Cyclic boronic acid esters useful as adjuvants in the treatment of bacterial infections

Abstract

The invention provides novel compounds of formula (I), stereoisomers and pharmaceutically acceptable salts thereof: (wherein: Q is a lipophilic, zinc chelating moiety which is selective for Zn2+ ions; L1 is a covalent bond or a C1-6 alkylene chain in which one or more -CH2- groups of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR4- (where R4 is H or C1-3 alkyl); Y is selected from the following groups: (II) and (III) (where each R5 is independently H or C1-3 alkyl; and R6 is H or C1-3 alkyl); L2 is a covalent bond or a C1-6 alkylene chain in which one or more -CH2- groups of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR7- (where R7 is H or C1-3 alkyl); R is -OH, -O-C1-6 alkyl, -O-(CH2)p-O-CO-C1-6 alkyl (where p is an integer of 1 or 2) or -O-CH(CH3)-O- CO-C1-6 alkyl; each R1 is independently selected from halogen and C1-3 alkyl; each R2 is independently selected from halogen and C1-3 alkyl; R3 is H or C1-3 alkyl; n is an integer of 0 or 1; and m is an integer of 0 or 1). The compounds according to the invention are selective zinc chelators that find use as adjuvants in the treatment of bacterial infections and / or bacterial biofilms that harbour such infections. For use in such treatment, the compounds are used in combination with an antibacterial agent, for example a β-lactam antibiotic. The invention further provides a triple combination therapy for use in the treatment of bacterial infections and / or bacterial biofilms which comprises co-administration of the compounds to a subject in combination with a β-lactam antibiotic and a serine β-lactamase inhibitor.

Description

[0001] Compounds

[0002] Field of the invention

[0003] The present invention relates to compounds which are selective zinc-chelators, to pharmaceutical compositions containing such compounds and their use in therapy.

[0004] In particular, the invention relates to such compounds for use as adjuvants in the treatment of bacterial infections and / or bacterial biofilms that harbour such infections. For use in such treatment, the compounds are used in combination with known antibacterial agents, for example antibiotics such as carbapenems.

[0005] The invention further relates to a triple combination therapy for use in the treatment of bacterial infections and / or bacterial biofilms which comprises co-administration of the compounds with other known adjuvants and antibacterial agents. In this triple combination therapy, the compounds are administered to a subject in combination with a P-lactam antibiotic and a serine p-lactamase inhibitor.

[0006] Background of the invention

[0007] The global increase in antimicrobial resistance is currently undermining our ability to treat bacterial infections and has become a critical public health threat worldwide. A cornerstone in treatment of serious and life-threatening infections caused by multidrugresistant (MDR) gram-negative bacterial pathogens such as Klebsiella pneumoniae and Escherichia coli has been the carbapenem p-lactam antibiotics, e.g. penicillins and carbapenems.

[0008] The major advantage of carbapenems has been their relative stability towards p-lactamases, such as the extended-spectrum p-lactamases (ESBLs) and AmpCs, which constitute common resistance mechanisms against p-lactams (Bush K., J Infect.

[0009] Chemother. (2013), 19(4): 549-59). However, we now observe a global increase in dissemination and diversity of p-lactamases (carbapenemases) with the ability to inactivate carbapenems (Logan, L. K. et al., J. Infect Dis. (2017), 215 (Suppl 1):S28-S36).

[0010] The impact of carbapenem-resistance is further illustrated in a European study in which carbapenem-resistance was shown to be the major contributor to the burden of infections by antibiotic-resistant bacteria in many countries (Cassini A. et al., Lancet Infectious diseases (2019), 19, 56-66). Moreover, a common feature of carbapenemase-producing gram-negative bacteria is MDR (multi-drug resistance), including resistance towards non-P-lactam antimicrobials, resulting in severely limited treatment options (Perez F, Expert Opin. Pharmacother. (2016), 17(6), 761-81).

[0011] An especially important disease area in which novel, more efficient bacterial enzymes play a vital role, is that of resistant microbes. Infectious diseases are a leading cause of death worldwide and account for millions of deaths annually including nearly two-thirds of all childhood mortality at less than 5 years of age. There is serious concern regarding new and re-emerging infectious diseases for which effective therapies are lacking (World Health Organization reports 2020 and 2021). The introduction of new, more potent derivatives of existing antibiotics provides only temporary solutions since existing resistance mechanisms rapidly adapt to accommodate the new derivatives (Theuretzbacher II. Curr. Opin. Pharmacol. (2011), 11, 433-438).

[0012] Although resistant gram-positive bacteria pose a significant threat, the emergence of multi-drug resistant (MDR) strains of common gram-negative pathogens such as Escherichia coli are of special concern. Pan-resistance or extreme drug resistance are now commonly used terms to describe clinically important isolates of Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae that are resistant to virtually all antibiotics (Bush K. et al., Clin. Microbiology Rev. (2020), 33, e00047-19).

[0013] There are few, if any, antimicrobial agents effective against gram-negative bacteria either in or entering phase I clinical trials that will address this critical need (Butler MS. et al., J. Antibiotics (2013), 66, 571-591). One important feature of bacteria, especially gramnegative bacteria, is that they have two cell membranes, one outer membrane that is more permeable and one inner cell membrane resembling an eucaryotic cell membrane. One important group of enzymes involved in antimicrobial resistance is the p-lactamases (Bush K. et al., Annu. Rev. Microbiol. (2011), 65, 455-478). They are excreted into the volume between these membranes (i.e. the periplasmic space) which is more accessible to drugs. The p-lactamases are divided into two main families and four classes, the serine P-lactamases (SBLs) and the metallo-p-lactamases (MBLs). The SBLs are classified as Ambler classes A, C and D. Examples of SBLs in class A are CepA, KPC-2, IMI-1 , SME-1, PC1, TEM-1, TEM-2, TEM-3, TEM-30, TEM-50, SHV-1, SHV-2, SHV-10, CTX-M-15, PER-1, VEB-1, PSE-1, CARB-3, and RTG-4. SBLs in Ambler class C include AmpC, CMY-1, ACT-1, FOX-1, MIR-1, GC1, CMY-10, CMY-19, and CMY-37, and those in Ambler class D include OXA-1, OXA-10, OXA-11, OXA-15, OXA-23, and OXA-48. The MBLs are classified in Ambler class B and include IMP, VIM, SPM, IND, NDM, DIM, GIM, SIM, AIM, CAU-1, GOB-1, FEZ-1, CcrA, IND-1, L1, CphA, Sfh-1, and ImiS.

[0014] The main distinction between SBLs and MBLs is that SBLs possess an active site serine hydroxy group, while MBLs require the presence of metal ions for activity, p-lactamases with carbapenemase activity have been identified in both of these families including SBLs such as KPC and OXA-48, and MBLs such as NDM, VIM and IMP. The recent introduction of serine carbapenemase inhibitors such as avibactam and vaborbactam used in combination with p-lactams has provided treatment options against serine carbapenemase-producing gram-negative pathogens.

[0015] Other SBL inhibitors with inhibitory activity against carbapenemases, such as relebactam and ETX2514, are either in late-stage development or early phase I clinical trials.

[0016] However, none of these p-lactamase inhibitors possess inhibitory activity against MBLs. Despite several reports of promising MBL inhibitors, including aspergillomarasmine A, dipicolinic acid derivatives, ANT431, bisthiazolodines, and bismuth antimicrobials, no selective and efficient inhibitors are close to market. Consequently, new treatment options for infections caused by MBL-producing gram-negatives, including NDM-producing Enterobacteriales, are urgently required.

[0017] The recent introduction of serine carbapenemase inhibitors such as avibactam, vaborbactam and relebactam used in combination with p-lactams provides treatment options against serine carbapenemase-producing gram-negative pathogens (Zhanel G.G., et al., Drugs (2018), 78, 65-98; H. Wright, H. etal, European Society of Clinical Microbiology and Infectious Diseases (2017), 23, 704-712). Unfortunately, none of these P-lactamase inhibitors possess inhibitory activity against MBLs.

[0018] The recent Italian outbreak of NDM-producing Enterobacteriaceae is significant due not only to its size but also the change in epidemiology of carbapenem-resistant Enterobacteriaceae (CRE) from endemic KPC-producing CRE to NDM-producing CRE and the subsequent reduction in treatment options (ECDC Report, Italian Outbreak NDM, 2018.2019, Stockholm). Consequently, new treatment options for infections caused by MBL-producing gram-negatives, including NDM-producing Enterobacteriales, are urgently required. Possible treatment options include cefiderocol (Zhanel, 2018) and the combination aztreonam-avibactam (Chew K.L. et al., Antimicrob. Agents Chemother. (2018), 62, e00414-18). Combinations of p-lactams and p-lactam enhancers such as zidebactam (Moya B., Antimicrob. Agents Chemother. (2019), 597, 63:e00128-19) have also shown promising activity. Moreover, several MBL inhibitors, including aspergillomarasmine A (King A.M., Nature. (2014), 510,503-6), dipicolinic acid derivatives (Chen A.Y. et al, J. Med Chem (2017), 60(17):7267-83), ANT431 (Everett M. et al., Antimicrob. Agents Chemother. (2018), 62:e00074-18), bisthiazolodines (Hinchliffe P, et al., Proc. Natl. Acad. Sci. USA (2016), 113(26), E3745-54) and bismuth antimicrobials (Wang R. et al., Nature Commun. (2018), 9(1):439) have been reported. Recently, VNRX- 5133 (taniborbactam), a dual SBL and MBL inhibitor has shown potent activity in combination with cefepime against MBL-producers (Hamrick J.C. et al., Antimicrob. Agents Chemother. (2020), 64, e01963-19.). However, taniborbactam in combination with cefepime, for example, has some weaknesses in having no effect against the MBL class IMP.

[0019] Thus, no selective and efficient MBL inhibitors are approved for clinical use (Bush K. et al., Clin. Microbiology Rev. (2020), 33, e00047-19). Metallo-p-lactamases (MBLs) belong to a large group of proteins only found in bacteria and, like penicillin-binding proteins (PBPs), have the ability to interact with p-lactams. Examples of PBPs and enzymes that bind p-lactams are MBLs, serine p-lactamase-like protein (LACTB), D,D-transferase, D-Ala(D,D)-carboxypeptidase, the D-Alanyl-D-alanine Dipeptidases VanA, VanX, VanY and others, as reviewed by Sauvage E. et al. in FEMS Microbiol. Rev. (2008), 32, 234-258. This class of proteins is only found in bacterial biology. Examples of compounds having affinity for PBPs are p-lactam antibiotics. p-Lactams have been the historical anchor of antibacterial chemotherapy and include penicillins, cephalosporins, monobactams and carbapenems (Bush Ketal., Annu. Rev. Microbiol. (2011), 65, 455-478). MBLs are emerging as one of the most clinically important family of p-lactamases (Patel et al., Front. Microbiol. (2013), 4, 48, Walsh et al., Int. J. Antimicrob. Agents (2010), S8-S14). The clinically most important MBLs, the IMP-, VIM-, GIM- and NDM-groups, are now widespread in a variety of gram-negative species. In particular, VIM- and NDM-enzymes have emerged as the dominant MBLs. The unprecedented global dissemination of NDM highlights the enormity of the problem. Since the first report in 2008, NDM has been identified in Australia, Africa, North America, Asia and many European countries (Johnson A.P. et al., J. Med. Microbiol. (2013), 62, 499-513). Worryingly, NDM is found in numerous gram-negative species and in the environment (Walsh T.R. et al., Lancet Infect. Dis. (2011), 11, 355-362).

[0020] Successful inhibitors of class A serine p-lactamases are clinically available, but lack inhibitory activity against MBLs (Drawz S.M. et al., Clin. Microbiol. Rev. (2010), 23, 160201). Inspired by the commercial success of the paradigm Augmentin (clavulanic acid - a suicide substrate for serine p-lactamases - and amoxicillin) several research groups have focused on similar approaches to develop inhibitors, but as yet no molecules that combine potency with activity against multiple MBL targets have reached clinical trials (Drawz S.M. et al., Antimicrob. Agents Chemother. 2014).

[0021] For the three clinically most threatening MBLs - the IMP, NDM and VIM groups - most inhibitors are reported for IMP-1, while few inhibitors are found for VIM-2 and NDM. For NDM, a natural fungal product, aspergillomarasmine A, has been identified as an MBL inhibitor and shown in vivo activity in mouse models (King A.M. et al., Nature (2014), 510, 503-506). However, relatively high doses of aspergillomarasmine A are required to reverse carbapenem resistance.

[0022] Other therapeutic options include the use of tri-p-lactam therapy incorporating a monobactam (Martinez, Future Med. Chem. (2012), 4(3), 347-59); however, the MICs are not impressive, and in vivo activity is severely compromised by the bacterial inoculum (Page et al., Antimicrob. Agents Chemother. (2011), 66, 867- 73).

[0023] Whereas in MBLs, where formation of a non-covalent reactive complex with the p-lactam is facilitated by Zn2+, distinct mechanisms exist for SBLs such as KPC and OXA-48, that utilise an active-site serine for hydrolysis. The serine-lactamases function by initially forming a covalently acylated enzyme. To date, a clinically available compound that inhibits both SBLs and MBLs has not yet been introduced.

[0024] An urgent medical challenge rising today is the increasing co-existence of MBLs and SBLs constitutively (constantly) expressed in the same pathogen. Thus, there is a need for inhibitors that simultaneously address MBLs and SBLs and which are capable of inhibiting a wide range of MBL isotypes (e.g. NDM, VIM and IMP) and at the same time SBLs (e.g. KPC, OXA and P99 / AmpC). However, developing such ultrabroad-spectrum inhibitors is highly challenging because MBLs and SBLs possess markedly different mechanisms of action in inactivating p-lactam antibiotics. The variety in structural topologies of these enzymes and their markedly different mechanisms of action makes it challenging to develop an inhibitor capable of tackling a wide range of different bacterial enzymes.

[0025] Important p-lactam antibiotics are the penicillins, cephalosporin and carbapenem classes. Many compounds have been reported as having MBL-inhibiting activities. In bacteria, zinc sensing is carried out by regulators of different families, including SmtB / ArsR, MerR, TetR, MarR, and the Fur family (Napolitano et al., Journal of Bacteriology (2012), 2426-2436). In some cases, the MBL inhibitors contain a Zn2+-binding group that can interact strongly with the central metal ion(s). Compounds which bind metal ions, so-called metal “chelators”, have been shown to affect bacterial biological mechanisms. The term "chelating" relates to the Greek term "chelos" denoting a crab claw binding an object via at least two contact points. A variety of substances are described in the prior art that are capable of chelating particular types of metal ions. Examples of metal chelators include compounds comprising variations of amino groups and hydroxyl groups, e.g. 1,10-phenanthroline, clioquinol, l,2-dimethyl-3-hydroxy-4-pyridinone (DMHP), 1 ,2-diethyl-3-hydroxy-4-pyridinone (DEHP), and deferasirox. In the prior art, metal chelators have been described as inhibitors of multiple diseases simultaneously, e.g. in WO 2006 / 117660, WO 2001 / 60349, US 6,410,570, and in WO 2009 / 140215.

[0026] Metal chelators have also been suggested as inhibitors of biofilm formation, e.g. in WO 2011 / 63394 and WO 2009 / 155088, or as antiviral agents, e.g. in WO 2004 / 71425 and WO 2006 / 43153. Attractive bacterial metal dependent targets involved in resistance mechanisms are known in the prior art to be inhibited by metal chelating agents. Three examples are the tightly regulated bacterial zinc uptake system Zur (Ellison et al. in PLOS ONE (2013), 8, e75389), biofilms (Conrady etal., PNAS (2008), 105 (49), 19456-19461) and the peptidase HmrA in MRSA. All these targets are inhibited by state of the art non-selective, toxic metal chelators like TPEN, EDTA or the phenanthrolines.

[0027] Biofilm formation is an important bacterial resistance mechanism that contributes to the growing therapeutic concern globally. Biofilms consist of extracellular polymeric substances (EPS). These are natural hydrophilic carbohydrate polymers of high molecular weight secreted by microorganisms into their environment, and determine the physiochemical properties of a biofilm. Biofilm formation with the nocosomial infections produced by multidrug-resistant gram-negative species producing metallo-p-lactamases, e.g. Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli, is problematic since they affect all traditional activity at hospitals, like surgery and wound healing. Of particular concern is MBL-producing Acinetobacter baumannii, as described by Peymani et al., Jpn. J. Infect. Dis. (2011), 64, 69-71. MBL-production was also found to be significantly higher in biofilm-positive isolates of Pseudomonas aeruginosa, as described by Heydari et al., Jundishapur J Microbiol. (2015), 8(3), e15514. Thus, there is a medical need for new drugs with acceptable toxicity and selectivity that are active against gram-negative bacteria harbouring MBL.

[0028] Zinc plays important roles in many disease states. For example, in hypoxic states related to cancer, zinc balance plays a role, e.g. zinc induces the accumulation of hypoxiainducible factor (H I F)-1 a, as discussed by Chun et al. in Biochemical and Biophysical Research Communications (2000), 268, 652-656. Zinc chelators may have a documented and significant effect on death of cancer cells as discussed by Ding et al., Cancer Letters (2008), 271, 251-259, by Lee et al. in Biochemical and Biophysical Research Communications (2007), 362, 766-772 and by Zuo in Journal of Cellular Biochemistry (2012), 113, 2567-2575. In the latter reference, derivatives of N,N-b / s(2-pyridyl-methyl)-amine, including the well described zinc chelators TPEN (J\I,N,N”, N”-tefra(2-pyridyl-methyl)-ethylene diamine) strongly induce cancer cell death related to cellular zinc depletion and destabilization of the X-linked inhibitor of apoptosis protein (XIAP). However, these chelating agents may not be used as therapeutic agents clinically because of high lipophilicity (logP) and high ability to penetrate normal cells, inducing an unacceptable general toxicity. These agents have no structural or molecular features providing biological selectivity for the target organ or disease area.

[0029] The most widely used groups of metal chelators are the so called amino-polycarboxylates, denoted hereinafter as APC chelators, comprising variations of aliphatic amino groups, hydroxyalkyl groups and carboxyalkyl groups. Common examples are aspergillomarasmine, ethylenediaminetetra-acetic acid (EDTA), ethylenediamine- / V, / \ / -diacetic-A / , / '-di-p-propionic acid (EDPA), diethylenetriamine pentaacetic acid (DTPA), trans-1,2-cyclohexane-diamine- / V, / \ / , / \ / ', / \ / -tetraacetic acid (CyDTA), carnosine, dihydroxyethylglycine (DHEG), 1 ,3-diamino-2-hydroxypropane- / V, / \ / , / \ / ', / \ / -tetraacetic (DTPA-OH), ethylenediamine- / V, / \ / -diacetic acid (EDDA), ethylenediamine- / V, / \ / -dipropionic acid (EDDP), / \ / -hydroxy-ethylenediamine- / \ / , / \ / ', / \ / -triacetic acid (EDTA-OH), / V, / V'-bis(2-hydroxybenzyl)ethylenediamine- / \ / , / \ / -diacetic acid (HBED), hexamethylene-1,6-diaminetetraacetic acid (HDTA), hydroxyethyliminodiacetic acid (HIDA), iminodiacetic acid (IDA), methyl-EDTA, nitrilotri acetic acid (NTA), nitrilotripropionic acid (NTP), triethylenetetraaminehexaacetic acid (TTHA), ethylenediamine-di(O-hydroxyphenylacetic acid) (EDDHA), ethyleneglycol bis(2-aminoethyl ether)- / V, / V, A / ', / V -tetraacetic acid (EGTA), trans-1,2-cyclohexanediaminetetraacetic acid (CDTA), N-(2-hydroxyethyl) ethylenedinitrilotriacetic acid (HEDTA), N-(2-hydroxyethyl) iminodiacetic acid (HEIDA), citric acid, 7, 19,30-trioxa-l,4, 10,13,16,22,27,33-octaazabicyclo[11,11,11] pentatriacontane (O-Bistren), penicillamine, chelators also comprising sulfur or phosphorus, e.g. diethyldithiocarbamate(DEDTC), 2,3-dimercapto-1-propanesulfonic acid (DMPS), ethylmaltol (EM), 4-(6-methoxy-8-quinaldinyl- aminosulfonyl)benzoic acid potassium salt (TFLZn), dithiozone, N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), ethylenediamine-N,N'-bis(methylphosphonic) acid (EDDPO), ethylenediaminetetra(methylenephosphonic) acid (EDTPO), nitrilotrimethylenphosphonic acid (NTPO), dimercaptosuccinic acid (DMSA), carboxymethyl-derivatives of 1,4,7-triaza-cyclononane or 1,4,7,10-tetra-aza-cyclododecane like DOTA, DO3A or NOTA, deferoxamine, dimercaprol, dimercaptosuccinic acid, and etidronic acid. The APC chelators are well known in the prior art, e.g. in Smith, R. M.; Martell, A. E. NIST Critically Selected Stability Constants of Metal Complexes, Version 2.0 U.S. Department of Commerce: Gaithersburg, MD, 1995. They are non-selective and often strong chelating agents with no molecular targeting moieties affecting their affinity for desired biological targets relevant for the disease. Thus, they usually also have antibacterial effect which is well described in the prior art, e.g. use of metal chelators against virus or bacteria in WO 2011 / 63394, WO 2004 / 71425, WO 2006 / 109069, WO 2001 / 60349, US 6,410,570, US 2003 / 0225155, WO 2006 / 43153 and WO 2006 / 43153.

[0030] Zinc chelators have also been suggested as antibacterial agents, e.g. in

[0031] WO 2009 / 140215, as inhibitors of biofilm formation, e.g. in WO 2011 / 63394 and

[0032] WO 2009 / 155088, or as antiviral agents, e.g. in WO 2004 / 71425 and WO 2006 / 43153, but in these cases the compounds lack biological or metal-chelating selectivity or have high solubility in fat, high logP, and are therefore often toxic to eukaryotic cells.

[0033] This lack of selectivity may lead to undesired toxicity of amino-polycarboxylate (APC) chelators and other biological effects when treating specific infections by a target organism in a host organism, e.g. when it is desirable to affect only specific microorganisms whilst a low toxicological effect on the host organism or other species (which are not a target for the treatment in question) is desired. One particularly serious adverse event exerted by non-selective chelating agents like the APC chelating agents are hemolytic effects even at low concentrations. A final report on the safety assessment of EDTA, calcium disodium EDTA, diammonium EDTA, dipotassium EDTA, disodium EDTA, TEA-EDTA, tetrasodium EDTA, tripotassium EDTA, trisodium EDTA, HEDTA, and trisodium HEDTA, ethylene diamine tetra acetic acid, EDTA, confirms that these have a swelling effect on erythrocytes after intravenous injections concentrations below 4 pM, eventually leading to haemolysis. This is well described in the prior art, e.g. by Witeska et al., in Turk. J. Vet. Anim. Sci. (2011), 35(2), 99-104, or by Igbokwe etal., Journal of basic and clinical physiology and pharmacology (2015), 26(2), 171-9. EDTA is also a potent anticoagulant because of its unselective metal chelation, resulting in chelation of e.g. Ca2+. However, this ability to bind calcium may lead to hypocalcemia, a deadly condition causing the US Food and Drug Administration (FDA) to withdraw the market approval of disodium EDTA because of the death of three patients related to hypocalcaemia, as reported by P. M. Wax, J. Med. Toxicol. (2013) 9:303-307. A report by R. S. Lanigan and T. Yamarik in International Journal of Toxicology, (2002), 21 (Suppl. 2), 95-142 concluded that EDTA, being a weaker chelating agent than other PACs, e.g. DTPA or DOTA, is a compound with biological effects incompatible with the demands for safe agents used clinically, especially for intravenous injection.

[0034] EDTA has recently been used as an adjuvant in a formulation denoted Elores or Sulbactomax comprising the p-lactam antibiotic ceftriaxone. Sulbactam, EDTA and ceftriaxone in combination administered intravenously have a good antibacterial effect, e.g. as described by Attili et al. in International Journal of Pharmaceutical Sciences and Research (2015), 6(6), 2569-2578. However, no description of studies of the haemolytic or anti-coagulant effects of the formulation are mentioned.

[0035] WO 2015 / 049546 and WO 2018 / 033719 describe new classes of selective zinc-chelating compounds. The chelator part of these compounds is highly selective for zinc (Zn2+ions) and the compounds are made hydrophilic by attaching a side chain, for example a carbohydrate-like moiety, which reduces the toxicity of the compounds.

[0036] WO 2018 / 033719 teaches that not only may a deficiency of zinc provoke the abnormal function of a cell, but also an excess of zinc may lead to abnormal cell functionality and unacceptable toxicity. Thus, there is a medical and technological need within this field for new therapeutic agents harnessing toxicity related to uncontrolled zinc depletion in healthy tissue and cells, whilst exerting the desired therapeutic effect. Many of the disease states in mammals related to metal imbalance are caused by enhanced metal concentration outside the eukaryotic cells in the extracellular space. Often the disease is related to excess metal outside the cells. Thus, there is a medical need for new metal chelating agents that have a low ability to pass eukaryotic cell membranes, and which at the same time have a high and selective affinity for the respective metal ions.

[0037] The compounds described in WO 2018 / 033719 are selective inhibitors of metallo-p-lactamases with no intrinsic antibiotic effects. They are used as adjuvants together with p-lactam antibiotics of the carbapenem class, exemplified by meropenem (MEM). When meropenem is used alone, many clinically isolated gram-negative bacteria with M BL as a resistance mechanism are shown to be resistant to the antibiotic, for example Klebsiella pneumoniae, E. coli and Pseudomonas aeruginosa. When MEM is used in combination with the compound of Example 26 of WO 2018 / 033719 against the same strains, MEM shows inhibitory effect - i.e. the sensitivity of these bacteria towards the antibiotic drug is reinstated. However, this combination has poor effect towards certain strains of Pseudomonas aeruginosa and many strains of Acinetobacter baumannii, and has no effect against bacteria which rely on the other main class of bacterial resistance enzymes, namely the SBLs.

[0038] An ongoing need thus exists for alternative zinc-chelating compounds and therapeutic treatments that are effective against bacterial infections, in particular against infections involving bacterial strains that have a defence mechanism to current treatments, such as those that rely on serine p-lactamase inhibitors (SBLs).

[0039] Summary of the invention

[0040] The inventors have now found a group of compounds having particularly advantageous properties as selective zinc-chelators. The compounds have an unprecedented ability to act as regulators of bacteria-specific enzymes, including both MBLs and SBLs, whilst having a low ability to penetrate mammalian cell membranes. As such, they can be safely used without toxicity to the living organism which is a host of the microorganisms.

[0041] In particular, the inventors have unexpectedly found that such compounds demonstrate improved properties over the zinc chelating compounds disclosed in WO 2018 / 033719 when used as adjuvants in the treatment of bacterial infections, in particular in the treatment of such infections that are associated with bacteria harbouring resistant enzymes, such as MBLs and SBLs. Specifically, the inventors have found that representative compounds in WO 2018 / 033719 display considerably higher minimal inhibitory concentrations (MIC) values than the compounds of the invention.

[0042] The inventors have also found that the combined use of the selective zinc-chelators, a p-lactam antibiotic, and a serine p-lactamase inhibitor in a single therapeutic regime provides significant additional advantages in the treatment of bacterial infections associated with MBLs and SBLs. The results provided herein evidence a ‘greater than expected’ effect associated with the combination of a selective zinc-chelator as disclosed herein, ceftriaxone (a p-lactam antibiotic which is a cephalosporin) and sulbactam (a serine p-lactamase inhibitor) against various bacterial strains harbouring resistance enzymes, such as Klebsiella pneumoniae ST147 (known to be associated with the two constitutively expressed resistant mechanisms NDM-1 and KPC-2), Klebsiella pneumoniae ST101 (OXA-48), Klebsiella pneumoniae K66-45 (NDM-1), Pseudomonas aeruginosa ST773, and Acinetobacter baumannii ST 1. Specifically, it has surprisingly been found that when the selective zinc-chelator and ceftriaxone are combined with sulbactam, the effect on resistant bacteria is even greater than the combination of the selective zinc-chelator and ceftriaxone. Furthermore, by analysing the microbiological test data, it has been found that the effect of the three agents together is not only additive, but synergistic, compared to the combination of the selective zinc-chelator and ceftriaxone. This finding is beyond expectation and supports a novel approach to the treatment of bacterial infections as herein described in which all three agents are co-administered as part of the same therapeutic regime. Additional results provided herein evidence synergy for other combinations of a selective zinc-chelator as disclosed herein, a p-lactam antibiotic and a serine p-lactamase inhibitor against a range of bacterial strains that harbour resistance enzymes.

[0043] This novel approach to the treatment of bacterial infections can be expected to extend to the combination of the following agents: (A) a selective zinc-chelator as defined herein; (B) a p-lactam antibiotic; and (C) a serine p-lactamase inhibitor. In addition to the treatment and / or prevention of bacterial infections, it is also proposed that this combination therapy may be used in the treatment or prevention of bacterial biofilms which harbour such infections, specifically in the disruption, removal or prevention of bacterial biofilms.

[0044] In one aspect the invention provides a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof:

[0045]

[0046] wherein:

[0047] Q is a lipophilic, zinc chelating moiety which is selective for Zn2+ions;

[0048] Li is a covalent bond or a C1-6 alkylene chain in which one or more -CH2- groups (e.g. all -CH2- groups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR4- (where R4is H or C1-3 alkyl, preferably H);

[0049] Y is selected from the following groups:

[0050]

[0051] (where each R5is independently H or C1-3 alkyl, preferably H; and

[0052] R6is H or C1-3 alkyl, preferably H);

[0053] L2 is a covalent bond or a C1-6 alkylene chain in which one or more -CH2- groups (e.g. all -CH2- groups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR7- (where R7is H or C1-3 alkyl, preferably H);

[0054] R is -OH, -O-C1-6 alkyl, -O-(CH2)P-O-CO-CI-6 alkyl (where p is an integer of 1 or 2) or -O-CH(CH3)-O-CO-CI-6 alkyl;

[0055] each R1is independently selected from halogen and C1-3 alkyl;

[0056] each R2is independently selected from halogen and C1-3 alkyl;

[0057] R3is H or C1-3 alkyl, preferably H;

[0058] n is an integer of 0 or 1 , preferably 0; and

[0059] m is an integer of 0 or 1, preferably 0. In another aspect, the invention provides a composition comprising a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0060] In another aspect, the invention provides a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof, for use in therapy.

[0061] In another aspect, the invention provides a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof, for use in a method of treatment of a bacterial infection in a subject, wherein said method comprises the step of administering to the subject a therapeutically effective amount of said compound in combination with (either simultaneously, separately or sequentially) at least one antibacterial agent.

[0062] In another aspect, the invention provides a pharmaceutical formulation comprising a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof, together with at least one antibacterial agent, and at least one pharmaceutically acceptable carrier, diluent or excipient.

[0063] In another aspect, the invention provides a kit comprising a first container containing a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof, and a second container containing an antibacterial agent.

[0064] In another aspect, the invention provides a method of treatment of a bacterial infection, said method comprising co-administration of an effective amount of each of the following agents to a subject in need thereof:

[0065] (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof;

[0066] (B) a p-lactam antibiotic; and

[0067] (C) a serine p-lactamase inhibitor.

[0068] In another aspect the invention provides (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof for use in the treatment of a bacterial infection in a subject by co-administration with:

[0069] (B) a p-lactam antibiotic; and

[0070] (C) a serine p-lactamase inhibitor. In another aspect the invention provides (B) a p-lactam antibiotic for use in the treatment of a bacterial infection in a subject by co-administration with:

[0071] (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof; and

[0072] (C) a serine p-lactamase inhibitor.

[0073] In another aspect the invention provides (C) a serine p-lactamase inhibitor for use in the treatment of a bacterial infection in a subject by co-administration with:

[0074] (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof; and

[0075] (B) a p-lactam antibiotic.

[0076] In another aspect the invention provides the use of (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of a bacterial infection in a subject by co-administration with (B) a p-lactam antibiotic; and (C) a serine p-lactamase inhibitor.

[0077] In another aspect the invention provides the use of (B) a p-lactam antibiotic in the manufacture of a medicament for use in the treatment of a bacterial infection in a subject by co-administration with (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof; and (C) a serine p-lactamase inhibitor.

[0078] In another aspect the invention provides the use of (C) a serine p-lactamase inhibitor in the manufacture of a medicament for use in the treatment of a bacterial infection in a subject by co-administration with (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof; and (B) a p-lactam antibiotic.

[0079] In another aspect the invention provides a pharmaceutical composition comprising:

[0080] (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof;

[0081] (B) a p-lactam antibiotic;

[0082] (C) a serine p-lactamase inhibitor; and

[0083] (D) one or more pharmaceutically acceptable carriers or excipients. In a further aspect the invention provides a kit comprising:

[0084] (i) a first container containing (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof;

[0085] (ii) a second container containing (B) a p-lactam antibiotic;

[0086] (iii) a third container containing (C) a serine p-lactamase inhibitor; and

[0087] (iv) optionally instructions for carrying out a method of treatment of a bacterial infection in a subject.

[0088] Detailed description of the invention

[0089] Definitions

[0090] Unless otherwise defined, all terms of the art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0091] As used herein, the term "alkyl" refers to a saturated hydrocarbon group and is intended to cover both straight-chained and branched alkyl groups. Examples of such groups include methyl, ethyl, n-propyl and iso-propyl.

[0092] As used herein, the term “halogen” or “halogen atom” refers to F, Cl, Br or I.

[0093] As used herein, the term "heteroaryl" refers to heterocyclic aromatic groups. Such groups may be monocyclic or bicyclic and contain at least one unsaturated heteroaromatic ring system. Where these are monocyclic, these comprise 5- or 6-membered rings which contain at least one heteroatom selected from nitrogen, oxygen and sulfur and contain sufficient conjugated bonds to form an aromatic system.

[0094] The compounds of the invention may contain one or more chiral centers and may therefore exist in different stereoisomeric forms. The term “stereoisomer” refers to compounds which have identical chemical constitution but which differ in respect of the spatial arrangement of the atoms or groups. Examples of stereoisomers are enantiomers and diastereomers. The term “enantiomers” refers to two stereoisomers of a compound which are non-superimposable mirror images of one another. The term “diastereoisomers” refers to stereoisomers with two or more chiral centres which are not mirror images of one another. The invention is considered to extend to diastereomers and enantiomers, as well as racemic mixtures.

[0095] The compounds herein described may be resolved into their enantiomers and / or diastereomers. For example, where these contain only one chiral center, these may be provided in the form of a racemate or racemic mixture (a 50:50 mixture of enantiomers) or may be provided as pure enantiomers, i.e. in the R- or S-form. Any of the compounds which occur as racemates may be separated into their enantiomers by methods known in the art, such as column separation on chiral phases or by recrystallization from an optically active solvent. Those compounds with at least two asymmetric carbon atoms may be resolved into their diastereomers on the basis of their physical-chemical differences using methods known perse, e.g. by chromatography and / or fractional crystallization, and where these compounds are obtained in racemic form, they may subsequently be resolved into their enantiomers.

[0096] The term “pharmaceutically acceptable salt” as used herein refers to any pharmaceutically acceptable organic or inorganic salt of any of the compounds herein described. A pharmaceutically acceptable salt may include one or more additional molecules such as counter-ions. The counter-ions may be any organic or inorganic group which stabilizes the charge on the parent compound. If the compound of the invention is a base, a suitable pharmaceutically acceptable salt may be prepared by reaction of the free base with an organic or inorganic acid. If the compound of the invention is an acid, a suitable pharmaceutically acceptable salt may be prepared by reaction of the free acid with an organic or inorganic base. Non-limiting examples of suitable salts are described herein.

[0097] The term “pharmaceutically acceptable” means that the compound or composition is chemically and / or toxicologically compatible with other components of the formulation or with the patient (e.g. human) to be treated.

[0098] By “a pharmaceutical composition” is meant a composition in any form suitable to be used for a medical purpose. The term "pro-drug" refers to a derivative of an active compound which undergoes a transformation under the conditions of use, for example within the body, to release an active drug. A pro-drug may, but need not necessarily, be pharmacologically inactive until converted into the active drug. As used herein, the term “pro-drug” extends to any compound which under physiological conditions is converted into any of the active compounds herein described. Suitable pro-drugs include compounds which are hydrolysed under physiological conditions to the desired molecule.

[0099] Pro-drugs may typically be obtained by masking one or more functional groups in the parent molecule which are considered to be, at least in part, required for activity using a pro-group. By “pro-group” as used herein is meant a group which is used to mask a functional group within an active drug and which undergoes a transformation, such as cleavage, under the specified conditions of use (e.g. administration to the body) to release a functional group and hence provide the active drug. Pro-groups are typically linked to the functional group of the active drug via a bond or bonds that are cleavable under the conditions of use, e.g. in vivo. Cleavage of the pro-group may occur spontaneously under the conditions of use, for example by way of hydrolysis, or it may be catalyzed or induced by other physical or chemical means, e.g. by an enzyme, by exposure to light, by exposure to a change in temperature, or to a change in pH, etc. Where cleavage is induced by other physical or chemical means, these may be endogenous to the conditions of use, for example pH conditions at a target tumor site, or these may be supplied exogenously.

[0100] As used herein, “treatment” includes any therapeutic application that can benefit a human or non-human animal (e.g. a non-human mammal). Both human and veterinary treatments are within the scope of the present invention, although primarily the invention is aimed at the treatment of humans. Treatment may be in respect of an existing disease or condition or it may be prophylactic.

[0101] As used herein, a “therapeutically effective amount” relates to an amount that will lead to the desired pharmacological and / or therapeutic effect, i.e. an amount of the agent which is effective to achieve its intended purpose. While individual patient needs may vary, determination of optimal ranges for effective amounts of the active agent is within the capability of one skilled in the art. Generally, the dosage regimen for treating a disease or condition with any of the compounds described herein is selected in accordance with a variety of factors including the nature of the medical condition and its severity. In one aspect, the invention relates to a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof:

[0102]

[0103] wherein:

[0104] Q is a lipophilic, zinc chelating moiety which is selective for Zn2+ions;

[0105] Li is a covalent bond or a C1-6 alkylene chain in which one or more -CH2- groups (e.g. all -CH2- groups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR4- (where R4is H or C1-3 alkyl, preferably H);

[0106] Y is selected from the following groups:

[0107]

[0108] (where each R5is independently H or C1-3 alkyl, preferably H; and

[0109] R6is H or C1-3 alkyl, preferably H);

[0110] L2 is a covalent bond or a C1-6 alkylene chain in which one or more -CH2- groups (e.g. all -CH2- groups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR7- (where R7is H or C1-3 alkyl, preferably H);

[0111] R is -OH, -O-C1-6 alkyl, -O-(CH2)P-O-CO-CI-6 alkyl (where p is an integer of 1 or 2) or -O-CH(CH3)-O-CO-CI-6 alkyl;

[0112] each R1is independently selected from halogen and C1-3 alkyl;

[0113] each R2is independently selected from halogen and C1-3 alkyl;

[0114] R3is H or C1-3 alkyl, preferably H;

[0115] n is an integer of 0 or 1 , preferably 0; and

[0116] m is an integer of 0 or 1, preferably 0. In the compounds of formula (I), Q is a zinc chelating moiety which is linked to a cyclic boronate ester moiety via a specifically defined linking moiety. A key structural feature of the compounds of the invention is the presence in the linking moiety of a nitrogen atom having a free lone pair and which is sufficiently separated from the boron atom in the boronate ester moiety. The nitrogen atom having a free lone pair may be provided in the linking moiety by either a secondary or tertiary amine group which is not adjacent to a carbonyl group, i.e. which does not form part of an amide group. In the compounds of formula (I), the nitrogen atom having a free lone pair is present in group Y. As can be seen, this nitrogen atom can form part of a piperazine ring which is positioned adjacent to a group -C(R5)2- (in which R5is H or C1-3 alkyl). Alternatively, it may be present as a secondary or tertiary amine which is flanked on both sides by a group -C(R5)2- (in which R5is H or C1-3 alkyl).

[0117] WO 2018 / 033719 discloses compounds in which a zinc chelating moiety is linked covalently to a cyclic boronate ester. However, in all of the compounds described in this earlier reference, the nitrogen in the linker part of the structure is adjacent to a carbonyl group, thus forming part of an amide rather than a secondary or tertiary amine.

[0118] Representative compounds disclosed in WO 2018 / 033719 display considerably higher minimal inhibitory concentrations (MIC) values than the compounds of the present invention.

[0119] Although not wishing to be bound by theory, a possible explanation for the observed effect of the compounds of the invention compared to those disclosed in WO 2018 / 033719 is that these include a nitrogen having a free lone pair that is not delocalised due to adjacent electron-withdrawing groups, thus allowing hydrogen-bonding to the active site of SBLs. In the compounds known in the art, the lone pair of the nitrogen is delocalised due to adjacent electron-withdrawing groups such as carbonyl or other electron deficient groups, thereby reducing or eliminating the ability of the nitrogen to take part in any hydrogenbonding.

[0120] In the compounds of formula (I), Q represents a lipophilic, zinc chelating moiety which is selective for Zn2+ions. As used herein, the term “chelating” refers to the coordination to, complexing of and / or binding of a metal, such as a metal ion, by at least two ligands, and the term “chelator” thus refers to a compound having the ability to coordinate to, complex and / or bind a metal, such as a metal ion, by at least two ligands. The term “ligand” refers to a moiety, such as a molecule, a part of a molecule, or a functional group, capable of coordinating to, complexing and / or binding a metal.

[0121] Q is a zinc chelator for chelating Zn2+. Chelators suitable for chelating zinc are known in the art and any such chelators may be employed as the zinc chelating moiety in the invention. Suitable for use in the invention are those chelators having a dissociation constant, denoted Kd.for the zinc chelating moiety with Zn2+which is less than or equal to 10'10M, preferably less than or equal to 10'12M, e.g. less than or equal to 10'14M.

[0122] Preferably, the zinc chelating moiety Q comprises one or more optionally substituted heteroaryl rings, preferably two or more optionally substituted heteroaryl rings, e.g. such heteroaryl rings in which each heteroaryl ring has at least one nitrogen atom in the ring structure. Examples of such heteroaryl rings include pyridine, e.g. unsubstituted pyridine.

[0123] In some embodiments, the zinc chelating moiety Q is derived from picolinic acid and its derivatives (e.g. from picoylamine). In some embodiments, the zinc chelating moiety comprises two or more (e.g. two, three or four) 2-pyridyl-methyl units.

[0124] Examples of the zinc chelating moiety Q include, but are not limited to, the following groups:

[0125]

[0126]

[0127] wherein

[0128] * denotes the point of attachment of the zinc chelating moiety to the linker group Li; and R’, where present, is H or C1-6 alkyl, preferably C1-3 alkyl, e.g. methyl.

[0129] In preferred embodiments, the zinc chelating moiety Q is one of the following groups:

[0130]

[0131] wherein * denotes the point of attachment of the chelating moiety to the linker group Li.

[0132] In some embodiments, Li is a covalent bond or a C1-4 alkylene chain in which one or more -CH2- groups (e.g. all -CH2- groups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR4- (where R4is H or C1-3 alkyl, preferably H).

[0133] In some embodiments, Li is a covalent bond or a C1-3 alkylene chain in which one or more -CH2- groups (e.g. all -CH2- groups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR4- (where R4is H or C1-3 alkyl, preferably H).

[0134] In some embodiments, Li is selected from the following: a covalent bond, -CH2-, -CH2-CH2-, -NR4-CH2-, -CO-, -CO-NR4-CH2-, and -CH2-CH2-NR4-CH2- (where R4is H orCi-3alkyl, preferably H). Examples of Li include the following: -CH2-, -CH2-CH2-, -CO- and -CO-NR4-CH2- (where R4is H or C1-3 alkyl, preferably H).

[0135] In some embodiments, Y is selected from the following groups:

[0136] < <

[0137]

[0138] In some embodiments, L2 is a covalent bond.

[0139] In some embodiments, R is -OH or -O-C1-6 alkyl, preferably -OH or-O-Ci-3 alkyl.

[0140] In some embodiments, R3is H. Examples of compounds of formula (I) according to the invention include the following, their stereoisomers and pharmaceutically acceptable salts:

[0141]

[0142]

[0143] As will be understood, the compounds described herein may exist in various stereoisomeric forms, including enantiomers, diastereomers, and mixtures thereof. The invention encompasses all optical isomers of the compounds described herein and mixtures of optical isomers. Hence, compounds that exist as diastereomers, racemates and / or enantiomers are within the scope of the invention. In particular, the invention extends to the enantiomers, diastereomers, and mixtures of diastereomers and / or enantiomers, of any of the compounds having a chiral centre.

[0144] In preferred embodiments, the compounds according to the invention include the following and their pharmaceutically acceptable salts:

[0145]

[0146]

[0147] Preferred compounds according to the invention include the following and their

[0148]

[0149] A preferred compound of formula (I) according to the invention is the following compound:

[0150]

[0151] and its pharmaceutically acceptable salts, in particular such a compound in the form of a disodium salt or calcium salt. The compounds according to the invention may be prepared from readily available starting materials using synthetic methods known in the art. For example, these may be prepared using methods analogous to those described in WO 2018 / 033719, the entire content of which is incorporated herein by reference. Non-limiting examples of procedures for obtaining the compounds according to the invention are shown in Examples 1-4.

[0152] The compounds of formula (I) may be resolved into their enantiomers and / or diastereomers. For example, where these contain only one chiral centre or axis, these may be provided in the form of a racemate or may be provided as pure enantiomers, i.e. in the R- or S-form. Any of the compounds of formula (I) which occur as racemates may be separated into their enantiomers by methods known in the art, such as column separation on chiral phases or by recrystallisation from an optically active solvent. Those compounds of formula (I) with at least two asymmetric centres or axes may be resolved into their diastereomers on the basis of their physical-chemical differences using methods known perse, e.g. by chromatography and / or fractional crystallisation, and where these compounds are obtained in racemic form, they may subsequently be resolved into the enantiomers.

[0153] Any of the compounds of formula (I) herein described may be provided in the form of a pharmaceutically acceptable salt. The compounds of formula (I) may be converted into a salt thereof, particularly into a pharmaceutically acceptable salt thereof with an inorganic or organic acid or base. Acids which may be used for this purpose include hydrochloric acid, hydrobromic acid, sulphuric acid, sulphonic acid, methanesulphonic acid, phosphoric acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid, maleic acid, acetic acid, trifluoroacetic acid and ascorbic acid. Bases which may be suitable for this purpose include alkali and alkaline earth metal hydroxides, for example sodium hydroxide, potassium hydroxide or cesium hydroxide, ammonia and organic amines such as diethylamine, triethylamine, ethanolamine, diethanolamine, cyclohexylamine and dicyclohexylamine. Procedures for salt formation are conventional in the art.

[0154] In one embodiment, the compounds of formula (I) may be formulated in the form of sodium, calcium or zinc salts. This may be advantageous in contributing to a balance of these ions in the body. Sources of calcium or zinc which can be used for this purpose include, but are not limited to, calcium or zinc chloride, calcium or zinc carbonate, calcium or zinc gluconate, and calcium or zinc-edetate. Preferred are calcium or zinc salts in clinical use and which are on the FDA GRAS list, for example any commercially available calcium gluconate or zinc gluconate salts of any of the compounds herein described.

[0155] A challenge related to many conventional antibiotics is their low oral bioavailability, i.e. a low percentage uptake from the gastrointestinal system to the blood following oral administration. The cause of this challenge is often related to a low logP or high watersolubility of the compound due to an excess of polar functional groups which form hydrogen bonds with the surrounding biological fluid. To address this challenge, hydrolytically or enzymatically cleavable derivatives of such polar functional groups may be used to eliminate the ability for hydrogen bonding and thus improve oral bioavailability. Such modified derivatives of any of the compounds of formula (I) herein described are examples of “prodrugs” that may be used in the invention.

[0156] Any of the compounds of formula (I) may be provided in the form of a “prodrug”. A wide variety of pro-groups suitable for masking functional groups in active compounds to provide prodrugs are well known in the art. For example, a hydroxy functional group may be masked as an ester, e.g. acetate esters, a phosphate ester, or a sulfonate ester which may be hydrolysed in vivo to provide the parent hydroxy group. An amide functional group may be hydrolysed in vivo to provide the parent amino group. A carboxyl group may be masked as an ester or amide which may be hydrolysed in vivo to provide the parent carboxyl group. Other examples of suitable pro-groups will be apparent to those of skill in the art.

[0157] The compounds of the invention may be present as an active ingredient in a desired composition, such as a dosage unit formulation, such as a pharmaceutically acceptable composition containing one or more conventional pharmaceutically acceptable carriers. As used herein, the term “composition” refers to a mixture, in any formulation, of one or more compounds according to the invention with one or more additional chemical components.

[0158] Hence, in another aspect, the invention provides a composition comprising a compound of formula (I) as disclosed herein, or a stereoisomer or pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier, diluent or excipient. The compositions of the invention are prepared from the compounds disclosed herein in substantially pure form. In some embodiments, the purity of the compound according to the invention used to formulate the composition is at least about 95%, such as at least 96%, 97%, 98%, or 99%. Preferably, the purity of the compound is at least 98%.

[0159] Conventional procedures and / or formulation techniques known in the art, e.g., conventional mixing, dissolving, suspending, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes, may be used to formulate the composition.

[0160] Procedures and techniques may be selected based on desired properties of the composition and / or based on the intended use of the composition, such as a route of administration. For example, a composition of the invention which is intended to be suitable for parenteral administration conveniently may comprise sterile aqueous solutions and / or suspensions of pharmaceutically active ingredients preferably made isotonic with the blood of the recipient generally using sodium chloride, glycerin, glucose, mannitol, sorbitol and the like. For example, a composition of the invention which is intended to be suitable for oral administration may, e.g., be in sterile purified stock powder form, preferably covered by an envelope or envelopes which may contain any of a number or adjuvants such as buffers, preservative agents, agents that promote prolonged or rapid release. For example, a composition of the invention which is intended to be suitable for local or topical administration may, e.g., comprise the therapeutic agent mixed with known suitable ingredients such as paraffin, Vaseline, cetanol, glycerol and the like, to form a suitable ointment or cream.

[0161] The compositions of the invention may be in the form of sterile aqueous solutions and / or suspensions of one or more compounds of the invention, aerosols, ointments and the like. The compositions may be in a sustained release form e.g. microparticles, nanoparticles, emulsions, nano-suspensions, lipid particles or oils. The compounds of the invention may also be used in formulations of ZnO nanoparticles described, for example, by Pati etal. in Nanomedicine (2014), 10(6), 1195-1208. In some embodiments, the compound or composition of the invention is coated onto a film, patch, or foil.

[0162] In some embodiments, the composition is formulated for a particular method of administration, such as intravenous, intracerebral, oral, parenteral, topical, subcutaneous administration or by inhalation. The compositions of the invention may further include one or more of any conventional, pharmaceutically acceptable excipients and / or carriers, e.g. solvents, fillers, diluents, binders, lubricants, glidants, viscosity modifiers, surfactants, dispersing agents, disintegration agents, emulsifying agents, wetting agents, suspending agents, agents that promote rapid onset of action, agents that promote prolonged duration of action, thickeners, buffers, pH modifiers, absorption-delaying agents, stabilisers, antioxidants, preservatives, antimicrobial agents, antibacterial agents, antifungal agents, chelating agents, adjuvants, sweeteners, aromas, and colouring agents.

[0163] Non-limiting examples of useful antioxidants include ascorbic acid, ascorbyl palmitate, ascorbyl stearate, BHA, BHT, citric acid, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate, onoisopropyl citrate, propyl gallate, EDTA, tartaric acid or any combination thereof.

[0164] Advantageously, the compositions may comprise solubilisers such as sodium taurocholate (NaTC), Labrasol, polyethylene glycol 400 (PEG 400), Transcutol P, propylene glycol, Gelucire 44 / 14, HCO-60, ethanol, Cremophor EL, Tween 80, and dimethylsulfoxide (DMSO), a- p- or y-cyclodextrins, preferably 2-hydroxypropyl-beta-cyclodextrin (2HP-beta-CyD), more preferably p-Cyclodextrin-sulfobutyl ether (Captisol).

[0165] Advantageously, the compositions may comprise one or more typical excipients approved by medical authorities like the FDA for oral and parenteral use for providing divalent metal ions like zinc and calcium, e.g. calcium gluconate (FDA Reference ID: 4112411) or zinc gluconate (FDA Code of Federal Regulations, Title 21, Volume 3, Revised as of April 1, 2020 CITE: 21CFR 182.8988) to mammals. Any other excipient for adjusting the calcium or zinc concentration in the formulation according to the invention may be used accordingly.

[0166] The compositions of the invention are preferably formulated prior to administration. The amount of the compound according to the invention present in the composition can vary. In typical embodiments, the amount of the compound of formula (I) according to the invention present in the composition is 0.05-99% by weight. In some embodiments, the amount of the compound according to the invention present in the composition is 0.05-50% by weight, such as 1-30%, such as 20-50%. In other embodiments, the amount of the compound according to the invention present in the composition is 30-70% by weight, such as 40-60%. In yet other embodiments, the amount of the compounds according to the invention present in the composition is 50-100% by weight, such as 50-70%, such as 50-80%, such as 60-98%, such as 70-95%, such as 80-99%, such as 95-100%.

[0167] Appropriate amounts may depend on the compound to be used, a precise condition to be treated using the composition, the age and / or weight of the subject to be treated, etc., and may be routinely determined by the skilled practitioner according to principles well known in the art.

[0168] Further, the composition of the invention is substantially free of contaminants or impurities. In some embodiments, the level of contaminants or impurities other than residual solvent in the composition is below about 5% relative to the combined weight of the compounds according to the invention and the intended other ingredients. In certain embodiments, the level of contaminants or impurities other than residual solvent in the composition is no more than about 2% or 1% relative to the combined weight of the compounds according to the invention and the intended other ingredients.

[0169] In some embodiments, the composition of the invention further comprises an antibacterial agent, such as an antibiotic, for example a p-lactam antibiotic.

[0170] In another aspect, the invention relates to a compound or composition as disclosed herein for use in therapy or for use as a medicament.

[0171] Thus, in this aspect, the invention relates to a compound of formula (I), or a stereoisomer or a pharmaceutically acceptable salt thereof, or a composition comprising said compound together with at least one pharmaceutically acceptable carrier, diluent, or excipient, for use in therapy.

[0172] Advantageously, the compounds of the invention may have a low logP value. A low logP value may, for example, lead to a lack of toxic effects and a low ability to pass eukaryotic cell membranes. This allows the use of the compounds and compositions of the invention for therapy and in methods of treatment in a controlled and safe manner.

[0173] Due to their strong ability to chelate metals, the compounds and compositions of the invention may be particularly useful for use in methods for the treatment of conditions, diseases and illnesses wherein down-regulation of metal ion levels may be useful.

[0174] Particularly, the compounds and compositions may be useful in the treatment of diseases or conditions associated with pathological extracellular metal ion levels, such as by removing excess metal ions, and in the treatment of bacterial infections, such as by acting as an adjuvant. As used herein, the term “adjuvant” means a substance that aids and / or enhances the effect of a drug and / or treatment.

[0175] Thus, in another embodiment, the invention relates to a compound or composition as disclosed above for use in a method for the treatment of a bacterial infection in a subject, wherein the method comprises the step of administering to the subject a therapeutically effective dose of said compound or composition in combination with at least one antibacterial agent.

[0176] The terms "treating" and "treatment" and “therapy” (and grammatical variations thereof) are used herein interchangeably, and refer to 1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in a subject who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder, including prevention of disease (i.e. prophylactic treatment, arresting further development of the pathology and / or symptomatology), or 2) alleviating the symptoms of the disease, or 3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an subject who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e. reversing the pathology and / or symptomatology). The terms may relate to the use and / or administration of medicaments, active pharmaceutical ingredients (API), food additives, food supplements, dietary supplements, nutritional supplements, over-the-counter (OTC) supplements, medical foods, and / or pharmaceutical grade supplements.

[0177] As used herein, the term “antibacterial agent” refers to both naturally occurring antibiotics, and agents synthesised or modified in the laboratory. Thus, the term encompasses all types of compounds having a bactericidal or bacteriostatic effect upon bacteria contacted by the compound. As used herein, the term "bactericidal" means having a destructive killing action upon bacteria. As used herein, the term "bacteriostatic" means having an inhibiting action upon the growth of bacteria. As used herein, the term “bacteria” refers to all bacterial organisms, including but not limited to both gram-positive and gram-negative bacteria.

[0178] As used herein, the term “bacterial infection” refers to the invasion of a host, such as a mammal, by pathogenic bacteria, and also encompasses the excessive growth of bacteria which are normally present in or on the body of the host. More generally, a bacterial infection can be any situation in which the presence of a bacterial population(s) is damaging to a host. Thus, a subject is “suffering” from a bacterial infection when excessive numbers of a bacterial population are present in or on their body, or when the effects of the presence of a bacterial population(s) is damaging the cells or other tissue of said subject.

[0179] Thus, in this aspect, the invention relates to a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof, or a composition comprising said compound together with at least one pharmaceutically acceptable carrier, diluent, and / or excipient, for use in a method for the treatment of a bacterial infection in a subject, wherein the method comprises the step of administering to the subject a therapeutically effective dose of said compound or composition in combination with at least one antibacterial agent.

[0180] The compounds and compositions of the invention may have the ability to affect bacteria-specific enzymes, such as p-lactamases, which may allow the compounds and compositions to affect metal homeostasis in bacteria without affecting a host that the bacteria are present in. In particular, the compounds and compositions surprisingly have been found to have the ability to efficiently regulate, such as inhibit, both bacterial metallo-P-lactamases (MBLs) and bacterial serine-p-lactamases (SBLs). This ability to act as regulators of both MBLs and SBLs gives the compounds and compositions of the invention an unprecedentedly wide scope of use.

[0181] A combination of a compound or composition of the invention with an antibacterial agent may result in a synergistic effect between the compound and the antibacterial agent, such as by the compound acting as an adjuvant by enhancing and / or aiding the effect of the antibacterial agent. The compounds and compositions of the invention may be used together with any known antibacterial agent. Their adjuvant effect may advantageously result in a lowering of the minimal inhibitory concentration (MIC) of the antibacterial agent, and may thus lower the amount of antibacterial agents, such as antibiotics, used in society. Further, their adjuvant effect may reinstall the sensitivity of resistant bacteria to antibacterial agents, such as antibiotics. Thus, the compounds of the invention may be a useful tool in the treatment of drug-resistant bacteria.

[0182] In some embodiments, the antibacterial agent is an antibiotic, such as a marketed antibiotic. Non-limiting examples of antibacterial agents include p-lactam antibiotics such as penicillins, e.g. ampicillin, cioxacillin, oxacillin, and piperacillin; cephalosporins and other cephems, e.g. cefaclor, cefamandole, cefazolin, cefoperazone, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, and cephalothin; carbapenems, e.g. imipenem and meropenem; penams; monobactams; penems; and clavams. Other antibacterial agents include glycopeptides, macrolides, quinolones, tetracyclines, aminoglycosides, tetracyclines, rifamycins, sulfonamides, trimethoprim, aminoglycosides, polymyxins, macrolides, chloramphenicol, oxazolidinones, glycopeptides, cycloserine, isoniazide, daptomycin, cyclosporine, phenazines, and derivatives thereof.

[0183] In some embodiments, the antibacterial agent is a p-lactam antibiotic, such as a carbapenem, cephalosporin, penicillin or oxapenem.

[0184] In some embodiments, the antibacterial agent is meropenem.

[0185] The bacterial infection may be associated with gram-positive bacteria. The bacterial infection may be associated with gram-negative bacteria. The bacterial infection may be associated with a combination of gram-negative and gram-positive bacteria. In some embodiments, the bacterial infection may be associated with bacteria that are resistant to one or more antibiotics, such as gram-positive and / or gram-negative bacteria that are resistant to one or more antibiotics.

[0186] In some embodiments, the bacterial infection is associated with bacteria harbouring p-lactamases. The bacterial infection may be associated with bacteria that comprise metallo-p-lactamases. The bacterial infection may be associated with bacteria that comprise serine-p-lactamases (SBLs). The bacterial infection may be associated with bacteria that comprise serine-p-lactamases and metallo-p-lactamases.

[0187] In some embodiments, the bacterial infection is associated with drug-resistant bacteria, such as multidrug-resistant bacteria.

[0188] In preferred embodiments, the bacterial infection is selected from the group comprising or consisting of nocosomial infections, complicated UTIs, hospital-acquired pulmonary infections (HAP), wound infections, muscle infections, and intraperitoneal infections. In this aspect, the patient group comprises patients having an undesired invasion or infection by one or more bacteria. In preferred embodiments, a subject is selected from this patient group. The subject may be a human or a non-human animal, preferably a warm-blooded animal, such as a human or non-human mammal, preferably a human patient. The subject may be male or female. In some embodiments, the subject is an adult (i.e. 18 years of age or older). In certain embodiments, the subject is geriatric. In certain embodiments, the subject is not geriatric. In certain embodiments, the subject is paediatric. In certain embodiments, the subject is not paediatric.

[0189] The compound or composition of the invention may be administered in combination with at least one antibacterial agent. As used herein, the term administration “in combination with” may mean that the compound or composition is administered simultaneously, intermittent, staggered, prior to, subsequent to, or combinations of these, with the administration of the antibacterial agent. The antibacterial agent may be provided the same formulation as said compound or composition, or in a separate formulation from said compound or composition.

[0190] As used herein, “subject” means any human or non-human animal, such as a mammal, selected for treatment or therapy, and encompasses, and may be limited to, “patient”. None of the terms should be construed as requiring the supervision (constant or otherwise) of a medical professional (e.g. physician, nurse, nurse practitioner, physician's assistant, orderly, clinical research associate, etc.) or a scientific researcher.

[0191] The compound or composition for use in a method for the treatment of a bacterial infection according to the invention will be administered to a subject in a therapeutically effective dose. As used herein, the term “therapeutically effective dose” means the amount of compound according to the invention which is effective for producing the desired therapeutic effect, such as the desired therapeutic effect of the compound, such as the desired therapeutic effect of the antibacterial agent, in a subject at a reasonable benefit / risk ratio applicable to any treatment. The therapeutically effective dosage amount may vary depending upon the route of administration and dosage form. Appropriate dosages may depend on the compound to be used, the stage of the condition, age and / or weight of the patient, etc. and may be routinely determined by the skilled practitioner according to principles well known in the art. When used in a method for the treatment of a bacterial infection, a suitable daily dosage of the compound according to the invention may range from about 0.1 to 100 mg / kg body weight, such as 15-20 mg / kg bodyweight, while the daily dosage of the antibacterial agent may typically range from 0.01-200 mg / kg body weight, such as from 1 to 500 mg / kg body weight, such as 4-50 mg / kg body weight. When used in a method for the treatment of a disease or condition associated with pathological extracellular metal ion levels, a suitable daily dosage of the compound according to the invention may range from about 0.5 to 100 mg / kg body weight, such as 1-30 mg / kg bodyweight.

[0192] As used herein, the terms “administer”, “administration”, and “administering” refer to (1) providing, giving, dosing and / or prescribing by either a health practitioner or their authorised agent or under their direction, or by self-administration, a formulation, preparation or composition according to the present disclosure, and (2) putting into, taking or consuming by the subject themselves, a formulation, preparation or composition according to the present disclosure.

[0193] The therapeutically effective dose can be administered in a single dose or in divided doses. The compound or composition according to the invention can be administered once, twice or more times a day, once every two days, once every three days, twice a week or once a week, or as deemed appropriate by a medical professional. In certain embodiments, the compound or composition according to the invention is administered once daily. In other embodiments, the compound or composition according to the invention is administered twice daily. In some embodiments, the dosage regimen is predetermined and the same for the entire patient group. In other embodiments, the dosage and the frequency of administration of treatment with the compound or composition according to the invention is determined by a medical professional, based on factors including, but not limited to, the stage of the disease, the severity of symptoms, the route of administration, the age, body weight, general health, gender and / or diet of the subject, and / or the response of the subject to the treatment.

[0194] In some embodiments, the therapeutically effective dose is administered at regular intervals. In other embodiments, the dose is administered when needed or sporadically. The compound or composition according to the invention may be administered by a medical professional or by self-administration. The compound or composition according to the invention may, depending on factors such as formulation and route of administration, be administered with food or without food. In some embodiments, the compound or composition according to the invention is administered at specific times of day. Preferred unit dosage formulations are those containing a therapeutically effective dose, as hereinbefore recited, or an appropriate fraction thereof, of a compound according to the invention. A composition for use in a method for the treatment of a bacterial infection or in a method for the treatment of a disease or condition associated with pathological extracellular metal ion levels may be presented in unit dosage form as a single dose wherein all active and inactive ingredients are combined in a suitable system and components do not need to be mixed before administration. Alternatively, a composition may be presented as a kit in which the drug, excipients and carriers are provided in two or more separate containers (e.g. ampules, vials, tubes, bottles or syringes) and need to be combined to form the composition to be administered.

[0195] In some embodiments, the treatment of a bacterial infection using a compound or composition of the invention may be sustained until the bacterial infection is cured, such as until a medical professional determines that no treatment is necessary. In some embodiments, the treatment of a disease or condition associated with pathological extracellular metal ion levels using a compound or composition according to the invention may be sustained until no further improvement can be expected, such as determined by a medical professional. In certain embodiments, the duration of the treatment with the compound or composition according to the invention is at least two weeks, at least one month, at least three months, such as three months, six months, nine months, a year, three years, five years. In other embodiments, the duration is determined by a medical professional, based on factors including but not limited to the nature and severity of symptoms, the route of administration, the age, body weight, general health, gender and / or diet of the subject, and / or the response of the subject to the treatment. In other embodiments, the compound or composition is administered chronically, such as in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect for an extended period of time. The latter may be particularly useful e.g. when the compound or composition of the invention is used prophylactically in the treatment of a disease or condition associated with pathological extracellular metal ion levels.

[0196] The compound or composition may advantageously be administered in a single dose to be taken at regular intervals, e.g. once or twice a day, once every 48 hours, or once every 72 hours. Sustained formulations may be given at longer intervals, e.g. 1 to 2 times a month or every three months. The precise dosage of the active compounds to be administered, the number of daily or monthly doses, and the length of the course of treatment may depend on a number of factors, but can readily be determined by those skilled in the art.

[0197] The compound or composition for use in a method for the treatment of a bacterial infection or in a method for the treatment of a disease or condition associated with pathological extracellular metal ion levels according to the invention may be administered locally or systemically. The administration may be performed using any suitable method known in the medicinal arts, including but not limited to, intracerebrally, parenterally, topically, pulmonary, orally, intraperitoneally, intravenously, intramuscularly, sublingually, subcutaneously, intrathecally, buccally, rectally, vaginally, occularly, nasally, transdermally, cutaneously, intracranially, intraarterially, or by inhalation.

[0198] In some embodiments, the compound or composition is administered orally. In some embodiments, the compound or composition is administered with a meal or before a meal.

[0199] In some embodiments, the compound or composition according to the invention is administered intravenously. In these embodiments, water is a particularly useful excipient. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions.

[0200] In the case of treatment of a bacterial infection, an especially attractive administration regime is continuous systemic administration of the compound combined with administration of the respective antibacterial agent. In the case of sepsis caused by resistant gram-negative bacteria, the compound may advantageously be intravenously continuously infused, while an antibacterial agent, e.g. a carbapenem (such as meropenem or imipenem), is administered as approved by the respective regulatory authorities. Continuous intravenous infusion of a compound according to the present invention may also be used simultaneously with other administration routes of antibacterial agents, e.g. by inhalation of a micronised formulation, by oral intake of a tableted antibiotic agent, or by topical administration of the drug.

[0201] Advantageously, the compound or composition for use in a method for the treatment of a bacterial infection or in a method for the treatment of a disease or condition associated with pathological extracellular metal ion levels is sterile. Sterilisation can be achieved by any suitable method, including but not limited to by applying heat, chemicals, irradiation, high pressure, filtration, or combinations thereof. In another aspect, the compounds of the invention are also useful against biofilm.

[0202] Biofilms comprise extracellular polymeric substances (EPS) that are natural hydrophilic carbohydrate polymers, forming extracellularly in a host organism. Since a key feature of the compounds of the invention is their low logP, they interact particularly well with EPS. Since the compounds and composition of the invention have a high efficacy against multidrug-resistant gram-negative species producing MBL, they can be used to reduce and / or prevent biofilm formation in infections caused by bacterial species producing MBL. Non-limiting examples include Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli.

[0203] The compounds and compositions of the invention may also be used against biofilm formation with gram-positive bacteria. Non-limiting examples of such bacteria include e.g. variants of Staphylococcus epidermidis and methicillin-resistant S. aureus (MRSA).

[0204] Species of gram-positive bacteria have been shown to be inhibited by zinc chelators of the APC class and TPEN, because of their zinc-dependent adhesion molecules mandatory for the biofilm formation, e.g. as described by Conrady etal., PNAS (2008), 105 (49), 19456-19461. The compounds of the invention show high metal chelating selectivity and low toxicity, rendering them promising candidates for use against biofilm.

[0205] Thus, in this aspect, the invention provides the use of a compound or a composition comprising said compound together with at least one pharmaceutically acceptable carrier, diluent, or excipient, for non-therapeutic removal, limitation and / or prevention of biofilm.

[0206] An example of such non-therapeutic purpose is treatment of medical devices, such as medical devices for use in a hospital.

[0207] In another aspect, the invention relates to a kit comprising:

[0208] (i) a first container containing a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof, or a composition comprising said compound together with at least one pharmaceutically acceptable carrier, diluent, or excipient; and

[0209] (ii) a second container containing an antibacterial agent.

[0210] The kit may contain one or more compounds according to the invention or a composition according to the invention. The composition of the invention may, in certain embodiments, be divided into more than one (the first) container. The kit may further contain instructions for storing, preparing, administering and / or using the composition resulting from the combination of the contents of the containers. The skilled person understands that the first and / or the second container may contain further contents, such as a solvent, for example.

[0211] In a further aspect, the invention provides a method of treating a bacterial infection in a human or non-human mammal, the method comprising the step of administering to the mammal a therapeutically effective dose of a compound or composition of the invention in combination with an antibacterial agent.

[0212] As described herein, the compounds of formula (I) may also be used in a combination therapy for the treatment of bacterial infections with other known adjuvants and antibacterial agents. As demonstrated herein, the effect of these agents in combination is synergistic. This finding is unexpected and provides additional advantages when all of these agents are used as part of the same therapeutic regime in the treatment of bacterial infections, particularly those associated with MBLs and SBLs.

[0213] In a further aspect the invention thus provides a method of treatment of a bacterial infection, said method comprising co-administration of an effective amount of each of the following agents to a subject in need thereof:

[0214] (A) a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof as herein described;

[0215] (B) a p-lactam antibiotic; and

[0216] (C) a serine p-lactamase inhibitor.

[0217] In such combination therapy, the first agent (A) is a selective zinc-chelator which is a compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof. All embodiments and features disclosed herein for this compound apply equally to this aspect of the invention.

[0218] In some embodiments, the second agent (B) is a p-lactam antibiotic selected from the group consisting of the penicillins, monobactams and carbapenems.

[0219] In some embodiments, the second agent (B) is a penicillin. In some embodiments, the penicillin may be selected from the group consisting of cioxacillin, dicloxacillin, flucioxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, pivampicillin, bacampicillin, metampicillin, talampicillin, hetacillin, epicillin, phenoxymethylpenicillin, benzylpenicillin, carboxypenicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, piperacillin and azlocillin. Any of these may be used in the form of a pharmaceutically acceptable salt.

[0220] In some embodiments, the penicillin is ampicillin, amoxicillin, temocillin, piperacillin, pivampicillin, or any pharmaceutically acceptable salt thereof. In some embodiments, the penicillin is ampicillin, amoxicillin, temocillin, piperacillin, or any pharmaceutically acceptable salt thereof. In some embodiments, the penicillin is amoxicillin or a pharmaceutically acceptable salt thereof.

[0221] In some embodiments, the penicillin is piperacillin, amoxicillin, ampicillin, ora pharmaceutically acceptable salt thereof. In some embodiments, the penicillin is piperacillin or amoxicillin, or a pharmaceutically acceptable salt thereof.

[0222] In some embodiments, the second agent (B) is a monobactam. Monobactams are monocyclic, bacterially-produced p-lactam antibiotics or chemically synthesised equivalents thereof.

[0223] Any known monobactam antibiotic may be used in the invention. Non-limiting examples of monobactams include aztreonam, aztreonam lysine, tigemonam, nocardicin A, tabtoxin, BAL 30072, SYN 2416 (BAL 19764), carumonam, AIC 499, BOS 228 (LYS 228), MC-1, and their pharmaceutically acceptable salts. Some of these monobactams are shown in the following table:

[0224]

[0225]

[0226] In some embodiments, the monobactam for use in the invention is aztreonam or a pharmaceutically acceptable salt thereof. Aztreonam is a synthetic version of a chemical obtained from the bacterium Chromobacterium violaceum.

[0227] In some embodiments, the second agent (B) is a carbapenem. Carbapenems are -lactam antibiotics which kill bacteria by binding to penicillin-binding proteins thus inhibiting bacterial cell wall synthesis. Examples of carbapenems that may be used in the invention include, but are not limited to, benapenem, biapenem, doripenem, ertapenem, imipenem, lenapenem, meropenem, panipenem, razupenem, tebipenem, tebipenem (e.g. tebipenem pivoxil), thienpenem (also known as thienamycin), tomopenem and derivatives thereof. Derivatives include prodrug forms such as any of those herein described (e.g. esters), and any pharmaceutically acceptable salts thereof.

[0228] In some embodiments, the carbapenem is selected from meropenem, doripenem, imipenem, tebipenem, and any derivatives thereof. In some embodiments, the carbapenem is meropenem or a derivative thereof. In some embodiments, the carbapenem is meropenem. Preferred derivatives of carbapenems include the pharmaceutically acceptable salts thereof, for example the sodium salts. In one embodiment, the carbapenem is meropenem in the form of its sodium salt.

[0229] In some embodiments, the second agent (B) for use in the invention is a p-lactam antibiotic which is a cephalosporin.

[0230] Cephalosporins are well known and documented in the art. Suitable examples include, but are not limited to, the compounds described in the following patent publications, the entire contents of which are incorporated herein by reference: US 2018 / 0064691, US 5,958,915, US 9,949,982, US 2015 / 374673, US 5,599,557, WO 00 / 65920,

[0231] WO 2010 / 136423, US 11,278,622, WO 2014 / 068388, and CA 2831421.

[0232] Non-limiting examples of cephalosporins for use in the invention include the following compounds, their pharmaceutically acceptable salts and prodrugs:

[0233] "

[0234]

[0235] "

[0236] " >

[0237] > >

[0238] >

[0239] <

[0240]

[0241]

[0242] >

[0243] > >

[0244] >

[0245]

[0246] In some embodiments, the cephalosporin is selected from the group consisting of cefepime, cefditoren pivoxil, cefixime, cefpodoxime, cefotaxime, ceftaroline, ceftazidime, ceftibuten, ceftobiprole, ceftolozane, ceftriaxone, cefotaxone, cephmetazole, cephalexin, cefiderocol, and their pharmaceutically acceptable salts.

[0247] In some embodiments, the cephalosporin is selected from the group consisting of consisting of cefepime, ceftaroline, ceftobiprole, ceftriaxone, cefotaxone, cephmetazole, cephalexin, cefiderocol, and their pharmaceutically acceptable salts. In some embodiments, the cephalosporin is selected from the group consisting of cefepime, ceftolozane, cefpodoxime, ceftaroline, ceftriaxone, cefiderocol, and their pharmaceutically acceptable salts.

[0248] In one embodiment, the cephalosporin is ceftriaxone or a pharmaceutically acceptable salt thereof.

[0249] The third agent (C) for use in the invention is a serine p-lactamase inhibitor. A serine p-lactamase inhibitor is a compound which inhibits the activity of at least one serine p-lactamase (SBL) in the Ambler classes A, C or D. Serine p-lactamases (SBLs) are characterised by an active site serine.

[0250] In some embodiments, the third agent (C) is one which inhibits at least one SBL in Ambler class A, for example CepA, KPC-2, IMI-1, SME-1, PC1, TEM-1, TEM-2, TEM-3, TEM-30, TEM-50, SHV-1, SHV-2, SHV-10, CTX-M-15, PER-1, VEB-1, PSE-1, CARB-3, or RTG-4. In some embodiments, the third agent (C) is one which inhibits at least one SBL in Ambler class C, for example AmpC, CMY-1, ACT-1, FOX-1, MIR-1, GC1, CMY-10, CMY-19, or CMY-37. In some embodiments, the third agent (C) is one which inhibits at least one SBL in Ambler class D, for example OXA-1, OXA-10, OXA-11, OXA-15, OXA-23, OXA-48.

[0251] In some embodiments, the serine p-lactamase inhibitor for use in the invention is a diaza-bicyclo-octanone (DBO) compound, a prodrug ora pharmaceutically acceptable salt thereof. Such compounds are well known and documented in the art. Suitable examples include, but are not limited to, any of the compounds described in the following patent publications, the entire contents of which are incorporated herein by reference:

[0252] WO 2020 / 030761 , WO 2018 / 208557, WO 2021 / 041616, US 2015 / 0374673,

[0253] US 10,722,521, WO 02 / 10172, US 9,695,122, WO 2016 / 157057, WO 2013 / 149121, WO 2018 / 053215, WO 2017 / 055922, WO 2019 / 145784, WO 2016 / 156348,

[0254] WO 2016 / 177862, WO 2018 / 141986, WO 2018 / 060484, WO 2014 / 091268,

[0255] WO 2016 / 116878, WO 2009 / 091856, WO 2013 / 038330 and WO 2013 / 030733. No n-limiting examples of diaza-bicyclo-octanone (DBO) compounds for use in the

[0256] invent ion include the following compounds, their pharmaceutically acceptable salts and prodrugs:

[0257] "

[0258] "

[0259]

[0260] <

[0261] <

[0262] &

[0263] <

[0264] <

[0265]

[0266] In one embodiment, the serine p-lactamase inhibitor for use in the invention is avibactam or a pharmaceutically acceptable salt thereof. In one embodiment, avibactam is provided in the form of the sodium salt. Salts of avibactam for use in the invention may be provided in any known polymorphic or pseudopolymorphic forms (i.e. “crystalline forms”) such as, but not limited to, the crystalline forms disclosed in WO 2011 / 042560, the entire content of which is incorporated herein by reference. In one embodiment, a salt of avibactam may be used in crystalline Form “A”, “B”, “C”, “D” or “E”, preferably in crystalline “Form B”. In one embodiment, the sodium salt of avibactam may be used in crystalline form “A”, “B”, “C”, “D” or “E”, preferably in crystalline “Form B”. Any crystalline form may be provided in anhydrous or in hydrated form.

[0267] In some embodiments, the serine p-lactamase inhibitor for use in the invention is a p-lactam.

[0268] In some embodiments, the serine p-lactamase inhibitor for use in the invention is a nonantibiotic p-lactam. As used herein, the term “non-antibiotic p-lactam” is intended to refer to a p-lactam compound having negligible intrinsic antimicrobial activity. Non-limiting examples of such compounds include clavulanic acid, sulbactam, tazobactam and enmetazobactam, their pharmaceutically acceptable salts or prodrugs thereof.

[0269] In some embodiments, the serine p-lactamase inhibitor for use in the invention is sulbactam, tazobactam or a pharmaceutically acceptable salt or prodrug thereof. In some embodiments, the serine p-lactamase inhibitor is sulbactam or a pharmaceutically acceptable salt thereof.

[0270] In some embodiments, the third agent (C) for use in the invention is a cyclic boronate ester (also referred to as a “cyclic boronic acid ester”) having serine p-lactamase inhibitory activity, a prodrug or a pharmaceutically acceptable salt thereof.

[0271] As will be understood, the cyclic boronate ester selected for use in any method of treatment, medical use, composition or combination according to the invention will differ from the first agent (A) (which comprises a cyclic boronate ester moiety). Preferably, the cyclic boronate ester for use as the third agent (C) is not a zinc chelator, i.e. it is not capable of chelating Zn2+ions.

[0272] Cyclic boronate esters having p-lactamase inhibitory activity are well known and documented in the art. Suitable examples include, but are not limited to, the compounds described in the following patent publications, the entire contents of which are incorporated herein by reference: WO 2018 / 005662, WO 2014 / 089365, WO 2012 / 021455, and WO 2015 / 191907.

[0273] Specific examples of cyclic boronate esters for use in the invention include the following compounds, th eir pharmaceutically acceptable salts and prodrugs:

[0274]

[0275] In some embodiments, the cyclic boronate ester for use in the invention is vaborbactam, taniborbactam, xeruborbactam or a pharmaceutically acceptable salt thereof. In one embodiment, the cyclic boronate ester for use in the invention is xeruborbactam or a pharmaceutically acceptable salt thereof. In embodiments of any of the methods, uses, combinations or pharmaceutical compositions herein described, the selective zinc-chelator (A) is the following compound, a stereoisomer, a pharmaceutically acceptable salt, or a prodrug thereof:

[0276]

[0277] and either:

[0278] the second agent (B) is amoxicillin, piperacillin, ora pharmaceutically acceptable salt thereof, and the third agent (C) is sulbactam, avibactam, tazobactam, or a pharmaceutically acceptable salt thereof; or

[0279] the second agent (B) is meropenem or a pharmaceutically acceptable salt thereof, and the third agent (C) is sulbactam, avibactam, or a pharmaceutically acceptable salt thereof; or the second agent (B) is cefepime, ceftolozane, cefiderocol, cefpodoxime, ceftriaxone, ceftaroline, or a pharmaceutically acceptable salt thereof, and the third agent (C) is avibactam, sulbactam, tazobactam, xeruborbactam, ora pharmaceutically acceptable salt thereof.

[0280] In preferred embodiments of any of the methods, uses, combinations or pharmaceutical compositions herein described, the selective zinc-chelator (A) is the following compound, a stereoisomer, a pharmaceutically acceptable salt, or a prodrug thereof:

[0281]

[0282] and

[0283] the second agent (B) is ceftriaxone or a pharmaceutically acceptable salt thereof, and the third agent (C) is sulbactam or a pharmaceutically acceptable salt thereof. Any of the compounds (A), (B) and (C) herein described may be provided in the form of a pharmaceutically acceptable salt. These compounds may be converted into a salt thereof, particularly into a pharmaceutically acceptable salt thereof with an inorganic or organic acid or base. Acids which may be used for this purpose include hydrochloric acid, hydrobromic acid, sulphuric acid, sulphonic acid, methanesulphonic acid, phosphoric acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid, maleic acid, acetic acid, trifluoroacetic acid and ascorbic acid. Bases which may be suitable for this purpose include alkali and alkaline earth metal hydroxides, for example sodium hydroxide, potassium hydroxide or cesium hydroxide, ammonia and organic amines such as diethylamine, triethylamine, ethanolamine, diethanolamine, cyclohexylamine and dicyclohexylamine. Procedures for salt formation are conventional in the art.

[0284] In one embodiment, the compounds (A), (B) or (C) may be formulated in the form of sodium, calcium or zinc salts. This may be advantageous in contributing to a balance of these ions in the body. Sources of calcium or zinc which can be used for this purpose include, but are not limited to, calcium or zinc chloride, calcium or zinc carbonate, calcium or zinc gluconate, and calcium or zinc-edetate. Preferred are calcium or zinc salts in clinical use and which are on the FDA GRAS list, for example any commercially available calcium gluconate or zinc gluconate salts of any of the compounds (A), (B) or (C) as herein described.

[0285] A challenge related to many conventional antibiotics is their low oral bioavailability, i.e. a low percentage uptake from the gastrointestinal system to the blood following oral administration. The cause of this challenge is often related to a low logP or high watersolubility of the compound due to an excess of polar functional groups which form hydrogen bonds with the surrounding biological fluid. To address this challenge, hydrolytically or enzymatically cleavable derivatives of such polar functional groups may be used to eliminate the ability for hydrogen bonding and thus improve oral bioavailability. Such modified derivatives of any of the compounds (A), (B) and (C) herein described are examples of “prodrugs” that may be used in the invention.

[0286] Any of the compounds (A), (B) and (C) herein described may be provided in the form of a “prodrug”. As defined herein, the term "prodrug" refers to a derivative of an active compound which undergoes a transformation under the conditions of use, for example within the body, to release an active drug. A prodrug may, but need not necessarily, be pharmacologically inactive until converted into the active drug. As used herein, the term “prodrug” extends to any compound which under physiological conditions is converted into any of the agents (A), (B) and (C) as herein described.

[0287] Suitable prodrugs of agents (A), (B) and (C) include compounds which are hydrolysed under physiological conditions to the desired molecule. Prodrugs may typically be obtained by masking one or more functional groups in the parent molecule which are considered to be, at least in part, required for activity using a pro-group. By “pro-group” as used herein is meant a group which is used to mask a functional group within an active drug and which undergoes a transformation, such as cleavage, under the specified conditions of use (e.g. administration to the body) to release a functional group and hence provide the active drug. Pro-groups are typically linked to the functional group of the active drug via a bond or bonds that are cleavable under the conditions of use, e.g. in vivo. Cleavage of the pro-group may occur spontaneously under the conditions of use, for example by way of hydrolysis, or it may be catalysed or induced by other physical or chemical means, e.g. by an enzyme, or by exposure to a change in pH, etc. Where cleavage is induced by other physical or chemical means, these may be endogenous to the conditions of use, for example pH conditions at a target site, or these may be supplied exogenously.

[0288] A wide variety of pro-groups suitable for masking functional groups in active compounds to provide prodrugs are well known in the art. For example, a hydroxy functional group may be masked as an ester, e.g. acetate esters, a phosphate ester, or a sulfonate ester which may be hydrolysed in vivo to provide the parent hydroxy group. An amide functional group may be hydrolysed in vivo to provide the parent amino group. A carboxyl group may be masked as an ester or amide which may be hydrolysed in vivo to provide the parent carboxyl group. Other examples of suitable pro-groups will be apparent to those of skill in the art.

[0289] In one embodiment, the compounds (A), (B) and (C) for use in the invention have a hydroxy functional group that can be derivatised to produce suitable prodrugs. For example, the hydroxy group in a parent molecule can be converted to an alkyl or aryl ester, a phosphate ester, or a sulfonate ester. Non-limiting examples of such derivatisation are illustrated below: parent parent

[0290] molecule molecule

[0291] parent

[0292] molecule

[0293]

[0294] In the parent molecule of the prodrug, Ri, R2 and R3 = H. In the corresponding prodrug, R1 , R2 and R3 may be a group exemplified by the following groups:

[0295]

[0296] wherein R4 to Rs are independently selected from H, C1-6 haloalkyl and C1-6 alkyl; and Li and L2 are linking groups, for example an optionally halogenated, straight chained or branched, C1-6 alkylene or C1-6 alkenylene group.

[0297] The combination of agents (A), (B) and (C) herein described finds use in therapy. The invention thus provides compositions and combinations of these agents as described herein for use as a medicament or for use in therapy.

[0298] The agents (A), (B) and (C), compositions containing such agents and combinations of these agents may be used in the treatment of a bacterial infection in a subject. The present invention therefore provides methods of treatment of bacterial infections in a subject in need thereof, which methods comprise administering to said subject an effective amount of such agents, compositions or combinations as described herein. The invention also provides agents, compositions or combinations as herein described for use in treating a bacterial infection. It also provides the use of agents, compositions or combinations as herein described in the manufacture of a medicament for use in the treatment of a bacterial infection. All features and embodiments disclosed herein relating to the use of the compound of formula (I) in the treatment of bacterial infections apply equally to these additional aspects of the invention.

[0299] In some embodiments relating to the combined use of agents (A), (B) and (C) herein described, the subject is a human or non-human mammal. The term “mammal” is used in its usual biological context and includes humans, dogs, cats, horses, cattle, dogs, cats, rats and mice. In further embodiments, the subject is a human. The term “bacterial infection” refers to the invasion of the host organism by one or more species of pathogenic bacteria. A bacterial infection will generally be understood to occur when the presence of the bacterial population causes damage or harm to the host organism, i.e. to the subject, for example damage or harm to the host’s cells, tissues or organs.

[0300] As used herein, the terms “treat, “treatment” or “treating” refer to administration of an agent or combination of agents for therapeutic or prophylactic purposes. Therapeutic treatment is effective to relieve, to some extent, one or more symptoms of the infection. It refers to administering treatment to a subject that is already suffering from an infection. In some embodiments, therapeutic treatment includes clinical cure, i.e. elimination of symptoms of an active infection, including elimination of the underlying cause of the infection (i.e. viable microbes involved in the infection). Prophylactic treatment refers to the treatment of a subject who is not yet infected, but who is susceptible to, or otherwise at risk of, an infection. Prophylactic treatment reduces the chances that the subject will develop an infection.

[0301] Bacterial infections that can be treated in accordance with the invention can comprise a wide spectrum of bacteria. Example organisms include gram-positive and gram-negative bacteria.

[0302] In some embodiments, the bacterial infection is associated with gram-positive and / or gram-negative bacteria. In some embodiments, the bacterial infection is associated with gram-negative bacteria.

[0303] In some embodiments, the infection is associated with gram-positive or gram-negative bacteria which are resistant to treatment with one or more conventional antibiotics when administered alone, particularly bacteria that are resistant to treatment with p-lactam antibiotics.

[0304] In some embodiments, the bacterial infection is associated with gram-positive or gramnegative bacteria which produce metallo-p-lactamases. In some embodiments, the bacterial infection is associated with gram-negative bacteria which produce metallo-p-lactamases. In some embodiments, the infection is associated with gram-negative multiresistant bacteria harbouring extended spectrum metallo-p-lactamases (ESBL). In some embodiments, the infection is associated with gram-positive or gram-negative bacteria which produce serine-p-lactamases. In some embodiments, the bacterial infection is associated with gram-negative bacteria which produce serine-p-lactamases.

[0305] The agents (A), (B) and (C), compositions containing them and combinations of such agents herein described find particular use in the treatment of bacterial infections caused by bacteria which are resistant to treatment with antibiotics when administered alone, particularly where the resistance is caused by SBLs. The agents, compositions and combinations are therefore useful in the elimination or reduction of antibiotic resistance, in particular in gram-negative bacteria. In particular, they are useful in eliminating or reducing resistance caused by SBLs.

[0306] In some embodiments, the agents (A), (B) and (C), compositions containing them and combinations of such agents herein described may be used in the treatment of a bacterial infection which occurs after a relapse following an antibiotic treatment. These can therefore be used in the treatment of a subject (e.g. a patient) who has previously received antibiotic treatment for the bacterial infection.

[0307] In some embodiments, the infection is caused by a bacteria selected from the group consisting of Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Aeromonas spp, Aeromones hydrophilia, Bacillus cereus Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Bacteroides thetaiotaomicron, Borrelia burgdorferi, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Burkholderia cepacia, Branhamella catarrhalis, Campylobacterfetus, Campylobacter jejuni, Campylobacter coli, Chryseobacterium indoIogenes Citrobacter freundii, Clostridium difficile, Corynebacterium diphtheriae, Corynebacterium ulcerans, Elizabethkingia meningoseptica, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Francisella tularensis, Gardnerella vaginalis, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, Kingella, Moraxella, Klebsiella pneumoniae, Klebsiella oxytoca, Legionella pneumophila, Listeria monocytogenes, Morganella morganii, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Moraxella, Mycobacterium leprae, Myroides odoratimimus, Neisseria gonorrhoeae, Neisseria meningitidis Pasteurella multocida, Pasteurella haemolytica, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonasfluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonasputida, Serratia marcescens, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Serratia marcescens, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saccharolyticus. Stenotrophomonas maltophilia, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Yersinia enterocolitica, and Yersinia pestis.

[0308] In some embodiments of the invention, the infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae, Acinetobacter baumanii, Pseuodomonas aeruginosa and Escherichia coli.

[0309] In some embodiments of the invention, the infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae K66-45, Klebsiella pneumoniae ST147, Klebsiella pneumoniae ST101, Klebsiella pneumoniae BAA1705, Acinetobacter baumannii ST15, Pseudomonas aeruginosa ST773, Pseudomonas aeruginosa ST111 , Escherichia coli ST2083 and Escherichia coli ST95.

[0310] In some embodiments of the invention, the infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae K66-45, Klebsiella pneumoniae ST147, Klebsiella pneumoniae ST101, Klebsiella pneumoniae BAA1705, Acinetobacter baumannii ST 1 , Acinetobacter baumannii ST2, Acinetobacter baumannii ST15, Acinetobacter baumannii ST25, Pseudomonas aeruginosa ST1047, Pseudomonas aeruginosa ST773, Pseudomonas aeruginosa ST111 , and Escherichia coli BAA2469.

[0311] In some embodiments of the invention, the bacterial infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae Kpn St147, Klebsiella pneumoniae ST101, Klebsiella pneumoniae K66-45 and Klebsiella pneumoniae BAA1705. In some embodiments, the agents (A), (B) and (C), compositions containing them and combinations of such agents herein described may be employed to treat a bacterial biofilm. As used herein, the term “bacterial biofilm” means a community of bacteria which are contained within an extracellular polymeric substance (EPS) matrix produced by the bacteria and attached to a body surface. Treatment of a bacterial biofilm may be effective to disrupt, remove or detach at least part of a bacterial biofilm. In some embodiments, treatment will be effective to eradicate a biofilm.

[0312] Preferred combinations of agents (A), (B) and (C) for use in the invention are those which demonstrate enhanced (e.g. synergistic) activity in any of the treatment methods herein described, for example in the treatment of a bacterial infection in a subject relative to the use of any one of the agents alone, preferably relative to the use of any two of the agents in combination. For example, the combinations of agents may demonstrate enhanced (e.g. synergistic) activity against one or more bacteria, such as Klebsiella pneumoniae ST147, Klebsiella pneumoniae ST101, and Klebsiella pneumoniae K66-45 (or any of the other bacterial strains against which the combinations of agents are tested in the examples), relative to the use of any one of the agents alone, preferably relative to the use of any two of the agents in combination. Evidence of synergy may include any one of the following: a faster cure rate, cure time or symptom improvement (e.g. improvement in at least one sign or key symptom of bacterial infection); and a reduction in the relapse rate of bacterial infection (i.e. the rate of reappearance of the infection after cessation of the treatment).

[0313] In the context of the susceptibility of a microorganism to an antimicrobial agent, synergy may be understood with reference to the definition set out by The European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) 2000 (see EUCAST Definitive Document E. Def 1.2: Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. Clin. Microbiol. Infect. 2000; 6(9): 503-508): FICI < 0.5; synergy.

[0314] In the combination therapy herein described, the agents (A), (B) and (C) are coadministered to the subject (e.g. a human patient). The term “co-administration” is used herein to refer to the delivery of two or more separate chemical entities in vivo. Coadministration includes the simultaneous delivery of separate agents; the simultaneous delivery of a combination of agents; as well as the delivery of one or two agents in combination followed by delivery of an additional agent or agents. In all cases, agents that are co-administered are intended to work in conjunction with each other, i.e. these will be present together in vivo, regardless of when or how they are actually administered, to achieve the desired therapeutic and / or prophylactic effect.

[0315] The agents for use in the invention may thus be administered simultaneously, separately or sequentially. Administration of each agent (whether separately or simultaneously) may be via any conventional administration route or routes, such as oral or parenteral administration.

[0316] The agents (A), (B) and (C) for use in the invention may be formulated together or they may be formulated separately. Where these are formulated separately, the agents (A), (B) and (C) may each be provided in a separate formulation. Alternatively, two of the agents may be provided in a single formulation and the third agent may be provided separately. In other words, the first agent (A) may be formulated with the second agent (B) and the third agent (C) may be formulated separately; or the first agent (A) may be formulated with the third agent (C) and the second agent (B) may be formulated separately; or the second agent (B) may be formulated with the third agent (C) and the first agent (A) may be formulated separately.

[0317] In some embodiments, the agents (A), (B) and (C) are administered simultaneously. In one such embodiment, administration may be achieved by combining the agents into a single dosage form, i.e. into the same pharmaceutical composition. Simultaneous administration may also be achieved by administering the agents in different pharmaceutical compositions. These may be administered via the same route, such as orally or parenterally (e.g. intravenously). In another embodiment the agents are administered via different routes, for example one or two of the agents may be administered orally and one or two of the agents may be administered intravenously.

[0318] In some embodiments, the agents are administered sequentially. In one embodiment, the agents are administered via the same route, such as orally or parenterally (e.g. intravenously). In another embodiment the agents are administered via different routes, for example one or two of the agents may be administered orally and one or two of the agents may be administered intravenously. Pharmaceutical compositions for use in the combination therapy comprise one or more of the agents (A), (B) and (C) as herein defined and one or more pharmaceutically acceptable carriers, diluents or excipients. By "pharmaceutically acceptable" is meant that the ingredients must be compatible with other ingredients of the composition as well as physiologically acceptable to the recipient. Pharmaceutical compositions may be formulated according to techniques and procedures well known in the art and widely described in the literature and may comprise any of the known carriers, diluents or excipients. Other ingredients may also be included, according to techniques well known in the art, for example stabilisers, preservatives, etc. The compositions may be in the form of sterile aqueous solutions and / or suspensions of the pharmaceutically active ingredients, aerosols, ointments and the like. The compositions may also be in a sustained release form, for example microparticles, nanoparticles, emulsions, nanosuspensions, lipid particles or oils. The agents for use in the invention may also be provided in formulations of ZnO nanoparticles as described, for example, by Pati et al. in Nanomedicine (2014), 10(6), 1195-1208. These may additionally be provided in the form of films, patches or folios having the selective zinc-chelator coated on the surface.

[0319] The compositions may be formulated according to techniques and procedures well known in the literature and may comprise any of known carriers, diluents or excipients. For example, compositions for use in the invention which are suitable for parenteral administration conveniently may comprise sterile aqueous solutions and / or suspensions of the active agent(s) preferably made isotonic with the blood of the recipient generally using sodium chloride, glycerin, glucose, mannitol, sorbitol and the like. In addition, the composition may contain any of a number of adjuvants, such as buffers, preservatives, dispersing agents, agents that promote rapid onset of action or prolonged duration of action.

[0320] Compositions suitable for oral administration may be in sterile purified stock powder form, preferably covered by an envelope or envelopes which may contain any of a number or adjuvants such as buffers, preservative agents, agents that promote prolonged or rapid release. Compositions suitable for local or topical administration may comprise the agent(s) mixed with known ingredients such as paraffin, vaseline, cetanol, glycerol and the like, to form suitable ointments or creams. The active agent(s) (A), (B) and (C) in any pharmaceutical composition may comprise from 0.05% to 99% by weight of the composition. An appropriate amount of the agent(s) may readily be determined by the skilled person.

[0321] In some embodiments, agents (A), (B) and (C) may be formulated in the same pharmaceutical composition. Such compositions form a further aspect of the invention. The invention thus provides a pharmaceutical composition comprising an effective amount of the first agent (A), second agent (B) and third agent (C) herein described, together with at least one pharmaceutically acceptable diluent or carrier. In some embodiments, each agent is present in the composition in a therapeutically effective amount. In some embodiments, each agent is present in a prophylactically effective amount. Such an amount may readily be determined by the skilled person, for example a physician (where the subject is a human patient).

[0322] In some embodiments, provided herein are pharmaceutical compositions in which the agents (A), (B) and (C) are present in synergistically effective amounts.

[0323] Any of the compositions described herein containing any of agents (A), (B) and (C) may be provided in unit dose form. By “unit dose form” is meant a composition containing one or more agents that is suitable for administration to a subject, preferably a human or nonhuman mammal, in a single dose. The preparation of a single or unit dose does not imply that it is administered once per day or once per course of therapy. It may be administered more than once per day (e.g. twice, thrice or more per day) and may be given more than once during the course of a therapy.

[0324] Administration of the agents (A), (B) and (C) herein described to a subject may be by any suitable method known in the medicinal arts, including intravenous, intracerebral, oral, parenteral, topical, subcutaneous, or inhalation. In the case of an antibacterial adjuvant, a suitable administration regime is continuous systemic administration of the adjuvant in combination with co-administration of the respective antibacterial agent(s). In the case of sepsis caused by resistant gram-negative bacteria, for example, the adjuvant may be intravenously continuously infused, while an antibacterial agent is administered as approved by the respective regulatory authorities.

[0325] In one embodiment, continuous intravenous infusion of any of the agents (A), (B) and (C) herein described may also be carried out simultaneously with administration of the other active agents via other administration routes, for example by inhalation, or by oral or topical administration. For inhalation, a micronized formulation of the agent is suitable. For oral administration, a tableted form of the agent is suitable.

[0326] It will be understood that any pharmaceutical composition containing an agent (A), (B) or (C) or any combination of such agents as herein described will be administered to the subject in an “effective amount”, i.e. in an amount that will elicit the biological or medical response of the patient that is being sought by the physician in the treatment as herein described, for example in an amount that will inhibit or eliminate bacterial growth. The “effective amount” may depend on factors such as the nature of the particular active agents, the choice of other non-active components, the severity of the condition, the timing and duration of the treatment, whether the treatment is intended to be therapeutic or prophylactic, etc. In one embodiment, the effective amount is a “therapeutically effective amount” for the alleviation, to some extent, of one or more symptoms of the infection or for clinical cure, i.e. elimination of symptoms of an active infection, including elimination of the underlying cause of the infection (i.e. viable microbes involved in the infection). In one embodiment, the effective amount is a “prophylactically effective amount” for prophylaxis of the symptoms of the condition being prevented.

[0327] In the case of an antibacterial agent or adjuvant to an antibacterial agent, this may be administered in a single dose to be taken at regular intervals e.g. once or twice a day, once every 48 hours or once every 72 hours. Sustained formulations may be given at longer intervals, e.g. 1 to 2 times a month or every three months.

[0328] Appropriate dosages of each agent (A), (B) and (C) to be administered will depend on the nature of the agent, the precise condition to be treated and its severity, the age and weight of the subject to which it is administered (e.g. a patient), the manner and schedule of administration (e.g. the number of daily or monthly doses and the length of the course of treatment) etc. and may be routinely determined by the skilled practitioner.

[0329] In some embodiments, the first agent (A) is administered in a dosage range from about 1 to 200 mg / kg body weight, for example from 1 to 100 mg / kg body weight, e.g. 5 to 70 mg / kg body weight, 5 to 50 mg / kg body weight, 10 to 70 mg / kg body weight, or 10 to 50 mg / kg body weight. Administration of the agent (A) can be via any of the accepted modes of administration including, but not limited to, intravenous, intracerebral, oral, parenteral, topical, subcutaneous, or inhalation. Oral or intravenous administration may be preferred. More preferably, the agent (A) may be administered intravenously.

[0330] In some embodiments, the second agent (B) is administered in a dosage range from about 1 to 200 mg / kg body weight, for example from 1 to 100 mg / kg body weight, e.g. 5 to 70 mg / kg body weight, 5 to 50 mg / kg body weight, 10 to 70 mg / kg body weight, or 10 to 50 mg / kg body weight. Administration of the agent (B) can be via any of the accepted modes of administration including, but not limited to, intravenous, intracerebral, oral, parenteral, topical, subcutaneous, or inhalation. Oral or intravenous administration may be preferred. More preferably, the agent (B) may be administered intravenously.

[0331] In some embodiments, the third agent (C) is administered in a dosage range from about 1 to 200 mg / kg body weight, for example from 1 to 100 mg / kg body weight, e.g. 1 to 70 mg / kg body weight, 1 to 50 mg / kg body weight, 5 to 30 mg / kg body weight, or 5 to 20 mg / kg body weight. Administration of the agent (C) can be via any of the accepted modes of administration including, but not limited to, intravenous, intracerebral, oral, parenteral, topical, subcutaneous, or inhalation. Oral or intravenous administration may be preferred. More preferably, the agent (C) may be administered intravenously.

[0332] Some embodiments of the invention include a kit comprising the agents (A), (B) and (C) as herein described. In such a kit, each agent may be provided in a separate container. In other embodiments, two of the agents may be provided in the same container and the third may be provided in a separate container. Each container may include a solid, solution or dispersion. Where any of the agents are solids intended for reconstitution, the kit may additionally contain a diluent suitable for preparation of the intended formulation for administration. Optionally, a kit may additionally contain instructions relating to the use of the agents in a method of treatment as herein described.

[0333] As herein described, the invention relates to a combination therapy which involves the coadministration of three agents, i.e. (A), (B) and (C). In some embodiments, the therapy may further comprise the step of administration to the subject of one or more additional antibacterial and / or adjuvant agents generally known and used in the art. However, in some embodiments, the therapy is a “triple combination” therapy in which only the three agents (A), (B) and (C) as herein described are employed as active agents, i.e. no additional antibacterial or adjuvant agents are required to treat and / or prevent the bacterial infection or the bacterial biofilm. In one set of embodiments, the methods of medical treatment according to the invention thus consist essentially of (e.g. consist of) the steps herein described. In another set of embodiments, the medical uses according to the invention consist essentially of (e.g. consist of) co-administration of (A), (B) and (C) as herein described. In another set of embodiments, the pharmaceutical compositions according to the invention consist essentially of (e.g. consist of) the agents (A), (B) and (C).

[0334] The invention shall not be limited to the shown embodiments and examples. While various embodiments of the present disclosure are described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications and changes to, and variations and substitutions of, the embodiments described herein will be apparent to those skilled in the art without departing from the disclosure. It is to be understood that various alternatives to the embodiments described herein can be employed in practicing the disclosure.

[0335] It is to be understood that every embodiment of the disclosure can optionally be combined with any one or more of the other embodiments described herein.

[0336] It is to be understood that each component, compound, or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more of each and every other component, compound, or parameter disclosed herein. It is further to be understood that each amount / value or range of amounts / values for each component, compound, or parameter disclosed herein is to be interpreted as also being disclosed in combination with each amount / value or range of amounts / values disclosed for any other component(s), compound(s), or parameter(s) disclosed herein, and that any combination of amounts / values or ranges of amounts / values for two or more component(s), compound(s), or parameter(s) disclosed herein are thus also disclosed in combination with each other for the purposes of this description. Any and all features described herein, and combinations of such features, are included within the scope of the present invention provided that the features are not mutually inconsistent.

[0337] The invention is described in more detail with reference to the following non-limiting examples and accompanying figures:

[0338] Fig. 1 a-e Isobolograms of combinations of meropenem and Ex. 1 compound on five different clinical isolates of resistant Gram-negative bacterial strains Fig. 2 Bacterial burden results from Example 7

[0339] Fig. 3 Results from time-kill experiment in Example 75 with meropenem at 8 pg / mL (MEM 8), meropenem at 8 pg / mL and Avibactam at 8 pg / mL (MEM / Avi 8) and a triple combination of meropenem (8 pg / mL), Avibactam (8 pg / mL) with Ex. 1 compound at 16 pg / mL (MEM / Avi / Ex. 1 16). The graph shows the CFU / mL over the course of 24 hours.

[0340] Examples

[0341] Examples 1-4: Synthesis

[0342] All experiments were conducted under N2 in anhydrous solvents unless noted otherwise The following compounds were prepared according to literature methods: tert-butyl (2-(bis(pyridin-2-ylmethyl)amino)ethyl)carbamate,1tert-butyl 2-((tert-butoxycarbonyl)oxy)-3-((S)-2-chloro-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1 ,3,2]dioxaborol-2-yl)ethyl)benzoate,2methyl 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinate,3(6-((Bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl methanesulfonate,38-((S)-2-chloro-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one.4All final products were obtained with a HPLC purity of >90 % @ 250 nm.

[0343] 1Eri Kawabata, Kazuya Kikuchi, Yasuteru llrano, Hirotatsu Kojima, Akira Odani, and Tetsuo Nagano. J. Am. Chem. Soc. 2005 727(3), 818-819. DOI: 10.1021 / ja044697q.2Scott J. Hecker, K. Raja Reddy, Olga Lomovskaya, David C. Griffith, Debora Rubio-Aparicio, Kirk Nelson, Ruslan Tsivkovski, Dongxu Sun, Mojgan Sabet, Ziad Tarazi, Jonathan Parkinson, Maxim Totrov, Serge H. Boyer, Tomasz W. Glinka, Orville A.

[0344] Pemberton, Yu Chen, and Michael N. Dudley. J. Med. Chem. 202063 (14), 7491-7507. DOI : 10.1021 / acs.jmedchem.9b01976.

[0345] 3Christian Schnaars, Geir Kildahl-Andersen, Anthony Prandina, Roya Popal, Sylvie Radix, Marc Le Borgne, TorGjoen, Adriana Magalhaes Santos Andresen, Adam Heikal, Ole Andreas 0kstad, Christopher Frohlich, 0rjan Samuelsen, Silje Lauksund, Lars Petter Jordheim, Pal Rongved, and Ove Alexander Hogmoen Astrand. ACS Infect. Dis. 20184 (9), 1407-1422. DOI: 10.1021 / acsinfecdis.8b00137.4Anete Parkova, Anka Lucic, Alen Krajnc, Jurgen Brem, Karina Calvopina, Gareth W. Langley, Michael A. McDonough, Peteris Trapencieris, and Christopher J. Schofield. ACS Infect. Dis. 20206 (6), 1398-1404. DOI: 10.1021 / acsinfecdis.9b00330.

[0346] Example 1 - Synthesis of (R)-3-(4-(((2-(bis(pyridin-2-ylmethyl)amino)ethyl)amino) methyl)benzamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic

[0347]

[0348] Example 1A- Synthesis of methyl 4-(((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(tert-butoxycarbonyl)amino)methyl)benzoate

[0349]

[0350] A solution of tert-butyl (2-(bis(pyridin-2-ylmethyl)amino)ethyl)carbamate (5.15 g, 15.1 mmol) in 250 mL dry DMF was cooled down on an ice bath under nitrogen. Sodium hydride (1.81 g 60% dispersion, 45.3 mmol) was added, followed by methyl 4-bromomethylbenzoate (3.80 g, 16.6 mmol). After 1 h, the reaction mixture was allowed to reach room temperature and stirred overnight. The reaction mixture was mixed with ethyl acetate (750 mL), washed with water (250 mL) and the aqueous phase re-extracted with ethyl acetate (250 mL). The combined extracts were washed with water (250 mL) and saturated NaCI (aq.), followed by drying over Na2SO4 (s.). Filtration and removal of solvent under reduced pressure gave 6.15 g crude product, which was purified on a dry column vacuum chromatography (DCVC) column, using silica as adsorbent and eluted with a 0%-5% gradient of methanol in dichloromethane. Fractions with product were combined and following evaporation of solvent gave 3.38 g product (46%) as a light brown oil.

[0351] 1H NMR (400 MHz, chloroform-d) 5H8.49 (m, 2H), 7.91 (d, J= 7.9 Hz, 2H), 7.40 (dd, J = 7.7 Hz, 1.8 Hz, 1H), 7.62 (m, 2H), 7.51-7.44 (m, 2H), 7.18-7.12 (m, 4H), 4.43-4.31 (m, 2H), 3.89-3.81 (m, 7H), 3.43-3.27 (m, 2H), 2.77-2.66 (m, 2H), 1.34 (br s, 9H).

[0352] The spectrum is compatible with restricted carbamate rotation.

[0353] MS (ESI, positive mode) mlz 491.3 [M+H]+.

[0354] Example 1B - Synthesis of 4-(((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(tert-butoxycarbonyl)amino)methyl)benzoic acid

[0355]

[0356] To an ice-cold solution of methyl 4-(((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(tert-butoxycarbonyl)amino)methyl)benzoate (460.4 mg, 0.938 mmol) in 15 mL tetrahydrofuran, water (15 mL) and lithium hydroxide monohydrate (157.9 mg, 3.76 mmol) was added. After stirring on an ice bath for 30 min, the mixture was allowed to stir at room temperature overnight, after which the reaction mixture was neutralized with 1 M HCI (aq.). After removal of volatiles under reduced pressure, the crude material was used “as is” in the subsequent coupling step.

[0357] Example 1C - Synthesis of tert-Butyl 3-(( / ?)-2-(bis(trimethylsilyl)amino)-2-((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate

[0358]

[0359] A flask containing tert-butyl 2-((tert-butoxycarbonyl)oxy)-3-((S)-2-chloro-2-((3aS,4S,6S,7aF?)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)benzoate (624.1 mg, 1.167 mmol) under a nitrogen atmosphere was dissolved in 20 mL tetrahydrofuran and cooled down to -85 °C. A solution of lithium bis(trimethylsilyl)amide, 1.0 M in tetrahydrofuran was added (1.3 mL, 1.3 mmol), and the solution was then allowed to slowly reach room temperature. Stirring was continued over night, after which volatiles were removed under reduced pressure. The crude product was stirred in n-heptane (50 mL) under nitrogen for 30 min and filtered through celite. The filter pad was washed with n-heptane (2 x 25 mL) and after removal of solvent under reduced pressure the resulting crude material was immediately reacted in the next step.

[0360] Example 1D - Synthesis of tert-butyl 3-(( / ?)-2-amino-2-((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate

[0361]

[0362] The crude material from the preceding step was dissolved in tetrahydrofuran (20 mL) and put under nitrogen atmosphere. Methanol (2 mL) was added, and the mixture was stirred at room temperature for 2 h. After removal of solvent, the crude material was immediately used “as is” in the following step.

[0363] Example 1E - Synthesis of tert-butyl 3-(( / ?)-2-(4-(((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(tert-butoxycarbonyl)amino)methyl)benzamido)-2-((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate

[0364]

[0365] To the acid prepared in Example 1B (est. 0.938 mmol) HATLI was added (388 mg, 1.02 mmol), followed by dichloromethane (20 mL). The resulting suspension was put under nitrogen atmosphere and cooled down to -20 °C. After dropwise addition of triethylamine (0.2 mL, 1.4 mmol), the mixture was stirred first at -20 °C for 30 min, and then 1 h at room temperature, before again cooling to -20 °C. The amine prepared in Example 1D (est.

[0366] 1.167 mmol) was then added dropwise as a solution in dichloromethane (10 mL), after which the mixture was slowly allowed to reach room temperature during overnight stirring. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (1 x 100 mL, 2 x 50 mL). The combined extracts were washed with saturated NaCI (aq.), dried over MgSCL (s), and filtered. Removal of solvent under reduced pressure gave 1.30 g crude material which was purified with DCVC, using Bondesil-C18-OH as packing material. The column was eluted with a stepwise gradient of methanol in water (50% -100%, in 10% increments). Pure fractions were collected, and after removal of solvent under reduced pressure the product was obtained as a pale yellow solid (352.6 mg, 39%). MS (ESI, positive mode) mlz 996.5 [M+Na]+.

[0367] Example 1F - Synthesis of ( / ?)-3-(4-(((2-(bis(pyridin-2-ylmethyl)amino)ethyl)amino) methyl)benzamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic acid disodium salt

[0368]

[0369] A solution in dry dichloromethane (25 mL) of the fully protected intermediate prepared in Example 1F (1.40 g, 1.44 mmol) was cooled down to -78 °C under nitrogen atmosphere. A solution of boron trichloride in dichloromethane (1.0 M, 7.0 mL, 7 mmol) was added dropwise over 10 min and afterwards stirred at -78 °C for 1 h. The resulting suspension was allowed to reach room temperature and quenched with the addition of 20 mL water. The mixture was transferred to a separatory funnel with 300 mL water, and washed 3x with diethyl ether (200 mL + 150 mL + 150 mL). The organic phases were back extracted with 50 mL water. Combined aqueous phases were neutralized with 40 mL 0.5 M NaOH (aq.) to pH 7-8. A colourless precipitate was filtered off and the mother liquor evaporated to give 2.24 g crude product. The crude material was purified on a large SPE column with 17 g Bondesil C18-OH material, packed by running through pure methanol, 1:1 methanol / water and 1 :9 methanol / water. Loading of material was done by first suspending it in 20 mL 1:9 methanol / water followed by addition of 20 mL 0.5 M NaOH, after which everything entered solution, followed by addition to the column under careful application of vacuum. Elution was done with 25 mL fractions, using the following scheme: 1:9 methanol / water (4 fractions), 2:8 methanol / water (4 fractions), 3:7 methanol / water (2 fractions), 4:6 methanol water (2 fractions). Fractions 5-10 were judged to contain product of acceptable purity and was collected. After removal of solvent under reduced pressure the product was obtained as a yellow solid (0.63 g, 70%)1H NMR (400 MHz, water-d2) bH8.25 (m, 2H), 7.62 (app. td, J= 7.8 Hz, 1.5 Hz, 2H), 7.56 (m, 2H), 7.27 (d, J= 7.9 Hz, 2H), 7.19-7.12 (m, 5H), 7.05 (m, 1H), 6.65 (t, J= 7.4 Hz, 1H), 3.63 (s, 4H), 3.47 (s, 2H), 3.21 (m, 1H), 3.04 (m, 1H), 2.92 (m, 1H), 2.58 (m, 2H), 2.37 (m, 2H).

[0370] MS (ESI, positive mode) m / z 566.3 [M+H]+.

[0371] Example 2 - Synthesis of ( / ?)-3-(4-((4-((6-((bis(pyridin-2-ylmethyl)amino)methyl) pyridin-3-yl)methyl)piperazin-1-yl)methyl)benzamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1 ,2]oxaborinine-8-carboxylic acid, disodium salt

[0372]

[0373] Example 2A - Synthesis of tert-Butyl 4-((6-((bis(pyridin-2- ylmethyl)amino)methyl)pyridin-3-yl)methyl)piperazine-1 -carboxylate

[0374]

[0375] (6-((Bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl methanesulfonate (5.55 g, 13.9 mmol, 1.1 eq.) was dissolved in MeCN (98 mL) and DMF (42 mL) and K2CO3 (5.25 g, 38.0 mmol, 3.0 eq.), KI (630 mg, 3.80 mmol, 0.30 eq.) and tert-butyl piperazine-1- carboxylate (2.41 g, 12.7 mmol, 1.0 eq.) were added. The pink solution was heated to 35 °C and stirred for 2 hours. The reaction mixture was then filtered, and the precipitate was washed with MeCN before concentration by rotary evaporator. The residue was dissolved in EtOAc (250 mL) and washed with brine (3x250 mL). The organic phase was dried over Na2SC>4, filtered and concentrated under reduced pressure. Purification by flash chromatography (silica gel, 99% CH2Ch / 1% MeOH to 90% CH2Cl2 / 10% MeOH) was performed and afforded the desired compound (2.81 g, 5.75 mmol, 45 %) as a brown solid after concentrating from EtOAc.

[0376] 1H NMR (400 MHz, DMSO-cfe): 6H 8.48 (d, J= 4.6 Hz, 2H), 8.38 (d, J= 1.5 Hz, 1H), 7.78- 7.74 (m, 2H), 7.67 (dd, J= 8.0 Hz, 2.0 Hz, 1H), 7.59 (d, J= 7.9 Hz, 2H), 7.55 (d, J= 8.0 Hz, 1H), 7.26-7.22 (m, 2H), 4.11-4.10 (m, 2H), 3.78-3.77 (m, 6H), 3.30-3.28 (m, 4H), 2.29- 2.27 (m, 8H), 1.38 (s, 9H).

[0377] Example 2B - Synthesis of 1-(5-(Piperazin-1-ylmethyl)pyridin-2-yl)-N,N-bis(pyridin- 2-ylmethyl)methanamine, trifluoroacetic acid salt

[0378]

[0379] tert-Butyl 4-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl)piperazine-1-carboxylate (2.81 g, 5.75 mmol, 1.0 eq.) was dissolved in CH2CI2(8.8 mL). TFA (8.8 mL) was added carefully. The solution was stirred for 1 hour at room temperature. The

[0380] reaction mixture was concentrated under reduced pressure. The residue was dissolved in MeOH and concentrated under reduced pressure again to remove excess TFA. This step was repeated two more times before a final concentration from CH2CI2to obtain the TFA salt of the desired compound as a dark oil. This crude material was used in the next step without further purification.

[0381] Example 2C - Synthesis of methyl 4-((4-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl)piperazin-1-yl)methyl)benzoate

[0382]

[0383] The trifluoroacetic acid salt of 1-(5-(Piperazin-1-ylmethyl)pyridin-2-yl)- / V, / \ / -bis(pyridin-2-ylmethyl)methanamine (2.89 g, 5.75 mmol, 1.0 eq.) was dissolved in MeCN (40 mL) and

[0384] DMF (17 mL). K2CO3(3.97 g, 28.8 mmol, 5.0 eq.), KI (0.286 mg, 1.73 mmol, 0.30 eq.)

[0385] and methyl 4-(bromomethyl)benzoate (1.45 g, 6.33 mmol, 1.1 eq.) were added. The

[0386] reaction mixture was heated to 35 °C and stirred for 2 hours. The reaction mixture was

[0387] then filtered and the precipitate was washed with MeCN. The combined filtrate and

[0388] washings were concentrated under reduced pressure. The obtained residue was

[0389] dissolved in EtOAc (200 mL) and washed with brine (3x200 mL), dried over Na2SO4,

[0390] filtered and concentrated under reduced pressure to give crude material. Recrystallization

[0391] (20 mL EtOAc + 2.5 mL n-heptane) was performed to obtain the desired compound (1.19 g, 2.22 mmol, 39 %) as a brown solid. Flash chromatography (silica gel, 98% CH2CI2 / 2% MeOH to 90% CH2CI2 / 10% MeOH) was performed on the filtrate from the recrystallization to obtain more of the desired compound (0.692 g, 1.29 mmol, 22 %) as a brown solid.

[0392] The two purification steps gave a total yield of 61 %.

[0393] 1H NMR (400 MHz, DMSO-cfe): bH8.48 (dd, J= 4.8 Hz, 0.8 Hz, 2H), 8.37 (d, J= 1.7 Hz,

[0394] 1H), 7.90 (d, J= 8.2 Hz, 2H), 7.78-7.74 (m, 2H), 7.65 (dd, J= 8.1 Hz, 2.1 Hz, 1H), 7.58 (d, J= 7.8 Hz, 2H), 7.54 (d, J= 7.9 Hz, 1H), 7.43 (d, J= 8.2 Hz, 2H), 7.25-7.22 (m, 2H), 3.84

[0395] (s, 3H), 3.78-3.77 (m, 6H), 3.52 (s, 2H), 3.45 (s, 2H), 2.48-2.24 (m, 8H). Example 2D - Synthesis of 4-((4-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl)piperazin-1 -yl)methyl)benzoic acid

[0396]

[0397] The methyl ester from Example 2C (1.17 g, 2.19 mmol, 1.0 equiv.) was dissolved in a mixture of THF (4 mL) and MeOH (2 mL) and cooled to 0 °C. LiOH H2O (0.183 g, 4.38 mmol, 2.0 equiv.) dissolved in water (4 mL) was added. The cooling bath was removed and the reaction mixture was stirred for 6h. 1M AcOH (aq.) (7 mL) was added until pH = 5. The volatiles were removed under reduced pressure. Water (20 mL) was added to the obtained residue, followed by extraction with 3:1 CHCh / iPrOH (4x40 mL). The combined organic phases were washed with brine (20 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure and the residue was purified by recrystallization from 1:3 MeOH / Et2O. The product (1.00 g, 1.92 mmol, 87%) was obtained as a dark yellow solid.

[0398] 1H NMR (400 MHz, DMSO-cfe): 6H 12.68 (broad s, 1H), 8.49 (dd, J= 4.7 Hz, 0.7 Hz, 2H), 8.40 (s, 1H), 7.90 (d, J= 8.1 Hz, 2H), 7.79-7.75 (m, 2H), 7.68-7.66 (m, 1H), 7.60-7.55 (m, 3H), 7.42 (d, J= 7.8 Hz, 2H), 7.26-7.23 (m, 2H), 3.78 (s, 6H), 3.57-3.34 (m, 4H), 2.68-2.34 (m, 8H).

[0399] Example 2E - Synthesis of tert-butyl 3-(( / ?)-2-(4-((4-((6-((bis(pyridin-2-ylmethyl) amino)methyl)pyridin-3-yl)methyl)piperazin-1-yl)methyl)benzamido)-2- ((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol- 2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate, hexafluorophosphoric acid salt

[0400]

[0401] The amide coupling was carried out as described in Example 1E, using the acid from Example 2D (0.362 g, 0.700 mmol, 1.0 eq.), amine from Example 1 D (est. 1.04 mmol, 1.5 equiv.), HATLI (0.292 g, 0.770 mmol, 1.1 equiv.), NEta (0.16 mL, 1.15 mmol, 1.6 equiv.) in CH2CI2 (15 mL). After purification by flash column chromatography (silica gel, 85% CH2CI2 / 5% MeOH / 10% n-heptane to 70% CH2CI2 / 20% MeOH / 10% n-heptane), the product was obtained as a white solid, both as a HPFe salt (0.144 g, 0.124 mmol, 18 %) and as the free base (0.207 g, 0.203 mmol, 29%). The NMR data of the latter is given.

[0402] 1H NMR (400 MHz, CDCI3): 5H8.52 (d, J = 4.5 Hz, 2H), 8.43 (d, J = 1.4 Hz, 1 H), 7.78 (dd, J= 7.8 Hz, 1.6 Hz, 1H), 7.73 (d, J= 8.0 Hz, 2H), 7.66-7.50 (m, 8H), 7.35 (d, J= 8.0 Hz, 2H), 7.27-7.23 (m, 1H), 7.15-7.11 (m, 2H), 4.29 (d, J= 8.0 Hz, 1H), 3.87-3.86 (m, 6H), 3.51-3.47 (m, 4H), 3.01-3.96 (m, 3H), 2.40-2.35 (m, 9H), 2.21-2.19 (m, 1H), 2.06-2.03 (m, 1H), 1.91-1.87 (m, 2H), 1.64 (d, J= 10.3 Hz, 1H), 1.58 (s, 9H), 1.40 (s, 3H), 1.37-1.25 (m, 12H), 0.90 (s, 3H).

[0403] Example 2F - Synthesis of ( / ?)-3-(4-((4-((6-((bis(pyridin-2-ylmethyl)amino)methyl) pyridin-3-yl)methyl)piperazin-1-yl)methyl)benzamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic acid, disodium salt

[0404]

[0405] The deprotection was carried out as described in Example 1 F. Starting from the fully protected free base from Example 2E (0.162 g, 0.159 mmol, 1.0 equiv.) and BCh (1.0 M in CH2CI2) (1.5 mL, 1.5 mmol, 9.0 equiv.) in CH2CI2 (5 mL), followed by purification under basic conditions, the disodium salt of the product (0.0806 g, 0.104 mmol, 65%) was obtained as a white solid.

[0406] 1H NMR (400 MHz, water-d2) 5H8.18 (d, J= 4.3 Hz, 2H), 8.01 (d, J= 1.5 Hz, 1H), 7.55-7.50 (m, 4H), 7.36 (dd, J= 8.0 Hz, 1.9 Hz, 1H), 7.30 (d, J= 7.9 Hz, 2H), 7.22 -7.18 (m, 3H), 7.07-7.00 (m, 3H), 6.89 (d, J= 6.9 Hz, 1H), 6.55-6.51 (m, 1H), 3.69-3.65 (m, 4H), 3.41 (s, 2H), 3.26 (s, 2H), 3.09-3.08 (m, 1H), 2.90 (dd, J= 15.9 Hz, 3.5 Hz, 1H), 2.73 (dd, J= 16.0 Hz, 5.4 Hz, 1H), 2.30-1.82 (m, 8H). MS (ESI, positive mode) mlz 347.7 [2M-2H2O+4H]4+, 712.3 [M+H]+, 734.3 [M+Na]+. Example 3 - Synthesis of ( / ?)-3-(4-((4-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinoyl)piperazin-1-yl)methyl)benzamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic acid, disodium salt

[0407]

[0408] Example 3A - Synthesis of 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid

[0409]

[0410] Methyl 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinate (10.9 g, 31.4 mmol, 1.0 equiv.) was dissolved in a mixture of THF (60 mL) and MeOH (30 mL) and cooled to 0 °C.

[0411] LiOH H2O (2.63 g, 62.8 mmol, 2.0 equiv.) dissolved in water (60 mL) was added. The cooling bath was removed and the reaction mixture was stirred overnight. 1M AcOH (aq.) (80 mL) was added until pH = 5. The volatiles were removed under reduced pressure. Water (120 mL) was added to the obtained residue, followed by extraction with 3:1 CHCh / iPrOH (4x200 mL). The combined organic phases were washed with brine (100 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure and the residue was purified by recrystallization from 1:2 MeOH / Et2O. The product (7.73 g, 23.1 mmol, 74%) was obtained as yellow crystals.

[0412] 1H NMR (400 MHz, DMSO-cfe): 6H 8.98 (d, J= 1.6 Hz, 1H), 8.51-8.49 (m, 2H), 8.24 (dd, J = 8.1 Hz, 2.1 Hz, 1H), 7.80-7.73 (m, 3H), 7.57 (d, J= 7.7 Hz, 2H), 7.27-7.24 (m, 2H), 3.88 (s, 2H), 3.81 (s, 4H). MS (ESI, positive mode) mlz 335.1 [M+H]+. Example 3B - Synthesis of tert-butyl 4-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinoyl)piperazine-1 -carboxylate, hexafluorophosphoric acid salt

[0413] Boc

[0414]

[0415] Carboxylic acid from Example 3A (2.04 g, 6.11 mmol, 1.0 eq.) and HATU (2.44 g, 6.42 mmol, 1.05 eq.) were mixed in DMF (40 mL) and cooled to 0 °C. NEta (1.3 mL, 9.32 mmol, 1.5 equiv.) was added dropwise and the solution was stirred for 30 min., followed by the addition of tert-butyl piperazine-1 -carboxylate (1.25 g, 6.71 mmol, 1.1 equiv.). The cooling bath was removed and the reaction mixture was stirred overnight. Volatiles were removed under reduced pressure and the residue was mixed with CH2CI2 (100 mL) and water (50 mL). The phases were separated and the aqueous phase was extracted with CH2CI2 (2x 50 mL). The combined organic phases were washed with brine (3x100 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (silica gel, 98% CH2Ch / 2% MeOH to 90% CH2Cl2 / 10% MeOH). The product (3.64 g, 5.61 mmol, 92%) was obtained as a beige solid.

[0416] 1H NMR (400 MHz, DMSO-cfe): 6H 8.58-8.57 (m, 3H), 7.89-7.83 (m, 3H), 7.63 (d, J= 8.1 Hz, 1H), 7.60 (d, J= 7.8 Hz, 2H), 7.38-7.35 (m, 2H), 4.10 (s, 6H), 3.60-3.37 (m, 8H), 1.41 (s, 9H). The resonance corresponding to the acidic proton was not observed.

[0417] 19F NMR (377 MHz): 5F-70.1 (d, J3IP,I9F = 709 Hz).

[0418] 31P NMR (162 MHz): 5P-144.2 (spt, Ji9F,3ip = 709 Hz).

[0419] Example 3C - Synthesis of (6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)(piperazin-1 -yl)methanone, hexafluorophosphoric acid / trifluoroacetic acid salt

[0420]

[0421] A solution of the protected secondary amine from Example 3B (3.02 g, 4.66 mmol, 1.0 equiv.) in CH2CI2 (30 mL) was cooled to 0 °C and TFA (30 mL) was added dropwise over 30 min. The cooling bath was removed and the solution was stirred for 2h additionally, before the volatiles were removed under reduced pressure. The residue was dissolved in MeOH (50 mL) and concentrated under reduced pressure again to remove excess TFA. This step was repeated two more times before a final concentration from CH2CI2 to obtain the TFA salt of the desired compound as a pale brown oil. The product was used in Example 4D without any further purification or characterization. Quantitative yield was assumed.

[0422] Example 3D - Synthesis of mMethyl 4-((4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinoyl) piperazin-1 -yl)methyl)benzoate

[0423]

[0424] A suspension of the product from Example 3C (est. 4.66 mmol, 1.0 equiv.), methyl (4-bromomethyl)benzoate (1.18 g, 5.15 mmol, 1.1 equiv.) and K2CO3 (4.83 g, 35.0 mmol, 7.5 equiv.) in DMF (70 mL) was stirred at RT for 4h. DMF was removed under reduced pressure and CH2CI2 (90 mL) was added. The suspension was stirred vigorously for 30 min. and filtrated to remove inorganic salts. The solids and the filter were washed with CH2CI2 (3x50 mL) and the washings were combined with the filtrate. CH2CI2 was then removed under reduced pressure and the residue was purified by flash column chromatography (silica gel, 98% CH2Ch / 2% MeOH to 90% CH2Cl2 / 10% MeOH). The product (1.97 g, 3.59 mmol, 77%) was obtained as a pale yellow solid.

[0425] 1H NMR (CDCI3): 5H8.57-8.53 (m, 3H), 7.98 (d, J= 8.1 Hz, 2H), 7.72 (dd, J= 8.1 Hz, 2.0 Hz, 1H), 7.67-7.63 (m, 3H), 7.54-7.52 (m, 2H), 7.39 (d, J= 8.1 Hz, 2H), 7.16-7.13 (m, 2H), 3.91-3.88 (m, 9H), 3.78-3.43 (m, 6H), 2.52-2.40 (m, 4H). Example 3E - Synthesis of 4-((4-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinoyl)piperazin-1 -yl)methyl)benzoic acid

[0426]

[0427] The methyl ester from Example 3D (2.39 g, 4.35 mmol, 1.0 equiv.) was dissolved in a mixture of THF (10 mL) and MeOH (5 mL) and cooled to 0 °C. LiOH H2O (0.363 g, 8.65 mmol, 2.0 equiv.) dissolved in water (10 mL) was added. The cooling bath was removed and the reaction mixture was stirred for 4h. 1M AcOH (aq.) (12 mL) was added until pH = 5. The volatiles were removed under reduced pressure. Water (40 mL) was added to the obtained residue, followed by extraction with 3:1 CHCh / iPrOH (4x80 mL). The combined organic phases were washed with brine (40 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure and the residue was purified by recrystallization from EtOH. The material was recrystallized three times in total. The product (1.65 g, 3.07 mmol, 70%) was obtained as pale yellow crystals.

[0428] 1H NMR (400 MHz, DMSO-cfe): 6H 12.88 (broad s, 1H), 8.50-8.48 (m, 3H), 7.91 (d, J= 8.2 Hz, 2H), 7.80-7.75 (m, 3H), 7.65 (d, J= 8.0 Hz, 1H), 7.58 (d, J = 7.7 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.26-7.23 (m, 2H), 3.83-3.81 (m, 6H), 3.64-3.34 (m, 6H), 2.50-2.38 (m, 4H).

[0429] Example 3F - Synthesis of tert-butyl 3-(( / ?)-2-(4-((4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinoyl)piperazin-1-yl)methyl)benzamido)-2-((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate, hexafluorophosphoric acid salt

[0430]

[0431] The amide coupling was carried out as described in Example 1E, using the acid from Example 4E (0.372 g, 0.693 mmol, 1.0 eq.), amine from Example 1 D (est. 1.02 mmol, 1.5 equiv.), HATLI (0.301 g, 0.790 mmol, 1.1 equiv.), NEt3(0.16 mL, 1.15 mmol, 1.6 equiv.) in CH2CI2 (15 mL). After multiple purifications by flash column chromatography (silica gel, eluting with either 95% CH2CI2 / 5% MeOH to 90% CH2CI2 / 10% MeOH or 85% CH2CI2 / 5% MeOH / 10% n-heptane to 75% CH2Ch / 15% MeOH / 10% n-heptane), the product (0.174 g, 0.147 mmol, 21%) was obtained as a white solid.

[0432] 1H NMR (400 MHz, CDCI3): 5H8.57-8.54 (m, 3H), 7.79 (dd, J= 7.8 Hz, 1.5 Hz, 1H), 7.76 (d, J= 8.2 Hz, 2H), 7.72 (dd, J= 8.2 Hz, 2.0 Hz, 1H), 7.67-7.63 (m, 3H), 7.59-7.50 (m, 4H), 7.37 (d, J= 8.2 Hz, 2H), 7.27-7.23 (m, 1H), 7.16-7.13 (m, 2H), 4.32-4.29 (m, 1H), 3.90-3.87 (m, 6H), 3.77-3.44 (m, 6H), 3.02-2.96 (m, 3H), 2.47-2.35 (m, 5H), 2.22-2.19 (m, 1H), 2.06-2.03 (m, 1H), 1.91-1.88 (m, 2H), 1.63 (d, J= 10.2 Hz, 1H), 1.56 (s, 9H), 1.46 (s, 3H), 1.38-1.30 (m, 12H), 0.90 (s, 3H). The resonance corresponding to the acidic proton could not be observed.

[0433] 19F NMR (377 MHz): 5F-70.1 (d, J3IP,I9F = 709 Hz).

[0434] 31P NMR (162 MHz): 5P-144.2 (spt, Ji9F,3ip = 709 Hz).

[0435] MS (ESI, positive mode) m / z [M+H]+.

[0436] Example 3G - Synthesis of (7?)-3-(4-((4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinoyl)piperazin-1-yl)methyl)benzamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1 ,2]oxaborinine-8-carboxylic acid, disodium salt

[0437]

[0438] The deprotection was carried out as described in Example 1 F. Starting from the fully protected compound from Example 4F (0.162 g, 0.137 mmol, 1.0 equiv.) and BCh (1.0 M in CH2CI2) (1.5 mL, 1.5 mmol, 11 equiv.) in CH2CI2 (4 mL), followed by purification under basic conditions, the disodium salt of the product (0.0464 g, 0.0590 mmol, 43%) was obtained as a white solid.

[0439] 1H NMR (400 MHz, water-d2) 5H8.21-8.17 (m, 3H), 7.58-7.48 (m, 5H), 7.36-7.34 (m, 3H), 7.23 (d, J= 8.1 Hz, 2H), 7.08 (dd, J = 6.8 Hz, 5.5 Hz, 2H), 7.02 (d, J= 6.2 Hz, 1H), 6.92 (d, J = 6.7 Hz, 1H), 6.56-6.52 (m, 1H), 3.76 (s, 6H), 3.59-3.46 (m, 4H), 3.15-3.10 (m, 3H), 2.91 (d, J= 15.6 Hz, 1H), 2.78 (dd, J= 15.6 Hz, 5.2 Hz, 1H), 2.47-2.27 (m, 4H).

[0440] MS (ESI, positive mode) m / z 354.7 [2M-2H2O+4H]4+.

[0441] Example 4 - Synthesis of ( / ?)-3-(4-(((2-(6-((bis(py ridin-2-ylmethyl)amino)methyl)nicotinamido)ethyl)amino)methyl)benzamido)-2-hydroxy- 3,4-dihydro-2H-benzo[e][1 ,2]oxaborinine-8-carboxylic acid, disodium salt

[0442]

[0443] Example 4A - Synthesis of tert-butyl (2-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido) ethylcarbamate, hexafluorophosphoric acid salt

[0444]

[0445] Carboxylic acid from Example 3A (2.10 g, 6.28 mmol, 1.0 eq.) and HATLI (2.51 g, 6.60 mmol, 1.05 eq.) were mixed in DMF (40 mL) and cooled to 0 °C. NEta (1.3 mL, 9.32 mmol, 1.5 equiv.) was added dropwise and the solution was stirred for 30 min., followed by the addition of tert-butyl piperazine-1 -carboxylate (1.10 g, 6.88 mmol, 1.1 equiv.). The cooling bath was removed and the reaction mixture was stirred overnight. Volatiles were removed under reduced pressure and the residue was mixed with CH2CI2 (100 mL) and water (50 mL). The phases were separated and the aqueous phase was extracted with CH2CI2 (2x 50 mL). The combined organic phases were washed with brine (3x100 mL) and dried over Na2SC>4. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (silica gel, 98% CH2Ch / 2% MeOH to 90% CH2Ch / 10% MeOH). The product was obtained as a yellow solid (3.10 g, 4.97 mmol, 79%).

[0446] 1H NMR (400 MHz, DMSO-cfe): bH8.92 (d, J= 1.5 Hz, 1H), 8.61 (t, J= 5.5 Hz, 1H), 8.53 (dd, J= 4.7 Hz, 0.6 Hz, 2H), 8.16 (dd, J= 8.0 Hz, 2.0 Hz, 1H), 7.83-7.79 (m, 2H), 7.67 (d, J= 8.2 Hz, 1H), 7.59 (d, J= 8.0 Hz, 2H), 7.32-7.29 (m, 2H), 6.92 (t, J= 5.8 Hz, 1H), 3.96-3.92 (m, 7H), 3.32-3.27 (m, 2H), 3.13-3.30 (m, 2H), 1.35 (s, 9H).

[0447] 19F NMR (377 MHz): 6F-70.1 (d, J3IP,I9F = 709 Hz).

[0448] 31P NMR (162 MHz): 6P-144.2 (spt, Ji9F,3ip = 709 Hz).

[0449] Example 4B - Synthesis of A / -(2-aminoethyl)-6-((bis(pyridin-2-ylmethyl)amino) methyl)nicotinamide

[0450]

[0451] A solution of the protected primary amine from Example 4A (2.40 g, 3.86 mmol, 1.0 equiv.) in CH2CI2 (25 mL) was cooled to 0 °C and TFA (25 mL) was added dropwise over 30 min. The cooling bath was removed and the solution was stirred for 2h additionally, before the volatiles were removed under reduced pressure. The obtained residue was suspended in CH2CI2 (100 mL), cooled to 0 °C and 1M NaOH (aq.) was added in portions until a pH of 14 was obtained (100 mL used in total). The organic phase cleared up completely during these additions. The phases were separated and the aqueous phase was extracted with CH2CI2 (2x50 mL). The combined organic phases were washed with brine (50 mL) and dried over Na2SC>4. The solvent was removed under reduced pressure and the product (0.960 g, 2.55 mmol, 66%) was obtained as an orange oil. The product was used in Example 5Cwithout any further purification or characterization.

[0452] Example 4C - Synthesis of methyl 4-(((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)ethyl)amino)methyl)benzoate

[0453]

[0454] A solution of primary amine from Example 4B (1.46 g, 3.87 mmol, 1.0 equiv.), methyl 4-formylbenzoate (0.505 g, 3.07 mmol, 0.8 equiv.) and AcOH (0.35 mL, 6.12 mmol, 1.6 equiv.) in EtOH (50 mL) was heated at reflux temperature for 3h. The reaction mixture was cooled to RT and NaBH(OAc)3 (2.66 g, 12.4 mmol, 3.2 equiv.) was added in one portion under a flow of N2. After 2h, more aldehyde (0.0667 g, 0.406 mmol, 0.1 equiv.) and more NaBH(OAc)3 (0.667 g, 3.15 mmol, 0.8 equiv.) were added. The suspension was stirred at RT overnight. EtOH was removed under reduced pressure and the obtained residue was suspended in CH2CI2 (100 mL). The suspension was stirred at RT for 30 min and filtered through a pad of Celite to remove insoluble material. The Celite and the insoluble material were washed with CH2CI2 (total 50 mL). The filtrate and the washings were combined and volatiles were removed under reduced pressure. The residue was purified by flash column chromatography (silica gel, 97% CH2Cl2 / 2% MeOH / 1% NEt3to 89% CH2CI2 / 10% MeOH / 1% NEt3). The product (1.45 g, 2.76 mmol, 80%) was obtained as a yellow semi-solid after repeated evaporation from toluene (3x 20 mL) and n-heptane (3x 20 mL).

[0455] 1H NMR (400 MHz, DMSO-cfe): 6H 8.93 (d, J = 2.0 Hz, 1H), 8.68 (t, J = 5.5 Hz, 1H), 8.49 (dd, J = 5.0 Hz, 0.9 Hz, 2H), 8.17 (dd, J = 8.2 Hz, 2.0 Hz, 1H), 7.92 (d, J = 8.1 Hz, 2H), 7.79-7.75 (m, 2H), 7.69 (d, J = 8.1 Hz, 1H), 7.59-7.53 (m, 4H), 7.26-7.22 (m, 2H), 3.94 (s, 2H), 3.84-3.79 (m, 9H), 3.46-3.43 (m, 2H), 2.82-2.79 (m, 2H). The resonance corresponding to the NH proton was not observed.

[0456] Example 4D - Synthesis of methyl 4-(((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)ethyl)(tert-butoxycarbonyl)amino)methyl)benzoate

[0457]

[0458] 5% NaHCOs (15 mL) was added to a solution of secondary amine from Example 3D (1.46 g, 2.79 mmol, 1.0 equiv.) and Boc2O (0.920 g, 4.22 mmol, 1.5 equiv.) in THF (15 mL).

[0459] The resulting two-phase system was stirred vigorously overnight. CH2CI2(50 mL) and water (50 mL) were added, the phases were separated and the aqueous phase was extracted with CH2CI2(3x25 mL). The combined organic phases were washed with brine (50 mL) and dried over Na2SC>4. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (silica gel, 97% CH2CI2 / 3% MeOH to 90% CH2CI2 / 10% MeOH). The product (1.21 g, 1.94 mmol, 69%) was obtained as a pale yellow solid.

[0460] 1H NMR (400 MHz, DMSO-cfe): 6H 8.89-8.86 (m, 1H), 8.66-8.63 (m, 1H), 8.50-8.48 (m, 2H), 8.15-8.10 (m, 1H), 7.90 (d, J= 8.2 Hz, 2H), 7.79-7.75 (m, 2H), 7.68 (d, J= 7.8 Hz, 1H), 7.57 (d, J= 7.8 Hz, 2H), 7.34 (d, J= 8.2 Hz, 2H), 7.26-7.23 (m, 2H), 4.49-4.47 (m, 2H), 3.83-3.78 (m, 9H), 3.40-3.30 (m, 4H), 1.31-1.24 (m, 9H).

[0461] Example 4E - Synthesis of 4-(((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)ethyl)(tert-butoxycarbonyl)amino)methyl)benzoic acid

[0462]

[0463] The methyl ester from Example 4D (1.19 g, 1.90 mmol, 1.0 equiv.) was dissolved in a mixture of THF (3 mL) and MeOH (1.5 mL) and cooled to 0 °C. LiOH H2O (0.130 g, 3.10 mmol, 1.6 equiv.) dissolved in water (3 mL) was added. The cooling bath was removed and the reaction mixture was stirred for 5h. 1M AcOH (aq.) (5 mL) was added until pH = 5. The volatiles were removed under reduced pressure. Water (20 mL) was added to the obtained residue, followed by extraction with 3:1 CHCh / iPrOH (4x40 mL). The combined organic phases were washed with brine (20 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure and the residue was dried by evaporation with toluene (3x10 mL) and n-heptane (3x25 mL). The product (1.14 g, 1.87 mmol, 98%) was obtained as a pale yellow solid.

[0464] 1H NMR (400 MHz, DMSO-cfe): 6H 12.87 (broad s, 1H), 8.91-8.88 (m, 1H), 8.69-8.64 (m, 1H), 8.51-8.49 (m, 2H), 8.16-8.11 (m, 1H), 7.90 (d, J= 8.2 Hz, 2H), 7.80-7.75 (m, 2H), 7.69 (d, J= 7.8 Hz, 1H), 7.58 (d, J= 7.9 Hz, 2H), 7.32 (d, J= 8.2 Hz, 2H), 7.27-7.23 (m, 2H), 4.49-4.47 (m, 2H), 3.85 (s, 2H), 3.79 (s, 4H), 3.41-3.30 (m, 4H), 1.33-1.26 (m, 9H).

[0465] Example 4F - Synthesis of 8-(( / ?)-2-amino-2-((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2,2-dimethyl-4H-benzo[d][1 ,3]dioxin-4-one

[0466]

[0467] A solution of 8-((S)-2-chloro-2-((3aS,4S,6S,7aF?)-3a,5,5-trimethylhexahydro-4,6- methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one (0.489 g, 1.16 mmol, 1.0 equiv.) in THF was cooled down to -100 °C under N2. LiHMDS (1.0 M in THF) (1.2 mL, 1.2 mmol, 1.0 equiv.) was added and the solution was stirred for 1 h before the temperature was gradually increased to -80 °C. After stirring for 2h at -80 °C, the solution was allowed to slowly reach room temperature overnight while stirring, after which volatiles were removed under reduced pressure. The crude product was stirred in n-heptane (25 mL) under N2 for 30 min and filtered through Celite. The filter pad was washed with n-heptane (2 x 25 mL) and after removal of solvent under reduced pressure the resulting crude material was immediately dissolved in THF (10 mL) under N2 and cooled to -20 °C. Methanol (0.10 mL, 2.47 mmol, 2.1 equiv.) was added and the mixture was stirred at -20 °C for 10 min. before the cooling bath was removed. After additionally 10 min., the solvent was removed under reduced pressure. The obtained residue was immediately used in the next step.

[0468] Example 4G - Synthesis of tert-butyl (2-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)ethyl)(4-((( / ?)-2-(2,2-dimethyl-4-oxo-4H-benzo[d][1,3]dioxin-8-yl)-1- ((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol- 2-yl)ethyl)carbamoyl)benzyl)carbamate

[0469]

[0470] The amide coupling was carried out as described in Example 1E, using the acid from Example 4E (0.493 g, 0.807 mmol, 1.0 eq.), amine from Example 4F (est. 1.16 mmol, 1.4 equiv.), HATLI (0.332 g, 0.874 mmol, 1.1 equiv.), NEta (0.18 mL, 1.15 mmol, 1.6 equiv.) in CH2CI2 (15 mL). Purification was carried out by dissolving the crude material in DMSO (5 mL) containing 2-3 drops of water. The solution was stirred at RT for 2 days, before it was added to water (100 mL) and extracted with CH2CI2 (3x50 mL). The combined organic phases were washed with brine (3x100 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography. After purification by flash column chromatography (silica gel, 95% CH2CI2 / 5% MeOH to 85% CH2CI2 / 15% MeOH), the product (0.388 g, 0.391 mmol, 48%) was obtained as a white solid.

[0471] 1H NMR (400 MHz, CDCI3): 8.96-8.75 (m, 1 H), 8.52 (d, J = 4.4 Hz, 2H), 8.07 (d, J = 7.7 Hz, 1H), 7.80 (d, J= 8.0 Hz, 1H), 7.74 (d, J= 8.2 Hz, 2H), 7.67-7.63 (m, 3H), 7.54-7.47 (m, 4H), 7.29-7.26 (m, 2H), 7.16-7.13 (m, 3H), 7.07-7.03 (m, 1H), 4.48 (s, 2H), 4.31 (d, J = 7.6 Hz, 1H), 3.91-3.86 (m, 6H), 3.75-3.34 (m, 4H), 3.19-3.17 (m, 1H), 3.04-2.89 (m, 2H), 2.39-2.33 (m, 1H), 2.15-2.11 (m, 1H), 2.03-2.00 (m, 1H), 1.88-1.83 (m, 2H), 1.66-1.64 (m, 6H), 1.44-1.22 (m, 16H), 0.88 (s, 3H).

[0472] Example 4H - Synthesis of ( / ?)-3-(4-(((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)ethyl)amino)methyl)benzamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic acid, disodium salt

[0473]

[0474] In Step 1 (acetonide hydrolysis) fully protected compound from Example 4G (0.364 g, 0.367 mmol, 1.0 equiv.) was dissolved in THF (2 mL). Water (2 mL), followed by LiOH H2O (0.0770 g, 1.836, 5.0 equiv.). The reaction mixture was stirred at RT for6h. 1 M AcOH (aq.) (4 mL) was added, followed by water (10 mL). The product was extracted by 3:1 CHC^iPrOH (3x10 mL). The combined extracts were washed with brine (10 mL) and dried over Na2SO4. The volatiles were removed, yielding hydrolysis product which was used directly in Step 2 (removal of boronic acid and amine protection groups). The residue was dissolved in acetone (8 mL), followed by the addition of MeB(OH)2(0.226 g, 3.78 mmol, 10 equiv.) and 0.12 M HCI (aq.) (8 mL). The reaction mixture was stirred at RT, with occasional removal of the stopper of the reaction flask. After 2 days at RT, volatiles were removed under reduced pressure (40 °C). 0.12 M HCI (aq.) (6 mL) was added and removed under reduced pressure at 40 °C. This procedure was repeated three times. For each time, an increasingly homogenous solution was obtained. The crude compound was further subjected to Step 3 (purification under alkaline conditions and isolation of the product as a disodium salt) which was carried out as described in Example 1F. The product (0.131 g, 0.171 mmol, 47% (from Step 1)) was obtained as a white solid.

[0475] 1H NMR (400 MHz, water-d2) 5H8.49 (d, J= 2.1 Hz, 1H), 8.21 (d, J= 4.6 Hz, 2H), 7.78 (dd, J= 8.2 Hz, 2.3 Hz, 1H), 7.59-7.55 (m, 2H), 7.49 (d, J= 8.2 Hz, 2H), 7.37-7.33 (m, 3H), 7.26 (d, J= 8.2 Hz, 2H), 7.10-7.03 (m, 3H), 6.89 (d, J= 7.1 Hz, 1H), 6.55-6.51 (m, 1H), 3.78-3.72 (m, 8H), 3.44-3.40 (m, 2H), 3.08-3.06 (m, 1H), 2.75-2.70 (m, 4H). The chemical shifts and resonances in the1H NMR spectrum are influenced by the concentration of the compound.

[0476] MS (ESI, positive mode) m / z 700.3 [M+H]+, 722.3 [M+Na]+.

[0477] Example 5 - Checkerboard microbiological testing of the selective zinc-chelator of Example 1 in combination with a carbapenem Example 5A - General experimental setup

[0478] Checkerboard broth microdilution assays were performed according to the guidelines of the Clinicial Microbiology Procedures Handbook, Fourth Edition, Chapter 5.16: Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods (ASM Press), and Clinical and Laboratory Standards Institute (CLSI) guidelines M100, 31st ed.

[0479] Performance Standards for Antimicrobial Susceptibility Testing.

[0480] Test and quality control strains were recovered from frozen storage (-80°C) by culturing on Nutrient Agar (NA) (Oxoid, #CM0003) and NA supplemented with 1 pg / mL meropenem for E. coli IR3 in an incubator set at 37°C for 18-24 h.

[0481] Meropenem was dissolved in sterile water at a concentration of 20.48 mg / mL, and further diluted in Mueller Hinton II Broth (Cation-Adjusted; CA-MHB) to provide 2* stocks of the final top test concentrations (1024, 256, and 8 pg / mL dependent on strain tested). Drug-free (98 pL) CA-MHB was dispensed to all wells in rows B to P of sterile 96-well flatbottom microdilution plates (Corning; #3370), whilst 196 pL of the 2* meropenem-containing broth was added to wells in row A. Serial two-fold dilutions were performed from row A to O, changing tips after each dilution step, to provide 2* concentrations of the final test concentration range.

[0482] The compound of Ex. 1 was dissolved in sterile-filtered 100% dimethyl sulfoxide (DMSO) at a concentration of 12.8 mg / mL to provide a 100* stock of the final top test concentration (128 pg / mL). A 96-well round-bottom microdilution plate (Corning; #3788) was used to prepare 100* stocks of the Ex. 1 compound assay concentration range by performing serial two-fold dilutions in sterile-filtered 100% DMSO from column 1 to 11, changing tips after each dilution step. Column 12 contained drug-free sterile-filtered 100% DMSO. 2 pL of each 100* stock of the Ex. 1 assay concentration range microplate was dispensed manually to appropriate wells of microplates containing 2* stocks of the meropenem assay concentration range.

[0483] Bacterial suspensions equivalent to a McFarland 0.5 standard were prepared for each test strain in 5 mL sterile phosphate-buffered saline (PBS) and diluted 1:150 in CA-MHB to provide an inoculum of approximately 1 x 106CFU / mL. 100 pL of each inoculum was dispensed into all wells, to bring the final inoculum to 5 x 105CFU / mL (range, 2-8 x 105 CFU / mL) and to establish the final test concentration ranges for the Ex. 1 compound and meropenem, and final DMSO concentration of 1% [v / v] per well. Assay plates were stored in an incubator set at 37°C for 20 h in air, along with a small (2-3 mL) aliquot of the growth medium to serve as a sterility control. An inoculum purity and viability check of the QC strain P. aeruginosa ATCC 27853, plus selected test strains, was performed on NA and incubated at 30°C for 18-24 h.

[0484] After incubation, assay plates were read visually and by spectrophotometer at an absorbance of 600 nm, and meropenem minimum inhibitory concentrations (MICs) as a single agent were recorded as the lowest concentration causing complete inhibition of growth as detected by the unaided eye. The lowest concentration of interaction in Ex. 1 compound and meropenem combination wells resulting in complete inhibition of growth was recorded as clear wells.

[0485] The fractional inhibitory concentration index (FICI) was determined for each clear well as described by Hall etal. (J. Antimicrob. Chemother. (1983): 11(5): 427-33) and calculated as (MICA combination / MICA alone) + (MICB combination / MICB alone). As the Ex. 1 compound exhibited no MIC as a single agent, the MICA alone value used to calculate the FICI was assumed as one doubling-dilution greater than the top test concentration (256 pg / mL). Interactions were defined as follows: FICI >4 = antagonism between Drug A and Drug B, FICI 0.5-2 = indifferent interaction Drug A and Drug B, FICI <0.5, synergism between Drug A and Drug B.

[0486] Example 5B - Results

[0487] Synergistic combinations (FICI <0.5) between Ex. 1 compound at 16 pg / mL combined with meropenem reduced the meropenem MIC by 512-fold, compared to the MIC of meropenem alone, to below the CLSI susceptible breakpoint (<1 pg / mL) against blaKPC-producing E. coli ATCC BAA-1705.

[0488] Interactions at 16 pg / mL Ex. 1 compound also reduced the meropenem MIC by 256-fold against blaNDM-1-producing E. coli IR3. Also at 16 pg / mL Ex. 1 compound, the meropenem MIC decreased by 128-fold to the Acinetobacter spp. CLSI susceptible breakpoint (<2 pg / mL) against blaNDM-1-producing A. baumannii AR088.

[0489] Against K. pneumoniae NCTC 13443 and A. baumannii AR033, Ex. 1 compound at 16 pg / mL in combination with meropenem reduced the meropenem MIC by 16- and 32-fold, respectively, compared to the meropenem MIC alone. There was one indifferent interaction between Ex. 1 compound and meropenem against A. baumannii AR0297.

[0490] The MIC of meropenem alone against the QC strain P. aeruginosa ATCC 27853 were within CLSI M100, 31st ed. range (0.125-1 pg / mL). Inoculum viability checks against the selected strains were all approximately the target range of 2-8 x 105CFU / mL.

[0491] P. aeruginosa ATCC 27853 viability counts could not be determined due to merging of colonies preventing accurate quantitation. Inoculum Viability Check showed that E. coli IR3 had a CFU / mL of 3.5 x 105, K. pneumoniae NCTC 13443 had a CFU / mL of 1.0 x 106and A. baumannii AR033 had a CFU / mL 1.0 x 106.

[0492] The results are summarized in Table 1 and 2. Table 1

[0493] " "

[0494]

[0495] Table 2

[0496] " "

[0497]

[0498] Example 6 - Checkerboard microbiological testing of the selective zinc-chelator of Example 1 in combination with a carbapenem

[0499] Example 6A - Isobolograms

[0500] The protocol in Example 5 was used to generate data from checkerboard broth microdilution assays for the combination of Ex. 1 compound and meropenem for test strains. The results were plotted in isobolograms where the MIC values were plotted with concentrations of Ex. 1 compound on the X axis and the concentrations of meropenem was plotted on the Y axis. Interactions occurred in CA-MHB. Combinations of Ex. 1 compound and meropenem were plotted as a dashed line. The hypothetical line of no interaction or synergy was plotted as a solid line between the maximum concentration on the two axes. The stronger the synergy in the antibacterial effect between Ex. 1 compound and meropenem, the closer the dotted line is to origin in the isobologram. The results are provided in Figures 1 a-e.

[0501] Example 7 - In vivo efficacy of the double combination of the selective zinc-chelator of Example 1 in combination with meropenem in a 9-hour model of K. pneumoniae ATCC BAA 1705 thigh infection in male CD1 mice

[0502] Protocol

[0503] A total of 73 CD1 male mice (Charles River Laboratories, UK), weighing 11 -15g on arrival was divided into groups 4 mice per group 9 groups including pre-treatment. Ex. 1 compound and meropenem (MEM) were separately dissolved in water for injection (WFI). A 50 pL / thigh intramuscular infection with K. pneumoniae ATCC BAA 1705 was injected into both thighs, giving 8 x 106CFU / mL, 4 x 105CFU per thigh, according to the schedule in Table 3.

[0504] Table 3

[0505]

[0506] Results

[0507] The Bacterial burden results are given in Figure 2. Comparison to meropenem monotherapy was made using Kruskal-Wallis with Conover-Inman (to make all pairwise comparisons between groups) tests in StatsDirect software v. 3.3.5.

[0508] Conclusion

[0509] In the present in vivo assessment of the efficacy of the compound of Ex. 1 , administration of the compound of Ex. 1 in combination with meropenem resulted in a log 2-2.5 reduction in average dose dependent reduction in thigh bacterial burden at all dose levels tested.

[0510] Examples 8-10: Triple combinations of the selective zinc-chelator compound of Example 1 with an antibiotic drug (a cephalosporin) and an inhibitor of serine-p-lactamases (SBLi)

[0511] Example 8 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTX) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae K66-45 and comparison with the triple combination of ceftriaxone, sulbactam and EDTA-disodium

[0512] The effect of the triple combination of Ex. 1 compound with ceftriaxone (CEFTX) and sulbactam (SUL) was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the p-lactamase NDM-1. Synergy was obtained at a concentration of 16 pg / mL of the compound of Ex. 1.

[0513] The experimental results from MIC determination with a triple combination of CEFTX, SUL and the zinc chelator (Ex. 1) are given in Tables 4-6. Two technical replicates were performed for each sample. An X in the table indicates bacterial growth, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTX are given in the top row left to right (row 1). Concentrations (pg / mL) of SUL are given in column 1. The zinc chelator (Ex. 1) or EDTA were added in fixed concentrations, at 16 pg / mL in tables 5 and 6. As a negative control, the effect of CEFTX, SUL and bacteria only was tested (Table 4). All wells contained bacterial growth medium (caMHB, 5.4 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the lowered CEFTX MIC values, showing synergy. Table 4 - No Ex. 1 or EDTA

[0514]

[0515] Table 5 -With Ex. 1, 16 pg / ml

[0516] >

[0517]

[0518] Table 6 - With EDTA, no Ex. 1 , 16 pg / ml

[0519] >

[0520]

[0521] Example 9 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTX) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST101 and comparison with the triple combination of ceftriaxone, sulbactam and EDTA-disodium

[0522] The effect of the triple combination of Ex. 1 compound with CEFTX and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST101, carrying genes encoding the P-lactamase OXA-48. Synergy was obtained at a concentration of 16 pg / mL of Ex. 1 compound. The experimental results from MIC determination with a triple combination of CEFTX, SUL and the zinc chelator (Ex. 1) are given in Tables 7-9. Two technical replicates were performed for each sample. An X in the table indicates bacterial growth, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTX are given in the top row left to right (row 1). Concentrations (pg / mL) of SUL are given in column 1. The zinc chelator (Ex. 1) compound or EDTA were added in fixed concentrations, at 16 pg / mL in tables 7 and 8. As a negative control, the effect of CEFTX, SUL and bacteria only was tested (Table 6). All wells contained bacterial growth medium (caMHB, 5.4 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the lowered CEFTX MIC values, showing synergy.

[0523] Table 7 - No Ex. 1 or EDTA

[0524]

[0525] Table 8 -With Ex. 1, 16 pg / ml

[0526] >

[0527]

[0528] Table 9 - With EDTA, 16 pg / ml

[0529] >

[0530]

[0531] Example 10 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTX) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST147 and comparison with the triple combination of ceftriaxone, sulbactam and EDTA-disodium

[0532] The effect of the triple combination of Ex. 1 compound with CEFTX and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the P-lactamases NDM-1 and KPC-2, constitutively expressed. Synergy was obtained at a concentration of 16 pg / mL of Ex. 1 compound.

[0533] The experimental results from MIC determination with a triple combination of CEFTX, SUL and the zinc chelator (Ex. 1) are given in Tables 10-12. Two technical replicates were performed for each sample. An X in the table indicates bacterial growth, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTX are given in the top row left to right (row 1). Concentrations (pg / mL) of SUL are given in column 1. The zinc chelator (Ex. 1) was added in fixed concentrations, at 16 and 8 p g / mL in tables 11 and 12 respectively. EDTA was added in fixed concentration, at 16 pg / mL in table 13. As a

[0534] negative control, the effect of CEFTX, SUL and bacteria only was tested (Table 10). All wells contained bacterial growth medium (caMHB, 5.4 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the lowered CEFTX MIC values, showing synergy.

[0535] Table 10 - No Ex. 1 or EDTA

[0536]

[0537] Table 11 - With Ex. 1, 16 pg / ml

[0538] >

[0539]

[0540]

[0541] Table 12 - With Ex. 1, 8 pg / ml

[0542] >

[0543]

[0544] Table 13 - With EDTA, no Ex. 1, 16 pg / ml

[0545] >

[0546]

[0547] Examples 11-74: Minimal Inhibitory Concentration (MIC) determination for p-lactam antibiotics in combination with the selective zinc chelator compound of Example 1 and a serine-p-lactamase inhibitor (sBLI) (checkerboard assays)

[0548] Materials and Methods:

[0549] The antibiotics and serine p-lactamase-inhibitors (sBLIs) were purchased from the following suppliers: Meropenem (MEM): AdooQ Bioscience; Piperacillin (PIP): Sigma-Aldrich; Amoxicillin (AMOX): Sigma-Aldrich; Cefepime (FEP): Sigma-Aldrich; Ceftaroline (CEFT): MedChem Express; Cefpodoxime (CEFP): MedChem Express; Ceftriaxone (CEFTR): Merck / Sigma-Aldrich; Cefiderocol (CEFDC); MedChem Express; Ceftolozane (CFLOZ): Boc Sciences; Sulbactam (SUL): Sigma-Aldrich; Avibactam (AVI): Biosynth; Tazobactam (TAZ): Sigma-Aldrich; Taniborbactam: Sigma-Aldrich; Durolbactam: Sigma-Aldrich / Targetmol; Xeruborbactam: MedChem Express. In each of Examples 11-74, the compound of Example 1 was employed as the zinc chelator. The following abbreviations are used in these examples: sBLI means serine-p-lactamase inhibitor; CFU means colony-forming unit, which is a group of microbes that grows from a single cell on a petri plate; DMSO means dimethyl sulfoxide; MHB means Mueller-Hinton broth; caMHB means cation-adjusted Mueller-Hinton broth; MIC means minimum inhibitory concentration - the lower the MIC, the more efficient the combination against the respective bacterial strain.

[0550] All examples demonstrated synergy between the zinc chelator and the other two components in the triple combination, meaning that the MIC for the double combination of the antibiotic drug and the serine-p-lactamase inhibitor alone was always higher in one or more wells than the triple combination of the zinc chelator + the antibiotic drug + the serine-p-lactamase inhibitor. Since neither the zinc chelator nor the sBLI have intrinsic antibiotic effect, the MIC value always means the MIC of the antibiotic component in the combination. The conclusion of synergy was made through visual reading of the bacterial growth in the wells in the combination experiment.

[0551] General protocol used in Examples 11-74 - Broth microdilution

[0552] Antimicrobial susceptibility testing (AST) by broth microdilution (MIC determination) was performed according to The European Committee on Antimicrobial Susceptibility Testing (ELICAST) guidelines (see Reading guide for broth microdilution Version 5.0, 2024:

[0553] htps: / / ww.eucast.org). Care was taken that a final bacterial inoculum of 3-7 x 105CFU / ml was used for each test condition (each well) and that final concentrations in each well of solvents used for making stock solutions of chemicals did not exceed concentrations having an effect on bacterial growth (e.g. DMSO).

[0554] For combinations containing meropenem as the antibiotic, Sensitive™ FINMER meropenem 64-0.03 pg / mL plates (Thermo Fischer Diagnostics custom plates; YFINMER) were used in most of the experiments. For other antibiotics (and for meropenem whenever a higher start concentration than 64 pg / mL was needed) sterile clear round bottom polystyrene (non-treated) 96-well microplates (Corning 3788) were used for making serial two-fold dilutions of the antibiotic in cation-adjusted Mueller Hinton II broth (caMHB; Thermo Scientific, T3462, containing 29.4 pM Zn2+or MILLIPORE, 90922-500G, containing 5.6 pM Zn2+), across the columns of the microtiter plate (64-0.03 pg / mL; columns 1-12). In the experiments using cefiderocol, iron-depleted caMHB was prepared by treating caMHB from Millipore with Chelex. Relevant cations were then re-added (5.6 pM Zn2+, 20-25 pg / mL CaCI2, 10-12 pg / mL MgCI2).

[0555] The checkerboard plates were all modified by adding a serine p-lactamase inhibitor (sBLI), at a fixed concentration of 16 or 8 pg / ml. Additionally, the zinc chelator was added at various fixed concentrations. Samples were mixed so that all plates contained a control containing antibiotic alone (no sBLI, no zinc chelator), a sample containing antibiotic and sBLI (double combination; no zinc chelator), and a sample containing antibiotic, sBLI and zinc chelator (triple combination). All test wells used in each plate contained growth medium (caMHB), and inoculated bacteria at a final inoculum between 3-7 x 105CFU / ml. After incubation at 37°C for 20 h, assay plates were assessed visually, and the minimum inhibitory concentration (MIC) was determined as the lowest concentration of antimicrobial agent that completely inhibited bacterial growth as detected by the unaided eye. Synergy for the triple combination (antibiotic, sBLI, and zinc chelator) was defined as when the MIC in any of the rows containing the zinc chelator in combination with antibiotic and sBLI (triple combination), was lower than for antibiotic alone and lower than for the antibiotic and sBLI double combination.

[0556] Example 11 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae K66-45

[0557] The effect of the triple combination of the zinc chelator with FEP and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the p-lactamase NDM-1 (Table 14). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0558] Experimental results from MIC determination with a triple combination of FEP, SUL and the zinc chelator are given in Table 14. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the FEP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0559] Table 14

[0560]

[0561] Example 12 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST147

[0562] The effect of the triple combination of the zinc chelator with FEP and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the p-lactamases NDM-1 and KPC-2 (Table 15). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0563] Experimental results from MIC determination with a triple combination of FEP, SUL and the zinc chelator are given in Table 15. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the FEP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0564] Table 15

[0565]

[0566]

[0567] Example 13 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST101

[0568] The effect of the triple combination of the zinc chelator with FEP and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST101, carrying genes encoding the p-lactamase OXA-48 (Table 16). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0569] Experimental results from MIC determination with a triple combination of FEP, SUL and the zinc chelator are given in Table 16. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the FEP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0570] Table 16

[0571]

[0572] Example 14 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and sulbactam (SUL) vs. Acinetobacter baumannii ST 1 The effect of the triple combination of the zinc chelator with FEP and SUL was tested on a clinical isolate of Acinetobacter baumannii, strain ST1, carrying genes encoding the -lactamases OXA-23, OXA-69 and ADC-191 (Table 17). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL and 8 pg / mL.

[0573] Experimental results from MIC determination with a triple combination of FEP, SUL and the zinc chelator are given in Table 17. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the FEP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0574] Table 17

[0575]

[0576] Example 15 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and sulbactam (SUL) vs. Acinetobacter baumannii ST2

[0577] The effect of the triple combination of the zinc chelator with FEP and SUL was tested on a clinical isolate of Acinetobacter baumannii, strain ST2, carrying genes encoding the p-lactamases NDM-5, OXA-23, OXA-66, ADC-73 and TEM-1 (Table 18). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0578] Experimental results from MIC determination with a triple combination of FEP, SUL and the zinc chelator are given in Table 18. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the cefepime MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0579] Table 18

[0580]

[0581] Example 16 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and sulbactam (SUL) vs. Pseudomonas aeruginosa ST 1047

[0582] The effect of the triple combination of the zinc chelator with FEP and SUL was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST1047, carrying genes encoding the P-lactamases IMP-1, OXA-10, OXA-488 and PDC-12 (Table 19). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0583] Experimental results from MIC determination with a triple combination of FEP, Sul and the zinc chelator are given in Table 19. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the FEP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 19

[0584]

[0585] Example 17 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and sulbactam (SUL) vs. Pseudomonas aeruginosa ST773

[0586] The effect of the triple combination of the zinc chelator with FEP and SUL was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the p-lactamases NDM-1 , OXA-395 and PDC-16 (Table 20). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL and 8 pg / mL.

[0587] Experimental results from MIC determination with a triple combination of FEP, SUL and the zinc chelator are given in Table 20. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the cefepime MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0588] Table 20

[0589]

[0590] Example 18 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and sulbactam (SUL) vs. Pseudomonas aeruginosa ST111

[0591] The effect of the triple combination of the zinc chelator with FEP and SUL was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST111 , carrying genes encoding the p-lactamases VIM-2, OXA-395 and PDC-3 (Table 21). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL and 8 pg / mL.

[0592] Experimental results from MIC determination with a triple combination of FEP, SUL and the zinc chelator are given in Table 21. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the FEP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0593] Table 21

[0594]

[0595] Example 19 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae K66-45

[0596] The effect of the triple combination of the zinc chelator with MEM and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the p- lactamase NDM-1 (Table 22). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0597] Experimental results from MIC determination with a triple combination of MEM, SUL and the zinc chelator are given in Table 22. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0598] Table 22

[0599]

[0600] Example 20 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST147

[0601] The effect of the triple combination of the zinc chelator with MEM and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the -lactamases NDM-1 and KPC-2 (Table 23). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL and 4 pg / mL.

[0602] Experimental results from MIC determination with a triple combination of MEM, SUL and the zinc chelator are given in Table 23. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0603] Table 23

[0604]

[0605] Example 21 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST101

[0606] The effect of the triple combination of the zinc chelator with MEM and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST101, carrying genes encoding the p-lactamase OXA-48 (Table 24). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL.

[0607] Experimental results from MIC determination with a triple combination of MEM, SUL and the zinc chelator are given in Table 24. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 24

[0608]

[0609] Example 22 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and sulbactam (SUL) vs. Acinetobacter baumannii ST 1

[0610] The effect of the triple combination of the zinc chelator with MEM and SUL was tested on a clinical isolate of Acinetobacter baumannii, strain ST1, carrying genes encoding the -lactamases OXA-23, OXA-69 and ADC-191 (Table 25). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL.

[0611] Experimental results from MIC determination with a triple combination of MEM, SUL and the zinc chelator are given in Table 25. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0612] Table 25

[0613]

[0614]

[0615] Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and sulbactam (SUL) vs. Acinetobacter baumannii ST2

[0616] The effect of the triple combination of the zinc chelator with MEM and SUL was tested on a clinical isolate of Acinetobacter baumannii ST2 carrying genes encoding the p- lactamases NDM-5, OXA-23, OXA-66, ADC-73, TEM-1 (Table 26). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL and 8 pg / mL.

[0617] Experimental results from MIC determination with a triple combination of MEM, SUL and the zinc chelator are given in Table 26. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0618] Table 26

[0619]

[0620]

[0621] Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and sulbactam (SUL) vs. Acinetobacter baumannii ST15

[0622] The effect of the triple combination of the zinc chelator with MEM and SUL was tested on a clinical isolate of Acinetobacter baumannii ST15 carrying genes encoding the p- lactamases NDM-1 OXA-51 , ADC-263 (Table 27). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL. Experimental results from MIC determination with a triple combination of MEM, SUL and the zinc chelator are given in Table 27. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0623] Table 27

[0624]

[0625] Example 25 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and sulbactam (SUL) vs. Pseudomonas aeruginosa ST773

[0626] The effect of the triple combination of the zinc chelator with MEM and SUL was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the P-lactamases NDM-1 , OXA-395 and PDC-16 (Table 28). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL and 4 pg / mL.

[0627] Experimental results from MIC determination with a triple combination of MEM, SUL and the zinc chelator are given in Table 28. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0628] Table 28

[0629]

[0630]

[0631] Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and sulbactam (SUL) vs. Pseudomonas aeruginosa ST111

[0632] The effect of the triple combination of the zinc chelator with MEM and SUL was tested on a clinical isolate of Pseudomonas aeruginosa ST111 carrying genes encoding the p- lactamases VIM-2, OXA-395, PDC-3 (Table 29). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL.

[0633] Experimental results from MIC determination with a triple combination of MEM, SUL and the zinc chelator are given in Table 29. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0634] Table 29

[0635]

[0636]

[0637] Example 27 - Minimal Inhibitory Concentration (MIC) determination of ceftolozane (CFLOZ) in combination with zinc chelator and tazobactam (TAZ) vs. Pseudomonas aeruginosa ST 1047

[0638] The effect of the triple combination of the zinc chelator with CFLOZ and TAZ was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST1047, carrying genes encoding the p-lactamases IMP-1, OXA-10, OXA-488 and PDC-12 (Table 30). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0639] Experimental results from MIC determination with a triple combination of CFLOZ, TAZ and the zinc chelator are given in Table 30. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CFLOZ are given in the top row left to right (row 0). TAZ and the zinc chelator were added in fixed concentrations (TAZ at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CFLOZ alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CFLOZ MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0640] Table 30

[0641]

[0642]

[0643] Minimal Inhibitory Concentration (MIC) determination of ceftolozane (CFLOZ) in combination with zinc chelator and tazobactam (TAZ) vs. Pseudomonas aeruginosa ST773 The effect of the triple combination of the zinc chelator with CFLOZ and TAZ was tested on a clinical isolate of Pseudomonas aeruginosa ST773 carrying genes encoding the p-lactamases NDM-1 , OXA-395, PDC-16 (Table 31). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0644] Experimental results from MIC determination with a triple combination of CFLOZ, TAZ and zinc chelator are given in Table 31. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CFLOZ are given in the top row left to right (row 0). TAZ and the zinc chelator were added in fixed concentrations (TAZ at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CFLOZ alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CFLOZ MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0645] Table 31

[0646]

[0647] Example 29 - Minimal Inhibitory Concentration (MIC) determination of ceftolozane (CFLOZ) in combination with zinc chelator and tazobactam (TAZ) vs. Klebsiella pneumoniae ST101

[0648] The effect of the triple combination of the zinc chelator with CFLOZ and TAZ was tested on a clinical isolate of Klebsiella pneumoniae ST101 carrying genes encoding the p-lactamase OXA-48 (Table 32). Synergy with the zinc chelator was obtained at the concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL. Experimental results from MIC determination with a triple combination of CFLOZ, TAZ and zinc chelator are given in Table 32. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CFLOZ are given in the top row left to right (row 0). TAZ and the zinc chelator were added in fixed concentrations (TAZ at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CFLOZ alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CFLOZ MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0649] Table 32

[0650]

[0651] Example 30 - Minimal Inhibitory Concentration (MIC) determination of ceftolozane (CFLOZ) in combination with zinc chelator and tazobactam (TAZ) vs. Klebsiella pneumoniae ST147

[0652] The effect of the triple combination of the zinc chelator with CFLOZ and TAZ was tested on a clinical isolate of Klebsiella pneumoniae ST147 carrying genes encoding the p-lactamases NDM-1 and KPC-2 (Table 33). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0653] Experimental results from MIC determination with a triple combination of CFLOZ, TAZ and zinc chelator are given in Table 33. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CFLOZ are given in the top row left to right (row 0). TAZ and the zinc chelator were added in fixed concentrations (TAZ at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CFLOZ alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CFLOZ MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0654] Table 33

[0655]

[0656] Example 31 - Minimal Inhibitory Concentration (MIC) determination of ceftolozane (CFLOZ) in combination with zinc chelator and tazobactam (TAZ) vs. Klebsiella pneumoniae K66-45

[0657] The effect of the triple combination of the zinc chelator with CFLOZ and TAZ was tested on a clinical isolate of Klebsiella pneumoniae K66-45 carrying genes encoding the -lactamase NDM-1 (Table 34). Synergy with the zinc chlelator was obtained at a concentration of 16 pg / mL.

[0658] Experimental results from MIC determination with a triple combination of CFLOZ, TAZ and zinc chelator are given in Table 34. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CFLOZ are given in the top row left to right (row 0). TAZ and the zinc chelator were added in fixed concentrations (TAZ at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CFLOZ alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CFLOZ MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 34

[0659]

[0660] Example 32 - Minimal Inhibitory Concentration (MIC) determination of ceftolozane (CFLOZ) in combination with zinc chelator and tazobactam (TAZ) vs. Klebsiella pneumoniae BAA 1705

[0661] The effect of the triple combination of the zinc chelator with CFLOZ and TAZ was tested on a clinical isolate of Klebsiella pneumoniae BAA 1705 carrying gene encoding the p-lactamase KPC-2 (Table 35). Synergy with the zinc chelator was obtained at the concentrations of 16 pg / mL, 8 pg / mL and 4 pg / m.

[0662] Experimental results from MIC determination with a triple combination of CFLOZ, TAZ and zinc chelator are given in Table 35. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CFLOZ are given in the top row left to right (row 0). TAZ and the zinc chelator were added in fixed concentrations (TAZ at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CFLOZ alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CFLOZ MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0663] Table 35

[0664]

[0665]

[0666] Minimal Inhibitory Concentration (MIC) determination of ceftolozane (CFLOZ) in combination with zinc chelator and tazobactam (TAZ) vs. Acinetobacter baumannii ST15

[0667] The effect of the triple combination of the zinc chelator with CFLOZ and TAZ was tested on a clinical isolate of Acinetobacter baumannii ST15 carrying genes encoding the p- lactamases NDM-1 OXA-51 , ADC-263 (Table 36). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0668] Experimental results from MIC determination with a triple combination of CFLOZ, TAZ and zinc chelator are given in Table 36. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CFLOZ are given in the top row left to right (row 0). TAZ and the zinc chelator were added in fixed concentrations (TAZ at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CFLOZ alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CFLOZ MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0669] Table 36

[0670]

[0671]

[0672] Minimal Inhibitory Concentration (MIC) determination of ceftolozane (CFLOZ) in combination with zinc chelator and tazobactam (TAZ) vs. Escherichia coli BAA 2469

[0673] The effect of the triple combination of the zinc chelator with CFLOZ and TAZ was tested on a clinical isolate of Escherichia coli BAA 2469 carrying genes encoding the - lactamase NDM-1 (Table 37). Synergy with the zinc chelator was obtained at the concentrations of 16 pg / mL and 8 pg / mL.

[0674] Experimental results from MIC determination with a triple combination of CFLOZ, TAZ and zinc chelator are given in Table 37. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CFLOZ are given in the top row left to right (row 0). TAZ and the zinc chelator were added in fixed concentrations (TAZ at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CFLOZ alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CFLOZ MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0675] Table 37

[0676]

[0677] Example 35 - Minimal Inhibitory Concentration (MIC) determination of cefiderocol (CEFDC) in combination with zinc chelator and Xeruborbactam vs. Pseudomonas aeruginosa ST 1047

[0678] The effect of the triple combination of the zinc chelator with CEFDC and Xeruborbactam was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST1047, carrying genes encoding the p-lactamases IMP-1, OXA-10, OXA-488 and PDC-12 (Table 38). Synergy for the zinc chelator was obtained at concentrations of 16 pg / m, 8 pg / mL and 4 pg / mL.

[0679] Experimental results from MIC determination with a triple combination of CEFDC, Xeruborbactam and the zinc chelator are given in Table 38. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFDC are given in the top row left to right (row 0).

[0680] Xeruborbactam and the zinc chelator were added in fixed concentrations (Xeruborbactam at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFDC alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+, 20-25 pg / mL CaCh, 10-12 pg / mL MgCh) and were inoculated with the test bacterium. The grey cells indicate the CEFDC MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0681] Table 38

[0682]

[0683] Example 36 - Minimal Inhibitory Concentration (MIC) determination of cefiderocol (CEFDC) in combination with zinc chelator and Xeruborbactam vs. Pseudomonas aeruginosa ST773

[0684] The effect of the triple combination of the zinc chelator with CEFDC and Xeruborbactam was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the p-lactamases NDM-1 , OXA-395 and PDC-16 (Table 39). Synergy for the zinc chelator was obtained at concentrations of 16 pg / m, 8 pg / mL and 4 pg / mL.

[0685] Experimental results from MIC determination with a triple combination of CEFDC, Xeruborbactam and the zinc chelator are given in Table 39. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth.

[0686] Concentrations (pg / mL) of CEFDC are given in the top row left to right (row 0).

[0687] Xeruborbactam and the zinc chelator were added in fixed concentrations (Xeruborbactam at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFDC alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+, 20-25 pg / mL CaCh, 10-12 pg / mL MgCh) and were inoculated with the test bacterium. The grey cells indicate the CEFDC MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0688] Table 39

[0689]

[0690] Example 37 - Minimal Inhibitory Concentration (MIC) determination of cefiderocol (CEFDC) in combination with zinc chelator and Xeruborbactam vs. Pseudomonas aeruginosa ST111

[0691] The effect of the triple combination of the zinc chelator with CEFDC and Xeruborbactam was tested on a clinical isolate of Pseudomonas aeruginosa ST111 carrying genes encoding the p-lactamases VIM-2, OXA-395, PDC-3 (Table 40). Synergy with the zinc chelator was obtained at concentrations of 16 pg / m, 8 pg / mL and 4 pg / mL.

[0692] Experimental results from MIC determination with a triple combination of CEFDC, Xeruborbactam and the zinc chelator are given in Table 40. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth.

[0693] Concentrations (pg / mL) of CEFDC are given in the top row left to right (row 0).

[0694] Xeruborbactam and the zinc chelator were added in fixed concentrations (Xeruborbactam at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFDC alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+, 20-25 pg / mL CaCh, 10-12 pg / mL MgCh) and were inoculated with the test bacterium. The grey cells indicate the CEFDC MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 40

[0695]

[0696] Example 38 - Minimal Inhibitory Concentration (MIC) determination of cefiderocol (CEFDC) in combination with zinc chelator and Xeruborbactam vs. Klebsiella pneumoniae K66-45

[0697] The effect of the triple combination of the zinc chelator with CEFDC and Xeruborbactam was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the p-lactamase NDM-1 (Table 41). Synergy with the zinc chlelator was obtained at concentrations of 16 pg / m, 8 pg / mL and 4 pg / mL.

[0698] Experimental results from MIC determination with a triple combination of CEFDC, Xeruborbactam and the zinc chelator are given in Table 41. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth.

[0699] Concentrations (pg / mL) of CEFDC are given in the top row left to right (row 0).

[0700] Xeruborbactam and the zinc chelator were added in fixed concentrations (Xeruborbactam at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFDC alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+, 20-25 pg / mL CaCh, 10-12 pg / mL MgCh) and were inoculated with the test bacterium. The grey cells indicate the CEFDC MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0701] Table 41

[0702]

[0703] Example 39 - Minimal Inhibitory Concentration (MIC) determination of cefiderocol (CEFDC) in combination with zinc chelator and Xeruborbactam vs. Klebsiella pneumoniae ST147

[0704] The effect of the triple combination of the zinc chelator with CEFDC and Xeruborbactam was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the p-lactamases NDM-1 and KPC-2 (Table 42). Synergy with the zinc chelator was obtained at concentrations of 16 pg / m, 8 pg / mL and 4 pg / mL.

[0705] Experimental results from MIC determination with a triple combination of CEFDC, Xeruborbactam and the zinc chelator are given in Table 42. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth.

[0706] Concentrations (pg / mL) of CEFDC are given in the top row left to right (row 0).

[0707] Xeruborbactam and the zinc chelator were added in fixed concentrations (Xeruborbactam at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFDC alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+, 20-25 pg / mL CaCh, 10-12 pg / mL MgCh) and were inoculated with the test bacterium. The grey cells indicate the CEFDC MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0708] Table 42

[0709]

[0710] Example 40 - Minimal Inhibitory Concentration (MIC) determination of cefiderocol (CEFDC) in combination with zinc chelator and Xeruborbactam vs. Acinetobacter baumannii ST25

[0711] The effect of the triple combination of the zinc chelator with CEFDC and Xeruborbactam was tested on a clinical isolate of Acinetobacter baumannii, strain ST25, carrying genes encoding the p-lactamases NDM-1, OXA-58, OXA-64, OXA-10, VEB-21 and ADC-26 (Table 43). Synergy with the zinc chelator was obtained at concentrations of 16 pg / m, 8 pg / mL and 4 pg / mL.

[0712] Experimental results from MIC determination with a triple combination of CEFDC, Xeruborbactam and the zinc chelator are given in Table 43. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth.

[0713] Concentrations (pg / mL) of CEFDC are given in the top row left to right (row 0).

[0714] Xeruborbactam and the zinc chelator were added in fixed concentrations (Xeruborbactam at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFDC alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+, 20-25 pg / mL CaCh, 10-12 pg / mL MgCh) and were inoculated with the test bacterium. The grey cells indicate the CEFDC MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0715] Table 43

[0716]

[0717] Example 41 - Minimal Inhibitory Concentration (MIC) determination of cefiderocol (CEFDC) in combination with zinc chelator and Xeruborbactam vs. Acinetobacter baumannii ST2

[0718] The effect of the triple combination of the zinc chelator with CEFDC and Xeruborbactam was tested on a clinical isolate of of Acinetobacter baumannii ST2 carrying genes encoding the p-lactamases NDM-5, OXA-23, OXA-66, ADC-73, TEM-1 (Table 44).

[0719] Synergy with the zinc chelator was obtained at concentrations of 16 pg / m, 8 pg / mL and 4 pg / mL.

[0720] Experimental results from MIC determination with a triple combination of CEFDC, Xeruborbactam and the zinc chelator are given in Table 44. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth.

[0721] Concentrations (pg / mL) of CEFDC are given in the top row left to right (row 0).

[0722] Xeruborbactam and the zinc chelator were added in fixed concentrations (Xeruborbactam at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFDC alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+, 20-25 pg / mL CaCh, 10-12 pg / mL MgCh) and were inoculated with the test bacterium. The grey cells indicate the CEFDC MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0723] Table 44

[0724]

[0725] Example 42 - Minimal Inhibitory Concentration (MIC) determination of cefpodoxime (CEFP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae K66-45

[0726] The effect of the triple combination of the zinc chelator with CEFP and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the p-lactamase NDM-1 (Table 45). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0727] Experimental results from MIC determination with a triple combination of CEFP, SUL and the zinc chelator are given in Table 45. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0728] Table 45

[0729]

[0730] Example 43 - Minimal Inhibitory Concentration (MIC) determination of cefpodoxime (CEFP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST147

[0731] The effect of the triple combination of the zinc chelator with CEFP and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the p-lactamases NDM-1 and KPC-2 (Table 46). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0732] Experimental results from MIC determination with a triple combination of CEFP, SUL and the zinc chelator are given in Table 46. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0733] Table 46

[0734]

[0735]

[0736] Example 44 - Minimal Inhibitory Concentration (MIC) determination of cefpodoxime (CEFP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST101

[0737] The effect of the triple combination of the zinc chelator with CEFP and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST101, carrying genes encoding the p-lactamase OXA-48 (Table 47). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0738] Experimental results from MIC determination with a triple combination of CEFP, SUL and the zinc chelator are given in Table 47. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0739] Table 47

[0740]

[0741] Example 45 - Minimal Inhibitory Concentration (MIC) determination of cefpodoxime (CEFP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae BAA 1705 The effect of the triple combination of the zinc chelator with CEFP and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain BAA 1705, carrying genes encoding the P-lactamase KPC-2 (Table 48). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0742] Experimental results from MIC determination with a triple combination of CEFP, SUL and the zinc chelator are given in Table 48. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0743] Table 48

[0744]

[0745]

[0746] Minimal Inhibitory Concentration (MIC) determination of cefpodoxime (CEFP) in combination with zinc chelator and sulbactam (SUL) vs. Escherichia coli BAA 2469

[0747] The effect of the triple combination of the zinc chelator with CEFP and SUL was tested on a clinical isolate of Escherichia coli BAA 2469 carrying genes encoding the p-lactamase NDM-1 (Table 49). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL.

[0748] Experimental results from MIC determination with a triple combination of CEFP, SUL and the zinc chelator are given in Table 49. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0749] Table 49

[0750]

[0751] Example 47 - Minimal Inhibitory Concentration (MIC) determination of cefpodoxime (CEFP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae K66-45

[0752] The effect of the triple combination of the zinc chelator with CEFP and SUL was tested on a clinical isolate of Klebsiella pneumoniae K66-45 carrying genes encoding the p-lactamase NDM-1 (Table 50). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0753] Experimental results from MIC determination with a triple combination of CEFP, SUL and the zinc chelator are given in Table 50. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 50

[0754]

[0755] Example 48 - Minimal Inhibitory Concentration (MIC) determination of cefpodoxime (CEFP) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST147

[0756] The effect of the triple combination of the zinc chelator with CEFP and SUL was tested on a clinical isolate of Klebsiella pneumoniae ST147 carrying genes encoding the p-lactamase NDM-1 and KPC-2 (Table 51). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0757] Experimental results from MIC determination with a triple combination of CEFP, SUL and the zinc chelator are given in Table 51. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFP are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0758] Table 51

[0759]

[0760] Example 49 - Minimal Inhibitory Concentration (MIC) determination of amoxicillin (AMOX) in combination with zinc chelator and sulbactam (SUL) vs. Pseudomonas aeruginosa ST111

[0761] The effect of the triple combination of the zinc chelator with AMOX and SUL was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST111 , carrying genes encoding the P-lactamases VIM-2, OXA-395 and PDC-3 (Table 52). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL.

[0762] Experimental results from MIC determination with a triple combination of AMOX, SUL and the zinc chelator are given in Table 52. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of AMOX are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of AMOX alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the AMOX MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0763] Table 52

[0764]

[0765] Example 50 - Minimal Inhibitory Concentration (MIC) determination of amoxicillin (AMOX) in combination with zinc chelator and sulbactam (SUL) vs. Escherichia coli BAA 2469

[0766] The effect of the triple combination of the zinc chelator with AMOX and SUL was tested on a clinical isolate of Escherichia coli BAA 2469 carrying genes encoding the p-lactamase NDM-1 (Table 53). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL.

[0767] Experimental results from MIC determination with a triple combination of AMOX, SUL and the zinc chelator are given in Table 53. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of AMOX are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of AMOX alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the AMOX MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0768] Table 53

[0769]

[0770] Example 51 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae K66-45

[0771] The effect of the triple combination of the zinc chelator with MEM and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the -lactamase NDM-1 (Table 54). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0772] Experimental results from MIC determination with a triple combination of MEM, AVI and the zinc chelator are given in Table 54. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0773] Table 54

[0774]

[0775] Example 52 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae ST147

[0776] The effect of the triple combination of the zinc chelator with MEM and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the p-lactamases NDM-1 and KPC-2 (Table 55). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0777] Experimental results from MIC determination with a triple combination of MEM, AVI and the zinc chelator are given in Table 55. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 55

[0778]

[0779]

[0780] Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and avibactam (AVI) vs. Acinetobacter baumannii ST15

[0781] The effect of the triple combination of the zinc chelator with MEM and AVI was tested on a clinical isolate of Acinetobacter baumannii ST15 carrying genes encoding the p-lactamases NDM-1 OXA-51 , ADC-263 (Table 56). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0782] Experimental results from MIC determination with a triple combination of MEM, AVI and the zinc chelator are given in Table 56. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0783] Table 56

[0784]

[0785] Example 54 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and avibactam (AVI) vs. Pseudomonas aeruginosa ST773

[0786] The effect of the triple combination of the zinc chelator with MEM and AVI was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the p-lactamases NDM-1 , OXA-395 and PDC-16 (Table 57). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0787] Experimental results from MIC determination with a triple combination of MEM, AVI and the zinc chelator are given in Table 57. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0788] Table 57

[0789]

[0790] Example 55 - Minimal Inhibitory Concentration (MIC) determination of meropenem (MEM) in combination with zinc chelator and avibactam (AVI) vs. Escherichia coli BAA 2469

[0791] The effect of the triple combination of the zinc chelator with MEM and AVI was tested on a clinical isolate of Escherichia coli BAA 2469 carrying genes encoding the p-lactamase NDM-1 (Table 58). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0792] Experimental results from MIC determination with a triple combination of MEM, AVI and the zinc chelator are given in Table 58. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of MEM are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of MEM alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the MEM MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0793] Table 58

[0794]

[0795] Example 56 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae K66-45

[0796] The effect of the triple combination of the zinc chelator with FEP and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the -lactamase NDM-1 (Table 59). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0797] Experimental results from MIC determination with a triple combination of FEP, AVI and the zinc chelator are given in Table 59. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the FEP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0798] Table 59

[0799]

[0800] Example 57 - Minimal Inhibitory Concentration (MIC) determination of cefepime (FEP) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae ST147

[0801] The effect of the triple combination of the zinc chelator with FEP and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the p-lactamases NDM-1 and KPC-2 (Table 60). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0802] Experimental results from MIC determination with a triple combination of FEP, AVI and the zinc chelator are given in Table 60. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of FEP are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of FEP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the FEP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 60

[0803]

[0804] Example 58 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae K66-45

[0805] The effect of the triple combination of the zinc chelator with CEFTR and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the P-lactamase NDM-1 (Table 61). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0806] Experimental results from MIC determination with a triple combination of CEFTR, AVI and the zinc chelator are given in Table 61. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0807] Table 61

[0808]

[0809] Example 59 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae ST147

[0810] The effect of the triple combination of the zinc chelator with CEFTR and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the P-lactamases NDM-1 and KPC-2 (Table 62). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0811] Experimental results from MIC determination with a triple combination of CEFTR, AVI and the zinc chelator are given in Table 62. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0812] Table 62

[0813]

[0814] Example 60 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and avibactam (AVI) vs. Pseudomonas aeruginosa ST773

[0815] The effect of the triple combination of the zinc chelator with CEFTR and AVI was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the p-lactamases NDM-1, OXA-395 and PDC-16 (Table 63). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL and 8 pg / mL.

[0816] Experimental results from MIC determination with a triple combination of CEFTR, AVI and the zinc chelator are given in Table 63. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0817] Table 63

[0818]

[0819]

[0820] Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and avibactam (AVI) vs. Escherichia coli BAA 2469

[0821] The effect of the triple combination of the zinc chelator with CEFTR and AVI was tested on a clinical isolate of Escherichia coli BAA 2469 carrying genes encoding the -lactamase NDM-1 (Table 64). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL.

[0822] Experimental results from MIC determination with a triple combination of CEFTR, AVI and the zinc chelator are given in Table 64. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0823] Table 64

[0824]

[0825] Example 62 - Minimal Inhibitory Concentration (MIC) determination of ceftaroline (CEFT) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae K66-45

[0826] The effect of the triple combination of the zinc chelator with CEFT and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the p-lactamase NDM-1 (Table 65). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0827] Experimental results from MIC determination with a triple combination of CEFT, AVI and the zinc chelator are given in Table 65. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFT are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFT alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFT MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 65

[0828]

[0829] Example 63 - Minimal Inhibitory Concentration (MIC) determination of ceftaroline (CEFT) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae ST147

[0830] The effect of the triple combination of the zinc chelator with CEFT and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the p-lactamases NDM-1 and KPC-2 (Table 66). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL, and 4 pg / mL.

[0831] Experimental results from MIC determination with a triple combination of CEFT, AVI and the zinc chelator are given in Table 66. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFT are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFT alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFT MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0832] Table 66

[0833]

[0834] Example 64 - Minimal Inhibitory Concentration (MIC) determination of ceftaroline (CEFT) in combination with zinc chelator and avibactam (AVI) vs. Klebsiella pneumoniae BAA 1705

[0835] The effect of the triple combination of the zinc chelator with CEFT and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain BAA 1705, carrying genes encoding the P-lactamase KPC-2 (Table 67). Synergy with the zinc chelator was obtained at a concentration of 8 pg / mL.

[0836] Experimental results from MIC determination with a triple combination of CEFT, AVI and the zinc chelator are given in Table 67. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFT are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFT alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFT MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0837] Table 67

[0838]

[0839] Example 65 - Minimal Inhibitory Concentration (MIC) determination of ceftaroline (CEFT) in combination with zinc chelator and avibactam (AVI) vs. Pseudomonas aeruginosa ST773

[0840] The effect of the triple combination of the zinc chelator with CEFT and AVI was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the P-lactamases NDM-1 , OXA-395 and PDC-16 (Table 68). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0841] Experimental results from MIC determination with a triple combination of CEFT, AVI and the zinc chelator are given in Table 68. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFT are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFT alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFT MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0842] Table 68

[0843]

[0844] Example 66 - Minimal Inhibitory Concentration (MIC) determination of ceftaroline (CEFT) in combination with zinc chelator and avibactam (AVI) vs. Escherichia coli BAA 2469

[0845] The effect of the triple combination of the zinc chelator with CEFT and AVI was tested on a clinical isolate of Escherichia coli BAA 2469 carrying genes encoding the p-lactamase NDM-1 (Table 69). Synergy with the zinc chelator was obtained at concentrations of 16 pg / mL, 8 pg / mL and 4 pg / mL.

[0846] Experimental results from MIC determination with a triple combination of CEFT, AVI and the zinc chelator are given in Table 69. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFT are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 8 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of CEFT alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFT MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0847] Table 69

[0848]

[0849] Example 67 - Minimal Inhibitory Concentration (MIC) determination of piperacillin (PIP) in combination with zinc chelator and avibactam (AVI) vs. Pseudomonas aeruginosa ST773

[0850] The effect of the triple combination of the zinc chelator with PIP and AVI was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the p-lactamases NDM-1 , OXA-395 and PDC-16 (Table 70). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0851] Experimental results from MIC determination with a triple combination of PIP, AVI and the zinc chelator are given in Table 70. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of PIP are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of PIP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 29.4 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the PIP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively. Table 70

[0852]

[0853]

[0854] Minimal Inhibitory Concentration (MIC) determination of piperacillin (PIP) in combination with zinc chelator and avibactam (AVI) vs. Pseudomonas aeruginosa ST111

[0855] The effect of the triple combination of the zinc chelator with PIP and AVI was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST111 , carrying genes encoding the p-lactamases VIM-2, OXA-395 and PDC-3 (Table 71). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0856] Experimental results from MIC determination with a triple combination of PIP, AVI and the zinc chelator are given in Table 71. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of PIP are given in the top row left to right (row 0). AVI and the zinc chelator were added in fixed concentrations (AVI at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of PIP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 29.4 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the PIP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0857] Table 71

[0858]

[0859] Example 69 - Minimal Inhibitory Concentration (MIC) determination of piperacillin (PIP) in combination with zinc chelator and tazobactam (AVI) vs. Pseudomonas aeruginosa ST773

[0860] The effect of the triple combination of the zinc chelator with PIP and tazobactam was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the p-lactamases NDM-1, OXA-395 and PDC-16 (Table 72). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0861] Experimental results from MIC determination with a triple combination of PIP, tazobactam and the zinc chelator is given in Table 72. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of PIP are given in the top row left to right (row 0). Avibactam and the zinc chelator were added in fixed concentrations (Tazobactam at 16 pg / mL in rows 2-5, zinc chelator at 16 pg / mL in row 3, at 8 pg / mL in row 4, and at 4 pg / mL in row 5, respectively). As a control, the effect of PIP alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 29.4 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the PIP MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0862] Table 72

[0863]

[0864] Example 70 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae K66-45

[0865] The effect of the triple combination of the zinc chelator with CEFTR and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain K66-45, carrying genes encoding the P-lactamase NDM-1 (Table 73). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0866] Experimental results from MIC determination with a triple combination of CEFTR, SUL and the zinc chelator are given in Table 73. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-3 and zinc chelator at 16 pg / mL in row 3.) As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0867] Table 73

[0868]

[0869] Example 71 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST147

[0870] The effect of the triple combination of the zinc chelator with CEFTR and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the P-lactamases NDM-1 and KPC-2 (Table 74). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0871] Experimental results from MIC determination with a triple combination of CEFTR, SUL and the zinc chelator are given in Table 74. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-3 and zinc chelator at 16 pg / mL in row 3.) As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0872] Table 74

[0873]

[0874] Example 72 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST773

[0875] The effect of the triple combination of the zinc chelator with CEFTR and SUL was tested on a clinical isolate of Pseudomonas aeruginosa, strain ST773, carrying genes encoding the p-lactamases NDM-1, OXA-395 and PDC-16 (Table 75). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0876] Experimental results from MIC determination with a triple combination of CEFTR, SUL and the zinc chelator are given in Table 75. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-3 and zinc chelator at 16 pg / mL in row 3.) As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0877] Table 75

[0878]

[0879] Example 73 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and sulbactam (SUL) vs. Klebsiella pneumoniae ST101

[0880] The effect of the triple combination of the zinc chelator with CEFTR and SUL was tested on a clinical isolate of Klebsiella pneumoniae, strain ST101, carrying genes encoding the P-lactamase OXA-48 (Table 76). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL.

[0881] Experimental results from MIC determination with a triple combination of CEFTR, SUL and the zinc chelator are given in Table 76. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-3 and zinc chelator at 16 pg / mL in row 3.) As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0882] Table 76

[0883]

[0884] Example 74 - Minimal Inhibitory Concentration (MIC) determination of ceftriaxone (CEFTR) in combination with zinc chelator and sulbactam (SUL) vs. Acinetobacter baumannii ST 1

[0885] The effect of the triple combination of the zinc chelator with CEFTR and SUL was tested on a clinical isolate of Acinetobacter baumannii, strain ST1, carrying genes encoding the P-lactamases OXA-23, OXA-69 and ADC-191 (Table 77). Synergy with the zinc chelator was obtained at a concentration of 16 pg / mL. Experimental results from MIC determination with a triple combination of CEFTR, SUL and the zinc chelator are given in Table 77. An X in rows 1-5 in the table indicates growth of the bacterial inoculum, while a blank cell indicates no growth. Concentrations (pg / mL) of CEFTR are given in the top row left to right (row 0). SUL and the zinc chelator were added in fixed concentrations (SUL at 16 pg / mL in rows 2-3 and zinc chelator at 16 pg / mL in row 3.) As a control, the effect of CEFTR alone was tested (row 1). All wells contained bacterial growth medium (caMHB, 5.6 pM Zn2+) and were inoculated with the test bacterium. The grey cells indicate the CEFTR MIC. For rows where the exact MIC could not be determined, the MIC was set at >64 pg / mL (black shading) or <0.03 pg / mL (light grey shading), respectively.

[0886] Table 77

[0887]

[0888] Example 75 - Time-kill experiment for the triple combination of meropenem (MEM), Avibactam (AVI) and the zinc chelator

[0889] The effect of the triple combination of the zinc chelator (compound of Ex. 1) with MEM and AVI was tested on a clinical isolate of Klebsiella pneumoniae, strain ST147, carrying genes encoding the p-lactamases NDM-1 and KPC-2. The kinetics of killing of the triple combination was determined by treating K. pneumoniae ST147 bacterial culture

[0890] (1 x 106CFU / ml) in caMHB (MILLIPORE, 90922-500G, containing 5.6 pM Zn2+). The bacterial culture was divided in three separate flasks and treated with either 8 pg / mL MEM, a combination of 8 pg / mL MEM and 8 pg / mL AVI or a triple combination of 8 pg / mL MEM, 8 pg / mL AVI plus the compound of Ex. 1 at 16 pg / mL and grown at 37°C with shaking (180 rpm). The cell number was determined over the course of 24 h (at 0, 2, 4, 8 and 24h) by making serial dilutions of culture in sterile PBS which was spotted and dried on square (10 x 10 cm) Luria-Bertani agar (Oxoid, Basingstoke, UK) plates before incubation at 37°C. The number of colonies were counted the next day and CFU / mL calculated. The limit of detection was set as one colony in 5 ml of undiluted culture, representing 100 CFU / ml. The results are shown in Fig. 3. The triple combination is reduced to a CFU / mL at the limit of detection (100 CFU / mL) after 24 hours.

Claims

Claims:

1. A compound of formula (I), a stereoisomer or pharmaceutically acceptable salt thereof:wherein:Q is a lipophilic, zinc chelating moiety which is selective for Zn2+ions;Li is a covalent bond or a C1-6 alkylene chain in which one or more -CH2- groups (e.g. all -CH2- groups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR4- (where R4is H or C1-3 alkyl, preferably H);Y is selected from the following groups:(where each R5is independently H or C1-3 alkyl, preferably H; andR6is H or C1-3 alkyl, preferably H);L2 is a covalent bond or a C1-6 alkylene chain in which one or more -CH2- groups (e.g. all -CH2- groups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR7- (where R7is H or C1-3 alkyl, preferably H);R is -OH, -O-C1-6 alkyl, -O-(CH2)P-O-CO-CI-6 alkyl (where p is an integer of 1 or 2) or -O-CH(CH3)-O-CO-CI-6 alkyl;each R1is independently selected from halogen and C1-3 alkyl;each R2is independently selected from halogen and C1-3 alkyl;R3is H or C1-3 alkyl, preferably H;n is an integer of 0 or 1, preferably 0; andm is an integer of 0 or 1, preferably 0.

2. A compound according to claim 1, wherein the zinc chelating moiety Q comprises one or more optionally substituted heteroaryl rings, preferably two or more optionally substituted heteroaryl rings, e.g. such heteroaryl rings in which each heteroaryl ring has at least one nitrogen atom in the ring structure (e.g. pyridine, especially unsubstituted pyridine).

3. A compound according to claim 2, wherein the zinc chelating moiety Q is derived from picolinic acid and its derivatives (e.g. from picoylamine), preferably wherein the zinc chelating moiety comprises two or more (e.g. two, three or four) 2-pyridyl-methyl units.

4. A compound according to claim 1, wherein the zinc chelating moiety Q is one of the following groups:wherein* denotes the point of attachment of the zinc chelating moiety to the linker group Li; and R’, where present, is H or C1-6 alkyl, preferably C1-3 alkyl, e.g. methyl.

5. A compound according to claim 1, wherein the zinc chelating moiety Q is one of the following groups:wherein * denotes the point of attachment of the chelating moiety to the linker group Li.

6. A compound according to any one of the preceding claims, wherein Li is a covalent bond or a C1-4 alkylene chain in which one or more -CH2- groups (e.g. all -Cogroups) of the alkylene chain are optionally replaced by a group independently selected from -CO- and -NR4- (where R4is H or C1-3 alkyl, preferably H).

7. A compound according to claim 6, wherein Li is selected from the following: a covalent bond, -CH2-, -CH2-CH2-, -NR4-CH2-, -CO-, -CO-NR4-CH2-, and-CH2-CH2-NR4-CH2- (where R4is H or C1-3 alkyl, preferably H).

8. A compound according to claim 6, wherein Li is selected from the following: -CH2-, -CH2-CH2-, -CO- and -CO-NR4-CH2- (where R4is H or C1-3 alkyl, preferably H).

9. A compound according to any one of the preceding claims, wherein Y is selected from the following groups:< <10. A compound according to any one of the preceding claims, wherein l_2is a covalent bond.

11. A compound according to any one of the preceding claims, wherein R is -OH or -O-C1-6 alkyl, preferably -OH or -O-C1-3 alkyl.

12. A compound according to any one of the preceding claims, wherein R3is H.

13. A compound according to claim 1 which is a compound selected from the following, a stereoisomer or pharmaceutically acceptable salt thereof:

14. A compound according to claim 1 which is a compound selected from the following, or a pharmaceutically acceptable salt thereof:

15. A compound according to claim 1 which is a compound selected from the following, or a pharmaceutically acceptable salt thereof:

16. A compound according to claim 1 which is:or a pharmaceutically acceptable salt thereof, preferably the disodium salt thereof.

17. A compound according to any one of claims 1 to 16 which is provided in the form of a disodium salt or calcium salt.

18. A pharmaceutical composition comprising a compound according to one of claims 1 to 17, together with at least one pharmaceutically acceptable carrier, diluent or excipient.

19. A compound according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18 for use in therapy.

20. A compound according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18, for use in a method of treatment of a bacterial infection in a subject, wherein said method comprises the step of administering to the subject a therapeutically effective amount of said compound or said pharmaceutical composition in combination with (either simultaneously, separately or sequentially) at least one antibacterial agent.

21. A method of treatment of a bacterial infection in a subject, said method comprising the step of administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 17 or a pharmaceutical composition according to claim 18 in combination with (either simultaneously, separately or sequentially) at least one antibacterial agent.

22. A compound or pharmaceutical composition for use, or method of treatment according to claim 20 or claim 21, wherein the bacterial infection is associated with grampositive and / or gram-negative bacteria which are resistant to one or more antibiotics, for example bacteria that are resistant to treatment with p-lactam antibiotics.

23. A compound or pharmaceutical composition for use, or method of treatment according to any one of claims 20 to 22, wherein the bacterial infection is associated with bacteria that comprise metallo-p-lactamases and / or serine-p-lactamases.

24. A compound or pharmaceutical composition for use, or method of treatment according to any one of claims 20 to 23, wherein the bacterial infection is caused by a bacteria selected from the group consisting of Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Aeromonas spp, Aeromones hydrophilia, Bacillus cereus Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Bacteroides thetaiotaomicron, Borrelia burgdorferi, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Burkholderia cepacia, Branhamella catarrhalis, Campylobacterfetus, Campylobacter jejuni, Campylobacter coli, Chryseobacterium indoIogenes Citrobacter freundii, Clostridium difficile, Corynebacterium diphtheriae, Corynebacterium ulcerans, Elizabethkingia meningoseptica, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Francisella tularensis, Gardnerella vaginalis, Haemophilus influenzae,Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, Kingella, Moraxella, Klebsiella pneumoniae, Klebsiella oxytoca, Legionella pneumophila, Listeria monocytogenes, Morganella morganii, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Moraxella, Mycobacterium leprae, Myroides odoratimimus, Neisseria gonorrhoeae, Neisseria meningitidis Pasteurella multocida, Pasteurella haemolytica, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonasfluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonasputida, Serratia marcescens, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Serratia marcescens, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saccharolyticus.Stenotrophomonas maltophilia, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Yersinia enterocolitica, and Yersinia pestis.

25. A compound or pharmaceutical composition for use, or method of treatment according to claim 24, wherein the bacterial infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae, Acinetobacter baumannii, Pseuodomonas aeruginosa and Escherichia coli, preferably wherein the bacterial infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae K66-45, Klebsiella pneumoniae ST147, Klebsiella pneumoniae ST101, Klebsiella pneumoniae BAA1705, Acinetobacter baumannii ST1, Acinetobacter baumannii ST2, Acinetobacter baumannii ST15, Acinetobacter baumannii ST25, Pseudomonas aeruginosa ST1047, Pseudomonas aeruginosa ST773, Pseudomonas aeruginosa ST111 , and Escherichia coli BAA2469.

26. A compound or pharmaceutical composition for use, or method of treatment according to any one of claims 20 to 25, wherein the subject is a human.

27. A compound or pharmaceutical composition for use, or method of treatment according to any one of claims 20 to 26, wherein the at least one antibacterial agent is provided in a separate formulation from the compound or pharmaceutical composition.

28. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 17 together with at least one antibacterial agent, and at least one pharmaceutically acceptable carrier, diluent or excipient.

29. A kit comprising:(i) a first container containing a compound according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18; and(ii) a second container containing an antibacterial agent.

30. A compound or pharmaceutical composition for use, method of treatment, pharmaceutical formulation or kit according to any one of claims 20 to 29, wherein said antibacterial agent is an antibiotic, preferably a p-lactam antibiotic.

31. A compound or pharmaceutical composition for use, method of treatment, pharmaceutical formulation or kit according to claim 30, wherein said antibiotic is selected from the group consisting of penicillins, cephalosporins and cephems, monobactams, carbapenems, penems, monobactams, clavams, glycopeptides, macrolides, quinolones, tetracyclines, aminoglycosides, tetracyclines, rifamycins, sulfonamides, trimethoprim, polymyxins, macrolides, chloramphenicol, oxazolidinones, glycopeptides, cycloserine, isoniazide, daptomycin, cyclosporine, phenazines, and derivatives thereof.

32. A compound or pharmaceutical composition for use, method of treatment, pharmaceutical formulation or kit according to claim 30, wherein said antibiotic is a p-lactam antibiotic selected from the group consisting of penicillins, monobactams and carbapenems.

33. A compound or pharmaceutical composition for use, method of treatment, pharmaceutical formulation or kit according to claim 32, wherein said p-lactam antibiotic is a carbapenem.

34. A compound or pharmaceutical composition for use, method of treatment, pharmaceutical formulation or kit according to claim 33, wherein the carbapenem isselected from the group consisting of benapenem, biapenem, doripenem, ertapenem, imipenem, lenapenem, meropenem, panipenem, razupenem, tebipenem, tebipenem, thienpenem, tomopenem and derivatives thereof, e.g. a pharmaceutically acceptable salt thereof.

35. A compound or pharmaceutical composition for use, method of treatment, pharmaceutical formulation or kit according to claim 34, wherein the carbapenem is meropenem or a pharmaceutically acceptable salt thereof.

36. A method of treatment of a bacterial infection, said method comprisingco-administration of an effective amount of each of the following agents to a subject in need thereof:(A) a selective zinc-chelator which is a compound according to any one of claims 1 to 17;(B) a p-lactam antibiotic; and(C) a serine p-lactamase inhibitor.

37. A method of treatment according to claim 36, wherein agent (B) is selected from the group consisting of penicillins, monobactams and carbapenems.

38. A method of treatment according to claim 37, wherein agent (B) is a penicillin.

39. A method of treatment according to claim 38, wherein the penicillin is selected from the group consisting of cioxacillin, dicloxacillin, fl ucloxacill in, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, pivampicillin, bacampicillin, metampicillin, talampicillin, hetacillin, epicillin, phenoxymethylpenicillin, benzylpenicillin, carboxypenicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, piperacillin, azlocillin, and their pharmaceutically acceptable salts, preferably wherein the penicillin is piperacillin, ampicillin, amoxicillin or a pharmaceutically acceptable salt thereof, more preferably wherein the penicillin is piperacillin, amoxicillin or a pharmaceutically acceptable salt thereof.

40. A method of treatment according to claim 37, wherein agent (B) is a monobactam.

41. A method of treatment according to claim 40, wherein the monobactam is selected from the group consisting of include aztreonam, aztreonam lysine, tigemonam, nocardicinA, tabtoxin, BAL 30072, SYN 2416 (BAL 19764), carumonam, AIC 499, BOS 228 (LYS 228), MC-1, and their pharmaceutically acceptable salts, preferably wherein the monobactam is aztreonam or a pharmaceutically acceptable salt thereof.

42. A method of treatment according to claim 37, wherein agent (B) is a carbapenem.

43. A method of treatment according to claim 42, wherein the carbapenem is selected from the group consisting of benapenem, biapenem, doripenem, ertapenem, imipenem, lenapenem, meropenem, panipenem, razupenem, tebipenem, tebipenem, thienpenem, tomopenem and derivatives thereof, e.g. a pharmaceutically acceptable salt thereof.

44. A method of treatment according to claim 43, wherein the carbapenem is meropenem or a pharmaceutically acceptable salt thereof.

45. A method of treatment according to claim 36, wherein agent (B) is a cephalosporin.

46. A method of treatment according to claim 45, wherein agent (B) is a cephalosporin selected from the following compounds, their pharmaceutically acceptable salts and47. A method of treatment according to claim 45, wherein agent (B) is a cephalosporin selected from the group consisting of cefepime, cefditoren pivoxil, cefixime, cefpodoxime, cefotaxime, ceftaroline, ceftazidime, ceftibuten, ceftobiprole, ceftolozane, ceftriaxone, cefotaxone, cephmetazole, cephalexin, cefiderocol, and their pharmaceutically acceptable salts.

48. A method of treatment according to claim 47, wherein agent (B) is a cephalosporin selected from the group consisting of consisting of cefepime, ceftaroline, ceftobiprole, ceftriaxone, cefotaxone, cephmetazole, cephalexin, cefiderocol, and their pharmaceutically acceptable salts, preferably a cephalosporin selected from the group consisting of cefepime, ceftolozane, cefpodoxime, ceftaroline, ceftriaxone, cefiderocol, and their pharmaceutically acceptable salts.

49. A method of treatment according to claim 47, wherein agent (B) is ceftriaxone or a pharmaceutically acceptable salt thereof.

50. A method of treatment according to any one of claims 36 to 49, wherein agent (C) is a serine p-lactamase inhibitor which inhibits at least one SBL in Ambler class A, for example CepA, KPC-2, IMI-1, SME-1, PC1, TEM-1, TEM-2, TEM-3, TEM-30, TEM-50, SHV-1, SHV-2, SHV-10, CTX-M-15, PER-1, VEB-1, PSE-1, CARB-3, or RTG-4; ora serine p-lactamase inhibitor which inhibits at least one SBL in Ambler class C, for example AmpC, CMY-1, ACT-1, FOX-1, MIR-1, GC1, CMY-10, CMY-19, or CMY-37, ora serine p-lactamase inhibitor which inhibits at least one SBL in Ambler class D, for example OXA-1 , OXA-10, OXA-11, OXA-15, OXA-23, OXA-48.

51. A method of treatment according to claim 50, wherein the serine p-lactamase inhibitor is a diaza-bicyclo-octanone (DBO) compound, a prodrug or a pharmaceutically acceptable salt thereof.

52. A method of treatment according to claim 51 , wherein said diaza-bicyclo-octanone (DBO) compound is selected from the following compounds, their pharmaceutically acceptable salts and prodrugs:

53. A method of treatment according to claim 51 , wherein said diaza-bicyclo-octanone (DBO) compound is avibactam or a pharmaceutically acceptable salt thereof, preferably the sodium salt of avibactam, for example crystal Form B of the sodium salt of avibactam.

54. A method of treatment according to any one of claims 36 to 50, wherein the serine P-lactamase inhibitor is a p-lactam.

55. A method of treatment according to claim 54, wherein the p-lactam is selected from the group consisting of clavulanic acid, sulbactam, tazobactam, enmetazobactam, their pharmaceutically acceptable salts and prodrugs thereof, preferably wherein the p-lactam is sulbactam, tazobactam or a pharmaceutically acceptable salt thereof.

56. A method of treatment according to claim 54, wherein the p-lactam is sulbactam or a pharmaceutically acceptable salt thereof.

57. A method of treatment according to any one of claims 36 to 50, wherein agent (C) is a cyclic boronate ester having serine p-lactamase inhibitory activity.

58. A method of treatment according to claim 57, wherein agent (C) is a cyclic boronate ester which inhibits at least one SBL in Ambler class A, for example CepA, KPC-2, IMI-1, SME-1, PC1, TEM-1, TEM-2, TEM-3, TEM-30, TEM-50, SHV-1, SHV-2, SHV-10, CTX-M-15, PER-1, VEB-1, PSE-1, CARB-3, or RTG-4; or a cyclic boronate ester which inhibits at least one SBL in Ambler class C, for example AmpC, CMY-1 , ACT-1 , FOX-1 , MIR-1, GC1, CMY-10, CMY-19, or CMY-37, or a cyclic boronate ester which inhibits at least one SBL in Ambler class D, for example OXA-1 , OXA-10, OXA-11 , OXA-15, OXA-23, OXA-48.

59. A method of treatment according to claim 57 or 58, wherein the cyclic boronate ester is selected from the following compounds, their pharmaceutically acceptable salts and prodrugs:

60. A method of treatment according to claim 59, wherein the third agent (C) is vaborbactam, taniborbactam, xeruborbactam or a pharmaceutically acceptable salt thereof, preferably xeruborbactam or a pharmaceutically acceptable salt thereof.

61. A method of treatment according to claim 36, wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said P-lactam antibiotic is amoxicillin, piperacillin, or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam, avibactam, tazobactam, or a pharmaceutically acceptable salt thereof;or wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is meropenem or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam, avibactam, or a pharmaceutically acceptable salt thereof;or wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is cefepime, ceftolozane, cefiderocol, cefpodoxime, ceftriaxone, ceftaroline, or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is avibactam, sulbactam, tazobactam, xeruborbactam, or a pharmaceutically acceptable salt thereof;preferably wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is ceftriaxone or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam.

62. A method of treatment according to any one of claims 36 to 61 , wherein the bacterial infection is associated with gram-positive or gram-negative bacteria, preferably gram-negative bacteria, more preferably wherein said bacteria are resistant to treatment with one or more antibiotics, particularly bacteria that are resistant to treatment with p-lactam antibiotics.

63. A method of treatment according to claim 62, wherein the bacterial infection is associated with gram-positive or gram-negative bacteria which produce metallo-p-lactamases and / or serine-p-lactamases, preferably gram-negative bacteria which produce serine-p-lactamases.

64. A method of treatment according to any one of claims 36 to 63, wherein the bacterial infection is caused by a bacteria selected from the group consisting of Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Aeromonas spp, Aeromones hydrophilia, Bacillus cereus Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Bacteroides thetaiotaomicron, Borrelia burgdorferi, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Burkholderia cepacia, Branhamella catarrhalis, Campylobacterfetus, Campylobacter jejuni, Campylobacter coli, Chryseobacterium indoIogenes Citrobacter freundii, Clostridium difficile, Corynebacterium diphtheriae, Corynebacterium ulcerans, Elizabethkingia meningoseptica, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Francisella tularensis, Gardnerella vaginalis, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, Kingella, Moraxella, Klebsiella pneumoniae, Klebsiella oxytoca, Legionella pneumophila, Listeria monocytogenes, Morganella morganii, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Moraxella, Mycobacterium leprae, Myroides odoratimimus, Neisseria gonorrhoeae, Neisseria meningitidis Pasteurella multocida, Pasteurella haemolytica, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonasfluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonasputida, Serratia marcescens, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Serratia marcescens, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saccharolyticus.Stenotrophomonas maltophilia, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Yersinia enterocolitica, and Yersinia pestis.

65. A method of treatment according to claim 64, wherein the bacterial infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae, Acinetobacter baumannii, Pseuodomonas aeruginosa and Escherichia coli, preferably wherein the bacterial infection is caused by a bacteria selected from the group consisting of Klebsiella pneumoniae K66-45, Klebsiella pneumoniae ST 147, Klebsiella pneumoniae ST101, Klebsiella pneumoniae BAA1705, Acinetobacter baumannii ST1, Acinetobacter baumannii ST2, Acinetobacter baumannii ST15, Acinetobacter baumannii ST25, Pseudomonas aeruginosa ST1047, Pseudomonas aeruginosa ST773, Pseudomonas aeruginosa ST111 , and Escherichia coli BAA2469.

66. A method of treatment according to any one of claims 36 to 65, wherein the subject is a human.

67. A selective zinc-chelator (A) which is a compound according to any one of claims 1 to 17 for use in the treatment of a bacterial infection in a subject by co-administration with (B) a p-lactam antibiotic as defined in any one of claims 36 to 49; and (C) a serine p-lactamase inhibitor as defined in any one of claims 36 and 50 to 60.

68. A p-lactam antibiotic (B) as defined in any one of claims 36 to 49 for use in the treatment of a bacterial infection in a subject by co-administration with (A) a selective zinc-chelator which is a compound according to any one of claims 1 to 17; and (C) a serine p-lactamase inhibitor as defined in any one of claims 36 and 50 to 60.

69. A serine p-lactamase inhibitor (C) as defined in any one of claims 36 and 50 to 60 for use in the treatment of a bacterial infection in a subject by co-administration with (A) a selective zinc-chelator which is a compound according to any one of claims 1 to 17; and (B) a p-lactam antibiotic as defined in any one of claims 36 to 49.

70. A selective zinc-chelator (A), a p-lactam antibiotic (B), or a serine p-lactamase inhibitor (C) for use according to any one of claims 67 to 69, wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is amoxicillin, piperacillin, or a pharmaceuticallyacceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam, avibactam, tazobactam, or a pharmaceutically acceptable salt thereof;or wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is meropenem or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam, avibactam, or a pharmaceutically acceptable salt thereof;or wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is cefepime, ceftolozane, cefiderocol, cefpodoxime, ceftriaxone, ceftaroline, or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is avibactam, sulbactam, tazobactam, xeruborbactam, or a pharmaceutically acceptable salt thereof;preferably wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is ceftriaxone or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam.

71. A selective zinc-chelator (A), a p-lactam antibiotic (B), or a serine p-lactamase inhibitor (C) for use according to any one of claims 67 to 70, wherein said bacterial infection is as defined in any one of claims 62 to 65.

72. A pharmaceutical composition comprising:(A) a selective zinc-chelator which is a compound according to any one of claims 1 to 17;(B) a p-lactam antibiotic as defined in any one of claims 36 to 49;(C) a serine p-lactamase inhibitor as defined in any one of claims 36 and 50 to 60; and(D) one or more pharmaceutically acceptable carriers or excipients.

73. A pharmaceutical composition according to claim 72, wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is amoxicillin, piperacillin, or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam, avibactam, tazobactam, or a pharmaceutically acceptable salt thereof;or wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is meropenem or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam, avibactam, or a pharmaceutically acceptable salt thereof;or wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is cefepime, ceftolozane, cefiderocol, cefpodoxime, ceftriaxone, ceftaroline, or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is avibactam, sulbactam, tazobactam, xeruborbactam, or a pharmaceutically acceptable salt thereof;preferably wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is ceftriaxone or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam.

74. A pharmaceutical composition according to claim 72 or 73 for use in the treatment of a bacterial infection, preferably for use in the treatment of a bacterial infection as defined in any one of claims 62 to 65.

75. A kit comprising:(i) a first container containing (A) a selective zinc-chelator which is a compound according to any one of claims 1 to 17;(ii) a second container containing (B) a p-lactam antibiotic as defined in any one of claims 36 to 49;(iii) a third container containing (C) a serine p-lactamase inhibitor as defined in any one of claims 36 and 50 to 60; and(iv) optionally instructions for carrying out a method of treatment of a bacterial infection in a subject, preferably a bacterial infection asdefined in any one of claims 62 to 65.

76. A kit according to claim 75, wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is amoxicillin, piperacillin, or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam, avibactam, tazobactam, or a pharmaceutically acceptable salt thereof;or wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is meropenem or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam, avibactam, or a pharmaceutically acceptable salt thereof;or wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is cefepime, ceftolozane,cefiderocol, cefpodoxime, ceftriaxone, ceftaroline, or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is avibactam, sulbactam, tazobactam, xeruborbactam, or a pharmaceutically acceptable salt thereof;preferably wherein said selective zinc-chelator is a compound according to claim 16, or a pharmaceutically acceptable salt thereof; said p-lactam antibiotic is ceftriaxone or a pharmaceutically acceptable salt thereof; and said serine p-lactamase inhibitor is sulbactam.

Images