Phorbol acetone co-crystal compounds, methods of making and methods of purifying the same

Abstract

This invention provides a phorbol acetone cocrystal compound, its preparation method, and a method for preparing a pure product, belonging to the field of industrial extraction technology of phorbol. In the phorbol acetone cocrystal compound, acetone and the C9 hydroxyl group of phorbol form a 1:1 hydrogen bond. The phorbol acetone cocrystal compound provided by this invention utilizes the property of phorbol itself to form a solvent compound. Through recrystallization, the resulting phorbol acetone cocrystal compound has a 1:1 hydrogen bond between acetone and the C9 hydroxyl group of phorbol, which facilitates precipitation under specific conditions, provides excellent impurity removal, and effectively improves the stability of the compound. While ensuring product purity, this makes the phorbol acetone cocrystal compound more suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of industrial extraction technology of phorbol, and particularly relates to phorbol acetone cocrystal compound and its preparation method and pure product preparation method. Background Technology

[0002] Phorbol is a tetracyclic diterpenoid compound with a complex structure and the molecular formula C60. 20 H 28 O6 is composed of a four-ring system of 5 / 7 / 6 / 3 rings. Due to the presence of five hydroxyl groups in its molecule, phorbolol exhibits strong polarity and is readily soluble in highly polar solvents such as methanol. Pharbolol is chemically very reactive and easily affected by factors such as heat, light, oxygen, acids, and alkalis, leading to reactions such as oxidation, esterification, and epimerization. In nature, phorbolol exists in the form of phorbolol esters, mainly distributed in plants such as croton, jatropha, euphorbia, and weeping poplar. Croton contains the highest content of phorbolol esters, reaching up to 2.6%.

[0003] Currently, the tumor-promoting activity of phorbol esters and its activation mechanism via protein kinase C (PKC) have become hot research topics. As an important intermediate in the synthesis of phorbol esters, phorbol can be artificially modified to obtain compounds with specific physiological activities, which have significant value in the pharmaceutical field. For example, prostratin, synthesized from phorbol, can induce a reduction in HIV receptors on the surface of target cells, thereby reducing the risk of HIV infection and clearing HIV viruses hidden within immune cells. Drugs containing prostratin have passed Phase II clinical trials in the United States.

[0004] Despite its significant medicinal value and broad market prospects, phorbol's high price poses a major obstacle to drug research. As a highly active compound and a precursor and raw material for many emerging drugs, the preparation of large-scale, high-purity phorbol has become an important research topic. In 2016, a US research team successfully synthesized phorbol through a 19-step reaction, but the synthesis process was lengthy and complex, with unsatisfactory yields, lacking industrialization potential. The purity and impurity control of phorbol are extremely stringent; therefore, obtaining large-scale, high-purity phorbol remains a crucial research objective.

[0005] Amorphous solids of phorbol typically exhibit poor stability, primarily for the following reasons: First, amorphous solids lack a long-range ordered crystal structure, resulting in weak intermolecular interactions. This makes them prone to movement or rearrangement under changing environmental conditions, increasing the likelihood of decomposition or degradation. Second, amorphous solids have a large specific surface area, increasing their contact area with the environment and making them susceptible to factors such as humidity, temperature, and oxygen, promoting oxidation, hydrolysis, and other chemical reactions, thus reducing their stability. Furthermore, amorphous solids have a higher energy state, placing them in a thermodynamically unstable state, making them more prone to phase transitions or chemical reactions. Their high hygroscopicity means that after absorbing moisture, water molecules act as reaction media, promoting chemical reactions and leading to the degradation or inactivation of phorbol. Finally, amorphous solids are more susceptible to deformation or breakage under mechanical stress, further contributing to chemical instability.

[0006] In summary, existing technologies for the preparation and purification of phorbol have many shortcomings, particularly in controlling stability and purity. The disordered structure, large specific surface area, high energy state, high hygroscopicity, and poor mechanical stability of the extracted amorphous phorbol solid all affect its performance in industrial preparation and applications. Therefore, improving the stability and purity of phorbol remains a crucial problem that urgently needs to be solved. Summary of the Invention

[0007] To address the above problems, the present invention provides a phorbol acetone cocrystal compound, wherein acetone and phorbol acetone form a hydrogen bond at the C9 position in a 1:1 ratio.

[0008] Preferably, the chemical structure of the phorbol acetone cocrystal compound is as follows:

[0009] .

[0010] Preferably, the crystallographic characteristics of the phorbol acetone eutectic compound include:

[0011] Orthorhombic crystal system, space group P2(1)2(1)2;

[0012] The cell parameters are a = 12.7363(4) Å, alpha = 90°, b = 18.3437(7) Å, beta = 90°, c = 9.6743(3) Å, gamma = 90°;

[0013] Number of molecules per unit lattice Z = 4;

[0014] The unit cell volume is 2260.22(13) A. 3 .

[0015] Furthermore, to address the aforementioned problems, the present invention also provides a method for preparing the phorbol acetone cocrystal compound as described above, comprising:

[0016] Adding a crystallization solvent to the phorbol product yields a phorbol solvent mixture;

[0017] After heating the phorbol solvent mixture until it is completely dissolved, a first stirring treatment is performed for 1 hour, followed by natural cooling to 30°C for 1 hour.

[0018] After a second stirring treatment for 1 hour, the solid precipitated and the temperature was lowered to 0°C.

[0019] After a third stirring treatment for 1 hour, the mixture was filtered to obtain the phorbol acetone cocrystal compound.

[0020] Preferably, the crystallization solvent includes at least one of acetone and a mixture of acetone and methanol;

[0021] Preferably, the ratio of the acetone-methanol mixture includes either 5:1 or 4:1;

[0022] Preferably, the temperature range of the heat treatment is 40℃-60℃;

[0023] Preferably, the temperature of the heat treatment is 50°C.

[0024] Preferably, before adding a crystallization solvent to the phorbol product to obtain a phorbol solvent mixture, the process further includes:

[0025] In a reaction vessel, an alkaline alcohol solution and croton oil are added and mixed to carry out a hydrolysis reaction, resulting in a hydrolysate mixture.

[0026] The hydrolyzed mixture was subjected to a first extraction treatment using a low-polarity solvent to obtain an alcohol layer extract;

[0027] The alcohol extract was subjected to a first column chromatography treatment to obtain a purified product.

[0028] The primary refined product is subjected to a second extraction with tetrahydrofuran to obtain a secondary refined product.

[0029] The secondary purified product was subjected to a second column chromatography treatment to obtain the tertiary purified product;

[0030] The tertiary purified product was purified by pulping using a poorly soluble solvent of phorbol to obtain the phorbol product.

[0031] Preferably, the method for preparing the alkaline alcohol solution includes:

[0032] The alkaline alcohol solution is obtained by adding an alcohol reagent to an alkaline substance and mixing the mixture.

[0033] Preferably, the alcohol reagent includes at least one of anhydrous ethanol and anhydrous methanol;

[0034] Preferably, the alkaline substance includes at least one selected from potassium carbonate, cesium carbonate, sodium hydroxide, and potassium hydroxide;

[0035] Preferably, the pH of the alkaline alcohol solution is 9-10;

[0036] Preferably, the alkaline alcohol solution is at least one of potassium carbonate and cesium carbonate.

[0037] Preferably, the step of adding an alkaline alcohol solution and croton oil to a reaction vessel and mixing them to carry out a hydrolysis reaction to obtain a hydrolyzed mixture includes:

[0038] The alkaline alcohol solution and croton oil are added to the reaction vessel to form a mixed solvent system;

[0039] The mixed solvent system is subjected to a hydrolysis reaction under stirring to clarify the turbid mixed solvent system. The pH is then adjusted to 6-7 with an acidic solvent to obtain the hydrolyzed mixture.

[0040] Preferably, the acidic solvent includes at least one of hydrochloric acid and sulfuric acid;

[0041] Preferably, in the reaction vessel, the volume ratio of the alkaline alcohol solution to the croton oil is (6-12):1;

[0042] Preferably, the hydrolysis reaction process also includes tracking the reaction using TLC.

[0043] Preferably, the first extraction process includes:

[0044] The low-polarity solvent is added to the hydrolysate mixture and stirred; after standing, the lower extract is obtained.

[0045] The lower alcohol layer stock solution was extracted twice more using the low-polarity solvent, and the extracts were combined to obtain the alcohol layer extract.

[0046] Preferably, the low-polarity solvent is petroleum ether;

[0047] Preferably, the first extraction process also includes TLC tracking.

[0048] Preferably, the first column chromatography treatment includes:

[0049] The alcohol layer extract was loaded using a wet method and eluted with a silica gel gradient to obtain the first filtrate;

[0050] The first filtrate is concentrated to obtain the primary refined product;

[0051] Preferably, the eluent for the gradient elution in the first column chromatography treatment is a methanol-dichloromethane system;

[0052] Preferably, the gradient elution step of the first column chromatography treatment includes:

[0053] Remove the impurity fraction obtained by eluting from 1:20 to 1:10 using a methanol-dichloromethane system;

[0054] The filtrate was collected by eluting from a methanol-dichloromethane system at a ratio of 1:5 to 1:3.

[0055] Preferably, the second extraction process includes:

[0056] A saturated saline solution is added to the primary refined product;

[0057] Repeated extraction was performed using tetrahydrofuran extraction until no product remained in the tetrahydrofuran layer.

[0058] All of the tetrahydrofuran layers were mixed and concentrated to obtain the secondary refined product;

[0059] Preferably, the second extraction process also includes TLC tracking.

[0060] Preferably, the second column chromatography process includes:

[0061] The secondary purified product was subjected to wet loading and silica gel gradient elution to obtain a second filtrate.

[0062] The second filtrate was concentrated to obtain the three-stage refined product;

[0063] Preferably, the eluent for the gradient elution in the second column chromatography treatment is a methanol-dichloromethane system;

[0064] Preferably, the gradient elution step of the second column chromatography treatment includes:

[0065] Remove the impurity fraction obtained by eluting from 1:20 to 1:10 using a methanol-dichloromethane system;

[0066] The filtrate was collected by eluting from a methanol-dichloromethane system at a ratio of 1:5 to 1:3.

[0067] Preferably, the step of using a sparingly soluble solvent of phorbol to purify the tertiary purified product by pulping includes:

[0068] In the three-stage refined product, a sparingly soluble solvent of phorbol alcohol is added to perform a first pulping, and the solid is obtained by filtration. Methanol is added to dissolve the solid, and insoluble substances are filtered out. After concentration, the first solid is obtained.

[0069] The first solid is added to a poorly soluble solvent of phorbol, and the mixture is pulped a second time. The solid is then filtered to obtain the second solid, which is dissolved in methanol and the insoluble substances are filtered out. The mixture is then concentrated to obtain the second solid, which is the phorbol product.

[0070] Preferably, the sparingly soluble solvent for phorbol is acetone;

[0071] Preferably, the duration of the first pulping is 3-4 hours; the temperature is -5±5℃;

[0072] Preferably, the duration of the second pulping is 2 hours, and the temperature is 0°C.

[0073] Furthermore, to address the aforementioned problems, this invention also provides a method for preparing pure phorbol alcohol. In the method for preparing the phorbol alcohol-acetone cocrystal compound as described above, after the third stirring treatment for 1 hour and filtration to obtain the phorbol alcohol-acetone cocrystal compound, the method further includes:

[0074] The phorbol acetone cocrystal compound was subjected to vacuum drying to remove crystalline acetone, yielding pure phorbol acetone.

[0075] Preferably, the drying temperature during the reduced pressure drying process is 30°C.

[0076] This invention provides a phorbol acetone cocrystal compound, its preparation method, and a method for preparing a pure product. In the phorbol acetone cocrystal compound, acetone and the C9 hydroxyl group of phorbol form a 1:1 hydrogen bond. The phorbol acetone cocrystal compound provided by this invention utilizes the property of phorbol itself to form a solvent compound. Through recrystallization, the resulting phorbol acetone cocrystal compound has a 1:1 hydrogen bond between acetone and the C9 hydroxyl group of phorbol, which allows for easier precipitation under specific conditions, excellent impurity removal, and effectively improved compound stability. While ensuring product purity, this makes the phorbol acetone cocrystal compound more suitable for industrial production. Attached Figure Description

[0077] Figure 1 This is a schematic flowchart of the method for preparing the phorbol acetone cocrystal compound of the present invention.

[0078] Figure 2 This is a schematic flowchart of an example of the method for preparing phorbol acetone cocrystal compound of the present invention (method for extracting phorbol acetone product);

[0079] Figure 3 This is a schematic diagram of the TLC results of the product after the hydrolysis reaction in Example 1 of the present invention;

[0080] Figure 4 This is a schematic diagram of the TLC results of the products after the first extraction treatment in Example 1 and Comparative Examples 1-2 of the present invention;

[0081] Figure 5 This is a schematic diagram of the HPLC detection results of phorbol alcohol after vacuum drying to remove the crystallization solvent in Example 4 of the present invention;

[0082] Figure 6 This is a schematic diagram of the mass spectrometry detection results of pure phorbolol in Example 4 of the present invention;

[0083] Figure 7 This is a schematic diagram of the 1H NMR spectrum of the pure phorbolol in Example 4 of the present invention;

[0084] Figure 8 This is a schematic diagram of the carbon NMR spectrum of the pure phorbolol in Example 4 of the present invention;

[0085] Figure 9 The absolute configuration of the phorbol acetone cocrystal compound prepared in Example 4 of this invention is shown below. Figure 9 -A), and the SC-XRD three-dimensional ellipsoid diagram ( Figure 9 -B);

[0086] Figure 10 This is a cell packing projection along the C-axis of the phorbol acetone eutectic compound prepared in Example 4 of the present invention.

[0087] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0088] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0089] In this embodiment of the application, a phorbol acetone cocrystal compound is provided, wherein acetone and phorbol acetone form a hydrogen bond at the C9 position in a 1:1 ratio.

[0090] It should be noted that a cocrystal is a crystal structure formed by two or more different compounds in a specific ratio through non-covalent interactions (such as hydrogen bonds, van der Waals forces, π-π interactions, etc.). These different compounds are called cocrystal formers. Cocrystals possess unique physical and chemical properties, and may exhibit different solubility, stability, melting point, and other characteristics compared to their individual components.

[0091] In phorbol acetone cocrystals, hydrogen bonding is a relatively weak non-covalent interaction that typically occurs between donor molecules containing hydrogen atoms (such as hydrogen atoms in hydroxyl groups) and acceptor molecules containing lone pairs of electrons (such as oxygen atoms in acetone). Hydrogen bonding plays a crucial role in stabilizing the cocrystal structure.

[0092] In its chemical structure, phorbol molecule has multiple hydroxyl (-OH) groups. The C9 hydroxyl group refers to the hydroxyl group located on the ninth carbon atom of the phorbol molecule's backbone.

[0093] As described above, acetone and phorbol form hydrogen bonds at the C9 position in a 1:1 ratio. This means that in the cocrystal, the C9 hydroxyl group of each phorbol molecule is bonded to one acetone molecule via hydrogen bonds. This specific ratio and bonding method contributes to the formation of a stable crystal structure.

[0094] Specifically, the formation of hydrogen bonds enables specific interactions between phorbol and acetone molecules, stabilizing the crystal structure of the cocrystal. This ordered hydrogen bond network may affect the physical properties of the cocrystal, such as melting point and solubility, making it more advantageous in applications. For bioactive compounds such as pharmaceuticals, the cocrystal form may improve their solubility and bioavailability, thereby enhancing their efficacy.

[0095] Amorphous solids of phorbol typically exhibit poor stability, which is closely related to their physical and chemical properties. The main reasons for the poor stability of amorphous phorbols are as follows: (1) Structural disorder: Amorphous solids lack long-range ordered crystal structures, and this disordered state makes the intermolecular interactions weak. This makes molecules more prone to movement or rearrangement when environmental conditions change, thus increasing the possibility of decomposition or degradation. (2) Larger surface area: Amorphous solids usually have a larger specific surface area, which increases the contact area with the environment, making them more susceptible to environmental factors such as humidity, temperature, and oxygen. This increased contact area promotes chemical reactions, such as oxidation and hydrolysis, thereby reducing their stability. (3) Energy state: Amorphous solids usually have a higher energy state and are in a thermodynamically unstable state. In contrast, crystalline solids tend to be more thermodynamically stable due to their lower free energy. This makes amorphous solids more prone to phase transitions (such as crystallization) or chemical reactions to achieve a more stable energy state. (4) Hygroscopicity: Due to the structural characteristics of amorphous solids, they often have high hygroscopicity. After absorbing water, water molecules can act as a reaction medium, promoting the chemical reaction and leading to the degradation or inactivation of phorbol. (5) Poor mechanical stability: Amorphous solids are more prone to deformation or breakage under mechanical stress (such as compression or stirring). This mechanical instability may further lead to its chemical instability.

[0096] The reason why acetone cocrystals can improve the stability of phorbol alcohol is mainly related to their specific physical and chemical structures. Compared with amorphous solids, cocrystals have a significant advantage in stability. This is because:

[0097] First, eutectic compounds are ordered crystal structures formed by two or more components through non-covalent interactions. This ordered crystal structure makes the molecular arrangement more stable and less susceptible to external environmental influences. In contrast, amorphous solids lack this long-range order, and their molecules are more prone to movement and rearrangement, resulting in poor stability.

[0098] In the eutectic compound, phorbol molecules are bound to acetone molecules through non-covalent interactions such as hydrogen bonds and van der Waals forces. These interactions effectively stabilize the phorbol molecules, reducing their degrees of freedom and thus lowering the likelihood of decomposition or degradation under changing environmental conditions.

[0099] Eutectic compounds are generally in a more thermodynamically stable state with lower free energy. This means that, under given temperature and pressure conditions, eutectic compounds are less likely to undergo chemical reactions or phase transitions than amorphous solids. This thermodynamic stability makes phorbol alcohol more stable in its eutectic form.

[0100] The crystalline structure of the eutectic compound results in a relatively small surface area, reducing the contact area with environmental humidity. This reduces its hygroscopicity, thereby minimizing the influence of water molecules on phorbol and preventing hydrolysis and other degradation reactions.

[0101] Eutectic compounds possess high mechanical strength and physical stability, making them resistant to breakage or deformation due to mechanical stress. This physical stability also helps maintain the chemical stability of phorbol, preventing chemical reactions caused by physical degradation.

[0102] Acetone, as a co-crystallizing agent, has the following advantages: First, acetone is a polar organic solvent that can effectively dissolve phorbol under heating conditions, facilitating the formation of the co-crystallized compound and allowing the product to precipitate easily upon cooling. Second, acetone is volatile and can be easily removed during co-crystallization, preventing residual contamination of the final co-crystallized compound. Third, acetone is relatively inert chemically and will not react adversely with phorbol, ensuring the purity and stability of the co-crystallized compound.

[0103] By forming a eutectic compound with acetone, phorbolol molecules can be stabilized in an ordered crystal structure, which greatly improves its chemical and physical stability. Compared with amorphous solids, the eutectic compound is more effective in resisting environmental changes, reducing hygroscopicity, and providing higher mechanical strength, thereby maintaining the stability and activity of phorbolol.

[0104] In summary, the phorbol acetone cocrystal compound, through its ordered crystal structure and well-defined stoichiometry, not only significantly improves the thermodynamic, thermal, and chemical stability of the compound, but also ensures its high purity through selective crystallization and reduced byproduct formation, making it more promising for applications in drug development and materials science.

[0105] Furthermore, the chemical structure of the phorbol acetone cocrystal compound is as follows:

[0106] .

[0107] Furthermore, the crystallographic characteristics of the phorbol acetone eutectic compound include: an orthorhombic crystal system with space group P2(1)2(1)2; cell parameters a=12.7363(4) Å, alpha = 90°, b = 18.3437(7) Å, beta = 90°, c=9.6743(3) Å, gamma = 90°; number of molecules per unit lattice Z=4; and cell volume of 2260.22(13) Å3.

[0108] The crystallographic features of the acetone cocrystals described above provide detailed geometric information about their crystal structure.

[0109] First, regarding crystal systems and space groups, the orthorhombic system is a common crystal system, characterized by all three crystal axes being perpendicular to each other and having unequal axis lengths. Space group P2(1)2(1)2: This is the space group of an orthorhombic system, indicating that the crystal structure possesses a certain degree of symmetry and periodicity. Space group P2(1)2(1)2 indicates that the crystal has two translational symmetry operations in each direction.

[0110] Secondly, regarding the cell parameters, a = 12.7363(4) Å, b = 18.3437(7) Å, c = 9.6743(3) Å: these are the side lengths of the cell. The three side lengths of the orthorhombic crystal system are not equal, namely a, b, and c. alpha = 90°, beta = 90°, gamma = 90°: this is a typical characteristic of the orthorhombic crystal system, where all interior angles are 90 degrees.

[0111] Secondly, the number of molecules per unit lattice, Z, Z=4: This means that each unit cell contains 4 independent molecules or molecular units. This helps in understanding the internal structure and molecular arrangement of each unit cell.

[0112] Regarding the cell volume, the cell volume is 2260.22(13) ų: The cell volume is an important parameter of crystal structure, reflecting the density of the crystal and the degree of compactness of the molecules in the cell. A larger cell volume usually indicates that there is more space between molecules, which may affect the physical properties of the crystal, such as solubility and stability.

[0113] In summary, the orthorhombic crystal system and specific space group (P2(1)2(1)2) indicate a high degree of symmetry in the crystal structure, which generally contributes to improved crystal stability. Highly symmetric structures are generally more likely to form stable lattices, contributing to enhanced thermodynamic stability of the compound. The cell parameters and number of molecules per unit lattice (Z=4) indicate four molecular units per cell, which likely means that the molecules are arranged in a regular pattern within the cell. This regular arrangement helps reduce molecular motion, improving the physical and chemical stability of the compound. The large cell volume (2260.22 ų) indicates a large intermolecular spacing. This larger intermolecular spacing helps reduce intermolecular interactions, thereby improving the solubility and bioavailability of the compound. Simultaneously, the larger spacing also helps reduce intermolecular stress, thus improving the mechanical stability of the crystal.

[0114] In summary, the crystallographic features of the acetone cocrystal, including its orthorhombic crystal system, specific space group (P2(1)2(1)2), well-defined unit cell parameters, number of molecules per unit lattice (Z=4), and large unit cell volume (2260.22 ų), collectively indicate that this compound possesses a highly ordered and stable crystal structure. Such a structure contributes to improved thermodynamic stability, physical stability, and purity of the compound, and may also enhance its solubility and bioavailability, giving it significant advantages in fields such as drug development and materials science.

[0115] In addition, refer to Figure 1 In this application embodiment, a method for preparing the phorbol acetone cocrystal compound as described above is also provided, comprising:

[0116] Step S100: Add a crystallization solvent to the phorbol product to obtain a phorbol solvent mixture;

[0117] Step S200: After heating the phorbol solvent mixture until it is completely dissolved, perform a first stirring treatment for 1 hour and then allow it to cool naturally to 30°C for 1 hour.

[0118] Step S300: Perform a second stirring treatment for 1 hour. After the solid precipitates, cool down to 0°C.

[0119] Step S400: Perform a third stirring treatment for 1 hour, and after filtration, the phorbol acetone cocrystal compound is obtained.

[0120] Furthermore, the crystallization solvent includes at least one of acetone and a mixture of acetone and methanol;

[0121] Furthermore, the ratio of the acetone-methanol mixture includes either 5:1 or 4:1;

[0122] Furthermore, the temperature range of the heat treatment is 40℃-60℃;

[0123] Furthermore, the temperature of the heat treatment is 50°C.

[0124] The aforementioned phorbol product is a crude phorbol product with a certain purity. This phorbol product can be crystallized using the above method to obtain a phorbol acetone cocrystal compound.

[0125] It should be noted that a conventional technique involves using ultrasound-assisted alcoholysis to extract phorbol. The main technical procedure involves mixing 1 mL of croton oil with an alkaline alcohol solution, ultrasonically extracting at 40°C for 30 min to obtain a supernatant containing phorbol, and then measuring the phorbol content after neutralization. This technique primarily focuses on screening the alkali and its dosage, followed by HPLC detection of the phorbol content in the hydrolysate to calculate the theoretical yield. However, since pure phorbol is not obtained, the true yield cannot be determined.

[0126] In this technology, the crude phorbol product was further crystallized. It should be noted that recrystallization is a purification technique, particularly in organic and medicinal chemistry, used to improve the purity of solid compounds. After obtaining the phorbol product, it was recrystallized to obtain an acetone cocrystal of phorbol. The purpose of recrystallization is to effectively remove impurities from phorbol, including residual solvents, other organic impurities, or inorganic salts, thereby obtaining a higher purity solvent cocrystal product. The single-crystal sample obtained in this embodiment has high crystal quality, which facilitates more accurate crystal structure analysis. High-purity single-crystal phorbol is more stable during storage and use, reducing adverse reactions or degradation caused by impurities. Obtaining the phorbol cocrystal through recrystallization not only ensures product quality but also provides a solid foundation for further research and applications.

[0127] Furthermore, after crystallization, phorbolol forms a eutectic compound with acetone, stabilizing the phorbolol molecules within an ordered crystal structure, which significantly improves its chemical and physical stability. Compared to amorphous solids, the eutectic compound is more effective at resisting environmental changes, reducing hygroscopicity, and providing higher mechanical strength, thus maintaining the stability and activity of phorbolol. This makes it more suitable for industrial production than crude or amorphous phorbolol products obtained through conventional techniques.

[0128] In the above steps, a crystallization solvent (specifically, acetone or a mixture of acetone and methanol) is added to the phorbol product to form a phorbol solvent mixture. This step is to dissolve the phorbol in preparation for the subsequent crystallization operation.

[0129] The phorbol solvent mixture is heated until completely dissolved (40°C-60°C, or 50°C in a preferred embodiment). This helps ensure complete dissolution of the phorbol, creating conditions for uniform crystallization.

[0130] First stirring treatment: After heating and dissolving, stir for 1 hour, then allow to cool naturally to 30°C. Stirring helps maintain the homogeneity of the solution and promotes a uniform temperature distribution.

[0131] Second stirring treatment: An additional hour of stirring is performed, followed by solidification and cooling to 0°C. This step aims to further promote crystallization by increasing the driving force of crystallization through cooling.

[0132] Third stirring treatment: Stirring was carried out for a final hour at 0°C, followed by filtration to collect the phorbol acetone cocrystal. This step aims to completely precipitate the cocrystal and separate the pure solid product by filtration.

[0133] The above-mentioned method for preparing the phorbol acetone cocrystal compound has the following advantages:

[0134] By employing optimized heating and cooling processes, the temperature during crystallization can be effectively controlled, which contributes to the formation of more stable and uniform crystals. Three consecutive stirring treatments ensure solution homogeneity and efficient crystal growth, resulting in better product quality. Using acetone or an acetone-methanol mixture as the crystallization solvent allows for the selection of the most suitable solvent system based on specific solubility and crystallization characteristics, improving the purity and yield of the eutectic compound. Stepwise cooling allows for more precise control of the crystallization rate and crystal size, contributing to the attainment of desired physical properties.

[0135] In summary, this processing method optimizes the preparation process of eutectic compounds by precisely controlling reaction conditions, and may have high efficiency and product quality.

[0136] It should be noted that after the extraction of phorbol, the final product of phorbol needs to be structurally identified in order to confirm that the obtained product is phorbol.

[0137] The structural identification of conventional phorbol products (crude and amorphous products) requires a combination of methods, such as nuclear magnetic resonance (NMR) spectra (e.g., 1H NMR, 1C NMR, and even 2D NMR such as HH-COSY), mass spectrometry, specific rotation, and CD, due to purity issues and the properties of the products themselves. However, the combination of NMR spectra and other methods results in high detection costs and complex analysis, making the results less clear and intuitive.

[0138] To address the aforementioned deficiencies, in this embodiment of the application, the chiral structural characteristics of the obtained phorbol acetone cocrystal compound are utilized. After obtaining the phorbol product, the phorbol acetone is further recrystallized to obtain the phorbol acetone cocrystal compound, and its absolute configuration can be characterized by XRD single-crystal diffraction.

[0139] Single-crystal X-ray diffraction (XRD) is a powerful tool for determining the absolute configuration and intermolecular interactions of organic compounds, and can provide precise three-dimensional structural information for phorbolol.

[0140] If the detected phorbolol chirality is correct, meaning there are no isomer impurities, the detection efficiency is greatly improved and the detection cost is reduced while ensuring the accuracy of structural identification.

[0141] refer to Figure 2 In this embodiment of the application, before adding a crystallization solvent to the phorbol product to obtain a phorbol solvent mixture, the process further includes:

[0142] Step S1: Add alkaline alcohol solution and croton oil to the reaction vessel, mix and carry out hydrolysis reaction to obtain hydrolysis mixture;

[0143] Step S2: The hydrolyzed mixture is subjected to a first extraction treatment using a low-polarity solvent to obtain an alcohol layer extract;

[0144] Step S3: Perform a first column chromatography treatment on the alcohol layer extract to obtain a first purified product;

[0145] Step S4: The primary refined product is subjected to a second extraction treatment using tetrahydrofuran to obtain a secondary refined product;

[0146] Step S5: Perform a second column chromatography treatment on the secondary purified product to obtain a tertiary purified product;

[0147] Step S6: The three-stage purified product is pulped and purified using a poorly soluble solvent of phorbol to obtain the phorbol product.

[0148] As mentioned above, croton oil is a toxic, viscous liquid extracted from croton seeds. It is usually light yellow to brown in color and is naturally transparent.

[0149] In conventional techniques, there is a method for extracting phorbol from Jatropha curcas seeds. Specifically, a large number of Jatropha curcas seeds are physically crushed and extracted multiple times. However, this method has significant limitations in raw materials, cannot yield large quantities of Jatropha curcas oil, and has low extraction efficiency, thus limiting its industrial-scale application.

[0150] To address this issue, croton oil is used as the raw material in this embodiment for the following reasons: Wide availability: As a commercially available product, croton oil has a relatively wide range of sources, which helps ensure a stable supply. Lower cost: Compared to extraction from Jatropha curcas seeds or croton seeds, using croton oil directly is less expensive, helping to reduce the overall extraction cost. Simple operation: Using croton oil simplifies the extraction process, making the entire extraction process easier and more convenient. High purity: By using croton oil and combining it with subsequent purification steps, high-purity (≥99%) phorbol extraction can be achieved, which is crucial for research and application in the pharmaceutical field. Suitable for industrial production: This method has high yield and high purity, is simple to operate, and is suitable for large-scale industrial production, a significant advantage compared to existing methods.

[0151] In summary, using croton oil directly as a raw material not only ensures a stable supply of raw materials and reduces costs, but also simplifies the operation process and improves extraction efficiency and purity, making it very suitable for the needs of industrial production.

[0152] The reaction vessel can be an industrial equipment such as a reaction kettle or reaction tank, or it can be a device specifically used for the extraction and purification of phorbol.

[0153] In step S1, the hydrolysis reaction is part of the phorbol extraction process. The purpose of the hydrolysis reaction is to convert the esters in croton oil into the corresponding alcohols and carboxylic acids, thereby releasing phorbol. Since phorbol mainly exists in nature in the form of esters, it needs to be converted into a free state that can be further extracted and purified through hydrolysis.

[0154] In the hydrolysis reaction, a basic alcohol solvent is used as the hydrolytic agent; for example, potassium carbonate can be added to anhydrous methanol. This alkaline environment is conducive to the hydrolysis of esters because the base can neutralize the acid generated during hydrolysis, driving the reaction toward the formation of alcohols.

[0155] The advantages of first treating croton oil with a basic alcohol solvent for hydrolysis are: using a basic alcohol solvent accelerates the hydrolysis reaction and improves the extraction efficiency of phorbol. The use of a basic alcohol solvent simplifies the hydrolysis process and reduces the need for subsequent neutralization and separation steps. By controlling the hydrolysis conditions, side reactions can be minimized, resulting in higher purity phorbol. After hydrolysis, phorbol is released from the esters and can then be further purified through extraction and purification steps.

[0156] Hydrolysis is a key step in the phorbol extraction process, which determines the efficiency of subsequent extraction and purification processes and the quality of the final product. It not only improves extraction efficiency but also helps to obtain high-purity phorbol.

[0157] As described above, column chromatography is a chromatographic technique that separates substances based on their different partition coefficients in the stationary and mobile phases. In the embodiments of this application, a stationary phase is used, and the eluent is used as the mobile phase. The purpose of the first column chromatography is to remove small polar impurities, obtaining a primary purified product through gradient elution. The second column chromatography further purifies the primary purified product; similarly, the purity of phorbolol can be optimized by changing the eluent ratio to obtain a product with higher purity.

[0158] As described above, extraction is a process that utilizes the difference in solubility of substances in two immiscible solvents to achieve separation. In the embodiments of this application, the first extraction uses a low-polarity solvent as the extractant to remove low-polarity impurities. Through the extraction action of the upper low-polarity solvent, impurities in croton oil that are soluble in low polarity are separated, while phorbol remains in the lower alcohol layer, resulting in an alcohol layer extract.

[0159] The second extraction, using tetrahydrofuran as the extractant, is performed after the first column chromatography process. Its purpose is to further remove impurities such as oils and fats, improve the purity of phorbol, and facilitate the extraction of a secondary refined product containing phorbol from the primary refined product.

[0160] The process employs alternating column chromatography and extraction steps to progressively improve the purity of phorbolol, with each step targeting different impurities. Column chromatography primarily targets impurities adsorbed by the stationary phase, while extraction targets impurities soluble in specific solvents, such as oils and fats. This stepwise separation method more effectively removes various types of impurities, avoiding the problem of incomplete impurity removal in a single step.

[0161] By employing two column chromatography and extraction processes, the purity of the final product can be significantly improved, meeting the high purity requirements of the pharmaceutical industry. Although there are multiple steps, each operation has a clear purpose and conditions, facilitating standardization and large-scale production. The process allows for adjustments to solvent ratios and operating conditions based on the specific characteristics of the raw materials, demonstrating good adaptability. Optimized steps and conditions allow for cost control while maintaining purity, improving the process's economic efficiency.

[0162] In summary, the two column chromatography and two extraction processes are designed to progressively improve the purity of phorbolol, ensuring that the final product meets the high standards of the pharmaceutical industry. This process design enables highly efficient and high-purity phorbolol extraction, providing strong technical support for industrial production.

[0163] Furthermore, this method of alternating extraction and column chromatography effectively removes different types of impurities, ensuring the purity of the final product meets industrial standards. While the alternating extraction and column chromatography steps may appear complex, each step has a clear purpose and conditions, facilitating standardization and large-scale production. The process allows for adjustments to solvent ratios and operating conditions based on the specific raw materials, exhibiting good adaptability and handling quality fluctuations across different batches. Optimized steps and conditions allow for cost control while maintaining purity, improving the process's economics. Compared to methods requiring large amounts of solvent, alternating extraction and column chromatography utilize solvent more efficiently, reducing overall solvent consumption. Column chromatography, especially with pre-packed columns, ensures reproducibility and optimized performance, making it suitable for large-scale production needs.

[0164] In summary, this method, which involves alternating extraction and column chromatography, may be more suitable for industrial production due to its advantages such as progressively increasing purity, simplified operation, high adaptability, cost-effectiveness, and suitability for large-scale production. However, its applicability still needs to be evaluated based on specific production conditions, raw material characteristics, and product quality requirements.

[0165] The above-mentioned pulping purification is a method for improving the purity of a target component in a solid powder or suspension. In the embodiments of this application, pulping purification is used in the purification process of substances, which can effectively remove impurities from the sample and improve the purity of the product. The principle of pulping purification in this technology mainly utilizes the difference in solubility of phorbolol in the solvent, and uses physical methods to induce phorbolol to precipitate from the solution, thereby achieving separation from other impurities. Through this pulping purification method, the purity of phorbolol can be effectively improved, impurities that may affect its efficacy and stability can be removed, and the quality and safety of the final product can be ensured.

[0166] In summary, the embodiments of this application achieve scaled-up extraction of phorbol by using pressed croton oil, enabling kilogram-level feeding and hundred-gram-level phorbol extraction. The combination of two extractions and two column chromatography processes greatly improves the purity of the product, achieving high yield, high purity, simple operation, and large-scale industrial production of phorbol.

[0167] Furthermore, the method for preparing the alkaline alcohol solution includes:

[0168] The alkaline alcohol solution is obtained by adding an alcohol reagent to an alkaline substance and mixing the mixture.

[0169] Furthermore, the alcohol reagent includes at least one of anhydrous ethanol and anhydrous methanol;

[0170] Furthermore, the alkaline substance includes at least one of potassium carbonate, cesium carbonate, sodium hydroxide, and potassium hydroxide.

[0171] Furthermore, the pH of the alkaline alcohol solution is 9-10. For example, the pH can be 9, 9.5, 10, etc.

[0172] Further, in step S1, an alkaline alcohol solution and croton oil are added to a reaction vessel and mixed to carry out a hydrolysis reaction, yielding a hydrolyzed mixture, comprising:

[0173] Step S11: Add the alkaline alcohol solution and croton oil to the reaction vessel to form a mixed solvent system;

[0174] Step S12: The mixed solvent system is subjected to a hydrolysis reaction under stirring to clarify the turbid mixed solvent system. The pH is then adjusted to 6-7 with an acidic solvent to obtain the hydrolyzed mixture. For example, the pH can be 6, 6.5, 7, etc.

[0175] Furthermore, the acidic solvent includes at least one of hydrochloric acid and sulfuric acid;

[0176] Furthermore, in the reaction vessel, the volume ratio of the alkaline alcohol solution to the croton oil is (6-12):1; for example, it can be 6:1, 8:1, 10:1, 12:1, etc.

[0177] Furthermore, the hydrolysis process also includes tracking the reaction using TLC.

[0178] As mentioned above, hydrolysis is a key step in the extraction of phorbol, and pH adjustment is crucial for the efficiency and selectivity of the reaction.

[0179] Ester compounds are more readily hydrolyzed under alkaline conditions. An alkaline methanol solution can provide suitable conditions for the hydrolysis reaction, promoting the breaking of ester bonds and releasing phorbol. pH significantly affects the hydrolysis rate. Maintaining a suitable pH allows for control of the reaction rate, preventing excessively fast or slow hydrolysis and ensuring smooth reaction progress. Excessively high or low pH values ​​can lead to unwanted side reactions that may affect the yield and purity of the target product. Adjusting the pH to 9-10 can reduce the likelihood of these side reactions. Phorbol is more stable within a specific pH range. Adjusting the pH can improve product stability and reduce decomposition or degradation.

[0180] As mentioned above, TLC (thin-layer chromatography) was used to monitor the hydrolysis reaction. TLC is a qualitative analytical technique used to detect and track the progress of organic reactions.

[0181] TLC can display changes in various components of a reaction mixture in real time, helping to understand the reaction progress. It is a simple and rapid analytical method that can be performed without complex instrumentation. Compared to other analytical techniques, TLC is less expensive and suitable for frequent use in industrial production. TLC provides intuitive results; researchers can observe the separation of target compounds and impurities through spot patterns on the chromatographic plate. By tracking the movement of spots using TLC, reaction conditions such as time, temperature, and pH can be adjusted to optimize reaction efficiency and product purity. Furthermore, TLC can help determine when to stop the reaction to prevent excessive hydrolysis or side reactions.

[0182] Furthermore, in step S2, the first extraction process includes:

[0183] Step S21: Add the low-polarity solvent to the hydrolysis mixture and stir to mix; after standing, the lower extract is obtained.

[0184] Step S22: The lower alcohol layer stock solution is extracted twice more using the low-polarity solvent, and the extracts are combined to obtain the alcohol layer extract.

[0185] Furthermore, the low-polarity solvent is petroleum ether;

[0186] Furthermore, the first extraction process also includes TLC tracking.

[0187] As mentioned above, the low-polarity solvent is petroleum ether. In the embodiments of this application, petroleum ether is used to stir and mix with the hydrolysis mixture to extract the low-polarity impurities in the hydrolysis mixture, thereby achieving the purpose of removing impurities.

[0188] Furthermore, in step S3, the first column chromatography treatment includes:

[0189] Step S31: The alcohol layer extract is loaded using a wet method and eluted with a silica gel gradient to obtain the first filtrate;

[0190] Step S32: Concentrate the first filtrate to obtain the primary refined product;

[0191] Furthermore, the eluent for the gradient elution in the first column chromatography treatment is a methanol-dichloromethane system;

[0192] Furthermore, the gradient elution step of the first column chromatography treatment includes:

[0193] Remove the impurity fraction obtained by eluting from 1:20 to 1:10 using a methanol-dichloromethane system;

[0194] The filtrate was collected by eluting from a methanol-dichloromethane system at a ratio of 1:5 to 1:3.

[0195] Furthermore, in step S4, the second extraction process includes:

[0196] Step S41: Add a saturated saline solution to the primary refined product;

[0197] Step S42: Repeated extraction is performed using tetrahydrofuran extraction until no product is found in the tetrahydrofuran layer;

[0198] Step S43: Mix all of the tetrahydrofuran layers and concentrate to obtain the secondary refined product;

[0199] Furthermore, the second extraction process also includes TLC tracking.

[0200] Furthermore, in step S5, the second column chromatography process includes:

[0201] Step S51: The secondary purified product is loaded with a wet sample and eluted with a silica gel gradient to obtain a second filtrate;

[0202] Step S52: Concentrate the second filtrate to obtain the three-stage refined product;

[0203] Furthermore, the eluent for the gradient elution in the second column chromatography treatment is a methanol-dichloromethane system;

[0204] Furthermore, the gradient elution step of the second column chromatography treatment includes:

[0205] Remove the impurity fraction obtained by eluting from 1:20 to 1:10 using a methanol-dichloromethane system;

[0206] The filtrate was collected by eluting from a methanol-dichloromethane system at a ratio of 1:5 to 1:3.

[0207] As described above, gradient elution is achieved by utilizing a methanol-dichloromethane mixed solvent system based on the adsorption characteristics of silica gel. The ratio of methanol to dichloromethane in the system gradually increases the polarity of the solvent, resulting in gradient elution. Specifically, a 1:20 ratio results in a higher dichloromethane content and thus lower polarity, while a 1:10 ratio results in a higher methanol content and thus higher polarity.

[0208] Further, in step S6, the three-stage purified product is purified by pulping using a poorly soluble solvent of phorbol to obtain the phorbol product, comprising:

[0209] Step S61: Add the sparingly soluble solvent of phorbol to the three-stage refined product, perform the first pulping, filter to obtain the solid, add methanol to dissolve, filter out insoluble substances, and concentrate to obtain the first solid.

[0210] Step S62: The first solid is added to the sparingly soluble solvent of phorbol alcohol, and pulped a second time. The solid is then filtered to obtain the solid, dissolved in methanol, and the insoluble substances are filtered out. After concentration, the second solid is obtained as the phorbol alcohol product.

[0211] Furthermore, the sparingly soluble solvent for phorbol is acetone;

[0212] Furthermore, the duration of the first pulping is 3-4 hours; the temperature is -5±5℃; for example, the duration of the first pulping can be 3 hours, 3.5 hours, 4 hours, etc.; the temperature can be -10℃, -5℃, 0℃, etc.

[0213] Furthermore, the duration of the second pulping is 2 hours; the temperature is 0°C.

[0214] Furthermore, this application embodiment also provides a method for preparing pure phorbol alcohol. In the method for preparing the phorbol alcohol-acetone cocrystal compound as described above, after step S400, which involves a third stirring treatment for 1 hour and filtration to obtain the phorbol alcohol-acetone cocrystal compound, the method further includes:

[0215] Step S500: The phorbol acetone cocrystal compound is subjected to vacuum drying to remove crystalline acetone, thereby obtaining pure phorbol acetone.

[0216] Furthermore, the drying temperature during the vacuum drying process is 30°C.

[0217] This process involves vacuum drying under reduced pressure to improve drying efficiency and rapidly remove crystalline acetone from the phorbol acetone co-crystal compound, thereby obtaining pure phorbol.

[0218] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0219] Example 1: In this example, the alcohol layer extract of phorbol was extracted.

[0220] Experimental methods: (1) Preparation of alkaline alcohol solution: 50L of anhydrous methanol and 2.5kg of potassium carbonate were added to a 50L reactor and stirred at 15℃ for 2 hours. After 2 hours, the mixture was filtered and the pH of the filtrate was measured to be within the range of 9-10. The filtrate was set aside for use. (2) Hydrolysis reaction: 40L of alkaline alcohol solution and 5kg of commercially pressed croton oil were added to a 50L reactor. At this time, the system was turbid. The hydrolysis reaction was carried out by stirring at 15℃ for 1 hour. The system gradually became clear and brown with stirring. During the process, the reaction was monitored by TLC. There were product points (refer to the reference). Figure 3 ,in Figure 3-A is a simulation diagram. Figure 3 -B is the actual thin-layer result). After the reaction, the pH was adjusted to 7-8 with hydrochloric acid to obtain the hydrolyzed mixture; the content of phorbol alcohol was 0.94% as determined by HPLC. The theoretical yield was 47g. (3) First extraction treatment: 10L of low polarity solvent (petroleum ether) was added, stirred for 20 minutes, and allowed to stand for separation. The upper petroleum ether layer was monitored by TLC (developing solvent: dichloromethane: methanol = 7:1) to be an impurity. The lower methanol layer (alcohol layer solution) was taken back to the reaction vessel. The extraction was repeated twice with 10L of petroleum ether. The lower layer was concentrated and dried to obtain the crude product, which is the alcohol layer extract (TLC results are referenced). Figure 4 , Figure 4 -A is a simulation diagram. Figure 4 -B represents the actual thin-layer result.

[0221] Comparative Example 1: In this comparative example, phorbol extract was extracted.

[0222] Experimental method: Basically the same as in Example 1, except that in step (3), the methanol in the hydrolysis mixture is concentrated to dryness, 10L of dichloromethane is added to dissolve it, and water is used as the extractant to extract the impurities. The extraction is repeated 4-5 times, and the dichloromethane layers are combined and concentrated to obtain the crude product. Some impurities are still not completely removed (see reference). Figure 4 , Figure 4 -C is a simulation diagram. Figure 4 -D represents the actual thin-layer result.

[0223] Analysis: Using water as the extractant results in low efficiency in removing impurities.

[0224] Comparative Example 2: In this comparative example, phorbol extract was extracted.

[0225] Experimental method: Basically the same as in Example 1, except that in step (3), the methanol in the hydrolysis mixture was concentrated to dryness, 20L of ethyl acetate was added to dissolve it, and water was used as the extractant to extract the impurities. The extraction was repeated 4-5 times, and the ethyl acetate layers were combined and concentrated to obtain the crude product. Some impurities were still not completely removed. In addition, the aqueous layer contained a small amount of product (see reference). Figure 4 , Figure 4 -E is a simulation diagram. Figure 4 -F represents the actual thin-layer result).

[0226] Analysis: Ethyl acetate has poor solubility, requires a large amount of ethyl acetate, and is prone to emulsification due to layering.

[0227] Example 2: In this example, phorbol was extracted.

[0228] Experimental method: In this embodiment, based on Example 1, the following steps are also included:

[0229] (4) First column chromatography treatment: The alcohol extract obtained above was filtered with silica gel. 2 kg of silica gel was placed in a Buchner funnel, and the crude product was dissolved in a small amount of dichloromethane and loaded onto the sample using the wet method.

[0230] Small polar impurities were removed by column elution with a methanol / dichloromethane gradient of 1 / 20 to 1 / 10. Once the impurities were basically removed, the target compound was flushed out with a methanol / dichloromethane gradient of 1 / 5 to 1 / 3. The filtrate containing the product was concentrated to dryness to obtain a primary purified product (530g). The content of phorbol was determined to be 8.1% by HPLC.

[0231] Comparative Example 3: In this comparative example, phorbol extract was extracted.

[0232] Experimental method: It is basically the same as in Example 2, except that in step (4), resin adsorption is used to remove impurities; the resin is pretreated with sodium hydroxide and hydrochloric acid and washed with ethanol; the alcohol layer extract is added to the resin and allowed to stand for adsorption for 24 hours. After 24 hours, the column is packed, ethanol and water are eluted, and the eluent is concentrated to obtain the product.

[0233] Results: The resin adsorption efficiency of phorbolol is not high, and a large amount of resin is required for long-term adsorption. Therefore, the impurity removal effect is not good, the loss is large, the obtained product fraction contains water, and it is easy to deteriorate when concentrated at high temperature, which limits the industrial scale-up.

[0234] Comparative Example 4: In this comparative example, phorbol extract was extracted.

[0235] Experimental method: basically the same as in Example 2, except that in step (4), silica gel adsorption is used to remove impurities. Silica gel column chromatography is performed using an ethyl acetate-petroleum ether system. 4 kg of silica gel is placed in a Buchner funnel, and a small amount of crude product is dissolved in dichloromethane and loaded onto the column using a wet method. The column is eluted with a gradient of eluent (ethyl acetate: petroleum ether = 1:10-1:5) to remove small polar impurities; after the impurities are basically removed, the column is washed with a gradient of eluent (ethyl acetate: petroleum ether = 1:3-2:1), the eluent is collected, and the residual product is eluted with methanol: dichloromethane = 1:3.

[0236] Results: The petroleum ether-ethyl acetate system requires an increased amount of silica gel. Due to the high solubility of this system, a large amount of solvent is needed for elution, and product residues still remain. Methanol-dichloromethane is required to further elute the residual products. The large amount of solvent used and waste liquid generated greatly increases the extraction cost and makes it unsuitable for industrial application.

[0237] Example 3: In this example, phorbol was extracted.

[0238] Experimental methods: In this embodiment, based on Example 2, the following steps are also included: (5) Second extraction treatment: 2L of saturated saline solution is added to the first purified product obtained above, and 500mL of tetrahydrofuran is extracted each time. TLC monitoring is performed until there is no product in the tetrahydrofuran layer; all tetrahydrofuran layers are mixed and concentrated to dryness to obtain the second purified product. (6) Second column chromatography treatment: The second purified product obtained above is filtered with silica gel. 1kg of silica gel is placed in a Buchner funnel, and a small amount of crude product is dissolved in dichloromethane and loaded onto the column by wet method. The column is eluted with a gradient of eluent (methanol:dichloromethane=1:20-1:10) to remove small polar impurities; after the impurities are basically removed, the target product is flushed out with eluent (methanol:dichloromethane=1:5-1:3), and the filtrate containing the product is concentrated to dryness to obtain the third purified product (82g). The content is 48.8% as determined by HPLC.

[0239] Comparative Example 5: In this comparative example, phorbol was extracted.

[0240] Experimental method: basically the same as in Example 3, except that in step (6), resin adsorption is used to remove impurities. The resin is pretreated with sodium hydroxide and hydrochloric acid and washed with ethanol; the secondary purified product is added to the resin and allowed to stand for adsorption for 24 hours. After 24 hours, the column is packed, eluted with ethanol and water, and the eluent is concentrated to obtain the product.

[0241] Results: The resin adsorption efficiency of phorbolol is not high, and a large amount of resin is required for long-term adsorption. Therefore, the impurity removal effect is not good, the loss is large, the obtained product fraction contains water, and it is easy to deteriorate when concentrated at high temperature, which limits the industrial scale-up.

[0242] Comparative Example 6: In this comparative example, phorbol was extracted.

[0243] Experimental method: basically the same as in Example 3, except that in step (6), silica gel adsorption is used to remove impurities. Silica gel column chromatography is performed using an ethyl acetate-petroleum ether system. 4 kg of silica gel is placed in a Buchner funnel, and a small amount of crude product is dissolved in dichloromethane and loaded onto the column using a wet method. The column is eluted with a gradient of eluent (ethyl acetate: petroleum ether = 1:10-1:5) to remove small polar impurities; after the impurities are basically removed, the column is washed with a gradient of eluent (ethyl acetate: petroleum ether = 1:3-2:1), the eluent is collected, and the residual product is eluted with methanol: dichloromethane = 1:3.

[0244] Results: The petroleum ether-ethyl acetate system requires an increased amount of silica gel. Due to the high solubility of this system, a large amount of solvent is needed for elution, and product residues still remain. Methanol-dichloromethane is required to further elute the residual products. The large amount of solvent used and waste liquid generated greatly increases the extraction cost and makes it unsuitable for industrial application.

[0245] Example 4: In this example, the preparation of pure phorbol acetone cocrystal compound and pure phorbol was carried out.

[0246] Experimental method: In this embodiment, based on Example 3, the following steps are also included: (7) Preparation of pure phorbol acetone cocrystal compound and pure phorbol acetone: The above-obtained three-stage purified product is added to 150 mL of acetone and pulped for the first time at 0°C for 3-4 hours, and a large amount of solid precipitates out; after 4 hours, the first solid is obtained by filtration, methanol is added to the first solid to dissolve it, insoluble matter is removed by filtration, the filtrate is concentrated to dryness, 150 mL of acetone is added, and pulping is performed for the second time at 0°C for 2 hours to obtain phorbol acetone product; the above phorbol acetone product is further purified as follows: 150 mL of crystallization solvent acetone is added to the phorbol acetone product to obtain phorbol acetone solvent mixture; the phorbol acetone solvent mixture is heated until completely dissolved, and then stirred for the first time for 1 hour, and then naturally cooled to 30°C for 1 hour; the second stirring is performed for 1 hour, and a large amount of solid precipitates out, and then the temperature is lowered to 0°C; the third stirring is performed for 1 hour, and after filtration, the phorbol acetone cocrystal compound is obtained.

[0247] Furthermore, the phorbol acetone cocrystal compound was dried at 25°C by forced air drying to remove free acetone, yielding 32g of white crystals, which is the pure phorbol acetone cocrystal compound. Additionally, the cocrystal compound was dried at 30°C under reduced pressure to remove crystalline acetone, thus obtaining pure phorbol.

[0248] Purity determined by HPLC is ≥99% (reference) Figure 5 4.3 g of pure phorbol was recovered from the crystallization mother liquor, and the crystallization recovery rate was calculated to be 88.2%.

[0249] Based on the theoretical yield calculation, the hydrolysis recovery rate of phorbol was 77.4%. Furthermore, after obtaining pure phorbol-acetone cocrystals and pure phorbol, the structures were characterized as follows:

[0250] (1) By mass spectrometry (reference) Figure 6 Molecular weight determination (methanol dissolution and injection; mass spectrometry shows the molecular weight of the main structure): High-resolution mass spectrometry shows a quasi-molecular ion peak at m / z 387.1840 [M-C3H6O+Na]. + (Calculated value: C) 20 H 28 O6,364.1885), the main structural molecular formula is determined to be: C 20 H 28 O6;

[0251] (2) By proton nuclear magnetic resonance (H-NMR) (400 M, reference) Figure 7 Confirm the structure: 1H-NMR (400 MHz, CD3OD)δ7.61 (m, 1H), 5.60 (d, J = 5.9, 1H), 4.05 (d, J = 10.2 Hz, 1H), 3.94 (m,2H), 3.16 (dd, J = 5.7, 5.7 Hz, 1H), 3.09 (m, 1H), 2.57-2.39 (m, 2H), 1.94(dq, J = 10.1, 6.5 Hz, 1H), 1.75 (dd, J = 3.0, 1.4 Hz, 3H), 1.26 (s, 3H),1.14 (s, 3H), 1.07 (d, J = 6.5 Hz, 3H), 0.73 (d, J = 5.4 Hz, 1H);

[0252] (3) Using carbon spectroscopy C-NMR (100M, reference) Figure 8 Structural assessment was conducted to confirm the structure. 13 C-NMR (101 MHz, CD3OD, 20 peaks total) δ209.4, 159.9, 140.5, 132.9, 129.8, 80.7, 78.4, 73.5,66.9, 61.8, 57.4, 44.8, 38.9, 37.2, 36.1, 25.7, 22.7, 16.4, 14.1, 8.8;

[0253] (4) The acetone solvation structure of its phorbol acetone cocrystal compound was confirmed by SC-XRD (see reference). Figure 9 and Figure 10 ): Orthorhombic crystal system, space group P2(1)2(1)2; cell parameters a=12.7363(4) Å, alpha = 90°, b =18.3437(7) Å, beta = 90°, c= 9.6743(3) Å, gamma = 90°; number of molecules per unit lattice Z=4; cell volume 2260.22(13) Å 3 .

[0254] Comparative Example 7: In this comparative example, phorbolol was purified.

[0255] Experimental method: It is basically the same as in Example 4, except that in step (7), 150 mL of petroleum ether is added, and the mixture is stirred at 0°C for 3-4 hours. A large amount of solid is precipitated, and the solid is obtained by filtration after 4 hours.

[0256] Results: Petroleum ether has low polarity and cannot dissolve, nor can it form solvent compounds. Some impurities were not removed and there was no purification effect.

[0257] Example 5: In this comparative example, phorbolol was purified.

[0258] Experimental method: It is basically the same as in Example 4, except that in step (7), the crystallization solvent is replaced with 150mL acetone / methanol=5 / 1, and heated to 40℃ to dissolve.

[0259] Result: The same target purity and yield can be achieved.

[0260] Comparative Example 9: In this comparative example, phorbolol was purified.

[0261] Experimental method: basically the same as in Example 4, except that in step (7), the mixture is heated to 70°C to dissolve.

[0262] Results: Trace degradation of phorbol was observed at 70℃.

[0263] Comparative Example 10: In this comparative example, phorbolol was purified.

[0264] Experimental method: It is basically the same as in Example 4, except that in step (7), after heating and dissolving at 50°C, the external temperature is lowered to 0°C and stirred for 1 hour to precipitate a large amount of solid. The product is obtained by filtration.

[0265] Result: Solvates were not fully formed, resulting in poor impurity removal.

[0266] Comparative Example 11: In this comparative example, phorbolol was purified.

[0267] Experimental method: basically the same as in Example 5, except that in step (8), phorbol product is taken and 4 times the volume of ethyl acetate-n-hexane solution is added (ethyl acetate:n-hexane = 1:4).

[0268] Result: No solid precipitation.

[0269] Comparative Example 12: In this comparative example, phorbolol was purified.

[0270] Experimental method: It is basically the same as in Example 5, except that in step (8), phorbol product is taken and 4 times the volume of dichloromethane-acetone solution is added (dichloromethane:acetone = 1:4).

[0271] Result: Amorphous solids precipitated, and the removal of impurities was ineffective.

[0272] Comparative Example 13: In this comparative example, phorbolol was purified.

[0273] Experimental method: basically the same as in Example 5, except that in step (8), phorbol product is taken and 4 times the volume of ethyl acetate-acetone solution is added (ethyl acetate:acetone = 1:4).

[0274] Result: Amorphous solids precipitated, and the removal of impurities was ineffective.

[0275] Experimental results and analysis:

[0276] (1) Statistical analysis was performed on the experimental conditions and the state, purity, and yield of the product / pure product in the aforementioned examples and comparative examples. The specific results are as follows:

[0277] Table 1. Comparison of crystallization conditions and results in the examples and comparative examples.

[0278] ;

[0279] In the table above, “real” represents an example, such as real 4, which is example 4, and “compare” represents a comparative example, such as “compare 7”, which is comparative example 7.

[0280] Analysis: From the data summarized in the table above, it can be seen that high-purity phorbol can be obtained by using Examples 4 and 5 of the present invention. The reason is that when the crystallization solvent is acetone or a mixture of acetone and methanol, the acetone eutectic compound to be obtained in this application can be formed, which has a good impurity removal effect.

[0281] (2) Conduct stability testing experiments:

[0282] Experimental method A: The products obtained in the above examples were used as test samples: Sample 1: phorbol acetone cocrystal compound; Sample 2: phorbol amorphous solid; They were stored at 25℃, 2-8℃, and -20℃ respectively, and the purity was tested at 3 days, 7 days, and 14 days.

[0283] Experimental results (B):

[0284] Table 2. Stability test at 25℃

[0285] ;

[0286] Table 3. Stability study of phorbol at 2-8℃

[0287] ;

[0288] Table 4. Stability study of phorbol at -20℃

[0289] ;

[0290] The experimental data above show the stability of phorbol acetone eutectic compound (sample 1) and phorbol amorphous solid (sample 2) under different temperature conditions.

[0291] Analysis of the data in the table above leads to the following conclusions:

[0292] 1) Sample 1 is a phorbol acetone cocrystal compound. At 25°C, Sample 1 exhibits very good stability, with its purity slightly decreasing from 99.7% to 99.5% before recovering to 99.6%. This indicates that the sample shows almost no significant purity change at room temperature. Between 2 and 8°C, the purity of Sample 1 fluctuates slightly but remains generally between 99.5% and 99.7%, demonstrating good stability. At -20°C, the purity of Sample 1 fluctuates less, slightly decreasing from 99.7% to 99.4% before recovering to 99.7%. This indicates that Sample 1 maintains very good stability even at lower temperatures.

[0293] 2) Sample 2 is an amorphous solid of phorbol alcohol. At 25℃, the purity of Sample 2 continuously decreased from 95.3% to 92.6%, showing a significant decrease in stability. Between 2 and 8℃, Sample 2 exhibited better stability at lower temperatures, with the purity gradually decreasing from 95.3% to 94.2%. At -20℃, although the purity of Sample 2 decreased slightly from 95.3% to 94.7%, the overall change was not significant, indicating relatively good stability.

[0294] 3) Sample 1 (cocrystal compound) showed superior stability compared to Sample 2 (amorphous solid). The purity of Sample 1 remained almost constant with very little fluctuation under all temperature conditions, indicating that the cocrystal morphology is beneficial to drug stability.

[0295] 4) Temperature has a greater impact on sample 2. The stability of sample 2 decreases rapidly at room temperature (25℃), while the stability improves at lower temperatures (2-8℃ and -20℃), but still gradually decreases.

[0296] Conclusion: Due to its high chemical stability at different temperatures, the morphology of phorbol acetone cocrystals can be determined, making them a superior form for drug storage and transportation. In contrast, while amorphous solids are relatively stable at lower temperatures, they exhibit poor stability at room temperature and may require more stringent temperature control, more demanding storage conditions, and more specific control measures to ensure their quality.

[0297] The above describes preferred embodiments and corresponding examples of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, including but not limited to adjustments in proportion, process, dosage and reaction vessel, all of which fall within the protection scope of the present invention.

Claims

1. A method for preparing a phorbol acetone cocrystal compound, characterized in that, include: A crystallization solvent is added to the phorbol product to obtain a phorbol solvent mixture; the crystallization solvent includes at least one of acetone and an acetone-methanol mixture; the ratio of the acetone-methanol mixture includes either 5:1 or 4:

1. After the phorbol solvent mixture is heated until completely dissolved, it is stirred for 1 hour and then cooled naturally to 30°C for 1 hour; the temperature range of the heating treatment is 40°C-60°C. After a second stirring treatment for 1 hour, the solid precipitated and the temperature was lowered to 0°C. After a third stirring treatment for 1 hour, the mixture was filtered to obtain the phorbol acetone cocrystal compound. In the phorbol acetone cocrystal compound, acetone forms a hydrogen bond with the C9 hydroxyl group of phorbol in a 1:1 ratio. The chemical structure of the phorbol acetone cocrystal compound is as follows: ； The crystallographic features of the phorbol acetone eutectic compound include: Orthorhombic crystal system, space group P2(1)2(1)2; The cell parameters are a = 12.7363(4) Å, alpha = 90°, b = 18.3437(7) Å, beta = 90°, c = 9.6743(3) Å, gamma = 90°; Number of molecules per unit lattice Z = 4; The unit cell volume is 2260.22(13) A. 3 .

2. The method for preparing the phorbol acetone cocrystal compound as described in claim 1, characterized in that, The temperature for the heat treatment is 50°C.

3. The method for preparing the phorbol acetone cocrystal compound as described in claim 1, characterized in that, Before adding a crystallization solvent to the phorbol product to obtain a phorbol solvent mixture, the process further includes: In a reaction vessel, an alkaline alcohol solution and croton oil are added and mixed to carry out a hydrolysis reaction, resulting in a hydrolysate mixture. The hydrolyzed mixture was subjected to a first extraction treatment using a low-polarity solvent to obtain an alcohol layer extract; The alcohol extract was subjected to a first column chromatography treatment to obtain a purified product. The primary refined product is subjected to a second extraction with tetrahydrofuran to obtain a secondary refined product. The secondary purified product was subjected to a second column chromatography treatment to obtain the tertiary purified product; The tertiary purified product was purified by pulping using a poorly soluble solvent of phorbol to obtain the phorbol product.

4. The method for preparing the phorbol acetone cocrystal compound as described in claim 3, characterized in that, The method for preparing the alkaline alcohol solution includes: The alkaline alcohol solution is obtained by adding an alcohol reagent to an alkaline substance and mixing them.

5. The method for preparing the phorbol acetone cocrystal compound as described in claim 4, characterized in that, The alcohol reagent includes at least one of anhydrous ethanol and anhydrous methanol.

6. The method for preparing the phorbol acetone cocrystal compound as described in claim 4, characterized in that, The alkaline substance includes at least one of potassium carbonate, cesium carbonate, sodium hydroxide, and potassium hydroxide.

7. The method for preparing the phorbol acetone cocrystal compound as described in claim 3, characterized in that, The pH of the alkaline alcohol solution is 9-10.

8. The method for preparing the phorbol acetone cocrystal compound as described in claim 3, characterized in that, The alkali in the alkaline alcohol solution is at least one of potassium carbonate and cesium carbonate.

9. The method for preparing the phorbol acetone cocrystal compound as described in claim 3, characterized in that, The process involves adding an alkaline alcohol solution and croton oil to a reaction vessel, mixing them, and carrying out a hydrolysis reaction to obtain a hydrolyzed mixture, comprising: The alkaline alcohol solution and croton oil are added to the reaction vessel to form a mixed solvent system; The mixed solvent system is subjected to a hydrolysis reaction under stirring to clarify the turbid mixed solvent system. The pH is then adjusted to 6-7 with an acidic solvent to obtain the hydrolyzed mixture.

10. The method for preparing the phorbol acetone cocrystal compound as described in claim 9, characterized in that, The acidic solvent includes at least one of hydrochloric acid and sulfuric acid.

11. The method for preparing the phorbol acetone cocrystal compound as described in claim 9, characterized in that, In the reaction vessel, the volume ratio of the alkaline alcohol solution to the croton oil is (6-12):

1.

12. The method for preparing the phorbol acetone cocrystal compound as described in claim 9, characterized in that, The hydrolysis process also includes tracking the reaction using TLC.

13. The method for preparing the phorbol acetone cocrystal compound as described in claim 3, characterized in that, The first extraction process includes: The low-polarity solvent is added to the hydrolysis mixture and stirred; after standing, the lower extract is obtained. The lower alcohol layer stock solution was extracted twice more using the low-polarity solvent, and the extracts were combined to obtain the alcohol layer extract.

14. The method for preparing the phorbol acetone cocrystal compound as described in claim 13, characterized in that, The low-polarity solvent is petroleum ether.

15. The method for preparing the phorbol acetone cocrystal compound as described in claim 13, characterized in that, The first extraction process also includes TLC tracking.

16. The method for preparing the phorbol acetone cocrystal compound as described in claim 3, characterized in that, The first column chromatography process includes: The alcohol layer extract was loaded using a wet method and eluted with a silica gel gradient to obtain the first filtrate; The first filtrate is concentrated to obtain the primary refined product.

17. The method for preparing the phorbol acetone cocrystal compound as described in claim 16, characterized in that, The eluent for the gradient elution in the first column chromatography treatment was a methanol-dichloromethane system.

18. The method for preparing the phorbol acetone cocrystal compound as described in claim 17, characterized in that, The gradient elution step of the first column chromatography treatment includes: Remove the impurity fraction obtained by eluting from 1:20 to 1:10 using a methanol-dichloromethane system; The filtrate was collected by eluting from a methanol-dichloromethane system at a ratio of 1:5 to 1:

3.

19. The method for preparing the phorbol acetone cocrystal compound as described in claim 4, characterized in that, The second extraction process includes: A saturated saline solution is added to the primary refined product; Repeated extraction was performed using tetrahydrofuran extraction until no product remained in the tetrahydrofuran layer. All of the tetrahydrofuran layers were mixed and concentrated to obtain the secondary refined product.

20. The method for preparing the phorbol acetone cocrystal compound as described in claim 19, characterized in that, The second extraction process also includes TLC tracking.

21. The method for preparing the phorbol acetone cocrystal compound as described in claim 3, characterized in that, The second column chromatography process includes: The secondary purified product was subjected to wet loading and silica gel gradient elution to obtain a second filtrate. The second filtrate is concentrated to obtain the three-stage refined product.

22. The method for preparing the phorbol acetone cocrystal compound as described in claim 21, characterized in that, The eluent for the gradient elution in the second column chromatography treatment is a methanol-dichloromethane system.

23. The method for preparing the phorbol acetone cocrystal compound as described in claim 21, characterized in that, The gradient elution step of the second column chromatography treatment includes: Remove the impurity fraction obtained by eluting from 1:20 to 1:10 using a methanol-dichloromethane system; The filtrate was collected by eluting from a methanol-dichloromethane system at a ratio of 1:5 to 1:

3.

24. The method for preparing the phorbol acetone cocrystal compound as described in claim 3, characterized in that, The process of purifying the three-stage purified product using a sparingly soluble solvent of phorbol to obtain the phorbol product comprises: In the three-stage refined product, a sparingly soluble solvent of phorbol alcohol is added to perform a first pulping, and the solid is obtained by filtration. Methanol is added to dissolve the solid, and insoluble substances are filtered out. After concentration, the first solid is obtained. The first solid is added to a poorly soluble solvent of phorbol, and the mixture is pulped a second time. The solid is then filtered to obtain the second solid, which is dissolved in methanol and insoluble substances are filtered out. The mixture is then concentrated to obtain the second solid, which is the phorbol product.

25. The method for preparing the phorbol acetone cocrystal compound as described in claim 24, characterized in that, The sparingly soluble solvent for phorbol is acetone.

26. The method for preparing the phorbol acetone cocrystal compound as described in claim 24, characterized in that, The duration of the first pulping is 3-4 hours; the temperature is -5±5℃.

27. The method for preparing the phorbol acetone cocrystal compound as described in claim 24, characterized in that, The second pulping process lasted for 2 hours at a temperature of 0°C.

28. A method for preparing pure phorbol alcohol, characterized in that, In the method for preparing the phorbol acetone cocrystal compound as described in claim 1, after the third stirring treatment for 1 hour and filtration to obtain the phorbol acetone cocrystal compound, the method further includes: The phorbol acetone eutectic compound was subjected to vacuum drying to remove crystalline acetone, yielding pure phorbol acetone.

29. The method for preparing pure phorbolol as described in claim 28, characterized in that, The drying temperature during the vacuum drying process is 30°C.

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