NZ199601A - Coadministration of a cephalosporin derivative and a penicillin - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number 1 99601 1 99601 FrimKy Do** Xl'JI: 7fc Compete Specification Fife Cl»* P.bW3tJ.?^3 PubliecUon Dstt l£5°l "(5'6'JLIL 1984 || ^ ^ (jVt rat? k# \"'''' '' ■' '■ ^ Under the provisions of Regulation 23. jl) the _ st , - Specification has been ante-dated JO <2 <S 19 j?}?.... 2.8 JAN\^ Divided from No.: 190797 Date; 22 June 1979 NEW ZEALAND PATENTS ACT, 1953 . I COMPLETE SPECIFICATION "COADMINISTRATION OF PENICILLANIC ACID 1,1-DIOXIDE WITH 7-(D-2- [ 1+-ETHYLPIPERAZIN-2,3-DI0NE-1-CARB0XAMID0 ]-2-[U-HYDROXYPHENYL] ACETAMIDO)-3-([1-METHYL-5-TETRAZOLYL]THIOMETHYL)-3-DESACETOXY-METHYLCEPHALOSPORANIC ACID" I/We, PFIZER INC., a corporation organized under the laws of the State of Delaware, United States of America, of 235 East U2nd Street, New York, State of New York, United States of America, hereby declare the invention for which Jc / we pray that a patent may be granted toxEoec/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - (followed by pace la) - la - 19960t One of the most well-known end widely used of Che "classes of antibacterial agents is the class known as the beta-lactam antibiotics. These compounds are characterized in that they have a nucleus consisting of a 2-azetidinone (beta-lactam) ring fused to either a thiazolidine or a dihydro—1,3—thiazine ring. When the nucleus contains a thiazolidine • ring, the compounds are usually referred to generically as penicillins, whereas when the nucleus contains a dihydrothiazine ring, the compounds are referred to as cephalosporins. Typical examples of penicillins which are conmonly used in clinical practice are benzylpenicillin / (penicillin G), phenoxynethylpenicillin (penicillin V), ampicillin atid I carbenicillin; typical examples of coeoo cephalosporins are cephalothin, cephale:dLn and cefazolin.

/ . However, despite the wide use ar.d wide acceptance of the fceta-lactara antibiotics as valuable chenotherapeutic agents, they suffer froa the major drawback that certain jzeabers are not active against certain 1 nicroorganisns- It is thought that in many instances this resistance of a particular microorganism to a given beta-lactara antibiotic results because 1 9 9601 enzymes which cleave the beta-lactam ring of penicillins and cephalosporins to give products which are devoid of antibacterial activity. However, certain substances have the ability to inhibit beta-lactamases, and when a beta-lactanase inhibitor is used in combination with a penicillin or cephalosporin it can increase or enhance the antibacterial effectiveness of the penicillin or cephalosporin against certain microorganisms. It is considered that there is an enhancement of antibacterial effectiveness when the antibacterial activity of a combination of a beta-lactanase inhibiting substance and a beta-lactam antibiotic is significantly greater than the sum of the antibacterial activities of the individual components.

Penicillanic acid 1,1-dioxide, its pharaaceutically-acceptable salts, i ^— - ■ and. its esters readily hydrolyzable in vivo are potent inhibitors of microbial beta-lactamases. In our New Zealand Patent Specification No. 190797 there is disclosed and claimed a method for increasing the effectiveness of the cephalosporin antibiotic, 7—(D—2—[4-ethylpiperaz in-2,3-dione—1-carboxamido]-2-[4-hydroxypheny1 ] — acetamido)-3-([l-methyl-5-tetra2olyl]thiomethyl)-3-desacet027Eethylcephalo-sporanic acid and its pharmaceutically-acceptable salts, using said penicillanic acid 1,1-dioxide, pharmaceutically acceptable salts thereof, or esters thereof readily hydrolyzable in vivo. Additionally, there are disclosed pharmaceutical compositions, useful for treating bacterial infections in masmals, which comprise penicillanic acid 1,1—dioxide or a pharmaceutical^ acceptable salt thereof or an ester thereof readily hydrolyzable in vjyo> and 7—(D-2-[4-ethylpiperazin-2,3-dione—l-carboxaaido]-2-[4-hydroxyphenyl]acetacido)-3—([l-methyl-5-tetrazolyl]thiomethy1)-3-desacetoxy-cethylcephalosporanic acid or a pharaaceutically-acceptable salt thereof. 1,1-Diorides of benzylpenicillln, phenoxynethylpenicillin and certain esters thereof have been disclosed in United States Patents 3,197,466 /' and 3,536,698, and in an article by Guddal et al., in Tetrahedron Letters, 199601 Ho. 9, 381 (1962). Harrison et al., in the Journal of the Cheaical Society (London), Perkin I, 1772 (1976), have disclosed a variety of penicillin 1,1-dioxides and 1-oxides, including methyl phthaliaidopenicil-lanate 1,1-dioxide, methyl 6,6-dibrocopenicillanate 1,1-dioxide, methyl peaicillanate 1-alpha-oxide, methyl penlcillanate 1-beta-oxide, 6,6— dibromopenicillanic acid 1-alpha-oxide and 6,6-dibromopenicillanic acid 1-beta—oxide.

According to the invention, there is provided a method of increasing the effectiveness of the beta-lactan antibiotic, 7-(D^-2-[4-ethylpiperazin-2,3-dione-l-carboxamido]—2-[4-hydroxyphenyllacetamido)-3-([l-methyl-5-tetrazolyl]thiomethyl)-3—desacetoxyaethylcephalosporanic acid, and the pharmaceutically-acceptable salts thereof, in a mamraalian subject. Said method comprises co-adninistering to said subject a beta- I lactam antibiotic effectiveness increasing amount of a compound of the formula 0 0 <oCH_ J N- V/ 0 'C00R-1 "(I) or a pharmaceutically-acceptable base salt thereof, wherein 3.^" is selected from the group consisting of hydrogen and ester-forming residues readily hydrolyzable in vivo.

To facilitate the method of the invention, there are provided pharmaceutical ccmpositions, useful for treating bacterial infections in n—rr.als. Said compositicns comprise r 7—(D-2-[4- ethylp iperazin-2,3-dione—l-carboxasido]-2-[4-hydroxyphen.y1]acetanido)—3— ([l-methyl-5—tetrazolyl]thiomethyl)-3-desacetcxymethylcephalosporanic acid or a pharmaceutically-acceptable salt thereof, and a compound of the foraula I or a pharsiaceutically-acceptable base salt thereof, vherein )— 199601 The tern "ester-forming residues readily hydrolyzable in vivo" i ! is here intended to refer to non-toxic ester residues which are rapidly A. J. P.^S. ! cleaved in mammalian blood or tissue, to release the corresponding free j acid (i.e. the compound of formula I, wherein R^" is hydrogen). Typical examples of such readily hydrolyzable ester-forming residues which can be- used for are alkanoyloxymethyl having from 3 to 8 carbon atoms, 1- / (alkanoylozcy) ethyl having from 4 to 9 carbon atoms, 1-nethyl-1-(alkanoyloxy)■ ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having i from 3 to 6 carbon atoms, l-(alko:tycarbonyloxy) ethyl having from 4 to 7 carbon atoms, l-methyl-l-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, l-(N-[alkox7carbonyl]amino)ethyl having from 4 to 10 carbon atoms, / • I ' 3-phthalidyl, 4-crotonolactonyl and gamma-butyrolacton-4-yl. ik<,e. of This invention relates to the^compounds of formula I, and throughout this specification, they are referred to as derivatives of penicillanic acid, which is represented by the structural formula H CH, S r CH, -(IV) '"h '/C00H 1 996011 In formula 17, broken line attachment of a substituent to the bicyclic nucleus indicates that the substituent is below the plane of the bicyclic nucleus. Such a substituent is said to be in the a-configuration. Conversely, solid line attachment of a substituent to the bicyclic nucleus indicates that the substituent is attached above the plane of the nucleus- This latter configuration is referred to as the (3-configura-tion.

Also in this specification reference is made to a derivative of cephalosporanic acid. Cepnalosporanic acid has the formula -(v) C00H In formula V, the hydrogen at C-6 is below the plane of the bicyclic nucleus. The derived term 3-desacetoxymethylcephalosporanic acid refers to the structure VI. 4-Crotonolactcnyl and Y~hutyrolacton-4-yl refer to structures VII and VIII, respectively. The wavy lines are intended to denote each of the two epimers and mixtures thereof. i (VII) (VIII) 199601 When R is an escer-foraing residue readily hydrolyzable in vivo in a coapound of formula I, it is a grouping which is notlonally derived iron an alcohol of the fornula R^-OH, such chat Che Eoiety COOR^" in such a coEpound Y . 1 or fornula I represents an ester grouping. Moreover, R is of such a nature' that hhe grouping COOR^* Is readily cleaved in vivo to liberate a free car- 1 boxy group (COOH). Thitt is to say, R is a group of the type chat when a compound of f omul a I, wherein R^" is an ester-forning residue readily hydroly^;- in vivo, is exposed to blood or tissue, the conpound of formula I, J. P. & wherein R^" is hydrogen, is readily produced. The groups are veil-known in the penicillin art. In cost instances they_iaiprove the absorption characteristics of the penicillin coapound. Additionally, R^" should be of such a nature that it iiroarts phar^iaceutically-acceptable properties to a compound of formula I, and it liberates pharr^ceutically-accsptable fragments when cleaved in vivo. 1 • " As. indicated above, the groups R are wall-kncwn and are readily .identified by those skilled in the penicillin art. Sea, for an'irsJ^y frir firmm OffrmltTiffliirgT-iphr-' ft* No 7,51 T^r^n-i Typical groups for R^" are 3-phthalidyl, 4-crotonolactonyl, Y-butyrolacron-4-yl and groups ox the formula R • 0 I » 5 - C-O-C-R R 0 I » 5 -C-O-C-O-R 4 IX R 0 I II 5 — c-nh-c-otrj A* • XI and - 6 t 99601 3 4 wherein R and R are each selected from the group consisting of hydrogen j and alkyl having from 1 to 2 carbon atoms, and R^ is alkyl having from 1 to 6 carbon atoms. However, preferred groups for R* are alkanoyloxy-methyl having from 3 to 8 carbon atoms, 1-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-l-(alkanoyloxy)ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkozycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-l-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)-amincmethyl having from 3 to 9 carbon atoms, 1-(N-1alkoxycarbonyl]amino)-ethyl having from 4 to 10 carbon atoms,^3-phthalidyl, 4-crotonolactonyl and if-butyrolactoii-4-yl.

H 3 CH„ r ^/CQOR1 -(ID a I Och3 T >CH.

'''"C00R1 (Ill) 7. * 199601 The compounds of formula I, wherein R"1" is as defined previously can be prepared by oxidation of either of the compounds of formula II or III, wherein R^ is as defined previously. A wide variety of oxidants known in the art for the oxidation of sulfoxides to sulfones can be used for this process. However, particularly convenient reagents are metal permanganates, such the alkali metal permanganates and the alkaline earth metal permanganates, and organic peroxy acids, such as organic peroxycarboxylic acids. Convenient individual reagents are sodium permanganate, potassium permanganate, 3-chloroperbenzoic acid and peracetic acid.

When a compound of the Formula II or III, wherein R"*" is as defined previously, is oxidized to the corresponding compound of the formula I using a metal permanganate, the reaction is usually carried out by treating the compound of the formula II or III with from 0.5 to about 5 molar equivalents of the permanganate, and preferably about 1 molar equivalent of the permanganate, in an appropriate solvent system. An appropriate solvent system is one that does not adversely interact with either the starting materials or the product, and water is commonly used. If desired, a co-solvent which is miscible with water but will not interact with the permanganate, such as tetrahydrofuran, can be added. The reaction is normally carried out at a temperature in the range from about -20° to about 50°C., and preferably at about 0°C. At about 0°C. the reaction is normally substantially complete within a short period, e.g. within one hour. Although the reaction can be carried out under neutral, basic or acid conditions, it is preferable to operate under substantially neutral conditions in order to avoid decomposition of the P -lactam ring system of the compound of the formula I. Indeed, it is often advantageous to buffer the pH of the reaction medium in the vicinity of neutrality. The product is recovered by conventional techniques. Any excess permanganate is usually decomposed using sodium bisulfite, and then if the product is out of solution, it is recovered by filtration. It is APR 195^1 separated from manganese dioxide by extracting it into an organic solvent and removing the solvent by evaporation. Alternatively, if the product is not out of solution at the end of the reaction, it is isolated by the ususal procedure of solvent extraction.

When a compoind of the formula II or III, wherein R^ is as previously defined, is oxidized to the corresponding compound of the formula I, using an organic peroxy acid, e.g., a peroxycarboxylic acid, the reaction is usually carried out by treating the compound of the formula II or III with from about 1 to about 4 molar equivalents, and preferably about 1.2 equivalents of the oxidant in a reaction-inert organic solvent. Typical solvents are chlorinated hydrocarbons, such as dichloromethane, chloroform and 1,2-dichloroethane; and ethers, such as diethyl ether, tetrahydrofuran and 1,2-dimethoxyethane. The reaction is normally carried out at a o o temperature of from about -20 to about 50 C., and preferably at about o o C. At about 25 C. reaction times of about 2 to about 16 hours are commonly used. The product is normally isolated by removal of the solvent by evaporation in vacuo. The product can be purified by conventional methods, well-known in the art.

When oxidizing a compound .of the "formula II or III to a compound of the formula I using ah organic peroxy acid, it is sometimes advantageous to add a catalyst such as a manganese salt, e.g. manganic acetylacetonate.

In like manner, compounds of the formula I, wherein R^" is as previously defined, can be prepared by oxidation of a compound of the formula 0^ , w„3 | I I —(xII) 'COOR1 wherein R"'' is as previously defined. This is carried out in exactly the same manner as described hereinbefore for oxidation of a compound of the formula II or III, excpet that twice as much oxidant is usually used. 1 99601 Compounds of the formula I, wherein R* is an ester-foraing residue readily hydrolyzable in vivo, can be prepared directly from the compound of formula I, wherein R^" is hydrogen, by esterification. The specific method chosen will depend naturally upon the. precise structure of the ester-forming residue, but an appropriate method will be readily selected by one skilled in the art. In the case wherein R^" is selected from the group consisting of 3-phthalidyl, 4-crotonolactonyl, ybutyro- 3 4 lacton-4-yl and groups of the formula IX, X.and XI, wherein R , R and R"* are as defined previously, they can be prepared by alkylation of the compound of formula I, wherein R^" is hydrogen, with a 3-phthalidyl halide, a 4-crotonolactonyl halide, a y~butyrolacton-4-yl halide or a compound of the formula ^ 3 3 RJ 0 R 0 R 0 1 II S 1 B 5 1 11 5 Q-C-O-C-R , Q-C-0-C-0-R or Q-C-NH-C-OR ■if .A4 A4 (XIII) (XIV) (X7) 3 4 5 wherein Q is halo, and R , R and R are as previously defined. The terms "halide" and "halo" are intended to mean derivatives of chlorine, bromine and iodine. The reaction is conveniently carried out by dissolving a salt of the compound of formula I, wherein R^" is hydrogen, in a suitable, i! 199601 j polar, organic solvent, such as INjN-diaethylformamide, and then adding J about one molar equivalent of the halide. When the reaction has proceeded essentially to completion, the product is isolated by standard techniques. It is often sufficient simply to dilute the reaction medium with an excess of water, and then extract the product into a water-inaniscible organic solvent and then recover same by solvent evaporation.

Salts of the starting material which are commonly used are alkali metal . i ' salts, such as sodium and potassium salt, and tertiary amine salts, such as triethylamine, 11-ethylpiperidine, N,N-dimethylaniline and N-nethylmorpholine salts. The reaction is run at a temperature in the range from about 0 to 100° C., and usually at about 25° C. The length of time needed to reach completion varies according to a variety of factors, such as the concentration of the reactants and the reactivity i of the reagents. Thus, when considering the halo compound, the iodide reacts faster than the bromide, which in turn reacts faster than the chloride. In fact, it is sometimes advantageous, when utilizing a thloro compound, to add up to one molar equivalent of an alkali metal iodide. This has the effect of speeding up the reaction. With full regard for the foregoing factors, reaction times of from about 1 to about 24 hours are commonly used.

. ' * J *J J ; Penicillanic acid la-oxide, the compound of the formula II, wherein is hydrogen, can be prepared by debromination of 6,6-dibromopenicillanic acid la-oxide. The debromination can be carried out using a conventional hydrogenolysis technique. Thus, a solution of 6,6-dibromopenicillanic acid la-oxide is stirred or shaken under an atmosphere of hydrogen, or hydrogen mixed with an inert diluent such as nitrogen or argon, in the presence of a catalystic amount of palladium-on-calcium carbonate catalyst. Convenient solvents for this debromination are lower-alkanols, such as methanol; ethers, such as tetrahydrofuran.and dioxan; low molecular weight esters, such as ethyl acetate and butyl acetate; water; and mixtures of these solvents. However, it is usual to choose conditions under which the dibromo compound is soluble. The hydrogenolysis is usually carried out at room temperature and at a pressure from about atmospheric pressure to about 50 p.s.i. The catalyst is usually present in an amount from about 10 percent by weight based on the dibromo compound, up to an amount equal in weight to the dibromo compound, although larger amounts can be used. The reaction commonly takes about one hour, after which the compound of the formula II, wherein R''" is hydrogen, is recovered simply by filtration followed by removal of the solvent in vacuo. 6,6-Dibromopenicillanic acid la-oxide is prepared by oxidation of 6,6-dibromopenicillanic acid with 1 equivalent of 3-chloroperbenzoic acid in tetrahydrofuran at 0-25°C. for ca. 1 hour, according to the procedure of Harrison et_ al., Journal of the Chemical Society (London) Perkin I, 1772 (1976). 6,6-Dibromopenicillanic acid is prepared by the method of Clayton, Journal of the Chemical Society (London), (C) 2123 (1969). 1 99601 Penicillanic acid l-^g-roxide, the compound of the formula III, wherein R^" is hydrogen, can be prepared by controlled oxidation of penicillanic acid. Thus, it can be prepared by treating penicillanic acid with one molar equivalent of -3-chloroperbenzoic acid in an inert solvent at about 0° C. for about one hour. Typical solvents which can be used include chlorinated hydrocarbons, such as chloroform and di-chloromethane; ethers, such as diethyl ether and tetrahydrofuran; and low molecular weight esters such as;ethyl acetate and butyl acetate. The product Is recovered by conventional techniques.

Penicillanic acid is prepared as described in British Patent No. 1,072,108.

Compounds of the formula II and III, wherein R^" is an ester- forming residue readily hydrolyzable in vivo, can be prepared directly from the compound of formula II or III, wherein R^" is hydrogen, by esterification, using standard procedures^ In the case wherein R*" is selected from the group consisting of 3-phthalidyl, 4-crotonolactonyl, 3 Y-butyrolacton-4-yl and groups of the fdrmula IX, X and XI, wherein R , 4 5' R and R are as defined previously, they can be prepared by alkylation of the appropriate compound of the formula II or III, wherein R^" is hydrogen, with 3-phthalidyl halide, 4-crotonolactonyl halide, a y-butyrolacton-4-yl halide, or a compound of the formula XIII, XIV or XV. The reaction is carried out in exactly the same manner as described previously for esterification of penicillanic acid 1,1-dioxide with a 3-p'nthalidyl halide, a 4-crotonolactonyl halide, a Y~butyrolacton-4-yl halide, or a compound of the formula XIII, XIV or XV.f 'h 199601 Alternatively, the compounds of the foraula II, wherein R^ is an ester-foralng residue readily hydrolyzable in. vivo, can be prepared by oxidation of the appropriate est.er of 6,6-dibromopenicillanic acid, followed by ! debromination. The esters of 6,6'-dibronopenicillanic acid are prepared from 6,6-dibronopeniclllanic acid by standard methods- The oxidation is carried out, for exaxaple, by oxidation with one aolar equivalent of 3-chloroperbenzoic acid, as described previously for the oxidation of 6,5-d±br0E0penicillanic acid ,'to 6,6—dibrouopenicillanic acid la-oxide; 'and the debromination is carried out as desc/ribed previously for the debromination of 6,6-dibromopenicillanic acid la-oxide.

•In like manner, the compounds of the formula III, vherein is an ester-forr.ing residue readily hydrolyzable in'vivo can be prepared by oxidation of the appropriate"ester of penicillanic acid. The latter compounds are readily prepared by esterification of penicillanic acid using standard methods. The oxidation is carried out, for exaspla, by oxidation with one nolar equivalent of 3-chloroperbenzoic acid, as described previously for the oxidation of penicillanic acid to penicillanic acid 13—oxide. i 16. 1 99601 In an alternate method, the compounds of formula I, wherein R is selected from the group consisting of hydrogen, and ester-forming residues readily hydrolyzable iri vivo, can be prepared by a two-step procedure which comprises the steps of: (a) contacting a compound selected from the group consisting of H- CH y 3 P- >.CH3 y-*-—Wn_i r COOR ,.(XVI)/ r~ CH_ ^"COOR1 (XVII) and base salts thereof with a reagent selected from the group consisting I of alkali metal permanganates, alkaline earth metal permanganates and organic peroxycarboxylic acids, to give a compound selected from the group consisting of X H V H ^ S - /r 0 N ;,CH, ^CH.

^//JCOOR H V° CH_ H. ""i COOR (XVIZT) (XIX) and base salts thereof, wherein X is selected from the group consisting of chloro, bromo, and iodo; and (b) dehalogenating the compound of the formula XVIII or XIX or mixture thereof.

A preferred way of carrying out step (b) comprises contacting the product of step (a) with hydrogen, in an inert solvent, at a pressure 2 in the range from about 1 to about 100 kg/cm , at a temperature in the 16. 1 99601 range from about 0 to 60° C., and at a pH in the range from about 4 to about 9, and in the presence of a hydrogenolysis catalyst. The hydrogenolysis catalyst is usually present in an acount from about 0.01 to about 2.5 weight-percent, and preferably froa about 0.1 to about 1.0 weight-percent, based.on the halo-sulfone. The preferred value for X is brorao, and the preferred reagents for carrying out step (a) are potassium permanganate and 3-chloroperbenzoic acid.

In a furtner alternate method, the cODpounds of forcula I, wherein H?" is selected from the group consisting of hydrogen, and ester-forming residues readily hydrolyzable in vivo, can be prepared by a further two-step procedure which comprises the steps of (c) contacting a compound of the formula 2 'i«'i Y H CH ' c vV 3 s y1 1 hcH k N 1 if A 3 . (XX) COOR1 0- or a base salt thereof with a reagent selected from the group consisting of alkali metal permanganates, alkaline earth metal permanganates and organic peroxycarboxylic acids, to give a compound of the formula 0 0 H % I _LK.»-P"3 -3\ ; ---(XXI) W n 199601 or a base salt thereof, wherein Y and Z are each selected from the group consisting of chloro, brono and iodo; provided that Y and Z are not both chloro and Y and Z are not both iodo; and (d) dehalogenating the compound of formula XXI.

A preferred way of carrying out step (]d) comprises contacting the product of step (c) with hydrogen, in an inert solvent, at a pressure / . in the range from about 1 to about 100 kg/cm , at a temperature in the range from about 0 to about 60° C,, and at a pH in the range from'about i 4 to about 9, and in the presence of a hydrogenolysis catalyst. The hydrogenolysis catalyst is usually present in an amount from about 0.01 to about 2.5 weight-percent, and preferably from about 0.1 to about 1.0 weight-percent, based on the dihalo-sulfone.

The preferred value for Y and Z is bromo, and the preferred reagents for carrying out step (c) are potassium permanganate and 3-chloroperbenzoic acid.

In the case wherein Y and Z are both chloro, the compound of formula XX is difficult to obtain. In the case wherein Y and Z are both iodo, step (c) of the above process proceeds inconveniently slowly. 1$ -■ o o / n < i J JOV i The compounds of formulas I, II and III, wherein R is hydrogen, are acidic and will form salts with basic agents. These salts can be prepared by standard techniques, such as contacting the acidic and basic components, usually in a 1:1 molar ratio, in an aqueous, non-aqueous or partially aqueous medium, as appropriate. They are then recovered by filtration, by precipation with a non-solvent followed by filtration, by evaporation of the solvent, or in the case of aqueous solutions, by lyophilization, as appropriate. Basic agents which are suitably employed in salt formation belong to both the organic and inorganic types, and they include ammonia, organic amines, alkali metal hydroxides, carbonates, bicarbonates, hydrides and alkoxides, as well as alkaline earth metal hydroxides, carbonates, hydrides and alkoxides. Representative examples of such bases are primary amines, such as n-propylamine, n-butylamine, aniline, cyclo-hexylamine, benzylamine and octylamine; secondary amines, such as diethylamine, morpholine, pyrrolidine and piperidine; tertiary amines, such as triethylamine, N-ethylpiperidine, N-methylmorpholine and 1,5-diazabicyclo/4.3.0/non-5-ene; hydroxides, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and barium hydroxide; alkoxides such as sodium ethoxide and potassium ethoxide; f I 1 99601 hydrides, such as calcium hydride and sodium hydride; carbonates, such as po tassiun carbonate and sodium carbonate; bicarbonatea, such as sodium bicarbo and potassium bicarbonate; and alkali metal salts of long-chain fatty acids, such as .sodium 2-ethylhexanoate-.

Preferred salts of the compounds of the formulas I, II and III are sodium, potassium and triethylamine salts.

The compounds of formula I, wherein R* is hydrogen or an ester-forming residue"Tead'Ily hydrolyzable in vivo, are antibacterial agents of nedium potency. The in vitro activity of the compound of the formula I, wherein R^" is hydrogen, can be demonstrated by measuring its minimum inhibitory concentrations (2-HC's) in meg/ml against a variety of microorganisms. The procedure which is followed is the one recommended by the International Collaborative Study on Antibiotic Sensitivity Testing % (Ericcson and Sherris, Acta. Pathologica et Kicrobiologia Scancinav, Supp. 217, Sections A and 3: 64-68 [1971]), and employs brain heart infusion , (SHI) agar and the inocula .replicating device. Overnight growth tubes are diluted 100 fold for use as the standard inoculum (20,000-10,000 cells in approximately 0.002 ml. are placed on the agar surface; 20 ml. of SHI agar/dish). Twelve 2 fold dilutions of the test compound are employed, with initial concentration of the test drug being 2C0 meg./ml. Single colonies are disregarded when reading plates after 18 hrs. at 37° C. The susceptibility (MIC) of the test organism is accepted as the lowest concentration of compound capable of producing complete inhibition of growth as •judged by the naked eye. MIC values for penicillanic acid 1,1-dioxide against several microorganisms are shown in Table I.

- - TABLE I In Vitro Antibacterial Activity of Penicillanic Acid 1,1-dioxide Microorganism MIC (meg./ml.) Staphylococcus aureus Streptococcus faecalis Streptococcus pyogenes Escherichia coli Pseudomonas aeruginosa Klebsiella pneumoniae Proteus mirabilis Proteus morgani Salmonella typhimurium Pasteurella multocida Serratia marcescens Enterobacter aerogenes Enterobacter clocae Citrobacter freundii Providencia staphylococcus epidermis Pseudomonas putida Hemophilus influenzae Neisseria gonorrhoeae 100 > 200 100 50 200 50 100 100 50 50 100 25 100 50 100 200 > 200 7 50 -a i The compounds of the formula I, wherein R is hydrogen or an ester-forming residue readily hydrolyzable ill vivo, are active as antibacterial agents in vivo. In determining such activity, acute experimental infection are produced in mice by the intraperitoneal inoculation of the mice with a standardized culture of the test organism suspended in 5 percent hog gastric mucin. Infection severity is standardized so that the mice receive one to ten times the dose of the organism (LD^^: the minimum inoculum of organism required to consistently kill 100 percent of the infected, non-treated control mice). The test compound is administered to the infected mice using a multiple dosage regimen. At the end of the test, the activity of a compound is assesed by counting the number of survivors among the treated animals and expressing the activity of a compound as the percentage of animals which survive.

The iri vitro antibacterial activity of the compound of the formula I wherein R1 is hydrogen makes it useful as an industrial antimicrobial, for example in water treatment, slime control, paint preservation and wood preservation, as well as for topical application as a disinfectant.

In the case of use of this compound for topical application, it is . often convenient to admix the active ingredient with a non-toxic carrier, such as vegetable or mineral oil or an emollient cream. Similarly, it can be dissolved or dispersed in liquid diluents or solvents such as water, alkanols, glycols or mixtures thereof. In most instances it is appropriate to employ concentrations of the active ingredient of from 0.1 percent to about 10 percent by weight, based on total composition.

The iri vivo activity of the compounds of formula I, wherein R^" is hydrogen or an ester-forming-residue readily hydrolyzable in vivo, makes them suitable for the control of bacterial infections in mammals, including man, by both the oral and parenteral modes of administration. The compounds will find use in the control of infections caused by susceptible bacteria in hyman subjects, e.g. infections caused by strains of Neisseria gonorrhoeae . 0.1 When considering therapeutic use of a compound of the formula I, or a salt thereof, in a mammal, particularly man, the compound can be administered alone, or it can be mixed with pharmaceutically acceptable carriers or diluents. They can be administered orally or parenterally, i.e. intramuscularly, subcutaneously or intraperitoneally. The carrier or diluent is chosed on the basis of the intended mode of administration. For example, when considering the oral mode of administration, an antibacterial penam compound of formula I can be used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like, in accordance with standard pharmaceutical practice. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility and stability of the active ingredient, as well as the dosage contemplated. However, pharmaceutical compositions containing an antibacterial agent of the formula I will likely contain from about 20% to about 95% of active ingredient. In the case of tablets and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavouring agents can be added. For parenteral administreation, which includes intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. 199601 "- . The antibacterial agents of formula I are of use in human subjects against susceptible organisms. The prescribing physician will ultinately determine the appropriate dose for a given human subject, and this can be expected to vary according to the age, weight, and response of the individual patient, as well as the nature and the severity of the patient's symptomsi The compounds of formula I will normally be used orally at dosages in the range from about 10 to about 200 mg. per kilogram of body weight pe!r day, and parenterally at dosages from about 10 to about 400 mg. per kilogram of body weight per day. These figures are illustrative only, however, and in some cases it nay be necessary to use dosages outside these limits.

However, as indicated hereinbefore, the compounds of the formula I, wherein R1 is hydrogen or an ester-forming residue readily hydrolyzable in vivo, are potent inhibitors of microbial beta-lactamases, and they increase the antibacterial effectiveness of 7*-(D-2-[4-ethylpiperazin-2,3-dione-l-carboxamido]—2—[4-hydroxyphenyl]acetamido)—3—([l-methyl-5-tetrazolyl] thio— methyl)—3—desacetoxymethylcephalosporanic acid and the pharmaceutically— acceptable salts thereof. The latter named compound is the compound of the formula Wv t 99601 HO <XXII) and methods for its preparation are described in United States Patent No. 4-,087,424 and Belgian patent specification 837,682. Whenever reference is made to the compound of formula XXII, said reference is also intended to embrace the pharmaceutically-acceptable salts thereof.

The manner in which the compounds of the formula I increase the effectiveness of the compound of formula XXII and the pharmaceutically-acceptable salts thereof can be appreciated by reference to experiments in which the MIC of the compound of the formula XXII alone, and the compound of the formula I wherein S.1" is hydrogen alone, are measured. These MIC1 s are then compared with the MIC values obtained with a combination of the compound of formula XXII and the compound of the formula I, vherein R1" is hydrogen.

When the antibacterial potency of the combination is significantly greater than would have been predicted from the potencies.of the individual compounds, this is considered to constitute enhancement of activity. The MIC values of combinations are measured using the method described by Barry and Sabath in "Manual of Clinical Microbiology", edited by Lenette, Spaulding and Truant, 2nd edition, 1974, American Society for Microbiology. 4 I 9 9601 The compounds of formula I are particularly useful for enhancing the antibacterial effectiveness of the compound of formula XZII against ampicillin-resistant strains of Escherichia coli and 3acteroides spp.

The compounds of the formula I, wherein is hydrogen or an ester-forming residue readily hydrolyzable in vivo, and salts thereof, enhance the antibacterial effectiveness of the compound of formula XXII in vivo. This is, they lower the amount of the coapound of formula XXII which is needed to protect mice against an otherwise lethal inoculum of certain beta-lactamase producing bacteria.

The ability of the compounds of•the formula I, wherein R1 is I hydrogen or an ester-forming residue readily hydrolyzable _in vivo, and salts thereof, to enhance the effectiveness of the compound of formulaXXII against beta-lactsmase-producing bacteria makes them valuable for coadministration with the compound of formula XXII in the treatment of 15 bacterial infections in mammals, particularly man. In the treatment of a bacterial infection, said compound of the formula I or salt thereof can be coiaingled with the compound of formula XXII, and the two agents thereby administered simultaneously. Alternatively, said compound of the formula' I or salt thereof can be administered as a separate agent during a course 20 of treatment with the compound of foraula XXII. In some instances it will be advantageous to pre—dose the subject with the compound of the formula I or salt thereof before initiating treatment with the compound of formula XXII. ab When a compound of formula I or salt thereof is used as a separate agent during a course of treatment with the compound of formula XXII, the compound of formula I can be administered orally or parenterally, which includes intramuscular, subcataneous and intraperitoneal use. For these purposes, the compound of formula I or salt thereof is administered preferably in formulation with standard pharmaceutical carriers or diluents. The methods of formulation discussed earlier for use of a compound of formula I as a single-entity antibacterial agent can be used.

When a compound of the formula I or salt thereof and the compound of formula XXII are to be co-mingled for co-administration to a mammal, it is also preferable to add a pharmaceutical carrier or diluent. Additionally, in this instance, it is preferable to prepare a formulation suitable for parenteral administration, since the compound of formula XXII is more effective when administered parenterally. Parenteral use includes intramuscular, subcutaneous and intraperitoneal use. For these purposes, the compound of formula I, or salt thereof, and the compound of formula XXII are co-formulated using the methods discussed earlier for preparation of a pharmaceutical composition of a compound of formula I suitable for parenteral use. A pharmaceutical composition comprising a pharmaceutically-acceptable carrier, the compound of the formula XXII, and a compound of the formula I or salt thereof will normally contain from about 5 to about 80 percent of the pharmaceutically-acceptable carrier by weight.

Although the prescribing physician will utlimately decide the dosages of the compound of formula XXII and a compound of formula I salt thereof, the ratio of the daily dosages of the two compounds will normally be in the range from about 1:6 to about 6:1 by weight, and preferably 1:2 to 2:1. Additionally, the daily parenteral dosage of each component will normally be in the range from about 100 mg. per kilogram of body weight, and preferably from about 10 to about 50 mg. per kilogram of body weight. The daily oral dosage of a compound of formula I or salt thereof will normally be about 5 to about 100 mg. per kilogram of body weight and preferably from about 10 to about 50 mg. per kilogram of body weight. These figures are illustrative only, however, and in some cases it may be neces- 1 9 96 0 1 The following examples are provided solely for the purpose of further illustration. Infrared (IR) spectra were measured as potassium bromide discs (XBr discs) or as Nujol culls, and diagnostic absorption bands are reported in wave numbers (cm ^)". Nuclear magnetic resonance spectra (N>IR) were measured at 60 MHz for solutions in deuterochloroform (CDCl^), perdeutero dimethyl sulfoxide (DMSO-d^) or deuterium oxide (D^O), and peak positions are expressed in parts per million (ppta) downfield from tetranethylsilane or sodium 2,2-dimethyl-2-silapentane-5-sulfonate. The I following abbreviations for peak shapes are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. ' •• n o 1 n < : J j U"J ' EXAMPLE I Penicillanic Acid 1,1-Dioxide To a solution of 6.51 g. (41 mmole) of potassium permanganate in 130 ml. of water and 4.95 ml. of glacial acetic acid, cooled to ca. 5°C., was added a cold (ca 5°C.) solution of 4.58 g. (21 mmole) of the sodium salt of penicillanic acid in 50 ml. of water. The mixture was stirred at ca. 5°C. for 20 minutes and then the cooling bath was removed. Solid sodium bisulfite was added until the color of the potassium permanganate had been discharged, and then the mixture was filtered. To the aqueous filtrate was added half its volume of saturated chloride solution, and then the pH was adjusted to 1.7. The acidic solution was extracted with ethyl acetate. The extracts were dried, and then evaporated in_ vacuo, to give 3.47 g. of the title product. The aqueous mother liquor was saturated with sodium chloride, and further extracted with ethyl acetate. The ethyl acetate solution was dried and evaporated in vacuo, to give a further 0.28 g. of product. The total yield was therefore 3.75 g. (78% yield). The NMR spectrum (DMSO-dg) of the product showed absorptions at 1.40 (s,3H), 1.50 (s,3H), 3.13 (d of d's, 1H, JL = 16Hz, 3^ = 2Hz), 3.63 (d of d's, 1H, J = 16 Hz, J2 = 4Hz), 4.22 (s,1H) and 5.03 (d of d's, 1H, - 4Hz, = 2Hz) ppm. •: o o / n * i •* r i ! \ / J yj ■j \ EXAMPLE 2 Benzyl Penicillanate 1,1-Dioxide To a stirred solution of 6.85 solution of 6.85 g. (24 mmole) of benzyl penicillanate in 75 ml. of ethanol-free chloroform, under nitrogen, in an ice-bath, was added in two portions, several minutes apart, 4.68 g. of 85% pure 3-chloroperbenzoic acid. Stirring was continued for 30 minutes in the ice-bath, and then for 45 minutes without external cooling. The reaction mixture was washed with aqueous alkali (ph 8.5), followed by saturated sodium chloride, and then it was dried and evaporated in_ vacuo to give 7.05 g. of residue. Examination of this residue showed it to be a 5.5:1 mixture of benzyl penicillanate 1-oxide and benzyl penicillanate 1,1-dioxide.

To a stirred solution of 4.85 g. of the above 5.5:1 sulfoxide-sulfone mixture in 50 ml. of. ethanol-free chloroform, .under nitrogen, was added 3.2 g. of 85% pure 30-chloroperbenzoic acid at room temperature. The reaction mixture was stirred for 2.5 hours, and then it was diluted with ethyl acetate. The resultant mixture was added to water at pH 8.0, and then the layers were separated. The organic phase was washed with water at pH 8.0, followed by saturated sodium chloride, and then it was dried using sodium sulfate. Evaporation of the solvent iri vacuo afforded 3.59 g. of the title compound. The NMR spectrum of the product (in CDCI3) showed absorptions at 1.28 (s,3H), 1.58 (s,3H), 3.42 (m,2H), 4.37 (s,lH), 4.55 (m,1H), 5.18 (q,2H, J = 12 Hz) and 7.35 (s,5H) ppm. i-96^ = i / s \J u .

EXAMPLE'3 Penicillanic Acid 1,1-Dioxide To a stirred solution of 8.27 g. of benzyl penicillanate 1,1-dioxide in a mixture of 40 ml. of methanol and 10 ml. of ethyl acetate was slowly added to 10 ml. of water, followed by 12 g. of 5% palladium-on-calcium carbonate. The mixture was shaken under an atmosphere of hydrogen, at 52 psi, for 40 minutes, and then it was filtered through supercel (a diatomaceous earth). The filter cake was washed with methanol, and with aqueous methanol, and the washings were added to the filtrate. The combined solution was evaporated in vacuo to remove the majority of the organic solvents and then the residue was partitioned between ethyl acetate and water at a pH of 2,8. The ethyl acetate layer was removed and the aqueous phase was further extracted with ethyl acetate The combined ethyl acetate solutions were washed with saturated sodium chloride solution, dried using sodium sulfate and then evaporated in vacuo The residue was slurred in a 1:2 mixture of ethyl acetate-ether, to give 2.37 g. of the title product having a melting point of 148-51°C. The ethyl acetate-ether mixture was evaporated giving a further 2.17 g. of product.

EXAMPLE 4 Pivaloyloxymethyl Penicillanate 1,1-Dloxide To 0.615 g. (2.41 mmole) of penicillanate acid 1,1-dioxide in 2 ml. of N,N-dimethylformamide was added 0.215 g. (2.50 mmole) of diisopropylethylamine followed by 0.365 ml. of chloromethyl pivalate. The reaction mixture was stirred at room temperature for 24 hours, and then it was diluted with ethyl acetate and water. The ethyl acetate layer was separated and washed three times with water and once with saturated sodium chloride solution. The ethyl acetate was then dried using anhydrous sodium sulfate, and evaporated in vacuo to give 0.700 g of the title product as a solid, mp 103-4°C. The NMR spectrum of the product (in CDCI^) showed absorptions at 1.27 (s, 9H), 1.47 (s, 3H), 1.62 (s, 3H), 3.52 (m, 2H), 4.47 (s, 1H), 4.70 (m, 1H), 5.73 (d, 1H, J = 6.0 Hz) and 5.98 (d, 1H, J = 6.0 Hz).

EXAMPLE 5 The procedure of Example 4 is repeated, except that the pivaloyoxy methyl chloride used therein is replaced by an equimolar amount of acetoxymethyl chloride, propionyloxymethyl chloride and hexanoyloxy-methyl chloride, respectively, to give: acetoxymethyl penicillanate 1,1-dioxide, propionyloxymethyl penicillanate 1,1-dioxide and hexanoyloxymethyl penicillanate 1,1-dioxide, respectively. 00 f?:< / 0 - .

EXAMPLE 6 3-Phthalidyl Penicillanate 1,1-Dioxide To 0.783 g. (3.36 mmole) of penicillanic acid 1,1-dioxide in 5 ml. of N,N-dimethylformamide was added 0.47 ml. of triethylamine followed by 0.715 g. of 3-bromophthalide, The reaction mixture was stirred for 2 hours at room temperature and then it was diluted with ethyl acetate and water. The pH of the aqueous phase was raised to 7.0 and the layers were separated. The ethyl acetate layer was washed successively with water and saturated sodium chloride solution, and then it was dried using sodium sulfate. The ethyl acetate solution was evaporated in_ vacuo leaving the title product as a white foam. The NMR spectrum of the product (in CDCI^) showed absorptions at (s, 6H), 3.43 (m, 1H), 4.45 (s, 1H), 4.62 (m, 1H), 7.40 and 7.47 (2s's, 1H) and 7.73 (m, 4h) ppm.

When the above procedure is repeated, except that the 3-bromophthalide is replaced by 4-bromocrotonolactone and 4-bromo-y-butyrolactone, respectively, this affords: 4-crotonolactonyl penicillanate 1,1-dioxide and y-butyrolacton-4-yl penicillanate, 1,1-dioxide, respectively. ■f 0'0/t ^ EXAMPLE 7 1-(Ethoxycarbonyloxy)ethyl Penicillanate 1,1-Dioxide A mixture of G.654 g. of penicillanic acid 1,1-dioxide, 0.42 ml. of triethylamine, 0.412 g. of 1-chloroethyl ethyl carbonate, 0.300 g. of sodium bromide and 3 ml. of t^, N_—d imethyl form amide was stirred at room, temperature for 6 days. It was then worked up by diluting it with ethyl acetate and water, and then the pH was adjusted to 8.5. The ethyl acetate layer was separated, washed three times with water, washed once with saturated sodium chloride, and then it was dried using anhydrous sodium sulfate. The ethyl acetate was removed by evaporation in vacuo leaving 0.390 g. of the title product as an oil.

The above product was combined with an approximately equal amount of similar material from a similar experiment. The combined product was dissolved in chloroform and 1 ml. of pyridine was added. The mixture was stirred at room temperature overnight and then the chloroform was removed by evaporation in vacuo. The residue was partitioned between ethyl acetate and water at pH 8. The separated and dried ethyl acetate was then evaporated in vacuo to give 150 mg. of the title product (yield ca 7%). The IR spectrum (film) of the product showed absorptions at 1805 and 1763 cm \ The NMR spectrum (CDCI^) showed absorptions at 1.43 (m, 12H), 3.47 (m, 2H), 3.9 (q, 2H, J =7.5 Hz), 4.37/m, 1H), 4,63 (m, lH) and' 6.77 (m, 1H) ppm. v)96Hf s * , * i EXAMPLE 8 The procedure of Example 7 is repeated, except that the 1-chloro-ethyl ethyl carbonate is replaced by an equimolar amount of the appropriate 1-chloroalkyl alkyl carbonate, 1-(alkanoyloxy)ethyl chloride or 1-methyl-l-(alkanoyloxy)ethyl chloride, to produce the following compounds: methoxycarbonyloxymethyl penicillanate 1,1-dioxide, ethoxycarbonyloxymethyl penicillanate 1,1-dioxide, isobutoxycarbonyloxymethyl penicillanate 1,1-dioxide, 1-(methoxycarbonyloxy)ethyl penicillanate 1,1-dioxide, 1-(butoxycarbonyloxy)ethyl penicillanate 1,1-dioxide, 1-(acetoxy)ethyl penicillanate 1,1-dioxide, 1-(butyryloxy)ethyl penicillanate 1,1-dioxide, 1-(pivaloyloxy)ethyl penicillanate 1,1-dioxide, 1-(hexanoyloxy)ethyl penicillanate 1,1-dioxide, 1-methyl-l-(acetoxy)ethyl penicillanate 1,1-dioxide and 1-methyl-l-(isobutyryloxy)ethyl penicillanate 1,1-dioxide, respectively.

OAMPr,i::_9_ H ^ ■ 1 y 70U ; The procedure of Example 4 is repeated, except that the chloromethyl pivalate is.replaced by an equimolar amount of benzyl bromide and 4-nitro-benzyl bromide, respectively, to produce benzyl penicillanate 1,1-dioxide and 4-nitrobenzyl penicillanate 1,1-dioxide, respectively.

EXAMPLE 10 Penicillanic Acid la-Oxide To 1.4 g. of prehydrogenated 5% palladium-on-calcium carbonate in 50 ml. of water was added a solution of 1.39 g. of benzyl 6,6-dibromo-penicillanate la-oxide in 50 ml. of tetrahydrofuran. The mixture was shaken under an atmosphere of hydrogen at ca. 45 p.s.i. and 25°C. for 1 hour, and then it was filtered. The filtrate was evaporated in vacuo to remove the bulk of the tetrahydrofuran, and then the aqueous phase was extracted with ether. The ether extracts were evaporated in vacuo to give 0.5 g. of material which appeared to be largely benzyl penicillanate la-oxide.

The above benzyl penicillanate la-oxide was combined with a further 2.0 g. of benzyl 6,6-d.ibromopenicillanate la-oxide and dissolved in 50 ml. of tetrahydrofuran. The solution was added to 4.0 g. of 5% palladium-on-calcium carbonate, in 50 ml. of water, and the resulting mixture was shaken under an atmosphere of hydrogen, at ca. 45 p.s.i. and 25°C. overnight. The mixture was filtered, and the filtrate was extracted with ether. The extracts were evaporated in vacuo, and the residue was purified by chromatography on silica gel, eluting with chloroform.

This afforded 0.50 g. of material.

The latter material was hydrogenated at ca. 45 p.s.i. at 25°C. in water-methanol (1:1) with 0.50 g. of 5% palladium-on-calcium carbonate for two hours. At this point, an additional 0.50 g. of 5% palladium-on-calcium carbonate was added and the hydrogenation was continued at 45 p.s.i.

ENrH.

J/Ai (*,15 APR 1982 - 3f> - | V O ' f, < ' ;; o 'j: and 25°C overnight. The reaction mixture was filtered, extracted with ether and the extracts were discarded. The residual aqueous phase was adjusted to pH 1.5 and then extracted with ethyl acetate. The ethyl acetate extracts were dried (Na^SO^) and then evaporated iii vacuo to give 0.14 g. of penicillanic acid la-oxide. The NMR spectrum (CDCI^/ DMSO-d^) showed absorptions at 1.4 (s, 3H), 1.64 (s, 3H), 3.60 (m, 2H), 4.3 (s, 1H) and 4.54 (m, 1H) ppm. The IR spectrum of the product (KBr disc) showed absorptions at 1795 and 174 5 cm EXAMPLE 11 Penicillanic Acid la-Oxide To 1.0 g. of prehydrogenated 5% palladium-on-clacium carbonate in 30 ml. of water is added a solution of 1.0 g. of 6,6-dibromopenicillanic acid la-oxide. The mixture is shaken under an atmosphere of hydrogen, at ca. 45 p.s.i. and 25°C, for one hour. The reaction mixture is then filtered and the filtrate is concentrated in vacuo.to remove the methanol. The residual aqueous phase is diluted with an equal volume of water, adjusted to pH 7, and washed with ether. The aqueous phase is then acidified to pH 2 with dilute hydrochloric acid and extracted with ethyl acetate. The ethyl acetate extracts are dried (Na^O^) and evaporated in vacuo to give penicillanic acid la-oxide. -■ 0<5 " ; „■ J -0 J ; EXAMPLE 12 Penicillanic Acid IB-Oxide To a stirred solution of 2.65 g. (12.7 mmole) of penicillanic acid in chloroform at 0°C. was added 2.58 g. of 85% pure 3-chloroperbenzoic acid. After 1 hour, the reaction mixture was filtered and the filtrate was evaporated in vacuo. The residue was dissolved in a small amount of chloroform. The solution was concentrated slowly until a precipitate began to appear. At this point the evaporation was stopped and the mixture was diluted with ether. The precipitate was removed by filtration, washed with ether and dried, to give 0.615 g. of penicillanic acid IB-oxide, m.p. 140-3°C. The IR spectrum of the product (CHCI^ solution) showed absorptions at 1775 and 1720 cm ^. The NMR spectrum (CDCI^/DMSO-dg) showed absorptions, at 1.35 (s, 3H) , 1.76 (s, 3H), 3.36 (m, 2H), 4.50 (s, 1H) and 5.05 (m, 1H) ppm. From the NMR spectrum, the product appeared to be ca. 90% pure.

Examination of the chloroform-ether mother liquor revealed that it contained further penicillanic acid IB-oxide, and also some penicillanic acid la-oxide.

EXAMPLE 13 Esterification of penicillanic acid la-oxide or penicillanic acid IB-oxide, as appropriate, with the requisite alkanoyloxy chloride, according to Example 5, provides the following compounds: acetoxymethyl penicillanate la-oxide, propionyloxymethyl penicillanate la-oxide, pivaloyoxymethyl penicillanate la-oxide, acetoxymethyl penicillanate IB-oxide, propionyloxymethyl penicillanate IB-oxide and pivaloyloxymethyl penicillanate IB-oxide, respectively.

EXAMPLE 14 Reaction of penicillanic acid la-oxide or penicillanic acid IB-oxide with 3-bromophthalide, 4-bromocrotonolactone or 4-bromo-y-butyrolactone, as appropriate, affords the following compounds: 3-phthalidyl penicillanate la-oxide 4-crotonolactonyl penicillanate la-oxide, 3-phthalidyl penicillanate IB-oxide, 4-crotonolactonyl penicillanate IB-oxide and y-butyrolactori-4-yl penicillanate IB-oxide, respectively. n,':V ! 1> ■ EXAMPLE 15 Reaction of penicillanic acid la-oxide or penicillanic acid lb-oxide, as appropriate, with the requisite 1-chloroalkyl alkyl carbonate, 1-(alkanoyloxy)ethyl chloride, N-(alkoxycarbonyl)amim methyl chloride or 1-(N-/alkoxycarbonyl/amino)ethyl chloride, according to the procedure of Example 7, provides the following compounds: 1-(ethoxycarbonyloxy)ethyl penicillanate la-oxide, methoxycarbonyloxymethyl penicillanate la-oxide, ethoxycarbonyloxymethyl penicillanate la-oxide, isobutoxycarbonyloxymethyl penicillanate la-oxide, 1-(methoxycarbonyloxy)ethyl penicillanate la-oxide, 1-(butoxycarbonyloxy)ethyl penicillanate la-oxide, 10(acetoxy)ethyl penicillanate la-oxide, 1-(butyryloxy)ethyl penicillanate la-oxide, 1-(pivaloyloxy)ethyl penicillanate la-oxide, l-(ethoxycarbonyloxy)ethyl penicillanate IB-oxide, methoxycarbonyloxymethyl penicillanate lB-oxide, ethoxycarbonyloxymethyl penicillanate■lB-oxide, isobutoxycarbonyloxymethyl penicillanate lB-oxide, 1-(methoxycarbonyloxy)ethyl penicillanate lB-oxide, 1-(butoxycarbonyloxy)ethyl penicillanate lB-oxide, 1-(acetoxy)ethyl penicillanate lB-oxide, 1-(butyryloxy)ethyl penicillanate lB-oxide, 1-(pivaloyloxy)ethyl penicillanate lB-oxide, 1 9 96 01t N-(methoxycarbonyl)aminometh,yl penicillanate la-roxide, t i N-(ethyloxycarbonyljaminomethyl penicillanate lavOxi.de, N-(hexyloxycarbanyl)aiainoniethyl penicillanate la-oxide, l-(N-[raethyloxycarbonyl]aiaino)ethyl penicillanate la-oxide, 1-(N-[isopropoxycarbonyl]pnino)ethyl penicillanate la-oxide, l-(tI-[hexyloxycarbonyl]anino)ethyl penicillanate la-oxide, N- (methoxycarbonyl)aminomethyl penicillanate IB—oxide, N-(ethoxycarbonyl)aninoiaethyl penicillanate IB—oxide, N-(hexyloxycarbonyl)aiainon!ethyl penicillanate lg-oxide, 1-(n-[aethoxycarbony1]a-rrfno)ethyl penicillanate IB—oxide, l-(N-[t-butoxycarbonyl]amino)ethyl penicillanate IB-oxide and 1- (N-[hexyloxycarbonyl]amino)ethyl penicillanate IB-oxide, respectively- w \ I EXAMPLE 16 Reaction of penicillanic acid la-oxide and penicillanic acid IB-oxide with benzyl bromide, according to the procedure of Example 4, produces benzyl penicillanate la-oxide and benzyl penicillanate lB-oxide, respectively.

In like manner, reaction of penicillanic acid la-oxide and penicillanic acid lB-oxide with 4-nitrobenzyl bromide, according to the procedure of Example 4, produces 4-nitrobenzyl penicillanate la-oxide and 4-nitrobenzyl penicillanate lB-oxide, respectively.

EXAMPLE 17 Penicillanic Acid 1,1-Dioxide To 2.17 g. (10 mmole) of penicillanic acid la-oxide in 30 ml. of ethanol-free chloroform at ca_. 0°C. is added 1.73 g. (10 mmole) of 3-chloroperbenzoic acid. The mixture is stirred for 1 hour at ca. 0°C. and then for an additional 24 hours at 25°C. The filtered reaction mixture is evaporated in vacuo to give penicillanic acid 1,1-dioxide. •; 0Q/.'p.< •' * ^ ■ EXAMPLE 18 The procedure of Example 17 is repeated, except that the penicillanic acid la-oxide used therein is replaced by: penicillanic acid lB-oxide, acetoxymethyl penicillanate la-oxide, propionyloxymethyl penicillanate la-oxide, pivaloyoxymethyl penicillanate la-oxide, acetoxymethyl penicillanate IB-oxide, propionyloxymethyl penicillanate lB-oxide, pivaloyloxymethyl penicillanate lB-oxide, 3-phthalidyl penicillanate la-oxide, 3-phthalidyl penicillanate lB-oxide, 1-(ethoxycarbonyloxy)ethyl penicillanate la-oxide, methoxycarbonyloxymethyl penicillanate la-oxide, ethoxycarbonyloxymethyl penicillanate la-oxide, isobutoxycarbonyloxymethyl penicillanate la-oxide, 1-(methoxycarbonyloxy)ethyl penicillanate la-oxide, 1-(butoxycarbonyloxy)ethyl penicillanate la-oxide, 1-(acetoxy)ethyl penicillanate la-oxide, 1-(butyryloxy) ethyl penicillanate la-oxide, 1-(pivaloyloxy)ethyl penicillanate la-oxide, l-(ethoxycarbonyloxy)ethyl penicillanate lB-oxide, methoxycarbonyloxymethyl penicillanate lB-oxide, ethoxycarbonyloxymethyl penecillanate lB-oxide, isobutoxycarbonyloxymethyl penicillanate lB-oxide, 1-(methoxycarbonyloxy)ethyl penicillanate lB-oxide, 1-(butoxycarbonyloxy)ethyl penicillanate lB-oxide, 1-(acetoxy)ethyl penicillanate lB-oxide, 1-(butyryloxy)ethyl penicillanate lB-oxide and 1-(pivaloyloxy)ethyl penicillanate lB-oxide, respectively. This affords: penicillanic acid 1,1-dioxide, acetoxymethyl penicillanate 1,1-dioxide, propionyloxymethyl penicillanate 1,1-dioxide, pivaloyoxymethyl penicillanate 1,1-dioxide, acetoxymethyl penicillanate 1,1-dioxide, propionyloxymethyl penicillanate 1,1-dioxide, pivaloyloxymethyl penicillanate 1,1-dioxide, 3-phthalidyl penicillanate 1,1-dioxide, 3-phthalidyl penicillanate 1,1-dioxide, 1-(ethoxycarbonyloxy)ethyl penicillanate 1,1-dioxide, methoxycarbonyloxymethyl penicillanate 1,1-dioxide, ethoxycarbonyloxymethyl penicillanate 1,1-dioxide, isobutoxycarbonyloxymethyl penicillanate 1,1-dioxide, 1-(methoxycarbonyloxy)ethyl penicillanate 1,1-dioxide, 1-(butoxycarbonyloxy)ethyl penicillanate 1,1-dioxide, l-(acetoxy)ethyl penicillanate 1,1-dioxide, 1-(butyryloxy)ethyl penicillanate 1,1-dioxide, 1-(pivaloyloxy)ethyl penicillanate 1,1-dioxide, 1-(ethoxycarbonyloxy)ethyl penicillanate 1,1-dioxide, methoxycarbonyloxymethyl penicillanate 1,1-dioxide, ethoxycarbonyloxymethyl penicillanate 1,1-dioxide, isobutoxycarbonyloxymethyl penicillanate 1,1-dioxide, 1-(methoxycarbonyloxy)ethyl penicillanate 1,1-dioxide, 1-(butoxycarbonyloxy)ethyl penicillanate 1,1-dioxide, 1-(acetoxy)ethyl penicillanate 1,1-dioxide, 1-(butyryloxy)ethyl penicillanate 1,1-dioxide and 1-(pivaloyloxy)ethyl penicillanate 1,1-dioxide, respectively.

APR / 982 - 44 - \ EXAMPLE 19 Oxidation of benzyl penicillanate la-oxide and benzyl penicillanate lB-oxide with 3-chloroperbenzoic acid, according to the procedure of Example 17, produces, in each case, benzyl penicillanate 1,1-dioxide.

In like manner, oxidation of 4-nitrobenzyl penicillanate la-oxide and 4-nitrobenzyl penicillanate lB-oxide with 3-chloroperbenzoic acid, according to the procedure of Example 17, produces 4-nitrobenzyl penicillanate 1,1-dioxide.

EXAMPLE 20 Penicillanic Acid 1,1-Dioxide Hydrogenolysis of 4-nitrobenzyl penicillanate 1,1-dioxide, according to the procedure of Example 3, affords penicillanic acid 1,1-dioxide.

EXAMPLE 21 Sodium Penicillanate 1,1-Dioxide To a stirred solution of 32.75 g. (0.14 mole) of penicillanic acid 1,1-dioxide in 450 ml. of ethyl acetate was added a solution of 25.7 g. of sodium 2-ethylhexanoate (0.155 mole) in 200 ml. of ethyl acetate. The resulting solution was stirred for 1 hour and then an additional 10% excess of sodium 2-ethylhexanoate in a small volume of ethyl acetate was added. Product immediately began to precipitate. Stirring was continued for 30 minutes and then the precipitate was removed by filtration. It was washed sequentially with ethyl acetate, with 1:1 ethyl acetate-ether and with ether. The solid was then dried over phosphorus pentoxide, at ca. 0.1 mm of Hg for 16 hours at 25°C., giving 36.8 g. of the title sodium salt, contaminated with a small amount of ethyl acetate. The ethyl acetate content was reduced by heating to 100 C. for 3 hours under vacuum. The lR spectrum of this i> o / fi t « r r, i , / final prcxiuct (BBr disc) showed absorptions at 1786 and 1608 cm \ The NMR spectrum (D2O) showed absorptions at 1.48 (s, 3H), 1.62 (s, 3H) , 3.35 (d of d's, 1H, = 16 Hz, = 2Hz), 3.70 (d of d's> lH, = 16 Hz, = 4 Hz), 4.25 (s, 1H) and 5.03 (d of d's, 1H, J^= 4Hz, J' = 2Hz) ppm.

The title sodium salt can also be prepared using acetone in place of ethyl acetate.

EXAMPLE 22 Penicillanic Acid 1,1-Dioxide To a mixture of 7,600 ml. of water and 289 ml. of glacial acetic acid was added, portionwise, 379.5 g. of potassium permanganate. This mixture was stirred for 15 minutes, and then it was cooled to 0°C.

To it was then added, with stirring, a mixture which had been prepared from 270 g. of penicillanic acid, 260 ml. of 4N sodium hydroxide and o 2,400 ml. of water (pH 7.2), and which had then been cooled to 8 C. o The temperature rose to 15 C. during this latter addition. The temperature of the resulting mixture was reduced to 5°C. and the stirring was continued for 3 0 minutes. To the reaction mixture was then added 142.1 g. of sodium bisulfite, in portions, during 10 minutes. The mixture was stirred for 10 minutes at 10°C., and then 100 g. of supercel (a diatomaceous earth) was added. After a further 5 minutes of stirring, the mixture was filtered. To the filtrate was added 4.0 liters of ethyl acetate, and then the pH of the aqueous phase was lowered to 1.55 using 6N hydrochloric acid. The ethyl acetate layer was removed and combined with several further ethyl acetate extracts. The combined organic layer was washed with water, dried (MgSO^) and evaporated almost to dryness ill vacuo. The slurry thus obtained was stirred with 700 ml. of ether at 10°C., for 20 minutes, and then the solid was collected by filtration. This afforded 82.6 g. (26% yield) of the title compound having a melting point of 154-155.5°C. (dec.). 19560 EXAMPLE 23 Pivaloyloxymethyl Penicillanate 1,1-Dioxide To a solution of 1.25 g. pivaloyoxymethyl penicillanate in 40 ml. of chloroform, cooled to ca. -15°C., was added 0.8 g. of 3-chloroperbenzoic acid. The mixture was stirred at ca. -15°C. for 20 minutes and then it was allowed to warm to room temperature. Analysis of the resulting solution by NMR indicated that it contained both the la-and lB-oxide.

The chloroform solution was concentrated to about 20 ml. and a further 0.8 g. of 3-chloroperbenzoic acid was added. This mixture was stirred overnight at room temperature, and then all the solvent was removed by evaporation in vacuo. The residue was redissolved in ca 4 ml. of dichloromethane and 0.4 m. of 3-chloroperbenzoic.acid was added. The mixture was stirred for 3 hours and then the solvent was removed by evaporation in_ vacuo. The residue was partitioned between ethyl acetate and water at pH 6.0, and sodium bisulfite was added until a test for the presence of peroxides was negative. The pH of the aqueous phase was raised to 8.0 and the layers were separated. The organic layer was washed with brine, dried using anhydrous sodium sulfate and evaporated in vacuo. The residue was dissolved in ether and reprecipitated by the addition of hexane. The resulting solid was recrystallized from ether to give 0.357 g. of the title compound.

The NMR spectrum of the product (CDCI^) showed absorptions at 1.23 (s, 9H), 1.50 (s, 2H), 1.67 (s, 3H), 3.28 (m, 2H), 4.45 (s, lH), 5.25 (m, lH) and 5.78 (m, 2H) ppm. - 4R - 4 199601 EXAMPLE 24 ; 3-?hthalidvl Penicilianace l.l-Dioxide To "a solution of 713 mg. of 3-phthalidyl penicillanate in 3' ml. •• of chloroform was added 0.430 g.-of 3-chloroperbenzoic acid at ca. 10" C.

The .mixture was stirred for 30 minutes and then a further 0.513 g. of 3- chloroperbenzoic acid was. added. The mixture was stirred for 4 hours at room temperature, and then the solvent was removed by evaporation in vacuo.

\ The residue was" partitioned between ethyl acetate and water at pK 6-0, and sodium bisulfite was added to decompose any remaining peracid. ThepH / • of the aqueous phase was raised to 8.8. The layers were separate.d and the organic phase was evaporated in vacuo. This affotded the title coapound as foan. The NMR spectrua (CDCl^) showed absorptions at 1.62 (m,6E), 3.3(ni,2H) 4.52 (p,lil), 5.23(m,lE) and-7.63 (tn,5E)ppa.

M 199601 EXAMPLE 25 2,2,2-Tric'nloroethyl Penicillanate 1,1-Dioxide To 100 ag. of 2,2,2-trichloroethyl penicilla.na.ce in a ssiall'voluae of chlorofom was added 50 mg- of- 3-chloroperbenzoic acid and the mixture was stirred for 30 adnutes. Examination of che reaction product at eliis point revealed that it was costly sulfoxide (The NMR spectrum (CDCl^) showed absorptions at 1.6 (s,3H), 1.77 (s,3H),•3.30(n,2H), A.65 (s,lH), 4.85 0n,2H) and 5.37 (n,lH)ppa.) A further 100 mg. of 3-chloroperbenzoic acid was added and the mixture was stirred overnight^' The solvent was then ratsoved by evaporation in vacuo, and the residue was partitioned between ethyl acetate and water at pH 6.0. Sufficient sodiuai bisulfite was added to decompose the excess peracid and then the pH was raised to 8.5. The organic phase was separated, washed with brine-and dried. Evaporation in vacuo afforded 65 ag. « of the title product. The NMR. spectrun (CDC1_) showed absorptions at 1.53 (s,2H), 1.72(s,3E), 3.47(a,2H), 4.5(s,lH), 4.6 (a,12) and 4.8 (n,2H)?pui. 50 \nm EXAMPLE 26 ' 4-Nitrobenzyl Penicillanate 1,1-Dioxide A solution of 4-nitrobenzyl penicillanate in chloroform was cooled to about 15°C. and 1 equivalent of 3-chloroperbenzoic acid was added. The reaction mixture was stirred for 20 minutes. Examination of the reaction mixture at this point by nuclear magnetic resonance spectroscopy revealed that it contained 4-nitrobenzyl penicillanate 1-oxide. A further 1 equivalent of 3-chloroperbenzoic acid was added and the reaction mixture was stirred for 4 hours. At this point a further 1 equivalent of 3-chloroperbenzoic acid was added and the reaction mixture was stirred overnight. The solvent was removed by evaporation, and the residue was partitioned!-between ethyl acetate and water at pH 8.5. The ethyl acetate layer was separated, washed with water, dried and evaporated to give the crude product. The crude product was purified by chromatography on silica gel, eluting with at 1:4 mixture of ethyl acetate/chloroform.

The NMR spectrum of the product (CDCI^) showed absorptions at 1.35 (s, 3H), 1.58 (s, 3H), 3.45 (m, 2H), 4.42 (s, lH), 4.58 Im, 2H), 5.30 (s, 2H) and 7.83 (q, 4H) ppm. ' 1 9 96 01.

EXAMPLE 27 Penicillanic Acid 1,1-Dioxide " ' To 0.54 g. of 4-nitrobenzyl penicillanate 1,1-dioxide in 30 ml. of methanol and 10 ol. of ethyl acetate was added 0.5.4 g. of 10Z palladium-on-carbon. The mixture was then shaken under an atmosphere of hydrogen at a pressure of about 50 psig. until hydrogen uptake ceased. The reaction mixture was filtered, and the solvent removed by evaporation. The residue was • * partitioned between.ethyl acetate and water at pH 8.5, and the water layer ' was removed. Fresh ethyl acetate was added and the pH was adjusted to 1.5. The ethyL^acatate layer was re-roved, washed with water and dried, and then it was evaporated in vacuo. This afforded 0.168 g. of the title, compound as a crystalline solid.

EXAMPLE 28 Penicillanic Acid l.l-Dioxida A stirred solution of 512 mg. of 4-nitrobenzyl penicillanate- 1,1-dioxide in a mixture of 5 ml. of acetonitrile and 5 ml. of water was cooled to 0° C. and then a. solution of 434 ng. of- sodium dithionite in 1.4 sal. of 1.0N_ sodium hydroxide was added porticnwise over several minutes. The reaction mixture was stirred for an additional 5 minutes and then it was diluted with ethyl acetate and water at pE 8.5. The ethyl acetate layer was removed and evaporated in vacuo giving 300 mg. of starring material. Fresh. ethyl acetate % was added to the aqueous phase and the pE was adjusted to 1.5. The ethyl acetate was removed, dried and evaporated in vacuo giving 50 mg. of the title compound. nl '.ia . ' ;; j "n s f 996 01 EXAMPLE 29 1 * 1-Mechyl-l'-(acetoxy)ethyl 'Penicillanate 1,1-Dioxide » . 0 To 2-33 g. of penicillanic acid 1,1-dioxide in 5 al..of JI^N-dimethyl-foraaaida was added 1.9 ml. of ethyldiisopropylanine, followed v*oy the dropwise addition of 1.37 g. of 1-methyl-l-(acetoxy)ethyl chloride, at ca. 20" C. The mixture was stirred at ambient temperature overnight and then- the. mixture was diluted with ethyl acetate and with'watetf. ' The layers were separated and the ethyl acetate, layer was washed with water"at pH 9. The ethjrl ac?taTe _ solucion was then dried (Na^SO^), and evaporated in vacuo leaving 1.65 g. of i ' crude product as an oil. The oil solidified on standing.in the refrigerator, and it was then recrystallized from a mixture of chloroform and ether giving material having a melting point of 90-92" C.

The HMR spectrum of Che crude product (CDCl^) showed absorptions at 1.5 (s, 3E), 1.61 (s, 3H), 1.85 (a, 3H), 1.93 (s, 33), 2-0.7 (s, 3H), 3.43 (a, 2H), 4.3 (s, IE) and 4.57 (a, IH) P?m- EXAMPLE' 30 The procedure of Example 29 is repeated, except that the 1-methyl— 1—(acetoxy)echyl chloride is replaced by the appropriate l-cjethyl-l-(alkanoyluA ethyl chloride, to produce the following compounds: 1-methyl-l-(propionyloxy)ethyl penicillanate 1,1-dioxide, 1-methyl-l-(pivaloyloxy)ethyl penicillanate 1,1-dioxide and 1-methy1—1-(hexanoyloxy)ethyl penicillanic acid 1,1-dioxide, respectively. 53 I ) ) 199601 EXAMPLE 31 Penicillanic'Acid 1,1-Dioxide To a stirred solution of 1.78 g. of penicillanic acid in water, at pH 7.5, was added 1.46 nl. of 402 peracstic acid, followed by an additional 2.94 ml. of 402 peracetic acid 30 minutes lacar. The reaction mixture was stirred for 3 days at room temperature and then it was diluted with ethyl acetate and water. Solid sodium bisulfite was added to decompose excess peracid, and then the pH was adjusted to 1.5. The ethyl acetate layer was removed, dried (Na^SO^) and evaporated in vacuo. The residue was a I3i2 mixture of penicillanic acid 1,1-dioxide and penicillanic acid 1-oxide. 54 ) .) o 19 9601 example '32 PivaloyloxysfitKyl Penicillanate 1,1-Dioxide ' A stirred solution of 595 ag. of pivaloyloxyaiethyl penicillanate l-oxide in 5 ml. of ethyl acetate was cooled to ca -15* C., and 5 ng. of manganic acetylacetonate was added. To the dark brown mixture thus obtained was added, during several minutes, 0.89 ml. of 40Z peracetic acid in snail .. amounts over several minutes. After 40 minutes the cooling bath was removed, and the mixture was stirred at ambient temperature for 3 days. The mixture was diluted with ethyl acetate and water at pH 8.5, and the ethyl acetate layer was removed, dried and evaporated in vacuo♦ This afforded 178 mg. of material which was shown by NMR spectroscopy to be a mixture of pivaloyoxy- i methyl penicillanate 1,1-dioxide and pivaloyloxyaethyl penicillinate 1—oxide.

The above material was redissolved in ethyl acetate and reoxidizsd using 0.9 ml. of peracetic acid and 5 mg. of manganic acetylacetonate, as described above, using a reaction time of 16 hours. The reaction mixtyre was worked u? as described above. This afforded 186 mg. of pivaloyloxymethyl penicillanate 1,1-dioxide. — 65 ) 199601, Example 33 I lf-(Ethoxycarbonyl)aininomethyl Penicillanate I 1,1-Dioxide I ' — To 615 mg. (2.41 mmole) of penicillanic acid 1,1-dioxide in 3 ml. of N.,N_dimethylformamide is added 215 mg. (2.50 mmole) of diisopropylethylamine, followed by 350 ng. (2.54 mmole) of N-(ethoxy- carbonyl)aminoraethyl chloride and 20 mg. of sodium iodide. The reaction mixture is stirred for id hours at ambient temperature, and then it is diluted with ethyl acetate and 10% aqueous sodium bicarbonate solution.

The ethyl acetate layer is removed and then it is washed with water. The ethyl acetate solution is dried using anhydrous sodium sulfate, and evaporated in" vacuo giving the title compound.

I . .

Example 34 The procedure of Example 33 is repeated, except that the IT—(ethoxyca.rbonyl)aminomethyl chloride is replaced by an equimolax amount of N-(siethoxycarhonyl)sminomethyl chloride, N—(isobutyloxycarbo— nyl)aminonethyl chloride, N-(hexyloxyCarbonyl)am±noiaethyl chloride, l-(N-[methoxycarbonyl]amino)ethyl chloride and 1-(N-Ihexyloxycarbcnyl] amino)ethyl chloride, respectively. This affords: N^(methoxycarbonyl)aminoraethyl penicillanate, 1,1-dioxide, ^-(isobutyloxycarbonyljatainomethyl penicillanate 1,1-dioxide, N-(hexyloxycarbonyl)aminomethyl penicillanate 1,1-dioxide, l-(N-[nethoxycarbonyl]amino)ethyl penicillanate 1,1-dioxide and l-(N-[hexyloxycarbonyl]amino)ethyl penicillanate 1,1-dioxide, respectively. 5b \ ) 199601 Example 35 Penicillanic Acid 1,1-Dioxide To 100 ml. of water was added 9.4 g. of 6-alpha-bromopeni- cillanic acid 1,1-dioxide, at 22° C., followed by sufficient 4N sodium hydroxide solution to achieve a stable pH of 7.3. To the resulting solution was added 2.25 g. of 5% palladiuin-on-carbon followed by 6.9 g. of dipotassium phosphate trihydrate. This mixture was then shaken under 2 an atmosphere'of hydrogen at a pressure varying from 3.5 to 1.8 kg/cm . i ■ - When hydrogen uptake ceased, the solids were removed by filtration, and the aqueous solution was covered with 100 ml. of ethyl acetate. The pH was slowly lowered from 5.0 to 1.5 with 6N-hydrochloric acid. The layers were separated, and the aqueous phase was extracted with further ethyl acetate. The combined ethyl acetate layers were washed with brine, dried using anhydrous magnesium sulfate and evaporated in vacuo ■ The ~~ residue was triturated under ether and then the solid material was collected by filtration. This afforded 4.5 g. (65% yield) of the title compound.

Analysis: -Calcd. for CgH^NO^S: C, 41-20; H, 4.75; N, 6.00; S, 13.75%. Found: C, 41.16; H, 4.81; N, 6.11; S, 13.51%. 57 199601 Example 36 Penicillanic Acid 1,1-Dioxide The ethyl acetate solution of 6,6-dibromopenicillanic acid 1,1-dioxide from Preparation TC was combined with 705 nil. of saturated sodium bicarbonate solution and 8.88 g. of 5% palladium-on-carbon catalyst.

The mixture was shaken under an atnosphere of hydrogen, at a pressure 2 of about 5 kg/ca for about 1 hour. The catalyst was removed by filtration and the pH of the aqueous phase of the-filtrate was adjusted to 1.2 with 6lT-hydrochloric acid. The aqueous phase was> saturated with sodium chloride. The layers were separated and the aqueous phase was extracted with further ethyl acetate (3 X 200 ml.). The combined ethyl acetate solutions were dried (MgSO^) and evaporated in vacuo to afford 33.5 g. (58Z yield from 6-aminopenicillanic acid) of penicillanic acid 1,1-dioxide. This product was dissolved in 600 ml. of ethyl acetate, Che solution was decolorized using activated carbon and' the solvent was removed by I . evaporation in vacuo. The product was washed with hexane. This afforded 31.0 g. of pure product. -5 Example' 37 Effect of Penicillanic Acid 1,1-Dioxide on the Antibacterial Activity of 7-(D-2-I4-ethylpiperazine-2,3-dione-l-carboxamido]- 2-[4-hydroxyphenyl]acetaraido)-3-([1-mcthy1-5-tetrazolyl]thiomethyl)*• 3-doBnce t.oxymethylccphnlospornnlc Acid The minimum inhibitory concentrations (HIC's) of penicillanic acid 1,1-dioxide (PA 1,1-dioxide) alone and 7-(D-2-I4-ethylpipcrazine-2,3-dione-l~carboxnnjido]-2-[4-hydroxy-phcny1]neetamido)-3-([l-methyl-5-tetrazolyl]thiomethyl)-3-desncetoxymethylcephalosporanic acid (T-1551) alone, against 30 strains of resistant Escherichia coli, were measured. These MIC's were then compared with the MIC values obtained with a combination of the two compounds. The results were as follows: i Table VI Microorganism No. of Strains Mode MIC of . T-1551 alone.

Mode MIC of PA 1,1-Dioxide alone Mode MIC's of T-1551 & PA 1,1-Dioxide in Combination Escherichia coll 100. 50 T-1551 PA 1,1-Dioxide 1.56 1.56 f 99601 The MIC's ware measured using the method recommended by the | International Collaborative Study on Antibiotic Sensitivity Testing j (Ericcson and Sherris, Acta. Pathologica et Microbiologica Scandivay, Supp. 217, Section B, 64 - 68 [1971]), which employed brain heart infusion agar and the inocular replicating device. Overnight growth tubes were diluted 10-fold for use as the standard inoculum. Twelve two-fold dilutions of the test, compound were employed with initial concentration of the test drug being 200 mcg/ml. Single colonies were disregarded when reading plates after 18 hours at 37° C, The MIC of the test compound is accepted as the lowest concentration of.compound capable of producing complete inhibition of growth as judged by the naked eye. The MIC's of combinations were measured using the method described by Barry and Sabath in "Manual of Clinical Microbiology", edited by, Lenette, Spaulding and Truant, 2nd edition, 1974, American Society for Microbiology. to r9960t PREPARATION A 6,6-Dibromopenicillanic Acid la-Oxide The title compound is prepared by oxidation of 6,6-dibromopenicillanic acid with 1 equivalent of 3-chloroperbenzoic acid in tetrahydrofuran at 0-25°c. for ca. 1 hour, according to the procedure of Harrison et al., Journal of the Chemical Society (London) Perkin I, 177 2 (1976). preparation b Benzyl 6,6-Dibromopenicillanate To a solution of 54 g. (0.165 mole) of 6,6-dibromopenicillanic acid in 3 50 ml. of N,N^-dimethylacetamide was added 22.9 ml. (0.165 mole) of triethylamine and the solution was stirred for 40 minutes.

Benzyl bromide (19,6 ml., 0.165 mole) was added and the resulting mixture was stirred at room temperature for 48 hours. The precipitated triethylamine hydrobromide was filtered off, and the filtrate was added to 1,500 ml. of ice-water, adjusted to pH2. The mixture was extracted with ether, and the extracts were washed successively with saturated sodium bicarbonate, water and brine. The dried (MgSO^) ether solution was evaporated in_ vacuo to give an off-white solid, which was recrystallized from isopropanol. This afforded 70.0 g. (95% yield) of the title compound m.p. 75-76°C. The IR spectrum (KBr disc) -showed absorptions at 1795 and 1740 cm 1. The NMR spectrum (CDCI^) showed absorptions at 1.53 (s, 3H) , 1.53 (s, 3H) , 1.58 (s, 3H) , 4.50 (s, 1H), 5.13 (s, 2H), 5.72 (s, lH) and 7.37 (s, 5H).ppm. 61 % .^.,15 APR 1982. ^ 4# *-! VV C 199601 PREPARATION C Benzyl 6,6-Dibromopenicillanate la-oxide To a stirred solution of 13.4 g. (0.03 mole) of benzyl 6,6-dibromo-penicillanate in 200 ml. of dichloromethane was added a solution of 6.12 g. (0.03 mole) of 3-chloroperbenzoic acid in 100 ml. of dichloromethane, at ca. 0°C. Stirring was continued for 1.5 hours at ca, 0°C. and then the reaction mixture was filtered. The filtrate was washed successively with 5% sodium bicarbonate and water, and then it was dried (Na2SO^). Removal of the solvent by evaporation in vacuo gave 12.5 g. of the title product as an oil. The oil was induced to solidfy by trituration under ether. Filtration then afforded 10.5 g. of benzyl 6,6-dibromopenicillanate la-oxide as a solid. The IR spectrum (CHCI^) showed absorptions at 1800 and 1750 cm \ The NMR spectrum of the product (CDCI^) showed absorptions at 1.3 (s, 3H), 1.5 (s, 3H), 4.5 (s, lH), 5.18 (s, 2H), 5.2 (s, 1 H) and 7.3 (s, 5H) ppm.

PREPARATION D 4- Nitrobenzyl Penicillanate Reaction of the triethylamine salt of penicillanic acid with 4-bitrobenzyl bromide; according to the procedure of Preparation B, affords 4-nitrobenzyl penicillanate. 159601 PREPARATION E 2,2,2-Trichloroethyl Penicillanate To 403 mg. of penicillanic acid in 10 ml. of dichloromethane was added 25 mg. of diisoporopylcarbodiimide followed by 0.19 ml. of 2,2,2-trichloroethanol. The mixture was stirred overnight and then the solvent was removed by evaporation in vacuo■ The crude product was purified by column chromatography using silica gel as the absorbent and chloroform as the eluant. mmt PREPARATION F-3-Phthalidyl Penicillanate To a solution of 506 mg. of penicillanic acid in 2 ml. of N,N-dimethylformamide was added 0,476 ml. of diisopropylethylamine followed by 536 mg. of 2-phthalidyl bromide. The mixture was stirred overnight and then it was diluted with ethyl acetate and water. The pH was adjusted to 3.0 and the layers were separated. The organic layer was washed with water, and then with water at pH 8.0, and then it was dried using anhydrous sodium sulfate. The dried ethyl acetate solution was evaporated ir^ vacuo giving 713 mg. of the title ester as an oil.. The NMR spectrum (CDCI^) showed absorptions at 1.62 (m, 6H), 3.3 (m, 2H), 4,52 (s, lH), 5.23 (Mc lH) and 7.63 (m, 5H).

IS APR 1982 t .> r> r a PREPARATION G Pivaloyloxymethyl penicillanate To 3.588 g. of 6,6-dibromopenicillanic acid in 10 ml. of N^N-dimethylformamide was added 1.8 ml. of diisopropylethylamine, followed by 1.40 ml. of chloromethyl pivalate. The mixture was stirred overnight and then it was diluted with ethyl acetate and water. The organic layer was removed and washed successively with water at pH 3.0 and water at pH 8.0. The ethyl acetate solution was dried (Na^SO^) and then evaporated in vacuo to give pivaloyloxymethyl 6,6-dibromopenicill-anate as an amber oil (3.1 g.) which slowly crystallized.

The above ester was dissolved in 100 ml. of methanol, and then 3.1 g. of 10% palladium-on-carbon and 1.31 g. of potassium bicarbonate in 20 ml. of water were added. The mixture was shaken under hydrogen at atmospheric pressure until hydrogen uptake ceased. The reaction mixture was filtered and the methanol was removed by evaporation in vacuo. The residue was partitioned between water and ethyl acetate at pH 8, and then the organic layer was removed. The latter was dried (Na2S04) and evaporated in vacuo to give 1.25 g. of the title compound. The NMR spectrum (CDCI^) showed absorptions at 1.23 (s, 9H), 1.5 (s, 3H), 1.67 (s, 3H), 3.28 (m, 2H), 4.45 (s, lH), 5.25 (m, 1H) and 5.78 (m, 2H) ppm. ) \ 99601 PREPARATION H 4-Nitro'oenzyl Penicillanate To a stirred solution of 2.14 g. of penicillanic acid ar.d 2.01 ml. of ethyldiisopropylaaine in 10 tal. of ^,N_-dinethylfor=aaiide was added dropwise 2.36 g. of 4-nitrobenzyl bromide, at ca. 20®C. The mixture was stirred ac ambient ^temperature overnight, and then it was diluted with ethyl acetate and water. The layers were separated and the ethyl acetate layer was washed with water at pH 2.5, followed by water at pH 8.5. The ethyl acetate solucion was then dried (Na2S0^) arid evaporated in vacuo leaving 3.36 g. of the title compound.

The spectrun of the product (in CDClO showed absorptions at 1.45 (s, 3H), 1.63 (s, 3E), 3.32 (m, 2H), 4.50 (s, 12), 5.23 (m, IE), 5.25 (s, 2H) and 7.85 (q, 4H) ppm. i Uo 1 99601 PREPARATION I i " | 6-alpha-Bromopenicillanic Acid 1,1-dioxide To a stirred mixture of 560 ml. of water, 300 ml', of di— chloromethane and 56.0 g. of 6-alpha-bromopenicillanic acid was added AN sodium hydroxide solution until a stable pH of 7,2 was achieved.

This required 55 ml. of sodium hydroxide. The mixture was stirred at pH 7.2 for 10 minutes and then it was filtered. The layers were separated and the organic phase was discarded. The aqueous phase was then poured rapidly, with stirring, into an oxidizing mixture which had been prepared as follows. ■' In a 3 liter flask was mixed 63.2 g. of potassium permanganate, 1,000 ml. of water and 48.0 g. of acetic acid. This mixture was stirred for 15 minutes at 20° C. and then it was cooled to 0° C.

After the 6-alpha-bromopenicillanic acid solution had been added to the oxidizing mixture, a cooling bath at -15° C. was maintained around the reaction mixture. The internal temperature rose to 15° C. and then fell to 5' C. over a 20 minute period. At this point, 30.0 g. of sodium metabisulfite was added with stirring over a 10 minute period at about 10° C. After a further 15 minutes, the mixture was filtered, and the pH of the filtrate was lowered to 1.2 by the addition of 170 ml. of 6N hydrochloric acid. The aqueous phase was extracted with chloroform, and then with ethyl acetate. Both the chloroform extracts and the ethyl acetate extracts were dried using anhydrous magnesium sulfate and then they were evaporated in vacuo. The chloroform solution afforded 10.0 g. (16% yield) of the title compound. The ethyl acetate solution afforded 57 g. of an oil, which was triturated under hexane. A white solid appeared. .It was filtered off, giving 41.5 g. (66% yield) of the title compound, mp 134° C. (dec.). a ) } 1 996 011 PREPARATION J -» 11 6,6-Dibromopenicillanic Acid To 500 ml. of dichloronethane cooled to 5° C, was added 119.9 g. of bromine, 200 ml. of 2,5Nsulfuric acid and 34,5 g. of sodium nitrite. To this stirred mixture was then added 54.0 g. of 6-aiainopenicillanic acid, portionwise over 30 minutes, with the ' . / temperature maintained from 4 to 10° c'. Stirring was continued for 30 minutes at 5° C,, and then 410 ml. of a 1.0M slution of sodium bisulfite was added dropwise at 5 to 10° C. during 20 minutes. The layers were separated and the aqueous layer was extracted twice with 150 ml. of dichloromethane. The original dichloromethane layer was combined with the two extracts to give a solution of 6,6-dibronopeni- I cillanic acid. This solution was used directly in Preparation K. .£# 1 PREPARATION K 6,6-Dibromopenicillanic Acid 1,1-Dioxide To the dichloromethane solucion of 6,6-dibromopenicillanic acid from Preparation. J was added 300 ml. of water, followed by the dropwise addition over a period of 30 minutes of 105 ml, of 3N sodium hydroxide. The pH stabilized at 7.0. The aqueous layer was removed and the organic layer was extracted with water (2 X 100 ml.). To the combined aqueous solutions was added, at -5° C,, a premised solution prepared from 59.25 g. of ;potassium permanganate, 18 ml. of coucen- I trated phosphoric acid and 600 121. of water, until the pink color of the permanganate persisted. The addition took 50 minutes and 500 ml. of oxidant were required. At this point, 500 i"!- ethyl acetate was added and then the pH was lowered to 1.23 by the addition of 105 ml. of 6N hydrochloric acid. Then 250 ml. of H sodium bisulfite was added during 10-15 minutes at ca. 10° C. During the addition of the sodium bisulfite solution, the pH was maintained at 1.25-1.35 using 6JJ hydrochloric acid. The aqueous phase was saturated with sodium chloride and the two phases were separated. The aqueous solution was extracted with additional ethyl acetate (2 X 150 ml.) and the combined ethyl acetate solutions were washed with brine and dried (MgSO^).

This afforded an ethyl acetate solution of 6,6—dibromopenicillanic acid 1,1-dioxide.

The 6,6-dibromopenicillanic acid 1,1-dioxide can be isolated by removal of the solvent in vacuo. A sample so isolated from an analgous preparation had a melting point of 201° C (dec.). The NMR spectrum (CDCl-/ DMS0-d,) showed absorptions at 9.35 (s,lH), 5.30 j o f . 1 (S,1H), A.42 (s,lH), 1.63 (s,3H) and 1.50 (s,3H)ppra. The 1R spectrum (KBr disc) showed absorptions at 38A6-2500, 1818, 175A, 13A2 and 1250-1110 ci*-1.;* - TO - I9960t

Claims (6)

WHAT WE CLAIM IS: p.-s-Ms "..A

1. A method of increasing the antibacterial effectiveness of 7-(D- • 2-{4-ethylpiperazin-2,3-dione-l-carboxamidoJ-2-[4-hydroxyphenylJacetamido)- | ; 3-([l-methyl-5-tetrazolyl]thiomethyl)-3-dcsacetoxymethylcephalosporanic ■ • Aon | acid, or a pharmaceutically-acceptable salt thereof, in a mammalian subject, which comprises co-administering therewith to said subject an effective amount of a compound of the formula H \? - /S-^ "N ' CH_ CH_ COOR . J. P. & S. or a pharmaceutically-acceptable base salt thereof, wherein R is selected from the group consisting of hydrogen and ester-forming residues readily hydrolyzable jin vivo. :

2. The method according to Claim 1, wherein is hydrogen.

3. A method according to Claim 1 , wherein R^ is selected from the group consisting of alkanoyloxymethyl having from 3 to 8 carbon atoms, 1-(alkanoyloxy)ethyl having from ^ to 9 carbon atoms, 1-methyl-l-(alkanoyloxy1)ethyl t having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, l-(alkoxycarbonyloxy)ethyl having from A to 7 carbon atoms, 1-methyl-l-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1- ' (N-[alkoxycarbony1]amino)ethyl having from A to 10 carbon atoms, 3- i phthalidyl, A-crotonolactonyl and gamma-butyrolacton-A-yl.

1^. The method according to Claim 3, wherein R^ is pivaloyloxy-incthyl. ? 1

5. The metliod according to Claim wherein R is l-(ethoxycarbonyl-oxy)ethyl.

6. A method as claimed in any one of the preceding claims when performed substantially as hereinbefore described. DATED TH.S 2^ DAY OF Oft A- J- PARK & SON 28 JAW _ ^ (W- HggENEP AGEMTS FOR THB APPLICANTS