Preparation method of Boc-Cys (S-Py)-OH

Abstract

The invention relates to a preparation method of Boc-Cys (S-Py)-OH, and belongs to the technical field of drug intermediate synthesis. Boc-Cys (S-Py)-OH is a key material required for synthesizing the entecatide hydrochloride, and in the prior art, the problems of few synthesis methods, high raw material price, many byproducts, low purity and the like exist, cysteine hydrochloride and dithiopyridine are mixed and stirred in an organic solvent to react to obtain an intermediate, the intermediate is subjected to t-butyloxycarboryl protection to obtain a crude product, and the crude product is subjected to post-treatment to obtain the entecatide hydrochloride. And recrystallizing the crude product by using a mixed solvent of alcohols and water, and drying to obtain the product Boc-Cys (S-Py)-OH white solid. According to the method provided by the invention, cheap and easily available cysteine hydrochloride is taken as a raw material, Boc-Cys (S-Py)-OH is obtained through two-step reaction, the route is simple, the product purity reaches 99% or above, the yield reaches 85% or above, and the method is suitable for industrial large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical intermediate synthesis, in particular to a preparation method of Boc-Cys(S-Py)-OH. Background Art

[0002] Parsabiv (etelcalcetide) is a calcium-sensitive receptor agonist used to treat secondary hyperparathyroidism (SHPT) in adults with chronic kidney disease after dialysis. It helps control the secretion of parathyroid hormone by regulating calcium ion metabolism in the body, thereby producing a positive therapeutic effect. Its structural formula is shown below:

[0003]

[0004] In the process of synthesizing etelkatide hydrochloride, Boc-Cys(S-Py)-OH (CAS 117310-07-3) is a key material. In the currently reported synthesis methods of Boc-Cys(S-Py)-OH, the main impurities are excess dithiodipyridine and by-product 2-mercaptopyridine. If dithiodipyridine remains in the product, it will participate in the reaction in the subsequent solid phase synthesis, thereby producing by-products, affecting the yield of the reaction and the purity of the product. Some methods use acetic acid as a reaction catalyst, which increases the cost and also affects the subsequent synthesis reaction. In the quality standard of the final product, the residual acetic acid content in the solvent needs to be detected. Therefore, how to improve the purity of Boc-Cys(S-Py)-OH in the process of synthesizing it and simplify the synthesis steps has become an urgent problem to be solved.

[0005] There are three main methods for preparing Boc-Cys(S-Py)-OH in the prior art:

[0006] (1) Using (Boc-Cys-OH)2 as a raw material, a reduction reaction is performed to obtain Boc-Cys-OH, which is then reacted with dithiodipyridine to generate a product; the post-treatment of this method adopts a column purification method, which is not suitable for scale-up and industrial production, and the raw material Boc-Cys-OH is rarely available on the market and is relatively expensive; the product is obtained by a reduction reaction using triphenylphosphine, ZrCl4 / NaBH4, etc., and the reaction is complicated and costly;

[0007]

[0008] (2) Using Boc-Cys-OR as a raw material, the product is obtained through a two-step reaction; the raw material Boc-Cys-OR is also relatively expensive, and the intermediate Boc-Cys(S-Py)-OR obtained by the reaction is an oily substance that needs to be purified by a column and is not suitable for industrial production and expansion;

[0009]

[0010] (3) The preparation of raw materials is relatively complicated and is not commercially available, which is also not suitable for industrial production scale-up.

[0011] Summary of the invention

[0012] In order to solve the above technical problems, the present invention provides a method for preparing Boc-Cys(S-Py)-OH. Cysteine ​​hydrochloride is used as a raw material, which is mixed with dithiodipyridine in an organic solvent to obtain an intermediate. The intermediate reacts with di-tert-butyl dicarbonate in the presence of a solvent so that the intermediate is protected by a tert-butyloxycarbonyl group to obtain a crude Boc-Cys(S-Py)-OH product. Finally, the crude product is recrystallized using a mixed solvent of alcohol and water to obtain a finished Boc-Cys(S-Py)-OH product which is a white solid.

[0013] The main purpose of the present invention is to provide a method for preparing Boc-Cys(S-Py)-OH, comprising the following steps:

[0014] Step S1, mixing cysteine ​​hydrochloride and dithiodipyridine in an organic solvent, and concentrating to remove the organic solvent after the reaction is completed to obtain an intermediate;

[0015] Step S2, reacting the intermediate with di-tert-butyl dicarbonate in the presence of a solvent to obtain an intermediate protected by tert-butyloxycarbonyl, i.e., a crude product of Boc-Cys(S-Py)-OH;

[0016] Step S3, recrystallizing the crude Boc-Cys(S-Py)-OH in a mixed solvent of alcohol and water to obtain the Boc-Cys(S-Py)-OH.

[0017] The reaction process involved in the present invention is as follows:

[0018]

[0019] Furthermore, in step S1, the reaction temperature is 0-30°C.

[0020] Preferably, the reaction temperature is 20-30°C.

[0021] Furthermore, step S1 also includes a step of pulping and purifying the intermediate, specifically adding ethers for suction filtration.

[0022] The pulping purification operation is simple and is more suitable for large-scale production compared with purification methods such as column purification involved in the prior art.

[0023] Furthermore, the ether is tertiary methyl ether or isopropyl ether.

[0024] Preferably, the ether is tertiary methyl ether.

[0025] Furthermore, in step S1, the molar ratio of the cysteine ​​hydrochloride to the disulfide dipyridine is 1:(1.0-2.0).

[0026] Furthermore, in step S2, the molar ratio of the di-tert-butyl dicarbonate to the intermediate is 1:(1.0-2.0).

[0027] Furthermore, in step S2, the solvent is tetrahydrofuran or dioxane.

[0028] Furthermore, in step S2, after the reaction is completed, hydrochloric acid is used to adjust the pH to an acidic environment.

[0029] Furthermore, the pH was adjusted to 2-3 using hydrochloric acid.

[0030] Furthermore, in step S3, the alcohol is methanol, ethanol or isopropanol.

[0031] Preferably, the alcohol is ethanol.

[0032] Furthermore, in step S3, the ratio of the alcohol to water is 80-95%.

[0033] Preferably, the ratio of the alcohol to water is 95%.

[0034] The type of alcohol reagent and the mixing ratio of alcohol and water affect the purity of the finished product.

[0035] Furthermore, in step S3, the recrystallization step is specifically as follows: heating to 70-80°C to dissolve, then cooling to 10-15°C, and continuously stirring at this temperature.

[0036] In one embodiment of the present invention, the stirring time is 2 hours.

[0037] Furthermore, after the stirring is completed, the product is filtered and washed, and dried under reduced pressure at 40-45° C. to obtain the finished product of Boc-Cys(S-Py)-OH.

[0038] Furthermore, the washing is performed twice, using a mixture of ethanol and water, and n-heptane respectively.

[0039] Furthermore, the Boc-Cys(S-Py)-OH finished product is a white solid.

[0040] Beneficial effects of the present invention:

[0041] The method provided by the invention uses cheap and easily available cysteine ​​hydrochloride as a raw material, obtains Boc-Cys(S-Py)-OH through a two-step reaction, has a simple route and simple operation, purifies the intermediate product in a pulping manner, does not require column passing, saves tedious operation process, and has a yield of more than 85%. A mixed solvent of alcohol and water is used to recrystallize the crude product, and the product purity reaches more than 99%, avoids the generation of excessive by-products in the reaction process that will affect subsequent solid-phase synthesis, and is suitable for industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0043] Figure 1 This is a graph showing the HPLC results of the finished product Boc-Cys(S-Py)-OH of Example 1 of the present invention;

[0044] Figure 2 The finished product Boc-Cys(S-Py)-OH of Example 1 of the present invention 1 H NMR result diagram;

[0045] Figure 3 This is a graph showing the HPLC results of the finished product Boc-Cys(S-Py)-OH of Example 3 of the present invention;

[0046] Figure 4 This is a graph showing the HPLC results of the finished product Boc-Cys(S-Py)-OH of Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0048] Example 1

[0049] (1) Add cysteine ​​hydrochloride (88 g, 0.5 mol, 1.0 equiv), dithiodipyridine (133 g, 0.6 mol, 1.2 equiv) and methanol (880 mL) to a 1 L four-necked reaction bottle, stir and react at 20-30° C. for 2 hours, concentrate to remove methanol, add tertiary methyl ether (880 mL) and filter to obtain a filter cake, i.e., an intermediate, which can be used directly without drying;

[0050] (2) The filter cake was added to a 1L four-necked bottle, and an aqueous solution of dioxane and sodium bicarbonate was added. A dioxane solution of Boc2O (131 g, 0.6 mol, 1.2 equiv) was added dropwise. After the addition was complete, the mixture was stirred at 20-30°C for 2 hours. After the reaction was completed, the pH was adjusted to 2-3 with hydrochloric acid, and water was added. A large amount of solid precipitated. The mixture was stirred at 20-30°C for 1 hour, and the crude product of Boc-Cys(S-Py)-OH was obtained by suction filtration.

[0051] (3) The crude product was washed with water and directly added to a 1L four-necked bottle without drying. A mixed solvent of 95% ethanol and water was added. The mixture was heated to 70-80°C to dissolve. The mixture was cooled to 10-15°C and stirred for 2 hours at this temperature. The mixture was filtered and washed once with a mixture of ethanol and water and once with n-heptane. The mixture was dried under reduced pressure at 40-45°C for 12 hours to obtain Boc-Cys(S-Py)-OH as a white solid with a weight of 142 g and a yield of 86%.

[0052] The purity of the prepared Boc-Cys(S-Py)-OH was detected by HPLC. Figure 1 As shown, the peak area accounts for 99.78%, that is, the purity of the finished Boc-Cys(S-Py)-OH is 99.78%.

[0053] 1 H NMR results are as follows Figure 2 As shown, 1 H NMR (d6-DMSO, δ): 12.8 (brs, 1H), 8.47 (t, 1H, d = 4.0Hz), 7.83-7.75 (m, 2H), 7.39 (d, 1H ,d=8.0Hz),7.28(t,1H,d=4.0Hz),4.13-4.19(m,1H),3.06-3.20(m,2H),1.39(s,9H).

[0054] Example 2

[0055] (1) Add cysteine ​​hydrochloride (88 g, 0.5 mol, 1.0 equiv), dithiodipyridine (165 g, 0.75 mol, 1.5 equiv) and methanol (880 mL) to a 1 L four-necked reaction bottle, and stir at 20-30° C. for 2 hours; concentrate to remove methanol, add tertiary methyl ether (880 mL) and filter to obtain a filter cake, i.e., an intermediate, which is used directly without drying;

[0056] (2) The filter cake was added to a 1L four-necked bottle, and dioxane and sodium bicarbonate aqueous solution were added. Boc2O (131 g, 0.6 mol, 1.2 equiv) in dioxane was added dropwise. After the addition was complete, the mixture was stirred at 20-30°C for 2 hours. After the reaction was completed, the pH was adjusted to 2-3 with hydrochloric acid, and water was added. A large amount of solid precipitated. The mixture was stirred at 20-30°C for 1 hour, and the crude product of Boc-Cys(S-Py)-OH was obtained by suction filtration.

[0057] (3) The crude product was washed with water and directly added to a 1L four-necked bottle without drying. A mixed solvent of 95% ethanol and water was added. The mixture was heated to 70-80°C to dissolve. The mixture was cooled to 10-15°C and stirred for 2 hours at this temperature. The mixture was filtered and washed once with a mixture of ethanol and water and once with n-heptane. The mixture was dried under reduced pressure at 40-45°C for 12 hours to obtain Boc-Cys(S-Py)-OH as a white solid. The weight was 145 g and the yield was 87.8%.

[0058] The purity of the prepared Boc-Cys(S-Py)-OH was detected by HPLC, and the peak area accounted for 99.25%, that is, the purity of Boc-Cys(S-Py)-OH was 99.25%.

[0059] Example 3

[0060] (1) Add cysteine ​​hydrochloride (88 g, 0.5 mol, 1.0 equiv), dithiodipyridine (165 g, 0.75 mol, 1.5 equiv) and methanol (880 mL) to a 1 L four-necked reaction bottle, and stir at 20-30° C. for 2 hours; concentrate to remove methanol, add isopropyl ether (880 mL) and filter to obtain a filter cake, i.e., an intermediate, which is used directly without drying;

[0061] (2) The filter cake was added to a 1L four-necked bottle, tetrahydrofuran and an aqueous solution of sodium bicarbonate were added, and a tetrahydrofuran solution of Boc2O (131 g, 0.6 mol, 1.2 equiv) was added dropwise. After the addition was complete, the mixture was stirred at 20-30°C for 2 hours. After the reaction was completed, most of the tetrahydrofuran was removed by concentration, and the pH was adjusted to 2-3 with hydrochloric acid. Water was added, and a large amount of solid precipitated. The mixture was stirred at 20-30°C for 1 hour, and the crude product of Boc-Cys(S-Py)-OH was obtained by suction filtration.

[0062] (3) Wash with water. Without drying, add directly to a 1L four-necked bottle, add a mixed solvent of 95% ethanol and water, raise the temperature to 70-80°C to dissolve, cool to 10-15°C, stir at this temperature for 2 hours, filter, wash once with a mixture of ethanol and water and once with n-heptane, and dry under reduced pressure at 40-45°C for 12 hours to obtain Boc-Cys(S-Py)-OH as a white solid, weighing 140 g, with a yield of 85%.

[0063] The purity of the prepared Boc-Cys(S-Py)-OH was detected by HPLC. Figure 3 As shown, the peak area accounts for 99.70%, that is, the purity of the finished Boc-Cys(S-Py)-OH is 99.7%.

[0064] Example 4

[0065] The preparation steps are the same as those in Example 1, except that in step (3), a mixed solvent of 80% ethanol and water is used to recrystallize the crude Boc-Cys(S-Py)-OH, and the purity of the obtained Boc-Cys(S-Py)-OH finished product is 98.1%.

[0066] Example 5

[0067] The preparation steps are the same as those in Example 1, except that in step (3), a mixed solvent of 90% methanol and water is used to recrystallize the crude Boc-Cys(S-Py)-OH, and the purity of the obtained Boc-Cys(S-Py)-OH finished product is 98.2%.

[0068] Comparative Example 1

[0069] (1) In a 1L four-necked reaction flask, cysteine ​​hydrochloride (88 g, 0.5 mol, 1.0 equiv), dithiodipyridine (218 g, 1.0 mol, 2.0 equiv), methanol (880 mL) and acetic acid (35 mL) were added and reacted at 20-30°C with stirring for 24 hours; methanol was removed by concentration, and ether (880 mL) was added and filtered to obtain a filter cake, i.e., an intermediate, which was used directly without drying;

[0070] (2) The filter cake was added to a 1L four-necked bottle, tetrahydrofuran and an aqueous solution of sodium bicarbonate were added, and a tetrahydrofuran solution of Boc2O (131 g, 0.6 mol, 1.2 equiv) was added dropwise. After the addition was complete, the mixture was stirred at 20-30°C for 2 hours. After the reaction was completed, most of the tetrahydrofuran was removed by concentration, and the pH was adjusted to 2-3 with hydrochloric acid. Water was added, and a large amount of solid precipitated. The mixture was stirred at 20-30°C for 1 hour, and the crude product of Boc-Cys(S-Py)-OH was obtained by suction filtration.

[0071] (3) The crude product was washed with water and directly added to a 1L four-necked bottle with a mixed solvent of 95% ethanol and water without drying. The mixture was heated to 70-80°C to dissolve, cooled to 10-15°C, stirred at this temperature for 2 hours, filtered, washed once with a mixture of ethanol and water and once with n-heptane, and dried under reduced pressure at 40-45°C for 12 hours to obtain Boc-Cys(S-Py)-OH as a white solid, weighing 140 g, with a yield of 85%.

[0072] The purity of the prepared Boc-Cys(S-Py)-OH was detected by HPLC. Figure 4 As shown, the peak area accounts for 95.67%, that is, the purity of Boc-Cys(S-Py)-OH is 95.67%.

[0073] This method was reported in the literature "A New Heterobifunctional Cross-linking Reagent for the Study of Biological Interactions between Proteins" (1981). Acetic acid is used as a catalyst, which affects the subsequent synthesis reaction and may cause instability in the amount of sodium bicarbonate added. In addition, the residual acetic acid content in the solvent needs to be tested in the quality standard of the final product.

[0074] Comparative Example 2

[0075] The preparation steps are the same as those in Example 1, except that in step (3), a mixed solvent of 90% isopropanol and water is used to recrystallize the crude Boc-Cys(S-Py)-OH, and the purity of the obtained Boc-Cys(S-Py)-OH finished product is 91%.

[0076] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing Boc-Cys(S-Py)-OH, characterized in that: The following steps are involved: Step S1, mixing cysteine ​​hydrochloride and dithiodipyridine in an organic solvent, and concentrating to remove the organic solvent after the reaction is completed to obtain an intermediate; Step S2, reacting the intermediate with di-tert-butyl dicarbonate in the presence of a solvent to obtain an intermediate protected by tert-butyloxycarbonyl, i.e., a crude product of Boc-Cys(S-Py)-OH; Step S3, recrystallizing the crude Boc-Cys(S-Py)-OH in a mixed solvent of alcohol and water to obtain the Boc-Cys(S-Py)-OH.

2. The preparation method according to claim 1, characterized in that: In step S1, the reaction temperature is 0-30°C.

3. The preparation method according to claim 1, characterized in that: Step S1 also includes a step of pulping and purifying the intermediate, specifically adding ethers for suction filtration.

4. The preparation method according to claim 3, characterized in that: The ethers are tertiary methyl ether or isopropyl ether.

5. The preparation method according to claim 1, characterized in that: In step S1, the molar ratio of the cysteine ​​hydrochloride to the disulfide dipyridine is 1:(1.0-2.0).

6. The preparation method according to claim 1, characterized in that: In step S2, the molar ratio of the di-tert-butyl dicarbonate to the intermediate is 1:(1.0-2.0).

7. The preparation method according to claim 1, characterized in that: In step S2, the solvent is tetrahydrofuran or dioxane.

8. The preparation method according to claim 1, characterized in that: In step S3, the alcohol is methanol, ethanol or isopropanol.

9. The preparation method according to claim 1, characterized in that: In step S3, the ratio of the alcohol to water is 80-95%.

10. The preparation method according to claim 1, characterized in that: In step S3, the recrystallization step is specifically as follows: heating to 70-80°C to dissolve, then cooling to 10-15°C, and continuously stirring at this temperature.

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