CA1119164A - PENICILLANIC ACID 1,1-DIOXIDES AS .beta.-LACTAMASE INHIBITORS - Google Patents

Abstract

Abstract of the Disclosure Penicillanic acid l,l-dioxide, and esters thereof readily hydrolyzable in vivo, are useful as antibacterial agents, and also for enhancing the effectiveness of several .beta.-lactam antibiotics against many .beta.-lactamase producing bacteria. Derivatives of penicillanic acid l,l-dioxide having the carboxy group protected by a conventional penicillin carboxy protecting group are useful intermediates to penicillanic acid l,l-dioxide.
Penicillanic acid l-oxides and certain esters thereof are useful chemical intermediates to penicillanic acid l,l-dioxide and its esters.

Description

~1191~

One of the most well-known and widely used class of antibacterial agents are the so-called ~-lactam antibiotics. These compounds are characterized in that they have a nucleus consisting of a 2-azetidinone (~-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 cephalos-porins. Typical examples of penicillins which are commonly used in clinical practice are benzylpenicillin ~penicillin G), phenoxymethylpenicillin (penicillin V), ampicillin and carbenicillin; typical examples of common cephalosporins are cephalothin, cephalexin and cefazolin.
However, despite the wide use and wide acceptance of theJB-lactam antibiotics as valuable chemotherapeutic agents, they suffer from the major drawback that certain members are not active against certain microorganisms.
It is thought that in many instances this resistance of a particular micro-',,~ ~

'` ~ 111~16g organism to a given B-lactam antibiotic results because the microorganism produces a ~-lactamase. The latter substances are enzymes which cleave the ~-lactam ring of penicillins and cephalosporins to give products whiCh are devoid o~ antibacterial activity. However~ Certain substances have the ability¦
to inhibit ~-lactamases, and when a ~-lactamase inhibitor is used in combina-tion with a penicillin or cephalosporin it can increase or enhance the anti-bacterial 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 B-lacta-mase inhibiting 8ubstance and a ~-lactam antibiotic is significantly greater than the sum of the antibacterial activities of the individual components.
Thus, according to the invention, there are provided certain new chemical compounds Which are new members of the class of antibiotics known l as the penicilliDs, and which are useful as antibacterial!agents. Nore specifically, these new penicillin compo~mds are penicillanic acid l,l-dioxide, ~and esters thereof readily hydrolyzable in vivo.
¦ Additionally~ penicillanic acid l,l-dioxide and its esters readily ¦hydrolyzable in vivo are potent inhibitors of microbial ~-lactamases.
¦ Accordingly~ there is also provided a method for enhancing the effectiveness lof ~-lactam antibiotics, using penicillanic acid l,l-dioxide and certain ,readily hydrolyzable esters thereof.
Still further~ there are provided derivatives of penicillanic acid dioxide having a carboxy protecting group~ said compounds being useful ¦a8 chemical intermediate8 for penicillanic acid l,l-dioxide.
'I - . , 111916~

Yet further, there are provided penicillanic acid l-oxides, and ¦certain esters thereof, as chemical intermediates to penicillanic acid 1,1-dioxide.
l,l-Dioxides of benzylpenicillin, phenoxymethylpenicillin and certain esters thereof have been disclosed in ~nited States Patents 3,197,466 and 3,536,698, and in an article by Guddal et al., in Tetrahedron Letters, No. 9, 381 ~1962). Harrison et al., in the Journal of the Chemical SocietY (Londonj, Perkin I, 1772 (1976), have disclosed a variety of penicillin l,l-dioxides and l-oxides, including methyl phthalimidopenicillanate l,l-dioxide, methyl 6,6-dibromopenicillanate l,l-dioxide, methyl penicillanate l~-oxide, methyl penicillanate lB-oxide, 6,6-dibromopenicillanic acid l-oxide and 6,6-dibromo-penicillanic acid lB-oxide.

Su~marY of the Invention According to the invention there are provided novel compounds of the lS formula . . . '.' -~ 1119164 ~ I

¦ and the pharmaceutically-acceptable base salts thereof, wherein Rl is selected ¦ from the group consisting of hydrogen, ester-forming residues readily ¦ hydrolyzable in vivo, and conventional penicillin carboxy protecting groups.
¦ The term "ester-forming residues readily hydrolyzable in v '~is here ¦ intended to refer to non-toxic ester residues which are rapidly cleaved in ¦ mammalian blood or tissue, to release the corresponding free 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 Rl are I alkanoyloxymethyl having from 3 to 8 carbon atoms, l-(alkanoyloxy)ethyl having 10 ¦ from 4 to 9 carbon atoms, l-methyl-l-(alkanoyloxy)ethyl having from 5 to 10 carbon a.oms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, ¦ l-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, l-methyl-l-(alkoxy-¦ carbonyloxy)ethyl having from 5 to 8 carbon atoms, 3-phthalidyl, 4-crotono-I lactonyi and y-butyrolacton-4-yl.
¦ The compounds of the formula I, wherein R is hydrogen or an ester-¦ forming residue readily hydrolyzable in vivo, are useful as antibacterial agent 3 and for enhancing the antibacterial activity of ~-lactam antibiotics. Said ¦ compounds of the formula I, wherein Rl is a penicillin carboxy protecting I group, are useful as chemical intermediates to the compound of the formula I, ¦ wherein R is hydrogen or an ester-forming residue readily hydrolyzable in vivo ¦ Typical carboxy protecting groups are benzyl and substituted benzyl, e.g.
¦ 4-nitrobenzyl.

ll ~119~.6 -. I
¦ A1BO accorBin~ to the invention thare are provided novel co~pounds .
of the fO--UL COOR1 . and oC~ 1 ~~~ (llr~

1. , I

_ ¦1 and the 6alts thereof, wherein Rl i8 a6 defined previsua1y. Said comFounds ~' ,il of the formulas II and III are intermediates to said compounds of the formula ~1 I. .
Il . . ' I Dctailed Description of the Invention ~ This invention relates to the novel compounds of formulas I, II and ¦~ III, and throu~hout this specification they are.referred to as derivatives of .

~¦ penicillanic acid, which is representad ~y the 6tructural formula F~ CR3 _--(IV~

COOH

9~
., .1 ¦ In formula IV, broken line attachment of a su~sti~uent to tbe bicyclic nucleus ¦
indicates that the substituent i9 below the plane ~f the bicyclic nucleus.
I Such a substituent i8 6aid to be in the ~-confi~uration. Conversely, solid ¦ line attachment of a 6ubstituent to the bicyclic nucleus indicates ~ha~ the 5. ¦¦ substituent is ateached above the plane of ~he nucleu6. This laeter config-¦l uration is referred to as the ~-configuration. -I Also in this 6pecification reference is made to certain derivatives of cephalosporanic acid, which ~las the ~ormula ' S, I

COOH
o l! In formula V, the hydrogen at C-6 is below the plane of the bicyclic nucleus.
¦ ~he derived terms desacetoxycephalosporanic acid and 3-desacetoxymethylcephalo-¦
¦ sporanic acid are u6ed to refer to the 6tructures VI and YII, respectively.

' H
FS ~ . ',~ S~

¦l O ~ CH3 O ~ ~
COOH COOH

¦I VI VII

Il 4-Crotonolactonyl and y-butyrolacton-4-yl refer to 15¦ 6tructure6 VIII~,and IX, respectively. The wavy line6 are ineended to denote each of the two epimers and mixtures thereof.

VIII

~ 6 1, . 1.

When Rl is an ester-forming residue readily hydrolyzable in vivo in a compGund of formula I, it is a grouping which is notionally derived from an alcohol of the formula Rl-OH, such that the moiety COOR in such a compound of formula I represents an ester grouping. Moreover, R is of such a nature that the grouping COORl is readily cleaved in in vivo to liberate a free car-boxy group (COO~). That is to say, R is a group of the type that when a compound of formula I, wherein R is an ester-forming residue readily hydrolyze d in VivoJ is exposed to mammalian blood or tissue, the compound of formula I, wherein R is hydrogen, is readily produced. The groups Rl are well-known in the penicillin art. In most instances they improve the absorption characteristics of the penicillin compound. Additionally, Rl should be of such a nature that it imparts pharmaceutically-acceptable properties to a compound of formula I, and it liberates pharmaceutically-acceptable fragments when cleaved in vivo. !
As indicated above, the groups R are well-known and are readily identified by those skilled in the penicillin art. See, for example, West German Offenlegungsschrift No. 2,517,316. Typical groups for R are 3-phthalidyl, 4-crotonolactonyl, y-butyrolacton-4-yl and groups of the formula - I-o-C-R5 and -C-O-C-O-R
l4 R4 X XI
.~ .

~ - 6a __ . ~

111~16~

wherein R3 and R4 are each selected from the group consisting of hydrogen and alkyl ha~ing from 1 to 2 carbon atoms, and R5 is alkyl having from 1 to 6 car-~bon atoms. However, preferred groups for Rl are alkanoyloxymethyl having from 3 to 8 carbon atoms, l-~alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-¦ methyl-l-(alkanoyloxy)ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxy-¦ methyl having from 3 to 6 carbon atoms, l-(alkoxycarbonyloxy)ethyl having from 14 to 7 carbon atoms, l-methyl-l-alkoxycarbonyloxy)ethyl having from 5 to 8 ~ '.; ' ~ ' ., . l I -6~-: :. , . :

~119164 The compounds of formula I, wherein R 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 ear*h 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 Rl is as defined previously, is oxidized to the corresponding compound of t~e formula I using a metal permanganate, the reaction is usually carried out by treating the compound of the formula II or III with from about 0.5 to about 5 molar equivalents of the permanganate, and preferably about`l molar equivalent of the permanganate, in an appropriate solvent system, An appropriate soIvent 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 B-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 ~==z:_.

1119~69~

separated from manganese dioxide by extracting lt 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 usual procedure of solvent extraction.
When a compound 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 temperature of from about -20 to about 50 C., and preferably at about 25C. 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-~nown in the art.
When oxidizing a compound of the formula II or III to a compound of the formula I using an organic peroxy acid, it is sometimes advantageous ¦to add a catalyst such as a manganese salt, e.g. msnganic acetylacetonate.
The compound of the formula I, wherein Rl is hydrogen, can also be obtained by~removal of the protecting group Rl from a compound of the formula I, wherein R is a penicillin carboxy protecting group. In this context, Rl can be any carboxy protecting group conventionally used in the penlcillin art to protect carboxy groups at the 3-position. The identity of the carboxy protecting group is not critical. The only requirements for the carboxy protecting group Rl are that: (l) it must be stable during -t '11~91Ç;4 oxidation of the compound of formula II or III; and (il) it must be removable from the compound of formula I, using conditions under which the ~-lactam re-mains substantially intact. Typical examples which can be used are the tetra-hydropyranyl group, the benzyl group, substituted benzyl groups (e.g. 4-nitro-benzyl), the benzylhydryl group, the 2,2,2-trichloroethyl group, the t-butyl group and the phenacyl group. See further: United States Patents 3,632,850 and 3,197,466; British Patent No. 1,041,985, Woodward et al., Journal of the American Chemical Society, 88, 852 (1966); Chauvette, Journal of Organic ChemistrY, 3~, 1259 (1971); Sheehan et al., Journal of Organic ChemistrY, 29, 2006 (1964); and "Cephalosporin and Penicillins, Chemistry and Biology", edited by H. E. Flynn, Academic Press, Inc., 1972. The penicillin carboxy protecting group is removed in conventional manner, having due regard for the lability of the ~-lactam ring system.
In like manner, compounds of the formula I, wherein R is as previously defined, can be prepared by oxidation of a compaund of the formula ~ 3 O r~ 1 ~
COOR

wherein R i8 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, except that twice as much oxidant is usually used.

.___ _ .~
- ' ' ~119164 Compounds of the formula I, wherein Rl is an ester-forming residue readily hydrolyzable in vivo, can be prepared directly from the com-pound of formula I, wherein X i6 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, y-butyrolacton-4-yl and groups of the formula X and Xl, wherein R3, R4 and R5 are as defined previously, they can be prepared by alkylation of the compound of formula I, wherein is hydrogen, with a 3-phthalidyl halide, a 4-crotonolactonyl halide, a y-butyrolacton-4-yl halide or a compound of the formula R3 o R3 0 Q-l-o-CeR5 and Q-C-O-C-O-R

XII XIII

wherein Q is halo, and R3, R4 and R5 are as previously defined. The terms "halide" and "halo" are intended to mean derivatives of chlorine, bromine and lS iodine. The reaction is conveniently carried out by dissolving a salt of the compound of formula I, wherein R is hydrogen, in a suitable, p~lar, organic solvent, such 8S N,N-dimethylformamide, and then adding about one molar equivalent of the halite. When the reaction has proceeded essentially to ¦¦ cot;~ tlon, the od=ct ls lsolated by ~tandard technlquell. It Is ofteD

. . ~

~ 9a -I , , 111916~
sufficient simply to dilute the reaction medium with an excess of water, and then extract the product into a water-immiscible organic solvent and then recover sa~e by solvent evaporation. Salts of the starting material which are commonly used are alkali metal salts, such as sodium and potassium salt, and tertiary amine saltæ, æuch as triethylamine, N-ethylpiperidine, N,N-- dimethylaniline and N-methylmorpholine salts. The reaction is run at a temperature in the range from about 0 to 100 C., and usually at about 25C.
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 of~
the reagentæ. 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 chloro compound, to add up to one molar equivalent of an alkali metal iodide. This has the effect of spçeding up the reaction. With full regard for the for,egoing factors, .
reaction times of from about l to about 24 houræ are commonly used.

_ _. ~

., Il I
9~
'I , .
., ,,11 , . ., !i 1Penicillanic acid l~-oxide, the compound of the formula II, wherein R is hydro~en, can be prepared by tebromination of 6,6-dibromopenicillanic l acit lu-oxide. The debromination can be carried o~t using a conventional l hydrogenolysis technique. ~hus, a solution of 6,6-dibro penicillanic acid 1~-oxide is stirred or shaken unter an atmosphere of hydrogen, or hydrogen mixed . with an inert diluent such as nitrogen or ar~on, in the presence of a catalytic amount of palladium-on-calcium carbonate catalyst. Convenient solvents for I this debromination are lower-alkanols, such as methanol; ethers, such as tetra-¦ hydrofuran and dioxan; low molecular weight esters, such as ethyl acetate andbutyl acetate; water; and mixtures of these solvents. However, it is usual to choose conditions under which the dibromo compound is soiuble. The hydro-¦¦ genolysis is usually carried out at room temperature and at a pressure from I¦ about atmospheric pressure to about 50 p.s.l. The catalyst is usually present I in an amount from about 10 percent by weight based on the tibromo compound,up to an amount equal in weight to the dibromo compound, although larger . l, amounts can be used. The reaction commonly talces about one hour, after which the compound of the formula II, wherein-R is hydrogen, is recovered simply I by filtration followed by removal of the solvent in vacuo.
25 I 6,6-Dibromopenicillanic acid l~-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 Societ~ (London) Perkin I, 1772 (1976)j.
6,6-Dibromopenicillanic acid is prepared by the method of Clayton, Journal of the ~ S5.~ (London), (C) 2123 (1969).

,,.-. 1. .

1!

.... .
, . . . . . ~. ~.. , . ~ . ~ . . .-, 1119164 `~
Penicillanic acid l~-oxide, the compound of the formula III, wherein Rl i6 hydrogen, can be prepared by controlled oxidation of peniclllanic acid.
~hus, it can be prepared by treating penicillanic acid with one molar equiva-lent 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 dichloromethane; 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 Rl is hydrogen, by esterification, using standard procedures. In the case wherein Rl is selected from the group consisting of 3-phthalidyl, 4-crotonolactonyl, y-butyrolacton-4-yl and groups of the formula X, and XI, wherein R3, R4 and R5 are as defined previously, they can be prepared by alkylation of the appropriate compound of the ormula II or III, wherein Rl is hydrogen, ~ith-a 3-phthalidyl halide, 4-crotonolactonyl halide, a y-butyrolacton-4-yl halide, or a compound of the formula XII or XIII. The reaction is carried out in exactly the same manner as described previously for esterification of penicillanic acid l,l-dioxide Wlth a 3-phthalidyl halide, a 4-crotonolactonyl halide, a y-butyrolacton-4-yl halide, or a compound of the formula XII or XIII.

_ ~ ternatively, the compou=do of the for=ula Il, whereiD R ~s a:
ester-forming residue readily hydrolyzable in vivo, can be prepared by oxida-tion of the appropriate ester of 6,6-dibromopenicillanic acid, followed by debromination. The esters of 6,6-dibromopenicillanic acid are prepared from 6,6-dibromopenicillanic acid by standard methods. The oxidation is carried out, for example, by oxidation with one molar equivalent of 3-chloroperbenzoic acid, as described previously for the oxidation of 6,6-dibromopenicillanic acid to 6,6-dibromopenicillanic acid l~-oxide; and the debromination is carried out as described previously for the debromination of 6,6-dibromopenicillanic acid l-oxide.
In like manner, the compounds of the formula III, wherein Rl is an ester-forming 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 example, by oxidation with one molar equivalent of 3-chloroperbenzoic acid, as described previously for the oxidation of penicillanic acid to penicillanic acid l~-oxide.
The compounds of the formula II, wherein R is a carboxy protecting group can be obtained in one of two ways. They can be obtained simply by taking penicilIanic acid l~-oxide and at"aching a carboxy protecting group thereto. Alternatively, they can be obtained by: (a) attaching a carboxy protecting group to 6,6-dibromopenicillanic acid; (b) oxidizino the protected ~ l ~
6,6-dibromopenicillanic acid to a protected 6,6-dibromopenicillanic acid 1~-oxide u6ing 1 molar equivalent of 3-chloroperbenzoic acid; and (c) debromina-ting the protected 6,6-dibromopenicillanic acid l~-oxide by hydrogenolysis.
¦ The compounds of the formula III, wherein Rl is a carboxy protecting group can be obtained simply by attaching a protecting group to penicillanic acid l~-oxide. Alternatively, they can be obtained by: (a) attaching a car-boxy protecting group to penicillanic acid; and (b)oxidizing the protected penicillanic acid using 1 molar equivalent of 3-chloroperbenzoic acid as pre-viou61y described. ' The compound~ of formulas I, II and III, wherein R is hydrogen, are acidic and will form salts with ba6ic agents. Such salts are considered to be within the scope of this invention. These 6alts 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 precipitation with a ¦ non-601vent'followed by filtration, by evaporation of the solvent, or in the ¦¦ case of aqueous solutions, by lyophilization, as appropriate. Basic agent~
¦! which are 6uitably employed in salt formation belong to both the organic and jl inorganic types, and they include ammonia, organic amines, alkali metal hy-20 ¦ droxide~, car~onates, bicarbonates, hyd~ides and alkoxides, as well as alkaline ¦ earth metal hydroxides, carbonates, hydrides and alkoxides. Representative ¦ example6 of 6uch bases are primary amines, such as n-propylamine, n-butylamine, aniline, cyclohexylamine, benzylamine.and octylamine; secondary amine~, 6~ch'as diethylamine, morpholine, pyrrolidine and 25 ¦ piperidinei tertiary amines, such a6 triethylamine, N-¦ ethylpiperidine, N-methylmorpholine and 1,S-diazabicyclo~4.3.0~non-5-ene; hy-¦ droxides, ~uch as sodium hydroxide, potassium hydroxide, ammonium hydroxide snd barium hydroxide; alkoxides, such' as ~odium ethoxide and pota6sium ethoxide ;

hydrides, such as calcium hydride and sodium hydride; carboDate~, such as po-tassium carbonate and sodium carbonate; bicarbonates, such as sodium bicarbona and potassium bicarbonate; and al~ali 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.
As indicated hereinbefore, the compounds of formula I, wherein R is hydrogen or an ester-forming residue readily hydrolyzable ln vivo, are anti-bacterial agents of medium 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 (MIC's) in mcg/ml against a variety of microorganisms. Tlle procedure which is followed is the one recommended by the International Collaborative Study on Antibiotic Sensitivity Testing (Ericcson and Sherris, Acta. Patholo~ica et Microb ologia Scandinav, Supp.
217, Sections A and B: 1-90 [1~70]), and employs brain heart infusion (BHI) agar and the inocula replicating device. Overnight growth tubes are diluted 100 fold for use as the standard inoculum t20,000-10,000 cells in approxi~ately 0.002 ml. are placed on the agar surface; 20 ml. of BHI agar/dish). Twelve 2 fold dilutions of the test compound are employed, with initial concentration of the test drug being 200 mcg./ml. Single colonie6 are disregarded when reading plates &fter 18 hrs. at 37 C. The susceptibility (MIC) of the test organism is accepted as the lowest concen-tration o$ compound capable of producing complete inhibition of growth as judged by the naked eye. MIC values for penicillanic acid l,l-dioxide agalnst several microorganisms are shown in Table I.

'.;`

9~
.
TABLE I

In Vitro Antibacterial Activity of Penicillanic Acid l.l-Dioxide . . '' ' '' _ Microorganism MIC (mc~./ml.) Staphylococcus aureus 100 Streptococcus faecalis>200 Streptococcus pyogenes100 Escherichia coli 5V
` Pseudomonas aeruginoSa200 Xlebsiella pneumoniae 50 Proteus mirabilis 100 Proteus morgani 100 ! Salmonella typhimurium50 ¦, Pasteurella multocida 50 15 I Serratia marcescens 100 I Enterobacter aerogenes25 ¦ Enterobacter clocae 100 ~`
¦ - Citrobacter freundii 50 Il Providencia 100 20 j Staphylococcus epitermis 200 Pseudomonas putida >200 I Hemophilus influenzae >50 ,¦1 Neisseria gonorrhoeae0.312 I, .

.

~, -I ~119~64 The compounds of the formula I, wherein Rl is hydrogen or an ester-forming residue readily hydrolyzable in vivo, are active as antibacterial agents in vivo. In determining such activity, acute experimental infections ¦ are produced in mice by the intraperitoneal inocùlation of the mice with a 1 standardized culture of the test organism suspended in 5 percent hog gastric l mucin. Infection severity is standardized so that the mice receive one to ten - ~ ¦ times the LDloo dose of the organism (LDloo: 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 assessed by counting the number of survivors among the treated animals and expressing the activity of a compound as the percentage of animals which sur-vive.
The in vitro antibacterial activity of the compound of the formula I
wherein R 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 ,- 20 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 st instances it is appropriate to employ concentrations of the active ingredient of from about 0.1 percent to about 10 percent by weight, based on total compositian.
The in vivo activity of the compounds of formula I, wherein Rl 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 J~,~iJ; will find use in the control of infections caused by susceptible bacteria ,3~ 30 in buman sub~ects, e.g. infections caused by strains of Neisseria gonorrhoeae.
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` ~ , 1119164 ¦ ~hen conslderlng therapeutic o~e of a co=pound o~ the or==1- 1, or a salt thereof, in a mammal, particularly man, the compound can be administered alone, or it can be mixet with pharmaceutically acceptable carriers or diluents Ihey can be administered orally or parenterally, i.e. intramuscularly, sub-cutaneously or intraperitoneally. The carrier or diluent is chosen on the basis of the intendet mode of administration. ~or example, when considering the oral mode of administration, an antibacterial penam cempound of this in-vencion can be used in the form of tablets, capsules, lozenges, troches, powderg, syrups, ellxiss, aqueous solutions and suspensions, and the like, in accordance with standard pharmaceutical practice. The proportional ratio of active in-gredient to earrier will naturally depend on the chemieal nature, solubility and stability of the aetive ingredient, as well as the dosage contemplated.
~owever, pharmaceutical compositions eontaining an antibacterial agent of tbe formula I will likely contain from about 20Zto about 95% of active ingredient.
In the ease of tabiets for oral use, earriers which are com~only used include laetose, sodium citrate and salts of phosphoric acid. Various disintegrants ! such as starch, and lubricatlng agents, such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration ll in eapsule form, useful diluents are lactose ant high molecular wei~ht poly-20 1l ethylene glycols. When aqueous suspensions are required for oral use, theactive ingredient is combined wlth emuls,ifying and suspending agencs. If teslred, certain 8weetening and/or flavorin~ agents can be added. ~or paren-eeral atministration, which includes intramuscular, intraperitoneal, sub-eutaneous and intravenous u6e, sterile solutions of the active lngredient 25 are u~ually prepared, and the pH of the solutions are 6uitably adjusted and buffered. For intravenous use, the total eoncentation of solutes should be controlled to render the preparation ~so~onie.

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- As lndicated earlier, the antibacterial agents of thi6 invention are of u6e in human subject6 again6t 6u6ceptible Organi8ms. The prescribing physician will ultimately determine the appropria~e dose for a given human 6ubject, and thi6 can be expected to Vary according to the age, weight~
and respon6e of the individu~l patient~ a6 well as the nature and the severity of the patient'6 6ymptoms. The compound6 of this invention will normally be u8ed orally at do3age~ in the range from about lO to about 200 mg. per kilogra of body weight per day, and parenterally at dosage6 from about 10 to about 400 mg. per"kilogram of body weight per day. These figures are illu6trative I only~ however~ and in some ca6e6 it may be neCessary to use dosages outside these limits.
However, as indicated hereinbefore, the compounds of the formula I, wherein R is hydrogen or an ester-forming residue readily hydrolyzable in vivo, are potent inhibitor6 of microbial ~-iactanà6e6, and they increase the anti-bacterial effectiveness of ~-lactam antibiotic6 (penicillin6 and cephalosporin~) agaln8t many microorganism6, particularly those whiCh produce a B-lactam.~qe.
The manner in which the said compounds of the formula I increase the effective~
ne8s of a ~-lactam antibiotic can be appreciated by reference to exp-:-;imonts i~
i! whiCh the MIC of a given antibiotic alone~ and a compound of the formula I
¦ alone~ are measured. The6e MIC's are then compared with the MIC values obtained with a combination of the given antibiotic and the compound of the formula I. When the antibacterial potency of the combination is significantly greater than would have been predicted from the potencies of the individual ompound8, thi8 is considered to constitute enhancement of activity. The MIC
values of combinations are measured u6ing the method de6cribed by Barry and Sabath in "Manual of Clinical Microbiology", edited by Lenette, Spaulding and Truant~ 2nd edition, 1974~ American Society for Micrnbiology.

.

Ii ¦ Results of experiments illuserating that penicillanic acit 1,1-¦ dioxide enhances the effectiveness of ampicillin are reported in ~able II.
From Table II, it can be seen that against 19 ampicillin-resistant strains of SaEhyl~coccus aur_~s, the mote ~IC of ampicillin, and of penicillanic acid l,l-dioxide, is 200 mcg./ml. Howèver, the mode MIC's of ampicillin and peni-cillanic acid l,l-dioxide in combination are 1.56 and 3.12 mcg./ml., respectively. Looked at another way, this means that whereas ampicillin alone has a mode MIC of 200 mcg.lml. against the 19 strains of StaPhylococcus .
aureus, its mode ~IC is reducet to 1;56 mcg./ml. in the presence of 3.12 mcg./
o~ penic~llanic acid l,l-dioxide. The other entries in Table II show enhance-ment of the antibacterial effectiveness of ampicillin against 26 ampicillin i resistant stra~ns of HaemoDhilus influenzae, 18 ampiciilin resistant strains of Klebsiella Dneumoniae and 15 6trains of the anerobe Baceeroides fra~ilis.
¦ Tables III, IV and V 6how enhancement of the antibacterial potency of benzyl-penicillin (penicillin G) carbenicillin (~-carboxybenzylpenicillin) and cefazolin, respectively, against strains of S. aureus, H. influenzae, ~- e~ d -c~r~

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The compounds of ~he for~ula I, wherein R is hydrogen or an ester-forming residue readily hydrolyzable in ~ enhance the antibacterial ef-fectiveness of ~-lactam antibiotics in vivo. This is, they lower the amount of the antibiotic which is needed to protect mice against an otherwise lethal inoculum of certain ~-lacta s se producing bacteria.
The ability of the compounds of the formula I, wherein R is hydroge or an ester-forming residue readily hydrolyzable in vivo, to enhance the effectiveness of a B-lactam antibiotic~against B-lactamase-producing bacteria makes them valuable for co-administration with ~-lactam antibiotics in the treatment of bacterial iafections in mammals, particularly man. In the treat-ment of a bacterial infection, the said compound of the formula I can be comingled with the ~-lactam antibiotic, and the two agents thereby administere 6imultaneously. Alternatively, the said compound of the formula I can be administered as a separate agent during a course of trea~ment with a ~-lactam antibiotic. In some instances it will be advantageous to pre-dose the subject with the compound of the formula I before initiating treatment with a B-lactam antibiotic.
When using penicillanic acid l,l-dioxide or an ester thereof readily hydrolyzable in vivo to enhance the effectiveness of ~-lactam antibiotic, it is administered preferably in formulation with standard pharmaceutical carriers or diluents. The methods of formulation discussed earlier for use of penicil-lanic acid l,l-dioxide or an ester thereof readily hydrolyzable in vivo as a 6ingle-entity antibacterial agent can be used when co-administration with another ~-lactam antibiotic is intended. A pharmaceutical composition com-pri~ing a pharmaceutically-acceptable carrier, a ~-lactam antibiotic and penicillanic acid l,l-dioxide or a readily hydrolyzable ester thereof will normally contain from abo~t 5 to about 80 percent of the pharmaceutically acceptable carrier by weight.
When using penicillanic acid l,l-dioxide or an ester thereof readily hydrolyzable in vivo in combination with another B-lactam antibiotic, the sulfone can be administered orally or parenterally, i.e. intramuscularly, sub-cutaneou~ly or intraperitoneally. Although the prescribing physician will . , , ` ` .

1119~64 .
ultimately decide the do~age to be used in a human subject, the ratio of the daily dosages o~ the penicillanic acid l,l-dioxide or ester thereof and the ~-lactam antibiotic will normally be in the range from about 1:3 to 3:1.
Additionally, when using penicillanic acid l,l-dioxide or an ester thereof readily hydrolyzable in vivo in combination with another ~-lactam antibiotic, the daily oral dosage of each component will normally be in the range from abo~ t 10 to about 200 mg. per kilogram of body wei~ht and the daily parenteral dosage of each component will normally be about 10 to about 400 mg. per kilo-gram of body weight. These figures are illustrative only, however, and in come cases it nay be nece~ary to use dosages outside these limits.
Typical B-lactam antibiotics with which penicillanic acid l,l-dioxid and its esters readily hydrolyzable in vivo can be co-administered are:
6-(2-phenylacetamido)penicil~anic acid, 6-(2-phenoxyacetamido)penicillanic acid, -6-(2-phenylpropionamido)penicillanic acid, 6-(D-2-aniino-2-phenylacetamido)penicillanic acid, 6-(D-2-amino-2-[4-hydroxyphenyl~acetamido)penicillanic acid, 6-(D-2-amino-2-[1,4-cyclohexadienyl~acetamido)penicillanic acid, 6-(1-aminocyclohexanecarboxamido)penicillanic acid, 6-(2-carboxy-2-phenylacetamido)penicillanic acid, 6-(2-carboxy-2-E3-thienyl]acetamido)penicillanic ?cid, 6-(D-2-[4-ethylpiperazin-2,3-dione-1-carboxamido~-2-phenylacetamido)penicil-lanic acid, 6-(D-2-[4-hydroxy-1,5-naphthyridine-3-carboxamido]-2-pllenylacetamido)-penlcillanic acid, 6-(p-2-s~lfo-2-phenylacetamido)penicillanic acid, 6-(D-2-sulfoamino-2-phenylacetamido)penici}lanic acid, 6-(D-2-[imldazolidin-2-one-1-carboxamido]-2-phenylacetamido)penicillanic acid, 6-(D-[3-methylsulfonylimidazolidin-2-one-1-carboxamido]-2-phenylacetamido)-penicillanic acid, 6-([hexahydro-lH-azepin-l-yl]methyleneamino)penicillanic acid, .. , . , . . ~,,, ~ ., ' :,- ~
:

11191~i4 ~¦ acetoxymethyl 6-(2-phenylacetamido)peniclllanate, acetoxymethyl 6-(D-2-amdno-2-phenylacetamido)penicillanate, acetoxymethyl 6-(D-2-amino-2-[4-hydroxyphenyl]acetamido)penicillanate, pivaloyloxymethyl 6-(2-phenylacetamido)penicillanate, .
S pivaloyloxymethyl 6-(D-2-amino-2-phenylacetamido)penicillanate, pivaloyloxymethyl 6-(D-2-amino-2-14-hydroxyphenyl]acetamido)penicillanate, l-(ethoxycarbonyloxy)ethyl 6-(2-phenylacetamido)penicillanate, ¦
¦1 l-(ethoxycarbonyloxy)ethyl 6-(D-2-amino-2-phenylacetamido)penicillanate, I l-(ethoxycarbonyloxy)ethyl 6-(D-2-amino-2-[4-hydroxyphenyllacetamido)-10 ¦ penicillanate, ~ -3-phthalidyl 6-(2-phenylacetamido)penicillanate, 3-phthalidyl 6-(D-2-amino-2-phenylacetamido)penicillanate, - ., Il 3-phthalidyl 6-(D-2-amino-2-[4-hydroxyphenyl]acetamido)penicillanate, ¦
j'l 6-(2-phenoxycarbonyl-2-phenylacetamido)penicillanic acid, j i, 15 li 6-~2-tolyloxycarbonyl-2-phenylacetamido)penicillanic:acid, 6-~2-~5-indanyloxycarbonyl]-2-phenylacetamido)penicillanic acit, j 6-(2-phenoxycarbonyl-2-13-thienyl~acetamido)penicillanic acid, t 6-(2-tolyloxycarbonyl-2-[3-thienyl]acetamido)penicillanic acid, ¦
¦ 6-(2-[5-indanyloxycarbonyl~-2-~3-thienyl]acetamido)penicillanic acid, 20 !1 6-(2,2-dimethyl-5-oxD-4-phenyl-1-imidazolidinyl?penicillanic acid, 7-(2-[2-thienyl]acetamido)cephalosporanic acid, . I
7-(2-[1-tetrazolyl~acetamido-3-(2-[5-methyl-1,3,4-thiadiazolyl]thiomethyl)-3- ..
¦ desacetoxymethylcephalosporanic acid, ¦ ;
7-(D-2-amino-2-phenylacetamido)desacetoxycephalosporanic acid, 7--methoxy-7-(2-[2-thienyl~acetamido)-3-carbamoyloxymethyl-3-desacetoxymethyl cephalosporanlc acid, 7-~2-cyanoacetamido)cephalosporanic acid, 7-(D-2-hydroxy-2-phenylacetamido)-3-(5-[l-methyltetrazolyl]tbiomethyl)-3-de~acetoxymethylcepbalosporanic acid, 7-(2-[4-pyridylthio~acetamido)cephalosporanic acid, 7-(D-2-a~ino-2-[1,4-cyclohexadienyl]acetamidojcephalosporanic acid, . ¦ 7_ ~e-2-amino-2-phenylacetamldo)cephalosporanic acid, and the pharmaceutically-acceptable salts thereof.
- 26 ~

.
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~1119164 As will be appreciated by one skilled in the art, some of the above ~-lactam compounds are effective when administered orally or parenterally, while others ar~ effective only when administered by the parenteral route.
IWhen penicillanic acid l,l-dioxide or an ester thereof readily hydrolyzable in vivo is to be used simultaneously (i.e. co-mingled) with a ~-lactam anti-biotic which is effective only on parenteral administration, a combination r formulation suitable for parenteral use will be required. When the penicillanic acid l,l-dioxide or ester thereof is to be used simultaneously (co-mingled) with a ~-lactam antibiotic which is effective orally or parenterally, combina- -' tions suitable for either oral or parenteral administration can be prepared.
Additionally, it is possible to administer preparations o ~hc penicillanic acid l,l-dioxide or ester thereof orally, while at the same time administering further ~-lactam antibiotic parenterally; and it is also possible to lladminister preparations of the penicillanic àcid l,l-dioxide or ester ~hereof parenterally, while at the same time administering the further ~-~actam antibiotic orally.
The following examples are provided solely for the purpose of further hllustration. Infrared (IR) spectra were measured as potassium bromide discs B KBr discs) or as Nujol~mulls, and diagnostic absorption bands are reported in ave numbers (cm ). Nuclear magnetic resonance spectra (NMR) were measured t 60 MHz for solution6 in deuterochloroform (CDCi3), perdeutero dimethyl sul-foxide (DMSO-d6) or deuterium oxide (D20), and peak positions are expressed in arts per million (ppm) downfield from tetramethylsilane or sodium 2,2-imethyl-2-silapentane-S-sulfonate. The following abbreviations for peak hapes are used: ~, singlet; d, dbublet; t, triplet; q, quartet, m, multiplet.
3~ ,, , TraJ~n1 a r k 111916g EX~PL~ 1 Penicillanic Acid l,l-Dioxide To a solution of 6.51 g. (41 mmole) of potassium permanganate I in 130 ml. of water and 4.95 ml. of glacial acetic acid, cooled to ca. 5 C., 5 ¦ was added a cold (ca. 5 C.) solution of 4.5B 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 i 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 sodium chloride solution, and then the pI~ 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 ln vacuo, to give a further 0.28 g. of i product. The total yield was therefore 3.75 g. ~78Z yield). The ~IR spectrum I (D~O-d6) of the product showed absorptions at 1.40 (s,3H)J 1.50 (s,3H)~
3.13 (d of d's, lH, Jl ~ 16Hz, J2 ~ 2~z), 3.63 (d of d's~ 1~, Jl D 16 Hz, ~! J2 ' 4Uz), 4..2 (s, lU) a~d 5.03 (d of d~s, lU, JI = 4Uz, J2 ~ Z~Z) ppm.

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,19~64 EXA~IPLE 2 !!
¦ Benzyl Penicillanate l,l-Dioxide ¦

¦ To à stirred solution of 6.85 g. (24 mmole) of benzyl penicillanate ¦¦ in 75 ml; of ethanol-free chloroform, urder nitrogen, in an ice-bath, was ¦1 added in two portions, several minutes apart, 4.78 g. of 85% pure 3-chloro- It ¦I perbenzoic acid. Stirring was continued for 30 minutes in the ice-bath, .-and then for 45 minutes without external cooling. The reaction mixture was ll washed with aqueous alkali (pH 8.5), followed by saturated sodium chloride, ~ Ii and then it was dried and evaporated in vacuo to give 7.05 g. of residue. ¦
10 Examination of this residue showed it to be a 5.5:1 mixture of benzyl penicil- ¦
¦ lanate l-oxide and benzyl penicillanate l,l-dioxide.
To a stirred solution of 4.85 g. of the above 5.5:1 sulfoxide-l¦ sulfone mixture in 50 ml. of ethanol-free ch~loroform, under nitro~en, was ¦¦ added 3.2 g. of 85% pure 3-chloroperbenzoic acid at room temperature. The 15 l¦ reaction mixture was stirred for 2.5 hours, and then it was diluted with ~¦ ethyl acetate. The re~ultant mlxture was added to water at pH 8.0, and then the layers were separated. The or~anic phase was washed with water at pH 8.0, ¦ followed by saturated sodium chloride, and then it was dried using sodium J .sulfate. Evaporation of the solvent in vacuo afforded 3.59 g. of the title 20 ¦~ compound. The N~iR spectrum of the produot (in CDC13) showed absorptions at ¦¦ 1.28 (6, 3H), 1.58 (s,3H), 3.42 (m,211), 4.37 (s,lH), 4,55 (m,lH), 5.18 J
l~ (q,2H, J ~ 12 Hz) and 7.35 (s,5H) ppm. .
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_29 Il .,' ;

11~9164 EXAMPLE 3 .
Penicillanic Acid l.l-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 ~1. of ethyl acetate was 5 slowly added 10 ml. of water, followed by 12 g. of 5% palladium-on-calcium carbonate. The mixture was shaken under an atmosphere of hydrogen, at r 52 psi, for 40 minutes, and then it was filtered through supercel (a diatomaceous earth). The filter cake was washed with methanol, and with agueous methanol, and the washings were added to the filtrate. The combined 10 solution was evaporated in vacuo to remove the ma~or$ty of the organic solvents and then the residue was partitioned between ethyl acetate and water I at a pH of 2.8. The ethyl acetate layer was r~moved and the aqueous phase ! was further extracted with ethyl acetate. ~le combined ethyl acetate solutlonsl were washed with saturated sodium chloride solut$on, dried using sodium sul-15 fate and then evaporated in vacuo. The residue was slurried in a 1:2 ~ixture . _ of ethyl acetate-ether, to ~lve 2037 g. of the tltle product havinP. a melting point of 148-51 C. The ethyl acetate-ether mixture was evaporated giving a further 2.17 g oE producc.

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Il . ', ¦ EXA~LE 4 Pivaloyloxymethyl Penicillanate -1 l-Dioxide .
. ' ' . .' I To 0.615 g. (2. 41 mmole? of penicillanic acid l,l-dioxide in

2 ml. of N,N-dimethylformamide was added 0.215 g. t2.50 mmole) of diiso-propylethylamine followed by 0.365 ml. of chloromethyl pivalate. The reaction mixture was stirred at roo~ 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 solution was then drled using anhydrous sodiu~ sulfate~
¦ and evaporated in vacuo to give 0.700 g. of the title product as a solid, I mp 103-4 C. The N~R spectrum of the product (in CDC13) showed absorptions ¦ at 1.27 (s, 9H), 1.47 (s, 3H), 1.62 (s, 3H), 3.52 (m, 2H), 4.47 (s, lH), ¦¦ 4.70 (m, lH), 5.73 (d, lH, J - 6.0 Hz) and 5.98 (d, lH, J - 6.0 Hz).

15 ¦¦ EXAMPLE 5 ! .

The procedure of Example 4 is repeated, except that the pivaloyoxy-¦ methyl chloride used therein is replaced by an equimolar amount of acetoxy-I methyl chloride propionyloxymethyl chloride and hexanoyloxymethyl chloride, .-.¦ respectively, to give:

acetoxymethyl'penicillanate l,l-dioxide, .j propionyloxymethyl penicillanate l,l-dioxide and hexanoyloxymethyl penicil~lanate l,l-dioxide, renpectively. : ¦

_ 31 _ 3l119~

EXIu'~E 6

3-Phthalidyl Penicillanate l,l-Dioxide ¦ To 0.783 g. (3.36 mmole) of penicillanic acid l,l-dioxide in 5 ml.
of N,N-dimethylformamlde was added 0.47 ml. of triethylamine followed by 0.715 g. of 3-bromophthalide. The reaction mlxture 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.
l The ethyl acetate layer was washed successively with water and saturated 10 ¦ 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 CDC13) showed absorpCions at 1.47 (s, 6H), 3.43 (m, lH), 4.45 (s, lH), 4.62 (m, lH~, 7.40 and l 7.47 (26's, lH) and 7.73 (m, 4H) ppm ' 15 ¦ When the above procedure is repeated, except that the 3-bromophthalid i6 replaced b!y 4-bromocrotonolactone and 4-bro~o-y- -butyrolactone, respectlvely, this affords: !

4-crotonolactonyl penicillanate l,l-dioxide.and . I ~-butyrolacton-4-yl penicillanate, 20 ¦ respect~ve y.~

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l-(Ethoxycarbonyloxy)ethyl Penicillanate l,l-Dioxide A mixture of 0.654 g. of penicillanic acid l,l-dioxide, 0.42 ml.
of triethylamine, 0.412 g. of l-chloroethyl ethyl carbonate, 0.300 g. of sodium bromide and 3 ml. of N,N-dimethylformamide 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 et~yl 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 (CDC13) showed absorptions at 1.43 (m, 12H), 3.47 (m, 2H), 3.9 (q, 2H, J = 7.5 Hz), 4.37/m, lH), 4.63 (m, lH) and 6.77 (m, lH) ppm.

ll l :1.119164 ~LE 8 ¦ ~

¦~he procedure o~ Example 7 is repeated, except that the l-chloroethy~ j ethyl.carbonate is replaced by an equimolar amount of the appropriate 1-, chloroalkyl alkyl carbonate, l-(alki~noyloxy~ethyl chloride or l-methyl-l-

5 1 (alkanoyloxy)ethyl chloride, to produce the following compounds:

methoxycarbonyloxymethyl penicillanate l,l-dioxide, ethoxycarbonyloxymethyl penicillanate .l,l-dioxide, isobutoxycarbonyloxymethyl penicillanate l,l-dioxide, l-(methoxycarbonyloxy)ethyl penicillanate l,l-tioxide, 10 1 l-(butoxycarbonyloxy)ethyl penicillanate l,l-dioxide, ¦l l-(acetoxy)ethyl penicillanate l,l-dioxide, .
~ (butyryloxy)ethyl penicillanate l,l-dioxide, ¦¦ l-(pivaloyloxy)ethyl penicillanate l,l-dioxide, ~ (hexanoyloxy)ethyl penicillanate l,l-dioxide, !_ ¦
¦ l-methyl-l-(acetoxy)ethyl penicillanate l,l-dioxide and l-methyl-l-(isobutyryloxy)ethyl penicillanate l,l-dioxide, ¦ ;

! respectively. I
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1119~6~

XA~LE 9 ¦ The procedure of Example 4 ls repeated, except that the chloromethyl pivalate is re?laced by an equimolar amount of benzyl bromide ant 4-nitrobenzy L
bromide, respectively, to produce benzyl penicillanate l,l-dioxide and 4_nitro benzyl penicillanate l,l-dioxide, respectively.

EXA~LE 10 Penicillanic Acid l~-Oxide Io 1~4 g. of prehydrogenated 5~ palladium-on-calcium carbo~ate in l 50 ml. of water was added a solution of 1.39 g. of benzyl 6,6-dibromopenicilla .
10 ¦ nate l~-oxide in 50 ml. of tetrahydrofuran. The ~ixture 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 ap-peared to be largely benzyl penicillanate l~-oxide.
The above benzyl penicillanate l~-oxide was combined with a further ¦¦ 2.0 g. of benzyl 6,6-tibromopenicillanate l~-oxide and dissolved in 50 ml. of ¦ tetrahydrofuran. The solution was added to 4.0 g. of 5% palladium-on-calcium .1 carbonate, in 50 ml. of water, and the res~ltin~ mixture was shaken under 20 ! an atmosphere of hydrogen, at ca. 45 p,s.i. and 25C. overnight. The mixture ¦ was flltered, and the filtrate was extracted with ether. Ihe extracts were evaporated in vacuo, and the residue was purified by chro~atog s phy 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.
1n water-methanol (1:1) with 0.50 g. of 5% palladium-on-calcium carbonate for ¦ 2 hours. At this point, an additional 0.50 g. of SZ palladium-on-calcium car-bonate was adted and the hydrogenatlon was continued at 45 p.s.i. and 25C.

' '''' '` ' . ~, 1~ .
~1 - 35 -~1.119164 overnigh~. The reac~ion =i~ore was fil~ered, ex~rac~ed ~i~h e~her and che ex-tracts were discarded. The residual aqueous phase was adjusted to pH 1.5 and then extracted with ethjl acetate. The ethyl acetate extracts were dricd (~a2SO4) and then evaporated in vacuo ~o give 0.14 g. of penicillanic acid ¦l-oxide. The NMR spectrum (CDC13/3MSO-d6~ showed absorptions at 1.4 (s, 3H), ¦1.64 (s, 3H), 3.60 (m, 2H), 4.3 (s, lH) and 4.54 (m, lH)ppm. The IR spectrum -of the product (KBr disc~ showed absorptlons at 1795 and 1745 cm l.

EX~U~LE 11 . . !~
Penicillanic Acid l~-Oxide -10 ¦ Io 1.0 g. of prehydrogenated 5% palladium-on-calcium carbonate in ¦30 ~1. of water is added a solution of l.O g. of 6,6-dibromopenicillanic acid jl~-oxide. The mixture is shaken under an atmosphere of hydrogen, at ca. 45 !j p.s.i. and 25C., for l hour. The reaction mixture is then-filtered and the ' l filtrate is concentrated in vacuo to remove the methanol. The residual -aqueous 15 jphase is diluted with an equal volume of water, adjusted to pH 7, and washed l¦with ether. The aqueous phase is then acidified to pH 2 with dilute hydro-¦Ichloric acid and extracted with ethyl acetate. The ethyl acetate extracts are ¦¦dried (Na2SO4) and evaporated in vacuo to give pe icillanic acid l-oxide.

~1 ' ' ' ' . ':;
,~

l - 36 -~,, 64 ~ i EXA~PLE lZ

Penicillanic Acid_18-Oxide . .
To a stirred soiution oE 2.65 g. tl2.7 mmole) of penicillanic acid in ¦ chloroform at 0C. was added 2.58 g. of 85% pure 3-chloroperbenzoic acit. I
After 1 hour, the reaction mixture was filtered and the filtrate was evaporated ¦ i in vacuo. The residue was dissolved in a small amount of chloroform. The solution was concentrated slowly until a precipitate be~an 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 18-oxide, m.p. 140-3C. The IR spectrum of the product (CHC13 solution) showed absorptions at 1775 and 1720 cm 1 The N~
¦Ispectrum (CDC13/DMSO-d6) showed absorptions at 1.35 (s, 3H), 1.76 (s, 3H), 3.36 ¦¦(m, 2H), 4.50 (~, lH) and 5.05 (m, lH~ppm. ~rom the NMR spectrum, the product l¦appeared to be ca. 90% pure. ~
lS I Examination of the chloroform-ether mother liquor revealed that it contained further penicillanic acid 18-oxide, and also some penicillanic acid l~-oxJde.

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~L119164 ; EXAMPLE 13 .

¦ Esteri~ication of penicillanic acid l~-oxide or penicillanic acid ¦lB-oxide, as appropriate, with the requisite alkanoyloxy chloride, according to Example 5, provides the following compounds: 1.

S acetoxymethyl penicillanate l-oxide, propionyloxymethyl penicillanate l~-oxide, pivaloyoxymethyl penicillanate l-oxide, acetoxymethyl penicillanate l~-oxide, propionyloxymethyl penicillanate l~-oxide ant pivaloyloxymethyl penicillanate lB-oxide, respectively.

1~ E.YAMPLE 14 ¦ Reactlon of penicillanic acid la-oxide or penicillanic acid lB-oxide with 3-bromophthalide, 4-bromocrotonolactone or 4-bro -y-butyrolactone, a~ appropriate, affords the following compound~:
¦¦ 3-phthalidyl penicillanate l~-oxide ¦1 4-crotonolactonyl penicillanate l-oxide, 3-phthalidyl penicillanate l~-oxide, ¦1 4-crotonolactonyl penlcillanate l~-oxide and 20 !I y-butyrolacton-4-yl penicillanate l~-oxide, respectlvely.

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i . EXAM~L~ 15 ., Reaction of penicillanic acid l-oxide or penicillanic acid 13-oxide, as appropriate, with the requisite l-chloroalkyl alkyl carbonate or l-(alkanoyl-l oxy)ethyl chloride, according to the procedure of ~xample 7, provides the fol-lowing compounds:

¦ l-(ethoxycarbonyloxy)ethyl penicillanate l-oxide, ¦ methoxycarbonyloxymethyl penicillanate l~-oxide, ¦ ethoxycarbonyloxymethyl penicillanate l-oxide, l isobutoxycarbonyloxymethyl penicillanate l~-oxide, .
lO ¦ l-(methoxycarbonyloxy)ethyl penicillanate l~-oxide, . I
¦ l-(butoxycarbonyloxy)ethyl penicillanate l~-oxide, : ¦ l-(acetoxy)ethyl penicillanate l~-oxide, l-(butyryloxy)ethyl penicillanate l~-oxide,`
¦ l-(pivaloyloxy)ethyl penicillanate l~-oxide, ~
15 ¦¦ l-(ethoxycarbonyloxy)ethyl penicillanate lB-oxide, I m-thoxycarbonyloxymethyl penicillanate lB-oxide, ¦ ethoxycarbonyloxymethyl penicillanate lB-oxide, . isobutoxycarbonyloxymetnyl penicillanaee lB-oxide, l l-(methoxycarbonyloxy)ethyl penicillanate lB-oxide, 20 ¦ l-(butoxycarbonyloxy)ethyl penicillante'l~-oxide, l-(acetoxy)ethyl penicillanate lB-oxite, ¦ l-(butyryloxy)ethyl penicillanate lB-oxide and ¦ l-(pivaloyloxy)ethyl penicillanate lB-oxide, respectively. `

~ 39~

I EXAMPLE 16 .

¦ Reaction of penicillanic acid l~-oxide and penicillanic acid lB-oxide with benzyl bromide, according to the procedure of Example 4, produces benzyl penicillanate l~-oxide and benzyl penicillanate lB-oxide, respectively.
¦ In like manner, reaction of penicillanic acid l~-oxide and penicillanic ¦acid lB-oxide with 4-nitrobenzyl bromide, according to the procedure of ~xample 4, produces 4-nitrobenzyl penicillanate ll_oxide and 4-nitrobenzyl penicillanate lB-oxide, respectively.

I EXA~MPLE 17 ! Penicillanic Acid l,l-Dloxide To 2.17 g. (10 mmole) of penicillanic acid l~-oxide in 30 ml. of ¦ethanol-free chloroform at ca. 0C. is added 1.73 g. (10 mmole) of 3-~hloroper-¦benzoic acid. The mixture i9 stirred for l hour at ca. 0C. and then for an ,ladditional 24 hours at Z5~C. The filtered reaction mixture is evaporated in 15 livacuo to give penicillanic acid l,l-dioxide.
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i EX~IPLE 18 . The procedure of Example 17 i6 repeated, except that the penicillanic t ¦acid l-oxide used therein is replaced by:
I . ,, ¦penicillanic acid lB-oxide, 5 1 acetoxymeehyl penicillanate la-oxide, }
¦propionyloxymethyl penicillanate la-oxide, -pivaloyoxymethyl penicillanate la-oxide, ¦acetoxymethyl penicillanate lB-oxide,-¦propionyloxymethyl penicillanate lB-oxide, -10 ¦pivaloyloxymethyl penicillanate lB-oxide, ¦3-phthalidyl penicillanate la-oxide, I3-phthalidyl penicillanate lB-oxide, ¦¦l-(ethoxycarbonyloxy)ethyl penicillanate la~oxide, !
¦¦~ethoxycarbonyIoxymethyl penicillanate l-oxide, 151~ethoxycarbonyloxymethyl penicillanate l-oxide, , iaobutoxycarbonyloxymethyl penicillanate l-oxide, ¦ l-(methoxycarbonyloxy)ethyl penicillanate l-oxide, I l-(butoxycarbonyloxy)ethyl penicillanate l-oxide, .1 l-~acetoxy)ethyl penicillanate l-oxide, 20!I l-(butyryloxy)ethyl penicillanate l-oxi'de, ¦ l-(pivaloyloxy)ethyl penicillanate l-oxide, l-(ethoxycarbonyloxy)ethyl penicillanate lB-oxide, methoxycarbonyloxymethyl penicillanate lB-oxide, ¦ ethoxycarbonyloxymethyl penlcillanate lB-oxide, 25 I i~obutoxycarbonyloxymethyl penicillanate lB-oxide, , l-(methoxycarbonyloxy~ethyl penicillanate lB-oxide, l-(butoxycarbonyloxy)ethyl penicillanate lB-oxide, ¦ I-(acetoxy)ethyl penicillanate lB-oxide, _.

: . i ;l l-(butyryloxy)ethyl penicillanate lB-oxide and .
l-(pivaloyloxy)ethyl penicillanate 13-oxide, .
respectively.` This affords:
. ' ,' , .. , penicillanic acid l,l-dioxide, ¦ acetoxymetnyl penicillanate l,l-dioxide, ropionyloxymethyl penicillanate l,l-dioxlde, pivaloyoxyme~hyl penicillanate l,l-dioxide, acetoxymethyl penicillanate l,l-dioxide7 l propionyloxymethyl penicillanate l,l-tioxide, 10 ¦ pivaloyloxymethyl penicillanate l,l-dioxide, 3-phtha1idyl penicillanate l,l-dioxide, I, 3-phthalidyl penicillanate l,l-dioxide, (ethoxycarbonyloxy)ethyl penicillanate l,l-dioxide, ~ methoxycarbonyloxymethyl penicillanate l,l-dioxide, -15 ¦1 ethoxycarbonyloxymethyl penicillanate l,l-dioxide, ¦ isobutoxycarbonyloxymethyl penicillanate l,l-dioxide, l-(methoxycarbonyloxy)ethyl penicillanate l,l-dioxide, l-(butoxycarbonyloxy)ethyl penicillanate l,l-dioxide, ~ l-(acetoxy)ethyl penicillanate l,l-dioxide, 20 ¦ l-(butyryloxy)éthyl penicillanate l,l-di,oxide, ¦ l-(pivaloyloxy)ethyl penicillanate l,l-dioxide, ¦ l-(ethoxycarbonyloxy)ethyl ?enicillanate l,l-dioxide, ¦ ;
¦ methoxycarbonyloxymethyl penicillanate l,l-dloxide, . ¦

. ~ ethoxycarbonyloxymethyl penicillanate l,l-dioxide, isobutoxycsrbonyloxymethyl penicillanate l,l-dioxide, l-(methoxycarbonyloxy)ethyl penicillanate l,l-dioxide, l-(butoxycarbonyloxy)ethyl penicillanate l,l-dioxide, l-(acetoxy)ethyl penicillanate l.l-dioxide, .
I¦ l-(bu~yryloxy)ethyl penicillanate l,l-dioxide and 30 1¦ l-(pivaloyloxy)ethyl penicillanate l,l-dioxide, respectively.

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EXA~PLE 19 Oxidacion of benzyl penicillanate l~-oxide and benzyl penicillanate 18-oxide with 3-chloroperbenzoic acid, according to the procedure of Example ¦ 17, produces, in each case, benzyl penicillanate l,l-dioxide.
¦In like manner, oxidation of 4-nitrobenzyl penicillanate l~-oxide t and 4-nitrobenzyl penicillanate l~-oxide with 3-chloroperbenzoic acid, accordin t to the procedure of ~xample 17, produces 4-nitrobenzyl penicillanate 1,1- ¦ r dioxide. ' . ' ' . .

- Penicillanic Acid l,l-Dioxide i aydrogenolysis of 4-nitrobenzyl penicillanate l,l-dioxide, according to the procedure of Example 3, affords penicillanic acid l,l-dioxide.

Sodium Penicillanate l,l-Dioxide I .
.
15 ¦ To a stlrred solution of 32.75 g. (0.14 mole) of penicillanic acid ¦ l,l-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 acetace. The resultin -j solution was stirred for 1 hour and then an addi'tional 10% excess of sodium 2-ethylhexanoate in a small volume of'ethyl acetate was added. Product im-', 20 mediately began to precipitate. Stirring was continued for 30 minutes and then the precipitate was removed by filtration. It was washed'sequentially wit t ethyl acetate, with 1:1 ethyl acetate-ether and with ether. The solid was then drled over phospnorus pentoxide, at ca. 0.1 r~m of Hg'for 16 hours at 25C., 1 giving 36.8 g. of the title sodium salt, contaminated with a small amount of 25 ¦ ethyl acetate. The ethyl acetate content was reduced by heating to 100C.
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for 3 hours under vacuum. The IR spectrum of this final product (KBr disc) . ~showed absorptions at 1786 and 1608 cm 1. The N~R spectrum lD20) showet ab-. s orptions at 1.48 (s, 3H), 1.62 ~s, 3H), 3.35 (d of d's, lH, J1~16Hz, J2=2Hz), ; 3.70 (d of t's, lH, J1~16Hz, J2~4Hz), 4.25 (s, lH) and 5.03 (t of d's, lH, S Jl-4Hz, J2~2Hz)ppm. .
The title sodium salt can also be prepared using acetone ln place of ethyl acetate.
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Penicillanic Acld l,l-Dioxide To a mixture of 7,600 ml. of water ant 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 hat been prepared from 270 g. of penicil-lanic acid, 260 ml. of 4~ sodium hydroxide and 2,400 ml. of water tPH 7.2), and which had then been cooled to 8q C. The temperature rose to 15 C. during ¦ thi6 latter addition. Tl~e temperature of the resulting mixture was reduced 10 ¦ to 5 C. and the stirrin~ was continued for 30 minutes. To the reaction l ¦
mixture was then added 142.1 g. of sodium bisulfite, in portions, during 10 j minutes. The mixture was stirred for 10 minutes at 10 C., and then 100 g;
of supercel (a diatomaceous earth) was addèd. After a further 5 minuces of Il stirring, the mixture was filtered. To the filtrate was added 4.0 liters of 15 ll ethyl acetate, and then the pH of the aqueous phase was lowered to 1.55 using ¦¦ 6N hydrochloric acid. Tbe ethyl acetate layer was removet and combined with ¦¦ several further ethyl acetate extracts. The combined organic layer was ¦ washed with water, dried t~gS04) and evaporated almost to dryness in vacuo The slurry thus obtained was stirred with 700 ml. of ether at 10 C., for 20 20 ¦ minutes, and then the solid was collected by filtration. This a'forded ¦ 82.6 g. (26% yield) of the title compound having a melting point of 154-155.5 ,1 C. ~-c.).

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t l Pivaloyloxymethyl Penicillanate l,l-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 tnen it was allowed to warm to room temperature. Analysis of the resulting solution by NMR indicated that it contained both the 1~- 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 dichloro-methane and 0.4 g. 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 anhydroussodium sulfate and evaporated ln vacuo. The residue was dissolved in ether an~
~ reprecipitated by the addition of hexane. The resulting solid was recrystal-lized from ether to give 0.357 g. of the title compound.
The NMR spectrum of the product (CDC13) showed absorptions at t 1.23 (s,9H), 1.50(s,3H), 1.67 (s,3H), 3.28 (m,2H), 4.45 (s,lH), 5.25 (m,lH) and 5.78 (m,2H)ppm.

~ gl~4 EX~MPL~ 24 3-PhthalidYl Penicillanate 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 pH 6.0, and sodium bisulfite was added to decompose any remQining peracid. The pH
of the aqueous phase was raised to 8.8. The layers were separated and the organic phase was evaporated in vacuo. This afforded the title compound as a foam. The NMR spectrum (CDC13) showed absorptions at 1.62 (m,6H), 3.3(m,2H), 4.52 (p,lH), 5.23(m,lH) and 7.63 (m,5~1)ppm.

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, .` ~ ' ' ' ' . . ,, ''' " :,' ' ' ' ' 2,2,2-Trichloroethyl Penicillanate l,l~Dioxide ., To 100 mg. of 2,2,2-trichloroethyl penicillanate in a s~all volume of chloroform was added 50 mg. of 3-chloroperbenzoic acid and the mixture was stirred for 30 minutes. Examination of the reaction plOdlCt aL ti~is point revealed that it was mostly sulfoxide (The NMR spectrum (CDCl3) showed absorptions at 1.6 (s,3H), 1.77 (s,3H), 3.38(m,2H), 4.65 (s,lil), 4.85 ~m,2H) and 5.37 ~m,lH)ppm.) A further 100 mg. of 3-chloroperbenzoic acid was added and the mixture was stirred overnight. The solvent was then removed by evaporation in vacuo, and the residue was partitioned between ethyl acetate and water at pH 6Ø Sufficient sodium bisulfite was added to decompose the excess peracid and then the pH was raised to 8.5. The organic phase was 6eparated, washed with brine and dried. Evaporation in vacuo afforded 65 mg.
of the title product. The NMR spectrum (CDC13) showed absorptions at 1.53 (8 , 3H), 1.72 (8 ? 3H), 3.47(m,2H), 4.5(s,lH), 4.6 (m,lH) and 4.8 (m,2H)ppm.
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¦ EXAMPLE 26 ¦ 4-Nitrobenzyl Penicillanate l,l-Dioxide r A solution of 4-nitrobenzyl penicillanate in chloroform was cooled to about 15 C. and l eguivalent 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 l-oxide. A further l 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-chloropèrbenzoic acid was added and the reaction mixture was stirred overnight. The solvent was re ved 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 ,~el, eluting with at 1:4 mixture lS of ethyl acetate/chloroform.
The NMR spectrum of the product (CDCl3) showed absorptions at 1.35 (s, 3H), 1.58 (6, 3H), 3.45 (m, 2H), 4.42 (s, lH), 4.58 (m, lH), 5.30 (s, 2H) and 7.83 (q, 4H)ppm , , . .

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Penicillanic Acid l,l-Dioxide To 0.54 g. of 4-nitrobenzyl penicillanate l,l-dioxide in 30 ml.
of methanol and 10 ml. of ethyl acetate was added 0.54 g. of 10% 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 mixtur~
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 re ved. Fresh ethyl aceta~e was added and the pH was adjusted to 1.5.
The ethyl;acetate layer was removed, washed with water and dried, and then it was evaporated ln vacuo. This afforded 0.168 g. of the title compound as a crystalline solid.

Penicillanic Acid l,l-Dioxide '-A stirred solution of 512 mg. of 4-nitrobenzyl penicillanate 1,1- t dioxide in a mixture-of 5 ml. of acetonitrile and 5 ml. of water was cooled to 0 C. and then a solution of 484 ~g. of sodium dithionite in 1.4 ml. of l.ON sodium hydroxide was added portionwise over several minutes. The reaction mixture was stirred for an additional 5 minutes and then it was diluted with ethyl acetate and water at pH 8.5. The ethyl acetate layer was removed and evaporated in ~acuo giving 300 mg. of starting material. ~resh ethyl acetate was added to the aqueous phase and the pH was adjusted to 1.5. The ethyl acetate was removed, drled and evaporated in vacuo giving 50 mg. of the title compound. ~
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..~ ' ' ' . 'l ~ ~1191~4 ' EXAM~LE 29 l-Methyl-l-(acetoxy)ethyl'Penicillanate l,l-Dioxide To 2.33 g. of penicillanic acid l,l-dioxide in 5 ml. of ~N-dimethyl-formamide was added 1.9 ml. of ethyldiisopropylamine, followed by the dropwise addition of 1.37 g. of l-methyl-l-(acetoxy)ethyl chloride, at ca 20 C. The ~ixture was stirred at ambient temperature overnight and then-the mixture was ' ;
diluted with ethyl acetate and with water. The layers were separated and ~;
- the ethyl acetate layer was washed with water~at pH 9. The ethyl acRta~
solution was then dried (Na2S04) and e~aporated in vacuo leaving 1.65 g. of 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 NMR spectrum of the crude product (CDC13) showed absorpt~ons at 1.5 (6, 3H), 1.62 (s, 3H), 1.85 (s, 3H), 1.93 (s, 3H), 2.07 (s, 3H), 3.43 (m, 2H), 4.3 (s, lH) and 4.57 (m, lH)PPm-. i EXA~LE 30 The procedure of Example 29 is repeated, except that the l-methyl-l-(acetoxy)ethyl chloride is replaced by the appropriate l-methyl-l-(alkanoylox ')~
ethyl chloride, to produce the following compounds:

l-methyl-l-(propionyloxy)ethyl penicillanate l,l-dioxide, l-methyl-l-(pivaloyloxy)ethyl penicillanate l,l-dioxide and l-methyl-l-(hexanoyloxy)ethyl penicillanic acid l,l-dioxide, respectively.

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Penicillanic Acid l,l-Dioxide To a stirred solution of 1.78 g. of penicillanic acid in water, at pH 7.5, was added 1.46 ml. of 40% peracetic acid, followed by an additional 2.94 ml. of 40% peracetic acid 30 minutes later. 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 peracidS and then the pH was adjusted to 1.5. The ethyl acetate layer was re ved, dried ~Na2S04) and evaporated in vacuo. The residue was a 3:2 mixture of penicillanic acid l,l-dioxide and penicil}anic acid l-oxide.
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Pivaloyloxymethyl Penicillanate l,l-Dioxide A stirred solution of 595 mg. of pivaloyloxymethyl penicillanate l-oxide in 5 ml. of ethyl acetate was cooled to ca -15 C., and 5 mg. of 5 ¦ manganic acetylacetonate was added. To the dark brown mixture thus obtained was added, during several minutes, 0.89 ml. of 40~ peracetic acid in small amounts over several minutes. After 40 minutes the cooling bath was removed, t and the mixture was stirred at ambient temperature for 3 days. The mixture l was diluted with ethyl acetate and water at pH 8.5, and the ethyl acetate 10 ¦ layer was removed, dried and evsporated in vacuo. This afforded 178 mg.

i of material which was shown by NMR spectroscopy to be a mixture of pivaloyoxy-methyl penicillanate l,l-dioxide and pivaloyloxymethyl penicillanate l-oxide.
The above material was redissolved in ethyl acetate and reoxidized l using 0.9 ml. of peracetic acid and 5 mg. of manganic acetylacetonate, as 15 1 described above, using a reaction time of 16 hours. The reaction mixture was worked up as described above. This afforded 186 mg. of pivaloyloxymethyl penicillanate l,l-dioxide.

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164 I i, . ', . '.
¦ PREPARATION A

¦ 6,6-Dibromopenicillanic Acid ln-Oxide . ' .
¦ The title compound is prepared by oxidation of 6,6-dibromopenicillanic ¦acid with 1 equivalent of 3-chloroperbenzoic acid in tetrahydrofuran at 0-25C. ' 5 ¦ for ca. 1 hour, according to the procedure of Harrison et al., Journal of the Chemical Society (London) Perkin I, 1772 tl976).

¦ PREPARATION B

Benzyl 6.6-Dibromopenicillanate To a solution of 54 g. (0.165 mole) of 6,6-dibromopenicillanic acid in 350 ml. of N,N-dimethylacetamide was added 22.9 ml. (0.165 mole) of triethyl;
amine and the solution was stirred for 40 minutes. Ben~yl bromide (19.6 ml., 0.165 mole) was added and the sesulting mixture was stirred at room temperature for 48 hourfi. .The precipitated triethylamine hydrobromide was fil~ered off, l and the filtrate was added to 1,500 ml. of ice-water, adjusted to pH 2. The 15 1 mLxture was extracted with ether, and the extracts were washed successively with saturated sodium bicarbonate, water and brine. The dried (MgS04) ether solu-. tion was evaporated in vacuo to give an off-white solid, which was recrys~al-I lized from isopropanol. This afforded 70.0 g. (957. yield) of the title compoundl m.p. 75-76C. The IR spectrum (KBr disc) showed absorptions at 1795 and 20 1 1740 cm 1. The N~ spectrum (CDC13) showsd absorptions at 1.53 (s, 3H), l.S8 ~ (o, 3H), 4. (s, lH~, 5.13 ~s, ZU), 5.72 (G, 13) aDd 7.37 (s, 53~pp~.

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¦ PREPAtATIOW C

Benzyl 6.6-Dibromopenicillanate l~-Oxide ¦¦ To a stirred solution of 13.4 g. (0.03 mole) of benzyl 6,~-dibromo-I 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.
¦I 0C. Stirring was continued for 1.5 hours at ca. 0C. and then the reaction i mixture was filtered. The filtrate was washed successively with SZ sodium bicarbonate and water, and then it w~s dried (~a2SO4). Removal of the solvent by evaporation in vacuo gave 12.5 g. of the title product as an oil. The oil was induced to solidi~y by trituration under ether. Filtration then afforded 10.5 g. of benzyl 6,6-dibromopenicillanate l~-oxide as a solid. Ihe IR spec-¦ trum (CHC13) showed absorptions at lôO0 and 1750 cm . The N~IR spectrum of the product (CDC13) showed absorptions at 1~3 (s, 3H), l.S (s, 3H), 4.5 (s, lH) 5.18 (s, 2H), 5.2 (s, lH) and 7.3 (s, 5H)ppm.

PREPARATION D

l4-Nitrobenzyl Penicillanate ¦Reaction of the triethylamine salt of penicillanic acid with 4-nitro-¦ benzyl bromide according to the procedure of Preparation B, affords 4-nitro-benzyl ptnl lllanate.
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- PREPARAl'ION E
222,2-Trichloroeth,yl Penicillanate To ~03 mg. of penicillanic acid in 10 ml. of dichloromethane was added 25 mg. of diisopropylcarbodiimide followed by O.l9 ml. of 2,2,2-trichloro~ethanol. The mixture was stirred overnight and then the solvent was removed by evaporation in vacuo. The crude product was purified by column chroma- :~
tography using eilica gel as the adsorbent and chloroform as the eluant.
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PREPARATION F
3-Phthalidyl Penicillanate , To a solution of 506 mg. of penicillanic acid in 2 ml. of N,N-dimethylormamide was added 0.476 ml. of diisopropyietbylamine followed by 5 536 mg. of 3-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 wasbed with water, and then with water at pH 8.0, and then it was dried using anhydrous sodium sulfate I The dried ethyl acetate solution was evaporated in vacuo giving 713 ~g. of the10 ¦title ester as an oil. The N~ spectrum (CDC13) showed absorptions at L
¦1.62 (m,6H), 3.3 (m,2H), 4.52 (s,lH), 5.23 (m,lH) and 7.63 (m,5H).

19~4 ~., PREPA~ATION 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 8Ø The ethyl acetat solution was dried (Na2SO4) and then evaporated in vacuo to give pivaloyloxy-methyl 6,6-dibromopenicillanate as an amber oil (3.1 g.) which slowly crystallized.
The above ester was dissolved in lO0 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 atmospher: c 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 (Na2SO4) and evaporated in vacuo to give 1.25 g. of the title compound. The NMR spectrum (CDC13) showed absorptions at 1.23 (s,gH), 1.5 (s,3H), 1.67 (s,3H), 3.28 (m,2H), 4.45 (s,lH), 5.25 (m,lH) and 5.78 (m,2H) 20 ppm. .

111~164 ` PREPARATION H
4-Nitrobenzyl Penicillanate To a stirred solution of 2.14 g. of penicillanic acid and 2.01 ml.
of ethyldiisopropylamine in 10 ml. of N,N-dimethylformamide was added dropwise 2.36 g. of 4-nitrobenzyl bromide, at ca. 20C. The mixture was stirred at 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 solution was then dried (Na2S04) and evaporated in vacuo leaving 3.36 g. of the title compound. -The NMR spectrum of the product (in CDC13) showed absorptions at 1.45 (s, 3H), 1.68 (s, 3H), 3.32 (m, 2H), 4.50 (s, lH), 5.23 (m, lH), 5.25 (s, 2H) and 7.85 (q, 4H) ppm.

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Claims (23)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a pharmaceutically active compound of the formula (I) or a pharmaceutically-acceptable salt thereof, wherein R6 is hydrogen or an ester-forming residue readily hydrolyzable in vivo, characterized in that it comprises (a) oxidizing a compound of the formula , or wherein R1 is hydrogen, an ester residue readily hydrolysable in vivo or a conventional penicillin carboxy protecting group, followed where necessary by removal of the carboxy protecting group; or (b) for the preparation of those compounds corresponding to formula (I) in which R6 represents a radical selected from the group consisting of 3-phthalidyl, 4-crotonolactonyl, .gamma.-butyrolacton-4-yl, and groups of the formula and wherein R3 and R4 are each hydrogen or alkyl of 1 to 2 carbon atoms, and R5 is alkyl of 1 to 6 carbon atoms, reacting a salt of penicillanic acid l,l-dioxide with 3-phthalidyl chloride, 3-phthalidyl bromide, 4-crotonolacton-4-yl chloride, 4-crotonolacton-4-yl bromide, .gamma.-butyrolacton-4-yl chloride, .gamma.-butyrolacton-4-yl bromide, or a compound of the formula or wherein Q is chloro or bromo, in a reaction-inert solvent, at a temperature in the range from 0 to 100°C.

2. A process for the preparation of a pharmaceutically-active compound of the formula or a pharmaceutically-acceptable salt thereof, wherein R6 is hydrogen or an ester-forming residue readily hydrolyzable in vivo, characterized in that it comprises oxidizing a compound of the formula , or wherein R1 is hydrogen, an ester-forming residue readily hydrolyzable in vivo, or a conventional penicillin carboxy protecting group, followed when necessary by removal of the carboxy protecting group.

3. A process according to claim 2, wherein the starting material is or

4. A process according to claim 2, wherein the starting material is

5. A process according to claim 2, wherein R1 is hydrogen or an ester-forming residue readily hydrolyzable in vivo.

6. A process according to claim 5, wherein the oxidation is carried out using a reagent selected from metal permanganates and organic peroxycarboxylic acids.

7. A process according to claim 6, wherein the oxidation is carried out using a metal permanganate, in a reaction-inert solvent, at a temperature in the range from about -20 to about 50°C.

8. A process according to claim 7, wherein said metal permanganate is potassium permanganate.

9. A process according to claim 8, wherein R1 is hydrogen.

10. A process according to claim 8, wherein R1 is pivaloyloxymethyl.

11. A process according to claim 6, wherein the oxidation is carried out using an organic peroxycarboxylic acid, in a reaction-inert solvent, at a temperature in the range from about -20 to about 50°C.

12. A process according to claim 11, wherein said peroxycarboxylic acid is peracetic acid.

13. A process according to claim 12, wherein the oxidation is carried out in the presence of a manganese salt.

14. A process according to claim 12, wherein R1 is hydrogen.

15. A process according to claim 12, wherein R1 is pivaloyloxymethyl.

16. A process for the preparation of a pharmaceutically active compound of the formula wherein R7 is selected from the group consisting of 3-phthalidyl, 4-crotono-lactonyl, .gamma.-butyrolacton-4-yl, and groups of the formula and wherein R3 and R4 are each hydrogen or alkyl of 1 to 2 carbon atoms, and R5 is alkyl of 1 to 6 carbon atoms, characterized in that it comprises reacting a salt of penicillanic acid 1,1-dioxide with 3-phthalidyl chloride, 3-phthalidyl bromide, 4-crotonolacton-4-yl chloride, 4-crotonolacton-4-yl bromide, .gamma.-butyrolacton-4-yl chloride, .gamma.-butyrolacton-4-yl bromide, or a compound of the formula or wherein Q is chloro or bromo, in a reaction-inert solvent, at a temperature in the range from 0 to 100°C.

17. A process according to claim 16, wherein a salt of penicillanic acid 1,1-dioxide is reacted with a compound of the formula Q.CH2.O.CO.R5.

18. A process according to claim 16, wherein a salt of penicillanic acid 1,1-dioxide is reacted with a compound of the formula Q.CH2.O.CO.C(CH3)3.

19. A process according to claim 16, 17 or 18, wherein said salt is an alkali metal salt.

20. A process according to claim 16, 17 or 18 wherein said salt is the sodium or potassium salt.

21. A process according to claim 16, 17 or 18, wherein said salt is a tertiary amine salt.

22. A process according to claim 16, 17 or 18 wherein said salt is the triethylamine, diisopropylethylamine, N-ethylpiperidine, N,N-dimethylaniline or N-methylmorpholine salt.

23. Compounds of the general formula (I) defined in claim 1, and their pharmaceutically acceptable salts, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.