NO135827B - - Google Patents

Description

The present invention relates to an analogue method for the production of synthetic compounds which are active as agents for the treatment of mastitis in cattle and as therapeutic agents for poultry and animals as well as humans, for the treatment of infectious diseases caused by Gram-positive bacteria and in particular Staphylococcus aureus. The method comprises the production of hitherto unknown 6-[5'-methyl-3'-phenylisothiazole-4<1->carboxamido]-penicillanic acids which may contain chlorine substituents on the benzene ring, as well as non-toxic, pharmaceutically usable salts thereof.

With the method according to the invention, it is possible to produce substances which are particularly useful for the treatment of infectious diseases caused by resistant bacterial strains, e.g. benzylpenicillin-resistant strains of Staphylococcus aureus (Micrococcus pyogenes var. aureus).

In the method according to the invention, compounds with the formula are produced

12 where R and R denote hydrogen or chlorine and salts thereof; the method is characterized in that 6-aminopenicillanic acid or a salt thereof is reacted at a temperature between -50°C and +50°C with at least one equivalent of an acid with the formula

or a reactive derivative thereof where R 1 and R 2 have the same meaning as stated above, after which the compound formed is possibly converted into a salt in a manner known per se.

The reactive derivatives listed above are well known

and includes the corresponding carboxylic acid bromides, acid anhydrides, including mixed anhydrides and especially mixed anhydrides which are prepared from stronger acids, such as lower aliphatic monoesters of carboxylic acid, alkyl or arylsulfonic acids or such acids as diphenylacetic acid. Other suitable reactive derivatives include the corresponding acid azide and the active esters or thioesters, e.g. with p-nitrophenol, 2,4-dinitrophenol, thiophenol, thioacetic acid. Alternatively, the free acid itself can be combined with 6-aminopenicillanic acid after first being reacted with N,N<1->dimethylchloroforminium chloride (cf. British patent document no. 1,008,170 and Novak and Weichet, Experientia XXI/6,

360 (1965)). 6-Aminopenicillaic acid can also be acylated with the free acid using enzymes or in the presence of an N,N'-carbonyldiimidazole or an N,N<1->carbonylditriazole (cf. South African patent preparation 63/2684), a carbodiimide reagent ( in particular N,N'-dicyclohexy 1-carbodiimide,, N,'N'-diisopropylcarbodiimide or N-cyclohexyl-N<1->(2-morpholinoethyl)-carbodiimide, cf. Sheehan and

Hess, J. Amer. Chem. Soc. 77, 1067 (1955), an alkynyl amine reagent;

cf. R. Buijle and H.G. Viehe, Angew. Chem. International Edition 3, 582 (1964), a ketenimine reagent, cf. C.L. Stevens and M.E. Monk,

J. Amer. Chem. Soc. 80, 4065 (1958) , or an isoxazolium salt reagent

cf. R.B. Woodward, R.A. Olofson, and H. Mayer, J. Amer. Chem. Soc. 83, 1010 (1961).

Another reactive derivative of the<s>acid that is useful

for the acylation of 6-aminopenicillanic acid, the corresponding azolide, i.e. an amide of the corresponding acid, whose amide nitrogen consists of

a quasi-aromatic five-membered ring containing at least two nitrogen atoms, e.g. imidazole, pyrazole", triazoles, benzimidazole, benzo-triazole and their substituted derivatives. As an example of a general

method for the preparation of an azolide, N,N'-carbonyl-diimidazole is reacted with a carboxylic acid in equimolar amounts at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inactive solvent to form a carboxylic acid-imidazolide in practically quantitative yield while releasing carbon dioxide and one mole of imidazole. Dicarboxylic acids give di-imidazolides. The by-product, imidazole, settles to the bottom and can be isolated and the imidazolide isolated, which is however not necessary. Procedures for carrying out these reactions for producing a penicillin and methods for isolating penicillins thus produced are well known.

The non-toxic pharmaceutically usable salts of penicillin obtained by the application of the present process include metal salts, such as sodium, potassium, calcium and aluminium, ammonium salts and substituted ammonium salts, e.g. salts of such non-toxic amines as trialkylamines including triethylamine, procaine, dibenzylamine, N-benzyl-3-phenethylamine, 1-ephenamine, N,N<*->dibenzylethylenediamine, dehydroabiethylamine, N,N'-bis-dehydroabiethylethylenediamine, N-lower alkyl piperidines, e.g. N-ethylpiperidine and other amines used to form salts with benzylpenicillin.

The acylation reaction can be carried out in an inactive organic solvent or in a mixture of such a solvent and water. Suitable inert organic solvents include acetone, tetrahydrofuran, dioxane, dimethylformamide, dimethylacetamide,

chlorinated aliphatic hydrocarbons, e.g. methylene chloride, dimethyl sulfoxide, methyl isobutyl ketone and dialkyl ethers of ethylene glycol or diethylene glycol. In certain cases it may be appropriate to add a solution of the acylating agent in a solvent such as benzene to a solution of a salt of 6-amino-penicillanic acid in an aqueous organic solvent, e.g. acetone-water. IN

in such cases, the reaction medium can be either single-phase or two-phase, depending on the relative proportions of water and acetone. Vigorous stirring is naturally preferable when a two-phase reaction medium is used.

Although the acylation reaction can be carried out within a rather wide reaction interval of -50°C to +50°C, it is however preferred to keep the reaction temperature between approx. -5°C and +15°C.

5-methyl-3-arylisothiazole-4-carboxylic acids used

as starting materials in the present invention, are prepared from known anilines using the following reaction core where AR - denotes the group

and R 1 and R 2 here have the same meaning as above and X 2 denotes iodine or bromine.

In another suitable method, the above-mentioned compound III is converted to the compound IV in the following way: In a further method, the compound I is first treated with BuLi and CHjI to form a and then reacted with CuCN to form the above-mentioned compound II.

In a modification of this method, benzonitrile is produced as starting material; to illustrate this, the production of 2,6-dichlorobenzonitrile can be mentioned:

It will also be possible to use the following method for the production of the desired 5-methyl-3-arylisothiazole-4-carboxylic acids:

Other methods for the production of the desired 5-methyl-3-arylisothiazole-4-carboxylic acids are those listed below, which are illustrated by the production of 5-methyl-3-phenylisothiazole-4-carboxylic acid:

Pharmacological investigations

In experiments carried out, the compound according to example 1, sodium 6-(5-methyl-3-phenylisothiazole-4-carboxamide)-penicillanate - briefly designated compound 1 - and sodium 6-[5-methyl-3-(261-dichloro -phenyl)isothiazole-4-carboxamide]-penicillanate - briefly designated compound 2 - compared to three isoxazolylpenicillins, oxacillin, cloxacillin and dicloxacillin which are known from the state of the art to be highly effective. The inhibitory effect (MIC) and the curative dose of CD^Q against a number of microorganisms were investigated. The results are tabulated below. The values in parentheses were obtained in another series of experiments.

Furthermore, the acid stability of compounds 1 and 2 in comparison with oxacillin and dicloxacillin was examined. The results of these investigations are listed below: Acid stability

The toxicological examination gave the following values for the above-mentioned compounds:

In the following, the invention will be further explained by examples where all melting points are uncorrected and all temperatures are stated in °C.

Example 1

Sodium 6-(5- methyl- 3- phenylisothiazole- 4- carboxamido)- penicillanate

A solution of 3.2 g (0.0136 mol) of 5-methyl-3-phenylisothiazole-4-carbonyl chloride in 5 ml of methylene chloride was added over 2 minutes to a rapidly stirred solution of 3 g (0.0138 mol) 6 -aminopenicillanic acid and 3.0 g (0.03 mol) triethylamine in 50 ml methylene chloride at 5-10°C. The reaction mixture was stirred for 1 hour at 15°C and extracted with water in portions of 50 ml. The combined aqueous extracts were washed with ether in portions of 50 ml, after which 100 ml of ethyl acetate was added and the pH value was

in

set to 2 with 10% hydrochloric acid. The ethyl acetate layer was isolated and the aqueous layer re-extracted with ethyl acetate in portions of 100 ml.

The combined ethyl acetate extracts were washed with 50 ml of water,

dried with sodium sulfate, filtered (an additional 50 mL of ethyl acetate was used to wash out the flask and filter) and treated with 4 mL of 39% sodium 24-ethyl hexanoate in methyl isobutyl ketone.

The clear solution was evaporated to a volume of approx. 200 ml, and

the isolated fine needles of sodium 6-(5-methyl-3-phenylisothiazole-4-carboxamido)-penicillanate (yield A) were collected by filtration.

Another yield (yield B) was obtained from the filtrate by addition of dry ether. Yield: A 197 g (33%), B 0.38 g (6%), melting point: A 184-190°C (decomposition), B 18O-190°C (decomposition) Xmax 266'5 mV (e 11* 200),<v>"|<0>p1780, 1665, 1615, 1540, 1410,

1330 cm<*1.>

Analysis calculated for C^gH^gNgO^E^Na H^O:

C: 49.88 H: 4.11 N: 9.19

Found: C: 50.31, 49.69 H: 4.91, 4.61 N: 9.02, 9.23.

This compound showed in vitro minimal inhibitory concentrations of 0.4 - 0.8 mcg/ml against Staphylococcus aureus Smith and at concentrations of 0.8 mcg/ml against benzylpenicillin-resistant Staphylococcus aureus BX 1633-2, and in mice against Staphylococcus aureus BX-1633-2 the compound showed a CD^Q of approx. 1.56 mg/kg by intramuscular administration. This compound was likewise very stable in aqueous acid with a half-life of 4 hours at a pH value of 2.0.

Example 2

Sodium 6-(3-p-chlorophenyl-5-methylisothiazole-4-carboxamido)-penicillanate

A solution of 620 mg (2.28 mmol) 3- p-chlorophenyl-5-methylisothiazole-4-carbonyl chloride in 20 ml of dry acetone at 0-2°C. The reaction mixture was stirred for 15 minutes at 10-15°C and washed out twice with ether in portions of 100 ml, after which 100 ml of ethyl acetate was added and the pH of the mixture was adjusted to 2 with dilute hydrochloric acid at 2°C while stirring. The organic layer was isolated and the aqueous layer extracted twice with ethyl acetate in 50 ml portions. The combined ethyl acetate extracts were washed twice with water in portions of 50 ml and dried with anhydrous sodium sulfate. The filtrate was concentrated to approx. 50 ml under reduced pressure at 30°C. The concentrate was treated with 1.2 ml of 39% sodium 2-ethylhexanoate. When treating the flask with a spatula for approx. After 30 minutes, the substance began to crystallize. The crystals of sodium 6-(3-p-chlorophenyl-5-methylisothiazole-4-carboxamido)-penicillanate were collected by filtration, washed with ethyl acetate and dried in vacuo over phosphorus pentoxide. The yield was 870 mg (80%). Melting point 192-197°C (during decomposition). ^Jåks 268 million

(e 14,400). v£jj£s<1>770, 1635, 1605, 1405 cm"1.

Analysis calculated for C19H17ClN304S2Na 2 3 H20:

C: 45.56 H: 4.02 N: 8.39

Found: C: 45.64, 45.68 H: 4.02, 4.19 N: 8.88, 8.73

This penicillin had in vitro minimal inhibitory concentrations of approx. 0.125 mcg/ml against Staphylococcus aureus Smith and of approx. 0.4 mcg/ml against benzylpenicillin-resistant Staphylococcus aureus BX-1633-2, and in mice the penicillin had a CD^Q value of approx. 18 mg/kg against both Staphylococcus aureus Smith and Staphylococcus aureus BX-1633-2.

This penicillin was also very stable in aqueous acid with a half-life of 4.3 hours at a pH value of 2 and at 37°C

Example 3

Sodium 6-(3-o-chlorophenyl-5-methylisothiazole-4-carboxamido)-penicillanate

A solution of 2.55 g (0 .0094 mol)

3-o-chlorophenyl-5-methylisothiazoleT4-carbonyl chloride in 30 ml of dry acetone at 5°C, and stirring was continued for 30 minutes at 10-15°C. The reaction mixture was washed out twice with ether in portions of 100 ml, after which 100 ml of ethyl acetate was added and the pH value was adjusted to 2 with dilute hydrochloric acid at 5°C. The ethyl acetate layer was isolated, while the aqueous layer was extracted with two 50 ml portions of ethyl acetate. The combined ethyl acetate extracts were washed with water, dried with anhydrous sodium sulfate and evaporated to 3/4 volume. The concentrate was then treated with 35% sodium 2-ethyl hexanoate to form a crystalline precipitate of sodium 6-(3-o-chlorophenyl-5-methylisothiazole-4-carboxamido)-penicillanate, which was collected; by filtering. When washing out the precipitate with acetone, it showed a tendency to dissolve in the solvent. The remaining precipitate "B" (1.3 g) was washed out with ethyl acetate. To the acetone solution, containing part of the sample, 5 parts by volume of ethyl acetate were added. The resulting solution was slightly concentrated to form 1.6 g of sodium 6-(3-b-chlorophenyl-5-methylisothiazole-4-carboxamido)-penicillanate crystals, designated "A". The crystal forms "A" and "B" were mutually different with respect to the infrared spectrum, but almost identical with respect to the microbiological effects. "B" was likewise recrystallized with acetone and ethyl acetate to give 0.6 g of crystals, which had an infrared spectrum identical to "A". The total yield was 2.2 g (50%).

"B": melting point 182-188°C (decomposition)vjjj£s>1760, 1650, 1595,

1530, 1405 cm"<1>. A JJ|° 258 mp (e9,100).

"A": melting point 182-188°C (decomposition) .v *®£s _ 3530, 3370, 1765,

1650, 1600, 1510, 1480, 1410 cm"<1>. X JJ|£S # 257.5 my (e9,400).

Analysis calculated for C19H17ClN304S2Na.2 3 H20:

C: 45.55 H: 4.02 N: 8.39

Found: C: 45.31, 45.66 H: 3.83, 4.16 N: 8.45, 8.95

The crystalline form "A" for this penicillin had

in vitro minimal inhibitory concentrations of approx. 0.4 mcg/ml against Staphylococcus aureus Smith and approx. 0.4 mcg/ml against benzylpenicillin-resistant Staphylococcus aureus BX-1633-2, and in mice the penicillin showed a CD^Q value of approx. 72 mg/kg against Staphylococcus aureus Smith and approx. 200 mg/kg against Staphylococcus aureus BX-1633-2. This penicillin was likewise very stable in aqueous acid with a half-life of 3.3-3.7 hours at a pH value of 2 and a temperature of 37°C.

Example 4

Sodium 6-[3-(2,4-dichlorophenyl)-5-methylisothiazole-4-carboxamido]-penicillanate

A solution of 1.1 g (0.0036 mol) 3-(2,4-dichlorophenyl)-5-methylisothiazole-4-carbonyl chloride in 15 ml of dry acetone at 5°C. The reaction mixture was stirred for 1 hour at 5°C. washed out with two portions of ether of 100 ml, 100 ml of ethyl acetate was added and adjusted to a pH value of 2.0 with 10% hydrochloric acid at 5°C. The ethyl acetate layer was isolated and the aqueous layer extracted with two 40 ml portions of ethyl acetate. The combined ethyl acetate extracts were washed with water in two 20 ml portions and dried with anhydrous sodium sulfate. The filtrate was evaporated to 50 ml under 40°C and under reduced pressure. The concentrate was treated with 1.7 ml of 35% sodium 2-ethylhexanoate and then with 100 ml of n-hexane to produce the product sodium 6-[3-(2,4-dichlorophenyl)-5-methylisothiazole-4- carboxamido]-penicillanate, which was collected by filtration and dried in vacuo over phosphorus pentoxide. The yield was 1.64 g (89%) with a melting point of 170-175°C during decomposition, £®£ s 1765, 1640, 1600, 1400 cm"<1>. X Jj2°s258 my (e 9,400).

Analysis calculated for C^gH^gCl2N304S2Na:

C: 44.89 H: 3.17 N: 8.27

Found: C: 46.14, 46.28 H: 4.06, 4.14 N: 7.23, 7.02

This product showed in vitro minimal inhibitory concentrations of approx. 0.4 mcg/ml against Staphylococcus aureus Smith and approx. 0.8 mcg/ml against benzylpenicillin-resistant Staphylococcus aureus BX-1633-2, and in mice by intramuscular administration a CD^ value of approx. 12 mg/kg against Staphylococcus aureus Smith and of approx. 50 mg/kg against Staphylococcus aureus BX-1633-2. This penicillin was likewise very stable in aqueous acid with a half-life of approx. 4.2 hours at a pH value of 2 and at 37°C.

Example 5

Sodium- 6-( 3- m- chlorophenyl- 5- methylisothiazole- 4- carboxamido)- penicillanate

There, 3-m-chlorophenyl-5-methylisothiazole-4-carboxylic acid with a melting point of 147-148°C was produced, which was converted to acid chloride with a boiling point of 137-139°C at a pressure of 0.6 mm; this substance was reacted with 6-amino-penicillanic acid to form sodium 6-(3-m-chlorophenyl-5-methylisothiazole-4-carboxamido)-penicillanate, which melted at 213-218°C with decomposition.

Analysis calculated for C19H17ClN304S2Na.l 1/2 H2<0:>

C: 45.55 H: 4.25 N: 8.39

Found: C: 45.37, 45.47 H: 4.33, 4.22 N: 8.46, 8.66.

This product showed in vitro minimal inhibitory concentrations of approx. 0.2-0.4 mcg/ml against Staphylococcus aureus Smith and of approx. 0.8 mcg/ml against benzylpenicillin-resistant Staphylococcus aureus BX-1633-2 and was very stable in aqueous acid with a half-life of approx. 4.9 hours at a pH value of 2 and at 37°C.

Example 6

Potassium- 6-( 5- methyl- 3- phenylisothiazole- 4- carboxamido)- penicillanate

A solution of 5-methyl-3-phenylisothiazole-4-carboxylic acid (0.01 mol) in a mixture of 20 ml of dimethylformamide and 35 ml of methylene chloride was cooled to -5°C. Ethyl chloroformate (0.01 mol) was added to this solution, whereby the temperature rose to -1°C. The reaction mixture was cooled and stirred for 30 minutes at -5°C. A solution of 6-aminopenicillanic acid (0.01 mol) in 25 ml of methylene chloride and triethylamine (0.02 mol) was added portionwise to the first reaction mixture at -2°C. The temperature rose to 0°C, and carbon dioxide evolution occurred for several minutes. The reaction mixture was stirred for 40 minutes at -5°C. The methylene chloride was removed by distillation under reduced pressure at 35°C, and the resulting mixture was diluted to 200 ml with dry ether and then filtered. Potassium 2-ethyl hexanoate (2 g) was dissolved in a small amount of ethyl acetate and added to the filtrate, whereupon a white, crystalline precipitate formed. This precipitate was collected by filtration, dissolved in 50 ml of water and acidified to a pH value of 1.8 with sulfuric acid. The aqueous solution was extracted with ether in 2 ml portions. The aqueous solution was then adjusted to a pH of 4.0 with sodium bicarbonate, filtered and extracted with four 20 ml portions of cold ethyl acetate. The ethyl acetate extract was dried over anhydrous sodium sulfate, and potassium 2-ethylhexanoate was added to precipitate the product. The product, the potassium salt of 6-(5-methyl-3-phenylisothiazole-4-carboxamido)-penicillanic acid, was collected by filtration and dried over P2°5°9 was found to melt at 180-190°C with decomposition.

Claims (1)

Analogous method for the production of hitherto unknown, therapeutically effective penicillins with the formula: where R 1 and R 2 denote hydrogen or chlorine, as well as salts thereof, characterized in that 6-aminopenicillanic acid or a salt thereof is reacted at a temperature between -50°C and +50°C with at least one equivalent of an acid with the formula: 1 2 or a reactive derivative thereof where R and R have the same meaning as stated above, after which the compound formed is optionally converted into a salt in a manner known per se.