Method for preparing carbon isotope labeled levodopa through asymmetric synthesis

Abstract

The invention discloses a method for preparing carbon isotope labeled levodopa through asymmetric synthesis. The method comprises the following steps: carrying out nucleophilic addition reaction on carbon isotope labeled CO2 and an aryl nucleophilic reagent to obtain carbon isotope labeled pepper acid; carrying out reduction and bromination reaction on piperic acid to synthesize a carbon isotope labeled aryl bromide fragment; the aryl bromide fragment and a chiral auxiliary Schiff base fragment are connected through an asymmetric alkylation reaction, and the carbon isotope labeled levodopa is obtained by removing an auxiliary and a protecting group from a connection product. According to the method, cheap and easily available raw materials are adopted, the cost is low, the yield is high, and the chiral center stereo enantioselectivity is high; according to the method, the L-DOPA can be accurately and stably labeled at the levodopa 3 site at a fixed point, the metabolic stability of the carbon isotope labeled L-DOPA is improved, the asymmetric synthesis of the carbon isotope labeled L-DOPA at the levodopa 3 site is completed for the first time, and a material support is provided for metabolism research related to the levodopa.

Description

Technical Field

[0001] This application relates to the field of levodopa synthesis technology, and in particular to a method for the asymmetric synthesis of carbon isotope-labeled levodopa. Background Technology

[0002] L-DOPA (levodopa) is a precursor to dopamine. It can cross the blood-brain barrier and be converted to dopamine in the central nervous system by dopa decarboxylase, thus improving Parkinson's disease symptoms. Levodopa was approved by the U.S. Food and Drug Administration (FDA) in 1970 for the treatment of Parkinson's syndrome. With the increasing aging of the global population, the market demand for levodopa is growing stronger. Utilizing isotope tracing technology, which offers unique advantages such as precise traceability, source tracking, and intuitive operation, to study the metabolic pathways and mechanisms of action of levodopa in vivo can promote the development of new levodopa formulations and their combination therapy with other drugs for Parkinson's disease. The structural formula of levodopa is as follows:

[0003]

[0004] The synthesis of levodopa is a classic and important topic in organic chemistry and the pharmaceutical industry. Its core challenge lies in how to efficiently and economically introduce chiral centers to obtain L-configuration products with high optical purity. Currently, methods for preparing levodopa include chemical synthesis, plant extraction, enzymatic catalysis, and microbial fermentation.

[0005] Traditional chemical synthesis methods primarily rely on the resolution of chiral sources or racemic derivatives extracted from natural products. For example, the Bucherer-Bergs hydrolysis method, a classic approach for synthesizing hydantoin (hydantoin), can be used to synthesize amino acids. Starting with veratral (3,4-dimethoxybenzaldehyde), it reacts with potassium cyanide (or sodium cyanide) and ammonium carbonate to generate a hydantoin derivative. Under alkaline conditions, hydrolysis yields racemic D,L-DOPA dimethyl ether. Using strong demethylating agents such as hydrobromic acid or boron tribromide, the methoxy group is converted to a phenolic hydroxyl group, yielding racemic D,L-DOPA. A chiral resolving agent, such as camphor sulfonic acid, is then used to separate the racemic D,L-DOPA, obtaining the desired L-configuration. This method is lengthy, uses highly toxic cyanide, requires resolution leading to low overall yield, high cost, and significant environmental pollution.

[0006] To overcome the drawbacks of separation, chemists have developed a variety of asymmetric catalytic synthesis methods.

[0007] a) Monsanto Process (1974 Nobel Prize in Chemistry): This is the most famous chemical synthesis method for the industrial production of L-DOPA. Developed under the leadership of William S. Knowles, it was the first asymmetric catalytic reaction successfully applied to industrial production. Using 3,4-dimethoxyphenylacrylic acid (or its amide or ester) as the starting material, the carbon-carbon double bond of the precursor is asymmetrically hydrogenated in the presence of a chiral phosphine-rhodium complex catalyst (such as DIPAMP-Rh). The hydrogenation product (protected L-DOPA) is then demethylated to obtain L-DOPA. This method exhibits high enantioselectivity, achieving an ee value (enantiomeric excess value) of over 95%, high yield, and high atom economy.

[0008] b) Sharpless asymmetric dihydroxylation: Starting with 3,4-dimethoxystyrene, using AD-mix reagents (containing chiral ligands such as sulfones of DHQD or DHQ, potassium osmium tetroxide, and an oxidizing agent), the double bond of an olefin can be enantioselectively converted to a vicinal diol. This vicinal diol can then undergo a series of transformations (such as oxidation, rearrangement, etc.) to produce L-DOPA.

[0009] Biosynthesis / biocatalysis: This method uses enzymes or whole microbial cells as catalysts. It has the advantages of mild conditions, strong stereospecificity (100% L-configuration), and environmental friendliness.

[0010] a) Enzymatic Catalysis: The most successful method uses tyrosine phenol lyase. Pyruvate, ammonia, and catechol (catechol) are synthesized in a single step under the catalysis of tyrosine phenol lyase. This is a one-step reaction with an extremely short process. It offers very high atom economy and almost 100% enantioselectivity. Challenges: Enzyme stability, cost, and substrate (catechol) supply.

[0011] b) Microbial fermentation: This method utilizes genetically engineered microorganisms (such as *E. coli* and yeast) as "cell factories" to produce L-DOPA through their own metabolic pathways. This involves introducing or overexpressing tyrosine phenol lyase genes into the microorganisms, enabling them to synthesize L-DOPA intracellularly using catechol and pyruvate as precursors. Alternatively, the tyrosine metabolic pathway of the microorganisms can be modified to accumulate large amounts of L-tyrosine, which can then be converted to L-DOPA by introducing exogenous enzymes. This method uses inexpensive carbon sources such as glucose as a starting point, has low cost potential, and aligns with green and sustainable development requirements. However, the metabolic pathway is complex, requiring advanced metabolic engineering techniques, and the product concentration and yield need further improvement.

[0012] There are few reports on the synthesis of carbon isotope-labeled levodopa. Generally, considering chemical stability, metabolic stability, and toxicological importance, aromatic ring labeling of levodopa is the best option. However, the synthetic route for benzene ring labeling is long and expensive. Therefore, the existing labeling sites are positions 1 and 2, because the synthesis of positions 1 and 2 is relatively easy. The synthesis of position 3 labeling is relatively difficult and also leads to higher costs. However, carbon isotope-labeled levodopa is mainly used for metabolic studies. Since the carbon at position 1 is a carboxyl functional group and the carbon at position 2 is attached to both amino and carboxyl groups, it is relatively easy to be metabolized during metabolism after labeling, which may lead to the inability to continue metabolic studies or inaccurate results.

[0013] Although carbon atoms directly attached to the aromatic ring are usually relatively stable and not easily detached during tracing or metabolism, there is currently no simple, low-cost, high-yield, and highly enantioselective method for synthesizing levodopa with third-site labeling. Summary of the Invention

[0014] The purpose of this application is to provide a novel method for the asymmetric synthesis of carbon isotope-labeled levodopa.

[0015] To achieve the above objectives, this application adopts the following technical solution:

[0016] This application discloses a method for the asymmetric synthesis of carbon isotope-labeled levodopa, comprising employing... 12 CO2, 13 CO2, 14 At least one of CO2 reacts with an aryl nucleophile in a nucleophilic addition reaction to obtain carbon isotope-labeled piperic acid; the carbon isotope-labeled piperic acid is then reduced and brominated to synthesize a carbon isotope-labeled aryl bromide fragment; the carbon isotope-labeled aryl bromide fragment and a chiral Schiff base fragment are linked by an asymmetric alkylation reaction, and the linking product is desorbed by removing the auxiliary agent and protecting group to obtain carbon isotope-labeled levodopa.

[0017] It should be noted that the preparation method of this application uses inexpensive and readily available materials, achieving high yield and significantly reducing material costs. It avoids the use of expensive metal catalysts and asymmetric ligands, employing inexpensive, sterically hindered chiral agents to control the construction of the chiral center, resulting in high stereoenantioselectivity. More importantly, the preparation method of this application, by using carbon isotope-labeled CO2, can accurately, site-specifically, and stably label L-DOPA at position 3, improving the metabolic stability of isotope-labeled L-DOPA. This application is the first to complete the asymmetric synthesis of L-DOPA with carbon isotope labeling at position 3, providing material support for L-DOPA-related metabolic research.

[0018] In one implementation of this application, the aryl bromide fragment is 3,4-methylenedioxybenzyl bromide.

[0019] In one implementation of this application, the structural formula of 3,4-methylenedioxybenzyl bromide is as follows.

[0020] .

[0021] In one implementation of this application, the chiral auxiliary Schiff base fragment is at least one of the compounds shown in the following structural formulas.

[0022] .

[0023] In one implementation of this application, the aryl nucleophile is at least one of the compounds shown in the following structural formulas.

[0024] ;

[0025] Where X is MgBr, MgCl, MgI, Li, Na, K, ZnBr, ZnCl or ZnI.

[0026] In one implementation of this application, the nucleophilic addition reaction includes stirring carbon isotope-labeled CO2 with an aryl nucleophile at -80℃ to -20℃ for 0.5h to 3h, then adding hydrochloric acid solution, heating to room temperature, adjusting the pH of the reaction system to less than or equal to 3 with hydrochloric acid, adding dichloromethane, extracting and separating the liquid, and concentrating under reduced pressure to obtain a white powder, namely piperic acid.

[0027] In one embodiment of this application, piperic acid is reduced and brominated to synthesize an aryl bromide fragment. Specifically, piperic acid and tetrahydrofuran are mixed with borane dimethyl sulfide at -80°C to -10°C, and the mixture is stirred at -80°C to -10°C for 1 h to 8 h. Methanol is added, the mixture is heated to room temperature, and concentrated under reduced pressure to obtain piperic alcohol. Phosphorus tribromide is added to the diethyl ether solution of piperic alcohol at -20°C to 10°C, and the mixture is stirred for 20 min to 2 h to quench the reaction. The liquid layer is separated, the aqueous layer is extracted with diethyl ether, the organic layers are combined, washed with brine, dried, and concentrated to obtain the aryl bromide fragment.

[0028] In one implementation of this application, the asymmetric alkylation reaction includes mixing tetrahydrofuran, diisopropylamine, and n-butyllithium at -80°C to -30°C to prepare a mixture, adding a chiral Schiff base fragment to the mixture, stirring for 20 min to 1 h, adding an aryl bromide fragment and hexamethylphosphoric triamine, stirring for 0.5 h to 3 h, then heating to room temperature to quench the reaction, extracting the reaction mixture with diethyl ether, combining the organic phases, washing, drying, concentrating, purifying by silica gel column chromatography, and eluting to obtain the ligation product.

[0029] In one implementation of this application, the ligation product is used to obtain levodopa by removing the auxiliaries and protecting groups. Specifically, the ligation product is mixed with sodium hydroxide, acetic acid, NH2OH·HCl, methanol, and chloroform. The mixture is stirred at room temperature for 12-30 hours, then concentrated, neutralized with hydrochloric acid, and extracted with diethyl ether. The organic layer is washed with water, dried with anhydrous sodium sulfate, filtered, and concentrated to recover the chiral auxiliaries. The combined aqueous layer is neutralized with sodium hydroxide to pH 10-11 and extracted with diethyl ether. The combined organic layer is washed, dried, filtered, and concentrated to obtain an amino ester. The amino ester, phenol, glacial acetic acid, and hydrochloric acid are mixed, and the mixture is heated under reflux for 24-48 hours and concentrated to obtain a light pink powder. The powder is dissolved with butyl acetate and extracted with water. The aqueous phase is adjusted to pH 5-6 with ammonia and sodium bisulfite is added. The mixture is then cooled to 5°C to precipitate a white crystalline product, namely levodopa.

[0030] In one implementation of this application, the carbon isotope-labeled CO2 is... 12 CO2, 13 CO2, 14 At least one of CO2.

[0031] In one implementation of the application, the chiral auxiliary Schiff base fragment is prepared by amidation and condensation reaction of L-camphor-10-sulfonyl chloride.

[0032] In one embodiment of this application, the amidation and condensation reaction includes: cooling a mixture of L-camphor-10-sulfonyl chloride and tetrahydrofuran to -10°C to 10°C, adding diisopropylamine, stirring at -10°C to 10°C for 0.5 h to 5 h, then concentrating, extracting with ethyl acetate, and sequentially washing, drying, filtering, and concentrating the organic layer to obtain camphor sulfonamide; mixing camphor sulfonamide, glycine tert-butyl ester, and toluene, refluxing the mixture at 100°C to 120°C for 24 h to 72 h, cooling, filtering, concentrating the filtrate, purifying it by silica gel column chromatography, and eluting to obtain the chiral auxiliary Schiff base fragment.

[0033] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:

[0034] The preparation method described in this application uses inexpensive and readily available raw materials to synthesize levodopa, resulting in low cost, high yield, and high stereoenantioselectivity of the chiral center. More importantly, the preparation method described in this application can accurately and stably label levodopa at position 3, improving the metabolic stability of the isotope-labeled L-DOPA. This application is the first to complete the asymmetric synthesis of levodopa with carbon isotope labeling at position 3, providing material support for levodopa-related metabolic research. Attached Figure Description

[0035] Figure 1 This is a liquid chromatogram of the L-DOPA reference standard in the embodiments of this application;

[0036] Figure 2 In the embodiments of this application, [3- 14 C] L-DOPA liquid chromatogram;

[0037] Figure 3 In the embodiments of this application, [3- 14 C] Figure of solid-state flare-electrochemical purity analysis results of L-DOPA;

[0038] Figure 4 This is the proton spectrum of L-DOPA in the embodiments of this application;

[0039] Figure 5 This is the carbon spectrum of L-DOPA in the embodiments of this application. Detailed Implementation

[0040] Existing carbon isotope labeling sites for levodopa are positions 1 and 2, as these are relatively easy to synthesize, while position 3 labeling is more difficult. Carbon isotope-labeled levodopa is mainly used for metabolic studies. Since the carbon at position 1 is a carboxyl functional group, and the carbon at position 2 is attached to both amino and carboxyl groups, the labeled carbon is easily metabolized during metabolism, potentially leading to discontinuation of metabolic studies or inaccurate results. Carbon atoms directly attached to the aromatic ring are generally more stable and less prone to detachment during tracing or metabolism. Therefore, research on the synthesis of isotope labeling at position 3 is of significant value and importance.

[0041] This application employs a chemical synthesis method, with a fixed and stable labeling at position 3. Starting from inexpensive and readily available source materials, L-DOPA is synthesized asymmetrically using chiral auxiliaries. This method exhibits high stereoenantiomerism selectivity and high yield, while avoiding the use of expensive metal catalysts and asymmetric ligands, thus significantly reducing material costs.

[0042] Specifically, the preparation method of this application includes: performing a nucleophilic addition reaction between carbon isotope-labeled CO2 and an aryl nucleophile to obtain carbon isotope-labeled piperic acid; synthesizing a carbon isotope-labeled aryl bromide fragment by reduction and bromination of the carbon isotope-labeled piperic acid; linking the carbon isotope-labeled aryl bromide fragment with a chiral Schiff base fragment via an asymmetric alkylation reaction; and obtaining carbon isotope-labeled levodopa by removing the auxiliary agent and protecting group from the linking product.

[0043] This application marks the first successful asymmetric synthesis of L-DOPA with carbon isotope labeling at position 3. The technical route is as follows:

[0044]

[0045] 1) Cold simulation (using conventional CO2, i.e., 12°C)

[0046] First, a conventional route was explored using common raw materials: using CO2 as the carbon source at position 3, nucleophilic addition with an aryl nucleophile (1) yielded piperic acid (2), which was then synthesized via reduction and bromination to form the aryl bromide fragment (3). On the other hand, starting with levorotatory camphor-10-sulfonyl chloride (4), a chiral Schiff base fragment (6) was prepared via amidation and condensation reactions. Next, the two fragments were linked via an asymmetric alkylation reaction, and the conventional asymmetric synthesis of levodopa was completed by removing the auxiliary agent and protecting group.

[0047] 2) 13 C mark

[0048] After cold simulation completes route exploration, isotope labeling synthesis will then begin. [With] 13 [C]-CO2, using the carbon source at position 3 as an isotope, undergoes nucleophilic addition with an aryl nucleophile to yield... 13 C-piperic acid (2), which was subsequently reduced and brominated. 13 The synthesis of the C-aryl bromide fragment (3) was carried out. On the other hand, starting from levorotatory camphor-10-sulfonyl chloride (4), a chiral Schiff base fragment (6) was prepared via amidation and condensation reactions. Then, the two fragments were linked via an asymmetric alkylation reaction, and by removing the auxiliary agent and protecting group, the [3- 13 Asymmetric synthesis of C]-levodopa.

[0049] 13 The technical approach for C-marking is as follows:

[0050]

[0051] 3) 14 C mark

[0052] by[ 14 C] Barium carbonate is an isotopic raw material, which is converted into [ 14 [C]-CO2, using the carbon source at position 3 as an isotope, undergoes nucleophilic addition with an aryl nucleophile to yield... 14 C-piperic acid (2), which was subsequently reduced and brominated. 14 The synthesis of the C-aryl bromide fragment (3) was carried out. On the other hand, starting from levorotatory camphor-10-sulfonyl chloride (4), a chiral Schiff base fragment (6) was prepared via amidation and condensation reactions. Then, the two fragments were linked via an asymmetric alkylation reaction, and by removing the auxiliary agent and protecting group, the [3- 14 Asymmetric synthesis of C]-levodopa.

[0053] 14 The technical approach for C-marking is as follows:

[0054]

[0055] Compared with existing synthesis methods, the preparation method of this application has the following advantages:

[0056] 1) This application is the first to complete the asymmetric synthesis of L-DOPA with carbon isotope labeling at position 3, providing material support for L-DOPA-related metabolic research;

[0057] 2) This application uses a chemical synthesis method, which can accurately and stably label L-DOPA at position 3, thereby improving the metabolic stability of isotope-labeled L-DOPA.

[0058] 3) Avoid using expensive metal catalysts and asymmetric ligands, and use inexpensive sterically hindered chiral agents to control the construction of chiral centers, resulting in high stereoenantioselectivity of chiral centers;

[0059] 4) Starting with inexpensive and readily available raw materials, the yield is high, which greatly reduces material costs.

[0060] The present application will be further described in detail below through specific embodiments. These embodiments are merely illustrative and should not be construed as limiting the present application. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other devices, materials, or methods. In some cases, certain operations related to the present application are not shown or described in the specification to avoid obscuring the core parts of the present application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; a complete understanding of the related operations can be obtained from the description in the specification and general technical knowledge in the art.

[0061] Example 1

[0062] The asymmetric preparation method of L-DOPA in this example is as follows:

[0063]

[0064] Step 1:

[0065] A thoroughly dried 100 mL single-necked round-bottom flask was filled with argon gas using a magnetic stirrer. Then, 1.0 mol / L aryl Grignard reagent (1) (50 mL, 50 mmol) was slowly added to the flask, and the flask was cooled to -20 °C (using a cryogenic circulating pump and ethanol bath condensation). Carbon dioxide (1.12 L, 50 mmol) was then slowly introduced into the condenser below the liquid level using a gas flow meter at a flow rate of 100 mL / min. After the gas was introduced, the reaction system was stirred for 1 h at -20 °C. Then, 10 mL of 2 N hydrochloric acid solution was added to the system. After the addition, the system was heated to room temperature in a water bath, the pH of the solution was tested, and the pH was adjusted to less than or equal to 3 using dilute hydrochloric acid solution. Then, 300 mL of dichloromethane was added, and the solution was transferred to a 500 mL separatory funnel. The extracted and concentrated white powder under reduced pressure was obtained, namely piperic acid (2) (7.89 g, 95%). 1 H NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H), 7.49 (dd, J = 8.2, 1.7Hz, 1H), 7.30 (d, J = 1.7 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.06 (s, 2H). 13 CNMR (100 MHz, DMSO-d6) δ 167.16, 151.66, 147.99, 125.49, 109.32, 108.59,102.46.

[0066] Step Two:

[0067] A thoroughly dried 500 mL single-necked round-bottom flask was fitted with a magnetic stir bar and filled with argon gas. Piperic acid (2) (4.15 g, 25 mmol) and tetrahydrofuran (100 mL) were then added to the flask, which was subsequently cooled to -20 °C (using a cryogenic circulating pump and ethanol bath condensation). Then, 2.0 mol / L borane dimethyl sulfide (31.25 mL, 62.5 mmol) was slowly added. The reaction system was stirred for 5 h at -20 °C. Subsequently, 10 mL of methanol was slowly added to the system. After the addition was complete, the system was heated to room temperature in a water bath, and concentrated under reduced pressure to obtain crude piperine.

[0068] At 0 °C, phosphorus tribromide (2.07 mL, 22 mmol) dissolved in ether (25 mL) was slowly added dropwise to a stirred solution of piperine (3.04 g, 20 mmol) in ether (30 mL) over 15 minutes. After stirring for another 30 minutes, water (50 mL) was added to quench the reaction, and the liquid layers were separated. The aqueous layer was extracted with ether (3 × 30 mL), and the combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the target product, aryl bromide fragment (3) (4.15 g, 97%), as a colorless solid that required no further purification. 1 H NMR (CDCl3, 400 MHz) δ 6.89-6.83 (m, 2H), 6.73 (dd, J = 7.6, 0.8 Hz, 1H), 5.96 (s, 2H), 4.45 (s, 2H). 13 CNMR (CDCl3, 100 MHz) δ = 148.1, 148.0, 131.7, 122.9, 109.6, 108.5, 101.5,34.3.

[0069] Step 3:

[0070] To a 50 mL flask containing tetrahydrofuran (5 mL) and diisopropylamine (0.73 mL, 5.6 mmol), n-butyllithium (2.2 M, 2.34 mL, 5.14 mmol) was added with stirring at -78 °C. The mixture was heated to 0 °C and reacted for 30 minutes, then cooled back to -78 °C. A Schiff base, i.e., the chiral Schiff base fragment (6) (2.00 g, 4.67 mmol), dissolved in tetrahydrofuran (5 mL), was added dropwise to the reaction system. The resulting mixture was stirred for another 30 minutes, followed by the addition of a tetrahydrofuran solution (5 mL) containing 3,4-methylenedioxybenzyl bromide, i.e., the aryl bromide fragment (3) (1.14 g, 5.30 mmol) and hexamethylphosphoric triamine (1.65 mL, 9.35 mmol). The reaction mixture was stirred for 1 hour, then heated to room temperature, and the reaction was quenched with water. The reaction mixture was extracted with diethyl ether, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate-n-hexane (1:6, containing 0.5% triethylamine) as the eluent, and finally a white crystal, namely the ligation product (7) (2.34 g, yield 89%), was obtained. 11H NMR δ 6.70 (s, 1H), 6.68 (d, J = 8.0Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 5.88 (s, 1H), 5.86 (s, 1H), 3.83 (dd, J =9.6, 2.8 Hz, 1H), 3.81 (septet, J = 6.8 Hz, 2H), 3.69 (d, J = 14.4 Hz, 1H),3.07 (dd, J = 13.2, 2.8 Hz, 1H), 2.87 (dd, J = 13.2, 9.6 Hz, 1H), 2.78 (d, J= 14.4 Hz, 1H), 2.43 (td, J = 13.2, 4.4 Hz, 1H), 2.15 (m, 1H), 1.83-1.70 (m,2H), 1.45-1.40 (m, 1H), 1.37 (s, 9H), 1.33 (d, J = 6.8 Hz, 6H), 1.31 (d, J =6.8 Hz, 6H), 1.06 (s, 3H), 1.03 (d, J = 17.2 Hz, 1H), 0.84-0.76 (m, 1H), 0.74(s, 3H); 13 13C NMR δ 181.3, 170.9, 147.0, 145.9, 132.2, 122.8, 110.4, 107.8,1,00.7, 80.6, 66.6, 55.0, 53.2, 48.1, 47.6, 43.4, 38.2, 34.8, 27.9, 27.1,26.9, 22.5, 22.1, 19.8, 19.2.

[0071] Step Four:

[0072] The linker product (7) (1.00 g, 1.79 mmol), sodium hydroxide (86 mg, 2.1 mmol), acetic acid (0.123 mL, 2.14 mmol), NH₂OH·HCl (149 mg, 2.14 mmol), methanol (10 mL), and chloroform (6 mL) were added to a 25 mL flask. The mixture was stirred at room temperature for 24 hours, then concentrated, neutralized with 2 N hydrochloric acid (5 mL), and extracted three times with diethyl ether. The organic layers were washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated to recover the chiral auxiliaries (510 mg, 91%). The combined aqueous layers were neutralized with sodium hydroxide to pH 10–11 and extracted with diethyl ether. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give amino ester (8) (420 mg, 89%), a pale yellow liquid. 1 H NMRδ 6.72 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 1.6 Hz, 1H), 6.63 (dd, J = 8.0, 1.6Hz, 1H), 5.91 (s, 2H), 3.52 (dd, J = 5.2, 8.0 Hz, 1H), 2.94 (dd, J = 13.6,5.2 Hz, 1H), 2.73 (dd, J = 13.6, 8.0 Hz, 1H), 1.44 (s, 9H); 13 C NMR δ 174.2, 147.6, 146.3, 131.1, 122.3, 109.6, 108.1, 100.8, 81.1, 56.3, 40.7, 28.0.

[0073] Step 5:

[0074] To a 50 mL brown flask, (8) (200 mg, 0.754 mmol), phenol (213 mg, 2.26 mmol), glacial acetic acid (0.130 mL, 2.26 mmol), and 6 N hydrochloric acid (10 mL) were added sequentially. The mixture was heated under reflux for 36 hours and then concentrated to obtain a light pink powder. The residue was dissolved in butyl acetate and extracted with water. The aqueous phase was adjusted to pH 5 with 28% ammonia and a trace amount of sodium bisulfite was added. The mixture was then cooled to 5 °C, and a white crystalline product (104 mg, 70%) precipitated, which was the preparation of levodopa in this example. The sample was analyzed by chiral HPLC using a chiral column, and the enantiomeric excess was 98.4%. 1HNMR (D2O) δ 6.88 (d, J = 8.7 Hz, 1H), 6.81 (s, 1H), 6.72 (d, J = 8.3 Hz, 1H), 3.91 (dd, J = 7.5, 5.5 Hz, 1H), 3.15 (dd, J = 14.8, 5.1 Hz, 1H), 2.98 (dd, J= 14.7, 7.9 Hz, 1H); 13 C NMR (D2O) δ 173.98, 144.21, 143.35, 127.54, 121.77, 116.90, 116.49, 55.95, 35.54.

[0075] Example 2

[0076] The preparation method of the chiral auxiliary Schiff base fragment (6) is as follows:

[0077]

[0078] Step 1:

[0079] Camphor sulfonyl chloride, namely L-camphor-10-sulfonyl chloride (4) (3.26 g, 13 mmol) and tetrahydrofuran (100 mL), were added to a 250 mL flask. The mixture was then cooled to 0 °C, and diisopropylamine (1.52 g, 15 mmol) was added to the reaction solution. The mixture was stirred at 0 °C for 1 hour, then concentrated, extracted with ethyl acetate, and the organic layer was washed with water, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain crude camphor sulfonamide (5), which can be used directly in the next step.

[0080] Step Two:

[0081] Camphor sulfonamide (5) (3.36 g, 10.6 mmol), glycine tert-butyl ester (3.47 g, 26.5 mmol), and toluene (50 mL) were placed in a two-necked round-bottom flask, and the reaction mixture was heated under reflux for 48 hours. After cooling, the mixture was filtered, and the filtrate was concentrated and purified by silica gel column chromatography using ethyl acetate-n-hexane (1:5) as the eluent to obtain a Schiff base, namely the chiral Schiff base fragment (6) (3.96 g, 79% yield). 1H NMR δ 3.94, 3.92 (ABq, J = 16.0 Hz, 2H), 3.77 (septet, J = 6.8 Hz, 2H), 3.70 (d, J = 14.4 Hz, 1H), 2.86 (d, J = 14.4 Hz, 1H), 2.61 (ddd, J = 12.2, 12.0, 4.4 Hz, 1H), 2.27 (ddd, J = 16.8, 3.6, 3.6Hz, 1H), 1.97-1.87 (m, 2H), 1.79 (d, J = 16.8 Hz, 1H), 1.56-1.54 (m, 1H),1.43 (s, 9H), 1.31 (d, J = 6.8 Hz, 6H), 1.30 (d, J = 6.8 Hz, 6H), 1.30-1.20(m, 1H), 1.16 (s, 3H), 0.83 (s, 3H); 13 C NMR δ 183.3, 169.2, 80.7, 55.1, 54.7,52.8, 48.0, 47.7, 43.8, 34.9, 27.9, 27.2, 27.0, 22.5, 21.9, 20.0, 19.4.

[0082] Example 3

[0083] This example [3- 13 The asymmetric preparation method of L-DOPA (C) is as follows:

[0084]

[0085] Step 1:

[0086] A thoroughly dry 50 mL single-necked round-bottom flask was filled with a magnetic stir bar and purged with argon gas. Then, 1.0 mol / L aryl Grignard reagent (1) (25 mL, 25 mmol) was slowly added to the flask, followed by cooling to -20 °C (using a cryogenic circulating pump and ethanol bath condensation). Then... 13Carbon dioxide (560 mL, 25 mmol) was slowly introduced into the condenser reaction flask below the liquid level using a gas flow meter at a flow rate of 50 mL / min. After the gas was introduced, the reaction system was stirred for 1 h at -20 °C. Then, 5 mL of 2 N hydrochloric acid solution was added to the system. After the addition, the system was heated to room temperature in a water bath, the pH of the solution was tested, and the pH was adjusted to less than or equal to 3 with dilute hydrochloric acid solution. Then, 100 mL of dichloromethane was added, the solution was transferred to a 500 mL separatory funnel, extracted, and concentrated under reduced pressure to obtain a white powder, i.e. 13 C-piperic acid (2) (3.86g, 93%).

[0087] Step Two:

[0088] Using a thoroughly dry 250 mL single-necked round-bottom flask, add a magnetic stir bar and fill with argon gas. Then add... 13 C-piperic acid (2) (2.07 g, 12.5 mmol) and tetrahydrofuran (50 mL) were added, and the round-bottom flask was then cooled to -20 °C (using a cryogenic circulating pump and ethanol bath condensation). Then, 2.0 mol / L borane dimethyl sulfide (15.6 mL, 31.25 mmol) was slowly added. The reaction system was stirred for 4 h at -20 °C. Subsequently, 10 mL of methanol was slowly added to the system. After the addition was complete, the system was heated to room temperature in a water bath and concentrated under reduced pressure to obtain the final product. 13 C-Piperol crude product.

[0089] At 0 °C, add to the stirring... 13 In a 20 mL solution of C-piperidine (1.52 g, 10 mmol) in diethyl ether, phosphorus tribromide (1.0 mL, 11 mmol) dissolved in 10 mL of diethyl ether was slowly added dropwise over 15 minutes. After stirring for another 30 minutes, water (30 mL) was added to quench the reaction, and the liquid layers were separated. The aqueous layer was extracted with diethyl ether (3 × 30 mL), and the combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the desired product. 13 C-aryl bromide fragment (3) (1.98 g, 92.5%), no further purification required.

[0090] Step 3:

[0091] To a 25 mL flask containing tetrahydrofuran (5 mL) and diisopropylamine (0.36 mL, 2.8 mmol), n-butyllithium (2.2 M, 1.17 mL, 2.57 mmol) was added with stirring at -78 °C. The mixture was heated to 0 °C and reacted for 30 minutes, then recooled to -78 °C. A solution of Schiff base (6) (1.00 g, 2.34 mmol) dissolved in tetrahydrofuran (5 mL) was added dropwise to the reaction system. The resulting mixture was stirred for another 30 minutes, followed by the addition of... 13 C-3,4-methylenedioxybenzyl bromide, i.e. 13 A 5 mL solution of C-aryl bromide fragment (3) (0.57 g, 2.65 mmol) and hexamethylphosphoric triamine (0.82 mL, 4.75 mmol) in tetrahydrofuran was prepared. The reaction mixture was stirred for 1 hour, then heated to room temperature and quenched with water. The reaction mixture was extracted with diethyl ether, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate-n-hexane (1:6, containing 0.5% triethylamine) as eluent, ultimately yielding white crystals. 13 C-linked product (7) (1.16 g, yield 88%).

[0092] Step Four:

[0093] Add to a 25 mL flask 13 C-linked product (7) (0.5 g, 0.9 mmol), sodium hydroxide (43 mg, 1.0 mmol), acetic acid (0.1 mL, 1.8 mmol), NH₂OH·HCl (75 mg, 1.07 mmol), methanol (5 mL), and chloroform (3 mL). The mixture was stirred at room temperature for 24 hours, then concentrated, neutralized with 2 N hydrochloric acid (3 mL), and extracted three times with diethyl ether. The organic layer was washed successively with water, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 13 C-amino ester (8) (206 mg, 87.3%).

[0094] Step 5:

[0095] Add the following to a 25 mL brown flask in sequence 13C-amino ester (8) (100 mg, 0.377 mmol), phenol (107 mg, 1.13 mmol), glacial acetic acid (0.10 mL, 1.8 mmol), and 6 N hydrochloric acid (5 mL). The mixture was heated under reflux for 24 hours and concentrated. The residue was dissolved in butyl acetate and extracted with water. The aqueous phase was adjusted to pH 5 with 28% ammonia and a trace amount of sodium bisulfite was added. The mixture was then cooled to 5 °C, and a white crystalline product precipitated, which is the [3-] in this example. 13 C] L-DOPA (50 mg, 67%).

[0096] Example 4

[0097] This example [3- 14 The asymmetric preparation method of L-DOPA (C) is as follows:

[0098]

[0099] Step 1:

[0100] Will Ba 14 CO3 (1.70 g, 8.5 mmol) was placed in a dry 250 mL round-bottom flask, and 50 mL of concentrated H2SO4 was placed in an attached constant-pressure separatory funnel. The H2SO4 was then added dropwise under vacuum to generate CO3. 14 CO2, collect carefully. 14 CO2 is used as a spare in the spiral assembly of the vacuum system.

[0101] Using a thoroughly dry 100 mL single-necked round-bottom flask, add a magnetic stir bar and fill with argon gas. Then, slowly add 8.5 mL (8.5 mmol) of 1.0 mol / L aryl Grignard reagent (1) to the flask. Transfer to a vacuum chamber with liquid nitrogen for later use. 14 CO2 was introduced into the reaction flask containing the aryl Grignard reagent (1), and the temperature was raised to -30°C using isopropanol and dry ice. The mixture was kept at -30 °C and reacted with stirring for 1-2 hours. Then, 5 mL of 2 N hydrochloric acid solution was added to the system, followed by 30 mL of dichloromethane. The solution was transferred to a 100 mL separatory funnel, extracted, and concentrated under reduced pressure to obtain the final product. 14 C-piperic acid (2) (1.34 g, 95%).

[0102] Step Two:

[0103] Using a thoroughly dry 100 mL single-necked round-bottom flask, add a magnetic stir bar and fill with argon gas. Then add... 14C-piperic acid (2) (1.0 g, 6.3 mmol) and tetrahydrofuran (20 mL) were reacted in a round-bottom flask cooled to -20 °C (using a cryogenic circulating pump and ethanol bath condensation). Then, 2.0 mol / L borane dimethyl sulfide (7.8 mL, 15.6 mmol) was slowly added. The reaction mixture was stirred for 4 h at -20 °C. Subsequently, 5 mL of methanol was slowly added to the system. After the addition was complete, the system was heated to room temperature in a water bath and concentrated under reduced pressure to obtain the final product. 14 C-Piperol crude product.

[0104] At 0 °C, add to the stirring... 14 In a 20 mL solution of C-piperol in diethyl ether, phosphorus tribromide (0.5 mL, 6.0 mmol) dissolved in 10 mL of diethyl ether was slowly added dropwise over 15 minutes. After stirring for another 30 minutes, water (10 mL) was added to quench the reaction, and the liquid layers were separated. The aqueous layer was extracted with diethyl ether (3 × 10 mL), and the combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain... 14 C-aryl bromide fragment (3) (0.96 g, 90.5%), no further purification required.

[0105] Step 3:

[0106] To a 25 mL flask containing tetrahydrofuran (5 mL) and diisopropylamine (0.36 mL, 2.8 mmol), n-butyllithium (2.2 M, 1.17 mL, 2.57 mmol) was added with stirring at -78 °C. The mixture was heated to 0 °C and reacted for 30 minutes, then recooled to -78 °C. A solution of Schiff base (6) (1.00 g, 2.34 mmol) dissolved in tetrahydrofuran (5 mL) was added dropwise to the reaction system. The resulting mixture was stirred for another 30 minutes, followed by the addition of... 14 C-3,4-methylenedioxybenzyl bromide, i.e. 14 A 5 mL solution of tetrahydrofuran containing C-aryl bromide fragment (3) (0.5 g, 2.34 mmol) and hexamethylphosphoric triamine (0.82 mL, 4.75 mmol) was prepared. The reaction mixture was stirred for 1 hour, then heated to room temperature and quenched with water. The reaction mixture was extracted with diethyl ether, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by short silica gel column chromatography to obtain the final product. 14 C-linked product (7) (1.13 g, yield 86%).

[0107] Step Four:

[0108] Add to a 25 mL flask 14C-linked product (7) (0.5 g, 0.9 mmol), sodium hydroxide (43 mg, 1.0 mmol), acetic acid (0.1 mL, 1.8 mmol), NH₂OH·HCl (75 mg, 1.07 mmol), methanol (5 mL), and chloroform (3 mL). The mixture was stirred at room temperature for 24 hours, then concentrated, neutralized with 2 N hydrochloric acid (3 mL), and extracted three times with diethyl ether. The organic layer was washed successively with water, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 14 C-amino ester (8) (211 mg, 89.5%).

[0109] Step 5:

[0110] Add the following to a 25 mL brown flask in sequence 14 C-amino ester (8) (100 mg, 0.377 mmol), phenol (107 mg, 1.13 mmol), glacial acetic acid (0.10 mL, 1.8 mmol), and 6 N hydrochloric acid (5 mL). The mixture was heated under reflux for 24 hours and concentrated. The residue was dissolved in ethyl acetate and extracted with water. The aqueous phase was adjusted to pH 5 with 28% ammonia and a trace amount of sodium bisulfite was added. The mixture was then cooled to 5 °C, and a white crystalline product precipitated, which is the [3-] in this example. 14 C] L-DOPA (53 mg, 71%).

[0111] After obtaining the experimental product, its structure was verified by high-performance liquid chromatography (HPLC) retention time. Specifically, the structure of [3- 14 [C] L-DOPA was subjected to HPLC chemical purity analysis, with levodopa reference standard (Macklin, L-DOPA purity: 99%) used as a control. Meanwhile, the [3- 14 C] L-DOPA was used for solid-flashover purity analysis. The liquid chromatogram of the reference standard is shown below. Figure 1 As shown, the [3- 14 The liquid chromatogram of C] L-DOPA is as follows Figure 2 As shown, [3- 14 C] The results of solid-state flare atomization purity analysis of L-DOPA are as follows: Figure 3 As shown.

[0112] The results showed that the retention time of the reference standard was 6.135 min, and the retention time of the experimental sample was 6.072 min. By using high-performance liquid chromatography (HPLC) with mixed injection and retention time, the obtained compound was confirmed to be [3- 14 [3-] L-DOPA. The [3-] were determined by high-performance liquid chromatography. 14The chemical purity of C] L-DOPA is 96.25%, [3- 14 The radiochemical purity of L-DOPA is 99.3%, and its specific activity is 51.20 mCi / mmol.

[0113] The L-DOPA prepared in Example 1 was analyzed by proton and carbon spectroscopy, and the results are as follows: Figure 4 and Figure 5 As shown. Figure 4 and Figure 5 The results showed that the NMR spectrum of the prepared L-DOPA was consistent with that reported in the literature, confirming that the structure of L-DOPA prepared by this method was correct.

[0114] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A method for asymmetric synthesis of carbon isotope-labeled levodopa, characterized in that: The process includes a nucleophilic addition reaction of carbon isotope-labeled CO2 with an aryl nucleophile to obtain carbon isotope-labeled piperic acid; the piperic acid is then reduced and brominated to synthesize a carbon isotope-labeled aryl bromide fragment; the aryl bromide fragment is then linked with a chiral Schiff base fragment via an asymmetric alkylation reaction, and the linked product is then desorbed by removing the auxiliary agent and protecting group to obtain carbon isotope-labeled levodopa.

2. The method according to claim 1, characterized in that: The aryl bromide fragment is 3,4-methylenedioxybenzyl bromide; Optionally, the structural formula of the 3,4-methylenedioxybenzyl bromide is as follows: 。 3. The method according to claim 1, characterized in that: The chiral auxiliary Schiff base fragment is at least one of the compounds shown in the following structural formulas. 。 4. The method according to claim 1, characterized in that: The aryl nucleophile is at least one of the compounds shown in the following structural formulas. ； Where X is MgBr, MgCl, MgI, Li, Na, K, ZnBr, ZnCl or ZnI.

5. The method according to any one of claims 1-4, characterized in that: The nucleophilic addition reaction includes stirring carbon isotope-labeled CO2 with an aryl nucleophile at -80℃ to -20℃ for 0.5h to 3h, then adding hydrochloric acid solution, heating to room temperature, adjusting the pH of the reaction system to less than or equal to 3 with hydrochloric acid, adding dichloromethane, extracting and separating the liquid, and concentrating under reduced pressure to obtain a white powder, namely piperic acid.

6. The method according to any one of claims 1-4, characterized in that: The synthesis of aryl bromide fragments from piperic acid via reduction and bromination reactions specifically includes: mixing piperic acid and tetrahydrofuran with borane dimethyl sulfide at -80℃ to -10℃, stirring and reacting at -80℃ to -10℃ for 1 h to 8 h, adding methanol, heating to room temperature, and concentrating under reduced pressure to obtain piperic alcohol; adding phosphorus tribromide to the diethyl ether solution of piperic alcohol at -20℃ to 10℃, stirring and reacting for 20 min to 2 h to quench the reaction, separating the liquid layer, extracting the aqueous layer with diethyl ether, combining the organic layers, washing with brine, drying, and concentrating to obtain the aryl bromide fragments.

7. The method according to any one of claims 1-4, characterized in that: The asymmetric alkylation reaction includes mixing tetrahydrofuran, diisopropylamine, and n-butyllithium at -80°C to -30°C to prepare a mixture, adding a chiral Schiff base fragment to the mixture, stirring for 20 min to 1 h, adding an aryl bromide fragment and hexamethylphosphoric triamine, stirring for 0.5 h to 3 h, then heating to room temperature to quench the reaction, extracting the reaction mixture with diethyl ether, combining the organic phases, washing, drying, concentrating, purifying by silica gel column chromatography, and eluting to obtain the ligation product.

8. The method according to any one of claims 1-4, characterized in that: The ligation product is used to obtain levodopa by removing the auxiliaries and protecting groups. Specifically, the ligation product is mixed with sodium hydroxide, acetic acid, NH2OH·HCl, methanol, and chloroform. The mixture is stirred at room temperature for 12-30 hours, then concentrated, neutralized with hydrochloric acid, and extracted with diethyl ether. The organic layer is washed with water, dried with anhydrous sodium sulfate, filtered, and concentrated to recover the chiral auxiliaries. The combined aqueous layer is neutralized with sodium hydroxide to pH 10-11 and then extracted with diethyl ether. The combined organic layer is washed, dried, filtered, and concentrated to obtain the amino ester. Amino ester, phenol, glacial acetic acid, and hydrochloric acid were mixed and heated under reflux for 24-48 hours, then concentrated to obtain a light pink powder. The powder was dissolved in butyl acetate and extracted with water. The aqueous phase was adjusted to pH 5-6 with ammonia and sodium bisulfite was added. The mixture was then cooled to 5 °C, and a white crystalline product, namely the L-DOPA, precipitated.

9. The method according to any one of claims 1-4, characterized in that: The carbon isotope-labeled CO2 is 12 CO2, 13 CO2, 14 At least one of CO2.

10. The method according to any one of claims 1-4, characterized in that: The chiral auxiliary Schiff base fragment was prepared by amidation and condensation reaction of L-camphor-10-sulfonyl chloride; Optionally, the amidation and condensation reaction includes cooling a mixture of L-camphor-10-sulfonyl chloride and tetrahydrofuran to -10°C to 10°C, adding diisopropylamine, stirring at -10°C to 10°C for 0.5 h to 5 h, then concentrating, extracting with ethyl acetate, and washing, drying, filtering, and concentrating the organic layer sequentially to obtain camphor sulfonamide; mixing camphor sulfonamide, glycine tert-butyl ester, and toluene, refluxing the mixture at 100°C to 120°C for 24 h to 72 h, cooling, filtering, concentrating the filtrate, purifying it by silica gel column chromatography, and eluting to obtain the chiral auxiliary Schiff base fragment.

Images