Preparation method of everolimus related substance F

Abstract

The invention discloses a preparation method of an everolimus related substance F, and belongs to the field of pharmaceutical chemicals. The preparation method of the everolimus related substance F comprises the following steps: dissolving everolimus in an ethanol solvent to obtain the everolimus related substance F under the action of oxygen, and combining silica gel column chromatography with semi-preparative separation and purification to obtain a target compound pure product. The invention provides the preparation method of the everolimus related substance F, the reaction conditions are mild, everolimus degradation impurities are generated, the process flow is simple, the product purity is high, and the quality control of everolimus and a preparation thereof is effectively improved.

Description

Technical Field

[0001] This invention relates to a method for preparing everolimus-related substance F, belonging to the field of pharmaceutical and chemical engineering. Background Technology

[0002] Everolimus (RAD001), also known as 43-O-(2-hydroxyethyl)-rapamycin, is a semi-synthetic derivative of sirolimus (rapamycin) and an inhibitor of the mammalian target of rapamycin (mTOR). Everolimus regulates protein synthesis and cell proliferation by forming a high-affinity complex with intracellular FK506-binding protein-12 (FKBP12), which then binds to and inhibits the function of the FRB domain of the mTOR protein.

[0003] Everolimus was originally developed by Novartis in Switzerland and first marketed in Sweden in 2003 as an immunosuppressant under the brand name Afinitor for the treatment of organ transplant rejection. It was first marketed in the United States in March 2009 as a kinase inhibitor for the treatment of advanced renal cell carcinoma. In addition, everolimus has been approved by the FDA for the treatment of advanced breast cancer, advanced pancreatic neuroendocrine tumors, advanced renal cell carcinoma, subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis, and renal angiomyolipoma with tuberous sclerosis. It is well-tolerated clinically, with mild adverse reactions after oral administration, which can be relieved by dose reduction or symptomatic treatment.

[0004] Everolimus is synthesized by reacting rapamycin with a protecting ethylene glycol trifluoromethanesulfonate via a monoetherification reaction under the action of an organic base to form an everolimus precursor, followed by desilylation protection. Everolimus belongs to the triene macrocyclic lactone class of compounds, with a complex structure containing numerous sensitive groups (such as olefin and ester bonds), exhibiting poor stability and readily generating degradation products during synthesis and storage. The quality of everolimus and its formulations is a crucial standard for drug quality, with strict requirements on its purity and impurities. Developing impurity reference standards is essential to ensuring the quality of everolimus and its formulations.

[0005] Currently, both the United States Pharmacopeia (USP) and the European Pharmacopeia (EPP) have technical specifications for the quality standards and testing methods of everolimus. The EPP specifies that related substance F (structural formula below) is one of the related substances in the reference standard, and its content must not exceed 0.2%. Therefore, to provide a related substance reference standard for the quality research of everolimus and its preparations, and to improve the quality standards of everolimus, it is necessary to provide a method for preparing related substance F of everolimus.

[0006] Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for preparing everolimus related substance F. This method is simple to operate, has mild reaction conditions, and yields related substance F with high purity, which can be used as an impurity reference for the testing of everolimus and its preparations.

[0008] The present invention achieves the above-mentioned technical problem through the following technical solution:

[0009] A method for preparing everolimus-related substance F includes the following steps:

[0010]

[0011] Specifically, the following steps are included:

[0012] (1) Dissolve everolimus in ethanol, heat to T1, introduce oxygen, keep warm and stir the reaction, after the reaction is complete, concentrate to remove the ethanol solvent, and obtain everolimus related substance F product; the T1 is 30-80℃.

[0013] (2) The required components were collected by silica gel column chromatography and then concentrated to dryness to obtain crude everolimus related substance F; further semi-preparative separation and purification were combined to obtain pure everolimus related substance F.

[0014] Furthermore, the temperature T1 is 60-70℃.

[0015] Furthermore, the ethanol has a water content of 40% to 0% by weight, preferably 10% to 0%.

[0016] Furthermore, the oxygen is a mixed gas containing 10%-100% oxygen, preferably 20%-100%.

[0017] Furthermore, the reaction time is 10-100 hours, preferably 60-80 hours.

[0018] Furthermore, the concentration temperature is 30°C to 60°C, and a rotary evaporator is used for concentration; more preferably, it is 35°C to 45°C.

[0019] Furthermore, the semi-preparation column used in the semi-preparation is one of C1, C4, C8 and C18 reversed silica gel; C18 reversed silica gel is particularly preferred.

[0020] Furthermore, the eluent used in the silica gel column chromatography is one or more organic solvents such as methanol, dichloromethane, acetone, ethyl acetate, n-heptane, and other alkanes; acetone-n-heptane is particularly preferred.

[0021] Furthermore, the eluent used in the semi-preparation is one of an aqueous organic solvent such as methanol, ethanol, isopropanol, or acetonitrile, with a water content of 10% to 50% by weight; particularly preferred is one of an aqueous organic solvent such as methanol or acetonitrile, with a water content of 30% to 40% by weight.

[0022] The HPLC method for detecting related substance F of everolimus is as follows:

[0023] The determination of related substances was performed according to the European Pharmacopoeia EP 10.3 Everolimus. The chromatographic conditions were as follows: Hypersil BDS column (3 mm × 250 mm, 5 μm); detection wavelength: 210 / 275 nm; column temperature: 50 ℃; injection volume: 10 μL; flow rate: 1.1 mL / min; mobile phase A was acetonitrile-0.27 g / L potassium dihydrogen phosphate solution (40:60, V / V); mobile phase B was acetonitrile, with gradient elution. The elution program is shown in Table 1.

[0024] Accurately weigh an appropriate amount of everolimus related substance F sample, dissolve it in acetonitrile, and quantitatively dilute it to a solution containing 3 mg per 1 mL. This solution is used as the test solution. Accurately measure 10 μL and inject it into the liquid chromatograph, recording the chromatogram. Calculate the purity of everolimus related substance F using the area normalization method. Figure 3 As shown.

[0025] Table 1 Gradient elution program

[0026]

[0027] The present invention has the following advantages:

[0028] The everolimus-related substance F prepared by this invention is one of the important impurities in the quality inspection of everolimus and its preparations. It can be used as an impurity control in the quality research of everolimus and its preparations to ensure the quality control of everolimus and its preparations. This invention provides a method for preparing everolimus-related substance F with mild reaction conditions, generating everolimus degradation impurities. The process is simple and the product has high purity. It can be used as an impurity control in the testing standards of everolimus finished products and preparations, effectively improving the accuracy of quality control of everolimus and its preparations. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings and examples. Confirmation of the structure of everolimus-related substances obtained by the present invention:

[0030] Figure 1 The everolimus-related substance F in Example 1 of this invention 1 H-NMR spectrum

[0031] Figure 2The everolimus-related substance F in Example 1 of this invention 13 C-NMR spectrum.

[0032] Figure 3 This is an HPLC chromatogram of everolimus-related substance F in Example 1 of the present invention. Detailed Implementation

[0033] The following provides specific embodiments of the present invention. These embodiments are merely exemplary and are not intended to limit the scope of the present invention as described herein. These embodiments are only used to illustrate the implementation methods of the present invention.

[0034] Example 1

[0035] Everolimus (9.58 g, 0.01 mol) and 200 mL of 95% ethanol (5% water content) were added to a 500 mL round-bottom flask. Pure oxygen (flow rate: 80 mL / min) was continuously introduced at 60 °C until the reaction was complete, and the mixture was magnetically stirred for 80 h. After the reaction was complete, the ethanol solvent was removed by concentration, and silica gel column chromatography was performed (stationary phase: 200-300 mesh silica gel, eluent: acetone: n-heptane = 1:3 v / v). The desired fraction was collected and concentrated to dryness at 35-40 °C to obtain 1.3 g of crude everolimus related substance F. The crude product was then purified by C18 reversed-phase preparative high-performance liquid chromatography (column: C18, 21 mm × 250 mm, 10 μm) with acetonitrile:water (6:4) as the eluent, yielding 0.9 g of pure everolimus related substance F with an HPLC purity of 98.5%. The purity of everolimus related substance F provided by this method reaches over 98%, which can be directly used as an impurity control in the testing standards of everolimus finished products and formulations, effectively improving the quality control of everolimus finished products and formulations.

[0036] HRMS: C 55 H 89 O 16 NNa[M+Na] + :1042.6061.

[0037] The NMR detection results are shown in Table 2. Figure 1 and Figure 2 As shown.

[0038] Table 2. Related substances of everolimus (F) 1 HNMR, 13 C10 NMR chemical shift data are attributed to a(d6-DMSO).

[0039]

[0040]

[0041] a1 H and 13 CNMR was measured at 500 MHz and 150 MHz, respectively.

[0042] Example 2

[0043] Everolimus (9.58 g, 0.01 mol) and 200 mL of 95% ethanol (5% water content) were added to a 500 mL round-bottom flask. The mixture was stirred magnetically at 60 °C with air purging (flow rate: 80 mL / min) for 80 h. After the reaction was complete, the ethanol solvent was removed by concentration, and the mixture was subjected to silica gel column chromatography (stationary phase: 200-300 mesh silica gel, eluent: acetone: n-heptane = 1:3 v / v). The desired fraction was collected and concentrated to dryness at 35-40 °C to obtain 0.74 g of crude everolimus related substance F. The crude product was then purified by C18 reversed-phase preparative high-performance liquid chromatography (column: C18, 21 mm × 250 mm, 10 μm) with acetonitrile:water (6:4) as the eluent, yielding 0.36 g of pure everolimus related substance F with an HPLC purity of 98.1%.

[0044] Example 3

[0045] Everolimus (2.87 g, 3 mmol) and 60 mL of 85% ethanol (15% water content) were added to a 250 mL round-bottom flask. The mixture was stirred magnetically with pure oxygen (flow rate: 80 mL / min) at 60 °C for 80 h. After the reaction was complete, the ethanol solvent was removed by concentration, and the mixture was subjected to silica gel column chromatography (stationary phase: 200-300 mesh silica gel, eluent: acetone: n-heptane = 1:3 v / v). The desired fraction was collected and concentrated to dryness at 35-40 °C to obtain 0.35 g of crude everolimus related substance F. The crude product was then purified by C18 reversed-phase preparative high-performance liquid chromatography (column: C8, 21 mm × 250 mm, 10 μm) with acetonitrile:water (7:3) as the eluent, yielding 0.15 g of pure everolimus related substance F with an HPLC purity of 97.2%.

[0046] Example 4

[0047] Everolimus (1 g, 1.25 mmol) and 20 mL of anhydrous ethanol were added to a 100 mL round-bottom flask. The mixture was stirred magnetically at 55 °C with air purging (flow rate: 60 mL / min) for 70 h. After the reaction was complete, the ethanol solvent was removed by concentration, and the product was subjected to silica gel column chromatography (stationary phase: 100-200 mesh silica gel, eluent: ethyl acetate: n-heptane = 1:1 v / v). The desired fraction was collected and concentrated to dryness at 35-40 °C to obtain 0.1 g of crude everolimus related substance F. The crude product was then purified by C18 reversed-phase preparative high-performance liquid chromatography (column: C18, 21 mm × 250 mm, 10 μm) with acetonitrile:water (7:3) as the eluent, yielding 35 mg of pure everolimus related substance F with an HPLC purity of 96.9%.

[0048] Example 5

[0049] Everolimus (2.87 g, 3 mmol) and 60 mL of 70% ethanol (30% water content) were added to a 250 mL round-bottom flask. A mixed gas containing 40% oxygen (V) was then introduced at 50 °C. 氩气 V 氧气 The reaction mixture was stirred magnetically at a ratio of 3:2 (flow rate: 40 mL / min) for 80 h. After the reaction was complete, the ethanol solvent was removed by concentration, and silica gel column chromatography was performed (stationary phase: 200-300 mesh silica gel, eluent: acetone: n-heptane = 1:3 v / v). The desired fraction was collected and concentrated to dryness at 35-40℃ to obtain 0.15 g of crude everolimus related substance F. The crude product was then purified by C18 reversed-phase preparative high-performance liquid chromatography (column: C18, 10 mm × 250 mm, 10 μm) with methanol:water (8:2) as the eluent to obtain 30 mg of pure everolimus related substance F with an HPLC purity of 95.8%.

[0050] Example 6

[0051] Everolimus (1.44 g, 1.5 mmol) and 30 mL of anhydrous ethanol were added to a 100 mL round-bottom flask. The mixture was stirred magnetically at 60 °C with air purging (flow rate: 80 mL / min) for 60 h. After the reaction was complete, the ethanol solvent was removed by concentration, and the mixture was subjected to silica gel column chromatography (stationary phase: 200-300 mesh silica gel, eluent: acetone: petroleum ether = 1:3 v / v). The desired fraction was collected and concentrated to dryness at 35-40 °C to obtain 0.12 g of crude everolimus related substance F. The crude product was then purified by C18 reversed-phase preparative high-performance liquid chromatography (column: C18, 10 mm × 250 mm, 10 μm) with methanol:water (8:2) as the eluent, yielding 40 mg of pure everolimus related substance F with an HPLC purity of 97.7%.

[0052] As the concentration of ethanol and the oxygen content increase, the content of everolimus-related substance F obtained from the reaction also increases.

[0053] Comparative Example 1

[0054] Everolimus (1 g, 1.25 mmol), 2,6-di-tert-butyl-p-cresol (BHT) (10 mg), and 20 mL of ethanol were added to a 100 mL round-bottom flask. The mixture was stirred magnetically (500 rpm) at 55 °C with air introduced (flow rate: 60 mL / min) for 70 h. HPLC analysis showed no everolimus-related substance F.

[0055] Comparative Example 2

[0056] Everolimus (1 g, 1.25 mmol) and 20 mL of isopropanol were added to a 100 mL round-bottom flask. The mixture was stirred magnetically (500 rpm) at 55 °C with air introduced (flow rate: 60 mL / min) for 70 h. No everolimus-related substance F was detected by HPLC.

[0057] Comparative Example 3

[0058] Everolimus (1 g, 1.25 mmol) and 20 mL of methanol were added to a 100 mL round-bottom flask. The mixture was stirred magnetically (500 rpm) at 55 °C with air introduced (flow rate: 60 mL / min) for 70 h. HPLC analysis showed no everolimus-related substance F.

[0059] Comparative Example 4

[0060] Everolimus (1 g, 1.25 mmol) and 20 mL of acetone were added to a 100 mL round-bottom flask. Air was introduced at 55 °C (flow rate: 60 mL / min), and the mixture was magnetically stirred (500 rpm) for 70 h. HPLC analysis showed that no everolimus-related substance F was detected.

[0061] As can be seen from the above comparative examples, everolimus can only be converted into everolimus-related substance F through oxygen reaction in the presence of the reaction substrate ethanol.

[0062] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing everolimus-related substance F, characterized in that, Includes the following steps:

2. The preparation method according to claim 1, characterized in that, Specifically, the following steps are included: (1) Dissolve everolimus in ethanol, heat to T1, introduce oxygen, keep warm and stir the reaction, after the reaction is complete, concentrate to remove ethanol solvent, and obtain everolimus related substance F product; the T1 is 30-80℃. (2) The required components were collected by silica gel column chromatography and then concentrated to dryness to obtain crude everolimus related substance F; further semi-preparative separation and purification were performed to obtain pure everolimus related substance F.

3. The preparation method according to claim 2, characterized in that, The T1 mentioned in step (1) is 60-70℃.

4. The preparation method according to claim 2, characterized in that, The reaction time described in step (1) is 10-100 h.

5. The preparation method according to claim 2, characterized in that, The concentration temperature in step (2) is 30℃~60℃.

6. The preparation method according to claim 2, characterized in that, In step (2), the semi-preparation column used is one of C1, C4, C8 and C18 reversed silica gel.

7. The preparation method according to claim 2, characterized in that, The eluent used in the silica gel column chromatography in step (2) is selected from methanol, dichloromethane, acetone, ethyl acetate, and n-heptane.

8. The preparation method according to claim 2, characterized in that, The eluent used in the semi-preparation in step (2) is an aqueous organic solvent, including methanol, ethanol, isopropanol or acetonitrile, with a water content of 10% to 50% by weight.

9. The preparation method according to claim 2, characterized in that, The oxygen mentioned in step (1) is pure oxygen or a mixture containing oxygen, wherein the volume percentage of oxygen in the mixture containing oxygen is 10%-100%.

10. The preparation method according to claim 2, characterized in that, The water content of the ethanol in step (1) is 40% to 0% by weight.

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