Anti-biofilm compositions and methods for using the same

Abstract

Disclosed herein are compounds (e.g., compounds of formula (I) or (II)). and compositions comprising the same. The disclosed compounds and compositions may be used in methods of preventing or treating an infection in a subject, or in methods of inhibiting biofilm formation or reducing the grow th of an established biofilm on a surface.

Description

[0001] ANTI-BIOFILM COMPOSITIONS AND METHODS FOR USING THE SAME

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to United States Provisional Application No. 63 / 691,960, filed September 06, 2024, the content of which is incorporated by reference in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (169852_00173.xml; Size: 2,830 bytes; and Date of Creation: September 4, 2025) are herein incorporated by reference in its entirety.

[0006] BACKGROUND OF THE INVENTION

[0007] Staphylococcus aureus is arguably one of the most problematic pathogens faced by modem healthcare systems today. S', aureus produces a diverse array of infections, many of which are recalcitrant to antimicrobials even in the absence of issues related to acquired resistance. A primary contributing factor to this recalcitrance is formation of a biofilm on both native tissues and indwelling medical devices. This is due to the fact that the biofilm confers a degree of intrinsic resistance that often necessitates surgical intervention to debride infected tissues and / or remove infected devices. For example, one study found that nearly half of patients with implanted orthopedic devices admitted to a hospital with S. aureus bacteremia had developed an implant-associated infection. Within a biofilm, S. aureus adopts a lifestyle characterized by limited metabolic activity’ and the emergence of persister cells, both of which limit the efficacy of host defenses and conventional antibiotics. Therefore, there remains a need for therapeutic agents and methods that specifically target biofilm formation and the biofilm lifestyle.

[0008] BRIEF SUMMARY OF THE INVENTION

[0009] Disclosed herein are compounds and derivatives thereof, compositions comprising the same, and methods for using the same, such as in methods for prevention or treatment of infections.

[0010] One aspect of the invention provides a compound of formula (I) or formula (II),

[0011] Page 1 of 47

[0012] QB\169852.00173\98287232. 1

[0013] formula (II) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein is an ethylene having E or Z conformation; and wherein

[0014] X1is independently O or NRA;

[0015] RAis hydrogen or C1-C3 alky l;

[0016] X2is C or N;

[0017] R1is hydrogen, naphthyl, phenyl optionally substituted with hydroxyl, or pyrazolyl optionally substituted with C1-C3 alkyl optionally substituted with heterocycloalkyl; and wherein

[0018] (i) X3is NRA, ring A is 3-pyridyl, R2is hydrogen, halogen, or phenyl; or

[0019] (ii) X3is O, ring A is 3-pyndyl or pyrazolyl optionally substituted with C1-C3 alkyl, R2is hydrogen; and with the proviso that:

[0020] X1and X2are not simultaneously' O and C.

[0021] Another aspect of the invention provides a pharmaceutical composition. The pharmaceutical composition comprises a compound as described herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0022] Another aspect of the invention provides a composition. The composition comprises the compound as described herein, a solvent, and a polymer.

[0023] Another aspect of the invention provides a method of preventing or treating an infection in a subject in need thereof. The method comprises administering a compound that binds to SarA, MsaB, or a combination thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof or a composition comprising the compound to the subject. In some embodiments, the compound is a compound as described herein.

[0024] Page 2 of 47

[0025] QB\169852.00173\98287232.1 Another aspect of the invention provides a method of inhibiting biofilm formation or reducing the growth of an established biofilm on a surface. The method comprises contacting a compound that binds to SarA, MsaB, or a combination thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, or a composition comprising the compound to the surface. In some embodiments, the compound is a compound as described herein.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0028] FIG. 1 shows multiple sequence alignment and ligand solvent accessible surface (SAS) prediction. Panel A: Contraint Based Alignment (COBALT) of SarA (SEQ ID NO: 1) and MsaB (SEQ ID NO: 2) shows that both proteins share similar protein motifs. Panel B: Surface representation depicting predicted SAS for 6998 when bound to SarA or MsaB, before (left panel) and after 300 ns simulation (right panel), shaded based high to low.

[0029] FIG. 2 shows the biofilm inhibition assay of 6998. Compound 6998 inhibited biofilm formation in a dose-dependent manner. The ICso was 8.87 pM for strain USA300 LAC strain and 3.0 pM for strain UAMS-1. Results represent the average and standard error of the mean from three biological replicates, each of which included three experimental replicates. The parent strain assayed in the presence of an equivalent amount of DMSO and its isogenic sarA, msa . and sarA / msaB mutants were used as controls.

[0030] FIG. 3 shows the biofilm eradication assay of 6998. Compound 6998 disrupted established biofilm in both strains USA300 LAC and UAMS-1 in a dose-dependent manner. Results represent the average and standard error of the mean from three biological replicates, each of which included three experimental replicates. The parent strain assayed in the presence of an equivalent amount of DMSO and its isogenic sarA, msaB, and sarA / msaB mutants were used as controls.

[0031] FIG. 4 shows the persister cells assay of 6998. The cells are grown up to the stationary growth phase (24 hours). Then, the cells are treated with compound 6998 (31.25 pM) for 3 hours. The cells are then harvested and treated with 10X MIC of daptomycin, 10X MIC of

[0032] Page 3 of 47

[0033] QB\169852.00173\98287232. 1 gentamicin, and 50X MIC of vancomycin. The antibiotic treatments are done for 24 hours. Afterward, the cells are harvested, and the remaining CFUs are counted. The persister fraction is calculated as a percentage compared to the number of CFUs before the antibiotic treatment is initiated. Compound 6998 treatment is done only on wild-type strain USA300 LAC and its isogenic sarA, msaB, and sarA'msaB mutants were used as controls and were not treated with 6998.

[0034] FIG. 5A shows the biofilm inhibition assay of 917. Compound 917 (amide derivative of 6998) inhibited biofilm formation in a dose-dependent manner. The IC50 was 10.6 pM for USA300 LAC strain. Results represent the average and standard error of the mean from three biological replicates, each of which included three experimental replicates.

[0035] FIG. 5B shows the biofilm eradication assay of 917. Compound 917 disrupted established biofilm in a dose-dependent manner. Results represent the average and standard error of the mean from three biological replicates, each of which included three experimental replicates. The parent strain assayed in the presence of an equivalent amount of DMSO and its isogenic sarA, msaB. and sarA / msaB mutants were used as controls.

[0036] FIG. 6 shows the biofilm prevention and eradication result comparison between compound 6998 and 917. Both compound 6998 and 917 showed similar ICso for preventing biofilm formation. Compound 917 showed increased biofilm disruption property' compared to 6998.

[0037] FIG. 7 shows the persister cells assay of 917. The cells are grown up to the stationary growth phase (24 hours). Then, the cells are treated with compound 917 (31.25 pM) for 3 hours. The cells are then harvested and treated with 10X MIC of daptomycin, 10X MIC of gentamicin, and 50X MIC of vancomycin. The antibiotic treatments are done for 24 hours. Afterward, the cells are harvested, and the remaining CFUs are counted. The persister fraction is calculated as a percentage compared to the number of CFUs before the antibiotic treatment is initiated. Compound 917 treatment is done only on wild-type strain USA300 LAC and its isogenic sarA, msaB, and sarA'msaB mutants were used as controls and were not treated with 917.

[0038] FIG. 8 shows the cytotoxicity' assay of 917 on mammalian cells. RAW 267.4 cells monolayers formed over 24 hours at 37°C in 5% CO2. The growth medium was replaced with Opti-MEM™ media containing the desired concentrations of 917 (200 pM, 100 pM, 50 pM, 25 pM, and 10 pM). These concentrations were selected to represent IX to 20X of the 917 IC50 value which prevent biofilm formation. The cells were allowed to grow for 6, 12, and 24 hours,

[0039] Page 4 of 47

[0040] QB\169852.00173\98287232. 1 after which cytotoxicity assays were performed using the LIVE / DEAD® Viability / Cytotoxicity Kit (Invitrogen) and the CyQUANT™ LDH Cytotoxicity Assay Kit (Invitrogen) according to the manufacturer’s protocol. Statistical analysis was done using a one-way ANOVA with Dunnett’s correction and no statistically significant difference were observed relative to media + DMSO controls.

[0041] FIG. 9 shows biofilm prevention by compound 846 in LAC and UAMS strains. 846 is the derivative of parent compound with substituted N in the tricycle ring to make the 6998 compound symmetrical. 846 inhibited biofilm formation with IC50 value of 24.69 pM.

[0042] FIG. 10 shows biofilm prevention by compound 873 in LAC and UAMS strains. 873 is the derivative of 846a with substituted amide group in the furanone ring. 873 inhibited biofilm formation with IC50 value of 2.44 pM and 12.37 pM in LAC and UAMS strains backgrounds respectively.

[0043] FIG. 11 shows biofilm eradication. Compound 873 did not disrupt mature biofilm formation, whereas parent Compound 6998 disrupted >50% mature biofilm.

[0044] FIG. 12 shows the thermal shift assay comparison in the presence of 2 mM compound. Protein thermal assay using GloMelt™ dye was used to determine the thermal stability of SarA and MsaB in the presence of test compounds as a ligand. Analysis of melting temperature (Tm) was performed using the Boltzmann method from a plot of fluorescence intensity7vs. temperature and the derivative method (from a plot of d(fluorescence) / dT vs. temperature).

[0045] FIG. 13 shows the thermal shift assay with 917 and the effect of 917 on (A) MsaB and (B) SarA thermal stability. The thermal stability of MsaB (40 pg) and SarA (40 pg) was analyzed using a fluorescence-based thermal shift assay with the GloMelt™ Thermal Shift Protein Stability Kit (Biotium) in the presence of varying concentrations of compound 917 (1.0 mM to 4 mM). The thermostability curves demonstrate that compound 917 has a dosedependent effect on the stability of both MsaB and SarA proteins. In the absence of compound 917, the melting temperatures (Tm) for MsaB and SarA were 78.4 °C and 56.18 °C, respectively. Upon addition of compound 917, the Tm shifts for MsaB ranged from 0.36 °C to 2.16 °C, while the Tm shifts for SarA ranged from 0.15 °C to 1.0 °C. These shifts indicate specific binding of compound 917 to MsaB and SarA, contributing to their altered thermal stability.

[0046] FIG. 14 shows the biofilm inhibition assay of 5718. Compound 5718 prevented biofilm with IC50 value of 17 uM, and also disrupted mature biofilm.

[0047] Page 5 of 47

[0048] QB\169852.00173\98287232. 1 FIG. 15 shows the thermal shift assay of 5718, which showed that compound 5718 binds with SarA. but not with MsaB.

[0049] FIG. 16 shows the biofilm inhibition assay of 5676.

[0050] FIG. 17 shows the biofilm inhibition assay of 5935.

[0051] FIG. 18 shows the biofilm inhibition assay of 6023.

[0052] FIG. 19A shows the biofilm eradication assay of 5676.

[0053] FIG. 19B shows the biofilm eradication assay of 5935.

[0054] FIG. 19C shows the biofilm eradication assay of 5718.

[0055] FIG. 19D shows the biofilm eradication assay of 6023.

[0056] FIG. 20A shows biofilm prevention of R969.

[0057] FIG. 20B shows biofilm prevention of R958.

[0058] FIG. 20C shows biofilm prevention of R971.

[0059] FIG. 20D shows biofilm prevention of R983.

[0060] FIG. 20E shows biofilm prevention of R981.

[0061] FIG. 20F shows biofilm prevention of R996.

[0062] FIG. 20G shows biofilm prevention of R997.

[0063] FIG. 20H shows biofilm prevention of R998.

[0064] FIG. 21 shows the structure of the compounds disclosed herein and their ICso values for biofilm inhibition. Mixtures of E / Z isomers of 6998 and 917 are obtained, with only one isomer shown in the figure.

[0065] FIG. 22 shows confocal images of biofilm treatment with 917. S. aureus strain USA300 LAC was treated with 100 pM (10X ICso), 50 pM (5X ICso), and 10 pM (IX ICso) of 917. Biofilms were stained with SYTO-9 and Toto-9. Viable bacteria and dead bacteria are indicated in the images. Images were obtained at 60x magnification with an oil immersion lens.

[0066] FIG. 23 shows the synergistic activity between 917 and vancomycin.

[0067] FIG. 24 show-s the dose-response curve showing inhibition of S. aureus biofilm formation by 917 in Tegaderm™ formulation.

[0068] FIG. 25 shows the dose-response curve showing inhibition of S. aureus biofilm formation by 917 in intraperitoneal formulation (PEG / NMP / EtOH / H2O at 50 / 12 / 10 / 28 v / v).

[0069] FIG. 26A shows the standard calibration curve generated to quantify compound 917, with absorbance measured at 515 nm.

[0070] FIG. 26B shows the release kinetics of 917 from bone-regenerating scaffold over 11 days.

[0071] Page 6 of 47

[0072] QB\169852.00173\98287232. 1 FIG. 27 shows the daily eluates of 917, indicating sustained in vitro release of 917 and biofilm prevention from bone-regenerating scaffold.

[0073] FIG. 28 shows the in vitro release of 917 and biofilm prevention from Carbopol®- based gel formulations.

[0074] DETAILED DESCRIPTION OF THE INVENTION

[0075] The present invention relates to compounds, compositions, and methods for treating infections, inhibiting biofilm formation and / or reducing the grow th of an established biofilm.

[0076] The term “biofilm” as used herein refers to an aggregate of microorganisms in which cells adhere to each other and / or to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance. A variety of microorganisms have the ability to form biofilms. Biofilms may be formed by bacteria, archaea, fungi, protozoa, algae, and combinations thereof. Non-limiting examples of bacteria that may form biofilms include Staphylococcus, Streptococcus, Peptostreptococcus, Corynebacterium, Clostridium, Listeria, Bacillus, Enter obacteriaceae. Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas , Bdellovibrio, and Legionella. Fungi that may form biofilms include, w ithout limit, Candida. For example, the biofilm may comprise bacteria such as be Corynebacterium spp., Escherichia coli, Enterococcus spp., Klebsiella spp., Legionella spp., Neisseria gonorrhoeae, Pseudomonas aeruginosa, Staphylococcus spp., methicillin-resistant Staphylococcus spp.. Streptococcus mutans, or Streptococcus sanguinis.

[0077] In some embodiments, the biofilm comprises Staphylococcus aureus, or methicillin- resistant Staphylococcus aureus (MRSA). In some embodiments, S. aureus adopts a lifestyle characterized by limited metabolic activity and the emergence of persister cells. Persister cells represent a subpopulation of bacterial cells that are less susceptible to conventional antibiotics. They are present in all populations of bacterial cells, including planktonic cultures, but are more prevalent in a biofilm.

[0078] Chemical Entities

[0079] The term "alkyl" refers to a straight-chain or branched alkyl radical in all of its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as Ci-Ci2-alkyl, Ci-Cio-alkyl, and Ci-Ce-alkyl, respectively.

[0080] The term "alkylene" refers to a diradical of straight-chain or branched alkyl group (e.g., a diradical of straight-chain or branched C1-C12 alkyl group). Exemplary alkylene groups include, but are not limited to -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, - CH2CH(CHS)CH2-, -CH(CH2CH3)CH2-, and the like.

[0081] Page 7 of 47

[0082] QB\169852.00173\98287232. 1 The term “ethylene” refers to a hydrocarbon containing a double bond and two carbon The ethylene may be optionally substituted and exist as a mixture of E and

[0083] Z (or cis and trans) stereoisomers.

[0084] The term "alkenyl" refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2- 6 carbon atoms, referred to herein as C2-Ci2-alkenyl, C2-Cio-alkenyl, and C2-C6-alkenyl, respectively.

[0085] The terms "alkoxy" or "alkoxyl" refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, tertbutoxy and the like.

[0086] The term "cycloalkyl" refers to a monovalent saturated or partially saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as "C4-8-cycloalkyl," derived from a cycloalkane. Unless specified otherwise, the cycloalkyl group is not substituted, i.e.. it is unsubstituted.

[0087] The term "heterocycloalkyl" (or "heterocyclyl") refers to a monovalent saturated or partially saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4- 6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and / or S.

[0088] The term "halo" refers to a halogen atom or halogen radical (e.g., -F, -Cl, -Br, or -I).

[0089] The term "haloalkyl" refers to an alkyl group that is substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like.

[0090] The term "aryl" refers to a carbocyclic aromatic group. The term "aryl" includes monocyclic ring systems, and polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. Unless specified otherwise, the aryl ring is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure. Representative aryl groups include phenyl, naphthyl, anthracenyl, 1.3 -benzodi oxolyl and the like.

[0091] The substituents on the aryl (e.g., phenyl) rings in the compounds of the disclosure may be at ortho-, meta-, or para- positions.

[0092] Page 8 of 47

[0093] QB\169852.00173\98287232.1 The term "heteroarvl" refers to an aromatic 5- to 10-membered ring structure, alternatively 6- to 10-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heteroaryl group can be specified using Cx-Cx nomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7 heteroaryl group refers to an aromatic 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The term "heteroaryl" includes monocyclic ring systems, and polycyclic ring systems having two or more heterocyclic rings in which two or more carbon or heteroatom are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is a heterocyclic aromatic group and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. Representative heteroaryl groups include pyrazolyl. pyridyl, quinolinyl, furanyl, thionyl, indolyl, and the like.

[0094] The term "pyridyl" refers to the radical . The term “3-pyridyl” refers to the radical

[0095] R /

[0096] The term "pyrazolyl" refers to the radical . The pyrazolyl radical may be optionally substituted with a substituent (e.g., an R group) as defined herein.

[0097] The term '‘optionally substituted” refers to zero, one or more carbon atoms in the group being independently substituted with one or more functional groups described herein.

[0098] The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term "stereoisomers" when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “E” or “Z”, "7?" or "S," or "+" or depending on the configuration of substituents around the stereogenic carbon atom and or the optical rotation observed. The present invention encompasses various stereo isomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated (±)" in nomenclature, but the skilled artisan will recognize that a structure may Page 9 of 47

[0099] QB\169852.00173\98287232.1 denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Also contemplated herein are compositions comprising, consisting essentially of, or consisting of an enantiopure compound, which composition may comprise, consist essentially of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g.. at least about 99% of an enantiomer of a given compound).

[0100] As used herein, "salt" refers to acid addition salts and basic addition salts. It may also refer to those salts that may be prepared in situ during the final isolation and purification of the compounds of the invention.

[0101] Examples of acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, malate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectinate, persulfate. 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, 72-toluenesul fonate and undecanoate. Also, the basic nitrogen-containing groups may be quatemized with such agents as lower alkyd halides such as, but not limited to, methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as, but not limited to, decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzy l and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid and such organic acids as acetic acid, fumaric acid, maleic acid, 4-methylbenzenesulfonic acid, succinic acid, and citric acid.

[0102] Basic addition salts may be prepared in situ during the final isolation and purification of compounds of this invention by reacting a carboxylic acid-containing moiety with a suitable base such as. but not limited to, the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as, but not limited to, lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine

[0103] Page 10 of 47

[0104] QB\169852.00173\98287232. 1 cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like. Other examples of organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

[0105] Compounds described herein may exist in unsolvated as well as solvated forms, including hydrated forms, such as hemi-hydrates. In general, the solvated fonns, with pharmaceutically acceptable solvents such as water and ethanol among others are equivalent to the unsolvated forms for the purposes of the invention.

[0106] Compounds

[0107] Disclosed herein includes a compound of formula (I) or formula (II), formula (II) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and wherein is an ethylene having E or Z conformation; wherein

[0108] X1is independently O or NRA;

[0109] RAis hydrogen or C1-C3 alkyd;

[0110] X2is C or N;

[0111] R1is hydrogen, naphthyl, phenyl optionally substituted with hydroxyl, or pyrazolyl optionally substituted with C1-C3 alky l optionally substituted with heterocycloalky l; and wherein

[0112] (i) X3is NRA, ring A is 3-pyridyl, R2is hydrogen, halogen, or phenyl; or

[0113] (ii) X3is O, ring A is 3-pyridyl or pyrazoly l optionally substituted with C1-C3 alkyl, R2is hydrogen; and with the proviso that:

[0114] X1and X2are not simultaneously O and C.

[0115] Page 11 of 47

[0116] QB\169852.00173\98287232.1 In some embodiments, the compound may be of formula (I). In some embodiments. X1may be NH. In some embodiments, X2is C. In some other embodiments. X2is N.

[0117] In some embodiments, the compound may be of formula (I). In some embodiments, X1may be O. In some embodiments, R1may be hydrogen, naphthyl, phenyl optionally substituted with hydroxyl, or pyrazolyl optionally substituted with C1-C3 alkyl; said C1-C3 alkyl may be optionally substituted with heterocycloalkyl. For example, in some embodiments, R1is hydrogen.

[0118] In some embodiments, the compound may be of formula (II). In some embodiments, X3may be NH and ring A may be 3-pyridyl. In some embodiment, R2may be halogen or phenyl.

[0119] In some embodiments, the compound may be

[0120] Page 12 of 47

[0121] QB\169852.00173\98287232. 1

[0122] solvate thereof.

[0123] In some embodiments, the compound may be or pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0124] In some embodiments, the compound may bind to SarA. In some embodiments, the compound may bind to MsaB. In some embodiments, the compound may bind to both SarA and MsaB. In some embodiments, binding of the compound to a protein may be evaluated by protein thermal shift assay. For example, Protein thermal assay using GloMelt™ dye may be used to determine the thermal stability of SarA and MsaB in the presence of the disclosed compound as a ligand. In some embodiments, analysis of melting temperature (Tm) may be performed using the Boltzmann method from a plot of fluorescence intensity vs. temperature and the derivative method (from a plot of d(fluorescence) / dT vs. temperature).

[0125] Page 13 of 47

[0126] QB\169852.00173\98287232.1 The disclosed compounds may exhibit one or more biological activities. In some embodiments, the disclosed compounds may inhibit the formation of biofilm. In some embodiments, the biofilm may comprise Staphylococcus aureus. In some embodiments, the disclosed compounds may inhibit biofilm formation by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than 250 pM, 200 pM, 150 pM, 125 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, 1 pM, 0.1 pM, 0.05 pM. 0.01 pM, 0.005 pM. 0.001 pM, or less, either alone or in combination with an additional antimicrobial agent. In some embodiments, the biofilm comprises MRSA, such as strain USA300 LAC. In some embodiments, the biofilm comprises MSSA, such as strain UAMS-1. While USA300 LAC and UAMS-1 are selected as model strains to assess the compounds described herein, the disclosed compounds may be effective against one or more strains of Staphylococcus aureus besides USA300 LAC and UAMS-1. The disclosed compounds may be effective against all, or substantially all, Staphylococcus aureus strains. Concentration ranges also are contemplated herein, for example, a concentration range bounded by end-point concentrations selected from 0.001 pM, 0.005 pM. 0.01 pM, 0.05 pM, 0.1 pM, 1.0 pM, 5.0 pM, 10 pM, 20 pM, 30 pM. 40 pM. 50 pM. 100 pM, 125 pM, 150 pM. 200 pM. and 250 pM.

[0127] In some embodiments, the disclosed compounds disrupt or eradicate established biofilm by reducing the growth of an established biofilm. In some embodiments, the biofilm may comprise Staphylococcus aureus. In some embodiments, the disclosed compounds may reduce the growth of an established biofilm by at least 50%. 60%. 70%. 80%. 90%. 95%, 96%, 97%, 98%, or 99% at a concentration of less than 250 pM, 200 pM, 150 pM, 125 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less, either alone or in combination with an additional antimicrobial agent. In some embodiments, the disclosed compounds may increase the amount of dead biofilm cells or decrease the amount of live biofilm cells by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than 250 pM, 200 pM, 150 pM, 125 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less, either alone or in combination with an additional antimicrobial agent. In some embodiments, the disclosed compound has an ICso value in inhibiting biofilm cells of less than 250 pM, 200 pM, 150 pM, 125 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less, either alone or in combination with an additional antimicrobial agent. In some embodiments, the additional antimicrobial agent comprises vancomycin. In some embodiments, the biofilm comprises

[0128] Page 14 of 47

[0129] QB\169852.00173\98287232. 1 MRSA, such as strain USA300 LAC. In some embodiments, the biofilm comprises MSSA, such as strain UAMS-1. While USA300 LAC and UAMS-1 are selected as model strains to assess the compounds described herein, the disclosed compounds may be effective against one or more strains of Staphylococcus aureus besides USA300 LAC and UAMS-1. The disclosed compounds may be effective against all, or substantially all, Staphylococcus aureus strains. Concentration ranges also are contemplated herein, for example, a concentration range bounded by end-point concentrations selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.05 pM. 0.1 pM, 1.0 pM, 5.0 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 100 pM, 125 pM, 150 pM, 200 pM, and 250 pM.

[0130] In some embodiments, the disclosed compounds reduce the growth of persister cells. In some embodiments, the persister cells may comprise Staphylococcus aureus. In some embodiments, the disclosed compounds may reduce the growth of persister cells by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than 250 pM, 200 pM, 150 pM, 125 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less, either alone or in combination with an additional antimicrobial agent. In some embodiments, the persister cells comprises MRSA. such as strain USA300 LAC. In some embodiments, the persister cells comprises MSSA, such as strain UAMS-1. While USA300 LAC and UAMS-1 are selected as model strains to assess the compounds described herein, the disclosed compounds may be effective against one or more strains of Staphylococcus aureus besides USA300 LAC and UAMS-1. The disclosed compounds may be effective against all, or substantially all. Staphylococcus aureus strains. Concentration ranges also are contemplated herein, for example, a concentration range bounded by end-point concentrations selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.05 pM, 0.1 pM, 1.0 pM, 5.0 pM, 10 pM, 20 pM, 30 pM. 40 pM, 50 pM, 100 pM, 125 pM. 150 pM, 200 pM, and 250 pM.

[0131] In some embodiments, the disclosed compound may be incorporated in a boneregenerating scaffold, such as bone filler matrices, while still retaining its biofilm inhibitory activity as described herein. In some embodiments, the compound released from the boneregenerating scaffold maintains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of its biofilm inhibitory activity for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, or 25 days post-loading.

[0132] Page 15 of 47

[0133] QB\169852.00173\98287232. 1 In some embodiments, the disclosed compound may achieve synergistic effects when administered in combination with one or more additional antimicrobial agents. For example, in some embodiments, the IC50 value in inhibiting biofilm cells of the compound disclosed herein decreases by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, or at least 300%, when administered in combination with one or more additional antimicrobial agents compared to when administered alone. In some embodiments, the additional antimicrobial agent comprises vancomycin.

[0134] Another aspect of the present invention provides a composition comprising the compound disclosed herein.

[0135] The composition may contain a solvent. Suitable solvents include, but are not limited to, a lactam (e.g., N-Methyl-2-pyrrolidone (NMP)), an organosulfur compound (e.g., dimethyl sulfoxide (DMSO)), and polyethylene glycol. The solvent may optionally contain a surfactant (e.g., polysorbate 80). In some embodiments, the solvent comprises N-Methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), polyethylene glycol, polysorbate 80 (e g., Tween® 80), ethanol, water, or a combination thereof.

[0136] The composition may contain a polymer. The polymer may form viscous gels with solvents such as DMSO and NMP and stabilizes hydrophobic compounds (e.g., drugs). The polymer may also provide bioadhesive properties, enhancing residence time on tissues and ensuring prolonged exposure of the compound. The polymer may be biocompatible. USP / NF compliant, and accepted in pharmaceutical applications. Suitable polymers include carbomers, such as Carbopol® polymers. Commercially available Carbopol® polymers include Carbopol® 980 NF, Carbopol® 971 NF, Carbopol® 974 NF, Carbopol® 1342 NF, Carbopol® 947P NF, and Carbopol® ETP 2020NF.

[0137] In some embodiments, the composition disclosed herein comprises a Carbopol® polymer, such as Carbopol® 980 NF or Carbopol® 971 NF, achieving a gel texture. The Carbopol®-based hydrogels may enable controlled and sustained local release of the compound at the infection site, maintaining therapeutic concentrations necessary for biofilm disruption while minimizing systemic toxicity.

[0138] In some embodiments, the compound in the composition and formulations disclosed herein retains its biofilm inhibitory activity as described herein. In some embodiments, the formulation inhibiting a biofilm may comprise a compound as disclosed herein (e.g. compound 917). a solvent (e.g.. NMP, DMSO), and a carbomer (e.g.. Carbopol 980 NF, Carbopol 971

[0139] Page 16 of 47

[0140] QB\169852.00173\98287232. 1 NF, Carbopol 974 NF, Carbopol 1342 NF. Carbopol 947P N, Carbopol ETP 2020NF). In some embodiments, the formulation inhibiting a biofilm may comprise a compound as disclosed herein, a solvent, and a polyethylene glycol (e.g. PEG400). In some embodiments, the formulation inhibiting a biofilm may comprise a compound as disclosed herein, a solvent, a polyethylene glycol, and a carbomer. In some embodiments, the formulation may comprise a compound as disclosed herein, a solvent, a polyethylene glycol, a carbomer, and surfactant (e.g.. polysorbate 80).

[0141] In some embodiments, the formulation inhibiting a biofilm may comprise between about 1.5% and about 3% solvent. In other embodiments, the formulation inhibiting a biofilm may comprise between about 1.5% and about 3% solvent.

[0142] In some embodiments, the formulation inhibiting a biofilm may comprise between about 0.4% and about 2% polymer (e.g., a carbomer). In other embodiments, the formulation inhibiting a biofilm may comprise between about 1.5% and about 3% solvent (e.g. NMP, DMSO). In further embodiments, the formulation inhibiting a biofilm may comprise between about 5% and about 50% polyethylene glycol. In other embodiments, the formulation inhibiting a biofilm may comprise about 10% ethanol and between about 37% to about 63% water. In some embodiments, the formulation inhibiting a biofilm may comprise about 1% surfactant (e.g., polysorbate 80). Exemplary' formulations are shown in Example 3, Table 3.

[0143] Pharmaceutical Compositions

[0144] Another aspect of the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises the compounds described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0145] The compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound

[0146] Page 17 of 47

[0147] QB\169852.00173\98287232. 1 chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.

[0148] In some embodiments, the compounds disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0. 1 to about 1000 mg / kg body weight (preferably about 0.5 to about 500 mg / kg body weight, more preferably about 50 to about 100 mg / kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.05 pM, 0.1 pM. 1.0 pM, 5.0 pM. 10 pM, 20 pM, 30 pM, 40 pM. 50 pM, 100 pM, 125 pM, 150 pM, 200 pM, and 250 pM.

[0149] It is understood by those skilled in the art that dosage amount will vary with the activity7of a particular inhibitor compound, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts. In general, a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect. If desired, an effective dose of such a compound, pharmaceutically acceptable salt thereof, or related composition may be administered in two or more sub-doses, administered separately over an appropriate period of time.

[0150] In some embodiments, a pharmaceutical composition comprising the compound of as disclosed herein and a pharmaceutically^ suitable carrier, diluent, or excipient is provided.

[0151] The pharmaceutical composition may include the compound in a range of about 0. 1 to 2000 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 1 to 100 mg. The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.5 to about 500 mg / kg body

[0152] Page 18 of 47

[0153] QB\169852.00173\98287232. 1 weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 50 to about 100 mg / kg body weight. In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, and 100 pM (e.g., 0.1 pM - 1.0 pM).

[0154] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0155] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0156] Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and

[0157] Page 19 of 47

[0158] QB\169852.00173\98287232. 1 Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0159] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, com starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

[0160] Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate. L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0161] The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0162] Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method know n in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0163] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0164] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.

[0165] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated

[0166] Page 20 of 47

[0167] QB\169852.00173\98287232. 1 dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

[0168] In some embodiments, the compound disclosed herein may be formulated for topical administration in Tegaderm™.

[0169] For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are in some embodiments applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

[0170] Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

[0171] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

[0172] Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g. , in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i. e. , by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

[0173] Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0174] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0175] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example

[0176] Page 21 of 47

[0177] QB\169852.00173\98287232. 1 water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0178] Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fdlers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch: or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well know n in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil. oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0179] Optionally, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders. Tn some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior to. concurrently with, or after administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds. In some embodiments, the disclosed pharmaceutical composition is formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders.

[0180] Methods of preparing pharmaceutical formulations or compositions include the step of bringing an inhibitor compound into association with a carrier and, optionally, one or more additional adjuvants or ingredients. For example, standard pharmaceutical formulation

[0181] Page 22 of 47

[0182] QB\169852.00173\98287232. 1 techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

[0183] Regardless of composition or formulation, those skilled in the art will recognize various avenues for medicament administration, together with corresponding factors and parameters to be considered in rendering such a medicament suitable for administration.

[0184] Methods

[0185] The disclosed compounds and pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treating a subj ect in need thereof. For example, in the methods of treating a subject in need thereof may include a subject having an infection.

[0186] Another aspect of the present invention provides a method for preventing or treating an infection in a subject. The method comprises administering a compound that binds to SarA, MsaB, or a combination thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof or a composition comprising the compound to the subject.

[0187] In some embodiments, the compound is the compound disclosed herein; and the composition is the pharmaceutical composition disclosed herein or the composition disclosed herein.

[0188] As used herein, the terms “treating'’ or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0189] A “subject in need thereof’ as utilized herein refers to a subject in need of treatment for an infection or disease or disorder associated with infection. In some embodiments, the infection may be associated with biofilm formation. In some embodiments, the subject may be suffering from or diagnosed with an infection or disease state in which treatment will inhibit or prevent biofilm formation, or disrupt / eradicate established biofilm. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. In some embodiments, the treated subject may be a mammalian subject, such as humans or other mammals. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs.

[0190] As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person.

[0191] Page 23 of 47

[0192] QB\169852.00173\98287232. 1 Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein.

[0193] In some embodiments, the infection may be a bacterial infection. In some embodiments, the bacteria include, but are not limited to. gram-positive bacteria, such as Staphylococci (e.g., Staph, aureus, Staph, epidermidis, Staph, saprophyticus, Staph, auricularis. Staph, capitis capitis, Staph, c. ureolyticus, Staph, caprae, Staph, cohnii cohnii, Staph, c. urealyticus, Staph, equorum, Staph, gallinarum, Staph, haemolyticus, Staph, hominis hominis, Staph, h. novobiosepticius. Staph, hyicus, Staph, intermedius, Staph, lugdunensis, Staph, pasteuri, Staph, saccharolyticus, Staph, schleiferi schleiferi, Staph, s. coagulans, Staph, sciuri. Staph, simulans, Staph, warneri and Staph, xylosus).

[0194] In some embodiments, the infection may be a Staphylococcus aureus infection. Infections, diseases, and disorders associated with Staphylococcus aureus may include, but are not limited to, bacteremia, infective endocarditis, skin and soft tissue infections (e.g., impetigo, folliculitis, furuncles, carbuncles, cellulitis, scalded skin syndrome, and others), osteomyelitis, septic arthritis, prosthetic device infections, pulmonary infections (e.g., pneumonia and empyema), gastroenteritis, meningitis, toxic shock syndrome, and urinary tract infections. For example, Staphylococcus aureus may cause infections involving bone and indwelling medical devices, such as orthopedic implants.

[0195] The methods disclosed herein may be applicable to different strains of Staphylococcus aureus. For example, in some embodiments, the infection may be a methicillin-resistant Staphylococcus aureus (MRSA) infection. In some embodiments, the infection may be a community-acquired MRSA (CA-MRSA) infection or a healthcare-acquired MRSA (HA- MRSA) infection. In some embodiments, MRSA infection comprises USA300 LAC. USA300 LAC was originally isolated from the Los Angeles County jail.

[0196] In some embodiments, the infection may be a methicillin-susceptible Staphylococcus aureus (MSSA) infection. In some embodiments, the infection may be associated with a clinical isolate. In some embodiments, the clinical isolate comprises strain UAMS-1. Strain UAMS1 was originally isolated from the bone of a patient suffering from osteomyelitis.

[0197] In some embodiments, the infection is associated with biofilm formation. In some embodiments, the biofilm comprises persister cells.

[0198] Page 24 of 47

[0199] QB\169852.00173\98287232. 1 In some embodiments, the infection may be chronic biofilm-associated infections, including but are not limited to osteomyelitis and non-healing wounds.

[0200] The compounds for use according to the methods of disclosed herein may be administered as a single compound or in combination with one or more additional agents. For example, a compound disclosed herein may be administered as a single compound or in combination with another agent that prevents or treats S. aureus associated infections, or that has a different pharmacological activity.

[0201] In some embodiments, the method comprises further administering an antimicrobial agent to the subject. A variety of antimicrobial agents may be included in the combination of the invention. For example, the antimicrobial agent may be antibiotic, an antifungal agent, an antiseptic, or a combination thereof.

[0202] In some embodiments, the antimicrobial agent may be an antibiotic. Non-limiting examples of suitable antibiotics include amino glycosides (such as, e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, and tobramycin); beta-lactams (i.e., penicillins such as amoxicillin, ampicillin, carbenicillin. cioxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V. piperacillin, and ticarcillin; cephalosporins such as cefadroxil cefazolin, cephalexin, cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, and ceftriaxone; carbecephems such as loracarbef; carbapenems such as certapenem. imipenem, and meropenem); glycopeptides (such as vancomycin, ramoplanin, teicoplanin, telavancin, bleomycin, ramoplanin, and decaplanin); macroglides (such as azithromycin, clarithromycin, dirithromycin, erythromycin, and troleandomycin; monobactam); polypeptides (such as actinomycin, bacitracin, colistin, and polymyxin B); quinolines (such as, ciprofloxacin, enoxacin. gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin); sulfonamides (such as co-trimoxazole, mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfis oxazole, trimethoprim, and trimethoprim-sulfamethoxazole); tetracyclines (such as demeclocy cline, doxycycline, minocycline, oxytetracycline, and tetracycline); and other antibiotics (e.g., rifamycins such as rifampin; lincosamides such as clindamycin and lincomycin; lipopeptides such as daptomycin; phenolics such as triclosan). In some embodiments, the antibiotic may be daptomycin, vancomycin, gentamicin, amphotericin B, clindamycin, dicloxicillin, minocycline, nafcillin, oxacillin, ramoplanin, rifampin, triclosan, or combinations thereof. In some embodiments, the method comprises further administering an antibiotic agent to the

[0203] Page 25 of 47

[0204] QB\169852.00173\98287232. 1 subject. In exemplary embodiments, the antibiotic may be daptomycin, vancomycin, or gentamicin.

[0205] In some embodiments, the antimicrobial agent may be an antifungal agent. Suitable antifungal agents include, without limit, allylamines (such as amorolfme, butenafme, naftifme, and terbinafme); antimetabolites (such as flucytosine); azoles (such as bifonazole, clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, ravuconazole, posaconazole, terconazole, and voriconazole); echinocandins (such as caspofungin. micafungin, and anidulafungin); mitotic inhibitors (such as griseofulvin); phenolics (such as triclosan); and polyenes (such as Amphotericin B, candicin, filipin, hamycin, natamycin, nystatin, and rimocidin).

[0206] In additional embodiments, the antimicrobial agent may be an antiseptic agent. Nonlimiting examples of suitable antiseptic agents include biguanides (such as alexidine, chlorhexidine, polyhexamethylbiguanide); dyes (such as genetian violet, methyl violet, methylene blue); metal ion salts or conjugates thereof (such as silver, silver sulfadiazone, zinc, copper, bismuth, gallium, iodine); phenolics (such as chloroxylenol, hexachlorophene, iodophene, triclosan. and thymol); and quaternary ammonium compounds (such as benzalkonium chloride (also called alkyldimethylbenzylammonium chloride), benzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium, cetrimide, didecyldimethylammonium chloride, dofanium chloride, domiphen bromide, methylbenzethonium chloride, tetraethylammonium bromide, and 3-(trimethyoxysilyl)-propyl dimethyl octadecyl ammonium chloride).

[0207] The concentration of the antimicrobial agent can and will vary depending upon the identity of the agent and the use of the combination. The concentration of the antimicrobial agent may range from very low levels (e.g., microorganism growth is not reduced to a substantial degree) to very high levels (i.e., it may exceed by 2- to 1000-fold the concentration of the agent that eradicates an equivalent free-floating population of the same microorganisms).

[0208] In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg. 10 mg, 12.5 mg, 15 mg. 17.5 mg. 20 mg. 22.5 mg. 25 mg. 27.5 mg. 30 mg. 32.5 mg, 35 mg. 37.5 mg, 40 mg. 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the

[0209] Page 26 of 47

[0210] QB\169852.00173\98287232. 1 subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg. 12.5 mg. 15 mg. 17.5 mg. 20 mg, 22.5 mg, 25 mg,

[0211] 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg,

[0212] 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and / or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg - 200 mg).

[0213] In some embodiments of the disclosed treatment methods, a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. In some embodiments, a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10. 20. 30. 40. 50. 60. 70. 80. 90. 100, 150. 200. 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. Minimal and / or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end-points any of these disclosed dose levels (e g, 500 - 2000 ng / kg body weight of the subject).

[0214] As used herein the term “effective amount'’ refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for treating an infection, such as a Staphylococcus aureus infection. The infection may be associated with biofilm formation.

[0215] An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the

[0216] Page 27 of 47

[0217] QB\169852.00173\98287232. 1 severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0218] A ty pical daily dose may contain from about 0.01 mg / kg to about 100 mg / kg (such as from about 0.05 mg / kg to about 50 mg / kg and / or from about 0. 1 mg / kg to about 25 mg / kg) of each compound used in the present method of treatment.

[0219] Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and / or about 25 mg. The term “unit dosage form” refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantify of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.

[0220] Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver.

[0221] The compounds and compositions disclosed herein may be administered in methods of treatment as known in the art. Accordingly, various such compounds and compositions can be administered in conjunction with such a method in any suitable way. For example, administration may comprise oral, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.

[0222] In some embodiments, the compound or the composition is administered through local delivery. In some embodiments, the compound or the composition may be administered topically. In some embodiments, the compound or the composition may be administered through injection. In some embodiments, the compound or the composition may be administered through an implantable system. In some embodiments, the compound or the composition may be administered through intraperitoneal administration.

[0223] Page 28 of 47

[0224] QB\169852.00173\98287232. 1 In some embodiments, the method comprises incorporating the compound or the composition in a bone-regenerating scaffold, such as bone fdler matrices.

[0225] Another aspect of the present invention provides a method for inhibiting biofilm formation or reducing the growth of an established biofilm on a surface. The method comprises contacting a compound that binds to SarA, MsaB, or a combination thereof, such as the compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; or the pharmaceutical composition disclosed herein with the surface.

[0226] In some embodiments, the biofilm comprises Staphylococcus aureus. In some embodiments, the biofilm may comprise MRSA, such as strain USA300 LAC. In some embodiments, the biofilm may comprise MSSA, such as strain UAMS-1.

[0227] In some embodiments, the biofilm comprises persister cells.

[0228] In some embodiments, the method comprises contacting an antibiotic agent with the surface. The antibiotic agent may be selected from daptomycin, vancomycin, and gentamicin.

[0229] The location of the biofilm grow th and / or formation can and will vary'. In some embodiments, the biofilm growth and / or formation may be on a surface or within a subject (e.g.. a veterinary subject, a health care patient, a health care worker, or a food production worker). For example, the microorganisms (or biofilm) may be on a squamous epithelial surface (e.g., skin surface), or a mucus membrane surface (e.g., nasal, respiratory', or alimentary' tract surfaces). Additionally, the microorganisms (or biofilm) may be on the surface of an internal organ or tissue of the subject, within an internal organ or tissue of the subject, or systemic to the subject. In other embodiments, the microorganisms (or biofilm) may be on a surface of an implanted medical device. In still further embodiments, the microorganisms (or biofilm) may be on or within a food product, or a piece of equipment used in the preparation of the food product, as detailed below.

[0230] In some embodiments, the biofilm growth and / or formation may be on a surface of an implantable medical device. For example, the implantable medical device may be a catheter. Non-limiting examples of suitable catheters include intravascular catheters (such as, e.g., arterial catheters, central venous catheters, hemodialysis catheters, peripheral and venous catheters), endovascular catheter microcoils, peritoneal dialy sis catheters, urethral catheters, and catheter access ports. In another embodiment, the implantable device may be a cardiac device. Suitable cardiac devices include, without limit, cardiac stents, defribrillators, heart valves, heart ventricular assist devices, OEM component devices, pacemakers, and pacemaker wire leads. In a further embodiment, the implantable medical device may be an orthopedic

[0231] Page 29 of 47

[0232] QB\169852.00173\98287232. 1 device. Non-limiting examples of suitable orthopedic devices include knee replacements, hip replacements, other joint replacements, spinal disc replacements, orthopedic pins, plates, screws, rods, and orthopedic OEM components. In yet other embodiments, suitable implantable medical devices include endotracheal tubes, nasogastric feeding tubes, gastric feeding tubes, synthetic bone grafts, bone cement, biosynthetic substitute skin, vascular grafts, surgical hernia mesh, embolic filter, ureter renal biliary’ stents, urethral slings, gastric bypass balloons, gastric pacemakers, insulin pumps, neurostimulators, penile implants, soft tissue silicone implants, intrauterine contraceptive devices, cochlear implants, and voice restoration devices.

[0233] In some embodiments, the biofilm growth and / or formation may on a surface or within a food product, or a piece of equipment used in the preparation of the food product. Nonlimiting examples of suitable food products include fresh or processed food. The food may be fresh, frozen, canned, dried, baked, fried, processed, fruit or fruit-based, vegetable or vegetable-based, grain or grain-based, cereal or cereal-based, nut or nut-based, dairy or dairybased, egg or egg-based, meat or meat-based, seafood or seafood-based, algae or algae-based, and so forth. Also included is any piece of equipment used in the preparation of a food product. Non-limiting examples of suitable equipment include washers, dryers, blenders, grinders, mixers, homogenizers, extruders, and the like.

[0234] Miscellaneous

[0235] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0236] As used herein, “about”, “approximately,” “substantially.” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0237] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of' should be interpreted as being “closed” transitional terms that do not permit the inclusion additional

[0238] Page 30 of 47

[0239] QB\169852.00173\98287232. 1 components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0240] The phrase “such as” should be interpreted as “for example, including.” Moreover the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0241] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g. , “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone. C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0242] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1 , 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0243] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0244] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate

[0245] Page 31 of 47

[0246] QB\169852.00173\98287232. 1 the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0247] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety’ herein.

[0248] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary’ skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0249] EXAMPLES

[0250] Example 1

[0251] An interdisciplinary program was established to target two key regulators of biofilm formation in S. aureus: MsaB and SarA. A small molecule, compound 6998, was discovered that binds both proteins, interferes with their DNA-binding properties, inhibits biofilm formation, and diminishes an established biofilm (FIGS. 1-4). Additional compounds with high docking scores for both MsaB and SarA were identified (Table 1). Compounds, such as 917, were synthesized to as a small molecule inhibitor of biofilm formation and biofilm- associated phenotypes such as formation of persister cells (Table 2 and FIG. 21). The thermal shift assay results in FIGS. 12, 13, and 15 demonstrate binding of some selected compounds with MsaB, SarA, or both. These compounds may be used alone or in combination with conventional antibiotics to overcome the therapeutic recalcitrance of biofilm-associated 5. aureus infections. FIGS. 5-6, 9-11, 14, 16-20 show biofilm inhibition and eradication results for exemplary compounds. In particular, compound 917 showed similar ICso for preventing biofilm formation compared to 6998. Compound 917 also showed increased biofilm disruption property compared to 6998. (FIGS. 5-7)

[0252] Page 32 of 47

[0253] QB\169852.00173\98287232. 1 Table 1. Docking scores for MsaB and SarA.

[0254] Table 2. Exemplary Compounds

[0255] Page 33 of 47

[0256] QB\169852.00173\98287232.1

[0257] Biofilm Formation Assay

[0258] Page 34 of 47

[0259] QB\169852.00173\98287232.1 Two different biofilm assays were performed, one measuring inhibition of biofdm formation (prophylactic use) and the other assessing disruption of an established biofdm (therapeutic use). A standard microtiter biofdm assay was used. The microtiter wells were first coated with 20% human plasma and then inoculated S. aureus normalized to ODeoo of 0.05 in biofdm medium (TSB supplemented with 0.5% glucose and 3% NaCl). For the inhibition assay, the compound was added at the time of inoculation with S', aureus cells. For the disruption assay, biofilms were first allowed to form in the absence of inhibitor, after which the medium was replaced with fresh biofdm medium containing the test compound. Biofdm biomass was quantified by standard crystal violet staining method. The half-maximal inhibitory concentration (IC50) for each analog was determined by testing a series of compound concentrations and performing dose-response curve analysis.

[0260] Protein Thermal Shift Assay

[0261] Protein thermal assay using GlomeltTM dye was used to determine the thermal stability of SarA and MsaB in the presence of test compounds as a ligand. Analysis of melting temperature (Tm) was performed using the Boltzmann method from a plot of fluorescence intensity vs. temperature and the derivative method (from a plot of d(fluorescence) / dT vs. temperature).

[0262] Cytotoxicity Assay

[0263] The cytotoxicity of 917 and its effects on mammalian cells was analyzed using RAW 267.4 cells (FIG. 8). Viability was assessed using a CyQUANT™ LDH Cytotoxicity’ Assay (Invitrogen) and LIVE / DEAD Viability / Cytotoxicity (Invitrogen). We confirmed that 917 does not exhibit any toxicity at a concentration of 200 uM, which is 20X of the IC50 value (10 uM) required to limit biofilm prevention (FIG. 2). Maximum LDH activity from the lysed cells of untreated controls samples was used to determine percent cytotoxity in the LDH cytotoxicity assay. Fluorescence at 530 nm (F(530)max) in untreated control samples labelled with calcein AM only, and fluorescence at 645 nm (F(645)max) in untreated control samples where all the cells are lysed and labelled with EthD-1 only were used to determine percent live cells, and percent dead cells respectively.

[0264] Synergy Testing by Checkerboard Biofilm Assay

[0265] Page 35 of 47

[0266] QB\169852.00173\98287232. 1 Synergy' between the compound and antibiotics against biofilms was evaluated using a checkerboard microdilution assay. Two-fold serial dilutions of the compound were combined with corresponding two-fold dilutions of antibiotics, ranging from inhibitory to sub-inhibitory concentrations, in 96-yvell plates. After incubation, biofilm formation was quantified, and the half-maximal inhibitory' concentration (ICso) of the compound yvas determined in the presence and absence of antibiotics. Sub-inhibitory antibiotic concentrations that reduced the ICso of the compound were considered to exhibit synergistic activity, yvhereas those that did not were classified as non-synergistic.

[0267] Confocal Microscopic Analysis of Biofilms

[0268] Biofilms were grown in 96-well Coming high-content imaging microplates in presence of 100 pM (lOxICso), 50 pM (5xICso), and 10 pM (IxICso) of compound for 24 h. Wells were yvashed three times yvith sterile PBS and stained with 50 pl of Syto-9 (1.3 pM) and Toto-3 (2.0 pM) prepared in Tris-buffer saline (50mM Tris, pH 7.5 + 0.9% NaCl). Images were acquired using a Zeiss 510 Meta confocal laser scanning microscope with a 60 x 1.4 oil DIC objective. Syto-9 was excited at 488 nm and detected at 515 ± 15 nm, while Toto-3 was excited at 633 nm and detected at 680 ± 30 nm. Z-stacks were collected at 1.0 pm intervals and processed using COMSTAT software to quantify total biomass, biofilm thickness, structural destabilization, dead cells, and extracellular DNA.

[0269] Example 2

[0270] FIG. 22 shows confocal Images of Biofilm Treatment with 917. S. aureus strain USA300 LAC was treated with 100 pM (10X ICso). 50 pM (5X IC50), and 10 pM (IX IC50) of 917. Biofilms were stained with SYTO-9 and Toto-9. Confocal Imaging and image analysis (ImageJ) showed that 917 significantly reduced biofilm formation, increased bacterial cell death within the biofilm, and disrupted biofilm structure, leading to a more fragmented and less developed biofilm. The effect of 917 is dose-dependent, with higher concentrations showing greater biofilm inhibition, structural destabilization, and increased bacterial cell death.

[0271] FIG. 23 shows the synergistic activity between 917 and Vancomycin. When combined yvith a sub-inhibitory' concentration of vancomycin, yvhich alone increased biofilm biomass to 200% of the untreated control, the ICso of 917 decreased to 5.52 pM from 9.2 pM. This result indicates synergistic activity between 917 and vancomycin. While vancomycin alone promotes

[0272] Page 36 of 47

[0273] QB\169852.00173\98287232. 1 biofilm at low doses, its combination with 917 significantly enhances the potency of 917, reducing the effective concentration needed for biofilm inhibition.

[0274] FIG. 24 shows the dose-response curve showing inhibition of S. aureus biofilm formation by 917 in Tegaderm™ formulation. Topical formulation of 917 in Tegaderm™ inhibited S. aureus biofilm development in vitro. 917 in Tegaderm™ formulation inhibited biofilm formation with an ICso value of 28.7 pM.

[0275] FIG. 25 shows the dose-response curve showing inhibition of S. aureus biofilm formation by 917 in intraperitoneal formulation (PEG / NMP / EtOH / ThO at 50 / 12 / 10 / 28 v / v). 917 Maintained its anti-biofilm activity when formulated for local delivery. 917 in IP Formulation inhibits biofilm formation with an IC50 value of 9.42 pM.

[0276] FIG. 26A shows the standard calibration curve generated to quantity’ compound 917, with absorbance measured at 515 nm. The calibration curve was linear over the concentration range of 6 to 400 pg / ml, with an R2value of 0.99929, indicating excellent linearity’. Boneregenerating scaffolds were incubated in 917 formulated in IP buffer for 2 hours. Based on the volume of formulation absorbed, each scaffold retained approximately 437.4 pg of 917.

[0277] Following loading, the scaffolds were placed in fresh IP buffer to allow passive elution of 917 over a period of 11 days. Each day, the IP buffer was collected to quantity the amount of 917 released and replaced with fresh buffer to continue the release study. The cumulative release of 917 over time was quantified using the standard curve and plotted to assess release kinetics. FIG. 26B shows the release kinetics of 917 from bone-regenerating scaffold over 11 days. This approach allows for incorporation of 917 in bone filler matrices.

[0278] The daily eluates of 917 were evaluated for their ability’ to prevent biofilm formation (FIG. 27). For these assays, one-fourth of the eluted concentration was used. The results demonstrated that 917 released up to day 8 retained significant anti-biofilm activity’. Eluates from days 1 to 4 showed a -90% reduction in biofilm formation, while eluates from days 5 to 8 continued to inhibit biofilm formation by over 50%. These findings indicate that 917 maintains potent biofilm-inhibitory activity’ for at least eight days post-loading, demonstrating the sustained in vitro release of 917 and biofilm prevention from bone-regenerating scaffold.

[0279] Compound 917 was also formulated in various Carbopol ©-based gels. Among the tested formulations, only Carbopol 980 NF (1% 917 + 3% NMP + 10% PEG400 + 1% w / w Carbopol 980 NF) and Carbopol 971 NF (1% 917 + 3% NMP + 10% PEG400 + 1% w / w Carbopol 971 NF) demonstrated favorable performance. Formulations prepared with these polymers — Formulation C 980NF and Formulation C 97 INF — were suspended in

[0280] Page 37 of 47

[0281] QB\169852.00173\98287232. 1 Simulated Wound Fluid (SWF), composed of 50% fetal bovine serum, l x PBS, and 5 mM dextrose. A release study was conducted over a period of two days, and the eluates were collected and tested for anti-biofilm activity (FIG. 28). The results showed that 917 released from both formulations significantly inhibited biofilm formation, achieving >80-90% reduction. These two formulations allow for administration of 917 in an injectable manner for local deliver}’ or topical application.

[0282] Formulating compound 917 in Carbopol® polymers offers several advantages for treating chronic biofilm-associated infections such as osteomyelitis and non-healing wounds. Carbopol®-based hydrogels enable controlled and sustained local release of 917 at the infection site, maintaining therapeutic concentrations necessary for biofilm disruption while minimizing systemic toxicity. These polymers also stabilize hydrophobic drugs like 917, which is only soluble in NMP or DMSO, and can form viscous gels compatible with such solvents. Their strong bioadhesive properties enhance residence time on tissues, ensuring prolonged exposure. Carbopol® products are biocompatible, USP / NF compliant, and widely accepted in pharmaceutical applications, making them suitable for regulatory development. Moreover, the viscosity and texture of Carbopol® gels can be tuned for various applications — from topical wound coverage to injectable or implantable systems for bone infections — while allowing for incorporation of co-therapies like vancomycin or gentamicin.

[0283] Example 3

[0284] Exemplary formulations may be prepared with components according to Table 3.

[0285] Table 3. Formulations with compound 917 that exhibit anti-biofilm activity.

[0286] Page 38 of 47

[0287] QB\169852.00173\98287232. 1

[0288] Page 39 of 47

[0289] QB\169852.00173\98287232. 1

Claims

CLAIMSI / We claim:

1. A compound of formula (I) or formula (II),or a pharmaceutically acceptable salt, hydrate, or solvate thereof, whereinX is an ethylene having E or Z conformation; and whereinXIis independently O or NRA;RAis hydrogen or C1-C3 alkyd;X2is C orN;R1is hydrogen, naphthyl, phenyl optionally substituted with hydroxyl, or pyrazolyl optionally substituted with C1-C3 alkyl optionally substituted with heterocycloalkyl; and wherein(i) X3is NRA, ring A is 3-pyridyl, R2is hydrogen, halogen, or phenyl; or(ii) X3is O, ring A is 3-pyridyl or pyrazolyl optionally substituted with C1-C3 alkyl, R2is hydrogen; and with the proviso that:X1and X2are not simultaneously O and C.

2. The compound of claim 1, wherein the compound is of formula (I).

3. The compound of claim 2, wherein X1is NH.

4. The compound of claim 3, wherein X2is C.Page 40 of 47QB\169852.00173\98287232.

15. The compound of claim 3, wherein X2is N.

6. The compound of claim 2, wherein X1is O.

7. The compound of any one of claims 1-6, wherein R1is hydrogen, naphthyl, phenyl optionally substituted with hydroxyl, or pyrazolyl optionally substituted with C1-C3 alkyl optionally substituted with heterocycloalkyl.

8. The compound of claim 7, wherein R1is hydrogen.

9. The compound of claim 1, wherein the compound is of formula (II).

10. The compound of claim 9, wherein X3is NH and ring A is 3-pyridyl.

11. The compound of claim 9 or claim 10. wherein R2is halogen or phenyl.

12. The compound of claim 1, wherein the compound isPage 41 of 47QB\169852.00173\98287232. 1QB\169852.00173\98287232. 1or pharmaceutically acceptable salt, hydrate, or solvate thereof.

13. The compound of claim 1, wherein the compound isor pharmaceutically acceptable salt, hydrate, or solvate thereof.

14. The compound of any one of claims 1-13, wherein the compound binds to SarA, MsaB, or a combination thereof.

15. A pharmaceutical composition comprising the compound according to any one of claims 1-14 and a pharmaceutically acceptable excipient, carrier, or diluent.Page 43 of 47QB\169852.00173\98287232.

116. A composition comprising the compound according to any one of claims 1-14, a solvent, and a polymer.

17. The composition of claim 16, wherein the solvent comprises N-Methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polysorbate 80, ethanol, water, or a combination thereof.

18. The composition of claim 16, wherein the polymer comprises a carbomer.

19. A method of preventing or treating an infection in a subject in need thereof, the method comprising administering a compound that binds to SarA, MsaB. or a combination thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof or a composition comprising the compound to the subject.

20. The method of claim 15, wherein the compound is the compound of any one of claims 1-14; and the composition is the pharmaceutical composition of claim 15, or the composition of any one of claims 16-18.

21. The method of claim 19 or 20, wherein the infection is a Staphylococcus aureus infection.

22. The method of any one of claims 19-21, wherein the infection is a methicillin- resistant Staphylococcus aureus (MRSA) infection.

23. The method of claim 22, wherein the MRSA infection is a USA300 LAC infection.

24. The method of any one of claims 19-21, wherein the infection is a methicillin- susceptible Staphylococcus aureus (MS SA) infection.

25. The method of claim 24, wherein the methicillin-susceptible Staphylococcus aureus (MSSA) infection is a UAMS-1 infection.Page 44 of 47QB\169852.00173\98287232.

126. The method of any one of claims 19-25, wherein the infection is associated with biofilm formation.

27. The method of claim 26, wherein the biofilm comprises persister cells.

28. The method of any one of claims 19-27, wherein the method comprises further administering an antibiotic agent to the subject.

29. The method of claim 28, wherein the antibiotic agent is selected from daptomycin, vancomycin, and gentamicin.

30. The method of any one of claims 19-29, wherein the compound or the composition is administered through local delivery.

31. The method of any one of claims 19-30, wherein the compound or the composition is administered topically.

32. The method of any one of claims 19-30, wherein the compound or the composition is administered through injection.

33. The method of any one of claims 19-30, wherein the compound or the composition is administered through an implantable system.

34. The method of any one of claims 19-30, wherein the compound or the composition is administered through intraperitoneal administration.

35. The method of any one of claims 19-29, comprising incorporating the compound or the composition in a bone-regenerating scaffold.

36. A method of inhibiting biofilm formation or reducing the growth of an established biofilm on a surface, the method comprising contacting a compound that binds to SarA, MsaB, or a combination thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof or a pharmaceutical composition comprising the compound to the surface.Page 45 of 47QB\169852.00173\98287232.

137. The method of claim 36, wherein the compound is the compound of any one of claims 1-14, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

38. The method of claim 36 or 37, wherein the biofilm comprises Staphylococcus aureus.

39. The method of any one of claims 36-38, wherein the biofilm comprises MRSA.

40. The method of claim 39, wherein the MRSA comprises USA300 LAC.

41. The method of any one of claims 36-38, wherein the biofilm comprises MSSA.

42. The method of claim 41, wherein the MSSA comprises UAMS-1.

43. The method of any one of claims 36-42, wherein the biofilm comprises persister cells.

44. The method of any one of claims 36-43, wherein the method comprises contacting an antibiotic agent with the surface.

45. The method of claim 44. wherein the antibiotic agent is selected from daptomycin, vancomycin, and gentamicin.

46. The method of any one of claims 19-45, wherein the compound is selected fromPage 46 of 47QB\169852.00173\98287232.1

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