CN118598950A - A preparation method of cyclosporine - Google Patents

Abstract

The invention discloses a preparation method of a cyclosporine, and relates to the technical field of chemical synthesis; the invention provides a preparation method of a pair of cyclosporins, which takes cyclosporin A as a raw material and synthesizes the cyclosporin A through acetyl protection, oxidation reaction, horner-Wittig reaction, reduction, dehydration and deacetylation reaction; the method can conveniently synthesize the Fu-cyclosporin, has mild reaction conditions, high yield and high purity, is favorable for obtaining the Fu-cyclosporin with more convenience and higher yield in commercial production, and has excellent application prospect.

Description

A preparation method of cyclosporine

Technical Field

The invention relates to the technical field of chemical synthesis, and in particular to a method for preparing cyclosporine.

Background Art

Voclosporin was designed and synthesized by Isotechnika Pharmaceuticals of Canada. Oral voclosporin was approved for marketing by the U.S. Food and Drug Administration (FDA) in 2021 under the trade name Lupkynis. This product can be used clinically to treat lupus nephritis. Compared with similar drugs, voclosporin has certain advantages and characteristics. It has higher immunosuppressive activity, and the immunosuppressive activity of trans-voclosporin is 1.5 times that of cis-voclosporin and 5 times that of cyclosporin A. Compared with cyclosporin A, it has fewer toxic side effects and significantly reduced nephrotoxicity. The drug has been widely used in the treatment of lupus nephritis and has broad market demand and development prospects.

The commonly used synthesis route of cyclosporine is shown in Figure 1. The route uses cyclosporine A as the starting material. First, the hydroxyl group of the amino acid at position 1 is protected with an acetyl group, then acetyl-cyclosporine A aldehyde is obtained by oxidation, and then the carbon chain is introduced by reacting with Wittig reagent, and finally the acetyl group is removed to obtain cyclosporine.

However, during the oxidation reaction, highly toxic and expensive osmium tetroxide is required. At the same time, allyl triphenylphosphonium bromide will produce triphenylphosphine after the reaction. Its polarity is similar to that of cyclosporine and it is difficult to purify. It requires multiple column chromatography with different polarities to separate it, resulting in a very low yield after the reaction, which seriously affects the operability and economic benefits of the reaction.

Therefore, in order to ensure the quality of commercial products and improve the reaction yield, the process route in the production process needs to be optimized.

Summary of the invention

In order to solve the above technical problems, the purpose of the present invention is to provide a method for preparing cyclosporine, which uses cyclosporine A as a raw material to conveniently synthesize cyclosporine, has mild reaction conditions, high yield and high purity, and effectively solves the problems of difficult purification, low yield, low operability and low economic benefit in the prior art.

The technical solution of the present invention to solve the above technical problems is as follows: a method for preparing cyclosporine is provided, and the preparation route thereof is:

;

The preparation method specifically comprises the following steps:

(1) Compound M0 is reacted with an acetylation agent under alkaline conditions to obtain compound M1;

(2) subjecting the compound M1 obtained in step (1) to an oxidation reaction with an oxidant to obtain a compound M2;

(3) subjecting the compound M2 obtained in step (2) to an oxidation reaction with an oxidant 2 to obtain a compound M3;

(4) reacting the compound M3 obtained in step (3) with a Horner-Wittig reagent under alkaline conditions to obtain compound 1a;

(5) subjecting the compound 1a obtained in step (4) to a selective reduction reaction with a reducing agent to obtain compound 1aa;

(6) subjecting the compound 1aa obtained in step (5) to a dehydrating reaction with a dehydrating agent to obtain a compound M4;

(7) Deacetylation of the compound M4 obtained in step (6) under alkaline conditions to obtain compound M5, i.e., cyclosporine.

Furthermore, in step (1), the alkaline solution is pyridine, and the acetylation agent is acetic anhydride; the molar ratio of compound M0, acetic anhydride and pyridine is 1:10:15.

Furthermore, in step (2), the oxidant 1 is potassium osmate dihydrate and N-methylmorpholine oxide; the molar ratio of compound M1, potassium osmate dihydrate and N-methylmorpholine oxide is 1:0.03:2.

Furthermore, in step (3), the second oxidant is sodium periodate; and the molar ratio of compound M2 to the second oxidant is 1:2.

Furthermore, in step (4), the alkali solution is potassium carbonate, the Horner-Wittig reagent is diethyl acetonyl phosphate, and the molar ratio of compound M3, diethyl acetonyl phosphate and potassium carbonate is 1:3:3.

Furthermore, in step (5), the reducing agent is sodium borohydride and cerium chloride heptahydrate; the molar ratio of compound 1a, sodium borohydride and cerium chloride heptahydrate is 1:2:2.

Furthermore, in step (6), the dehydrating agent is p-toluenesulfonic acid monohydrate; and the molar ratio of compound 1aa to p-toluenesulfonic acid monohydrate is 1:0.3.

Furthermore, in step (7), the alkali solution is potassium carbonate; and the molar ratio of compound M4 to potassium carbonate is 1:8.

Furthermore, in step (1), the reaction is carried out at -10-35°C for 1-3 days.

Furthermore, in steps (2) and (3), the reaction is carried out at 0-25°C for 5-24 hours.

Furthermore, in step (4), the reaction is carried out at 0-25 °C for 3 days.

Furthermore, in step (5), the reaction is carried out at 0-25°C for 3-5 hours.

Furthermore, in step (6), the reaction is carried out at 0-120°C for 1-3 h.

Furthermore, in step (7), the reaction is carried out at 0-25°C for 1-3 days.

Specifically, the preparation method of cyclosporine provided by the present invention comprises the following steps:

(1) Compound M0, pyridine and DMAP were dissolved in dichloromethane, stirred for 0.5 h to 0 °C under ice bath conditions, then acetic anhydride was dissolved in dichloromethane and slowly added dropwise to the reaction system, stirred to room temperature, and stirred at 35 °C for 3 days. After the reaction was complete, water was added and stirred for 0.5 h, and the mixture was allowed to stand for stratification. The aqueous layer was extracted with dichloromethane three times, and the organic phases were combined and then washed with 1 N hydrochloric acid, water, saturated sodium bicarbonate solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound M1;

(2) Compound M1 and potassium osmate dihydrate were dissolved in acetone and water, stirred for 0.5 h in an ice bath until the temperature reached 0 °C, and then N-methylmorpholine oxide was dissolved in water and slowly added dropwise to the reaction system in batches. After the addition was completed, the mixture was returned to room temperature and stirred for 36 h. After the reaction was complete after spot plate detection, most of the acetone was removed by vacuum concentration, and then sodium bisulfite solution was added and stirred for 0.5 h. The mixture was extracted with ethyl acetate three times, and the organic phases were combined and washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound M2;

(3) Compound M2 was dissolved in methanol and stirred for 0.5 h in an ice bath. Then sodium periodate was dissolved in water and slowly added dropwise to the reaction system. After the addition was completed, the temperature was naturally restored to room temperature. The reaction was stirred for 24 h. After the reaction was complete, the plate was tested for completion, the mixture was filtered through diatomaceous earth and washed with a small amount of methanol. Saturated sodium thiosulfate solution was added to the filtrate and stirred for 0.5 h to quench the reaction. Most of the methanol was removed by vacuum concentration. If solids precipitated, they were filtered through diatomaceous earth again and washed with a small amount of methanol. Most of the methanol was removed by vacuum concentration. The solids were extracted with ethyl acetate three times. The organic phases were combined and washed with water and saturated brine in turn. The organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound M3.

(4) Compound M3 was dissolved in tetrahydrofuran, stirred in an ice bath for 0.5 h, potassium carbonate was added, and then diethyl acetonyl phosphate was added to the reaction system in batches. The temperature was raised to room temperature and the reaction was allowed to react for 3 days. After the reaction was complete, the tetrahydrofuran was removed by vacuum concentration, and the compound was dissolved in a mixed solution of ethyl acetate and water. The mixture was allowed to stand for stratification, and the aqueous layer was extracted with ethyl acetate for 3 times. The organic phases were combined, washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a pale yellow oily crude product. Finally, the compound was separated and purified by column chromatography to obtain compound 1a.

(5) Compound 1a was dissolved in anhydrous methanol, stirred at 0 °C for 0.5 h, cerium chloride heptahydrate was added, and then sodium borohydride was slowly added to the reaction system. The mixture was slowly stirred until the reaction was allowed to react at room temperature for 0.5 h. After the reaction was complete by spot plate detection, water was added and stirred for 0.5 h. The mixture was concentrated in vacuo to remove most of the methanol. The aqueous layer was extracted three times with ethyl acetate, and the organic phases were combined and washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 1aa.

(6) Under argon atmosphere, compound 1aa was dissolved in toluene, p-toluenesulfonic acid was added, and the mixture was refluxed and stirred at 120 °C for 2 h. After the reaction was complete by spot plate detection, the reaction product was cooled to room temperature, washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a yellow foamy solid crude product; finally, the product was separated and purified by column chromatography to obtain compound M4;

(7) Compound M4 was dissolved in methanol, and water was added. Potassium carbonate was then added and stirred for 2 days. After the reaction was complete by spot plate detection, most of the methanol was removed by vacuum concentration. The aqueous layer was extracted three times with ethyl acetate. The organic phases were combined and washed with 0.5 N hydrochloric acid, water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude cyclosporine. Finally, the crude product was separated and purified by column chromatography to obtain compound M5, i.e., cyclosporine.

The present invention has the following beneficial effects:

1. The present invention uses cyclosporine A as a raw material, and synthesizes cyclosporine through acetyl protection, oxidation reaction, Horner-Wittig reaction, reduction, dehydration and deacetylation reaction. The method of the present invention can conveniently synthesize cyclosporine, with mild reaction conditions, high yield and high purity, which is conducive to obtaining cyclosporine more conveniently and at a higher yield in commercial production, and has excellent application prospects.

2. The present invention uses K ₂ OsO ₄ ·2H ₂ O, which is safer and more readily available, in the oxidation reaction, and the reaction yield is 88%; the Horner-Wittig reagent diethyl acetone phosphate, which has mild reaction conditions and by-products that are easily soluble in water and removed, is used to couple the intermediate 1a with acetyl CsA aldehyde, and the yield can reach 87%; and acetyl cyclosporine can also be obtained by selectively reducing the carbonyl group with sodium borohydride NaBH ₄ and cerium chloride heptahydrate CeCl ₃ ·7H ₂ O and dehydrating with p-toluenesulfonic acid, and the yields of both steps are above 80%. The optimized route has mild reaction conditions, simple operation, and convenient purification, and the total reaction yield is 28%, which is twice as high as that of the original route.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a commonly used synthesis route of cyclosporine;

FIG2 is a ¹ H-NMR spectrum of compound M1;

FIG3 is a ¹ H-NMR spectrum of compound M2;

FIG4 is a ¹ H-NMR spectrum of compound M3;

FIG5 is a ¹ H-NMR spectrum of compound 1a;

FIG6 is a ¹ H-NMR spectrum of compound 1aa;

FIG7 is a ¹ H-NMR spectrum of compound M4;

FIG8 is a ¹ H-NMR spectrum of compound M5.

DETAILED DESCRIPTION

The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

Example 1

A method for preparing cyclosporine comprises the following steps:

(1) Compound M0 (cyclosporine A; 50 g, 41.6 mmol), pyridine (51 mL, 622 mmol) and DMAP (1.014 g, 8.32 mmol) were dissolved in dichloromethane (260 mL), stirred for 0.5 h to 0 °C under ice bath conditions, then acetic anhydride (39 mL, 416 mmol) was dissolved in dichloromethane (55 mL) and slowly added dropwise to the reaction system, stirred to room temperature, and stirred at 35 °C for 3 d. After the reaction was complete by spot plate detection, water (200 mL) was added and stirred for 0.5 h. The mixture was allowed to stand for stratification, and the aqueous layer was extracted with dichloromethane three times. The organic phases were combined and then washed with 1 N hydrochloric acid, water, saturated sodium bicarbonate solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a white foamy solid, i.e., compound M1 (acetylcyclosporine A; 46.1 g, yield 89%).

The ¹ H-NMR spectrum of compound M1 is shown in Figure 2, and the data are as follows:

¹ HNMR (400 MHz, CDCl ₃ ) δ 8.54 (d, J = 9.7 Hz, 1H), 8.03 (d, J = 6.9 Hz, 1H), 7.47 (dd, J = 16.3, 8.5 Hz, 2H), 5.67 ( dd, J = 11.1, 4.3 Hz, 1H), 5.51 (s,2H), 5.38 (dd, J = 11.8, 3.9 Hz, 1H), 5.32 – 5.11 (m, 5H), 5.01 – 4.93 (m, 2H) ,4.88 – 4.72 (m, 2H), 4.64 (d, J = 13.7 Hz, 1H), 4.42 (m, J = 14.1, 7.4 Hz, 1H), 3.44 (s, 3H), 3.26 – 3.18 (m, 10H), 3.08 (s, 3H), 2.70 – 2.61 (m, 7H), 2.42(m, J = 9.9, 6.6 Hz, 1H ), 2.24 – 2.04 (m, 6H), 2.00 (s, 4H), 1.70 (p, J = 4.0,3.5 Hz, 6H), 1.60 – 1.56 (m, 4H), 1.49 – 1.37 (m, 3H), 1.31 (d, J = 7.2 Hz, 5H), 1.26 (d, J = 7.0 Hz, 8H), 1.05 – 1.01 (m, 5H), 1.00 – 0.96 (m, 8H), 0.94 (dd, J = 6.6, 2.8 Hz, 8H), 0.86 (m, J = 10.4, 6.7, 3.0 Hz, 22H), 0.79 (d, J = 6.6Hz , 7H).

(2) Compound M1 (46.1 g, 41.4 mmol) and potassium osmate dihydrate (345 mg, 1.1 mmol) were dissolved in acetone (400 mL) and water (100 mL), and stirred for 0.5 h to 0 °C under ice bath conditions. Then, N-methylmorpholine oxide (9.7 g, 82.8 mmol) was dissolved in water (10 mL) and slowly added dropwise to the reaction system in batches. After the addition was completed, the mixture was returned to room temperature and stirred for 36 h. After the reaction was complete by spot plate detection, most of the acetone was removed by vacuum concentration, and sodium bisulfite solution was added and stirred for 0.5 h. The mixture was extracted with ethyl acetate three times, and the organic phases were combined and washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a white foamy solid, namely compound M2 (acetylcyclosporine A vicinal diol; 42 g, yield 88%).

The ¹ H-NMR spectrum of compound M2 is shown in FIG3 , and the data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.54 (d, J = 9.7 Hz, 1H), 8.03 (d, J = 6.9 Hz, 1H), 7.47 (dd, J = 16.3, 8.5 Hz, 2H), 5.67 (dd, J = 11.1, 4.3 Hz, 1H), 5.51 (s,2H), 5.38 (dd, J = 11.8, 3.9 Hz, 1H), 5.31 – 5.22 (m, 2H), 5.14 (dd, J = 8.5 , 5.5Hz, 1H), 4.96 (t, J = 10.9 Hz, 2H), 4.86 – 4.72 (m, 2H), 4.64 (d, J = 13.7 Hz, 1H), 4.41 (q, J = 7.3 Hz, 1H), 3.44 (s, 2H), 3.26 – 3.19 (m, 8H), 3.08 (s, 2H), 2.69 – 2.62 (m, 6H), 2.23 – 2.04 (m, 5H), 2.00 (s, 4H), 1.68 (m, J = 6.9, 6.2,3.1 Hz, 6H), 1.60 – 1.56 (m, 3H), 1.31 (d, J = 7.2 Hz, 4H), 1.26 (d, J = 7.0 Hz, 6H), 1.04 (d, J = 6.5 Hz, 3H), 1.00 – 0.96 (m, 6H), 0.94 (dd, J = 6.6, 2.8 Hz, 6H), 0.86 (m, J = 10.4, 6.7, 3.0 Hz, 18H), 0.79 (d, J = 6.6 Hz, 5H).

(3) Compound M2 (41 g, 32.06 mmol) was dissolved in methanol (400 mL) and stirred for 0.5 h in an ice bath. Then, sodium periodate (10.29 g, 48.1 mmol) was dissolved in water (100 mL) and slowly added dropwise to the reaction system. After the addition was completed, the temperature was naturally restored to room temperature. The reaction was stirred for 24 h. After the reaction was complete, the filtrate was filtered with diatomaceous earth and washed with a small amount of methanol. Saturated sodium thiosulfate solution (20 mL) was added to the filtrate and stirred for 0.5 h to quench the reaction. Most of the methanol was removed by vacuum concentration. Solids precipitated and were filtered again with diatomaceous earth, washed with a small amount of methanol, and most of the methanol was removed by vacuum concentration. The solids were extracted with ethyl acetate three times. The organic phases were combined and washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a white foamy solid, namely compound M3 (acetylcyclosporine A aldehyde; 34.5 g, yield 85%).

The ¹ H-NMR spectrum of compound M3 is shown in FIG4 , and the data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.59 (s, 1H), 8.53 (d, J = 9.7 Hz, 1H), 7.96 (d, J = 6.9 Hz, 1H), 7.48 (dd, J = 18.3, 8.4 Hz, 2H), 5.66 (dd, J = 11.2, 4.2 Hz,1H), 5.57 – 5.46 (m, 2H), 5.31 (dd, J = 12.2, 3.7 Hz, 1H), 5.16 (m, J = 17.8 ,9.7, 4.9 Hz, 2H), 5.03 – 4.93 (m, 2H), 4.83 (p, J = 6.8 Hz, 1H), 4.72 (t, J = 9.6Hz, 1H), 4.64 (d, J = 13.9 Hz, 1H), 4.40 (p, J = 7.1 Hz, 1H), 3.45 (s, 3H), 3.28(d, J = 1.4 Hz, 6H), 3.20 (s, 4H), 3.07 (s, 3H), 2.65 (d, J = 8.8 Hz, 6H), 2.47 –2.33 (m, 3H), 2.19 – 2.12 (m, 2H), 1.98 (s, 4H), 1.69 (q, J = 3.6 Hz, 6H), 1.30 (d, J = 7.1 Hz, 4H), 1.27 – 1.24 (m, 5H), 1.07 (d, J = 6.9 Hz, 3H), 1.01 (dd, J =8.3, 6.6 Hz, 6H), 0.95 (m, J = 11.5, 6.6, 3.5 Hz, 14H), 0.90 – 0.79 (m , 21H),0.77 (d, J = 6.5 Hz, 3H).

(4) Compound M3 (20 g, 16.23 mmol) was dissolved in tetrahydrofuran (100 mL), stirred in an ice bath for 0.5 h, potassium carbonate (6.73 g, 48.68 mmol) was added, and then diethyl acetonyl phosphate (9.45 g, 48.68 mmol) was added to the reaction system in batches. The temperature was raised to room temperature and the reaction was allowed to react for 3 d. After the reaction was complete by spot plate detection, the tetrahydrofuran was removed by vacuum concentration, and the compound was dissolved in a mixed solution of ethyl acetate (100 mL) and water (100 mL). The mixture was allowed to stand for stratification, and the aqueous layer was extracted with ethyl acetate for 3 times. The organic phases were combined, washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a pale yellow oily crude product. Finally, the product was separated and purified by column chromatography (dichloromethane: methanol = 50:1) to obtain a white foamy solid, i.e., compound 1a (acetylcyclosporine A allylic ketone; 18 g, yield 87%).

The ¹ H-NMR spectrum of compound 1a is shown in FIG5 , and the data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.52 (d, J = 9.7 Hz, 1H), 8.02 (d, J = 6.9 Hz, 1H), 7.51 (dd, J = 31.9, 8.3 Hz, 2H), 6.76 (dt, J = 15.0, 7.2 Hz, 1H), 5.91 (d, J =15.8 Hz, 1H), 5.67 (dd, J = 11.1, 4.3 Hz, 1H), 5.59 – 5.47 (m, 2H), 5.33 – 5.25(m, 2H), 5.18 – 5.10 (m, 1H), 4.96 (dd, J = 10.5, 4.6 Hz, 2H), 4.83 (p, J = 7.1Hz, 1H), 4.72 – 4.60 (m, 2H), 4.40 (t, J = 7.1 Hz, 1H), 3.45 (s, 3H), 3.31 –3.18 (m, 10H), 3.10 (s, 3H), 2.66 (d, J = 9.7 Hz, 6H), 2.24 (d, J = 14.7 Hz, 5H), 2.19 – 2.08 (m, 4H), 2.01 (s, 4H), 1.67 (s, 6H), 1.30 (d, J = 7.2 Hz, 4H), 1.26 (d, J = 7.0 Hz, 4H), 1.22 – 1.15 (m, 2H), 1.06 – 1.01 (m, 6H), 1.00 – 0.97 (m,7H), 0.94 (d, J = 6.6 Hz, 6H), 0.87 (m, J = 10.1, 5.2, 3.3 Hz, 20H), 0.81 (dd, J =6.7, 3.5 Hz, 6H).

(5) Compound 1a (10 g, 7.86 mmol) was dissolved in anhydrous methanol (50 mL), stirred at 0 °C for 0.5 h, cerium chloride heptahydrate (5.85 g, 15.7 mmol) was added, and then sodium borohydride (595 mg, 15.7 mmol) was slowly added to the reaction system. The mixture was slowly stirred to room temperature for 0.5 h. After the reaction was complete by spot plate detection, water (20 mL) was added and stirred for 0.5 h. The mixture was concentrated in vacuo to remove most of the methanol. The aqueous layer was extracted with ethyl acetate three times, and the organic phases were combined and washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a white foamy solid, namely compound 1aa (acetylcyclosporine A allyl alcohol; 9 g, yield 90%).

The ¹ H-NMR spectrum of compound 1aa is shown in FIG6 , and the data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.60 (d, J = 9.7 Hz, 1H), 8.04 (d, J = 6.8 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.51 (d , J = 7.7 Hz, 1H), 5.66 (dd, J = 11.2, 4.2Hz, 1H), 5.61 – 5.46 (m, 3H), 5.40 (dd, J = 12.5, 3.8 Hz, 1H), 5.33 – 5.24 ( m,2H), 5.14 (dd, J = 8.1, 5.7 Hz, 1H), 4.95 (dd, J = 10.0, 7.4 Hz, 2H), 4.83 (p, J =7.0 Hz, 1H), 4.69 – 4.55 (m, 2H), 4.38 (p, J = 7.0 Hz, 1H), 4.08 (dd, J = 8.3,6.1 Hz, 1H), 3.48 (s , 3H), 3.27 (d, J = 9.6 Hz, 6H), 3.21 (s, 3H), 3.13 (s,3H), 2.65 (d, J = 8.5 Hz, 6H), 2.43 (m, J = 10.4, 6.7 Hz, 1H), 2.33 – 2.20 (m,2H), 2.19 – 2.08 (m, 2H), 2.00 (s, 5H), 1.92 (d, J = 9.9 Hz, 2H), 1.70 (dd, J =14.3, 7.1 Hz, 3H), 1.62 (s, 8H), 1.30 (d, J = 7.2 Hz, 4H), 1.26 (d, J = 7.0 Hz, 4H) , 1.20 (d, J = 6.1 Hz, 4H), 1.06 – 0.93 (m, 18H), 0.92 – 0.88 (m, 5H), 0.88– 0.76 (m, 19H).

(6) Under argon atmosphere, compound 1aa (5 g, 3.92 mmol) was dissolved in toluene (5 g, 3.92 mmol), p-toluenesulfonic acid (224 mg, 1.18 mmol) was added, and the mixture was refluxed and stirred at 120 °C for 2 h. After the reaction was complete by spot plate detection, the reaction product was cooled to room temperature, washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a yellow foamy solid crude product; finally, it was separated and purified by column chromatography (petroleum ether: acetone = 3:1) to obtain a white foamy solid, i.e., compound M4 (acetylcyclosporine; 4 g, yield 81%).

The ¹ H-NMR spectrum of compound M4 is shown in FIG7 , and the data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.57 (dd, J = 9.7, 6.5 Hz, 1H), 8.03 (dd, J =9.3, 6.9 Hz, 1H), 7.51 (dd, J = 9.2, 2.8 Hz, 1H), 7.44 (t, J = 8.1 Hz, 1H), 6.54– 6.21 (m, 1H), 5.97 – 5.84 (m, 1H), 5.67 (dd, J = 11.2, 4.2 Hz, 1H), 5.52 (d , J = 2.7 Hz, 2H), 5.34 (dd, J = 11.3, 3.5 Hz, 1H), 5.29 – 5.22 (m, 1H), 5.14 (d, J =7.2 Hz, 1H), 5.00 – 4.91 (m, 3H), 4.88 – 4.70 (m, 2H), 4.64 (d, J = 13.8 Hz,1H), 4.40 (m, J = 7.1, 2.9 Hz, 1H), 3.44 (s, 3H), 3.26 (d, J = 5.8 Hz, 3H), 3.20(t, J = 4.2 Hz, 7H), 3.08 (s, 3H), 2.66 (d , J = 8.8 Hz, 7H), 2.16 (m, J = 15.0,8.2, 4.1 Hz, 3H), 2.03 (t, J = 6.3 Hz, 6H), 1.68 (s, 7H), 1.31 (d, J = 7.2 Hz, 4H), 1.25 (d, J = 6.9 Hz, 4H), 1.06 – 0.96 (m, 14H), 0.94 (dd, J = 6.6, 2.7 Hz ,9H), 0.89 – 0.82 (m, 18H), 0.79 (dd, J = 6.7, 2.5 Hz, 6H).

(7) Compound M4 (25 g, 19.9 mmol) was dissolved in methanol (250 mL), and water (100 mL) was added. Potassium carbonate (22 g, 159.2 mmol) was then added and stirred for 2 d. After the reaction was complete by spot plate detection, most of the methanol was removed by vacuum concentration. The aqueous layer was extracted three times with ethyl acetate. The organic phases were combined and washed with 0.5 N hydrochloric acid, water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude cyclosporine. Finally, the crude product was separated and purified by column chromatography (1. toluene: ethyl acetate = 3:2, 2. dichloromethane: methanol = 100:1, 3. petroleum ether: acetone = 3:1) to obtain a white foamy solid, i.e., compound M5, i.e., cyclosporine (16.5 g, yield 68%).

The ¹ H-NMR spectrum of compound M5 is shown in FIG8 , and the data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.96 (dd, J = 29.3, 9.6 Hz, 1H), 7.66 (dd, J =16.4, 7.4 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H) , 7.16 (dd, J = 18.8, 7.9 Hz, 1H), 6.43 (m, J = 110.8, 17.0, 10.4 Hz, 1H), 6.03 – 5.94 (m, 1H), 5.68 (dd, J = 10.9, 4.2 Hz , 1H), 5.47 (t, J = 6.8 Hz, 1H), 5.32 (m, J = 12.2, 8.9, 4.0 Hz, 1H), 5.14– 5.09 (m, 1H), 5.09 – 5.01 (m, 3H), 5.00 – 4.90 (m, 2H), 4.81 (p, J = 6.9 Hz,1H), 4.72 (dd, J = 14.0, 1.8 Hz, 1H), 4.64 (t, J = 9.1 Hz, 1H), 4.50 (m, J = 7.4,4.7 Hz, 1H), 3.51 (d, J = 2.3 Hz, 3H), 3.39 (d, J = 3.7 Hz, 3H), 3.25 – 3.19 (m,5H), 3.10 (d, J = 2.8 Hz, 5H), 2.72 – 2.66 (m, 6H), 2.41 (m, J = 9.5, 6.5 Hz,1H), 2.17 – 1.96 (m, 6H), 1.84 – 1.66 (m, 8H), 1.61 (m, J = 11.9, 4.9 Hz, 2H ),1.34 (dd, J = 7.3, 2.2 Hz, 4H), 1.26 – 1.23 (m, 4H), 1.18 (s, 4H), 1.06 (dd, J =6.5, 2.0 Hz, 3H), 1.03 – 0.97 ( m, 9H), 0.94 (t, J = 6.1 Hz, 8H), 0.88 – 0.78(m, 19H), 0.73 (dd, J = 18.3, 6.6 Hz, 3H).

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A method for preparing cyclosporine, characterized in that the preparation route is: ; The preparation method specifically comprises the following steps: (1) Compound M0 is reacted with an acetylation agent under alkaline conditions to obtain compound M1; (2) subjecting the compound M1 obtained in step (1) to an oxidation reaction with an oxidant to obtain a compound M2; (3) subjecting the compound M2 obtained in step (2) to an oxidation reaction with an oxidant 2 to obtain a compound M3; (4) reacting the compound M3 obtained in step (3) with a Horner-Wittig reagent under alkaline conditions to obtain compound 1a; (5) subjecting the compound 1a obtained in step (4) to a selective reduction reaction with a reducing agent to obtain compound 1aa; (6) subjecting the compound 1aa obtained in step (5) to a dehydrating reaction with a dehydrating agent to obtain a compound M4; (7) Deacetylation of the compound M4 obtained in step (6) under alkaline conditions to obtain compound M5, i.e., cyclosporine. 2. The method for preparing cyclosporine according to claim 1, characterized in that in step (1), the alkali solution is pyridine, and the acetylation agent is acetic anhydride; and the molar ratio of the compound M0, acetic anhydride and pyridine is 1:10:15. 3. The method for preparing cyclosporine according to claim 1, characterized in that in step (2), the oxidant 1 is potassium osmate dihydrate and N-methylmorpholine oxide; and the molar ratio of the compound M1, potassium osmate dihydrate and N-methylmorpholine oxide is 1:0.03:2. 4. The method for preparing cyclosporine according to claim 1, characterized in that, in step (3), the second oxidant is sodium periodate; and the molar ratio of the compound M2 to the second oxidant is 1:2. 5. The method for preparing cyclosporine according to claim 1, characterized in that in step (4), the alkali solution is potassium carbonate, the Horner-Wittig reagent is diethyl acetonyl phosphate, and the molar ratio of the compound M3, diethyl acetonyl phosphate and potassium carbonate is 1:3:3. 6. The method for preparing cyclosporine according to claim 1, characterized in that in step (5), the reducing agent is sodium borohydride and cerium chloride heptahydrate; and the molar ratio of compound 1a, sodium borohydride and cerium chloride heptahydrate is 1:2:2. 7. The method for preparing cyclosporine according to claim 1, characterized in that in step (6), the dehydrating agent is p-toluenesulfonic acid monohydrate; and the molar ratio of the compound 1aa to p-toluenesulfonic acid monohydrate is 1:0.3. 8. The method for preparing cyclosporine according to claim 1, characterized in that in step (7), the alkali solution is potassium carbonate; and the molar ratio of the compound M4 to potassium carbonate is 1:8.