NL8601842A - CEPH-3-EM-CARBONIC ACID DERIVATIVES, PROCESS FOR THE PREPARATION THEREOF, CONTAINING PHARMACEUTICAL PREPARATIONS THEREOF, PROCESS FOR THE PREPARATION OF THE latter, AND PROCESS FOR THE TREATMENT OF PATIENTS LIKING THOSE LIKE THOSE LIKE THIS. - Google Patents

Description

% NL 33,554 dJ / vdM ^ - 1 -

Ceph-3-em-carboxylic acid derivatives, method for the preparation thereof, pharmaceutical compositions containing such compounds, method for the preparation of the latter, and method for the treatment of patients suffering from bacterial diseases.

The present invention relates to a new ceph-3-em-4-carboxylic acid derivative, a process for the preparation thereof, pharmaceutical preparations containing such a compound as active ingredient, a process for the preparation of such pharmaceutical preparations, and a method of treating patients suffering from bacterial diseases.

A large number of experiments have been carried out in the field of the semi-synthetic β-lactam derivatives in order to prepare compounds with a broad or special antimicrobial spectrum. A significant group of these compounds includes a substituted or unsubstituted isoxazole group attached via an acetamido group to the carbon atom at position 6 of 6-amino-penicillanic acid and 15 to the carbon atom at position 7 of 7-amino-cephalosporanoic acid, respectively . Several representatives of this group of compounds exhibit significant anti-microbial activity.

Active molecules are known which have been developed into medicaments and which include a substituted isoxazol-4-yl-acetamido group at position 6 of the penicillin moiety. Molecules of this type are described in U.S. Pat. Nos. 2,996,501 and 3,239,507 and in French Pat. No. BSM No. 6432.

US Patent No. 4,394,504 describes penicillin and cephalosporin derivatives which, in addition to a 3-amino-isoxazol-5-yl-acetamido group, also have an alkoxyimino group at the α position, with respect to the isoxazole ring. The compounds disclosed in this U.S. Patent 30 have significant activity against staphylococcal strains.

8601342 *% - 2 -

German Offenlegungsschrift No. 2,155,081 describes penicillin and cephalosporin derivatives containing (3-substituted phenyl) isoxazol-5-yl acetamido groups. However, none of the actually described and tested phenyl-5 isoxazole derivatives show any significant anti-microbial activity over the test microorganisms used.

Furthermore, German Offenlegungsschrift 2,409,949 discloses isoxazol-5-yl-acetamido derivatives. However, the iso-10xazole ring of these derivatives carries other substituents in place of the phenyl substituent.

The chemotherapeutic and pharmacological effects of a portion of the compounds described in this German Offenlegungsschrift were determined. However, the tested molecules failed to have excellent antibacterial activity over the test microorganisms used.

The MIC values of some compounds have also been described. However, these values do not reach the efficacy indispensable for an active ingredient selected to be developed into a drug.

The present invention now aims to provide new ceph-3-em-4-carboxylic acid derivatives with excellent pharmacological properties.

To this end, according to one aspect of the present invention, 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl -ceph-3-em-4-carboxylic acid of formula 1 of the formula sheet, as well as pharmaceutically-logically acceptable salts thereof.

It was surprisingly found that the new 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph -3-em-4-carboxylic acid of formula 1 and its pharmacologically acceptable salts exhibit a very high and useful anti-bacterial activity. This has been confirmed by chemotherapeutic research and pharmacological tests. Although the new compound is 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl- 8601842 4 * - 3 - eeph-3-em-4-carboxylic acid falls under the general formula of German Offenlegungsschrift 2,409,949, its preparation is not described in this Offenlegungsschrift, nor are its physicochemical data and characteristic constants and MIC-5 values described.

According to a further aspect of the present invention, there is provided a process for the preparation of 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazole -5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid of formula 10 1 and its pharmacologically acceptable salts, the process characterized by a) converting 3-chloro-isoxazole-acetic acid of formula 2 of the formula sheet , or a reactive derivative thereof with 7-amino-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -15 thiomethyl-ceph-3-em-4-carboxylic acid of formula 3 from the formula sheet and isolating the thus obtained 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) - thiomethyl-ceph-3-em-4-carboxylic acid of formula 1 of the formula sheet from the reaction mixture in a manner known per se and, if desired, converting the latter compound into a pharmacologically acceptable salt thereof by known methods and isolating the salt from the reaction mixture o p a manner known per se; or b) reacting 1-methyl-1H-1,2,3,4-tetrazol-5-yl-thiol of formula 4 of the formula sheet with 7- (3-chloro-isoxazol-5-yl) -acetamido -cephalosporanic acid and isolating the 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl thus obtained -ceph-30 3-em-4-carboxylic acid of formula 1 of the formula sheet in a manner known per se and, if desired, converting the latter compound into a pharmacologically acceptable salt thereof, by known methods and isolating the salt from the reaction mixture in a manner known per se.

According to step a) of the process according to the invention, 3-chloro-isoxazole acetic acid of formula 2 or a reactive derivative thereof is reacted with 7-amino-3- (1-methyl-δ o ü ί 3 42 Τ · ν - 4 - 1Η -1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid of formula 3. The reaction can be carried out in a solvent, preferably in an aqueous acetone medium, or in a water-immiscible organic solvent. The reaction 5 is carried out with stirring and the reaction time is usually from 30 minutes to 3 hours.

According to process variant b) of the process of the present invention, 1-methyl-1H-1,2,3,4-tetrazol-5-yl-thiol of formula 4 is reacted with 7- (3-chloro-10-isoxazole-5-) yl) -acetamido-cephalosporanic acid.

The reaction is carried out in a solvent, preferably in a mixture of water and acetone, at 30-90 ° C, preferably at a temperature between 60-65 ° C, at a pH in the range of 4.5-6, 0. The reaction time is usually 15 2-10 hours *

The antibacterial activity of 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph-3- em-4-carboxylic acid is detected by the MIC values of this compound. The compound 20 iS ': tested against a number of test microorganisms and as reference compounds. use commercially available medicines. The results are summarized in Table Ά.

»

TABLE A

Test microorganism compound with Cefa-Cefu-Cefoxitin formula 1 lexin roxim

Bordetella bronchiseptica 12.5 500 500 100

Escherichia 30 coli K 12 0.5 6.2 2 1.6

Escherichia coli 6 R 25 15.6 4 1.6 E. coli poliresistens 100 500 250 100 35 Staphylococcus aureus SMITH 0.06 0.8 0.8 1.2 8601842

Sr 'w - 5 - TABLE A (continued) test microorganism compound with Cefa-Cefu-Cefoxitin formula 1 lexin roxim

Staphylococcus 5 epidermidis ATCC

1-12228 0.03 0.8 0.8 0.6 E. coli R-222 0.5 6.2 4 3.1 E. coli R-15 1.6 6.2 4 3.1

Klebsiella 10 ATCC 10,031 0.16 6.2 0.08 12.5

Proteus vulgaris XL 100 500 62.5 3.1

Pseudomonas pyocyanea NTCT

10,490 100 500 500 100 15 Salmonella typhimurium 0.5 6.2 4 3.1

Bacillus substilis ATCC 6633 0.015 0.3 0.1 0.1

Staphylococcus 20 aureus 1110 1.25 62.5 125 25

Staphylococcus aureus 53 0.25 2.5 1.6 12.5

Streptococcus faecalis 50 250 500 100 25 Steptococcus haemoliticus Pneumo 0.03 0.31 0.008 0.6

Steptococcus haemoliticus A 118 0.06 0.31 - 0.3

Steptococcus 30 haemoliticus Robb 0.03 1.25 0.003 1.2

Absorption tests have surprisingly revealed that the compound of formula 1, which can be considered a second generation cephalosporin derivative in view of the MIC values, unlike other antibiotics belonging to this group, is an absorp possesses a index of index which permits the use of this compound for the preparation of pharmaceutical preparations suitable for oral administration. The results are set forth in Table B.

ί 1 * ί λ '* ^ α v £ * λ y - 6 - ρ · ο .. EDc0 mg / kg absorption index = - ,, ... . s.c. EDca mg / kg (in mice p.o.) 50 µ y

TABLE B

absorption index 5 active ingredient ^ Proteus

Smith typhi XL

compound of formula t 20 1-2 1-2

Cefuroxime 30 07 001 4 10 Cefoxitin 7-8 0.001 /

Cefamandol 5 20 /

Cefalexin 1 1 /

Cefotaxime 0.001 0.001 0.001

Acute toxicological tests show that in the separate acute dose, which is characteristic of 8-lactams, the compound of formula 1 is non-toxic. An i.p. or p.o. dose of 2500 mg / kg body weight did not cause any death in mice.

LD ^ q - 2500 mg / kg body weight.

Pharmacokinetic tests have also been carried out to determine the absorption of the compound of formula 1. The tests were performed on mice of a CFLP strain by i.p. and p.o. administration. 20 mice were treated simultaneously with an aqueous solution of the compound of formula 1 at an appropriate concentration.

Dose: i.p .: in the case of 20 mg / kg body weight, 0.1 ml of a solution at a concentration of 4 mg / ml; p.o .: in the case of 80 mg / kg body weight, 0.4 ml of a solution with a concentration of 4 mg / ml. The average weight of the mice was 20 g. At some point, the mice were allowed to bleed to death by decapitation (blood clotting is prevented by the addition of heparin).

8601342. c κ »- 7 -

The active substance in the blood is microbiologically determined (test ant organism:

Bacillus subtilis ATCC 6633). The results are summarized in Table C.

5 TABLE C

a) i.p. administration (20 mg / kg body weight) time (hours) blood level (mg / 1) 0.15 1/23 10 0.30 -2.6 0.45 1.23 1 0 b) po administration (80 mg / kg body weight ) time (hours) blood level 15 (mg / 1) 0.15 0.35 0.30 0.70 0.45 0.58 1 0.46 20 1.5 0.22 2 0

The absorption of the compound of formula 1 has also been investigated in beagle dogs. The test compound was administered p.o. and i.m. (in the case of i.m. administration 20 mg / kg body weight, with p.o administration 80 mg / kg body weight). Bacillus subtilis ATCC 6633 was used as the test microorganism. Blood was obtained through a venous puncture. The results are shown in Table D.

BSO1 $ 42 -8-

TABLE D

a) im administration (20 mg / kg body weight) time blood level (hours) (mg / 1) 5 0.5 54.6 - 55.6 1 29.7 - 31.1 1.5 10.6 2 5.0 - 5.1 3 1.82- 1.88 10 5 0.34 b) po administration (80 mg / kg body weight) time blood level (hours) (mg / 1) 0.5 0.63 - 0.645 15 1 0.645- 0.67 1/5 3.1 2 5.7 - 5.8 3 7.5. 5 ^ 2.78 The concentration course formed in the blood after PO administration shows that the compound of formula 1 is suitable for the preparation of pharmaceutical preparations for oral administration.

The binding capacity of the compound of formula 1 to serum protein has been determined. Two methods were used for this; one of these methods is based on equilibrium dialysis / Scholten: Antibiot. Chemother. No. 12, p.

103 (1963) _7 and the other on the so-called "large plate" method / F. Kawanagh: Analytical Microbiol. Acad Press N.Y.

30 (1963) _7 · The degree of binding to serum protein of the compound of formula 1 is 50-70%. This value also contributes to its suitability for oral use.

8601842 -9-.

- τ

The in vivo activity of the compound of formula 1 has been determined on mice of a CFLP strain. In order to determine the ED ^ 0 value, the mice were injected i.p. infected with 6-8 hours of cultures of the ant organism; the dose is 5 x 10 LD100. One hour after the infection, the animals were treated with the aqueous solution of the test compound. Commercially available drugs were used as the reference compound. The results were evaluated using the Litchfield-Wilcoxon method. In accordance with the known data for 8-lactarnen characteristic data, it was found that as a result of the treatment carried out with the compound of formula 1, in the case of an infection with Staphylococcus or Proteus, the number of live animals remained virtually unchanged.

In order to demonstrate that the compound of formula 1 is more active than the compounds of German Offenlegungsschrift No. 2,409,949, the following tests were carried out in substantially the same manner as described above.

20 TABLE E

Comparison of ED ^ g values, expressed in mg / kg.

Animal: mice of the CFLP strain of both species with an average weight of 20 g.

Bacteria used for infection: see table.

Infection method: i.p., in 2.5% mucin.

effective treatment Staphylococcus aureus SMITH

constituent mode ED50 ^ 30 compound of 0.23 formula 1 s'c * (0.06 - 0.8} po 4.3 (0.6 - 28.0) compound H * sc (15.9'-38, 2} 35 compound F ** po (40If'3 142.7) 860.1 342-10 * sodium 7- / (3-chloroisoxazol-5-yl) acetamido / cephalosporanate.

** sodium-7- / ~ (3-chloroisoxazol-5-yl) acetamido7-3- / (5-methyl-1,3,4-thiadiazol-2-yl) thiomethyl7-ceph-3-em-4-5 carboxylate.

As a further aspect of the present invention, there are provided pharmaceutical compositions which, as active ingredient, comprise the compound 7- (3-chloro-isoxazol-5-yl) -acetamido-5- (1-methyl-1H-1,2,3 4-Tetrazol-5-yl) -thio-10 methyl-ceph-3-em-4-carboxylic acid of formula 1, or containing a pharmacologically acceptable salt thereof in admixture with suitable inert solid or liquid pharmaceutical carriers.

The above-mentioned pharmaceutical preparations 15 can be prepared by methods which are known per se in the pharmaceutical industry.

Further details of the present invention can be found in the following examples, without limiting the scope of the present invention to these examples.

EXAMPLE I

Preparation of 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph-3-em -4-carboxylic acid and its sodium salt.

4.99 g (30.9 mmol) of 3-Chloro-isoxazol-5-yl-acetic acid and 7.07 g (33.99 mmol) of phosphorus pentachloride were dissolved in 120 ml of anhydrous benzene with continuous stirring. The reaction mixture was left overnight at room temperature and then at 60 ° C for one hour.

The benzene was distilled off, the residue was worked up several times with anhydrous petroleum ether, decanted and evaporated until it was free from petroleum ether. The residue was dissolved in 40 ml of anhydrous acetone and the solution thus obtained was added at 0-5 ° C with continuous stirring for 50 min to a solution of 6.75 g (20.6 mmol) 7-amino-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl - 8601342 i - 11 - thiomethyl-ceph-3-em-4-carboxylic acid, 5.2 g (61.8 mmol) sodium bicarbonate , 144 ml of water and 103 ml of acetone. After the addition was complete, the reaction mixture was stirred at 0-5 ° C for one hour and at room temperature for 2 hours, while the pH was adjusted to 7-8 by addition of a saturated sodium bicarbonate solution. The mixture was acidified to a pH value of 1.5-2 by addition of dilute sulfuric acid and then extracted three times with 100 ml of ethyl acetate each time. The ethyl acetate extracts were dried over magnesium sulfate and the solvent was removed.

The evaporated residue was treated with diethyl ether, filtered, washed and dried. There was thus obtained 3.5 g of 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph -3-em-4-carboxylic acid.

The above compound was dissolved in anhydrous acetone and a solution of an equivalent amount of anhydrous sodium acetate in 10% methanol was added. The solution was stirred at 20 ° C for an hour and diluted with an equal volume of diethyl ether. The sodium salt of 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph-3- em-4-carboxylic acid was thus precipitated.

Characteristic data 1 H NMR: (DMSO-dg) 25 3.52 (ABq, 2H, H-2), 3.90 (s, 3H, N-CH3) 3.97 (s, 2H, CH2CO), 4.35 ( ABq, 2Ξ, H-10), 4.97 (d, 1H, H-6), 5.52 (q, 1H, H-7), 6.67 (s, 1H, H-Ar), 9.25 ( d, 1H, C7-NH).

Mp: 150-155 ° C (decompn.).

EXAMPLE II

Preparation of 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph-3-em- 4-carboxylic acid and its sodium salt.

10.68 g (0.04 mol) of pentachlorophenol and 8.32 g (0.04 mol) of dicyclohexylcarbodiimide were dissolved in 230 ml of anhydrous dichloromethane, followed by 6.4 g (0.04 mol) of 3-chloroisoxazole-5 ethyl acetic acid was added. The reaction mixture S301S42 Ό - 12 - was stirred at room temperature for one day and the precipitated dicyclohexylcarbodiimide was filtered off. The solution of pentachlorophenyl ester in dichloromethane thus obtained was added at 0 ° C with continuous stirring to a solution of 13.2 g (0.04 mol) of 7-amino-3- (1-methyl-1H-1,2,3) 4-tetrazol-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid, 16.8 ml (0.12 mol) of triethylamine and 120 ml of dichloromethane. After the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred at this temperature for one hour. After the reaction was complete, the dichloromethane was distilled off in vacuo, the residue was dissolved in aqueous ethyl acetate, acidified with 2N sulfuric acid to pH 2, the phases were separated, the organic layer was dried over magnesium sulfate and. Evaporated The residue was triturated with diethyl ether, filtered, washed and dried over phosphorus pentoxide in vacuo at room temperature. The product thus obtained was converted into its sodium salt, as described in Example I. 12 g of the sodium salt were obtained, in a yield of 61% (the yield of the free acid was 63.8%).

EXAMPLE III

Preparation of 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-25-5-yl) -thiomethyl-ceph-3-em -4-carboxylic acid and its sodium salt.

0.084 g (1 mmol) of sodium bicarbonate and 0.415 g (1 mmol) of 7- (3-chloro-isoxazol-5-yl) -acetamidocephalosporanic acid were dissolved in a mixture of 17 ml of water and 8 ml of acetone. 0.14 g (1.2 mmol) of 1-methyl-1H-1,2,3,4-tetrazol-5-yl-thiol was added to the solution and the reaction mixture was allowed to stand at 60-65 ° C for 8 hours, adjusting the pH to 5.0-5.5 by adding a saturated sodium bicarbonate solution.

After the reaction was completed, the pH of the reaction mixture was adjusted to 2 using 3 N hydrochloric acid. The mixture was extracted with ethyl acetate, the organic phase evaporated and the residue triturated with ether.

8601842 * e c - 13 -

There was thus obtained 0.35 g of the desired free acid in a yield of 74.5%.

The free acid thus obtained was converted into its sodium salt according to the procedure described in Example 5 I.

10 3 013 4 2

Claims (4)

1 CH 1 -CO-NH-1 k = k o 'J-nL U-CH-S-C \ O J \ 7 Cl COOH 1 H 3 2 n; V-ch-cooh xox H2N “I- {N-N 3 y o I '' Nr COOH I ch3 N-N 2. Process for the preparation of 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph -3-em-4-carboxylic acid of formula 1 and its pharmacologically acceptable salts, characterized 10.oor a) reacting 3-chloro-isoxazole-acetic acid of formula 2 of the formula sheet, or a reactive derivative thereof with 7-amino-3 - (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl'-ceph-3-em-4-carboxylic acid of formula 3 from the formula sheet and isolating the thus obtained 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph-3-em-4 - carboxylic acid of formula 1 from the reaction mixture in a manner known per se and, if desired, converting the latter compound into a pharmacologically acceptable salt thereof by known methods and isolating the salt from the reaction mixture in itself known manner; or b) reacting 1-methyl-1H-1,2,3,4-tetrazol-5-yl-thiol of formula 4 of the formula sheet with 7- (3-chloro-isoxazol-5-yl) ) -acetamido-cephalosporanoic acid and isolation of the 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazol-5-yl) thus obtained -thiomethyl-ceph-3-em-4-carboxylic acid of formula 1 in a manner known per se and, if desired, converting the latter compound into a pharmacologically acceptable salt thereof by known methods and isolating the salt from the reaction mixture in a manner known per se. Process according to process variant a) of claim 2, characterized by using as the reactive derivative of the 3-chloroisoxazole acetic acid of the formula II the chloride or the pentachlorophenyl ester thereof. 4. Pharmaceutical preparation, which as active BSO 1342 4S? - 15 - component 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (T-methyl-1H-1,2,3,4-tetrazol-5-yl) -thiomethyl-ceph-3- em-4-carboxylic acid of formula 1 or a pharmacologically acceptable salt thereof, in admixture with suitable inert solid or liquid pharmaceutical carriers. Process for the preparation of pharmaceutical preparations according to claim 4, characterized by mixing 7- (3-chloro-isoxazol-5-yl) -acetamido-3- (1-methyl-1H-1,2,3 4-tetrazol-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid of formula 1 or a pharmacologically acceptable salt thereof with suitable inert solid or liquid pharmaceutical carriers. Method for the treatment of patients suffering from bacterial diseases, characterized by using a composition according to claim 4 for the treatment. 20 8j * Λ * s -> T Λ 0Λ / 1 NL 33554-Kp / cs Belongs to O.A. in the name of Biogal Gyógyszergyar in Debrecen, Hungary. FORMULA SHEET CL 4 HS - n 'ch3 8801842