Field of the art

[0001] The present invention relates generally to compounds capable of inhibiting the activity of EptA and increasing the sensitivity of bacterial cells to cationic antimicrobial peptides. The invention also relates to uses of such compounds in the inhibition of bacterial growth and bacterial infections and in the treatment and prevention of bacterial infections and diseases associated with such infections.

Background

[0002] The increasing incidence of multidrug resistant and extreme-drug resistant bacteria is a significant global health problem. As pathogens develop and acquire resistance to multiple antibiotics, the prospect of infections that are untreatable with current antibiotics and antimicrobial agents is growing, leading to increased morbidity, mortality rates and economic costs to health systems.

[0003] Neisseria gonorrhoeae is a Gram-negative pathogen of humans causing the sexually transmitted infection gonorrhoea with a world-wide yearly estimate of more than 78 million infections. It causes both symptomatic and (frequent) asymptomatic infections at genital and extra-genital sites in both men and women that can have serious consequences for the reproductive and general health of both sexes. In addition, Neisseria gonorrhoeae can also be transmitted to newborns by infected mothers during vaginal delivery, resulting in ophthalmia neonatorum. Repeated Neisseria gonorrhoeae infections can also facilitate transmission or acquisition of the human immunodeficiency virus (HIV).

[0004] Empiric monotherapy has been used to treat gonorrhea over the past 50 years, which has steadily resulted in the emergence of multidrug resistant strains which are resistant to all classes of antibiotics used in therapy. More recently, the emergence of extreme-drug resistant isolates that are resistant to the standard dual antibiotic therapy (azithromycin and ceftriaxone) have been sporadically detected world-wide.

[0005] Accordingly there is a recognised urgent need for the development of novel approaches to the treatment and prevention of infections and diseases caused by bacterial pathogens. In particular the World Health Organisation has urged the research and development of novel therapies to stem the spread and improve the treatment of multi- and extreme-drug resistant infections. [0006] One approach that has gained momentum in the last decade, is the development of anti-virulence compounds designed to inhibit a key determinant in pathogenesis thus enabling the natural immune responses of the host to clear the infection. One potential target for such compounds is the virulence factor EptA (formerly termed lipid A phosphoethanolamine transferase LptA) which catalyses the transfer of phosphoethanolamine (PEA) from phosphatidylethanolamine (PtdE) to lipid A at the 1 and/or 4’ head group positions.

[0007] EptA is required for multiple aspects of Neisseria gonorrhoeae pathogenesis including colonization, inflammation and survival in neutrophils. EptA mutants of GC are attenuated in female mouse models of colonization of the urogenital tract and are unable to colonise the urethra of male humans. The loss of PEA headgroups on lipid A increases susceptibility to complement-mediated killing mechanisms in serum, and reduces the cytokine response by the TLR-4/MD-2 pathways. EptA mutants are sensitive to cationic antimicrobial peptides such as cathelicidins and LL-37 in macrophages and neutrophils. EptA therefore represents a good potential target for the development of novel anti- virulence compounds.

Summary

[0008] The present inventors have identified novel EptA inhibiting compounds. As exemplified herein, the inventors have developed small molecule inhibitors of EptA that are shown to inhibit EptA in bacterial cells, increasing sensitivity of the cells to cationic antimicrobial peptides (CAMPs).

[0009] In a first aspect, the present invention provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

Formula (I) wherein:

- represents a single bond or a double bond;

A is independently selected from the group consisting of C, CH, N, NH, O and S, where appropriate;

X is selected from the group consisting of CH, S, SO, SO ₂ , NH, NR ³ , NHCO and CONH; m is an integer from 0 to 5;

Y is absent or represents a carbonyl (C=O) group;

Z is absent or represents an NH group;

R ¹ and R ² are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted cycloalkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted amino, optionally substituted amido, optionally substituted acyl, optionally substituted sulfonyl, optionally substituted aryl or optionally substituted heteroaryl; and R ³ is optionally substituted alkyl.

[0010] In particular embodiments, the dashed bonds are each double bonds and each recitation of A is N, such that the compound has the structure of Formula (la):

Formula (la) wherein variables X, m, Y, Z, R ¹ and R ² have the definitions above.

[0011] In particular embodiments, Z is NH in the compound of Formula (la), such that the compound has the structure of Formula (lb):

Formula (lb) wherein variables X, m, Y, R ¹ and R ² have the definitions above.

[0012] In particular embodiments, X is S, m is 1 and Y represents carbonyl in the compound of Formula (lb), such that the compound has the structure of Formula (Ic):

Formula (Ic) wherein variables R ¹ and R ² have the definitions above. [0013] In particular embodiments, R ¹ is an optionally substituted aryl group in the compound of Formula (Ic), such that the compound has the structure of Formula (Id):

Formula (Id) wherein the variable R ⁴ is an optional substituent on the aryl group.

[0014] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0015] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0016] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0017] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0018] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0019] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0020] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0021] In particular embodiments, the compound of the first aspect is an inhibitor of EptA activity. In particular embodiments, the compound sensitises a bacterial cell expressing EptA to a cationic antimicrobial peptide.

[0022] A second aspect of the present invention provides a pharmaceutical composition comprising one or more compounds according to the first aspect, optionally in combination with one or more pharmaceutically acceptable carriers, excipients and/or adjuvants.

[0023] A third aspect of the present invention provides a method for sensitising a bacterial cell expressing EptA to a cationic antimicrobial peptide, comprising exposing the bacterial cell to one or more compounds of the first aspect.

[0024] Typically the bacteria is a pathogenic bacteria. The bacteria may be a multidrug resistant or extreme-drug resistant bacteria. In an exemplary embodiment, the bacteria is Neisseria gonorrhoeae.

[0025] In a particular embodiment, the bacteria is a pathogenic bacteria residing in a subject organism, the method comprising administering the one or more compounds to the subject. The cationic antimicrobial peptide may be endogenous to the subject, optionally produced by macrophages, neutrophils and/or epithelial cells of the subject. Alternatively, the cationic antimicrobial peptide may be exogenous to the subject, wherein the method further comprises administering to the subject an effective amount of a cationic antimicrobial peptide.

[0026] A fourth aspect of the present invention provides a method for inhibiting a bacterial infection or for treating or preventing a disease caused by or associated with said bacterial infection, the method comprising administering to a subject in need thereof one or more compounds of the first aspect, wherein the bacteria causing the infection expresses EptA, and wherein the one or compounds render the bacteria more susceptible to a cationic antimicrobial peptide than in the absence of the one or more compounds.

[0027] The cationic antimicrobial peptide may be endogenous to the subject, optionally produced by macrophages, neutrophils and/or epithelial cells of the subject.

[0028] The cationic antimicrobial peptide may be exogenous to the subject, and the method may further comprise administering to the subject an effective amount of a cationic antimicrobial peptide.

[0029] The bacteria may be a multidrug resistant or extreme-drug resistant bacteria. In an exemplary embodiment, the bacteria is Neisseria gonorrhoeae.

[0030] A fifth aspect of the present invention provides a method for inhibiting a bacterial infection or for treating or preventing a disease caused by or associated with said bacterial infection, wherein the bacteria causing the infection expresses EptA, and wherein the method comprises administering to a subject in need thereof one or more compounds of the first aspect.

[0031] A sixth aspect of the present invention provides a method for inhibiting the growth of a bacterial cell expressing EptA, comprising exposing the bacterial cell to an effective amount of one or more compounds of the first aspect.

[0032] Also provided herein are uses of compounds of the first aspect in the manufacture of medicaments for inhibiting bacterial infections or for treating or preventing diseases caused by or associated with said bacterial infections.

Brief description of the drawings

[0033] Aspects and embodiments of the present invention are described herein, by way of non-limiting example only, with reference to the following drawings.

[0034] Figure 1 : Total association of N. gonorrhoeae strain FA1090 in the presence and absence of compounds with RAW murine macrophages expressed as percentage survival relative to the appropriate control. Percentage survival of strain FA0190ΔeptA was calculated relative to wild-type (WT). Percentage survival of WT with compounds 3, 4 and 5 were calculated relative to the WT in the presence of 1% DMSO which is the carrier for the compounds. Statistical analysis was performed using Dunn’s multiple comparisons test against appropriate controls. (Error bars = SEM; *p<0.05, **p<0.01).

[0035] Figure 2: Representative high-resolution negative-ion MALDI-TOF mass spectra of intact LOS from FA1090 treated with no compound (NC), and from FA1090 incubated with compound 3, 4, 5. Regions of the spectra from m/z 1600 to 2000 containing prompt fragment ions for lipid A (labelled in red front) are presented with m/z values detected for monoisotopic peaks. Peaks for sodiated species are marked with a single asterisk.

[0036] Figure 3: Graphs of average percentages of the areas of the negative-ion prompt fragment ion peaks in MALDI-TOF MS for (A) lipid A with PEA, (B) diphosphoryl (2P) lipid A, and (C) 2P lipid A and 2P PEA lipid A. In Panel A, the bars represent diphosphoryl 2P PEA lipid A and 3P PEA lipid A ions, prominent fragment ion peaks at m/z 1737.1 for 3P PEA lipid A - H ₄ P ₂ O ₇ , fragment ion peaks at m/z 1614.1 for 2P-lipid A-H ₃ PO ₄ , and peaks for these fragment ions but also with sodium adducts. The data represent six spectra for each LOS sample, FA1090 with no compound (NC), and FA1090 incubated with compound 3, 4, or 5. A total of 9 peaks per LOS (each including areas for “M, M+l, and M+2” resolved isotope peaks) were analyzed. Error bars = SD. *P < 0.05, ***p <0.001.

[0037] Figure 4 : ELISA was used to quantify TNFa levels in supernatants from human THP-1 cells that were incubated for 18 h with LOS (100 ng/mL) purified from compound treated and untreated FA1090 (NC) and the FA1090ΔeptA mutant. Treatment with compound 5 significantly reduced the induction of TNFa by FA1090 LOS compared with the untreated LOS (p<0.001), whereas bacteria treated with either 3 or 4 had no effect on the induction of TNFa by LOS. The lipid Afrom FA1090ΔeptA induced significantly less TNFa than wild-type (p<0.05). Results are expressed as the mean ± SD of six biological replicates with statistical analysis performed using one-way analysis of variance with Bonferroni’s post hoc tests. *p<0.05, ***p<0.001.

[0038] Figure 5: Analysis of binding to purified EptA by (STD)-NMR spectroscopy. Panel A shows the data generated for 1, where the bottom plot shows the aromatic region off- resonance spectrum from the (STD)-NMR experiment in green and the top plot shows the same region of the STD difference spectrum in black. Panel B, shows the same data but for 5. In each case the off-resonance experiment contains resonances that are consistent with the structure of the compound, which verifies the structure and confirms that it is present and soluble under the experimental conditions.

[0039] Figure 6: Identification of EptA inhibitors increasing the susceptibility of N. gonorrhoeas strain FA1090 to LL-37. (A) LL-37 kill-curves of WT FA1090 and ΔeptA. The data express as percentage of survival after 90 minutes of incubation. (B) A total of thirty-two compounds (50 pM) elaborated from the lead compound 5 (Boxed in black) were screened for the ability to increase the susceptibility of WT FA1090 to killing by LL-37. Red bars: WT strain was incubated with 50 pM of each compound and 3.2 pM LL-37. White bars: WT strain was incubated with only 50 pM of each compound or 2.5% DMSO (Inhibitor solvent). P values (**P < 0.01, **** p < 0.0001) were determined for WT with compounds alone in relation to WT with 2.5% DMSO using one-way ANOVA with Dunnett test. (C) The lead six compounds with cut-off threshold of 50% of survival and without inhibitory effects on the WT FA1090 in the first screen (Boxed in blue in B) were used to determine the minimum effective concentration (MEC90) in presence of 3.2 pM LL-37. Error bars are a mean ± SEM of three biological repeats.

[0040] Figure 7: EptA inhibitors had no demonstrated inhibitory effects on the FA1090 \eptA strain. The FA1090 \ep/A strain was exposed to MEC90 of the lead compounds (Figure 6C) using the same conditions described previously in LL-37 killing assay. Error bars are a mean ± SEM of three biological repeats. No significance, one-way ANOVA with Dunnett test.

[0041] Figure 8: Macrophage bactericidal assay of gonococcal strains in presence of EptA inhibitors. The WT FA 1090 and \eplA strains were pre-treated with compounds or 1% DMSO (inhibitor solvent) for three hours and exposed to RAW 264.7 murine macrophages for 1 hour as described previously. The results are expressed as the percentage of survival. The 1% DMSO-treated WT FA1090 (A) and WT FA1090 (B) set as 100% of survival, respectively. Error bars are a mean ± SEM of three to five biological repeats, with each having two technical repeats. Statistical significances (****, p < 0.0001; ns, no significance) were determined by one-way ANOVA with Dunnett test.

[0042] Figure 9: Mean PEA modification of V. gonorrhoeae FA1090 treated with different EptA inhibitors. The FA 1900 AeptA mutant had no PEA modification. The mean of four biological repeats of compound-treated samples were compared with the untreated WT using a one-way ANOVA with Dunnett’ s multiple comparisons test. Samples that were significantly different are shown with an asterisk (*** = <0.01, **** = < 0.001). [0043] Figure 10: The EptA inhibitors increase the susceptibility of WHO gonococcal strains to LL-37. LL-37 kill-curves of WHO F, WHO G, WHO P, and their AeptA mutant strains with or without 2.5% DMSO (inhibitor solvent) or 100 pM compounds. Error bars are a mean ± SEM of three to six biological repeats. Statistical significances (*P < 0.05, **P < 0.01, ***P < 0.001, 0.0001) were determined by two-way ANOVA with Dunnett test.

[0044] Figure 11: The compounds 127 and 128 increase the susceptibility of intracellular AMS- and MDR-isolates to killing by murine macrophages. WT and AeptA strains were exposed to the murine RAW 264.7 macrophages for 30 minutes (MOI = 40) and was subsequently incubated with 100 pg/mL gentamicin for 1 hour to remove external bacteria. The the monolayer of cells was washed with PBS before adding the new media containing 75pM 128 or 50pM 127. 1% DMSO was also used for every assay as the solvent control of compounds. The macrophage cells were continuously incubated until 6 hours after infection that were then lysed by 1% saponin for 10 minutes before enumerating the bacterial CFU/mL. The results are expressed as the percentage of intracellular survival. The WT and DMSO-treated samples set as 100%. Error bars are a mean ± SEM of three biological repeats. Statistical significances (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) were determined by two-way ANOVA with Dunnett test.

[0045] Figure 12: LL-37 kill-curves of N. gonorrhoeas. LL-37 kill-curves of WT strain FA1090 were conducted with either 100 pM compounds (128 or 127) or 2.5% DMSO in presence or absence of 1 % HEC. AeptA strain was used as the negative control for every assay. The results are expressed as percentage of survival after 90 minutes of incubation. Untreated samples (FA1090 and AeptA) were set as 100% survival. Error bars are a mean ± SEM of three biological repeats. Statistical significances (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) were determined by two-way ANOVA with Dunnett test.

[0046] Figure 13 : Compound therapy during gonococcal infection in the mouse model of vaginal infection reduces the inflammatory response as measured by neutrophil influx. Panel A. Treatment plan of the mice used in this study. Premarin was administered to halt estrous. WT strain FA1090 at a cfu of 2x106 cells were used to inoculate female mice at day 0. 1% HEC gel containing lOOpM of EptA compound 128 or placebo gel with no compound was administered daily. Swabs for bacterial counts and neutrophil counts were taken daily. Panel B. Kaplan Meyer plot showing the prevalence of gonococcal carriage in mice over 5 days in the treated (1% HEC gel containing lOOpM of EptA compound A (red)) and untreated groups (1% HEC gel (black)). Panel C. Polymorphonuclear neutrophil counts daily. No difference in bacterial burden was noted between treated and untreated mice. A significant difference in PMN count was detected between treatment groups. Paired time points were compared between groups using a repeated measures ANOVA followed by a Bonferroni post-hoc analysis with multiple comparisons. In both sets of analyses, P < 0.05 will be considered significant (**).

Detailed Description

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0048] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0049] In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0050] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0051] The term "optionally" is used herein to mean that the subsequently described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiments in which the event or circumstance occurs as well as embodiments in which it does not. [0052] As used herein the term "antimicrobial activity" refers to the ability of any agent that, alone or in combination with another agent such as an antibiotic, is capable of killing or inhibiting the growth of one or more species of microorganism, typically bacteria.

[0053] As used herein the term "cationic antimicrobial peptide" means a basic, amphipathic peptide displaying antibacterial activity. Typically cationic antimicrobial peptides act by inserting into the membrane of a bacterial cell, destabilizing the cell membrane and lysing the cell. As used herein "cationic antimicrobial peptide" includes within its scope molecules of the innate immune system of a host organism produced by macrophages, neutrophils and epithelial cells in response to infection or injury, as well as synthetically produced cationic peptides and cationic peptides of microbial origin which have antibacterial activity.

[0054] As used herein the term "sensitivity" is used in its broadest context to refer to the ability of a bacterial cell to survive exposure to a cationic antimicrobial peptide which acts, at least in part, to inhibit the growth of the bacterial cell, kill the bacterial cell or inhibit one or more cellular functions of the bacterial cell. The terms "sensitivity" and "susceptibility" may be used interchangeably herein, as may the terms "sensitive" and "susceptible".

[0055] As used herein the term "exposing" means generally bringing into contact with. Typically direct exposure refers to administration of a compound or agent to a bacterial cell or otherwise bringing the bacterial cell into contact with the compound or agent itself. Indirectly "exposing" a bacteria or bacterial cell to a compound or agent includes the administration of the compound or agent to a subject in or on which the bacteria resides, and may be causing an infection. Thus, in the present disclosure the terms "exposing", "administering" and "delivering" and variations thereof may, in some contexts, be used interchangeably.

[0056] The term "inhibiting" and variations thereof such as "inhibition" and "inhibits" as used herein in relation to bacterial growth and bacterial infections means complete or partial inhibition of growth of a bacteria and/or development, establishment, growth or spread of a bacterial infection. Inhibition may be permanent or temporary. The inhibition may be to an extent (in magnitude and/or spatially), and/or for a time, sufficient to produce the desired effect. Further, such inhibition may be direct or indirect. By indirect inhibition is meant that the compound or agent may affect the expression or activity of molecules which in turn impacts growth of a bacteria and/or development, establishment, growth or spread of a bacterial infection. [0057] As used herein the terms "treating" and "preventing" and grammatical equivalents refer to any and all uses which remedy infection or disease, prevent the establishment of an infection or disease, or otherwise prevent, hinder, retard, or reverse the progression of an infection or disease, or at least one symptom thereof. Thus the term "treating" is to be considered in its broadest context. For example, treatment does not necessarily imply that a subject is treated until total recovery.

[0058] The term "subject" as used herein refers to mammals and includes humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs), performance and show animals (e.g. horses, livestock, dogs, cats), companion animals (e.g. dogs, cats) and captive wild animals. Preferably, the mammal is human or a laboratory test animal. Even more preferably, the mammal is a human.

[0059] As used herein the term "effective amount" includes within its meaning a non-toxic but sufficient amount of an agent to provide the desired effect. The exact amount required will vary from subject to subject depending on factors such as the species of microorganisms being treated, the extent, severity and/or age of the biofilm being treated, whether the biofilm is surface-associated or suspended, the particular agent(s) being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

[0060] The enzyme lipooligosaccharide (LOS) phosphoethanolamine transferase A (EptA) is responsible for the addition of phosphoethanolamine (PEA) to lipid A and this modification is essential for bacterial resistance to cationic antimicrobial peptides. The present inventors have developed a series of small molecule inhibitors of EptA, which bind to EptA, inhibit addition of phosphoethanolamine to LOS and, as exemplified herein, can sensitise Neisseria gonorrhoeae to killing by cationic antimicrobial peptides produced by macrophages.

[0061] In one aspect, provided herein are compounds of Formula (I) and pharmaceutically acceptable salts thereof:

Formula (I) wherein:

- represents a single bond or a double bond;

A is independently selected from the group consisting of C, CH, N, NH, O and S, where appropriate;

X is selected from the group consisting of CH, S, SO, SO ₂ , NH, NR ³ , NHCO and CONH; m is an integer from 0 to 5;

Y is absent or represents a carbonyl (C=O) group;

Z is absent or represents an NH group;

R ¹ and R ² are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted cycloalkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted amino, optionally substituted amido, optionally substituted acyl, optionally substituted sulfonyl, optionally substituted aryl or optionally substituted heteroaryl; and R ³ is optionally substituted alkyl.

[0062] In some embodiments, the dashed bonds both represent single bonds. In some embodiments, the dashed bonds both represent double bonds. In some embodiments, one dashed bond represents a single bond and the other dashed bond represents a double bond.

[0063] In some embodiments, each recitation of A represents a heteroatom. In an embodiment, each recitation of A is N.

[0064] In particular embodiments, the dashed bonds are each double bonds and each recitation of A is N, such that the compound has the structure of Formula (la):

Formula (la) wherein variables X, m, Y, Z, R ¹ and R ² have the definitions above.

[0065] In particular embodiments, Z is NH in the compound of Formula (la), such that the compound has the structure of Formula (lb): Formula (lb) wherein variables X, m, Y, R ¹ and R ² have the definitions above.

[0066] In some embodiments, X represents a group containing S atom. In an embodiment, X represents S. In another embodiment, X represents SO. In another embodiment, X represents SO ₂ . In some embodiments, X represents a group containing an N atom. In an embodiment, X represents NH. In another embodiment, X represents NR ³ and R ³ is an optionally substituted alkyl group. In another embodiment, X represents NHCO. In another embodiment, X represents CONH. In a preferred embodiment, X is S. In another preferred embodiment, X is SO. In yet another preferred embodiment, X is SO ₂ .

[0067] In some embodiments, m represents an integer from 0 to 5. In an embodiment, m is 0. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In a preferred embodiment, m is 1.

[0068] In some embodiments, Y is absent. In some embodiments, Y represents a carbonyl (C=O) group.

[0069] In some embodiments, Z is absent. In some embodiments, Z represents an NH group. [0070] In some embodiments, Y is absent and Z represents an NH group, such that the compound of Formula (I) contains an amino functional group. In some embodiments, Y represents a carbonyl group and Z is absent, such that the compound of Formula (I) contains a carbonyl functional group. In some embodiments, Y represents a carbonyl group and Z represents NH, such that the compound of Formula (I) contains an amido functional group. [0071] In some embodiments, the dashed bonds each represent double bonds and each recitation of A is N, X is S, m is 1, Y represents a carbonyl (C=O) group and Z represents an NH group

Formula (Ic) wherein variables R1 and R2 have the definitions above.

[0072] In some embodiments, R ¹ is an optionally substituted aryl group in the compound of Formula (Ic), such that the compound has the structure of Formula (Id):

Formula (Id) wherein the variable R ⁴ is an optional substituent on the aryl group.

[0073] In some embodiments of compounds of the Formula (I), (la), (lb), (Ic) or (Id), R ¹ is an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group, each of which may be optionally substituted one or more times.

[0074] In some embodiments, R ¹ is an alkyl group. In some embodiments, R ¹ is a C ₁ to C12 alkyl group. In another embodiment, R ¹ is a methyl group. In another embodiment, R ¹ is an ethyl group. In another embodiment, R ¹ is a propyl group. In an embodiment, R ¹ is an optionally substituted alkyl group. In another embodiment, R ¹ is an optionally substituted methyl group. In another embodiment, R ¹ is an optionally substituted ethyl group. In some embodiments, R ¹ is an ethyl group substituted with a heterocyclic group. In a preferred embodiment, R ¹ is a morpholinoethyl group. In another preferred embodiment, R ¹ is a hydroxyethyl group. In another preferred embodiment, R ¹ is a methoxyethyl group.

[0075] In some embodiments, R ¹ is a cycloalkyl group. In another embodiment, R ¹ is a C ₃ - C ₈ cycloalkyl group. In a preferred embodiment, R ¹ is a cyclopentyl or cyclohexyl group. In another embodiment, R ¹ is an optionally substituted cycloalkyl group. In another embodiment, R ¹ is an optionally substituted C ₃ -C ₈ cycloalkyl group. In another embodiment, R ¹ is an unsubstituted cycloalkyl group. In another embodiment, R ¹ is an unsubstituted C ₃ - C ₈ cycloalkyl group. In a preferred embodiment, R ¹ is an optionally substituted cyclopentyl group. In a preferred embodiment, R ¹ is an unsubstituted cyclopentyl group.

[0076] In some embodiments, R ¹ is a heterocyclyl group. In another embodiment, R ¹ is a saturated heterocyclyl group. In another embodiment, R ¹ is an unsaturated heterocyclyl group. In an embodiment, R ¹ is a 5-membered heterocyclyl group. In an embodiment, R ¹ is a 6-membered heterocyclyl group. In an embodiment, R ¹ is a morpholino group. In an embodiment, R ¹ is an optionally substituted morpholino group. In an embodiment, R ¹ is a piperazinyl group. In an embodiment, R ¹ is an optionally substituted piperazinyl group. In an embodiment, R ¹ is a piperdinyl group. In an embodiment, R ¹ is an optionally substituted piperidinyl group. [0077] In some embodiments, R ¹ is an aryl group. In an embodiment, R ¹ is a C ₆ -C ₁₀ aryl group. In an embodiment, R ¹ is a monocyclic aryl group. In an embodiment, R ¹ is a bicyclic aryl group. In another embodiment, R ¹ is a fused bicyclic aryl group. In another embodiment, R ¹ is a phenyl group. In another embodiment, R ¹ is a naphthyl group. In an embodiment, R ¹ is an optionally substituted phenyl group. In another embodiment, R ¹ is an optionally substituted naphthyl group. In a preferred embodiment, R ¹ is a substituted phenyl group. In a preferred embodiment, R ¹ is a phenyl group substituted with one or more groups. In a preferred embodiment, R ¹ is a phenyl group substituted with one or more instances of halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, aryl, nitro, cyano, acyl, amino, hydroxyl or sulfonyl. In a preferred embodiment, R ¹ is a phenyl group substituted with an amino group. In a preferred embodiment, R ¹ is a phenyl group substituted with an amino group that is optionally substituted. In a preferred embodiment, R ¹ is a phenyl group substituted with an amino group that is substituted with one or more alkyl groups. In a preferred embodiment, R ¹ is a phenyl group substituted with an amino group that is substituted with one or more straight chain alkyl groups. In a preferred embodiment, R ¹ is a phenyl group substituted with an amino group that is substituted with an alkyl group further bearing an aryl group. In a preferred embodiment, R ¹ is a phenyl group substituted with an amino group that is substituted with an optionally substituted benzyl group. In a preferred embodiment, R ¹ is a phenyl group substituted with an amino group that is a benzyl optionally substituted with one or more alkyl groups.

[0078] In some embodiments, R ¹ is a heteroaryl group. In an embodiment, R ¹ is a heteroaryl group containing one or more heteroatoms selected from sulphur, nitrogen and oxygen. In another embodiment, R ¹ is a heteroaryl group containing 1 heteroatom. In another embodiment, R ¹ is a heteroaryl group containing 2 heteroatoms. In another embodiment, R ¹ is a heteroaryl group containing one or more nitrogen atoms. In another embodiment, R ¹ is a heteroaryl group containing one nitrogen atom. In another embodiment, R ¹ is a heteroaryl group containing two nitrogen atoms. In another embodiment, R ¹ is a 5-membered heteroaryl group. In another embodiment, R ¹ is a 6-membered heteroaryl group. In another embodiment, R ¹ is a 10-membered heteroaryl group. In a preferred embodiment, R ¹ is 5- membered heteroaryl group containing one nitrogen atom. In another preferred embodiment, R ¹ is a 5-membered heteroaryl group containing two nitrogen atoms. In another preferred embodiment, R ¹ is a 5-membered heteroaryl group containing a sulphur atom. In another preferred embodiment, R ¹ is a 6-membered heteroaryl group containing 1 nitrogen atom. In a preferred embodiment, R ¹ is a 6-membered heteroaryl group containing 2 nitrogen atoms. In a preferred embodiment, R ¹ is a 10-membered heteroaryl group containing 1 nitrogen atom. In a preferred embodiment, R ¹ is an indolyl group. In another preferred embodiment, R ¹ is a protected indolyl group. In another preferred embodiment, R ¹ is a pyridinyl group. In another preferred embodiment, R ¹ is a pyrimidinyl group. In another preferred embodiment, R ¹ is a quinolinyl group. In another preferred embodiment, R ¹ is a thienyl group. In a preferred embodiment, R ¹ is a heteroaryl group that is optionally substituted. For example, R ¹ is an optionally substituted indolyl, pyridinyl, pyrazolyl, pyrimidinyl, quinolinyl or thienyl group.

[0079] In some embodiments of compounds of the Formula (I), (la), (lb), (Ic) or (Id), R ² is an alkyl group, a cycloalkyl group, an arylalkyl group, an amino group or an aryl group, each of which may be optionally substituted by one or more groups, which may be the same or different.

[0080] In some embodiments, R ² is an alkyl group. In some embodiments, R ² is a C ₁ to C ₁₂ alkyl group. In another embodiment, R ² is a methyl group. In another embodiment, R ² is an ethyl group. In another embodiment, R ² is a propyl group. In an embodiment, R ² is an optionally substituted alkyl group. In another embodiment, R ² is an optionally substituted methyl group. In another embodiment, R ² is an optionally substituted ethyl group. In some embodiments, R ² is an ethyl group substituted with a heterocyclic group. In a preferred embodiment, R ² is a morpholinoethyl group. In another preferred embodiment, R ² is a hydroxyethyl group. In another preferred embodiment, R ² is a methoxyethyl group. In another embodiment, R ² is an ethyl group substituted with an amino moiety.

[0081] In some embodiments, R ² is a cycloalkyl group. In some embodiments, R ² is a C ₃ - C ₈ cycloalkyl group. In a preferred embodiment, R ² is a cyclopentyl or cyclohexyl group. In another embodiment, R ² is an optionally substituted cycloalkyl group. In another embodiment, R ² is an optionally substituted C ₃ -C ₈ cycloalkyl group. In another embodiment, R2 is an unsubstituted cycloalkyl group. In another embodiment, R ² is an unsubstituted C ₃ - C ₈ cycloalkyl group. In a preferred embodiment, R ² is an optionally substituted cyclopentyl group. In a preferred embodiment, R ² is an unsubstituted cyclopentyl group.

[0082] In some embodiments, R ² is a heterocyclyl group. In another embodiment, R ² is a saturated heterocyclyl group. In another embodiment, R ² is an unsaturated heterocyclyl group. In an embodiment, R ² is a 5-membered heterocyclyl group. In an embodiment, R ² is a 6-membered heterocyclyl group. In an embodiment, R ² is a morpholino group. In an embodiment, R ² is an optionally substituted morpholino group. In an embodiment, R ² is a piperazinyl group. In an embodiment, R ² is an optionally substituted piperazinyl group. In an embodiment, R ² is a piperdinyl group. In an embodiment, R ² is an optionally substituted piperidinyl group.

[0083] In some embodiments, R ² is an alkylaryl group. In some embodiments, R ² is an alkylaryl group where the alkylene moiety contains 1 to 4 carbon atoms in the chain. In some embodiments, R ² is an alkylaryl group where the aryl group contains 6 to 10 carbon atoms. In some embodiments, R ² is an alkylaryl group where the alkylene moiety has 1 carbon atom. In some embodiments, R ² is an alkylaryl group where the alkylene moiety has 2 carbon atoms. In some embodiments, R ² is an alkylaryl group where the alkylene moiety is a methylene moiety. In some embodiments, R ² is an alkylaryl group where the alkylene group is an ethylene moiety. In some embodiments, R ² is an alkylaryl group where the aryl group has 6 carbon atoms. In some embodiments, R ² is an alkylaryl group where the aryl moiety is a phenyl moiety. In some embodiments, R ² is a benzyl group. In some embodiments, R ² is a phenethyl group. In some embodiments, R ² is an optionally substituted benzyl group. In some embodiments, R ² is an optionally substituted phenethyl group.

[0084] In some embodiments, R ² is an amino group. In some embodiments, R ² is an optionally substituted amino group.

[0085] In some embodiments, R ² is an aryl group. In an embodiment, R ² is a Ce-Cio aryl group. In an embodiment, R ² is a monocyclic aryl group. In an embodiment, R ² is a bicyclic aryl group. In another embodiment, R ² is a fused bicyclic aryl group. In another embodiment, R ² is a phenyl group. In another embodiment, R ² is a naphthyl group. In an embodiment, R ² is an optionally substituted phenyl group. In another embodiment, R ² is an optionally substituted naphthyl group. In a preferred embodiment, R ² is a substituted phenyl group. In a preferred embodiment, R ² is a phenyl group substituted with one or more groups. In a preferred embodiment, R ² is a phenyl group substituted with one or more instances of halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, aryl, nitro, cyano, acyl, amino, hydroxyl or sulfonyl. In some embodiments, R ² is a heteroaryl group.

[0086] In some embodiments, R ² is a heteroaryl group. In an embodiment, R ² is a heteroaryl group containing one or more heteroatoms selected from sulphur, nitrogen and oxygen. In another embodiment, R ² is a heteroaryl group containing 1 heteroatom. In another embodiment, R ² is a heteroaryl group containing 2 heteroatoms. In another embodiment, R ² is a heteroaryl group containing one or more nitrogen atoms. In another embodiment, R ² is a heteroaryl group containing one nitrogen atom. In another embodiment, R ² is a heteroaryl group containing two nitrogen atoms. In another embodiment, R ² is a 5-membered heteroaryl group. In another embodiment, R ² is a 6-membered heteroaryl group. In another embodiment, R ² is a 10-membered heteroaryl group. In a preferred embodiment, R ² is 5- membered heteroaryl group containing one nitrogen atom. In another preferred embodiment, R ² is a 5-membered heteroaryl group containing two nitrogen atoms. In another preferred embodiment, R ² is a 5-membered heteroaryl group containing a sulphur atom. In another preferred embodiment, R ² is a 6-membered heteroaryl group containing 1 nitrogen atom. In a preferred embodiment, R ² is a 6-membered heteroaryl group containing 2 nitrogen atoms. In a preferred embodiment, R ² is a 10-membered heteroaryl group containing 1 nitrogen atom. In a preferred embodiment, R ² is an indolyl group. In another preferred embodiment, R ² is a protected indolyl group. In another preferred embodiment, R ² is a pyridinyl group. In another preferred embodiment, R ² is a pyrimidinyl group. In another preferred embodiment, R ² is a quinolinyl group. In another preferred embodiment, R ² is a thienyl group. In a preferred embodiment, R ² is a heteroaryl group that is optionally substituted. For example, R ² is an optionally substituted indolyl, pyrazolyl, pyridinyl, pyrimidinyl, quinolinyl or thienyl group.

[0087] "Alkyl" refers to a monovalent alkyl groups that may be straight chained or branched, and preferably have from 1 to 10 carbon atoms, or more preferably 1 to 6 carbon atoms. Examples of such groups include methyl, ethyl, n-isopropyl, iso-propyl, n-butyl, iso- butyl, n-hexyl, and the like.

[0088] "Cycloalkyl" refers to a saturated monocyclic or fused or spiro polycyclic carbocycle preferably containing from 3 to 12 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the link, unless otherwise specified. Monocyclic cycloalkyl groups include cyclopentyl and cyclohexyl, bicyclic cycloalkyl groups include decalin and polycyclic cycloalkyl groups include adamantane. Preferred cycloalkyl groups are C ₃ to C ₉ cycloalkyl groups.

[0089] "Alkenyl" refers to a monovalent aliphatic carbocyclic group having at least one carbon-carbon double bond and which may be straight chained or branched, preferably having from 2 to 10 carbon atoms. Examples of such groups include a vinyl or ethenyl group (-CH=CH ₂ ), n-propenyl (-CH ₂ CH=CH ₂ ), iso-propenyl (-C(CH ₃ )=CH ₂ ), but-2-enyl (- CH ₂ CH=CHCH ₃ ), and the like.

[0090] "Alkynyl" refers to a monovalent aliphatic carbocyclic group having at least one carbon-carbon triple bond and which may be straight chained or branched, preferably having from 2 to 10 carbon atoms. Examples of such groups include an acetylene or ethynyl group (-C=CH), propargyl (-CH ₂ OCH), and the like. [0091] "Aryl" refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl, anthracenyl), preferably having from 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracenyl and the like.

[0092] "Arylalkyl" refers to an -alkylene- aryl group having 1 to 10 carbon atoms in the alkylene chain and 6 to 10 carbon atoms in the aryl moiety. Examples of arylalkyl groups in benzyl, phenethyl and the like.

[0093] "Heteroaryl" refers to a monovalent aromatic heterocyclic group which fulfils the Htickel criteria for aromaticity (i.e. contains 4n + 2TI electrons) and preferably has from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur within the ring (and includes oxides of sulfur, selenium and nitrogen). Such heteroaryl groups can have a single ring (e.g. pyridyl, pyrrolyl or N-oxides thereof or furyl) or multiple condensed rings (e.g. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl).

[0094] "Heterocyclyl" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 4 to 12 carbon atoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur, oxygen, selenium or phosphorus within the ring. The most preferred heteroatom is nitrogen.

[0095] Further examples of heterocyclyl and heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4, 5, 6, 7 tetrahydrobenzo [b] thiophene, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiazolidine, thiophene, benzo [b] thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, triazole, and the like.

[0096] "Alkoxy" and "aryloxy" refers to the groups "-O-alkyl" and "-O-aryl", respectively, wherein the alkyl and aryl groups are described above.

[0097] "Halogen" refers to the groups fluoro, chloro, bromo and iodo.

[0098] " Amino" refers to a group derived from ammonia and having a nitrogen centre substituted with one or more hydrocarbyl groups. An amino group may be optionally substituted. [0099] Amino groups and other nitrogen heteroatoms may be protected with a protecting group. A nitrogen protecting group prevents the nitrogen moiety reacting during a further derivatisation step or other reaction. A nitrogen protecting group can be readily removed, when desired. Examples of suitable nitrogen protecting groups that may be used include formyl, trityl, phthalimido, acetyl, trichloroacetyl, chloroacetyl, bromoacetyl, iodoacetyl; urethane-type blocking groups such as benzyl oxy carbonyl (‘CBz’), 4- phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4- fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 3 -chlorobenzyloxycarbonyl, 2- chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 3- bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, t- butoxy carbonyl (‘tBoc’), 2-(4-phenyl)-isopropoxycarbonyl, 1,1-diphenyleth-l- yloxycarbonyl, 1,1-diphenylprop-l-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl, 2-(p- toluyl)-prop-2-yloxy-carbonyl, cyclopentanyloxy-carbonyl, 1- methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl, 1- methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl, 2-(4-toluylsulfono)- ethoxycarbonyl, 2-(methylsulfono)ethoxycarbonyl, 2-(triphenylphosphino)- ethoxycarbonyl, fluorenylmethoxycarbonyl ('Fmoc'), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, l-(trimethylsilylmethyl)prop- 1-enyloxycarbonyl, 5-benzisoxalymethoxy carbonyl, 4-acetoxybenzyloxycarbonyl, 2,2,2 -trichloroethoxycarbonyl, 2-ethynyl-2- propoxycarbonyl, cyclopropylmethoxycarbonyl, 4-(decyloxy)benzyloxycarbonyl, isobomyloxycarbonyl, 1 -piperidyloxycarbonyl and the like; benzoylmethylsulfono group, 2-nitrophenylsulfenyl, diphenylphosphine oxide, and the like. The actual nitrogen protecting group employed is not critical so long as the derivatised nitrogen group is stable to the condition of subsequent reaction(s) and can be selectively removed as required without substantially disrupting the remainder of the molecule including any other nitrogen protecting group(s). Further examples of these groups are found in: Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, Second edition; Wiley-Interscience: 1991; Chapter 7; McOmie, J. F. W. (ed.), Protective Groups in Organic Chemistry, Plenum Press, 1973; and Kocienski, P. J., Protecting Groups, Second Edition, Thieme Medical Pub., 2000. [0100] The term "optionally substituted" as used throughout the specification denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more non-hydrogen substituent groups. In certain embodiments the substituent groups are one or more groups independently selected from the group consisting of halogen, =0, =S, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkenyl, haloalkynyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxy alkyl, alkyloxycycloalkyl, alkyloxyheterocycloalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkyloxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl and carbonyl. [0101] In some embodiments each optional substituent is independently selected from the group consisting of: halogen, =0, =S, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, COOH, SH, and acyl.

[0102] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0103] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0104] In an exemplary embodiment, the compound of Formula (I) has the structure:

[0105] The term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the above-identified compounds, and include pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of compounds of Formula (I) may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic, arylsulfonic. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995. In the case of agents that are solids, it is understood by those skilled in the art that the inventive compounds, agents and salts may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the present invention and specified formulae.

[0106] Compounds of the present invention are demonstrated herein to render bacterial cells expressing EptA more sensitive or susceptible to the activity of cationic antimicrobial peptides in the presence of a compound of the invention than in the absence of the compound. Accordingly, the present invention contemplates uses of compounds of the invention in the inhibition of the growth of bacteria and the inhibition, treatment or prevention of bacterial infections and diseases associated with bacterial infections, where the bacteria express EptA.

[0107] Provided herein are methods for sensitising a bacterial cell expressing EptA to a cationic antimicrobial peptide, comprising exposing the bacterial cell to one or more compounds of the invention.

[0108] Also provided herein are methods for inhibiting bacterial infections or for treating or preventing diseases caused by or associated with bacterial infections, where the bacteria express EptA, wherein one or more compounds of the invention are administered to a subject, rendering the bacteria more sensitive or susceptible to cationic antimicrobial peptides than in the absence of the one or more compounds. Inflammation may be observed as a symptom of a bacterial infection in a subject. Alternatively, inflammation may be a symptom of a condition that is caused by or associated with a bacterial infection. The present inventors believe that the compounds discussed herein can reduce inflammation associated with a bacterial infection.

[0109] Also provided herein are methods for inhibiting the growth of bacterial cells that EptA, comprising exposing the bacterial cells to an effective amount of one or more compounds of the invention.

[0110] The bacteria and bacterial cells against which the compounds and methods of the invention can be employed may be any bacteria or bacterial cell that expresses EptA. Typically the bacteria is an animal pathogen, more typically a human pathogen. The bacteria or bacterial cell may display resistance to one or more antibiotics or antimicrobial agents. In exemplary embodiments, the bacteria is Neisseria gonorrhoeae. However the scope of the invention is not so limited and the compounds and methods may be employed against any bacteria known to express EptA, including, for example but not limited to, N. meningitidis, Escherichia coli, Shigella flexneri, Acetobacter baumannii, Klebsiella pneumonia, Salmonella enterica, Pseudomonas aeruginosa, Vibrio cholera and Helicobacter pylori.

[0111] Compounds and methods of the invention may be employed to increase the sensitivity or susceptibility of an EptA-expressing bacteria to a cationic antimicrobial peptide produced by a host organism in which the bacteria is residing, typically as a pathogen causing infection and/or disease in the host organism. Thus, embodiments of the present invention contemplate administering one or more compounds of the invention to a subject having a bacterial infection, wherein the one or more compounds sensitise the bacteria causing the infection to endogenous cationic antimicrobial peptides produced by the immune system of the subject. Such endogenous cationic antimicrobial peptides may be produced by, for example, macrophages, neutrophils, mast cells or epithelial cells. Exemplary endogenous cationic antimicrobial peptides include cathelicidins (e.g. LL-37), defensins such as a-defensins and P-defensins, and hepcidin.

[0112] Alternatively or in addition, compounds of the invention may be employed to increase the sensitivity or susceptibility of an EptA-expressing bacteria to a cationic antimicrobial peptide exogenous to the subject to which the one or more compounds are administered. Such cationic antimicrobial peptides include synthetic peptides or peptides of microbial or fungal origin such as, for example, polymyxins including polymyxin B and polymyxin E (colistin). Thus, embodiments of the invention provide methods wherein one or more cationic antimicrobial peptides is administered in conjunction with the one or more compounds of the invention. The one or more cationic antimicrobial peptides may be administered to the subject prior to, concomitantly with, or subsequent to the one or more compounds of the invention, and may be administered by the same or different routes.

[0113] Compounds of the invention and compositions comprising such compounds may be administered to subjects in need thereof via any convenient or suitable route such as by parenteral (including, for example, intraarterial, intravenous, intramuscular, subcutaneous), topical (including dermal, transdermal, subcutaneous, etc), oral, nasal, mucosal (including sublingual), or intracavitary routes. The preferred route of administration will depend on a number of factors including the nature and extent of the infection or disease to be treated and the desired outcome. The most advantageous route for any given circumstance can be determined by those skilled in the art. For example, in circumstances where it is required that appropriate concentrations of the desired agent are delivered directly to the site in the body to be treated, administration may be regional rather than systemic. Regional administration provides the capability of delivering very high local concentrations of the desired agent to the required site and thus is suitable for achieving the desired therapeutic or preventative effect whilst avoiding exposure of other organs of the body to the compound and thereby potentially reducing side effects.

[0114] Compounds may also be used in coating or impregnating medical devices and materials, including medical and surgical equipment and implantable medical devices, including but not limited to drainage catheters (e.g. urinary catheters), venous catheters, cannulas such as drug-pump related delivery cannulas, dialysis equipment, stents, pacemakers, contact lenses, hearing-aids, percutaneous glucose sensors, prostheses such as artificial joints, hearts, heart valves or other organs, medical fixation devices (e.g. rods, screws, pins, plates and the like), dressings, bandages and patches.

[0115] For the applications contemplated herein compounds of the invention are typically formulated into a suitable pharmaceutical composition. Thus compositions may be formulated in a variety of forms including solutions, suspensions, emulsions, and solid forms and are typically formulated so as to be suitable for the chosen route of administration, for example as an injectible formulations suitable for parenteral administration, capsules, tablets, caplets, elixirs for oral ingestion, in an aerosol form suitable for administration by inhalation (such as by intranasal inhalation or oral inhalation), or ointments, creams, gels, or lotions suitable for topical administration. Compositions typically also include carriers, diluents, excipients and/or adjuvants. Suitable carriers, diluents, excipients and adjuvants are known to those skilled in the art. The carriers, diluents, excipients and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20 ^th Edition, Williams& Wilkins, Pennsylvania, USA. The carrier will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case.

[0116] For administration as an injectable solution or suspension, non-toxic parenteral acceptable diluents or carriers can include Ringer's solution, medium chain triglyceride (MCT), isotonic saline, phosphate buffered saline, ethanol and 1 ,2 propylene glycol. Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.

[0117] Suitable adjuvants can include preservatives, wetting agents, emulsifying agents, emollients, emulsifiers, thickening agents, buffering agents and dispersing agents.

[0118] Methods for preparing parenteral administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein. The composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silica ceoussilicas, and other ingredients such as lanolin, may also be included.

[0119] Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, com starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include com starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propylparaben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

[0120] Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof. Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly- vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate. Polyoxyethylene sorbitan mono-or di-oleate, - stearate or -laurate and the like. Emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

[0121] If desired, and for more effective distribution, the compounds of the invention can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.

[0122] Compounds of the invention may be administered in combination with one or more antibiotics or antimicrobial agents. Where multiple agents are to be administered, each agent in the combination may be formulated into separate compositions or may be co-formulated into a single composition. If formulated in different compositions the compositions may be co-administered. By “co-administered” is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes, or sequential administration by the same or different routes. By “sequential” administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two compositions. The compositions may be administered in any order. [0123] Those skilled in the art will appreciate that the aspects and embodiments described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0124] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the present application. Further, the reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0125] The present disclosure will now be described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the disclosure.

Examples

[0126] The following examples are illustrative of the disclosure and should not be construed as limiting in any way the general nature of the disclosure of the description throughout this specification.

General methods

Bacterial strains and growth conditions

[0127] Gonococcal strains were cultured under aerobic conditions with 5% CO ₂ at 37°C on GC agar (GCA) or GC broth (GCB) (Oxoid) supplemented with 0.4% glucose, 0.01% glutamine, 0.2 mg of cocarboxylase per litre, and 5 mg of Fe(NO3)3 per litre. The EptA mutant of strain FA1090, F A1090ΔeptA.- :aphA-3 , was constructed as follows. pCMK519 containing an internal fragment of eptA (Piek et al., 2014) was digested with Hindi and ligated with Smal digested pUC18K to create pCMK594 containing the eptA::aphA-3 cassette. The eptA::aphA-3 cassette was transformed into N. gonorrhoeae FA1090 using a chemical transformation method. N. gonorrhoeae FA1090 (Opa +; Pili +) was grown on gonococcal agar plates (GCA) (Oxoid; Thermo Fisher Scientific) supplemented with 1% (v/v) Deakin modified isovitalex (DMIV) and PxB 32 pg/mL (Sigma-Aldrich). N. gonorrhoeae FA1090 AeptA(-)::aphA-3 (Opa +; Pili +) was grown on GCA supplemented with 1% (v/v) DMIV and kanamycin 50 μg/mL (Sigma- Aldrich). The 2008 World Health Organisation (WHO) reference panel of MDR-isolates was used to screen for the compounds (N. gonorrhoeae 31536-31544) (Unemo et al., 2016, J Antimicrob Chemother 71:3096- 3108).

Microdilution assays to determine the effectiveness of small molecule inhibitors

[0128] The minimum inhibitory concentration (MIC) of small molecule inhibitors of EptA were tested against strains F Al 090 and FA1090ΔeptA using a 96-well plate-based microdilution format. Gonococcal strains were grown to mid-log phase in 100 ml in 250 ml glass screw capped flasks in a shaking incubator at 220 rpm at 37 °C for 4 hrs. Aliquots were harvested and equilibrated to an OD560 of 0.4 (2 x 108 cells per ml). Compounds were resuspended in DMSO to a starting concentration of 200 mM which resulted in a final concentration of 5% DMSO in all biological assays. To determine the MIC of the compound alone, 2 x 108 cells per ml of strain FA1090 in 100 pl were mixed with 100 pl of diluted compound. The plates were sealed with sealing foils and incubated at 37 °C for 3 hrs with intermittent shaking every 5 secs in a BioRad XMark (Biorad). At time points zero and 3 hr, 10 pl was removed and spotted onto GCA plates which were incubated for 24 hrs at 37 °C and then were counted for colony forming units per ml. To determine if DMSO had an effect on bacterial growth, 2 x 108 cells per ml were incubated with a final maximum concentration of 5% DMSO in GC broth. To determine whether the compounds could sensitise bacteria to polymixin B (PxB), the assay was modified to contain the same bacterial cell concentration incubated with a sub-inhibitory concentration of PxB (32 pg/ml for FA1090 and 1 pg/ml for FA1090AeptA) that did not affect bacterial cell growth. Added to this was 20 pl of the compound at 2-fold below the established MIC of the compound (termed the maximum effective concentration (MEC) of compound). To examine the relative potency of the compounds that showed the ability to sensitize the bacteria to PxB, the concentration of the compound was kept constant and the concentration of PxB was serially diluted between concentrations of 32 pg/ml to 1 pg/ml.

Preparation of intact LOS for analysis by MALDI-TOF MS

[0129] Lipooligo saccharide (LOS) was extracted and purified by a modification of the hot phenol-water method (Apicella et al., 1994). For MALDI-TOF MS analysis intact LOS was deposited on top of a pre-spotted matrix layer using a procedure previously described (John et al., 2016). Briefly, a solution of THAP (200 mg/ml in methanol; Sigma-Aldrich) with a solution of nitrocellulose transblot membrane (15 mg/ml in acetone/isopropanol, 1:1; Bio- Rad) were mixed in a 3:1 (v/v) ratio. Aliquots of this matrix solution (~1 μl) were spotted on a sample plate. Aliquots of suspensions of intact LOS (4 to 10 mg/ml in methanol/water, 1:3, containing 5 mM EDTA) were desalted with cation exchange beads (Dowex 50WX8- 200, ammonium form) and the desalted LOS aliquots mixed 9:1 (v/v) with 100 mM dibasic ammonium citrate, and then -1 pl was deposited on air-dried matrix spots. Specifically, for the LOS of -4000 MW examined in this study, this amounted to -1-2.5 nmols loaded on each spot.

Negative-ion MALDI-TOF MS of intact LOS

[0130] MALDI-TOF MS was performed on a Synapt G2 high definition mass spectrometer (HDMS) (Waters Corporation, Manchester, UK) with an orthogonal TOF mass analyzer in “sensitivity mode.” The Nd:YAG laser was operated at a wavelength of 355 nm with a 200 Hz firing rate. In general, spectra were acquired for -1 misn, with a scan duration of 1.0 sec. Spectra were digitally smoothed and baseline-corrected using MassLynx 4.1 software. The instrument was calibrated using the mass for the monoisotopic (M-H)- ions for bovine insulin at m/z 5728.5931, insulin B-chain at m/z 3492.6357, renin substrate at m/z 1756.9175, and angiotensin II at m/z 1044.5267 (all from Sigma- Aldrich).

Quantitative analysis of phosphorylated hexaacylated lipid A ions in spectra of intact LOS [0131] The phosphoethanolaminylation and phosphorylation of the lipid A from strain FA1090 after treatment of the bacteria with small molecules was determined. This type of analysis is enabled by observation of prompt Y-type reducing terminal fragment ions for the lipid A moieties of interest including those that contained up to 3 phosphates and 1 phosphoethanolamine (PEA) moiety in MALDI TOF MS analysis. The MALDI spectra obtained on the Synapt G2 HDMS system were of high -resolution, enabling detection of monoisotopic peaks for the lipid A species. Areas of the most abundant negative-ion peaks for hexaacylated lipid A were determined for 6-7 LOS spectra for each sample after using standard conditions for smoothing and correcting the baseline of the spectra using Mass Lynx Version 4.1 (Waters Corporation) software. Areas were included for the three most abundant isotopic peaks (M, M+l, and M+2) of the species listed in Table 1. Average ion abundances were calculated for peaks for hexaacylated lipid A containing 2 or 3 phosphates (2P, 3P) with or without PEA relative to the ion abundances for all quantified peaks for phosphorylated hexaacylated lipid A. The ion abundance for peaks at m/z 1614.1 and 1737.1 were tabulated as fragment ions arising from facile loss of H ₃ PO ₄ (98.0 Da) from 2P lipid A, and H4P2O7 (177.9 Da) from 2P PEA lipid A, respectively, as previously described (John et al., 2009). Differences in ion abundance for peaks of interest were analyzed by ANOVA with a Bonferroni post-/ test to determine the significance of levels of ions in LOS from each of the treated samples compared to the vehicle-only controls.

Table 1. Phosphorylated Hexaacylated LA Negative Ions

Whole bacterial cell lipid A mass spectrometry

[0132] Mass spectrometry was performed as described previously (Larrouy-Maumus et al., 2016). Prior to mass spectrometry analysis, the 2, 5-dihydroxybenzoic acid (DHB) matrix was used at a final concentration of 10 mg/ml in chloroform/methanol (CHCL/MeOH) in a ratio 90:10 v/v. 0.5 pL of bacteria solution and 0.5 pl of the matrix solution were deposited on the target, mixed with a micropipette and dried gently under a stream of air. After optimization, this solvent system and ratio were selected in order to selectively ionize lipid A. MALDLTOF MS analysis was performed on a 4800 Proteomics Analyzer (with TOF-TOF Optics, Applied Biosystems) using the reflectron mode. Samples were analyzed operating at 20 kV in the negative ion mode using an extraction delay time set at 20 ns. Typically, spectra from 500 to 2000 laser shots were summed to obtain the final spectrum. All experiments were carried out in four independent bacterial cultures and in three technical replicates. The negative control consists of 0.5 pl of double distilled water and 0.5 pL of the matrix solution. Mass spectrometry data were analyzed using Data Explorer version 4.9 from Applied Biosystems. For all bacterial species, the negative mass spectrum was scanned between m/z 1000 and 2200.

Effect of inhibition ofEptA on TNFa expression induced in THP-1 cells by LOS.

[0133] Human THP-1 monocytic cells were cultured in RPMI 1640 with 10% FBS (growth medium) at 37°C in a 5% CO ₂ atmosphere. The cells were activated in growth medium containing 10 ng/mLphorbol myristate acetate (PMA, Sigma- Aldrich, St. Louis, MO, USA) for 18 h. After removing media and washing with PBS, the differentiated cells were trypsinized (1%) and seeded in 96-well plates (1 x 10 ⁴ cells per well in growth medium). The cells were treated with 100 ng/mL of LOS and incubated at 37°C in 5% CO ₂ for 18 h. The cell culture supernatants were then transferred to another 96-well plate and frozen at - 80°C. For detection of TNFa in aliquots of the supernatants, ELISA (ThermoFisher) assays were performed per the instructions of the vendor using a Thermomax (Molecular Devices, Sunnyvale, CA, USA) plate reader with detection at 450 nm.

RAW macrophage cell assays

[0134] RAW 264.7 murine macrophages (RAW) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), and 1 x GlutaMAX™ at 37 °C + 5% CO ₂ . All tissue culture media and supplements were produced by Gibco (Thermo Fisher Scientific).

Activity of Inhibitors in the RA W macrophage cell line

[0135] Cytotoxicity - RAW macrophages were grown to confluence (l x 10 ⁶ cells/mL) in 24 well tissue culture plates. Wells were washed with PBS and incubated at 37 °C + 5% CO ₂ with DMEM containing either 200 pM or 300 pM of the inhibitor suspended in 100% DMSO. The final concentration of DMSO in wells containing inhibitor was 4% (v/v). Control wells contained cells exposed to DMEM only, and DMEM containing 4% (v/v) DMSO. Lactate dehydrogenase (LDH) release was measured after 24 hours using the Cytotoxicity Detection Kit ^PLUS (LDH) (Roche; Sigma- Aldrich) as per the manufacturer’s instructions. A total of three biological repeats comprising two technical repeats were performed.

Bacterial assays

[0136] RAW macrophages were grown as above in 24 well tissue culture plates. N. gonorrhoeae grown on solid media for no longer than 14 hours was harvested and resuspended in gonococcal broth supplemented with 1% (v/v) DMIV and NaHCO ₃ to an optical density of OD490 of 0.04 ± 0.005. Bacteria were pre-incubated in glass conical flasks at 37 °C with agitation for a period of 4 hours prior to addition of either compound 3, 4, or 5 suspended in 100% DMSO to a final concentration of 50 pM of compound. The final concentration of DMSO in the DMSO control flask and flasks containing inhibitor was 1% (v/v). Bacteria were returned to the incubator for another 3 hours prior to infection of RAW cells. N. gonorrhoeas were diluted into DMEM containing inhibitor or DMSO at a final concentration of 50 pM to obtain a multiplicity of infection ranging from 0.5 - 5. Bacteria were centrifuged onto a RAW macrophage monolayer at 1400 rpm for 2 minutes prior to incubation at 37 °C + 5% CO ₂ for 15 minutes. RAW macrophages were then washed three times with sterile PBS to remove non-adherent bacteria and lysed with DMEM + 1% saponin (Sigma- Aldrich) for 10 minutes. The number of cell-associated bacteria were enumerated by viable count. A minimum of three biological repeats comprising two technical repeats were performed. One way analysis of variance (ANOVA) Kruskal-Wallis test with Dunn’s multiple comparisons were performed using GraphPad Prism version 7.0d for Mac OS X (GraphPad Software, La Jolla California USA, www.graphpad.com).

Mouse model

[0137] Thirty-two female BALB/c mice (n = 8 mice/group) in the diestrus stage of the estrous cycle were treated with three doses of 0.5 mg of Premarin (a pharmacological grade source of estradiol) every other day for a total of three doses (days -2, day. 0 and day +2). Antibiotics (streptomycin and trimethoprim) were also given starting on day -2. On day 0, mice were randomized into 4 groups of 8 mice- each, anesthetized and inoculated vaginally with 30 pL of test and control compounds. Test compounds were given at a concentration of 100 pM to increase gonococcal susceptibility to CAMPs in vitro. Thirty seconds later, mice were inoculated vaginally with 10 pL of a PBS suspension containing 10 ⁴ -10 ⁶ colonyforming units (CFU) of the gonococcal challenge strain. Vaginal mucus was quantitatively cultured daily for gonococcal on days 1 through 5 post-inoculation and results expressed as number of CFU/ml of vaginal swab suspension. The limit of detection of 20 CFU was used for cultures that do not yield Ng. The duration of colonization between test and control groups was plotted as Kaplan Meier colonization curves and analyzed by the Mantel-Cox (Log-Rank) test. The number of CFU recovered over time were compared between groups using a repeated measures ANOVA followed by a Bonferroni post-hoc analysis with multiple comparisons. In both sets of analyses, p < 0.05 is considered significant. Example 1 — EptA inhibitors

[0138] Using saturation-transfer difference (STD)-NMR, a library of 1137 compounds was screened in mixtures containing up to six individual compounds against the soluble domain of EptA. The compounds were ranked based on the intensity of the signals in the screening experiments as described above and validated by first recording (STD)-NMR experiments on single compounds against the soluble domain of EptA, and subsequently by recording both (STD)-NMR and Carr-Purcell-Meiboom-Gill (CPMG)-NMR experiments against the full-length EptA. The compounds were ranked on the basis of the magnitude of the STD and CPMG effects measured in the NMR experiments.

[0139] The top fifteen ranked compounds, by STD/CPMG-NMR were then tested against nine MDR gonococcal strains using a filter disc assay in the presence of the sub -inhibitory concentration of 48 pg/ml PxB. Compounds were screened for their ability to increase sensitivity of Neisseria gonorrhoeae strain FA1090 to PxB using a broth microdilution assay as described above. Complementing this, STD/CPMG-NMR binding to full length-EptA was performed with selected compounds based on a selectivity threshold of 20-fold in the bacterial PxB sensitivity screen. Compound 1 was selected for further analysis and modification.

Compound 1

[0140] Briefly, a library of variants of compound 1 was prepared systematically by modifying the substituent on the nitrogen atom of the amide functional group (compounds 2-45). Other variants of compound 1 were produced by replacing the amide functional group with the corresponding amine (i.e. removing the carbonyl group, compounds 46-55). Any potential coupling effects of the cyclopentyl group attached to the imidazoyl nitrogen atom and the sulfur-containing functional group was investigated, with compounds having a sulfone (compounds 56-71), sulfoxide (compounds 73-76) or thioether (compounds 75-124) functional group.

[0141] Compounds 2 to 124 were prepared by SYNthesis Pharm. The compounds were characterised by NMR spectroscopy, using either CDCh or DMSO- ₆ as solvent. [0142] The MIC of compounds 1 and 2 to 124 against wild type Neisseria gonorrhoeae strain FA1090 was determined as described above. MICs are presented in Tables 2 to 6.

[0143] A supplementary screen to test “off target” cytotoxicity examined the effect of the compound on the mutant strain FA1090ΔeptA (this mutant was found to be resistant to 1 pg/ml PxB). Compounds which increased sensitivity of FA1090ΔeptA in the presence of 1 pg/ml PxB were considered to be “off target” as these compounds had a mode of action independent of EptA expression. From these screens a total of 44 compounds were considered to be “on target” for EptA inhibition (Tables 2 to 6). In comparison to compound 1 which reduced the MIC of strain FA 1090 2-fold from 64 pg/ml to 32 pg/ml (at a concentration of 200 pM), 26 compounds reduced the MIC of strain FA 10902-fold from 64 pg/ml to 32 pg/ml, 14 compounds reduced the MIC 4-fold, and 4 compounds reduced the MIC 8-fold.

[0144] Four compounds, 2, 3, 4 and 5, exhibited the least bactericidal activity at 100 pM after 3 hrs incubation and of these compounds 3, 4 and 5 reduced the PxB resistance by 4- fold from wild-type levels. The structures of compounds 3, 4 and 5 are shown below.

Table 2: PxB sensitivity to wild-type strain FA1090 (64 pg/mL in broth) in the presence of compounds of Formula (I): e to

Table 3: PxB sensitivity to wild-type strain FA1090 (64 pg/mL in broth) in the presence of compounds of Formula (I): Table 4: PxB sensitivity to wild-type strain FA1090 (64 pg/mL in broth) in the presence of compounds of Formula (I):

Table 5: PxB sensitivity to wild-type strain FA1090 (64 pg/mL in broth) in the presence of compounds of Formula (I):

Table 6: PxB sensitivity to wild-type strain FA1090 (64 pg/mL in broth) in the presence of compounds of Formula (I):

Example 2 - Pre-treatment of bacterial cells with compound 3,4, or 5 increases killing of N. gonorrhoeae by RAW macrophage cell lines

[0145] EptA mutants of N. gonorrhoeae are known to be highly sensitive to killing by macrophages due to the presence of defensins and cathelcidins in these cells. The compounds 3, 4 and 5 were shown to have no cytotoxicity towards macrophages at 300 pM using a lactate dehydrogenase activity assay (data not shown). Based on this output bacteria were pre-incubated with each compound at 50 pM and then exposed to the RAW macrophage cell line and total association was measured after 15 min. The viability of FA I 090Ac in the presence of RAW macrophages was significantly reduced by 2.4-fold compared to the wild-type control (Dunn’s multiple comparisons test, p=0.0025) (Figure 1). Bacteria treated with either compound 3, 4 or 5 also showed reduced viability compared to wild-type of 1.9-fold, 2.6-fold, and 2.2-fold respectively (Dunn’s multiple comparison test, p=0.0260, p=0.0018; p=0.0078).

Example 3 - Reduction of PEA decoration of lipid A from N. gonorrhoeae treated with compound 5

[0146] To confirm that the compounds were directly inhibiting EptA in bacteria and resulting in the PxB -sensitivity phenotype, strain FA1090 was grown in the presence of either compound 3, 4 or 5 and the abundance of lipid A phosphorylation assessed by mass spectrometry of LOS extracted from broth grown bacteria incubated at mid-log phase with 50 pM compound for 3 hours (Table 1, Figure 1). Compound 5 significantly reduced the amount of PEA-decorated lipid A in the LOS relative to the LOS in control untreated bacterial cells and those bacterial cells treated with compounds 3 and 4 (Figure 3, Panel A). As the level of phosphoethanolaminylation was reduced in compound 5-treated bacteria, the relative abundance of 2P phosphoforms increased (Figure 3, Panels B and C).

[0147] To further verify the reduction of PEA decoration of lipid A, the same purified lipid A extractions were used to induce cytokine responses from THP-1 cells (Figure 4). The LOS from FA 1090ΔeptA and 5-treated bacteria induced significantly less TNFα expression by the THP-1 cells than untreated LOS (p<0.05 and p<0.001, respectively) while LOS from compound 3 and 4-treated bacteria were not significantly different from LOS from untreated bacteria.

Example 4 - Binding affinity of compound 5 for EptA

[0148] The binding of compound 5 to EptA was investigated by recording (STD)-NMR and CPMG experiments using a similar approach that was used to elucidate compound 1 and to gain insight into any improvement in binding (Figure 5). Compound 5 showed a significant increase in the intensity of the peaks in the STD difference spectrum, having an average STD intensity of 4.6% in comparison to the compound 1, which showed an average STD intensity of 0.4% using the same experimental parameters.

Example 5 - Effectiveness of compound 5 in reducing the PxB MIC for MDR-N. gonorrhoeae

[0149] Since the original screen to detect compound 5 was performed using the antibiotic sensitive strain FA 1090, the effectiveness of compound 5 to reduce the PxB MIC in MDR- N. gonorrhoeae was tested. In all instances, 50 pM of compound 5 was able to reduce the MIC for PxB in MDR isolates by 2-fold (see Table 7). To determine whether the reduction of PxB MIC by compound 5 had a synergistic or antagonistic effect on killing with other antibiotics, the assays were repeated with sub -inhibitory concentrations of penicillin, azithromycin and ceftriaxone which are used as first-in-line therapies. No change in the effectiveness of these antibiotics was observed indicating there is no antagonism with commonly used therapies (data not shown).

Table 7: MIC of PxB (pg/ml) of MDR-N gonorrhoeae in the presence of compound 5 in the broth microdilution assay.

PenLS = penicillin less susceptible, QLS = quinolones less susceptible, TRNG = tetracycline resistant N. gonorrhoeae, CMRP= chromosomally mediated resistant penicillin, QHLR= quinolone high level resistance, CroNS = ceftriaxone non-susceptible, PPNG = penicillinase producing N. gonorrhoeae, QRNG = quinolone resistant N. gonorrhoeae, SpecR = spectinomycin resistant, AzithR = azithromycin resistant. All genotypes are described in Unemo et al., 2016, J Antimicrob Chemother. 71:3096-3108). Example 6 — Further compounds based on compound 5 increase sensitivity to human defensin LL-37 phenocopying the EptA mutant phenotype

[0150] A further library of 55 variants based on compound 5 was produced (Tables 8, 9, 10). These were prepared by SYNthesis Pharm. The compounds were characterised by ¹ H NMR spectroscopy, using either CDC1 ₃ or DMSO- ₆ as solvent. The compounds were tested using the microdilution method for their ability to sensitize strain FA1090 to 32 pg/ml PxB. From this list, 32 candidates were identified that were better than the starting material, compound 5. The potency of these 32 candidates were ranked by examining the ability of the compounds to sensitize the bacteria to human defensin LL-37 at a concentration of 3.2 pM (Figure 6). To establish the conditions for screen of compounds, we first conducted the kill-curves of WT FA 1090 and AeptA to the LL-37 using the micro-dilution assay in 96- wells format. As expected, AeptA strain was more sensitive to the LL-37 when compared to the WT strain (Figure 6A). LL-37 at the concentration of 3.2 pM was selected for the screen of compounds as this concentration showed greater than 90% inhibition of survival of AeptA strain while not significantly affecting survival of WT FA1090.

[0151] The parent compound 5 at 50 pM concentration showed a little effect in assisting killing of WT bacteria by LL-37 (Figure 6B). Notably, a total of fifteen compounds significantly increased the susceptibility of strain FA1090 to LL-37 with 50% cut-off of survival after 90 minutes of incubation. However, in the absence of LL-37, nine compounds (147, 148, 125, 145, 126, 173, 174, 172) displayed the bactericidal effects on WT FA1090 compared to DMSO-treated control, indicating that they were off-target. These compounds were not used for further investigation. We thereby focused on the six compounds (164, 128, 127, 142, and 146) which had no demonstrated inhibitory effects on the survival of WT strain.

[0152] Next, titration experiments were performed to determine the minimal effective concentration of compound that results in 90% killing in the presence of 3.2 pM LL-37 (MEC90). All five compounds showed a dose-dependent effect on boosting the sensitization of strain FA1090 to LL-37 (Figure 7C). Among the tested compounds, 142 was the most effective compound which had the lowest MEC90 with a value of 25 pM. The compounds 164, 127, and 146 resulted in a MEC90 of 50 pM, followed by 128 with a value of 100 pM. All these compounds at the MEC90 had no bactericidal effects on the WT and AeptA strains (Figure 7), confirming their specificity on the enzyme EptA. Table 8: PxB sensitivity to wild-type strain FA1090 (64 pg/mL in broth) in the presence of compounds of Formula (I):

Table 9: PxB sensitivity to wild-type strain FA1090 (64 pg/mL in broth) in the presence of compounds of Formula (I): e y

Table 10: PxB sensitivity to wild-type strain FA 1090 (64 g/mL in broth) in the presence of compounds of Formula (I):

*NE: No effect, meaning that these compounds did not sensitize bacteria to PxB.

Example 7 - Further compounds based on compound 5 increase sensitivity to human RAW macrophages phenocopying the Ept A mutant phenotype

[0153] Before use in RAW macrophages, compounds 164, 128, 127, 142, and 146 were tested for cytotoxicity in the LDH assay (Table 11).

Table 11: EptA inhibitors assist the killing of N. gonorrhoeae FA1090 in association with murine RAW 264.7 macrophages.

*CC ₂₀ : Concentration of compound required to inhibit 20% viability of murine RAW 264.7 macrophages. MEC ₅₀ : Half minimal effective concentration of compound resulted in approximately 50% of bacterial killing in association with murine RAW 264.7 macrophages that normalized to the WT FA1090 treated with 1% DMSO (inhibitor solvent). The values of CC ₂₀ and MEC ₅₀ were obtained from three biological repeats, with each having two technical repeats. TI (Therapeutic index) = CC ₂₀ / MEC ₅₀ .

[0154] Serial two-fold dilutions of compound beginning at 2-fold below the CC ₂₀ was used to expose the wild-type bacteria FA1090 for three hours before exposure to RAW macrophages. The minimal effective concentration of compound that resulted in 50% clearance in macrophages (MEC ₅₀ ), was determined. As shown in the Table 9 and Figure 8 A, five compounds (164, 128, 127, 146) had the MEC ₅₀ value ranging from 50 pM to 75 pM. Exceptionally, the compound 142 was the least effective compound with a value of 125 pM. Overall, the compounds 128 and 127 were the lead compounds in macrophage assays that the MEC ₅₀ showed a 4-fold lower than the cytotoxicity CC ₂₀ value. Additionally, these compounds showed no inhibiting effects on the killing of AeptA strain by macrophages (Figure 8B), demonstrating that they do not affect to the phagocytic capacity of macrophages.

Example 8 - Further compounds based on compound 5 decrease PEA decoration on lipid A of bacteria

[0155] Mass spectroscopy of whole bacteria was used to determine the relative abundance of PEA-decorated lipid A from bacteria exposed to related compounds 125, 126, 128, 127 and unrelated compounds 134, 139, 151, and 141. Example 9 — Compounds 127 and 128 increase susceptibility of multi-drug isolates to killing by LL-37 human defensin

[0156] To further explore if EptA inhibitors increase the LL-37 susceptibility of others gonococcal strains, including MDR isolates, we conducted the LL-37 kill-curves of WHO F (AMS; WT MtrCDE and PorBlb), WHO G (MDR; Over-expressed MtrCDE efflux pump and WT PorBlb), WHO P (MDR; Over-expressed MtrCDE efflux pump and decreased influx of antimicrobials through the porin PorBlb), and their \eplA strains in presence of the lead compounds 127 and 128.

[0157] As expected, all \eplA strains were rapidly killed by LL-37 with the MIC decreasing by 2- to 4-fold when compared to WT WHO gonococcal strains (Figure 10). Notably, the addition of 127 and 128 significantly increased the LL-37 sensitivity of WHO F (MIC = 6.4 pM) and WHO G (MIC = 12.8 pM) in a dose-dependent manner, that are comparable to those found in their \eplA strains (MIC = 3.2 pM for WHO F \eplA and MIC = 6.4 pM for WHO G ΔeptA). These compounds were also able to increase the LL-37 susceptibility of WHO P by 2-fold. No significant inhibitory effects of 127 and 128 were observed in both the WT and \eplA strains when bacteria were incubated with only compounds. The results again provide the confirmation that our compounds are specific to the enzyme EptA.

Example 10 — Compounds 127 and 128 increases the rate of clearance of pre-infected murine macrophages

[0158] An assay was designed to determine whether compounds 127 and 128 could induce clearance of pre-infected macrophages. In this model, the RAW macrophages are exposed to bacteria for 30 mins and are internalised by the murine macrophages. External bacteria are killed by gentamycin which is thoroughly washed away before the compounds are added to the external supernatant. The mixture is further incubated and the counts of internalised bacteria are enumerated at the end of the assay. Both 127 and 128 reduced the bacterial load to levels similar for the EptA mutant of the WT FA 1090, WHO F and the MDR isolates WHO G and WHO P (Figure 11).

Example 11 — Compounds 127 and 128 can be formulated in hydroxyethylcelluose without loss of function

[0159] The aim of this study was to assess the in vivo efficacy of EptA inhibitors using a mouse model of vaginal infection. Hydroxy-ethylcellulose (HEC) was selected as delivery vehicle of compounds because it is widely used as gelling agent in vaginal lubricants. We first performed LL-37 kill-curves of WT strain FA1090 and with or without 1% HEC to test if the potency of compounds (128 and 127) is affected when they are suspended in HEC. 1% HEC did not affect growth of the bacteria compared to control (Figure 12A and B). LL-37 displayed bactericidal activity against both WT FA1090 and \eplA in a dosedependent manner in the presence of 1% HEC gel indicating that HEC does not interfere the activity of the compounds or LL-37.

Example 12 — Compounds 128 is functional in a mouse model of vaginal gonorrhoea infection

[0160] To establish the mouse model of vaginal infection, 2xl0 ⁶ CFU of WT strain FA1090 were used to inoculate intravaginally female mice. The infected mice were then treated daily with 1% HEC or 1% HEC gel containing 100 pM 128. As shown in Panel A of Figure 13, 25 to 75% decrease in bacterial colonization were observed for the control group (1% HEC) on days 4 to 6 of infection. No decline in bacterial recovery were recorded from the mice treated with compound 128. Of note, the number of PMNs influx to the infection site in mice treated with 128 were significantly lower than the control group on days 3 to 5 of infection. These results implied that the treatment scheme of compound 128 used in this study could reduce the inflammatory response triggered by bacteria, but not bacterial colonization. This reflects the same phenotype of the EptA mutant which will colonise mouse vaginal tracts but result in inflammation.