# Patent

## Title
Beta-lactamase inhibitors

## Abstract

Aryl substituted diazabicyclooctanes (DBO) compounds that inhibit β-lactamases of class A, class C or class D and potentiate β-lactam antibiotics are disclosed. In particular, this disclosure provides DBO compounds that, when used in the disclosed Synergy MIC Assay with a β-lactam antibiotic at a fixed concentration have an MIC of 8 μg/mL or less against one or more isogenic β-lactamase expressing bacterial strains.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/618,119, filed Mar. 30, 2012, incorporated herein by reference, and U.S. Provisional Application No. 61/792,672, filed Mar. 15, 2013, also incorporated herein by reference. This application is also related to U.S. patent application Ser. Nos. 13/853,443, 13/853,498 and 13/853,506, filed Mar. 29, 2013, the contents of which are incorporated herein by reference in their entirety.


TECHNICAL FIELD
This invention is directed to β-lactamase inhibitors (BLI) or compositions comprising β-lactamase inhibitors.
BACKGROUND
Bacterial resistance to β-lactam antibiotics is most commonly mediated by inactivation by β-lactamases. There is an urgent need for novel BLIs that are effective in inhibiting β-lactamases thus, slowing or preventing the degradation of the β-lactam antibiotic and restoring β-lactam antibiotic susceptibility to β-lactamase producing bacteria.
SUMMARY OF INVENTION
Aryl substituted diazabicyclooctanes (DBO) compounds that potentiate (i.e. make more potent) β-lactam antibiotics are disclosed. In particular, this invention provides DBO compounds that inhibit β-lactamases of class A, C and D, such as KPC-2, CTX-M-15, SHV-12 and P99 AmpC and potentiate a β-lactam antibiotic as evidenced by the Synergy MIC (sMIC) assay described in Example 23. Unexpectedly, many of these compounds are better inhibitors of OXA-15 β-lactamase than previously reported diazabicyclooctanes. These compounds have potential as better inhibitors of the β-lactamase enzyme.
The present invention provides, in one aspect, compounds of chemical Formula (I), or a pharmaceutically acceptable salt thereof wherein the compound of Formula I, when used in the Synergy MIC Assay of Example 23 with an antibiotic selected from CXA-101 (Ceftolozane) or ceftazidime at a fixed concentration of 4 μg/mL, has an MIC of 8 μg/mL or less against one or more isogenic β-lactamase expressing bacterial strains from Table X.




    
    
        wherein
        Z is a five-membered heteroaryl ring containing from one to four hetero atoms selected from N, O or S;
        R is selected
        from
    
    






    
    
        and
        R1 is selected from:
        
            a. hydrogen,
            b. cyano,
            c. (C1-C3)-unsubstituted alkyl,
            d. (C1-C3)-alkyl substituted with at least one substituent selected from hydroxyl,
        
        
    
    






    
    
        
        
             wherein each of R2, R3 and R4 is independently selected from hydrogen, (C1-C3)-alkyl, aminoalkyl, aminocycloalkyl, or hydroxyalkyl,
            e.
        
        
    
    






wherein R5 is selected from H or aminoalkyl,

    
    
        
        
            f.
        
        
    
    






    
    
        
        
            g. amino,
            h. (C1-C3)alkylamino, or
            i. a 4-6 membered heterocyclyl containing 1-2 heteroatoms selected from O and N, or NX, wherein X is H,
        
        
    
    






    
    
        provided that the compound of Formula (I) is not a compound wherein:
        (1) Z is imidazol-2-yl, R1* is hydrogen and R* is —OSO3H;
        (2) Z is 1,2,4-triazol-5-yl, R1* is methyl and R* is —OSO3H;
        (3) Z is 5-oxo-1,3,4-oxadiazol-2-yl, R1* is hydrogen or piperidin-4-yl, and R* is —OSO3H;
        (4) Z is 1,2,3-triazol-4-yl, R1* is methyl and R* is —OSO3H;
        (5) Z is pyrazol-3-yl, R1* is hydrogen or methyl, and R* is —OSO3H; or
        (6) Z is oxazol-2-yl, R1* is hydrogen and R* is —OSO3H;
        wherein Table X is:
    
    













TABLE X









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









In another aspect, the BLIs of the invention can be used in conjunction with a β-lactam antibiotic for evaluating BLIs for treatment in humans.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows Table I, Representative Compounds of The Invention.
FIG. 1B continues Table I.
FIG. 2 shows Table II, the Synergy MIC of representative compounds of Formula I against a panel of isogenic strains expressing β-lactamases.
FIG. 3 shows Table III, an assay to determine inhibition kinetics of representative compounds of Formula I for the KPC-2 β-lactamase.



DETAILED DESCRIPTION
Definitions
Molecular terms, when used in this application, have their common meaning unless otherwise specified.
The term “alkyl” is defined as a linear or branched, saturated radical having one to about twenty carbon atoms unless otherwise specified. Preferred alkyl radicals are “lower alkyl” radicals having one to about five carbon atoms. Examples of alkyl groups include, without limitation, methyl, ethyl, tert-butyl, isopropyl, and hexyl. A subset of the term alkyl is “(C1-C3)-unsubstituted alkyl” which is defined as an alkyl group that bears no substituent groups. Examples of (C1-C3)-unsubstituted alkyl groups include methyl, ethyl, propyl and isopropyl. It is understood that if a (C1-C3)-alkyl is “substituted” that one or more hydrogen atoms is replaced by a substitutent.
The term amino denotes a NH2 radical
The term “aminoalkyl” denotes an alkyl in which one or more of the alkyl hydrogen atoms has been replaced by an amino group.
The term “aminocycloalkyl” denotes a cycloalkyl in which one of the cycloalkyl hydrogen atoms has been replaced by an amino group.
The term “cycloalkyl” or “cycloalkyl ring” is defined as a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to twelve ring members. In a preferred embodiment, a cycloalkyl is a ring system having three to seven ring members. Examples of a cycloalkyl group include, without limitation, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl.
The term “heteroaryl” or “heteroaryl ring” denotes an aromatic ring which contain one to four hetero atoms or hetero groups selected from O, N, S. Examples of 5-membered heteroaryl groups include, without limitation, furanyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl, thidiazolyl, oxadiazolyl, triazolyl and tetrazolyl.
The term “heterocyclyl,” “heterocyclic” or “heterocyclyl ring” is defined as a saturated or partially unsaturated ring containing one to four hetero atoms or hetero groups selected from O, N, or NX, wherein X is H,




in a single or fused heterocyclic ring system having from three to twelve ring members unless otherwise specified. In a preferred embodiment, a heterocyclyl is a ring system having four to six ring members. Examples of a heterocyclyl group include, without limitation, azetidinyl, morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl.

The term “hydroxyalkyl” denotes an alkyl 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
    
    






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

The term “therapeutically-effective dose” and “therapeutically-effective amount” refer to an amount of a compound that prevents the onset, alleviates the symptoms, stops the progression of a bacterial infection, or results in another desired biological outcome such as, e.g., improved clinical signs or reduced/elevated levels of lymphocytes and/or antibodies. The term “treating” or “treatment” is defined as administering, to a subject, a therapeutically-effective amount of one or more compounds both to prevent the occurrence of an infection and to control or eliminate an infection. Those in need of treatment may include individuals already having a particular medical disease as well as those at risk for the disease (i.e., those who are likely to ultimately acquire the disorder).
The term “subject,” as used herein, refers to a mammal, a plant, a lower animal, or a cell culture. In one embodiment, a subject is a human or other animal patient in need of antibacterial treatment.
The term “administering” or “administration” and the like, refers to providing the compound of Formula Ito the subject in need of treatment. Preferably the subject is a mammal, more preferably a human.
The functional classification of β-lactamases and terms “class A”, “class C”, and “class D” β-lactamases are understood by one of skill in the art and are described in “Updated Functional Classification of β-Lactamases”, Bush, K.; Jacoby, G. A.; Antimicrob. Agents Chemother. 2010, 54, 969-976, herein incorporated by reference.
The salts of the compounds of the invention include acid addition salts and base addition salts. In a one embodiment, the salt is a pharmaceutically acceptable salt of the compound of Formula I. The term “pharmaceutically acceptable salts” embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds of the invention may be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Examples of appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include, without limitation, formic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic, galactic, and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts of compounds of the invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine and procaine. All of these salts may be prepared by conventional means from the corresponding compound of the invention by treating, for example, the compound of the invention with the appropriate acid or base.
The compounds of the invention can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or non-racemic mixtures thereof. The compounds of the invention can be utilized in the present invention as a single isomer or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment with an optically active acid or base. Examples of appropriate acids include, without limitation, tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. The mixture of diastereomers can be separated by crystallization followed by liberation of the optically active bases from the optically active salts. An alternative process for separation of optical isomers includes the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers. Still another available method involves synthesis of covalent diastereoisomeric molecules by treating compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to obtain the enantiomerically pure compound. The optically active compounds of the invention can likewise be obtained by utilizing optically active starting materials. These isomers may be in the form of a free acid, a free base, an ester or a salt.
The invention also embraces isolated compounds. An isolated compound refers to a compound which represents at least 10%, such as at least 20%, such as at least 50% and further such as at least 80% of the compound present in the mixture. In one embodiment, the compound, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound exhibits detectable (i.e. statistically significant) activity when tested in conventional biological assays such as those described herein.
β-Lactamase Inhibitors (BLIs)
The present invention provides, in one aspect, compounds of chemical Formula (I), or a pharmaceutically acceptable salt thereof wherein the compound of Formula I, when used in the Synergy MIC Assay of Example 23 with an antibiotic selected from ceftolozane or ceftazidime at a fixed concentration of 4 μg/mL, has an MIC of 8 μg/mL or less against one or more isogenic β-lactamase expressing bacterial strains from Table X.




The group Z of Formula I is a five-membered heteroaryl ring containing from one to four hetero atoms selected from N, O and S. In one aspect group Z is a five membered heteroaryl ring containing from one to three heteroatoms selected from N, O, or S.
Substituent R of Formula I is selected from




In a preferred embodiment, R is





The group R1 of Formula I is selected from:

    
    
        a. hydrogen,
        b. cyano,
        c. (C1-C3)-unsubstituted alkyl,
        d. (C1-C3)-alkyl substituted with at least one substituent selected from hydroxyl,
    
    






wherein each of R2, R3 and R4 is independently selected from hydrogen, (C1-C3)-alkyl, aminoalkyl, aminocycloalkyl, or hydroxyalkyl,

    
    
        e.
    
    






wherein R5 is selected from H or aminoalkyl,

    
    
        f.
    
    






    
    
        g. amino,
        h. (C1-C3)alkylamino, or
        i. a 4-6 membered heterocyclyl containing 1-2 heteroatom moieties selected from O and N, or NX, wherein X is H,
    
    






    
    
        provided that the compound of Formula (I) is not a compound wherein:
        (1) Z is imidazol-2-yl, R1* is hydrogen and R* is —OSO3H;
        (2) Z is 1,2,4-triazol-5-yl, R1* is methyl and R* is —OSO3H;
        (3) Z is 5-oxo-1,3,4-oxadiazol-2-yl, R1* is hydrogen or piperidin-4-yl, and R* is —OSO3H;
        (4) Z is 1,2,3-triazol-4-yl, R1* is methyl and R* is —OSO3H;
        (5) Z is pyrazol-3-yl, R1* is hydrogen or methyl, and R* is —OSO3H; or
        (6) Z is oxazol-2-yl, R1* is hydrogen and R* is —OSO3H.
    
    


The isogenic stains are selected from Table X











TABLE X









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









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




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.
Table 1 provides representative compounds of the invention, particularly compounds of Formula II (See FIG. 1).
In another aspect, the invention provides compounds of Formula A-I or pharmaceutically-acceptable salts thereof:




The group Z of Formula A-I is a five-membered heteroaryl ring containing from one to four hetero atoms selected from N, O and S. In one aspect group Z is a five membered heteroaryl ring containing from one to three heteroatoms selected from N, O, or S.
Substituent R* of Formula A-I is selected from




In a preferred embodiment, R* is




The group R1* of Formula A-I is selected from:

    
    
        a. hydrogen;
        b.
    
    






    
    
        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 or (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;
        c.
    
    






    
    
        wherein R8* is selected from NH2,
    
    






wherein each of R4*, R5*, R6*, and R7* is as described previously and each of R9, R10, and R11 is independently selected from hydrogen or (C1-C6)-alkyl, provided that at least one of R9, R10, and R11 is hydrogen;

    
    
        d. amino;
        e.
    
    






    
    
        Z is selected from CR12R13 or NR14,
        each of R12 and R13 is independently selected from H, NH2 and
    
    






wherein each of R4*, R5*, R6*, and R7*, is as described previously,

    
    
        alternatively, R12 and R13 together with the carbon to which they are attached, form a cycloalkyl or heterocyclyl ring containing 4-6 ring members,
        R14 is selected from H and
    
    






each of R15, R16 and R17 is independently selected from hydrogen, (C1-C6)-alkyl, aminoalkyl, aminocycloalkyl, and hydroxyalkyl, provided that at least one of R15, R16 and R17 is hydrogen,

    
    
        R18 is selected from NH2 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;
        f.
    
    






    
    
        wherein R19 is selected from NH2 and
    
    






wherein each of R4*, R5*, R6*, and R7* is as described previously,

    
    
        R20 is selected from amino and hydroxyl, and
        w is selected from 0 and 1;
        g.
    
    






    
    
        h.
    
    






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

    
    
        i.
    
    






    
    
        wherein R21 is selected from NH2, —NH(C1-C3)-alkyl and
    
    






wherein each of R4*, R5*, R6*, and R7* is as described previously,

    
    
        s selected from 0 and 1, and
        v is selected from 0, 1, 2, and 3;
        j.
    
    






    
    
        k.
    
    






    
    
        wherein M is selected from NR22, CR23R24 and O,
        wherein R22 is H or
    
    






wherein each of R15, R16, K and R17 is as described previously,

    
    
        each of R23 and R24 is independently selected from H, NH2 and
    
    






wherein each of R4*, R5*, R6* and R7* is as described previously, and

    
    
        u is selected from 0, 1 and 2;
        l.
    
    






    
    
        m.
    
    






    
    
        n.
    
    






    
    
        o. (C1-C3)-unsubstituted alkyl;
        p. and
    
    






    
    
        provided that the compound of Formula (A-I) is not a compound wherein:
        (1) Z is imidazol-2-yl, R1* is hydrogen and R* is —OSO3H;
        (2) Z is 1,2,4-triazol-5-yl, R1* is methyl and R* is —OSO3H;
        (3) Z is 5-oxo-1,3,4-oxadiazol-2-yl, R1* is hydrogen or piperidin-4-yl, and R* is —OSO3H;
        (4) Z is 1,2,3-triazol-4-yl, R1* is methyl and R* is —OSO3H;
        (5) Z is pyrazol-3-yl, R1* is hydrogen or methyl, and R* is —OSO3H; or
        (6) Z is oxazol-2-yl, R1* is hydrogen and R* is —OSO3H.

Use of the Compounds of Formulas I, A-I and II

    
    


In one aspect of the invention the compounds of Formulas I, A-I and II are effective in inhibiting β-lactamases expressed by isogenic strains. In one aspect of the invention, the β-lactamases are selected from class A, class C or class D β-lactamases. Class A β-lactamases for example, include, but are not limited to, TEM, SHV, CTX-M, KPC, GES, VEB, SME, and GEX. In a preferred aspect of the invention, the compounds of the invention inhibit KPC β-lactamases. More preferably the compounds of the invention inhibit KPC-2 or KPC-3 β-lactamases. 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 are OXA β-lactamases.
In one aspect of the invention, the β-lactamases expressed by isogenic strains are selected from KPC, CTX-M, SHV, OXA or AmpC β-lactamases. In another aspect of the invention, the isogenic strains that express β-lactamases are prepared according to the method described in Example 22. In another aspect of the invention the isogenic strains are those described in Table X.
Unless otherwise indicated, the activity of the BLI compounds can be described by the MIC value obtained from a Synergy MIC assay (e.g. as described herein). The lower the sMIC 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 data supports that the compounds of Formulas I, A-I and II potentiate (i.e. make more potent) the activity of the β-lactam antibiotic against beta-lactamase producing strains by inhibiting the β-lactamase.
In one aspect of the invention, the BLI activity is measured by the Synergy MIC Assay described in Example 23 as the sMIC. The sMIC gives the value required for the BLI to potentiate the activity of 4 μg/mL of CXA-101 (Ceftolozane) or ceftazidime to inhibit the growth of β-lactamase producing bacteria. Growth is 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.
Preferably, the Synergy MIC is 8 μg/mL or less. In a more preferred aspect of the invention, the Synergy MIC is 4 to 8 μg/mL. In an even more preferred aspect of the invention, the Synergy MIC is 1 to 2 μg/mL. In a still more preferred aspect of the invention, the Synergy MIC is 0.2 to 0.5 μg/mL. Synergy MICs for representative compounds of the invention are described in Table II (See FIG. 2). It will be understood by one of skill in the art that the growth inhibition of the isogenic β-lactamase producing strains can also be measured by a checkerboard synergy assay like that disclosed in International Patent Application Number WO 2008039420 or a standard BLI potentiation assay using a fixed concentration of BLI.
The compounds of the present invention have a broad spectrum of activity across a wide variety of β-lactamase producing bacteria. It was surprisingly found that the compounds of the present invention are active in potentiating activity of β-lactam antibiotics, in particular, Ceftolozane, against strains expressing class D β-lactamases, in particular the OXA-15 β-lactamase. Currently marketed BLIs inhibit most of the class A β-lactamases, but poorly inhibit class A KPC and class C β-lactamases and have variable success in inhibiting penicillinase and carbapenemase-type class D β-lactamases. The compounds of the present invention are active against 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). This increased activity against the class D β-lactamase is critical because differential effectiveness against different types of β-lactamase producing bacteria is necessary in order to effectively use β-lactam antibiotics to treat resistant strains of bacteria (vide infra).
In one embodiment, the compounds of Formulas I, A-I and II are as active, or more active against bacterial strains that express KPC β-lactamase than the most structurally similar compound Avibactam (comparator compound CCC). Compounds that are as active or more active than Avibactam are, for example, compounds 101, 102, 103, 200, 201, 203, 204, 205, 206, 300, 301, 302 and 401.
In one embodiment, the compounds of Formulas I, A-I and II are as active against bacterial strains that express CTX-M-15 β-lactamase than the most structurally similar compound Avibactam (comparator compound CCC). Compounds that are as active as Avibactam are, for example, compounds 101, and 300.
In one embodiment, the compounds of Formulas I, A-I and II are as active against bacterial strains that express SHV-12 β-lactamase than the most structurally similar compound Avibactam (comparator compound CCC). Compounds that are as active as Avibactam are, for example, compounds 103, 200, 300, 301, 302, 401, and 402.
In one embodiment, the compounds of Formulas I, A-I and II are as active against bacterial strains that express P99 β-lactamase than the most structurally similar compound Avibactam (comparator compound CCC). Compounds that are as active as Avibactam are, for example, compounds 101, 204, 206, 300, 301, and 500.
In one embodiment, the compounds of Formulas I, A-I and II are unexpectedly more active against bacterial strains that express OXA-15 β-lactamase than the most structurally similar compound Avibactam (comparator compound CCC). Compounds that are more active than Avibactam are, for example, compounds 200, 201, 204, 205, 206, 300, 301, and 302.
In another embodiment, the compounds of Formulas I, A-I and 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 Formulas I, A-I and II was measured according to the procedure outlined in Example 24. The compounds of Formulas I, A-I and II have a high binding affinity for the β-lactamase enzyme.
In one embodiment the compounds of Formulas I, A-I and II have a binding affinity of 1000-5000 mM−1s−1. Compounds that have a binding affinity of 1000-5000 mM−1s−1 are, for example, compounds 102 and 103 (Table III).
In one embodiment the compounds of Formulas I, A-I and II have a binding affinity of 100-999 mM−1s−1. Compounds that have a binding affinity of 100-999 mM−1s−1 are, for example, compounds 101, 200, 201, 204, 205, 206, 300, 301, 302, 400, 401, 402, and 500 (Table III).
In one embodiment the compounds of Formulas I, A-I and II have a binding affinity of 1-99 mM−1s−1. Compounds that have a binding affinity of 1-99 mM−1s−1 are, for example, compounds 202 and 203 (Table III).
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 III, See FIG. 3)
In another aspect, the compounds of the present invention are useful in evaluating BLIs for treatment in humans. Compounds of Formulas I, A-I and II when used in the Synergy MIC Assay of Example 23 with an antibiotic selected from ceftolozane or ceftazidime at a fixed concentration of 4 μg/mL, that have a sMIC value of 8 μg/mL or less against at least one isogenic β-lactamase expressing strain of Table X are useful candidates for human treatment against β-lactamase producing bacteria Inhibition of β-lactamases with BLIs slows or prevents degradation of β-lactam antibiotics and restores β-lactam antibiotic susceptibility to β-lactamase producing bacteria. Thus these compounds in conjunction with a β-lactam antibiotic are useful in treating bacterial infections in humans caused or exacerbated by β-lactamase producing bacteria.
In one aspect of the invention, the bacterial infection is caused by class A, class C or class D β-lactamase producing bacteria.
Representative Gram-negative pathogens known to express β-lactamases include, but are not limited to Acinetobacter spp. (including Acinetobacter baumannii), Citrobacter spp., Escherichia spp. (including Escherichia coli), Haemophilus influenzae, Morganella morganii, Pseudomonas aeruginosa, Klebsiella spp. (including Klebsiella pneumoniae), Enterobacter spp. (including Enterobacter cloacae and Enterobacter aerogenes), Pasteurella spp., Proteus spp. (including Proteus mirabilis), Serratia spp. (including Serratia marcescens), and Providencia spp. Bacterial infections can be caused or exacerbated by Gram-negative bacteria including strains which express β-lactamases that may confer resistance to penicillins, cephalosporins, monobactams and/or carbapenems. The co-administration of a novel BLI that inhibits these β-lactamases with a β-lactam antibiotic could be used to treat infections caused β-lactam resistant bacteria.
β-Lactam antibiotics that may be co-administered with compounds of Formula I include, but are not limited to cephalosporins (e.g. Ceftolozane), carbapenem (e.g. Meropenem), monobactam (e.g. Aztreonam), and penicillin (e.g. Piperacillin) classes of antibiotics.
Another object of the invention is pharmaceutical compositions or formulations comprising compounds of Formulas I, A-I and II, 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 compositions can comprise one or more of the compounds disclosed herein, preferably a compound of Formula I, A-I or 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 and includes tablet binders, lubricants, flavoring agents, preservatives, wetting agents, emulsifying agents, and dispersing agents. Compounds of the present invention preferably a compound of Formula I, A-I or II in conjunction with a β-lactam antibiotic, can be, for example:

    
    
        1. in the form of tablets, capsules, elixirs, suspensions, syrups, wafers, pills powders, granules and the like,
        2. dissolved or suspended in any of the commonly used intravenous fluids (e.g. physiological saline or Ringer's solution) and administered by infusion,
        3. in the form of 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,
        4. in the form of microencapsulating matrices of the drug in biodegradable polymers such as polylactide-polyglycolide,
        5. dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5% glucose,
        6. in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders,
        7. 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,
        8. in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery.
    
    


In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof a therapeutically-effective amount of the pharmaceutical composition comprising a compound of Formula I and a β-lactam antibiotic.
In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof, a therapeutically-effective amount of a β-lactam antibiotic in conjunction with a compound of Formula I, A-I or II.
In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection in a subject comprising the steps of
a. administering to the subject a compound of Formula I, A-I or II; and
b. administering to a subject a therapeutically-effective amount of a β-lactam antibiotic.
In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection in a subject comprising the steps of
a. administering to a subject a therapeutically-effective amount of a β-lactam antibiotic; and
b. administering to the subject a compound of Formula I, A-I or II.
The pharmaceutical compositions, preferably a compound of Formula I, A-I or 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.
Actual dosage levels of active ingredients in the pharmaceutical compositions of one or more compounds according to Formula I, A-I or II, preferably a compound of Formula I, A-I or 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 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 when the composition comprises a compound of Formula I, A-I or II and a β-lactam antibiotic, both the compound of Formula I, A-I or 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 Formula I, A-I or II, preferably in conjunction with a β lactam antibiotic. An effective dose of a compound of Formula I, A-I or II is generally between 125 mg/day to 2000 mg/day.
In one embodiment, a β-lactam antibiotic and a compound of Formula I, A-I or II are administered in ratio of 1:4 to 8:1 antibiotic:Formula I, A-I or II compound. In one embodiment the ratio is 1:4. In another embodiment the ratio is 3:4. In another embodiment the ratio is 5:4. In another embodiment the ratio is 7:4. In another embodiment the ratio is 1:2. In another embodiment the ratio is 3:2. In another embodiment the ratio is 5:2. In another embodiment the ratio is 7:2. In another embodiment the ratio is 1:3. In another embodiment the ratio is 2:3. In another embodiment the ratio is 4:3. In another embodiment the ratio is 5:3. In another embodiment the ratio is 7:3. In another embodiment the ratio is 1:2. In another embodiment the ratio is 3:2. In another embodiment the ratio is 5:2. In another embodiment the ratio is 7:2. In another embodiment the ratio is 1:1. In another embodiment the ratio is 2:1. In another embodiment the ratio is 3:1. In another embodiment the ratio is 4:1. In another embodiment the ratio is 5:1. In another embodiment the ratio is 6:1. In another embodiment the ratio is 7:1. In another embodiment the ratio is 8:1. It will be understood by one of skill in the art that the β-lactam antibiotic and compound of Formula I, A-I or 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 Formula I, A-I or 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 Formula I, A-I or II or vice versa.
One or more compounds of Formulas I, A-I and II, preferably a compound of Formula I, A-I or II in conjunction with a β-lactam antibiotic, can be administered as a single daily dose or in multiple doses per day.
One or more compounds of Formulas I, A-I and II, preferably a compound of Formula I, A-I or II in conjunction with a β-lactam antibiotic, may be administered according to this method until the bacterial infection is eradicated or reduced.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
Preparation of Compounds of Formulas I, A-I and II
A compound of Formulas I, A-I and 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
        sat.=saturated
        rt=room temperature
        TBAF=tetrabutylammonium fluoride
        TBS=t-butyldimethylsilyl
        TES=triethylsilyl
        TEA=triethylamine
        TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy, free radical
        THF=tetrahydrofuran
        TFA=trifluoroacetic acid
        TMS=trimethylsilyl
        TLC=thin layer chromatography
        VAP=Ventilator-Associated Pneumonia
    
    


The compounds of Formulas I, A-I and II can be prepared from intermediates 1 or 7, according to the following reaction schemes and examples, or modifications thereof, using readily available starting materials, reagents and conventional synthetic procedures including, for example, procedures described in U.S. Pat. No. 7,112,592 and WO2009091856. As depicted in Scheme 1, compound 3 can be synthesized following standard heterocyclic ring formation chemistry under appropriate reaction conditions from ester intermediate 1, or its corresponding derivatives, such as carboxylic acid derivative 2a and aldehyde derivative 2b (see, e.g., 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, hereafter Walker). Appropriate functional group protections for the R group are needed when necessary




Alternatively, compound 3 can be synthesized from intermediate 7 as shown in Scheme 2. Monocyclic ester intermediate 7 can be converted to 8 under standard Mitsunobu reaction conditions. Compound 9 can then be prepared following standard heterocyclic ring formation chemistry under appropriate reaction conditions from ester intermediate 8, or its corresponding derivatives (see, e.g., Walker.
It may be necessary to protect certain functionalities in the molecule depending on the nature of the R1 group. Protecting these functionalities should be within the expertise of one skilled in the art. See, e.g. P. G. M. Wuts and T. W. Greene, Protective Groups in Organic Synthesis, Fourth Edition, John Wiley and Sons, 2006, hereafter Greene.
Deprotection of the N-Ns group in compound 9 provides compound 10, which can be converted to compound 11 by treatment with diphosgene. Compound 3 can be obtained upon deprotection of the N-Boc group from compound 11 under appropriate conditions, such as 4M HCl in dioxane, and subsequent treatment with base, such as NEt3. Alternatively, deprotection of the N-Boc and N-Ns groups in compound 11 under appropriate conditions provides bis-amine derivative 12, which can then be cyclized to form compound 3 by treatment with diphosgene or triphogene, under appropriate conditions.




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




EXAMPLES
The specific examples which follow illustrate the synthesis of certain compounds. The methods disclosed may be adopted to variations in order to produce compounds of Formulas I, A-I and II, but not otherwise specifically disclosed. Further, the invention includes variations of the methods described herein to produce the compounds of Formulas I, A-I and II that would be understood by one skilled in the art based on the instant invention.
All temperatures are understood to be in Centigrade (C) when not specified. The nuclear magnetic resonance (NMR) spectral characteristics refer to chemical shifts (γ) expressed in parts per million (ppm) versus tetramethylsilane (TMS) as reference standard. The relative area reported for the various shifts in the proton NMR spectral data corresponds to the number of hydrogen atoms of a particular functional type in the molecule. The nature of the shifts as to multiplicity is reported as broad singlet (br s), broad doublet (br d), singlet (s), multiplet (m), doublet (d), quartet (q), doublet of doublet (dd), doublet of triplet (dt), and doublet of quartet (dq). The solvents employed for taking NMR spectra are DMSO-d6 (perdeuterodimethysulfoxide), D2O (deuterated water), CDCl3 (deuterochloroform) and other conventional deuterated solvents. The prep-HPLC conditions are: Waters SunFire® C18 (30×100 mm, 5 μm OBD) column; flow rate: 30-80 mL/minutes, ELSD or Mass-triggered fraction collection; sample loading: Each injection loading varied from 30-300 mg for different crude samples depending on their solubility and purity profiles; Solvent system using ammonium formate buffer: solvent A: water with 20 mM ammonium formate, solvent B: 85% of acetonitrile in water with 20 mM ammonium formate. Solvent system using NH4HCO3 buffer: solvent A: water with 10 mM NH4HCO3, solvent B: acetonitrile. Solvent system using NH4OH buffer: solvent A: water with 0.1% NH4OH, solvent B: acetonitrile with 0.1% NH4OH.
Example 1
Synthesis of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (Intermediate Compound 1)




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




n-BuLi was added dropwise to a solution of TMSCHN2 (690 mL, 1.38 mol) in dry THF (3 L) (600 mL, 1.5 mol) at −78° C., and the mixture was stirred at −78° C. for 30 min. 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 mM The reaction mixture was then quenched with sat. NH4Cl solution, and extracted with DCM three times. The combined organic layer was concentrated in vacuum and the crude product was purified by silica gel column chromatography (3:1 petroleum ether:EtOAc) to afford (S)-ethyl 2-((tert-butoxycarbonyl)amino)-6-diazo-5-oxohexanoate (262 g, 75%) as a yellow solid.
A solution of (S)-ethyl 2-((tert-butoxycarbonyl)amino)-6-diazo-5-oxohexanoate (350 g, 1.18 mol) in DCM (1500 mL) was added to a solution of Rh2(OAc)4 (3.5 g, 7.9 mmol) in DCM (750 mL) at 0° C. The reaction was then stirred at 20° C. overnight and then concentrated in vacuum. The crude sample was purified by silica gel column chromatography (5:1 petroleum ether/EtOAc) to afford (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (175.9 g, 55%) as a yellow oil.
Method B:




t-BuOK (330 g, 2.9 mol) was added to a solution of trimethylsulfoxonium iodide (750 g, 3.5 mol) in dry DMSO (3 L) and the mixture was stirred at rt for 1 h. (S)-1-tert-Butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate (900 g, 3.5 mol) was added and the mixture was stirred at rt for 2-3 hrs. Water was added to quench the reaction and the mixture was extracted with EtOAc 5 times. The combined organic layer was concentrated in vacuum and the crude sample was purified by silica gel column chromatography (1:1 petroleum ether/EtOAc then 1:10 MeOH/DCM) to afford sulfoxonium ylide intermediate (977 g, 80%) as a white solid.
A solution of sulfoxonium ylide intermediate (156 g, 0.446 mol) and [Ir(COD)Cl]2 (3 g, 4.46 mmol) in toluene(4 L) was degassed by bubbling nitrogen through the solution for 10 minutes. The reaction mixture was heated to 80-90° C. for 2-3 hrs and then cooled to 20° C. Then toluene was concentrated in vacuum, the residue was purified by silica gel column chromatography (10:1 to 3:1 gradient 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 solution of (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (250 g, 0.92 mol) in EtOH (1500 mL) at −40° C. The reaction mixture was then stirred at −40° C. for 0.5 hr then quenched with 10% HOAc solution. After diluting with water, the mixture was extracted with DCM three times. The combined organic layer was concentrated in vacuum and purified by silica gel column chromatography (1:1 petroleum ether/EtOAc) to afford (2S,5S)-1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (205 g, 80%) as a yellow oil.
Step 3: Synthesis of (2S,5R)-1-tert-butyl 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate




A solution of 2-nitrobenzene-1-sulfonyl chloride (500 g, 2.26 mol) in pyridine (1500 mL) was added dropwise to a solution of O-benzylhydroxylamine hydrochloride (400 g, 2.51 mol) in pyridine (1500 mL) at 0° C. The reaction mixture was then stirred at 20° C. overnight. The mixture was concentrated in vacuum, diluted with DCM and washed with HCl (10%) three times. The combined organic layer was concentrated in vacuum and re-crystallized with DCM to afford N-(benzyloxy)-2-nitrobenzenesulfonamide (485 g, 62.6%) as a yellow solid.
To a solution of N-(benzyloxy)-2-nitrobenzenesulfonamide (212 g, 0.69 mol) in THF (1000 mL) was added (2S,5S)-1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (171 g, 0.63 mol) and PPh3 (275 g, 1.05 mol), followed by dropwise addition of a solution of DEAD (195 g, 1.12 mol) in THF (500 mL). The mixture was then stirred at 20° C. overnight. The reaction mixture was then concentrated in vacuum and purified by silica gel column chromatography (3:1 petroleum ether/EtOAc) to afford (2S,5R)-1-tert-butyl 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate (283.8 g, 80%) as a yellow oil.
Step 4: Synthesis of (2S,5R)-1-tert-butyl 2-ethyl 5-((benzyloxy)amino)piperidine-1,2-dicarboxylate




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




TFA (600 mL) was added to a solution of (2S,5R)-1-tert-butyl 2-ethyl 5-((benzyloxy)amino)piperidine-1,2-dicarboxylate (263 g, 0.7 mol) in DCM (600 mL) at 20° C. The mixture was stirred at rt overnight and then concentrated in vacuum. The crude product was adjusted to pH 10 with sat. NaHCO3 solution, and then extracted with DCM three times. The combined organic layer was concentrated in vacuum and purified by silica gel column chromatography (20:1 DCM/MeOH) to afford (2S,5R)-ethyl 5-((benzyloxy)amino)piperidine-2-carboxylate (184.9 g, 95%) as a yellow oil.
Step 6: Synthesis of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate




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




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




LiBH4 (0.54 g, 24.67 mmol) was added to a solution of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (5 g, 16.44 mmol) in MeOH (50 mL) at −10° C. After 15 min, another portion of LiBH4 (0.54 g, 24.67 mmol) was added and the mixture was stirred at −10 to 0° C. for 4-5 h. The reaction mixture was carefully quenched by addition of sat. NaH2PO4 (50 mL) at 0° C. The mixture was diluted with water (20 mL) and extracted with DCM three times. The combined organic layer was concentrated and purified by silica gel column chromatography (gradient elution 0-100% petroleum ether/EtOAc, then 0-2% MeOHEtOAc) 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 portioned to a solution of (2S,5R)-6-(benzyloxy)-2-(hydroxymethyl)-1,6-diazabicyclo[3.2.1]octan-7-one (7.8 g, 30 mmol) and 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione (7.0 g, 30 mmol) in DCM (100 mL) at 0° C. The mixture was stirred at 0° C. for 2 h, and filtered through Celite®. The filtrate was dried over Na2SO4 and concentrated to afford (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde (7.0 g, 90%) as a yellow oil. ESI-MS (EI+, m/z): 261.1. 1H-NMR (500M, CDCl3): 9.74 (s, 1H), 7.45-7.36 (m, 5H), 5.07 (d, J=11.5 Hz, 1H), 4.92 (d, J=11.5 Hz, 1H), 3.89 (d, J=8.0 Hz, 1H), 3.27 (m, 1H), 3.21-3.05 (m, 1H), 2.56 (d, J=12.0 Hz, 1H), 2.20-2.15 (m, 1H), 2.05-2.01 (m, 1H), 1.95-1.93 (m, 1H), 1.49-1.46 (m, 1H).
Example 4
Synthesis of (2S,5R)-2-(isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 101)




Step 1: Synthesis of (E)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1f]octane-2-carbaldehyde oxime




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. The reaction mixture was then concentrated and the residue was diluted with DCM (25 mL), washed with water (3×), saturated sodium chloride, dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (gradient elution 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]+.
Step 2-4: Synthesis of (2S,5R)-6-(benzyloxy)-2-(isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-7-one




Step 2-3
NCS (290 mg, 2.17 mmol) and pyridine (2 drops) were added to a solution of (E)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde oxime (560 mg, 2.04 mmol) in dry DCM (15 mL). The reaction mixture was stirred at rt for about 3-4 hrs. Ethynyltrimethylsilane (240 mg, 2.44 mmol) was added, then a solution of DIPEA (290 mg, 2.24 mmol) in dry DCM (5 mL) was added slowly over a 0.5 h period. After addition, the reaction mixture was stirred at rt overnight. The reaction mixture was then washed with 10% citric acid, water, saturated sodium chloride (10 mL), dried and concentrated. The residue was purified by silica gel column chromatography (10:1 petroleum ether/EtOAc) to afford (2S,5R)-6-(benzyloxy)-2-(5-(trimethylsilyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (300 mg, 41%) as a colorless oil. ESI-MS (EI+, m/z): 372 [M+H]+.
Step 4
CsF (230 mg, 1.512 mmol was added to a solution of (2S,5R)-6-(benzyloxy)-2-(5-(trimethylsilyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (187 mg, 0.504 mmol) in CH3CN/EtOH (15 mL, 3:1). After stirring at rt for 2 hrs, the reaction mixture was concentrated and the residue was dissolved in DCM (25 mL), washed with water (3×), and saturated sodium chloride, then, dried, and concentrated. The residue was purified by silica gel column chromatography (6:1 petroleum ether/EA) to afford (2S,5R)-6-(benzyloxy)-2-(isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (116 mg, 77%) as a white solid. ESI-MS (EI+, m/z): 300 [M+H]+.
Step 5-6: Synthesis of (2S,5R)-2-(isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate




A mixture of (2S,5R)-6-(benzyloxy)-2-(isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (196 mg, 0.656 mmol) and 10% Pd/C (80 mg) in THF (20 mL) was stirred at rt for 1.5 h under H2. The reaction mixture was then filtered and concentrated. The residue was washed with Et2O (3×) and dried under high vacuum to afford (2S,5R)-6-hydroxy-2-(isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (130 mg, 98%), which was in the next step directly. ESI-MS (EI+, m/z): 210 [M+H]+.
A mixture of (2S,5R)-6-hydroxy-2-(isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (130 mg, 0.621 mmol) and SO3.Py complex (495 mg, 3.11 mmol) in dry pyridine (5.5 mL) was stirred at rt for 3 hrs. The reaction mixture was then concentrated in vacuum and the residue was redissolved in aqueous NaH2PO4 (1.5 M, 20 mL). Tetra-butylammonium hydrogensulphate (320 mg) was added. The mixture was stirred at rt for 20 min, and extracted with DCM (4×). The combined organic layer was dried and concentrated and the residue was purified by prep-HPLC using ammonium bicarbonate buffer to afford ammonium (2S,5R)-2-(isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (10 mg, 13%) as a white solid. ESI-MS (EI−, m/z): 288 [M]−. 1H NMR (500 MHz, D2O) δ 8.06 (s, 1H), 6.53 (s, 1H), 4.66 (m, 1H), 4.14 (m, 1H), 3.12-3.08 (m, 1H), 2.92 (d, J=12.5 Hz, 1H), 2.24-2.21 (m, 1H), 2.11-2.08 (m, 2H), 1.87-1.86 (m, 1H).
Example 5
Synthesis of (2S,5R)-2-(5-cyanoisoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 102)




Step 1: Synthesis of 3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)isoxazole-5-carbonitrile




2-Chloroacrylonitrile (1.08 g, 12.4 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-N-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbimidoyl chloride (1.9 g, 6.2 mmol) in DCM (19 mL) at −5° C., followed by dropwise addition of TEA (1.25 g, 1.24 mmol). The mixture was stirred at −5° C. for 2 hrs and was then quenched with 10% critic acid (20 mL). The organic phase was separated, washed with 10% critic acid, H2O, and saturated sodium chloride, then concentrated. The residue was purified by silica gel column chromatography (gradient elution 15:1 to 4:1 petroleum ether/EtOAc) to afford 3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazole-5-carbonitrile (325 mg, 16%) as a white solid. 1H-NMR (500 MHz, CDCl3): δ 7.45-7.35 (m, 5H), 7.04 (s, 1H), 5.09 (d, J=11.3 Hz, 1H), 4.94 (d, J=11.3 Hz, 1H), 4.64 (d, J=7.0 Hz, 1H), 3.34 (s, 1H), 2.90 (br d, J=12.0 Hz, 1H), 2.54 (d, J=12.0 Hz, 1H), 2.40-2.35 (m, 1H), 2.31-2.25 (m, 1H), 2.14-2.11 (m, 1H), 1.80-1.74 (m, 1H).
Step 2: Deprotection of O-Bn group using BCl3 
BCl3 (1M in DCM, 1.6 mL) was added to a solution of 3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazole-5-carbonitrile (104 mg, 0.32 mmol) in dry CH2Cl2 (9 mL) at −78° C. The mixture was stirred −78° C. for 2 hrs and quenched carefully with MeOH (2 mL). The solvents were removed in vacuum and the residue was partitioned between EtOAc and water. The organic phase was separated, washed with brine, dried over Na2SO4, and concentrated to afford 3-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazole-5-carbonitrile (45 mg, 60%), which was used directly in the next step. ESI-MS (EI+, m/z): 235.1 [M+H]+.
Step 3: Synthesis of (2S,5R)-2-(5-cyanoisoxcizol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate
A mixture of 3-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazole-5-carbonitrile (45 mg, 0.19 mmol) and SO3.Py complex (260 mg, 1.6 mmol) in dry pyridine (10 mL) was stirred at rt for 3 hrs. The reaction mixture was then concentrated in vacuum and the residue was redissolved in aqueous NaH2PO4 (1.5 M, 5 mL). Tetrabutylammonium hydrogensulphate (80 mg) was added. The mixture was stirred at rt for 20 minutes, then extracted with EtOAc (4×). The combined organic layer was dried and concentrated. The residue was purified by prep-HPLC using ammonium formate buffer to afford ammonium (2S,5R)-2-(5-cyanoisoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (11 mg, 17%) as a white solid. ESI-MS (EI−, m/z): 313.1 [M−H]−. 1H-NMR (500 MHz, D2O): δ 7.32 (s, 1H), 4.69 (m, 1H, overlapped with D2O peak), 4.14 (br s, 1H), 3.12 (br d, J=12.0 Hz, 1H), 2.89 (d, J=12.5 Hz, 1H), 2.29-2.25 (m, 1H), 2.14-2.07 (m, 2H), 1.88-1.84 (m, 1H).
Example 6
Synthesis of (2S,5R)-2-(5-carbamoylisoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 103)




Step 1
Propiolamide (0.30 g, 4.37 mmol) was added to a solution of (2S,5R,Z)-6-(benzyloxy)-N-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carb imido yl chloride (3.64 mmol) in dry DCM (10 mL), followed by the addition of TEA (0.56 mL, 4.0 mmol) in DCM (2 mL) over a 30 minute period. The reaction mixture was stirred at rt overnight. Then the mixture was diluted with EtOAc, washed with water and saturated sodium chloride. The combined organic layer was dried over Na2SO4, concentrated and purified by silica gel column chromatography (gradient elution 5:1 to 1:2, petroleum ether/EtOAc containing 1% TEA) to afford 3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazole-5-carboxamide (0.4 g, 32%) as a yellow solid. ESI-MS (EI+, m/z): 343 [M+H]+.
Step 2
A mixture of 3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazole-5-carboxamide (0.25 g, 0.73 mmol) and 10% Pd/C (0.12 g) in THF (8 mL) was stirred at rt under H2 atmosphere for 2 hrs. The reaction mixture was then filtered and concentrated to afford 3-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazole-5-carboxamide (0.17 g, 90%) as a light yellow solid, which was used directly in the next step. ESI-MS (EI+, m/z): 253 [M+H]+.
Step 3
To a solution of 3-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazole-5-carboxamide (0.165 g, 0.65 mmol) in dried pyridine (2 mL) was added SO3.Py (0.73 g, 4.58 mmol). The mixture was stirred at rt for 2 hrs. The pyridine was evaporated under vacuum and the residue was purified by prep-HPLC using ammonium hydroxide buffer to give ammonium (2S,5R)-2-(5-carbamoylisoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (70 mg, 33%) as a light yellow solid. ESI-MS (EI−, m/z): 331 [M−H]+.
Step 4: Na Resin Exchange
Ammonium (2S,5R)-2-(5-carbamoylisoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (40 mg) was dissolved in a minimum amount of water and acetone (0.5 mL/0.5 mL) and passed through a column of 1 g of DOWEX 50Wx8 Na+ resin (the resin was pre-washed with HPLC grade of water to neutral) and eluted with HPLC grade water to afford sodium (2S,5R)-2-(5-carbamoylisoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (30 mg, 75%) as a white solid after lyophilization. ESI-MS (EI−, m/z): 331.0 [M−H]−; 1H-NMR (500 MHz, D2O): δ 7.02 (s, 1H), 4.69 (m, 1H, overlapped with D2O peak), 4.13 (br s, 1H), 3.12 (br d, J=13.5 Hz, 1H), 2.93 (d, J=12.5 Hz, 1H), 2.25-2.23 (m, 1H), 2.12-2.04 (m, 2H), 1.86-1.83 (m, 1H).
Example 7
Synthesis of (2S,5R)-2-(5-methyl-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 200)







Step 1
HATU (8.3 g, 21.7 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylic acid (5.0 g, 18.1 mmol) and tert-butyl hydrazinecarboxylate (3.5 g, 27.2 mmol) in DCM (60 mL) at 0° C., followed by the addition of DIPEA (7.0 g, 54.3 mmol). The resulting mixture was stirred at 0° C. for 1 h. The reaction mixture was then diluted with DCM, washed with water (2×), and saturated sodium chloride, then dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (gradient elution 10:1 to 4:1 petroleum ether/EtOAc) to afford tert-butyl 2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbonyl)hydrazinecarboxylate (6 g, 85%). ESI-MS (EI+, m/z): 391 [M+H]+.
Step 2
TFA (15 mL) was added dropwise to a solution of tert-butyl 2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbonyl)hydrazinecarboxylate (3 g, 7.7 mmol) in DCM (30 mL) at 0° C. The resulting mixture was stirred at rt for 1 h, and then concentrated. The residue was further dried under high vacuum and washed with Et2O (3×) to afford (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbohydrazide TFA salt (3 g, 93%) as a white solid. ESI-MS (EI+, m/z): 291 [M+H]+.
Step 3
HATU (0.90 g, 2.48 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbohydrazide TFA salt (1.0 g, 2.48 mmol), acetic acid (0.12 g, 2.07 mmol) and DIPEA (1.1 g, 8.3 mmol) in dry DMF (10 mL) at 0° C. The reaction mixture was stirred at 0° C. for 1 h. The mixture was then quenched with saturated sodium chloride (50 mL) and exacted with EtOAc (3×). The combined organic layer was washed with saturated sodium chloride (2×), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 10:1 to 1:10 petroleum ether/EtOAc) to afford (2S,5R)—N′-acetyl-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbohydrazide (0.6 g, 73%). ESI-MS (EI+, m/z): 333 [M+H]+.
Step 4
(CF3SO2)2O (0.50 mL) was slowly added to a solution of (2S,5R)—N′-acetyl-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbohydrazide (0.5 g) and pyridine (0.5 mL) in dry DCM (10 mL) at −10° C. The reaction mixture was stirred at 0° C. for 1 h and then quenched carefully with sat. NaHCO3. The organic layer was separated and the aqueous layer was exacted with EtOAc (3×). The combined organic layer was dried over Na2SO4, concentrated and purified by silica gel column chromatography (gradient elution 10:1 to 1:1 petroleum ether/EtOAc) to afford (2S,5R)-6-(benzyloxy)-2-(5-methyl-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (0.3 g, 63%) as a slight yellow solid. ESI-MS (EI+, m/z): 315.0 [M+H]+.
Step 5
A mixture of ((2S,5R)-6-(benzyloxy)-2-(5-methyl-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (0.2 g, 0.6 mmol) and 10% Pd/C (0.2 g) in THF (20 mL) was stirred under H2 atmosphere at rt for 3 hrs. The reaction mixture was then filtered and concentrated to afford (2S,5R)-6-hydroxy-2-(5-methyl-1,3,4-oxadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (0.13 g, 92%) as a white solid, which was used directly in the next step. ESI-MS (EI+, m/z): 225 [M+H]+.
Step 6
To a solution of (2S,5R)-6-hydroxy-2-(5-methyl-1,3,4-oxadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (130 mg, 0.5 mmol) in dry pyridine (2 mL) was added SO3.Py (46 mg, 2.9 mmol). The mixture was stirred at rt for 3 h and then concentrated under vacuum. The residue was then redissolved in aqueous NaH2PO4 (1.5 M, 10 mL). Tetra-butylammonium hydrogensulphate (236 mg, 0.69 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 5:1 DCM/acetone) to afford tetrabutylammonium (2S,5R)-2-(5-methyl-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (120 mg, 38%) as a colorless oil. ESI-MS (EI−, m/z): 303.0 [M−H]−. 1H-NMR (500 MHz, CDCl3): δ4.65 (br d, J=7.5 Hz, 1H), 4.39 (br s, 1H), 3.35-3.24 (m, 9H), 2.84 (d, J=12 Hz, 1H), 2.54 (s, 3H), 2.33-2.21 (m, 2H), 2.21-2.16 (m, 1H), 2.03-1.97 (m, 1H), 1.75-1.64 (m, 8H), 1.48-1.43 (m, 8H), 1.02-0.99 (m, 12H).
Step 7: Sodium Resin Exchange
Tetrabutylammonium (2S,5R)-2-(5-methyl-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (120 mg) was dissolved in a minimum amount of water and acetone (0.5 mL:0.5 mL) and passed through a column of 10 g of DOWEX 50Wx8 Na+ resin (the resin was pre-washed with HPLC grade of water to neutral) and eluted with HPLC grade water to afford sodium (2S,5R)-2-(5-methyl-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (70 mg, 98%) after lyophilization as a white solid. ESI-MS (EI−, m/z): 303.1 [M−H]−. 1H-NMR (500 MHz, D2O): δ 5.02-5.00 (br d, J=7.0 Hz, 1H), 4.45 (s, 1H), 3.46-3.43 (br d, J=12.5 Hz, 1H), 3.19 (d, J=12.0 Hz, 1H), 2.76 (s, 3H), 2.55-2.51 (m, 1H), 2.46-2.37 (m, 2H), 2.24-2.17 (m, 1H).
Example 8
Synthesis of (2S,5R)-2-(5-ethyl-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 201)




Sodium (2S,5R)-2-(5-ethyl-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate was obtained by a similar method as Step 3-7 of Example 7, using propionic acid in place of HOAc in Step 3. After lyophilization a white solid was obtained (59 mg, 90%). ESI-MS (EI−, m/z): 317.1 [M−H]−. 1H-NMR (500 MHz, D2O): δ 4.84 (d, J=7.0 Hz, 1H), 4.28 (br s, 1H), 3.28-3.26 (br d, J=12.0 Hz, 1H), 3.01 (d, J=12.0 Hz, 1H), 2.95 (q, J=7.8 Hz, 2H), 2.38-2.34 (m, 1H), 2.27-2.21 (m, 2H), 2.06-2.00 (m, 1H), 1.36 (t, J=7.8 Hz, 3H).
Example 9
Synthesis of (2S,5R)-2-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 202)







Step 1-4
Tetrabutylammonium (2S,5R)-2-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (450 mg) was obtained by a similar method as Steps 3-6 of Example 7 using 2-((tert-butyldimethylsilyl)oxy)acetic acid in place of HOAc in Step 3. ESI-MS (EI−, m/z): 433 [M−H]−.
Step 5
To a solution of tetrabutylammonium (2S,5R)-2-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (450 mg, 0.7 mmol) in dry THF (40 mL) was added Et3N-3HF (340 mg, 2.1 mmol). The resulting mixture was stirred at rt overnight, then concentrated. The residue was purified by silica gel column chromatography (3:1 acetone/ethanol) to afford tetrabutyl ammonium (2S,5R)-2-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (160 mg, 73%) as a white solid. ESI-MS (EI−, m/z): 319 [M−H]−.
Step 6
Tetrabutyl ammonium (2S,5R)-2-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (160 mg) was dissolved in a minimum amount of water and acetone (0.5 mL/0.5 mL) and passed through a column of 10 g of DOWEX 50Wx8 Na+ resin (the resin was pre-washed with HPLC grade of water to neutral) and eluted with HPLC grade water to provide sodium (2S,5R)-2-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (130 mg) after lyophilization. The sample was then purified by prep-HPLC using ammonium formate buffer to afford ammonium (2S,5R)-2-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (30 mg, 27%) as a white solid. ESI-MS (EI−, m/z): 319 [M−H]−. 1H-NMR (500M, D2O): δ 4.79 (s, 2H), 4.80-4.75 (m, 1H), 4.20 (br s, 1H), 3.20 (br d, J=12 Hz, 1H), 2.93 (d, J=12.5 Hz, 1H), 2.29-2.20 (m, 1H), 2.19-2.13 (m, 2H), 1.97-1.95 (m, 1H).
Example 10
Synthesis of (2S,5R)-2-(5-(methylamino)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 203)







Following the above synthetic scheme and procedures described for similar transformations in other examples, (2S,5R)-2-(5-(methylamino)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (23 mg) was obtained as TFA salt. ESI-MS (EI+, m/z): 320.2. 1H NMR (300 MHz, D2O) δ 4.64 (d, J=6.3 Hz, 1H), 4.19 (br s, 1H), 3.22-3.18 (m, 1H), 3.03-2.99 (m, 1H), 2.93 (s, 3H), 2.31-1.79 (m, 4H).
Example 11
Synthesis of (2S,5R)-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 204)







Step 1: Synthesis of tert-butyl 4-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazinecarbonyl)piperidine-1-carboxylate
HATU (3.3 g, 8.7 mmol) and tert-butyl 4-(hydrazinecarbonyl)piperidine-1-carboxylate (2.29 g, 9.42 mmol) were added to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxylic acid (2.0 g, 7.25 mmol) in CH2Cl2 (50 mL) at 0° C. DIPEA (2.8 g, 21.8 mmol) was then added and the reaction mixture was stirred at 0° C. for 12 hrs. The reaction mixture was then washed with water and saturated sodium chloride and dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (10:1 DCM/MeOH) to give tert-butyl 4-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazinecarbonyl)piperidine-1-carboxylate (2.2 g, 62%) as a white solid. ESI-MS (EI+, m/z): 502.2 [M+H]+.
Step 2: Synthesis of tert-butyl-4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)piperidine-1-carboxylate
Pyridine (2.6 mL, 35.0 mmol) was added to a solution of tert-butyl-4-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazinecarbonyl)piperidine-1-carboxylate (901 mg, 1.8 mmol) in DCM (200 mL). Then, (CF3SO2)2O (2.6 mL, 9.0 mmol) was added slowly at −10° C. The reaction mixture was stirred at rt for 3 hrs then saturated NaHCO3 was added very slowly at −10° C. The organic layer was separated and the aqueous layer was exacted with EtOAc (3×). The combined organic layer was dried over Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography (2:1 EtOAc/hexanes) to give tert-butyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)piperidine-1-carboxylate (719 mg, 82%) as a slight yellow solid. ESI-MS (EI+, m/z): 484.2 [M+H]+.
Step 3: Synthesis of (2S,5R)-6-(benzyloxy)-2-(5-(piperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one
CF3COOH (5 mL) was slowly added to a solution of tert-butyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)piperidine-1-carboxylate (719 mg, 1.5 mmol) in DCM (20 mL) at 0° C. The mixture was stirred at 0° C. for 3 hrs then, the solvent was concentrated in vacuum to afford (2S,5R)-6-(benzyloxy)-2-(5-(piperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (988 mg) as a brown oil, which was used directly in the next step. ESI-MS (EI+, m/z): 384.1 [M+H]+.
Step 4: Synthesis of (2S,5R)-6-(benzyloxy)-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one
K2CO3 (621 mg, 4.5 mmol) was added to a solution of crude (2S,5R)-6-(benzyloxy)-2-(5-(piperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (988 mg) in acetone (30 mL). Me2SO4 (105 μl, 1.6 mmol) was slowly added at 0° C. The mixture was stirred at rt for 6 h, then concentrated and purified by reverse phase column chromatography (gradient elution 0-80% of acetonitrile in water) to give (2S,5R)-6-(benzyloxy)-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (198 mg, 33% for two steps) as a yellow solid. ESI-MS (EI+, m/z): 398.3 [M+H]+.
Step 5: Synthesis of (2S,5R)-6-hydroxy-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one
To a solution of (2S,5R)-6-(benzyloxy)-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (198 mg, 0.50 mmol) in THF (20 mL) was added 10% Pd/C (200 mg). The mixture was stirred under H2 atmosphere at rt for 3 hrs. The reaction mixture was filtered and concentrated to afford (2S,5R)-6-hydroxy-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (151 mg, 97%), which was used directly in the next step. ESI-MS (EI+, m/z): 308.1 [M+H]+.
Step 6: Synthesis of (2S,5R)-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-6-sulfonic acid
To a solution of (2S,5R)-6-hydroxy-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (151 mg, 0.5 mmol) in dry pyridine (10 ml) was added SO3.Py (400 mg, 2.5 mmol). The mixture was stirred under N2 atmosphere at rt for 8 hrs. The pyridine was evaporated under vacuum and the residue was purified by prep-HPLC using ammonium formate buffer to give (2S,5R)-2-(5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-6-sulfonic acid (40 mg, 20%). ESI-MS (EI−, m/z): 386.1 [M−H]−. 1H-NMR (500 MHz, D2O): δ 4.76 (d, J=7.0 Hz, 1H), 4.23 (t, J=6.5 Hz, 1H), 4.19 (s, 1H), 4.07 (t, J=6.0 Hz, 1H), 3.61-3.46 (m, 1H), 3.26-3.06 (m, 3H), 2.93-2.90 (m, 1H), 2.82 (s, 3H), 2.36-2.26 (m, 3H), 2.19-2.13 (m, 2H), 2.01-1.91 (m, 3H).
Example 12
Synthesis of (2S,5R)-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 205)







Step 1
DIPEA (105 g, 0.81 mol) was added to a solution of tert-butyl piperazine-1-carboxylate (25.0 g, 0.134 mol) in DCM (250 mL) at 0° C., followed by the addition of triphosgene (92 g, 0.27 mol) in portions over a 40 min time period. The reaction mixture was stirred for at rt for 3 hrs, filtered and concentrated to afford 1-tert-butyl 4-trichloromethyl piperazine-1,4-dicarboxylate (50 g) as an oil. A solution of 1-tert-butyl 4-trichloromethyl piperazine-1,4-dicarboxylate (50 g, 0.145 mol) in THF (50 mL) was added dropwise over a 30 minute period to a solution of hydrazine hydrate (18 mL, 0.434 mol) in THF (150 mL). The reaction mixture was stirred at rt for 2 hrs, diluted with saturated sodium chloride (50 mL) and exacted with EtOAc (3×). The combined organic layer was washed with saturated sodium chloride (2×), dried with Na2SO4, and concentrated. The residue was purified by crystallization (3:1 petroleum ether/EtOAc) to obtain of tert-butyl 4-(hydrazinecarbonyl)piperazine-1-carboxylate, (13 g, 40% for two steps). ESI-MS (EI+, m/z): 245 [M+H]+. 1H-NMR (500 MHz, CDCl3): δ5.92 (s, 1H), 3.45-3.35 (m, 8H), 1.41 (s, 9H).
Step 2
DIPEA (3.7 g, 10 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxylic acid (1.45 g, 5.3 mmol) and tert-butyl 4-(hydrazinecarbonyl)piperazine-1-carboxylate, (1.6 g, 6.6 mmol) in dry DMF (50 mL) at 0° C., followed by the addition of HATU (1.45 g, 5.3 mmol). The reaction mixture was stirred at rt overnight. The mixture was diluted with water (200 mL), and the resulting precipitation was collected by filtration, rinsed with water, and then recrystallized (3:1 petroleum ether/EtOAc) to afford tert-butyl 4-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbonyl)hydrazinecarbonyl)piperazine-1-carboxylate (1.4 g, 54%), ESI-MS (EI+, m/z): 503 [M+H]+.
Step 3
Pyridine (2.8 mL, 36.0 mmol was added to a solution of tert-butyl 4-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazine-carbonyl)piperazine-1-carboxylate (903 mg, 1.8 mmol) in DCM (200 mL). (CF3SO2)2O (2.8 ml, 9.0 mmol) was then added slowly at −10° C. The reaction mixture was stirred at rt for 3 hrs. Saturated NaHCO3 was added at −10° C. very slowly. The combined organic layer was separated and the aqueous layer was exacted with EtOAc (3×). The combined organic layer was dried over Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography (2:1 EtOAc/hexanes) to give tert-butyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)piperazine-1-carboxylate (723 mg, 83%) as a slight yellow solid. ESI-MS (EI+, m/z): 485.2 [M+H]+.
Step 4: Synthesis of (2S,5R)-6-(benzyloxy)-2-(5-(piperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one
CF3COOH (5 mL) was slowly added at 0° C. to a solution of tert-butyl 4-(5-((2S,5R)-6-(benzyl-oxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)piperazine-1 carboxylate (723 mg, 1.5 mmol) in DCM (20 mL). The mixture was stirred at 0° C. for 3 hrs, then, the solvent was concentrated to afford (2S,5R)-6-(benzyloxy)-2-(5-(piperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (997 mg) as a brown oil, which was used directly in the next step. ESI-MS (EI+, m/z): 385.1 [M+H]+.
Step 5: Synthesis of (2S,5R)-6-(benzyloxy)-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one
Et3N (631 μL, 4.5 mmol) was added to a solution of crude (2S,5R)-6-(benzyloxy)-2-(5-(piperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (997 mg) in DMF (15 mL). Then, MeI (106 μL, 1.6 mmol) was slowly added at 0° C. The mixture was stirred at rt for 6 hrs then, the mixture was concentrated and purified by reverse phase column chromatography (gradient elution, 0 to 80% of acetonitrile in water) to give (2S,5R)-6-(benzyloxy)-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (202 mg, 34% for two steps) as a yellow solid. ESI-MS (EI+, m/z): 399.3 [M+H]+.
Step 6: Synthesis of (2S,5R)-6-hydroxy-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6diaza-bicyclo[3.2.1]octan-7-one
To a solution of (2S,5R)-6-(benzyloxy)-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (202 mg, 0.51 mmol) in THF (150 mL) was added 10% Pd/C (300 mg). The mixture was stirred under H2 atmosphere at rt for 3 hrs then filtered and concentrated to afford (2S,5R)-6-hydroxy-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (154 mg, 98%), which was used directly in the next step. ESI-MS (EI+, m/z): 309.1 [M+H]+.
Step 5: Synthesis of (2S,5R)-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate
To a solution of (2S,5R)-6-hydroxy-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (154 mg, 0.5 mmol) in dried pyridine (10 mL) was added SO3.Py (400 mg, 2.5 mmol). The mixture was stirred under N2 atmosphere at rt for 8 hrs. The pyridine was evaporated under vacuum at 40° C., then, the mixture was concentrated and purified by pre-HPLC using ammonium formate buffer to give (2S,5R)-2-(5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (45 mg, 23%). ESI-MS (EI−, m/z): 387.1 [M−H]−. 1H-NMR (500 MHz, D2O): δ 4.62 (d, J=7.0 Hz, 1H), 4.16 (br s, 1H), 3.89 (br s, 4H), 3.31 (br s, 4H), 3.16 (br d, J=12.0 Hz, 1H), 2.95 (d, J=12.0 Hz, 1H), 2.82 (s, 3H), 2.20-2.17 (m, 1H), 2.12-2.05 (m, 2H), 1.94-1.87 (m, 1H).
Example 13
Synthesis of (2S,5R)-2-(5-(morpholin-2-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 206)







Following the above synthetic scheme and procedures described for similar transformations in other examples, (2S,5R)-2-(5-(morpholin-2-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (22 mg) was obtained as TFA salt. ESI-MS (EI+, m/z): 376.15. 1H NMR (300 MHz, D2O) δ 5.32 (dd, J=9.7, 3.1 Hz, 1H), 4.82 (d, J=5.7 Hz, 1H), 4.24-4.12 (m, 2H), 4.07-3.98 (m, 1H), 3.78-3.73 (m, 1H), 3.61-3.54 (m, 1H), 3.44-3.28 (m, 2H), 3.25-3.19 (m, 1H), 2.95 (d, J=12.4 Hz, 1H), 2.36-2.07 (m, 3H), 1.99-1.92 (m, 1H).
Example 14
Synthesis of (2S,5R)-2-(5-morpholino-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 207)







Step 1
To a solution of tert-butyl 2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazinecarboxylate (0.78 g, 2.0 mmol) in dioxane (10 mL) was added 4M HCl in dioxane (5 mL) at 0° C. The reaction mixture was stirred at rt for 1 h, then, Et2O (10 mL) was added. The solid precipitation was collected by filtration, washed with Et2O (2×), and dried under vacuum to provide (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbohydrazide hydrochloride (0.65 g, 99%), which was used directly in the next step. ESI-MS (EI+, m/z): 291.19 [M+H]+.
Step 2
Morpholine-4-carbonyl chloride (297 mg, 1.99 mmol) was added dropwise over a period of 10 minutes to a solution of DMAP (24 mg, 0.199 mmol) and pyridine (0.16 mL, 1.99 mmol) in dry DCM (5 mL). The mixture was stirred at 0° C. for 20 minutes then, a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbohydrazide hydrochloride (0.65 g, 1.99 mmol) in dry DCM (2.0 mL) was added over a period of 10 minutes. The mixture was stirred at 0° C. for 15 min and was then allowed to warm to rt and was stirred overnight. The reaction mixture was diluted with DCM (10 mL), and washed with 1N HCl (10 mL), and saturated sodium chloride (10 mL), then was dried over Na2SO4 and concentrated. The crude product was purified by silica gel column chromatography (0 to 1:100 MeOH/DCM, containing 1% TEA) to give N′-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)morpholine-4-carbohydrazide (0.38 g, 47%) as a white solid. ESI-MS (EI+, m/z): 404.19 [M+H]+.
Step 3
Pyridine (2.0 mL) was added to a solution of N′-(2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)morpholine-4-carbohydrazide (0.38 g, 0.94 mmol) in dry DCM (10 mL). (CF3SO2)2O (2.0 mL) was then added slowly at −10° C. The reaction mixture was stirred at rt for 0.5 h then saturated NaHCO3 was added at −10° C. very slowly. The organic layer was separated and the aqueous layer was exacted with EtOAc (3×). The combined organic layer was dried with Na2SO4, concentrated, and purified by silica gel column chromatography (0 to 1:50 MeOH/DCM) to give (2S,5R)-6-(benzyloxy)-2-(5-morpholino-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (0.12 g, 33%). ESI-MS (EI+, m/z): 386 [M+H]+.
Step 4
A mixture of (2S,5R)-6-(benzyloxy)-2-(5-morpholino-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (0.12 g) and 10% Pd/C (70 mg) in THF (5 mL) was stirred at rt under H2 atmosphere for 1-2 hrs. The reaction mixture was then filtered and concentrated to afford (2S,5R)-6-hydroxy-2-(5-morpholino-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (83 mg, 90%), which was used directly in the next step. ESI-MS (EI+, m/z): 296 [M+H]+.
Step 5
To a solution of (2S,5R)-6-hydroxy-2-(5-morpholino-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (83 mg, 0.28 mmol) in dried pyridine (2 mL) was added SO3.Py (224 mg, 1.41 mmol). The mixture was stirred at rt for 3 hrs. then the pyridine was evaporated under vacuum. The residue was purified by prep-HPLC using ammonium formate buffer to give ammonium (2S,5R)-2-(5-morpholino-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (70 mg) as a white solid. ESI-MS (EI−, m/z): 374.0 [M−H]−; 1H-NMR (500 MHz, D2O): δ 4.63-4.61 (m, 1H), 4.17 (m, 1H), 3.77-3.75 (m, 4H), 3.47-3.45 (m, 4H), 3.16 (d, J=12.0 Hz, 1H), 2.95 (d, J=12.5 Hz, 1H), 2.21-2.17 (m, 1H), 2.09 (m, 2H), 1.95-1.90 (m, 1H).
Step 6: Resin Exchange
Ammonium (2S,5R)-2-(5-morpholino-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (70 mg) was dissolved in a minimum amount of water and passed through a column of 2 g of DOWEX 50Wx8 Na+ resin (the resin was pre-washed with HPLC grade of water to neutral) and eluted with HPLC grade water to afford sodium (2S,5R)-2-(5-morpholino-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (65 mg) as a white solid after lyophilization. ESI-MS (EI−, m/z): 374.0 [M−H]−; 1H-NMR (500 MHz, D2O): δ 4.71 (d, J=7.0 Hz, 1H), 4.27 (m, 1H), 3.87-3.85 (−m, 4H), 3.56-3.54 (m, 4H), 3.27-3.25 (m, 1H), 3.05 (d, J=12.0 Hz, 1H), 2.31-2.29 (m, 1H), 2.20-2.17 (m, 2H), 2.03-2.00 (m, 1H).
Example 15
Synthesis of (2S,5R)-7-oxo-2-(1,3,4-thiadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 300)




Step 1: Synthesis of (2S,5R)-6-(Benzyloxy)-N′-formyl-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbohydrazide
To a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxylic acid (1.0 g, 6.0 mmol) in THF (30 mL) was added 1,1′-carbonyldiimidazole (1.6 g, 7.2 mmol) at 0° C. The reaction mixture was stirred at rt for 1 h, then, formohydrazide (1.1 g, 18.1 mmol) was added rapidly at rt. The reaction mixture was stirred at rt for 3 hrs and then diluted with EtOAc (100 mL). The combined organic layer was washed with saturated sodium chloride (10 mL), dried over Na2SO4, and concentrated to afford (2S,5R)-6-(benzyloxy)-N′-formyl-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbohydrazide (1.6 g), which was used directly in the next step. ESI-MS (EI+, m/z): 319.1 [M+H]+.
Step 2: Synthesis of (2S,5R)-6-(benzyloxy)-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one
Lawesson's reagent (2.40 g, 6.0 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-N′-formyl-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbohydrazide (1.6 g, 6.0 mmol) in THF (30 mL) at rt. The mixture was stirred at rt for 8 hrs., then the solvent was concentrated and the residue was purified by silica gel column chromatography (1:6 to 1:1 EtOAc/hexanes) to give (2S,5R)-6-(benzyloxy)-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (568 mg, 30%) as a yellow solid. ESI-MS (EI+, m/z): 317.1 [M+H]+. 1H-NMR (400 MHz, CDCl3): δ 9.12 (s, 1H), 7.38-7.29 (m, 5H), 5.02 (d, J=8.8 Hz, 1H), 4.89-4.83 (m, 2H), 3.28 (m, 1H), 2.86-2.83 (m, 1H), 2.70-2.54 (m, J=2H), 2.29-2.24 (m, 1H), 2.17-2.13 (m, 1H), 2.07-1.79 (m, 1H).
Step 3: Synthesis of (2S,5R)-6-hydroxy-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one
BCl3 (1 M in CH2Cl2, 9.0 mL, 9.0 mmol) was added dropwise to a solution of (2S,5R)-6-(benzyloxy)-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (560 mg, 1.8 mmol) in dry CH2Cl2 (20 mL) at −78° C. The mixture was stirred under a N2 atmosphere at 0° C. for 6 hrs., then, MeOH (9 mL) was slowly added dropwise at −78° C. The solvents were evaporated under vacuum at 0° C. to give (2S,5R)-6-hydroxy-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (329 mg, 81%) as a yellow solid, which was used directly in the next step. ESI-MS (EI−, m/z): 227.1 [M+H]−.
Step 4: Synthesis of tetrabutylammonium (2S,5R)-7-oxo-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate
To a solution of crude (2S,5R)-6-hydroxy-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (329 mg) in dried pyridine (15 mL) was added SO3.Py (1.2 g, 7.0 mmol). The mixture was stirred under N2 atmosphere at rt for 6 hrs., then the pyridine was evaporated under vacuum at 40° C. The residue was redissolved in aqueous NaH2PO4 (1.5 M, 50 mL) and tetra-butylammonium hydrogensulphate (609 mg, 1.9 mmol) was added. The mixture was stirred at rt for 20 min, and extracted with EtOAc (4×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (10:1 to 2:1 DCM/acetone) to give tetrabutylammonium (2S,5R)-7-oxo-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (313 mg, 41% for two steps) as a yellow solid. ESI-MS (EI−, m/z): 305.0 [M−H]−. 1H-NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 4.85 (d, J=5.6 Hz, 1H), 4.38 (s, 1H), 3.32-3.24 (m, 9H), 2.75-2.64 (m, 2H), 2.31-1.29 (m, 3H), 1.83-1.91 (m, 1H), 1.71-1.65 (m, 8H), 1.50-1.43 (m, 8H), 1.02 (t, J=5.6 Hz, 12H).
Step 6: Resin Exchange
Tetrabutylammonium (2S,5R)-7-oxo-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (140 mg) was dissolved in a minimum amount of HPLC grade water ( ̃10 mL) and passed through a column of 10 g of DOWEX 50WX 8 Na+ resin (the resin was pre-washed with >0.5 L of HPLC grade water to neutral) and eluted with HPLC grade water to afford sodium (2S,5R)-7-oxo-2-(1,3,4-thiadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate after lyophilization as a white solid (68 mg). ESI-MS (EI−, m/z): 305.0 [M−H]−. 1H-NMR (400 MHz, CDCl3): δ 9.49 (s, 1H), 5.00 (d, J=5.6 Hz, 1H), 4.20 (br s, 1H), 3.19 (br d, J=9.6 Hz, 1H), 2.92 (d, J=12.0 Hz, 1H), 2.55-2.51 (m, 1H), 1.26-1.15 (m, 2H), 1.97-1.90 (m, 1H).
Example 16
Synthesis of (2S,5R)-2-(5-(aminomethyl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 301)







Step 1
HATU (4.9 g, 12.9 mmol) and tert-butyl 2-hydrazinyl-2-oxoethylcarbamate (2.2 g, 11.4 mmol) were added to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxylic acid (3 g, 10.8 mmol) in CH2Cl2 (50 mL) at 0° C. DIPEA (4.2 g, 32.6 mmol) was then added and the reaction mixture was stirred at 0° C. for 12 hrs. The reaction mixture was then washed with water, and saturated sodium chloride, dried over Na2SO4, and concentrated. The crude sample was purified by silica gel column chromatography (1 to 10% MeOH/DCM) to give tert-butyl (2-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbonyl)hydrazinyl)-2-oxoethyl)carbamate (3.5 g, 62%) as a white solid. ESI-MS (EI+, m/z): 448.2 [M+H]+.
Step 2
Lawesson's reagent (0.68 g, 1.68 mol) was added to a solution of tert-butyl 2-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazinyl)-2-oxoethylcarbamate (0.50 g, 1.12 mmol) in THF (30 mL). The reaction mixture was heated at 70° C. for 0.5 h. The solution was cooled down to rt and saturated NaHCO3 was added. The organic layer was separated and the aqueous layer was exacted with EtOAc (2×). The combined organic layer was dried over Na2SO4, and concentrated. The crude sample was dissolved in THF (100 mL), and Hg(OAc)2 (1.68 g) was added. The mixture was stirred at rt overnight, filtered and concentrated. The residue was dissolved in EtOAc (100 mL), stirred for 30 min, filtered and concentrated. The crude material was purified by silica gel column chromatography (20% to 50% EtOAc/petroleum ether) to give tert-butyl ((5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)methyl)carbamate (230 mg, 45%) as a white solid. ESI-MS (EI+, m/z): 446.2 [M+H]+.
Step 3
To a solution of tert-butyl ((5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)methyl)carbamate (400 mg, 0.89 mmol) in THF (60 mL) was added 10% Pd(OH)2C (3 g). The mixture was stirred under H2 atmosphere at rt for 3 hrs. The reaction mixture was filtered and concentrated to afford tert-butyl ((5-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)methyl)carbamate as a yellow solid, which was directly used in the next step. ESI-MS (EI+, m/z): 356.1 [M+H]+.
Step 4
To a solution of tert-butyl ((5-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)methyl)carbamate in dry pyridine (3 mL) was added SO3.Py (990 mg, 6.23 mmol). The mixture was stirred at rt for 3 hrs., then the pyridine was evaporated under vacuum at 25° C. The residue was redissolved in aqueous NaH2PO4 (1.5 M, 20 mL), then tetrabutylammonium hydrogensulphate (150 mg) 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 (10:1 to 1:1 DCM/acetone) to give tetrabutylammonium (2S,5R)-2-(5-(((tert-butoxycarbonyl)amino)methyl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (100 mg, 20% in 3 steps) as a light yellow solid. ESI-MS (EI−, m/z): 434.1 [M−H]−.
Step 5
Tetrabutylammonium (2S,5R)-2-(5-(((tert-butoxycarbonyl)amino)methyl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (95 mg, 0.14 mmol) was dissolved in a minimum amount of HPLC grade water ( ̃10 mL) and passed through a column of 16 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. Sodium (2S,5R)-2-(5-(((tert-butoxycarbonyl)amino)methyl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (53 mg, 80%) was obtained after lyophilization as a white solid. ESI-MS (EI−, m/z): 434.0 [M−H]−. 1H-NMR (500 MHz, D2O) δ 4.86 (d, J=6.5 Hz, 1H), 4.55 (s, 2H), 4.12 (s, 1H), 3.10-3.13 (m, 1H), 2.87-2.89 (m, 1H), 2.39-2.43 (m, 1H), 2.07-2.12 (m, 2H), 1.84-1.86 (m, 1H), 1.31 (s, 9H).
Step 6
TFA (0.30 mL) was added to a mixture of sodium (2S,5R)-2-(5-(((tert-butoxycarbonyl)amino)methyl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (50 mg) in dry DCM (0.80 mL) at 0° C. The reaction mixture was stirred at 0° C. for 2-3 hrs and then diluted with ether ( ̃15 mL). The precipitate was collected via centrifugation, washed with ether (3×) and dried under high vacuum to afford (2S,5R)-2-(5-(aminomethyl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (30 mg) as TFA salt. ESI-MS (EI+, m/z): 336.1. 1H NMR (300 MHz, D2O) δ 4.97 (br d, J=6.2 Hz, 1H), 4.68 (s, 2H), 4.19 (br s, 1H), 3.21 (br d, J=13.5 Hz, 1H), 2.94 (d, J=12.2 Hz, 1H), 2.54-2.49 (m, 1H), 2.30-2.07 (m, 2H), 1.98-1.87 (m, 1H).
Example 17
Synthesis of (2S,5R)-7-oxo-2-(5-(piperazin-1-yl)-1,3,4-thiadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 302)







Step 1
TEA (2.1 mL, 0.015 mol) was added to a solution of tert-butyl piperazine-1-carboxylate (1.86 g, 0.01 mol) and di-(1H-imidazol-1-yl)methanethione (2.67 g, 0.015 mol) in THF (30 mL) at rt. The reaction mixture was stirred at rt for 2 hrs., then hydrazine hydrate (0.73 mL, 0.015 mol) was added. The resultant solution was stirred at rt for another 2 hrs. The reaction mixture was quenched with saturated sodium chloride (15 mL) and extracted with EtOAc (3×). The combined organic layer was dried over Na2SO4, concentrated and recrystallized (3:1 petroleum ether/EtOAc) to afford tert-butyl 4-(hydrazinecarbonothioyl)piperazine-1-carboxylate (2.2 g, 85%) as a light yellow solid.
Step 2
DIPEA (1.75 mL, 10.8 mmol) was added to a solution of tert-butyl 4-(hydrazinecarbonothioyl)piperazine-1-carboxylate (0.95 g, 3.6 mmol), (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carboxylic acid (1.0 g, 3.6 mmol) and HATU (1.51 g, 3.98 mmol) in dry DMF (15 mL) and the resulting reaction mixture was stirred at rt for 1 h. The reaction mixture was quenched with water (10 mL), and extracted with EtOAc (3×). The combined organic layer was dried over Na2SO4, then concentrated and purified by silica gel column chromatography (1:50 MeOH/DCM) to afford tert-butyl 4-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazinecarbonothioyl)piperazine-1-carboxylate (0.92 g, 49%) as a light yellow solid. ESI-MS (EI+, m/z): 519.2 [M+H]+.
Step 3
A solution of tert-butyl 4-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazinecarbonothioyl)piperazine-1-carboxylate (0.66 g, 1.27 mmol) and Lawesson's reagent (0.36 g, 0.89 mmol) in THF (15 mL) was heated at 66° C. for 1 h, then cooled down to rt Hg(OAc)2 (0.4 g) was added and the mixture was stirred at rt overnight. The solution was then concentrated and the residue was purified by silica gel column chromatography (1:2 EtOAc/petroleum ether) to give tert-butyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)piperazine-1-carboxylate (0.15 g, 24%) as a light yellow solid. ESI-MS (EI+, m/z): 501 [M+H]+.
Step 4
A mixture of tert-butyl 4-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)piperazine-1-carboxylate (180 mg, 0.36 mmol) and 10% Pd(OH)2 (1.8 g) in THF (10 mL) was stirred under H2 at rt for 8.5 hrs. The reaction mixture was then filtered and concentrated to give tert-butyl 4-(5-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)piperazine-1-carboxylate (133 mg, 90%) as a white solid. ESI-MS (EI+, m/z): 411.17 [M+H]+.
Step 5
A mixture of tert-butyl 4-(5-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)piperazine-1-carboxylate (133 mg, 0.324 mmol) and SO3.Py (0.258 g, 1.62 mmol) in dry pyridine (1 mL) was stirred at rt for 2 hrs, and then concentrated under vacuum. The residue was redissolved in aqueous NaH2PO4 (1.5 M, 20 mL) and tetrabutylammonium hydrogensulphate (130 mg, 0.38 mmol) was added. The mixture was stirred at rt for 20 min, and extracted with EtOAc (4×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (5:1 DCM/acetone) to give tetrabutylammonium (2S,5R)-2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (100 mg, 42%) as a white solid. ESI-MS (EI−, m/z): 489.0 [M−H]−.
Step 6: Resin Exchange: sodium (2S,5R)-2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate
Tetrabutylammonium (2S,5R)-2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (100 mg) was dissolved in a minimum amount of HPLC grade water and acetone (1 mL/1 mL) and passed through a column of 5 g of DOWEX 50WX 8 Na+ resin (the resin was pre-washed with HPLC grade water) and eluted with HPLC grade water to give sodium (2S,5R)-2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (60 mg, 90%) as a white solid after lyophilization. ESI-MS (EI−, m/z): 489.0 [M−H]−. 1H-NMR (500 MHz, D2O): δ 4.24 (s, 1H), 3.62 (m, 4H), 3.56-3.54 (m, 4H), 3.22 (d, J=12.5 Hz, 1H), 3.03 (d, J=12.0 Hz, 1H), 2.46-2.42 (m, 1H), 2.18-2.11 (m, 2H), 1.98-1.93 (m, 1H), 1.47 (s, 9H).
Step 6
TFA (0.20 mL) was added to a mixture of sodium (2S,5R)-2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate after lyophilization (33 mg, 0.064 mmol) in dry DCM (0.60 mL) at 0° C. The reaction mixture was stirred at 0° C. for 2-3 hrs and then diluted with ether ( ̃10 mL). The precipitate was collected via centrifugation, washed with ether (3×) and dried under high vacuum to afford (2S,5R)-7-oxo-2-(5-(piperazin-1-yl)-1,3,4-thiadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (10 mg) as aTFA salt. ESI-MS (EI+, m/z): 391.1.
Example 18
Synthesis of (2S,5R)-2-(3-methyl-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 400)




Step 1
DIPEA (0.137 mL, 0.787 mmol) was added to a solution of ethyl 5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxylate (145 mg, 0.525 mmol) and (Z)—N′-hydroxyacetimidamide (38.9 mg, 0.525 mmol) in DCM (2.6 mL), followed by addition of HATU (239 mg, 0.630 mmol). The reaction mixture was stirred at rt for 45 minutes and then concentrated to provide (2S,5R)-6-(benzyloxy)-N—((Z)-1-(hydroxyimino)ethyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxamide (174 mg, 100%), which was used directly in the next step. ESI-MS (EI+, m/z): 333.5.
Step 2
A solution of (2S,5R)-6-(benzyloxy)-N—((Z)-1-(hydroxyimino)ethyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxamide (174 mg, 0.524 mmol) in DMF (2 mL) was heated at 80° C. for 2-3 hrs. The solvent was then concentrated in vacuum and the residue was purified by silica gel column chromatography (0 to 5% MeOH/DCM) to provide (2S,5R)-6-(benzyloxy)-2-(3-methyl-1,2,4-oxadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (130 mg, 79%) as a white solid.
Step 3
BCl3 (1M DCM solution, 2.5 mL, 2.5 mmol) was added dropwise to a solution of (2S,5R)-6-(benzyloxy)-2-(3-methyl-1,2,4-oxadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (130 mg, 0.414 mmol) in dry DCM (12 mL) at −78° C. The mixture was stirred at −78° C. to 0° C. for 4 hrs. The reaction mixture was then quenched with MeOH (2 mL). The solvent was removed and the residue was purified by silica gel column chromatography (0 to 10% MeOH/DCM) to afford (2S,5R)-6-hydroxy-2-(3-methyl-1,2,4-oxadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (30 mg, 32%).
Step 4
To a solution of (2S,5R)-6-hydroxy-2-(3-methyl-1,2,4-oxadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (30 mg, 0.13 mmol) in dry pyridine (3 mL) was added SO3.Py (85 mg, 0.54 mmol). The mixture was stirred at rt overnight and then concentrated under vacuum. The residue was redissolved in aqueous NaH2PO4 (1.5 M, 15 mL) and tetrabutylammonium hydrogensulphate (200 mg, 0.58 mmol) was added. The mixture was stirred at rt for 20 min, and extracted with 10% MeOH/DCM (3×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (gradient elution 10:1 to 1:1 DCM/acetone, containing 0.25% of NEt3) to afford tetrabutylammonium (2S,5R)-2-(3-methyl-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (25 mg, 34%).
Step 5
Tetrabutylammonium (2S,5R)-2-(3-methyl-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (25 mg) was dissolved in a minimum amount of water and acetone (0.5 mL/0.5 mL) and passed through a column of 20 g of DOWEX 50Wx8 Na+ resin (the resin was pre-washed with HPLC grade of water to neutral) and eluted with HPLC grade water to afford sodium (2S,5R)-2-(3-methyl-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (14 mg, 93%) after lyophilization as a white solid.
ESI-MS (EI+, m/z): 305.1. 1H NMR (300 MHz, D2O) δ 4.80 (d, J=6.8 Hz, 1H), 4.18 (br s, 1H), 3.25-3.21 (m, 1H), 2.95-2.89 (m, 1H), 2.36 (s, 3H), 2.32-2.03 (m, 3H), 1.99-1.77 (m, 1H).
Example 19
Synthesis of ethyl 5-((2S,5R)-7-oxo-6-(sulfooxy)-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxylate (Compound 402)




Step 1
EDC (0.916 g, 4.78 mmol) was added to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylic acid (1.2 g, 4.34 mmol) and HOBt (0.732 g, 4.78 mmol) in DMF (12 mL) at rt. The mixture was stirred at rt for 0.5 h, then ethyl 2-(hydroxyamino)-2-iminoacetate (0.689 g, 5.21 mmol) was added and the mixture was stirred for another 0.5 h at rt. The reaction mixture was then heated to 100° C. under microwave for 80 minutes. The solvent was removed under vacuum and the residue was purified by silica gel column chromatography (0 to 5% MeOH/DCM) to afford ethyl 5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxylate (0.85 g, 53%). ESI-MS (EI+, m/z): 373.4.
Step 2
To a solution of ethyl 5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxylate (323 mg, 0.867 mmol) in MeOH (6.0 mL) and THF (3.0 mL) was added 10% Pd/C (18.46 mg, 0.017 mmol). The reaction mixture was stirred under hydrogen balloon at rt for 1 h. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel column chromatography (0 to 5% MeOH/DCM) to afford ethyl 5-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxylate (85 mg, 35%).
Step 3
To a solution of ethyl 5-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxylate (85 mg, 0.301 mmol) in dry pyridine (5 mL) was added SO3.Py (240 mg, 1.506 mmol). The mixture was stirred at rt overnight and then concentrated under vacuum. The residue was redissolved in aqueous NaH2PO4 (1.5 M, 15 mL). and tetra-butylammonium hydrogensulphate (200 mg, 0.58 mmol) was added. The mixture was stirred at rt for 20 minutes, then was extracted with 10% MeOH/DCM (3×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (10:1 to 1:1 DCM/acetone, containing 0.25% of NEt3) to afford tetrabutylammonium (2S,5R)-2-(3-(ethoxycarbonyl)-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (94 mg, 51%).
Step 4
Tetrabutylammonium (2S,5R)-2-(3-(ethoxycarbonyl)-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (94 mg) was dissolved in a minimum amount of water and acetone (1.0 mL/1.0 mL) and passed through a column of 20 g of DOWEX 50Wx8 Na+ resin (the resin was pre-washed with HPLC grade of water to neutral) and eluted with HPLC grade water to afford sodium (2S,5R)-2-β-(ethoxycarbonyl)-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (58 mg, 94%) after lyophilization as a white solid. ESI-MS (EI+, m/z): 363.2. 1H NMR (300 MHz, D2O) δ 4.93 (d, J=6.9 Hz, 1H), 4.46 (q, J=7.2 Hz, 2H), 4.21 (br s, 1H), 3.34-3.19 (m, 1H), 3.07-2.97 (m, 1H), 2.45-2.07 (m, 1H), 2.05-1.86 (m, 3H), 1.34 (t, J=7.2 Hz, 3H).
Example 20
Synthesis of (2S,5R)-2-(3-carbamoyl-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 401)




Step 1
Ammonia in MeOH solution (2.3 mL, 16.11 mmol) was added to a solution of ethyl 5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxylate (600 mg, 1.61 mmol) in isopropanol (8 mL) and the reaction mixture was heated at 40° C. for 1 h. The solvent was removed under vacuum and the residue was purified by silica gel column chromatography (0 to 10% MeOH/DCM) to afford 5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxamide (220 mg, 40%). ESI-MS (EI+, m/z): 344.3.
Step 2
To a solution of 5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxamide (220 mg, 0.641 mmol) in MeOH (4.0 mL) and THF (12.0 mL) was added 10% Pd/C (13.6 mg, 0.013 mmol). The reaction mixture was stirred under hydrogen balloon for 1 h at rt, then filtered and concentrated to afford 5-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxamide ( ̃150 mg, 93%), which was used directly in the next step.
Step 3
To a solution of 5-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,2,4-oxadiazole-3-carboxamide ( ̃150 mg, 0.5 mmol) in dry pyridine (5 mL) was added SO3.Py (510 mg, 3.20 mmol). The mixture was stirred at rt overnight and then concentrated under vacuum. The residue was redissolved in aqueous NaH2PO4 (1.5 M, 15 mL) and tetrabutylammonium hydrogensulphate (400 mg, 2.38 mmol) was added. The mixture was stirred at rt for 20 min, and extracted with 10% MeOH/DCM (3×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (10:1 to 1:1 DCM/acetone, containing 0.25% of NEt3) to afford tetrabutylammonium (2S,5R)-2-(3-carbamoyl-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (22 mg, 8%).
Step 4
Tetrabutylammonium (2S,5R)-2-(3-carbamoyl-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (22 mg, 0.038 mmol) was dissolved in a minimum amount of water and acetone (0.5 mL/0.5 mL) and passed through a column of 20 g of DOWEX 50Wx8 Na+ resin (the resin was pre-washed with HPLC grade of water to neutral) and eluted with HPLC grade water to afford sodium (2S,5R)-2-β-carbamoyl-1,2,4-oxadiazol-5-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (12 mg, 89%) after lyophilization as a white solid. ESI-MS (EI+, m/z): 334.1. 1H NMR (300 MHz, D2O) δ 4.90 (d, J=7.0 Hz, 1H), 4.20 (br s, 1H), 3.-3.23 (m, 1H), 3.00-2.93 (m, 1H), 2.45-2.07 (m, 3H), 2.03-1.95 (m, 1H).
Example 21
Synthesis of (2S,5R)-7-oxo-2-(1,2,4-thiadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (Compound 500)







Step 1: Synthesis of (2S,5R)-5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid
LiOH.H2O (2.7 g, 64 mmol) was added to a solution of (2S,5R)-1-tert-butyl 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate (9.0 g, 16 mmol) in THF (60 mL) and H2O (60 mL) at rt. The mixture was stirred at rt for 36 hrs., then the reaction mixture was adjusted to pH 4 with 1N HCl, and extracted with EtOAc (3×). The combined organic layer was washed with saturated sodium chloride, dried over Na2SO4 and concentrated to give (2S,5R)-5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (8.2 g, 95%) as a yellow solid. ESI-MS (EI+, m/z): 558 [M+Na]+.
Step 2: Synthesis of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-carbamoyl-piperidine-1-carboxylate
A mixture of (2S,5R)-5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (8.56 g, 16 mmol), EDCI (3.2 g, 16 mmol), HOBt (2.2 g, 16 mmol), NH4Cl (1.7 g, 32 mmol), and DIPEA (4.0 g, 32 mmol) in dry DMF (40 mL) was stirred at rt overnight. The mixture was diluted with EtOAc (300 mL), and washed with water (3×). The organic layer was dried over Na2SO4 and concentrated to give (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-carbamoyl-piperidine-1-carboxylate as yellow solid (7.4 g, 86%). ESI-MS (EI+, m/z): 557 [M+Na]+.
Step 3: Synthesis of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-carbamothioylpiperidine-1-carboxylate
A mixture of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-carbamoyl-piperidine-1-carboxylate (2.2 g, 4.0 mmol) and Lawesson's reagent (1.3 g, 3.2 mmol) in dry THF (40 mL) was heated at 50° C. under N2 atmosphere for 1 h. The solvent was removed, and the residue was dissolved in DCM (100 mL). NaOH solution (0.1 N, 100 mL) was added and the mixture was stirred at rt for 10 minutes. The organic phase was separated, dried, and concentrated. The residue was purified by silica gel column chromatography (2:1 petroleum ether/EtOAc) to give (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-carbamothioylpiperidine-1-carboxylate (1.6 g, 75%) as white solid. ESI-MS (EI+, m/z): 573 [M+Na]+.
Step 4: Synthesis of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-((E)-(dimethylamino)methylenecarbamothioyl)piperidine-1-carboxylate
A mixture of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-carbamothioylpiperidine-1-carboxylate (1.65 g, 3 mmol) and 1,1-dimethoxy-N,N-dimethylmethanamine (1.08 g, 9.0 mmol) in dry dioxane (20 mL) was stirred at 45° C. overnight. The reaction mixture was concentrated under vacuum and the residue was purified by silica gel column chromatography (1:1 petroleum ether/EtOAc) to give (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-((E)-(dimethylamino)methylenecarbamothioyl)piperidine-1-carboxylate (1.3 g, 72%) as a yellow solid. ESI-MS (EI+, m/z): 606 [M+H]+.
Step 5: Synthesis of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate
A solution of pyridine (320 mg, 4.0 mmol) in CH3OH (10 mL) was added to a mixture of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-((E)-(dimethylamino)methylenecarbamothioyl)piperidine-1-carboxylate (1.2 g, 2.0 mmol) in EtOH (20 mL), followed by addition of (aminooxy)sulfonic acid (270 mg, 2.4 mmol). The mixture was stirred at rt for 2 hrs. The solvent was removed, and the residue was purified by silica gel column chromatography (3:1 petroleum ether/EtOAc) to (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate (520 mg, 48%) as a yellow solid. ESI-MS (EI+, m/z): 598 [M+Na]+.
Step 6: Synthesis of (2S,5R)-tert-butyl 5-(benzyloxyamino)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate
To a mixture of (2S,5R)-tert-butyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate (1.17 g, 2.0 mmol) in dry DMF (30 mL) at rt was added LiOH.H2O (640 mg, 16.0 mmol), followed by addition of 2-mercaptoacetic acid (920 mg, 10.0 mmol). The mixture was stirred at 30° C. for 24 hrs., then the mixture was diluted with EtOAc (200 mL), washed with water (4×), dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (3:1 petroleum ether/EtOAc) to give (2S,5R)-tert-butyl 5-(benzyloxyamino)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate (560 mg, 75%) as an oil. ESI-MS (EI+, m/z): 413 [M+Na]+.
Step 7: Synthesis of (2S,5R)-tert-butyl 5-(benzyloxy(chlorocarbonyl)amino)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate
Diphosgene (360 mg, 1.8 mmol) was added to a solution of (2S,5R)-tert-butyl 5-(benzyloxyamino)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate (468 mg, 1.2 mmol) in dry DCM (20 mL) at 0° C. followed by addition of TEA (360 mg, 3.6 mmol). The mixture was stirred at 0° C. for 2 hrs and then quenched with saturated NaHCO3 (20 mL). The reaction mixture was then extracted with DCM (3×) and the combined organic layer was dried over Na2SO4 and concentrated to afford (2S,5R)-tert-butyl 5-(benzyloxy(chlorocarbonyl)amino)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate, which was directly used in the next step. ESI-MS (EI+, m/z): 445 [M+H]+.
Step 8: Synthesis of (3R,6S)-6-(1,2,4-thiadiazol-5-yl)piperidin-3-yl(benzyloxy)carbamic chloride
(2S,5R)-tert-butyl 5-(benzyloxy(chlorocarbonyl)amino)-2-(1,2,4-thiadiazol-5-yl)piperidine-1-carboxylate was dissolved in 4 N HCl in dioxane (10 mL). The mixture was stirred at rt for 0.5 h and then concentrated to provide the crude ((3R,6S)-6-(1,2,4-thiadiazol-5-yl)piperidin-3-yl)(benzyloxy)carbamic chloride HCl salt, which was directly used in the next step. ESI-MS (EI+, m/z): 445 [M+H]+.
Step 9: (2S,5R)-6-(benzyloxy)-2-(1,2,4-thiadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-7-one
To a mixture of ((3R,6S)-6-(1,2,4-thiadiazol-5-yl)piperidin-3-yl)(benzyloxy)carbamic chloride HCl salt in dry DCM (15 mL) at 0° C. was added TEA (606 mg, 6.0 mmol). The mixture was stirred at rt for 2 hrs and then concentrated. The residue was purified by silica gel column chromatography (3:1 petroleum ether/EtOAc) to give (2S,5R)-6-(benzyloxy)-2-(1,2,4-thiadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (190 mg, 51%) as a yellow solid. ESI-MS (EI+, m/z): 317 [M+H]+, 1HNMR (500 M, CDCl3): δ 8.62 (s, 1H), 7.46-7.37 (m, 5H), 5.09 (d, J=11.2 Hz, 1H), 4.92 (d, J=11.2 Hz, 1H), 4.90 (br d, J=5.5 Hz, 1H), 3.35 (m, 1H), 2.88 (br d, J=6.0 Hz, 1H), 2.79 (d, J=12.0 Hz, 1H), 2.31-2.28 (m, 2H), 2.21-2.11 (m, 1H), 1.98-1.90 (m, 1H).
Step 10: Synthesis of (2S,5R)-6-hydroxy-2-(1,2,4-thiadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one
BCl3 (1 M in DCM, 3.0 mL, 3.0 mmol) was added dropwise to a solution of (2S,5R)-6-(benzyloxy)-2-(1,2,4-thiadiazol-5-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (190 mg, 0.60 mmol) in dry DCM (20 mL) at −78° C. The mixture was stirred at 0° C. for 6 hrs., then, MeOH (3 mL) was added slowly at −78° C. The solvents were evaporated under vacuum to give (2S,5R)-6-hydroxy-2-(1,2,4-thiadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (110 mg, 80%) as a yellow solid, which was directly used in the next step. ESI-MS (EI−, m/z): 227.1 [M−H]−.
Step 11: Synthesis of tetrabutylammonium (2S,5R)-7-oxo-2-(1,2,4-thiadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate
To a solution of crude (2S,5R)-6-hydroxy-2-(1,2,4-thiadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one (110 mg) in dry pyridine (5 mL) was added SO3.Py (0.40 g, 2.3 mmol). The mixture was stirred at rt for 6 hrs and then concentrated under vacuum.
The residue was redissolved in aqueous NaH2PO4 (1.5 M, 10 mL).
Tetrabutylammonium hydrogensulphate (203 mg, 0.6 mmol) was added. The mixture was stirred at rt for 20 min, and extracted with EtOAc (4×). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (gradient elution 10:1 to 2:1 DCM/acetone) to give tetrabutylammonium (2S,5R)-7-oxo-2-(1,2,4-thiadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate as a yellow solid (104 mg, 40% for two steps). ESI-MS (EI−, m/z): 305.0 [M−H]−.
Step 13: Resin Exchange, synthesis of sodium (2S,5R)-7-oxo-2-(1,2,4-thiadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate
Tetrabutylammonium (2S,5R)-7-oxo-2-(1,2,4-thiadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (104 mg) was dissolved in a minimum amount of HPLC grade water ( ̃5 mL) and passed through a column of 10 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 provide sodium (2S,5R)-7-oxo-2-(1,2,4-thiadiazol-5-yl)-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (56 mg, 90%) was obtained after lyophilization as a white solid. ESI-MS (EI−, m/z): 305.0 [M−H]−. 1H-NMR (500 MHz, CDCl3): δ 8.68 (s, 1H), 4.96 (d, J=6.0 Hz, 1H), 4.16 (s, 1H), 3.23 (br d, J=12.0 Hz, 1H), 3.02 (d, J=12.0 Hz, 1H), 2.39-2.35 (m, 1H), 1.30-2.22 (m, 1H), 2.15-2.09 (m, 1H), 1.88-1.81 (m, 1H).
Example 22
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′-cgcatatgactettectttttcaatattattg-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 (pBR322 ΔNdeI ΔTEM-1::cat Seq. ID 5) which retains both the pBR322 tetracycline resistance cassette, tetA, and the plasmid origin of replication but the blaTEM-1 gene was replaced by the cat gene.
Using this engineering strategy a number of plasmids producing β-lactamase genes from different classes (see below) were generated using synthetic genes with an engineered NdeI restriction site at the 5′ end and BamHI restriction site at the 3′ end of each gene (GenScript). Both the synthetic β-lactamase genes and cat gene were ligated into the NdeI/BamHI sites of the pBR322 ΔNdeI PCR product and transformed into electrocompetent E. coli ElectroMax DH10B (Invitrogen/Life Technologies). E. coli DH10B harboring the recombinant plasmids were selected on LB agar (supplemented with 25 μg/mL tetracycline) and single isolated colonies were then inoculated into 5 mL LB media (supplemented with 25 μg/mL tetracycline), and incubated at 37° C. with aeration (250 rpm) for 18 hrs. The cultures were frozen back at −80° C. in 20% glycerol. The DNA sequence of the cloned β-lactamase genes was confirmed. The β-lactamase gene expression in the recombinant E. coli strains was driven by the blaTEM-1 promoter in the pBR-CBST plasmid and was characterized by MIC profiling of the E. coli recombinant strains against comparator β-lactam/BLI combinations in broth microdilution assay.











TABLE X









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









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


ttcttgaagacgaaagggcctcgtgatacgcctatttttatataggttaa


 


tgtcatgataataatggtttacttagacgtcaggtggcacttttcgggga


 


aatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatat


 


gtatccgctcatgagacaataaccctgataaatgcttcaataatattgaa


 


aaaggaagagtcatATGGAGAAAAAAATCACTGGATATACCACCGTTGAT


 


ATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGC


 


TCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAA


 


AGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATT


 


CTTGCCCGCCTGATGAATGCTCATACGGAATTTCGTATGGCAATGAAAGA


 


CGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCC


 


ATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGAT


 


TTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGA


 


AAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCT


 


CAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAAT


 


ATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGCAAATATTATACGCA


 


AGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCT


 


GTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGC


 


GATGAGTGGCAGGGCGGGGCGTAAGTGGCAGGGCGGGGCGTAAGGCGCGC


 


CATTTAAATGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGggatccctg


 


tcagaccaagtttactcatatatactttagattgatttaaaacttcattt


 


ttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacca


 


aaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaa


 


aagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctg


 


cttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatc


 


aagagctaccaactctttttccgaaggtaactggcttcagcagagcgcag


 


ataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaa


 


gaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccag


 


tggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaaga


 


cgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtg


 


cacacagcccagcttggagcgaacgacctacaccgaactgagatacctac


 


agcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggac


 


aggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagct


 


tccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacc


 


tctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagccta


 


tggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctg


 


gccttttgctcacatgttctttcctgcgttatcccctgattctgtggata


 


accgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacg


 


accgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcg


 


gtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgca


 


ctctcagtacaatctgctctgatgccgcatagttaagccagtatacactc


 


cgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaaca


 


cccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacag


 


acaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgt


 


catcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcg


 


tgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgag


 


tttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaa


 


gggcggttttttcctgtttggtcactgatgcctccgtgtaagggggattt


 


ctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgat


 


acgggttactgatgatgaacatgcccggttactggaacgttgtgagggta


 


aacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggt


 


caatgccagcgcttcgttaatacagatgtaggtgttccacagggtagcca


 


gcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgact


 


tccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgtt


 


gttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctc


 


gcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagccta


 


gccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggac


 


ccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgc


 


gatggatatgttctgccaagggttggtttgcgcattcacagttctccgca


 


agaattgattggctccaattcttggagtggtgaatccgttagcgaggtgc


 


cgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgc


 


aacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaa


 


cccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcg


 


gtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagc


 


tgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgc


 


gggcatcccgatgccgccggaagcgagaagaatcataatggggaaggcca


 


tccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggcc


 


gccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcggg


 


accagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaa


 


gcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaa


 


atgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaa


 


gacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaagg


 


agctgactgggttgaaggctctcaagggcatcggtcgacgctctccctta


 


tgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgag


 


caccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtc


 


ccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatg


 


agcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatata


 


ggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtc


 


cggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcg


 


atagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcg


 


gtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcat


 


atagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacg


 


atatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctac


 


agcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatt


 


tcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccg


 


cattaaagcttatcgatgataagctgtcaaacatgagaa


 


pBR-CBST-KPC-2


SEQ ID 6


ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatg


 


tcatgataataatggtttcttagacgtcaggtggcacttttcggggaaat


 


gtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgta


 


tccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa


 


ggaagagtcatATGTCACTGTATCGCCGTCTAGTTCTGCTGTCTTGTCTC


 


TCATGGCCGCTGGCTGGCTTTTCTGCCACCGCGCTGACCAACCTCGTCGC


 


GGAACCATTCGCTAAACTCGAACAGGACTTTGGCGGCTCCATCGGTGTGT


 


ACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAG


 


CGCTTCCCACTGTGCAGCTCATTCAAGGGCTTTCTTGCTGCCGCTGTGCT


 


GGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACG


 


GCAAAAATGCGCTGGTTCCGTGGTCACCCATCTCGGAAAAATATCTGACA


 


ACAGGCATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGTGA


 


TAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGGCCGGGC


 


TGACGGCCTTCATGCGCTCTATCGGCGATACCACGTTCCGTCTGGACCGC


 


TGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGATGCGCGCGATACCTC


 


ATCGCCGCGCGCCGTGACGGAAAGCTTACAAAAACTGACACTGGGCTCTG


 


CACTGGCTGCGCCGCAGCGGCAGCAGTTTGTTGATTGGCTAAAGGGAAAC


 


ACGACCGGCAACCACCGCATCCGCGCGGCGGTGCCGGCAGACTGGGCAGT


 


CGGAGACAAAACCGGAACCTGCGGAGTGTATGGCACGGCAAATGACTATG


 


CCGTCGTCTGGCCCACTGGGCGCGCACCTATTGTGTTGGCCGTCTACACC


 


CGGGCGCCTAACAAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGC


 


GGCTAGACTCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAAggatccc


 


tgtcagaccaagtttactcatatatactttagattgatttaaaacttcat


 


ttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgac


 


caaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtag


 


aaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc


 


tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgga


 


tcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgc


 


agataccaaatactgtccttctagtgtagccgtagttaggccaccacttc


 


aagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacc


 


agtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaa


 


gacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg


 


tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct


 


acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgg


 


acaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggag


 


cttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgcca


 


cctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcc


 


tatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgc


 


tggccttttgctcacatgttctttcctgcgttatcccctgattctgtgga


 


taaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaa


 


cgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatg


 


cggtattttctccttacgcatctgttgcggtatttcacaccgcatttggt


 


gcactctcagtacaatctgctctgatgccgcatagttaagccagtataca


 


ctccgctatcgctacgtgactgggtcatggctgcgccccgacacccgcca


 


acacccgctgacgcgccctgacgggcttgtctgctcccggcatccgctta


 


cagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcac


 


cgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtgg


 


tcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgtt


 


gagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgt


 


taagggcggttttttcctgtttggtcactgatgcctccgtgtaaggggga


 


tttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcac


 


gatacgggttactgatgatgaacatgcccggttactggaacgttgtgagg


 


gtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcag


 


ggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtag


 


ccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctg


 


acttccgcgtttccagactttacgaaacacggaaaccgaagaccattcat


 


gttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcg


 


ctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagc


 


ctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccag


 


gacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcgga


 


cgcgatggatatgttctgccaagggttggtttgcgcattcacagttctcc


 


gcaagaattgattggctccaattcttggagtggtgaatccgttagcgagg


 


tgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcga


 


cgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgc


 


caacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatca


 


gcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttga


 


agctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaa


 


cgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaagg


 


ccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcg


 


gccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggc


 


gggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccg


 


caagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccg


 


aaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaa


 


gaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccgga


 


aggagctgactgggttgaaggctctcaagggcatcggtcgacgctctccc


 


ttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgtt


 


gagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaaca


 


gtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctc


 


atgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgat


 


ataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgc


 


gtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtag


 


tcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaa


 


gcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacg


 


catatagcgctagcagcacgccatagtgactggcgatgctgtcggaatgg


 


acgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcc


 


tacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttag


 


atttcatacacggtgcctgactgcgttagcaatttaactgtgataaacta


 


ccgcattaaagcttatcgatgataagctgtcaaacatgagaa


 


pBR-CBST-CTX-M-15


SEQ ID 7


ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatg


 


tcatgataataatggtttcttagacgtcaggtggcacttttcggggaaat


 


gtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgta


 


tccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa


 


ggaagagtcatATGGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATC


 


TGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGACGA


 


TGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTG


 


GCGATGAATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCGC


 


GTTCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGC


 


CGACGTTAAACACCGCCATTCCGGGCGATCCGCGTGATACCACTTCACCT


 


CGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGG


 


CGACAGCCAACGGGCGCAGCTGGTGACATGGATGAAAGGCAATACCACCG


 


GTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGAT


 


AAAACCGGCAGCGGTGGCTATGGCACCACCAACGATATCGCGGTGATCTG


 


GCCAAAAGATCGTGCGCCGCTGATTCTGGTCACTTACTTCACCCAGCCTC


 


AACCTAAGGCAGAAAGCCGTCGCGATGTATTAGCGTCGGCGGCTAAAATC


 


GTCACCGACGGTTTGTAAggatccctgtcagaccaagtttactcatatat


 


actttagattgatttaaaacttcatttttaatttaaaaggatctaggtga


 


agatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcg


 


ttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgaga


 


tcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgc


 


taccagcggtggtttgtttgccggatcaagagctaccaactctttttccg


 


aaggtaactggcttcagcagagcgcagataccaaatactgtccttctagt


 


gtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacat


 


acctcgctctgctaatcctgttaccagtggctgctgccagtggcgataag


 


tcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgca


 


gcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaa


 


cgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgcc


 


acgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggt


 


cggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatc


 


tttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttg


 


tgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggc


 


ctttttacggttcctggccttttgctggccttttgctcacatgttctttc


 


ctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtga


 


gctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgag


 


cgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgt


 


gcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgat


 


gccgcatagttaagccagtatacactccgctatcgctacgtgactgggtc


 


atggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggc


 


ttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccggga


 


gctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcag


 


ctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgc


 


ctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtct


 


ggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtc


 


actgatgcctccgtgtaagggggatttctgttcatgggggtaatgatacc


 


gatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatg


 


cccggttactggaacgttgtgagggtaaacaactggcggtatggatgcgg


 


cgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatac


 


agatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatcc


 


ggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaa


 


acacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgtttt


 


gcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaac


 


cagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacg


 


atcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgt


 


gcggctgctggagatggcggacgcgatggatatgttctgccaagggttgg


 


tttgcgcattcacagttctccgcaagaattgattggctccaattcttgga


 


gtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtg


 


gcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatag


 


ggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcgg


 


cataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggta


 


agagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctg


 


cctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcga


 


gaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagc


 


aagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgctt


 


ctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgaggg


 


cgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctc


 


cagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctg


 


tcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatag


 


tcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaag


 


ggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcc


 


cagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcat


 


gcaaggagatggcgcccaacagtcccccggccacggggcctgccaccata


 


cccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttc


 


cccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgc


 


cggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggt


 


gtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgag


 


caggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtg


 


cgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtga


 


ctggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtac


 


cggcataaccaagcctatgcctacagcatccagggtgacggtgccgagga


 


tgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttag


 


caatttaactgtgataaactaccgcattaaagcttatcgatgataagctg


 


tcaaacatgagaa


 


pBR-CBST-SHV-12


SEQ ID 8


ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatg


 


tcatgataataatggtttcttagacgtcaggtggcacttttcggggaaat


 


gtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgta


 


tccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa


 


ggaagagtcatATGCGTTATATTCGCCTGTGTATTATCTCCCTGTTAGCC


 


ACCCTGCCGCTGGCGGTACACGCCAGCCCGCAGCCGCTTGAGCAAATTAA


 


ACAAAGCGAAAGCCAGCTGTCGGGCCGCGTAGGCATGATAGAAATGGATC


 


TGGCCAGCGGCCGCACGCTGACCGCCTGGCGCGCCGATGAACGCTTTCCC


 


ATGATGAGCACCTTTAAAGTAGTGCTCTGCGGCGCAGTGCTGGCGCGGGT


 


GGATGCCGGTGACGAACAGCTGGAGCGAAAGATCCACTATCGCCAGCAGG


 


ATCTGGTGGACTACTCGCCGGTCAGCGAAAAACACCTTGCCGACGGCATG


 


ACGGTCGGCGAACTCTGCGCCGCCGCCATTACCATGAGCGATAACAGCGC


 


CGCCAATCTGCTGCTGGCCACCGTCGGCGGCCCCGCAGGATTGACTGCCT


 


TTTTGCGCCAGATCGGCGACAACGTCACCCGCCTTGACCGCTGGGAAACG


 


GAACTGAATGAGGCGCTTCCCGGCGACGCCCGCGACACCACTACCCCGGC


 


CAGCATGGCCGCGACCCTGCGCAAGCTGCTGACCAGCCAGCGTCTGAGCG


 


CCCGTTCGCAACGGCAGCTGCTGCAGTGGATGGTGGACGATCGGGTCGCC


 


GGACCGTTGATCCGCTCCGTGCTGCCGGCGGGCTGGTTTATCGCCGATAA


 


GACCGGAGCTAGCAAGCGGGGTGCGCGCGGGATTGTCGCCCTGCTTGGCC


 


CGAATAACAAAGCAGAGCGCATTGTGGTGATTTATCTGCGGGATACCCCG


 


GCGAGCATGGCCGAGCGAAATCAGCAAATCGCCGGGATCGGCGCGGCGCT


 


GATCGAGCACTGGCAACGCTAAggatccctgtcagaccaagtttactcat


 


atatactttagattgatttaaaacttcatttttaatttaaaaggatctag


 


gtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagtt


 


ttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttctt


 


gagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca


 


ccgctaccagcggtggtttgtttgccggatcaagagctaccaacttttta


 


ccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttct


 


agtgtagccgtagttaggccaccacttcaagaactctgtagcaccgccta


 


catacctcgctctgctaatcctgttaccagtggctgctgccagtggcgat


 


aagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggc


 


gcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagc


 


gaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagc


 


gccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcag


 


ggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggt


 


atctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattt


 


ttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgc


 


ggcctttttacggttcctggccttttgctggccttttgctcacatgttct


 


ttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgag


 


tgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagt


 


gagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatc


 


tgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctct


 


gatgccgcatagttaagccagtatacactccgctatcgctacgtgactgg


 


gtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacg


 


ggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccg


 


ggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagg


 


cagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtc


 


tgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatg


 


tctggcttctgataaagcgggccatgttaagggcggttttttcctgtttg


 


gtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgat


 


accgatgaaacgagagaggatgctcacgatacgggttactgatgatgaac


 


atgcccggttactggaacgttgtgagggtaaacaactggcggtatggatg


 


cggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaa


 


tacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcaga


 


tccggaacataatggtgcagggcgctgacttccgcgtttccagactttac


 


gaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgt


 


tttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattct


 


gctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacagg


 


agcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcg


 


ccgcgtgcggctggagatggcggacgcgatggatatgttctgccaagggt


 


tggtttgcgcattcacagttctccgcaagaattgattggctccaattctt


 


ggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgag


 


gtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggta


 


tagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgagg


 


cggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctg


 


gtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctac


 


ctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaag


 


cgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgcc


 


agcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctg


 


cttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcga


 


gggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcg


 


ctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcac


 


ctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacga


 


tagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctc


 


aagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagca


 


gcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtg


 


catgcaaggagatggcgcccaacagtcccccggccacggggcctgccacc


 


atacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatc


 


ttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtgg


 


cgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacg


 


ggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagc


 


gagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgg


 


gtgcgcatagaaattgcatcaacgcaatatagcgctagcagcacgccata


 


gtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggca


 


gtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccg


 


aggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcg


 


ttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataa


 


gctgtcaaacatgagaa


 


pBR-CBST-P99


SEQ ID 9


ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatg


 


tcatgataataatggtttcttagacgtcaggtggcacttttcggggaaat


 


gtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgta


 


tccgctcatgagacaataacctgataaatgcttcaataatattgaaaaag


 


gaagagtcatATGATGAGAAAATCCCTTTGCTGCGCCCTGCTGCTCGGCA


 


TCTCTTGCTCTGCTCTCGCCACGCCAGTGTCAGAAAAACAGCTGGCGGAG


 


GTGGTCGCGAATACGATTACCCCGCTGATGAAAGCCCAGTCTGTTCCAGG


 


CATGGCGGTGGCCGTTATTTATCAGGGAAAACCGCACTATTACACATTTG


 


GCAAGGCCGATATCGCGGCGAATAAACCCGTTACGCCTCAGACCCTGTTC


 


GAGCTGGGTTCTATAAGTAAAACCTTCACCGGCGTTTTAGGTGGGGATGC


 


CATTGCTCGCGGTGAAATTTCGCTGGACGATGCGGTGACCAGATACTGGC


 


CACAGCTGACGGGCAAGCAGTGGCAGGGTATTCGTATGCTGGATCTCGCC


 


ACCTACACCGCTGGCGGCCTGCCGCTACAGGTACCGGATGAGGTCACGGA


 


TAACGCCTCCCTGCTGCGCTTTTATCAAAACTGGCAGCCGCAGTGGAAGC


 


CTGGCACAACGCGTCTTTACGCCAACGCCAGCATCGGTCTTTTTGGTGCG


 


CTGGCGGTCAAACCTTCTGGCATGCCCTATGAGCAGGCCATGACGACGCG


 


GGTCCTTAAGCCGCTCAAGCTGGACCATACCTGGATTAACGTGCCGAAAG


 


CGGAAGAGGCGCATTACGCCTGGGGCTATCGTGACGGTAAAGCGGTGCGC


 


GTTTCGCCGGGTATGCTGGATGCACAAGCCTATGGCGTGAAAACCAACGT


 


GCAGGATATGGCGAACTGGGTCATGGCAAACATGGCGCCGGAGAACGTTG


 


CTGATGCCTCACTTAAGCAGGGCATCGCGCTGGCGCAGTCGCGCTACTGG


 


CGTATCGGGTCAATGTATCAGGGTCTGGGCTGGGAGATGCTCAACTGGCC


 


CGTGGAGGCCAACACGGTGGTCGAGGGCAGCGACAGTAAGGTAGCACTGG


 


CGCCGTTGCCCGTGGCAGAAGTGAATCCACCGGCTCCCCCGGTCAAAGCG


 


TCCTGGGTCCATAAAACGGGCTCTACTGGCGGGTTTGGCAGCTACGTGGC


 


CTTTATTCCTGAAAAGCAGATCGGTATTGTGATGCTCGCGAATACAAGCT


 


ATCCGAACCCGGCACGCGTTGAGGCGGCATACCATATCCTCGAGGCGCTA


 


CAGTAAggatccctgtcagaccaagtttactcatatatactttagattga


 


tttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttg


 


ataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcg


 


tcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct


 


gcgcgtaatctgctgcttgcaacaaaaaaaccaccgctaccagcggtggt


 


ttgtttgccggatcaagagctaccaactctttttccgaaggtaactggct


 


tcagcagagcgcagataccaaatactgtccttctagtgtagccgtagtta


 


ggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgct


 


aatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccg


 


ggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga


 


acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccga


 


actgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaag


 


ggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag


 


cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgt


 


cgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag


 


gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc


 


ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccc


 


tgattctgtggataaccgtattaccgcctttgagtgagctgataccgctc


 


gccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaa


 


gagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcaca


 


ccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaa


 


gccagtatacactccgctatcgctacgtgactgggtcatggctgcgcccc


 


gacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccg


 


gcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtca


 


gaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagct


 


catcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcg


 


tccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaa


 


gcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccg


 


tgtaagggggattttctgttcatgggggtaatgataccgatgaaacgaga


 


gaggatgctcacgatacgggttactgatgatgaacatgcccggttactgg


 


aacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagaga


 


aaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgt


 


tccacagggtagccagcagcatcctgcgatgcagatccggaacataatgg


 


tgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccg


 


aagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtc


 


gcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggc


 


aaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgc


 


acccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgct


 


ggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcat


 


tcacagttctccgcaagaattgattggctccaattcttggagtggtgaat


 


ccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctc


 


catgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcc


 


tacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcg


 


ccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcg


 


agcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacag


 


catggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatca


 


taatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtag


 


cccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaa


 


acgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaaga


 


ttccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaag


 


cggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgag


 


ttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgcccc


 


gcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggt


 


cgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtag


 


gttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggaga


 


tggcgcccaacagtcccccggccacggggcctgccaccataccacgccga


 


aacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtg


 


atgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgcc


 


ggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgcca


 


tgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactggg


 


cggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaa


 


ttgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgc


 


tgtcggaatggacgatatcccgccgaggcccggcagtaccggcataacca


 


agcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagc


 


gcattgttagatttcatacacggtgcctgactgcgttagcaatttaactg


 


tgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgag


 


aa


 


pBR-CBST-OXA-15


SEQ ID 10


ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatg


 


tcatgataataatggtttcttagacgtcaggtggcacttttcggggaaat


 


gtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgta


 


tccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa


 


ggaagagtcatATGGCAATCCGAATCTTCGCGATACTTTTCTCCATTTTT


 


TCTCTTGCCACTTTCGCGCATGCGCAAGAAGGCACGCTAGAACGTTCTGA


 


CTGGAGGAAGTTTTTCAGCGAATTTCAAGCCAAAGGCACGATAGTTGTGG


 


CAGACGAACGCCAAGCGGATCGTGCCATGTTGGTTTTTGATCCTGTGCGA


 


TCGAAGAAACGCTACTCGCCTGCATCGACATTCAAGATACCTCATACACT


 


TTTTGCACTTGATGCAGGCGCTGTTCGTGATGAGTTCCAGATTTTTCGAT


 


GGGACGGCGTTAACAGGGGCTTTGCAGGCCACAATCAAGACCAAGATTTG


 


CGATCAGCAATGCGGAATTCTACTGTTTGGGTGTATGAGCTATTTGCAAA


 


GGAAATTGGTGATGACAAAGCTCGGCGCTATTTGAAGAAAATCGACTATG


 


GCAACGCCGGTCCTTCGACAAGTAATGGCGATTACTGGATAGAAGGCAGC


 


CTTGCAATCTCGGCGCAGGAGCAAATTGCATTTCTCAGGAAGCTCTATCG


 


TAACGAGCTGCCCTTTCGGGTAGAACATCAGCGCTTGGTCAAGGATCTCA


 


TGATTGTGGAAGCCGGTCGCAACTGGATACTGCGTGCAAAGACGGGCTGG


 


GAAGGCCGTATGGGTTGGTGGGTAGGATGGGTTGAGTGGCCGACTGGCTC


 


CGTATTCTTCGCACTGAATATTGATACGCCAAACAGAATGGATGATCTTT


 


TCAAGAGGGAGGCAATCGTGCGGGCAATCCTTCGCTCTATTGAAGCGTTA


 


CCGCCCAACCCGGCAGTCAACTCGGACGCTGCGCGATAAggatccctgtc


 


agaccaagtttactcatatatactttagattgatttaaaacttcattttt


 


aatttaaaaggatctaggtgaagatccttttgataatctcatgaccaaaa


 


tcccttaacgtgagattttcgttccactgagcgtcagaccccgtagaaaa


 


gatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgct


 


tgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaa


 


gagctaccaactctttttccgaaggtaactggcttcagcagagcgcagat


 


accaaatactgtccttctagtgtagccgtagttaggccaccacttcaaga


 


actctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtg


 


gctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacg


 


atagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgca


 


cacagcccagcttggagcgaacgacctacaccgaactgagatacctacag


 


cgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacag


 


gtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttc


 


cagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctc


 


tgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatg


 


gaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggc


 


cttttgctcacatgttctttcctgcgttatcccctgattctgtggataac


 


cgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgac


 


cgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggt


 


attttctccttacgcatctgtgcggtatttcacaccgcatttggtgcact


 


ctcagtacaatctgctctgatgccgcatagttaagccagtatacactccg


 


ctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacc


 


cgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagac


 


aagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtca


 


tcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtg


 


aagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtt


 


tctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagg


 


gcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttct


 


gttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatac


 


gggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaa


 


caactggcggtatggatgcggcgggaccagagaaaaatcactcagggtca


 


atgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagc


 


agcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttc


 


cgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgt


 


tgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgc


 


gtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagc


 


cgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggaccc


 


aacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcga


 


tggatatgttctgccaagggttggtttgcgcattcacagttctccgcaag


 


aattgattggctccaattcaggagtggtgaatccgttagcgaggtgccgc


 


cggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaac


 


gcggggaggcagacaaggtatagggcggcgcctacaatccatgccaaccc


 


gttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtc


 


cagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgt


 


ccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcggg


 


catcccgatgccgccggaagcgagaagaatcataatggggaaggccatcc


 


agcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgcc


 


atgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggacc


 


agtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcg


 


acaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatg


 


acccagagcgctgccggcacctgtcctacgagttgcatgataaagaagac


 


agtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagc


 


tgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgc


 


gactcctgcattaggaagcagcccagtagtaggagaggccgagagcaccg


 


ccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccg


 


gccacggggcctgccaccatacccacgccgaaacaagcgctcatgagccc


 


gaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgc


 


cagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcg


 


tagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagt


 


ggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcgg


 


acagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagc


 


gctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatc


 


ccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcat


 


ccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcata


 


cacggtgcctgactgcgttagcaatttaactgtgataaactaccgcatta


 


aagcttatcgatgataagctgtcaaacatgagaa






Example 23
Synergy MIC Assay
The synergy MIC (sMIC) assay determines the concentration of the BLI required to potentiate the activity of a fixed concentration of a β-lactam antibiotic against β-lactamase producing bacterial strains. The experimental protocol was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines with modifications as described below (CLSI guidelines can be derived from the CLSI document M07-A9 published in January 2012: “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Ninth Edition”). The assay is set-up by serially diluting the BLI across 11 of the 12 wells in each row of a 96-well broth microdilution assay plate, adding the β-lactam at a fixed concentration to all wells in the assay plate, inoculating the assay plate with bacterial strains, and determining the lowest concentration of BLI required to inhibit overnight bacterial growth. Bacterial growth in the 12th well of the assay plate, which contains the β-lactam at a fixed concentration but does not contain any BLI, demonstrates that the bacterial strains are resistant to the β-lactam antibiotic (e.g. ceftolozane) at the fixed concentration of 4 μg/mL.
To prepare for MIC testing, frozen glycerol stocks of 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 tetracycline supplemented LB plates containing engineered strains. The engineered strain material was suspended in CAMHB (supplemented with tetracycline at 25 μg/mL) in 14 mL culture tubes. All strains were incubated at 37° C. with aeration (200 rpm) for 2 hrs until the OD600 was ≧0.1.
The two compound components of the assay were each prepared in CAMHB and added to the 96-well broth microdilution assay plates. 50 μL, of the BLI was added to each well of the assay plate in 2-fold dilutions with final concentrations ranging from 128 to 0.13 μg/mL. 25 μL, of the β-lactam was added to all wells in the broth microdilution plates at a final concentration of 4 μg/mL. Inoculum cultures were prepared by standardizing the primary cultures to OD600=0.1 and then adding 20 μL of the adjusted primary culture per 1 mL CAMHB for clinical strains or CAMHB (supplemented with tetracycline at 100 μg/mL) for isogenic strains, so that the final inoculum density was  ̃105 colony forming units per milliliter. Diluted inoculum cultures were used to inoculate 25 μL per well in 96-well broth microdilution assay plates. The final volume of each well was 100 μL, and contained a BLI at different concentrations, a β-lactam at 4 μg/mL concentration, the bacterial culture at an OD600 of approximately 0.001 and when necessary tetracycline at 25 ug/mL.
Interpreting the sMIC Data:
Plates were incubated for 18-20 hours at 37° C. with aeration (200 rpm). Following incubation, growth was confirmed visually placing plates over a viewing apparatus (stand with a mirror underneath) and then OD600 was measured using a SpectraMax 340PC384 plate reader (Molecular Devices, Sunnyvale, Calif.). Growth was defined as turbidity that could be detected with the naked eye or achieving minimum OD600 of 0.1. sMIC values were defined as the lowest concentration producing no visible turbidity.
The sMIC values represent the amount of BLI required to potentiate the activity of 4 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 II.
Example 24
Inhibition Kinetics
Inhibition or inactivation of KPC-2 by test inhibitors was assessed using 100 μM nitrocefin (NCF) as a reporter substrate. Assays were performed in 1×PBS pH 7.4, 0.1 mg/ml BSA, in 96-well half area plates, 50 μl reaction volume. NCF was dissolved in DMSO and diluted in assay buffer. Test inhibitors were dissolved in water or DMSO and serially diluted in the assay with final concentrations between 2000-0.195 μM.
The enzyme activity in the presence of varying concentrations of test inhibitor was determined by monitoring the hydrolysis of NCF spectrophotometrically at 486 nm, for 5 minutes, 25° C., using a SpectraMax Plus384 microplate reader with SoftMax Pro software (Molecular Devices). Data analysis was performed using GraphPad Prism (GraphPad Software, Inc.).
Progress curves were fit to a first-order rate decay equation (Eq. 1) to determine kobserved (kobs).
kobs vs. inhibitor concentration [I] curves were then fit to Eq.2 to determine the inhibitor dissociation constant (K) and the first order rate constant of enzyme inactivation at infinite inhibitor concentration (kinact). Table III shows kinetics results from representative test compounds. A larger kinact K ratio indicates a more effective enzyme inactivator.

Yt=V0*(1−e(−kobs*t))/kobs  Eq. 1

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

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

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

Biological data using the procedures described herein are shown for certain compounds in U.S. patent application Ser. Nos. 13/853,443, 13/853,498 and 13/853,506, the contents of which are incorporated herein by reference in their entirety.

## Claims

1. A process for preparing a compound of Formula (A-I):



wherein
Z is selected from the group consisting of 1,3,4-oxadiazole, 1,2,4-oxadiazole, 1,3,4-thiadazole and 1,2,4-thiadazole;
R* is —OSO3H; and
R1* is selected from:
a. hydrogen;
b.







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 or (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;

c.







wherein R8* is selected from NH2,








wherein each of R4*, R5*, R6*and R7* is as described previously and each of R9, R10, and R11 is independently selected from hydrogen or (C1-C6)-alkyl, provided that at least one of R9, R10, and R11 is hydrogen;

d. amino;
e.







wherein Z* is selected from CR12R13 or NR14,
each of R12 and R13 is independently selected from H, NH2 and








wherein each of R4*, R5*, R6*and R7* is as described previously, alternatively, R12 and R13 together with the carbon to which they are attached, form a cycloalkyl or heterocyclyl ring containing 4-6 ring members,
R14 is selected from H and







wherein each of R15, R16, and R17 is independently selected from hydrogen, (C1-C6)-alkyl, aminoalkyl, aminocycloalkyl, and hydroxyalkyl, provided that at least one of R15, R16 and R17 is hydrogen,


R18 is selected from NH2 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;

f.







wherein R19 is selected from NH2 and







wherein each of R4*,


R5*, R6*and R7* is as described previously,
R20 is selected from amino and hydroxyl, and
w is selected from 0 and 1;

g.






h.






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

i.







wherein R21 is 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;

j.






k.







wherein M is selected from NR22, CR23R24 and O,
wherein R22 is H or







wherein each of R15, R16 and R17 is as described previously,


each of R23 and R24 is independently selected from H, NH2 and







wherein each of R4*, R5*, R6* and R7* is as described previously, and


u is selected from 0, 1 and 2;

l.






m.






n.






o. (C1-C3)-unsubstituted alkyl; and
p.





the process comprising the steps of:
(a) reacting compound 1 with lithium hydroxide to produce compound 2a:





or
reacting compound 1 with lithium borohydride, followed by TEMPO-mediated oxidation to produce compound 2b:




(b) converting the carboxylic acid of 2a or the aldehyde of 2b to a heterocyclic ring to produce compound 3:




(c) debenzylating compound 3 to produce compound 4:





and
(d) reacting intermediate 4 with SO3.pyridine complex to produce compound 5:

2. The process of claim 1, wherein compound of Formula (A-I) is the compound:




and
wherein
step (a) comprises reacting compound 1 with lithium hydroxide in THF/H2O to produce compound 2a; and
step (b) comprises the steps of:
(1) reacting compound 2a with tert-butyl hydrazinecarboxylate to produce the compound:






(2) reacting the product of step (1) with trifluoroacetic acid to produce the compound:






(3) reacting the product of step (2) with N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) to produce the compound:






(4) reacting the product of step (3) with triflic anhydride to produce compound:






step (c) comprises the step of reacting the product of step (b) with Pd/C and H2 to produce the compound:






and

step (d) comprises reacting the product of step (c) with SO3.pyridine complex to produce the compound:

3. The process of claim 1, wherein compound 1 is produced by a process comprising the steps of:
(1) reacting the compound:




with the compound:




to produce the compound:




(2) reacting the product of step (1) with lithium hydroxide monohydrate and 2-mercaptoacetic acid to produce the compound:




(3) deprotecting the product of step (2) to produce the compound:





and
(4) reacting the product of step (3) with triphosgene such that compound 1 is formed.