EP0436568A1 - Dihydroxyaryl 4-substituted monocarbams as antibiotics - Google Patents

Abstract

Monocarbames (analogs of 2-oxoazetidine) having a dihydroxyaryl group in position 4 of carbon, having an antimicrobial activity. The compounds of the present invention include compounds of formula (I) or a pharmaceutically acceptable salt, wherein R1 is an acyl group derived from a carboxylic acid; R2 hydrogen or methyl; and R3 hydrogen, K or any other pharmaceutically acceptable cation.

Description

DIHYDROXYARYL 4-SUBSTITUTED MONOCARBAMS AS ANTIBIOTICS

BACKGROUND OF THE INVENTION

This invention concerns novel monobactams which having a C-4 methyloxy substituent containing a hydroxy substituted aryl group. These compounds have enhanced antibacterial activity versus certain gram-negative bacteria such as Pseudomonas aeruginosa.

INFORMATION DISCLOSURE

A general background to β-lactam antibiotics can be found in a paper prepared from the Proceedings of the Third International Symposium, arranged by the Fine Chemicals and Medicinals Group of the Industrial Division of the Royal Society of Chemistry entitled "Recent Advances in the Chemistry of β-Lactam Antibiotics" (July 4, 1984).

Analogs of 2-oxoazetidine derivatives having antimicrobial activity are known in the art. Takeda, European Patent Applications 53-815 and 53-816. Squibb, U.S. Patent 4,478,749 and European Patent Application 76-7582A. Among the known monobactam analogs are those containing an O-substituted 2-aminothiazolyl-2-hydroxyiminoacetamido group on the C-3 position of the monobactam ring. Roussel UCLAF, European Patent Application 114-128-A. Various substituents have been described for the C-4 position of the monobactam ring. Takeda Patent Application EP 53-816.

SUMMARY OF THE INVENTION

The present invention concerns novel dihydroxyaryl substituted 2-oxoazetidine analogs and their use as microbial growth inhibitors. Novel intermediates and processes are also disclosed.

As illustrated in the formula chart, the present invention provides for both "racemic" mixtures and optically active isomers of compounds of Formula I wherein:

R₁ is a stable linking group such as a carbonyl, a C₁-C₁₀ alkyl which can contain heteroatoms (N, O or S), carbonyl groups, or C₆-C₁₂ aryl which can also contain heteroatoms (N, O or S) or carbonyl groups;

R₂ is hydrogen or methyl;

R₃ is K, hydrogen or other pharmaceutically acceptable cations. A C₁-C₁₀ alkyl can include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and isomeric forms thereof and can be substituted with fluoro, chloro or bromo groups. A C₆-C₁₂ aryl can include phenyl, α-naphthyl, β-naphthyl, m- methylphenyl, p-trifluoromethylphenyl and the like. The aryl groups can also be substituted with one to 3 hydroxy, C₁-C₃ alkoxy, C₁-C₃ alkyl, trifluoromethyl, fluoro, chloro, or bromo groups.

The choice of the R₁ group is only limited by the requirement that it be of a sufficiently stable nature to effectively bind the dihydroxy substituted aryl group to the hydroxy methyl group at the C-4 position as shown in Formula I.

The most preferred R₁ substituents are shown in Formula II.

The scope of this invention includes the pharmaceutically acceptable salts of the disclosed compounds. Such salts include the following pharmaceutically acceptable cations but are not limited to these: alkali metal ions such as potassium, sodium, lithium, alkaline earth metal ions such as magnesium or calcium and ammonium ions such as ammonium, tetralkylammonium and pyridinium.

It will be apparent to those skilled in the art that compounds of the invention herein described may contain several chiral carbons. All of the optically active, enantiomorphic and sterioisomeric forms are included within the scope of this invention. The invention also includes both the individual isomers and mixtures. Specifically the azetidines of this invention (Formula I) have chiral carbon atoms at positions C-3 and C-4 of the β-lactam ring. The preferred form is cis at centers 3 and 4 and the most preferred is 3(S) and 4(S) with regard to orientation at C-3 and C-4. The phrase "C-3 and C-4 cis isomers" means that the substituents at C-3 and C-4 are both oriented on the same side of the β-lactam ring.

Compounds of this invention are tested for in vitro antimicrobial activity using standard testing procedures such as the determination of minimum Inhibitory concentration (MIC) by agar dilution as described in "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically" (MFT) published January 1983 by the National Committee for Clinical Laboratory Standards, 771 East Lancaster Avenue, Villanova, PA, USA, 19084. Briefly, MIC values are determined in unsupplemented Mueller Hinton Agar (MHA). The compounds tested are diluted serially into molten MHA at 47ºC. The agar is poured into petri dishes and allowed to harden. The various bacterium used for testing are grown overnight on MHA at 35ºC and transferred to Trypticase soy broth (TSB) until a turbidity of 0.5 McFarland standard is obtained. The bacterium are diluted one to twenty in TSB and inoculated on the plates (1μl using a Steers replicator). The plates are incubated at 35ºC for 20 hours and the MIC is read to be the lowest concentration of drug that completely inhibits visible growth of the bacterium. The MIC test results of two compounds of this invention are found in Table 1.

DETAILED DESCRIPTION

A process for making compounds of Formula I wherein K₁ Is either of the formulas shown as Formula II is illustrated in Scheme 1.

The starting material for the synthesis of monobactams 3a and 3b is alcohol 1. This alcohol can be prepared as follows:

Preparation of Alcohol 1 (Scheme 1) cis-(±)-3-[2[(2-t-Butoxycarbonyl- amino-4-thiazolyl]-2-[(1-t-butoxycarbonylmethoxy)imino]]acetamido-4- hydroxymethyl-2-azetidinone.

To a stirred solution of zinc chloride (23.2 g) in anhydrous tetrahydrofuran (300 ml) at 0º C is added sodium borohydride (13.8 g) and the mixture is allowed to warm to room temperature and is stirred overnight. To the mixture is added cis-(±)-4-(methoxycarbonyl)-3- [((phenylmethoxy)carbonyl)amino]-2-azetidinone (39.2 g) and the reaction mixture is slowly heated to 65º C and stirred at that temperature for 2 hrs. The reaction mixture is cooled to 0° C and 6 N hydrochloric acid (200 ml) is added dropwise with stirring. The mixture is poured into ethyl acetate (1.0 1) and the organic layer is taken. The aqueous layer is saturated with sodium chloride and re- extracted with ethyl acetate (200 ml). The combined organic layer is washed with water (200 ml) and with 200 ml of brine twice and dried over anhydrous sodium sulfate. The solvent is concentrated under reduced pressure to afford a yellow oil which is purified by column chromatography on silica gel (ethyl acetate as eluent) to obtain a white solid, cis-(±)-4-(Hydroxymethyl)-3-[((phenylmethoxy)-carbonyl- )amino]-2-azetidinone.

To a stirred solution of this white solid, (19.5 g) in methanol (150 ml) is added palladium black (7.6 g) slurried in ethanol (25 ml) and the reaction mixture is stirred under 1 atm of hydrogen gas for 24 hrs. Toluene (100 ml) is added to the reaction mixture and it is stirred for 15 mins. The solid material is filtered and the filtrate solution is concentrated under reduced pressure to obtain 3-amino-4- hydroxymethyl-2-azetidinone which is used directly for the next step. The compound obtained above Is dissolved in methylene dichloride (200 ml) and dimethylformamide (500 ml) and cooled in the ice bath. To this cooled solution, 2-[(2-t-butoxycarbonylamino)-4-thiazolyl]- [(1-t-butoxycarbonylmethoxy)imino]-carboxylic acid (23.8 g) is added followed by dicyclohexylcarbodiimide (12.6 g) and 1-hydroxybenzotriazole (4.2 g). The reaction mixture is stirred for 3 hrs at 0º C. The precipitated solid Is filtered and the filtrate solution is partitioned between ethyl acetate (2.5 1) and water (1.0 1). The organic layer is taken and the aqueous layer Is washed with 500 ml of ethyl acetate twice. The combined organic layer is washed with aqueous sodium bicarbonate followed by brine and dried over anhydrous sodium sulfate. It is filtered and the filtrate solution is concentrated under reduced pressure and the residual material is chromatographed on silica gel eluting with hexane:ethyl acetate/1:1 followed by ethyl acetate alone to obtain 14.1 g of the alcohol 1.

Physical characteristics are as follows:

MP: 195ºC. (decomp.).

1H NMR (δ, CDCl₃) 8.4, 8.2, 7.35, 6.45, 5.5, 4.68, 4.2-3.7, 1.6, 1.50, 1.44.

The preferred process for Step 1 of Scheme 1 is that of Hassner, A. and V. Alexanian: Direct room temperature esterification of carboxylic acids, Tetrahedron Lett. 4475-4478, 1978. In this process approximately molar quantities of the desired acid, alcohol, and a carbodiimide, such as dicyclohexylcarbodiimide, and a catalytic amount, usually 0.1 equivalent, of an appropriate amine, such as N,N- dimethylaminopyridine, are combined in a suitable solvent, such as dimethylformamide, acetonitrile, or tetrahydrofuran and stirred, preferably, at ambient temperature. The time of reaction is variable from 0.5-24 hrs being required, although usually 3-4 hrs is sufficient. The dicyclohexyl urea formed during the reaction is removed by filtration. The esters 2 are isolated from the filtrate by extractive procedures and chromatography.

Step 2 of Scheme 1 embodies sulfonation followed by removal of blocking groups in a two step group.

The sulfonation step is carried out by dissolving or suspending the amide in a suitable solvent such as dimethylformamide or methylene dichloride and 1-3 equivalents of a sulfonating agent added. The preferred reagent is dimethylformamide-sulfur trioxide complex usual ly used as an approximately 1.0 molar solution in dimethyl formamide, K. Hofman and G. Simchen, Synthesis, 699-700 (1979). Pyridine-SO₃ complex is also operative. When the reaction is complete, the reaction mixture is diluted with water or phosphate buffer, n-tetrabutylammonium bisulfate added, and the sulfonated azetidine is extracted with a water immiscible solvent, such as methylene dichloride.

Blocking groups on R(2a or 2b, as shown in Scheme 1) are generally removed by treatment with acid. In the preferred method an excess of trifluoroacetic acid is added to a solution of the substrate while stirring in an ice bath. The residue resulting from evaporation of the solvent affords the n-tetrabutylammonium salt which is dissolved in water (a small volume of methanol may be added to hasten solution) and passed through a column of Dowex-50 resin in the K⁺ form. The column is washed with water. This eluant, now as the potassium salt, is passed over a column of HP-20 resin (Mitsubishi Chemical Industries). This column is eluted and combined which results in the obtention of monobactam 3a or 3b (Scheme 1).

Preferably, the o-benzyl groups are removed by catalytic hydrogenolysis over Pd. The preferred process is dependent upon the blocking group that is present.

When carbonate is substituted at Bn with phenylmethoxycarbonyl (Cbz), the substrate is dissolved in a suitable solvent such as tetrahydrofuran, ethyl acetate, dimethyl formamide, methanol, or ethanol. Hydrogenolysis in the presence of a hydrogenation catalyst, such as palladium black or palladium on a support, such as carbon, is accomplished by shaking or stirring in an atmosphere of hydrogen, preferably of less than 3 ata. When the reaction is completed, the catalyst is removed by filtration and the filtrate contains a solution of amine. If Bn is t-butoxycarbonyl, this blocking group must be removed by acid, such as trifluoroacetic acid, and the amine isolated by evaporation of the reaction mixture. If B_n is 9-fluorenylmethoxycarbonyl, the amine may be obtained by treatment with an organic base, such as piperidine or morpholine. Isolation of amine then may require chromatography. Amines need not be isolated.

The compounds of Formula I have broad spectrum antimicrobial activity. They are useful as surface sterilants and as additives to products where microbial populations are sought to be limited, e.g., animal feed. The compounds of Formula I are effective for treating bacterial Infections in mammals, including humans.

Various compositions of the present invention are presented for administration to humans and animals in unit dosage forms, such as granules, sterile parenteral solutions or suspensions, eye drops, solutions or suspensions, and water-in-oil emulsions containing suitable quantities of compounds of Formula I.

For parenteral administration, fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved In the vehicle. In preparing solutions the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder Is then sealed in the vial and an accompanying vial of water for injection is supplied to reconstitute the liquid prior to use. Parenteral suspensions can be prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

The term "unit dosage form", as used in the specification, refers to physically discrete units suitable as unitary dosages for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce the desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular effect to be achieved and (b) the limitations inherent In the art of compounding such an active material for use in humans and animals, as disclosed in detail in this specification, these being features of the present invention. Examples of suitable unit dosage forms in accord with this invention are powder packets, granules, cachets, ampoules, vials, segregated multiples of any of the foregoing, and other forms as herein described.

An effective quantity of the compound is employed in treatment. The dosage of the compound for treatment depends on many factors that are well known to those skilled in the art. They include for example, the route of administration and the potency of the particular compound. A dosage schedule for humans having an average weight of 70 kg is from about 50 to about 3000 mg of compound in a single dose, administered parenterally or in the compositions of this invention, are effective for treating bacterial infections. More specifically, the single dose is from about 100 mg to 2000 mg of compound. More specifically, the single dose is from about 100 mg to about 2000 mg of compound. It is expected that the dosages can be given one to four times per day.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, practice the present invention to its fullest extent. The following detailed examples describe how to prepare the various compounds and/or perform the various processes of the invention and are to be construed as merely illustrative, and not limitations of the preceding disclosure in any way whatsoever. Those skilled in the art will promptly recognize appropriate variations from the procedures both as to reactants and as to reaction conditions and techniques.

Example 1 Preparation of Monobactam: [3-[[(2-amino-4-thiazolyl)-

[(carboxymethoxy)imino]acetyl]amino]-4-oxo-1-sulfo-2- azetidinyl]methyl ester, 3,4-dihydroxy-, Benzoic acid, monopotassium salt.

The subject compound was prepared as described below and as shown in Scheme 2.

The 3.4-Di(phenylmethoxy)benzoic acid (3) was prepared by adding 5.0 g (0.26 mol) of ethyl 3,4-dihydroxybenzoate and 200 mL of acetone to a three-necked round-bottomed flask. While stirring 25.0 g (0.18 mol) of potassium carbonate, 2.0 g (0.012 mol) of potassium iodide, and 14.4 g (0.084 mol) of benzyl bromide were added to the solution. The solution was heated to reflux for 2.5 hr. The reaction was monitered by TLC (10:1 chloroform-methanol). After the solution cooled to room temperature, the solids were filtered and washed with acet one. The solvent was distilled under vacuum. The reside was partitioned between 70 mL of methylene chloride and 30 mL of water. The methylene chloride was washed with 20 mL of water, dried over sodium sulfate, and concentrated under vacuum. The excess benzyl bromide was distilled under vacuum at 90º C. A yield of 7.84 g (82.6%) of acid 2 was obtained.

Ethyl 3,4-di(phenylmethoxy)benzoate (7.8 g, 0.022 nol) was added to 78 mL of 95% aqueous ethanol and 39 mL of water. Sodium hydroxide (3.44 g, 0.087 mol) was added to the solution. The solution was warmed to 55º C. and became clear after 15 mln. The solution was heated at 55º C. for 2 hr. The ethanol was distilled under vacuum and a white precipitate formed. Warm water (120 mL) was added and upon shaking the precipitate gradually went into solution. The solution was cooled in an ice bath and acidified to pH 2 with 6N HCl. A white precipitate formed and was removed by filtration. A yield of 12.38 g of white solid was obtained after drying under vacuum. ¹³C nmr (MeOD) : δ70.0, 70.3 (OCH₂), 113.4, 115.1 , 123.5-128.4 (8 peaks), 136.7, 137.0, 147.7, 152.2, 166.9 (aromatic).

The alcohol (1) (1.00 g, 2 mmol) was dissolved In 25 mL of di- methylformamide. Acid (2) (1.34 g, 4 mmol), 0.824 g (4 mmol) of dicyclohexylcarbodiimide, and 0.049 g (0.4 mmol) of 4-dimethylamino- pyridine were added. The solution was stirred overnight. The precipitate was removed by filtration and the solvent was distilled under vacuum. The residue was taken up in 50 mL of methylene chloride and 100 mL of ethyl ether. The solution was washed with 30 mL of 2N HCl, 30 mL of water, then two 30 mL portions of 10% aqueous potassium bicarbonate, dried over sodium sulfate, and concentrated under vacuum. The residue was dissolved in 75 mL of warm acetone and a small amount of precipitate removed. The acetone was distilled under vacuum. The residue was chromatographed over 35 g of silica gel eluting with 2:1 chloroform-ethyl acetate. A yield of 0.198 g (12.1%) of azetidinone ester (3) was obtained.

¹³C nmr (MeOD): δ 28 (CH₃), 52.8 (C-4), 59.2 (C-3), 65.8 (OCH₃) , 71.3, 71.7, 72.0 (-C- 2, OCH₂), 114.2, 115.0, 116.6, 128, 138, 142, 149, 151 (aromatic), 161.0, 163.2, 166.9, 170.1 (C=O).

MS: Exact mass calcd for C₁₄H₄₆N₅O₁₁S₁ [M·=K]⁺: 816.2914.

Found: 816.2903.

Ester (3) (0.300 g, 0.37 mmol) was dissolved in 0.4 mL (0.4 mmol) of 1 M solution of dimethylformamide-sulfur trioxide (DMF-SO₃) in DMF. The reaction solution was stirred for 1 hr. The reaction was monitored by TLC (10:1 chloroform-methanol). Thereafter, 0.4 mL of 1M solution of DMF-SO₃ In DMF was added and solution stirred for 1 hr. The reaction solution was added to 50 mL of 0.5 M aqueous KH₂PO₄ solution. Addition of 0.250 g (0.74 mmol) of Bu₄NHSO₄ resulted in a clear solution. This solution was washed with 4-30 mL portions of methylene chloride. The methylene chloride solution was dried over sodium sulfate and concentrated under vacuum. The residue was dis- solved in 10 mL of dimethylformamide and 0.150 g of Pd black added. The resulting suspension was stirred for 2 hrs under H₂ delivered via a balloon. The Pd black was removed by filtration and washed with dimethylformamide. The dimethylformamide was distilled under vacuum. The residue was dissolved in 6 mL of trifluoroacetic acid while cooling in ice. The solution was cooled in an ice bath for 10 min and then stirred at room temperature for 1 hr. The solution was poured into 200 mL of 1:1 Skellysolve B-ethyl acetate. A fine white precipitate formed and was removed by filtration, washed with a small amount of ethyl acetate and dried under vacuum with no heat applied. A yield of 0.192 g of white solid was obtained. This solid was dissolved in water and methanol and passed over 20 mL of Dowex 50 K⁺. Fractions containing UV activity were combined and passed over 40 mL of HP-20. The column was eluted with 200 mL of water, 200 mL of 5% aqueous acetonitrile, 200 mL of 10% aqueous acetonitrile, and then 200 mL of 20% aqueous acetonitrile. Fractions were monitered via dipped-disc assay versus Escherichia coli and HPLC (ODS C-18 column, 30% acetonitrile-70% 0.1 M Bu₄NHSO₄ solution, 1.25 mL/min, 254 nm). On the basis of the results, fractions 2-20 were combined and lyophilized to yield 0.156 g of a white monobactam product (4).

MS: Exact mass calcd for C₁₈H₁₆N₂O₁₂S₂K₂ [M·+K]⁺: 635.9511. Found: 635.9525.

In vitro and in vivo antibacterial testing of the monobactam is shown in Tables 1 and 2. In vitro antibacterial testing showed substantial in vitro activity versus Ps. aeruginosa UC 9191.

Example 2 Preparation of Monobactam: [3-[[(2-amino-4-thiazolyl)[(carboxymethoxy)imino]acetyl]amino]-4-oxo-1-sulfo- 2-azetidinyl]methyl]ester, N-(3,4-dihydroxybenzoyl)-, Glycine, monopotas ium salt. The subject compound was prepared as described below and shown in Scheme 3.

The preparation of acid (1) was performed by adding glycine, ethyl ester hydrochloride (1.218 g, 8.7 mmol) and 1.3 mL (9.6 mmol) of triethylamine to 36 mL of acetonitrile. Water (1.8 mL) was added to dissolve the reactants. To this solution was added 2.9 g (8.7 mmol) of 3,4-di(phenylmethoxy)benzoic acid. Acetonitrile (50 mL) was added to dissolve the solids. Dicyclohexyldicarbodiimide (1.79 g, 8.7 mmol) was added. The reaction solution was stirred at room temperature and was monitered by TLC (2:1 Skellysolve B-ethyl acetate). TLC at 2.5 hr. showed trace amounts of starting material. The precipitate was removed by filtration. The solvent was distilled under vacuum and the residue was dissolved in methylene chloride. The solution was washed with 5% aq. KHCO₃, dried over sodium sulfate, and concentrated under vacuum. The crude product was chromatographed over silica gel eluting with 7:1 chloroform-ethyl acetate. A yield of 1.955 g (53.7%) of ester was obtained. The ester (1.955 g , 4.7 mmol) was suspended in 25 mL of 95% ethanol. Water (11.3 mL) and 0.744 g (18.6 mmol) of sodium hydroxide were added to the suspension. The mixture was heated to 50º C for 1 hr. The solution became clear and TLC (2:1 Skellysolve B-ethyl acetate) indicated the reaction was complete. The ethanol was dissolved under vacuum. The solids were dissolved In 100 mL of warm water. The slightly hazy solution was filtered to remove the solids. The solution was acidified with 4N HCl resulting in a white precipitate. This solution was cooled in ice for 20 min. The solids were removed by filtration and dried under vacuum at 60º C to yield 1.74 g (95.6%) of acid (2). mp: 171- 172º C.

Alcohol (2) (1.00 g, 2 mmol) was dissolved in 40 mL of tetra- hydrofuran (THF). To this solution were added 0.430 g (2 mmol) of DCC, 0.820 g (2 mmol) of acid (1), and 0.025 g (0.2 mmol) of DMAP. This solution was stirred overnight. The solvent was then distilled under vacuum. Acetone was added. The resulting precipitate was removed by filtration. The acetone was distilled under vacuum. The crude material was chromatographed over silica gel eluting with 20:1 chloroform-methanol. A yield of 0.688 g (40.8%) of acetidinone ester (3) was obtained.

₁₃C NMR (d-DMSO) : δ 28.0 (CH3), 47.7 (CH₂NH), 52 (C-4), 57 (C- 3), 65 (CH₂O), 70.1, 70.5 (CH₂O) , 71 (OCH₂C=O) , 81.4, 81.6 (-C-), 113.7, 121.3, 127.7, 128.0, 128.6, 137.2, 140-170 11 peaks (aromatic and C=O).

MS: Exact mass calcd for C₄₃H₄₉N₆O₁₂S₁ [M-+H]⁺: 873.3129.

Found: 873.3110.

Ester (3) (0.600 g, 0.708 mmol) was dissolved in 1.4 mL of 1M DMF-SO₃ in DMF. The reaction solution was stirred for 1 hr at RT. The reaction was monitered by TLC (10:1 chloroform-methanol) and trace starting material remained at 1 hr. To this solution was added 100 mL of 1M K₂HPO₄ solution. To the resulting cloudy solution was added 0.482 g(1.416 mmol) of Bu₄NHSO₄. The solution was extracted with 4-60 mL portions of methylene chloride. The organic solution was dried over sodium sulfate and concentrated under vacuum. The residue was dissolved in 20 mL of DMF. Pd black (0.300 g) was added. The reaction solution was stirred for 2 hrs under H₂ delivered by balloon. The catalyst was removed by filtration and washed with DMF. The filtrate was concentrated under vacuum and the residue was dissolved in 12 mL of trifluoroacetic acid. The solution was stirred in ice for 10 min then stirred at RT for 50 min. The solution was added to 400 mL of 1:1 ethyl ether-Skellysolve B. The resulting precipitate was removed by filtration and dried under vacuum. A white solid (0.450 g) was obtained. This material was passed over 20 mL of Dowex 50 K⁺ to obtain the potassium salt. The fractions containing uv activity were combined and chromatographed over 80 mL of HP-20 resin eluting with 400 mL of water, 400 mL of 5% aq. acetonitrile, 400 mL of 10% aq. acetonitrile, and 400 mL of 20% aq. acetonitrile. The fractions were assayed by HPLC (ODS C-18 column, 30% acetonitrile-70% 0.1M Bu₄NHSO₄ solution, 1.25 mL/min, 254 nm) and by dipped-disc assay versus Esherichia coli. On the basis of the results fractions 6-20 were combined and lyophilized to yield 0.254 g of white monobactam product (4).

MS: Exact mass was calcd for C₂₀H₁₉N₆O₁₃S₂K₂ [M·+K]⁺: 692.9726.

Found: 692.9747.

In vitro and in vivo antibacterial testing of the monobactam is shown in Tables 1 and 2. Both in vitro and in vivo testing showed the Example 2 monobactam to have substantial activity versus Ps. aeruginosa UC 9191. ¹

Claims

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ is a carbonyl group, a C₁-C₁₀ alkyl group, a C₁-C₁₀ alkyl group having substituted heteroatoms, carbonyl groups, C₆-C₁₂ aryl or C₆- C₁₂ aryl having substituted heteroatoms or carbonyl groups;

R₂ is hydrogen or methyl; and

R₃ Is hydrogen, K or other acceptable pharmaceutical cations.

2. The compound according to Claim 1 wherein R₁ is:

0

a. -C- or

0 0

b. -C-CH₂-NH-C-.

3. The compound according to Claim 2 wherein R₂ is hydrogen.

4. The compound according to Claim 3 wherein R₃ is K⁺.

5. The compound according to Claim 4 which is :

a. [3-[[(2-amino-4-thiazolyl)[(carboxymethoxy)imino]acetyl]- amino]-4-oxo-1-sulfo-2-azetldinyl]methyl ester, 3,4-dihydroxy-, Ben¬zoic acid, monopotassium salt, or

b. [3-[[(2-amino-4-thiazolyl)[(carboxymethoxy)imino]acetyl]- amino]-4-oxo-1-sulfo-2-azetidinyl]methyl]ester, N-(3,4-dihydroxybenzoyl)-, Glycine, monopotassium salt.

6. A method for treating bacterial infections in mammals, including humans comprising:

administering a pharmaceutically effective amount of a compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein:

R₁ is a carbonyl group, a C₁-C₁₀ alkyl group, a C₁-C₁₀ alkyl group having substituted heteroatoms, carbonyl groups, C₆ -C ₁₂ aryl or C ₆ -

C₁₂ aryl having substituted heteroatoms or carbonyl groups;

R₂ is hydrogen or methyl; and

R₃ is hydrogen, K or other acceptable pharmaceutical cations.

7. The method according to Claim 6 wherein R₁ is:

8. The method according to Claim 7 wherein R₂ is hydrogen.

9. The method according to Claim 8 wherein R₃ is K.

10. The method according to Claim 9 wherein said compound is

a. [3-[[(2-amino-4-thiazolyl)[(carboxymethoxy)imino]acetyl]- amino]-4-oxo-1-sulfo-2-azetidinyl]methyl ester, 3,4-dihydroxy-, Benzoic acid, monopotassium salt, or

b. [3-[[(2-amino-4-thiazolyl)[(carboxymethoxy)imino]acetyl]- amino]-4-oxo-1-sulfo-2-azetidinyl]methyl]ester, N-(3,4-dihydroxybenzoyl)-, Glycine, monopotassium salt.

11. The method according to Claim 6 wherein said compound is administered in a unit dosage form for parenteral administration.