Preparation method of daptomycin beta isomer

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a preparation method of a daptomycin beta isomer. The invention discloses a preparation method of a daptomycin beta isomer, which comprises the following steps: S1, dissolving a crude product containing the daptomycin beta isomer to obtain a loading solution; s2, separating and purifying the loading solution through preparative chromatography to obtain a primary preparation solution; s3, concentrating the primary preparation solution to obtain a primary concentrated solution, and separating and purifying the primary concentrated solution through preparative chromatography to obtain a secondary preparation solution; s4, concentrating the secondary preparation solution to obtain a secondary concentrated solution, and separating and purifying the obtained secondary concentrated solution through preparative chromatography to obtain a third preparation solution; and S5, sequentially desalting, concentrating and freeze-drying the tertiary preparation solution to obtain the high-purity daptomycin beta isomer. The method disclosed by the invention is rapid, simple, convenient and low in cost, and the purity of the obtained daptomycin beta isomer is higher than 99%.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and specifically relates to a method for preparing the β isomer of daptomycin. Background Technology

[0002] Daptomycin is a cyclic lipopeptide antibiotic produced by fermentation of *Streptomyces rosesporus*. It was approved for marketing in September 2003 for the treatment of complicated skin and soft tissue infections. In vitro studies have shown that daptomycin exhibits rapid in vitro bactericidal activity against a variety of drug-resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae, thus attracting widespread attention. Daptomycin was initially discovered and developed by Lilly in the early 1980s, and trials conducted in the early 1990s involved hundreds of subjects. Lilly reported good efficacy when using the compound to treat bloodstream infections, endocarditis, and complicated skin and soft tissue infections. In 1997, Cubist Pharmaceuticals acquired the exclusive global rights to develop, manufacture, and market daptomycin from Eli Lilly. After six years of research and development, the U.S. FDA approved Cubicin (daptomycin injection) in 2003 for the treatment of complicated skin and soft tissue infections (cSSSI) in adults caused by Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci, as well as bacteremia and right endocarditis caused by Staphylococcus aureus.

[0003] Although daptomycin is now produced industrially through fermentation, its complex chemical structure means that variations in production, purification, transportation, and storage conditions can lead to the generation of impurities, affecting product quality and therapeutic efficacy. To evaluate drug safety and the biological impact of impurities, the structures of these impurities must be confirmed. Furthermore, the source of these impurities must be specified when applying for daptomycin registration; therefore, research on these impurities is crucial. According to my country's quality standards for injectable daptomycin, the impurities requiring attention include: RS-1, RS-2, RS-3, daptomycin lactone hydrolysate, daptomycin β-isomer, RS-5 / 6, RS-7, RS-7a, dehydrated daptomycin, and RS-8a.

[0004] Existing literature rarely covers extraction methods for daptomycin isomers. For example, international patent WO2001053330 discloses a purification method for the daptomycin main peak and the structures of 11 impurities, but does not specify the preparation methods for these impurities. As one of the important impurities of daptomycin, convenient and efficient preparation of daptomycin isomers and research on their stability and bioactivity are of great significance for improving the quality of daptomycin. Summary of the Invention

[0005] The primary objective of this invention is to provide a method for preparing daptomycin isomers, which can obtain high-purity daptomycin β isomers, facilitating the study of daptomycin β isomers.

[0006] Therefore, the present invention provides the following technical solution.

[0007] One aspect of the present invention provides a method for preparing daptomycin β isomer, the method comprising the following steps:

[0008] S1: Dissolve the crude product containing the daptomycin β isomer to obtain the loading solution;

[0009] S2: The sample solution is purified by preparative chromatography to obtain a preparative solution;

[0010] S3: The primary preparation solution is concentrated to obtain a primary concentrate, and the obtained primary concentrate is purified by preparative chromatography to obtain a secondary preparation solution;

[0011] S4: The secondary preparation solution is concentrated to obtain a secondary concentrate, and the obtained secondary concentrate is purified by preparative chromatography to obtain a tertiary preparation solution;

[0012] S5: The three preparation solutions are sequentially desalted, concentrated, and freeze-dried to obtain high-purity daptomycin β isomer.

[0013] In a preferred embodiment of the present invention, in step S1, the purity of daptomycin β isomer in the crude product containing daptomycin β isomer is less than 5%.

[0014] In a preferred embodiment of the present invention, in step S1, the loading solution is an aqueous solution containing crude daptomycin β isomer, and the concentration of the loading solution is 50 mg / ml.

[0015] In a preferred embodiment of the present invention, in step S2, the chromatographic column used for the preparation chromatography is C8 or C18; wavelength: 214 or 223 nm; column temperature: 20-30℃; flow rate: 70 ml / min.

[0016] In a preferred embodiment of the present invention, in step S2, the mobile phase is a mixture of acetonitrile and salt solution with a pH value between 2 and 4.5.

[0017] In a preferred embodiment of the present invention, in step S3, the chromatographic column used for the preparation chromatography is C18, C18HC, C18HCE or C18YE; wavelength: 214 or 223 nm; column temperature: 20-30℃; flow rate: 70 ml / min.

[0018] In a preferred embodiment of the present invention, in step S3, the mobile phase is a mixture of acetonitrile and salt solution with a pH value between 5 and 7.

[0019] In a preferred embodiment of the present invention, in step S4, the chromatographic column used for the preparation chromatography is a C8 or C18 (250×50mm id, Wavelength: 214 or 223 nm; Column temperature: 20-30℃; Flow rate: 70 ml / min.

[0020] In a preferred embodiment of the present invention, in step S4, the mobile phase is a mixture of acetonitrile and salt solution with a pH value between 2 and 4.5.

[0021] In a preferred embodiment of the present invention, the volume ratio of acetonitrile to salt solution is 20:80 to 50:50; the concentration of salt solution is 5-30 mmol / L.

[0022] In a preferred embodiment of the present invention, the salt is selected from one or a mixture of two or more of ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, and ammonium acetate.

[0023] In a preferred embodiment of the present invention, the concentration method is rotary evaporation at a temperature of 35-40°C.

[0024] In a preferred embodiment of the present invention, in step S5, the desalting method is column concentration desalting.

[0025] Another aspect of the present invention provides a daptomycin β isomer prepared according to the preparation method described above, characterized in that the purity of the daptomycin β isomer is greater than 99%.

[0026] By employing the above technical solution, the present invention has at least the following advantages:

[0027] This invention provides a rapid, simple, and low-cost process for preparing high-purity daptomycin β isomers using preparative chromatography. The invention employs preparative chromatography to purify crude daptomycin β isomer samples. After three separation and purification processes using this system, high-purity daptomycin β isomers with a purity exceeding 99% can be obtained. The method of this invention is simple, inexpensive, and yields high purity and high efficiency.

[0028] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0029] Figure 1 The high-performance liquid chromatogram of the daptomycin β isomer obtained in Example 1 is shown below.

[0030] Figure 2 This is a structural diagram of the daptomycin β isomer obtained in Example 1;

[0031] Figure 3 The daptomycin β isomer obtained in Example 1 1 H NMR spectrum;

[0032] Figure 4 The daptomycin β isomer obtained in Example 1 13 C NMR spectrum;

[0033] Figure 5 The DEPT 90° spectrum of the daptomycin β isomer obtained in Example 1;

[0034] Figure 6 The DEPT 135° spectrum of the daptomycin β isomer obtained in Example 1;

[0035] Figure 7 The HSQC spectrum of the daptomycin β isomer obtained in Example 1;

[0036] Figure 8 The HMBC spectrum of the daptomycin β isomer obtained in Example 1;

[0037] Figure 9 The daptomycin β isomer obtained in Example 1 1 H- 1 H COSY spectrum. Detailed Implementation

[0038] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] Example 1:

[0042] The crude daptomycin containing the β isomer was dissolved in water to obtain a loading solution with a concentration of 50 mg / ml.

[0043] The obtained sample solution was purified by preparative chromatography using a Hanbang NP-7000 series chromatographic column (250×50 mm i.d., 10 μm); wavelength: 223 nm; column temperature: 25℃ room temperature; flow rate: 70 mL / min; mobile phase: acetonitrile and 10 mmol / L ammonium dihydrogen phosphate solution, pH = 2.5; elution ratio: 30% acetonitrile isocratic. The chromatographic peak of daptomycin β isomer was collected and detected by high performance liquid chromatography. A first-stage preparative solution was obtained, in which the purity of daptomycin β isomer was 80%.

[0044] The obtained primary preparation solution was rotary evaporated at 35℃ until sample precipitate, yielding a primary concentrate. This primary concentrate was used as a loading solution for preparative chromatography purification. The chromatographic column was a C18HCE (250×50mm id, 10μm); wavelength: 223nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: acetonitrile and 10mmol / L ammonium dihydrogen phosphate solution, pH=5.5; elution conditions: 0-30min, 20-50% acetonitrile. The chromatographic peak of the daptomycin β isomer was collected and detected by high-performance liquid chromatography (HPLC). A secondary preparation solution was obtained, in which the purity of the daptomycin β isomer was 94.2%.

[0045] The obtained secondary preparation solution was rotary evaporated at 35℃ until sample precipitate was obtained, yielding a secondary concentrated solution. This secondary concentrated solution was used as a loading solution for preparative chromatography purification. The chromatographic column was a C18 (250×50mm id, 10μm); wavelength: 223nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: acetonitrile and 10mmol / L ammonium dihydrogen phosphate solution, pH=2.5; elution conditions: isocratic with 35% acetonitrile; the chromatographic peak of the daptomycin β isomer was collected and detected by high performance liquid chromatography. A tertiary preparation solution was obtained, in which the purity of the daptomycin β isomer was 99.33%.

[0046] The obtained three preparation solutions were sequentially desalted by column loading, the eluted fractions were collected, and finally the solid daptomycin β isomer was obtained by vacuum freeze drying.

[0047] Table 1 below shows the C1H NMR spectral assignments of the daptomycin β isomer.

[0048] Table 1. Carbon-hydrogen spectrum assignment of daptomycin β isomer

[0049]

[0050]

[0051] like Figure 1 As shown, the purity of the daptomycin β isomer was confirmed to be 99.31% after detection by high performance liquid chromatography.

[0052] like Figures 3 to 9 As shown, NMR analysis confirmed that the impurity was the β isomer of daptomycin.

[0053] Example 2:

[0054] The crude daptomycin containing the β isomer was dissolved in water to obtain a loading solution with a concentration of 50 mg / ml.

[0055] The obtained sample solution was purified by preparative chromatography using a Hanbang NP-7000 series chromatographic column (C8, 250×50 mm i.d., 10 μm); wavelength: 214 nm; column temperature: 25℃; flow rate: 70 mL / min; mobile phase: acetonitrile and 15 mmol / L sodium dihydrogen phosphate solution, pH = 3; elution ratio: 33% acetonitrile isocratic. The chromatographic peak of daptomycin β isomer was collected and detected by high performance liquid chromatography (HPLC). A preparative solution was obtained, in which the purity of daptomycin β isomer was 79%.

[0056] The obtained primary preparation solution was rotary evaporated at 35℃ until sample precipitate, yielding a primary concentrate. This concentrate was used as the loading solution for preparative chromatography purification. The chromatographic column was a C18HC (250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: acetonitrile and 15mmol / L sodium dihydrogen phosphate solution, pH=6; elution conditions: 0-30min, 18-45% acetonitrile. The chromatographic peak of the daptomycin β isomer was collected and detected by high-performance liquid chromatography (HPLC). A secondary preparation solution was obtained, in which the purity of the daptomycin β isomer was 93.9%.

[0057] The obtained secondary preparative solution was rotary evaporated at 35℃ until sample precipitate, yielding a secondary concentrated solution. This secondary concentrated solution was used as the loading solution for preparative chromatography purification. The chromatographic column was a C18 (250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: acetonitrile and 15mmol / L sodium dihydrogen phosphate solution, pH=3; elution conditions: isocratic elution with 33% acetonitrile. The chromatographic peak of the daptomycin β isomer was collected and detected by high-performance liquid chromatography. A tertiary preparative solution was obtained, in which the purity of the daptomycin β isomer was 99.27%.

[0058] The obtained three preparation solutions were sequentially desalted by column loading, the eluted fractions were collected, and finally the solid daptomycin β isomer was obtained by vacuum freeze drying.

[0059] The purity of the daptomycin β isomer was confirmed to be 99.33% by high performance liquid chromatography.

[0060] Nuclear magnetic resonance analysis confirmed that the impurity was the β isomer of daptomycin.

[0061] Example 3:

[0062] The crude daptomycin containing the β isomer was dissolved in water to obtain a loading solution with a concentration of 50 mg / ml.

[0063] The obtained sample solution was purified by preparative chromatography using a Hanbang NP-7000 series chromatographic column (C18, 250×50 mm i.d., 10 μm); wavelength: 214 nm; column temperature: 25℃ room temperature; flow rate: 70 mL / min; mobile phase: acetonitrile and 20 mmol / L potassium dihydrogen phosphate solution, pH = 3.3; elution ratio: 37% acetonitrile isocratic. The chromatographic peak of daptomycin β isomer was collected and detected by high performance liquid chromatography (HPLC). A preparative solution was obtained, in which the purity of daptomycin β isomer was 80%.

[0064] The obtained primary preparation solution was rotary evaporated at 35℃ until sample precipitate, yielding a primary concentrate. This primary concentrate was used as a loading solution for preparative chromatography purification. The chromatographic column was a C18YE (250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: acetonitrile and 20mmol / L potassium dihydrogen phosphate solution, pH=5.5; elution conditions: 0-30min, 20-45% acetonitrile. The chromatographic peak of the daptomycin β isomer was collected and detected by high-performance liquid chromatography (HPLC) to obtain the secondary preparation solution, in which the purity of the daptomycin β isomer was 93.8%.

[0065] The obtained secondary preparation solution was rotary evaporated at 35℃ until sample precipitated, yielding a secondary concentrated solution. This secondary preparation solution was then used as a loading solution for preparative chromatography purification (column: C18 (250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: acetonitrile and 20mmol / L potassium dihydrogen phosphate solution, pH=3.3; elution conditions: 37% acetonitrile isocratic). The chromatographic peak of the daptomycin β isomer was collected and detected by high-performance liquid chromatography. The resulting tertiary preparation solution had a purity of 99.27% ​​for the daptomycin β isomer.

[0066] The obtained three preparation solutions were sequentially desalted by column loading, the eluted fractions were collected, and finally the solid daptomycin β isomer was obtained by vacuum freeze drying.

[0067] The purity of the daptomycin β isomer was confirmed to be 99.23% by high performance liquid chromatography.

[0068] Nuclear magnetic resonance analysis confirmed that the impurity was the β isomer of daptomycin.

[0069] Comparative Example 1:

[0070] The crude daptomycin containing the β isomer was dissolved in water to obtain a loading solution with a concentration of 50 mg / ml.

[0071] The obtained sample solution was purified by preparative chromatography using a Hanbang NP-7000 series chromatographic column (C18AQ-L, 250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: ethanol and 20mmol / L sodium chloride solution, pH=6.3; elution ratio: 50% ethanol isocratic. The chromatographic peak of daptomycin β isomer was collected and detected by high performance liquid chromatography. A first-stage preparative solution was obtained, in which the purity of daptomycin β isomer was 8.2%.

[0072] The obtained primary preparation solution was rotary evaporated at 35℃ until sample precipitate was obtained, yielding a primary concentrate. This primary concentrate was used as a loading solution and purified by preparative chromatography. The chromatographic column was X4 (250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: ethanol and 20mmol / L sodium chloride solution, pH=3.3; elution conditions: 0-30min, 30-65% ethanol. The chromatographic peak of the daptomycin β isomer was collected and detected by high performance liquid chromatography (HPLC) to obtain the secondary preparation solution, in which the purity of the daptomycin β isomer was 10.1%.

[0073] The obtained secondary preparation solution was rotary evaporated at 35℃ until sample precipitated, yielding a secondary concentrated solution. This secondary preparation solution was then used as a loading solution for preparative chromatography purification (column: C18AQ-L (250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: ethanol and 20mmol / L sodium chloride solution, pH=6.3; elution conditions: 50% ethanol isocratic). The chromatographic peak of the daptomycin β isomer was collected and detected by high performance liquid chromatography. The resulting tertiary preparation solution had a purity of 13.6% for the daptomycin β isomer.

[0074] Therefore, it is evident that daptomycin β isomers with acceptable purity cannot be obtained after three preparations.

[0075] Comparative Example 2:

[0076] The crude daptomycin containing the β isomer was dissolved in water to obtain a loading solution with a concentration of 50 mg / ml.

[0077] The obtained sample solution was purified by preparative chromatography using a Hanbang NP-7000 series chromatographic column (C18HCE, 250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: methanol and 20mmol / L ammonium formate solution, pH=6.3; elution ratio: 52% methanol isocratic. The chromatographic peak of daptomycin β isomer was collected and detected by high performance liquid chromatography (HPLC). A first-stage preparative solution was obtained, in which the purity of daptomycin β isomer was 8.5%.

[0078] The obtained primary preparation solution was rotary evaporated at 35℃ until sample precipitate was obtained, yielding a primary concentrate. This primary concentrate was used as a loading solution for preparative chromatography purification. The chromatographic column was X4 (250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: methanol and 20mmol / L ammonium formate solution, pH=3.3; elution conditions: 0-35min, 35-68% methanol. The chromatographic peak of the daptomycin β isomer was collected and detected by high-performance liquid chromatography (HPLC) to obtain the secondary preparation solution, in which the purity of the daptomycin β isomer was 9.1%.

[0079] The obtained secondary preparation solution was rotary evaporated at 35℃ until sample precipitate was obtained, yielding a secondary concentrated solution. This secondary preparation solution was then used as a loading solution for preparative chromatography purification (column: C18HCE (250×50mm id, 10μm); wavelength: 214nm; column temperature: 25℃; flow rate: 70ml / min; mobile phase: methanol and 20mmol / L ammonium formate solution, pH=6.3; elution conditions: 52% methanol isocratic). The chromatographic peak of the daptomycin β isomer was collected and detected by high performance liquid chromatography. A tertiary preparation solution was obtained, in which the purity of the daptomycin β isomer was 12.6%.

[0080] Therefore, it is evident that daptomycin β isomers with acceptable purity cannot be obtained after three preparations.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a daptomycin β-isomer, characterized in that, The method includes the following steps: S1: Dissolve the crude product containing the daptomycin β isomer to obtain the loading solution; S2: The sample solution is purified by preparative chromatography to obtain a preparative solution; S3: The primary preparation solution is concentrated to obtain a primary concentrate, and the obtained primary concentrate is purified by preparative chromatography to obtain a secondary preparation solution; S4: The secondary preparation solution is concentrated to obtain a secondary concentrate, and the obtained secondary concentrate is purified by preparative chromatography to obtain a tertiary preparation solution; S5: The three preparation solutions are sequentially desalted, concentrated, and freeze-dried to obtain high-purity daptomycin β isomer.

2. The preparation method according to claim 1, characterized in that, In step S1, the purity of the daptomycin β isomer in the crude product containing the daptomycin β isomer is less than 5%; The loading solution is an aqueous solution containing crude daptomycin β isomer, and the concentration of the loading solution is 50 mg / ml.

3. The preparation method according to claim 1, characterized in that, In step S2, the chromatographic column used for the preparation chromatography is C8 or C18; Wavelength: 214 or 223 nm; Column temperature: 20-30℃; Flow rate: 70 ml / min; The mobile phase is a mixture of acetonitrile and salt solution with a pH value between 2 and 4.

5.

4. The preparation method according to claim 1, characterized in that, In step S3, the chromatographic column used for the preparation chromatography is C18, C18HC, C18HCE, or C18YE; Wavelength: 214 or 223 nm; Column temperature: 20-30℃; Flow rate: 70 ml / min; The mobile phase is a mixture of acetonitrile and salt solution with a pH value between 5 and 7.

5. The preparation method according to claim 1, characterized in that, In step S4, the chromatographic column used for the preparative chromatography is a C8 or C18 column (250×50mm id, 10μm). ); Wavelength: 214 or 223 nm; Column temperature: 20-30℃; Flow rate: 70 ml / min; The mobile phase is a mixture of acetonitrile and salt solution with a pH value between 2 and 4.

5.

6. The preparation method according to any one of claims 3 to 5, characterized in that, The volume ratio of acetonitrile to salt solution is 20:80 to 50:50; the concentration of salt solution is 5-30 mmol / L.

7. The preparation method according to claim 6, characterized in that, The salt is selected from one or a mixture of two or more of the following: ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, and ammonium acetate.

8. The preparation method according to claim 1, characterized in that, The concentration method is rotary evaporation at a temperature of 35-40℃.

9. The preparation method according to claim 1, characterized in that, In step S5, the desalting method is column concentration desalting.

10. The daptomycin β-isomer prepared by the preparation method according to any one of claims 1 to 9, characterized in that, The purity of the daptomycin β isomer is greater than 99%.

Images