WO2024081714A2 - GroEL INHIBITORS FOR ANTIBIOTIC APPLICATIONS - Google Patents

GroEL INHIBITORS FOR ANTIBIOTIC APPLICATIONS Download PDF

Info

Publication number
WO2024081714A2
WO2024081714A2 PCT/US2023/076571 US2023076571W WO2024081714A2 WO 2024081714 A2 WO2024081714 A2 WO 2024081714A2 US 2023076571 W US2023076571 W US 2023076571W WO 2024081714 A2 WO2024081714 A2 WO 2024081714A2
Authority
WO
WIPO (PCT)
Prior art keywords
aryl
substituted
heteroaryl
compound
alkyl
Prior art date
Application number
PCT/US2023/076571
Other languages
French (fr)
Other versions
WO2024081714A3 (en
Inventor
Steven Michael Johnson
Original Assignee
The Trustees Of Indiana University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of Indiana University filed Critical The Trustees Of Indiana University
Publication of WO2024081714A2 publication Critical patent/WO2024081714A2/en
Publication of WO2024081714A3 publication Critical patent/WO2024081714A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C233/65—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/70—Nitro radicals
    • C07D307/71—Nitro radicals attached in position 5
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • ESKAPE pathogens an acronym that stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter species.
  • trypanosomatids including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania species – which cause Human African Trypanosomiasis (HAT), Chagas disease, and the Leishmaniases, respectively – threaten over 1 billion people worldwide.
  • All organisms have one GroEL or HSP60 homolog that performs canonical protein folding housekeeping functions – such is the case for E. coli and the ESKAPE bacteria – while others, like M. tuberculosis and T. brucei, contain additional chaperonin isoforms that appear to perform non-canonical functions that are not well understood. While the non-canonical isoforms appear important for optimal cell survival, the canonical chaperonin isoforms are essential for survival under all conditions; thus, this class of molecular machines represent excellent targets for antibiotic development.
  • the present disclosure is directed to inhibitors of GroEL and HSP60 chaperonins in E. coli and the ESKAPE bacteria, M. tuberculosis, and T.
  • a compound is of the formula (IA) or (IIA) or a pharmaceutically acceptable salt thereof, wherein: X is O or S; R 1 is halo, C1-C8 alkyl, C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, - C(O)R A , -OR A ,-SR A or -NHR A , or one or more R 1 in combination with the atoms to which each is attached combine to form a C9-C14 aryl or C9-C12 bicyclic heteroaryl, wherein each hydrogen atom in C 6 -C 10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, C 1 -C 8 alkyl, -C(O)C 2 -C 6 alkenylene-phenyl, C9-C12 aryl and C9-C12 bicyclic heteroaryl is
  • a compound is of the formula (I) (II) wherein R 1 is H, halo, C6-C10 aryl, C5-C10 heteroaryl, substituted C6-C10 aryl, substituted C5-C10 heteroaryl, phenoxy, C 1 -C 8 alkyl, or R 1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein said substituted C6-C10 aryl and substituted C 5 -C 10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO 2 ; R 2 is C6-C8 aryl, C5-C8 heteroaryl, substituted C6-C10 aryl, substituted C5-C10 heteroaryl, wherein said substituted C 6 -C 10 aryl and substituted C 5 -C 10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2;
  • the present invention re directed to inhibitors of the GroEL/ES and HSP60/10 chaperonin systems and their use as new antibiotic candidates against a variety of infectious organisms.
  • analogs have been identified that can selectively kill Escherichia coli, the ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae), and Trypanosoma brucei relative to human cell lines in cell culture.
  • an inhibitor of the GroEL/ES and HSP60/10 chaperonin systems wherein the compound has the general structure of formula I: wherein R 3 is C5-C8 aryl, C5-C8 heteroaryl, a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R 3 substituents is optionally substituted with halogen, -OR 5 , nitro or -NR 5 R 6 ; R 15 and R 16 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, or bicyclic C6- C10 heteroaryl; or a pharmaceutically acceptable salt thereof, optionally wherein R 15 and R 16 are each independently hydrogen or -CH3.
  • an inhibitor of the GroEL/ES and HSP60/10 chaperonin systems wherein the compound has the general structure of formula I: wherein R 11 is halo; R 12 is -NO2; and R 15 and R 16 are each independently hydrogen or C1-C6 alkyl.
  • the disclosure relates to a compound or a pharmaceutically acceptable salt thereof, having the formula II wherein R 10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R 10 substituent is optionally substituted with halo, phenoxy, -OR 5 , -NR 5 R 6 , -S(O)2NR 5 R 6 , or –NHSO2R 7 ; R 5 and R 6 are each independently hydrogen, C 1 -C 6 alkyl, C 6 -C 10 aryl, or bicyclic C 6 -C 10 heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, aryl, and heteroaryl is optionally substituted with halogen or –OC 1 -C 6 alkyl; and R 7 is C5-C6 aryl, or C5-C16 heteroaryl wherein
  • an inhibitor of the GroEL/ES and HSP60/10 chaperonin systems has the structure of Compound 49 Compound 50.
  • a method of inhibiting the GroEL/ES and HSP60/10 chaperonin systems is provided, wherein the method comprising contacting said system with a compound disclosed herein.
  • a method of inhibiting the growth of T. brucei and/or treating a T. brucei infection is provided wherein a composition comprising a compound of the present invention is administered to a subject in need of said treatment.
  • BRIEF DESCRIPTION OF THE DRAWINGS Fig.1 provides a general synthetic protocol for the synthesis of compound 23 analogs.
  • Fig.2 presents data on folding assays for compound 23 and its analogs. Fig.2 compares IC50 results for 60 minutes pre-incubation with compounds for the Rho assay with that of the MDH assay.
  • Fig.3 is a graph comparing FHC cell viability CC 50 values to EC 90 values for actively- replicating M. tuberculosis with compound 23 analogs. The light circle data point represents compound 23, and the dark circle represents compound 50.
  • purified polypeptide is used herein to describe a polypeptide that has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.
  • isolated requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
  • the term “treating” includes alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
  • an "effective" amount or a "therapeutically effective amount" of a drug/cell therapy refers to a nontoxic but enough of the drug/cell therapy to provide the desired effect.
  • the amount that is “effective” will vary from subject to subject or even within a subject overtime, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • the term "patient” without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans receiving a therapeutic treatment in the presence or absence of a physician’s supervision.
  • the term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter.
  • alkyl refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-30 for straight chains, C 3-30 for branched chains), and more preferably 20 or fewer.
  • alkyl as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.
  • C x-y or “C x -C y ”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain.
  • Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal.
  • a C1-6 alkyl group for example, contains from one to six carbon atoms in the chain.
  • aryl as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon.
  • the ring is a 5- to 7-membered ring, more preferably a 6-membered ring.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
  • heteroaryl and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • heteroaryl and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • the terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • heterocyclyl and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.
  • polycyclyl refers to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”.
  • Each of the rings of the polycycle can be substituted or unsubstituted.
  • each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.
  • halogen or “halo” includes bromo, chloro, fluoro, and iodo.
  • haloalkyl refers to an alkyl radical bearing at least one halogen substituent, for example, chloromethyl, fluoroethyl or trifluoromethyl and the like.
  • C 1 -C n alkyl wherein n is an integer, as used herein, represents a branched or linear alkyl group having from one to the specified number of carbon atoms.
  • C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
  • aryl refers to a mono- or multi-cyclic carbocyclic ring system having one or more aromatic rings including, but not limited to, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, anthracenyl and the like.
  • Optionally substituted aryl includes aryl compounds having from zero to four substituents, and “substituted aryl” includes aryl compounds having one to three substituents, wherein the substituents include hydroxyl, C1- C 4 alkyl, halo or amino substituents.
  • polyaromatic hydrocarbon refers to a multi-cyclic carbocyclic ring system comprising two or more aromatic rings (selected from aryl and heteroaryl ring structures), and including but not limited to napthalene, fluorene, phenanthrene, pyrene, fluoranthene, chrysene, triphenylene, perylene, acridine; 2,2’ dipyridyl; 2,2’ biquinoline; 9-anthracenecarbonitrile; dibenzothiophene; 1,10’-phenanthroline; 9’ anthracenecarbonitrile; and anthraquinone.
  • Substituted polyaromatic hydrocarbon includes polyaromatic hydrocarbon compounds having one to three substituents, wherein the substituents include aryl, heteraryl, hydroxy, C1-C4 alkyl, halo, -CN, or amino substituents.
  • the term “heterocyclic group” refers to a mono- or multi-cyclic carbocyclic ring system containing one or more heteroatoms wherein the heteroatoms are selected from the group consisting of oxygen, sulfur, and nitrogen.
  • heteroaryl refers to a mono- or multi-cyclic carbocyclic ring system having one or more aromatic rings containing one or more heteroatoms (such as O, N and S) and includes, but is not limited to, furyl, thienyl, pyridyl and the like.
  • the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, —OCO—CH 2 —O- alkyl, —OP(O)(O-alkyl)2 or —CH2—OP(O)(O-alkyl)2.
  • “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.
  • the term “alkyl” refers to saturated aliphatic groups, including but not limited to C 1 -C 10 straight-chain alkyl groups or C 1 -C 10 branched-chain alkyl groups.
  • the “alkyl” group refers to C1-C6 straight-chain alkyl groups or C1-C6 branched- chain alkyl groups.
  • alkyl group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups.
  • alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3- octyl or 4-octyl and the like.
  • alkyl may be optionally substituted.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes 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., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic mo
  • compositions and methods of the present disclosure may be utilized to treat an individual in need thereof.
  • the individual is a mammal such as a human, or a non-human mammal.
  • the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • the aqueous solution is pyrogen-free, or substantially pyrogen-free.
  • the excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like.
  • the composition can also be present in a transdermal delivery system, e.g., a skin patch.
  • the composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the disclosure.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • a pharmaceutically acceptable carrier including a physiologically acceptable agent, depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system.
  • the pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the disclosure.
  • Liposomes for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and eth
  • a pharmaceutical composition can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin).
  • the compound may also be formulated for inhalation.
  • a compound may be simply dissolved or suspended in sterile water.
  • compositions suitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients.
  • active compound such as a compound of the disclosure
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient.
  • capsules including sprinkle capsules and gelatin capsules
  • cachets pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth)
  • lyophile powders,
  • compositions or compounds may also be administered as a bolus, electuary or paste.
  • solid dosage forms for oral administration capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like)
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6)
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • 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 may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • microorganisms Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.
  • isotonic agents such as sugars, sodium chloride, and the like into the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • the rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals.
  • biocompatible polymers including hydrogels
  • biodegradable and non-degradable polymers can be used to form an implant for the sustained release of a compound at a particular target site.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • terapéuticaally effective amount is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with a compound of the disclosure. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).
  • a suitable daily dose of an active compound used in the compositions and methods of the disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the active compound may be administered two or three times daily.
  • the active compound will be administered once daily.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.
  • compounds of the disclosure may be used alone or conjointly administered with another type of therapeutic agent.
  • the present disclosure includes the use of pharmaceutically acceptable salts of compounds of the disclosure in the compositions and methods of the present disclosure.
  • contemplated salts of the disclosure include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.
  • contemplated salts of the disclosure include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L- lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.
  • contemplated salts of the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.
  • contemplated salts of the disclosure include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane
  • the pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared.
  • the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • X is O or S. In some embodiments X is O. In some embodiments X is S. In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0 and the phenyl ring is unsubstituted. In some embodiments, n is 1. In certain embodiments when n is 1, R 1 is optionally substituted phenoxy. In some embodiments, n is 2.
  • n is 2 and the two R 1 in combination with the atoms to which each is attached combine to form a C 9 - C 14 aryl (e.g., ) or C 9 -C 12 bicyclic heteroaryl, each of which may be optionally substituted, for example by a heteroaryl.
  • R 1 is halo, C 1 -C 8 alkyl, C 6 -C 10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, -C(O)R A , -OR A ,-SR A or -NHR A .
  • one or more R 1 in combination with the atoms to which each is attached combine to form a C 9 -C 14 aryl or C9-C12 bicyclic heteroaryl.
  • each hydrogen atom in C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, C 1 -C 8 alkyl, -C(O)C 2 -C 6 alkenylene-phenyl, C 9 -C 12 aryl and C9-C12 bicyclic heteroaryl is optionally substituted by halo, cyano, or -NO2.
  • R 2 is C 6 -C 10 aryl or 5- to 12-membered heteroaryl, wherein each hydrogen atom in C6-C10 aryl and 5- to 12-membered heteroaryl is optionally substituted by halo, hydroxy, or -NO 2 .
  • R 2 may be phenyl optionally substituted by alkoxy, ester, halo such as chloro, etc.
  • R 2 is 5- to 12-membered heteroaryl, for example , each of which may be optionally substituted.
  • R 11 is nitro or -N(H)COR 3 .
  • R 12 is H or -C(O)C1-C6 alkyl (e.g, -C(O)ethyl).
  • R 13 is H or C 1 -C 6 alkyl (e.g., methyl).
  • R 14 is H, C6-C10 aryl (e.g., phenyl), or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C 6 -C 10 aryl and 5 to 12-membered heteroaryl is optionally substituted by halo, 5 to 12-membered heteroaryl, -NHC(O)C1-C6 alkyl, C1-C6 alkoxy, -C(O)C 1 -C 6 alkoxy, -R A , -OR A , -SR A , -C(O)R A or -NHR A .
  • C6-C10 aryl e.g., phenyl
  • R 14 is H, C6-C10 aryl (e.g., phenyl), or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C 6 -C 10 aryl and 5 to 12-membered heteroaryl is optionally substituted by halo, 5 to 12-membere
  • each R A is C1-C6 alkyl (e.g., methyl), C6-C10 aryl, C2-C6 alkenyl- C 6 -C 10 aryl, -C(O)C 1 -C 6 alkyl, C 1 -C 6 alkoxy (e.g., methoxy), or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C 1 -C 6 alkoxy, or 5 to 12-membered heteroaryl is optionally substituted by nitro or halo; or two R A combine to form C4-C6 cycloalkyl optionally substituted by phenyl, for example if R 14 is phenyl, two R A can combine with the phenyl to form .
  • C1-C6 alkyl e.
  • R 3 is C 6 -C 10 aryl, 5 to 12-membered heteroaryl, or C 9 -C 12 bicyclic aryl, wherein each hydrogen atom in C6-C10 aryl, 5 to 12-membered heteroaryl, and C9- C 12 bicyclic aryl is optionally substituted by halo, C 1 -C 6 alkyl, or nitro.
  • MMV Malaria Venture
  • the compounds of the present disclosure have utility as inhibitors of M. tuberculosis and T. brucei parasites.
  • a composition comprising any of analogs 39-52 of Table 1, and in one embodiment compounds 49 and 50, are used as potent and selective inhibitors against T. brucei.
  • Compounds 49 and 50 and functional derivatives thereof have more potent and selective activity against T. brucei than Suramin and Nifurtimox - the first line treatments used for treating HAT. These compounds are anticipated to have additional applications against other infectious organisms.
  • Analogs were prepared in which the left-hand side of the molecule was retained along with the amide linker and the aryl on the right side of the compound was varied in an effort to modulate the reactivity – with the exception of 38 (the parent amine starting material) and 51 and 52 (both contained unsubstituted phenyl rings).
  • Analog 49 included a para-nitrofluoro ring in an effort to increase the efficiency of the nucleophilic aromatic substitution reaction with GroEL cysteines.
  • Analog 50 included a nitrofuran group as we previously reported this to be efficacious in GroEL-targeting antibacterials due to its ability to act as a prodrug and become activated via nitroreductases. The general synthesis of these analogs is presented in Figure 1, which resulted in the generation of 15 additional analogs for testing (see Table 1 for the structures of the different analogs).
  • Table 1 Dose-response results for the compound 23 analogs tested in the GroEL/ES-mediated refolding assays and native reporter enzyme counter-screens. Biochemical Assay IC 50 ( ⁇ M) Overall, the compound 23 analogs were less potent at inhibiting E. coli GroEL refolding functions (Table 1), with the exception of 49, which was ⁇ 2-fold more potent than 23.
  • IC 50 values for these analogs exhibited high correlations between the two GroEL-mediated client protein refolding assays and were inactive in the native MDH and Rho reporter counter-screens (with the exception of compound 50 in the native Rho counter-screen), supporting that inhibitors were functioning against the GroEL/ES-mediated refolding cycle and were not false-positives of the MDH and Rho reporter enzymes.
  • Compound 50 was also tested with the E.
  • Table 2 Dose-response testing of the compound 23 analogs in cell viability assays. Presented are antibiotic efficacy against actively replicating T. brucei (EC 50 ) parasites and M. tuberculosis, E. coli, and the ESKAPE bacteria (EC90) and cytotoxicity (CC50) to human colon (FHC) and intestine (FHs 74Int) cell lines.
  • Table 3 Dose-response results for the nitrofuran analogs tested in the GroEL/ES- mediated refolding assays, native MDH reporter enzyme counter-screen, and T. brucei and human intestine and colon cell viability assays.
  • a compound of the formula (I) (II) wherein R 1 is H, halo, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, phenoxy, C 1 -C 8 alkyl, or R 1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R 1 substituents is optionally substituted with halogen, -OR 5 , -NR 5 R 6 , -S(O)2NR 5 R 6 , or –NHSO2R 7 ; R 2 is C 5 -C 8 aryl, C 5 -C 8 heteroaryl,
  • a compound of the formula (I) (II) wherein R 1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R 1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl; R 3 is C5-C6 aryl, C5-C6 heteroaryl, wherein the C5-C6 aryl, and C5-C6 heteroaryl further comprises at least one -NO 2 and one halo substituent; and R 15 and R 16 are each independently hydrogen or -CH3; or a pharmaceutically acceptable salt thereof.
  • a compound of the formula (I) (II) wherein R 1 is H; R 3 is R 11 is halo; R 12 is NO2; and R 15 and R 16 are each independently hydrogen or C 1 -C 6 alkyl; or a pharmaceutically acceptable salt thereof, optionally wherein R 15 is F and R 16 is each independently hydrogen or C 1 -C 3 alkyl.
  • the disclosure relates to a compound or a pharmaceutically acceptable salt thereof, having the formula II wherein R 10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R 10 substituent is optionally substituted with halo, phenoxy, -OR 5 , -NR 5 R 6 , -S(O)2NR 5 R 6 , CO2(C1-C4 alkyl), -C(O)NH2 or –NHSO2R 7 ; R 5 and R 6 are each independently hydrogen, C 1 -C 6 alkyl, C 6 -C 10 aryl, or bicyclic C 6 -C 10 heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, aryl, and heteroaryl is optionally substituted with halogen or –OC 1 -C 6 alkyl; and R 7 is C
  • a compound having the general structure of wherein R 1 is halo or -OH; R 15 is H or C 1 -C 6 alkyl; X is S or O, or a pharmaceutically acceptable salt thereof.
  • a compound having the formula wherein X is S or O, and R 1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1- C 8 alkyl, or R 1 in combination with the atom to which it is attached forms a C 9 -C 12 bicyclic aryl or C9-C12 bicyclic heteroaryl; or a pharmaceutically acceptable salt thereof.
  • a compound having the formula wherein R 1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R 1 in combination with the atom to which it is attached forms a C 9 -C 12 bicyclic aryl or C 9 -C 12 bicyclic heteroaryl; or a pharmaceutically acceptable salt thereof.
  • R 1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R 1 in combination with the atom to which it is attached forms a C 9 -C 12 bicyclic aryl or C 9 -C 12 bicyclic heteroaryl; or a pharmaceutically acceptable salt thereof.
  • the compound or a pharmaceutically acceptable salt thereof selected from the group consisting
  • a pharmaceutical composition comprising any of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof, and at least one diluent, carrier or excipient.
  • a method of treating a microbial infection comprising administering to a subject in need of such treatment an effective amount of at least one compound as disclosed herein, or a pharmaceutically acceptable salt thereof.
  • R 2 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C10 aryl, substituted C5-C10 heteroaryl, wherein said substituted C 5 -C 10 aryl and substituted C 5 -C 10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2;
  • the compound used in embodiment 1 has the general structure of formula I: wherein R 3 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C 5 -C 10 aryl and substituted C 5 -C 10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof, optionally wherein R 15 and R 16 are each independently hydrogen or -CH3.
  • the compound used in embodiment 1 or 2 has the general structure of formula I: wherein R 11 is halo; R 12 is -NO2; R 16 is C1-C6 alkyl; and R 15 is H.
  • R 11 is halo
  • R 12 is -NO2
  • R 16 is C1-C6 alkyl
  • R 15 is H.
  • the compound used in any one of embodiments 1-3 is provided wherein R 16 is -CH2CH3.
  • the compound used in embodiment 1 has the structure of formula II (II) wherein R 10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, substituted C 5 -C 8 aryl, substituted C 5 -C 8 heteroaryl, substituted C 9 -C 12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, substituted C 9 -C 12 bicyclic aryl or substituted C 9 -C 12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo, phenoxy, -NR 5 R 6 , -OR 5 , -S(O) 2 NR 5 R 6 , or –NHSO 2 R 7 ; R 5 and R 6 are each independently hydrogen, C 1 -C 6 alkyl, C 6 -C 10 , C
  • the compound used in embodiment 5 or 6 is provided wherein said compound is Compound 49 Compound 50.
  • a pharmaceutical composition is provided comprising a compound of any one of embodiments 1-7 and a pharmaceutically acceptable carrier.
  • a method of inhibiting the GroEL/ES and HSP60/10 chaperonin system in a microorganism is provided wherein said method comprises contacting said microorganism with the pharmaceutical composition of embodiment 8.
  • a method of inhibiting the growth of Trypanosoma brucei and/or treating a Trypanosoma brucei infection is provided, said method comprising administering compound of Compound 49 Compound 50. to a subject in need of treatment.
  • EXAMPLE 1 Abbreviations: The examples described herein use materials, including but not limited to, those described by the following abbreviations known to those skilled in the art:
  • Reverse-phase high-performance liquid chromatography was performed using a Waters 1525 binary pump, 2489 tunable UV/Vis detector (254 and 280 nm detection), and 2707 autosampler.
  • samples were chromatographically separated using a Waters XSelect CSH C18 OBD prep column (part number 186005422, 130 ⁇ pore size, 5 ⁇ m particle size, 19x150 mm), eluting with a H2O:CH3CN gradient solvent system.
  • DH5 ⁇ and BL21 (DE3) E. coli cells were purchased from New England Biolabs.
  • the Mycobacterium tuberculosis proliferation assay used an H37Rv strain that researchers at the Seattle Children’s Research Institute (Seattle, WA, USA) previously engineered to express a codon-optimized mCherry fluorescent protein (TOPred). Trypanosoma brucei Plimmer and Bradford parasites (Lister 427 VSG 221 [TetR T7RNAP] transgenic bloodstream form) were obtained from the ATCC (PRA-383).
  • FHs 74 Int (CCL-241) cells and FHC (CRL-1831) cells were obtained from the ATCC.
  • E. coli GroEL and GroES Purification E. coli GroEL was expressed from a trc-promoted and Amp(+) resistance marker plasmid in DH5 ⁇ E. coli cells. GroES was expressed from a T7-promoted and Amp(+) resistance plasmid in E. coli BL21 (DE3) cells. Transformed colonies were plated onto Ampicillin-treated LB agar and incubated for 24 h at 37°C.
  • the combined suspension was lysed by sonication, the lysate was centrifuged at 14,000 rpm, and the clarified lysate was passed through a 0.45 ⁇ m filter (Millipore).
  • Anion exchange purification The filtered lysate was loaded onto a GE HiScale Anion exchange column (Q Sepharose fast flow anion exchange resin) that was equilibrated with 2 column volumes of Buffer A.
  • the loaded column was washed with 10% Buffer B (50 mM Tris.HCl, pH 7.4, 1 M NaCl) for 3 CVs when purifying GroES and 28% Buffer B for 3 CVs when purifying GroEL, then bound protein was eluted with 3 CVs of 10-50% gradient flow of Buffer B when purifying GroES and 3 CVs of 28-60% gradient flow of Buffer B when collecting GroEL.
  • Buffer B 50 mM Tris.HCl, pH 7.4, 1 M NaCl
  • Protein-containing fractions as identified by SDS-PAGE, were collected, spin concentrated using a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore), and dialyzed overnight with 10 kDa SnakeSkinTM dialysis tubing (Thermo Scientific) at 4°C in 2 L of 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl solution (Dialysis Buffer). Size exclusion chromatography: The dialyzed protein was loaded onto a Superdex 200 column (HiLoad 26/600, GE) column that was equilibrated with 2 column volumes of SEC Buffer (50 mM Tris-HCl, pH 7.4 and 150 mM NaCl).
  • SEC Buffer 50 mM Tris-HCl, pH 7.4 and 150 mM NaCl
  • the loaded column was eluted with 1 column volume of 100% SEC Buffer and the column was washed with 2 CVs of the same solution.
  • Protein-containing fractions as identified by SDS-PAGE, were collected, spin concentrated using a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore). The final protein concentration was determined by Coomassie Protein Assay Kit (Thermo Scientific). Batches of protein for testing were stored at 4°C for up to six weeks and then discarded.
  • dMDH Denatured MDH
  • the binary complex solutions were prepared by adding the dMDH stock to GroEL (or HSP60) in folding buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl 2 , and 1 mM DTT), followed by addition of GroES (or HSP10).
  • the binary complex stocks were prepared immediately prior to dispensing into the assay plates and had final protein concentrations of 83.3 nM GroEL (Mr 800 kDa) or HSP60 (Mr 400 kDa), 100 nM GroES or HSP10 (Mr 70 kDa), and 20 nM dMDH in folding buffer.
  • ATP initiation stock ATP solid was diluted into folding buffer to a final concentration of 2.5 mM.
  • Quench solution contained 600 mM EDTA (pH 8.0).
  • the MDH enzymatic assay stock consisted of 20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, and 1 mM DTT).
  • Assay protocol First, 30 ⁇ L aliquots of the GroEL/ES-dMDH or HSP60/10-dMDH binary complex stocks were dispensed into clear, 384-well polystyrene plates. Next, 0.5 ⁇ L of the compound stocks (10 mM to 4.6 ⁇ M, 3-fold dilutions series in DMSO) were added by pin- transfer (V&P Scientific).
  • the chaperonin-mediated refolding cycles were initiated by addition of 20 ⁇ L of ATP stock (reagent concentrations during refolding cycle: 50 nM GroEL or HSP60, 60 nM GroES or HSP10, 12 nM dMDH, 1 mM ATP, and compounds of 100 ⁇ M to 46 nM, 3- fold dilution series), and the refolding reactions incubated at 37°C.
  • the incubation times were determined from refolding time-course control experiments until they reached ⁇ 90% completion of refolding of the denatured MDH – generally ⁇ 15-40 min for GroEL/ES, and ⁇ 40- 60 min for HSP60/10.
  • MDH enzymatic assay stock (20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer, 50 mM Tris pH 7.4, 50 mM KCl, 1 mM DTT), and followed by measuring the NADH absorbance in each well at 340 nm using a Molecular Devices, SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD + does not).
  • a 340 nm measurements were recorded at 0.5 minutes (start point) and at successive time points until the amount of NADH consumed reached ⁇ 90% (end point, generally between 20-35 minutes). The differences between the start and end point A 340 values were used to calculate the % inhibition of the GroEL/ES or HSP60/10 machinery by the compounds.
  • IC50 values for the test compounds were obtained by plotting the % inhibition results in GraphPad Prism and analyzing by non-linear regression using the log (inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC 50 values obtained from at least four replicates. Counter-screening compounds for inhibition of native MDH enzymatic activity.
  • Reagent preparations and assay protocol This assay was performed as described above for the GroEL/ES-dMDH refolding assay; however, the assay protocol differed in the sequence of compound addition to the assay plates.
  • the refolding reactions were allowed to proceed for 45- 50 min at 37°C in the absence of test compounds (nearly complete refolding of MDH occurs), then quenched with the EDTA stock. Compounds were then pin-transferred into the plates after the EDTA quenching step; thus, compounds’ effects are only possible by inhibiting the fully refolded MDH reporter substrate.
  • MDH enzymatic activity of the refolded MDH was initiated by addition of 20 ⁇ L MDH enzymatic assay stock (20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer(50 mM Tris pH 7.4, 50 mM KCl, 1 mM DTT), and followed by measuring the NADH absorbance in each well at 340 nm using a Molecular Devices SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD+ does not). A340 nm measurements were recorded at 0.5 minutes (start point) and at successive time points until the amount of NADH consumed reached ⁇ 90% (end point, generally between 15-30 minutes).
  • IC 50 values for the test compounds were obtained by plotting the % inhibition results in GraphPad Prism and analyzing by non-linear regression using the log (inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC50 values obtained from at least four replicates. Evaluating compounds for inhibition in the GroEL/ES-dRho refolding assay.
  • Reagent preparation For this assay, five primary reagent stocks were prepared: 1) GroEL/ES- dRho binary complex stock; 2) ATP initiation stock; 3) Enzyme solution; 4) Formaldehyde quench solution; 5) Fe(NO 3 ) 3 assay stock.
  • Denatured Rho (dRho) was prepared by 3-fold dilution of Rho (Roche product #R1756, stock diluted to 10 mg/ml with H2O) with denaturant buffer (12 M Urea, 50 mM Tris, pH 7.4, and 10 mM DTT). Rho was completely denatured by incubating at room temperature for 40 min.
  • the binary complex solution was prepared by slowly adding the dRho stock to a stirring solution of concentrated GroEL in modified folding buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, 5 mM Na2S2O3 and 1 mM DTT) and then incubated at room temperature for 10 min. The solution was centrifuged at 16,000 x g for 5 minutes, and the supernatant was collected and added to a solution of GroES in modified folding buffer to give final protein concentrations of 100 nM GroEL, 120 nM GroES, and 80 nM dRho in modified folding buffer. The binary complex stock was prepared immediately prior to use.
  • modified folding buffer 50 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, 5 mM Na2S2O3 and 1 mM DTT
  • ATP solid was diluted into modified folding buffer to a final concentration of 2.0 mM.
  • the thiocyanate enzymatic assay stock was prepared to contain 70 mM KH2PO4, 80 mM KCN, and 80 mM Na2S2O3 in water.
  • the formaldehyde quench solution contained 30% formaldehyde in water.
  • the ferric nitrate reporter stock contained 8.5% w/v Fe(NO3)3 and 11.3% v/v HNO3 in water.
  • Assay protocol First, 10 ⁇ L aliquots of the GroEL/ES-dRho complex stock was dispensed into clear, 384-well polystyrene plates.
  • Reagent preparations and assay protocol Reagents were identical to those used in the GroEL/ES-dRho refolding assay described above; however, the assay protocol differed in the sequence of compound addition to the wells. Compounds were pin-transferred after the 60-minute incubation for the refolding cycle, but prior to the addition of the thiocyanate enzymatic assay stock.
  • E. coli were grown with LB medium and all ESKAPE bacteria were grown in Mueller-Hinton Broth 2, Cation-Adjusted (CA-MHB) medium (Millipore), with all liquid cultures supplemented with 20-25 mg/L Ca2+ and 10-12.5 mg/L Mg 2+ to mimic physiological free concentrations of these cations.
  • CA-MHB Cation-Adjusted
  • General Assay Protocol Stock bacterial cultures were streaked onto LB or CA-MHB agar plates and grown overnight at 37°C.
  • EC90 values for the test compounds were obtained by plotting the OD 600 results in GraphPad Prism and analyzing by non-linear regression using the [agonist] vs. response - Find ECanything equation. Results presented represent the averages of EC90 values obtained from at least four replicates. Evaluating compounds for inhibition of M. tuberculosis proliferation.
  • M. tuberculosis strain H37Rv
  • M. tuberculosis was grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 10% v/v oleic acid, and albumin dextrose catalase (OADC) supplement (Becton Dickinson) (7H9-Tw-OADC).
  • Stock bacterial cultures were inoculated in a startup culture and grown to a logarithmic phase of OD 590 ⁇ 0.7. This was sub-cultured in fresh media (1:10 dilution) and grown to an OD5901.0, then diluted again into fresh media to achieve a final OD 590 reading of 0.04 (just prior to dispensing into plates).
  • Compound plates were prepared by adding 4 ⁇ L of compound stocks to 96 ⁇ L of fresh medium in 96-well plates. Aliquots of the diluted Mtb cultures (100 ⁇ L) were then added to the compound plates, which were incubated in sealed bags at 37oC for 5 days.
  • brucei cell viability assay in 384-well plate format. Briefly, 55 ⁇ L of 2000 parasites/mL (110 parasites/well) of Trypanosoma brucei brucei (strain BF427) in HMI-9 medium were dispensed in to clear, 384-well polystyrene plates (BRAND cell culture grade plates, 781980). Plates were sealed with "Breathe Easy” oxygen permeable membranes (Diversified Biotech) and incubated at 37°C, 5% CO2 for 24 h.
  • EC50 and EC90 values for the test compounds were obtained by plotting the % Alamar Blue reduction results in GraphPad Prism and analyzing by non-linear regression using the [agonist] vs. response - Find ECanything equation.
  • DMSO was used as negative control, and pentamidine, suramin, and nifurtimox (drugs used to treat HAT) were used as positive controls. Results presented represent the averages of EC 50 and EC 90 values obtained from at least four replicates.
  • FHs 74 Int intestine and FHC colon cells were maintained in Hybri-care Medium ATCC 46-X supplemented with 30ng/ml epidermal growth factor (EGF) and 10% FBS.
  • FHC cells were maintained in DMEM: F-12 media, supplemented with 10mM HEPES (final concentration 25 mM), 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005mg/ml transferrin, 100 ng/ml hydrocortisone, and 10% FBS (Sigma, F2242).
  • the Alamar Blue reporter reagents were then added to a final concentration of 10%, the plates incubated at 37°C and 5% CO2, and sample fluorescence (535 nm excitation, 590 nm emission) was read using a Molecular Devices FlexStation II 384-well plate reader (readings taken between 4-24 h of incubation so as to achieve signals in the 30-60% range for conversion of resazurin to resorufin).
  • Cell viability was calculated as per vendor instructions (Thermo Fisher - Alamar Blue cell viability assay manual).
  • Cytotoxicity CC50 values for the test compounds were obtained by plotting the % resazurin reduction results in GraphPad Prism and analyzing by non- linear regression using the log(inhibitor) vs. response (variable slope) equation. Results presented represent the averages of CC 50 values obtained from at least four replicates for FHs 74 Int small intestine cells and four replicates for FHC colon cells. Control compounds, calculation of IC 50 values, and statistical considerations.
  • DMSO was used as a negative control, and a panel of our previously discovered and reported chaperonin inhibitors were used as positive controls: e.g., compounds 8, 9, and 18 from Johnson et.
  • IC 50 results reported are averages of values determined from individual dose-response curves in assay replicates as follows: 1) Individual IC50 values from assay replicates were first log-transformed and the average log(IC 50 ) values and standard deviations (SD) calculated; 2) Replicate log(IC50) values were evaluated for outliers using the ROUT method in GraphPad Prism (Q of 10%); and 3) Average IC 50 values were then back- calculated from the average log(IC50) values.
  • results were represented as 0.1 log units higher than the maximum concentrations tested (i.e.1.7, 1.9, 2.1, and 2.5 – or 53, 79, 126, and 316 ⁇ M, respectively) so as not to overly bias comparisons because of the unavailability of definitive values for these inactive compounds.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Disclosed herein are new inhibitors of GroEL/ES and HSP60/10 chaperonin systems that serve as new antibiotic candidates against a variety of infectious organisms. The identified compounds can selectively kill Escherichia coli, the ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae), and Trypanosoma brucei parasites relative to human cell lines in cell culture.

Description

GroEL INHIBITORS FOR ANTIBIOTIC APPLICATIONS RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/415,519, October 12, 2022, the entire disclosure of which is incorporated herein by reference. GOVERNMENT SUPPORT CLAUSE This invention was made with government support under AI151532 and GM120350 awarded by National Institutes of Health. The Government has certain rights in the invention. BACKGROUND Globally, millions of people die every year due to complications involving infections from antibiotic-resistant bacteria. Of these infections, the most common organisms are from Mycobacterium tuberculosis (Mtb) and a group of bacteria known as the ESKAPE pathogens (an acronym that stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter species). Furthermore, infections by trypanosomatids including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania species – which cause Human African Trypanosomiasis (HAT), Chagas disease, and the Leishmaniases, respectively – threaten over 1 billion people worldwide. For HAT, ~10,000 new cases are reported each year across countries of Sub-Sarahan Africa. Unfortunately, as the need for new antibiotics to combat these infections continues to increase with each passing year, industrial antibiotic discovery and development programs are drying up and those that remain largely explore drugs targeting previously exploited antibiotic classes and targets. However, mechanistically unique antibiotic candidates targeting new pathways and proteins may be better suited to address antibiotic resistance. Towards this goal, potential antibiotic targets that are currently not the focus of any drug on the market are GroEL and HSP60 chaperonin systems. GroEL and HSP60 chaperonins are complex, oligomeric proteins that are upregulated in the cell under stressful conditions and help to prevent the misfolding and aggregation of other proteins. All organisms have one GroEL or HSP60 homolog that performs canonical protein folding housekeeping functions – such is the case for E. coli and the ESKAPE bacteria – while others, like M. tuberculosis and T. brucei, contain additional chaperonin isoforms that appear to perform non-canonical functions that are not well understood. While the non-canonical isoforms appear important for optimal cell survival, the canonical chaperonin isoforms are essential for survival under all conditions; thus, this class of molecular machines represent excellent targets for antibiotic development. The present disclosure is directed to inhibitors of GroEL and HSP60 chaperonins in E. coli and the ESKAPE bacteria, M. tuberculosis, and T. brucei parasites. SUMMARY In certain aspects, a compound is of the formula (IA) or (IIA)
Figure imgf000003_0001
or a pharmaceutically acceptable salt thereof, wherein: X is O or S; R1 is halo, C1-C8 alkyl, C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, - C(O)RA, -ORA,-SRA or -NHRA, or one or more R1 in combination with the atoms to which each is attached combine to form a C9-C14 aryl or C9-C12 bicyclic heteroaryl, wherein each hydrogen atom in C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, C1-C8 alkyl, -C(O)C2-C6 alkenylene-phenyl, C9-C12 aryl and C9-C12 bicyclic heteroaryl is optionally substituted by halo, cyano, or -NO2; R2 is C6-C10 aryl or 5- to 12-membered heteroaryl, wherein each hydrogen atom in C6- C10 aryl and 5- to 12-membered heteroaryl is optionally substituted by halo, hydroxy, or -NO2; R11 is nitro or N(H)COR3; R12 is H or C(O)C1-C6 alkyl; R13 is H or C1-C6 alkyl; R14 is H, C6-C10 aryl, or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C6-C10 aryl and 5 to 12-membered heteroaryl is optionally substituted by halo, 5 to 12- membered heteroaryl, -NHC(O)C1-C6 alkyl, C1-C6 alkoxy, -C(O)C1-C6 alkoxy, -RA, -ORA, - SRA, -C(O)RA or -NHRA; each RA is individually C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C1-C6 alkoxy, or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C1-C6 alkoxy, or 5 to 12- membered heteroaryl is optionally substituted by nitro or halo; or two RA combine to form C4- C6 cycloalkyl optionally substituted by phenyl; and R3 is C6-C10 aryl, 5 to 12-membered heteroaryl, or C9-C12 bicyclic aryl, wherein each hydrogen atom in C6-C10 aryl, 5 to 12-membered heteroaryl, and C9-C12 bicyclic aryl is optionally substituted by halo, C1-C6 alkyl, or nitro; and n is 0, 1, 2, or 3. In some aspects, a compound is of the formula
Figure imgf000004_0001
(I) (II) wherein R1 is H, halo, C6-C10 aryl, C5-C10 heteroaryl, substituted C6-C10 aryl, substituted C5-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein said substituted C6-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R2 is C6-C8 aryl, C5-C8 heteroaryl, substituted C6-C10 aryl, substituted C5-C10 heteroaryl, wherein said substituted C6-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R3 is C6-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl, C9-C12 bicyclic heteroaryl, substituted C6-C8 aryl, substituted C5-C8 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C6-C10 aryl, substituted C5-C10 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; and R15 and R16 are each independently hydrogen or C1-C6 alkyl; or a pharmaceutically acceptable salt thereof. In accordance with some embodiments the present invention re directed to inhibitors of the GroEL/ES and HSP60/10 chaperonin systems and their use as new antibiotic candidates against a variety of infectious organisms. As disclosed herein analogs have been identified that can selectively kill Escherichia coli, the ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae), and Trypanosoma brucei relative to human cell lines in cell culture. Inhibition of the GroEL/ES and HSP60/10 chaperonin systems of the infectious organisms is believed to prevent the chaperonin systems from performing their biological functions of assisting other proteins to fold to their functional forms, resulting in disruption of protein homeostasis and death of the cells. In some embodiments, an inhibitor of the GroEL/ES and HSP60/10 chaperonin systems is provided wherein the compound has the general structure of formula I:
Figure imgf000005_0001
wherein R3 is C5-C8 aryl, C5-C8 heteroaryl, a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R3 substituents is optionally substituted with halogen, -OR5, nitro or -NR5R6; R15 and R16 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, or bicyclic C6- C10 heteroaryl; or a pharmaceutically acceptable salt thereof, optionally wherein R15 and R16 are each independently hydrogen or -CH3. In some embodiments, an inhibitor of the GroEL/ES and HSP60/10 chaperonin systems is provided wherein the compound has the general structure of formula I:
Figure imgf000005_0002
wherein
Figure imgf000005_0003
R11 is halo; R12 is -NO2; and R15 and R16 are each independently hydrogen or C1-C6 alkyl. In other embodiments, the disclosure relates to a compound or a pharmaceutically acceptable salt thereof, having the formula II
Figure imgf000006_0001
wherein R10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R10 substituent is optionally substituted with halo, phenoxy, -OR5, -NR5R6, -S(O)2NR5R6, or –NHSO2R7; R5 and R6 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, or bicyclic C6-C10 heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, aryl, and heteroaryl is optionally substituted with halogen or –OC1-C6 alkyl; and R7 is C5-C6 aryl, or C5-C16 heteroaryl wherein one or more of the hydrogen atoms of the C5-C6 aryl, or C5-C16 heteroaryl is optionally substituted with halo, -OR5 or nitro; or a pharmaceutically acceptable salt thereof, optionally wherein R5 and R6 are each independently hydrogen or -CH3. In accordance with some embodiments, an inhibitor of the GroEL/ES and HSP60/10 chaperonin systems has the structure of
Figure imgf000006_0002
Compound 49 Compound 50. In accordance with one embodiment a method of inhibiting the GroEL/ES and HSP60/10 chaperonin systems is provided, wherein the method comprising contacting said system with a compound disclosed herein. In one embodiment a method of inhibiting the growth of T. brucei and/or treating a T. brucei infection is provided wherein a composition comprising a compound of the present invention is administered to a subject in need of said treatment. BRIEF DESCRIPTION OF THE DRAWINGS Fig.1 provides a general synthetic protocol for the synthesis of compound 23 analogs. Reagents and conditions: a) X = Cl: Cs2CO3, anhydrous CH3CN; b) X = OH: SOCs2, 60°C, 1 h, then concentrate and add arylamine, Cs2CO3, anhydrous CH3CN. Fig.2 presents data on folding assays for compound 23 and its analogs. Fig.2 compares IC50 results for 60 minutes pre-incubation with compounds for the Rho assay with that of the MDH assay. Fig.3: is a graph comparing FHC cell viability CC50 values to EC90 values for actively- replicating M. tuberculosis with compound 23 analogs. The light circle data point represents compound 23, and the dark circle represents compound 50. DETAILED DESCRIPTION DEFINITIONS In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below. The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent but is not intended to limit any value or range of values to only this broader definition. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values. As used herein, the term "purified" and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment. As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative definition. The term "purified polypeptide" is used herein to describe a polypeptide that has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates. The term "isolated" requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated. As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans. As used herein, the term "treating" includes alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. As used herein an "effective" amount or a "therapeutically effective amount" of a drug/cell therapy refers to a nontoxic but enough of the drug/cell therapy to provide the desired effect. The amount that is "effective" will vary from subject to subject or even within a subject overtime, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. As used herein the term "patient" without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans receiving a therapeutic treatment in the presence or absence of a physician’s supervision. The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer. Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc. The term “Cx-y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A C1-6 alkyl group, for example, contains from one to six carbon atoms in the chain. The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7. As used herein, the term “halogen” or “halo” includes bromo, chloro, fluoro, and iodo. The term “haloalkyl” as used herein refers to an alkyl radical bearing at least one halogen substituent, for example, chloromethyl, fluoroethyl or trifluoromethyl and the like. The term “C1-Cn alkyl” wherein n is an integer, as used herein, represents a branched or linear alkyl group having from one to the specified number of carbon atoms. Typically C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like. As used herein the term “aryl” refers to a mono- or multi-cyclic carbocyclic ring system having one or more aromatic rings including, but not limited to, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, anthracenyl and the like. “Optionally substituted aryl” includes aryl compounds having from zero to four substituents, and “substituted aryl” includes aryl compounds having one to three substituents, wherein the substituents include hydroxyl, C1- C4 alkyl, halo or amino substituents. The term “polyaromatic hydrocarbon” refers to a multi-cyclic carbocyclic ring system comprising two or more aromatic rings (selected from aryl and heteroaryl ring structures), and including but not limited to napthalene, fluorene, phenanthrene, pyrene, fluoranthene, chrysene, triphenylene, perylene, acridine; 2,2’ dipyridyl; 2,2’ biquinoline; 9-anthracenecarbonitrile; dibenzothiophene; 1,10’-phenanthroline; 9’ anthracenecarbonitrile; and anthraquinone. Substituted polyaromatic hydrocarbon includes polyaromatic hydrocarbon compounds having one to three substituents, wherein the substituents include aryl, heteraryl, hydroxy, C1-C4 alkyl, halo, -CN, or amino substituents. The term “heterocyclic group” refers to a mono- or multi-cyclic carbocyclic ring system containing one or more heteroatoms wherein the heteroatoms are selected from the group consisting of oxygen, sulfur, and nitrogen. As used herein the term “heteroaryl” refers to a mono- or multi-cyclic carbocyclic ring system having one or more aromatic rings containing one or more heteroatoms (such as O, N and S) and includes, but is not limited to, furyl, thienyl, pyridyl and the like. As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, —OCO—CH2—O- alkyl, —OP(O)(O-alkyl)2 or —CH2—OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted. As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched-chain alkyl groups. Preferably, the “alkyl” group refers to C1-C6 straight-chain alkyl groups or C1-C6 branched- chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3- octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted. The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes 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., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. 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, aromatic and non-aromatic substituents of organic compounds. 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 may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For the sake of brevity, the disclosures of the publications cited in this specification, including patents, are herein incorporated by reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, or other publication that is herein incorporated by reference, the definition set forth in this section prevails over the definition incorporated herein by reference. As used herein and in the appended clauses, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the clauses may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of clause elements, or use of a “negative” limitation. As used herein, the terms “including,” “containing,” and “comprising” are used in their open, non-limiting sense. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Except as otherwise noted, the methods and techniques of the present embodiments are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp.360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001. Chemical nomenclature for compounds described herein has generally been derived using the commercially-available ACD/Name 2014 (ACD/Labs) or ChemBioDraw Ultra 13.0 (Perkin Elmer). As used herein and in con ructures depicting the various
Figure imgf000012_0005
embodiments described herein, , each represent a point of covalent attachment of the chemical group or chemical structure in which the identifier is shown to an adjacent chemical group or chemical structure. For example, in a hypothetical chemical structure A-B, where A and B are joined by a covalent bond, in some embodi of A-B d fin d b th r r h mi l tr t r A n b r r nted by
Figure imgf000012_0004
Figure imgf000012_0001
, or , where each of “-*”, “-**”, and “ ” represents a bond to A and the point of covalent bond attachment to B. Alternatively, in some embodim n of
Figure imgf000012_0003
A-B defined b the rou or chemical structure B can be re resented by
Figure imgf000012_0002
represents a bond to B and the point of covalent bond attachment to A. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the chemical groups represented by the variables are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace compounds that are stable compounds (i.e., compounds that can be isolated, characterized, and tested for biological activity). In addition, all subcombinations of the chemical groups listed in the embodiments describing such variables are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination of chemical groups was individually and explicitly disclosed herein. PHARMACEUTICAL COMPOSITIONS The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment. A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the disclosure. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the disclosure. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. A pharmaceutical composition (or preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste. To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), 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 may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue. For use in the methods of this disclosure, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site. Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with a compound of the disclosure. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference). In general, a suitable daily dose of an active compound used in the compositions and methods of the disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present disclosure, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily. The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general. In certain embodiments, compounds of the disclosure may be used alone or conjointly administered with another type of therapeutic agent. The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the disclosure in the compositions and methods of the present disclosure. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L- lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d- glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts. The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. EMBODIMENTS In certain aspects, a compound is of formula (IA) or (IIA)
Figure imgf000022_0001
or a pharmaceutically acceptable salt thereof. In certain embodiments, X is O or S. In some embodiments X is O. In some embodiments X is S. In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0 and the phenyl ring is unsubstituted. In some embodiments, n is 1. In certain embodiments when n is 1, R1 is optionally substituted phenoxy. In some embodiments, n is 2. In some embodiments, n is 2 and the two R1 in combination with the atoms to which each is attached combine to form a C9- C14 aryl (e.g., ) or C9-C12 bicyclic heteroaryl, each of which may be optionally substituted, for example by a heteroaryl. In some embodiments, R1 is halo, C1-C8 alkyl, C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, -C(O)RA, -ORA,-SRA or -NHRA. In some embodiments, one or more R1 in combination with the atoms to which each is attached combine to form a C9-C14 aryl or C9-C12 bicyclic heteroaryl. In certain embodiments, each hydrogen atom in C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, C1-C8 alkyl, -C(O)C2-C6 alkenylene-phenyl, C9-C12 aryl and C9-C12 bicyclic heteroaryl is optionally substituted by halo, cyano, or -NO2. In some embodiments, R2 is C6-C10 aryl or 5- to 12-membered heteroaryl, wherein each hydrogen atom in C6-C10 aryl and 5- to 12-membered heteroaryl is optionally substituted by halo, hydroxy, or -NO2. For example R2 may be phenyl optionally substituted by alkoxy, ester, halo such as chloro, etc. In some embodiments, R2 is 5- to 12-membered heteroaryl, for example
Figure imgf000022_0002
, each of which may be optionally substituted. In some embodiments, R11 is nitro or -N(H)COR3. In some embodiments, R12 is H or -C(O)C1-C6 alkyl (e.g, -C(O)ethyl). In some embodiments, R13 is H or C1-C6 alkyl (e.g., methyl). In some embodiments, R14 is H, C6-C10 aryl (e.g., phenyl), or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C6-C10 aryl and 5 to 12-membered heteroaryl is optionally substituted by halo, 5 to 12-membered heteroaryl, -NHC(O)C1-C6 alkyl, C1-C6 alkoxy, -C(O)C1-C6 alkoxy, -RA, -ORA, -SRA, -C(O)RA or -NHRA. In some embodiments, each RA is C1-C6 alkyl (e.g., methyl), C6-C10 aryl, C2-C6 alkenyl- C6-C10 aryl, -C(O)C1-C6 alkyl, C1-C6 alkoxy (e.g., methoxy), or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C1-C6 alkoxy, or 5 to 12-membered heteroaryl is optionally substituted by nitro or halo; or two RA combine to form C4-C6 cycloalkyl optionally substituted by phenyl, for example if R14 is phenyl, two RA can combine with the phenyl to form
Figure imgf000023_0001
. In some embodiments, R3 is C6-C10 aryl, 5 to 12-membered heteroaryl, or C9-C12 bicyclic aryl, wherein each hydrogen atom in C6-C10 aryl, 5 to 12-membered heteroaryl, and C9- C12 bicyclic aryl is optionally substituted by halo, C1-C6 alkyl, or nitro. Initial screening of a small library of 960 compounds received from the Medicines for Malaria Venture (MMV) foundation, identified 37 initial GroEL inhibitor hits. These initial 37 compounds served as a basis for the development 14 analogs of compound 23, having the structure:
Figure imgf000023_0002
. Compounds were screened for killing of E. coli, the ESKAPE bacteria, M. tuberculosis, and T. brucei parasites in liquid culture. In accordance with one embodiment the compounds of the present disclosure have utility as inhibitors of M. tuberculosis and T. brucei parasites. In accordance with one embodiment a composition comprising any of analogs 39-52 of Table 1, and in one embodiment compounds 49 and 50, are used as potent and selective inhibitors against T. brucei. Compounds 49 and 50 and functional derivatives thereof have more potent and selective activity against T. brucei than Suramin and Nifurtimox - the first line treatments used for treating HAT. These compounds are anticipated to have additional applications against other infectious organisms. Developing analogs of compound 23 Compound 23 was chosen as the model for analogs because of its potency inhibiting actively-replicating M. tuberculosis and GroEL folding functions. Additionally, in 2013, Wilson et al. found this compound to have whole cell antituberculosis activity, with evidence supporting inhibition of Pks13 – an essential step in the process of synthesizing mycolic acids, a part of the M. tuberculosis cell wall that is imperative in the pathogenesis of the bacteria. Analogs were prepared in which the left-hand side of the molecule was retained along with the amide linker and the aryl on the right side of the compound was varied in an effort to modulate the reactivity – with the exception of 38 (the parent amine starting material) and 51 and 52 (both contained unsubstituted phenyl rings). Analog 49 included a para-nitrofluoro ring in an effort to increase the efficiency of the nucleophilic aromatic substitution reaction with GroEL cysteines. Analog 50 included a nitrofuran group as we previously reported this to be efficacious in GroEL-targeting antibacterials due to its ability to act as a prodrug and become activated via nitroreductases. The general synthesis of these analogs is presented in Figure 1, which resulted in the generation of 15 additional analogs for testing (see Table 1 for the structures of the different analogs).
Table 1: Dose-response results for the compound 23 analogs tested in the GroEL/ES-mediated refolding assays and native reporter enzyme counter-screens. Biochemical Assay IC50 (µM)
Figure imgf000025_0001
Overall, the compound 23 analogs were less potent at inhibiting E. coli GroEL refolding functions (Table 1), with the exception of 49, which was ~2-fold more potent than 23. Of particular significance was IC50 values for these analogs exhibited high correlations between the two GroEL-mediated client protein refolding assays and were inactive in the native MDH and Rho reporter counter-screens (with the exception of compound 50 in the native Rho counter- screen), supporting that inhibitors were functioning against the GroEL/ES-mediated refolding cycle and were not false-positives of the MDH and Rho reporter enzymes. Compound 50 was also tested with the E. coli NfsB nitroreductase, resulting in a GroEL/ES-dMDH refolding IC50 value of 13 uM – slightly more potent than when tested without the NfsB nitroreductase (17 uM IC50), suggesting it may act through a pro-drug mechanism. The compound 23 analogs were all tested in the T. brucei, ESKAPE and Mtb bacterial proliferation assays and human cell viability counter-screens. Most analogs were less toxic against human cells, though compounds 49 and 50 had comparable cytotoxicity to compound 23 (Table 2). Compounds 39 and 50 were exceptionally potent at killing T. brucei parasites with high selectivity compared to cytotoxicity to human colon and intestine cells. While most compounds were inactive against the ESKAPE bacteria, parent compound 23 and nitrofuran analog 50 were particularly potent against M. tuberculosis (EC90 = 4.1 μM and 0.20 μM, respectively), with weak-moderate activity against E. coli, S. aureus, and E. faecium. Further study is needed to determine the mechanism of this inhibition, as compound 50 was largely inactive against the other Gram-negative KAPE bacteria. As nitrofuran analog 50 was potent and selective for killing both T. brucei and M. tuberculosis, a panel of 14 additional nitrofuran-containing amide analogs based on formula II were synthesized as described in Figure 1 and evaluated for their ability to inhibit GroEL/ES- mediated folding functions (in the absence and presence of E. coli NfsB nitroreductase to examine their pro-drug potential via metabolizing the nitrofurans to active metabolites), their efficacy at killing T. brucei parasites, and cytotoxicity to human colon and intestine cells – results are presented in Table 3. All compounds were stronger GroEL/ES inhibitors in the presence of NfsB, suggesting they can function as pro-drugs activated by nitroreductases. All compounds were more potent than Nifurtimox at inhibiting GroEL/ES folding functions and killing T. brucei parasites, with four proving more potent at killing T. brucei parasites than Suramin (54.60, 63, and 65).
Table 2: Dose-response testing of the compound 23 analogs in cell viability assays. Presented are antibiotic efficacy against actively replicating T. brucei (EC50) parasites and M. tuberculosis, E. coli, and the ESKAPE bacteria (EC90) and cytotoxicity (CC50) to human colon (FHC) and intestine (FHs 74Int) cell lines.
Figure imgf000027_0001
Table 3: Dose-response results for the nitrofuran analogs tested in the GroEL/ES- mediated refolding assays, native MDH reporter enzyme counter-screen, and T. brucei and human intestine and colon cell viability assays. Results for HAT therapeutics Nifurtimox and Suramin are shown for comparison. GroEL/ES-dMDH refolding results are shown from testing in the absence and presence of E. coli NfsB nitroreductase.
Figure imgf000028_0001
In accordance with one embodiment, a compound of the formula
Figure imgf000029_0001
(I) (II) is provided wherein R1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R1 substituents is optionally substituted with halogen, -OR5, -NR5R6, -S(O)2NR5R6, or –NHSO2R7; R2 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C10 aryl, substituted C5-C10 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R3 is C5-C8 aryl, C5-C8 heteroaryl, a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein each of the C5-C8 aryl, C5-C8 heteroaryl, a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl groups further comprises at least one -NO2 and one halo substituent; R5 and R6 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, or bicyclic C6-C10 heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, aryl, and heteroaryl is optionally substituted with halogen or –OC1-C6 alkyl; R7 is C5-C6 aryl, or C5-C16 heteroaryl wherein one or more of the hydrogen atoms of the C5-C6 aryl, or C5-C16 heteroaryl is optionally substituted with halo, -OR5 or -NO2; and R15 and R16 are each independently hydrogen or -CH3; or a pharmaceutically acceptable salt thereof, optionally wherein R5 and R6 are each independently hydrogen or -CH3. In accordance with one embodiment, a compound of the formula
Figure imgf000029_0002
(I) (II) is provided wherein R1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl; R3 is C5-C6 aryl, C5-C6 heteroaryl, wherein the C5-C6 aryl, and C5-C6 heteroaryl further comprises at least one -NO2 and one halo substituent; and R15 and R16 are each independently hydrogen or -CH3; or a pharmaceutically acceptable salt thereof. In accordance with one embodiment, a compound of the formula
Figure imgf000030_0001
(I) (II) is provided wherein R1 is H; R3 is
Figure imgf000030_0002
R11 is halo; R12 is NO2; and R15 and R16 are each independently hydrogen or C1-C6 alkyl; or a pharmaceutically acceptable salt thereof, optionally wherein R15 is F and R16 is each independently hydrogen or C1-C3 alkyl. In another embodiment, the disclosure relates to a compound or a pharmaceutically acceptable salt thereof, having the formula II
Figure imgf000030_0003
wherein R10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R10 substituent is optionally substituted with halo, phenoxy, -OR5, -NR5R6, -S(O)2NR5R6, CO2(C1-C4 alkyl), -C(O)NH2 or –NHSO2R7; R5 and R6 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, or bicyclic C6-C10 heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, aryl, and heteroaryl is optionally substituted with halogen or –OC1-C6 alkyl; and R7 is C5-C6 aryl, or C5-C16 heteroaryl wherein one or more of the hydrogen atoms of the C5-C6 aryl, or C5-C16 heteroaryl is optionally substituted with halo, -OR5 or nitro; or a pharmaceutically acceptable salt thereof, optionally wherein R5 and R6 are each independently hydrogen or -CH3. In one embodiment a compound having the general structure of
Figure imgf000031_0001
is provided wherein R1 is halo or -OH; R15 is H or C1-C6 alkyl; X is S or O, or a pharmaceutically acceptable salt thereof. In accordance with one embodiment a compound having the formula
Figure imgf000031_0002
is provided wherein X is S or O, and R1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1- C8 alkyl, or R1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl; or a pharmaceutically acceptable salt thereof. In accordance with one embodiment a compound having the formula
Figure imgf000031_0003
is provided wherein R1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl; or a pharmaceutically acceptable salt thereof. The compound or a pharmaceutically acceptable salt thereof, selected from the group consisting
Figure imgf000031_0004
Figure imgf000032_0001
Figure imgf000033_0001
A pharmaceutical composition comprising any of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof, and at least one diluent, carrier or excipient. A method of treating a microbial infection comprising administering to a subject in need of such treatment an effective amount of at least one compound as disclosed herein, or a pharmaceutically acceptable salt thereof. In embodiment 1 a compound having the formula:
Figure imgf000033_0002
(I) (II) wherein R1 is H, halo, C5-C10 aryl, C5-C10 heteroaryl, substituted C5-C10 aryl, substituted C5-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R2 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C10 aryl, substituted C5-C10 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R3 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl, C9-C12 bicyclic heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R15 and R16 are each independently hydrogen or C1-C6 alkyl; or a pharmaceutically acceptable salt thereof, optionally wherein R5 and R6 are each independently hydrogen or -CH3 is used to treating a microbial infection, including inhibiting the growth of Mycobacterium tuberculosis or T. brucei and/or treating Mycobacterium tuberculosis T. brucei infection. In embodiment 2, the compound used in embodiment 1 has the general structure of formula I:
Figure imgf000034_0001
wherein R3 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof, optionally wherein R15 and R16 are each independently hydrogen or -CH3. In embodiment 3, the compound used in embodiment 1 or 2 has the general structure of formula I:
Figure imgf000035_0001
wherein
Figure imgf000035_0002
R11 is halo; R12 is -NO2; R16 is C1-C6 alkyl; and R15 is H. In embodiment 4, the compound used in any one of embodiments 1-3 is provided wherein
Figure imgf000035_0003
R16 is -CH2CH3. In embodiment 5, the compound used in embodiment 1 has the structure of formula II
Figure imgf000035_0004
(II) wherein R10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo, phenoxy, -NR5R6, -OR5, -S(O)2NR5R6, or –NHSO2R7; R5 and R6 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, bicyclic C6-C10 heteroaryl, substituted C1-C6 alkyl, substituted C6-C10 aryl, or substituted bicyclic C6-C10 heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl and or substituted bicyclic C6-C10 heteroaryl comprise 1 to 5 substituents selected from halo, -NO2 and –OC1-C6 alkyl; and R7 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof In embodiment 6, the compound used in embodiment 5 is provided wherein R10 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, comprise 1 to 5 substituents selected from halo, phenoxy, -NR5R6, and -OR5; and R5 and R6 are each independently hydrogen or -CH3. In embodiment 7, the compound used in embodiment 5 or 6 is provided wherein said compound is
Figure imgf000036_0001
Compound 49 Compound 50. In embodiment 8, a pharmaceutical composition is provided comprising a compound of any one of embodiments 1-7 and a pharmaceutically acceptable carrier. In embodiment 9, a method of inhibiting the GroEL/ES and HSP60/10 chaperonin system in a microorganism is provided wherein said method comprises contacting said microorganism with the pharmaceutical composition of embodiment 8. In embodiment 10, a method of inhibiting the growth of Trypanosoma brucei and/or treating a Trypanosoma brucei infection is provided, said method comprising administering compound of
Figure imgf000036_0002
Compound 49 Compound 50. to a subject in need of treatment. EXAMPLE 1 Abbreviations: The examples described herein use materials, including but not limited to, those described by the following abbreviations known to those skilled in the art:
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
General synthetic methods. Unless otherwise stated, all chemicals were purchased from commercial suppliers and used without further purification. Reaction progress was monitored by thin-layer chromatography on silica gel 60 F254 coated glass plates (EM Sciences). Flash chromatography was performed using a Biotage Isolera One flash chromatography system and eluting through Biotage KP-Sil Zip or Snap silica gel columns for normal-phase separations (hexanes:EtOAc gradients), or Snap KP-C18-HS columns for reverse-phase separations (H2O:MeOH gradients). Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed using a Waters 1525 binary pump, 2489 tunable UV/Vis detector (254 and 280 nm detection), and 2707 autosampler. For preparatory HPLC purification, samples were chromatographically separated using a Waters XSelect CSH C18 OBD prep column (part number 186005422, 130 Å pore size, 5 μm particle size, 19x150 mm), eluting with a H2O:CH3CN gradient solvent system. Linear gradients were run from either 100:0, 80:20, or 60:40 A:B to 0:100 A:B (A = 95:5 H2O:CH3CN, 0.05% TFA; B = 5:95 H2O:CH3CN, 0.05% TFA. Products from normal-phase separations were concentrated directly, and reverse-phase separations were concentrated, diluted with H2O, frozen, and lyophilized. For primary compound purity analyses (HPLC-1), samples were chromatographically separated using a Waters XSelect CSH C18 column (part number 186005282, 130 Å pore size, 5 μm particle size, 3x150 mm), eluting with the above H2O:CH3CN gradient solvent systems. For secondary purity analyses of final test compounds (HPLC-2), samples were chromatographically separated using a Waters XBridge C18 column (either part number 186003027, 130 Å pore size, 3.5 μm particle size, 3x100 mm, or part number 186003132, 130 Å pore size, 5.0 μm particle size, 3x100 mm), eluting with a H2O:MeOH gradient solvent system. Linear gradients were run from either 100:0, 80:20, 60:40, or 20:80 A:B to 0:100 A:B (A = 95:5 H2O:MeOH, 0.05% TFA; B = 5:95 H2O:MeOH, 0.05% TFA). All test compounds were found to be >95% in purity from both RP-HPLC analyses, with the exceptions of 3 (90%), 10 (93%), 12 (N/A as the compound has no chromophore), 17 (89%), 22 (86%), 32 (93%), and 37 (94%). Mass spectrometry data were collected using an Agilent analytical LC-MS at the IU Chemical Genomics Core Facility (CGCF). 1H-NMR and 13C-NMR spectra were recorded on either Bruker 600 MHz or 300 MHz spectrometers. Chemical shifts are reported in parts per million and calibrated to the d6-DMSO solvent peaks at 2.50 ppm (1H) and 39.51 ppm (13C). The general amide coupling procedure to give parent inhibitor 23 and analogs is presented below, followed by characterization data for each analog. General procedure for the amide coupling to give inhibitor 23 and analogs. To stirring mixtures of either aniline or ethyl 2-amino-5-carbamoyl-4-methylthiophene- 3-carboxylate (1 eq.) in anhydrous acetonitrile were added the respective R-COCl reagents (1 eq.) and Cs2CO3 (1.3 eq.). Note that for any analogs where the R-CO2H starting materials were only commercially available, the acids were first converted to the acid chlorides by stirring in thionyl chloride at 60°C for 1 h, then concentrating. The reactions were allowed to stir at room temperature overnight, then diluted with water/DMSO to solubilize, and flash chromatographic purification (reverse-phase with a water:MeOH gradients) afforded the products as solids after lyophilization. If necessary, products were further purified by preparatory RP-HPLC (water:CH3CN and/or water:MeOH gradients), concentrated, and lyophilized. Analog characterization data. (38) Ethyl 2-amino-5-carbamoyl-4-methylthiophene-3-carboxylate. Commercially obtained starting material. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.27 (t, J=7.2 Hz, 3 H) 2.47 (s, 3 H) 4.20 (q, J=7.2 Hz, 2 H) 7.07 (br. s., 2 H) 7.64 (s, 2 H); 13C-NMR (150 MHz, d6-DMSO) δ 14.29, 16.05, 59.18, 105.46, 113.06, 139.82, 164.38, 164.83, 164.99; MS (ESI) C9H11N2O3S [M-H]- m/z expected = 227.1, observed = 226.9. HPLC-1 = 98%, HPLC-2 = >99%. (39) Ethyl 5-carbamoyl-2-(2-fluorobenzamido)-4-methylthiophene-3-carboxylate. Yield = 22%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.34 (t, J=7.2 Hz, 3 H), 2.56 (s, 3 H), 4.36 (q, J=7.0 Hz, 2 H), 7.41-7.60 (m, 4 H), 7.70-7.79 (m, 1 H), 8.06 (t, J=7.5 Hz, 1 H), 12.28 (d, J=10.6 Hz, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 14.00, 15.47, 61.01, 114.41, 116.69, 116.85, 118.96, 119.03, 124.05, 125.52, 131.64, 135.44, 135.49, 136.94, 148.05, 159.18, 159.94, 160.84, 164.22, 165.01; MS (ESI) C16H14FN2O4S [M-H]- m/z expected = 349.1, observed = 349.0. HPLC-1 = >99%, HPLC-2 = >99%. (40) Ethyl 5-carbamoyl-2-(4-fluorobenzamido)-4-methylthiophene-3-carboxylate. Yield = 11%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.36 (t, J=7.0 Hz, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.0 Hz, 2 H), 7.41-7.61 (m, 4 H), 8.00 (dd, J=7.7, 5.5 Hz, 2 H), 12.07 (br. s., 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 14.01, 15.41, 61.14, 114.36, 116.30, 116.44, 123.72, 128.26, 130.13, 130.20, 136.94, 148.62, 162.36, 164.02, 164.22, 165.48, 165.69; MS (ESI) C16H14FN2O4S [M- H]- m/z expected = 349.1, observed = 349.0. HPLC-1 = >99%, HPLC-2 = >99%. (41) Ethyl 5-carbamoyl-2-(2-chlorobenzamido)-4-methylthiophene-3-carboxylate. Yield = 6%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.30 (t, J=7.2 Hz, 3 H), 2.55 (s, 3 H), 4.30 (q, J=7.2 Hz, 2 H), 7.47-7.59 (m, 3 H), 7.59-7.69 (m, 2 H), 7.79 (d, J=7.3 Hz, 1 H), 11.71 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 13.92, 15.24, 61.02, 115.20, 123.81, 127.82, 130.04, 130.29, 130.51, 132.94, 133.05, 137.05, 146.99, 163.16, 164.13, 164.71; MS (ESI) C16H14ClN2O4S [M- H]- m/z expected = 365.0, observed = 365.0. HPLC-1 = >99%, HPLC-2 = >99%. (42) Ethyl 5-carbamoyl-2-(3-chlorobenzamido)-4-methylthiophene-3-carboxylate. Yield = 29%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.37 (t, J=7.2 Hz, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.1 Hz, 2 H), 7.52 (br. s., 2 H), 7.64-7.69 (m, 1 H), 7.76-7.80 (m, 1 H), 7.86-7.89 (m, 1 H), 7.93 (t, J=1.8 Hz, 1 H), 12.00 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 13.98, 15.35, 61.16, 114.86, 123.95, 125.91, 127.21, 131.23, 132.83, 133.82, 133.93, 137.04, 147.99, 162.14, 164.16, 165.17; MS (ESI) C16H14ClN2O4S [M-H]- m/z expected = 365.0, observed = 365.0. HPLC-1 = >99%, HPLC-2 = >99%. (43) Ethyl 5-carbamoyl-2-(4-chlorobenzamido)-4-methylthiophene-3-carboxylate. Yield = 11%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.36 (t, J=7.2 Hz, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.0 Hz, 2 H), 7.52 (br. s., 2 H), 7.71 (d, J=8.1 Hz, 2 H), 7.94 (d, J=8.1 Hz, 2 H), 12.08 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 14.01, 15.41, 61.16, 114.50, 123.84, 129.19, 129.37, 130.45, 136.96, 137.98, 148.45, 162.40, 164.19, 165.45; MS (ESI) C16H14ClN2O4S [M-H]- m/z expected = 365.0, observed = 365.0. HPLC-1 = >99%, HPLC-2 = >99%. (44) Ethyl 5-carbamoyl-2-(3,4-dichlorobenzamido)-4-methylthiophene-3-carboxylate. Yield = 9%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.37 (t, J=7.0 Hz, 3 H), 2.56 (s, 3 H), 4.38 (q, J=7.0 Hz, 2 H), 7.53 (br. s., 2 H), 7.88 (dd, J=8.4, 2.2 Hz, 1H), 7.93 (d, J=8.4 Hz, 1 H), 8.13 (d, J=1.8 Hz, 1 H), 11.97 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 14.46, 15.82, 61.86, 115.65, 124.61, 127.88, 129.95, 132.02, 132.54, 132.80, 136.32, 137.51, 148.18, 161.97, 164.60, 165.52; MS (ESI) C16H13Cl2N2O4S [M-H]- m/z expected = 399.0, observed = 398.9. HPLC-1 = 97%, HPLC-2 = 96%. (45) Ethyl 5-carbamoyl-4-methyl-2-(3-methylbenzamido)thiophene-3-carboxylate. Yield = 6%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.37 (t, J=67.0 Hz, 3 H), 2.42 (s, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.1 Hz, 2 H), 7.45-7.57 (m, 4 H), 7.72 (br. s., 1 H), 7.75 (s, 1 H), 12.04 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 14.00, 15.42, 20.93, 61.12, 114.18, 123.82, 124.29, 127.76, 129.16, 131.66, 133.75, 137.00, 138.75, 148.67, 163.45, 164.25, 165.39; MS (ESI) C17H17N2O4S [M-H]- m/z expected = 345.1, observed = 345.0. HPLC-1 = >99%, HPLC-2 = >99%. (46) Ethyl 5-carbamoyl-4-methyl-2-(4-methylbenzamido)thiophene-3-carboxylate. Yield = 6%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.36 (t, J=7.2 Hz, 3 H), 2.41 (s, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.2 Hz, 2 H), 7.43 (m, J=8.1 Hz, 2 H), 7.50 (br. s., 2 H), 7.83 (m, J=8.1 Hz, 2 H), 12.07 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 14.01, 15.44, 21.12, 61.11, 113.9, 123.54, 127.25, 128.80, 129.79, 136.96, 143.55, 148.92, 163.19, 164.26, 165.55; MS (ESI) C17H17N2O4S [M-H]- m/z expected = 345.1, observed = 345.0. HPLC-1 = >99%, HPLC-2 = >99%. (47) Ethyl 5-carbamoyl-4-methyl-2-(3-nitrobenzamido)thiophene-3-carboxylate. Yield = 4%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.38 (t, J=7.2 Hz, 3 H), 2.56 (s, 3 H), 4.39 (q, J=7.1 Hz, 2 H), 7.54 (br. s., 2 H), 7.94 (t, J=8.1 Hz, 1 H), 8.34 (d, J=7.7 Hz, 1 H), 8.53 (d, J=8.4 Hz, 1 H), 8.68 (s, 1 H), 12.11 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 13.97, 15.33, 61.20, 115.35, 122.23, 124.25, 127.37, 131.03, 133.30, 133.53, 137.05, 147.80, 148.03, 161.85, 164.12, 165.04; MS (ESI) C16H14N3O6S [M-H]- m/z expected = 376.1, observed = 375.9. HPLC-1 = >99%, HPLC-2 = >99%. (48) Ethyl 5-carbamoyl-4-methyl-2-(4-nitrobenzamido)thiophene-3-carboxylate. Yield = 3%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.37 (t, J=7.0 Hz, 3 H), 2.57 (s, 3 H), 4.39 (q, J=7.2 Hz, 2 H), 7.55 (br. s., 2 H), 8.18 (d, J=8.8 Hz, 2 H), 8.46 (d, J=8.8 Hz, 2 H), 12.16 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 14.02, 15.38, 61.22, 115.21, 124.23, 124.32, 128.97, 137.00, 137.24, 147.82, 149.92, 161.96, 164.11, 165.25; MS (ESI) C16H14N3O6S [M-H]- m/z expected = 376.1, observed = 376.0. HPLC-1 = >99%, HPLC-2 = 99%. (49) Ethyl 5-carbamoyl-2-(2-fluoro-5-nitrobenzamido)-4-methylthiophene-3-carboxylate. Yield = 68%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.35 (t, J=7.2 Hz, 3 H), 2.55 (s, 3 H), 4.36 (q, J=7.0 Hz, 2 H), 7.54 (br. s., 2 H), 7.77 (t, J=9.9 Hz, 1 H), 8.51-8.60 (m, 1 H), 8.74 (dd, J=6.1, 2.8 Hz, 1 H), 12.33 (d, J=9.9 Hz, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 13.98, 15.42, 61.14, 115.02, 118.75, 118.92, 120.53, 120.44, 124.54, 127.06, 130.15, 130.22, 136.97, 14.36, 147.44, 158.18, 162.18, 163.91, 164.07, 164.95; MS (ESI) C16H13FN3O6S [M-H]- m/z expected = 394.1, observed = 393.9. HPLC-1 = >99%, HPLC-2 = 97%. (50) Ethyl 5-carbamoyl-4-methyl-2-(5-nitrofuran-2-carboxamido)thiophene-3- carboxylate. Yield = 22%. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.39 (t, J=7.0 Hz, 3 H), 2.56 (s, 3 H), 4.40 (q, J=7.0 Hz, 2 H), 7.57 (br. s., 2 H), 7.68 (d, J=3.7 Hz, 1 H), 7.87 (d, J=4.0 Hz, 1 H), 12.08 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 13.96, 15.38, 61.33, 113.84, 115.24, 118.65, 124.82, 137.00, 145.66, 146.79, 151.71, 153.17, 163.97, 164.85; MS (ESI) C14H12N3O7S [M-H]- m/z expected = 366.0, observed = 366.0. HPLC-1 = 97%, HPLC-2 = 98%. (51) 2-fluoro-5-nitro-N-phenylbenzamide. Yield = 89%. 1H NMR (300 MHz, DMSO-d6) δ ppm 7.08-7.21 (m, 1 H), 7.38 (t, J=7.9 Hz, 2 H), 7.63-7.78 (m, 3 H), 8.46 (ddd, J=9.1, 4.3, 3.0 Hz, 1 H), 8.54 (dd, J=5.9, 2.9 Hz, 1 H), 10.68 (s, 1 H); 13C-NMR (75 MHz, d6-DMSO) δ 117.89, 118.22, 119.89, 124.30, 125.75, 125.82, 126.03, 127.94, 128.08, 138.45, 143.78, 143.81, 160.56, 160.65, 184.09; MS (ESI) C13H8FN2O3 [M-H]- m/z expected = 259.1, observed = 259.0. HPLC-1 = >99%, HPLC-2 = 97%. (52) 2,3,4,5,6-pentafluoro-N-phenylbenzamide. Yield = 79%. 1H NMR (300 MHz, DMSO- d6) δ ppm 7.12-7.25 (m, 1 H), 7.40 (t, J=7.9 Hz, 2 H), 7.60-7.74 (m, 2 H), 11.00 (s, 1 H); 13C- NMR (75 MHz, d6-DMSO) δ 119.62, 124.79, 129.12, 137.90, 154.87; MS (ESI) C13H5F5NO [M-H]- m/z expected = 286.0, observed = 286.0. HPLC-1 = >99%, HPLC-2 = >99%. General procedure for the amide coupling to give nitrofuran inhibitor analogs 53-66. To stirring mixture of the respective arylamine (1 eq.) in dichloromethane was added 5- nitro2-furoyl chloride (1.1 eq.) followed by pyridine (1.2 eq.), then the reactions were left to stir at RT overnight. The following day, the reactions were concentrated and flash chromatographic purification over silica (hexanes:EtOAc gradient) afforded the products as solids. If necessary, products were further purified by preparatory RP-HPLC (water:CH3CN gradient), concentrated, and lyophilized. (53) N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-5-nitrofuran-2- carboxamide. Yield = 86%. 1H NMR (600 MHz, DMSO-d6) δ ppm 2.27 (s, 3 H), 6.04 (s, 1 H), 7.41 (d, J=8.1 Hz, 2 H), 7.51 (d, J=7.7 Hz, 2 H), 7.56 (d, J=9.5 Hz, 2 H), 7.61 (d, J=3.7 Hz, 1 H), 7.83 (d, J=3.3 Hz, 1 H), 10.45 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 17.48, 38.09, 113.43, 116.94, 119.00, 127.67, 129.18, 129.61, 130.83, 131.73, 133.00, 133.63, 133.93, 136.45, 147.48, 151.77, 154.94; MS (ESI) C20H13Cl2N3O4 [M-H]- m/z expected = 428.0210, observed = 428.0172. HPLC-1 = 98%, HPLC-2 = 97%. (54) (E)-5-nitro-N-(4-(3-(3-nitrophenyl)acryloyl)phenyl)furan-2-carboxamide. Yield = 10%. 1H NMR (600 MHz, DMSO-d6) δ ppm 7.71 (d, J=2.9 Hz, 1 H), 7.76 (t, J=7.9 Hz, 1 H), 7.82 - 7.89 (m, 2 H), 7.98 (d, J=8.1 Hz, 2 H), 8.19 (d, J=15.4 Hz, 1 H), 8.28 (d, J=8.1 Hz, 3 H), 8.35 (d, J=7.7 Hz, 1 H), 8.80 (br. s., 1 H), 10.95 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 113.45, 117.18, 119.98, 122.95, 124.67, 130.04, 130.37, 133.03, 135.13, 136.67, 141.11, 142.57, 147.45, 148.45, 151.92, 154.92, 187.52; MS (ESI) C20H13N3O7 [M-H]- m/z expected = 406.0681, observed = 406.0649. HPLC-1 = 98%, HPLC-2 = 97%. (55) 5-nitro-N-(4-phenoxyphenyl)furan-2-carboxamide. Yield = 92%. 1H NMR (600 MHz, DMSO-d6) δ ppm 7.01 (d, J=8.1 Hz, 2 H), 7.06 (m, J=8.4 Hz, 2 H), 7.13 (t, J=7.3 Hz, 1 H), 7.39 (t, J=7.5 Hz, 2 H), 7.63 (d, J=3.3 Hz, 1 H), 7.75 (m, J=8.4 Hz, 2 H), 7.82 (d, J=3.3 Hz, 1 H), 10.67 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 113.49, 116.45, 118.25, 119.16, 122.47, 123.29, 130.04, 133.46, 147.96, 151.74, 152.98, 154.44, 156.95; MS (ESI) C17H12N2O5 [M-H]- m/z expected = 323.0673, observed = 323.0652. HPLC-1 = >99%, HPLC-2 = >99%. (56) 5-nitro-N-(4-(phenylamino)phenyl)furan-2-carboxamide. Yield = 83%. 1H NMR (600 MHz, DMSO-d6) δ ppm 6.81 (t, J=7.2 Hz, 1 H), 7.06 (d, J=7.7 Hz, 2 H), 7.09 (d, J=8.4 Hz, 2 H), 7.22 (t, J=7.5 Hz, 2 H), 7.60 (d, J=6.2 Hz, 3 H), 7.77 - 7.85 (m, 1 H), 8.18 (s, 1 H), 10.50 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 113.54 (s, 1 C) 116.03, 116.38, 117.03, 119.49, 122.06, 129.18, 130.11, 140.20, 143.51, 148.32, 151.66, 154.08; MS (ESI) C17H13N3O4 [MH]+ m/z expected = 324.0979, observed = 324.0914. HPLC-1 = 98%, HPLC-2 = >99%. (57) N-(4-acetamidophenyl)-5-nitrofuran-2-carboxamide. Yield = 48%. 1H NMR (600 MHz, DMSO-d6) δ ppm 2.03 (s, 3 H), 7.55 - 7.60 (m, 2 H), 7.60 - 7.63 (m, 1 H), 7.64 (d, J=8.1 Hz, 2 H), 7.76 - 7.85 (m, 1 H), 9.96 (s, 1 H), 10.57 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 23.92, 113.49, 116.28, 119.24, 121.18, 132.83, 136.01, 148.08, 151.71, 154.31, 168.11; MS (ESI) C13H11N3O5 [M-H]- m/z expected = 288.0626, observed = 288.0609. HPLC-1 = >99%, HPLC-2 = >99%. (58) N-(4-methoxyphenyl)-5-nitrofuran-2-carboxamide. Yield = 78%. 1H NMR (600 MHz, DMSO-d6) δ ppm 3.75 (s, 3 H), 6.96 (d, J=8.4 Hz, 2 H), 7.59 (d, J=3.7 Hz, 1 H), 7.64 (d, J=8.4 Hz, 2 H), 7.81 (d, J=3.7 Hz, 1 H), 10.52 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 55.21, 113.51, 113.94, 116.17, 122.32, 130.73, 148.18, 151.66, 154.25, 156.15; MS (ESI) C12H10N2O5 [M-H]- m/z expected = 261.0517, observed = 261.0496. HPLC-1 = 98%, HPLC-2 = >99%. (59) methyl 4-(5-nitrofuran-2-carboxamido)benzoate. Yield = 21%. 1H NMR (600 MHz, DMSO-d6) δ ppm 3.86 (s, 3 H), 7.66 - 7.72 (m, 1 H), 7.81 - 7.85 (m, 1 H), 7.92 (d, J=8.1 Hz, 2 H), 8.00 (d, J=8.1 Hz, 2 H), 10.91 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 52.01, 113.41, 117.10, 119.99, 125.14, 130.23, 142.31, 147.44, 151.91, 154.87, 165.72; MS (ESI) C13H10N2O6 [M-H]- m/z expected = 289.0466, observed = 289.0460. HPLC-1 = 97%, HPLC-2 = >99%. (60) N-(3-chloro-4-(4-chlorophenoxy)phenyl)-5-nitrofuran-2-carboxamide. Yield = 93%. 1H NMR (600 MHz, DMSO-d6) δ ppm 6.97 (m, J=8.4 Hz, 2 H), 7.24 (d, J=8.8 Hz, 1 H), 7.42 (m, J=8.4 Hz, 2 H), 7.64 (d, J=3.7 Hz, 1 H), 7.74 (d, J=9.2 Hz, 1 H), 7.83 (d, J=2.9 Hz, 1 H), 8.06 (s, 1 H), 10.83 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 113.47, 116.94, 118.58, 120.97, 122.20, 122.29, 124.89, 126.97, 129.88, 135.48, 147.16, 147.47, 151.83, 154.67, 155.77; MS (ESI) C17H10Cl2N2O5 [M-H]- m/z expected = 390.9894, observed = 390.9882. HPLC-1 =97%, HPLC-2 = >99%. (61) 5-nitro-N-(quinolin-6-yl)furan-2-carboxamide. Yield = 41%. 1H NMR (600 MHz, DMSO-d6) δ ppm 7.52 (dd, J=8.1, 4.0 Hz, 1 H), 7.71 (d, J=3.3 Hz, 1 H), 7.85 (d, J=3.3 Hz, 1 H), 7.99 - 8.09 (m, 2 H), 8.36 (d, J=8.4 Hz, 1 H), 8.46 (s, 1 H), 8.79 - 8.87 (m, 1 H), 10.94 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 113.49, 116.85, 117.14, 121.94, 124.13, 128.09, 129.60, 135.77, 145.15, 147.72, 149.73, 151.88, 154.91; MS (ESI) C14H9N3O4 [M-H]- m/z expected = 282.0520, observed = 282.0514. HPLC-1 = >99%, HPLC-2 = 99%. (62) N-(4-(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-5-nitrofuran-2-carboxamide. Yield = 74%. 1H NMR (600 MHz, DMSO-d6) δ ppm 7.52 (dd, J=8.1, 4.0 Hz, 1 H), 7.71 (d, J=3.3 Hz, 1 H), 7.85 (d, J=3.3 Hz, 1 H), 8.00 - 8.08 (m, 2 H), 8.36 (d, J=8.4 Hz, 1 H), 8.46 (s, 1 H), 8.80 - 8.89 (m, 1 H), 10.94 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 113.46, 117.49, 120.19, 121.49, 121.73, 121.87, 122.30, 124.62, 126.53, 134.89, 138.75, 138.88, 141.94, 147.12, 151.96, 153.39, 155.06, 167.61; MS (ESI) C18H10ClN3O4S2 [M-H]- m/z expected = 429.9728, observed = 429.9709. HPLC-1 = >99%, HPLC-2 = >99%. (63) N-(9H-fluoren-3-yl)-5-nitrofuran-2-carboxamide. Yield = 46%. 1H NMR (600 MHz, DMSO-d6) δ ppm 3.96 (s, 2 H), 7.30 (t, J=7.3 Hz, 1 H), 7.38 (t, J=7.3 Hz, 1 H), 7.57 (d, J=7.3 Hz, 1 H), 7.67 (d, J=3.3 Hz, 1 H), 7.73 (d, J=8.4 Hz, 1 H), 7.83 (d, J=3.3 Hz, 1 H), 7.86 (d, J=7.3 Hz, 1 H), 7.90 (d, J=8.1 Hz, 1 H), 8.05 (s, 1 H), 10.70 (s, 1 H); 13C-NMR (150 MHz, d6- DMSO) δ 36.54, 113.51, 116.45, 117.48, 119.48, 119.73, 120.19, 125.08, 126.45, 126.80, 136.76, 137.62, 140.75, 143.01, 143.73, 148.03, 151.79, 154.50; MS (ESI) C18H12N2O4 [M-H]- m/z expected = 319.0724, observed = 319.0718. HPLC-1 = 99%, HPLC-2 = 98%. (64) N-(4-(benzo[d]thiazol-2-ylthio)phenyl)-5-nitrofuran-2-carboxamide. Yield = 69%. 1H NMR (600 MHz, DMSO-d6) δ ppm 7.34 (t, J=7.5 Hz, 1 H), 7.45 (t, J=7.7 Hz, 1 H), 7.70 (d, J=4.0 Hz, 1 H), 7.84 (d, J=8.4 Hz, 4 H), 7.93 (d, J=7.7 Hz, 1 H), 7.97 (d, J=7.7 Hz, 2 H), 10.92 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 113.46, 117.10, 121.34, 121.77, 121.82, 123.45, 124.45, 126.45, 134.78, 136.42, 140.47, 147.49, 151.90, 153.49, 154.89, 169.75; MS (ESI) C18H11N3O4S2 [M-H]- m/z expected = 396.0118, observed = 396.0095. HPLC-1 = >99%, HPLC-2 = >99%. (65) N-(2,3-di(furan-2-yl)quinoxalin-6-yl)-5-nitrofuran-2-carboxamide. Yield = 60%. 1H NMR (600 MHz, DMSO-d6) δ ppm 6.69 - 6.77 (m, 4 H), 7.74 (d, J=3.7 Hz, 1 H), 7.86 (d, J=3.7 Hz, 2 H), 7.90 (d, J=8.4 Hz, 1 H), 8.06 - 8.17 (m, 2 H), 8.63 (s, 1 H), 11.12 (s, 1 H); 13C-NMR (150 MHz, d6-DMSO) δ 112.15, 112.23, 112.54, 113.02, 113.49, 116.88, 117.29, 125.12, 129.25, 137.30, 139.80, 140.49, 141.03, 142.52, 144.75, 145.01, 147.45, 150.38, 151.94, 155.14; MS (ESI) C21H12N4O6 [M-H]- m/z expected = 415.0684, observed = 415.0672. HPLC-1 = >99%, HPLC-2 = 96%. (66) 5-nitro-N-phenylfuran-2-carboxamide. Yield = 86%. 1H NMR (600 MHz, DMSO-d6) δ ppm 7.16 (t, J=7.3 Hz, 1 H), 7.39 (t, J=7.7 Hz, 2 H), 7.64 (d, J=3.7 Hz, 1 H), 7.73 (d, J=7.7 Hz, 2 H), 7.82 (d, J=3.7 Hz, 1 H), 10.62 (s, 1 H); 13C-NMR (7150 MHz, d6-DMSO) δ 113.46, 116.50, 120.71, 124.57, 128.82, 137.79, 147.93, 151.76, 154.59; MS (ESI) C11H8N2O4 [M-H]- m/z expected = 231.0411, observed = 231.0395. HPLC-1 = >99%, HPLC-2 = >99%. General materials for biochemical & cell-based experiments. DH5α and BL21 (DE3) E. coli cells were purchased from New England Biolabs. The Mycobacterium tuberculosis proliferation assay used an H37Rv strain that researchers at the Seattle Children’s Research Institute (Seattle, WA, USA) previously engineered to express a codon-optimized mCherry fluorescent protein (TOPred). Trypanosoma brucei Plimmer and Bradford parasites (Lister 427 VSG 221 [TetR T7RNAP] transgenic bloodstream form) were obtained from the ATCC (PRA-383). FHs 74 Int (CCL-241) cells and FHC (CRL-1831) cells were obtained from the ATCC. E. coli GroEL and GroES Purification E. coli GroEL was expressed from a trc-promoted and Amp(+) resistance marker plasmid in DH5α E. coli cells. GroES was expressed from a T7-promoted and Amp(+) resistance plasmid in E. coli BL21 (DE3) cells. Transformed colonies were plated onto Ampicillin-treated LB agar and incubated for 24 h at 37°C. Cells were then grown at 37°C in Ampicillin-treated LB medium until an OD600 of ~0.5 was reached, then were induced with 0.8 mM IPTG and continued to grow for 2-2.5 h at 37°C. The cultures were centrifuged at 8,000 rpm and the cell pellets were collected and re-suspended in Buffer A (50 mM Tris-HCl, pH 7.4, and 20 mM NaCl) supplemented with half a PierceTM protease inhibitor tablet, EDTA-free (Thermo Scientific). The combined suspension was lysed by sonication, the lysate was centrifuged at 14,000 rpm, and the clarified lysate was passed through a 0.45 μm filter (Millipore). Anion exchange purification: The filtered lysate was loaded onto a GE HiScale Anion exchange column (Q Sepharose fast flow anion exchange resin) that was equilibrated with 2 column volumes of Buffer A. The loaded column was washed with 10% Buffer B (50 mM Tris.HCl, pH 7.4, 1 M NaCl) for 3 CVs when purifying GroES and 28% Buffer B for 3 CVs when purifying GroEL, then bound protein was eluted with 3 CVs of 10-50% gradient flow of Buffer B when purifying GroES and 3 CVs of 28-60% gradient flow of Buffer B when collecting GroEL. Protein-containing fractions, as identified by SDS-PAGE, were collected, spin concentrated using a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore), and dialyzed overnight with 10 kDa SnakeSkin™ dialysis tubing (Thermo Scientific) at 4°C in 2 L of 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl solution (Dialysis Buffer). Size exclusion chromatography: The dialyzed protein was loaded onto a Superdex 200 column (HiLoad 26/600, GE) column that was equilibrated with 2 column volumes of SEC Buffer (50 mM Tris-HCl, pH 7.4 and 150 mM NaCl). The loaded column was eluted with 1 column volume of 100% SEC Buffer and the column was washed with 2 CVs of the same solution. Protein-containing fractions, as identified by SDS-PAGE, were collected, spin concentrated using a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore). The final protein concentration was determined by Coomassie Protein Assay Kit (Thermo Scientific). Batches of protein for testing were stored at 4°C for up to six weeks and then discarded. Determination of compounds’ refolding inhibition in dMDH reporter assay Reagent preparation: For these assays, four primary reagent stocks were prepared: 1) GroEL/ES-dMDH or HSP60/10-dMDH binary complex stock; 2) ATP initiation stock; 3) EDTA quench stock; 4) MDH enzymatic assay stock. Denatured MDH (dMDH) was prepared by 2-fold dilution of MDH (5 mg/ml, soluble pig heart MDH from Roche, product #10127248001) with denaturant buffer (7 M guanidine-HCl, 200 mM Tris, pH 7.4, and 50 mM DTT). MDH was completely denatured by incubating at room temperature for 60 min. The binary complex solutions were prepared by adding the dMDH stock to GroEL (or HSP60) in folding buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, and 1 mM DTT), followed by addition of GroES (or HSP10). The binary complex stocks were prepared immediately prior to dispensing into the assay plates and had final protein concentrations of 83.3 nM GroEL (Mr 800 kDa) or HSP60 (Mr 400 kDa), 100 nM GroES or HSP10 (Mr 70 kDa), and 20 nM dMDH in folding buffer. For the ATP initiation stock, ATP solid was diluted into folding buffer to a final concentration of 2.5 mM. Quench solution contained 600 mM EDTA (pH 8.0). The MDH enzymatic assay stock consisted of 20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, and 1 mM DTT). Assay protocol: First, 30 µL aliquots of the GroEL/ES-dMDH or HSP60/10-dMDH binary complex stocks were dispensed into clear, 384-well polystyrene plates. Next, 0.5 µL of the compound stocks (10 mM to 4.6 µM, 3-fold dilutions series in DMSO) were added by pin- transfer (V&P Scientific). The chaperonin-mediated refolding cycles were initiated by addition of 20 µL of ATP stock (reagent concentrations during refolding cycle: 50 nM GroEL or HSP60, 60 nM GroES or HSP10, 12 nM dMDH, 1 mM ATP, and compounds of 100 μM to 46 nM, 3- fold dilution series), and the refolding reactions incubated at 37°C. The incubation times were determined from refolding time-course control experiments until they reached ~90% completion of refolding of the denatured MDH – generally ~15-40 min for GroEL/ES, and ~40- 60 min for HSP60/10. Next, the assays were quenched by addition of 10 µL of the EDTA stock, to final concentration of 100 mM. Enzymatic activity of the refolded MDH was initiated by addition of 20 µL MDH enzymatic assay stock (20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer, 50 mM Tris pH 7.4, 50 mM KCl, 1 mM DTT), and followed by measuring the NADH absorbance in each well at 340 nm using a Molecular Devices, SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD+ does not). A340 nm measurements were recorded at 0.5 minutes (start point) and at successive time points until the amount of NADH consumed reached ~90% (end point, generally between 20-35 minutes). The differences between the start and end point A340 values were used to calculate the % inhibition of the GroEL/ES or HSP60/10 machinery by the compounds. IC50 values for the test compounds were obtained by plotting the % inhibition results in GraphPad Prism and analyzing by non-linear regression using the log (inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC50 values obtained from at least four replicates. Counter-screening compounds for inhibition of native MDH enzymatic activity. Reagent preparations and assay protocol: This assay was performed as described above for the GroEL/ES-dMDH refolding assay; however, the assay protocol differed in the sequence of compound addition to the assay plates. The refolding reactions were allowed to proceed for 45- 50 min at 37°C in the absence of test compounds (nearly complete refolding of MDH occurs), then quenched with the EDTA stock. Compounds were then pin-transferred into the plates after the EDTA quenching step; thus, compounds’ effects are only possible by inhibiting the fully refolded MDH reporter substrate. Next, enzymatic activity of the refolded MDH was initiated by addition of 20 µL MDH enzymatic assay stock (20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer(50 mM Tris pH 7.4, 50 mM KCl, 1 mM DTT), and followed by measuring the NADH absorbance in each well at 340 nm using a Molecular Devices SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD+ does not). A340 nm measurements were recorded at 0.5 minutes (start point) and at successive time points until the amount of NADH consumed reached ~90% (end point, generally between 15-30 minutes). Compounds were tested in 8-point, 3-fold dilution series (62.5 μM to 29 nM during the reporter reaction) in clear, flat-bottom 384-well microtiter plates. DMSO was used as negative control, and previously discovered native MDH inhibitors were used as positive controls. IC50 values for the test compounds were obtained by plotting the % inhibition results in GraphPad Prism and analyzing by non-linear regression using the log (inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC50 values obtained from at least four replicates. Evaluating compounds for inhibition in the GroEL/ES-dRho refolding assay. Reagent preparation: For this assay, five primary reagent stocks were prepared: 1) GroEL/ES- dRho binary complex stock; 2) ATP initiation stock; 3) Enzyme solution; 4) Formaldehyde quench solution; 5) Fe(NO3)3 assay stock. Denatured Rho (dRho) was prepared by 3-fold dilution of Rho (Roche product #R1756, stock diluted to 10 mg/ml with H2O) with denaturant buffer (12 M Urea, 50 mM Tris, pH 7.4, and 10 mM DTT). Rho was completely denatured by incubating at room temperature for 40 min. The binary complex solution was prepared by slowly adding the dRho stock to a stirring solution of concentrated GroEL in modified folding buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, 5 mM Na2S2O3 and 1 mM DTT) and then incubated at room temperature for 10 min. The solution was centrifuged at 16,000 x g for 5 minutes, and the supernatant was collected and added to a solution of GroES in modified folding buffer to give final protein concentrations of 100 nM GroEL, 120 nM GroES, and 80 nM dRho in modified folding buffer. The binary complex stock was prepared immediately prior to use. For the ATP initiation stock, ATP solid was diluted into modified folding buffer to a final concentration of 2.0 mM. The thiocyanate enzymatic assay stock was prepared to contain 70 mM KH2PO4, 80 mM KCN, and 80 mM Na2S2O3 in water. The formaldehyde quench solution contained 30% formaldehyde in water. The ferric nitrate reporter stock contained 8.5% w/v Fe(NO3)3 and 11.3% v/v HNO3 in water. Assay protocol: First, 10 µL aliquots of the GroEL/ES-dRho complex stock was dispensed into clear, 384-well polystyrene plates. Next, 0.5 µL of the compound stocks (10 mM to 4.6 µM, 3- fold dilutions in DMSO) were added by pin-transfer. The chaperonin-mediated refolding cycle was initiated by addition of 10 µL of ATP stock (reagent concentrations during refolding cycle: 50 nM GroEL, 60 nM GroES, 40 nM dRho, 1 mM ATP, and compounds of 250 µM to 114 nM, 3-fold dilution series). After incubating for 45 minutes at 37°C for the refolding cycle, 30 µL of the thiocyanate enzymatic assay stock was added and incubated for 60 min at room temperature for the refolded rhodanese enzymatic reporter reaction. The rhodanese-catalyzed thiosulfate- cyanide reaction was quenched by adding 10 µL of the formaldehyde quench solution, and then 40 µL of the ferric nitrate reporter stock was added to quantify the amount of thiocyanate produced during the enzymatic reporter reaction, which is proportional to the amount of dRho refolded by GroEL/ES. After incubating at room temperature for 15 min, the absorbance by Fe(SCN)3 was measured at 460 nm using a Molecular Devices, SpectraMax Plus384 microplate reader. A second set of baseline control plates were prepared analogously, but without binary solution, to correct for possible interference from compound absorbance or turbidity. IC50 values for the test compounds were obtained by plotting the A460 results in GraphPad Prism and analyzing by non-linear regression using the log(inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC50 values obtained from at least four replicates. Counter-screening compounds for inhibition of native Rho enzymatic activity. Reagent preparations and assay protocol: Reagents were identical to those used in the GroEL/ES-dRho refolding assay described above; however, the assay protocol differed in the sequence of compound addition to the wells. Compounds were pin-transferred after the 60-minute incubation for the refolding cycle, but prior to the addition of the thiocyanate enzymatic assay stock. Thus, the refolding reactions were allowed to proceed for 60 min at 37°C in the absence of test compounds, but the enzymatic activity of the refolded rhodanese reporter enzyme was monitored in the presence of test compounds (inhibitor concentration range during the enzymatic reporter reaction is 100 µM to 46 nM – 3-fold dilutions). IC50 values for the rhodanese reporter enzyme were determined as described above. Results presented represent the averages of IC50 values obtained from at least three replicates. Bacterial Proliferation Assays. Bacterial Strains: NEB 5-alpha Escherichia coli (a derivative of DH5α E. coli, New England Biolabs #C2987H); Enterococcus faecium - (Orla-Jensen) Schleifer and Kilpper-Balz strain NCTC 7171 (ATCC #19434); Staphylococcus aureus - Rosenbranch strain Seattle 1945 (ATCC #25923); Klebsiella pneumoniae - (Schroeter) Trevisan strain NCTC 9633 (ATCC #13883); Acinetobacter baumannii - Bouvet and Grimont strain 2208 (ATCC 19606); Pseudomonas aeruginosa - (Schroeter) Migula strain NCTC 10332 (ATCC #10145); Enterobacter cloacae - E. cloacae, subsp. cloacae (Jordan) Hormaeche and Edwards strain CDC 442-68 (ATCC #13047). Growth Media: E. coli were grown with LB medium and all ESKAPE bacteria were grown in Mueller-Hinton Broth 2, Cation-Adjusted (CA-MHB) medium (Millipore), with all liquid cultures supplemented with 20-25 mg/L Ca2+ and 10-12.5 mg/L Mg2+ to mimic physiological free concentrations of these cations. General Assay Protocol: Stock bacterial cultures were streaked onto LB or CA-MHB agar plates and grown overnight at 37°C. Fresh aliquots of cation supplemented media were inoculated with single bacterial colonies and the cultures were grown overnight at 37°C with shaking (240 rpm). The following morning, cultures were diluted 10-fold into fresh media and grown at 37°C until bacteria had reached mid-log phase growth (OD600 ~ 0.4-0.6). The cultures were then diluted into fresh media to achieve final CFU/mL of 8.33 x105 CFU/mL. Aliquots of these diluted cultures (30 μL) were added to clear, flat-bottom 384-well polystyrene plates that were previously stamped with 0.5 μL of test compounds in 20 μL of media (yielding initial density of 5 x 105 CFU/mL). All compounds were tested in dose-response with concentration ranges during the proliferation assays from 100 μM to 46 nM (3-fold dilution series). A second set of baseline control plates were prepared analogously, but without any bacteria added, to correct for possible compound absorbance and/or precipitation. Plates were sealed with "Breathe-Easier" oxygen permeable membranes (Diversified Biotech) and left to incubate at 37°C without shaking (stagnant assay) for 20-24 hours. Plates were then read at 600 nm using a Molecular Devices SpectraMax Plus384 microplate reader. EC90 values for the test compounds were obtained by plotting the OD600 results in GraphPad Prism and analyzing by non-linear regression using the [agonist] vs. response - Find ECanything equation. Results presented represent the averages of EC90 values obtained from at least four replicates. Evaluating compounds for inhibition of M. tuberculosis proliferation. M. tuberculosis (strain H37Rv) was grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 10% v/v oleic acid, and albumin dextrose catalase (OADC) supplement (Becton Dickinson) (7H9-Tw-OADC). Stock bacterial cultures were inoculated in a startup culture and grown to a logarithmic phase of OD590 ~0.7. This was sub-cultured in fresh media (1:10 dilution) and grown to an OD5901.0, then diluted again into fresh media to achieve a final OD590 reading of 0.04 (just prior to dispensing into plates). Compound plates were prepared by adding 4 µL of compound stocks to 96 µL of fresh medium in 96-well plates. Aliquots of the diluted Mtb cultures (100 µL) were then added to the compound plates, which were incubated in sealed bags at 37ºC for 5 days. All compounds were first evaluated in singlicate at a single concentration of 200 µM, with compounds showing >50% inhibition re-evaluated in dose- response format (inhibitor concentration range of 200 μM to 390 nM – 2-fold dilutions) to determine EC50 values. After 5 days, OD590 values were read and % growth inhibition for each well was calculated. EC90 values for the test compounds were obtained by plotting the OD590 results in GraphPad Prism and analyzing by non-linear regression using the [agonist] vs. response - Find ECanything equation. Results presented represent the averages of EC90 values obtained from at least duplicate experiments. T. brucei cell viability assay protocol. Test compounds were evaluated using a T. brucei cell viability assay in 384-well plate format. Briefly, 55 μL of 2000 parasites/mL (110 parasites/well) of Trypanosoma brucei brucei (strain BF427) in HMI-9 medium were dispensed in to clear, 384-well polystyrene plates (BRAND cell culture grade plates, 781980). Plates were sealed with "Breathe Easy" oxygen permeable membranes (Diversified Biotech) and incubated at 37°C, 5% CO2 for 24 h. Next, 1 µL of the compound stocks (10 mM to 4.6 µM, 3-fold dilutions in DMSO) were pre-diluted by pin-transfer into 20 µL HMI-9 medium, then 5 µL of these diluted compounds were added to the parasite assay plates to give an inhibitor concentration range of 42 µM to 19 nM during the assay (the final DMSO concentration of 0.42% was maintained during the assay). Parasites were incubated for an additional 48 h at 37°C and 5% CO2. Cell viability was then measured by adding 10 µL of Alamar Blue reagent to give 10% v/v in the assay. Plates were incubated at 37°C and 5% CO2 and sample fluorescence (535 nm excitation, 590 nm emission) monitored over time using a Molecular Devices FlexStation II 384-well plate reader, and cell viability was calculated as per vendor instructions. EC50 and EC90 values for the test compounds were obtained by plotting the % Alamar Blue reduction results in GraphPad Prism and analyzing by non-linear regression using the [agonist] vs. response - Find ECanything equation. DMSO was used as negative control, and pentamidine, suramin, and nifurtimox (drugs used to treat HAT) were used as positive controls. Results presented represent the averages of EC50 and EC90 values obtained from at least four replicates. Evaluating compounds for effects on human intestine and colon cell viability. Evaluation of compound cytotoxicity to FHs 74 Int intestine and FHC colon cells was performed using Alamar Blue-based viability assays. FHs 74 Int cells were maintained in Hybri-care Medium ATCC 46-X supplemented with 30ng/ml epidermal growth factor (EGF) and 10% FBS. FHC cells were maintained in DMEM: F-12 media, supplemented with 10mM HEPES (final concentration 25 mM), 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005mg/ml transferrin, 100 ng/ml hydrocortisone, and 10% FBS (Sigma, F2242). All assays were carried out in 384-well plates (BRAND cell culture grade plates, 781980). Cells at 80% confluence were harvested and diluted in growth medium, then 45 µL of the FHs 74 Int cells (1,500 cells/well) or FHC cells (1,500 cells/well) were dispensed per well, and plates were sealed with "Breathe Easy" oxygen permeable membranes (Diversified Biotech) and incubated at 37°C, 5% CO2, for 24 h. The following day, 1 µL of the compound stocks (10 mM to 4.6 µM, 3-fold dilutions in DMSO) were pre-diluted by pin-transfer into 25 µL of the relevant growth mediums. Then, 15 µL aliquots of the diluted compounds were added to the cell assay plates to give inhibitor concentration ranges of 100 μM to 46 nM during the assay (final DMSO concentration of 0.1% was maintained during the assay). Plates were sealed with "Breathe Easy" oxygen permeable membranes and incubated for an additional 48 h at 37°C and 5% CO2. The Alamar Blue reporter reagents were then added to a final concentration of 10%, the plates incubated at 37°C and 5% CO2, and sample fluorescence (535 nm excitation, 590 nm emission) was read using a Molecular Devices FlexStation II 384-well plate reader (readings taken between 4-24 h of incubation so as to achieve signals in the 30-60% range for conversion of resazurin to resorufin). Cell viability was calculated as per vendor instructions (Thermo Fisher - Alamar Blue cell viability assay manual). Cytotoxicity CC50 values for the test compounds were obtained by plotting the % resazurin reduction results in GraphPad Prism and analyzing by non- linear regression using the log(inhibitor) vs. response (variable slope) equation. Results presented represent the averages of CC50 values obtained from at least four replicates for FHs 74 Int small intestine cells and four replicates for FHC colon cells. Control compounds, calculation of IC50 values, and statistical considerations. For the chaperonin-mediated biochemical assays, DMSO was used as a negative control, and a panel of our previously discovered and reported chaperonin inhibitors were used as positive controls: e.g., compounds 8, 9, and 18 from Johnson et. al 2014 and Abdeen et. al 2016; suramin and compound 2h-p from Abdeen et. al 2016; compounds 20R, 20L, and 28R from Abdeen et. al 23. All IC50 results reported are averages of values determined from individual dose-response curves in assay replicates as follows: 1) Individual IC50 values from assay replicates were first log-transformed and the average log(IC50) values and standard deviations (SD) calculated; 2) Replicate log(IC50) values were evaluated for outliers using the ROUT method in GraphPad Prism (Q of 10%); and 3) Average IC50 values were then back- calculated from the average log(IC50) values. For compounds where log(IC50) values were greater than the maximum compound concentrations tested (i.e. >1.6, >1.8, >2.0, and >2.4 – or >42, >63, >100, and >250 μM, respectively), results were represented as 0.1 log units higher than the maximum concentrations tested (i.e.1.7, 1.9, 2.1, and 2.5 – or 53, 79, 126, and 316 μM, respectively) so as not to overly bias comparisons because of the unavailability of definitive values for these inactive compounds.

Claims

WHAT IS CLAIMED IS: 1. A compound of the formula (IA) or (IIA)
Figure imgf000055_0001
or a pharmaceutically acceptable salt thereof, wherein X is O or S; R1 is halo, C1-C8 alkyl, C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, - C(O)RA, -ORA,-SRA or -NHRA, or one or more R1 in combination with the atoms to which each is attached combine to form a C9-C14 aryl or C9-C12 bicyclic heteroaryl, wherein each hydrogen atom in C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, C1-C8 alkyl, -C(O)C2-C6 alkenylene-phenyl, C9-C12 aryl and C9-C12 bicyclic heteroaryl is optionally substituted by halo, cyano, or -NO2; R2 is C6-C10 aryl or 5- to 12-membered heteroaryl, wherein each hydrogen atom in C6- C10 aryl and 5- to 12-membered heteroaryl is optionally substituted by halo, hydroxy, or -NO2; R11 is nitro or N(H)COR3; R12 is H or C(O)C1-C6 alkyl; R13 is H or C1-C6 alkyl; R14 is H, C6-C10 aryl, or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C6-C10 aryl and 5 to 12-membered heteroaryl is optionally substituted by halo, 5 to 12- membered heteroaryl, -NHC(O)C1-C6 alkyl, C1-C6 alkoxy, -C(O)C1-C6 alkoxy, -RA, -ORA, - SRA, -C(O)RA or -NHRA; each RA is individually C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C1-C6 alkoxy, or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C1-C6 alkoxy, or 5 to 12- membered heteroaryl is optionally substituted by nitro or halo; or two RA combine to form C4- C6 cycloalkyl optionally substituted by phenyl; R3 is C6-C10 aryl, 5 to 12-membered heteroaryl, or C9-C12 bicyclic aryl, wherein each hydrogen atom in C6-C10 aryl, 5 to 12-membered heteroaryl, and C9-C12 bicyclic aryl is optionally substituted by halo, C1-C6 alkyl, or nitro; and n is 0, 1, 2, or 3.
2. A compound of the formula
Figure imgf000056_0001
(I) (II) wherein R1 is H, halo, C6-C10 aryl, C5-C10 heteroaryl, substituted C6-C10 aryl, substituted C5-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein said substituted C6-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R2 is C6-C8 aryl, C5-C8 heteroaryl, substituted C6-C10 aryl, substituted C5-C10 heteroaryl, wherein said substituted C6-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R3 is C6-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl, C9-C12 bicyclic heteroaryl, substituted C6-C8 aryl, substituted C5-C8 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C6-C10 aryl, substituted C5-C10 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; and R15 and R16 are each independently hydrogen or C1-C6 alkyl; or a pharmaceutically acceptable salt thereof.
3. The compound of claim 1 or 2 having the general structure of formula I:
Figure imgf000056_0002
wherein R3 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof.
4. The compound of any one of claims 1-3, having the general structure of formula I:
Figure imgf000057_0001
or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000057_0002
R11 is halo; R12 is -NO2; R16 is C1-C6 alkyl; and R15 is H.
5. The compound of claim 3, wherein
Figure imgf000057_0003
R11 is F; and R16 is -CH2CH3.
6. A compound or a pharmaceutically acceptable salt thereof, having the formula of
Figure imgf000057_0004
wherein R10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo, phenoxy, -NR5R6, -OR5, -S(O)2NR5R6, or –NHSO2R7; R5 and R6 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, bicyclic C6-C10 heteroaryl, substituted C1-C6 alkyl, substituted C6-C10 aryl, or substituted bicyclic C6-C10 heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl and or substituted bicyclic C6-C10 heteroaryl comprise 1 to 5 substituents selected from halo, -NO2 and –OC1-C6 alkyl; and R7 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof
7. The compound of claim 6, wherein R10 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, comprise 1 to 5 substituents selected from halo, phenoxy, -NR5R6, and -OR5; and R5 and R6 are each independently hydrogen or -CH3.
8 The compound of claim 1 or 2, wherein said compound is
Figure imgf000058_0001
Compound 49 Compound 50.
9. A pharmaceutical comprising a compound of any one of claims 1-8 and a pharmaceutically acceptable carrier.
10. A method of inhibiting the GroEL/ES and HSP60/10 chaperonin system in a microorganism said method comprising contacting said microorganism with the pharmaceutical composition of claim 8.
11. A method of inhibiting the growth of Trypanosoma brucei and/or treating a Trypanosoma brucei infection, said method comprising administering compound of
Figure imgf000059_0001
Compound 49 Compound 50. to a subject in need of treatment.
12. A method of inhibiting the growth of Trypanosoma brucei and/or treating a Trypanosoma brucei infection, said method comprising administering a therapeutically effective amount of a compound of any one of claims 1-8 to a subject in need of treatment.
PCT/US2023/076571 2022-10-12 2023-10-11 GroEL INHIBITORS FOR ANTIBIOTIC APPLICATIONS WO2024081714A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263415519P 2022-10-12 2022-10-12
US63/415,519 2022-10-12

Publications (2)

Publication Number Publication Date
WO2024081714A2 true WO2024081714A2 (en) 2024-04-18
WO2024081714A3 WO2024081714A3 (en) 2024-07-18

Family

ID=90670319

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/076571 WO2024081714A2 (en) 2022-10-12 2023-10-11 GroEL INHIBITORS FOR ANTIBIOTIC APPLICATIONS

Country Status (1)

Country Link
WO (1) WO2024081714A2 (en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2016261223A1 (en) * 2015-05-08 2018-01-04 Oklahoma Medical Research Foundation Nitrofuran derivatives that induce apoptosis in breast cancer cells by activating protein expression

Also Published As

Publication number Publication date
WO2024081714A3 (en) 2024-07-18

Similar Documents

Publication Publication Date Title
FI56531C (en) FRAMEWORK FOR THE PROTECTION OF ISOTIOCYANOBENSTIAZOLER WITH ANTI-MICROBICID NETWORK
US8859798B2 (en) Isothiocyanate compounds, pharmaceutical compositions, and uses thereof
US20190389819A1 (en) Processes for preparing oxathiazin-like compounds
CN101959429A (en) Agonists for antimicrobial peptide systems
JPS62161728A (en) Antibacterial
BR112015000128A2 (en) Internal parasitism insect control agent
JP2008545788A (en) Of parasites in animals by N-[(phenyloxy) phenyl] -1,1,1-trifluoromethanesulfonamide and N-[(phenylsulfanyl) phenyl)]-1,1,1-trifluoromethanesulfonamide derivatives control
LT3918B (en) 2,4- and 2,5- substituted pyridin-n-oxides, methods for the production thereof, pharmaceutical compositions and preparation the same
RU2413513C2 (en) Anthelminthic composition of quaternary phosphonium salt and substituted dinitrobenzofuroxane
RU2019132396A (en) MITORIBOSCINS: THERAPEUTIC AGENTS BASED ON MITOCHONDRIA TARGETED TO CANCER CELLS, BACTERIA AND PATHOGENIC YEAST
KR102679975B1 (en) Compounds for reducing lipotoxic damage
Fan et al. Nosiheptide analogues as potential antibacterial agents via dehydroalanine region modifications: semi-synthesis, antimicrobial activity and molecular docking study
WO2023272571A1 (en) Medical use of 2,3-epoxy succinyl derivative
WO2024081714A2 (en) GroEL INHIBITORS FOR ANTIBIOTIC APPLICATIONS
GB2132617A (en) Propynlaminothiazole derivatives
US4021571A (en) Substituted phenylisothioureas and processes for their preparation and use
EP4186503A1 (en) Anti-inflammatory composition comprising benzofuran-based n-acylhydrazone derivatives
Cai et al. Design and synthesis of novel anti-multidrug-resistant staphylococcus aureus derivatives of glycyrrhetinic acid by blocking arginine biosynthesis, metabolic and H2S biogenesis
US20050143346A1 (en) Composition for treating cancer containing n,n-dimethylphytosphingosine
DE69519581T2 (en) GUANIDYLMETHYL CYCLOHEXANCARBONIC ACID ESTER DERIVATIVES
Melville The chemistry of biotin
CN112920168B (en) New application of talpimod and derivative or pharmaceutically acceptable salt thereof and mycobacterium tuberculosis inhibitor
Ghochikyan et al. Study of the biological properties of semi-and thiosemicarbazones of carbonyl derivatives of 4-butanolides
RU2294324C1 (en) N-chloroacetyl-5-bromoanthranilic acid sodium salt eliciting hemostatic effect
US7547713B2 (en) O-pyridinequinone derivatives, the composition containing the derivatives, the process for preparation of the derivatives and the use of the derivatives

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23878182

Country of ref document: EP

Kind code of ref document: A2