CZ2001277A3 - Novel C2 S/O- and S/N-formaldehyde acetal derivatives of carbapenem-3-carboxylic acids and their use as antibiotics and {beta}-lactamase inhibitors - Google Patents

Abstract

Compounds of Formula I wherein R 1 is hydrogen hydroxymethyl or 1-hydroxyethyl, R 2 is hydrogen or methyl and R 3 is a pharmaceutically acceptable moiety that is bound to the rest of the molecule by a single oxygen-carbon bond or a simple nitrogen-carbon bond and a pharmaceutically acceptable salt thereof acceptable salts, ester and amide derivatives are broad-spectrum antibiotics and β-lactamase inhibitors.

Description

Novel C ² S / O- and S / N-formaldehyde acetal derivatives of carbapenem-3-carboxylic acids and their use as antibiotics and β-lactamase inhibitors

Technical field

The present invention relates to novel C ² S / O- and S / N-formaldehyde acetal derivatives of carbapenem-3-carboxylic acids and their use as antibiotics and β-lactamase inhibitors.

BACKGROUND OF THE INVENTION

The prior art is cited with reference to European patent applications EP 0 079 244 A, EP 0 168 707 A and EP 0 169 410 A. There are described compounds which are structurally similar to the compounds of the present invention. However, the compounds disclosed in these patent applications are either antibiotics having only activity against β-lactamases or antibiotics having only antibacterial activity.

SUMMARY OF THE INVENTION

The present invention relates to novel C ² S / O- and S / N-formaldehyde acetal derivatives of carbapenem-3-carboxylic acids of formula I

where • · · · φ • • • • • • • • • • · · · φ φ · φ φ

R ¹ is a hydrogen atom, a hydroxymethyl group or a 1-hydroxyethyl group, R ² is a hydrogen atom or a methyl group, and R ³ is a pharmaceutically acceptable group that binds to the remainder of the molecule by a simple oxygen-carbon bond or a single nitrogen-carbon bond is selected from the group consisting of substituted or unsubstituted alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, N-heterocyclyl group, heterocyclyloxy, heterocyclylcarbonyloxy heterocyklylthiokarbonyloxyskupina, acyloxy, thioacyloxyskupina, alkoxycarbonyloxy, carbamoyloxy, thiocarbamoyloxy, heterocyklyloxykarbonyloxyskupina, heterocyklyloxythiokarbonyloxyskupina, Nheterocyklykarbamoyloxyskupina, N heterocyklylthiokarbamoyloxyskupina, heterocyclylcarbonylamino, heterocyklylthiokarbonylaminoskupina , heterocyclyloxycarbonylamino, acylamino, alkoxycarbonylamino, alkoxythiocarbonylamino INA thioacyklaminoskupina, N heterocyklylkarbamoylaminoskupina, N heterocyklylthiokarbamoylaminoskupina ureido thiokarbamoylaminoskupina, imidoylamínoskupina, guanidino, N-heterocyclyl-alkoxycarbonylamino, N-heterocyklylalkylthiokarbonylaminoskupina and N-sulfonylamino where the previous alkyl, alkenyl, alkynyl, acyl, thioacyl or the imidoyl moiety of the molecule contains 1 to 6 carbon atoms and the heterocyclyl residue is monocyclic or bicyclic and contains 3 to 10 ring atoms, one or more of which is selected from the group consisting of oxygen, sulfur and nitrogen, and wherein the substituents of the above R groups can be: alkyl, acyl, thioacyl, heterocyclyl, hydroxyl, hydroxyalkyl, alkoxy, hydroxyalkoxy, aminoalkoxy, amidinoalkoxy, guanidinoalkoxy, acyloxy, hetero ·

cyklyloxyskupina, alkylheterocyklyloxyskupina, hydroxyalkylheterocyklyloxyskupina, aminoalkylheterocyklyloxyskupina, carbamoyl alkyl group, alkylcarbamoyl, dialkylcarbamoyl, carbamoyloxy, alkylcarbamoyloxy, dialkylcarbamoyloxy, thiocarbamoyl group, alkylthiocarbamoyl group, dialkylthiokarbamoylová group, thiocarbamoyloxy, alkylthiokarbamoyloxyskupina, dialkylthiokarbamoyloxyskupina, mercapto, alkylthio, hydroxyalkylthioskupina, aminoalkylthioskupina, 'monoalkylaminoalkylthioskupina, dialkylaminoalkylthioskupina, amidinoalkylthioskupina, acylthio, heterocyclylthio, alkylheterocyklylthioskupina, hydroxyalkylheterocyklylthioskupina, aminoalkylheterocyklylthioskupina, karbamoylthioskupina, monoalkylkarbamoylthioskupina, dialkylkarbamoylthioskupina, thiokarbamoylthioskupina, alkylthiokarbamoylthioskupina, dialkylcarbamoyl thio, amino, monoalkylamino, hydroxyalkylamino, aminoalkylamino, dialkylamino, oxo, oximino or an alkylimino group, imidoylaminoskupina, alkylimidoylaminoskupina, dialkylimidoylaminoskupina, trialkylammonium group, cycloalkylamino, heterocyclylamino, alkylheterocyklylaminoskupina, heterocyclylcarbonylamino, alkylheterocyklylkarbonylaminoskupina, acylamino, amidino, monoalkylamidinoskupina, dialkylamidinoskupina, guanidino, alkylguanidinoskupina, dialkylguanidinoskupina ureido thiokarbamoylaminoskupina, alkylkarbamoylaminoskupina, thiokarbamoylaminoskupína , alkylthiocarbamoylamino, nitro, chloro, bromo, fluoro, iodo, azido, cyano, alkylsulfinyl, alkylsulfonyl, sulfonamido, sulfamoyloxy, alkylsulfamoyloxy, alkylsulfonyloxy, alkylsulfonyloxy, sulfo,

N, N-dialkylcarboxamido or carboxy wherein the substituents are independently occurring one or more times and their alkyl moiety contains 1 to 6 carbon atoms and wherein the heteroeyl moiety is monocyclic or bicyclic and contains 3 to 10 ring members of which one or more is selected from the group consisting of oxygen, sulfur and nitrogen, and these compounds and their pharmaceutically acceptable salts, esters and amides are useful as antibiotics and as β-lactamase inhibitors.

The pharmaceutically acceptable R ³ groups that are bonded via a single oxygen-carbon bond or a single nitrogen bond are common groups, for example, in the field of β-lactam antibiotics or β-lactamase inhibitors. These groups are described, for example, in MS Sassiver, A. Lewis, Advances in Applied Microbiology, Ed. D. Perlman, Academic Press NY (1970) or in a number of patents, for example US Pat. 5,096,899.

The term "pharmaceutically acceptable salt" as used herein means non-toxic acid and base salts and zwitterionic salts. Base salts include inorganic salts such as sodium, potassium, magnesium and calcium or ammonium salts, and salts with non-toxic amines such as trialkylamines, alkanolamines, arginine, or cyclic amines such as piperazine, procaine and other amines used to form carboxylic acid salts. . Acid salts include salts with inorganic acids such as chloride, sulfate, phosphate and the like, and salts with organic acids such as acetate, maleic, citrate, succinate, ascorbate, lactate, fumarate, tartrate and oxalate, and other organic acid salts used to form salts with amines.

Pharmaceutically useful ester and amide derivatives serve as prodrugs that hydrolyze in the body to form antibiotics. They are preferably administered orally because the hydrolysis in principle takes place under the influence of digestive enzymes. If hydrolysis occurs in the blood, administration outside the digestive system may be used in some cases. Examples of pharmaceutically acceptable ester and amide derivatives include physiologically hydrolyzable esters and amides known and used with penicillin and cephalosporin e.g. Advances in Drug Res. 17, 197 (1988). Said ester and amide derivatives are prepared by conventional techniques known to those skilled in the art.

The compounds of the present invention have several asymmetric centers and can exist in several stereochemical forms. The invention encompasses mixtures of isomers as well as individual stereoisomers. Most preferred compounds of formula I have the IR, 5S and 6S substituted carbapenem core configuration and the 1'R or 1'S configuration of the 6- (1-hydroxyethyl) side chain. It may be asymmetric carbon atoms in R ^3rd The invention includes compounds having the R and S configurations at the R ³ substituent.

The invention also relates to methods for preparing compounds of formula I, pharmaceutical compositions containing them, and methods of treatment comprising administering said compounds and compositions wherein an antibiotic effect is indicated.

The terminology of the compounds of this group can be based on the root of the word "carbapenem", which is a trivial and simple way of nomenclature (used in the general description). Alternatively, these compounds may also be described by the Chemical Abstracts nomenclature, which is more suitable for describing the individual compounds of this group, the Chemical Abstracts nomenclature.

5 ^ 1

Therefore, the carbapenem nomenclature of the Chemical Abstracts nomenclature is used in the examples

Classical β-lactam antibiotics, such as penicillins or cephalosporins, become partially ineffective in treating infectious diseases because they are resistant to bacteria. In addition to the natural resistance of some bacteria, many species of pathogenic microorganisms have acquired resistance after long-term use of antibiotics in bulk. For example, most penicillin-resistant Staphylococcus aureus species, and many gram-negative bacteria, such as Enterobacter cloacae, Pseudomnas aeruginosa or even Escherichia coli, have acquired resistance to cephaloscorin.

Therefore, there is still a need for novel antibiotics, especially anitbiotics with a broad spectrum of activity or orally active antibiotics.

Among β-lactams, carbapenems are the most potent group of compounds. They are also active against most strains that are resistant to penicillin and cephalosporin. However, the currently used carbapenems are administered parenterally because they do not have sufficient oral activity. Lack of oral activity is known in the art and described, for example, in Infection 14, (1986), Appendix 2, page 115.

Thus, it is an object of the present invention to provide a new class of carbapenem antibiotics with a very broad antibacterial spectrum and oral activity.

Compounds of the above formula I of the following scheme:

are generally prepared according to

Removal of a protecting group

Intermediate 3 is prepared by reacting a starting material 1 which contains a leaving group X in which R ¹ and R ^{2 are} as defined above with HS / O- or HS / N-formaldehyde acetals of HS-CH ₂ -R ³ suitably in the presence of a base, wherein R ³ is as defined above.

Due to the high reactivity of the HS / O- or HS / N-acetals of formaldehyde, especially in the presence of a base, the leaving group X is not a very critical factor. The reaction can be carried out with a wide variety of leaving groups X. Examples of such leaving groups X are alkoxy groups such as methoxy, alkylsulfonyloxy or arylsulfonyloxy groups such as methylsulfonyloxy group, trifluoromethylsulfonyloxy group and p-toluenesulfonyloxy group or diaryloxyphenoxy group or dialkoxyphenoxy group.

Examples of methods for preparing the starting material 1 are described in Heterocycles 1984, 21, 29-40, or Tetrahedron Lett. 1980, 21, 4221-4224.

In process 1 → 3, inorganic or organic bases such as potassium carbonate or cesium or tertiary amines such as triethylamine and pyridine or suitably sterically hindered bases such as diisopropylethylamine and 2,6-dimethylpyridine can be used.

Due to the high reactivity of the above-mentioned HS / O- or HS / N-acetal formaldehyde, the reaction temperature varies considerably. Process 1 -> 3 is suitably carried out at a temperature between -70 ° C and room temperature. Non-polar or polar solvents such as methylene chloride or acetonitrile, or suitably Ν, Ν-dimethylformamide, are suitable. Process 1-3 can also be carried out by phase transfer catalysis using, for example, water, a non-polar solvent such as carbon tetrachloride or methylene chloride, and a phase transfer catalyst such as tetrabutylammonium bromide.

Process 1 → 3 can also be carried out using a previously formed salt, suitably an alkali or alkaline earth salt, or the tetraalkylammonium salt of HS / O- or HS / N-acetal formaldehyde 2.

In the case of inorganic salts, the process 1 - 3 is suitably carried out without an additional base in a polar solvent such as Ν, Ν-dimethylformamide. For more soluble tetraalkylammonium salts, a less polar solvent such as tetrahydrofuran is suitable.

The protecting groups Y in the starting material 1 and in the intermediate 3 are readily removable radicals which are known and commonly used in organic syntheses. Protecting groups of this type can be found, for example, in Gunda I. Georg " The Organic Chemistry of .beta.-Lactams, VCH Publishers UK, Cambridge, 1993, pages 23-29.

Removal of the protecting group 3- < 1 > yields the free carboxylic acid or the corresponding inorganic salt. In some cases, the selected substituent R ³ 'may be modified simultaneously (during the process 3 -> I). An example of such a case is the simultaneous reduction «

• · * · · · · ··· ··· · ·

2-azidoethoxy R ³ to 2-aminoethoxy during deprotection of compound 3 (Y = p-nitrobenzyl) by catalytic hydrogenolysis.

A prerequisite for the preparation of a compound of formula I is the availability of the corresponding HS / O- and HS / N-formaldehyde acetals 2. It has been found that the groups of simple HS / O formaldehyde acetals (R ³ = unsubstituted alkoxy) and HS / N-formaldehyde acetals (R ³ = unsubstituted acylamino), not yet known. Therefore, it is also an object of the present invention to prepare novel suitable formaldehyde derivatives 2.

2-Alkoxyalkylthiocarbapenems are described in European patent application EP 0 010 317. They are prepared from known unsubstituted 2-alkoxyalkanethioles as starting materials. 2-Alkoxymethylthiocarbapenems, however, are not available in this way since the necessary starting materials, HS / O-formaldehyde acetals (2, R ³ = unsubstituted alkoxy) of 1 to 6 carbon atoms in their alkoxy moiety, are a class of compounds which have not been known hitherto. The group of these substances was considered too unstable for practical use (Houben-Weyl, Methoden der Org. Chemie, Volume E 14a / l, G. Thieme, Stuttgart, NY 1991, page 793). This text speaks against the above 2-alkoxymethanthioles and the solution of the present invention which leads to 2-alkoxymethylthiocarbapenem-3-carboxylic acids.

Although previously a compound with a boiling point of 52 ° C / 2 kPa (15 mm) was mislabeled as methoxymethanethiol (Chem. Zentralbl. 1912, 1192), we did not find any later references to the use of this reagent in Chemical Abstracts. Methoxymethanethiol was not available as described, but was prepared in a very different manner (4 → 6- 6 or 5 → 6 → 2). In fact, methoxymethanethiol is much more volatile than the compound described and. :::. :::

• 9 99 9 999 9999999 · 99 999 99 999 has a boiling point of 51 ° C at atmospheric pressure! Since the corresponding HS / O-acetal of formaldehyde was not available, 2-alkoxymethylthiocarbapenems I (R ³ = methoxy) were never prepared and were therefore not described in Chemical Abstracts.

Similarly, the unsubstituted HS / N-acetals of formaldehyde 2 (R ³ = unsubstituted acylamino) are an unknown group of compounds. Since reagents 2 (R ³ = acylamino) are needed for the preparation of acylaminomethylthiocarbapenems I (R ³ = acylamino), these compounds are not available in the prior art. Therefore, 2-acylaminomethylthiocarbapenems have not been described in Chemical Abstracts.

The novel HS / O- or HS / N-acetals of formaldehyde 2 are readily prepared by the following procedures:

O

X 3 H ₃ C 5 S-CH ₂ -R

Cl-CH ₂ -R ³ + H ₃ C-4 bK

Hydrolysis τ

hs-ch ₂ -r ³ wherein R ³ is as defined above. Starting compounds 4 and 5 are known and prepared according to known methods or are commercially available, such as chloromethyl methyl ether (4, R ³ = OCH ³ ) or N-hydroxymethyl acetamide (5, R ³ = HN-CO-CH 3). Process 4 → 6 is conveniently carried out in a polar or non-polar solvent such as acetonitrile, ether or chloroform, at -70 ° C to room temperature, reaction 5 → 6 can also be carried out in a solvent, or suitably without solvent (using excess thioacetic acid) at -30 ° C to +60 ° C. Alternatively, another alkali metal or alkaline earth metal thioacetate may be used in place of commercially available potassium thioacetate.

The hydrolysis process 6 → 2 can be carried out under basic or acidic conditions using, for example, an alkali metal or alkaline earth metal hydroxide or alkoxide, suitably sodium hydroxide or sodium methoxide in a polar solvent such as water, acetonitrile or methanol. Preferred acidic conditions in process 6 → 2 use strong acids, suitably hydrochloric acid, in a polar solvent such as water or methanol. The hydrolysis is suitably performed at a temperature of -30 ° C to room temperature. The HS / O- or HS / N-acetals of formaldehyde 2 can be isolated free or as alkali metal, alkaline earth metal or tetraalkylammonium salts.

As already mentioned, currently used carbapenems do not have sufficient oral activity. The oral activity of the compounds of the formula I according to the invention originates from the novel S / O or S / N-acetal formaldehyde. Similar formaldehyde derivatives, i.e. 0/0 formaldehyde acetals, have led to an increase in the oral absorbability of penicillin, for example as pivaloyloxymethyl penicillin esters (see Merck Index, 11th Edition, 7484, page 1193). A strong influence of the substituent bridge length is reported for cephalosporins (Journ. Antibiot. 1993, 46, 177), where intensive research has focused on bioavailability. According to this work, the formaldehyde acetal S / S derivative is the best of the other fourteen compounds. Unfortunately, due to the non-polar nature of the S / S acetal group, activity against Pseudomnas aeruginosa (inherent to the aminothiazolcephalosporin group) was greatly reduced.

In the field of carbapenems, oral activity has also been reported for 2-S / S formaldehyde acetal derivatives in European patent application 0 481 511 A2, which states that a methylene group is suitable as a bridge between two sulfur atoms (to achieve oral activity for this class of antibiotics) .

However, S / O- or N / O-formaldehyde acetals have never been mentioned and no data have been reported on their oral bioavailability or their antibacterial activity. Compared to the above-mentioned 2-S / S-acetals of formaldehyde, the compounds of formula I of the present invention are more polar and therefore also active against clinically important pathogenic Pseudomnas aeruginosa.

Compared to other 2-alkoxyalkylthiocarbapenems or 2-acylaminoalkylthiocarbapenems with a longer bridge length of their alkylene moiety, the compounds of formula I are far more preferred due to their oral absorbability.

In the general description of the present invention, R ¹ is hydrogen, hydroxymethyl or 1-hydroxyethyl, R ² is hydrogen or methyl, and R ³ is a pharmaceutically acceptable group that binds to the rest of the molecule by a simple oxygen-carbon or nitrogen- carbon and is selected from the group consisting of substituted or unsubstituted alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, N-heterocyclyl, heterocyclyloxy, heterocyclylcarbonyloxy heterocyklylthiokarbonyloxyskupina, acyloxy, thioacyloxyskupina, alkoxycarbonyloxy, carbamoyloxy, thiocarbamoyloxy, heterocyklyloxykarbonyloxyskupina, heterocyklyloxythiokarbonyloxyskupina, N heterocyklykarbamoyloxyskupina, N-heterocyclylthiocarbamoyloxy, heterocyclylcarbonylamino, heterocyclylthiocarbonylamino, heterocyclyloxycarbonylamino, acyclamino, alkoxycarbonylamino and, alkoxythiokarbonylaminoskupina, thioacylaminoskupina, N heterocyklylkarbamoylaminoskupina, N heterocyklylthiokarbamoylaminoskupina ureido thiokarbamoylaminoskupina, imidoylaminoskupina, guanidino, N-heterocyklylalkoxy13 carbonylamino, N-heterocyklylalkylthiokarbonylaminoskupina and N-sulfonylamino where the previous alkyl, alkenyl, alkynyl, acyl, thioacyl or imidoyl molecule part it contains 1 to 6 carbon atoms and the heterocyclyl residue is monocyclic or bicyclic and contains 3 to 10 ring members, one or more of which is selected from the group consisting of oxygen, sulfur and nitrogen and wherein the substituent of the above R group may be: alkyl group, acyl, thioacyl, heterocyclyl, hydroxyl, hydroxyalkyl, alkoxy, hydroxyalkoxy, aminoalkoxy, amidinoalkoxy, guanidinoalkoxy, acyloxy, heterocyclyloxy acyloxy,, alkylheterocyklyloxyskupina, hydroxyalkylheterocyklyloxyskupina, aminoalkylheterocyklyloxyskupina, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, carbamoyloxy, alkylcarbamoyloxy, dialkylcarbamoyl oxy gr on, thiocarbamoyl group, alkylthiokarbamo.vlová group dialkylthiokarbamoylová group, thiocarbamoyloxy, alkylthiokarbamoyloxyskupina, dialkylthiokarbamoyloxyskupina, mercapto, alkylthio, hydroxyalkylthioskupina, aminoalkylthioskupina, monoalkylaminoalkylthioskupina, dialkylaminoalkylthioskupina, amidinoalkylthioskupina, acylthio, heterocyclylthio, alkylheterocyklylthioskupina, hydroxyalkylheterocyklylthioskupina, aminoalkylheterocyklylthioskupina, karbamoylthioskupina, monoalkylkarbamoylthioskupina, dialkylkarbamoylthioskupina, thiokarbamoylthioskupina, alkylthiokarbamoylthioskupina, dialkylkarbamoylthioskupina, amino, monoalkylamino, hydroxyalkylamino, amino, alkylamino, dialkylamino, oxo, oximino or an alkylimino group, imidoylaminoskupina, alkylimidoylaminoskupina, dialkylimidoylaminoskupina, tetraalkylammonium cykloalkylaminoskupi14 on, heterocyclylamino alkylheterocyklylaminoskupina, heterocyclylcarbonylamino, alkylheterocyklylkarbonylaminoskupina, acylamino, amidino, monoalkylamidinoskupina, dialkylamidinoskupina, guanidino, alkylguanidinoskupina, dialkylguanidinoskupina ureido thiokarbamoylaminoskupina, alkylkarbamoylaminoskupina, thiokarbamoylaminoskupina, alkylthiokarbamoylaminoskupina, nitro, chlorine, bromine, fluorine, iodine, azido, cyano, alkylsulfinyl, alkylsulfonyl, sulfonamido, sulfamoyloxyskupina, alkylsulfamoyloxyskupina, alkylsulfonyloxy or sulfo, sulfoxy, carboxamido, N-monoalkylkarboxamidoskupina, N, N-dialkylkarboxamidoskupina or carboxy where the subst ituents independently occur one or more times and their alkyl moiety contains 1 to 6 carbon atoms and wherein the heteroeyl moiety is monocyclic or bicyclic and contains 3 to 10 ring members, one or more of which is selected from the group consisting of oxygen, sulfur and nitrogen.

A preferred class of compounds of formula I are compounds wherein R ¹ is hydrogen, hydroxymethyl or 1-hydroxyethyl group, R ² is a hydrogen atom or a methyl group and R ³ is a substituted or unsubstituted alkoxy, heterocyclyloxy, acyloxy, carbamoyloxy, N-heterocyclyl group, , acylamino, carbamoylamino, imidoylamino, wherein the above alkyl, acyl, thioacyl or imidoyl group contains 1 to 3 carbon atoms and the heterocyclyl group is monocyclic and contains 3 to 6 ring members of which one or more atoms are selected from the group consisting of: an oxygen atom, a sulfur atom and a nitrogen atom; and wherein the substituents of the above R groups may be: alkyl, acyl, thioacyl, heterocyclyl, hydroxyl, hy15; Hydroxyalkyl, alkoxy, hydr oxyalkoxy, aminoalkoxy, amidinoalkoxyskupina, guanidinoalkoxyskupina, acyloxy, heterocyclyloxy, alkylheterocyklyloxyskupina, hydroxyalkylheterocyklyloxyskupina, aminoalkylheterocyklyloxyskupina, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, carbamoyloxy, alkylcarbamoyloxy, dialkylcarbamoyloxy, thiocarbamoyl group, alkylthiocarbamoyl group, dialkylthiokarbamoylová group, thiocarbamoyloxy, alkylthiokarbamoyloxyskupina, dialkylthiokarbamoyloxyskupina, mercapto, alkylthio hydroxyalkylthioskupina, aminoalkylthioskupina, monoalkylaminoalkylthioskupina, dialkylaminoalkylthioskupina, amidinoalkylthioskupina, acylthio group, heterocyclic thio, alkylheterocyklylthioskupina, hydroxyalkylheterocyklylthioskupina, aminoalkylheterocyklylthioskupina, karbamoylthioskupina, monoalkylkarbamoylthioskupina, dialkylkarbamoylthioskupina, thiokarbamoylthioskupina, alkylthiokarbamoyl thio, dialkylkarbamoylthioskupina, amino, monoalkylamino, hydroxyalkylamino, aminoalkylamino, dialkylamino, oxo, oximino, or an alkylimino group, imidoylaminoskupina, alkylimidoylaminoskupina, dialkylimidoylaminoskupina, tetraalkylammonium, cycloalkylamino, heterocyclylamino, alkylheterocyklylaminoskupina, heterocyclylcarbonylamino, alkylheterocyklylkarbonylaminoskupina, acylamino, amidino, monoalkylamidinoskupina, dialkylamidinoskupina, guanidino, alkylguanidino, dialkylguanidino, carbamoylamino, thiocarbamoylamino, alkylcarbamoylamino, thiocarbamoylamino, alkylthiocarbamoylamino, nitro, chloro, bromo, fluoro, iodo, sulfonyl, cyano, sulfonyl, cyano, sulfonyl, cyano, sulfonyl, cyano

pine, sulfamoyloxy, alkylsulfamoyloxy, alkylsulfonyl oxy or sulfo, sulfoxy, carboxamido, N-monoalkylcarboxamido, N, N-dialkylcarboxamido or carboxy, wherein the substituents independently of one another are present one or more times up to 6 and their alkyl groups and wherein the heterocyclic group is monocyclic and contains 3 to 6 ring atoms of which one or more is selected from the group consisting of oxygen, sulfur and nitrogen.

A particularly preferred class of compounds of formula I of the present invention are those wherein R ¹ is 1-hydroxyethyl, R ² is methyl and R ³ is substituted alkoxy, acylamino, alkylcarbamoylamino, alkoxycarbamoylamino, N-heterocyclyl and imidoylamino, wherein the above alkyl a group, acyl or imidoyl group in the molecule contains 1 to 3 carbon atoms and the heterocyclyl group is monocyclic and contains 3 to 6 ring atoms of which one or more is selected from the group consisting of: oxygen, sulfur and nitrogen; and wherein the substituents of the above R ³ are basic groups such as amino, alkylamino, dialkylamino, imidoyl, amidino and guanidino. In this case, the compounds of formula I of the present invention are very polar and may exist in the form of a zwitterion. Therefore, these compounds are particularly active against gram-negative bacteria including Pseudomnas aeruginosa.

The selection of compounds of formula I according to the present invention shows high antibacterial activity in a 10 µg disc sensitivity test at the following inhibitory diameters: Staph. aureus (9-39mm), E. coli (27-34mm), E. cloacae (23-27mm) and Ps. aeruginosa (13-26 mm). These data correspond to the values of clinically available carbapenem as described in Journ. Antimicrob. Chemotherapy 24 (1989) Suppl. A, 253.

Therefore, these novel antibiotics are active against a number of bacterial pathogens, including, for example, Gram-positive and Gram-negative bacteria such as Staphylococcus aureus, Escherichia coli, Enterobacter cloacae Enterococcus and Pseudomnas aeruginosa.

Surprisingly, it was found that the compounds of the present invention strongly inhibit bacterial β-lactamases isolated from Enterobacter cloacae and Escherichia coli. Furthermore, all tested Gram-negative pathogens resistant to ceftazidime were susceptible to the combination of ceftazidime and the compound of the present invention.These compounds are therefore very potent β-lactamase inhibitors. Very high β-lactamase inhibitor activity of the compounds of the present invention has also been observed in isolated bacterial enzymes in a nitrocefin assay (R. Reimer, Methodicum Chimicum: Antibiotics, Vitamins and Hormons; F. Korte, M. Goto, eds., Thieme , Stuttgart, 1977, p. 11, E. Wasielewski, Arzneimittel, Vol. 4; Chemotherapeutica, Part 1, Verlag Chemie, Weinheim 1972).

Representative compounds of Formula I of the invention show high blood levels in mice following oral treatment of 25 mg / kg, indicating that the compounds are absorbed by the oral route.

Thus, an object of the present invention is a novel class of carbapenem antibiotics and β-lactamase inhibitors that is important in veterinary and human medicine and in non-viable systems. The high and broad spectrum antibacterial activity and β-lactamase inhibitory activity of the compounds of the invention, together with their oral activity, could not be expected from this prior art.

The novel compounds of the present invention are valuable antimicrobial agents that are active against most Gram-positive and Gram-negative pathogens including also most bacteria that are resistant to penicillin and cephalosporin and are anaerobic.

Particularly suitable bactericides are the free acids, and in particular the alkali metal and alkaline earth metal salts or zwitterions thereof, and can be used for the removal of pathogens from dental and medical devices for the removal of microorganisms and for therapeutic purposes in human and veterinary medicine. For the latter purpose, pharmaceutically acceptable salts known to those skilled in the art for the administration of penicillins and cephalosporins are used. These salts may be used together with pharmaceutically acceptable liquid and solid ingredients to provide suitable unit dosage forms such as pills, tablets, capsules, suppositories, syrups, elixirs and the like, which may be prepared by methods known to those skilled in the art. known.

The novel compounds are valuable antibiotics against most pathogenic bacteria and are therefore used in human and veterinary medicine. They can be used as antibacterial drugs for the treatment of infections caused by Gram-positive and Gram-negative bacteria, for example, Staphylococcus aureus, Escherichia coli, Klebsiela pneumoniae, Bacilus subtilis, Salmonella tyfosa, Enterobacter cloacae, Enterococcus, Pseudomnas aeruginosa and Bacteria.

The antibacterial agents may further be used as additives to animal feed, for example for feed preservation and as disinfectants. For example, they may be used in aqueous formulations at concentrations of 0.1 to 100 parts antibiotic / million • 0 0 0 · * 0 0 0 0 * 0 0 · 0 000 0 000 • 0 0 0 000 • 000 »000 0 0 • 0 000 000 ···· 00 »· 00 000 00 000 parts of a solution for destroying and inhibiting the growth of harmful bacteria on a treatment device and as bactericides in industrial applications, such as water-based paints and soft water in paper mills, to inhibit the growth of harmful bacteria .

The products of the present invention can be used alone or together with other active ingredients of many other pharmaceutical preparations. These preparations can be used in the form of capsules or tablets, powders or liquid solutions or suspensions or elixirs. They may be administered orally, intravenously or intramuscularly.

The compositions are preferably administered in a form suitable for absorption through the gastrointestinal tract. Tablets and capsules for oral administration may be in unit dosage form and may contain conventional pharmaceutical excipients such as binding agents, for example syrups, acacia, gelatin, sorbitol, or polyvinylpyrrolidone, fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine, lubricants such as magnesium stearate, talc, polyethylene glycol or silica, disintegrants such as starch or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated according to methods well known to those skilled in the art. Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs, and the like, or may be in the form of dry products, for example, for conditioning prior to administration with water or other suitable ingredients. Liquid preparations of this type may contain additives known to those skilled in the art, such as suspending agents, for example sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum gel stearate or hydrogenated edible oils, for example ·· · Almond oil, fractionated coconut oil, oil esters, propylene glycol or ethyl alcohol, preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid. Suppositories contain suppository bases known to those skilled in the art, for example, cocoa butter or other glycerides.

Formulations for injection may be presented in unit dosage form or in multi-dose containers together with preservatives. The formulations may be in the form of suspensions, solutions or emulsions in oily or aqueous additives, and may contain formulatory agents such as suspending agents, stabilizers and / or dispersing agents. Alternatively, the active ingredient may be in powder form suitable for preparation of the composition in a suitable excipient just before use, for example in sterile pyrogen-free water.

The formulations may also be in a form suitable for absorption through the nasal mucosa and esophagus or bronchial tissue and may be in the form of powders or liquid sprays or inhalants, suckers, throat rubs, and so on. For eye and ear medicaments, the compositions may be used in the form of individual capsules in liquid or semi-liquid form, or may be used in the form of drops and so on. Topical application forms can be prepared in a suitable hydrophobic vehicle such as ointments, creams, lotions, lotions, powders and so on.

The compositions of the present invention may contain, in addition to additives, other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, viscosity or flavor control agents and the like.

44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 · 4

The compositions of the present invention may also contain, in addition to additives, enzyme inhibitors, for example cilastatin (Merck Index, 11th edition 2275), to enhance the therapeutic effect.

Further, the compositions may contain one or more active antibacterial ingredients to provide a broader antibiotic range. Examples of these additional active ingredients are antibiotics, preferably β-lactam antibiotics, for example penicillins such as Ampicillin or Amoxycillin or cephalosporins such as Cefalexin, Cefachlor or Ceftazidime. With these added conventional β-lactam antibiotics, the active ingredient of the present invention acts as an antibacterial agent and as an β-lactamase inhibitor.

For veterinary purposes, the formulations may be formulated, for example, as intramammary formulations, either in a long-acting or rapid-release vehicle. The dose to be administered depends greatly on the condition of the patient to be treated and on his weight and the manner and frequency of administration. Generally, the daily dose will contain about 10 to 200 mg of active ingredient / kg body weight of the patient in the case of one or more administrations per day. The preferred daily dose for an adult is 20 to 120 mg active ingredient / kg body weight.

The compositions of the present invention can be administered in various unit dosage forms, for example, solid or liquid dosage forms, which can be administered orally. The preparation may contain 0.1 to 99% of active ingredient per unit dose, either in solid or liquid form. A range of 10 to 60% is preferred. The formulations generally contain 15 to 1500 mg of active ingredient, but it is usually preferred to use an amount of 250 g to 1000 mg per dose. In the case of parenteral administration, the normal dose is the pure compound dissolved in sterile water or in the form of a soluble powder which may be dissolved.

The examples below illustrate the products, processes, compositions and methods of treatment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Methoxymethanthiol

To 7.7 ml of a 5.2N aqueous sodium hydroxide solution (40 mmol) at 0 ° C was added 2.40 g (20 mmol) of methoxymethylthioacetate with stirring. After 30 minutes, 4.0 mL (20 mmol) of a 5N aqueous hydrogen chloride solution was added to the resulting yellow solution, whereupon an oily layer separated. After the aqueous layer was saturated with sodium chloride at 0 ° C, the oil layer was separated, dried over magnesium sulfate, filtered, and the filtrate was distilled at normal pressure to give a colorless liquid, mp 51 ° C. NMR spectrum in deuterochloroform 2.0 (t, 1H, J = 12 Hz), 3.4 (s,

3H), 4.8 (d, 2H, J = 12Hz) ppm.

Alternatively, the biphasic mixture is extracted with deuterochloroform. The resulting solution was dried over magnesium sulfate, filtered and stored in a refrigerator. It contains pure title compound (1.17 g, 80%). This solution was extracted with 7.5 mL (15 mmol) of 2N sodium hydroxide solution and the extract was immediately lyophilized under high vacuum to give a colorless solid sodium methoxymethylthiolate.

• ·

Example 2

2- (Azidoethyloxy) methanethiol

Hydrogen chloride gas was introduced at -10 ° C to a mixture of 2-azidoethane (2.0 g, 23 mmol) and trioxane (0.74 g, 8.2 mmol). After 1.5 hours the solid liquefies. After flushing the apparatus with nitrogen, the mixture contains (2-azidoethyl) chloromethyl ether. NMR (CDCl3): 3.5 (m, 2H), 3.9 (m, 2H), 5.5 (s, 2H) ppm.

(2-Azidoethyl) chloromethyl ether (crude product, 23 mmol) was added at 0 ° C to a stirred suspension of 2.63 g (23 mmol) of potassium thioacetate in 7.5 mL of dry ether. The mixture was allowed to stir at room temperature overnight. Insoluble materials were filtered off and the filtrate was evaporated in vacuo to give an orange liquid. This was distilled under vacuum (0.3 Pa) using a short path distillation apparatus and a safety shield. Pure (2-azidoethoxy) methylthio acetate, b.p. 80-90 ° C / 0.3 Pa, was obtained as a pale yellow liquid in a yield of 65%. NMR spectrum in deuterochloroform: 2.38 (s, 3H), 3.36 (m, 2H), 3.61 (m, 2H), 5.10 (s, 2H) ppm.

A solution of 848 mg (4.84 mmol) of (2-azidoethoxy) methylthioacetate in 5 mL of tetrahydrofuran was added dropwise to the stirred solution of 121 N aqueous sodium hydroxide solution (121 mL, 24.2 mmol) at 0 ° C. The solution obtained is washed with 100 ml of ether and then acidified at 0 ° C with 19.4 ml of a 1.0N aqueous hydrochloric acid solution to pH 6. The aqueous solution is extracted twice with 50 ml of ether. The combined ether layers were dried over magnesium sulphate and the solvent evaporated in vacuo (1.7 kPa). The crude product obtained is purified by chromatography on silica gel (200 - 63 .mu.m, 23

g) eluting with hexane-ether (9: 1) to give the colorless title compound in a yield of 47% after drying under vacuum (1.7). Β 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · NMR spectrum in deuterochloroform: 2.0 (t, 2H, = 10 Hz),

3.4 (t, 2H, J = 6Hz), 3.7 (t, 2H, J = 6Hz), 4.8 (d, 2H, J =

Hz) ppm.

Example 3 (2-Azido-1,1-dimethylethoxy) methanethiol

To a stirred suspension of 310 mg (13 mmol) of sodium hydride in 5 ml of dry tetrahydrofuran at 0 ° C was added a solution of 1.15 g (10 mmol) of 1-azido-2-methyl-2-propanol in 2 ml of tetrahydrofuran. When hydrogen evolution ceased, 3 mL of dry hexamethylphosphoric triamide and 1.14 mL (15 mmol) of chloromethyl methyl ether were added to the light yellow solution at 0 ° C and the mixture was stirred at room temperature overnight. The reaction mixture was poured into 40 mL of 10% aqueous sodium chloride solution and extracted twice with 150 mL and 50 mL of ether. The combined ether layers were washed twice with 50 ml of 10% sodium chloride and 50 ml of saturated sodium chloride solution. The ether layer was dried over magnesium sulfate and the solvent was evaporated in vacuo (1.7 kPa) to give 1.41 g (89%) of formaldehyde - ((2-azido-1,1-dimethylethyl) methyl acetal. NMR spectrum in deuterochloroform). 1.28 (s, 6H); 3.22 (s, 2H);

3.39 (s, 3H), 4.74 (s, 2H), ppm.

To a stirred solution of 1.35 g (8.47 mmol) of formaldehyde - ((2azido-1,1-dimethylethyl) methyl acetal in 5 mL of dry dichloromethane at 0 ° C was added 3.64 mL (3.64 mmol) of chloride solution. The reaction mixture was stirred at room temperature for 3 hours to give a solution of (2-azido-1,1-dimethylethyl) chloromethyl ether NMR spectrum in deutrochloroform: 1.36 (s, 6H), 3.24 (ABkv, 2H). 5.60 (s, 2H) ppm.

To a suspension of powdered solid potassium thioacetate (1.28 g, 11.3 mmol) in dry dichloromethane (8 mL) at 0 ° C was added 8.0 ° C. Ml of the above solution containing 1,23 g (7,53 mmol) of (2azido-1,1-dimethylethyl) chloromethyl ether and obtained the suspension was stirred overnight at room temperature. The mixture was diluted with dichloromethane (80 ml), washed with water (3 x 30 ml) and brine (30 ml). The organic layer was dried over magnesium sulfate and the solvent was evaporated in vacuo to give 1.30 g (85%) of crude (2-azido-1,1-dimethylethoxy) methylthioacetate. This was purified by silica gel column chromatography (40-60 pm) using hexane-ether (4: 1) as eluent to give 0.81 g (53%) of pure (2-azido-1,1-dimethylethoxy). methyl thioacetate as a colorless liquid. Deuterochloroform NMR Spectrum: 1.28 (s, 6H), 2.37 (s, 3H), 3.21 (s, 2H), 5.02 (s, 2H).

To a stirred 0.1 N sodium hydroxide solution (25 mL, 2.5 mmol) was added 102 mg (0.5 mmol) of (2-azido-1,1-dimethylethoxy) methylthioacetate dissolved in 0.5 mL at 0 ° C. tetrahydrofuran. The reaction mixture was stirred at 0 ° C for 90 minutes. The solution was washed with 15 mL of ether and then acidified at 0 ° C with 1.8 mL (1.8 mmol) of 1N hydrochloric acid to pH 6-7. The solution was stirred at 0 ° C for 30 minutes and then transferred to a separatory funnel. After stirring for 3 minutes, the organic layer was removed and the aqueous layer was extracted twice with 8 ml of ether each time. The combined ether layers were dried over magnesium sulfate and the solvent was evaporated in vacuo (1.7 kPa) to give 67 mg (83%) of the title compound as a light yellow liquid. NMR spectrum in deuterochloroform: 1.28 (s, 6H), 2.18 (t, 1H, J = 10 Hz), 3.23 (s, 2H), 4.73 (d, 2H, J = 10 Hz) ) ppm.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · »8 ·· ·

Example 4

Ν- (Mercaptomethyl) acetamide

13.4 g (0.15 mol) of N- (hydroxymethyl) -acetamide and 14.3 g (0.188 mol) of thiol acetic acid are heated at 40 ° C for 3 days. The resulting mixture is chromatographed on silica gel (63-200 µπ, 700 g) eluting with toluene-ethyl acetate 4: 1 to 1: 1 to give pure crystalline acetamidomethylthio acetate (14.2 g, 64%), m.p. 93-94 ° C. C.

Acetamidomethylthioacetate (1.47 g, 10 mmol) was dissolved in 2.0 N hydrogen chloride in dry methanol (1.8 mL) and the solution was maintained at room temperature for 3.5 hours. The solution was neutralized to pH 7 with 2N sodium methoxide solution in 1.8 ml dry methanol. The precipitated sodium chloride is filtered off and the filtrate is evaporated in vacuo. The residue was purified by silica gel chromatography (63-200 µm, 30 g) using ethyl acetate to give 0.70 g (67%) of the pure title compound. This was maintained at -30 ° C under an argon atmosphere. NMR spectrum in deuterochloroform: 1.95 (s, 3H), 2.38 (t, 1H, J = 9 Hz),

4.28 (dd, 2H, J = 9 Hz); 6.81 (broad s, 1H) ppm.

Example 5

2-Azido-N- (mercaptomethyl) acetamide

A mixture of 1.40 g (14 mmol) of 2-azido-acetamide, 1.40 g (14 mmol) of 30% aqueous (methanol-free) formaldehyde and 0.28 ml (0.28 mmol) of a 1.0 N aqueous potassium hydroxide solution was added. Stir 4 hours at 0 ° C. To the reaction mixture was added a 1.0N aqueous hydrogen chloride solution (pH 7) and the resulting mixture was evaporated in vacuo. The residue is suspended in 50 ml of ethyl acetate, the solution is dried over magnesium sulphate, filtered and the filtrate is evaporated to a volume of 5 ml. Purification by chromatography on silica gel (63-200 μπι, 18 g) eluting with ethyl acetate gave 2-azido-N- (hydroxymethyl) acetamide as a colorless oil in a yield of 90%. NMR spectrum in deuterochloroform: 3.6 (broad signal, 1H), 4.0 (s, 2H), 4.8 (d, 2H, J = 6 Hz), 7.3 (broad signal, 1H) ppm.

To 650 mg (5 mmol) of 2-azido-N- (hydroxymethyl) -acetamide at -10 ° C was added 635 mg (5 mmol) of oxalyl chloride. After 10 minutes gas evolution ceased. The mixture was diluted with deuterochloroform. NMR (CDCl3): 4.1 (s, 2H), 5.2 (d, 2H, J = 10 Hz), 7.3 (broad signal, 1H) ppm. NMR corresponds to 2-azido-N (chloromethyl) acetamide (75% yield).

To a solution of crude 2-azido-N- (chloromethyl) -acetamide (715 mg) in 4 mL of deutrochloroform at 0 ° C was added 520 mg (4.6 mmol) of potassium thioacetate (520 mg, 4.6 mmol) and the suspension was Stir overnight at room temperature. The mixture is diluted with 50 ml of chloroform and the solution is then washed twice with 15 ml of water and 10% brine each. The organic layer was dried over magnesium sulfate and the solvent was evaporated in vacuo to give 530 mg of a non-crystalline solid. This was purified by silica gel chromatography (63-200 μπι) eluting with toluene-ethyl acetate (2: 1) to give 400 mg (56%) of pure (2-azidoacetamido) methylthio acetate. NMR spectrum in deuterochloroform: 2.4 (s, 3H), 4.0 (s, 2H), 4.7 (d, 2H, J = 7 Hz), 7.2 (broad signal, 1H) ppm.

mg (0.28 mmol) of pure (2-azidoacetamido) methylthioacetate was dissolved in 0.6 ml (1.1 mmol) of 1.8N hydrogen chloride in dry methanol and the solution was stirred at room temperature for 6 hours. Neutralize with 0.46 mL (1.03 mmol) of 2.2N sodium methoxide in dry methanol (pH 6). The precipitated sodium chloride is filtered off and the filtrate is diluted with 0.7 ml of perdeuterodimethylsulfoxide and then the methanol is evaporated off under vacuum (2 kPa) and finally under a high vacuum of 0.13 Pa. The perdeuterodimethylsulfoxide solution is maintained at -80 ° C. According to NMR spectroscopy, this is the title compound (56% yield, determined with 10 μΐ of benzene as internal standard). NMR spectra in perdeuterodimethylsulfoxide: 2.9 (broad signal, 1H), 3.95 (s, 2H), 4.38 (dd, 2H), 8.9 (broad signal, 1H) ppm.

Example 6 1-Ethyl-4- (mercaptomethyl) piperazine-2,3-dione

To a solution of 2.13 g (15 mmol) of 1-ethylpiperazine-2,3-dione (2.13 g, 15 mmol) in 30% aqueous (free of methanol) formaldehyde (15 mmol) was added 150 mg (2.7 mmol). mmol) of potassium hydroxide and the mixture is stirred at 50 ° C for 7 days. Neutralize 50 μ 7. of a 5N aqueous hydrochloric acid solution to pH 7. The mixture is evaporated in vacuo (2 kPa) and then dried under a high vacuum of 0.13 Pa to give 1-ethyl-4- (hydroxymethyl) -piperazine-2, 3-Dione as a colorless solid in 100% yield. NMR spectrum in deuterochloroform: 1.15 (broad signal, 1H), 1.15 (t, 3H, J = 7 Hz), 3.47 (kv, 2H, J = 7 Hz), 3.54 (m, 2H), 3.68 (m, 2H), 4.88 (s, 2H) ppm.

To 156 mg (0.906 mmol) of 1-ethyl-4- (hydroxymethyl) -piperazine-2,3-dione at -10 ° C was added 78 μΐ (0.906 mmol) of oxalyl chloride. The mixture was stirred at -10 ° C for 2 hours. The gas stops evolving after 30 minutes. The reaction mixture was dried under high vacuum to give 1- (chloromethyl) -4-ethyl-piperazine-2,3-dione in 100% yield. NMR spectrum in deuterochloroform: δ, 15 (t, 3H, J = 7 Hz), 3.42 (qu, 2H, J = 7 Hz), 3.65 (broad signal, 4H), 5.30 (s, 2H) ppm.

To a solution of crude 1- (chloromethyl) -4-ethyl-piperazine-2,3-dione (170 mg, 0.9 mmol) in 1 mL deuterochloroform was added 123 mg (1.08 mmol) with stirring at 0 ° C. solid potassium thioacetate. The reaction mixture was allowed to stir at room temperature overnight. The mixture is centrifuged and the supernatant solution is collected. The remaining solid (potassium chloride) was washed with 2 ml of deuterochloroform and the organic solutions were combined and the solvent was evaporated in vacuo. The residue was purified by silica gel chromatography (6 g, 63-200 μπι) using chloroform-methanol (19: 1) as eluent to give 1- (acetylthiomethyl) -4-ethyl-piperazine-2,3-dione as a white solid. solids (total yield 44%). NMR Specter in deuterochloroform: 1.22 (t, 3H, J = 7Hz), 2.44 (s, 3H), 3.48 (kv, 2H, J = 7Hz), 3.4-3.7 (m, 4H), 4.95 (s, 2H) ppm.

mg (0.32 mmol) of 1- (Acetylthiomethyl) -4-ethylpiperazine-2,4-dione was dissolved in 0.56 ml (1.2 mmol) of a 1.95N solution of hydrogen chloride in methanol and the solution was allowed to stir at room temperature for 6 hours . The mixture is neutralized at 0 ° C with 0.54 ml (1.2 mmol) of a 2.24N solution of sodium methoxide in dry methanol to pH 6. The precipitated sodium chloride is filtered off and the filtrate is evaporated in vacuo and the residue is dried under vacuum at 0, 13 Pa. Purify by silica gel chromatography (2.0 g, 63-200 μπι) eluting with chloroform-methanol (19: 1) to give the title compound as a colorless solid in 80% yield. NMR spectrum in deuterochloroform: 1.23 (t, 3H, J = 7 Hz), 2.43 (broad signal, 1H) 3.55 (kv, 2H, J = 7 Hz), 3.67 (broad s,

4H), 4.60 (broad s, 2H) ppm.

Example 7 (4R, 5S, 6S) -6 - ((1'R) -Hydroxyethyl) -3- (methoxymethylthio) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene Sodium or potassium 2-carboxylate (Ia)

p-Nitrobenzyl- (4R, 5S, 6S) -6 - ((1'R) -hydroxyethyl) -3- (methoxymethylthio) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2 -en-2-carboxylate

To a solution of 892 mg (1.5 mmol) of p-nitrobenzyl (4R, 5R, 6S) -3 - (difenyioxyfosfinoyloxy) -6- ((1 ¹ R) -hydroxyethyl) -4-methyl-7-oxo-azabicyclo [ 3,2,0] Hept-2-ene-2-carboxylates in 15 ml of dry dimethyl formamide at -50 ° C is added a solution of 152 mg (1.95 mmol) of methoxymethanethiol in 3 ml of deuterochloroform and then 334 μΐ (1.95) mmol) of diisopropylethylamine. The reaction mixture was allowed to warm to 0 ° C. After 2 hours at 0 ° C, the reaction mixture is diluted with 300 mL of ethyl acetate and the solution is left at room temperature for 5 minutes. This solution is washed successively with 125 ml of a 10% aqueous potassium carbonate solution, three times with 100 ml of water and 100 ml of brine. The organic layer was dried over magnesium sulfate and the solvent was evaporated in vacuo. The residue was purified by silica gel chromatography (50 g, 63-200 pm), eluting with toluene-ethyl acetate (2: 1) and (1: 1) to give the title compound as a solid. A pale yellow, non-crystalline solid in a yield of 78%. IR in dichloromethane: 3600, 3050, 2900, 1770, 1710, 1605,

1520, 1345, 1210, 1135, 1080 cm ¹ .

(4R, 5S, 6S) -6 - ((1'R) -Hydroxyethyl) -3- (methoxymethylthio) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-en-2- potassium carboxylate

To 750 mg of 10% palladium on carbon, prehydrogenated at 0 ° C in a biphasic mixture of 30 ml of ethyl acetate and 85 mg (0.85 mmol) of potassium bicarbonate in 12 ml of water, was added a solution of 479 mg (1.13 mmol) of p- nitrobenzyl- (4R, 5S, 6S) -6 ((1'R) -hydroxyethyl) -3- (methoxymethylthio) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-en-2 -carboxylate in 10 ml of ethyl acetate. The mixture is hydrogenated at normal pressure and at 0 ° C. After 70 minutes the uptake of hydrogen (70 ml) was greatly retarded. An additional 150 mg of catalyst was added and hydrogenation was continued for another 100 minutes. An additional 50 ml of hydrogen was consumed. The catalyst is filtered off, washed with 5 ml of ethyl acetate and 2 ml of water and the filtrate is transferred to a separatory funnel. The aqueous layer was removed and the organic layer was extracted with a solution of 28 mg (0.28 mmol) of potassium bicarbonate in 3 mL of water. The combined aqueous solutions were evacuated to remove residual ethyl acetate and then lyophilized at -30 ° C and under high vacuum (0.13 Pa) to give the title compound as a white powder in 50% yield. UV spectrum in water = 2992 nm (ε = 8000). NMR spectrum in deuterated water (Me ₂ SiCD ₂ CD ₂ COONa internal standard): 1.21 (d, 3H, J = 7 Hz), 1.31 (d, 3H, J = 6 Hz), 3.43 (s ,

3H), 3.45 (m, 1 Η), 3.52 (m, 1 Η), 4.2 5.03 (ABq, J = 8 Hz, _S-CH2 -O) ppm.

4.3 (m, 2H), 4.76 and

Example 8 (4R, 5S, 6S) -3 - ((2-Aminoethoxy) methylthio) - ((1'R) -hydroxyethyl) 4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylic acid (Ib)

o-ch ₂ -ch ₂ -nh _2- p-Nitrobenzyl- (4R, 5S, 6S) -3 - ((2-azidoethoxy) methylthio) -6 ((1'R) -hydroxyethyl) -4-methyl-7- oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

Following the procedure described in Example 7, using (2-azidoethoxy) methanethiol, after purification by chromatography, eluting with toluene / ethyl acetate (1: 1), the p-nitrobenzyl ester was prepared in 80% yield as a pale yellow non-crystalline solid. IR in dichloromethane: 3600, 3050, 2900, 2100 (N ₃ ), 1770, 1710, 1610, 1520, 1350, 1210, 1140, 1085 cm ^-1 .

o-ch / oh ^

T ^CH >

COOH ·

(4R, 5S, 6S) -3 - ((2-Aminoethoxy) methylthio) -6 - (( ¹ R) -hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2 -en-2-carboxylic acid (zwitterionic form)

700 mg of 10% palladium on carbon was prehydrogenated at 0 ° C in a biphasic mixture of 30 ml of ethyl acetate and 15 ml of water and 422 mg (0.884 mmol) of p-nitrobenzyl- (4R, 5S, 6S) solution was added via syringe at 0 ° C. -3 - ((2-azidoethoxy) methylthio) -6 - ((1'R) -hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-en-2- of carboxylate in 10 ml of ethyl acetate. After 65 minutes of hydrogenolysis at 0 ° C and normal pressure, hydrogen uptake (70 mL) slowed. An additional 100 mg of catalyst was added and hydrogenation was continued at 0 ° C for an additional 75 minutes. An additional 35 ml of hydrogen was absorbed. The catalyst was filtered off, washed with 5 ml of ethyl acetate and 3 ml of water and the filtrate was transferred to a separatory funnel. The aqueous layers were combined and the organic layer was extracted with 3 mL cold water. The combined aqueous solutions were evacuated to remove residual ethyl acetate and then lyophilized at -30 ° C under high vacuum (0.13 Pa) to give the title compound as a white powder in 68% yield. UV spectrum in water: kmax 292 nm (E = 8000). NMR spectrum in deuterated water (Me ₃ SiCD ₂ CD ₂ COONa internal standard): 1.21 (d, 3H, J = 7 Hz), 1.30 (d, 3H, J = 6)

Hz), 3.23 (m, 2H), 3.48 (dd, 1H), 3.55 (m, 1H) 3.70 and 3.93 (2m, 2H), 4.26 (complex signal) 2H), 4.78 and 5.17 (ABq, 2H, J = 11 Hz, _S-CH2 -O) ppm.

Example 9 (4R, 5S, 6S) -3 - ((2- (Formimidoylamino) ethoxy) methylthio) - ((1 ' R) hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept -2-en-2-carboxylic acid (Ic) (zwitterionic form) o-ch ₂ -ch ₂ -nh-ch = nh

To a solution of 4.7 mg (13.8 pmol) of (4R, 5S, 6S) -3 - ((2-aminoethoxy) methylthio) - ((1'R) -hydroxyethyl) -4-methyl-7-oxo- of 1-azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid in 0.18 ml of water at 0 ° C is added a solution of 83 µl (41 pmol) followed by 4.5 mg (41 pmol) of solid ethylformimide hydrochloride . After 30 minutes at 0 ° C, an additional 55 µL (28 pmol) of 0.5 N sodium bicarbonate and 3.0 mg (28 pmol) of ethylformimide hydrochloride (pH 8) were added and the reaction mixture was stirred at 0 ° C for 60 minutes. Finally, 9 µl (45 pmol) of 0.5 M potassium carbonate solution was added and the mixture was stirred at 0 ° C for 30 minutes.

The solution was purified at 0 ° C by slow filtration through an ion exchange column containing Dowex 50W x 4 (0.5 g, Na + -cycle) using water as eluent.

Collect 12 fractions (0.5 mL) and examine by thin layer chromatography (reverse phase silica gel RP-18, water-acetonitrile (3: 1)). Fractions containing the product were combined and acetonitrile was evaporated in a high vacuum. lyophilized at 0.13 Pa to give the title compound in 31% yield as a colorless amorphous solid UV spectrum in water: λ max = 2992 nm (E = 8,000) NMR spectrum in deuterated water (internal standard) Me ₃ SiCD ₂ CD ₂ COONa): 1.21 (d, 3H, J = 7Hz), 1.30 (d, 3H, J = 6Hz), 3.4-4.0 (m, 6H), 4.2-4.3 (m, 2H), 4.71 and 5.17 (ABkv, 2H, J = 7 Hz), 7.8 (d, 1H, J = Hz).

XO-CH ₂ -CH ₂ -NH ₂

COOH

Example 10 (4R, 5S, 6S) -3 - ((2-Amino-1,1-dimethylethoxy) methylthio) - ((1 ' R) hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2. 0] hept-2-ene-2 carboxylic acid (Id)

p-Nitrobenzyl- (4R, 5S, 6S) -3 - ((2-azido-1,1-dimethylethoxy) methylthio) -6 - ((1 ' R) -hydroxyethyl) -4-methyl-7-oxo-1 -azabicyclo [3.2.0] hept-2-ene-2-carboxylate

Following the procedure described in Example 7, using (2-azido-1,1-dimethylethoxy) methanethiol, after purification by chromatography eluting with toluene / ethyl acetate (2: 1) the title pnitrobenzyl ester was obtained in 72% yield as a pale yellow non-crystalline solids. IR spectrum in dichloromethane: 3600, 3025, 2990, 2105 (N _3), 1775, 1710, 1610, 1525,

1350, 1210, 1135, 1055 cm ^-1 .

(4R, 5S, 6S) -3 - ((2-amino-1,1-dimethylethoxy) methylthio) -6 ((1'R) -hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo 2,0] hept-2en-2-carboxylic acid (zwitterionic form)

Following the procedure described in Example 8, hydrogenolysis of p-nitrobenzyl- (4R, 5S, 6S) -3 - ((2-azido-1,1-dimethyl-ethoxy) methylthio) -6 - ((1'R) -hydroxyethyl) 4-Methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate affords the title compound in 28% yield as a colorless lyophilized powder. UV spectrum in water: kmax = 292 nm (E = 8000).

Example 11 (4R, 5S, 6S) -3- (Acetamidomethylthio) - ((1'R) -Hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-en-2- sodium or potassium carboxylate (Ie)

H ₃ C

nh-co-ch ₂

COONa / K

Nit-Nitrobenzyl- (4R, 5S, 6S) -3- (acetamidomethylthio) -6- ((1'R) hydroxyethyl) -4-methyl-7-oxo φ Nit Nit Nit Nit Nit Nit Nit Nit Nit Nit Nit Nit -1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

Following the procedure described in Example 7, using N- (mercaptomethyl) acetamide, purification by chromatography eluting with ethyl acetate gave the title p-nitrobenzyl ester in 36% yield as a pale yellow non-crystalline solid. IR in dichloromethane: 3600 (OH), 3430 (NH), 3050, 2950, 1770 (β-lactate C = O), 1705 (ester C = O) 1675 (amide I), 1605, 1525 (NO ₂ ), 1505 (amide II), 1350 (NO ₂ ), 1210, 1135 cm ^-1 .

NH-CO-CH ₃ n -h-co-ch ₃ (4R, 5S, 6S) -3- (acetamidomethylthio) - ((1'R) -hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2. Potassium Hept-2-ene-2-carboxylate

Following the procedure described in Example 7, the p-nitrobenzyl ester was hydrogenolyzed and lyophilized to give the title compound as a white powder in 54% yield. UV spectrum ve

water: K_ 2 _x 94 nm (ε = 8,000). NMR spectrum in deuterated  water (internal standard Me ₃ CD ₂ CD ₂ COONa): 1.31 (d, 3H, J = 7Hz) 3.30 (d, 3H, J = 6Hz) 2.01 (s, 3H), 3.42-3, 48 (2m, 2H) , 20 -

4.25 (2m, 2H), 4.36 and 4.66 (ABq, 2H, J = 14 Hz, _S-CH2 -N) ppm.

• · <

• · · · · · ·

Example 12 (4R, 5S, 6S) -3 - ((2-Aminoacetamido) methylthio) -6 - ((1'R) -hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept -2-ene-2-carboxylic acid (If)

p-Nitrobenzyl- (4R, 5S, 6S) -3 - ((2-azidoacetamido) methylthio) -6 ((1 ' R) -hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2 en-2-carboxylate

Following the procedure described in Example 7, using 2-azido-N (mercaptomethyl) -acetamide, after purification by chromatography, eluting with toluene-ethyl acetate (1: 1) and ethyl acetate, the title p-nitrobenzyl ester in the form of light yellow non-crystalline solids in 63% yield. IR spectrum in dichloromethane: 3600 (OH), 2900 (NH), 2100 (N ₃ ), 1770 (β-lactam)

C = O), 1705 and 1695 (ester and amide I), 1600 (C = C), 1520 (NO ₂ and amide II), 1350 NO, 1205, 1130 cm ^-1 .

NH-COCH ₂ -N ₃

nh-co-ch ₂ -nh ₂

O

COOH

·· * · • »· · • · 9 9 * 9- 9 9 9 9 9 • · 9 9 9 9 9 9 99 999 99 999

(4R, 5S, 6S) -3 - ((2-aminoacetamido) methylthio-6 - ((1 ¹ R) -hydroxyethyl) - 4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylic acid (zwitterionic form)

Following the procedure described in Example 8, the p-nitrobenzyl ester f

nydrogenolysed and then lyophilized to give the title compound as a white powder in 53% yield. UV spectrum in water: 1 _max 292 nm (E = 8000). NMR spectrum in deuterated water (internal standard Me ₃ CD ₂ CD ₂ COONa): 1.21 (d, 3H, J = 7 Hz), 1.30 (d, 3H, J = 6 Hz), 3.5 (complex signal, 2H),

3.77 (s, 2H), 4.25 (complex signal, 2H), 4.41 and 4.72 (ABkv,

2H, J = 14 Hz, _S-CH2 -N) ppm.

Example 13 (4R, 5S, 6S) -3 - ((2,3-dioxo-4-ethyl-piperazinyl) methylthio) - 6 ((R ¹ R) hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] Sodium or potassium hept-2 en-2-carboxylate (Ia)

p-Nitrobenzyl- (4R, 5S, 6S) -3 - ((2,3-dioxo-4-ethyl-piperazinyl) methylthio) -6 - (( ¹ R) hydroxyethyl) -4-methyl-7-oxo -1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate

X solution 118 mg (0.2 mmol) of p-nitrobenzyl- (4R, 5R, 6S) -3- (diphenyloxyphosphinoyloxy) -β - ((1'R) -hydroxyethyl) -4-methyl-7-oxo-1azabicyclo [ 3.2.0] Hept-2-ene-2-carboxylate in 1.5 ml of dry dimethylformamide is added at -50 ° C a solution of 49 mg (0.26 mmol) of 1-ethyl-4- (mercaptomethyl) piperazine-2, 3-dione in 1.2 ml deuterochloroform and then 44 μΐ (0.26 mmol) diisopropylethylamine.

• · · · · · · · · · · · · · · · · · · · · · · · · ·

The reaction mixture was allowed to warm to 0 ° C. After 3 hours at 0 ° C, the reaction mixture is diluted with 50 mL of ethyl acetate and the solution is left at room temperature for 5 minutes. This solution is washed successively with 20 ml of 10% aqueous potassium carbonate solution and 20 ml of brine. The organic layer was dried over magnesium sulfate and the solvent was evaporated in vacuo. The residue was purified by silica gel chromatography (4 g, 63-200 μπι) eluting with chloroform-methanol (9: 1) to give the title p-nitrobenzyl ester as a pale yellow non-crystalline solid in 89% yield. IR spectrum in dichloromethane: 3600, 3050, 2900, 1770, 1710, 1685, 1605, 1520, 1345, 1200, 1135 cm ^-1 .

(4R, 5S, 6S) -3 - ((2,3-dioxo-4-ethyl-piperazinyl) methylthio) -6 ((1'R) hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2] Potassium Hept-2 en-2-carboxylate

To 90 mg of 10% palladium on carbon, prehydrogenated at 0 ° C in a biphasic mixture of 4 mL of ethyl acetate and 10.6 mg (0.106 mmol) of potassium bicarbonate in 3 mL of water, was added a solution of 76 mg (0.14 mmol) of p. -nitrobenzyl- (4R, 5S, 6S) -3 ((2,3-dioxo-4-ethylpiperazinyl) methylthio) -6 ((1'R) -hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [ 3.2.0] hept-2-ene-2-carboxylate in 4 ml ethyl acetate. The mixture is then hydrogenated at normal pressure and 0 ° C. After 70 minutes the uptake of hydrogen (10.8 mL) was greatly slowed. An additional 30 mg of catalyst was added and hydrogenation was continued for a further 45 minutes. An additional 6.6 ml of hydrogen was consumed. The catalyst is filtered off, washed with 2 ml of ethyl acetate and 1 ml of water and the filtrate is transferred to a separatory funnel. The aqueous layer was separated and the organic layer was extracted with a solution of 3.6 mg (0.036 mmol) of sodium bicarbonate in 1 mL of water. The combined aqueous solutions were evacuated to remove residual ethyl acetate and then lyophilized at -30 ° C under high vacuum (0 mm Hg) to give the pure title compound as a white powder in 65% yield. UV spectrum in water λ _max = 292 nm (ε = 8000), 222 nm (ε = 11200). NMR spectrum in deuterated water (internal standard Me ₃ CD ₂ CD ₂ COONa): 1.1 - 1.3 (m, 6H), 1.30 (d, 3H, J = 6 Hz), 3.50 (m, 6H), 3.4-3.8 (m, 8H), 4.18 (m, 1H), 4.25 (m, 1H), 4.30 and 5.30 (ABkv, 2H, J = 14 Hz) , N-CH ₂ -S) ppm.

Example 14

Biological activity

1. In vitro antibacterial activity

Table I shows the inhibition diameter in mm after testing 10 µg of representative antibiotics (plate test results). The tests are performed in sterile polypropylene dishes (8.5 cm diameter) containing 10 ml of Difco Nutrient Agar. Inhibition is recorded after 18 hours at 37 ° C (inoculum of about 10 ^-5 cells).

Table I

[0057] Ib Ic Ie If Staph. aureus DSM 1104 39 39 36 33 Staph. aureus resistant 35 35 32 30 Staph. aureus 25768 28 26 27 Mar: 25 Staph. aureus Innsbruck 9 8 - 12 Eschericha coli DSM 1103 30 3 0 32 27 Mar:

• ·

lb Ic Ie If Eschericha coli TEM 1 33 33 34 33 Enterobacter cloacae DSM 30054 26 25 27 Mar: 23 Enterococcus 21 21 18 20 May Pseudomonas aer. DSM 1117 27 Mar: 25 16 18 Pseudomonas aer. resistant 17 13 - 14

2. β-lactamase inhibitory activity against isolated (cell-free) enzymes

Table II shows the β-lactamase inhibitory activity (mol per liter) as determined by the nitrocefin method. IC ₅₀ values are determined in 1 cm UV cell at 37 ° C after 15 minutes of preincubation of enzyme and inhibitor.

Table II

IC ₅₀ (mol / liter)

Compound Penicillinase from E. coli TEM 1 β-Lactamase from Enterobac1 ter cloacae Ia 2x10 4x10 ' lb 5,5x10 8,5x10 Ic 3x10 6x10

3. β-lactamase inhibitory activity against resistant bacteria

Table III shows the antibacterial activity (MIC, pg per ml) of ceftazidime (CAZ) without and when incubated with a representative compound Ia (potassium salt).

Table III

strain of bacteria enzyme CAZ (pg / ml) CAZ + Ia (pg / ml) E. cloacae EB 131 Type 1 Ceph'ase > 64 2 + 1 K. pneumoniae KL 140 CAZase > 64 2 + 1 K. pneumoniae KL 141 CAZase > 64 64 + 0.12 E. coli EC 227 CAZase > 64 0.25 + 0.5 E. coli EC 228 CAZase 16 1 + 0.5

strain of bacteria enzyme CAZ (pg / ml) CAZ + Ia (pg / ml) E. coli EC 225 TEM-5 64 2 + 0.5

4. Stability in phosphate buffer

Table IV shows the hydrolysis half-lives (in hours) of representative compounds in physiological phosphate buffer at 7.4 and 37 ° C as determined by the UV method.

compound half-life (hours) Ia 50 [0057] Ib 50 Ic 35 Id 50 Ie 40 lf 16 42

5. Oral activity

Table V shows the plasma level and half-life in mice following oral administration of compound Ia (potassium salt) of the invention (dose 25 mg per kg).

Table V ⁶

compound Plasma level (pg / ml) 10 minutes after application Approximate serum half-life (min) Ia 5.5 25

6. Cytotoxicity »Table VI shows the cytotoxicity of Saccharomyces cerevisiae representative compounds (1.0 mg) as determined by the agar diffusion method. Sterile 8.5 cm polypropylene dishes containing 10 ml of yeast and yeast agar are used. Cytotoxicity was recorded after 16 hours incubation at 30 ° C (inoculum of about 10 ⁵ ) cells.

• ·

Table VI

Compound Inhibitory diameter (mm) lb 0 Ie 0 lf 0

Example 15

Manufacture of pharmaceutical preparations

A single dosage form is prepared by mixing 60 mg of (4R, 5S, 6S) -3 - ((2-aminoethoxy) methylthio) - ((1'R) -hydroxyethyl) 4-methyl-7-oxo-1-azabicyclo [3.2. 0] hept-2-en-2-carboxylic acid (1b) with 20 mg of lactose and 5 mg of magnesium stearate and 85 mg of the mixture is transferred to gelatin capsule 3. Similarly, if less or more active ingredients are used, prepare other dosage forms and fill in No. 3 gelatin capsules. Similarly, larger gelatin capsules as well as compressed tablets and pills can also be made. The following examples represent the preparation of pharmaceutical preparations.

(4R, 5S, 6S) -3 - ((2-Aminoethoxy) methylthio) ((1'R) -hydroxyethyl) 4-methyl-7-oxo-1-aza-

bicyclo [3.2.0] hept-2-ene-2-carboxylic acid (1b) 120 mg Cornstarch 6 mg Magnesium stearate 232 mg Calcium hydrogen phosphate 192 mg Lactose 250 mg

The active ingredient is mixed with dibasic calcium phosphate, lactose and about half the amount of corn starch and coarse sieved. It is dried under high vacuum and sieved again through a sieve with a mesh size of 1.00 mm (sieve No. 16). Add the residue and compress the mixture per mg and a diameter of about 1.27 corn starch and magnesium stearates to obtain tablets each having a weight of 800 mm (0.5 in).

Parenteral solution

(4R, 5S, 6S) -3 - ((2-Aminoethoxy) methylthio) ((1'R) -hydroxyethyl) 4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene- 2-carboxylic acid (1b) 250 mg Sterile water (to be added from a separate vial by syringe just before use) 4 ml

(4R, 5S, 6S) -3 - ((2-Aminoethoxy) methylthio) ((1'R) -hydroxyethyl) 4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene ocular solution -2-carboxylic acid (1b) 50 mg Hydroxypropylmethylcellulose 5 mg

Sterile water (to be added from a separate ampoule with a syringe just before use) 1 ml

(4R, 5S, 6S) -3 - ((2-Aminoethoxy) methylthio) ((1'R) -hydroxyethyl) 4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene -2-carboxylic acid (1b) 50 mg Benzalkonium chloride 0.1 mg

Sterile water (to be added from a separate ampoule using a syringe just before use) 1 ml

Topically cream or ointment (4R, 5S, 6S) -3 - ((2-Aminoethoxy) methylthio) ((1'R) -hydroxyethyl) 4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2 -en-2-carboxylic acid (1b) Polyethylene glycol 4000

0 mg 40 mg

Polyethylene glycol 400 1.0 g

The active ingredient in the formulation above may be admixed alone or with other biologically active ingredients, for example, other antibacterial agents such as penicillins or cephalosporins, or other therapeutic agents such as probenicide.

It should be noted that the description and examples are given for illustration only and do not limit the scope of the present invention and that other embodiments falling within the scope of the present invention will be apparent to those skilled in the art.

Claims (12)

Compounds of structural formula I and their pharmaceutically acceptable salts, ester and amide derivatives, wherein R ¹ is a hydrogen atom, a hydroxymethyl group or a 1-hydroxyethyl group, R ² is a hydrogen atom or a methyl group, and R ³ is a pharmaceutically acceptable group which is bonded to the remainder of the molecule via a single oxygen-carbon bond or a single nitrogen-carbon bond and which is selected from the group consisting of substituted or unsubstituted: alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, N-heterocyclyl, heterocyclyloxy, heterocyclylcarbonyloxy, heterocyclylcarbonyloxy, heterocyclylcarbonyloxy, heterocyclylcarbonyloxy , thioacyloxy, alkoxycarbonyloxy, carbamoyloxy, thiocarbamoyloxy, heterocyclyloxycarbonyloxy, heterocyclyloxythiocarbonyloxy, N-heterocyclycarbamoyloxy, Nheterocyclylthiocyclylthiocyclylthiocyclylthiocarbonylcyclylthiocyclylthiocyclylthiocyclylthiocyclylthiocyclylthiocarbonyl rbonylaminoskupina, heterocyklyloxykarbonylaminoskupina, acylamino, alkoxycarbonylamino, alkoxythiokarbonylaminoskupina, thioacyklaminoskupina, heterocyklylkarbamoylaminoskupina N, N-heterocyklylthiokarbamoylaminoskupina ureido thiokarbamoylaminoskupina, imidoylaminoskupina, guanidino, N-heterocyklylalkoxykarbonylaminoskupina, N-heterocyclyl-alkylthiokar- bonylaminoskupina and N-sulfonylamino where the above alkyl, an alkenyl group, an alkynyl group, an acyl group, a thioacyl group or an imidoyl group which is part of the molecule contains 1 to 6 carbon atoms and the heterocyclyl group is monocyclic or bicyclic and contains 3 to 10 ring atoms of which one or more is selected from an oxygen atom, a sulfur atom and a nitrogen atom, and wherein the substituents of the above R ³ group may be: alkyl, acyl, thioacyl, heterocyclyl, hyd roxylová, hydroxyalkyl, alkoxy, hydroxyalkoxy, aminoalkoxy, amidinoalkoxyskupina, guanidinoalkoxyskupina, acyloxy, heterocyclyloxy, alkylheterocyklyloxyskupina, hydroxyalkylheterocyklyloxyskupina, aminoalkylheterocyklyloxyskupina, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, carbamoyloxy, alkylcarbamoyloxy, dialkylcarbamoyloxy, thiocarbamoyl group, alkylthiocarbamoyl group, dialkylthiokarbamoylová group, thiocarbamoyloxy, alkylthiokarbamoyloxyskupina, dialkyl thiocarbamoyloxy, mercapto, alkylthio, hydroxyalkylthioskupina, aminoalkylthioskupina, monoalkylaminoalkylthioskupina, dialkylaminoalkylthioskupina, amidinoalkylthioskupina, acylthio, heterocyclylthio, alkylheterocyklylthioskupina, hydroxyalkylheterocyklylthioskupina, aminoalkylheterocyklylthioskupina, karbamoylthioskupina, monoalkylkarbamoylthioskupina, dialkylk arbamoylthioskupina, thiokarbamoylthioskupina, alkylthiokarbamoylthioskupina, dialkylcarbamoyl thio, amino, monoalkylamino, hydroxyalkylamino, aminoalkylamino, dialkylamino, oxo, oximino or an alkylimino group, imidoylaminoskupina, alkylimidoylaminoskupina, dialkylimidoylaminoskupina, trialkylammonium group, cykloalkylaminoskupi49 on, heterocyclylamino alkylheterocyklylaminoskupina, heterocyclylcarbonylamino, alkylheterocyklylkarbonylaminoskupina, acylamino, amidino, monoalkylamidinoskupina , dialkylamidinoskupina, guanidino, alkylguanidinoskupina, dialkylguanidinoskupina ureido thiokarbamoylaminoskupina, alkylkarbamoylaminoskupina, thiokarbamoylaminoskupina, alkylthiokarbamoylaminoskupina, nitro, chlorine, bromine, fluorine, iodine, azido, cyano, alkylsulfinyl, alkylsulfonyl, sulfonamido, sulfamoyloxyskupina alk ylsulfamoyloxy, alkylsulfonyloxy or sulfo, sulfoxy, carboxamido, N-monoalkylcarboxamido, N, N-dialkylcarboxamido or carboxy wherein the substituents are independently occurring one or more times and their alkyl portions contain 1 to 6 carbon atoms and wherein the heterocyclic group is monocyclic or bicyclic and contains 3 to 10 ring atoms of which one or more selected from the group consisting of: an oxygen atom, a sulfur atom, and a nitrogen atom. Compounds according to claim 1, wherein R ¹ is hydrogen, hydroxymethyl or 1-hydroxyethyl, R ² is hydrogen or methyl and R ³ is selected from the group consisting of substituted or unsubstituted alkoxy, heterocyclyloxy, acyloxy, carbamoyloxy , N-heterocyclyl, acylamino, carbamoylamino, imidoylamino, wherein the above alkyl, acyl, thioacyl, or imidoyl moiety of the molecule contains 1 to 3 carbon atoms and the heterocyclyl group is monocyclic and has 3 to 6 atoms in a ring of which one or more is selected from the group consisting of: oxygen, sulfur and nitrogen, and wherein the substituents of the above R ³ groups may be: alkyl, acyl, thioacyl, heterocyclyl, hydroxyl, hydroxyalkyl, alkoxy, hydroxyalkoxy, aminoal alkoxy, amidinoalkoxyskupina, guanidinoalkoxyskupina, acyloxy group, heterocyclic oxy gr on, alkylheterocyklyloxyskupina, hydroxyalkylheterocyklyloxyskupina, aminoalkylheterocyklyloxyskupina, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, carbamoyloxy, alkylcarbamoyloxy, dialkylcarbamoyloxy, thiocarbamoyl group, alkylthiocarbamoyl group, dialkylthiokarbamoylová group, thiocarbamoyloxy, alkylthiokarbamoyloxyskupina, dialkyl thiocarbamoyloxy , mercapto, alkylthio, hydroxyalkylthioskupina, aminoalkylthioskupina, monoalkylaminoalkylthioskupina, dialkylaminoalkylthioskupina, amidinoalkylthioskupina, acylthio, heterocyclylthio, alkylheterocyklylthioskupina, hydroxyalkylheterocyklylthioskupina, aminoalkylheterocyklylthioskupina, karbamoylthioskupina, monoalkylkarbamoylthioskupina, dialkylkarbamoylthioskupina, thiokarbamoylthioskupina, alkylthiokarbamoylthioskupina, dialkylk arbamoylthioskupina, amino, monoalkylamino, hydroxyalkylamino, aminoalkylamino, dialkylamino, oxo, oximino, or an alkylimino group, imidoylaminoskupina, alkylimidoylaminoskupina, dialkylimidoylaminoskupina, trialkylammonium group, cycloalkylamino, heterocyclylamino, alkylheterocyklylaminoskupina, heterocyclylcarbonylamino, alkylheterocyklylkarbonylaminoskupina, acylamino, amidino, monoalkylamidinoskupina, dialkylamidinoskupina, guanidino, alkylguanidinoskupina, dialkylguanidinoskupina ureido thiokarbamoylaminoskupina, alkylkarbamoylaminoskupina, thiokarbamoylaminoskupina, alkylthiokarbamoylaminoskupina, nitro, chlorine, bromine, fluorine, iodine, azido, cyano, alkylsulfinyl, alkylsulfonyl, alkyl group, sulfonamido, sulfamoyloxyskupina, alkylsulfamoyloxyskupina, alkylsulfonyloxy or sulfo, sulfoxy, carboxy amido, N-monoalkylcarboxamido, Ν, N-dialkylcarboxamido or a carboxyl group wherein the substituents are independently occurring one or more times and their alkyl groups contain 1 to 6 carbon atoms and wherein the heterocyclic group is monocyclic and contains 3 to 6 ring atoms, wherein one or more is selected from the group consisting of: an oxygen atom, a sulfur atom, and a nitrogen atom. Compounds according to claim 1, wherein R ¹ is hydrogen, hydroxymethyl or 1-hydroxyethyl, R ² is hydrogen or methyl and R ³ is selected from the group consisting of: substituted alkoxy, acylamino, N-heterocyclyl and imidoylamino, wherein the above alkyl, acyl or imidoyl moiety of the molecule contains 1 to 3 carbon atoms and the heterocyclyl group is monocyclic and contains 3 to 6 ring members of which one or more is selected from the group consisting of: it consists of: an oxygen atom, a sulfur atom and a nitrogen atom and wherein the substituents of the abovementioned R ³ groups are basic groups such as amino, alkylamino, dialkylamino, imidoylamino, amidino and guanidino, wherein the alkyl, amidino and imidoyl groups contain 1 to 3 atoms carbon. Compounds according to claim 1, wherein R ¹ is 1-hydroxyethyl, R ² is methyl and R ³ is selected from the group consisting of OCH, NH OCH ₂ CH ₂ NH ₂ OCH ₂ CH, NH-CH O II NH-C-CH NH-C-CH, -NH, CH, IA oc-ch ₂ -nh ₂ CH, N -CH ₂ -CH CH, ¹⁴ O-C-CH AND CH, o-ch-ch ₂ -nh ₂ CH, A pharmaceutical composition comprising an antibacterially effective amount of at least one compound according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier or diluent. A method of preparing a composition according to claim 5, wherein an antibacterially effective amount of at least one compound according to any one of claims 1 to 4 is added to a pharmaceutically acceptable carrier or diluent. Use of at least one compound according to any one of claims 1 to 4 for the preparation of a pharmaceutical composition for inhibiting bacteria in a patient in need of such treatment. A pharmaceutical composition comprising an effective amount of at least one compound according to any one of claims 1 to 4 for inhibiting β-lactamase, a common β-lactam antibiotic, and a pharmaceutically acceptable carrier or diluent. A process for preparing a composition according to claim 8, wherein an effective amount of at least one compound according to any one of claims 1 to 4 and a conventional β-lactam antibiotic is added to a pharmaceutically acceptable carrier or diluent. Use of at least one compound according to any one of claims 1 to 4 and a conventional β-lactam antibiotic for the preparation of a pharmaceutical composition for inhibiting β-lactamase in a patient in need of such treatment. A process for the preparation of a compound according to claims 1 to 4, characterized by reacting a compound of the formula wherein R ¹ and R ² are as defined in claim 1, X is a leaving group and Y is a conventional carboxyl protecting group, with a thiol of the formula HS-CH ₂ -R ³ or an organic or inorganic salt, wherein R ³ is as defined claim 1. A process for the preparation of compounds according to claims 1 to 4, characterized in that the compound of the formula is reacted with a deprotecting agent, wherein R ¹ , R ² and R ³ are as defined in claim 1 and Y is a conventional carboxyl protecting group.