Preparation method of C isotope labeled phenylalanine or phenylglycine derivative
Through a series of chemical reactions, the halogenated compound with a benzene ring is used to interact with C-13 labeled potassium cyanide to solve the problem of the C-13 isotope at the α position of DL-phenylalanine and DL-phenylglycine in the prior art. The problem of lack of labeling was eliminated, and high-yield C-13 labeled synthesis was achieved.
Patent Information
- Application Number
- CN202411419940.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-12
- Publication Date
- 2025-01-14
AI Technical Summary
There is no method specifically for C-13 isotope labeling at the alpha position for DL-phenylalanine and DL-phenyglycine.
Through a series of chemical reactions, including cyanation reaction, reduction reaction, addition reaction and hydrolysis reaction, the halide with benzene ring interacts with C-13-labeled potassium cyanide to gradually synthesize C-13-labeled benzene. Alanine or phenylglycine derivatives and make sure the C-13 label is in the alpha position.
The synthesis of C-13-labeled DL-phenylalanine and DL-phenylglycine derivatives with high yields was achieved. The C-13 label is located at the α position, and the yield can reach about 40%.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of isotope labeling, and particularly relates to a method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative. Background Art
[0002] DL-phenylalanine is one of the essential amino acids for the human body and belongs to the aromatic amino acid family. Most of it is oxidized into tyrosine by phenylalanine hydroxylase in the body, and together with tyrosine, it synthesizes important neurotransmitters and hormones, and participates in the body's sugar metabolism and fat metabolism. It is a kind of α-amino acid, among which the optical isomer with biological activity is L-phenylalanine, with a specific rotation of -35.1°. Phenylalanine cannot be synthesized naturally in the human body and animals, and is an important component of compound amino acid infusion; used in medicine, it is an intermediate of amino acid anticancer drugs such as phenylproline and formic acid sarcolytic, and is also a raw material for the production of adrenaline, thyroxine and melanin.
[0003] DL-phenylglycine and its derivatives are important pharmaceutical intermediates, commonly used in the synthesis of β-lactam antibiotics such as ampicillin, cephalexin, cephradine, etc., and also used in the synthesis of polypeptide hormones and various pesticides. DL-phenylglycine is also a metabolite in breast milk during the lactation period from W2 to W4. Since phenylglycine is optically active, the left-handed and right-handed products have different effects, and the left-handed L-phenylglycine is mostly used in the production of medicines and pesticides.
[0004] Currently, there are no patents or literature related to DL-phenylalanine and DL-phenylglycine labeled with C-13 at the α position. Summary of the invention
[0005] The invention discloses a method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative. The method can obtain the C isotope-labeled phenylalanine or phenylglycine derivative with a higher yield, and the C isotope is labeled at the α position.
[0006] The technical solution of the present invention is as follows:
[0007] A method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative comprises the following steps:
[0008] (1) a halogenated compound containing a benzene ring is subjected to a cyanation reaction with C isotope-labeled potassium cyanide to obtain a C isotope-labeled cyano compound containing a benzene ring;
[0009] (2) the C isotope-labeled benzene ring-containing cyano compound undergoes a reduction reaction with a reducing agent to obtain a C isotope-labeled aldehyde compound;
[0010] (3) the C isotope-labeled aldehyde compound undergoes an addition reaction with potassium cyanide and ammonia water to obtain a C isotope-labeled aminobenzonitrile compound;
[0011] (4) hydrolyzing the C isotope-labeled aminobenzonitrile compound, and obtaining a C isotope-labeled phenylalanine or phenylglycine derivative through post-treatment;
[0012] The reaction formula is as follows:
[0013]
[0014] Wherein, n is 0 or 1, X is bromine or I, and *C is C13 or C14, more preferably C13.
[0015] Preferably, when n=0, in step (1), the cyanation reaction is carried out under the action of CuI, the solvent of the cyanation reaction is DMF, the reaction temperature is 120-140° C., and the reaction time is 12-24 h;
[0016] When n=1, in step (1), the cyanation reaction is carried out under the action of tetrabutylammonium bromide, and the solvent of the cyanation reaction is a mixed system of water and DCM.
[0017] Preferably, in step (2), the reducing agent is DIBAL-H, and the solvent for the reduction reaction is a mixture of anhydrous ether and n-hexane;
[0018] Alternatively, the reducing agent is Raney nickel, and the solvent for the reduction reaction is a mixture of water and formic acid.
[0019] Preferably, in step (3), the addition reaction is carried out under the action of ammonium chloride;
[0020] The solvent for the addition reaction is ethanol or methanol.
[0021] Preferably, in step (4), the hydrolysis reaction is carried out under acidic conditions, and the acid used in the acidic conditions is concentrated hydrochloric acid and / or acetic acid.
[0022] Preferably, in step (5), the post-treatment is: dissolving the crude product in water, adding LiOH to adjust the pH to 6-7, precipitating the solid, filtering and drying to obtain the C isotope-labeled phenylalanine or phenylglycine derivative.
[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0024] The present invention uses a simple halogenated compound with a benzene ring as a starting material, and sequentially undergoes cyanation reaction, reduction reaction, addition reaction and hydrolysis reaction to synthesize DL-phenylalanine-2-13C and DL-phenylglycine-2-13C with high yield (relative to C-13 potassium cyanide), and the C isotope is labeled at the α position, and the yields of both can reach about 40%. DETAILED DESCRIPTION
[0025] Example 1C-13 Synthesis of Labeled DL-Phenylalanine
[0026] The reaction formula is as follows:
[0027]
[0028] Step 1 Synthesis of phenyl(acetonitrile-1-13C) (Condition 1)
[0029] At room temperature, add 13C potassium cyanide (195mg, 3mmol, 1eq) and tetrabutylammonium bromide (165mg, 0.51mmol, 0.17eq) to a 50mL single-mouth round-bottom flask, add water (6mL) to dissolve, and then add DCM (6mL). Finally, add benzyl bromide (872mg, 5.1mmol, 1.7eq) dropwise. After the addition, replace with nitrogen, stir at room temperature overnight, and the system is colorless and clear. Add water (10mL) to the reaction solution and then extract with DCM (10mL*4), combine the organic phases, dry and concentrate with anhydrous magnesium sulfate, column chromatography: EA:HEX=1:100~1:10, elution to obtain 230mg of colorless oil. The calculated yield is 64.90%.
[0030] Step 2 Synthesis of Phenyl(Acetaldehyde-1-13C)
[0031] In a 25mL single-mouth bottle, add anhydrous ether and n-hexane 1:1 (5mL) to the compound phenyl (acetonitrile-1-13C) (230mg, 1.95mmol, 1eq), replace with nitrogen, cool to -40℃, then slowly add DIBAL-H (2.4mL, 2.4mmol, 1.2eq) dropwise, and stir at this temperature for 1h after adding. Add 3mL of 10% citric acid aqueous solution dropwise at -40℃, quench the reaction, warm the reaction solution to room temperature, and then slowly add 10% citric acid solution dropwise to pH=3-4, the system is basically clear, extract with ether (20mL*3), combine the organic phases, wash with saturated brine (20mL), dry with anhydrous sodium sulfate, and concentrate in an ice-water bath to obtain a crude oily substance. Column chromatography: n-pentane: ether = 5:1-5:3, eluted to obtain 226mg of yellow oily substance. The calculated yield is 95.83%.
[0032] Step 3 Synthesis of aminophenylpropionitrile-2-13C
[0033] In a 25mL single-mouth vacuum bottle, add ammonia water (2mL) to KCN (126mg, 1.93mmol, 1.05eq), then add ammonium chloride (207mg, 3.86mmol, 2eq) to dissolve, replace with nitrogen, cool in an ice-water bath, add phenyl (acetaldehyde-1-13C) (226mg, 1.85mmol, 1eq) in ethanol (1.5mL) dropwise, stir for 5min after addition, warm to room temperature and stir overnight. Concentrate the reaction solution, add 5mL of water to the mother liquor, extract with EA (10mL*5), combine the organic phases, dry and concentrate column chromatography: EA:HEX=1:10~1:1, elute to obtain 170mg of yellow oil, and the calculated yield is 61.91%.
[0034] Step 4Synthesis of DL-phenylalanine-2-13C
[0035] In a 25mL single-mouth vacuum bottle, add concentrated hydrochloric acid (3mL) to IM-3 (170mg, 1.15mmol, 1eq). Solid precipitates. Connect a condenser, heat to 110°C under nitrogen protection, and reflux with stirring for 20h. Concentrate the reaction solution to obtain a yellow solid. Use a small amount of methanol to dry the water. Pump it out with an oil pump for 30min to obtain 273mg of a crude yellow solid. After dissolving the crude product in water, add 5% LiOH aqueous solution to adjust the pH to 5.48, filter and dry to obtain 190mg of a white solid with a purity of more than 98%. 13 C abundance is above 99%. Calculated yield: 98.99%. Total yield: 38.11% (based on 13C potassium cyanide).
[0036] Comparative Example 1: Synthesis of C-13 labeled DL-phenylalanine
[0037] Step 1 Synthesis of phenyl(acetonitrile-1-13C) (condition 2)
[0038] At room temperature, add 13C potassium cyanide (195 mg, 3 mmol, 1 eq) to a 50 mL single-mouth round-bottom flask, add water (6 mL) to dissolve, and then add DCM (6 mL). Finally, add benzyl bromide (872 mg, 5.1 mmol, 1.7 eq) dropwise. After the addition, replace with nitrogen, stir at room temperature overnight, and the system is colorless and clear. Add water (10 mL) to the reaction solution and then extract with DCM (10 mL*4). Combine the organic phases, dry and concentrate over anhydrous magnesium sulfate, and column chromatography: EA:HEX=1:100~1:10, eluting to obtain 50 mg of colorless oil. Yield: 14.11%.
[0039] The difference between this condition and Example 1 is that tetrabutylammonium bromide is not added, and the results show that the yield is greatly reduced.
[0040] Example 2C-13 Synthesis of Labeled DL-Phenylalanine
[0041] The reaction formula is as follows:
[0042]
[0043] Step 1 Synthesis of 1-13C-benzonitrile
[0044] Add CuI (1.676 g, 8.8 mmol, 1.1 eq) to a 50 mL single-mouth round-bottom flask, connect a condenser and a tee, and dry it at 500 ° C for 20 min with a hot air gun under oil pump vacuum. After cooling to room temperature, add 13C potassium cyanide (535 mg, 8 mmol, 1 eq) and DMF (16 mL) under nitrogen protection, and finally add iodobenzene (1.92 mL, 16 mmol, 2 eq) dropwise to it. After the addition, strictly replace the nitrogen, and heat to 130 ° C, reflux and stir for 20 h. After the reaction solution is cooled to room temperature, drop it into 40 mL of ice water, stir for 10 min, then filter with diatomaceous earth, wash the filter residue alternately with water and EA, separate the mother liquor, extract the aqueous phase with EA (15 mL * 5), combine the organic phases, wash the organic phase with saturated brine (20 mL * 2), dry and then concentrate, mix the crude product and column chromatography. EA:HEX=1:100-1:10, 742 mg of white solid was obtained by elution. Yield: 89.08%.
[0045] Step 2 Synthesis of 1-13C-benzaldehyde (condition 1)
[0046] In a 50mL single-necked bottle, compound 1-13C-benzonitrile (742mg, 7.12mmol, 1eq), water (4mL) were added, and then Raney nickel (800mg) was added. Finally, formic acid (15mL) was dropped. The system slightly exothermic, a condenser was connected, and the temperature was raised to 100°C under nitrogen protection. Stir for 2h, and then Raney nickel (800mg) was added after cooling. After the addition, the temperature was continued to rise to 100°C and the reaction was reacted for 2h. The reaction solution was cooled to room temperature, stirred in an ice-water bath for 10 min, filtered with diatomaceous earth, and the filter residue was washed alternately with water and ether. The mother liquor was cooled in an ice-water bath, and then a saturated sodium bicarbonate solution was added thereto to adjust the pH to 6. The aqueous phase was extracted with ether (20 mL*5), the organic phases were combined, dried, and concentrated in an ice-water bath. The concentrate was directly wet-loaded on a column chromatography with n-pentane: ether = 100:1-50:1 to obtain 640 mg of an oily substance with a yield of 83.84%.
[0047] Step 3 Synthesis of aminophenylacetonitrile-2-13C
[0048] In a 48mL sealed tube, potassium cyanide (319mg, 4.89mmol, 1.1eq) and ammonium chloride (238mg, 4.44mmol, 1eq) were mixed and then concentrated ammonia (5mL) was added under an ice-water bath. After the addition was complete, the temperature was raised to room temperature and stirred for 10min. After the solid was completely dissolved, a methanol (5mL) solution of 1-13C-benzaldehyde (640mg, 5.97mmol, 1eq) was added dropwise under an ice-water bath. After the addition was complete, the temperature was raised to room temperature and stirred overnight. The reaction solution was transferred to a 100mL single-mouth round-bottom flask, concentrated to remove methanol, water (16mL) was added to the mother liquor, and then extracted with methanol: dichloromethane = 1:10 (15mL*5), the organic phases were combined, dried, and concentrated to obtain 585mg of crude product.
[0049] Step 4Synthesis of DL-phenylglycine-2-13C
[0050] To the crude aminophenylacetonitrile-2-13C (585 mg) was added 10 mL of acetic acid and 10 mL of concentrated hydrochloric acid, the temperature was raised to 105°C under nitrogen protection, and refluxed with stirring for 5 h. The reaction solution was cooled to room temperature and concentrated in a 48°C water bath, and the water was dried with 10 mL of ethanol. 8 mL of water was added to the concentrated solid, and 5% LiOH was added in an ice water bath to adjust the pH to 6-7. The solid was precipitated, filtered and dried to obtain 500 mg of a white solid with a purity of more than 98%. 13 C abundance is above 99%, and the combined yield of Step 3 and Step 4 is 55.00%. The total yield is 41.07% (calculated as 13C potassium cyanide).
[0051] Comparative Example 2
[0052] Step 2 Synthesis of 1-13C-benzaldehyde (condition 2)
[0053] In a 25mL single-mouth bottle, add anhydrous ether and n-hexane 1:1 (5mL) to compound 1-13C-benzonitrile (250mg, 2.4mmol, 1eq), replace with nitrogen, cool the low-temperature freezer to -40℃, then slowly add DIBAL-H (2.9mL, 2.8mmol, 1.2eq) to it, and stir at this temperature for 1h after adding. Add 3mL of 10% citric acid aqueous solution at -40℃, quench the reaction, warm the reaction solution to room temperature, and then slowly add 10% citric acid solution to it until pH=3~4, the system is basically clear, extract with ether (20mL*3), combine the organic phases, wash with saturated brine (20mL), dry with anhydrous sodium sulfate, and concentrate in an ice-water bath to obtain a crude oily substance. Column chromatography: n-pentane: ether = 100:1~50:1, eluted to obtain 38mg of yellow oily substance. Yield: 14.78%.
[0054] The conditions were similar to those of Example 1, where DIBAL-H was used as a reducing agent in Step 2 of the synthesis of DL-phenylalanine-2-13C. As a result, the yield was significantly reduced.
Claims
1. A method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative, characterized in that: The following steps are involved: (1) a benzene ring-containing halogenated compound is subjected to a cyanation reaction with C isotope-labeled potassium cyanide to obtain a C isotope-labeled benzene ring-containing cyano compound; (2) the C isotope-labeled benzene ring-containing cyano compound undergoes a reduction reaction with a reducing agent to obtain a C isotope-labeled aldehyde compound; (3) the C isotope-labeled aldehyde compound undergoes an addition reaction with potassium cyanide and aqueous ammonia to obtain a C isotope-labeled aminobenzonitrile compound; (4) hydrolyzing the C isotope-labeled aminobenzonitrile compound, and obtaining a C isotope-labeled phenylalanine or phenylglycine derivative through post-treatment; The reaction formula is as follows: Wherein, n is 0 or 1, X is bromine or I, and *C is C13 or C14.
2. The method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative according to claim 1, characterized in that: When n=0, in step (1), the cyanation reaction is carried out under the action of CuI, the solvent of the cyanation reaction is DMF, the reaction temperature is 120-140° C., and the reaction time is 12-24 h; When n=1, in step (1), the cyanation reaction is carried out under the action of tetrabutylammonium bromide, and the solvent of the cyanation reaction is a mixed system of water and DCM.
3. The method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative according to claim 1, characterized in that: In step (2), the reducing agent is DIBAL-H, and the solvent for the reduction reaction is a mixture of anhydrous ether and n-hexane; Alternatively, the reducing agent is Raney nickel, and the solvent for the reduction reaction is a mixture of water and formic acid.
4. The method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative according to claim 1, characterized in that: In step (3), the addition reaction is carried out under the action of ammonium chloride; The solvent for the addition reaction is ethanol or methanol.
5. The method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative according to claim 1, characterized in that: In step (4), the hydrolysis reaction is carried out under acidic conditions, and the acid used in the acidic conditions is concentrated hydrochloric acid and / or acetic acid.
6. The method for preparing a C isotope-labeled phenylalanine or phenylglycine derivative according to claim 1, characterized in that: In step (5), the post-treatment is as follows: after dissolving the crude product in water, adding LiOH to adjust the pH to 6-7, precipitating the solid, filtering and drying to obtain the C isotope-labeled phenylalanine or phenylglycine derivative.