DK146764B - ALKALIMETAL CLAVULANATE USED AS INTERMEDIATE IN THE PREPARATION OF CLAVULANIC ACID OR OTHER DERIVATIVES THEREOF, AND MIXTURE USED FOR THE PREPARATION OF CLAVULANIC ACID OR DERIVATIVES THEREOF - Google Patents

Description

in 146764

The invention relates to a novel alkali metal clavulate for use as an intermediate in the preparation of cla-vulanoic acid or other derivatives thereof. This alkali metal clavulanate according to the invention is peculiar in that it is lithiumel avulanate.

The invention also relates to a composition for use in the preparation of clavulanic acid or derivatives thereof, and according to the invention this composition is characterized by containing at least 98% lithium clavulanate. As pure lithium clavulanate as possible, ie with a maximum of 2% impurities, is preferred for the preparation of clavulanic acid.

Fermentation of Streptomyces clavuligerus, in particular the strain NRRL 3585, causes the formation of various antibiotic substances and GB Patent No. 1,315,577 discloses the cultivation of said strain to produce antibiotics A 16886 I and A 16886 II.

The organism may additionally form the antibiotic (2R, 5R, Z) -3- (2-hydroxyethylidene) -7-oxo-4-oxa-1-azabicyclo [3,2,0] -heptane-2-carboxylic acid of formula I :

In CH-OH

H

H * COOH

and this compound is referred to herein as clavulanic acid. Thus, the invention relates to the lithium salt of this compound.

Clavulanic acid and various acids and salts thereof are described in Danish Patent Application No. 1672/75, and here also is described how some cleaning of the material can be done.

The best method of purification is based on forming a benzyl ester salt from a salt of clavulanic acid, which is then purified in a conventional manner, and then the acid is released from it. It has been found that by reductive cleavage of benzylelavulanate, 10-15% of an inactive isomer is formed. Thus, the best material obtainable by this known purification method contains 10-15% of the undesirable isomer; material obtained according to said application and without the esterification process contains at least 1/3 impurities.

It is an object of the invention to provide a clavulanic acid compound which, when used as an intermediate in a purification process, can lead to clavulanic acid or various derivatives thereof in a form which is substantially purer than can be obtained by the aforementioned prior art. Surprisingly, it has been found that this can be achieved by the lithium salt, which has the unparalleled property of being heavily soluble in water, while easily precipitating in high purity, after which it can be converted to clavulanic acid or other derivatives of clavulanic acid. . As indicated above, lithium clavulanate is preferably prepared in a purity of at least 98%.

As mentioned, it is surprising that precisely the lithium salt is readily prepared in such high purity and thus can lead to very pure other clavulanic acid compounds. Namely, from Examples 15 and 17 of the aforementioned DK patent application 1672/75, it is known to apply a crude culture filtrate containing crude clavulanic acid to a column of a weakly basic ion exchange resin and then elute with NaCl. Thereby, no crystallization of sodium cla-vulanate occurs, but this salt is obtained by evaporation of the eluate. Hereby no appreciable separation from impurities is obtained, and according to said example 17, the evaporated product has a purity of approx. 1/3 of the best of the purities indicated in the application examples. By contrast, if sodium chloride is replaced by lithium chloride as the eluent from the column of ion exchange resin salt with clavulanic acid, crystalline lithium clavulanate is formed which precipitates and is thus separated from impurities. The other alkali metals do not form such crystalline salts with clavulanic acid.

Examples of impure salts which, in addition to crude clavulanic acid, can form the starting material for the preparation of lithium clavulanate are alkali metal salts such as the sodium, potassium and lithium salts; alkaline earth metal salts such as the calcium, magnium and barium salts; the ammonium salt; and salts with organic bases such as salts derived from primary, secondary, tertiary or N-quaternary amines, for example, mono-, di- or tri-alkylammonium salts such as the methyl ammonium salt and the triethylammonium salt, and heterocyclic base salts such as the piperidinium salt.

The salts with inorganic bases and most of the salts with organic bases are generally more stable in aqueous solution than free clavulanic acid. The salts can exist in the form of solvates, ie. with crystal water or another crystallization solvent.

From lithium clavulanate according to the invention and with a purity of 98% or more, ie having a content of impurities and / or isomers originating from the production of not more than 2% by weight, several other clavulanate salts have been prepared in crystalline form. These salts were substantially pure as evidenced by the molar extinction coefficients, measured at 259 + 1 nm in 0.1M aqueous sodium hydroxide, of at least 16,200. The values 24 for the molar rotation, [M] +, in aqueous solution were at least + 137 ° + 5 °. The free acid prepared from the salts has exhibited 1% an extinction coefficient, ^ cm in 0.1M aqueous sodium hydroxide at 259 nm, of 590 or greater, and a specific optical rotation [α] ² in dimethyl sulfoxide of ca. + 54 °. It will be appreciated that the production of material having this degree of purity makes it possible to use the products in pharmaceutical and veterinary preparations, and also that such purity is highly desirable when using the materials as intermediates.

The term "purity" in this specification refers to the percentage of clavulanic acid and / or salt thereof, expressed in relation to the total amount of dry matter present by weight, but without regard to bound water or other bound solvents.

Clavulanic acid and its salts have antibacterial activity against a variety of Gram-negative and Gram-positive microorganisms, are stable against the action of 3-lactamases produced by Gram-positive and Gram-negative organisms, and have the ability to inhibit 3-lactamase enzymes produced by Gram-positive and Gram-negative bacteria. In addition, a number of class I enzymes are inhibited.

Thus, clavulanic acid and its salts have the ability to protect B-lactamase-sensitive 3-lactam antibiotics from hydrolysis induced by 3-lactamase, and it is therefore of interest for use with 3-lactam antibiotics which are sensitive to B-lactamases from both Gram-positive and Gram-negative organisms.

It is generally preferred to use salts of cla-vulanoic acid with a purity of at least 98%, i.e. with less than 2% impurities or isomers derived from the production other than solvents, together with a broad-spectrum 8-lactam antibiotic.

Preparation of Lithium Clavulanate

Clavulanic acid and its salts can be recovered from a fermentation medium produced by culture as disclosed in British Patent Specification No. 1,315,177 to a strain of Streptomyces clavuligerus, e.g. other S-lactam antibiotics. Conventional technique is difficult, in particular because of the uniform behavior of the various B-lactam carboxylic acids present, such as the aforementioned antibiotics A 16,886 I and II. Extraction of the antibiotic is greatly facilitated by converting clavulanic acid or its salts to lithium clavulanate and precipitating the latter, usually in crystalline form. Such precipitation may be accomplished, possibly due to a surprisingly high affinity of clavulan ions to lithium ions, with no or no significant simultaneous precipitation of impurities, especially other 8-lactams. In addition, by direct isolation of the salt while avoiding conversion of clavulanic acid to organic derivatives such as esters with subsequent genome formation to the acid, for example by a reduction technique, clavulanic acid is converted to isomers.

It should be noted that in fermentation media and other solutions at approximately neutral pH, clavulanic acid and salts formed with one or more cations exist in equilibrium, and isolation processes will frequently be performed on clavulanic acid and / or salts thereof, depending on pH and other conditions. In general, clavulanic acid and its salts are rather unstable in aqueous solution outside the pH range of 5.5 to 8, and during the processes described below it is desirable to maintain the pH within this range, preferably close to ca. pH 6.5 unless otherwise stated.

5 148764

In the preparation of lithium clavulanate, clavulanic acid, usually crude clavulanic acid in a crude fermentation medium or crude, is reacted from the lithium-salt different salt of clavulanic acid with an aqueous xonic lithium compound to produce an aqueous solution containing lithium clavulanate, which is then precipitated and then precipitated. resolution.

In general, the ionic lithium compound will be a salt. It is preferred to use lithium chloride, but also lithium bromide, lithium iodide or lithium sulfate as well as lithium carboxylates such as the acetate, propionate, formate, benzoate or lactate are suitable. The choice of salt may be influenced by other materials present, and if, for example, the initially present clavulanic acid salt is the barium salt, it may be preferable to use lithium sulfate to effect a prior precipitation of barium sulfate prior to the precipitation of lithium clavulanate.

Generally, it is preferred that the concentration of lithium clavulanate prior to precipitation is at least 0.1% by weight, preferably at least 2%, and higher concentrations such as up to 12% or even up to 20% by weight, of course, yield greater percent recoveries.

The initially present salt of clavulanic acid which needs purification may be, for example, an alkali metal salt such as the sodium salt or potassium salt or even the lithium salt if present as a quantitatively minor component of the clavulanic acid material, or it may be an alkaline earth metal salt such as calcium, barium or the magnesium salt or a salt having an organic base as described above, or a salt formed with a basic ion exchange resin.

The fact that lithium clavulanate readily precipitates in high purity can be utilized in several different ways.

Salting out of lithium clavulanate

A sufficient amount of water-soluble lithium salt, especially that used to form lithium clavulanate, may be introduced into the aqueous solution containing lithium clavulanate; thereby raising the concentration of lithium ions so that the solubility product of lithium clavula night at the given temperature is greatly exceeded. As the clavulanate is less soluble at lower temperatures, it is usually appropriate to decrease the temperature of the solution to bring the precipitate to maximum, e.g., to about 0-5 ° C.

In such a salting out, the concentration of the ionic lithium compound in the aqueous solution containing the lithium clavulanate is preferably in the range of 4M to 10M, but concentrations up to saturation are useful; a particularly preferred range is 5M to 8M.

It may be advantageous after harvesting the first yield of lithium clavulanate to concentrate further and harvest a next yield.

Conversion of other clavulanates to lithium clavulanate

The solution containing lithium clavulanate may be formed by dissolving a salt of clavulanic acid other than the lithium salt, e.g., the sodium, potassium, magnium, barium, calcium or ammonium salt, in an aqueous solution and incorporating therein a water-soluble lithium salt such as lithium chloride. In the case of the barium salt, use of a high concentration of lithium chloride may result in some simultaneous precipitation of barium chloride with lithium clavulanate. However, the barium chloride can be easily eliminated by redissolving the mixture in water and adding lithium sulfate to precipitate barium sulfate which can be removed, for example, by filtration, followed by addition of lithium chloride to precipitate pure lithium clavulanate.

Formation of lithium clavulanate on an ion exchange resin Particularly pure lithium clavulanate is obtained if the salt of clavulanic acid used as a starting material is a salt with a basic ion exchange resin and it is contacted with an aqueous solution of a lithium salt to produce an aqueous solution of lithium salt. . The resin will normally be used in the form of a column to which impure clavulanic acid and / or a salt thereof is applied, and from which an aqueous solution of lithium clavulanate is eluted using an aqueous solution of a water-soluble lithium salt such as lithium chloride. The resin will usually be washed, eg with water, before elution.

The resin will generally contain amino groups or tertiary amino groups (weakly basic) or quaternary ammonium groups (strongly basic). The resin may be, for example, a polystyrene resin, polyacrylic resin, epoxy-polyamine resin, phenolic-polyamine resin or cross-linked dextran resin, and it may be macroretic or microretic.

The term "resin" is used herein for practical reasons to include cellulose derivatives and the aforementioned dextran derivatives derived from naturally occurring polymers. Typical weakly basic ion exchange resins are (R i, "Amberlite" IRA68 (microretric; polyacrylate cross-linked with divinylbenzene; tertiary amino groups) and "Amberlite" ILA93 (macroretic; polystyrene cross-linked with divinylbenzene) tertiary amine and tertiary amine and tertiary amine groups). .a. "Zerolit" ^ FF and "Zerolit" FF (iP) (negotiated by Zerolit Co. Ltd.).

Advantageously, basic ion exchange resins are in salt form when contacted with an impure clavulanic acid and / or a salt thereof; The anion is preferably the same as in the lithium salt used as the eluent, suitably the chloride ion, but different anions can also be used without decisive adverse effects.

The concentration of the aqueous lithium salt used as the eluent is preferably in the range of 0.02M to 8M, but. however, the lower concentrations give very thin solutions of lithium clavulanate and make the subsequent precipitation more difficult. Generally, it is preferred to use concentrations in the range 0.5 to 2.5M.

Although adsorption / elution techniques are conventionally carried out in such a way that the desired product is subjected to separation of the chromatographic type from other adsorbed materials, it has been found that the subsequent step of precipitation is so effective in separating lithium clavulanate from undesirable impurities. it is usually preferable to substantially strip the column using relatively high concentrations of lithium salt in the eluent. This gives a narrow band of clavulanate on the column which can be eluted to a relatively small volume of eluate, thereby facilitating subsequent precipitation.

The eluate will normally contain the lithium salt, for example lithium chloride at a concentration in the range 0.5 to 2.5M, while as mentioned before the salting effect is most effective at concentrations in the range 5M to 10M. It is therefore preferred to concentrate the eluate, for example by evaporation in vacuo to ca. 1/5 of the volume. The solubility of lithium clavulanate at ca. 20 ° C in various concentrations of aqueous lithium chloride is shown in the table below:

Table 1

Molarity of Solubility of Lithium Clavula

LiCl 2 night, mg / ml (approximate) 2.5 23.5 3.75 10.2 5.0 4.1 6.25 1.8 7.5 0.8

The aforementioned step of concentration is preferred over the addition of additional lithium salt, since lithium clavulanate is also concentrated and losses in the mother liquor are therefore reduced.

In order to reduce the elution of adsorbed impurities from the resin, it may be advantageous in the eluant to incorporate a high concentration water miscible organic solvent. It is also possible, after elution in the absence of such a solvent, to add this to the eluate to precipitate eluted impurities, after which the precipitate thus formed is separated before further treatment. The solvent may be, for example, a ketone such as acetone, an alcohol such as methanol, ethanol, isopropyl alcohol or ethylene glycol, an ether such as dioxane or tetrahydrofuran or a substituted amide, sulfide oxide solvent such as dimethylformamide or dimethylsulfoxide. Generally, alcohols such as solvents are preferred, for example ethanol or isopropyl alcohol.

U6764 9

For use in such separation from undesirable impurities, the preferred concentration of the alcohol in the eluent or eluate, after addition of the alcohol, is 70 to 97% by volume.

Precipitation of lithium clavulanate by a water-miscible solvent

If the concentration of the water-miscible organic solvent and lithium salts in the procedure just described is too high, the lithium clavulanate may be precipitated prematurely.

It is possible to precipitate lithium clavulanate from an aqueous solution using very high concentrations of such solvents, and this denotes an alternative precipitation method which utilizes the advantages of the described selective precipitation of lithium clavulanate. thus, clavulanic acid and / or a salt thereof can be contacted with a relatively low concentration of lithium salt, either by elution from a column or by dissolving the salts in a single solution, after which the desired precipitation can be carried out without concentration by adding water-miscible solvent. Thus, for example, concentrations of alcohol of at least 90 volumes, preferably at least 95%, are effective in precipitating lithium clavulanate. It may be necessary to harvest a first yield of lithium clavulanate and then concentrate in vacuo, e.g. to 1/4 volume, to obtain a second yield.

Preparation of a salt of clavulanic acid with an ion exchange resin.

The salt of clavulanic acid with a basic ion exchange resin may be formed by contact between the resin and a fermentation medium containing clavulanic acid and / or a salt thereof, thereby saving an adsorption / elution step. Solid material is usually removed in advance, for example, by filtration or centrifugation. This possibility exists because of the remarkable purification that occurs by precipitation with an ionic lithium compound. After removal of solids, the nutrient medium can be treated with adsorbent charcoal to adsorb clavulanic acid and / or the salt thereof; this helps separate other salts from the clavulanate and avoid excessive loading of the basic ion exchange resin with undesirable ionic material.

In general, the clarified medium can be passed through a charcoal bed, for example in a column, preferably using just enough charcoal to adsorb all the desired clavulanic acid and / or salt thereof, usually in a ratio of approx. 1 volume charcoal to 3-10 volumes clarified medium. Ordinary charcoal is suitable and it is not necessary to use highly activated material.

The charcoal can then be stripped with an aqueous water miscible solvent, for example a ketone such as methyl ethyl ketone, methyl isobutyl ketone or preferably acetone, advantageously at a concentration of from 30 to 95% ketone, preferably 50 to 70%. Prior to stripping, the charcoal is preferably washed, for example, with water to remove residual components from the medium.

Another variation of the process just described consists in preparing a resin salt of clavulanic acid as described above and eluting it with a salt other than a lithium salt, e.g., sodium salt, potassium salt, magnesium salt or calcium salt, e.g., a chloride or acetate, or a ammonium salt or pyridinium salt, for example ammonium formate or acetate or pyridine hydrochloride, to form an aqueous solution of the corresponding clavulanate; excess water-soluble lithium salt can then be added to the eluate and lithium clavulanate precipitated as previously described.

Formation of lithium clavulanate and other clavulanates by extraction of a phenolic solution of clavulanic acid

An aqueous solution containing lithium clavulanate can also be obtained by extracting a phenolic solution of clavulanic acid with an aqueous solution of lithium hydroxide, and then precipitating lithium clavulanate is carried out as described above, preferably after removal of the remaining phenolic solvent by extraction of the aqueous solution with aqueous solution. solvent such as ether, chloroform or carbon tetrachloride.

This technique can also be used to prepare clavulanic acid salts other than the lithium salt by extracting the phenolic solution with a suitable base, for example alkaline earth metal hydroxide such as calcium or barium hydroxide. Any other precipitate formed, e.g., barium sulfate, should be removed prior to the precipitation of the clavulanate salt and this can then be isolated, for example, by freeze drying. Purification by conversion to the lithium salt can then be performed as described above.

Generally, the extraction of the phenolic solvent is carried out by titrating the aqueous phase with a base to obtain a pH of approx. 6.5.

The phenolic extract can be prepared by extracting with an phenolic solvent an aqueous eluate from a charcoal or resin adsorbate of the kind described above, usually after concentration of the eluate and optionally after precipitation of undesirable organic impurities by the addition of one or more water-miscible organic solvents. and / or removing such impurities by extraction with a water-immiscible solvent.

Thus, in this type of procedure, it may be desirable to concentrate the eluate from either the charcoal or a resin by evaporation under reduced pressure. In general, the treatment during the purification is preferably carried out at a pH in the range 6.0-7.0, e.g. 6.5, to minimize decomposition. The eluate can then be purified by precipitation of unwanted material with a water-miscible ketone such as acetone, preferably so that the ketone concentration reaches 50 to 90% by volume, advantageously approx. 85%. The pH of this step is preferably about 6.5 and if the aqueous liquid already contains water-miscible ketone, it is preferably removed to facilitate the pH measurement. The pH can be adjusted by the addition of a base, for example, an alkali metal hydroxide such as sodium hydroxide.

Further purification can be performed with a solvent at extraction step to remove undesirable components, e.g. by concentrating and controlling the pH of the filtrate from the ketone precipitate to ca. 4 by the addition of a mineral acid such as sulfuric acid or hydrochloric acid, and extraction with n-butanol or a higher molecular weight liquid alcohol. Practically 1 to 8 volumes of solvent can be used.

After this extraction, the aqueous phase is preferably concentrated at ca. pH 6.5, and it can be further purified by extraction of the desired antibiotic into a phenolic solvent, e.g., the compound itself phenol or a cresol, preferably after reducing the pH to ca. 4 with a mineral acid. The phenolic solvent advantageously contains a base such as Ν, Ν-dimethylaniline and a water-immiscible solvent such as chloroform or carbon tetrachloride. The extraction is advantageously carried out several times using approx. 2/3 volume of solvent for each extraction. The extracts can then be combined and water may be added, preferably approx. 1/15 of the solvent volume, to form a separate phase. The antibiotic can then be back-extracted by adding to the aqueous layer of a base, preferably an alkali metal hydroxide such as lithium hydroxide, or an alkaline earth metal hydroxide such as barium hydroxide or calcium hydroxide, to a pH of about 6.5. The aqueous layer is separated from the phenolic layer and the back extraction process is advantageously repeated, after which the aqueous extracts are combined. After separation of all precipitates other than clavulanate, e.g. barium sulfate, any remaining phenolic solvent may be removed from the aqueous solution by extraction with a water immiscible solvent such as ether, chloroform or carbon tetrachloride, and to recover the salt of the antibiotic, the aqueous phase may be freeze dried or spray dry at pH 6.5.

Further purification can be performed by conventional techniques such as chromatography, in particular using such materials as "Sephadex" ® (a cross-linked dextran). Thus, at this stage, the antibiotic which will normally take the form of a salt, e.g. barium salt, can be applied to a column of "Sephadex", e.g. "Sephadex" G 15, and eluted with water, combining the fractions containing substantial antibiotic activity to subsequent extracting a salt, for example, by freeze-drying.

Conversion of lithium clavulanate to clavulanic acid and other salts

Purified lithium clavulanate prepared as described above can be converted to other salts by ion exchange processes, for example by means of an ion exchange resin. Thus, for example, the aqueous lithium salt can be applied to a cation exchange resin, for example "Bio Rad AG50X8" in cation form, the cation being the one desired in the clavulanic acid salt, eg sodium or potassium, followed by elution, eg with water.

Free clavulanic acid can be formed by acidification, for example to pH approx.

2.6, of an aqueous solution of the lithium salt, preferably of high ionic strength, for example, saturated with sodium chloride or ammonium sulfate in the presence of a water-immiscible solvent for clavulanic acid, for example an ester solvent such as ethyl acetate. If necessary, the aqueous phase can be extracted with additional solvents and the extracts combined. In general, any acid which gives a sufficiently low pH value will be suitable for the acidification, for example a mineral acid such as hydrochloric acid. The solvent can then be removed to produce the free acid, usually in the form of an oil.

The dissolution of the free acid in the water-immiscible solvent can be used to prepare many different salts by extraction with an aqueous solution of the base and isolation of the salt therefrom. It may be necessary to filter solid material from the aqueous phase before proceeding to isolate the salt.

Since the free acid is relatively unstable, it should preferably be used as soon as possible after its formation, for example for the preparation of salts or other derivatives.

The invention will be explained in detail below, partly by an example of the preparation of the starting material for lithium clavulanate, partly by some examples of its preparation and partly by examples of the use of the lithium salt as an intermediate.

Clavulanic acid content in process fluids and solids is measured here by:

Ultraviolet Spectroscopy

Aqueous solutions of clavulanic acid and its salts exhibit very low UV absorptions at more than 230 nm and, for example, the molar extinction coefficient ε at 280 nm is approx. 60. However, when dissolved in alkali, intense UV absorption develops at 259 + 1, and it can be used to determine clavulanic acid and / or its salts. For such determinations, solids are accurately weighed and dissolved in thin sodium hydroxide (0.1M) to produce a known volume of solution corresponding to ca. 0.01 mg / ml clavulanic acid. The optical density of the solution at an absorption maximum at or about

259 nm was measured on a spectrophotometer; solid impurities can be calculated assuming that ε for pure clavulanic acid is 16,700.

Molar extinction coefficients can be calculated from E the extinction coefficients of a 1% solution in a 1 cm cell. Similarly, process liquids, if necessary after removal of organic solvents, were precisely diluted with dilute sodium hydroxide to give similar concentrations of alkali and clavulanic acid, from which the clavulanic acid concentration in the original liquid was determined in the manner described above. Values for crude solids and process fluids were corrected for the absorption of impurities by solutions of the same concentration in water.

Starting material 1 a) Graft culture development 10 ml of sterile distilled water was added to a 14 day old slurry culture on malt / yeast extract agar of Streptomyces cla-vuligerus NRRL 3585 and a suspension was prepared.

A 1.5 ml aliquot of this suspension was used to inoculate 150 ml of a medium containing (expressed in% v / r, i.e., that the ratio of units of solids to liquids is as between g and ml):% v / r sucrose 2.0 distillers' solubles 1.5 yeast extract 0.5 K2 HPO4 0.02 tryptone 0.5 glycerol 1.0 and water to 100% in a 2 liter long necked, round, flat bottom flask.

This flask was incubated at 26 ° C for 48 hours at 220 rpm on a rotary shaker with a 5 cm stroke. 150 ml of this seed culture was used to inoculate 4 liters of a medium containing: U6764 15% v / r soybean flour 2.1 distillers' solubles 0.52 casein hydrolyzate 0.52

FeSO4, 7H20 0.01 soluble starch 4.7 glucose 0.78 and water to 100% in a 5 liter large fermentation vessel with aeration (0.75 v / v / min) and kept at 28 ° C with stirring (750 rpm ) for 20 hours.

b) Fermentation 7.5 liters of the 20 hour old inoculum of paragraph (a) above was seeded in 150 liters of a medium containing:% soluble starch 4.7 soybean flour 2.1 distillers' solubles 0.52 casein hydrolyzate 0, 52 glucose 0.78

FeSO4, 7H20 0.01 polyglycol (P.2000 from Dow Chemical Co.) 0.05 and water to 100% in a 220 liter container and fermented here for 90 hours at 28 ° C under aeration (2 vol / vol / min) and stirring (350 rpm).

Preparation of the barium salt

A fermented medium obtained as described above is purified as follows: 135 liters of fermentation medium of pH 6.25 were clarified on a centrifuge to produce a supernatant in an amount of 112 liters and of pH 6.3. This was passed through a column of 25 liters of charcoal and the column was washed with 50 liters of water. Gently poured 10 liters of acetone into the top of the bed and elution commenced. This was followed by 60 liters of 90% aqueous acetone. The eluate was collected in fractions (1x10 liter, 2x25 liter).

Each fraction was distilled under reduced pressure to remove acetone. The remaining aqueous solution was adjusted to pH 6.0 with 1M sodium hydroxide. The fractions were then combined and further evaporated to 2.9 liters, transferred to a container and regulated up to 3.3 liters with boiler washing fluids. 17 liters of acetone and 500 g of filter aid "Celite" ® 535 were added to the concentrate with vigorous stirring.

The resulting suspension was filtered and the cake washed with 4 liters of 85% aqueous acetone.

The filtrate and washings were combined and the combination was evaporated under reduced pressure to 4.0 liters. The pH was adjusted from 5.65 to 6.0 with 1M sodium hydroxide and evaporation continued until the volume was 1.0 liter. The resulting concentrate was acidified to pH 4.0 with 20% sulfuric acid and washed with 4 x 750 ml and 1 x 500 ml butan-1-ol. Dissolved butan-1-ol was precipitated by the aqueous phase under vacuum.

After being adjusted to pH 4.2 with 20% sulfuric acid, the concentrate was extracted three times with a mixture of 265 ml of liquefied phenol B.P. (ie British Pharmacopoeia grade), 75 ml of carbon tetrachloride and 25 ml of Ν, Ν-dimethylaniline. The pH was adjusted to 4.2 for each extract. The combined solvent extracts were stirred with 250 ml of water and the pH was adjusted to 6.5 with 70 ml of saturated aqueous barium hydroxide. After separation of the phases, the solvent was re-extracted with 200 ml of water and 7 ml of saturated aqueous barium hydroxide.

The combined aqueous phases were washed with 3 x 200 ml diethyl ether, reduced in vacuo to 200 ml and lyophilized to 29.4 g light brown solid. E ^ = 152. Purity below 25%.

Example 1 i) Preparation of the potassium salt a) 8.83 g of crude barium salt (purity <25%) from starting material 1 was added to 50 ml of saturated aqueous solution of (NH 2 SO 2).

50 ml of ethyl acetate was added and the mixture was stirred. A pH test instrument was inserted into the mixture and the pH was adjusted from 6.8 to 2.6 with approx. 15 ml of 1M H2SO4 · The aqueous solution was separated from the ethyl acetate and stirred again with a fresh portion of ethyl acetate, 50 ml. The two ethyl acetate extracts were combined, 100 ml of distilled water was added and the mixture was stirred in the presence of the pH sample instrument. Approx. 40 ml of saturated solution of a calcium hydroxide to bring the pH of the mixture to 6.6. The aqueous solution was separated from the ethyl acetate, filtered through filter aid and lyophilized to give 1.44 g of solid calcium salt.

b) The solid from (a) was dissolved in 15 ml of distilled water, filtered through a millipore filter and applied to a column containing "Sephadex" ® G15, packed in water to a bed of about height. 150 cm and a diameter of 2.5 cm. Eluted with distilled water and 20 ml fractions were collected. The fractions were determined by thin layer chromatography (tic; cellulose, "Eastman" ® "Kodak" ® 6065 plates; solvent acetonitrile / water 7: 3) by overlaying with nutrient agar containing Staphylococcus aureus. Fractions 33-37 were combined and lyophilized to give 490 mg. The solid was kept above room for 60 hours. The calcium salt from section (b) of this example had the following characteristics: pKa. The pKa value for the corresponding acid was found to be approx.

2.4 by potentiometric titration of the salt.

Optical rotation. [a] D at 22 ° C: + 44.9 ° (concentration 0.287 g / 100 ml water).

UV spectrum. A sample of 0.00148 g which had been dissolved in 100 ml of 0.1 M NaOH exhibited absorption maximum, λ, at 258 nm with an E 550th

IR spectrum. The infrared spectrum of a sample in nujol exhibited absorption maxima at wave numbers (cm j as follows: 3300 s, br 2330 w 1788 s 1692 m 1604 s 1404 s 1305 s 1190 m 1118 m 1082 m 1060 m 1042 m 1012 m 992 m 968 m 892 m 848 w 790 w 740 m 654 w 146764 18 (s, m, br and w denote strong, medium, wide and weak intensity, respectively).

The full spectrum is shown in Figure 3 of the drawing.

NMR spectrum. The proton-nuclear magnetic resonance spectrum in the form of dissolution of the sample in heavy water exhibited groups of peaks (τ values) centered at.ca. 4.31, 5.10, 5.85, 6.46 and 6.91.

Thin Layer Chromatography. Portions of the sample, dissolved in water, were applied at the beginning of either "Eastman" - "Kodak" cellulose TL plates (with plastic backing EK 6065) or "Eastman" - "Kodak" silica ka plates (with plastic backing, EK 6060 ). The plates were developed with solvent at room temperature and then air dried and overlaid with nutrient agar containing Staphylococcus aureus. Rf values, calculated as the distance from the beginning to the center of each zone of inhibited bacterial growth, divided by the distance from the beginning to the solvent front, are given below for five systems.

Solvent Carrier Rf propan-1-ol / water 7: 3 cellulose 0.60 butan-1-ol / acetic acid / water 3: 1: 1 "0.64 acetonitrile / water 7: 3" 0.68

Acetonitrile / water / propan-2-ol 1: 1: 1 "0.87 butanol-1-ol / acetic acid / water 3: 1: 1 silica 0.63

Papirionoforese. Portions of the sample were subjected to ionophoresis on Whatman® 541 paper for 1 hour with 400 volts applied over 20 cm. The activity, found by superimposing the air-dried paper with nutrient agar containing Staphylococcus aureus, had a 4.5 cm cyanocobalamin mobility toward the anode at pH 4.8 (0.01M acetate), pH 6.9 (0, 1M phosphate) and pH 9.5 (0.01M pyrophosphate).

ii) Preparation of the lithium salt

1 g of the calcium salt (E ^ 590) prepared as described above in (i) (b) was dissolved in 10 ml of water and 10 ml of a saturated solution of lithium chloride in water was added. Crystallization occurred without scraping or grafting. After cooling to 0 ° C

The crystals were filtered and washed with 5 ml of ethanol, 5 ml of acetone and 2 x 5 ml of diethyl ether. The crystals were dried under reduced pressure to 0.1 mm Hg over silica gel for 2 hours to give 495.5 mg of solid lithium salt. This salt had purity above 95% and the following characteristics:

Elemental analysis. Values found (mean values in parentheses): C 45.5, 45.8 (45.65); H 3.8, 3.8 (3.8); N 7.0, 7.2 (7.1); Li 3.2%. Sulfur was not detected by a method specified by B.D. Cheronis and J.B. Entrikin (1947) in Semimicko Qualitative Analysis page 93, Crowell, New York. The composition CgHgNOgLi, 1/4 H2O requires C 45.84; H, 4.06; N, 6.68; Li 3.31%.

The value for the Li analysis stated above, 3.2%, was determined by atomic absorption spectrophotometry. Sulfated ash was 26.8%, calculated as LI2SO4 equivalent to 3.38% lithium.

pK. The pKa value for the corresponding acid was found to be approx. 2.3 by potentiometric titration of the salt.

Optical rotation. The value of a 0.145% v / r aqueous solution at 24 ° C was + 66.0 °.

Ultraviolet spectrum. The UV absorption spectrum of a 0.00091% solution in 0.1M sodium hydroxide had absorption maximum (λλ) at 258 nm with an E₂ value of 788.

max 1 c

Infrared spectrum.The nujol IR spectrum exhibited absorption peaks (cm 1) at approximately: 3420 (m) 1402 (s) 1200 (w) 1026 (m) 880 (w) 3012 (w) 1338 (m) 1129 (m) 992 (m) 850 (w) 1765 (s) 1325 (s) 1101 (m) 976 (m) 734 (m) 1683 (s) 1300 (m) 1062 (m) 950 (s) 708 (w) 1618 (s) 1224 (w) 1048 (s) 900 (m)

The full spectrum is shown in Figure 1 of the drawing.

NMR spectrum. A proton-nuclear magnetic resonance spectrum at 100 MHz for the lithium salt in solution in heavy water exhibited peaks (τ values with multiples and coupling constants, Hz, in parentheses), centered at ca. 4.26 (d, 3), 5.05 (t, 8), 5.06 (s), 5.81 (d, 8), 6.43 (dd, 3 and 17) and 6.89 (d) , 17).

U6764 20

Here, s, d, dd, t and m denote singlet, doublet, double doublet, triplet and multiplet respectively.

iii) Recrystallization of the lithium salt 0.1 g of the lithium salt prepared as described above under (ii) was dissolved in 1.0 ml of water and carefully diluted with 19 ml of isopropanol. The product crystallized slowly at 0 ° C and was harvested in two yields by boiling under reduced pressure to 5 ml for the second yield. The crystals of the lithium salt were dried over silica gel in vacuo for 3 days.

Yield 1. 20.0 mg Xmax 259 nm, E 2 = 814 in sodium hydroxide solution (0.1M) at 10 pg / ml.

Elemental Analysis

Calculated for C CHgNO ^ Li, 1/4 I ^O: C 45.84 H 4.06 N 6.68

Found: C 46.2 H 4.10 46.0 3.85 N 6.8 6.7%.

Yield 2. 67.0 mg. ^ max 259 nm, E ^ = 800 in sodium hydroxide (0, IN) at 10 µg / ml.

Elemental Analysis

Calcd for CgHgNOgLi, 1 / 4H2O: C 45.84 H 4.06 N 6.68 Li 3.31

Found: C 46.15 H 3.9 N 6.65 Li 3.4% (sulfated ash) C 46.5 H 4.0 N 6.5

Example 2 30 g of the barium salt (E (= 274; purity <45%, prepared as starting material 1) were dissolved in 40 ml of water and 300 ml of saturated ammonium sulfate solution was added. The pH of the solution was adjusted to 2.3 with 23.0 ml of 20% sulfuric acid and then extracted with 2 x 300 ml of ethyl acetate. 200 ml of water was added to the combined extracts. The mixture was stirred vigorously and 89.3 ml of 1M sodium hydroxide solution was added until the pH reached 6.8. The aqueous phase was separated and evaporated to 33 ml under reduced pressure. 770 ml of butan-1-ol was added to the aqueous concentrate and then mixed, heated to 40 ° C and shaken vigorously. Insoluble U6764 21 material was filtered off and re-extracted with water / butan-l-ol 1:23 (volume ratio) until all dissolved. The combined solutions were cooled to 4 ° C overnight. The crystalline solid formed was collected by filtration, washed with butan-1-ol and acetone and air dried to 3.34 g of the sodium salt (E + = 648).

2.92 g of this sodium salt was dissolved in 20 ml of water and filtered. The filtrate was stirred at 0 ° C while introducing 20 ml of lithium chloride solution (saturated at 20 ° C) over 5 minutes. Stirring and cooling were continued for one hour, after which the crystals were added by filtration, washed with 20 ml of ethanol, 2 x 20 ml of acetone and 2 x 25 ml of diethyl elastomer and air dried to give 2,275 g of the lithium salt as white, long-lasting flat prisms (E ^ = 770). Purity above 90%.

Example 3 7.99 g of crude barium salt (E ^ 288), purity <45%, prepared as starting material 1, dissolved in 60 ml of water and filtered. The filtrate was treated portionwise with 4.0 g of lithium sulfate with stirring at room temperature until no reaction of barium appeared on an outer test plate with sodium rhodionate. The suspension was cleared by centrifugation and the supernatant was decanted and boiled to ca. 35 ml under reduced pressure. 9.0 g of lithium chloride was added portionwise with stirring and cooling; after 1 hour at 0 ° C, the lithium salt was added by filtration, washed with 10 ml of ethanol, 2 x 25 ml of acetone and 2 x 20 ml of diethyl ether and air dried in the filter funnel to give 1,590 g of white prisms (E + = 790). The purity of the lithium clavulanate was over 95%.

Application Example A

Preparation of the sodium salt 3.5 g of the lithium salt prepared as in Example 1 (ii) was dissolved in 20 ml of distilled water and applied to a column containing 50 ml of "Bio-Rad" AG50x8 cation exchange resin (Na +; 200-400 mesh size). The elution was done with water and 8 ml fractions were collected. The fractions were determined after applying portions to paper by overlaying with nutrient agar containing Staphylococcus aureus. Active fractions (4 to 13) were combined and lyophilized.

The freeze-dried solid was dissolved in distilled water to 19 ml of solution, shaken with 450 ml of butan-1-ol and heated on a water bath until the solution was almost clear. The hot solution was filtered through a sinter to remove yellow solid and the filtrate was kept at 4 ° C for 60 hours. The crystals thus formed were filtered, washed with 2 x 10 ml of butan-1-ol and then with 2 x 10 ml of acetone and dried under reduced pressure at 40 ° C for one hour to 2.18 g of solid. The solid was recrystallized from water / butan-1-ol 1: 23.3 as described in Example 2. The crystals were dried under reduced pressure over silica gel at 44 ° C for 1 1/2 hours to give 1.8 g of solid sodium salt.

The solid was hygroscopic. It was painted with a bump in a mortar and allowed to absorb atmospheric water at 18 ° C. After recording approx. 22% by weight of water reached equilibrium.

This salt had the following characteristics:

Elemental analysis of the equilibrated moist solid: Found (quoting means in parentheses) C 33.3, 33.2 (33.25); H 4.6, 4.6 (4.6); N, 4.6, 4.7 (4.65); Na 7.3 (by absorption spectrophotometry), 7.9 (calculated from sulfated ash); water 21.95%. The composition CgHgO ^N Na, 41 ^O requires C 32.76; H, 5.46; N, 4.78; Na 7.8; water 24.57%.

Metal analysis 1) Found by atomic absorption spectrophotometry: Na 7.3 + 0.2% (this is the value stated in the elemental analysis).

2) Assuming the sulfated ash is Na 2 SO 4, the Na content was calculated to be 7.9%.

Optical rotation. [a] The D value for a 0.134% v / r aqueous solution at 24 ° C was + 47 °.

Ultraviolet spectrum. The UV absorption spectrum of a 0.00098% solution in 0.1M NaOH exhibited absorption maximum (λ_ =) at 258 nm with a value of 555.

Infrared spectrum. The nujol IR spectrum exhibited absorption peaks (cm

U6764 23 3400 s 1592 s 1288 m 1080 w 986 s 850 w 3300 s 1396 s 1206 w 1060 m 967 m 802 w 1792 s 1348 m 1190 m 1048 m 945 w 753 m 1690 s 1310 s 1138 m 1015 s 902 m 1665 m 1302 sh 1120 m 998 m 880 w

The full spectrum is shown in Figure 2 of the drawing.

NMR spectrum. The proton magnetic resonance spectrum from a solution in heavy water exhibited groups of peaks (τ values) centered at ca. 4.28, 5.07, 5.82, 6.44 and 6.88.

Application Example B

Preparation of the Potassium Salt 3.0 g of the lithium salt prepared as described in Example 1 (ii) was dissolved in 100 ml of water and passed through a 450 ml large column of "Dowex" 50W x 2 potassium ion exchange resin ("Dowex" is one in Denmark registered trademark). A course of 150 ml was discarded. The next 400 ml of eluate was collected and evaporated to 15 ml under reduced pressure. 340 ml of butan-1-ol was added and the mixture heated and shaken well.

Some insoluble solid was filtered off. The filtrate was evaporated under reduced pressure to 200 ml and then stored at 4 ° C overnight. The crystalline precipitate was filtered, washed with 2 x 10 ml of butan-1-ol, 2 x 50 ml of acetone and 2 x 50 ml of diethyl ether and finally dried in a vacuum desiccator at room temperature. Yield 2.34 g of potassium salt (E? - = 704). The value of 1 1 for E1 was determined by dissolving 7.1 mg of the potassium salt in 100 ml of water. This solution was diluted 1:10 with 0.1M sodium hydroxide to a final solution of 7.1 µg / ml.

Elemental analysis. Found (with mean values in parentheses): C 40.0, 40.14 (40.07); H 3.5, 3.55 (3.53); N 6.0, 5.82 (5.91); K (by absorption spectrophotometry) 16.0 (by determination of sulfated ash) 15.9; water 1.75, 1.95 (1.85)%. The composition CgHgNO4 K, 1/41 O requires C 39.7; H 3.5; N, 5.8; K 16.2; water 1.96%.

23 °

Optical rotation. The value of [α] β for a 0.276% s v / r aqueous solution was 58.4 °.

146764 24

The calcium, barium, and magnesium salts of clavulanic acid were prepared from lithium clavulanate in a similar manner and found to have E-values of 530, 576, and 713, respectively.

Application Example C

Preparation of free clavulanic acid 500 mg of the lithium salt of clavulanic acid, prepared as in Example 1 (ii), was partitioned between 10 ml of ethyl acetate and 10 ml of saturated aqueous sodium chloride. 1 ml of 2N hydrochloric acid was added and the mixture was shaken briefly. The aqueous phase was separated and washed with 10 ml of ethyl acetate and the combined organic extracts washed with 15 ml of saturated aqueous sodium chloride. The resulting organic solution was dried over sodium sulfate and evaporated almost to dryness to give 352 mg of the free acid as an oil containing ca. 0.5 mole of ethyl acetate.

The compound had the following characteristics: 24 o [a] + = 54.5 (c = 1.0 in DMSO); λ of a 0.00098% s L JD max solution in aqueous 0.1N NaOH is 258 nm (E + = 590); nujol infrared spectrum exhibits peaks at 3350, 1790 and 1722 cm cm *; NMR peaks (DMSO-dg) include χ 4.31 (d, 3 Hz), 4.99 (s), 5.23 (t, 7 Hz), 5.97 (d , 7 Hz), 6.37 and 6.93 (dd, 3 and 17 Hz; d, 17 Hz); peaks centered at χ 8.82, 8.00 and 5.95 showed that the sample contained about 0.5 These values indicate that the sample contained at least 85% by weight of clavulanic acid.

Application Example D

Preparation of the ammonium salt

A 240 ml column of "Dowex" ® 50W was converted to the ammonium form by treatment with ammonium sulfate and washed free of sulfate with water. 1.0 g of the lithium salt of clavulanic acid, prepared as in Example 1 (ii), was dissolved in 15 ml of water and applied to the column and it was developed with water. 25 ml fractions were taken and tested for UV absorption in 0.1N sodium hydroxide. The active fractions (Nos. 4-7) were combined and evaporated to near dryness (2 ml) under reduced pressure and 85 ml of n-butanol was added. The mixture was carefully distilled at 25 ° C under 0.1 mm Hg pressure until a crystalline material precipitated. The ammonium salt was added by filtration, washed with very little ethanol and acetone and finally washed with diethyl ether and dried at 0.1 mm Hg for 6 hours to give 0.54 g of sooty white crystals. = + 60.1 ° (c = 0.39% in water); λ (0.1N sodium hydroxide, 8.8 µg / ml) 258 nm (E +: 745); Nujol IR spectra include peaks at 3360, 1780, 1700 and 1580 cm 1; τ values (5% D 2 O) include 4.27 (d, 3Hz), 5.08 (s), 5.09 (t, 7Hz), 5.84 (d, 7Hz), 6.43 (dd) , 17Hz, 3Hz) and 6.89 (d, 17Hz).

Calcd for C 9 H 9 NO 3 NH 4: C 44.4 H 5.6 N 13.0 Found: C 44.4 H 5.6 N 13.3%.

By Karl Fischer analysis traces of water (0.6%) were found.

Application Example E

Preparation of the methylamine salt

A 200 ml column of "Amberlite" ® IR-120 H + was converted to the methyl ammonium form by treatment with 0.5M methylamine solution in water. It was washed to neutrality with water and 3.0 g of methylammonium chloride was introduced into 10 ml of water. The column was washed free of chloride with water and then ready for use.

1.50 g of the lithium salt of clavulanic acid, prepared as in Example 1 (ii), was dissolved in 15 ml of water and introduced at the top of the column. The column was developed in water and 25 ml fractions were collected.

Fractions # 3-7 were combined (161 ml with washings) and evaporated at 25 ° C / 1.0 mm Hg to ca. 2 ml, then 200 ml of n-butanol were added. The clear solution was evaporated to 20 ml under similar conditions, thereby crystallizing. The crystals were harvested by filtration after one hour at 2 ° C, washed with 2 x 15 ml diethyl ether and dried for 3 hours at 1 mm Hg to 1.2 g of the methylamine salt as clusters 2 3 of white elongated prisms. [α] = + 56.1 ° (c = 0.23% in υ 1

water); λ (0.1N sodium hydroxide, 9.5 µg / ml) 260 nm (E, = max -L

584); The nujol IR spectrum includes peaks at 2500, 1790, 1692, 1632, and 1576 cm S S S τ values (8% D₂O) include 6.40 and 6.86 δ (dd, 17Hzf 3Hz; d, 17Hz), 4 , 24 (d, 3Hz), 5.06 (t, 7Hz), 5.78 (d, 7Hz), 5.08 (s), 7.42 (s).

Calculated for C CHH₂N₂O: c 47 ° H 6.1 N 12.2 Found:. C 46.7 H 6.1 N 12.5%.

Application Example F

Preparation of the piperidine salt

A column of ion exchange resin (200 ml, from "Bio-Rad" Laboratories, AG 50W x 2, 100-200 mesh H + mold) was converted to the piperidine urea form with a solution of 75 ml piperidine in 1500 ml water. The resin was washed to neutrality with water and treated with 3 g of piperidinium chloride in 10 ml of water. The column was washed free of chloride with water and ready for use.

1.50 g of lithium clavulanate prepared as in Example 1 (ii) was introduced at the top of the column into 15 ml of water and the column was developed in water, taking 25 ml of large fractions. Fractions 3 to 6 were combined with washing liquids to make up 172 ml.

The solution was evaporated to near dryness at 35 ° C / 1.0 mm Hg and pure toluene was added. The oily suspension was evaporated to dryness under reduced pressure as above and the crystalline solid thus obtained was triturated with 90 ml of ethyl acetate. The crystalline piperidinium salt was added by filtration and washed with 3 x 30 ml of ethyl acetate and residual solvent removed at 0.1 mm Hg over 3 hours, yielding 1.775 g of slightly sooty white prisms. [α] D = + 42.2 ° (c = 0.35% in water); λ = 0.1 N sodium hydroxide, 10 µg / ml) 259.5 nm (E + = 474); Nujol IR spectrum includes peaks at 3380, 2540, 1782, 1682 and 1608 cm t-values (8% D 2 O) include 6.90 (d, 17Hz), 6.44 (dd, 3Hz and 17Hz); 4.28 (d, 3Hz), 5.08 (s), 5.84 (d, 8Hz), 5.08 (t, 7Hz), 6.84 (complex multiplet), 8.0-8.5 ( complex multiplied).

Calculated for C₂ ^H₂NN₂ ° 5'® H₂O: C C, 9H 7.4 N 9.5

Found: C 52.8 H7.2 N 9.3%.

27

14676A

Application Example G

Preparation of the triethylamine salt

A column of ion exchange resin ("Bio-Rad" 50W) as described in Application Example F was converted to the triethylammonium form with a solution of triethylamine in water (0.5N, 1.5 liters) and washed to neutral with water. A solution of 3 g of triethylammonium chloride in 15 ml of water was introduced into the column, the column was washed free of chloride with water and ready for use.

Then 1.5 g of lithium clavulanate, prepared as in Example 1 (ii), was added to the top of the column in 15 ml of water and the column was developed in water, taking 25 ml of large fractions. Fractions 4-9 were combined and together with the washing liquids amounted to 175 ml.

The solution was boiled under reduced pressure (35 ° C / 1.0 mm Hg) to give an oil which was boiled three times with toluene under the same conditions. The resulting crystals were broken up under 50 ml of ethyl acetate, filtered, washed twice with 20 ml of diethyl ether and freed of residual solvent in a desiccator for 3 hours at 0.1 mm Hg, yielding 1.588 g of the triethyl ammonium salt as weak. o dirty white prisms, [α] = +44.3 (c = 0.22% in water); λ d 2 max (0.1N sodium hydroxide, 9.7 µg / ml) 258 nm (E + = 485); Nujol IR spectrum includes peaks at 3250, 2080, 1784, 1700 and 1640 cm 2; τ values (10% D 2 O) include 6.41 and 6.87 (dd, 17Hz, 3Hz; d, 17Hz), 4.26 (d, 3Hz), 5.07 (t, 7Hz), 5.78 ( d, 7Hz), 5.07 (s), 8.74 (t, 7Hz) and 6.78 (q, 7Hz).

Calcd. For C 14 H 24 N 2 O 5,1 / 4H 2 O: C 55.2 H8.0 N9.2 Found: C 55.2 H 7.9 N 9.2%.