New 2-oxo-1-azetidine sulfonic acid derivatives with an aminoalkyl substituted "anti" (E-isomer) oxyimino group in the acylamino substituent at the 3 position of the monobactam ring. These compounds are potent inhibitors of bacterial .beta.-lactamases. These compounds can be used in combination with .beta.-lactam antibiotics to increase the effectiveness of the .beta.-lactam antibiotics in fighting infection caused by .beta.-lactamase producing bacteria.

BACKGROUND OF THE INVENTION 
This invention relates to new 2-oxo-1-azetidine sulfonic acid derivatives 
which are of value for use in combination with .beta.-lactam antibiotics 
to increase their effectiveness in infection caused by .beta.-lactamase 
producing bacteria. 
The most important bacterial resistance to .beta.-lactam antibiotics is the 
degradation of the .beta.-lactam nucleus by production of .beta.-lactamase 
enzyme. The apparently endless capacity of .beta.-lactamases to develop 
the ability to degrade the commercially used penicillins and 
cephalosporins, has led to the alternative strategy of seeking inhibitors 
to block their action. When a .beta.-lactamase inhibitor is used in 
combination with a .beta.-lactamase-susceptible .beta.-lactam antibiotic, 
the effectiveness of the .beta.-lactam antibiotic is increased or 
enhanced. Such an effect is known as synergy. Synergy is deemed to be 
exhibited by a combination of .beta.-lactamase inhibitor and a 
.beta.-lactam antibiotic when the antibacterial activity of the 
combination is significantly greater than the sum of the antibacterial 
activities of the individual components. 
The present invention provides certain novel 2-oxo-1-azetidine sulfonic 
acid derivatives which are potent inhibitors of bacterial 
.beta.-lactamases, particularly against class C .beta.-lactamases 
(cephalosporinase). U.S. Pat. No. 4,775,670 issued Oct. 4, 1988 to Sykes 
et al. discloses the discovery of 2-oxo-1-azetidine sulfonic acid salts as 
antibacterial agents. One member from this series, called aztreonam is a 
known antibiotic. Several publications [(e.g., Antimicrobial Agents and 
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 .beta.-lactamase inhibitory properties. 
Aztreonam is a monocyclic .beta.-lactam having a sulfonic acid substituent 
in the 1-position and a (substituted oxyimino) acylamino group in the 
3-position. The orientation of the "substituted oxyimino" group in 
aztreonam is in the "Syn" form (Z-isomer). 
The present inventors found that by introducing an aminoalkyl substituted 
"anti" (E-isomer) oxyimino group in the acylamino substituent at 
3-position of the monobactam ring, it is possible to obtain a monobactam 
compound having remarkable .beta.-lactamase inhibitory activity, 
particularly against class C .beta.-lactamases (cephalosporinase). 
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 .beta.-lactamase 
inhibitory activity, particularly against class C .beta.-lactamases 
(cephalosporinases). 
It is a further object of the invention to provide pharmaceutical 
compositions comprising a .beta.-lactamase inhibitor of this invention in 
combination with a .beta.-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 
.beta.-lactamase (cephalosporinase) producing bacteria in mammalian 
subjects, particularly in humans. 
Accordingly, this invention provides novel 2-oxo-1-azetidine sulfonic acid 
derivatives having the formula (I) 
##STR1## 
and the pharmaceutically acceptable salts thereof, wherein 
R.sub.1 is selected from a 5-membered heterocyclic ring containing from 1 
to 4 of any one or more of the heteroatoms selected from O, S and N. 
R.sub.2 is selected from any one of the following groups: 
##STR2## 
X is O, S or NH M is hydrogen or a pharmaceutically acceptable salt 
forming cation. 
The present inventors found that the "anti" (E-isomer) orientation of the 
oxyimino group (.dbd.N--OR.sub.2) in the formula (I) provides excellent 
.beta.-lactamase inhibitory activity and superior synergy in combination 
with a .beta.-lactam antibiotic against class C .beta.-lactamase 
(cephalosporinase) producing gram-negative bacteria, including Pseudomonas 
aeruginosa. 
The present inventors also found that the inhibitory activity against 
isolated .beta.-lactamase and the synergy with a .beta.-lactam antibiotic 
is greatly influenced by the nature of the heterocyclic ring represented 
by R.sub.1 and the nature of the substituent in the oxime fragment 
represented by R.sub.2. 
Thus, thiophene is the preferred 5-membered heterocyclic ring as R.sub.1 
and amino(C.sub.1-6) alkyl is the preferred group for R.sub.2. 
Furthermore, amino(C.sub.1-6) alkyl may optionally be substituted by 
(C.sub.1-6) alkyl, hydroxy (C.sub.1-6) alkyl, amino (C.sub.1-6) alkyl, 
amino, hydroxy, guanidino, amidino, guanidino (C.sub.1-6) alkyl, amidino 
(C.sub.1-6) alkyl. 
DETAILED DESCRIPTION OF THE INVENTION 
The .beta.-lactamase inhibitors of this invention are the compounds having 
the formula (I) 
##STR3## 
The present .beta.-lactamase inhibitors of the invention are effective in 
enhancing the antimicrobial activity of .beta.-lactam antibiotics, when 
used in combination to treat a mammalian subject suffering from a 
bacterial infection caused by a .beta.-lactamase producing microorganism. 
Examples of antibiotics which can be used cojointly with the compounds of 
the present invention are commonly used penicillins such as amoxicillin, 
aspoxicillin, ampicillin, azlocillin, mezlocillin, apalcillin, hetacillin, 
bacampicillin, carbenicillin, sulbenicillin, ticarcillin, piperacillin, 
mecillinam, pivmecillinam, methicillin, ciclacillin, talampicillin, 
oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, 
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, cefpimizole, cefuzonam, cefclidin, 
cefixime, ceftibuten, cefdinir, cefpodoxime proxetil, cefteram pivoxil, 
cefetamet pivoxil, cefcapene pivoxil, cefditoren pivoxil, cefepime, 
cefoselis, cefluprenam; commonly used carbapenems such as imipenem, 
meropenem, panipenem, biapenem and the like; commonly used monobactams 
such as aztreonam and carumonam and salts thereof. Furthermore, the 
.beta.-lactamase inhibitors of the present invention can be used in 
combination with another .beta.-lactamase inhibitor to enhance the 
antimicrobial activity of any of the above mentioned .beta.-lactam 
antibiotics. For example, the inhibitors of this invention can be combined 
with piperacillin/tazobactam combination; ampicillin/sulbactam 
combination; amoxicillin/clavulanic acid combination; 
ticarcillin/clavulanic acid combination, cefoperazone/sulbactam 
combination, and the like. 
R.sub.1 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. 
Preferably, R.sub.1 in the formula (I) is thiophene, furan, pyrrole, 
1-methyl pyrrole, 2-aminothiazole. 
Even more preferably, R.sub.1 is thiophene. 
R.sub.2 in the formula (I) is amino(C.sub.1-6) alkyl which may optionally 
be substituted by any one of the following groups, such as (C.sub.1-6) 
alkyl, hydroxy (C.sub.1-6) alkyl, amino (C.sub.1-6) alkyl, amino, amidino, 
hydroxy, guanidino, amidino (C.sub.1-6) alkyl, guanidino (C.sub.1-6) alkyl 
and the like. The substitution could be at the carbon atom or at the 
nitrogen atom of the amino group. Preferred examples of R.sub.2 are the 
following: 
##STR4## 
where X=O, S or NH. 
Illustrative of (C.sub.1-6)alkyl are linear or branched alkyl groups 
including methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, 
t-butyl, pentyl and hexyl groups. 
The amino groups mentioned above may remain as free amino group or it may 
form salts with inorganic acids or organic acids or it may form a 
zwitterion (inner salt) by interaction with the hydrogen atom of the 
sulfonic acid group [i.e., when M is hydrogen in the formula (I)]. 
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) 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 acid salts include lysine, arginine, ornithine, 
histidine and the like. 
Moreover, when M is hydrogen in the formula (I) it can form a zwitterion 
(inner salt) by interacting with the amino group present in the molecule 
of formula (I) or with a basic nitrogen atom other than amino group 
present in the molecule. 
The present invention encompasses all the possible stereoisomers as well as 
their racemic or optically active mixtures. 
A variety of protecting groups conventionally used in the .beta.-lactam art 
to protect the amino groups present in the formula (I) can be used. While 
it is difficult to determine which amino-protecting groups should be used, 
the major requirement for such a group is that it can be removed without 
cleaving the .beta.-lactam ring and without reducing the double bond of 
the oxyimino group (.dbd.N--OR.sub.2) 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 
amino-protecting groups are: trityl, tert-butoxycarbonyl, formyl, and the 
like. 
The compounds of this invention having the formula (I) can be prepared 
using a variety of well known procedures as shown below: 
##STR5## 
Each procedure utilizes as a starting material the known azetidine of the 
formula 
##STR6## 
Azetidines of the formula (III) are well known in the literature; see, for 
example, the United Kingdom patent application no. 2,071,650 published 
Sep. 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 
##STR7## 
in the presence of a coupling agent. 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. Suitable reaction temperature may range from -20.degree. 
C. to 50.degree. C., preferably from 0.degree. C. to room temperature, 
even more preferably at room temperature. Reaction time may range from 1 
hour to 24 hours, preferably from 4 hours to 12 hours. 
The reaction of an acid of 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 monoalkyl carbonic acid; mixed anhydrides with an 
aliphatic carboxylic 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, dibenzyloxyphosphoric 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 amides 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, mesylphenyl, pyranyl, pyridyl, 
piperidyl and 8-quinolylthio esters. Additional examples of activated 
esters are esters with a N-hydroxy compound e.g., 
N,N-dimethylhydroxylamine, 1-hydroxy-2(1H)pyridone, N-hydroxy succinimide, 
N-hydroxy-phthalimide, 1-hydroxy-1H-benzotriazole, 
1-hydroxy-6-chloro-1H-benzotriazole, 1,1'-bis [(6-trifluoro 
methyl)benzotriazolyl]oxalate (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-cyclohexyl-N'-morpholinoethylcarbodiimide, 
N'-cyclohexyl-N'-(4-diethylaminocyclohexyl) carbodiimide 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, 
dimethylformamide, 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, tributylamine, 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 
##STR8## 
with a hydroxylamine derivative (or a salt thereof) of formula 
EQU R.sub.2 --O--NH.sub.2 (IX) 
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). 
##STR9## 
with the alcohol (R.sub.2 --OH, X) under Mitsunobu conditions (PPh.sub.3 
/DEAD/THF) will provide the compounds of formula (I). 
Alternatively, the compounds of formula (I) can be prepared by reacting the 
hydroxyimino derivative (VIII) (process E) with a compound of the formula 
(XI). 
EQU R.sub.2 --X (XI) 
wherein X is a leaving group such as halogen, trifluoroacetate, 
alkylsulfonate, arylsulfonate or other activated esters of alcohols. 
The compounds of formula (I) which has a sulfo group (SO.sub.3 H) 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. Acid ion 
exchange resins can also be used. Solvents may 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) 
##STR10## 
with the alcohol R.sub.2 --OH (X) under standard Mitsunobu conditions 
(PPh.sub.3 /DEAD/THF; D. L. Hughes, The Mitsunobu Reaction in Organic 
Reactions; P. Beak et al., Eds; John Wiley & Sons, Inc: New York, Vol. 42, 
pp. 335-656, 1992). 
R.sub.1 has the same definition as defined before. R.sub.3 is a protective 
group for the carboxyl group. The protective groups for said carboxyl 
group include all groups generally usable as carboxyl-protecting groups in 
the field of .beta.-lactam compound and organic chemistry, for example, 
methyl, ethyl, propyl, isopropyl, allyl, t-butyl, benzyl, p-methoxybenzyl, 
p-nitrobenzyl, benzhydryl, methoxymethyl, ethoxymethyl, acetoxymethyl, 
pivaloyloxymethyl, trityl, 2,2,2-trichloroethyl, .beta.-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 R.sub.3 can be effected by selective 
application of a per se known method such as the method involving a base, 
the method involving the use of palladium tetrakis. 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 of alkaline earth metals 
(e.g., calcium, magnesium, etc.) or organic bases such as metal alkoxides, 
organic amines, quarternary ammonium salts or basic ion exchange resins, 
etc. 
The reaction temperature is about 0.degree. to 80.degree. C. more 
preferably about 10.degree. to 40.degree. 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, toluene) 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 R.sub.2 --X (XI) 
wherein X is a leaving group such as halogen, trifluoroacetate, 
alkylsulfonate, arylsulfonate or other activated esters of alcohols. 
In another approach, the intermediate (II) can be prepared by reacting a 
keto acid compound of formula (VII) with a hydroxylamine derivative (or 
it's salt) of formula R.sub.2 --O--NH.sub.2 (IX) using conventional 
procedures; see for example, EP 0251,299 (Kaken). 
The acids useful for removing the amino-protecting group in the final step 
of the preparation of compound of the formula (I) are formic acid, 
trichloroacetic acid, trifluoroacetic acid, hydrochloric acid, hydrobromic 
acid, hydroiodic acid, hydrofluoric 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 adversely 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) can be purified by standard procedures well-known 
in the art such as crystallization and chromatography over silica gel or 
HP-20 column. 
Typical solvates of the compounds of formula (I) may include water as water 
of crystallization and water miscible solvents like methanol, ethanol, 
acetone, dioxane or acetonitrile. 
Under the scope of this invention are included compounds containing 
variable amounts of water produced by a process such as lyophilization or 
crystallization from solvents containing water as a co-solvent. 
Under the scope of the present invention are also included compounds 
containing variable amounts of acids, such as formic acid, trifluoroacetic 
acid, and the like which are used to remove amino-protecting groups. 
Favourable pharmaceutically acceptable salts of the compounds of formula 
(I) are sodium, potassium and calcium. 
As already mentioned, the oxyimino group, i.e., .dbd.N--OR.sub.2 in the 
formula (I) in it's `anti` orientation provides excellent synergy with a 
.beta.-lactam antibiotic against class C .beta.-lactamase 
(cephalosporinase) producing gram-negative bacteria including P. 
aeruginosa. Thus this invention includes only those compounds having the 
formula (I) in which the oxyimino group (.dbd.N--OR.sub.2) is specifically 
in the `anti` orientation (E-isomer) as shown in the formula (I). 
Furthermore, the inhibitory activity against the isolated .beta.-lactamase 
(cephalosporinase) and the synergy with a .beta.-lactam antibiotic against 
cephalosporinase producing gram-negative bacteria are greatly influenced 
by the nature of the heterocyclic ring represented by R.sub.1 and the 
nature of the substituent in the oxime fragment represented by R.sub.2. 
Thiophene is the choice of heterocycle as R.sub.1. 
In the formula (I), R.sub.2 is amino(C.sub.1-6) alkyl. The amino(C.sub.1-6) 
alkyl may optionally be substituted by (C.sub.1-6) alkyl, hydroxy 
(C.sub.1-6) alkyl, amino (C.sub.1-6) alkyl, hydroxy, amino, amidino, 
guanidino, amidino (C.sub.1-6) alkyl, guanidino (C.sub.1-6) alkyl. 
More preferably, R.sub.2 is selected from the following groups: 
##STR11## 
X is O, S or NH. 
The compounds of the present invention including the pharmaceutically 
acceptable salts thereof are inhibitors of bacterial .beta.-lactamases 
particularly of cephalosporinases and they increase the antibacterial 
effectiveness of .beta.-lactamase susceptible .beta.-lactam 
antibiotics--that is, they increase the effectiveness of the antibiotic 
against infections caused by .beta.-lactamase (cephalosporinase) producing 
gram-negative bacteria including Pseudomonas aeruginosa. This makes the 
compounds of formula (I) and said pharmaceutically acceptable salts 
thereof valuable for co-administration with .beta.-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 .beta.-lactam antibiotic, and 
the two agents thereby administered simultaneously. Alternatively, the 
said 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, orally, 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 .beta.-lactam antibiotic in association with one or 
more pharmaceutically acceptable excipients and/or carriers. 
The pharmaceutical compositions of the invention are usually prepared 
following conventional methods and are administered in a pharmaceutically 
suitable form. For instance, solutions for intravenous injection or 
infusion may contain as 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 of formula (I) and the .beta.-lactam 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 of formula (I) 
in association with a .beta.-lactam antibiotic 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 .beta.-lactam antibiotic, diluents, e.g., 
lactose, dextrose, saccharose, cellulose, corn starch 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. 
In most instances, an effective .beta.-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 .beta.-lactamase inhibitor 
of the present invention and the .beta.-lactam antibiotic with which it is 
being administered will normally be in the range of 1:20 to 20:1, 
preferably in the range of 1:8 to 8:1, even more preferably in the range 
of 1:4 to 4:1. 
Test for Antibacterial Activity: 
The compounds of the present invention in combination with ceftazidime and 
ceftazidime alone 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 (e.g., 
ceftazidime) alone, the MICs of ceftazidime in combination with reference 
compounds particularly aztreonam (Ref. compd. I) and the MICs of the 
.beta.-lactamase inhibitors (10 .mu.g/ml) of the present invention in 
combination with ceftazidime were determined with the same 
.beta.-lactamase producing bacteria. After incubation in Mueller-Hinton 
Broth (Difco) at 37.degree. C. for 18 h, the bacterial suspension was 
diluted and about 10.sup.5 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.degree. C. on the lowest combinations of 
drug that inhibited visible growth of bacteria. 
Test for .beta.-Lactamase Inhibitory Activity: 
The inhibitory activities of present compounds (.beta.-lactamase 
inhibitors) against cephalosporinase from P. aeruginosa 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. 
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. 
##STR12## 
TABLE 1 
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.beta.-Lactamase inhibitory activity of the prepared compounds 
Compound 
R.sub.1 R.sub.2 /Orientation of OR.sub.2 
M IC.sub.50, .mu.M 
__________________________________________________________________________ 
Reference Compound I (Aztreonam) 
H 0.13 # 
- Reference Compound II 
#STR14## 
H 0.8 ## 
- Compound 1 
#STR16## 
H 0.02 # 
- Compound 2 
#STR18## 
H 0.02 # 
- Compound 3 
#STR20## 
H 0.4 ## 
- Compound 4 
#STR22## 
H 0.065 
- Compound 5 
#STR24## 
H 0.1 ## 
- Compound 6 
#STR26## 
H 0.1 ## 
- Compound 7 
#STR28## 
H 0.08 # 
- Compound 8 
#STR30## 
H 0.3 ## 
- Compound 9 
#STR32## 
H 0.08 # 
- Compound 10 
#STR34## 
H 0.08 # 
- Compound 11 
#STR36## 
H 0.2 ## 
- Compound 12 
#STR38## 
H 1.09## 
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TABLE 2 
__________________________________________________________________________ 
.sup.1 H NMR spectra of some representative compounds 
Compd. no. 
Solvent 
.delta. (ppm) 
__________________________________________________________________________ 
2 DMSO-d.sub.6 
9.29(d, 1H, J=8.3Hz); 7.92(dd, 1H, J=0.9 and 5.0Hz), 7.77(dd, 
1H, J=0.9 and 4.0Hz); 7.71 
(br, s, 3H); 7.21(dd, 1H, J=4.2 and 4.8Hz); 4.49(dd, 1H, J=2.7 and 
8.2Hz); 4.39(t, 2H, J=6.0 
Hz); 3.57-3.94(m, 1H); 2.85-3.04(br, t, 2H); 1.93-2.19(br, m, 2H); 
1.42(d, 3H, J=6.13Hz). 
4 DMSO-d.sub.6 9.31(d, 1H, J=8.3Hz); 7.93(dd, 1H, J=0.9 and 5.0Hz); 
7.75(dd, 1H, J=0.9 and 3.07Hz); 7.20 
(dd, 1H, J=0.9 and 4.2Hz); 4.80-5.35(br, m, exchangeable with D.sub.2 
O); 4.52(dd, 1H, J=2.6 and 
8.2Hz); 4.13-4.40(br, s, 2H); 3.75-3.90(m, 1H); 2.60-3.40(m, 3H); 
1.42(d, 3H, J=6.1Hz). 
5 DMSO-d.sub.6 9.25(d, 1H, J=8.2Hz); 7.91(dd, 1H, J=0.9 and 4.9Hz); 
7.78(dd, 1H, J=0.9 and 3.1Hz); 7.21 
(dd, 1H, J=0.9 and 4.9Hz); 5.80-6.40(br, m, 6H, exchangeable with 
D.sub.2 O); 4.50(dd, 1H, J=2.6 
and 8.1Hz); 4.22(d, 2H); 3.75-3.90(m, 1H); 2.90-3.40(m, 3H); 1.50-1.90 
(m, 2H); 1.43(d, 3H, J= 
6.1Hz). 
6 DMSO-d.sub.6 9.19(d, 1H, J=8.1Hz); 7.60-8.10(m, 8H); 7.20(t, 1H, 
J=3.9Hz); 4.52(dd, 1H, J=2.68 and 8 20 
Hz); 4.40(br, s, 1H); 3.70-3.85(m, 1H); 3.50-3.63(m, 1H); 2.70-2.90(br 
, s, 2H); 1.53-1.80(br, m, 
4H); 1.42(d, 3H, J=6.1Hz). 
7 DMSO-d.sub.6 9.37(d, 1H, J=8.32Hz); 8.10-8.50(br), 7.94(dd, 1H, J=0.9 
and 5. 1Hz); 7.89(dd, 1H, J=0.9 
and 4Hz); 7.20(dd, 1H, J=4.02 and 5.1Hz); 4.55-4.65(br, m, 2H); 
4.49(dd, 1H, J=2.4 and 8.3 
Hz); 3.86(m, 1H); 3.43-3.58(br, m, 2H); 3.20-3.40(br, m, 4H); 1.42(d, 
3H, J=6.1Hz). 
8 DMSO-d.sub.6 9.16(d, 1H, J=8.2Hz); 7.85-8.10(m, 5H); 7.20(dd, 1H, 
J=4.0 and 5.0Hz); 4.50-4.60(m, 2H); 
3.65-3.90(m, 3H); 3.20-3.50(m, 2H); 1.45(d, 3H, J=6.1Hz). 
11 DMSO-d.sub.6 9.18(two sets of d, 1H, J=8.2Hz); 7.60-8.10(m, 5H); 
7.20(dd, 1H, J=4.0 and 5.0Hz); 4.48-4.75 
(m, 2H); 3.66-3.72(m, 1H); 3.19(d, 2H, J=5.4Hz); 1.40(t, 6H, J=6.1Hz). 
12 DMSO-d.sub.6 9.08(two sets of d, 1H, J=8.1Hz); 7.90-8.00(m, 6H); 
7.13-7.20(m, 5H); 4.60-4.70(br, m; 1H); 
4.51(dd, 1H, J=2.5 and 8.15Hz); 3.77-3.89(m, 1H); 3.50-3.65(br, m, 
2H); 3.18-3.20(br, m, 2H); 
1.43(d, 3H, J=6.07Hz). 
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TABLE 3 
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Antibacterial activity of ceftazidime with compounds (.beta.-lactamase 
inhibitor) 
__________________________________________________________________________ 
MIC of ceftazidime(.mu.g/ml) 
with Ref compd. I 
Organism alone (Aztreonam) with Ref compd. II with compd 1 
__________________________________________________________________________ 
E. cloacae 40054 &gt;32 &gt;32 &lt;0.25 &lt;0.25 
E. cloacae MNH-2 &gt;32 &gt;32 1.0 1.0 
E. cloacae P 99 &gt;32 &gt;32 32 1.0 
E. aerogenes S-95 &gt;32 &gt;32 1.0 &lt;0.25 
E. aerogenes 41006 &gt;32 &gt;32 0.5 0.5 
C. freundii CT-76 &gt;32 &gt;32 8.0 32 
C. freundii 44032 &gt;32 &gt;32 0.5 0.5 
M. morganii 36010 &gt;32 &gt;32 16 4.0 
M. morganii 36014 &gt;32 &gt;32 &lt;0.25 &lt;0.25 
M. morganii 36030 &gt;32 &lt;0.25 4.0 &lt;0.25 
P. aeruginosa L46004 &gt;32 &gt;32 &gt;32 8.0 
P. aeruginosa 46012 &gt;32 &gt;32 &gt;32 8.0 
P. aeruginosa 46017 &gt;32 &gt;32 32 2.0 
P. aeruginosa 46220 DR-2 16 16 8.0 1.0 
P. aeruginosa 46220 DR-2-1 &gt;32 &gt;32 &gt;32 1.0 
P. aeruginosa CT-122 16 16 8.0 4.0 
P. aeruginosa CT-137 8.0 16 8.0 2.0 
P. aeruginosa CT-144 32 &gt;32 32 2.0 
P. aeruginosa PAO 303 carb-4 32 32 1.6 2.0 
P. aeruginosa sp 2439 Wt. &gt;32 &gt;32 &gt;32 4.0 
P. aeruginosa M 1405 &gt;32 &gt;32 &gt;32 8.0 
P. aeruginosa M2297 &gt;32 &gt;32 &gt;32 4.0 
P. aeruginosa AU-1 &gt;32 &gt;32 -- 8.0 
P. aeruginosa AU-5 &gt;32 1.0 -- 4.0 
P. aeruginosa AU-8 &gt;32 &gt;32 -- 16 
P. aeruginosa AU-10 &gt;32 &gt;32 -- 4.0 
__________________________________________________________________________ 
Organism alone 
with comp. 2 
with compd. 3 
with compd. 8 
with compd. 9 
with compd. 11 
__________________________________________________________________________ 
E. cloacae 40054 &gt;32 &lt;0.25 0.5 &lt;0.25 &lt;0.25 0.5 
E. cloacae MNH-2 &gt;32 2.0 4.0 2.0 2.0 4.0 
E. cloacae P 99 &gt;32 2.0 2.0 1.0 1.0 2.0 
E. aerogenes S-95 &gt;32 1.0 1.0 0.5 0.5 1.0 
E. aerogenes 41006 &gt;32 1.0 2.0 2.0 1.0 4.0 
C. freundii CT-76 &gt;32 &gt;32 8.0 8.0 &gt;32 &gt;32 
C. freundii 44032 &gt;32 0.5 1.0 1.0 0.5 2.0 
M. morganii 36010 &gt;32 8.0 4.0 8.0 1.0 8.0 
M. morganii 36014 &gt;32 &lt;0.25 &lt;0.25 &lt;0.25 &lt;0.25 &lt;0.25 
M. morganii 36030 &gt;32 &lt;0.25 &lt;0.25 &lt;0.25 &lt;0.25 &lt;0.25 
P. aeruginosa L46004 &gt;32 32 16 32 32 16 
P. aeruginosa 46012 &gt;32 16 16 32 32 16 
P. aeruginosa 46017 &gt;32 8.0 8.0 16 16 8.0 
P. aeruginosa 46220DR-2 16 1.0 1.0 1.0 1.0 1.0 
P. aeruginosa 46220 DR-2-1 &gt;32 2.0 2.0 4.0 16 4.0 
P. aeruginosa CT-122 16 8.0 8.0 8.0 8.0 8.0 
P. aeruginosa CT-137 8.0 2.0 4.0 4.0 4.0 4.0 
P. aeruginosa CT-144 32 4.0 4.0 8.0 8.0 8.0 
P. aeruginosa PAO 303 carb-4 32 2.0 4.0 8.0 8.0 8.0 
P. aeruginosa sp 2439 Wt. &gt;32 16 8.0 16 32 16 
P. aeruginosa M 1405 &gt;32 16 8.0 32 &gt;32 16 
P. aeruginosa M2297 &gt;32 8.0 8.0 16 32 16 
P. aeruginosa AU-1 &gt;32 16 16 32 32 16 
P. aeruginosa AU-5 &gt;32 8.0 8.0 4.0 4.0 8.0 
P. aeruginosa AU-8 &gt;32 32 32 -- -- -- 
P. aeruginosa AU-10 &gt;32 4.0 4.0 -- -- -- 
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