Multiple mechanisms of LPXC inhibitors potentiate potency versus neisseria gonorrhoeae

Abstract

The present disclosure relates to phenyl isoxazole compounds, compositions, and methods for inhibiting LpxC activity. The disclosed compounds and compositions can be useful in, for example, the treatment of infections associated with LpxC activity (e.g., gram -negative bacterial infection). This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Description

Attorney Docket No.37759.0695P1MULTIPLE MECHANISMS OF LPXC INHIBITORS POTENTIATE POTENCY VERSUS NEISSERIA GONORRHOEAECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U. S. Application No. 63 / 774984, filed on March 20, 2025, and U. S. Application No. 63 / 945,417, filed on December 19, 2025, the contents of which are incorporated herein by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under BX000727 awarded by the US Department of Veterans Affairs. The government has certain rights in the invention.BACKGROUND

[0003] Gonorrhea is a growing public health threat due to an increase in multidrug resistant (MDR) strains. The WHO estimates there were 82.4 million new gonorrhea cases in 2020 worldwide (WHO. 2021. Global progress report on HIV, viral hepatitis and sexually transmitted infections, 2021. Geneva). A total of 601,319 cases of gonorrhea among all age groups were reported to the CDC in 2023, making it the second most commonly reported STI in the United States (CDC. 2023. Sexually Transmitted Diseases Surveillance, 2023). Due to the occurrence of asymptomatic infections, reported rates reflect screening levels as well as incidence.Furthermore, MDR gonorrhea may be globally underestimated because most of the reports come from wealthier countries, which have the minority of gonorrhea cases.

[0004] Development of new therapeutics for unique targets is an urgent need. A number of small molecule LpxC inhibitors targeting the active site of the LpxC enzyme (UDP-3-O-(R-3-hydroxymyristoyl)- / V-acetylglucosamine deacetylase), which catalyzes the rate-limiting second step in lipid A biosynthesis in Gram-negative bacteria, have been developed (lackman JE, et al., 1999. Biochemistry 38: 1902-11). Inhibition of LpxC is intended to halt the biosynthesis of lipid A, which anchors the lipooligosaccharide (LOS) or lipopolysaccharide (LPS) to the outer membrane. This kills Neisseria gonorrhoeae because it is incapable of surviving without LOSAttorney Docket No.37759.0695P1(Raetz CR, et al., 2002 Annu Rev Biochem 71:635-700; van der Ley P, et al., 2003J Endotoxin Res 9:124-8; Kalinin DV, et al., 2016 Curr Top Med Chem 16:2379-430). LOS as an endotoxin is the major causation of the inflammation that occurs in gonococcal sequelae (Beutler B. 2004 Mol Immunol 40:845-59; Liu M, et al., 2014 J Immunol 192:1768-77) (7, 8), such as pelvic inflammatory disease. Thus, while inhibiting the LpxC enzyme can be bactericidal by reducing the levels of LOS, it should also reduce the occurence of serious symptoms induced by inflammation (McGee ZA, et al., 1992 Microb Pathog 12:333-41; Fujita K, et al., 2022 J Antibiot (Tokyo) 75:136-145; Yang Y, et al., 2020 J Reprod Immunol 142:103192).

[0005] LpxC inhibitors were initially discovered in the mid-1980s. The initial interest spurred by the great need for new antimicrobial agents waned primarily due to lack of potency o Pseudomonas aeruginosa and enteric bacteria (Erwin AL. 2016. Cold Spring Harb Perspect Med 6). Interest was revitalized when researchers at the University of Washington and Chiron collaborated in the development of LpxC inhibitors that were more potent for P. aeruginosa in the early 2000s (Erwin AL. 2016. Cold Spring Harb Perspect Med 6; Krause KM, et al., 2019 Antimicrob Agents Chemother 63:e00977-19). It was previously reported that two of the methylsulfone hydroxamic acid LpxC inhibitors developed by scientists at Pfizer were bactericidal for human challenge and MDR strains of N. gonorrhoeae (John CM, et al., 2018 J Antimicrob Chemother 73:2064-2071). However, these inhibitors have high rates of clearance and protein binding, low solubility (Montgomery JI, et al., 2012 J Med Chem 55:1662-70) (15), and a hydroxamate head group moiety, which is theorized to contribute to cardiotoxicity via nonspecific metal ion chelation (Fujita K, et al., 2022 J Antibiot (Tokyo) 75:98-107; Shen S, Kozikowski AP. 2016. ChemMedChem 11:15-21). Two LpxC inhibitors, ACHN-975 (Achaogen) and RC-01 (Reci da Therapeutics), which both have hydroxamate-containing head groups, reached clinical trials but these were halted after Phase 1 due to safety concerns (Cohen F, et al., 2019 ChemMedChem 14: 1560-1572; Medicine UNLo. 2013. NCT01870245. https: / / clinicaltrials.gov / study / NCT01870245. Accessed 3 / 21 / 2025; Medicine UNLo. 2019. NCT03832517 https: / / clinicaltrials.gov / study / NCT03832517. Accessed 3 / 21 / 2025) (18-20). Importantly, since then two LpxC inhibitors, TP0586532 and LPC-233, that do not cause cardiovascular toxicity in animal studies have been reported (Ushiyama F, et al., 2021 Bioorg Med Chem 30:115964; Zhao J, et al., 2023 Sci Transl Med 15:eadf5668) (21, 22).Attorney Docket No.37759.0695P1

[0006] Accordingly, there remains a need for compounds that inhibit LpxC activity with improved specificity and potency and improved toxicity profiles, and methods of making and using same.SUMMARY

[0007] In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the invention, in one aspect, relates to phenyl isoxazole compounds, compositions, and methods for inhibiting LpxC activity. The disclosed compounds and compositions can be useful in, for example, the treatment of infections associated with LpxC activity (e.g., gram-negative bacterial infection).

[0008] Thus, disclosed are compounds having a structure represented by a formula:wherein n is an integer selected from 1 and 2; wherein Z1is selected from -N- and -CH-; wherein Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b-; wherein R10is selected from hydrogen and C1-C4 alkyl; wherein each of R11aand R11bis independently selected from hydrogen and methyl; and wherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof.

[0009] Also disclosed are pharmaceutical compositions comprising a therapeutically effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0010] Also disclosed are methods of inhibiting LpxC activity in a cell, the method comprising contacting the cell with an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof.Attorney Docket No.37759.0695P1

[0011] Also disclosed are methods for inhibiting LpxC activity in a subject, the method comprising administering to the subject an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof.

[0012] Also disclosed are methods for treating a Gram-negative bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof.

[0013] Also disclosed are kits comprising a disclosed compound, or a pharmaceutically acceptable salt thereof, and one or more selected from: (a) an iron chelator; (b) an antibacterial agent; (c) instructions for administering the compound in connection with a Gram-negative bacterial infection; and (d) instructions for treating a Gram-negative bacterial infection.

[0014] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.BRIEF DESCRIPTION OF THE FIGURES

[0015] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

[0016] FIG. 1 shows representative in silico docking models of FA1090 LpxC enzyme generated by AlphaFold with compounds LI, PF, and CHIR-090.

[0017] FIG. 2 shows representative data illustrating the bactericidal activity of increasing concentrations (0.008, 0.04, 0.2, 1.0, 5.0 and 25 pg / mL) of PF, LI, L2, L3, and L4 LpxC inhibitors for FA1090, F89, and H041 strains of N. gonorrhoeae.Attorney Docket No.37759.0695P1

[0018] FTG.3A and FIG. 3B show representative data for LDH of ME-180 and THP-1 cells after 24 hour treatment with PF, LI, or L3 and hemolysis levels of sheep red blood cells treated with PF, LI, or L3 for 1 hour.

[0019] FIG. 4 shows representative data illustrating the bactericidal assay with FA1090 and increasing concentrations (0.2, 1.0, 5.0 and 25 pg / mL) of PF, LI, and L3 in the presence of a confluent monolayer of Endl / E6E7, ME-180, or THP-1 cells.

[0020] FIG. 5 shows representative data illustrating the bactericidal activity of increasing concentrations (0.2, 1.0, 5.0 and 25 pg / mL) of PF, LI and L3 LpxC inhibitors for sialylated FA1090, F89, and H041 bacteria.

[0021] FIG. 6 shows representative data for ELISA of TNF-a released from activated THP-1 cells after 18 hour treatment with FA1090, F89, or H041 and PF, LI, or L3.

[0022] FIG. 7 shows representative data illustrating for ELISA of 2C7 monoclonal antibody binding to FA1090 after 4 hour treatment with LI at 0.2 or 1.0 pg / mL concentrations.

[0023] FIG. 8 shows representative data for the results of bactericidal assays with H041 treated with PF, LI, and CHIR with or without 50 pg / mL PApN.

[0024] FIG. 9 shows representative spectral scans of antibiotics in the presence of FeC13 from 300-700 nm in 2-nm increments (3:1).

[0025] FIG. 10 shows representative growth curves of FA1090, F89, and H041 treated with vehicle, DFO, LI, and / or PF over 8 hours.

[0026] FIG. 11A and FIG. 11B show a schematic illustrating resistant mutant development that reveal alternative mechanisms of resistance to LI and PF.

[0027] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0028] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.Attorney Docket No.37759.0695P1

[0029] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0030] While aspects of this disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of this disclosure can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order.Accordingly, where a method claim does not specifically state in the claims or description that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0031] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior invention. Further, stated publication dates may be different from actual publication dates, which can require independent confirmation.A. DEFINITIONS

[0032] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, forAttorney Docket No.37759.0695P1example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like.

[0033] As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.”

[0034] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0035] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0036] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight ofAttorney Docket No.37759.0695P1component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0037] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0038] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0039] As used herein, the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0040] As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g, a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is aAttorney Docket No.37759.0695P1human. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).

[0041] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0042] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.

[0043] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0044] As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of theAttorney Docket No.37759.0695P1treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[0045] As used herein, “dosage form” means a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. A dosage form can comprise a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, in combination with a pharmaceutically acceptable excipient, such as a preservative, buffer, saline, or phosphate buffered saline. Dosage forms can be made using conventional pharmaceutical manufacturing and compounding techniques. Dosage forms can comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjustment agents (e.g, hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha- tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxy cholate), solution and / or cryo / lyo stabilizers (e.g, sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g, polydimethylsilozone), preservatives (e.g, thimerosal, 2-phenoxy ethanol, EDTA), polymeric stabilizers and viscosity-adjustment agents (e.g, polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). A dosage form formulated for injectable use can have a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, suspended in sterile saline solution for injection together with a preservative.Attorney Docket No.37759.0695P1

[0046] As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0047] As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc ), brief or detailed protocols for using the kit, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.

[0048] As used herein, the terms “therapeutic agent” include any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and / or physiologic effect by local and / or systemic action. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14thedition), the Physicians' Desk Reference (64thedition), and The Pharmacological Basis of Therapeutics (12thedition), and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. For example, the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, butAttorney Docket No.37759.0695P1not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) including both double- and single-stranded molecules, gene constructs, expression vectors, antisense molecules and the like), small molecules (e.g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas. The term "therapeutic agent" also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or pro- drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

[0049] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0050] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimedAttorney Docket No.37759.0695P1compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0051] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption.Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by fdtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0052] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety isAttorney Docket No.37759.0695P1actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more -OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more -CO(CH2)8CO- moi eties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

[0053] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (z.e., further substituted or unsubstituted).

[0054] In defining various terms, “A1,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0055] The term “aliphatic” or “aliphatic group,” as used herein, denotes a hydrocarbon moiety that may be straight-chain ( / .<?., unbranched), branched, or cyclic (including fused, bridging, and spirofused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.Attorney Docket No.37759.0695P1

[0056] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, / / -propyl, isopropyl, / / -butyl, isobutyl, s-butyl, / -butyl, / / -pentyl, isopentyl, -pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfooxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also be a Cl alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, Cl -CIO alkyl, and the like up to and including a C1-C24 alkyl.

[0057] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. Alternatively, the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g. fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted with two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “aminoalkyl” specifically refers to an alkyl group that is substituted with one or more amino groups. The term “hydroxyalkyl” specifically refers to an alkyl group that is substituted with one or more hydroxy groups. When “alkyl” is used in one instance and a specific term such as “hydroxyalkyl” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like.

[0058] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substitutedAttorney Docket No.37759.0695P1alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0059] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. For example, the cycloalkyl group and heterocycloalkyl group can be substituted with 0, 1, 2, 3, or 4 groups independently selected from C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4 alkoxy, -NH2, (C1-C4) alkylamino, (C1-C4)(C1-C4) dialkylamino, ether, halogen, -OH, C1-C4 hydroxyalkyl, -NO2, silyl, sulfo-oxo, -SH, and C1-C4 thioalkyl, as described herein.

[0060] The term “polyalkylene group” as used herein is a group having two or more CH2 groups linked to one another. The polyalkylene group can be represented by the formula — (CH2)a —, where “a” is an integer of from 2 to 500.

[0061] The terms “alkoxy” and “alkoxy!” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as — OA1where A1is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a poly ether such as — OA1— OA2or — OA1— (OA2)a — OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are alkyl and / or cycloalkyl groups.

[0062] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A’A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl,Attorney Docket No.37759.0695P1alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0063] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. For example, the cycloalkenyl group and heterocycloalkenyl group can be substituted with 0, 1, 2, 3, or 4 groups independently selected from C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4 alkoxy, C2-C4 alkenyl, C3-C6 cycloalkenyl, C2-C4 alkynyl, aryl, heteroaryl, aldehyde, –NH2, (C1-C4) alkylamino, (C1-C4)(C1-C4) dialkylamino, carboxylic acid, ester, ether, halogen, -OH, C1-C4 hydroxyalkyl, ketone, azide, -NO2, silyl, sulfo-oxo, -SH, and Cl-C4 thioalkyl, as described herein.

[0064] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0065] The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy,Attorney Docket No.37759.0695P1alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0066] The term “aromatic group” as used herein refers to a ring structure having cyclic clouds of delocalized 7t electrons above and below the plane of the molecule, where the n clouds contain (4n+2) n electrons. A further discussion of aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497, incorporated herein by reference. The term “aromatic group” is inclusive of both aryl and heteroaryl groups.

[0067] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, — NEE, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” In addition, the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carbon-carbon bond. For example, biaryl can be two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0068] The term “aldehyde” as used herein is represented by the formula — C(O)H.Throughout this specification “C(O)” or “CO” is a short hand notation for a carbonyl group, i.e., C=O.

[0069] The terms “amine” or “amino” as used herein are represented by the formula — NA1A2, where A1and A2can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is — NH2.

[0070] The term “alkylamino” as used herein is represented by the formula — NH(-alkyl) where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropyl ami no group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, and the like.Attorney Docket No.37759.0695P1

[0071] The term “dialkylamino” as used herein is represented by the formula — N(-alkyl)2 where alkyl is a described herein. Representative examples include, but are not limited to, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

[0072] The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

[0073] The term “ester” as used herein is represented by the formula — OC(O)A1or — C(O)OA1, where A1can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula — (A1O(O)C-A2-C(O)O)a— or — (A1O(O)C-A2-OC(O))a —, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.

[0074] The term “ether” as used herein is represented by the formula A1OA2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term “polyether” as used herein is represented by the formula — (A1O-A2O)a—, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0075] The terms “halo,” “halogen,” or “halide” as used herein can be used interchangeably and refer to F, Cl, Br, or I.

[0076] The terms “pseudohalide,” “pseudohalogen,” or “pseudohalo” as used herein can be used interchangeably and refer to functional groups that behave substantially similar to halides. Such functional groups include, by way of example, cyano, thiocyanate, azido, trifluorom ethyl, trifluoromethoxy, perfluoroalkyl, and perfluoroalkoxy groups.

[0077] The term “heteroalkyl” as used herein refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein theAttorney Docket No.37759.0695P1nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0078] The term “heteroaryl” as used herein refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. The heteroaryl group can be substituted or unsubstituted. The heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl groups can be monocyclic, or alternatively fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further not limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzoftZjoxazolyl, benzo[ ]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[l,2-b]pyridazinyl, imidazo[l,2-a]pyrazinyl, benzofc] [1, 2, 5]thiadiazolyl, benzo[c][l,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0079] The terms “heterocycle” or “heterocyclyl” as used herein can be used interchangeably and refer to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term is inclusive of, but not limited to, “heterocycloalkyl,” “heteroaryl,” “bicyclic heterocycle,” and “polycyclic heterocycle.” Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2, 3 -oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2, 3 -thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridazine, pyrazine, triazine, including 1,2,4-triazine and 1,3,5-triazine, tetrazine, including 1,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be a C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10Attorney Docket No.37759.0695P1heterocyclyl, C2-C11 heterocyclyl, and the like up to and including a C2-C18 heterocyclyl. For example, a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, and the like. Alternatively, for example, a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.

[0080] The term “bicyclic heterocycle” or “bicyclic heterocyclyl” as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, or wherein an aromatic ring is fused with a non-aromatic ring. Bicyclic heterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[l,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-l,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, lH-pyrazolo[4,3-c]pyridin-3-yl; 1H-pyrrolo[3,2-b]pyridin-3-yl; and lH-pyrazolo[3,2-b]pyridin-3-yl.

[0081] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi-and tricyclic ring systems. The heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0082] The term “hydroxy” or “hydroxyl” as used herein is represented by the formula — OH.

[0083] The term “ketone” as used herein is represented by the formula A1C(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.Attorney Docket No.37759.0695P1

[0084] The term “azide” or “azido” as used herein is represented by the formula — N3.

[0085] The term “nitro” as used herein is represented by the formula — NO2.

[0086] The term “nitrile” or “cyano” as used herein is represented by the formula — CN or — C=N.

[0087] The term “silyl” as used herein is represented by the formula — SiA^A3, where A1, A2, and A3can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0088] The term “sulfo-oxo” as used herein is represented by the formulas — S(O)A — S(O)2A1, — OS(O)2A1, or — OS(O)2OA1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.Throughout this specification “S(O)” is a shorthand notation for S=O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2A1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfone” as used herein is represented by the formula A1S(O)2A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by the formula A1S(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0089] The term “thiol” as used herein is represented by the formula — SH.

[0090] “R1,” “R2,” “R3,” “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.Attorney Docket No.37759.0695P1

[0091] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogen of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.Combinations of substituents envisioned by this invention are those that result in the formation of stable or chemically feasible compounds. In is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0092] The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain aspects, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0093] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)o 4R0; -(CH2)o 4OR0; -0(CH2)o-4R°, -O-(CH2)O4C(O)ORO; -(CH2)O 4CH(ORO)2; -(CH2)O4SRO; -(CH2)O4Ph, which may be substituted with R°; -(CH2)o 40(CH2)o iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)o^O(CH2)o i -pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)O-4N(R°)2; -(CH2)O-4N(R0)C(0)R°; -N(R°)C(S)R°; -(CH2)O-4N(RO)C(O)NR°2; -N(RO)C(S)NR°2; -(CH2)O4N(RO)C(O)OR°; - N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)o-iC(0)R°; -C(S)R°; -(CH2)o 4C(O)ORO; -(CH2)O^C(0)SR°; -(CH2)o^C(0)OSiR°3; -(CH2)o4OC(O)R°; -OC(0)(CH2)O -4SR-, SC(S)SR°; -(CH2)Q-ISC(O)RO; -(CH2)o4C(O)NR°2; -C(S)NR°2; -C(S)SR°; -(CH2)O4OC(O)NRO2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)RO; -C(NOR°)R°; -(CH2)o 4SSR0, -(CH2)O-IS(0)2R0; -(CH2)(wS(0)20Ro; -(CH2)o ^OS(O)2R°; -S(O)2NR°2; -(CH2)O 4S(O)RO: -N(RO)S(O)2NR°2; -N(RO)S(O)2R°; -N(OR°)R°; -C(NH)NRO2; -P(O)2R°; -P(O)R°2; -OP(O)RO2; -OP(O)(OR°)2; SiR°3; -(Ci-4 straight or branched alkylene)O-N(R°)2; or -(Ci-4 straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH2Ph, -0(CH2)oAttorney Docket No.37759.0695P1iPh, -CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0094] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o-2R*, -(haloR*), -(CH2)o-20H, -(CH2)o2OR*, -(CH2)o-2CH(OR*)2; -O(haloR’), -CN, -N3, -(CH2)o2C(O)R", -(CH2)o2C(O)OH, -(CH2)o2C(O)OR*, - (CH2)O2SR*, -(CH2)O2SH, -(CH2)O2NH2, -(CH2)O2NHRe, -(CH2)O2NR*2, -NO2, -SiR*3, - OSiR*3, -C(O)SR*, - (Ci^i straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Ci-4 aliphatic, -CH2Ph, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0095] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*,=NNHS(O)2R*, =NR*, =N0R*, -O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, wherein each independent occurrence of R is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0- 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*2)2 3O-, wherein each independent occurrence of R is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0096] Suitable substituents on the aliphatic group of R* include halogen, - R*, -(haloR*), -OH, -OR’, -O(haloR’), -CN, -C(O)OH, -C(O)ORe, -NH2, -NHR’, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with oneAttorney Docket No.37759.0695P1or more halogens, and is independently Ci^i aliphatic, -CH2Ph, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0097] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R1', -NRT, -C(O)Rf, -C(O)ORf, -C(O)C(O)Rt, -C(O)CH2C(O)Rt, -S(O)2Rt, -S(O)2NRf2, -C(S)NRt2, -C(NH)NRt2, or -N^) 8(0)^; wherein each R* is independently hydrogen, Ci-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R\ taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0098] Suitable substituents on the aliphatic group of R:are independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci^i aliphatic, -CH2Ph, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0099] The term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, and brosylate.

[0100] The terms “hydrolysable group” and “hydrolysable moiety” refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions. Examples of hydrolysable residues include, without limitation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, “Protective Groups in Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience, 1999).

[0101] The term “organic residue” defines a carbon containing residue, z.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen,Attorney Docket No.37759.0695P1nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited to alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

[0102] A very close synonym of the term “residue” is the term “radical,” which as used in the specification and concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, a 2,4-thiazolidinedione radical in a particular compound has the structure:Oregardless of whether thiazolidinedione is used to prepare the compound. In some embodiments the radical (for example an alkyl) can be further modified (z.e., substituted alkyl) by having bonded thereto one or more “substituent radicals.” The number of atoms in a given radical is not critical to the present invention unless it is indicated to the contrary elsewhere herein.

[0103] “Organic radicals,” as the term is defined and used herein, contain one or more carbon atoms. An organic radical can have, for example, 1-26 carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In a further aspect, an organic radical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen bound to at least some of the carbon atoms of the organic radical. One example of an organic radical that comprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthyl radical. In some embodiments, an organic radical can contain 1-10 inorganic heteroatoms bound thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organic radicals include but are not limited to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino, di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkyl carb oxami de, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or substitutedAttorney Docket No.37759.0695P1heterocyclic radicals, wherein the terms are defined elsewhere herein. A few non-limiting examples of organic radicals that include heteroatoms include alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals, dimethylamino radicals and the like.

[0104] “Inorganic radicals,” as the term is defined and used herein, contain no carbon atoms and therefore comprise only atoms other than carbon. Inorganic radicals comprise bonded combinations of atoms selected from hydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, and halogens such as fluorine, chlorine, bromine, and iodine, which can be present individually or bonded together in their chemically stable combinations. Inorganic radicals have 10 or fewer, or preferably one to six or one to four inorganic atoms as listed above bonded together. Examples of inorganic radicals include, but not limited to, amino, hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonly known inorganic radicals. The inorganic radicals do not have bonded therein the metallic elements of the periodic table (such as the alkali metals, alkaline earth metals, transition metals, lanthanide metals, or actinide metals), although such metal ions can sometimes serve as a pharmaceutically acceptable cation for anionic inorganic radicals such as a sulfate, phosphate, or like anionic inorganic radical. Inorganic radicals do not comprise metalloids elements such as boron, aluminum, gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gas elements, unless otherwise specifically indicated elsewhere herein.

[0105] Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.

[0106] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in usingAttorney Docket No.37759.0695P1racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0107] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Ingold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0108] Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically labeled or isotopically substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,13N,18O,170,33S,18F and36C1, respectively. Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this invention. Certain isotopically labeledAttorney Docket No.37759.0695P1compounds of the present invention, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays.Tritiated, z. e?.,3H, and carbon-14, i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.

[0109] The compounds described in the invention can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates. Unless stated to the contrary, the invention includes all such possible solvates.

[0110] The term “co-crystal” means a physical association of two or more molecules which owe their stability through non-covalent interaction. One or more components of this molecular complex provide a stable framework in the crystalline lattice. In certain instances, the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g. “Crystal Engineering of the Composition of Pharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?” Almarasson, O., et. al., The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p-toluenesulfonic acid and benzenesulfonic acid.

[0111] It is also appreciated that certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form.Attorney Docket No.37759.0695P1Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. As another example, pyrazoles can exist in two tautomeric forms, Afl-unsubstituted, 3-A3and A1-unsubstituted, 5-A3as shown below.A4N-NHUnless stated to the contrary, the invention includes all such possible tautomers.

[0112] It is known that chemical substances form solids, which are present in different states of order which are termed polymorphic forms or modifications. The different modifications of a polymorphic substance can differ greatly in their physical properties. The compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.

[0113] In some aspects, a structure of a compound can be represented by a formula:which is understood to be equivalent to a formula:Rn(a)pn(d)wherein n is typically an integer. That is, R" is understood to represent five independent substituents, RZ7(a), R”(b), R"(c), R"(d), R"(e). By “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance R”(a)is halogen, then R"(b)is not necessarily halogen in that instance.

[0114] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as AldrichAttorney Docket No.37759.0695P1Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N. J ), Fisher Scientific (Pittsburgh, Pa ), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and supplemental volumes (Elsevier Science Publishers, 1989);Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0115] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0116] Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This conceptAttorney Docket No.37759.0695P1applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0117] It is understood that the compositions disclosed herein have certain functions.Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.B. COMPOUNDS

[0118] In one aspect, the invention relates to compounds that inhibit LpxC activity. The disclosed compounds can be useful in, for example, the treatment of an infection associated with LpxC activity (e.g., gram-negative bacterial infection).

[0119] It is contemplated that each disclosed derivative can be optionally further substituted. It is also contemplated that any one or more derivative can be optionally omitted from the invention. It is understood that a disclosed compound can be provided by the disclosed methods. It is also understood that the disclosed compounds can be employed in the disclosed methods of using.1. STRUCTURE

[0120] In one aspect, disclosed are compounds having a structure represented by a formula:wherein n is an integer selected from 1 and 2; wherein Z1is selected from -N- and -CH-; wherein Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b-; wherein R10is selected from hydrogen and C1-C4 alkyl; wherein each of R11aand R11bis independently selected from hydrogen and methyl; and wherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -Attorney Docket No.37759.0695P1NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof.

[0121] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.[00122J In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0123] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0124] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.Attorney Docket No.37759.0695P1

[0125] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0126] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0127] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0128] In various aspects, the compound has a structure represented by a formula:wherein each of Q1, Q2, and Q3is independently selected from -N= and -C(R20)=; wherein each occurrence of R20is independently selected from hydrogen, halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof.

[0129] In various aspects, the compound has a structure represented by a formula:Attorney Docket No.37759.0695P1or a pharmaceutically acceptable salt thereof.

[0130] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0131] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0132] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0133] In various aspects, the compound is selected from:Attorney Docket No.37759.0695P1or a pharmaceutically acceptable salt thereof.

[0134] In various aspects, the compound is selected from:Attorney Docket No.37759.0695P1or a pharmaceutically acceptable salt thereof.

[0135] In one aspect, n is 1 or 2. In a further aspect, n is 1. In a still further aspect, n is 2.a. Q1, Q2, AND Q3GROUPS

[0136] In one aspect, each of Q1, Q2, and Q3is independently selected from -N= and -C(R20)=. In a further aspect, each of Q1, Q2, and Q3is -N=. In a still further aspect, each of Q1, Q2, and Q3is -C(R20)=b. Z1GROUPS

[0137] In one aspect, Z1is selected from -N- and -CH-. In a further aspect, Z1is -N-. In a still further aspect, Z1is -CH-.c. Z2GRO PS

[0138] In one aspect, Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b- In a further aspect, Z2is selected from -NR10-, -O-, -S-, and -SO2-. In a still further aspect, Z2is selected from -NR10-, -O-, -S-, and -CR11aR11b- In yet a further aspect, Z2is selected from -NR10-, -O-, -SO2-, and -CR11aR11b- In an even further aspect, Z2is selected from -NR10-, -S-, -SO2-, and -CR11aR11b- In an even still further aspect, Z2is selected from -O-, -S-, -SO2-, and -CR11aR11b- In yet an even further aspect, Z2is selected from -NR10-, -O-, and -S-. In a further aspect, Z2is selected from -NR10-, -O-, and -SO2-. In a still further aspect, Z2is selected from -NR10-, -O-, and -CR11aR11b- In yet a further aspect, Z2is selected from -NR10--S-, and -SO2-. In an even further aspect, Z2is selected from -NR10-, -S-, and -CR11aR11b- In an even still further aspect, Z2is selected from -NR10-, -SO2-, and -CR11aR11b- In yet an even further aspect, Z2is selected from -O-, -S-, and -SO2-. In a further aspect, Z2is selected from -O—, -S-, and -CR11aR11b- In a still further aspect, Z2is selected from -O-, -SO2-, and -Attorney Docket No.37759.0695P1CRllaRllb- In yet a further aspect, Z2is selected from -S-, -SO2-, and -CR11aR11b- In an even further aspect, Z2is selected from -NR10- and -O-. In an even still further aspect, Z2is selected from -NR10- and -S-. In yet an even further aspect, Z2is selected from -NR10- and -SO2-. In a further aspect, Z2is selected from -NR10- and -CR11aR11b- In a still further aspect, Z2is selected from -O- and -S-. In yet a further aspect, Z2is selected from -O- and -SO2-. In an even further aspect, Z2is selected from -O- and -CR11aR11b- In an even still further aspect, Z2is selected from -S- and -SO2-. In yet an even further aspect, Z2is selected from -S- and -CR11aR11b- in a further aspect, Z2is selected from -SO2- and -CR11aR11b- In a still further aspect, Z2is -NR10-. In yet a further aspect, Z2is -O-. In an even further aspect, Z2is -S-. In an even still further aspect, Z2is -SO2-. In yet an even further aspect, Z2is CRllaRllb.

[0139] In various aspects Z2is selected from -NR10- and -O-. In a further aspect, Z2is selected from -N(CH3)- and -O-.d. R10GROUPS

[0140] In one aspect, R10is selected from hydrogen and C1-C4 alkyl. In a further aspect, R10is selected from hydrogen, methyl, ethyl, propyl, and isopropyl. In a yet further aspect, R10is selected from hydrogen, methyl, and ethyl. In a still further aspect, R10is selected from hydrogen and methyl.

[0141] In various aspects, R10is C1-C4 alkyl. In a further aspect, R10is selected from methyl, ethyl, propyl, and isopropyl. In a yet further aspect, R10is selected from methyl and ethyl. In a still further aspect, R10is methyl.

[0142] In various aspects, R10is hydrogen.e. R11AAND R11BGROUPS

[0143] In one aspect, each of R11aand R11bis independently selected from hydrogen and methyl. In a further aspect, Rllais hydrogen and Rllbis methyl. In a still further aspect, Rllais methyl and Rllbis hydrogen. In yet a further aspect, each of R11aand R11bis hydrogen. In an even further aspect, each of R11aand R11bis methyl.f. R20GROUPSAttorney Docket No.37759.0695P1

[0144] In one aspect, each occurrence of R20is independently selected from hydrogen, halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C 1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, -Br, -CN, -NH2, -OH, -NO2, methyl, ethyl, propyl, isopropyl, ethenyl, propenyl, isopropenyl, -CH2F, -CHF2, -CF3, -CH2CI, -CHCh, -CCI3, -CH2CH2F, -CH2CHF2, -CH2CF3, -CH2CH2CI, -CH2CHCI2, -CH2CCI3, -CH2CH2CH2F, -CH2CH2CHF2, -CH2CH2CF3, -CH2CH2CH2CI, -CH2CH2CHCI2, -CH2CH2CCI3, -CH(CH3)CH2F, -CH(CH3)CHF2, -CH(CH3)CF3, -CH(CH3)CH2C1, -CH(CH3)CHCh, -CH(CH3)CC13, CH2CN, CH2CH2CN, CH2CH2CH2CN, CH(CH3)CH2CN, CH2OH, CH2CH2OH, -CH2CH2CH2OH, -CH(CH3)CH2OH, -OCH2F, -OCHF2, -OCF3, -OCH2CI, -OCHCh, -OCC13, -OCH2CH2F, -OCH2CHF2, -OCH2CHF3, -OCH2CH2C1, -OCH2CHC12, -OCH2CC13, -OCH2CH2CH2F, -OCH2CH2CHF2, -OCH2CH2CF3, -OCH2CH2CH2C1, -OCH2CH2CHC12, -OCH2CH2CC13, -OCH(CH3)CH2F, -OCH(CH3)CHF2, -OCH(CH3)CF3, -CH(CH3)CH2C1, -OCH(CH3)CHC12, -OCH(CH3)CC13, -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)CH3, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -NHCH(CH3)CH3, -N(CH3)2, -N(CH3)(CH2CH3), -N(CH3)(CH2CH2CH3), -N(CH2CH3)2, -N(CH2CH3) (CH2CH2CH3), -N(CH2CH2CH3)2, -N(CH(CH3)CH3)2, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, and -CH(CH3)CH2NH2. In a still further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, -CN, -NH2, -OH, -NO2, methyl, ethyl, ethenyl, -CH2F, -CHF2, -CF3, -CH2CI, -CHCh, -CC13, -CH2CH2F, -CH2CHF2, -CH2CF3, -CH2CH2CI, -CH2CHCI2, -CH2CC13, -CH2CN, -CH2CH2CN, -CH2OH, -CH2CH2OH, -OCH2F, -OCHF2, -OCF3, -0CH2CI, -OCHCh, -OCC13, -OCH2CH2F, -OCH2CHF2, -OCH2CHF3, -OCH2CH2C1, -OCH2CHCI2, -OCH2CC13, -OCH3, -OCH2CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -N(CH3)(CH2CH3), -N(CH2CH3)2, -CH2NH2, and -CH2CH2NH2. In yet a further aspect, each occurrence of R20is independently selected from hydrogen, -F, -CN, -NH2, -OH, -NO2, methyl, -CH2F, -CHF2, -CF3, -CH2CI, -CHCh, -CCI3, -CH2CN, -CH2OH, -OCH2F, -OCHF2, -OCF3, -OCH2C1, -OCHCh, -OCCI3, -OCH3, -NHCH3, -N(CH3)2, and -CH2NH2.

[0145] In various aspects, each occurrence of R20is independently selected from hydrogen, halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, and C2-C4 alkenyl. In a further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, -Br, -CN, -NH2, -OH, -Attorney Docket No.37759.0695P1NCh, methyl, ethyl, propyl, isopropyl, ethenyl, propenyl, and isopropenyl. In a still further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, -CN, -NH2, -OH, -NO2, methyl, ethyl, ethenyl. In yet a further aspect, each occurrence of R20is independently selected from hydrogen, -F, -CN, -NH2, -OH, -NO2, and methyl.

[0146] In various aspects, each occurrence of R20is independently selected from hydrogen, halogen, -CN, -NH2, -OH, -NO2, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 haloalkoxy. In a further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, -Br, -CN, -NH2, -OH, -NO2 -CH2F, -CHF2, -CF3, -CH2CI, -CHCI2, -CC13, -CH2CH2F, -CH2CHF2, -CH2CF3, -CH2CH2CI, -CH2CHCI2, -CH2CCI3, -CH2CH2CH2F, -CH2CH2CHF2, -CH2CH2CF3, CH2CH2CH2CI, CH2CH2CHCI2, CH2CH2CCI3, CH(CH3)CH2F, CH(CH3)CHF2, CH(CH3)CF3, -CH(CH3)CH2C1, -CH(CH3)CHC12, -CH(CH3)CC13, -CH2CN, -CH2CH2CN, -CH2CH2CH2CN, -CH(CH3)CH2CN, -OCH2F, -OCHF2, -OCF3, -OCH2CI, -OCHC12, -OCC13, -OCH2CH2F, -OCH2CHF2, -OCH2CHF3, -OCH2CH2C1, -OCH2CHC12, -OCH2CC13, -OCH2CH2CH2F, -OCH2CH2CHF2, -OCH2CH2CF3, -OCH2CH2CH2C1, -OCH2CH2CHC12, -OCH2CH2CCI3, -OCH(CH3)CH2F, -OCH(CH3)CHF2, -OCH(CH3)CF3, -CH(CH3)CH2C1, -OCH(CH3)CHCb, and -OCH(CH3)CCh. In a still further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, -CN, -NH2, -OH, -NO2, -CH2F, -CHF2, -CF3, -CH2CI, -CHCI2, -CC13, -CH2CH2F, -CH2CHF2, -CH2CF3, -CH2CH2CI, -CH2CHCI2, -CH2CCI3, -CH2CN, -CH2CH2CN, -OCH2F, -OCHF2, -OCF3, -OCH2CI, -OCHCb, -OCCI3, -OCH2CH2F, -OCH2CHF2, -OCH2CHF3, -OCH2CH2CI, -OCH2CHCI2, and -OCH2CCI3. In yet a further aspect, each occurrence of R20is independently selected from hydrogen, -F, -CN, -NH2, -OH, -NO2, -CH2F, -CHF2, -CF3, -CH2CI, -CHCI2, -CCI3, -CH2CN, -OCH2F, -OCHF2, -OCF3, -OCH2CI, -OCHCI2, and -OCCI3.

[0147] In various aspects, each occurrence of R20is independently selected from hydrogen, -NH2, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, each occurrence of R20is independently selected from hydrogen, -NH2,-NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -NHCH(CH3)CH3, -N(CH3)2, -N(CH3)(CH2CH3), -N(CH3)(CH2CH2CH3), -N(CH2CH3)2, -N(CH2CH3) (CH2CH2CH3), -N(CH2CH2CH3)2, -N(CH(CH3)CH3)2, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, and -CH(CH3)CH2NH2. In a still further aspect, each occurrence of R20is independently selected from hydrogen, -NH2, -NHCH3, -NHCH2CH3, -N(CH3)2, -N(CH3)(CH2CH3), -N(CH2CH3)2, -CH2NH2, and -Attorney Docket No.37759.0695P1CH2CH2NH2. In yet a further aspect, each occurrence of R20is independently selected from hydrogen, -NH2, -NHCH3, -N(CH3)2, and -CH2NH2.

[0148] In various aspects, each occurrence of R20is independently selected from hydrogen, -OH, C1-C4 hydroxyalkyl, and C1-C4 alkoxy. In a further aspect, each occurrence of R20is independently selected from hydrogen, -OH, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH(CH3)CH2OH, -OCH3, -OCH2CH3, -OCH2CH2CH3, and -OCH(CH3)CH3. In a still further aspect, each occurrence of R20is independently selected from hydrogen, -OH, -CH2OH, -CH2CH2OH, -OCH3, and -OCH2CH3. In yet a further aspect, each occurrence of R20is independently selected from hydrogen, -OH, -CH2OH, and -OCH3.

[0149] In various aspects, each occurrence of R20is independently selected from hydrogen, halogen, and C1-C4 alkyl. In a further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, -Br, methyl, ethyl, propyl, and isopropyl. In a still further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, methyl, and ethyl. In yet a further aspect, each occurrence of R20is independently selected from hydrogen, -F, and methyl.

[0150] In various aspects, each occurrence of R20is independently selected from hydrogen and halogen. In a further aspect, each occurrence of R20is independently selected from hydrogen, -F, -Cl, and -Br. In a still further aspect, each occurrence of R20is independently selected from hydrogen, -F and -Cl. In yet a further aspect, each occurrence of R20is independently selected from hydrogen and -F.

[0151] In various aspects, each occurrence of R20is independently selected from hydrogen and C1-C4 alkyl. In a further aspect, each occurrence of R20is independently selected from hydrogen, methyl, ethyl, propyl, and isopropyl. In a still further aspect, each occurrence of R20is independently selected from hydrogen, methyl, and ethyl. In yet a further aspect, each occurrence of R20is independently selected from hydrogen and methyl.

[0152] In various aspects, each occurrence of R20is hydrogen.g. AR1GROUPS

[0153] In one aspect, Ar1is selected from C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4Attorney Docket No.37759.0695P1aminoalkyl. In a still further aspect, Ar1is selected from C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0 or 1 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Ar1is selected from C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with a group independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (Cl-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In an even further aspect, Ar1is selected from C6 aryl and a 5- to 6-membered heteroaryl, and is further unsubstituted.

[0154] As detailed herein, Ar1contains two points of attachment to the remainder of the compound:Thus, as shown, Ar1is substituted. In various aspects, however, Ar1is further substituted with 0, 1, 2, or 3 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. As would readily be appreciated by one of skill in the art, when Ar1is further substituted with 0 of these groups, Ar1is said to be further unsubstituted.

[0155] In various aspects, Ar1is a C6 aryl substituted with 0, 1, or 2 independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C 1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a still further aspect, Ar1is a C6 aryl substituted with 0 or 1 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Ar1is a C6 aryl substituted with a group independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino,Attorney Docket No.37759.0695P1(C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In an even further aspect, Ar1is a C6 aryl and is further unsubstituted.

[0156] In various aspects, Ar1is a 5- to 6-membered heteroaryl substituted with 0, 1, or 2 independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, Cl-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxy alkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. Examples of 5- to 6-membered heteroaryl include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyrrolyl, furanyl, thiophenyl. In a still further aspect, Ar1is a 5- to 6-membered heteroaryl substituted with 0 or 1 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Ar1is a 5- to 6-membered heteroaryl substituted with a group independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In an even further aspect, Ar1is a 5- to 6-membered heteroaryl and is further unsubstituted.

[0157] In various aspects, Ar1is a 5- to 6-membered heteroaryl selected from imidazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridinyl, and triazinyl, substituted with 0, 1, or 2 independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a still further aspect, Ar1is a 5- to 6-membered heteroaryl selected from imidazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridinyl, and triazinyl, substituted with 0 or 1 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Ar1is a 5- to 6-membered heteroaryl selected from imidazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridinyl, and triazinyl, substituted with a group independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, Cl-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxy alkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In an even further aspect,Attorney Docket No.37759.0695P1Ar1is a 5- to 6-membered heteroaryl selected from imidazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridinyl, and triazinyl, and is further unsubstituted.2. EXAMPLE COMPOUNDS

[0158] In one aspect, a compound can be present as one or more of the following structures:or a pharmaceutically acceptable salt thereof.C. METHODS OF MAKING A COMPOUND

[0159] The compounds of this invention can be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature, exemplified in the experimental sections or clear to one skilled in the art. For clarity, examples having a single substituent are shown where multiple substituents are allowed under the definitions disclosed herein.Attorney Docket No.37759.0695P1

[0160] Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in the following Reaction Schemes, as described and exemplified below. In certain specific examples, the disclosed compounds can be prepared by Routes I-IV, as described and exemplified below. The following examples are provided so that the invention might be more fully understood, are illustrative only, and should not be construed as limiting.1. ROUTE I

[0161] In one aspect, imidazole compounds can be prepared as shown below.SCHEME 1A.

[0162] Compounds are represented in generic form, wherein R is a C1-C4 alkyl, wherein PG is an alcohol protecting group (e.g., tert-butyl, tetrahydropyran), and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.SCHEME IB.DIBAIH4 (2.5 eq), pTSA, CH2Cl2-78°C, 2.5 hAttorney Docket No.37759.0695P1oTHPO, Oxaldehyde (7.5 eq),NH4OH, MeOH, RT, 16 h1.8

[0163] In one aspect, compounds of type 1.8, and similar compounds, can be prepared according to reaction Scheme IB above. Thus, compounds of type 1.6 can be prepared by a protection reaction of an appropriate substituted alcohol, e.g., 1.5 as shown above. Appropriate alcohols are commercially available or prepared by methods known to one skilled in the art. The protection reaction is carried out in the presence of an appropriate protecting reagent, e.g, dihydropyran, in the presence of an appropriate catalyst, e.g., para-toluene sulfonic acid, in an appropriate solvent, e.g., di chloromethane. Compounds of type 1.7 can be prepared by a reduction reaction of an appropriate ester, e.g, 1.6 as shown above. Appropriate esters are commercially available or prepared by methods known to one skilled in the art. The reduction reaction is carried out with an appropriate reducing agent, e.g., diisobutylaluminium hydride, in an appropriate solvent, e.g, ether, at an appropriate temperature, e.g, -78°C, for an appropriate amount of time, e.g., 2.5h. Compounds of type 1.8 can be prepared by a cyclization reaction of an appropriate aldehyde, e.g, 1.7 as shown above. Appropriate aldehydes are commercially available or prepared by methods known to one skilled in the art. The cyclization reaction is carried out with an appropriate reagent, e.g., oxaldehyde, in the presence of ammonium hydroxide, in an appropriate solvent, e.g, methanol, at an appropriate temperature, e.g, room temperature, for an appropriate amount of time, e.g., 16h. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 1.1, 1.2, and 1.3), can be substituted in the reaction to provide phenyl isoxazole compounds similar to Formula 1.4.2. ROUTE II

[0164] In one aspect, phenyl isoxazole compounds can be prepared as shown below.Attorney Docket No.37759.0695P1SCHEME 2A.2.42.5

[0165] Compounds are represented in generic form, wherein X is a suitable leaving group (e.g., tosylate, triflate, iodide, bromide, chloride), wherein R is a C1-C4 alkyl, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.SCHEME 2B.o i. Diethyl oxalate (1.5 eq),NaH (2.5 eq), Toluene, 50°C, 2 h.2.6NH2OH HCI NaBH4(1.5 eq) MeOH 80°C, 1hAttorney Docket No.37759.0695P1TsCI (1.5 eq), TEA (3.0 eq), DCM, RT, 2 h;2.102.9

[0166] In one aspect, compounds of type 2.8, and similar compounds, can be prepared according to reaction Scheme 2B above. Thus, compounds of type 2.7 can be prepared by a nucleophilic acyl substitution of an appropriate ketone, e., 2.6 as shown above. Appropriate ketones are commercially available or prepared by methods known to one skilled in the art. The nucleophilic acyl substitution reaction is carried out with an appropriate dialkyl oxylate, e.g., diethyl oxylate, in the presence of an appropriate base, e.g., sodium hydride, in an appropriate solvent, e.g., toluene, at an appropriate temperature, e.g., 50°C, for an appropriate amount of time, e.g., 2h. Compounds of type 2.8 can be prepared by a cyclization reaction of an appropriate P-ketoester, e.g., 2.7 as shown above. Appropriate -ketoesters are commercially available or prepared by methods known to one skilled in the art. The cyclization reaction is carried out in the presence of hydroxyl amine hydrochloride, in an appropriate solvent, e.g., toluene, at an appropriate temperature, e.g., 80°C, for an appropriate amount of time, e.g., Ih. Compounds of type 2.9 can be prepared by a reduction reaction of an appropriate ester, e.g., 2.8 as shown above. Appropriate esters are commercially available or prepared by methods known to one skilled in the art. The reduction is carried out with an appropriate reducing agent, e.g., sodium borohydride, in an appropriate solvent, e.g., methanol. Compounds of type 2.10 can be prepared by a substitution reaction of an appropriate alcohol, e.g., 2.9 as shown above.Appropriate alcohols are commercially available or prepared by methods known to one skilled in the art. The substitution reaction is carried out with an appropriate regent, e.g., para-toluene sulfonyl chloride, in the presence of an appropriate base, e.g., tri ethyl amine, in an appropriate solvent, e.g., dichloromethane, at an appropriate temperature, e.g., room temperature, for an appropriate amount of time, e.g., 2h. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 2.1, 2.2, 2.4, and 2.4), can be substituted in the reaction to provide phenyl isoxazole compounds similar to Formula 2.5.Attorney Docket No.37759.0695P13. ROUTE III

[0167] In one aspect, phenyl isoxazole compounds can be prepared as shown below.SCHEME 3A.NN

[0168] Compounds are represented in generic form, wherein X and Y are independently suitable leaving groups (e.g., tosylate, triflate, iodide, bromide, chloride), wherein B is a boron moiety, wherein PG is an alcohol protecting group (e.g., tert-butyl, tetrahydropyran), and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.SCHEME 3B.TsOjl NaH (1.5 eq).N H DMF, O-rt, 2 hN-^NAttorney Docket No.37759.0695P1

[0169] In one aspect, compounds of type 3.6, and similar compounds, can be prepared according to reaction Scheme 3B above. Thus, compounds of type 3.6 can be prepared by a substitution reaction of an appropriate substituted imidazole, e.g., 3.4 as shown above, and an appropriate isoxazole, e.g., 3.5 as shown above. Appropriate imidazoles and appropriate isoxazoles are commercially available or prepared by methods known to one skilled in the art. The substitution reaction is carried out in the presence of an appropriate base, e.g., sodium hydride, in an appropriate solvent, e.g., dimethylformamide, at an appropriate temperature, e.g., 0°C to room temperature, for an appropriate amount of time. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 3.1 and 3.2), can be substituted in the reaction to provide phenyl isoxazole compounds similar to Formula 3.3.4. ROUTE IV

[0170] In one aspect, phenyl isoxazole compounds can be prepared as shown below.SCHEME 4A.4.24.14.3

[0171] Compounds are represented in generic form, wherein X is a suitable leaving group (e.g., tosylate, triflate, iodide, bromide, chloride), wherein B is a boron moiety, wherein PG is an alcohol protecting group (e.g., tert-butyl, tetrahydropyran), and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.Attorney Docket No.37759.0695P1THPO / ,i. Pd(dppf)CI2-DCM(0.2eq), K2CO3(3.0 eq), 1,4-dioxane: water=4:1; 80°C, N2, ii. P-TsOH- H2O (3.0 eq), MeOH,rt, 2 h.

[0172] In one aspect, compounds of type 4.6, and similar compounds, can be prepared according to reaction Scheme 4B above. Thus, compounds of type 4.6 can be prepared by a Suzuki coupling reaction of an appropriate aryl halide, e.g, 4.4 as shown above, and an appropriate boronic ester or boronic acid, e.g., 4.5 as shown above. Appropriate aryl halides and appropriate boronic esters or boronic acids are commercially available or prepared by methods known to one skilled in the art. The Suzuki coupling reaction is carried out in the presence of an appropriate catalyst, e.g., l,l'-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane, in the presence of an appropriate base, e.g., potassium carbonate, in an appropriate solvent, e.g., dioxane and water, at an appropriate temperature, e.g., 80°C.Subsequent deprotection of the alcohol with an appropriate cleaving agent, e.g., para-toluene sulphonic acid, in an appropriate solvent, e.g., methanol, at an appropriate temperature, e.g., room temperature, for an appropriate amount of time, e.g., 2h. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 4.1 and 4.2), can be substituted in the reaction to provide phenyl isoxazole compounds similar to Formula 4.3.Attorney Docket No.37759.0695P1D. PHARMACEUTICAL COMPOSITIONS

[0173] In one aspect, the invention relates to pharmaceutical compositions comprising the disclosed compounds and products of disclosed methods. That is, a pharmaceutical composition can be provided comprising an effective amount of at least one disclosed compound, at least one product of a disclosed method, or a pharmaceutically acceptable salt, thereof, and a pharmaceutically acceptable carrier. In one aspect, the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of at least one disclosed compound; or a pharmaceutically acceptable salt, thereof.

[0174] Thus, in one aspect, disclosed are pharmaceutical compositions comprising an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0175] In one aspect, disclosed are pharmaceutical compositions comprising an effective amount of a compound having a structure represented by a formula:wherein n is an integer selected from 1 and 2; wherein Z1is selected from -N- and -CH-; wherein Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b-; wherein R10is selected from hydrogen and C1-C4 alkyl; wherein each of R11aand R11bis independently selected from hydrogen and methyl; and wherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0176] In various aspects, the pharmaceutical composition further comprising an effective amount of an iron chelator. In a further aspect, the iron chelator is selected from deferoxamine (DFO), deferasirox, and deferiprone, or a combination thereof. In a still further aspect, the iron chelator is deferoxamine (DFO).Attorney Docket No.37759.0695P1

[0177] In a further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount. In a still further aspect, the pharmaceutical composition comprises a compound that is a product of a disclosed method of making.

[0178] In a further aspect, the pharmaceutical composition comprises a disclosed compound. In a yet further aspect, the pharmaceutical composition comprises a product of a disclosed method of making.

[0179] In a further aspect, the pharmaceutical composition is used to treat a mammal. In a further aspect, the mammal is a human.

[0180] In certain aspects, the disclosed pharmaceutical compositions comprise the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0181] As used herein, the term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium, manganese (-ic and -ous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N, N’ -dibenzyl ethylenediamine, di ethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-Attorney Docket No.37759.0695P1ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

[0182] As used herein, the term “pharmaceutically acceptable non-toxic acids,” includes inorganic acids, organic acids, and salts prepared therefrom, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

[0183] In practice, the compounds of the invention, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, and / or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and / or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

[0184] Thus, the pharmaceutical compositions of this invention can include a pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of the compounds of the invention. The compounds of the invention, or pharmaceutically acceptableAttorney Docket No.37759.0695P1salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.

[0185] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0186] In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.

[0187] A tablet containing the composition of this invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0188] The pharmaceutical compositions of the present invention comprise a compound of the invention (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.Attorney Docket No.37759.0695P1

[0189] Pharmaceutical compositions of the present invention suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.Further, a preservative can be included to prevent the detrimental growth of microorganisms.

[0190] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0191] Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.

[0192] Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0193] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can beAttorney Docket No.37759.0695P1included to render the formulation isotonic with the blood of the intended recipient.Compositions containing a compound of the invention, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0194] In the treatment conditions that require inhibiting LpxC activity, an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day and can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg / kg per day; more preferably 0.5 to 100 mg / kg per day. A suitable dosage level can be about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg / kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. This dosing regimen can be adjusted to provide the optimal therapeutic response.

[0195] It is understood, however, that the specific dose level for any particular patient will depend upon a variety of factors. Such factors include the age, body weight, general health, sex, and diet of the patient. Other factors include the time and route of administration, rate of excretion, drug combination, and the type and severity of the particular disease undergoing therapy.

[0196] The present invention is further directed to a method for the manufacture of a medicament for inhibiting LpxC activity (e.g., gram-negative bacterial infection) in mammals (e.g., humans) comprising combining one or more disclosed compounds, products, or compositions with a pharmaceutically acceptable carrier or diluent. Thus, in an aspect, the invention relates to a method for manufacturing a medicament comprising combining at least one disclosed compound or at least one disclosed product with a pharmaceutically acceptable carrier or diluent.

[0197] The disclosed pharmaceutical compositions can further comprise other therapeutically active compounds, which are usually applied in the treatment of the above mentioned pathological conditions.Attorney Docket No.37759.0695P1

[0198] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.E. METHODS OF INHIBITING LPXC ACTIVITY IN A CELL

[0199] In one aspect, disclosed are methods of inhibiting LpxC activity in a cell, the method contacting the cell with an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof.

[0200] Thus, in one aspect, disclosed are methods of inhibiting LpxC activity in a cell, the method comprising contacting the cell with an effective amount of a compound having a structure represented by a formula:wherein n is an integer selected from 1 and 2; wherein Z1is selected from -N- and -CH-; wherein Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b-; wherein R10is selected from hydrogen and C1-C4 alkyl; wherein each of R11aand R11bis independently selected from hydrogen and methyl; and wherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof.

[0201] In various aspects, the cell is mammalian. In a still further aspect, the cell is human.

[0202] In various aspects, the cell has been isolated from a human prior to the administering step.

[0203] In various aspects, the contacting is ex vivo. In a further aspect, the contacting is in vitro.

[0204] In various aspects, contacting is via administration to a subject. In a further aspect, the mammal has been diagnosed with a need for inhibiting LpxC activity prior to theAttorney Docket No.37759.0695P1administering step. In an even further aspect, the mammal has been diagnosed with a need for treatment of a Gram-negative bacterial infection associated with LpxC activity.F. METHODS OF INHIBITING LPXC ACTIVITY IN A SUBJECT

[0205] In one aspect, disclosed are methods of inhibiting LpxC activity in a subject in need thereof, the method comprising administering to the subject an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof.

[0206] Thus, in one aspect, disclosed are methods of inhibiting LpxC activity in need thereof, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula:Nwherein n is an integer selected from 1 and 2; wherein Z1is selected from -N- and -CH-; wherein Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b-; wherein R10is selected from hydrogen and C1-C4 alkyl; wherein each of R11aand R11bis independently selected from hydrogen and methyl; and wherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof

[0207] In various aspects, the subject is a mammal. In a still further aspect, the subject is a human.

[0208] In various aspects, the subject has been diagnosed with an infection associated with LpxC activity prior to the administering step.

[0209] In various aspects, the subject has been diagnosed with a need for modifying LpxC activity prior to the administering step.

[0210] In various aspects, the subject has been diagnosed with a need for treatment of a Gram-negative bacterial infection associated with LpxC activity prior to the administering step.Attorney Docket No.37759.0695P1

[0211] In various aspects, the method further comprising the step of identifying a subject in need of treatment of a Gram-negative bacterial infection associated with LpxC activity.G. METHODS OF TREATING A GRAM-NEGATIVE BACTERIAL INFECTION IN A SUBJECT

[0212] In one aspect, disclosed are methods of treating a Gram-negative bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof.

[0213] Thus, in one aspect, disclosed are methods of treating a Gram-negative bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula:Nwherein n is an integer selected from 1 and 2; wherein Z1is selected from -N- and -CH-; wherein Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b-; wherein R10is selected from hydrogen and C1-C4 alkyl; wherein each of R11aand R11bis independently selected from hydrogen and methyl; and wherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof.

[0214] In various aspects, the Gram-negative bacterial infection is due to a Gram-negative bacteria selected from Acinetobacter spp., Aeromonas spp., Bordetella spp., Citrobacter spp., Enterobacter spp., Escherichia spp., Haemophilus spp., Klebsiella spp., Moraxella spp., Neisseria spp., Proteus spp., Pseudomonas spp., Salmonella spp., Shigella spp., Stenotrophomonas spp., Vibrio spp., and Yersinia spp. In a further aspect, the Gram-negative bacterial infection is due to Neisseria spp. In a further aspect, the Gram-negative bacterial infection is due to Neisseria gonorrhoeae.Attorney Docket No.37759.0695P1

[0215] In various aspects, the effective amount is a therapeutically effective amount. In a further aspect, the effective amount is a prophylactically effective amount.

[0216] In various aspects, the subject is a mammal. In a further aspect, the mammal is a human.

[0217] In various aspects, the subject has been diagnosed with a need for treatment of the Gram-negative bacterial infection prior to the administering step.

[0218] In various aspects, the subject is at risk for developing the Gram-negative bacterial infection prior to the administering step.

[0219] In various aspects, the method further comprising the step of identifying a subject in need of treatment of the Gram-negative bacterial infection.

[0220] In various aspects, the method further comprising administering to the subject an effective amount of an iron chelator.

[0221] In various aspects, the compound and the iron chelator are administered simultaneously. In a further aspect, the compound and the iron chelator are co-formulated. In a still further aspect, the compound and the iron chelator are not co-formulated. In yet a further aspect, the compound and the iron chelator are administered sequentially.

[0222] In various aspects, the iron chelator is selected from deferoxamine (DFO), deferasirox, and deferiprone, or a combination thereof. In a further aspect, the iron chelator is deferoxamine (DFO).

[0223] In various aspects, the method further comprising administering to the subject an effective amount of an antibacterial agent.

[0224] In various aspects, the compound and the antibacterial agent are administered simultaneously.

[0225] In various aspects, the compound and the antibacterial agent are co-formulated.

[0226] In various aspects, the compound and the antibacterial agent are not co-formulated.

[0227] In various aspects, the compound and the antibacterial agent are administered sequentially.

[0228] In a further aspect, the antibacterial agent is selected from amoxicillin, ampicillin, azithromycin, aztreonam, azlocillin, bacitracin, carbenicillin, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cefdinir, cefditorin, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime,Attorney Docket No.37759.0695P1chloramphenicol, cilastin, ciprofloxacin, clarithromycin, clavulanic acid, clinafloxacin, clindamycin, clofazimine, cioxacillin, colistin, dalbavancin, dalfopristin, daptomycin, demeclocycline, dicloxacillin, dirithromycin, doxycycline, erythromycin, enrofloxacin, enoxacin, enviomycin, ertepenem, ethambutol, flucioxacillin, fosfomycin, furazolidone, gatifloxacin, gentamicin, imipenem, isoniazid, kanamycin, linezolid, lomefloxacin, loracarbef, mafenide, moxifloxacin, meropenem, metronidazole, mezlocillin, minocycline, mupirocin, nafcillin, nalidixic acid, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oritavancin, oxytetracycline, penicillin, piperacillin, platensimycin, polymixin B, quinupristin, retapamulin, rifabutin, rifampin, rifapentine, roxithromycin, sparfloxacin, spectinomycin, sulbactam, sulfacetamide, sulfamethizole, sulfamethoxazole, teicoplanin, telithromycin, telavancin, temafloxacin, tetracycline, tedolizid, thioacetazone, thioridazine, ticarcillin, tinidazole, tobramycin, torezolid, tosufloxacin, trimethoprim, troleandomycin, trovafloxacin, and vancomycin.H. KITS[00229J In one aspect, disclosed are kits comprising a disclosed or a pharmaceutically acceptable salt thereof, and one or more selected from: (a) an iron chelator; (b) an antibacterial agent; (c) instructions for administering the compound in connection with a Gram-negative bacterial infection; and (d) instructions for treating a Gram-negative bacterial infection.

[0230] In one aspect, disclosed are kits comprising a compound having a structure represented by a formula:wherein n is an integer selected from 1 and 2; wherein Z1is selected from -N- and -CH-; wherein Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b-; wherein R10is selected from hydrogen and C1-C4 alkyl; wherein each of R11aand R11bis independently selected from hydrogen and methyl; and wherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -Attorney Docket No.37759.0695P1NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof, and one or more selected from: (a) an iron chelator; (b) an antibacterial agent; (c) instructions for administering the compound in connection with a Gram-negative bacterial infection; and (d) instructions for treating a Gram-negative bacterial infection.

[0231] In various aspects, the compound and the iron chelator are co-packaged. In a further aspect, the compound and the iron chelator are co-formulated.

[0232] In various aspects, the compound and the antibacterial agent are co-packaged. In a further aspect, the compound and the antibacterial agent are co-formulated.

[0233] All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the event of an inconsistency between the present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls.I. EXAMPLES

[0234] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0235] The Examples are provided herein to illustrate the invention, and should not be construed as limiting the invention in any way. Examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any way.

[0236] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art fromAttorney Docket No.37759.0695P1consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0237] Described herein are a series of novel LpxC inhibitors with a non-hydroxamate head group designed to inhibit the N. gonorrhoeae LpxC enzyme. The head group consists of a 2-(LS'-hydroxymethyl)- imidazole zinc chelator, which has been shown to be a potent driver for binding of the LpxC enzyme active site that minimized the cardiovascular toxicity (Fujita K, et al., 2022 J Antibiot (Tokyo) 75:136-145; Fujita K, et al., 2022 J Antibiot (Tokyo) 75:98-107; Ushiyama F, et al., 2021 Bioorg Med Chem 30:115964; Yamada Y, et al., 2020 J Med Chem 63:14805- 14820). The previously described compound with the 2-(15-hydroxymethyl)- imidazole head group, TP0586532, was designed for optimal binding to P. aeruginosa LpxC and the tail group for minimal protein binding (Ushiyama F, et al., 2021 Bioorg Med Chem 30:115964). Typically, LpxC inhibitors contain in addition to a zinc-chelating head group, a lengthy connector to fill the hydrophobic tunnel, and a tail group to fill the distal end that is solvent exposed (Ushiyama F, et al., 2021 Bioorg Med Chem 30: 115964; Barb AW, et al., 2007 Proc Natl Acad Sci U S A 104: 18433-8). Similarities and differences in the homology of LpxC from P. aeruginosa and N. gonorrhoeae led us to maintain the oxazole-phenyl moiety of TP0586532 but shorten the moiety meant to occupy the hydrophobic tunnel. The morpholine group of CHIR-090 was added to the tail because upon binding, it can form a boat conformation that contributes to tight binding (Barb AW, et al., 2007 Proc Natl Acad Sci U S A 104:18433-8).1. CHEMISTRY MATERIALS AND METHODSa. LPXC INHIBITORS

[0238] The strategies used for the synthesis of the LI, L2, L3, and L4 LpxC inhibitors by SYNthesis Med Chem (San Diego, CA, USA) are outlined in the Schemes 5 to 8.Scheme 5.THPOz,pTSA, CH2Cl2Attorney Docket No.37759.0695P1 i. DIBAIH4 (2.5 eq), -78°C, 2.5 hii. Oxaldehyde (7.5 eq), NH4OH, MeOH, RT, 16 h;Scheme 6i. Diethyl oxalate (1.5 eq), NaH (2.5 eq), Toluene, 50°C, 2 h.ii. NH2OH HCI (3 eq), 80°C, 1hScheme 7NaH (1.5 eq)* DMF, O-rt, 2 hTHPO / ,.Attorney Docket No.37759.0695P1THPOz,i. Pd(dppf)CI2-DCM(0.2eq), K2CO3(3.0 eq), 1,4-dioxane: water=4:1; 80°C, N2, ii. P-TsOH- H2O (3.0 eq), MeOH,rt, 2 h.

[0239] Briefly, synthesis of the inhibitors was achieved by developing the imidazole containing head group and adding the adjacent 5-phenyl isoxazol-3-yl.

[0240] The halogen substituent on the phenyl participated in Suzuki-Miyaura coupling with pinacolborane (4, 4, 5, 5-Tetramethyl-l, 3, 2- dioxaborolane). This coupling facilitated the addition of a phenyl and a nitrogen containing heterocyclic tail group. Purity was verified by HPLC and structure was confirmed by 'HNMR and LCMS.2. EXEMPLARY COMPOUNDS

[0241] A list of exemplary compounds prepared using the synthetic methods as detailed herein is shown in Table 1 below. PF-04753299 and CHIR-090 were obtained from Sigma-Aldrich, St. Louis, MO, USA.Attorney Docket No.37759.0695P1Compound Structure^oL2 / =n AAN. N^Z V / VNHO'' XL3[ N 0HO"' XX- / K \ IL # — A / / — / ) — \,L4 V^ZN—HO'' XJO PF-04753299 u fjO' | OH |?HO"NYCHIR-090oHUL.TPI Ob. MOLECULAR DOCKING OF LPXC INHIBITORS TO ALPHAFOLD GENERATED LPXC ENZYME

[0242] The LpxC enzyme (UDP-3-O-acyl-A-acetylglucosamine deacetylase) from F Al 090 (Uniprot: Q5F569) was retrieved from AlphaFold 3 (Google, DeepMind, London, UK) in PDBAttorney Docket No.37759.0695P1format (UniProt: Q5F569)(Jumper J, et al., 2021 Nature 596:583-589; Varadi M, et al., 2024 Nucleic Acids Res 52: D368-D375). AlphaFold is a multicomponent Al model that uses protein sequences to predict protein structures with high fidelity (Abramson J, et al., 2024 Nature 630:493-500). Since addition of hydrogen atoms is not a priority for AlphaFold, UCSF ChimeraX was utilized to add hydrogen atoms and make other corrections. The structure was imported to UCSF ChimeraX, where Dock Prep was used to delete solvents, delete noncomplexed ions, standardize, replace incomplete side chains using Dundrack rotamer library (Shapovalov MV, Dunbrack RL, Jr. 2011 Structure 19:844-58), and add hydrogens and charges (Pettersen EF, et al., 2004 J Comput Chem 25: 1605-12). Lastly, the modified structure was saved as a PDB file and uploaded to Swissdock (SIB Swiss Institute of Bioinformatics, Lausanne, CH) to perform docking with the AutoDock Vina docking algorithm, which maintains flexible ligand and protein side chains, and add a zinc ion (Zn2+) to the active site (Trott O, Olson AJ. 2010 J Comput Chem 31:455-61; Bugnon M, et al., 2024 Nucleic Acids Res 52: W324-W332; Grosdidier A, et al., 2011 Nucleic Acids Res 39: W270-7; Eberhardt J, et al., 2021 J Chem Inf Model 61:3891-3898). Determining the active site zinc location was done by comparison to the well defined A. aeolicus LpxC enzyme crystal structure complexed with CHIR-090 (PDB file: 2JT2). The appropriate complexed FA1090 LpxC enzyme with the zinc ion was saved as a PDB file using ChimeraX. The complexed enzyme was uploaded again to Swissdock’ s Autodock Vina platform. LpxC inhibitor structures were written as SMILES and docking was tested with sampling exhaustivity set to 4. The value of the greatest docking affinity for each inhibitor with the head group positioned in the LpxC active site was reported.3. BIOLOGICAL MATERIALS AND METHODS:a. BACTERIAL STRAINS[00243J N. gonorrhoeas strain FA1090 has been widely used in laboratory experiments in vitro and in animal models, and in experimental human studies. The 1-81-S2 FA1090 variant was isolated from a human challenge study (Seifert HS, et al., 1994. J Clin Invest 93:2744-9). WHO Y and X, referred to as F89 and H041, respectively, exhibit extensive antibiotic resistance to the expanded-spectrum cephalosphorins, ceftriaxone and cefixime (Unemo M, et al., 2012 Antimicrob Agents Chemother 56:1273-80; Unemo M, et al., 2016 J Antimicrob Chemother 71:3096-3108). In 2010, F89 was isolated from the urethra of a patient in France (Unemo M, etAttorney Docket No.37759.0695P1al., 2012 Antimicrob Agents Chemother 56:1273-80). H041 was isolated from athroat sample of a patient in Japan in 2009 (Ohnishi M, et al., 2011 Emerg Infect Dis 17: 148-9).b. BACTERICIDAL ASSAYS[00244J Gonococci were grown overnight on Difco GC agar (Becton, Dickinson, Sparks, MD, USA) containing 1% BBL IsoVital eX Enrichment (Becton Dickinson) in a 37°C and 5% CO2incubator. After suspending in PBS the absorbance at 595 nm was used to serially dilute stocks. Aliquots of the diluted bacterial suspension were added to RPMI-1640 medium containing 10% FBS (both from Gibco, ThermoFisher Scientifific, Pittsburgh, PA, USA) in a cell culture-treated 96-well plate (Corning, CellBIND). Increasing concentrations of the LpxC inhibitors in RPMI medium or vehicle only, which was 1% DMSO in RPMI, were added to plates for total reaction volumes of 200 pL / well containing approximately 4,000 CFUs. Plates were covered and gently shaken before incubating at 37°C in a 5% CO2incubator for 4 hours. For colony counting aliquots of test concentrations were plated on GC agar plates containing 1% IsoVitaleX in quadruplicates. Agar plates were incubated overnight and then the colonies were enumerated. Survival was expressed as the percentage of bacteria detected as CFUs from aliquots of LpxC inhibitor-treated bacteria compared to the CFUs in aliquots of the vehicle only treated bacterial suspensions. To determine the effect of LOS sialylation on bactericidal potency, bacteria were grown overnight on agar containing 10 pg / mL CMP -NANA (Sigma-Aldrich).c. CYTOTOXICITY OF LPXC INHIBITORS FOR HUMAN CELLS

[0245] ME-180 and activated THP-1 cells were seeded into 96-well plates (Coming, CellBIND) at 104cells / well, in Opti-MEM (Gibco) containing 1% FBS. The THP-1 cells had been activated overnight with 10 ng / mL phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich). LpxC inhibitors in PBS or DMSO-containing vehicle were added once the cells had become adherent. The plates were shaken gently and incubated at 37°C and 5% CO2for 18 hours. A Cyquant LDH Kit (Invitrogen Thermo-Fisher) was used to quantify the LDH released as per the manufacturer guidelines. An Epoch plate reader was used to detect 490-680nm. LDH release was expressed as percentage of positive controls treated with Triton X-100 Lysis Buffer.Attorney Docket No.37759.0695P1d. HEMOLYSIS ASSAY

[0246] LpxC inhibitors and washed pooled sheep red blood cells (10% vol / vol, Rockland, Philadelphia, PA, USA) diluted in PBS to 2% were added to 96-well plates (Corning, CellBIND). The plates were covered, mixed gently, and then incubated for 1 hour at 37°C and 5% CO2. The plate was centrifuged at 500 x g for 10 min and supernatants were added to a fresh plate as previously described (Witherell KS, et al., 2021 Sci Rep 11:2151). An Epoch plate reader was used to measure 405nm. Results were reported as % hemolysis, calculated by: % hemolysis = [(405nm Abs of RBCs in peptide solution - 405 nm Abs of RBCs in PBS) / (405 nm Abs of RBCs in 0.1% Triton X-100 -405 nm Abs of RBCs in PBS)] x 100%. Values of <10% were defined as non-hemolytic (Amin K, Dannenfelser RM. 2006 J Pharm Sci 95: 1173-6).e. MIC AND CHECKERBOARD ASSAYS

[0247] Bacteria grown overnight on GC agar were suspended in GC broth supplemented with 1% Isovitalex. Bacterial suspensions were diluted based on the absorbance at 595 nm and added to 96-well plates (Falcon 96-well, non-treated, flat-bottom microplate 351172) with 4xl06CFU / well (Jacobson RK, et al., 2019 J Microbiol Methods 157:93-99). LpxC inhibitors or DMSO-containing vehicle were diluted in PBS and added to quadruplicate wells. Plates were covered, gently shaken, and incubated at 37°C in a 5% CO2incubator for 24 hours. After incubation, the absorbance was measured using an Epoch plate reader (Biotek Instruments, Winooski, Vermont, USA) with path length correction. This protocol was followed for checkerboard assays in which DFO (Sigma-Aldrich) solubilized in PBS was added (83). Results were validated by 3 independent experiments. For checkerboard assays, FICI was calculated by the equation FICI = and synergism was defined as having a FICI value below 0.5(Odds FC. 2003 J Antimicrob Chemother 52:1).f. BACTERICIDAL ASSAYS IN THE PRESENCE OF HUMAN CELLS

[0248] Bacterical assays in the presence of human cells were carried out as previously described utilizing strain (John CM, et al., 2024 Microbiol Spectr 12:e0099724). Endl / E6E7 (ATCC, Manassas, VA, USA) cells were cultured in Keratinocyte Serum Free Media with supplements (K-SFM, ThermoFisher). ME- 180 and THP-1 (both from ATCC) cells wereAttorney Docket No.37759.0695P1cultured in RPMI-1640 containing 10% FBS. THP-1 cells were activated by overnight incubation with 10 ng / mL PMA. The Endl / E6E7, ME-180, or activated THP-1 cells were added to a 96-well plate (Coming, CellBIND) at 30,000 cells / well to produce confluent monolayers. Aliquots of LpxC inhibitors or DMSO-containing vehicle and 4,000 N. gonorrhoeas in media were added to cells with total reaction volume of 200 pL. After 4 hours of incubation, human cells were lysed with 1% saponin and incubated for 15 minutes. Aliquots from each test concentration were plated in quadruplicate on GC agar plates, which were incubated overnight and then the bacteria enumerated.g. EFFECT OF LPXC INHIBITORS ON TNF-A INDUCTION BY N.GONORRHOEAE INFECTION OF THP-1 CELLS

[0249] THP-1 cells were activated by overnight incubation with 10 ng / mL PMA. Once the cells had attached after incubation at 37°C and 5% CO2 for approximately 2 hours, aliquots of a suspension of FA1090 N. gonorrhoeas in cell culture medium was added to achieve a moi of 1 after which aliquots of solutions of LpxC inhibitors were added. Plates were mixed gently and then incubated for 18 hours. Supernatants were isolated and frozen down at -80°C until analysis. The human TNF-a Uncoated ELISA kit (Invitrogen, ThermoFisher) was used for detection and results were quantified by an Epoch plate reader at 450 nm.h. EFFECT OF LPXC INHIBITION ON LOS BIOSYNTHESIS

[0250] A whole cell ELISA to quantify LOS biosynthesis was performed as previously described (Abdillahi H, Poolman JT. 1988 Microb Pathog 4:27-32; Estabrook MM, et al., 2004 J Immunol 172:3784-92). Bacterial suspensions of FA1090 grown overnight were made in 2 mL GC broth and treated with PF and LI at 37 °C and 5% CO2for 4 hours. 96-well ELISA plates (Corning, Costar EIA / RIA Plate 3590) were coated in triplicate wells with 100 pl of bacteria suspended in PBS (OD = 1 at 595 nm) and incubated overnight. After washing and blocking plates with casein (0.33% casein in PBS with 0.15% Tween-20; casein solution), plates were treated with 2C7 mouse monoclonal IgG3 and then anti-mouse IgG3 antibody (Rockland Immunochemicals, Limerick, PA, USA) labeled with alkaline phosphatase, both in casein solution, at 37 °C for 1 hour. The 2C7 epitope encompasses lactosyl moi eties on both heptose I and II of the inner core of the gonococcal LOS and is expressed by FA1090 (Gulati S, et al.,Attorney Docket No.37759.0695P12019 PLoS Biol 17:e3000323; Gulati S, et al., 2019 Front Immunol 10:321). Subsequently wells were incubated with p-nitrophenyl phosphate substrate (1 mg / ml), reactions stopped with 50 pL NaOH and plates analyzed on an Epoch plate reader. Absorbance (405 nm) of PBS-only control wells was subtracted.i. EFFECT OF EFFLUX PUMP INHIBITOR ON POTENCY OF LPXC INHIBITORS

[0251] To test the effect of an efflux pump inhibitor, aliquots of a solution of phenylalaninearginine 0-naphthylamide dihydrochloride (PA0N, Sigma-Aldrich, P4157) in RPMI-1640 cell culture medium were added to bacteria to produce a concentration of 50 pg / mL (Tomaras AP, et al., 2014 mBio 5:e01551— 14) along with the LpxC inhibitors or DMSO-containing vehicle that were incubated for 4 hours using the bactericidal protocol as described above.j. ANALYSIS OF IRON BINDING TO LPXC INHIBITORS

[0252] The binding affinity of LI, L3, PF, and CHIR-090 LpxC inhibitors, deferoxamine, ceftriaxone, and chloramphenicol to FeCl3(Sigma-Aldrich) was compared using spectral scans from wavelengths 300-700 nm, with 2-nm increments on an Epoch plate reader (Chan DCK, et al., 2020 Antimicrob Agents Chemother 64). For this assay 100 pL of 4 mM of each of the compounds (all from Sigma-Aldrich excluding Ll-4) in DMSO and 100 pL of 12 mM FeCl3in water were added to a clear 96-well plate (Falcon 96-Well, Non-Treated, Flat-Bottom Microplate 351172).k. GROWTH ASSAYS

[0253] FA1090, F89, and H041 were grown overnight on GC agar plates containing 1% IsoVital ex (BBL). Colonies were added to GC broth containing 1% IsoVital ex and the absorbance at 595 nm was used to dilute the bacterial suspensions to approximately 3 x 106CFU / mL. Then 2 mL of bacterial suspensions and aliquots of LpxC inhibitors with or without 50 pM DFO were added to snap cap tubes which were incubated at 37°C and 5% CO2. LpxC inhibitor concentrations were 0.04 pg / mL LI and 0.002 pg / mL PF for FA1090, 0.256 pg / mL LI and 0.008 pg / mL PF for F89, and 0.256 pg / mL LI and 0.032pg / mL PF for H041. To quantify the bacteria, 100 pL of suspensions was removed from cultures after 0, 2, 4, 6, and 8 hours. The bacterial suspensions were diluted in GC broth and then plated on GC agar plates containing 1%Attorney Docket No.37759.0695P1Isovitalex. Plates were incubated overnight and then the colonies were enumerated. According to the convention, synergism in the time course assays was defined as differences between results with the combination treatment and the single agent of > 2 log₁₀ (Doem CD. 2014 J Clin Microbiol 52:4124-8).l. SPONTANEOUS MUTATION RATE OF FA1090 TO LI AND PF

[0254] F Al 090 grown overnight was added to D-PBS and diluted so 2.5 × 108CFU were plated on GC agar containing 1% IsoVitaleX and increasing concentrations of PF or LI. Plates were incubated for 48 hours and then enumerated.m. DEVELOPMENT AND GENOMIC ANALYSIS OF LPXC INHIBITOR RESISTANT GONOCOCCI DERIVED BY SPONTANEOUS MUTATION

[0255] FA1090 1-81-S2 was grown overnight, suspended in PBS, and diluted to roughly 5 × 107CFU / mL. Bacterial suspensions were added to GC agar containing 1% IsoVitalex and the LpxC inhibitors at the MIC and twice the MIC, and incubated overnight. When growth was observed, colonies were streaked onto GC agar and incubated overnight. These colonies were suspended in PBS, diluted, and rechallenged with the LpxC inhibitor at the same concentration of previously observed growth and twice that concentration (Abdellati S, et al., 2024 Sci Rep 14: 1179). This process was repeated until FA1090 was resistant at 16- and 8-fold higher levels than the MIC for LI and PF, respectively. This process was also carried out with FA1090 grown on GC agar containing a DMSO vehicle. The resulting strains grown on LI, PF, and DMSO will be referred to as FA1090 SOI (SOI), FA1090 SO4 (SO4), and FA1090 SOV (SOV). SOI, SO4, and SOV were grown on GC agar overnight and then added to PBS. 5 × 108CFU were pelleted by centrifugation at 10,000 x for 30 seconds. Supernatants were aspirated and the Invitrogen PureLink Genomic DNA Mini Kit was used to extract genomic DNA. Instructions for Gramnegative bacteria extraction was followed per the manufacturer’s guidelines. Purity and genomic DNA concentration was determined by UV-vis spectroscopy. For the transformations, 1 pg gDNA from SOI, SO4, and SOV was added to naive FA1090 in GC broth with 1% isovitalex and incubated at 37°C for 6 hours with shaking at 100 RPM. Resulting bacterial suspensions were plated on GC agar either containing the DMSO vehicle, 0.8 pg / mL LI, or 0.16 pg / mL PF. After 24-hour incubation, colonies were enumerated and isolated. Genomic DNA from theAttorney Docket No.37759.0695P1transformants was isolated as described above. Bacterial genome sequencing and assembly was performed by Plasmidsaurus using Oxford Nanopore Technology and custom analysis. FASTA files from the alignment were uploaded to PubMLST using the Neisseria isolates Genome Comparator Plugin. The reference genome used was FA1090 (accession: NC_002946).n. STATISTICAL ANALYSES

[0256] Graphpad Prism 10.3 (GraphPad Software, Boston MA) and SigmaPlot version 12.5 (Systat Software, San Jose, CA) were used for statistical analysis. Multigroup comparisons were performed using a one-way analysis of variance (ANOVA) with Tukey multiple comparison post hoc tests. Statistical analysis on the growth curve assay was performed using a two-way ANOVA with Tukey ’s multiple comparisons test, with a single pooled variance. Significance was defined as P < 0.05 for all comparisons.

[0257] In this study, it was found that the most potent of the novel inhibitors was effective at killing A, gonorrhoeae in multiple conditions in vitro. However, in comparison to historical LpxC inhibitors with hydroxamate head groups, PF-04753299 (PF) and CHIR-090 (CHIR), unexplained differences in potency were observed. The explorations of differences in the iron binding and membrane permeabilization capacity of the inhibitors were reported, which could affect their relative potency for / V. gonorrhoeae. The results, furthermore, reveal a potential new pathway for development of combination therapy for gonorrhea, an approach that could lead to greater activity for a wider range of MDR isolates and organisms, and could reduce the occurrence of resistance.4. RESULTSa. MOLECULAR DOCKING WITH ALPHAFOLD-GENERATED LPXC ENZYME FROM FA 1090 REVEALED VARIABLE BINDING OF INHIBITORS

[0258] Four novel LpxC inhibitors were synthesized using the strategies shown in Scheme 5 - 8. Molecular docking of the gonococcal LpxC enzyme to the four LpxC inhibitor structures shown in Table 1 was used to determine theoretical binding affinities and identify similarities relative to bactericidal potency. The crystal structure of the gonococcal LpxC enzyme has not been solved, therefore, AlphaFold 3 was utilized to generate a model of the 3-dimensional structure of the protein. A positively-charged zinc ion was added that is present in the active siteAttorney Docket No.37759.0695P1along with hydrogens and charges to best resemble those that inhibitors would be expected to encounter in vivo. As shown in FIG. 1, the docking simulations resembled those of previously solved LpxC enzyme and inhibitor complexes (Barb AW, et al., 2007 Proc Natl Acad Sci U S A 104:18433-8), which typically also correlated with the most favorable docking score. These complex conformations consisted of the zinc chelator, either the hydroxamate or the imidazole, occupying the active site and within close proximity to the positively-charged zinc ion. The distal phenyl groups occupied the hydrophobic passage. Lastly, the LpxC inhibitors that consist of a tail group, namely the novel series and CHIR, typically exhibited the morpholine in the chair conformation corking the active site of the enzyme.

[0259] Refering to FIG. 1, In silico docking models of FA1090 LpxC enzyme generated by AlphaFold is shown. Docking simulations of LI, PF, and CHIR-090 were performed by Autodock Vina platform. Not shown are docking simulations of L2, L3, and L4, which were similar to LI.

[0260] The dissociation constants for the novel LpxC inhibitors and CHIR were similar in the range of 200-1000 nM, with L4 exhibiting the most favorable value (Table 2). The highest dissociation constant of the LpxC inhibitors was for PF calculated at 2514 nM. Antibiotics ceftriaxone and chloramphenicol were employed as negative controls, exhibiting dissociation constants of 5383 nM and 23128 nM, respectively. Whereas binding affinity is not the sole determinant of bactericidal potency, and in silico molecular docking simulations have limitations in calculating affinities, the data suggest that comparatively the inhibitors exhibit promising affinity for the gonococcal LpxC enzyme.Table 2. In silico Binding Affinities for FA1090 LpxC*Free energy DissociationAntibiotic(kcal / mol) constant (nM)LI -9.07 416L2 -9.03 444L3 -8.52 1015L4 -9.38 252PF-04753299 -7.96 2514CHIR-090 -8.75 699Attorney Docket No.37759.0695P1Free energy DissociationAntibiotic(kcal / mol) constant (nM)Ceftriaxone -7.49 5383Chlorampenicol -6.59 23128*Autodock Vina expresses binding scores as Free Energy (AG). Relative dissociation constant Kd) was calculatedusing the Gibbs-Helmholtz equation AG= RTln(Kd).Assuming 37.5°C.b. PF AND L 1 WERE THE MOST POTENT LPXC INHIBITORS IN BACTERICIDAL ASSAYS

[0261] The bactericidal potency of the four novel LpxC inhibitors and inhibitor PF was determined in the presence of 10% serum in the 4 hour bactericidal assay (FIG.2). Of the four LpxC inhibitors, LI and L3 were the most potent, with LI being significantly more potent to the human challenge and MDR strains than L3. The difference of potency between LI and L3 highlight the implications of relatively minor structural changes, since the only difference between them is the para versus meta orientation of the morpholino group on the distal phenol. PF was significantly more potent than LI against all strains in the bactericidal assays.Intriguingly, these data contrast with PF having a far lower binding affinity than the novel LpxC inhibitors in the in silico molecular docking assay. L2 and L4 were excluded from the rest of the analyses to focus on the two compounds, LI and L3, that were most potent in vivo.

[0262] Refering to FIG.2, bactericidal activity of increasing concentrations (0.008, 0.04, 0.2, 1.0, 5.0 and 25 pg / mL) of PF, LI, L2, L3, and L4 LpxC inhibitors for FA1090, F89, and H041 strains of N. gonorrhoeae is shown. All assays were performed in RPMI-1640 medium containing 10% FBS for 4 h. Bars represent the mean ± SEM of results from three experiments that were each performed in duplicate.c. LPXC INHIBITORS CAUSED LIMITED CYTOTOXICITY AND NO HEMOLYSIS

[0263] The cytotoxicity of the LpxC inhibitors was tested by quantification of lactate dehydrogenase (LDH) release from human cells and hemolysis of sheep red blood cells. LDH release was measured after treatment of cervical epithelial ME- 180 cells and activated THP-1Attorney Docket No.37759.0695P1monocytic cells with 25 or 50 pg / mL PF, LI, and L3 (FIG. 3). Treatment of THP-1 cells with PF at 50 pg / mL was significantly more cytotoxic than LI at 50 pg / mL (P < 0.05). Hemolysis levels for PF, LI, and L3 at 25 pg / mL and 50 gg / mL were well below the 10% threshold for being considered non-hemolytic (Amin K, Dannenfelser RM. 2006 J Pharm Sci 95:1173-6). The results indicate that minimal cytotoxicity should be expected in vivo.

[0264] Refering to FIG. 3, LDH of ME-180 and THP-1 cells (top panels) after 24 hour treatment with PF, LI, or L3 is shown. LDH release was normalized to cells treated with 0.1% Triton X-100 and PBS for 1 hour and expressed as a percentage. Bars represent SEM of three experiments performed in triplicate (*P < 0.05). Hemolysis levels (bottom panel) of sheep red blood cells treated with PF, LI, or L3 for 1 hour. Hemolysis was normalized to cells treated with 0.1% Triton X-100 and PBS. Bars represent the mean ± SEM of three experiments performed in triplicate (***P < 0.001).d. EFFICACY OF LPXC INHIBITORS IN 24-HOUR MIC ASSAYS DIFFERED FROM IN SILICO BINDING AFFINITY[00265J The MIC values for the most potent novel inhibitor, LI, versus human challenge and MDR strains were below 1 pg / mL, suggesting promising efficacy (Table 3). Furthermore, both PF and LI had MICs for human challenge and MDR strains that were 50-fold lower than levels that induced the observed cytoxicity. The MIC values of PF for the three N. gonorrhoeae strains were lower by a factor of 8-32 fold compared to LI in the 24 hour MIC assay. This result is in contrast with the in silico data, which showed that PF had less affinity for the LpxC enzyme compared to LI (Table 2 and Table 3). Furthermore, the MIC of PF for N. gonorrhoeae was more than 3 orders of magnitude lower than that of CHIR, whereas the theoretical relative binding affinity of CHIR for the N. gonorrhoeae LpxC enzyme was more favorable than that of PF. In the 24 hour MIC assays, PF was also significantly more potent than LI versus F89.However, the relative difference in potency for H041 bacteria between PF and LI was less in the 24 hour MIC assay compared to the difference observed in the 4 hour bactericidal testing (FIG.2). Because the measured MIC after 24 hr was significantly different than that seen in the bactericidal killing assay after 4 hrs (see FIG.2 vs Table 3), the results suggest that the bactericidal activity of the LpxC inhibitors is through depletion of LOS overtime.Attorney Docket No.37759.0695P1Table 3. MIC values* in 24 hour assaysTreatmentBacterial strainLI PF-04753299 CHIR-090 FA1090 0.040 0.002 5F89 0.256 0.008 25H041 0.256 0.032 50*MICs = pg / mLe. POTENCY OF LPXC INHIBITORS IN THE PRESENCE OF HUMAN CELLS

[0266] The 4 hour bactericidal assay was performed with FA1090 and inhibitors LI, L3, and PF in the presence of human Endl / E6E7 endocervical epithelial cells, ME- 180 cervical epithelial cells, and activated THP-1 monocytes to determine bactericidal potency ex vivo. After incubation, the cells were lysed to facilitate quantification of both intracellular and extracellular bacteria (FIG. 4). In the presence of all three cell types, PF at 0.2 pg / mL was more potent than L3, and PF was more potent than LI in the presence of Endl / E6E7 cells (P < 0.05). In the presence of ME-180 and THP-1 cells, LI compared to L3 at 0.2 pg / mL was significantly more potent (both P < 0.001). The results demonstrated that all three LpxC inhibitors were bactericidal for N. gonorrhoeae during the process of infecting human cells.

[0267] Refering to FIG. 4, Bactericidal assay with FA 1090 and increasing concentrations (0.2, 1.0, 5.0 and 25 pg / mL) of PF, LI, and L3 in the presence of a confluent monolayer of Endl / E6E7, ME-180, or THP-1 cells is shown. Cells were lysed with 1% saponin after 4 hour treatment to survey potential intracellular bacteria. Bars represent the mean ± SEM of results from three experiments that were each performed in duplicate (*P < 0.05, **P < 0.01, and ***P < 0.001).

[0268] These results were compared to the bactericidal assay without human cells (FIG. 2).There were no significant differences between the percent survival of bacteria with or without ME-180 cells. The presence of Endl / E6E7 cells significantly reduced the susceptibility of gonococci to LI, PF, and L3 (0.2 pg / mL LI P < 0.0001; 1 pg / mL LI P < 0.0001; 0.2 pg / mL PF P < 0.0001; and 0.2 pg / mL L3 P < 0.05). The presence of THP-1 cells on the activity of the LpxC inhibitors had variable effects. The THP-1 cells did not significantly affect the potency of LI, but their presence increased the potency of PF at 0.2 pg / mL (P < 0.05) and decreased the potency of L3 at 0.2 pg / mL (P < 0.01).Attorney Docket No.37759.0695P1f. STALYLATTON OF BACTERIA MINIMALLY REDUCED EFFICACY OF LPXC INHIBITORS

[0269] Human challenge and MDR WHO strains of N. gonorrhoeae were grown overnight on GC agar containing CMP -NANA to sialylate the bacteria, which were subsequently treated with LI, L3, and PF in the presence of serum (FIG. 5). Both PF and LI were more potent than L3 for FA1090 and H041 at 0.2 pg / mL, the lowest concentration tested (all P < 0.001).

[0270] At the three highest concentrations tested, sialylation did not alter the potency of the LpxC inhibitors compared with non-sialylated bacteria (FIG.2 and FIG.5). However, at the lowest concentration tested, 0.2 pg / mL, significantly more sialylated FA1090 survived when treated with L3 (P < 0.001) compared to non-sialylated FA1090, and the percent survival of sialylated versus non-sialylated H041 was significantly greater when the bacteria were treated with PF (P < 0.01).

[0271] Refering to FIG. 5, bactericidal activity of increasing concentrations (0.2, 1.0, 5.0 and 25 pg / mL) of PF, LI and L3 LpxC inhibitors for sialylated FA1090, F89, and H041 bacteria is shown. For sialylation, bacteria were plated overnight on GC agar with 1% Isovitalex and 10 pg / mL CMP-NANA. All assays were performed in RPMI-1640 medium containing 10% FBS for 4 h. Bars represent the mean ± SEM of results from three experiments that were performed in duplicate (*P < 0.05, **P < 0.01, and ***P < 0.001).g. LPXC INHIBITORS REDUCED THE INDUCTION OF TNF-A BY GONOCOCCAL INFECTION OF THP-1 CELLS

[0272] The effect of treatment with the LpxC inhibitors was analyzed on the induction of TNF-a caused by strain FA1090 infection of the THP-1 human monocytic cells using an ELISA (FIG. 6). PF and LI at 1 pg / mL significantly reduced TNF-a expression by THP-1 cells in the presence of FA1090 (both P < 0.001). PF, LI, and L3 at 5 and 25 pg / mL significantly reduced the TNF-a expression in the presence of FA1090, F89 or H041. The result showing that PF was more potent than LI versus FA1090 and F89 in the MIC assay was reflected by the significantly greater reduction in TNF-a expression by PF at 1 pg / mL compared to LI or L3 for both strains.

[0273] Refering to FIG. 6, ELISA of TNF-a released from activated THP-1 cells after 18 hour treatment with FA1090, F89, or H041 and PF, LI, or L3 is shown. Black lines and asterisksAttorney Docket No.37759.0695P1denote significant differences between the effect of treatment compared to the control whereas grey lines and asterisks denote significant differences of the paired treatments. White bars represent 18 hour treatment with bacteria and vehicle control (0.5% DMSO in PBS). Bars represent the mean ± SEM of three experiments performed in triplicate (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).h. LPXC INHIBITORS REDUCED THE EXPRESSION OF LOS

[0274] A whole-cell ELISA utilizing the 2C7 antibody, which binds to the 2C7 epitope of gonococcal LOS that is expressed by FA1090 (Gulati S, et al., 2019 PLoS Biol 17:e3000323; Gulati S, et al., 2019 Front Immunol 10:321), was used to quantify the inhibition of lipid A biosynthesis in the strain. The levels observed are expected to reflect LOS expressed on the outer membrane of the bacteria. In comparison to untreated controls, the expression of LOS by FA1090 treated with PF and LI for 4 hours showed a dose-response reduction in LOS expression that was significantly reduced by approximately 35% with a 1 pg / ml dose of either inhibitor (FIG. 7).[00275J Referring to FIG. 7, ELISA of 2C7 monoclonal antibody binding to FA1090 after 4 hour treatment with LI at 0.2 or 1.0 pg / mL concentrations is shown. The white bar represents 2C7 binding to LOS of FA1090 treated with vehicle-only. Bars represent the mean ± SEM of two experiments performed in triplicate (**P < 0.01 and ***P < 0.001).i. AN EFFLUX PUMP INHIBITOR INCREASED THE POTENCY OF LI AND PF

[0276] The effect of adding the efflux pump inhibitor, PAβN, to bactericidal assays was tested with the LpxC inhibitors PF, LI, and CHIR. The H041 isolate was specifically tested because it is known to be highly drug resistant due to its up-regulation of efflux pumps (29). Both LI and PF were significantly more potent for H041 in the presence of PAβN, whereas differences in potency of CHIR for H041 with or with out PAβN did not achieve statistical significance (FIG. 8).

[0277] Refering to FIG. 8, the results of bactericidal assays with H041 treated with PF, LI, and CHIR with or without 50 pg / mL PAβN is shown. Bars represent the mean ± SEM of results from three experiments that were each performed in duplicate (*P < 0.05 and **P < 0.01).Attorney Docket No.37759.0695P1j. PF AND CHIR LPXC INHIBITORS BOUND INORGANIC IRON

[0278] Without wishing to be bound by theory, iron binding by the LpxC inhibitors could be a second, bacteriostatic mechanism contributing to their potency since some LpxC inhibitors have been shown to bind zinc, and the LpxC enzyme can bind either iron or zinc, which are essential nutrients for N. gonorrhoeae that are of limited availability in their human hosts (Jackman JE, et al., 1999 Biochemistry 38:1902-11; Yamada Y, et al., 2020 J Med Chem 63:14805-14820; McClerren AL, et al., 2005 Biochemistry 44:16574-83; Barb AW, Zhou P. 2008 Curr Pharm Biotechnol 9:9-15; Cornelissen CN. 2018 Pathog Dis 76; Gattis SG, et al., 2010 J Biol Chem 285:33788-96). PF and CHIR, but not LI bound to iron as determined by the shift in spectral scan (FIG. 9). Evidence of iron binding by the LpxC inhibitors was obtained by observing the formation of colored complexes that absorb visible wavelengths, which is a natural property of iron and other transition metals (Chan DCK, et al., 2020 Antimicrob Agents Chemother 64). Chloramphenicol, ceftriaxone, and deferoxamine (DFO) were used as reference controls due to their known iron binding properties (Chan DCK, et al., 2020 Antimicrob Agents Chemother 64). Chloramphenicol and DFO served as negative and positive controls, respectively. Ceftriaxone bound to iron intermediately to the iron binding capacity of chloramphenicol and DFO.

[0279] Refering to FIG. 9, spectral scan of antibiotics in the presence of FeCl3 from 300-700 nm in 2-nm increments (3:1) is shown. FeCl3 was solubilized in water and antibiotics were solubilized in DMSO. DFO and chloramphenicol were the positive and negative controls, respectivelyk. COMBINATION TREATMENT OF LPXC INHIBITORS AND DFO IN 24 HOUR CHECKERBOARD ASSAYS WAS NOT SYNERGISTIC

[0280] Treatment of FA1090 with LI and DFO in GC broth supplemented with IsoVitalex in single time point 24 hour checkboard assays lowered the MIC compared to treatment with LI or DFO alone (Table 4). Intermediately, the MIC values of CHIR with DFO, were respectively lowered slightly with a fractional inhibitory concentration index (FICI) value of 1. The MIC values of PF with DFO remained the same in combination treatment compared to the single agent treatment. Based on the guidelines for defining synergism in single-time point checkboardAttorney Docket No.37759.0695P1assays, FICI must be equal to or below 0.5 (Odds FC. 2003 J Antimicrob Chemother 52:1), which was not achieved by any of the combination treatments shown here.Table 4. MIC values of FA1090 in checkerboard assaysMIC MIC of LpxC inhibitorCompound FICI (pg / mL) and DFO (pg / mL)DFO 35LI 0.04 0.02 and 6.25 0.75PF-04753299 0.002 0.002 and 25 2CHIR-090 5 2.5 and 12.5 11. COMBINATION TREATMENT WITH LPXC INHIBITORS AND DFO SHOWED PROMISING RESULTS TN 8 HOUR GROWTH CURVES

[0281] To further explore the interaction between the LpxC inhibitors, iron chelation, and N. gonorrhoeae beyond simply determining MIC values of the combinations, assays of growth in liquid GC broth after 2, 4, 6 and 8 hour time periods were performed. The growth assays were performed with all three strains to reflect potential differences in susceptibility. Over the 8 hours, DFO and LpxC inhibitors inhibited the division rate of FA1090, F89, and H041 (FIG. 10). DFO treatment significantly reduced the growth of FA1090 relative to the vehicle at 6 and 8 hours (both P < 0.0001). DFO treatment of F89 also significantly reduced growth relative to the vehicle-only treated controls at 8 hours (P < 0.001). However, the results showed that the DFO treatment of H041 did not significantly reduce growth compared to vehicle treated controls. Comparisons of LI alone and PF alone compared to the combination of LI or PF plus DFO revealed significant differences in treatment of FA1090 at 6 and 8 hours and for H041 at 8 hours (FIG. 10). The killing of LI and PF with DFO in comparison to LI or PF alone exhibited synergism as defined by differences > 2 log₁₀ (Doem CD. 2014 J Clin Microbiol 52:4124-8).

[0282] Refering to FIG. 10, growth curves of FA1090, F89, and H041 treated with vehicle, DFO, LI, and / or PF over 8 hours is shown. Bacteria were grown in 5-mL snap cap tubes containing GC broth with 1% IsoVitaleX and sampled every 2 hours. Bars represent the mean ± SEM of three experiments (*P < 0.1, **P < 0.01, ***P < 0.001; ****P < 0.0001).Attorney Docket No.37759.0695P1m. DEVELOPMENT OF LPXC INHIBITOR RESISTANT GONOCOCCI AND GENOMIC ANALYSIS

[0283] Because it was noted that resistance could be impacted by efflux pump inhibitors, mutants that had elevated MICs were attempted to be isolated. When naive bacteria were plated directly on antibiotic containing media, colonies were failed to be isolated after 48 hrs of incubation. However, serial passage of FA1090 on increasing concentrations allowed us to isolate colonies that grew well on elevated concentrations of the inhibitors.

[0284] To determine if this growth was due to a selection of a spontaneous mutation, DNA was isolated from a colony that grew on elevated levels of PF and LI, and then used this DNA to perform a transformation with selection at 4 times the MIC. The data in Table 4 demonstrate that colonies were able to be generated at a frequency at least 3 logs higher than the background mutation frequency. Without wishing to be bound by theory, this data strongly indicates that resistance is arising through a heritable change.

[0285] In order to better understand the genetic basis of this resistance, genomic DNA sequencing was performed on about 10 of the transformants for each antibiotic. DNA sequence analysis indicated that all transformants retained the native IpxC sequence, suggesting the target of the antibiotic was not involved in the acquired resistance. An impact was observed on resistance using an efflux pump inhibitor and, therefore, the DNA sequence was analyzed of the MTR region (MtrR, MtrA, MtrCDE, MacA-MacB, and Nor M) and found that no variations in sequence were identified. Because the number of identified SNPs among all of the strains sequenced was high, a specific sequence change could not be correlated with elevated resistance.

[0286] Referring to FIG. 11 A and FIG. 1 IB, resistant mutant development reveals alternative mechanisms of resistance to LI and PF. FIG. 11A shows mutants resistant to PF and LI of FA1090 were developed by serial passage on antibiotic containing agar. FIG. 11B shows genomic DNA from the mutants was extracted and analyzed by nanopore sequencing. Mutant DNA was added to naive bacteria and plated on GC agar.5. DISCUSSION

[0287] The development of an inhibitor of the LpxC deacetylase enzyme from N. gonorrhoeae was focused on as a potential treatment for gonorrhea. MICs of the LpxC inhibitors for three isolates of N. gonorrhoeae were determined and bactericidal assays were performedAttorney Docket No.37759.0695P1including those with ex vivo conditions because using MIC values alone can be misleading when aiming to translate to clinical applications (Belanger CR, Hancock REW 2021. Nat Protoc 16:3761-3774). The conditions tested in the bactericidal assays included the presence of serum, human cells, and sialylated bacteria. PF was more potent than LI either with or without the addition of serum in the 4-hour assays, however LI exhibited significant potency for the three gonococcal strains with MIC values of 0.256 pg / mL or below and bactericidal 4 hour assay MBC90 values of 1 pg / mL for FA1090 and F89 and 5 pg / mL for H041. It was also shown that LI reduced the synthesis of LOS, the mechanism that is the basis for development of LpxC inhibitors.

[0288] In the presence of endocervical epithelial cells, the LpxC inhibitors had significantly decreased potency, which was expected because antibiotics can be hindered kinetically by off-target binding to secreted, cell surface, and intracellular proteins. Instances where increased or sustained potency in the presence of human cells was observed, as with the monocytes and the cervical epithelial cells, respectively, could be attributed to greater susceptibility of gonococci to LpxC treatment due to increased shedding of peptidoglycan, LOS, and outer membrane vesicles by the bacteria while initiating infection (Quillin SJ, Seifert HS. 2018 Nat Rev Microbiol 16:226-240). Since the gonococcus relies more heavily on LOS and outer membrane biosynthesis during invasion, LpxC inhibition potentially could be more efficacious during this process. Additionally, supplementation of the GC growth media with sialic acid, which A. gonorrhoeas pirates from human hosts during infection to sialylate its LOS, had relatively little effect on the potency of the LpxC inhibitors. The induction of TNF-a in epithelial cells, monocytes, and other cells such as macrophages primarily through the interaction of LOS with TLR4 is a well characterized inflammatory response to gonococcal infection (McGee ZA, et al., 1992 Microb Pathog 12:333-41; Pridmore AC, et al., 2003 Infect Immun 71:3901-8). Treatment of THP-1 monocytic cells with human challenge and MDR strains of N. gonorrhoeae and PF, LI, or L3 all significantly reduced TNF-a after 18 hours. Overall, these data show that PF, LI, and L3 were efficacious in various representative ex vivo conditions.

[0289] Subtle differences in the LpxC enzyme can contribute to significant differences in potency of LpxC inhibitors (Barb AW, et al., 2007 Biochemistry 46:3793-802). In silico docking studies were performed to compare the relative affinities of the LpxC enzyme to the novel LpxC inhibitor series, PF, and CHIR. A recent study correlated gepotidacin resistance with structuralAttorney Docket No.37759.0695P1changes in GyrA and ParC caused by mutations in N. gonorrhoeae isolates, which were characterized with AlphaFold (David A, et al., 2024 J Antimicrob Chemother 79:2221-2226). Since the crystal structure for the N. gonorrhoeae LpxC enzyme has not been solved, AlphaFold and semblance to the homologous LpxC enzyme from Aquifex aeolicus whose crystal structure has been solved was relied on (Barb AW, et al., 2007 Proc Natl Acad Sci U S A 104: 18433-8). Interestingly, although it was found that the novel LpxC inhibitors and CHIR had higher affinity than PF for the LpxC enzyme in silico the pattern of relative binding affinities was not well correlated with relative differences in potency observed in bactericidal potency. Therefore, other potential interactions of the LpxC inhibitors with N. gonorrhoeae were explored that could affect the relative potency observed with N. gonorrhoeae.

[0290] While docking programs have become a mainstay in many drug development projects, docking scores can be misleading (Chen YC. 2015 Trends Pharmacol Sci 36:78-95). For example, CHIR binding to LpxC in A. aeolicus is thought to occur via a rapid and reversible first step and a slower, irreversible, and tightly bound second step, a two-step mechanism which is not reflected in the docking that was performed (McClerren AL, et al., 2005 Biochemistry 44:16574-83). Thus, docking scores are presented herein are as one comparative tool.

[0291] Up-regulation of efflux pumps is theorized to contribute to LpxC inhibition resistance (Caughlan RE, et al., 2012 Antimicrob Agents Chemother 56:17-27; Tomaras AP, et al., 2014 mBio 5:e01551— 14). The effect of addition of an efflux inhibitor, PApN, was tested and found that it increased the potency of both LI and PF for strain H041. However, it was found that PAβN did not significantly potentiate the activity of CHIR. This apparent discrepancy could be because H041 is so much less susceptible to the bactericidal effects of CHIR that small differences in potency in the presence of PaPN were unable to be detected.

[0292] Differences in potency between the LpxC inhibitors potentially could be due to differences in membrane permeability. Moller et al., explored the possibility that because LpxC inhibitors such as CHIR and PF have amphipathic characteristics, depolarization of the membrane could be an additional bactericidal mechanism, like the mechanism for polymyxin B. However, they found no depolarization of E. coli by the LpxC inhibitors tested using the fluorescence probe DiSC3 (Moller AM, et al., 2024 J Biol Chem 300:107143). The multidrug efflux pump system (MtrCDE) is the most important pump affecting antibiotic resistance (Ayala JC, et al., 2022. Microbiology (Reading) 168). Furthermore, since the DNA sequence of theAttorney Docket No.37759.0695P1MTR region was conserved in all of the stable mutations obtained after DNA transformation, it suggests that entry or efflux of the inhibitors occurs via another pathway.

[0293] The LpxC enzyme has been found to contain a Zn2+molecule in the active site and LpxC inhibitors have been shown to complex with the Zn2+when bound to the enzyme (Jackman JE, et al., 1999 Biochemistry 38:1902-11; Yamada Y, et al., 2020 J Med Chem 63:14805-14820; McClerren AL, et al., 2005 Biochemistry 44:16574-83; Barb AW, Zhou P. 2008 Curr Pharm Biotechnol 9:9-15; Cornelissen CN. 2018 Pathog Dis 76) (3, 24, 30-32). Interestingly, LpxC purified from E. coli has been found to bind either Fe2+or Zn2+(Gattis SG, et al., 2010 J Biol Chem 285:33788-96). Furthermore, pirating metal ions including Fe from the metal binding proteins of their host or other bacteria is essential for the survival of N gonorrhoeae, which do not encode siderophores (Cornelissen CN. 2018 Pathog Dis 76). Therefore, the possibility that iron binding was a bacteriostatic mechanism that facilitated killing by PF was next explored, whereas LI activity reflected solely LpxC inhibition. It was initially determined that the hydroxamate based inhibitors bind to iron by a spectral scan. To explore how iron chelation could play a role in bactericidal capability a potent iron chelator, DFO, was added to checkerboard and growth curve assays. None of the LpxC inhibitors showed synergism with DFO in checkerboard assays, however combination treatment with LI and DFO exhibited a reduction in their respective MIC values. Without wishing to be bound by theory, it was then postulated that the addition of DFO to PF treatment had no effect because PF already utilizes iron binding as a mechanism of killing. To test this hypothesis further the growth curve assays were performed with LI and PF with and without DFO. Overall, the differences in potency in the growth curve assays between the LpxC inhibitors alone and the synergistic potency observed of the LpxC Inhibitors with DFO potentially could be attributed to multiple factors. One possibility is that DFO could increase the permeability of the bacterial membrane by binding ions in solution (Bellotti D, Remelli M. 2021 Molecules 26), thereby aiding uptake.

[0294] Differences in iron acquisition systems among the WHO strains has been observed (El-Rami FE, et al., 2019 Mol Cell Proteomics 18:127-150). It was found that FA1090 and F89 were more sensitive than H041 to the same concentration of DFO in media. Intriguingly, FA1090 and F89 also were more susceptible than H041 to PF. However, F89 sensitivity to LI resembled that of H041. The correlation between strain-specific sensitivity to iron chelationAttorney Docket No.37759.0695P1resembling sensitivity to PF further suggests that PF utilizes a second unexploited iron binding mechanism.

[0295] Explanations for the synergy observed between LpxC inhibition and DFO could be attributed to biological factors affecting the bacteria. N. gonorrhoeae is an iron scavenger that must regulate its iron storage and iron acquisition systems proficiently to survive and evade the immune system in vivo (Cornelissen CN. 2018 Pathog Dis 76; Zughaier SM, et al., 2014 PLoS One 9:e87688). Additionally, iron directly affects a multitude of transcriptional regulators in Gram-negative bacteria (Ezraty B, Barras F. 2016 FEMS Microbiol Rev 40:418-35).Intriguingly, gonococcal sensitivity to antibiotics that are subject to efflux can be decreased by iron limiting conditions, which reduces the expression of the iron uptake regulator Fur (Ezraty B, Barras F. 2016 FEMS Microbiol Rev 40:418-35; Mercante AD, et al., 2012 Antimicrob Agents Chemother 56:1491-501). Fur acts by a multistep mechanism by repressing mpeR expression, which in turn increases the expression of MtrR that represses the mtrCDE operon. Greater expression of mtrCDE operon would increase transcription of efflux pumps, which can decrease antibiotic sensitivity.

[0296] Some groups have suggested that iron can be sequestered intracellularly or by the pilus of N. gonorrhoeae (Hu LI, et al., 2022. PLoS Pathog 18:e 1010561). Future studies could include testing pil+ and pil- variants of N gonorrhoeae against the LpxC inhibitors and iron limiting growth conditions to determine if this plays a significant role in susceptibility. N. gonorrhoeae genes for rapid bacterial growth are preferentially expressed under iron limitation conditions (Ducey TF, et al., 2005 J Bacteriol 187:4865-74). Iron starvation conditions have been shown to induce the output of larger outer membrane vesicles (OMV) in N. gonorrhoeae (Placzkiewicz J, et al., 2023 Sci Rep 13:18733). Furthermore, iron starvation generally induces a stressful environment for the bacteria, through oxidative stress, which has been well documented to induce OMV production in Gram-negative bacteria (Mozaheb N, Mingeot-Leclercq MP. 2020 Front Microbiol 11:600221). The necessity for increased production of the outer membrane, such as during bacterial replication and production of OMVs, could potentially sensitize the bacteria to LpxC inhibitors.

[0297] Based on the data presented here, combination treatment of LpxC inhibitors with DFO may be a promising strategy. Utilizing DFO in combination treatment with other antibacterial agents has been explored in a limited manner (Chan DCK, et al., 2020 AntimicrobAttorney Docket No.37759.0695P1Agents Chemother 64; Erinmez M, Zer Y. 2024 Adv Clin Exp Med 33:491-497). Ironically, depending on the infection and bacteria, there are examples of iron chelation decreasing (Ando Y, et al., 1988 Hokkaido Igaku Zasshi 63:207-12) and promoting bacterial growth (Collins HL, et al., 2002 J Immunol 168:3458-63; Chan GC, et al., 2009 Hemoglobin 33:352-60).Differences between the N. gonorrhoeae strains in response to DFO treatment were observed. DFO is a semi-synthetic drug derived from the bacterial siderophore desferrioxamine B that was originally discovered in Streptomyces pilosus (Bellotti D, Remelli M. 2021 Molecules 26).Desferrioxamine B is a xenosiderophore for N. gonorrhoeae and the exact nature of the interaction of its derivative DFO with TV. gonorrhoeae is unknown (Cornelissen CN. 2018 Pathog Dis 76; Mickelsen PA, Sparling PF. 1981 Infect Immun 33:555-64).

[0298] Our results showing that LpxC inhibition is facilitated by combination with iron chelation for N. gonorrhoeae are aligned with strategies that are being actively pursued to reduce the development of resistance. The simultaneous use of two agents that act by different mechanisms delivered in a single dosage form or administered separately over the same time period has been used with great success (Geddes AM, et al., 2007 Int J Antimicrob Agents 30 Suppl 2: S109-12; Wesolek JL, et al., 2022 Am J Emerg Med 56:178-182; Masters PA, et al., 2003 Arch Intern Med 163:402-10). Perhaps of greatest relevance is what was the first line treatment for gonorrhea in many countries for more than a decade - administration of the third generation cephalosporin, ceftriaxone, along with azithromycin, the latter a bacteriostatic macrolide antibiotic that inhibits bacterial protein synthesis.

[0299] Overall, the differences in potency between the LpxC inhibitors and the synergism with DFO and the LpxC inhibitors specifically with N. gonorrhoeae could be attributed to a multitude of factors. Therefore, resistance was induced to LI and PF in FA1090, which was only achievable by continual passaging. To determine if resistance was heritable, naive FA1090 with the mutant DNA were incubated, which resulted in resistance to both LpxC inhibitors. The resistant mutants were then sequenced with Oxford Nanopore and no alterations to IpxC or mtrCDE were discovered. While sequencing with this method is typically coupled with MiSeq, recent studies suggest that with proper assembly, the latest generation Nanopore sequencing can be an accurate predictor of MDR determinants in N. gonorrhoeae (Phillips LT, et al., 2023 J Infect Dis 228: 1179-1188; Golparian D, Unemo M. 2022 Expert Rev Mol Diagn 22:29-48). Furthermore, all 20 strains sequenced did not contain mutations in the IpxC, MtrR, MtrA,Attorney Docket No.37759.0695P1MtrCDE, MacA-MacB, or Nor A loci. These data are encouraging for the development of LpxC inhibitors for treating MDR N. gonorrhoeae because spontaneous mutation rates are very low and resistance via a known MDR determinant, namely MTR, was not discovered.

[0300] Multiple groups have developed mutants of other species of Gram-negative bacteria that were resistant to LpxC Inhibitors (Erwin AL. 2016. Antibacterial drug discovery targeting the lipopolysaccharide biosynthetic enzyme LpxC. Cold Spring Harb Perspect Med 6).Intriguingly, few groups have shown resistance that was attributed to mutations in the LpxC gene (Caughlan RE, et al., 2012 Antimicrob Agents Chemother 56:17-27; Tomaras AP, et al., 2014 mBio 5:e01551— 14; Clements JM, et al., 2002 Antimicrob Agents Chemother 46:1793-9). The very low spontaneous mutation rates was observed for FA 1090 grown with LpxC inhibitors at 4X the MIC are consistent with previous reports of Gram-negative bacteria susceptibility to historical LpxC inhibitors (Erwin AL. 2016. Antibacterial drug discovery targeting the lipopolysaccharide biosynthetic enzyme LpxC. Cold Spring Harb Perspect Med 6; Barb AW, et al., 2007 Biochemistry 46:3793-802; Zeng D, et al., 2013 J Biol Chem 288:5475-86). Pathways for resistance development potentially could be attributed to differences in permeability as discussed above, and mutations in the fabZ gene, which is theorized to increase the concentration of LpxC substrates, have been reported by other groups (Clements JM, et al., 2002 Antimicrob Agents Chemother 46: 1793-9; Zeng D, et al., 2013 J Biol Chem 288:5475-86). Verifying genomic resistance determinants of LpxC resistant mutants by sequencing will be part of future studies.

[0301] Our results support the concept that an LpxC inhibitor with a non-hydroxymate head group, such as LI, has promise as a treatment of N. gonorrhoeae infections that would be expected to have a low potential for inducing bacterial resistance. It was observed that iron chelation with DFO was bactericidal for N. gonorrhoeae and that the zinc-binding ability of LpxC inhibitors also could facilitate binding to iron. The differences in the theoretical binding affinities of the inhibitors for LpxC that was derived from in silico modeling and their relative potency in vitro suggests that other differences, such as in iron binding capacity and or membrane permeability, may contribute to their bactericidal activity. Although the off-target binding of LpxC inhibitors that is dependent on chelation has been linked with adverse cardiovascular effects (Fujita K, et al., 2022 J Antibiot (Tokyo) 75:98-107; Shen S, Kozikowski AP. 2016 ChemMedChem 11: 15-21), It was shown that the combination of an LpxC inhibitorAttorney Docket No.37759.0695P1with a iron chelator could have synergistic anti-N. gonorrhoeae activity. The findings support further exploration of an iron chelator such as DFO in combination with an antimicrobial agent such as a specific LpxC inhibitor for gonorrhea.J. REFERENCES

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Claims

Attorney Docket No.37759.0695P1CLAIMSWhat is claimed is:A compound having a structure represented by a formula:Ar1bk Nwherein n is an integer selected from 1 and 2;wherein Z1is selected from -N- and -CH-;wherein Z2is selected from -NR10-, -O-, -S-, -SO2-, and -CR11aR11b-;wherein R10is selected from hydrogen and C1-C4 alkyl;wherein each of R11aand R11bis independently selected from hydrogen and methyl; andwherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl,or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein n is 1.

3. The compound of claim 1, wherein n is 2.

4. The compound of claim 1, wherein Z1is -N-.

5. The compound of claim 1, wherein Z1is -CH-.Attorney Docket No.37759.0695P16. The compound of claim 1, wherein Z2is selected from -NR10- and -O-.

7. The compound of claim 1, wherein Z2is selected from -N(CH3)- and -O-.

8. The compound of claim 1, wherein Ar1is selected from a C6 aryl and a 5- to 6-membered heteroaryl, and is further unsubstituted.

9. The compound of claim 1, wherein Ar1is a C6 aryl substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, Cl-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl.

10. The compound of claim 1, wherein Ar1is a C6 aryl substituted and is further unsubstituted.

11. The compound of claim 1, wherein Ar1is a 5- to 6-membered heteroaryl substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl.

12. The compound of claim 1, wherein Ar1is a 5- to 6-membered heteroaryl and is further unsubstituted.

13. The compound of claim 1, wherein Ar1is a 5- to 6-membered heteroaryl selected from imidazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridinyl, and triazinyl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxy alkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl.

14. The compound of claim 1, wherein Ar1is a 5- to 6-membered heteroaryl selected from imidazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridinyl, and triazinyl, and is further un substituted.

15. The compound of claim 1, wherein the compound has a structure represented by a formula:Attorney Docket No.37759.0695P1or a pharmaceutically acceptable salt thereof.

16. The compound of claim 1, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

17. The compound of claim 1, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

18. The compound of claim 1, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.Attorney Docket No.37759.0695P119. The compound of claim 1, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

20. The compound of claim 1, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

21. The compound of claim 1, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

22. The compound of claim 1, wherein the compound has a structure represented by a formula:wherein each of Q1, Q2, and Q3is independently selected from -N= and -C(R20)=;Attorney Docket No.37759.0695P1wherein each occurrence of R20is independently selected from hydrogen, halogen, - CN, -NH2, -OH, -NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl,or a pharmaceutically acceptable salt thereof.

23. The compound of claim 22, wherein each occurrence of R20is independently selected from hydrogen, halogen, and C1-C4 alkyl.

24. The compound of claim 22, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

25. The compound of claim 22, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

26. The compound of claim 22, wherein the compound has a structure represented by a formula:Attorney Docket No.37759.0695P1or a pharmaceutically acceptable salt thereof27. The compound of claim 22, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

28. The compound of claim 1, wherein the compound is selected from:or a pharmaceutically acceptable salt thereof.Attorney Docket No.37759.0695P129. The compound of claim 1, wherein the compound is selected from:or a pharmaceutically acceptable salt thereof.

30. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 29, further comprising an effective amount of an iron chelator.

32. The pharmaceutical composition of claim 31, wherein the iron chelator is selected from deferoxamine (DFO), deferasirox, and deferiprone, or a combination thereof.Attorney Docket No.37759.0695P133. The pharmaceutical composition of claim 31, wherein the iron chelator is deferoxamine (DFO).

34. A method of inhibiting LpxC activity in a cell, the method comprising contacting the cell with an effective amount of the compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof.

35. The method of claim 34, wherein the cell is mammalian.

36. The method of claim 34, wherein the cell is human.

37. The method of claim 34, wherein the cell has been isolated from a human prior to the administering step.

38. The method of claim 34, wherein contacting is via administration to a subject.

39. The method of claim 38, wherein the subject has been diagnosed with a need for inhibition of LpxC activity prior to the administering step.

40. The method of claim 38, wherein the subject has been diagnosed with a need for treatment of a Gram-negative bacterial infection associated with LpxC activity.

41. A method for inhibiting LpxC activity in a subject, the method comprising administering to the subject an effective amount of the compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof.

42. The method of claim 41, wherein the subject has been diagnosed with an infection associated with LpxC activity prior to the administering step.

43. The method of claim 41, wherein the subject has been diagnosed with a need for modifying LpxC activity prior to the administering step.

44. The method of claim 41, wherein the subject has been diagnosed with a need for treatment of a Gram-negative bacterial infection associated with LpxC activity prior to the administering step.Attorney Docket No.37759.0695P145. The method of any one of claims 41 to 44, further comprising the step of identifying a subject in need of treatment of a Gram -negative bacterial infection associated with LpxC activity.

46. A method for treating a Gram-negative bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof.

47. The method of claim 46, wherein the Gram-negative bacterial infection is due to a Gramnegative bacteria selected from Acinetobacter spp., Aeromonas spp., Bordetella spp., Citrobacter spp., Enterobacter spp., Escherichia spp., Haemophilus spp., Klebsiella spp., Moraxella spp., Neisseria spp., Proteus spp., Pseudomonas spp., Salmonella spp., Shigella spp., Stenotrophomonas spp., Vibrio spp., and Yersinia spp.

48. The method of claim 46, wherein the Gram-negative bacterial infection is due to Neisseria spp..

49. The method of claim 46, wherein the Gram-negative bacterial infection is due to Neisseria gonorrhoeae.

50. The method of any one of claims 46 to 49, wherein the effective amount is a therapeutically effective amount.

51. The method of one of claims 46 to 49, wherein the effective amount is a prophylactically effective amount.

52. The method of one of claims 46 to 51, wherein the subject is a mammal.

53. The method of claim 52, wherein the mammal is a human.

54. The method of one of claims 46 to 53, wherein the subject has been diagnosed with a need for treatment of the Gram-negative bacterial infection prior to the administering step.

55. The method of one of claims 46 to 53, wherein the subject is at risk for developing the Gram-negative bacterial infection prior to the administering step.Attorney Docket No.37759.0695P156. The method of one of claims 46 to 55, further comprising the step of identifying a subject in need of treatment of the Gram-negative bacterial infection.

57. The method of one of claims 46 to 56, further comprising administering to the subject an effective amount of an iron chelator.

58. The method of claim 57, wherein the compound and the iron chelator are administered simultaneously.

59. The method of claim 58, wherein the compound and the iron chelator are co-formulated.

60. The method of claim 58, wherein the compound and the iron chelator are not coformulated.

61. The method of claim 57, wherein the compound and the iron chelator are administered sequentially.

62. The method of any one of claims 57 to 61, wherein the iron chelator is selected from deferoxamine (DFO), deferasirox, and deferiprone, or a combination thereof.

63. The method of any one of claims 57 to 61, wherein the iron chelator is deferoxamine (DFO).

64. The method of one of claims 46 to 63, further comprising administering to the subject an effective amount of an antibacterial agent.

65. The method of claim 64, wherein the compound and the antibacterial agent are administered simultaneously.

66. The method of claim 65, wherein the compound and the antibacterial agent are coformulated.

67. The method of claim 65, wherein the compound and the antibacterial agent are not coformulated.Attorney Docket No.37759.0695P168. The method of claim 64, wherein the compound and the antibacterial agent are administered sequentially.

69. The method of any one of claims 64 to 68, wherein the antibacterial agent is selected from amoxicillin, ampicillin, azithromycin, aztreonam, azlocillin, bacitracin, carbenicillin, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cefdinir, cefditorin, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, chloramphenicol, cilastin, ciprofloxacin, clarithromycin, clavulanic acid, clinafloxacin, clindamycin, clofazimine, cloxacillin, colistin, dalbavancin, dalfopristin, daptomycin, demeclocycline, dicloxacillin, dirithromycin, doxycycline, erythromycin, enrofloxacin, enoxacin, enviomycin, ertepenem, ethambutol, flucloxacillin, fosfomycin, furazolidone, gatifloxacin, gentamicin, imipenem, isoniazid, kanamycin, linezolid, lomefloxacin, loracarbef, mafenide, moxifloxacin, meropenem, metronidazole, mezlocillin, minocycline, mupirocin, nafcillin, nalidixic acid, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oritavancin, oxytetracycline, penicillin, piperacillin, platensimycin, polymixin B, quinupristin, retapamulin, rifabutin, rifampin, rifapentine, roxithromycin, sparfloxacin, spectinomycin, sulbactam, sulfacetamide, sulfamethizole, sulfamethoxazole, teicoplanin, telithromycin, telavancin, temafloxacin, tetracycline, tedolizid, thioacetazone, thioridazine, ticarcillin, tinidazole, tobramycin, torezolid, tosufloxacin, trimethoprim, troleandomycin, trovafl oxaci n, and vancomycin.

70. A kit comprising the compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, and one or more selected from:(a) an iron chelator;(b) an antibacterial agent;(c) instructions for administering the compound in connection with a Gram-negative bacterial infection; and(d) instructions for treating a Gram-negative bacterial infection.

71. The kit of claim 70, wherein the compound and the iron chelator are co-packaged.Attorney Docket No.37759.0695P1 72. The kit of claim 70, wherein the compound and the iron chelator are co-formulated.

73. The kit of claim 70, wherein the compound and the antibacterial agent are co-packaged.

74. The kit of claim 70, wherein the compound and the antibacterial agent are co-formulated.

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