IL21947A - 6-alpha-amino acetamino-thiopenicillanic acid - Google Patents

Description

This invention relates to new synthetic compounds of value as antibacterial agents, as nutritional supplements in animal feeds, as agents for the treatment of mastitis in cattle and as therapeutic agents in poultry and animals, including man, in the treatment especially of infectious diseases caused by Gram-positive bacteria. More particularly, this invention relates to 6-(a-aminophenylaceta-mido)-, 6-(a-aminonaphthylacetamido)- and 6-i a-amlno-a-(2- or 3-thienyl)acetamido7thiopenicillanic acids and the salts thereof. In another aspect, this invention relates to 6-aminothiopenicillanlc acid which is useful as an intermediate in the preparation of the thiopenicillins of this invention and other thiopenicillins, e.g., described in United States Patent No. 2,751,378. The intermediate, 6-aminothiopenicillanic acid, is acylated to the desired thiopenicillins according to procedures known in the art, e.g., the acid chloride and mixed anhydride procedures which are described in many United States and foreign patents such as listed in the following discussion of the invention.

Antibacterial agents such as benzylpenicillln have proved highly effective in the past in the therapy of infections due to Gram-positive bacteria but such agents suffer from the serious drawbacks of being unstable in aqueous acid, e.g., upon oral administration, and of being ineffective against numerous so-called resistant strains of bacteria.

It is an object of the present invention to provide compounds useful in the treatment of infections caused by Gram-positive bacteria, including particularly resistant strains of bacteria, e.g., benzyl-penicillin-resistant strains of Staphylococcus aureus (Micrococcus pyogenes var. aureus ) . It is a further object of the present invention, to provide compounds which, in addition to their potent anti-bacterial activity, exhibit resistance to destruction by acid. It is a still further object of the present invention to ro idia ' intermediate useful for the preparation of thiopenicillins.

The objects of the present invention have been achieved by the provision of 6-aminothiopenicillanic acid and by the provision of a member selected from the group consisting of acids having the formula g - SH II 0 wher ein R is hydrogen or a member selected from the group consisting of radicals of the formulae 1 wherein R , R and ^ are each a member selected from the group consisting of hydrogen, chloro, bromo, fluoro, iodo, nitro, sulfamyl, trifluoromethyl, (lower )alkyl, ( lowerJalkoxy, (lower)alkylthio, di( lower)alkylarnino, ( lower)alkanoylamino, ( lower)alkanoyl, (lower)alkanoyloxy, (lower)alk lsulfonyl, henyl and benzyl, wherein 4 5 p and R are each a member selected from the group consisting of hydrogen, chloro, bromo, fluoro, iodo, nitro, ( lower)alkoxy, phenoxy, benzyloxy, trifluoromethyl, 6 7 (lower)alkanoylamino and (lower)alkyl; and wherein R , R and R are each a member selected from the group consisting of hydrogen, chloro, bromo, fluoro, iodo, nitro ( lower)alkyl, ( lower)alkoxy, phenyl, phenoxy, benzyloxy, ( lower)alkylthio, di( lower}alkylamlno, ( lower)alkanoylamino, (lower)alkanoyl, ( lower)alk lsulfonyl, cycloalkyl radicals having from 5 to 7 carbon atoms inclusive, cy-cloalkoxy radicals having from 5 to 7 carbon atoms inclusive; and the pharmaceutically acceptable nontoxic salts thereof. The pharmaceutically acceptable nontoxic salts include nontoxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and substituted ammonium salts, e.g., salts of such nontoxic amines as trialkylamines, including triethylamine, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, 1-ephenamine, N, ' -dibenzylethylene-diamlne, dehydroabietylamine, N, f-bis-dehydroabietylethylenediamine, N-(lower)alkylpiperidines, (e.g., N-ethylpiperidine ) and other amines which have been used to form salts with benzylpenicillin. Also included within the scope of this aspect of the present invention are easily hydrolyzed esters which are converted to the free thio acid form by chemical or enzymatic hydrolysis.

It is apparent that 6-aminothiopenicillanic acid may be readily converted to any of the pharmaceutically acceptable nontoxic salts described above, or to any of the other salts commonly formed by organic acids, i.e., magnesium, iron, lead, tin, copper, nickel, cobalt, mercury, and the like. The salts of 6-aminothiopenicillanic acid are also, therefore, considered to be within the scope of this invention, and all references to 6-aminothiopenicillanic acid herein are intended to include the salts thereof.

The term "( lower)alkyl" as used herein means both straight and branched chain aliphatic hydrocarbon radicals having from 1 to 10 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, amyl and hexyl. Similarly, where the term "(lower)" is used as part of the description of another group, e.g., "( lower)alkoxy", it refers to the alkyl portion of such group which is therefore as described above in connection with " (lower)alkyl" . To illustrate groups including (lower)alkyl groups, it is pointed out that lower alkoxy includes such radicals as methoxy, ethoxy ^ and isopropoxy; ( lower)alkanoyl includes such radicals as acetyl, propionyl and butyryl; (lower) alkylsulfonyl includes such radicals as ciethylsulfonyl, ethylsulfonyl and hexysulfonyl ; di(lower) alk lamino includes such radicals as dimethylamino, dieth lamino and e h lmeth lamino.

In those compounds where R is other than hydrogen the o-carbon atom of the acyl group (to which the amino group is attached) is an asymmetric carbon atom. Thus, the compounds of this invention can therefore exist in two optically active isomeric forms (the D- and L-diastereoisomers) , as well as in a mixture of the two optically active forms. All such isomeric forms of the compounds are included within the scope of the present inven io .

It should be noted in connection with the foregoing consideration of the diastereoisomers of this invention that many isomers other than t - e two caused by the asymmetric carbon of the side chain are possible due to the presence of asymmetric carbon atoms in the 6-aminopenicillanic acid nucleus. Such additional isomers, however, are not presently significant since 6-aminopenicillanic acid which is the product of fermentation processes is consistently of one configuration and such 6-aminopenicillanic acid is presently used in the production of the compounds of this invention.

The compounds of the present invention exist in tautomeric forms which can be represented as follows: 0 C ITCH - σ - SH (I 0 (thiol form) (VI) OH tt (thione form) For this reason, the products of the present invention are described by the use of the term "thio" , e.g., the compound of formulae V and VI wherein R corresponds to the formula and R , R and R are hydrogen, is termed "6-(a-aminophenyl-acetamido) thiopenicillanic acid"* A preferred group of compounds of formula I are those in which at least one substituent of the phenyl, naphthyl or thienyl radicals is hydrogen; thus the phenyl, naphthyl or thienyl moiety of such preferred compounds can be represented by the formulae wherein R , R , R , R and each have the meaning set forth above. Of these preferred compounds, a preferred subgroup is that wherein R are selected from th© group consisting of hydrogen, (lower )alkoxy, chloro and phenyl.

Another preferred group of compounds are those wherein R is of the formula (XI) and wherein R , R and a are each a member selected from the group consisting of hydrogen, ehloro, bromo, fluoro, iodo, nitro, sulfamyl, trifluororaethyl, (lower)alky1, . (lower)alkoxy, di( lower)alkylamino, (lower)alkanoylamino, (lower)alkanoyl and (lower)alkanoyloxy.

The compounds of the present invention are prepared from the corresponding pericillins, i.e., the penicillins of the formula (XII), 0 » GH - COOH wherein R is other than hydrogen. Such penicillins can be prepared by a variety of methods presently known in the art and described in many United States and foreign patents, e.g.. United States Patents No. 2,751,378, No. 2,941,995, ho. 2,996,501, Ho. 2,985,648, Ho. 3,035,047, Ho. 3,080,356 and British Patents No. 877,120, No. 870,395, Ko. 891,279 and in other scientific literature, e.g., the article entitled "Derivatives of 6-Aainopenicillanic Acid. I. Partially Synthetic Penicillins Prepared from a-Aryloxylalkanoic Acids" by Perron et el., Vol. 82, J.A.C.3., pp. 3934-3938 (I960).

To prepare the compounds of the present invention, the amino group of the starting penicillin is first protected with a protecting group such as described in United States Patent No. 2,985,648, and then an active acylating derivative of the protected starting penicillin such as an anhydride or an acid chloride is prepared and reacted with a source of sulfhydryl groups, e.g., hydrogen sulfide, sodium hydrosulfide or potassium hydrosulfide. In a preferred process for the preparation of the compounds of the present invention, a mixed anhydride (for instance, the mixed anhydride with ethoxy- or isobutoxy-carbonic acid), of a penicillin as described above is prepared according to the procedures illustrated in the examples below and in United States Patent No. 2,751,378. Such mixed anhydride is then reacted with an alkali salt of hydrogen sulfide, e.g., sodium hydrogen sulfide (NaHS), and thereafter the protecting group is removed by catalytic hydrogenation to produce the compounds of the present invention, which may then be isolated and purified according to the procedures which are known and described in the prior art.

Functional equivalents of the foregoing mixed anhydride of penicillins may also be used. One such functional equivalent is the azolide corresponding to the penicillins of formula XII above, i.e., an amide of the corresponding penicillin whose nitrogen is a member of a quasi-aromatic five-membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, Ν,Ν'-carbonyldiimidazole is reacted with a carboxylic acid (penicillin in the acid form) in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethyIformamide or a similar inert solvent to form the carboxylic acid imldazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole. Dicarboxylic acids yield diimidazolides . The by-product, imidazole, precipitates and may be separated and the imidazolide isolated but this 1B not essential.

Thus, an elegant procedure for preparing a compound of the present invention by way of a mixed anhydride with ethoxy- or isobutoxy-carbonic acid comprises mixing substantially stoichiometric equivalents of an acid (of formula XII above) having a protected amino group, isobutyl chloroformate (or ethyl chloroformate) and if desired, an acid binding agent such as a tertiary hydro-carbonyl- or aliphatic amine (slight excess), e.g., 2, 6-lutidine or triethylamine, in an anhydrous, inert and preferably water-miscible solvent such as dimethylformamide and if desired, 2 ml. pure, dry acetone for a short period of time, e.g., about 5 to 60 minutes, in the cold, e.g., at about 5°C. To this solution of the mixed anhydride, there is then added a chilled suspension of sodium hydrogen sulfide in an inert anhydrous solvent (e.g. dimethylformamide). The reaction mixture is stirred for a period of an hour or so to form the substituted ammonium salt of the desired product. The mixture may then, if desired, be extracted at alkaline pH (such as pH 8; aqueous sodium bicarbonate may be used, for example, if necessary to adjust the pH) with a water-immiscible solvent such as ether to remove unreacted starting materials. The product in the aqueous phase is then converted to the free acid, preferably in the cold under a layer of ether by the addition of dilute mineral acid, e.g., 5 N H2S04 to pH 2. The free acid is then extracted into a water-immiscible, neutral organic solvent such as ether, and the extract is washed with water quickly in the cold, if desired, and then dried, as with anhydrous Na2S0ij, or MgSOj The product in the ethereal extract in its free acid form is then converted to any desired metal or amine salt by treatment with the appropriate base, e.g., a free amine such as procaine base or a solution of potassium 2-ethylhexanoate in dry n-butanol. These salts are usually insoluble in solvents such as ether and can be recovered in pure form by simple filtration. Thereafter, the protecting group is removed by hydrogenation in the presence of palladium.

Since some of the antibiotic substances obtained by the process of this invention are relatively unstable compounds which readily undergo chemical changes resulting in the loss of antibiotic activity, it is desirable to choose reaction conditions which are sufficiently moderate to avoid their decomposition. The reaction conditions chosen will, of course, depend largely upon the reactivity of the chemical reagent being used. In most instances, a compromise has to be made between the use of very mild conditions for a lengthy period and the use of more vigorous conditions for a shorter time with the possibility of decomposing some of the antibiotic substance.

The temperature chosen for the process of preparation of the compounds of the present invention should, in general, not exceed 30°C and in many cases, a suitable temperature is ambient temperature. In addition to the use of aqueous media for the reaction, use can be made of organic solvents which do not contain reactive hydrogen atoms. Examples of such inert solvents are di-methyl ormamide, dlmethylacetamide , methylene dlchloride, chloroform, acetone, methyl isobutyl ketone, and dioxane. Vigorous stirring is, of course, advisable when more than one phase is present, e.g., solid and liquid, or two liquid phases.

At the conclusion of the reaction, the products are isolated if desired, by the techniques used with phenoxyethylpenicillin, benzylpenicillin, and phenoxymethylpenicillin. Thus, the product can be extracted into diethyl ether or n-butanol at acid pH and then recovered by lyophilization or by conversion to a solvent-insoluble salt, as by neutralization with an n-butanol solution of potassium 2-ethylhexanoate, or the product can be precipitated from aqueous solution as a water-insoluble salt of an amine or recovered directly by lyophilization, preferably in the form of a sodium or potassium salt. When formed as the triethylamine salt, the product is converted to the free acid form and thence to other salts in the manner used with phenoxyethylpenicillin, benzylpeni-cillin and other penicillins. Thus, treatment of euch a triethylamine compound in water with sodium hydroxide converts it to the sodium salt, and the triethylamine may be removed by extraction, as with toluene. Treatment of the sodium salt with strong aqueous acid converts the compound to the acid form, which can be converted to other amine salts, e.g., procaine, by reaction with the amine base. Salts so formed are isolated by lyophilization or, if the product is insoluble, by filtration. A particularly elegant method of isolating the product as a crystalline potassium salt comprises extracting the product from an acidic, aqueous solution (e.g., pH 2) into diethyl ether, drying the ether and adding at least one equivalent of a solution of potassium 2-ethylhexanoate (e.g., 0.373 gm./ml.) in dry n-butanol. The potassium salt forms, precipitates, usually in crystalline form, and is collected by filtration or decantation. . μ An alternative method for the preparation of the com-pounds the present inventlonVcomprises acylating 6-aminothio-penicillanic acid according to procedures which are known in the prior art, e.g., the acid chloride and mixed anhydride procedures which are described in United States and foreign patents such as are listed above. 6-Aminothiopenicillanic acid can be prepared from 6-aminopenicillanic acid by first protecting the amino group of 6-aminopenicillanic acid with a protecting group such as described in United States Patent No. 2, 985, 6 8, then forming a mixed anhydride of such protected 6-aminopenicillanic acid by reacting the protected 6-aminopenicillanic acid with an ester of chlorocarbonlc acid, e.g., ethyl chlorocarbonate and then reacting such mixed anhydride with sodium hydrogen sulfide and thereafter removing the protecting group by catalytic hydrogenation. Alternatively, the 6-aminothiopenicillanic acid may be prepared from benzylthiopeni-cillin which is described in United States Patent No. 2, 751, 378, by enzymatic hydrolysis of such benzylthiopenicillin by the methods described in Union of South Africa Patent No. 60/1493 and Belgium Patent No. 586, 922. Additional examples of the organisms useful in the enzymatic hydrolysis process described in the foregoing patents are the following specimens of Escherichia coll on deposit with the American Type Culture Collection in Washington, reaction of 6-aminothiopenicillanic acid with a chloride hydrochloride of the appropriate -phenyl, naphthyl or thienyl glycine. The phenyl, naphthyl or thienyl substituted glycyl chloride hydrochloride may be formed by reacting Ο.Ο32 mole of the corresponding a-phenyl, naphthyl or thienyl glycine, suspended in methylene chloride and cooled to below 0°C, with 0.048 mole of phosphorus pentachloride . The reaction mixture is stirred while maintaining a temperature of about 0°C or below. Additional methylene chloride may be added to thin the mixture. A few drops (about 10) of di-methylf rmamide are added to the mixture. The a-phenyl, naphthyl or thienyl glycyl chloride hydrochloride forms as a precipitate and is recovered by filtration. About 0.028 mole of the product is then added to a solution of 0.019 mole of 6-aminothiopenicil-lanic acid in a mixture of acetone and water at a pH of 2-2 and maintained at a temperature of 0°C or below. The solution is then stirred for a few minutes after completion of the addition of the chloride hydrochloride and the pH is adjusted to 4-5. The acetone is removed, the solution is filtered to remove any insoluble material, and the filtrate is cooled to about 5°C, layered with about one-fourth volume of methyl isobutyl ketone, and acidified to pH 2 while stirring the mixture. The methyl isobutyl ketone layer is separated and discarded. The aqueous layer is then cooled to about 0°C, and extracted with 0.012 mole of dioctyl sodium sulfosuccinate in methyl isobutyl ketone. The extract is filtered and neutralized to a pH of 5-6 at which point a precipitate of the desired 6-substituted thiopenicillanic acid is formed.

The compounds of the present invention are useful in the treatment of infections caused by Gram-positive bacteria, including particularly the resistant strains of bacteria, e.g., penicillin-resistant strains of Staphylococcus aureus (Micrococcus pyogenes var. aureus ) . In addition, the compounds of the present invention, in addition to their potent antibacterial activity, exhibit resistance to destruction by acid.

In the treatment of bacterial infections in man, the compounds of this invention are administered orally or parenteral-ly, in accordance with conventional procedures for antibiotic administration, in an amount of from about 5 to 60 mg./kg./day, and preferably about 20 mg./kg./day in divided dosage, e.g., three or four times a day. They are administered in dosage units containing, for example, 125, or 250, or 500 mg. of active ingredient with suitable physiologically acceptable carriers or excipients. The dosage units can be in the form of liquid preparations such as solutions, dispersions, or emulsions, or in solid form such as tablets, capsules, etc.

The following examples will serve to illustrate this invention without limiting it thereto.

Example 1 Preparation of 6-Aminothiopenicillanic Acid A fermentation broth is prepared by the fermentation of Escherichia coll under submerged aerobic conditions according to conventional procedures and found to contain 5, 270 penicillin amidase units per ml.

A solution of 2.5 gm. calcium nitrate dihydrate dissolved in 6 ml. of water is added to 1 liter of such fermentation broth. After mixing, the broth is filtered, and the filtered mat washed with 150 ml. water. The filtered mat is then suspended in 200 ml. of water to which is added 5 ml. of toluene, and the suspension is stirred for 5 hours. After stirring, the suspension is filtered and the collected solid materials washed with 100 ml. of water. The filtrate is stirred with 1 gm. activated carbon (Darco KB) and 1.2 ml. of "Quaternary Ammonium Salt Mixture No. I". 'Quaternary Ammonium Salt Mixture No. I" is commercially available from Armour & Company of Chicago, Illinois, under the trademark of "Arquad 16-50" and is a liquid quaternary ammonium salt mixture containing, by weight, about h5 hexadecyltrimethylammonium chloride, about i octadecyltrimethylammonium chloride, about 2% octadecenyltrimethyl-ammonium chloride, about 35$ isopropanol, about lk°J> water, and about 1$ sodium chloride.

After thorough mixing, all solid materials are removed from the mixture by filtration, and there is obtained 280 ml. of an essentially pure aqueous solution of penicillin amidase assaying I95O penicillin amidase units per ml. and containing about \0 of the penicillin amidase originally present in the broth.

Benzylthiopenicillin ( 8 g .) is prepared by the procedure described in United States Patent No. 2, 751, 578 and added to 400 ml. of penicillin amidase solution, prepared according to the procedure described above. This suspension is maintained at pH 8.0 and 35°C for 4 hours, during which time the enzyme brings about the enzymatic hydrolysis of the benzylthiopenicillin. The benzylthiopenicillin and phenylacetate (produced during the hydrolysis) are removed at pH 2 and 5°C with methyl isobutyl ketone. After extraction, a portion of the liquor ( 250 ml.) is adjusted to pH 7 with sodium hydroxide and vacuum concentrated to 20 ml. The concentrate is adjusted to pH 4 with hydrochloric acid ( 6N ) and cooled to 5°C and allowed to stand for 20 hours at 5°C, during which time crystallization occurs. The crystals are filtered, washed with water ( 10 ml.) and then dry acetone. The 6-aminothio-penicillanic acid is recovered, weighs 0. 29 gm. and the presence of the - Q ~ SH group and β-lactam ring is confirmed by infrared analysis .

Example 2 Preparation of 6- (ct-Aminophenylacetamido ) - thiopenlcillanic Acid 6-(a-Carbobenzyloxyaminophenylacetamido)penicillanic acid which is obtained by the reaction of equivalent quantities of 6-(a-aminophenylacetamldo)penicillanic acid and benzyl chlorocar-bonate in aqueous sodium hydroxide is dissolved in dimethylforma-mide ( 125 ml.) and cooled to 5°C. 2, 6-Lutidine ( 2. 2 gm.) and ethyl chloroformate (2.2 gm.) are added dropwise to the solution. After stirring for 15 minutes, a mixture of sodium hydrosulfide (4.5 gm.) in dimethyl ormamide (50 ml.) is added in one portion. The solution turns brown and is stirred for 30 minutes and then is poured into water (1000 ml.). The pH is adjusted to 2 with dilute sulfuric acid. The 6-(a-carbobenzyloxyaminophenylacetamido)thio-penicillanic acid is extracted into ether and dried over anhydrous magnesium sulfate. Potassium 2-ethylhexanoate (4 gm. ) is added and potassium 6-(a-carbobenzyloxyaminophen lacetamido ) thiopenicil-lanate precipitates. The salt is dissolved in ethyl acetate and all traces of water are removed. The residue is slurried with Skellysolve B, filtered, and dried. The potassium 6-(a-carbo-benzyloxyaminophenylacetamido)thiopeniclllanate weighs gm., is found to have a melting point of greater than 100°C with decomposition, and to contain the β-lactam ring as shown by infrared analysis.

Potassium 6- (a-carbobenzyloxyaminophenylacetamido ) thio-penicillanate (5 gm.) is dissolved in water (75 ml.). Palladium ( 5 gm. of J0$) is added and the mixture hydrogenated at 50 psi for 30 minutes. The pH is adjusted to 2 with dilute sulfuric acid, and the palladium collected by filtration. Sodium hydroxide {10%) is added to the filtrate until the pH is adjusted to 5. The 6-(a-aminophenylacetamido)thiopenicillanic acid is recovered and found to contain the β-lactam ring as shown by infrared analysis, and to inhibit Staph, aureus Smith at concentrations of 0.001 percent by weight.

Example 3 Preparation of d, l-6- a-Amino- (2-thienyl)acetamido7thiopenicillanic Acid a- ( 2-Thienyl)glycine 5 .0 gm., Ο.Ο32 mole is suspended in 35 ml. of methylene chloride and cooled to -7°C with stirring and in the presence of calcium chloride desiccant. Phosphorus pentachloride ( 10 gm., 0.048 mole) is added in one portion causing the temperature to rise to -3°C. The mixture is stirred for one-half hour while maintaining the temperature at -5°C to 0°C, 15 ml. of methylene chloride is added, and stirring at -5°C to 0°C is continued for another one-half hour. Then, 10 drops of dimethylfor-mamide is added and another 20 ml. of methylene chloride is added to thin the mixture. The mixture is stirred rapidly and a purple slurry results. The product, d, 1-a- ( 2-thienyl)glycyl chloride hydrochloride, is collected by filtration under reduced pressure, washed three times with methylene chloride and dried iri vacuo over P2O5. The weight of the product is 5 .9 gm. which represents a 72 yield.

The pH of a solution of 6-aminothiopenicillanic acid (O.OI9 mole) in 80 ml. of water is adjusted to 6.3 by the addition of a lO by weight solution of NaOH with stirring. The solution is then diluted with 0 ml. of acetone and cooled to -5°C The pH of the solution is then adjusted quickly to 2. 5 by the addition of 6N HCl to the well-stirred mixture. To this solution is added d, l- -(2-thienyl)glycyl chloride hydrochloride ( 5. 9 gm., 0.028 mole) over a 20-minute period, the pH being kept at 2 to 2.5 by intermittent additions of 10 NaOH. The solution is stirred for 5 minutes after completion of the addition of the chloride hydrochloride and the pH is then adjusted to 4.8 by the addition of 10 NaOH. The acetone is removed under reduced pressure at 3O 0 to °C and the solution is filtered to remove Insoluble material.

The filtrate, having a pH of 4 , is cooled to 5°C, adjusted to a pH of 7 by the addition of 10 NaOH, layered with 20 ml. of methyl isobutyl ketone and acidified to pH 2 with 0 H2S04 . Upon removal of the methyl isobutyl ketone layer, the color of the remaining aqueous layer clarifies. This aqueous layer is extracted again with 20 ml. of methyl isobutyl ketone and the extracts are discarded The aqueous layer is then cooled to 0°C and extracted at pH 2 with a 10 solution containing dioctyl sodium sulfosuccinate ( 5.3 gm., 0 .012 mole) in methyl isobutyl ketone. The methyl isobutyl ketone extract is separated by decanting, filtered through anhydrous a SOlj and concentrated slightly by evaporation under reduced pressure at 30°C. It is then neutralized to pH 5 .3 by the addition of triethylamine . The solid product which forms is collected by filtration under reduced pressure, slurried with ether, refiltered and dried in vacuo over P2O5 . The product, d, l-6- -amino( 2-thienyl acetamido7thiopenicillanic acid, is found to contain the β-lactam structure as shown by infrared analysis, and to inhibit Staph. aureus Smith at a concentration of 0 .001 percent by weight.

Example 4 In the general procedure of Example 3 , the a- ( 2-thienyl)-glycine is replaced by 0 .032 mole of a- ( 3-thienyl) glycine, a- ( 5-ethyl-2-thienyl)glycine, a- ( -me thy 1-2-thienyl )glycine , a- ( 5-t-butyl-2-thienyl)glycine, a- ( 2 ,5 -dimethyl-j3-thlenyl)glyclne, a- ( 5-chloro-2-thienyl)glycine, a- ( -bromo-2-thieny 1 )glyc ine , a-(5-phenyl-3-chloro-2-thienyl)glycine, a- (3, 5-dimethyl-2-thienyl)glycine, a- (5-cyclohex 1-2-thienyl)glycine, a-(5-diethylamino-2-thienyl)glycine, a-( -methylsulfonyl-2-thienyl)glycine, a-(3-ethylthio-2-thienyl)glycine, and a-( -cycloheptyloxy-2-thienyl)glycine, respectively, to produce d, 1-6-^ -amino- (3-thienyl )acetamido/thiopenicillanic acid, 6- a-amino-(5-ethy1-2-thienyl)acetamido_7thiopenicillanic acid, 6-^-amino-(5-methy1-2-thienyl)acetamido7thiopenicillanic acid, 6- a-amino-(5-t-buty1-2-thienyl)acetamido7thiopenicillanic acid, 6-^-amino-( , 5-dlmethy1-3-thienyl)acetamido/thiopenicillanic acid, 6-i a-amino-(5~chloro-2-thienyl) acetamido_7thiopenicillanic acid, 6-Ja-amino-(5-bromo-2-thienyl)acetamido7thiopenicillanic acid, 6- a-amino-(5-phenyl-3-chloro-2-thienyl)acetamido7thiopenicillanic acid 6-^a-amino- (3, 5-dimethy1-2-thienyl)acetamido thiopenicillanic acid, 6-^-amino- (5-cyclohexy1-2-1hieny1 ) ce tamido_7thiopenicillanic acid, 6- -amino-(5-diethylamino-2-thienyl)acetamido_7thiopenicillanic aci 6-/^a-amino-(4-methyls lfony1-2-thienyl )acetamido thiopenicillanic acid, 6- a-amino-(3-ethylthio-2-thienyl)acetamldoJ7thiopenicillanic acid, 6- a-amino-(4-cycloheptyloxy-2-thienyl)acetamido_7thiopeniclllanic acid, respectively, which are isolated and found to contain the β-lactam ring as shown by infrared analysis, and to inhibit Staph. aureus Smith at concentrations of 0.001 percent by weight.

Example 5 Preparation of D- ( - ) -6-Aminophenylacetamldo)- thiopeniclllanlc acid D_ (-)-a-phenylglycine ( 25 .0 gm., 0. 165 mole -153°) is suspended in methylene dichloride ( 500 ml.) and phosphorus pentachloride ( 65 .0 gm., 0.30 mole) is added to the suspension at 3°C. After 10 minutes, dimethylformamide (2 ml.) is added to the reaction mixture which is then stirred for 10 minutes in an ice-bath and thereafter for 2 hours at room temperature. The resulting product, D-(-)-a-phenylglycylchloride hydrochloride, is collected by filtration, washed with methylene dichloride and ether, dried in vacuo over P2O5 for 2 hours and thereafter found to weigh 19. gm. 6-Aminothiopenicillanic acid (0 .019 mole) is dissolved in water (80 ml.) and the pH of the solution is adjusted to 6.3 by the addition of a 10$ by weight solution of sodium hydroxide with stirring. The solution is then diluted with acetone (^40 ml.) and cooled to -5°C. The pH of the solution is then adjusted quickly to 2.5 by the addition of 6 N hydrochloric acid to the well-stirred mixture. To this solution is added D-(-) -a-phenylglycyl chloride hydrochloride (0 .028 mole) over a 20-minute period, the pH being kept at 2 to 2. 5 by intermittent additions of 10$ sodium hydroxide. The solution is stirred for 5 minutes after completion of the addition of the chloride hydrochloride and the pH is then adjusted to 4 . 8 by the addition of 10$ sodium hydroxide. The acetone is removed under reduced pressure at J50-J5 °C, and the solution is filtered to remove insoluble material. The filtrate, having a pH of 4, is cooled to 5°C, adjusted to a pH of 7 by the addition of $ sodium hydroxide, layered with methyl isobutyl ketone (20 ml.), and acidified to pH 2 with 20$ sulfuric acid. Upon removal of the methyl isobutyl ketone layer, the color of the remaining aqueous * layer clarifies. This aqueous layer is extracted again with methyl isobutyl ketone (20 ml.) and the extracts are discarded. The aqueou layer is then cooled to 0°C and extracted at pH 2 with a 10$ solution containing dioctyl sodium sulfosuccinate (5.3 gm., 0.012 mole) in methyl isobutyl ketone. The methyl isobutyl ketone extract is separated by decanting, filtered through anhydrous sodium sulfate and concentrated slightly by evaporation under reduced pressure at 30°C. It is then neutralized to pH 5.3 by the addition of tri-ethylamine. The solid product which forms is collected by filtra-tion under reduced pressure, slurried with ether, refiltered, and dried in vacuo over phosphorus pentoxide. The product, D-(-)-6-(a-aminophenylacetarnido)thiopenlcillanic acid, is found to contain the β-lactam ring as shown by infrared analysis, and to inhibit Staph, aureus Smith at a concentration of 0.001 precent by weight.

Example 6 In the general procedure of Example 5, the D(-)-a-phenylglycine is replaced by 0.165 mole of -p-chlorophenylglycine, a-p-methoxyphenylglycine, L-(+)-a-phen lgl cine, a- -diethylaminophen lglyc ine, α- -tri luoromethylphenylglycine, a-2, -dibromophenylglycine , a-2-nitrophenylglycine, a-3-methylphenyl lycine , a- -sulfamylphenylglycine, α-2-iodophen lglycine, α- -t-but lphen lglyc ine, α-2-acetamidophenylglycine, α-5-ni rophenyl lycine, α-3,4-dimethoxyphenylglycine, a-4-dimethylaminophenylglycine, a-2,4-dichlorophenylglycine, a-4-isopropylphenylglycine, a-3-bromophenylglycine, a-jj-iodophenylglycine, a-2-diethylaminophen lglycine, α-2-trifluoromethylphen lglyc ine, α-4-fluorophenylglycine, a-5,4,5-trifluoromethylphenylglycine, respectively, to produce 6-(a-amino-p-chlorophenylaeetamido) hiopenicillanic acid, 6-(a-amino-p-methoxyphenylacetamido) hiopenicillanic acid, 6-^-(+)-a-aminophenylacetamido7thiopenicillanic acid, 6-( -amino-4-diethylaminophenylacetamido) hiopenicillanic acid, 6-(a-amino-4~trifluoromethylphenylace amido) thiopenicillanic acid, 6- (a-amino--2,4--dibromophenylacetamido) thiopenicillanic acid, 6- (a-amino-2-nitrophenylacetamido) thiopenicillanic acid, 6-(a-amino-3-methylphenylacetamido) thiopenicillanic acid, 6-(a-amino-4-sulfam lphenylace amido) hiopenicillanic acid, 6-(a-amino-2-iodophen lacetamido) thiopenicillanic acid, 6-(a-amino-4-t-butylphenylacetamido) thiopenicillanic acid, 6-(a-amlno-2-acetamidophenylacetamido) hiopenicillanic acid, 6-(a-amino-3-nitrophen lacetamido) thiopenicillanic acid, 6-(a-amino-3,4-dimethoxyphenylacetamido) thiopenicillanic acid, 6-(a-amino-4-dimethylaminophenylacetamido) thiopenicillanic acid, 6-(a-amino-2,4-dichlorophen lacetamido ) thiopenicillanic acid, 6-(a-amino-4~isoprop lphen lacetamido) thiopenicillanic acid, 6-(a-amino-3-bromophenylacetamido) thiopenicillanic acid, 6-(a-amino-5-iodophenylacetamido) thiopenicillanic acid, 6- (a-amino-2-diethylaminophenylacetamido ) thiopenicillanic acid, 6-(a-amino-2-trifluorome th Iphenylacetamido) thiopenicillanic acid, 6-(a-amino-4-fluorophenylace tamido) thiopenicillanic acid, and 6-(a-amino-5, , 5-trifluorometh lphenylace tamido) thiopenicillanic acid, respectively, which are isolated and found to contain the β-lactam ring as shown by infrared analysis and to inhibit Staph. aureus Smith at concentrations of 0.001 percent by weight.

Example 7 In the general procedure of Example 5, the D(-)-a-phenylglycine is replaced by 0.165 mole of a- (1-naphth 1)glyc ine, a- ( 2-naphthyl )glycine , a- ( l-chloro-2-naphthyl )glycine , a- (2-me thy1-7-naphthy1) lycine , a-(6-nitro-l-naphthyl)glycine, a- (2, 7-dibromo-3-naphthyl)glycine , a- (4-trifluoromethyl-l-naphthyl)glyc ine, a- (8-iodo-l-naphthy1 )glycine a-(l-methoxy-2-naphthyl)glycine, and ct-(4-acetamido-l-naphthyl )glycine, respectively, to produce 6-/ -amino-(l-naphthyl)acetamido7thiopenicillanic acid, 6-^/ -amino- (2-naphthyl) ce tamido7thlopenicillanic acid, 6- a-atnino- ( l-chloro-2-naphthyl)acetamido7thiopenicillanic acid, 6-J -amino-(2-methyl-7-naphthyl)acetamido7thiopenicillanic acid, 6-^-amino-(6-nitro-l-naphthyl)acetamido7thiopenicillanic acid, 6- a-amino-(2,7-dibromo-3-naphthyl)acetamido7thiopenicillanic acid, 6-^-amino- (4-trifluorome thyl-l-naphthyl)acetamido7thiopenicillanic acid, e-^ a-amino- S-lodo-l-naphthylJacetamido/thiopenicillanic acid, 6- a-amino- ( 1-methoxy-2-naphthy1 )acetamido/thiopenicillanic ac id , 6-j5-amino-(4-acetamido-l-naphthyl)acetamido7thiopenicillanic acid, respectively, which are isolated and found to contain the β-lactam ring as shown by infrared analysis, and to inhibit Staph, aureus Smith at concentrations of 0.001 percent by weight.

Claims (1)

1. HAVING particularly described and ascertained the nature of our said invention and in what manner the same is to be we declare that what we claim A compound selected from the group consisting of acids having the formula wherein R is hydrogen or a member selected from the group consisting of radicals of the formulae 1 2 wherein R R and are each a member selected from the group consisting of 4 5 and wherein R and each a member selected from the group consisting of nitro 6 7 and and wherein R and R are each a member selected from the group consisting of cycloalkyl radicals having from 5 to 7 carbon atoms cycloalkoxy radicals having from 5 to 7 carbon atoms and the pharmaceutically able nontoxic salts acid and the acceptable nontoxic salts thiopenicillanic acid and the pharmaceutically acceptable nontoxic salts lace tamido thiopenic illanic acid and the pharmaceutically acceptable nontoxic salts and the pharmaceutically acceptable nontoxic salts 4 therapeutic composition useful in the treatment of bacterial infections in unit dosage form comprising from 125 to 500 of a according to Claim 1 or a pharmaceutically acceptable nontoxic salt A compositio according to Claim wherein said compound is thiopenicillanic acid and the pharmaceutically acceptable nontoxic salts composition according to Claim wherein said compound is thiopenicillanic acid and the acceptable nontoxic salts A composition according to Claim wherein said compound is thiopenicillanic acid and the pharmaceutically acceptable nontoxic salts A composition according to Claim wherein said compound is thiopenicillanic acid and the pharmaceutically acceptable nontoxic salts Dated this 19th For the insufficientOCRQuality