60.84,55.92,55.83,54.91,52.14,42.26,41.34,40.04,29.15,25.37,24.27,21.65.

[0053] 1H NMR spectrum 1 ¹H NMR analysis revealed that the two pairs of diastereomers of 2-amino-dihydrobutylbenazine exhibited significant common spectral features in key structural units: two sets of singlet signals (δ 6.7 and 6.6 ppm) were observed in the benzene ring region, suggesting the presence of two aromatic protons in different chemical environments. This phenomenon may be due to the symmetry difference of the benzene ring substituents, which caused the protons to split into two equivalent systems; the characteristic peak of methoxy (-OCH3) appeared in singlet form at δ 3.8 ppm, and its chemical shift was consistent with the typical characteristics of ortho-substituted aromatic rings, and no coupling splitting phenomenon was observed, indicating that this substituent was in a chemical environment with higher symmetry; the methyl proton (-CH3) at the end of the molecule showed a characteristic double peak signal (δ 0.93 ppm), suggesting that there was significant spatial coupling between the methyl group and the adjacent methine proton. Furthermore, it is noteworthy that the two pairs of diastereomers exhibit significant differences in the methylene (-CH2-) and methine (-CH-) regions near the three chiral centers. Their complex splitting patterns can be attributed to variations in coupling constants and electronic microenvironment perturbations caused by differences in spatial configuration between the diastereomers. 13In 12C NMR spectroscopy characterization, both pairs of diastereomers of 2-amino-dihydrobutylbenazine exhibited 19 characteristic signals (excluding solvent peaks) precisely corresponding to the 19 carbon atoms of the molecule. Spectroscopic analysis showed that the three characteristic carbon signals (around δ147, 130, and 110 ppm) in the benzene ring substitution region maintained high consistency across the two pairs of isomers, consistent with the electronic structure characteristics of the rigid aromatic system in the molecule. Notably, the chemical shifts of the remaining carbon atoms exhibited shift differences of 1–5 ppm between the different diastereomers. This phenomenon can be attributed to the influence of specific chiral centers in the stereoisomers on the stereochemical environment of neighboring carbon atoms. Systematic analysis revealed that the splitting patterns, shift characteristics, and relative intensities of all carbon signals highly matched the complex stereochemical structure and multi-substituted aromatic system of the target molecule.

[0054] This invention provides an in-depth analysis of the efficient separation mechanism of two pairs of diastereomers of 2-amino-dihydrobutylbenazine. Based on the principle of chiral environment regulating NMR behavior, under achiral conditions, protons at the same position in an enantiomer mixture exhibit identical chemical shifts due to being in an isotropic magnetic environment. In stark contrast, due to the anisotropic magnetic shielding effect caused by differences in spatial configuration, protons at the same position in a diastereomer mixture typically exhibit significantly split multiplying signals in the proton NMR spectrum. Experimental results... 1 H NMR characterization results ( Figure 5 The results show that the two pairs of diastereomers of 2-amino-dihydrobenzonazine exhibit independent single-peak signals in the two characteristic protons of the benzene ring region. It should be noted that if diastereomers coexist in the system, theoretical predictions suggest that multiple splitting signals should be observed. The excellent resolution and complete peak shape of the single-peak signals in this experiment fully verify that the separation method developed in this invention can effectively separate the α- and β-type diastereomers of 2-amino-dihydrobenzonazine, and the stereochemical purity of the obtained diastereomers meets the technical requirements for structural characterization.

[0055] The α-configuration of dihydro-2-amino-2-dihydro-2-amino ... 18 F]5 exhibited an elution peak at 16 minutes during the semi-preparative HPLC purification process. The entire radiosynthesis process took approximately 100 minutes, with an uncorrected radiochemical yield of approximately 10%. The target radioproduct was verified to have radiochemical purity (>99%) by analytical HPLC, and its retention time (5.6 minutes) was consistent with that of the non-radioactive control compound 5. Figure 7a) This fully confirms 18 The accuracy of the F-labeling strategy and the radiochemical homogeneity of the products were assessed. The chemical structure of the non-radioactive control compound 5 was confirmed by electrospray ionization mass spectrometry. Figure 7 b; ESI-MS: m / z [M+H] + calcd for C 22 H 36 FN2O2 + (379.53, found 379.50). The successful establishment of this radiolabeling method lays the technical foundation for the subsequent development of novel PET probes based on the molecular backbone of α-type 2-amino-dihydrobutenazine.

[0056] Example 3

[0057] Synthesis and purification of 2-amino-dihydrobutenazine: The synthetic route of 2-amino-dihydrobutenazine is as follows... Figure 1 As shown, butylbenazine (TBZ, compound 1) is converted to 2-amino-dihydrobutylbenazine (2-NH2-DTBZ, compound 2) by Borch reduction ammoniation in the presence of ammonium acetate and sodium cyanoborohydride.

[0058] (1) Chemical synthesis: Bubenazine (2g, 6.3mmol), ammonium acetate (4.86g, 63mmol) and sodium cyanoborohydride (0.8g, 12.6mmol) were dissolved in 50mL of anhydrous methanol, and 3g of molecular sieve was added. The mixture was stirred continuously at room temperature for 24 hours. After the reaction, the reaction solution was diluted with 100 mL of methanol and filtered. The methanol solvent was removed by concentration under reduced pressure, and the residue was dissolved in 50 mL of water. The aqueous phase was adjusted to pH < 4 with concentrated hydrochloric acid, followed by extraction with diethyl ether (100 mL × 2). After discarding the organic phase, the aqueous phase was adjusted to pH > 10 with 5M sodium hydroxide solution and extracted again with diethyl ether (100 mL × 2). The ether extracts were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain the crude product.

[0059] (2) Separation and Purification: 300-400 mesh dry silica gel was wet-packed into a 4 cm diameter glass chromatography column. During packing, a rubber mallet was used to gently tap the column wall to remove air bubbles, ensuring dense packing and forming a stable silica gel column system with a diameter-to-height ratio of approximately 1:6. Approximately 2 g of the crude 2-NH2-DTBZ obtained from the previous synthesis was completely dissolved in 2 mL of anhydrous methanol and slowly loaded onto the silica gel surface along the column wall. A layer of defatted cotton was then placed over the silica gel surface to effectively prevent eluent erosion and column surface disturbance. A dichloromethane-methanol (9:1, v / v) mixture was used as the elution system, with an elution flow rate of approximately 4 mL / min. Eluent was collected in conical flasks at approximately 30 mL / s, and the separation process was monitored in real-time using thin-layer chromatography (TLC). Finally, the target components were differentially collected based on the TLC results, achieving effective separation of the diastereomers of 2-NH2-DTBZ. TLC analysis showed that the separation effect of α-type 2-amino-dihydrobutanazine was consistent with that in Example 1.

[0060] Example 4

[0061] Synthesis and purification of 2-amino-dihydrobutenazine: The synthetic route of 2-amino-dihydrobutenazine is as follows... Figure 1 As shown, butylbenazine (TBZ, compound 1) is converted to 2-amino-dihydrobutylbenazine (2-NH2-DTBZ, compound 2) by Borch reduction ammoniation in the presence of ammonium acetate and sodium cyanoborohydride.

[0062] (1) Chemical synthesis: Bubenazine (2g, 6.3mmol), ammonium acetate (5.4g, 70mmol) and sodium cyanoborohydride (0.4g, 6.3mmol) were dissolved in 50mL of anhydrous methanol, and 3g of molecular sieve was added. The mixture was stirred continuously at room temperature for 24 hours. After the reaction, the reaction solution was diluted with 100 mL of methanol and filtered. The methanol solvent was removed by concentration under reduced pressure, and the residue was dissolved in 50 mL of water. The aqueous phase was adjusted to pH < 4 with concentrated hydrochloric acid, followed by extraction with diethyl ether (100 mL × 2). After discarding the organic phase, the aqueous phase was adjusted to pH > 10 with 5M sodium hydroxide solution and extracted again with diethyl ether (100 mL × 2). The ether extracts were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain the crude product.

[0063] (2) Separation and Purification: 300-400 mesh dry silica gel was wet-packed into a 4 cm diameter glass chromatography column. During packing, a rubber mallet was used to gently tap the column wall to remove air bubbles, ensuring dense packing and forming a stable silica gel column system with a diameter-to-height ratio of approximately 1:8. Approximately 2 g of the crude 2-NH2-DTBZ obtained from the previous synthesis was completely dissolved in 2 mL of anhydrous methanol and slowly loaded onto the silica gel surface along the column wall. A layer of defatted cotton was then placed over the silica gel surface to effectively prevent eluent erosion and column surface disturbance. A dichloromethane-methanol (11:1, v / v) mixture was used as the elution system, with an elution flow rate of approximately 6 mL / min. Eluent was collected in conical flasks at approximately 30 mL / s, and the separation process was monitored in real-time using thin-layer chromatography (TLC). Finally, the target components were differentially collected based on the TLC detection results, achieving effective separation of the diastereomers of 2-NH2-DTBZ. TLC analysis showed that the separation effect of α-type 2-amino-dihydrobutanazine was consistent with that in Example 1.

[0064] Comparative Example 1

[0065] Synthesis and purification of 2-amino-dihydrobenazine: Compared with Example 1, in this comparative example, 300-400 mesh dry silica gel was wet-packed into a 4 cm diameter glass chromatography column. During the purification process, a rubber mallet was used to gently tap the column wall to remove air bubbles, ensuring dense packing and forming a stable silica gel column system with a diameter-to-height ratio of approximately 1:3. Approximately 2 g of the crude 2-NH2-DTBZ obtained from the previous synthesis was completely dissolved in 2 mL of anhydrous methanol and slowly loaded onto the silica gel surface along the column wall. A layer of defatted cotton was then placed over the silica gel surface to effectively prevent eluent erosion and column surface disturbance. A dichloromethane-methanol (10:1, v / v) mixture was used as the elution system, with an elution flow rate of approximately 5 mL / min. Eluent was collected in Erlenmeyer flasks at approximately 30 mL intervals, and the separation process was monitored in real-time by thin-layer chromatography (TLC). Experimental results are as follows: Figure 8 As shown, effective separation of the α- and β-type diastereomers of 2-amino-dihydrobutanazine was not achieved, and two characteristic fractions (I and II) were obtained. Fraction I contained impurities from the chemical synthesis reaction. Figure 8 b), fraction II is a mixture of α-type and β-type isomers ( Figure 8 c; ESI-MS: m / z [M+H] + calcd for C 19 H 31 N2O2 + 319.46, found 319.50).

[0066] Comparative Example 2

[0067] Synthesis and purification of 2-amino-dihydrobenazine: Compared with Example 1, in this comparative example, 300-400 mesh dry silica gel was wet-packed into a 4 cm diameter glass chromatography column using a rubber mallet to gently tap the column wall to remove air bubbles, ensuring dense packing and forming a stable silica gel column system with a diameter-to-height ratio of approximately 1:7. Approximately 2 g of the crude 2-NH2-DTBZ obtained from the aforementioned synthesis was completely dissolved in 2 mL of anhydrous methanol and slowly loaded onto the silica gel surface along the column wall. A layer of defatted cotton was then placed over the silica gel surface to effectively prevent eluent erosion and column surface disturbance. A dichloromethane-methanol (20:1, v / v) mixed solvent was used as the elution system, with an elution flow rate controlled at approximately 5 mL / min. Eluent was collected in Erlenmeyer flasks at approximately 30 mL / s, and the separation process was monitored in real-time using thin-layer chromatography (TLC). Experimental results are as follows: Figure 9 As shown, effective separation of the α- and β-type diastereomers of 2-amino-dihydrobutanazine was not achieved, and two characteristic fractions (I and II) were obtained. Fraction I contained impurities from the chemical synthesis reaction. Figure 9 b), fraction II is a mixture of α-type and β-type isomers ( Figure 9 c; ESI-MS: m / z [M+H] + calcd for C 19 H 31 N2O2 + 319.46, found 319.50).

[0068] Comparative Example 3

[0069] Synthesis and purification of 2-amino-dihydrobenazine: Compared with Example 1, in this comparative example, 300-400 mesh dry silica gel was wet-packed into a 4 cm diameter glass chromatography column. During the purification process, a rubber mallet was used to gently tap the column wall to remove air bubbles, ensuring dense packing and forming a stable silica gel column system with a diameter-to-height ratio of approximately 1:7. Approximately 2 g of the crude 2-NH2-DTBZ obtained from the previous synthesis was completely dissolved in 2 mL of anhydrous methanol and slowly loaded onto the silica gel surface along the column wall. A layer of defatted cotton was then placed over the silica gel surface to effectively prevent eluent erosion and column surface disturbance. An ethyl acetate-methanol (5:1, v / v) mixture was used as the elution system, with an elution flow rate of approximately 5 mL / min. Eluent was collected in Erlenmeyer flasks at approximately 30 mL intervals, and the separation process was monitored in real-time by thin-layer chromatography (TLC). Experimental results are as follows: Figure 10 As shown, the α- and β-type diastereomers of 2-amino-dihydrobutanazine are completely inseparable, yielding two characteristic fractions (I and II). Fraction I is an impurity from the chemical synthesis reaction. Figure 10 b), fraction II is a mixture of α-type and β-type isomers ( Figure 10c; ESI-MS: m / z [M+H] + calcd for C 19 H 31 N2O2 + 319.46, found 319.50).

[0070] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing α-type 2-amino-dihydrobutanalazine, characterized in that, Includes the following steps: Step 1) React the raw materials containing butylbenazine, ammonium acetate and sodium cyanoborohydride to obtain a reaction mixture; perform post-treatment on the reaction mixture to obtain a crude product; Step 2) The crude product from Step 1 is subjected to column chromatography to separate α-type 2-amino-dihydrobenzenarazine; the stationary phase used in the column chromatography is silica gel; the stationary phase diameter-to-height ratio is 1:6-8, and a mixture of dichloromethane and methanol is used as the elution system, with a volume ratio of dichloromethane to methanol of 9-11:1; the elution flow rate is 4-6 mL / min.

2. The method for preparing α-type 2-amino-dihydrobutanalazine according to claim 1, characterized in that, In step one), the molar ratio of butylbenazine:ammonium acetate:sodium cyanoborohydride is 1-2:9-11:1-2. Preferably, the raw materials in step one further include a solvent, wherein the solvent comprises at least one of methanol, ethanol, and propanol; Preferably, in the raw materials of step one), the mass fraction of butylbenazine is 2-6%.

3. The method for preparing α-type 2-amino-dihydrobutanalazine according to claim 1, characterized in that, The method for post-processing the reaction mixture includes: diluting and filtering the reaction mixture with a solvent; removing the solvent from the filtrate, dissolving the residue in water to obtain a reaction product solution; adjusting the pH of the reaction product solution to <4, and then extracting with diethyl ether; adjusting the pH of the raffinate to >10, and then extracting with diethyl ether; combining the diethyl ether extracts, washing and drying to obtain the crude product.

4. The method for preparing α-type 2-amino-dihydrobutanalazine according to claim 1, characterized in that, In step two), the separation product was monitored by thin-layer chromatography during column chromatography, and α-type 2-amino-dihydrobutanazine was separated.

5. A method for preparing radioisotope-labeled α-type 2-amino-dihydrobenzoxazine, characterized in that, Includes the following steps: α-type 2-amino-dihydrobutanazine was prepared by the method described in any one of claims 1 to 4. The α-type 2-amino-dihydrobutanazine is labeled with a radioactive isotope, and a group containing the radioactive isotope is attached to the amino group of the α-type 2-amino-dihydrobutanazine to obtain the radioactively labeled α-type 2-amino-dihydrobutanazine.

6. The method for preparing radioisotope-labeled α-amino-dihydrobutanazine according to claim 5, characterized in that, The radioactive isotope is 18 F.

7. The method for preparing radioisotope-labeled α-amino-dihydrobutanazine according to claim 5, characterized in that, The labeling reaction of α-type 2-amino-dihydrobenzidine with a radioactive isotope includes: 18 F - The mixture was reacted with 1,3-bis(toluenesulfonyloxy) at a constant temperature of 80–100 °C for 10–30 minutes, and then reacted with the α-type 2-amino-dihydrobutanazine at 110–150 °C for 15–35 minutes to obtain radioisotope-labeled α-type 2-amino-dihydrobutanazine.

8. Radioisotope-labeled α-amino-dihydrobutanazine prepared by any one of claims 5 to 7.

9. The use of the radioisotope-labeled α-amino-dihydrobutenazine of claim 8 in the preparation of products for diagnosing diseases related to type 2 vesicular monoamine transporters.

10. The application according to claim 9, characterized in that, The disease associated with the type 2 vesicular monoamine transporter is Parkinson's disease.

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