SMTP production method

Abstract

The purpose of the present invention is to provide a means for obtaining SMTP from a fermentation product including SMTP via a method that can easily be scaled up; to provide an SMTP crystallization method; or to provide a means for obtaining SMTP via a method including SMTP crystallization. The present invention provides an SMTP production method including: a step I for putting a Stachybotrys microspora fermentation product in contact with an aqueous solvent having a pH of 9 or greater and obtaining an extract liquid including SMTP; a step II for supplying the extract liquid obtained in step I for purification and obtaining a purified product; and a step III for crystallizing the SMTP included in the purified product obtained in step II in a crystallization solvent including one or more selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl-tert-butyl ether, and acetonitrile. Alternatively, provided is an SMTP production method including a step for crystallizing SMTP having a purity of 70% or greater in a solvent including one or more selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl-tert-butyl ether, and acetonitrile. Alternatively, provided is an SMTP production method including putting a Stachybotrys microspora fermentation product in contact with an aqueous solvent having a pH of 9 or greater and obtaining an extract liquid including SMTP.

Description

SMTP manufacturing method

[0001] The present invention relates to a method for producing SMTP, and in some embodiments, to the purification, extraction, or crystallization of SMTP. More specifically, some embodiments of the present invention relate to a simplified purification process of SMTP from fermentation broth by chromatographic methods, solvent extraction, and crystallization, which allows for the isolation of highly pure SMTP. The present invention also relates to a process for preparing SMTP that is scalable for commercial production.

[0002] SMTP (Stachybotrys Microspora Triprenyl Phenol) is a general term for a group of triprenylphenols produced by the fungus Stachybotrys Microspora, and consists of a chroman lactam structure, an isoprene side chain, and an N-linked side chain. One of its analogs, SMTP-7, is widely known to be effective in, for example, a cerebral infarction model caused by thrombus or embolism (Patent Document 1). SMTP-7 also has, for example, antioxidant and anti-inflammatory effects (Patent Document 2).

[0003] In Patent Documents 1 and 2, therapeutic applications of SMTP-7 are presented, but the purification process of SMTP is mentioned only briefly. In principle, the procedure for recovery of SMTP from the fermentation broth is based on the application of organic solvent extraction of SMTP from the fermentation broth and the use of an HPLC purification method that can only be performed on a small scale.

[0004] U.S. Patent No. 5,999,233 and U.S. Patent No. 5,999,233 disclose a selective production method for SMTP. These patents also disclose the following purification method: extracting the fermentation broth with methanol; subsequently concentrating the extract by spin distillation, followed by extraction with ethyl acetate; subsequently dehydrating the solution with anhydrous sodium sulfate, filtering, concentrating, and drying; subsequently dissolving the solidified material in methanol and purifying it using a reversed-phase packing; subsequently extracting the target product with ethyl acetate and other steps to obtain the target product.

[0005] Patent Documents 3 and 5 report extracting the culture filtrate, rather than the fermentation broth, with an organic solvent. However, our evaluation showed that this procedure did not allow recovery of even half of the product, as most of the SMTP was retained intracellularly and very little in the supernatant.

[0006] In reports using solvent extraction, methanol, which is a Class II solvent, is mainly used, and solvent extraction is performed using only one of EtOAc or acetic acid. All of these extractions use large amounts of solvent, about 100 times or more, which are difficult to handle, generate a lot of waste, and are difficult to scale up.

[0007] After extraction, the solvent and water are distilled before further purification begins, a laborious, energy- and time-consuming process. When an extraction process is performed after the distillation or concentration process and before purification using the methods of U.S. Patent Nos. 5,629,999 and 5,629,999 (HPLC) or 5,629,999 and 5,629,999 (silica gel), the solvents used are primarily methanol, acetonitrile, hexane, and chloroform. These solvents are Class II. Because the silica gel used cannot be regenerated, significant waste is generated, increasing environmental impact and costs. Furthermore, when fine powders such as silica gel are used in purification, there is a concern that they may be easily inhaled by workers.

[0008] In the case of HPLC purification methods, only small amounts of samples can be handled and the desired purity cannot be achieved in one cycle, so the purification process is repeated with different solvent systems (Patent Document 4). Therefore, scale-up under these conditions is not easy, and the environment, health, and safety (EHS) are seriously compromised.

[0009] Furthermore, it is known that crystallization of SMTP is difficult (Non-Patent Document 1), and purification including crystallization is not described in any of Patent Documents 1-8.

[0010] International Publication No. 2010 / 110026 International Publication No. 1998 / 56940 Patent No. 4257026 International Publication No. 2022 / 171151 Japanese Patent Publication No. 2004 / 224737 Chinese Patent No. 105153200 International Publication No. 2012 / 115209 Chinese Patent No. 106946910

[0011] Bo Wang and Yiqing Lin, Absolute configuration determination of SMTP-7 via microcrystal electron diffraction (MicroED). Chem. Commun., 2022, 58, 13071-13074 DOI: 10.1039 / D2CC05218K

[0012] An object of the present invention is to obtain SMTP from a fermentation product containing SMTP by a method that can be easily scaled up.

[0013] Alternatively, the present invention aims to provide a method for crystallizing SMTP, or to provide a means for obtaining SMTP by a method including crystallization of SMTP.

[0014] The following inventions are provided. [1] A method for producing SMTP, comprising the following steps I to III: I. A step of contacting a fermentation product of Stachybotrys microspora with an aqueous solvent having a pH of 9 or higher to obtain an extract containing SMTP (Stachybotrys Microspora Triprenyl Phenol); II. A step of purifying the extract obtained in step I to obtain a purified product; and III. A step of crystallizing the SMTP contained in the purified product obtained in step II in a crystallization solvent containing one or more selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl tert-butyl ether, and acetonitrile. [2] The production method according to [1], wherein in step I, the pH of the fermentation product is set to 11 or higher. [3] The production method according to [1] or [2], wherein in step II, purification is performed using an aromatic synthetic adsorbent. [4] The method according to any one of [1] to [3], wherein Step II comprises the following Steps II-1 to II-3: II-1. Adjusting the pH of the extract obtained in Step I to 9 to 11; II-2. Adsorbing the SMTP in the extract whose pH has been adjusted in Step II-1 onto an aromatic synthetic adsorbent; and II-3. Eluting the SMTP adsorbed onto the aromatic synthetic adsorbent in Step II-2 with a solvent containing a hydrophilic organic solvent. [5] The method according to any one of [1] to [4], further comprising the following Steps II'-1 to II'-3 after Step II and before Step III: II'-1. Concentrating the purified product obtained in Step II; II'-2. Extracting an organic phase containing SMTP from the concentrate obtained in Step II'-1 with a water-immiscible organic solvent; and II'-3. Treating the extract obtained in Step II'-2 with activated carbon. [6] The method according to [5], wherein the extraction in Step II'-2 is carried out in the presence of a water-soluble salt. [7] The method according to any one of [1] to [6], wherein the crystallization solvent in Step III contains acetone, ethyl acetate, and methyl tert-butyl ether, and the volume ratio of acetone, ethyl acetate, and methyl tert-butyl ether in the crystallization solvent (acetone:ethyl acetate:methyl tert-butyl ether) is 0 to 4:0 to 2:4 to 20.[8] The production method according to any one of [1] to [7], further comprising the following step III' after step III: III'. A step of recrystallizing the SMTP crystallized in step III. [9] A method for producing SMTP, comprising a step of crystallizing SMTP in a crystallization solvent containing one or more selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl tert-butyl ether, and acetonitrile.

[10] The production method according to any one of [1] to [9], wherein SMTP is obtained with a purity of 90% or more.

[11] A method for producing SMTP, comprising contacting a fermentation product of Stachybotrys microspora with an aqueous solvent having a pH of 9 or higher to obtain an extract containing SMTP.

[12] The production method according to any one of [1] to

[11] , wherein the SMTP is SMTP-7.

[0015] The present invention may also include the following: 1) Extraction of SMTP from fermentation broth is accomplished without the use of organic solvents, achieving a very high efficiency of at least 90%. 2) This is achieved by utilizing the Bronsted-Lowry acid-base theory. 3) A strong base is used to deprotonate both the carboxylic acid and phenolic groups, converting the carboxylic acid and / or phenolic functionalities to their respective salt forms. A pH greater than 9.0 (preferably pH 10-13) is used to achieve greater than 90% extraction recovery of SMTP by aqueous methods. 4) The extracted sample is then filtered and subjected to a highly environmentally friendly organic solvent purification process without evaporation or extraction, reducing energy consumption, waste, and production costs. 5) Purification is achieved by adsorption onto a synthetic adsorbent, such as HP20SS, followed by elution with an aqueous Class III solvent to remove impurities with higher and lower polarities than SMTP. The proportion of analog impurities is also reduced, improving purity to about 70% to about 85% r.p. 6) The resin used in the purification process is subsequently regenerated and reused, unlike the silica gel method, which is a single-use method. 7) Because the purified pool has a high SMTP concentration, extraction of SMTP is performed using a small amount of Class III solvent, such as EtOAc, during solvent distillation. This makes the treatment process easier and allows for efficient scalability of the process. 8) To achieve the desired purity, the extracted solution can be treated with activated carbon to enhance the crystallization process and therefore the purification process. The purity of SMTP is increased to approximately 85% to over 98% r.p. 9) The crystallization process allows for selective crystallization of SMTP, as important analogs are retained in the mother liquor. The application of crystallization shortens the process and utilizes limited solvent to provide large quantities of SMTP. 10) This method is very robust, as it has been tested as follows: i) Extraction of SMTP from fermentation broth (120 L) yielded SMTP with an extraction efficiency of approximately 96%. ii) Several resin purification test runs were performed using 1.2 L of resin, with the purity of the desired product being approximately 84-88% rp (the desired level). The process recovery was also high at approximately 95%.iii) The crystallization process also proceeds smoothly, even in a test run of about 60 g, providing the desired SMTP at an r.p. of over 98% and a recovery of about 78%. iv) The robustness of the crystallization process was also tested using a mother liquor sample with a relative purity of 56% r.p., and crystallization occurred to yield SMTP with a relative purity of 97%.

[0016] Effect of pH, temperature, and time on SMTP extraction efficiency. High extraction efficiencies were obtained at pH 11 and 12. Effect of acetone to MTBE ratio on SMTP recovery. Recovery increased with decreasing dissolving solvent (acetone) ratio and increased with increasing anti-solvent (MTBE) ratio. Effect of acetone to MTBE ratio on SMTP purity. Purity increased with increasing dissolving solvent (acetone) ratio and decreased with increasing anti-solvent (MTBE) ratio. Graphical representation of flux for all runs. Graphical representation of flux versus MTBE / SMTP-7 ratio with varying acetone ratio. Filtration rate increased with decreasing anti-solvent (MTBE) ratio and increased anti-solvent (acetone) ratio. HPLC chart of filtrate from alkaline extraction of broth. Highly polar impurities are removed during the column treatment step, leaving the desired product in a more concentrated form that is easily extracted with organic solvent after evaporation of the organic solvent. Using a higher proportion of organic solvent would result in a colored band co-eluting with the desired band, necessitating the use of a larger amount of carbon to stabilize the crystallization process, which would be detrimental to the purification process. HPLC chart of the resin-purified sample. HPLC chart of the crystallized sample. HPLC chart of the recrystallized sample. Recrystallization from acetone and a mixture of ethyl acetate and MTBE improved the purity to approximately 99.9%.

[0017] One embodiment of the present invention relates to a method for obtaining highly pure crystalline SMTP. Those skilled in the art will recognize that there are various variables that can be adjusted during the chromatographic procedure of the present invention. Such variables, such as the type of resin, packing, washing and elution conditions, e.g., ionic strength, buffer composition, pH, temperature, addition of one or more organic solvents, etc., are easily adjusted in the art to establish optimal conditions.

[0018] In the following, when an object is described as "including..." or "having...", it means that the object may have other features, and each of these features can be arbitrarily replaced with "consisting of...", "consisting only of...", etc. "Consisting of..." or "consisting only of..." means that the object does not have any other unspecified features.

[0019] In the present invention, "purity" (sometimes referred to as relative purity) (% or % r.p.) refers to the proportion of the target substance (e.g., SMTP) in the substance (typically a compound) obtained by the method of the present invention or each step in the method of the present invention. Purity can be measured by methods known to those skilled in the art. Taking SMTP as an example, purity can be calculated using the area percentage value of SMTP obtained by HPLC measurement under appropriate conditions. When referring to the purity of SMTP in the present invention, it can be said that "SMTP of a predetermined purity or higher" or the like, but this can be appropriately replaced with "a composition containing SMTP at a predetermined purity or higher," "SMTP as a composition of a predetermined purity or higher," etc.

[0020] In the present invention, "recovery rate" (sometimes referred to as "yield") (%) refers to the ratio of a target substance (e.g., SMTP) in the materials obtained by the method of the present invention or each step of the method of the present invention to the target substance in the materials obtained by the method of the present invention or each step of the method of the present invention, relative to the target substance in the materials used in the method of the present invention or each step of the method of the present invention. The recovery rate can be measured by methods known to those skilled in the art. Taking SMTP as an example, the recovery rate can be calculated using the area percentage value of SMTP obtained by HPLC measurement under appropriate conditions (this value can be multiplied by the volume of the recovered solution to determine the SMTP content in the recovered solution). In addition, in the present invention, the recovery rate for each step is sometimes referred to as "step recovery rate."

[0021] <Method for producing SMTP> A first embodiment of the present invention relates to a method for producing SMTP, comprising the following steps: I. a step of contacting a fermentation product of Stachybotrys microspora with an aqueous solvent having a pH of 9 or higher to obtain an extract containing SMTP; II. a step of purifying the extract obtained in step I to obtain a purified product; and III. a step of crystallizing the SMTP contained in the purified product obtained in step II in a crystallization solvent containing one or more selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl tert-butyl ether, and acetonitrile.

[0022] <Method for Purifying SMTP> The second embodiment of the present invention relates to a method for purifying SMTP. This purification method may include the same steps as the above-described method for producing SMTP.

[0023] [SMTP] SMTP (Stachybotrys Microspora Triprenyl Phenol) is a collective term for a group of triprenylphenols produced by the fungus Stachybotrys Microspora, and unless otherwise specified, refers to a group of compounds represented by the following general formula I:

[0024]

[0025] In one embodiment, the SMTP is SMTP-7, as shown in Formula II.

[0026] According to the production method of the present invention, SMTP can be obtained with a purity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more, preferably 96% or more or 97% or more, more preferably 98% or more, and even more preferably 98.5% or more, 99% or more, or 99.5% or more. A purity of 90% or more can be useful in the production of pharmaceuticals.

[0027] [Step I] Extraction of SMTP from Stachybotrys microspora fermentation product Step I involves aqueous extraction of SMTP from a culture of Stachybotrys microspora. In a preferred embodiment of Step I, a very high SMTP extraction efficiency (i.e., recovery rate) of at least 90% (more preferably 93% or more, even more preferably 96% or more, and particularly preferably 98% or more) is achieved. In a preferred embodiment, the extract obtained in Step I does not contain any methyl esterified SMTP.

[0028] (Stachybotrys microspora fermentation product) The fermentation product of Stachybotrys microspora may typically contain a culture medium, a mixture of the culture medium and cells (suspension), or a cell lysate obtained by inoculating a suitable raw material (e.g., a medium) with Stachybotrys microspora and fermenting the mixture.

[0029] Some embodiments of the present invention may further comprise, before step I, step i of fermenting Stachybotrys microspora to obtain a fermentation product of Stachybotrys microspora.

[0030] (pH) The extraction in step I is achieved by utilizing the Bronsted-Lowry acid-base theory using a predetermined aqueous solvent. The aqueous solvent used in step I may contain a base. The base may be any base that can adjust the pH of the Stachybotrys microspora fermentation product to 9 to 13, and examples thereof include alkali metal hydroxides such as NaOH and KOH; alkali metal carbonates and bicarbonates such as sodium carbonate and sodium bicarbonate; alkaline earth metal hydroxides such as calcium hydroxide; ammonia and ammonium salts (e.g., NH ４ The aqueous solvent may be an aqueous solution of amines such as NaOH (NaOH), ethanolamine, etc. In a preferred embodiment, NaOH is used. The pH of the aqueous solvent is preferably 9.5 or higher, more preferably 10 or higher, even more preferably 11 or higher, and particularly preferably 12 or higher.

[0031] In one preferred embodiment of the extraction method, the pH of the suspension of Stachybotrys microspora fermentation product is directly adjusted to 9 to 13 by contacting with an aqueous solvent. In Step I, the pH of the Stachybotrys microspora fermentation product is preferably adjusted to 9.0 or higher, more preferably 9.5 or higher, even more preferably 10 or higher, particularly preferably 11 or higher, and most preferably 12 or higher. The pH of the Stachybotrys microspora cell suspension is approximately 7. However, adjusting the pH to within the above range is believed to improve the efficiency of extraction of SMTP from the Stachybotrys microspora fermentation product, because it is believed that SMTP can be converted into a salt form of the counter ion (e.g., carboxylic acid groups and phenol groups are deprotonated) and the extraction of hydrophobic impurities derived from the fungus body (which may be unsuitable for purification in Step II, described below) is reduced. On the other hand, the pH of the Stachybotrys microspora fermentation product is preferably 13 or lower. The above pH values ​​may be arbitrarily combined to represent a pH range.

[0032] In one embodiment, the extraction in step I is carried out without using an organic solvent such as methanol, acetone, methyl ethyl ketone, ethyl acetate, etc. By adjusting the pH to within the above range and performing aqueous extraction, the volume of the extraction liquid can be reduced compared to when an organic solvent such as methanol is used, making the extraction more scalable (for example, the amount of Stachybotrys microspora fermentation product used is not limited as long as the desired SMTP is obtained. The amount may be, for example, 300 L or less, 500 L or less, or 1000 L or less, or may include a larger range. On the other hand, the extraction can also be carried out on a small scale, and the amount may be, for example, 1 mL or more, or may include a smaller range. The production method of the present invention can be carried out even when these amounts of Stachybotrys microspora fermentation product are used).

[0033] Extraction Process Step I may include further agitating the slurry following pH adjustment, which facilitates dissolution of the SMTP in the aqueous solution.

[0034] Step I may also include treating the Stachybotrys microspora fermentate prior to extraction. The treatment may include, for example, one or more of acidifying the suspension, adding a filter aid, and filtering the solution to obtain a cell cake. If step I includes these treatments, the cake is subsequently extracted with an aqueous solvent to obtain an extract containing SMTP.

[0035] Filtration can be carried out according to conventional methods, for example, using a filter medium or filter aid, which can remove solids. Examples of filter aids include diatomaceous earth, filter sand (e.g., manganese sand, manganese zeolite, activated carbon, anthracite, ceramic sand), perlite, cellulose, etc. Filter aids can be used alone or in combination of two or more. In a preferred embodiment, perlite is used as the filter aid.

[0036] The aqueous extraction of step I can be carried out at a temperature of, for example, 0°C to 20°C or higher, preferably 30°C or higher, more preferably 40°C or higher, while it can be carried out at a temperature of, for example, 70°C to 100°C or lower, preferably 60°C or lower, more preferably 50°C or lower.

[0037] The aqueous extraction of step I can also be carried out with or without heating, which can result in efficient extraction of SMTP within 30 minutes. In step I, the extracted solution can be separated by any conventional filtration method, such as a filter press, and is preferably washed with an aqueous solvent as described above to obtain a process recovery rate of greater than 90%.

[0038] The duration of the aqueous extraction in step I may be, for example, 5 minutes or more, preferably 10 minutes or more, preferably 20 minutes or more, preferably 30 minutes or more, while it may be 12 hours or less, 6 hours or less, 3 hours or less, or 1 hour or less.

[0039] [Step II] Step II is a step of purifying the extract obtained in Step I. The purification in Step II may be carried out by any method suitable for purifying SMTP, and can be carried out, for example, using an appropriate adsorbent.

[0040] (Aromatic synthetic adsorbents) Purification can be carried out using aromatic synthetic adsorbents. Aromatic synthetic adsorbents have a porous structure, i.e., they have many fine voids either inside or on the surface of the adsorbent, or both. Synthetic adsorbents can adsorb or desorb various organic substances from a solution through physical interactions between the pore surfaces of the resin and the adsorbent. Examples of aromatic synthetic adsorbents include particulate resins having a network structure containing vinyl monomers such as styrene and vinylpyrrolidone and crosslinkable monomers such as divinylbenzene. In one embodiment, the aromatic synthetic adsorbent does not contain electron-withdrawing groups such as aryl or bromine atoms. In a preferred embodiment, the aromatic synthetic adsorbent is a styrene-divinylbenzene synthetic adsorbent.

[0041] The aromatic synthetic adsorbent may be commercially available, for example, Duolite manufactured by Diamond Shamrock Chemical Co. ＴＭ S-30, ES33, S-37, S-382, S-861, S-587, S-761; Amberlite manufactured by Rohm and Haas ＴＭ XAD-2, XAD-4, XAD-7, XAD-8, XAD-16, XAD-1180, XAD-2000, XAD-2010, etc.; Mitsubishi Chemical Industries Co. , Ltd. Diaion made by ＴＭ HP-10, HP-20, HP-21, HP20SS, HP-40, etc.; Sepabeads manufactured by Dow Chemical Co., Ltd. ＴＭSP-850, etc.; Dowex XUS-40323, XUS-40285, etc.; KS, HS, AF, L-1, Polyclar SB-100, Polyclar Super R, Polyclar 10 (PVPP polyvinylpolypyrrolidone) manufactured by Hokuetsu Carbon Industry Co., Ltd.; and Toyo HW-40 manufactured by Toyo Soda Kogyo Co., Ltd. In one embodiment, the aromatic synthetic adsorbent is selected from styrene divinylbenzene synthetic adsorbents HP-20, HP-21, and HP20SS.

[0042] The purity of SMTP in the purified product obtained in step II is preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, and particularly preferably 85% or more, 86% or more, 87% or more, or 88% or more.

[0043] [Step II-1] Step II may include Step II-1, in which the pH of the extract obtained in Step I is adjusted. Adjusting the extract to a predetermined pH increases the charge capacity and reduces the amount of acid required for protonating the salt form of SMTP (to the acidic form). In Step II-1, the pH of the extract is preferably adjusted to 9 or higher, more preferably 10 or higher, and even more preferably 10.5 or higher. This is because a pH below 9 impairs the dissolution of SMTP in aqueous solution, leading to precipitation, which may reduce the adsorption of SMTP on the resin in steps subsequent to Step II and result in column blockage. Furthermore, a pH below 9 also reduces the desired purification efficiency.

[0044] The pH adjustment in step II-1 is typically carried out by slowly adding an acid. The acid may be any acid commonly used for adjusting pH. However, when calcium ions are present in the solution, H is preferably added because the formation of insoluble calcium sulfate may impair the charge capacity and the number of cycles of resin reuse. ２ SO ４ Therefore, in one embodiment, the pH adjustment in step II-1 is carried out by using H ２ SO４ This is done using acids other than

[0045] [Step II-2] Step II may include Step II-2, in which the SMTP in the extract whose pH has been adjusted in Step II-1 is adsorbed onto an aromatic synthetic adsorbent. In a preferred embodiment, Step II-2 improves the purity of SMTP to 70% to 80% or more (about 85%), and the resulting pool band concentration is about 15 g / L.

[0046] [Step II-3] Step II may include Step II-3, in which the SMTP adsorbed on the aromatic synthetic adsorbent in Step II-2 is eluted. In Step II-3, an elution solvent containing a hydrophilic organic solvent can be used. Examples of hydrophilic organic solvents include alcohols such as methanol; ketones such as acetone; and nitriles such as acetonitrile. The hydrophilic organic solvent may contain one of these alone, or may contain two or more of these. In a preferred embodiment, the hydrophilic organic solvent contains acetone.

[0047] When acetone is used, the concentration of acetone in the hydrophilic organic solvent is preferably 60-75 v / v %. When a lower alcohol such as methanol or acetonitrile is used, the concentration of the lower alcohol in the hydrophilic organic solvent is preferably 80-90 v / v %. Organic solvent concentrations higher than these levels should be avoided to prevent less polar impurities from eluting with the desired SMTP and impairing the crystallization process.

[0048] In step II-3, isocratic elution or gradient elution can be performed using a mixed solvent containing 0 to 90 v / v % acetone as the elution solvent. The mixed solvent may contain, for example, an aqueous solution of an inorganic acid such as sulfuric acid, hydrochloric acid, or nitric acid. The pH of these aqueous inorganic acid solutions is preferably 1 to 7, more preferably 1 to 3. When gradient elution is performed, the content and pH of the organic solvent can be changed during elution.

[0049] In step II-3, for example, the eluate may be divided into appropriate fractions, and necessary fractions may be pooled based on the amount and relative purity of the target SMTP analyzed in each fraction, thereby achieving a high recovery rate while satisfying the desired purity.

[0050] In a preferred embodiment, SMTP having a relative purity of more than 80% can be obtained by the operations up to step II-3.

[0051] Furthermore, the aromatic synthetic adsorbent used in the purification can be regenerated or reused, which makes the method of the present invention different from methods using silica gel, which is a single-use method.

[0052] [Step II-2'] The production method of the present invention may further include a step II-2' after step II-2 and before step II-3, in which impurities not adsorbed to the aromatic synthetic adsorbent are removed. In step II-2', impurities having a higher or lower polarity than SMTP are mainly removed. In a preferred embodiment of step II-2', the ratio of impurities is reduced, and the purity of SMTP is improved to about 70% to about 85% r.p.m. Step II-2' may include a step II-2'-1 in which the aromatic synthetic adsorbent is washed with water. The amount of water used in step II-2'-1 may be equal to or greater than the total volume of the column used, and preferably is at least twice the total volume of the column used.

[0053] Step II-2' may include Step II-2'-2, which is subsequent to Step II-2'-1, of treating the aromatic synthetic adsorbent with an acidic aqueous solution. The main purpose of Step II-2'-2 is to convert the carboxylate group of SMTP into a carboxylic acid form. Therefore, the pH of the acidic aqueous solution is preferably 1 to 5, and more preferably 4.7 or less, which is lower than the pKa of the carboxylic acid group. The acid used to adjust the pH of this acidic aqueous solution may be any acid commonly used for adjusting pH, such as H ２ SO ４ According to Step II-2'-2, before Step II-3, the polarity of the impurities and SMTP is ensured and relaxed to enable selective elution of highly polar impurities.

[0054] Step II-2' may include, following Step II-2'-2, Step II-2'-3, in which highly polar impurities are removed using a mixture of an organic solvent and water. In Step II-2'-3, the organic solvent may be, for example, acetone, methanol, or the like. Furthermore, when acetone is used, the concentration of the organic solvent in the mixture is preferably 55 v / v % or less, more preferably 52 v / v % or less, and when methanol is used, it is preferably 80 v / v % or less, more preferably 70 v / v % or less. These solvent ratios are suitable for selectively removing highly polar impurities without impairing the recovery of SMTP. The mixture may also include an acidic aqueous solution.

[0055] [Step II'] The production method of the present invention may further include, after Step II and before Step III, Step II'-1 of concentrating the purified product obtained in Step II, Step II'-2 of extracting an organic phase containing SMTP from the concentrate obtained in Step II'-1 using a water-immiscible organic solvent, and Step II'-3 of treating the extract obtained in Step II'-2 with activated carbon. In a preferred embodiment, Steps II'-1 to II'-3 result in a process recovery rate of SMTP of more than 95%.

[0056] Step II'-1 is a step of concentrating the purified product obtained in Step II. This facilitates the treatment process and enables efficient scale-up of the process. Concentration of the purified product may include, for example, evaporating the elution solvent used in Step II. In Step II'-1, the purified product is preferably concentrated to 30 v / v % or more, more preferably 35 v / v % or more, even more preferably 40 v / v % or more, and particularly preferably 42 v / v % or more.

[0057] Step II'-2 is a step of extracting an organic phase containing SMTP from the concentrate obtained in Step II'-1 using a water-immiscible organic solvent. The water-immiscible organic solvent may be a Class 3 solvent specified in the "Guideline for Residual Solvents in Pharmaceuticals (ICH Q3C)," and can be arbitrarily selected from, for example, ethyl acetate, butyl acetate, propyl acetate, methyl tert-butyl ether, diethyl ether, butanol, cyclohexane, heptane, hexane, chloroform, dichloromethane, and mixtures thereof.

[0058] In a preferred embodiment, the extraction in step II'-2 is carried out in the presence of a water-soluble salt. The presence of the water-soluble salt can achieve good phase separation. The water-soluble salt may be an alkali halide or an alkaline earth halide, and examples thereof include NaCl and KCl. From the viewpoint of achieving efficient phase separation, the amount of the water-soluble salt used is preferably about 0.5 w / v % or more in the concentrate, more preferably 1 w / v % or more, and even more preferably 2 w / v % or more. An example of a preferred combination of a water-immiscible organic solvent and a water-soluble salt is a combination of ethyl acetate and NaCl.

[0059] A water-soluble salt may be added to the purified product obtained in step II before the concentration in step II'-1. This allows for the separation of SMTP as a separable oil having a higher density than water, thereby reducing the amount of organic solvent-water waste mixture. In this case, following concentration, the concentrate is diluted with a water-immiscible organic solvent. Furthermore, the organic phase may be separated and washed with an aqueous salt solution containing a water-soluble salt to promote dehydration. From the viewpoint of achieving efficient dehydration, the concentration of the water-soluble salt in the aqueous salt solution is preferably at least 2 w / v % or more, more preferably 3 w / v % or more, even more preferably 4 w / v % or more, and particularly preferably 5 w / v % or more. Furthermore, from the viewpoint of increasing the crystallization efficiency in step III, washing is preferably performed twice.

[0060] Step II'-3 is a step of treating the extract obtained in Step II'-2 with activated carbon. Step II'-3 removes impurities, further improving the crystallization process in Step III. Furthermore, in a preferred embodiment, Step II'-3 increases the purity of SMTP to about 85% to 98% r.p. or higher. The activated carbon treatment may be performed, for example, via a batch method, a carbon filter treatment method, or the like. From the perspective of scale-up, the use of a carbon filter is preferred, as it is expected to improve the treatment process. The activated carbon used preferably has a carbon content of 1 wt / w% or more, more preferably 1.5 wt / w% or more, and even more preferably 1.8 wt / w% or more, based on the weight of SMTP, but may also have a carbon content of 5 wt / w% or less.

[0061] [Step III] Step III is a step in which the SMTP contained in the purified product obtained in Step II or the SMTP contained in the activated carbon-treated product obtained in Step II'-3 is crystallized in a predetermined crystallization solvent. In the crystallization process, related impurities that could not be removed in the stages up to Step III are retained in the mother liquor, thereby enabling selective crystallization of SMTP. Crystallization of SMTP can be confirmed by any method known to those skilled in the art. Such a method may be, for example, observation using a polarizing microscope or confirmation of peaks derived from crystals by X-ray diffraction.

[0062] (Crystallization Solvent) The crystallization solvent refers to a solvent including a dissolving solvent (also called a good solvent) and a poor solvent used in crystallization. The crystallization solvent may include, for example, one or more solvents selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl tert-butyl ether (MTBE), and acetonitrile. The crystallization solvent may include one of these solvents alone, or may include two or more of these solvents.

[0063] Step III may include dissolving the purified product obtained in Step II or the activated carbon-treated product obtained in Step II'-3 in a dissolution solvent and a poor solvent. This solution will be referred to as a solution of SMTP in the following description of Step III and Step IV.

[0064] The dissolution solvent may be, for example, one or more selected from acetone, ethyl acetate, 2-butanone, and acetonitrile, and preferably one or two selected from ethyl acetate and acetone. The amount of the dissolution solvent used may be 0.5 v / w % or more, preferably 1 v / w % or more, more preferably 1.3 v / w % or more, and may be 4 v / w % or less, based on the weight of the SMTP to be crystallized.

[0065] The ratio of the volume of the dissolving solvent to the weight of the crystallized SMTP (volume of the dissolving solvent (mL) / weight of SMTP (g)) is preferably 0.5 to 3.6 in consideration of the purity of the resulting SMTP, and more preferably 0.5 to 1.6 in consideration of the recovery rate of SMTP.

[0066] The anti-solvent may be, for example, one or more selected from diisopropyl ether, diethyl ether, and methyl tert-butyl ether, preferably methyl tert-butyl ether. The amount of the anti-solvent used may be 2 v / w % or more, preferably 3 v / w % or more, based on the weight of the SMTP to be crystallized, and may be 25 v / w % or less, preferably 20 v / w % or less.

[0067] The ratio of the volume of the anti-solvent to the weight of the crystallized SMTP (anti-solvent volume (mL) / SMTP weight (g)) is preferably 4 to 20 in consideration of the purity of the resulting SMTP, and more preferably 6 to 20 in consideration of the recovery rate of SMTP.

[0068] In a preferred embodiment, the crystallization solvent comprises acetone, ethyl acetate, and methyl tert-butyl ether, and further has the following composition: Acetone: 0 to 4 v / w %, more preferably 0.5 to 4 v / w %, and even more preferably 0.5 to 3.6 v / w %, based on the weight of the SMTP to be crystallized Ethyl acetate: 0 to 2 v / w %, based on the weight of the SMTP to be crystallized Methyl tert-butyl ether: 4 to 20 v / w %, more preferably 4 to 19.9 v / w %, based on the weight of the SMTP to be crystallized

[0069] In another preferred embodiment, the crystallization solvent contains acetone, ethyl acetate, and methyl tert-butyl ether, and the volume ratio of acetone, ethyl acetate, and methyl tert-butyl ether in the crystallization solvent (acetone:ethyl acetate:methyl tert-butyl ether) is 0-4:0-2:4-20, more preferably 0.5-4:0-2:4-20, and even more preferably 0.5-3.6:0-2:4-19.9. Note that a volume ratio of 0 means that the component is not contained (the same applies hereinafter).

[0070] In another preferred embodiment, the crystallization solvent contains acetone, ethyl acetate, and methyl tert-butyl ether, and has the following composition: Acetone: 0 to 10 v / v % relative to the total amount of the crystallization solvent, more preferably 0 to 8 v / v % Ethyl acetate: 0 to 20 v / v % relative to the total amount of the crystallization solvent, more preferably 2 to 15 v / v % Methyl tert-butyl ether: 70 to 95 v / v % relative to the total amount of the crystallization solvent, more preferably 80 to 90 v / v %

[0071] By using the crystallization solvents having the above-described compositions, it becomes possible to crystallize SMTP, and furthermore, the crystal size of the resulting SMTP can be increased. Small crystal sizes result in dense cakes during filtration, significantly increasing the filtration time (significantly decreasing the filtration rate), so larger crystal sizes are preferred. Furthermore, larger crystal sizes are preferred because they provide better washability when using a washing solution to wash away impurities from the mother liquor adhering to the crystals. Crystal size can be measured by any method known to those skilled in the art, such as using a polarizing microscope or an optical microscope.

[0072] Alternatively, the crystallization solvent may contain acetone and methyl tert-butyl ether, and the volume ratio of acetone to methyl tert-butyl ether (acetone:methyl tert-butyl ether) in the crystallization solvent may be 0 to 1.5:6 to 20, more preferably 0.3 to 1.3:8 to 16, and even more preferably 0.4 to 0.9:10 to 16. Alternatively, the crystallization solvent may have the following composition: Acetone: 0 to 1.5 v / w %, more preferably 0.3 to 1.3 v / w %, and even more preferably 0.4 to 0.9 v / w %, based on the weight of the SMTP to be crystallized Methyl tert-butyl ether: 6 to 20 v / w %, more preferably 8 to 16 v / w %, and even more preferably 10 to 16 v / w %, based on the weight of the SMTP to be crystallized By using a crystallization solvent having a composition within these ranges, the recovery rate as well as the purity of the resulting SMTP can be improved.

[0073] (Crystallization Process) Step III may further include concentrating the solution containing the purified product obtained in Step II or the activated carbon-treated product obtained in Step II'-3 before dissolving it in a crystallization solvent. The concentration may be, for example, by evaporating and removing the extraction solvent (e.g., EtOAc) to a concentration of 3 v / w % or less, preferably 2 v / w % or less, more preferably 1.6 v / w % or less, based on the weight of SMTP.

[0074] In step III, dissolving the purified product obtained in step II or the activated carbon-treated product obtained in step II'-3 in a dissolution solvent and a poor solvent may involve heating the crystallization solvent. This is because heating may promote dissolution. When the crystallization solvent is heated, the temperature may be 35°C or higher, preferably 40°C or higher, or may be 60°C or lower, preferably 55°C or lower, and more preferably 50°C or lower.

[0075] Step III may further comprise maintaining the solution of SMTP at 40° C. or higher, preferably 45° C. or higher, more preferably 50° C. or higher, for 1 hour or longer, preferably 2 hours or longer. Step III may further comprise stirring the solvent.

[0076] Subsequently, step III may further include maintaining the SMTP solution at, for example, 50° C. or lower, 45° C. or lower, 40° C. or lower, or 35° C. or lower, preferably 30° C. or lower or room temperature, for 5 hours or longer, preferably 10 hours or longer, or 15 hours or longer. Note that the temperature does not need to be constant and may, for example, be gradually decreased.

[0077] Step III may include adding seed crystals of SMTP to the solution of SMTP. Adding seed crystals of SMTP can promote the crystallization of SMTP. The amount of seed crystals of SMTP added may be, for example, 0.01 to 5 wt % based on the weight of the solution of SMTP.

[0078] Step III may further include adding a poor solvent to the solution of SMTP. This allows for good nucleation. When a poor solvent is further added, the concentration of the poor solvent in the solution of SMTP is preferably 4 to 23 wt %, more preferably 4 to 20 wt %.

[0079] [Step III'] The production method of the present invention may further include a step III' after step III and before step IV, in which the SMTP crystallized in step III is recrystallized. In a preferred embodiment, the purity of the resulting SMTP is 99.5% or higher after recrystallization. The recrystallization method may be the same as that described in the section on step III, independent of step III.

[0080] In the preferred embodiment shown in this example, scale-up of the crystallization process has been extensively tested and shown to yield the desired product in greater than 99% purity.

[0081] [Step IV] The method of the present invention may further include Step IV, in which the SMTP crystallized in Step III is recovered. Recovery of the crystallized SMTP can be carried out by a method known to those skilled in the art, and may include separation, washing, drying, etc. of the crystallized SMTP. Separation of the crystallized SMTP can be carried out using a method known to those skilled in the art, such as filtration or centrifugation. Washing can be carried out, for example, using a mixture of the same dissolution solvent and anti-solvent used in Step III.

[0082] A third embodiment of the present invention relates to a method for producing SMTP, comprising the steps of: contacting a fermentation product of Stachybotrys microspora with an aqueous solvent having a pH of 9 or higher to obtain an extract containing SMTP.

[0083] The features in the third embodiment may be the same as the features in the SMTP extraction process described above, and can be selected arbitrarily.

[0084] According to the third embodiment, SMTP can be obtained from the fermentation product of Stachybotrys microspora with a recovery rate of 80% or more, in a preferred embodiment with a recovery rate of 85% or more, in a more preferred embodiment with a recovery rate of 90% or more, in an even more preferred embodiment with a recovery rate of 93% or more, in a most particularly preferred embodiment with a recovery rate of 96% or more, and in a preferred embodiment with a recovery rate of 98% or more. Also, according to the third embodiment, SMTP can be obtained with a purity of 50% or more, in a preferred embodiment with a purity of 55% or more, in a more preferred embodiment with a purity of 60% or more, in an even more preferred embodiment with a purity of 65% or more, and in a particularly preferred embodiment with a purity of 70% or more.

[0085] A fourth embodiment of the present invention relates to a method for producing SMTP or a method for producing crystals of SMTP, comprising the following steps: crystallizing SMTP in a crystallization solvent comprising one or more selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl tert-butyl ether, and acetonitrile.

[0086] In the fourth embodiment, the purity of the SMTP used is not particularly limited as long as SMTP crystals can be obtained. For example, SMTP with a purity of 70% or more can be used, preferably SMTP with a purity of 75% or more, 80% or more, or 85% or more, and more preferably SMTP with a purity of 90% or more, 95% or more, or 98% or more. The SMTP may be prepared to a predetermined purity or higher by any method. Furthermore, the SMTP that can be used in this embodiment preferably does not contain a methyl esterified product. The type and other forms of the SMTP can be selected as described above.

[0087] The features of the crystallization step in the fourth embodiment can be selected arbitrarily, similar to the features of the crystallization step described above. The fourth embodiment may further include a step of recrystallizing the crystallized SMTP. The features of the recrystallization step may be the same as the features of the recrystallization step described above, and can be selected arbitrarily.

[0088] <Method for extracting SMTP> The fifth embodiment of the present invention relates to a method for extracting SMTP. The crystallization method of the present invention may optionally have the same features as the features of the above-mentioned SMTP extraction step.

[0089] <Method for Crystallizing SMTP> The sixth embodiment of the present invention relates to a method for crystallizing SMTP. The crystallization method of the present invention may optionally have the same features as the features of the crystallization step described above.

[0090] A seventh embodiment of the present invention is described as a method for producing or purifying SMTP, comprising the steps of: a. extraction of SMTP under basic conditions and filtering the fermentation broth, b. optional pH adjustment of the filtrate from step (a) by adding acid, c. purification of SMTP by loading the filtrate from step (a) or step (b) onto a reversed phase resin followed by elution, d. recovery of purified SMTP from the reversed phase chromatographic eluate, e. concentration of the reversed phase chromatographic eluate from step (d) by evaporation to minimize the amount of organic solvent, f. extraction of the concentrate from step (e) with a water-immiscible organic solvent, g. optional addition of salt to step (f) to improve liquid-liquid phase separation, h. washing the organic phase from step (g) with brine to reduce water content and high polarity solvent, i. The process comprises the steps of: treating the organic phase from step (h) with activated carbon (either batch, column, or filter type) to enhance the crystallization process; j. concentrating the solution from step (i) followed by crystallization from EtOAc and / or acetone and MTBE; k. adding high purity seed crystals at elevated temperatures to step (j) to improve particle size and the crystallization process; l. cooling the slurry once sufficient nucleation has occurred to increase recovery; m. separating the desired product from the slurry by any of the known filtration methods, rinsing, and drying.

[0091] Abbreviations used in the examples PTFE: Polytetrafluoroethylene membrane filter MTBE: Methyl tert-butyl ether MeOH: Methanol EtOAc: Ethyl acetate

[0092] In this example, the recovery rate (%) is a value obtained by analyzing the recovered solution in each step by HPLC, multiplying the quantitative value (concentration) by the volume of the recovered solution to determine the SMTP content, and calculating the ratio of this to the amount of SMTP used in each step. Also, in this example, the purity (relative purity) (% or % r.p.) is a value obtained from the area % of HPLC.

[0093] Example 1: Scale-up Implementation 1. Extraction and Filtration of SMTP-7 Solution from Fermentation Broth Fermentation of SMTP-7 was carried out in a 200 L fermentor. After the fermentation process was completed, the pH of the fermentation broth was adjusted from approximately 7 (neutral) to approximately 11.5 by adding 9 M NaOH solution. The broth (111 L) was then diluted two-fold by adding an equal volume of water. The diluted fermentation broth (222 L, 965 g of SMTP-7) was stirred at 40°C or 50°C for 30 minutes or 1 hour, after which a filter aid, perlite (3% w / v broth, 3.6 kg), was added, and the slurry was mixed for approximately another 10 minutes. The biomass was separated by a filter press method and then rinsed with a basic aqueous solution (pH 11.0, 60 L). The extraction efficiency of SMTP-7 was estimated to be 930 g out of the 965 g in the initial fermentation broth, or 96.5%, recovered in the filtrate, indicating that the purity of SMTP was approximately 70% r.p. (Figure 6). Furthermore, HPLC results indicated that no methyl esterified SMTP was detected (Figure 6). This result demonstrated that the developed aqueous extraction method for SMTP is highly efficient, even on an industrial scale. The purity of SMTP remained unchanged before and after the extraction process, indicating that all SMTP analogs were obtained with similar extraction efficiencies. Therefore, this method is not limited to SMTP-7 but applies to the SMTP class of compounds.

[0094] The pH of the fermentation broth was adjusted to various pH values ​​from 5 to 12, and similar extractions were performed. The pH of the cell suspension was approximately 7, and at this pH, the proportion of SMTP in the supernatant was approximately 20%, which further decreased to approximately 11% when the pH was lowered to approximately 5. When the pH was further increased to 11, the SMTP in the supernatant increased to approximately 93%. When the pH was further increased to approximately 12, the extraction recovery improved to approximately 99% (Figure 1).

[0095] 2. Purification by Synthetic Aromatic Adsorbent a) Aqueous Acetone Elution System The pH of the SMTP-7 filtrate (4 L, 27.2 g SMTP-7) obtained by the above method was adjusted to pH 10.5 by adding acid. This solution was filtered and then loaded onto a column packed with 1.2 L DIAION HP20SS (Mitsubishi Chemical Co., Ltd.) at a flow rate of less than 2 head volumes / hour. After loading, the resin was washed with 2 BV of water (twice the head volume of the column) and then with H. ２ SO ４ The resin was washed with 2 B.V. of aqueous acid (pH 1.5). This was followed by treatment with 2 B.V. of 20% acetone-water to swell the resin, followed by treatment with 4 B.V. of 52% acetone-water to remove highly polar impurities. The desired SMTP-7 band was then eluted with 3 B.V. of 68% acetone-water. The desired band was determined by HPLC / UV and pH monitoring methods. This procedure led to a recovery of approximately 96% (approximately 26 g SMTP-7) and a relative purity of 88% r.p. (Figure 7).

[0096] b) Aqueous MeOH elution system: This method also provides the desired level of purification, however, esterification of small amounts of SMTP-7 during workup results in additional impurities in the final product. Therefore, the acetone-water system was preferred.

[0097] Extraction and Carbon Treatment Method: Three resin purification runs were performed, and the resulting pool (75.54 g SMTP-7, 6.2 L) was used in this extraction step. Acetone was distilled by evaporation until approximately 2.6 L of a 42% v / v concentrate was obtained. To this, approximately 1.3 L of ethyl acetate (17 v / w) and NaCl (51.6 g, 2% w / v concentrate) were added and mixed for approximately 10 minutes. The bottom aqueous phase was removed, and the organic phase was washed twice with approximately 5% w / v aqueous NaCl (630 mL x 2). The recovery of SMTP-7 from the evaporation and solvent extraction steps was 98% (approximately 74 g SMTP-7).

[0098] After sampling, the residual ethyl acetate extract (72.9 g, SMTP-7) was passed through a B47-R53SP Zeta carbon filter (3M Corporation) with a total carbon content of approximately 1.8% w / w based on the weight of SMTP-7, resulting in a process recovery of 98% (71.5 g SMTP-7). The resulting solution was then used in the following crystallization process.

[0099] 3. Crystallization Step: SMTP-7 / Acetone / Ethyl Acetate / MTBE (60 g / 80 mL / 363 mL / 887 mL). The carbon-treated solution (1.09 L, 60 g of SMTP-7) obtained by the above process was filtered through 1 μm PTFE and concentrated by evaporation (target 1.6-3 w / w) until a semi-solid paste (approximately 96 g) was obtained. To this paste, acetone (80 mL, 1.33 v / w) was added and mixed at approximately 40-50°C, followed by the addition of MTBE (213 mL, 3.54 v / w). The solution was mixed until complete dissolution was achieved, and more MTBE (674 mL, 11.2 v / w) was added. When the temperature reached approximately 50°C, SMTP-7 seed crystals (120 mg, 0.2% w / w) were added, and mixing was continued for approximately 2 hours while maintaining the temperature above 50°C. The slurry temperature was cooled to about 45° C. and mixing was continued for 4 hours. The slurry was cooled to room temperature and mixing was continued for 14 hours.

[0100] The desired product was filtered and washed with approximately 50 mL of 1:10 acetone / MTBE. The resulting crystals were dried under reduced pressure to give 47 g of SMTP-7 crystals (99.2% rp, 78% step recovery). The overall recovery rate for this purification process was approximately 70%.

[0101] Example 2. Crystallization: SMTP-7 / Acetone / Ethyl Acetate / MTBE (22 g / 0 mL / 53 mL / 322 mL) The HP20SS purified sample was evaporated and extracted in a manner similar to that described in Example 1, up to the "Extraction and Carbon Treatment Method" section. The ethyl acetate extract (23.2 g, SMTP-7) was batch treated with approximately 5% w / w of SG280P activated carbon (Futamura Chemical Co.) based on the weight of SMTP-7. The slurry was mixed for 30 minutes, filtered through PTFE (1.0 μm), rinsed with ethyl acetate, and concentrated to yield 33.5 g of wet SMTP-7 (21.5 g, net). Ethyl acetate (53 mL, 2.4 v / w) was added to this paste and mixed at approximately 40-50°C, followed by the addition of MTBE (322 mL, 14.6 v / w). Once a temperature of approximately 50°C was achieved, SMTP-7 seed crystals (64.5 mg, 0.29% w / w) were added and mixing was continued for approximately 2 hours while maintaining the temperature above 50°C. The slurry was then cooled to approximately 45°C and mixing was continued for 4 hours. The slurry was cooled to room temperature and mixing was continued for 14 hours. The desired product was filtered and rinsed with approximately 43 mL of 1 / 10 EtOAc / MTBE. The resulting crystals were dried under reduced pressure to yield 18.9 g of SMTP-7 crystals (98.8% rp, 88.2% process recovery). The crystal particle size was very small (less than 2 μm). The filtration rate (flux, 104 LMH) was very low, which was due to the formation of small crystals.

[0102] Example 3. Crystallized SMTP-7 / Acetone / Ethyl Acetate / MTBE (22 g / 40 mL / 12 mL / 360 mL) The HP20SS purified sample was evaporated and extracted in a manner similar to that described in Example 1, up to the "Extraction and Carbon Treatment Method" section. The ethyl acetate extract (24 g, SMTP-7) was batch treated with approximately 5% w / w of SG280P activated carbon (Futamura Chemical Co.) based on the weight of SMTP-7. The slurry was mixed for 30 minutes, filtered through PTFE (1.0 μm), rinsed with ethyl acetate, and concentrated to yield 35 g of wet SMTP-7 (21.85 g, net). Acetone (40 mL, 1.8 v / w) was added to this paste and mixed at approximately 40-50°C, followed by the addition of MTBE (350 mL, 16.3 v / w). Once a temperature of approximately 50°C was achieved, SMTP-7 seed crystals (64.5 mg, 0.29% w / w) were added, and mixing was continued for approximately 2 hours while maintaining the temperature above 50°C. The slurry temperature was cooled to approximately 45°C, and mixing was continued for 4 hours. The slurry was cooled to room temperature, and mixing was continued for 14 hours. The desired product was filtered and rinsed with approximately 43 mL of 1 / 10 acetone / MTBE. The resulting crystals were dried under reduced pressure to yield 16.5 g of SMTP-7 crystals (99.4% rp, 75.5% process recovery). The crystals had a large particle size (5-15 μm). The filtration rate (flux, 781 LMH) for this run was approximately 7 times faster than in Example 2. The enhanced flux was due to the larger particle size of the crystals from the acetone / EtOAc / MTBE system.

[0103] Example 4. Crystallized SMTP-7 / Acetone / Ethyl Acetate / MTBE (22 g / 40 mL / 12 mL / 360 mL) The HP20SS purified sample was evaporated and extracted in a manner similar to that described in Example 1, up to the "Extraction and Carbon Treatment Method" section. The ethyl acetate extract (21.85 g, SMTP-7) was batch treated with approximately 5% w / w of SG280P activated carbon (Futamura Chemical Co.) based on the weight of SMTP-7. The slurry was mixed for 30 minutes, filtered through PTFE (1.0 μm), rinsed with ethyl acetate, and concentrated to yield 35 g of wet SMTP-7 (21.85 g, net). To this paste, acetone (10 mL, 0.46 v / w) and acetone (30 mL, 1.5 v / w total) were added and mixed at approximately 40-50°C, followed by the addition of MTBE (350 mL, 16.3 v / w). Once a temperature of approximately 50°C was achieved, SMTP-7 seed crystals (64.5 mg, 0.29% w / w) were added, and mixing was continued for approximately 2 hours while maintaining the temperature above 50°C. The slurry temperature was cooled to approximately 45°C, and mixing was continued for 4 hours. The slurry was cooled to room temperature, and mixing was continued for 14 hours. The desired product was filtered and rinsed with approximately 43 mL of 1 / 10 acetone / MTBE. The resulting crystals were dried under reduced pressure to yield 16.1 g of SMTP-7 crystals (99.52% r.p., 73.7% process recovery). The crystals had a large particle size (5-15 μm). The filtration rate (flux, 2083 LMH) for this run was approximately 20 times faster than in Example 2. The enhanced flux was due to the larger particle size of the crystals from the acetone / EtOAc / MTBE system.

[0104] Example 5. Study of the composition of the crystallization solvent A test similar to that in Example 1 was conducted by changing the ratio of acetone to MTBE in the solvent used for crystallization. The relationship between the ratio of acetone or MTBE in the solvent used for crystallization and the recovery rate or purity of the resulting SMTP-7 crystals is shown in Figure 2 (recovery rate) or Figure 3 (purity). Furthermore, the relationship between the ratio of acetone or MTBE in the solvent used for crystallization and the filtration rate (flux) of the resulting SMTP-7 crystals is shown in Figures 4-5.

Claims

1. A method for producing SMTP, comprising the following steps I to III: I. a step of contacting a fermentation product of Stachybotrys microspora with an aqueous solvent having a pH of 9 or higher to obtain an extract containing SMTP (Stachybotrys Microspora Triprenyl Phenol); II. a step of purifying the extract obtained in step I to obtain a purified product; and III. a step of crystallizing the SMTP contained in the purified product obtained in step II in a crystallization solvent containing one or more selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl tert-butyl ether, and acetonitrile.

2. The method of claim 1, wherein in step I, the pH of the fermented product is adjusted to 11 or higher.

3. The method according to claim 1, wherein purification is carried out using an aromatic synthetic adsorbent in step II.

4. The production method according to claim 3, wherein step II comprises the following steps II-1 to II-3: II-1. A step of adjusting the pH of the extract obtained in step I to 9 to 11; II-2. A step of adsorbing the SMTP in the extract whose pH has been adjusted in step II-1 onto an aromatic synthetic adsorbent; and II-3. A step of eluting the SMTP adsorbed onto the aromatic synthetic adsorbent in step II-2 with a solvent containing a hydrophilic organic solvent.

5. The production method according to claim 1, further comprising the following steps II'-1 to II'-3 after step II and before step III: II'-1. A step of concentrating the purified product obtained in step II; II'-2. A step of extracting an organic phase containing SMTP from the concentrate obtained in step II'-1 using a water-immiscible organic solvent; and II'-3. A step of treating the extract obtained in step II'-2 with activated carbon.

6. The method according to claim 5, wherein the extraction in step II'-2 is carried out in the presence of a water-soluble salt.

7. The method according to claim 1, wherein the crystallization solvent in step III contains acetone, ethyl acetate, and methyl tert-butyl ether, and the volume ratio of acetone, ethyl acetate, and methyl tert-butyl ether in the crystallization solvent (acetone:ethyl acetate:methyl tert-butyl ether) is 0-4:0-2:4-20.

8. The method of claim 1, further comprising the following step III' after step III: III': a step of recrystallizing the SMTP crystallized in step III.

9. A method for producing SMTP, comprising the step of crystallizing SMTP in a crystallization solvent containing one or more selected from the group consisting of ethyl acetate, butyl acetate, acetone, 2-butanone, diisopropyl ether, diethyl ether, methyl tert-butyl ether, and acetonitrile.

10. The method according to any one of claims 1 to 9, wherein SMTP is obtained with a purity of 90% or more.

11. A method for producing SMTP, comprising the step of contacting a fermentation product of Stachybotrys microspora with an aqueous solvent having a pH of 9 or higher to obtain an extract containing SMTP.

12. The method according to any one of claims 1 to 9 or 11, wherein the SMTP is SMTP-7.

Images