US20140323459A1

US20140323459A1 - Isoxazole beta-lactamase inhibitors

Info

Publication number: US20140323459A1
Application number: US14/260,184
Authority: US
Inventors: Yu Gui Gu; Yong He; Ning Yin; Dylan C. Alexander; Jason B. Cross; Chester A. Metcalf, III; Robert Busch
Original assignee: Cubist Pharmaceuticals LLC; Calixa Therapeutics Inc
Current assignee: Merck Sharp and Dohme LLC; Calixa Therapeutics Inc
Priority date: 2012-03-30
Filing date: 2014-04-23
Publication date: 2014-10-30
Also published as: US20130345190A1; CN104334555A; TW201343646A; IL234845A0; SG11201406120SA; EP2831069A1; US8927724B2; MX2014011825A; EP2831069B1; AR090589A1; US20150038479A1; AU2013237869A1; CA2869051A1; KR20150003777A; CO7091182A2; JP2015512419A; EP2831069A4; WO2013149136A1

Abstract

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.

Description

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/618,127, filed Mar. 30, 2012, and U.S. Provisional Application No. 61/790,248, filed Mar. 15, 2013. The entire contents of these applications are incorporated herein by reference in their entireties.

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 infections caused by 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.
wherein
R is selected from
and
R¹is selected from:

- a.

- - wherein R²is selected from

- - wherein each of R³, R⁴and R⁵is independently selected from hydrogen, (C₁-C₃)-alkyl, aminoalkyl, aminocycloalkyl, and hydroxyalkyl, and n is selected from 1, 2 and 3;
- b.

- wherein R⁶is selected from
  - H and

- C.

- - wherein R⁷is selected from H, (C₁-C₃)-unsubstituted alkyl, amino-(C₂-C₃)-alkyl, amino cyclo alkyl, hydroxyalkyl,
  - and

- - wherein each of p and q is independently selected from 1 and 2.

In another aspect, the invention provides compounds of chemical Formula (A-I) or a pharmaceutically acceptable salt thereof, which are BLIs and are useful in combination with β-lactam antibiotics for the treatment of bacterial infections.
wherein
R* is selected from
and
R¹* is selected from:

- a.

- - wherein R²* is selected from

- - R³* is selected from hydrogen, (C₁-C₃)-alkyl, aminoalkyl, aminocycloalkyl, hydroxyalkyl,

- - each of R⁴*, R⁵*, R⁶* and R⁷* is independently selected from hydrogen, (C₁-C₆)-alkyl, aminoalkyl, aminocycloalkyl, and hydroxyalkyl, provided that at least one of R⁴*, R⁵*, R⁶* and R⁷* is hydrogen,
  - n is selected from 1, 2, 3 and 4, and
  - m is selected from 1, 2 and 3;
- b.

- - wherein R⁸is selected from —NH(C₁-C₃)-alkyl and

- - wherein each of R⁴*, R⁵*, R⁶* and R⁷* is as described previously;
- c.

- - wherein Z is selected from CR⁹R¹⁰and NR¹¹,
  - each of R⁹and R¹⁰is independently selected from H, NH₂, —NH(C₁-C₃)—alkyl and

- - wherein each of R⁴*, R⁵*, R⁶* and R⁷* is as described previously,
  - alternatively, R⁹and R¹⁰together with the carbon to which they are attached, form a cycloalkyl or heterocyclyl ring containing 4-6 ring members,
  - R¹¹is selected from H and

- - each of R¹², R¹³and R¹⁴is independently selected from hydrogen, (C₁-C₆)-alkyl, aminoalkyl, aminocycloalkyl, and hydroxyalkyl, provided that at least one of R¹², R¹³and R¹⁴is hydrogen,
  - R¹⁵is selected from NH₂and

- - wherein each of R⁴*,
  - R⁵*, R⁶* and R⁷* is as described previously,
  - each of p* and q* is independently selected from 0, 1, 2 and 3,
  - T is selected from NH and O,
  - t is selected from 0, 1, 2, 3, and 4, and
  - each of r and y is independently selected from 0 and 1;
- d.

- - wherein R¹⁶is selected from NH₂, —NH(C₁-C₃)-alkyl and

- - wherein each of R⁴*, R⁶* and R⁷* is as described previously,
  - s is selected from 0 and 1, and,
  - v is selected from 0, 1, 2, and 3;
- e.

- - wherein R¹⁸is selected from NH₂and

- - wherein each of R⁴*, R⁵*, R⁶* and R⁷* is as described previously,
  - R¹⁷is selected from amino and hydroxyl, and
  - w is selected from 0 and 1;
- f.

- g.

- - wherein M is selected from NR¹⁹, CR²⁰R²¹and O,
  - wherein R¹⁹is selected from H and

- - where each of R¹², R¹³and R¹⁴is as described previously,
  - each of R²⁰and R²¹is independently selected from H, NH₂and

- - wherein each of R⁴*, R⁶* and R⁷* is as described previously, and
  - u is selected from 0, 1 and 2; and
- h.

In one embodiment, the invention provides use of a compound of Formula I for inhibiting β-lactamases.
In one embodiment, the invention provides use of a compound of Formula A-I for inhibiting β-lactamases.
In one embodiment, the invention provides compounds of Formula I with high binding affinity for β-lactamase enzymes.
In one embodiment, the invention provides compounds of Formula A-I with high binding affinity for β-lactamase enzymes.
In one embodiment, the present invention also provides antibacterial compositions comprising compounds of Formula I and at least one β-lactam antibiotic.
In one embodiment, the present invention also provides antibacterial compositions comprising compounds of Formula A-I and at least one β-lactam antibiotic.
In one 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 one embodiment, the present invention provides pharmaceutical compositions comprising compounds of Formula A-I and at least one β-lactam antibiotic and methods of use thereof.
In one embodiment, the invention provides methods of use of the compounds of Formula I to treat bacterial infections in a subject.
In one embodiment, the invention provides methods of use of the compounds of Formula A-I to treat bacterial infections in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show Table I, Representative Compounds of Formula A-II

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

FIGS. 3A-3B show Table III, the synergy MIC of representative compounds of Formula II-A 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 II-A for the KPC-2 β-lactamase.

FIGS. 5A-5B show Table V, Synergy MIC of Comparator Compounds Against a Panel of Isogenic and Clinical Strains Expressing β-lactamases

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 NH₂radical.
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)NHR⁵represent 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 Functinal 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.
Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, and ³⁵S. In one embodiment, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet another embodiment, substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
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:
Substituent R of Formula I is selected from
In a preferred embodiment, R is
The group R¹of Formula I is selected from:

- a.

- - wherein R²is selected from

- - wherein R⁶is selected from
  - H and

- c.

- - wherein R⁷is selected from H, (C₁-C₃)-unsubstituted alkyl, amino-(C₂-C₃)-alkyl, amino cyclo alkyl, hydroxyalkyl, and

- - wherein each of p and q is independently selected from 1 and 2.

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 R¹is selected from —CH₂NH₂, —CH₂CH₂NH₂, —CONH(CH₂)₂NH₂,
In one embodiment of the invention, R¹is selected from
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, the compound is of Formula II and R is —OSO₃H and R¹is —CH₂NH₂.
In another embodiment of the invention, the compound is of Formula II and R is —OSO₃H and R¹is —CH₂CH₂NH₂.
In another embodiment of the invention, the compound is of Formula II and R is —OSO₃H and R¹is —CONH(CH₂)₂NH₂.
In another embodiment of the invention, the compound is of Formula II and R is —OSO₃H and R¹is
In another embodiment of the invention, the compound is of Formula II and R is —OSO₃H and R¹is
In another embodiment of the invention, the compound is of Formula II and R is —OSO₃H and R¹is
In another embodiment of the invention, the compound is of Formula II and R is —OSO₃H and R¹is
In another embodiment of the invention, the compound is of Formula II and R is —OSO₃H and R¹is
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 R¹and R, compounds of Formula I may exist in a salt or zwitterionic form.
In one aspect, the invention provides compounds of Formula A-I or pharmaceutically-acceptable salts thereof:
Substituent R* of Formula A-I is selected from
In a preferred embodiment, R* is
The group R¹* is selected from:

- a.

- - wherein R²* is selected from

- - wherein R⁸is selected from —NH(C₁-C₃)-alkyl and

- - wherein each of R⁴*, R⁶* and R⁷* is as described previously,
  - alternatively, R⁹and R¹⁰together with the carbon to which they are attached, form a cycloalkyl or heterocyclyl ring containing 4-6 ring members,
  - R¹¹is selected from H and

- - wherein each of R⁴*, R⁵*,
  - R⁶* and R⁷* is as described previously,
  - each of p* and q* is independently selected from 0, 1, 2 and 3,
  - T is selected from NH and O,
  - t is selected from 0, 1, 2, 3, and 4, and
  - each of r and y is independently selected from 0 and 1;
- d.

- - wherein R¹⁶is selected from NH₂, —NH(C₁-C₃)-alkyl and

- - wherein each of R⁴*, R⁵*, R⁶* and R⁷* is as described previously,
  - s is selected from 0 and 1, and,
  - v is selected from 0, 1, 2, and 3;
- e.

- - wherein R¹⁸is selected from NH₂and

- g.

In one aspect of the invention R¹* is selected from
In one embodiment of the invention R¹* is selected from
In one embodiment of the invention, the compounds of the invention are of the stereochemistry disclosed in Formula A-II.
In another embodiment of the invention, R* and R¹* are chosen from the substituents listed in Table I (See FIG. 1). Preferred compounds of Formula A-I are
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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) are effective in inhibiting β-lactamase. In one aspect of the invention the compounds of Table I are effective β-lactamase inhibitors. In one aspect the compound
is effective in inhibiting β-lactamase. In one aspect the compound
is effective in inhibiting β-lactamase
When used in combination with β-lactam antibiotics, the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) inhibit β-lactamases selected from class A, class C or class D 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. In one aspect of the invention the compounds of Formula A-I inhibit β-lactamases selected from class A, class C or class D β-lactamases. In one aspect of the invention the compounds of Formula II inhibit β-lactamases selected from class A, class C or class D β-lactamases. In one aspect of the invention the compounds of Formula A-II inhibit β-lactamases selected from class A, class C or class D β-lactamases. In one aspect of the invention the compounds of Table I inhibit β-lactamases selected from class A, class C or class D β-lactamases. In one aspect of the invention the compound of Formula
inhibits β-lactamases selected from class A, class C or class D β-lactamases. In one aspect of the invention the compound of Formula
inhibits 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 (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) inhibit KPC β-lactamases. In a preferred aspect of the invention, the compounds of Formula I inhibit KPC β-lactamases. In a preferred aspect of the invention, the compounds of Formula A-I inhibit KPC β-lactamases. In a preferred aspect of the invention, the compounds of Formula II inhibit KPC β-lactamases. In a preferred aspect of the invention, the compounds of Formula A-II inhibit KPC β-lactamases. More preferably the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) inhibit KPC-2 or KPC-3 β-lactamases. More preferably the compounds of Formula I inhibit KPC-2 or KPC-3 β-lactamases. More preferably the compounds of Formula A-I inhibit KPC-2 or KPC-3 β-lactamases. More preferably the compounds of Formula II inhibit KPC-2 or KPC-3 β-lactamases. More preferably the compounds of Formula A-II inhibit KPC-2 or KPC-3 β-lactamases. In one aspect of the invention, the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) inhibit KPC-2 or KPC-3 β-lactamases in clinical strains (FIG. 2, Table II). 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). In one aspect of the invention, the compounds of Formula A-I inhibit KPC-2 or KPC-3 β-lactamases in clinical strains (FIG. 2, Table II). In one aspect of the invention, the compounds of Formula II inhibit KPC-2 or KPC-3 β-lactamases in clinical strains (FIG. 2, Table II). In one aspect of the invention, the compounds of Formula A-II 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. In a preferred aspect of the invention, the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) inhibit OXA-15 β-lactamases. In a preferred aspect of the invention, the compounds of Formula I inhibit OXA-15 β-lactamases. In a preferred aspect of the invention, the compounds of Formula A-I inhibit OXA-15 β-lactamases. In a preferred aspect of the invention, the compounds of Formula II inhibit OXA-15 β-lactamases. In a preferred aspect of the invention, the compounds of Formula A-II inhibit OXA-15 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 of 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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, the sMIC value for the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) is 8 μg/mL or less. In a more preferred aspect of the invention, the sMIC value for the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) is 4 μg/mL to 8 μg/mL. In an even more preferred aspect of the invention, the sMIC value for the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) is 1 to 2 μg/mL. In a still more preferred aspect of the invention, the sMIC value for the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 value for the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) is 8 μg/mL or less. In a more preferred aspect of the invention, the MIC value for the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) is 4 to 8 μg/mL. In an even more preferred aspect of the invention, the MIC value for the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) is 1 to 2 μg/mL. In a still more preferred aspect of the invention, the MIC value for the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) is 0.2 μg/mL to 0.5 μg/mL.
The compounds of the present invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) are active in potentiating activity of β-lactam antibiotics, in particular, Ceftolozane, against strains expressing class D β-lactamase OXA-15 β-lactamase. Currently marketed BLIs inhibit most of the class A β-lactamases, but poorly inhibit class A KPCβ-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 (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 against bacterial strains that express the class D cephalosporinase OXA-15 are, for example, compounds 603, 604, 611, 614, 618.
In one embodiment, the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 Avibactam for at least one, bacterial strain that expresses KPC β-lactamase and/or show a better spectrum of activity against bacterial strains that express KPC β-lactamases than Avibactam are, for example, compounds 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, and 627.
In another aspect of the invention, the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) have high 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) was measured according to the procedure outlined in Example 37. The compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) have a high binding affinity for the β-lactamase enzyme.
In one embodiment the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) have a binding affinity of 1000-5000 mM⁻¹s⁻¹. Compounds that have a binding affinity of 1000-5000 mM⁻¹s⁻¹are, for example, compound 604 and 608 (Table IV).
In one embodiment the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) have a binding affinity of 100-999 mM⁻¹s⁻¹. Compounds that have a binding affinity of 100-999 mM⁻¹s⁻¹are, for example, compounds 601, 603, 605, 606, 607, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, and 627 (Table IV).
In one embodiment the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) have a binding affinity of 1-99 mM⁻¹s⁻¹. Compounds that have a binding affinity of 1-99 mM⁻¹s⁻¹are, for example, 602 (Table IV).
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).
The compounds of the invention were also shown to be better BLIs than other comparator compounds as shown in FIG. 5.

Pharmaceutical Compositions Comprising the Compounds of The Invention and Use Thereof

Another object of the invention is pharmaceutical compositions or formulations comprising compounds the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), or salts thereof, preferably further comprising a β-lactam antibiotic. In one embodiment of the invention is pharmaceutical compositions or formulations comprising compounds of Formula I, or salts thereof, preferably further comprising a β-lactam antibiotic. In one embodiment of the invention is pharmaceutical compositions or formulations comprising compounds of Formula A-I, or salts thereof, preferably further comprising a β-lactam antibiotic. In one embodiment of the invention is pharmaceutical compositions or formulations comprising compounds of Formula II, or salts thereof, preferably further comprising a β-lactam antibiotic. In one embodiment of the invention is pharmaceutical compositions or formulations comprising compounds of Formula A-II, or salts thereof, preferably further comprising a β-lactam antibiotic. In one embodiment of the invention is pharmaceutical compositions or formulations comprising compounds of Table I. In one embodiment of the invention is pharmaceutical compositions or formulations comprising compounds of Formula
or salts thereof, preferably further comprising a β-lactam antibiotic. In one embodiment of the invention is pharmaceutical compositions or formulations comprising compounds of Formula
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 (e.g. one or more compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II, 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 (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) preferably a compound of Formula A-I or Formula A-II, 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 propylpara-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.
For intravenous (IV) use, the pharmaceutical composition (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) preferably a compound of Formula A-I or Formula A-II, 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 (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) preferably a compound of Formula A-I or Formula A-II, 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 A-I or Formula A-II 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 A-I or Formula A-II 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 A-I or Formula A-II 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 A-I or Formula A-II 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 A-I or Formula A-II 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 A-I or Formula A-II 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 A-I or Formula A-II 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 art-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 the invention, preferably a compound of Formula A-I or Formula A-II 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 the invention, preferably a compound of Formula A-I or Formula A-II 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 the invention, preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) in conjunction with a β-lactam antiobiotic. When a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) is administered in conjunction with a β-lactam antiobiotic, the compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) and the β-lactam antiobiotic can be administered at the same time or different times. When the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) and the β-lactam antiobiotic 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 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 the pharmaceutical composition comprising a compound of Formula A-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 the pharmaceutical composition comprising a compound of Formula II, 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 the pharmaceutical composition comprising a compound of Formula A-II, 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II). 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 Formula I. 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 Formula A-I. 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 Formula II. 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 Formula A-II. 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 Table I. 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 Formula
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 Formula
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 the invention; and
- b. administering to the subject a therapeutically-effective amount of a β-lactam antibiotic.

In one embodiment the compound in step a is a compound of Formula I. In one embodiment the compound in step a is a compound of Formula A-I. In one embodiment the compound in step a is a compound of Formula II. In one embodiment the compound in step a is a compound of Formula A-II. In one embodiment the compound in step a is a compound of Table I. In one embodiment the compound in step a is a compound of Formula A-II. In one embodiment the compound in step a is a compound of Formula
In one embodiment the compound in step a is a compound of Formula
In one embodiment, the β-lactam antibiotic in step b is Ceftolozane or Ceftazidime. In one embodiment the compound in step a is a compound of Formula
and the β-lactam antibiotic in step b is Ceftolozane. In one embodiment the compound in step a is a compound of Formula
and the β-lactam antibiotic in step b is Ceftolozane.
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 therapeutically-effective amount of a β-lactam antibiotic; and
- b. administering to the subject a compound of the invention.

In one embodiment the compound in step b is a compound of Formula I. In one embodiment the compound in step b is a compound of Formula A-I. In one embodiment the compound in step b is a compound of Formula II. In one embodiment the compound in step b is a compound of Formula A-II. In one embodiment the compound in step b is a compound of Table I. In one embodiment the compound in step b is a compound of Formula
In one embodiment the compound in step b is a compound of Formula
In one embodiment, the β-lactam antibiotic in step a is Ceftolozane or Ceftazidime. In one embodiment the compound in step b is a compound of Formula
and the β-lactam antibiotic in step a is Ceftolozane. In one embodiment the compound in step a is b compound of Formula
and the β-lactam antibiotic in step a is Ceftolozane. 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 A-I or Formula A-II in conjunction with a β-lactam antibiotic. In one embodiment the compound is of Formula in
conjunction with a β-lactam antibiotic, preferably Ceftolozane or Ceftazidime, or compositions thereof. In one embodiment the compound is of Formula
in conjunction with a β-lactam antibiotic, preferably Ceftolozane or Ceftazidime, or compositions thereof. 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 opthalmic 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II in conjunction with a β-lactam antibiotic, are administered intravenously, subcutaneously or orally. In one embodiment for administering one or more compounds according to the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or A-II 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. In still another aspect, the bacterial infection is caused by a bacteria that produces multiple β-lactamases. Bacteria that produce multiple β-lactamases may produce β-lactamases of the same class or of different classes (e.g class A and class A or class A and class C or class A and class D etc).
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 BLI that inhibits these β-lactamases with a β-lactam antibiotic could be used to treat infections caused by 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 administered concurrently with compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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, Clclacillin, Talampicillin, Aspoxicillin, Oxacillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Nafcillin and Pivampicillin.
The pharmaceutical compositions, preferably a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably compound of Formula A-I or Formula A-II 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 of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably compound of Formula A-I or Formula A-II in conjunction with a β-lactam antibiotic, may 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, liquid or dry powder inhaler.
Actual dosage levels of active ingredients in the pharmaceutical compositions of one or more compounds according to the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) and a β-lactam antibiotic, both the compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) and the β-lactam antibiotic are active compounds.
The method comprises administering to the subject an effective dose of one or more compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably in conjunction with a β lactam antibiotic. An effective dose of a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II). 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II). In another embodiment, the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) are administered in ratio of 1:4 to 8:1 antibiotic:compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II). 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) or vice versa.
One or more compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II in conjunction with a β-lactam antibiotic, is administered as a single dose per day. In another embodiment, one or more compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I of Formula A-II in conjunction with a β-lactam antibiotic is administered as two equal doses per day. In another embodiment, the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II in conjunction with a β-lactam antibiotic is administered in three equal doses per day. In another embodiment, the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 of the invention and the β-lactam antibiotic 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II in conjunction with a β-lactam antibiotic, are administered for 7 to 56 days. In another embodiment, one or more compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II in conjunction with a β-lactam antibiotic, are administered for 7 to 28 days. In a further embodiment, one or more compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II 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 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II and at least 1 β-lactam antibiotic or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II and at least 1 cephalosporin antibiotic or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition comprising a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II and Ceftolozane or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition comprising a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II and at least 1 carbapenem antibiotic or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition comprising a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II and at least 1 monobactam antibiotic or a pharmaceutically acceptable salt thereof.
The embodiments described herein provide compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II that are novel and active β-lactamase inhibitors. Other embodiments described herein provide novel compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II in conjunction with β-lactam antibiotics for treatment of infections. Further embodiments described herein provide novel compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II that show unexpected activity against β-lactamases that other compounds in the class do not have.

Preparation of Compounds of the Invention

A compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 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
- Py=pyridine
- sat.=saturated
- rt=room temperature
- TBAF=tetrabutylammonium fluride
- 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, 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. Compound 3 can be synthesized following standard isoxazole ring formation chemistry from the chlorooxime intermediate 2d, which can be prepared from ester intermediate 1 (see, e.g., Abele, E.; Lukevics, E. Heterocycles 2000, 53, 2285-2336; Barr, L.; Lincoln, S. F.; Easton, C. J. Chemistry- A European Journal 2006, 12, 8571-8580; Walker, D. G.; Brodfuehrer, P. R.; Brundidge, S. P. Shih, K. M.; Sapino, C. Jr. J. Org. Chem. 1988, 53, 983-991 and references cited therein).
It may be necessary to protect certain functionalities in the molecule depending on the nature of the R¹group. 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.
The benzylic ether protecting group in 3 can be removed via standard hydrogenolysis conditions, such as, but not limited to, Pd/H₂in MeOH or THF or by acid-catalysed hydrolysis, such as, but not limited to, BCl₃in 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 an 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 then be converted to a sodium salt by cation exchange. When a protecting group(s) is 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 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 (y) 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 NH₄HCO₃buffer: 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 I) c

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

Method A

n-BuLi (600 mL, 1.5 mol) was added dropwise to a solution of TMSCHN₂(690 mL, 1.38 mol) in dry THF (3 L) 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 (3×). The combined organic layer was concentrated under reduced pressure 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 0° C. solution of Rh₂(OAc)₄(3.5 g, 7.9 mmol) in DCM (750 mL). The reaction mixture was then stirred at 20° C. overnight and then concentrated under reduced pressure. The crude sample was purified by silica gel column chromatography (5:1 petroleum ether/EtOAc) to afford (5)-1-ten-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×). The combined organic layer was concentrated under reduced pressure 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. The toluene was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (10:1 to 3: lgradient elution 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-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate

NaBH₄(36 g, 1.0 mol) was added in portions to a−40° C. solution of (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (250 g, 0.92 mol) in EtOH (1500 mL). 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 (3×). The combined organic layer was concentrated under reduced pressure 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 0° C. solution of O-benzylhydroxylamine hydrochloride (400 g, 2.51 mol) in pyridine (1500 mL). The reaction mixture was allowed to warm to room temperature then was stirred at 20° C. overnight. The mixture was concentrated under reduced pressure, diluted with DCM and washed with HCl (10%, 3×). The combined organic layer was concentrated under reduced pressure 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 under reduced pressure 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 saturated sodium chloride (3×), concentrated under reduced pressure 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 under reduced pressure. The crude product was adjusted to pH 10 with sat. NaHCO₃solution then extracted with DCM (3×). The combined organic layer was concentrated under reduced pressure 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 0° C. 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). The reaction mixture was allowed to warm to rt. After stirring at rt overnight, the reaction mixture was washed with H₃PO₄(10%), sat. NaHCO₃and saturated NaCl. The combined organic layer was concentrated under reduced pressure 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 (400Mz, 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-carbaldehyde (Intermediate Compound 2b)

LiBH₄(0.54 g, 24.67 mmol) was added to a −10° C. 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). 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 (3×). The combined organic layer was concentrated and purified by silica gel column chromatography (gradient elution 0-100% EtOAc/petroleum ether, 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 0° C. 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). The mixture was stirred at 0° C. for 2 h, and filtered through Celite®. The filtrate was dried over Na₂SO₄and 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 3

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 Na₂SO₄and 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 4

Synthesis of (2S,5R)-2-(5-(aminomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 601)

Step 1: NCS (295 mg, 2.2 mmol) was added to a solution of 6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbaldehyde oxime (560 mg, 2.0 mmol) in dry DCM (15 mL) at rt. Pyridine (one drop) was then added, and the reaction mixture was stirred at rt for 18 hrs. The solution was evaporated to afford (2S,5R)-6-(benzyloxy)-N-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbimidoyl chloride, which was directly used in the next step. ESI-MS (EI⁺, m/z): 274 [M-Cl]⁺.
Step 2: tert-Butyl prop-2-yn-1-ylcarbamate (0.37 g, 2.4 mmol) was added to the solution of (2S,5R)-6-(benzyloxy)-N-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbimidoyl chloride (˜2.0 mmol) in dry DCM (20 mL), followed by the addition of TEA (0.31 mL, 2.2 mmol) in dry DCM (2.0 mL) over a period of 30 minutes. The reaction mixture was stirred at rt overnight, then the mixture was diluted with EtOAc, washed with water and saturated sodium chloride. The organic layer was dried over Na₂SO₄, concentrated and purified by silica gel column chromatography (1:2 petroleum ether/EtOAc) to afford tert-butyl ((3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)isoxazol-5-yl)methyl)carbamate (180 mg, 21% for two steps) as a white solid. ESI-MS (EI⁺, m/z): 429.1 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃): δ 7.45-7.35 (m, 5H), 6.24 (s, 1H), 5.09 (d, J=11.5 Hz, 1H), 4.93 (d, J=11.5 Hz, 1H), 4.58 (d, J=7.5 Hz, 1H), 4.43 (m, 2H), 3.32 (s, 1H), 2.86-2.84 (m, 1H), 2.68 (d, J=11.5 Hz, 1H), 2.34-2.30 (m, 1H), 2.23-2.18 (m, 1H), 2.10-2.04 (m, 1H), 1.82-1.78 (m, 1H), 1.45 (s, 9H).
Step 3: To a solution of tert-butyl (3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazol-5-yl)methylcarbamate (210 mg, 0.5 mmol) in THF (5 mL) was added 10% Pd/C (100 mg). The reaction mixture was then filtered and concentrated to afford tert-butyl ((3-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)isoxazol-5-yl)methyl)carbamate (180 mg, 99%) as a light yellow solid, which was used directly in the next step. ESI-MS (EI⁺, m/z): 339.1 [M+H]⁺.
Step 4: To a solution of tert-butyl (3-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazol-5-yl)methylcarbamate (180 mg, 0.5 mmol) in dry pyridine (2.5 mL) was added SO₃.Py (480 mg, 3.0 mmol). The mixture was stirred at rt for 3 hrs and then concentrated under reduced pressure. The residue was re-dissolved in aqueous NaH₂PO₄(1.5 M, 20 mL) then tetrabutylammonium hydrogensulphate (230 mg, 0.67 mmol) was added. The mixture was stirred at rt for 20 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 3:1 DCM/acetone) to afford tetrabutylammonium (2S,5R)-2-(5-(((tert-butoxycarbonyl)amino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (180 mg, 54%) as a white solid. ESI-MS (EI⁻, m/z): 417.0 [M−H]⁻. ¹H NMR (500 MHz, CDCl₃): δ 6.28 (s, 1H), 4.97 (bs, 1H), 4.54 (d, J=7 Hz, 1H), 4.44-4.30 (m, 2H), 4.35 (m, 1H), 3.35-3.28 (m, 8H), 3.19-3.17 (m, 1H), 2.76 (d, J=11.5 Hz, 1H), 2.36-2.32 (m, 1H), 2.27-2.24 (m, 1H), 2.18-2.12 (m, 1H), 1.87-1.81 (m, 1H), 1.71-1.65 (m, 8H), 1.50-1.47 (m, 17H), 1.01 (t, J=7.0 Hz, 12H).

Step 5, Resin Exchange

Tetrabutylammonium (2S,5R)-2-(5-(tert-butoxycarbonylaminomethyl)-isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo [3.2.1]octan-6-yl sulfate (180 mg, 0.27 mmol) was dissolved in a minimum amount of HPLC grade water (˜10 mL) and passed through a column of 20 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-(5-(((tert-butoxycarbonyl)amino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (109 mg, 92%) was obtained after lyophilization as a white solid. ESI-MS (EI⁻, m/z): 417.0 [M−H]⁻. ¹H NMR (500 MHz, D₂O): δ6.32 (s, 1H), 4.61 (d, J=6.5 Hz, 1H), 4.32 (s, 2H), 4.13 (m, 1H), 3.10-3.08 (m, 1H), 2.91 (d, J=12.5 Hz, 1H), 2.21-2.17 (m, 1H), 2.09-2.02 (m, 2H), 1.86-1.82 (m, 1H), 1.34 (s, 9H).
Step 6: TFA (0.40 mL) was added to a 0° C. solution of sodium (2S,5R)-2-(5-(tert-butoxycarbonylaminomethyl)-isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (54 mg, 0.12 mmol) in dry DCM (1.2 mL). The reaction mixture was stirred at 0° C. for 30 minutes to 1 h and then diluted with diethyl ether. The precipitate was collected via centrifugation, washed with ether (3×) and further dried under high vacuum to provide (2S,5R)-2-(5-(aminomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate as a TFA salt (˜30 mg). ESI-MS (EI⁺, m/z): 319.2. The TFA salt (˜30 mg) was further purified by prep-HPLC using ammonium formate buffer to provide (2S,5R)-2-(5-(aminomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (601, 10 mg, 26%) as a white solid. ESI-MS (EI⁺, m/z): 319.21. ¹H NMR (300 MHz, D₂O) δ 6.61 (s, 1H), 4.68-4.65 (m, 1H), 4.35 (s, 2H), 4.17-4.14 (m, 1H), 3.14-3.10 (m, 1H), 2.95-2.91 (m, 1H), 2.31-1.80 (m, 4H).

Example 5

Synthesis of (2S,5R)-2-(5-(guanidinomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 603)

Step 1: Synthesis of 1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne

Di-tert-butyl (1H-pyrazol-1-yl)methanediylidenedicarbamate (1.71 g, 5.5 mmol) was added to a 0° C. solution of prop-2-yn-1-amine (275 mg, 5.0 mmol) and TEA (1.5 g, 15 mmol) in MeOH (40 mL). The reaction mixture was stirred at 0° C. for 3 hrs and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (1:10 EtOAc/Hexanes) to give 1-(2,3-bis(tert-butoxycarbonyl)guanidino) prop-2-yne (1.4 g, 93%) as a white solid. ESI-MS (EI⁺, m/z): 298.1 [M+H]⁺.

Step 2: Synthesis of (2S,5R)-6-(benzyloxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one

1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne (362 mg, 1.22 mmol) was added to the solution of (2S,5R)-6-(benzyloxy)-N-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbimidoyl chloride (473 mg, 1.53 mmol) in dry DCM (20 mL) at rt, followed by dropwise addition of TEA (0.16 g, 1.53 mmol). The mixture was stirred at rt for 2 hrs and then concentrated. The residue was purified by silica gel column chromatography (gradient elution 1:15 to 1:5 EtOAc/Hexanes) to give (2S,5R)-6-(benzyloxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (190 mg, 22% for two steps) as a white solid. ESI-MS (EI⁺, m/z): 571.2 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃): δ 11.49 (s, 1H), 8.86 (bs, 1H), 7.47-7.34 (m, 5H), 6.32 (s, 1H), 5.09 (d, J=11.5 Hz, 1H), 4.93 (d, J=12.0 Hz, 1H), 4.87-4.76 (m, 2H), 4.59 (d, J=7.0 Hz, 1H), 3.36 (s, 1H), 2.87-2.85 (m, 1H), 2.70 (d, J=12 Hz, 1H), 2.35-2.30 (m, 1H), 2.24-2.18 (m, 1H), 2.10-2.05 (m, 1H), 1.81-1.78 (m, 1H), 1.50 (s, 18H).

Step 3: Synthesis of (2S,5R)-6-(hydroxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one

To a solution of (2S,5R)-6-(benzyloxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (240 mg, 0.42 mmol) in THF (20 mL) was added 10% Pd/C (120 mg). The mixture was stirred under H₂atmosphere at rt for 1.5 hrs. The reaction mixture was then filtered and concentrated to afford (2S,5R)-6-(hydroxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (200 mg, 99%) as white solid, which was directly used in the next step. ESI-MS (EI⁺, m/z): 481.2 [M+H]⁺.

Step 4: Synthesis of tetrabutylammonium(2S,5R)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate

To a solution of (2S,5R)-6-(hydroxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (200 mg, 0.42 mmol) in dry pyridine (2.5 mL) was added SO₃.Py (400 mg, 2.5 mmol). The mixture was stirred at rt for 3 hrs then concentrated under reduced pressure. The residue was re-dissolved in aqueous NaH₂PO₄(1.5 M, 20 mL). Tetrabutylammonium hydrogensulphate (200 mg, 0.58 mmol) was added. The mixture was stirred at rt for 20 minutes then was 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 give tetrabutylammonium (2S,5R)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (220 mg, 66%) as a white solid. ESI-MS (EI⁻, m/z): 559.0 [M−H]⁻. ¹H NMR (500 MHz, CDCl₃): δ 11.48 (s, 1H), 8.78 (bs, 1H), 6.34 (s, 1H), 4.77-4.76 (m, 2H), 4.55 (d, J=7.5 Hz, 1H), 4.35 (m, 1H), 3.38-3.28 (m, 8H), 3.20-3.18 (m, 1H), 2.78 (d, J=11.5 Hz, 1H), 2.37-2.30 (m, 1H), 2.28-2.24 (m, 1H), 2.17-2.11 (m, 1H), 1.88-1.82 (m, 1H), 1.71-1.63 (m, 8H), 1.50-1.44 (m, 26H), 1.01 (t, J=7.0 Hz, 12H).
Step 5: TFA (2.20 mL) was added to a 0° C. solution of tetrabutylammonium (2S,5R)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (465 mg, 0.60 mmol) in dry DCM (6.60 mL). The reaction mixture was allowed to warm to rt. The reaction mixture was stirred at rt for 2 hrs and then diluted with ether. The precipitate was collected via centrifugation, washed with diethyl ether (3×) and further dried under high vacuum. The crude TFA salt was purified by prep-HPLC using ammonium formate buffer to provide (2S,5R)-2-(5-(guanidinomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (603, 60 mg, 20%) as a white solid. ESI-MS (EI⁺, m/z): 361.2. ¹H NMR (300 MHz, D₂O/DMSO-d6) δ 6.58 (s, 1H), 4.74-4.71 (m, 1H), 4.67 (s, 2H), 4.23 (br s, 1H), 3.22-3.17 (m, 1H), 2.99 (d, J=12.0 Hz, 1H), 2.36-2.26 (m, 1H), 2.24-2.10 (m, 2H), 2.02-1.91 (m, 1H).

Example 6

Synthesis of (2S,5R)-2-(5-(2-aminoethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 602)

Following Steps 1-6 in Example 4, replacing tert-butyl prop-2-yn-1-ylcarbamate in Step 2 with tert-butyl but-3-yn-1-ylcarbamate; (2S,5R)-2-(5-(2-aminoethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate TFA salt (602, 8 mg) was obtained as a light yellow solid. ESI-MS (EI⁺, m/z): 333.2. ¹H NMR (300 MHz, D₂O) δ 6.39 (s, 1H), 4.62 (d, J=6.8 Hz, 1H), 4.16-4.12 (m, 1H), 3.32 (t, J=7.1 Hz, 3H), 3.25-3.04 (m, 4H), 2.97-2.93 (m, 1H), 2.30-1.79 (m, 4H).

Example 7

Synthesis of (2S,5R)-2-(5-(2-guanidinoethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 604)

Following Steps 1-5 in Example 5, replacing prop-2-yn-1-amine in Step 1 with but-3-yn-1-amine; (2S,5R)-2-(5-(2-guanidinoethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (604, 48 mg) was obtained as a light yellow solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 375.1. ¹H NMR (300 MHz, D₂O/DMSO-d6) δ 6.33 (s, 1H), 4.62 (d, J=6.6 Hz, 1H), 4.14 (br s, 1H), 3.51 (t, J=6.4 Hz, 2H), 3.15-3.07 (m, 1H), 3.05 (t, J=6.4 Hz, 2H), 2.88 (d, J=6.4 Hz, 1H), 2.26-1.99 (m, 3H), 1.85-1.80 (m, 1H).

Example 8

Synthesis of (2S,5R)-7-oxo-2-(5-(piperidin-4-yl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 606)

Following Steps 1-6 in Example 4, replacing tert-butyl prop-2-yn-1-ylcarbamate in Step 2 with tert-butyl 4-ethynylpiperidine-1-carboxylate, (2S,5R)-7-oxo-2-(5-(piperidin-4-yl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (606, 35 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 371.3.

Example 9

Synthesis of (2S,5R)-2-(5-(1-carbamimidoylpiperidin-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicycl[3.2.1]octan-6-yl hydrogen sulfate (Compound 607)

Following Steps 1-5 in Example 5, replacing prop-2-yn-1-amine in Step 1 with 4-ethynylpiperidine; (2S,5R)-2-(5-(1-carbamimidoylpiperidin-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate TFA salt (607, 32 mg) was obtained. ESI-MS (EI⁺, m/z): 415.2.

Example 10

Synthesis of (2S,5R)-2-(5-((2-aminoethyl)carbamoyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 605)

Following Steps 1-6 in Example 4, replacing tert-butyl prop-2-yn-1-ylcarbamate in Step 2 with tert-butyl (2-propiolamidoethyl)carbamate, (2S,5R)-2-(5-((2-aminoethyl)carbamoyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate TFA salt (605, 20 mg) was obtained as a light yellow solid. ESI-MS (EI⁺, m/z): 376.1. ¹H NMR (300 MHz, D₂O) δ 7.05 (s, 1H), 4.70 (m, 1H, overlapped with D₂O peak) 4.16 (br s, 1H), 3.66 (t, J=5.8 Hz, 2H), 3.19 (t, J=5.8 Hz, 2H), 3.16-3.08 (m, 1H), 2.93 (d, J=12.0 Hz, 1H), 2.31-2.24 (m, 1H), 2.16-2.04 (m, 2H), 1.94-1.85 (m, 1H).

Example 11

Synthesis of (2S,5R)-2-(5-((2-guanidinoethyl)carbamoyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 608)

Following Steps 1-5 in Example 5, replacing prop-2-yn-1-amine in Step 1 with N-(2-aminoethyl)propiolamide; (2S,5R)-2-(5-((2-guanidinoethyl)carbamoyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (608, 25 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 418.12. ¹H NMR (300 MHz, D₂O) δ 4.79-4.76 (m, 1H), 4.16 (br s, 1H), 4.47-4.32 (m, 4H), 4.17-4.11 (m, 2H), 3.19-3.16 (m, 1H), 2.92 (d, J=12.0 Hz, 1H), 2.32-2.22 (m, 1H), 2.20-2.06 (m, 2H), 1.97-1.88 (m, 1H).

Example 12

Synthesis of (2S,5R)-7-oxo-2-(5-(2-(piperidin-4-ylamino)ethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 609)

Synthetic Scheme:

Procedures and Characterization:

Step 1: Synthesis of tert-butyl 4-(but-3-ynylamino)piperidine-1-carboxylate

A mixture of but-3-yn-1-amine hydrochloride (26.4 g, 0.25 mol), K₂CO₃(17.4 g, 0.13 mol), and tert-butyl 4-oxopiperidine-1-carboxylate (41.8 g, 0.21 mol) in MeOH (500 mL) was stirred at rt for 5 hrs. Then, NaBH(OAc)₃(133.6 g, 0.63 mol) was added and the suspension was stirred at rt for 17 hrs. The crude reaction mixture was used directly in the next step. ESI-MS (EI⁺, m/z): 253.2 [M+H]⁺.

Step 2: Synthesis of tert-butyl 4-(but-3-ynyl(tert-butoxycarbonyl)amino)piperidine-1-carboxylate

The reaction mixture in Step 1 was cooled to 0° C. and Et₃N (88.0 mL, 0.63 mol) and (Boc)₂O (91.6 g, 0.42 mol) were added. The solution was allowed to warm to rt, then was stirred at rt for 17 hrs. The reaction mixture was concentrated, and then EtOAc (800 mL) was added. The organic layer was washed with saturated sodium chloride (3×), dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 0˜10% EtOAC/petroleum ether) to afford tert-butyl 4-(but-3-ynyl(tert-butoxycarbonyl)amino)piperidine-1-carboxylate (66.0 g, 90%) as a colorless oil. ESI-MS (EI⁺, m/z): 353.2 [M+H]⁺.
Step 3-6: Following Steps 2-5 in Example 5, replacing 1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne in Step 2 with tert-butyl 4-(but-3-yn-1-yl(tert-butoxycarbonyl)amino)piperidine-1-carboxylate; (2S,5R)-7-oxo-2-(5-(2-(piperidin-4-ylamino)ethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (609, 220 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 416.4. ¹H NMR (300 MHz, D₂O) δ 6.29 (s, 1H), 4.59 (d, J=6.3 Hz, 1H), 4.12 (s, 1H), 3.39 (d, J=13.3 Hz, 2H), 3.10-2.91 (m, 9H), 2.23-1.95 (m, 5H), 1.87-1.76 (m, 1H), 1.54-1.40 (m, 2H).

Example 13

Synthesis of (2S,5R)-2-(5-(2-((1-carbamimidoylpiperidin-4-yl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 610)

Synthetic Scheme:

Procedures and Characterization:

Step 1: Synthesis of piperidin-4-one

A mixture of tert-butyl 4-oxopiperidine-1-carboxylate (10.0 g, 50 mmol), TFA (20 mL) and CH₂Cl₂(100 mL) was stirred at rt for 1 h. The reaction mixture was then concentrated to afford piperidin-4-one (15.0 g, 100%) as a colorless oil. ¹H NMR: (500 MHz, DMSO-d₆): δ 2.51 (m, 4H), 3.43 (m, 4H), 9.03 (m, 2H).

Step 2: Synthesis of di-tert-butyl (4-oxopiperidin-1-yl)methylenedicarbamate

(Z)-di-tert-butyl (1H-imidazol-1-yl)methylenedicarbamate (16.3 g, 52.5 mmol) was add to a 0° C. solution of piperidin-4-one (15 g, 50 mmol) and Et₃N (25.4 g, 251 mmol) in MeOH (200 mL). The reaction mixture was allowed to warm to rt then was stirred at rt for 17 hrs. The reaction mixture was concentrated, and then EtOAc (300 mL) was added. The organic layer was washed with saturated sodium chloride (3×), dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 0˜20% EtOAc/petroleum ether) to afford di-tert-butyl (4-oxopiperidin-1-yl)methylenedicarbamate (12.8 g, 75%) as a white solid. ESI-MS (EI⁺, m/z): 342.1 [M+H]⁺.

Step 3: Synthesis of di-tert-butyl(4-(but-3-ynylamino)piperidin-1-yl)methanediylidenedicarbamate

A mixture of but-3-yn-1-amine hydrochloride (3.0 g, 28.4 mmol), K₂CO₃(2.0 g, 14.2 mmol), MeOH (150 mL), di-tert-butyl (4-oxopiperidin-1-yl)methylene-dicarbamate (8.1 g, 23.7 mmol) and 4 Å molecular sieve (6.0 g) was stirred at rt for 3 hrs. Then, NaBH(OAc)₃(15.1 g, 71.1 mmol) was added, and the suspension was stirred at 70° C. for 1 h. The reaction mixture was filtered and concentrated to afford di-tert-butyl(4-(but-3-ynylamino)piperidin-1-yl)methanediylidenedicarbamate as a yellow oil (˜15.0 g), which was used directly in the next step.

Step 4: Synthesis of di-tert-butyl (4-(but-3-ynyl(tert-butylcarbamate))piperidin-1-yl)methanedi-ylidenedicarbamate

A mixture of di-tert-butyl(4-(but-3-ynylamino)piperidin-1-yl) methanediylidenedicarbamate (˜15.0 g), di-tert-butyl dicarbonate (10.3 g, 47.4 mmol), aq. NaHCO₃(60 mL) in THF (120 mL) was stirred at rt for 2 hrs. The reaction mixture was then concentrated and extracted with EtOAc (3×). The combined organics were dried (Na₂SO₄) and concentrated. The residue was purified by silica gel column chromatography (gradient elution 0˜20% EtOAc/petroleum ether) to afford di-tert-butyl(4-(but-3-ynyl(tert-butylcarbamate))piperidin-1-yl)methanedi-ylidenedicarbamate as a colorless oil (7.1 g, 60%). ESI-MS (EI⁺, m/z): 495.3 [M+H]⁺.

Step 5-8

Following Steps 2-5 in Example 5, replacing 1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne in Step 2 with tert-butyl (1-(N,N′-bis(tert-butoxycarbonyl)carbamimidoyl)piperidin-4-yl)(but-3-yn-1-yl)carbamate;(2S,5R)-2-(5-(2-((1-carbamimidoylpiperidin-4-yl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (610, 502 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 458.0. ¹H NMR (300 MHz, D₂O) δ 6.35 (s, 1H), 4.59 (d, J=7.0 Hz, 1H), 4.12 (s, 1H), 3.85 (d, J=14.5 Hz, 2H), 3.40-3.30 (m, 3H), 3.20-2.90 (m, 6H), 2.20-1.75 (m, 6H), 1.62-1.46 (m, 2H).
The compounds described in Examples 14-33 were prepared as described in the reation schemes following similar procedures of Examples 1-13

Example 14

Synthesis of (2S,5R)-2-(5-((R*)-2-guanidino-1-hydroxyethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 611)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((R*)-2-guanidino-1-hydroxyethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (611, 46 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 391.0 . ¹H NMR (300 MHz, D₂O) δ 6.46 (s, 1H), 5.04 (t, J=5.2 Hz, 1H), 4.62 (d, J=6.7 Hz, 1H), 4.11 (s, 1H), 3.56 (d, J=5.2 Hz, 2H), 3.08 (d, J=11.6 Hz, 1H), 2.85 (d, J=12.1 Hz, 1H), 2.24-2.14 (m, 1H), 2.12-2.00 (m, 2H), 1.86-1.76 (m, 1H).

Example 15

Synthesis of (2S,5R)-2-(5-((S*)-2-guanidino-1-hydroxyethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 612)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((S*)-2-guanidino-1-hydroxyethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (612, 72 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 391.0. ¹H NMR (300 MHz, D₂O) δ 6.47 (s, 1H), 5.04 (t, J=5.4 Hz, 1H), 4.63 (d, J=6.2 Hz, 1H), 4.12 (s, 1H), 3.65-3.49 (m, 2H), 3.09 (br d, J=12.7 Hz, 1H), 2.87 (d, J=12.2 Hz, 1H), 2.20-1.98 (m, 3H), 1.86-1.78 (m, 1H).

Example 16

Synthesis of (2S,5R)-2-(5-((1-guanidinocyclopropyl)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 613)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1-guanidinocyclopropyl)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (613, 28 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 401.3. ¹H NMR (300 MHz, D₂O) δ 6.35 (s, 1H), 4.61 (d, J=6.6 Hz, 1H), 4.12 (br s, 1H), 3.11-3.09 (m, 1H), 3.08-2.98 (m, 2H), 2.89 (d, J=12.0 Hz, 1H), 2.20-2.00 (m, 3H), 1.89-1.78 (m, 1H), 0.97-0.95 (m, 4H).

Example 17

Synthesis of (2S,5R)-2-(5-((1-methylguanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 614)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1-methylguanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (614, 12 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 375.1. ¹H NMR (300 MHz, D₂O with coupe of drops of DMSO-d6) δ 6.55 (s, 1H), 4.73 (s, 2H), 4.64 (d, J=6.0 Hz, 1H), 4.14 (br s, 1H), 3.15-3.07 (m, 1H), 3.06 (s, 3H), 2.89 (d, J=12.2 Hz, 1H), 2.26-2.20 (m, 1H), 2.15-2.09 (m, 2H), 1.92-1.85 (m, 1H).

Example 18

Synthesis of (2S,5R)-2-(5-(3-guanidinopropyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 615)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(3-guanidinopropyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (615, 18 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 389.1. ¹H NMR (300 MHz, D₂O with couple of drops of DMSO-d6) δ 6.28 (s, 1H), 4.57 (d, J=6.5 Hz, 1H), 4.11 (s, 1H), 3.17 (t, J=6.7 Hz, 2H), 3.07 (br d, J=11.5 Hz, 1H), 2.88 (d, J=12.4 Hz, 1H), 2.83 (t, J=6.7 Hz, 2H), 2.22-1.75 (m, 6H).

Example 19

Synthesis of (2S,5R)-2-(5-((1s,3R)-3-aminocyclobutyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 616)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1s,3R)-3-aminocyclobutyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (616, 7 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 359.0. ¹H NMR (300 MHz, DMSO) δ 6.43 (s, 1H), 4.46 (d, J=6.1 Hz, 1H), 4.02 (br s, 1H), 3.75-3.68 (m, 1H), 3.51-3.45 (m, 1H), 2.94-2.85 (m, 1H), 2.72-2.60 (m, 3H), 2.36-2.30 (m, 2H), 2.15-1.95 (m, 1H), 1.99-1.90 (m, 2H), 1.78-1.72 (m, 1H).

Example 20

Synthesis of (2S,5R)-2-(5-((1s,3R)-3-guanidinocyclobutyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 617)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1s,3R)-3-guanidinocyclobutyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (617, 37 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 401.0. ¹H NMR (300 MHz, DMSO) δ 8.11 (br s, 1H), 7.14 (brs, 4H), 6.41 (s, 1H), 4.45 (d, J=6.3 Hz, 1H), 4.02 (br s, 2H), 3.40-3.30 (m, 1H), 2.91 (br d, J=10.3 Hz, 1H), 2.80-2.63 (m, 3H), 2.30-2.10 (m, 3H), 2.00-1.85 (m, 2H), 1.82-1.66 (m, 1H).

Example 21

Synthesis of (2S,5R)-2-(5-(azetidin-3-ylmethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 618,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(azetidin-3-ylmethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (618, 75 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 359.0. ¹H NMR (300 MHz, D₂O) δ 6.23 (s, 1H), 4.56 (d, J=6.6 Hz, 1H), 4.15-4.08 (m, 2H), 3.93-3.82 (m, 2H), 3.44 (dd, J=14.3, 7.2 Hz, 1H), 3.28 (dt, J=15.9, 7.9 Hz, 1H), 3.10-3.03 (m, 3H), 2.87 (d, J=12.1 Hz, 1H), 2.21-1.94 (m, 3H), 1.84-1.76 (m, 1H).

Example 22

Synthesis of (2S,5R)-2-(5-((1-carbamimidoylazetidin-3-yl)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 619,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1-carbamimidoylazetidin-3-yl)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (619, 45 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 401.0. ¹H NMR (300 MHz, DMSO) δ 7.28 (br s, 4H), 6.36 (s, 1H), 4.45 (d, J=6.8 Hz, 1H), 4.18 (t, J=8.4 Hz, 2H), 4.02 (br s, 1H), 3.86-3.71 (m, 2H), 3.16 (d, J=7.0 Hz, 2H), 3.09-3.04 (m, 1H), 2.90 (br d, J=8.7 Hz, 1H), 2.67 (d, J=11.8 Hz, 1H), 2.16-2.09 (m, 1H), 2.00-1.90 (m, 2H), 1.80-1.72 (m, 1H).

Example 23

Synthesis of (2S,5R)-2-(5-(2-((2-aminoethyl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 620)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(2-((2-aminoethyl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (620, 20 mg, as formate salt) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 376.3. ¹H NMR (300 MHz, D₂O) δ 8.32 (s, 1H), 6.35 (s, 1H), 4.59 (d, J=6.5 Hz, 1H), 4.11 (br s, 1H), 3.34 (t, J=6.8 Hz, 2H), 3.30-3.13 (m, 6H), 3.11-3.03 (m, 1H), 2.91 (d, J=12.2 Hz, 1H), 2.21-1.98 (m, 3H), 1.88-1.78 (m, 1H).

Example 24

Synthesis of (2S,5R)-2-(5-(2-((2-guanidinoethl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 621,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(2-((2-guanidinoethyl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (612, 15 mg, as formate salt) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 418.0. ¹H NMR (300 MHz, D₂O) δ 8.28 (s, 1H), 6.30 (s, 1H), 4.55 (d, J=5.9 Hz, 1H), 4.07 (br s, 1H), 3.40 (t, J=5.9 Hz, 2H), 3.25 (t, J=6.1 Hz, 2H), 3.14-3.01 (m, 5H), 2.87 (d, J=12.1 Hz, 1H), 2.16-2.09 (m, 1H), 2.07-1.92 (m, 2H), 1.83-1.74 (m, 1H).

Example 25

Synthesis of (2S,5R)-2-(5-((azetidin-3-ylamino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 622,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((azetidin-3-ylamino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (622, 267 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 374.0. ¹H NMR (400 MHz, D₂O) δ 6.55 (s, 1H), 4.66 (d, J=6.6 Hz, 1H), 4.29-4.10 (m, 5H), 4.10-3.98 (m, 2H), 3.12 (d, J=12.0 Hz, 1H), 2.91 (d, J=12.1 Hz, 1H), 2.30-2.18 (m, 1H), 2.18-1.99 (m, 2H), 1.97-1.78 (m, 1H).

Example 26

Synthesis of (2S,5R)-2-(5-(2-(azetidin-3-ylamino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 623,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(2-(azetidin-3-ylamino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (623, 41 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 388.0.

Example 27

Synthesis of (2S,5R)-2-(5-((azetidin-3-yloxy)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 624)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((azetidin-3-yloxy)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (624, 349 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 375.2. ¹H NMR (300 MHz, D₂O) δ 6.51 (s, 1H), 4.64 (s, 2H), 4.62 (d, J=7.2 Hz, 1H), 4.56-4.52 (m, 1H), 4.20 (dd, J=12.5, 6.7 Hz, 2H), 4.11 (br s, 1H), 3.93 (dd, J=12.3, 5.2 Hz, 2H), 3.08 (br d, J=12.4 Hz, 1H), 2.87 (d, J=12.2 Hz, 1H), 2.22-2.14 (m, 1H), 2.12-2.00 (m, 2H), 187-1.78 (m, 1H).

Example 28

Synthesis of (2S,5R)-2-(5-(1-methylpyridin-1-ium-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (Compound 625)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(1-methylpyridin-1-ium-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (625, 20 mg) was obtained as a white solid. ESI-MS (EI⁻, m/z): 379.0. ¹H NMR (400 MHz, d6-DMSO) δ 9.15 (d, J=5.2 Hz, 2H), 8.60 (d, J=5.2 Hz, 2H), 7.86 (s, 1H), 4.66 (d, J=5.6 Hz, 1H), 4.36 (s, 3H), 4.07 (br s, 1H), 3.00-2.98 (m, 1H), 2.79 (d, J=12.2 Hz, 1H), 2.24-2.20 (m, 1H), 2.10-2.00 (m, 2H), 1.82-1.78 (m, 1H).

Example 29

Synthesis of (2S,5R)-2-(5-(1,1-dimethylpiperidin-1-ium-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (Compound 626)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(1,1-dimethylpiperidin-1-ium-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (626, 86 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁻, m/z): 399.2. ¹H NMR (400 MHz, CF₃COOD): δ 7.24 (s, 1H), 6.03 (d, J=6.8 Hz, 1H), 5.44 (br s, 1H), 4.76-4.72 (m, 1H), 4.51 (d, J=12.2 Hz, 1H), 4.26-4.12 (m, 4H), 3.99-3.92 (m, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.26-3.22 (m, 1H), 3.21-2.85 (m, 7H).

Example 30

Synthesis of (2S,5R)-7-oxo-2-(5-(piperidin-4-ylmethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 627)

Synthetic Scheme:

Characterization:

(2S,5R)-7-oxo-2-(5-(piperidin-4-ylmethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (627, 31 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 387.0.

Example 31

Synthesis of (2S,5R)-2-(5-(azetidin-3-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 628)

Characterization:

(2S,5R)-2-(5-(azetidin-3-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (628, 15 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 345.0. ¹H NMR (300 MHz, D₂O) δ 6.48 (s, 1H), 4.62 (d, J=5.2 Hz, 1H), 4.47-4.22 (m, 5H), 4.15-4.12 (m, 1H), 3.17-3.03 (m, 1H), 2.91 (d, J=12.0 Hz, 1H), 2.24-2.17 (m, 1H), 2.12-2.00 (m, 2H), 1.90-1.80 (m, 1H). CB-606,122.

Example 32

Synthesis of (2S,5R)-2-(5-(2-(azetidin-3-yl)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 629)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(2-(azetidin-3-yl)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (629, 34 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z): 373.0. ¹H NMR (300 MHz, DMSO) δ 8.33 (br s, 2H), 6.33 (s, 1H), 4.45 (d, J=6.4 Hz, 1H), 4.01 (br s, 1H), 3.92 (t, J=9.0 Hz, 2H), 3.61 (t, J=9.0 Hz, 2H), 3.40-3.26 (m, 1H), 2.94-2.88 (m, 1H), 2.85-2.70 (m, 4H), 2.16-2.08 (m, 1H), 2.06-1.87 (m, 3H), 1.84-1.67 (m, 1H).

Example 33

Synthesis of (2S,5R)-7-oxo-2-(5-(pyrrolidin-3-ylmethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 630)

Synthetic Scheme:

Characterization:

(2S,5R)-7-oxo-2-(5-(pyrrolidin-3-ylmethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (630, 9 mg) was obtained as a white solid after prep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁻, m/z): 371.0. ¹H NMR (300 MHz, D₂O) δ 6.29 (s, 1H), 4.59 (d, J=6.6 Hz, 1H), 4.12 (br s, 1H), 3.43 (dd, J=7.6, 5.0 Hz, 1H), 3.39-3.29 (m, 1H), 3.26-3.14 (m, 1H), 3.12-3.03 (m, 1H), 2.97-2.85 (m, 4H), 2.74-2.61 (m, 1H), 2.20-2.00 (m, 4H), 1.87-1.78 (m, 1H), 1.74-1.60 (m, 1H).

Example 34

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′-cgggatccctagagaataggaacttcgg-3′, SEQ ID 4) of the resistance gene. The two PCR products, pBR322 ΔNdeI and cat were ligated together generating pBR-CBST (pBR322ΔNdeI 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 pBR322ΔNdeI 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
β-Lactamase	Name & SEQ. ID of	β-		Number of
Expressing	plasmids producing	Lactamase	Species Origin of	β-Lactamase
Strain	β-Lactamase	Class	β-Lactamase Gene	Gene Sequence

KPC-2	pBR-CBST-KPC-2	A	K. pneumoniae	EU784136
	SEQ ID
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

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)

	pBR-CBST-cat
	SEQ ID 5
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGGAGAAAAAAATCACTG

	GATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTT

	TGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTT

	CAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGC

	ACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAA

	TGCTCATACGGAATTTCGTATGGCAATGAAAGACGGTGAGCTGGTG

	ATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAA

	CTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCG

	GCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAA

	AACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCG

	TCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGT

	GGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGCAAA

	TATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGG

	TTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAA

	TGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAGTG

	GCAGGGCGGGGCGTAAGGCGCGCCATTTAAATGAAGTTCCTATTCC

	GAAGTTCCTATTCTCTAGggatccctgtcagaccaagtttactcat

	atatactttagattgatttaaaacttcatttttaatttaaaaggat

	ctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa

	cgtgagttttcgttccactgagcgtcagaccccgtagaaaagatca

	aaggatcttcttgagatcctttttttctgcgcgtaatctgctgctt

	gcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggat

	caagagctaccaactctttttccgaaggtaactggcttcagcagag

	cgcagataccaaatactgtccttctagtgtagccgtagttaggcca

	ccacttcaagaactctgtagcaccgcctacatacctcgctctgcta

	atcctgttaccagtggctgctgccagtggcgataagtcgtgtctta

	ccgggttggactcaagacgatagttaccggataaggcgcagcggtc

	gggctgaacggggggttcgtgcacacagcccagcttggagcgaacg

	acctacaccgaactgagatacctacagcgtgagctatgagaaagcg

	ccacgcttcccgaagggagaaaggcggacaggtatccggtaagcgg

	cagggtcggaacaggagagcgcacgagggagcttccagggggaaac

	gcctggtatctttatagtcctgtcgggtttcgccacctctgacttg

	agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaa

	aaacgccagcaacgcggcctttttacggttcctggccttttgctgg

	ccttttgctcacatgttctttcctgcgttatcccctgattctgtgg

	ataaccgtattaccgcctttgagtgagctgataccgctcgccgcag

	ccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagag

	cgcctgatgcggtattttctccttacgcatctgtgcggtatttcac

	accgcatttggtgcactctcagtacaatctgctctgatgccgcata

	gttaagccagtatacactccgctatcgctacgtgactgggtcatgg

	ctgcgccccgacacccgccaacacccgctgacgcgccctgacgggc

	ttgtctgctcccggcatccgcttacagacaagctgtgaccgtctcc

	gggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcg

	cgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattc

	acagatgtctgcctgttcatccgcgtccagctcgttgagtttctcc

	agaagcgttaatgtctggcttctgataaagcgggccatgttaaggg

	cggttttttcctgtttggtcactgatgcctccgtgtaagggggatt

	tctgttcatgggggtaatgataccgatgaaacgagagaggatgctc

	acgatacgggttactgatgatgaacatgcccggttactggaacgtt

	gtgagggtaaacaactggcggtatggatgcggcgggaccagagaaa

	aatcactcagggtcaatgccagcgcttcgttaatacagatgtaggt

	gttccacagggtagccagcagcatcctgcgatgcagatccggaaca

	taatggtgcagggcgctgacttccgcgtttccagactttacgaaac

	acggaaaccgaagaccattcatgttgttgctcaggtcgcagacgtt

	ttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcat

	tctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaa

	cgacaggagcacgatcatgcgcacccgtggccaggacccaacgctg

	cccgagatgcgccgcgtgcggctgctggagatggcggacgcgatgg

	atatgttctgccaagggttggtttgcgcattcacagttctccgcaa

	gaattgattggctccaattcttggagtggtgaatccgttagcgagg

	tgccgccggcttccattcaggtcgaggtggcccggctccatgcacc

	gcgacgcaacgcggggaggcagacaaggtatagggcggcgcctaca

	atccatgccaacccgttccatgtgctcgccgaggcggcataaatcg

	ccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagc

	cgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgc

	ctggacagcatggcctgcaacgcgggcatcccgatgccgccggaag

	cgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaa

	cgccagcaagacgtagcccagcgcgtcggccgccatgccggcgata

	atggcctgcttctcgccgaaacgtttggtggcgggaccagtgacga

	aggcttgagcgagggcgtgcaagattccgaataccgcaagcgacag

	gccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatg

	acccagagcgctgccggcacctgtcctacgagttgcatgataaaga

	agacagtcataagtgcggcgacgatagtcatgccccgcgcccaccg

	gaaggagctgactgggttgaaggctctcaagggcatcggtcgacgc

	tctcccttatgcgactcctgcattaggaagcagcccagtagtaggt

	tgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaagga

	gatggcgcccaacagtcccccggccacggggcctgccaccataccc

	acgccgaaacaagcgctcatgagcccgaagtggcgagcccgatctt

	ccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgt

	ggcgccggtgatgccggccacgatgcgtccggcgtagaggattcac

	aggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaa

	gtagcgaagcgagcaggactgggcggcggccaaagcggtcggacag

	tgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagc

	gctagcagcacgccatagtgactggcgatgctgtcggaatggacga

	tatcccgcaagaggcccggcagtaccggcataaccaagcctatgcc

	tacagcatccagggtgacggtgccgaggatgacgatgagcgcattg

	ttagatttcatacacggtgcctgactgcgttagcaatttaactgtg

	ataaactaccgcattaaagcttatcgatgataagctgtcaaacatg

	agaa

	pBR-CBST-KPC-2
	SEQ ID 6
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGTCACTGTATCGCCGTC

	TAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGC

	CACCGCGCTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAA

	CAGGACTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCT

	CAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTG

	CAGCTCATTCAAGGGCTTTCTTGCTGCCGCTGTGCTGGCTCGCAGC

	CAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGCAAAA

	ATGCGCTGGTTCCGTGGTCACCCATCTCGGAAAAATATCTGACAAC

	AAGGCATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAG

	TGATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCG

	GCCGGGCTGACGGCCTTCATGCGCTCTATCGGCGATACCACGTTCC

	GTCTGGACCGCTGGGAGCTGGAGCTGACTCCGCCATCCCAGGCGAT

	GCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACAAA

	AACTGACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTT

	TGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGC

	GCGGCGGTGCCGGCAGACTGGGCAGTCGGAGACAAAACCGGAACCT

	GCGGAGTGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCAC

	TGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCGGGCGCCTAAC

	AAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCGGCTAGAC

	TCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAAggatccctgtc

	agaccaagtttactcatatatactttagattgatttaaaacttcat

	ttttaatttaaaaggatctaggtgaagatcctttttgataatctca

	tgaccaaaatcccttaacgtgagttttcgttccactgagcgtcaga

	ccccgtagaaaagatcaaaggatcttcttgagatcctttttttctg

	cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcgg

	tggtttgtttgccggatcaagagctaccaactctttttccgaaggt

	aactggcttcagcagagcgcagataccaaatactgtccttctagtg

	tagccgtagttaggccaccacttcaagaactctgtagcaccgccta

	catacctcgctctgctaatcctgttaccagtggctgctgccagtgg

	cgataagtcgtgtcttaccgggttggactcaagacgatagttaccg

	gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagc

	ccagcttggagcgaacgacctacaccgaactgagatacctacagcg

	tgagctatgagaaagcgccacgcttcccgaagggagaaaggcggac

	aggtatccggtaagcggcagggtcggaacaggagagcgcacgaggg

	agcttccagggggaaacgcctggtatctttatagtcctgtcgggtt

	tcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg

	gggcggagcctatggaaaaacgccagcaacgcggcctttttacggt

	tcctggccttttgctggccttttgctcacatgttctttcctgcgtt

	atcccctgattctgtggataaccgtattaccgcctttgagtgagct

	gataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtga

	gcgaggaagcggaagagcgcctgatgcggtattttctccttacgca

	tctgtgcggtatttcacaccgcatttggtgcactctcagtacaatc

	tgctctgatgccgcatagttaagccagtatacactccgctatcgct

	acgtgactgggtcatggctgcgccccgacacccgccaacacccgct

	gacgcgccctgacgggcttgtctgctcccggcatccgcttacagac

	aagctgtgaccgtctccgggagctgcatgtgtcagaggttttcacc

	gtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcg

	tggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtcca

	gctcgttgagtttctccagaagcgttaatgtctggcttctgataaa

	gcgggccatgttaagggcggttttttcctgtttggtcactgatgcc

	tccgtgtaagggggatttctgttcatgggggtaatgataccgatga

	aacgagagaggatgctcacgatacgggttactgatgatgaacatgc

	ccggttactggaacgttgtgagggtaaacaactggcggtatggatg

	cggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcg

	ttaatacagatgtaggtgttccacagggtagccagcagcatcctgc

	gatgcagatccggaacataatggtgcagggcgctgacttccgcgtt

	tccagactttacgaaacacggaaaccgaagaccattcatgttgttg

	ctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctc

	gcgtatcggtgattcattctgctaaccagtaaggcaaccccgccag

	cctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtg

	gccaggacccaacgctgcccgagatgcgccgcgtgcggctgctgga

	gatggcggacgcgatggatatgttctgccaagggttggtttgcgca

	ttcacagttctccgcaagaattgattggctccaattcttggagtgg

	tgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtg

	gcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggt

	atagggcggcgcctacaatccatgccaacccgttccatgtgctcgc

	cgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaa

	gttaggctggtaagagccgcgagcgatccttgaagctgtccctgat

	ggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcat

	cccgatgccgccggaagcgagaagaatcataatggggaaggccatc

	cagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcgg

	ccgccatgccggcgataatggcctgcttctcgccgaaacgtttggt

	ggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccg

	aataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagc

	ggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctac

	gagttgcatgataaagaagacagtcataagtgcggcgacgatagtc

	atgccccgcgcccaccggaaggagctgactgggttgaaggctctca

	agggcatcggtcgacgctctcccttatgcgactcctgcattaggaa

	gcagcccagtagtaggttgaggccgttgagcaccgccgccgcaagg

	aatggtgcatgcaaggagatggcgcccaacagtcccccggccacgg

	ggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaa

	gtggcgagcccgatcttccccatcggtgatgtcggcgatataggcg

	ccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtc

	cggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgta

	gtcgatagtggctccaagtagcgaagcgagcaggactgggcggcgg

	ccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaatt

	gcatcaacgcatatagcgctagcagcacgccatagtgactggcgat

	gctgtcggaatggacgatatcccgcaagaggcccggcagtaccggc

	ataaccaagcctatgcctacagcatccagggtgacggtgccgagga

	tgacgatgagcgcattgttagatttcatacacggtgcctgactgcg

	ttagcaatttaactgtgataaactaccgcattaaagcttatcgatg

	ataagctgtcaaacatgagaa

	pBR-CBST-CTX-M-15
	SEQ ID 7
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGGAATCTGTTAAATCAG

	CGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTG

	CGGAAAAGCACGTCAATGGGACGATGTCACTGGCTGAGCTTAGCGC

	GGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATT

	GCTCACGTTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACAGC

	TGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAA

	CACCGCCATTCCGGGCGATCCGCGTGATACCACTTCACCTCGGGCA

	ATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCG

	ACAGCCAACGGGCGCAGCTGGTGACATGGATGAAAGGCAATACCAC

	CGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTG

	GGGGATAAAACCGGCAGCGGTGGCTATGGCACCACCAACGATATCG

	CGGTGATCTGGCCAAAAGATCGTGCGCCGCTGATTCTGGTCACTTA

	CTTCACCCAGCCTCAACCTAAGGCAGAAAGCCGTCGCGATGTATTA

	GCGTCGGCGGCTAAAATCGTCACCGACGGTTTGTAAggatccctgt

	cagaccaagtttactcatatatactttagattgatttaaaacttca

	tttttaatttaaaaggatctaggtgaagatcctttttgataatctc

	atgaccaaaatcccttaacgtgagttttcgttccactgagcgtcag

	accccgtagaaaagatcaaaggatcttcttgagatcctttttttct

	gcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcg

	gtggtttgtttgccggatcaagagctaccaactctttttccgaagg

	taactggcttcagcagagcgcagataccaaatactgtccttctagt

	gtagccgtagttaggccaccacttcaagaactctgtagcaccgcct

	acatacctcgctctgctaatcctgttaccagtggctgctgccagtg

	gcgataagtcgtgtcttaccgggttggactcaagacgatagttacc

	ggataaggcgcagcggtcgggctgaacggggggttcgtgcacacag

	cccagcttggagcgaacgacctacaccgaactgagatacctacagc

	gtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgga

	caggtatccggtaagcggcagggtcggaacaggagagcgcacgagg

	gagcttccagggggaaacgcctggtatctttatgtcctgtcgggtt

	tcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg

	gggcggagcctatggaaaaacgccagcaacgcggcctttttacggt

	tcctggccttttgctggccttttgctcacatgttctttcctgcgtt

	atcccctgattctgtggataaccgtattaccgcctttgagtgagct

	gataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtga

	gcgaggaagcggaagagcgcctgatgcggtattttctccttacgca

	tctgtgcggtatttcacaccgcatttggtgcactctcagtacaatc

	tgctctgatgccgcatagttaagccagtatacactccgctatcgct

	acgtgactgggtcatggctgcgccccgacacccgccaacacccgct

	gacgcgccctgacgggcttgtctgctcccggcatccgcttacagac

	aagctgtgaccgtctccgggagctgcatgtgtcagaggttttcacc

	gtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcg

	tggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtcca

	gctcgttgagtttctccagaagcgttaatgtctggcttctgataaa

	gcgggccatgttaagggcggttttttcctgtttggtcactgatgcc

	tccgtgtaagggggatttctgttcatgggggtaatgataccgatga

	aacgagagaggatgctcacgatacgggttactgatgatgaacatgc

	ccggttactggaacgttgtgagggtaaacaactggcggtatggatg

	cggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcg

	ttaatacagatgtaggtgttccacagggtagccagcagcatcctgc

	gatgcagatccggaacataatggtgcagggcgctgacttccgcgtt

	tccagactttacgaaacacggaaaccgaagaccattcatgttgttg

	ctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctc

	gcgtatcggtgattcattctgctaaccagtaaggcaaccccgccag

	cctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtg

	gccaggacccaacgctgcccgagatgcgccgcgtgcggctgctgga

	gatggcggacgcatggatatgttctgccaagggttggtttgcgcat

	tcacagttctccgcaagaattgattggctccaattcttggagtggt

	gaatccgttagcgaggtgccgccggcttccattcaggtcgaggtgg

	cccggctccatgcaccgcgacgcaacgcggggaggcagacaaggta

	tagggcggcgcctacaatccatgccaacccgttccatgtgctcgcc

	gaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaag

	ttaggctggtaagagccgcgagcgatccttgaagctgtccctgatg

	gtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatc

	ccgatgccgccggaagcgagaagaatcataatggggaaggccatcc

	agcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggc

	cgccatgccggcgataatggcctgcttctcgccgaaacgtttggtg

	gcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccga

	ataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcg

	gtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacg

	agttgcatgataaagaagacagtcataagtgcggcgacgatagtca

	tgccccgcgcccaccggaaggagctgactgggttgaaggctctcaa

	gggcatcggtcgacgctctcccttatgcgactcctgcattaggaag

	cagcccagtagtaggttgaggccgttgagcaccgccgccgcaagga

	atggtgcatgcaaggagatggcgcccaacagtcccccggccacggg

	gcctgccaccatacccacgccgaaacaagcgctcatgagcccgaag

	tggcgagcccgatcttccccatcggtgatgtcggcgatataggcgc

	cagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtcc

	ggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtag

	tcgatagtggctccaagtagcgaagcgagcaggactgggcggcggc

	caaagcggtcggacagtgctccgagaacgggtgcgcatagaaattg

	catcaacgcatatagcgctagcagcacgccatagtgactggcgatg

	ctgtcggaatggacgatatcccgcaagaggcccggcagtaccggca

	taaccaagcctatgcctacagcatccagggtgacggtgccgaggat

	gacgatgagcgcattgttagatttcatacacggtggcctgactgcg

	ttagcaatttaactgtgataaactaccgcattaaagcttatcgatg

	ataagctgtcaaacatgagaa

	pBR-CBST-SHV-12
	SEQ ID 8
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGCGTTATATTCGCCTGT

	GTATTATCTCCCTGTTAGCCACCCTGCCGCTGGCGGTACACGCCAG

	CCCGCAGCCGCTTGAGCAAATTAAACAAAGCGAAAGCCAGCTGTCG

	GGCCGCGTAGGCATGATAGAAATGGATCTGGCCAGCGGCCGCACGC

	TGACCGCCTGGCGCGCCGATGAACGCTTTCCCATGATGAGCACCTT

	TAAAGTAGTGCTCTGCGGCGCAGTGCTGGCGCGGGTGGATGCCGGT

	GACGAACAGCTGGAGCGAAAGATCCACTATCGCCAGCAGGATCTGG

	TGGACTACTCGCCGGTCAGCGAAAAACACCTTGCCGACGGCATGAC

	GGTCGGCGAACTCTGCGCCGCCGCCATTACCATGAGCGATAACAGC

	GCCGCCAATCTGCTGCTGGCCACCGTCGGCGGCCCCGCAGGATTGA

	CTGCCTTTTTGCGCCAGATCGGCGACAACGTCACCCGCCTTGACCG

	CTGGGAAACGGAACTGAATGAGGCGCTTCCCGGCGACGCCCGCGAC

	ACCACTACCCCGGCCAGCATGGCCGCGACCCTGCGCAAGCTGCTGA

	CCAGCCAGCGTCTGAGCGCCCGTTCGCAACGGCAGCTGCTGCAGTG

	GATGGTGGACGATCGGGTCGCCGGACCGTTGATCCGCTCCGTGCTG

	CCGGCGGGCTGGTTTATCGCCGATAAGACCGGAGCTAGCAAGCGGG

	GTGCGCGCGGGATTGTCGCCCTGCTTGGCCCCAATAACAAAGCAGA

	GCGCATTGTGGTGATTTATCTGCGGGATACCCCGGCGAGCATGGCC

	GAGCGAAATCAGCAAATCGCCGGGATCGGCGCGGCGCTGATCGAGC

	ACTGGCAACGCTAAggatccctgtcagaccaagtttactcatatat

	actttagattgatttaaaacttcatttttaatttaaaaggatctag

	gtgaagatcctttttgataatctcatgaccaaaatcccttaacgtg

	agttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg

	atcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaa

	acaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaag

	agctaccaactctttttccgaaggtaactggcttcagcagagcgca

	gataccaaatactgtccttctagtgtagccgtagttaggccaccac

	ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcc

	tgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgg

	gttggactcaagacgatagttaccggataaggcgcagcggtcgggc

	tgaacggggggttcgtgcacacagcccagcttggagcgaacgacct

	acaccgaactgagatacctacagcgtgagctatgagaaagcgccac

	gcttcccgaagggagaaaggcggacaggtatccggtaagcggcagg

	gtcggaacaggagagcgcacgagggagcttccagggggaaacgcct

	ggtatctttatagtcctgtcgggtttcgccacctctgacttgagcg

	tcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaac

	gccagcaacgcggcctttttacggttcctggccttttgctggcctt

	ttgctcacatgttctttcctgcgttatcccctgattctgtggataa

	ccgtattaccgcctttgagtgagctgataccgctcgccgcagccga

	acgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcc

	tgatgcggtattttctccttacgcatctgtgcggtatttcacaccg

	catttggtgcactctcagtacaatctgctctgatgccgcatagtta

	agccagtatacactccgctatcgctacgtgactgggtcatggctgc

	gccccgacacccgccaacacccgctgacgcgccctgacgggcttgt

	ctgctcccggcatccgcttacagacaagctgtgaccgtctccggga

	gctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgag

	gcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacag

	atgtctgcctgttcatccgcgtccagctcgttgagtttctccagaa

	gcgttaatgtctggcttctgataaagcgggccatgttaagggcggt

	tttttcctgtttggtcactgatgcctccgtgtaagggggatttctg

	ttcatgggggtaatgataccgatgaaacgagagaggatgctcacga

	tacgggttactgatgatgaacatgcccggttactggaacgttgtga

	gggtaaacaactggcggtatggatgcggcgggaccagagaaaaatc

	actcagggtcaatgccagcgcttcgttaatacagatgtaggtgttc

	cacagggtagccagcagcatcctgcgatgcagatccggaacataat

	ggtgcagggcgctgacttccgcgtttccagactttacgaaacacgg

	aaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgc

	agcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctg

	ctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgac

	aggagcacgatcatgcgcacccgtggccaggacccaacgctgcccg

	agatgcgccgcgtgcggctgctggagatggcggacgcgatggatat

	gttctgccaagggttggtttgcgcattcacagttctccgcaagaat

	tgattggctccaattcttggagtggtgaatccgttagcgaggtgcc

	gccggcttccattcaggtcgaggtggcccggctccatgcaccgcga

	cgcaacgcggggaggcagacaaggtatagggcggcgcctacaatcc

	atgccaacccgttccatgtgctcgccgaggcggcataaatcgccgt

	gacgatcagcggtccagtgatcgaagttaggctggtaagagccgcg

	agcgatccttgaagctgtccctgatggtcgtcatctacctgcctgg

	acagcatggcctgcaacgcgggcatcccgatgccgccggaagcgag

	aagaatcataatggggaaggccatccagcctcgcgtcgcgaacgcc

	agcaagacgtagcccagcgcgtcggccgccatgccggcgataatgg

	cctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggc

	ttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccg

	atcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgaccc

	agagcgctgccggcacctgtcctacgagttgcatgataaagaagac

	agtcataagtgcggcgacgatagtcatgccccgcgcccaccggaag

	gagctgactgggttgaaggctctcaagggcatcggtcgacgctctc

	ccttatgcgactcctgcattaggaagcagcccagtagtaggttgag

	gccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatg

	gcgcccaacagtcccccggccacggggcctgccaccatacccacgc

	cgaaacaagcgctcatgagcccgaagtggcgagcccgatcttcccc

	atcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcg

	ccggtgatgccggccacgatgcgtccggcgtagaggattcacagga

	cgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtag

	cgaagcgagcaggactgggcggcggccaaagcggtcggacagtgct

	ccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgcta

	gcagcacgccatagtgactggcgatgctgtcggaatggacgatatc

	ccgcaagaggcccggcagtaccggcataaccaagcctatgcctaca

	gcatccagggtgacggtgccgaggatgacgatgagcgcattgttag

	atttcatacacggtgcctgactgcgttagcaatttaactgtgataa

	actaccgcattaaagcttatcgatgataagctgtcaaacatgagaa

	pBR-CBST-P99
	SEQ ID 9
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGATGAGAAAATCCCTTT

	GCTGCGCCCTGCTGCTCGGCATCTCTTGCTCTGCTCTCGCCACGCC

	AGTGTCAGAAAAACAGCTGGCGGAGGTGGTCGCGAATACGATTACC

	CCGCTGATGAAAGCCCAGTCTGTTCCAGGCATGGCGGTGGCCGTTA

	TTTATCAGGGAAAACCGCACTATTACACATTTGGCAAGGCCGATAT

	CGCGGCGAATAAACCCGTTACGCCTCAGACCCTGTTCGAGCTGGGT

	TCTATAAGTAAAACCTTCACCGGCGTTTTAGGTGGGGATGCCATTG

	CTCGCGGTGAAATTTCGCTGGACGATGCGGTGACCAGATACTGGCC

	ACAGCTGACGGGCAAGCAGTGGCAGGGTATTCGTATGCTGGATCTC

	GCCACCTACACCGCTGGCGGCCTGCCGCTACAGGTACCGGATGAGG

	TCACGGATAACGCCTCCCTGCTGCGCTTTTATCAAAACTGGCAGCC

	GCAGTGGAAGCCTGGCACAACGCGTCTTTACGCCAACGCCAGCATC

	GGTCTTTTTGGTGCGCTGGCGGTCAAACCTTCTGGCATGCCCTATG

	AGCAGGCCATGACGACGCGGGTCCTTAAGCCGCTCAAGCTGGACCA

	TACCTGGATTAACGTGCCGAAAGCGGAAGAGGCGCATTACGCCTGG

	GGCTATCGTGACGGTAAAGCGGTGCGCGTTTCGCCGGGTATGCTGG

	ATGCACAAGCCTATGGCGTGAAAACCAACGTGCAGGATATGGCGAA

	CTGGGTCATGGCAAACATGGCGCCGGAGAACGTTGCTGATGCCTCA

	CTTAAGCAGGGCATCGCGCTGGCGCAGTCGCGCTACTGGCGTATCG

	GGTCAATGTATCAGGGTCTGGGCTGGGAGATGCTCAACTGGCCCGT

	GGAGGCCAACACGGTGGTCGAGGGCAGCGACAGTAAGGTAGCACTG

	GCGCCGTTGCCCGTGGCAGAAGTGAATCCACCGGCTCCCCCGGTCA

	AAGCGTCCTGGGTCCATAAAACGGGCTCTACTGGCGGGTTTGGCAG

	CTACGTGGCCTTTATTCCTGAAAAGCAGATCGGTATTGTGATGCTC

	GCGAATACAAGCTATCCGAACCCGGCACGCGTTGAGGCGGCATACC

	ATATCCTCGAGGCGCTACAGTAAggatccctgtcagaccaagttta

	ctcatatatactttagattgatttaaaacttcatttttaatttaaa

	aggatctaggtgaagatcctttttgataatctcatgaccaaaatcc

	cttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaa

	gatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc

	tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgc

	cggatcaagagctaccaactCtttttccgaaggtaactggcttcag

	cagagcgcagataccaaatactgtccttctagtgtagccgtagtta

	ggccaccacttcaagaactctgtagcaccgcctacatacctcgctc

	tgctaatcctgttaccagtggctgctgccagtggcgataagtcgtg

	tcttaccgggttggactcaagacgatagttaccggataaggcgcag

	cggtcgggctgaacggggggttcgtgcacacagcccagcttggagc

	gaacgacctacaccgaactgagatacctacagcgtgagctatgaga

	aagcgccacgcttcccgaagggagaaaggcggacaggtatccggta

	agcggcagggtcggaacaggagagcgcacgagggagcttccagggg

	gaaacgcctggtatctttatagtcctgtcgggtttcgccacctctg

	acttgagcgtcgatttttgtgatgctcgtcaggggggcggagccta

	tggaaaaacgccagcaacgcggcctttttacggttcctggcctttt

	gctggccttttgctcacatgttctttcctgcgttatcccctgattc

	tgtggataaccgtattaccgcctttgagtgagctgataccgctcgc

	cgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcgg

	aagagcgcctgatgcggtattttctccttacgcatctgtgcggtat

	ttcacaccgcatttggtgcactctcagtacaatctgctctgatgcc

	gcatagttaagccagtatacactccgctatcgctacgtgactgggt

	catggctgcgccccgacacccgccaacacccgctgacgcgccctga

	cgggcttgtctgctcccggcatccgcttacagacaagctgtgaccg

	tctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaa

	acgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagc

	gattcacagatgtctgcctgttcatccgcgtccagctcgttgagtt

	tctccagaagcgttaatgtctggcttctgataaagcgggccatgtt

	aagggcggttttttcctgtttggtcactgatgcctccgtgtaaggg

	ggatttctgttcatgggggtaatgataccgatgaaacgagagagga

	tgctcacgatacgggttactgatgatgaacatgcccggttactgga

	acgttgtgagggtaaacaactggcggtatggatgcggcgggaccag

	agaaaaatcactcagggtcaatgccagcgcttcgttaatacagatg

	taggtgttccacagggtagccagcagcatcctgcgatgcagatccg

	gaacataatggtgcagggcgctgacttccgcgtttccagactttac

	gaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcag

	acgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtga

	ttcattctgctaaccagtaaggcaaccccgccagcctagccgggtc

	ctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaa

	cgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgc

	gatggatatgttctgccaagggttggtttgcgcattcacagttctc

	cgcaagaattgattggctccaattcttggagtggtgaatccgttag

	cgaggtgccgccggcttccattcaggtcgaggtggcccggctccat

	gcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgc

	ctacaatccatgccaacccgttccatgtgctcgccgaggcggcata

	aatcgccgtgacgatcagcggtccagtgatcgaagttaggctggta

	agagccgcgagcgatccttgaagctgtccctgatggtcgtcatcta

	cctgcctggacagcatggcctgcaacgcgggcatcccgatgccgcc

	ggaagcgagaagaatcataatggggaaggccatccagcctcgcgtc

	gcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccgg

	cgataatggcctgcttctcgccgaaacgtttggtggcgggaccagt

	gacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagc

	gacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccga

	aaatgacccagagcgctgccggcacctgtcctacgagttgcatgat

	aaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcc

	caccggaaggagctgactgggttgaaggctctcaagggcatcggtc

	gacgctctcccttatgcgactcctgcattaggaagcagcccagtag

	taggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgc

	aaggagatggcgcccaacagtcccccggccacggggcctgccacca

	tacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccg

	atcttccccatcggtgatgtcggcgatataggcgccagcaaccgca

	cctgtggcgccggtgatgccggccacgatgcgtccggcgtagagga

	ttcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggc

	tccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcg

	gacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcat

	atagcgctagcagcacgccatagtgactggcgatgctgtcggaatg

	gacgatatcccgcaagaggcccggcagtaccggcataaccaagcct

	atgcctacagcatccagggtgacggtgccgaggatgacgatgagcg

	cattgttagatttcatacacggtgcctgactgcgttagcaatttaa

	ctgtgataaactaccgcattaaagcttatcgatgataagctgtcaa

	acatgagaa

	pBR-CBST-OXA-15
	SEQ ID 10
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGGCAATCCGAATCTTCG

	CGATACTTTTCTCCATTTTTTCTCTTGCCACTTTCGCGCATGCGCA

	AGAAGGCACGCTAGAACGTTCTGACTGGAGGAAGTTTTTCAGCGAA

	TTTCAAGCCAAAGGCACGATAGTTGTGGCAGACGAACGCCAAGCGG

	ATCGTGCCATGTTGGTTTTTGATCCTGTGCGATCGAAGAAACGCTA

	CTCGCCTGCATCGACATTCAAGATACCTCATACACTTTTTGCACTT

	GATGCAGGCGCTGTTCGTGATGAGTTCCAGATTTTTCGATGGGACG

	GCGTTAACAGGGGCTTTGCAGGCCACAATCAAGACCAAGATTTGCG

	ATCAGCAATGCGGAATTCTACTGTTTGGGTGTATGAGCTATTTGCA

	AAGGAAATTGGTGATGACAAAGCTCGGCGCTATTTGAAGAAAATCG

	ACTATGGCAACGCCGGTCCTTCGACAAGTAATGGCGATTACTGGAT

	AGAAGGCAGCCTTGCAATCTCGGCGCAGGAGCAAATTGCATTTCTC

	AGGAAGCTCTATCGTAACGAGCTGCCCTTTCGGGTAGAACATCAGC

	GCTTGGTCAAGGATCTCATGATTGTGGAAGCCGGTCGCAACTGGAT

	ACTGCGTGCAAAGACGGGCTGGGAAGGCCGTATGGGTTGGTGGGTA

	GGATGGGTTGAGTGGCCGACTGGCTCCGTATTCTTCGCACTGAATA

	TTGATACGCCAAACAGAATGGATGATCTTTTCAAGAGGGAGGCAAT

	CGTGCGGGCAATCCTTCGCTCTATTGAAGCGTTACCGCCCAACCCG

	GCAGTCAACTCGGACGCTGCGCGATAAggatccctgtcagaccaag

	tttactcatatatactttagattgatttaaaacttcatttttaatt

	taaaaggatctaggtgaagatcctttttgataatctcatgaccaaa

	atcccttaacgtgagttttcgttccactgagcgtcagaccccgtag

	aaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaat

	ctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgt

	ttgccggatcaagagctaccaactctttttccgaaggtaactggct

	tcagcagagcgcagataccaaatactgtccttctagtgtagccgta

	gttaggccaccacttcaagaactctgtagcaccgcctacatacctc

	gctctgctaatcctgttaccagtggctgctgccagtggcgataagt

	cgtgtcttaccgggttggactcaagacgatagttaccggataaggc

	gcagcggtcgggctgaacggggggttcgtgcacacagcccagcttg

	gagcgaacgacctacaccgaactgagatacctacagcgtgagctat

	gagaaagcgccacgcttcccgaagggagaaaggcggacaggtatcc

	ggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca

	gggggaaacgcctggtatctttatagtcctgtcgggtttcgccacc

	tctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggag

	cctatggaaaaacgccagcaacgcggcctttttacggttcctggcc

	ttttgctggccttttgctcacatgttctttcctgcgttatcccctg

	attctgtggataaccgtattaccgcctttgagtgagctgataccgc

	tcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaa

	gcggaagagcgcctgatgcggtattttctccttacgcatctgtgcg

	gtatttcacaccgcatttggtgcactctcagtacaatctgctctga

	tgccgcatagttaagccagtatacactccgctatcgctacgtgact

	gggtcatggctgcgccccgacacccgccaacacccgctgacgcgcc

	ctgacgggcttgtctgctcccggcatccgcttacagacaagctgtg

	accgtctccgggagctgcatgtgtcagaggttttcaccgtcatcac

	cgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtg

	aagcgattcacagatgtctgcctgttcatccgcgtccagctcgttg

	agtttctccagaagcgttaatgtctggcttctgataaagcgggcca

	tgttaagggcggttttttcctgtttggtcactgatgcctccgtgta

	agggggatttctgttcatgggggtaatgataccgatgaaacgagag

	aggatgctcacgatacgggttactgatgatgaacatgcccggttac

	tggaacgttgtgagggtaaacaactggcggtatggatgcggcggga

	ccagagaaaaatcactcagggtcaatgccagcgcttcgttaataca

	gatgtaggtgttccacagggtagccagcagcatcctgcgatgcaga

	tccggaacataatggtgcagggcgctgacttccgcgtttccagact

	ttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtc

	gcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcg

	gtgattcattctgctaaccagtaaggcaaccccgccagcctagccg

	ggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggac

	ccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcgg

	acgcgatggatatgttctgccaagggttggtttgcgcattcacagt

	tctccgcaagaattgattggctccaattcttggagtggtgaatccg

	ttagcgaggtgccgccggcttccattcaggtcgaggtggcccggct

	ccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcg

	gcgcctacaatccatgccaacccgttccatgtgctcgccgaggcgg

	cataaatcgccgtgacgatcagcggtccagtgatcgaagttaggct

	ggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtca

	tctacctgcctggacagcatggcctgcaacgcgggcatcccgatgc

	cgccggaagcgagaagaatcataatggggaaggccatccagcctcg

	cgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatg

	ccggcgataatggcctgcttctcgccgaaacgtttggtggcgggac

	cagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgc

	aagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcg

	ccgaaaatgacccagagcgctgccggcacctgtcctacgagttgca

	tgataaagaagacagtcataagtgcggcgacgatagtcatgccccg

	cgcccaccggaaggagctgactgggttgaaggctctcaagggcatc

	ggtcgacgctctcccttatgcgactcctgcattaggaagcagccca

	gtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgc

	atgcaaggagatggcgcccaacagtcccccggccacggggcctgcc

	accatacccacgccgaaacaagcgctcatgagcccgaagtggcgag

	cccgatcttccccatcggtgatgtcggcgatataggcgccagcaac

	cgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtag

	aggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatag

	tggctccaagtagcgaagcgagcaggactgggcggcggccaaagcg

	gtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaac

	gcatatagcgctagcagcacgccatagtgactggcgatgctgtcgg

	aatggacgatatcccgcaagaggcccggcagtaccggcataaccaa

	gcctatgcctacagcatccagggtgacggtgccgaggatgacgatg

	agcgcattgttagatttcatacacggtgcctgactgcgttagcaat

	ttaactgtgataaactaccgcattaaagcttatcgatgataagctg

	tcaaacatgagaa

	pBR-CBST-KPC-4
	SEQ ID 11
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGTCACTGTATCGCCGTC

	TAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGC

	CACCGCGCTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAA

	CAGGACTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCT

	CAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTG

	CAGCTCATTCAAGGGCTTTCTTGCTGCCGCTGTGCTGGCTCGCAGC

	CAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGCAAAA

	ATGCGCTGGTTCGGTGGTCACCCATCTCGGAAAAATATCTGACAAC

	AGGCATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGT

	GATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGG

	CCGGGCTGACGGCCTTCATGCGCTCTATCGGCGATACCACGTTCCG

	TCTGGACCGCTGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGAT

	GCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACAAA

	AACTGACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTT

	TGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGC

	GCGGCGGTGCCGGCAGACTGGGCAGTCGGAGACAAAACCGGAACCT

	GCGGAGGGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCAC

	TGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCGGGCGCCTAAC

	AAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCGGCTAGAC

	TCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAAggatccctgtc

	agaccaagtttactcatatatactttagattgatttaaaacttcat

	ttttaatttaaaaggatctaggtgaagatcctttttgataatctca

	tgaccaaaatcccttaacgtgagttttcgttccactgagcgtcaga

	ccccgtagaaaagatcaaaggatcttcttgagatcctttttttctg

	cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcgg

	tggtttgtttgccggatcaagagctaccaactctttttccgaaggt

	aactggcttcagcagagcgcagataccaaatactgtccttctagtg

	tagccgtagttaggccaccacttcaagaactctgtagcaccgccta

	catacctcgctctgctaatcctgttaccagtggctgctgccagtgg

	cgataagtcgtgtcttaccgggttggactcaagacgatagttaccg

	gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagc

	ccagcttggagcgaacgacctacaccgaactgagatacctacagcg

	tgagctatgagaaagcgccacgcttcccgaagggagaaaggcggac

	aggtatccggtaagcggcagggtcggaacaggagagcgcacgaggg

	agcttccagggggaaacgcctggtatctttatagtcctgtcgggtt

	tcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg

	gggcggagcctatggaaaaacgccagcaacgcggcctttttacggt

	tcctggccttttgctggccttttgctcacatgttctttcctgcgtt

	atcccctgattctgtggataaccgtattaccgcctttgagtgagct

	gataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtga

	gcgaggaagcggaagagcgcctgatgcggtattttctccttacgca

	tctgtgcggtatttcacaccgcatttggtgcactctcagtacaatc

	tgctctgatgccgcatagttaagccagtatacactccgctatcgct

	acgtgactgggtcatggctgcgccccgacacccgccaacacccgct

	gacgcgccctgacgggcttgtctgctcccggcatccgcttacagac

	aagctgtgaccgtctccgggagctgcatgtgtcagaggttttcacc

	gtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcg

	tggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtcca

	gctcgttgagtttctccagaagcgttaatgtctggcttctgataaa

	gcgggccatgttaagggcggttttttcctgtttggtcactgatgcc

	tccgtgtaagggggatttctgttcatgggggtaatgataccgatga

	aacgagagaggatgctcacgatacgggttactgatgatgaacatgc

	ccggttactggaacgttgtgagggtaaacaactggcggtatggatg

	cggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcg

	ttaatacagatgtaggtgttccacagggtagccagcagcatcctgc

	gatgcagatccggaacataatggtgcagggcgctgacttccgcgtt

	tccagactttacgaaacacggaaaccgaagaccattcatgttgttg

	ctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctc

	gcgtatcggtgattcattctgctaaccagtaaggcaaccccgccag

	cctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtg

	gccaggacccaacgctgcccgagatgcgccgcgtgcggctgctgga

	gatggcggacgcgatggatatgttctgccaagggttggtttgcgca

	ttcacagttctccgcaagaattgattggctccaattcttggagtgg

	tgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtg

	gcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggt

	atagggcggcgcctacaatccatgccaacccgttccatgtgctcgc

	cgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaa

	gttaggctggtaagagccgcgagcgatccttgaagctgtccctgat

	ggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcat

	cccgatgccgccggaagcgagaagaatcataatggggaaggccatc

	cagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcgg

	ccgccatgccggcgataatggcctgcttctcgccgaaacgtttggt

	ggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccg

	aataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagc

	ggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctac

	gagttgcatgataaagaagacagtcataagtgcggcgacgatagtc

	atgccccgcgcccaccggaaggagctgactgggttgaaggctctca

	agggcatcggtcgacgctctcccttatgcgactcctgcattaggaa

	gcagcccagtagtaggttgaggccgttgagcaccgccgccgcaagg

	aatggtgcatgcaaggagatggcgcccaacagtcccccggccacgg

	ggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaa

	gtggcgagcccgatcttccccatcggtgatgtcggcgatataggcg

	ccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtc

	cggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgta

	gtcgatagtggctccaagtagcgaagcgagcaggactgggcggcgg

	ccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaatt

	gcatcaacgcatatagcgctagcagcacgccatagtgactggcgat

	gctgtcggaatggacgatatcccgcaagaggcccggcagtaccggc

	ataaccaagcctatgcctacagcatccagggtgacggtgccgagga

	tgacgatgagcgcattgttagatttcatacacggtgcctgactgcg

	ttagcaatttaactgtgataaactaccgcattaaagcttatcgatg

	ataagctgtcaaacatgagaa

	pBR-CBST-DHA-1
	SEQ ID 12
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGAAAAAATCGTTATCTG

	CAACACTGATTTCCGCTCTGCTGGCGTTTTCCGCCCCGGGGTTTTC

	TGCCGCTGATAATGTCGCGGCGGTGGTGGACAGCACCATTAAACCG

	CTGATGGCACAGCAGGATATTCCCGGGATGGCGGTTGCCGTCTCCG

	TAAAGGGTAAGCCCTATTATTTCAATTATGGTTTTGCCGATATTCA

	GGCAAAACAGCCGGTCACTGAAAATACACTATTTGAGCTCGGATCT

	GTAAGTAAAACTTTCACAGGTGTGCTGGGTGCGGTTTCTGTGGCGA

	AAAAAGAGATGGCGCTGAATGATCCGGCGGCAAAATACCAGCCGGA

	GCTGGCTCTGCCGCAGTGGAAGGGGATCACATTGCTGGATCTGGCT

	ACCTATACCGCAGGCGGACTGCCGTTACAGGTGCCGGATGCGGTAA

	AAAGCCGTGCGGATCTGCTGAATTTCTATCAGCAGTGGCAGCCGTC

	CCGGAAACCGGGCGATATGCGTCTGTATGCAAACAGCAGTATCGGC

	CTGTTTGGTGCTCTGACCGCAAACGCGGCGGGGATGCCGTATGAGC

	AGTTGCTGACTGCACGCATCCTGGCACCGCTGGGGTTATCTCACAC

	CTTTATTACTGTGCCGGAAAGTGCGCAAAGCCAGTATGCGTACGGT

	TATAAAAACAAAAAACCGGTCCGCGTGTCGCCGGGACAGCTTGATG

	CGGAATCTTACGGCGTGAAATCCGCCTCAAAAGATATGCTGCGCTG

	GGCGGAAATGAATATGGAGCCGTCACGGGCCGGTAATGCGGATCTG

	GAAATGGCAATGTATCTCGCCCAGACCCGCTACTATAAAACCGCCG

	CGATTAACCAGGGGCTGGGCTGGGAAATGTATGACTGGCCGCAGCA

	GAAAGATATGATCATTAACGGTGTGACCAACGAGGTCGCATTGCAG

	CCGCATCCGGTAACAGACAACCAGGTTCAGCCGTATAACCGTGCTT

	CCTGGGTGCATAAAACGGGCGCAACAACTGGTTTCGGCGCCTATGT

	CGCCTTTATTCCGGAAAAACAGGTGGCGATTGTGATTCTGGCGAAT

	AAAAACTACCCGAATACCGAAAGAGTCAAAGCTGCACAGGCTATTT

	TGAGTGCACTGGAATAAggatccctgtcagaccaagtttactcata

	tatactttagattgatttaaaacttcatttttaatttaaaaggatc

	taggtgaagatcctttttgataatctcatgaccaaaatcccttaac

	gtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaa

	aggatcttcttgagatcctttttttctgcgcgtaatctgctgcttg

	caaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatc

	aagagctaccaactctttttccgaaggtaactggcttcagcagagc

	gcagataccaaatactgtccttctagtgtagccgtagttaggccac

	cacttcaagaactctgtagcaccgcctacatacctcgctctgctaa

	tcctgttaccagtggctgctgccagtggcgataagtcgtgtcttac

	cgggttggactcaagacgatagttaccggataaggcgcagcggtcg

	ggctgaacggggggttcgtgcacacagcccagcttggagcgaacga

	cctacaccgaactgagatacctacagcgtgagctatgagaaagcgc

	cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc

	agggtcggaacaggagagcgcacgagggagcttccagggggaaacg

	cctggtatctttatagtcctgtcgggtttcgccacctctgacttga

	gcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaa

	aacgccagcaacgcggcctttttacggttcctggccttttgctggc

	cttttgctcacatgttctttcctgcgttatcccctgattctgtgga

	taaccgtattaccgcctttgagtgagctgataccgctcgccgcagc

	cgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagc

	gcctgatgcggtattttctccttacgcatctgtgcggtatttcaca

	ccgcatttggtgcactctcagtacaatctgctctgatgccgcatag

	ttaagccagtatacactccgctatcgctacgtgactgggtcatggc

	tgcgccccgacacccgccaacacccgctgacgcgccctgacgggct

	tgtctgctcccggcatccgcttacagacaagctgtgaccgtctccg

	ggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgc

	gaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattca

	cagatgtctgcctgttcatccgcgtccagctcgttgagtttctcca

	gaagcgttaatgtctggcttctgataaagcgggccatgttaagggc

	ggttttttcctgtttggtcactgatgcctccgtgtaagggggattt

	ctgttcatgggggtaatgataccgatgaaacgagagaggatgctca

	cgatacgggttactgatgatgaacatgcccggttactggaacgttg

	tgagggtaaacaactggcggtatggatgcggcgggaccagagaaaa

	atcactcagggtcaatgccagcgcttcgttaatacagatgtaggtg

	ttccacagggtagccagcagcatcctgcgatgcagatccggaacat

	aatggtgcagggcgctgacttccgcgtttccagactttacgaaaca

	cggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttt

	tgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcatt

	ctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaac

	gacaggagcacgatcatgcgcacccgtggccaggacccaacgctgc

	ccgagatgcgccgcgtgcggctgctggagatggcggacgcgatgga

	tatgttctgccaagggttggtttgcgcattcacagttctccgcaag

	aattgattggctccaattcttggagtggtgaatccgttagcgaggt

	gccgccggcttccattcaggtcgaggtggcccggctccatgcaccg

	cgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaa

	tccatgccaacccgttccatgtgctcgccgaggcggcataaatcgc

	cgtgacgatcagcggtccagtgatcgaagttaggctggtaagagcc

	gcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcc

	tggacagcatggcctgcaacgcgggcatcccgatgccgccggaagc

	gagaagaatcataatggggaaggccatccagcctcgcgtcgcgaac

	gccagcaagacgtagcccagcgcgtcggccgccatgccggcgataa

	tggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaa

	ggcttgagcgagggcgtgcaagattccgaataccgcaagcgacagg

	ccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatga

	cccagagcgctgccggcacctgtcctacgagttgcatgataaagaa

	gacagtcataagtgcggcgacgatagtcatgccccgcgcccaccgg

	aaggagctgactgggttgaaggctctcaagggcatcggtcgacgct

	ctcccttatgcgactcctgcattaggaagcagcccagtagtaggtt

	gaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggag

	atggcgcccaacagtcccccggccacgggcctgccaccatacccac

	gccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttcc

	ccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtgg

	cgccggtgatgccggccacgatgcgtccggcgtagaggattcacag

	gacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagt

	agcgaagcgagcaggactgggcggcggccaaagcggtcggacagtg

	ctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgc

	tagcagcacgccatagtgactggcgatgctgtcggaatggacgata

	tcccgcaagaggcccggcagtaccggcataaccaagcctatgccta

	cagcatccagggtgacggtgccgaggatgacgatgagcgcattgtt

	agatttcatacacggtgcctgactgcgttagcaatttaactgtgat

	aaactaccgcattaaagcttatcgatgataagctgtcaaagatgag

	aa

	pBR-CBST-ADC-33
	SEQ ID 13
	ttcttgaagacgaaagggcctcgtgatacgcctatttttataggtt

	aatgtcatgataataatggtttcttagacgtcaggtggcacttttc

	ggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

	ttcaaatatgtatccgctcatgagacaataaccctgataaatgctt

	caataatattgaaaaaggaagagtcatATGCGATTTAAAAAAATTT

	CTTGTCTACTTTTATCCCCGCTTTTTATTTTTAGTACCTCAATTTA

	TGCGGGCAATACACCAAAAGACCAAGAAATTAAAAAACTGGTAGAT

	CAAAACTTTAAACCGTTATTAGAAAAATATGATGTGCCAGGTATGG

	CTGTGGGTGTTATTCAAAATAATAAAAAGTATGAAATGTATTATGG

	TCTTCAATCTGTTCAAGATAAAAAAGCCGTAAATAGCAGTACCATT

	TTTGAGCTAGGTTCTGTCAGTAAATTATTTACCGCGACAGCAGGTG

	GATATGCAAAAAATAAAGGAAAAATCTCTTTTGACGATACGCCTGG

	TAAATATTGGAAAGAACTAAAAAACACACCGATTGACCAAGTTAAC

	TTACTTCAACTCGCGACGTATACAAGTGGTAACCTTGCCTTGCAGT

	TTCCAGATGAAGTAAAAACAGACCAACAAGTTTTAACTTTTTTCAA

	AGACTGGAAACCTAAAAACTCAATCGGTGAATACAGACAATATTCA

	AATCCAAGTATTGGCCTATTTGGAAAGGTTGTGGCTTTGTCTATGA

	ATAAACCTTTCGACCAAGTCTTAGAAAAAACAATTTTTCCGGCCCT

	TGGCTTAAAACATAGCTATGTAAATGTACCTAAGACCCAGATGCAA

	AACTATGCATTTGGTTATAACCAAGAAAATCAGCCGATTCGAGTTA

	ACCGCGGCCCACTCGATGCCGCCCCTGCGTATGGCGTCAAATCGAC

	ACTACCCGACATGTTGAGTTTTATTCATGCCAACCTTAACCCACAG

	AAATATCCGGCTGATATTCAACGGGCAATTAATGAAACACATCAAG

	GGCGCTATCAAGTAAATACCATGTATCAGGCACTCGGTTGGGAAGA

	GTTTTCTTATCCGGCAACGTTACAAACTTTATTAGACAGTAATTCA

	GAACAGATTGTGATGAAACCTAATAAAGTGACTGCTATTTCAAAGG

	AACCTTCAGTTAAGATGTACCATAAAACTGGCTCAACCAACGGTTT

	CGGAACGTATGTAGTGTTTATTCCTAAAGAAAATATTGGCTTAGTC

	ATGTTAACCAATAAACGTATTCCAAATGAAGAGCGCATTAAGGCAG

	CTTATGCTGTGCTGAATGCAATAAAGAAATAAggatccctgtcaga

	ccaagtttactcatatatactttagattgatttaaaacttcatttt

	taatttaaaaggatctaggtgaagatcctttttgataatctcatga

	ccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccc

	cgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgc

	gtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtgg

	tttgtttgccggatcaagagctaccaactctttttccgaaggtaac

	tggcttcagcagagcgcagataccaaatactgtccttctagtgtag

	ccgtagttaggccaccacttcaagaactctgtagcaccgcctacat

	acctcgctctgctaatcctgttaccagtggctgctgccagtggcga

	taagtcgtgtcttaccgggttggactcaagacgatagttaccggat

	aaggcgcagcggtcgggctgaacggggggttcgtgcacacagccca

	gcttggagcgaacgaacctacaccgaactgagatacctacagcgtg

	agctatgagaaagcgccacgcttcccgaagggagaaaggcggacag

	gtatccggtaagcggcagggtcggaacaggagagcgcacgagggag

	cttccagggggaaacgcctggtatctttatagtcctgtcgggtttc

	gccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggg

	gcggagcctatggaaaaacgccagcaacgcggcctttttacggttc

	ctggccttttgctggccttttgctcacatgttctttcctgcgttat

	cccctgattctgtggataaccgtattaccgcctttgagtgagctga

	taccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagc

	gaggaagcggaagagcgcctgatgcggtattttctccttacgcatc

	tgtgcggtatttcacaccgcatttggtgcactctcagtacaatctg

	ctctgatgccgcatagttaagccagtatacactccgctatcgctac

	gtgactgggtcatggctgcgccccgacacccgccaacacccgctga

	cgcgccctgacgggcttgtctgctcccggcatccgcttacagacaa

	gctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgt

	catcaccgaaacgcgcgaggcagctgaggtaaagctcatcagcgtg

	gtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagc

	tcgttgagtttctccagaagcgttaatgtctggcttctgataaagc

	gggccatgttaagggcggttttttcctgtttggtcactgatgcctc

	cgtgtaagggggatttctgttcatgggggtaatgataccgatgaaa

	cgagagaggatgctcacgatacgggttactgatgatgaacatgccc

	ggttactggaacgttgtgagggtaaacaactggcggtatggatgcg

	gcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgtt

	aatacagatgtaggtgttccacagggtagccagcagcatcctgcga

	tgcagatccggaacataatggtgcagggcgctgacttccgcgtttc

	cagactttacgaaacacggaaaccgaagaccattcatgttgttgct

	caggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgc

	gtatcggtgattcattctgctaaccagtaaggcaaccccgccagcc

	tagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggc

	caggacccaacgctgcccgagatgcgccgcgtgcggctgctggaga

	tggcggacgcgatggatatgttctgccaagggttggtttgcgcatt

	cacagttctccgcaagaattgattggctccaattcttggagtggtg

	aatccgttagcgaggtgccgccggcttccattcaggtcgaggtggc

	ccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtat

	agggcggcgcctacaatccatgccaacccgttccatgtgctcgccg

	aggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagt

	taggctggtaagagccgcgagcgatccttgaagctgtccctgatgg

	tcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcc

	cgatgccgccggaagcgagaagaatcataatggggaaggccatcca

	gcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggcc

	gccatgccggcgataatggcctgcttctcgccgaaacgtttggtgg

	cgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaa

	taccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcgg

	tcctcgccgaaaatgacccagagcgctgccggcacctgtcctacga

	gttgcatgataaagaagacagtcataagtgcggcgacgatagtcat

	gcccgcgcccaccggaaggagctgactgggttgaaggctctcaagg

	gcatcggtcgacgctctcccttatgcgactcctgcattaggaagca

	gcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaat

	ggtgcatgcaaggagatggcgcccaacagtcccccggccacggggc

	ctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtg

	gcgagcccgatcttccccatcggtgatgtcggcgatataggcgcca

	gcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccgg

	cgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtc

	gatagtggctccaagtagcgaagcgagcaggactgggcggcggcca

	aagcggtcggacagtgctccgagaacgggtgcgcatagaaattgca

	tcaacgcatatagcgctagcagcacgccatagtgactggcgatgct

	gtcggaatggacgatatcccgcaagcggcccggcagtaccggcata

	accaagcctatgcctacagcatccagggtgacggtgccgaggatga

	cgatgagcgcattgttagatttcatacacggtgcctgactgcgtta

	gcaatttaactgtgataaactaccgcattaaagcttatcgatgata

	agctgtcaaacatgagaa

Example 35

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, Eschericia 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 μg/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 to 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 ˜10⁵colony 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 BLIcompound 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 36

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 12^thwell 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, Eschericia 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 ˜10⁵colony 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 μg/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 37

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 k_observed(k_obs).
k_obsvs. 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 (k_inact). Table IV shows kinetics results from representative test compounds. A larger k_inact/K ratio indicates a more effective enzyme inactivator.
Y _t =V ₀*(1−e ^(−k ^obs*^t))/k _obs Eq. 1
Where Y is the absorbance at time t, V₀is the uninhibited enzyme velocity, k_obsis the observed rate constant of the enzyme inactivation.
k _obs =k _inact *[I]/([I]+K(1+S/K _m)) Eq. 2
Where S is the NCF concentration, K_mis the KPC-2 K_mfor NCF

Claims

1-50. (canceled)

51. An antibiotic composition comprising ceftolozane and a compound of formula CCC:

52. The composition of claim 51, wherein the composition is formulated for intravenous delivery upon reconstitution.

53. The composition of claim 51, wherein the composition is in a unit dosage form comprising 100-1000 mg of the compound of formula (CCC).

54. The composition of claim 51, wherein the composition is in a unit dosage form comprising ceftolozane and the compound of formula (CCC) in a ratio between 1:4 and 8:1.

55. The unit dosage form of claim 54 comprising 100-1000 mg of the compound of formula (CCC).

56. The composition of claim 51, wherein the composition is a reconstituted composition obtained by a process comprising dissolving a composition comprising the ceftolozane and the compound of formula (CCC) in a pharmaceutically appropriate carrier to obtain the reconstituted composition.

57. A method of treating an infection comprising the administration of a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane and a compound of formula CCC:

58. The method of claim 57, wherein the composition is formulated for intravenous delivery upon reconstitution.

59. The method of claim 57, wherein the pharmaceutical composition is administered in three equal doses per day.

60. The method of claim 57, wherein the therapeutically effective amount comprises 100-1000 mg of the compound of formula (CCC) per unit dose.

61. The method of claim 57, wherein the therapeutically effective amount comprises ceftolozane and the compound of formula (CCC) in a ratio between 1:4 and 8:1.

62. The method of claim 57, wherein:

a. the therapeutically effective amount comprises ceftolozane and the compound of formula (CCC) in a ratio of 1:4 to 8:1;

b. the therapeutically effective amount comprises 100-1000 mg of the compound of formula (CCC) per unit dose; and

c. the pharmaceutical composition is administered in three equal doses per day.

63. The method of claim 57, wherein the composition has a MIC₉₀of about 8 μg/mL or less.

64. The method of claim 57, wherein the composition has a MIC₅₀of about 2 μg/mL or less.

65. The method of claim 57, wherein the infection is caused by a cephalosporin resistant pathogen.

66. The method of claim 57, wherein the infection is selected from the group consisting of intra-abdominal infection, urinary tract infection, and pneumonia.

67. The method of claim 57, wherein the infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae, Eschericia coli, Enterobacter spp, Citrobacter spp, and Pseudomonas aeruginosa.

68. A method of treating an infection caused by a bacteria selected from the group consisting of Klebsiella pneumoniae, Eschericia coli, Enterobacter spp, Citrobacter spp, and Pseudomonas aeruginosa, the method comprising the administration of a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane and a compound of formula CCC:

69. The method of claim 68, wherein:

a. the therapeutically effective amount comprises ceftolozane and the compound of formula (CCC) in a ratio of 1:4 to 8:1; and

b. the therapeutically effective amount comprises 100-1000 mg of the compound of formula (CCC) per unit dose.

70. The method of claim 68, wherein the infection is selected from the group consisting of intra-abdominal infection, urinary tract infection, and pneumonia.