Technical Field

This invention relates to novel compounds, pharmaceutical compositions thereof, and their use in therapy, particularly in the treatment of bacterial infections, such as bacterial infections caused by bacteria which are resistant to beta-lactam antibiotics.

Background

Antimicrobial drug resistance is a global health emergency, threatening advances in many areas of medicine including surgery, cancer chemotherapy, organ transplantation and survival of pre-term infants (O’Neill, 2014; Rossolini et al., 2014). The most prevalent and important resistance determinant is the beta-lactamase enzyme, which hydrolyses members of the beta-lactam class of antibiotic (e.g. penicillin, ampicillin, cephalothin) and thereby prevents engagement with their therapeutic target the penicillin-binding proteins (PBPs) (Kong et al., 2010; Fernandes et al., 2013). Of particular concern are the extended-spectrum beta-lactamases (ESBLs) such as the CTX-M class, which are able to cleave a wide range of clinically-relevant beta-lactam antibiotics (Bush, 2013; Canton, 2012; Shaikh et al., 2015).

Urinary tract infections (UTIs) are the most prevalent type of bacterial infection globally. These infections have a high rate of recurrence and can also lead to serious invasive infections such as sepsis, particularly in the elderly (Flores-Mireles et al., 2015; Peach et al. 2016). E. coli is the most common causative organism (-75% cases), of which -50% are resistant to beta-lactam antibiotics due to beta-lactamase expression (Flores-Mireles et al., 2015; Tandogdu et al., 2016).

As a consequence of the high rate of beta-lactam resistance in UTI pathogens, second-line, broad- spectrum antibiotics such as ciprofloxacin are increasingly used therapeutically (Kabbani, 2018; Scheld, 2003). Unfortunately, broad-spectrum antibiotics are associated with disruption to the beneficial bacteria that colonise the gastro-intestinal tract and other surfaces, known as the microbiota (Becattini et al., Stewardson et al. 2015; Dethlefsen et al., 201 1 ; Sullivan et al., 2001 ; Jernberg et al., 2001). This disruption can lead to serious secondary infections by antibiotic-resistant bacteria such as Clostridium difficle or fungi such as Candida albicans, leading to colitis and thrush respectively (Becattini et al., 2016; Brown et al., 2013). This is because antibiotics target conserved processes in bacteria such as cell wall, protein, DNA or RNA biosynthesis, which not only occur in the pathogens that cause infection but also in the members of the microbiota (Chellat et al., 2016; Lewis, 2013).

An additional complication associated with some second-line therapeutics such as ciprofloxacin is host toxicity. Ciprofloxacin holds two black box warnings, one for increased risk of tendinitis and tendon rupture and one for exacerbation of muscle weakness in Myasthenia gravis sufferers (FDA highlights of prescribing information: Cipro). Additionally, in 2015, the FDA officially recognized fluoroquinolone-associated disability (FQAD) as a syndrome. FQAD describes a range of disabling and potentially permanent side effects including disturbances of tendons, joints, muscles, nerves, the nervous system and induction of type 2 diabetes (FDA updates warnings for fluoroquinolone antibiotics; Marchant, 2018). As a result, strategies with the potential to mitigate host toxicity by reducing exposure to ciprofloxacin are needed.

Given the drawbacks associated with broad-spectrum antibiotics, efforts have been made to limit their use (Kabbani et al., 2018; Marchant, 2018; Dingle et al. , 2017). However, these efforts have had limited success with usage rates increasing globally, particularly in low-middle income countries (Klein, 2018). In part, this is due to a lack of access to fast and efficient diagnostic techniques and the need to respond quickly to serious bacterial infections with effective and cost-efficient treatment regimens that target a wide range of different bacterial pathogens (O’Neill, 2015). There is, therefore, a pressing need to develop new therapeutics that kill a broad range of different pathogens without damaging the host microbiota.

Expression of beta-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to beta-lactam antibiotics. Therefore, there remains a need for a novel therapeutic approach for treating bacterial infections, particularly bacterial infections which are caused by bacteria which express the beta-lactamase enzyme.

The present invention addresses this need.

Summary of Invention

The invention provides a compound of formula (I):

^{A— B} (I)

wherein A comprises a beta-lactam cleavable motif and B comprises a bactericidal fluoroquinolone motif, wherein A and B are joined by a cleavable linker such that cleaving of A leads to cleavage of the cleavable linker and release of B; or a pharmaceutically acceptable salt thereof.

Suitably the invention provides a compound of formula (la), or a pharmaceutically acceptable salt thereof, as defined below.

Also provided is a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use as a medicament, in particular, for use in the treatment or prevention of bacterial infection. Suitably, the bacterial infection is caused by bacteria that express beta-lactamase, or the bacterial infection is caused by de-colonisation of an individual who is colonised by a pathogen that expresses the beta-lactamase.

Also provided are pharmaceutical compositions containing a compound of formula (I), or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable carrier or excipient.

Also provided are processes for preparing compounds of formula (I) and novel intermediates of use in the preparation of compounds of formula (I).

Brief Description of Figures

Figure 1 shows activity of pro-drug 41 against recombinant DNA gyrase ± recombinant CTX-M-15 beta-lactamase. DNA was separated by agarose gel electrophoresis, gel bands corresponding to supercoiled DNA were quantified and normalised to no gyrase and gyrase only activity. Gyr, DNA gyrase; PD, pro-drug; CTX, CTX-M-15. Error bars represent SEM (n = 4); prodrug vs prodrug + CTX- M-15 was analysed by unpaired f-test, p = 0.0004 (GraphPad Prism 7.03).

Figure 2 shows antibacterial activities for pro-drug 41 (grey circles) and ciprofloxacin (V) (black squares) against CFT073 E.coli cells WT and expressing empty plasmid (pEMP), CTX-M-1 (pCTX), NDM1 (pNMD1) and KPC (pKPC). (A) Dose-response curves, each point represents the mean ± SEM, n = 3.

Figure 3 shows dose response curves for pro-drug 41 (grey circles) and ciprofloxacin (V) (black squares) against six uropathogenic E.coli clinical isolates. Each point represents mean ± SEM, n = 3.

Figure 4 shows the antibacterial activities for pro-drug 41 (circles) and ciprofloxacin (V) (squares) against E. faecalis (A) ATCC29212 and (B) GW01.

Figure 5 shows the antibacterial activities for pro-drug 41 against E. coli CFT073 expressing (A) empty plasmid (pEMP) and (B) CTX-M-15 (pCTX) with (triangles) and without (circles) serum.

Figure 6 shows survival of CFT073 pEMP (open circle) and pCTX (filled circle) with no treatment (black), ciprofloxacin (V) (78 nM) (light grey) or pro-drug 41 (78 nM) (dark grey).

Detailed Description of Invention

The invention relates to compounds which are antibiotic pro-drugs which comprise a bactericidal fluoroquinolone motif, such as those found in fluoroquinolone antibiotics such as ciprofloxacin, and a beta-lactam cleavable motif. The pro-drugs of the invention are bactericidal only after activation by beta-lactamase, after which the bactericidal fluoroquinolone motif is released, in accordance with the following proposed mechanism (by reference to example structures):

Without wishing to be bound by theory, it is thought that the beta-lactam cleavable motif selectively targets bacteria which express beta-lactamase. Since beta-lactamase is prevalent in certain bacteria (such as those bacteria which cause infections), but is much less prevalent within the microbiota, the pro-drug comprising the beta-lactam cleavable motif selectively targets the bacteria which express beta-lactamase over those which do not. Thus, disruption to beneficial bacteria which is associated with broad spectrum antibiotics such as ciprofloxacin is avoided. In turn, the prevalence of serious secondary infections such as those mentioned in the Background section is reduced. The compounds of the invention provide selective targeting of drug-resistant bacteria (such as beta- lactamase expressing bacteria) without disrupting or selecting for resistance within the microbiota, thus reducing the rate of secondary infections and subsequent antibiotic use.

Suitably the beta-lactam cleavable motif is a carrier moiety, and delivers the fluoroquinolone motif containing moiety (such as an antibiotic, for example ciprofloxacin) to cells which express beta- lactamase.

In an embodiment, the compounds of formula (I) (and formulae (la), (lb) and (lc)) are stable and not bactericidal when intact, but enable the release of the fluoroquinolone motif containing moiety (such as ciprofloxacin) upon activation of beta-lactamase.

Definitions

The term‘alkyl’ as used herein, such as in Ci-i ₂ alkyl, Ci- ₆ alkyl, Ci- ₄ alkyl or Ci- ₃ alkyl, whether alone or forming part of a larger group such as an Oalkyl group (e.g. OCi-i ₂ alkyl), is a straight or a branched fully saturated hydrocarbon chain containing the specified number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, /so-propyl, n-butyl, /so-butyl, sec-butyl, tert- butyl and n-pentyl, sec-pentyl and 3-pentyl, hexyl (all isomers thereof), heptyl (all isomers thereof), octyl (all isomers thereof), nonyl (all isomers thereof), decyl (all isomers thereof), undecyl (all isomers thereof) and dodecyl (all isomers thereof). Reference to“propyl” includes n-propyl and /so-propyl, and reference to“butyl” includes n-butyl, isobutyl, sec-butyl and tert- butyl. Examples of Oalkyl groups include the OCi-i ₂ alkyl groups methoxy, ethoxy, propoxy (which includes n-propoxy and iso- propoxy), butoxy (which includes n-butoxy, iso- butoxy, sec-butoxy and fe/f-butoxy), pentoxy (all isomers thereof), hexoxy (all isomers thereof), heptoxy (all isomers thereof), octoxy (all isomers thereof), nonoxy (all isomers thereof), decoxy (all isomers thereof), undecoxy (all isomers thereof) and dodecoxy (all isomers thereof). The term‘alkylene’ as used herein, such as in Co- ₃ alkyleneC _3-6 cycloalkyl is a bifunctional straight or a branched fully saturated hydrocarbon chain containing the specified number of carbon atoms. Examples of Co- ₃ alkylene groups are where the group is absent (i.e. Co), methylene (Ci), ethylene (C ₂ ) and propylene (C ₃ ).

The term‘cycloalkyl’ as used herein, such as in C _3-6 cycloalkyl is a fully saturated hydrocarbon ring containing the specified number of carbon atoms. Examples of cycloalkyl groups include the C ₃ - _6C ydoalkyl groups cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term ‘heterocycloalkyl’ as used herein, such as in C _3-6 heterocycloalkyl is a fully saturated hydrocarbon ring containing the specified number of carbon atoms wherein at least one of the carbon atoms is replaced by a heteroatom such as N, S or O. Examples of C _3-6 heterocycloalkyl include those comprising one nitrogen atom such as containing one heteroatom (i.e. nitrogen) or containing two heteroatoms (e.g. two nitrogen atoms or one nitrogen atom and one oxygen atom). Particular examples of C _3-6 heterocycloalkyl comprising one nitrogen atom include pyrrolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholinyl and thiomorpholinyl.

The term‘halo’ or‘halogen’ as used herein, refers to fluorine (fluoro), chlorine (chloro), bromine (bromo) or iodine (iodo). Particular examples of halo are chlorine and bromine, especially bromine.

The term‘haloalkyl’ as used herein, such as in C _M2 haloalkyl is a straight fully saturated hydrocarbon chain containing the specified number of carbon atoms and at least one halogen atom, such as fluoro or chloro, especially fluoro. An example of haloalkyl is CF ₃ .

The term ‘haloalkoxy’ as used herein, such as in OCi-i ₂ haloalkyl is a straight fully saturated hydrocarbon chain containing the specified number of carbon atoms and at least one halogen atom, such as fluoro or chloro, especially fluoro, which is attached via the oxygen atom. An example of haloalkyl is OCF ₃ .

The term‘heteroaryl’ as used herein refers to 5- or 6-membered aromatic rings containing at least one heteroatom (e.g. nitrogen, oxygen or sulphur). Exemplary heteroaryl rings include furanyl, thiophenyl, pyrrolidinyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrimidinyl or pyrazinyl. Exemplary heteroaryl groups are furanyl and thiophenyl. The heteroaryl ring is optionally substituted, as defined herein.

The term‘beta-lactam cleavable motif as used herein means a moiety which comprises the following motif: The beta-lactam ring is capable of being cleaved (as shown above) by the beta-lactamase enzyme, for example by hydrolysis. The hydrolysis may occur by hydrolysis through OH-containing amino acid groups such as serine on the beta-lactamase enzyme, or, for metallo-beta-lactamases (e.g. NDM1), the hydrolytic activity is from water and is mediated by a zinc metal ion bound in the enzyme active site. Beta-lactam cleavable motifs are found in the beta-lactam class of broad-spectrum antibiotics and include antibiotics such as cephalosporin, pencillin, monobactams and carbapenems, and derivatives thereof.

For example, the cephalosporin family of antibiotics comprises the following moiety:

The pencillin family of antibiotics comprises the following moiety:

The monobactams family of antibiotics comprises the following moiety:

The carbapenems family of antibiotics comprises the following moiety:

In one embodiment, A comprises the following structure:

Suitably, A comprises the following structure:

wherein:

R is selected from the group consisting of Ci-i ₂ alkyl, OCi-i ₂ alkyl, C _M2 haloalkyl, OC _M2 haloalkyl, NR ₂ R ₃ , (CH ₂ )o-iphenyl, (CH ₂ ) _O -I naphthyl, (CH ₂ ) _O -I heteroaryl, (CH ₂ )o-iC _3-6 cycloalkyl and (CH ₂ ) _O -IC ₃ - 6heterocycloalkyl;

R ₂ is selected the group consisting of H, Ci- ₃ alkyl, Ci- ₃ haloalkyl, (CH ₂ ) _O -I phenyl, naphthyl and (ChhK ₁ heteroaryl; and

R ₃ is selected from H and Ci- ₃ alkyl; or

R ₂ and R ₃ join such that, together with the nitrogen atom to which they are attached, they form a C _3-6 heterocycloalkyl; wherein any of the the phenyl, naphthyl, heteroaryl, cycloalkyl or heterocycloalkyl groups (such as phenyl or heteroaryl group) are optionally substituted by at least one (such as one) of the group consisting of Ci- ₆ alkyl, OCi- ₆ alkyl, Ci- ₆ haloalkyl, OCi- ₆ haloalkyl, Co- ₃ alkyleneC _3-6 cycloalkyl, OCo- ₃ alkyleneC _3-6 cycloalkyl, Co- ₃ alkyleneC _3-6 heterocycloalkyl, OCo- ₃ alkyleneC _3-6 heterocycloalkyl, CN, halo, NR ₂ R ₃ , nitro, phenyl, Ophenyl, heteroaryl and O-heteroaryl, such as Ci- ₄ alkyl, OCi- ₄ alkyl, Ci- ₂ haloalkyl, halo, nitro, phenyl or Ophenyl, wherein any of the second phenyl, heteroaryl, cycloalkyl or heterocycloalkyl substituent groups is optionally substituted by Ci- ₄ alkyl, OCi- ₄ alkyl, Ci- ₄ haloalkyl, OCi- ₄ haloalkyl, halo, CN or nitro; wherein optionally one or more carbons (for example 1 , 2 or 3 carbons, suitably 1 or 2, in particular 1) in an alkyl chain is/are replaced by a heteroatom selected from O, N, S(0) _p wherein p is 0, 1 or 2, (for example a CH ₂ group is replaced with O, or with NH, or with S, or with SO ₂ or a -CH ₃ group at the terminus of the chain or on a branch is replaced with OH or with NH ₂ ).

Suitably, R is selected from the group consisting of Ci-i ₂ alkyl, OCi-i ₂ alkyl, Ci-i ₂ haloalkyl, OC ₁ - i ₂ haloalkyl, (CH ₂ ) _O -I phenyl, (CH ₂ ) _O -I naphthyl, (CH ₂ ) _O -I heteroaryl, (CH ₂ )o-iC _3-6 cycloalkyl and (CH ₂ )o- iC _3-6 heterocycloalkyl, such as Ci-i ₂ alkyl, (CH ₂ ) _O -I phenyl, (CH ₂ ) _O -I naphthyl, (CH ₂ ) _O -I heteroaryl and (CH ₂ )o-iC _3-6 cycloalkyl; wherein the phenyl, heteroaryl, cycloalkyl or heterocycloalkyl group is optionally substituted by at least one (such as one) of the group consisting of Ci- ₆ alkyl, OCi- ₆ alkyl, Ci- ₆ haloalkyl, halo, nitro, phenyl, Ophenyl, wherein the phenyl ring is optionally substituted by Ci- ₄ alkyl, OCi- ₄ alkyl, Ci- ₄ haloalkyl, OCi- ₄ haloalkyl, halo, CN or nitro, such as the phenyl ring is not substituted.

Suitably, A comprises the following structure:

wherein:

R is selected from the group consisting of Ci-salkyl, (CFhjo-i phenyl, (CFhjo-i naphthyl, (CFhjo- ₁ heteroaryl and C _3-6 cycloalkyl; and wherein the phenyl, heteroaryl or naphthyl group (e.g. the phenyl or heteroaryl group) is optionally substituted by Ci- ₄ alkyl, OCi- ₄ alkyl, Ci- ₂ haloalkyl, halo, nitro, phenyl or Ophenyl.

Suitably, R is Ci-salkyl such as methyl, n-propyl or n-pentyl, e.g. methyl.

Alternatively, R is (CFhjo-i phenyl and the phenyl group is unsubstituted.

Suitably, R is (CFhjo-i phenyl, wherein the phenyl group is substituted by Ci- ₄ alkyl, OCi- ₄ alkyl, Ci- ₂ haloalkyl, halo, nitro, phenyl or Ophenyl.

Suitably, the phenyl group is substituted in the para position relative to the amide to which the phenyl group is joined.

Suitably, the phenyl group is substituted in the meta position relative to the amide to which the phenyl group is joined.

Suitably, the phenyl group is substituted by Ci- ₄ alkyl such as methyl. Suitably, the phenyl group is substituted by OCi- ₄ alkyl such as OMe. Suitably, the phenyl group is substituted by Ci- ₂ haloalkyl such as CF ₃ . Suitably, the phenyl group is substituted by halo such as F, Cl or Br, e.g. Cl or Br e.g. Br. Suitably, the phenyl group is substituted by nitro. Suitably, the phenyl group is substituted by phenyl. Suitably, the phenyl group is substituted by Ophenyl.

Suitably, R is phenyl. Alternatively, R is CFhphenyl.

Suitably, R is (CFhjo-i heteroaryl wherein the heteroaryl group is unsubstituted.

Alternatively, R is (CFhjo-i heteroaryl wherein the heteroaryl group is substituted by Ci- ₄ alkyl, OC ₁ - ₄ alkyl, Ci- ₂ haloalkyl, halo, nitro, phenyl or Ophenyl.

Suitably, the heteroaryl group is substituted by Ci- ₄ alkyl such as methyl. Suitably, the heteroaryl group is substituted by OCi- ₄ alkyl such as OMe. Suitably, the heteroaryl group is substituted by Ci- ₂ haloalkyl such as CF ₃ . Suitably, the heteroaryl group is substituted by halo such as F, Cl or Br, e.g. Cl or Br e.g. Br. Suitably, the heteroaryl group is substituted by nitro. Suitably, the heteroaryl group is substituted by phenyl. Suitably, the heteroaryl group is substituted by Ophenyl.

Suitably, the heteroaryl group a 5-membered heteroaromatic ring such as furanyl or thiophenyl. More suitably, the heteroaryl group is unsubstituted furanyl. More suitably, the heteroaryl group is thiophenyl substituted by bromo.

When R is a heteroaryl group, such as a 5-membered ring system such as a furanyl or thiophenyl, R may be attached as follows: wherein X is a heteroatom such as S or O.

Suitably, R is heteroaryl. Alternatively, R is CFhheteroaryl. Suitably R is furanyl.

Suitably, R is naphthyl. Alternatively, R is CFhnaphthyl. The naphthyl group may be unsubstituted.

Alternatively, the naphthyl group may be substituted. Suitably, the naphthyl group is substituted by Ci- ₄ alkyl such as methyl. Suitably, the naphthyl group is substituted by OCi- ₄ alkyl such as OMe. Suitably, the naphthyl group is substituted by Ci- ₂ haloalkyl such as CF ₃ . Suitably, the naphthyl group is substituted by halo such as F, Cl or Br, e.g. Cl or Br e.g. Br. Suitably, the naphthyl group is substituted by nitro. Suitably, the naphthyl group is substituted by phenyl. Suitably, the naphthyl group is substituted by Ophenyl.

Suitably, R is C _3-6 cycloalkyl such as cyclohexyl. Suitably, the cycloalkyl group is unsubstituted.

Alternatively, the cycloalkyl group is substituted. Suitably, the cycloalkyl group is substituted by Ci- ₄ alkyl such as methyl. Suitably, the cycloalkyl group is substituted by OCi- ₄ alkyl such as OMe. Suitably, the cycloalkyl group is substituted by Ci- ₂ haloalkyl such as CF ₃ . Suitably, the cycloalkyl group is substituted by halo such as F, Cl or Br, e.g. Cl or Br e.g. Br. Suitably, the cycloalkyl group is substituted by nitro. Suitably, the cycloalkyl group is substituted by phenyl. Suitably, the cycloalkyl group is substituted by Ophenyl.

The term‘bactericidal fluoroquinolone motif as used herein means a moiety which comprises the following motif:

and has bactericidal activity following cleavage of A, cleavage of the cleavable linker and release of B, but has no bactericidal activity when connected to portion A. Fluoroquinolone motifs are found in the fluoroquinolone class of antibiotics and antibiotics such as ciprofloxacin, which has the following structure:

Other antibiotics which comprise a fluoroquinolone motif include oxolinic acid, rosoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, sitafloxacin, prulifloxacin, besifloxacin. Thus, suitably, B, when released is selected from ciprofloxacin, oxolinic acid, rosoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, sitafloxacin, prulifloxacin and besifloxacin, suitably ciprofloxacin.

Suitably, B comprises the following structure:

The term‘cleavable linker’ connects A and B and as used herein, means a connecting moiety in which one or more bonds are capable of being broken (i.e. cleaved) upon hydrolytic cleavage of the beta-lactam cleavable motif, as shown above. The bond that is cleaved may be connected directly to the antibiotic (for example see formula (lc)) or it the bond may be connected to another group (such as a -C(=0)0- group) which is present on the antibiotic being released (see formulae (la) or (lb)).

In an embodiment, the cleavable linker comprises a methylene (CH2) group. Suitably, the linker is CH ₂ , CH ₂ 0C(=0), CH ₂ NHC(=0), such as CH ₂ 0C(=0).

Suitably, the compound of formula (I) is a compound of formula (la):

wherein R is as defined herein; or a pharmaceutically acceptable salt thereof.

Suitably, R is selected from the group consisting of methyl, furanyl, phenyl and Chhphenyl wherein the phenyl group of Chhphenyl is substituted in the para position by fluoro, or is substituted in the meta position by a group selected from methyl, halo, OCi- ₄ alkyl and nitro.

In one embodiment, R is methyl. In a second embodiment, R is furanyl. In a third embodiment R is phenyl. In a fourth embodiment R is Chhphenyl wherein the phenyl group of Chhphenyl is substituted in the para position by fluoro, or is substituted in the meta position by a group selected from methyl, halo, OCi- ₄ alkyl and nitro.

Suitably, R is Chhphenyl and the phenyl group is substituted in the para position by fluoro.

Suitably, R is Chhphenyl and the phenyl group is substituted in the meta position by methyl.

Suitably, R is Chhphenyl and the phenyl group is substituted in the meta position by OCi- ₄ alkyl such as OMe.

Suitably, R is Chhphenyl and the phenyl group is substituted in the meta position by nitro.

In one embodiment, the compound of formula (la) is:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of formula (la) is:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of formula (la) is:

or a pharmaceutically acceptable salt thereof.

Reference to the phenyl group being substituted in the para position is taken to mean:

wherein X is the defined substituent.

Reference to the phenyl group being substituted in the meta position is taken to mean:

wherein X is the defined substituent.

Alternatively, the compound of formula (I) is a compound of formula (lb):

wherein R is as defined herein;

or a pharmaceutically acceptable salt thereof.

Alternatively, the compound of formula (I) is a compound of formula (lc):

wherein R is as defined herein; or a pharmaceutically acceptable salt thereof.

More suitably, the compound of formula (I) is selected from the group consisting of the following compounds:

or a pharmaceutically acceptable salt thereof. Suitably, the compound is not Ceph-C:

or a pharmaceutically acceptable salt thereof. Suitably, the compound is not:

or a pharmaceutically acceptable salt thereof. Suitably, the compound is not:

or a pharmaceutically acceptable salt thereof.

It will be appreciated that for use in medicine the salts of the compounds of formula (I) should be pharmaceutically acceptable. Non-pharmaceutically acceptable salts of the compounds of formula (I) may be of use in other contexts. Suitable pharmaceutically acceptable salts will be apparent to those skilled in the art. Pharmaceutically acceptable salts include those described by Berge et al. (1977). Such pharmaceutically acceptable salts include acid and base addition salts. Pharmaceutically acceptable acid additional salts may be formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid. Other salts e.g. oxalates or formates, may be used, for example in the isolation of compounds of formula (I) and are included within the scope of this invention.

Certain compounds of the compounds of formula (I) may form acid or base addition salts with one or more equivalents of the acid or base. The present invention includes within its scope all possible stoichiometric and non-stoichiometric forms.

The compounds of formula (I) may be prepared in crystalline or non-crystalline form and, if crystalline, may optionally be solvated, e.g. as the hydrate. This invention includes within its scope stoichiometric solvates (e.g. hydrates) as well as compounds containing variable amounts of solvent (e.g. water).

It is to be understood that the present invention encompasses all isomers of formula (I) and their pharmaceutically acceptable derivatives, including all geometric, tautomeric and optical forms, and mixtures thereof (e.g. racemic mixtures). Where additional chiral centres are present in compounds of formula (I), the present invention includes within its scope all possible diastereoisomers, including mixtures thereof. The different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric syntheses.

The present disclosure includes all isotopic forms of the compounds of the invention provided herein, whether in a form (i) wherein all atoms of a given atomic number have a mass number (or mixture of mass numbers) which predominates in nature (referred to herein as the“natural isotopic form”) or (ii) wherein one or more atoms are replaced by atoms having the same atomic number, but a mass number different from the mass number of atoms which predominates in nature (referred to herein as an“unnatural variant isotopic form”). It is understood that an atom may naturally exist as a mixture of mass numbers. The term“unnatural variant isotopic form” also includes embodiments in which the proportion of an atom of given atomic number having a mass number found less commonly in nature (referred to herein as an“uncommon isotope”) has been increased relative to that which is naturally occurring e.g. to the level of >20%, >50%, >75%, >90%, >95% or >99% by number of the atoms of that atomic number (the latter embodiment referred to as an "isotopically enriched variant form"). The term“unnatural variant isotopic form” also includes embodiments in which the proportion of an uncommon isotope has been reduced relative to that which is naturally occurring. Isotopic forms may include radioactive forms (i.e. they incorporate radioisotopes) and non-radioactive forms. Radioactive forms will typically be isotopically enriched variant forms.

An unnatural variant isotopic form of a compound may thus contain one or more artificial or uncommon isotopes such as deuterium ( ² H or D), carbon-11 ( ¹¹ C), carbon-13 ( ¹³ C), carbon-14 ( ¹⁴ C), nitrogen-13 ( ¹³ N), nitrogen-15 ( ¹⁵ N), oxygen-15 ( ¹⁵ 0), oxygen-17 ( ¹⁷ 0), oxygen-18 ( ¹⁸ 0), phosphorus- 32 ( ³² P), sulphur-35 ( ³⁵ S), chlorine-36 ( ³⁶ CI), chlorine-37 ( ³⁷ CI), fluorine-18 ( ¹⁸ F) iodine-123 ( ¹²³ l), iodine-125 ( ¹²⁵ l) in one or more atoms or may contain an increased proportion of said isotopes as compared with the proportion that predominates in nature in one or more atoms.

Unnatural variant isotopic forms comprising radioisotopes may, for example, be used for drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Unnatural variant isotopic forms which incorporate deuterium i.e ² H or D may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half- life or reduced dosage requirements, and hence may be preferred in some circumstances. Further, unnatural variant isotopic forms may be prepared which incorporate positron emitting isotopes, such as ¹¹ C, ¹⁸ F, ¹⁵ 0 and ¹³ N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

In one embodiment, the compounds of the invention are provided in a natural isotopic form.

In one embodiment, the compounds of the invention are provided in an unnatural variant isotopic form. In a specific embodiment, the unnatural variant isotopic form is a form in which deuterium (i.e. ² H or D) is incorporated where hydrogen is specified in the chemical structure in one or more atoms of a compound of the invention. In one embodiment, the atoms of the compounds of the invention are in an isotopic form which is not radioactive. In one embodiment, one or more atoms of the compounds of the invention are in an isotopic form which is radioactive. Suitably radioactive isotopes are stable isotopes. Suitably the unnatural variant isotopic form is a pharmaceutically acceptable form. In one embodiment, a compound of the invention is provided whereby a single atom of the compound exists in an unnatural variant isotopic form. In another embodiment, a compound of the invention is provided whereby two or more atoms exist in an unnatural variant isotopic form.

Unnatural isotopic variant forms can generally be prepared by conventional techniques known to those skilled in the art or by processes described herein e.g. processes analogous to those described in the accompanying Examples for preparing natural isotopic forms. Thus, unnatural isotopic variant forms could be prepared by using appropriate isotopically variant (or labelled) reagents in place of the normal reagents employed in the Examples. Since the compounds of formula (I) are intended for use in pharmaceutical compositions it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions.

General Synthetic Routes

The compounds of the invention may be prepared using the following general methods.

Scheme 1: Synthesis of compounds of formula (!)

A— LG + XO— B - - A— B

(VI) (III) (I)

Compounds of formula (I) may be accessed by a final stage coupling step between A and B as shown in Scheme 1 wherein A and B comprise groups which can react with each other under particular conditions which are known to the skilled person. For example, A may comprise a leaving group (LG), wherein LG is defined elsewhere herein, and B may comprise an activated acid moiety (such as when X is a metal ion such as Li ⁺ , Na ⁺ or K ⁺ ).

Scheme 2: Synthesis of compounds of formula (la)

Compounds of formula (la) may be prepared according to Scheme 2. Compounds of formula (VI) (wherein LG is a leaving group such as OAc, halo such as iodo, and other leaving groups defined herein) can be prepared in three steps from commercially available compounds of formula (VIII) such as 7-ACA. Compounds of formula (VIII) undergo coupling with an ester or activated acid to give compounds of formula (VII). Protection of the carboxylic acid for example as the tert- butyl ester may be performed using non-basic carboxylic acid protection conditions such as using tert- butyl 2,2,2- trichloroacetimidate (TBTA) to give compounds of formula (VI). The leaving group in compounds of formula (VI) may be exchanged for another leaving group, depending on reactivity and coupling conditions. For example, an OAc leaving group in compounds of formulae (VIII) and (VII) may be exchanged for another leaving group such as halo (e.g. iodo) by iodination at the 3’-position with TMSI to give further compounds of formula (VI).

Ciprofloxacin (V) may be protected using standard nitrogen protecting groups known to the person skilled in the art such as Boc to give compounds of formula (IV) (wherein P is the protecting group), which in turn may be converted to the sodium salt under basic conditions, such as 0.1 M NaOH in methanol. Coupling of compounds of formula (VI) and formula (III) may be performed via a substitution reaction using any standard conditions known to the person skilled in the art such as stirring in a 3: 1 mixture of 1 ,4-dioxane:DMF at room temperature to give compounds of formula (la- P). Any residual protecting groups may be removed under standard deprotection conditions known to the person skilled in the art to give compounds of formula (la). For example, a Boc group may be removed under acidic conditions, such as a 1 : 1 mixture of TFA in DCM.

Scheme 3: Synthesis of compounds of formula (lb)

Compounds of formula (lb) may be prepared according to Scheme 3, and according to methods known in the art (Zhao et al., 2006). P is a suitable carboxylic acid protecting group. Hydrolysis of the acetyl group in compounds of formula (VIII) (7-ACA) under basic conditions such as 10 M NaOH in methanol at -20 °C liberates the OH group, the amino group may undergo an amide coupling reaction with an activated acid (such as an acid chloride, anhydride or activated ester), and protection of the carboxylic acid moiety can be performed using conditions known to the person skilled in the art, for example using diphenyldiazomethane, to provide compounds of formula (VII) from compounds of formula (VIII). The OH group in compounds of formula (VII) is primed for reaction with compound (V) using a reagent such as CIC(=0)OCHCICCl ₃ to provide compounds of formula (IX). Coupling of compounds of formula (IX) with compound (V) for example in the presence of N- methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) gives compounds of formula (X), and deprotection of the carboxylic acid group for example using TFA in anisole provides compounds of (lb). Scheme 4: Synthesis of compounds of formula (lc)

Compounds of formula (lc) can be prepared according to Scheme 4 and according to methods known in the art (Zhao et al. , 2006). P is a suitable carboxylic acid protecting group. Coupling of compounds of formula (VII) with (V) for example, in the presence of N-methyl-N-trimethylsilyl- trifluoroacetamide (MSTFA) in a 1 :1 mixture of DMF/1 ,4-dioxane, followed by removal of protecting group P gives the compounds of formula (lc).

Thus, in an embodiment of the invention, there is provided a process for the preparation of a compound of formulae (la), (lb) or (lc) or a pharmaceutically acceptable salt thereof which comprises reacting a compound of formula (II):

wherein LG is as defined herein; or a salt thereof; with ciprofloxacin or a protected derivative thereof, or a salt thereof.

Suitably, there is provided a process for the preparation of the compound of formula (la) or a salt, such as a pharmaceutically acceptable salt thereof, which comprises reacting a compound of formula (II):

wherein LG and R are as defined herein; or a salt thereof; with ciprofloxacin or a protected derivative thereof, or a salt thereof. In particular, ciprofloxacin is protected on the terminal nitrogen atom of the piperazine ring, e.g. with a Boc, acetyl, benzyl, tosyl, carbamate or para-methoxybenzyl group, such as with a Boc group.

Suitably, R is as defined above for formula (la).

Intermediates of the invention

The present invention also relates to novel intermediates in the synthesis of compounds of formula (la) such as compounds of formula (II) to (VIII). Particular intermediates of interest are those of the following general formulae, wherein the variable groups and associated preferences are as defined previously for compounds of formula (I):

wherein R is as defined elsewhere herein and LG is a leaving group such as OCi-4alkyl, 0C(=0)Ci- ₄ alkyl, 0C(=0)NH ₂ , halo,

e.g. 0C(=0)CH ₃ ;

or a salt thereof.

The present invention provides compounds of formula (II) as described in the Table below: Table 1. Compounds of formula (II)

Particular intermediates of the invention are as follows:

or a salt thereof. Suitably, compounds of formula (II) are:

or a salt thereof.

Therapeutic methods

Compounds of formula (I) (including compounds of formulae (la), (lb) and (lc)) have utility as antibiotics for use in the treatment of bacterial infections.

Thus there in one embodiment, there is provided a compound of the formula (I), or a pharmaceutically acceptable salt thereof, for use as a medicament.

The invention also provides the compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of bacterial infection.

The invention also provides a method of treating or preventing bacterial infection by administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Additionally provided is the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of bacterial infection.

In an embodiment, the bacterial infection is caused by bacteria that express beta-lactamase.

Suitably, bacteria is selected from the group consisting of Escherichia coli, Klebsiella species such as Klebsiella pneumoniae, Pseudomonas species such as Pseudomonas aeruginosa, Acinetobacter species such as Acinetobacter baumanii and Mycobacterium tuberculosis.

Suitably, the bacterial infection is selected from urinary-tract infections, lung infections in patients with cystic fibrosis, lung infections in patients who are ventilated, and bloodstream infections.

In another embodiment, the bacterial infection is caused by de-colonisation of an individual who is colonised by a pathogen that expresses the beta-lactamase.

Suitably, the pathogen is selected from Escherichia coli, Klebsiella species such as Klebsiella pneumoniae, Pseudomonas species such as Pseudomonas aeruginosa and Acinetobacter species such as Acinetobacter baumanii and Mycobacterium tuberculosis.

Suitably, the individual is de-colonised of multi-drug resistant E. coli from the intestinal tract of individuals with recurrent urinary-tract infection, or beta-lactamase expressing Pseudomonas aeruginosa from the lungs of individuals with cystic fibrosis. The term ‘treatment’ or‘treating’ as used herein includes the control, mitigation, reduction, or modulation of the disease state or its symptoms.

The term‘prophylaxis’ or‘preventing’ is used herein to mean preventing symptoms of a disease or disorder in a subject or preventing recurrence of symptoms of a disease or disorder in an afflicted subject and is not limited to complete prevention of an affliction.

Suitably the subject is a mammal, in particular the subject is a human.

Pharmaceutical Compositions

For use in therapy the compounds of the invention are usually administered as a pharmaceutical composition. The invention also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt optionally in combination with one or more pharmaceutically acceptable diluents or carriers.

In one embodiment, there is provided a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of a disease or disorder as described herein.

In a further embodiment, there is provided a method for the prophylaxis or treatment of a disease or disorder as described herein, which comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

The invention also provides the use of a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of a disease or disorder as described herein.

The compounds of formula (I) or their pharmaceutically acceptable salts thereof may be administered by any convenient method, e.g. by oral, parenteral, buccal, sublingual, nasal, rectal or transdermal administration, and the pharmaceutical compositions adapted accordingly.

The compounds of formula (I) or their pharmaceutically acceptable salts thereof which are active when given orally can be formulated as liquids or solids, e.g. as syrups, suspensions, emulsions, tablets, capsules or lozenges.

A liquid formulation will generally consist of a suspension or solution of the active ingredient (such as a compound of formula (I) or a pharmaceutically acceptable salt thereof) in a suitable liquid carrier(s) e.g. an aqueous solvent such as water, ethanol or glycerine, or a non-aqueous solvent, such as polyethylene glycol or an oil. The formulation may also contain a suspending agent, preservative, flavouring and/or colouring agent. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations, such as magnesium stearate, starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures, e.g. pellets containing the active ingredient (such as a compound of formula (I) or a pharmaceutically acceptable salt thereof) can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), e.g. aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.

Typical parenteral compositions consist of a solution or suspension of the active ingredient (such as a compound of formula (I) or a pharmaceutically acceptable salt derivative thereof) in a sterile aqueous carrier or parenterally acceptable oil, e.g. polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.

Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active ingredient in a pharmaceutically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a disposable dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas e.g. air, or an organic propellant such as a fluoro-chloro- hydrocarbon or hydrofluorocarbon. Aerosol dosage forms can also take the form of pump-atomisers.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles where the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

Compositions suitable for transdermal administration include ointments, gels and patches.

Suitably, the composition is in unit dose form such as a tablet, capsule or ampoule.

The composition may for example contain from 0.1 % to 100% by weight, for example from 10 to 60% by weight, of the active material, depending on the method of administration. The composition may contain from 0% to 99% by weight, for example 40% to 90% by weight, of the carrier, depending on the method of administration. The composition may contain from 0.05 mg to 2000 mg, for example from 1.0 g to 500 mg, of the active material, depending on the method of administration. The composition may contain from 50 mg to 1000 mg, for example from 100 mg to 400 mg of the carrier, depending on the method of administration. The dose of the compound used in the treatment or prophylaxis of the aforementioned disorders will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses may be 0.05 mg to 1000 mg, more suitably 1.0 mg to 500 mg, and such unit doses may be administered more than once a day, for example two or three a day. Such therapy may extend for a number of weeks or months.

The invention provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with a second or further pharmaceutically acceptable active ingredient or ingredients.

The invention provides a compound of formula (I), for use in combination with a second or further pharmaceutically acceptable active ingredient or ingredients.

When the compounds are used in combination with other therapeutic agents, the compounds may be administered separately, sequentially or simultaneously by any convenient route.

Suitably, the second or further pharmaceutically acceptable active ingredient is an antibiotic, such as aminoglycosides (such as gentamicin, tobramycin or amikacin), beta-lactams (such as penicillin, amoxicillin or imipenem) or metronidazole.

Some of the combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. The individual components of combinations may also be administered separately, through the same or different routes.

When a compound of formula (I) or a pharmaceutically acceptable salt thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

The compounds of the invention may be expected to:

• not be toxic to broad spectrum bacteria at < 100 uM;

• be efficiently hydrolysed by beta-lactamase; and

• be able to cross the bacterial outer membrane. The invention may be defined by the following clauses:

1. A compound of formula (I):

^{A— B} (I)

wherein A comprises a beta-lactam cleavable motif and B comprises a bactericidal fluoroquinolone motif, wherein A and B are joined by a cleavable linker such that cleaving of A leads to cleavage of the cleavable linker and release of B; or a pharmaceutically acceptable salt thereof.

2. The compound according to clause 1 wherein A comprises the following structure:

3. The compound according to clause 2 wherein A comprises the following structure:

wherein:

R is selected from the group consisting of Ci-i ₂ alkyl, OCi-i ₂ alkyl, C _M2 haloalkyl, OC _M2 haloalkyl, NR ₂ R ₃ , (CH ₂ )o-iphenyl, (CH ₂ ) _O -I naphthyl, (CH ₂ ) _O -I heteroaryl, (CH ₂ )o-iC _3-6 cycloalkyl and (CH ₂ ) _O -IC ₃ - 6heterocycloalkyl;

R ₂ is selected the group consisting of H, Ci- ₃ alkyl, Ci- ₃ haloalkyl, (Chhjo-i phenyl, naphthyl and (Chhjo- 1 heteroaryl; and

R ₃ is selected from H and Ci- ₃ alkyl; or

R ₂ and R ₃ join such that, together with the nitrogen atom to which they are attached, they form a C ₃ - ₆ heterocycloalkyl; wherein any of the the phenyl, naphthyl, heteroaryl, cycloalkyl or heterocycloalkyl groups (such as phenyl or heteroaryl group) are optionally substituted by at least one (such as one) of the group consisting of Ci- ₆ alkyl, OCi- ₆ alkyl, Ci- ₆ haloalkyl, OCi- ₆ haloalkyl, Co- ₃ alkyleneC _3-6 cycloalkyl, OCo- ₃ alkyleneC _3-6 cycloalkyl, Co- ₃ alkyleneC _3-6 heterocycloalkyl, OCo- ₃ alkyleneC _3-6 heterocycloalkyl, CN, halo, NR ₂ R ₃ , nitro, phenyl, Ophenyl, heteroaryl and O-heteroaryl, such as Ci- ₄ alkyl, OCi- ₄ alkyl, Ci- 2haloalkyl, halo, nitro, phenyl or Ophenyl, wherein any of the second phenyl, heteroaryl, cycloalkyl or heterocycloalkyl substituent groups is optionally substituted by Ci-4alkyl, OCi-4alkyl, Ci-4haloalkyl, OCi-4haloalkyl, halo, CN or nitro; wherein optionally one or more carbons (for example 1 , 2 or 3 carbons, suitably 1 or 2, in particular 1) in an alkyl chain is/are replaced by a heteroatom selected from O, N, S(0) _p wherein p is 0, 1 or 2, (for example a CH ₂ group is replaced with O, or with NH, or with S, or with SO ₂ or a -CH ₃ group at the terminus of the chain or on a branch is replaced with OH or with NH ₂ ).

4. The compound according to clause 3 wherein A comprises the following structure:

wherein:

R is selected from the group consisting of Ci-salkyl, (ChhKi phenyl, (ChhKi naphthyl, (ChhK 1 heteroaryl and C3-6cycloalkyl; and wherein the phenyl, heteroaryl or naphthyl group (e.g. the phenyl or heteroaryl group) is optionally substituted by Ci-4alkyl, OCi-4alkyl, Ci-2haloalkyl, halo, nitro, phenyl or Ophenyl.

5. The compound according to clause 3 or 4 wherein R is Ci-salkyl such as methyl, n-propyl or n-pentyl, e.g. methyl.

6. The compound according to clause 3 or 4 wherein R is (ChhKi phenyl and the phenyl group is unsubstituted.

7. The compound according to clause 3 or 4 wherein R is (ChhKi phenyl, wherein the phenyl group is substituted by Ci-4alkyl, OCi-4alkyl, Ci-2haloalkyl, halo, nitro, phenyl or Ophenyl.

8. The compound according to clause 7 wherein the phenyl group is substituted in the para position relative to the amide to which the phenyl group is joined.

9. The compound according to clause 7 wherein the phenyl group is substituted in the meta position relative to the amide to which the phenyl group is joined.

10. The compound according to any one of clauses 7-9 wherein the phenyl group is substituted by Ci-4alkyl such as methyl.

11. The compound according to any one of clauses 7-9 wherein the phenyl group is substituted by OCi-4alkyl such as OMe. 12. The compound according to any one of clauses 7-9 wherein the phenyl group is substituted by Ci-2haloalkyl such as CF ₃ .

13. The compound according to any one of clauses 7-9 wherein the phenyl group is substituted by halo such as F, Cl or Br, e.g. Cl or Br e.g. Br.

14. The compound according to any one of clauses 7-9 wherein the phenyl group is substituted by nitro.

15. The compound according to any one of clauses 7-9 wherein the phenyl group is substituted by phenyl.

16. The compound according to any one of clauses 7-9 wherein the phenyl group is substituted by Ophenyl.

17. The compound according to any one of clauses 3,4 or 6-16 wherein R is phenyl.

18. The compound according to any one of clauses 3, 4 or 6-16 wherein R is CFhphenyl.

19. The compound according to clause 3 or 4 wherein R is (CFhKi heteroaryl wherein the heteroaryl group is unsubstituted.

20. The compound according to clause 3 or 4 wherein R is (CFhKi heteroaryl wherein the heteroaryl group is substituted by Ci-4alkyl, OCi-4alkyl, Ci-2haloalkyl, halo, nitro, phenyl or Ophenyl.

21. The compound according to clause 20 wherein the heteroaryl group is substituted by halo such as F, Cl or Br, e.g. Cl or Br e.g. Br.

22. The compound according to any one of clauses 3, 4 or 19-21 wherein R is heteroaryl.

23. The compound according to any one of clauses 3, 4 or 19-21 wherein R is CFhheteroaryl.

24. The compound according to clause 3 or 4 wherein R is naphthyl.

25. The compound according to clause 3 or 4 wherein R is CFhnaphthyl.

26. The compound according to clauses 24 or 25 wherein the naphthyl group is substituted by Ci-4alkyl, OCi-4alkyl, Ci-2haloalkyl, halo, nitro, phenyl or Ophenyl.

27. The compound according to clause 3 or 4 wherein R is C3-6cycloalkyl such as cyclohexyl.

28. The compound according to any one of clauses 1-27 wherein B comprises the following structure:

29. The compound according to any one of clauses 1-28 wherein the cleavable linker comprises a methylene (CH2) group.

30. The compound according to clause 29 wherein the linker is CH2, CH ₂ 0C(=0), CH ₂ NHC(=0), such as CH ₂ 0C(=0).

31. The compound according to any one of clauses 1-30 which is a compound of formula (la):

wherein R is as defined in any one of clauses 3-27; or a pharmaceutically acceptable salt thereof.

32. The compound according to any one of clauses 1-30 which is a compound of formula (lb):

wherein R is as defined in any one of clauses 3-27; or a pharmaceutically acceptable salt thereof.

33. The compound according to any one of clauses 1-30 which is a compound of formula (lc):

wherein R is as defined in any one of clauses 3-27;

or a pharmaceutically acceptable salt thereof.

34. The compound according to clause 31 which is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

35. A compound of formula (II):

wherein R is as defined in any one o

OC(=0)Ci- ₄ alkyl, 0C(=0)NH ₂ , halo,

e.g. 0C(=0)CH ₃ ;

or a salt thereof.

36. The compound according to clause 35 which is selected from the group consisting of:

or a salt thereof.

37. The compound according to any one of clauses 1-34 for use as a medicament.

38. The compound according to any one of clauses 1-34 for use in the treatment or prevention of bacterial infection.

39. The compound for use according to clause 38 wherein the bacterial infection is caused by bacteria that express beta-lactamase.

40. The compound for use according to clause 39 wherein the bacteria is selected from the group consisting of Escherichia coli, Klebsiella species such as Klebsiella pneumoniae, Pseudomonas species such as Pseudomonas aeruginosa, Acinetobacter species such as Acinetobacter baumanii and Mycobacterium tuberculosis.

41. The compound for use according to any one of clauses 38-40 wherein the bacterial infection is selected from urinary-tract infections, lung infections in patients with cystic fibrosis, lung infections in patients who are ventilated, and bloodstream infections.

42. The compound for use according to clause 38 wherein the bacterial infection is caused by de-colonisation of an individual who is colonised by a pathogen that expresses the beta-lactamase.

43. The compound for use according to clause 42 wherein the pathogen is selected from Escherichia coli, Klebsiella species such as Klebsiella pneumoniae, Pseudomonas species such as Pseudomonas aeruginosa, Acinetobacter species such as Acinetobacter baumanii and Mycobacterium tuberculosis.

44. The compound for use according to clause 42 or clause 43 wherein the individual is de- colonised of multi-drug resistant E. coli from the intestinal tract of individuals with recurrent urinary- tract infection, or beta-lactamase expressing Pseudomonas aeruginosa from the lungs of individuals with cystic fibrosis.

45. A pharmaceutical composition comprising the compound according to any one of clauses 1- 34 optionally in combination with one or more pharmaceutically acceptable diluents or carriers. 46. The pharmaceutical composition according to clause 45 which comprises a second or further pharmaceutically acceptable active ingredient.

47. The compound for use according to any one of clauses 37-44 in combination with a second or further pharmaceutically acceptable active ingredient.

48. The pharmaceutical composition according to clause 46 or the compound for use according to clause 47 wherein the second or further pharmaceutically acceptable active ingredient is an antibiotic, such as aminoglycosides (such as gentamicin, tobramycin or amikacin), beta-lactams (such as penicillin, amoxicillin or imipenem) or metronidazole.

49. A process for the preparation of a compound of formula (la), (lb) or (lc) as defined in any one of clauses 31-33 or a pharmaceutically acceptable salt thereof which comprises reacting a compound of formula (II):

wherein LG is as defined in clause 35; or a salt thereof; with ciprofloxacin or a protected derivative thereof, or a salt thereof.

The invention is further exemplified by the following non-limiting examples.

EXAMPLES

Abbreviations used herein are defined below. Any abbreviations not defined are intended to convey their generally accepted meaning.

Table 2. Abbreviations

Ac acetyl

aq aqueous

Ar aryl

Boc te/f-butyloxycarbonyl

br broad

tBu tert- butyl

C ciprofloxacin

c.f.u. colony-forming unit

cm centimetre d doublet

DCM dichloromethane

DNA Deoxyribonucleic acid

DMF N,N-dimethylformamide

DMSO (-d6) dimethyl sulfoxide (hexadeuterodimethyl sulfoxide)

D ₂ O deuterium oxide

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EST electrospray ionisation, positive mode

Et ethyl

9 grams

His histidine

HRMS high resolution mass spectrometry

h(s) hour(s)

hz hertz

IPTG isopropyl beta-D-1-thiogalactopyranoside

IR infra red

K kelvin

kHz kilohertz

(M+H) ⁺ protonated molecular ion

M molar concentration

m multiplet

MIC minimum inhibitory concentration

ml_ / ml millilitre

mM / mmol millimole

mm millilitre

Me methyl

MHz megahertz

min(s) minute(s)

mg milligram

mp melting point

ms millisecond

m/z mass-to-charge ratio

nm nanometre

nM nanomolar

NMR nuclear magnetic resonance (spectroscopy)

OD optical density

ppm parts per million q quartet

RT / rt room temperature

r.p.m. revolutions per minute

s second(s)

satd saturated

SEM standard error of the mean

t triplet

TFA trifluoroacetic acid

TMS trimethylsilyl

THF tetrahydrofuran

UV ultraviolet

v/v volume/volume

ug microgram

uM micromolar

uL microlitre

us microsecond

w/v weight/volume

WT wild type

xg times gravity

°c degrees Celsius

General Methods

General procedures for the preparation of beta-lactam analogues Method A

7-ACA (1 equiv) was dissolved in sat. NaHCC>3 (aq) and acetone added, followed by acid chloride (1.2 or 2 equiv). The reaction was stirred at room temperature for 30 min then washed with EtOAc. The aqueous layer was acidified to pH 2 with 1 M HCI and extracted with DCM (x3). The organic extracts were combined, dried over Na2SC>4, evaporated and the resulting solid triturated with ice- cold Et2<D (unless otherwise stated) to afford the product.

Method B

7-ACA (1 equiv) and acid chloride (2 equiv) were dissolved in EtOAc and heated to reflux for 30 min. After cooling to room temperature, aniline (1.3 - 3 equiv) was added and stirred for 1 h before the reaction mixture was diluted with 3% NaHCOs (aq). The aqueous layer was separated and the organic layer washed with 3% NaHCOs (aq) (x2). The aqueous layers were combined, washed with EtOAc and acidified to pH 2 with 1 M HCI. The desired product was isolated as described. Method C

Carboxylic acid (0.735 mmol, 1.0 eq.) was added to a flame dried microwave vial under Argon. DCM (3 ml_) was added, followed by oxalyl chloride (0.075ml_, 0.885 mmol, 1.2 eq) and DMF (1 drop). The reaction mixture was stirred at room temperature under Argon for 18 hours. The solvent was removed under reduced pressure to afford the crude product. The resulting crude material was used directly for the next step without further purification.

Method D

Acid chloride (0.735 mmol, 2.0 eq.) was dissolved in EtOAc (3 ml_) and 7-aminocephalosporanic acid (100 mg, 0.367 mmol, 1.0 eq) was added, and the reaction mixture was then heated at reflux for 30 minutes. After cooling to room temperature, aniline (0.10 ml_, 1.10 mmol, 3.0 eq.) was added and the reaction mixture was stirred for 1 hour. The reaction mixture was washed with 3% wt/v aqueous NaHCC>3 solution (3 x 15 ml_). The aqueous extracts were combined and acidified to pH 2 with 1 M HCI. If a precipitate resulted, the solid was collected by vacuum filtration and dried under vacuum to give the crude product. If no precipitate resulted or the precipitate is too fine to be collected by filtration, the resulting mixture was extracted with DCM, dried over NaaSCU and concentrated to dryness under reduced pressure to afford the crude product.

Recombinant beta-lactamase protein

Amp-C

Recombinant Amp-C protein was purchased from Abeam (ab104926) and used without further purification.

CTX-M-15

Recombinant CTX-M-15 was expressed with an /V-terminal His6-tag in SoluBL21 (DE3) cells and purified as described previously (Cahill, S. T. et ai 2017). Briefly, bacterial cells were grown to an OD _6OO of 0.6-0.8 in 2x YT media at 37 °C with shaking. CTX-M-15 expression was induced with 0.5 mM IPTG, after which cells were grown at 18 °C for a further 16 h. Cells were harvested by centrifugation at 6,500 xg for 10 mins at 4 °C before the pellet was resuspended in 50 mM Hepes (pH 7.5) with 400mM NaCI and complete EDTA-free protease inhibitor (Roche). Cells were homogenised and lysed via two passages through a cell disruptor (constant systems) at 30 kpsi. The resulting cell lysate was centrifuged at 100,000 xg for 1 hour at 4°C before incubation with 4 ml of Ni-NTA bead slurry (Qiagen) for 1.5 hours with 10 mM imidazole to prevent non-specific binding. Ni- NTA resin bound to protein was washed with 50 mM Hepes pH 7.5, 400 mM NaCI, 10 mM imidazole followed by 50 mM Hepes pH 7.5, 300 mM NaCI, 20 mM imidazole and eluted after 5 minute incubation with 50 mM Hepes pH 7.5, 200 mM NaCI, 400 mM imidazole. The HiS ₆ tag was cleaved and removed as described previously using 3C protease. ⁸ Bacterial Strains

Table 3. Bacterial strains and characteristics relevant to this study

Transformation of E. coli with plasmids

Plasmids were introduced via heat shock into chemically competent DH5-alpha and via electroporation into E. coli CFT073 following published protocols (Sambrook et al. , 2001).

Bacterial cultures

The bacterial strains and plasmids used in this study are detailed in Table 3. E. coli was grown in Mueller-Hinton Broth (MHB) or Lennox Broth (LB) at 37 °C, with shaking (180 r.p.m.) overnight. Where required, culture medium was supplemented with Chloramphenicol (25ug ml ^-1 ).

Determination of MIC

MIC assays were performed in 96-well thermo scientific Nuclon Delta Surface plates and MICs were determined in accordance with the broth microdilution protocol (Wiegand et al., 2008). Strains grown to stationary-phase in MHB or LB were adjusted to Oϋboo nm of 0.05 in MHB and supplemented with a range of concentrations (two-fold dilution series) of ciprofloxacin or test compounds. After static, aerobic incubation at 37 °C for 18 h, the MIC was determined as the lowest concentration of antibiotic that inhibited visible bacterial growth. OD595 measurements were recorded on a BioRad 1 Mark Microplate Reader. Measurements were background corrected against the no-inocula control and normalized to the no-drug control.

Determination of bactericidal activity

Stationary-phase bacteria were inoculated into 3 ml MHB containing no antibiotic, ciprofloxacin (78 nM) or prodrug (78 nM), to give an Oϋboo nm of 0.1. Cultures were subsequently incubated at 37 °C with shaking (180 r.p.m.) and bacterial viability determined by c.f.u. counts. All compound concentrations were at 2.5 ^c MIC of ciprofloxacin, which has been shown previously to be bactericidal.

Recombinant beta-lactamase assay

Each compound was incubated for 1 hour at room temperature in 100 mM NaPCU (pH 7) ± 0.5 M NaOH. The wavelength at which the difference between un-hydrolysed and hydrolysed compound was the greatest was selected (typically 263 nm) and a compound concentration vs absorbance standard curve was generated. Compounds, typically 250 mM in assay buffer (100 mM NaPCU (pH 7)), were dispensed into a Corning 96-well UV transparent flat bottom plate. Recombinant Amp-C in assay buffer was added immediately before incubation in the microplate reader at 37 °C. UV absorbance was measured every 20 s for 1 h on a BMG LABTECH SPECTROstarNano. Kinetic parameters were determined by half-life analysis.

NMR beta-lactamase hydrolysis assay

Overnight bacterial cultures (5 - 25 ml) were pelleted by centrifugation at 3,200 x g for 20 min at 4 °C. Pellets were washed twice with 100 mM NaP0 ₄ (pH 7.0), 10 mM MgCh and cultures were corrected to an Oϋboo nm of 2.5 in 100 mM NaP0 ₄ (pH 7.0), 10 mM MgCh, 10% (v/v) deuterated water. Compounds were prepared in deuterated DMSO and added to 700ul of culture to give a final compound concentration of 100uM and incubated at room temperature for 1 h.

¹ H NMR spectrum were collected at 298 K on a Bruker 500MHz AVANCE III HD spectrometer running TopSpin3.2 and equipped with a z-gradient bbfo/5mm tuneable SmartProbe and a GRASP II gradient spectroscopy accessory providing a maximum gradient output (100%) of 53.5G/cm (5.35G/cmA). ¹ H water suppression spectra (Hwang et al. 1995) were collected using the Bruker pulse program zgesgp at a frequency of 500.13MHz with a spectral width of 10 kHz (centred on 4.705 ppm) and 65536 data points. A relaxation delay of 1s was employed along with square shaped 180o selective pulses of 2 ms (Squa100.1000 from Bruker library) and gradients pulses of 1 ms. The strength of the first pair of gradient pulses was 31 % and the second pair 1 1 %. All gradient pulses were smoothed-square shaped (SMSQ10.100 from Bruker library) and after each application a recovery delay of 200us used. 64 transients were collected after 4 dummy scans. The data was processed using 65536 data points applying an exponential function with a line broadening of 0.3 Hz. Integration of peaks corresponding to the intact and hydrolysed products was performed using MestReNova 8.0 and used to determine the percentage hydrolysis.

Recombinant DNA gyrase assay

E. coli gyrase supercoiling assay (Inspiralis) was performed according to the manufacturer’s instructions with the addition of 6 nM recombinant CTX-M-15 where required. Samples were loaded on a 1 % (w/v) agarose gel prepared with T ris/Borate/EDTA (TBE) or T ris/Acetate/EDTA (TAE) buffer. Electrophorese was carried out at 100 V for 90 minutes. The gel was then stained with SYBR Safe DNA gel stain (Invitrogen) (1 : 1000 dilution in TBE) for 30 min. Visualization was performed using a Syngene Gel Doc system. Quantification of gel bands corresponding to supercoiled DNA was performed using ImageJ 1.52a. Values were background corrected against the no-gyrase control and normalized to gyrase only activity. Gyrase activity values reflects the mean of three or four independent replicates ± SEM.

Synthesis of Compounds

(6/?,7/?)-3-(Acetoxymethyl)-7-(2-(4-bromothiophen-2-yl)ac etamido)-8-oxo-5-thia-1

azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (8). 2-(4-Bromothiophen-2-yl)acetic acid (203 mg, 0.92 mmol), EDC.HCI (193 mg, 1.01 mmol) and 7-ACA (250 mg, 0.92 mmol) were suspended in DMF (8 ml) and stirred at room temperature for 48 h (Quotadamo et al. 2016). The resulting mixture was filtered and the filtrate diluted with H2O and extracted with EtOAc (x3). The organic extracts were combined, washed with 1 M LiCI (aq) and brine, and dried over Na ₂ S0 ₄ . Solvent was removed under reduced pressure and the resulting oil triturated with Et ₂ 0. The precipitate was collected by vacuum filtration and washed with DCM to afford the product as beige amorphous solid (36 mg, 8%). IR (solid): u _max 3273, 3101 , 2837, 1774, 1748, 1707, 1662, 1539, 1223 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 9.15 (d, J = 8.1 Hz, 1 H), 7.51 (d, = 1.2 Hz, 1 H), 6.93 (s, 1 H), 5.68 - 5.59 (m, 1 H), 5.06 (d, J = 4.8 Hz, 1 H), 5.00 (d, J = 12.6 Hz, 1 H), 4.70 (d, J = 12.6 Hz, 1 H), 3.78 (d, J = 2.6 Hz, 2H), 3.58 (d, J = 18.0 Hz, 1 H), 3.42 (d, J = 18.7 Hz, 1 H), 2.02 (s, 3H). ¹³ C NMR (101 MHz, DMSO) d 170.3, 169.4, 162.8, 139.1 , 128.5, 122.8, 107.7, 59.0, 57.2, 35.6, 25.4, 20.6. HRMS (ESI ⁺ ): calcd for C ₁₆ H ₁₇ BrN ₂ 0 ₆ S ₂ (M + H) ⁺ 496.9453, found 496.9479.

(6/?,7/?)-3-(Acetoxymethyl)-8-oxo-7-(2-(thiophen-3-yl)ace tamido)-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (9). 3-Thiopheneacetic acid (104 mg, 0.74 mmol), oxalyl chloride (76 uL, 0.88 mmol) and DMF (1 drop) were reacted in DCM (3 ml_) according to method C. The resulting acid chloride and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (100 uL) according to method B. The aqueous layer was extracted with DCM (x3) and the organic layers were combined, dried over Na ₂ S0 ₄ and evaporated. The resulting solid was triturated with ice-cold DCM to afford the product as an off-white amorphous solid (48 mg, 33%). IR (solid): u _max 3284, 1751 , 1730, 1651 , 1621 , 1536, 1241 cm- ¹ . ¹ H NMR (400 MHz, DMSO- cfe) d 8.95 (d, J = 8.3 Hz, 1 H), 7.46 (dd, J = 4.9, 3.0 Hz, 1 H), 7.26 (dd, J = 2.9, 1.0 Hz, 1 H), 7.03 (dd, J = 4.9, 1.2 Hz, 1 H), 5.46 (dd, J = 8.3, 4.8 Hz, 1 H), 4.99 (d, J = 1 1.9 Hz, 1 H), 4.93 (d, J = 4.8 Hz, 1 H), 4.73 (d, J = 1 1.9 Hz, 1 H), 3.61 - 3.49 (m, 2H), 3.45 (d, J = 17.2 Hz, 1 H), 3.19 (d, J = 17.3 Hz, 1 H), 2.00 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.6, 170.5, 163.3, 162.8, 135.8, 135.6, 128.6, 125.7, 122.3, 64.7, 58.5, 57.2, 36.4, 25.1 , 20.8. HRMS (ESF): calcd for C^H^NaOeSa (M + H) ⁺ 397.0528, found 397.0540. (6/?,7/?)-3-(Acetoxymethyl)-8-oxo-7-(2-phenylacetamido)-5-th ia-1 -azabicyclo[4.2.0]oct-2-ene- 2-carboxylic acid (11 ). 7-ACA (100 mg, 0.35 mmol) and phenylacetyl chloride (97 uL, 0.73 mmol) were reacted in satd NaHCOs (aq) (10 ml_) and acetone (5 ml_) according to method A to afford the product as a white amorphous solid (45 mg, 31 %). IR (solid): i _½ax 3254, 3034, 1778, 1737, 1707, 1654, 1536, 1223 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.71 (br s, 1 H), 9.10 (d, J = 8.3 Hz, 1 H), 7.35 - 7.18 (m, 5H), 5.68 (dd, J = 8.3, 4.8 Hz, 1 H), 5.08 (d, J = 4.8 Hz, 1 H), 5.00 (d, J = 12.8 Hz, 1 H), 4.68 (d, J = 12.8 Hz, 1 H), 3.66 - 3.47 (m, 4H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO) d 170.9, 170.2, 164.7, 162.8, 135.8, 129.0, 128.2, 126.54, 126.48, 123.1 , 62.7, 59.1 , 57.4, 41.6, 25.5, 20.6. HRMS (ESI ⁺ ): calcd for C ₁₈ H ₁₉ N ₂ 0 ₆ S (M + H) ⁺ 391.0964, found 391.0972.

(6/?,7/?)-3-(Acetoxymethyl)-8-oxo-7-(2-(p-tolyl)acetamido )-5-thia-1 -azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (12). 4-Methylphenylacetic acid (11 1 mg, 0.74 mmol), oxalyl chloride (76 uL, 0.88 mmol) and DMF (1 drop) were reacted in DCM (3 ml_) according to method C. The resulting acid chloride and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (100 uL) according to method B. The resulting precipitate was collected by vacuum filtration and washed with DCM to afford the product as a white amorphous solid (63 mg, 42%). IR (solid): U _max 3261 , 3045, 1778, 1752, 1707, 1655, 1536, 1223 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.66 (br s, 1 H), 9.04 (d, J = 8.3 Hz, 1 H), 7.15 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 5.67 (dd, J = 8.3, 4.8 Hz, 1 H), 5.08 (d, J = 4.8 Hz, 1 H), 5.00 (d, J = 12.8 Hz, 1 H), 4.69 (d, J = 12.8 Hz, 1 H), 3.62 (d, J = 18.1 Hz, 1 H), 3.54 - 3.41 (m, 3H), 2.26 (s, 3H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO) d 171.1 , 170.2, 164.8, 162.8, 135.5, 132.7, 128.9, 128.8, 62.7, 59.1 , 57.4, 41.2, 25.5, 20.63, 20.57. HRMS (ESI ⁺ ): calcd for C ₁₉ H ₂ I N ₂ 0 ₆ S (M + H) ⁺ 405.1120, found 405.11 19.

(6/?,7/?)-3-(Acetoxymethyl)-7-(2-(4-fluorophenyl)acetamid o)-8-oxo-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (13). 4-Fluorophenylacetyl chloride (98 uL, 0.74 mmol) and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (100 uL) according to method B. The resulting mixture was cooled to 4 °C and the precipitate collected by vacuum filtration and washed with ice-cold DCM to afford the product as a white amorphous solid (61 mg, 41 %). IR (solid): u _max 3273, 1763, 1736, 1659, 1532, 1215 cm ^·1 . ¹ H NMR (400 MHz, DMSO-cfe) d 13.67 (br s, 1 H), 9.10 (d, J = 8.2 Hz, 1 H), 7.30 (dd, J = 8.4, 5.6 Hz, 2H), 7.12 (app t, J = 8.8 Hz, 2H), 5.67 (dd, = 8.1 , 4.8 Hz, 1 H), 5.08 (d, J = 4.8 Hz, 1 H), 5.00 (d, J = 12.8 Hz, 1 H), 4.69 (d, J = 12.8 Hz, 1 H), 3.65 - 3.46 (m, 4H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.8, 170.1 , 164.6, 162.8, 131.9, 130.9, 130.8, 126.4, 123.3, 1 15.0, 114.8, 62.7, 59.1 , 57.4, 40.6, 25.5, 20.5. HRMS (ESF): calcd for C ₁₈ H ₁₈ FN ₂ 0 ₆ S (M + H) ⁺ 409.0870, found 409.0864.

(6/?,7/?)-3-(Acetoxymethyl)-7-(2-(4-chlorophenyl)acetamid o)-8-oxo-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (14). Hexanoyl chloride (139 mg, 0.74 mmol) and 7- ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (75 uL) according to method B. The resulting precipitate was collected by vacuum filtration and washed with ice-cold DCM to afford the product as a cream amorphous solid (104 mg, 67%). IR (solid): i _½ax 3265, 3056, 1778, 1748, 1707, 1643, 1536, 1223 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.69 (br s, 1 H), 9.13 (d, J = 8.2 Hz, 1 H), 7.36 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H), 5.67 (dd, J = 8.2, 4.8 Hz, 1 H), 5.08 (d, J = 4.8 Hz, 1 H), 5.00 (d, J = 12.8 Hz, 1 H), 4.68 (d, J = 12.8 Hz, 1 H), 3.65 - 3.45 (m, 4H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.6, 170.2, 164.6, 162.8, 134.8, 131.3, 130.9,

128.2, 62.7, 59.1 , 57.4, 40.8, 25.5, 20.6. HRMS (ESI ⁺ ): calcd for C ₁₈ H ₁₈ CIN ₂ 0 ₆ S (M + H) ⁺ 447.0394, found 447.0414.

(6/?,7/?)-3-(Acetoxymethyl)-7-(2-(4-bromophenyl)acetamido )-8-oxo-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (15). 3-Bromophenylacetyl chloride (171 mg, 0.74 mmol) and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (75 uL) according to method B. The resulting precipitate was collected by vacuum filtration and washed with ice-cold DCM to afford the product as a cream amorphous solid (131 mg, 76%). IR (solid): t _½ax 3265, 3060, 1782, 1748, 1707, 1651 , 1543, 1223 crrr ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.66 (br s, 1 H), 9.14 (d, J = 8.2 Hz, 1 H), 7.50 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 5.67 (dd, J = 8.2, 4.8 Hz, 1 H), 5.08 (d, J = 4.8 Hz, 1 H), 5.00 (d, J = 12.8 Hz, 1 H), 4.68 (d, J = 12.8 Hz, 1 H), 3.65 - 3.45 (m, 4H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.6, 170.2, 164.7, 162.8, 135.2,

131.3, 131.1 , 126.3, 123.4, 1 19.7, 62.7, 59.1 , 57.4, 40.8, 25.5, 20.6. HRMS (ESI ⁺ ): calcd for C ₁₈ H ₁₈ BrN ₂ 0 ₆ S (M + H) ⁺ 469.0069, found 469.0076.

(6/?,7/?)-7-(2-([1 ,1'-Biphenyl]-4-yl)acetamido)-3-(acetoxymethyl)-8-oxo-5-thia -1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (16). 4-Biphenylacetic acid (156 mg, 0.736 mmol), oxalyl chloride (76 uL, 0.88 mmol) and DMF (1 drop) were reacted in DCM (3 ml_) according to method C. The resulting acid chloride and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (100 uL) according to method B. The resulting precipitate was collected by vacuum filtration and washed with ice-cold Et ₂ 0 to afford the product as a white amorphous solid (1 12 mg, 65%). IR (solid): i _½3c 3302, 1756, 1737, 1654, 1621 , 1536, 1237 crrr ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 9.02 (d, J = 8.3 Hz, 1 H), 7.65 (d, J = 7.2 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H), 7.45 (app t, J = 7.6 Hz, 2H), 7.41 - 7.30 (m, 3H), 5.48 (dd, J = 8.3, 4.8 Hz, 1 H), 5.00 (d, J = 1 1.9 Hz, 1 H), 4.94 (d, J = 4.8 Hz, 1 H), 4.75 (d, J = 12.0 Hz, 1 H), 3.62 (d, J = 13.9 Hz, 1 H), 3.54 (d, J = 13.9 Hz, 1 H), 3.46 (d, J = 17.2 Hz, 1 H), 3.20 (d, J = 17.2 Hz, 1 H), 2.00 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.9, 170.4, 163.4, 162.8, 139.9, 138.3, 135.7, 135.2, 129.6, 128.8, 127.2, 126.5, 126.5, 64.7, 58.5, 57.2, 41.2, 25.1 , 20.7. HRMS (ESI ⁺ ): calcd for C ₂₄ H ₂₃ N ₂ 0 ₆ S (M + H) ⁺ 467.1277, found 467.1287.

(6/?,7/?)-3-(Acetoxymethyl)-8-oxo-7-(2-(4-phenoxyphenyl)a cetamido)-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (17). 4-Phenoxyphenylacetic acid (170 mg, 0.74 mmol), oxalyl chloride (76 uL, 0.88 mmol) and DMF (1 drop) were reacted in DCM (3 ml_) according to method C. The resulting acid chloride and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (75 uL) according to method B. The aqueous layer was extracted with DCM (x3) and the organic layers were combined, dried over Na2SC>4 and evaporated. The resulting solid was precipitated from hot DCM to afford the product as a cream amorphous solid (24 mg, 14%). IR (solid): i _½ax 3280, 1774, 1730, 1655, 1532, 1223 crrr ¹ . ¹ H NMR (400 MHz, DMSO- cfe) d 9.05 (d, J = 8.3 Hz, 1 H), 7.37 (app t, J = 7.9 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H), 7.12 (t, J = 7.4 Hz, 1 H), 7.04 - 6.89 (m, 5H), 5.57 (dd, J = 8.2, 4.8 Hz, 1 H), 5.03 - 4.95 (m, 2H), 4.72 (d, J = 12.4 Hz, 1 H), 3.59 - 3.46 (m, 3H), 3.33 (d, J = 17.7 Hz, 1 H), 2.01 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 171.0, 170.3, 163.7, 163.2, 156.9, 155.2, 131.0, 130.6, 130.0, 123.2, 1 18.6, 1 18.3, 63.7, 58.8,

57.3, 40.7, 25.3, 20.6. HRMS (ESI ⁺ ): calcd for C ₂₄ H ₂₃ N ₂ O ₇ S (M + H) ⁺ 483.1226, found 483.1212.

(6/?,7/?)-3-(acetoxymethyl)-8-oxo-7-(2-(m-tolyl)acetamido )-5-thia-1 -azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (18). 3-Methylphenylacetic acid (1 11 mg, 0.74 mmol), oxalyl chloride (76 pl_, 0.88 mmol) and DMF (1 drop) were reacted in DCM (3 ml_) according to method C. The resulting acid chloride and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (75 uL) according to method B. The aqueous layer was extracted with DCM (x3) and the organic layers were combined, dried over Na2SC>4 and evaporated. The resulting solid was triturated with DCM to afford the product as a cream amorphous solid (36 mg, 24%). IR (solid): i _ax 3288, 1726, 1662, 1625, 1526, 1223 crrr ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 9.00 (d, J = 8.3 Hz, 1 H), 7.17 (app. t, J = 7.5 Hz, 1 H), 7.12 - 6.98 (m, 3H), 5.51 (dd, J = 8.1 , 4.7 Hz, 1 H), 5.05 - 4.91 (m, 2H), 4.74 (d, J = 12.1 Hz, 1 H), 3.56 - 3.41 (m, 3H), 3.26 (d, J = 17.5 Hz, 1 H), 2.27 (s, 3H), 2.01 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 171.0, 170.5, 163.5, 163.3, 137.2, 135.8, 129.7, 128.1 , 127.1 , 126.1 ,

64.3, 58.6, 57.3, 41.5, 25.2, 21.0, 20.7. HRMS (ESI ⁺ ): calcd for C ₁₉ H ₂ I N ₂ 0 ₆ S (M + H) ⁺ 405.1120, found 405.1127.

(6/?,7/?)-3-(Acetoxymethyl)-8-oxo-7-(2-(3-phenoxyphenyl)a cetamido)-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (19). 3-Phenoxyphenylacetic acid (170 mg, 0.74 mmol), oxalyl chloride (76 uL, 0.88 mmol) and DMF (1 drop) were reacted in DCM (3 ml_) according to method C. The resulting acid chloride and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (75 uL) according to method B. The aqueous layer was extracted with DCM (x3) and the organic layers were combined, dried over Na ₂ S0 ₄ and evaporated. The resulting solid was precipitated from hot DCM to afford the product as a cream amorphous solid (22 mg, 12%). IR (solid): i _ax 3280, 3042, 1771 , 1726, 1659, 1528, 1226 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 9.04 (d, J = 8.2 Hz, 1 H), 7.39 (app t, J = 7.9 Hz, 2H), 7.31 (t, J = 7.9 Hz, 1 H), 7.13 (t, J = 7.4 Hz, 1 H), 7.07 - 6.95 (m, 4H), 6.87 (dd, J = 8.1 , 2.2 Hz, 1 H), 5.55 (dd, J = 8.1 , 4.8 Hz, 1 H), 5.02 - 4.94 (m, 2H), 4.71 (d, J = 12.3 Hz, 1 H), 3.59 - 3.45 (m, 3H), 3.30 (d, J = 17.7 Hz, 1 H), 2.01 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.6, 170.3, 163.6, 163.1 , 156.6, 156.5, 138.0, 130.0, 129.69, 124.2, 123.3, 1 19.2, 1 18.6, 1 16.7, 63.7, 58.8, 57.2, 41.4, 25.3, 20.6. HRMS (ESF): calcd for C ₂₄ H ₂₃ N ₂ 0 ₇ S (M + H) ⁺ 483.1226, found 483.1233. (6/?,7/?)-3-(Acetoxymethyl)-7-benzamido-8-oxo-5-thia-1-azabi cyclo[4.2.0]oct-2-ene-2- carboxylic acid (20).7-ACA (100 mg, 0.37 mmol) and benzoyl chloride (51 uL, 0.44 mmol) were reacted in satd NaHCC>3 (aq) (10 mL) and acetone (5 ml_) according to method A to afford the product as a white amorphous solid (75 mg, 55%). IR (solid): i _½ax 3250, 1774, 1752, 1710, 1651, 1520, 1223 cm ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.69 (brs, 1H), 9.41 (d, J= 8.0 Hz, 1H), 7.91 (d, J= 7.7 Hz, 2H), 7.57 (t, J= 7.3 Hz, 1H), 7.48 (app t, J= 7.5 Hz, 2H), 5.88 (dd, = 8.1, 4.8 Hz, 1H), 5.19 (d, J = 4.8 Hz, 1 H), 4.99 (d, J= 12.8 Hz, 1H), 4.70 (d, J= 12.7 Hz, 1H), 3.64 (d, J= 18.0 Hz, 1H), 3.50 (d, J= 18.0 Hz, 1 H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.2, 166.9, 164.0, 162.8, 133.0, 131.8, 128.3, 127.7, 123.1, 62.7, 59.8, 57.6, 25.5, 20.5. HRMS (ESI ⁺ ): calcd for C^H^NaOeS (M + H) ⁺ 377.0807, found 377.0807.

(6/?,7/?)-3-(Acetoxymethyl)-7-(4-methylbenzamido)-8-oxo-5 -thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (21).7-ACA (100 mg, 0.37 mmol) and 4-methyl benzoyl chloride (97 uL, 0.74 mmol) were reacted in satd NaHCC>3 (aq) (10 mL) and acetone (5 mL) according to method A to afford the product as a white amorphous solid (39 mg, 26%). I R (solid): i _½ax 3258, 1774, 1730, 1648, 1525, 1223 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 9.29 (d, =8.1 Hz, 1H), 7.83 (d, =7.8Hz, 2H), 7.29 (d, J= 7.9 Hz, 2H), 5.81 (dd, J= 8.1, 4.7 Hz, 1H), 5.14 (d, J= 4.8 Hz, 1H), 4.99 (d, J= 12.5 Hz, 1H), 4.72 (d, J= 12.5 Hz, 1H), 3.59 (d, J= 17.7 Hz, 1H), 3.43 (d, J= 17.7 Hz, 1H), 2.37 (s, 3H), 2.03 (s, 3H). HRMS (ESI ⁺ ): calcd for C ₁₈ H ₁₉ N ₂ 0 ₆ S (M + H) ⁺ 391.0964, found 391.0972.

(6/?,7/?)-3-(Acetoxymethyl)-7-(4-methoxybenzamido)-8-oxo- 5-thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (22).7-ACA (100 mg, 0.37 mmol) and 4-methoxybenzoyl chloride (100 uL, 0.74 mmol) were reacted in EtOAc (5 mL) prior to the addition of aniline (100 uL) according to method B. The aqueous layer was extracted with DCM (x3) and the organic layers were combined, dried over Na2SC>4 and evaporated. The resulting solid was triturated with ice-cold Et2<D to afford the product as a white amorphous solid (36 mg, 24%). IR (solid): i _½ax 3254, 1774, 1752, 1705, 1640, 1528, 1223 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 9.22 (d, J= 8.1 Hz, 1H), 7.92 (d, J= 8.9 Hz, 2H), 7.01 (d, J= 8.9 Hz, 2H), 5.84 (dd, J= 8.1, 4.8 Hz, 1H), 5.16 (d, J= 4.8 Hz, 1H), 4.98 (d, J= 12.7 Hz, 1H), 4.70 (d, J= 12.7 Hz, 1H), 3.82 (s, 3H), 3.62 (d, J= 17.9 Hz, 1H), 3.46 (d, J= 17.9 Hz, 1H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.2, 166.3, 164.1, 162.9, 162.1, 129.7, 125.1, 113.6, 62.9, 59.7, 57.7, 55.4, 25.5, 20.6. HRMS (ESI ⁺ ): calcd for C ₁₈ H ₁₉ N ₂ 0 ₇ S (M + H) ⁺ 407.0913, found 407.0919.

(6/?,7/?)-3-(Acetoxymethyl)-7-(4-nitrobenzamido)-8-oxo-5- thia-1-azabicyclo[4.2.0]oct-2-ene- 2-carboxylic acid (23).7-ACA (100 mg, 0.37 mmol) and 4-nitrobenzoyl chloride (136 mg, 0.74 mmol) were reacted in satd NaHCC>3 (aq) (10 mL) and acetone (5 mL) according to method A. Several drops of MeOH were added prior to the addition of ice-cold Et2<D to afford the product as a white amorphous solid (54 mg, 35%). IR (solid): u _max 3265, 3064, 2971, 1785, 1748, 1711, 1640,

1595, 1524, 1223 cm ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.72 (s, 1H), 9.80 (d, J= 7.8 Hz, 1H), 8.34 (d, J= 8.8 Hz, 2H), 8.13 (d, J= 8.8 Hz, 2H), 5.88 (dd, J= 7.8, 4.7 Hz, 1H), 5.22 (d, J= 4.8 Hz, 1H), 5.00 (d, J= 12.8 Hz, 1H), 4.71 (d, J= 12.8 Hz, 1H), 3.66 (d, J= 17.9 Hz, 1H), 3.51 (d, J= 18.0 Hz, 1H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.7, 166.0, 164.0, 163.3, 149.9, 139.0, 129.8, 124.1, 63.2, 60.4, 58.0, 26.0, 21.0, 15.6. HRMS (ESI ⁺ ): calcd for C^H^NsOsS (M + H) ⁺ 422.0658, found 422.0660.

(6/?,7/?)-3-(Acetoxymethyl)-7-(furan-2-carboxamido)-8-oxo -5-thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (24).7-ACA (100 mg, 0.37 mmol) and 2-furoyl chloride (73 uL, 0.74 mmol) were reacted in satd NaHCC>3 (aq) (10 ml_) and acetone (5 ml_) according to method A to afford the product as a white amorphous solid (30 mg, 22%). IR (solid): i _½ax 3243, 1793, 1718, 1710, 1632, 1595, 1223 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.71 (br s, 1H), 9.29 (d, J= 8.2 Hz, 1H), 7.90 (d, J = 1.7 Hz, 1 H), 7.36 (d, J = 3.5 Hz, 1 H), 6.65 (dd, J = 3.5, 1.7 Hz, 1 H), 5.81 (dd, J = 8.2, 4.8 Hz, 1 H), 5.16 (d, J= 4.8 Hz, 1H), 4.99 (d, J= 12.8 Hz, 1H), 4.70 (d, J= 12.8 Hz, 1H), 3.64 (d, J= 18.0 Hz, 1H), 3.50 (d, J = 18.0 Hz, 1H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 170.7, 164.4, 163.3, 158.4, 146.8, 146.5, 115.4, 112.4, 63.2, 59.6, 58.1, 26.0, 21.0. HRMS (ESI ⁺ ): calcd for C ₁₅ H ₁₅ N ₂ 0 ₇ S (M + H) ⁺ 367.0600, found 367.0609.

(6/?,7/?)-3-(Acetoxymethyl)-7-(cyclohexanecarboxamido)-8- oxo-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (25). 7-ACA (100 mg, 0.37 mmol) and cyclohexanecarbonyl chloride (60 uL, 0.44 mmol) were reacted in satd NaHCOs(aq) (10 ml_) and acetone (5 ml_) according to method A to afford the product as a white amorphous solid (15 mg, 11%). IR (solid): i _ax 3261, 2926, 2851, 1778, 1737, 1711, 1648, 1532, 1215 crrr ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.67 (brs, 1H), 8.71 (d, J= 8.2 Hz, 1H), 5.63 (dd, J= 8.2, 4.8 Hz, 1H), 5.07 (d, J= 4.8 Hz, 1H), 4.99 (d, J= 12.8 Hz, 1H), 4.67 (d, J= 12.8 Hz, 1H), 3.61 (d, J= 18.1 Hz, 1H), 3.46 (d, J = 18.0 Hz, 1H), 2.34 - 2.24 (m, 1H), 2.03 (s, 3H), 1.76 - 1.57 (m, 5H), 1.39 - 1.14 (m, 5H). HRMS (ESI ⁺ ): calcd for C ₁₇ H ₂₃ N ₂ 0 ₆ S (M + H) ⁺ 383.1277, found 383.1264.

(6/?,7/?)-7-Acetamido-3-(acetoxymethyl)-8-oxo-5-thia-1-az abicyclo[4.2.0]oct-2-ene-2- carboxylic acid (26).7-ACA (500 mg, 1.84 mmol) was suspended in H ₂ 0 (8 ml_), NaHCC>3 (387 mg, 4.60 mmol) was added and the resulting mixture stirred at room temperature for 10 min before being cooled to 0 °C. Acetic anhydride (347 uL, 0.368 mmol) in acetone (10 ml_) was added and the reaction stirred at 0 °C for 30 min. Acetone was removed under reduced pressure, the resulting material was diluted in H ₂ 0 and neutralised with satd NaHCC>3 (aq). The aqueous solution was washed with EtOAc, acidified to pH 2 with 1 M HCI and extracted with EtOAc (x3). The organic layers were combined, washed with brine, dried over Na ₂ SC>4 and evaporated to afford the product as a colourless foam (471 mg, 81%). IR (solid): i _ax 3317, 2937, 1771, 1718, 1755, 1625, 1528, 1219 cm ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.67 (brs, 1H), 8.84 (d, J = 8.4 Hz, 1H), 5.68 (dd, J= 8.3, 4.9 Hz, 1H), 5.08 (d, J= 4.9 Hz, 1H), 5.00 (d, J= 12.8 Hz, 1H), 4.68 (d, J= 12.8 Hz, 1H), 3.63 (d, J = 18.0 Hz, 1 H), 3.48 (d, J= 18.1 Hz, 1H), 2.03 (s, 3H), 1.91 (s, 3H). ¹³ C NMR (101 MHz, DMSO) d 170.2, 170.1 , 165.0, 162.9, 126.4, 123.4, 62.7, 59.0, 57.4, 22.1 , 20.6. HRMS (ESI ⁺ ): calcd for C ₁₂ H ₁₅ N ₂ 0 ₆ S (M + H) ⁺ 337.0470, found 337.0479.

(6/?,7/?)-3-(Acetoxymethyl)-7-butyramido-8-oxo-5-thia-1 -azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid (27). Butyryl chloride (76 uL, 0.74 mmol) and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (100 uL) according to method B. The aqueous layer was extracted with DCM (x3) and the organic layers were combined, dried over Na ₂ S0 ₄ and evaporated. The resulting solid was triturated with ice-cold DCM to afford the product as a white amorphous solid (15 mg, 12%). IR (solid): i _ax 3265, 2960, 1774, 1751 , 1715, 1654, 1539, 1223 crrr ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 8.74 (d, J = 8.2 Hz, 1 H), 5.61 (dd, J = 7.9, 4.8 Hz, 1 H), 5.04 (d, J = 4.7 Hz, 1 H), 4.99 (d, J = 12.6 Hz, 1 H), 4.69 (d, J= 12.6 Hz, 1 H), 3.57 (d, J = 17.8 Hz, 1 H), 3.39 (d, J = 17.7 Hz, 1 H), 2.21 - 2.13 (m, 2H), 2.02 (s, 3H), 1.52 (app h, J = 7.2 Hz, 2H), 0.86 (t, J = 7.4 Hz, 3H). HRMS (ESI ⁺ ): calcd for C ₁₄ H ₁₉ N ₂ 0 ₆ S (M + H) ⁺ 343.0964, found 343.0959.

(6/?,7/?)-3-(Acetoxymethyl)-7-hexanamido-8-oxo-5-thia-1 -azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid (28). Hexanoyl chloride (103 uL, 0.74 mmol) and 7-ACA (100 mg, 0.37 mmol) were reacted in EtOAc (5 ml_) prior to the addition of aniline (100 uL) according to method B. The resulting precipitate was collected by vacuum filtration and washed with ice-cold DCM to afford the product as a white amorphous solid (78 mg, 57%). IR (solid): i _½ax 3283, 3183 2930, 1774, 1752, 1711 , 1651 , 1536, 1223 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 13.65 (br s, 1 H), 8.78 (d, J = 8.2 Hz, 1 H), 5.67 (dd, J = 8.2, 4.8 Hz, 1 H), 5.08 (d, J = 4.8 Hz, 1 H), 4.99 (d, J = 12.8 Hz, 1 H), 4.68 (d, J = 12.8 Hz, 1 H), 3.62 (d, J = 18.1 Hz, 1 H), 3.48 (d, J = 18.1 Hz, 1 H), 2.23 - 2.13 (m, 2H), 2.03 (s, 3H), 1.56 - 1.46 (m, 2H), 1.32 - 1.18 (m, 4H), 0.86 (t, J = 7.0 Hz, 3H). ¹³ C NMR (101 MHz, DMSO-cfe) d 173.0, 170.1 , 164.9, 162.8, 126.4, 123.2, 62.7, 59.0, 57.5, 34.6, 30.7, 25.5, 24.8, 21.8, 20.5, 13.8. HRMS (ESI ⁺ ): calcd for C ₁₆ H ₂₃ N ₂ 0 ₆ S (M + H) ⁺ 371.1277, found 371.1290.

3-(Acetoxymethyl)-7-(2-(4-(ferf-butyl)phenyl)acetamido)-8 -oxo-5-thia-1 -azabicyclo[4.2.0]oct- 2-ene-2-carboxylic acid (29). 4-te/f-Butylphenylacetic acid (141 mg, 0.733 mmol) was subjected to Method C to afford the crude product as a yellow gum. The resulting crude material was then subjected to Method D and the resulting mixture was extracted with DCM (3 x 15 ml_). The organic layers were combined, dried over Na ₂ S0 ₄ , and concentrated to dryness under reduced pressure to give the crude product. A few drops of ice cold DCM were added and the precipitate was washed thoroughly with ice cold DCM and dried under vacuum to give the product as a white solid (18.9 mg, 12%). Mp 115.0-1 17.0 °C; ¹ H NMR (400 MHz, CDCh) d 7.44-7.38 (m, 2H), 7.25 - 7.19 (m, 2H), 6.17 (d, J = 8.9 Hz, 1 H), 5.88 (dd, J = 9.0, 4.7 Hz, 1 H), 5.11 (d, J = 13.8 Hz, 1 H), 5.01 (d, J = 4.8 Hz, 1 H), 4.94 (d, J = 13.8 Hz, 1 H), 3.72-3.63 (m, 2H), 3.58 (d, J = 18.5 Hz, 1 H), 3.39 (d, J = 18.6 Hz, 1 H), 2.12 (s, 3H), 1.34 (s, 9H); ¹³ C NMR (101 MHz, Chloroform-d) d 171.9, 171.1 , 165.3, 162.7, 150.9, 130.4, 129.3, 127.6, 126.4, 125.3, 63.2, 59.3, 57.7, 42.9, 34.7, 31.5, 26.5, 20.9; IR v _m crrr ¹ 3277, 2962, 1774, 1751 , 1711 , 1655, 1534, 1224; HRMS (ESI ⁺ ): m/z Calcd for C ₂₂ H ₂₇ N ₂ O ₆ S (M + H ⁺ ): 447.1590. Found: 447.1601.

3-(Acetoxymethyl)-8-oxo-7-(2-(o-tolyl)acetamido)-5-thia-1 -azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid (30). o-Tolylacetic acid (1 11 mg, 0.739 mmol) was subjected to Method C to afford the crude product as yellow liquid. The resulting crude material was subjected to Method D and the resulting precipitate was collected by vacuum filtration and dried under vacuum to give the product as a yellow solid (8.6 mg, 6%). The filtrate was washed with DCM (3 x 15 ml_). The organic layers were combined, dried over Na ₂ SC> ₄ , and concentrated to dryness under reduced pressure to give the crude product. A few drops of ice cold DCM were then added and the precipitate was washed thoroughly with ice cold DCM and dried under vacuum to give the product as a white solid (34.4 mg, 23%). Mp 158.5-161.0 °C; ¹ H NMR (400 MHz, Methanol-d ₄ ) d 7.27-7.08 (m, 4H), 5.76 (d, J = 4.8 Hz, 1 H), 5.1 1 (d, J = 13.2 Hz, 1 H), 5.08 (d, J = 4.9 Hz, 1 H), 4.84 (d, J = 13.1 Hz, 1 H), 3.66 (d, J = 18.3 Hz, 1 H), 3.67 - 3.60 (m, 2H), 3.48 (d, J = 18.3 Hz, 1 H), 2.32 (s, 3H), 2.06 (s, 3H); ¹³ C NMR (101 MHz, Methanol-ck) d 174.4, 172.4, 166.2, 164.7, 138.2, 134.8, 131.3, 131.2, 128.3, 127.6,

127.1 , 126.9, 64.3, 60.7, 59.0, 40.8, 27.2, 20.6, 19.9; IR cm ¹ 3263, 1774, 1751 , 171 1 , 1657, 1534, 1230; HRMS (ESF): m/z Calcd for C ₁₉ H ₂ I N ₂ 0 ₆ S (M + H ⁺ ): 405.1 120. Found: 405.1 132.

3-(Acetoxymethyl)-7-(2-(3-chlorophenyl)acetamido)-8-oxo-5 -thia-1 -azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (31 ). 3-Chlorophenylacetic acid (125 mg, 0.733 mmol) was subjected to Method C to afford the crude product as yellow liquid. The resulting crude material was subjected to Method D and the resulting precipitate was collected by vacuum filtration and dried under vacuum to give the product as a pale pink solid (89.5 mg, 57%). Mp 164.7-166.3 °C; ¹ H NMR (400 MHz, Methanol-ck) d 7.41-7.21 (m, 4H), 5.73 (d, J = 4.8 Hz, 1 H), 5.1 1 (d, J = 13.1 Hz, 1 H), 5.07 (d, J = 4.8 Hz, 1 H), 4.84 (d, J = 13.1 Hz, 1 H), 3.67 (d, J = 18.3 Hz, 1 H), 3.63 (d, J = 14.3 Hz, 1 H), 3.57 (d, J = 14.3 Hz, 1 H), 3.48 (d, J = 18.3 Hz, 1 H), 2.06; ¹³ C NMR (101 MHz, Methanol-d ₄ ) d 173.8, 172.4,

166.1 , 164.7, 138.7, 135.3, 131.0, 130.3, 128.7, 128.1 , 127.5, 127.1 , 64.3, 60.7, 58.9, 42.6, 27.2, 20.6; IR V _max /crrr ¹ 3277, 1777, 1750, 1710, 1663, 1546, 1221 ; HRMS (ESI ): m/z Calcd for C ₁₈ H ₁₆ CIN ₂ 0 ₆ S (M - H ⁺ ): 423.0418. Found: 423.0420.

3-(Acetoxymethyl)-7-(2-(3-methoxyphenyl)acetamido)-8-oxo- 5-thia-1 -azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (32). 3-Methoxyphenylacetyl chloride (0.1 15 ml_, 0.738 mmol) was subjected to Method D and the resulting mixture was extracted with DCM (3 x 15 ml_) and the organic layers were combined and dried over Na ₂ S0 ₄ . The solvent was removed under reduced pressure to give the crude product. A few drops of MeOH was then added and the product was precipitate from cold Et ₂ 0. The precipitate was collected by vacuum filtration and washed thoroughly with ice cold Et ₂ 0 and dried under vacuum to give the product as a pale yellow solid (61.8 mg, 40%). Mp 155.5-156.8 °C; ¹ H NMR (400 MHz, Methanol-d ₄ ) d 7.26-7.17 (m, 1 H), 6.93 - 6.85 (m, 2H), 6.80 (ddd, J = 8.3, 2.6, 1.0 Hz, 1 H), 5.74 (d, J = 4.8 Hz, 1 H), 5.11 (d, J = 13.1 Hz, 1 H), 5.06 (d, J = 4.9 Hz, 1 H), 4.83 (d, J= 13.1 Hz, 1 H), 3.78 (s, 3H), 3.66 (d, J= 18.3 Hz, 1H), 3.60 (d, J= 14.1 Hz, 1H), 3.53 (d, J= 14.1 Hz, 1 H), 3.47 (d, J= 18.3 Hz, 1H), 2.06 (s, 3H). ¹³ C NMR (101 MHz, Methanol-d ₄ ) d 173.1, 171.0,

164.8, 163.3, 159.9, 136.4, 129.1, 126.2, 125.6, 121.1, 114.2, 112.3, 62.9, 59.2, 57.5, 54.2, 41.8,

25.8, 19.2; IR v _m crrr ¹ 3275, 1774, 1750, 1710, 1666, 1535, 1222; HRMS (ESI): m/z Calcd for C ₁₉ H ₁₉ N ₂ 0 ₇ S (M - H ⁺ ): 419.0913. Found: 419.0907.

3-(Acetoxymethyl)-7-(2-(3-bromophenyl)acetamido)-8-oxo-5- thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (33). 3-Bromophenylacetic acid (158 mg, 0.735 mmol) was subjected to Method C to afford the crude product as yellow liquid. The resulting crude material was subjected to Method D and the resulting precipitate was collected by vacuum filtration and dried under vacuum to give the product as an off white solid (124 mg, 72%). Mp 173.0-175.0 °C; ¹ H NMR (400 MHz, DMSO-cfe) d 13.72 (s, 1H), 9.19 (d, J = 8.3 Hz), 7.53-7.48 (m, 1H), 7.48-7.42 (m, 1H), 7.32-7.25 (m, 2H), 5.69 (dd, =8.3, 4.8 Hz, 1H), 5.09 (d, =4.8Hz, 1H), 5.01 (d, = 12.8 Hz, 1H), 4.69 (d, J = 12.8 Hz, 1 H), 3.63 (d, J= 18.1 Hz, 1H), 3.60 (d, J= 14.0 Hz, 1H), 3.52 (d, J= 14.0 Hz, 1H), 3.50 (d, J= 18.1 Hz, 1 H), 2.04 (s, 3H); ¹³ C NMR (101 MHz, DMSO-cfe) d 170.9, 170.7, 165.1, 163.2, 139.0, 132.2, 130.9, 129.9, 128.6, 126.7, 124.0, 121.9, 63.2, 59.6, 57.8, 41.5, 26.0, 21.1; IR v _m cm ¹ 3275, 1775, 1751, 1711, 1663, 1546, 1225; HRMS (ESI): m/z Calcd for C ₁₈ H ₁₆ BrN ₂ 0 ₆ S (M - H ⁺ ): 466.9912. Found: 466.9920.

3-(Acetoxymethyl)-7-(2-(naphthalen-2-yl)acetamido)-8-oxo- 5-thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (34). 2-Naphthaleneacetic acid (137 mg, 0.736 mmol) was subjected to Method C to afford the crude product as a yellow gum. The resulting crude material was subjected to Method D and the resulting precipitate was collected by vacuum filtration and dried under vacuum to give the product as a white solid (74.3 mg, 46%). Mp 177.0-178.5 °C; ¹ H NMR (400 MHz, DMSO-cfe) d 13.71 (s, 1H), 9.22 (d, J= 8.3 Hz, 1H), 7.97-7.81 (m, 3H), 7.81-7.74 (m, 1H), 7.59- 7.37 (m, 3H), 5.71 (dd, =8.4, 4.6 Hz, 1H), 5.09 (d, =4.8Hz, 1H), 5.00 (d, = 12.8 Hz, 1H), 4.68 (d, J= 12.8 Hz, 1 H), 3.76 (d, J= 14.0 Hz, 1H), 3.67 (d, J= 14.1 Hz, 1H), 3.62 (d, J= 18.1 Hz, 1H), 3.50 (d, J= 18.1 Hz, 1H), 2.03 (s, 3H); ¹³ C NMR (101 MHz, DMSO-cfe) d 171.4, 170.7, 165.2, 163.3,

133.9, 133.4, 132.3, 128.1, 128.00, 127.96, 127.9, 127.8, 126.8, 126.6, 126.1, 123.8, 63.2, 59.6,

57.9, 42.2, 26.0, 21.1; IR v _m crrr ¹ 3254, 3046, 1782, 1749, 1710, 1663, 1536, 1228; HRMS (ESF): m/z Calcd for C ₂₂ H ₂₀ N ₂ O ₆ NaS (M + Na ⁺ ): 463.0940. Found: 463.0935.

3-(Acetoxymethyl)-8-oxo-7-(2-(3-(trifluoromethyl)phenyl)a cetamido)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (35).3-(Trifluoromethyl)phenylacetic acid (150 mg, 0.735 mmol) was subjected to Method C to afford the crude product as a yellow gum. The resulting crude material was subjected to Method D and the resulting precipitate was collected by vacuum filtration and dried under vacuum to give the product as a white solid (120 mg, 72%). Mp 170.6- 171.6 °C; ¹ H NMR (400 MHz, Methanol-d ₄ ) d 7.69-7.47 (m, 4H), 5.74 (d, J= 4.8 Hz, 1H), 5.11 (d, J = 13.2 Hz, 1 H), 5.07 (d, J= 4.8 Hz, 1H), 4.84 (d, J= 13.1 Hz, 1H), 3.73 (d, J= 14.4 Hz, 1H), 3.67 (d, J = 14.6 Hz, 1 H), 3.66 (d, J = 18.2 Hz, 1 H), 3.47 (d, J = 18.2 Hz, 1 H), 2.06 (s, 3H); ¹³ C NMR (101 MHz, Methanol-ck) d 173.7, 172.4, 166.0, 164.7, 137.8, 134.1 , 130.3, 127.7, 127.2, 126.98, 126.94, 124.79, 124.76, 64.3, 60.7, 58.9, 42.6, 27.2, 20.6; IR v _m crrr ¹ 3277, 1783, 1749, 171 1 , 1657, 1547, 1223, 11 19; HRMS (ESI ⁺ ): m/z Calcd for C^H^NaOeNaSFs (M + Na ⁺ ): 481.0657. Found: 481.0666.

3-(Acetoxymethyl)-7-(2-(3-nitrophenyl)acetamido)-8-oxo-5- thia-1 -azabicyclo[4.2.0]oct-2-ene- 2-carboxylic acid (36). 3-Nitrophenylacetic acid (133 mg, 0.734 mmol) was subjected to Method C to afford the crude product as a brown solid. The resulting crude material was subjected to Method D and the resulting precipitate was collected by vacuum filtration and dried under vacuum to give the product as a yellow solid (94.3 mg, 59%). Mp 105.0-107.0 °C; ¹ H NMR (400 MHz, Methanol-^) d 8.29-8.24 (m, 1 H), 8.20-8.14 (m, 1 H), 7.78-7.73 (m, 1 H), 7.63-7.56 (m, 1 H), 5.76 (d, J = 4.8 Hz, 1 H), 5.13 (d, J = 13.2 Hz, 1 H), 5.09 (d, J = 4.8 Hz, 1 H), 4.86 (d, J = 13.2 Hz, 1 H), 3.81 (d, J = 14.6 Hz, 1 H), 3.75 (d, J = 14.6 Hz, 1 H), 3.68 (d, J = 18.3 Hz, 1 H), 3.50 (d, J = 18.3 Hz, 1 H), 2.08 (s, 3H); ¹³ C NMR (101 MHz, DMSO-cfe) d 170.7, 164.9, 163.3, 148.1 , 138.4, 136.5, 130.2, 124.2, 122.1 , 63.3, 59.6, 57.80, 41.3, 26.0, 21.1 ; IR v _m crrr ¹ 3266, 1766, 1735, 1710, 1648, 1526, 1351 , 1232; HRMS (ESP): m/z Calcd for C^H^NsCbNaS (M + Na ⁺ ): 458.0634. Found: 458.0624. ferf-Butyl (6/?,7/?)-7-acetamido-3-(acetoxymethyl)-8-oxo-5-thia-1 -azabicyclo[4.2.0]oct-2-ene-

2-carboxylate (37). Compound 26 (75 mg, 0.24 mmol) was dissolved in DCM (2 ml_), tert- butyl 2,2,2-trichloroacetimidate (170 uL, 0.96 mmol) was added and the reaction heated to 60 °C for 24 h. After cooling to room temperature the reaction was diluted with MeOH. Solvent was removed under reduced pressure and the resulting solid triturated with cold DCM. The solute was loaded directly onto a 10g SNAP KPSil column and purified by column chromatography (0 - 10% MeOH in DCM) to afford the product as a cream glassy solid (82 mg, 93%). IR (thin film): i _½ax 3291 , 2982, 1774, 1718, 1670, 1528, 1368, 1223 cm- ¹ . ¹ H NMR (400 MHz, CDCb) d 6.39 (d, J = 9.0 Hz, 1 H), 5.84 (dd, J = 9.0, 4.9 Hz, 1 H), 5.09 (d, J = 13.3 Hz, 1 H), 4.95 (d, J = 4.9 Hz, 1 H), 4.80 (d, J = 13.2 Hz, 1 H), 3.55 (d, J = 18.4 Hz, 1 H), 3.36 (d, J = 18.4 Hz, 1 H), 2.08 (s, 3H), 2.06 (s, 3H), 1.53 (s, 9H). ¹³ C NMR (101 MHz, CDCb) d 170.7, 170.4, 164.9, 160.5, 127.5, 123.8, 84.0, 63.3, 59.4, 57.5, 27.9, 26.6, 23.0, 20.9. HRMS (ESP): calcd for C^HasNaOeS (M + H) ⁺ 393.1096, found 393.1088.

7-(4-(ferf-Butoxycarbonyl)piperazin-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo-1 ,4-dihydroquinoline-

3-carboxylic acid (38). Ciprofloxacin (500 mg, 1.51 mmol) was dissolved in 1 M NaOH (aq) (5 ml_) and THF (10 ml_) added, followed by the drop-wise addition of B0C2O (360 mg, 1.66 mmol) in THF (10 ml) and stirred at room temperature for 16 h. Solvent was removed under reduced pressure and the resulting material diluted in H2O and neutralised with satd NH4CI (aq). The precipitate was collected by vacuum filtration and washed with H2O to afford the product as a white amorphous solid (502 mg, 77%). IR (solid): O _max 2971 , 1733, 1688, 1629, 1249 cm ^·1 . ¹ H NMR (400 MHz, CDCb) d 14.95 (s, 1 H), 8.78 (s, 1 H), 8.05 (d, J = 12.9 Hz, 1 H), 7.37 (d, J = 7.1 Hz, 1 H), 3.73 - 3.62 (m, 4H), 3.53 (tt, J = 7.3, 4.0 Hz, 1 H), 3.34 - 3.25 (m, 4H), 1.50 (s, 9H), 1.43 - 1.37 (m, 2H), 1.24 - 1.17 (m, 2H). ¹³ C NMR (101 MHz, CDCh) d 167.1 , 154.7, 153.1 , 147.7, 1 13.0, 1 12.7, 108.5, 105.1 , 80.5, 35.4, 28.6, 8.4. HRMS (ESI ⁺ ): Calcd for C22H27N3O5F (M+H) ⁺ 432.1935, Found: 432.1951.

Sodium 7-(4-(ferf-butoxycarbonyl)piperazin-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo-1 ,4- dihydroquinoline-3-carboxylate (39). Compound 38 (105 g, 0.240 mmol) was suspended in MeOH (2.44 ml_), 0.1 M NaOH (aq) (2.44ml_) was added and the reaction mixture stirred at 30 °C for 30 min. Solvent was removed under reduced pressure and resulting material suspended in H2O (5 uL) and EtOH (5 ml_) and evaporated to dryness (x3). Then, the solid was suspended in DCM and evaporated to afford the product as a cream amorphous solid (11 1 mg, quant.). IR (solid): i _ax 1617, 1478, 1242 crrr ¹ . ferf-Butyl (6/?,7/?)-7-acetamido-3-(((7-(4-(ferf-butoxycarbonyl)piperaz in-1 -yl)-1 -cyclopropyl-6- fluoro-4-oxo-1 ,4-dihydroquinoline-3-carbonyl)oxy)methyl)-8-oxo-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylate (40). Compound 37 (198 mg, 0.63 mmol) was dissolved in DCM (8 ml_) and TMSI (1 17 uL, 0.82 mmol) added drop-wise. The reaction mixture was stirred in the dark for 2 h at room temperature, then diluted with DCM and washed with 10% (wt/v) Na2SC>3 (aq). The organic layer was dried over Na2SC>4 and evaporated. The resulting yellow glassy solid (120 mg, 0.31 mmol) and compound 39 (100 mg, 0.26 mmol) were suspended in anhydrous 1 ,4- dioxane (3.5 ml_) and DMF (1.15 ml_) was added drop-wise. The reaction mixture was stirred in the dark for 2 h before the solvent was removed under a stream of N2. The resulting material was dissolved in minimal DCM and loaded directly onto a 10 g SNAP Ultra cartridge and purified by column chromatography (0 - 6% MeOH in DCM) to afford the product as a pale yellow glassy solid (108 mg, 52%). IR (solid): i _½ax 2974, 1782, 1685, 1618, 1250 cm- ¹ . ¹ H NMR (400 MHz, DMSO-cfe) d 8.86 (d, J = 8.5 Hz, 1 H), 8.44 (s, 1 H), 7.80 (d, J = 13.3 Hz, 1 H), 7.47 (d, J = 7.4 Hz, 1 H), 5.71 (dd, J = 8.4, 4.9 Hz, 1 H), 5.15 - 5.09 (m, 2H), 4.86 (d, J = 13.1 Hz, 1 H), 3.72 - 3.62 (m, 3H), 3.58 - 3.50 (m, 4H), 3.25 - 3.16 (m, 4H), 1.91 (s, 3H), 1.49 (s, 9H), 1.43 (s, 9H), 1.31 - 1.25 (m, 2H), 1.13 - 1.05 (m, 2H).

(6/?,7/?)-7-Acetamido-3-(((1 -cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1 -yl)-1 ,4- dihydroquinoline-3-carbonyl)oxy)methyl)-8-oxo-5-thia-1 -azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid (41 ). Compound 40 (15 mg, 0.02 mmol) was dissolved in anhydrous DCM (0.3 ml_) and cooled to 0 °C. Anhydrous anisole (3 drops) was added followed by the drop-wise addition of TFA (0.3 ml_). The reaction mixture was stirred at 0 °C for 30 min, warmed to room temperature and stirred for a further 40 min. Solvent was removed under a stream of N2 and the resulting gum triturated with ice-cold EtOAc. The precipitate was collected, diluted in H2O and DCM and basified to pH 9 with 3% NaHCCh (aq). The aqueous phase was separated and loaded directly onto a 12 g SNAP KP-C18-HS cartridge and purified by reverse-phase column chromatography (0 - 100% MeCN in H2O). Fractions containing product were freeze-dried to afford the product as a white solid (2.5 mg, 23%). ¹ H NMR (500 MHz, D ₂ 0) d 8.58 (s, 1 H), 7.73 (d, J = 13.0 Hz, 1 H), 7.46 (d, J = 7.2 Hz, 1H), 5.58 (d, J= 4.6 Hz, 1H), 5.11 (d, = 12.6 Hz, 1H), 5.07 (d, =4.7Hz, 1H), 4.81 (d, = 12.6 Hz, 1H), 3.68-3.31 (m, 11H), 2.00 (s, 3H), 1.27 (d, =6.9Hz, 2H), 1.06 (d, J= 4.3 Hz, 2H). HRMS (ESI ⁺ ): calcd for C ₂₇ H ₂₉ FN ₅ O ₇ S (M + H) ⁺ 586.1772, found 586.1794.

Compounds 42, 43 and 44 were prepared via the same synthetic route and experimental procedure as described for compound 41.

(6R,7R)-7-(2-([1,1'-biphenyl]-4-yl)acetamido)-3-(((1-cycl opropyl-6-fluoro-4-oxo-7-(piperazin-1- yl)-1,4-dihydroquinoline-3-carbonyl)oxy)methyl)-8-oxo-5-thia -1-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid (42). ¹ H NMR (500 MHz, DMSO-d6) d 9.09 (d, J= 8.4 Hz, 1H), 8.12 (s, 1H), 7.93 (d, J= 13.1 Hz, 1 H), 7.72-7.51 (m, 4H), 7.49-7.18 (m, 5H), 5.59 (dd, J= 8.3, 4.7 Hz, 1H), 5.33 (d, J= 12.6 Hz, 1 H), 5.03 (d, J= 4.8 Hz, 1H), 4.77 (d, J= 12.5 Hz, 1H), 3.83 (dt, J= 7.3, 3.5 Hz, 1H), 3.68 - 3.47 (m, 4H), 3.47 - 3.35 (m, 4H), 3.27 - 3.12 (m, 4H), 1.35- 1.27 (m, 1 H), 1.25 - 1.14 (m, 2H), 1.07-0.89 (m, 2H).

(6R,7R)-3-(((1 -cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1 -yl)-1 ,4-dihydroquinoline-3- carbonyl)oxy)methyl)-7-(furan-2-carboxamido)-8-oxo-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid (43). ¹ H NMR (500 MHz, Deuterium Oxide) d 8.58 (s, 1H), 7.72 (d, J= 13.0 Hz, 1 H), 7.46 (d, J= 7.2 Hz, 1H), 5.58 (d, J= 4.6 Hz, 1H), 5.10 (d, J= 12.6 Hz, 1H), 5.07 (d, J= 4.7 Hz, 1 H), 4.80 (d, J= 12.6 Hz, 1H), 3.63 (d, J= 17.8 Hz, 1H), 3.58-3.52 (m, 1H), 3.52-3.45 (m, 4H), 3.45 - 3.35 (m, 5H), 1.29 - 1.23 (m, 2H), 1.06 (dd, J = 6.1, 3.0 Hz, 2H).

(6R,7R)-3-(((1 -cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1 -yl)-1 ,4-dihydroquinoline-3- carbonyl)oxy)methyl)-7-(2-(3-nitrophenyl)acetamido)-8-oxo-5- thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (44). ¹ H NMR (500 MHz, DMSO-cfe) d 9.23 (d, J= 8.1 Hz, 1H), 8.69 (s, 1H), 8.22-8.18 (m, 1H), 8.15-8.10 (m, 1H), 7.97 (d, ³ _H ,F = 13.0 Hz, 1H), 7.79 (d, ⁴ _H ,F = 13.2 Hz, 1H), 7.76 - 7.71 (m, 1 H, ArH), 7.66 - 7.59 (m, 1 H), 7.42 (d, J = 6.9 Hz, 1 H), 5.67 - 5.63 (m, J = 5.7 Hz, 1 H), 5.23 (d, J= 13.0 Hz, 1H), 5.08 (d, J = 4.8 Hz, 1H), 4.87 (d, J= 13.2 Hz, 1H), 3.87-3.82 (m, 1 H), 3.81 -3.55 (m, 4H), 3.54-3.45 (m, 4H), 3.45-3.39 (m, 4H), 1.22-1.17 (m, 2H), 1.11 - 1.03 (m, 2H); HRMS (ESI ⁺ ): m/z Calcd for C ₃₃ H ₃₂ FN ₆ O ₉ S (M+H ⁺ ): 707.1936. Found: 707.1938

Ceph-C was prepared by analogous methods as described above.

Biological Examples

The compounds of the invention have the following properties: have low or no intrinsic bactericidal activity;

are susceptible to hydrolysis by beta-lactamase leading to bactericidal activity.

In order to increase selectivity towards beta-lactamase expressing bacteria, portion A should have low or no intrinsic bactericidal activity and should have beta-lactamase hydrolytic activity. Following exposure of the compound of formula (I) to beta-lactamase, the beta-lactam motif in A undergoes hydrolysis such that the beta-lactam is cleaved, the cleavable linker is cleaved, and B is released in active form.

The suitability of side chain R for use in beta-lactamase targeting pro-drugs of formula (la) was investigated and the data is presented below (sections 1 to 6).

1. MIC measurements

The A portion was first assessed for its bactericidal activity against one or two different bacterial strains (CFT073 and DH5-alpha), with or without a plasmid (pSU18), and with or without a beta- lactamase being present (CTX-M-1 or TEM-116). The results are presented in Table 4.

Table 4. Summary of compound structure and MIC data. Antibacterial activities (MIC) against CFT073 + pSU18 ± CTX-M-1 and DH5-alpha ± TEM-116

The group OAc was used to replace portion B (the bactericidal fluoroquinolone motif e.g. ciprofloxacin). All compounds tested in at least one of the two assays, or both assays, had little to no bactericidal activity, as shown by the MIC values of >10 microM such as >50 microM.

For all compounds tested in the DH5-alpha assay, a higher MIC value was determined for the E. coli strain expressing TEM-1 16 than the strain not expressing beta-lactamase, indicating hydrolytic activity by the beta-lactamase and hydrolytic degradation of the compound of formula (II).

Following initial assessment of compound activity in the laboratory E. coli strain DH5-alpha, a number of portion A compounds (with an OAc group in place of portion B) was tested against uropathogenic E. coli strain CFT073 in order to assess the activity of the beta-lactams against disease-relevant pathogenic bacteria. This strain was isolated from the blood of a patient with acute pyelonephritis, is devoid of all virulence plasmids commonly associated with uropathogenic strains and proved genetically tractable for manipulation (Welch et ai. 2002; Mobley et ai. 1990). The plasmid pSU18, either empty or encoding for beta-lactamase was introduced into CFT073, enabling comparison of compound activity in CFT073 and CFT073 + pSU18 ± beta-lactamase. The primary beta-lactamases used in this work were from the CTX-M class. CTX-M are able to cleave a wide range of clinically-relevant beta-lactam antibiotics (Canton, R. et ai. 2012).

MIC values were determined for a number of portion A compounds against CFT073 + pSU18 ± CTX- M-1 (Table 4). For all the compounds tested, the MIC values for CFT073 + pSU18 were within 2-fold of those determined against DH5-alpha. Furthermore, all compounds tested against CFT073 + pSU18 ± CTX-M-1 had little to no bactericidal activity, and for certain compounds a higher MIC value was determined for CFT073 + pSU18 + CTX-M-1 than CFT073 + pSU18 - CTX-M-1 , indicating hydrolytic activity by the beta-lactamase and hydrolytic degradation of the compound of formula (II).

Compounds with a MIC value of £50 uM against non-beta-lactamase expressing bacteria are less preferred as portion A, for example see compounds 7, 8, 9, 11 and 29.

Based on these results, a number of compounds appeared to be promising starting points for portion A.

2. Hydrolysis Studies

As well as having low or no intrinsic bactericidal activity, it is important that once in the bacterial cell which expresses beta-lactamase, the compounds are hydrolysed by beta-lactamase.

The susceptibility to beta-lactamase mediated hydrolysis was assessed by determining the physiological efficiency (/c _cat /K _m ) of hydrolysis by recombinant AmpC protein as well as a whole-cell beta-lactamase hydrolysis assay which was used to measure the magnitude of beta-lactamase mediated hydrolysis in cell. The results are presented in Table 5. Table 5. Summary of compound structure and biological data. AmpC hydrolytic efficiency and percentage hydrolysis in CFT073 + CTX-M-1 cells. In the AmpC-assay: + indicates a hydrolytic efficiency of >0.0-<1.0 microM/S ¹ ; ++ indicates a hydrolytic efficiency of 1.0-<5.0; +++ indicates a hydrolytic efficiency of 5.0-<10.0; and ++++ indicates a hydrolytic efficiency of >10.0. In the CFT073 NMR assay: + indicates a %hydrolysis of 0.0-<15%; ++ indicates a %hydrolysis of 15-<40%; +++ indicates a %hydrolysis of 40-<60%; ++++ indicates a %hydrolysis of >60%.

wherein ND means“not determined”; ^a Percentage hydrolysis determined by w hole-cell NMR assay.

All compounds tested showed hydrolytic activity to varying extents in one or both assays. Following an initial assessment of the susceptibility to beta-lactamase mediated hydrolysis using recombinant Amp-C protein, the hydrolytic activity of the compounds of formula (II) was assessed in a whole cell NMR assay. For the majority of the compounds tested, at least a low level of hydrolysis was observed after 60 minutes. Suitably, the compounds of the invention have a hydrolytic activity in one or both assays, particularly in the whole cell NMR assay, of at least 15%. Most suitably, the compounds of the invention have a hydrolytic activity in one or both assays, particularly in the whole cell NMR assay, of >60%. Thus, compounds 13, 18, 20, 24, 26, 31 , 32, 33 and 36 were deemed particularly suitable for portion A.

3. Cell permeability

The cell permeability of the compounds is thought to be the limiting factor in hydrolytic activity; if the compounds are not able to permeate the cells, a lower level of hydrolytic activity in a whole cell assay would be expected.

To address the question of compound permeability, a whole-cell beta-lactamase hydrolysis assay was used to measure the magnitude of beta-lactamase mediated hydrolysis in cell. Hydrolytic decomposition of beta-lactam rings is associated with changes in ¹ H NMR signals. Using ¹ H NMR spectroscopy the real-time monitoring of beta-lactam hydrolysis by whole bacterial cells can be achieved. As hydrolysis was occurring in cell, only compounds with sufficient intracellular accumulation and beta-lactamase activity were hydrolysed. The hydrolysis of 16, 17 and cephalothin 7 in DH5-alpha ± TEM-1 16 and DH5-alpha ± CTX-M-1 was evaluated (Table 6).

Table 6. Percentage hydrolysis of 7, 16 and 17 by DH5-alpha cells ± beta-lactamase in whole-cell NMR hydrolysis assay.

The results show that Compound 16 was highly hydrolysed (69% after 90 minutes) in DH5-alpha expressing the TEM-1 16 beta-lactamase (Table 6) but only <15% hydrolysed after 60 minutes in CFT073 expressing the CTX-M-1 beta-lactamase (Table 5). The results also show that Compound 17 was highly hydrolysed (53% after 90 minutes) in DH5-alpha expressing the TEM-1 16 beta- lactamase (Table 6) but only <15% hydrolysed after 60 minutes in CFT073 expressing the CTX-M- 1 beta-lactamase (Table 5). It is thought that the lack of hydrolytic activity observed for many of the compounds in Table 5 (<15%) could be a result of poor intracellular accumulation in CFT073 E. coli. To test this hypothesis, hydrolysis in DH5-alpha expressing CTX-M-1 was determined for 7, 16 and 17 (Table 6).

Complete hydrolysis was observed after 60 minutes for compounds 7 and 16 suggesting that the lack of hydrolysis observed in CFT073 + CTX-M-1 was not due to the inability of CTX-M-1 to hydrolyse this chemotype. Almost complete (95%) hydrolysis was observed was observed after 60 minutes for compound 17 also suggesting that the lack of hydrolysis observed in CFT073 + CTX-M- 1 was not due to the inability of CTX-M-1 to hydrolyse this chemotype. Instead it is likely in all cases that either due to poor membrane permeability or increased efflux activity, and that lipophilic analogues were unable to engage with CTX-M-1 in CFT073.

Given the large variation between bacterial cell types, it could be expected that membrane permeability and efflux activity will be variable between bacterial types. Thus, the extent of measured hydrolysis would be expected to vary between bacterial types. Portion A of the compounds of formula (I) thus may be tailored to different bacterial types within the family of bacteria that express beta- lactamase, of which there are also many types, which provides further antibacterial selectivity. Suitably, portion A motifs can be selected for certain bacterial cell types using assays similar to those disclosed herein.

Those compounds with good hydrolytic activity in vivo in Table 5 are also expected to have good cell permeability since they must enter the bacterial cell periplasm to be hydrolysed by the beta- lactamase.

4. Prodrug design

Compounds 16, 24, 26 and 36 were selected for the synthesis of compounds of formula (I) due to their high MIC values and high hydrolytic activity in whole CFT073 or DH5-alpha cells (see Tables 5 and 6). Compounds 24, 26 and 36 are especially preferred since they exhibit 15-<40% hydrolysis in the CFT073 + pSU 18 + CTX-M-1 NMR assay (Table 5).

5. In vitro DNA gyrase activity

Members of the fluoroquinolone antibiotic family, such as Ciprofloxacin (V), target the type II topoisomerase enzymes, DNA gyrase and topoisomerase IV. Inhibition of these enzymes results in the arrest of DNA replication and transcription preventing bacterial cell growth. Following preparation of compound 41 (a compound of the invention wherein R is methyl), it was investigated whether the intact prodrug would not inhibit DNA gyrase or topoisomerase IV but beta-lactamase triggered hydrolysis would result in the release of free ciprofloxacin (V) capable of engaging these targets. The ability of compound 41 to inhibit recombinant DNA gyrase enzyme activity both in the absence and presence of beta-lactamase CTX-M-15 was tested. Compound 41 was incubated with relaxed pBR322 plasmid DNA with and without recombinant CTX-M-15 and recombinant DNA gyrase. As expected, inhibition of DNA gyrase by pro-drug 41 was not observed in the absence of CTX-M-15. However in the presence of CTX-M-15, 1 mM 41 was capable of reducing DNA gyrase activity by >50% (Figure 1). These results confirmed that beta-lactamase-specific hydrolysis of 41 results in liberation of active antibiotic capable of DNA gyrase inhibition in vitro.

6. Selective pro-drug activity against uropathogenic E.coii expressing beta-lactamase

Next, the activity of pro-drug 41 was evaluated in whole bacterial cells. MIC values for 41 and ciprofloxacin (V) were determined in E. coli CFT073 + pSU18 ± CTX-M-1. To assess the activity of 41 in the presence of a range of disease-relevant beta-lactamases, MIC determination was also performed in CFT073 strains expressing New Delhi metal lo-beta-lactamase 1 (NDM1) and Klebsiella pneumoniae carbapenemase (KPC) beta-lactamase (Figure 2 and Table 7). NDM1 is an example of an increasingly prevalent beta-lactamase that is capable of hydrolyzing carbapenems, usually considered the last line of defense against beta-lactamase expressing bacteria (Khan et ai. 2017). KPC, a class A beta-lactamase, is the most common carbapenemase globally (Yigit et ai. 2001).

Table 7. Antibacterial activities for 41 and (V) against CFT073 E.coii cells WT and expressing empty plasmid (pEMP), CTX-M-1 (pCTX), NDM1 (pNMD1) and KPC (pKPC).

As expected, the MIC value determined for ciprofloxacin was consistent across all tested strains at 31 nM. The MIC determined for compound 41 in E. coli CFT073 WT and expressing empty plasmid (pEMP) was 310 nM. This represents a 10-fold decrease in activity compared to ciprofloxacin (V) in the absence of beta-lactamase. In E. coli CFT073 strains expressing CTX-M-1 , NDM1 and KPC the MIC value for 41 was 63 nM, only 2-fold higher than ciprofloxacin (V). These data show efficient beta-lactamase mediated pro-drug cleavage and active antibiotic release, resulting in arrest of bacterial cell growth at comparable concentrations to that with free ciprofloxacin.

Similar results were obtained for compounds of formula (I) 42, 43 and 44 as shown in Table 8. Table 8. Structural and Antibacterial activities for compounds 42, 43 and 44, as well as 41 and ciprofloxacin (V) against E.coli cells CFT073 + pSU18 ± CTX-M-1.

*n=1 data

The MIC value for ciprofloxacin (V) was consistent across all tested strains at 31 nM. The MICs determined for compounds 42, 43 and 44 in E. coli CFT073 WT and expressing empty plasmid (pEMP) were 625 nM, 625 nM and 1250 nM respectively. This represents a greater than 20-fold decrease in activity compared to ciprofloxacin (V) in the absence of beta-lactamase. In E. coli CFT073 strains expressing CTX-M-1 the MIC value for compounds 42, 43 and 44 was 312 nM, 156 nM and 313 nM. Compounds 41 , 43 and 44 are of particular interest since the compounds are at least 4-fold more active in the presence of beta-lactamase (CFT073 + pSU18 + CTX-M-1) compared to in the absence of beta-lactamase (CFT073 + pSU18). These data show efficient beta- lactamase mediated compound cleavage and active antibiotic release, resulting in arrest of bacterial cell growth at comparable concentrations to that with free ciprofloxacin.

The activity of compound 41 compared to ciprofloxacin was profiled further in six independently- isolated uropathogenic E.coli clinical isolates expressing CTX-M-15 obtained from Charing Cross Hospital, Imperial College NHS Trust. Three of the strains were ciprofloxacin sensitive (EC11 , EC16 and EC17) and three were ciprofloxacin resistant (EC12, EC13 and EC19). Activity of compound 41 was confirmed in the three ciprofloxacin sensitive bacterial strains, whilst no arrest in bacteria growth was observed for either ciprofloxacin (V) or compound 41 in EC12, EC13 or EC19 (Figure 3). These results provide evidence for the clinical utility of 41 and indicate that the antibacterial activity of 41 observed in beta-lactamase expressing strains is mediated through liberated ciprofloxacin. The gut microbiota includes both Gram-negative bacteria such as E. coli and Gram-positive organisms such as E. faecalis. Since compound 41 was inactive against E. coli (Table 7), the potential clinical value of the prodrug was examined by testing its activity against two representative E. faecalis strains that did not express b-lactamase. Prodrug 41 showed reduced activity compared to ciprofloxacin, indicating that the pro-drug approach could minimize undesirable damage to the microbiota caused by fluoroquinolones (Figure 4). The activity of 41 against CFT073 pEMP or pCTX- M-1 in the presence of human serum, which can modulate drug activity via protein-binding and also contains esterases that have the potential to activate the prodrug by cleaving the ester linkage, was also assessed. Data from MIC assays performed in the presence of human serum were equivalent to those obtained in the absence of serum (Figure 5). Combined, these findings provided further confidence in the selectivity of prodrug 41 and its stability in the host environment.

Finally, the ability of compound 41 to kill bacteria, rather than arrest growth was evaluated. Survival of E. coli CFT073 pEMP and pCTX with no treatment, ciprofloxacin (V), and 41 were determined over time by CFU counts (Figure 6). After 6 hours incubation with pro-drug 41 there was >100-fold greater killing of E. coli expressing CTX-M-1 , compared with bacteria that did not express the enzyme. Killing activity of 41 in E. coli expressing CTX-M-1 was comparable to free ciprofloxacin, whilst growth comparable to no treatment controls was detected for 41 in CFT073 expressing empty plasmid. These findings demonstrate that it is possible to selectively target beta-lactamase- producing bacteria using our pro-drug approach, without affecting bacteria that do not produce beta- lactamase.

Conclusions

A number of beta-lactam-fluoroquinolone antibiotic pro-drugs have been synthesised and evaluated for biological activity.

Overall, the activity of compounds 41-44, in particular compounds 41 , 43 and 44, as shown in Tables 7 and 8 and Figure 4 is consistent with: 1) permeability to pathogenic gram-negative bacteria; 2) a low-level of antibacterial activity for the intact pro-drug; 3) beta-lactamase mediated intracellular release of ciprofloxacin upon cleavage of the beta-lactam; 4) activity against a broad range of beta- lactamases; and 5) undesirable damage to the microbiota caused by fluoroquinolones is minimised.

It is expected that compounds of formula (II) which have a MIC value of >100 uM in all assays in Table 4 (DH5-alpha ± TEM-1 16; CFT073 + pSU 18 ± CTX-M-1), are efficiently hydrolysed by beta- lactamase (Table 5), and are able to cross the bacterial outer membrane (which is a pre-requisite for in vivo hydrolysis by beta-lactamases) would result in the formation of prodrugs which have similar properties to compounds 41-44, particularly compounds 41 , 43 and 44. Thus, Compounds 13, 18, 20, 24, 31 , 32 and 33 in particular may be expected to produce prodrugs after reaction with ciprofloxacin (V) which are selective for beta-lactamase bacteria. These studies demonstrate that the compounds of the invention can harness resistance as a therapeutic opportunity to selectively kill antibiotic-resistant bacteria. In the case of fluoroquinolones, increasing the selectivity profile has two major advantages. First, maintenance of the microbiota leading to reduced secondary infection rate and subsequent antibiotic use. Second, a decreased side-effect profile due to minimized exposure of host cells to fluoroquinolone antibiotics. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota.

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