Method for purifying brassinolide

Abstract

The present application relates to a method for purifying brassinolide, comprising the following steps: (I) derivatization: reacting brassinolide and aryl boronic acid in a solvent, filtering and crystallizing to obtain a brassinolide aryl borate ester compound; (II) deprotection: oxidizing the brassinolide aryl borate ester compound by hydrogen peroxide under alkaline conditions to remove aryl boronic acid, so that the brassinolide aryl borate ester compound becomes free brassinolide; (III) recrystallization: crystallizing the free brassinolide after deprotection using an ethanol-water mixed solvent to obtain purified brassinolide.

Description

Technical Field

[0001] This invention relates to a method for purifying brassinolide, and more specifically, to a method for removing unknown impurities from brassinolide. Background Technology

[0002] Brassinolide is a multifunctional plant growth regulator that is widely found in plant organs such as pollen, seeds, stems, and leaves. Compared with the five previously discovered plant growth regulators, namely auxin, gibberellic acid, cytokinin, abscisic acid, and ethephon, it has more diverse functions. It can not only promote growth but also promote flowering and fruit setting, increase fruit setting rate, promote fruit enlargement, and improve yield and quality. It requires a small amount of dosage but has a high effect, and is known as the sixth type of plant hormone.

[0003] Brassinolides are highly effective, broad-spectrum plant growth regulators. Currently, the main types registered for use in my country are 24-epibrassinolide, 24-epibrassinolide-triepibrassinolide (containing two isomers: 24-epibrassinolide and 22,23,24-epibrassinolide), 28-homobrassinolide, and 28-ephomobrassinolide, with the following structures:

[0004]

[0005] Currently, the purity of brassinolide obtained through artificial synthesis is low and difficult to meet the requirements, so it needs to be purified. Summary of the Invention

[0006] The inventors of this application discovered during the synthesis of brassinolide that the compound contained various unknown impurities in liquid chromatography, and these impurities could not be effectively removed by more than two crystallizations.

[0007] The present invention aims to provide a purification method for brassinolide, which can effectively remove the above-mentioned unknown impurities contained in brassinolide and obtain high-purity brassinolide.

[0008] Specifically, the present invention relates to:

[0009] (1) A method for purifying brassinolide, comprising the following steps:

[0010] (I) Derivatization: Brassinolide and arylboronic acid are dissolved in a solvent and reacted, then crystallized and filtered to obtain brassinolide arylboronic acid ester compound.

[0011] (II) Deprotection: The above brassinolide arylboronic acid ester compound is oxidized with hydrogen peroxide under alkaline conditions to remove arylboronic acid, so that the brassinolide arylboronic acid ester compound becomes free brassinolide.

[0012] (III) Recrystallization: The free brassinolide after deprotection is recrystallized using a solvent to obtain purified brassinolide.

[0013] (2) The purification method according to (1) above is characterized in that the brassinolide is one or a mixture of any two or more of 24-epibrassinolide, 22,23,24-triepibrassinolide, 24-epibrassinolide·triepibrassinolide, 28-homobrassinolide, and 28-epibhomobrassinolide, preferably 24-epibrassinolide, 22,23,24-triepibrassinolide or 24-epibrassinolide·triepibrassinolide.

[0014] (3) The purification method according to (2) above is characterized in that the arylboronic acid in step (I) is phenylboronic acid or naphthaleneboronic acid, preferably phenylboronic acid; more preferably, the amount of arylboronic acid added in step (I) is more than 2.0 molar equivalents of brassinolide, preferably 2.2-2.6 molar equivalents.

[0015] (4) The purification method according to any one of (1) to (3) above is characterized in that the solvent in step (I) is methanol, ethanol or tert-butanol, preferably methanol.

[0016] (5) The purification method according to any one of (1) to (4) above is characterized in that the amount of solvent added in step (I) is 5-60 times the mass of brassinolide, preferably 10-20 times the mass.

[0017] (6) The purification method according to any one of (1) to (5) above is characterized in that the solvent for deprotection in step (II) is methanol, ethanol or ethylene glycol, preferably ethylene glycol.

[0018] (7) The purification method according to any one of (1) to (6) above is characterized in that the base to be deprotected in step (II) is an alkali metal carbonate or sodium hydroxide, preferably sodium hydroxide.

[0019] (8) The purification method according to (7) above is characterized in that the mass concentration of the alkali is 1%-20%, preferably 5%.

[0020] (9) The purification method according to any one of (1) to (8) above is characterized in that the mixed solvent of recrystallization of ethanol and water in step (III) preferably has a mass ratio of ethanol to water of about 1:1.4.

[0021] (10) The purification method according to any one of (1) to (9) above is characterized in that the purity of the unknown impurities in the purified brassinolide is less than 0.1%. Detailed Implementation

[0022] The method for purifying brassinolide of the present invention includes derivatization, deprotection and crystallization steps.

[0023] The derivatization step (I) involves reacting brassinolide with arylboronic acid to generate an ester compound.

[0024] In some specific embodiments, arylboronic acids include, but are not limited to, phenylboronic acid or naphthaleneboronic acid, with phenylboronic acid being preferred.

[0025] In some specific embodiments, brassinolide is 22,23,24-triepiraminlide, arylboronic acid is phenylboronic acid, and the esterification reaction of the derivatization step is shown below:

[0026]

[0027] In other specific embodiments, the brassinolide is 24-epibrassinolide, the arylboronic acid is phenylboronic acid, and the esterification reaction of the derivatization step is shown below:

[0028]

[0029] In a more specific embodiment, the above reaction is performed as follows, but not limited to: Phenylated acid is added to a reaction flask, dissolved completely in methanol, and then 22,23,24-triepiramin lactone or 24-epiramin lactone is added. The mixture is heated and stirred. After the reaction is completed by monitoring in an evaporative photoreactor, the mixture is cooled and filtered to obtain 22,23,24-triepiramin lactone phenylboronic acid ester or 24-epiramin lactone phenylboronic acid ester compound.

[0030] In some more specific embodiments, in the derivatization step, the amount of arylboronic acid added relative to brassinolide is 2 molar equivalents or more, preferably 2.2-2.6 molar equivalents, to ensure that brassinolide can be completely esterified.

[0031] In some more specific embodiments, methanol, ethanol, or tert-butanol are used as solvents in the derivatization step, with methanol being preferred. The amount of methanol added is 5-60 times by weight relative to brassinolide, preferably 20 times by weight.

[0032] Currently, boric acid derivatization is used in the detection and analysis of brassinolide, but there is no report on the method of first esterifying and then deprotecting the brassinolide by deesterification to remove impurities, especially impurities with a retention time of 7.9 min.

[0033] Step (II) is the deprotection step, which involves removing the arylboronic acid from the brassinolide arylboronic acid ester compound obtained above to generate free brassinolide.

[0034] In some specific embodiments, brassinolide is 22,23,24-triepiraminlide, arylboronic acid is phenylboronic acid, and the deprotection is carried out by the following reaction:

[0035]

[0036] In other specific embodiments, brassinolide is 24-epibrassinolide, the arylboronic acid is phenylboronic acid, and the deprotection is performed by the following reaction:

[0037]

[0038] In a more specific embodiment, the above reaction is performed as follows, but not limited to: A solvent and a base are added to the above brassinolide phenylboronic acid ester compound, the mixture is stirred and heated, hydrogen peroxide is slowly added dropwise, and after the reaction of the raw materials is completed, water is added dropwise, and the mixture is cooled and filtered.

[0039] In some more specific embodiments, methanol, ethanol, or ethylene glycol is used as a solvent in the deprotection reaction. When ethylene glycol is used as a solvent, the conversion rate of the reaction intermediate is higher than that of methanol and ethanol, reaching more than 90%. Therefore, ethylene glycol is preferred as a solvent.

[0040] In some more specific embodiments, an alkali metal carbonate or sodium hydroxide is used as the base in the deprotection reaction. Compared to alkali metal carbonates, sodium hydroxide requires less solvent and produces a better reaction, making it preferred. Too high a concentration of sodium hydroxide can lead to violent decomposition of hydrogen peroxide, posing a safety hazard, while too low a concentration will reduce reaction efficiency. Therefore, an aqueous solution of sodium hydroxide with a mass concentration of 1%-20%, more preferably 5%, is preferred.

[0041] In some more specific embodiments, the crystallization step (III) uses an ethanol-water mixture as the crystallization solvent. If the proportion of ethanol in the mixed solvent is too high, the crystallization yield will decrease; if the proportion is too low, impurities may be unacceptable, affecting product quality. Preferably, the mass ratio of ethanol to water is 1:1 to 1:2, more preferably about 1:1.4.

[0042] The purity of unknown impurities in 22,23,24-epibrassinolide and 24-epibrassinolide obtained after purification through the above three steps is less than 0.1%.

[0043] Specific examples of brassinolide include, but are not limited to, 24-epibrassinolide, 22,23,24-triepibrassinolide, 24-epibrassinolide·triepibrassinolide, 28-homobrassinolide or 28-epibrassinolide, or any mixture of two or more thereof, preferably 24-epibrassinolide, 22,23,24-triepibrassinolide, 24-epibrassinolide·triepibrassinolide or mixtures thereof.

[0044] Since 24-epibrina·triepiramin lactone contains two components, 24-epibrina lactone and 22,23,24-epibrina lactone, with the mass fraction of 22,23,24-epibrina lactone ≥60% and the mass fraction of 24-epibrina lactone ≥30%, after purifying the two components separately according to the above method, they are then mixed according to their respective mass fractions to obtain purified 24-epibrina·triepiramin lactone. The purity of unknown impurities in the purified 24-epibrina·triepiramin lactone is less than 0.1%.

[0045] Example

[0046] The preferred embodiments of the method of the present invention will be described below with reference to specific examples. However, the present invention is not limited to these embodiments. Any modifications and changes that do not depart from the spirit and scope of the present invention shall fall within the scope of the present invention.

[0047] Example 1: Purification of 22,23,24-triepasisinolide

[0048] (1) Derivatization reaction

[0049]

[0050] 15.5 g of phenylboronic acid and 520 g of methanol were added to a reaction flask at room temperature and stirred until completely dissolved. Then, 26.5 g of 22,23,24-triepasisinolide was added, and the mixture was heated to 50-55 °C and stirred for 1 h. The reaction was monitored using an evaporative light detector. After the 22,23,24-triepasisinolide had reacted completely, the mixture was cooled to 10-15 °C, filtered, and the solid was 22,23,24-triepasisinolide phenylboronic acid ester.

[0051] Evaporative light detector conditions: Kromasil 100-5C18 column, mobile phase acetonitrile:water (pH adjusted to 3.5-4.0 with formic acid) = 65:35 (V:V), column temperature 30℃, detector temperature 100℃, flow rate 1.0 mL / min, gas flow rate 2.2 mL / min, splitless mode.

[0052] The detection results of the above derivatization of three different batches of raw material 22,23,24-triepasisinolide are shown in Table 1. Although the purity of the derivatized 22,23,24-triepasisinolide phenylboronic acid derivatives varied due to the different batches of raw materials, the purity of unknown impurities with retention times of 3.5-3.9 min and 7.9 min after derivatization was less than 0.1%.

[0053] Table 1

[0054]

[0055] (Note: Impurities with a retention time of 9.0 min are known impurities, and those with a retention time of 9.9 min are 22,23,24-triepiramin lactone.)

[0056] The component with a retention time of 12.7 min was 24-epibrassinolide; the rest were unknown impurities.

[0057] (2) Deprotection reaction

[0058]

[0059] 24.4 g of the above-mentioned 22,23,24-triepasisinolide phenylboronic acid ester was added to a reaction flask, followed by 500 g of ethylene glycol and 120 g of 5% NaOH aqueous solution. After stirring at 20-25°C for 1 h, the temperature was raised to 30-40°C, 2 drops of defoamer were added, and then 61.2 g of 25% H2O2 was slowly added dropwise. The reaction of the raw materials was monitored by high performance liquid chromatography until it was complete. After the intermediate boronic acid ester was monitored by evaporation light detector and no longer changed, 590 g of water was added dropwise, and the temperature was lowered to 20°C for filtration. The solid was 22,23,24-triepasisinolide.

[0060] High performance liquid chromatography (HPLC) conditions: C18 column, mobile phase acetonitrile:water (adjusted to pH 3.7–4.1 with formic acid) = 80:20 (V:V), column temperature 30℃, flow rate 1.5 mL / min, detection wavelength 222 nm.

[0061] Evaporative light detector conditions: Kromasil 100-5C18 column, mobile phase acetonitrile:water (pH adjusted to 3.5-4.0 with formic acid) = 65:35 (V:V), column temperature 30℃, detector temperature 100℃, flow rate 1.0 mL / min, gas flow rate 2.2 mL / min, splitless mode.

[0062] (3) recrystallization

[0063] Dissolve 5g of the deprotected wet product from step (II) in 40g of ethanol, heat to reflux until the solid is completely dissolved, slowly add 56g of water and introduce seed crystals, stir, slowly cool to 35-40℃, filter, and the solid is 22,23,24-triepiramin lactone.

[0064] The purity was determined and calculated using high-performance liquid chromatography (HPLC), and the results are shown in Table 2. Table 2 shows that the purity of the unknown impurities obtained by recrystallization at retention times of 4.1–5.0 min and 11.1 min was less than 0.1%.

[0065] Table 2

[0066]

[0067] (Note: Impurities with a retention time of 9.0 min are known impurities, and those with a retention time of 9.9 min are 22,23,24-triepiramin lactone.)

[0068] The substance with a retention time of 12.7 min is 24-triepirocin lactone; the rest are unknown impurities.

[0069] Preparation of the derivatization solution:

[0070] (1) Phenylboronic acid methanol solution: Weigh 100 mg of phenylboronic acid into a 50 mL volumetric flask, dilute to volume with methanol, and sonicate until completely dissolved.

[0071] (2) Preparation of the reference solution: Accurately weigh 30-40 mg of the reference standard using weighing paper and place it in a 50 mL volumetric flask. Add 10 mL of pure methanol and 20 mL of phenylboronic acid methanol solution, sonicate until completely dissolved, derivatize at 50 °C for 2 h, cool to room temperature, and dilute to volume with methanol. Detect by liquid chromatography. Perform two parallel determinations and calculate the RSD between parallel response values ​​to be ≤0.5%.

[0072] (3) Sample solution preparation: Accurately weigh 30-40 mg of sample using weighing paper and place it in a 50 mL volumetric flask. Add 10 mL of pure methanol and 20 mL of phenylboronic acid methanol solution, sonicate until completely dissolved, derivatize at 50 °C for 2 h, cool to room temperature, and then dilute to volume with methanol. Detect by liquid chromatography. Perform two parallel determinations and calculate the RSD between parallel response values ​​to be ≤0.5%.

[0073] High performance liquid chromatography (HPLC) conditions: C18 column, mobile phase acetonitrile:water (adjusted to pH 3.7–4.1 with formic acid) = 80:20 (V:V), column temperature 30℃, flow rate 1.5 mL / min, detection wavelength 222 nm.

[0074] Example 2 Purification of 24-epibrassinolide

[0075] (1) Derivatization reaction

[0076]

[0077] 5.59 g of phenylboronic acid and 240 g of methanol were added to a reaction flask at room temperature and stirred until completely dissolved. Then, 12 g of 24-epibrassinolide was added, and the mixture was heated to 50-55 °C and stirred for 1 h. The reaction was monitored using an evaporative light detector. After the 24-epibrassinolide had reacted completely, the mixture was cooled to 0-5 °C, filtered, and the solid was 24-epibrassinolide phenylboronic acid ester.

[0078] Evaporative light detector conditions: Kromasil 100-5C18 column, mobile phase acetonitrile:water (pH adjusted to 3.5-4.0 with formic acid) = 65:35 (V:V), column temperature 30℃, detector temperature 100℃, flow rate 1.0 mL / min, gas flow rate 2.2 mL / min, splitless mode.

[0079] (2) Deprotection reaction

[0080]

[0081] 24.4 g of the derivatized 24-epibrassinolide phenylboronic acid ester was added to a reaction flask, followed by 500 g of ethylene glycol and 120 g of 5% NaOH aqueous solution. The mixture was stirred at 20-25°C for 1 h, then heated to 30-40°C. Two drops of defoamer were added, and 61.2 g of 25% H₂O₂ was slowly added dropwise. The reaction was monitored by high-performance liquid chromatography (HPLC) to ensure complete reaction, and the purity of the intermediate boronic acid ester was monitored by an evaporative light detector to ensure it was less than 1.5%. After the reaction was complete, 590 g of water was added dropwise, and the mixture was cooled to 20°C and filtered. The solid was 24-epibrassinolide.

[0082] High performance liquid chromatography (HPLC) conditions: C18 column, mobile phase acetonitrile:water (pH adjusted to 3.7–4.1 with formic acid) = 80:20 (V:V), column temperature 30℃, flow rate 1.5 ml / min, detection wavelength 222 nm.

[0083] Evaporative light detector conditions: Kromasil 100-5C18 column, mobile phase acetonitrile:water (pH adjusted to 3.5-4.0 with formic acid) = 65:35 (V:V), column temperature 30℃, detector temperature 100℃, flow rate 1.0 mL / min, gas flow rate 2.2 mL / min, splitless mode.

[0084] (3) recrystallization

[0085] Dissolve 5g of the deprotected wet product from step (II) in 40g of ethanol, heat to reflux until the solid is completely dissolved, slowly add 56g of water and introduce seed crystals, stir, slowly cool to 35-40℃, filter, and the solid is 24-epibrassinolide.

[0086] The purity was determined and calculated using high-performance liquid chromatography (HPLC) derivatization (the derivatization process was the same as in Example 1), and the results are shown in Table 3. Table 3 shows that, through derivatization-deprotection-recrystallization, the purity of the unknown impurities at retention times of 4.6 min, 7.9 min, and 8.7 min was less than 0.1%.

[0087] Table 3

[0088]

[0089] (Note: The retention time of 9.9 min is for 22,23,24-triepiramin lactone, and the retention time of 11.6 min is for 24-...)

[0090] Epibrassinolide isomers; the one with a retention time of 12.7 min is 24-epibrassinolide, and the rest are unknown impurities.

[0091] High performance liquid chromatography (HPLC) conditions: C18 column, mobile phase acetonitrile:water (adjusted to pH 3.7–4.1 with formic acid) = 80:20 (V:V), column temperature 30℃, flow rate 1.5 mL / min, detection wavelength 222 nm.

[0092] Comparative Example 1: Purification of crude triepasisinolide by direct crystallization

[0093] (1) Preparation of crude 22,23,24-triepasisinolide

[0094] Add 6.1 mL of 30% H₂O₂ and 50 mL of CH₂Cl₂ to the reaction flask, cool to 0 °C, and add 38.1 mL of trifluoroacetic anhydride dropwise below 0 °C. After the addition is complete, stir for 10 min. Dissolve 2.5 g of 22,23,24-triepiramin lactone intermediate (a mixture of (22S,23S,24R)-2a,3a,22,23-tetrahydroxy-5a-ergosterane-6-one and (22R,23R,24R)-2a,3a,22,23-tetrahydroxy-5a-ergosterane-6-one in a mass ratio of 2:1) in 150 mL of CH₂Cl₂ and add it to the above reaction flask. Stir at room temperature for 2-6 h, add 25 mL of water and continue stirring for 10 min, then let stand and separate the layers. The organic phase was washed twice with 25 mL of saturated Na2CO3 and then twice with 25 mL of saturated NaHSO3. The solvent was removed by rotary evaporation of the organic phase and crystallized with ethyl acetate to obtain 2.07 g of crude 22,23,24-triepiramin lactone, which was dried at 70-75 °C.

[0095] (2) First recrystallization

[0096] 110g of crude 22,23,24-triepasisinolide obtained by the above method was dissolved in 660g of methanol. The mixture was heated to reflux until the crude product was completely dissolved, stirred for 30min, cooled to 20-25℃, and filtered. The solid was the first recrystallized 22,23,24-triepasisinolide.

[0097] (3) Second recrystallization

[0098] Add 4g of the first recrystallized 22,23,24-triepasisinolide to a reaction flask, add 116g of ethyl acetate, heat to reflux until completely dissolved, stir for 30min, cool to 20-25℃, filter, and the solid is the second recrystallized 22,23,24-triepasisinolide.

[0099] When purifying crude 22,23,24-triepastragalin lactone by direct crystallization, methanol was used for the first recrystallization. Then, different solvents (such as ethyl acetate, ethanol, acetonitrile, methanol, ethanol / ethyl acetate, and ethanol / water) and different solvent ratios (such as ethanol and ethanol / water) were used for the second recrystallization. The purity of the crystallized product was calculated by liquid phase derivatization detection (the derivatization detection process is the same as in Example 1). The results are shown in Table 4.

[0100] Secondary recrystallization can effectively remove known impurities with a retention time of 9.0 min, but unknown impurities with retention times of 3.9 min, 4.1 min, 4.7 min and 7.9 min have no purification effect after secondary recrystallization.

[0101] Table 4

[0102]

[0103] (Note: Impurities with a retention time of 9.0 min are known impurities, and those with a retention time of 9.9 min are 22,23,24-triepiramin lactone.)

[0104] The component with a retention time of 12.7 min was 24-epibrassinolide; the rest were unknown impurities.

[0105] High performance liquid chromatography (HPLC) conditions: C18 column, mobile phase acetonitrile:water (adjusted to pH 3.7–4.1 with formic acid) = 80:20 (V:V), column temperature 30℃, flow rate 1.5 mL / min, detection wavelength 222 nm.

[0106] The purification method of the present invention is also applicable to the purification of other brassinolide compounds such as 28-homocysinolide and 28-epiohomocysinolide.

Claims

1. A method for purifying brassinolide, characterized in that, Includes the following steps: (I) Derivatization: Brassinolide and arylboronic acid are dissolved in a solvent and reacted, then crystallized and filtered to obtain brassinolide arylboronic acid ester compound. (II) Deprotection: The above brassinolide arylboronic acid ester compound is oxidized with hydrogen peroxide under alkaline conditions to remove arylboronic acid, so that the brassinolide arylboronic acid ester compound becomes free brassinolide. (III) Recrystallization: The free brassinolide after deprotection is recrystallized using a solvent to obtain purified brassinolide.

2. The purification method according to claim 1, characterized in that, The brassinolide is one or a mixture of any two or more of the following: 24-epibrassinolide, 22,23,24-triepiraminide, 24-epibrassinolide·triepiraminide, 28-homopiraminide, and 28-epiraminhobrassinolide.

3. The purification method according to claim 2, characterized in that, The brassinolide is 24-epibrassinolide, 22,23,24-triepibrassinolide, or 24-epibrassinolide·triepibrassinolide.

4. The purification method according to any one of claims 1 to 3, characterized in that, In step (I), the arylboronic acid is phenylboronic acid or naphthaleneboronic acid; the amount of arylboronic acid added in step (I) is more than 2.0 molar equivalents of brassinolide.

5. The purification method according to claim 4, characterized in that, In step (I), the arylboronic acid is phenylboronic acid.

6. The purification method according to claim 4, characterized in that, In step (I), the amount of arylboronic acid added is 2.2-2.6 molar equivalents of brassinolide.

7. The purification method according to any one of claims 1 to 3, characterized in that, In step (I), the solvent is methanol, ethanol, or tert-butanol.

8. The purification method according to claim 7, characterized in that, In step (I), the solvent is methanol.

9. The purification method according to any one of claims 1 to 3, characterized in that, In step (I), the amount of solvent added is 5-60 times the mass of brassinolide.

10. The purification method according to claim 9, characterized in that, In step (I), the amount of solvent added is 10-20 times the mass of brassinolide.

11. The purification method according to any one of claims 1 to 3, characterized in that, In step (II), the solvent used for deprotection is methanol, ethanol, or ethylene glycol.

12. The purification method according to claim 11, characterized in that, In step (II), the solvent used for deprotection is ethylene glycol.

13. The purification method according to any one of claims 1 to 3, characterized in that, In step (II), the base used for deprotection is an alkali metal carbonate or sodium hydroxide.

14. The purification method according to claim 13, characterized in that, In step (II), the base used for deprotection is sodium hydroxide.

15. The purification method according to claim 13, characterized in that, The mass concentration of the alkali is 1%-20%.

16. The purification method according to claim 15, characterized in that, The mass concentration of the alkali is 5%.

17. The purification method according to any one of claims 1 to 3, characterized in that, In step (III), the recrystallization solvent is a mixture of ethanol and water, with a mass ratio of ethanol to water of 1:1 to 1:

2.

18. The purification method according to claim 17, characterized in that, In step (III), the recrystallization solvent is a mixture of ethanol and water, with a mass ratio of ethanol to water of 1:1.

4.

19. The purification method according to any one of claims 1 to 3, characterized in that, The purity of unknown impurities in the purified brassinolide was less than 0.1%.

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