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

Abstract

The invention discloses a deuterium-labeled rapamycin stable isotope compound and a preparation method thereof, and the preparation method includes: at room temperature and under nitrogen protection, deuterated methanol D4 and trifluoroacetic acid are added to the dichloromethane solution of rapamycin, and heavy water is added after the stirring reaction; organic phase and heavy water phase are separated, and the organic phase is washed with a saturated solution of heavy water of sodium bicarbonate and sodium chloride successively, and dichloromethane is removed under reduced pressure to obtain a crude product; the crude product is successively separated by silica gel column chromatography and purified by high performance liquid chromatography. The present invention uses a simple one-step reaction to synthesize rapamycin D3, and the utilization rate of stable isotope atoms is high. Trifluoroacetic acid is used as a catalyst and at a suitable temperature and time (especially for secondary reaction under the same conditions), and it is ensured that the product does not contain non-isotopic rapamycin initiators; its process is simple, and the product is easy to purify and the chemical purity and isotopic abundance are extremely high, which can meet the requirements as a standard reagent, and the use value is high.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis of stable isotope standards and relates to a stable isotope-labeled rapamycin-D3 (Rapamycin-D3) and a preparation method thereof, and specifically relates to a method for synthesizing rapamycin-D3 (Rapamycin-D3) by using a stable isotope-labeled raw material deuterated methanol-D4 and a non-isotope rapamycin. Background Art

[0002] Rapamycin (RPM), also known as sirolimus, is a new type of macrolide potent immunosuppressant; its molecular formula is C 51 H 79 NO 13 , molecular weight 914.18, CAS#52123-88-9; easily soluble in methanol, ethanol, dichloromethane, chloroform, ethyl acetate and butyl acetate, soluble in acetonitrile, poorly soluble in n-hexane, ether and petroleum ether, insoluble in water.

[0003] In 1999, the U.S. FDA approved rapamycin as a new clinical anti-renal transplant rejection drug. Rapamycin was approved for marketing by the European Medicines Agency (EMA) in 2001 and by the China Food and Drug Administration (CFDA) in 2010. Research in authoritative journals such as Cell, Blood and Gut showed that rapamycin also has new potential for treating difficult and complicated diseases such as diabetes, obesity, Parkinson's disease and pancreatic cancer, indicating that rapamycin has a broad future application prospect.

[0004] 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.

[0005] Stable isotope-labeled rapamycin (such as rapamycin-D3) is indispensable for investigating the metabolic pathways of non-deuterated rapamycin in the body and evaluating its mechanism of action, and is an important tool for clinical testing and drug research of rapamycin.

[0006] In the prior art, the existing stable isotope-labeled rapamycin is mainly rapamycin-D3. The main problem is that the product generally contains non-isotopic rapamycin. Many manufacturers have specifically noted this in their products. For example, Toronto Research Chemicals of Canada notes its product as follows: Rapamycin-d3 (contains d0) Technical Grade. This 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, there are few public reports on the specific production process of stable isotope-labeled rapamycin.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides a deuterium-labeled rapamycin stable isotope compound and a preparation method thereof.

[0009] To achieve the above object, the technical solution of the present invention is as follows:

[0010] A method for preparing a deuterium-labeled rapamycin stable isotope compound comprises the following steps:

[0011] S1. At room temperature and under nitrogen protection, deuterated methanol-D4 and trifluoroacetic acid (TFA) are added to the dichloromethane solution of rapamycin in sequence, and after the reaction is complete with stirring, heavy water is added;

[0012] S2, separating the organic phase and the heavy water phase, then washing the organic phase with a saturated heavy water solution of sodium bicarbonate and a saturated heavy water solution of sodium chloride in sequence, and then removing dichloromethane under reduced pressure to obtain a crude product;

[0013] S3. Separate the crude product by silica gel column chromatography and purify by high performance liquid chromatography to obtain a deuterium-labeled rapamycin stable isotope compound.

[0014] Specifically, in the above technical scheme, the synthesis route is as follows:

[0015]

[0016] In the above technical solution, in step S1, the usage ratio of rapamycin, deuterated methanol-D4 and trifluoroacetic acid is 300 mg: 3-4 ml: 1-1.5 ml.

[0017] Specifically, in the above technical solution, corresponding to 300 mg of rapamycin, when the amount of trifluoroacetic acid used is greater than 1.5 ml, it will lead to an increase in by-products, thereby resulting in a decrease in yield.

[0018] In the above technical solution, in step S1, the usage ratio of rapamycin and dichloromethane is 300 mg: 50-65 ml.

[0019] Specifically, in the above technical solution, corresponding to 300 mg of rapamycin, the amount of dichloromethane used is greater than 65 ml, which will slow down the reaction rate and is not conducive to the generation of high-purity deuterium-labeled rapamycin stable isotope compounds.

[0020] In the above technical solution, in step S1, the speed and time of the stirring reaction are 320-450 rpm and 72-96 h respectively.

[0021] Specifically, in the above technical solution, if the stirring reaction time is greater than 96 hours, it will lead to an increase in by-products and thus a decrease in yield.

[0022] In the above technical solution, in step S1, the volume of heavy water added is 0.45-0.6 times that of dichloromethane.

[0023] In the above technical solution, in step S2, the organic phase is washed with a saturated heavy water solution of sodium bicarbonate and a saturated heavy water solution of sodium chloride in sequence until the pH value is 7.

[0024] Furthermore, in the above technical solution, in step S3, the liquid phase used in the silica gel column chromatography separation is n-hexane-ethyl acetate in a volume ratio of 1:2.

[0025] Furthermore, in the above technical solution, in step S3, the step of HPLC purification specifically includes:

[0026] The product separated by silica gel column chromatography was dissolved in dimethylformamide and then injected. The mobile phase A was deionized water and the mobile phase B was acetonitrile. After all the samples were injected, they were rinsed with 30-90v% acetonitrile for 20 minutes.

[0027] Specifically, in the above technical solution, in step S3, in the high performance liquid chromatography purification, the high performance liquid chromatography column is: Prep C18, 30*150mm, 5μm, OBD TM .

[0028] Furthermore, in the above technical solution, step S3 further includes:

[0029] Before separation by silica gel column chromatography and purification by high performance liquid chromatography, the crude product was dissolved in a dichloromethane solution, and steps S1 and S2 were repeated in sequence to perform a secondary reaction under the same conditions.

[0030] Another aspect of the present invention provides a deuterium-labeled rapamycin stable isotope compound, which is prepared by the above 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 rapamycin and a method for synthesizing the same. Non-isotopic rapamycin is used as a raw material, and deuterated methanol-D4 with an isotope abundance of more than 99.8% is used as an isotope labeling source. Rapamycin-D3 is synthesized by a simple one-step reaction, and the utilization rate of stable isotope atoms is high. At the same time, trifluoroacetic acid is used as a catalyst and a suitable reaction temperature and time (especially a secondary reaction is used under the same reaction conditions) can ensure that the prepared rapamycin-D3 product does not contain non-isotopic rapamycin starting materials, thereby avoiding quantitative errors and even qualitative errors caused by standard reagents containing non-isotopic rapamycin on the market during use as standard reagents;

[0034] 2) The preparation method of the deuterium-labeled rapamycin 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 isotopic 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 rapamycin, 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 rapamycin stable isotope compound (rapamycin-D3) prepared in Example 1 of the present invention;

[0036] Figure 2 This is a high performance liquid chromatogram of the deuterium-labeled rapamycin stable isotope compound (rapamycin-D3) 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] The present invention provides a method for preparing a deuterium-labeled rapamycin stable isotope compound (rapamycin-D3), and the synthesis route thereof is as follows:

[0045]

[0046] Example 1

[0047] A method for preparing a deuterium-labeled rapamycin stable isotope compound (rapamycin-D3), comprising the following steps:

[0048] S1. At room temperature (25°C) and under nitrogen protection, add 3.0 ml of deuterated methanol-D4 (CD3OD) and 1.0 ml of trifluoroacetic acid (TFA) to a dichloromethane solution (60 ml) containing 300 mg of rapamycin in sequence, stir the reaction at 400 rpm for 72 h, and then add 30 ml of heavy water;

[0049] S2, standing to separate the organic phase and the heavy water phase, then washing the organic phase with 30 ml of a saturated heavy water solution of sodium bicarbonate and 30 ml of a saturated heavy water solution of sodium chloride in sequence until neutral (pH=7), and then removing dichloromethane under reduced pressure to obtain a crude product 1 (302 mg);

[0050] S3, 302 mg of crude product 1 was added to 60 ml of dichloromethane, and steps S1 and S2 were repeated in sequence, and a secondary reaction was carried out under the same conditions (stirring reaction for 72 hours) to obtain crude product 2 (297 mg), and the crude product 2 was then separated by silica gel column chromatography and eluted with n-hexane-ethyl acetate (volume ratio of 1:2) to obtain a primary purified product (231 mg), and finally the primary purified product was further purified by high performance liquid chromatography. The specific process is as follows: the primary purified product (231 mg) was dissolved in 5 ml of dimethylformamide (DMF), and a Gilson GX-281 high performance liquid chromatograph was selected, and the liquid chromatography column was Prep C18, 30*150mm, 5μm, OBD TM , mobile phase A and mobile phase B are deionized water and acetonitrile respectively. After all the primary purified materials are added, they are rinsed with 30-90v% acetonitrile for 20 min to obtain 165 mg of deuterium-labeled rapamycin stable isotope compound (rapamycin-D3, white solid).

[0051] Example 2

[0052] A method for preparing a deuterium-labeled rapamycin stable isotope compound (rapamycin-D3), comprising the following steps:

[0053] S1. At room temperature (25°C) and under nitrogen protection, add 3.0 ml of deuterated methanol-D4 (CD3OD) and 1.5 ml of trifluoroacetic acid (TFA) to a dichloromethane solution (60 ml) containing 300 mg of rapamycin in sequence, stir the reaction at 400 rpm for 72 h, and then add 30 ml of heavy water;

[0054] S2, standing to separate the organic phase and the heavy water phase, then washing the organic phase with 30 ml of a saturated heavy water solution of sodium bicarbonate and 30 ml of a saturated heavy water solution of sodium chloride in sequence until neutral (pH=7), and then removing dichloromethane under reduced pressure to obtain a crude product 1 (295 mg);

[0055] S3, add 295 mg of crude product 1 to 60 ml of dichloromethane, repeat steps S1 and S2 in sequence, and react twice under the same conditions (stirring reaction for 72 hours) to obtain crude product 2 (283 mg), and then separate the crude product 2 by silica gel column chromatography and elute with n-hexane-ethyl acetate (volume ratio of 1:2) to obtain a primary purified product (217 mg), and finally further purify the primary purified product by high performance liquid chromatography. The specific process is as follows: the primary purified product (217 mg) is dissolved in 5 ml of dimethylformamide (DMF), and a Gilson GX-281 high performance liquid chromatograph is selected, and the liquid chromatography column is Prep C18, 30*150mm, 5μm, OBD TM, mobile phase A and mobile phase B are deionized water and acetonitrile respectively. After all the primary purified materials are added, they are rinsed with 30-90v% acetonitrile for 20 min to obtain 128 mg of deuterium-labeled rapamycin stable isotope compound (rapamycin-D3, white solid).

[0056] Example 3

[0057] A method for preparing a deuterium-labeled rapamycin stable isotope compound (rapamycin-D3), comprising the following steps:

[0058] S1. At room temperature (25°C) and under nitrogen protection, add 3.0 ml of deuterated methanol-D4 (CD3OD) and 1.0 ml of trifluoroacetic acid (TFA) to a dichloromethane solution (60 ml) containing 300 mg of rapamycin in sequence, stir at 350 rpm for 72 h, and then add 30 ml of heavy water;

[0059] S2, standing to separate the organic phase and the heavy water phase, then washing the organic phase with 30 ml of a saturated heavy water solution of sodium bicarbonate and 30 ml of a saturated heavy water solution of sodium chloride in sequence until neutral (pH=7), and then removing dichloromethane under reduced pressure to obtain a crude product 1 (301 mg);

[0060] S3, 301 mg of crude product 1 was added to 60 ml of dichloromethane, and steps S1 and S2 were repeated in sequence, and a secondary reaction was carried out under the same conditions (stirring reaction for 72 hours) to obtain crude product 2 (296 mg), and the crude product 2 was then separated by silica gel column chromatography and eluted with n-hexane-ethyl acetate (volume ratio of 1:2) to obtain a primary purified product (229 mg), and finally the primary purified product was further purified by high performance liquid chromatography. The specific process is as follows: the primary purified product (229 mg) was dissolved in 5 ml of dimethylformamide (DMF), and a Gilson GX-281 high performance liquid chromatograph was selected, and the liquid chromatography column was Prep C18, 30*150mm, 5μm, OBD TM , mobile phase A and mobile phase B are deionized water and acetonitrile respectively. After all the primary purified materials are added, they are rinsed with 30-90v% acetonitrile for 20 min to obtain 160 mg of deuterium-labeled rapamycin stable isotope compound (rapamycin-D3, white solid).

[0061] Example 4

[0062] A method for preparing a deuterium-labeled rapamycin stable isotope compound (rapamycin-D3), comprising the following steps:

[0063] S1. At room temperature (25°C) and under nitrogen protection, add 3.0 ml of deuterated methanol-D4 (CD3OD) and 1.0 ml of trifluoroacetic acid (TFA) to a dichloromethane solution (60 ml) containing 300 mg of rapamycin in sequence, stir the mixture at 400 rpm for 96 h, and then add 30 ml of heavy water;

[0064] S2, standing to separate the organic phase and the heavy water phase, then washing the organic phase with 30 ml of a saturated heavy water solution of sodium bicarbonate and 30 ml of a saturated heavy water solution of sodium chloride in sequence until neutral (pH=7), and then removing dichloromethane under reduced pressure to obtain a crude product 1 (292 mg);

[0065] S3, 292 mg of crude product 1 was added to 60 ml of dichloromethane, and steps S1 and S2 were repeated in sequence, and a secondary reaction was carried out under the same conditions (stirring reaction for 96 hours) to obtain crude product 2 (286 mg), and the crude product 2 was then separated by silica gel column chromatography and eluted with n-hexane-ethyl acetate (volume ratio of 1:2) to obtain a primary purified product (211 mg), and finally the primary purified product was further purified by high performance liquid chromatography. The specific process is as follows: the primary purified product (211 mg) was dissolved in 5 ml of dimethylformamide (DMF), and a Gilson GX-281 high performance liquid chromatograph was selected, and the liquid chromatography column was Prep C18, 30*150mm, 5μm, OBD TM , mobile phase A and mobile phase B were deionized water and acetonitrile respectively. After all the primary purified material was added, it was rinsed with 30-90v% acetonitrile for 20 min to obtain 119 mg of deuterium-labeled rapamycin stable isotope compound (rapamycin-D3, white solid).

[0066] Example 5

[0067] A method for preparing a deuterium-labeled rapamycin stable isotope compound (rapamycin-D3), comprising the following steps:

[0068] S1. At room temperature (25°C) and under nitrogen protection, add 3.0 ml of deuterated methanol-D4 (CD3OD) and 1.0 ml of trifluoroacetic acid (TFA) to a dichloromethane solution (60 ml) containing 300 mg of rapamycin in sequence, stir the reaction at 400 rpm for 72 h, and then add 30 ml of heavy water;

[0069] S2, standing to separate the organic phase and the heavy water phase, then washing the organic phase with 30 ml of a saturated heavy water solution of sodium bicarbonate and 30 ml of a saturated heavy water solution of sodium chloride in sequence until neutral (pH=7), and then removing dichloromethane under reduced pressure to obtain a crude product 1 (302 mg);

[0070] S3. The crude product 1 (302 mg) was separated by silica gel column chromatography and eluted with n-hexane-ethyl acetate (volume ratio 1:2) to obtain a primary purified product (238 mg). Finally, the primary purified product was further purified by high performance liquid chromatography. The specific process is as follows: the primary purified product (238 mg) was dissolved in 5 ml of dimethylformamide (DMF), and a Gilson GX-281 high performance liquid chromatography was selected. The liquid chromatography column was Prep C18, 30*150mm, 5μm, OBD TM , mobile phase A and mobile phase B were deionized water and acetonitrile respectively. After all the primary purified material was added, it was rinsed with 30-90v% acetonitrile for 20 min to obtain 171 mg of deuterium-labeled rapamycin stable isotope compound (rapamycin-D3, white solid).

[0071] Example 6

[0072] A method for preparing a deuterium-labeled rapamycin stable isotope compound (rapamycin-D3), comprising the following steps:

[0073] S1. At room temperature (25°C) and under nitrogen protection, add 3.0 ml of deuterated methanol-D4 (CD3OD) and 3.0 ml of trifluoroacetic acid (TFA) to a dichloromethane solution (60 ml) containing 300 mg of rapamycin in sequence, stir the mixture at 400 rpm for 72 h, and then add 30 ml of heavy water;

[0074] S2, standing to separate the organic phase and the heavy water phase, then washing the organic phase with 30 ml of a saturated heavy water solution of sodium bicarbonate and 30 ml of a saturated heavy water solution of sodium chloride in sequence until neutral (pH=7), and then removing dichloromethane under reduced pressure to obtain a crude product 1 (289 mg);

[0075] S3, 289 mg of crude product 1 was added to 60 ml of dichloromethane, and steps S1 and S2 were repeated in sequence, and a secondary reaction was carried out under the same conditions (3.0 ml of CD3OD and 3.0 ml of TFA were added, and the reaction was stirred at 400 rpm for 72 hours) to obtain crude product 2 (276 mg), and the crude product 2 was separated by silica gel column chromatography and eluted with n-hexane-ethyl acetate (volume ratio of 1:2) to obtain a primary purified product (178 mg), and finally the primary purified product was further purified by high performance liquid chromatography. The specific process is as follows: the primary purified product (178 mg) was dissolved in 5 ml of dimethylformamide (DMF), and a Gilson GX-281 high performance liquid chromatography was selected, and the liquid chromatography column was Prep C18, 30*150mm, 5μm, OBD TM, mobile phase A and mobile phase B are deionized water and acetonitrile respectively. After all the primary purified materials are added, they are rinsed with 30-90v% acetonitrile for 20 min to obtain 99 mg of deuterium-labeled rapamycin stable isotope compound (rapamycin-D3, white solid).

[0076] Example 7

[0077] A method for preparing a deuterium-labeled rapamycin stable isotope compound (rapamycin-D3), comprising the following steps:

[0078] S1. At room temperature (25°C) and under nitrogen protection, add 3.0 ml of deuterated methanol-D4 (CD3OD) and 1.0 ml of trifluoroacetic acid (TFA) to a dichloromethane solution (60 ml) containing 300 mg of rapamycin in sequence, stir the reaction at 400 rpm for 72 h, and then add 30 ml of heavy water;

[0079] S2, standing to separate the organic phase and the heavy water phase, and then removing dichloromethane from the organic phase under reduced pressure to obtain a crude product 1 (298 mg);

[0080] S3, 298 mg of crude product 1 was added to 60 ml of dichloromethane, and steps S1 and S2 were repeated in sequence, and a secondary reaction was carried out under the same conditions (stirring reaction for 72 hours) to obtain crude product 2 (285 mg), and the crude product 2 was separated by silica gel column chromatography and eluted with n-hexane-ethyl acetate (volume ratio of 1:2) to obtain a primary purified product (149 mg), and finally the primary purified product was further purified by high performance liquid chromatography. The specific process is as follows: the primary purified product (149 mg) was dissolved in 5 ml of dimethylformamide (DMF), and a Gilson GX-281 high performance liquid chromatograph was selected, and the liquid chromatography column was Prep C18, 30*150mm, 5μm, OBD TM , mobile phase A and mobile phase B are deionized water and acetonitrile respectively. After all the primary purified materials are added, they are rinsed with 30-90v% acetonitrile for 20 min to obtain 42 mg of deuterium-labeled rapamycin stable isotope compound (rapamycin-D3, white solid).

[0081] Figure 1 and Figure 2They are respectively the hydrogen nuclear magnetic resonance spectrum (using deuterated chloroform CDCl3 as solvent, obtained by JEOL-400MHz nuclear magnetic resonance instrument) and the HPLC chromatogram (the sample was dissolved in acetonitrile, the mobile phase A was deionized water containing 0.1% formic acid, the mobile phase B was acetonitrile containing 0.1% formic acid, in Shimadzu Nexera X2, the HPLC column was SunFire C18, 4.6*50mm, 3.5μm; the flow rate was 1.0ml / min through the HPLC column; the flushing gradient was: 10-95% mobile phase B for the first 6min, and then 95% mobile phase B for 6min; the detection was carried out by evaporative light scattering detector ELSD to obtain the HPLC chromatogram of isotope-labeled rapamycin-D3).

[0082] from Figure 1 It can be seen that there is no methoxy absorption peak at the chemical shift of 3.13ppm, indicating that the structure is rapamycin-D3, which does not contain any non-isotopic rapamycin; Figure 2 It can be seen that the purity of the prepared deuterium-labeled rapamycin stable isotope compound (rapamycin-D3) sample reaches 99.2%.

[0083] The yields of rapamycin-D3 of Examples 1-5 were calculated respectively, and the chemical purity and isotopic abundance of the prepared deuterium-labeled rapamycin stable isotope compound (rapamycin-D3) were detected by high performance liquid chromatography and mass spectrometry respectively. The results are shown in Table 1 below.

[0084] Table 1 Results of yield, chemical purity and isotopic abundance of the products of each example

[0085]

[0086] As can be seen from Table 1, the amount of trifluoroacetic acid in Example 2 was increased to 1.5 ml, resulting in a decrease in the yield of the product to 42%; the amount of deuterated methanol-D4 in Example 3 was increased to 4.0 ml, which did not have much effect on the yield of the product, 52%; the reaction time in Example 4 was extended to 96 h, resulting in a decrease in the yield of the product to 39%; in Example 5, only one reaction was carried out, and although the yield was increased to 56%, the product contained 4.7% of non-isotopic rapamycin, resulting in a decrease in purity to 94.5% and a decrease in isotopic abundance to 95.1%; in Example 6, the amount of trifluoroacetic acid was increased to 3.0 ml, resulting in a decrease in the yield of the product to 33%; in Example 7, the post-reaction washing did not reach neutrality, and the reaction by-products increased, resulting in a decrease in the yield of the product to 14%.

[0087] 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.

[0088] 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 rapamycin stable isotope compound, characterized in that: The following steps are involved: S1. At room temperature and under nitrogen protection, deuterated methanol-D4 and trifluoroacetic acid are added to the dichloromethane solution of rapamycin in sequence, and after stirring for complete reaction, heavy water is added; The dosage ratio of rapamycin, deuterated methanol-D4 and trifluoroacetic acid is 300 mg: 3-4 ml: 1-1.5 ml; and / or, the dosage ratio of rapamycin to dichloromethane is 300 mg: 50-65 ml; S2, separating the organic phase and the heavy water phase, then washing the organic phase with a saturated heavy water solution of sodium bicarbonate and a saturated heavy water solution of sodium chloride in sequence, and then removing dichloromethane under reduced pressure to obtain a crude product; S3, separating the crude product by silica gel column chromatography and purifying by high performance liquid chromatography to obtain a deuterium-labeled rapamycin stable isotope compound; Step S3 also includes: Before separation by silica gel column chromatography and purification by high performance liquid chromatography, the crude product was dissolved in a dichloromethane solution, and steps S1 and S2 were repeated in sequence to perform a secondary reaction under the same conditions.

2. The method for preparing a deuterium-labeled rapamycin stable isotope compound according to claim 1, characterized in that: In step S1, The speed and time of the stirring reaction are 320-450 rpm and 72-96 h respectively; And / or, the added volume of the heavy water is 0.45-0.6 times that of the dichloromethane.

3. The method for preparing a deuterium-labeled rapamycin stable isotope compound according to claim 1, characterized in that: In step S2, the organic phase is washed with a saturated solution of sodium bicarbonate in heavy water and a saturated solution of sodium chloride in heavy water in sequence until the pH value is 7.

4. The method for preparing a deuterium-labeled rapamycin stable isotope compound according to any one of claims 1 to 3, characterized in that: In step S3, The liquid phase used in the silica gel column chromatography separation is n-hexane-ethyl acetate in a volume ratio of 1:

2.

5. The method for preparing a deuterium-labeled rapamycin stable isotope compound according to any one of claims 1 to 3, characterized in that: In step S3, the high performance liquid chromatography purification step specifically includes: The product separated by silica gel column chromatography was dissolved in dimethylformamide and then injected. The mobile phase A was deionized water and the mobile phase B was acetonitrile. After all the samples were injected, they were rinsed with 30-90v% acetonitrile for 20 minutes.

6. The method for preparing a deuterium-labeled rapamycin stable isotope compound according to claim 5, characterized in that: In step S3, In the HPLC purification, the HPLC column is: Prep C18, 30*150mm, 5μm, OBD TM .

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