IL43913A - Preparation of (-(d-(alpha-amino-alpha-p-hydroxyphenyl)-acetamido)-3-(1,2,3-triazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid and salts thereof - Google Patents

Abstract

1460914 Cephalosporin derivatives BRISTOL-MYERS CO 21 Dec 1973 [26 Dec 1972] 59332/73 Heading C2C The novel compound I having the D-(-)-configuration in the 7-side chain and salts and esters thereof are prepared by reacting a compound II or an ester or salt thereof with an acylating derivative of an acid III which is in the D-(-)-form (B is an amino-protecting group) followed by removal of the protecting group and optional esterification or salt formation. Potassium 1,2,3-triazole-5-thiolate is prepared by reaction of benzoyl isothiocyanate with diazomethane under nitrogen to give 5-benzamido- 1,2,3-thiadiazole, refluxing this with sodium hydroxide to give 1,2,3-triazole-5-thiol and reaction of this with alcoholic potassium hydroxide. 7-Amino-3-(1,2,3-triazol-5-yl-thio-methyl) - 3 - cephem - 4 - carboxylic acid is prepared by reaction of 7-aminocephalosporanic acid with 5-mercapto-1,2,3-triazole or its potassium salt. Salts and esters of the 7-amino-3-(1,2,3- triazol - 5 - ylthiomethyl) - 3 - cephem - 4 - carboxylic acid are prepared similarly. Sodium D - (-) - N - (2 - methoxycarbonyl - 1 - methylvinyl) - α - amino - α - (4 - hydroxyphenyl)- acetate is prepared by reaction of D-(-)-2-(phydroxyphenyl)glycine with sodium hydroxide followed by methyl acetoacetate. D-α-t-Butoxycarbonylamino - α - (p - hydroxyphenyl)acetic acid is prepared by reaction of D-(-)-p-hydroxyphenylglycine with magnesium oxide, then with t-butoxycarbonyl azide. Pharmaceutical compositions used as antibiotics contain a compound I together with a suitable carrier or diluent . Reference has been directed by the Comptroller to Specification 1,363,833. [GB1460914A]

Description

-( »3D *ορΐΝ-π»π- P- α-ΐ3»βκ-α )-P7-7 nsain naan -oBS-3- *noIR»n( b»κ-5- ι»κ *TB-3, 2~1 )~3- i"t»D«DSR The pre ar ion of 7^$^a-t_dno^^ ao©tamido7-3-(1»2,3-triazol-5-yl)thlom©thyl-3-O0phem- 4-carboxylic acid and salts horoof BRISTOL-MYERS COMPANY 0: 42073 43913/2 This invention relates to a novel process for the preparation of 3-thiolated-7-acylamindocephalosporanic acid derivatives having the D-(-) configuration in the side chain represented by. the structural formula and pharmaceutically acceptable salts thereof.

The pharmaceutically acceptable salts referred to above include the nontoxic carboxylic acid salts, e.g. nontoxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and salts with nontoxic amines, e.g. trialkylamines , procaine, dibenzylamine, N-benzyl-£-phenethyla-mine, 1-ephenamine, N, ' -dibenzylethylenediamine, N-alkylpiperi-dine and other amines which have been.used to form salts of penicillins. Also included within the definition of pharmaceutical ly acceptable salts are the nontoxic acid addition salts (amine salts), e.g. salts with mineral acids such as hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric and salts with organic acids such as maleic, acetic, citric, oxalic, succinic, benzoic, tartaric, fumaric, mandelic, ascorbic and malic. 43913/2 There is extensive literature relating to the cephalosporin antibiotics. The more relavant prior art in this field is given below.

The preparation of various 7-[a-amino-arylacetamido] -cephalosporanic acids and the corresponding desacetoxy compounds in which aryl represents unsubstituted or substituted phenyl or 2- or 3-thienyl is described, for example, in British specifications 985,747; 1,017,624; 1,054,806 and 1,123,333, in Belgian Patent 696,026, in U.S. Patents 3, 311, 621; 3 , 352, 858; 3,489,750; 3,489,751; 3,489,752 and 3,518,260, in Japanese Patent 16871/66, by Spencer et al., J\_ Med. Chem. , 9 (5) , 746-750 (1966) and by Kurita et al., J. Antibiotics (Tokyo) (A) 19, 243-249 (1966) and in U.S. Patent 3,485,819.

Netherlands Patents 68/11676 and 68/12382 and U.S. Patents 3,489,750, 3,489,751 and 3,489,752 disclose ring-substituted cephaloglycins .

Various 7- [a-amino-arylacetamido] cephalosporins in which one hydrogen of the a-amino group is replaced by a carbonyl group which is attached in turn to another moiety have been reported. The earliest were the cephaloglycin and cephalexin precursors in which use was made of a common peptide blocking group such as carbobenzyloxy as illustrated by U.S. Patent 3,364,212, Belgian Patent 675,298, South African Patent 67/1260 and Belgian patent 696,026. Related compounds include those of U.S. Patents 3,303,193, 3,311,621 and 3,518,260.

Various cephalosporins, including cephalosporin C on occasion, have been reacted with nucleophilic, aromatic mercaptans to produce compounds having the structure In U.S. Patent 3,278,531 Ar is phenyl or certain substituted phenyls or certain aromatic heterocyclic rings. Similar nucleophiles, e.g. 2-mercaptopyrimidines, are disclosed in. U.S. 3,261,832 and Great Britain 1,101,422 and UlS. 3,479,350 and U. S. 3,502,665. Additional nucleophiles of this type were disclosed in Belgium 714,518, in Canada 818,501, in Great Britain 1,187,323 and in U.S.; 3,516,997 which includes the compound named cefazolin, which has a tetrazoiyTacetyl sidechain on the 7-amino group and a 5-methyl-thiadiazolylthio methyl group at the 3-position and is described at some length in the scientific literature, e.g. in Antimicrobial Agents and Chemotherapy - 1969, American Society for Microbiology, Bethesda, Maryland at pages 236-243 and in J. Antibiotics (Japan) 23(3) , 131-148 (1970) .

Replacement of the 3-acetoxy group of a cephalosporin by various heterocyclic thiols has been disclosed in U.S. Patent 3,563,983 and in South African Patent 70/2290 where the side-chains were, for example, 7-a-aminophenylacetamido and typical heterocyclic thiols were 2-methyl-l, 3,4rthiadiazole-5-thiol 43913/2 and 1-methyl-l , 2 , 3 , 4-tetrazole-5-thiol.

The novel process provided by the present invention for the preparation of a compound having the D-(-) configuration in the side chain of the formula and pharmaceutically acceptable salts thereof; comprise reacting a silyl derivative of a compound of the formul II with an acylating derivative of the formula III 43913/2 to produce after removal of the amino-protective grou a compound of formula I or a pharmaceutically acceptable salt thereof and, If desired, either before or after removal of the blocking groups (a) converting by methods known per se the product in the form of the fee acid thereof to a corresponding pharmaceutically acceptable salt thereof or (b) converting by methods known per se the product in the form of a salt thereof to the corresponding free acid compound or another pharmaceutically acceptable salt thereof.

The 3-thiolated-7-aminocephalosporanic acid starting compound of formula II may be prepared by displacement of the 3-acetoxy group of 7-aminocephalosporanie acid with 5-mercapto-l,2,3-triazole or a salt thereof. The displacement of an ester group with a thiol group is a known reaction and is preferably accomplished in aqueous solution with heating.

The above-mentioned starting compound is converted prior to the acylation reaction to a silylated derivative thereof. The procedures for preparing the silyl derivatives are disclosed in the literature and are well-known to those skilled in the art of penicillin and cephalosporin chemistry, such as described, e.g., in U.S. Patent 3,249,622. The silyl ester group is removed following the acylation reaction by hydrolysis or alcoholysis. 43913/2 Prior to the acylation reaction the amino group of the. p-hydroxyphenylglycine acylating agent is protected by a proton, in the compound of the formula After the cylation coupling reaction, the amino-protecting proton is removed by methods known per se to form the desired product of formula I, e.g. by neutralization, etc.

If desired, the phenolic hydroxy group of the acylating agent III may also be protected during the acylation steps, e.g. as with an easily removable protecting group such as O-benzyl, O-benzyloxycarbonyl or trimethylsilyl , but generally the protection of this group is not essential. Obviously other functionally equivalent blocking groups for the hydroxy group can be used and such groups are considered within the scope of this invention.

Acylation of a 7-amino group of a cephalosporin is a well-known reaction and many functional equivalents of formula III have been commonly used as acylating agents for primary amino groups. Examples of such acylating derivatives of the free acid include the corresponding acid anhydrides, mixed anhydrides, e.g. alkoxyformic anhydrides, acid halides, acid azides, active esters and active thioesters.

The p-hydroxyphenylglycine chloride hydrochloride is a particularly preferred acylating agent which also serves a dual function of carboxyl activation and amino protection. 43913/2 The particular process conditions, e.g. temperature, solvent, reaction time, etc. selected for the coupling reaction are determined by the nature of the acylation method used and are known to those skilled in the art. Generally it is useful tcS add an organic tertiary amine, e.g. triethylamine, N, N-dimethylaniline, ethylpiperidine, 2,6-lutidine or quinoline, to serve as a proton acceptor or salt-forming agent.

The compounds obtained by the process of the present invention may be isolated in any of the ways customarily employed for the isolation of similar cephalosporins. Thus, the product may be obtained as the neutral molecule, although this is probably more accurately represented as the zwitterion, or it may be isolated as a salt. Formation or the desired pharmaceutically acceptable carboxylic acid or acid addition salt is carried out by known methods, e.g. reaction of the acid with an appropriate base or acid.

At the conclusion of the acylation reaction the product obtained may be converted (before or after removal of the amino-protecting group) by methods known per se to another desired product of formula I. Thus, the compound of formula I in the form of the free acid or a salt thereof may be converted by known methods to a corresponding pharmaceutically acceptable salt thereof. Similarly, the product of formula I in the form of a salt thereof may be converted to the free acid product or another pharmaceutically acceptable salt by known methods. 43913/2 The preparation of the acylating agent of formula III, i.e. D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride, is described in Israel Patent Specification No. S077. which provides a process comprising the consecutive steps of (1) reacting D- (-) -2- (p-hydroxyphenyl) glycine with an excess of phosgene with heating in a suitable substantially anhydrous inert organic solvent as defined herein to form in solution the anhydride of the formula 0 removing excess phosgene from the reaction mixture; adding an excess of HCl gas to the cooled reaction mixture 43913/2 (4) recovering the desired D- (- ) -2- (p-hydroxyphenyl ) glycyl chloride hydrochloride.

In a preferred embodiment of the process described and claimed in Israel Patent Specification No . «_ί#77, the D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride is prepared by a process comprising the consecutive steps of (1) reacting D- (-) -2- (p-hydroxyphenyl) glycine having a particle size of less than 200 mesh with at least 1.6 moles of phosgene per mole of D- (-) -2- (p-hydroxyphenyl) glycine in substantially anhydrous dioxane with heating for the minimum time necessary to form the anhydride of the formula (2) removing excess phosgene from the reaction mixture as rapidl as possible after formation of the anhydride; (3) adding a large excess of HCl gas to the reaction mixture at a temperature in the range of about 0-5°C. for a period of time sufficient to form D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride; and (4) recovering the product from the reaction mixture.

The above process may be represented by the following flow diagram.

It can be seen from the above diagram that the process involves formation of an N-carboxyanhydride (Leuch's anhydride) by reaction of the p-hydroxyphenylglycine with phosgene and then conversion of the anhydride to the desired acid chloride hydrochloride by treatment with gaseous hydrogen chloride.

The Leuch's anhydride intermediate is found to gradually decompose in the presence of phosgene and. hence it is advantageous for maximum yields to have the reaction between the phosgene and p-hydroxyphenylglycine proceed as rapidly as possible and to remove excess phosgene from the reaction mixture as soon as possible after formation of the anhydride intermediate.

The amino acid, i.e. D- (-) -2- (p-hydroxyphenyl) glycine, and phosgene are reacted in an anhydrous inert organic solvent. Suitable solvents are those anhydrous organic solvents which are (1) substantially chemically inert toward the p-hydroxyphenylglycine and phosgene, (2 ) solvents for the Leuch's anhydride intermediate and (3) substantially non-solvents for the acid chloride hydrochloride end-product. Examples of 1 suitable solvents include dioxane, acetonitrile and tetrahydro- \ 2 furan. '. The solvent preferred for maximum yields is dioxane 3 since in addition to the above properties it has a boiling. 4 point sufficient to enable the reaction mixture to be heated 5 to the preferred temperatures as described below. A 6 substantially anhydrous solvent is used as the phosgene is 7 sensitive to moisture. We prefer to use solvents having a 8 moisture content of ½ 0.02% water, most preferably those 9 having a moisture content of ^ 0.01%. 10 For maximum yields, the reaction rate of the phosgene 11 addition step is maximized by proper adjustment of such factors 2 as particle size and concentration of the p-hydroxyphenyl- 13 glycine, temperature of the reaction mixture and concentration l^ and rate of addition of phosgene. The D- (-) -2- (p-hydroxyphenyl) - 15 glycine is preferably ground and screened to a finely divided ^ state so as to increase the surface area. Best results have ^ been achieved when the p-hydroxyphenylglycine has a particle l8 size of less than 200 mesh, i.e. the amino acid is ground so 19 that less than 3% is retained on a 200 mesh screen. An excess 20 of phosgene is used to decrease reaction time. The phosgene 21 is preferably used in an amount of at least 1.6 to 2.0 moles per mole of amino acid. The addition of phosgene to the p- hydroxyphenylglycine results in an exothermic reaction. To 2 increase the reaction rate and minimize decomposition, however, 25 the reaction mixture is preferably stirred and heat is added. 26 The most advantageous results have been obtained when the 27 phosgene is added as rapidly as possible with heating to about 28 60-80° C. for the minimum time necessary to form the anhydride. 29 Use of rapid phosgene addition and higher temperatures of about 30 60-80° C. for short time periods, e.g. 5 minutes, is found to be particularly advantageous for obtaining good yields when the reaction is conducted on a large scale. Best, results have been obtained when a fairly concentrated suspension of amino acid is employed, most preferably a concentration of about 10 g. amino acid per 80 - 100 ml. solvent.

Complete solubility of the amino acid is indicative of a complete reaction in the phosgene addition step. The reaction may also be monitored by appropriate techniques, e.g. thin layer chromatography, so as to indicate the minimum reaction time required for formation of the anhydride. By use of the preferred reaction conditions discussed above, practically quantitative yields of anhydride may be produced.

Since the Leuch's anhydride is found to gradually decompose in the presence of phosgene as mentioned previously, excess phosgene is preferably removed as rapidly as possible after completion of the phosgene addition step. Suitable methods for phosgene removal include purging with dry nitrogen gas and vacuum withdrawal.

Prior to addition of the gaseous HCl, it is found advantageous but not essential to remove a portion of the organic solvent, preferably by vacuum concentration. The solution may be concentrated so as to preferably remove up to a maximum of about 50-60% of the original solvent volume.

Since the chloride hydrochloride product generally is at least partially soluble in the reaction solvent, it has been found desirable to add an antisolvent prior to the HCl addition step so as to maximize recovery of the crystalline product.

Suitable antisolvents include such inert organic solvents as chlorinated hydrocarbons, e.g. methylene chloride, chloroform 1 or ethylene dichloride and aromatic hydrocarbons, e.g. benzene, 2 xylene or toluene. The most preferred antisolvents are toluene 3 and methylene chloride. Most advantageous results have been k obtained when the dioxane reaction mixture is concentrated to 5 a volume of up to about 50-60% of the original volume and 6 sufficient toluene or methylene chloride antisolvent added so 7 as to result in a solution having a dioxane :antisolvent 8 volume-volume ratio of about 7:3. 9 To convert the Leuch 1 s anhydride intermediate to the 10 desired chloride hydrochloride product, an excess of gaseous 11 hydrogen chloride is added. To increase the solubility of the 12 HCl, the anhydride solution is cooled, preferably to a 13 temperature in the range of about 0-5° C. Best results have l^ been obtained with a dioxane solvent when a large excess of HCl is used so as to completely protonate the dioxane. If ^ insufficient HCl is used, a solvate of the acid chloride ^ hydrochloride may form. This solvate may be used to acylate 18 in situ the 6-amino or 7-amino group of a penicillin or 19 cephalosporin nucleus but has been found to have poor storage > 20 stability. Accordingly, any solvate which forms is preferably 21 converted to the unsolvated acid chloride hydrochloride by 22 such procedures as (1) further gassing with additional HCl 23 or (2) filtering out the solvate and reslurrying in dry 24 methylene chloride. 25 g Recovery of the (p-hydroxyphenyl^ glycyl chloride hydro- 2γ chloride is carried out according to conventional procedures. 28 During or following HCl addition, seed crystals of the desired 2 product are preferably added to induce crystallization. 30 Progress of the HCl addition step may be followed by periodic testing with thin layer chromatography until conversion to the , j acid chloride hydrochloride is indicated. After sufficient HCl has been added, the solution is allowed to gradually warm to room temperature so as to allow slow crystallization and forma-tion of the desired heavy dense crystalline product. Chilling of the crystal slurry should be avoided so as to prevent solvate formation.

The crystalline product is filtered, washed, e.g. with toluene, dioxane, methylene chloride, and dried to give yields of up to about 95% of excellent product when the preferred reaction conditions are followed.

The D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride may be used to acylate the 6-amino or 7-amino* groups of 6-aminopenicillanic acid or 7-aminopenicillanic acid or derivatives thereof, e.g. derivatives of 7-aminocephalosporanic acid in which the acetoxy group is replaced by other nucleophiles by methods known in the literature. 43913/2 The pharmaceutically active compounds obtained by the process of the, present invention exhibit highly desirable solubility in water, are efficiently absorbed on oral administration to provide relatively high and prolonged blood levels and are potent antibacterial agents useful in the treatment of infectious diseases in poultry and animals, including man, caused by many Gram-positive and Gram-negative bacteria. The active compounds are also of value as nutritional supplements in animal feeds and as agents for the treatment of mastitis in cattle.

The medicaments provided by the process of the present invention may be formulated as pharmaceutical compositions comprising, in addition to the active ingredient, a pharmaceutically acceptable carrier or diluent. The compounds may be administered both orally and parenterally. The pharmaceutical preparations may be in solid form such as capsules, tablets or dragees, or in liquid form such as solutions, suspensions or emulsions.

In the treatment of bacterial infections in man, the compounds of this invention may be administered parenterally in an amount of from about~5 to-200: mg; kg;/day 'and preferably about~5 to -- 20 mg./kg./day in divided dosage, e.g. 3 to 4 times a day. They are administered in dosage units containing e.g. 125, 250 or 500 mg. of active ingredient with suitable physiologically acceptable carriers or excipients. 43913/2 The compounds obtained by the process of the present invention have surprisingly been found to possess potent antibacterial activity and at the same time excellent absorption upon oral administration. The combination of the particular D-(-) p-hydroxyphenylglycine sidechain on the 7-amino group and the particular l,2,3-triazol-5-ylthioraethyl group at the 3-position of the cephalosporin molecule has unexpectedly resulted in the highly advantageous properties of the compounds obtained by the process of the present invention.

STARTING MATERIALS Synthesis of potassium l,2,3-triazole-5-thiolate The synthesis of ...the thiol was accomplished by a procedure essentially identical to that described in the literature [j. Goerdler and G. Gnad,Chem. Ber. Q, l6l8 (1966)] 5-Benzamido-l .2. -thladiazole ; To a stirred solution of benzoylisothiocyanate (50.6 g., . 310 mmoles) in commercial anhydrous ether (400 ml.), maintained at ' 0° and in a nitrogen atmosphere, was added dropwise with vigorous ' stirring, Ο.685 N ethereal diazomethane (453 ml., 310 mmoles). When the addition was completed, the mixture was stirred for 1 hour at 0°, the solid' was collected by filtration and dried in vacuo. The melting point of the crude material (23.3 g.) thus obtained was observed somewhere in the region 232 to 257°. Goerdler reported M.p. 267° for the pure material. A small second crop (2.1 g.) was ob- tainted by evaporation of the mother liquor in vacuo. The total yield was therefore 0#. l.2. -Trl3zole-5-thiol.

A solution of the above benzamido compound (8.2 g., 40 ramoles) in 2N sodium hydroxide (80 ml., l6o mmoles) was heated under reflux temperature in a nitrogen atmosphere for 24 hours. The so- lution was cooled to 0° in ice, and concentrated hydrochloric acid (26 ml.) was added, while a continuous "stream of nitrogen was passed through the solution. The benzoic acid which precipitated was removed by filtration. The filtrate was immediately extracted with ethyl acetate, the extract was washed with saturated salt solution, dried over magnesium sulfate and then evaporated in vacuo . The viscous oil which remained was immediately evapora-tively distilled in vacuo (70-75°/0.001 mm.) to give an oil. (2.84g., 70 ) which solidified (m.p. 52-59°; Goerdler reported m.p. 60°) spontaneously.

Potassium 1.2.VTriazole-5-thiolate To a solution of the above thiol (2.84 g., 28.1 mmoles) in absolute ethanol (28 ml.) was added 1.93 N alcoholic potassium hydroxide solution (14.5 ml.). The solution was then diluted with anhydrous ether until crystallization of the salt was completed. The solid was collected by..filtration, washed with ether, and dried in vacuo. The salt obtained in this manner (3.65 g.» $) . had m.p. 225° with decomposition.

It is important to note that the conversion of the benzamido thladiazole to the triazole thiol is kno'wn to proceed vla.5-aTnino-l,2,5-thiadiazole CG. Goerdler and G. Gnad, Chem. Ber. £2, 1618 (1966) ] . ' 5-ATnino-l,2,3-thiadiazole can be prepared by an alternative route not involving dlazomethane [D.L. Pain and R. Slack, J. Chem. Soc. 5166 (1965)] - HC02Et 1 7-Amlno-3- (l , 2 .3-triazol-5-ylthlomethyl) -3-cephem-4-carboxyllc 2 acid. (II). 3 The reactions were conducted under a nitrogen atmosphere A in a reaction vessel protected from light. The water and 5 phosphate buffer were gassed vigorously with nitrogen prior 6 to use to displace oxygen. 7 5-Aminp-l,2,3-thiadiazole (10.3 g., 0.102 mole) was added 8 to a solution of 8. l6 g. of sodium hydroxide in 100 ml. of water. The mixture was heated rapidly to reflux and then re- 10 fluxed for 10 in. to rearrange 5-amino-l,2,3~thiadiazole to H 5"-mercapto-l,2,3-triazole . To the reaction mixture containing 12 5~niercapto-l,2,3-triazole cooled in an ice bath was added 13 1100 ml. of ice cold 0.1M pH 6.4 phosphate buffer. The 1 solution, which was at pH 10.5 » was adjusted to pH 8.5 with 15 2$ phosphoric acid. 7-Aminocephalosporanic add (21.8 g., 16 0.08 mole) was added and the mixture heated at 50° for 4 hours ΐγ The clear solution was cooled in an ice bath and adjusted to 18 PH ^ .5 with cone. HC1. The precipitated product was collected 19 by filtration, washed with water and air dried; l6.2 g. 20 The crude product ( 15 .2 g.) was brought into solution 1 with 600 ml. of methanol and 40 ml. of cone. HC1. After 2 carbon treatment the solution was diluted with 1.5 1. of 3 ice water and extracted once with ethyl acetate. The aqueous phase was concentrated at reduced pressure to remove methanol. 5 The cold aqueous concentrate was adjusted slowly to pH 4.0 6 with 20 sodium hydroxide causing crystallization of the 7 product. The product was collected by filtration, washed 8 with water and methanol and dried in. vacuo over phosphorus Anal. Calcd.: for c10HnN5¾S2: c> 38.42; H, 3.55; N, 22.40.

Found : C, 38.27, 38.26; H, 3.76, 3.40; N, 2¾D2, 21.00; HgO, 1.70.

- Purification of 7-Amino- -(l.2. -trlazol-5-ylthlomethyl)-3- cephem-4-carboxylic acid (II) .

Crude 7-amino-3-(l,2,3-triazol-5-ylthiomethyl)-3- cephem-4-carboxylic acid (l6.1 g.) containing approximately 20 mole fo of 7-aminocephalosporanic acid as an Impurity, was " brought into solution with 600 ml. of methanol and 40 ml. of cone. HC1. After carbon treatment, the solution was diluted with 1.5 1. of ice water and extracted once with ethyl acetate. The aqueous phase was concentrated at reduced pressure to remove methanol. The cold aqueous concentrate was then adjusted slowly to pH 4.0 with 20 sodium hydroxide causing the product to crystallize. The product was collected by filtration, washed with water and methanol and dried in vacuo over, phosphorus pent oxide; 11.4 g. The NMR spectrum indicated that this product contained abo t 7 mole # of 7- aminocephalosporanic acid as an impurity.

The above purification procedure was repeated on 11.4 g. of the product using 425 ml. of methanol, 28 ml. of cone. HC1 and 1 1, of ice water yielding 8.0 g. of product. The NMR spectrum was fully consistent for the desired product and indicated no trace of 7~aminocephalosporanic acid as an impurity.

Anal. Calcd. for c10Hi5N5°5S2: c'' 38.42; H, 3.55; N, 22.40. ound: C 06 8 H 6 . . .·■ V'.v 7-Amlno^ -[S-(l.2. trlazole-5-yl)-thiomethyl1-3-cephem-4- carboxylic acid. (IlV Ten grams (0.075 mole) of 5-mercapto-l,2,3-triazole potassium salt was added to a stirred slurry of 19 g. (0.07 mole) of purified 7~aminocephalosporanic acid and 5.9 g. (0.07 mole of NaHCO^ in 3 0 ml. of 0.1M phosphate buffer (pH 6.4) and the mixture heated and stirred at 55° C. for 3 1/2 hours under a nitrogen atmosphere. The resulting solution was cooled to 22° C. and the pH adjusted to 5.5 with 4C$ Η,ΡΟ^. The resulting precipitate was filtered off, washed with cold water (50 ml.) \ and air dried. The yield of 7-amino-3-[S-(l,2,3-triazole- 5-yl)-thiomethyl3-3-cephem-4- carboxylic acid was 8 g., dec. ,...-. pt. 230° C. IR analysis showed some decomposition of the β- ' lactam ring but it was used "as is" for the next step.

Anal. Calcd. for c10HnN °3S2: C' 38.39 H, 3.54.

Found : C, 38.36; H, 3.78. 7-Amino- -(l .2.3- trlazol- -ylthloTriethyl)- -cephem-4- carboxylic ' acid (II).

' Two hundred seventy-two g. (l.O mole) of 7~amino- cephalosporanic acid was suspended in 3000 ml. of 0.1M phosphate buffer, pH 6.4, and 150 ml. of methyl isobutyl ketone followed by 84 g. (1.0 mole) of sodium bicarbonate (Note: The sodium bicarbonate was added in portions). Then 143 g. (1.0 mole) of 5~mercapto-l(H) -l,2,3~triazole potassium salt was added and the mixture stirred at 55° C. ± 1° C. under a nitrogen atmosphere for 4 hours. After 1 hr. the pH was readjusted to 6.4 by addition of a small amount of 40# H^PO^. At the end of the 4 hr. heating period, 50 g. of "Dare o KB" decolorizing ("Celite") pad. The pad was washed with 3 x 100 ml. water. the pH of the combined filtrates was adjusted. while hot to 4.5 by slow addition of 6 N HCl. After cooling 30 min. at 0° C, the crude product was collected by filtration, washed with 2 x 200 ml. cold water followed by 2 x 1000 ml. methanol and air dried.

The crude product was suspended in 000 ml. of 50 methanol-water and 300 g. (1.5 mole) of jD-toluenesulfonic acid was added. It was stirred for 15 min. and then 50 g. of "Darco KB" decolorizing charcoal was added. After stirring for 15 min. at 22° C, the. slurry was filtered through a "Celite"' pad and the pad washed with 2 x 100 ml. of 50$ methanol-water.

The pH of the combined filtrates was adjusted to.4.0 by addition of approximately 210 ml. of triethylamine . After cooling at 0° C. for 1 hour the product was collected by filtration, washed with 2.x 400 ml. 50 methanol-water and then 2 x 1000 ml. . methanol. It was air dried.

This material was suspended in 2000 ml. water and 84 g. ( 1 mole) of sodium bicarbonate was added. After stirring for io min. at 22° C, 50 g. of "Darco KB" charcoal was added and, after stirring for 15 min. at 22° C, the slurry was filtered through a "Celite" pad. It was washed with 2 x 100 ml. water and the pH of the combined filtrates was adjusted to 3.5 by slow addition of 6 N HCl. After stirring for 10 min. at 22° C, it was cooled to 0° C. for 1 hr. The product was collected by filtration, washed with 2 x 200 ml. cold water and 2.x 1000 ml. acetone. After drying over ?2 5 n'a vacuum desiccator for Ψ 14 hr., at room temperature, the yield was 100 g; dec. pt. 230° C. 43913/2 .

■Anal. Calcd. for C.AH_ -N_0_S-«l/2 Ho0: C, 37.51; 1U 11 3 j d d H, 3.75; , 21.68.

KF (HpO), 2.8 Found: C, 37.78; H, 3.69; N, 20.42.

KF (H20), 2.46 The following examples are given in illustration of, but not in limitation of, the present invention. All temperatures are in degrees Centigrade. 7-Aminoeephalosporanic acid is abbreviated as 7-ACA, methyl isobutyl ketone as MIBK and tetra-hydrofuran as THF. "Skellysolve B" is a petroleum ether fraction of B.P. 60-68° C. consisting essentially of n-hexane. • 1 Example 7-rD-g-Amlno-g-f D-hvdroxyohenyl)acetamldo]- ~ rs-' f1.2."¾- trlazole-5-γΐ thiomethyl3-3-ceohem-4-carboxylic acid To a stirred suspension of 95. g. (0.3 mole) of 7-amino-3-(l;,2, 3-triazol-5-ylthiomethyl) -3-cephem- - carboxylic acid in l400 ml. of .dry CHgClg is added 81 ml. (0.58 mole) of triethylamine followed by 50 ml. (0.39 mole) of N,N~dimethylaniline. After cooling to 12° C., 76.2 ml. (0.6 mole) of trimethylchlorosilane is added over a 15 min. period. After another 15 min. the mixture is slowly brought to reflux and, after 5 min. refluxing, the almost clear solution is cooled to 5° C. where 0.35^ mole of D-(-)-a- amino-g- (p-hydroxyphenyl) -acetyl chloride hydrochloride is added in portions over a 30 min. period. The mixture is stirred 1 hr. at 5° C. and 1 hr. with the ice bath removed (temp. max'. 18° C). One liter of water is added and, with good stirring, the pH is slowly adjusted to 2.2 with 20$ NaOH.

The aqueous phase is separated and the CHgClg layer and gummy ppt . ' discarded. Under a" layer of ether (l liter), the pH of the solution i . · Is adjusted to 2 with good stirring. The aqueous phase is stirred 15 min. with 20 g. of "Darco KB" (carbon), filtered /a . . and the pH, under a Iyer ( 1 liter) of fresh ether is adjusted to 4. After seeding and stirring 1 hr. at 20° C , the crystals are filtered off, washed well with water, then acetone and air dried. After vacuum drying over * there is obtained solid - [D-g-amino-g- (p-hydroxyphenyl)acetamido]-3~ [S- ( 1,2, 3- triazole-5~yl) -thiomethyl] -3-cephem 4-carboxylic acid. 43913/2 Example 2 Sodium 7-[P- f -amino-q-p-hydroxyphenylacetamldo) 1-3- [S-( 1.2.3-triazole-5-yl)-thio~ethyl1-3-cephem-4-carboxylate To a stirred aqueous suspension of the zwitterionic form of 7-[D-(g-amino-g-p-hydroxyphenylacetamido) 1-3"[S-(l,2,3-triazole-5-yl)-methyl]-2-cephem- -carboxylic acid (0.8 mmole) is added 1 N aqueous sodium hydroxide at room temperature until a clear solution (pH 10.8) is obtained. This solution is immediately freeze-dried to give impure, solid sodium 7- [D- (a-amino-a-p-hydroxyphen lacetamido) ]-3~[S-( 1,2,3"triazol-5-yl) thiomethyl]-3-cephem- -carboxylate .

Example 3 7-.D- (-) - -Amino- - f o-hvdroxyohenyl)acetamidol -3-[S-( 1.2.3-triazole- -yl) -thiorneth 13-3-cephem-^-carboxylic acid To a stirred suspension of 16 g„ (0.05 mole) of 7-amino-3-[S-(l,2,3-trlazole-5-yl)-thiomethy¾.]-3-cephem-4-carboxylic acid, in 150 ml. of methylene chloride is added 3.5 ml. (0.092 mole) of triet ylamine, 15 ml. (Q.118 mole) of Ν,Ν-dimethylanillne and, with cooling (7° C to 15° C.) 19. 1 ml. (0.15 mole) of trimethylchlorosilane . After 15 min. cooling the mixture is refluxed for 25 min., cooled to 5° C. and Ο.θβΐ mole of D-(-)-a~amino-a-(p-hydroxyphenyl)-acetylchloride hydrochloride is added. The mixture is stirred at 10° C. to 12° C. for 1 hour and then 150 ml. of water was added and, after 15 min. stirring, the mixture is filtered, the aqueous phase separated, the pH adjusted to 2 with 20 NaOH, carbon ( 10 g. of "Darco KB") added and, after 15 min. stirring, the mixture is filtered. The pale yellow solution is layered with ether (150 ml.) and, with goqd stirring, the pH is adjusted to 4 with 20 NaOH. The aqueous phase is separated and filtered and 0 ml. of acetonltrile added to it. Scratching or seeding induces crystallization and gives solid 7-[l>- (-)-a-amino-a-(p-hydroxyphenyl)-acetamido]-3-[S- ( 1, 2, 3-triazole-5-yl) -thio-meth l]-3-cephem-4-carboxylic acid. . . . 4 Fxample ft , Purification of 7-[D- -amino-a- (p-hydroxyphenyl^acetamido]-^- .(,1 ,2.3-triazol-5-ylthlomethyl) -3-cephem-4-carboxylic acid (methanol crystallization) .

Four hundred mg. of amorphous 7-[D-a-am.ino-a-(p-hydroxy- phenyl)acetamido]-3-(l,2,3-triazol-5-ylthiomethyl)-3-cephem- 4-carboxylic acid was dissolved in warm 95 methanol-HgO (20 ml.), filtered and scratched. After four hours the white crystalline material was collected by filtration, washed with methanol "and air dried. After further drying over there was obtained 100 mg; dec. pt. 190° -220° C. indefinite. The NMR spectra was consistent for the desired structure and indicated about one mole of methanol present.

Anal. Ca led. for C^gH^NgO^: C, 1*6.65; H, 3.91 J N, l8.l4.

Found : C, 44.23; H, 4.64; N, .15.35.

(Uncorrected for 5.99$ found by Karl Fisher method, also uncorrected for methanol) .

Example 10 Prepa ation of 7- [.D-a-amlno-a- ( o-hydroxyphenyl acetamido]-3-' (l .2.3-triazol- -ylthiomethyl)-3-ceohem- -carboxylic acid via the acid chloride hydrochloride.

To a stirred slurry of 6.26 g. (0.02 mole) of 7-amino-3- (l,2,3-triazole-5-yl) thiomethyl-3-cephem- -carboxylic acid in 150 ml. of CHgClg'was added 5.6 ml. (0.04 mole) of triethyl-. amine, 5 ml. of Ν,Π-dimethylaniline and 7-62 ml. (0.06 mole) of chlorotrimethylsilane, dropwise and then the mixture was slowly brought to reflux and refluxed for about one and one half hours. An almost clear solution was obtained. Next, 4.44 . 0.02 mole , of D- - -2- 4-h drox phen l) l cine acid 43913/2 a JO min. period. After 2 hours stirring at 22° C, 20 ml. of methanol was added and after >0 min..50 ml. of water was added with good stirring. The pH was then adjusted to 2.1 with 20 NaOH and the solids filtered off. The aqueous laye was then separated and stirred 10 min. with 2 g. of. decolorizing carbon ( "Darco-KB") , filtered arid the pH adjusted to 4 with 20 NaOH under a 50 ml. layer of ether. The aqueous phase was separated and an equal volume of methanol added and the solution seeded. After standing overnight at about 10° C. there was obtained 400 mg. of 7- [D-a-amino-a- (p-hydroxyphenyl)acetamido]"5-(lj2J3-triazol-5-ylthiomethyl)-3-cephem- -carbox lic acid.

Example £β Preparation of D- (-) -2- (p-hydroxyphenyl) lycyl chloride hydrochloride 10.0 g. (^0.06 mole) of D- (-) -2- (p-hydroxyphenyl) glycine was slurried in 100 ml. of dioxane. The slurry was stirred and COClj (phosgene) was passed in while the slurry temperature was held at 50-58° C. The C0C12 was passed in for a total time of 3.5 hours. A yellow solution was obtained.

The solution was purged with nitrogen to expel the excess COClj. HC1 gas was bubbled through the solution for 2.5 hours. The solution was stirred and a small amount was diluted with some ether to obtain some crystals which were added to the batch as seed. The solution was stirred at 20-25° C. for 16 hours. The resulting slurry of crystalline D-(-)-2- p-hydroyxphenyl glycyl chloride hydrochloride was filtered to collect the product. The filter-cake was washed with dioxane and methylene chloride and then dried in a vacuum desiccator over P2°5* T^e Y^e^ of the tit e product was 7.3 g. The IR spectrum indicated that excellent product was obtained. Elemental Analysis: CI H N Theory 31.93 43.14 09 37 Found 31.96 42.46 22 56 Acid Chloride Assay Acid Chloride 98.6% Free COOH None Free HCl None Example ]Ll Preparation of Mono-Dioxane Solvate of D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride 20 g. of D- (-) -2- (p-hydroxyphenyl) glycine was slurried in 200 ml. dioxane. Phosgene gas was passed in for a period of 3.75 hours and the reaction mixture was heated up during this period to a temperature of about 50° C. The yellow solution and the presence of a single zone by thin layer chromatography (TLC) indicated that the reaction had reached completion. The solution was purged with and cooled to about 6° C. HC1 gas was added for about 130 minutes and the temperature was maintained at between -3° C. and 6° C.

Seed crystals were added to the solution approximately 90 minutes after the start of HCl addition. The solution was allowed to warm up to about 15° C. and stirred overnight. A heavy slurry of crystals formed. The system was slowly placed under vacuum whereupon HCl was removed. The slurry was stirred at room temperature for several hours, filtered, washed with dioxane and methylene chloride and dried in a vacuum desiccator over P2°5 to ^el^ 26.4 g. of white product. The product was found to be the monodioxane solvate of D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride.

Assay Acid Chloride 62% COOH 4.5 Free HCl 0.4 Dioxane 29.5 Example jl8 K Preparation of Acetonitrile Solvate of D- (-) -2- (p-hydroxyphenyl) -qlycyl chloride hydrochloride 10.0 g. D-(-) -2-(p-hydroxyphenyl) glycine was slurried in 100 ml. acetonitrile. The suspension was stirred and phosgene was added for a period of about 25 minutes. The reaction mixture was then heated to about 46° C. with continued addition of COClj for another 2 hours. The solution was cooled to about 1° C. and stirred for about 15 minutes. The solution was then allowed to warm up to room temperature and additional COClj was added with heating to 46° C. until a total of 193 g.

COCI2 had been used. Excess COCI2 was removed by purging with and the solution was chilled. HCl gas was added and the solution was seeded with crystals of p-hydroxyphenylglycyl chloride HCl. After HCl addition for 5.75 hours at about 4-6° C, there was added 25 ml. acetonitrile and the crystals which had formed began to dissolve. The reaction mixture was stirred for several hours, seeded and crystals formed.

The mixture was stirred overnight, filtered, washed with acetonitrile and methylene chloride and dried to yield 4.2 g.

The IR indicated the product was the acetonitrile solvate of by gas liquid chromatography (G.L.C.) showed 15.6% acetonitrile content which is in excellent agreement with the theoretical percentage of 15.6 for the solvate.

Example JL9 Preparation of D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride using finely divided amino acid 10.0 g. of D- (-) -2- (p-hydroxyphenyl) glycine having a particle size of less than 200 mesh was slurried in 100 ml. dioxane.

Phosgene was passed in for 10 minutes and then the reaction mixture was heated up to 64° C. with additional phosgene addition until a total of 1.8 moles C0C12 had been used per mole of amino acid. After completion of the reaction and expulsion of the excess COCl2, the solution was concentrated under vacuum. Toluene (25 ml.) was added and the solution was brought to a volume of 80 ml. with dioxane. The solution was chilled and slowly gassed with HCl for approximately 1 hour. Seed crystals were added and the reaction mixture was stirred and allowed to warm to room temperature. After continued stirring for several hours, the crystals were filtered, washed and dried over P205 to yield 11.65 g. (87.5%) of the title product.

Preparation of D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride usincr 2.0 moles phosgene per mole amino acid 100 g. D- (-) -2- (p-hydroxyphenyl) glycine screened to 200 mesh was slurried in 1000 ml. dioxane. C0C12 was added for about 15 minutes (123 g.) and the mixture heated to about 62° C. until the reaction was shown by TLC to be essentially complete. Excess C0C12 was removed with N2 and the solution was concentrated under vacuum to a volume of about 550 ml. To this concentrated solution was added 250 ml. toluene.

The solution was cooled to 5° C. and HCl gas (190 g.) bubbled in slowly at 0-5°C. for 2 hours. After stirring at 3° C. for 20 minutes, the reaction mixture was seeded and stirred for several more hours to form a heavy slurry. The slurry was filtered, washed with dioxane-toluene and methylene chloride and dried over ^2°5 to form 112 g* (84·2%) of the title product. 11 1 Example ti 2 Preparation of D- (-) -2- (p-hydroxyphenyl) glycyl chloride 3 hydrochloride A. Preparation of Touc 's Anhydridp o n- (-) -2- (o-hvdroxv- 5 phenyl) glycine 6 100 g. of D- (-) -2- (p-hydroxyphenyl) glycine (screened so that 7 less than 3% was retained on a 200 mesh screen) was slurried 8 in 1000 ml. dioxane. Phosgene was added for 25 minutes 9 with heating up to about 80° C. whereupon an orange solution 10 was produced. Excess phosgene was removed by house vacuum and 11 the reaction mixture concentrated under vacuum to a volume 2 of approximately 420 ml. Dioxane was added to give a volume 13 of 500 ml. TLC indicated the presence of only Leuch's I'* anhydride in the reaction mixture. 1C5 B. Preparation of chloride hydrochloride using methylene l6 chloride - dioxane mixed solvent ^ To 125 ml. of the Leuch's anhydride solution from Step A was added 62 ml. methylene chloride and 13 ml. dioxane. The solution ^ was stirred, chilled to 5° C. and 50 g. HC1 gas added over a 40 20 minute period at 0 - 6° C. After stirring and seeding, a slurry 21 of crystals formed. The reaction mixture was allowed to warm to 22 room temperature and stirred overnight. The crystals were washed 23 twice with 200 ml. dioxane-methylene chloride and 300 ml. ^ methylene chloride and dried in a vacuum desiccator over 1?2Q5 25 to give 28.4 g. of title product (85.5% yield). 26 Assay 27 Acid chloride = 92.9% 28 Free COOH = 2.6% 29 Free HCl = 0.6% 3° Dioxane = 1.06% 1 C. Preparation of chloride hydrochloride using toluene- { 2 dioxane mixed solvent 3 The procedure of Part B was repeated except that 65 ml. 4 toluene was substituted for the 62 ml. methylene chloride used 5 therein. There was produced 28.7 g. (86.7% yield) of dried 6 product which was characterized by IR as being the desired 7 chloride hydrochloride. 8 Assay 9 Acid chloride = 93.9% 0 Free COOH = 3.6% 1 Free HCl = 0.5% 2 D. Preparation of chloride hydrochloride via dioxane solvate 3 To 125 ml. of the Leuch's anhydride solution from Step A was added 12 ml. dioxane and 65 ml. toluene. The solution was 15 chilled to 0° C. and 26 g. HCl added over a 30 minute period 1°" at 2° C. After stirring and allowing the solution to warm ^ to room temperature, a heavy slurry of crystals formed which were found by microscopic examination to be needles of the dioxane solvate of D- (-) -2- (p-hydroxyphenyl) glycyl chloride HCl. 20 An additional 60 g. HCl gas was added as the temperature was 21 gradually lowered to 7° C. and the solvate crystals were 22 seen to break up. Upon seeding, the desired chloride hydrochlor23 ide crystals formed and after filtering, washing and drying, 2U there was produced 29.7 g. (89.5% yield) of product. IR 25 O indicated little or no COOH and good )* . 26 C - Cl. 7 Assay 28 Acid chloride = 90.4% 29 COOH = 4.4% 30 HCL = 1.4% Dioxane = 1.49% Exampleβ$.

Preparation of D- (-) -2- (p-hydroxyphenyl) qlycyl chloride hydrochloride , using slower HCl addition and dioxane-toluene mixed solvent To 125 ml. of Leuch's anhydride solution prepared according to Example 21A was added 13 ml. dioxane and 70 ml. toluene. The solution was stirred, chilled to 0°C. and gassed at 0-3° C. with 50 g. HCl over a 75 minute period. After stirring and seeding the chilled solution, a heavy slurry of crystals formed which when filtered, washed and dried gave 31.3 g. (95.5% yield) of excellent chloride hydrochloride product. Assay Acid chloride = 92.3% COOH = 5.9% HCl » 1.6% Dioxane = 1.12% Example Preparation of D- (-) -2- (p-hydroxyphenyl) qlycyl chloride hydrochloride using dioxane solvent .12- The procedure of Example was repeated except that (1) 10 ml. dioxane was added instead of the mixture of dioxane and toluene, (2) 70 g. HCl gas was used instead of 50 g. and (3) the solution was gassed for about 3 hours instead of 75 minutes. There was produced 22.7 g. (68.5% yield) of dried product which was identified by IR as being the desired chloride hydrochloride.

Claims (4)

CLAIMS t

1. A process for the preparation of a compound having the D-(-) configuration in the side chain of the formula I and pharmaceutically acceptable salts thereof; which process comprises reacting a silyl derivative of a compound of the formula II with an acylating derivative of the formula to produce, after removal of the proton from the amino group, a compound of formula I or a pharmaceutically acceptable salt thereof and, if desired, either before or after removal of the silyl blocking groups (a) converting by methods known per se the product in the form of the free acid thereof to a corresponding pharmaceutically acceptable salt thereof or (b) converting by methods known per se the product in the' form of a salt thereof to the corresponding free acid compound or another pharmaceutically acceptable salt thereof.

2. A process according to Claim 1, wherein the acylating derivative of formula III in Claim 1 is prepared by the consecutive steps of: 1) reacting D- (-) -2- (p-hydroxyphcnyl) glycine with an excess of phosgene with heating in a suitable substantially anhydrous inert organic solvent as defined herein to form in solution the anhydride of the formula 2) removing excess phosgene from the reaction mixture; 3) adding an excess of HCl gas to the cooled reaction mixture 4) recovering the desired D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride. 43913/3

3. The process according to Claim 1 wherein the acylating derivative of formula III in Claim 1 is prepared by the consecutive steps of (1) reacting D- (-) -2- (p-hydroxyphenyl) glycine having a particle size of less than 200 mesh with at least 1.6 moles of phosgene per mole of D-(-) -2- (p-hydroxyphenyl) glycine in substantially anhydrous dioxane with heating from about 60-B0°C. for the minimum time necessary to form the anhydride of the formula (2) ' removing excess phosgene from the reaction mixture as rapidly as possible after formation of the anhydride; (3) adding a large excess of HCl gas to the reaction mixture at a temperature in the range of about 0 - 5° C. for a period of time sufficient to form D- (-) -2- (p-hydroxyphenyl) glycyl chloride hydrochloride; and recovering the product from the reaction mixture

4. A compound of formula I in Claim 1 or a salt thereof when prepared according to the process of any of Claims 1-3.