CA1222951A - Pencillin derivatives and process for preparation of the same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A pharmaceutical composition useful for treating bacterial infections in mammals which comprises (A) a B-lactam antibiotic and (B) a penicillin derivative represented by the following formula

Description

9~1 'i'll iS i llVellt iOII :relc~ec; Lo l~f:'ll i{' i 1. L Ll~
(I('I`iV(ItiVOS ~ Oat-L:iC~ ar to a ~ lc~rnl-lC~`lltiC.-IL
o~itLoll ~IseLul l~or tLeatinCJ b.lCter:i~ Cti()l~S il~
~ nILI15 which comprises sclid peniclllir) ~el-ivativos.
Tl~is ~pplication a divisional application copendincJ
~pplication No. 429,938 filed June 8, 1983.

Of the commercially available antibiotics, lo ~-lactam type antibiotics having a ~-lactam ring, namely penicillins and cephalosporins, are best known and frequently used. Although widely used as useful chemotherapeutic drugs, the ~-lactam type antibiotics can not achieve satisfactory effects against some types of microorganisms because of resistance of the micro-organism to the ~-lactam type antibiotics. The resistance thereof are usually attributable to B-lactamase produced by the microorganism. The ~-lactamase is an enzyme which acts to cleave the ~-lactam ring of the ~-lactam type antibiotic, thereby causing the antibiotic to lose its antimicrobial activity. For this reason, the action o ~-lactamase must be eliminated or inhibited so as to enable the ~-lactam type antibiotic to produce satis-factory effects. The elimination or inhibition of the ~-lactamase activity can be achieved by ~-lactamase inhibitors, which are used conjointly with the ~-lactam type antibiotic to increase the antimicrobial activity of the antibiotic.

-lZZ295~

The present invention provides pha~ceutical coml~o~itior~ having excellent ~-lactam~se inhibitory ~ction, and in particular compositions combined with ~-lactam type antibiotics, which there is an increase in the anti-bacterial activity of the antibiotics.

In copending application No. 429,938 there are disclosed in claim 1 penicillin derivatives represented by the formula N N
Cllz N ~ ~1 ~ ~ -CI~ COOR ( I ) O 1 `COOR3 wllerein Rl and R2 are each the same or differetlt and reyresent hydrogen, Cl 18 alkyl, mononitro-substituted ' ben~yl or group for forming a pharmaceutically acceptable salt an~ R3 is selected from the group consistin~ of : , hydrogen, Cl_6 alkyl, C2_7 alkoxymethyl, C3_8 alkyl-carbonyloxymethyl, C4 9 alkylcarbonyloxyethyl, (C5 7 ' ~ ' .."~

''.,- ~ -' -': ~

''~ `- ;

122295~

cyclo~.lllcyl)(:.lrbollyloxyllletllyl, Cg_14 be~lzylcarL~ yloxy-alkyl, C3 ~ alkoxycarbonyl~lethyl, C4 9 alkoxycarbonyl-etl~yl, yLltllalidyl, crotonolacton-4-yl, y-butyrolacton-~-yl, llalogenated Cl 6 alkyl substituted with l to 3 halogen atoms, Cl 6 alkoxy- or nitro-su~stituted or ullsu~stituted benzyl, benzhydryl, tetrahydropyranyl, dimetllylaminoethyl, dimethylchlorosilyl, trichlorosilyl, (5-su~stituted Cl 6 alkyl or phenyl or unsubstituted-2-oxo-1,3-dioxoden-4-yl)methyl, C8 13 benzoyloxyalkyl and lo group ~or forming a pharmaceutically acceptable salt., The penicillin derivatives of the copending application are all novel compounds and have ~-lactamase inhibitory properties, hence useful as ~-lactamase inhibitory agents.
The present invention provides phanmaceutical compositions having excellent ~-lactamase inhibitory action, and in particular compositions combined with ~-lactam type antibiotics, which there is an increase in the anti-bacterial activity of the antibiotics.
It has thus been found that the penicillin derivatives of the copending application when used in combination with a known ~-lactam type antibiotic, can increase the antimicrobial activity of the ~-lactam type antibiotic.
Examples of antibiotics which can be used conjointly with the compounds of the copending application are ~-lactam antibiotics which exhibit antibacterial action against gram-positive or gram-negative bacteria and which --3cl--12~2951 comlllollly used p~nicillins such as ampicillin, amo~:icillill, hetacillin, ciclacillin, mecillinam, carbenicillin, sulbenicillin, tricarcillin, 12~951 piperacillin, apalcillin, methicillin, mezlocillin and salts thereof; esters of penicillins such as bacampicillin, carindacillin, talampicillin, carfecillin and pivmecillinam; cephalosporins such as cephaloridine, cephalothin, cephapirin, cephacetrile, cefazolin, cephalexin, cefradine, cefotiam, cefal~ndole, cefuroxime, cefoxitin, cefmetazole, cefsulodin, cefoperazone, cefotaxime, ceftizoxime, cefmenoxime, latamoxef, cefaclor, cefroxadine, cefatrizine, cefadroxil, cephaloglycin, and lo salts thereof. The R-lactam antibiotics are usually used in an amount of about 0.1 to about 10 parts by weight, preferably about 0.2 to about 5 parts by weight, per part by weight of the compound of the invention.
Examples of the groups represented by Rl and lS R2 in the formula (I) include; Cl 18 alkyl such as methyl, ethyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, octadecyl and like straight- or branched-chain alkyl; and mononitro-substituted benzyl such as o-nitrobenzyl, p-nitrobenzyl and the like.
Examples of the groups represented by R3 in the formula (I) include; Cl 6 alkyl such as methyl, ethyl, propyl, isopropyl, tert-butyl, pentyl, hexyl and like straight- or branched-chain alkyl; C2_7 alkoxymethyl such as methoxymethyl, ethoxymethyl, propyloxymethyl, isoyropyloxymethyl, butoxymethyl and hexyloxymethyl;
C3 8 alkylcarbonyloxymethyl such as methylcarbonyloxy-methyl, ethylcarbonyloxymethyl, butylcarbonyloxymethyl and hexylcarbonyloxymethyl; C4 9 alkylcarbonyloxyethyl such as methylcarbonyloxyethyl, ethylcarbonyloxyethyl, butylcarbonyloxyethyl and pivaloyloxyethyl; (C5 7 cycloalkyl)carbonyloxymethyl such as cyclopentyl-carbonyloxymethyl, cyclohexylcarbonyloxymethyl and cycloheptylcarbonyloxymethyl; Cg 14 benzylcarbonyloxy-alkyl such as benzylcarbonyloxymethyl, benzylcarbonyloxy-ethyl, benzylcarbonyloxypropyl and benzylcarbonyloxybutyl;
C3 8 alkoxycarbonylmethyl such as methoxycarbonylmethyl, ethoxycarbonylmethyl, propyloxycarbonylmethyl and hexyloxycarbonylmethyl; C4 9 alkoxycarbonylethyl such as methoxycarbonylethyl, ethoxycarbonylethyl, propyloxy-~: carbonylethyl, butoxycarbonylethyl and hexyloxycarbonyl-~: ethyl; halogenated Cl 6 alkyl substituted with 1 to 3 halogen atoms such as chloromethyl, 2,2-dibromoethyl and trichloroethyl; Cl 6 alkoxy- or nitro-substituted / 20 or unsubstituted benzyl such as p-methoxybenzyl, p-ethoxybenzyl, o-nitrobenzyl and p-nitrobenzyl;
(5-substituted C1 6 alkyl or phenyl or unsubstituted-

2-oxo-1,3-dioxoden-4-yl)methyl such as (2-oxo-1,3-dioxoden-4-yl)methyl, (5-methyl-2-oxo-1,3-dioxoden-4-yl)-methyl and (5-phenyl-2-oxo-1,3-dioxoden-4-yl)methyl;
~, : ' . - .
.~ . .
.
- . .
,: .
~ . ' ~ . .
- ~

lZ2~951 C8 13 ~enzoyloxyalkyl such as benzoylox~nethyl, benzoyloxyethyl, benzoyloxypropyl and benzoyloxybutyl;
etc. The ester residues represented by R3 in the formula (I) include both carboxyl-protecting groups S acceptable in the synthesis of penicillin compounds and pharmaceutically acceptable ester residues.
A pharmaceutically acceptable ester having such residue is an ester which is easily hydrolyzed in vivo and which is a non-poisonous ester capable of rapidly decomposing in the blood or tissue of humans, thereby producing the corresponding acid of the formula ~I) in which R3 is hydrogen atom. Generally in the synthesis of penicillin compounds, ester-protecting groups are used in the art to protect penicillin carboxyl groups or other carboxyl groups. While it is difficult to determine which ester-protecting group should be used, consideration is usually given to select esters in which the protecting group per se is sufficiently stable in the reaction and which does not permit cleavage of the ~-lactam ring in removal of the ester-protecting groups. Most commonly used as such ester-protecting groups are p-nitrobenzyl group, benzhydryl group, trichloroethyl group, trichlorosilyl group, tetrahydropyranyl group, etc.
Examples of the pharmaceutically acceptable ester groups are phthalidyl, crotonolacton-4-yl, y-butyro-' ' ' . `

~22Z95 lac~on-4-yl, (2-oxo-1,3-dioxoden-4-yl)methyl, etc.
Examples of the group for forming a pharma-ceutically acceptable salt represented by Rl, R2 and R3 in the ~ormula (I) include; sodium, potassium, lithium, Or like alkali metal atoms; calcium, ma~nesium or like alkaline earth metal atoms; cyclohexylamine, trimethyl-amine, diethanolamine or like the organic amine residues;
alginine, lysine or like basic amino acid residues;
ammonium residues, etc.
The penicillin derivatives having the formula (I) can be prepared according to the reaction equation given below.

O O

CH3 3tep (A) O `COOR4 (II) N N

CH2-N ~ \ COOR5 ?
CH3 ooR6 Step (B) O I ~COOR4 (I-a) ~f; `
- 8 - 12X295~

N = N

CH 2 - N~CoO Rl ~ CH COOR2 0 "COOR

(I) In the foregoing formulae, Rl, R2 and R3 are as defined above, R4 represents penicillin carboxyl-protecting group and R5 and R6 represent hydrogen, Cl 8 alkyl or mononitro-substituted benzyl.
Examples of the penicillin carboxyl protecting group expressed by R4 include known groups such as those described in Japanese Unexamined Patent Publication No.81380/1974 and H.E. Flynn, "Cephalosporins and Penicillins, Chemistry and Biology" (published in 1972 by Acade~ic Press). Specific examples thereof are ethyl, propyl, tert-butyl, trichloroethyl and like substituted or unsubstituted alkyl groups; benzyl, diphenyl methyl, p-nitrobenzyl and like substituted or unsubstituted aralkyl groups; acetoxymethyl, benzyloxymethyl and like acyloxyalky~ groups, methoxy-methyl and like alkoxyalkyl groups; and other groups such as tetrahydropyranyl, dimethylaminoethyl, dimethyl-dichlorosilyl, trichlorosilyl and like groups.
The steps (A) and (B) of the foregoing process ,: .

.

9 12~29~

will be described below in detail.
Step (A) A penicillanic acid derivative of the formula (II) is reacted with an acetylene derivative of the formula (III) to provide a compound of the formula (I-a).
The reaction is conducted in a suitable solvent by reacting a known penicillanic acid derivative of the formula (~I) with a known acetylene derivative of the formula (III) in an amount of about 1 to about 50 moles, preferably about 1 to about 10 moles, per mole of the derivative of the formula (II).
The solvents useful in the reaction are not particularly limited and include any of those which do not adversely affect the reaction. Specific examples of the solvents are benzene, toluene, xylene and like aromatic hydrocarbons, acetone and like polar organic solvents; etc. These solvents are used singly or in mixture. The reaction proceeds usually at a temperature of between about 50C and a boiling point of the solvent or at a temperature of less than 200C
in a sealed reactor, and goes to completion in about 2 to about 72 hours.
Depending upon the kind of the penicillin carboxyl-protecting group represented by R4, the com-pounds of the formula (I-a) obtained in step (A) may be f~ `',`.
122295l esters oE the penicillin derivatives of the present invention having the formula (I). The compaunds of the formula (I-a) are preferably subjected to de-esterification to form a derivative of the ormula (I) in the form of free acid which, in turn, is converted into a pharmaceutically acceptable salt or ester thereof as in the following step (B). The c~mpound of the formula (I-a) can also be made into an ester of the formula (I) by the conventional ester interchange reaction in the step (B).
Step (B) The compound of the formula (I-a) is subjected to de-esterification without or after isolation from the reaction mixture obtained in step (A), whereby a penicillin derivative of the formula (I) is obtained in the form of free acid.
. , As the de-esterification method, reduction, hydrolysis, treatment with an acid and like method can be employed for converting the carboxyl-protecting group to carboxyl group. For example, if the carboxyl-protecting group is an active ester, the reaction frequently proceeds.
with ease under mild hydrolysis conditions or by merely bringing the ester into contact with water. The reduction method is employed when the carboxyl-protecting group is trichloroethylbenzyl, p-nitrobenzyl, diphenyl methyl or . ~?

122i~95~

tlle lilce. Treatment with an acid is a~op~e~ wlle[l the carboxyl-protecting group is 4-methoxybenzyl, tert-butyl, trityl, diphenylmethyl, methoxymethyl, tetrahydropyranyl or the like.
The reduction can be conducted by treating the ester of the formula (I-a) with a mixture of (a) zinc, zinc-amalgam or like metal and/or chromium chloride, chromium acetate or like chromium salt and (b) formic acid, acetic acid or like acid. Alternatively, the reduction can be conducted with use of a catalyst in hydrogen atomosphere in a solvent. Examples of the catalysts are platinum, platinum oxide, palladium, palladium oxide, palladium-barium sulfate, palladium-calcium carbonate, palladium-carbon, nickel oxide, Raney-nickel, etc. The solvents are not particularly limited so far as they do not adversely affect the reaction, and include methanol, ethanol and like alcohols; tetrahydrofuran, dioxane and like ethers;
ethyl acetate and like esters; acetic acid and like fatty acids; and a mixture of these organic solvents and water.
The acids useful for eliminating the carboxyl-protecting group of the ester of the formula (I-a) are formic acid, acetic acid and like lower fatty acids;
trichloroacetic acid, trifluoroacetic acid and like ' .
~ .

1 2 - 122295~

~rihalogenate~ acetic acids; hydrochloric acid, hydro-fluoric acid and like hydrohalogenic acidsi p-toluene-sulfonic acid, trifluoromethane-sulfonic acid and like organic sulfonic acids; and a mixture of these.
In this reaction, when the acid used is in a liquid state and acts also as a solvent, it is not necessary to use other solvents. However, dimethylformamide, dichloromethane, chloroform, tetrahydrofuran, acetone and like solvents which do not adversely affect the reaction may be used.
The penicillin derivative of the present invention having the formula (I) in the form of free acid can be transformed by the salt-forming reaction or esterification commonly employed in the art into a pharmaceutically acceptable salt or ester as contemplated.
If the ester residue is, for example,

3-phthalidyl, crotonolacton-4-yl, y-butyrolacton-4-yl - or like group, the penicillin derivative of the formula-(I-a) can be alkylated by using 3-halogenated phthalide, ~0 4-halogenated crotonolactone, 4-halogenated-y-butyrolactone or the like. Suitable halogens of the foregoing halides include chlorine, bromine, iodine, etc. The reaction is carried out by dissolving the salt of the penicillin derivative of the formula (I-a) -in N,N-dimethylformamide or like suitable polar organic ,'' ' . ' .
~ , `

t - 13- 12~9Sl solven~ and ad~itlg an approximately e(lui~llolecular amount of a halide to the solution. The reaction tc~nperature ranges from about 0 to about 100C, preferably from about 15 to about 35C. Suitable salts of the penicillin derivative to be used in the esterification are salts of sodium, potassium or like alkali metals; salts of tri-ethylamine, ethyldiisopropylamine, N-ethylpiperidine, N,N-dimethylaniline, N-methylmorpholine or like tertiary amines, etc. After completion of the reaction, the contemplated product can be easily separated by the con-ventional method and also can be purified, when required, by recrystallization, thin layer chromatography, column chromatography or like method.

The compound of the formula (II) to be used as the starting material in the step (A) is a novel compound undisclosed in literature and can be synthesized by the method described in applicants Canadian Patent Application No. 426,531 filed April 22, 1983. The disclosed method comprises the steps of reacting a metal azide with a known derivative of penicillanic acid of the formula CH3 (V) N ~
O~ "COOR

`''' 122~95~

wllerein X represents chlorine atom or bromine atom and R4 represents penicillin carboxyl-protecting group, oxydizing the reaction mixture and subjecting the resulting compound to de-esterification.
The foregoing method will be described below in detail. The reaction between the compound of the formula (V) and the metal azide is conducted in a suitable solvent by using the metal azide in an amount of about 1 to about 50 moles, preferably about 1 to about 10 moles, per mole of the compound of the formula (V). Examples of the metal azides which can be used include those commonly used, such as sodium azide, potassium azide and like azides of alkali metals, and barium azide and like azides of alkaline earth metals.
Useful solvents are not particularly limited as far as they do not adversely affect the reaction. Examples of useful solvents are dimethylformamide, ethyl acetate, acetone, dichloromethane, tetrahydrofuran, dioxane, methanol, ethanol and like organic solvents. These organic solvents can be used singly or in mixtures.
Also a mixture of such solvent and water is usable.
The reaction proceeds at a temperature of usually about -20 to about 100C, preferably about 0 to about 100C.
The resulting product can be used in subsequent oxidation without isolation, or alternatively after isolation and purification by a conventional method. The oxidation subsequent to the azide-forming reaction is conducted by using an oxidizing agent commonly employed such as permanganic acid, periodic acid, peracetic acid, performic acid, trifluoroperacetic acid, perbenzoic acid, m-chloroperbenzoic acid, hydrogen peroxide, etc.
The oxidizing agent can be used in large excess, and may be employed preferably in an amount of about 1 to about 2 moles per mole of the starting compound. The oxidation is carried out usually in a suitable solvent. Useful solvents include any of those which do not adversely affect the oxidation reaction such as chloroform, pyridine, tetrahydrofuran, dioxane, methylene chloride, carbon tetrachloride, acetic acid, formic acid, dimethyl-formamide, water, etc. The oxidation is performed ata temperature which is not particularly limited but generally ranges from room temperature to cooling tem-perature, preferably about 0 to about 30C.
The compound thus obtained is subjected to de-esterification whereby the compound of the formula (II) can be produced. The de-esterification is effected under the same conditions as shown in the reaction scheme of the step (B). The process for preparing the compound of the formula (II) is described in detail in reference 25 e:~amples to be set forth later.

_ 16 -The penicillin derivative of fomlula is moxed with a suitable antibiotic subst~nce to form a prepara~ion which is orally or parenterally administered. Alternatively, the present compound and a suitable antibiotic can be separately administered.
Thus the derivatives of the formula (I) can be used for treating infectious disease of human beings and other animals.
The composition of the present invention may be made into tablets, pills, capsules, granules, powders, syrups, lozenges, solutions, suspensions, etc. for oral administration and aqueous, suspending or water-soluble preparations for intravenous, subcutaneous or intramuscular injections.
Carriers useful in formulating the preparations are those commonly used pharmaceutically acceptable non-toxic carriers such as gelatin, lactose, starch, magnesium stearate, talc, vegetable oil, animal oil, polyalkylene glycol, etc. The carrier may be used with other additives such as diluents, binders, buffer agents, preservatives, glazes, disintegrators, coating agents, etc.
The daily dose of the preparation can be appropriately determined and is not particularly limited.
Preferably the daily dose is such that the total amount of the present compound and ~-lactam antibiotic is about t. ~ t 12~29Sl 1 to a~out 200 mg/Kg body weight for oral admillistration and about 1 to about 100 mg/Kg body weight for parenteral administration.
The present invention will be described below in more detail with reference to examples given below.
Reference Example 1 Preparation of benzhydryl 2~-azidomethyl-2-methylpenam-3a-carboxylate An aqueous solution of 5.00 g of sodium azide and 53 ml of water was added to a solution of benzhydryl 2~-chloromethyl-2~-methylpenam-3a-carboxylate (5.13 g) in dimethylformamide (155 ml). The mixture was stirred at room temperature for 4 hours. The resulting reaction mixture was poured into cooled water and the mixture was - 15 extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried over magnesium sulfate and concentrated to provide 4.87 g of the contemplated product as oil in 93 % yield.
Infrared absorption spectrumi tNuiol)(a trademark) v max (cm 1): 2120, 1812, 1765 Nuclear magnetic resonance spectrum (CDC13) (ppm) : 1.30 (3H, s), 3.25 (2H, m), 3.42 (lH, d), 3.63 (lH, d), -

4.75 (lH, s), 4.76 (lH, m), 7.00 (lH, s), 7.40 (10~, s) 12~2951 ReEerence ~xample 2 Preparation of benzhydryl 2R-azidomethyl-2a-methylpenam-3c~-carboxylate-1,l-dioxide ~ 7.03 g quantity of benzhydryl 2~-azidomethyl-

5 2c~-methylpenam-3a-carboxylate was dissolved in 40 ml of water and 240 ml of of acetic acid. To the solution was added 6.02 g of potassium permanganate over a period of more than 1 hour. The mixture was stirred at room temperature for 2.5 hours. Iced water was added to the 10 reaction mixture to form a precipitate which was filtered off and washed with water. The resulting solids were dissolved in ethyl acetate and the solution was washed with an aqueous solution of sodium hydrogencarbonate, dried over magnesium sulfate and concentrated, giving 15 5.48 g of the contemplated compound in 72 ~ yield.
Infrared absorption spectrum (nujol) v max (cm 1): 2120, 1812, 1765 Nuclear magnetic resonance spectrum (CDCl) ~ (ppm): 1.18 (3H, s), 3.50 (2H, d), 3.72 (lH, d), 3.93 (lH, d), 4.60 (lH, m), 4.65 (lH, s), 7.00 (lH, s), 7.36 (lOH, s) Reference Example 3 Preparation of p-nitrobenzyl 2~-azidomethyl-2a-25 methylpenam-3-carboxylate .

- 19 - i2 ~ 9~

The procedure of Reference Example 1 was repeated with the exception of using as the starting material p-nitrobenzyl 2~-chloromethyl-2a-methylpenam-3~-carboxylate, affording the above contemplated compound.
Infrared absorption spectrum (KBr) ~ max (cm 1): 2120, 1798, 1760 Nuclear magnetic resonance spectrum (CDC13) (ppm): 1.40 (3H, s), 3.12 (lH, dd), 3.50 (2H, s), 3.62 (lH, dd), 4.83 (lH, s), 5.29 (2H, s), 5.36 (lH, dd), 7.56 (2H, d), 8.26 (2H, d) Reference Example 4 Preparation of p-nitrobenzyl 2~-azidomethyl-2a-methylpenam-3a-carboxylate-1,1-dioxide The procedure of Reference Example 2 was followed with the exception of using as the starting material p-nitrobenzyl 2~-azidomethyl-2a-methylpenam-3a-carboxylate, giving the above contemplated compound.
0 Infrared absorption spectrum (KBr) v max (cm 1): 2120, 1770 Nuclear magnetic resonance spectrum (CDC13) (ppm) : 1.42 (3H, s), 3.45-3.60 (2H, m), 3.75 (lH, d), 3.96 (lH, d), 4.56-4.75 (lH, m), 4.64 (lH, s), ~;?

- 20 _ ~222 951 5.33 (2H, s), 7.56 (211, d), 8.26 (2H, d) Example 1 Preparation of benzhydryl 2~-(4,5-dimethoxy-carbonyl-1,2,3-triazol-1-yl)methyl-2-methylpenam-3a-carboxylate-l,l-dioxide~ (Compound 1) A mixture of 0.870 g of benzhydryl 2~-azido-methyl-2a-methylpenam-3-carboxylate-1,1-dioxide and 0.618 g of dimethylacetylene-dicarboxylate was stirred in 15 ml of benzene with reflux in nitrogen atmosphere for 18 hours. The solvent was removed by distillation.
The residue was chromatographed on silica gel column - with ethyl acetate-chloroform (1:3) as eluate, giving 0.495 g of the contemplated product as light yellow crystals in 44 % yield which melts at 75 to 77C.
Infrared absorption spectrum (KBr) v max (cm 1): 1800, 1735 Nuclear magnetic resonance spectrum (CDC13) ~ (ppm) : 1.20 (3H, s), 3.48 (2H, t), 3.97 (3H, s), 3.98 (3H, s), 4.59 (lH, m), 4.95 (lH, s), 5.26 (2H, s), 6.97 (lH, s), 7.36 (lOH, s) ; -.~

~, , ~ . .

; ' : ' -- . ,:
.. :

~ - 21 _ 12X2 9 51 I

Exam~e 2 1 Preparation of sodium 2~-(4,5-dimethoxycarbonyl-1,2,3-I triazol-l-yl)methyl-2a-methylpenam-3~-carboxylate-l,l-dioxide (Compound 2) Hydrogenation was conducted at ordinary pressure and room temperature by using 100 ml of tetrahydrofuran, 100 ml of water, 116 mg of benzhydryl ! 2~-(4,5-dimethoxycarbonyl-1,2,3-triazol-1-yl)-methyl-2a-methylpenam-3~-carboxylate-1,1-dioxide, 58 mg of 10 % palladium charcoal and 17 mg of sodium bicarbonate.
After the absorption of the hydrogen was completed, the reaction mixture was filtered and the tetrahydrofuran was removed from the filtrate at reduced pressure by distillation. The residue was washed with chloroform and the aqueous solution was concentrated at reduced pressure. The aqueous mixture was washed with chloro-form and was chromatographed with a column of high porous polymer and eluted with water-10 % acetone in water (gradient). The liquid thus obtained was freeze-dried to obtain 53 mg of the contemplated product as white powder in 60 % yield. The white powder decomposed at over 165C. -Infrared absorption spectrum (KBr) v max (cm 1): 1785, 1735, 1630 2 2 - lZX~951 Nuclear magnetic resonance spectrum ~D20) (ppm) : 1.41 (3H, s), 3.40 (lH, ~d), 3.80 (lH, dd), 3.98 (3H, s), 4.05 (3H, s), 4.51 (lH, s) 5.03 (lH, dd), 5.48 (2~1, d) Example 3 Preparation of p-nitrobenzyl 2B-(4,5-diethoxycarbonyl-1,2,3-triazol-1-yl)methyl-2a-methylpenam-3~-carboxylate-l,l-dioxide~(Compound 3) The same procedure as in Example 1 was repeated with the exception of using as the starting material p-nitrobenzyl 2R-azidomethyl-2a-methylpenam-3a-carboxylate-l,l-dioxide, producing the above product.
Infrared absorption spectrum (KBr) lS v max (cm 1): 1805, 1735 Nuclear magnetic resonance spectrum (CDC13) (ppm) : 1.33-1.48 (9H, m), 3.30-3.72 (2H, m), 4.42 (2H, q), 4.45 (2H, q), 4.64-4.71 (lH, m), 5.02 (lH, s), 5.22-5.40 (4H, m), 7.55 (2H, d), 8.24 (2H, d) Example 4 Preparation of 2~-(4,5-diethoxycarbonyl-1,2,3-triazol-l-yl~methyl-2a-methylpenam-3a-carboxylic acid-l,l-dioxide (Compound 4) and sodium salt thereof (Compound 5) i2~951 _ 23 -A 3.5 g quantity of p-nitrobenzyl ~-(4,5-diethoxycarbonyl-1,2,3-triazol-1-yl)methyl-2~-methylpenam-3a-carboxylate-1,1-dioxide was hydrogenated at an initial pressure o~ 3 atm. by using 1.1 g of sodium hydrogencarbonate and 0.6 g of 10 % palladium charcoal in 90 ml of ethyl acetate and ~0 ml of water.
In one hour, the reaction mixture was sedimented and the aqueous layer was separated. The aqueous layer was washed twice with ether and its pH was adjusted to 1.7 with diluted hydrochloric acid. The aqueous solution was extracted with ethyl acetate and dried over magnesium sulfate. The solvent was removed at reduced pressure by distillation, whereby 2.2 g of 2~-(4,5-diethoxycarbonyl-1,2,3-triazol-1-yl)methyl-2a-methylpenam-3a-carboxylic acid in the form of amorphous crystals in 83 % yield.
A 170 mg quantity of 2~-(4,5-diethoxycarbonyl-1,2,3-triazol-1-yl)methyl-2a-methylpenam-3~-carboxylic acid was dissolved in a solution of 84 mg g of sodium hydrogencarbonate in 10 ml of water.
solution was purified with an MCI gel (a trademark for a product of Mitsubishi Kasei Co., Ltd., Japan). The liquid thus obtained was freeze-dried, giving 130 mg of amorphous product in 73 % yield which~melts at 155 to 160C (~ecomposition). - -- 24 - i2~951 rared a~sorption spectrum (KBr) v max (cm ): 1785, 1735, 1630 Nuclear magnetic resonance spectrum (D20) ~ (ppm) : 1.32-1.49 (9H, m), 3.34-3.82 (2H, m), 4.35-4.65 (5H, m), 5.01-5.07 (lH, m), 5.47 (2H, s) 5.03 (lH, dd), 5.48 (2H, d) Example 5 10 Preparation of 2~-(4,5-dimethoxycarbonyl-1,2,3-triazol-l-yl)methyl-2a-methylpenam-3a-carboxylic acid-l,l-dioxide (Compound 6) Hydrogenation was carried out in 100 ml of tetrahydrofuran and 100 ml of water at room temperature by using 116 mg of benzhydryl 2~-(4,5-dimethoxycarbonyl-1,2,3-triazol -1-yl)methyl-2a-methylpenam-3a-carboxylate-l,l-dioxide, 58 mg of 10 % palladium charcoal and 17 mg of sodium hydrogencarbonate, After completion of absorption of hydrogen, the reaction liquid was filtered and the tetrahydrofuran was distilled off from the filtrate at r~duced pressure. The residue was washed with chloroform. After adjusting its pH to 1.5 to 1.7 with diluted hydrochloric acid, the aqueous solution was washed with chloroform, extracted with ethyl acetate and dried over magnesium sulfate. The ethyl acetate .

12~951 2s -was distilled off at reduced pressure, and tlle residue was dissolved in water. The solution was freeze-dried to provide 83 mg of amorphous product in 76 ~ yield which has a melting point of 135 to 145C tdecomposition).
Infrared absorption spectrum tKBr) v max tcm 1): 1805, 1735 Nuclear magnetic resonance spectrum tCDC13 + DMS0-d6) tppm): 1.55 t3H, s), 3.2-3.6 t2~1, m), 3.96 (3H, s), 4.02 (3H, s), 4.68 (lH, s), 4.6-4.8 (lH, m), 5.35 (2H, s) Example 6 Preparation of chloromethyl 2~-(4,5-dimethoxycarbonyl-1,2,3-triazol -1-yl)methyl-2a-methylpenam-3~-carboxylate-l,l-dioxide tCompound 7) A 1.25 g quantity of sodium hydrogencarbonate and 0.133 g of tetrabutylammonium hydrogensulfate were added with stirring at less than 10C to 1.61 g of 2~-t4,5-dimethoxycarbonyl-1,2,3-triazol -1-yl)methyl-2a-methylpenam-3-carboxylic acid-l,l-dioxide, 8 ml of dichloromethane and 8 ml of water. To the mixture was dropwise added at the same temperature 0.74 g of chloromethyl chlorosulfonate. The mixture was stirred at room temperature for 30 minutes. The organic layer was separated, washed once with water and dried over 122295~

- magnesium sulfate. The solvent was removed by distil-lation at reduced pressure to obtain as the residue 1.5 g of amorphous product in 83 % yield.
Infrared absorption spectrum (KBr) v max (cm~l): 1805, 1735 Nuclear magnetic resonance spectrum (CDC13) (ppm) : 1.55 (3H, s), 3.2-3.8 (2H, m), 3.99 (3H, s), 4.03 (3H, s~, 4.6-4.8 (lH, m), 5.01 (lH, s), 5.32 (2H, d), 5.62 (lH, d), 5.81 (lH, d) Example 7 Preparation of iodomethyl 2~-(4,5-dimethoxycarbonyl-1,2,3-triazol -1-yl)methyl-2-methylpenam-3-carboxylate-l,l-dioxide (Compound 8) A 1.05 g quantity of chloromethyl 2~-(4,5-dimethoxycarbonyl-1,2,3-triazol -1-yl)methyl-2-methyl-penam-3a-carboxylate-1,1-dioxide, 0.52 g of sodium iodide and 1.8 ml of acetone were stirred at room temperature for 18 hours. To the reaction mixture was added 1.5 ml of water and the resulting mixture was adjusted to a pH
- of 7 to 8 with an aqueous solution of sodium hydrogen-carbonate. Thereto was added 1.5 ml of water and the mixture was decolorized with an aqueous solution of 0.5 M sodium thiosulfate. The mixture was extracted .: ,~ ' ' .
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witl~ dicloromethane, washed with water and dried over magnesium sulfate. The solvent was distilled off at reduced pressure to produce 1.1 g of amorphous product in 78 % yield.
Infrared absorption spectrum (KBr) v max (cm 1): 1800, 1730 Nuclear magnetic resonance spectrum (CDC13) (ppm): 1.56 (3H, s), 3.2-3.8 (2H, m), 3.99 (3H, s), 4.03 (3H, s), 4.6-4.8 (lH, m), 4.97 (lH, s), 5.30 (2H, d`), 5.85 (iH, d), 5.96 (lH, d) Example 8 Preparation of p-nitrobenzyl 2R-(4,5-dimethoxycarbonyl-1, 2, 3-triazol -1-yl)methyl-2-methylpenam-3a-carboxylate-l,l-dioxide (Compound 9) A 3.5 g quantity of p-nitrobenzyl 2~-azido-methyl-2~-methylpenam-3-carboxylate-1,1-dioxide and 4.8 g of dimethylacetylenedicarboxylate in 80 ml of absolute benzene were refluxed under nitrogen atmosphere for 18 hours. The solvent was distilled off at reduced pressure to provide 4.7 g of the contemplated compound.
Infrared absorption spectrum (KBr) v max (cm 1): 1~05, 1735 ~'~
, :1~22951 _ 28 -Nuclear magnetic resonance spectrum (CDC13) (ppm) : 1.43 (3H, s), 3.2-3.4 (2~1, m), 3.96 (3H, s), 3.99 (3~1, s), 4.64-4.76 (lH, m), 4.98 (lH, s), 5.04-5.44 (4H, m), 7.56 (2H, d), 8.23 (2H, d~
Example 9 Preparation of : p-nitrobenzyl 2~-(4,5-di-n-butoxy-carbonyl-1,2,3-triazol -1-yl)methyl-2-methylpenam-3a-carboxylate-l,l-dioxide (Compound 10); p-nitrobenzyl 2~-(4,5-di-isobutoxycarbonyl-1,2,3-triazol -l-yl)methyl-2a-methylpenam-3a-carboxylate-1,1-dioxide (Compound 11);
p-nitrobenzyl 2~-(4,5-di-n-tetradecyloxycarbonyl-1,2,3-triazol -l-yl)methyl-2a-methylpenam-3a-carboxylate-1,1-dioxide (Compound 12); p-nitrobenzyl 2~-(4,5-di-n-octadecyloxycarbonyl-1,2,3-triazol -1-yl)methyl-2a-methylpenam-3-carboxylate-1,1-dioxide (Compound 13), p-nitrobenzyl 2~-[4,5-di-(p-nitrobenzyloxy)carbonyl-1,2,3-triazol -1-yl]methyl-2a-methylpenam-3a-carboxylate-l,l-dioxide (Compound 14).
The foregoing compounds were prepared in the -same manner as in Example 1.
Compound 10 Infrared absorption spectrum (NaCl) v max (cm 1): 1805, 1730 ~ -' ,d- ';

12Z295~
_ 29 -Nuclear magnetic resonance spectrum (CDC13) (ppm) : 0.96 (6H, t), 1.43 (3EI, s), 1.2-2.0 (8H, m), 3.3-3.7 (2H, m), 4.2-4.5 (4H, m), 4.6-4.7 (lH, m), 5.02 (lH, s), 5.0-5.4 (4~S, m), 7.54 (2H, d), 8.24 (2H, d) Compound 11 Infrared absorption spectrum (NaCl) v max (cm 1): 1805, 1735 Nuclear magnetic resonance spectrum (CDC13) ~PPm? : 0.98 (6H, d), 1.00 (6H, d), 1.43 (3H, s), 1.8-2.3 (2H, m), 3.3-3.7 (2H, m), 4.15 (4ES, d), 4.6-4.7 (lH1 m), 5~02 (lH, s), 5.0-5.4 (4H, m), 7.55 (2H, d), 8.24 (2H, d) Compound 12 Infrared absorption spectrum (KBr) ~ max (cm 1): 1800, 1730 Nuclear magnetic resonance spectrum (CDC13) (ppm) : 0.70-1.90 (57H m), 3.44-3.60 ~2H,m), 4.22-4.45 (4H, m), 4.58-4.70 (lH, m), 5.03 (lH, s), 4.95-5.40 (4H, m), 7.54 (2H, d), ` 8.24 (2H, d) ... . .

~, '. .
:

Compound 13 Infrared absorption spectrum (KBr) v max (cm ): 1800, 1730 Nuclear magnetic resonance spectrum (CDC13) ~ (ppm) : 0.70-1.90 (73H m), 3.44-3.60 (2H,m), 4.22-4.45 (4H, m), 4.54-4.68 (lH, m), 5.03 (lH, s), 4.80-5.40 (4H, m), 7.53 (2H, d), 8.24 (2H, d) Compound 14 Infrared absorption spectrum (KBr) ~- v max (cm l): 1800, 1735 Nuclear magnetic resonance spectrum (CDC13) ~ (ppm) : 1.46 (3H, s), 3.42-3.56 (2H, m), 4.58-4.70 (lH, m), 4.88 (lH, s), 5.31 (4H, s), 5.46 (4H, s), 7.42-7.66 (6H, m), 8.05-8.30 (6H, m) ~` 20 Example 10 ~ Preparation of tri-sodium 2~-(4,5-dicarboxy-1,2,3-`~ triazol -l-yl)methyl-2-methylpenam-3a-carboxylate-l,l-dioxide (Compound 15) The above compound with a melting point of ;~ 25 over 203C (decomposition) was produced in the same ; manner as in Example 4 from Compound 14.
. ",. .
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1;~ 5~

Infrared absorp~ion spectrum (KBr) v max (cm 1): 1780, 1720 Nuclear magnetic resonance spectrum (D20) ~ (ppm) : 1.42 (3H, s), 3.25-3.82 (2H, m), 4.50 (lH, s), 4.95-5.04 (lH, m) 5.52 (2H, dd) The compounds of the present invention obtained above were checked for pharmacological activity in the followin~ manner.
(1) ~-lactamase inhibitory activity The compounds of the present invention obtained in the examples were tested by p~ Stat method (Journal of Pharmaceutical Science, Vol. 61, No.10, pp 1954 to 1958, published in 1972) for inhibitory activity against penicillinase (~-lactamase) from Bacillus SP.
The .test results revealed that the Compound 2 had a 50 %-~-lactamase inhibitory concentration (IC50) of 3 X 10 7M and Compound 15 had an IC50 f 2.3 x 10 6M.
The other derivatives obtained in the other examples were found to have similar values in IC50.
(2) Antibacterial activity (synergistic effects attainable by the combined use of the present `
compounds and ampicillin) The compounds of the present invention and ampicillin, each singly used, were checked for minimal ~j. .
122~951 ~nhibitory concentration (MIC) against the bacteria listed in Table l given below in which the bacteria in the list were cultivated at 37C for 20 hours by the standard method of Japan Society of Chemotherapy (Chemotherapy, Vol. 29, No.l p76 to 79). The MICs of ampicillin as combined with the present compounds (10 ~g/ml) were also measured in the same manner against the same bacteria. Mueller Hinton Broth (Difco) was used as the growth medium and Mueller Hinton Agar (Difco) as the medium measuring the MIC.
Table 1 below shows the results.
The bacteria used in the tests are those heretofore known and preserved in a depository, and are all offered by Dr. Nishino at Department of Microbiology, Kyoto College of Pharmacy except P. vulgaris.

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Given below are examples of preparation of ttle present antibacterial compositions.
Preparation Example 1 Ampicillin 200 mg Compound 2 200 mg Lactose 100 mg Crystalline cellulose 57 mg Magnesium stearate3 mg Total 560 mg (amount per capsule) The above ingredients are formulated in the proportions listed above into a capsule.
Preparation Example 2 Amoxycillin 100 mg lS Compound 5 70 mg .
Lactose 330 mg Corn starch . 490 mg Hydroxypropyl methyl cellulose 10 mg Total 1000 mg ;.' (amount per dose) .
The above ingredients are formulated in the proportions listed above into granules. .
Preparation Example 3 Pivmecillinam 70 mg "
Compound 15 70 mg '`'-122~951 Lactose 33 mg Crystalline cellulose 15 mg Magnesium stearate 3 mg Talc 4 mg Corn starch 15 mg Hydroxypropyl methyl cellulose 10 mg Total 220 mg (amount per tablet) The above ingredients are formulated in the proportions listed above into a tablet.
Preparation Example 4 Compound 5 120 mg .
Hydroxypropyl cellulose 3 mg Corn starch 25 mg .
Magnesium stearate 2 mg --Total 150 mg . (amount per tablet) The above ingredients are formulated in the . .
proportions listed above into a tablet.
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Claims (29)

1. THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
   PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
   
   . A pharmaceutical composition useful for treating bacterial infections in mammals which comprises (A) a B-lactam antibiotic and (B) a compound of the formula wherein Rl and R2 are each the same or different and represent hydrogen, Cl 18 alkyl, mononitro-substituted benzyl or group for fonning a pharmaceutically acceytable salt and R3 is selected from the group consisting of hydrogen, Cl-6 alkyl, C2-7 alkoxymethyl, C3-8 alkylcarbonyloxymethyl, C4-9 alkylcarbonyloxyethyl, (C5-7 cycloalkyl)carbonyloxymethyl, C9 14 benzyl-carbonyloxyalkyl, C3-8 alkoxycarbonylmethyl, C4-9 alkoxycarbonylethyl, phthalidyl, crotonolacton-4-yl, y-butyrolacton-4-yl, halogenated Cl-6 alkyl substituted with 1 to 3 halogen atoms, Cl-6 alkoxy- or nitro-substituted or unsubstituted benzyl, benzhydryl, tetra-hydropyranyl, dimethylaminoethyl, dimethylchlorosilyl, trichlorosilyl, (5-substituted Cl-6 alkyl or phenyl or unsubstituted-2-oxo-1,3-dioxoden-4-yl)methyl, C8-13 benzoyloxyalkyl and group for forming a pharmaceutically acceptable salt, the weight ratio of (A)/(B) being 0.1 to 10, said B-lactam antibiotics being selected from the group consisting of penicillins such as ampicillin, amoxicillin, hetacillin, ciclacillin, mecillinam, carbenicillin, sulbenicillin, ticarcillin, piperacillin, apalcillin, methicillin, mezlocillin, bacampicillin, carindacillin, talampicillin, carfecillin and pivmecillinam; cephalosporins such as cephaloridine, cephalothin, cephapirin, cephacetrile, cefazolin, cephalexin, cefradine, cefotiam, cefamandole, cefuroxime, cefoxitin, cefmetazole, cefsulodin, cefoperazone, cerfotaxime, ceftizoxime, cefmenoxime, latamoxef, cefaclor, cefroxadine, cefatrizine, cefadroxil and cephaloglycin; and pharmaceutically accepted salts thereof.
2. 2. A composition as defined in claim 1 wherein R3 is C2-7 alkoxymethyl.
3. 3. A composition as defined in claim 1 wherein R3 is C3-8 alkylcarbonyloxymethyl, C4 9 alkylcarbonyl-oxyethyl, (C5-7 cycloalkyl)carbonyloxymethyl, C9-14 benzylcarbonyloxyalkyl or C8-13 benzoyloxyalkyl.
4. 4. A composition as defined in claim 1 wherein R3 is C3-8 alkoxycarbonylmethyl or C4-8 alkoxycarbony-lethyl.
5. 5. A composition as defined in claim 1 wherein R3 is phthalidyl.
6. 6. A composition as defined in claim 1 wherein R3 is crotonolacton-4-yl and y-bu-tyrolacton-4-yl.
7. 7. A composition as defined in claim 1 wherein R3 is (5-substituted Cl-6 alkyl or phenyl or unsubstituted -2-oxo-1,3-dioxoden-4-yl)methyl.
8. 8. A composition as defined in claim 1 wherein R3 is a group for forming a pharmaceutically acceptable salt.
9. 9. A composition as defined in claim 1 wherein R3 is Cl-6 alkyl or halogenated Cl-6 alkyl substituted with 1 to 3 halogen atoms, Cl-6 alkoxy- or nitro-substituted or unsubstituted benzyl, benzhydryl, tetra-hydrophranyl, dimethylchlorosilyl and trichlorosilyl.
10. 10. A composition as defined in claim 8 wherein the group for forming a pharmaceutically acceptable salt represented by R3 is alkali metal atom, alkaline earth metal atom, organic amine residue, basic amino acid residue or ammonium residue.
11. 11. A composition as defined in claim 1 wherein R1 and R2 are hydrogen.
12. 12. A composition as defined in claim 1 wherein R1 and R2 are C1-18 alkyl.
13. 13. A composition as defined in claim 1 wherein R1 and R2 are mononitro-substituted benzyl.
14. 14. A composition as defined in claim 1 wherein R1l and R2 are groups for forming a pharmaceutically acceptable salt.
15. 15. A composition as defined in claim 14 wherein the group for forming a pharmaceutically acceptable salt represented by R1 and R2 is alkali metal atom, alkaline earth metal atom, organic amine residue, basic amino acid residue or ammonium residue.
16. 16. A composition as defined in claim 1 in which the penicillin derivative is 2.beta.-(4,5-dimethoxy-carbonyl-1,2,3-triazol-1-yl)methyl-2.alpha.-methylpenam-3.alpha.-carboxylate-1,1-dioxide.
17. 17. A composition as defined in claim 1 in which the penicillin derivative is 2.alpha.-(4.5-dimethoxycarbonyl-1,2, 3-triazol-1-yl)methyl-2a-methylpenam-3.alpha.-carboxylate-1.1-dioxide.
18. 18. A composition as defined in claim 1 in which the penicillin derivative is 2.beta.-(4.5-diethoxycarbonyl-1,2,3-triazol-1-yl)methyl-2.alpha.-methylpenam-3a-carboxylate-1,1-dioxide.
19. 19. A colllposition as defined in claim 1 in which the penicillin derivative is 2.beta.-(4,5-diethoxycarbonyl-1,2,3 -triazol-l-yl)methyl-2.alpha.-methylpenam-3.alpha.-carboxylic acid-l, l-dioxide.
20. 20. A composition as defined in claim 1 in which the penicillin derivative is 2.beta.-(4,5-dimethoxycarbonyl-1,2, 3-triazol-1-yl)methyl-2.alpha.-methylpenam-3.alpha.-carboxylic acid-l, l-dioxide.
21. 21. A composition as defined in claim 1 in which the penicillin derivative is 2.beta.-(4,5-dimethoxycarbonyl-1,2, 3-triazol-1-yl)methyl-2.alpha.-methylpenam-3.alpha.-carboxylate-1,1-dioxide.
22. 22. A composition as defined in claim 1 in which the penicillin derivative is 2.beta.-(4,5-dimethoxycarbonyl-1,2, 3-triazol-1-yl)methyl-2.alpha.-methylpenam-3a-carboxylate-1,1-dioxide.
23. 23. A composition as defined in claim 1 in which the penicillin derivative is 2.beta.-(4,5-dimethoxycarbonyl-1,2, 3-triazol-1-yl)methyl-2.alpha.-methylpenam-3a-carboxylate-1,1-dioxide.
24. 24. A composition as defined in claim 1 in which the penicillin derivative is p-nitrobenzyl 2.beta.-(4,5-di-n-butoxy-carbonyl-1,2,3-triazol-1-yl)methyl-2.alpha. -methylpenam-3.alpha.-carboxylate-1,1-dioxide.
25. 25. A composition as defined in claim 1 in which the penicillin derivative is p-nitrobenzyl 2.beta.-(4,5-di-isobutoxycarbonyl-1,2,3-triazol-1-yl)methyl-2.alpha.-methylpenam-3.alpha.-carboxylate-1,1-dioxide.
26. 26. A composition as defined in claim 1 in which thc penicillin derivative is p-nitrobenzyl 2.beta.-(4,5-di-n-tetradecyloxycarbonyl-1,2,3-triazol-1-yl)methyl-2.alpha.-methylpenam-3.alpha.-carboxylate-1,1-dioxide.
27. 27. A composition as defined in claim 1 in which the penicillin derivative is p-nitrobenzyl 2.beta.-(4,5-di-n-octadecyloxycarbonyl-1,2,3-triazol-1-yl)methyl-2.alpha.-methylpenam-3.alpha.-carboxylate-1,1-dioxide.
28. 28. A composition as defined in claim 1 in which the penicillin derivative is p-nitrobenzyl 2.beta.-[4,5-di-(p-nitrobenzyloxy)carbonyl-1,2,3-triazol-1-yl]methyl-2.alpha.-methylpenam-3.alpha.-carboxylate-1.1-dioxide.
29. 29. A composition as defined in claim 1 in which the penicillin derivative is tri-sodium 2.beta.-(4,5-dicarboxy-1,2,3-triazol-1-yl)methyl-2.alpha.-methylpenam-3.alpha.-carboxylate-l,l-dioxide.