Method for detecting nebivolol hydrochloride intermediate and isomer thereof

Abstract

The invention discloses a method for detecting a nebivolol hydrochloride intermediate, namely 6-fluoro-3, 4-dihydro-alpha-[[(phenyl methyl) amino] methyl]-2H-1-benzopyran-2-methanol and an isomer of the nebivolol hydrochloride intermediate, namely 6-fluoro-3, 4-dihydro-alpha-[[(phenyl methyl) amino] methyl]-2H-1-benzopyran-2-methanol. The method can effectively elute, separate and quantitatively detect each component through optimization of chromatographic conditions, is high in sensitivity, strong in specificity and high in accuracy, and provides accurate, rapid and effective quality control for nebivolol hydrochloride products and a preparation process.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting a Nibivolol hydrochloride intermediate and its isomers, in particular to a method for detecting 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol and its isomers. BACKGROUND

[0002] Chiral drug molecules contain one or more chiral centers in their structure, which is a branch of drugs. The interaction between chiral drugs of different configurations and receptors is different, which makes the physiological activity of enantiomers different. At the same time, most endogenous ligands are chiral themselves, such as hormones, neurotransmitters, and neuropeptides, which also show specific selectivity in drug absorption. For example, the analgesic effect of naproxen, an anti-inflammatory analgesic drug, is mainly produced by S-naproxen, which is 35 times stronger than the R-isomer. For example, thalidomide, the R-isomer has analgesic effect, and the S-isomer has teratogenic effect, that is, one enantiomer has therapeutic effect, and the other has toxic side effect. Therefore, the U.S. Food and Drug Administration (FDA) has clearly stipulated that for drugs with chiral centers, the stereo enantiomers must be clearly known.

[0003]

[0004] Nibivolol hydrochloride is a selective β-receptor blocker with vasodilating effect developed by Johnson Company in the United States. The drug for treating primary hypertension is a racemic body in which (R,S,S,S)-enantiomer and (S,R,R,R)-enantiomer are mixed in equal amounts. Among them, (R,S,S,S)-enantiomer is a β-receptor blocker, and (S,R,R,R)-enantiomer can reduce peripheral vascular resistance, and the combination of the two is more effective in reducing blood pressure in clinical practice.

[0005]

[0006]

[0007] 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol is one of the key intermediates for synthesizing Nibivolol hydrochloride. In order to obtain Nibivolol hydrochloride with the target configuration, it is particularly important to control its chirality, and there is no effective detection method at present. SUMMARY

[0008] Invention purposes: The present application aims to provide a detection method for effectively eluting, separating and quantitatively detecting 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol intermediate of nebivolol hydrochloride and isomers thereof.

[0009] Technical solutions: The detection method for nebivolol hydrochloride intermediate and isomers thereof provided by the present application, wherein the nebivolol hydrochloride intermediate is enantiomers of SS configuration and RR configuration, and the structures are as follows:

[0010]

[0011] The structures of the isomers are as follows:

[0012]

[0013] The detection method comprises the following steps:

[0014] (1) Preparing a sample: preparing the sample to a set concentration by using a diluent;

[0015] (2) Detection: adopting normal phase chromatography and a chiral chromatographic column, and using a low alkyl alcohol-n-hexane solution added with an alkaline charge transfer agent as a mobile phase to perform elution;

[0016] (3) Analysis: recording a chromatogram and calculating the content of the isomers.

[0017] In the step (1), the prepared sample comprises a test solution and a positioning solution, and the specific preparation method is as follows:

[0018] a. The test solution: 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol intermediate is dissolved and diluted to 2.0-3.0 mg / ml, specifically 2.5 mg / ml, by using a diluent.

[0019] b. The positioning solution: 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol of four configurations is respectively dissolved and diluted to a set concentration, specifically 1 mg / ml, by using a diluent.

[0020] Preferably, the diluent in the step (1) is a low alkyl alcohol.

[0021] Further preferably, the diluent is methanol.

[0022] Preferably, the chromatographic column in the step (2) uses a silica gel coated with amylose-tris(3,5-dimethylphenylcarbamate) as a filler.

[0023] Further preferably, the size of the chromatographic column is 4.6 x 250 mm, 5 μm.

[0024] Still further preferably, the chromatographic column is a CHIRALPAK AD-H chromatographic column.

[0025] The CHIRALPAK AD-H chromatographic column contains a hydrophilic amylose structure and an amide structure in the stationary phase, and the 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-l-benzopyran-2-methanol structure contains a secondary amino group, which can form a hydrogen bond with the amide bond in the structure of the stationary phase.

[0026] Preferably, the volume fraction of the lower alkyl alcohol in the mobile phase of step (2) is not higher than 15%.

[0027] Further preferably, the volume fraction of the lower alkyl alcohol in the mobile phase is not higher than 10%.

[0028] Preferably, the lower alkyl alcohol in step (2) is ethanol, isopropanol.

[0029] Further preferably, the lower alkyl alcohol is isopropanol.

[0030] Preferably, the basic charge-migrating agent in step (2) is diethylamine, triethylamine, ethanolamine, diethanolamine.

[0031] Further preferably, the basic charge-migrating agent is ethanolamine.

[0032] Preferably, the volume of the basic charge-migrating agent added in the mobile phase of step (2) is 0.1% to 0.5% of the volume of the lower alkyl alcohol.

[0033] Further preferably, the volume of the basic charge-migrating agent added in the mobile phase is 0.2% to 0.4% of the volume of the lower alkyl alcohol, specifically any volume fraction between 0.2% and 0.4%, not limited to 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%.

[0034] Preferably, the mobile phase of step (2) is n-hexane-0.2% (V / V) ethanolamine isopropanol solution = 90-10 (V / V), and the specific preparation method is to first prepare a 0.2% (V / V) ethanolamine isopropanol solution, and then mix n-hexane with the 0.2% (V / V) ethanolamine isopropanol solution according to a ratio of 90:10 (V / V) to obtain the mobile phase.

[0035] Because 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol is a weakly basic compound, it can interact with exposed silanol groups in the stationary phase of a chiral column, resulting in poor peak shape and difficulty in elution. Therefore, adding a certain proportion of basic charge-shifting agent to the mobile phase to cover the silanol groups on the surface of the stationary phase changes the polarity of the mobile phase, thereby improving the peak shape.

[0036] Preferably, the detection wavelength in step (2) is 260-290 nm, the mobile phase flow rate is 0.8-1.2 ml / min, and the column temperature is 20-30 °C.

[0037] Further preferred, the detection wavelength is 280 nm, the mobile phase flow rate is 1.0 ml / min, and the column temperature is 25 °C.

[0038] Preferably, the sample solution injection volume in step (2) is 2 to 10 μl.

[0039] Further preferably, the sample solution injection volume for detection is 10 μl.

[0040] Preferably, the content of the isomer in step (3) is calculated by the area percentage method.

[0041] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0042] The detection method designed in this invention targets the four configurations of 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol, an intermediate of nebivolol hydrochloride. It has high sensitivity, strong specificity, and high accuracy, and can effectively elute, separate, and quantify each component, providing accurate, rapid, and effective quality control for nebivolol hydrochloride products and their preparation process. Attached Figure Description

[0043] Figure 1 The HPLC chromatogram for Example 1 is shown below.

[0044] Figure 2 The HPLC chromatogram for Example 2 is shown below.

[0045] Figure 3 The HPLC chromatogram for Example 3 is shown below.

[0046] Figure 4 The HPLC chromatogram of Example 4;

[0047] Figure 5 This is the HPLC chromatogram of the positioning solution in Example 5;

[0048] Figure 6 This is the HPLC chromatogram of the RR configuration test solution in Example 5;

[0049] Figure 7 This is the HPLC chromatogram of the SS configuration test solution in Example 5. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the embodiments.

[0051] Example 1

[0052] 1. Experimental Methods

[0053] (1) Chromatographic conditions were set as follows: the high performance liquid chromatograph was an Agilent 1260 liquid chromatograph, the column was a CHIRALPAKAD-H (4.6×250mm 5μm); the mobile phase was n-hexane-0.2% ethanolamine ethanol solution-isopropanol = 35-55-10 (V / V / V), isocratic elution for 25 min; the injection volume was 10 μl, the column temperature was 35℃, the flow rate was 0.8 ml / min, and the UV detection wavelength was 280 nm.

[0054] (2) Preparation of localization solution: Take appropriate amounts of 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol of each of the four configurations, add methanol and sonicate to dissolve, and quantitatively dilute to prepare a mixed solution containing about 1 mg of each per ml.

[0055] (3) Determination: Inject the localization solution and record the chromatogram.

[0056] 2. Experimental Results

[0057] Depend on Figure 1 As can be seen, the chromatographic column used in this invention exhibits strong chiral recognition of the intermediate 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol. Among the four isomers, the RS and SR isomers show no separation tendency, while the RR and SS isomers are well separated from the RS and SR isomers. Furthermore, the peak shapes are good, indicating that ethanolamine is a suitable basic charge-shifting agent.

[0058] Example 2

[0059] 1. Experimental Methods

[0060] (1) Set the chromatographic conditions: the mobile phase was n-hexane-0.2% ethanolamine ethanol solution = 90-10 (V / V), and the elution was isocratic for 40 min; the other chromatographic parameters were the same as in Example 1.

[0061] (2) Preparation of localization solution: Same as in Example 1.

[0062] (3) Determination: Inject the localization solution and record the chromatogram.

[0063] 2. Experimental Results

[0064] Depend on Figure 2 It is evident that changing the mobile phase ratio to n-hexane-0.2% ethanolamine ethanol solution = 90-10 (V / V) resulted in a separation trend between the RS and SR isomers, with a resolution of 1.14. Furthermore, the RR and SS isomers were well separated from the RS and SR isomers, but the SS isomer was more strongly retained with a broader peak shape. This indicates that reducing the alcohol ratio and increasing the n-hexane ratio can improve the separation effect between the RS and SR isomers.

[0065] Example 3

[0066] 1. Experimental Methods

[0067] (1) Chromatographic conditions were set as follows: the high performance liquid chromatograph was an Agilent 1260 liquid chromatograph, the column was a CHIRALPAKAD-H (4.6×250mm 5μm); the mobile phase was n-hexane-0.4% ethanolamine isopropanol solution = 85-15 (V / V), isocratic elution for 45 min; the injection volume was 10 μl, the column temperature was 25℃, the flow rate was 1.0 ml / min, and the UV detection wavelength was 280 nm.

[0068] (2) Preparation of localization solution: Take appropriate amounts of 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol of each of the four configurations, add methanol and sonicate to dissolve, and quantitatively dilute to prepare a mixed solution containing about 1 mg of each per ml.

[0069] (3) Determination: Inject the localization solution and record the chromatogram.

[0070] 2. Experimental Results

[0071] Depend on Figure 3 As can be seen, the four isomers were well separated, with a minimum resolution of 2.52. Combined with the detection results from Examples 1, 2, and 3, this indicates that the strong hydrogen bonding in the ethanol solution weakens the stereochemical recognition of the RS and SR isomers on the chromatographic column, resulting in poor separation.

[0072] Example 4

[0073] 1. Experimental Methods

[0074] (1) Set the chromatographic conditions: the mobile phase was n-hexane-0.4% ethanolamine isopropanol solution = 90-10 (V / V), and the isocratic elution was performed for 45 min; the other chromatographic parameters were the same as in Example 2.

[0075] (2) Preparation of localization solution: Same as in Example 1.

[0076] (3) Determination: Inject the localization solution and record the chromatogram.

[0077] 2. Experimental Results

[0078] Depend on Figure 4 It can be seen that the separation degree between the RS and SR configuration isomers is 2.93, which is 0.41 higher than that in Example 3. This indicates that the separation effect of the four configuration isomers is better when the mobile phase is n-hexane-0.4% ethanolamine isopropanol solution = 90-10 (V / V).

[0079] Example 5

[0080] 1. Experimental Methods

[0081] (1) Set the chromatographic conditions: the mobile phase was n-hexane-0.2% ethanolamine isopropanol solution = 90-10 (V / V), and the elution was isocratic for 45 min; the other chromatographic parameters were the same as in Example 2.

[0082] (2) Preparation of localization solution: Same as in Example 1.

[0083] Preparation of test solution: Dissolve and dilute the intermediate 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol (i.e., a pair of enantiomers with SS and RR configurations) with diluent to a concentration of 2.5 mg / ml.

[0084] (3) Determination: Inject the positioning solution and the test solution separately and record the chromatograms. Calculate the content of isomers in the test sample using the area percentage method.

[0085] 2. Experimental Results

[0086] Depend on Figure 5 As can be seen, the separation degree between the RS configuration and the SR configuration isomer is 2.94, which is not significantly different from that in Example 4. Therefore, the volume fraction of ethanolamine in the mobile phase is preferably 0.2%. Figure 6 and Figure 7 The images show typical spectra of the RR and SS type test samples, respectively, with good separation between the peaks of each isomer and the main peak.

[0087] Further methodological validation of the detection method designed in this invention showed that the resolution between 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol and its isomers and adjacent peaks reached baseline separation, and the injection precision of the detection results was good. The specific validation results are as follows:

[0088]

[0089]

[0090] As can be seen from the above methodological validation results, the method designed in this invention for the detection of the chirality of 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol and its isomers is highly specific and has good chromatographic condition robustness. It can be used for product quality control of 6-fluoro-3,4-dihydro-α-[[(phenylmethyl)amino]methyl]-2H-1-benzopyran-2-methanol and process quality control of nebivolol hydrochloride preparation.

Claims

1. A method for detecting nebivolol hydrochloride intermediates and their isomers, wherein the nebivolol hydrochloride intermediates are enantiomers of SS and RR configurations, with the following structures: The structure of the isomer is as follows: Its features are, Includes the following steps: (1) Sample preparation: Prepare the sample to the set concentration using the diluent; (2) Detection: Normal phase chromatography and chiral column were used, with a low alkyl alcohol-n-hexane solution containing a basic charge migration agent as the mobile phase for elution; (3) Analysis: Record the chromatogram and calculate the content of isomers.

2. The detection method according to claim 1, characterized in that, The diluent mentioned in step (1) is a lower alkyl alcohol.

3. The detection method according to claim 2, characterized in that, The diluent is methanol.

4. The detection method according to claim 1, characterized in that, The chromatographic column described in step (2) is packed with silica gel coated with amylose-tris(3,5-dimethylphenylcarbamate).

5. The detection method according to claim 4, characterized in that, The chromatographic column has a size of 4.6 × 250 mm and a diameter of 5 μm.

6. The detection method according to claim 1, characterized in that, In the mobile phase described in step (2), the volume fraction of lower alkyl alcohols is no more than 15%.

7. The detection method according to claim 1, characterized in that, The lower alkyl alcohols mentioned in step (2) are ethanol and isopropanol.

8. The detection method according to claim 1, characterized in that, The alkaline charge transfer agent mentioned in step (2) is diethylamine, triethylamine, ethanolamine, or diethanolamine.

9. The detection method according to claim 1, characterized in that, In the mobile phase described in step (2), the volume of the basic charge transfer agent added is 0.1% to 0.5% of the volume of the lower alkyl alcohol.

10. The detection method according to claim 1, characterized in that, The detection wavelength in step (2) is 260-290 nm, the mobile phase flow rate is 0.8-1.2 ml / min, and the column temperature is 20-30 °C.

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