NL2022854B1 - Prodrug inhibitors - Google Patents

Abstract

Provided herein are compounds useful as metallo-p-lactamase (MBL) inhibitors. The 5 compounds have a formula A-B, where A is a p-lactam antibiotic moiety comprising a bridging methylene (-CH2-) covalently attached to -B; and Bis a latent MBL inhibitor. Also provided are formulations comprising such compounds; as well as such compounds or formulations for use as a medicament. The compounds and formulations may be used in the treatment of antibiotic resistance, bacterial infection. The compounds and formulations 10 may be used in the inhibition of a bacterial MBL.

Description

Prodrug inhibitors

[0001] This invention relates to compounds that are useful as prodrug inhibitors of bacterial metallo-B-lactamase enzymes. Also provided are methods of producing these compounds and uses of these compounds.

BACKGROUND

[0002] Bacterial infections are typically treated with antibiotics. For the treatment of Gram-negative infections, B-lactam antibiotics represent an important class of therapeutic molecules. Mechanistically, once a B-lactam antibiotic passes the outer membrane, it inhibits the transpeptidase activity utilized by the bacterium in the final step of peptidoglycan synthesis. This interferes with cell wall formation, leading to bacterial cell lysis.

[0003] Worryingly, Gram-negative pathogens are increasingly becoming resistant to B- lactams via the acquisition of genes that code for so called metallo-B-lactamase enzymes (MBLs).

[0004] The MBLs are collectively classified as class B beta-lactamases encompassing a number of plasmid-encoded enzymes including: imipenemase (IMP), Verona integron- encoded metallo-B-lactamase (VIM), Sao Paulo metallo-B-lactamase (SPM), German imipenemase (GIM), Seoul imipenemase (SIM), New Delhi metallo-B-lactamase (NDM), and Dutch imipenemase (DIM). Notably, each of these MBLs have been detected in Gram- negative pathogens and are of considerable clinical concern (Bebrone et al., Metallo-beta- lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily, Biochem Pharmacol, 2007, 74, 1686-1701; Somboro et al., Diversity and Proliferation of Metallo-beta-Lactamases: a Clarion Call for Clinically Effective Metallo- beta-Lactamase Inhibitors, App! Environ Microbiol, 2018, 84). Pathogens that express MBLs are able to catalyze the hydrolysis of nearly all available p-lactam antibiotics and are thus extremely worrisome (Walsh et al., Metallo-beta-lactamases: the quiet before the storm?, Clin Microbiol Rev, 2005, 18, 306-325). Much effort has been spent in trying to develop MBL inhibitors to block their hydrolytic ability as a means of resensitizing MBL- expressing pathogens to conventional B-lactams antibiotics (Tehrani et a/., beta- lactam/beta-lactamase inhibitor combinations: an update. Medchemcomm, 2018, 9, 1439- 1456; Linciano et al., Ten Years with New Delhi Metallo-beta-lactamase-1 (NDM-1}: From Structural Insights to Inhibitor Design, ACS Infect Dis, 2018). However, no such MBL inhibitors have been granted approval for therapeutic use in humans (Queenan et al., Carbapenemases: the versatile beta-lactamases, Clin Microbiol Rev, 2007, 20, 440-458;

Bush et al., What we may expect from novel antibacterial agents in the pipeline with respect to resistance and pharmacodynamic principles, J Pharmacokinet Pharmacodyn, 2007, 44, 113-132).

[0005] The increasing global prevalence of MBLs presents a major threat to world health, affecting both community and hospital environments (Kelly ef a/., Carbapenem-resistant Enterobacteriaceae in the community: a scoping review, Int J Antimicrob Agents, 2017, 50, 127-134; Potter et al., The rapid spread of carbapenem-resistant Enterobacteriaceae, Drug Resist Updat, 2016, 29, 30-46; Gupta et al., Carbapenem-resistant Enterobacteriaceae: epidemiology and prevention, Clin infect Dis, 2011, 53, 60-67; Falagas et al., Deaths attributable to carbapenem-resistant Enterobacteriaceae infections, Emerg Infect Dis, 2014, 20, 1170-1175). Of particular concern is the rise in infection rates for carbapenem- resistant Enterobacteriaceae (CRE), including MBL-positive infections, for which mortality rates of up to 50% have been reported (van Duin ef al., Carbapenem-resistant Enterobacteriaceae: a review of treatment and outcomes, Diagn Microbiol Infect Dis, 2013, 75, 115-120; Kim et al., Update on the Epidemiology, Treatment, and Outcomes of Carbapenem-resistant Acinetobacter infections, Chonnam Med J, 2014, 50, 37-44). In response, the World Health Organization (WHO) recently listed multi-drug resistant CRE as one of their “critical priority pathogens” for which novel antibiotics are urgently needed (World Health Organization, 2017, Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics, Geneva, Switzerland). Over the past decade, the number of drug-resistant bacterial infections reported has dramatically increased, affecting millions of patients worldwide. Such infections may lead to prolonged hospitalization, increased disability leave, rising health care costs, and higher rates of mortality (Wang et al., Increased prevalence of carbapenem resistant Enterobacteriaceae in hospital setting due to cross-species transmission of the bla NDM-1 element and clonal spread of progenitor resistant strains, Front Microbiol, 2015, 6, 595; Pittet ef a/., Infection control as a major World Health Organization priority for developing countries, J Hosp infect, 2008, 68, 285-292). Thus, there is a need to provide therapies that reduce B-lactam resistance in Gram-negative pathogens.

[0006] An object of the invention is to provide compounds that are useful as inhibitors of one or more MBLs, which may reduce or overcome resistance to B-lactam antibiotics.

BRIEF SUMMARY OF THE DISCLOSURE

[0007] The invention provides compounds that are useful as prodrug inhibitors of bacterial metallo-B-lactamase enzymes (MBLs). In particular, the compounds comprise a

B-lactam antibiotic moiety and a latent MBL inhibitor. Hydrolysis of the B-lactam ring, for example by a metallo-p-lactamase enzyme, results in a double bond migration within the prodrug molecules leading to release and activation of the latent MBL inhibitor. Many MBL inhibitors function by binding to the zinc ion(s) in the MBL enzyme active site. Such MBL inhibitors often also bind other metals and in doing so can also interfere with the activities of other metallo enzymes. In the invention here disclosed, the activity of the MBL inhibitor is masked in an inactive prodrug form until it engages its target. Such an approach has the capacity to limit possible off-target effects of the MBL inhibitor.

[0008] In accordance with the present invention there is provided in a first aspect a compound of formula I: A—B (0) wherein: A is a B-lactam antibiotic moiety comprising A’-CH2-; and B is selected from: R7 0 ORs a or ng Xe Re nee A | i " Ny, | Rs Rs Ry N° AANA Ra Ri Ris | Riz Ris R23 = HN Rie AA 0 ~ Rao HN R22

I oO R21 or 0 wherein: X, is selected from -S-, -O-, -OC(O)-, -S(O)- and -S(O)z-; Xz is selected from -S-, -OC(O)-, -S(O)- and -S(O).-; Xs is selected from -S-, -O-, -OC(O)-, -S(O}- and -S(O)--; Y; is selected from -N=, or -C(Rs)=; Y2 is selected from -N=, or -C(R49)=; Ys is selected from -N=, or -C(R44)=; Ys is selected from -N=, or -C(R1s)=; each of R2, Rj, Rs, Rs, Ry, Rs, Rs, Rig, R41, Riz, R14, Ris, Ras, R47, Ris, Ris, Raz, Raq and Ry; are independently selected from hydrogen, halo, cyano, nitro, hydroxy, substituted or unsubstituted (C:-Cs)alkyl, substituted or unsubstituted aryl, said one or more optional substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, sulfonamide, NHz, NH(C+-C2 alkyl), N(C+-C; alkyl)2, methoxy, ethoxy; each of Rs and R13 are independently selected from H and (C+-C4)alkyl; and R22 is an aryl or heteroaryl, optionally substituted by one or more substituents independently selected from hydrogen, halo, cyano, nitro, hydroxy, NHC(O)H, NHC(O) (C+-C4)alkyl, and (C+-C4)alkyl optionally substituted by one or more substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, NH2, NH(C+-C» alkyl), N(C+-C:2 alkyl)2, methoxy, ethoxy, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.

[0009] B may be a latent MBL inhibitor, e.g. B may represent a latent Zn?* chelator.

[0010] In accordance with the present invention there is provided in a second aspect a formulation comprising compound of the invention and optionally a pharmaceutically acceptable carrier.

[0011] A third aspect provides a compound or formulation of the invention for use as a medicament.

[0012] A fourth aspect provides a compound or formulation of the invention for use in the treatment of antibiotic resistance. The treatment may further comprise the administration of another active agent.

[0013] A fifth aspect provides a compound or formulation of the invention for use in the treatment of a bacterial infection. The compound or formulation may be for use in the treatment of a bacterial infection, by combined, sequential or separate administration with an antibacterial agent. The antibacterial agent may be a p-lactam antibiotic.

[0014] A sixth aspect provides a method of inhibiting a bacterial metallo-p-lactamase in vitro or in vivo, comprising administration to a cell of an effective amount of a compound of the invention or formulation of the invention.

[0015] A seventh aspect provides a use of a compound of the invention or formulation of the invention for the inhibition of a bacterial metallo-B-lactamase.

[0016] An eighth aspect provides a method of treating a bacterial infection in a patient, comprising administering to the patient an effective amount of a compound of the invention or formulation of the invention.

[0017] The invention will now be described further by reference to the following examples and figures. These are not intended to be limiting but merely exemplary of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 provides a representation of an inactive metallo-B-lactamase inhibitor 5 linked to an exemplary beta-lactam core. The metallo-B-lactamase inhibitor is not active, as the functional group essential for metallo-B-lactamase binding is covalently linked to the beta-lactam. Figure 2 shows a schematic overview of the activation of a metallo-B-lactamase inhibitor via the hydrolysis of a metallo-B-lactamase inhibitor prodrug.

Figure 3 shows the mechanism of action for the release of an active metallo-f3- lactamase inhibitor (R?) from an inactive cephalosporin core.

Figure 4 shows a reaction scheme for the synthesis of a compound of the invention.

Figure 5 shows a reaction scheme for the synthesis of another compound of the invention.

Figure 6 shows a reaction scheme for the synthesis of a compound of the invention.

Figure 7 shows a reaction scheme for the synthesis of compounds of the invention.

Figure 8 shows a reaction scheme for the synthesis of compounds of the invention.

DETAILED DESCRIPTION

[0019] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0020] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments.

The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0021] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0022] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

DEFINITIONS

[0023] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

[0024] The invention concerns amongst other things the treatment of a disease. The term “treatment”, and the therapies encompassed by this invention, include the following and combinations thereof: (1) hindering, e.g. delaying initiation and/or progression of, an event, state, disorder or condition, for example arresting, reducing or delaying the development of the event, state, disorder or condition, or a relapse thereof in case of maintenance treatment or secondary prophylaxis, or of at least one clinical or subclinical symptom thereof; (2) preventing or delaying the appearance of clinical symptoms of an event, state, disorder or condition developing in an animal (e.g. human) that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; and/or (3) relieving and/or curing an event, state, disorder or condition (e.g., causing regression of the event, state, disorder or condition or at least one of its clinical or subclinical symptoms, curing a patient or putting a patient into remission). The benefit to a patient to be treated may be either statistically significant or at least perceptible to the patient or to the physician. It will be understood that a medicament will not necessarily produce a clinical effect in each patient to whom it is administered; thus, in any individual patient or even in a particular patient population, a treatment may fail or be successful only in part, and the meanings of the terms “treatment”, “prophylaxis” and “inhibitor” and of cognate terms are to be understood accordingly. The compositions and methods described herein are of use for therapy and/or prophylaxis of the mentioned conditions.

[0025] The term “prophylaxis” includes reference to treatment therapies for the purpose of preserving health or inhibiting or delaying the initiation and/or progression of an event, state, disorder or condition, for example for the purpose of reducing the chance of an event, state, disorder or condition occurring. The outcome of the prophylaxis may be, for example, preservation of health or delaying the initiation and/or progression of an event, state, disorder or condition. It will be recalled that, in any individual patient or even in a particular patient population, a treatment may fail, and this paragraph is to be understood accordingly.

[0026] The term “inhibit” (and “inhibiting”) includes reference to delaying, stopping, reducing the incidence of, reducing the risk of and/or reducing the severity of an event, state, disorder or condition. Inhibiting an event, state, disorder or condition may therefore include delaying or stopping initiation and/or progression of such, and reducing the risk of such occurring. The products of the disclosure may be used to inhibit one or more metallo-B-lactamases (MBLs) and thereby aid in clearing bacterial infection and/or other events, disorders and/or conditions which are disclosed herein. For example, the compounds of the invention may release an MBL inhibitor.

[0027] An “inhibitor” is a molecule that binds to an enzyme and decreases its activity. An “irreversible inhibitor” is an inhibitor where the binding involves a chemical reaction, e.g. formation of a covalent bond between the molecule and enzyme. The compounds of the invention may release an MBL inhibitor.

[0028] A “metallo-B-lactamase enzyme” (MBL) is an enzyme that catalyzes the hydrolysis of a broad range of B-lactam drugs including carbapenems and cephalosporins. Exemplary MBL enzymes include BCI, CcrA, IMP, VIM, SPM, GIM, NDM, CphA, ImiS, L1 and FEZ. A more complete list of MBLs can be found in [doi:10.1128/AEM.00698-18]. Active MBL enzymes typically require one or two zincs in the active site. The compounds of the invention represent prodrugs which may be hydrolysed by an MBL, releasing an inhibitor that binds the zinc ion(s) in the active site of an MBL, thereby inhibiting the MBL.

[0029] A “B-lactam antibacterial agent” or ““B-lactam antibiotic” is an antibiotic agent that comprises a beta-lactam ring in its molecular structure. Exemplary p-lactam antibacterial agents include carbapenems, cephalosporins, carbacephems and penicillins such as ureidopenicillins. Non limiting examples of suitable B-lactam antibacterial agents include carbapenems (e.g. meropenem, faropenem, imipenem, ertapenem, doripenem, panipenem/betamipron and biapenem as well as razupenem, tebipenem, lenapenem and tomopenem), ureidopenicillins (e.g. piperacillin), carbacephems (e.g. loracarbef) and cephalosporins (e.g. cefpodoxime, ceftazidime, cefotaxime, ceftriaxone, ceftobiprole, and ceftaroline). Further examples of suitable B-lactam antibacterial agents include temocillin, piperacillin, cefpodoxime, ceftazidime, cefotaxime, ceftriaxone, meropenem, faropenem, imipenem, loracarbef, ceftobiprole and ceftaroline.

[0030] The terms “alkyl” as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The term includes reference to, for example, methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec- butyl or tert-butyl), pentyl, hexyl and the like. In particular, alkyl may be a “C4-Cs alkyl”, i.e. an alkyl having 1, 2, 3 or 4 carbon atoms; or a “C+-Cs alkyl”, i.e. an alkyl having 1, 2, 3,4, 5 or 6 carbon atoms; or a “C+-Cs alkyl”, i.e. an alkyl having 1, 2 or 3 carbon atoms. The term “lower alkyl” includes reference to alkyl groups having 1, 2, 3 or 4 carbon atoms.

[0031] The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkyl, as exemplified, but not limited, by -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[0032] The term “cycloalkyl” as used herein includes reference to an alicyclic moiety having 3, 4, 5 or 6 carbon atoms. The group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

[0033] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of at least one carbon atoms and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH- CH2-0-CHs3, -CH2-CH2-NH-CHa, -CH2-CH2-N(CH3)-CHs3, -CH2-S-CH2-CHs, -CH2-CH2,-S(O)- CHs, -CH2-CH2-S(0)2-CHs, -CH=CH-O-CH3, -Si(CHs)s, -CH2-CH=N-OCH3, -CH=CH- N(CHs3)-CHs3, O-CHs, -0-CH2-CHs and —CN. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH: and —CH>-O-Si(CHs)s. Similarly, the term

“heteroalkylene” by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CHz- and —CH2-S- CH2-CH2-NH-CHz-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O):R’- represents both -C{O)sR’- and —-R’C(0)z-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R’, -C(O)NR’, -NR'R’, -OR’, -SR, and/or -SO2R'. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R” are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR’R’” or the like.

[0034] The term "heterocycloalkyl" as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, axygen, phosphorus and sulphur. For example, a heterocycloalkyl may comprise 3, 4, or 5 ring carbon atoms and 1 or 2 ring heteroatoms selected from nitrogen and oxygen. The group may be a polycyclic ring system but more often is monocyclic. This term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolizidinyl and the like.

[0035] The terms “halo” or “halogen" as used herein includes reference to F, Cl, Br or |, for example F, Cl or Br. In a particular class of embodiments, halogen is F or Cl, of which F is more common.

[0036] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “haloalkyl” refers to an alkyl group where one or more hydrogen atoms are substituted by a corresponding number of halogens. For example, the term “halo(C:+- C4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like.

[0037] The term “alkoxy” as used herein include reference to -O-alkyl, wherein alkyl is straight or branched chain and comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1, 2, 3 or 4 carbon atoms, e.g. 1, 2 or 3 carbon atoms. This term includes reference to, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert- butoxy, pentoxy, hexoxy and the like. The term “lower alkoxy” includes reference to alkoxy groups having 1, 2, 3 or 4 carbon atoms.

[0038] The term “haloalkoxy” as used herein refers to an alkoxy group where one or more hydrogen atoms are substituted by a corresponding number of halogens.

[0039] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) which are fused together or linked covalently. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4- oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5- thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 8-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. “Arylene” and “heteroarylene” refers to a divalent radical derived from a aryl and heteroaryl, respectively.

[0040] Each of the above terms (e.g., “alkyl,” “cycloalkyl,” “heteroalkyl,” “aryl” and “heteroaryl”}, unless otherwise noted, are meant to include both substituted and unsubstituted forms of the indicated radical. Where a substituent is R-substituted (e.g. an R*substituted alkyl, where “X” is an integer), the substituent may be substituted with one or more R groups as allowed by chemical valency rules where each R group is optionally different (e.g. an R*substituted alkyl may include multiple R* groups wherein each R* group is optionally different). Certain examples of substituents for each type of radical are provided below.

[0041] The term “substituted” as used herein in reference to a moiety means that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described substituents. Unless otherwise specified, exemplary substituents include —OH, -CN, -NHz, =O, -halo, -C+-Cs alkyl, -C2-Cs alkenyl, -C+-Ce haloalkyl, -C+-Cs haloalkoxy and-C2-Ce haloalkenyl, -C+-Cs alkylcarboxylic acid (e.g. -CH3COOH or -COOH). Where the substituent is a -C+-Cs alkyl or -C+-Cs haloalkyl, the C4-Cs chain is optionally interrupted by an ether linkage (-O-) or an ester linkage (-C(O)O-). Exemplary substituents for a substituted alkyl may include —OH, -CN, -NH>, =O, -halo, -CO:H, -C+-Cs haloalkyl, -C1-Cs haloalkoxy and-Cz-Cshaloalkenyl, -C+-Cs alkylcarboxylic acid (e.g. -CH3sCOOH or -COOH). For example, exemplary substituents for an alkyl may include —OH, -CN, -NHz, =O, -halo.

[0042] It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible. For example, amino or hydroxy groups with free hydrogen may be unstable if bound to carbon atoms with unsaturated (e.g. olefinic) bonds. Additionally, it will of course be understood that the substituents described herein may themselves be substituted by any substituent, subject to the aforementioned restriction to appropriate substitutions as recognised by the skilled person.

[0043] Where steric issues determine placement of substituents on a group, the isomer having the lowest conformational energy may be preferred.

[0044] Where a compound, moiety, process or product is described as “optionally” having a feature, the disclosure includes such a compound, moiety, process or product having that feature and also such a compound, moiety, process or product not having that feature. Thus, when a moiety is described as “optionally substituted”, the disclosure comprises the unsubstituted moiety and the substituted moiety.

[0045] Where two or more moieties are described as being “independently” or “each independently” selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties.

[0046] The term “pharmaceutically acceptable” as used herein includes reference to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This term includes acceptability for both human and veterinary purposes.

[0047] The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids ar bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0048] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.

The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0049] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention.

Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0050] Certain compounds of the present invention possess asymmetric carbon atoms (optical centres) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those which are known in the art to be too unstable to synthesize and/or isolate.

[0051] The symbol denotes a point of attachment of a moiety to the remainder of a compound.

[0052] The term “prodrug” as used herein represents compounds which are transformed in vivo to the parent compound or other active compound, for example, by hydrolysis in blood. An example of such a prodrug is a pharmaceutically acceptable ester of a carboxylic acid. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed, Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; H Bundgaard, ed, Design of Prodrugs, Elsevier, 1985; and Judkins, et al. Synthetic Communications, 26(23), 4351-4367 (1996); and The organic chemistry of drug design and drug action by Richard B Silverman in particular pages 497 to 546; each of which is incorporated herein by reference. Compounds of the invention may represent prodrugs (e.g. comprising a latent MBL inhibitor), where hydrolysis of a B- lactam results in release and activation of the latent MBL inhibitor.

[0053] The term “pharmaceutical formulation” as used herein includes reference to a formulation comprising at least one active compound and optionally one or more additional pharmaceutically acceptable ingredients, for example a pharmaceutically acceptable carrier. Where a pharmaceutical formulation comprises two or more active compounds, or comprises at least one active compound and one or more additional pharmaceutically acceptable ingredients, the pharmaceutical formulation is also a pharmaceutical composition. Unless the context indicates otherwise, all references to a “formulation” herein are references to a pharmaceutical formulation.

[0054] The term “product” or “product of the invention” as used herein includes reference to any product containing a compound of the present invention. In particular, the term product relates to compositions and formulations containing a compound of the present invention, such as a pharmaceutical composition, for example.

[0055] The term “therapeutically effective amount” as used herein refers to an amount of adrug, or pharmaceutical agent that, within the scope of sound pharmacological judgment, is calculated to (or will) provide a desired therapeutic response in a mammal (animal or human). The therapeutic response may for example serve to cure, delay the progression of or prevent a disease, disorder or condition.

COMPOUNDS

[0056] In one aspect, the invention provides compounds of formula | as previously described or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

[0057] A may be a cephalosporin moiety or a carbapenem moiety. A may be a cephalosporin moiety. A may be a carbapenem moiety.

[0058] A may be:

Ry

JN © ©

Z R+ is substituted or unsubstituted -C+-Csalkyl aryl, substituted or unsubstituted -C+-C4alkyl cycloalkyl, substituted or unsubstituted -C+-Caalkyl heteroaryl, substituted or unsubstituted -C4-Csaminoalkyl aryl, substituted or unsubstituted -C:-C4aminoalkyl cycloalkyl, substituted or unsubstituted -C+-C4aminoalkyl heteroaryl; said optional substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, NHz, NH(C+-C:2 alkyl), N(C+-C2 alkyl)z, methoxy, ethoxy. Y is -S-, -S(O)-, -S(0O)2, -O-, or -CHz-. Z is -C(O)OH, -C(O)O(C+- Ca, alkyl), tetrazole, hydroxamic acid, amide, sulfonamide, phosphate, or sulfate.

[0059] R: may be a substituted or unsubstituted -C+-Caalkyl aryl. Ry may be a substituted or unsubstituted -C+-C4alkyl cycloalkyl. Ry may be a substituted or unsubstituted -C:-C4alkyl heteroaryl. Ri may be a substituted or unsubstituted -C+- C4aminoalkyl aryl. Ry may be a substituted or unsubstituted -C:-C4aminoalkyl cycloalkyl. R1 may be a substituted or unsubstituted -C:-C4aminoalkyl heteroaryl. When substituted, the substituents may be independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy. Ri may be -C+-C4alkyl aryl, -C+-Caalkyl cycloalkyl, -C4-Caalkyl heteroaryl, -C+- C4aminoalkyl aryl, -C1-C4aminoalkyl cycloalkyl, -Cs-Csaminoalkyl heteroaryl. Ri may be - C+-Caalkyl aryl. Ry may be -C4-C4alkyl cycloalkyl. Ri may be -C4-Caalkyl heteroaryl. Rj may be -Ci-Csaminoalkyl aryl. R1 may be -C+-C4aminoalkyl cycloalkyl. R1 may be -C+- C4aminoalkyl heteroaryl. Ry may be -C4-Caalkyl phenyl. R+ may be phenacyl.

[0060] Y may be -S-, -S(O)-, -S(0)2, -O-. Y may be -S-, -S(O)- or -S(O)2. Y may be -S-. Y may be -S(O)-. Y may be -S(O)2. Y may be -O-. Y may be -CH-.

[0061] Z may be -C(O)OH, -C(O)O(C+-Cs alkyl), or tetrazole. Z may be -C(O)OH, - C(O)O(C:-Ca alkyl). Z may be -C(O)OH. Z may be -C{O)O(C+-C4 alkyl), e.g. -C(O)OCH:. Z may be tetrazole. Z may be hydroxamic acid. Z may be amide. Z may be sulphonamide. Z may be phosphate. Z may be sulfate.

[0062] B may be a a latent MBL inhibitor and / or B may represent a latent Zn2* chelator.

[0063] B may be:

0 ORs TX R2 Rs Rs R4 Xi is selected from -S-, -O-, -OC(O)-, -S(O)- and -S(O)2-. X: may be -S- or -O-. X: may be -S-. Xi may be -O-. Xi may be -OC(O)-. X; may be -S(O)-. Xi may be -S(O)--.

[0064] Each of Rs, Rs, Rs and Rs are independently selected from hydrogen, halo, cyano, nitro, hydroxy, substituted or unsubstituted (C:-C4)alkyl, substituted or unsubstituted aryl, said one or more optional substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, sulfonamide, NH», NH(C+-C- alkyl), N(C+-C: alkyl)., methoxy, ethoxy, or aryl. Said optional substituents may be independently selected from oxo, halo, cyano, nitro, hydroxy, or carboxy. Each of Rz, Rs, Rs and Rs may be independently selected from hydrogen, halo, or (C:-C4)alkyl. At least 2 of R2, Rs, Rs and Rs may be hydrogen. At least three of R2, Rs, Rs and Rs may be hydrogen. Each of R2, Rs, Rs and Rs may be hydrogen.

[0065] Rs is selected from H and (C+-Ca)alkyl. Rs may be H. Rs may be (C+-C4)alkyl.

[0066] In an embodiment, X: may be -S- or -O- (e.g. -S-), Rs may be H and at least 2 (e.g. 3 or 4) of Rz, R3, Rs and Rs may be hydrogen.

[0067] B may be: R7

R Ar 8

A AN DL Ys Riz R11 Xz is selected from -S-, -OC{(O)-, -S(O)- and -S(O)2-. Xz may be -S-. Xz may be -OC(O)-. Xz may be -S(O)-. X2 may be -S(O)z-.

[0068] Y: is selected from -N=, or -C{Rs)=. Y: may be -N=. Y: may be -C(Rg)=. Y2is selected from -N=, or -C(R19)=. Y2 may be -N=. Y2 may be -C(R+)=. For example, Y+ may be -C(Rs)= and Y2 may be -C(R+9)=.

[0069] Each of Rs, Rs, Rs, R+, R11 and R42 are independently selected from hydrogen, halo, cyano, nitro, hydroxy, substituted or unsubstituted (C+-Ca)alkyl, substituted or unsubstituted aryl, said one or more optional substituents independently selected from

0x0, halo, cyano, nitro, hydroxy, carboxy, sulfonamide, NH», NH{C+4-C:2 alkyl), N(C+-C> alkyl)2, methoxy, ethoxy, or aryl. Said optional substituents may be independently selected from oxo, halo, cyano, nitro, hydroxy, or carboxy. Each of R7, Rs, Re, Rig, Rts and Riz may be independently selected from hydrogen, halo, or {C+1-C4)alkyl. At least 2, 3, or 4 of Ry, Rs, Re, Rig, R11 and R12 may be hydrogen. Rs and Rig may be hydrogen. Ry, Rs, R+: and R42 may be hydrogen. Each of Ry, Rs, Re, R+, R11 and R42 may be hydrogen.

[0070] B may be: Dd

IS | Ris | Xs is selected from -S-, -O-, -OC(O)-, -S(O)- and -S(O)z-. Xs may be -S- or -O-. Xz may be -S-. Xs may be -O-. X3 may be -OC(O)-. Xs may be -S(O)-. Xs may be -S(O)z-.

[0071] Y: is selected from -N=, or -C{R+4)=. Y3 may be -N=. Ys may be -C(R1)=. Ya is selected from -N=, or -C(R15)=. Y4 may be -N=. Y4 may be -C(R+4)=. For example, Y3 may be -C(R14)= and Y4 may be -C(R1s)=.

[0072] R:: is selected from H and (C+-Cs)alkyl. Riz may be H. Ris may be (C+-Cs)alkyl.

[0073] Each of R+, Ris and Rss are independently selected from hydrogen, halo, cyano, nitro, hydroxy, substituted or unsubstituted (C+-Ca)alkyl, substituted or unsubstituted aryl, said one or more optional substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, sulfonamide, NH», NH(C+-C- alkyl), N(C+-C: alkyl)., methoxy, ethoxy, or aryl. Said optional substituents may be independently selected from oxo, halo, cyano, nitro, hydroxy, or carboxy. Each of Rs, Ris and Ris may be independently selected from hydrogen, halo, or (C:-Ca)alkyl. At least 2 of R14, Ris and Ris may be hydrogen. Ris and Ris may be hydrogen. Ris may be hydrogen. Each of R+4, Ris and Ris may be hydrogen.

[0074] B may be: R17 Ris HN Ris 0 x R20 Oo R21

[0075] Each of R+, Ris, Ris, R2o and Ra: are independently selected from hydrogen,

halo, cyano, nitro, hydroxy, substituted or unsubstituted (C1-C4)alkyl, substituted or unsubstituted aryl, said one or more optional substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, sulfonamide, NH2, NH(C+-C; alkyl), N(C+4-C2 alkyl)2, methoxy, ethoxy, or aryl. Said optional substituents may be independently selected from oxo, halo, cyano, nitro, hydroxy, or carboxy. Each of R47, Ris, Rig, R2o and R21 may be independently selected from hydrogen, halo, or (C:-C4)alkyl. At least 2 (e.g. at least 3) of R17, Ris, Rig and R2o may be hydrogen. Each of R+7, Ris, Rie and R2o may be hydrogen. R21 may be a substituted or unsubstituted aryl. Raz; may be an aryl substituted by a sulphonamide; e.g. aryl-S(0O)2NR2R2s. Each of R24 and Rss are selected from H and (C:- Cyalkyl. For example each of R24 and Ras may be (C4-Cq)alkyl, e.g. methyl.

[0076] B may be: Ras

A

S “ HN R22

I R22 is an aryl or heteroaryl, optionally substituted by one or more substituents independently selected from hydrogen, halo, cyano, nitro, hydroxy, NHC(O)H, NHC(O) (C+-Cyalkyl, and (C+-C4)alkyl optionally substituted by one or more substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, NHz, NH(C+-C; alkyl), N{C+-C: alkyl), methoxy, ethoxy. R22 may be an optionally substituted aryl. R22 may be a para substituted aryl. The substituent may be NHC(O)H or NHC(O) (C+-Ca)alkyl; for example the substituent may be NHC(O) (C+-C4)alkyl (e.g. NC(O)CHa.

[0077] Rais selected from hydrogen, halo, cyano, nitro, hydroxy, substituted or unsubstituted (C+-C4)alkyl, substituted or unsubstituted aryl, said one or more optional substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, sulfonamide, NHz, NH(C+-C: alkyl), N(C+-C- alkyl), methoxy, ethoxy, or aryl. Said optional substituents may be independently selected from oxo, halo, cyano, nitro, hydroxy, or carboxy. Rss may be selected from hydrogen, halo, or (C:-C4)alkyl. R23 may be hydrogen.

[0078] B may be selected from:

R7 2 | Rg R130 0 R17 Rus

S R og Ro Nl 1 HN Rig 0 | os oO na Riz Rio ks Ris ht Roo R14 i O Rie , oO Ro , and Ros

A S AN

R 0 HN—¢” 2 No | 0 , or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0079] B may be selected from: R7 w= | Rg R130 0 Riz Ris

S R Ny Rg NEN 1 HN Rig O | 8 0 SN. R42 Rio ht Ris he Roo R1 , © Rie and O R21 ‚ or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0080] B may be selected from: R7 R R430 © NA | 8 13

S R ny Ry AN 14 © 3 Riz Rio ht Ris R11 and © Rie , or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0081] In an embodiment, the compound is selected from:

FTN FN i yn, i N, en, x f N NX 7 \ Nan 27 NH 3 OQ, OH nd . or NH 8 EEN OTN ö TY ToL bd & N EN 5 , NN > N ~. Sa 3 ~ ee Le 0 Ho G | bo oA u LL ed wl Ng O07 "OH a 07 OH EE

H Ory

NEN O ~ 1B NLs Cotas 0 i A 2 FN HOO B Ng, é 3 a } 5 0 OH yy 0 Sed AN A FEN if . DENN i 7 Q rt 5 1 is f NF a N a a ~ a 7 EN Oo A ©” OM ’ 5 © LER ein hy HN NX © HN NN i Q ! Ne 0 EN TD AND a N ps Qo Ey 0 Nyy ~ 0 ro a i 0 OH ij & BR Oo OH { - i IS = oF & A Nar or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0082] In embodiments, the compounds of the invention represent prodrugs which may be hydrolysed by an MBL, releasing an MBL inhibitor, e.g. a zinc chelator that binds the zinc ion(s) in the active site of an MBL, thereby inhibiting the MBL.

[0083] Exemplary compounds may be made in accordance with the methods of synthesis illustrated in the examples. In addition, as the skilled person will appreciate, these methods and the methods illustrated in the reaction schemes illustrated in figures 4 to 8 may be readily adapted to provide other compounds of the present disclosure.

USES

[0084] Compounds provided herein represent prodrug of inhibitors of MBL. These prodrugs represent molecules with two moieties, an MBL inhibitor moiety and a B-lactam moiety. The MBL inhibitor moiety is linked to a B-lactam moiety, e.g. cephalosporin or carbapenem as indicated in compounds of the invention and disclosure. Upon hydrolysis of the beta-lactam core by beta-lactamase enzymes, specifically metallo-beta-lactamases,

a bond rearrangement leads to the release of the active MBL inhibitor, as illustrated in figures 2 and 3. Formation of the hydrolysed B-lactam byproduct and the release of the MBL inhibitor have been confirmed for exemplary compounds by NMR (detection of the vinylic CHz specific to the B-lactam byproduct) as well as by MS (direct detection of ions with the expected mass of pseudomolecular ions of both the B-lactam byproduct and released MBL inhibitor).

[0085] In the prodrug state the latent MBL inhibitor is linked (e.g. covalently) to a B- lactam moiety via a functional group essential for its activity. In this way the conjugated MBL inhibitors are inactive until released, for example because a covalent bond between the functional group and a B-lactam moiety inactivates the functional group. The advantage of this prodrug strategy is local release of the inhibitor at the active site of the target enzyme, as the MBL inhibitor is in its latent (prodrug) form until it is released by hydrolytic activity of the MBL on the B-lactam moiety.

[0086] Many MBL inhibitors operate by binding with zinc ion(s) present in the active site of the MBLs. These MBL inhibitors either strip the zinc ions from the active site or bind tightly to the zinc ions rendering the MBLs inactive. The use of MBL inhibitors that utilize zinc binding motifs have been described elsewhere (see, for example, WO 2014/198849 A1, WO 2017/093727 A1). As zinc is an essential metal in many processes and enzymes throughout the human body, systemic exposure to potent zinc chelating agents can be expected to lead to a large variety of off target effects. In contrast to the known agents, which provide systemic exposure to active inhibitors, the approach described here provides the release of MBL inhibitors, including those with zinc chelating motifs, only at the target MBL enzyme.

[0087] Without wishing to be bound by any theory, it is believed that another advantage of the prodrug strategy provided by the present compounds it that it can allow the use of certain MBL inhibitor moieties that might be rapidly inactivated if directly administered as active compounds. For example, the present approach may be compatible with the use of thiol containing compounds, such as the compound present in example 2, that would otherwise readily form disulfides, inactivating the inhibitor before reaching its target.

[0088] Compounds and formulations of the invention are accordingly useful as medicaments. For example, a compound or formulation of the invention may be provided for use as a medicament.

[0089] A compound or formulation of the invention may be used in the treatment of antibiotic resistance. The treatment may further comprise the administration of another active agent. In this context, the other active agent may itself be a prodrug (i.e. a compound transformed in vivo to the parent compound or other active compound), or the other active agent may be active in the form it is administered. The other active agent may be a a p-lactam antibacterial agent or analogue thereof. Where the treatment comprises the administration of another active agent, the treatment may be by combined, sequential or separate administration of the present compound or formulation and the other active agent. For example, the treatment may comprise combined administration of the present compound and the other active agent. For example, the treatment may comprise administration of the present compound followed by administration of the other active agent.

[0090] A compound or formulation of the invention may be used in the treatment of a bacterial infection. The treatment may further comprise the administration of another active agent. In this context, the other active agent may itself be a prodrug (i.e. a compound transformed in vivo to the parent compound or other active compound), or the other active agent may be active in the form it is administered. The other active agent may be a a B-lactam antibacterial agent or analogue thereof. Where the treatment comprises the administration of another active agent, the treatment may be by combined, sequential or separate administration of the present compound or formulation and the other active agent. For example, the treatment may comprise combined administration of the present compound and the other active agent. For example, the treatment may comprise administration of the present compound followed by administration of the other active agent.

[0091] Also provided is a method of of treating a bacterial infection in a patient, comprising administering to the patient an effective amount of a compound of the invention or formulation of the invention. The method may further comprise the administration of another active agent. In this context, the other active agent may itself be a prodrug (i.e. a compound transformed in vivo to the parent compound or other active compound), or the other active agent may be active in the form it is administered. The other active agent may be a B-lactam antibacterial agent or analogue thereof. Where the treatment comprises the administration of another active agent, the treatment may be by combined, sequential or separate administration of the present compound or formulation and the other active agent. For example, the method may comprise combined administration of the present compound and the other active agent. For example, the method may comprise administration of the present compound followed by administration of the other active agent.

[0092] Compounds of the invention administered in combination with appropriate B- lactam antibiotics can be applied for the treatment of bloodstream infections, central nervous system infections, respiratory tract infections, complicated skin/skin structure infections, complicated intra-abdominal infections and complicated Urinary-tract Infections.

[0093] ‘Where the compounds are used fur the treatment of a bacterial infection, the bacterial infection may be caused by Gram-negative or Gram-positive bacteria. For example, the bacterial infection may ba caused by bacleria from ons or more {(2.¢g. atleast one) of the foliowing families: Clostridium, Pseudomonas, Escherichia, Klebsielia, Enterococcus, Enterobacter, Serratia, Stenotrophomonas, Aeromonas, Morganells, Yersinia, Salmonella, Proteus, Pasteurella, Haemophilus, Citrobacter, Burkholderia, Brucella, Moraxella, Mycobacterium, Streptococcus or Staphviococcus. Particular examples include Clostridium, Pseudomonas, Escherichia, Klebsielia, Enterococcus, Enterobacter, Streptococcus and Siaphylococous. The bacterial infection may, for example, be caused by one or more bacteria selected from Moraxsita catarrhalis, Brucella abortus, Burkholderia cepacia, Citrobacter species, Escherichia coli, Haemophilus Frisumonia, Kiebsiella Pneumonia, Pasteurella multocida, Proteus mirabilis, Salmonella typhimurium, Clostridium difficile, Yersinia enterocolitica Mycobacterium tuberculosis, Staphylococcus aureus, group B streptococcal, Streptococcus Fneumonia, and Streptococcus pyogenas, e.g. from E coli and K, pneumoniae.

[0094] Methods are provided for inhibiting bacterial metallo-B-lactamase in vitro or in vivo, comprising administration to a cell of an effective amount of a compound of the invention or formulation of the invention. a compound of the invention or formulation of the invention for the inhibition of a bacterial metallo-B-lactamase.

[0095]

FORMULATIONS AND ADMINISTRATION

[0096] Compounds of the invention may be administered orally, topically, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation. The compounds may be administered in the form of pharmaceutical preparations comprising the compound either as a free compound or, for example, a pharmaceutically acceptable non-toxic organic or inorganic acid or base addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.

[0097] Typically, therefore, the pharmaceutical compounds of the invention may be administered orally, topically, or parenterally (“parenterally” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion) to a host to obtain a protease-inhibitory effect. In the case of larger animals, such as humans, the compounds may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers.

[0098] Actual dosage levels of active ingredients in the pharmaceutical formulations and pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0099] In the treatment, prevention, control, amelioration, or reduction of risk of conditions which require inhibition of MBL activity, an appropriate dosage level may generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. The dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. For oral administration, the compositions may be provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0,

75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0 and

1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, e.g. once or twice per day. The dosage regimen may be adjusted to provide the optimal therapeutic response.

[00100] According to a further aspect of the invention there is thus provided a pharmaceutical formulation or composition including a compound of the invention, optionally in admixture with a pharmaceutically acceptable adjuvant, diluents or carrier.

[00101] Pharmaceutical formulations or compositions of this invention for parenteral injection may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[00102] These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Inhibition of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol or phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars or sodium chloride, for example.

Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents (for example, aluminium monostearate and gelatine) which delay absarption.

[00103] In some cases, in arder to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[00104] Injectable depot forms may be made by forming microencapsule matrices of the drug in biodegradable polymers, for example polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.

[00105] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants, such as glycerol; d} disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds; 9) wetting agents, such as cetyl alcohol and glycerol monostearate; h) absorbents, such as kaolin and bentonite clay and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.

[00106] Oral formulations may contain a dissolution aid. Examples of dissolution aids include nonionic surface active agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g. sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and alkyamine oxides; bile acid and salts thereof (e.g. chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and salts thereof, and glycine or taurine conjugate thereof); ionic surface active agents, such as sodium laurylsulfate, fatty acid soaps, alkylsufonates, alkylphosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface active agents, such as betaines and aminocarboxylic acid salts.

[00107] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and/or in delayed fashion. Examples of embedding compositions include polymeric substances and waxes.

[00108] The active compounds may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[00109] The active compounds may be in finely divided form, for example it may be micronized.

[00110] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,

propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and traganacanth and mixtures thereof.

[00111] Compositions for rectal or vaginal administration may be in the form of suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[00112] Dosage forms for topical administration of a compound of this invention include powders, sprays, creams, foams, gels, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

[00113] Insofar as they do not interfere with the activity of the compounds, the formulations according to the present subject matter may contain other active agents intended, in particular, for use in treating a bacterial infection. Exemplary other active agents include, without limitation, a 3-lactam antibacterial agent or analogue thereof, such as carbapenems, cephalosporins, carbacephems, penicillins (e.g. ureidopenicillins). Non limiting examples of suitable B-lactam antibacterial agents include carbapenems (e.g. meropenem, faropenem, imipenem, ertapenem, doripenem, panipenem/betamipron and biapenem as well as razupenem, tebipenem, lenapenem and tomopenem), ureidopenicillins (e.g. piperacillin), carbacephems (e.g. loracarbef) and cephalosporins (e.g. cefpodoxime, ceftazidime, cefotaxime, ceftriaxone, ceftobiprole, and ceftaroline). Specific examples of suitable B-lactam antibacterial agents include, for example, temocillin, piperacillin, cefpodoxime, ceftazidime, cefotaxime, ceftriaxone, meropenem, faropenem, imipenem, loracarbef, ceftobiprole and ceftaroline.

[00114] The formulations according to the present subject matter may also contain inactive components. Suitable inactive components are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological

Bases of Therapeutics, 8"Ed., Gilman et al, Eds. Pergamon Press (1990), and Remington’s Pharmaceutical Sciences, 17! Ed., Mack Publishing Co., Easton, Pa. (1990), both of which are incorporated by reference herein in their entirety.

[00115] The formulations may be used in combination with an additional pharmaceutical dosage form to enhance their effectiveness in treating any of the disorders described herein. In this regard, the present formulations may be administered as part of a regimen additionally including any other pharmaceutical and/or pharmaceutical dosage form known in the art as effective for the treatment of any of these disorders.

ASSAYS

[00116] Compounds of the invention can be assessed for biological activity using any suitable assay that would be known to the person skilled in the art. Exemplary assays that are useful for the assessment of compounds of the invention are provided in the following paragraphs. In the assays described below, it will be appreciated that the compound to be tested (e.g. a compound of the invention) may be referred to as a “compound” or “(pro- drug) inhibitor”.

[00117] Enzyme inhibition assay A. Compounds of the invention were tested for their inhibitory activity against representative MBLs including NDM-1, VIM-2 and IMP-28 using the chromogenic substrate nitrocefin (S.S. van Berkel, et al., J. Med. Chem., 2013, 56 (17), 6945-6953). Assay buffer was 50 mM HEPES pH 7.2, supplemented with 1 pM ZnSO4 and 0.01% triton X-100. Briefly, on a flat-bottom polystyrene 96-well microplate NDM-1 (6 nM), VIM-2 {8 nM) or IMP-28 (1 nM) were incubated with various concentrations of test compounds for 15 min at 25 °C. Nitrocefin (10 pM, —2xKm) was added to the wells and absorption at 492 nm was immediately monitored on a TECAN Spark microplate reader over 30-40 cycles. The initial velocity data were used for ICsp curve-fitting using GraphPad Prism 7. All the compounds were tested in 3 independent replicates.

[00118] Enzyme inhibition assay B. Compounds of the invention were tested for their inhibitory activity against representative MBLs including NDM-1, VIM-2 and IMP-28 using the fluorescent substrate FC-5 (S.S. van Berkel, et al, J. Med. Chem., 2013, 56 (17), 6945-6953). Assay buffer was 50 mM HEPES pH 7.2, supplemented with 1 pM ZnSO4 and 0.01% triton X-100. Briefly, on a black half-area flat-bottom polystyrene 96-well microplate NDM-1 (50 pM}, VIM-2 {100 pM) or IMP-28 (60 pM) were incubated with various concentrations of test compounds for 15 min at 25 °C. FC-5 (Kn-concentrations of

0.5 uM for NDM-1 and VIM-2 and 16 uM for IMP-28) was added to the wells and fluorescence (Aex = 380 nm and Aem = 460 nm) was immediately monitored on a TECAN Spark microplate reader over 30-40 cycles. The initial velocity data were used for ICso determination. It is preferable to test compounds with enzyme inhibition assay B, rather than enzyme inhibition assay A, as assay B requires the use of lower amounts of enzyme and substrate.

[00119] Determination of minimum inhibitory concentration (MIC). Compounds of the invention were tested for their antibacterial activity using broth microdilution method and following the clinical and laboratory standards institute (CLSI) guidelines. The detailed procedure can be found in K.H.M.E. Tehrani and N.I. Martin, ACS Infect Dis. 2017 Oct 13;3(10):711-717, in particular in the section on page 714 titled “MIC Determinations and Synergy Assays”.

[00120] /n vitro synergy assay. Following the same general procedure for MIC determination described above, on a polypropylene 96-well plate, 25 uL of selected B- lactam antibiotics were added via serial dilution. Then 25 pL of the test compounds were added to all the wells followed by adding 50 uL of bacterial suspension. Thus, MIC of the B-lactam antibiotics was determined in the presence and absence of test compounds. To determine a synergistic relationship between B-lactam antibiotics and the test compounds, fractional inhibitory concentration index was calculated using the following formula: MIC of test compound in combination MIC of antibiotic in combination FICI = + _ MIC of test compound in alone MIC of antibiotic alone FICI < 0.5 was considered to indicate a synergistic relationship.

EXAMPLES

[00121] Example 1: Synthesis of 6R,7R}-3-(((2-carboxyphenyl}thiojmethyl)-8-ox0-7- (2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

H N S O. „OH 6 ALs L © | Oo“ OH F# The reaction scheme followed for the synthesis of this compound is illustrated in figure 4.

[00122] (6R,7R)-3-(acetoxymethyl}-8-oxo-7-(2-phenylacetamido)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (1.28 mmol, 500 mg, 1 eq) and sodium hydrogen carbonate (1.28 mmol, 108 mg, 1 eq) were added to 10 mL of water and stirred to make a clear solution. Thiosalicylic acid (1.28 mmol, 198 mg, 1 eq) was then added and the mixture was heated at 65 °C for 6 h during which pH was maintained neutral by dropwise addition of 1 M NaOH. The reaction mixture was then cooled to room temperature and washed with ethyl acetate (2x30 ml). The aqueous phase was acidified to pH 3 by 1 M HCI, extracted with ethyl acetate (3x30 mL}, washed with brine, dried and concentrated under vacuum. The residue was solidified after adding a few drops of methanol, then filtered and recrystallized from water/methanol to afford 258 mg of a pale yellow solid (Yield: 42%). 1H NMR (DMSO-d6): ò 9.07 (1H, d, J = 8.4 Hz, aromatic H),

7.84 (dd, J =7.8, 1.6 Hz, 1H), 7.47 (m, 1H), 7.38 (d, J = 8.0, 1H), 7.18-7.28 (m, 5H), 5.61 (dd, J=8.4,4.7 Hz, 1H), 5.07 (d, J = 4.8 Hz, 1H), 4.09 (d, J = 11.8 Hz, 1H), 3.98 (d, J =

11.8 Hz, 1H), 3.68 (d, J = 17.8 Hz, 1H), 3.44-3.55 (m, 3H).

[00123] Example 2: Synthesis of (6R,7R)-8-ox0-7-(2-phenylacetamido}-3-((quinolin-8- ylthio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid Cry ts N= S oO 0” "OH The reaction scheme followed for the synthesis of this compound is illustrated in figure 5.

[00124] Intermediate 2: 4-methoxybenzyl (BR,7 R)-8-0x0-7-(2-phenylacetamido)-3- {(quinolin-8-ylthioymethyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 0] 0 Oo OD, 4-methoxybenzyl (6R,7 R)-3-(chloromethyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1- azabicyclo-[4.2.0]oct-2-ene-2-carboxylate (GCLE, 486 mg, 1.0 mmol, 1 eq) was dissolved in DMF (5 mL) and sodium iodide (165 mg, 1.1 mmol, 1.1 eq) is added. After stirring for 1 hour at room temperature, 8-thioquinoline hydrochloride (238 mg, 1.2 mmol, 1.2 eq) and sodium bicarbonate (202 mg, 2.4 mmol, 2.4 eq) were added and the mixture was stirred at room temperature for 4 hours. The mixture was partitioned between DCM and water, the aqueous layer was extracted with DCM and the combined organic layers were washed with Brine, dried over sodium sulfate and concentrated. The crude product was purified by column chromatography (DCM/EtOAc 95:5 to 8:2) yielding 500 mg yellowish powder

(84%). *H NMR (400 MHz, CDCls) 8 8.95 (dd, J= 4.2, 1.3 Hz, 1H), 8.15 (dd, J=8.2, 1.5 Hz, 1H), 7.64 (d, J=8.1 Hz, 1H), 7.55 -= 7.42 (m, 2H), 7.42 7.22 (m, 7H), 6.82 (d, J= 8.5 Hz, 2H), 6.05 (d, J=9.1 Hz, 1H), 5.74 (dd, J= 9.1, 4.8 Hz, 1H), 5.15 - 4.94 (m, 2H), 4.81 (d, J=4.8 Hz, 1H), 4.45 (d, J= 12.9 Hz, 1H), 3.95 (d, J= 12.9 Hz, 1H), 3.77 (s, 3H), 3.72 (d, J=18.3 Hz, 1H), 3.69 - 3.56 (m, 2H), 3.49 (d, J= 18.3 Hz, 1H).

[00125] Intermediate 2 (273 mg, 0.45 mmol) was cooled on ice and dissolved in a mixture of TFA/anisole (5:1, 12 mL). The mixture was stirred for 30 minutes at 0°C and 30 minutes at room temperature until TLC (DCM/EtOAc 9:1) showed complete conversion. The mixture was concentrated to about 5 mL, precipitated with diethyl ether and washed with diethyl ether. The crude product was purified by preparative HPLC yielding 60 mg yellowish powder. ’H NMR (400 MHz, DMSO-ds) 6 9.11 (d, J = 8.3 Hz, 1H), 8.90 (dd, J =

4.2, 1.6 Hz, 1H), 8.39 (dd, J=8.3, 1.5 Hz, 1H), 7.78 (d, J= 7.9 Hz, 1H), 7.69 — 7.57 (m, 2H), 7.54 (t, J=7.7 Hz, 1H), 7.36 — 7.14 (m, 5H), 5.63 (dd, J= 8.3, 4.7 Hz, 1H), 5.07 (d, J = 4.8 Hz, 1H), 4.31 (d, J= 12.4 Hz, 1H), 4.11 (d, J= 12.4 Hz, 1H), 3.80-3.45 (m, 4H).

[00126] Example 3: Synthesis of (6R,7R)}-3-({{6-carboxypicolinoyl)oxy)methyl)-8-oxo- 7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

H Cry ts © ju AN O~0 0 Oo“ OOH |] ON Ss 0

OH The reaction scheme followed for the synthesis of this compound is illustrated in figure 6.

[00127] Intermediate 3: 2-(4-methoxybenzyl) 6-(((6R,7R)-2-(((4- methoxybenzyl)oxy)carbonyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct- 2-en-3-yl)methyl) pyridine-2,6-dicarboxylate

H Os © I = 0._.0 0 Oo 0 | ~N PMB x= 0 9 pMB

[00128] 4-methoxybenzyl (6R,7R)-3-{chloromethyl)-8-ox0-7-(2-phenylacetamido)-5-thia-1-

azabicyclo-[4.2.0]oct-2-ene-2-carboxylate (GCLE, 650 mg, 1.3 mmol, 1 eq) was dissolved in DMF (10 mL) and sodium iodide (220 mg, 1.45 mmol, 1.1 eq) was added. After stirring for 1 hour at room temperature, a premixed suspension of 6-(((4- methoxybenzyl}oxy)carbonyl)picolinic acid (400 mg, 1.3 mmol, 1 eq) and sodium bicarbonate (340 mg, 4 mmol, 3 eq) in DMF (5 mL) was added and the mixture was stirred overnight at room temperature. The mixture was concentrated, extracted from water with EtOAc and the combined organic phases washed with Brine, dried over sodium sulfate and concentrated. The crude product was purified by column chromatography (petroleum ether/EtOAc 1:1 to 1:3) yielding 545 mg white powder (57%).

[00129] Intermediate 3 (170 mg, 0.23 mmol) was dissolved in DCM (6 mL). Anisole (0.40 mL) was added and the mixture was cooled to 0°C. Trifluoroacetic acid (0.95 mL) was added dropwise over a period of 10 minutes and the mixture was stirred for 2 hours at 0°C. The mixture was concentrated, washed with cold diethyl ether and filtered. The crude compound was purified by preparative HPLC yielding 20 mg white powder. "H NMR (600 MHz, DMSO-ds) 6 9.16 (d, J=8.3 Hz, 1H), 8.26 (d, J=7.7 Hz, 2H), 8.20 (d, J= 7.8 Hz, 1H), 7.40 — 7.17 (m, 5H), 5.82 — 5.62 (m, 1H), 5.33 (d, J=12.7 Hz, 1H), 5.14 (d, J= 4.6 Hz, 1H), 5.08 (d, J = 12.7 Hz, 1H), 3.80 — 3.65 (m, 2H), 3.60-3.36 (m, 2H).

[00130] Example 4: Synthesis of (6R,7R)-3-(((3-(4-(N,N-dimethylsulfamoyl)phenyl)-7- isopropyl-1H-indole-2-carbonyl}oxy)methyl}-8-oxo-7-(2-phenylacetamido)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

0. 0 'S “TN ze N, H (0 Cri NLs O 0 0” "OH The reaction scheme followed for the synthesis of this compound is illustrated in figure 7.

[00131] Intermediate 4: 4-methoxybenzyl (6R,7R)-3-(((3-(4-(N,N- dimethylsulfamoyl)phenyl)-7-isopropyl-1H-indole-2-carbonyhoxy)methyl)-8-0x0-7-{2- phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate eN 0 'S

TN 2 4 A) NLs 0 0 oo PMB

[00132] 4-methoxybenzyl (6R,7R)-3-(chloromethyl}-8-oxo-7-(2-phenylacetamido)-5-thia-1- azabicyclo-[4.2.0]oct-2-ene-2-carboxylate (GCLE, 100 mg, 0.20 mmol, 1.1 eq) is dissolved in DMF (2.5 mL) and sodium iodide (34 mg, 0.23 mmol, 1.2 eq) is added. After stirring for 1 hour at room temperature, 3-(4-(N,N-dimethylsulfamoyl)phenyl)-7-isopropyl-1H-indole-2- carboxylic acid (SC-73, 70 mg, 0.18 mmol, 1 eq) and sodium bicarbonate (30 mg, 0.36 mmol, 3 eq) are added and the mixture is stirred overnight at room temperature. The mixture is filtered over cotton, concentrated and absorbed on silica. The crude product was purified by column chromatography (petroleum ether/EtOAc 1:1 yielding 71 mg white powder (47%)

[00133] Intermediate 4 (26 mg) is dissolved in DCM (1.5 mL). Anisole (0.20 mL) is added and the mixture is cooled to 0°C. Trifluoroacetic acid (0.45 mL) is added dropwise over a period of 10 minutes and the mixture is stirred for 2 hours at 0°C. The mixture is concentrated and the crude compound is purified by preparative HPLC yielding 12 mg off- white powder. ’'H NMR (500 MHz, DMSO-ds) ò 11.83 (s, 1H), 9.02 (d, J = 8.4 Hz, 1H), 7.81 (d, J= 8.5 Hz, 2H), 7.74 (d, J= 8.5 Hz, 2H), 7.34 — 7.20 (m, 7H), 7.15 — 7.08 (m, 1H), 5.69 (dd, J=8.3,4.8 Hz, 1H), 5.27 (d, J= 12.9 Hz, 1H), 5.01 (d, J= 4.9 Hz, 1H), 4.93 (d, J=

12.9 Hz, 1H), 3.81 -3.71 (m, 1H), 3.57 (d, J = 13.9 Hz, 1H), 3.50 (d, J = 13.9 Hz, 1H),

3.35-3.22 (m, 2H), 2.69 (s, 6H), 1.30 (dt, J= 7.3, 3.7 Hz, 6H).

[00134] Example 5: Synthesis of (6R,7R)-3-(((3-(4-(1H-pyrazol-1-yl)phenyl)-7- isopropyl-1H-indole-2-carbonyl}oxy)methyl}-8-oxo-7-(2-phenylacetamido)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

NH | (2) NLs 0 Oo 0” "OH The reaction scheme followed for the synthesis of this compound is illustrated in figure 7.

[00135] Intermediate 5: 4-methoxybenzyl (6R,7R)-3-(((3-(4-((1H-pyrazol-1- yhmethyl)phenyl)-7-isopropyl-1H-indole-2-carbonyl)oxy)methyl)-8-oxo-7-(2- phenylacetamido)}-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-carboxylate “Ln Oy ts o 0 0 o PMB

[00136] 4-methoxybenzyl (6R,7 R)-3-(chloromethyl}-8-ox0-7-(2-phenylacetamido)-5-thia-1- azabicyclo-[4.2.0]oct-2-ene-2-carboxylate (GCLE, 153 mg, 0.32 mmol, 1.5 eq) is dissolved in DMF (5 mL) and sodium iodide (48 mg, 0.32 mmol, 1.5 eq) is added. After stirring for 1 hour at room temperature, 3-(4-((1H-pyrazol-1-yhmethyhphenyl})-7-isopropyl-1H-indole-2- carboxylic acid (SC-79, 75 mg, 0.21 mmol, 1 eq) and sodium bicarbonate (56 mg, 0.66 mmol, 3.2 eq) are added and the mixture is stirred overnight at room temperature. The mixture is filtered over cotton, concentrated and absorbed on silica. The crude product was purified by column chromatography (petroleum ether/EtOAc 2:1 to 2:3) yielding 100 mg white powder (62%).

[00137] Intermediate 5 (50 mg) is dissolved in DCM (2.5 mL). Anisole (0.40 mL) is added and the mixture is cooled to 0°C. Trifluoroacetic acid (0.9 mL) is added dropwise over a period of 10 minutes and the mixture is stirred for 2 hours at 0°C. The mixture is concentrated and the crude compound is purified by preparative HPLC yielding 25 mg off- white powder. 'H NMR (500 MHz, DMSO-cs) 8 11.56 (s, 1H), 9.14 (d, J= 8.3 Hz, 1H), 7.86 (dd, J= 2.3, 0.6 Hz, 1H), 7.51 (dd, J= 1.8, 0.6 Hz, 1H), 7.41 (d, J= 8.2 Hz, 2H), 7.34 —

7.18 (m, 10H), 7.08 — 7.02 (m, 1H), 6.33 —- 6.27 (m, 1H), 5.72 (dd, J = 8.3, 4.8 Hz, 1H),

5.41 (s, 2H), 5.14 (d, J= 13.0 Hz, 1H), 4.98 (d, J= 4.9 Hz, 1H), 4.93 (d, J= 13.0 Hz, 1H),

3.79 (s, 2H), 3.77 = 3.71 (m, 1H), 3.60 (d, J = 13.9 Hz, 1H), 3.52 (d, J= 13.9 Hz, 1H), 3.10 (s, 2H), 1.39 — 1.13 (m, 6H).

[00138] General procedure for the synthesis of ester linked MBL prodrugs. © (ii) RCOOH, NaHCO; © I Oo 9 Oo o

PMB PMB 4-methoxybenzyl (6R,7R)-3-(chloromethyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1- azabicyclo-[4.2.0]oct-2-ene-2-carboxylate (GCLE, 1.1 eq) is dissolved in DMF and sodium iodide (1.1 eq) is added. After stirring for 1 hour at room temperature, the carboxylic acid (RCOOH, 1 eq) and sodium bicarbonate (3 eq) are added and the mixture is stirred overnight at room temperature. The mixture is filtered over cotton, concentrated and the PMB-protected intermediate is purified by column chromatography.

[00139] General procedure for the deprotection of ester linked MBL prodrugs. 4 TL (i) PhOCHs, DCM 4 TL Nc © R -— Nc © R 0 A TT (i) TFA, 2 h, 0 °C 0 A TT © 0 © ©” OH ©

PMB The PMB-protected intermediate is dissolved in DCM and anisole is added. The mixture is cooled to 0°C. Trifluoroacetic acid is added dropwise over a period of 10 minutes and the mixture is stirred for 2 hours at 0°C. The mixture is concentrated and the crude compound is purified by preparative HPLC. Example 6: Assessment of the synergistic activity of Examples 1-3

[00140] Performed as described at 0083-0084. The MBL prodrugs were tested against a variety of MBL-producing bacteria. For MIC evaluation the prodrugs were serially diluted two-fold from 256 pg/mL. No growth inhibition was observed up to this concentration. Subsequently, the compounds were tested at 64 pg/mL in the presence of varying concentrations of meropenem. From this the fractional inhibitory concentration (FIC) was calculated according to the formula in 0084. The MIC values obtained from the synergy assay in the presence or absence of examples 1-3 are presented in Table 1. Between brackets the factor improvement is indicated.

[00141] Table 1. Minimum inhibitory concentration (MIC) values of meropenem in Hg/mL in the presence or absence of examples 1-3 tested at 64 pg/mL. The factor improvement is given between brackets. Meropenem | Meropenem MBL Meropenem | +example 2 | + example 3 K. Pneumoniae JS022 NDM-1 16 1.0 (16) 1.0 (16) K. Pneumoniae RC0048 VIM-1 64 4.0 (16) 8.0 (8) K. Pneumoniae RCO021 VIM-1 16 0.5 (32) 1.0 (16) K. Pneumoniae NRZ-293 VIM-2 4 0.13 (32) 0.25 (16) K. Pneumoniae JS265 IMP-28 2 0.13 (16) 0.25 (8) P. Aeruginosa NRZ-8418 NDM-1 64 32.0 (2) 32.0 (2) P. Aeruginosa 581 VIM-2 16 8.0 (2) 2.0(8) P. Aeruginosa 2018-009 IMP-13 32 32.0(1) 32.0 (1) E. Coli 2018-015 NDM-1 8 0.5 (16) 0.5 (16) E. Coli 1322 VIM-2 4 0.25 (16) 0.25 (16) During the development of compound 2, where 8-thioquinoline (8-SH-Q) is conjugated to the cephalosporin core, the activity of the structural analogue 8-hydroxyquinoline (8-OH-Q) was evaluated as well. Both 8-hydroxyquinoline and 8-thioquinoline were evaluated for their antibacterial and synergistic effects. From the MIC data it becomes clear that 8- hydroxyquinoline has an MIC value of 64 ug/mL against K. Pneumoniae strains, whereas 8-thioquinoline showed no growth inhibition up to 256 pg/mL. To evaluate their synergistic effects with meropenem, the compounds were tested at concentrations below their MIC and FIC values were calculated. As shown for a VIM-1 producing K. Pneumoniae strain, only 8-thioquinoline significantly reduced the MIC of meropenem and no synergy was observed for 8-hydroxyquinoline

[00142] Table 2. Comparison of the inhibitory and synergistic activity of 8- hydroxyquinoline and 8-thioquinoline. MIC values are given in ug/mL. The factor improvement is given between brackets. 32 pg/mL + 16 pg/mL + on | hi meropenem meropenem vw a

64 pg/mL + 32 pg/mL + neva wen | Cy | en. meropenem meropenem

[00143] In order to confirm the interaction of the compounds with the target enzymes, enzyme inhibition experiments were performed as described under 0082. The inhibition data obtained for examples 1-3 and reference compounds and known zinc binders dipicolinic acid (DPA) and ethylenediaminetetraacetic acid (EDTA) is presented in table 2.

[00144] Table 3. IC50 values in micromolar (UM) for examples 1-3 and reference compounds EDTA and DPA, tested against representative enzymes of the three most important classes of metallo-beta-lactamases IMP, NDM and VIM. Data was obtained with Enzyme inhibition assay A IMP-28 NDM-1 VIM-2 Example 1 >200 124.83+554 106.57 £1.56 Example 2 5.36 +£0.18 465+0.44 4.55+0.27 Example 3 20.02 + 1.81 8.94 + 0.55 9.17 £0.68 DPA 28.88 £ 5,83 10.00 £0.13 10.42 + 0.81 EDTA 204.70 +9.85 1.26 + 0.03 0.94 + 0.03

[00145] Table 4. IC50 values in nanomolar (nM) for examples 4-5 and the corresponding unconjugated MBL inhibitors SC-73 and SC-79, tested against representative enzymes of the three most important classes of metallo-beta- lactamases IMP, NDM and VIM.

Data was obtained with Enzyme inhibition assay B

IMP-28 NDM-1 VIM-2

Example 4 | 130.92 + 11.29 225+028 0.173 + 0.061

Example 5 56.62 £8.99 1.92+066 0.179 + 0.051

SC-73 1762+1.03 0.142+0.008 0.136 £0.005

SC-79 086+0.97 0.257 +0.011 0.061 + 0.004

Claims (33)

CONCLUSIONS A compound of formula I: A-B (D wherein A is a B-lactame antibiotic group comprising a bridging methylene (-CH> -) covalently bonded to -B; and B a latent metallo-f-lactamase ( MBL) inhibitor selected from: Ry © ORs A R40 0 XA R; A 2 Ka Yy Ys $ Y Rs R3 Raz NS AN Ra, Fi y Reg: Rar Rig Raa A $ l no © ~~: HN Rizo 0 8 No C Ra i or OQ ‚where Xi is selected from -S-, -O-, -OC {O) -, -S (O) -, and -S ( O):-; X2 is selected from -S-, -OC (O) -, -S (O) -, and -S (O) 2-; A3 is selected from -S-, -O-, -OC (O) -, -S (O) -, and -S (O) 2-; Y is selected from -N = or -C (Ro) =; Y is selected from -N = or -C (R10) =; Ys is selected from -N = or -C (R14} =; Ya is selected from -N = or -C (R13) =; wherein each of Ra, Rs, R4, Rs, R7, Rs, Rs, Rio, Ri, Riz, Ris, Ris, Ris, R17, Ris, Ris, Rao, R21, and R23 is independently selected from hydrogen , halo, cyan; nitro, hydroxy, substituted or unsubstituted (C 1 -C 4) alkyl, substituted or unsubstituted aryl, said one or more substituents being independently selected or from oxo, halo, cyano, nitro, hydroxy, carboxy, sulfonamide, NH 2 , NH (C 1 -C 6 alkyl), N (C 1 -C 4 alkyl) 2, methoxy, ethoxy, or aryl; each R5 and R15 is independently selected from H and (C1-Cy) alkyl; and R22 is an aryl or a heteroaryl optionally substituted by one or more substituents independently selected from hydrogen, halo, cyano, nitro, hydroxy, NHC (O) H, NHC (O) (C1-Cyalkyl, and ( C: -Ca) alkyl, optionally substituted by one or more substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, NH 2, NH (CC: alkyl), N (C: -C2 alkyl) 2, methoxy, ethoxy, or a pharmaceutically acceptable salt, a pharmaceutically acceptable stereoisomer, a pharmaceutically acceptable solvate, or a pharmaceutically acceptable prodrug thereof. A compound according to claim 1, wherein A is a cephalosporin group or a carbapenem group. A compound according to claim 1 or claim 2, wherein A is a cephalosporin group. A compound according to any one of the preceding claims, wherein A is: Ry fn $ Ir 3 10 4 wherein: R; substituted or unsubstituted -C1 -C4 alkylaryl, substituted or unsubstituted -C1 -C4 alkylcycloalkyl, substituted or unsubstituted -C1 -C4 alkylheteroaryl, substituted or unsubstituted -C1 -C4 aminoalkylaryl, substituted or unsubstituted -C1-C4 aminoalkylcycloalkyl, substituted or unsubstituted -C1-C4 aminoalkylheteroaryl, said optional substituents independently selected from oxo, halo, cyano, nitro, hydroxy, carboxy, NH, NH (C1-C> alkyl), N (C1-C: alkyl) s, methoxy, ethoxy; Y is -S-, -S (O) -, -S (O) 2, -O-, or -CH: -; and Z is -C (O) OH, -C (O) YO (C-C4 alkyl), tetrazole, hydroxamic acid, amide, sulfonamide, phosphate, or sulfate. A compound of claim 4, wherein R 1 is -C 1 -C 4 alkylphenyl, optionally phenacyl. A compound according to claim 4 or claim 5, wherein Y represents -S-. A compound according to any one of claims 4 to 6, wherein Z is -C (O) OH. A compound according to any one of the preceding claims, wherein each of Ra, R1, R4, Rs, Ry, Rs, Ro, Rig, Rit, Riz, Ris, Ris, Ris, Ri7, Ris, Ris, Reo, R21 and Ros are equal is to H. A compound according to any one of the preceding claims, wherein each of R5 and R13 is H. A compound according to any one of the preceding claims, wherein B is equal to: R7 A Ra Rya0 Q R17 Ris and 5 Ria R ht Rg i Xx HN 19h 0 O _ Row Ryo A Ris ht R20 Ris i Q Ris, or 0 R21 . A compound according to any one of the preceding claims, wherein B is: Rz NA | te ny Re Rig Rip Ry ‚ A compound according to any one of claims 1 to 10, wherein B is: R450 Q Fy. N Xe 14 o Jl oe SS Rec 5 Q Ris i A compound according to any one of claims 1 to wherein B is: R17 Ris HN Rig 40 Dn Rao 0 R24 / A compound according to any one of claims 1 to 9, wherein B is: Has A 5 Vb ze Ro: 3 HN— g u IN o 9. A compound according to any one of the preceding claims, selected from: STR FN oF XD ee oF AND Nanasan 5. # A, ‚A ee 7 \‚ EE a OON SS PNN o pp oF Y oe y Pe Net NT Od a "~~ es 3 SEEN a B me A vere ie Von we I ¢ Sd X: i Ret SNH a a. oO Ng as xs N ve J hl LD Ay ul haa Ry 7 Na Sd ii GT OH NH OT ts = © KA = Od 0 Tr 1 0 Oo HNL Oo ““ OH Fr ONE oH G nd wed H \ TN HH Fy H fmm N is FERNY MLS Nes Mn i Mi N ri i ¥ HY sd No J © oN © 0 fo A Ns 3 A ST Nz Se I 0 To OA \ © gi {3 AN © OH Ton TN \} 0 ”" OH Yellow Sy \ 'or \ "amg or a pharmaceutically acceptable salt, solvate, or prodrug thereof. A formulation comprising a compound according to any preceding claim, as well as optionally a pharmaceutically acceptable carrier. A formulation according to claim 16, further comprising an additional active agent. A formulation according to claim 17, wherein the additional active agent is a β-lactame antibiotic, or a pharmaceutically acceptable salt or prodrug thereof. A formulation according to claim 17 or claim 18, wherein the additional active agent is at least one carbapenem, at least one cephalosporin, at least one carbacephem, at least one penicillin, or at least one ureidopenicillin. A compound according to any one of claims 1 to 15, or a formulation according to any one of claims 16 to 19, for use as a medicament]. A compound according to any one of claims 1 to 15, or a formulation according to any one of claims 16 to 19, for use in the treatment of an antibiotic resistance. The compound or formulation of claim 21, wherein the antibiotic resistance is provided by a bacterial metallo-β-lactamase. A compound according to any one of claims 1 to 15, or a formulation according to any one of claims 16 to 19, for use in the treatment of a bacterial infection. The compound or formulation of claim 23, wherein the treatment further comprises administering an additional active agent. A compound or formulation according to claim 24, wherein the additional active agent is a β-lactame antibiotic, or a pharmaceutically acceptable salt or prodrug thereof; optionally wherein the additional active agent is at least one carbapenem, at least one cephalosporin, at least one carbacephem, at least one penicillin, or at least one ureidopenicillin. A compound or formulation according to claim 24 or claim 25, wherein the additional active agent is provided as a separate dosage form from the compound or formulation. A compound according to any one of claims 1 to 15 for use in the treatment of a bacterial infection, by means of a combined, sequential, or separate administration with an antibacterial agent. A compound of claim 27, wherein the antibacterial agent is a β-lactame antibiotic, or a pharmaceutically acceptable salt or prodrug thereof; optionally wherein the antibacterial agent is at least one carbapenem, at least one cephalosporin, at least one carbacephem, at least one penicillin, or at least one ureidopenicillin. A method of inhibiting a bacterial metallo-β-lactamase in vitro or in vivo, administering to a cell an effective amount of a compound according to any one of claims 1 to 15, or a formulation according to any one of claims 1 to 15. claims 16 to 19. Use of a compound according to any one of claims 1 to 15, or a formulation according to any one of claims 16 to 19, for the inhibition of a bacterial metallo-β-lactamase. A method of treating a bacterial infection in a patient, comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 15, or a formulation according to any one of claims 16 to 19 . The method of claim 31, wherein the method further comprises a combined, sequential, or separate administration of an effective amount of an antibiotic agent. The method of claim 32, wherein the antibiotic agent is a β-lactame antibiotic, or a pharmaceutically acceptable salt or prodrug thereof; optionally wherein the antibacterial agent is at least one carbapenem, at least one cephalosporin, at least one carbacephem, at least one penicillin, or at least one ureidopenicillin.