SE416472B - PROCEDURE FOR PREPARATION OF 7- (ALFA-AMINO-P-HYDROXIFENYLACETAMIDO) -3- (1,2,3-TRIAZOLYLTYOMETHYL) -3-CEFEM-4-CARBOXYL ACID - Google Patents

Description

NH 2 or an acylating or activated derivative thereof, wherein the NH 2 group. _ _ 2 is Cloudy in a suitable manner if required, and that any protective group is subsequently removed.

Of the preferred compounds of the invention may be mentioned: 7- (p-hydroxy-α-aminophenylacetamido) -3- (1,2,3-triazol-4-ylthiomethyl) -3-cephem-4-carboxylic acid, 7- (p- hydroxy-α-aminophenylaethamido) -5- (4-methyl-1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid.

The antibacterial compounds of the invention have surprisingly advantageous properties compared to related compounds.

For example, the compound 7- (α-amino-p-hydroxyphenylacetamido) -3- (1,2,3-triazol-4-ylthiomethyl) -β-cephem-4-carboxylayra (compound 60771) is advantageous in comparison with the compound 7- (a -aminophenylacetamido) -3- (1,2,3-triazol-4-ylthiomethyl) -3-cephem-4-carboxylic acid (Compound 60222) when it is desired to produce higher maximum serum concentrations in mice and especially higher serum half-lives. As a result, the activity that can be observed after both oral and subcutaneous administration to mice is significantly improved, as evidenced by lower EDSO.

The results obtained are shown in Tables A and B. 7302579-3 3. TABLE A _ Serum concentration in mice United, m, Éïïåïšäïïa 'Éäåïååmmøn (ng / m) "alïåïå'fšs" id 60771 20 mg / kg p. °. H 52 '.'62 60222 20 Ing / kg p.o. 13 48 60771 20 mg / kg s.c. 54. 71 60222 20: ng / kg s.c. 35 '34 Tmmm.B Study regarding protection of mice Association Test organism Method of administration §Q5 ° fi ggzkg) 60771 E. 0011 12140 5.0. <0.8, 0.8 60222 E. coli 12140 _ s.c. <3, 3.6 60771 E. 0011 12140 13.0. <0.8, 1.2 60222 E. 0011 121140 pd). 412,5, 1! - 60771 K. pneumoníae 6.0. 0.6, 0.6 4200 - 60222 K. pneumoniae s.c. 4.8 4200 60771 K. pneumcniae p.o. 0.4, 0.5 11200 60222 K. pneumoniae p.o. 12.5 4200 7 _ Compound 60771 is also. advantageous in Comparison with 7- (01-amino-p-hydroxyphenylacetamido) -3- (5-methyl-1,2,2-triazol-6-ylthylmethyl) -3-cephem-4-carboxylic acid (Compound 61529), since it has significantly lower ED5O in the protection sample. The results obtained are shown in Table C. x Ü nozsfze-s 4 TABLE C Study regarding protection of mice Association Test organism Method of administration §Q5O § @ g¿k52 60771 E. e ° 11 12140 s.e. 61529 E. 0011 12140 8.0. 10.7 60771 E. coli 12140 p.o. (0.8, 1.2 61529 E. coli 12140 po 7.5 60771 K. pneumoniae sc 0.6, 0.6 4200 261529 K. pneumoniae sc 8.6 4200 60771 K. pneumcniae po 0.4, 0, 4200 61529 K. pneumoniae po 16 4200, when c111 is the compound 7α (β-amino-p-hydroxy-phenylalecamide) -3- (4-methyl-1,2,3-triazol-5-ylthiomethyl) -3-cephem -4-carboxylic acid (61775) advantageous in Comparison with 7- (α-aminophenylacetamido) -3- (4-methyl-1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (61348), as this compound has lower ED50 1 protection samples.Recent results are shown in Table D.'Engenggg Testorganism 61775 61348 51775: 61348 51775 61348 - 1 51775 61348 E. 0011 12140 E. 0011 12140 E. 0011 12140 E. coli 12140 K. pneumoniae 4200 K. pneumoniae 4200 K. pneumoniae 4200 TABLE D 2 §§gdium regarding protection of mice Aaminiscracionssäzt §g5o g @ g¿k51_ se 1.8 s.§. 42, 18,2 po 3,6, 3 po 50, 19 sc 2.8 sc 20 po 5.2 146 7 K. pneumoniae 4200 po 10 5 7302579-3 Furthermore, the compound is 7- (2-amino-p-hydroxyphenylaoetamido) -3- (1-methyl-1,2,3 -triaz ol-5-ylythiomethyl) -3-olefin-4-carboxylic acid (60876) advantageous in Comparison with 7- (α-aminophenylagetamido) -3- (1-methyl-1,2,3-triazol-5-yltiomethyl) -3 -cephem-4-carboxylic acid (60637), as it has lower ED50 and higher maximum serum levels and half-lives, as shown in Tables E and F. respectively. regarding protection of mice Association Test organism Method of administration 60876 E. 6611 12140 3.0. 60637 E. coli 12140 s.o. 60876 E. coli 12140 p.o. 60637 E. coli 12140 p.o. 60876 K. pneumoniae s.c. 4200 60637 K. pneumoniae s.c. 4200 60876 K. pneumoniae p.o. 4200 60637 K. pneumoniae p.o.

TABLE F Serum concentration in mice Administra- Maximum Compound Qgâ tion method concentration §ug¿m1) 60876 20 mg / kg p.o. 14 60637 20 mg / kg p.o. 1.8 60876 20 mg / kg s.c. 44 60637 20 mg / kg s.c. 21 x Too short to be meaningful.

For comparison, related compounds from Swedish patent application 5262/70 were tested, namely 7- (M-aminophenylaoethamido) -3- (2-methyl-1,3,4-triadiazol-5-ylthiomethyl) -3-cephan-4- carboxylic acid (Compound No. 44065) and 7- (vraminophenylacetamido) -3- (1-methyl-tetrazol-5-ylthiomethyl) -3-cephan-4-carboxylic acid (Compound No. 4629) and the compounds 7- (p-hydroxy-acetamide). Mraminophenylacetamido) cephalosporanic acid (Compound No. 60875) and 7 '(β-hydroxy-draminophenylacetamido) -acetoxycephalosporanic acid (Compound No. 60524) from the American Halveri period (min. 48 * x 48 12 752 and the results obtained are summarized in the following table G. 0 TABLE G Studio for protection of mice Association Test organism Method of administration §§5o (mg / kg) ÄU065 E. coli l2lÄ0 sc U UU065 E. coli 12140 po ll üüO65 K. Päeumoniae sc 29, U _ Ä200 I U4065 K. pneumoniae po 55 _ 4200 U5029 E. C011 12130 SC šlß, 1,4 H6029 E. coli 12140 po 6, l0 Ä6029 K. pneumoniae sc 8, ll ^ 0200 ü60 29 K. pneumoniae p.o. 9, 13 11200 60875 E. coli l2lü0 s.c. 3.5 50875 E. cøli 12140 p.0. _ 18 60875 K. pneumoniae s.c. 9.2 U200 60875 K. pneumoniae p.o. 17 N200 50523 E. C011 12150 s.c. 32 60520 E. coli l2lU0 p.O. Å 25 6052Ä K. pneumoniae s.c. 56 U200 6052H K. pneumoniae p.o. 33 U200 It appears from this table that in comparison with the compounds given therein, the compounds Nos. 60771, 60876 and 61775 which are prepared according to the invention show a better effect, which is evident from lower ED50 values.

In general, it can be said that the compounds of the present invention are superior in that they allow lower protective doses con / or higher and prolonged serum concentrations.

The compounds of the invention can be prepared by aylating the 7-amino group of the appropriate 7-amino-3-heterocyclic thiomethylcephalosporin nucleus with a p-hydroxyphenylglycine. It is desirable to protect the amino group on the glycine component with a protecting group which can be easily removed, such as t-butoxycarbonyl, benzyl-Xicarb ° 1 Y1 = Uri-chloroethoxycarbonyl or any similar protecting group. which was normally used in peptide syntheses before acylation. To effect acylation, the carboxyl group in the acylating agent can be activated by transfer to the acid chloride or to a mixed anhydride with e.g. a lower alkyl chloroformate. The carboxyl group can also be activated by transfer to 2,4-dinitrophenyl or N-hydroxysuccinimidoyl esters. If an ester of the cephalosporin nucleus is used, e.g. benzhydryl, t-butyl, trichlorethyl or a benzyl ester, the protected phenylglycine compound can be coupled directly to the 7-amino group using a carbodiimide, such as the dicyclohexylcarbodiimide. Alternatively, the protected phenylglycine compound can be activated for condensation with the appropriate cephalosporin nucleus by first reacting it with the carbonyl diimidazole or its equivalent.

The compounds of the present invention may also be prepared by reacting a compound which differs from compounds of formula I in that the 3-substituent represents acetoxymethyl instead of R-thiomethyl with a mercaptoheterocyclic compound. The reaction is preferably carried out at almost neutral pH. The solvent is preferably water or a mixture of water and acetone. The reaction can be carried out at temperatures from about room temperature to the boiling point of the solvent, the reaction time varying with the temperature, the solvent and the reactants. The reaction product is isolated by carefully acidifying the reaction mixture and extracting it with a suitable organic solvent. The d-amino group of the phenylglycine compound used as a starting material should be protected with a gruPD that can be easily removed, such as t-butoxycarbonyl, carbobenzyloxy or trichloroethoxycarbonyl. The regrouping position is then carried out, after which the protecting group is removed in a conventional manner.

The starting materials used in the regrouping in the 3-position of a 7-acylated cephalosporinic acid are described in the literature or obtained in a known manner. The starting materials used in aylation in the 7-position of a 5-heterocyclic thiomethyl nucleus are prepared by displacing the 3-acetoxy group of an alkali metal salt of 7-aminocephalosporinic acid (7-AGA) with an alkali metal salt of the heterocyclic thiol hot aqueous aoetone.

Because the 7-acetamido group contains an asymmetric α-carbon atom, optical isomers exist. The D-isomer is preferred, but the L-isomer and the racemic mixture also fall within the scope of the invention. The compounds are transferred to injectable or oral preparations in the same manner as other cephalosporin antibiotics. They are administered by injection or orally to prevent and treat bacterial infections in doses that vary with the nature and severity of the infection and the age, weight and condition of the patient being treated. The compounds in which Y represents NH 2 are generally administered either orally or parenterally. Due to the presence of both an amino group and a carboxylic acid group in some of the cephalosporin compounds of the present invention, it is possible to prepare both acidic methods by standard methods. salts and base salts of pharmaceutically acceptable non-toxic acids and bases as well as the zwitterionic forms of the compounds. When the salts are obtained, they are easily converted to zwitterions in a known manner. Salts of compounds which contain only an acidic function are also prepared by the use of pharmaceutically acceptable non-toxic bases. All of these salts are within the scope of the present invention.

The following examples are intended to illustrate the methods and compounds of the invention.

EXAMPLE 1 7- (α-Amino-p-hydroxyphenylacetamido) -3- (1,2,3-triazol-4-ylthiomethyl) -3-cephem-4-carboxylic acid A stirred solution of Nt-butoxycarbonyl-p-hydroxyphenylglycine (10 .75 g, 0.0375 mol) in dry tetrahydrofuran (150 ml) was added with triethylamine (5.2 ml, 0.0375 mol). The mixture was cooled to -10 ° and then isobutyl chloroformate (,, 92 mL, 0.0375 mol) was added dropwise over a 10 minute period. The reaction mixture was stirred at -10 ° for 70 minutes and then a cold solution of 7-ACA (10.0 g, 0.0375 mol) in 50% aqueous tetrahydrofuran (140 ml) and triethylamine (6.75, 0.048 mol) was added. ) for a period of 15 minutes. The reaction mixture was stirred at -5 ° to 0 ° for 1 hour and at room temperature for 2 hours. The organic solvents were evaporated and water (150 ml) was added to the aqueous residue.

The solution was extracted with ethyl acetate and the aqueous phase was separated, covered with freshly prepared ethyl acetate, acidified to pH 2.8 and filtered. The phases were separated and the acidic solution was re-extracted with ethyl acetate. The extracts of the acidified aqueous solution were combined, dried and evaporated to give the N-butoxycarboxyl derivative of 7- (α-amino-p-hydroxyphenylacetamido) cephalosporanic acid. A mixture of the indicated product (3.0 g, 0.00493 mol) in 30 ml of a phosphate buffer of pH 6.4 was treated with NaHCO 3 (1, 085 g, 0.023233 mol) and then with 4-mercapto-1,2,3-triazole (0.748 g, 0.0074 mol). The solution was heated to 70 ° and stirred at 70 ° C for 2.75 hours. The solution was cooled, filtered and acidified to pH 2.5 to give a residue. The solvents were decanted and the residue was washed with water. The product was dissolved in ethyl acetate, washed with water, dried and evaporated to give the N-protected product, which was again precipitated from a mixture of acetone and chloroform.

The protected product was stirred at 0 to 5 ° with a mixture of trifluoroacetic acid and anisole in a ratio of 9: 1 for 70 minutes. The solvents were evaporated and the residue was poured with rapid stirring into ether (350 ml). The solid was collected, dissolved in water and stirred with a basic ion exchange resin (Amberlite IE-45, an anionic polystyrene amine resin) until the value became constant.

The resin was filtered off and the aqueous solution was lyophilized to give the product.

Calculated for C 18 H 18 N 6 O 5 S 2.2 H 2 O: C, 43.57; H, 4.45; N, 16.86; Found: C, 43.67; H, 4.14; N, 16.62.

EXAMPLE 2 7- (Do-amino-p-hydroxyphenylacetamido) -3- (4-methyl-1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid 5-benzamido-4-methyl-1 , 2,3-thiadiazole [6.60 3, 0.03 mol, prepared as described in Berichte 22, 1629 (1966)] was added with 60 ml of 2 N NaOH and the solution was heated under reflux overnight.

After about 25 hours of heating, the reaction mixture was cooled with ice and 120 ml of 2N HCl was added with stirring. The mixture was then allowed to stand in the refrigerator over the weekend. The precipitated solid (benzoic acid) was then filtered off while the mixture was still cold. The solid was washed with a small amount of water and the pH of the filtrate was adjusted to 3.0 with 10% NaOH. The aqueous solution was extracted with ethyl acetate four times. The extracts were dried and filtered. Sodium 2-ethylhexanoate was added until precipitation ceased. The solid sodium salt of 5-mercapto-4-methyl-1,2,3-triazole was filtered off and then dried under vacuum.

A stirred suspension of 6.78 g (0.013 mol) of 7- (at-butoxycarbonylamino-p-hydroxyphenylacetamido) -cephalosporanic acid and 5.82 g (0.02 mol) of the sodium salt of 5-mercapto-4-methyl 1,2,3-Triazole In 100 ml of phosphate buffer with a pH of 6.4 was added with 1.09 g (0.013 mol) of NaHCO 3. The mixture was heated at 70 ° for 4 1/2 hours and the reaction was monitored by TLC. The mixture was allowed to cool and the rubber formed was stirred with water and any insoluble substances were filtered off.

The solution was covered with ethyl acetate and refrigerated overnight.

Then the layers were separated and the aqueous layer was acidified to pH 3.0 with BN HCl. Medium acetate was then extracted several times and the extracts were dried and evaporated. The resulting rubber was triturated with ether to give 2.69 g of solid.

Small amounts were then reprecipitated twice from a mixture of methanol and ether and then the desired material was precipitated with petroleum ether. The resulting solid (1.75 g) was chromatographed on 100 g of silica gel using 90: 10: 3 chloroform: methanolic formic acid.

The gay column 0.80 g of t-butoxycarbonyl derivative of the title compound.

The indicated derivative (0.8O g, 0.00139 mol) was placed in a small round bottom flask with a drying tube and immersed in an ice bath. About 10 ml of cold trifluoroacetic acid was added and the mixture was stirred in the cold for about 15 minutes, after which the mixture was evaporated for 15 minutes. The residue was poured into about 100 ml of ether and the resulting solid was collected and dissolved in about 15 ml of water. It was then stirred with IR 45 Amberlite resin, a basic polystyrene ion exchange resin (washed 3 times), until the solution reached a pH of about 5.5. The resin was filtered off and the solution was lyophilized overnight to give the title product.

Calculated for C 19 H 20 N 6 O 5 S 2. H, 4.89; N, 15.97; Found = c, 43.75; H, 4.80; N, 15.27.

EXAMPLE 3 When the t-butoxycarbonyl derivative of 7- (α-amino-p-hydroxyphenylacetamido) cephalosporanic acid is reacted with the appropriate mercaptoheterocyclic compound according to the procedure of Example 1, the following compounds are obtained: 7- ) -3- (1-methyl-1,2,3-triazol-4-ylthiomethyl) -3-cephem-4-carboxylic acid. 7- (α-amino-p-hydroxyphenylacetamido) -3- (1-methyl-1,2,3-triazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid EXAMPLE 4. The compounds described in the examples 1-3, can also be prepared by reacting an appropriate 7-amino-3-heterocyclic thiomethyl-5-cephem-4-carboxylic acid with the mixed anhydride formed of Nt-butoxycarbonyl-p-hydroxyphenylglycine and isobutyl-. ... ..., ...._...., _,., ..._ .. H _ _ 10 15 20 25 30 35 40 7302579-3 ll chloroformate or trichloroacetyl chloride. The N-protecting group is removed with trifluoroacetic acid or glacial acetic acid H01.

EXAMPLE Q. A pharmaceutical composition for parenteral administration can be prepared by dissolving 500 mg of sodium 7- (α-amino-β-hydroxyphenylacetamido) -3- (1,2,3-triazol-4-ylthiomethyl) -3 -cephem-4-carboxylate in 2 ml of sterile water or normal saline. Any of the other cephalosporins listed can be used in a similar manner.

EXAMPLE 6. A capsule for oral administration may be prepared by mixing 500 mg of a cephalosporin compound of the type indicated, 250 mg of lactose and 75 mg of magnesium stearate. The other specified products can be prepared in a similar way.

EXAMPLE 7. 7- (Chramino-p-hydroxyphenylacetamido) -3- (1,2,3-triazol-N-ythiomethyl) -3-cephem-U-carboxylic acid To a solution of N-hydroxysuccinimidyl-M- (t-butoxycarbonyl amino) -mr (β-hydroxyphenyl) acetic acid (3.64 g, 0.01 mol) in dry pyridine (30 ml) containing triethylamine (1.1 g, 0.011 mol) was added trimethylsilyl chloride (1.19 3, 0.011 mol) ~ The reaction mixture was stirred at room temperature for one hour and then filtered to remove. precipitated salt. The filtrate was added directly to a solution of 7-amino-3- (1,2,3-triazol-4-ylthiomethyl) -3-cephem-1-carboxylic acid (1.3 g, 0.01 mol) in dry pyridine (30 ml) containing dry 0.02 mol) and formamide (2.2 ml). The reaction mixture was stirred at room temperature for 3.5 hours and then filtered to remove precipitated salt. The filtrate was slowly poured into ether (600 ml) and the resulting product which precipitated as its triethylamine salt was collected and dried. The salt was dissolved in water, and the pH was adjusted to 7 with NaHCO 3 solution and filtered by means of filter aids. The filtrate was layered with ethyl acetate and the pH was adjusted to 1 with 3N HCl. A small amount of insoluble material was removed by filtration. The triethylamine (2.02 g, organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined ethyl acetate solutions were washed with water, dried and evaporated in vacuo to 1/6 of the volume. The residue was slowly poured into petroleum ether with stirring. and the precipitated solid was recovered and dried (2.6, H1, 2%).

The above protected product (1, μg, 2U mmcl) was added to an ice-cooled solution of trifluoroacetic acid (5 ml) containing anisole (1 ml). The reaction mixture was stirred while cooling to 0 ° 730 for 3 hours. The trifluoroacetic acid was removed in vacuo and the resulting residue was triturated with ether to give a solid trifluoroacetate salt. The salt was dissolved in water (100 ml), filtered through a filter aid and the pH was adjusted to 4 with basic ion exchange resin (Amberlite IR-45, an anionic polystyrene amine resin). The solution was lyophilized, which gave the zwitterion of the title compound (700 mg, 66.6%). IR and elemental analysis were similar to those of the unuctuct prepared in Example 1.

Claims (3)

A process for the preparation of a compound of the formula _______. ~ ________ S Ho - cHcoNH - “___ T / _.- I NH» 2 o = ._._. N / cnzs R åoon wherein R represents 1,2,3-Triezolyl, 1-methyl-1,2,3-triazolyl or H-methyl-1,2,3-triazolyl, characterized in that an acetoxymethylcephalosporin compound of the formula S 1 NH 2 -H 2 'I l an 5. 1. wherein R 1 represents HO-4 / "* - CHCO, wherein the NH 2 group is protected in a suitable manner, if necessary, or H, is reacted with a heterocyclic thiol compound of the formula HS -R, wherein R is as defined, and that, when R1 is H, the resulting compound is acylated with a p-hydroxyphenylacetic acid compound of the formula Ho- <2 É> -cacoo fl \ - in NH2 or an acylating or activated derivative thereof, wherein the NH 2 group is suitably protected if required, and that any protecting group is subsequently removed. ~ A process according to claim 1 for the preparation of 7-GX-amino-p-hydroxyphenylacetamido) -3- (1,2,3-triazol-4-ylthiomethyl) -3-cephem-β-carboxylic acid, characterized therefrom, reacting the Nt-butoxycarbonyl derivative of 7- (béamino-p-hydroxyphenylacetamido) cephalosporanic acid with N-mercapto-1,2,3-triazole, after which the protecting group is removed with trifluoroacetic acid. A process according to claim 1 for the preparation of 7- (06 amino-p-hydroxyphenylacetamido) -3 '(H-methyl-1,2,3-triazol-5-ylthiomethyl) -3-cephem-U-carboxylic acid, This results in the reaction of the Nt-butoxycarbonyl derivative of 7- (Q-amino-p-hydroxyphenylacetamido] cephalosporanic acid with H-methyl-5-mercapto-1,2,3-triazole, after which the protecting group is removed with trifluoroacetic acid H. Process according to claim 1 for the preparation of 7- (® * amino-p-hydroxyphenylacabamido) -3- (1-methyl-1,2,3-triazol-5-ylthiomethyl) -3-cephem -H-carboxylic acid, characterized in that the Nt-butoxycarbonyl derivative of 7- (O 1 -amino-p-hydroxyphenylacetamidonephalosporanic acid is reacted with 1-methyl-5-mercapto-1,2,3-triazole, was after the protecting group is removed with trifluoroacetic acid BEFORE PUBLICATIONS: Sweden patent applications 5 262/70, 7317197-7 US 3,489,752