Chiral amine reagent, preparation method thereof and application of chiral amine reagent in chiral drug resolution and analysis

Abstract

The invention discloses a chiral amine reagent and a preparation method thereof as well as a method for analyzing and splitting a chiral drug, the chiral amine reagent is APMA # imgabs0 # or d5-APMA # imgabs1 #, and the configuration of a carbon atom connected with an amino group on a piperidine ring in the structural formula is an S configuration. The APMA and d5-APMA provided by the invention can be used for analysis and detection of chiral drugs, 3-aminocyclohexyl-phenyl methane ketone (APMA) and the d5-APMA in a stable isotope form thereof are independently or jointly used as a pair of isotope labeling reagents and are subjected to condensation reaction with the chiral drugs to obtain derivatives, enantiomers of the chiral drugs can be effectively split, and the chiral drugs can be analyzed and detected. And an easy-to-ionize group is introduced, so that the mass spectrum response in an ESI positive ion mode is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chiral drug detection, and particularly relates to a chiral amine reagent, a preparation method thereof, and an application in chiral drug resolution and analysis. Background Art

[0002] There are significant differences in the pharmacological activities, metabolic processes, and toxicities of different enantiomers of chiral drugs in vivo. Eventually, the therapeutic effects of the enantiomers are very different, and one of the enantiomers may have side effects or even toxicity. Therefore, in the physicochemical property analysis of drugs, the separation technology of chiral drugs plays an important role.

[0003] Chemical derivatization can achieve the effective separation of R / S chiral drugs on a chromatographic column, and can also achieve the signal enhancement of chiral drugs and a lower background signal to realize the trace detection of drugs. Therefore, the combination of chemical derivatization and chromatographic technology is a relatively common chiral drug detection technology at present. Although the existing derivatization reagents can improve the sensitivity of chiral drug detection, there is still a matrix effect. The introduction of isotope internal standards can effectively correct the results of mass spectrometry analysis, but the types of isotope internal standards are limited and the prices are expensive.

[0004] In summary, the development of new chiral drug derivatization reagents is of great significance for reducing the detection cost, improving the sensitivity of R / S chiral drugs in mass spectrometry, and quantitative detection. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to improve the sensitivity of chiral drug detection and resolve chiral drugs.

[0006] The present invention solves the above technical problems by the following technical means:

[0007] A chiral amine reagent, the structural formula of which is one of the following structures:

[0008]

[0009] Among them, the configuration of the carbon atom connected to the amino group on the piperidine ring in the structural formula is the S configuration.

[0010] The present invention also provides a preparation method of the chiral amine reagent, comprising the following steps:

[0011] S1. React benzoic acid or deuterated benzoic acid with oxalyl chloride in a solvent;

[0012] S2. React the product obtained in S1 with (S)-3-Boc-aminopiperidine in the presence of a base and a solvent;

[0013] S3. React the product obtained in S2 with trifluoroacetic acid in a solvent to obtain the chiral amine reagent.

[0014] Preferably, in S1, the dosage ratio of benzoic acid or deuterated benzoic acid to oxalyl chloride is 500 mg: 0.42 mL; the mass ratio of (S)-3-Boc-aminopiperidine used in S2 to benzoic acid or deuterated benzoic acid used in S1 is 1:1; in S3, the dosage ratio of the product obtained in S2 to trifluoroacetic acid is 500 mg: 1.6 mL.

[0015] Preferably, in S1, during the reaction process, it includes reacting for half an hour under ice bath conditions and then reacting at room temperature for 4 hours; in S2, the reaction includes reacting for half an hour under ice bath conditions and then reacting at room temperature for 1 hour; in S3, the reaction includes reacting at room temperature for 1 hour; in S1, the solvent is dichloromethane; in S2, the solvent is dichloromethane and the base is triethylamine; in S3, the solvent is dichloromethane.

[0016] The present invention also provides an application of the chiral amine reagent in chiral drug analysis and / or resolution.

[0017] The present invention also provides a method for analyzing and / or resolving chiral drugs with the chiral amine reagent, including the following steps: using the chiral amine reagent as a labeling reagent to react with the chiral drug to obtain a derivative; then detecting the derivative by LC-MS.

[0018] Preferably, the chiral drug is a chiral carboxylic acid drug.

[0019] Preferably, the chiral drug is one or a mixture of more than one of R-mandelic acid (R-MA), S-mandelic acid (S-MA), R-2-phenylpropionic acid (R-2-PPA), S-2-phenylpropionic acid (S-2-PPA), R-ibuprofen (R-IBU), S-ibuprofen (S-IBU), R-naproxen (R-NAP), and S-naproxen (S-NAP).

[0020] Preferably, mix the solution of the chiral drug with triphenylphosphine, 2,2'-dithiopyridine, and the chiral amine reagent, and react at 30 - 55 °C for 30 - 60 minutes to complete the labeling to obtain a derivative.

[0021] Preferably, using the chiral amine reagent as a labeling reagent to react with the chiral drug to obtain a derivative; then detecting the derivative by LC-MS; or, using the chiral amine reagent as a labeling reagent to react with the chiral drug to obtain a derivative A, and using the chiral amine reagent as a labeling reagent to react with the chiral drug to obtain a derivative B as an internal standard; mixing derivative A and derivative B and then performing LC-MS detection.

[0022] Preferably, during LC-MS detection, the chromatographic column model is Thermo Scientific TM Accucore TM C18, with a specification of 2.1×150mm, 2.6μm. The column temperature is 30°C. An aqueous solution of 0.1% formic acid and acetonitrile are used as mobile phase A and mobile phase B respectively for chromatographic detection. The chromatographic gradient is as follows: 0 - 5 min, 25% B by volume; 5 - 22 min, 25 - 55% B by volume; 22 - 24 min, 55 - 85% B by volume; 24 - 26 min, 85% B by volume; 26 - 28 min, 85 - 25% B by volume; 28 - 30 min, 25% B by volume; and the flow rate is 0.3 mL / min.

[0023] The advantages of the present invention are as follows:

[0024] 1. The APMA and d 5 -APMA provided by the present invention can be used for the analysis and detection of chiral drugs. By using 3-aminocyclohexyl-phenylmethanone (APMA) and its stable isotope form d 5 -APMA alone or in combination as a pair of isotope-labeled reagents to undergo a condensation reaction with chiral drugs, the resulting derivatives introduce easily ionizable groups, greatly improving the mass spectrometry response in the ESI positive ion mode.

[0025] 2. The present invention not only greatly improves the detection sensitivity of chiral drugs but also successfully separates their enantiomers in chromatography.

[0026] 3. The present invention provides a method for preparing APMA and d 5 -APMA, and the preparation process is simple and easy to operate. Description of the Drawings

[0027] Figure 1 It is the NMR spectrum of APMA prepared in Example 1 of the present invention;

[0028] Figure 2 It is the NMR spectrum of d 5 -APMA prepared in Example 1 of the present invention;

[0029] Figure 3 It is the secondary mass spectrum of the labeled products of different chiral drugs labeled with APMA and d 5 -APMA in Example 2 of the present invention;

[0030] Figure 4 It is the chromatogram of the separation of the products of different chiral drugs before and after labeling with APMA in Example 3 of the present invention;

[0031] Figure 5 This is the chromatogram of the product after chiral drug is labeled with APMA under changed chromatographic conditions in Example 3 of the present invention;

[0032] Figure 6 This is for APMA and d 5 -APMA in Example 3 of the present invention, which is the chromatogram of the labeled product after simultaneous labeling. Detailed implementation manners

[0033] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] The test materials and reagents used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0035] For those not specifying specific technologies or conditions in the embodiments, they can all be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications.

[0036] It should be noted that the chemical reagents used in the present invention are commercially available reagents. The LC-MS analysis of the present invention is carried out on a Shimadzu MS-8050 mass spectrometer (Shimadzu, Japan), equipped with an electrospray ionization source (ESI) (Turbo Ion Spray) and a Shimadzu LC-30AD UPLC system. The chromatographic column model is Thermo Scientific TM Accucore TM C18 (2.1×150 mm, 2.6 μm).

[0037] The chiral drugs in the embodiments of the present invention include a mixture of R-mandelic acid (R-MA), S-mandelic acid (S-MA), R-2-phenylpropionic acid (R-2-PPA), S-2-phenylpropionic acid (S-2-PPA), R-ibuprofen (R-IBU), S-ibuprofen (S-IBU), R-naproxen (R-NAP), and S-naproxen (S-NAP) mixed in equal mass ratios.

[0038] Example 1

[0039] Synthesis of the reagent APMA and d 5 -APMA of the present invention

[0040] Dissolve benzoic acid (500 mg) in dichloromethane (5 mL), slowly add oxalyl chloride (0.42 mL) dropwise, react in an ice bath for half an hour and then transfer to room temperature for 4 hours. After the reaction is completed, evaporate to dryness, dissolve with dichloromethane (3 mL) and add (S)-3-Boc-aminopiperidine (500 mg), then slowly add triethylamine (0.7 mL) dropwise. After the addition is complete, react in an ice bath for half an hour and then transfer to room temperature for 1 h. After the reaction is completed, add 1 mL of water for quenching, add ethyl acetate and saturated brine (volume ratio 1:2) for extraction, and dry the organic layer with anhydrous sodium sulfate. Purify by column chromatography (petroleum ether / ethyl acetate, volume ratio 2:1) to obtain a white intermediate.

[0041] Dissolve the obtained intermediate (500 mg) in 10 mL of dichloromethane, slowly add trifluoroacetic acid (1.6 mL) dropwise, react at room temperature for 1 h. After the reaction is completed, evaporate to dryness and extract with dichloromethane. Purify by a chromatography column (dichloromethane / methanol, volume ratio 2:1) to obtain the product APMA The configuration of the carbon atom on the piperidine ring connected to the amino group in the structural formula is the S configuration, and its NMR spectrum is as Figure 1 shown;

[0042] Replace the initial substrate in the above steps with deuterated benzoic acid to prepare d 5 -APMA The configuration of the carbon atom on the piperidine ring connected to the amino group in the structural formula is the S configuration, and its NMR spectrum is as Figure 2 shown.

[0043] Example 2

[0044] APMA / d 5 Chemical labeling of APMA / d

[0045] Take the ACN solution of the above chiral drug (the concentration of each drug in the solution is 10 μg / mL, 5 μL), and successively add the catalyst triphenylphosphine TPP (100 mmol / L, 16 μL) and 2,2'-dithiopyridine DPDS (100 mmol / L, 16 μL) dissolved in acetonitrile. APMA or d 5-APMA (100 mmol / L, 6 μL), and then acetonitrile was added to make the total volume of the labeling reaction 200 μL. The chiral carboxylic acid drug derivatives could be obtained by reacting at 35 °C for 30 minutes. The chiral drugs selected were chiral carboxylic acid drugs, and both R and S configurations were added simultaneously, with a ratio of 1:1, including R-mandelic acid (R-MA), S-mandelic acid (S-MA), R-2-phenylpropionic acid (R-2-PPA), S-2-phenylpropionic acid (S-2-PPA), R-ibuprofen (R-IBU), S-ibuprofen (S-IBU), R-naproxen (R-NAP), and S-naproxen (S-NAP).

[0046] The labeling reaction formula is as follows:

[0047]

[0048] Among them, R is phenyl, one of; R 1 is hydroxyl or methyl, and the * marked is chiral.

[0049] Figure 3 The R / S configuration mixtures of MA, 2-PPA, IBU, and NAP were effectively labeled by APMA and d 5 -APMA, and the corresponding secondary mass spectra of the labeled products were obtained. It can be seen from the figure that different labeled products can produce characteristic secondary fragments to assist in qualitative analysis.

[0050] Example 3

[0051] Separation of R / S configurations

[0052] The LC-ESI-MS analysis of the chiral carboxylic acid drug derivatives labeled by chiral amine APMA and d 5 -APMA and the underivatized carboxylic acid drugs in Example 2 were carried out on a Shimadzu MS-8050 mass spectrometer (Shimadzu, Japan), equipped with an electrospray ion source and a Shimadzu LC-30AD UPLC system. The mass spectrometry parameters of the chiral carboxylic acid drug derivatives and the underivatized chiral carboxylic acid drugs were optimized in the positive ion mode and the negative ion mode respectively to achieve the best analysis performance, and the analysis mode was the MRM mode. The MRM parameters were optimized by direct injection to obtain the best analysis performance. The chromatographic column model was ThermoScientific TM Accucore TMC18 (2.1×150 mm, 2.6 μm), the column temperature was 30 °C. An aqueous solution of formic acid with a volume fraction of 0.1% (phase A) and acetonitrile (phase B) were used as the mobile phase for chromatographic analysis. The optimized chromatographic gradient was: 0 - 5 min, 25% B by volume; 5 - 22 min, 25 - 55% B by volume; 22 - 24 min, 55 - 85% B by volume; 24 - 26 min, 85% B by volume; 26 - 28 min, 85 - 25% B by volume; 28 - 30 min, 25% B by volume; the flow rate was 0.3 mL / min. Figure 4 It is the chromatogram for the resolution of chiral carboxylic acid drugs by APMA. It can be seen from the figure that the R / S configurations of the four chiral drugs were effectively resolved with good resolution. 5 -APMA has the same physical and chemical properties as APMA, and its resolution chromatogram is exactly the same as that of APMA; if the chromatographic conditions are changed, the resolution effect is not very ideal, such as Figure 5 shown, the chromatographic conditions: the column model is Thermo Scientific TM Accucore TM C18 (2.1×150 mm, 2.6 μm), the column temperature was 35 °C, and the chromatographic gradient was: 0 - 3 min, 10% B, 3 - 15 min, 10% - 60% B, 15 - 26 min, 60% - 80% B, 26 - 28 min, 80% - 10% B, where the percentages are volume fractions; the flow rate was 0.35 mL / min. The chiral amine APMA and the chiral carboxylic acid drug derivatives labeled with d 5 -APMA in Example 2 were mixed in a molar ratio of 1:1 and then detected according to the above chromatographic conditions with good resolution. Figure 6 It is the chromatogram for the separation of the labeled products labeled with APMA (shown in red) and d 5 -APMA (shown in blue). It can be seen from the figure that the chromatographic separation and chromatographic retention behavior of the heavily and lightly labeled products were basically the same.

[0053] Example 4

[0054] Labeling efficiency

[0055] The experimental results of Example 2 showed that chiral carboxylic acid drugs could be well labeled by APMA. By detecting the change in the chromatographic peak area before and after the labeling of chiral carboxylic acid drugs, the derivatization efficiency of the APMA labeling reaction was investigated. The results showed that the labeling efficiency was > 95% (as shown in Table 1), indicating a high labeling efficiency. The sensitivity is the concentration at which the signal-to-noise ratio is equal to 3. The results showed that after chiral carboxylic acid drugs were labeled by APMA, a significant improvement in sensitivity was achieved (as shown in Table 2).

[0056] Table 1. Efficiency of APMA labeling chiral carboxylic acid drugs

[0057]

[0058] Table 2. Comparison of the sensitivity of chiral carboxylic acid drugs before and after APMA labeling

[0059]

[0060] d 5 The physicochemical properties of -APMA and APMA are the same, and the labeling results are consistent with those of APMA under the same labeling reaction conditions.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chiral amine reagent, characterized in that: Its structural formula is one of the following structures: Wherein, the configuration of the carbon atom connected to the amino group on the piperidine ring in the structural formula is S configuration.

2. A method for preparing a chiral amine reagent as claimed in claim 1, characterized in that: The following steps are involved: S1. reacting benzoic acid or deuterated benzoic acid and oxalyl chloride in a solvent; S2, reacting the product obtained in S1 with (S)-3-Boc-aminopiperidine in the presence of a base and a solvent; S3, reacting the product obtained in S2 with trifluoroacetic acid in a solvent to obtain the chiral amine reagent.

3. The method for preparing a chiral amine reagent according to claim 2, characterized in that: In S1, the usage ratio of benzoic acid or deuterated benzoic acid and oxalyl chloride is 500 mg:0.42 mL; the mass ratio of (S)-3-Boc-aminopiperidine used in S2 to benzoic acid or deuterated benzoic acid used in S1 is 1:1; in S3, the usage ratio of the product obtained in S2 to trifluoroacetic acid is 500 mg:1.6 mL.

4. The method for preparing a chiral amine reagent according to claim 2, characterized in that: In S1, the reaction process includes reacting under ice bath conditions for half an hour and then reacting at room temperature for 4 hours; in S2, the reaction includes reacting under ice bath conditions for half an hour and then reacting at room temperature for 1 hour; in S3, the reaction includes reacting at room temperature for 1 hour; in S1, the solvent is dichloromethane; in S2, the solvent is dichloromethane and the base is triethylamine; in S3, the solvent is dichloromethane.

5. Use of the chiral amine reagent according to claim 1 in the analysis and / or separation of chiral drugs.

6. A method for analyzing and / or resolving chiral drugs using the chiral amine reagent according to claim 1, characterized in that: The following steps are involved: A chiral amine reagent is used as a labeling reagent to react with a chiral drug to obtain a derivative; and then the derivative is detected using LC-MS.

7. The method for analyzing and / or separating chiral drugs according to claim 6, characterized in that: The chiral drug is a chiral carboxylic acid drug.

8. The method for analyzing and / or separating chiral drugs according to claim 6, characterized in that: The solution of the chiral drug is mixed with triphenylphosphine, 2,2'-dithiodipyridine and a chiral amine reagent, and the mixture is reacted at 30-55° C. for 30-60 minutes to complete the labeling and obtain a derivative.

9. The method for analyzing and / or separating chiral drugs according to claim 6, characterized in that: Chiral amine reagent As a labeling reagent, it reacts with chiral drugs to obtain derivatives; then the derivatives are detected by LC-MS; or, with chiral amine reagents As a labeling reagent, it reacts with chiral drugs to obtain derivative A, and chiral amine reagent It is a labeling reagent, reacts with a chiral drug to obtain a derivative B used as an internal standard; derivative A and derivative B are mixed and then detected by LC-MS.

10. The method for analyzing and / or separating chiral drugs according to any one of claims 6 to 9, characterized in that: In the LC-MS detection process, the column model is Thermo Scientific TM Accucore TM C18, specification is 2.1×150mm, 2.6μm, the chromatographic column temperature is 30℃, 0.1% formic acid aqueous solution and acetonitrile are used as mobile phase A and mobile phase B respectively for chromatographic detection, the chromatographic gradient is: 0-5min, volume fraction 25% B; 5-22min, volume fraction 25-55% B; 22-24min, volume fraction 55-85% B; 24-26min, volume fraction 85% B; 26-28min, volume fraction 85-25% B; 28-30min, volume fraction 25% B; the flow rate is 0.3mL / min.

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