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WO2016007837A1 - Inhibiteurs de la tolérance aux antibiotiques à base d'hétéroaryle bicyclique à liaison carbonyle - Google Patents

Inhibiteurs de la tolérance aux antibiotiques à base d'hétéroaryle bicyclique à liaison carbonyle Download PDF

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Publication number
WO2016007837A1
WO2016007837A1 PCT/US2015/039911 US2015039911W WO2016007837A1 WO 2016007837 A1 WO2016007837 A1 WO 2016007837A1 US 2015039911 W US2015039911 W US 2015039911W WO 2016007837 A1 WO2016007837 A1 WO 2016007837A1
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Prior art keywords
heterocycle
carbocycle
yloxy
alkyl
pyridin
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Robert Zahler
Xinxing Tang
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Spero Therapeutics Inc
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Spero Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

Definitions

  • the present disclosure includes carbonyl linked bicyclic heteroaryl compounds and related compounds and methods of using such compounds.
  • the disclosure provides methods of using the compounds in treating acute and chronic bacterial infections.
  • MvfR The ligand activated transcriptional regulator, plays a central role in controlling the pathology of acute bacterial infections, and the shift of Gram-negative bacteria from acute to chronic infection. MvfR inhibitors reduce virulence of Pseudomonas aeruginosa, a Gram-negative bacterial species, and reduce the formation of antibiotic resistant Pseudomonas strains in vitro.
  • this disclosure includes compounds of Formula (I) and the pharmaceutically acceptable salts, hydrates, solvates and prodrugs thereof.
  • Q is selected from:
  • Each of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 , Y 8 , and Y 9 is independently selected from N, CH, and
  • Each of Y 10 and Y 11 is independently selected from N and C.
  • At least one of Y 1 , Y 2 , Y 3 , or Y 4 is C(R 2 ).
  • At least one of Y 6 , Y 7 , Y 8 , or Y 9 is C(R 2 ).
  • W is C and, when X is a bond or -C(R la )(R lb )-, W is additionally selected from N.
  • Z is selected from S, O, N, N(H) and NCd-Cs-alkyl), wherein the alkyl portion of Z, if present, is optionally substituted.
  • Each " represents independently a single or a double bond, wherein at least one of the bonds between Y 10 and Y 5 or W and Y 5 is a double bond.
  • X is selected from a bond, -S-, -0-, -N(R 6 )-, and -C(R la )(R lb )-.
  • Each R la and each R lb is independently selected from hydrogen, halogen, -OH, -NH 2 , -CN, -Ci-C6-alkyl, -C 2 -C6 -alkenyl, -C 2 -C6-alkynyl, -(Ci-C 4 -alkylene)-(heterocycle), -(C 2 -C -alkenylene)- (heterocycle), -(C 2 -C 4 -alkynylene)-heterocycle, -(Ci-C 4 -alkylene)-(carbocycle), -(C 2 -C -alkenylene)- (carbocycle), and -(C 2 -C -alkynylene)-(carbocycle), or any R la and R lb bound to the same carbon atom are optionally taken together with the carbon atom to form an optionally substituted carbocycle or heterocycle.
  • Each R 2 is independently selected from halogen, -OH, -NH 2 , -CN, -Ci-C 6 -alkyl, -C 2 -C 6 - alkenyl, -C 2 -C6-alkynyl, -(C 0 -C -alkylene)-(heterocycle), -(C 2 -C -alkenylene) -(heterocycle), -(C 2 -C - alkynylene) -heterocycle), -(C 0 -C -alkylene)-(carbocycle), -(C 2 -C -alkenylene)-(carbocycle), and -(C 2 -C - alkynylene) -(carbocycle), or any two R 2 are optionally taken together with the carbon atoms to which they are bound to form an optionally substituted carbocycle or heterocycle;
  • R 3 is selected from hydrogen, -Ci-C 6 -alkyl, -C 2 -C 6 -alkenyl, -C 2 -C 6 -alkynyl, -(C 0 -C 4 - alkylene) -(heterocycle), -(C 2 -C 4 -alkenylene) -(heterocycle), -(C 2 -C 4 -alkynylene)-heterocycle), -(C 0 -C 4 - alkylene) -(carbocycle), -(C 2 -C 4 -alkenylene)-(carbocycle), and -(C 2 -C 4 -alkynylene)-(carbocycle).
  • R 4 is selected from hydrogen, -CN, OH, NH 2 , C0 2 H, -C(0)NH 2 , -d-Cs-alkyl, -C 2 -C 6 - alkenyl, -C 2 -C6-alkynyl, -(C 0 -C 4 -alkylene)-(heterocycle), -(C 2 -C 4 -alkenylene)-(heterocycle), -(C 2 -C - alkynylene)-heterocycle), -(C 0 -C 4 -alkylene)-(carbocycle), -(C 2 -C -alkenylene)-(carbocycle), and -(C 2 -C - alkynylene) -(carbocycle) .
  • R 5 is selected from hydrogen, -CN, -OH, -NH 2 , -C(0)NH 2 , -d-C 6 -alkyl, -C 2 -C 6 -alkenyl, -(C 0 -C 4 -alkylene)-(heterocycle), -(C 2 -C 4 -alkenylene)-(heterocycle), -(C 0 -C 4 -alkylene)-(carbocycle), and - (C 2 -C 4 -alkenylene)-(carbocycle).
  • Each R 6 is independently selected from hydrogen, Ci-C 3 -alkyl, and Ci-C 3 -fluoroalkyl.
  • Each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, heterocycle or carbocycle portion of any R la , R lb , R 2 , R 3 , R 4 and R 5 is optionally and independently substituted.
  • the disclosure further includes a method of treating a bacterial infection in a subject, comprising administering a therapeutically effective amount of a compound, salt, solvate, or hydrate of Formula (I) or a prodrug thereof to a subject in need of such treatment.
  • the compound of Formulae (I) may be administered as the only active agent or may be administered together with one or more additional active agents.
  • Form (I) encompasses all compounds that satisfy Formula (I), including any enantiomers, racemates and stereoisomers, as well as all pharmaceutically acceptable salts, solvates, and hydrates of such compounds.
  • “Formula (I)” includes all subgeneric groups of Formula (I) unless clearly contraindicated by the context in which this phrase is used.
  • Compounds of Formula (I) include all compounds of Formula (I) having isotopic substitutions at any position.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium and isotopes of carbon include n C, 13 C, and 14 C.
  • An "active agent” means a compound (including a compound disclosed herein), element, or mixture that when administered to a subject, alone or in combination with another compound, element, or mixture, confers, directly or indirectly, a physiological effect on the subject.
  • the indirect physiological effect may occur via a metabolite or other indirect mechanism.
  • a dash (“-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent.
  • -C(0)NH 2 is attached through carbon of the keto C(O) group.
  • An "aliphatic group” is a hydrocarbon group having the indicated number of carbon atoms in which the carbon atoms are covalently bound in single, double or triple covalent bonds in straight chains, branched chains, or non-aromatic rings. Aliphatic groups may be substituted.
  • Alkyl is a branched or straight chain saturated aliphatic hydrocarbon group, having the specified number of carbon atoms, generally from 1 to about 8 carbon atoms.
  • the term Ci-C 6 -alkyl as used herein indicates an alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms.
  • Other embodiments include alkyl groups having from 1 to 6 carbon atoms, 1 to 4 carbon atoms or 1 or 2 carbon atoms, e.g. Ci-C 8 -alkyl, Ci-C 4 -alkyl, and Ci-C 2 -alkyl.
  • alkyl examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl, and sec-pentyl.
  • alkenyl is a branched or straight chain aliphatic hydrocarbon group having one or more double carbon-carbon bonds that may occur at any stable point along the chain, having the specified number of carbon atoms.
  • alkenyl include, but are not limited to, ethenyl and propenyl.
  • alkynyl is a branched or straight chain aliphatic hydrocarbon group having one or more triple carbon-carbon bonds that may occur at any stable point along the chain, having the specified number of carbon atoms.
  • alkynyl include, but are not limited to, ethynyl and propynyl.
  • Alkoxy is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes by an oxygen bridge (-0-).
  • alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3- pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3- methylpentoxy.
  • Alkylthio indicates an alkyl group as defined above attached through a sulfur linkage, i.e. a group of the formula alkyl-S-. Examples include ethylthio and pentylthio.
  • Alkanoyl is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes through a carbonyl C(O) bridge.
  • the carbonyl carbon is included in the number of carbons, that is C 2 alkanoyl is a CH 3 C(0)- group.
  • Alkylester is an alkyl group as defined herein covalently bound to the group it substitutes by an ester linkage.
  • the ester linkage may be in either orientation, e.g., a group of the formula -OC(0)-alkyl or a group of the formula -C(0)0-alkyl.
  • Aryl indicates aromatic groups containing only carbon in the aromatic ring or rings. Typical aryl groups contain 1 to 3 separate, fused, or pendant rings and from 6 to about 18 ring atoms, without heteroatoms as ring members. When indicated, such aryl groups may be further substituted with carbon or non-carbon atoms or groups.
  • Aryl groups include, for example, phenyl, naphthyl, including 1- naphthyl, 2-naphthyl, and bi-phenyl.
  • a "carbocycle” is a monocyclic or bicyclic saturated, partially unsaturated, or aromatic ring system in which all ring atoms are carbon. Usually each ring of the carbocycle contains from 4-6 ring atoms and a bicyclic carbocycle contains from 7 to 10 ring atoms, but some other number of ring atoms may be specified. Unless otherwise indicated, the carbocycle may be attached to the group it substitutes at any carbon atom that results in a stable structure. When indicated the carbocyclic rings described herein may be substituted at any carbon atom if the resulting compound is stable. Examples of carbocycles include phenyl, naphthyl, tetrahydronaphthyl, cyclopropyl, cyclohexyl, and cyclohexenyl.
  • Cycloalkyl is a saturated hydrocarbon ring group, having the specified number of carbon atoms.
  • Monocyclic cycloalkyl groups typically have from 3 to about 8 carbon ring atoms or from 3 to 6 (3, 4, 5, or 6) carbon ring atoms.
  • Cycloalkyl substituents may be pendant from a substituted nitrogen, oxygen, or carbon atom, or a substituted carbon atom that may have two substituents may have a cycloalkyl group, which is attached as a spiro group.
  • Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • Halo or “halogen” indicates any of fluoro, chloro, bromo, and iodo.
  • Haloalkyl indicates both branched and straight-chain alkyl groups having the specified number of carbon atoms, substituted with 1 or more halogen atoms, up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and penta-fluoroethyl.
  • Haloalkoxy indicates a haloalkyl group as defined herein attached through an oxygen bridge (oxygen of an alcohol radical).
  • heterocycle indicates a monocyclic saturated, partially unsaturated, or aromatic ring containing from 1 to 4 heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a bicyclic saturated, partially unsaturated, or aromatic heterocycle containing at least 1 heteroatom chosen from N, O, and S in one of the two rings of the two ring system and containing up to about 4 heteroatoms independently chosen from N, O, and S in each ring of the two ring system.
  • each ring of the heterocycle contains from 4-6 ring atoms but some other number of ring atoms may be specified.
  • the heterocycle may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.
  • the heterocycles described herein may be substituted on carbon, sulfur, or nitrogen atom if the resulting compound is stable. It is preferred that the total number of heteroatoms in a heterocycle is not more than 4 and that the total number of S and O atoms in a heterocycle is not more than 2, more preferably not more than 1.
  • heterocycles include, pyridyl, indolyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl,
  • benz[b]thiophenyl isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl, dihydroisoindolyl, 5,6,7, 8-tetrahydroisoquinoline, pyrazolyl, pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl.
  • a heterocycle is chosen from pyridinyl, pyrimidinyl, furanyl, thienyl, and pyrrolyl.
  • heterocycles include, but are not limited to, phthalazinyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzoisoxolyl,
  • Heteroaryl is a stable monocyclic aromatic ring having the indicated number of ring atoms which contains from 1 to 4, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5- to 7-membered aromatic ring which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon.
  • Monocyclic heteroaryl groups typically have from 5 to 7 ring atoms.
  • bicyclic heteroaryl groups are 9- to 10-membered heteroaryl groups, that is, groups containing 9 or 10 ring atoms in which one 5- to
  • 7-member aromatic ring is fused to a second aromatic or non-aromatic ring.
  • the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heteroaryl group is not more than 2. It is particularly preferred that the total number of S and O atoms in the aromatic heterocycle is not more than 1.
  • heteroaryl groups include, but are not limited to, oxazolyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, thiazolyl, thienylpyrazolyl, thiophenyl, triazolyl, benzo[i ]oxazolyl, benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxadiazolyl, dihydrobenzodioxynyl, furanyl, imidazolyl, indolyl, and isoxazolyl.
  • mono- and/ or di-alkylamino indicates secondary or tertiary alkyl amino groups, wherein the alkyl groups are independently chosen alkyl groups, as defined herein, having the indicated number of carbon atoms. The point of attachment of the alkylamino group is on the nitrogen. Examples of mono- and di-alkylamino groups include ethylamino, dimethylamino, and
  • substituted means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded.
  • an oxo group substitutes aromatic moieties, the corresponding partially unsaturated ring replaces the aromatic ring.
  • a pyridyl group substituted by oxo is a pyridone.
  • a stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture and subsequent formulation into an effective therapeutic agent.
  • substituents are named into the core structure. For example, it is to be understood that when aminoalkyl is means the point of attachment of this substituent to the core structure is in the alkyl portion and when alkylamino means the point of attachment is a bond to the nitrogen of the amino group.
  • Suitable groups that may be present on a "substituted" or “optionally substituted” position include, but are not limited to, e.g., halogen; cyano; -OH; -NH 2 ; nitro; azido; alkanoyl (such as a C 2 -C 6 alkanoyl group); C(0)NH 2 ; alkyl groups (including cycloalkyl groups) having 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 8, or 2 to about 6 carbon atoms; alkoxy groups having one or more oxygen linkages and from 1 to about 8, or from 1 to about 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those having one or more thioether linkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms; alkylsulfiny
  • "optionally substituted” includes one or more substituents independently chosen from halogen, hydroxyl, amino, cyano, -CHO, -C0 2 H, -C(0)NH 2 , Ci-C 6 -alkyl, C 2 -C 6 -alkenyl, Ci-C 6 -alkoxy, C 2 -C 6 -alkanoyl, Ci-C 6 -alkylester, (mono- and di-Ci-C 6 -alkylamino)C 0 -C 2 - alkyl, Ci-C 2 -haloalkyl, and Ci-C 2 haloalkoxy.
  • a "dosage form” means a unit of administration of an active agent.
  • dosage forms include tablets, capsules, injections, suspensions, liquids, emulsions, creams, ointments, suppositories, inhalable forms, transdermal forms, and the like.
  • compositions are compositions comprising at least one active agent, such as a compound or salt, solvate, or hydrate of Formula (I) or a prodrug thereof, and at least one other substance, such as a carrier.
  • Pharmaceutical compositions optional contain one or more additional active agents.
  • pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturing practice) standards for human or non-human drugs.
  • “Pharmaceutical combinations” are combinations of at least two active agents which may be combined in a single dosage form or provided together in separate dosage forms with instructions that the active agents are to be used together to treat a disorder, such as a Gram-negative bacterial infection.
  • “Pharmaceutically acceptable salts” includes derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof.
  • the salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two.
  • salts of the present compounds further include solvates of the compounds and of the compound salts.
  • Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, H0 2 C-(CH 2 ) n -C0 2 H where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in Remington's Pharmaceutical Sciences
  • carrier applied to pharmaceutical compositions/combinations of the disclosure refers to a diluent, excipient, or vehicle with which an active compound is provided.
  • a "pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition/ combination that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable excipient” as used in the present application includes both one and more than one such excipient.
  • a "subject” is a human or non-human animal in need of medical treatment. Medical treatment can include treatment of an existing condition, such as a disease or disorder, prophylactic or preventative treatment, or diagnostic treatment. In some embodiments the subject is a human patient.
  • Providing means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.
  • Treatment includes providing a compound of this disclosure such as a compound of any of Formulae (I), either as the only active agent or together with at least one additional active agent sufficient to: (a) inhibiting the disease, i.e. arresting its development; and (b) relieving the disease, i.e., causing regression of the disease and in the case of a bacterial infection to eliminate the infection in the subject.
  • Treating and “treatment” also means providing a therapeutically effective amount of a compound of the disclosure as the only active agent or together with at least one additional active agent to a subject having or susceptible to a bacterial infection.
  • prolactic treatment includes administering an amount of a compound of the disclosure sufficient to significantly reduce the likelihood of a disease from occurring in a subject who may be predisposed to the disease but who does not have it.
  • a "therapeutically effective amount" of a pharmaceutical composition/ combination is an amount effective, when administered to a subject, to provide a therapeutic benefit such as an amelioration of symptoms, e.g., an amount effective to decrease the symptoms of a bacterial infection and/ or effect a cure. In certain circumstances a subject suffering from a microbial infection may not present symptoms of being infected. Thus a therapeutically effective amount of a compound is also an amount sufficient to significantly reduce the detectable level of microorganism in the subject's blood, serum, other bodily fluids, or tissues.
  • the disclosure also includes, in certain embodiments, using compounds of the disclosure in prophylactic treatment and therapeutic treatment.
  • a "therapeutically effective amount” is an amount sufficient to significantly decrease the treated subject's risk of contracting a bacterial infection.
  • prophylactic treatment may be administered when a subject will knowingly be exposed to infectious microbes.
  • a significant reduction is any detectable negative change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p ⁇ 0.05.
  • prodrug refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Nature Reviews Drug Discovery 2008, 7, 255; Current Topics in Medicinal Chemistry 2011, 11, 2265; and Molecules 2008, 13, 519).
  • a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as Ci-C 8 -alkyl, (C 2 -Ci 2 alkyl)carbonyloxymethyl, l-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1 -methyl- 1- (alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1 -(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-l- (alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, l-(N-(alkyl), (C 2 -Ci 2 alkyl)carbonyloxymethyl, l-(alkylcarbonyloxy)ethyl
  • a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (Ci-C 6 -alkyl)carbonyloxymethyl, l-((Ci-C 6 -alkyl)carbonyloxy)ethyl, l-methyl-l-((Ci-C 6 - alkyl)carbonyloxy)ethyl, (Ci-C6-alkoxy)carbonyloxymethyl, N-(Ci-C6-alkoxy)carbonylaminomethyl, succinoyl, (d-Cs-alkyftcarbonyl, -P(0)(OH) 2 , -P(0)(OH)(0-C !
  • glycosyl the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate
  • alpha-aminoacyl the alpha-aminoacyl is derived from the natural L-aminoacids.
  • a prodrug can be formed, for example, by creation of an amide or carbamate, an as (C 1 -C6- alkyl)carbonyloxymethylcarbonyl derivative, a l-((Ci-C6-alkyl)carbonyloxyethylcarbonyl) derivative, an (oxodioxolenyl)methyl derivative, a N-Mannich base, imine or enamine.
  • a secondary amine can be metabolically cleaved to generate a bioactive primary amine, or a tertiary amine can metabolically cleaved to generate a bioactive primary or secondary amine.
  • Formulae (I) include all subformulae thereof.
  • the compounds of any of Formulae (I) may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.
  • These compounds can be, for example, racemates or optically active forms.
  • these compounds can additionally be mixtures of diastereomers.
  • compounds having asymmetric centers it should be understood that all of the optical isomers and mixtures thereof are encompassed.
  • compounds with carbon-carbon double bonds may occur in Z- and E-forms, with all isomeric forms of the compounds being included in the present disclosure.
  • single enantiomers i.e., optically active forms, can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates.
  • Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example using a chiral HPLC column.
  • Y 2 and Y 4 are independently selected from CH and C(R 2 );
  • Y 3 is selected from N, CH and C(R 2 );
  • Y 5 is selected from N and C(R 2 );
  • Y 2 is selected from CH, C(halo) and C(CN);
  • Y 3 is selected from N, CH and C(halo);
  • Y 4 is selected from CH, C(d-C 4 alkyl) and C(C 3 -C 6 cycloalkyl);
  • Y 5 is selected from N, C(CN), C(C ! -C 4 alkyl) and C(C 3 -C 6 cycloalkyl), wherein any alkyl or cycloalkyl in Y 4 or Y 5 is optionally and independently substituted with halo or hydroxyl.
  • Y 2 is selected from CH, C(C1), C(F), and C(CN);
  • Y 3 is selected from N, CH and C(C1);
  • Y 4 is selected from CH, C(CH(CH 3 ) 2 OH) and C(l-hydroxycycloprop-l-yl);
  • Y 5 is selected from N, C(CN), C(CH(CH 3 ) 2 OH) and C(l-hydroxycycloprop-l-yl).
  • X is selected from -S-, -NH- and -CH 2 .
  • Each R la is independently selected from hydrogen and hydroxyl; and each R lb is hydrogen.
  • Each of Y 6 , Y 7 , Y 8 , and and Y 9 is independently selected from CH and C(R 2 ).
  • Y 8 is a carbon atom substituted with a substituent selected from halo, -R 6 -heteroaryl, and -R 6 -aryl, wherein R 6 is selected from -0-, -CH 2 - and -CF 2 -; and the heteroaryl or aryl moiety in Y 8 is optionally substituted with one or more substituents independently selected from halo, cyano and Ci-C 4 alkyl.
  • the heteroaryl or aryl is a phenyl, pyridine, pyrimidine, or pyrazine, each of which may be optionally substituted, for example with one or more halo, cyano and C 1 -C4 alkyl.
  • Y 8 is a carbon atom substituted with a substituent selected from chloro, pyridin-3- yloxy, pyridin-3-ylmethyl, difluoro(pyridin-3-yl)methyl, pyrimidin-2-yloxy, pyrazin-2-yloxy, and phenoxy, wherein any ring moiety in a substituent on Y 8 is optionally substituted with one or two substituents independently selected from fluoro, chloro, cyano, and methyl.
  • Each R 3 if present, is independently selected from hydrogen, methyl, ethyl and -CH 2 -C(CH 3 ) 2 OH.
  • R 4 if present, is hydrogen.
  • Q is selected from 6-chloro-lH-indazol-3-yl, 6-chloro-l-methylindazol-3-yl, 6- chloro-lH-indol-3-yl, 6-chloro-l-methylindol-3-yl, 6-(pyridin-3-yloxy)-lH-pyrrolo[2,3-b]pyridin-3-yl, 6- (pyridin-3-ylmethyl)-lH-pyrrolo[2,3-b]pyridin-3-yl, 6-(pyridin-3-yloxy)imidazo[l,5-a]pyridin-l-yl, 7- (pyridin-3-yloxy)imidazo[l,2-a]pyridin-3-yl), 3-(pyridin-3-yloxy)imidazo[l,5-a]pyrimidin-8-yl, 6- (difluoro(pyridin-3 -yl)methyl)- 1 H
  • W is C and, when X is a bond or -C(R la )(R lb )- and Y 5 is CH or C(R 2 ), W is additionally selected from N.
  • X is CH 2 and R la and R lb are both hydrogen.
  • (xvii) is S and R la and R lb are both hydrogen.
  • the disclosure includes any and all combinations of conditions (i) to (xvii) that result in a stable compound of Formula (I). In any of the conditions (i) to (xvii) where a variable of Formula (I) is selected from a list of moieties, the disclosure also includes individual and subsets of moieties from that list in both that condition and in combination with any and all combinations of the other of conditions (i) to (xiii).
  • the disclosure also includes combination comprising individual or subsets of moieties from one of conditions (i) to (xvii) combined with individual or subsets of moieties from one or more other of conditions (i) to (xvii) and optionally in combination with additional conditions (i) to (xvii).
  • the disclosure includes a compound wherein X is -S- (an individual moiety in condition (v)); Y 8 is a carbon atom substituted with a substituent selected from pyridin-3-yloxy, pyridin-3-ylmethyl and pyrimidin-2-yloxy, wherein any ring moiety in a substituent on Y 8 is optionally substituted with one or two substituents independently selected from fluoro, chloro, cyano, and methyl (a subset of the moieties set forth in condition (x)); and each R 3 , if present, is independently selected from hydrogen, methyl, ethyl and -CH 2 -C(CH 3 ) 2 OH (the entirety of condition (xi)).
  • Formula I includes the following subformulas in which Q, R la , R lb , and R 2 carry any of the defi
  • the disclosure includes prodrugs of Formula I.
  • the disclosure includes prodrugs of the following formulas:
  • each of the variables e.g. Y ⁇ Y 11 , R la , R lb , and R 5 may carry any of the definitions set forth in this disclosure.
  • L 1 is a bond or a Ci-C 6 -alkyl, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl linker, and one or more methylene units in the alkyl, alkenyl, or alkynyl portion are optionally and independently replaced with O , S, N(R 6 ) , S(O) or S(0) 2 ;
  • L 2 is a Ci-C6-alkyl, C 2 -C 6 alkenyl, or C 2 -Cealkynyl linker, and one or more methylene units in the alkyl, alkenyl, or alkynyl portion are optionally and independently replaced with O , S, N(R 6 ) , S(O) or S(0) 2 ; and
  • PD is a water-solublizing prodrug well known in the art such as but not limited to P(0)(OH) 2 or an alpha-aminoacyl, wherein the alpha-aminoacyl is derived from the natural L-aminoacids.
  • the disclosure includes a method of treating a bacterial infection in a subject by administering an effective amount of one or more compounds of the disclosure to a subject at risk for a bacterial infection or suffering from a microorganism infection.
  • Treatment of human patients is particularly contemplated. However, treatment of non-human subjects is within the scope of the disclosure.
  • the disclosure includes treatment or prevention of microbial infections in fish, amphibians, reptiles or birds, but a preferred embodiment of the disclosure includes treating mammals.
  • the bacterial infection or antibiotic-tolerant infection is caused by a Gram-negative bacterium.
  • the microbial infection is the result of a pathogenic bacterial infection.
  • pathogenic bacteria include, without limitation, bacteria within the genera Aerobacter, Aeromonas, Acinetobacter, Agrobacterium, Bacillus, Bacteroides, Bartonella, Bordetella, Brucella, Burkholderia Calymmatobacterium, Campylobacter, Citrobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Hafnia, Helicobacter, Klebsiella, Legionella, Listeria, Morganella, Moraxella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Treponema, Xanthomonas, Vibrio, and Yersinia.
  • Such bacteria include Vibrio harveyi, Vibrio cholerae, Vibrio parahemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum, Pseudomonas aeruginosa, Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni, Mycobacterium tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus epidermidis, and Staphylococcus aureus
  • the gram-negative bacterium is a Pseudomonas, e.g., P.
  • the gram-negative bacterium is Burkholderia species.
  • the gram-negative bacterium is Acinetobacter, e.g., A. baumannii.
  • the gram-negative bacterium is an Enterobacteriaceae, e.g., Klebsiella pneumonia, e.g., Escherichia coli, e.g., Enterobacter cloacae, e.g., Serratia marcescens, e.g., Salmonella typhimurium, e.g., Shigella dysenteriae, e.g., Proteus mirabilis, e.g., Citrobacter freundii, e.g., Yersinia pestis.
  • Enterobacteriaceae e.g., Klebsiella pneumonia, e.g., Escherichia coli, e.g., Enterobacter cloacae, e.g., Serratia marcescens, e.g., Salmonella typhimurium, e.g., Shigella dysenteriae, e.g., Proteus mirabilis,
  • the infection is a polymicrobial infection, e.g., an infection comprising more than one organism.
  • the infection comprises at least one of the organisms listed above, e.g., one or more oi Pseudomonas, e.g., P aeruginosa, Kelbsiella, e.g., Klebsiella pneumoniae, and/or Acinetobacter, e.g., A. baumannii.
  • the methods further include administering an additional active agent in combination with a compound of the disclosure, such as an antibiotic selected from the group consisting of: beta-lactams such as penicillins, cephalosporins, carbacephems, cephamycins,
  • an antibiotic selected from the group consisting of: beta-lactams such as penicillins, cephalosporins, carbacephems, cephamycins,
  • carbapenems monobactams, quinolones including fluoroquinolones and similar DNA synthesis inhibitors, tetracyclines, aminoglycosides, macrolides, glycopeptides, chloramphenicols, glycylcyclines, lincosamides, lipopeptides, and oxazolidinones.
  • the bacterial infection is an upper respiratory tract infection, pneumonia, a systemic infection, sepsis and septic shock, a urinary tract infection, a gastrointestinal infection, endocarditis, a bone infection, central nervous system infections such as meningitis, or an infection of the skin and soft tissue.
  • the subject is a mammal, e.g., a human or non-human mammal.
  • the methods include treating one or more cells, e.g., cells in a culture dish.
  • the present disclosure features a method of treating a Gram-negative infection in a subject, the method comprising administering to said subject in need of such treatment a therapeutically effective amount of a compound described herein.
  • the Gram-negative infection is caused by Pseudomonas aeruginosa.
  • the disclosure includes treating an infection caused by Gram positive bacteria, such as Staphylococcus epidermidis and Staphylococcus aureus.
  • the subject is a trauma patient or a burn patient suffering from a burn or skin wound.
  • the present disclosure features a method of reducing bacterial tolerance in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound described herein.
  • the method further includes identifying said subject suffering from an infection with bacteria tolerant to antimicrobial therapy.
  • the disclosure includes methods of treatment in which a compound or composition of the disclosure is administered orally, topically, parenterally, or inhaled.
  • a compound of the disclosure may be administered about 1 to about 5 times per day. Daily administration or post-periodic dosing may be employed. Frequency of dosage may also vary depending on the compound used, the particular disease treated and the bacteria causing the disease. It will be understood, however, that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • the disclosure also includes methods of treating bacterial infection in plants and fungal crops (e.g. mushrooms) comprising contacting a compound of the Formula I with a plant or fungal organism.
  • fungal crops e.g. mushrooms
  • the plant may be an agricultural crop plant, such as a tobacco plant or a tomato plant .
  • Pseudomonas species also can be involved in agricultural damage.
  • Some of Pseudomonas syringae's numerous pathovars can be plant pathogens.
  • P. syringae pathovar tabacii, phaseolicola, and tomato are pathogenic to plants.
  • tolaasii are pathogens of cultivated mushrooms.
  • the compounds of this disclosure can are useful for reducing or eliminating the virulence of such Pseudomonal pathovars to plant and fungal crops.
  • the disclosure includes the process for making a pharmaceutical composition containing at least one compound of Formula I.
  • An embodiment comprises mixing one or more of the present compounds and an optional pharmaceutically acceptable carrier; and includes those compositions resulting from such a process, which process includes conventional pharmaceutical techniques.
  • compositions of the disclosure include ocular, oral, nasal, transdermal, topical with or without occlusion, intravenous (both bolus and infusion), inhalable, and injection (intraperitoneally, subcutaneously, intramuscularly, intratumorally, or parenterally) formulations.
  • the composition may be in a dosage unit such as a tablet, pill, capsule, powder, granule, liposome, ion exchange resin, sterile ocular solution, or ocular delivery device (such as a contact lens and the like facilitating immediate release, timed release, or sustained release), parenteral solution or suspension, metered aerosol or liquid spray, drop, ampoule, auto-injector device, or suppository; for administration ocularly, orally, intranasally, sublingually, parenterally, or rectally, or by inhalation or insufflation.
  • a dosage unit such as a tablet, pill, capsule, powder, granule, liposome, ion exchange resin, sterile ocular solution, or ocular delivery device (such as a contact lens and the like facilitating immediate release, timed release, or sustained release), parenteral solution or suspension, metered aerosol or liquid spray, drop, ampoule, auto-injector device, or suppository; for administration
  • the dosage form containing the composition of the disclosure contains an effective amount of the active ingredient necessary to provide a therapeutic effect by the chosen route of administration.
  • the composition may contain from about 5,000 mg to about 0.5 mg (preferably, from about 1 ,000 mg to about 0,5 mg) of a compound of the disclosure or salt form thereof and may be constituted into any form suitable for the selected mode of administration.
  • the pharmaceutical composition may be a parenteral formulation suitable for parenteral administration via injection or infusion.
  • a parenteral formulation may consist of the active ingredient dissolved in or mixed with an appropriate inert liquid carrier.
  • Acceptable liquid carriers usually comprise aqueous solvents and other optional ingredients for aiding solubility or preservation.
  • aqueous solvents include sterile water, Ringer's solution, or an isotonic aqueous saline solution.
  • Other optional ingredients include vegetable oils (such as peanut oil, cottonseed oil, and sesame oil), and organic solvents (such as solketal, glycerol, and formyl).
  • a sterile, non-volatile oil may be employed as a solvent or suspending agent.
  • the parenteral formulation is prepared by dissolving or suspending the active ingredient in the liquid carrier whereby the final dosage unit contains from 0.005 to 10% by weight of the active ingredient.
  • Other additives include preservatives, isotonizers, solubilizers, stabilizers, and smoothing agents.
  • injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.
  • the pharmaceutical composition of this disclosure may be a composition formulated for administration directly to the lungs by inhalation, such as an aerosol formulation.
  • aerosol drug delivery systems include, for example, a unit dose dry-powder inhaler, a dry powder pulmonary device, a pressurized metered dose inhaler, a metered-dose inhaler, a nebulizer, and the like.
  • compositions of this disclosure may be formulated for oral administration.
  • Compositions of the disclosure suitable for oral administration include solid forms such as pills, tablets, caplets, capsules (each including immediate release, timed release, and sustained release formulations), granules and powders; and, liquid forms such as solutions, syrups, elixirs, emulsions, and suspensions.
  • compositions of this disclosure may be formulated for topical administration, including topical administration to the eye.
  • topical administration including topical administration to the eye.
  • the composition is preferably in the form of an ophthalmic composition.
  • the ophthalmic compositions are preferably formulated as eye -drop formulations and filled in appropriate containers to facilitate administration to the eye, for example a dropper fitted with a suitable pipette.
  • MvfR is a LysR-type transcriptional regulator that directs 4-hydroxy-2-alkylquinolines (HAQs) synthesis, including that of its ligands,
  • HHQ 4-hydroxy-2-heptylquinoline
  • PQS 3,4-dihydroxy-2-heptylquinoline
  • MvfR promotes the production of HAQs by binding to and activating the pqs operon, which encodes enzymes for HAQ synthesis.
  • Anthranilic acid (AA) derived from the phnAB, kynABU, and trpEG pathways, is the precursor for HAQs.
  • Pqs A encodes an anthranilate -coenzyme A ligase, which activates anthranilic acid and catalyzes the first committed step to HAQ production.
  • PqsB and PqsC are unknown, though both show homology to acyl-carrier-proteins and both are required for HHQ and PQS production.
  • PqsD is a condensing enzyme that along with PqsA has been shown to be necessary and sufficient for the production of 2,4-dihydroxyquinoline (DHQ), a molecule whose biological role has yet to be determined.
  • DHQ 2,4-dihydroxyquinoline
  • PqsE encodes for a putative hydrolase, and while the protein is not required for the synthesis of HAQs, it is necessary for pyocyanin production.
  • Step A 6-Chloro-lH-indazole.
  • DMA dimethyl methacrylate
  • N 2 H 4 .H 2 0 25 mL, 99% wt.
  • the mixture was placed in a sealed tube and stirred at r.t. for 1 hr, then heated to 100 °C and stirred at that temperature for 48 hr.
  • the resulting mixture was cooled to r.t., poured into ice water, and extracted with EtOAc.
  • Step B 6-Chloro-3-iodo-lH-indazole.
  • I 2 10 g, 40 mmol
  • KOH well-powdered KOH
  • the reaction mixture was stirred at r.t. for 3 hr, then poured into a pre-cooled solution of aq.Na 2 S 2 0 3 and extracted with EtOAc.
  • the combined organic layers were washed with aq. LiCl (10%wt), dried over anhydrous Na 2 S0 4 , and concentrated under reduced pressure.
  • Step C Methyl 6-chloro-lH-indazole-3-carboxylate.
  • Step D 6-Chloro-lH-indazole-3-carboxylic acid.
  • MeOH 90 mL
  • THF 30 mL
  • NaOH 2.4 g, 60 mmol
  • Step E 6-Chloro-N-methoxy-N-methyl-lH-indazole-3-carboxamide.
  • 6- chloro-lH-indazole-3-carboxylic acid 1 g, 5 mmol
  • THF 50 mL
  • ⁇ , ⁇ - dimethylhydroxylamine hydrochloride 0.58 g, 6 mmol
  • pyridine 2.1 mL, 26 mmol
  • EDCI 1.73 g, 9 mmol
  • Step F l-(6-chloro-lH-indazol-3-yl)ethanone.
  • a mixture of the above crude 6- chloro-N-methoxy-N-methyl-lH-indazole-3-carboxamide (1.2 g, 5 mmol) in dry THF (60 mL) at -5 °C was added dropwise MeMgBr (7 mL, 21 mmol, 3 M in THF) over a period of 10 min. After addition was complete, the mixture was stirred at 0°C for 2 hr, thenslowly poured into a pre-cooled solution of NH 4 C1 with vigorous stirring and extracted with EtOAc. The combined organic layers were dried over anhydrous Na 2 S0 4 and concentrated under reduced pressure. The residue was purified by flash column
  • Step G 2-bromo-l-(6-chloro-lH-indazol-3-yl)ethanone.
  • l-(6-chloro- lH-indazol-3-yl)ethanone 100 mg, 0.5 mmol
  • EtOAc 5 mL
  • CuBr 2 279 mg, 1.25 mmol
  • the reaction mixture was stirred at reflux for 2 hr, then cooled to r.t. and filtered through Celite. The filtrate was washed with a solution of aq.
  • Step H 2-(5-Chloro-lH-benzo[d]imidazol-2-ylthio)-l-(6-chloro-lH-indazol-3- yljethanone.
  • 2-bromo-l-(6-chloro-lH-indazol-3-yl)ethanone 70 mg, 0.25 mmol
  • DMF 3 mL
  • 5-chloro-lH-benzo[d]imidazole-2-thiol 70 mg, 0.375 mmol
  • K 2 C0 3 138 mg, 1 mmol
  • Step C 2-(2-(6-chloro-lH-indol-3-yl)-2-oxoethylthio)-lH-benzo[d]imidazole-5- carbonitrile.
  • 2-mercapto-lH-benzo[d]imidazole-5-carbonitrile 88 mg, 0.5 mmol
  • DMF 8 mL
  • K 2 C0 3 138 mg, 1 mmol
  • 2-chloro-l-(6-chloro-lH-indol-3-yl)ethanone 114 mg, 0.5 mmol.
  • the reaction mixture was stirred at r.t. for 1 hr, then quenched with aq.
  • Step A 6-Chloro-lH-indazole.
  • DMA dimethyl methacrylate
  • N 2 H 4 .H 2 0 25 mL, 99 wt
  • the reaction mixture was stirred at r.t. for 1 hr then at 100 °C for 48 hr.
  • the resulting mixture was cooled to r.t., poured into ice water and extracted with EtOAc.
  • the combined organic layers were washed with brine, dried over anhydrous Na 2 S0 4 and concentrated under reduced pressure.
  • the residue was purified by flash column chromatography to give 6-chloro-lH-indazole (3.2 g, 42% yield) as pale yellow solid.
  • LC-MS m/z 153 (M+H) + .
  • Step B 6-Chloro-3-iodo-lH-indazole.
  • I 2 10 g, 40 mmol
  • KOH powdered KOH
  • the reaction mixture was stirred at r.t for 3 hr, poured into a cooled solution of aq. Na 2 S 2 0 3 and extracted with EtOAc.
  • the combined organic layers were washed with aq. LiCl (10%wt), and concentrated under reduced pressure.
  • Step C Methyl 6-chloro-lH-indazole-3-carboxylate.
  • Step E 6-Chloro-N-methoxy-N-methyl-lH-indazole-3-carboxamide.
  • 6- chloro-lH-indazole-3-carboxylic acid 1 g, 5 mmol
  • THF 50 mL
  • ⁇ , ⁇ - dimethylhydroxylamine hydrochloride 0.58 g, 6 mmol
  • pyridine 2.1 mL, 26 mmol
  • EDCI 1.73 g, 9 mmol
  • Step F l-(6-Chloro-lH-indazol-3-yl)ethanone.
  • a solution of the above crude 6- chloro-N-methoxy-N-methyl-lH-indazole-3-carboxamide (1.2 g, 5 mmol) in dry THF (60 mL) at -5 °C was added dropwise MeMgBr (7 mL, 21 mmol, 3 M in THF) over 10 min. After addition, the mixture was stirred at 0°C for 2 hr, then slowly poured into an ice -cooled solution of aq. NH 4 C1 with vigorous stirring. The resulting mixture was extracted with EtOAc, and the combined organic layers were concentrated under reduced pressure.
  • Step G l-(6-Chloro-l-methyl-lH-indazol-3-yl)ethanone.
  • l-(6-chloro- lH-indazol-3-yl)ethanone 390 mg, 2 mmol
  • DMF 5 mL
  • K 2 C0 3 552 mg, 4 mmol
  • Mel 312 mg, 2.2 mmol
  • the resulting mixture was stirred at r.t for 4 hr, then poured into ice- water with vigorous stirring and extracted with EtOAc. The combined organic layers were washed with aq. LiCl (10%wt) and concentrated under reduced pressure.
  • Step H 2-bromo-l-(6-chloro-l-methyl-lH-indazol-3-yl)ethanone.
  • l-(6- chloro-l-methyl-lH-indazol-3-yl)ethanone 104 mg, 0.5 mmol
  • EtOAc 5 mL
  • CuBr 2 245 mg, 1.1 mmol
  • the reaction mixture was stirred at reflux for 2 hr, then cooled to r.t. and filtered through Celite. The filtrate was washed with a solution of aq.
  • Step I l-(6-Chloro-l-methyl-lH-indazol-3-yl)-2-(5-chloro-lH-benzo[d]imidazol-2- ytihiojethanone (Compound 5)
  • DMF dimethyl methyl
  • 5-chloro-lH-benzo[d]imidazole- 2-thiol 110 mg, 0.6 mmol
  • K 2 C0 3 138 mg, 1 mmol
  • Step A 5-(Pyridin-3-yloxy)picolinonitrile.
  • a mixture of 5-fluoropicolinonitrile (1.22g, 10 mmol) and pyridin-3-ol (0.95 g, 10 mmol) in DMF (20 mL) was added K 2 C0 3 (2.07 g, 15 mmol).
  • the reaction mixture was stirred at r.t. for 2 hr, then quenched with water (10 mL) and extracted with EtOAc. The combined organic layers were dried over anhydrous Na 2 S0 4 and concentrated under reduced pressure.
  • the residue was purified by flash column chromatography to give 5-(pyridin-3- yloxy)picolinonitrile (1.6 g, 84% yield) as light yellow oil.
  • LC-MS m/z 198(M+H) + .
  • Step B (5-(Pyridin-3-yloxy)pyridin-2-yl)methanamine.
  • Step C N-((5-(pyridin-3-yloxy)pyridin-2-yl)methyl)formamide.
  • Amixture of (5-(pyridin- 3-yloxy)pyridin-2-yl)methanamine (600 mg, 3 mmol) in formic acid (6 mL) was heated at 110 °C for 12 hr, then cooled to 0 °C, quenched with aq. NH 4 OH (25%wt) and extracted with DCM. The combined organic layers were dried over anhydrousNa 2 S0 4 and concentrated under reduced pressure.
  • Step D.6-(Pyridin-3-yloxy)imidazo[l,5-a]pyridine To a mixture of N-((5-(pyridin-3- yloxy)pyridin-2-yl)methyl)formamide (460 mg, 2 mmol) in toluene (10 mL) at 0 °C was added POCl 3 (612 mg,4 mmol). The reaction mixture was stirred at 100 °C for 12 hr, then cooled to 0 °C, quenched with aq. NH 4 OH (25%wt) to pH9 and extracted with DCM. The combined organic layers were dried over anhydrous Na 2 S0 and concentrated under reduced pressure.
  • Step E 2-chloro-l-(6-(pyridin-3-yloxy)imidazo[l,5-a]pyridin-l-yl)ethanone.
  • 6-(pyridin-3-yloxy)imidazo[l,5-a]pyridine 210 mg, 1 mmol
  • A1C1 3 801 mg, 6 mmol
  • chloroacetyl chloride 678 mg, 6 mmol
  • the reaction mixture was stirred at 60 °C for 2 hr, then cooled to 0 °C, quenched with aq.NaOAc (1 N) to pH 9, and extracted with EtOAc.
  • Step F 2-(5-chloro-lh-benzo[d]imMazol-2-ylthio)-l-(6-(pyridin-3-yloxy)imidazo[l,5- a]pyridin-l-yl)ethanone.
  • a mixture of 2-chloro-l-(6-(pyridin-3-yloxy)imidazo[l,5-a]pyridin-l- yl)ethanone (57 mg, 0.2 mmol), 5-chloro-lH-benzo[d]imidazole-2-thiol (37 mg, 0.2 mmol), and potassium carbonate (55 mg, 0.4 mmol) in DMF was stirred at r.t.
  • Step A 2-Fluoro-4-(pyridin-3-yloxy)benzaldehyde.
  • DMSO DMSO
  • K 2 C0 3 3.3 g, 24 mmol
  • pyridin-3-ol 1.9 g, 20 mmol.
  • the reaction mixture was stirred at r.t. overnight, then slowly poured into ice -water with vigorous stirring and extracted with EtOAc. The combined organic layers were washed with brine twice, dried over anhydrous Na 2 S0 4 and concentrated under reduced pressure.
  • Step B 6-(Pyridin-3-yloxy)-lH-indazole.
  • 2-fluoro-4-(pyridin-3- yloxy)benzaldehyde (2.17 g, 10 mmol) in dioxane (5 mL) in a sealed tube at 10 °C was added dropwise N 2 H 4 .H 2 0 (5 mL, 99%wt).
  • N 2 H 4 .H 2 0 5 mL, 99%wt.
  • the reaction mixture was stirred at r.t. for 0.5 hr, then stirred 120 °C for 48 hr.
  • the resulting mixture was cooled to r.t. and concentrated under reduced pressure. The residue was partitioned between water and EtOAc.
  • Step D Methyl 6-(pyridin-3-yloxy)-lH-indazole-3-carboxylate.
  • Step E 6-(Pyridin-3-yloxy)-lH-indazole-3-carboxylic acid.
  • MeOH methyl 6- (pyridin-3-yloxy)-lH-indazole-3-carboxylate (540 mg, 2 mmol) in MeOH (30 mL) at r.t.
  • THF 10 mL
  • NaOH 400 mg, 10 mmol
  • H 2 0 10 mL
  • the reaction mixture was stirred at reflux for 5 hr, then cooled to r.t. and concentrated under reduced pressure to a volume of about 10 mL.
  • the resulting mixture was acidified to pH 5-6 with cone. HC1 at 0 °C.
  • Step F N-methoxy-N-methyl-6-(pyridin-3-yloxy)-lH-indazole-3-carboxamide.
  • 6-(pyridin-3-yloxy)-lH-indazole-3-carboxylic acid 500 mg, 2 mmol
  • THF 30 mL
  • N,0-dimethylhydroxylamine hydrochloride 233 mg, 2.4 mmol
  • pyridine 821 mg, 10.4 mmol
  • EDCI 691 mg, 3.6 mmol
  • Step H 2-Bromo-l-(6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone hydrobromide.
  • l-(6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone 50 mg, 0.2 mmol
  • a solution of Br 2 42 mg, 0.26 mmol
  • aq. HBr 1 mL, 48 wt.
  • Step I 2-((5-chloro-lH-benzo[d]imidazol-2-yl)thio)-l-(6-(pyrMin-3-yloxy)-lH-indazol- 3-yl)ethanone.
  • 2-bromo-l-(6-(pyridin-3-yloxy)-lH-indazol-3- yl)ethanone hydrobromide salt 66 mg, 0.2 mmol
  • DMF 4-mL
  • 5-chloro-lH- benzo[d]imidazole-2-thiol 37 mg, 0.2 mmol
  • K 2 C0 3 (276 mg, 2 mmol
  • Step A l-(l-Methyl-6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone.
  • Step B 2-Bromo-l-(l-methyl-6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone hydrobromide.
  • l-(l-methyl-6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone 54 mg, 0.2 mmol
  • a solution of Br 2 42 mg, 0.26 mmol
  • aq. HBr (1 mL, 48%).
  • the reaction mixture was stirred at r.t.
  • Step C 2-(5-Chloro-lH-benzo[d]imidazol-2-ylthio)-l-(l-methyl-6- ⁇ yridin-3-yloxy)- lH-indazol-3-yl)ethanone .
  • Step A 2-Chloro-l-(6-(pyridin-3-yloxy)-lH-indol-3-yl)ethanone.
  • 6-(pyridin-3-yloxy)-lH-indole 200 mg, 0.95 mmol
  • A1C1 3 126 mg, 0.95 mmol
  • 2-chloroacetyl chloride 0.06 mL, 0.76 mmol
  • Step B 2-(5-Chloro-lH-benzo[d]imidazol-2-ylthio)-l-(6-(pyridin-3-yloxy)-lH-indol-3- yljethanone.
  • 2-chloro-l-(6-(pyridin-3-yloxy)-lH-indol-3-yl)ethanone 45 mg, 0.16 mmol
  • DMF 3 mL
  • 5-chloro-lH-benzo[d]imidazole-2-thiol 30 mg, 0.16 mmol
  • K 2 C0 3 66 mg, 0.48 mmol
  • Step A Ethyl 4-(l-methyl-6-(pyridin-3-yloxy)-lH-indol-3-yl)-4-oxobutanoate.
  • l-methyl-6-(pyridin-3-yloxy)-lH-indole 300 mg, 1.3 mmol
  • A1C1 3 621 mg, 2.6 mmol
  • ethyl 4-chloro-4- oxobutanoate (0.37 mL, 2.6 mmol) in anhydrous DCM (1 mL).
  • Step B 4-(l-Methyl-6-(pyridin-3-yloxy)-lH-indol-3-yl)-4-oxobutanoic acid.
  • ethyl 4-(l-methyl-6-(pyridin-3-yloxy)-lH-indol-3-yl)-4-oxobutanoate 100 mg, 0.28 mmol
  • THF 2 mL
  • NaOH 34 mg, 0.85 mmol
  • H 2 0 2 mL
  • the reaction mixture was stirred at r.t. for 2 hr, then adjusted to pH 6 with aq. HCl (IN) and extracted with EtOAc.
  • Step C N-(2-amino-4-chlorophenyl)-4-(l-methyl-6-(pyridin-3-yloxy )-lH-indol-3-yl)-4- oxobutanamide and N-(2-amino-5-chlorophenyl)-4-(l-methyl-6-(pyridin-3-yloxy)-lH-indol-3-yl)-4- oxobutanamide.
  • Step D 3-(5-chloro-lH-benzo[d]imidazol-2-yl)-l-(l-methyl-6-(pyridin-3-yloxy)-lH- indol-3-yl)propan-l-one.
  • Step A 4-Chloro-2-(2,5-dimethyl-lH-pyrrol-l-yl)pyridine.
  • 4- chloropyridin-2-amine (2.56 g, 20 mmol) in toluene (50 mL) were added hexane-2,5-dione (2.7 g, 24 mmol) and TPSA (0.1 g).
  • TPSA 0.1 g
  • the reaction mixture was stirred at reflux for 12 hr, then cooled to r.t. and diluted with EtOAc. The resulting mixture was washed with water twice, dried over anhydrous Na 2 S0 4 and concentrated under reduced pressure.
  • Step B 2-(2,5-Dimethyl-lH-pyrrol-l-yl)-4-(pyridin-3-yloxy)pyridine.
  • pyridin-3-ol 1.4 g, 15 mmol
  • t-BuOK 2.2 g, 20 mmol
  • Step C 4-(Pyridin-3-yloxy)pyridin-2-amine.
  • 2-(2,5-dimethyl-lH-pyrrol- l-yl)-4-(pyridin-3-yloxy)pyridine (1.32 g, 5 mmol) at 0 °C in MeOH (50 mL) was added cone.
  • HC1 (10 mL).
  • the resulting solution was stirred reflux for 16 hr, then concentrated under reduced pressure to give crude 4-(pyridin-3-yloxy)pyridin-2-amine HC1 salt (1.2 g, quant, crude yield) as yellow solid which was used directly in the next step without any further purification.
  • LC-MS m/z 188 (M+H) + .
  • Step D 7-(Pyridin-3-yloxy)imidazo[l,2-a]pyridine.
  • DCM dimethylethyl sulfoxide
  • 2-chloroacetaldehyde 2.4 g, 12.3 mmol, 40%wt in water.
  • the reaction was stirred vigorously at r.t. for 16 hr.
  • the organic layer was separated, and the aq. layer was extracted with DCM.
  • the combined organic layers were washed with brine, dried over anhydrous Na 2 S0 4 , and concentrated under reduced pressure.
  • Step A 2-chloro-l-(6-(pyridin-3-yloxy)-lH-indol-3-yl)ethanone.
  • 6- (pyridin-3-yloxy)-lH-indole 200 mg, 0.95 mmol
  • A1C1 3 190 mg, 1.43 mmol
  • 2-chloroacetyl chloride 0.11 mL, 1.43 mmol
  • the mixture was stirred at 0 °C for 3 hr, then quenched with aq.
  • Step B 2-(5-Chloro-7-(2-hydroxypropan-2-yl)-lH-benzo[d]imidazol-2-ylthio)-l-(6- (pyridin-3-yloxy)-lH-indol-3-yl)ethanone.
  • Step A l-ethyl-6-(pyridin-3-yloxy)-lH-indole.
  • Ste/7 B 2-Chloro-l-(l-ethyl-6-(pyridin-3-yloxy)-lH-indol-3-yl)ethanone.
  • l-ethyl-6-(pyridin-3-yloxy)-lH-indole 150 mg, 0.63 mmol
  • A1C1 3 126 mg, 0.95 mmol
  • 2-chloroacetyl chloride 107 mg, 0.95 mmol
  • Step C 2-(5-Chloro-lH-benzo[d]imMazol-2-ylthio)-l-(l-ethyl-6-(pyridin-3-yloxy)-lH- indol-3-yl)ethanone .
  • 2-chloro-l-(l-ethyl-6-(pyridin-3-yloxy)-lH-indol-3-yl)ethanone 40 mg, 0.13 mmol
  • Step A l-(l-Ethyl-6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone.
  • a mixture of 1-(1- ethyl-6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone (260 mg, 1.03 mmol), iodoethane (160 mg, 1.03 mmol), and K 2 C0 3 (284 mg, 2.05 mmol) in DMF (5 mL) was stirred at r.t. for 1 hr, then diluted with EtOAc and washed with aq. LiCl (10 wt).
  • Step B 2-Chloro-l-(l-ethyl-6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone.
  • a mixture of l-(l-ethyl-6-(pyridin-3-yloxy)-lH-indazol-3-yl)ethanone (220 mg, 0.782 mmol), and TEA (103 mg, 1.02 mmol) in DCM (lOmL) was cooled to -5°C, followed by addition of TMSOTf (348 mg, 1.54 mmol). The mixture was stirred at that temperature for 2 hr, followed by addition of NCS (104 mg, 0.782 mmol) in one portion.
  • Step C 2-(6-Chloro-lH-benzo[d]imidazol-2-ylthio)-l-(l-ethyl-6-(pyridin-3-yloxy)- 1H- indazol-3-yl)ethanone .
  • 2-chloro-l-(l-ethyl-6-(pyridin-3-yloxy)-lH-indazol-3- yl)ethanone 45mg, 0.143 mmol
  • 5-chloro-lH-benzo[d]imidazole-2-thiol 26 mg, 0.143 mmol
  • DMF 5 mL
  • K 2 C0 3 48 mg, 0.286 mmol
  • Step A 6-(Pyrazin-2-yloxy)-lH-indole.
  • a mixture of lH-indol-6-ol (300 mg, 2.25 mmol) and 2-chloropyrazine (284 mg, 2.48 mmol) and K 2 C0 3 (1.47 g, 4.51 mmol) in DMF (10 mL) was stirred at 100 °C for 1 hr, then diluted with water and extracted with EtOAc. The combined organic layers were washed with S. aq. LiCl, dried over anhydrous Na 2 S0 4 , and concentrated under reduced pressure.
  • Step B 2-Chloro-l-(6-(pyrazin-2-yloxy)-lH-indol-3-yl)ethanone.
  • a mixture of 6- (pyrazin-2-yloxy)-lH-indole (100 mg, 0.47 mmol) and A1C1 3 (315 mg, 2.37 mmol) in anhydrous DCM (5 mL) was stirred at 0 °C for 10 min, followed by slow addition of 2-chloroacetyl chloride (0.2 mL, 2.37 mmol). The reaction mixture was stirred at r.t. for 3 hr, then poured into ice-water and extracted with DCM.
  • Step A 6-Chloro-3H-imidazo[4,5-c]pyridine-2-thiol.
  • THF tetrahydrofuran
  • di-(lH-imidazol-l- yl)methanethione 534 mg,3 mmol.
  • the reaction mixture was stirred at 80 °C for 4 hr, then diluted with water and extracted with EtOAc. The combined organic layers were dried over anhydrous Na 2 S0 4 and concentrated under reduced pressure. The residue was purified by flash column chromatography to give 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (200 mg, 54 % yield).
  • Step B 2-(6-Chloro-3H midazo[4,5-c]pyrMin-2-ylthio)-l-(5-(pyridin-3-yloxy)-lH- indol-3-yl)ethanone.
  • 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol 184 mg,l mmol
  • DMF DMF
  • K 2 C0 3 276 mg, 2 mmol
  • 2-chloro-l-(5-(pyridin-3-yloxy)-lH-indol-3- yl)ethanone 286 mg, 1 mmol.
  • the reaction mixture was stirred at r.t.
  • Step A Methyl 4-oxo-4-(l-(phenylsulfonyl)-6-(pyridin-3-yloxy)-lH-indol-3-yl) butanoate.
  • l-(phenylsulfonyl)-6-(pyridin-3-yloxy)-lH-indole 455 mg, 1.3 mmol
  • A1C1 3 621 mg, 2.6 mmol
  • a solution of methyl 4-chloro-4-oxobutanoate (0.37 mL, 2.6 mmol) in anhydrous DCM (1 mL).
  • Step B 4-Oxo-4-(6-(pyridin-3-yloxy)-lH-indol-3-yl)butanoic acid.
  • a mixture of methyl 4-oxo-4-(l-(phenylsulfonyl)-6-(pyridin-3-yloxy)-lH-indol-3-yl)butanoate (120 mg, 0.28 mmol) in THF (2 mL) was added a solution of NaOH (34 mg, 0.85 mmol) in H 2 0 (2 mL). The reaction mixture was stirred at r.t. for 2 hr, then adjusted to pH 6 with aq. HCl (IN) and extracted with EtOAc.
  • Step C N-(2-amino-4-chlorophenyl)-4-oxo-4-(6-(pyridin-3-yloxy)-lH-indol-3-yl) butanamide and N-(2-amino-5-chlorophenyl)-4-oxo-4-(6-(pyridin-3-yloxy)-lH-indol-3-yl) butanamide.
  • Step D 3-(5-chloro-lH-benzo[d]imidazol-2-yl)-l-(6-(pyridin-3-yloxy)-lH-indol-3-yl) propan-l-one.
  • a mixture of N-(2-amino-4-chlorophenyl)-4-oxo-4-(6-(pyridin-3- yloxy)-lH-indol-3- yl)butanamide (43 mg, 0.10 mmol) in AcOH (2 mL) was refluxed for 2 hr, then cooled down and concentrated under reduced pressure.
  • Step B (E)-2-(3-nitro-4-(2-(pyrrolidin-l-yl)vinyl)phenoxy)pyrimidine.
  • 2-(4-methyl-3-nitrophenoxy)pyrimidine 3 g, 13 mmol
  • DMF-DMA dimethyl acetal
  • Step C 6-(Pyrimidin-2-yloxy)-lH-indole.
  • EtOAc a mixture of (E)-2-(3-nitro-4-(2- (pyrrolidin-l-yl)vinyl)phenoxy)pyrimidine (4 g) in EtOAc (20 mL) was added Pd/C (10 wt, 0.4 g). The reaction mixture was stirred at r.t. under H 2 atmosphere overnight then filtered. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography
  • Step D 2-Chloro-l-(6-(pyrimidin-2-yloxy)-lH-indol-3-yl)ethanone.
  • 6-(pyrimidin-2-yloxy)-lH-indole 100 mg, 0.474 mmol
  • A1C1 3 189 mg, 1.442 mmol
  • 2-chloroacetyl chloride 160 mg, 1.422 mmol
  • the reaction mixture was stirred at r.t. for 16 hr, then quenched with water and extracted with DCM (20 mL). The organic layer was separated, washed with brine, dried over anhydrous Na 2 S0 4 and concentrated under reduced pressure to give the crude product (160 mg) as yellow solid which was directly used in the next step without any further purification.
  • Step E 2-(5-chloro-lH-benzo[d]imidazol-2-ylthio)-l-(6-(pyrimidin-2-yloxy)-lH -indol- 3-yl)ethanone2.
  • 2-chloro-l-(6-(pyrimidin-2-yloxy)-lH-indol-3-yl)ethanone 60 mg, 0.18 mmol
  • 5-chloro-lH-benzo[d]imidazole-2-thiol 60 mg, 0.33 mmol
  • K 2 C0 3 149 mg, 1.08 mmol
  • Test compounds were dissolved in DMSO at 10 mM and stored at -20 °C until needed. Test concentrations from 0.0032 to 31.6 micromolar (topseal) or 0.001 micromolar to 31.6 micromolar (breathable seal) were used for each compound. Seven test concentrations were used for each compound in the topseal assay and eight test concentrations were used for each compound in the breathable seal assay.
  • CFU Colony Forming Units
  • the topseal assay plate was sealed and incubated at 37°C under shaking (-700 rpm) using a microtiter shaker for 24 hours.
  • the breathable seal assay the plate was sealed with a breathable seal, covered with a lid, and incubated at 37°C under shaking (-700 rpm) using a microtiter shaker for 24 hours.
  • the plate was centrifuged at 4,000g for 40 minutes at room temperature, 100 ⁇ / ⁇ of the supernatant was transferred to a 96-microtiter PS flat-bottom plate, and absorbance at 690 nm was determined using SPECTROstar® Nano microplate reader.
  • the level of pyocyanin in the presence of test compound was expressed as percentage of inhibition with respect to the control. Curve fitting and IC 50 determination were carried out using a four-parameter logistic model using GraphPad Prism v.5.
  • Pyocyanin inhibition data obtained in the topseal assay is provided in Table 1.
  • Pyocyanin inhibition data obtained in the breathable assay is provided in Table 2.
  • Three stars (***) indicates an IC 50 ⁇ 0.1 ⁇
  • two stars (**) indicates 0.1 ⁇ ⁇ IC 50 ⁇ 1.0 ⁇
  • one star (*) indicates 1.0 ⁇ ⁇ IC 50 ⁇ 10.0 ⁇
  • no stars beside a compound number indicates that compound has an IC 50 > 10 ⁇ .
  • Table 1 Topseal data
  • Test compounds were dissolved in DMSO at 10 mM and stored at -20 °C until needed. Seven test concentrations from 0.0032 micromolar to 31.6 micromolar were used for each compound.
  • Colony Forming Units (CFU)/mL of the overnight culture and T 0 culture were determined by a series of 1 :10 serial dilutions in sterile saline. 4x20 ⁇ L of each dilution is seeded on LB agar plates and incubated at 35 ⁇ 2 °C for 20 hours. CFU/mL was determined from the dilution that gives 2 to 50 colonies/ 20 ⁇ .
  • the plate is sealed and incubated at 37°C for 24 hours under shaking (-700 rpm) using a microtiter shaker. At the end of the incubation period, 150 ⁇ ⁇ ⁇ of the deep well plate culture were transferred in a new non-sterile plate, and 150 ⁇ ⁇ ⁇ of methanol containing tetra-deuterated PQS (D4- PQS) and 2% acetic acid were added to each well. The plate was sealed, shaken at high speed for 5 minutes and centrifuged at 4,000g for 40 minutes at room temperature. 100 ⁇ ⁇ ⁇ of the supernatant was then transferred to glass vials (vials crimp 0.2 mL) and kept at 4°C until LC-MS/MS analysis.
  • D4- PQS tetra-deuterated PQS
  • PQSs quantification is carried out by analyzing samples in discrete batches together with spiked standards and blank samples.
  • Calibration curves were constructed from PQS standards, and respective deuterated PQS is used as an Internal Standards (IS) to calculate the concentration of analytes in the sample and improve the precision of the assay.
  • IS Internal Standards
  • the back-calculated concentrations of the calibration standards from the calibration curve shall be within ⁇ 20% of the nominal values, the range of the analytical method is determined, and the lower and upper limit of quantification specified.
  • Liquid chromatography separations were performed using an Agilent HP 1100 system (Agilent Technologies) coupled with a CTC PAL Autosampler (CTC Analytics AG). Chromatographic retention is obtained using a monolithic column (Chromolith® RP-18, 50 x 4.6 mm).
  • the solvent system consists of water containing 0.1 % (v/v) formic acid (A) and acetonitrile containing 0.1 % (v/v) formic acid (B).
  • the gradient elution profile is chosen as follows: 0 min: 70% A (1500 ⁇ / ⁇ ), 0.3 min: 70% A (1500 ⁇ / ⁇ ), 1.3 min: 5% A (2500 ⁇ / ⁇ ), 1.6 min: 5% A (2500 ⁇ , ⁇ ).
  • MS/MS analysis is performed with an API4000 series mass spectrometer (AB SciexTM) operating in Multiple Reaction Monitoring (MRM) mode and equipped with a TIS ion source.
  • the specific ions monitored were PQS (m z 260 -> m z 175) and D4-PQS (m z 264 -> m z 179).
  • the computer systems used in this study to acquire and quantify data may be an AnalystTM vl.5.
  • PQS 24 hour inhibition data is provided in Table 3.
  • IC 50 of less than 10 micromolar in the Pyocyanin inhibition assay were tested in the PQS 24 hour inhibition assay.
  • Three stars (***) indicates an IC 50 ⁇ 1.0 ⁇
  • two stars (**) indicates 1.0 ⁇ ⁇ IC 50 ⁇ 5.0 ⁇
  • one star (*) indicates 5.0 ⁇ ⁇ IC 50 ⁇ 10.0 ⁇
  • no stars beside a compound number indicates that compound has an IC 50 > 10 ⁇ .
  • concentration range with 7 test concentrations was chosen for each test compound.
  • concentration ranges for the PQS and HHQ assays were from 0.03 to 31.6 ⁇ .
  • Compounds were prepared in solvent, usually DMSO, at either 200 or 500 times the final test concentration.
  • a 0.5 mL aliquot of bacterial suspension was transferred from each 15 mL glass tube, after shaking, to a 2 mL polypropylene vial.
  • 0.5 mL of methanol-containing D4-PQS (deuterated-PQS), D4-HHQ (deuterated-HHQ) and 2% acetic acid were added to each 2 mL vial and each vial was vigorously shaken.
  • the vials were then centrifuged for 5 minutes at 12,000g, and 200 ⁇ of supernatant from each vial is transferred to a glass vial (vials crimp 0.2mL), and the sealed vials were kept at 4°C until LC-MS/MS analysis was performed.
  • HAQs quantification is carried out analyzing samples in discrete batches together with spiked standards and blank samples. Calibration curves were constructed from HAQs standards, and respective deuterated forms were used as Internal Standards (IS), to calculate the concentration of analytes in the sample and improve the precision of the assay. The back-calculated concentrations of the calibration standards from the calibration curve are within ⁇ 20% of the nominal values, the range of the analytical method was determined, and the lower and upper limit of quantification specified.
  • Liquid chromatography separations were performed using Agilent HP1100 system (Agilent Technologies) coupled with a CTC PAL Autos ampler (CTC Analytics AG). Chromatographic retention is obtained using a monolithic column (Chromolith® RP-18, 50 x 4.6 mm).
  • the solvent system consists of water containing 0.1 % (v/v) formic acid (A) and acetonitrile containing 0.1 % (v/v) formic acid (B).
  • the gradient elution profile wass chosen as follows: 0 min: 70% A (1500 ⁇ ), 0.3 min: 70% A (1500 ⁇ 7 ⁇ ), 1.3 min: 5% A (2500 ⁇ ), 1.6 min: 5% A (2500 ⁇ 7 ⁇ ).
  • the MS/MS analysis was performed with a API4000 series mass spectrometer (AB SciexTM) operating in Multiple Reaction Monitoring (MRM) mode and equipped with a TIS ion source.
  • the specific ions monitored were PQS (m/z 260 -> m/z 175), D4-PQS (m/z 264 -> m/z 179), HHQ (m/z 244 -> m/z 159) and D4-HHQ (m/z 248 -> m/z 163).
  • PQS and HHQ in presence of different test compound concentrations were expressed as percentage of inhibition of the basal level in control samples.
  • PQS 6 hour inhibition data is provided in Table 4.
  • HHQ 6 hour inhibition data is provided in Table 5.
  • IC 50 of less than 1 micromolar in the Pyocyanin inhibition assay were tested in the PQS and HHQ 6 hour inhibition assays.
  • Three stars (***) indicates an IC 50 ⁇ 1.0 ⁇
  • two stars (**) indicates 1.0 ⁇ ⁇ IC 50 ⁇ 5.0 ⁇
  • one star (*) indicates 5.0 ⁇ ⁇ IC 50 ⁇ 10.0 ⁇
  • no stars beside a compound number indicates that compound has an IC 50 > 10 ⁇ .

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Abstract

L'invention concerne des composés et des compositions pharmaceutiques de ces composés utiles pour traiter des infections bactériennes chroniques et aiguës. Certains de ces composés sont des composés de Formule générale (I) ou un sel pharmaceutiquement acceptable ou un promédicament de ceux-ci; Q, dans la formule, étant choisi parmi a), b), c) et d). Certains composés de cette invention sont des inhibiteurs de MvfR. Les inhibiteurs de MvfR réduisent la formation de souches bactériennes tolérantes aux antibiotiques et sont utilisés pour traiter des infections bactériennes Gram négatif et réduire la virulence de Pseudomonas aeruginosa. L'invention concerne également des méthodes de traitement d'infections bactériennes chez un sujet, notamment les infections par Pseudomonas aeruginosa .
PCT/US2015/039911 2014-07-11 2015-07-10 Inhibiteurs de la tolérance aux antibiotiques à base d'hétéroaryle bicyclique à liaison carbonyle Ceased WO2016007837A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020007938A1 (fr) 2018-07-03 2020-01-09 Helmholtz-Zentrum für Infektionsforschung GmbH Agonistes inverses de pqsr
WO2021136803A1 (fr) 2020-01-02 2021-07-08 Helmholtz-Zentrum für Infektionsforschung GmbH Nouveaux agonistes inverses de pqsr
WO2021136805A1 (fr) 2020-01-02 2021-07-08 Helmholtz-Zentrum für Infektionsforschung GmbH Nouveaux agonistes inverses de pqsr
US11827627B2 (en) 2021-06-04 2023-11-28 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels
US11834441B2 (en) 2019-12-06 2023-12-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4918079A (en) * 1987-10-02 1990-04-17 Beecham Group P.L.C. Imidazole derivatives as 5-HT3 receptor antagonists
US20120101096A1 (en) * 2009-03-20 2012-04-26 University of Georgia Foundation, Inc. Compounds and Methods for Treating Mammalian Gastrointestinal Microbial Infections
US20140066454A1 (en) * 2011-02-22 2014-03-06 Institut National De La Recherche Scientifique Antibiotic Tolerance Inhibitors
WO2014176258A1 (fr) * 2013-04-23 2014-10-30 The General Hospital Corporation Composés utilisés en tant qu'inhibiteurs de la tolérance aux antibiotiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4918079A (en) * 1987-10-02 1990-04-17 Beecham Group P.L.C. Imidazole derivatives as 5-HT3 receptor antagonists
US20120101096A1 (en) * 2009-03-20 2012-04-26 University of Georgia Foundation, Inc. Compounds and Methods for Treating Mammalian Gastrointestinal Microbial Infections
US20140066454A1 (en) * 2011-02-22 2014-03-06 Institut National De La Recherche Scientifique Antibiotic Tolerance Inhibitors
WO2014176258A1 (fr) * 2013-04-23 2014-10-30 The General Hospital Corporation Composés utilisés en tant qu'inhibiteurs de la tolérance aux antibiotiques

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020007938A1 (fr) 2018-07-03 2020-01-09 Helmholtz-Zentrum für Infektionsforschung GmbH Agonistes inverses de pqsr
US11883387B2 (en) 2018-07-03 2024-01-30 Helmholtz-Zentrum für Infektionsforschung GmbH PqsR inverse agonists
US11834441B2 (en) 2019-12-06 2023-12-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US11919887B2 (en) 2019-12-06 2024-03-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US12247021B2 (en) 2019-12-06 2025-03-11 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
WO2021136803A1 (fr) 2020-01-02 2021-07-08 Helmholtz-Zentrum für Infektionsforschung GmbH Nouveaux agonistes inverses de pqsr
WO2021136805A1 (fr) 2020-01-02 2021-07-08 Helmholtz-Zentrum für Infektionsforschung GmbH Nouveaux agonistes inverses de pqsr
US12528783B2 (en) 2020-01-02 2026-01-20 Helmholtz-Zentrum Fur Infektionsforschung Gmbh PqsR inverse agonists
US11827627B2 (en) 2021-06-04 2023-11-28 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels
US12258333B2 (en) 2021-06-04 2025-03-25 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels

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