Manufacturing method of smtp

Abstract

To provide means for obtaining SMTP from a fermentation product containing SMTP by a method that is easy to scale up, to provide a method for crystallizing SMTP, or to provide means for obtaining SMTP by a method including crystallization of SMTP.SOLUTION: Provided is a method for producing SMTP, including: a step I 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; a step II of purifying the extract obtained in the step I to obtain a purified product; and a step III of crystallizing the SMTP contained in the purified product obtained in the 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.SELECTED DRAWING: None

Description

[Technical Field]

[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. [Background technology]

[0002] SMTP (Stachbotrys Microspora Triprenyl Phenol) is a general term for a group of triprenylphenols produced by the fungus Stachybotrys Microspora. They consist 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 models of cerebral infarction caused by thrombus or embolism (Patent Document 1). SMTP-7 also has antioxidant and anti-inflammatory effects (Patent Document 2).

[0003] Although the therapeutic applications of SMTP-7 are described in Patent Documents 1 and 2, the purification process of SMTP is only briefly mentioned. 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] Patent Documents 3 and 4 disclose a method for selectively producing SMTP. These patent documents also disclose the following purification methods: The fermentation broth is extracted with methanol. The extract is then concentrated by spin distillation and then extracted with ethyl acetate. The solution is then dehydrated using anhydrous sodium sulfate, filtered, concentrated, and dried. The solidified material is then dissolved in methanol and purified using a reversed-phase packing. The target product is then extracted with ethyl acetate and another step is performed 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, since 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 only one of EtOAc or acetic acid is used for solvent extraction. 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 Patent Documents 4 and 6 (HPLC) or Patent Documents 7 and 8 (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, a large amount of waste is generated, increasing the environmental impact and costs. Furthermore, when fine powders such as silica gel are used for 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 sample can be handled and the desired purity cannot be achieved in one cycle, so the purification process must be repeated with different solvent systems (Patent Document 4). Therefore, scaling up under these conditions is very difficult, and the environment, health, and safety (EHS) are greatly 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. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2010 / 110026 [Patent Document 2] International Publication No. 1998 / 56940 [Patent Document 3] Patent No. 4257026 [Patent Document 4] International Publication No. 2022 / 171151 [Patent Document 5] Japanese Patent Application Laid-Open No. 2004 / 224737 [Patent Document 6] Chinese Patent No. 105153200 [Patent Document 7] International Publication No. 2012 / 115209 [Patent Document 8] Chinese Patent No. 106946910 [Non-patent literature]

[0011] [Non-Patent Document 1] 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 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[0014] The following inventions are provided: [1] A method for producing SMTP, comprising the following steps I to III: I. A step of contacting the fermentation product of Stachybotrys microspora with an aqueous solvent having a pH of 9 or higher to obtain an extract containing SMTP (Stachbotrys Microspora Triprenyl Phenol); II. 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 [1], wherein in step I, the pH of the fermented product is set to 11 or higher. [3] The method according to [1] or [2], wherein purification is carried out using an aromatic synthetic adsorbent in step II. [4] The method according to any one of [1] to [3], wherein step II includes 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 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 using 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 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 [5], wherein the extraction in step II'-2 is carried out in the presence of a water-soluble salt. [7] The crystallization solvent in Step III comprises acetone, ethyl acetate, and methyl tert-butyl ether; The production method according to any one of [1] to [6], wherein 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 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 comprising the steps 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; How SMTP is produced.

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

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

[12] The 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) The extraction of SMTP from the fermentation broth is carried out without the use of organic solvents, achieving a very high efficiency of at least 90%. 2) The above is achieved by utilizing the Bronsted-Lowry acid-base theory. 3) Converting the carboxylic acid and / or phenolic functionalities to their respective salt forms using a strong base that deprotonates both the carboxylic acid and phenolic groups. To achieve greater than 90% extraction recovery of SMTP by aqueous methods, a pH greater than 9.0 (preferably pH 10-13) is used. 4) The extracted sample is then filtered and subjected to a purification process using organic solvents without evaporation or extraction, reducing energy consumption, waste and production costs, making it very environmentally friendly. 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 approximately 70% to approximately 85% rp. 6) The resin used in the purification process is then regenerated and reused, unlike the silica gel process, which is a single-use method. 7) Because the purified pool band has a high SMTP concentration, extraction of SMTP is performed using a small amount of Class III solvent, such as EtOAc, during solvent distillation, which makes the treatment process easier and allows for efficient scalability of the process. 8) To achieve the desired purity, the extract solution can be treated with activated carbon to enhance the crystallization process and therefore the purification process. The purity of SMTP can be increased to approximately 85% to over 98% rp. 9) The crystallization process allows for selective crystallization of SMTP, as important analogs are retained in the mother liquor. The application of crystallization makes the process shorter and utilizes limited solvent to provide large amounts of SMTP. 10) This method is very robust as it has been tested as follows: i) Extraction of SMTP from the fermentation broth (120 L) yields SMTP with an extraction efficiency of approximately 96%. ii) Several resin purification test runs were performed using 1.2 L of resin, and the purity of the desired product was approximately 84-88% rp (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 with an rp of over 98% and a recovery of about 78%. iv) The robustness of the crystallization process is also tested using a mother liquor sample with a relative purity of 56% rp, and crystallization occurs to give SMTP with a relative purity of 97%. [Brief explanation of the drawings]

[0016] [Figure 1] Effects of pH, temperature, and time on the extraction efficiency of SMTP. High extraction efficiency was obtained at pH 11 and 12. [Figure 2] Effect of the ratio of acetone to MTBE on the recovery of SMTP. The recovery rate increased with decreasing the ratio of the dissolving solvent (acetone) and with increasing the ratio of the antisolvent (MTBE). [Figure 3] The effect of the ratio of acetone to MTBE on the purity of SMTP. The purity increases with increasing ratio of the dissolving solvent (acetone) and decreases with increasing ratio of the anti-solvent (MTBE). [Figure 4] Graphical representation of flux for all runs. [Figure 5]Graphical representation of flux versus MTBE / SMTP-7 ratio for varying acetone ratio. Filtration rate increased with decreasing antisolvent (MTBE) ratio and increased with increasing dissolving solvent (acetone) ratio. [Figure 6] HPLC analysis of the filtrate from alkaline extraction of the broth. Polar impurities are removed during the column process, leaving the desired product in a more concentrated form that is easier to extract with organic solvent after evaporation of the organic solvent. Using a higher proportion of organic solvent results in a colored band co-eluting with the desired band, necessitating the use of larger amounts of carbon to stabilize the crystallization process, which is detrimental to the purification process. [Figure 7] HPLC chart of resin purified sample. [Figure 8] HPLC chart of crystallized sample. [Figure 9] HPLC chart of the recrystallized sample. Recrystallization from acetone and a mixture of ethyl acetate and MTBE improved the purity to approximately 99.9%. DETAILED DESCRIPTION OF THE INVENTION

[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 readily adjusted in the art to establish optimal conditions.

[0018] In the following, when "comprises...", "has...", etc. are used, it means that other features may be included, and each of these features can be arbitrarily replaced with "consisting of...", "consisting only of...", etc. "consisting of..." or "consisting only of..." means that no other unspecified features are included.

[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 the case of SMTP as an example, it 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 expressed as "SMTP with a predetermined purity or higher", etc., but this can be appropriately replaced with "a composition containing SMTP with a predetermined purity or higher", "SMTP as a composition with a predetermined purity or higher", etc.

[0020] In the present invention, "recovery rate" (sometimes denoted as "yield") (%) refers to the proportion of the target substance (e.g., SMTP) in the substance obtained by the method of the present invention or each step in the method of the present invention to the target substance in the substance used in the method of the present invention or each step in the method of the present invention. The recovery rate can be measured by methods known to those skilled in the art. Taking the case of SMTP as an example, it can be calculated using the area percentage value of SMTP obtained by HPLC measurement under appropriate conditions (the content of SMTP in the recovered solution can be obtained by multiplying this by the volume of the recovered solution). In the present invention, the recovery rate for each step may also be denoted as "step recovery rate".

[0021] <Method for Producing SMTP> The first embodiment of the present invention relates to a method for producing SMTP, which includes 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 subjecting the extract obtained in step I to purification to obtain a purified product; and III. A step of crystallizing 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 (Stachbotrys Microspora Triprenyl Phenol) is a general term for a group of triprenyl phenols produced by the fungus Stachbotrys Microspora, and refers to a group of compounds represented by the following general formula I unless otherwise specified.

[0024] [Chemical Formula] R = H, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group

[0025] In one aspect, SMTP is SMTP-7 represented by Formula II. [Chemical Formula]

[0026] According to the production method of the present invention, SMTP is 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 may be useful in the production of pharmaceuticals.

[0027] [Step I] Extraction of SMTP from the Fermentation Product of Stachbotrys Microspora 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 higher, even more preferably 96% or higher, and especially preferably 98% or higher) 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 comprise a culture medium, a mixture of 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] An embodiment 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 capable of adjusting the pH of the Stachybotrys microspora fermentation product to 9 to 13, such as an aqueous solution of an alkali metal hydroxide (e.g., NaOH, KOH); an alkali metal carbonate or bicarbonate (e.g., sodium carbonate or sodium bicarbonate); an alkaline earth metal hydroxide (e.g., calcium hydroxide); ammonia and ammonium salts (e.g., NH4OH); or an amine (e.g., ethanolamine). 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 Stachybotrys microspora fermentation suspension is directly adjusted to 9 to 13 by contacting with an aqueous solvent. In step I, the pH of the Stachybotrys microspora fermentation suspension 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. Adjusting the pH to within the above range is believed to improve the extraction efficiency of SMTP from the Stachybotrys microspora fermentation suspension, possibly by converting SMTP into the salt form of the counter ion (e.g., by deprotonating the carboxylic acid group and phenol group) and reducing the extraction of hydrophobic impurities derived from the fungus cells (which may be unsuitable for purification in step II, described below). On the other hand, the pH of the Stachybotrys microspora fermentation suspension is preferably adjusted to 13 or lower. Any combination of the above pH values ​​may be expressed as 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, or ethyl acetate. By adjusting the pH to within the above range and performing aqueous extraction, the volume of the extraction solution can be reduced compared to when an organic solvent such as methanol is used, making the extraction more scalable (e.g., the amount of Stachybotrys microspora fermentation product used is not limited as long as the desired SMTP is obtained. The amount can be, for example, 300 L or less, 500 L or less, or 1000 L or less, or even larger. On the other hand, the extraction can also be carried out on a small scale, and the amount can be, for example, 1 mL or more, or smaller. The production method of the present invention can be carried out even when using these amounts of Stachybotrys microspora fermentation product).

[0033] (Extraction process) Step I may include further agitating the slurry following pH adjustment, which will facilitate dissolution of the SMTP into 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 in 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, or 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] [Process 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, for example, by using an appropriate adsorbent.

[0040] (aromatic synthetic adsorbent) Purification can be carried out using an aromatic synthetic adsorbent. Aromatic synthetic adsorbents have a porous structure, i.e., they have numerous 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.; Diaion manufactured by Mitsubishi Chemical Industries Co., Ltd. TMHP-10, HP-20, HP-21, HP20SS, HP-40, etc.; Sepabeads manufactured by Dow Chemical Co., Ltd. TM SP-850, etc.; Dowex XUS-40323, XUS-40285, etc.; KS, HS, AF, L-1 manufactured by Hokuetsu Carbon Industry Co., Ltd.; Polyclar SB-100, Polyclar Super R, Polyclar 10 (PVPP polyvinylpolypyrrolidone) manufactured by ISP; 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] [Process II-1] Step II may include step II-1, in which the pH of the extract obtained in step I is adjusted. Adjusting the pH to a predetermined value increases the charge capacity and reduces the amount of acid required for protonating the salt form of SMTP (to the acid 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 performed by the gradual addition of an acid. The acid may be any acid commonly used for pH adjustment, but H2SO4 should not be used when calcium ions are present in the solution because the formation of insoluble calcium sulfate can impair the charge capacity and the number of resin reuse cycles. Therefore, in some embodiments, the pH adjustment in step II-1 is performed using an acid other than H2SO4.

[0045] [Process 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] [Process 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 solvents alone, or may contain two or more of these solvents. 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 co-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] According to 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] [Process 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 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% rp. 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 follows step II-2'-1 and involves treating the aromatic synthetic adsorbent with an acidic aqueous solution. The main purpose of step II-2'-2 is to convert the carboxylate groups of SMTP to carboxylic acid forms. 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 H2SO4. In step II-2'-2, the polarity of the impurities and SMTP is ensured and the solution is moderated before step II-3 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. The organic solvent in Step II-2'-3 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. When methanol is used, the concentration 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 contain an acidic aqueous solution.

[0055] [Process 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 step 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 of 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, the concentrate is diluted with a water-immiscible organic solvent following concentration. Furthermore, the organic phase may be separated and washed with an aqueous salt solution containing the water-soluble salt to promote dehydration. From the viewpoint of efficient dehydration, the concentration of the water-soluble salt in the aqueous salt solution is preferably at least 2 wt / v% or more, more preferably 3 wt / v% or more, even more preferably 4 wt / v% or more, and particularly preferably 5 wt / v% or more. Furthermore, from the viewpoint of increasing the efficiency of crystallization 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. In a preferred embodiment, Step II'-3 increases the purity of SMTP to about 85% to 98% rp or higher. The activated carbon treatment may be carried out, for example, via a batch method or a carbon filter treatment method. 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] [Process 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 as observation using a polarizing microscope or X-ray diffraction.

[0062] (crystallization solvent) The crystallization solvent includes 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 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 is referred to as a solution of SMTP in the following description of Steps III and IV.

[0064] The dissolution solvent may be, for example, one or more selected from acetone, ethyl acetate, 2-butanone, and acetonitrile, 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, while it may be 25 v / w% or less, preferably 20 v / w% or less.

[0067] The ratio of the volume of the antisolvent to the weight of the SMTP to be crystallized (antisolvent 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: 0-2 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. A volume ratio of 0 means that the component is not contained (the same applies hereinafter).

[0070] In another preferred embodiment, the crystallization solvent comprises acetone, ethyl acetate, and methyl tert-butyl ether and has the following composition: Acetone: 0 to 10 v / v % of 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 % based on the total amount of the crystallization solvent, more preferably 80 to 90 v / v %

[0071] By using the crystallization solvents with the above-mentioned 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 that adhere 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 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 in the crystallization solvent (acetone:methyl tert-butyl ether) 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 purity of the resulting SMTP as well as the recovery rate are improved.

[0073] (crystallization process) Step III may further comprise 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 the 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 60°C or lower, preferably 55°C or lower, and more preferably 50°C or lower.

[0075] Step III may further comprise maintaining the SMTP solution 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 comprise 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 to be added may be, for example, 0.01 to 5 w / w% 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 w / w%, more preferably 4 to 20 w / w%.

[0079] [Process 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 recrystallization results in a purity of 99.5% or higher of the resulting SMTP. 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] [Process IV] The method of the present invention may further comprise a step IV of recovering the SMTP crystallized in step III. The recovery of the crystallized SMTP may be carried out by a method known to those skilled in the art, and may include separation, washing, drying, etc. of the crystallized SMTP. The separation of the crystallized SMTP may be carried out by a method known to those skilled in the art, such as filtration or centrifugation. Washing may be carried out, for example, using a mixture of the same dissolving solvent and antisolvent as used in step III.

[0082] A third embodiment of the present invention relates to a method for producing SMTP, comprising the steps of: A process of contacting the 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, and in a preferred embodiment, SMTP can be obtained with a recovery rate of 85% or more, more preferably, 90% or more, even more preferably, 93% or more, especially, 96% or more, and especially, 98% or more. Furthermore, according to the third embodiment, SMTP can be obtained with a purity of 50% or more, a preferred embodiment, 55% or more, a more preferred embodiment, 60% or more, an even more preferred embodiment, 65% or more, and especially, 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 steps of: 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. SMTP may be prepared by any method to a predetermined purity or higher. Furthermore, the SMTP that can be used in this embodiment preferably does not contain a methyl esterified product. The type and other forms of SMTP can be selected as described above.

[0087] Each feature of the crystallization process in the fourth embodiment can be arbitrarily selected in the same manner as each feature of the crystallization process described above. Further, the fourth embodiment may further include a step of recrystallizing the crystallized SMTP. Each feature of the recrystallization process may be the same as each feature of the recrystallization process described above and can be arbitrarily selected.

[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 arbitrarily have features similar to each feature in the above-described extraction step of SMTP.

[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 arbitrarily have features similar to each feature in the above-described crystallization process.

[0090] The seventh embodiment of the present invention is described as a method for manufacturing or purifying SMTP including the following steps: a. A step of extracting SMTP under basic conditions and filtering the fermentation broth. [[ID= 20]]b. An optional step of adjusting the pH of the filtrate from step (a) by adding an acid. [[ID=2 2]]c. A step of purifying SMTP by loading the filtrate from step (a) or step (b) onto a reverse-phase resin and subsequently eluting it. d. A step of recovering the purified SMTP from the eluate of the reverse-phase chromatography. e. A step of concentrating the eluate of the reverse-phase chromatography from step (d) by evaporation to minimize the amount of the organic solvent. f. A step of extracting the concentrate from step (e) with an organic solvent immiscible with water. g. An optional step of adding a salt to step (f) to improve the liquid-liquid phase separation. h. A step of washing the organic phase from step (g) with brine to reduce the water content and the highly polar solvent. i. Treatment of the organic phase from step (h) with activated carbon (either batch, column or filter type) to enhance the crystallization process. j. Concentration of the solution from step (i) followed by a step of crystallization from EtOAc and / or acetone and MTBE. k. Adding high purity seed crystals to step (j) at elevated temperature to enhance particle size and the crystallization process. l. Once sufficient nucleation has occurred, cooling the slurry to increase recovery. m. The desired product is separated from the slurry by any known filtration method, rinsed and dried. [Example]

[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 %rp) is a value obtained from the area % of HPLC.

[0093] Example 1: Scale-up run 1. Extraction and Filtration of SMTP-7 Solution from Fermentation Broth Fermentation of SMTP-7 was carried out in a 200 L fermenter. 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 an additional approximately 10 minutes. The biomass was separated by a filter press 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, or 96.5%, of the initial 965 g in the fermentation broth, indicating that the purity of SMTP-7 was approximately 70% rp (Figure 6). Furthermore, the HPLC results showed that no methyl esterified forms of SMTP were 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 efficiency. Therefore, this method is not limited to SMTP-7, but covers the entire SMTP class of compounds.

[0094] The pH of the fermentation broth was adjusted to various pH values ​​between 5 and 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%. This decreased to approximately 11% when the pH was lowered to approximately 5. When the pH was further increased to 11, the SMTP content in the supernatant increased to approximately 93%. When the pH was further increased to approximately 12, the extraction recovery rate improved to approximately 99% (Figure 1).

[0095] 2. Purification using synthetic aromatic adsorbents 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 bed volumes per hour. After loading, the resin was washed with 2 BV of water (twice the bed volume of the column) followed by 2 BV of aqueous acid solution of H2SO4 (pH 1.5). The resin was then treated with 2 BV of 20% acetone-water to swell the resin, followed by 4 BV of 52% acetone-water to remove highly polar impurities. The desired SMTP-7 band was then eluted with 3 BV of 68% acetone-water. The desired band was determined by HPLC / UV and pH monitoring. This procedure led to a recovery of about 96% (about 26 g SMTP-7) and a relative purity of 88% rp (FIG. 7).

[0096] b) Aqueous MeOH elution system This method also provides the desired level of purification, but 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 Methods 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. 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 through the evaporation and solvent extraction steps was 98% (approximately 74 g SMTP-7).

[0098] After sampling, the remaining 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 until a semi-solid paste (approximately 96 g) was obtained (target: 1.6-3 w / w). Acetone (80 mL, 1.33 v / w) was added to this paste and mixed at approximately 40-50 °C. MTBE (213 mL, 3.54 v / w) was then added. 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 h while maintaining the temperature above 50 °C. The slurry temperature was cooled to approximately 45 °C, and mixing was continued for 4 h. The slurry was then cooled to room temperature, and mixing was continued for 14 h.

[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 yield 47 g of SMTP-7 crystals (99.2% rp, 78% process 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 / 5 3mL / 322mL) The HP20SS purified sample was evaporated and extracted as 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 SG280P activated carbon (Futamura Chemical Co.) based on the weight of SMTP-7. The slurry was mixed for 30 min, 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 the 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 h 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 vacuum to give 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. Crystallization of SMTP-7 / acetone / ethyl acetate / MTBE (22 g / 40 mL / 1 2mL / 360mL) 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 SG280P activated carbon (Futamura Chemical Co.) based on the weight of SMTP-7. The slurry was mixed for 30 min, 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 h 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 vacuum to yield 16.5 g of SMTP-7 crystals (99.4% rp, 75.5% process recovery). The crystal particle size was large (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. Crystallization of SMTP-7 / acetone / ethyl acetate / MTBE (22 g / 40 mL / 1 2mL / 360mL) 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 SG280P activated carbon (Futamura Chemical Co.) based on the weight of SMTP-7. The slurry was mixed for 30 min, filtered through a PTFE (1.0 μm) filter, 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. After mixing at approximately 40–50°C, MTBE (350 mL, 16.3 v / w) was added. Once a temperature of approximately 50°C was obtained, 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 vacuum to yield 16.1 g of SMTP-7 crystals (99.52% rp, 73.7% process recovery). The crystal particle size was large (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. Examination of the composition of crystallization solvent The same test as in Example 1 was carried out by varying 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). 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. 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 according to claim 1, wherein in step I, the pH of the fermented product is adjusted to 11 or higher.

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

4. The method according to claim 3, wherein step II includes 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 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 using 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. 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 crystallization solvent of Step III comprises acetone, ethyl acetate, and methyl tert-butyl ether; 2. The method according to claim 1, wherein 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, after step III, the following step III': III'. Recrystallizing the SMTP crystallized in step III.

9. 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; How SMTP is manufactured.

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

11. The method comprises contacting a fermentation product of Stachybotrys microspora with an aqueous solvent having a pH of 9 or higher to obtain an extract containing SMTP. How SMTP is manufactured.

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

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