TWI291952B - Purification of lipstatin - Google Patents

Description

1291952 V. INSTRUCTIONS (1) '~~----一· | Ben & Ming is a method for purifying ribastatin. More specifically I The present invention relates to a method for combining a liquid-liquid I stroke method in the form of a dual flow extraction method with a re-extraction method in the form of a countercurrent extraction method, as a single high separation from the crude product of ribavirin The method of yield and high purity of Ribs i. Ribsstatin is a key intermediate in the preparation of tetrahydroribastatin (THL, Or listat), which is suitable for the prevention and treatment of diseases associated with obesity. Ribsstatin, its mashing method, known from the microbial isolation method and its hydrogenation method of tetrahydroribastatin, is described, for example, in the US Patent No. 4, 5 98, 08 9. Ribsstatin is as follows:

A method for preparing a crude product of ribavirin is also described in the European patent application N 〇 · 9 6 1 0 6 5 9 8 . The method comprises aerobic culture by an actinomycete microorganism, such as Streptomyces toxytricini, which is produced in an aqueous medium substantially free of fats and oils and comprising a suitable carbon and gas source, and an inorganic salt. Ribsstatin, until the beginning of the long-term @ is over, and has produced sufficient cell quality. Then in the nutrient solution 12919952 5, invention instructions (2) together add linoleic acid and antioxidants, and need to choose the octanoic acid, and N-methyl decyl-L-leucine, or preferably add L-white Amino acid. After the fermentation is completed, the fermentation broth is extracted. The resulting crude production of ribastatin can be further concentrated and purified, for example, by the chromatography described in U.S. Patent No. 4, 5 98, 〇89. The prior art method for purifying the crude product of ribavirin is characterized by using multi-stage chromatography or liquid-liquid extraction to increase the concentration of ribsstatin' combined multi-stage chromatography to produce pure lining. Bustin. These methods are typically applied to isolate the lysate metabolites on a laboratory scale. However, these methods are generally not suitable for economical large-scale production.

Attempts have been made to purify the crude ribavirin by distillation, but it has not been successful. Ribstatin maintains several hours of stability at 60 t, but because of the low vapor pressure (7 x W mbar), vacuum distillation (less than 2 * bar) cannot be performed at this temperature. At higher temperatures, 1 statin releases carbon dioxide and degrades. The crude ribastatin product can also be purified by crystals. However, it is required to be high and the crystallization yield is extremely dependent on the low quality of the crude ribavirin product, and the yield is also low. Ribs's low temperature crystallization technology equipment at a temperature below -200 °C, the quality of the crude product. In particular, the ribastatin obtained by the crystallization method provides a novel, simple statin crude product which has a high yield and a low quality of the crude product. The invention is based on the crude product of ribastatin: and the method of low cost, which can be used for purifying ribastatin from ribb and southern purity.

Page 5 1291952 V. Description of the invention (3) a) Extraction of ribastatin from a non-polar solvent selected from the group consisting of: aliphatic or aromatic hydrocarbons by liquid-liquid extraction to: carboxylic acid, alcohol, 0 - single-substituted single or aromatic solvent in the presence of ethylene glycol, glycol or bipolar aprotic solvent, then I b) dilute the polar solvent phase with water, or change the two ratios and re-extract! Bustastatin to fresh non-polar solvents. The following definitions are intended to illustrate and define the meaning and scope of the various terms used in the description of the invention herein. "Double flow extraction method' refers to a countercurrent extraction of two solvents. The extraction method of this extraction method is as follows: | — ► light phase outlet (residual liquid) + impurity. I extraction zone extraction solvent feed washing zone scrubbing solvent - ► heavy phase outlet (extraction) in the extraction unit Two solvents with low miscibility are introduced at the top and bottom. The mixture to be separated (feed) can be fed at any point, but if it is fed in combination with the washing solvent, it is preferably fed in the middle of the extraction unit. In the above figure, 12919952 5. Inventive Note (4) The extraction solvent and extract of the extraction method contain higher density, soluble i (heavy phase), while the washing solvent and the residual liquid respectively contain lower density. Dissolve! (light phase). "Countercurrent extraction method π - word means that the substance to be extracted (feed) is added together with the solvent to be used. At this time, the amount of the compound to be purified is not as good as the double flow direction extraction method because the washing zone is omitted. The extraction method is carried out as follows: ——►light phase outlet (extraction) feed + solvent - extraction solvent - η_ ——► heavy phase outlet (residual liquid) A solvent is added to the top and bottom of the extraction unit. Feeding and solvent with low miscibility. The extraction solvent at one end is rich in the target outflow column (extract). At the other end, the solvent (residual) for sending the target substance after feeding is sent out. The column is characterized by a high-density solvent (heavy phase) for the feed and the raffinate, and a low-density solvent (light phase) for the extractant and the extract, respectively. A substance | extracted form used to dissolve 1291952 5, invention description (5) π ribs statin crude product" The term refers to the separation of cells from the fermentation process, biomass, extraction and concentration to obtain ribavirin and The crude product of impurities. The present invention relates to a method of combining two liquid-liquid extraction methods for separating high-purity and virtually all-receiving ribastatin from the crude ribavirin product.

In general, the method for purifying ribavirin from a crude ribastatin product comprises a liquid-liquid extraction method for extracting ribastatin from a non-polar solvent selected from a lipid or an aromatic hydrocarbon. From a polar solvent of a carboxylic acid, an alcohol, a 0-monosubstituted mono or oligoethylene glycol, a glycol or a bipolar aprotic solvent, then dilute the polar solvent phase with water, or change the two ratios, and again! Ribastatin is added to fresh, non-polar solvents. | i Liquid-liquid extraction is carried out in the following solvent systems: Non-polar solvents 丨 are selected from: aliphatic or aromatic hydrocarbons. Preferred aliphatic hydrocarbons are (: 5-(:8 aliphatic hydrocarbons; more preferably &::6-(:7 aliphatic hydrocarbons such as hexane or heptane. The aromatic hydrocarbons may be selected from benzene, which are visible: It is required to be substituted with 1 to 3 methyl groups. Preferred aromatic hydrocarbons are benzene and toluene. The polar solvent may be selected from a carboxylic acid such as formic acid or q-C3 alkyl carboxylic acid.

Such as: acetic acid or propionic acid, or alcohol, such as: sterol, ethanol, propanol, or 0-: monosubstituted mono or oligoethylene glycol, such as: ethylene glycol monomethyl ether or (: 2- (: 3 a diol such as ethylene glycol or 1,3-propanediol, or a mercapto alcohol or a tetrahydrofurfuryl alcohol. Bipolar aprotic solvents include, for example, dimethyl decylamine, N-methylpyrrolidone, dimethyl Solvents such as arsenic, acetonitrile, cyclobutyl sulphate, succinyl or the like. Preferred polar solvents are water-soluble organic carboxylic acids, alcohols or 0-monosubstituted mono- or polyethylene glycols, such as acetic acid, decyl alcohol or ethylene glycol. Alcohol monoterpene ether, especially preferably acetic acid. Polar solvents can be used directly or mixed with water.

Page 8 1291952 V. Description of invention (6)

In addition to the 丄Ξ:: the non-polar impurities of the crude product are removed in the first extraction stage = excluded in the second extraction stage. When the polar solution U ribastatin is used, the exclusion of the pole two: miscellaneous ... non-polar - agent extraction ΐϊ ΐϊ ΐϊ ΐϊ 中 中 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次 两次It is a method of adding polar water to dilute a polar solvent or changing the ratio of ribs to change the distribution of ribavirin. κ 极性 极性 Ϊ Ϊ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 Statin, preferably used in the first step > a valley non-polar solvent. Dry % / ~ the same = in the specific embodiment, the first step of the solution - liquid extraction method for the polar solvent contains about} to about 2 〇% water, more preferably about 5% water. Special:: In a preferred embodiment, the non-polar solvent is a lipid system. In the preferred embodiment of the invention, heptane, the first step of the liquid_beer One method is the double flow extraction method, and the ribastatin is extracted with a polar solvent. The crude material is ribsstatin crude, and one of the solvents can be used to dilute the (heavy) phase extract ribastatin. Non-polar impurities such as: fatty acids or esters remain in the non-polar (light) phase. The distribution coefficient of statin and impurities depends on the concentration, the ratio of the two solvents, the theoretical coefficient of the extraction system, and the ratio of the feed to the solvent, so it must be adjusted according to the appropriate method. TC Luo (Lo), Baird (Bairds) ), MHI, M, Ha fez, c nanson (1983) · / glutinous stroke method manual (Handbook of 1291952 five, invention description (7) extraction), New York Willie ( WUey, heart and stomach Y〇rk). The dependence of the distribution coefficient on the concentration is strongly changed with the type of polar solvent used in the extraction system. The aqueous acetic acid has excellent results in achieving high concentration, so there is a high amount of material passing material and the minimum amount of material used. Solvents. For extraction, a continuous extraction apparatus such as a mixer clarifier or an extraction column such as a pulsed sieve plate extractor or a stirred column may be used. In a preferred embodiment, re-extraction Previously (second purification step) dilute the polar solvent to about 70% to 90%, preferably about 80%. In a preferred embodiment of the invention, the second purification step is re-extracted = countercurrent extraction, and Bustastat extraction into non-polar ^ At this time, it can be used directly for the first time extraction fish extracts required;. J

The double flow direction extraction method & J ribsstatin feed, if the second extraction is carried out by the continuous K extraction method, the first extraction of two /;, L bustin. Ribsstatin is extracted to a non-polar (light lining remains in the polar (heavy) phase. ... phase and polar miscellaneous = several amino acid analogs of ribastatin are formed during leaven. N_mercapto) leucine as a side chain and by-product 2 contains other N-methyl mercapto amino acids. One of the by-products is a class; the product = ribavirin. This by-product of the crude product of ribastatin: J is 3% relative to ribsstatin. Purification "statin :: The method is very important. Otherwise, the subsequent hydrogenation will be similar to the methyl methionine in the crude product of the ruthenium; the purification of the crude statin. Unless it is oxidized into the corresponding

O:\62\62173.PTD Page 10 1291952 I I. Description of invention (8) i, otherwise it is not possible to directly use crystallization or extraction to exclude ribavirin similar to methionine I to purify Ribs He Ding. This oxidation process is carried out according to conventional method I, for example, by using peracetic acid in acetic acid. Similar to 曱 曱 曱 丨 丨 丨 丨 可 可 可 可 氧化 氧化 氧化 氧化 氧化 氧化 氧化 氧化 氧化

CH, C0-0-0H 〇=s=〇

If it is desired to oxidize to yam, the amount of the oxidizing agent is erbene-like methionine or the molar amount of 1. 5 equivalents, and if it is to be oxidized into maple, it is used twice. A large excess should be avoided to prevent oxidative decomposition of ribavirin. The oxidation process is carried out at 0 ° to 50 ° C, preferably at room temperature; the reaction is completed in a few minutes. The solvent used in the oxidation method is not critical, and one of the solvents used in the extraction purification method is preferably used in the oxidation method. Ribastatin, which is similar to methionine, can be oxidized in the crude ribastatin step or after the first extraction step. The oxidation method is preferably carried out using a heptane solution containing a high concentration of the crude ribavirin, and the resulting mixture is used for the first extraction and purification.

Page 11 1291952

I V. Description of invention (9) Feed of the steps. After the oxidation of the j 1 impurity, in the second extraction! If the extraction purification method uses I, the first method is used directly as a second! Similar to the thiol amide in the best embodiment i The method of dicing is to separate and purify from heptane by double-flow I 95% acetic acid/heptane, then acetic acid, and counter-current heptane from heptane: Aqueous acetic acid-I-Busstatin crude product (including A One of the thioacids is oxidized by a by-product. The heptane is similar to the methionine in the polar phase. The aqueous solution is removed from the aqueous acetic acid/heptane system. The distribution between busbutatin is 'distilled from the crude product of ribavirin to the extraction method and dilute the ribavirin extraction solution with water. Then use concentrated ribastatin. This preferred heptane ten non-polar impurity is the first extraction of statin as a polar 〇 to countercurrent extraction in the addition of water. Therefore, the crude product of Ribstin is purified to about 80% reduction or crystallization. The method is illustrated in the following figure. The purified ribastatin I---- Polarity in water ▲ Water second extraction Geng burning - ► Aqueous acetic acid + polar impurities

Page 12 I29l952 I V. INSTRUCTIONS (10) 1 As described above, according to any of the above = conversion to tetrahydroribastatin ('Osri"::: hydrogenation can be carried out by the method known per se by hydrogen itself Ribsstatin can be based on: the method of collar 08", in the two touch: the catalyst used in the case of the case is Pd / C, the oxidation of nU is carried out. The solvent can not be heavy [optimal solution: f At this time, hydrogenation is used directly. The solvent containing the ribavirin; the following:: solvent of the step: Other suitable solvents are, for example, low-carbon alcohols such as or gas, a hospital. The best hydrogenation Under low hydrogen milling and chambers, such as: the second invention also included in the above extraction method, the hydroquinone thus 笱 Φ k from Ding + ribastatin, forming tetraploid ribastatin. Preferably, the reaction is carried out using a hydrogenation catalyst containing a noble metal at 25 and 0.5 to 5 bar. * (For example: = ribsstatin can be purified and isolated by crystallization. It is preferred to use non-polar / squeezing such as: hydrogenation and crystallization by calcination or heptane. In summary, the invention relates to a method as defined above, which comprises : a) oxidize ribastatin similar to methionine, b) extract ibsstatin crude product with a flow of about 95% acetic acid / heptane, c) dilute ribastatin solution with water to about 8 〇% acetic acid; and d) counter-current extraction of the solution with heptane. Following this process, the hydrogenated ribastatin forms tetrahydroribastatin, and the crystallization of tetrahydroribastatin is continued as needed. The invention also includes a method of preparing tetrahydroribastatin comprising:

Page 13

1291952 V. INSTRUCTIONS (11) a) Oxidation of ribastatin similar to methionine b) extraction of crude ribastatin by a flow of about 95% acetic acid/heptane; c) dilution of water by Ribs The butyl solution is about 8 〇% acetic acid; and d) the solution is extracted countercurrently with heptane, and then e) hydrogenated ribavirin forms tetrahydroribastatin. Further, the present invention relates to the use of the preparation of ribastatin and tetrahydroribastatin by the above method. The following examples illustrate preferred embodiments of the invention, but do not limit the use of the invention.

Example Example 1:

The crude product of ribavirin obtained by separation of the biomass from the fermentation process and extraction is concentrated. The substance used contained 58% (w/w) ribavirin (HPLC analysis) and 0.9% (w/w) ribavirin similar to methionine (iH-NMR analysis). a · Oxidation of ribastatin similar to ribastatin: Dilute 5 5.7 bristastine crude product with 7 7 ml of heptane. 21 2 μl of 37% peracetic acid in acetic acid solution was added. The mixture was stirred at room temperature for 3 minutes.

b. Double flow extraction with heptane/9 5% aqueous acetic acid: To simulate an extractor with 7 separation stages, 7 separation funnels were used. The mixture obtained by the oxidation method described in item a was divided into 6 portions, each of which was fed from the fourth separation funnel (s f -4 ). Starting from the s f _ 4 feed, 5 〇 liters of heptane and 100 ml of 95% aqueous acetic acid were added, which corresponds to a ratio of 1 /2 for the heptane/aqueous acetic acid. The mixture was shaken and layered. Move the upper layer to the next separation

Page 14 1291952 V. INSTRUCTIONS (12) Funnel, the lower layer is moved to the previous separation funnel - at the beginning, fresh solvent is used and then gradually replaced by the solvent phase from the previous and next separation funnels. When all the separation funnels were filled in this way, 'Gengyuan was added to the first separation funnel, and only the seventh acetic acid was added to the separation funnel. Collect the extract containing ribavirin. The ribavirin in the raffinate was traced by thin layer chromatography to keep the content below 1 gram per liter' and the pre-calculated two ratios and concentrations were controlled. After feeding all the crude products of ribastatin, add these two clean dissolves.

Agent' until all the ribsstatin is washed out of the system. This extraction method is described below: _ heptane _ heptane + #polar by-product (residual liquid) heptane ribastatin crude product

I ▼ sf-3 sf-4 sf-5 I sf-2 ◄---! Ribastatin is contained in aqueous acetic acid (extract) sf-6 sf-7 A 95% acetic acid

95% aqueous acetic acid μ was evaporated under reduced pressure to remove the solvent, and the extract was concentrated to a volume of about 6 〇 〇. The water content measured by the Karl Fischer method was 3.5%. C • Countercurrent extraction: In order to simulate an extractor with 7 separation stages, 7 separate funnels were used again.

1291952 V. INSTRUCTIONS (13) Distillate (600 ml) from item b was divided into 6 portions (1 ml each), and each was added to the 7th separation funnel together with 30 ml of water. Add 130 ml of heptane to the first branch == i bucket. A continuous method like b) simulates the extraction of line 77, which moves the upper layer to the next separation funnel and the lower layer to the previous separation funnel. The ribavirin in the raffinate was traced by thin layer chromatography to keep the content below 1 gram per liter to control the pre-calculated two ratios and the concentration of . After the extract from the first extraction step was fed, both solvents were added until all the ribavirins were washed out. The following figure says; ^ extraction method: e s heptane

Heptane τ

Sf-4 sf-5 sf-6 polar enamel (residual liquid) Ribastatin (extracted liquid) containing bellulidine in heptane. Ribastatin in aqueous acetic acid (first extract) Liquid) + water

Aqueous acetic acid The pure ribastatin (extract) contained in heptane was collected and concentrated under reduced pressure to a volume of • 2 50 ml. This solution contains 3 5 3 g of pure ribavirin with a purity of 90% (w / w) (IIP L C ). Calculated according to the content of ribastatin in the pure product,

Page 16 1291952 V. Description of the invention (14) | The yield of ribavirin content relative to the crude product is equivalent to 98% P d. Hydrogenation method: 250 ml of purified ribavixine in stainless steel 蚤I In the case of 25 I and 5 bar pressure, a total of 6 hours of hydrogenation was carried out using 3.5 grams of 5% Pd/C. The catalyst was filtered off and the filtrate was evaporated. The residue was dissolved in 700 ml of heptane to crystallize the tetrahydroribastatin at Ot:. The product was filtered, washed with cold gent, and dried under vacuum at Torr. 28 g of tetrahydroribastatin was obtained with a purity of 丨9 6% (W/W) (HPLC). The yield of ribastatin relative to the pure product is 丨85/. The purity of tetrahydroribastatin after recrystallization in oneself was 1.97% (w/w) (HPLC). Further Example 2: It is known to use a 7-stage mixed is clarifier system for continuous extraction using the same solvent i as the example. The ribastatin crude product used was 65% pure (w/w), which contained 0.14% (w/w) of ribavirin similar to guanidine thioacetate. a. Double flow extraction using heptane / 95% aqueous acetic acid: 100 kg of the crude S-bisbistine was diluted with ι 〇〇 heptane and used as a solution. The mixer clarifier was filled with heptane and 95% aqueous acetic acid, 1/2 U/V). When extracting and purifying, add 3? 5 burning per hour, 75 liters of 95% aqueous acetic acid and 15 liters of ribastatin crude product solution (1) added Gengyuan' in the 4th addition of ribastatin solution, f Mixer adds aqueous acetic acid. Collect the amount of impurities contained in the extract of bustadine. Thin layer chromatography was used to trace the amount of rag in the residual liquid and keep it below 1 gram of the mother. After all the crude ribsatin 3, continue to feed the two solvents until all the ribsstatin is washed out

1291952 V. Description of invention (15). A sample of the extract (lower layer) was concentrated and found to have a purity of 71% (w/w) of ribavirin. A total of 1,500 kg of extract was obtained with a water content of 4 6% (Karl Fisher method). b. Oxidation of ribavirin similar to methionine. Add 6 ml of a 37% acetic acid solution of acetic acid to the extract from the previous extraction, and the mixture was stirred at room temperature for 30 minutes. c. Countercurrent extraction: Add 280 liters of water to adjust the water content to 2%. The solution was extracted with 6 liters of heptane because the oil of the ribastatin was separated after the addition of water. The extract was separated and the latter was added to the continuously extracted heptane phase. In order to continuously extract the residual aqueous ibexatin-containing acetic acid, the same mixer clarifier as the first extraction method was used. For continuous extraction, Nissho added 50 liters of heptane per hour to the first branch, and added 5 liters of aqueous acetic acid containing ribastatin to the seventh mixer per hour. The heptane extract was added to the extract obtained once, and the solvent was distilled off under reduced pressure, and the solution was concentrated to a volume of about 1000 liters. The sample was concentrated to dryness to yield ritastatin with a purity of 86% (w/w). d. Hydrogenation: The resulting solution was hydrogenated in three equal portions. Hydrogenation is carried out in a stirred hydrogenator of a lining glass mat. Use 2 kg 5% Pd/C per batch. After 1 hour, the hydrogenation was carried out to a hydrogen pressure of 5 bar. The catalyst was filtered off, and the solvent was distilled off to distill the filtrate. The dried sample product contains 90% (w/w) tetrahydroribastatin (the quality is higher than the purified ribavirin, because ribastatin contains "transformable by-products, which have different positions Or an isomer containing only one C=C double bond, which is also converted to tetrahydroribastatin after hydrogenation. Add 600 kg of Gengzhuo' to make tetrahydroribastatin to 〇. : Crystallization. Filter out the product to cool

Page 18 1291952 V. Description of invention (16) Gengyuan washing, drying under reduced pressure. One batch produced 17 kg of 97% (w/w) tetrahydroribastatin. Its yield relative to ribastatin in the crude ribastatin product is equivalent to 76% tetrahydroribastatin. * Example 3: Separation of biomass from the fermentation process and concentration of the crude ribavirin obtained after extraction. This material contains 62% (w/w) ribastatin and 〇·i% (w/w) ribastatin like methionine. a. Oxidation of ribavirin similar to methionine: 7 5 ml of heptane, 53 clibetstatin crude product. 24 μl of 37% peracetic acid in acetic acid solution was added, and the mixture was stirred at room temperature for 3 minutes. ^Double-flow extraction using heptane/1% aqueous sterol: Example 1 was used to simulate continuous extraction with a seperate, 7 separation funnel system. In the future, d) the mixture of the rolling method is divided into 6 parts, and the funnel is fed. Add 100 ml of heptane to the first 丨Φ + injury from the 4th branch of the 1st to the 7th separation: the bucket to =L is equivalent to 1 0/1. Collect the extract (the upper layer, containing two of the sterols). When all the crude ribsstatin products were sent to the Dingzhi Geng fresh solvent, until all the 1 sceptin was washed "·in and out, then add two new solvent to remove the solvent and concentrate to a volume of 丨i 0 ml. " The extract is steamed under reduced pressure c. • Heptane/10% aqueous sterol is used to carry out the extraction method, and the liquid is occupied by eight occupants, and he is drawn to the method of stroke: it will be taken from the top, and the liquid knives will be obtained. Into 6 injuries. The same extraction method as described above was used, but the two comparison examples were different. The hoff funnel system is fed into the funnel. Add 100 ml of heptane to the first = all from the 4th branch, add 1 0 0%

Page 19 1291952

V. INSTRUCTIONS (17) Aqueous Examples of alcohols). Freshly soluble and dissolved in methanol to the 7th separation funnel, the heptane/recognition 1 / Ί A in - / , the situation / water methanol + two compared to 1 / 1. Collecting the stroke fluid (the lower layer of water containing ribsstatin), after all the crude ribastatin products were fed, add m and all the ribsstatin was washed out. The extract was added to a volume of 250 ml. ..., d. Extract ribastatin batchwise in heptane: add water to the extract from c) = ', the whole water content to 5〇%. The mixture was extracted with 2 g of each of 600 ml of heptane. The fluid is concentrated by distillation to remove the solvent. Obtained 34 g of purity 86% (w/w)

Ribsstatin. Based on the content of ribastatin in the pure product, the yield of ribavirin relative to the crude product was equivalent to 89%. e. Hydrogenation: Similar to the example of hydrazine and crystallization. The tetrahydroribastatin has a yield of 9% by weight relative to the libastatin in the pure product. Example 4: The crude ribastatin product also from Example 3 was purified using the same 7-stage extraction apparatus as described in Example 3. Alternatively, 丨〇% aqueous ethylene glycol monomethyl ether and heptane are used as a solvent. Compared with Example 3, the concentration and the two examples were unchanged. The purity of the purified ribavirin sample was 8〇% (w/w). The hydrogenation and crystallization of tetrahydroribastatin were carried out in accordance with the foregoing examples. A tetrahydroribastatin having a purity of 96% (w/w) was obtained. Example 5: square; purified ribsstatin and 0.6% (w/w) ribastatin-like ribastatin crude product on the Kuehni column .

Page 20 1291952

Claims (1)

Gang j No. 89102668 Ms VI. Patent Application Range 1. A method for purifying lipstatin from a crude product of ribavirin comprising: a) performing a liquid-liquid extraction method selected from a lipid system or In a non-polar solvent of an aromatic hydrocarbon, the ribastatin is extracted into a polar solvent selected from the group consisting of a carboxylic acid, an alcohol, a 0-monosubstituted mono or polyethylene glycol, a glycol or a bipolar aprotic solvent. Then b) dilute the polar solvent phase with water, or change the two ratios and extract the ribstatin into a fresh non-polar solvent. 2. The method of claim 1, wherein the polar solvent of step a) comprises from about 1 to about 20% water. 3. The method of claim 1 or 2, wherein the polar solvent of step a) comprises about 5% water. 4. The method according to item 1 of the patent application, wherein the non-polar solvent is a aliphatic hydrocarbon. 5. The method of claim 4, wherein the non-polar solvent is heptane. 6. The method of claim 1, wherein the polar solvent is a water-soluble phase carboxylic acid, an alcohol, or a mono- or mono-oligoethylene glycol. 7. The method according to claim 6, wherein the polar solvent is acetic acid, methanol or ethylene glycol monomethyl ether. 8. The method of claim 1, wherein the liquid-liquid extraction method defined in step a) of claim 1 is a dual flow extraction method, and the ribavirin is extracted into a polar solvent. . 9. According to the method of claim 1 of the scope of patent application, in which re-extraction O:\62\62173-960823.ptc Page 23 1291952 Case No. 89102668 Λ_ 曰 Amendment 6. Before applying for a patent, the polar solvent is diluted with water to about 90% to about 70%. 10. The method according to claim 9 wherein the polar solvent is diluted with water to about 80 ° / 〇 1 1 according to the method of claim 1 of the patent application, wherein step b in the first claim The re-extraction method defined is countercurrent extraction, and the ribstatin is extracted into a non-polar solvent. A method according to the first aspect of the patent application, wherein a by-product of propyl citrate similar to methionine is oxidized prior to extraction. 1 3. According to the method of claim 1 of the patent application, which subsequently hydrogenates the ribastatin to form tetrahydroribastatin. 1 4. The method according to claim 13 wherein the hydrogenation reaction is carried out at 25 ° C under a low hydrogen pressure using a hydrogenation catalyst containing a noble metal. 1 5. The method of claim 1, wherein a) oxidizing ribavirin like methionine, b) extracting crude ribastatin by a double flow of about 9.5 % acetic acid / heptane , c) diluting the ribavirin solution with water to about 80% acetic acid; and d) extracting the solution countercurrently with heptane. It is subsequently hydrogenated and subsequently subjected to tetrahydrogen including: 16. The method according to claim 15 of the patent application = statin forms tetrahydroribastatin. 1 7. Crystallization of bustastatin according to the method of claim 16 of the patent application. Stastatin crude product 18. A method for preparing tetrahydroribastatin, a) oxidizing ribastatin like methionine b) in a double flow of about 9.5 % acetic acid / heptane O:\62\62173-960823.ptc Page 24 1291952 _ Case No. 89102668_Yearly revision _ Six, the scope of application for patent c) Add water to dilute ribastatin solution to about 80% acetic acid; and d) The solution was refluxed to extract the solution, followed by e) hydrogenation of ribavirin to form tetrahydroribastatin, f) crystallizing tetrahydroribastatin. O:\62\62173-960823.ptc Page 25