WO1998047895A1 - 2-OXO-1-AZETIDINE SULFONIC ACID DERIVATIVES AS POTENT β-LACTAMASE INHIBITORS - Google Patents

Abstract

This invention relates to novel 2-oxo-1-azetidine sulfonic acid derivatives of formula (I) which are of value for use in combination with β-lactam antibiotics to increase their effectiveness against infections caused by β-lactamase producing bacteria. In said formula, (I) R1 is a 5-membered heterocyclic ring esp. thiophene, 2-amino thiazole. R2 is a moiety containing hydroxypyridone or N-hydroxypyridone.

Description

2-OXO-1-AZETID1NE SULFONIC ACID DERIVATIVES

AS POTENT β-LACTAMASE INHIBITORS

BACKGROUND OF THE INVENTION:

This invention relates to novel 2-oxo-1-azetidine sulfonic acid

derivatives which are of value for use in combination with β-lactam antibiotics

to increase their effectiveness in infection caused by β-lactamase producing

bacteria.

Of the commercially available β-lactam antibiotics, penicillins and

cephalosporins are best known and frequently used. Although widely used as

useful chemotherapeutic agents, enzymatic inactivation of β-lactam

antimicrobial agents has been an obstacle to the treatment of infection for as

long as these agents have been used. The production of enzymes that

degrade the β-lactam containing antimicrobial agents - penicillins and

cephalosporins - is an important mechanism of bacterial resistance, thereby

causing the antibiotic to lose it's antimicrobial activity. A novel approach to

countering these bacterial enzymes is the delivery of a β-lactam antimicrobial

agent together with an enzyme inhibitor. When a β -lactamase inhibitor is

used in combination with a β-lactam antibiotic, it can increase or enhance the

antibacterial effectiveness of the antibiotic against certain microorganisms.

The present invention provides certain novel 2-oxo-1-azetidine sulfonic

acid derivatives which are potent inhibitors of bacterial β-lactamases, particularly against class C β-lactamases (cephalosporinase). Aztreonam (US

pat. 4,775,670) is a known monobactam antibiotic. Several publications [(e.g.,

Antimicrobial Agents of Chemotherapy, vol. 22, pp. 414-420, 1982;

Chemotherapy, vol. 30, pp. 398-407 (1984); J. Antibiotics, vol. 35, no. 5, pp.

589-593 (1982); J. Antibiotics, vol. 43, no. 4, pp. 403-410 (1990)] suggest that

aztreonam possesses β-lactamase inhibitory properties.

SUMMARY OF THE INVENTION:

It is an object of the present invention to provide novel and new 2-oxo-

1-azetidine sulfonic acid derivatives having β-lactamase inhibitory activity,

particularly against class C β-lactamases (cephalosporinase).

It is a further object of the invention to provide pharmaceutical

compositions comprising a β-lactamase inhibitor of this invention in

combination with a β-lactam antibiotic and a pharmaceutically acceptable

carrier or diluent.

It is an additional object of the invention to provide an improved method

for the treatment of bacterial infections caused by class C β-lactamase

(cephalosporinase) producing bacteria in mammalian subjects, particularly in

humans.

Accordingly, this invention provides novel 2-oxo-1-azetidinesulfonic

acid derivatives having the formula (I)

and the pharmaceutically acceptable salts thereof,

wherein R, is a 5-membered heterocyclic ring and R₂ is selected from any one

of the following groups:

(a) -(CH₂)_n- where n = 1 or 2, and X = NH, N-OH or

HO pharmaceutically acceptable salts thereof;

eptable , phenyl

stituted)

(0)„

, N-OH or y acceptable

n = 2 or 3

, N-OH or y acceptable

(e)

le

(f) wherein X = NH; Y is selected

-Y— (CH₂)_n— from a 5-membered heterocyclic ring such as

HO oxadiazole, thiadiazole, o isoxazole, isothiazole and thiazole. n = 1 , 2, 3, or 4;

(g) wherein X = NH; Y is selected from a 5-membered heterocyclic ring such as

oxadiazole, thiadiazole, isoxazole, isothiazole and thiazole. n = 1, 2, 3, or 4; M is hydrogen or a pharmaceutically acceptable salt forming cation.

The present inventors found that the oxyimino group, i.e. =N-OR₂ in

formula (I) while in the 'anti' orientation provides excellent synergy with a β -

lactam antibiotic against class C β-lactamase (cephalosporinase) producing

microorganisms. In particular, they show markedly superior synergy in

combination with cephalosporins (e.g., ceftazidime) against Pseudomonas

aeruginosa.

The present inventors also found that the inhibitory activity against isolated β-lactamase (e.g., cephalosporinase from P. aeruginosa 46012) and

the synergy with a β-lactam antibiotic e.g., ceftazidime is greatly influenced by

the nature of the heterocyclic ring represented by R, and the nature of the

substituent in the oxime fragment represented by R₂.

Thus, thiophene is the preferred 5-membered heterocyclic ring as R,

and hydroxy pyridone including N-hydroxy pyridone is the preferred 6-

membered heterocyclic ring [attached through a spacer to the oxygen atom;

items (a) to (g)] as one of the components represented by R₂.

DETAILED DESCRIPTION OF THE INVENTION:

The β-lactamase inhibitors of this invention are the compounds having

the formula (I)

The present β-lactamase inhibitors of the invention are effective in

enhancing the antimicrobial activity of β-lactam antibiotics, when used in

combination to treat a mammalian subject suffering from a bacterial infection

caused by a β-lactamase producing microorganism. Examples of antibiotics

which can be used in combination with the compounds of the present

invention are commonly used penicillins such as amoxicillin, ampicillin, azlocillin, mezlocillin, apalcillin, hetacillin, bacampicillin, carbenicillin,

sulbenicillin, ticarcillin, piperacillin, mecillinam, pivmecillinam, methicillin,

ciclacillin, talampicillin, aspoxicillin, oxacillin, cloxacillin, dicloxacillin,

flucloxacillin, naficillin, pivampicillin; commonly used cephalosporins such as

cephalothin, cephaloridine, cefaclor, cefadroxil, cefamandole, cefazolin,

cephalexin, cephradine, cefuroxime, cefoxitin, cephacetrile, cefotiam,

cefotaxime, cefsulodin, cefoperazone, ceftizoxime, cefmenoxime,

cefmetazole, cephaloglycin, cefonicid, cefodizime, cefpirome, ceftazidime,

ceftriaxone, cefpiramide, cefbuperazone, cefozopran, cefepime, cefoselis,

cefluprenam, cefuzonam, cefpimizole, cefclidin, cefixime, ceftibuten, cefdinir,

cefpodoxime proxetil, cefteram pivoxil; cefetamet pivoxil, cefcapene pivoxil,

cefditoren pivoxil; commonly used carbapenem antibiotics such as imipenem,

meropenem, biapenem, panipenem and the like; commonly used

monobactams such as aztreonam and carumonam and salts thereof.

Furthermore, the β-lactamase inhibitors of the present invention can be

used in combination with another β-lactamase inhibitor to enhance the

antimicrobial activity of any of the above mentioned β-lactam antibiotics. For

example, the inhibitors of this invention can be combined with

piperacillin/tazobactam combination; ampicillin/sulbactam combination;

amoxycillin/clavulanic acid combination; ticarcillin/clavulanic acid combination;

cefoperazone/sulbactam combination, and the like.

R, in the formula (I) is a 5-membered heterocyclic ring containing from 1 to 4 heteroatoms independently selected from the group consisting of O, S

and N.

Preferred heterocyclic rings are:

Preferably, R, in the formula (I) is thiophene and 2-aminothiazole;

Even more preferably R, is thiophene.

R₂ in the formula (I) is selected from any one of the following groups:

l )

(0)_n , N-OH or acceptable

, N-OH or y acceptable

le

(f) -Y— (CHJ.- wherein X = NH; Y is selected from a 5 membered

HO heterocyclic ring, such as oxadiazole, thiadiazole,

O isoxazole, isothiazole and thiazole. n = 1. 2, 3, or 4;

from a such xazole,

Examples of 5-membered heterocyclic ring represented by "Y" include

oxadiazoles, isoxacoles, isothiazoles, thiaxoles and thiadiazoles.

Examples of the group for forming a pharmaceutically acceptable salt

represented by M in the formula (I) include the inorganic base salts,

ammonium salts, organic base salts, basic amino acid salts. Inorganic bases

that can form the inorganic base salts include alkali metals (e.g., sodium,

potassium, lithium) and alkaline earth metals (e.g., calcium, magnesium);

organic bases that can form the organic base salts include cyclohexylamine,

benzylamine, octylamine, ethanolamine, diethanolamine, diethylamine, triethylamine, procaine, morpholine, pyrrolidine, piperidine, N-ethylpiperidine,

N-methylmorpholine; basic amino acids that can form the basic amino acid

salts include lysine, arginine, omithine and histidine.

As will be appreciated by one skilled in the art, the compounds of .

formula (I) containing an acidic hydrogen atom other than the SO₃H group at

N-1 position are capable of forming salts with basic groups as mentioned

earlier. Such salts with pharmaceutically acceptable bases are included in the

invention.

Moreover, when M is hydrogen in the formula (I) it can form a zwitterion

by interacting with a basic nitrogen atom present in the molecule of formula

(I)-

A variety of protecting groups conventionally used in the β-lactam art

to protect the OH groups present in the items (a) to (g) can be used. While it

is difficult to determine which hydroxy-protecting group should be used, the

major requirement for such a group is that it can be removed without cleaving

the β-lactam ring and the protecting group must be sufficiently stable under

the reaction conditions to permit easy access to the compound of formula (I).

Examples of most commonly used hydroxy-protecting groups are:

diphenylmethyl, 4-methoxybenzyl, allyl, etc.

The compounds of this invention having the formula (I) can be

prepared using a variety of well known procedures as shown below: Process A:

Process B:

(IV) (III) (V)

R,

(VI) (i) Process C:

(VII) (III) (VI)

Process D:

(VI) ( III )

* (« ) Process E:

R*

( III ) ( I )

Each procedure utilizes as a starting material the known azetidine of the formula

Azetidines of the formula (III) are well known In the literature; see for

example the United Kingdom patent application no. 2,071 ,650 published September 23, 1981 ; J. Org. chem., vol. 47, pp. 5160-5167, 1982. In a preferred procedure the compounds of the formula (I) can be prepared by reacting azetidines of the formula (III) with compounds of the

formula

R in the presence of a coupling agent. R_λ and R₂ have the same meaning as

described before. It is preferable to first treat the compound of formula III with

one equivalent of a base; e.g. tributylamine or trioctylamine or sodium

bicarbonate. Preferably the reaction is run in the presence of a substance

capable of forming a reactive intermediate in situ, such as N-

hydroxybenzotriazole and a catalyst such as dimethylaminopyridine, using a

coupling agent such as dicyclohexylcarbodiimide. Exemplary solvents which

can be used for the reaction are dimethylformamide, tetrahydrofuran, dichloromethane or mixtures thereof.

The reaction of an acid formula (II) or a salt thereof, and a (3S)-3-

amino-2-oxo-1-azetidinesulfonic acid salt of formula (III) proceeds most readily

if the acid of formula (II) is in activated form. Activated forms of carboxylic

acids are well known in the art and include acid halides, acid anhydrides

(including mixed acid anhydrides), activated acid amides and activated acid

esters.

To be more concrete, such reactive derivatives are:

(a) Acid anhydrides:

The acid anhydrides include, among others, mixed anhydride with a

hydrohaloic acid e.g. hydrochloric acid, hydrobromic acid; mixed anhydrides

with a monalkyl carbonic acid; mixed anhydrides with an aliphatic carboxlic

acid, e.g., acetic acid, pivalic acid, valeric acid, isopentanoic acid, trichloroacetic acid; mixed anhydrides with an aromatic carboxylic acid e.g.,

benzoic acid; mixed anhydride with a substituted phosphoric acid e.g.,

dialkoxyphosphoric acid, dibenzyloxphosphoric acid, diphenoxyphosphoric

acid; mixed anhydride with a substituted phosphinic acid e.g.,

diphenylphosphinic acid, dialkylphosphinic acid; mixed anhydride with

sulfurous acid, thiosulfuric acid, sulfuric acid, and the symmetric acid

anhydride.

(b) Activated amides:

The activated amides include amies with pyrazole, imidazole, 4-substituted

imidazoles, dimethylpyrazole, triazole, benzotriazole, tetrazole, etc.

(c) Activated esters:

The activated esters include, among others, such esters as methyl, ethyl,

methoxymethyl, propargyl, 4-nitrophenyl, 2,4-dinitrophenyl, trichlorophenyl,

pentachlorophenyl, mesylpnehyl, pryanyl, pyridyl, piperidyl and 8-quinolythio

esters. Additional examples of activated esters are esters with an N-hydroxy

compound e.g., N,N-dimethylhydroxylamine, 1-hydroxy-2(1H)pyridone, N-

hydroxy succinimide, N-hydroxyphtyalimide, 1-hydroxy-1 H-benzotriazole, 1-

hydroxy-6-chloro-1 H-benzotriazole, 1 , 1 '-bis[6-triforomethyl)

benzotriazolyljoxlate (BTBO) N-ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline,

and the like.

Appropriate reactive derivatives of organic carboxylic acids are

selected from among such ones as mentioned above depending on the type of the acid used. When a free acid is used as the acylating agent, the

reaction is preferably carried out in the presence of a condensing agent.

Examples of the condensing agent are N,N'-dicyclohexylcarbodiimide, N-

cyclohexy-N'-morpholinoethylcarbodiimide, N-cyclohexyl-N'-(4-

diethylaminocyclohexyl) carbodiimmide and N-ethyl-N'-(3-

dimethylaminopropyl) carbodiimide.

The acylation reaction is usually carried out in a solvent. The solvent

includes water, acetone, dioxane, acetonitrile, methylene chloride, chloroform,

dichloroethane, tetrahydrofuran, ethyl acetate, dimethylforamide, pyridine and other common organic solvents inert to the reaction.

The acylation reaction can be carried out in the presence of an

inorganic base such as sodium hydroxide, sodium carbonate, potassium

carbonate or sodium hydrogen carbonate or an organic base such as

trimethylamine, triethylamine, tributylamien, N-methylmorpholine, N-

methylpiperidine, N,N-dialkylaniline, N,N-dialkylbenzylamine, pyridine,

picoline, lutidine, 1 ,5-diazabicyclo [4.3.0] non-5 ene, 1 ,4-diazabicyclo [2.2.2]

octane, 1 ,8-diazabicyclo [5.4.4] undecene-7, tetra-n-butylammonium

hydroxide. The reaction is usually conducted under cooling or at room

temperature.

The amides of formula v, which result from the coupling of acid IV (or a

salt thereof) and a (3S)-3-amino-2-oxo-1-azetidinesulfonic acid salt of formula

(III) can be oxidized to the corresponding ketoamide of formula VI (Process B). A wide variety of oxidation procedures may be used e.g., potassium

nitrosodisulfonate in water (or a mixed aqueous solvent), selenium dioxide in

dioxane; use of metal catalysts in the presence of a suitable co-oxidant.

Alternatively, the ketoamide (VI) can be prepared (Process C) by

coupling the keto acid (VII) with (3S)-3-amino-2-oxo-1-azetidinesulfonic acid of

formula III (or a salt thereof).

The compounds of this invention of formula (I) can also be prepared by

reacting a ketoamide (VI) (Process B or Process C) having the formula

with a hydroxylamine derivative (or a salt thereof) of formula

R₂-O-ΝH₂ ... (IX)

wherein R and R₂ have the same meaning as described before.

Alternatively, the ketoamide (VI) can be reacted with hydroxylamine

hydrochloride to provide the hydroxyimino derivative (VIII) (Process D).

Coupling of the hydroxyimino derivative of formula (VIII).

with the alcohol (R₂-OH, X) under Mitsunobu conditions (PPh₃/DEAD/THF) will

provide the compounds of formula (I). R₂ has the same meaning as described

before.

Alternatively, the compounds of formula (I) can be prepared by reacting

the hydroxyimino derivative (VIII) (Process E) with a compound of the formula,

R₂-X (XI) wherein X is a leaving group such as halogen, trifluoroacetate,

alkylsulfonate, arylsulfonate or other activated esters of alcohols.

Wherein R₂ has hereinbefore been defined.

The compounds of formula (I) which has a sulfo group (SO₃H) at N-1

position can generally react with a base to form a salt thereof. Therefore, the

compound (I) may be recovered in the form of a salt and such salt may be

converted into the free form or to another salt. And, the compound (I)

obtained in the free form may be converted into a salt.

The present invention also covers the compound (I) in a

pharmaceutically acceptable salt form. For conversion of the compound

obtained in the salt form into the free form, the method using an acid can be

used. Usable acids depend on the kind of protective group and other factors.

The acids include, for example, hydrochloric acid, sulfuric acid, phosphoric

acid, formic acid, acetic acid, trifluoroacetic acid, p-toluenesulfonic acid,

among others. Acid ion exchange resins can also be used. Solvents to be

used include hydrophilic organic solvents such as acetone, tetrahydrofuran, methanol, ethanol, acetonitrile, dioxane, dimethylformamide, dimethyl

sulfoxide, water and mixed solvents thereof.

Compounds of formula (II) are novel compounds and as such form an

integral part of this invention. The compounds of formula (II) can be prepared

by reacting an intermediate of formula (XII).

With the alcohol R₂-OH (X) under standard Mitsunobu conditions

(PPH₃/DEAD/THF; D.L. Hughes, The Mitsunobu Reactions in Organic

Reactions; P. Beak et al., Eds.: John Wiley & Sons, Inc.; New York, vol. 42,

pp. 335-656, 1992).

R has the same definition as defined before. R₃ is a protective group

for the carboxyl group. The protective groups for said carboxyl group include

all groups generally useable as carboxyl-protecting groups in the field of β-

lactam compound and organic chemistry, for example, methyl, ethyl, propyl,

isopropyl, allyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, benzhydryl,

methoxymethyl, ethoxymethyl, acetoxy methyl, pivaloyloxymethyl, trityl, 2,2,2-

trichloroethyl, β-iodoethyl, t-butyldimethylsilyl, dimethylsilyl, acetylmethyl,

among others.

The selection of the said protective group should be in such a way which at the end of the above described reaction sequence can be cleaved

from the carboxyl group under conditions that do not alter the rest of the

molecule. Preferred protective groups are methyl, ethyl, allyl.

The removal of protective groups R₃ can be effected by selective

application of a per se known method such as the method involving the use of

an acid, one using a base, the method involving the use of palladium tetrakis.

The method involving the use of an acid employs according to the type of

protective group and other conditions, inorganic acid such as hydrochloric

acid, phosphoric acid; organic acid like formic acid, acetic acid, trifluoroacetic

acid, acidic ion exchange resins and so on. The method involving the use of a

base employs, according to the type of protective group and other conditions,

inorganic bases such as the hydroxides or carbonates of alkali metals (e.g.,

sodium, potassium etc.) or alkaline earth metals (e.g., calcium, magnesium,

etc.) or organic bases such as metal alkoxides, organic amines, quarternary

ammonium salts or basic ion exchnage resins, etc.

The reaction termperature is about 0^° to 80^'C, more preferably about

10^' to 40^'C. The reaction is usually carried out in a solvent. As the solvent,

organic solvents such as ethers (e.g., dioxane, tetrahydrofuran, diethyl ether),

esters (e.g., ethyl acetate, ethyl formate), halogenated hydrocarbons (e.g.,

chloroform, methylene chloride), hydrocarbons (e.g., benzene, tolune) and

amides (e.g. dimethylformamide, dimethylacetamide) and a mixture thereof

are used. Alternatively, the intermediate of formula (II) can be prepared by

reacting the compound of formula (XII) with a compound of formula, R₂-X (XI)

wherein X is a leaving group such as halogen, trifluoroacetate, alkylsulfonate,

arylsulfonate or other activated esters of alcohols.

R₂ has the same meaning as defined before.

In another approach, the intermediate (II) can be prepared by reacting

a keto acid compound of formula (VII) with a hydroxylamine derivative (or its

salt) of formula, R₂-O-NH₂ (IX) using conventional procedures; see for

example, EP 0251 ,299 (Kaken); Tokkai Hei 6-263766 (Kyorin, Sept. 20,

1994).

The acids useful for eliminating the hydroxy-protecting group present in

the items (a) to (g) in the final step of the preparation of compound of the

formula (I) are formic acid, trichloroacetic acid, trifluoroacetic acid,

hydrochloric acid, trifluoromethanesulfonic acid or the like. When the acid is

used in a liquid state, it can act also as a solvent or an organic solvent can be

used as a co-solvent. Useful solvents are not particularly limited as far as

they do not adverely affect the reaction. Examples of useful solvents are

anisole, trifluoroethanol, dichloromethane and like solvents.

The 2-oxo-1 -azetidinesulfonic acid derivatives of the present invention

having the formula (I) in which M is hydrogen can be purified by standard

procedures well known in the art such as crystallization and chromatography

over silica gel or HP-20 column. The present invention encompasses all the possible stereoisomers as

well as their racemic or optically active mixtures.

Typical solvates of the compounds of formula (I) may include water as

water of crystallization and water miscible solvents like methanol, ethanol,

acetone or acetonitrile. Compounds containing variable amounts of water

produced by a process such as lyophilization or crystallization from solvents

containing water are also included under the scope of this invention.

The β-lactamase inhibitors of this invention of formula (I) are acidic and

they will form salts with basic agents. It is necessary to use a

pharmaceutically acceptable non-toxic salt. However, when M is hydrogen

and when there is an acidic hydrogen in the R₂ residue as exemplified by N-

OH, the compound of the formula (I) is diacid and can form disalts. In the

latter case, the two cationic counterions can be the same or different. Salts of

the compounds of formula (I) can be prepared by standard methods known in

the β-lactam literature. Typically, this involves contacting the acidic and basic

components in the appropriate stoichiometric ratio in an inert solvent system

which can be aqueous, non-aqueous or partially aqueous, as appropriate.

Favourable pharmaceutically-acceptable salts of the compounds of

formula (I) are sodium, potassium and calcium.

The compounds of the present invention including the

pharmaceutically-acceptable salts thereof are inhibitors of bacterial β-

lactamases particularly of cephalosporinases (class C enzyme) and they increase the antibacterial effectiveness of β-lactamase susceptible β-lactam

antibiotics - that is, they increase the effectiveness of the antibiotic against

infections caused by β-lactamase (cephalosporinase) producing

microorganisms, e.g. Pseudomonas aeruginosa, in particular. This makes the

compounds of formula (I) and said pharmaceutically acceptable salts thereof

valuable for co-administration with β-lactam antibiotics in the treatment of

bacterial infections in mammalian subjects, particularly humans. In the

treatment of a bacterial infection, said compound of the formula (I) or salt can

be mixed with the β-lactam antibiotic, and the two agents thereby

administered simultaneously. Alternatively, the compound of formula (I) or

salt can be administered as a separate agent during a course of treatment

with the antibiotic.

The compounds of the invention can be administered by the usual

routes. For example, parenterally, e.g. by intravenous injection or infusion,

intramuscularly, subcutaneously, intraperitoneally; intravenous injection or

infusion being the preferred. The dosage depends on the age, weight and

condition of the patient and on the administration route.

The pharmaceutical compositions of the invention may contain a

compound of formula (I) or a pharmaceutically acceptable salt thereof, as the

active substance mixed with a β-lactam antibiotic in association with one or

more pharmaceutically acceptable excipients and/or carriers.

Alternatively, the pharmaceutical compositions of the invention may contain a compound of formula (I) mixed with a β-lactam antibiotic in

association with a salt forming basic agent, e.g. NaHCO₃ or Na₂CO₃ in an

appropriate ratio.

The pharmaceutical compositions of the invention are usually prepared

following conventional methods and are adminstered in a pharmaceutically

suitable form. For instance, solutions for intravenous injection or infusion may

contain a carrier, for example, sterile water or, preferably, they may be in the

form of sterile aqueous isotonic saline solutions.

Suspensions or solutions for intramuscular injections may contain,

together with the active compound and the antibiotic, a pharmaceutically

acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g.

propylene glycol.

For oral mode of administration a compound of this invention can be

used in the form of tablets, capsules, granules, powders, lozenges, troches,

syrups, elixirs, suspensions and the like, in accordance with the standard

pharmaceutical practice. The oral forms may contain together with the active

compound of this present invention and a β-lactam antibiotic, diluents, e.g.

lactose, dextrose, saccharose, cellulose, cornstarch, and potato starch;

lubricants e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or

polyethylene glycols; binding agents e.g. starches, arabic gums, gelatin,

methylcellulose, carboxymethyl cellulose, disaggregating agents, e.g. a

starch, alginic acid, alginates, sodium starch glycolate, effervescing mixtures; dyestuffs; sweetners; wetting agents e.g., lecithin, polysorbates,

laurylsulphates and pharmacologically inactive substances used in

pharmaceutical formulations.

As already said, the oxyimino fragment i.e., =N-OR₂ in the formula (I) in

its 'anti' orientation provides excellent synergy with a β-lactam antibiotic

against class C β-lactamase (cephalosporinase) producing microorganisms,

P. aeruginosa, in particular. Thus this invention includes only those

compounds having the formula (I) in which the oxyimino group (=N-OR₂) is

specifically in the 'anti' orientation as shown in (I). Furthermore, the inhibitory

activity against the isolated β-lactamase (e.g., cephalosporinase from P.

aeruginosa 46012) and the synergy with a β-lactam antibiotic is greatly

influenced by the nature of the heterocyclic ring represented by R, and the

nature of the substituent in the oxime fragment represented by R₂.

Thus, thiophene and 2-aminothiazole are the preferred 5-membered

heterocyclic rings as R, and hydroxypyridone including N-hydroxypyridone as

represented by

HO where X = NH and N-OH

O

is the preferred 6-membered heterocyclic ring as one of the components

represented by R₂. Furthermore, in the above formula when X is N-OH the

compounds of formula (I) may have the following keto and enol tautomeric isomers; the keto - form being the preferred one.

Keto-isomer Enol-isomer

In most instances, an effective β-lactamase inhibiting dose of a

compound of formula (I) or a pharmaceutically acceptable salt thereof, will be

a daily dose in the range from about 1 to about 500 mg/kg of body weight

orally, and from about 1 to about 500 mg/kg of body weight parenterally.

However, in some cases it may be necessary to use dosages outside these

ranges. The weight ratio of the β-lactamase inhibitor of the present invention

and the β-lactam antibiotic with which it is being administered will normally be

in the range of 1 :20 to 20:1.

TEST FOR ANTIBACTERIAL ACTIVITY:

The compounds of the present invention in combination with

ceftazidime were tested for minimal inhibitory concentration (MIC) against the

bacteria listed in Table 3, according to the microbroth dilution method

described below. The MICs of the antibiotics (ceftazidime) alone, the MICs of

ceftazidime in combination with reference compounds particularly aztreonam

(ref. compd. I) and the MICs of the β-lactamase inhibitors (10 μg/ml) of the

present invention in combination with ceftazidime were determined with the same β-lactamase producing bacteria. After incubation in Mueller-Hinton

Broth (Difco) at 37° C for 18 h, the bacterial suspension was diluted and about

10⁵ CFU/ml was applied to the drug-containing Mueller-Hinton Broth in each

well of 96 well plate. The MICs were recorded after 18 h of incubation at 37°

C on the lowest combinations of drug that inhibited visible growth of bacteria.

Test for β-Lactamase Inhibitory Activity

The inhibitory activities of the present compounds (β-lactamase

inhibitors) against cephalosporinase was measured by spectrophotometric

rate assay using 490 nM and using nitrocefin as a substrate (J. Antimicrob.

chemother., vol. 28, pp 775-776, 1991). Table 1 shows the results.

Table 1

Table 1 (continued)

Table 1 (continued)

Table 1 (continued)

The following examples are provided to demonstrate the operability of the

present invention. The structures of the compounds were established by the

modes of synthesis and by extensive high field nuclear magnetic resonance

spectral techniques. Preparation of Compound 1 (Example 1)

Step l

Ethyl (E)-2-(2-thienyl)-2-(hydroxyimino) acetate

To a solution of ethyl 2-oxo-2-(2-thienyl) acetate (41 gm, 0.223 moles)

in ethanol (350 ml) was added hydroxylamine hydrochloride (23.2 gm, 0.334

moles) followed by pyridine (21.6 ml, 0.267 moles) and the mixture was

heated at 40-45° C overnight. Solvent was removed under reduced pressure.

Ethyl acetate (120 ml) was added and the mixture was cooled to 0° C; the

precipitated solid was collected by filtration (10 gm). The mother liquor was

concentrated under reduced pressure and the residue was taken in ether (400

ml); a stream of hydrogen chloride gas was bubbled through the solution for

35 min, stirred at room temp, for 0.5 hr. After removal of the solvent, the

precipitated solid was collected by filtration and washed thoroughly with ether

to give an additional amount of the product. Ethyl (E)-2-(2-thienyl)-2-

(hydroxyimino) acetate was obtained as white crystalline solid (38 gm, 92%

yield).

Step 2

Ethyl (E)-2-(2-thieny lϊ-2-|ϊ1.5-dibenzhydryloxy-4-pyridon-2-yl methoxy) imino] acetate

A solution of 1 ,5-dibenzhydryloxy-2-hydroxymethyl-4-pyridone (EP

0251 299) (46.7 gm) in dimethylformamide (200 ml) was gently heated until

the solution became clear. After cooling the solution to room temperature, 19.0 gm of ethyl (E)-2-(2-thienyl)-2-(hydroxyimino) acetate and 25.0 gm of

triphenyl phosphine was added. To this mixture was added diethyl

azodicarboxylate (15 ml) dropwise and the mixture was reacted at room

temperature for 2 hours under stirring. DMF was removed under reduced

pressure and the residue was taken in ethyl acetate (300 ml), washed

successively with water and brine and dried to obtain 40 gm of the above

identified compound.

NMR (DMSO-d₆): 51.30 (t, 3H), 4.35 (q, 2H), 5.00 (br, s, 2H), 6.00 (s, 1 H),

6.35 and 6.40 (2s, 2H), 7.20-7.45 (m, 11 H), 7.72 (d, 1 H), 7.77 (s, 1 H), 7.98 (d,

1 H).

Step 3

(E)-2-(2-thienyl)-2-f(1.5-dibenzhydryloxy-4-pyridon-2-yl methoxy) imino] acetic acid A mixture of ethyl (E)-2-(2-thienyl)-2-[(1 ,5-dibenzhydryloxy-4-pyridon-2-

yl methoxy) imino] acetate (from step 2, 17.74 gm) in methanol (350 ml) and

THF (75 ml) was stirred at room temperature until the reaction mixture

became clear. To this mixture an aqueous solution of NaOH (1.58 gm

dissolved in 100 ml of water) was added dropwise over 20 min, and the

mixture was stirred at room temp, for 5 hr. After completion of the reaction,

solvent was removed under reduced pressure. The residue was diluted with

water (350 ml), cooled in an ice-bath and carefully acidified by dropwise

addition of dilute hydrochloric acid (3.5 ml of cone. HCI dissolved in 15 ml of water) with vigorous stirring. At pH 2~3, fine white solid started to precipitate

out. To the mixture chloroform (500 ml) was added and partitioned. The

aqueous layer was separated out and reextracted with CHCI₃ (2 x 200 ml).

The combined organic layer was concentrated to dryness. The solid thus

obtained was suspended in ether (100 ml), stirred for 30 min, filtered off and

washed thoroughly with ether (2 x 30 ml). The solid was dried over P₂O₅ under

vacuum to obtain 17.5 gm of the above identified product.

NMR (DMSO-d₆): δ4.96 (br, s, 2H), 6.01 (s, 1H), 6.37 and 6.40 (2s, 2H),

7.20-7.50 (m, 1 H), 7.70 (d, 1 H), 7.80 (s, 1 H), 7.95 (d, 1 H).

Step 4

(3S)-trans-3-[(E)-2-(2-thienyl)-2-{(1.5-dibenzhydryloxy-4-Pyridon-2-yl methoxy) imino}acetamido]-4-methy l-2-oxoazetidine-1 -sulfonic acid- potassium salt

A mixture of (3S)-trans-3-amino-4-methyl-2-oxoazetidine-1 -sulfonic acid

[1.37 gm, J. Org. Chem.. 47, 5160, (1982)], (E)-2-(2-thienyl)-2-[(1 ,5-

dibenzhydryloxy-4-pyridon-2-ylmethoxy) imino] acetic acid (step 3, 4.89 gm),

DCC (1.73 gm) and 1-hydroxybenzotriazole (1.13 gm) in DMF (50 ml) was

stirred under N₂ at room temperature for 30 min, KHCO₃ (0.762 gm) was

added in one portion and the mixture was stirred at room temperature for 24

hr. The solid was filtered off and the filtrate was evaporated under reduced

pressure to remove DMF. The crude product was taken in THF (50 ml) and

cooled to -30° C. The solid was filtered off and the filtrate was evaporated to

dryness. The residue (7.5 gm) was dissolved in 10 ml of methanol and diluted with ether with stirring at room temperature. The separated fine solid was

collected by filtration. The filtrate was concentrated again to give a foam

which was dissolved in minimum amount of methanol and diluted with ether.

The separated solid was collected by filtration and air dried. Total mass of the

product was 6.0 gm, which was used for the next step.

Step 5

(3S)-trans-3-[(E)-2-(2-thienyl)-2-{(1.5-dihydroxy-4-pyridon-2-ylmethoxy) imino}acetamido]-4-methyl-2-oxoazetidine-1 -sulfonic acid 25.0 gm of (3S)-trans-3-[(E)-2-(2-thienyl)-2-{(1 ,5-dibenzhydryloxy-4-

pyridon-2-yl methoxy) imino} acetamido]-4-methyl-2-oxoazetidine-1 -sulfonic

acid, potassium salt (from step 4) was taken in 40 ml of dry methylene

chloride and anisole mixture (1 :1) and cooled to -10 C, trifluoroacetic acid (46

ml) was added dropwise over 10 min, an additional amount of methylene

chloride (20 ml) was added and the mixture was stirred at -10° C for 2 hr. All

the volatile solvents were removed under reduced pressure and the residue

was digested with ether and the solvent was decanted off. The residue was

finally digested with ethyl acetate and the off-white precipitated solid was

collected by filtration. The solid thus obtained was crystallized from methanol-

water to provide 8.0 gm of the target compound as white powder; m.p. 170^*C

(decomp.) C, H analysis: Calcd, C, 40.67; H, 3.41 ; N, 11.86

Found, C, 40.14; H, 3.46; N, 11.44

NMR (DMSO-d₆): δ 1.41 (d, 3H, J = 6.15 Hz), 3.78-3.82 (m, 1 H), 4.49 (dd, 1 H,

J = 2.68 Hz and 8.25 Hz), 5.58 (s, 2H), 7.10 (s, 1H), 7.27 (dd, 1 H, J = 4.0 Hz

and 5.0 HZ), 7.82 (dd, 1 H, J = 1.0 Hz and 4.0 Hz), 8.01 (dd, 1H, J = 1.0 Hz

and 5.0 Hz), 8.28 (s, 1 H), 9.40 (d, J = 8.25 Hz).

Preparation of Compound 14 (Example 2)

Step l

Ailyl (E)-2-(2-thienyl)-[(5-benzhydryloxy-4-pyranon-2-yl methoxy) imino]acetate

To an ice cooled solution of allyl (E)-2 (2-thienyl)-2-(hydroxyimino)

acetate (1.0 gm, 4.734 mmol), 5-benzhydryloxy-2-(hydroxymethyl) pyran-4-

one (1.46 gm, 4.734 mmol) and triphenylphosphine (1.24 gm, 4.734 mmol) in

dry TFH (30 ml) under nitrogen was added dropwise diethyl azodicarboxylate

(820 μl, 5.208 mmol). The reaction mixture was stirred at room temperature

overnight and concentrated under reduced pressure to afford the crude as

yellow gum (4.93 gm). The product was purified by silica gel column

chromatography using hexane-ethyl acetate (2:1) as eluant to provide the title

compound as a gummy foam in 47% yield (1.11 gm).

¹H NMR (DMSO-d₆): δ 8.15 (s, IH); 8.01 (dd, IH, J = 0.85 Hz and 5.0 Hz); 7.76

(dd, 1 H, J= 1.0 Hz and 4.0 Hz); 7.23-7.46 (m, 11 H); 6.48 (s, IH); 6.47 (s, 1 H);

5.91-6.11 (m, 1 H); 5.28-5.43 (m, 2H); 5.25 (s, 2H); 4.84 (d, 2H,J = 5.51 Hz) Step 2

Sodium (E)-2-(2-thienyl)-2-[(5-benzhydryloxy-4-pyranon-2-yl methoxy)imino] acetate

A solution of allyl (E)-2-(2-thienyl)-2-[5-benzhydrxloxy-4-pyranon-2-yl

methoxy)imino] acetate (from Step 1 , Example 2, 2.09 gm, 4.17 mmol), in a

mixture of methylene chloride and ethyl acetate (25 ml : 55 ml) was treated

with sodium 2-ethylhexanoate (693 mg, 4.17 mmol), triphenylphosphine (109

mg, 0.417 mmol) and Pd (PPh₃)₄ (193 mg, 0.167 mmol) and the mixture was

stirred at room temperature for 5 hrs. The resulting precipitate was filtered,

washed with a mixture of ether-ethyl acetate (1 :1) and dried in vacuo to afford

a white solid 2.0 gm, 99% yield).

¹H NMR (DMSO-d₆); δ 8:10 (s, 1H); 7.72 (dd, 1 H, J = 0.9 Hz and 5.0 Hz); 7.61

(dd, 1H, J = 1.0 Hz and 4.0 Hz); 7.24-7.45 (m, 10H); 7.10 (dd, 1H, J = 4.0 Hz

and 5.0 Hz); 6.47 (s, 1 H); 6.34 (s, 1H); 4.96 (s, 2H).

Step 3

(E)-2-(2-Thienyl)-2-[(5-benzhydryloxy-4-pyridon-2-yl methoxy)imino] acetic acid

A suspension of sodium (E)-2-(2-thienyl)-2-[(5-benzhydryloxy-4-

pyranon-2-yl methoxy) imino] acetate (from Step 2, Example 2, 1.07 gm, 2.07

mmol) in 30% NH₄OH (25 ml) in a steel bomb was heated at 90^' C for 1 hr.

On cooling to room temp. N₂ gas was bubbled through and the brown solution

was cooled to 0^°C and the pH was carefully adjusted to ~ 2.0 with 50% HCI. The precipitated solid was filtered off, washed with water, ethyl acetate,

hexane and dried successively to give a beige solid in 60% yield (610 mg).

¹H NMR (DMSO-d₆): δ 7.89 (dd, 1 H, J = 0.8 and 5.0 Hz); 7.76 (dd, 1 H, J = 1.0

and 4.0 Hz); 7.69 (s, 1 H); 7.16-7.61 (m, 11 H); 6.61 (s, 1 H); 6.58 (s, 1 H); 5.16

(s, 2H).

Step 4

(3S)-trans-3-r(E⁾-2-(2-thieny)-2-{5-benzhydryloxy-4-pyridon-2-yl methoxy) imino} acetamido]-4-methyl-2-oxoazetidine-1 -sulfonic acid, potassium salt

A mixture of (3-S)-trans-3-amino-4-methyl-2-oxoazetidine-1 -sulfonic

acid, potasssium salt [373 mg, 1.71 mmol, J. Orα. Chem.. 47. 5160 (1982)],

(E)-2-(2-thienyl)-2-[(5-benzhydryloxy-4-pyridon-2-yl methoxy)imino] acetic acid

(Step 3, Example 2, 656 mg, 1.425 mmol), DCC (294 mg, 1.425 mmol) and 1-

hydroxybenzotriazole (193 mg, 1.425 mmol) in dry DMF (40 ml) was stirred

under N₂ at room temp, for 20 hrs, filtered and the filtrate was concentrated to

dryness under reduced pressure to give a gum which was dissolved in a

mixture of acetonitrile-water (7:3) and freeze dried to give a brown fluffy mass

(1.21 gm). The product was purified over a HP-20 column using acetonitrile-

water as eluant. The appropriate fractions were collected and freeze dried to

afford the title compound as a light brown fluffy solid in 63% yield (590 mg).

¹H NMR (DMSO-d₆): δ 9.34 (d, 1 H, J =*8.3 Hz); 8.12 (s, 1 H); 7.97 (dd, 1 H, J =

1.0 Hz and 5.0 Hz); 7.75 (dd, 1 H, J = 1.0 Hz and 3.8 Hz); 7.21-7.52 (m, 11 H);

7.06 (s, 1H); 6.76 (s, 1H); 5.37 (s, 2H); 4.47 (dd, 1H, J = 2.7 Hz and 8.3 Hz); 3.76-3.81 (m, 1 H); 1.40 (d, 3H, J = 6.14 Hz).

Step 5

(3S)-trans-3-[(E)-2-(2-thienyl)-2-{(5-hydroxy-4-pyridon-2-yl methoxy)imino} acetamido]-4-methy l-2-oxoazetidine-1 -sulfonic acid_f potassium salt

A suspension of (3S)-trans-3-[(E)-2-(2-thienyl)-2-{(5-benzhydryloxy-4-

pyridon-2-yl methoxy)imino} acetamido]-4-methyl-2-oxoazetidine-1 -sulfonic

acid, potassium salt (from Step 4, Example 2, 570 mg, 0.863 mmol) in dry

anisole (1ml) at - 10^'C was treated with trifluoroacetic acid (1.33 ml) and

stirred for 2 hr. The reaction mixture was concentrated under reduced

pressure to give a gum which was triturated with diethyl ether followed by a

mixture of ethyl acetate-ether (5:1) to give a light brown solid (440 mg).

Purification over a HP-20 column using acetone-water 1 :10) as eluant and

after freeze drying of the appropriate fractions, the title compound was

obtained as a pale yellow fluffy solid, 226 mg (53% yield).

¹H NMR (DMSO-d₆): δ 9.36 (br, s, 1 H); 7.93 (dd, 1 H, J = 0.9 Hz and 5.0 Hz);

7.77 (dd, 1 H, J = 0.9 Hz and 4.0 Hz); 7.53 (br, s, 1 H); 7.21 (dd, 1H, J = 4.0 Hz

and 5.0 Hz); 6.46 (br, s, 1 H); 5.20 (s, 2H); 4.49 (s, 1 H); 3.81-3.86 (m, 1 H);

1.41 (d, 3H, J = 6.2 Hz). Table 2 H NMR spectra of some representative compounds

Compd No Solvent δ (ppm)

3 L O 7 71-7 79 (m, 2H), 7 56 (s, IH), 7 15 (s, I H), 6 60 (s, IH), 5 37 (s, 2H), 4 54 (d, IH), 4 23-

4 34 (m, I H), 1 53 (d, 3H, J = 6 0 Hz)

DMSO-d₆ 9 32 (d, I H, J = 8 3 Hz), 8 18 (s, IH), 8 02 (d, IH, J = 4 1 Hz), 7 70-7 80 (m, IH), 7 26 (t, IH,

J = 5 0 Hz), 6 95 (s, IH), 5 83 (q, IH, J = 7 0 Hz), 4 40-4 48 ( , IH), 3 70-3 80 (br, m, IH),

1 66 (d, 3H, J = 7 0 Hz), 1 38 (d, 3H, J = 5 9 Hz) DMSO-dβ 9 33 (br, s, I H), 7 95 (d, IH, J = 4 7 Hz), 7 78 (t, IH, J = 3 2 Hz), 7 50 (s, IH), 7 22 (t, IH, J =

4 5 Hz), 6 36 (d, IH, J = 2 0 Hz), 5 75-5 95 (m, 2H), 5 03-5 15 (m, 2H), 4 41 (br, s, IH), 3 75-

3 83 (m, IH), 2 55-2 85 (m, 2H), 1 37 (d, 3H, J = 5 95 Hz)

DMSO-d₆ 9 41 (br, s, IH), 7 92-7 98 (m, I H), 7 73-7 80 (m, IH), 7 40-7 58 ( , 2H), 7 10-7 30 (m, 4H),

6 93 (s, IH), 6 3 1 (s, IH), 4 40-4 48 (m, IH), 3 75-3 90 (m, IH), 1 39 (d, 3H, J = 6 1 Hz)

DMSO-d₆ 9 35 (d, I H, J = 8 4 Hz), 7 97 (s, I H), 7 88 (dd, I H, J = 1 0 Hz and 5 0 Hz), 7 80 (dd, IH, J =

1 0 Hz and 4 0 Hz), 7 20 (dd, I H, J = 4 0 Hz and 5 0 Hz), 7 05 (s, IH), 4 50 (dd, IH, J = 2 6

Hz and 8 2 Hz), 4 46 (t, 2H, J = 6 0 Hz), 3 94 (s, 2H), 3 83 (m, IH), 3 00 (t, 2H, J = 6 0 Hz),

1 41 (d, 3H, J = 6 0 Hz)

Table 2 (continued) . _

Compd no Solvent δ (ppm)

^~8 DMSO-dβ 9 35-9 45 (m, I H), 7 50-7 95 (m, 3H), 7 12-7 20 (m, IH), 6 81 (s, IH), 4 35-4 70 (m, 4H),

4 08-4 20 (m, I H), 3 79-3 89 (m, I H), 2 98-3 55 (m, 2H), 1 42 (d, 3H, J = 6 1 Hz)

9 DMSO-dβ 9 15 (br, s, I H), 7 79-7 84 (m, 2H), 7 56-7 66 (m, 3H), 7 10-7 15 (m, 2H), 6 96-7 05 (m, IH),

6 67 (s, IH), 5 1 1 (s, 2H), 4 40-4 46 (m, 3H), 3 78-3 84 (m, IH), 3 60-3 70 (br, m, 2H), 1 40 (d, 3H, J = 6 2 Hz)

10 DMSO-d₆ 8 75 (br, s, IH), 7 75-7 90 (m, 5H), 7 58 (s, I H), 7 10-7 18 (m, 2H), 6 52 (s, IH), 5 33 (s, 2H),

4 36-4 50 (m, 3H), 3 53-3 85 (m, 3H), 1 40 (d, 3H, J = 6 0 Hz)

1 1 DMSO-dβ 8 10 (br, s, IH), 7 96 (d, IH, J = 5 0 Hz), 7 90 (br, s, IH), 7 74 (d, IH, J = 3 2 Hz), 7 24 (s,

I H), 7 16 (t, I H, J = 5 0 Hz), 6 04 (s, I H), 5 14 (s, 2H), 4 28-4 52 (m, 3H), 3 89-3 98 (m, I H),

3 56 (br, s, 2H), 1 96 (s, 3H), 1 41 (d, 3H, J = 6 1 Hz)

12 DMSO-de 9 30 (br, s, IH), 8 89 (br, t, IH), 7 84-7 90 (m, 2H), 7 68 (s, IH), 7 05-7 30 (m, 3H), 6 70 (s,

IH), 6 65 (s, IH), 5 08 (s, 2H), 4 40-4 50 (m, 3H), 3 80-3 90 (m, IH), 3 58-3 70 (m, 2H), 1 41

Table 2 (continued)

Compd no Solvent δ (ppm)

13 DMSO-dβ 9 30 (br, s, IH), 8 58 (br, s, IH), 7 83-7 88 (tn, 2H), 7 76 (dd, IH, J = 1 0 Hz and 4 0 Hz), 7 38

(s, IH), 7 15 (dd, I H, J = 4 0 Hz and 5 0 Hz), 4 47 (br, s, IH), 4 39 (t, 2H, J = 5 3 Hz), 3 80-

3 84 (m, IH), 3 55-3 67 (m, 2H), 1 40 (d, 3H, J = 6 2 Hz)

15 DMSO-dβ 9 28 (br, d, IH), 7 68 (s, IH), 6 90 (s, IH), 6 59-6 66 (m, 2H), 6 10 (dd, IH, J = 1 0 Hz and 2 0

Hz), 5 19 (s, 2H), 4 42 (br, s, IH), 3 80-3 85 (m, IH), 3 50 (s, 3H), 1 39 (d, 3H, J = 6 0 Hz)

16 DMSO-dβ 9 3 (d, IH, J = 8 0 Hz), 7 80-7 93 (m, 4H), 7 16-7 40 (m, 3H), 6 89 (s, I H), 5 21 (s, 2H),

4 48-4 65 (m, 4H), 4 05-4 12 (m, IH), 3 74-3 82 (m, IH), 3 50-3 70 (m, I H), 1 27 (d, 3H, J = 6 1 Hz)

TABLE 3 ANTIBACTERIAL ACTIVITY OF CEFTADIZ1ME WITH COMPOUNDS (β-LACTAMASE INHIBITOR)

MICo ceftazidime (μg/ml)

Organism alone with with with with with with

Ref Compd I Ref Compd II Ref Compd III Compd Compd 2 Compd 3

(Aztreonam)

E cloacae 40054 >32 >32 32 10 10 10 10

E cloacae MNH-2 >32 >32 >32 >32 20 40 20

E cloacae P99 >32 >32 16 >32 >32 >32 32

E aerogenes S-95 >32 32 80 40 10 40 05

E aerogenes 41006 >32 >32 >32 >32 ~ >32 >32

M morganii 36014 32 — <025 <025 <025 <025 <025

M morganii 36030 >32 <025 <025 <025 <025 <025 <025

P aeruginosa L46004 >32 >32 >32 >32 16 32 32

P aeruginosa 46012 (R) >32 >32 >32 32 40 16 80

P aeruginosa 46017 >32 >32 >32 32 40 16 40 4^

P aeruginosa 46220 DR-2 32 80 16 <025 <025 <025 10 l\}

P aeruginosa 46220 DR-2- 1 >32 >32 >32 16 <025 10 20

P aeruginosa CT- 122 32 16 16 80 40 80 80

P aeruginosa CT- 137 16 16 32 40 20 40 40

P aeruginosa CT- 144 >32 >32 >32 80 10 20 40

P aeruginosa PAO 303 carb-4 32 32 32 40 40 40 20

P aeruginosa sp 2439 Wt >32 >32 >32 32 40 80 40

P fluorescens sp 5953 >32 >32 >32 >32 40 80 80

P aeruginosa M 1405 >32 >32 >32 32 20 80 32

P aeruginosa M 2297 >32 >32 >32 32 32 16 32

P aeruginosa AU-1 >32 >32 16 40 80 80

P aeruginosa AU-5 >32 <025 80 20 20 80

P aeruginosa AU-7 >32 >32 16 40 80 80

P aeruginosa AU-8 >32 >32 80 80 80

P aeruginosa AU-10 32 16 80 40 40 40

TABLE 3 (continued) ANTIBACTERIAL ACTIVITY OF CEFTADIZIME WITH COMPOUNDS (β-LACTAMASE INHIBITOR)

MIC of ceftazidime (μg/ml)

Organism alone with with with with with

Ref Compd I Compd 7 Compd 8 Compd 9 Compd 10

(Aztreonam)

E cloacae 40054 >32 >32 <025 05 10 <025

E cloacae MNH-2 >32 >32 >32 >32 80 80

E cloacae P 99 >32 >32 32 >32 >32 >32

E aerogenes S-95 >32 32 10 05 10 10

E aerogenes 41006 >32 >32 32 >32

M morganii 36014 32

M morganii 36030 >32 <025 <025 20 20 <025

P aeruginosa L 46004 >32 >32 80 16 80 32

P aeruginosa 46012 (R) >32 >32 80 32 10 10

P aeruginosa 46017 >32 >32 80 32 10 05 4^

P aeruginosa 46220 DR-2 32 80 10 10 05 05

P aeruginosa 46220 DR-2- 1 >32 >32 05 20 <025 <025

P aeruginosa CT- 122 32 16 40 80 20 20

P aeruginosa CT-137 16 16 20 20 10 10

P aeruginosa CT- 144 >32 >32 20 80 05 10

P aeruginosa PAO 303 carb-4 32 32 20 20 20

P aeruginosa sp 2439 Wt >32 >32 20 80 20 40

P fluorescens sp 5953 >32 >32 80 80 10 10

P aeruginosa M 1405 >32 >32 80 16 05 10

P aeruginosa M 2297 >32 >32 80 32 32 32

P aeruginosa AU-1 >32 >32 80 80 20 40

P aeruginosa AU-5 >32 <025 10 20

P aeruginosa AU-7 >32 >32 80 80 20 40

P aeruginosa AU-8 >32 >32 80 80 80 80

P aeruginosa AU-10 32 16 40 80 20 2.0

Claims

CLAIMSWe claim:

1. A compound of formula (I)

wherein (I)

R, is a 5-membered heterocyclic ring containing 1 to 4 heteroatoms

independently selected from O, S and N;

R₂ is selected from the group consisting of:

(a) salts

(b)

, N-OH or y acceptable

le

(e)

(f) bered 1 to 4 lected

M is hydrogen or a pharmaceutically acceptable salt forming non-toxic

cation; wherein the oxyimino fragment (= N-OR₂) in formula (I) is in the 'anti'

orientation.

2. The compound according to claim 1 where the R, is selected from

the group consisting of thiophene and 2-aminothiazole.

3. The compound according to claim 2, wherein R₁ is thiophene.

4. The compound according to claim 1 where R₂ is hydroxypyridone

and its derivatives as represented by the following formula:

wherein X = NH, N-OH or pharmaceutically acceptable salts

thereof.

5. The compound according to claim 4, wherein when X is N-OH, R₂ is

a keto or enol tantomeric isomer of the formula:

Keto-isomer Enol-isomer

6. The compound according to claim 5, wherein R₂ is a keto tautomeric

isomer.

7. The compound according to claim 1 , wherein Y is selected from the

group consisting of oxadiazoles, thiaediazoles, isoxazoles, isothiazoles, and thiazoles.

8. The compound according to claim 1 , wherein said compound

contains variable amounts of water.

9. The compound according to claim 8, wherein said variable amounts

of water result from lyophilization, crystallization or column purification.

10. A pharmaceutical composition suitable for the treatment of

bacterial infections in mammals comprising the compound described in claim

1 or a pharmaceutically acceptable salt thereof and a pharmaceutically

acceptable carrier.

11. A pharmaceutical composition suitable for the treatment of bacterial

infections comprising the compound according to claim 1 or a

pharmaceutically acceptable salt thereof, a ╬▓-lactam antibiotic and a

pharmaceutically acceptable carrier.

12. A ╬▓-lactamase inhibitor for an antibiotic containing a ╬▓-lactam

antibiotic, which comprise as an active ingredient the compound described in claim 1 or a pharmaceutically acceptable salt thereof.

13. An antibiotic comprising the compound described in claim 1 or a

pharmaceutically acceptable salt thereof and a ╬▓-lactam antibiotic.

14. The pharmaceutical composition according to claim 11 , wherein

said ╬▓-lactam antibiotic is selected from the group consisting of penicillins,

cephalosporeins, carbapenems and monobactams.

15. The pharmaceutical composition according to claim 14, wherein

said penicillins are selected from the group consisting of amoxicillin, ampicillin,

azolcillin, mezlocillin, apalcillin, hetacillin, bacampicillin, carbenicillin,

sulbenicillin, ticarcillin, piperacillin, mecillinam, pivmecillinam, methicillin,

ciclacillin, talampicillin, aspoxicillin, oxacillin, cloxacillin, dicloxacillin,

flucoxacillin, nafcillin and pivampicillin.

16. The pharmaceutical composition according to claim 14, wherein

said cephalosporins are selected from the group consisting of cephalothin,

cephaloridine, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin,

cephradin, cefuroxime, cefoxitin, cephacetrile, cefotiam, cefotaxime,

cefsulodin, cefoperazone, ceftizoxime, cefmenoxime, cefmetazole,

cephalogylcin, cefonicid, cefodizime, cefpirome, ceftazidime, ceftriaxone, cefpiramide, cefbuperazone, cefuzonam, cefpimizole, cefozopran, cefepime,

cefoselis, cefluprenam, cefclidin, cefixime, ceftibuten, cefdinir, cefpoxime

proxetil, cefteram pivoxil, cefetamet, pivoxil, cefcapene pivoxil and cefditoren

pivoxil.

17. The pharmaceutical composition according to claim 14 wherein

said carbapenems are selected from the group consisting of imipenem,

meropenem, biapenem and panipenem and said monobactams are selected

from the group consisting of aztreonam and carumonam.

18. The pharmaceutical composition according to claim 14, wherein

said compound and a ╬▓-lactam antibiotic are contained in the range of 1 :20 to

20:1 weight ratio.

19. The method for inhibiting a ╬▓-lactamase inactivation of an antibiotic

containing a ╬▓-lactam antibiotic, which comprises administering to a patient an

effective amount of the compound described in claim 1 or a pharmaceutically

acceptable salt thereof.

20. The method for treating bacterial infections comprising

administering an effective amount of ╬▓-lactam antibiotic and a compound

according to claim 1 or a pharmaceutically salt thereof to a subject in need of such treatment.

21. The method of according to claim 20 wherein the ╬▓-lactam antibiotic and said compound are administered simultaneously.

22. The method of according to claim 20 wherein the ╬▓-lactam antibiotic

and said compound are administed separately.

23. A pharmaceutical composition comprising the compound according

to claim 1 in combination with a mixture of a ╬▓-lactamase inhibitor and a ╬▓-

lactam antibiotic.

24. The composition according to claim 23, wherein said mixture is

selected from the group consisting of piperacillin/tazobactam,

ampicillin/sulbactam, amoxycillin/clavulanic acid, ticarcillin/clavulanic acid and

cefoperazone/sulbactam.

25. A method for treating a bacterial infection comprising administering

an antibacterial effective amount of a compound according to claim 1 in

combination with a mixture of a ╬▓-lactamase inhibitor and a ╬▓-lactam antibiotic

to a patient in need of such treatment.

26. The method according to claim 25, wherein said mixture is

selected from the group consisting of piperacillin/tazobactam,

ampicillin/sulbactam, amoxycillin/clavulanic acid, ticarcillin/clavulanic acid and

cefoperazone/sulbactam.