FR2591598A1 - New ceph-3-em-4-carboxylic acid derivatives, their preparation and medicaments containing them - Google Patents

Abstract

Ceph-3-em-4-carboxylic acid derivatives, medicaments containing them and processes for their preparation; these derivatives are 7-[(3-chloroisoxazol-5-yl)acetamido]-3-[(1-methyl-1H-1,2,3,4-tetrazol- 5-yl)thiomethyl]ceph-3-em-4-carboxylic acid of formula I: and its salts which are suitable in pharmacology; medicaments showing in particular a high activity against a wide spectrum of microorganisms and processes for preparing these derivatives and medicaments are also described.

Description

 The present invention relates to new ceph-3-em-4-carboxylic acid derivatives, a process for their preparation and medicaments containing them.

 According to one of its aspects, the invention provides new compounds consisting of 7- (3-chloro isoxazole-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-) acid. tetrazole-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid and its salts suitable for pharmacology.

ou un de ses dérivés réactifs avec l'acide 7-amino-3 (l-méthyl-lH-1,2,3,4-tétrazole-5-yl)-thiométhyl-ceph- 3-em-4-carboxylique de formule III

et à isoler l'acide 7-(3-chloro-isoxazole-5-yl)-acéta mido-3-(1-méthyl-lH-1,2,3,4-tétrazole-5-yl)-thiométhyl- ceph-3-em-4-carboxylique de formule I

ainsi obtenu du mélange réactionnel de façon connue en soi et, si on le désire, transformer celui-ci en un de ses sels convenant en pharmacologie selon des procédés connus, et à isoler le sel du mélange réactionnel de façon connue en soi ; ou
b) faire réagir le l-méthyl-lH-1,2,3,4-tétrazole-5-yl-thiol de formule IV

avec l'acide 7-(3-chloro-isoxazole-5-yl)-acétamidocéphalosporanique et à isoler l'acide 7-(3-chloro-iso xazole-5-yl)-acétamido-3-(1-méthyl-lH-1,2,3,4-tétrazole- 5-yl)-thiométhyl-ceph-3-em-4-carboxylique de formule I ainsi obtenu de façon connue en soi et, si on le désire, transformer celui-ci en un de ses sels convenant en pharmacologie selon des procédés connus et isoler le sel du mélange réactionnel de façon connue en soi.According to another aspect, the invention provides a process for the preparation of 7- (3-chloro isoxazole-5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-) acid. tetrazole-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid and its salts suitable for pharmacology which comprises the steps of
a) reacting the 3-chloro-isoxazoleacetic acid of formula II

or one of its derivatives reactive with 7-amino-3 (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thiomethyl-ceph- 3-em-4-carboxylic acid of formula III

and isolating 7- (3-chloro-isoxazole-5-yl) -aceta mido-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thiomethyl- ceph acid -3-em-4-carboxylic of formula I

thus obtained from the reaction mixture in a manner known per se and, if desired, transforming it into one of its salts suitable in pharmacology according to known methods, and isolating the salt from the reaction mixture in a manner known per se; or
b) reacting l-methyl-1H-1,2,3,4-tetrazole-5-yl-thiol of formula IV

with 7- (3-chloro-isoxazole-5-yl) -acetamidocephalosporanic acid and to isolate 7- (3-chloro-iso xazole-5-yl) -acetamido-3- (1-methyl-1H acid -1,2,3,4-tetrazole- 5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid of formula I thus obtained in a manner known per se and, if desired, convert it to a of its salts suitable in pharmacology according to known methods and isolating the salt from the reaction mixture in a manner known per se.

 According to method a) of the method of the present invention, reacting 3-chloro-isoxazoleacetic acid of formula II or one of its reactive derivatives with 7-amino-3- (1-methyl-1H-1 acid) , 2,3,4-tetrazole-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid of formula III.

The reaction can be carried out in a solvent, preferably an aqueous medium containing acetone or in an organic solvent immiscible with water. The reaction is carried out with stirring and the reaction time is generally between 30 minutes and 3 hours.

 According to method b) of the process of the present invention, the 1-methyl-1H-1,2,3,4-tetrazole-5-yl-thiol of formula IV is reacted with 7- (3-chloro acid isoxazole-5-yl) -acetamido-cephalosporanique.

 The reaction is carried out in a solvent, preferably in a mixture of water and acetone, between 30 and 90 "C - preferably at a temperature between 60" C and 65 "C - at a pH in the range of 4 , 5 to 6.0 The reaction time is generally between 2 and 10 hours.

 In the field of semi-synthetic derivatives of the 0-lactam type, a large number of experiments have been carried out to prepare compounds having a broad or specific antimicrobial spectrum. An important group of said compounds comprises a substituted or unsubstituted isoxazole group attached by an acetamido group respectively to the carbon atom 6 of 6-aminopenicillanic acid and to the carbon atom 7 of 7 amino-cephalosporanic acid. Several representative examples of the compounds in this group have a notable antimicrobial effect.

 Molecules which are effective as medicaments are known which comprise an isoxazole-4-yl-acetamido group substituted in the 6 position of the penicillin fragment.

Molecules of this type are described in the patents
US No. 2,996,501 and 3,239,507 and in French Patent No. "BSM No." 6432.

 U.S. Patent No. 4,394,504 describes derivatives of penicillin and cephalospozin which include, in addition to a 3-amino-is-s - le-5-yl-acetamido group, an alkoxyimino group in the $ position with respect to the isoxazole cycle The compounds described in this US patent have a notable activity against strains of Staphylococcus.

 German patent application DOS No. 2,155,081 describes penicillin and cephalosporin derivatives including (3-substituted phenyl) isoxazole-5-yl-acetamido groups. However, none of the phenylisoxazole derivatives actually described and tested exhibits any notable antimicrobial activity against the test microorganisms used.

 German patent application DOS No. 2,409,949 further describes isoxazole-5-yl-acetamido type derivatives. The isoxazole ring of said derivatives however carries other substituents instead of the phenyl substituent.

 The chemotherapeutic and pharmacological effects of part of the compounds described in said DOS application have been determined. However, the molecules studied did not exhibit remarkable antibacterial activity against the test microorganisms used.

 The MICs of certain compounds are also indicated. These values do not, however, reach the activity essential for an active ingredient to be chosen for the production of a medicament.

 One of the aims of the present invention is to prepare new ceph-3-em-4-carboxylic acid derivatives having remarkable pharmacological properties.

 The Applicant has discovered that, surprisingly, the new acid 7- (3-chloro-isoxazole-5-yl) acetamido-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl ) -thio-methyl-ceph-3-em-4-carboxylic of formula I and its salts suitable in pharmacology have a very high and useful antibacterial activity. This effect has been confirmed by chemotherapy selection tests and pharmacological tests. Although the new compound that is 7- (3-chloro-isoxazole-5-yl) acetamido-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) acid - thio-methyl-ceph-3-em-4-carboxylic is covered by the general formula of the German patent application DOS No. 2,409,949, its preparation was not described in said application and its physicochemical values, its constant characteristics and MICs are not mentioned.

 The antibacterial activity of 7- (3 chloro-isoxazole-5-yl) -acetamldo-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thiomethyl-ceph acid -3-em-4-carboxylic acid is established by the MICs of this compound. The compound has been studied against a number of test microorganisms and commercial drugs have been used as reference compounds. The results are collated in Table I.

Table I
Microorganism Composed of Cefalexin Cefuroxime Cefoxitin test formula I
Bordetella bronchiseptica 12.5 500 500 100
Escheridia coli K 12 0.5 6.2 2 1.6
Escheridia coli 6 R 25 15.6 4 1.6
E. coli polyresistens 100 500 250 100
Staphylococcus aureus SMITH 0.06 0.8 0.8 1.2
Staphylococcus epidermidis ATCC
I-12228 0.03 0.8 0.8 0.6
E. coli R-222 0.5 6.2 4 3.1 continued from Table I
Microorganism Composed of Cefalexin Cefuroxime Cefoxitin test formula I
E. coli R-15 1.6 6.2 4 3.1
Klebsiella
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
Salmonella typhimurium 0.5 6.2 4 3.1
Bacillus substilis
ATCC 6633 0.015 0.3 0.1 0.1
Staphylococcus 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
Streptococcus haemolyticus
Pneumo 0.03 0.31 0.008 0.6
Streptococcus haemolyticus A 118 0.06 0.31 - 0.3
Streptococcus haemolyticus Robb 0.03 1.25 0.003 1.2
The absorption tests revealed that, surprisingly, the compound of formula I, which can be considered as a second generation cephalosporin derivative because of the MIC values, present, unlike the other antibiotics belonging to this group, an absorption index which makes it possible to use it for the preparation of pharmaceutical compositions suitable for oral administration.

The results are shown in Table II.

Absorption index DE50p.o. mg / kg
(po in mice) DE50s.c. mg / kg
ED50 sc mg / kg
Table II
Absorption index
Active ingredient S. aur. Salm. Proteus
Smith typhi xl
Compound of formula I 20 1-2 1-2
Cefuroxime 30 0.001 4
Cefoxitin 7-8 0.001
Cefamandole 5 20 /
Cefalexin 1 1
Cefotaxime 0.001 0.001 0.001
The acute toxicity tests show that at the single acute dose characteristic of p-lactams, the compound of formula I is not toxic. One ip dose

or p.o. of 2,500 mg / kg of body weight did not cause the death of any mouse.

LD50 = 2,500 mg / kg body weight.

 Pharmacokinetic tests were also carried out to determine the absorption of the compound of formula I. The tests were carried out on mice of CFLP strain by i.p. administration. and PO..

Twenty mice were treated simultaneously with aqueous solutions of the compound of formula I at an appropriate concentration. Dosage: i.p. : in the case of 20 mg / kg of body weight, 0.1 ml of a solution having a concentration of 4 mg / ml; p.o.: in the case of 80 mg / kg of body weight, 0.4 ml of a solution having a concentration of 4 mg / ml. The average weight of the mice is 20 g. At a given point, the mice are bled by decapitation (the blood clotting is inhibited by the addition of heparin).

 The blood concentration of the active ingredient is determined according to a microbiological method (test microorganism: Bacillus subtilis ATCC 6633).

The results are shown in Table III.

Table III
a / ip administration (20 mg / kg bodyweight
body)
Time (hours) Blood concentration (mg / l) 0.15 1.23 0.30 2.6 0.45 1.23 1 0
Table III (continued)
b / po administration (80 mg / kg body weight
body)
Time (hours) Blood concentration
(mg / l) 0.15 0.35 0.30 0.70 0.45 0.58 1 0.46 1.5 0.22 2 0
The absorption of the compound of formula I has also been studied in Beagle dogs. A po and im administration of the test compound was carried out (in the case of im administration 20 mg / kg body weight, for po administration 80 mg / kg body weight). As test microorganism
Bacillus subtilis ATCC 6633. Blood is drawn by venipuncture. The results are shown in Table IV.

Table IV
a / im administration (20 mg / kg body weight
body)
Time (hours) Blood concentration
(mg / l) 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 5 0.34
b / po administration (80 mg / kg body weight
body)
Time (hours) Blood concentration (mg / l) 0.5 0.63 - 0.645 1 0.645 - 0.67 1.5 3.1 2 5.7 - 5.8 3 7.5 5 2.78
The blood penetration curve for po administration indicates that the compound of formula
It is suitable for the preparation of pharmaceutical compositions for oral administration.

 The binding of the compound of formula I to the serum proteins was determined. Two methods were used; one of the said methods was based on balanced dialysis (Schloten: Antibiot. Chemoter.

 No. 12 page 103 (1964)) and the other was the so-called "big box" method (F. Kawanagh: Analytical Microbiol.

Acad. Press N.Y. (1963)). The level of binding to serum proteins of the compound of formula I is 50-70%.

This value also confirms that it is suitable for oral administration.

 The in vivo activity of the compound of formula I was determined on mice of CFLP strain. To determine the ED50, the mice are infected i.p.

with cultures of 6 to 8 hours of the microorganism the dose used is 10 x DL100. One hour after infection, the animals are treated with the aqueous solution of the compound studied. As a reference, commercial drugs are used. The results are evaluated according to the Litchfield-Wilcoxon method. According to the characteristic values of the p-lactams of the prior art, it has been found that under the effect of the treatment carried out with the compound of formula I, in the case of infections with Staphylococcus or Proteus, the number of living animals remained practically unchanged 48 hours after treatment.

 To establish that the compound of formula I is more active than the compounds of German patent application DOS No. 2,409,949, the following tests were carried out practically as described above.

Table V
Comparison of ED50 expressed in mg / kg.

Test animals: CFLP strain mouse from both
sexes weighing on average 20 g.

 Bacteria used for infection: see table.

 Route of infection: i.p. in 2.5% mucin.

Active ingredient Method of treatment Staphylococcus aureus SMITH
ED50 in mg / kg
Composed of sc 0.23 formula I (0.06 - 0.8)
in 4.3
(0.6 - 28.0)
Compound H * sc 24.6
(15.9 - 38.2)
Compound F ** in. 75.7
(40.2 - 142.7) * 7-E 3-chloro-isoxazole-5-yl) acetamido] cephalosporanate
sodium ** 7-Ee3-chloro-isoxazole-5-yl) acetamido] -3 - [(5-methyl- 1, 3, 4-thiadiazole-2-yl) thiomethyl] -ceph-3-em-4- because-
sodium boxylate.

 According to another aspect, the invention provides pharmaceutical compositions comprising as active product the acid 7- (3-chloro-isoxazole-5-yl) -acetamido-5 (1-methyl-1H-1,2,3,4 -tetrazole-5-yl) -thiomethyl-ceph3-em-4-carboxylic or a salt thereof suitable in pharmacology in admixture with suitable inert solid or liquid pharmaceutical carriers.

 The above pharmaceutical compositions can be prepared according to methods known per se from the pharmaceutical industry.

The invention is illustrated in more detail by the following nonlimiting examples
Example 1
Preparation of 7- (3-chloro-isoxazole-5-yl) acetamido-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thio- methyl-ceph- acid 3-em-4-carboxylic acid and its sodium salt
4.99 g (30.9 mmol) of 3-chloroisoxazole-5-yl-acetic acid and 7.07 g (33.99 mmol) of phosphorus pentachloride are dissolved in 120 ml of anhydrous benzene with constant stirring. The reaction mixture is left to stand at room temperature overnight and at 60 ° C. for a further 1 hour. The benzene is distilled off, the residue is treated several times with anhydrous petroleum ether, decanted and evaporated until elimination of the petroleum ether. The residue is dissolved in 40 ml of anhydrous acetone and the solution thus obtained is added between 0 and 5 "C with constant stirring over 50 minutes to a solution of 6.75 g ( 20.6 mmol) of 7-amino-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid, 5.2 g (61.8 mmol) of sodium bicarbonate, 144 ml of water and 103 ml of acetone. After completion of the addition, the reaction mixture is stirred between 0 and 5 "C for 1 hour and then at room temperature for 2 hours, adjusting the pH between 7 and 8 by adding a saturated solution of sodium bicarbonate.

The mixture is acidified to a pH of 1.5 to 2 by addition of dilute sulfuric acid and extracted three times with each time 100 ml of ethyl acetate. The extracts are dried in ethyl acetate over magnesium sulfate and the solvent is removed.

 The evaporation residue is treated with diethyl ether, filtered, washed and dried.

3.5 g of 7- (3-chloro-isoxazole 5-yl) -acetamido-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thiomethyl acid are thus obtained. -ceph-3-em-4-carboxylic.

 The above compound is dissolved in anhydrous acetone and a solution of an equivalent amount of anhydrous sodium acetate in 10% methanol is added. The solution is stirred at 20 "C for 1 hour and diluted with an equal volume of diethyl ether. The sodium salt of 7- (3-chloro-isoxazole-5-yl) acetamido-3- (1 -methyl-1H-1,2,3,4-tetrazole-5-yl) -thio-methyl-ceph-3-em-4-carboxylic precipitate.

Characteristic values
H-NMR: (DMSO-d6)
3.52 (ABq, 2H, H-2), 3.90 (s, 3H, N-CH3),
3.97 (s, 2H, CH2CO), 4.35 (ABq, 2H, 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).

m.p. : 150-155 "C (decomposition).

Example 2
Preparation of 7- (3-chloro-isoxazole-5-yl) acetamido-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thio- methyl-ceph- acid 3-em-4-carboxylic acid and its sodium salt
10.68 g (0.04 mole) of pentachlorophenol and 8.32 g (0.04 mole) of dicyclohexylcarbodiimide are dissolved in 230 ml of anhydrous dichloromethane and then 6.4 g (0.04 mole) of acid 3 are added. -chloro-isoxazole-5-yl-acetic. The reaction mixture is stirred at room temperature for one day and the precipitated dicyclohexylcarbodiimide is filtered off. The solution of pentachlorophenyl ester in dichloromethane thus obtained is added at 00C with constant stirring to a solution of 13.2 g (0 , 04 mole) 7-amino-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid, 16 , 8 ml (0.12 mole) of triethylamine and 120 ml of dichloromethane. After the addition is complete, the reaction mixture is allowed to warm to room temperature and stirred at this temperature for 1 hour. After completion of the reaction, the dichloromethane is removed by vacuum distillation, the residue is dissolved in aqueous ethyl acetate, acidified to pH 2 with 2 N sulfuric acid, the phases are separated, the mixture is dried. organic layer over magnesium sulphate and evaporated. The residue is triturated with diethyl ether, filtered, washed and dried at ordinary temperature over phosphorus pentoxide under vacuum. The product thus obtained is converted into the sodium salt as described in Example 1. 12 g of the sodium salt are obtained, yield 61% (the yield of free acid is 63.8%).

Example 3
Preparation of acid 7- (3-chloro-isoxazole-5-yl) - acetamido-3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thio- methyl-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-isoxazole 5-yl) -acetamidocephalosporanic acid are dissolved in a mixture of 17 ml of water and 8 ml of acetone is added to the solution 0.14 g (1.2 mmol) of 1-methyl-1H-1,2,3,4-tetrazole-5-yl-thiol, the reaction mixture is left to stand at 60-65 "C for 8 hours, then the pH is adjusted to 5.0-5.5 by adding a saturated solution of sodium bicarbonate
After completion of the reaction, the pH of the reaction mixture is adjusted to 2 with 3N hydrochloric acid. The mixture is extracted with ethyl acetate, the organic phase is evaporated and the residue is triturated with l 'ether. 0.35 g of the desired free acid is thus obtained.

Yield: 74.5%.

 The free acid thus obtained is converted into the sodium salt according to the process described in Example 1.

Claims (5)

 1. New compounds characterized in that they consist of 7- (3-chloro-isoxazole-5-yl) -acid tamido-3- (1-methyl-1H-1,2,3,4-tetrazole acid -5-yl) -thiomethyl- ceph-3-em-4-carboxylic acid of formula I

 and its salts suitable for pharmacology.  2. Process for the preparation of 7 (3-chloro-isoxazole-5-yl) -acetamido-3- (1-methyl-1H- 1,2,3,4-tetrazole-5-yl) -thiomethyl acid -ceph-3-em-4-carboxylic of formula I and its salts suitable in pharmacology, characterized in that it comprises the stages consisting in  a) reacting the 3-chloro-isoxazoleacetic acid of formula II

 or one of its derivatives reactive with 7-amino-3 (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thiomethyl-ceph 3-em-4-carboxylic acid of formula III

 and to isolate the acid 7- (3-chloro-isoxazole-5-yl) -acetamido3- (1-methyl-1H-1,2,3,4-tetrazole-5-yl) -thiomethyl-ceph3-em- 4-carboxylic acid of formula I thus obtained from the reaction mixture in a manner known per se and, if desired, transform it into a salt suitable in pharmacology according to known methods and isolate the salt from the reaction mixture in a manner known per se ; or  b) reacting l-methyl-1H-1,2,3,4-tetrazole5-yl-thiol of formula IV

 with 7- (3-chloro-isoxazole-5-yl) -acetylamidocephalosporanic acid and isolate 7- (3-chloro-isoxazole-5-yl) -acetamido-3- (1-methyl-1H-1 acid) , 2,3,4-tetrazole 5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid of formula I thus obtained in a manner known per se and, if desired, convert it into a suitable salt pharmacology according to known methods and isolating the salt from the reaction mixture in a manner known per se.  3. Method according to step a) of claim 2, characterized in that it comprises the use as reactive derivative of 3-chloro-isoxazole-acetic acid of formula II, the chloride or the pentachlorophenyl ester of this one.  4. Pharmaceutical compositions characterized in that they contain as active product the acid 7- (3-chloro-isoxazole-5-yl) -acetamido-3- (1-methyl-1H- 1,2,3,4- tetrazole-5-yl) -thiomethyl-ceph-3-em-4-carboxylic of formula I or a salt thereof suitable in pharmacology in admixture with suitable inert solid or liquid pharmaceutical carriers.  5. Method for the preparation of pharmaceutical compositions according to claim 4, characterized in that it comprises the mixture of 7- (3-chloro isoxazole-5-yl) -acetamido-3- (1-methyl-1H) acid -1,2,3,4-tetrazole-5-yl) -thiomethyl-ceph-3-em-4-carboxylic acid of formula I or of a salt thereof suitable for pharmacology with inert solid or liquid pharmaceutical carriers appropriate.