NO159797B - ANALOGY PROCEDURE FOR THE PREPARATION OF NEW THERAPEUTIC ACTIVE CEPHALOSPORINALS. - Google Patents

Abstract

Compounds of the general formula <IMAGE> [wherein X represents the 1,2,5,6- tetrahydro-2-methyl-5,6-dioxo-as- triazin-3-yl group, the 2,5-dihydro-6- hydroxy-2-methyl-5-oxo-as-triazin-3- yl group of the 1,4,5,6-tetrahydro-4- methyl-5,6-dioxo-as-triazin-3-yl group] and readily hydrolysable esters, readily hydrolysable ethers and salts thereof and hydrates of these compounds have broad-spectrum anti-bacterial activity.

Description

The present invention relates to an analog method

in the preparation of therapeutically active cephalosporin salts of general formula I

Examples of salts with formula I are alkali metal salts, such as sodium and potassium salts, ammonium salts, alkaline earth metal

salts such as calcium salts, salts with organic bases such as salts with amines, e.g. salts with N-ethyl-piperidine,

procaine, dibenzylamine, N,N'-dibenzylethylethylenediamine, alkylamines or dialkylamines, as well as salts with amino acids, such as e.g. salts with arginine or lysine. As can be seen from formula I, the salts are disalts, as the second salt formation occurs in connection with the hydroxy group on the 2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl group.

The salts of formula I are hydrated, whereby salt hydrates are formed. Hydration can occur during manufacture or with time as a result of the hydroscopic properties of the initially anhydrous product.

As can be seen from formula I, the cephalosporin salts are present

which is produced according to the invention in the syn-isomeric form:

A preferred product is (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-(Z-methoxyimino)-acetamido]-3-/[2,5-dihydro-6-hydroxy- 2-Methyl-5-oxo-as-triazin-3-yl)thio]-methyl/-8-oxo-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid disodium salt -trihydrate. The cephalosporin salts are produced according to the invention by employing a compound of general formula

wherein X is the 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-as-triazin-3-yl group, respectively its corresponding tautomeric form, 2,5-dihydro-6-hydroxy-2- the methyl-5-oxo-as-triazin-3-yl group

in the syn-isomeric form with at least two equivalents of a base and isolates the formed salt in the presence of water or atmospheric moisture as a salt hydrate.

The starting compound with formula II can again be prepared by using a compound with general formula III

wherein X has the above meaning,

R means a cleavable protecting group and the carboxyl group can be present in protected form,

in the syn-isomeric form clefts of the protecting group R, and optionally also a possibly present carboxy protecting group. This method is the subject of the divided patent application 86.5360.

The carboxyl groups present in the compound of formula III may optionally be protected, e.g. by esterification with an easily cleavable ester, such as the silyl ester, e.g. the trimethyl silyl ester. The above-mentioned easily hydrolyzable esters also come into consideration. The carboxy group can also be protected by salt formation with an inorganic or tertiary organic base, such as triethylamine. Possible R protecting groups are e.g. acidic hydrolytically cleavable protective groups, such as e.g. t-butoxycarbonyl or trityl, or also basic hydrolytically cleavable protecting groups, such as e.g. trifluoroacetyl. Preferred R protecting groups are chloro-, bromo- and iodoacetyl, especially chloroacetyl. The latter protective groups can be removed by treatment with thiourea.

The compounds of formula III can e.g. is produced by N-acylation of the corresponding 7-amino compounds by reacting a compound of general formula IV

in which X has the meaning stated above and the carboxy group and/or the amino group can be present in protected form,

with an acid of general formula

in which R has the above meaning,

in the syn-isomeric form or with a reactive functional derivative of this acid and cleaves off a possibly present carboxy protecting group.

The carboxy group present in the 7-amino compound of formula IV may optionally be protected in the same way as described above for the preparable starting compound of formula III. The amino group of the compound of formula IV can e.g. be protected with a silyl protecting group such as trimethylsilyl.

As reactive functional derivatives of acids with formula V, e.g. halides, i.e. chlorides, bromides and fluorides, azides, anhydrides, especially mixed anhydrides with stronger acids, reactive esters, e.g. N-hydroxy-succinimide esters, and amides, e.g. imidazolides, in consideration.

The reaction of the 7-amino compound of formula IV with the acid of formula V or with a reactive functional derivative thereof can be carried out in a manner known per se. This is how you can e.g. condensing a free acid of formula V with one of the aforementioned esters corresponding to formula IV by means of a carbodiimide, such as dicyclohexylcarbodiimide in an inert solvent such as ethyl acetate, acetonitrile, dioxane, chloroform, methylene chloride, benzene or dimethylformamide, and then cleaving off the ester group. Instead of kårbo-diimides, oxazolium salts can also be used as condensation agents, such as e.g. N-ethyl-5-phenyl-isoxazolium-3'-sulfonate.

According to another embodiment, a salt of an acid with formula IV is employed, e.g. a trialkylammonium salt such as the triethylammonium salt, with a reactive functional derivative of an acid of formula V as mentioned above in an inert solvent, e.g. one of the above mentioned.

According to another embodiment, an acid halide, preferably the chloride of an acid of formula V is reacted with the amine of formula IV. The reaction preferably takes place in the presence of an acid-binding agent, e.g. in the presence of aqueous alkali, preferably caustic soda, or also in the presence of an alkali metal carbonate, such as potassium carbonate, or in the presence of a lower alkylated amine, such as triethylamine. The solvent used is preferably water, possibly in a mixture with an inert organic solvent, such as tetrahydrofuran or dioxane. It can also be worked in an aprotic organic solvent such as e.g. dimethylformamide, dimethyl sulfoxide or hexamethylene phosphoric acid triamide. When using silylated starting compounds of formula IV, work is carried out in an alcohol-free medium.

The reaction of the 7-amino compound of formula IV with the acid of formula V or a reactive functional derivative thereof can conveniently be carried out at temperatures between approx.

-40°C and room temperature, for example at approx. 0-10°C.

For the preparation of the starting compound of formula II, as mentioned above, the amino protecting group R in a starting compound of formula III is cleaved off. Protective groups that can be split off by acid hydrolysis are preferably removed using a lower alkane carboxylic acid, which may optionally be halogenated. In particular, formic acid or trifluoroacetic acid is used. The temperature is usually room temperature, although slightly elevated or slightly lowered temperatures can also be used, e.g. in the area from approx. 0°C - +40°C. Alkaline-cleavable protective groups are generally hydrolysed with dilute aqueous lye at 0°C - 30°C. Chloroacetyl, bromoacetyl and iodoacetyl protecting groups can be removed with the help of tlourea in an acidic, neutral or alkaline medium at approx. 0-30°C. Hydrogenolytic cleavage (e.g. cleavage of benzyl) is unsuitable here, as the oxime function is reduced to an amino group by hydrogenolysis.

After cleavage of the amino protecting group R in a starting compound of formula III, a carboxy protecting group possibly present in the reaction product can, if desired, be cleaved off. When the protecting group forms a silyl group (silyl ester), this group can be cleaved particularly easily by treating the reaction product with water. Lower alkanoyloxyalkyl, alkoxycarbonyloxyalkyl, lactonyl, alkoxymethyl and alkanoylaminomethyl esters are preferably cleaved enzymatically with the aid of a suitable esterase (at approx. 20-40°C). When the carboxyl group has been protected by salt formation (e.g. with triethylamine, the splitting off of the salt-forming protecting group can take place by treatment with acid. As an acid, e.g. hydrochloric acid, sulfuric acid, phosphoric acid or citric acid can be used.

The carboxy protecting group can be removed in the same way as just described before the removal of the protecting group

R.

The preparation according to the invention of the cephalosporin salts of formula I takes place in a manner known per se by reacting the carboxylic acid of formula II with at least two equivalents of the desired base, suitably in a solvent such as water in an organic solvent such as ethanol, methanol, acetone or others. When two equivalents of base are used, salt formation also takes place on the tautomeric enol form (2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-treiazin-3-yl-group X), whereby a disalt is formed . The temperature is not important, it is usually at room temperature, but can also be slightly above or below, approximately in the range from 0°C - +50°C.

Formed salts of formula I can of course be transferred in the usual way into other salts of formula I with other meanings of M (see the following example).

The production of the hydrates usually occurs automatically during production or as a result of the hygroscopic properties of an initially anhydrous product. For the targeted production of a hydrate, a completely or partially anhydrous product (carboxylic acid with formula II, or salt with formula I) can be exposed to a moist atmosphere, e.g. at approx. +10°C - +40°C.

The 7-amino compounds of formula IV used above can be prepared starting from a compound of formula VIII

in which Y forms a leaving group with a thiol of general formula VII

in which X has the above meaning.

As leaving group Y in a compound of formula VIII, for example, the halogens, e.g. chlorine, bromine or iodine, acyloxy groups, e.g. lower alkanoyloxy groups such as acetoxy, lower alkyl or arylsulfonyloxy groups such as mesyloxy or tosyloxy or the azido group in question.

The reaction of the compound of formula VIII with the thiol of formula VII can take place in a manner known per se, e.g. at a temperature of between approx. 40 - 80°C, suitable at approx. 60°C, in water or a buffer solution with a pH of approx. 6-7, preferably 6.5.

The cephalosporin salts produced according to the present invention are antibiotically, particularly bactericidally active. They possess a rich spectrum of activity against Gram-positive and Gram-negative microorganisms, which include ε-lactamase-producing staphylococci and various 3-lactamase-producing Gram-negative bacteria, such as e.g. pseudomonas aeruginosa, haemophilus influenzae, escherichia coli, serratia marcescens, proteus and klebsiella species.

The cephalosporin salts produced according to the present invention can be used for the treatment and prophylaxis of infectious diseases. For an adult, a daily dose of approx. 0.lg - 2g in consideration. The parenteral administration of the compounds produced according to the invention is particularly preferred.

To demonstrate the antimicrobial activity of the aforementioned products, the following representative was tested: Product A: (6R,7R)-7-[2-(2-amino-4-thiazolyl9-2-(Z- methoxyimino)acetamido]-3-/[2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl)-thio]-methyl/-8-oxo-5-thia- 1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid disodium salt. 3.5 H 2 O. ("trihydrate")

Activity in vivo

Groups of 5 mice are infected with an aqueous suspension of Escherichia coli intraperitoneally. Three times, i.e. 1 hour, 2 1/2 hours and 4 hours after the infection, the test substance is applied in physiological saline solution. The number of surviving animals is determined on the 4th day. Different dosages are applied and by interpolation the dose at which 50% of the test animals survive is determined (CD50, mg/kg).

The products produced according to the invention can be used as medicines, e.g. in the form of pharmaceutical preparations, which contain them or their salts in admixture in a pharmaceutical, organic or inorganic inert carrier material suitable for enteral or parenteral administration, such as e.g. water, gelatin, gum arabic, milk sugar, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols, vaseline, etc. The pharmaceutical preparations can be in solid form, e.g. as tablets, dragées, suppositories, capsules or in liquid form, e.g. as solutions, suspensions or emulsions. If necessary, they are sterilized and/or contain auxiliary substances such as preservatives, stabilisers, cross-linking or emulsifying agents, salts for changing the osmotic pressure, anesthetics or puffs. They may also contain other therapeutically valuable substances. They are preferably considered for parenteral administration and for this purpose they are preferably prepared as lyophilisates or dry powders for dilution with common agents such as water or isotonic saline solutions.

Example

Preparation of the disodium salt "trihydrate" of (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-(Z-methoxyimino)acetamido]-3-/

[(2,5-dihydro^6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl)thio]-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct -2-ene-2-carboxylic acid

6.0 g (6R,7R)-7-[2-(2-mino-4-thiazolyl)-2-(-methoxyimino)acetamido]-3-/[(2,5-dihydro-6-hydroxy-2 -methyl-5-oxo-as-triazin-3-yl)thio]-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid is suspended in a mixture of 40 ml of acetone and 40 ml of water and reacted with 20 ml of a 2-n solution of 2-ethylcaproic acid sodium salt in acetic ester. 50 ml of acetone is added to the resulting orange-coloured solution, whereby a brown resin precipitates, which is separated by means of filtration. The yellow filtrate is stirred for 30 minutes, whereby the disodium salt crystallizes. Add 50 ml of acetone in portions to the mixture and store overnight in a refrigerator. The crystallisate is filtered off with suction and washed successively with an acetone-water mixture 85:15, pure acetone and low-boiling petroleum ether and dried overnight in a vacuum at 40°C. The title substance is obtained as beige crystals. [a]^<0> = -144° (c = 0.5 in water). The nuclear resonance spectrum and the microanalysis correspond to the listed structure. The formed disodium salt contains 3.5 mol of hydrated water per mole of substance and is then referred to as "trihydrate".

The starting product used (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-(Z-methoxyimino)acetamido]-3/[(2,5-dihydro-6-hydroxy-2 -methyl-5-oxo-as-triazin-3-yl)-thio)methyl/-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid can be prepared as follows : 22.24 g of 2-(2-chloroacetamido-thiazol-4-yl)-2-(Z-methoxyimino)-acetic acid are suspended in 240 ml of methylene chloride. 13.39 ml of triethylamine is added to this suspension, whereby a light brown solution is formed. This solution is cooled to 0-5°C and 16.72 g of phosphorus pentachloride is added, stirred for 5 minutes at 0-5°C and 20 minutes without cooling. The yellow solution is evaporated in vacuo at 35°C. The residue that remains after evaporation is shaken twice with n-heptane and the latter is decanted off. The resinous residue is treated with 240 ml of tetrahydrofuran and the undissolved triethylamine hydrochloride is filtered off. The yellow filtrate contains the acid chloride. 22 g of (7R)-7-amino-3-desacetoxy-3-[(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl)-thio]-cephalosporanic acid are suspended in a mixture of 300 ml of water and 300 ml of tetrahydrofuran. 2-n caustic soda is added dropwise to the suspension under a vigorous nitrogen supply using an autotitrator, until a brownish-red solution with pH 8 is formed. This is cooled to 0-5°C and added dropwise over the course of 15 minutes to the above-prepared solution of the acid chloride in tetrahydrofuran. It is then stirred for 1 2 1/2 hours at 25°C. The pH of the acylation mixture is kept constant at 8 during the addition of 2-n caustic soda. The practically black solution is liberated under vacuum at 40° C. from tetrahydrofuran. Then 100 ml of 2-n sulfuric acid is added. The thereby precipitated substance is sucked out, washed with water and sucked out vigorously. The moist, brown filter mass is dissolved in 1 1.5 1 acetone. The dark solution is filtered over "Hyflo" from some darkest undissolved material, carbon is added and stirred for 30 minutes, and filtered again over "Hyflo". The orange-red filtrate is dried over sodium sulfate, evaporated in vacuo and evaporated with vinegar. This precipitates a black resin, which is filtered off and removed. The 2-phase filtrate, which still contains water, is azeotropically distilled three times with benzene in vacuum at 40°C. The thereby precipitated substance is filtered off with suction and dried in a vacuum at 40°C. The latter is stirred twice with respectively 1 1 acetone, whereby a brown resin remains, which is discarded. The combined orange colored acetone extracts are evaporated in vacuum at 40°C to approx. 150 ml, whereby a brown resin is filtered off and discarded. The filtrate is added to 1 1 of acetic acid and evaporated in vacuum at 40°C. The thereby precipitated substance is filtered off with suction, washed with vinegar and then with ether. [(6R,7R)-7-/2-[2-(2-chloro-acetamido)-4-thiazolyl]-2-(Z-methoxyimino)acetamido/-3-/

[(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-az-trlazin-3-yl)tlo]-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct -2-ene-2-carboxylic acid, fraction I: This fraction I can then be used directly for the production of the desired end product.

The acetic ester mother liquor is strongly evaporated in vacuo at 40°C, diluted with ether and the precipitated substance filtered off with suction [(6R,7R)-7-(2-[2-(2-chloroacetamido)-4-thiazolyl]-2-(Z-methoxyimino) )acetamido/-3-/[(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl-)thio]methyl-8-oxo-5-thia-1- azablcyklo [4.2.0]oct-2-ene-2-carboxylic acid, fraction II: a light beige, amorphous acid], thin-layer chromatographically somewhat purer than fraction

IN].

To prepare the disodium salt, dissolve 3.5 g of acid (fraction II). 1 a mixture of 20 ml of acetone and 11 ml of water. To the solution is added 7 ml of a 2-n solution of 2-ethylcaproic acid sodium salt in acetic ester, whereby the disodium salt crystallizes. Now add 25 ml of acetone in portions and store the mixture for 2 hours in a deep freezer. The crystallisate is then filtered off with suction, washed successively with 25 ml of an ice-cold acetone-water mixture (80:20), pure acetone and low-boiling petroleum ether and dried overnight in a high vacuum at 40 °C. The disodium salt of (6R,7R)-7-/2-[2-(2-chloroacetamido)-4-thiazolyl]-2-(Z-methoxyimino)-acetamido/-3-/[(2,5-dihydro -6-hydroxy-2-methyl-5-oxo-az-triazin-3-yl)thio]-methyl/-8-oxo-5-tla-1-azabicyclo[4.2.0]-oct-2-ene- 2-carboxylic acid as pale yellow crystals. Ccx]<2>^0 = -142.7° (c = 1 in water). The nuclear resonance spectrum and the microanalysis correspond to the listed structure.

15.3 g (6R,7R)-7-(2-[2-(2-chloroacetamido)'-4-thiazolyl]-2-(Z-methoxyimino)acetamido/-3-/[(2,5-dlhydro -6-hydroxy-2-methyl-5-oxo-az-triazin-3-yl)-thio]methyl/-8-oxo-5-thia-1-azabi-cyclo[4.2.0]oct-2-ene -2-carboxylic acid (fraction I, see below) is suspended together with 5 g of thiourea in 150 ml of water. During vigorous nitrogen addition and stirring, the pH is adjusted with saturated sodium hydrogen carbonate solution to 6.8-7.0, whereby an orange colored solution is produced. By means of an autofilter while adding sodium hydrogen carbonate solution, the pH of the reaction solution is kept constant at 6.8 during 6 hours. A further 2.5 g of thiourea is then added and the solution is stirred for a further 13 hours, whereby the pH is kept at 6.8 by the addition of saturated sodium bicarbonate solution. The red solution is then stored overnight in a refrigerator, whereby it darkens. The pH of this solution is adjusted by adding 100 µg of formic acid to 2.0-2.5, whereby the substance precipitates out. This suction is drained, washed with 100 ml of 10# formic acid. The mother liquor is discarded. The brown filter mass is suspended in 200 ml of water and the pH is adjusted with triethylamine, whereby a brown solution is formed. This solution is stirred for 30 minutes with 2 g of activated charcoal, filtered from the charcoal, and the still brown filtrate is adjusted to pH 3.5 with 100 µg mmauric acid with vigorous stirring. The thus precipitated substance is filtered off with suction, washed with 50 ml of 10% formic acid and discarded. The dark yellow filtrate is adjusted to pH 2-2.5 with 100#-Ig of formic acid, whereby the substance precipitates out. This is suctioned, washed with ice water and dried. One obtains (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-(Z-meifesymino)-acetamido]-3-/[(2,5-dihydro-6-hydroxy -2-methyl-5-oxo-as-triazln-3-yl)thio]-methyl-8-oxo-5-thia-1-azablocyclo[4.2.0]

oct-2-ene-2-carboxylic acid, which is reacted further without further purification.

Claims (2)

1. Analogous process for the preparation of a therapeutically active cephalosporin salt of the general formula I wherein M means the cation from a pharmaceutically acceptable base in hydrated form, as well as in the syn form, characterized by reacting a compound of the general formula II in which X means the group 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-as-triazin-3-yl, respectively the group 2,5-dihydro-6-hydroxy-2-methyl-5- oxo-as-triazin-3-yl, in the synisomeric form, with at least two equivalents of a base, and isolates the formed salt in the presence of water or of atmospheric moisture as a salt hydrate. 2. Process according to claim 1 for the preparation of (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-(Z-methoxyimino^acetamido]-3- J_[(2 ,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl)thio]-methyl/-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct- 2-ene-2-carboxylic acid disodium salt trihydrate, characterized in that a base is used which emits sodium ions and the salt formed is isolated as an approximate trihydrate.