A deuterium-labeled everolimus stable isotope compound and preparation method thereof

Abstract

The invention discloses a deuterium-labeled stable isotope compound of everolimus and a preparation method thereof, the preparation method comprising: heating and heat-insulating a toluene solution of rapamycin, then adding dropwise a toluene solution containing 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and an organic base, stirring and heat-insulating the reaction and then cooling naturally, and post-processing to obtain tert-butyldimethylsilyloxy-everolimus-d4, wherein the organic base is one of N,N-diisopropylethylamine, triethylamine, pyridine and 2,6-lutidine; at 0°C and under nitrogen protection, hydrochloric acid solution is added to a methanol solution of tert-butyldimethylsilyloxy-everolimus-d4, and the reaction solution is reprocessed after the stirring reaction to obtain the product. The present invention ensures that the product does not contain other impurities and non-isotopic everolimus that are difficult to separate by adding an organic base and 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 simultaneously.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis of stable isotope standards, and relates to stable isotope-labeled Everolimus-D4 and a preparation method thereof, and specifically relates to a method for synthesizing Everolimus-D4 by using a stable isotope-labeled raw material, deuterated ethylene glycol-D4, and non-isotopic rapamycin as starting materials. Background Art

[0002] Everolimus is a new generation of macrolide immunosuppressant and anti-tumor drug developed by Novartis. It is derived from 42-OH of rapamycin to 42-O-(2-hydroxyethyl), so everolimus is also called 42-O-(2-hydroxyethyl)-rapamycin. Its molecular formula is C 53 H 83 NO 14 , molecular weight is 958.24, CAS#159351-69-6, and its structural formula is as follows:

[0003]

[0004] Everolimus is mainly used clinically to prevent rejection after kidney and heart transplantation. Currently, it can also be used to treat patients with advanced renal cancer who have used two vascular endothelial growth factor receptor kinase inhibitors, sunitinib and sorafenib, with mild side effects. Compared with rapamycin, the hydroxyethyl structure of everolimus increases water solubility, improves oral bioavailability, significantly improves pharmacological properties, reduces adverse reactions, and has a higher therapeutic index and human tolerance.

[0005] Stable isotope tracer technology is widely used in clinical pharmacokinetic studies; gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry online technology is used to detect labeled and unlabeled drugs in biological samples, with high test specificity and sensitivity.

[0006] Stable isotope-labeled everolimus (such as everolimus-D4) is indispensable for investigating the metabolic pathways of non-deuterated everolimus in the body and evaluating its mechanism of action, and is an important tool for clinical testing and drug research of everolimus.

[0007] In the prior art, the existing stable isotope labeled everolimus is mainly Everolimus-D4. At present, the production process of this product is rarely published; however, the main problem of foreign products is that the products generally contain up to about 10% impurities or a small amount of non-isotopic Everolimus in the products. Some manufacturers have specially noted this in their products, such as: Toronto Research Chemicals Company in Canada notes its products as follows: Everolimus-d4 (~90%), and Cayman Chemicals Company in the United States also notes its products as follows: ≥99% deuterated forms (d1-d4), ≤1% d0, which greatly limits the role of the product as a stable isotope standard, and it is even unqualified for many high-standard and high-precision studies; in addition, the specific production process of stable isotope labeled everolimus is rarely reported publicly.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] In view of the deficiencies in the prior art, the present invention provides a deuterium-labeled everolimus stable isotope compound and a preparation method thereof.

[0010] To achieve the above object, the technical solution of the present invention is as follows:

[0011] A method for preparing a deuterium-labeled everolimus stable isotope compound comprises the following steps:

[0012] S1. Under nitrogen protection, heat the toluene solution of rapamycin to 40-60° C. and keep warm, then add dropwise the toluene solution containing 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and an organic base, stir and heat, keep warm for 16-32 hours, then cool naturally to room temperature, and then post-treat the reaction solution to obtain tert-butyldimethylsilyloxy-everolimus-d4;

[0013] S2. In a 0°C ice-water bath and under nitrogen protection, add the hydrochloric acid solution to the methanol solution of tert-butyldimethylsilyloxy-everolimus-d4, stir and react for 0.5-3h, and then re-treat the reaction solution to obtain a deuterium-labeled everolimus stable isotope compound;

[0014] In step S1, the organic base is at least one of N,N-diisopropylethylamine, triethylamine, pyridine and 2,6-lutidine.

[0015] In the above technical solution, in step S1, corresponding to 790 mg of rapamycin, the added amounts of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and the organic base are 275-350 mg and 0.2-0.5 ml, respectively.

[0016] Furthermore, in the above technical solution, in step S1, the toluene solution containing 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and the organic base is added 2-6 times, and the interval time between each addition is 45-75 min.

[0017] In the above technical solution, in step S2, corresponding to 600 mg of tert-butyldimethylsilyloxy-everolimus-d4, the molar concentration and added volume of the hydrochloric acid solution are 0.75-1.2 mol / L and 0.8-1.25 ml, respectively.

[0018] Furthermore, in the above technical solution, before step S1, it also includes:

[0019] S0: Using deuterated ethylene glycol-D4 as raw material, 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 was prepared.

[0020] In detail, in the above technical solution, step S0 specifically includes:

[0021] S01: In a 0°C ice-water bath and under nitrogen protection, a tetrahydrofuran solution of potassium tert-butoxide and a tetrahydrofuran solution of tert-butyldimethylsilyl chloride were added dropwise to a tetrahydrofuran solution of deuterated ethylene glycol-D4, and then the ice-water bath was removed. The reaction solution was stirred for 2-4 hours and naturally warmed to room temperature, diluted with water, and extracted with ether. The organic phase was taken and washed with a saturated aqueous NaCl solution. After removing the organic solvent under reduced pressure, it was separated by silica gel column chromatography and eluted with n-hexane-ether in a volume ratio of 2:1 to obtain 2-(tert-butyldimethylsilyloxy)ethanol-d4;

[0022] S02: In a 0°C ice-water bath and under nitrogen protection, add N,N-diisopropylethylamine and trifluorosulfonic anhydride dropwise to a n-hexane solution of 2-(tert-butyldimethylsilyloxy)ethanol-d4 in sequence, stir for 1-3 hours, remove the organic solvent under reduced pressure, separate by silica gel column chromatography and elute with n-hexane-ether in a volume ratio of 4:1 to obtain 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4.

[0023] Specifically, in the above technical solution, in step S01, corresponding to 2.22 g of deuterated ethylene glycol-D4, the added amounts of potassium tert-butoxide and tert-butyldimethylchlorosilane are 3.77 g and 4.56 g, respectively.

[0024] Specifically, in the above technical solution, in step S01, corresponding to 80 ml of the reaction solution, 45-60 ml of water is added to dilute it, and then ether is added to extract 2-5 times, adding 45-60 ml of ether each time, and the organic phases are combined and washed with saturated NaCl aqueous solution.

[0025] Specifically, in the above technical solution, in step S02, corresponding to 1.80 g of 2-(tert-butyldimethylsilyloxy)ethanol-d4, the added amounts of N,N-diisopropylethylamine and trifluorosulfonic anhydride are 1.42 g and 2.82 g respectively.

[0026] Furthermore, in the above technical solution, in step S1, the post-processing step specifically includes:

[0027] After diluting with 10 wt % citric acid aqueous solution, extract with ethyl acetate 2-4 times, wash the organic phase obtained by combining with saturated sodium bicarbonate aqueous solution and saturated NaCl aqueous solution respectively, remove the organic solvent under reduced pressure, separate by silica gel column chromatography and elute with ethyl acetate-n-hexane in a volume ratio of 1:1.

[0028] Further, in the above technical solution, in step S2, the reprocessing step specifically includes:

[0029] After diluting with water, extract with ethyl acetate 2-4 times, wash the organic phase obtained by combining with saturated sodium bicarbonate aqueous solution and saturated NaCl aqueous solution respectively, remove the organic solvent under reduced pressure, dissolve the product in ethyl acetate and then add n-hexane dropwise until a white solid precipitates, then place the turbid liquid at 0°C for 12-20h, and finally filter the precipitate and wash it with n-hexane 2-5 times.

[0030] Another aspect of the present invention further provides a deuterium-labeled stable isotope compound of everolimus, which is prepared by the above-mentioned preparation method and has a molecular structure as shown below:

[0031]

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] (1) The present invention provides stable isotope-labeled everolimus and a synthesis method thereof, wherein at least one of N,N-diisopropylethylamine, triethylamine, pyridine and 2,6-lutidine is used as an organic base, and 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 is added dropwise to a non-isotopic rapamycin solution simultaneously. Under appropriate reaction temperature and time, it is ensured that the prepared everolimus-D4 product does not contain other impurities that are difficult to separate and non-isotopic everolimus, thereby avoiding quantitative errors or even qualitative errors caused by everolimus standard reagents containing impurities or non-isotopic everolimus on the market during use as standard reagents;

[0034] 2) The preparation method of the deuterium-labeled everolimus stable isotope compound provided by the present invention has a simple reaction process, and the product is easy to separate and purify. The chemical purity and isotope abundance of the obtained product are respectively above 99% and 99% atom, which meets the requirements of being used as a standard reagent for quantitative detection of everolimus, has high use value, and has good economic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of the deuterium-labeled everolimus stable isotope compound (everolimus-D4) prepared in Example 1 of the present invention;

[0036] Figure 2 This is a high performance liquid chromatogram of the deuterium-labeled everolimus stable isotope compound (everolimus-D4) prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0038] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0039] In the examples, unless otherwise specified, all the means used are conventional means in the art.

[0040] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0041] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0042] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0043] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0044] Its synthetic route is as follows:

[0045] Example 1

[0046] A method for preparing a deuterium-labeled everolimus stable isotope compound (everolimus-D4), the synthesis route of which is as follows:

[0047]

[0048] The specific steps include:

[0049] S0, at 0 ℃ (ice-water bath) and under nitrogen protection, to a tetrahydrofuran solution (60 ml) containing 2.664 g of deuterated ethylene glycol-D4, a tetrahydrofuran solution (18 ml) containing 4.524 g of potassium tert-butoxide (tBuOK) and a tetrahydrofuran solution (18 ml) containing 5.472 g of tert-butyldimethylsilyl chloride (TBDMS-Cl) were added dropwise, then the ice-water bath was removed, stirred for 3 hours and naturally warmed to room temperature, diluted with 60 ml of water, extracted three times with ether (60 ml each time), and the mixture was combined with The organic phase was washed with a saturated aqueous NaCl solution, and the organic solvent was removed under reduced pressure to obtain a crude product 1 (2.568 g). The crude product 1 was separated by silica gel column chromatography and eluted with n-hexane-ether (volume ratio 2:1) to obtain 1.80 g of 2-(tert-butyldimethylsilyloxy)ethanol-d4 (colorless liquid); 1.42 g of 2-(tert-butyldimethylsilyloxy)ethanol-d4 was added dropwise to a n-hexane solution (40 ml) containing 1.80 g of 2-(tert-butyldimethylsilyloxy)ethanol-d4 at 0°C (ice-water bath) and under nitrogen protection. N, N-diisopropylethylamine and 2.82g trifluoromethanesulfonic anhydride were stirred at 0°C (ice-water bath) for 1.5h, and the organic solvent was removed under reduced pressure to obtain a crude product 2 (1.72g). The crude product 2 was then separated by silica gel column chromatography and eluted with n-hexane-ether (volume ratio 4:1) to obtain 1.30g of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 (colorless liquid);

[0050] S1. Under nitrogen protection, a toluene solution (10 mL) containing 790 mg of rapamycin was stirred and heated to 50°C, and then a toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.45 mL of N,N-diisopropylethylamine (DIPEA) was added dropwise. After stirring at 50°C for 1 hour, a toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.45 mL of N,N-diisopropylethylamine (DIPEA) was added dropwise again. This addition was repeated, and a total of 1.2 g of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 1.8 mL of DIPEA were added. N, N-diisopropylethylamine (DIPEA), stirred and heated, kept at 50°C for 22 hours under nitrogen protection, and then naturally cooled to room temperature (25°C), diluted with 25mL of citric acid aqueous solution (10wt%), and then extracted with ethyl acetate 3 times (25mL each time), and the organic phase obtained by the combination was washed with saturated sodium bicarbonate aqueous solution (25mL) and saturated NaCl aqueous solution (25mL) respectively, and the organic solvent was removed under reduced pressure to obtain a crude product 3 (842mg), which was then separated by silica gel column chromatography and eluted with ethyl acetate-n-hexane in a volume ratio of 1:1 to obtain 600mg of tert-butyldimethylsilyloxy-everolimus-d4 (viscous grayish white oil);

[0051] S2. In a 0°C ice-water bath and under nitrogen protection, add 1 mL of hydrochloric acid solution (1 mol / L) to a methanol solution (5 mL) containing 600 mg of tert-butyldimethylsilyloxy-everolimus-d4, and stir to react for 1.5 h (0°C ice-water bath and under nitrogen protection), then add 25 mL of water to dilute and extract with ethyl acetate 3 times (25 mL each time), and wash the organic phase obtained with saturated sodium bicarbonate aqueous solution (25 mL) and saturated NaCl aqueous solution ( The crude product 4 (530 mg) was dissolved in ethyl acetate (3 mL) and then 10 mL of n-hexane was added dropwise until a white solid precipitated. The turbid solution was then placed in a 0°C refrigerator for 16 h. Finally, the precipitate was filtered and washed 3 times with n-hexane (5 mL each time) to obtain 510 mg of deuterium-labeled everolimus stable isotope compound (everolimus-D4, white solid).

[0052] Example 2

[0053] A method for preparing a deuterium-labeled everolimus stable isotope compound (everolimus-D4), the preparation process of which is similar to that of Example 1, except that:

[0054] In step S1, a toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.30 mL of 2,6-lutidine is added to the toluene solution of rapamycin. After being stirred at 50° C. for 1 h, the addition is repeated, and a total of 1.2 g of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 1.2 mL of 2,6-lutidine are added. The masses of the crude product 3 and tert-butyldimethylsilyloxy-everolimus-d4 obtained are 827 mg and 565 mg, respectively.

[0055] In step S2, 1 mL of hydrochloric acid solution (1 mol / L) is added to a methanol solution (5 mL) containing 565 mg of tert-butyldimethylsilyloxy-everolimus-d4, and the masses of the crude product 4 and everolimus stable isotope compound (everolimus-D4) finally obtained are 511 mg and 475 mg, respectively.

[0056] Example 3

[0057] A method for preparing a deuterium-labeled everolimus stable isotope compound (everolimus-D4), the preparation process of which is similar to that of Example 1, except that:

[0058] In step S1, a toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.21 mL of pyridine is added to the toluene solution of rapamycin. After being stirred at 50° C. for 1 h, the addition is repeated, and a total of 1.2 g of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.84 mL of pyridine are added. The masses of the crude product 3 and tert-butyldimethylsilyloxy-everolimus-d4 obtained are 815 mg and 481 mg, respectively.

[0059] In step S2, 1 mL of hydrochloric acid solution (1 mol / L) is added to a methanol solution (5 mL) containing 481 mg of tert-butyldimethylsilyloxy-everolimus-d4, and the masses of the crude product 4 and everolimus stable isotope compound (everolimus-D4) finally obtained are 433 mg and 411 mg, respectively.

[0060] Example 4

[0061] A method for preparing a deuterium-labeled everolimus stable isotope compound (everolimus-D4), the preparation process of which is similar to that of Example 1, except that:

[0062] In step S1, a toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.36 mL of triethylamine is added to the toluene solution of rapamycin. After being stirred at 50° C. for 1 h, the addition is repeated, and a total of 1.2 g of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 1.44 mL of triethylamine are added. The masses of the crude product 3 and tert-butyldimethylsilyloxy-everolimus-d4 obtained are 796 mg and 343 mg, respectively.

[0063] In step S2, 1 mL of hydrochloric acid solution (1 mol / L) is added to a methanol solution (5 mL) containing 343 mg of tert-butyldimethylsilyloxy-everolimus-d4, and the masses of the crude product 4 and everolimus stable isotope compound (everolimus-D4) finally obtained are 309 mg and 292 mg, respectively.

[0064] Example 5

[0065] A method for preparing a deuterium-labeled everolimus stable isotope compound (everolimus-D4), the preparation process of which is similar to that of Example 1, except that:

[0066] In step S1, the toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.45 mL of N,N-diisopropylethylamine (DIPEA) was added to the toluene solution of rapamycin at different times, and a total of 1.5 g of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 2.25 mL of N,N-diisopropylethylamine (DIPEA) were added. The masses of the crude product 3 and tert-butyldimethylsilyloxy-everolimus-d4 obtained were 824 mg and 589 mg, respectively;

[0067] In step S2, 1 mL of hydrochloric acid solution (1 mol / L) is added to a methanol solution (5 mL) containing 589 mg of tert-butyldimethylsilyloxy-everolimus-d4, and the masses of the crude product 4 and everolimus stable isotope compound (everolimus-D4) finally obtained are 530 mg and 503 mg, respectively.

[0068] Example 6

[0069] A method for preparing a deuterium-labeled everolimus stable isotope compound (everolimus-D4), the preparation process of which is similar to that of Example 1, except that:

[0070] In step S1, the toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.45 mL of N,N-diisopropylethylamine (DIPEA) was added to the toluene solution of rapamycin at different times, and a total of 0.9 g of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 1.35 mL of N,N-diisopropylethylamine (DIPEA) were added. The masses of the crude product 3 and tert-butyldimethylsilyloxy-everolimus-d4 obtained were 817 mg and 515 mg, respectively;

[0071] In step S2, 1 mL of hydrochloric acid solution (1 mol / L) is added to a methanol solution (5 mL) containing 515 mg of tert-butyldimethylsilyloxy-everolimus-d4, and the masses of the crude product 4 and everolimus stable isotope compound (everolimus-D4) finally obtained are 463 mg and 439 mg, respectively.

[0072] Example 7

[0073] A method for preparing a deuterium-labeled everolimus stable isotope compound (everolimus-D4), the preparation process of which is similar to that of Example 1, except that:

[0074] In step S1, the toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.45 mL of N,N-diisopropylethylamine (DIPEA) was added to the toluene solution of rapamycin at different times, and a total of 0.6 g of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.9 mL of N,N-diisopropylethylamine (DIPEA) were added. The masses of the crude product 3 and tert-butyldimethylsilyloxy-everolimus-d4 obtained were 809 mg and 433 mg, respectively;

[0075] In step S2, 1 mL of hydrochloric acid solution (1 mol / L) is added to a methanol solution (5 mL) containing 433 mg of tert-butyldimethylsilyloxy-everolimus-d4, and the masses of the crude product 4 and everolimus stable isotope compound (everolimus-D4) finally obtained are 390 mg and 369 mg, respectively.

[0076] Example 8

[0077] A method for preparing a deuterium-labeled everolimus stable isotope compound (everolimus-D4), the preparation process of which is similar to that of Example 1, except that:

[0078] In step S1, a toluene solution (1 mL) containing 300 mg of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and 0.45 mL of N,N-diisopropylethylamine (DIPEA) was added to the toluene solution of rapamycin once (without repeated addition), and the masses of the crude product 3 and tert-butyldimethylsilyloxy-everolimus-d4 obtained were 795 mg and 254 mg, respectively;

[0079] In step S2, 1 mL of hydrochloric acid solution (1 mol / L) is added to a methanol solution (5 mL) containing 254 mg of tert-butyldimethylsilyloxy-everolimus-d4, and the masses of the crude product 4 and everolimus stable isotope compound (everolimus-D4) finally obtained are 229 mg and 217 mg, respectively.

[0080] Figure 1 and Figure 2 They are respectively the nuclear magnetic resonance hydrogen spectrum (using deuterated chloroform CDCl3 as solvent, obtained by JEOL-400MHz nuclear magnetic resonance instrument) and the high performance liquid chromatogram (the sample is dissolved in acetonitrile, the mobile phase A is deionized water containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, in Shimadzu Nexera X2, the high performance liquid column is SunFire C18, 4.6*50mm, 3.5μm; the flow rate is 1.0ml / min through the high performance liquid column; the flushing gradient is: 10-95% mobile phase B for the first 6min, and then 95% mobile phase B for 6min; the detection is carried out by evaporative light scattering detector ELSD to obtain the high performance liquid chromatogram of isotope-labeled everolimus-D4).

[0081] from Figure 1 It can be seen that there is no absorption peak of ethylene glycol group at the chemical shift of 3.82ppm to 3.86ppm, indicating that the structure is Everolimus-D4, which does not contain any non-isotopic Everolimus; Figure 2 It can be seen that the purity of the prepared deuterium-labeled everolimus stable isotope compound (everolimus-D4) sample reaches 99.38%.

[0082] The yields of everolimus-D4 of Examples 1-8 were calculated respectively, and the chemical purity and isotopic abundance of the prepared deuterium-labeled everolimus stable isotope compound (everolimus-D4) were detected by high performance liquid chromatography and mass spectrometry, respectively. The results are shown in Table 1 below.

[0083] Table 1 Results of yield, chemical purity and isotopic abundance of the products of each example

[0084]

[0085] As can be seen from Table 1, the organic base in Example 2 was changed from N, N-diisopropylethylamine to 2,6-lutidine, the ratio of all reagents remained unchanged, and the yield of the product was reduced to 57%; the organic base in Example 3 was changed from N, N-diisopropylethylamine to pyridine, the ratio of all reagents remained unchanged, and the yield of the product was reduced to 50%; the organic base in Example 4 was changed from N, N-diisopropylethylamine to triethylamine, the ratio of all reagents remained unchanged, and the yield of the product was reduced to 35%; the organic base in Example 5 was N, N-diisopropylethylamine to triethylamine, the ratio of all reagents remained unchanged, and the yield of the product was reduced to 35%. In Example 1, the four times of feeding were changed to five times, and the yield remained basically unchanged at 61%; in Example 6, the organic base was N, N-diisopropylethylamine, and the four times of feeding in Example 1 were changed to three times, and the yield of the product was reduced to 53%; in Example 7, the organic base was N, N-diisopropylethylamine, and the four times of feeding in Example 1 were changed to two times, and the yield of the product was reduced to 45%; in Example 8, the organic base was N, N-diisopropylethylamine, and the four times of feeding in Example 1 were changed to one time, and the yield of the product was reduced to 26%. The chemical purity and isotopic abundance of the final products in all examples met the qualified requirements.

[0086] The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent.

[0087] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a deuterium-labeled stable isotope compound of everolimus, characterized in that: The following steps are involved: S1. Under nitrogen protection, heat the toluene solution of rapamycin to 40-60° C. and keep warm, then add dropwise the toluene solution containing 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and an organic base, stir and heat, keep warm for 16-32 hours, then cool naturally to room temperature, and then post-treat the reaction solution to obtain tert-butyldimethylsilyloxy-everolimus-d4; S2. In a 0°C ice-water bath and under nitrogen protection, add the hydrochloric acid solution to the methanol solution of tert-butyldimethylsilyloxy-everolimus-d4, stir and react for 0.5-3h, and then re-treat the reaction solution to obtain a deuterium-labeled everolimus stable isotope compound; In step S1, the organic base is at least one of N,N-diisopropylethylamine, triethylamine, pyridine and 2,6-lutidine; In step S1, the toluene solution containing 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and an organic base is added 2-6 times, and the interval time of each addition is 45-75min; In step S1, corresponding to 790 mg of rapamycin, the added amounts of 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4 and the organic base are 275-350 mg and 0.2-0.5 ml, respectively; in step S2, corresponding to 600 mg of tert-butyldimethylsilyloxy-everolimus-d4, the molar concentration and added volume of the hydrochloric acid solution are 0.75-1.2 mol / L and 0.8-1.25 ml, respectively.

2. The method for preparing the deuterium-labeled everolimus stable isotope compound according to claim 1, characterized in that: Before step S1, the method further includes: S0: using deuterated ethylene glycol-D4 as a raw material to prepare 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4; Wherein, step S0 specifically includes: S01: In a 0°C ice-water bath and under nitrogen protection, a tetrahydrofuran solution of potassium tert-butoxide and a tetrahydrofuran solution of tert-butyldimethylsilyl chloride were added dropwise to a tetrahydrofuran solution of deuterated ethylene glycol-D4, and then the ice-water bath was removed. The reaction solution was stirred for 2-4 hours and naturally warmed to room temperature, diluted with water, and extracted with ether. The organic phase was taken and washed with a saturated aqueous NaCl solution. After removing the organic solvent under reduced pressure, it was separated by silica gel column chromatography and eluted with n-hexane-ether in a volume ratio of 2:1 to obtain 2-(tert-butyldimethylsilyloxy)ethanol-d4; S02: In a 0°C ice-water bath and under nitrogen protection, add N,N-diisopropylethylamine and trifluorosulfonic anhydride dropwise to a n-hexane solution of 2-(tert-butyldimethylsilyloxy)ethanol-d4 in sequence, stir for 1-3 hours, remove the organic solvent under reduced pressure, separate by silica gel column chromatography and elute with n-hexane-ether in a volume ratio of 4:1 to obtain 2-(tert-butyldimethylsilyloxy)ethyl trifluoromethanesulfonate-d4.

3. The method for preparing the deuterium-labeled everolimus stable isotope compound according to claim 2, characterized in that: In step S01, corresponding to 2.22 g of deuterated ethylene glycol-D4, the added amounts of potassium tert-butoxide and tert-butyldimethylsilyl chloride are 3.77 g and 4.56 g respectively; And / or, corresponding to 80 ml of the reaction solution, add 45-60 ml of water to dilute, then add ether to extract 2-5 times, adding 45-60 ml of ether each time, and then combine the organic phases and wash with saturated NaCl aqueous solution.

4. The method for preparing the deuterium-labeled everolimus stable isotope compound according to claim 2, characterized in that: In step S02, Corresponding to 1.80 g of 2-(tert-butyldimethylsilyloxy)ethanol-d4, the added amounts of N,N-diisopropylethylamine and trifluorosulfonic anhydride were 1.42 g and 2.82 g, respectively.

5. The method for preparing the deuterium-labeled everolimus stable isotope compound according to any one of claims 1 to 4, characterized in that: In step S1, the post-processing steps specifically include: After diluting with 10 wt % citric acid aqueous solution, extract with ethyl acetate 2-4 times, wash the organic phase obtained by combining with saturated sodium bicarbonate aqueous solution and saturated NaCl aqueous solution respectively, remove the organic solvent under reduced pressure, separate by silica gel column chromatography and elute with ethyl acetate-n-hexane in a volume ratio of 1:

1.

6. The method for preparing the deuterium-labeled everolimus stable isotope compound according to any one of claims 1 to 4, characterized in that: In step S2, the reprocessing step specifically includes: After diluting with water, extract with ethyl acetate 2-4 times, wash the organic phase obtained by combining with saturated sodium bicarbonate aqueous solution and saturated NaCl aqueous solution respectively, remove the organic solvent under reduced pressure, dissolve the product in ethyl acetate and then add n-hexane dropwise until a white solid precipitates, then place the turbid liquid at 0°C for 12-20h, and finally filter the precipitate and wash it with n-hexane 2-5 times.

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