JPS6127398B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to separating cephalosporin C as an adduct with an organic base from an aqueous solution containing cephalosporin C, and to producing 7-aminocephalosporanic acids from the adduct. Cephalosporin C and 7- derived therefrom
Cephalosporanic acid is famous and very important as a key compound for producing cephalosporin antibiotics. One of the objects of this invention is to industrially advantageously separate cephalosporin C and deacetoxycephalosporin C from a fermentation liquid containing cephalosporin C and deacetoxycephalosporin C. Another object is to provide novel N-acylcephalosporins C and adducts thereof with organic bases. Another object is to industrially advantageously obtain 7-aminocephalosporanic acids from the novel adducts of N-acylcephalosporin C and organic bases. As a method for separating cephalosporin C, U.S. Patent No. 3,467,654 discloses that acetone is added to the fermentation broth of cephalosporin C to precipitate impurities, then cephalosporin C is adsorbed from the solution onto an anion exchange resin, and cephalosporin C is removed from the resin using an acidic buffer. It states that it will elute. Also, special public service in 1977
No. 61494 describes a method for extracting cephalosporin C by acylating the amino group of the side chain with a halogen-containing fatty acid, adding quinoline, and precipitating the halogen-containing acylated product of cephalosporin C as quinolinic acid at around pH 3. This latter method is a considerably improved method for recovering the target product from a conventional aqueous solution containing cephalosporin C, but when the concentration of cephalosporin C is 1% or less, no precipitation of quinoline salt is observed; It cannot be said that this method is satisfactory in terms of yield. The inventor of this invention discovered that since the concentration of cephalosporin C in a solution obtained by industrial fermentation production of cephalosporin C is about 0.3%, it is possible to produce a solution with good yield even from a solution containing cephalosporin C at a low concentration. This invention was arrived at as a result of various studies on methods for economically recovering cephalosporins C. In addition, we have discovered an industrially advantageous method for producing 7-aminocephalosporanic acids through a series of steps. Thus, the present invention provides an aqueous solution containing cephalosporin C having the general formula R ₁ -Y-R ₂ -COOH (wherein R ₁ is a lower alkyl group, Y is a sulfur atom or a sulfinyl group, and R ₂ is a lower alkylene group). An aqueous solution containing N-acylcephalosporin C is prepared by reacting the reactive derivative of the acid used in the reaction, and then quinoline or isoquinoline is added, and an acidifying agent is added to adjust the pH to about 2.0 to 3.5, and inorganic substances are added as necessary. A method for extracting cephalosporin C is provided which comprises adding a salt and separating the adduct of N-acyl cephalosporin C. In this invention, the cephalosporin C-containing aqueous solution contains cephalosporin C and/or deacetoxycephalosporin C at a low concentration, e.g.
Aqueous solutions containing 0.3% or more and about 8% can be used. Such an aqueous solution includes a fermented solution of cephalosporin C or deacetoxycephalosporin C that has undergone partial purification that is commonly carried out in the technical field, such as overtreatment, acetone treatment, ion exchange resin treatment, etc., and a partial concentration treatment. Contains things. However, if the concentration of cephalosporin C is too high, the adduct of N-acyl cephalosporin C and the organic base obtained in the subsequent treatment becomes muddy and difficult to separate and recover, which is not preferable. On the other hand, if the concentration is too low, the recovery yield of cephalosporin C tends to decrease. Further, concentrating the fermentation liquid as described above requires a large amount of heat source and labor, and is also accompanied by the risk of decomposition of cephalosporin C, which is not desirable from both a technical and economic point of view. Therefore, in the method of the present invention, substances that adversely affect acylation contained in the fermentation liquid, such as monoamino monocarboxylic acids, are removed by partial purification, or the fermentation liquid is used as it is, or one that has been subjected to minimal concentration. Favorable results can be obtained by doing so. Such an aqueous solution containing cephalosporin C is reacted with a reactive derivative of an acid having the general formula R ₁ -Y-R ₂ -COOH (each symbol in the formula has the same meaning as above). The lower alkyl group for R ₁ in this general formula includes a straight chain or alkyl group having 1 to 5 carbon atoms. The lower alkylene group for R ₂ includes a straight chain or branched alkylene group having 1 to 3 carbon atoms. Specific examples of such acids include those represented by the following chemical formula. Furthermore, as the reactive derivative of the above acid, one having the ability to sufficiently acylate the side amino group of cephalosporin C at a temperature of about 0 to 30°C even at a low concentration of cephalosporin C should be used. is preferred. In addition, the selection of the reactive derivative is determined based on economic considerations. Thus, the preferred reactive derivatives used in the present invention are acid anhydrides formed by two molecules of the acid represented by the above general formula;
The acid shown in the above general formula, acetic acid, propionic acid,
Lower fatty acids such as α-methylpropionic acid, butyric acid, α-ethylbutyric acid, valeric acid, pivalic acid, 2-ethylhexanoic acid, halocarbonic acid alkyl esters such as methyl chlorocarbonate, butyl chlorocarbonate, isobutyl chlorocarbonate, etc. mixed acid anhydrides. That is, the acid to be the raw material for the mixed acid anhydride exhibits a dissociation constant that is at least one-half or less, preferably about one-fifth, of the dissociation constant of the acid represented by the above general formula. Acids are a preferred example.
Other acid halides and the like may also be used. This acylation reaction takes place at a temperature of about 0 to 30°C and a pH of about 7 to
11, more preferably about 8 to 9.5. Since the pH tends to decrease during the acylation reaction, it is best to maintain the desired pH by adding a base or by adding a buffer. The amount of the acylating agent to be used varies depending on the type of fermentation liquid, but for example, in the case of a fermentation liquid that has been partially purified using an ion exchange resin, good results can be obtained if it is used in an amount of about 1.5 to 3.0 molar equivalents per mole of cephalosporin C contained therein. For fermentation liquors purified with the acetone moiety, it would be necessary to use about 4 to 7 molar equivalents. N-acylcephalosporin C produced here
are treated with quinoline or isoquinoline and precipitated from aqueous solution. This organic base may be a single substance or a mixture, and does not need to be very pure. The amount added is about 2 to 5 mol per 1 mol of N-acylcephalosporin C. After addition, use an acidifying agent such as sulfuric acid, hydrochloric acid, nitric acid, or phosphoric acid.
Adjust the pH to 2.0-3.5 and heat the mixture to 0-30℃, usually 10
After stirring for about 1 hour at ~20°C, N-acylcephalosporin C and the organic base precipitate. This substance is considered to be an adduct and is usually crystalline. At this time, if the product is difficult to precipitate due to the concentration in the solution or the relationship with other components in the solution, inorganic salts such as sodium sulfate or sodium chloride may be added as appropriate. This may then be separated by conventional separation means such as centrifugation, filtration, etc., washed with water, then with an organic solution such as ethyl acetate, and dried. The thus obtained adduct of N-acylcephalosporin C and an organic base is converted into 7-
led to cephalosporanic acids. That is, the adduct separated first is protected by converting its side chain and carboxyl group at the 4-position into an easily hydrolyzable ester or mixed acid anhydride. As a reagent for this purpose, the general formula

_[ Formula _{] (} In _the formula, (may form a ring with the phosphorus halogen compound) or the general formula

Examples include silicon halogen compounds represented by the formula: (wherein X is a halogen atom, R ₅ is a lower alkyl group, lower alkoxy group, or halogen atom, and R ₆ and R ₇ are lower alkyl groups or lower alkoxy groups). Among these phosphorus or silicon halogen compounds, specific examples of preferred compounds include:

【formula】

[Formula] CH ₃ OPCl ₂ , C ₂ H ₅ OPCl ₂ , C ₄ H ₉ OPCl ₂ ,
CH ₃ PCl ₂ , C ₄ H ₉ PCl ₂ ; (CH ₃ ) ₃ SiCl, CH ₃
(CH ₃ O) ₂ SiCl, (CH ₃ O) ₃ SiCl, CH ₃ O (CH ₃ )
SiCl, ( _CH3 ) _{2SiCl2 , ( CH3O} ) _2SiCl2 , _CH3
Examples include ( _CH3O ) _SiCl2 . Among these, more preferable compounds are:

【formula】

[Formula] (CH ₃ ) ₃ Cl, CH ₃ (CH ₃ O) ₂ SiCl, CH ₃ O (CH ₃ ) ₂ SiCl,
Examples include (CH ₃ O) ₃ SiCl. In addition, carbon halogen compounds such as COCl ₂ and CH ₃ COCl may be used as the reagent. In short, in this invention, the protection of the carboxyl group of N-acylcephalosporin C is carried out to prevent it from participating in the subsequent iminohalide formation reaction and iminoether formation reaction. The type of group is not particularly limited. However, it is desirable to select one from the above in consideration of easy hydrolyzability, cost of reagents, ease of treatment, etc. This reaction for protecting carboxyl groups involves the addition of an organic base such as triethylamine, quinoline, pyridine, dimethylaniline, and their congeners under anhydrous conditions in an inert organic solvent such as methylene chloride, ethylene chloride, or chloroform. will be carried out. The above reaction product is then reacted with an iminohalide forming agent such as phosphorus pentachloride, phosphorus oxychloride, or phosgene. Through this reaction, the acid amide bonds at the 7- and 5'-positions of N-acylcephalosporin C are converted to iminohalides. Thereafter, lower alcohols such as methanol, propanol, butanol, amyl alcohol, ethylene glycol, propylene glycol, ethylene chlorohydrin, and alkoxyethanol are added to the reaction solution. The iminohalide is thereby converted into an iminoether. In the above two reactions, the reaction solution in which the carboxyl group has been protected is used as it is. The iminoether obtained above is subjected to a hydrolysis reaction with water and is led to the desired 7-cephalosporanic acid. This hydrolysis is carried out under acidic conditions, but preferably at pH 2 to 3 when N-acylcephalosporin C is used as the raw material. In the case of N-acyl deacetoxycephalosporin C, the pH may be about 1. By adjusting the hydrolyzed reaction solution to the isoelectric point of 7-cephalosporanic acids, the desired substance can be obtained as a precipitate. This precipitate is separated and dried according to a conventional method. By the above method, the desired substance can be obtained in extremely high yield and purity. Next, the method of the present invention will be explained with reference to Examples, but the present invention is not limited thereto. Example 1 Approximately 0.2 g of sodium borate was added to 20 ml of "resin eluent" containing 30 mg of cephalosporin C (by UV quantitative method) per ml, and the pH was adjusted to 9.0 with dilute sodium hydroxide solution, and hybaric acid was added to this. Add 5 ml of an ethyl acetate solution of a mixed acid anhydride made from chloride and the N-methylformoline salt of methylthioacetic acid (containing 2.5 equivalents per mole of cephalosporin C) and stir vigorously at 0-10°C. The reaction solution is reacted for about 1 hour while adjusting the pH to 9.0 with diluted sodium hydroxide solution. When the reaction ends
TLC (benzene:acetic acid:pyridine:water=15:
3:10:12, the coloring agent is confirmed by heating after spraying the azide iodide solution, and the pH is adjusted to 5 to 6 with dilute sulfuric acid. Remove the organic layer and dilute the aqueous layer to 30 ml (2% solution in terms of cephalosporin C). Add 0.43 ml of quinoline (5 equivalents per mole of cephalosporin C) to 15 ml of this solution, and add dilute sulfuric acid to pH 3.0 while stirring at 10°C.
Adjust to. Crystals will soon begin to precipitate. After stirring for 1 hour,
The crystals are collected, washed with a small amount of ice water, further washed with ethyl acetate, and dried overnight in a vacuum dryer. IR 1790cm ^-1 (β-lactam), UVλ _nax 264m
μ. 450 mg (97% purity by UV quantitative method). Yield 98
% Example 2 Take 15 ml each of the reaction solution from a 3% solution of cephalosporin C (2% solution in terms of cephalosporin C) carried out according to Example 1, make solutions diluted as below, and add 0.43 ml of quinoline to each. (5 equivalents per mole of cephalosporin C) is added and treated according to Example 1. (The numbers in the box indicate the values after diluting with 5% saline.)

[Table] Example 3 Approximately 0.2 g of disodium phosphate was added to 20 ml of "resin eluent" containing 15 mg of cephalosporin C (by UV quantitative method) per ml, and the pH was adjusted to 9.0 with dilute sodium hydroxide solution. To this was added 5 ml of an ethyl acetate solution of a mixed acid anhydride (containing 2.5 equivalents per mole of cephalosporin C) prepared from potassium methylthioacetate, propionic acid chloride, and a trace amount of N-methylformoline, and the mixture was heated at 0 to 10°C. Stir vigorously. The reaction solution is dilute sodium hydroxide solution.
Incubate for 1 hour while adjusting the pH to 9.0. The reaction mixture is adjusted to pH 5-6. Separate the organic layer and dilute the aqueous layer to 30 ml with 5% saline (1% solution in terms of cephalosporin C). Add 0.22 ml of isoquinoline (5 equivalents per mole of cephalosporin) to 15 ml of this solution, and adjust the pH to 3.0 with dilute sulfuric acid while stirring at 10°C. Crystals will soon begin to precipitate. After stirring for 1 hour, the crystals are collected, washed with a small amount of ice water, then with ethyl acetate, and dried in vacuo overnight. N- with a purity of 97% (by UV quantitative method)
228 mg (yield 97%) of an isoquinoline adduct of methylthioacetyl-cephalosporin C was obtained. UV
λ _nax is 263 mμ, and the remaining 15 ml of the reaction solution is diluted to 30 ml (0.5% solution in terms of cephalosporin C) with 5% saline, 0.22 ml of quinoline is added, the pH is adjusted to 3.0, and the mixture is stirred at 5 to 10°C. Crystals will soon precipitate.
After 1 hour, the crystals are collected, washed with ice water and then ethyl acetate, and dried in vacuo. A quinoline adduct of N-methylthioacetyl-cephalosporin C with a purity of 97%.
Obtained 226 mg. Example 4 Approximately 0.04 g of sodium borate was added to 10 ml of "resin eluent" whose concentration was adjusted to contain cephalosporin C at a ratio of 10 mg/ml, and the pH was adjusted to 9.0 with dilute sodium hydroxide solution. Add 3 ml of an ethyl acetate solution of a mixed acid anhydride (containing 2.5 equivalents per mole of cephalosporin C) prepared from potassium methylthioacetate, trace amounts of N-methylformoline and acetyl chloride, and stir vigorously at 10-20°C. The reaction solution is reacted for about 1 hour while adjusting the pH to 9.0 with diluted sodium hydroxide solution. Add 0.1 ml of quinoline to the reaction mixture, and adjust the pH to 3.0 with 10% phosphoric acid while stirring. Crystals will soon begin to precipitate. After stirring for about 1 hour, the crystals are collected, washed with a small amount of ice water, then with ethyl acetate, and dried in vacuo overnight. N-methylthioacetyl-cephalosporin C quinoline adduct 145 mg (purity 98
%) was obtained. IR and UV were consistent with the standard. Using acetic anhydride instead of the above mixed acid anhydride,
When N-acetylation was carried out, no crystals of the quinoline adduct were obtained. Example 5 In place of the mixed acid anhydride of methylthioacetic acid and acetic acid in Example 4, an ethyl acetate solution of an acid anhydride prepared from methylthioacetyl chloride and N-methyl-morpholine salt of methylthioacetic acid was used. 150 mg of quinoline adduct of N-methylthioacetylcephalosporin C (purity 98%) was obtained. Example 6 Instead of the mixed acid anhydride of methylthioacetic acid and acetic acid in Example 4, an ethyl acetate solution of a mixed acid anhydride made from potassium ethylthioacetate, propionic acid chloride, and a trace amount of N-methylmorpholine was used. , according to Example 4 to obtain 125 mg (purity 96%) of a quinoline adduct of N-ethylthioacetyl-cephalosporin C. IR 1790cm ^-1 , UVλ _nax 263mμ. Example 7 Instead of the mixed acid anhydride of methylthioacetic acid and acetic acid in Example 4, (a) a mixed acid anhydride made from isopropylthioacetic acid potassium salt, pivalic acid chloride, and a trace amount of N-methylmorpholine, (b) Mixed acid anhydride made from isobutylthioacetic acid potassium salt, pivalic acid chloride, and a trace amount of N-methylformoline; (c) Mixed acid anhydride made from α-methylthiopropionate potassium salt, propionic acid chloride, and a trace amount of N-methylmorpholine. The mixture was treated according to Example 4 using a methyl acetate solution of each acid anhydride (b) a mixed acid anhydride made from potassium methylsulfinyl acetate, pivalic acid chloride, and a trace amount of N-methylmorpholine. , respectively obtained the following: (a) 108 mg of quinoline adduct of N-isopropylthioacetyl-cephalosporin C (purity 95%)
UVλ _nax 263 mμ, (b) N-isobutylthioacetyl-cephalosporin C quinoline adduct 105 mg (purity 96%),
UVλ _nax 263 mμ, (c) N-α-methylthiopropionyl-cephalosporin C quinoline adduct 134 mg (purity 97
%) UVλ _nax 263 mμ, (d) N-methylsulfenylacetyl-cephalosporin C quinoline adduct 138 mg (purity 94
%) UVλ _nax 263mμ. Example 8 Instead of the mixed acid anhydride of methylthioacetic acid and acetic acid in Example 4, (a) a mixed acid anhydride made from β-methylthiopropionate potassium salt, pivalic acid chloride, and a trace amount of N-methylthiomorpholine (b) ) β-ethylthiopropionate potassium salt and a mixed acid anhydride prepared from pivalic acid chloride and a trace amount of N-methylmorpholine, respectively, using ethyl acetate solutions, treated according to Example 4 to produce the following, respectively. I got something. (a) 100 mg of quinoline adduct of N-β-methylthiopropionyl-cephalosporin C (purity 96
%) IR 1790cm ^-1 , UVλ _nax 263mμ, (b) N-β-ethylthiopropionyl-cephalosporin C quinoline adduct 82mg (purity 96%)
IR 1790cm ^-1 , UVλ _nax 263mμ. Example 9 0.63 g of dried quinoline adduct of N-methylthioacetyl-cephalosporin C was added to 0.3 g of triethylamine and 0.4 g of dimethylaniline to 10 ml of dry methylene chloride, and a methylene chloride solution containing 0.43 g of trimethylsilyl chloride was prepared with stirring at 0°C. drip. After about 30 minutes, cool the clear solution to -30℃ and add 0.5g of Pcl ₅ .
Add fine powder and continue stirring at -20°C to -5°C for 2 hours. Cool again to -30°C, add 3 ml of anhydrous isobutanol dropwise, stir at -30°C to -10°C for 2 hours, and then keep at -20°C overnight. Add 3 ml of ice water to this and adjust the pH
Stir for 30 minutes while adjusting with ammonium carbonate salt to maintain the temperature between 2.0 and 2.5. Then, gradually raise the pH to the isoelectric point of PH3.5, and leave it in an ice room overnight. The precipitated crystals are centrifuged, washed with a small amount of cold 60% acetone water and then with acetone, and dried. 7-Aminocephalosporanic acid 0.23g (84%)
I got it. Purity 98% (by UV quantitative method). IR 1800
cm ^-1 , UVλ _nax 262mμ. Example 10 In place of trimethylsilyl chloride in Example 9, (a) 0.5 g of 2-chloro-1,3,2-dioxaphosphorane, (b) 2-chloro-4-methyl-1,
Example 9 Using 0.55 g of 3,2-dioxaphosphorane, (c) 0.63 g of trimethoxysilyl chloride, and 0.5 g of (d) dimethylmethoxysilyl chloride, respectively.
Processed according to (a) 0.22g, (b) 0.24g, (c) 0.23g,
(d) 7-aminocephalosporanic acid was obtained with a respective yield of 0.23 g. Example 11 Instead of isobutyl alcohol in Example 9, (a) anhydrous methanol and (b) n-butanol were used, and treated according to Example 9, resulting in (a) 0.25 g, (b)
7-aminocephalosporanic acid was obtained with a respective yield of 0.21 g. Example 12 Approximately 0.08 g of sodium borate is added to 20 ml of "resin eluent" whose concentration has been adjusted to contain cephalosporin C at a rate of 10 mg/ml, and the pH is adjusted to 9.0 with dilute sodium hydroxide solution. To this solution was added 5 ml of an ethyl acetate solution of a mixed acid anhydride (containing 4 equivalents per mole of cephalosporin C) made from potassium methylthioacetate, a trace amount of N-methylmorpholine, and ethyl chlorocarbonate, and the mixture was stirred vigorously at 0 to 5°C. do. The reaction solution was adjusted to pH with dilute sodium hydroxide solution.
Let it react for about 1 hour while adjusting the temperature to 9.0. Add 0.2ml of quinoline to the reaction solution and add while stirring.
Adjust to PH3.0. After saturating with sodium chloride and stirring for about 1 hour, the crystals are collected, washed successively with ice water and ethyl acetate, and dried. N-methylthioacetyl-cephalosporin C
260 mg of quinoline adduct (purity 97%) was obtained.

Claims (1)

[Scope of Claims] 1. In an aqueous solution containing cephalosporin C, a compound of the general formula R ₁ -Y-R ₂ -COOH (in the formula, R ₁ is a lower alkyl group, Y is a sulfur atom or a sulfinyl group, and R ₂ is a lower alkylene group) A reactive derivative of the acid represented by is reacted to obtain an aqueous solution containing N-acylcephalosporin C, then quinoline or isoquinoline is added, and an acidifying agent is added to adjust the pH to about 2.0 to 3.5 and as necessary. 1. A method for extracting cephalosporin C, which comprises adding a corresponding inorganic salt to separate an adduct of N-acyl cephalosporin C.