WO2025264672A1 - Inhibiteurs de l'enzyme de résistance acquise à la colistine (mcr-1) et leurs procédés d'utilisation - Google Patents

Inhibiteurs de l'enzyme de résistance acquise à la colistine (mcr-1) et leurs procédés d'utilisation

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Publication number
WO2025264672A1
WO2025264672A1 PCT/US2025/033970 US2025033970W WO2025264672A1 WO 2025264672 A1 WO2025264672 A1 WO 2025264672A1 US 2025033970 W US2025033970 W US 2025033970W WO 2025264672 A1 WO2025264672 A1 WO 2025264672A1
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methyl
chlorophenyl
biphenyl
cdd
group
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Inventor
Timothy Palzkill
Martin Matzuk
Srinivas CHAMAKURI
Isabel MATERON
Moh SATTAR
Zhizeng SUN
Sreenivasa Ramana
Nicholas MSZAR
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Baylor College of Medicine
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Baylor College of Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C

Definitions

  • Polymyxins are cationic polypeptides that act by binding to the lipid A moiety of bacterial lipopolysaccharide (LPS) and subsequently disrupting the bacterial membrane.
  • LPS bacterial lipopolysaccharide
  • Acquired and chromosome-encoded resistance to colistin has been reported among Gram-negative bacteria and some species, such as Neisseria spp., are intrinsically resistant to colistin.
  • the most common mechanism of acquired resistance involves modification of the LPS component of the outer membrane. Specifically, resistance occurs due to modification of the 1 and 4’ phosphate groups of lipid A to neutralize the negative charge and reduce binding of the positively charged colistin.
  • the phosphates are modified with 4-amino-arabinose by the aminoarabinose transferase ArnT or by addition of phosphoethanolamine (PEA) by PEA transferase enzymes.
  • PEA phosphoethanolamine
  • Chromosome encoded and acquired resistance to polymyxins is associated with mutations found in genes for two-component regulatory systems and result in - 1 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) expression of the transferase enzymes that modify LPS.
  • ArnT is a membrane protein with a periplasmic domain and is a glycosyltransferase.
  • the PEA transferases also have a membrane-spanning domain and a periplasmic catalytic domain.
  • the catalytic domain of the NmEptA and EptC PEA transferases have a similar structure and are members of the sulfatase group with a fold similar to alkaline phosphatase.
  • MCR-1 plasmid- encoded LPS-modifying enzyme
  • the disclosure provides a compound of Formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof, wherein R 1 , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , L 1 , L 2 , and X 1 are defined elsewhere herein: .
  • composition comprising at least one compound of the carrier or excipient.
  • disclosure provides a method of treating, preventing, and/or ameliorating a bacterial infection in a subject.
  • the method comprises administering to the subject at least one compound of the disclosure, or a pharmaceutical composition thereof, and at least one polymyxin antibiotic, or an analogue or derivative thereof.
  • FIG.1 shows the structure of lipid A of E. coli showing reaction catalyzed by PEA transferases such as MCR-1.
  • FIGs.2A-2B show the structure of NmEptA solved by X-ray crystallography.
  • FIG 2A shows a ribbon diagram of NmEptA.
  • Dodecyl- ⁇ -D-maltoside (DDM) shown as spheres.
  • FIG. 2B shows the active site with DDM. Key residues are labeled.
  • the catalytic zinc is shown as a sphere.
  • Helices PH2 and PH2’ are at the border of the active site and membrane domain.
  • FIG.3 shows the DECL process.
  • FIG.4 shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) of purified fractions of full length MCR-1 (62 kDa).
  • FIG.5A shows schematic of enzyme-catalyzed removal of phosphoethanolamine - 3 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) from fluorescently labeled phosphotidylethanolamine substrate (NDB-PEA) to create product NDB-DG.
  • FIG.5B shows thin-layer chromatography (TLC) of reaction products. Positions of NBD-PEA and NDB-DG are labeled. CT is a no enzyme control TLC.
  • FIG.6 shows compounds initially identified compounds CDD-1794 and CDD-1938.
  • FIGs.7A-7F show spot test for E. coli growth with colistin and CDD-1938. Overnight (O/N) cultures of E.
  • FIG.7B shows 25 ⁇ M CDD-1938 with no colistin.
  • FIG.7C shows .0.8 ⁇ g/ml colistin, no CDD-1938.
  • FIG.7D shows 0.8 ⁇ g/ml colistin with 3.1 ⁇ M CDD-1938.
  • FIG.7E shows 0.8 ⁇ g/ml colistin with 6.3 ⁇ M CDD-1938.
  • FIG.7F shows 0.8 ⁇ g/ml colistin with 12.5 ⁇ M CDD-1938.
  • FIGs.8A-8B docking results of CDD-1794 and CDD-1938 to MCR-1 homology model.
  • FIG.8A shows CDD-1794 docked in MCR-1 model.
  • CDD-1794 is in a pocket adjacent to the active site Zn and catalytic Thr285.
  • FIG.8B shows CDD-1938 docked in MCR-1 homology model. Without being bound by theory, CDD-1938 is postulated to occupy the same site as CDD-1794.
  • FIG.9 shows structure-activity relationships for CDD-1938. Schematic of the DECL hit based on DNA sequencing. The linker, cycle 1, building block position (C1). Cycle 2 (C2) and 3 (C3) building block positions are shown.
  • B-M are molecular analogs of CDD-1938.
  • FIG.10 shows colistin MIC values for E. coli expressing MCR-1 in the presence of inhibitor compounds of the disclosure. MICs shown on Y-axis in log2 scale. Compound concentrations indicated on X-axis. Compounds are indicated in the inset.
  • FIG.11 shows spot test assay for CDD compounds against MCR-1 mediated colistin resistant clinical isolates. The top row shows the spot test for agar plates containing 0.8 ⁇ g/ml colistin.
  • the bottom two rows show spot test results using agar plates containing 0.8 mg/ml colistin and 12.5 ⁇ M CDD compound. Each strain was tested in duplicate. The spots on each plate are a non-diluted overnight culture on the far right and 10-fold serial dilutions proceeding right to left. The strains corresponding to each row of spots are labeled. WT-1 and WT-2 represent the duplicates of E. coli XL1-Blue containing MCR-1. The clinical strains and duplicates are labeled. Each column of plates corresponds to the compound tested as indicated below the columns. The columns are arranged from most potent compound on the right to least potent on the left.
  • FIG.12 shows structure-activity relationships for analogs based on the high potency - 4 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) CDD-2901 and CDD-2847 compounds. Compound names and potency based on comparison to colistin alone in the spot assay are shown. Cytotoxicity as indicated by IC 50 for HepG2 cells is shown for select compounds.
  • FIG.13 shows a summary of structure-activity relationships based on analogs of CDD-1794.
  • FIG.14 Catalytically functional MCR-1 is necessary for protection against colistin in E. coli. Time-kill curve of E.
  • FIGs.15A-15D Structures of DNA-encoded chemical library (DECL) hits and analogs with activity against MCR-1. Activity of the compounds with or without colistin against E. coli/WT MCR-1 growth was tested using the spot assay and minimum inhibitory concentration (MIC) method, respective values determined at 12.5 ⁇ g/mL compound concentration are indicated under each compound structure. For the spot assay method, colistin was set at 0.32 ⁇ g/mL and scored based on the fold difference (X) between growth with colistin alone vs colistin/compound combination; while for MIC, colistin was tested from 0-32 ⁇ g/mL and 12.5 ⁇ g/mL compound.
  • MIC minimum inhibitory concentration
  • FIG.15A Structure of DECL screening hit CDD-1938. This racemic mixture was identified through affinity-based selection of a DECL of 2 million compounds against purified WT MCR-1 His-tagged enzyme.
  • FIG.15B Structure of DECL screening hit CDD-3358. This compound was also identified through affinity-based selection of a DECL of 2 million compounds against purified WT MCR-1 enzyme, and a truncated version of the CDD-3358 compound, CDD-1794, was synthesized and tested due to difficulties in resynthesizing CDD-3358.
  • FIG.15C Structures of potent inhibitor analogs derived from the original DECL hit, CDD-1938.
  • FIG.15D Structure of a potent inhibitor analog derived from the original DECL truncated hit, CDD-1794. This lead inhibitor evolved from a series of analogs that were - 5 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) optimized by modifying the C1, C2 and C3 building blocks to enhance bioactivity of CDD- 1794.
  • FIGs.16A-16B MCR-1 protects E.
  • FIG.16A DNA- encoded chemical library (DECL) screening hits block the catalytic activity of MCR-1 and resensitize bacteria to colistin killing. Spot assay experiments for E. coli containing wild- type MCR-1 (1) and T285A MCR-1 (2) expressed from a plasmid also conferring chloramphenicol (Cm) resistance were performed. E.
  • coli strains were grown overnight in LB media containing Cm (12.5 ⁇ g/mL), normalized, diluted (10-1 through 10-6) and spotted on LB media containing Cm (12.5 ⁇ g/mL): media control, or LB + Cm with a sub-inhibitory concentration of colistin sodium methane sulfonate (0.32 ⁇ g/mL), or with a high (12.5 ⁇ M) concentration of inhibitor only and with both 0.32 ⁇ g/mL colistin and low (3.125 ⁇ M), medium (6.25 ⁇ M) or high (12.5 ⁇ M) concentrations of inhibitors CDD-1938 and CDD- 1794. All inhibitor only controls were similar to the representative inhibitor, CDD-1938, and media only controls shown in the chart.
  • FIG.17 Optimized inhibitors show improved potency against MCR-1 and resensitize bacteria to colistin killing. Spot assay experiments for E.
  • E. coli containing wild-type MCR-1 (1) and T285A MCR-1 (2) expressed from a plasmid also conferring chloramphenicol (Cm) resistance were performed.
  • E. coli strains were grown overnight in LB media containing Cm (12.5 ⁇ g/mL), normalized, diluted (10-1 through 10-6) and spotted on LB media containing Cm (12.5 ⁇ g/mL): media control, or LB + Cm with a sub-inhibitory concentration of colistin sodium methane sulfonate (0.32 ⁇ g/mL), or with a high (12.5 ⁇ M) concentration of inhibitor only and with both 0.32 ⁇ g/mL colistin and high (12.5 ⁇ M) concentrations of inhibitors: CDD-2750, CDD-2847, CDD-3002, CDD-2902, CDD-3003, and CDD-3356.
  • FIGs.18A-18B Colistin MIC values for E. coli expressing MCR-1 in the absence and presence of inhibitor compounds.
  • FIG.18A Colistin sulfate MIC values in the absence and presence of inhibitor compounds from screening hits and their optimized analogs for E.
  • FIG. 18B A graphic representation of the MICs shown on the Y-axis in log2 scale and compound concentrations are indicated on the X-axis. Compounds tested are indicated in the inset. Error bars represent standard error mean.
  • FIG.19 MCR-1 inhibitors are active against Enterobacterales clinical isolates containing MCR-1 and MCR-2.
  • FIGs.20A-20B Time-kill curve for E.
  • FIG.20A Time-kill analysis of various treatments with inhibitor CDD-3003 as described in the inset.
  • FIG.20B Time-kill analysis of various treatments with inhibitor CDD-3356 as described in the inset.
  • Unidirectional error bars for clarity, represent the standard error mean.
  • FIGs.21A-21C Comparison of MCR-1 and its variants using AlphaFold2.
  • FIG.22 Catalytically functional MCR-1 is necessary for protection against colistin in E. coli. Time-kill curve of E.
  • FIG.23 Optimized inhibitor shows activity against select mcr-1+ E. coli clinical isolates and partially resensitizes these bacteria to colistin killing. Spot assay experiments for laboratory E. coli containing wild-type MCR-1 (1) and T285A MCR-1 (2) expressed from a plasmid also conferring chloramphenicol (Cm) resistance as controls and mcr-1+ E. coli clinical isolates were performed. E. coli clinical isolates: #107 (3), #109 (4), #240 (5), #6770 (6), #9824 (7), #9991 (8) and #10618 (9) were obtained.
  • the laboratory strains were grown overnight in LB media containing Cm (12.5 ⁇ g/mL) and E. coli clinical isolates were grown in LB media; all strains were normalized, diluted (10-1 through 10-6) and spotted on LB agar containing Cm (12.5 ⁇ g/mL) or LB agar as a media control, or LB + Cm with a sub- inhibitory concentration of colistin sodium methane sulfonate (0.32 ⁇ g/mL), or with a high (12.5 ⁇ M) concentration of inhibitor only and with both 0.32 ⁇ g/mL colistin and high (12.5 ⁇ M) concentrations of inhibitors, CDD-2750. All media only and inhibitor only controls were similar among the clinical isolates shown in the chart.
  • FIGs.24A-24B Time-kill curve analysis for E. coli expressing WT MCR-1 against 4 ⁇ g/mL colistin sulfate (CS) in the presence and absence of the racemic mixture, CDD-3366, and its S-enantiomer, CDD-3356.
  • FIG.24A Various concentrations of racemic mixture, CDD-3366, combined with 4 ⁇ g/mL CS were tested against E.
  • FIG.24B Various concentrations of S-enantiomer, CDD-3356, combined with 4 - 8 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) ⁇ g/mLCS were tested against E. coli/WT MCR-1 in CAMH + Cm (12.5 ⁇ g/mL) grown at 37°C for 4.5 hours. Data represents one independent experiment performed in duplicate. Unidirectional error bars, for clarity, represent the standard error mean.
  • FIGs.25A-25B Structural comparison of MCR-1, NmEptA and PmrC.
  • FIG.25B Superposition of the Alphafold2 predicted full-length MCR-1 from E. coli and PmrC from A.
  • FIG.26 Analog series of chemotype 1, CDD-1938, showing varied bioactivity. Spot assay score defines the difference in growth between colistin-only treatment vs colistin/MCR-1 inhibitor treatment; it is described as a fold difference. Data shown here are representative of multiple (2-4) experiments. Where there are ranges, both values were identified several times in multiple experiments.
  • FIG.27 Analog series of chemotype 2, CDD-1794, showing varied bioactivity.
  • a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) - 9 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range.
  • the statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise.
  • Polymyxin E also known as colistin
  • polymyxin B are the most studied and clinically used forms. They act predominantly by targeting the anionic lipid A component of the lipopolysaccharide (LPS) present in the outer membrane (OM) of Gram- negative bacteria causing membrane destabilization, OM and inner membrane (IM) permeability, leakage of cellular contents, and ultimately cell death.
  • LPS lipopolysaccharide
  • OM outer membrane
  • IM inner membrane
  • colistin resistance has already been identified in carbapenemase-producing, multidrug-resistant (MDR) or extensively drug-resistant (XDR) Gram-negative bacteria on the CDC and WHO priority threat lists. Colistin resistance is primarily mediated through a reduction of the electrostatic attraction between colistin and the Gram-negative OM.
  • intrinsic polymyxin resistance is associated with the constitutive expression of chromosomal genes encoding transferase enzymes, such as ArnT, EptA (also known as PmrC), and EptC.
  • transferase enzymes such as ArnT, EptA (also known as PmrC), and EptC.
  • Aminoarabinose transferase, ArnT modifies the 1’- and 4’- anionic phosphate groups of the glucosamine moieties of lipid A in LPS with cationic 4- amino-4-deoxy-L-arabinose (L-Ara4N) and the phosphoethanolamine (pEtN) transferase, EptA, modifies the phosphate with a cationic pEtN.
  • mcr-1 an ortholog of eptA (pmrC)
  • pmrC ortholog of eptA
  • pEtN transferase confers colistin resistance through the addition of pEtN from phosphatidylethanolamine, a naturally occurring phospholipid, to the 1’- or 4’- phosphate group of lipid A component of LPS as it is trafficked through the IM on the way to the OM.
  • MDR multidrug-resistant
  • mcr-1 + strains and restore colistin efficacy such as CRISPR/CAS-based tools to eliminate mcr-1 strains, mcr-1 plasmid conjugation inhibitors, MCR inhibitors, novel polymyxin derivatives, combination therapy using various antibacterial agents and non-antibiotic compounds.
  • CRISPR/CAS-based tools to eliminate mcr-1 strains, mcr-1 plasmid conjugation inhibitors, MCR inhibitors, novel polymyxin derivatives, combination therapy using various antibacterial agents and non-antibiotic compounds.
  • Described herein is an approach to extend the efficacy of colistin using combination therapy with a synergizing bioactive partner, in this case, an inhibitor of the important resistance determinant MCR-1.
  • a synergizing bioactive partner in this case, an inhibitor of the important resistance determinant MCR-1.
  • MCR-1 the important resistance determinant
  • a DNA-encoded chemical libraries DECL
  • DECLs consist of very large collections of compounds, each coupled to distinctive DNA tags serving as amplifiable identification barcodes, which permit the retrieval and identification of binding compounds after screening.
  • the colistin/inhibitor combination operates through self-promoted uptake of colistin as previously described, facilitating the entry of the lipophilic inhibitor across the OM and into the periplasm where it reaches the enzyme target, MCR-1, located in the IM.
  • MCR-1 has been shown to protect the IM and confer resistance to polymyxins by modifying high levels of LPS with pEtN at the IM, thus repelling colistin from the IM.
  • the inhibitor blocks MCR-1 activity allowing colistin to then, permeabilize the IM, trigger bacterial lysis and cause death.
  • the anti-infective efficacy of the two antibiotic/inhibitor - 13 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) combinations was also assessed using TKCs, where the rate of bactericidal killing was followed over a range of inhibitor concentrations and fixed colistin concentration.
  • the TKC data for E. coli/MCR-1 indicate that both chemotypes, at their most effective concentrations (12.5 ⁇ M), in combination with colistin, achieve complete bacterial killing by as early as 1.5 hours (CDD-3356/colistin) or 1 hour (CDD-3003/colistin) upon administration, which is up to 2.5 hours or 4 hours earlier to killing with colistin alone (FIGs.20A-20B).
  • CDD-3356 and CDD-3003 both display a dose-dependent behavior, although it is more pronounced for CDD-3356.
  • the best effective inhibitor concentrations, 6.25 and 12.5 ⁇ M, combined with colistin are close to the goal to develop small-molecule compounds that inhibit MCR-1 in the 1 ⁇ g/mL (2 ⁇ M) range and effectively kill E. coli/MCR-1.
  • the antibacterial activity of these compounds also extends to MDR mcr-1+ K. pneumoniae clinical isolates by reducing growth by 16-fold (CDD-3356) or by 8-fold (CDD-3003), as measured by the microdilution method (Table 9).
  • Inhibitors within the CDD-3003 chemotype series combined with colistin although cytotoxic, also demonstrated inhibitory growth effects ranging from 10 2 -10 5 -fold against mcr-2+ E. coli and multiple mcr- 1+ E. coli clinical strains and, at best, 10-fold against mcr-1+ K. pneumoniae compared to colistin alone as measured using the spot assay (FIG.19).
  • the lower range observed in MIC and spot assay values for the colistin/inhibitor combination among some of the clinical isolates tested, particularly K. pneumoniae may be due to poor compound penetration.
  • acyl refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom.
  • the carbonyl carbon atom is bonded to a hydrogen forming a "formyl” group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like.
  • An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group.
  • An acyl group can include double or triple bonds within the meaning herein.
  • An acryloyl group is an example of an acyl group.
  • An acyl group can also include heteroatoms within the meaning herein.
  • a nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein.
  • Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like.
  • alkenyl refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms.
  • alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms.
  • alkoxy refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein.
  • An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple - 15 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) bonds, and can also include heteroatoms.
  • an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.
  • alkyl refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms.
  • straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.
  • branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
  • alkyl encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl.
  • Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • alkynyl refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms.
  • alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to – C ⁇ CH, -C ⁇ C(CH3), -C ⁇ C(CH2CH3), -CH2C ⁇ CH, -CH2C ⁇ C(CH3), and -CH2C ⁇ C(CH2CH3) among others.
  • antibiotic refers to a compound or composition that kills or reduces the viability of a microorganism or inhibits the growth or proliferation of a microorganism.
  • amine refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like.
  • amino group refers to a substituent of the form -NH2, - NHR, -NR 2 , -NR 3 + , wherein each R is independently selected, and protonated forms of each, - 16 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) except for -NR3 + , which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine.
  • An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group.
  • An "alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.
  • aralkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
  • Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.
  • Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
  • aryl refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring.
  • Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.
  • the term “bacteria”, as used herein, refers to both prokaryotic and archaebacteria cellular organisms. “Bacteria” are unicellular microorganisms which have cell walls but lack organelles and an organized nucleus, including some that can cause disease.
  • bacterial colony refers to growths of bacteria accumulated in distinct segments on solid materials or surfaces including agar plates, which in some cases includes cells from bacterial infection of a subject or host. A bacterial colony is derived from a common mother cell.
  • biofilm refers to a chemical matrix produced to protect a bacteria or microorganism population. Biofilms are generally characterized as adhering to a surface and having a thin, slimy texture. Biofilms may grow in or on medical devices or prosthetic joints, or at other sites in tissue and organs.
  • composition or “pharmaceutical composition” refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a - 17 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) patient or subject.
  • Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • cycloalkyl refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • the terms "effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result.
  • haloalkyl group includes mono-halo alkyl groups, poly- halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl - 18 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro.
  • haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like.
  • heteroaryl refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members.
  • a heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure.
  • a heteroaryl group designated as a C 2 -heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.
  • a C 4 -heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth.
  • Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolin
  • heteroarylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.
  • heterocyclylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein.
  • heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.
  • heterocyclyl refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S.
  • a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof.
  • heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members.
  • a heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two - 20 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.
  • a C 4 -heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms.
  • a heterocyclyl ring can also include one or more double bonds.
  • a heteroaryl ring is an embodiment of a heterocyclyl group.
  • the phrase "heterocyclyl group" includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein.
  • the phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein.
  • Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquino
  • substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein.
  • hydrocarbon or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups.
  • (C 1 -C 4 )hydrocarbyl means the hydrocarbyl group can be methyl (C 1 ), ethyl (C2), propyl (C3), or butyl (C4), and (C0-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group.
  • the term "independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise.
  • the phrase "X 1 , X 2 , and X 3 are independently selected from noble - 21 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) gases" would include the scenario where, for example, X 1 , X 2 , and X 3 are all the same, where X 1 , X 2 , and X 3 are all different, where X 1 and X 2 are the same but X 3 is different, and other analogous permutations.
  • the term “infection” as used herein refers to the onset of a disease by one or more pathogenic bacteria, including but not limited to Gram negative bacteria.
  • the term "monovalent” as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.
  • organic group refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a human.
  • pharmaceutically acceptable refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate).
  • Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic,
  • Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
  • the term "pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function.
  • Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injuri
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and - 23 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydro
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions.
  • the "pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound(s) described herein.
  • Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
  • the term “prosthesis” as used herein refers to an artificial body part.
  • room temperature refers to a temperature of about 15 °C to 28 °C.
  • solvent refers to a liquid that can dissolve a solid, liquid, or gas.
  • Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.
  • substantially refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%.
  • substantially free of can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less.
  • substantially free of can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.
  • substituted as used herein in conjunction with a molecule or an organic - 24 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms.
  • functional group or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group.
  • substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups.
  • a halogen e.g., F, Cl, Br, and I
  • an oxygen atom in groups such as hydroxy groups, al
  • Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R) 2 , CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R) 2 , SR, SOR, SO 2 R, SO 2 N(R) 2 , SO 3 R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0- 2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
  • treatment or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound or compounds as described herein (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein or a symptom of a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, or the symptoms of a condition contemplated herein.
  • the compound of Formula (I) is a compound of Formula (Ia): of is a compound of certain embodiments, the compound of Formula (I) is a compound of In certain embodiments, the compound of Formula (I) is a compound of In certain embodiments, the compound of Formula (I) is a compound of Formula (If): (If).
  • the compound of Formula (I) is a compound of Formula (Ia- certain embodiments, the compound of Formula (I) is a compound of Formula certain embodiments, the compound of Formula (I) - 30 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) is a compound of Formula In certain embodiments, the compound of Formula (I) is a compound of In certain embodiments, the compound of Formula (I) is a compound of 1). In certain embodiments, the compound of Formula (I) is a : certain embodiments, the compound of Formula (I) is a compound of Formula (Id-1): (Id-1).
  • the compound of Formula (I) is a compound of Formula certain embodiments, the compound of Formula (I) is a compound of In certain embodiments, the compound of Formula (I) is a compound of In certain - 31 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) embodiments, the compound of Formula (I) is a compound of Formula (If-1): (If-1).
  • the compound of Formula (I) is a compound of Formula (If- , wherein R 5a , R 5b , R 5c , R 5d , and R 5e are of H, halogen, CN, NO 2 , OR A , , substituted C6-C10 aryl, and optionally substituted C 2 -C 10 heteroaryl.
  • R 5a , R 5b , R 5d , and R 5e are each independently selected from of H, ha A A B logen, CN, NO2, OR , N(R )(R ), optionally substituted C 1 -C 6 alkyl, optionally substituted C 6 -C 10 aryl, and optionally substituted C2-C10 heteroaryl.
  • at least one of R 5a , R 5b , R 5c , R 5d , and R 5e is phenyl.
  • the phenyl in R 5a , R 5b , R 5c , R 5d , or R 5e is substituted with at least one selected from the group consisting of OH, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 alkyl, and C1-C6 haloalkyl.
  • R 5a is H. In certain embodiments, R 5a is CH3. In certain embodiments, R 5a is Ph. In certain embodiments, R 5a . In certain embodiments, R 5a . In certain embodiments, R 5a . In certain embodiments, R 5a is . In certain embodiments, R 5a .
  • R 5b is H. In certain embodiments, R 5b is CH3. In certain embodiments, R 5b is Ph. In certain embodiments, R 5b . In certain embodiments, R 5b . In certain embodiments, R 5b . In certain embodiments, R 5b In certain embodiments, . In certain embodiments, R 5c is H. In certain embodiments, R 5c is embodiments, R 5c is Ph. In certain embodiments, R 5c is . In certain embodiments, R 5c . In certain embodiments, R 5c is . In certain embodiments, R 5c . In certain embodiments, certain embodiments, certain embodiments, R 5d is H.
  • R 5d is CH 3 . In certain embodiments, R 5d is Ph. In certain embodiments, R 5d . In certain embodiments, R 5d . In certain embodiments, R 5d . In certain embodiments, R 5d . In certain embodiments, R 5e R 5e is CH 3 . In R 5e is Ph. In certain embodiments, R 5e . In certain embodiments, . In certain embodiments, R 5e . In certain embodiments, R 5e is - 33 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) .
  • R 6a , R 6b , R 6c , R 6d , and R 6e are each independently selected of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C2-C6 haloalkyl, optionally substituted C6-C10 aryl, optionally - 34 - 55708207.3
  • R 6a , R 6b , R 6c , R 6d , and R 6e are each independently selected of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C2-C6 haloalkyl, optionally substituted C6-C10 aryl, optionally - 34 - 55708207.3
  • R 6a is H. In certain embodiments, R 6a is F. In certain embodiments, R 6a is Cl. In certain embodiments, R 6b is H. In certain embodiments, R 6b is F. In certain embodiments, R 6b is Cl. In certain embodiments, R 6c is H. In certain embodiments, R 6c is F. In certain embodiments, R 6c is Cl. In certain embodiments, R 6d is H. In certain embodiments, R 6d is F. In certain embodiments, R 6d is Cl. In certain embodiments, R 6e is H. In certain embodiments, R 6e is F. In certain embodiments, R 6e is Cl.
  • one of R 6a , R 6b , R 6c , R 6d , and R 6e is F or Cl.
  • R 7a , R 7b , R 7c , and R 7d is H. In certain embodiments, at least two of R 7a , R 7b , R 7c , and R 7d are H. In certain embodiments, at least three of R 7a , R 7b , R 7c , and R 7d are H. In certain embodiments, each of R 7a , R 7b , R 7c , and R 7d are H. In certain embodiments, R 7e is H. In certain embodiments, R 7e is CH3. In certain embodiments, R 7e is CH2OCH3. In certain embodiments, R 7e is . In certain R 7e . In R 7f is H.
  • R 7f is CH 3 . In certain embodiments, R 7f is CH2OCH3. In certain embodiments, R 7f is . In certain . In R 8 is phenyl optionally substituted with at least one halogen. In certain embodiments, R 8 is phenyl. In certain embodiments, R 8 is 4-chlorophenyl. In certain embodiments, R 8 is 4-fluorophenyl. In certain embodiments, L 3 is a bond. In certain embodiments, L 3 is –(CH2)-. In certain embodiments, L 3 is –(CHCH 3 )-. In certain embodiments, L 3 is –[CH(CH 2 OCH 3 )]CH 2 - . In certain embodiments, L 3 .
  • R 9 is optionally substituted indolyl. In certain embodiments, R 9 is optionally substituted naphthyl. In certain embodiments, the phenyl is optionally substituted with at least one C 1 -C 6 haloalkyl. In certain embodiments, the haloalkyl is CF3. In certain embodiments, R 9 is . In certain embodiments, R 9 9 . In certain embodiments, R is - 41 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) . is H. In certain embodiments, the combination of L 3 -R 9 is . In certain embodiments, the combination of L 3 -R 9 is .
  • the combination of L 3 -R 9 In certain embodiments, the combination of L 3 -R 9 . In certain embodiments, the combination of L 3 -R 9 . In certain embodiments, the combination of L 3 -R 9 . In certain embodiments, the combination of L 3 -R 9 . In certain embodiments, the combination of L 3 -R 9 . In certain embodiments, the combination of L 3 -R 9 In certain embodiments, the In certain embodiments, the certain embodiments, the combination of L 3 -R 9 is certain embodiments, the In certain embodiments, the - 42 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) certain embodiments, the combination of L 3 -R 9 is . In certain embodiments, the combination of L 3 -R 9 .
  • the combination of L 3 -R 9 In certain embodiments, the certain embodiments, the combination of L 3 -R 9 is certain embodiments, the In certain embodiments, the combination of L 3 -R 9 .
  • the compound is selected from the group consisting of: 2-(4-(4-chlorophenyl)thiazol-2-yl)-1-(cyclopropyl(phenyl)methyl)-N-methyl-1H- benzo[d]imidazole-5-carboxamide; (R)-2-(4-(4-chlorophenyl)thiazol-2-yl)-1-(cyclopropyl(phenyl)methyl)-N-methyl- 1H-benzo[d]imidazole-5-carboxamide; (S)-2-(4-(4-chlorophenyl)thiazol-2-yl)-1-(cyclopropyl(phenyl)methyl)-N-methyl-1H- benzo[d]imidazole-5-carboxamide; 1-benzyl-N-methyl-2-(4-phenylthiazol-2-yl)-1H-benzo[d]imidazole-5-carboxamide; 2-(4-(4-chlorophen
  • At least one additional agent suitable for treating, preventing, and/or ameliorating a bacterial infection is selected from the group consisting of a polymyxin, aminoglycoside, ⁇ -lactam (e.g., penicillin, cephalosporin, or carbapenem), monobactam, fluoroquinolone, sulfonamide, tetracycline, and macrolide.
  • the additional agent suitable for treating, preventing, and/or ameliorating a bacterial infection is at least one selected from the group consisting of colistin (polymyxin E), polymyxin B, amikacin, ampicillin, amoxicillin, aminoglycoside, azithromycin, aztreonam, carbapenem, cefepime, cefiderocol, cefotaxime, ceftriaxone, ceftaroline, ceftazidime, ceftobiprole, ceftolozane, ciprofloxacin, clindamycin, dalbavancin, daptomycin, doxycycline, ertapenem, fluoroquinolone, gentamicin, imipenem, levofloxacin, linezolid, meropenem, minocycline, mupirocin, oritavancin, piperacillin, streptogramin, sulbactam,
  • colistin
  • compositions containing the compound(s) described herein include a pharmaceutical composition comprising at least one compound as described herein and at least one pharmaceutically acceptable carrier.
  • the composition is formulated for an administration route such as oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal, intravesical, intrapulmonary, intraduodenal, - 57 - 55708207.3
  • a pharmaceutical composition comprising a compound provided herein and further comprising a pharmaceutically acceptable excipient.
  • Preparation of Compounds Compounds described herein can be prepared by the general schemes described herein, using the synthetic method known by those skilled in the art. The following examples illustrate non-limiting embodiments of the compound(s) described herein and their preparation.
  • the compounds described herein can possess one or more stereocenters, and each stereocenter can exist independently in either the (R) or (S) configuration.
  • compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein.
  • Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a mixture of one or more isomer is utilized as the therapeutic compound described herein.
  • compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers.
  • Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
  • the methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound(s) described herein, as well as metabolites and active metabolites of these compounds having the same type of activity.
  • Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like.
  • the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol.
  • the - 58 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) compounds described herein exist in unsolvated form.
  • the compound(s) described herein can exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
  • compounds described herein are prepared as prodrugs.
  • a “prodrug” refers to an agent that is converted into the parent drug in vivo.
  • a prodrug upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
  • sites on, for example, the aromatic ring portion of compound(s) described herein are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway.
  • the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
  • Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2 H, 3 H, 11 C, 13 C, 14 C, 36 Cl, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O, 17 O, 18 O, 32 P, and 35 S.
  • isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies.
  • substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
  • substitution with positron emitting isotopes, such as 11 C, 18 F, 15 O and 13 N is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
  • the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
  • the compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley - 59 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4 th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemi st ry 4th Ed.
  • Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed.
  • each protective group is removable by a different means.
  • Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal.
  • protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions.
  • Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile.
  • Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.
  • carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc.
  • Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while co- - 60 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) existing amino groups are blocked with fluoride labile silyl carbamates.
  • Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts.
  • an allyl-blocked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups.
  • Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.
  • blocking/protecting groups may be selected from allyl, benzyl (Bn), benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), methyl, ethyl, t-butyl, t- butyldimethylsilyl (TBDMS), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), t-butyloxycarbonyl (Boc), para-methoxybenzyl (PMB), triphenylmethyl (trityl), acetyl, and fluorenylmethoxycarbonyl (FMOC).
  • Bn benzyl
  • Cbz benzyloxycarbonyl
  • Alloc allyloxycarbonyl
  • the disclosure provides a method of treating, preventing, and/or ameliorating a bacterial infection in a subject.
  • the method comprises administering to the subject at least one compound of the disclosure or the pharmaceutical composition of the disclosure.
  • the method comprises administering to the subject at least one polymyxin antibiotic, or an analogue or derivative thereof.
  • the disclosure provides a method of sensitizing a bacterial colony to at least one antibiotic polypeptide.
  • the method comprises contacting the bacterial colony with at least one compound of the disclosure or the pharmaceutical composition of the disclosure.
  • antibiotic polypeptide is colistin (polymyxin E).
  • polymyxin antibiotic is polymyxin B.
  • the antibiotic polypeptide is a defensin.
  • the antibiotic polypeptide comprises about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, or 500 amino acids.
  • the antibiotic polypeptide is a defensin.
  • the - 61 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) antibiotic polypeptide has a charge of about +1, +2, +3, +4, +5, +10, +15, +20, +25, or +30 at a pH of about 7.4.
  • the bacterial infection or bacterial colony comprises a bacterial species selected from the group consisting of: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdenensis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus warnerii, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus pettenkoferi, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Group C streptococci, Streptococcus constellatus, Enterococcus faecalis, Enterococcus faecium, Corynebacterium jeikeium, Lactobacillus acidophilus, Listeria monocytogenes, Escher
  • the method further comprises administering at least one additional antibiotic.
  • the additional antibiotic is at least one selected from the group consisting of a aminoglycoside, ⁇ -lactam (e.g., penicillin, cephalosporin, or carbapenem), monobactam, fluoroquinolone, sulfonamide, tetracycline, and macrolide.
  • the additional antibiotic is at least one selected from the group consisting of amikacin, ampicillin, amoxicillin, aminoglycoside, azithromycin, aztreonam, carbapenem, cefepime, cefiderocol, cefotaxime, ceftriaxone, ceftaroline, ceftazidime, ceftobiprole, ceftolozane, ciprofloxacin, clindamycin, dalbavancin, daptomycin, doxycycline, ertapenem, fluoroquinolone, gentamicin, imipenem, levofloxacin, linezolid, meropenem, minocycline, mupirocin, oritavancin, piperacillin, streptogramin, sulbactam, tedizolid, telavancin, tigecycline, ticarcillin, tobramycin, trimethoprim/sul
  • the bacterial infection is a persistent or antibiotic resistant - 62 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) bacterial infection.
  • formation of a bacterial biofilm is at least partially prevented or inhibited.
  • the infection occurs on a prosthesis or an implant.
  • the prosthesis selected from the group consisting of a knee prosthesis, a hip prosthesis, elbow prosthesis, ankle prosthesis, shoulder prosthesis, and spine prosthesis.
  • the subject is a mammal. In certain embodiments, the mammal is a human.
  • the methods described herein include administering to the subject a therapeutically effective amount of at least one compound described herein, which is optionally formulated in a pharmaceutical composition.
  • a therapeutically effective amount of at least one compound described herein present in a pharmaceutical composition is the only therapeutically active compound in a pharmaceutical composition.
  • the method further comprises administering to the subject an additional therapeutic agent that treats a disease state.
  • administering the compound(s) described herein to the subject allows for administering a lower dose of the additional therapeutic agent as compared to the dose of the additional therapeutic agent alone that is required to achieve similar results in treating a disease state in the subject.
  • the compound(s) described herein enhance(s) the activity of the additional therapeutic compound, thereby allowing for a lower dose of the additional therapeutic compound to provide the same effect.
  • the compound(s) described herein and the therapeutic agent are co-administered to the subject.
  • the compound(s) described herein and the therapeutic agent are coformulated and co-administered to the subject.
  • the subject is a mammal.
  • the mammal is a human Combination Administration and Treatment
  • a synergistic effect is observed when a compound as described herein is administered with one or more additional therapeutic agents or compounds.
  • a synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-E max equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol - 63 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) Pharmacol.114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul.22:27-55).
  • Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination.
  • the corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.
  • Administration/Dosage/Formulations The regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after the onset of a disease state. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions described herein to a patient may be carried out using known procedures, at dosages and for periods of time effective to treat a disease state in the patient.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a disease state in the patient.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a non-limiting example of an effective dose range for a therapeutic compound described herein is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or - 64 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • compositions described herein are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • the compositions described herein are administered to the - 65 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) patient in dosages that range from one to five times per day or more.
  • the compositions described herein are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
  • the compound(s) described herein for administration may be in the range of from about 1 ⁇ g to about 10,000 mg, about 20 ⁇ g to about 9,500 mg, about 40 ⁇ g to about 9,000 mg, about 75 ⁇ g to about 8,500 mg, about 150 ⁇ g to about 7,500 mg, about 200 ⁇ g to about 7,000 mg, about 350 ⁇ g to about 6,000 mg, about 500 ⁇ g to about 5,000 mg, about 750 ⁇ g to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the dose of a compound described herein is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound described herein used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the compound(s) described herein can be administered to a - 66 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) subject in an amount ranging from about 0.01 mg/kg to about 200 mg/kg, or about 0.5 mg/kg to about 190 mg/kg, or about 0.75 mg/kg to about 180 mg/kg, or about 1 mg/kg to about 170 mg/kg, or about 1.5 mg/kg to about 160 mg/kg, or about 2 mg/kg to about 150 mg/kg, or about 2.5 mg/kg to about 140 mg/kg, or about 3 mg/kg to about 130 mg/kg, or about 3.5 mg/kg to about 120 mg/kg, or about 4 mg/kg to about 110 mg/kg, or about 4.5 mg/kg to about 100 mg/kg, or about 5 mg/kg to about 95 mg/kg, or about 5.5 mg/kg to about 90 mg/kg, or about 6 mg/kg to about 85 mg/kg, or about 6.5 mg/kg to about 80 mg
  • the compound(s) described herein can be administered to a subject in an amount that is less than, equal to, or greater than about 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 12 mg/kg, 14 mg/kg, 16 mg/kg, 18 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg
  • a composition as described herein is a packaged pharmaceutical composition
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like.
  • other active agents e.g., other analgesic agents.
  • the compounds for use in the compositions described herein can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • transdermal e.g., sublingual, lingual, (trans)buccal, (trans)urethral
  • vaginal e.g., trans- and perivaginally
  • intravesical, intrapulmonary, intraduodenal, intragastrical intrathecal
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions described herein are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compound(s) described herein can be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropyl methylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropyl methylcellulose
  • fillers e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate
  • the tablets may be coated using suitable methods and coating materials such as OPADRYTM film - 68 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) coating systems available from Colorcon, West Point, Pa. (e.g., OPADRYTM OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRYTM White, 32K18400).
  • suitable methods and coating materials such as OPADRYTM film - 68 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) coating systems available from Colorcon, West Point, Pa. (e.g., OPADRYTM OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRYTM White, 32K18400).
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorbic acid.
  • Compositions as described herein can be prepared, packaged, or sold in a
  • Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.
  • Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, dispersing agents, surface-active agents, disintegrating agents, binding agents, and lubricating agents.
  • Suitable dispersing agents include, but are not limited to, potato starch, sodium starch glycollate, poloxamer 407, or poloxamer 188.
  • One or more dispersing agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more dispersing agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Surface-active agents include cationic, anionic, or non-ionic surfactants, or combinations thereof.
  • Suitable surfactants include, but are not limited to, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, carbethopendecinium bromide, cetalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylpyridine chloride, didecyldimethylammonium chloride, dimethyldioctadecylammonium bromide, dimethyldioctadecylammonium chloride, domiphen - 69 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) bromide, lauryl methyl gluceth-10 hydroxypropyl dimonium chloride, tetramethylammonium hydroxide, thonzonium bromide, stearalkonium chloride, octenidine dihydrochloride, olaflur, N-oleyl-1,3
  • One or more surfactants can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more surfactants can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Suitable diluents include, but are not limited to, calcium carbonate, magnesium carbonate, magnesium oxide, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate, Cellactose ® 80 (75 % ⁇ - lactose monohydrate and 25 % cellulose powder), mannitol, pre-gelatinized starch, starch, sucrose, sodium chloride, talc, anhydrous lactose, and granulated lactose.
  • One or more diluents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more diluents can each be individually present in the composition in an amount of at least, greater than, or less than - 70 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Suitable granulating and disintegrating agents include, but are not limited to, sucrose, copovidone, corn starch, microcrystalline cellulose, methyl cellulose, sodium starch glycollate, pregelatinized starch, povidone, sodium carboxy methyl cellulose, sodium alginate, citric acid, croscarmellose sodium, cellulose, carboxymethylcellulose calcium, colloidal silicone dioxide, crosspovidone and alginic acid.
  • One or more granulating or disintegrating agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more granulating or disintegrating agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Suitable binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, anhydrous lactose, lactose monohydrate, hydroxypropyl methylcellulose, methylcellulose, povidone, polyacrylamides, sucrose, dextrose, maltose, gelatin, polyethylene glycol.
  • One or more binding agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more binding agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Suitable lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, hydrogenated castor oil, glyceryl monostearate, glyceryl behenate, mineral oil, polyethylene glycol, poloxamer 407, poloxamer 188, sodium laureth sulfate, sodium benzoate, stearic acid, sodium stearyl fumarate, silica, and talc.
  • One or more lubricating agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more lubricating agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Tablets can be non-coated or they may be coated using known methods to achieve - 71 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient.
  • a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.
  • tablets may be coated using methods described in U.S. Patent Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets.
  • Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Tablets can also be enterically coated such that the coating begins to dissolve at a certain pH, such as at about pH 5.0 to about pH 7.5, thereby releasing a compound as described herein.
  • the coating can contain, for example, EUDRAGIT ® L, S, FS, and/or E polymers with acidic or alkaline groups to allow release of a compound as described herein in a particular location, including in any desired section(s) of the intestine.
  • the coating can also contain, for example, EUDRAGIT ® RL and/or RS polymers with cationic or neutral groups to allow for time controlled release of a compound as described herein by pH-independent swelling.
  • Parenteral Administration For parenteral administration, the compounds as described herein may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
  • Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain - 72 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) alcohol diluent or dispersant, such as such as lauryl, stearyl, or oleyl alcohols, or similar alcohol.
  • Additional dosage forms suitable for use with the compound(s) and compositions described herein include dosage forms as described in U.S. Patents Nos.6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include dosage forms as described in PCT Applications Nos.
  • Controlled Release Formulations and Drug Delivery Systems can be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use with the method(s) described herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the dosage forms to be used can be provided as slow or controlled- release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions described herein.
  • single unit dosage - 73 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, that are adapted for controlled-release are encompassed by the compositions and dosage forms described herein.
  • Most controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
  • Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance.
  • controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects.
  • Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.
  • Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
  • controlled-release component is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient.
  • the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma - 74 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound described herein depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a disease state in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a compound described herein can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different.
  • a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the administration of the compound(s) described herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”).
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 - 75 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a maintenance dose is administered if necessary.
  • the dosage or the frequency of administration, or both is reduced to a level at which the improved disease is retained.
  • patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
  • the compounds described herein can be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED 50 .
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • the libraries were incubated with MCR-1 and the complexes were captured using Nickel-beads and washed. Bound small molecules were released and bound again to fresh MCR-1 protein for two additional rounds of enrichment.
  • the DNA section of the conjugates was amplified by PCR and subjected to deep sequencing. Bioinformatics analysis was used to reveal the sequences most highly represented after the binding selection as described in Faver, John C., et al. "Quantitative comparison of enrichment from DNA-encoded chemical library selections.” ACS combinatorial science 21.2 (2019): 75-82. Because the barcode DNA sequence encodes the small molecule building blocks added, it reveals the structure of library members that bound the target protein.
  • CDD-2901, CDD-2750, CDD-3002, CDD-2918, CDD- 2902, CDD-3019, and CDD-2847 (FIGs.9-10). All of these compounds showed significant activity by the spot assay (>100x) and, for some, MIC against E. coli XL1-Blue encoding MCR- 1 (FIGs.9-10). Docking CDD-1794 and CDD-1938 to an MCR-1 homology model- The structure of full-length MCR-1 is not known.
  • a homology model was constructed based on the structure of NmEptA, which has 37% sequence identity - 77 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) to MCR-1.
  • Autodock Vina was used to computationally dock CDD-1794 and CDD-1938 to the MCR-1 homology model (FIGs.8A-8B).
  • the highest ranked pose of each compound is bound in a similar pocket near the catalytic Thr285 residue.
  • the compounds are predicted to bind in a similar position as where dodecyl- ⁇ -D-maltoside was found in the NmEptA structure and was proposed as the phosphatidylethanolamine binding site.
  • CDD compounds for cytotoxicity versus human cells. An important question is whether the compounds that have activity against strains expressing MCR-1 are cytotoxic to human cells. This was addressed in cytotoxicity testing using human HepG2 cells. A set of potent compounds including CDD-2750, CDD-2901, CDD-2847, and CDD-2902 was tested. A HepG2 cell line was used to screen for in vitro metabolism-associated drug toxicity using cell viability assays that measure the number of live cells after a 24-hour incubation with increasing concentrations (0-100 ⁇ M) of compounds. A CellTiter-Glo ⁇ Luminescent Cell Viability Assay was used to determine the number of viable cells in culture. Bacterial strains and expression vectors E.
  • MCR-1-StrepII Strep-tag II-fused MCR-1
  • Pfu polymerase Invitrogen
  • MCR-1-StrepII-pTP470 an isopropyl- ⁇ -d-thiogalactopyranoside (IPTG)-inducible trc promoter
  • Strep-tag II at the C-terminus of MCR-1 was used to monitor the expression of MCR-1 in E. coli by immunoblotting with anti-Strep tag II antibody, it was not used in this study.
  • the Strep tag II-modified MCR-1 plasmid construct was shown to provide colistin resistance to E. coli XL1-Blue cells.
  • mcr-1 was cloned between NdeI and XhoI restriction sites of a modified pET28a vector (Novogen), in which the thrombin recognition sequence was replaced with the tobacco etch virus (TEV) protease recognition sequence and 2 extra codons for histidine residues were inserted before the TEV protease recognition sequence to increase binding affinity of His-tagged target proteins to the HisTrap - 78 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) columns.
  • TEV tobacco etch virus
  • the clinical isolates (independently determined to have plasmid-encoded antibiotic resistance genes and microdilution MICs based on CLSI testing standards where the resistance cut-off is 4 ⁇ g/mL) were obtained from the Center for Disease Control & Prevention (CDC)/US Food & Drug Administration (FDA) Antibiotic Resistance (AR) Isolate Bank panel entitled “Isolates with new or novel antibiotic resistance (NEW)”.
  • the strains in the panel are as follows: K.
  • coli cells containing the MCR-1 expression plasmid were grown at 37°C to an OD600 of 0.8 in LB medium containing 25 ⁇ g/ml kanamycin. Expression of the His-MCR-1 protein was induced with 0.5 mM IPTG at 18°C for 16 h.
  • the cells were pelleted and suspended in lysis buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) and lysed using a French press and a short period of sonication. The cell debris was removed by centrifugation at 12,000 ⁇ g for 30 min, and the membranes were isolated by ultracentrifugation at 170,000xg for 1.5 hours at 4°C.
  • His-MCR-1 was solubilized from the membranes by incubating the isolated membranes at 4°C overnight with gentle shaking with 20 mM n-dodecyl- ⁇ -d-maltopyranoside (DDM) in 20 mM HEPES, pH 7.4, 300 mM NaCl, 1 ⁇ EDTA-free protease inhibitor cocktail (Gendepot).
  • DDM n-dodecyl- ⁇ -d-maltopyranoside
  • HEPES pH 7.4
  • 300 mM NaCl 1 ⁇ EDTA-free protease inhibitor cocktail
  • Solubilized His-MCR-1 was purified by metal-chelating chromatography by loading the supernatant from the ultracentrifugation onto a 1-mL HisTrap FF column (GE - 79 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) Healthcare), which had been equilibrated by Buffer A (20 mM HEPES, pH 7.4, 300 mM NaCl, 20 mM imidazole, 0.5 mM DDM, 1 mM phenylmethylsulfonyl fluoride [PMSF]).
  • Buffer A (20 mM HEPES, pH 7.4, 300 mM NaCl, 20 mM imidazole, 0.5 mM DDM, 1 mM phenylmethylsulfonyl fluoride [PMSF]).
  • Buffer A After washing with 5 column volumes (CVs) of Buffer A, the bound proteins were eluted with 0 to 100% Buffer B (20 mM HEPES, pH 7.4, 300 mM NaCl, 500 mM imidazole, 0.5 mM DDM, 1 mM PMSF) over 10 CVs. Fractions containing His-MCR-1 were pooled, concentrated, and buffer exchanged to Buffer A with a 50-kDa cut-off Amicon Ultra-15 centrifugal filter unit (EMD Millipore).
  • Buffer B 20 mM HEPES, pH 7.4, 300 mM NaCl, 500 mM imidazole, 0.5 mM DDM, 1 mM PMSF
  • the His-tag was cleaved by incubating with TEV protease for 16 hours at 4°C at a ratio of 1:50, and the TEV protease was removed by incubating with Ni-Sepharose 6 fast-flow resin (GE Healthcare).
  • MCR-1 was further purified by gel filtration chromatography using a Superdex 200 Increase GL 10/300 sizing column (GE Healthcare) with 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM DDM, 1 mM PMSF as running buffer. Fractions containing MCR-1 were pooled and concentrated.
  • the DNA-encoded chemical library (DECL) was mixed with 1 or 5 ⁇ M of His-MCR- 1 in 200 ⁇ L of selection buffer ([20 mM HEPES pH 7.5, 134 mM potassium acetate, 10 mM imidazole, 1 mM TCEP, 8 mM sodium acetate, 0.8 mM magnesium acetate, 1 mM CHAPS, 4 mM sodium chloride, and 0.1 mg/mL Sheared Salmon Sperm DNA]).
  • selection buffer [20 mM HEPES pH 7.5, 134 mM potassium acetate, 10 mM imidazole, 1 mM TCEP, 8 mM sodium acetate, 0.8 mM magnesium acetate, 1 mM CHAPS, 4 mM sodium chloride, and 0.1 mg/mL Sheared Salmon Sperm DNA]
  • NTC non-target control
  • sterile Luria Bertani (LB) Becton Dickinson agar plates supplemented with 12.5 ⁇ g/mL chloramphenicol (for plasmid maintenance) were prepared containing: 1) subinhibitory concentration (0.32 ⁇ g/mL) of colistin sodium methane sulfonate (CMS) antibiotic only (adjusted for colistin base using an adjustment factor of 2.56X in water); 2) high inhibitor compound concentration (12.5 ⁇ M) only and 3) no antibiotic/inhibitor, and test plates containing: 4) low inhibitor concentration (3.25 ⁇ M) and CMS antibiotic (0.32 ⁇ g/mL); 5) medium inhibitor concentration (6.25 ⁇ M) and CMS antibiotic (0.32 ⁇ g/mL), and 6) high inhibitor concentration (12.5 ⁇ M) and CMS antibiotic (0.32 ⁇ g/mL).
  • CMS colistin sodium methane sulfonate
  • Dilutions were transferred to a 96 well plate and a multichannel pipette was used to transfer 7 ⁇ L of dilutions, prepared as biological triplicates, onto each of the control and test plates. Spots were allowed to dry by absorption, and plates were inverted and incubated at 37oC overnight. Plates, without the lid, were photographed and growth was visually confirmed. Viability was our endpoint. Growth on the test plates was compared to growth on the control plates. Growth was defined as the presence of single colonies or overall growth coverage within the spot. Clinical isolates were sub-cultured from -80oC freezer stocks onto 5% sheep blood agar plates and spot assays were prepared the same as the laboratory strains.
  • the colistin MIC was defined as the lowest concentration of colistin ⁇ inhibitor that inhibited visible growth of bacterial culture after overnight incubation. No inoculum controls were included.
  • Time-kill curve assays Initially, growth curves were carried out to confirm that strains would reach a stable early to mid-log phase by 4-5 hours and inoculum was adjusted accordingly.
  • Time-kill curve (TKC) assays were performed in a 96-well format with a final volume of 100 ⁇ L per well. Briefly, E.
  • Thin-layer chromatography was performed on silica gel 60 F254 plates (E. Merck). Non-UV active compounds were visualized on thin-layer chromatography (TLC) using one of the following stains: KMnO 4 , ninhydrin, p-anisaldehyde, 2,4-DNP, or bromocresol green.
  • TLC thin-layer chromatography
  • Liquid chromatography-mass spectrometry (LC-MS) used for data collection was a 1290 Infinity Series liquid chromatography with a 6150 mass spectrometer system (Agilent).
  • the solid phase column was an Agilent Eclipse Plus C18, 2.1 mm ⁇ 50 mm (8 ⁇ m), and the mobile phase solvents were A: 0.05% formic acid in water and B: 5% water in acetonitrile. Peak detection was done at 254 and 230 nm.
  • the high-resolution mass spectrometry (HRMS) data were acquired on a Thermo Q Exactive Orbitrap MS coupled with a Thermo Vanish UHPLC.
  • the Zorbax XDB-C18 (4.6 mm ⁇ 50 mm, 3 ⁇ m) was used for separation and kept at 40 °C.
  • the flow rate used was 0.3 mL/min with a gradient ranging from 2 to 95% aqueous acetonitrile containing 0.1% formic acid in a 7 min run.
  • Q Exactive MS was operated in - 83 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) positive mode with electrospray ionization.
  • the lock mass at m/z 371.1012 for positive mode was used as a reference ion during acquisition. All compounds are >95% pure by HPLC.
  • NMR spectra were collected using a Bruker 600 or 151 MHz NMR for 1 H and 13 C, respectively. All NMR chemical shifts were referenced to the residual nondeuterated solvent as an internal standard.
  • the procedure involves adding the CellTiter-Glo ⁇ reagent to the cells whereby it lyses the cell membranes to release ATP, the reagent inhibits endogenous ATPases and provides luciferin and luciferase needed to measure ATP using a bioluminescent reaction.
  • a CLARIOstar plate reader was used to detect the signal and measure the ATP in viable cells.
  • the CellTiter-Blue ⁇ Cell Viability Assay which uses an optimized reagent containing resazurin that can enter living cells where it is reduced to the fluorescent resorufin product; this conversion is proportional to the number of metabolically active, viable cells present in a culture.
  • the HepG2 cells in the assay plate are incubated at 37°C for 24 hours. The signal is measured using a standard multi-well fluorometer.
  • % viability is plotted as a function of compound concentration (uM), and non-linear regression (Log(concentration)-normalized cell viability) is used to calculate the half-maximal inhibitory concentration (IC 50 ) with Prism 10.0.
  • IC 50 cut-off values compounds retaining 100X antimicrobial activity at a concentration equal to 10% of the IC50 were considered to have a low cytotoxicity profile.
  • Example 1 DNA encoded library (DECL) screening for MCR-1 inhibitors
  • DECL DNA-encoded chemical library technology
  • An innovative aspect of the present disclosure is the use of DNA-encoded chemical library technology (DECL) to explore chemical space to identify small molecule high affinity - 84 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) binders for the clinically important MCR-1 mobile colistin resistance enzyme for discovery of novel inhibitors.
  • the DECL approach involves the creation of libraries of drug-like molecules covalently attached to a unique DNA barcode that enables identification of binders for a target in a pool of millions of compounds (FIG.3).
  • the conjugates are constructed in sequential cycles of chemical attachment of drug-like fragments (chemical building blocks) to a molecular scaffold concomitantly encoded through the ligation of a unique DNA barcode.
  • the libraries can be screened against a tagged version of the protein target to identity molecules with high affinity, which are isolated though the pull-down of the tag from solution.
  • Next-generation DNA sequencing allows the determination of the DNA barcode sequences isolated and, thus, the structures of the high-affinity compounds.
  • the DECL approach was applied to identify small molecule inhibitors of the OXA-48 carbapenemase, NDM-1 metallo- ⁇ -lactamase, and SARS CoV-2 Mpro protease.
  • the present example applies a similar strategy to identify inhibitors of MCR-1.
  • Example 2 Crystal structure of the catalytic domain of MCR-1
  • the MCR-1 protein is predicted to consist of a membrane-spanning domain and a periplasmic catalytic domain.
  • the X-ray crystal of the catalytic domain (cMCR) was determined at 1.32 ⁇ resolution.
  • cMCR-1 is a zinc metallo-enzyme that assumes a ⁇ / ⁇ / ⁇ fold characteristic of the alkaline phosphatase superfamily.
  • an active-site zinc in an analogous position as the catalytic zinc in alkaline phosphatase may stabilize the alkoxide form of Thr285 for nucleophilic attack on the phosphate of the phosphatidylethanolamine substrate to create an intermediate with Thr285 linked to PEA.
  • Binding of lipid A in an appropriate position for nucleophilic attack on the lipid A 1’ or 4’ phosphate on the phosphate of the Thr285-PEA intermediate could then transfer the group to lipid A.
  • Example 3 MCR-1 expression reduces E. coli susceptibility to polymyxins - 85 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) If Thr285 is the catalytic nucleophile, mutation of this residue would be expected to greatly decrease MCR-1 function. This was tested by mutating this residue to alanine in a plasmid encoding the full-length mcr-1 gene and measuring the minimum inhibitory concentration (MIC) of colistin and polymyxin B.
  • MIC minimum inhibitory concentration
  • the protein was expressed in E. coli and a cell lysate was solubilized using 30 mM dodecyl- ⁇ -D- maltoside (DDM) detergent, centrifuged, and the supernatant was filtered and subjected to affinity purification using a nickel-sepharose resin.
  • DDM dodecyl- ⁇ -D- maltoside
  • the His-tag was removed by with TEV protease and separated from MCR-1 on a nickel-charged resin.
  • the MCR-1 protein was then further purified by size exclusion chromatography.
  • CDD-1938 The DNA is attached via a linker to the C1 position while the C2 position branches from C1 and C3 branches from the C2 position.
  • the CDD-1938 compound that was synthesized off-DNA and shows activity against E. coli expressing MCR-1 contains C1, C2, and C3 substituents.
  • CDD-1938 is a racemic mixture with a chiral center at the phenyl substitution on the piperazine ring on C1. Both enantiomers of CDD-2778 (R-enantiomer) grew up to a 100-fold lower dilution than the colistin only control, which reflects a 10-fold increase in potency compared to CDD-1938, while E.
  • CDD-2932 shows a 100-fold loss in potency in the spot assay compared to the CDD-2750 parent compound, indicating the C3 group makes important contributions to binding.
  • CDD-2889 which retains the C3 phenyl but is lacking the isopropyl group also showed a 100-fold decrease in potency relative to the CDD-2750 parent, revealing the importance of the isopropyl group for inhibition.
  • the original CDD-1938 hit compound and the more potent CDD-2750 analog contain a halide (Cl) appended to the phenyl group at the C1 position.
  • CDD-2835 is CDD-2750 lacking the halide. This compound showed a 100-fold loss in potency relative to CDD-2750, indicating the importance of the halide for potency.
  • the Cl in CDD-2750 was replaced with fluorine (F) to create CDD-3228, which displayed similar potency in the spot assay as CDD- 2750.
  • the Cl was also replaced by F in CDD-2901, which has a benzyl rather than a pyridine ring at C2, to create CDD-3002.
  • CDD-3002 showed similar potency as CDD-2901 in the spot assay.
  • both of the F-containing compounds exhibited similar potency as the parent compounds while the lack of a halide decreased potency, indicating the importance of Cl or F at this position.
  • the C3 moiety is critical for inhibition and the isopropyl group appended to the phenyl at C3 is important.
  • Different substituents were also examined on the C3 phenyl group. Notably, the addition of a hydroxyl at the 2 position and a trifluoromethoxy group at the 5 position of the phenyl ring enhanced potency.
  • the CDD-2847 and CDD-2902 compounds containing these changes exhibited a 10-fold increase in potency compared to the parent compounds CDD-2750 and CDD-2901. E.
  • CDD-2847 and CDD-2902 are the most potent antibacterial compounds identified in the SAR studies herein. - 88 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) As described above, all of the analog compounds were tested against E. coli expressing MCR-1 using the spot test shown in FIG.7.
  • MICs minimum inhibitory concentrations
  • the colistin MIC in the absence of compounds is 4.0 ⁇ g/ml in all experiments (FIG.10).
  • the colistin MIC is 2 ⁇ g/ml at 6.25 ⁇ M CDD-1938, 1 ⁇ g/ml at 50 ⁇ M, and 0.5 ⁇ g/ml at 100 ⁇ M compound.
  • CDD-2750 which is 10-fold more potent than CDD-1938 in the spot assay
  • the colistin MIC is 0.125 ⁇ g/ml at 6.25 ⁇ M compound and 0.0156 ⁇ g/ml at 50 ⁇ M compound.
  • the colistin MIC is 0.0156 ⁇ g/ml at 6.25 ⁇ M compound and 0 ⁇ g/ml at 25 ⁇ M compound.
  • CDD-2901 has similar MIC values as CDD- 2847 (FIG.10).
  • CDD-2847 and CDD-2901 are very potent compounds as measured by MIC.
  • CDD- 2847 As seen in FIG.11, the most potent compound against the clinical strains is CDD- 2847, which also displayed high potency by spot and MIC versus the laboratory strain of E. coli.
  • CDD-2847 showed a 10000-fold effect compared to the colistin only control for all of the E. coli MCR-1 clinical strains but showed a 10-fold effect versus the K. pneumoniae strain.
  • CDD-3019, CDD-2902, and CDD-2918 also showed 100-1000x activity against the E. coli strains and 10x activity versus K. pneumoniae.
  • CDD-3002, CDD-2750, and CDD-2901 showed progressively less activity towards E. coli and no activity against the K. pneumoniae strain.
  • CDD-3083 is CDD-2901 with a piperidine ring replacing the pyrazine ring. The spot test, however, showed CDD-3083 has no activity.
  • CDD-3124 is CDD-2901 with a pyrrolidine ring replacing the pyrazine but it also shows no activity in the spot test. Compounds with various substituents on the nitrogen of the pyrazine ring were examined.
  • CDD-3259 is CDD- 2901 with a pyridine ring attached to the pyrazine nitrogen. The spot test revealed CDD-3259 retains activity but is 100-fold less potent compared to CDD-2901.
  • CDD-3295 is CDD-2901 with a guanidinium group at the pyrazine nitrogen. Interestingly, the spot test showed this compound is only 10-fold less potent than CDD-2901 and thus shows 100x activity compared to the colistin only control.
  • Exemplary colistin sulfate minimum inhibitory concentration (MIC) data Cmpd Colistin sulfate MIC ( ⁇ g/mL) - 90 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) concentration E. coli XL1Blue + K. pneumoniae AR E.
  • MIC minimum inhibitory concentration
  • pneumoniae used LB media, and for E. coli it used LB+ chloramphenicol (12.5 ⁇ g/mL) to maintain the plasmid carrying the wild type mcr-1 or T285A mcr-1 mutant gene.
  • the 96-well culture plates were grown for 20.5 hrs at 35°C with 200 revolutions per minute (RPM) orbital shaking.
  • NA Not available
  • ND Not determined
  • ( ) unclear if there was growth, difficult to read; data obtained using microdilution method.
  • Table 6 Exemplary colistin sulfate minimum inhibitory concentration (MIC) data Colistin sulfate MIC ( ⁇ g/mL) Compound E. coli XL1Blue + WT MCR-1 E.
  • Example 6 Testing of certain exemplary compounds for cytotoxicity versus human cells Based on the cytotoxicity results, a structure-activity study was performed to reduce the toxicity of antibiotic compounds. It was hypothesized that the basicity of the secondary - 91 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) amine on the pyrazine ring may contribute to toxicity and therefore synthesized a series of morpholines with oxygen replacing the nitrogen. Table 7 summarizes the HepG2 cellular assay results.
  • Compound CDD-3212 is a morpholine derivative of CDD-2902.
  • CDD-3213 and CDD-3239 are morpholine derivatives of CDD-2901 and CDD-3002 and the spot assay shows a >100-fold decrease in potency.
  • CDD-3240 and CDD-3084 are morpholine derivatives of CDD-3019 and CDD-2901 but also lacking the halide. Spot assay results showed both of these compounds exhibit a >100-fold decrease in potency. Cytotoxicity assays of CDD-3084 with HepG2 cells showed low toxicity with an IC 50 > 100 ⁇ M, suggesting the morpholine modification reduces cytotoxicity.
  • the spot assay indicated that, although it is less potent than CDD-2901, it has 10-100X activity compared to the colistin only control.
  • the IC50 for HepG2 cells is >100 ⁇ M, suggesting this is a candidate for further study.
  • CDD-3003 is the methylated version CDD-3002 and the spot assay revealed it has 100x activity compared to the colistin control.
  • the IC 50 for HepG2 cells is 51 ⁇ M, indicating relatively low toxicity.
  • CDD-3337 is the methylated version of CDD-3228 and the spot assay showed it has 100x activity compared to the colistin control. Interestingly, it has similar activity as the parent CDD-3228.
  • CDD-3350 is the methylated version of CDD-3227, which displayed 100x activity compared to the colistin control in the spot assay. CDD-3350 also showed 100x activity relative to the colistin control. However, the IC 50 of CDD-3350 for HepG2 cells is 28 ⁇ M, indicating moderate toxicity. Based on potency and toxicity, CDD-3003 and CDD-3737 are the top candidates for advancement from the CDD-1938 series. In addition, cytotoxicity assays with human HepG2 cells showed only modest toxicity for CDD-3356 with an IC50 of 53 ⁇ M.
  • coli MCR-1 plays a central role in lipid A modification in E. coli and confers colistin resistance.
  • WT wild type
  • T285A MCR-1 a catalytically inactive mutant expressed from a plasmid were grown in rich media with and without colistin antibiotic over 6 hours up to 24 hours. It was shown that WT MCR-1 de-sensitizes E. coli to various concentrations of colistin, while the lack of a catalytically functional MCR-1 results in the sensitization of E. coli to colistin (FIG. 14 and FIG.22). WT and mutant strains grew similarly without antibiotics (FIG.14).
  • DNA-encoded chemical libraries consisting of a total of 2 billion compounds were screened against purified N-terminal 6xHis-tagged full-length E. coli WT MCR-1.
  • DNA-encoded small molecule/protein complexes were captured using nickel beads - 93 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) and washed. Bound small molecules were released and bound again to fresh MCR-1 protein for two additional rounds of enrichment. After the final elution, the DNA section of the conjugates was amplified by PCR and subjected to deep sequencing. Bioinformatics analysis was used to identify the sequences most highly represented after the binding selection.
  • the spot assay results showed CDD-1938 and CDD-1794 reduced the growth of E. coli/MCR-1 in combination with colistin (FIG.16A).
  • the presence of increasing concentrations of CDD-1938 resulted in progressively greater susceptibility of E. coli/MCR-1; however, a dose-dependent susceptibility was not observed for the concentrations of CDD-1794 tested.
  • the viability of E. coli/MCR-1 and E. coli/T285A on control and test plates was evaluated. For example, on the plates containing the highest inhibitor concentration (12.5 ⁇ M) and 0.32 ⁇ g/mL colistin, E.
  • coli/MCR-1 grew to a 10-fold lower dilution compared to growth on plates containing 0.32 ⁇ g/mL colistin with no inhibitor (FIG.16A). This difference in growth between treatments was scored as a 10X effect for the inhibitors CDD-1938 or CDD-1794, which showed similar results. It was confirmed that MCR-1 was the target of the compounds by testing against E. coli with the catalytically inactive MCR-1 T285A mutant, where the expression of the mutant MCR-1 fails to protect E. coli from colistin. Unlike with E. coli/MCR-1, no effect of the compounds was observed in combination with colistin against E.
  • Example 10 Optimization and prioritization of certain exemplary chemotype I MCR-1 inhibitors The development of compounds that inhibit MCR-1 and show bioactivity in the 1 ⁇ g/mL range in the colistin MIC assay were sought. These potencies would compare favorably with the potencies observed for beta-lactamase inhibitors in clinical use.
  • both chemotype CDD-1938 and CDD-1794 were synthesized and tested for bioactivity as described above.
  • the SAR approach focused on modifying and testing the DECL cycle building blocks for each compound; in this case, both chemotype CDD-1938 and CDD-1794 consisted of three cycle building blocks (BB) each, which were designated as C1 BB, C2 BB and C3 BB (FIGs.15A-15B).
  • Compound CDD-1938 is a racemic mixture with a chiral center at the phenyl substitution on the piperazine ring on the C1 BB (FIG.15A). Both enantiomers were synthesized and tested for activity.
  • CDD-1938 and its more potent analog, CDD-2750 contain a halide appended to the phenyl group at the C1 BB (FIG.15A and FIG.15C).
  • CDD-2835 is CDD-2750 lacking the halide (FIG.26). This compound showed a 100-fold loss in potency relative to CDD-2750, revealing the importance of the halide for inhibitor bioactivity.
  • the chlorine in CDD-2750 was also replaced with fluorine to create CDD-3228, which exhibited similar potency in the spot assay as CDD-2750 (FIG.26).
  • the absence of the linker improved the potency of the inhibitor, and the stereochemistry of the chiral carbon of the piperazine ring did not noticeably impact inhibitor potency.
  • CDD-2901 The pyridine ring in CDD-2750 was replaced with a benzyl ring to create CDD-2901 (FIG.15C) and the spot assay showed no change in potency against E. coli/MCR-1, suggesting the nitrogen was not important for inhibition (FIG.17).
  • the Cl was substituted by an F in CDD-2901, which possesses a benzyl rather than a pyridine ring at C2, to create CDD-3002 (FIG.17C).
  • CDD-3002 displayed similar potency to CDD-2901 based on the spot assay (FIG.17).
  • CDD-2847 and CDD-2902 are the most potent compounds that were identified in the SAR studies for chemotype CDD-1938.
  • Example 11 Activity of certain exemplary chemotype 1 MCR-1 inhibitors against colistin resistant clinical isolates
  • the spot assay was next used to examine the activity of a subset of compounds against a set of five clinically relevant isolates that were obtained from the CDC & FDA Antibiotic Resistance Isolate Bank. These isolates include MDR strains that are validated for - 97 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) the presence of the MCR-1 or MCR-2 colistin resistance genes.
  • the MCR-2 variant shares 81% amino acid identity with MCR-1.
  • coli strains were tested using spot assays with 0.32 ⁇ g/mL CMS and 12.5 ⁇ M compound (FIG.19).
  • a total of seven chemotype 1 compounds were evaluated including CDD-2901, CDD-2750, CDD-3002, CDD-2918, CDD-2902, CDD-3019 and CDD-2847. All of these compounds had been shown to have significant activity in the spot assay ( ⁇ 100X) (FIG.17 and FIG.26) and, for some, by MIC against E. coli/MCR-1 (FIGs.18A-18B).
  • the most potent compound against clinical strains is CDD-2847, which also displayed the highest potency by spot and MIC versus the E. coli/MCR-1 laboratory strain.
  • CDD-2847 showed a 10,000-fold effect compared to the colistin only control for all the E. coli/MCR-1 clinical strains; however, it exhibited a 10-fold effect against the K. pneumoniae strain. Without wishing to be bound by any theory, this may be due to its difficult-to-permeate capsule and the presence of efflux pumps such as the OqxA pump.
  • the low sub-inhibitory concentration of colistin at 0.32 ⁇ g/mL, in combination with the inhibitor, may also affect its efficacy in killing K. pneumoniae. It is hypothesized that killing will be more effective when colistin concentration is increased.
  • CDD-3019, CDD-2902 and CDD-2918 also showed >1000-fold activity against all five of the E.
  • CDD-3002, CDD-2750 and CDD-2901 displayed progressively less activity towards E. coli and no activity against the K. pneumoniae strain.
  • these three latter compounds have an isopropyl on the C3 BB compared to the C3 BB on the former three compounds, which possess the -OH and -OCF3 combination.
  • these compounds displayed potent activity against E. coli clinically relevant strains, but to a lesser degree against K. pneumoniae clinical isolates.
  • CDD-2750, in combination with colistin was tested, as described above, against seven E.
  • Example 12 Cytotoxicity profile of certain exemplary chemotype 1 MCR-1 inhibitors The potential for cytotoxicity was examined by testing a set of potent compounds including CDD-2750, CDD-2901, CDD-2847 and CDD-2902 against the human HepG2 cell line. The viability assays revealed that the compounds show significant cytotoxicity against the HepG2 cells with IC 50 values ranging from 14-21 ⁇ M (Table 8).
  • Cytotoxicity of enhanced potency MCR-1 inhibitors measured against human and mouse hepatocytes IC 50 ( ⁇ M) Compound Primary human Primary mouse HepG2 hepatocytes hepatocytes hepatocytes CDD-2750 21.41 8.63 ⁇ 10 CDD-2901 15.85 ND ⁇ 10 CDD-2847 15.63 9.81 ⁇ 10 CDD-2902 14.02 ND ⁇ 10 Compounds were incubated at different concentrations (0-100 ⁇ M) with human HepG2 and primary human hepatocytes and incubated at room temperature for 24 hours. Viability of cells was determined using the CellTiterGlo assay. Primary human hepatocytes (PHH) were cryopreserved, 100-donor mixed gender.
  • Example 13 Structure-activity studies to reduce toxicity and maintain potency Based on the results described elsewhere herein, additional structure-activity studies were performed to reduce the cytotoxicity of the compounds. It was hypothesized that the basicity of the secondary amine on the piperazine ring contributes to the toxicity and therefore a series of morpholines were synthesized with oxygen replacing the nitrogen at position 1 (FIG.26). Compound CDD-3212 is a morpholine derivative of CDD-2902 (FIG. 26).
  • CDD-3213 and CDD-3239 are morpholine analogs of CDD-2901 and CDD-3002 (FIG.26) and the spot assay revealed a >100-fold reduction in potency (FIG.26).
  • compound CDD-3240 a morpholine derivative of CDD-2902 with the exception that the - OH and -OCF3 substituents are flipped in position on the C3 BB and the chlorine is substituted for a F on the C1 BB
  • CDD-3084 Cytotoxicity assays of CDD-3084 with HepG2 cells showed low toxicity with an IC50 > 100 ⁇ M, indicating the morpholine modification reduces cytotoxicity (FIG.26); however, potency was lost and thus, the morpholine analogs were eliminated as candidates. Other modifications on the C1 BB were also assessed.
  • CDD-3083 is CDD-2901 with a piperidine ring replacing the piperazine ring (FIG.26). The spot test results showed the ring substitution in CDD-3083 led to loss of potency, suggesting the substitution of the nitrogen at the 1 position with a carbon is not well tolerated with respect to inhibitor potency, like the oxygen in the morpholine analogs.
  • CDD-3124 is CDD-2901 with a pyrrolidine ring in place of the piperazine, and it showed no activity in the spot test (FIG.26), indicating the presence of a nitrogen in this N1 position is important for activity. Compounds with various substituents on the nitrogen at position 1 of the piperazine ring were also evaluated.
  • CDD-3259 is CDD-2901 with a pyridine ring attached to the piperazine nitrogen (FIG.26). The spot assay showed CDD-3259 retains some activity but is 100-fold less potent compared to CDD-2901.
  • CDD-3295 is CDD-2901 with a guanidine group at the piperazine N1 (FIG.26).
  • CDD-3260 is CDD-3002 with the fluorobenzene ring at the 2-position of the piperazine ring rather than the 1-position (FIG.26). This substitution caused a >10 reduction in activity and was toxic based on the IC 50 of 19 ⁇ M for HepG2 cells, supporting the idea that the basicity of a free piperazine secondary amine contributes to toxicity.
  • CDD-3035 is 10- to 100-fold more active than the colistin-only control, but it was 10-fold less active than its parent compound.
  • the IC50 of CDD-3035 for HepG2 cells was >100 ⁇ M, indicating that methylation of the piperazine N1 greatly reduces cytotoxicity.
  • CDD-3003 (FIG.15C) is the methylated version of CDD-3002 (FIG.15C) and the spot assay showed it has 100-fold activity compared to the colistin-only control (FIG.17)
  • the IC50 for CDD-3003 for HepG2 cells is 64 ⁇ M, indicating relatively low toxicity.
  • CDD-3337 (FIG.26) is the methylated version of CDD-3228 (FIG.
  • CDD-3350 (FIG.26) is CDD-3337 with -OH and -OCF3 instead of isopropyl on C3 BB, which displayed 1000-fold activity relative to the colistin-only control in the spot assay.
  • the IC 50 of CDD-3350 for HepG2 cells is 28 ⁇ M, pointing to moderate toxicity and suggesting the -OH and -OCF3 on the C3 BB play a role in toxicity.
  • CDD-3003 was selected as the lead candidate for the chemotype CDD-1938 series.
  • Example 14 Optimization and prioritization of MCR-1 chemotype 2 lead inhibitors
  • the original DECL hit for CDD-3358 contained a DNA molecule attached via a linker to the C1 BB.
  • the C2 and C3 substituents branch from the C1 BB.
  • CDD-1794 which lacks the DNA linker, ethyl cyclopentane of the C2 group and halide appended to the C3 phenyl group. Focus was placed on modification of CDD-1794, which in combination with colistin exhibits activity against E. coli/MCR-1 in the spot and MIC assays (FIGs.16A-16B).
  • CDD-3359 was synthesized to examine the effect of the addition of an ethyl indole to the C1 BB.
  • E. coli/MCR-1 grew to a 10-fold lower dilution on plates containing colistin with 12.5 ⁇ M of CDD-3359 compared to the colistin-only control, indicating that this addition results in no change to potency compared to CDD-2832 or CDD-1794 (FIG.27).
  • a different addition a chiral ethylnaphthalene at the C1 BB of CDD-2832 to create CDD-3367.
  • CDD-3357 was synthesized by attaching an ethyl methyl trifluoromethyl benzene ring to the C1 BB in the R-configuration. CDD-3357 showed a similar potency to CDD-3367 (FIG.27). It is unclear if this result indicates that stereochemistry at the chiral center of the two substituents does not matter or if it is the result of the size and nature of the substituents.
  • CDD-3356 (FIG.15D), which is an S- enantiomer with a chiral center at the CH2 of the benzyl substituent on the C1 BB.
  • this addition increased the potency of the inhibitor by 10-fold compared to CDD-2833 (FIG. 27) or any of the other inhibitors tested, suggesting the addition of cyclopropane enhances potency in conjunction with the benzyl ring.
  • This expanded chemical space at the C2 BB may increase the permeability characteristics of the inhibitor or improve binding to MCR-1.
  • CDD-3356 cytotoxicity assays with human HepG2 cells displayed only modest toxicity for CDD-3356 with an IC 50 of 53 ⁇ M versus 44 ⁇ M for CDD-3366, and it is difficult to synthesize the S- and R-enantiomers of CDD-3366.
  • the Cl in CDD-3356 was replaced with an F to create CDD-3682, which exhibited a slight decrease in potency in the spot assay compared to CDD-3356, indicating that Cl exhibits improved activity at the 4-position of the C3 phenyl ring.
  • CDD-3356 is a lead candidate for the CDD-1794-related (chemotype 2) series.
  • Example 15 Activity of certain exemplary lead MCR-1 inhibitors against colistin resistant Klebsiella pneumoniae The MIC assay was used to examine the activity of CDD-3356 and CDD-3003 (Table 9) versus a colistin resistant clinical strain of Klebsiella pneumoniae known to contain mcr-1 and a laboratory strain of E. coli expressing WT MCR-1 or catalytically inactive T285A MCR-1.
  • MIC assays were performed by broth microdilution with 2-fold dilutions of colistin sulfate antibiotic and 0, 6.25, 12.5 and 25 ⁇ M compound.
  • the colistin MIC for mcr-1 + K. pneumoniae was 8 ⁇ g/mL.
  • the colistin MIC was reduced by 16-fold to 0.5 ⁇ g/mL (Table 9), indicating that the inhibitor blocks the colistin resistance mechanism encoded by mcr-1.
  • CDD-3003 was also able to reduce the colistin MIC, but less so with an 8-fold reduction at 12.5 ⁇ M (Table 9).
  • CDD-3356 Based on the MIC data, CDD-3356 displays progressively more activity against K. pneumoniae mcr-1 + as its concentration increases when combined with colistin. For colistin/CDD-3003, an abrupt change in colistin MIC occurs at 12.5 ⁇ M, but not at lower inhibitor concentrations. Four-fold less CDD-3356 is needed to re-sensitize K. pneumoniae mcr-1 + at an equivalent level compared to CDD-3003 suggesting CDD-3356 is the more potent inhibitor against this isolate. K.
  • Optimized inhibitors resensitize a Klebsiella pneumoniae clinical isolate and laboratory E. coli carrying wildtype MCR-1 in a dose-dependent manner to colistin killing Colistin sulfate MIC ( ⁇ g/mL) Cmpd K. pneumoniae AR E. coli XL1Blue + WT E.
  • bacterial - 104 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) growth ends at 4 hours, while killing occurs at 5 hours post-treatment with 4 ug/mL colistin and 3.125 or 1.56 ⁇ M CDD-3003 (FIG.20A).
  • Complete killing of E. coli/MCR-1 with the combination treatment occurs by 1.5 hours for CDD-3356 when using 12.5 ⁇ M of the compound, which is at least 2.5 hours before killing occurs with colistin alone.
  • Colistin with CDD-3356 concentrations of 1.56 ⁇ M through 6.25 ⁇ M show varying inhibitory effects with killing between 3 and 4 hours (FIG.20B).
  • the TKC data support that CDD-3003 and CDD- 3356 achieve a similar killing endpoint for E. coli/MCR-1 as seen in the spot assay (FIG.17).
  • a similar response was observed in the colistin MIC results for the K. pneumoniae mcr-1+ strain in the presence of CDD-3003 or CDD-3356 (Table 9).
  • Embodiment 2 provides the compound of Embodiment 1, wherein the compound of Formula (I) is selected from the group consisting of: , . the compound of Formula (I) is selected from the group consisting of: R 4 2), 2).
  • R 1 is selected from the group consisting of: - 106 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) , wherein R 5a , R 5b , R 5c , R 5d , selected from the group consisting of H, halogen, , substituted C 1 -C 6 alkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl.
  • Embodiment 5 provides the compound of Embodiment 4, wherein at least one of R 5a , R 5b , R 5c , R 5d , and R 5e is phenyl, optionally wherein the phenyl is substituted with at least one selected from the group consisting of OH, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 1 -C 6 alkyl, and C1-C6 haloalkyl.
  • Embodiment 6 provides the compound of Embodiment 4 or 5, wherein R 5a , R 5b , R 5c , R 5d , and R 5e , if present, are each independently selected from the group consisting of H, CH3, .
  • Embodiment 10 provides the compound of Embodiment of 9, wherein R 6a , R 6b , R 6c , R 6d , and R 6e are each independently selected from the group consisting of H, F, and Cl, optionally wherein one of R 6a , R 6b , R 6c , R 6d , and R 6e is F or Cl.
  • Embodiments 1-12 provides the compound of any one of Embodiments 1-12, which is selected from the group consisting of: 4-(3-(4-chlorophenyl)-4-((3-(2-isopropylphenyl)pyridin-4-yl)methyl)piperazine-1- carbonyl)-N-methylbenzamide; - 108 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) (S)-4-(3-(4-chlorophenyl)-4-((3-(2-isopropylphenyl)pyridin-4-yl)methyl)piperazine-1- carbonyl)-N-methylbenzamide; (R)-4-(3-(4-chlorophenyl)-4-((3-(2-isopropylphenyl)pyridin-4-yl)methyl)piperazine- 1-carbonyl)-N-methylbenzamide; 2-(4-fluorophenyl)-1-((2
  • Embodiment 15 provides the compound of Embodiment 14, wherein at least one of the following applies: (a) at least one of R 7a , R 7b , R 7c , and R 7d is H; (b) at least two of R 7a , R 7b , R 7c , and R 7d are H; (c) at least three of R 7a , R 7b , R 7c , and R 7d are H; and (d) each of R 7a , R 7b , R 7c , and R 7d are H.
  • Embodiment 16 provides the compound of Embodiment 14 or 15, wherein each occurrence of R 7e and R 7f is independently selected from the group consisting of H, CH 3 , .
  • Embodiments 14-16 wherein R 8 is phenyl optionally substituted with at least one halogen.
  • Embodiment 18 provides the compound of any one of Embodiments 14-17, wherein R 8 is selected from the group consisting of phenyl, 4-chlorophenyl, and 4-fluorophenyl.
  • Embodiment 19 provides the compound of any one of Embodiments 14-18, wherein - 114 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) L 3 is selected from the group consisting of a bond, -(CH2)-, -(CHCH3)-, - .
  • Embodiment 21 provides the compound of Embodiment 20, wherein the phenyl, naphthyl, or indolyl is optionally substituted with at least one C1-C6 haloalkyl, optionally wherein the haloalkyl is CF 3 .
  • Embodiment 22 provides the compound of any one of Embodiments 14-21, wherein , , .
  • R 10a and R 10b are each independently selected from the group consisting of H, C1-C6 alkyl, and optionally substituted C 3 -C 6 cycloalkyl, optionally wherein R 10a and R 10b are each - 115 - 55708207.3 Attorney Docket No.046641-7064WO1(00175) independently selected from the group consisting of H, CH3, .
  • Embodiment 27 provides a pharmaceutical composition comprising at least one compound of any one of Embodiments 1-26 and a pharmaceutically acceptable carrier or excipient.
  • Embodiment 28 provides the pharmaceutical composition of Embodiment 27, further comprising at least one additional agent suitable for treating, preventing, and/or ameliorating a bacterial infection.
  • Embodiment 29 provides the pharmaceutical composition of Embodiment 28, wherein the at least one additional agent suitable for treating, preventing, and/or ameliorating a - 118 - 55708207.3
  • Attorney Docket No.046641-7064WO1(00175) bacterial infection is selected from the group consisting of a polymyxin, aminoglycoside, ⁇ - lactam (e.g., penicillin, cephalosporin, or carbapenem), monobactam, fluoroquinolone, sulfonamide, tetracycline, and macrolide.
  • Embodiment 30 provides the pharmaceutical composition of Embodiment 28 or 29, wherein the additional agent suitable for treating, preventing, and/or ameliorating a bacterial infection is at least one selected from the group consisting of colistin (polymyxin E), polymyxin B, amikacin, ampicillin, amoxicillin aminoglycoside, azithromycin, aztreonam, carbapenem, cefepime, cefiderocol, cefotaxime, ceftriaxone, ceftaroline, ceftazidime, ceftobiprole, ceftolozane, ciprofloxacin, clindamycin, dalbavancin, daptomycin, doxycycline, ertapenem, fluoroquinolone, gentamicin, imipenem, levofloxacin, linezolid, meropenem, minocycline, mupirocin, oritavancin, piperacillin
  • Embodiment 31 provides a method of treating, preventing, and/or ameliorating a bacterial infection in a subject, the method comprising administering to the subject: (a) at least one compound of any one of Embodiments 1-26 or the pharmaceutical composition of any one of Embodiments 27-30; and (b) at least one polymyxin antibiotic, or an analogue or derivative thereof.
  • Embodiment 32 provides a method of sensitizing a bacterial colony to at least one antibiotic polypeptide, the method comprising contacting the bacterial colony with at least one compound of any one of Embodiments 1-26, or the pharmaceutical composition of any one of Embodiments 27-30.
  • Embodiment 33 provides the method of Embodiment 31 or 32, wherein the antibiotic peptide is at least one selected from the group consisting of a polymyxin antibiotic and a defensin, optionally wherein the polymyxin antibiotic is selected from the group consisting of colistin (polymyxin E) and polymyxin B.
  • the antibiotic peptide is at least one selected from the group consisting of a polymyxin antibiotic and a defensin, optionally wherein the polymyxin antibiotic is selected from the group consisting of colistin (polymyxin E) and polymyxin B.
  • Embodiment 34 provides the method of any one of Embodiments 31-33, wherein the bacterial infection or bacterial colony comprises a bacterial species selected from the group consisting of: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdenensis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus warnerii, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus pettenkoferi, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Group C streptococci, Streptococcus constellatus, Enterococcus faecalis, Enterococcus faecium, Corynebacterium jeikeium, Lac
  • Embodiment 35 provides the method of any one of Embodiments 31-34, wherein the method further comprises administering at least one additional antibiotic.
  • Embodiment 36 provides the method of Embodiment 35, wherein the additional antibiotic is at least one selected from the group consisting of a aminoglycoside, ⁇ -lactam (e.g., penicillin, cephalosporin, or carbapenem), monobactam, fluoroquinolone, sulfonamide, tetracycline, and macrolide.
  • ⁇ -lactam e.g., penicillin, cephalosporin, or carbapenem
  • monobactam e.g., fluoroquinolone, sulfonamide, tetracycline, and macrolide.
  • Embodiment 37 provides the method of Embodiment 35 or 36, wherein the additional antibiotic is at least one selected from the group consisting of amikacin, aminoglycoside, azithromycin, aztreonam, carbapenem, cefepime, ceftriaxone, ceftaroline, ceftazidime, ceftobiprole, ceftolozane, ciprofloxacin, clindamycin, dalbavancin, daptomycin, doxycycline, ertapenem, fluoroquinolone, gentamicin, imipenem, levofloxacin, linezolid, meropenem, minocycline, mupirocin, oritavancin, piperacillin, streptogramin, tedizolid, telavancin, tigecycline, ticarcillin, tobramycin, trimethoprim/sulfamethoxazole, and vancomycin
  • Embodiment 38 provides the method of any one of Embodiments 31 and 33-37, wherein the bacterial infection is a persistent or antibiotic resistant bacterial infection.
  • Embodiment 39 provides the method of any one of Embodiments 31-38, wherein formation of a bacterial biofilm is at least partially prevented or inhibited.
  • Embodiment 40 provides the method of any one of Embodiments 31 and 33-39, wherein the infection occurs on a prosthesis or an implant.
  • Embodiment 41 provides the method of Embodiment 40, wherein the prosthesis selected from the group consisting of a knee prosthesis, a hip prosthesis, elbow prosthesis, ankle prosthesis, shoulder prosthesis, and spine prosthesis.
  • Embodiment 42 provides the method of any one of Embodiments 31 and 33-41, wherein the subject is a mammal.
  • Embodiment 43 provides the method of any one of Embodiments 31 and 33-42, wherein the subject is a human.
  • the terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application.

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Abstract

Selon certains aspects, la divulgation concerne des inhibiteurs de l'enzyme de résistance acquise à la colistine (MCR) (MCR-1), des compositions pharmaceutiques de ceux-ci, et des procédés d'utilisation de ceux-ci pour traiter, prévenir et/ou améliorer des infections bactériennes et/ou sensibiliser des bactéries à des antibiotiques à base de polymyxines.
PCT/US2025/033970 2024-06-17 2025-06-17 Inhibiteurs de l'enzyme de résistance acquise à la colistine (mcr-1) et leurs procédés d'utilisation Pending WO2025264672A1 (fr)

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DATABASE PUBCHEM COMPOUND 5 December 2007 (2007-12-05), XP093386351, Database accession no. 21527900 *
MUCCIOLI GIULIO G., FAZIO NICOLA, SCRIBA GERHARD K. E., POPPITZ WOLFGANG, CANNATA FABIO, POUPAERT JACQUES H., WOUTERS JOHAN, LAMBE: "Substituted 2-Thioxoimidazolidin-4-ones and Imidazolidine-2,4-diones as Fatty Acid Amide Hydrolase Inhibitors Templates", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 49, no. 1, 1 January 2006 (2006-01-01), US , pages 417 - 425, XP093386345, ISSN: 0022-2623, DOI: 10.1021/jm050977k *
NADIR UPENDER K., VIJAYA KRISHNA R.: "Facile cleavage of i N /i arylsulfonyl bond of i N /i ‐arylsulfonylimidazolidinone with magnesium in methanol", JOURNAL OF HETEROCYCLIC CHEMISTRY, WILEY-BLACKWELL PUBLISHING, INC., US, vol. 41, no. 5, 1 September 2004 (2004-09-01), US , pages 737 - 739, XP093386346, ISSN: 0022-152X, DOI: 10.1002/jhet.5570410514 *

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