A method for determining the absolute configuration of oseltamivir

Abstract

This invention relates to a method for determining the absolute configuration of oseltamivir. The method involves obtaining the NMR spectrum of the oseltamivir derivative after the reaction of oseltamivir with a chiral carboxylic acid using nuclear magnetic resonance spectroscopy (NMR spectroscopy). The absolute configuration of oseltamivir is then determined by resolving the NMR spectrum, primarily through the NOESY spectrum. This method is simple to operate, convenient to use, and can be widely applied in the pharmaceutical industry.

Description

Technical Field

[0001] This invention belongs to the technical field of absolute configuration determination of chiral substances, and specifically relates to a method for determining the absolute configuration of oseltamivir. Background Technology

[0002] Chirality is a ubiquitous phenomenon in nature, caused by the asymmetry of compound molecules. Many drug targets in the human body (such as various receptors, enzymes, and proteins) are composed of L-amino acids. When chiral drugs interact with drug targets, stereoselectivity occurs, and different enantiomers may produce drastically different pharmacological activities, physiological activities, toxicities, and metabolic effects in vivo. For example, thalidomide, a drug used to treat postpartum discomfort in the "seal baby" incident, has a potent R isomer, while the S isomer not only lacks sedative effects but also has strong teratogenic effects on the fetus. Therefore, the absolute configurational identification of chiral molecules is crucial for the development of chiral drugs.

[0003] Currently, methods for determining the structure of chiral compounds mainly include specific rotation, optical rotational dispersive spectroscopy, circular dichroism spectroscopy, X-ray diffraction, chiral HPLC, and nuclear magnetic resonance. Specific rotation analysis has high requirements for product purity, sample weight, and solvent; when the sample contains impurities, the analytical results are often unreliable. Optical rotational dispersive spectroscopy requires sample amounts in the milligram range and sample purity higher than 95%, and can only be applied to chiral molecules that absorb in the visible or ultraviolet regions, generally with a ring structure. Furthermore, this method is empirical and subject to a certain probability of error. Circular dichroism spectroscopy, similar to optical rotational dispersive spectroscopy, also has requirements for the sample itself, relies on empirical judgment, and requires a sample purity greater than 95%. X-ray diffraction requires obtaining crystals of sufficient size and regular shape, which often necessitates repeated experiments to find suitable conditions. Nuclear magnetic resonance (NMR) is a new method developed in recent years for determining the absolute configuration of chiral compounds. It is non-destructive and rapid. However, one-dimensional NMR analysis methods are difficult to distinguish due to severe signal overlap. Therefore, various two-dimensional NMR techniques and methods have been developed based on this.

[0004] Oseltamivir phosphate (OT) is a neuraminidase inhibitor antiviral drug used to treat and prevent avian influenza, H1N1 influenza A virus, and influenza B virus. Oseltamivir has three chiral centers and seven opposing chiral isomers. These chiral isomers are structurally and physically similar to oseltamivir, potentially affecting product quality. Currently, there are no reports on using nuclear magnetic resonance (NMR) spectroscopy to determine the absolute configuration of oseltamivir phosphate. The main problem in determining the absolute configuration of oseltamivir phosphate lies in the severe signal overlap in the NMR spectra of the prepared samples, making them difficult to distinguish. Summary of the Invention

[0005] This invention overcomes the deficiencies in the prior art and provides a method for determining the absolute configuration of oseltamivir.

[0006] The specific technical solution is as follows:

[0007] This invention provides a method for determining the absolute configuration of oseltamivir. The method involves obtaining the nuclear magnetic resonance spectrum of the oseltamivir derivative after the reaction of oseltamivir with a chiral carboxylic acid using nuclear magnetic resonance spectroscopy, and determining the absolute configuration of oseltamivir by resolving the nuclear magnetic resonance spectrum.

[0008] Furthermore, the nuclear magnetic resonance spectrum includes 1 H NMR, 13 C NMR, DEPT, 1 H- 1 H COSY, HSQC, HMBC, and NOESY spectra.

[0009] Furthermore, the absolute configuration of the oseltamivir derivative is determined by the NOESY spectrum in the nuclear magnetic resonance spectrum.

[0010] Furthermore, the absolute configuration of the oseltamivir derivative is determined by the correlation signal of the hydrogen atom on its chiral carbon atom in the NOESY spectrum.

[0011] Furthermore, the chiral carboxylic acid is selected from one of R-tetrahydrofuranic acid, S-tetrahydrofuranic acid, R-mandelic acid, S-mandelic acid, R-methoxyphenylacetic acid, S-methoxyphenylacetic acid, N-acetyl-D-glutamic acid or N-acetyl-L-glutamic acid, preferably R-tetrahydrofuranic acid.

[0012] Furthermore, the oseltamivir is selected from one of oseltamivir phosphate, oseltamivir sulfate, oseltamivir nitrate, oseltamivir carboxylic acid, and oseltamivir sulfonate, preferably oseltamivir phosphate.

[0013] Furthermore, the structure of the oseltamivir derivative is shown below:

[0014]

[0015] The oseltamivir derivative contains four chiral centers with absolute configurations of (3R, 4R, 5S, 18R).

[0016] Furthermore, the structure of the oseltamivir is as follows:

[0017]

[0018] Oseltamivir contains three chiral centers with absolute configurations of (3R, 4R, 5S).

[0019] The present invention also provides an oseltamivir derivative, which is prepared by reacting oseltamivir with a chiral carboxylic acid. The nuclear magnetic resonance spectrum of the oseltamivir derivative is obtained by nuclear magnetic resonance spectroscopy, and the absolute configuration of oseltamivir is determined by resolving the nuclear magnetic resonance spectrum.

[0020] Furthermore, the absolute configuration of the oseltamivir derivative is determined by the NOESY spectrum in the nuclear magnetic resonance spectrum.

[0021] Furthermore, the absolute configuration of the oseltamivir derivative is determined by the correlation signal of the hydrogen atom on its chiral carbon atom in the NOESY spectrum.

[0022] Furthermore, the oseltamivir is selected from one of oseltamivir phosphate, oseltamivir sulfate, oseltamivir nitrate, oseltamivir carboxylic acid, and oseltamivir sulfonate, preferably oseltamivir phosphate.

[0023] Furthermore, the chiral carboxylic acid is selected from one of R-tetrahydrofuranic acid, S-tetrahydrofuranic acid, R-mandelic acid, S-mandelic acid, R-methoxyphenylacetic acid, S-methoxyphenylacetic acid, N-acetyl-D-glutamic acid or N-acetyl-L-glutamic acid, preferably R-tetrahydrofuranic acid.

[0024] Furthermore, the structure of the oseltamivir derivative is shown below:

[0025]

[0026] The oseltamivir derivative contains four chiral centers with absolute configurations of (3R, 4R, 5S, 18R).

[0027] Furthermore, the structure of the oseltamivir is as follows:

[0028]

[0029] Oseltamivir contains three chiral centers with absolute configurations of (3R, 4R, 5S).

[0030] The present invention also provides a method for preparing oseltamivir derivatives, the specific steps of which are as follows: oseltamivir free base, organic solvent and catalyst are mixed, and under nitrogen protection at 5-15°C, a chiral carboxylic acid solution treated with acyl halide reagent is added dropwise, and the reaction is carried out at 10-20°C. After washing, drying, filtration, concentration and column chromatography separation and purification, oseltamivir derivatives are obtained.

[0031] Further, the organic solvent is selected from one of dichloromethane, toluene, tetrahydrofuran, chloroform, acetonitrile, acetone, methyl isobutyl ketone, isopropyl acetate, methyl ethyl ketone, methyl tert-butyl ether, 1,4-dioxane, xylene, diphenyl ether, methyl cyclopentyl ether, methanol, ethanol, and isopropanol, preferably dichloromethane. In one embodiment of the present invention, the organic solvent is dichloromethane.

[0032] Further, the catalyst is selected from one of triethylamine, 4-dimethylaminopyridine, 2-methylaminopyridine, 2,4-dimethylpyridine, 3,5-dimethylpyridine, and 4-methylpyridine, preferably triethylamine. In one embodiment of the present invention, the catalyst is triethylamine.

[0033] Further, the reaction time is 1 to 6 hours, for example, 1, 2, 3, 4, 5, or 6 hours, preferably 2 to 3 hours. In one embodiment of the present invention, the reaction time is 2 to 3 hours.

[0034] Furthermore, the method for preparing the oseltamivir free base involves mixing a pharmaceutically acceptable salt of oseltamivir, an organic solvent, and an alkaline reagent, reacting the mixture at 20–30°C, and then extracting, washing, drying, filtering, and concentrating the mixture to obtain the oseltamivir free base.

[0035] Furthermore, in the method for preparing the oseltamivir free base, the pharmaceutically acceptable salt of oseltamivir is selected from one of oseltamivir phosphate, oseltamivir sulfate, oseltamivir nitrate, oseltamivir carboxylate, and oseltamivir sulfonate, preferably oseltamivir phosphate. In one embodiment of the present invention, the pharmaceutically acceptable salt of oseltamivir is oseltamivir phosphate.

[0036] Furthermore, in the method for preparing the oseltamivir free base, the organic solvent is selected from ethyl acetate, methanol, acetonitrile, or acetone. In one embodiment of the present invention, the organic solvent is ethyl acetate.

[0037] Furthermore, in the method for preparing the oseltamivir free base, the alkaline reagent is selected from sodium carbonate, sodium hydroxide, sodium bicarbonate, potassium carbonate, or potassium hydroxide, preferably sodium carbonate.

[0038] Furthermore, in the method for preparing oseltamivir free base, the weight percentage of the alkaline reagent is 1-15%, preferably 5-12%, and more preferably 8-11%.

[0039] In one embodiment of the present invention, the alkaline reagent is 10% sodium carbonate.

[0040] Furthermore, in the method for preparing the free oseltamivir base, the alkaline reagent adjusts the pH of the reaction solution to 7.0–8.0, for example, pH values ​​of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.

[0041] Furthermore, in the method for preparing oseltamivir free base, the reaction time is 10–60 min, for example, the reaction time is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 min. In one embodiment of the present invention, the reaction time is 20 min.

[0042] Furthermore, the specific treatment method for the chiral carboxylic acid solution treated with the acyl halide reagent is as follows: the chiral carboxylic acid and organic solvent are mixed, and the acyl halide reagent is added dropwise at 20-30°C under nitrogen protection. The temperature is raised to 40-45°C for reaction, and the solution is concentrated to obtain the chiral carboxylic acid solution treated with the acyl halide reagent.

[0043] Further, the chiral carboxylic acid is selected from one of R-tetrahydrofuranic acid, S-tetrahydrofuranic acid, R-mandelic acid, S-mandelic acid, R-methoxyphenylacetic acid, S-methoxyphenylacetic acid, N-acetyl-D-glutamic acid, or N-acetyl-L-glutamic acid, preferably R-tetrahydrofuranic acid. In one embodiment of the present invention, the chiral carboxylic acid is R-tetrahydrofuranic acid.

[0044] Further, the organic solvent used to mix with the chiral carboxylic acid is selected from one of dichloromethane, toluene, tetrahydrofuran, chloroform, acetonitrile, acetone, methyl isobutyl ketone, isopropyl acetate, methyl ethyl ketone, methyl tert-butyl ether, 1,4-dioxane, xylene, diphenyl ether, methyl cyclopentyl ether, methanol, ethanol, and isopropanol, preferably dichloromethane. In one embodiment of the present invention, the organic solvent is dichloromethane.

[0045] Furthermore, the reaction time after heating to 40-45°C is 1-6 hours, for example, 1, 2, 3, 4, 5, or 6 hours, preferably 2-3 hours. In one embodiment of the present invention, the reaction time is 2-3 hours.

[0046] Furthermore, the acyl halide reagent is selected from one of thionyl chloride, oxalyl chloride, phosphorus trichloride, or phosphorus oxychloride, preferably thionyl chloride. In one embodiment of the present invention, the acyl halide reagent is thionyl chloride.

[0047] This invention utilizes nuclear magnetic resonance spectroscopy to obtain the nuclear magnetic resonance spectrum of oseltamivir derivatives after the reaction of oseltamivir with chiral carboxylic acids. The absolute configuration of the oseltamivir derivatives is determined by resolving the NOESY spectrum, thereby confirming the absolute configuration of oseltamivir. This method is simple to operate and convenient to use, and can be widely applied in the pharmaceutical industry. Attached Figure Description

[0048] Figure 1 The reaction steps for preparing OT-YS are shown.

[0049] Figure 2 The diagram shows the structural formula of OT-YS.

[0050] Figure 3 The image shown is a high-resolution mass spectrum of OT-YS (ESI positive ion mode).

[0051] Figure 4 The image shown is a high-resolution mass spectrum of OT-YS (ESI negative ion mode).

[0052] Figure 5 The image shown is of OT-YS. 1 H NMR spectrum.

[0053] Figure 6 The image shown is of OT-YS. 13 C NMR spectrum

[0054] Figure 7 The image shown is the DEPT135 spectrum of OT-YS.

[0055] Figure 8 The image shown is of OT-YS. 1 H- 1 H COSY spectrum

[0056] Figure 9 The HSQC spectrum of OT-YS is shown below.

[0057] Figure 10 The NOESY spectrum of OT-YS is shown. Detailed Implementation

[0058] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0059] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0060] Example 1: Preparation of oseltamivir derivative OT-YS

[0061] Add 5.0 g of oseltamivir phosphate and 50 mL of ethyl acetate to a single-necked flask. Adjust the pH to neutral with 10% sodium carbonate solution at room temperature and stir for 20 min. Allow the mixture to stand and separate into layers. Add 50 mL of ethyl acetate to the aqueous phase for extraction. Combine the organic phases, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate for 1 h, filter, and concentrate to obtain the free oseltamivir base.

[0062] Add R-tetrahydrofuranic acid (2.2 eq) and 20 ml DCM to a three-necked flask. Under nitrogen protection, control the temperature at 20–30 °C and add thionyl chloride (2.4 eq) dropwise. After the addition is complete, raise the temperature to 40–45 °C and maintain the temperature for 2–3 h. Monitor the reaction progress with TLC. After the reaction is complete, evaporate to dryness under reduced pressure. Take 40 ml of DCM to dryness once, stop the concentration, and dissolve in 20 ml of DCM to obtain the standby solution.

[0063] Add oseltamivir free base, 20 ml DCM, and triethylamine to another three-necked flask. Cool to 5–15 °C under nitrogen protection. Add the above-mentioned mixed solution dropwise. After the addition is complete, keep the temperature at 10–20 °C for 2–3 h. Monitor the reaction progress by TLC. After the reaction is complete, add 15 ml of water to wash. Allow to stand and separate the layers. Dry the organic phase with anhydrous sodium sulfate, filter, evaporate the organic phase under reduced pressure, and separate and purify by column chromatography to obtain the oseltamivir derivative OT-YS.

[0064] The above reaction steps are as follows Figure 1 As shown, the structural formula of the prepared oseltamivir derivative OT-YS is as follows: Figure 2 As shown.

[0065] Example 2 Data Analysis

[0066] 2.1 High-resolution mass spectrometry (HRMS)

[0067] The OT-YS sample was dissolved in methanol and detected using an Agilent 290-6545B liquid chromatography-quadrupole time-of-flight mass spectrometer with ESI ionization. The results are shown in Table 1. Figure 3 and Figure 4 As shown.

[0068] Table 1 High-resolution mass spectrometry analysis data of the samples

[0069]

[0070] 2.2 Nuclear Magnetic Resonance Spectroscopy (NMR)

[0071] The OT-YS sample was dissolved in deuterated chloroform, loaded into an NMR tube, and measured separately. 1 H NMR, 13 C NMR, DEPT135 1 H- 1 H COSY, HSQC, and NOESY spectra, the results are as follows Figures 5-10 As shown.

[0072] Combination 1 H NMR, 13 C NMR, 1 H- 1 The H COSY and HSQC spectra can be used to assign hydrogen and carbon atoms in the sample structure, and the results are shown in Tables 2 and 3.

[0073] As shown in Table 2 and Figures 5-7 As shown, the signal peak (δ) after deducting deuterated chloroform is... H 7.22, δ C 77.37, δ C 77.05, δ C After 76.73), 1 The H NMR spectrum showed that the sample structure contained 19 groups of hydrogen signals, totaling 34 hydrogen atoms; 13 The C10 NMR spectrum showed that the sample structure contained 20 groups of carbon signals, among which δ C A signal value of 30.56 is doubled, indicating that it is a peak formed by the overlap of two sets of carbon signals. Therefore, 13 The 13C NMR spectrum indicates that the sample structure contains 21 carbons; the DEPT135 spectrum shows that the sample structure contains 6 positive peaks and 10 negative peaks, among which, the positive peaks contain δ C The signal value of 30.56 is double, indicating that it is a peak of overlapping two sets of carbon signals. Therefore, DEPT135 indicates that the sample structure contains 7 -CH2 structures and 10 -CH3 or -CH structures. The above conclusion is consistent with the information of the OT-YS structure.

[0074] As shown in Table 3 and Figure 10As shown, the NOESY spectrum reveals correlation signals between the hydrogen at positions 5 and 18, 3 and 5, and 4 and 2', but no correlation between the hydrogen at position 4 and 18. This indicates that the hydrogen at positions 5 and 18 are on the same side, the hydrogen at positions 3 and 5 are on the same side, the hydrogen at positions 4 and 2' are on the same side, and the hydrogen at positions 4 and 18 are on opposite sides. Given that the absolute configuration of the chiral carboxylic acid reagent tetrahydrofuranose is R, it can be deduced that the absolute configuration of the hydrogen at position 3 on OT-YS is R, the absolute configuration of the hydrogen at position 4 is R, and the absolute configuration of the hydrogen at position 5 is S.

[0075] Table 2OT-YS 1 H NMR and 13 C NMR spectral data

[0076]

[0077]

[0078] Table 3 OT-YS 1 H- 1 H COSY and NOESY spectral data

[0079]

[0080] 2.3 Conclusion

[0081] Analysis of the NMR spectrum of OT-YS confirmed that its structure is (3R, 4R, 5S)-4-acetamido-3-(pent-3-yloxy)-5-(R)-tetrahydrofuran-2-carboxamido)cyclohexyl-1-en-1-carboxylic acid ethyl ester. This indicates that the structure of the oseltamivir phosphate test sample used is (3R, 4R, 5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester, with a chiral center of (3R, 4R, 5S).

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining the absolute configuration of oseltamivir, characterized in that, The determination method is to obtain the nuclear magnetic resonance spectrum of the oseltamivir derivative after the reaction of oseltamivir with a chiral carboxylic acid using nuclear magnetic resonance spectroscopy, and then determine the absolute configuration of oseltamivir by resolving the nuclear magnetic resonance spectrum to determine the absolute configuration of oseltamivir. The chiral carboxylic acid is selected from one of R-tetrahydrofuranic acid, S-tetrahydrofuranic acid, R-mandelic acid, S-mandelic acid, R-methoxyphenylacetic acid, or S-methoxyphenylacetic acid; The absolute configuration of the oseltamivir derivative is determined by the NOESY spectrum in the nuclear magnetic resonance spectrum.

2. The determination method according to claim 1, characterized in that, The absolute configuration of the oseltamivir derivative is determined by the correlation signal of the hydrogen atom on its chiral carbon atom in the NOESY spectrum.

3. The determination method according to claim 1, characterized in that, The chiral carboxylic acid is R-tetrahydrofuran carboxylic acid.

4. The determination method according to claim 1, characterized in that, The oseltamivir mentioned is selected from one of oseltamivir phosphate, oseltamivir sulfate, oseltamivir nitrate, oseltamivir carboxylic acid, and oseltamivir sulfonate.

5. The determination method according to claim 4, characterized in that, The oseltamivir mentioned is oseltamivir phosphate.

6. The determination method according to any one of claims 1 to 5, characterized in that, The structure of the oseltamivir derivative is shown in the figure below: 。 7. An oseltamivir derivative, characterized in that, The oseltamivir derivative is prepared by reacting oseltamivir with a chiral carboxylic acid. The nuclear magnetic resonance spectrum of the oseltamivir derivative is obtained by nuclear magnetic resonance spectroscopy. The absolute configuration of oseltamivir is determined by resolving the nuclear magnetic resonance spectrum. The chiral carboxylic acid is R-tetrahydrofuran carboxylic acid; The structure of the oseltamivir derivative is shown in the figure below: 。 8. The oseltamivir derivative according to claim 7, characterized in that, The oseltamivir mentioned is selected from one of oseltamivir phosphate, oseltamivir sulfate, oseltamivir nitrate, oseltamivir carboxylic acid, and oseltamivir sulfonate.

9. The oseltamivir derivative according to claim 8, characterized in that, The oseltamivir mentioned is oseltamivir phosphate.

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