Chiral analysis method of semeglutide series intermediates

Abstract

The invention discloses a chiral analysis method of a series of semeglutide intermediates, the analysis method is used for rapidly determining the chiral purity of a series of semeglutide intermediates, a high performance liquid chromatography (HPLC) is combined with a chiral column, and chromatographic conditions and elution procedures are optimized, so that the analysis method is simple and convenient to operate, rapid, high in universality, high in sensitivity, high in sensitivity and high in accuracy. According to the method, the chromatographic conditions and the elution procedure are optimized, the chiral separation effect and resolution are improved, efficient and accurate chiral separation and analysis of the semeglutide series intermediates shown in the formula 1 are achieved, and the method is suitable for chiral quality control of the semeglutide series intermediates shown in the formula 1.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry, relates to the analysis of semaglutide intermediates, and in particular to a chiral analysis method for rapidly determining the chiral purity of a series of semaglutide intermediates. Background Art

[0002] Semaglutide is a glucagon-like peptide receptor agonist, namely GLP-1 receptor agonist. The human body's GLP-1 is a peptide hormone secreted by intestinal L cells. Its mechanism of action is to act on pancreatic β cells, promote the synthesis and secretion of insulin, stimulate the proliferation and differentiation of pancreatic β cells, inhibit the apoptosis of pancreatic β cells, increase the number of pancreatic β cells, and protect the function of the pancreas. It can also act on pancreatic α cells, inhibit the release of glucagon, and reduce the release of glycogen. It acts on pancreatic δ cells, promotes the secretion of somatostatin, and participates in inhibiting the secretion of glucagon. It enhances insulin sensitivity, slows down gastric emptying, acts on the hypothalamus to suppress appetite, and reduces weight. Through these actions, it achieves the effect of lowering blood sugar. Semaglutide connects the lysine position at position 26 of the peptide chain to an 18-carbon fatty diacid side chain or its derivative (referred to as the semaglutide side chain, as shown in the following compound 1), which can mediate and promote the strong binding of semaglutide to albumin and reduce the renal clearance rate. Compared with liraglutide with a C16 side chain, the affinity of the semaglutide side chain to albumin is enhanced by 5 to 6 times. Binding to albumin can increase the molecular weight of the product, avoid rapid clearance by the kidneys, prevent metabolic degradation, and prolong the half-life in the body.

[0003] Therefore, as an important hypoglycemic and weight-loss drug, semaglutide uses multiple chiral amino acids in its preparation process, resulting in multiple chiral centers in its molecular structure. In the preparation process of semaglutide, chiral amino acids may undergo racemization, and ensuring the chiral purity of the intermediate is crucial to the quality and efficacy of the final drug.

[0004] Therefore, developing an accurate and efficient chiral analysis method is of great significance for monitoring the chiral purity of semaglutide intermediates.

[0005] At present, chiral analysis methods mainly include nuclear magnetic resonance, circular dichroism, polarimetry, high performance liquid chromatography, etc. Except for high performance liquid chromatography or gas chromatography, other methods have disadvantages such as high cost, low instrument versatility, and low sensitivity, which limit their application in industrial production. There are currently no reports on high performance liquid chromatography or gas chromatography for semaglutide series intermediates. All semaglutide series intermediates have chiral isomers, and chiral purity is an important indicator for intermediate quality control. The development of a universal chiral purity inspection method improves the convenience of intermediate quality control during the production process. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a chiral analysis method for a series of semaglutide intermediates. The analysis method of the present invention is used to quickly determine the chiral purity of a series of semaglutide intermediates. High performance liquid chromatography (HPLC) is combined with a chiral column. By optimizing the chromatographic conditions and elution procedures, efficient and accurate chiral separation and analysis of the series of semaglutide intermediates are achieved. The method has the advantages of fast analysis speed, high accuracy, simple operation, etc.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a chiral analysis method for a series of semaglutide intermediates, wherein the chiral analysis method is to use high performance liquid chromatography combined with a chiral column for analysis;

[0009] The structural formula of the semaglutide series intermediates is shown in Formula I:

[0010] In the formula, R1 is hydrogen, C1-C4 alkyl, ethoxy, tert-butyloxy, benzyl, tert-butylmethoxy or tert-butylethoxy, and R2 is Fmoc or Cbz; R3 is hydrogen, C1-C4 alkyl, and R4 is hydrogen, C1-C4 alkyl, tert-butyl formate, tert-butyl acetate, tritylformamide, tritylacetamide or p-tert-butylphenyl ether.

[0011] As a preferred solution of the present invention, the chiral column is a silica gel surface coated with cellulose-tris (3, 5-dimethylphenylcarbamate) as a filler.

[0012] As a preferred embodiment of the present invention, the specific steps of the analysis method include:

[0013] 1) Prepare the sample: take the semaglutide intermediate and add the diluent to prepare a test solution;

[0014] 2) Sample injection analysis: Take the test solution obtained in step 1) and inject it into the high performance liquid chromatograph for analysis, and record the chromatogram.

[0015] As a preferred embodiment of the present invention, in step 1), the concentration of the test solution is 0.5 mg / mL-3.0 mg / mL, and the diluent is 80%-100% ethanol.

[0016] As a preferred embodiment of the present invention, in step 2), the chromatographic conditions of the high performance liquid chromatography are: column temperature of 20°C-45°C, flow rate of 0.5mL / min-2.0mL / min, detection wavelength of 205nm-285nm, and injection volume of 5μL-50μL.

[0017] As a preferred embodiment of the present invention, the column temperature is 25°C-40°C, the flow rate is 0.8mL / min-1.5mL / min, and the detection wavelength is 210nm-254nm.

[0018] As a preferred embodiment of the present invention, the mobile phase of the HPLC is composed of mobile phase A and mobile phase B, and isocratic or gradient elution is performed, the mobile phase A is a phosphate or acetate buffer, and the mobile phase B is acetonitrile.

[0019] As a preferred embodiment of the present invention, the molar concentration of the buffer salt in the mobile phase A is 5 mmol / L-50 mmol / L.

[0020] As a preferred embodiment of the present invention, during isocratic elution, the ratio of mobile phase A to mobile phase B is 70:30-30:70.

[0021] As a preferred embodiment of the present invention, the gradient elution program is: 0 min, 70% mobile phase A, 30% mobile phase B; 15 min, 15% mobile phase A, 85% mobile phase B; 20 min, 15% mobile phase A, 85% mobile phase B; 20.1 min, 70% mobile phase A, 30% mobile phase B; 25 min, 70% mobile phase A, 30% mobile phase B.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The present invention uses high performance liquid chromatography combined with a chiral column to achieve efficient and accurate chiral separation and analysis of a series of semaglutide intermediates.

[0024] 2) The present invention improves the effect and resolution of chiral separation by optimizing the chromatographic conditions and elution procedure.

[0025] 3) The analytical method of the present invention is simple, rapid, and versatile, and is suitable for the chiral quality control of the semaglutide series intermediates shown in Formula 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 is the chromatogram of Example 1.

[0028] Figure 2 This is the chromatogram of Example 2.

[0029] Figure 3 This is the chromatogram of Example 3.

[0030] Figure 4 This is the chromatogram of Example 4.

[0031] Figure 5 This is the chromatogram of Example 5.

[0032] Figure 6 This is the chromatogram of Example 6.

[0033] Figure 7 This is the chromatogram of Example 7.

[0034] Figure 8 It is the chromatogram of Example 8. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0036] The structural formula of the semaglutide series intermediates described in the present invention is shown in Formula I:

[0037] In the formula, R1 is hydrogen, C1-C4 alkyl, ethoxy, tert-butyloxy, benzyl, tert-butylmethoxy or tert-butylethoxy, and R2 is Fmoc or Cbz; R3 is hydrogen, C1-C4 alkyl, and R4 is hydrogen, C1-C4 alkyl, tert-butyl formate, tert-butyl acetate, tritylformamide, tritylacetamide or p-tert-butylphenyl ether.

[0038] In the present invention, the chromatographic conditions are respectively:

[0039] The column temperature is 20℃-45℃, the flow rate is 0.5mL / min-2.0mL / min, the detection wavelength is 205nm-285nm, and the injection volume is 5μL-50μL.

[0040] More preferably, the column temperature is 25°C-40°C, the flow rate is 0.8mL / min-1.5mL / min, and the detection wavelength is 210nm-254nm.

[0041] The mobile phase consists of mobile phase A and mobile phase B, and isocratic or gradient elution is performed. The mobile phase A is a phosphate or acetate buffer, and the mobile phase B is acetonitrile.

[0042] Preferably, the molar concentration of the buffer salt in the mobile phase A is 5 mmol / L-50 mmol / L.

[0043] More preferably, mobile phase A is phosphate buffer, and the buffer concentration is 10 mmol / L-30 mmol / L.

[0044] Preferably, during isocratic elution, the ratio of mobile phase A to mobile phase B is 30-45:55-70.

[0045] The "%" appearing in the present invention refers to the volume fraction.

[0046] Example 1

[0047] This embodiment provides a chiral analysis method for a semaglutide intermediate, wherein the compound is as shown in Formula I, R1 is a methyl group, R2 is Fmoc, R3 is an ethyl group, and R4 is a methyl group.

[0048] Diluent: 95% ethanol, test solution: 1.0 mg / mL, injection volume: 5 μL.

[0049] Chromatographic conditions and injection method:

[0050] Inject sample according to the following chromatographic conditions and record the chromatogram;

[0051] Chromatographic column: CHIRALCEL OD-RH 4.6mm×250mm, 5μm, flow rate: 1.0mL / min, column temperature: 30℃, wavelength: 210nm, mobile phase A: 25mmol / L mixed solution of potassium dihydrogen phosphate and potassium hydrogen phosphate, mobile phase B: acetonitrile

[0052] Elution gradient: See Table 1.

[0053] Chromatography Figure 1 The peak at 12.291 minutes is the L configuration, and the peak at 12.576 minutes is its enantiomer, and the separation degree between the two is 1.65.

[0054] Table 1. Elution gradient

[0055] Time / min Mobile phase A / % Mobile phase B / % 0 70 30 15 15 85 20 15 85 20.1 70 30 25 70 30

[0056] Example 2

[0057] This embodiment provides a chiral analysis method for a semaglutide intermediate, wherein the compound is as shown in Formula I, R1 is tert-butylmethoxy, R2 is Fmoc, R3 is methyl, and R4 is tert-butyloxy.

[0058] Diluent: anhydrous ethanol, test solution 3.0 mg / mL, injection volume: 10 μL.

[0059] Chromatographic conditions and injection method:

[0060] Inject sample according to the following chromatographic conditions and record the chromatogram;

[0061] Chromatographic column: CHIRALCEL OD-RH 4.6mm×250mm, 5μm, flow rate: 0.9mL / min, column temperature: 35℃, wavelength: 210nm, mobile phase A: 10mmol / L diammonium phosphate solution, mobile phase B: acetonitrile

[0062] Elution gradient: isocratic elution A:B=45:55.

[0063] Chromatograms see Figure 2 The peak at 10.910 minutes is the L configuration, and the peak at 9.622 minutes is its enantiomer, and the separation degree between the two is 2.88.

[0064] Example 3

[0065] This embodiment provides a chiral analysis method for a semaglutide intermediate, wherein the compound is as shown in Formula I, R1 is hydrogen, R2 is Fmoc, R3 is hydrogen, and R4 is tert-butyl acetate.

[0066] The diluent was anhydrous ethanol, the test solution was 0.5 mg / mL, and the injection volume was 5 μL.

[0067] Chromatographic conditions and injection method:

[0068] Inject sample according to the following chromatographic conditions and record the chromatogram;

[0069] Chromatographic column: CHIRALCEL OD-RH 4.6mm×250mm, 5μm, flow rate: 1.0mL / min, column temperature: 30℃, wavelength: 210nm, mobile phase A: 20mmol / L potassium dihydrogen phosphate solution, mobile phase B: acetonitrile, elution gradient: isocratic elution A:B=45:55.

[0070] Chromatograms see Figure 3 The peak at 21.545 minutes is the L configuration, and the peak at 13.811 minutes is its enantiomer, and the separation degree between the two is 8.28.

[0071] Example 4

[0072] This embodiment provides a chiral analysis method for a semaglutide intermediate, wherein the compound is as shown in Formula I, R1 is tert-butyloxy, R2 is Cbz, R3 is methyl, and R4 is tert-butyloxy.

[0073] The diluent is anhydrous ethanol, the test solution is 2.0 mg / mL, and the injection volume is 5 μL.

[0074] Chromatographic conditions and injection method:

[0075] Inject sample according to the following chromatographic conditions and record the chromatogram;

[0076] Chromatographic column: Ultimate Cellu-DR 4.6mm×250mm, 5μm, flow rate: 1.0mL / min, column temperature: 30℃, wavelength: 210nm, mobile phase A: 10mmol / L sodium acetate solution, mobile phase B: acetonitrile, elution gradient: isocratic elution A:B=35:65.

[0077] Chromatograms see Figure 4 The peak at 9.802 minutes is the L configuration, and the peak at 7.639 minutes is its enantiomer, and the separation degree between the two is 5.29.

[0078] Example 5

[0079] This embodiment provides a chiral analysis method for a semaglutide intermediate, wherein the compound is as shown in Formula I, R1 is a benzyl group, R2 is Fmoc, R3 is hydrogen, and R4 is tert-butyl acetate.

[0080] The diluent is anhydrous ethanol, the test solution is 1.5 mg / mL, and the injection volume is 20 μL.

[0081] Chromatographic conditions and injection method:

[0082] Inject sample according to the following chromatographic conditions and record the chromatogram;

[0083] Chromatographic column: CHIRALCEL OD-RH 4.6mm×250mm, 5μm, flow rate: 0.8mL / min, column temperature: 30℃, wavelength: 210nm, mobile phase A: 10mmol / L sodium dihydrogen phosphate solution, mobile phase B: acetonitrile, elution gradient: isocratic elution A:B=45:55.

[0084] Chromatograms see Figure 5 The peak at 18.001 minutes is the L configuration, and the peak at 16.759 minutes is its enantiomer, and the separation degree between the two is 1.66.

[0085] Example 6

[0086] This embodiment provides a chiral analysis method for a semaglutide intermediate, wherein the compound is as shown in Formula I, R1 is a methyl group, R2 is Fmoc, R3 is hydrogen, and R4 is a tritylcarboxamide group.

[0087] The diluent is anhydrous ethanol, the test solution is 2.5 mg / mL, and the injection volume is 10 μL.

[0088] Chromatographic conditions and injection method:

[0089] Inject sample according to the following chromatographic conditions and record the chromatogram;

[0090] Chromatographic column: CHIRALCEL OD-RH 4.6 mm × 250 mm, 5 μm, flow rate: 0.8 mL / min, column temperature: 30 ° C, wavelength: 254 nm, mobile phase A: 15 mmol / L potassium dihydrogen phosphate solution, mobile phase B: acetonitrile, elution gradient: see Table 1.

[0091] Chromatograms see Figure 6 The peak at 15.908 minutes is the L configuration, and the peak at 14.880 minutes is its enantiomer, and the separation degree between the two is 1.70.

[0092] Example 7

[0093] This embodiment provides a chiral analysis method for a semaglutide intermediate, wherein the compound is as shown in Formula I, R1 is an isobutyl group, R2 is Fmoc, R3 is hydrogen, and R4 is a p-tert-butylphenyl ether group.

[0094] The diluent is anhydrous ethanol, the test solution is 1.5 mg / mL, and the injection volume is 10 μL.

[0095] Chromatographic conditions and injection method: Inject samples according to the following chromatographic conditions and record the chromatogram;

[0096] Chromatographic column: Ultimate Cellu-DR 4.6mm×250mm, 5μm, flow rate: 1.0mL / min, column temperature: 25℃, wavelength: 220nm, mobile phase A: 25mmol / L ammonium acetate solution, mobile phase B: acetonitrile, elution gradient: isocratic elution A:B=40:60.

[0097] Chromatograms see Figure 7 The peak at 13.288 minutes is the L configuration, and the peak at 10.986 minutes is its enantiomer, and the separation degree between the two is 3.58.

[0098] Example 8

[0099] This embodiment provides a chiral analysis method for a semaglutide intermediate, wherein the compound is as shown in Formula I, R1 is isopropyl, R2 is Fmoc, R3 is hydrogen, and R4 is tert-butyl acetate.

[0100] The diluent is sewage ethanol, the test sample solution is 2.0 mg / mL, and the injection volume is 5 μL.

[0101] Chromatographic conditions and injection method:

[0102] Inject sample according to the following chromatographic conditions and record the chromatogram;

[0103] Chromatographic column: Ultimate Cellu-DR 4.6mm×250mm, 5μm, flow rate: 1.0mL / min, column temperature: 25℃, wavelength: 210nm, mobile phase A: 15mmol / L diammonium dihydrogen phosphate solution, mobile phase B: acetonitrile, elution gradient: isocratic elution A:B=45:55.

[0104] Chromatograms see Figure 8 The peak at 11.170 minutes is the L configuration, and the peak at 9.671 minutes is its enantiomer, and the separation degree between the two is 2.56.

[0105] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A chiral analysis method for a semaglutide series of intermediates, characterized in that: The chiral analysis method is to use high performance liquid chromatography combined with a chiral column for analysis; The structural formula of the semaglutide series intermediates is shown in Formula I: In the formula, R1 is hydrogen, C1-C4 alkyl, ethoxy, tert-butyloxy, benzyl, tert-butylmethoxy or tert-butylethoxy, and R2 is Fmoc or Cbz; R3 is hydrogen, C1-C4 alkyl, and R4 is hydrogen, C1-C4 alkyl, tert-butyl formate, tert-butyl acetate, tritylformamide, tritylacetamide or p-tert-butylphenyl ether.

2. The chiral analysis method of a semaglutide series intermediate according to claim 1, characterized in that: The chiral column is a silica gel surface coated with cellulose-tris (3, 5-dimethylphenyl carbamate) as a filler.

3. A chiral analysis method for a semaglutide series intermediate according to claim 1 or 2, characterized in that: The specific steps of the analysis method include: 1) Prepare the sample: take the semaglutide intermediate and add the diluent to prepare a test solution; 2) Sample injection analysis: Take the test solution obtained in step 1) and inject it into the high performance liquid chromatograph for analysis, and record the chromatogram.

4. The chiral analysis method of a semaglutide series intermediate according to claim 3, characterized in that: In step 1), the concentration of the test solution is 0.5 mg / mL-3.0 mg / mL, and the diluent is 80%-100% ethanol.

5. The chiral analysis method of a semaglutide series intermediate according to claim 3, characterized in that: In step 2), the chromatographic conditions of the HPLC are: column temperature of 20°C-45°C, flow rate of 0.5mL / min-2.0mL / min, detection wavelength of 205nm-285nm, and injection volume of 5μL-50μL.

6. The chiral analysis method of a semaglutide series intermediate according to claim 5, characterized in that: The column temperature is 25°C-40°C, the flow rate is 0.8mL / min-1.5mL / min, and the detection wavelength is 210nm-254nm.

7. The chiral analysis method of a semaglutide series intermediate according to claim 3, characterized in that: The mobile phase of the high performance liquid chromatography is composed of mobile phase A and mobile phase B, and isocratic or gradient elution is performed. The mobile phase A is a phosphate or acetate buffer, and the mobile phase B is acetonitrile.

8. The chiral analysis method of a semaglutide series intermediate according to claim 7, characterized in that: The molar concentration of the buffer salt in mobile phase A is 5 mmol / L-50 mmol / L.

9. The chiral analysis method of a semaglutide series intermediate according to claim 7, characterized in that: During isocratic elution, the ratio of mobile phase A to mobile phase B is 70:30-30:

70.

10. The chiral analysis method of a semaglutide series intermediate according to claim 7, characterized in that: The gradient elution program was as follows: 0 min, 70% mobile phase A, 30% mobile phase B; 15 min, 15% mobile phase A, 85% mobile phase B; 20 min, 15% mobile phase A, 85% mobile phase B; 20.1min, 70% mobile phase A, 30% mobile phase B; 25min, 70% mobile phase A, 30% mobile phase B.

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