DK164225B - CEPHALOSPORINE COMPOUNDS FOR USING NUTRITIONAL SUPPLEMENTS IN ANIMAL FEED - Google Patents

Description

in

DK 164225 B

The present invention relates to novel synthetic cephalosporin compounds which are valuable as nutritional supplements in animal feed.

More particularly, the present invention relates to the condensation products of aldehydes, different from formaldehyde and acetaldehyde, with 3-thiolated cephalosporins which in 7 style have an α-aminophenylacetamido substituent substituted in the benzene ring with a para-hydroxy group.

Derivatives of various α-aminocephalosporins with nitrosubstituted heterocyclic scaffolds are disclosed in U.S. Patent No. 3,647,781. Reaction products of various α-aminocephalosporins with formaldehyde are disclosed in South African Patent No. 72/8475, with acetaldehyde in South African Patent No. 72/8474, and with various aldehydes and ketones in South African Patent No. 72/8476.

15 Derivatives of cephalosporins which in the 7-position acylamino group have an α-amino group which has been reacted with an aldehyde (but limited to methyl or acetoxymethyl in 3-way) are disclosed in U.S. Patent No. 3,880,842, U.S. U.S. Patent No. 3,887,546 and Farmdoc 49804W.

The reaction products of acetone with various α-aminocephalospores are disclosed in the patent literature as follows 1) with cephaloglycine, in United States Patent No. 3,303,193, 2) with cephalexin, in U.S. Pat. 1,314,758 and U.S. Patent No. 3,780,028, 3) with 7- [α-amino- (2'-thienyl) acetamido] cephalosporanoic acid, in U.S. Patent No. 3,311,621, 4) with certain ring-substituted cephaloglycines and U.S. Patent Nos. 3,464,985; and 5) with certain ring-substituted cephalexins and cephaloglycins, in U.S. Patent Nos. 3,489,750, 3,489,751, and 3,489,752.

30 The reaction products known from United States Patent Nos. 3,489,751 and 3,489,752 are stated, among other things, to be nutritional supplements in animal feed. For such use, however, it is essential that the reaction products are rapidly hydrolyzed in the body to restore the original cephalosporin. Compared to these known reaction products, the condensation products of the invention, as indicated below, possess a surprising advantage.

More distantly related compounds are the intermediates produced when a cephalosporin nucleus, e.g. 7-ACA or 7-ADCA, are acylated

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2 with a reactive derivative of an α-amino acid wherein the α-amino group has been protected by prior reaction with a β-diketo compound such as methyl-acetoacetate, methyl-acetoacetamide or acetyl acetone, examples of which are found in Farmdoc 22850W and 60669 V.

In the penicillin area, the penicillins containing an α-amino group in the 7-acylamido substituent, e.g. amnicillin, and is reacted with ketones and aldehydes, apparently first disclosed in United States Patent Nos. 3,198,804 and 3,198,788. Similar reaction products prepared from different such penicillins by reaction 10 with the same or different aldehydes and ketones were later described in United States Patent Nos. 3,230,214, 3,316,247 (diketones), 3,325,479 (diketones) , 3,489,746, 3,549,746, 3,558,602, 3,635,953, 3,641,000, 3,647,781 (which includes some cephalosporins), 3,725,389, 3,780,028, 3,784,562 (diketone), 3,886,140, 3,888,848, 3,905,955 and 3,904,604, and British Patent Specification No. 1,267,936.

Thus, the present invention relates to condensation products of the general formula 20a = 7 in H0 - \ - / 1 I / S in - HN H-if] ®2-S- * 3 COOR1 wherein A is hydrogen, hydroxy, methyl or methoxy, R 1 is hydrogen, sodium or potassium, 2 R is 2-furyl, 2-furan-5-sulfonyl, phenyl-2-sulfonic acid, 4-methoxyphenyl-3-sulfonic acid, 4-hydroxyphenyl-3-sulfonic acid , 2-carboxymethoxyphenyl, 4-carboxymethoxyphenyl, 3-hydroxy-4-carboxyphenyl, 4- (2'-carboxy) vinyl-phenyl, carboxylic, 2-carboxyphenyl, 3-carboxyphenyl, methanesulfonic acid or methanedensulfonic acid, and R is 1 , 2,3-triazol-5-yl, tetrazol-5-yl, 1,2,4-thiadiazol-5-yl, 1,3,4-thiadiazol-2-yl, 1,3,4-oxadiazol-2 -yl or 1,2,4-triazol-5-yl wherein each of these groups is unsubstituted or substituted by 1 or 3

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2 alkyl groups having 1 to 4 carbon atoms.

In the preferred embodiments, the carbon atom D configuration is associated with the benzene ring (para-11) relative to the hydroxyl group.

Thus, the sodium * or potassium salt of the present condensation products of formula I is the equimolar condensation product of a) a 2 2 aldehyde of the general formula R -CHO wherein R is as defined above, with b) an amphoteric 3-thiolated cephalosporin which contains an α-substituted α-aminoacetatamido group in 7-step 11 at a time and in its 10 zwitterion form has a water solubility of less than 125 mg / ml.

The cephalosporin compounds of the invention give by hydrolysis amphoteric cephalosporins of formula II

A O

15 ^ = \ II ^ s \ Η0Λ_ / f Π — i Ί

Ah, II

COOR1 20 1 3 wherein A, R and R have the above meanings.

The compounds of the present invention exhibit desirable solubility, stability and absorption. The preferred species 25 hydrolyze rapidly and completely in the body to recover the original amphoteric cephalosporin of formula II; this is not the case with similar derivatives prepared from formaldehyde, acetaldehyde or acetone, such as e.g. those of the United States Pat.

3,489,751 and 3,489,752 known reaction products in that they completely hydrolyze, but at an undesirably lower rate. Thus, the compounds of the present invention solve the problems due to the high pH instability and the frequent relative insolubility in the zwitterionic form of the amphoteric cephalosporins of formula II.

In the preferred embodiments of the present invention, the aldehyde has an acid function in the form of

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4

ISLAND

II / O H-C 2 SO 3 Na 5 5-Formyl-2-furanesulfonic acid sodium salt,

CKO

10 kff o-benzaldehyde sulfonic acid,

CHO

15 (TS

[IUo3h AND 3 4-methoxybenzaldehyde-3-sulphonic acid,

20 CHO

\ ^ - SO3H

CH

4-hydroxybenzaldehyde-3-sulfonic acid,

CHO

»(Y ** O

0CH2c00H

35 o- and p-formylphenoxyacetic acid,

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5

COOH

/ V-CH

CHO

5-formyl-salicylic acid,

CHO

A.

V

, n r,,, CH = CHCOCH

10 p-formyl-cinnamic acid, AND-COOH 15 gyoxylic acid,

<Jho

acoustic phthalaldehyde,

CHO

“Ό Ύ

COOH

30-p-formylbenzoic acid, OHC - CH 2 SO 3 Na acetaldehyde sulfonic acid sodium salt or OHC - CH (SO 3 Na) 2 acetaldehyde sulfonic acid sodium salt.

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6

Further preferred embodiments of the present invention are those forms of formula I wherein A is hydrogen, R is derived from one of the above aldehydes and is preferably especially 5

N-N -N

- / ° \ —i ^ 0vv-SO, Na s3 is I lira, I! I I l | or \\ | Γ 3, and RJ is As / ffl3 or '~ \ J1

10 H

Amphoteric 3-thiolated cephalosporins, which in their zwitterion form 15 have a water solubility of less than about 125 mg / ml, are clearly not suitable as nutritional supplements for animal feed and are also usually not converted to ordinary soluble sodium salts (unlike their non-amphoteric counterparts). (such as cephalothin, etc.) because the required pH is so high as to cause decomposition, and furthermore, the free amino group existing at the pH value is presumed to catalyze decomposition.

The cephalosporins of the invention of formula I H O

V = \ I II

I H S 1 HN -S> V-H 'J-CH 2 -S-R 3

Rs 0 COOR1 30 wherein 12 3 A, R, R and R have the above meanings can be prepared by a) treating an aqueous suspension of an amphoteric cephalo-sporin with the above formula II, or a solvate or hydrate thereof with an aldehyde of formula R -CHO wherein R is as defined above and a sufficient amount of water-soluble sodium or potassium base to raise the pH of the reaction mixture to between

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7, 5.5 and 8, to form the desired compound I as the salt in solution, and then b) recovering the compound I as the salt from the solution.

The compounds of formula I have an asymmetry center at the carbon atom which is connected to 2 nitrogen atoms. Thus, compounds of formula I may exist in the form of the DL mixture or as the individual D or L-1 summers.

The cephalo-sporin starting material used in step a) in the above process can be any form of the 10 amphoteric cephalosporin of formula II, including the free acidic zwitter ion or a hydrate or solvate of this zwitterion.

The concentration of amphoteric cephalosporin starting material is not critical and good results have been obtained with concentrations between about 25 and 300 mg of cephalosporin starting material per day. ml of solvent. The starting material is preferably ground and sieved to a finely divided state, preferably in particular to a particle size of less than 0.127 mm (200 mesh) to increase the surface area and rate of conversion.

The amphoteric cephalosporin starting material is slurried in water to form an aqueous slurry. An alternative to using an aqueous slurry in step a) would be to suspend the cephalosporin starting material in an organic solvent, which is 1) a solvent for all the potassium salt final product, 2) miscible with the aldehyde, 3) chemically inert to cephalosporin. the starting material and the final product; and 4) easily removable from the final product such as by mild drying. Examples of organic solvents that could be used are dimethyl sul foxide and dimethyl formamide. However, due to the difficulty of removing residual organic solvent from the alkali metal salt in the product, the starting material is preferably provided as an aqueous slurry.

After obtaining the cephalosporin starting material in slurry, the desired alkali metal salt of formula I is formed in solution by the addition of the aldehyde and an amount of water-soluble all potassium base, preferably a sodium or potassium base, sufficient to increase the pH of the reaction mixture to between As the pH increases until it is within this range, the alkali metal salt of the reaction product is formed by the amphoteric cephalosporin and the aldehyde and dissolves.

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8

The temperature at which step a) is carried out is not critical. The reaction can be carried out at room temperature; but higher or lower temperatures can be used and temperatures in the range of 50 to 60 ° C are preferred.

About 1 mole of aldehyde is required per mole of cephalosporin starting material, but the aldehyde is preferably added in some excess relative to the necessary theoretical amount to ensure complete reaction, i.e. a small molar excess. The most preferred ratio of aldehyde to cephalosporin starting material is about 1.3 to 1.4: 1, and 10 is often preferred 1: 1.

The sodium or potassium base can be any water-soluble base capable of 1) providing sodium or potassium ions, and 2) increasing the pH of the reaction mixture to between about 5.5 and 8, preferably especially about 6, 2 to 7.2. Preferred bases 15 are sodium or potassium hydroxide, because of their desirable solubility properties. The R3-S portion of compound I can decompose at high pH. For this reason, the base is added to the reaction mixture in such a way that the pH is not allowed to rise above about 8. Preferably, the base is used in the form of an aqueous solution and slowly added to the reaction mixture with stirring until the reaction turns out to be completely by pH measurement and by forming a solution or almost a solution. The amount of base used is not critical, but preferably about 1 mole base is used. mole of cephalosporin starting material.

For best results, the solution obtained at the end of step a) is filtered to remove solid impurities before the recovery step b). Before filtration, the solution may optionally be carbon treated with activated carbon to aid in the removal of all colored impurities.

The desired product of formula I is then recovered from aqueous or non-aqueous solution, such as by precipitation or lyophilization. Precipitation of the alkali metal salt can be carried out by adding an organic solvent in which the desired salt is insoluble, i.e. an anti-solvent. Examples of such anti-solvent agents are isopropanol, n-propanol, t-butanol and acetonitrile.

The solvent for use in the precipitation step should be one which can be easily removed from the final product under conditions which will not result in any appreciable decomposition of the alkali metal salt. That

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9 most preferred anti-solvent is isopropanol. The anti-solvent may be added to the solution resulting from step a) or, alternatively and preferably, the solution containing the desired alkali metal salt is added with stirring to a large excess of the anti-solvent. All the potassium salt of formula I is then recovered by filtration, washed with a suitable organic solvent, e.g. isopropanol, and dried by covalent processes, e.g. vacuum drying at 50 to 56 ° C for 24 to 48 hours or air drying at 60 ° C for 48 hours. As an alternative method for recovering the final product 10 by precipitation, the salt of formula I can also be recovered by lyophilizing the solution prepared in step a).

An alternative process for preparing the compounds of formula I comprises: a) forming a slurry of the amphoteric cephalosporin or a solvate or hydrate thereof in a suitable inert organic solvent, wherein this solvent is a solvent for the triethylamine salt of the aldehyde reaction product of the amphoteric cephalosporin and a non-solvent for the alkali metal salt of formula I; b) treating the slurry with the aldehyde and sufficient triethylamine to form the triethyl amine salt of the aldehyde reaction product of the amphoteric cephalosporin in solution; I from the solution by the addition of a solvent-soluble sodium or potassium base.

The starting material is slurried in an inert organic solvent which is a solvent for the triethylamine salt or aldehyde reaction product of the amphoteric cephalosporin, but which is a non-solvent for the desired alkali metal salt of formula I. Preferably, the solvent selected for step a) removable from the final product under conditions which will not result in any significant decomposition of the final product. Suitable solvents for step a) can be determined by simple experiments.

The slurry formed in step a) is then treated with an aldehyde, preferably with a molar excess and most preferably with from about 1.3 to 1.4 moles of aldehyde per minute. mole of cephalosporin starting material, and sufficient triethylamine to form the triethylamine salt of the cyclic reaction product of the aldehyde and the amphoteric cephalosporin in solution. The reaction mixture is preferably stirred for at least about 30

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10 minutes to ensure complete turnover. The amount of triethylamine used is not critical, but preferably about 1 mole is used. mole of cephalosporin starting material. The reaction in step b) is conveniently carried out at room temperature, but temperatures higher or lower than this can be selected with the expected decrease or increase in turnover time, respectively.

After forming a solution or nearly a solution in step b), the reaction mixture is preferably treated and filtered as in the above-described procedure.

The desired all potassium metal salt of formula I can then be recovered from the solution in step) by the addition of a solvent-soluble sodium or potassium salt. The preferred salts are sodium or potassium salts of organic acids having between about 2 and 18 carbon atoms, e.g. solvent-soluble salts of such acids as 2-ethyl-hexanoic acid, capric acid, oleic acid, glycolic acid, propionic acid, acetic acid etc.

Preferred salts for the methanol solvent system are sodium or potassium 2-ethyl hexanoate, preferably especially solutions of these salts in a methanol miscible organic solvent such as isopropanol. The most preferred alkali metal salts are solutions of sodium or potassium 2-ethylhexanoate in isopropanol. The alkali metal salt is added, preferably slowly and with stirring, in sufficient quantity to obtain the maximum amount of precipitate from the solution. After complete precipitation has occurred, the reaction mixture is stirred, preferably for at least about 1 hour, and then filtered. The precipitate is washed out with an appropriate organic solvent, e.g. methanol, and dried by conventional methods, e.g. vacuum drying at 50 to 56 ° C for 24 to 48 hours or air drying at 60 ° C for 48 hours.

The second procedure can also be followed without the use of the triethyl-30 amine in step b). In this modified process, the slurry of the amphoteric cephalosporin or solvate or hydrate thereof, preferably the methanol or propylene glycol solvate or hydrate form and especially preferably the methanol solvate, is suspended in an inert organic solvent which is a non-solvent for the product of formula I , preferably methanol, and the slurry is then treated with the aldehyde, preferably in a molar excess, and a solvent-soluble sodium or potassium base, this base being added in an amount sufficient to increase the pH of the reaction mixture.

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11 to between about 5.5 and 8. It is preferred to use as the bases the sodium or potassium salts mentioned above as being preferred by the second process. In the modified process, the cephalosporin starting material dissolves and the insoluble alkali metal salt is precipitated almost immediately. Since a solution is not obtained upon completion of this reaction, the reaction mixture is preferably stirred and heated to about 45 to 50 ° C for a period of up to several hours to ensure maximum yields of the final product. The solid product is removed by filtration, washed and dried to give the desired salt of formula I.

The alkali metal salts of the present invention can be used as nutritional supplements which have acceptable solid state thermal stability, high water solubility, satisfactory water stability and excellent in vivo and in vitro antibacterial activity against many different Gram-positive and Gram-negative bacteria.

The sodium and potassium salts of formula I can be dissolved in water to form relatively concentrated solutions with at least 250 mg / ml activity. Concentrations with 250 mg / ml activity (pH 5.7 - 6.8) of these salts have acceptable water-stable ether.

Activities of the compounds of formula I are substantially equivalent to those of the original amphoteric cephalosporin of formula II.

The compounds of formula I are valuable nutritional supplements for animal feed due to the combination of high stability, good oral absorption and rapid biological hydrolysis in animals as well as low mean preventive dose of PDgg. These effects and, surprisingly, in relation to the prior art are shown below.

The following describes methods for preparing starting materials.

30

Process for Preparation of 7- [Da-Amino-α (ph, hydroxyphenyl) acetamidol-3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid, methanol solvate Into a three-necked flask equipped with a reflux condenser, a stirrer and thermometer set above, a thoroughly mixed mixture of 8.36 g (0.05 mole) of D - (-) - p-hydroxyphenylglycine and 3.02 g (0.075 mol) of magnesium oxide in 120 ml of 50% aqueous dioxane. The mixture was stirred for 1 hour and then treated with 10.74 g (0.075 mol) of t-butane.

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12 oxycarbonylazide. The mixture was stirred and then heated to 45-50 ° C for 17 hours under N 2. The solution was diluted with 400 ml of HgO and extracted twice with 300 ml of ethyl acetate. The aqueous phase was acidified with 10% citric acid solution to pH 4 and saturated with NaCl. The aqueous mixture was extracted with 3 x 400 ml of ethyl acetate. The solution was dried over Na 2 SO 4 and the solvent was evaporated. The residue was triturated with Skellysolve B to give D-α-t-butoxycarbonylamino-α- (p-hydroxyphenyl) acetic acid as a solid weighing 10.4 g (78.5%).

To a slurry of 7-amino-3- (1,2,3-triazol-5-ylthiomethyl) -3-10 cephem-4-carboxylic acid (6.0 g, 19.0 mmol) in 100 ml of dry methylene chloride was added. The mixture was stirred and heated under reflux for 4 hours, at which time a clear solution was obtained. The solvent was evaporated and the residue was evaporated off. the oil was exposed to high vacuum overnight at room temperature.

The foamy residue was dissolved in 85 ml of dry THF (tetrahydrofuran) and cooled to about -15 ° C before introduction into the subsequent reaction mixture.

D-α-t-butoxycarbonylamino-α- (4-hydroxyphenyl) acetic acid (4.4 g, 16.5 mmol) was dissolved in 145 ml of dry THF. The solution was stirred and cooled to 20 to -20 ° C. N-methylmorpholine (1.6 g, 16 mmol) and isobutyl chloroformate (2.3 g, 16.8 mmol) were added sequentially at such a rate that the temperature of the mixture did not exceed about -10 ° C. The resulting mixture was then stirred for 20 minutes at -12 to -15 ° C. It was then cooled to -20 ° C and the THF solution of silylated 7-amino-3- (1,2,3-25 triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid was added at once. The temperature rose to about -12 ° C. Outer cooling is stopped until the temperature rises to 0 ° C. At this point, an ice bath was placed and the mixture was stirred for 3 hours at 2 to 3 ° C. This was followed by a period of 1 hour without external cooling, where the temperature rose to 20 ° C.

A total of 30 ml of methanol was added and stirring was continued for 15 minutes at room temperature. After evaporation of the solvents under reduced pressure, the residue was slurried in 300 ml of ethyl acetate. The suspended solid was filtered off (11.8 g). The ethyl acetate solution was extracted 3 times with NaHCO 3 (5%) solution. The combined sodium hydrogen carbonate extracts were cooled in an ice bath, separated by ethyl acetate and acidified to a pH of 2.5 with 42.5% HgPO 2.

The phases were shaken and then separated. The ethyl acetate solution was then dried by passing it through sodium sulfate and evaporating.

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13 then pedes to about 15 to 20 ml. This solution was then added dropwise to unstirred, in an Erlenmeyer flask containing cyclohexane (corresponding to 400 ml). After stirring for half an hour, the precipitated solid collected by filtration. The combined solid 7- [D-α-t-but-oxycarbonylamino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid was air dried. It weighed 1.75 g.

7- [Dat-butoxycarbonylamino-t- (p-hydroxyphenyl) -acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid, 3.5 g, was dissolved in 80 g. ml of HCOOH, 98-100%, and stirred for 2 hours at room temperature. HClOH 10 was evaporated under reduced pressure (aspirator bath temperature not above 40 ° C) and finally azeotroped 3 times with 30 ml of toluene. The solid was dried overnight under high vacuum over P20g. A total amount of 3.5 g of foam was obtained. The foam, 2 g, was stirred with 300 ml of 1 O: CH 2 OH (8: 2). The solvent was filtered from some solid (0.3 g), charcoal treated with 700 mg of Darko KB, filtered through diatomaceous earth ("Celite") and lyophilized to give 0.9 g of crude 7- [D- < * amino-a- (p -hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid. Ti 1 crystallization was used in the following procedure. A slurry of 0.2 g of the crude material in 6 ml of 99% methanol was heated in a test tube 20 for boiling. The heating is immediately interrupted and the melt triturated with grain. The melt solidified to a crystalline mass. In this way, a total amount of 0.211 g of 7- [Da-amino-α- (phydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid was obtained from 0.400 g of raw material. The material was dried at 56 ° C / 0.1 mm over P20g for 20 hours, melting point> 200 ° C, decomposition. IR and NMR are consistent with the structure. NMR showed the presence of 1/3 mol of CH 2 OH.

Analysis for C18H18NgO5S2, H2O, 1 / 3CH30H:

Calculated: C, 44.83; H, 4.38; N, 17.10; S, 13.09 Found: C, 43.97; H, 4.36; N, 15.84; S 6.18.

A total amount of 6.5 g (11.55 mmol) of 7- [Dat-butoxycarbonylamino-θ'- (p-hydroxyphenyl) acetamidoj -3- (1,2,3-triazol-5-yl thiomethyl) - 3-cephem-4-carboxylic acid was dissolved in 175 ml of 98-100% formic acid under anhydrous conditions. The mixture was stirred at room temperature for 2.5 hours. Part of the solution, 125 ml, was evaporated under reduced pressure to an amber 35 oil. The oil was then azeotroped 3 times with 70 ml of toluene under reduced pressure. The residue was slurried in an 80:20 H 2 -CH 2 OH solution (700 mL) and stirred for 0.5 h until the most solid dissolved, then filtered. The filtrate was treated with 1.5 g ("Darko") charcoal for about 20 14

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minutes. The charcoal was filtered through a "Celite" layer. The solution was then lyophilized in 9 separate 100 ml round bottom flasks. The freeze-dried material weighed 2.415 g. It was recrystallized in 0.200 g portions as described above to give a total amount of 0.923 g of 7- [Da-5 amino-α- (p-hydroxyphenyl) acetamido] -3- (1, 2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid. NMR was consistent and showed the presence of 1/3 mol of CH 3 OH.

Analysis for C ^ gN ^S₂O, I / 3CH3OH:

Calculated: C, 44.83; H, 4.38; N, 17.10; S, 13.09 Found: C, 45.77; 44.36; H 4.44, 4.34; N, 16.61, 16.52; S, 13.01, 13.01.

Process for the preparation of crystalline methanol solvate of 7---ami no-cr- (p-hydroxyphenyl) acetamido -3- (1,2,3-triazol-5-yl thiomethyl) -3-cephem-4-carboxylic acid 1. 50 g of 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] - 3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid is slurried in 250 ml of 95% v / v methanol / water (95% methanol) solution at 22 to 25 ° C. .

2. Concentrated hydrochloric acid is added with rapid stirring to a pH of 1.3 to 1.5. A solution or almost a solution is obtained.

3. Adjust the pH to 1.7 with triethylamine.

4. Add 7.5 g of activated charcoal ("Darco G-60") and slurry for 0.5 hour.

5. The charcoal is removed by filtration and washed with 75 ml of methanol which is added to the filtrate. Steps 2, 3 and 4 should be completed within 5 hours.

6. The combined washout and filtrate from step 5 is stirred rapidly. Triethylamine is added over a 5 minute period until pH 4.5. Crystallization begins after about 1 to 3 minutes.

The mixture is slurried for 1 hour.

7. The crystals are collected by filtration, washed with 100 ml of methanol and vacuum dried at 56 oC for 24 hours. Bio-yield 75 - 90%; bio sample = 850-900 µg / mg; NMR-IR = consistent for 1 mole of methanol; % water, KF = 2-4.0.

35

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15

Process for Preparation of Crystalline 1,2-Propylene Glycol Solvate of 7-ΓΒ-tt-Amino-α- (p-hydroxyphenylacetamidol-3- (1,2,3-triazol-5-yl-thiomethyl) -3-cephem-4 -carboxylic acid 1. 25 g of the above-prepared methanol solvate of 7- [Da-5-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-Cephem-4-carboxylic acid is slurried in 150-200 ml of 75% v / v propylene glycol aqueous solution at 20-25 ° C.

2. Concentrated hydrochloric acid is added up to a pH of 1 to 1.2 to give a solution or almost a solution.

10 3. Tri ethyl amine (TEA) is added slowly with rapid stirring to obtain a pH of 1.7 to 1.8.

4. 5 g of "Darco G-60" are added and the mixture is slurried for 0.5 hour. The charcoal is removed by filtration (filtration is carried out slowly, an 18.5 cm SS No. 576 paper is proposed). The charcoal filter cake is washed out with 40 ml of 75% 15 v / v propylene glycol aqueous solution. The detergent is added to the filtrate.

Steps 2, 3 and 4 above should be completed within 5 hours.

5. Triethylamine is added to the rapidly stirred filtrate leachate mixture from step 4 until the pH = 4.5 over a 10 minute 20 period. Crystals form after about 1 to 3 minutes. The mixture is slurried for 1 hour.

6. The propylene glycol solvate crystals of 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylth in methyl) -3-cephem-4 carboxylic acid is collected by filtration. Filtration is carried out slowly (a 12.5-15.0 cm 25 SS No. 604 paper is proposed). The crystals are washed successively with 50 ml of 75% propylene glycol, 50 ml of methanol and 50 ml of acetone and vacuum-dried at 56 ° C for 24 hours. Biological yield: 80-95%.

Properties of 7-β-ftamino-e- (p-hydroxyphenyl) acetamidol-3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid, propylene glycol solvate a. Bio sample = 850-900 g / mg.

b. IR NMR = consistent for a structure containing 1.0 - 1.5 moles of propylene glycol (14-19% propylene glycol). Apparently no loss of the 3-triazole side chain.

35 c.% Water, K.F. = 1-3.0.

d. Crystal morphology = 100% crystalline (microcrystals, triangular).

e. Melting point = 182-184 ° C (Decomposition, hot table).

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16 f- [α] β5 (0 = 1%; 1N-HCl) = + 53 °.

gr Water solubility = about 10 mg / ml in water at 23 ° C.

h. Loss of bioactivity upon standing at elevated temperatures: 100 ° C, 24 hours = <6%; 48 hours = <12%; 56 ° C, 1 month = <10%.

5

Process for preparing crystalline methanol solvate from crystalline propylene glycol solvate

The propylene glycol solvate of 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (50 g) as above, slurried in 250 ml of 95% (v / v) methanol-aqueous solution at 22-25 ° C. Concentrated HCl is added with rapid stirring to a pH of 1.1-1.5, whereupon a solution or nearly a solution is obtained. The pH is adjusted to 1.7 with triethylamine and 7.5 g of activated charcoal is added with slurry for 0.5 hour.

The charcoal is removed by filtration and washed with 75 ml of methanol. The leach solution is then added to the filtrate. (The steps from adding HC1 to this point should be completed within 5 hours). The combined leachate and filtrate are stirred rapidly and triethylamine is added over a 5 minute period until a pH of 4.5 is reached. Crystallization begins after about 1-3 minutes. The mixture is slurried for 1 hour and the crystals removed by filtration, washed with 100 ml of methanol and vacuum dried at 56 ° C for 24 hours. Bio-yield 75 -90%; bio sample = 850-900 increase / mg. NMR-IR = consistent for 1 mole of methanol; % H2 O, K.F. = 2-4.0.

25

Process for the preparation of crystalline 7-rD-α-amino-α- (p-hydroxyphenyl) acetamidol-3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid, sesgui hydrate 7- [Da-amino no-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-30 ylthiomethyl) -3-cephem-4-carboxylic acid, methanol solvate (15 g) was suspended. in 60 ml of water. The pH was raised to 6.5 by the addition of 4N NaOH and the mixture was passed through a sieve with a mesh width of 0.127 mm (200 mesh). The reaction mixture was slurried at room temperature for 2 hours during which time the pH was maintained at 6.5. The crystals were removed by filtration, washed with 20 ml of water and air-dried at 37 ° C for 24 hours to give 11.5 g of the crystalline product mentioned in the title. Bio-sample = 924 µg / mg (mean% HgO, K.F. = 5.26). NMR and IR were consistent with the proposed structure and showed that the product

DK 164225 B

17 contained no methanol, but actually contained a trace propylene glycol.

Process for the preparation of crystalline 7-rD-tt-amino-α- (p-hydroxyphenyl) acetamidol-3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-5 carboxylic acid, sesquihydrate and formation of other crystalline hydrates 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-carboxylic acid, methanol solvate (0.127 10.0 g), which is substantially free of propylene glycol, is slurried in 30-40 ml of deionized water at ambient room temperature (20-25 ° C) to form an aqueous slurry of pH value 3-4. NaOH (40¾) is added slowly with rapid stirring to raise the pH to 6.3-6.7. The mixture is slurried at pH 6.3-6.7 for 2 hours. The crystals are removed by filtration, washed with water and air-dried at room temperature for 24 hours to give 75-80% by weight yield of 15,950-1000 Mg / mg crystals of 7- [Da-amino-α- (phydroxyphenyl) acetamido] - 3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid dihydrate. IR and NMR analyzes were consistent with the proposed structure and showed that the product contained no methanol but did indeed contain a trace of propylene glycol. HgO, K.F. - 6.56.

A sample of the crystalline dihydrate was air-dried at 37 ° C for 24 hours to give the crystalline sesquihydrate of 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3- triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid. HgO, K.F. * 4.26.

Another sample of the dihydrate was air-dried at 45 ° C for 24 hours to form the crystalline sesquihydrate. HgO, K.F. = 5.5.

A sample of the dihydrate was air-dried at 56 ° C for 24 hours to give the crystalline monohydrate of 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazole) 5-ylthiomethyl) -3-cephem-4-carboxylic acid. HgO, K.F. = 4.38 (theoretical% HgO for monohydrate = 3.75).

A sample of the dihydrate was vacuum dried over P20g at room temperature for 24 hours to give the crystalline hemihydrate of 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazole (5-ylthiomethyl) -3-cephem-4-carboxylic acid. HgO, K.F. = 2.63 (theoretical% HgO for hemihydrate -1.91).

A sample of the dihydrate was vacuum dried at 56 ° C for 24 hours to form the crystalline hemihydrate. HgO, K.F. = 1.6 - 2.0.

DK 164225 B

18 D - (-) - 2- (3'-methyl-4'-hydroxyphenyl) glycyl chloro d, hydrochloro d is prepared in a high degree of purity and high yield by the following procedure:

About 0.06 moles of D - (-) - 2- (3, -methyl-4H-hydroxyphenyl) glycine 5 are suspended in 100 ml of dioxane. The slurry is stirred and COC12 (phosgene) is sent while the slurry temperature is maintained at 50-58 ° C. COC12 is sent in a total time of 3.5 hours. A yellow solution is obtained. The solution is purified with nitrogen to expel the excess CO12. HCl gas is bubbled through the solution for 2.5 hours. The solution is stirred and a small amount is diluted with some ether to give some crystals which are added to the whole portion as grain. The solution is stirred at 20-25 ° C for 16 hours. The resulting slurry of crystalline D - (-) - 2- (3'-methyl-4, -hydroxyphenyl) glycyl chloride, hydrochloride is filtered to collect the product. The filter cake is washed with dioxane and methylene chloride and then dried in a vacuum desiccator over P 2 and to give about 7 g of D - (-) - 2- (3β-methyl-4, -hydroxyphenyl) glycyl chloride, hydrochloride.

2- (3'-methyl-4'-hydroxyphenyl) glycine A solution of 59.02 g (0.6 mol) of 75% glyoxylic acid in 100 ml of water was added to a slurry of 54.6 g (0.5 mol) 2-methyl phenol and 140 ml of concentrated ammonium hydroxide in 400 ml of water at room temperature. The temperature of the mixture rose to 37 ° C. The mixture was stirred at room temperature for 65 hours. The solution, which initially had a pH of 10.1, was adjusted to pH 6.8 with 6N hydrochloric acid, causing the product to crystallize. The product was collected by filtration, washed with water and dried in vacuo over phosphorus pentoxide to give 31.5 g (34.8%) of 2- (3'-methyl-4'-hydroxyphenyl) glycine; decomposition 196-199 ° C. The infrared and the nuclear magnetic resonance spectrum were consistent for the desired product. Reference: Belgian Patent Specification No. 774,029 (Farmdoc 27, 122T), which indicates melting point 205-207 ° C.

Analysis for C

Calculated: C, 59.66; H 6.13; N, 7.73.

Found: C, 57.68; H, 6.23; N 7.47, H 2 O, 2.34 Found, corrected for 2.34% water: C 59.06; H, 6.12; N, 7.67.

DK 164225B

19 D, L-N-chloroacetyl-2- (3'-methyl-4'-hydroxyphenyl) quinoline

A slurry of 20.2 g (0.112 mol) of D, L-2- (3'-methyl-4'hydroxy-phenyl) glycine in 175 ml of water was adjusted to pH 10.3 with 20% sodium hydroxide, resulting in a solution. The solution was cooled in an ice bath. Chloroacetic anhydride (38.2 g, 0.224 mol) was added at once and the pH of the reaction mixture was maintained at pH 10 by the addition of 20% sodium hydroxide until no further pH change was recorded. The reaction mixture was stirred for a further 10 minutes in the cold. The reaction mixture was then acidified to pH 2.0 with 6N hydrochloric acid, causing the product to crystallize. The product was collected by filtration, washed with water and air-dried. Recrystallization from 200 ml of hot water gave 13.7 g (47.4%) of D, L-N-chloroacetyl-2- (3'-methyl-4'-hydroxyphenyl) glycine. The infrared and the nuclear magnetic resonance spectrum were consistent for the desired product.

Analysis for CjjH₂NNO4Cl, H2O:

Calculated: C, 47.92; H, 5118; N, 5.081.

Found: C, 48.11; H, 5.16; N, 5.15.

D - (-) - N-chloroacetyl-2- (3'-methyl-4'-hydroxyphenyl) glycine D, LN-chloroacetyl-2- (3'-methyl-4'-hydroxyphenyl) glycine (5.0 g 0.0194 mol) and L-ephenamine acetate (6.1 g, 0.0213 mol) were dissolved in 50 ml of isopropyl alcohol by heating on a steam bath. Water (50 ml) was added and upon cooling, the L-ephenamine salt was crystallized. The salt was collected by filtration and air-dried.

The salt was slurried in 30 ml of water and 50 ml of methylene chloride and the mixture was adjusted to pH 10.0 with 20% sodium hydroxide. The phases were separated and the aqueous phase was extracted twice more with methylene chloride.

The aqueous solution was then adjusted to pH 2.0 with 6N hydrochloric acid, causing the product to crystallize. The product was collected by filtration and dried in vacuo over phosphorus pentoxide to give 0.9 g (36.1%) of D - (-) - N-chloroacetyl-2- (3-methyl-4H-hydroxyphenyl) glycine; m.p. 170 DEG-172 DEG C., ars 185.9 DEG (Ol, 95% EtOH). The infrared and nuclear magnetic resonance spectrum were consistent for the desired product.

DK 164225 B

20

Analysis for C

Calculated: C, 51.27; H, 4,696; N, 5,436.

Found: C, 51.21; H, 4.77; N, 5.29.

1,2-diphenyl-2-methylaminoethanol, usually called ephenamine 5 (according to the Federal Register, June 7, 1951) has the structure

/ V-ch - CH - {\ A

\ // \ i \ _J

io OH “CH3

The compound is also called N-methyl-1,2-diphenyl-2-hydroxyethylamine or α, β-diphenyl-β-hydroxy-N-methylethylamine or 1,2-diphenyl-2-methylamino-1-ethanol.

This method utilizes only the levo-erythrosis isomer. Methods for its preparation and reaction with penicillin G are described in U.S. Patent Nos. 2,645,638 (V.V. Young) and 2,768,081 (F.H. Buckwalter). In the last patent, 20 previous literature is reviewed, just as it is done in W.B. Wheatley et al., J. Org. Chem., 18 (11), 1564-1571 (1953). This compound was used to cleave racemic phenoxymethylpenicillin by Sheehan et al., J. Am. Chem. Soc., 81, 3089-3094 (1959); see especially p. 3091.

D - (-) - 2- (3-methyl-4'-hydroxyphenyl) glycine D - (-) - N-chloroacetyl-2- (3'-methyl-4'-hydroxyphenyl) glycine (11, 1 g, 0.0431 mol) was combined with 100 ml of 2N hydrochloric acid and the mixture was heated under reflux for 1.5 hours. The solution cooled and the pH was adjusted to 5.0 with 20% sodium hydroxide causing the product to crystallize. The product was collected by filtration, washed with water and dried in vacuo over phosphorus pentoxide to give 7.4 g (94.7%) of D - (-) - 2- (3'-methyl-4'-hydroxyphenyl) glycine, decomposition 205 -209 ° C, [α] D = 152.6 ° (C = 1.1 N HCl). The infrared and nuclear magnetic resonance spectrum were consistent for the desired product.

Analysis for C

Calculated: C, 59.66; H 6.13; N, 7.73.

Found: C, 58.62; H, 5.49; N, 7.78; H2 O, 1.46.

Found, corrected for 1.46% water: C 59.48; H, 5.41; N, 7.84.

DK 164225B

21 D - (-) - N-t-butoxycarbonyl-2- (3'-methyl-4'-hydroxyphenyl) glycine

To a slurry in 200 ml of HgO-dioxane (1: 1) of 7.2 g (0.0397 mol) of D - (-) - 2- (3'-methyl-4'-hydroxyphenyl) glycine and 3.2 g (0.08 mol) of powdered magnesium oxide, which was stirred at room temperature, was added dropwise 5 9.7 g (0.068 mol) of t-butoxycarbonylazide. The reaction mixture was then heated to 42-45 ° C under a nitrogen atmosphere for 19 hours. The mixture was then diluted with 100 ml of ice water. The solution was phase separated with ethyl acetate and filtered to remove any insoluble material which had been separated. The aqueous phase of the filtrate was separated and extracted 2 10 times more with ethyl acetate. The aqueous solution was then adjusted to pH 5.0 with 42% phosphoric acid and extracted 5 times with ethyl acetate. The combined organic extracts were washed 3 times with water, dried over sodium sulfate and the solvent removed at reduced pressure leaving an oil. The oil was dried in vacuo over phosphorus pentoxide resulting in 10.6 g (95%) of D - (-) - N-t-butoxycarbonyl-2- (3'-methyl-4'-hydroxyphenyl) glycine. The infrared spectrum was consistent for the desired structure.

7-β-2-t-butoxycarbonylamino (3'-methyl-4'-hydroxyphenyl) acetamidol-3-20 (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carbox. y1 acid A. Mixed anhydride

A solution of 2.8 g (0.01 mol) of D - (-) - Nt-butoxycarbonyl-2- (3'-methyl-4'-hydroxyphenyl) glycine in 100 ml of tetrahydrofuran was cooled to 25-15 ° C in a ice-salt-acetone bath. N-methylmorpholine (1.01 g, 0.01 mole) was added followed by 1.37 g (0.01 mole) of isobutyl chloroformate and the reaction mixture was stirred at -15 to -20 ° C for 8 minutes. A precipitate of N-methylmorpholine, hydrochloride is excreted immediately.

B. Coupling 7-Amino-3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (3.1 g, 0.01 mole) was suspended in 100 ml of water and 1.01 g (0.01 mole) of N-methylmorpholine was added. A complete solution was not obtained. Then 1,1,3,3-tetramethylguanidine was added dropwise to this stirred slurry until a clear solution was obtained. The solution was then cooled to 4 ° C in an ice bath and added to the mixed anhydride solution at -15 ° C. The cooling bath was removed and the mixture stirred for 1.5 hours. The tetrahydrofuran was then removed at reduced pressure and the aqueous concentrate

DK 164225 B

22 was separated by ethyl acetate. The aqueous phase was adjusted to pH 2-, O with 42% phosphoric acid, causing the insoluble material to separate. The solid was removed by filtration. The aqueous phase of the filtrate was separated and extracted twice more with ethyl acetate.

The combined organic extracts were washed twice with water, dried over sodium sulfate and the solvent removed under reduced pressure leaving a viscous oil residue. The oil was triturated with "Skelly-solve B" which contained a small amount of diethyl ether to give a solid. This solid product was collected by filtration, washed with "Skellysolve B" and air dried. A TLC (silica gel; solvent, 97: 3 acetone acetic acid) of the product showed that the material was a mixture of the desired product and of the side chain acid.

This material was then stirred with 100 ml of anhydrous diethyl ether for 1 hour. The insoluble solid was collected by filtration and air dried to give 0.95 g (16.4%) of 7- [D-2-t-butoxycarbonylamino- (3'-methyl-4'-hydroxyphenyl) acetamido] - 3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid. A TLC (silica gel; solvent 97: 3 acetone acetic acid) showed that this sample contained only a trace amount of the side chain acid. The infrared spectrum was consistent for the desired product.

7-rD-2-t-butocycarbonyl yl amino- (3 H -methyl-4 H -hydroxyphenyl) yl) acetamido-3- (1,2,3-triazol-5-yl) thi-methyl-3-cephem-4-carboxylic acid A. Mixed anhydride A solution of 14.06 g (0.05 mole) of D - (-) - N-butoxycarbonyl-2- (3'-methyl) 4'-hydroxyphenyl) glycine in 500 ml of tetrahydrofuran was cooled to -15 ° C in an ice-salt-acetone bath. Nmethylmorpholine (5.06 g, 0.05 mol) was added followed by 6.83 g (0.05 mol) of isobutyl chloroformate and the reaction mixture was stirred at -13 to -17 ° C for 10 minutes. A precipitate of 30 N-methylmorpholine, hydrochloride is immediately excreted.

B. She's starving

A mixture of 8.0 g (0.025 mole) of 7-amino-3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid, 7.0 ml (0.05 mole) ) triethylamine, 6.9 ml (0.0545 mol) of Ν, Ν-dimethylaniline, 9.5 ml (0.075 mol) of chlorotrimethylsilane and 650 ml of methylene chloride were heated under reflux for 3 hours. An additional 1.6 ml of chlorotrimethylsilane was added after the first hour of reflux. The cloudy solution was cooled and the methylene chloride

DK 164225 B

23 were removed at reduced pressure. The residue was taken up in 500 ml of tetrahydrofuran. Coupling 5 The silylester solution was cooled to 10-15 ° C in an ice bath and added at once to the mixed anhydride solution, maintained at -15 ° C.

The cooling bath was removed and the reaction mixture stirred for 2 hours. Then, 500 ml of water was added and the tetrahydrofuran was removed under reduced pressure causing a gummy oil to separate. The oil 10 was extracted into ethyl acetate and the phases separated. The aqueous phase (pH = 3.8) was phase separated with ethyl acetate causing some solid to separate. This precipitate was removed by filtration and the aqueous phase of the filtrate was separated. The aqueous solution was adjusted to pH 2.3 with 42% phosphoric acid and extracted again with ethyl-15 acetate. The combined ethyl acetate extracts were washed 3 times with water, dried over sodium sulfate and the solvent removed at reduced pressure leaving a viscous oil. The oil was stirred with 100 ml of anhydrous diethyl ether for 1.5 hours to give a solid. The product was filtered, washed sparingly with anhydrous diethyl ether and air dried. A TIC (silica gel; solvent, 99: 1 acetone acetic acid) of the product showed that the material was a mixture of the desired product, the side chain acid and a third unidentified component.

The material was again stirred in 100 ml of anhydrous diethyl ether for 1.5 hours.

The insoluble solid was collected by filtration and air dried to give 10.7 g (74.4%) of 7- [D-2-t-butoxycarbonylamino- (3β-methyl-4β-hydroxyphenyl) acetamido] -3 - (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid. A TLC (silica gel; solvent 99: 1 acetone-acetic acid) showed that this sample contained only a trace amount of the side chain acid and a third unidentified component. The infrared spectrum was consistent for the desired product, 7-rD-2-t-butoxycarbonylamino- (3'-methyl-4H-hydroxy-phenylPacetamido1-3- (1,2,3-triazol-5-yl) omethyl) -3-cephem-4-carboxylic acid 35 A. Mixed anhydride

A solution of 12.2 g (0.0434 mol) of D ~ (~) -Nt-butoxycarbonyl-2- (3'-methyl-4, -hydroxyphenyl) glycine in 425 ml of tetrahydrofuran was cooled to -15 ° C in an ice-cream. salt-acotone bath. Nmethylmorpholine (4.35 g, 0.0434 mol)

DK 164225B

24 was added followed by 5.87 g (0.0434 mol) of isobutyl chloroformate and the reaction mixture was stirred at -15 ° C for 10 minutes. A precipitate of N-methylmorpholine, hydrochloride is excreted immediately.

5 B. They are light starters

A mixture of 6.8 g (0.0217 mole) of 7-amino-3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid, 6.02 ml (0.043 mole) of triethylamine , 6.0 ml (0.0473 mol) of N, N-dimethylaniline, 8.26 ml (0.0651 mol) of chlorotriethylsilane and 550 ml of methylene chloride were kept under reflux for 2 hours. The cloudy solution was cooled and the methylene chloride removed at reduced pressure. The residue was taken up in 425 ml of tetrahydrofuran.

C. Coupling 15 The silylester solution was cooled to 10 ° C in an ice bath and added at once to the mixed anhydride solution maintained at -15 ° C. The cooling bath was removed and the reaction mixture stirred for 2 hours. Then, 425 ml of water was added and the mixture was stirred for 5 minutes. The tetrahydrofuran was removed at reduced pressure, causing a rubbery oil to separate. The oil was extracted into ethyl acetate and some insoluble solid which was separated was removed by filtration. The aqueous phase of the filtrate was separated, adjusted to pH 2.0 with 42% phosphoric acid and extracted twice more with ethyl acetate. The combined ethyl acetate extracts were washed 3 times with water, dried over sodium sulfate and the solvent was removed at reduced pressure leaving a viscous oil. The oil was dissolved in 30 ml of acetone and this solution was added dropwise to a thoroughly stirred 300 ml volume of anhydrous diethyl ether. The mixture was stirred at room temperature for 2.5 hours. The insoluble solid which is separated was collected by filtration and air-dried. A TLC (silica gel, solvent 99: 1 acetone acetic acid) of the product showed that the material was a mixture of the desired product, the side chain acid and a third unidentified component. The solid was again stirred in 80 ml of anhydrous diethyl ether for 1 hour. The insoluble material was collected by filtration, washed sparingly with anhydrous diethyl ether and air-dried to give 5.8 g (46.5%) of 7- [D-2-t-butoxycarbonylamino- (3'-methyl-4'- hydroxyphenyl) acetamino] -3- (1,23-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid. A TLC (silica gel; solvent 99: 1 acetone acetic acid) showed that this sample contained only one side chain acid.

DK 164225 B

25 the acid and a third unidentified component. The infrared spectrum was consistent for the desired product.

7-rD-2-amino- (3β-methyl-4β-hydroxyphenyl) acetamidol-3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid 7- [D 2-t-Butoxycarbonylamino- (3'-methyl-4'-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (0 (95 g, 0.002 mol) was added to 15 ml of cold tri-fluoroacetic acid and the solution was stirred for 15 minutes. The trifluoroacetic acid solution was then poured into 10 200 ml of 2: 1 "Separate lysol ve B" anhydrous diethyl ether and the mixture cooled.

The precipitated trifluoroacetic acid salt of the product, 7- [D-2-amino- (3'-methyl-4'-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem 4-Carboxylic acid, collected by filtration, was washed out with 2: 1 "Shell Lysolve B" anhydrous diethyl ether and dried in vacuo over 15 phosphorus pentoxide resulting in 0.75 g of substance.

The salt (0.75 g) was slurried in 50 ml of water, 25 ml of "Amberiite LA-1" resin acetate form (25% in methyl isobutyl ketone) and 25 ml of methyl isobutyl ketone and stirred at room temperature for 2 hours.

The phases were separated and the methyl isobutyl ketone layer was extracted once with water. The aqueous phases were combined and then extracted 8 times with diethyl ether. The aqueous phase was filtered and the solvent removed at reduced pressure. The residue was triturated with methyl isobutyl ketone to give a solid. The substance was collected by filtration, washed with methyl isobutyl ketone and acetone and dried at 65 ° C in vacuo over phosphorus pentoxide to give 0.36 g (59.0%) of 7- [D-2-amino- (3'-methyl) -4'-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid; decomposition> 150 ° C. The infrared and nuclear magnetic resonance spectrum were consistent for the desired product.

30 "LA-R resin is a mixture of secondary amines wherein each secondary amine has the general formula R 1 CH 3 (CH 2) 10 CH 2 NH 2 R 2 R 3 wherein each of R 1, R 2 and R 3 is a monovalent aliphatic hydrocarbon radical wherein R1, R2 and R3 together contain from 11 to 14 carbon atoms.

DK 164225B

26 atoms. This particular blend of secondary amines, sometimes referred to herein as "Liquid Amine Blend # II", is a clear amber liquid having the following physical properties: Viscosity at 25 ° C of 70 3 centipoise; density at 20 ° C of 0.826 g / cm; refractive index at 25 ° C of 1.4554; distillation interval at 10 mm Hg, up to 170 ° C - 0.5%, 170-220 ° C - 3%, 220-230 ° C - 90% and above 230 ° C - 6.5%.

7- [D-2-amino- (3'-methyl-4'-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid is also called BL-S638.

2- (3'-methoxy-4'-hydroxyphenyl) quinoline

A solution of 59.2 g (0.6 mole) of 75% glyoxylic acid in 100 ml of water was added to a slurry of 62.07 g (0.5 mole) of 2-methoxyphenol and 140 ml of concentrated ammonium hydroxide in 400 ml of water at room temperature. The temperature of the mixture rose to 35 ° C. The mixture was stirred at room temperature for 65 hours. The product which crystallized was collected by filtration, washed with water, then acetone and dried in vacuo over phosphorus pentoxide to give 57.4 g (58.2%) of 2- (3'-methoxy-4'-hydroxyphenyl) - glycine; decomposition 218-220 ° C (literature 240 ° C). The infrared and the nuclear magnetic resonance spectrum were consistent for the desired product.

Analysis for C

Calculated: C, 54.82; H, 5.62; N, 7.10

Found: C, 53.77; H, 5.91; N, 6.97; H2 O, 1.13

Found, corrected for 1.13% HgO: C, 54.38; H, 5.85; N, 7.05.

Reference: B. Block, Z. Physiol. Chem., 98, 226 (1917).

Cleavage of 2- (3β-methoxo-4β-hydroxyphenyl) ql.ycin A. Methyl 2- (3'-methoxy-4'-hydroxyphenyl) glycinate A cooled slurry of 94 g (0.476 mol) 2 - (3'-methoxy-4'-hydroxyphenyl) glycine in 500 ml of absolute methanol was flowed at high speed with HCl vapor for 20 minutes. First, a clear solution was obtained and then crystalline product was excreted in greater quantity. After 20 hours, the methyl ester hydrochloride was filtered and then scrubbed sparingly with methanol; 99.6 g after air-drying. A cooled solution of the hydrochloride in 800 ml of water was adjusted to pH 8 (NaOH) resulting in a crystalline precipitate of the ester as a free base; 81.3 g.

The IR and NMR spectra were consistent.

27

DK 164225 B

Analysis for C

Calculated: C, 56.86; H, 6.20; N, 6.63.

Found: C, 56.46; H, 6.28; N, 6.55; Wfl 0.59.

B. D- (-) -2- (37-Methoxy-4-hydroxyphenyl) glycine

A mixture of 50 g (0.237 mol) of methyl 2- (3'-methoxy-4- (hydroxy-phenyl) glycinate, 19 ml (0.333 mol) of acetic acid and 1 liter of i-PrOH (isopropyl alcohol)) was heated to boil resulting Dibenzoyl-d-tartaric acid, monohydrate (89.2 g, 0.237 mol) was added under good stirring and then the mixture was heated to reflux. The salt soon began to crystallize. After cooling in an ice bath, the precipitate was collected by filtration. The filtrate was concentrated to about 1/3 of its original volume, resulting in a small second portion of salt; total yield of both portions 54.1 g after air drying (solid A, see below).

The filtrate was concentrated to remove solvent. The viscous residue was combined with 300 ml of 1N HCl and the mixture was extracted with 400 ml of CHCl 3. The CHClg phase was extracted twice with 100 ml 20 portions of 1N HCl. The combined HCl extracts were briefly concentrated to remove residual CHCl 3 and heated under reflux for 1 hour. The solution was concentrated to a small volume, causing the amino acid HCl salt to crystallize. The product was collected by filtration and recrystallized from 50 ml of 1N HCl. A solution of the product in 200 ml of water was adjusted to pH 4.5 (NaOH). The mixture was approximately heated to boiling and allowed to cool to precipitate D - (-) - 2- (3, -methoxy-4'-hydroxyphenyl) glycine as fluffy needle-like crystals. After cooling overnight, the product was collected by filtration, washed sparingly with water and methanol and dried at 40 ° C; 8.7 g, [a] p 4-136.5 ° (C = 1, 1N HCl). The IR and NMR spectra were fully consistent.

Analysis for C

calculated; C, 50.23; H, 6.09; N, 6.51; 8.37.

Found: C, 50.43; H, 6.23; N, 6.51; f, 8.95.

C. L - (+) - 2- (3'-methoxy-4'-hydroxyphenyl) glycine

Solid A (see above) (54.1 g) is heated in 300 ml of 1N HCl and 500 ml of CHCl 3 with good shaking. The salt is not immediately dissolved in this system, 35

DK 164225 B

28, therefore, CHC13 was separated as well as possible and 300 ml of MIBK was added under good shaking. The MIBK phase was extracted with an additional 200 ml of 1N HCl in 3 portions. The combined and filtered HCl extracts were briefly concentrated to remove residual solvents and heated under reflux for 1 hour to hydrolyze the ester. The reaction mixture was concentrated to a small volume. After cooling in an ice bath, the crystalline amino acid HCl salt was collected by filtration.

The salt was recrystallized from 75 ml of 1N HCl and dissolved in 500 ml of water under heating, the solution filtered blank and adjusted to pH-10 value 4.5 (NaOH), causing the zwitterion to crystallize. The mixture was heated to boiling, filtered and left to cool to precipitate the crystalline product, L - (+) - 2- (3'-methoxy-4'-hydroxyphenyl) glycine. The product was collected by filtration, washed sparingly with water and methanol, and dried at 40 ° C; 9.6 g, [a] j-j = 15 + 127.2 ° (C = 1, 1N HCl). The IR and NMR spectra were consistent.

Analysis for C

Calculated: C, 50.23; H, 6.09; N, 6.51; H 2 O, 8.37.

Found: C, 50.53; H, 6.06; N, 6.62; H 2 O, 7.46.

Sodium D-N- (2-methoxycarbonyl-1-methylvinyl-α-amino-α- (3'-methoxy-4'-hydroxyphenyl) acetate

To a stirred solution of 3.02 g (0.078 mole) of NaOH in 320 ml of methanol is added 0.08 mole of D - (-) - 2- (3'-methoxy-4H-hydroxyphenyl) glycine and the resulting mixture is heated under reflux while a solution of 9.6 ml (0.088 mol) of methyl acetoacetate in 80 ml of methanol is added over a 30 minute period. After a further 30 minutes of reflux, the methanol is distilled off while toluene is added at the same rate so that almost the same volume of content is maintained. When the content temperature reaches 100 ° C, the slurry is cooled in 30 ice water for 4 hours, filtered, washed well with toluene, air dried and vacuum dried over P 2 O 5 to constant weight to form solid sodium DN- (2-methoxycarbonyl-1-methyl) vinyl) -α-amino no-α- (3 '-methoxy-4'-hydroxyphenyl) acetate.

D - (-) - 2- (3'-methoxy-4'-hydroxyphenyl) g1.ycyl chloride, hydrochloride is prepared in a high degree of purity and high yield by the following procedure:

About 0.06 moles of D - (-) - 2- (3'-methoxy-4'-hydroxyphenyl) glycine

DK 164225B

29 is suspended in 100 ml of dioxane. The slurry is stirred and COC12 (phosgene) is sent while maintaining the slurry temperature at 50 - 58 ° C. COC12 is sent in a total time of 3.5 hours. A yellow solution is obtained. The solution is purified with nitrogen to expel the excess COCl 2, 5 HCl gas is bubbled through the solution for 2.5 hours. The solution is stirred and a small amount is diluted with some ether to give some crystals which are added to the whole portion as grain. The solution is stirred at 20-25 ° C for 16 hours. The resulting slurry of crystalline D - (-) - 2- (3'-methoxy-4'-hydroxyphenyl) glycyl chloride, hydrochloride is filtered to collect the product. The filter cake is washed with dioxane and methylene chloride and then dried in a vacuum desiccator over P 2 O 5 to give about 7 g of D - (-) - 2- (3'-methoxy-4'hydroxyphenyl) glycyl chloride, hydrochloride.

D - (-) - N- (t-butoxycarbonyl) -2- (3 H -methoxy-4'-hydroxyphenylglycine)

A mixture of 8.6 g (0.04 mole) of D - (-) - 2- (3'-methoxy-4'-hydroxy-phenyl) glycine, 3.2 g (0.08 mole) of magnesium oxide, 9, 7 g (0.068 mol) of t-butoxycarbonyl azide and 240 ml of 1: 1 dioxane water were stirred and kept heated at 45-50 ° C for 20 hours under a nitrogen atmosphere. The cooled reaction mixture was diluted with 240 ml of ice water, filtered and extracted once with ethyl acetate. The acidified (pH 2) aqueous phase was extracted 5 times with ethyl acetate. The combined and dried (Na 2 SO 4) ethyl acetate extracts were concentrated to remove solvent at reduced pressure resulting in the product as a viscous oil; 6.3 g.

Cleavage of 2- (3-methoxy-4-hydroxyphenyl) yl Iglycine A. (-) - 2- (3-methoxy-4-hydroxyphenylylglycine, d-10-camphor sulfonate) A mixture of 5 , 0 g of 2- (3-methoxy-4-hydroxyphenyl) glycine, 50 ml of glacial acetic acid and 2.5 ml of H2 O were heated to form a solution The hot solution blank was triturated and left at room temperature for 18 hours to crystallize resulting in 1 78 g of the salt Recrystallization from 15 ml of acetic acid followed by drying at 40 ° C gave 1.54 g of (-) - 2- (3-methoxy-4-hydroxyphenyl) glycine, d-10-camphor sulfonate, partial decomposition 164 -170 ° C, then decomposing 175-180 ° C, M [.alpha. -35.3 "(Cl, H2 O).

The combined acetic acid filtrates were concentrated to a small

DK 164225 B

30 volumes at reduced pressure. The crystalline solid was filtered off and recrystallized from 15 ml of acetic acid, after drying at 40 ° C, another portion of product; 2.48 g, decomposition 164-170 ° C, ["]" - -32.2 "(C = 1, H2 O).

B. (-) - 2- (3-methoxy-4-hydroxyphenyl) glycine

A 1.2 g portion of the first portion of (-) - 2- (3-methoxy-4-hydroxy-phenyl) glycine, d-10-camphor sulfonate was dissolved in 12 ml of water and the solution adjusted to pH 4.5 with dilute aqueous NH 2 OH. The mixture 10 was heated to form a solution. The solution was allowed to cool first at room temperature, then at 5 ° C to crystallize the amino acid. The product was filtered off, washed sparingly with a few drops of water and methanol and dried at 40 ° C to give 0.37 g of (-) - 2- (3-methoxy-4-hydroxyphenyl) glycine; decomposition 210-212 ° C, [α] p 4 = -137.6 ° (C = 1, 1N HCl).

Analysis for Cg HnNO4:

Calculated: C, 54.82; H, 5.62; N, 7.10.

Found: C, 54.37; H, 5.90; N, 7.21; HgO, 0.71.

C. (-) - 2- (3-Methoxy-4-h, ydroxyphenyl) glycine

A mixture of 200 g of 2- (3-methoxy-4-hydroxyphenyl) glycine, hydrate, 260 g of d-10 camphorsulfonic acid, 2 l of glacial acetic acid and 100 ml of water was heated to form a solution and blank filtered. The solution was seeded with crystal grain and allowed to stand at room temperature for 3 days to crystallize the salt; 76 g. The salt was recrystallized from 400 ml of acetic acid; 71.0 g.

The filtrate from the first portion of salt was concentrated to about half its original volume and the product was allowed to crystallize; 118.5 g. The product was recrystallized from 500 ml of acetic acid resulting in, after air drying, 105.2 g of (-) - 2- (3-methoxy-4-hydroxyphenyl) glycine, d-10-camphor sulfonate.

Concentrated NH 4 OH was added dropwise with good stirring to a solution of 71.0 g of the camphor sulfonate salt in 150 ml of water plus 150 ml of methanol. The mixture, which soon became very viscous, was heated to 50 ° C and the dropwise addition of NH 2 OH continued to pH 4.5.

After ice cooling, the product was filtered off, washed sparingly with cold 1: 1 MeOH water and cold methanol, resulting in drying in a vacuum oven at 40 ° C, 34.5 g of (-) - 2- (3-methoxy-4-hydroxyphenyl) glycine;

DK 164225 B

31 decomposes 204-206 ° C, [α] D = -132.4 ° (Ol, 1N HCl).

Analysis for CgH2NO4 .HgO:

Calculated: C, 50.23; H, 6.09; N, 6.51.

Found: C, 49.96; H, 6.12; N, 6.61.

(-) - 2- (3,4-dihydroxyphenyl) glycine

A mixture of 9.2 g of (-) - 2- (3-methoxy-4-hydroxyphenyl) glycine and 50 ml of 48% hydrobromic acid was heated at reflux for 4 hours. The solution was cooled in ice resulting in a crystalline precipitate which was collected by filtration and dried at 40 ° C; yield 1.8 g, m.p. 248-250 ° C decomposition with prior dark staining over ca. 200 ° C,

Wf- -42.1 · (Ol, H2 O).

Analysis for CgHgNO4, HBr, 1/2 H2 O:

Calculated: C, 35.18; H, 4.06; N, 5.13; H₂O, 3.30 Found: C, 35.26; H 4.01, N 5.32; H 2 O, 3.20.

It was concluded that this substance was a 3: 1 composition of (-) - and (+) - isomers of 2- (3,4-dihydroxyphenyl) glycine, HBr.

The filtrate was concentrated to a small volume. The crystalline mass which is separated is collected by filtration (the filtrate is retained). The crude product was recrystallized from 20 ml of acetic acid. The product was washed on the filter with methyl isobutyl ketone and anhydrous ether to give 3.0 g of white crystalline (-} - 2- (3,4-dihydroxyphenyl) glycine, hydrobromide; mp 106-109 ° C, [α] + = - 85.0 ° (Ol, H 2 O).

Analysis for CgHgNO4, HBr, H2O: Calculated: C, 34.06; H, 4.29; N, 4.97; HgO, 6.37.

Found: C, 43.07; H, 4.17; N, 4.99; HgO, 6.80.

The acetic acid filtrate was concentrated to remove solvent. To the oily residue was added 75 ml of methyl isobutyl ketone and the product was allowed to crystallize first at room temperature and then in an ice bath. The product was filtered off, washed with methyl isobutyl ketone and anhydrous ether to give an additional 1.5 g of white crystalline (-) - 2- (3,4-dihydroxyphenyl) glycine, hydrobromide; mp 106-108 ° C, [<* 3 p , 5 ° (Ol, H 2 O).

Analysis for CgHgNO4, HBr, H2O: Calculated: C, 34.06; G, 4.29; N, 4.97; H 2 O, 6.39.

Found: C, 33.79; H, 4.31; N, 4.94; H 2 O, 7.08.

The filtrate from the crude HBr salt was concentrated to remove solvent. The crystalline residue was dissolved in 8 ml of water, and

DK 164225 B

The 32 solution was adjusted to pH 4.5 with concentrated aqueous NH 4 OH causing the zwitterion to crystallize. The mixture was diluted with an equal amount of methanol and cooled in ice for 1 1/2 hours. The product, (-) - 2- (3,4-dihydrophenyl) glycine, was filtered off, washed sparingly with 1: 1 MeOH-H2 O and MeOH, and dried for 2 hours at 40 ° C; yield 0.536 g, indistinct decomposition 234-238 ° C with prior dark staining over ca. 220 ° C, [α] p2 = -158.2 ° (C = 1, 1N HCl).

Analysis for CgHgNO4:

Calculated: C, 52.46; H, 4.95; N, 7.65.

Found: C, 51.82; H, 5.03; N, 7.75.

7-rD-tt-amino- (3 H -methoxy-4 H -hydroxyphenyl) acetamidol-3- (1,2,3-triazol-4-ylmethyl) -3-cephem-4-carboxylic acid

A mixture of 4.27 g (0.01365 mol) of 7-amino-3- (1,2,3-triazol-4-ylthiomethyl) -3-cephem-4-carboxylic acid, 6.64 g (0.041 mol) 1,1,1,3,3,3-hexamethyldisilazane and 200 ml of methylene chloride were heated under reflux for 4 hours to give a clear solution. After standing overnight at room temperature, the solvent was removed at reduced pressure. The residue was dissolved in 150 ml of tetrahydrofuran, the solution was cooled to 0 to 5 ° C before use.

N-methylmorpholine (3.06 mL, 0.0273 mol) and 3.48 mL (0.0273 mol) of isobutyl chloroformate were added to a solution of 8.1 g (0.0273 mol) of D - (-) - N- ( t-butoxycarbonyl) -2- (3-methoxy-4'-hydroxyphenyl) glycine in 300 ml of tetrahydrofuran at -15 ° C. The mixture was stirred at -15 ° C for 6 minutes to form the mixed anhydride.

The tetrahydrofuran solution of the silyl ester at 0 to 5 ° C was added to the mixed anhydride at -15 ° C. After 10 minutes, the cooling bath was removed and the mixture was stirred for another 2.5 hours. Water (250 ml) was added to the reaction mixture and the mixture was concentrated at reduced pressure to remove most of the tetrahydrofuran. The aqueous concentrate was acidified with 42% phosphoric acid and extracted 3 times with ethyl acetate. The mixture was filtered during the first extraction to remove a small amount of insoluble matter. The combined ethyl acetate extracts were washed once with water, dried (Na 2 SO 4) and concentrated to dryness. The residue was triturated with anhydrous ether to give 2.8 g of solid.

A solution of the solid (2.8 g) and 50 ml of 97% formic acid was stirred at room temperature for 2 hours. The formic acid was distilled off at reduced

DK 164225 B

33 pressure. The residue was azeotroped with toluene to completely remove formic acid. The residue was triturated with wet ethyl acetate resulting (after drying for 2 hours in vacuo at 65 ° C over phosphorus pentoxide) 1.42 g of 7- [Da-amino- (3'-methoxy-4'-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-4-5 ylthiomethyl) -3-cephem-4-carboxylic acid; decomposition gradually over about 165 ° C. The IR and NMR spectra were consistent. 7- [D-α-amino- (3'-methoxy-4'-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-4-ylthiomethyl) -3-cephem-4-carboxylic acid is also called BL-S689.

The compounds of the present invention are prepared by the reaction of an aldehyde having the structure R -CHO (wherein R is as previously defined) with a cephalosporin having the general formula HO ~ Z VcH-C-NH-1-

W IW2 β-n \ J-ch2-s-e3 II

0 COOR1 20 1 3 wherein A, R and R have the meanings previously stated. In the preferred embodiments, the carbon atom attached to the α-amino group has D-configuration. The latter compounds of formula II are prepared by the usual, and often specific, procedures set forth in the following U.S. Patent Nos. 3,641,021, 3,899,394, 3,855,213 and 3,867,380, South African Patent No. 73 / 4055, Belgian Patents Nos. 776,222 (Farmdoc 38983T) and 810,477 (Farmdoc 57268V), West German Patents Nos. 2,364,192 (Farmdoc 49048V) and 2,500,386 (Farmdoc 49692W), Belgian Patent Nos. 814,727 (Farmdoc 82562V) and West German Patent No. 2,404,592 (Farmdoc 57268V).

An alternative method for preparing the amphoteric cephalosporins used as starting materials herein is the use of the appropriate p-hydroxy-2-phenylglycine (which may contain an additional substituent and wherein the α-amino group is suitably protected by acylation in a conventional manner) instead of the side chain acid, e.g. 2-phenyl-glycine or tetrazole-acetic acid, which has heretofore been used as in U.S. Patent Nos. 3,813,388, 3,759,904, and 3,850,916 to prepare either 3-thiolated cephalosporins or 7-substituted cephalospores.

DK 164225 B

34 spanoic acids in which the 3-acetoxy group is then replaced by the desired thiol. U.S. Patent Nos. 3,757,012 and 3,757,015.

Reference below to BL-S640 refers to 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) 3-cephem-4-5 carboxylic acid, which also called cefatrizine. Reference to the propylene glycolate thereof refers to the 1,2-propylene glycol solvate described above. Reference to BL-S643 refers to 7- [D-ft-amino-α- (β, -hydroxy-phenyl) acetamido] -3- (2-methyl-1,3,4-thi adi-azol-5-yl) thi (methyl) -3-cephem-4-carboxylic acid, also known as cefaparol.

The following examples are given to illustrate but not limit the present invention, the preferred embodiments being described.

35

DK 164225 B

Example 1 - 0-4 - f 5 '- rt-1 N - in D, L -> XC ^ -H j * in ch 2 -s-v / H 0 iogNa f 10

To 0.34 ml of furfural (2-furfuraldehyde) dissolved in 10 ml of water was added with stirring 2 g (1 equivalent) of the methanol solvate of 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3- (l, 2,3triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid. 4N sodium hydroxide was added with rapid stirring to maintain the pH of 6.2 to 6.5. The resulting solution was allowed to stand at room temperature for 1 hour and lyophilized to give the product sodium salt as a solid.

Properties of the substance: IR-NMR consistent for product.

Β-lactam intact.

Triazole intact.

Example 2

The procedure of Example 1 is repeated except that the methanol solvate starting material used therein is replaced with an equimolar amount of 7- [Da-amino-α- (p-hydroxyphenyl) acetamido] -3-cephem-4-carboxylic acid , sesquihydrate. Sodium salt is prepared which is identical to that obtained in Example 1. '·. 1 35

DK 164225 B

36

Example 3

Preparation of BL-S1052, the sodium salt of the reaction product of 2-furfuraldehyde and BL-S640 Formula, BL-SI052

f / S. I-k

H \ κΝ-1 I. h2 °

10 / C X-N ^^ i-CHgS-L N

HX (Γ T

zrr ^ COONa

_ISLAND

15

Process 1) 2 7/10 ml (3.13 g, 1.1 equivalent) of 2-furfuraldehyde are dissolved in 50 ml of water at 20 to 30 ° C with rapid stirring.

2) 15 g of BL-S640 methanol adduct is added over a 10 minute 20 interval with simultaneous addition of 40% sodium hydroxide to pH 5.5-6.0 (pH should not exceed 6.5). A light orange colored or near solution is obtained.

3) The solution is filtered through appropriate filters to remove particles, pyrogens and bacteria. Steps 1 and 2 together should be completed within 2 hours.

4) Lyophilize for 48 hours, then leave the solids under vacuum at 50-56 ° C for 24 hours. The resulting solid is BL-S1052 (BL-S-1052 can also be obtained from the solution in step 3 by precipitation from 15 to 20 volumes of sterile isopropanol).

30

Properties of BL-S1052 1) Bio-sample * 775-800 β $ / π \ 9.

2) IR NMR = a) Well defined, consistent.

b) At approximately 60 mg / ml in D 2, 2 products appear, 40% cyclic adduct, 60% non-cyclic adduct.

c) β-lactam and 3-triazole are intact.

3) Solubility = greater than 400 mg / ml.

DK 164225 B

37 4) Paper strip chromatography = single zone by for BL-S640.

(Concentration = 0.2 mg / ml).

5) Liquid chromatography (concentration = 1 mg / ml).

5 Time in% free BL-S640 hours present 0 69.2 1 74.2 10 2 94.5 6) Analytical data

Found On a dry basis Theoretical% H2 O KF 5.35 15% C 47.45 50.2 49.2% H 3.72 3.4 3.39% N 14.45 15.29 14.95% S 10.34 10.92 11.4% Ash as Nn 2.18 2.51 4.1 20 —i 38

DK 164225 B

- VD (Λ ΙΛ LTi tn CUOOHOJ H ΙΛ LA in i '* - ·>. R »\ OOOO OJOCJ ^ OHCJOHOCJlTNlPiinOCM Η tA OJ CM OJ tn CO i — i H ri IAA Λ pq <—i O' P- ___; _

4J I (0 I

U 1 Γ5

+ j t H

m in S

Λ I O I W I in i

c! O VO t n m LTV LA

.¾ i - = f o o h cm oj la in in tn 3 coooocMO ^ -oooowoHOCJinininoaj c i m oj <M cm tn

in ιό Η Η H

-Hl pq A A A

£ I

o i

U 1 4J I Ai i <D I CU I

m i-- i. * I U) I • Η I • P I O 1 ri l λ i t> - σ \ o tn <θ ',

• Hl ^ J-NNtOVOC ^ HHIAS ^ OinAHtONON

u 1 coommoootnHt-C'-HOH.d-oininin into iniAvovo mininvo cntncninco ovowto cr \ nnc ^ <jiCnHO \ HwnHC? iHO \ ain (j> cr \ * * * * rn tn - = f tn oj ίη · = ΐ ' · ι- = ρ · ·· = ί -.} = - = ί- · Ρ - = 3 ·· · ί = -: 3 - 2 = ·· = ί "

I 1 I 1 I I I I I I I I I I I I I I I

ooooooooooooooooooo

Hhhi-IHHHHHHHr-iiHiHiHr-IHHH

£ to • H £ i ρω d Jh Φ wi ω tn tn o; ω

Pi .c ti) r <\ Ι <Λ i ·. i tu 0 1 + 1 '3 (1 \ (l Λ * Ί J i'l l,' id w -.1 V,> -l Η -p · ι-ι Φ a> m to ω cd Q) £ o) <X <up H dd \ ioo ω a> ω -ri t: to in pq pq “S -ri .c; -h -ri r-ι -ri c to cd ddp ω + Up C il -.- i c. -ri <uooo O no to two to to a> ι-i O o .o ut W) oddd POPPPPP- P fi £ dt) 0 P u OO o P> 3 <U OJ a) (U (UC-H-HPPP <P-iPi d iH Η. H (UCCP ^ P · ιPιί-ιP ^ Φr ^ HQ) ω · r ^ OQJ £ OOa c ppppp OOGC £ £ <ii.ddddd * ooCLiPi ....

P r- | »I * C -P -P -P

• +3 · · · · · cd · · · · P ^ Ph to tu c C C

tococococococococoMM- ^^ ΡΟΟϋΟ-, εοωωω 39

DK 164225 B

* 45% AAB + 5% serum + 50% substrate listed above.

** Dilute the substrate culture after standing overnight.

1) Adjusted for 79.5% BL-S640 content. In other words, the numerical values are lowered, ie. improved. This adjustment was also made in the test experiments listed below or, alternatively, a larger amount of weight was used to provide equivalent dosing.

Mice levels after IM administration of 10 mg / kq body weight 10 Blood concentrations / µg / ml)

Number of hours after administration

Compound mice 0.25 0.5 1 1.5 BL-S1052 16 15.1 15.1 11.5 7.8 15 BL-S640 32 15.7 13.2 9.5 6.8

The compounds were prepared in 0.01% phosphate buffer. BL-S640 was used by default for all compounds.

20

Mouse level concentrations after PQ administration of 100 mq / kq body weight

Blood concentrations (µg / ml)

Number of hours after administration

Compound mice 0.5 1 2 3.5 25

BL-S1052 16 45.2 48.1 31.9 14.4 BL-S640 32 53.4 45.4 27.2 10.7 1 35

The compounds were prepared in Tween®-CMC. BL-S640 was used by default for all compounds.

DK 164225 B

40

Urinary recovery following IM administration of 50 and 10 mg / kg to rats

Percentage of administered dose recovered hours after Dose Number of administration 5 Compound (mg / kg) rats 0-6 6-24 0-24 BL-S1052 50 4 40.3 2.1 42.4 10 3 21.7 2.4 24 , 1 10 BL-S640 50 7 44.5 2.7 47.2 10 7 24.3 1.4 25.7

The compounds were prepared in 0.01% phosphate buffer. BL-S640 15 was used as standard for all compounds.

Paper chromatograms were obtained by chromatography on rat urine collected between 0 and 2 and between 2 and 4 hours after IM administration of BL-S1052 and BL-S640 to detect abtibiotically active metabolites using "decreasing" chromatography with "system # 9" (butyl acetate: n-butanol: glacial acetic acid: water = 80: 15: 40: 24). Two identical spots were observed in all cases, except in the case of the standard (which had not been administered to the rat and provided a single identical spot for each of BL-S640 and BL-S1052). This indicated complete hydrolysis of the derivative of the original compound BL-S640 and its putative metabolite.

41

DK 164225 B

Example 4

The compounds of formulas HO ^ h O

5 ΗΟ- ^ Λ-έ-c s \ = ΛΑ. J-r-f X [j—? C-H JJ J_CH -S-il χΝ Μ · X COONa 10

_ H O

15 H0 /> i-C S

Wi. X if

pJ 0 T H

'COOKa 20 1 = /? 8 H0 / y \ v_i — c s

25 V = /, HN ^^ N-pi I II

C-H CH2-S-kN ^ N

i 0 I H

COOIla 1 35 is prepared by replacing the cefatrizine in the method of Example 3 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

42

DK 164225 B

Example 5

The compounds of formulas H 0 Η <Ηθ ^ ^ Ν-τ-γΝ r— n-ch, o COONa

H ° \ H O

/ = \ I II

HCK \ / M-c s 15 li "" CH? _ COONa CH,

20 \ 3 H O

/ = \ I II

H ° \ v // C \ -C

^ ιΓηΊ * f, —r ° “5 T“ “^ Η 'and 25 O COONa CH, 0

3 \ _ H O

30 Η0-Οϊ ^ _Γ / Ί =] - r-3

| "H 0j-N- ^ LcH2-S-lU? H

COONa

ISLAND

is prepared by replacing the cefatrizine in the method of Example 3 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

35

DK 164225B

43

Example 6

The compounds of the formulas

5 H O

hohT> c c c

JPF

_ i Ch2 "s" \ n ^ N

io F \ i I

o COONa CH, 1 == / ^

H0V_ H O

“Oii ^ n

f / -Nv ^ J-CHg-S

F = \ 0 T I

O COONa ΟΚ Ο H- “/

H O

20 RO- / \ -C - C ς

"χι ^ Ί p-jH

f 0 ^ Nx ^ L-CHa-s "^ N''N and 0 COONa CH-25 CH-0

_ H O

^ JcL-Λ jn?

30? -H i-N ^ jJ-CH -S-k „/ N

1 0 T I

- \ COONa CH,

O J

1- / 35 is prepared by replacing the cefatrizine in the method of Example 3 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

DK 164225 B

44

Example 7

Reaction product of BL-S640 and 5-formyl-2-furansulfonic acid sodium salt (BL-S1027)

Formula, BL-S1027 5 ~ rv? · / · „

H-N N - r i-N

10 / o / -1

H i M'SQ ^ Na COONa H

Process for Preparation 1) 9.0 g of 5-formyl-2-furanesulfonic acid sodium salt (EC-S

y T

20 is suspended in 75 ml of water at 60 ° C.

2) 20 g (1 equivalent) of BL-S640-1,2-propylene glycol adduct is sprinkled with rapid stirring over a 10 minute period while simultaneously adjusting the pH to 6-7.0. A solution or near solution is formed.

3) The solution is stirred at 55-50 ° C, pH 5-7.0 for 10 minutes and then cooled to 4-8 ° C. A small amount of precipitate is formed.

4) The mixture is stirred for 15 minutes at 4-8 ° C and then filtered.

5) The filtrate is added over a 5 minute interval to 1 liter of isopropanol with rapid stirring. A heavy precipitate forms. The mixture is slurried for 5 minutes.

5) The solids are removed by filtration, washed with 200 ml of isopropanol and vacuum dried at 56 ° C for 24 hours.

7) The solids are dissolved in 60 ml of 4-6 ° C water (a small amount of 35 insoluble matter is removed by filtration). The filtrate is lyophilized for 24 hours.

Yield: 23 g, bio-yield = 89%.

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45

Properties of BL-S1Q27 1) - Bio sample as BL-S640 = 724 µg / mg (theoretical = 750 Mg / ml).

2) Solubility => 500 mg / ml.

3) IR NMR: Consistent for structure. At 70 mg / ml, the compound exists as 80% cyclic, 20% non-cyclic derivative.

4) Paper strip and liquid chromatography indicate that at 0.2 and 1.0 mg / ml aqueous solution, the product exists as free BL-S640.

5) MIC bacterial spectrum is equal to the spectrum of BL-S640.

6) Oral and intramuscular rat blood concentrations are equal to the concentration of BL-S640.

7) A solution of this product in water at a concentration of 25,000 Mg / ml was stable (maintained full bioactivity) for at least 3 days at room temperature and for 14 days at 6 ° C (refrigerator), thus providing a solution which is suitable for oral use.

15

Analytical data

Found On a dry basis Theoretical 20% H2 O 5.14 C 40.15 42.1 43.0 H 3.10 2.9 2.96 N 12.39 13.05 13.1 S 13.6 14.35 14, 95 25 Ash as Na 5.86 6.16 6.8 for di-Na

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46

Antibiotic activity in nutrient substrate MIC (/ µg / ml)

Sodium salt * Cepha-5 Organism for example 7 BL-S6402 lotin _3

Str. pneumoniae * (10) ** A9585 0.03 0.13 0.03 10 Str. pyogenesis (10 ~ 3) ** A9604 0.03 0.06 0.03 -4 S. aureus Smith (10) A9537 0.27 0.5 0.13 15 -4 S. aureus (10) + 50% serum A9537 2 4 0.5 S. aureus (10-3) 20 BX1633 A9606 0.5 0.5 0.13 S. aureus (10 ”2) BX1633 A9606 4 4 0.25 25 S. aureus (10 ~ 3)

Meth-Res A15097 4-8 8-16 1-16

Sole. enteritidis (10 ~ 4) A9531 0.25 0.25 0.25 E. coli Ouhl (10 "4) A15119 1 1 16 E. coli (10" 4) 35 A9675 4 4 32 47

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Antibiotic activity in nutrient substrate MIC (/ µg / ml)

Sodium salt1 Cepha-5 Organism for example 7 BL-S640 ^ lotin -4 K. pneumoniae (10) A9977 0.5 0.5 1 -4 10 K. pneumoniae (10) A15130 1 2 32

Pr. mirabilis (10 ~ ^) A9900 0.5 0.5 1 15 -4

Pr. morganii (10) A15153 32 63> 125 -4

Ps. aeruginosa (10) 20 A9843A> 125> 125> 125 -4

Ser. marcescens (10) A20019> 125> 125> 125 25 Ent. cloacae (10 ~ ^) A9656> 125> 125> 125

Ent. cloacae (10 ~ ^) A9657 1 1 8 30 -4

Ent. cloacae (10) A9659 32 63> 125 35

DK 164225 B

48 * 45% AAB + 5% serum + 50% substrate listed above.

** Dilute the substrate culture after standing overnight.

* These figures were recalculated on the basis that there was only 65% BL-S640 in the sample; in other words, the numerical values were lowered, ie. improved.

BL-S640 = 7- [D-α-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid.

Mice levels after oral administration of 100 mg / kq 10 body weight

Bl od concentrate in on (/ zg / ml) hours after administration Compound 0.5 1 2 3.5 15 Compound from Example 7 45 36.6 20.4 10.4 BL-S6401 50.5 39 24.5 9.6

Compound 20 from Example 7 54.6 53.5 27.6 8.2 BL-S6401 57.4 47 29.5 10.9

The compounds were prepared Tween® - carboxymethyl cellulose and 8 mice 25 were used for each compound.

* BL-S640, 7- [D-ot-amino-α- (p-hydroxyphenyl) acetamido] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid was used as a standard. 1 2 3 4 5 6 BL-S1027 is the water-soluble derivative which forms when BL-S640 2 is reacted with furfural sodium sulfonate. The bio-strength is 724 µg / mg of BL-3 S640 activity.

4

Aqueous solutions containing 250, 25 and 10 mg of BL-S640-5 activity / ml as BL-S1027 can be stored under cooling (4 ° C) or at 6 room temperature for at least 24 hours without any significant loss in activity.

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49

Mouse blood concentrations following intramuscular administration of 10 mg / kg 1 squirrel weight

Hours after administration Compound 0.25 0.5 1 1.5 5 ----......-.............-.......- .. ................-..........

Compound of Example 7 15.1 14.8 11.9 8 14.6 15.4 11.7 8.3 10 BL-S64Q1 16.3 13.5 9.4 6.3 * BL-S640, 7- [Da-amino-β- (p-hydroxyphenyl) acetaminodo] -3- (1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid was used as a standard.

The compounds were dissolved in 0.01% phosphate buffer.

Example 8 Reaction product of BL-S640 and o-formylphenoxyacetic acid (BL-S1048)

H O

/ Π \ I N

ΗΟ-γ · C

25 Htl N -, - I-H

JL "^ COON.t, 'f, ^ VOCH COONil 2 30

Process for Preparation 1) 375 mg of o-formylphenoxyacetic acid

CHO

35 i

-COOH -OCH

DK 164225 B

50 is slurried in 10 ml of water and 4N NaOH is added with rapid stirring until pH-host is 7.0. A solution is obtained.

2) 2 g (1 equivalent) BL-S640, methanol adduct is added with rapid stirring over a 10 minute interval with the simultaneous addition of 5 4N NaOH to a pH of 6-6.5. A solution or near solution is obtained.

3) The solution is filtered and left at ambient room temperature for 0.5 hour.

4) The solution is lyophilized for 24 hours to give product 10 as a solid.

Properties of solid product 1) IR NMR (70 mg / ml) consistent for structure, triazole and β-lactam intact.

2) Paper strip and liquid chromatography at 0.2 and 1 mg / ml shows that BL-S640 is completely free of the aldehyde.

Analytical data

Found On dry basis Theoretical 20% water 4.06 C 47.15 49.2 50.1 H 3.79 3.5 3.86 N 11.44 11.93 13.0 S 8.87 9.27 9, 91 25 Ash as Na 5.55 5.78 6.47 3) Solubility in water => 400 mg / ml.

30

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51 Λ)

CO ΙΟ ΙΛ ΙΛ Lf \ [A

- = f O O Η OJ HLHinm LT \ H OOOCUOAJ. = rOOOJOHOOJUOir \ iriOCCJ CO (Λ W W W ΙΛ

i> H t — I H

• J A A Λ

CQ

-Μ d k.-V 4J k w · - · J3 ^ n ιλ ro in tn 3 Hojooojin oo in a w ^ · - *. W 'S. H OOOCMO- = i- ^ rOH ^ COHOCJLntAinrHCO

Cn c3 CO m OJ CCJ OJ K \ C 3 I rk rk rk • k.> VJ A Λ iij m c s g 2 --— 3 k 4-1 a; <u O CO A A in cn w O O H CO OJlALncn Lf \ · * to oooojo-d-coooojoHoojintninooj

“I ΓΑ OJ OJ OJ iA

Tj t-3 Η H. H

q CQ A A A

• Η Λ • Η -Ρ c __; _ <

C'- CP \ O rA <CA

in λ · n mo co σ \ rk rk in c · - γα o in mh co n oc COOfAlAOOOrAikt'-n-rkOrk -.- Otninin in co in in \ o co in in co OiinocLnoDOvococo cn oc cn (n cn cn η ση h cn tn h cn h ca oj cn on cn <<<; <<<<; '<<<! <«<<<<<<<<< * * * *

ΙΛ ΙΛ 4T 4Γ Λ W fA43-43-.si -. = R.sf.sj -. = * -. = R. = R.3 -. = R. = R II 1 I l I i 1 1 I l III 1 1 III

OOOOOOOOOOOOOOOOOOO

iH i — Ir — I rk rk i — Irkrkrkrkrkrkr — I rk rk rk rk rk -Ί Π —i \ d i. µ

rt) U> <D

in ίο (Λ ia ro nk · Xj fA tA I -H wo i- · »45 no co co xi about: CD 3 v (O Ή rk +5 -H <DC) '0 <1 O) d οι G m ; <x <d -p i-4 λ ej- -ri -ri ooa> <d cd • kc co + pq ro s ή r, · η · η η · η c η ο3 da C ω k 3 cc -π r. -π <dao υ O bD ww co co co cu h) OOA d> i) oddd

£ 03: 3333 + 5 GSdbOdkOOO

3 >> 0 <D0a> <D £ -k-k33kkkdiHHr-l QJDakkkkkCDHrkCUCD-kOOGooO C 3 3 3 3 3 O O C "C £ G d

Cu * ddddd * ooQ.Q <....

k rk. . . S-, 4-5 4J 4->

* 4J. · ·. · D · * · .kktOOCCC

ro: n ro ro to c, o ro ro free ro v o, ex c_. co ro m rk

DK 164225 B

52 * 45% MB + 5% serum + 50% substrate listed above.

** Dilute the substrate charcoal after standing overnight.

1) Adjusted for 68% BL-S640 content.

2) Adjusted for 67% BL-S640 content. In other words, the numerical 5 values are lowered, ie. improved. This adjustment was also made in the test experiments listed below, or alternatively a larger amount of weight was used to provide equivalent dosing.

Mouse blood concentrations after IM administration of 10 mq / kg body weight 10 B1 od od concentrate! oner / zg / ml)

Number of hours after administration

Compound mice 0.25 0.5 1 1.5 BL-S1027 16 15.4 15.1 11.8 8.2 15 BL-S1048 16 14.8 14.1 10.3 7 BL-S640 32 15.7 13.2 9.5 6.8

The compounds were prepared in 0.01% phosphate buffer. BL-S640 20 was used as standard for all compounds.

Mouse blood concentrations after IM administration of 10 mq / kg body weight

Blood concentrations pg / ml)

Number of hours after administration 25 Compound mice 0.5 1 2 3.5 BL-S1027 16 49.8 45.1 24 9.3 BL-S1048 16 46.5 44.6 28.1 12.8 BL-S640 32 53 , 4 45.4 27.2 10.7 30 ..........................-........... ..............----------------

The compounds were prepared in Tween®-CMC. BL-S640 was used by default for all compounds.

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53

Urinary recovery following IM administration of 50 and 10 mg / kg to rats

Percentage of administered dose recovered hours after Dose Number of administration 5 Compound (mg / kg) rats 0-6 6-24 0-24 BL-S1027 50 4 39.6 2.6 42.2 10 4 30.1 1.7 31 , 8 10 BL-S1048 50 4 32.3 2.4 34.7 10 4 21.7 2 23.7 BL-S640 50 7 44.5 2.7 47.2 10 7 24.3 1.4 25, 7 15 --------------------------------------------.... ............-.....

The compounds were prepared in 0.01% phosphate buffer. BL-S640 was used by default for all compounds.

20 Paper chromatograms were obtained after chromatography on rat urine collected between 0 and 2 and between 2 and 4 hours after IM administration (at 10 mg / kg and at 50 mg / kg) of BL-S1027, 1048 and 640 for the detection of antibiotic active metabolites using "decreasing" chromatography with system # 9 (butyl acetate: n-25 butanol rice acetic acid: 40 = 80: 15: 40: 24). Two identical spots were observed in all cases except the standard one (not from the animal), which showed a single identical spot. This indicates complete hydrolysis of the 2 derivatives to the original compound (BL-S640).

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54

Example 9

Reaction product of BL-S640 and o-benzaldehyde sulfonic acid H 0 5 HO-V V-C-C Η K w W <T μκ / κ .-?

CH Λ I

j ^^ J ~ S0-Na coONa ^

Process for Preparation 1) 870 mg of o-benzaldehyde sulfonic acid sodium salt was dissolved in 10 ml of water.

2) 2 g of BL-S640 methanol adduct (1 equivalent) was added with rapid stirring over a 10 minute interval with the simultaneous addition of 4N NaOH to a pH of 6.5. A solution was obtained.

3) The solution was filtered, allowed to stand at room temperature for 1 hour and lyophilized for 24 hours to give the desired product as a solid.

Properties of Product 1) IR-NMR = well defined, consistent for structure.

2) Solubility Ί water => 400 mg / ml.

25 3) Analytical data

Found On dry basis Theoretical% water 3.49 C 41.88 43.4 43.3 30 H 3.41 3.3 3.25 N 11.53 12.1 12.35 S 13.74 14.25 14, 24 Ashes as Na 5.45 5.63 6.77 35 ------------------------------------ ----------------------------

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55 4) Bio-sample = 700 μg / mg.

Δ IR - NMR = a) Intact β-lactam and triazole moiety.

b) Compound does not appear to be a cyclic adduct.

6) Paper Strip Chromatography: There appears to be a double zone at R ^ for BL-S640.

7) Liquid chromatography

Time in hours% free BL-S640 10 0 64.0 1 70.5 2 88.0 8) Water solubility = greater than 400 mg / ml.

15 9) Number, this product is called BL-S1055.

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56

O ΙΛ K \ ΙΛ rO rO

^ ΟΟΉ r — T ri lil LO LO LO

VØ Η ** V K K ·. ft \

COOOOOJOOJOJOOCUOHOVOLOrOrOOfO I H C \ J LQ LO VO

(-¾ iH

^ W A

Γ1 ε \ &> 3

-P A

(0 SH. ---—-—- 4-1 Vj

W H

Λ S.

3

W

tn tn -.

5 H LO

7 * - LO H fO IO

gj .LOOOHlO ro IOLO LO

• w | _p \ ·. * »*»

c OOOOCJOCVJJ-Or-l ^ f-OHOaJLOlOLOOfO

. i [ro C \ J CVJ CM LO

W Η Η H

ε ά Λ? m

S4 W

-P

Λί (1) α to Μ ~~ '~ "to -Η 4-1 Ο η IS- (Ο Ο ΓΟ <d ΟΛ 1ζ in ^ J- Nb-LOLO CTiH r4 inisioo lAlOHLO S-CTi

4J COOrOIOOOOrOHiS-C'-HOrH- = i-OlOLOLO

c lololololovol.oiololooniog \ locoovovo \ .o

ss CTiOtACTiOOiHOrlOi ^ HOr-I ^ WOOO

* * * * ιο ιο 4 · -ϊ ro cm ro. =} - ^ - 3 * ^ i -. = i- ^ i -. = j -. = r. = i - = j - = r -3-

I I I I I 1 I I I I I I I I I I I I I

ooooooooooooooooooo ^ r— (i — I i — 1 i— (i — I i — ti — I <—I i — 1 i — I i — I — I I— {i — I i — ί i — ί itT i — rii to ε • H 3 C d to CO 0 0) bO w ro ro PS to d .3 rO ro i -H to

O * $ 44 IS. LO VO -d Ό d O

0 to -rj O i-l r-r 1-1 -f — I f, J 0 1 /) ') U) r.t (!) I! io: -:: -: <n c <· i «ι ·.« ·. i ·. t π o <n o ø ·. i i: ω (ij iu; ·; ·, i jl \ -, 1 -i i, -ι ·, i o m ø ut d die d 3 d c ·,) d ·,! 0 o o o O bD to two w to two 0 * 3 o O jo ci ho o ej d d

E02333344 EEdbD3dOOO

3> 5 0 0 0 0 0 d -d -H 3 3 d d d 3 H r-t r-t ØQ, dddddØHrHØø-r4 0øE000 d 3 3 3 3 3 o o d d E E d

Di.ddddd * 000, Cl <....

d i — I ·. · D 44 44 -P

• 44. · · · · D. . . . d d ω 0 d d d.

æcowcococooocowpjbd ^ PiCL.PitoutJU

DK 164225 B

57 * 45% AAB + 5% serum + 50% substrate listed above.

** Dilute the substrate culture after standing overnight.

1) Adjusted for 65% BL-640 content.

In other words, the numerical values are lowered, ie. improved.

This adjustment was also made in the test experiments listed below, or alternatively a larger amount of weight was used to provide equivalent dosing.

Mice levels after IM administration of 10 mg / kg body weight 10 B1 od concentrations Mg / ml) hours after administration Compound 0.25 0.5 1 1.5 BL-S10551 10.3 9.9 7.6 6.5 15 BL-S640 12.7 11.5 8 5.7

The compounds were prepared in 0.01% phosphate buffer. Values are mean numbers easily from 3 experiments.

20 1BL-S640 was used as standard.

Mouse blood concentrations after PO administration of 100 mg / kg body weight

Blood concentrations / µg / ml 25 hours after administration

Compound 0.5 1 2 3.5 BL-S10551 38.2 35.7 22.0 11.3 BL-S640 48.7 42.4 24.0 10.1 30 .......... ..-.........--------------------------------

The compounds were prepared in Tween®-CMC. Values are the mean number of 2 experiments.

35 * BL-S640 was used as standard.

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58

Urinary recovery after IM administration of 50 and 10 mq / kq to rats

Percentage of dose administered recovered hours after Number of administration 5 Compound rats 0-6 6-24 0-24 BL-S10551 3 36.6 2.4 39 BL-S640 4 44.2 3.3 47.5 10

The compounds were prepared in 0.01% phosphate buffer.

* BL-S640 was used as standard.

Paper chromatograms were obtained by rat urine chromatography, which collected between 0 and 2 and between 2 and 4 hours after IM 15 administration of BL-S1055 and 640 for the detection of antibiotically active metabolites, using "declining" chromatography with system no.

9 (butyl acetate: n-butanol: glacial acetic acid: H 2 O * 80: 15: 40: 24). A single identical spot was observed in all cases. This indicates complete hydrolysis of the derivative to the original compound (BL-S64Q).

20

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59

Comparative oral PD PD values of BL-S640 derivatives in mice PD50 (mg / kg / treatment) * BL-S BL-S BL-S BL-S cepha-5 Organism 640 1027 1048 1055 lexin E. coli 1.4 - 3.1 1.8 13 A15119 4.1 - 1.6 1.6 13 10 P. mirabilis 7.2 6.3 4.7 8.2 66 A9900 9.6 2.7 3.6 8 , 2 55 15 P. mirabilis 2.4 1.8 2.4 1 50 A9707 K. pneumoniae 1.6 - 0.7 1.6 43 A9977 20 S. pyogenes 0.4 - 0.2 0.4 11 A9604 * Mice were treated at 1 and 3.5 hours after exposure.

25

Comparative IH PD5Q values of BL-S640 derivatives in mice PD50 (mg / kg / treatment) * BL-S BL-S BL-S BL-S cepha-30 Organism 640 1027 1048 1055 lexin P. mirabilis - .. ....

A9900 1.6 1.6 1.6 1.6 50 35 ----------------------------------- --------------..............

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60

Example 10 BL-S1027, the reaction product of BL-S640 and 5-formyl-2-furanesulfonic acid Structure BL-S1027

ISLAND

5 ho / AJ-J

> —PA Ί jn ”(D, L ·) —--- ^ K ^^ J-CHgS-Λ ^ Η

10 ^ J> C00Ha H

SO, Na $

Preparation of BL-S1027 1) 9 g of 5-formyl-2-furanesulfonic acid sodium salt for HCl-SO 2 -SO 3 Na 2 are rapidly stirred in 75 ml water at 60-65 ° C. Most of the solids dissolve.

2} 20 g of BL-S640 propylene glycol adduct are sprinkled with rapid stirring over a 10 minute interval, simultaneously adjusting the pH-25 to 6.5-7.0 with 40% NaOH (the pH should not rise above 7.2 ). A solution or near solution is formed.

3) The solution of pH 6.5-7.0 is stirred at 55-60 ° C for 15 minutes and cooled to 4-8 ° C. Stir at 4-8 ° C for 10 minutes.

4) Filter to remove a small amount of insoluble matter.

5) Add the filtrate + (see Note 1) to 1 liter of isopropanol with rapid stirring. An amorphous precipitate is formed. Stir for 5 minutes.

6) The solids are removed by filtration, washed with 100 ml of isopropanol and vacuum dried at 50-56 ° C for 18 hours.

7) The solids are dissolved in 60 ml of water and cooled to 4-6 ° C. A very small amount of precipitate can be formed and removed by coarse filtration.

8) The filtrate is filtered at ambient temperature through appropriate filters to remove particles, pyrogens and bacteria.

DK 164225B

61

Steps 7 and 8 should be completed within 4 hours.

9 / Sterilize lyophilized for 48 hours and then heat at 50-56 ° C under vacuum for 18-24 hours. Yield of BL-S1027 is about 22-44 g.

5 + Note 1, Step 5: If 18.7 g of BL-S640 methanol adduct is used in place of the 20 g of BL-S640 propylene glycol adduct, the filtrate can be cooled to 4-6 ° C, coarse filtered and lyophilized directly as described in steps 8 to 9.

10

Properties of BL-S1027 1) Bio sample = 724 µg / mg.

2) Bio-yield = 85 - 95% 3) IR - NMR = a) consistent for structure, b) about 80% cyclic, 20% in non-cyclic D20 at 70 mg / ml, c) about 0, 05 moles of isopropanol.

4) Paper strip chromatography (0.2 mg / ml) = 100% BL-S640, no sign of derivative.

5) Liquid chromatography (1 mg / ml in phosphate buffer pH = * 7) 0 hour = 85% free BL-S640 0.5 hour = 94% free BL-S640 1.0 hour = 100% free BL -S640.

6) Water solubility => 400 mg / ml.

25 7) Analytical data

Found On a dry basis Theoretical% H 2 0 KF 5.14% C 40.15 42.1 43.0% H 3.10 2.9 2.96 30% N 12.39 13.05 13.1% S 13.6 14.33 14.95% ash as Na 5.86 6.16 6.5 8) Mouse body concentrations = oral and intramuscular concentrations approximately equal to the concentrations of BL-S640 propylene glycol adduct. BL-S640 appeared in urine with no evidence of the derivative.

9) (a) Stability of solids: less than 10% loss for 1 month at 56 ° C.

~ b) Stability of solutions: less than 10% loss at 250, 25 and 10 mg / ml of BL-S640 activity for at least 24 hours at room temperature.

62

DK 164225 B

Example 11 5

ISLAND

Ηο- (δνίΗ "α" ΗΗ \ f Ί H n ii! + 2 tea + l ch * s —vw

10 2 o · I I

C ° 2H H

Cl8Hi8N6 ° 5S2 'Molecular = 462'50 15 [j Π + KEH -—-> OHC-SO 2 Na 20

ISLAND

II

H0- <JO) - H ~ 1 ir '- [H-N - Jjr- ~ Kyy ~ CE 2 ~ S ~ -

25 (H CO 2 K

Γ SO, Na;>

30 C25H17N608S3NaK

35

DK 164225 B

63

Materials 1 ~ g (0.00217 mol) BL-S640 429 mg (0.00217 mol) 5-furfural sulfonic acid Na salt 217 mg (0.00434 mol) triethylamine 1.2 g (0.00659 mol) potassium 2-ethylhexanoate (KEH).

Course of action

To a slurry of 1 g of BL-S640 in 8 ml of methanol was added 217 mg of triethylamine (TEA) and 429 mg of 5-furfural sulfonic acid. The mixture 10 was heated to reflux and soon all solids dissolved.

The solution was heated for 3 minutes and 1.2 g of KEH was added. A yellow salt was separated and collected and washed with methanol. The salt was dried over PgOjj in a desiccator. Yield 800 mg.

Analysis for C 23 H 17 N 6 ° 8S 3 Na 2 4H 2 O: Calculated: C, 37.54; H, 3.41; N, 11.42 Found: C, 37.30; H, 3.31; N, 10.18

Melting point> 140 and decomposition.

The same reaction was carried out as above except that sodium 2-ethylhexanoate was used instead of KEH. The salt was not precipitated by methanol, so the ole solution was filtered and the filtrate was diluted with isopropylal carbon. The solid precipitate was collected and washed with isopropyl alcohol. The salt was hygroscopic, which is why it was dried in vacuo over P20g. Yield: 800 mg, melting point> 140 ° C decomposition.

Analysis for C ^H₂ ^N₂O₂S₂Na ,,:

Calculated: C, 42.65; H, 2.78; N, 13.65. Found: C, 41.03; H, 3.94; N, 10.54.

The sample was soluble in water. The solution showed a pH of 6.5.

Paper strip test in buffer and human serum was performed. The sample was found to exhibit only one stain corresponding to BL-S640. Both samples showed a solubility of> 100 mg / ml at pH 6.5.

Paper strip chromatography showed that the above compound was completely hydrolyzed to BL-S640 in mixed human serum at pH 7 and 0.1 M phosphate buffer at pH 7. Paper strip bioautographs were obtained as follows. Samples were dissolved at a concentration of 500 µg / ml in 0.1 M phosphate buffer of pH 7 and at a concentration of 500 µg / ml in mixed human serum at pH 7. A 10 µl sample of BL-S640 and of the aforementioned product was applied to one spot

DK 164225 B

64 12.7 mm (1/2 inch) strip "What # 1" paper and developed at 27 ° C for 16 hours in a system of butyl acetate: N-butanol: acetic acid: water (80: 15: 40: 24 ). The strips were air dried and placed on an agar plate grafted with B.subtilis A.T.C.C. 6633. At 30-minute, 5-hour, 3-hour, and 6-hour intervals, a single zone of Rf value 0.3 was detected for all samples.

High-pressure liquid chromatography at a concentration of 1 mg / ml showed complete hydrolysis to the original BL-S640 after 1/2 hour. Samples were chromatographed on a µ Bondapak C18 (Waters Associates) column, using a 0.01 M sodium acetate pH 4 / methanol 8/2 moving phase at a flow rate of 0.52 ml / minute. Detection was carried out at using UV at 354 μ. Samples were dissolved in the moving phase concentration, 2 μΐ injected, and BL-S640 eluted at 18 minutes and the aldehyde at 6 minutes. Quantification of BL-S640 and 15 5-formylfuran sulfonic acid was performed. using p-toluenesulfonic acid as an internal standard.

Example 12

Compounds of the general formula 20 .o-fyLJ, * aTX XcH sjp £

25 L / γ ^ T

R1 in H

COONa wherein R is phenyl-2-sulfonic acid, 4-methoxyphenyl-3- sulfonic acid, 4-hydroxyphenyl-3-sulfonic acid, 2-carboxymethoxyphenyl, 4-carboxymethoxyphenyl, 3-hydroxy-4-carboxyphenyl, 4- (2 -carboxy) vinyl-phenyl, carboxyl, 2-carboxyphenyl, 3-carboxyphenyl, methanesulfonic acid or methanedisulphonic acid are prepared by substituting the 5-formyl-2-furanesulfonic acid in the procedure of Example 7 with an equimolar weight amount of the corresponding aldehyde which has the general formula R -CHO.

65

DK 164225 B

Example 13

The compounds of the general formulas

110 i, O

w »By t <1 η— I J-N. ^ J-CDI-S-I. / Ά H2 0 I 11] 0 COONa.

i c H3C \ h 0 15 ν-λ1 11 HO- / 'C-— c s w; J-rf ί jf? '20 ^ '' y cvs'1 "20 COONa

CH3O H O

Ho / \ c — C s \ = / h1v "Ί Π |! | '

25 C-H I 11 J- CH2-S-k.KN

R2 ° H

COOUa .0.

Wherein R is: J_l_ is prepared by replacing the cefatrizine in the procedure of Example 7 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

35

Example 14

The compounds of the general formulas H 0 66

DK 164225 B

'"^ O-JcS-τΛ 5-1'-»; igH 0ΛΝΧ ^ -0Η2-3 Ύ "COONa 10“ S -' / Ti Ί Ti -? - ch3

15 C

COONa

CH

ν 'H O

20 / = \ I II

Η0Λ \ // 'l? YY

®υην “π 1 ri, - ~ rcH5 I CH0-S and R8 ° COONa 25 CH-

^ \ _ H O

/ = \ I II

HO-v Λ - ccq 3o jv "~ CH3 Ί;" o ^ y -'- v5- ^ 8 COO ?: a 35 wherein R2 is: "jfjp is prepared by the replacement of the cafatrizin of the procedure of Example 7 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

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Example 15

The compounds of the general formulas

H O

67 "^ irj-τ-Λ η- 9 ^ J-n-CHp-s

i2 0 1 I

COONa CH

10 HO H H

> = \ i II

HO — L · Λ-C C g '' ass. J'-j-f '^ I i

l'H / -Mv ^ -ch -s N ^ N 'r2 0 I I

15 COONa CH3 CH-, Ϋ Η o π and 20 J-ν 'J ~ CH? -S-ArKH and Η2 ° I 1 COONa CH3 CH, 0

or 5 \ H O

} = \ 1 11

K0 - \\ // 'ΐ f Λ' ') π—; H

HN ^ / n i \ I J 1 'C-H \ .lk Λ— CH2-S-ktK'!

R2 ° in I

30 COONa CH3

ISLAND

wherein 35 is prepared by replacing the cefatrizine in the procedure of Example 7 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

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68

Example 16

The compounds of formulas HO h O

5 Ho> y} -c s

R loo * a H

10

Vx? il HO- ^ Vc-C g 15 wjk j-ri ί m? C-H U j-CI ^ -SV and

<K I H

COONa CH3 ° \ h i! 20 I 11 \ = / ^ k J-rY Ί jpf

l'H CH2 ~ S

wherein R is phenyl-2-sulfonic acid, 4-methoxyphenyl-3-sulfonic acid, 4-hydroxyphenyl-3-sulfonic acid, 2-carboxymethoxyphenyl, 4-carboxy-methoxyphenyl, 3-hydroxy-4-carboxyphenyl, respectively. 4- (2'-carboxy) vinylphenyl, carboxyl, 2-carboxyphenyl, 3-carboxyphenyl, methanesulfonic acid and methanesulfonic acid are prepared by replacing the 5-formyl-2-furanesulfonic acid in the procedure of Example 7 with an equimolar weight loss of the corresponding aldehyde having the formula R -CHO.

Example 17

The compounds of the formulas

H O

69

DK 164225 B

H0Oi Jn-r / iS n n-ch3 f / ο ^ Νγί-ΟΗ2-3_Ι ^ Ν R COONa

H0 \ H O

) = \ i li H0 _ (\ // - <r-c s HN ^ / N-r-I 'Ί' ll-? "Η J-IU CH.-S—

Å8 o T

COONa CH,

\ 3 H O

? _H CH -s · —Il /> N and 0X Y 2 ^ COONa

CH = O

V-Λ H ° ί "11

* 2 Ox T

R I

COONa 2 70

DK 164225 B

wherein R is phenyl-2-sulfonic acid, 4-methoxyphenyl-3-sulfonic acid, 4-hydroxyphenyl-3-sulfonic acid, 2-carboxymethoxyphenyl, 4-carboxy-methoxyphenyl, 3-hydroxy-4-carboxyphenyl, 4- (2, respectively). '-carboxy) vinyl phenyl, carboxyl, 2-carboxyphenyl, 3-carboxyphenyl, methanesulfonic acid and methanesulfonic acid are prepared by replacing the 5-formyl-2-furanesulfonic acid in the procedure of Example 7 with an equimolar weight of about 10 corresponding aldehyde having the formula R -CHO.

Example 18

The compounds of the formulas

H O

71

DK 164225 B

Jr. '? 9 J - CHp-S,

R2 ° I

COONa CH-

H0X H O

l'H VN.

R2 1 I

COONa CH ^ CH, V-λ H '°' ^ ti-r-Λ jnr? "H CH ° g R2 0 1 i COONa CII5 CH-2.0) = \" __n

Η ° Λ_JC '£ / S - [N

hn ^ / t'-rn I I! {, if R 1 ΓΗ COONa Ltl3

DK 164225 B

Wherein R is phenyl-2-sulfonic acid, 4-methoxyphenyl-3-sulfonic acid, 4-hydroxyphenyl-3-sulfonic acid, 2-carboxymethoxyphenyl, 4-carboxy-methoxyphenyl, 3-hydroxy-4-carboxyphenyl, 4- (2) '-carboxy) vinylphenyl, carboxyl, 2-carboxyphenyl, 3-carboxyphenyl, methanesulfonic acid and methanesulfonic acid are prepared by substituting the 5-formyl-2-furanesulfonic acid in the procedure of Example 7 with an equimolar weight of about 1 - 2 corresponding to aldehyde having the formula R -CHO.

Example 19 Preparation of BL-S1057, the sodium salt of the reaction product BL-S643 and 2-furaldehyde Formula, BL-S1057

J \ H II

H0 \ == / f Ί S N-

lf / \ 0 ^ -NN ^ CII2S— \ s / ~ aS

) —O COONa

ISLAND

Process 1) 1 g of BL-S643.3H2O is slurried in 10 ml of water at 40-45 ° C.

2) 0.25 g (1.25 equivalents) of 2-furaldehyde is added.

3) 1N sodium hydroxide is added with rapid stirring until pH 7-7.5. A solution or near solution is obtained in less than 5 minutes.

4) The solution is cooled to 22-24 ° C and appropriately filtered to remove particles, bacteria and pyrogens.

5) Lyophilize under sterile conditions for 48 hours to obtain BL-S1057 powder (BL-S1057 can also be obtained by precipitation from the sterile solution in step 4 with 15 to 20 volumes of sterile isopropanol).

35 Properties of BL-S1057 1) Bio sample (using cefatrizine as the standard) = 759 Mg / mg.

2) IR = intact β-lactam; well defined.

3) NMR = a) well defined; consistent

DK 164225 B

73 b) about 75% cyclic adduct (60 mg / ml in D 2 O).

4) · Solubility => 400 mg / ml in water.

5) Paper strip chromatography = mainly 1 zone at R 2 value for BL-S643 at 37 ° C at 0 and 1 hour (concentration = 0.2 mg / ml).

6) Liquid chromatography = 100% hydrolyzed in buffer of pH 6.1 within 1 hour at room temperature (concentration = 1 mg / ml).

7) Melting point = 180 ° C (shrinkage) - 200 ° C decomposition.

Analytical data 10 Found on a dry basis Theoretical% H2 O KF 5.18% C 45.91 48.4 48.4% H 4.05 3.7 3.37% N 10.81 11.4 11.8 15% S 14.92 15.73 16.48% ash

After 3.84 4.03 3.88

Example 20 BL-S1058, the reaction product of BL-S643 and sodium 5-formyl-2-furanesulfonic acid Formula, BL-S1058

'—F HN .N — i — r η N-N

A oJ— \\ ^ Λ— CH S JLcH3, 5H2 ° C00Na SO, I'Ia 5

Preparation of BL-S1058 1) 0.45 g of 5-formyl-2-furanesulfonic acid sodium salt 35 µl-SO Na). Island Island

DK 164225 B

74 is slurried in 10 ml of water at 40-45 ° C. A solution or nearly solution is obtained. 2) 1 g of BL-S643.3H2O is sprayed over a 10 minute period with rapid stirring and simultaneous addition of 1 N sodium hydroxide to a pH of 7-7.5. A solution or near solution is obtained within 5 minutes.

3) Cool to 20-23 ° C and filter the solution through appropriate filters to remove particles, bacteria and pyrogens.

4) Lyophilia is performed for 48 hours. The resulting powder is 10 BL-S1058 (sterile BL-S-1058 can also be obtained from the solution in step 3 by precipitation from 15-20 volumes of sterile isopropanol).

Properties of BL-S1058 1) Bio sample (using cefatrizine as the standard) = 585 µg / mg.

2) IR - NMR = a) well defined; consistent b) β-lactam and 3-side chain intact c) 90% cyclic adduct (at about 60 mg / ml in D 2 O).

3) Paper strip chromatography = mainly 1 zone at R 2 value for BL-S643 at 0 and 1 hour at 37 ° C (concentration is 0.2 20 mg / ml). There is evidence of another zone.

4) Liquid chromatography (1 mg / ml in pH 6 buffer).

Time in hours% hydrolyzed 0 25.4 25 1 46.5 2 59.0

Analytical data

Found On a dry basis Theoretical 30% H2 O KF 10.17% C 36.5 40.5 41.6% H 2.89 2.4 2.74% N 8.52 9.49 10.01% S 17.09 18.98 18.6 35% Ash as

Na 7.04 7.93 6.63

DK 164225 B

75 V 'C £> rfl fA m Ή lp ΙΓΝ U V O O \ S \ W ^^

to 'wOOOOOH ^ f ^ rOfH ^ O- ^ OvOLA

X) I H ^

in PQ A

w σ c ___—- • H --—- -----------—- u fy c

-H A ~ N

(M

C —- <U ^ OD VD 00 • U, -, ΙΠγΗΟΙΛ m Λ ._ je gooocxj in η inin • H ti WOOOOOO- = fCMOHC \ JOCMOVpin

U X. I H CM

0 ~ lJ Ή

• O or A

ΓΊ U

™ H

'T »r V £) in J ____——-- CQ ----—-—-

never zL

c x vo oo 2 o o o in cm inin ^ WOOO00H ^ 3-CMOH ^ -O- = rOVDcn ω Λ η a ^ a

to Q A

J * to -----------—--—— v o

• H

^ t— od om <4j in ^ rN-f'-vovoODr-iiHinr —- noinfn c co ommooo κ \ h c- t- rt o <in \ o in m co co in in ό σ \ in σ \ in co ODotcnCAC ^ CArH (AH OC C70 ι-1 CT \ rj C \ * * * * ^^ (Λ K \ ~ ~ (OvCMrn- = J- ~ - = r ~~ · ^ p "ll III 1 I ll I 1 1 1 1 ooooooooooooooo i-Hr- | r-lrHr — li — lr-lr — Ir-tr-Hi — I r — r—): • rl 3 c; ^

rj (DO

hfi co n rn K to U .C fA tA I --- (.

O '* * - A Vi. VO CO -C Ό ™

O to T-l o rH IH - »-> -Η Ο o to W

rj a> t: in X X Ο η m r «3 · η · η O

• η γ; ω - + - cq cq 2 -H jo a · η h -h c

CO C 3 C C -H C

ouDtotf> «wwa) t ':> 22-9 3,« S.0 3 3 3 3 3A - 3>, (D (D <DO o CHH 3 3 H 3 "<dac cccc <u —ι h oo- HO o 333352 oocc 6 ε * a.rtrjrtnJrt - oocun, C r — 1 ...

...... · '·· £ * jr g __re in m to co in co co Μ ΙΟ X o. A.

DK 164225 B

76 0 te ---- yeah,

island C

ΤΓ tu ftj S ΓΑ u * 2 - O tn

r VO -Π B

"K ΐΛ ΙΛ 4- W tn> i oj co tn tn J rH H S Cn 0

PQ Λ O CM

s * 4 * £

2 - \ tu 4J

5 _ / "c 3 d; s- -n tu S ^ ^ ΓΛ tu 0 σ» W o MC <uc —- ^ co ic jj -S te 03, CM CJ te μ E m KQ c B \ to pzA o ω C tn H - \ I ra nl ^ pi co in in o pq os ® ^ -ι-l I CM CM te // - \ at 10 n pq A f co \\ - o £ ® "1 \ r Si> ______I us

M-V ^ S

CU ° \ ° · te 5 s T V * * § cn in cc - S te m 'o in p = v te o n.

ra co in tn cT vo o ^ mco, l CM ΓΟ Η / IC te te ^ ^ τί l = / .I «tu n ^ PQ a I XN ^ 0 0 p 1 ΊΓ - w £ 1 5-1 cn ou 2 (U «L Ό ,, te -π w 7, Ό M + i S II * · fij te ø m ^ te te> 4J te 1 {j \ K cn ø., '-H to N σ \ tete ø ø :> te o in an un co ω o; u · η • HO vo CO vo 2 'i tn> Π cm ον σν σ \ 2 aa · η ø λ:

o <<<<^ ffipQ Pi te f-J

2 te C (U Pi te LO 0) te te £ Ό te • Η teM raraamnj 2 tn -u s-ι tete ra ω ef IIII Ζί 2> —1 —t 0 tn ^ oooo 0X0 3> oo te P h h2Sh ° ^ o tete (o ® qj,, ____> _, ___ in -O tete Pi te tn .p tac CC ø cc S + Pi 0 te -H Øte C 4J -p) te P |

Gο G tn J-J ..v> „v Pi ø .Q

,, 1 n .O m o, i \ o c ø ti li Ml! (il id -i — i '/' tu tn øøø te "0 Pi te Pi Pi Pite> ^ V, ocwc o o te .h

0 o \ o «-Ι 0 ø IW IH c -Η -H

O V V <u in C 4-> 0 + J

w l? to island Cn ø v jo »ία 22 2 2 +> cc island island-i

pririrt O -HØ Pi Pi øCn-H

ca.tete 00 S04J

Oi-lrHrHriJUc 4J4J

ε o O O <P |>, P | 0 0 · & -P 0 o o '' · · # ti P U t—) i— »p (44> .4J4JinCO« -l 0 c c c ί m ikø co w w ω „„ - * * iH ro% 77

Paper chromatograms were obtained after rat urine chromatography collected between 0 and 2 and between 2 and 4 hours after IM administration of BL-S1057, 1058 and 643 for the detection of antibiotically active metabolites using "decreasing" chromatography with system no.

9 (butyl acetate: n-butanol lacetic acid HgO 2 O: 15:40:24). A single iden- tical spot was observed in all cases. This indicates complete hydrolysis of the 2 derivatives to the original compound (BL-S643).

Example 21 10 Compounds of Formula -C S _ 15 v === / hN ^^ N | in [2 O] CH 2'S_kS / L ^ COONa wherein R is phenyl-2-sulfonic acid, 4-methoxyphenyl-3-sulfonic acid, 4-hydroxyphenyl-3-sulfonic acid, 2-carboxymethoxyphenyl, 4-carboxy-methoxyphenyl, respectively 3-Hydroxy-4-carboxyphenyl, 4- (2'-carboxy) vinyl phenyl, carboxyl, 2-carboxyphenyl, 3-carboxyphenyl, methanesulfonic acid and methanesulfonic acid are prepared by substituting 5-formyl-2-furansul the phonic acid of the method of Example 20 with an equimolar weight amount of the ten 1 to 2 corresponding aldehyde having the formula R -CHO.

78

DK 164225 B

Example 22

The connections with the formulas

5 HO ^ _ h O

no ^ ~ \ - c— c s

\ = / hN ^^^ N-II

f "1 ^ N ^ -CTg-e-lL JLcHj

R2 ° I

10 COONa

15 H3Cv H O

y \ * 11

\ = / HNX ^ N —- f N-N

| ZH0CH2-S A CH5 09

R2 ° I

20 COONa

CHJ ° V H O

ΗοΌ-ί-! .S

'-' HNX ^ N —- f> N-N

25 | "H CH2-S -A J ~ Ch O 'J * S ^ COONa 2/0. O.

Wherein R is ~ || Na or Na 2 Na is prepared by replacing the cefaparol in the procedures of Examples 19 and 20 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

35 79

Example 23

The compounds of the formulas

H O

/ = \ i II

Η0Λ \ // 'f / Y

r ~ in? h <f Y 2 n

K I I

COONa 'CHj

HAY

ηο ^ Ϊ) -Η / s.

VhnxA_ Tr-jT

COONa I

CH3

CH

- Ηΐγ / N- | -Y \ "-n C-H J II i 'a σ ^ -Νγ ^ ν8 -%>

COONa I

CH-CH, 0

\ _ H O

H0HO ~ i — f γγ

N- 'HN ^ XN-j-f> N-N

C-H 1 H li

I-, N

π I I

COONa '.

O i i “7) wherein R2 is ~~ f \ 1] or ~ | f ^ S ° 3Wa

DK 164225 B

80 is prepared by replacing the cephaparol in the procedures of Examples 19 and 20 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

Example 24

The compounds of formulas Η0Ό-Ή / s ντπ. jnr COONa

HAY

\ _ H O

HO · \ c-c

^ N- | —r'N H-W

| 'H 0J_n - ^ - ch2-s -A J- ch,

R2 U T 0 D

COONa CH,

Yi_ H O

a

- 'HN ^ / N —- N-N

| 'H 07 ^ γίθΗ2-3- ^ 0ΑθΗ3 and COONa CH, 0

_ H O

Η0-Ο-ί- ""

X- 'ilN ^ / N —- f] Π-N

li11 / - »vJ-CI ^ -S

R ^ I

COONa 81 o 0 wherein R 2 is - / or SO - Na 5 is prepared by replacing the cefaparol in the methods of Examples 19 and 20 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

Example 25

The compounds of the formulas

H O

- \ I II ch m-l Ve-c «3 '^ ~ v ~ C _J ~ h” ^ 2 (r 2 n COOIfa

HAY

\ _ H O

HO- ^ Vc-C CH

\ u i i / S. i 3

- HNV ^ N — i-f N K-H

'C oV, J - ir T ri COONa CH,

\ p, H O

r>

'-' HN ^ / N — i-p V N-N

? "H J-N. /> LciI0-S-L jJ oq

^ 20X

COONa

DK 164225 B

82 CH, 0

3 \ _ H O

_> K

'-' HN ^ / N —- p j N-N

5? "Η J-N ^^^ l-CHp-S — k Yj j, 2 <r Y 2 COONa

/ N °

, Λ -1 fl —Π I-SO, Na 10 wherein R is | or J 2 is prepared by replacing the cephaparol in the methods of Examples 19 and 20 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

Example 26

The compounds of the formulas

H O

'- ^ HlL, / N-i-N-C-CR'? 'H ^ -n ^ y ^ L-ch -s-Jn ^ 2 cr y d s ^ COONa

K0V H O

) = \ i II

HO - \\ Λ "C ~ C q / K — r — f'O \," "CH3 1;"

R I

COONa

DK 164225 B

83 CH,

\ 5 H O

/ = \ i II

HO- / v / C C c

^ B »v / i-I-f 'l N-C-CH

See "COONa CH-, 0

3 \ H O

10 M i ii HO-u Λ-C - C ’'irT0" 3 I / 7 - ^ - CH "-S—>

* 2 Y * S

η I

2 COONa 2 - Π Π, Il II in SO, Na wherein R is) J or | 3 20 is prepared by replacing the cefaparol in the procedures of Examples 19 and 20 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

DK 164225 B

84

Example 27

The compounds of formulas H O

* "^ OJ-S-tVS; = r ^ C-H ^ i_CH2-S-4H-N-UI3,

'2 O T

R I

COONa

HAY

10) = \ j il? "H J-nLxI-CH, -S ^^ Ν · ° Η3 o 'γ *"

· H O

/ = \ I II

HO - (\ // ~ Q "C C

20 CH2-S- ^ n ^ N "CH5 ° 9

R2 ° T

COONa CH, 0 25) = \ J »

v // Λ λ__N - N

N- 'HN. XN I i i? ”H / ~ nL ^ -CH2-S-

i20 T.

COONa "Tf" 0 "" - SO, Na wherein R2 is [| _ |] or [j_ | J? Is prepared by replacing the cefaparol in the procedures of Examples 19 and 20 with an equimolar weight amount of the corresponding amphoteric cephalosporin.

35

Claims (115)

85 UIV 104 ^ 9 D 1. Cephalosporin Compounds for Use as Nutritional Supplements in Animal Feeds, CHARACTERIZED BY THEIR GENERAL FORMULA 5 A X_ H O '- y \ = · / Wherein H is N, hydroxy, methyl or methoxy, R * is hydrogen, sodium or potassium, 10 HN N-1-R 2 R 2 is 2-furyl, 2-furan-5-sulfonic acid, phenyl-2-sulfonic acid, 4-methoxy-phenyl-3-sulfonic acid, 4-hydroxyphenyl-3-sulfonic acid, 2-carboxymethoxy-phenyl, 4-carboxymethoxyphenyl, 3 -hydroxy-4-carboxyphenyl, 4- (2'-carboxy) vinyl phenyl, carboxyl, 2-carboxyphenyl, 3-carboxyphenyl, methanesulfonic acid or methanesulfonic acid, and R 1 is 1,2,3-triazole 5-yl, tetrazol-5-yl, 1,2,4-thiadiazol-5-yl, 1,3,4-thiadiazol-2-yl, 1,3,4-oxadiazol-2-yl or 1,2 , 4-triazol-5-yl, wherein each of these groups is unsubstituted or substituted by 1 or 2 alkyl groups having 1 to 4 carbon atoms. A compound according to claim 1, characterized in that R 2 is 2-furyl. A compound according to claim 1, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to any one of claims 1-3, characterized in that R * is sodium. A compound according to any one of claims 1-3, characterized in that R * is potassium. 6. A compound according to claim 1, characterized in that it has the general formula AX_H O 'o ^ U-S 5 \ = y | In S | -H HK K-f I L-NxJ-CH2-S-i M O7 I, N P COOH1 R2 H 10 wherein 1 2 A, R and R have the meanings specified in claim 1. A compound according to claim 6, characterized in that R 2 is 2-furyl. A compound according to claim 6, characterized in that R 2 is 2-pyran-5-sulfonic acid. A compound according to any one of claims 6-8, characterized in that R * is sodium. A compound according to any one of claims 6-8, characterized in that R * is potassium. 11. A compound according to claim 6, characterized in that it has the general formula 25. -HO HO - ^^ Vc-i WII 3 -N Nc4i I 1 30 «2 h -Nv ^ -CHa-S- ^ NRO '- i Ψ COOR1 IH 35 wherein 1 2 R and R have the meanings specified in claim 1. A compound according to claim 11, characterized in that R 2 is 2-furyl. 87 A compound according to claim 11, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to claim 12 or 13, characterized in that it has a D configuration and that R * is sodium. A compound according to claim 12 or 13, characterized in that it has D-configuration and that R1 is potassium. A compound according to claim 12 or 13, characterized in that it has a D-configuration and that R * is hydrogen. A compound according to claim 1, characterized in that it has the general formula A \ _ H O 15 // \ I II HO - <^ J> -C-C N-N-CH3 HN N-r-j / S ^ | Y / lY7s'V ”20 cooir wherein 1 2 A, R and R have the meanings specified in claim 1. A compound according to claim 17, characterized in that R 2 is 2-furyl. A compound according to claim 17, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to any one of claims 17-19, characterized in that R 1 is sodium. A compound according to any one of claims 17-19, characterized in that R * is potassium. A compound according to claim 17, characterized in that it has the general formula 35 | W I I / S N-N-CH, H 2 v / N-s 2 il COOR1 wherein 1 2 R and R have the meanings specified in claim 1. A compound according to claim 22, characterized in that R 2 is 2-furyl. A compound according to claim 22, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to claim 22, characterized in that it has D-configuration and that R 1 is sodium. A compound according to claim 22, characterized in that it has D-configuration and that R1 is potassium. A compound according to claim 22, characterized in that it has a D configuration and that R 1 is hydrogen. A compound according to claim 1, characterized in that it has the general formula 25 \ H O \ = z / I I / S. N-N ao-i-i i i i 'i i2 c ° o., I l 3 35 89 wherein 1 2 A, R and R have the meanings specified in claim 1. A compound according to claim 28, characterized in that R 2 is 2-furyl. A compound according to claim 28, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to any one of claims 28-30, characterized in that R 1 is sodium. A compound according to any one of claims 28-30, characterized in that R 1 is potassium. A compound according to claim 28, characterized in that it has the general formula HtVKTY L Γ "" in "COOR1 I 20 ch3 wherein 1 2 R and R have the meanings specified in claim 1. A compound according to claim 33, characterized in that R 2 is 2-furyl. The compound of claim 33, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to claim 34 or 35, characterized in that it has a D configuration and R 1 is sodium. A compound according to claim 34 or 35, characterized in that it has a D configuration and that R1 is potassium. A compound according to claim 34 or 35, characterized in that it has a D-configuration and that R * is hydrogen. 39. A compound according to claim 1, characterized in that it has the general formula. 5 HO- / \ -C-C _ —Π j-L | -xr> - · αίο-ΰ-Λ) -CIU lv o 'In COORl 10 wherein 1 2 A, R and R have the meanings specified in claim 1. A compound according to claim 39, characterized in that R 1 is 2-furyl. A compound according to claim 39, characterized in that R 1 is 2-furan-5-sulfonic acid. A compound according to any one of claims 39-41, characterized in that R * is sodium. A compound according to one or more of claims 39-41, characterized in that R 1 is potassium. A compound according to claim 39, characterized in that it has the general formula 25 N-N hn \ / M-r ~ f Ί li 1 c-π y -'ch3 30 R2 0 T! 0 COOIr · wherein R and R have the meanings specified in claim 1. The compound of claim 44, characterized in that R 1 is 2-furyl. The compound of claim 44,. CHARACTERISTICS: R 2 is 2-furan-5-sulfonic acid. A compound according to claim 45 or 46, characterized in that it has a D-configuration and R * is sodium. A compound according to claim 45 or 46, characterized in that it has a D configuration and that R1 is potassium. A compound according to claim 45 or 46, characterized in that it has a 5-configuration and R 1 is hydrogen. 50. A compound according to claim 1, characterized in that it has the general formula A 10 f13 in i .s L_f, HN N-1-S N 1 | H H 15 la (f T C O OR1 wherein 1 2 A, R and R have the meanings specified in claim 1. A compound according to claim 50, characterized in that R 2 is 2-furyl. The compound of claim 50, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to any one of claims 50-52, characterized in that R * is sodium. A compound according to any one of claims 50-52, characterized in that R * is potassium. The compound of claim 50, characterized in that it has the general formula 30 Ho-fyLj W / II s in I IN N - N f-N ^ C-M I I 35 ^ 2 j COOR1 DK 164225 B 92 wherein 1 2 R and R have the meanings specified in claim 1. A compound according to claim 55, characterized in that R 2 is 2-furyl. 57. A compound according to claim 55, characterized in that R 2 is 2-furan-5-sulfonic acid. 58. A compound according to claim 56 or 57, characterized in that it has D-configuration and that R is sodium. A compound according to claim 56 or 57, characterized in that it has a D configuration and that R * is potassium. A compound according to claim 56 or 57, characterized in that it has a D-configuration and that R * is hydrogen. 61. A compound according to claim 1, characterized in that it has the general formula 15 \ H O -c Jr in N-C-CH-3 ao r j ii 3? H _CHp-S-A N L · s J 'ν' COOR1 wherein 1 2 A, R and R have the meanings specified in claim 1. A compound according to claim 61, characterized in that R 2 is 2-furyl. The compound of claim 61, characterized in that R 2 is 2-furan-5-sulfonic acid. 64. A compound according to any one of claims 61-63, characterized in that R * is sodium. A compound according to any one of claims 61-63, characterized in that R * is potassium. 66. A compound of claim 61, characterized in that it has the general formula 93 HO -f \ -l_I 5 \ = y II s. NQ-CH R and R have the meanings specified in claim 1. 67. A compound according to claim 66, characterized in that R 1 is 2-furyl. 68. A compound according to claim 66, characterized in that R 1 is 2-furan-5-sulfonic acid. 69. A compound according to claim 67 or 68, characterized in that it has a D configuration and that R1 is sodium. 70. A compound according to claim 67 or 68, characterized in that it has a D configuration and that R * is potassium. A compound according to claim 67 or 68, characterized in that it has a D configuration and that R 1 is hydrogen. 72. A compound according to claim 1, characterized in that it has the general formula 25 A \ HO \ = zJ II 3. Γ1 - N 30 / N-1 - (Ί II A T1 "COOH1 wherein 1 2 A, R and R have the meanings set forth in claim 1. The compound of claim 72, characterized in that R 2 is 2-furyl. 74. A compound according to claim 72, characterized in that R 2 is 2-furan-5-sulphonic acid. 75. A compound according to any one of claims 72-74, characterized in that R * is sodium. A compound according to any one of claims 72-74, characterized in that R * is potassium. A compound according to claim 72, characterized in that it has the general formula 10 HO-fVi_I 15 w 11 v J * - ^ - | Λ Ni | the meanings specified in claim 1. A compound according to claim 77, characterized in that R 2 is 2-furyl. 79. A compound according to claim 77, characterized in that R 2 is 2-furan-5-sulphonic acid. 80. A compound according to claim 78 or 79, characterized in that it has a D configuration and that R * is sodium. 81. A compound according to claim 78 or 79, characterized in that it has a D configuration and that R 1 is potassium. 82. A compound according to claim 78 or 79, characterized in that it has a D configuration and that R * is hydrogen. 83. A compound of claim 1, characterized in that it has the general formula 95 A 9. I_N. CH 2 -s-1 J L · f f Y COOR1 CH, 10 ^ wherein 1 2 A, R and R have the meanings specified in claim 1. 84. A compound according to claim 83, characterized in that R 2 is 2-furyl. A compound according to claim 83, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to any one of claims 83-85, characterized in that R 1 is sodium. 87. A compound according to any one of claims 83-85, characterized in that R * is potassium. 88. A compound according to claim 83, characterized in that it has the general formula !! "YyLY \ = y i i / S —n HN Ά -— r I r y * T {^ 30 R2 ° I Ί I COOR1 CH3 35 wherein 1 2 R and R have the meanings specified in claim 1. A compound according to claim 88, characterized in that R 2 is 2-furyl. A compound according to claim 88, characterized in that R 2 is 2-furan-5-sulfonic acid. 9l ·. A compound according to claim 89 or 90, characterized in that it has a D configuration and that R * is sodium. A compound according to claim 89 or 90, characterized in that it has D-configuration and that R1 is potassium. 93. A compound according to claim 89 or 90, characterized in that it has a D configuration and that R 1 is hydrogen. 94. A compound according to claim 1, characterized in that it has the general formula 10. s COOR-1 · 20 wherein 1 2 A, R and R have the meanings specified in claim 1. 95. A compound according to claim 94, characterized in that R 2 is 2-furyl. 96. A compound according to claim 94, characterized in that R 2 is 2-furan-5-sulfonic acid. A compound according to any one of claims 94-96, characterized in that R * is sodium. A compound according to any one of claims 94-96, characterized in that R * is potassium. 99. A compound according to claim 94, characterized in that it has the general formula 35. N_u η% 4ν ~ Π '\ J i I i - N \ i ^^ ch2 "s- \ JJ-CH3 R2 0X j COOR1 wherein R and R have the meanings specified in claim 1. A compound according to claim 99, characterized in that R 2 is 2-furyl. A compound according to claim 99, characterized in that R 2 is 2-furan-5-sulfonic acid. 102. The compound of claim 100 or 101 is 101, characterized in that it has a D configuration and that R 1 is sodium. 103. A compound according to claim 100 or 101, characterized in that it has a D configuration and that R1 is potassium. 104. A compound according to claim 100 or 101, characterized in that it has a D configuration and that R 1 is hydrogen. 105. The sodium or potassium salt of the compound of claim 1, characterized in that it is the salt of the equimolar condensation product of a) an aldehyde which is different from formaldehyde or acetaldehyde, with b) an amphoteric 3-thiolated cephalosporin, as in The 3-membered 11-membered group has the -CHg-SR group wherein R is 1,2,3-triazol-5-yl unsubstituted or substituted with 1 or 2 alkyl groups of 1-4 carbon atoms, and which 3-thiolcrete cephalosporin contains an α-substituted α-amino-acetamido group at the 7-position and in its zwitterion form has a water solubility of less than 125 mg / ml. 106. The sodium or potassium salt of the compound of claim 1, characterized in that it is the salt of the equimolar condensation product 2 of a) an aldehyde having the general formula R -CHO wherein R is as defined in claim 1, with b) an amphoteric 3-thiolated cephalosporin, 3 which at the 3-position has the group -CH2-SR, wherein R is 1,2,3-triazol-5-35 yl unsubstituted or substituted by 1 or 2 alkyl groups having 1-4 carbon atoms and which 3-thiolated cephalosporin contains an α-substituted α-aminoacetamido group at the 7-position and in its zwitterionic form has a water solubility of less than 125 mg / ml. DK 164225 B 98 107. A compound according to claim 106, characterized in that it is 2-furarr-5-sulphonic acid. 5 10 15 20