This invention was made with support from the NIH (grant GM59957) and the NSF (grant MCB-9734484). The U. S. Government has certain rights in the invention.

FIELD OF THE INVENTION The invention relates to certain (3-lactam analogs and compositions containing them.

The invention also relates to the use of (3-lactam analogs to inhibit (3-lactamases and to treat ß-lactam-antibiotic-resistant bacterial infections.

BACKGROUND Bacterial resistance to antibiotics has raised fears of an approaching medical catastrophe (Neu, Science, 257,1064-1073 (1992)). Evolutionary selection and genetic transformation have made this problem pressing. Most antibiotic drugs are derivatives of naturallyoccurringbactericides (Davies, Science, 264,375-382 (1994)), and many resistance mechanisms evolved long ago. Human use of antibiotics has refined these mechanisms and promoted their spread through gene transfer (Davies, Science, 264,375-382 (1994)). A resistance mechanism originating in one species of bacteria can be expected to spread throughout the biosphere.

Bacterial adaptations to (3-lactam drugs (e. g., amoxicillin, cephalothin, clavulanate, aztreonam) are among the best studied and most pernicious forms of antibiotic resistance.

(3-lactams target enzymes that are unique to bacteria and are thus highly selective. They have been widely prescribed. In the absence of resistance, p-lactams are the first choice for treatment in 45 of 78 common bacterial infections (Goodmafz & Gilman's The Pharmacological Basis of Therapeutics (Hardman et al., eds., McGraw-Hill, New York, 1996)). The evolution of resistance to these drugs has raised the cost of antibiotic therapy and reduced its effectiveness, leading to increased rates of morbidity and mortality.

(3-lactam antibiotics inhibit bacterial cell wall biosynthesis (Tomasz, Rev. Infect. Dis., 8, S270-S278 (1986)). The drugs form covalent complexes with a group of transpeptidases/carboxypeptidases called penicillin binding proteins (PBPs). PBP inactivation disrupts cell wall biosynthesis, leading to self-lysis and death of the bacteria.

Bacteria use several different mechanisms to escape from (3-lactam drugs (Sanders, ClinicallnfectiousDisease, 14,1089-1099 (1992) ; Li et al., Antimicrob.Agents Chemother., 39,1948-1953 (1995)). The most widespread is the hydrolysis of p-lactams by p-lactamase enzymes.

Partly in response to the emergence and spread of p-lactamases, many lactam derivatives have been developed; over forty are currently used in clinical practice. These

analogs preserve the p-lactam core of the drug but explore diverse functionality off the C6 (7) position of the four-membered ring, in what will be referred to as the Rl side chain (see Figure 1 and below). The different R, side chains confer different pharmacological profiles, different bacterial spectra of action, and different levels of resistance to ß-lactamases.

Whereas early penicillins and cephalosporins, such as penicillin G and cephalothin (Figure 1), are rapidly inactivated by p-lactamases, later agents, such as cloxacillin and ceftazidime, are much less susceptible to these enzymes, though not to broad-spectrum mutant ß- lactamases or to hyper-produced p-lactamases.

Thus, there is a need for inhibitors of (3-lactamases. Such inhibitors would allow current (3-lactam antibiotics to work against bacteria where (3-lactamases provide the dominant resistance mechanism.

SUMMARY OF THE INVENTION The invention provides a method of treating a ß-lactam-antibiotic-resistant bacterial infection. The method comprises administering to an animal suffering from such an infection an effective amount of a (3-lactam antibiotic and an effective amount of a compound of the following formula: wherein: R2 is H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkyl-cycloalkyl, heteroalkyl- cycloalkyl, alkyl-heterocycloalkyl, heteroalkyl-heterocycloalkyl, alkenyl, heteroalkenyl, cyclic alkene, heterocyclic alkene, alkyl-cyclic alkene, heteroalkyl-cyclic alkene, cyclic alkene-alkyl, cyclic alkene-heteroalkyl, alkyl-heterocyclic alkene, heterocyclic alkene-alkyl, heterocyclic alkene-heteroalkyl, heteroalkyl-heterocyclic alkene, alkyl-O-cyclic alkene, alkyl- O-heterocyclic alkene, alkyl-S-cyclic alkene, alkyl-S-heterocyclic alkene, or each R2 may be unsubstituted or substituted with one or more R3 groups ;

each R3 is independently alkyl, heteroalkyl, cyclic alkene, cyclic alkene substituted with one or more R4 groups, heterocyclic alkene, heterocyclic alkene substituted with one or more R4 groups, halogen,-NHz, =NH, =N, =N-OH, =O,-OH,-O-C (O) H,-O-alkyl,-COOH, -(CH2)n-COOH, =CH-(CH2)m-COOH, -CN, =N-O-CH3, =N-O-C(CH3)2-COOH, =N-O- C (CH3) 2-C (O)-O-alkyl,-(CH2) m-NH2, =C (COOH)-C (O)-NH2,-C (O)-O-alkyl,-C (O)-O-cyclic alkene,-S-alkyl,-S03H, or-SO2-CH3 ; each R4is independently alkyl, halogen, =NH, -NH2, -(CH2)m-NH2, =O, -OH, -(CH2)m- OH, -COOH, -(CH2)m-COOH, -C(=O)NH2, -SO3H, or -SO2-CH3; R5 is cyclic alkene or heterocyclic alkene, each of which may be unsubstituted or substituted with one or more R4 groups ; R6 is alkyl or heteroalkyl, each of which maybe unsubstituted or substituted with one or more R4 groups ; R7 is H or R7 is alkyl or heteroalkyl, each of which may be unsubstituted or substituted with one or more R4 groups ; m is 1-4; and n is 0-2; or a pharmaceutically-acceptable salt thereof.

The invention also provides a method of inhibiting a (3-lactamase. The method comprises contacting the ß-lactamase with an effective amount of a compound of the following formula : wherein: R2 is H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkyl-cycloalkyl, heteroalkyl- cycloalkyl, alkyl-heterocycloalkyl, heteroalkyl-heterocycloalkyl, alkenyl, heteroalkenyl, cyclic alkene, heterocyclic alkene, alkyl-cyclic alkene, heteroalkyl-cyclic alkene, cyclic alkene-alkyl, cyclic alkene-heteroalkyl, alkyl-heterocyclic alkene, heterocyclic alkene-alkyl, heterocyclic alkene-heteroalkyl, heteroalkyl-heterocyclic alkene, alkyl-O-cyclic alkene, alkyl- O-heterocyclic alkene, alkyl-S-cyclic alkene, alkyl-S-heterocyclic alkene, or

each R2 may be unsubstituted or substituted with one or more R3 groups ; each R3 is independently alkyl, heteroalkyl, cyclic alkene, cyclic alkene substituted with one or more R4 groups, heterocyclic alkene, heterocyclic alkene substituted with one or more R4 groups, halogen, -NH2, =NH, =N, =N-OH, =O,-OH,-O-C (O) H,-O-alkyl,-COOH, -(CH2) m-COOH, =CH-(CH2) m-COOH,-CN, =N-O-CH3, =N-O-C (CH3) 2-COOH, =N-O- C (CH3) 2-C(O)-O-alkyl, -(CH2)m-NH2, =C (COOH)-C (O)-NH2,-C (O)-O-alkyl,-C (O)-O-cyclic alkene, -S-alkyl, -SO3H, or -SO2-CH3 ; each R4 is independently alkyl, halogen, =NH, -NH2, -(CH2)m-NH2, =O, -OH, -(CH2)m- OH,-COOH,-(CH2) m-COOH,-C (=O) NH2,-SO3H, or-SO2-CH3 ; Rs is cyclic alkene or heterocyclic alkene, each of which may be unsubstituted or substituted with one or more R4 groups ; R6 is alkyl or heteroalkyl, each of which may be unsubstituted or substituted with one or more R4 groups ; R7 is H or R7 is alkyl or heteroalkyl, each of which may be unsubstituted or substituted with one or more R4 groups ; m is 1-4; and n is 0-2; or a pharmaceutically-acceptable salt thereof.

The invention further provides a pharmaceutical composition. The composition comprises a pharmaceutically-acceptable carrier, a (3-lactam antibiotic and a compound of the following formula : wherein: R2 is H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkyl-cycloalkyl, heteroalkyl- cycloalkyl, alkyl-heterocycloalkyl, heteroalkyl-heterocycloalkyl, alkenyl, heteroalkenyl, cyclic alkene, heterocyclic alkene, alkyl-cyclic alkene, heteroalkyl-cyclic alkene, cyclic alkene-alkyl, cyclic alkene-heteroalkyl, alkyl-heterocyclic alkene, heterocyclic alkene-alkyl, heterocyclic alkene-heteroalkyl, heteroalkyl-heterocyclic alkene, alkyl-O-cyclic alkene, alkyl- O-heterocyclic alkene, alkyl-S-cyclic alkene, alkyl-S-heterocyclic alkene, or

each R2 may be unsubstituted or substituted with one or more R3 groups ; each R3 is independently alkyl, heteroalkyl, cyclic alkene, cyclic alkene substituted with one or more R4 groups, heterocyclic alkene, heterocyclic alkene substituted with one or more R4 groups, halogen,-NH2, =NH, =N, =N-OH, =O,-OH,-O-C (O) H, -O-alkyl, -COOH, -(CH2)m-COOH, =CH-(CH2)m-COOH, -CN, =N-O-CH3, =N-O-C (CH3) 2-COOH, =N-0- C (CH3) 2-C (O)-O-alkyl, -(CH2)m-NH2, =C (COOH)-C (O)-NH2, -C (O)-O-alkyl,-C (O)-O-cyclic alkene,-S-alkyl,-S03H, or -SO2-CH3 ; each R4is independently alkyl, halogen, =NH, -NH2, -(CH2)m-NH2, =O, -OH, -(CH2)m- OH,-COOH,- (CH2) m-COOH,-C (=O) NH2,-SO3H, or-SO2-CH3 ; R5 is cyclic alkene or heterocyclic alkene, each of which may be unsubstituted or substituted with one or more R4 groups ; R6 is alkyl or heteroalkyl, each of which may be unsubstituted or substituted with one or more R4 groups ; R7 is H or R7 is alkyl or heteroalkyl, each of which may be unsubstituted or substituted with one or more R4 groups ; m is 1-4; and n is 0-2; or a pharmaceutically-acceptable salt thereof.

The invention provides another pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically-acceptable carrier and a compound of the following formula: wherein: R2 is cycloalkyl, heterocycloalkyl, alkyl-cycloalkyl, heteroalkyl-cycloalkyl, alkyl- heterocycloalkyl, heteroalkyl-heterocycloalkyl, cyclic alkene, heterocyclic alkene, alkyl- cyclic alkene, heteroalkyl-cyclic alkene, cyclic alkene-alkyl, cyclic alkene-heteroalkyl, alkyl- heterocyclic alkene, heterocyclic alkene-alkyl, heterocyclic alkene-heteroalkyl, heteroalkyl-

heterocyclic alkene, alkyl-O-cyclic alkene, alkyl-O-heterocyclic alkene, alkylene-S-cyclic alkene, or alkyl-S-heterocyclic alkene, wherein each cycloalkyl, heterocycloalkyl, alkyl- cycloalkyl, heteroalkyl-cycloalkyl, alkyl-heterocycloalkyl, heteroalkyl-heterocycloalkyl, cyclic alkene, heterocyclic alkene, alkyl-cyclic alkene, heteroalkyl-cyclic alkene, cyclic alkene-alkyl, cyclic alkene-heteroalkyl, alkyl-heterocyclic alkene, heterocyclic alkene-alkyl, heterocyclic alkene-heteroalkyl, heteroalkyl-heterocyclic alkene, alkyl-O-cyclic alkene, alkyl- O-heterocyclic alkene, alkyl-S-cyclic alkene, alkyl-S-heterocyclic alkene comprises two or more rings ; or R2 is each R2 may be unsubstituted or substituted with one or more R3 groups ; each R3 is independently alkyl, heteroalkyl, cyclic alkene, cyclic alkene substituted with one or more R4 groups, heterocyclic alkene, heterocyclic alkene substituted with one or more R4 groups, halogen,-NH2, =NH, =N, =N-OH, =O,-OH,-O-C (O) H,-O-alkyl,-COOH, <BR> <BR> <BR> -(CH2) m-COOH, =CH-(CH2) m-COOH,-CN, =N-O-CH3, =N-O-C (CH3) 2-COOH, =N-O- C (CH3)2-C(O)-O-alkyl, -(CH2)m-NH2, =C (COOH)-C (O)-NH2,-C (O)-O-alkyl,-C (O)-O-cyclic alkene,-S-alkyl,-S03H, or-SO2-CH3 ; each R4 is independently alkyl, halogen, =NH, -NH2, -(CH2)m-NH2, =O, -OH, -(CH2)m- OH,-COOH,-(CH2) m-COOH,-C (=O) NH2,-SO3H, or-SO2-CH3; R5 is cyclic alkene or heterocyclic alkene, each of which may be unsubstituted or substituted with one or more R4 groups ; R6 is alkyl or heteroalkyl, each of which may be unsubstituted or substituted with one or more R4 groups ; R7 is H or R7 is alkyl or heteroalkyl, each of which may be unsubstituted or substituted with one or more R4 groups ; m is 1-4; and n is 0-2 ; or a pharmaceutically-acceptable salt thereof.

Finally, the invention provides a compound of the following formula:

wherein: R2 is cycloalkyl, heterocycloalkyl, alkyl-cycloalkyl, heteroalkyl-cycloalkyl, alkyl- heterocycloalkyl, heteroalkyl-heterocycloalkyl, cyclic alkene, heterocyclic alkene, alkyl- cyclic alkene, heteroalkyl-cyclic alkene, cyclic alkene-alkyl, cyclic alkene-heteroalkyl, alkyl- heterocyclic alkene, heterocyclic alkene-alkyl, heterocyclic alkene-heteroalkyl, heteroalkyl- heterocyclic alkene, alkyl-O-cyclic alkene, alkyl-O-heterocyclic alkene, alkylene-S-cyclic alkene, or alkyl-S-heterocyclic alkene, wherein each cycloalkyl, heterocycloalkyl, alkyl- cycloalkyl, heteroalkyl-cycloalkyl, alkyl-heterocycloalkyl, heteroalkyl-heterocycloalkyl, cyclic alkene, heterocyclic alkene, alkyl-cyclic alkene, heteroalkyl-cyclic alkene, cyclic alkene-alkyl, cyclic alkene-heteroalkyl, alkyl-heterocyclic alkene, heterocyclic alkene-alkyl, heterocyclic alkene-heteroalkyl, heteroalkyl-heterocyclic alkene, alkyl-O-cyclic alkene, alkyl- O-heterocyclic alkene, alkyl-S-cyclic alkene, alkyl-S-heterocyclic alkene comprises two or more rings; or R2 is each R2 may be unsubstituted or substituted with one or more R3 groups ; each R3 is independently alkyl, heteroalkyl, cyclic alkene, cyclic alkene substituted with one or more R4 groups, heterocyclic alkene, heterocyclic alkene substituted with one or more R4 groups, halogen,-NH2, =NH, =N, =N-OH, =O,-OH,-O-C (O) H,-O-alkyl,-COOH, -(CH2)m-COOH, =CH-(CH2)m-COOH, -CN, =N-O-CH3, =N-O-C (CH3) 2-COOH, =N-O- C (CH3) 2-C (O)-O-alkyl,-(CH2) m-NH2, =C (COOH)-C (O)-NH2,-C (O)-O-alkyl,-C (O)-O-cyclic alkene,-S-alkyl,-S03H, or-SO2-CH3 ; each R4 is independently alkyl, halogen, =NH,-NH2,- (CH2)m-NH2, =O, -OH, -(CH2)m- OH, -COOH, -(CH2)m-COOH, -C(=O)NH2, -SO2H, or -SO2-CH3 ; R5 is cyclic alkene or heterocyclic alkene, each of which may be unsubstituted or substituted with one or more R4 groups ;

R6 is alkyl or heteroalkyl, each of which maybe unsubstituted or substituted with one or more R4 groups ; R7 is H or R7 is alkyl or heteroalkyl, each of which may be unsubstituted or substituted with one or more R4 groups ; m is 1-4; and n is 0-2; or a pharmaceutically-acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Structures of several clinically-used penicillins (I-V) and cephalosporins (VI-VIII) showing the Rl side chains.

Figure 2. Diagram illustrating B-lactam hydrolysis by serine B-lactamases.

Figure 3. Comparison of the structures of a deacylation high-energy intermediate of a penicillin in a serine B-lactamase and the transition state analog formed by an acylaminomethaneboronic acid and the same enzyme.

Figures 4A-B. Stereoviews of 2Fo-Fc electron density of the refined models for complexes of compound 9 (Figure 4A) and compound 11 (Figure 4B) with the B-lactamase AmpC. The density is contoured at 1 sigma (s). These figures were generated using Turbo (available from Université Aix-Marseille II, Marseille, France).

Figures 5A-B. Diagrams illustrating keypolar interactions observed between AmpC and compound 9 (Figure 5A) and compound 11 (Figure 5B). These figures were generated with MidasPlus [Ferrin et al., J. Mol. Graph, 6: 13-27 (1988)].

Figure 6. Diagram illustrating overlay of the structure of cloxacillin in complex with the AmpC mutant enzyme Q120L/Y150E and of the transition-state analog 9 in complex with wild-type AmpC.

Figure 7 : Diagram illustrating general scheme for synthesis of acylaminomethaneboronic acids. Me = methyl, BuLi = butyl lithium, TMS = tetramethyl silane.

DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS As used herein, the following terms have the following meanings: "Alkyl"means a straight-chain or branched-chain alkyl containing 1-10 carbon atoms (e.g,-CH3,-CH2-CH3,-CH2-CH (CH3)-CH2-CH2-CH3).

"Heteroalkyl"means an alkyl as defined above containing one or more atoms of S, N or O in the chain (e. g.,-CH2-O-CH2-CH3,-CH2-NH2-CH2-CN,-CH2-N-(CH3) 2,-CH2-S- CH2-CN, -C(CH3)2-CH2-O-C(CH3)3, -CH2-S-(CH2)3-CH3).

"Alkenyl"means a straight-chain or branched-chain alkenyl containing 2-10 carbon atoms and at least one double bond (e. g.,-CH=CH-CH3 and-CH2-CH2-CH=CH2).

"Heteroalkenyl"means an alkenyl as defined above containing one or more atoms of S, N or O in the chain (e. g.,-CH2-S-CH2-CH=CH2).

"Cycloalkyl"means a structure containing from 1 to 3 rings, each ring containing from 4 to 7 carbon atoms (e. g.,-C4H7,-C5H9,-C6Hl,,-C7Hl3).

"Heterocyloalkyl"means a cycloalkyl as defined above wherein at least one carbon atom of at least one of the ring (s) has been replaced by S, N or O, such as: "Cyclic alkene"means a structure containing from 1 to 3 rings, each ring containing from 4 to 7 carbon atoms, and at least one double bond. One, two or all three of the rings may be aromatic. Representative cyclic alkenes include-C6H5 and "Heterocyclic alkene"means a cyclic alkene as defined above wherein at least one carbon atom of at least one of the ring (s) has been replaced by S, N or O. Representative heterocyclic alkenes include: "Alkyl-cycloalkyl"means an alkyl having attached thereto a cycloalkyl (e. g,-CH2- C5H9).

"Heteroalkyl-cycloalkyl"means a heteroalkyl having attached thereto a cycloalkyl (e.g.,-CH2-CH2-O-CH2-C5Hg).

"Alkyl-heterocycloalkyl"means an alkyl having attached thereto a heterocycloalkyl.

"Heteroalkyl-heterocycloalkyl"means a heteroalkyl having attached thereto a heterocycloalkyl.

"Alkyl-cyclic alkene"means an alkyl having attached thereto a cyclic alkene (e. g.,- CH2-C6H5)

"Heteroalkyl-cyclic alkene"means a heteroalkyl having attached thereto a cyclic alkene (e. g.,-CH2-O-CH2-C6H5).

"Cyclic alkene-alkyl"means a cyclic alkene having attached thereto an alkyl (e. g.,- C6H4-CH3) "Cyclic alkene-heteroalkyl"means a cyclic alkene having attached thereto a heteroalkyl (e. g,-C6H4-CH2-O-CH3).

"Alkyl-heterocyclic alkene"means an alkyl having attached thereto a heterocyclic alkene.

"Heterocyclic alkene-alkyl"means a heterocyclic alkene having attached thereto an alkyl.

"Heterocyclic alkene-heteroalkyl"means a heterocyclic alkene having attached thereto a heteroalkyl.

"Heteroalkyl-heterocyclic alkene"means a heteroalkyl having attached thereto a heterocyclic alkene.

"Alkyl-O-cyclic alkene"means an alkyl attached to a cyclic alkene by means of an oxygen atom (e. g,-CH2-O-C6H5 and-CH (CH3)-O-C6H5).

"Alkyl-O-heterocyclic alkene"means an alkyl attached to a heterocyclic alkene by means of an oxygen atom.

"Alkyl-S-cyclic alkene"means an alkyl attached to a cyclic alkene by means of a sulfur atom (e. g,-CH2-S-C6H5 and-CH (CH3)-S-C6H5).

"Alkyl-S-heterocyclic alkene"means an alkyl attached to a heterocyclic alkene by means of a sulfur atom.

"Halogen"means fluoro, chloro, bromo or iodo.

All of the above terms include all possible isomers.

Preferred compounds of formula (1) are those wherein the portion of the compound is the Rl side chain of a (3-lactam antibiotic. As used herein,"Rl side chain of a p-lactam antibiotic"means the side chain attached to the four-membered ring of p-lactam antibiotics and which contains the amide functionality common to most p-lactam antibiotics (see Figure 1). (3-lactam antibiotics include cephalosporins, penicillins, monobactams, carbapenems, carbacephems, and others. The RI side chains of penicillin G, penicillin V, phenethicillin, nafcillin, cloxacillin, cephalothin, cephapirin and ceftazidime are illustrated in Figure 1. Additional (3-lactam antibiotics include ampicillin, amoxicillin, aztreonam, bacampicillin, carbenicillin, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefdinir, cefepime, cefixime, cefmenoxime, cefinetazole, cefonicid,

cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefpodoxime, cefprozil, cefroxadine, cefsulodin, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, celesticetin, cephalexin, cephaloglycin, cephaloridine, cephalosporin C, cephamycins, cephradine, ceprozil, geocillin, imipenem, loracarbef, moxalactam, mezlocillin, penamecillin, penicillin BT, penicillin N, penicillin O, penicillin S, piperacillin, and ticarcillin. The structures of (3-lactam antibiotics are known (see, e. g., The Merck INdex (10th ed., Merck & Co., Inc., Rahway, N. J., 1983) andPhysicians'DeskReference (53rd ed., Medical Economics Co., Inc., Montvale, N. J., 1999)), and the structures of their Rl side chains can be readily identified. The preferred compounds of formula (1) wherein

is the Rl side chain of a (3-lactam are: Additional preferred compounds of formula (1) are those wherein R2 is a cyclic alkene. The preferred compound wherein R2 is a cyclic alkene is:

Further preferred compounds of formula (1) are those wherein R2 is Most preferred of these compounds are :

The compounds of formula (1) can be synthesized as described below (see Example 1). Unless otherwise noted, the various chemicals used in the synthesis described below are available from commercial sources including Aldrich Chemical, Milwaukee, WI, Lancaster Synthesis, Windham, NH, TCI America, Portland, OR, Sigma Chemical Co., St. Louis, MO, Acros Organics, Pittsburgh, PA, Chemservice Inc., West Chester, PA, BDH hie., Toronto, Canada, Fluka Chemical Corp., Ronkonkoma, NY, Pfaltz & Bauer, Inc., Waterbury, CT, Avocado Research, Lancashire, UK, Crescent Chemical Co., Hauppauge, NY, Fisher Scientific Co., Pittsburgh, PA, Fisons Chemicals, Leicestershire, UK, ICN Biomedicals, Inc., Costa Mesa, CA, Pierce Chemical Co., Rockford, IL, Riedel de Haen AG, Hannover, Germany, Wako Chemicals USA, Inc., Richmond, VA, Maybridge Chemical Co. Ltd., Cornwall, UK, Trans World Chemicals, Inc., Rockville, MD, Apin Chemicals Ltd., Milton Park, UK, and Parish Chemical Co., Orem, UT. Some of the compounds of formula (1) can also be synthesized as described in U. S. Patent No. 5,574,017, the complete disclosure of which is incorporated herein by reference.

The compounds of formula (1) may contain an acidic or basic functional group and are, thus, capable of forming phannaceutically-acceptable salts with pharmaceutically- acceptable acids and bases. The term"pharmaceutically-acceptable salts"refers to the relatively non-toxic, inorganic and organic acid and base addition salts of compounds of formula (1). These salts can be prepared by reacting the purified compound with a suitable acid or base. Suitable bases include the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, ammonia, or a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. Representative acid addition salts include the hydrobromide, hydrochloride, sulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylat, citrate, maleate, fumarate, succinate, tartrate, napthalate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.

The compounds of formula (1), and the pharmaceutically-acceptable salts thereof, are inhibitors of (3-lactamases. Assays for the inhibition of p-lactamase activity are well known

in the art. For instance, the ability of a compound to inhibit (3-lactamase activity in a standard enzyme inhibition assay may be used (see, e. g., Example 1 below and M. G. Page, Biochem J. 295 (Pt. 1) 295-304 (1993)). ß-lactamases for use in such assays may be purified from bacterial sources or, preferably, are produced by recombinant DNA techniques, since genes and cDNA clones coding for many a-lactamases are known. See, e. g., S. J. Cartwright and S. G. Waley, Biochem J. 221,505-512 (1984). Alternatively, the sensitivity of bacteria known, or engineered, to produce a P-lactamase to an inhibitor may be determined (see Example 1 below). Other bacterial inhibition assays include agar disk diffusion and agar dilution. See, e. g., W. H. Traub & B. Leonhard, Chemotherapy 43,159-167 (1997). Thus, a P-lactamase can be inhibited by contacting the p-lactamase enzyme with an effective amount of a compound of formula (1) or by contacting bacteria that produce the ß-lactamase enzymes with an effective amount of a compound of formula (1) so that the the (3-lactamase in the bacteria is contacted with the inhibitor. The contacting may take place in vitro or in vivo."Contacting"means that the (3-lactamase and the inhibitor are brought together so that the inhibitor can bind to the ß-lactamase. Amounts of a compound of formula (1) effective to inhibit a (3-lactamase may be determined empirically, and making such determinations is within the skill in the art. Inhibition includes both reduction and elimination of p-lactamase activity.

The compounds offormula (1), and the pharmaceutically-acceptable salts thereof, can be used to treat ß-lactam-antibiotic-resistant bacterial infections."ß-lactam-antibiotic- resistant bacterial infection"is used herein to refer to an infection caused by bacteria resistant to treatment with one or more (3-lactam antibiotics due primarily to the action of a ß- lactamase. Resistance to (3-lactam antibiotics can be determined by standard antibiotic sensitivity testing. The presence of (3-lactamase activity can be determined as is well known in the art (see above). Alternatively, and preferably, the sensitivity of a particular bacterium to the combination of a compound of formula (1), or a pharmaceutically-acceptable salt thereof, and a (3-lactam antibiotic can be determined by standard antibiotic sensitivity testing methods.

To treat a a-lactam resistant bacterial infection, an animal suffering from such an infection is given an effective amount of a compound of formula (1), or a pharmaceutically- acceptable salt thereof, and an effective amount of a p-lactam antibiotic. The compound of formula (1), or a phannaceutically-acceptable salt thereof, and the (3-lactam antibiotic may be given at different times or given together. When administered together, they may be contained in separate pharmaceutical compositions or they may be in the same pharmaceutical composition.

Many suitable (3-lactam antibiotics are known (see above). ß-lactam antibiotics are effective (in the absence of resistance) against a wide range of bacterial infections. These

include those caused by both gram-positive and gram-negative bacteria, for example, bacteria of the genus Staphylococcus (such as Staphylococcus aureus and Staphylococcus epidermidis), Streptococcus (such as Streptococcus agalactine, Streptococcus penumoniae and Streptococcusfaecalis), Micrococcus (such as Micrococcus luteus), Bacillus (such as Bacillus subtilis), Listerella (such as Listerella monocytogenes), Escherichia (such as Escherichia coli), Klebsiella (such as Klebsiella pneumoniae), Proteus (such as Proteus mirabilis and Proteus vulgaris), Salmonella (such as Salmonella typhosa), Shigella (such as Shigella sonnei), Enterobacter (such as Enterobacter aerogenes and Enterobacter Cloacae), Serratia (such as Serratia marcescens), Pseudomonas (such as Pseudomonas aeruginosa), Acinetobacter such as Acinetobacter anitratus), Nocardia (such as Nocardia autotrophica), and Mycobacterium (such as Mycobacterium fortuitum). Effective doses and modes of administration of a-lactam antibiotics are known in the art or maybe determined empirically as described below for the compounds of formula (1).

To treat an animal suffering from a (3-lactam-antibiotic-resistant bacterial infection, an effective amount of a compound of formula (1), or a pharmaceutically-acceptable salt thereof, is administered to the animal in combination with a (3-lactam antibiotic. Effective dosage forms, modes of administration and dosage amounts of a compound of formula (1), maybe determined empirically, and making such determinations is within the skill of the art.

It is understood by those skilled in the art that the dosage amount will vary with the activity of the particular compound employed, the severity of the bacterial infection, the route of administration, the rate of excretion of the compound, the duration of the treatment, the identity of any other drugs being administered to the animal, the age, size and species of the animal, and like factors well known in the medical and veterinary arts. In general, a suitable daily dose will be that amount which is the lowest dose effective to produce a therapeutic effect. The total daily dosage will be determined by an attending physician or veterinarian within the scope of sound medical judgment. If desired, the effective daily dose of a compound of formula (1), or a pharmaceutically-acceptable salt thereof, maybe administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day. Treatment of a (3-lactam-antibiotic-resistant bacterial infection according to the invention, includes mitigation, as well as elimination, of the infection.

Animals treatable according to the invention include mammals. Mammals treatable according to the invention include dogs, cats, other domestic animals, and humans.

Compounds of formula (1) or pharmaceutically-acceptable salts thereof, may be administered to an animal patient for therapy by any suitable route of administration, including orally, nasally, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. The preferred routes of administration are orally and parenterally.

While it is possible for the active ingredient (s) (one or more compounds of formula (1), or pharmaceutically-acceptable salts thereof, alone or in combination with a 3-lactam antibiotic) to be administered alone, it is preferable to administer the active ingredient (s) as a pharmaceutical formulation (composition). The pharmaceutical compositions of the invention comprise the active ingredient (s) in admixture with one or more pharmaceutically- acceptable carriers and, optionally, with one or more other compounds, drugs or other materials. Each carrier must be"acceptable"in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

Pharmaceutical formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. Regardless of the route of administration selected, the active ingredient (s) are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

The amount of the active ingredient (s) which will be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration and all of the other factors described above. The amount of the active ingredient (s) which will be combined with a carrier material to produce a single dosage form will generally be that amount of the active ingredient (s) which is the lowest dose effective to produce a therapeutic effect.

Methods of preparing pharmaceutical formulations or compositions include the step of bringing the active ingredient (s) into association with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient (s) into association with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of the active ingredient (s). The active ingredient (s) may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient (s) is/are mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethyl-

cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin ; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. hi the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolat or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient (s) moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient (s) therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and maybe of a composition that they release the active ingredient (s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient (s) can also be in microencapsulated form.

Liquid dosage forms for oral administration of the active ingredient (s) include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient (s), the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in

particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active ingredient (s), may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing the active ingredient (s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active ingredient (s). Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration ofthe active ingredient (s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient (s) may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to the active ingredient (s), excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active ingredient (s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of the active ingredient (s) to the body. Such dosage forms can be made by dissolving, dispersing or otherwise incorporating the active ingredient (s) in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux

of the active ingredient (s) across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the active ingredient (s) in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise the active ingredient (s) in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the active ingredient (s), it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the active ingredient (s) then depends upon its/their rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of parenterally-administered active ingredient (s) is accomplished by dissolving or suspending the active ingredient (s) in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the active ingredient (s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the active ingredient (s) to polymer, and the nature of the particular polymer employed, the rate of release of the active ingredient (s) can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the active ingredient (s) in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for inj ection, immediately prior to use.

Extemporaneous injection solutions and suspensions maybe prepared from sterile powders, granules and tablets of the type described above.

The pharmaceutical compositions of the present invention may also be used in the form of veterinary formulations, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules or pellets for admixture with feed stuffs, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension or, when appropriate, by intramammary injection where a suspension or solution is introduced into the udder of the animal via its teat; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally, for example, as a pessary, cream or foam.

EXAMPLES Example 1: Preparation And Characterization Of Acylaminomethaneboronic Acids Despite intense study, it has been difficult to understand how the seemingly differing R, side chains of ß-lactams contribute to ß-lactam-ß-lactamase recognition. The problem lies in the covalent bond that (3-lactams form with Group I and Group II (3-lactamases. In these enzymes, the catalytic serine attacks the lactam bond to form an acyl adduct (Figure 2); this step is rapid for P-lactamases. The acyl adduct is then hydrolyzed in a second step; deacylation is rate-determining for Group I (3-lactamases and for (3-lactam inhibitors or poor substrates for both classes of enzyme. The covalent nature of the acyl-adduct, and its rapid formation, means that binding is effectively irreversible, and that binding equilibria are not available through steady-state kinetics. This precludes easy energetic analyses, which assume reversible equilibria. In P-lactamases, Km values are convolutions of irreversible acylation and deacylation rates with reversible on and off rates, and IC50 values are often dominated by rates of deacylation. Rarely do they reflect binding energies or inhibitor-enzyme complementarity in any simple way (Matagne et al., Biochem J, 265: 131-46 (1990); Galleni et al., Biochem. Pharmacol., 49: 1171-1178 (1995)). Indeed, both structural (Maveyraud et al., J. Am. Chem. Soc., 120: 9748-9752 (1998); Patera et al., J. Am. Chel7l. Soc., 122: 10504- 10512 (2000)) and stability (Beadle et al., Protein Sci., 8: 1816-24 (1999)) studies have suggested that some (3-lactam inhibitors of P-lactamases fit the enzyme poorly-their inhibitory properties derive from their ability to form the acyl adduct and then block attack by the hydrolytic water. On the other hand, some R, side chains undoubtedly fit the enzymes well. Which ones do so, and how well they do so, has been difficult to determine.

To investigate the energetic bases of (3-lactam functional group recognition by (3- lactamases, acylaminomethaneboronic acids that bear the R, side chains of eight characteristic penicillins and cephalosporins (Figure 1), as well as four other analogs, were synthesized. Boronic acids are transition-state analog inhibitors of Group I and Group II B- lactamases [Beesley et al., Biocheni. J., 209: 229-233 (1983); Crompton, Biochem J., 251: 453-459 (1988); Strynadlca et al., Nat. Struct. Biol., 3: 688-95 (1996); Weston et al., J.

Med. Chem, 41: 4577-4586 (1998); Usher et al., Biochemistry, 37: 16082-16092 (1998); Powers et al., Protein Sci., 8: 2330-7 (1999); Ness et al., Biochemistry, 39: 5312-5321 (2000)].

Unlike (3-lactams, they form reversible adducts with these enzymes. Binding energies thus can be calculated directly from K, values. By comparing the affinities of different acylaminomethaneboronic acids, it can be determined what the different R, side chains contribute to binding to a given (3-lactamase. By comparing the affinities to a Group I ß- lactamase, AmpC, and a Group II (3-lactamase, TEM-1, differential recognition between

characteristic representatives of the two most widespread classes of (3-lactamases can be investigated.

To give the binding energies a molecular context, the structures of two of the inhibitors in their complexes with AmpC p-lactamase were determined by X-ray crystallography. Comparing one of these structures with that of its (3-lactam counterpart in complex with AmpC (Patera et al., J. Am. Chem. Soc., 122: 10504-10512 (2000)) allowed investigation of how interactions with the R, side chain differ between the acylated ground state and the presumptive deacylation high-energy intermediate. Comparing these transition- state analog complexes with the B-lactam/AmpC acyl-adducts suggests how recognition changes between the ground state and deacylation high-energy intermediates along the reaction path. These structures also give a molecular context to the interaction energies and may guide the design of further anti-resistance compounds in this series.

To explore the application of acylaminomethaneboronic acids in reversing lactamase-basedresistance, their abilityto act synergisticallywith p-lactams against resistant, pathogenic bacteria was investigated. Two of the inhibitors were tested in these assays.

A. Procedures 1. Synthesis and Analysis of Compounds 'H-and'3C-NMR spectra were recorded on a Brucker DPX-200 MHz spectrometer and a Brucker AMX-400 MHz spectrometer, respectively: chemical shifts are reported in J values from tetramethyl silane (TMS) as internal standard (s singlet, d doublet, t triplet, br broad). Coupling constants (J) are given in Hz. Mass spectra were performed on a Finnigan MAT-SSQ 710A mass spectrometer and a Hewlett Packard 5872 (EI, 70 eV) mass spectrometer. Elemental analyses were determined with a Carlo Erba Elemental Analyzer model 1106; elemental analyses for the compounds were within i 0.5 % of the theoretical values. IR spectra were recorded with a Perkin Elmer 1600 series FTIR spectrometer; IR signals reported refer to C-O amide stretching and N-H amide stretching respectively.

Solvents were dried and distilled before use; glassware was dried at 110 °C for 40 min. All reactions were conducted under inert atmosphere unless otherwise specified. The common synthetic procedure is presented in Figure 7.

Pinacol chloromethaneboronate (1')(Whiting,?e<7/:eonZee.

32: 1503-1506 (1991)). Butyl lithium in hexane (2.5 M, 9.2 ml, 23 mmol) was added dropwise to a stirred solution of bromochloromethane (1.5 ml, 23 mmol) and tri-tert- butylborate (2.3 ml, 21 mmol) in anhydrous tetrahydrofuran (THF) (25 ml) at-78°C under nitrogen flow; the resulting mixture was allowed to react for 1 hour (h). Thereafter, the reaction was quenched at-78 °C with trimethylsylil chloride (3.2 ml, 25.2 mmol), and the temperature gradually raised to room temperature (RT). After 16 h a solution of pinacol (2.7

g, 23 mmol) in ethyl ether (10 ml) was added dropwise and the mixture was stirred for an additional3 h. The solution was diluted with water (20 ml) and ethyl ether (10 ml) and the aqueous phase extracted with ethyl ether (3 x 10 ml); the combined organic phases were washed with brine and dried (MgSO4). After removal of the solvent under reduced pressure, the oily residue was distilled in vacuo (bp 55-56°C/2 mmHg) to yield 1 (76%) as a colorless oil.'H-NMR (CDCl3) : # 1. 31 (12H, s, CH3), 2.97 (2H, s, CH2) ; 13C-NMR (CDCl3) : 8 25.4, 85. 3; MS, m/z : 178-176 (M+), 163-161 (basepeak), 145,136-134,120-118,105-103,85,59, 43.

Pinacol N, N'-diformamidomethaneboronate (2) (Versleiien et al., Tetrahedron Letters 36: 2109-2112 (1995)). A solution of 1 (836 mg, 4.73 mmol) in anhydrous CH3CN (2 ml) was added to a solution of sodium difonnylamide (540 mg, 5.68 mmol) in anhydrous CH3CN (2 ml) and the mixture stirred for 3 h at 80°C. The white precipitate (NaCI) was centrifuged off, the solution concentrated and the residue distilled under reduced pressure to give 2 as a clear dense oil (73%), bp 83 °C/1. 5-10-2 mmHg.'H- NMR (CDC13) : b 1.25 (12H, s, CH3), 3.19 (2H, s, CH2), 8.86 (2H, s, COH) ; MS, mlz : 213 (M+), 198,183,155 (base peak), 114,126,97 86.

Pinacol formamidomethaneboronate (3) (Versleijen et al., Tetrahedron Letters 36: 2109-2112 (1995)). Compound 2 (233 mg, 1.09 mmol) was dissolved in anhydrous methanol (MeOH) (1.2 ml) and stirred at RT for 1 h, until a gas liquid chromatography (GLC) analysis showed disappearance of the starting material; the solution was concentrated in vacuo and triturated with CH2Cl2 The viscous oily residue crystallized overnight in the refrigerator (4°C) to give 3 (99%) as a white solid, mp 55-56°C. IR (KBr) 1629,3375 cm-1 ;'H-NMR (CDCl3) : # 1.32 (12H, s, CH3), 2.93 (2H, d, J4.5, CH2), 5.70 (1H, br, NH), 8. 20 (1H, s, CHO) ; 13C-NMR (DMSO) : 6 24. 7,82.3,162.2; MS, mlz : 185 (M+), 170, 154,127 (base peak), 86,70,59,43.

Pinacol bis- (trimethylsylil)-aminomethaneboronate (4) (Lebarbier et al., Synthesis, pp. 1371-1374 (1996)). Lithium hexamethyldisilazane in THF (1.0 M, 4.6 ml, 4.6 mmol) was added dropwise to a solution of 1 (800 mg, 4.6 mmol) in anhydrous ethyl ether (6 ml) cooled at-78 °C under nitrogen. After stirring for 10 minutes (min) at-78°C, the cooling bath was removed and the solution stirred for 2 h at RT. The inorganic precipitate (Lid) was centrifuged, the supernatant concentrated under reduced pressure, and the oily residue was distilled under reduced pressure (bp 80-81 ° C/1 mmHg) to give 4 (57%) as a colorless oil.'H-NMR (CDCl3) : os 0.10 (18H, s, SiCH3), 1.26 (12H, s, CH3), 2.47 (2H, s, CH2) ; MS, m/z : 301 (M+), 286,228,186 (base peak), 170,112,73,59.

Pinacol acetamidomethaneboronate (5) (Matteson et al., Organometallics. 8: 726-729 (1989)). Acetic acid (36.4 1L, 0.63 mmol) and acetic anhydride (165 uL, 1.75 mmol) were added at-10°C to a solution of 4 (150 mg, 0.5 mmol) in

anhydrous ethyl ether (Et2O) (3 ml) under inert atmosphere. After 1.5 h the solvent was evaporated in vacuo, and the residue crystallized from Et2O/pentane to give 5 (60%) as a white solid, mp 108 °C. IR (KBr) 1619,3370 cm-1 ;'H-NMR (CDCl3) : os 1.26 (12H, s, CH3), 2.07 (3H, s, COACH3), 2.56 (2H, d, J4. 4, CH2), 6.97 (1H, br, NH) ; 13C-NMR (DMSO): 324.2, 25.1,79.3,174.3; MS, only+ : 199 (M+), 184,169,141,140 (base peak), 100,99,84,83,74, 55.

Reaction of pinacol bis-ftrimethylsyliD-aminomethaneboronate(4 with acyl chlorides: general procedure (Matteson et al., Organometallics, 3: 1284 (1984)).

A solution of anhydrous methanol in THF (1 mmol, 2.5M) was added at-10°C to a solution of 4 (1 mmol) in THF under nitrogen. The cooling bath was removed, the reaction mixture stirred for 1 h at RT and then cooled again to-10°C. A solution of the acyl derivative (1 mmol) in THF was slowly added and allowed to react for the reported time until a GLC analysis showed total disappearance of 4. The solvent was evaporated in vacuo and the residue purified by crystallization.

Pinacol phenvlacetamidomethaneboronate (6). Phenylacetylchloride was allowed to react with 4 for 1 h; thereafter, the solvent was evaporated and the viscous oily residue crystallized from hexane to give 6 (91 % yield) as a pale yellow solid, mp 114 ° C.

IR (KBr) 1615,3169 cm-1 ;'H-NMR (CDCl3) : oe 1.27 (12H, s, CH3), 2.59 (2H, d, J2. 6, BCH2), 3.68 (2H, s, PhCH2), 6.97 (1H, br, NH), 7.24-7.44 (5H, m, aromatic) ;'3C-NMR (DMSO): # 24. 9,38.4,80.4,126.8,128.4,128.9,134.7,174.0; MS, mlz : 275 (M+), 260,217, 176,160,142,91 (base peak), 83.

Pinacol phenoxvacetamidomethaneboronate (7). After 1 h at RT, the solvent was evaporated and the residue crystallized from pentane to yield 7 (80%) as a white solid, mp 60°C. IR (KBr) 1630, 3454 cm-1 ;'H-NMR (CDCl3) : # 1. 31 (12H, s, CH3), 2.92 (2H, d, J4.5, BCH2), 4.55 (2H, s, OCH2), 6.77 (1H, br, NH), 6.92-7.10 (3H, m, aromatic), 7.30-7.40 (2H, m, aromatic) ; 13C-NMR (DMSO): # 24.7,66.1,82.3,114.8,121.2,129.4, 157.6,169.0; MS, m/z : 291 (M+), 276,233 (base peak), 176,98,94,77.

Pinacol [ (2-phenoxypropanoyl) amino] methaneboronate (8). After 1 h at RT, the solvent was evaporated to give a white solid residue which was triturated with n-pentane to afford 8 (89%), mp. 78°C. IR (KBr) 1621,3186 ;'H-NMR (CDC13) : J 1.28 (12H, s, CCH3), 1.61 (3H, d, J 6. 8, CHCH3), 2.83 (2H, d, J 4. 5, CH2), 4.73 (1H, q, J 6. 8, CHCH3), 6.65 (1H, br, NH), 6.91-7.08 (3H, m, aromatic), 7.27-7.38 (2H, m, aromatic) ; 13C- NMR (DMSO): # 18.6,24.6,73.0,82.4,115.4,121.1,129.4,157.1,172.3; MS, mlz : 305 (M+), 290,247 (base peak), 184,121,112,83,77.

Pinacol [[(3-(2-chlorophenvi)-5-methyl-4-isoxazolyi)carbonyl]amino] methaneboronate (9). After 1 h at RT, the solvent was evaporated and the residue crystallized from pentane to afford 9 (92%) as white crystalline solid, mp 91 °C. IR (KBr)

1622,3060 ;'H-NMR (CDC13) : 61. 21 (12H, s, CH3), 2.74 (2H, d, J3.8, CH2), 2.81 (3H, s, Chug), 5.59 (1H, br, NH), 7.40-7.51 (4H, m, aromatic) ; 13C-NMR (DMSO): 6 12. 2,24.7,82.0, 111.8,127.1,127.4,129.6,131.4,131.6,132.6,159.5,161.9,170.5; MS, m/z : 377 (M-'), 341 (base peak), 318, 241, 215,178,111,83.

Pinacol [(2-ethoxy-l-naphthoyi)amino]methaneboronic acid (10).

After 16 h at RT, the solvent was removed affording a viscous residue, which was purified by silica column chromatography (elution : ethyl ether/ethyl acetate 1 : 1 v/v and then methanol) to give the free boronic acid 10 (57%) as a white solid, mp. 150°C (dec). IR (KBr) 1630,3250 ;'H-NMR (DMSO): os 1.32 (3H, t, J6.7, CH 4.20 (2H, d, J6. 7, OC_2), 7.20-8.00 (9H, m, aromatic, NH, OH) ; 13C-NMR (DMSO): os 14.9, 64.8,64.9,115.4,122.5,123.7,124.5,126.6,127.7,128.2,129.8,13 1.1,152.2,166.5; MS, mlz : 273 (M+), 229,199,171,170,155,142,127,115 (base peak), 89,88.

Pinacol a-thienylacetamidomethaneboronate (11). After 1 h at RT, the solvent was removed and the residue crystallized from ethyl ether/pentane yielding 7 (62%) as a white solid, mp. 86-87°C. IR (KBr) 1619,3169 ;'H-NMR (CDC13) : os 1.28 (12H, s, CH3), 2.66 (2H, d, J3. 1, BCH2), 3.87 (2H, s, ArCH2), 6.10 (1H, br, NH), 6.97-7.03 (2H, m, aromatic), 7.28 (1H, dd, J5. 0,1.5, S-CH) ; 13C-NMR (DMSO): os 24.8,33.6,81.3,125.2, 126.4,126.6,136.2,172.0; MS, m/z : 281 (M+), 266,223,182,166,142,97 (base peak), 83, 55.

Pinacol chloroacetamidomethaneboronate (12). After 1.5 h at RT, the solvent was evaporated in vacuo affording 12 as a clear viscous oil (100%), which was used without further purification. 1H-NMR (CDCl3) : J 1.31 (12H, s, CH3), 2.90 (2H, d, J4. 4, BCH2), 4.09 (2H, s, CH2Cl), 6.75 (1H, br, NH). 13C-NMR (CDCl3) : # 25. 5,43.1,84.9,167.6.

MS, mlz : 235-233 (M+), 220-218, 198,177-175,136-134,119-117 (base peak), 98,83,55.

Pinacol (4-pyridil)thioacetamidomethaneboronate hydrochloride (13).

A solution of 4-mercaptopyridine (31 mg, 0.28 mmol) in anhydrous dimethylfuran (DMF) (0.5 ml) was added under vigorous stirring to a solution of 12 (66 mg, 0.28 mmol) in anhydrous DMF (2 ml). After 3.5 h at 100°C, the solvent was evaporated under reduced pressure. The residue was crystallized from ethyl ether/pentane to give 13 as a white crystalline solid (90%), mp 186°C dec. IR (KBr) 1665,3283 and 2550 (N+-H stretching); 'H-NMR (DMSO): # 1. 16 (12H, s, Chug), 2.52 (2H, d, J4. 3, BCH2), 4.08 (2H, s, SCHZ), 7.85 (2H, dd, J7. 0,1.4, H (3), H (5) aromatic), 8.52 (1H, br, NH), 8.62 (2H, dd, J7. 0,1.2, H (2), H (6) aromatic) ; 13C-NMR (DMSO): # 24. 6,33.1,82.8,122.3,140.4,160.8,167.0; MS, m/z: 308 (M) +, 293,250,209,198,152,140,125 (base peak), 111,98,83.

Pinacol [[2-amino-a-[1-(e-butoxycarbonyl)-1-methylethoxyimmo]- 4-thiazoleacetyl amino] methaneboronate (14). Triethylamine (184 µL, 1.32 mmol) and isobutylchloroformate (171 I1L, 1.32 mmol) were added to a solution of (Z)-2-amino-a- [l-

(tert-butoxycarbonyl)-l-methylethoxyimino]-4-thiazoleacetic acid (438 mg, 1.32 mmol) in anhydrous THF (30 ml) at 0°C and allowed to react under inert atmosphere for 40 min.

Thereafter, a solution of 4 (400 mg, 1.32 mmol) in anhydrous THF (4 ml), previously treated with anhydrous methanol (1.32 mmol,) was added at the same temperature. After 20 min the temperature was raised to RT and the mixture allowed to react for 1 h. The white precipitate (ammonium chloride) was centrifuged, and the supernatant was evaporated under reduced pressure. The residue was crystallized from CH2Cl2/pentane to give 14 as a white crystalline solid (80%), mp 188-190°C dec. IR (KBr) 1630,1724,3322 ; tH-NMR (CDC13) : zu 1.32 (12H, s, CH3), 1.47 (9H, s, C (CH3)3), 1.57 (6H, s, CH3CCO), 1.81 (2H, br, NH2), 3.00 (2H, d, J4. 4, C_2NH), 6.98 (1H, tJ4.4, NH), 7.08 (1H, s, aromatic) ;"C-NMR (CDC'3) : os 24.4, 25.6,28.8,82.6,83.5,85.0,112.0,143.3,149.6,163.7,170.2,174.5 ; MS, m/z : 468 (M+), 453,410,367,354,309,285,251,226,209,184,142,126 (base peak), 98,83,59.

Pinacol [ [2-amino-α-(1-carboxy-1-methylethoxyimino)-4- thiazoleacetvl] amino] methaneboronate trifluoroacetic salt (15). Compound 14 (200 mg, 0.43 mmol) was dissolved in trifluoroacetic acid (2.5 ml); after 50 min the excess was evaporated under reduced pressure to give a dense oil which was crystallized from CH2Cl2/n- hexane as a yellowish crystalline solid (80%), mp 161 °C dec. IR: 1661,3286.'H-NMR (CDCl3) : # 1. 32 (12H, s, CH3), 1.69 (6H, s, CH3CCO), 2.99 (2H, d, J4.3, CH2), 5.76 (3H, b, NH3+), 7.31 (1H, s, aromatic), 7.92 (1H, t, J4. 3, NH), 8.24 (1H, b, COOH). 13 C-NMR 6 (CDC13) : 24.2,25.4,85.1,85.8,110. 9, 133.4,142.2,160.1,170.9,176.3. MS, m/z : 412 (M+), 397,354,310,295,268,252,229,226,221,198,185,153,129,125,103, 98 (base peak), 83,69,59.

2. Enzyme Purification AmpC fromEscherichia coli was expressed andpurifiedto homogeneityas described (Usher et al., Biochemistry, 37: 16082-16092 (1998)). The TEM-1 gene was amplified from pBR322 by PCR and expressed from a pAlterEx II plasmid (Promega, Madison, WI) from transformed E. coli JM109 cells. TEM-1 was expressed and purified using a procedure described in Vanhove et al., Biochem J, 321: 413-7 (1997).

3. Enzyme Inhibition Assays The pinacol esters of the acylaminomethaneboronic acids were hydrolyzed to the free acids by dissolving them in 50 mM phosphate buffer at pH 7.0 (Kettner et al., J. Biol. Chers., 259: 15106-14 (1984)), at a concentration of 10 mM; more dilute stocks (1 mM to 100 I1M) were subsequently prepared as necessary. Kinetic measurements with AmpC were performed using cephalothin as a substrate (Weston et al., J. Med. Chem, 41: 4577-4586 (1998)).

Reactions were initiated by the addition of 1.5 nM enzyme. No pre-incubation effect was

detected for any compound, consistent with earlier studies (Weston et al., J. Med. Chem, 41: 4577-4586 (1998); Waley, Biochem. J., 205: 631-633 (1982)). IC50 values were determined at 100, uM substrate concentration.

TEM-1 enzyme assays used 100, uM furylacryloylamidopenicillanic acid as substrate, monitoring absorbance changes at 340 nm on an Hewlett-Packard HP8453 spectrophotometer. Reactions were initiated with addition of 0.3 nM enzyme, using the same buffer as in the AmpC assays.

The K ; values for compounds 3-14 were obtained by comparison of progress curves in the presence and absence of inhibitor (Waley, Bioche7n. J, 205: 631-633 (1982)). Sufficient inhibitor was used to give at least 50% inhibition. This method correlates well with full K analysis through coupled substrate and inhibitor concentration variation (Weston et al., J.

Med. Chem, 41: 4577-4586 (1998)). For compound 9, a Ki of 170A10 nM, consistent with the value of 160 nM determined by progress curve analysis, was also determined by Lineweaver-Burk analysis of multiple substrate and inhibitor concentrations (data not shown).

The selectivity of compounds 3 and 15 for (3-lactamases was determined bymeasuring their activity against a-chymotrypsin (bovine pancreatic), (3-trypsin (bovine pancreatic), and elastase (porcine pancreatic), all from Sigma (St. Louis, MO). Substrates for a-chymotrypsin (N-benzoyl-L-tyrosine ethyl ester, BTEE) and P-trypsin (N-benzoyl-L-arginine ethyl ester, BAEE) were also purchased from Sigma. The elastase substrate used (elastase substrate 1, MeOSuc-Ala-Ala-Pro-Val-pNA) was purchased from Calbiochem (San Diego, CA).

Substrates were diluted from 10 mM dimethylsulfoxide (DMSO) stock solutions, and all reactions were performed in 50 mM potassium phosphate buffer, pH 7.0,25°C. For a- chymotrypsin, 140, uM of BTEE was used, the reactions were initiated by addition of 5, uL of a 0.1 mg/ml enzyme stock solution, and monitored at 260 nm. For (3-trypsin, 200, uM of BAEE was used, the reactions were initiated by the addition of 5, uL of a 0.2 mg/ml enzyme stock solution, and monitored at 260 nm. For elastase, 640 MM of elastase substrate was used, the reactions were initiated by the addition of 30, uL of a 0.2 mg/ml enzyme stock solution, and monitored at 385 nm. Initial rate fits to the absorbance data for the first 150 seconds of each reaction were used to determine reaction velocities.

4. Crystal Growth and Structure Determination Co-crystals of 9 and 11 were grown by vapor diffusion in hanging drops equilibrated over 1.7 M potassium phosphate buffer (pH 8.7) using microseeding techniques. The initial concentration of protein in the drop was 95, uM, and the concentrations of each inhibitor were 586, uM. The inhibitors were added to the crystallization drop in a 2% DMSO, 1.7 M potassium phosphate buffer (pH 8.7) solution. Crystals appeared within 3-5 days after equilibration at 23 °C.

Data were collected on the DND-CAT beam line (5IDB) of the Advanced Photon Source at Argonne National Lab at 100 K using a 162 mm Mar CCD detector. Prior to data collection, crystals were immersed in a cryoprotectant solution of 20% sucrose, 1.7M potassium phosphate, pH 8.7, for about 20 seconds, then flash cooled in liquid nitrogen.

Each data set was measured from a single crystal.

Reflections were indexed, integrated and scaled using the HKL program suite (Otwinowski et al., Methods Enzymol., 276: 307-326 (1997)) (Table 3). The space group was C2, with two AmpC molecules in the asymmetric unit. Each AmpC molecule contained 358 residues. The structure was determined by molecular replacement using an AmpC/boronic acid complexed structure (Powers et al., Protein Sci., 8: 2330-7 (1999)) with inhibitor and water molecules removed, as the initial phasing model. The model was refined using the maximum likelihood target in CNS and included a bulk solvent correction (Brunger et al., Acta CrystallogrD Biol Crystallogr, 54: 905-21 (1998)). Sigma A-weighted electron density maps were calculated using CNS, and manual rebuilding was done in the program O (Jones et al., Acta Cryst., A47 : 110-119 (1991)). The inhibitor was built into the observed difference density in each active site of the asymmetric unit, and the structure of the complex was further refined using CNS (Table 3). All atoms of inhibitor 9 were refined with an occupancy of 1.0. All atoms of inhibitor 11 were refined with an occupancy of 1.0, except for atoms of the thiophene ring (C9, C10, Cl 1 and S 1) which were refined with an occupancy of 0.5 for each of the two possible conformations.

5. Microbiology Compounds 10 and 15 were tested for synergy with 13-lactams against pathogenic bacteria that are sensitive to (3-lactams and to pathogenic bacteria that are resistant to B- lactams through production of either Group It or Group I B-lactamases. Bacterial strains tested included: Enterobacter cloacae EB5 (p-lactamase negative), Enterobacter cloacae 265A (Group I p-lactamase hyper-producer), and Staphylococcus aureus V41 (Group II B- lactamase producer). All bacterial strains are available from Jesus Blazques and Fernando Baquero, Servicio de Microbiologia, Hospital Ramon y Cajal, National Institute o f Health, Madrid, Spain. Minimum inhibitory concentration (MIC) values were determined with Mueller-Hinton Broth II using the microdilution method according to the guidelines of the National Conznzitteefor Clihical Laboratory Standards,"Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically,"Approved Standard M7-A4, volume 17 (National Committee for Clinical Laboratory Standards, Villanova, PA, 1997).

Cefotaxime and ceftazidime were the B-lactams used with E. cloacae, and amoxicillin was used with S. aureus assays. Checkerboard assays were performed to study the synergistic effects.

Disk diffusion plate assays were performed as follows. E. cloacae EB5 and E. cloacae 265A were each grown to log-phase and then diluted in TY broth to a turbidity equivalent of McFarland 1. The cultures were further diluted 100-fold into melted TY agar medium and allowed to solidify in Falcon 150 25 mm plates. The plates were then spotted with ceftazidime (25 llg per disk for the EB5 plate, 50 pg per disk for the 265A plate) and 100 llg of 15 and 10. After overnight incubation at 35°C, the zones of inhibition were imaged.

B. RESULTS 1. Synthesis Six acylaminomethaneboronic acids (6,7,8,9,10,11,12) were synthesized through nitrogen insertion with LiN [Si (CH3) 3] on chloromethylboronic acid pinacol ester 1, followed by deprotection with equimolar methanol, and finally condensation with an acylchloride (see Figure 7). The yield of these 2-step syntheses varied from 62 to 92%. Slight changes to this general scheme were performed to synthesize 5,13 and 15. Deprotection of 4 was performed with acetic acid; reaction with acetic anhydride gave 5 in 60% yield. The condensation of compound 12 with 4-mercaptopyridine gave 13 in 90% yield. Compound 14 was obtained by preactivating (Z)-2-amino-a- [1- (tert-butoxycarbonyl)-1-metliylethoxyimino]-4- thiazoleacetic acid as a mixed anhydride, which was subsequently reacted with the deprotected 4. The tert-butoxycarbonyl group of 14 was deprotected with trifluoroacetic acid (TFA) to give 15, with an overall three-step reaction yield of 64%. This general synthetic scheme seems well suited to attaching the Rt side chains of p-lactams to the boronic acid group.

2. Binding Constants The differential affinities of the acylaminomethaneboronic acids allowed the contributions of the Rl side chain of (3-lactams to molecular recognition by the various enzymes to be determined. Against the Group I P-lactamase AmpC, the K ; values spanned a 1000-fold range, from 20 nM to 19, uM (Table 1). Comparing the minimal amide side chain of 3 (K ; 4.8, uM) to methylboronic acid (K, 1 mM) or to boric acid (K ; 2.8 mM), suggests that the amide group itself contributes 3.2 kcal/mol to binding in this series.

Comparing the affinity of 3 to compounds with more elaborate side chains, such as the ceftazidime analog 15 (K ; 20 nM), suggests that variations distal to the amide group can contribute at least 4.0 kcal/mol further interaction energy with AmpC.

The acylaminomethaneboronic acids bound less tightly to the Group II (3-lactamase TEM-1 than they did to AmpC (Tablel). Against TEM-1, K ; values varied from 0.39, uM to 162, uM. These values were eight-to forty-fold (typically twenty-fold) worse (higher) than

with AmpC. As with AmpC, compound 15, bearing the ceftazidime side chain, was the most active compound against TEM-1 (K ; 0. 39, uM).

3. Selectivity Testing Compound 3, bearing the minimal amide side chain and compound 15, bearing the relatively elaborate ceftazidime side chain, were tested for p-lactamase selectivity versus the serine proteases a-chymotrypsin, (3-trypsin, and elastase (Table 2). Compound 3 showed no activity up to 100, uM against any of the proteases. Compound 15 had an IC50 of 82 nM for AmpC and a projected ICso of 2 mM for a-chymotrypsin, 450 µM for elastase, and no measurable activity against p-trypsin. These assays were performed at similar [substrate]/K", ratios for each enzyme.

4. X-RavCrvstallographicStructureDetermination The structures of both 9 and 11 in complex with the Group I B-lactamase AmpC were determined to 1.75 A and 1.90 A resolution, respectively (Table 3). The location of the inhibitor in each complex was unambiguously identified in the initial Fo-Fc difference maps when contoured at a level of 3 sigma (s). Simulated annealing omit maps of the refined models agreed well with the placement of the inhibitors in the active sites (not shown).

The quality of each of the models was analyzed with the program Procheck (Laskowski et al., J. Appl. Cryst., 26: 283-291 (1993)). For the model of the complex of 9 with AmpC, 92.7% of the non-proline, non-glycine residues were in the most favored region of the Ramachandran plot (7.3% in the additionally allowed region), and for the complex of 11 with AmpC, 91.7% of the non-proline, non-glycine residues were in the most favored region (8. 1% in the additionally allowed region). The structures have been deposited with the PDB as 1FSY (complex with 9) and 1FSW (complex with 11).

In both structures electron density is observed connecting Oy of the catalytic Ser64 to the boron atom of the inhibitors (Figure 4). The geometry around the boron is tetrahedral, as expected. The 01 of the boronic acid is within good hydrogen-bonding distance of the backbone nitrogens of Ser64 and Ala318, and also the backbone oxygen of Ala318 (Table 4). These interactions are highly conserved in p-lactamase structures with transition-state analogs (Usher et al., Biochemistry, 37: 16082-16092 (1998); Powers et al., Protein Sci., 8: 2330-7 (1999); Lobkovsky et al., Biochemistry, 33: 6762-72 (1994); Strynadka et al., Nat.

Struct. Biol., 3: 688-95 (1996); Ness et al., Biochemistry, 39: 5312-5321 (2000)). The 02 of the boronic acid, which probably represents the position of the deacylating water in the high- energy intermediate (Patera et al., J Am. Chem. Soc., 122: 10504-10512 (2000); Powers et al., Proteirz Sci., 8: 2330-7 (1999)), hydrogen-bonds with the putative catalytic base Tyrl50 (Lobkovsky et al., Biochemistry, 33: 6762-72 (1994); Dubus et al., Proteins, 25: 473-485

(1996)) (Talbe 4). Two well-ordered water molecules are also observed in the region of the tetrahedral center of each complex, as seen in a previous structure of AmpC in complex with a boronic acid inhibitor (Powers et al., Protein Sci., 8: 2330-7 (1999)). The first water (Wat402) interacts with 02 of the boronic acid, Oyl of Thr316, and another water molecule (Wat506). The second water (Wat403) interacts with Wat402, as well as with Odl of Asn346 andNnl of Arg349. In the complex with 9, Wat403 is also within hydrogen bonding distance to another water molecule (Wat549).

In the crystal structures, the amide groups in the acylaminomethaneboronic acids are placed close to where the analogous R, side chain amide is placed in the structures of complexesbetweenß-lactams andß-lactamases (Pateraetal., J. Am. Chem. Soc., 122: 10504- 10512 (2000); Oefner et al., Nature, 343: 284-288 (1990); Strynadka et la., Nature, 359: 700-5 (1992). The amide groups in both the transition-state analog structures and the (3-lactam acyl- adducts make similar interactions with the enzymes. In the structure of 11 with AmpC, the carbonyl oxygen (06) of the amide group interacts with N62 of Asn 152 (2.8 A) and NE2 of Glnl20 (2.8 A, Table 4); both residues are completely conserved among Group I B- lactamases. In the structure of 9 with AmpC, only the interaction between Nb2 of Asnl52 and 06 is seen (2.5 A). Glnl20 appears unable to hydrogen bond with 06 due to a steric conflict that would occur with the chlorine atom of the inhibitor; instead, this residue has rotated by 119° around #2, away from the chlorine atom. Additionally, in the complex with 9, an interaction is observed between Ala3180 and N4 (3.2 A) (Table 4).

In the complex of 11 with AmpC, the unique part of the RI side chain (the thiophene ring) appears to make few interactions with AmpC. Difference density suggests that this ring can assume two conformations; they differ from each other by a 180 degree rotation around the C7-C8 bond. In each of the conformations, the atoms to which the thiophene is nearest are Thr319C and Asn343Nd2; the distances range from 3.4-3.5 A. The nearest atom to the sulfur of the thiophene ring in one conformation is the CB of Ale318 (3.7 A) ; there is also a water molecule (Wat745) 3.4 A away from the sulfur in this conformation. In the second conformation, the sulfur is nearest to Ne2 of Glnl20 (4.3 A) and is close to two water molecules (Wat744,3.8 A and Wat743, 3.9 A).

In the complex of 9 with AmpC, O10 of the isoxazole ring interacts with a water molecule (Wat569, 3.1 A). The exocyclic methyl group (C9) packs against the aryl ring of Tyr221. The chlorobenzyl ring of the inhibitor is located in a hydrophobic pocket formed by residues Leul 19 and Leu293. The Cß of Ala318 also contributes to burial of this ring. The distances range from 3.6 A (from C18 to CB of Ala318) to 4.3 A (from C15 to Cd2 of Leul 19 and to Cdl of Leu293). This ring is also near residues Asn289 and Asn343 and appears to make van der Waals interactions with these residues. As mentioned above, the chlorine atom is placed near Glnl20 (3.8 A).

5. Microbiology The minimum inhibitory concentration (MIC) values of ceftazdime and cefotaxime against the EB5 strain of E. cloacae, which does not produce a Group I B-lactamase, were 0.4 , ug/ml and 0.25, ug/ml, respectively. Administered alone, neither 10 nor 15 had measurable activity against this strain, neither was synergy observed with either ceftazidime or cefotaxime against this B-lactamase negative strain. Similarly, compound 15 had no activity by itself against S. aureas strain V41; the MIC of compound 10 alone was 128 ug/ml against this strain. Against strain 265A of E. cloacae, which hyper-produces a Group I B-lactamase, the MIC values of ceftazidime and cefotaxime rose to 265 and 1281lg/ml, respectively. Both compounds 10 and 15 showed synergy with these B-lactams against this B-lactamase producing strain of E. cloacae. Compound 15 reduced the MIC of ceftazidime by 256-fold at 32 pg/ml of the inhibitor (data not shown), and reduced the MIC of cefotaxime by 128-fold at the same concentration (Table 5). Both inhibitors also showed synergy with amoxicillin against S. aureus expressing a Group IT P-lactamase (Table 5).

Disk diffusion plate assays were performed to study the effects of compounds 10 and 15 on the efficacy of the p-lactam ceftazidime. As expected, the plate containing E. cloacae that does not produce a (3-lactamase showed a large inhibition halo surrounding the disks that contained ceftazidime (data not shown). The disks which contained compound 10 or 15 had no effect on the inhibition halo of ceftazidime, nor did they show any inhibition halos of their own (data not shown). The plate containing E. cloacae that hyperproduce a Group I 3- lactamase showed greatly reduced inhibition halos surrounding the disks containing ceftazidime, and in contrast to the previous plate, the inhibition halos in the regions between the two disks were substantially increased (data not shown). This increase in the size of the inhibition halos between the two disks indicates that each compound had a synergistic effect when coupled with ceftazidime.

C. DISCUSSION Acylaminomethaneboronic acids span five orders of magnitude in affinity for AmpC, from a dissociation constant of 2.8 mM for boric acid itself to 20 nM for the ceftazidime analog 15 (Table 1). This suggests that the Rl side chains of (3-lactams can make considerable contributions to affinity for p-lactamases. The result is unexpected, if only because the effect of R, side chains on affinity has previously been largely unknown. The compounds appear to be selective for p-lactamases, especially Group I p-lactamases resembling AmpC, showing little affinity for serine proteases that are known to be inhibited by peptide boronic acids (Kettner et al., Biochemistry, 27: 7682-8 (1988)) (Table 2). This is consistent with the specific recognition of the Rl side chains by serine P-lactamases. The differential energies between compounds allow the observed interactions in the crystal

structures of two of these inhibitors to be interpreted and, by analogy, those observed in other ß-lactam complexes. Additionally, comparing the x-ray crystal structure of a transition-state analog complex with that of an acyl-enzyme intermediate suggests how recognition of the Rl side chain changes between the transition state and the acylated ground state in Group I (3-lactamases.

An important contribution to affinity comes from the amide group common to all the inhibitors. Comparing compound 3 to methylboronic acid suggests that this amide contributes 3.2 kcal/mol to the free energy of binding. This amide group represents the C6 (7) R1 amide that is ubiquitous among (3-lactams (Figure 1). In previous structures with B- lactams, the amide oxygen of the side chain has been observed to hydrogen bond to the side chain of the conserved Asnl52 (Asnl32 in Group II ß-lactamases). The amide nitrogen of the R, side chain has been observed to hydrogen bond to the main chain carbonyl of residue 318 in Group I (3-lactamases (237 in Group Il (3-lactamases) (Patera et al., J. Am. Chem. Soc., 122: 10504-10512 (2000); Lobkovsky et al., Biochemistry, 33: 6762-72 (1994); Oefner et al., Nature, 343: 284-288 (1990); Strynadka et al., Nature, 359: 700-5 (1992); Chen et al., J. Mol.

Biol., 234: 165-78 (1993)). In the x-ray crystal structures of 9 and 11 in complex with AmpC, the Rl amide hydrogen bonds to Asnl52 (Figure 5, Table 4). The hydrogen bonding interaction with Ale318 is observed in the structure of AmpC with 9 but not with 11. In other complexes, the hydrogen bond between the ligand amide nitrogen and the backbone carbonyl of residue 318/327 may not be as well conserved structurally as that of Asn 152/Asn 132 with the ligand amide oxygen (Patera et al., J. Am. Chem. Soc., 122: 10504-10512 (2000); Strynadka et al., Nature : Structural Biology (1996)).

The distal parts of the Rl side chain, which have been a principlefoci of design and modification of ß-lactam antibiotics, also contribute to binding affinity. Dissociation constants vary from 700 nM for 7, which bears the penicillin V side chain, to 20 nM for 15, which bears the ceftazidime side chain (Table 1). By comparison to the acetamido side chain compound 5 (K ; 18 uM), the contribution to affinity for each group can be determined. For instance, the ceftazidime side chain contributes 4.0 kcal/mol in differential affinity compared to 5. Intriguingly, a-lactam side chains that are associated with inhibitors of AmpC do not necessarily have higher affinities than boronic acids bearing substrate side chains. For instance, cloxacillin is an inhibitor of AmpC, whereas ceftazidime is a substrate for the enzyme, albeit a poor one; nevertheless, the ceftazidime analog 15 binds ten-fold better to the enzyme than does the cloxacillin analog 9. Similarly, compound 11, which bears the side chain of the very good substrate cephalothin, binds only two-fold less well than the cloxacillin analog 9 but more than ten-fold worse than the ceftazidime analog 15.

Although Group I and Group II ß-lactamases are mechanistically related, the two enzyme groups have different substrate preferences and inhibitor profiles. To investigate

differential recognition between these two classes of p-lactamases, the affinity of several of the acylaminomethaneboronic acid inhibitors was determined for the characteristic Group II ß-lactamase, TEM-1. Overall, there is a monotonic relationship between the affinity of acylaminomethaneboronic acids for AmpC and for TEM-1, with the affinities for TEM-1 being 8-40-fold worse. TEM-1, traditionally known as a penicillinase, does not appear to be more selective for RI side chains associated with penicillins than does the cephalosporinase AmpC for the side chains of R, side chains associated with cephalosporins (Table 1). To the extent that Group I and Group II (3-lactamases are selective for cephalosporins and penicillins (Matagne et al., Biochem J, 265: 131-46 (1990)), respectively, this does not seem to be due to differences in the R1 side chains associated with each of these classes of drugs.

The greater affinity of the acylaminomethaneboronic acids for AmpC versus TEM-1 suggests that the Rl side chain contributes more to recognition in Group I (3-lactamases than it does in Group II (3-lactamases. This would be consistent with mutational and inhibition studies in these enzymes. Whereas the R,-amide recognition residue Asnl32 of Group II p- lactamases can be substituted with an aspartate or a serine with little loss of enzyme activity (Osuna et al., J. Biol. Chem., 270: 775-780 (1995)), the analogous N152D AmpC mutant enzyme loses four orders of magnitude of activity (Dubus et al., Biochemistry, 34: 7757-7764 (1995)). Also, the C3 (4) carboxylate, on the other side of the (3-lactam ring, contributes stronglyto recognition of ß-lactams by Group II (3-lactamases but not to Group I B-lactamases (Varetto et al., Biochem. J., 278: 801-7 (1991)). The importance of this carboxylate is reinforced by a comparison of the activity against TEM-1 of compound 6 with that of an analogous boronic acid, (lR)-l-phenylacetamido-2- (3-carboxyphenyl)-ethylboronic acid.

The latter compound has the same R, side chain as 6 but also has a carboxylate resembling the C3 carboxylate in penicillins (Ness et al., Biochemistry, 39: 5312-5321 (2000)). The carboxylate-containing boronic acid has a K ; of 5.9 nM against TEM-1, whereas compound 6 has a Kj of 13.8 juM against TEM-1. Binding for Group I ß-lactamases may be strongly influenced by interactions in the R, side chain, whereas high affinity in Group It p-lactamases may depend more on interactions spread across the entire p-lactam molecule.

Boronic acids mimic the tetrahedral geometry of the transition state of Group I ß- lactamases (Patera et al., J. Am. Chem. Soc., 122: 10504-10512 (2000); Waley, Biochem. J., 205: 631-633 (1982); Powers et al., Protein Sci., 8: 2330-7 (1999)), and it is interesting to compare their placement of the Rl side chains with that of the p-lactam acyl adduct structures. When the structure of the AmpC/9 complex is overlapped with that of a mutant AmpC (Q120L/Y150E) bound to cloxacillin (Patera et al., J. Am. Chem. Soc., 122: 10504- 10512 (2000)) in an acyl adduct, it is observed that the two Rl side chains are placed similarly in both structures (Figure 6). The root mean square deviation (RMSD) between the two side chains is 0.9 A, and most of the interactions are maintained. The greatest

positional differences are at the amide nitrogen at the beginning of the Rl side chain, but even here the transition-state analog and the acyl-adduct maintain the same interactions. On the part of the enzyme, there is little reorganization in the active site region. The RMSD between the Ca atoms of conserved residues in the active sites (Ser64, Lys67, residue 150, Asnl52, Tyr221, Lys315, and Ala318) of transition-state structure and the acyl-enzyme structure is 0.17 A (0.44 A for all atoms of the above residues except residue 150, whose identity differs between the mutant and wild-type enzymes). Two inferences can be made based on this comparison. First, at least for the cloxacillin group, the presence of the rest of the (3-lactam has little effect on the positioning of the Rl side chain. Second, the similarity of the acyl- adduct ground state to the transition-state analog suggests that progress along the reaction requires little reorganization in the R, side chain or in the residues with which it interacts.

There may be little differential stabilization between the acylated ground state and the transition state in the region of the R, side chain.

Where the transition-state analog complexes differ most from the (3-lactam acylated ground-state complexes is in the region of the tetrahedral center. In moving from the planar ester center to the tetrahedral boronic acid, a hydrogen bond is gained between the 02 hydroxyl of the boronic acid and the hydroxyl of Tyr 15 0. The 02, which appears to represent the position of the deacylating water in the high-energy intermediate (Powers et al., Protein Sci., 8: 2330-7 (1999)), hydrogen-bonds with Wat402. This water is conserved in native (Usher et al., Biochemistry, 37: 16082-16092 (1998)), acyl-enzyme (Patera et al., J. Am.

Chem. Soc., 122: 10504-10512 (2000)), and transition-state analog structures (Powers et al., Protein Sci., 8: 2330-7 (1999)), and may identify the region from where the deacylating water attacks the acyl-adduct (Patera et al., J. Am. Chem. Soc., 122: 10504-10512 (2000)). The O1 atom of the boronic acid, which represents the position of what was the lactam carbonyl oxygen in the acyl-enzyme intermediate (Patera et al., J. Am. Chem. Soc., 122: 10504-10512 (2000)), moves to pick up a hydrogen bond with the backbone oxygen of Ala318 in the tetrahedral adduct. This may be consistent with the status of this oxygen as a hydroxyl in the high-energy intermediate (Usher et al., Biochemistry, 37 : 16082-16092 (1998); Powers et al., Protein Sci., 8: 2330-7 (1999)). The overall picture that emerges is that, in moving from an acylated ground state to a transition-state analog complex, structural change is largely localized to the transition from a planar to a tetrahedral center in the ligand itself.

Once very effective, third-generation cephalosporins, such as ceftazidime and cefotaxime, have become largely useless against hospital pathogens such as E. cloacae because of the hyper-production of Group I B-lactamases. Given the high affinity of compounds 10 and 15 in enzyme assays, it seemed worthwhile to investigate their ability to reverse this resistance. Both inhibitors were synergistic when used in combination with ceftazidime (data not shown) and cefotaxime (Table 5). At high concentrations of 15, the

MIC values of these antibiotics were reduced by two-orders of magnitude, close to the levels of non-resistant strains. The synergistic effect was perhaps shown most compellingly in the disk diffusion assays. Against non-resistant E. cloacae, compounds 10 and 15 had no obvious effect, whereas against resistant strains of the same bacteria, these compounds showed an unmistakable synergy. Both inhibitors were also active against an isolate of S. aureus expressing a Group II (3-lactamase, although synergistic effects were less dramatic.

The ability of 10 and 15 to reverse ß-lactamase-based resistance, especially against the nosocomial pathogen E. cloacae, suggests that these compounds maybe useful leads for the design of new agents to reverse bacterial resistance to (3-lactams.

The X-ray crystal structures of AmpC with 9 and 11 may guide further inhibitor design. In the complex of 9 with AmpC, non-polar complementarity is achieved through interactions with residues Leul 19, Leu293 and Ala318. The methyl group of the isoxazole ring of 9 forms van der Waals interactions with the conserved Tyr221. The function of this residue is unknown, but it often forms aromatic polar or stacking interactions with substrates (Patera et al., J. Am. Chem. Soc., 122: 10504-10512 (2000)) and inhibitors (Patera et al., J.

Am. Chem. Soc., 122: 10504-10512 (2000); Powers et al., Proteill Sci., 8: 2330-7 (1999)). In both complexes, the RI side chains only fill part of the enzyme cleft, leaving uncomplemented polar residues such as Aspl23, Arg204, and Ser212. Interactions with some of these residues may help explain the increased affinity of 15, which is the most polar and the most active of the compounds tested. Indeed, compound 14, the neutral analog of 15, is less active than 15 (Table 1), suggesting that the Rl carboxylic acid of this compound may make favorable interactions with enzyme residues. Intriguingly, Ser212 is just proximal to a site of a tandem insertion in a related Group I B-lactamase that leads to a mutant enzyme resistant to ceftazidime (Crichlow et al., Biochemistty, 38: 10256-61 (1999)), the (3-lactam analog of 15. It may be possible to further improve polar complementarity in this series to increase inhibition and specificity.

The reversible binding of the acylaminomethaneboronic acids allows us to explore the energetic bases for recognition of the R, side chain of p-lactams by (3-lactamases. These and related analogs are accessible synthetically and may provide useful tools for studying recognition and mechanism in P-lactamases. Several of the compounds are highly active, selective, and reverse resistance to (3-lactams in pathogenic bacteria. Coupling interaction energies with structure and synthesis may guide the design of anti-resistance agents in this series.

Table 1: Ki(µM) values of acylaminomethaneboronic acids against AmpC and TEM-1. compoundß-lactam analog side chain (R7) K, (lJiM) vs AmpC 3, 3 3--H 4.8 38 5----CH3 18.5 162 6 penicillinG ! ng 0.57 13.8 7 penicill ! nv 0.70 n. ma. 0 8 phenethicillin 0. 30 n. ma. k 9 cloxacillin 0. 150 6.8 ci N O Nuo) \ 10 nafcillin X 0. 033 n. ma. 11 cephalothin 0. 32 6.5 %, 12----CH2CI 0. 24 n. ma 13 cephapirin NaS'* 0. 175 n ma Ili N 14---o 0. 070 n. ma. N HZN'S H2N s N 15 ceftazidime a 0.020 0.39 0 N r boronic acids with no amide side chain K, (iM) vs AmpC Ki (pM) vs TEM-1 Boric acid HO SOH 2,800 1,500 OH ,-OH Oh OH Compounds 6 to 10 bear side chains common to the penicillins ; compounds 11 to 14 bear side chains common to the cephalosporins. 7 is common to other ß-lactams : cefoxitin, nitrocefin, and cephaloridine. a. Not measured.

Table 2 : Selectivity of 3 and 15 for AmpC and TEM-1 (3-lactamases versus serine proteases.

Enzyme C50(µM) for 3 IC50 (µM) for 15 AmpC 17 0.081 TEM-1 247 2.4 a-chymotrypsin >2,000'' 2,000 P-trypsin >400a >400a elastase >400a 450 a. No inhibition was observed at 100 µM. The IC50 values assume that inhibition was no greater than 20% at this concentration. b. No inhibition was observed at 500 uM. The Cgo values assume that inhibition was no greater than 20% at this concentration.

Table 3: Data collection and refinement statistics.

9/AmpC Complex 11/AmpC Complex Cell constants (A ; °) a=118. 36 b=77. 57 a=118.11 b=77.83 c=97.46; ß=116. 18 c=97.32; 6=115. 83 Resolution (A) 1.75 1.90 Unique reflections 79,142 58, 505 Total reflections 287,631 176,832 Rmerge (%) 6.4 (13.9) a 6.3 (17. 2) a Completeness (%) 98.6 (94.6) a 93.6 (94. 5)a <I>/<#1> 12.9 14.2 Resolution range for refinement (A) 20-1.75 (1.79-1.75 A) a 20-1. 90 (1.94-1.90 A) a Number of protein residues 716 716 Number of water molecules 499 415 RMSD bond lengths (A) 0.014 0.013 RMSD bond angles (°) 1.781 1.719 R-factor (%) 19.6 19.4 Rfree (%) 21.8b 22.7b Average B-factor, protein atoms (Å2) 30.0c 32.0c Average B-factor, protein atoms (Å2, 29.9 31.8 A monomer only)) Average B-factor, inhibitor atams (Å2) 40.6c 41.0c Average B-factor, inhibitor atoms (Å2, 36.8 39.9 A monomer only) a Values in parentheses are for the highest resolution shell used in refinement. b Rfre was calculated with 10% of reflections set aside randomly. c Values cited were calculated for both molecules in the asymmetric unit.

Table 4: Interactions in Complexed and Native AmpC B-lactamase

(9) (11) Distance(A) Interaction 9/AmpCa 11/AmpCa Nativeb S64N-01 2.8 3.0 Npc A318N-01 2.8 2.8 Npc A3180-01 2.7 2.8 Npc Y1500H-02 2.6 2.6 Npc Wat402-02 2.9 2.9 Npc Y150OH-K315N# 3.0 2.9 2.5 Y150OH-S64OY 2.9 2.8 3.2 Y150OH-K67N# 3. 4 3.4 3.1 K67Ng-A2200 2.9 2.8 3.5 K67N#-S64OY 2.7 2.7 3.5 Wat402-T316OY1 2.9 2.9 3.8 Wat402-Wat403 2.7 2.9 Npc Wat403-N346O#1 2.7 2.6 Npc Wat403-R349N#1 3.1 3.0 Np° A3180-N4 3.3 4.3 Npc N152N#2-O6 2.5 2.8 Npc Q120Ne2-06 5.2 2.9 Npc N152O#1-K67N# 2. 7 2.6 2.7 N152N#2-Q120O#1 6. 6 2.7 3.0 Wat569-O10 3. 1 Npc Npc Wat867-06 2.9 Npc Npc a. Distances are for monomer 1 of the asymmetric unit. b. Distances are for monomer 2 of the asymmetric unit. c. Not present. d. In the native structure, Wat402 is called Wat387.

Table 5: Synergy of compounds 10 and 15 with B-lactams against B-lactamase-producing bacteria.

E. c/oacae 265A S. aureus V41 cefotaxime 10 cefotaxime 15 amoxicillin 10 amoxicillin 15 MIC (µg/ml) (µg/ml) MIC (µg/ml) (µg/ml) MIC (µg/ml) (µg/ml) MIC (µg/ml) (µg/ml) 128 0 128 0 64 0 64 0 64 2 64 2 64 2 64 2 64 4 64 4 32 4 64 4 64 8 16 8 32 8 32 8 32 16 2 16 16 16 32 16 16 32 1 32 8 32 32 32 8 64 0. 5 64 2 64 16 64 4 128 0.5 128 0 128 8 128