Purification method of oxamacycline

Abstract

The invention provides a purification method of omalonic acid, which comprises the following steps: 1) preparing a solution of an omalonic acid crude product by using a solvent, optionally adjusting the pH value of the solution to be neutral by using acid or an aqueous solution thereof, and filtering to obtain a filtrate; (2) purifying the filtrate obtained in the step (1) by using a reversed-phase column chromatography to obtain purified ombracin; wherein the chromatographic conditions of the reversed phase column chromatography are as follows: a filler is monodisperse polystyrene / divinyl benzene microspheres; the particle size of the microspheres is 3-50 [mu] m, and / or the aperture of the microspheres is # imgabs0 #, the purity of a pure product of the omalonicycline obtained by the purification method is greater than 98%, the isomer is less than 1.5%, the purification yield is high and stable, the purification period is short, and the purification method has good feasibility and practicability and is suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug purification, and particularly relates to a method for purifying omadacycline. Background Art

[0002] Omadacycline (CAS: 389139-89-3, with the structure shown below), also known as omarcycline, is a new semi-synthetic tetracycline developed by Paratek Pharmaceuticals in the United States. It was derived from minocycline by Honeyman et al. through a two-step method and is the first amide-methyltetracycline variety to successfully enter clinical application. Omadacycline exerts its effect through the classical mechanism of tetracycline antibiotics, specifically binding to the A site of the 30S subunit of the bacterial ribosome, inhibiting the normal binding of aminoacyl-tRNA to this site, causing peptide chain elongation to terminate, blocking protein synthesis, and thus producing antibacterial effects. It has a broad antibacterial spectrum, no cross-resistance with other types of antibiotics, and has good antibacterial activity against common clinical Gram-positive bacteria, Gram-negative bacteria, atypical pathogens, anaerobes, etc. It even has good antibacterial activity against drug-resistant strains such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, penicillin-resistant and multi-drug-resistant Streptococcus pneumoniae; at the same time, its intravenous injection and oral tablets (trade name ) were approved for marketing by the US Food and Drug Administration (FDA) in October 2018, mainly used for the treatment of community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections in adults. In addition, the indications of omadacycline tosylate for the treatment of community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections were officially approved by the National Medical Products Administration (NMPA) in 2021.

[0003]

[0004] Currently, the existing research on omadacycline mainly focuses on its synthesis method, crystal form, and medical uses, etc. There is relatively little research on its separation and purification, and the separation and purification methods are relatively fixed (mainly crystallization method and high-performance liquid chromatography method).

[0005] CN111484424A discloses a method for purifying omadacycline crude product by crystallization using a dichloromethane / acetone mixed solvent. However, this patent document only describes the purification effect on the C-4 position β-epimer impurity and does not describe whether the α-epimer as the target product after purification meets the qualified standard; in addition, the amount of organic solvent used in this method is large, the content of isomer impurities is high (1.9%), the yield of the target product is low, and the purification time is long (about 18 h).

[0006] CN118745136A discloses a method for crystallizing and purifying omadacycline crude product using a mixed solvent of acetonitrile / phosphate buffer solution. However, this patent document only describes the purification effect on C-4 β-epimer impurities and other related impurities (omadacycline-4-one), and does not describe whether the α-epimer, which is the target product before and after purification, meets the qualified standards. In addition, the contents of C-4 β-epimer and other related impurities before and after purification by this method have no obvious change (the purification efficiency cannot be improved), and the content of other related impurities after purification also exceeds 0.5%, resulting in a relatively low total purity of the target product.

[0007] CN115667210A discloses a method for purifying crude omadacycline free base, which purifies omadacycline crude product by preparative high performance liquid chromatography. Although the purity of the product after purification reaches more than 98% and the β-isomer is about 1.71%, the purification time of this method is long (about 6.1 h) and the yield is low (only 66.65%), so it is not suitable for industrial production. Summary of the Invention

[0008] Problems to be Solved by the Invention

[0009] Aiming at the above-mentioned defects existing in the prior art, the purpose of the present invention is to provide a method for purifying omadacycline, which can further reduce the isomer content in the omadacycline product, so as to provide a method for purifying omadacycline with simple process, short production cycle, high yield, good product quality and suitable for industrial production.

[0010] Solutions for Solving the Problems

[0011] In order to solve the above technical problems, the present invention provides a method for purifying omadacycline, which comprises the following steps:

[0012] 1) Prepare a solution of omadacycline crude product using a solvent, and optionally adjust the pH value of the solution to neutral using an acid or its aqueous solution, then filter to obtain a filtrate;

[0013] 2) Purify the filtrate obtained in step 1) by reversed-phase column chromatography to obtain purified omadacycline;

[0014] Among them, the chromatographic conditions of the reversed-phase column chromatography include:

[0015] The packing material is monodisperse polystyrene / divinylbenzene microspheres; the particle size of the microspheres is 3 - 50 μm, and / or the pore size of the microspheres is

[0016] Preferably, the HPLC purity of the omadacycline crude product is 50 - 80%.

[0017] Preferably, the solution is prepared under stirring conditions.

[0018] Preferably, the solvent is an alcohol, preferably methanol.

[0019] Preferably, the mass ratio of the omadacycline crude product to the solvent is 1:(1 - 10), preferably 1:(2 - 5).

[0020] Preferably, the acid is an organic acid, preferably acetic acid.

[0021] Preferably, the volume percentage of the acid relative to its aqueous solution is 5% v / v - 20% v / v, preferably 10% v / v.

[0022] Preferably, the particle size of the microspheres is 10 μm.

[0023] Preferably, the pore size of the microspheres is

[0024] Preferably, the chromatographic conditions of the reversed-phase column chromatography further include: the elution mode is isocratic elution.

[0025] Preferably, the chromatographic conditions of the reversed-phase column chromatography further include: the eluent is a binary system of acetonitrile - aqueous acid solution, preferably a binary system of acetonitrile - aqueous acetic acid solution, more preferably a binary system of acetonitrile - 0.2% v / v aqueous acetic acid solution, and further preferably a binary system of acetonitrile - 0.2% v / v aqueous acetic acid solution with a volume ratio of 6:94.

[0026] Preferably, the chromatographic conditions of the reversed-phase column chromatography further include: the flow rate of sample loading is 0.05 - 5 L / min, preferably 3 - 5 L / min, more preferably 4 L / min.

[0027] Preferably, the chromatographic conditions of the reversed-phase column chromatography further include: the flow rate of elution is 0.05 - 20 L / min, preferably 5 - 14 L / min, more preferably 14 L / min.

[0028] Preferably, before purification, it further includes the step of equilibrating the chromatographic column with the eluent.

[0029] Preferably, after purification, it further includes the step of regenerating the chromatographic column with the regeneration solution.

[0030] More preferably, the regeneration solution is acetonitrile - 0.2% v / v acetic acid water or methanol - acetic acid.

[0031] Further preferably, the regeneration solution is acetonitrile - 0.2% v / v acetic acid water with a volume ratio of 20:80 or methanol - acetic acid with a volume ratio of 95:5.

[0032] In addition, the present invention also provides a use of monodisperse polystyrene / divinylbenzene microspheres in purifying tetracycline compounds, wherein the particle size of the microspheres is 3-50 μm, and / or the pore size of the microspheres is

[0033] Preferably, the particle size of the microspheres is 10 μm.

[0034] Preferably, the pore size of the microspheres is

[0035] Preferably, the tetracycline compound is omadacycline.

[0036] Effects of the Invention

[0037] The purification method of the present invention does not require steps such as extraction and crystallization, avoiding the large use of toxic organic solvents, and the packing used can be reused, simplifying the operation and greatly reducing the production cost, and has good application value. In addition, it is proved by scale-up experiments that the purity of the omadacycline pure product obtained by the purification method of the present invention is greater than 98%, the isomer is less than 1.5%, the purification yield is high and stable, the purification period is short (about 70 min), and it has good feasibility and practicability, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the SEM diagram of the chromatography column packing in the embodiment of the present invention;

[0039] Figure 2 It is the high performance liquid chromatography diagram collected during the purification of the crude omadacycline in Example 1 of the present invention;

[0040] Figure 3 It is the enlarged high performance liquid chromatography analysis diagram after the purification of the crude omadacycline in Example 1 of the present invention;

[0041] Figure 4 It is the high performance liquid chromatography analysis diagram before the purification of the crude omadacycline in Example 2 of the present invention;

[0042] Figure 5 It is the high performance liquid chromatography analysis diagram after the purification of the crude omadacycline in Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0043] The following will detail various exemplary embodiments, features and aspects of the present invention. The special term "exemplary" here means "serving as an example, embodiment or illustration". Any embodiment described as "exemplary" here does not have to be construed as superior to or better than other embodiments.

[0044] In addition, for a better illustration of the present invention, numerous specific details are given in the following detailed embodiments. Those skilled in the art should understand that the present invention can still be implemented without some specific details. In other instances, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail so as to highlight the gist of the present invention.

[0045] Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the systematic errors inevitable in industrial production.

[0046] In this specification, the numerical range expressed as "numerical value A to numerical value B" refers to the range including the endpoint numerical values A and B.

[0047] In this specification, the term "optionally" means that the subsequent described event or situation may or may not occur, and this description includes the occurrence and non-occurrence of the described event or situation.

[0048] In this specification, the contents of omadacycline and its isomers are data directly read and calculated based on the HPLC chromatogram obtained by HPLC detection. In the present invention, the contents of omadacycline and its isomers are obtained by integrating omadacycline and its isomers in the HPLC chromatogram.

[0049] Examples

[0050] The following will describe the implementation schemes of the present invention in detail in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0051] The high-performance liquid chromatography analysis method in the following examples is as follows: using octadecylsilane-bonded silica gel as the filler (Welch Ultimate XB-C18, 4.6×250 mm, 5 μm or a chromatographic column with equivalent efficiency); using 0.1 mol / L potassium dihydrogen phosphate buffer solution (weigh 13.6 g of potassium dihydrogen phosphate, add 900 mL of water to dissolve it, adjust the pH to 6.5 with 5 mol / L sodium hydroxide, add water to 1000 mL, add 0.5 g of tetrabutylammonium hydrogen sulfate and 0.4 g of disodium ethylenediaminetetraacetate respectively, dissolve, mix well, filter, and ultrasonicate) as mobile phase A, and acetonitrile as mobile phase B, and perform elution according to the gradient shown in Table 1, with a flow rate of 1.2 mL / min; the column temperature is 35°C; the detection wavelength is 280 nm; the temperature of the injection tray is 5°C; the injection volume is 5 μL.

[0052] Table 1:

[0053]

[0054] Since a saline mobile phase is used in the HPLC analysis process, the fluctuation of salt concentration will change the ionic strength of the mobile phase, thereby affecting the retention behavior of the analyte, and causing the peak time of the chromatographic peak corresponding to the analyte to fluctuate. Therefore, in the following embodiments, the chromatographic peak corresponding to omadacycline (reference substance) is used as the reference peak to calculate the relative retention time of the chromatographic peak corresponding to the β-isomer (target analyte). Since the reference substance and the target analyte are operated under the same conditions, the influence of system fluctuations (such as flow rate, temperature, salt concentration changes, etc.) on the two is synchronous, and the relative retention time can offset these fluctuations. By calculating the relative retention time, the retention time change problem caused by the mobile phase containing salt or other system fluctuations can be effectively reduced.

[0055] The polymer microspheres TMXT-10 (the matrix is ​​a monodisperse microsphere of polystyrene divinylbenzene, with a particle size of 10 μm and a pore size of ) was purchased from Suzhou Nanotech Co., Ltd., and its scanning electron microscope (SEM) image is shown in Figure 1 shown.

[0056] Embodiment 1:

[0057] Take 22.25 g of crude omadacycline (solid content of about 45 wt%), add 25 mL of methanol, and then adjust the pH value of the solution to 7.0 with 10% acetic acid. Visually observe that the sample is completely dissolved, add methanol to make the volume to 50 mL, filter after the solution is clarified, and collect the filtrate for later use.

[0058] A 50×250 mm chromatography column was used, and polymer microspheres (TMXT-10, produced by Suzhou Nano-Tech Co., Ltd.) were used as the chromatography column filler, with a column volume of 491 mL. 37 mL of the above filtrate was loaded into the chromatography column, with a loading capacity of 15 g / L and a loading flow rate of 80 mL / min. Acetonitrile-0.2% v / v acetic acid water (v / v=6:94) was used as the eluent for isocratic elution of 10 bv (bed volume, column volume), with an elution flow rate of 94 mL / min, and the solution of the target peak (such as Figure 2 As shown), acetonitrile-0.2% v / v acetic acid water (v / v=20:80) was used as the regeneration solution to wash 4 bv. The component liquids that met the requirements were collected and analyzed by high performance liquid chromatography ( Figure 3 ), the purity of omadacycline in the eluate is greater than 90%, and the yield is greater than 94.5%.

[0059] Among them, the chromatographic peak information of the purified omadacycline sample in this example is shown in Table 2.

[0060] Table 2:

[0061] Peak Number Retention Time (min) Area Area % Corresponding Substance 1 7.054 3026 0.032 2 8.341 199 0.002 3 8.871 861 0.009 4 9.718 387 0.004 5 9.94 709 0.008 6 10.163 1175 0.012 7 10.619 1259 0.013 8 10.942 2029 0.022 9 11.664 797 0.008 10 13.368 2948 0.031 11 13.6 836 0.009 12 14.001 294 0.003 13 14.869 1714 0.018 14 15.234 3667 0.039 15 16.017 185 0.002 16 16.287 577 0.006 17 17.037 799518 8.495 β-Isomer 18 17.779 8931 0.095 19 18.32 847 0.009 20 18.735 1426 0.015 21 19.708 968 0.01 22 20.324 19669 0.209 23 20.904 10746 0.114 24 21.264 8498991 90.307 Omadacycline 25 22.529 28852 0.307 26 22.979 9928 0.105 27 24.604 5074 0.054 28 26.015 5598 0.059

[0062] Wherein, taking the chromatographic peak corresponding to omadacycline as the reference peak, within the range of ±10%, the relative retention time of the chromatographic peak corresponding to the β-isomer is 0.80.

[0063] Embodiment 2:

[0064] Take 1 kg of crude omadacycline, add 5.0 kg of methanol, stir until the sample is completely dissolved visually, filter after the solution is clear, and collect the filtrate (its HPLC analysis chart is as follows Figure 4 as shown) for later use.

[0065] A 600×265mm chromatography column and polymer microspheres (TMXT-10, produced by Suzhou Nanotech Co., Ltd.) were used as the chromatography column filler, and the column volume was 74.89L. The above filtrate was loaded into the chromatography column, the loading capacity was 13g / L, and the loading flow rate was 4.0L / min. Acetonitrile-0.2% v / v acetic acid water (v / v=6:94) was used as the eluent for isocratic elution, the elution flow rate was 14.0L / min, and the elution time was 67.4min. The solution of the target peak was collected in sections, and then methanol-acetic acid (v / v=95:5) was used as the regeneration liquid to rinse 12.6bv. The component liquids that met the requirements were summarized and analyzed by high performance liquid chromatography ( Figure 5 ), the purity of omadacycline after purification is greater than 98%, and the isomer is less than 1.5%.

[0066] Among them, the chromatographic peak information of the omadacycline sample before purification in this example is shown in Table 3.

[0067] Table 3:

[0068] Peak Number Retention Time (min) Area Area % Corresponding Substance 1 8.857 4680 0.0819 2 9.696 4294 0.0751 3 10.937 1943 0.0340 4 14.048 3929 0.0687 5 14.983 2020954 35.3534 β-Isomer 6 19.043 3438267 60.1471 Omadacycline 7 26.248 4700 0.0822 8 33.090 122193 2.1376 9 34.851 111335 1.9476 10 35.972 1460 0.0255 11 39.746 2673 0.0468

[0069] Wherein, taking the chromatographic peak corresponding to omadacycline as the reference peak, within the range of ±10%, the relative retention time of the chromatographic peak corresponding to the β-isomer was 0.79.

[0070] The information of each chromatographic peak in the purified omadacycline sample of this example is shown in Table 4.

[0071] Table 4:

[0072] Peak Number Retention Time (min) Area Area % Corresponding Substance 1 5.017 3804 0.0300 2 13.691 145056 1.1444 β-Isomer 3 14.255 7776 0.0613 4 15.427 13974 0.1102 5 16.317 6923 0.0546 6 16.896 12454865 98.2590 Omadacycline 7 21.768 3691 0.0291 8 24.253 3895 0.0307 9 25.066 4698 0.0371 10 28.096 30861 0.2435

[0073] Wherein, taking the chromatographic peak corresponding to omadacycline as the reference peak, within the range of ±10%, the relative retention time of the chromatographic peak corresponding to the β-isomer was 0.81.

[0074] Comparative Example 1:

[0075] Compared with Example 1, the difference lies in that the packing material of the chromatography column is C18 silica gel.

[0076] The experimental results of each example and comparative example are shown in Table 5.

[0077] Table 5:

[0078]

[0079] It should be noted that although the technical solutions of the present invention are introduced by specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

[0080] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is also not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to technologies in the market, or to enable other ordinary skill in the art to understand the disclosed embodiments.

Claims

1. A method for purifying omadacycline, characterized in that: The method comprises the following steps: 1) preparing a solution of the crude omadacycline product using a solvent, optionally adjusting the pH value of the solution to neutral using an acid or an aqueous solution thereof, and filtering to obtain a filtrate; 2) purifying the filtrate obtained in step 1) by reverse phase column chromatography to obtain purified omadacycline; Wherein, the chromatographic conditions of the reverse phase column chromatography include: The filler is monodisperse polystyrene / divinylbenzene microspheres; The particle size of the microspheres is 3 to 50 μm, and / or the pore size of the microspheres is 2. The purification method according to claim 1, characterized in that The HPLC purity of the crude omadacycline is 50-80%.

3. The purification method according to claim 1 or 2, characterized in that The solvent is alcohol, preferably methanol.

4. The purification method according to any one of claims 1 to 3, characterized in that The mass ratio of the crude omadacycline to the solvent is 1:(1-10), preferably 1:(2-5).

5. The purification method according to any one of claims 1 to 4, characterized in that The acid is an organic acid, preferably acetic acid; and / or, The volume percentage of the acid relative to its aqueous solution is 5% v / v to 20% v / v, preferably 10% v / v.

6. The purification method according to any one of claims 1 to 5, characterized in that The particle size of the microspheres is 10 μm; and / or, The pore size of the microspheres is 7. The purification method according to any one of claims 1 to 6, characterized in that The chromatographic conditions of the reverse phase column chromatography also include: The elution mode was isocratic.

8. The purification method according to any one of claims 1 to 7, characterized in that The chromatographic conditions of the reverse phase column chromatography also include: The eluent is an acetonitrile-aqueous acid binary system, preferably an acetonitrile-acetic acid binary system, more preferably an acetonitrile-0.2% v / v acetic acid binary system, further preferably an acetonitrile-0.2% v / v acetic acid binary system in a volume ratio of 6:

94.

9. The purification method according to any one of claims 1 to 8, characterized in that The chromatographic conditions of the reverse phase column chromatography also include: The flow rate of loading is 0.05 to 5 L / min, preferably 3 to 5 L / min, more preferably 4 L / min; and / or, The elution flow rate is 0.05 to 20 L / min, preferably 5 to 14 L / min, more preferably 14 L / min.

10. Use of monodisperse polystyrene / divinylbenzene microspheres in purifying tetracycline compounds, characterized in that: The particle size of the microspheres is 3 to 50 μm, preferably 10 μm; and / or, The pore size of the microspheres is Preferably Preferably, the tetracycline compound is omadacycline.

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