DK157195B - PENICILLANIC ACID-1,1-DIOXIDE DERIVATIVES USED AS INTERMEDIATES IN THE PREPARATION OF 6-AMINOPENICILLANIC ACID-1,1-DIOXIDES - Google Patents

Description

DK 157195 B

One of the most well-known and widely used classes of antibacterial agents is the class known as β-lactam antibiotics. These compounds are characterized in that they have a core consisting of a 2-azetidinone ring (β-lactam ring) fused to either a thiazolidine ring or a dihydro-1,3-thiazine ring.

When the core consists of a thiazolidine ring, the compounds are usually referred to as the common name penicillins, while when the core contains a dihydrothiazine ring, they are referred to as cephalosporins. Typical examples of penicillins commonly used in clinical practice are benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), ampicillin and carbenicillin. Typical examples of common cephalosporins are cephalothin, cephalexin and cephazoline.

However, despite the widespread use and widespread recognition of β-lactam antibiotics as valuable chemotherapeutic agents, they suffer from the significant disadvantage that certain compounds are not active against certain microorganisms. It is believed that this resistance of a particular microorganism to a given β-lactam antibiotic is due to the microorganism producing a β-lactamase. These substances are enzymes that cleave the β-lactam ring of penicillins and cephalosporins 25 to form products which have no antibacterial effect. However, certain substances have the ability to inhibit β-lactamases, and when a β-lactamase inhibitor is used in combination with a penicillin or cephalosporin, it may increase the antibacterial effects of penicillin or cephalosporin against certain microorganisms. It is considered to be an enhancement of antibacterial efficacy when the antibacterial activity of a combination of a β-lactamase inhibitor and a β-lactam antibiotic is significantly greater than the sum of the antibacterial activities of the individual components.

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The present invention relates to penicillanic acid-1,1-dioxide derivatives for use as intermediates in the preparation of 6-aminopenicillanic acid 1,1-dioxide.

5 In Volume 76 (January to June 1972) of the Chemical Abstracts Chemical Substances Index, there is an article under the heading "4-Thia-1-a2a-bicyclo [3.2.0] -hepta-ne-2-carboxylic acid, 6- amino-3,3-dimethyl-7.oxo, 4,4-dioxide ". The latter name is of course another name for 6-aminopenicillanic acid 1,1-dioxide. The index refers to Abstract No. 153735η, which is a summary of DE Publication No. 2,140,119. However, Abstract No. 153735η any reference to penicillin-1,1-dioxides. DE Publication 15, No. 2,140,119 discloses a novel method for the oxidation of penicillin derivatives (e.g., 6-aminopenicillanic acid) to the corresponding 1-oxide. It is stated that the latter process produces penicillin-1-oxides (e.g., 6-aminopenicillanic acid-1-oxide) which are not contaminated with the corresponding 1,1-dioxides (e.g.

6-amino-penicillanic acid l, l-dioxide). There is no other mention of penicillin-1,1-dioxides in DE Public Liaison No. 2,140,119.

.1,1-Dioxides of benzylpenicillin, phenoxymethylpenicillin and certain esters thereof have been disclosed in U.S. Patents 3,197,466 and 3,536,698 and in an article by Guddal et al., Tetrahedron Letters, No. 9, 381 (1962). Harrison et al. have described in the Journal of the Chemical Society (London), Perkin I, 1772 (1976) a large number of pencillin-1,1-dioxides, including methylphthalimido-penicillanate-1,1-dioxide and methyl-6,6 -dibrompenicillanat-1, l-dioxide. U.S. Patent 3,544,581 discloses 6-amino-penicillinanoic acid 1-oxide. Chaikovskaya et al., Antibiotiki, 13, 155 (1968), 35 state that benzylpenicillin-1,1-dioxide has been found to be inactive when tested for β-lactamase inhibitory activity against E. coli.

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According to the present invention, novel penicillanic acid 1,1-dioxide derivatives are provided for use as intermediates in the preparation of 6-aminopenicillanic acid 1,1-dioxide, these compounds being characterized by the formula

H H

Wherein R 1 represents hydrogen, benzyl or pivaloylmethyl, and R 2 represents hydrogen, benzyloxycarbonyl or 2,2,2-trichloroethoxycarbonyl, with the proviso that R 1 and R 2 are not both hydrogen, or a site thereof.

The above-mentioned 6-ominopenicillanic acid 1,1-dioxide is useful as shown in the biological test below, for enhancing the antibacterial effect of β-lactam antibiotics.

The β-lactamase inhibitors, which can be prepared from the intermediates of the invention, have the formula

H H

30 ”AJ_ü ^ 3 JZÎ_h« 3 1111

O COOH

or are pharmacologically acceptable acid addition and basal 4

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six salts thereof. Both the intermediates of the invention and the final products will be designated in the present description. as derivatives of penicillanic acid represented by the structural formula 5 Ç CH, -s y 3 I | (111)

COOH

In the above formulas, binding of a substituent to the bicyclic nucleus by means of a broken line indicates that the substituent is below the plane of the bicyclic nucleus. Such a substituent is said to be in α configuration. Conversely, binding of a substituent to the bicyclic nucleus by means of a fully drawn union indicates that the substituent is attached to the nucleus over its plane. The latter configuration is termed β-20 configuration.

The invention relates to compounds of formula I wherein R 1 is hydrogen or benzyl or pi-valoylmethyl and R 2 is hydrogen or benzyloxycarbonyl or 2,2,2-trichloroethoxycarbonyl, although R 1 and O 2 cannot both be hydrogen. See also: U.S. Patent Nos. 3,632,850 and 3,197,466; British Patent No. 1,041,985, Woodward et al., Journal of the American Chemical Society, £ 8, 852 (1966); Chauvet tea. Journal of Organic Chemistry, £ 3, 1259 (1971); 30sheehan et al., Journal of Organic Chemistry, 29, 2006 (1964); and "Cephalosporins and Penicillins, Chemistry and Biology", published by H.E. Plynn, Academie Press,

Inc., 1972.

By using the compounds of the invention, the final product is obtained simply by removing the protecting group from a compound of the formula

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Wherein R 1 is a carboxy protecting group mentioned in claim 1 and R 2 is hydrogen. Obviously, the protecting group is removed in the usual way for the group in question, although conditions compatible with the β-lactam ring system must be selected. Conditions compatible with the β-lactam ring system are well known to those skilled in the art.

If R 1 represents a benzyl group (or a substituted benzyl group, especially 4-nitrobenzyl), then a catalyst can be suitably removed by catalytic hydrogenation. In this case, a solution of said compound wherein R 1 is benzyl (or substituted benzyl) is stirred or shaken in an inert solvent under a hydrogen atmosphere, or an atmosphere of hydrogen mixed with an inert diluent such as nitrogen or argon, in the presence of a catalytic amount of a hydrogenation catalyst. Suitable solvents for this hydrogenation are lower alkanols such as methanol, ethers such as tetrapyran and dioxane, lower molecular weight esters such as ethyl acetate and butyl acetate, water, and mixtures of these solvents. However, it is common to choose conditions under which the starting material is soluble. The hydrogenation is usually carried out at a temperature in the range of from 0 to about 25. 60 ° C and at a pressure in the range of approx. 1 to approx.

100 kg / cm 2. The catalysts used in this hydrogenation reaction are the type known in the art of this type of conversion, and typical examples are the noble metals such as nickel, palladium, platinum and rhodium. The catalyst is usually present in an amount of approx. 0.01 to approx. And preferably from about 2.5 wt.%. 0.1 to approx. 1.0 wt.%, Based on the compound of formula I. It is often convenient to apply the catalyst to an inert carrier. A particularly suitable catalyst is palladium applied to an inert carrier such as carbon. Furthermore, it is customary to 6

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buffer the reaction mixture to operate at a pH in the range of approx. 4 to 9, and preferably from 6 to 8. Borate and phosphate buffers are usually used. The reaction typically takes approx. For about 1 hour, then the desired final product is simply recovered by filtration followed by removal of the solvent in vacuo.

Another particularly valuable protecting group for R1 is the 2,2,2-trichloroethyl group. This group 10 can be removed by treating the compound of formula I wherein R 1 is 2,2,2-trichloroethyl, with zinc dust in acetic acid, formic acid or a phosphate buffer according to known methods. See also: Woodward et al., Journal of the American Chemical Society, 88, 852 (1966); Pike et al., Journal of Organic Chemistry, 4, 3552 (1969); Just et al. Synthesis, 457 (1976).

The desired final product, namely 6-aminopenicilanoic acid-1,1-dioxide, can be purified, if desired, by well known methods, e.g. recrystallization or chromatography.

The compounds of formula I wherein R 1 is a carboxy protecting group and R 2 is hydrogen can be prepared from a compound of formula 25 H O, R2-NH Ξ = and 3 —-- r 'V' jZi __> C “3 <IV! Wherein R7 is a common carboxy protecting group, as mentioned above, and R2 is a common amino protecting group, using a method which simply includes removing said 35 amino protecting group. For R2, a large number of groups known in the art can be used

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7 Λ for protecting amino groups. The principal requirements for R are: (i) the group must decrease the nucleophilicity of the nitrogen atoms to which it is attached, to such an extent that it is substantially unaffected during the oxidation of the sulfide group in the thiazolidine ring to a sulfone group, and ( ii) the protecting group R2 must be removable under conditions that do not adversely affect the β-lactam ring in the compound of formula I. In the context of the invention, the amino protecting group R 2 may be benzyloxycarbonyl or 2,2,2-trichloroethoxycarbonyl.

Removal of the protecting group R2 from a compound of formula IV wherein R7 is a conventional carboxy protecting group and R2 is benzyloxycarbonyl can be readily carried out by a conventional hydrogenation reaction. This can be carried out by the method previously described for removing the benzyl group from a compound of formula I wherein R 1 is benzyl (or substituted benzyl) and R 2 is H. Removal of the trichloroethoxycarbonyl group from a compound of formula IV, wherein R7 is a conventional carboxy protecting group and R2 is trichloroethoxycarbonyl may be carried out by reduction with zinc dust. The conditions previously discussed for removal of the trichloroethyl group from the compound of formula I wherein R 1 is trichloroethyl may be used for this purpose.

As will be apparent to those skilled in the art, in the case where R 7 is benzyl (or substituted benzyl) and R 2 is benzyloxycarbonyl, it is possible to effectively remove R 7 and R 2 in a single step by hydrogenation 30 to produce the desired final product. Similarly, R7 as trichloroethyl and R2 as trichloroethoxycarbonyl can be effectively removed in a single step from a compound of formula iV by zinc dust reduction to obtain said final product.

The compound of formula IV wherein R7 is a conventional carboxy protecting group and R2 is a conventional carboxy protecting group

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ventional amino protecting group, may be prepared from the corresponding compound of formula V

H h 5 R2NH Ξ Ξ s ^ CH3 i -r 7 coor '10 by oxidation. A large number of oxidants known in the art for the oxidation of sulfides to sulfones can be used for these processes. Particularly useful reagents, however, are alkali metal permits, e.g. potassium permanganate, and organic peroxides, for example, 3-chloro-perbenzoic acid. The latter reagent is a particularly suitable oxidant.

When a compound of formula V wherein R 7 and R 2 are as previously defined is oxidized to the corresponding compound of formula IV using 3-chloroperbenzoic acid, the reaction is usually carried out by treating compound V with from ca. 2 to 4 molar equivalents, and preferably approx. 2.2 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 usually carried out at a temperature of approx. 0e to approx. 80 ° C and preferably. at about. 25 ° C. At about. Reaction times of about 25 ° C are usually used. 2 to approx. 16 hours. The product is normally isolated by removing the solvent by evaporation in vacuo. The product can be purified by conventional methods well known in the art.

The compounds of formula V are either known compounds prepared in accordance with published

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9 methods, or they are analogous to known compounds prepared by analogous methods. In general, the compounds of formula V are simply prepared by linking the protecting groups R7 and R2 5 to the well-known intermediate 6-aminopenicillanic acid. Groups R7 and R2 can be linked by the known group of known methods, taking due account of the stability of the β-lactam ring system. In many cases, the order of association of R7 and R2 is not critical.

The desired final product, 6-aminopenicillanic acid 1,1-dioxide, can be prepared by removing the protecting group R2 from a compound of the invention of the formula 15HH0, r2-nh ^ l = ^ s \ .o'CK3 Γ ( VI) I CH3 XN-

20 ° COOH

ISLAND

wherein R 2 is benzyloxycarbonyl or 2,2,2-trichloroethoxycarbonyl. The group R2 is removed in the same manner as previously described for removal of R2 from a compound of formula IV.

The compounds of formula VI wherein R 2 is a usual amino protecting group can be prepared by oxidation of a compound of formula 30

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10

H H

2 = = S, xCH, R -SH; yf 3 Γ rCH3 5 / \ (VII) Ο ΌΟΟΗ wherein R2 is a conventional amino protecting group. This oxidation is carried out in exactly the same manner as previously described for the oxidation of a compound of formula v to IV.

The compounds of formula VII are prepared from 6-aminopenicillanic acid by attaching the protecting group thereto. The protecting group is associated in the usual manner, taking due account of the lability of the β-lactam ring system.

The compound of the invention, wherein R1 is the hydrogen, may form salts with basic reagents.

Such salts are considered to be within the scope of the present invention. These salts can be prepared by standard techniques such as joining the acidic and basic components, usually in stoichiometric ratio, in an aqueous, non-aqueous or even aqueous medium, as appropriate. The salts are then isolated by filtration, by precipitation with a non-solvent followed by filtration, by evaporation of the solvent, or in the case of aqueous solutions by lyophilization, as appropriate. Suitable basic reagents for the salt formation belong to the organic as well as the inorganic types and include ammonia, organic amines, alkali metal hydroxides, carbonates, bicarbonates, hydrides and alkoxides as well as alkaline earth metal hydroxides, carbonates, hydrides and alkoxides. Representative 35 examples of such bases are primary amines such as n-11

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propylamine, n-butylamine, cyclohexylamine, benzylamine and octylamine, secondary amines such as diethylamine, morpholine, pyrrolidine and piperidine, tertiary amines such as triethylamine, N-ethylpiperidine, N-methylmorpholine and 5,5,5-diazabicyclo [4.3.0] non- -one, hydroxides such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and barium hydroxide, alkoxides such as sodium ethoxide and potassium methoxide, hydrides such as calcium hydride and sodium hydride, carbonates such as potassium carbonate and sodium carbonate and sodium carbonate, bicarbonate and bicarbonate of long chain fatty acids such as sodium 2-ethyl hexanoate.

Preferred base salts of the compound of formula I wherein R 1 is hydrogen are sodium, potassium and triethylamine salts.

Furthermore, the compounds of the invention can form acid addition salts. Such salts come within the scope of the invention and are prepared by standard penam compound technology. Examples of acid addition salts which are particularly valuable are hydrochloride, hydrobromide, phosphate, perchlorate, citrate, tartrate, pamoate, glutarate and 4-toluenesulfonate salts.

As indicated above, 6-aminopenicillanic acid-1,1-dioxide and pharmacologically acceptable salts (the final product) thereof are inhibitors of microbial β-lactamases and increase the antibacterial efficacy of β-lactam antibiotics (penicillins and cephalosporins) against many microorganisms. producing a β-30 lactamase. The manner in which the end product enhances the efficacy of a β-lactam antibiotic in vitro can be assessed by assay, thereby measuring the MIC (Minimum Inhibitory Concentration) of a given antibiotic alone and of said end product alone. These MIC-35 values are then compared to MIC values obtained with a combination of the given antibiotic and final product.

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12 appeared. When the antibacterial effect of the combination is significantly greater than one would have predicted from the effect of the individual compounds, this is regarded as a disruption of the activity. MIC values of 5 combinations are measured using the procedure described by Barry and Sabath in "The Manual of Clinical Microbiology" published by Lenette, Spaulding and Truant, 2nd edition, 1974, American Society for Microbiology .

10 6-aminopenicillanic acid-1,1-dioxide and pharmacologically acceptable salts thereof reduce the antibacterial efficacy of penicillin G against anaerobic bacteria such as Bacteroides spp. and the efficacy of ampicillin against resistant strains of Staphy-15 lococcus aureus.

Furthermore, as in the below biological assay, one can measure the inhibitory effect of the final product on the hydrolysis of ampicillin by β-lactomas from ampicillin-resistant microorganisms.

The ability of the final product to enhance the efficacy of a β-lactam antibiotic against certain β-lactamase-producing bacteria makes them valuable for co-administration with certain β-lactam antibiotics in the treatment of bacterial infections in mammals, especially humans. In treating a bacterial infection, the final product can be mixed with the β-lactam antibiotic and the two agents thereby administered simultaneously. Alternatively, the final product may be administered as a separate agent during treatment with a β-lactam antibiotic.

The following examples serve to further illustrate the invention. Infrared (IR) spectra were measured as potassium bromide slices (KBr slices) and diagnostic absorption bands are given in bale length (cm-1). Nuclear magnetic resonance spectra (NMR) were measured at 35MHz for deutero-chloroform (CDCl3) or deuterium oxide (D20) solutions and peak positions are expressed in

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13 parts per million (ppm) "downfield" relative to tetramethylsilane or sodium 2,2-dimethyl-2-silapentane-5-sulfonate. The following abbreviations are used for top forms: s, singlet; d, doublet; t, triplet; q, the quarter; m, multiplied.

Example 1

Benzyl-6-benzyloxycarbonylaminopenicillanat-l, l-dioxide.

a) 6-Benzyloxycarbonylaminopenicillanic acid.

To a suspension of 108 g of 6-aminopenicillanic acid in 200 ml of water was added a small amount of ice. The suspension was stirred mechanically in an ice bath, adjusting the pH to 7.3 using 6 N sodium hydroxide.

To the thus prepared mixture was added 200 ml of acetone plus a small amount of additional ice. Then followed by the addition of 86 ml of benzyl chloroformate in acetone in two portions with approx. 5 minutes apart. The pH was maintained in the range of 6.5 to 7.0 by the addition of an additional 6 N sodium hydroxide. The mixture was stirred for approx. 45 minutes at which the pH was adjusted to 7.0. The reaction mixture was washed twice with ethyl acetate and then an additional amount of fresh ethyl acetate was added to the aqueous phase. The pH was adjusted to 2.7 and the layers separated. The ethyl acetate layer was washed with sodium chloride solution, dried using anhydrous sodium sulfate and evaporated in vacuo. This gave 188 g of the title compound, contaminated with a small amount of solvent. The NMR spectrum of the product (in CDCl3) showed absorptions at 7.32 (s), 30.05-5.26 (m), 5.08 (s), 4.41 (s), 1.63 (s) and 1.54 (s) ppm.

b) Benzyl-6-benzyloxycarbonylaminopenicillanate.

To a solution of 188 g of 6-benzyloxycarbonyl-aminopenicillanic acid (from a) in 300 ml of N, N-dimethylformamide was added 82.7 ml of diisopropylethylamine. This caused a solid to precipitate and there remained

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14 added an additional 100 ml of Ν, Ν-dimethylformamide. To this mixture was added 57 ml of benzyl bromide and the resulting mixture was stirred overnight at room temperature under nitrogen. The solids present were removed by filtration and discarded. The filtrate was divided into two halves and each half was combined with 600 ml of water and 500 ml of ethyl acetate. The pH was adjusted to 3.0 and the ethyl acetate layers were removed and combined. The resulting ethyl acetate solution 10 was washed with 500 ml of water at pH 2.9. The ethyl acetate solution was then washed twice with 400 ml of water adjusted to pH 8.1. Finally, the ethyl acetate solution was washed with 400 ml of sodium chloride solution and dried using anhydrous sodium sulfate. Evaporation of the dried solution in vacuo afforded 230.7 g of the title compound as an amber oil. The NMR spectrum of the product (in CDCl3) showed absorptions at 7.29 (S, 10H), 5.88-5.25 (m, 2H), 5.08 (s, 2H), 4.43 (s, 1H) , 1.57 (S, 3H) and 1.38 (s, 3H) ppm.

C) Benzyl-6-benzyloxycarbonylaminopenicillanate-1,1-dioxide.

To a solution of 217.8 g of benzyl-6-benzyl-oxycarbonylaminopenicillanate (from b) in 450 ml of methylene chloride was added 250 g of m-chloroperbenzoic acid with stirring over 1 hour. The reaction mixture was allowed to warm to room temperature and stirring was continued overnight. Then, the solid was removed by filtration and discarded, and the filtrate was evaporated to dryness in vacuo. The resulting solid 30 was partitioned between 500 ml of ethyl acetate and 500 ml of water and the pH was adjusted to 7.4 using saturated sodium bicarbonate solution. The ethyl acetate layer was removed and added to 400 ml of water and the pH was adjusted to 8.2 with saturated sodium bicarbonate solution. This resulted in the formation of an emulsion which was divided into two equal parts. To each part was set

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15 200 ml of saturated sodium chloride solution and 200 ml of ethyl acetate. This caused the emulsion to be broken and the ethyl acetate layers removed and united. The resulting ethyl acetate solution was washed with 200 ml of sodium chloride solution and triturated with anhydrous sodium sulfate. Evaporation of the terraced ethyl acetate top solution in vacuo yielded 168 g of crude product. This crude product was slurried in methanol and the solid material removed by filtration. This gave 70 g of the title compound in substantially pure form. The methanol mother liquors were evaporated to dryness and a ring © amount of e-methanol was added to the residue. This mixture was cooled, resulting in an additional amount of solid. This solid was isolated by filtration to obtain an additional 7 g of the title compound in substantially pure form.

The NMR spectrum (in CDCl3) showed absorptions at 7.29 (S, 10H), 6.22 (d, 1H, J = 10Hz), 5.77 (dd, 1H, J = 4Hz, J2 = 10Hz), δ , 20-5.05 (m, 4H), 4.70 (d, 1H, 20 J = 4Hz), 4.48 (s, 1H), 1.49 (s, 3H) and 1.24 (s, 3H) ppm.

Example 2 6- (Benzyloxycarbonylamino) penicillanic acid 1,1-dioxide-536 mg 6- (benzyloxycarbonylamino] penicillanic acid was dissolved in a mixture of 10 ml of water and 10 ml of acetone by means of 1 N sodium hydroxide solution. The pH of the solution was 6 A mixture of 484 mg of potassium permanganate and 625 mg of potassium dihydrogen phosphate was crushed in a mortar, whereupon the crushed material was dissolved in 30 ml of water to form an oxidizing solution. The oxidizing solution was cooled to about 0 ° C. and then stirred, while stirring, to the solution of the penicillin compound at about 0 ° C. Stirring was continued for 5 minutes, then the bath was removed and the mixture was stirred for a further 5 minutes.

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16 minutes. At this time, an equal volume of ethyl acetate was added to the reaction mixture and the pH was adjusted to 7.5. Solid sodium bisulfite was added keeping the pH at 7.5, 5 until the brown color disappeared. The pH was lowered to 1.5. The ethyl acetate layer was removed, washed with saturated sodium chloride solution, dried (Na2SC> 4) and evaporated in vacuo. There was thus obtained 442 mg (75% yield) of 6-benzyloxycarbonylamino) penicillanic acid 1,1-dioxide.

The IR spectrum showed absorptions at 1795, 1712, 1312 and 1163 cm -1. The NMR spectrum (CDCl3) showed absorptions at 7.32 (s, 5H), 6.37 (d, 1H, J = 10.5Hz), 5.83 (dd, 1H, J = 4, and 10.5 Hz) , 5.13 (s, 2H), 4.81 (d, 1H, J = 4 Hz), 4.49 (s, 1H), 1.56 (s, 3H) and 1.41 (s, 3H) ppm.

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Example 3

Benzyl 6-aminopenicillanate-l, l-dioxide.

To 1.23 g (2.4 mmol) of benzyl 6- (2,2,2-trichloroethoxycarbonylamino) penicillanate-1,1-dioxide in 20 ml of tetrahydrofuran was added 2.0 g of zinc dust followed by 4 , 0 ml of 1 M potassium dihydrogen phosphate with vigorous stirring. Stirring was continued for 1 hour and then an additional 2.0 g of zinc dust and 4.0 ml of 1 M potassium dihydrogen phosphate were added. After an additional 1 hour stirring, the reaction mixture was filtered and the filtrate was evaporated in vacuo keeping the pH within the range of 6.5 to 7.0. The aqueous residue was diluted with an additional amount of water, and the product was extracted with ethyl acetate. The ethyl acetate was washed with water and then dried using sodium sulfate. Evaporation of the dried ethyl acetate solution in vacuo afforded the title compound.

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Example 4

Pivaloyloxymethyl 6-aminopenicillanate-l, 1-dioxide.

a) Pivaloyloxymethyl-6-benzyloxycarbonylaminopenicilanate.

To a stirred solution of 3.50 g of 6-benzyloxycarbonylaminopenicillanic acid in 15 ml of N, N-dimethylformamide was added 1.30 g of diisopropylethylamine followed by 1.55 g of chloromethyl pivalate and 50 mg of sodium iodide at ca. 0 ° C. The reaction mixture was stirred at ca. 0 ° C for 30 minutes-10 hours and then at room temperature for 24 hours. The reaction mixture was then diluted with ethyl acetate and water and the pH of the aqueous phase was adjusted to 7.5. 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 dried using anhydrous sodium sulfate and evaporated in vacuo to give the title compound.

b) Pivaloyloxymethyl-6-aminopenicillanate-1,1-dioxide;

To a suspension of 2.0 g of 5% palladium-p-carbon in 20 ml of ethyl acetate was added to a solution of 2.0 g of pivaloyloxymethyl-6-benzyloxycarbonylaminopenicillanate-1,1-dioxide in 10 ml of ethyl acetate. The mixture was shaken under a hydrogen atmosphere at a pressure of approx. 50 p.s.i.g. for 30 minutes. The mixture was filtered and the solvent removed by evaporation in vacuo to give the title compound.

The product was redissolved in a small volume of ethyl acetate and a 0.5 M solution of 4-toluenesulfonic acid was added dropwise slowly, thereby precipitating the 304-toluenesulfonate salt of the title compound. It was isolated by filtration.

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Example 5

Pivaloyloxymethyl-6-benzyloxycarbonylaminopenicillanate-1,1-dioxide.

To 4.65 g of pivaloyloxymethyl-6-benzyloxycarbonylpenicillanate (see Ex. 4) in 15 ml of dichloromethane was added 4.00 g of 3-chloroperbenzoic acid at 0 ° C. The reaction mixture was stirred at 0 ° C for 1 hour and then at 25 ° C for 24 hours. The solid was removed by filtration and the filtrate was evaporated in vacuo.

The residue was partitioned between ethyl acetate and water at pH

7.5, and the ethyl acetate layer was removed. The ethyl acetate layer was dried and concentrated to dryness in vacuo to give the title compound.

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Application Example 1 a) Sodium 6-aminopenicillanate-1,1-dioxide.

To a suspension of 4.7 g of 5% palladium-carbon in 10 ml of water was added a solution of 4.7 g of benzyl-6-benzyloxycarbonylaminopenicillanate-1,1-dioxide (see Example 1). in 50 ml of ethyl acetate. The mixture was hydrogenated at a pressure of 58-44 p.s.i.g. for 15 minutes. Then 2.5 ml of 2N sodium hydroxide was added to the mixture and the resulting mixture was filtered. The aqueous phase was removed, its pH was adjusted to 5.3 and it was then lyophilized. This gave 1.19 g of the title compound. The IR spectrum (KBr disk) showed absorptions at 1810, 1610, 1320 and 1120 cm -1. The NMR spectrum (in D 2 O) showed absorption at 5.01 (d, 1H, 30 J = 4Hz), 4.87 (d, 1H, J = 4Hz), 4.24 (s, 1H), 1.55 ( s, 3H) and 1.42 (s, 3H) ppm.

b) 6-Aminopenicillanic acid 1,1-dioxide.

To a solution of 2.70 g (0.01 mole) of sodium 6-aminopenicillanate-1,1-dioxide in 30 ml of water was added 5 ml of 35 N hydrochloric acid. The mixture was concentrated to a small volume in vacuo and then the solid which formed

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19 precipitated, isolated by filtration. This achieved the zwitterion specified in the heading.

Application Example 2 6-Aminopenicillanic acid 1,1-dioxide.

A solution of 442 mg of 6- (benzyloxycarbonylamino) penicillanic acid -1,1-dioxide (see Ex. 2) in 10 ml of ethyl acetate was added to a slurry of 442 mg of 10% palladium palladium. carbon in 5 ml of water. The mixture was shaken under a hydrogen atmosphere for 30 minutes at ca. 50 psig. whereupon the catalyst was removed by filtration. The pH of the filtrate was adjusted to 5.2 using 1N sodium hydroxide solution, after which the aqueous phase was removed and lyophilized. There was obtained 150 mg of 6-aminopenicillanic acid 1,1-dioxide as the sodium salt (48% yield). The NMR spectrum (D20) showed absorptions at 5.0 (q, 2H), 4.25 (s, 1H), 1.50 (s, 3H) and 1.35 (s, 3H) ppm along with peaks for degradation products.

Application Example 3 6-Aminopenicillanic acid 1,1-dioxide.

By hydrogenating the benzyl ester of 6-amino penicillanic acid 1,1-dioxide (see Example 3) using the procedure of Application Example 1, 6-aminopenicillanic acid 1,1-dioxide was obtained in the form of the sodium salt. The sodium salt can be converted to the corresponding 30-switter ion using the method of the same example.

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DK 15 719 5 B

BIOLOGICAL TESTING OF 6-AMINOPENICILLANIC ACID-1,1-DIOXIDE a) Structures Q, o 5 NH2 ^) I> ch3 o5 "* - · N-1,

COOH

6-aminopenicillanic acid 1,1-dioxide.

a? ? CH-C-NH ^ Sv'1 CH3 «Η- Ί- <TCH3 2 ^ -N-

15 O tOOH

ampicillin (b) Testing procedure

Cell-free β-lactamase enzymes were extracted from 20 ampicillin-resistant microorganisms and measured by these for a 15-20 minute period of hydrolysis rate for ampicillin using microiodometry according to Novick (Biochemical Journal, 83, 236, 1962), while maintaining the hydrolysis medium. at 37 ° C and a pH-25 value of 6.5. The initial concentration of ampicillin was 33 mol / ml. Further, by the above procedure, the rate of hydrolysis of ampicillin for the β-lactamase was measured in the presence of 6-aminopenicillanic acid 1,1-dioxide, with the initial concentration of this compound in the hydrolysis medium being either 16.5 or -1 33 pmol / ml.

The function of 6-aminopenicillanic acid 1,1-dioxide as a β-lactamase inhibitor was determined by comparing the hydrolysis rates of ampicillin, respectively, without 35 and in the presence of 6-aminopenicillanic acid 1,1-dioxide, the results are shown in Table I,

DK 157195 B

The ability of the subject compound is calculated as 100x (l - (ratio of initial hydrolysis rates of ampicillin, respectively, with and without the presence of 6-aminopenicillanic acid-1,1-dioxide)).

C) Results __TABLE I_

Microorganism Staminé Inhibition of 10 6-aminopenicillanic acid 1,1-dioxide ___ at 16.5 mol / ml at 33 mol / ml

Staphylococcus 01A400 74 95 aureus 15 Escherichia 51A129 70 89 coli

Klebsiella 53A079 49 76 pneumoniae____ d) Conclusion

It can be deduced from the results in Table I that 6-aminopenicillanic acid β1-dioxide is an effective β-lactase inhibitor against the strains of Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia tested.

As a result, it can be predicted that 6-aminopenicillanic acid 1,1-dioxide will be useful in enhancing the antibacterial action of ampicillin against infections in humans with Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

Claims (3)

1. Penicillanic acid 1,1-dioxide derivatives for use as starting material in the preparation of 6-aminopenicillanic acid 1,1-dioxide characterized in that the penicillanic acid 1,1-dioxide derivatives have the formula 5 HH% f * CH 10 oJ ^ -V13! (I) COORX 4 wherein R1 represents hydrogen, benzyl or pivaloylmethyl, and R2 represents hydrogen, benzyloxycarbonyl or 2,2,2-trichloroethoxycarbonyl, with the proviso that R1 and R2 are not both hydrogen or a site thereof. A compound according to claim 1, characterized in that R 1 is hydrogen and R 2 is benzyloxycarbonyl or 2,2,2-trichloroethoxycarbonyl. A compound according to claim 1, characterized in that R 1 is benzyl and R 2 is benzyloxycarbonyl.