1,2,4-OXADIAZOLE AND 1,2,4-THIADIAZOLE BETA-LACTAMASE INHIBITORS

β-Lactamase inhibitor compounds (BLIs) are disclosed, including compounds that have activity against class A, class C or class D β-lactamases. Methods of manufacturing the BLIs, and uses of the compounds in the preparation of pharmaceutical compositions and antibacterial applications are also disclosed.

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Description
RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/618,136, filed Mar. 30, 2012. The entire content of this application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This disclosure is directed to β-lactamase inhibitors (BLIs) which are effective as inhibitors of β-lactamases and, when used in combination with β-lactam antibiotics are useful in the treatment of bacterial infections. The compounds when combined with a β-lactam antibiotic are effective in treating bacteria that are resistant to β-lactam antibiotics due to the presence of β-lactamases. Pharmaceutical compositions comprising such compounds, methods of using such compounds, and processes for preparing such compounds are also disclosed.

BACKGROUND

Bacterial resistance to β-lactam antibiotics, especially in Gram-negative bacteria, is most commonly mediated by β-lactamases. β-lactamases are enzymes that catalyze the hydrolysis of the β-lactam ring, which inactivates the antibacterial activity of the β-lactam antibiotic and allows the bacteria to become resistant. Inhibition of the β-lactamase with a BLI slows or prevents degradation of the β-lactam antibiotic and restores β-lactam antibiotic susceptibility to β-lactamase producing bacteria. Many of these β-lactamases are not effectively inhibited by BLIs currently on the market rendering the β-lactam antibiotics ineffective in treating bacteria that produce these β-lactamases. There is an urgent need for novel BLIs that inhibit β-lactamases that are not effectively inhibited by the current clinical BLIs (e.g. KPC, class C and class D β-lactamases) and that could be used in combination with β-lactam antibiotics to treat infections caused by β-lactam resistant bacteria.

SUMMARY OF INVENTION

The present invention provides, in one aspect, compounds of chemical formula (I), or pharmaceutically-acceptable salts thereof, which are BLIs and are useful in combination with β-lactam antibiotics for the treatment of bacterial infections.

A compound of Formula (I) or a pharmaceutically acceptable salt thereof:

    • wherein

Z is selected from a 1,2,4-oxadiazole or a 1,2,4-thiadiazole;

R is selected from

R1 is selected from:

    • a. hydrogen,
    • b.

      • wherein R2 is selected from

      • wherein each of R3, R4 and R5 is independently selected from hydrogen, (C1-C3)-alkyl, aminoalkyl, aminocycloalkyl, or hydroxyalkyl, and n is selected from 1, 2 or 3,
    • c. amino,
    • d.

      • wherein R6 is selected from H, (C1-C3)-unsubstituted alkyl, amino-(C2-C3)-alkyl, aminocycloalkyl, hydroxyalkyl,

      • and each of p and q is independently selected from 1 or 2; and
    • e. —CH2(R7)CH2NH2
      • wherein R7 is selected from amino or hydroxyl.

In another aspect, the invention provides use of a compound of Formula I for inhibiting β-lactamases.

In yet another aspect, the invention provides compounds of Formula I with high binding affinity for β-lactamase enzymes.

In a further aspect, the present invention also provides antibacterial compositions comprising compounds of Formula I and at least one β-lactam antibiotic.

In an even further embodiment, the present invention provides pharmaceutical compositions comprising compounds of Formula I and at least one β-lactam antibiotic and methods of use thereof.

In a still further aspect, the invention provides methods of use of the compounds of Formula I to treat bacterial infections in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Table I, Representative Compounds of Formula II

FIGS. 2a-2b show Table II, Standard BLI potentiation MIC assay against a panel of isogenic and clinical strains expressing β-lactamases.

FIG. 3 shows Table III, the synergy MIC of representative compounds of Formula I against a panel of isogenic and clinical strains expressing β-lactamases.

FIG. 4 shows Table IV, an assay to determine inhibition kinetics of representative compounds of Formula I for the KPC-2 β-lactamase.

 DETAILED DESCRIPTION Definitions

Molecular terms, when used in this application, have their common meaning unless otherwise specified.

The term “alkyl” is defined as a linear or branched, saturated radical having one to about twenty carbon atoms unless otherwise specified. Preferred alkyl radicals are “lower alkyl” radicals having one to about five carbon atoms. Examples of alkyl groups include, without limitation, methyl, ethyl, tert-butyl, isopropyl, and hexyl. A subset of the term alkyl is “(C1-C3)-unsubstituted alkyl” which is defined as an alkyl group that bears no substituent groups. Examples of (C1-C3)-unsubstituted alkyl groups include methyl, ethyl, propyl and isopropyl. It is understood that if a (C1-C3)-alkyl is “substituted” that one or more hydrogen atoms is replaced by a substitutent.

The term amino denotes a NH2 radical

The term “aminoalkyl” denotes an alkyl in which one or more of the alkyl hydrogen atoms has been replaced by an amino group.

The term “aminocycloalkyl” denotes a cycloalkyl in which one of the cycloalkyl hydrogen atoms has been replaced by an amino group.

The term “cycloalkyl” or “cycloalkyl ring” is defined as a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to twelve ring members. In a preferred embodiment, a cycloalkyl is a ring system having three to seven ring members. Examples of a cycloalkyl group include, without limitation, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl.

The term “hydroxyalkyl” denotes an alkyl radical in which one or more of the alkyl hydrogen atoms has been replaced by a hydroxyl group.

It will be understood by one of skill in the art that a

or -
denote the point of attachment of a substituent group where indicated. For example

or —C(O)NHR5

represent that the point of attachment of the amide moiety is at the carbonyl carbon.

The functional classification of β-lactamases and terms “Class A”, “Class C”, and “Class D” β-lactamases are understood by one of skill in the art and are described in “Updated Functional Classification of β-Lactamases”, Bush, K.; Jacoby, G. A.; Antimicrob. Agents Chemother. 2010, 54, 969-976, herein incorporated by reference.

The salts of the compounds of the invention include acid addition salts and base addition salts. In a one embodiment, the salt is a pharmaceutically acceptable salt of the compound of Formula I. The term “pharmaceutically acceptable salts” embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds of the invention may be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Examples of appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include, without limitation, formic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic, galactic, and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts of compounds of the invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine and procaine. All of these salts may be prepared by conventional means from the corresponding compound of the invention by treating, for example, the compound of the invention with the appropriate acid or base.

The compounds of the invention can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or non-racemic mixtures thereof. The compounds of the invention can be utilized in the present invention as a single isomer or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment with an optically active acid or base. Examples of appropriate acids include, without limitation, tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. The mixture of diastereomers can be separated by crystallization followed by liberation of the optically active bases from the optically active salts. An alternative process for separation of optical isomers includes the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers. Still another available method involves synthesis of covalent diastereoisomeric molecules by treating compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to obtain the enantiomerically pure compound. The optically active compounds of the invention can likewise be obtained by utilizing optically active starting materials. These isomers may be in the form of a free acid, a free base, an ester or a salt.

The invention also embraces isolated compounds. An isolated compound refers to a compound which represents at least 10%, such as at least 20%, such as at least 50% and further such as at least 80% of the compound present in the mixture. In one embodiment, the compound, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound exhibits detectable (i.e. statistically significant) activity when tested in conventional biological assays such as those described herein.

β-Lactamase Inhibitors (BLIs)

In one aspect, the invention provides compounds of Formula I or pharmaceutically-acceptable salts thereof:

The substituent Z of Formula I is selected from a 1,2,4-oxadiazole or a 1,2,4-thiadiazole. In one aspect of the invention the substituent

is selected from

wherein X is as described previously. In another embodiment of the invention,

is selected from

Substituent R of Formula I is selected from

In a preferred embodiment, R is

The group R1 is selected from:

    • a. hydrogen,
    • b.

      • wherein R2 is selected from

      • wherein each of R3, R4 and R5 is independently selected from hydrogen, (C1-C3)-alkyl, aminoalkyl, aminocycloalkyl, or hydroxyalkyl, and n is selected from 1, 2 or 3,
    • c. amino,
    • d.

      • wherein R6 is selected from H, (C1-C3)-unsubstituted alkyl, amino-(C2-C3)-alkyl, aminocycloalkyl, hydroxyalkyl,

      • and each of p and q is independently selected from 1 or 2; and
    • e. —CH2(R7)CH2NH2
      • wherein R7 is selected from amino or hydroxyl.

In one aspect of the invention n is 1. In another aspect of the invention n is 2. In another aspect of the invention n is 3.

In one aspect of the invention R1 is selected from H, NH2, or

In one embodiment of the invention, the compounds of the invention are of the stereochemistry disclosed in Formula II.

In another embodiment of the invention, Z, R and R1 are chosen from the substituents listed in Table I (See FIG. 1)

Preferred compounds of Formula I are the compounds:

It will be understood by one of skill in the art that depending on the nature of R1 and R, compounds of Formula I may exist in a salt or zwitterionic form.

Enzyme Inhibition and Binding Affinity

The compounds of Formula I are effective in inhibiting β-lactamase. When used in combination with β-lactam antibiotics, the compounds of Formula I potentiate the activity of the β-lactam antibiotic against microorganisms that are normally resistant to β-lactam antibiotics due to the presence of a β-lactamase or multiple β-lactamases.

In one aspect of the invention the compounds of Formula I inhibit β-lactamases selected from class A, class C or class D β-lactamases. Class A β-lactamases for example, include, but are not limited to, TEM, SHV, CTX-M, KPC, GES, VEB, SME, and GEX. In a preferred aspect of the invention, the compounds of the invention inhibit KPC β-lactamases. More preferably the compounds of the invention inhibit KPC-2 or KPC-3 β-lactamases. In one aspect of the invention, the compounds of Formula I inhibit KPC-2 or KPC-3 β-lactamases in clinical strains (FIG. 2, Table II). Class C β-lactamases for example, include, but are not limited to chromosomal AmpCs, and plasmid based ACC, DHA, CMY, FOX, ACT, MIR, LAT, MOX β-lactamases. Class D β-lactamase enzymes, for example, include, but are not limited to oxacillinases or OXA β-lactamases.

Unless otherwise indicated, the activity of the BLI compounds can be described by the MIC value obtained from a Synergy MIC assay or a BLI potentiation assay (e.g as described herein), both of which are run in the presence a β-lactam. The lower the sMIC or MIC value the more active the BLI, regardless of the mechanism of action of the BLI compound (e.g., including inhibition of β-lactamases by the BLI or any other mechanism of action or combination of mechanisms of action). The sMIC and BLI potentiation assay data supports that the compounds of Formula I potentiate (i.e. make more potent) the activity of the β-lactam antibiotic against β-lactamase producing strains by inhibiting the β-lactamase.

In one embodiment, the BLI activity is measured by growth inhibition of a β-lactamase producing bacterial strains in a Synergy MIC (sMIC) assay. Preferably, sMIC is 8 μg/mL or less. In a more preferred aspect of the invention, the sMIC is 4 μg/mL to 8 μg/mL. In an even more preferred aspect of the invention, the ssMIC is 1 to 2 μg/mL. In a still more preferred aspect of the invention, the sMIC is 0.2 to 0.5 μg/mL. Synergy MICs for representative compounds of the invention are described in Table III (See FIG. 3). It will be understood by one of skill in the art that the growth inhibition of β-lactamase producing strains can also be measured by a checkerboard synergy assay like that disclosed in International Patent Application Number WO 2008/039420 or a standard BLI potentiation assay using a fixed concentration of BLI.

In one embodiment, the BLI activity is measured by growth inhibition of a β-lactamase producing bacterial strains in a standard BLI potentiation assay using a fixed concentration of BLI. Preferably, the MIC is 8 μg/mL or less. In a more preferred aspect of the invention, the MIC is 4 to 8 μg/mL. In an even more preferred aspect of the invention, the MIC is 1 to 2 μg/mL. In a still more preferred aspect of the invention, the MIC is 0.2 to 0.5 μg/mL.

The compounds of the present invention have a broad spectrum of activity across a wide variety of β-lactamase producing bacteria. It was surprisingly found that the compounds of the present invention are active in potentiating activity of β-lactam antibiotics, in particular, Ceftolozane, against strains expressing class D β-lactamases, in particular the OXA-15 β-lactamase. Currently marketed BLIs inhibit most of the class A β-lactamases, but poorly inhibit class A KPC β-lactamases and class C β-lactamases and have variable success in inhibiting penicillinase and carbapenemase-type class D β-lactamases. The compounds of the present invention are active against a wide variety of bacterial strains that express class A and C β-lactamases and also, surprisingly are active against bacterial strains that express the class D cephalosporinase OXA-15 (Tables II and III). This increased activity against the class D β-lactamase is critical because differential effectiveness against different types of β-lactamase producing bacteria is necessary in order to effectively use β-lactam antibiotics to treat resistant strains of bacteria (vide infra).

In one embodiment, the compounds of Formula I are unexpectedly more active against bacterial strains that express OXA-15 β-lactamases than the most structurally similar compound, Avibactam (comparator compound CCC). Compounds that are more active than Avibactam are, for example, compounds 801, 802, 804, and 805.

In one embodiment, the compounds of Formula I are unexpectedly more active against and/or show broader spectrum of activity against bacterial strains that express KPC β-lactamases than the most structurally similar compound, Avibactam. Compounds that are more active than, and/or show a better spectrum of activity than Avibactam are, for example, compounds 801, 802, 804, and 805.

In another aspect of the invention, the compounds of Formula I have higher binding affinity for the β-lactamase enzyme. Consequently these compounds are better inhibitors of the β-lactamase enzyme. The inhibition kinetics of the compounds of Formula I was measured according to the procedure outlined in Example 14. The compounds of Formula I have a high binding affinity for the β-lactamase enzyme.

In one embodiment the compounds of Formula I have a binding affinity of 1000-5000 mM−1s−1.

In one embodiment the compounds of Formula I have a binding affinity of 100-999 mM−1s−1. Compounds that have a binding affinity of 100-999 mM−1 s−1 are, for example, compounds 801, 802, 804, 805, and 806 (Table IV).

In one embodiment the compounds of Formula I have a binding affinity of 1-99 mM−1s−1.

It was surprisingly found that the compounds of the present invention have a higher binding affinity for the β-lactamase enzyme than the closest structural comparator Avibactam (Table IV, See FIG. 4).

Pharmaceutical Compositions Comprising the Compounds of Formula I and Use Thereof

Another object of the invention is pharmaceutical compositions or formulations comprising compounds of Formula I, or salts thereof, preferably further comprising a β-lactam antibiotic.

The pharmaceutical compositions can be formulated for oral, intravenous, intramuscular, subcutaneous or parenteral administration for the therapeutic or prophylactic treatment of diseases, such as bacterial infections. Preferably, the pharmaceutical composition is formulated for intravenous administration.

The pharmaceutical preparations disclosed herein may be prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent or eliminate infection (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. and Goodman and Gilman's “The Pharmaceutical Basis of Therapeutics,” Pergamon Press, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of the methods for administering various antimicrobial agents for human therapy).

The pharmaceutical compositions can comprise one or more of the compounds disclosed herein, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, in association with one or more nontoxic, pharmaceutically-acceptable carriers and/or diluents and/or adjuvants and/or excipients. As used herein, the phrase “pharmaceutically-acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Non-limiting examples of carriers and excipients include corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The compositions may contain croscarmellose sodium, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid.

Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicone fluid, talc, waxes, oils and colloidal silica.

Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. It may also be desirable to add a coloring agent to make the dosage form more aesthetic in appearance or to help identify the product.

For oral or parenteral administration, compounds of the present invention preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like. The compositions comprising a compound of this invention may contain from about 0.1% to about 99% by weight of the active compound, such as from about 10% to about 30%.

For oral use, solid formulations such as tablets and capsules are useful. Sustained release or enterically coated preparations may also be devised. For pediatric and geriatric applications, one embodiment provides suspensions, syrups and chewable tablets. For oral administration, the pharmaceutical compositions are in the form of, for example, a tablet, capsule, suspension or liquid.

The pharmaceutical compositions may be made in the form of a dosage unit containing a therapeutically-effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, the tablets and capsules which can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica, or talc; disintegrants, for example, potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally are in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs, preparations of the invention may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Non-limiting examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

For intravenous (IV) use, the pharmaceutical composition, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline or Ringer's solution. Intravenous administration may be accomplished by using, without limitation, syringe, mini-pump or intravenous line.

Pharmaceutical compositions of this invention for parenteral injection comprise pharmaceutically-acceptable aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, benzyl alcohol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils (such as corn oil or 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. The compositions can include various buffers.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. They may also contain taggants or other anti-counterfeiting agents, which are well known in the art. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, and phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride. 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.

Injectable depot forms can be made by forming microencapsulating matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. Such forms may include forms that dissolve or disintegrate quickly in the oral environment. In such solid dosage forms, the active compound preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be mixed with at least one inert, pharmaceutically-acceptable excipient or carrier. Suitable excipients include, for example, (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders such as cellulose and cellulose derivatives (such as hydroxypropylmethylcellulose, hydroxypropylcellulose, and carboxymethylcellulose), alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as sodium starch glycolate, croscarmellose, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents such as paraffin; (f) absorption accelerators such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate, fatty acid esters of sorbitan, poloxamers, and polyethylene glycols; (h) absorbents such as kaolin and bentonite clay; (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (j) glidants such as talc, and silicone dioxide. Other suitable excipients include, for example, sodium citrate or dicalcium phosphate. The dosage forms may also comprise buffering agents.

Solid dosage forms, including those of tablets, dragees, capsules, pills, and granules, can be prepared with coatings and shells such as functional and aesthetic enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and colorants. They may also be in a form capable of controlled or sustained release. Examples of embedding compositions that can be used for such purposes include polymeric substances and waxes.

The pharmaceutical compositions can be delivered using controlled (e.g., capsules) or sustained release (e.g., bioerodable matrices) delivery systems. Exemplary delayed release delivery systems for drug delivery that are suitable for administering the pharmaceutical compositions are described in U.S. Pat. No. 4,452,775 (issued to Kent), U.S. Pat. No. 5,039,660 (issued to Leonard), and U.S. Pat. No. 3,854,480 (issued to Zaffaroni).

In some cases, in order to prolong the effect of the drug, it may be desirable to slow the absorption of the drug following subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. Amorphous material may be used alone or together with stabilizers as necessary. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form.

Alternatively, delayed absorption of a parenterally administered drug form can be accomplished by dissolving or suspending the drug in an oil vehicle.

For intramuscular preparations, a sterile formulation of compounds, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, or suitable soluble salt forms thereof, for example hydrochloride salts, can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5% glucose. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g., an ester of a long chain fatty acid such as ethyl oleate.

A dose of an intravenous, intramuscular, or parental formulation of compounds, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, may be administered as a bolus or by slow infusion. A bolus is a dose that is administered in less than 30 minutes. In one embodiment, a bolus is administered in less than 15 or less than 10 minutes. In another embodiment, a bolus is administered in less than 5 minutes. In yet another embodiment, a bolus is administered in one minute or less. An infusion is a dose that is administered at a rate of 30 minutes or greater. In one embodiment, the infusion is one hour or greater. In another embodiment, the infusion is substantially constant.

For topical use the pharmaceutical compositions, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient.

For application to the eyes or ears, the pharmaceutical composition can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.

For rectal administration, the pharmaceutical compositions, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, polyethylene glycol or a suppository wax or other glyceride that are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Alternatively, the pharmaceutical compositions can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery. In another embodiment, the unit dosage form of compounds, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be a solution of one or more compounds, or salts thereof, in a suitable diluent, in sterile hermetically sealed ampoules or sterile syringes. The concentration of the compounds, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, in the unit dosage may vary, e.g. from about 1 percent to about 50 percent, depending on the compound used and its solubility and the dose desired by the physician. If the compositions contain dosage units, each dosage unit can contain from 1-500 mg of the active material. For adult human treatment, the dosage employed can range from 5 mg to 10 g, per day, depending on the route and frequency of administration.

The pharmaceutical compositions disclosed herein can be placed in a pharmaceutically acceptable carrier and are delivered to a recipient subject (e.g., a human) in accordance with known methods of drug delivery. In general, the methods of delivering the pharmaceutical compositions in vivo utilize ar-recognized protocols for delivering the agent with the only substantial procedural modification being the substitution of the compounds of the present invention for the drugs in the art-recognized protocols. Likewise, methods for using the claimed compositions for treating cells in culture, for example, to eliminate or reduce the level of bacterial contamination of a cell culture, utilize art-recognized protocols for treating cell cultures with antibacterial agent(s) with the only substantial procedural modification being the substitution of the compounds of the present invention, preferably in combination with a β-lactam antibiotic for the drugs in the art-recognized protocols.

Exemplary procedures for delivering an antibacterial agent are described in U.S. Pat. Nos. 6,468,967; 6,852,689; and 5,041,567, issued to Rogers and in PCT patent application number EP94/02552 (publication no. WO 95/05384), the disclosures of which are incorporated herein by reference in their entirety. In one embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, or pharmaceutical compositions thereof are administered orally, rectally or via injection (intravenous, intramuscular or subcutaneous). In another embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, or pharmaceutical compositions thereof are administered orally, rectally or via injection (intravenous, intramuscular or subcutaneous) to treat an infection caused by β-lactam resistant bacteria. In another embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, or pharmaceutical compositions thereof are administered orally to treat an infection caused by β-lactamase producing bacteria. As used herein, the phrases “therapeutically-effective dose” and “therapeutically-effective amount” refer to an amount of a compound that prevents the onset, alleviates the symptoms, stops the progression of a bacterial infection, or results in another desired biological outcome such as, e.g., improved clinical signs or reduced/elevated levels of lymphocytes and/or antibodies. The term “treating” or “treatment” is defined as administering, to a subject, a therapeutically-effective amount of one or more compounds both to prevent the occurrence of an infection and to control or eliminate an infection. Those in need of treatment may include individuals already having a particular medical disease as well as those at risk for the disease (i.e., those who are likely to ultimately acquire the disorder). The term “subject,” as used herein, refers to a mammal, a plant, a lower animal, or a cell culture. In one embodiment, a subject is a human or other animal patient in need of antibacterial treatment.

The term “administering” or “administration” and the like, refers to providing the compound of Formula I to the subject in need of treatment. Preferably the subject is a mammal, more preferably a human. The present invention comprises administering the compound of Formula I in conjunction with a β-lactam antibiotic. When a compound of Formula I is administered in conjunction with a β-lactam antibiotic, the compound of Formula I and the β-lactam antibiotic can be administered at the same time or different times. When the compounds of Formula I and the β-lactam antibiotic are administered at the same time, they can be administered as a single composition or pharmaceutical composition or they can be administered separately. It is understood that when a compound of Formula I is administered in conjunction with a β-lactam antibiotic, that the active agents can be administered in a single combination or in multiple combinations. For example, when administered by IV, the compound of Formula I can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion, then a β-lactam antibiotic can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Conversely the β-lactam antibiotic can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion, then a compound of Formula I can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Alternatively, a pharmaceutical composition comprising a compound of Formula I and a β-lactam antibiotic can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion.

In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof a therapeutically-effective amount of the pharmaceutical composition comprising a compound of Formula I and a β-lactam antibiotic.

In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof, a therapeutically-effective amount of a β-lactam antibiotic in conjunction with a compound of claim 1.

In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection in a subject comprising the steps of

    • a. administering to the subject a compound of Formula I; and
    • b. administering a therapeutically-effective amount of a β-lactam antibiotic.

In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection in a subject comprising the steps of

    • a. administering a therapeutically-effective amount of a β-lactam antibiotic; and
    • b. administering to the subject a compound of Formula I.

In one embodiment, the invention provides a method for treating an infection in a subject by administering a therapeutically-effective amount of one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, or compositions thereof. In one embodiment, the method comprises administering to a subject in need thereof a pharmaceutical composition comprising at least one of the compounds described herein, preferably a compound of Formula I in conjunction with a β-lactam antibiotic. In one embodiment, the pharmaceutical composition can comprise any one of the compounds described herein as the sole active compound or in combination with another compound, composition, or biological material. The compound may be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally, or by an implanted reservoir, external pump or catheter. The compound may be prepared for ophthalmic or aerosolized uses. The compounds of the present invention can be administered as an aerosol for the treatment of pneumonia or other lung-based infections. In one embodiment, the aerosol delivery vehicle is an anhydrous or dry powder inhaler. One or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, or pharmaceutical compositions thereof also may be directly injected or administered into an abscess, ventricle or joint. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, cisternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion. In one embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, are administered intravenously, subcutaneously or orally. In one embodiment for administering one or more compounds according to Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic to a cell culture, the one or more compounds may be administered in a nutrient medium.

In one embodiment, one or more compounds according to Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, may be used to treat a subject having a bacterial infection in which the infection is caused or exacerbated by any type of bacteria, such as Gram-negative bacteria. In one aspect of the invention, the bacterial infection is caused by β-lactam resistant bacteria. In one aspect the bacterial infection is caused by β-lactamase producing bacteria. In another aspect the bacterial infection is caused by class A, class C or class D β-lactamase producing bacteria. In another aspect the bacterial infection is caused by class A β-lactamase producing bacteria. In another aspect the infection is caused by class C β-lactamase producing bacteria. In still another aspect the infection is caused by class D β-lactamase producing bacteria. In still another aspect the infection is caused by KPC β-lactamase producing bacteria. In still another aspect the infection is caused by OXA β-lactamase producing bacteria.

Representative Gram-negative pathogens known to express β-lactamases include, but are not limited to Acinetobacter spp. (including Acinetobacter baumannii), Citrobacter spp., Escherichia spp. (including Escherichia coli), Haemophilus influenzae, Morganella morganii, Pseudomonas aeruginosa, Klebsiella spp. (including Klebsiella pneumoniae), Enterobacter spp. (including Enterobacter cloacae and Enterobacter aerogenes), Pasteurella spp., Proteus spp. (including Proteus mirabilis), Serratia spp. (including Serratia marcescens), and Providencia spp. Bacterial infections can be caused or exacerbated by Gram-negative bacteria including strains which express β-lactamases that may confer resistance to penicillins, cephalosporins, monobactams and/or carbapenems. The co-administration of a novel BLIs that inhibits these β-lactamases with a β-lactam antibiotic could be used to treat infections caused β-lactam resistant bacteria.

In one aspect of the invention the infection is caused by a β-lactamase producing bacteria selected from Acinetobacter spp, Citrobacter spp, Escherichia coli, Enterobacter cloacae), Haemophilus influenzae, Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens, and Klebsiella pneumoniae,

β-Lactam antibiotics that may be co-administered with compounds of Formula I include, but are not limited to cephalosporin, carbapenem, monobactam, penem and penicillin classes of antibiotics.

In one embodiment of the invention, the β-lactam antibiotic is a cephalosporin. Examples of cephalosporins include, but are not limited to, Cefacetrile (cephacetrile), Cefadroxil (cefadroxyl), Cefalexin (cephalexin), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium), Cefaloridine (cephaloradine), Cefalotin (cephalothin), Cefapirin (cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (cephazolin), Cefradine (cephradine), Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefinetazole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil (cefproxil), Cefuroxime, Cefuzonam, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, Cefinenoxime, Cefodizime, Cefotaxime, Cefpimizole, Cefpodoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, Ceftizoxime, Ceftriaxone, Cefoperazone, Ceftazidime, Cefclidine, Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome, Cefaclomezine, Cefaloram, Cefaparole, Cefcanel, Cefedrolor, Cefempidone, Cefetrizole, Cefivitril, Cefmatilen, Cefinepidium, Cefovecin, Cefoxazole, Cefrotil, Cefsumide, Ceftaroline, Ceftioxide, Cefuracetime, cefbuperazone, cefminox, ceforanide, cefotiam, cefpiramide, cefsulodin, ceftobiprole latamoxef, loracarbef and Ceftolozane. In one embodiment the cephalosporin is Ceftolozane or Ceftazidime.

In one embodiment of the invention, the β-lactam antibiotic is a carbapenen. Examples of carbapenem antibiotics include, but are not limited to, Imipenem, Imipenem/Cilastatin, Biapenem, Doripenem, Meropenem, Ertapenem and Panipenem. In one embodiment the Carbapenem is Imipenem/Cilastatin or Meropenem.

In one embodiment of the invention, the β-lactam antibiotic is a monobactam. Examples of monobactam antibiotics include, but are not limited to Aztreonam, Tigemonam, Carumonam, BAL30072 and Nocardicin A.

In one embodiment of the invention, the β-lactam antibiotic is a penem.

In one embodiment of the invention, the β-lactam antibiotic is a penicillin Examples of penicillin antibiotics include, but are not limited to

Amoxicillin, Ampicillin, Azlocillin, Mezlocillin, Apalcillin, Hetacillin, Becampicillin, Carbenicillin, Sulbenicillin, Ticarcillin, Piperacillin, Azlocillin, Mecillinam, Pivmecillinam, Methicillin, Ciclacillin, Talampicillin, Aspoxicillin, Oxacillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Nafcillin and Pivampicillin.

The pharmaceutical compositions, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be used to treat a bacterial infection of any organ or tissue in the body caused by β-lactam resistant bacteria, preferably, Gram-negative β-lactam resistant bacteria. These organs or tissue include, without limitation, skeletal muscle, skin, bloodstream, kidneys, heart, lung and bone. For example, a pharmaceutical composition comprising at least one compound of Formula (I), preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be administered to a subject to treat, without limitation, skin and soft tissue infections (e.g., complex skin infections), bacteremia, intra-abdominal infections and urinary tract infections (e.g., cUTI). In addition, a compound of Formula (I) may be used to treat community acquired respiratory infections, including, without limitation, otitis media, sinusitis, chronic bronchitis and pneumonia (including community-acquired pneumonia, hospital-acquired pneumonia and ventilator associated pneumonia), including pneumonia caused by drug-resistant Pseudomonas aeruginosa. At least one compound of Formula (I), preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be administered to a subject to treat mixed infections that comprise different types of Gram-negative bacteria, or which comprise both Gram-positive and Gram-negative bacteria. These types of infections include intra-abdominal infections and obstetrical/gynecological infections. At least one compound of Formula (I), preferably a compound of Formula I in conjunction with a β-lactam antibiotic, may also be administered to a subject to treat an infection including, without limitation, endocarditis, nephritis, septic arthritis, intra-abdominal sepsis, bone and joint infections and osteomyelitis. At least one compound of Formula (I), preferably compound of Formula I in conjunction with a β-lactam antibiotic, or pharmaceutical compositions thereof, may also be directly injected or administered into an abscess, ventricle or joint. Pharmaceutical compositions administered as an aerosol for the treatment of pneumonia or other lung-based infections. In one embodiment, the aerosol delivery vehicle is an anhydrous, liquid or dry powder inhaler.

Actual dosage levels of active ingredients in the pharmaceutical compositions of one or more compounds according to Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, may be varied so as to obtain a therapeutically-effective amount of the active compound(s) to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The effective amount can be determined as described herein. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. In one embodiment, the data obtained from the assays can be used in formulating a range of dosage for use in humans. It will be understood by one of skill in the art that the when the composition comprises a compound of Formula I and a β-lactam antibiotic, both the compound of Formula I and the β-lactam antibiotic are active compounds.

The method comprises administering to the subject an effective dose of one or more compounds of Formula I, preferably in conjunction with a β lactam antibiotic. An effective dose of a compound of Formula I is generally between 125 mg/day to 2000 mg/day. In one embodiment, an effective dose is from about 0.1 to about 100 mg/kg of one or more compounds of Formula I or pharmaceutically acceptable salts thereof. In one embodiment, the dose is from about 0.1 to about 50 mg/kg of one or more compounds of Formula I or pharmaceutically acceptable salts thereof. In another embodiment, the dose is from about 1 to about 25 mg/kg of one or more compounds of Formula I or pharmaceutically acceptable salts thereof. In another embodiment, the dose is from about 1 to about 12 mg/kg of one or more compounds of Formula I. In another embodiment, the dose is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mg/kg of one or more compounds of Formula I. In another embodiment, the compounds of Formula I are administered to a human at a dose of 100 mg to 1000 mg per dose up to four times per day. In another embodiment, the compounds of Formula I are administered to a human at a dose of 125 mg to 750 mg per dose up to four times per day. In another embodiment, the compounds of Formula I are administered to a human at a dose of 250 mg to 500 mg per dose up to four times a day. An effective dose for cell culture is usually between about 0.1 and about 1000 μg/mL. In one embodiment, the effect dose for cell culture is between about 0.1 and about 200 μg/mL.

In one embodiment, a β-lactam antibiotic and a compound of Formula I are administered in ratio of 1:4 to 8:1 antibiotic:Formula I compound. In one embodiment the ratio is 1:4. In another embodiment the ratio is 3:4. In another embodiment the ratio is 5:4. In another embodiment the ratio is 7:4. In another embodiment the ratio is 1:2. In another embodiment the ratio is 3:2. In another embodiment the ratio is 5:2. In another embodiment the ratio is 7:2. In another embodiment the ratio is 1:3. In another embodiment the ratio is 2:3. In another embodiment the ratio is 4:3. In another embodiment the ratio is 5:3. In another embodiment the ratio is 7:3. In another embodiment the ratio is 1:2. In another embodiment the ratio is 3:2. In another embodiment the ratio is 5:2. In another embodiment the ratio is 7:2. In another embodiment the ratio is 1:1. In another embodiment the ratio is 2:1. In another embodiment the ratio is 3:1. In another embodiment the ratio is 4:1. In another embodiment the ratio is 5:1. In another embodiment the ratio is 6:1. In another embodiment the ratio is 7:1. In another embodiment the ratio is 8:1. It will be understood by one of skill in the art that the β-lactam antibiotic and compound of Formula I can be administered within the range of ratios provided regardless of the method of drug delivery. It will also be understood by one of skill in the art that the β-lactam antibiotic and compound of Formula I can be administered within the range of ratios provided together, for example, in a pharmaceutical composition, or sequentially, i.e. the β-lactam antibiotic is administered, followed by administration of a compound of Formula I or vice versa.

One or more compounds of Formula I may also be administered in the diet or feed of a patient or animal. If administered as part of a total dietary intake, the amount of compound employed can be less than 1% by weight of the diet, such as no more than 0.5% by weight. The diet for animals can be normal foodstuffs to which the compound can be added or it can be added to a premix.

One or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, can be administered as a single daily dose or in multiple doses per day. In one embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, is administered as a single dose per day. In another embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic is administered as two equal doses per day. In another embodiment, the compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic is administered in three equal doses per day. In another embodiment, the compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic is administered in four equal doses per day. The treatment regime may require administration over extended periods of time, e.g., for several days or for from two to four weeks. The amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the infection, the age and general health of the patient, the tolerance of the patient to the compound and the microorganism or microorganisms involved in the infection. The treatment regimen for one type of infection may differ greatly from the treatment regimen of another infection. For example, one type of infection may require administration via intravenous administration once daily, while another infection may require a treatment regimen of multiple dosing orally.

One or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, may be administered according to this method until the bacterial infection is eradicated or reduced. In one embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, are administered for a period of time from 3 days to 6 months. In another embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, are administered for 7 to 56 days. In another embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, are administered for 7 to 28 days. In a further embodiment, one or more compounds of Formula I, preferably a compound of Formula I in conjunction with a β-lactam antibiotic, are administered for 7 to 14 days. Compounds of the present invention may be administered for a longer or shorter time period if it is so desired.

Other embodiments of the invention include:

A pharmaceutical composition comprising a compound of Formula I and at least 1 β-lactam antibiotic or a pharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of Formula I and at least 1 cephalosporin antibiotic or a pharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of Formula I and Ceftolozane antibiotic or a pharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of Formula I and at least 1 carbapenem antibiotic or a pharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of Formula I and at least 1 monobactam antibiotic or a pharmaceutically acceptable salt thereof.

The embodiments described herein provide compounds of Formula I that are novel and active β-lactamase inhibitors. Other embodiments described herein provide novel compounds of Formula I in conjunction with β-lactam antibiotics for treatment of infections. Further embodiments described herein provide novel compounds of Formula I that show unexpected activity against β-lactamases that other compounds in the class do not have.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Preparation of Compounds of Formula I

A compound of formula (I) can be prepared by a variety of synthetic routes, including synthetic schemes described herein. These synthetic routes can be applied to large scale synthesis with appropriate adjustment of reaction sequence, reaction conditions, isolation/purification methods and choice of solvents which are environmentally friendly and cost-effective.

The following abbreviations have the following meanings unless otherwise indicated. Abbreviations not defined below have their generally accepted meaning.

    • Bn=benzyl
    • Boc=tert-butoxycarbonyl
    • Boc2O=di-tert-butyldicarbonate
    • Burgess reagent=methyl N-triethylammoniumsulfonyl)carbamate
    • CDI=carbonyldiimidazole
    • CFU=colony-forming units
    • CLSI=Clinical Laboratory Standards Institute
    • cSSSI=complicated skin and skin structure infections
    • DBU=1,8-diazabicyclo[5.4.0]undec-7-ene
    • DCM=dichloromethane
    • DEAD=diethyl azodicarboxylate
    • DIAD=diisopropyl azodicarboxylate
    • DIPEA=diisopropylethylamine
    • DMF=N,N-dimethylformamide
    • DMAc=N,N-dimethylacetamide
    • DMSO=dimethyl sulfoxide
    • EDCI=1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide
    • ELSD=evaporative light scattering detector
    • EtOAc=ethyl acetate
    • ESI-MS=electrospray ionization mass spectrometry
    • Fmoc=Fluorenylmethyloxycarbonyl
    • HAP=Hospital-Acquired Pneumonia
    • HATU=2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
    • HCl=hydrochloride
    • HOBt=1-hydroxybenzotrizole
    • Hrs=hours
    • HPLC=high performance liquid chromatography
    • Hunig's base=N,N-Diisopropylethylamine
    • Lawesson's reagent=2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide
    • MIC=minimum inhibitory concentration
    • mL=milliliter
    • MS=mass spectrometry
    • MRSA=methicillin-resistant Staphylococcus aureus
    • NMR=nuclear magnetic resonance
    • Ns=nitrobenzenesulfonyl
    • Pa=Pseudomonas aeruginosa
    • Prep=preparative
    • Ppm=parts per million
    • sat.=saturated
    • rt=room temperature
    • TBAF=tetrabutylammonium fluoride
    • TBS=t-butyldimethylsilyl
    • TES=triethylsilyl
    • TEA=triethylamine
    • TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy, free radical
    • THF=tetrahydrofuran
    • TFA=trifluoroacetic acid
    • TMS=trimethylsilyl
    • TLC=thin layer chromatography
    • VAP=Ventilator-Associated Pneumonia

The compounds of Formula (I) can be prepared from intermediate 1 or 7, according to the following reaction schemes and examples, or modifications thereof, using readily available starting materials, reagents and conventional synthetic procedures including, for example, procedures described in U.S. Pat. No. 7,112,592 and WO2009/091856. As depicted in Scheme 1, compound 3 can be synthesized following standard heterocyclic ring formation chemistry under appropriate reaction conditions from ester intermediate 1, or its corresponding derivatives, such as carboxylic acid derivative 2a and aldehyde derivative 2b (see, e.g., Jakopin, Z.; Dolenc, M. S. Curr. Org. Chem. 2008, 12, 850-898, hereafter Jakopin; Walker, D. G.; Brodfuehrer, P. R.; Brundidge, S. P. Shih, K. M.; Sapino, C. Jr. J. Org. Chem. 1988, 53, 983-991 hereafter Walker and references cited therein).

It may be necessary to protect certain functionalities in the molecule depending on the nature of the R1 group. Protecting these functionalities should be within the expertise of one skilled in the art. See, e.g. P. G. M. Wuts and T. W. Greene, Protective Groups in Organic Synthesis, Fourth Edition, John Wiley and Sons, 2006, hereafter Greene.

Alternatively, compound 3 can be synthesized from intermediate 7 as shown in Scheme 2. Monocyclic ester intermediate 7 can be converted to 8 under standard Mitsunobu reaction conditions. Compound 9 can then be prepared following standard heterocyclic ring formation chemistry under appropriate reaction conditions from ester intermediate 8, or its corresponding derivatives (see, e.g., Jakopin and Walker and references cited therein). Deprotection of N-Ns group in compound 9 provides compound 10, which can be converted to compound 11 by treating with diphosgene. Compound 3 can be obtained upon deprotection of N-Boc group from compound 11 under appropriate conditions, such as 4M HCl in dioxane, and subsequent treatment with base, such as NEt3. Alternatively, deprotection of N-Boc and N-Ns groups in compound 11 under appropriate conditions provides bis-amine derivative 12, which can then be cyclized to form compound 3 by treatment with diphosgene or triphogene, under appropriate conditions.

The benzylic ether protecting group in 3 can be removed via standard hydrogenolysis conditions, such as, but not limited to, Pd/H2 in MeOH or THF or by acid-catalyzed hydrolysis, such as, but not limited to, BCl3 in DCM to provide the hydroxy-urea intermediate 4, which can be used directly in the next step without further purification. Sulfation of 4 can be achieved by treatment with a sulfating reagent, such a, but not limited to, SO3.pyridine complex, in an appropriate solvent, such as pyridine, DMF or DMAc at a temperature of 0-80° C., preferable at room temperature. Compound 5 can then be isolated and purified via conventional methods. For example, 5 can be purified by standard reverse phase prep-HPLC using appropriate buffer system, i.e. ammonium formate buffer. In some cases, 5 can be purified by normal phase silica gel chromatography after converting to an appropriate salt form, such as sulfate tetrabutyl ammonium salt. The tetrabutyl ammonium salt can be converted to a sodium salt by cation exchange. When protecting group(s) are present in the sidechain (i.e. Boc or Fmoc for amine and guanidine protection, TBS or TES for alcohol protection, etc), a deprotection step is needed to convert 5 to its final product 6, which can be purified by reverse phase prep-HPLC using the conditions mentioned above. For example, for N-Boc deprotection, 5 can be treated with an acid, such as TFA, in an appropriate solvent, such as DCM at a temperature of 0-30° C., preferable at 0° C. to rt to give 6. For an O-TBS, or O-TES deprotection, a fluoride reagent such as HF.pyridine, HF.NEt3, or TBAF can be used. For an Fmoc deprotection, amines, such as diethylamine, DBU, piperidine, etc can be used.

EXAMPLES

The specific examples which follow illustrate the synthesis of certain compounds. The methods disclosed may be adopted to variations in order to produce compounds of Formula (I), but not otherwise specifically disclosed. Further, the disclosure includes variations of the methods described herein to produce the compounds of Formula (I) that would be understood by one skilled in the art based on the instant disclosure.

All temperatures are understood to be in Centigrade (C) when not specified. The nuclear magnetic resonance (NMR) spectral characteristics refer to chemical shifts (γ) expressed in parts per million (ppm) versus tetramethylsilane (TMS) as reference standard. The relative area reported for the various shifts in the proton NMR spectral data corresponds to the number of hydrogen atoms of a particular functional type in the molecule. The nature of the shifts as to multiplicity is reported as broad singlet (br s), broad doublet (br d), singlet (s), multiplet (m), doublet (d), quartet (q), doublet of doublet (dd), doublet of triplet (dt), and doublet of quartet (dq). The solvents employed for taking NMR spectra are DMSO-d6 (perdeuterodimethysulfoxide), D2O (deuterated water), CDCl3 (deuterochloroform) and other conventional deuterated solvents. The prep-HPLC conditions are: Waters SunFire® C18 (30×100 mm, 5 μm OBD) column; flow rate: 30-80 mL/minute, ELSD or Mass-triggered fraction collection; sample loading: Each injection loading varied from 30-300 mg for different crude samples depending on their solubility and purity profiles; Solvent system using ammonium formate buffer: solvent A: water with 20 mM ammonium formate, solvent B: 85% of acetonitrile in water with 20 mM ammonium formate. Solvent system using NH4HCO3 buffer: solvent A: water with 10 mM NH4HCO3, solvent B: acetonitrile. Solvent system using NH4OH buffer: solvent A: water with 0.1% NH4OH, solvent B: acetonitrile with 0.1% NH4OH.

Example 1 Synthesis of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (Intermediate Compound 1)

Step 1: Synthesis of (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate Method A:

n-BuLi was added dropwise to a solution of TMSCHN2 (690 mL, 1.38 mol) in dry THF (3 L) (600 mL, 1.5 mol) at −78° C., and the mixture was stirred at −78° C. for 30 minutes. The mixture was then transferred to a solution of (S)-1-tert-butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate (300 g, 1.17 mol) in dry THF (3 L) via cannula, and the mixture was stirred at −78° C. for 30 minutes. The reaction mixture was then quenched with sat. NH4Cl solution, and extracted with DCM three times. The combined organic layer was concentrated in vacuum and the crude product was purified by silica gel column chromatography (3:1 petroleum ether:EtOAc) to afford (S)-ethyl 2-((tert-butoxycarbonyl)amino)-6-diazo-5-oxohexanoate (262 g, 75%) as a yellow solid.

A solution of (S)-ethyl 2-((tert-butoxycarbonyl)amino)-6-diazo-5-oxohexanoate (350 g, 1.18 mol) in DCM (1500 mL) was added to a solution of Rh2(OAc)4 (3.5 g, 7.9 mmol) in DCM (750 mL) at 0° C. The reaction was then stirred at 20° C. overnight and then concentrated in vacuum. The crude sample was purified by silica gel column chromatography (5:1 petroleum ether/EtOAc) to afford (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (175.9 g, 55%) as a yellow oil.

Method B:

t-BuOK (330 g, 2.9 mol) was added to a solution of trimethylsulfoxonium iodide (750 g, 3.5 mol) in dry DMSO (3 L) and the mixture was stirred at rt for 1 h. (S)-1-tert-Butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate (900 g, 3.5 mol) was added and the mixture was stirred at rt for 2-3 hrs. Water was added to quench the reaction and the mixture was extracted with EtOAc 5 times. The combined organic layer was concentrated in vacuum and the crude sample was purified by silica gel column chromatography (1:1 petroleum ether/EtOAc then 1:10 MeOH/DCM) to afford sulfoxonium ylide intermediate (977 g, 80%) as a white solid.

A solution of sulfoxonium ylide intermediate (156 g, 0.446 mol) and [Ir(COD)Cl]2 (3 g, 4.46 mmol) in toluene(4 L) was degassed by bubbling nitrogen through the solution for 10 minutes. The reaction mixture was heated to 80-90° C. for 2-3 hrs and then cooled to 20° C. Then toluene was concentrated in vacuum, the residue was purified by silica gel column chromatography (gradient elution 10:1 to 3:1 petroleum ether/EtOAc) to afford (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (140 g, 57.8%) as a yellow oil.

Step 2: Synthesis of (2S,5S)-1-ten-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate

NaBH4 (36 g, 1.0 mol) was added in portions to a solution of (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (250 g, 0.92 mol) in EtOH (1500 mL) at −40° C. The reaction mixture was then stirred at −40° C. for 0.5 hr then quenched with 10% HOAc solution. After diluting with water, the mixture was extracted with DCM three times. The combined organic layer was concentrated in vacuum and purified by silica gel column chromatography (1:1 petroleum ether/EtOAc) to afford (2S,5S)-1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (205 g, 80%) as a yellow oil.

Step 3: Synthesis of (2S,5R)-1-tert-butyl 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate

A solution of 2-nitrobenzene-1-sulfonyl chloride (500 g, 2.26 mol) in pyridine (1500 mL) was added dropwise to a solution of O-benzylhydroxylamine hydrochloride (400 g, 2.51 mol) in pyridine (1500 mL) at 0° C. The reaction mixture was then stirred at 20° C. overnight. The mixture was concentrated in vacuum, diluted with DCM and washed with HCl (10%) three times. The combined organic layer was concentrated in vacuum and re-crystallized with DCM to afford N-(benzyloxy)-2-nitrobenzenesulfonamide (485 g, 62.6%) as a yellow solid.

To a solution of N-(benzyloxy)-2-nitrobenzenesulfonamide (212 g, 0.69 mol) in THF (1000 mL) was added (2S,5S)-1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (171 g, 0.63 mol) and PPh3 (275 g, 1.05 mol), followed by dropwise addition of a solution of DEAD (195 g, 1.12 mol) in THF (500 mL). The mixture was then stirred at 20° C. overnight. The reaction mixture was then concentrated in vacuum and purified by silica gel column chromatography (3:1 petroleum ether/EtOAc) to afford (2S,5R)-1-tert-butyl 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate (283.8 g, 80%) as a yellow oil.

Step 4: Synthesis of (2S,5R)-1-tert-butyl 2-ethyl 5-((benzyloxy)amino)piperidine-1,2-dicarboxylate

LiOH.H2O (95 g, 2.3 mol) and 2-mercaptoacetic acid (124 g, 1.3 mol) were added to a solution of (2S,5R)-1-tert-butyl 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate (251 g, 0.45 mol) in DMF (1200 mL). The reaction mixture was then stirred at 20° C. overnight. The reaction mixture was diluted with water and extracted with EtOAc (3×). The combined organic layer was washed with brine (3×), concentrated in vacuum and purified by silica gel column chromatography (3:1 petroleum ether/EtOAc) to afford (2S,5R)-1-tert-butyl 2-ethyl 5-((benzyloxy)amino)piperidine-1,2-dicarboxylate (122.9 g, 85%) as a yellow solid.

Step 5: Synthesis of (2S,5R)-ethyl 5-((benzyloxy)amino)piperidine-2-carboxylate

TFA (600 mL) was added to a solution of (2S,5R)-1-tert-butyl 2-ethyl 5-((benzyloxy)amino)piperidine-1,2-dicarboxylate (263 g, 0.7 mol) in DCM (600 mL) at 20° C. The mixture was stirred at rt overnight and then concentrated in vacuum. The crude product was adjusted to pH 10 with sat. NaHCO3 solution, and then extracted with DCM three times. The combined organic layer was concentrated in vacuum and purified by silica gel column chromatography (20:1 DCM/MeOH) to afford (2S,5R)-ethyl 5-((benzyloxy)amino)piperidine-2-carboxylate (184.9 g, 95%) as a yellow oil.

Step 6: Synthesis of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate

Triphosgene (21.3 g, 72 mmol) was added in portions to a solution of (2S,5R)-ethyl 5-((benzyloxy)amino)piperidine-2-carboxylate (50 g, 0.18 mol) and DIPEA (128 mL, 0.72 mol) in DCM (2000 mL) at 0° C. After stirring at 20° C. overnight, the reaction mixture was washed with H3PO4 (10%), sat. NaHCO3 and saturated NaCl. The combined organic layer was concentrated in vacuum and purified by silica gel column chromatography (3:1 petroleum ether/EtOAc) to afford (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (27.4 g, 50%) as a yellow solid. 1H NMR (400 Mz, CDCl3): δ 7.43-7.36 (m, 5H), 5.06 (d, J=11.4 Hz, 1H), 4.90 (d, J=11.4 Hz, 1H), 4.24 (q, J=7.1 Hz, 2H), 4.11-4.08 (m, 1H), 3.32-3.31 (m, 1H), 3.08-3.05 (m, 1H), 2.93 (d, J=11.9 Hz, 1H), 2.14-2.05 (m, 2H), 2.05-2.00 (m, 1H), 1.71-1.63 (m, 1H), 1.29 (t, J=7.1 Hz, 3H).

Example 2 Synthesis of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylic acid (Intermediate Compound 2a)

LiOH (1.2 g, 29.6 mmol) was added to a solution of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (9 g, 29.6 mmol) in THF/H2O (3:1, 240 mL). The mixture was then stirred at rt overnight. The reaction mixture was washed with EtOAc twice, then the aqueous solution was adjusted pH 2-3 with 1N HCl. The resulting mixture was extracted with DCM three times, and the combined organic layer was dried over saturated Na2SO4 and concentrated in vacuum to provide (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylic acid (7.0 g, 77.7%), which was directly used in the next step without further purification. ESI-MS (EI+, m/z): 277.31. 1H NMR (300 MHz, CDCl3) δ 7.49-7.29 (m, 5H), 5.06 (d, J=11.4 Hz, 1H), 4.91 (d, J=11.4 Hz, 1H), 4.15-4.10 (m, 1H), 3.36-3.34 (m, 1H), 3.15-3.11 (m, 1H), 2.83 (d, J=11.8 Hz, 1H), 2.32-2.15 (m, 1H), 2.11-2.01 (m, 2H), 1.74-1.56 (m, 1H).

Example 3 Synthesis of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde (intermediate compound 2b)

LiBH4 (0.54 g, 24.67 mmol) was added to a solution of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (5 g, 16.44 mmol) in MeOH (50 mL) at −10° C. After 15 minutes another portion of LiBH4 (0.54 g, 24.67 mmol) was added and the mixture was stirred at −10 to 0° C. for 4˜5 h. The reaction mixture was carefully quenched by addition of sat. NaH2PO4 (50 mL) at 0° C. The mixture was diluted with water (20 mL) and extracted with DCM three times. The combined organic layer was concentrated and purified by silica gel column chromatography (gradient elution 0-100% petroleum ether/EtOAc, then 0˜2% MeOH/EtOAc) to give (2S,5R)-6-(benzyloxy)-2-(hydroxymethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (3.8 g, 88%) as a white solid. ESI-MS (EI+, m/z): 263.1. 1H-NMR (500M, CDCl3): 7.44-7.35 (m, 5H), 5.05 (d, J=11.5 Hz, 1H), 4.90 (d, J=11.5 Hz, 1H), 3.73-3.69 (m, 1H), 3.61-3.58 (m, 2H), 3.33 (m, 1H), 3.01 (br d, J=12.0 Hz, 1H), 2.91 (m, 1H), 2.03-1.95 (m, 2H), 1.58-1.54 (m, 1H), 1.39-1.24 (m, 1H).

TEMPO (48 mg, 0.3 mmol) was added in portions to a solution of (2S,5R)-6-(benzyloxy)-2-(hydroxymethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (7.8 g, 30 mmol) and 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione (7.0 g, 30 mmol) in DCM (100 mL) at 0° C. The mixture was stirred at 0° C. for 2 h, and filtered through Celite®. The filtrate was dried over Na2SO4 and concentrated to afford (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde (7.0 g, 90%) as a yellow oil. ESI-MS (EI+, m/z): 261.1. 1H-NMR (500M, CDCl3): 9.74 (s, 1H), 7.45-7.36 (m, 5H), 5.07 (d, J=11.5 Hz, 1H), 4.92 (d, J=11.5 Hz, 1H), 3.89 (d, J=8.0 Hz, 1H), 3.27 (m, 1H), 3.21-3.05 (m, 1H), 2.56 (d, J=12.0 Hz, 1H), 2.20-2.15 (m, 1H), 2.05-2.01 (m, 1H), 1.95-1.93 (m, 1H), 1.49-1.46 (m, 1H).

Example 4 Synthesis of (E)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde oxime (intermediate compound 2c)

A solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde (510 mg, 1.96 mmol), hydroxylamine hydrochloride (158 mg, 2.27 mmol) and pyridine (621 mg, 7.85 mmol) in EtOH (15 mL) was stirred at rt for 2 hrs. Then, the reaction mixture was concentrated and the residue was diluted with DCM (25 mL), washed with water (3×), and saturated sodium chloride, dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (3:1 to 3:2 petroleum ether/EtOAc) to afford (E)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde oxime (228 mg, 42%) as a white solid. ESI-MS (EI+, m/z): 276 [M+H]+.

Example 5 Synthesis of (2S,5R)-2-(1,2,4-oxadiazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 804)

Step 1: A mixture of (2S,5R)-1-tert-butyl 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido) piperidine-1,2-dicarboxylate (5 g, 8.9 mmol), and LiOH.H2O (1.5 g, 35.5 mmol) in THF (30 mL) and H2O (100 mL) was stirred at rt for 24 hrs. 1M HCl was then added to adjust the pH to 5˜6, following by the addition of EtOAc (300 mL). The mixture was washed with saturated sodium chloride (5×), dried over Na2SO4, and concentrated. The residue was washed with petroleum ether/EtOAc (95:5, 200 mL) to give (2S,5R)-5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (4.75 g, 99%) as a pale yellow solid. ESI-MS (EI+, m/z): 436.0 [M+H-100]+.

Step 2: NH4Cl (1.39 g, 26.18 mmol), HOBt (3.53 g, 26.18 mmol), EDCI (5 g, 26.18 mmol), and DIPEA (8.44 g, 65.45 mmol) were added to a solution of (2S,5R)-5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (10 g, 18.7 mmol) in DMF (90 mL) The mixture was stirred at rt for 17 hrs, then, EtOAc (500 mL) was added. The mixture was washed with saturated sodium chloride (6×), dried over Na2SO4, and concentrated. The residue was washed with petroleum ether/EtOAc (95:5, 200 mL) to give (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-carbamoylpiperidine-1-carboxylate (9.9 g, 99%) which was used in the next step. ESI-MS (EI+, m/z): 535.0 [M+H]+.

Step 3: Pyridine (2 mL) was added to a solution of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-carbamoylpiperidine-1-carboxylate (1.0 g, 1.87 mmol) in dry THF (40 mL). Then, Tf2O (1.0 mL) was slowly added over 5 minutes. The mixture was stirred at 0° C. for 1 h and then EtOAc (30 mL) was added. The mixture was washed with saturated sodium chloride (4×), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 0˜40% petroleum ether/EtOAc) to give (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-cyanopiperidine-1-carboxylate (0.76 g, 79%) as a yellow solid ESI-MS (EI+, m/z): 539.0 [M+Na]+.

Step 4: A solution of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-cyanopiperidine-1-carboxylate (3.1 g, 6.008 mmol), NH2OH.HCl (400 mg, 12 mmol), and TEA (12.136 g, 120.160 mmol) in MeOH (30 mL) and EtOH (30 mL) was stirred at 70° C. for 17 hrs. EtOAc (300 mL) was then added and the mixture was washed with saturated sodium chloride (3×), dried over Na2SO4, and concentrated to give (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-(N-hydroxycarbamimidoyl)piperidine-1-carboxylate (3.4 g, 99%) as a pale yellow solid, which was used directly in the next step. ESI-MS (EI+, m/z): 550.2 [M+H]+.

Step 5: A mixture of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-(N-hydroxycarbamimidoyl)piperidine-1-carboxylate (3.0 g, 5.46 mmol), trimethoxymethane (60 mL), and PPTS (0.08 g) was stirred at 70° C. for 4 hrs. The solvent was then removed under vacuum. The residue was purified by silica gel column chromatography (gradient elution 0˜35% petroleum ether/EtOAc) to give (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-(1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (2.1 g, 69%) as a yellow solid. ESI-MS (EI+, m/z): 582 [M+Na]+.

Step 6: A mixture of (2S,5R)-tert-butyl5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-(1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (1.5 g, 2.683 mmol), HSCH2COOH (1.48 g, 16.1 mmol), LiOH.H2O (1.13 g, 26.83 mmol) in DMF (50 mL) was stirred at 30° C. for 17 hrs. EtOAc (150 mL) was then added and the mixture was washed with water (2×), and saturated sodium chloride (2×), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 0˜50% petroleum ether/EtOAc) to give (2S,5R)-tert-butyl 5-(benzyloxyamino)-2-(1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (700 mg, 63%) as a pale yellow solid. ESI-MS (EI+, m/z): 375.0 [M+H]+.

Step 7: TEA (405 mg, 4.0 mmol) and diphosgene (514 mg, 2.6 mmol) were added to a solution of (2S,5R)-tert-butyl 5-(benzyloxyamino)-2-(1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (750 mg, 2.0 mmol) in DCM (70 mL). The mixture was stirred at 0° C. for 3 hrs. Then, the reaction mixture was washed with saturated sodium chloride (2×), dried over Na2SO4, and concentrated to give (2S,5R)-tert-butyl 5-(benzyloxy(chlorocarbonyl)amino)-2-(1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (1.6 g), which was used directly in the next step. ESI-MS (EI+, m/z): 437[M+H]+.

Step 8: A mixture of (2S,5R)-tert-butyl 5-(benzyloxy(chlorocarbonyl)amino)-2-(1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (1.6 g), and 4 N HCl in dioxane (18 mL), was stirred at rt for 2 hrs. The solvent was then removed under vacuum to give (3R,6S)-6-(1,2,4-oxadiazol-3-yl)piperidin-3-yl(benzyloxy)carbamic chloride (1.4 g) as a white solid, which was used directly in the next step. ESI-MS (EI+, m/z): 301.0.

Step 9: To a solution of (3R,6S)-6-(1,2,4-oxadiazol-3-yl)piperidin-3-yl(benzyloxy)carbamic chloride (1.4 g) in DCM (40 mL) was added TEA until the pH was adjusted to pH to 8˜9. The mixture was stirred at rt for 2 hrs then, the solvent was removed and the residue was purified by silica gel column chromatography (gradient elution 0˜4% petroleum ether/EtOAc) to give the desired product (2S,5R)-6-(benzyloxy)-2-(1,2,4-oxadiazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (400 mg, 67% for 3 steps) as a white solid. ESI-MS (EI−, m/z): 301.2 [M+H]+.

Step 10: BCl3 (1M, 3.34 mL, 3.33 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-2-(1,2,4-oxadiazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (200 mg, 0.67 mmol) in dry DCM (45 mL) at −78° C. The mixture was stirred at 0° C. for 2 hrs., then it was cooled to −78° C. and quenched with MeOH (8 mL). The solvent was removed by vacuum to give (2S,5R)-6-hydroxy-2-(1,2,4-oxadiazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (180 mg) as a white solid, which was used directly in the next step. ESI-MS (EI+, m/z): 209 [M−H]+.

Step 11: To a solution of (2S,5R)-6-hydroxy-2-(1,2,4-oxadiazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (180 mg crude from above) in dry pyridine (4 mL) was added SO3.Py (608 mg). The mixture was stirred at rt for 3 hrs and then concentrated under vacuum. The residue was re-dissolved in aqueous NaH2PO4 (1.5 M, 50 mL) and then tetrabutylammonium hydrogensulphate (970 mg) was added. The mixture was stirred at rt for 30 minutes and then extracted with EtOAc (3×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (gradient elution 0 to 25% EtOAc:Acetone) to afford tetrabutylammonium (2S,5R)-2-(1,2,4-oxadiazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (280 mg, 45% for 2 steps) as a white solid. ESI-MS (EI−, m/z): 289.0 [M−H]−.

Step 12, Resin Exchange: Tetrabutylammonium (2S,5R)-2-(1,2,4-oxadiazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (280 mg) was dissolved in a minimum amount of HPLC grade water (˜3 mL) and passed through a column of 8 g of DOWEX 50WX 8 Na+ resin (the resin was pre-washed with >200 mL of HPLC grade water) and eluted with HPLC grade water to provide sodium (2S,5R)-2-(1,2,4-oxadiazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (180 mg, 90%) as a white solid after lyophilization. ESI-MS (EI−, m/z): 289.1 [M−H]−. 1H-NMR (500 MHz, D2O): δ 9.23 (s, 1H), 4.75 (d, J=7.5 Hz, 1H), 4.20 (br s, 1H), 3.18 (m, 1H), 2.99 (d, J=12 Hz, 1H), 2.32-2.27 (m, 1H), 2.21-2.14 (m, 2H), 1.97-1.92 (m, 1H).

Example 6 Synthesis of (2S,5R)-2-(5-amino-1,2,4-oxadiazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 805)

Step 1:

a) NCS (0.76 g, 5.72 mmol) was added to a solution of (E)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbaldehyde oxime (1.5 g, 5.45 mmol) in DCM (10 mL) then one drop of pyridine was added. The mixture was stirred at rt for 18 hrs, then the mixture was concentrated under reduced pressure and dried under high vacuum. The crude product was dissolved in absolute EtOH (10 mL).

b) In a separate flask, guanidine hydrochloride (1.04 g, 10.9 mmol) was mixed with EtONa (16% in EtOH, 4.63 g, 10.9 mmol) at rt, and the solid was filtered off.

The solution of a) was added to the filtrate of b) at rt. The mixture was stirred at rt overnight, then, the reaction mixture was concentrated, diluted with water (20 mL), and extracted with EtOAc (3×). The combined organic layer was dried over Na2SO4 and concentrated. The residue was purified by prep-HPLC using ammonium formate buffer to afford (2S,5R)-2-(5-amino-1,2,4-oxadiazol-3-yl)-6-(benzyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one (120 mg, 7%) as a white solid. ESI-MS (EI+, m/z): 316 [M+H]+.

Step 2. BCl3 (1M, 5.08 ml, 5.08 mmol) was added to a solution of (2S,5R)-2-(5-amino-1,2,4-oxadiazol-3-yl)-6-(benzyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one (160 mg, 0.508 mmol) in dry DCM (8 mL) at −78° C. The mixture was stirred under N2 atmosphere at 0° C. for 2 hrs then, it was cooled to −78° C. and quenched with MeOH (1.0 mL). The solvent was removed by vacuum to afford (2S,5R)-2-(5-amino-1,2,4-oxadiazol-3-yl)-6-hydroxy-1,6-diaza-bicyclo[3.2.1]octan-7-one (110 mg) as a white solid, which was used directly in the next step. ESI-MS (EI+, m/z): 226 [M+H]+.

Step 3: To a mixture of (2S,5R)-2-(5-amino-1,2,4-oxadiazol-3-yl)-6-hydroxy-1,6-diaza-bicyclo[3.2.1]octan-7-one (110 mg, 0.49 mmol) and SO3.Py (389 mg, 2.44 mmol) was added dry pyridine (2 mL) under N2. The mixture was stirred at rt for 2.5 hrs and then concentrated under vacuum to afford (2S,5R)-2-(5-amino-1,2,4-oxadiazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl hydrogen sulfate, which was used for next step directly. ESI-MS (EI−, 304, m/z): [M−H]−.

Step 4: Crude (2S,5R)-2-(5-amino-1,2,4-oxadiazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl hydrogen sulfate was purified by Prep-HPLC using ammonium formate buffer to afford (2S,5R)-2-(5-amino-1,2,4-oxadiazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl aminooxy sulfonate (25 mg, 20% of two steps). ESI-MS (EI−, m/z): 304 [M−H]−. 1H-NMR (500 MHz, D2O): δ 4.42 (d, J=8.0 Hz, 1H), 4.12 (s, 1H), 3.12-3.09 (m, 1H), 3.00 (d, J=15 Hz, 1H), 2.11-1.96 (m, 3H), 1.86-1.79 (m, 1H).

Example 7 Synthesis of (2S,5R)-7-oxo-2-(5-(piperidin-4-yl)-1,2,4-oxadiazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 806)

Step 1: A solution of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-(N-hydroxycarbamimidoyl)piperidine-1-carboxylate (3.12 g, 5.7 mmol), 1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidine-4-carboxylic acid (2 g, 5.7 mmol), HATU (3.23 g, 8.55 mmol) and DIPEA (1.50 g, 11.44 mmol) in DMF (50 mL) was stirred at rt for 1 h. EtOAc (150 mL) was then added and the mixture was washed with saturated sodium chloride (3×), dried over Na2SO4, and concentrated. The residue was dissolved in dioxane (50 mL) and heated at 90° C. for 17 hrs. The mixture was then concentrated under vacuum and the residue was purified by silica gel column chromatography (gradient elution 0˜45% petroleum ether/EtOAc) to give (2S,5R)-tert-butyl 2-(5-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-1,2,4-oxadiazol-3-yl)-5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1-carboxylate (1.2 g, 24%) as a yellow solid. ESI-MS (EI+, m/z): 765 [M+H-100]+.

Step 2: A mixture of (2S,5R)-tert-butyl 2-(5-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-1,2,4-oxadiazol-3-yl)-5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1-carboxylate (200 mg, 0.231 mmol), HSCH2COOH (128 mg, 1.386 mmol) and LiOH.H2O (97 g, 2.310 mmol) in DMF (5.0 mL) was stirred at 30° C. for 17 hrs. EtOAc (50 mL) was then added and the organic layer was washed with water (2×), and saturated sodium chloride (2×), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 0˜50% petroleum ether/EtOAc) to give (2S,5R)-tert-butyl 2-(5-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-1,2,4-oxadiazol-3-yl)-5-(benzyloxyamino) piperidine-1-carboxylate (40 mg, 25%) as a yellow oil. ESI-MS (EI+, m/z): 680 [M+H]+

Step 3: Diphosgene (144 mg, 0.727 mmol) was added to a solution of (2S,5R)-tert-butyl 2-(5-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-1,2,4-oxadiazol-3-yl)-5-(benzyloxyamino)piperidine-1-carboxylate (380 mg, 0.559 mmol) and TEA (113 mg, 1.118 mmol) in DCM (5.0 mL). The mixture was stirred at 0° C. for 1 h, then, DCM (20 mL) was added and the mixture was washed with saturated sodium chloride (2×), dried over Na2SO4, and concentrated to give (2S,5R)-tert-butyl 2-(5-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-1,2,4-oxadiazol-3-yl)-5-(benzyloxy(chlorocarbonyl)amino)piperidine-1-carboxylate (400 mg), which was directly used in the next step. ESI-MS (EI+, m/z): 742 [M+H]+.

Step 4: A mixture of (2S,5R)-tert-butyl 2-(5-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-1,2,4-oxadiazol-3-yl)-5-(benzyloxy(chlorocarbonyl)amino)piperidine-1-carboxylate (˜400 mg) and 4 N HCl/dioxane (5.0 mL) was stirred at rt for 2 hrs. The solvent was then removed under vacuum to give (9H-fluoren-9-yl)methyl 4-(3-((2S,5R)-5-(benzyloxy(chlorocarbonyl)amino)piperidin-2-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (400 mg) as a white solid. ESI-MS (EI+, m/z): 642 [M+H]+.

To a solution of crude (9H-fluoren-9-yl)methyl 4-(3-(((2S,5R)-5-(benzyloxy(chlorocarbonyl)amino)piperidin-2-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (400 mg) in DCM (25 mL) was added TEA until the pH was adjusted to pH 8˜9. The mixture was stirred at rt for 2 hrs then, the solvent was removed. The residue was purified by silica gel column (gradient elution 0˜45% petroleum ether/EtOAc) to give (9H-fluoren-9-yl)methyl 4-(3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (100 mg, 30% for 3 steps) as a white solid. ESI-MS (EI−, m/z): 606 [M+H]+.

Step 5: BCl3 (850 μL, 0.85 mmol, 1 M in DCM) was added to a solution of (9H-fluoren-9-yl)methyl 4-(3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (100 mg, 0.17 mmol) in dry DCM (10 mL) at −78° C. The mixture was stirred under N2 atmosphere at 0° C. for 6 hrs, cooled to −78° C., then MeOH (1 mL) was slowly added. The solvents were evaporated under vacuum at 0° C. to give (9H-fluoren-9-yl)methyl 4-(3-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (97 mg) as a yellow solid, which was used directly in the next step. ESI-MS (EI+, m/z): 516.3 [M+H]−.

Step 6: To a solution of crude (9H-fluoren-9-yl)methyl 4-(3-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (97 mg) in dry pyridine (2 mL) was added SO3.Py (140 mg, 0.85 mmol). The mixture was stirred at rt for 6 hrs and then concentrated under vacuum. The residue was re-dissolved in aqueous NaH2PO4 (1.5 M, 10 mL) then tetrabutylammonium hydrogensulphate (75 mg) was added. The mixture was stirred at rt for 20 minutes, then extracted with EtOAc (3×). The combined organic layer was dried and concentrated. The residue was purified by silica gel column chromatography (gradient elution 10:1 to 2:1 DCM/acetone) to give tetrabutylammonium (9H-fluoren-9-yl)methyl 4-(3-((2S,5R)-7-oxo-6-(sulfooxy)-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate sulfate as a white solid (84 mg, 61% for two steps). ESI-MS (EI−, m/z): 594.1 [M−H]−.

Step 7: Et2NH (0.5 mL, 5.0 mmol)) was added to a solution of tetrabutylammonium (9H-fluoren-9-yl)methyl 4-(3-((2S,5R)-7-oxo-6-(sulfooxy)-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate sulfate (84 mg, 0.1 mmol) in dry DCM (10 mL. The mixture was stirred at rt for 12 hrs and the solvents were evaporated under vacuum. The residue was purified by prep-HPLC to afford (2S,5R)-7-oxo-2-(5-(piperidin-4-yl)-1,2,4-oxadiazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl hydrogen sulfate (9.0 mg). ESI-MS (EI+, m/z): 374.15 [M+H]+. 1H NMR (300 MHz, D2O) δ 4.61 (d, J=6.0 Hz, 1H), 4.12 (br s, 1H), 3.50-3.33 (m, 3H), 3.15-3.05 (m, 3H), 2.91 (d, J=12.2 Hz, 1H), 2.35-1.72 (m, 8H).

Example 8 Synthesis of (2S,5R)-2-(1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 801)

Step 1: DIPEA (5.8 mL, 36.2 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxylic acid (5.0 g, 18.1 mmol), EDCI (5.2 g, 27.2 mmol), HOBT (3.7 g, 27.2 mmol) and NH4Cl (1.94 g, 36.2 mmol) in DMF (60 mL) at rt. The reaction mixture was stirred for 17 hrs, then diluted with ice water (100 mL) and extracted with EtOAc (3×). The combined organic layer was dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (2:1 EtOAc/petroleum ether) to afford (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxamide (4.0 g, 80%) as a white solid. ESI-MS (EI+, m/z): 276 [M+H]+.

Step 2. Dimethoxy-N,N-dimethylmethanamine (0.97 mL, 7 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxamide (1.38 g, 5 mmol) in 1,4-dioxane (20 mL) at rt. The mixture was stirred at 45-50° C. for 2 hrs under vacuum to remove the methanol formed during the reaction. Then, the reaction mixture was concentrated under vacuum, and the residue was washed with Et2O (2×) and dried to give (2S,5R,Z)-6-(benzyloxy)-N-((dimethylamino)methylene)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxamide (1.33 g, 80%) as a white solid. (ESI-MS (EI+, 331, m/z): [M+H]+.

Step 3: (2S,5R,Z)-6-(benzyloxy)-N-((dimethylamino)methylene)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxamide (1.33 g, 4.03 mmol) in EtOH (22 mL) was treated with pyridine (0.64 ml, 8.06 mmol), followed by the addition of a solution of HOSA (547 mg, 4.84 mmol) in MeOH (4.5 mL). The resulting mixture was stirred at rt for 30 minutes and then concentrated under vacuum. The residue was dissolved in DCM/THF (1:2, 30 mL) and washed with water (10 mL). The organic layer was dried over Na2SO4, and concentrated. The residue was re-dissolved in EtOH (22 mL) and 2N HCl (8.0 mL). The mixture was heated at reflux for 20 minutes, concentrated and extracted with DCM (2×). The crude material was purified by prep-HPLC to give (2S,5R)-6-(benzyloxy)-2-(1,2,4-oxadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (50 mg, 4.1%) as a light yellow oil. ESI-MS (EI+, 301, m/z): [M+H]+. 1H-NMR (500 MHz, CDCl3): δ 8.44 (s, 1H), 7.45-7.35 (m, 5H), 5.09 (d, J=11.5 Hz, 1H), 4.94 (d, J=11.5 Hz, 1H), 4.85 (d, J=8.0 Hz, 1H), 3.36 (m, 1H), 3.01 (d, J=12.0 Hz, 1H), 2.81 (d, J=12.0 Hz, 1H), 2.37-2.35 (m, 1H), 2.25-2.21 (m, 1H), 2.01-2.00 (m, 1H), 1.85-1.84 (m, 1H).

Step 4: BCl3 (1M in DCM, 1.2 mL, 1.2 mmol) was added to (2S,5R)-6-(benzyloxy)-2-(1,2,4-oxadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (50 mg, 0.167 mmol) in DCM (10 mL) at −78° C. The mixture was warmed to 0° C. and stirred for 2 hrs. The reaction was then quenched by the addition of MeOH (1 mL) and the resulting solution was concentrated under vacuum to give (2S,5R)-6-hydroxy-2-(1,2,4-oxadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (34 mg, 98%) as a white solid, which was used directly in the next step. ESI-MS (EI+, 211, m/z): [M+H]+.

Step 5: A mixture of (2S,5R)-6-hydroxy-2-(1,2,4-oxadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (48 mg, 0.228 mmol) and SO3.Py (182 mg, 1.14 mmol) in dry pyridine (2 mL) was stirred at rt for 2.5 hrs. The reaction mixture was then concentrated in vacuum and the residue was re-dissolved in aqueous NaH2PO4 (1.5 M, 10 mL). Tetrabutylammonium hydrogensulphate (105 mg) was added, the mixture was stirred at rt for 15 minutes, and then extracted with EtOAc (4×). The combined organic layer was dried and concentrated. The residue was purified by silica gel column chromatography (gradient elution 10:1 to 1:1 DCM/acetone) to give tetrabutylammonium (2S,5R)-2-(1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (50 mg, 41%) as a white solid. ESI-MS (EI−, 289, m/z): [M−H]−.

Step 6: Tetrabutylammonium (2S,5R)-2-(1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (50 mg) was dissolved in a minimum amount of HPLC grade water (˜1 mL) and passed through a column of 2 g of DOWEX 50WX 8 Na+ resin (the resin was pre-washed with >0.5 L of HPLC grade water) and eluted with HPLC grade water to afford sodium (2S,5R)-2-(1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (21 mg, 72%) as a white solid after lyophilization. ESI-MS (EI−, m/z): 289 [M−H]−. 1H-NMR (500 MHz, CDCl3): δ 8.76 (s, 1H), 4.92 (d, J=7.5 Hz, 1H), 4.24 (s, 1H), 3.29 (d, J=12.5 Hz, 1H), 2.98 (d, J=12.5 Hz, 1H), 2.42-2.38 (m, 1H), 2.29-2.20 (m, 2H), 2.0-1.97 (m, 1H).

Example 9 Synthesis of (2S,5R)-2-(3-amino-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl tetrabutylaminooxy sulfonate (Compound 802)

Step 1: Hydroxylamine (50% in water, 3.6 mL, 0.059 mol) was added to cyanamide (50% in water, 3.24 g, 0.077 mol) in methanol (100 mL). The mixture was heated to reflux for 4.5 hrs and concentrated to remove methanol/water, followed by co-evaporation with methanol (2×) to remove residual water to obtain 1-hydroxyguanidine (3.0 g, 68%) as a light yellow solid.

Step 2: A solution of (2S,5R)-6-(benzyloxy)-2-(5-(piperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (2.0 g, 7.25 mmol), EDCI (1.53 g, 7.98 mmol) and HOBT (1.08 g, 7.98 mmol) in DMF (15 mL) was stirred at rt for 0.5 h. 1-Hydroxyguanidine (0.653 g, 7.98 mmol) was then added and the reaction mixture was stirred for an additional 0.5 h. The resulting solution was treated under microwave at 100° C. for 1.5 hrs. The mixture was poured into water and extracted with EtOAc (2×). The combined organic layers were then washed with water (20 mL), and saturated sodium chloride (20 mL), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (2:1 EtOAc/petroleum ether) to give (2S,5R)-2-(3-amino-1,2,4-oxadiazol-5-yl)-6-(benzyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one (1.0 g, 44%). (ESI-MS (EI+, m/z): 316 [M+H]+.

Step 3: BCl3 (1M, 15.87 mL, 15.87 mmol) was added to a solution of (2S,5R)-2-(3-amino-1,2,4-oxadiazol-5-yl)-6-(benzyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one (1.0 g, 3.17 mmol) in dried DCM (20 mL) at −78° C. The mixture was stirred under N2 atmosphere at 0° C. for 2 hrs, cooled to −78° C. and quenched with MeOH (2 mL). The solvent was removed under vacuum to give ((2S,5R)-2-(3-amino-1,2,4-oxadiazol-5-yl)-6-hydroxy-1,6-diaza-bicyclo[3.2.1]octan-7-one (700 mg), which was used in the next step directly. ESI-MS (EI+, m/z): 226 [M+H]+.

Step 4: A mixture of ((2S,5R)-2-(3-amino-1,2,4-oxadiazol-5-yl)-6-hydroxy-1,6-diaza-bicyclo[3.2.1]octan-7-one (700 mg, 3.11 mmol) and SO3.Py (1.48 g, 9.33 mmol) in dry pyridine (5 mL) was stirred at rt for 2.5 hrs. The reaction mixture was then concentrated under vacuum. then re-dissolved in aqueous NaH2PO4 (1.5 M, 30 mL). Tetrabutylammonium hydrogensulphate (1.16 g) was added, the mixture stirred at rt for 15 minutes, and then extracted with EtOAc (4×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (gradient elution 10:1 to 5:1 DCM/acetone) to afford tetrabutylammonium (2S,5R)-2-(3-amino-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (1.7 g) as a white solid. (ESI-MS (EI−, 304, m/z): [M−H]−. 1H-NMR (500 MHz, DMSO-d6): δ 6.4 (s, 2H), 4.48 (d, J=8.0 Hz, 1H), 3.67 (s, 1H), 3.24-3.14 (m, 8H), 3.00 (d, J=12 Hz, 1H), 2.24 (d, J=11.5 Hz, 1H), 2.2-2.06 (m, 3H), 1.87-1.82 (m, 1H), 1.59-1.53 (m, 8H), 1.35-1.27 (m, 8H), 0.93-0.82 (m, 12H).

Step 5: Tetrabutylammonium (2S,5R)-2-(3-amino-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate was further purified by prep-HPLC using ammonium formate buffer to afford (2S,5R)-2-(3-amino-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate. ESI-MS (EI+, m/z): 306.1. 1H NMR (300 MHz, D2O) δ 4.19 (br s, 1H), 3.27-3.23 (m, 1H), 3.04-2.97 (m, 1H), 2.39-1.66 (m, 4H).

Example 10 Synthesis of (2S,5R)-7-oxo-2-(3-(piperidin-4-yl)-1,2,4-oxadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 803)

Step 1: CDI (511.3 mg, 3.1 mmol) was added to a solution of crude (2S,5R)-6-(benzyloxy)-2-(5-(piperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (726 mg, 2.6 mmol) in DMF (15 mL). The mixture was stirred at rt for 1 h, then, (E)-tert-butyl 4-(N′-hydroxycarbamimidoyl)piperidine-1-carboxylate (631.8 mg, 2.6 mmol) was added at rt. The mixture was stirred at rt for 2 hrs, and then stirred at 50° C. for another 6 hrs. The mixture was diluted with EtOAc (150 mL) and washed with 1 M HCl (2×), water (2×), and saturated sodium chloride (2×), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 to 1:1 EtOAc/hexanes) to give tert-butyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (968 mg, 76%) as a white solid. ESI-MS (EI+, m/z): 506.2 [M+Na]+. 1H-NMR (400 MHz, CDCl3): δ 7.47-7.36 (m, 5H), 4.99-4.94 (m, 2H), 4.73 (d, J=6.0 Hz, 1H), 3.93 (d, J=9.6 Hz, 1H), 3.72 (s, 1H), 3.08-2.90 (m, 4H), 2.72 (d, J=9.6 Hz, 1H), 2.15 (dd, J=12.0, 5.2 Hz, 1H), 2.09-1.93 (m, 5H), 1.85-1.79 (m, 1H), 1.59-1.50 (m, 2H), 1.40 (s, 9H).

Step 2: CF3COOH (4 mL) was added to the solution of tert-butyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (960 mg, 2.0 mmol) in 16 mL of CH2Cl2 at 0° C. The mixture was stirred at 0° C. for 2 hrs, then concentrated to give (2S,5R)-6-(benzyloxy)-2-(3-(piperidin-4-yl)-1,2,4-oxadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (1.1 g) as a brown oil, which was used directly in the next step. ESI-MS (EI+, m/z): 384.2 [M+H]+.

Step 3: DIPEA (1.6 mL, 10.0 mmol) was slowly added dropwise to a solution of (2S,5R)-6-(benzyloxy)-2-(3-(piperidin-4-yl)-1,2,4-oxadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (1.1 g, 2.0 mmol) in THF (50 mL) at 0° C. The mixture was stirred under N2 atmosphere at 0° C. for 15 minutes, then, Fmoc-OSu (2.4 g, 7.2 mmol) was added and the mixture was stirred at rt. for 8 hrs. The mixture was concentrated and the residue was purified by silica gel column chromatography (gradient elution 1:6 to 1:2 EtOAc/hexanes) to give (9H-fluoren-9-yl)methyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (1.0 g, 86%) as a white solid.

Step 4: BCl3 (8.5 ml, 8.5 mmol; 1 M in CH2Cl2) was added dropwise to a solution of (9H-fluoren-9-yl)methyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (1.0 g, 1.7 mmol, 1.0 eq.) in dried CH2Cl2 (20 mL) at −78° C. The mixture was stirred under N2 atmosphere at 0° C. for 6 hrs. Then, the reaction mixture was cooled to −78° C. and MeOH (2 mL) was added dropwise. The solvents were evaporated under vacuum at 0° C. to give ((9H-fluoren-9-yl)methyl 4-(5-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (970 mg) as a yellow solid, which was used directly in the next step. ESI-MS (EI−, m/z): 516.3 [M+H]−.

Step 5: To a solution of ((9H-fluoren-9-yl)methyl 4-(5-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (970 mg) in dry pyridine (15 mL) was added SO3.Py (1.4 g, 8.5 mmol). The mixture was stirred at rt for 6 h and then concentrated under vacuum. The resulting residue was then re-dissolved in aqueous NaH2PO4 (1.5 M, 100 mL) and Tetrabutylammonium hydrogensulphate (746 mg) was added. The mixture was stirred at rt for 30 minutes, then extracted with EtOAc (4×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (gradient elution 10:1 to 2:1 CH2Cl2/acetone) to afford tetrabutylammonium (9H-fluoren-9-yl)methyl 4-(5-((2S,5R)-7-oxo-6-(sulfooxy)-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate sulfate as a white solid (1.0 g, 71% for two steps). ESI-MS (EI−, m/z): 594.1 [M−H]−.

Step 6: Et2NH (6.1 mL, 60.0 mmol) was added to a solution of tetrabutylammonium (9H-fluoren-9-yl)methyl 4-(5-((2S,5R)-7-oxo-6-(sulfooxy)-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate sulfate (1.0 g, 1.2 mmol) in dried CH2Cl2 (30 mL). The mixture was stirred under N2 atmosphere at rt. for 12 hrs, then evaporated under vacuum. The residue was purified by prep-HPLC using the ammonium formate conditions. ESI-MS (EI+, m/z): 314.2. 1H NMR (300 MHz, D2O) δ 4.82 (d, J=8.0 Hz, 1H), 4.17 (br s, 1H), 3.47-3.45 (m, 2H), 3.25-3.12 (m, 3H), 2.96-2.92 (m, 1H), 2.39-1.94 (m, 8H).

Example 11 Construction of Isogenic β-lactamase Strains

A set of β-lactamase expressing isogenic E. coli strains was constructed by cloning a β-lactamase gene into a customized derivative of pBR322 (GenBank Accession Number J01749) and transforming the engineered plasmids into E. coli. The NdeI restriction site within the plasmid backbone of pBR322 was removed to generate pBR322 ΔNdeI. The pBR322 ΔNdeI vector itself, minus the blaTEM-1 gene, was amplified using two primers: (1) pBR-Pbla 5′-cgcatatgactcttcctttttcaatattattg-3, SEQ ID 1, a primer with an engineered NdeI restriction site at the 3′ end of the blaTEM-1 promoter and (2) pBR-vec-1 5′-gcggatccctgtcagaccaagtttactc-3′, SEQ ID 2, a primer with an engineered BamHI restriction site at the 3′ end of the blaTEM-1 open reading frame. The chloramphenicol resistance gene, cat, was generated by PCR amplification from pKD3 (GenBank Accession Number AY048742) using primers with an engineered NdeI restriction site at the 5′ end (Pbla-cat 5′-gccatatgatggagaaaaaaatcactgg-3′, SEQ ID 3) and an engineered BamHI restriction site at the 3′ end (Vec-1-cat 5′-cgggactccctagaagaataggaacttcgg-3′, SEQ ID 4) of the resistance gene. The two PCR products, pBR322 ΔNdeI and cat were ligated together generating pBR-CBST (pBR322 ΔNdeI ΔTEM-1::cat Seq. ID 5) which retains both the pBR322 tetracycline resistance cassette, tetA, and the plasmid origin of replication but the blaTEM-1 gene was replaced by the cat gene.

Using this engineering strategy a number of plasmids producing β-lactamase genes from different classes (see below) were generated using synthetic genes with an engineered NdeI restriction site at the 5′ end and BamHI restriction site at the 3′ end of each gene (GenScript). Both the synthetic β-lactamase genes and cat gene were ligated into the NdeI/BamHI sites of the pBR322 ΔNdeI PCR product and transformed into electrocompetent E. coli ElectroMax DH10B (Invitrogen/Life Technologies). E. coli DH10B harboring the recombinant plasmids were selected on LB agar (supplemented with 25 μg/mL tetracycline) and single isolated colonies were then inoculated into 5 mL LB media (supplemented with 25 μg/mL tetracycline), and incubated at 37° C. with aeration (250 rpm) for 18 hrs. The cultures were frozen back at −80° C. in 20% glycerol. The DNA sequence of the cloned β-lactamase genes was confirmed. The β-lactamase gene expression in the recombinant E. coli strains was driven by the blaTEM-1 promoter in the pBR-CBST plasmid and was characterized by MIC profiling of the E. coli recombinant strains against comparator β-lactam/BLI combinations in broth microdilution assay.

GenBank  Accession Number of β-Lactamase Name & SEQ. ID of β- Species Origin β-Lactamase Expressing plasmids producing Lactamase of β-Lactamase Gene Strain β-Lactamase Class Gene Sequence KPC-2 pBR-CBST-KPC-2 A K pneumoniae EU784136 SEQID 6 CTX-M-15 pBR-CBST-CTX-M-15 A K pneumoniae JF775516 SEQ ID 7 SHV-12 pBR-CBST-SHV-12 A K pneumoniae AY008838 SEQ ID 8 P99 AmpC pBR-CBST-P99 AMPC C E. cloacea XO7274 SEQ ID 9 OXA-15 pBR-CBST-OXA-15 D P. aeruginosa PAU63835 SEQ ID 10 KPC-4 pBR-CBST-KPC-4 A K pneumoniae EU447304 SEQ ID 11 DHA-1 pBR-CBST-DHA-1 C K pneumoniae AY585202 SEQ ID 12 ADC-33 pBR-CBST-ADC-33 C A.baumannii EU687478 SEQ ID 13 pBR-CBST-cat  SEQ ID 5 ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggt ttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttcta aatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaa aaggaagagtcatATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCG TAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGA TATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACAT TCTTGCCCGCCTGATGAATGCTCATACGGAATTTCGTATGGCAATGAAAGACGGTGAGCTGGTGAT ATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTG GAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGG TGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTG GGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCAC TATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGC CGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCA GGGCGGGGCGTAAGTGGCAGGGCGGGGCGTAAGGCGCGCCATTTAAATGAAGTTCCTATTCCGAAG TTCCTATTCTCTAGggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaa aaggatctaggtgaagatccatttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgct accagcggtggtttgtttgccggatcaagagctaccaactctattccgaaggtaactggcttcagcagagcgcagataccaa atactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaat cctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcg cagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagc gtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggaga gcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcga tttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgct ggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgatac cgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctcctt acgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtata cactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgt ctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccga aacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccag ctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtc actgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacggg ttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaa aatcactcagggtcaatgccagcgcttcgtttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgca gatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgtt gttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaa ggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccga gatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctc cgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggc ccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccat gtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatc cttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagc gagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatg ccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattc cgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccgg cacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggag ctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagta ggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcc tgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgata taggcgccagcaaccgcacctgtggcgccggtgstgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtgg tcgccatgatcgcgtagtcatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgct ccgagaacgggtgcgcatagaaattgcatcaacgcatataggctagcagcacgccatagtgactggcgatgctgtcggaatg agacgatatcccgcaagaggcccggcagtaccggcataccaagcctatgcctacagcatccagggtgacggtgccgaggatg acgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagct tatcgatgataagctgtcaaacatgagaa pBR-CBST-KPC-2  SEQ ID 6 ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttc ttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaataca ttcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagag tcatATGTCACTGTATCGCCGTCTAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGC CACCGCGCTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGGACTTTGGCGGCTCCATCGG TGTGTACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTG CAGCTCATTCAAGGGCTTTCTTGCTGCCGCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGA CACACCCATCCGTTACGGCAAAAATGCGCTGGTTCCGTGGTCACCCATCTCGGAAAAATATCTGACAAC AGGCATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGTGATAACGCCGCCGCCAATTTGTT GCTGAAGGAGTTGGGCGGCCCGGCCGGGCTGACGGCCTTCATGCGCTCTATCGGCGATACCACGTTCCG TCTGGACCGCTGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGATGCGCGCGATACCTCATCGCCGCG CGCCGTGACGGAAAGCTTACAAAAACTGACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTT TGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGCGCGGCGGTGCCGGCAGACTGGGC AGTCGGAGACAAAACCGGAACCTGCGGAGTGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCAC TGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCGGGCGCCTAACAAGGATGACAAGCACAGCGAGGC CGTCATCGCCGCTGCGGCTAGACTCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAAggatccctgtc agaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggt gaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaag atcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtgg tttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcct tctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacca gtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgg gctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatg agaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagg gagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgat gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgc tcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgc agccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgt gcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgcta tcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccgg catccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgag gcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagt ttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcct ccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatga tgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcag ggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaaca taatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggt cgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgc cagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcg tgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattg attggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatg caccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccga ggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgt ccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatca taatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataat ggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgca agcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtccta cgagttgcatgataaagaagacagtcataagttgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggt tgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggcc gttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccata cccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccag caaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgat cgcgtagtcgatagtggctcaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgg gtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatc ccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagc gcattgttagatttcatacaggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgat aagctgtc pBR-CBST-CTX-M-15 SEQ ID 7 ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttc ttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaataca ttcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagag tcatATGGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTG CGGAAAAGCACGTCAATGGGACGATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATA ACGTGGCGATGAATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACAGC TGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCATTCCGGGCGATCCGC GTGATACCACTTCACCTCGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCG ACAGCCAACGGGCGCAGCTGGTGACATGGATGAAAGGCAATACCACCGGTGCAGCGAGCATTCAGGCTG GACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCGGCAGCGGTGGCTATGGCACCACCAACGATATCG CGGTGATCTGGCCAAAAGATCGTGCGCCGCTGATTCTGGTCACTTACTTCACCCAGCCTCAACCTAAGG CAGAAAGCCGTCGCGATGTATTAGCGTCGGCGGCTAAAATCGTCACCGACGGTTTGTAAggatccctgt cagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttt tgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatct tcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagcc gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcc agtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccac gcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggg ggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggg ggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctt tcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgacc gagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcac accgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgac tgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttaca gacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggta aagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagc gttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggg gatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccg gttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccag cgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagg gcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgtttt gcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgg gtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctgg agatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaat tcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgca acgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaat cgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcg tcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaagg ccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctc gccgaaacgtttggtggcggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccga tcatcgtcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataa agaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaggagctgactgggttgaaggctctcaagggc atcggtcgacgtctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgc aaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagc gctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcg ccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtgg ctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattg catcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggc agtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcattgttagatttcaac acggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaa pBR-CBST-SHV-12  SEQ ID 8 ttcttgaagacgaaagggcctcgtgatacgcctattatataggttaatgtcatgataataatggtactta gacgtcaggtggcacattcggggaaatgtgcgcggaacccctatttgatatttactaaatacattcaaat atgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGC GTTATATTCGCCTGTGTATTATCTCCCTGTTAGCCACCCTGCCGCTGGCGGTACACGCCAGCCCGCAGCC GCTTGAGCAAATTAAACAAAGCGAAAGCCAGCTGTCGGGCCGCGTAGGCATGATAGAAATGGATCTGGCC AGCGGCCGCACGCTGACCGCCTGGCGCGCCGATGAACGCTTTCCCATGATGAGCACCTTTAAAGTAGTGC TCTGCGGCGCAGTGCTGGCGCGGGTGGATGCCGGTGACGAACAGCTGGAGCGAAAGATCCACTATCGCCA GCAGGATCTGGTGGACTACTCGCCGGTCAGCGAAAAACACCTTGCCGACGGCATGACGGTCGGCGAACTC TGCGCCGCCGCCATTACCATGAGCGATAACAGCGCCGCCAATCTGCTGCTGGCCACCGTCGGCGGCCCCG CAGGATTGACTGCCTTTTTGCGCCAGATCGGCGACAACGTCACCCGCCTTGACCGCTGGGAAACGGAACT GAATGAGGCGCTTCCCGGCGACGCCCGCGACACCACTACCCCGGCCAGCATGGCCGCGACCCTGCGCAAG CTGCTGACCAGCCAGCGTCTGAGCGCCCGTTCGCAACGGCAGCTGCTGCAGTGGATGGTGGACGATCGGG TCGCCGGACCGTTGATCCGCTCCGTGCTGCCGGCGGGCTGGTTTATCGCCGATAAGACCGGAGCTAGCAA GCGGGGTGCGCGCGGGATTGTCGCCCTGCTTGGCCCGAATAACAAAGCAGAGCGCATTGTGGTGATTTAT CTGCGGGATACCCCGGCGAGCATGGCCGAGCGAAATCAGCAAATCGCCGGGATCGGCGCGGCGCTGATCG AGCACTGGCAACGCTAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcattataattta aaaggatctaggtgaagatccatttgataatctcatgaccaaaatcccttaacgtgagattcgaccactgagcgtcagacccc gtagaaaagatcaaaggatcacttgagatcattattctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc ggtggatgatgccggatcaagagctaccaactattaccgaaggtaactggcttcagcagagcgcagataccaaatactgtcca ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagt ggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggct gaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaa agcgccacgcacccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagatc cagggggaaacgcctggtatcatatagtcctgtcgggatcgccacctctgacttgagcgtcgatattgtgatgctcgtcaggg gggcggagcctatggaaaaacgccagcaacgcggccatttacggacctggccattgctggccattgctcacatgactacctgc gttatcccctgattctgtggataaccgtattaccgcattgagtgagctgataccgctcgccgcagccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattactccttacgcatctgtgcggtatttcacaccgcatttg gtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggc tgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtga ccgtctccgggagctgcatgtgtcagaggattcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgt ggtcgtgaagcgattcacagatgtctgcctgacatccgcgtccagctcgttgagtactccagaagcgttaatgtctggcactg ataaagcgggccatgttaagggcggattacctgtaggtcactgatgcctccgtgtaagggggatactgacatgggggtaatga taccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgagtgagggtaaac aactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgtt ccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagacttta cgaaacacggaaaccgaagaccattcatgagagctcaggtcgcagacgattgcagcagcagtcgcttcacgttcgctcgcgta tcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacc cgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgactgccaaggg aggatgcgcattcacagttctccgcaagaattgattggctccaattcaggagtggtgaatccgttagcgaggtgccgccggca ccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatc catgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggt aagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcc cgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagc gcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgag ggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgaccc agagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcc caccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagca gcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccgg ccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatg tcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggac gggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcgg acagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctg tcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgcc gaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcatt aaagcttatcgatgataagctgtcaaacatgagaa pBR-CBST-P99 SEQ ID 9 ttcttgaagacgaaagggcctcgtgatacgcctattatataggttaatgtcatgataataatggtacttaga cgtcaggtggcacattcggggaaatgtgcgcggaacccctatttgatatttactaaatacattcaaatatgt atccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGATGAGAA AATCCCTTTGCTGCGCCCTGCTGCTCGGCATCTCTTGCTCTGCTCTCGCCACGCCAGTGTCAGAAAAACAGC TGGCGGAGGTGGTCGCGAATACGATTACCCCGCTGATGAAAGCCCAGTCTGTTCCAGGCATGGCGGTGGCCG TTATTTATCAGGGAAAACCGCACTATTACACATTTGGCAAGGCCGATATCGCGGCGAATAAACCCGTTACGC CTCAGACCCTGTTCGAGCTGGGTTCTATAAGTAAAACCTTCACCGGCGTTTTAGGTGGGGATGCCATTGCTC GCGGTGAAATTTCGCTGGACGATGCGGTGACCAGATACTGGCCACAGCTGACGGGCAAGCAGTGGCAGGGTA TTCGTATGCTGGATCTCGCCACCTACACCGCTGGCGGCCTGCCGCTACAGGTACCGGATGAGGTCACGGATA ACGCCTCCCTGCTGCGCTTTTATCAAAACTGGCAGCCGCAGTGGAAGCCTGGCACAACGCGTCTTTACGCCA ACGCCAGCATCGGTCTTTTTGGTGCGCTGGCGGTCAAACCTTCTGGCATGCCCTATGAGCAGGCCATGACGA CGCGGGTCCTTAAGCCGCTCAAGCTGGACCATACCTGGATTAACGTGCCGAAAGCGGAAGAGGCGCATTACG CCTGGGGCTATCGTGACGGTAAAGCGGTGCGCGTTTCGCCGGGTATGCTGGATGCACAAGCCTATGGCGTGA AAACCAACGTGCAGGATATGGCGAACTGGGTCATGGCAAACATGGCGCCGGAGAACGTTGCTGATGCCTCAC TTAAGCAGGGCATCGCGCTGGCGCAGTCGCGCTACTGGCGTATCGGGTCAATGTATCAGGGTCTGGGCTGGG AGATGCTCAACTGGCCCGTGGAGGCCAACACGGTGGTCGAGGGCAGCGACAGTAAGGTAGCACTGGCGCCGT TGCCCGTGGCAGAAGTGAATCCACCGGCTCCCCCGGTCAAAGCGTCCTGGGTCCATAAAACGGGCTCTACTG GCGGGTTTGGCAGCTACGTGGCCTTTATTCCTGAAAAGCAGATCGGTATTGTGATGCTCGCGAATACAAGCT ATCCGAACCCGGCACGCGTTGAGGCGGCATACCATATCCTCGAGGCGCTACAGTAAggatccctgtcagaccaagatactcata tatactttagattgatttaaaacttcattataatttaaaaggatctaggtgaagatccatttgataatctcatgaccaaaatcc cttaacgtgagattcgaccactgagcgtcagaccccgtagaaaagatcaaaggatcacttgagatcattattctgcgcgtaatc tgctgcttgcaaacaaaaaaaccaccgctaccagcggtggatgatgccggatcaagagctaccaactattaccgaaggtaactg gcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgc ctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagac gatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccg aactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggca gggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctct gacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcc tggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagt gagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtatt ttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagcc agtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggc ttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcacc gaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccag ctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcac tgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttac tgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcac tcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaa cataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggt cgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgcca gcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcg gctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggc tccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcga cgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataa atcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcg tcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggcc atccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccg aaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatc gtcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagac agtcataagtgcggcgacgatagtcatccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcga cgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggt gcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccg aagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggcc acgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcg agcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgct agcagcacgccatagtgactggcgatgactgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcc tatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgtta gcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaa pBR-CBST-OXA-15 SEQ ID 10 ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagac gtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgta tccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGGCAATCCGAAT CTTCGCGATACTTTTCTCCATTTTTTCTCTTGCCACTTTCGCGCATGCGCAAGAAGGCACGCTAGAACGTTCTGA CTGGAGGAAGTTTTTCAGCGAATTTCAAGCCAAAGGCACGATAGTTGTGGCAGACGAACGCCAAGCGGATCGTGC CATGTTGGTTTTTGATCCTGTGCGATCGAAGAAACGCTACTCGCCTGCATCGACATTCAAGATACCTCATACACT TTTTGCACTTGATGCAGGCGCTGTTCGTGATGAGTTCCAGATTTTTCGATGGGACGGCGTTAACAGGGGCTTTGC AGGCCACAATCAAGACCAAGATTTGCGATCAGCAATGCGGAATTCTACTGTTTGGGTGTATGAGCTATTTGCAAA GGAAATTGGTGATGACAAAGCTCGGCGCTATTTGAAGAAAATCGACTATGGCAACGCCGGTCCTTCGACAAGTAA TGGCGATTACTGGATAGAAGGCAGCCTTGCAATCTCGGCGCAGGAGCAAATTGCATTTCTCAGGAAGCTCTATCG TAACGAGCTGCCCTTTCGGGTAGAACATCAGCGCTTGGTCAAGGATCTCATGATTGTGGAAGCCGGTCGCAACTG GATACTGCGTGCAAAGACGGGCTGGGAAGGCCGTATGGGTTGGTGGGTAGGATGGGTTGAGTGGCCGACTGGCTC CGTATTCTTCGCACTGAATATTGATACGCCAAACAGAATGGATGATCTTTTCAAGAGGGAGGCAATCGTGCGGGC AATCCTTCGCTCTATTGAAGCGTTACCGCCCAACCCGGCAGTCAACTCGGACGCTGCGCGATAAggatccctgtc agaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgat aatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgag atcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagct accaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccac cacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgt gtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccag cttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcg gacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtc ctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaa cgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataac cgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag agcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctga tgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctg acgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggtt ttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgt tcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggtttttt cctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcac gatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccag agaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgc agatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgt tgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaa ccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgcc gcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattg attggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcac cgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggc ataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatgg tcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaagg ccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgcc gaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatc gtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaaga cagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcg acgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggt gcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccga agtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccac gatgcgtccggcgtagaggattcacaggacggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagca ggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctacagc acgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgccta cagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaa ctgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaa pBR-CBST-KPC-4  SEQ ID 11 ttcttgaagacgaaagggcctcgtgatacgcctattatataggttaatgtcatgataataatggtacttag acgtcaggtggcacattcggggaaatgtgcgcggaacccctatttgatatttactaaatacattcaaatat gtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGTCAC TGTATCGCCGTCTAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGCCACCGCGCTGACC AACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGGACTTTGGCGGCTCCATCGGTGTGTACGCGATGGA TACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCT TTCTTGCTGCCGCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGC AAAAATGCGCTGGTTCGGTGGTCACCCATCTCGGAAAAATATCTGACAACAGGCATGACGGTGGCGGAGCT GTCCGCGGCCGCCGTGCAATACAGTGATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGG CCGGGCTGACGGCCTTCATGCGCTCTATCGGCGATACCACGTTCCGTCTGGACCGCTGGGAGCTGGAGCTG AACTCCGCCATCCCAGGCGATGCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACAAAAACT GACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTTTGTTGATTGGCTAAAGGGAAACACGACCG GCAACCACCGCATCCGCGCGGCGGTGCCGGCAGACTGGGCAGTCGGAGACAAAACCGGAACCTGCGGAGGG TATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCACTGGGCGCGCACCTATTGTGTTGGCCGTCTACAC CCGGGCGCCTAACAAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCGGCTAGACTCGCGCTCGAGG GATTGGGCGTCAACGGGCAGTAAggatccctgtcagaccaagatactcatatatactttagattgatttaaaacttcattataat ttaaaaggatctaggtgaagatccatttgataatctcatgaccaaaatcccttaacgtgagattcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcacttgagatcattattctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc ggtggatgatgccggatcaagagctaccaactattaccgaaggtaactggcttcagcagagcgcagataccaaatactgtccact agtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggct gctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacgg ggggttcgtgcacacagcccagcaggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacg cttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaa acgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggag cctatggaaaaacgccagcaacgcggcctattacggacctggccattgctggccattgctcacatgactacctgcgttatcccct gattctgtggataaccgtattaccgcattgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgag cgaggaagcggaagagcgcctgatgcggtattactccttacgcatctgtgcggtatacacaccgcataggtgcactctcagtaca atctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgcc aacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatg tgtcagaggattcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagat gtctgcctgacatccgcgtccagctcgttgagtactccagaagcgttaatgtctggcactgataaagcgggccatgttaagggcg gattacctgtaggtcactgatgcctccgtgtaagggggatactgacatgggggtaatgataccgatgaaacgagagaggatgctc acgatacgggttactgatgatgaacatgcccggttactggaacgagtgagggtaaacaactggcggtatggatgcggcgggacca gagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgaccacagggtagccagcagcatcctgcgatgc agatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgt tgctcaggtcgcagacgattgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaac cccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccg cgtgcggctgctggagatggcggacgcgatggatatgactgccaagggaggatgcgcattcacagactccgcaagaattgattgg ctccaattcaggagtggtgaatccgttagcgaggtgccgccggcaccattcaggtcgaggtggcccggctccatgcaccgcgacg caacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatc gccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatcatgaagctgtccctgatggtcgtcatc tacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccag cctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtt tggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgct ccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcata agtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctc ccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaa ggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcga gcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtc cggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgg gcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgcca tagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcat ccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtga taaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaa pBR-CBST-DHA-1 SEQ ID 12 ttcttgaagacgaaagggcctcgtgatacgcctattatataggttaatgtcatgataataatggtacttagac gtcaggtggcacattcggggaaatgtgcgcggaacccctatttgatatttactaaatacattcaaatatgtat ccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGAAAAAATCGT TATCTGCAACACTGATTTCCGCTCTGCTGGCGTTTTCCGCCCCGGGGTTTTCTGCCGCTGATAATGTCGCGGC GGTGGTGGACAGCACCATTAAACCGCTGATGGCACAGCAGGATATTCCCGGGATGGCGGTTGCCGTCTCCGTA AAGGGTAAGCCCTATTATTTCAATTATGGTTTTGCCGATATTCAGGCAAAACAGCCGGTCACTGAAAATACAC TATTTGAGCTCGGATCTGTAAGTAAAACTTTCACAGGTGTGCTGGGTGCGGTTTCTGTGGCGAAAAAAGAGAT GGCGCTGAATGATCCGGCGGCAAAATACCAGCCGGAGCTGGCTCTGCCGCAGTGGAAGGGGATCACATTGCTG GATCTGGCTACCTATACCGCAGGCGGACTGCCGTTACAGGTGCCGGATGCGGTAAAAAGCCGTGCGGATCTGC TGAATTTCTATCAGCAGTGGCAGCCGTCCCGGAAACCGGGCGATATGCGTCTGTATGCAAACAGCAGTATCGG CCTGTTTGGTGCTCTGACCGCAAACGCGGCGGGGATGCCGTATGAGCAGTTGCTGACTGCACGCATCCTGGCA CCGCTGGGGTTATCTCACACCTTTATTACTGTGCCGGAAAGTGCGCAAAGCCAGTATGCGTACGGTTATAAAA ACAAAAAACCGGTCCGCGTGTCGCCGGGACAGCTTGATGCGGAATCTTACGGCGTGAAATCCGCCTCAAAAGA TATGCTGCGCTGGGCGGAAATGAATATGGAGCCGTCACGGGCCGGTAATGCGGATCTGGAAATGGCAATGTAT CTCGCCCAGACCCGCTACTATAAAACCGCCGCGATTAACCAGGGGCTGGGCTGGGAAATGTATGACTGGCCGC AGCAGAAAGATATGATCATTAACGGTGTGACCAACGAGGTCGCATTGCAGCCGCATCCGGTAACAGACAACCA GGTTCAGCCGTATAACCGTGCTTCCTGGGTGCATAAAACGGGCGCAACAACTGGTTTCGGCGCCTATG TCGCCTTTATTCCGGAAAAACAGGTGGCGATTGTGATTCTGGCGAATAAA AACTACCCGAATACCGAAAGAGTCAAAGCTGCACAGGCTATTTTGAGTGC ACTGGAATAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaag gatctaggtgaagatccatttgataatctcatgaccaaaatcccttaacgtgagattcgaccactgagcgtcagaccccgta gaaaagatcaaaggatcacttgagatcctattactgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcg gtggatgatgccggatcaagagctaccaactattaccgaaggtaactggcttcagcagagcgcagataccaaatactgtc cactagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacca gtggctgctgccagtggcgataagtcgtgtcttaccgggaggactcaagacgatagttaccggataaggcgcagcggtcg ggctgaacggggggacgtgcacacagcccagcaggagcgaacgacctacaccgaactgagatacctacagcgtgag ctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggaga gcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcga tttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctg gccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctc gccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacg catctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactcc gctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgc tcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacg cgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctc gttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatg cctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactga tgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatca ctcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatcc ggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgc tcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaac cccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgag atgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctcc gcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcc cggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccg ttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagcc gcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggccgcaacgcgggcatcccga tgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagc ccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaag gcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggt cctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaaagaagacagtcatagtgcg gcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctc tcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatgg tgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgct catgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtgg cgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcg atagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggt gcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgat atcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatg acgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagctta pBR-CBST-ADC-33 SEQ ID 13 ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggca cttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtcatATGCGATTTAAAAAAATTTCTTGTCTAC TTTTATCCCCGCTTTTTATTTTTAGTACCTCAATTTATGCGGGCAATACACC AAAAGACCAAGAAATTAAAAAACTGGTAGATCAAAACTTTAAACCGTTAT TAGAAAAATATGATGTGCCAGGTATGGCTGTGGGTGTTATTCAAAATAAT AAAAAGTATGAAATGTATTATGGTCTTCAATCTGTTCAAGATAAAAAAGC CGTAAATAGCAGTACCATTTTTGAGCTAGGTTCTGTCAGTAAATTATTTAC CGCGACAGCAGGTGGATATGCAAAAAATAAAGGAAAAATCTCTTTTGACG ATACGCCTGGTAAATATTGGAAAGAACTAAAAAACACACCGATTGACCAA GTTAACTTACTTCAACTCGCGACGTATACAAGTGGTAACCTTGCCTTGCAG TTTCCAGATGAAGTAAAAACAGACCAACAAGTTTTAACTTTTTTCAAAGA CTGGAAACCTAAAAACTCAATCGGTGAATACAGACAATATTCAAATCCAA GTATTGGCCTATTTGGAAAGGTTGTGGCTTTGTCTATGAATAAACCTTTCG ACCAAGTCTTAGAAAAAACAATTTTTCCGGCCCTTGGCTTAAAACATAGCT ATGTAAATGTACCTAAGACCCAGATGCAAAACTATGCATTTGGTTATAAC CAAGAAAATCAGCCGATTCGAGTTAACCGCGGCCCACTCGATGCCGCCCC TGCGTATGGCGTCAAATCGACACTACCCGACATGTTGAGTTTTATTCATGC CAACCTTAACCCACAGAAATATCCGGCTGATATTCAACGGGCAATTAATG AAACACATCAAGGGCGCTATCAAGTAAATACCATGTATCAGGCACTCGGT TGGGAAGAGTTTTCTTATCCGGCAACGTTACAAACTTTATTAGACAGTAAT TCAGAACAGATTGTGATGAAACCTAATAAAGTGACTGCTATTTCAAAGGA ACCTTCAGTTAAGATGTACCATAAAACTGGCTCAACCAACGGTTTCGGAA CGTATGTAGTGTTTATTCCTAAAGAAAATATTGGCTTAGTCATGTTAACCA ATAAACGTATTCCAAATGAAGAGCGCATTAAGGCAGCTTATGCTGTGCTG AATGCAATAAAGAAATAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttca tttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgag cgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagat accaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgct aatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataag gcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatac ctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtc ggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctg acttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagt gagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcg gtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaag ccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctg acgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccg tcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttca tccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcct gtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcac gatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcggg accagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatc ctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaag accattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgct aaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggac ccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttg cgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccatt caggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctac aatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagtt aggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgca acgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacg ccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcg ggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgc tccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaag acagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagg gcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgc cgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccac gccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccag caaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgcc atgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtg ctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgt cggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgac ggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaacta ccgcattaaagcttatcgatgataagctgtcaaacatgagaa Nucleotide Sequences of pBR-CBST Plasmids (Containing β-Lactamase or cat Genes) Used in the E. coli Isogenic Strains (relevant restriction sites are underlined; β-lactamase sequences in all caps, tetA sequence is in italics)

Example 12 Standard BLI Potentiation MIC Assay

The ability of compounds to potentiate the activity of β-lactams was demonstrated by determining the minimum inhibitory concentrations (MIC) of β-lactam and BLI compound combinations against various β-lactamase producing bacterial strains using the broth microdilution method. The experimental protocol was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines with modifications as described below (CLSI guidelines can be derived from the CLSI document M07-A9 published in January 2012: “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Ninth Edition”).

To prepare for MIC testing, frozen glycerol stocks of clinical isolates (Klebsiella pneumoniae, Escherichia coli, Enterobacter spp, Citrobacter spp, or Pseudomonas aeruginosa) were used to streak for isolated colonies on rich, non-selective, tryptic soy agar containing 5% sheep's blood (TSAB). Frozen glycerol stocks of laboratory engineered, isogenic E. coli strains, which contain cloned β-lactamase expressing plasmids were used to streak for isolated colonies on rich, selective LB agar supplemented with 25 μg/mL tetracycline to maintain the plasmid. All strains were incubated at 37° C. for 18-24 hrs.

On the day of testing, primary cultures were started by scraping off 5-10 colonies from the TSAB plates containing clinical strains or the tetracycline supplemented LB plates containing engineered strains. The clinical strain material was suspended in ˜5 mL of cation adjusted Mueller Hinton Broth (CAMHB) in 14 mL culture tubes. The engineered strain material was suspended in CAMHB (supplemented with 25 ug/mL tetracycline) in 14 mL culture tubes. All strains were incubated at 37° C. with aeration (200 rpm) for ˜2 hrs until the optical density at 600 nm (OD600) was ≧0.1.

The two compound components of the assay were each diluted in CAMHB and added to the 96-well broth microdilution assay plates. 50 μL of the β-lactam was added to each well of the assay plate in 2-fold dilutions with final concentrations ranging from 128-0.13 μg/mL. 25 μL of the BLI compound was added to all wells in the broth microdilution plates at a final concentration of 4 μg/mL. Inoculum cultures were prepared by standardizing the primary cultures to OD600=0.1 and then adding 20 μL of the adjusted primary culture per 1 mL CAMHB for clinical strains or CAMHB (supplemented with tetracycline at 100 μg/mL) for engineered strains, so that the final inoculum density was ˜105 colony forming units per milliliter. Diluted inoculum cultures were used to inoculate 25 μL per well in 96-well broth microdilution assay plates. The final volume of each well was 100 μL and contained a β-lactam at different concentrations, a BLI compound at 4 μg/mL concentration, the bacterial culture at an OD600 of approximately 0.001 and when necessary tetracycline at 25 μg/mL.

Plates were incubated for 18-20 hours at 37° C. with aeration (200 rpm). Following incubation, growth was confirmed visually placing plates over a viewing apparatus (stand with a mirror underneath) and then OD600 was measured using a SpectraMax 340PC384 plate reader (Molecular Devices, Sunnyvale, Calif.). Growth was defined as turbidity that could be detected with the naked eye or achieving minimum OD600 of 0.1. MIC values were defined as the lowest concentration producing no visible turbidity.

MIC values of representative compounds are shown in Table II.

Example 13 Synergy MIC (sMIC) Assay

The synergy MIC (sMIC) assay determines the concentration of the BLI required to potentiate the activity of a fixed concentration of a β-lactam antibiotic against β-lactamase producing bacterial strains. The experimental protocol was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines with modifications as described below (CLSI guidelines can be derived from the CLSI document M07-A9 published in January 2012: “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Ninth Edition”). The assay is set-up by serially diluting the BLI across 11 of the 12 wells in each row of a 96-well broth microdilution assay plate, adding the β-lactam at a fixed concentration to all wells in the assay plate, inoculating the assay plate with bacterial strains, and determining the lowest concentration of BLI required to inhibit overnight bacterial growth. Bacterial growth in the 12th well of the assay plate, which contains the β-lactam at a fixed concentration but does not contain any BLI, demonstrates that the bacterial strains are resistant to the β-lactam antibiotic (e.g ceftolozane) at the fixed concentration of 4 μg/mL.

To prepare for MIC testing, frozen glycerol stocks of clinical isolates (Klebsiella pneumoniae, Escherichia coli, Enterobacter spp, Citrobacter spp, or Pseudomonas aeruginosa) were used to streak for isolated colonies on rich, non-selective, tryptic soy agar containing 5% sheep's blood (TSAB). Frozen glycerol stocks of laboratory engineered, isogenic E. coli strains, which contain cloned β-lactamase expressing plasmids were used to streak for isolated colonies on rich, selective LB agar supplemented with 25 μg/mL tetracycline to maintain the plasmid. All strains were incubated at 37° C. for 18-24 hrs.

On the day of testing, primary cultures were started by scraping off 5-10 colonies from the TSAB plates containing clinical strains or the tetracycline supplemented LB plates containing engineered strains. The clinical strain material was suspended in ˜5 mL of cation adjusted Mueller Hinton Broth (CAMHB) in 14 mL culture tubes. The engineered strain material was suspended in CAMHB (supplemented with tetracycline at 25 μg/mL) in 14 mL culture tubes. All strains were incubated at 37° C. with aeration (200 rpm) for ˜2 hrs until the OD600 was ≧0.1.

The two compound components of the assay were each prepared in CAMHB and added to the 96-well broth microdilution assay plates. 50 μL of the BLI was added to each well of the assay plate in 2-fold dilutions with final concentrations ranging from 128 to 0.13 μg/mL. 25 μL of the β-lactam was added to all wells in the broth microdilution plates at a final concentration of 4 μg/mL. Inoculum cultures were prepared by standardizing the primary cultures to OD600=0.1 and then adding 20 μL of the adjusted primary culture per 1 mL CAMHB for clinical strains or CAMHB (supplemented with tetracycline at 100 μg/mL) for isogenic strains, so that the final inoculum density was ˜105 colony forming units per milliliter. Diluted inoculum cultures were used to inoculate 25 μL per well in 96-well broth microdilution assay plates. The final volume of each well was 100 μL and contained a BLI at different concentrations, a β-lactam at 4 μg/mL concentration, the bacterial culture at an OD600 of approximately 0.001 and when necessary tetracycline at 25 ug/mL.

Interpreting the sMIC Data:

Plates were incubated for 18-20 hours at 37° C. with aeration (200 rpm). Following incubation, growth was confirmed visually placing plates over a viewing apparatus (stand with a mirror underneath) and then OD600 was measured using a SpectraMax 340PC384 plate reader (Molecular Devices, Sunnyvale, Calif.). Growth was defined as turbidity that could be detected with the naked eye or achieving minimum OD600 of 0.1. sMIC values were defined as the lowest concentration producing no visible turbidity.

The sMIC values represent the amount of BLI required to potentiate the activity of 4 mg/ml of CXA-101 (Ceftolozane) or ceftazidime to inhibit the growth of the β-lactamase producing bacteria.

sMIC values of representative compounds are shown in Table III.

Example 14 Inhibition Kinetics

Inhibition or inactivation of KPC-2 by test inhibitors was assessed using 100 μM nitrocefin (NCF) as a reporter substrate. Assays were performed in 1×PBS pH 7.4, 0.1 mg/ml BSA, in 96-well half area plates, 50 μl reaction volume. NCF was dissolved in DMSO and diluted in assay buffer. Test inhibitors were dissolved in water or DMSO and serially diluted in the assay with final concentrations between 2000-0.195 μM.

The enzyme activity in the presence of varying concentrations of test inhibitor was determined by monitoring the hydrolysis of NCF spectrophotometrically at 486 nm, for 5 minutes, 25° C., using a SpectraMax Plus384 microplate reader with SoftMax Pro software (Molecular Devices). Data analysis was performed using GraphPad Prism (GraphPad Software, Inc.).
Progress curves were fit to a first-order rate decay equation (Eq. 1) to determine kobserved (kobs).
kobs vs. inhibitor concentration [I] curves were then fit to Eq. 2 to determine the inhibitor dissociation constant (K) and the first order rate constant of enzyme inactivation at infinite inhibitor concentration (kinact). Table IV shows kinetics results from representative test compounds. A larger kinact/K ratio indicates a more effective enzyme inactivator.


Yt=V0*(1−e(−kobs*t))/kobs  Eq. 1

Where Y is the absorbance at time t, V0 is the uninhibited enzyme velocity, kobs is the observed rate constant of the enzyme inactivation.


kobs=kinact*[I]/([I]+K(1+S/Km))  Eq. 2

Where S is the NCF concentration, Km is the KPC-2 Km for NCF.

Claims

1-37. (canceled)

38. A pharmaceutical composition comprising ceftolozane or a pharmaceutically acceptable salt thereof and a compound of Formula (I) or a pharmaceutically acceptable salt thereof: and wherein R6 is selected from H, (C1-C3)-unsubstituted alkyl, amino-(C2-C3)-alkyl, aminocycloalkyl, hydroxyalkyl,

wherein
Z is selected from a 1,2,4-oxadiazole or a 1,2,4-thiadiazole;
R is selected from
R1 is selected from:
hydrogen,
wherein R2 is selected from
wherein each of R3, R4 and R5 is independently selected from hydrogen, (C1-C3)-alkyl, aminoalkyl, aminocycloalkyl, or hydroxyalkyl, and n is selected from 1, 2 or 3,
amino,
and each of p and q is independently selected from 1 or 2; and
—CH2(R7)CH2NH2
wherein R7 is selected from amino or hydroxyl.

39. The pharmaceutical composition of claim 38 wherein Z—R1 is wherein X is selected from O or S and wherein R and R1 are as previously described.

40. The pharmaceutical composition of claim 38 wherein Z—R1 is wherein X is selected from O or S and wherein R and R1 are as previously described.

41. The pharmaceutical composition of claim 38 wherein the compound of Formula (I) exhibits a binding affinity for the KPC-2 β-lactamase enzyme of at least 250 mM−1s−1.

42. The pharmaceutical composition of claim 38 wherein the compound is selected from the group consisting of Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII) and Formula (VIII), and pharmaceutically acceptable salts thereof:

43. The pharmaceutical composition of claim 38 wherein the compound or a pharmaceutically acceptable salts thereof has the Formula:

44. The pharmaceutical composition of claim 38 wherein the compound or a pharmaceutically acceptable salts thereof has the Formula:

Patent History
Publication number: 20150031660
Type: Application
Filed: Oct 2, 2014
Publication Date: Jan 29, 2015
Inventors: Yu Gui Gu (Acton, MA), Yong He (Bedford, MA), Ning Yin (Lexington, MA), Dylan C. Alexander (Watertown, MA), Jason B. Cross (Acton, MA), Chester A. Metcalf, III (Needham, MA), Robert Busch (Wakefield, MA)
Application Number: 14/505,450
Classifications
Current U.S. Class: Additional Hetero Ring (514/202)
International Classification: C07D 471/08 (20060101); A61K 31/439 (20060101); A61K 31/4545 (20060101); A61K 31/546 (20060101);