WO2008094507A2 - Novel fusion compounds - Google Patents

Novel fusion compounds Download PDF

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Publication number
WO2008094507A2
WO2008094507A2 PCT/US2008/001092 US2008001092W WO2008094507A2 WO 2008094507 A2 WO2008094507 A2 WO 2008094507A2 US 2008001092 W US2008001092 W US 2008001092W WO 2008094507 A2 WO2008094507 A2 WO 2008094507A2
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WO
WIPO (PCT)
Prior art keywords
compound
reca
sulfur
nitrogen
oxygen
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PCT/US2008/001092
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French (fr)
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WO2008094507A3 (en
Inventor
Tsvetelina Lazarova
Michael Thormann
Michael Almstetter
Guillaume Cottarel
Steven Wayne Riesinger
Original Assignee
Cellicon Biotechnologies, Inc.
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Publication of WO2008094507A2 publication Critical patent/WO2008094507A2/en
Publication of WO2008094507A3 publication Critical patent/WO2008094507A3/en

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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
    • 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
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D499/21—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring with a nitrogen atom directly attached in position 6 and a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
    • C07D499/44—Compounds with an amino radical acylated by carboxylic acids, attached in position 6
    • C07D499/46—Compounds with an amino radical acylated by carboxylic acids, attached in position 6 with acyclic hydrocarbon radicals or such radicals substituted by carbocyclic or heterocyclic rings, attached to the carboxamido radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals

Definitions

  • the present invention relates to novel compounds derived from compounds having antibiotic activities.
  • the invention further provides compositions comprising a compound of this invention and the use of such compositions in methods of treating diseases and conditions beneficially treated by anti-infective agents, particularly those relating to the management of bacterial infection.
  • the present invention provides compounds, compositions comprising the compounds, and methods of treating and preventing disease by administration of the compounds and compositions thereof.
  • the invention also relates to methods of making the compounds/compositions of the invention as well.
  • the invention provides a compound that comprises a recA inhibitor compound bonded to a second therapeutic agent, particularly a compound having antibiotic activity.
  • the second therapeutic agent having antibiotic activity can be, for example, any compound with antibiotic activity, including those delineated herein (e.g., any antibiotic, a second recA inhibitor).
  • the compound is that wherein the RecA inhibitor and the second compound are bonded via a linker moiety; wherein the linker moiety comprises between 2-15 atoms selected from carbon, nitrogen or sulfur; wherein the second compound is a quinolone antibiotic; or wherein the second compound is a macrocyclic antibiotic.
  • the invention relates to a compound of Formula Al : R a '-L-A bl (Al) wherein,
  • R a ⁇ is a RecA inhibitor compound
  • L is a linker
  • a bl is an antibiotic compound; or acid addition salt, solvate, hydrate or polymorph thereof.
  • antibiotic agent refers to an agent that inhibitis and/or stops growth an/or proliferation of one or more species of microorganism (e.g., bacteria or fungus).
  • An antibiotic agent may display activity in vitro (e.g., when contacted with cells in culture), in vivo (e.g., when administered to a subject at risk of or suffering from an infection), or both.
  • antibiotic agent may be used to refer to bactericidal agents (i.e., agents that kill bacteria) and/or bacteriostatic agents (i.e., agents that inhibit or stop bacterial growth or proliferation but does not kill the cells).
  • bactericidal agents i.e., agents that kill bacteria
  • bacteriostatic agents i.e., agents that inhibit or stop bacterial growth or proliferation but does not kill the cells.
  • the invention contemplates bactericidal antibiotics and bacteriostatic antibiotics. Bactericidal antibiotics kill bacteria; bacteriostatic antibiotics slow the growth or reproduction of bacteria.
  • aryl used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members.
  • aryl may be used interchangeably with the term “aryl ring”.
  • the aryl may be optionally substituted as delineated herein.
  • disease or “disorder” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ; including those delineated herein.
  • “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • an "effective amount" of a biologically and/or pharmacologically active agent is an amount sufficient to achieve a desired biological and/or pharmacological effect when delivered to a cell or organism according to a selected administration form, route, and/or schedule.
  • the absolute amount of a particular agent that is effective may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the target tissue, etc.
  • an "effective amount” may be administered in a single dose, or may be achieved by administration of multiple doses.
  • an effective amount of an antibiotic agent may be an amount sufficient to achieve one or more of the following: (i) inhibit microbial growth in culture or in vivo; (ii) reduce the severity of or prevent one or more symptoms or signs of an infection; (iii) significantly reduce the risk of recurrence of an infection; (iv) significantly reduce the risk of a clinically significant infection in a subject who has been exposed to an infectious agent, etc.
  • an effective amount of a potentiating agent may be an amount sufficient to achieve the same level of antibiotic activity with a particular antibiotic agent as is achieved when that antibiotic agent is administered at its conventional dose, in circumstances where the antibiotic agent is administered at a reduced dose as compared with its conventional dose.
  • infection refers to the invasion of a host, whether the host is a vertebrate, invertebrate, fish, plant, bird, or mammal, by pathogenic microbes, e.g., bacteria.
  • pathogenic microbes e.g., bacteria.
  • the term encompasses excessive growth of microbes that are normally present in or on the body of a mammal or other organism. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host organism.
  • an organism is "suffering" from a microbial infection when excessive numbers of a microbial population are present in or on the organism's body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of an organism.
  • the agents and compositions of certain embodiments of the invention are also useful in treating microbial growth or contamination of cell cultures or other media, or inanimate surfaces or objects, and nothing herein should limit the invention to treatment of higher organisms, except when explicitly so specified in the claims.
  • agent or entity is “isolated” if it is separated from at least some materials or components with which it is associated in nature or when initially generated. In general, such separation involves activity of the hand of man. In aspects, the chemical compounds delineated herein are isolated.
  • linker or “L” refers to a divalent chemical moiety containing atoms selected from carbon, nitrogen or sulfur.
  • the linker contains between 2- 15 atoms, or in another aspect 2-10 atoms, each independently selected from carbon, nitrogen or sulfur atoms; in another aspect the linker is a divalent alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, wherein any carbon atom is replaced with a heteroatom selected from N, O, S, or P, and any atom is optionally substituted with H, alkyl, oxo, hydroxyl, alkoxy, thioalkoxy, NH 2 , NRR, S(O), S(O)2, C(O)OR, OC(O)OR, OC(O)NR, RNC(O)NR or C(O)NRR, wherein R is H, optionally substituted alkyl (e.g., alkyl substituted with R*, R 0 , etc.), or any "R" group defined herein (e.g., R 3 , R*, R 0 , etc.);
  • Linkers utilized in the invention include cleavable and non-cleavable linker.
  • Cleavable linkers may be cleaved by enzymes in a subject.
  • Typical cleavable linkers include, but are not limited to, esters, amides, phosphonates, sulfonates, carbon esters, phosphorus esters, and sulfur esters.
  • MIC minimum inhibitory concentration
  • MIC values may be for example, the concentration of agent that inhibits visible growth or may be expressed as MIC 5 O, MIC 90 or MIC 99 values i.e., the concentration of an agent that reduces bacterial proliferation to 50% or less, 10% or less, or 1% or less, respectively, of the control value that would occur in the absence of the agent.
  • MIC can be measured by a variety of methods, including automated and non-automated methods. Suitable methods are described in publications of the Clinical Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), as set forth in NCCLS: Performance Standards documents.
  • potentiate means to enhance or increase at least one biological effect or activity of a biologically and/or pharmacologically active agent so that either (i) a given concentration or amount of the agent results in a greater biological effect or activity when the agent is potentiated than the biological effect or activity that would result from the same concentration or amount of the agent when not potentiated; or (ii) a lower concentration or amount of the agent is required to achieve a particular biological effect or activity when the agent is potentiated than when the agent is not potentiated; or (iii) both (i) and (ii).
  • the biological effect or activity may be, for example, the ability to catalyze or inhibit one or more chemical reactions, the ability to activate or inhibit a biological or biochemical pathway, the ability to reduce or inhibit microbial proliferation, the ability to kill a microorganism, etc.
  • An agent whose presence potentiates another agent may be referred to as a "potentiating agent".
  • proliferation refers to an increase in microbial number. Since bacterial proliferation, rather than mere increase in cell mass without cell division, is usually of primary concern, and since under most circumstances of interest herein proliferation is accompanied by an increase in microbial biomass, the term “growth” is generally understood to mean “proliferation”, and the two terms are used interchangeably herein although it is recognized that different assays may measure either or both of these parameters. For example, optical density reflects biomass and does not specifically reflect cell number, whereas an assay based on detecting colonies formed from individual cells reflects cell number rather than biomass.
  • purified refers to agents or entities that have been separated from most of the components with which they are associated in nature or when originally generated. In general, such purification involves action of the hand of man. Purified agents or entities may be partially purified, substantially purified, or pure. Such agents or entities may be, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% pure; or X% pure wherein X is any number between 0-100%, inclusive.
  • quinolone antibiotic means an agent containing a quinolone or a naphthyridine nucleus with any of a variety of different side chains and substituents as known and understood in the art and that displays inhibitory activity towards one or more microbial species, e.g., various quinolone carboxylic acids.
  • quinolone antibiotic encompasses isolated enantiomers, salts, hydrates, solvates, or the free base form of any quinolone antibiotic.
  • an agent is a RecA inhibitor if one or more RecA activities is reduced in the agent's presence as compared with its absence, or if the level or amount of RecA protein or gene product is reduced in the agent's presence as compared with its absence.
  • RecA inhibitors act directly on RecA in that they physically interact with RecA. In other embodiments, inhibitors act indirectly on RecA.
  • agents that inhibit one or more activities of RecA, and/or that inhibit RecA expression levels may be useful in accordance with the present invention.
  • Exemplary RecA activities that may be inhibited include, but are not limited to, DNA binding, monomer interaction, helicase activity, filament formation, ATP binding and/or hydrolysis, co-protease activity (e.g., toward LexA and/or UmuD), recombinase activity, and replication function.
  • inventive RecA inhibitors inhibit one or more such activities with an IC 50 below about 100 ⁇ g /ml, 50 ⁇ g /ml, 15 ⁇ g /ml; 10 ⁇ g /ml; 5 ⁇ g /ml, 3 ⁇ g /ml, or 1 ⁇ g /ml, or below X ⁇ g /ml, wherein X is a number between 0 and 100, inclusive.
  • desirable RecA ATPase inhibitors may even have an ICs 0 well below 1 ⁇ g/ml, or even below 500 ng/ml, 100 ng/ml, 50 ng/ml, 30 ng/ml,. 25 ng/ml, 20 ng/ml, 15 ng/ml, 10 ng/ml, 5 ng/ml, 1 ng/ml, or less.
  • RecA inhibitors are broad spectrum in that they inhibit RecA (or the relevant RecA homolog) from more than one different microbial source. In some embodiments, however, the RecA inhibitors (which may be broad spectrum with regard to microbes) do not inhibit RecA (or the relevant RecA homolog) from one or more higher organisms (e.g., mammals, humans). For example, in some embodiments, inventive RecA inhibitors do not inhibit RAD51.
  • the present invention provides RecA inhibitors that inhibit the RecA ATPase activity. For example, the present invention demonstrates that a variety of compounds inhibit RecA ATPase activity in an in vitro luciferase assay (see e.g., Examples herein). The present invention provides the compounds (including portions thereof) depicted herein as RecA inhibitors with the indicated IC 5 oS in the in vitro luciferase assay.
  • inventive RecA inhibitors that bind directly to RecA bind to a site comprised of amino acid residues including R85, F270, Y271 , K310, and/or R324. According to the present invention, this site can be found on the outer surface of RecA, as positioned in a filament. According to the present invention, hinokiflavone may bind to this RecA site. Further according to the present invention, compounds that compete with hinokiflavone for binding to RecA may be desirable RecA inhibitors., inventive RecA inhibitors do not bind to the RecA ATP binding site. In certain embodiments, inventive RecA inhibitors bind two or more different sites on the RecA protein.
  • RecA inhibitors are small molecule agents, typically having some cyclic character (e.g., including one or more aryl rings). Certain RecA inhibitors according to the present invention are flavones or isoflavones; certain RecA inhibitors according to the present invention are bisflavones. In some embodiments of the invention, the RecA inhibitor is or includes hinokiflavone.
  • a small molecule is understood in the art to be an organic molecule that is less than about kilodaltons (Kd) in size. In some embodiments, the small molecule is less than about 3 Kd, 2 Kd, or 1 Kd. In some embodiments, the small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, small molecules are non-polymeric. In some embodiments, small molecules are not amino acids. In some embodiments, small molecules are not nucleotides. In some embodiments, small molecules are not saccharides.
  • a "subject”, as used herein, is an individual to whom an agent is to be delivered, e.g., for experimental, diagnostic, and/or therapeutic purposes.
  • Subjects of interest herein include animals, particularly agriculturally significant animals or companion animals (e.g., ruminants, cows, steer, sheep, goats, horses, swine, dogs, cats, rabbits, birds, fish, etc.), zoo animals (giraffe, bears, zebra, lions, tigers, etc.), reptiles, laboratory animals (e.g., mice, rats), mammals, primates, or humans.
  • animals particularly agriculturally significant animals or companion animals (e.g., ruminants, cows, steer, sheep, goats, horses, swine, dogs, cats, rabbits, birds, fish, etc.), zoo animals (giraffe, bears, zebra, lions, tigers, etc.), reptiles, laboratory animals (e.g., mice, rats), mammals, primates,
  • a “sublethal" concentration of an antibiotic refers to a concentration that is less than the MIC of the antibiotic. In certain embodiments of the invention a sublethal concentration is not sufficient to significantly reduce the growth rate (proliferation) of a microbial cell, e.g., the growth rate is reduced by less than 20%, preferably less than 10%. Such a concentration is referred to herein as a “non- inhibiting concentration”.
  • a “lethal" concentration of an antibiotic is one that is equal to or greater than the MIC and would ultimately result in microbial death and complete or essentially complete sterilization of a culture medium containing the microbe if continued indefinitely assuming that no resistant strains arise during the incubation period.
  • compounds of the invention may contain "optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • SC(S)SR 0 -(CH 2 )( M OC(O)NR° 2 ; -C(O)N(OR O )R°; -C(O)C(O)R 0 ; -C(O)CH 2 C(O)R 0 ; -C(NOR°)R°; -(CH 2 )o- 4 SSR°; -(CH 2 )( M S(O) 2 R°; -(CH 2 )( M S(O) 2 OR O ; -(CH 2 ) O _ 4 OS(O) 2 R°; -S(O) 2 NR° 2 ; -(CH 2 )( M S(O)R°; -N(R°)S(O) 2 NR° 2 ; -N(R°)S(O) 2 R°; -N(OR°)R°; -C(NH)NR° 2 ; -P(O) 2 R 0 ; -P(O)R
  • Suitable monovalent substituents on R° are independently halogen, -(CH 2 )o_ 2 R*, -(haloR*), -(CH 2 )o- 2 OH, -(CH 2 )o- 2 OR # , -(CH 2 ) 0 - 2 CH(OR*) 2 ; -O(haloR # ), -CN, -N 3 , -(CH 2 )o- 2 C(0)R # , -(CH 2 ) 0 - 2 C(O)OH, -(CH 2 )o_ 2 C(O)OR # , -(CH 2 )O -2 SR', -(CH 2 ) O-2 SH, -(CH 2 )o_ 2 NH 2 , -(CH 2 ) 0-2 NHR # , -(CH 2 ) 0 _ 2 NR # 2 ,
  • R * is selected from hydrogen, Ci_ 6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0_4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted” group include: - O(CR * 2 ) 2 _ 3 O-, wherein each independent occurrence of R * is selected from hydrogen, Ci_ 6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of R include halogen, -R', -(haloR'), -OH, -OR', -O(haloR'), -CN, -C(O)OH, -C(O)OR', -NH 2 , -NHR', -NR' 2 , or -NO 2 , wherein each R' is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C ⁇ aliphatic, -CH 2 Ph, -0(CH 2 )o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R + , -NR + 2 , -C(O)R + , -C(O)OR + , -C(O)C(O)R + , -C(O)CH 2 C(O)R + , -S(O) 2 R + , -S(O) 2 NR + 2 , -C(S)NR + 2 , -C(NH)NR + 2 , or -N(R + )S(O) 2 R + ; wherein each R + is independently hydrogen, Ci_ 6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R + , taken together with their intervening
  • Suitable substituents on the aliphatic group of R + are independently halogen, -R', -(haloR'), -OH, -OR', -O(haloR'), -CN, -C(O)OH, -C(O)OR', -NH 2 , -NHR', -NR* 2 , or -NO 2 , wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_4 aliphatic, - CH 2 Ph, -0(CH 2 )o_iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • treating refers to the provision of any type of medical or surgical management to a subject. Treating can include, but is not limited to, administering a pharmaceutical composition to a subject. Treating is typically undertaken in an effort to alter the course of a disease, disorder, or undesirable condition in a manner beneficial to the subject.
  • the effect of treating can generally include reversing, alleviating, reducing, delaying the onset of, inhibiting the progression of, and/or reducing the likelihood of occurrence of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder or condition.
  • a composition of this invention can be administered to a subject who has developed an infection or is at increased risk of developing an infection relative to a member of the general population.
  • a composition of this invention can be administered prophylactically, i.e., before development of any symptom or manifestation of a condition. Typically in this case the subject will be at risk of developing the condition.
  • the composition can be administered prior to exposure of the subject to an infectious agent or prior to the occurrence of a pathogenic event.
  • stable compounds refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., formulation into therapeutic products, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds, treating a disease or condition responsive to therapeutic agents).
  • a salt of a compound of this invention is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group.
  • the compound is a pharmaceutically acceptable acid addition salt.
  • Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic, lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids.
  • inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid
  • organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, as
  • Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenyl acetate, pheny
  • prodrug means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of this invention.
  • Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms.
  • Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds of any one of the formulae disclosed herein that comprise moieties such as amides, esters, carbamates, carbonates, ureides, and phosphate analogues.
  • Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs”.
  • hydrate means a compound which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
  • solvate means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, ethanol, methanol, dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces.
  • RecA protein is a key sensor and activator in response to DNA damage and plays a major role in inducing the SOS response pathway following such damage. It is known that RecA is also involved in other cellular processes in addition to recombination and DNA damage repair.
  • RecA acts as both a recombinase and co-protease. In the latter function, it activates the SOS response by cleaving the LexA repressor protein, which results in the induction of genes that are repressed by LexA under normal conditions.
  • SOS genes, and UmuD a sub-unit of polymerase IV, also involved in the SOS response are induced (Courcelle & Hanawalt, Annu Rev Genet. 37:61 1-646, 2003; Sutton et al., Annu Rev Genet. 34:479, 2000).
  • RecA Another fundamental role of RecA is to maintain the integrity of the genetic material.
  • the binding of RecA to single-stranded DNA regions that block replication forks serves as the sensor that replication is blocked and maintains the integrity of the replication fork itself until replication can resume (Courcelle & Hanawalt, Annu Rev Genet. 37:611-646, 2003).
  • the RecA protein is highly conserved and is cross species functional.
  • recA homologs from Yersinia pestis, Bacillus anthracis and M. tuberculosis have been shown to complement the E. coli recA- mutation (Suchkov & Mishan'kin, MoI Gen Mikrobiol Virusol. 5:34, 1989; Ko et al., J Bacteriol. 184:3917, 2002; Nair & Steyn, J Gen Microbiol. 137:2409, 1991 ).
  • inhibitors of RecA identified using the RecA protein from one species would be expected to show activity in a wide variety of bacteria.
  • the RecA protein has many functional features that present points of intervention to inhibit its activity in accordance with the present invention. Multiple alignment of the sequence reveals a canonical structure of RecA-like proteins consisting of distinct segments or motifs. These segments or modules are highly conserved and have been assigned functional roles based on genetic, biochemical and structural studies. Such modules are involved in DNA damage recognition and binding, monomer interaction, filament formation, helicase motifs, ATP binding and hydrolysis, recombination, replication and co-protease activity. Mutational studies have identified residues that are critical to these processes.
  • the Glyl57 change generates a constitutive co-protease form of RecA and results in a lower survival in response to UV treatment, a phenotype itself associated to a low recombination competent form of RecA.
  • the present invention makes use of this information to precisely map regions of RecA to be targeted for compound discovery, e.g., using computational approaches (see below).
  • RecX also called OraA
  • RecX is an inhibitor of RecA for both recombinase and co-protease activities
  • RecX appears to inhibit the ATP ase activity of RecA.
  • Genetic and biochemical evidence identifies sites of interaction between RecA and LexA, suggesting that amino acids at positions 67, 154-157, 229 and 243 are responsible at least in part for the binding to LexA (VanLook et al., J MoI Biol. 333:345, 2003).
  • amino acid changes at positions 122-123 and 150-161 dramatically decrease the ability of mutant cells ability to survive in response to UV radiation treatment.
  • a domain in RecA that likely forms part of the co-protease substrate binding site has also been identified (Nastri et al., MoI Microbiol. 25:967, 1997).
  • Exemplary structural classes of antibiotics for use in combination with RecA inhibitors that provide compounds of according to the present invention include, but are not limited to, quinolones (e.g., cinoxacin, nalidixic acid, pipemidic acid, cipro floxiacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and sparfloxacin), fluoroquinolones, ⁇ -lactams (penicillins, e.g., ampicillin, ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate, lanate, piperacillin, piperacillin/tazoba, ctam, pencillin G, pencillin V, azlocillin, carbenicillin, mezlocillin, ticarcillin, ticarcillin/clavulanate, or ce
  • quinupristin/dalfopristin and pristinamycin 2,4-diaminopyrimidines, nitrofurans (e.g., metronidazole and tinidazole), sulfonamides (para-aminobenzoic acid, pyrimethamine, sulfadiazine, sulfamethoxazole, sulfapyridine, sulfisoxazole, and trimethoprim), sulfones, rifabutins, dapsones, peptides, glycopeptides (e.g., teicoplanin, vancomycin), lipopeptides (e.g., daptomycin), cephamycins (e.g., cefotetan and cefoxitin), clofazimine monobactams (e.g., aztreonam), amidonopenicillins (e.g., mecillinam), spectin
  • Exemplary mechanistic classes of antibiotics for use in combination with RecA inhibitors according to the present invention include, but are not limited to, those that inhibit protein synthesis, cell wall synthesis, DNA replication, transcription, and/or cell division. It will be appreciated that biological and biochemical pathways are not mutually exclusive and that some biological or biochemical pathways may be considered to be subsets or sub-pathways of other biological or biochemical pathways.
  • Mechanisms of action more specifically include, but are not limited to, inhibiting protein synthesis (e.g., by binding ribosomal RNA or proteins, blocking tRNA binding to ribosome-mRNA complex, inhibiting peptidyl transferase), inhibiting or interfering with synthesis of a cell wall component (e.g., inhibition of peptidoglycan synthesis, disruption of peptidoglycan cross-linkage, disruption of movement of peptidoglycan precursors, disruption of mycolic acid or arabinoglycan synthesis), cell membrane disruption, inhibiting or interfering with nucleic acid synthesis or processing, acting as "antimetabolites" and either inhibiting an essential bacterial enzyme or competing with a substrate of an essential bacterial enzyme, inhibiting or interfering with cell division.
  • protein synthesis e.g., by binding ribosomal RNA or proteins, blocking tRNA binding to ribosome-mRNA complex, inhibiting peptidyl transferase
  • a cell wall component e.
  • Quinolone antibiotics are compounds that contain a quinolone or a naphthyridine nucleus with any of a variety of different side chains and substituents. Numerous modifications of the originally identified core structures have been made resulting in a large number of compounds with activity against differing groups of bacteria. Quinolone antibiotics are described, e.g., in Ronald, A.R. and Low, D. E., (eds.), "Fluoroquinolone Antibiotics", Birkhauser Verlag, Basel, 2003; DaSilva, et al., Curr Med Chem 10(l):21, 2003; Van Bambeke et al., Clin Microbiol. Infect., 1 1 :256, 2005; U.S. Pat.
  • quinolone antibiotics include, but are not limited to, any of the antibacterial agents disclosed in the foregoing references including, but not limited to, ciprofloxacin, oxolinic acid, cinoxacin, flumequine, miloxacin, rosoxacin, pipemidic acid, norfloxacin, enoxacin, moxifloxacin, gatifloxacin, ofloxacin, lomefloxacin, temafloxacin, fleroxacin, pefloxacin, amifloxacin, sparfloxacin, levofloxacin, clinafloxacin, nalidixic acid, enoxacin, grepafloxacin, levofloxacin, lomefloxacin norfloxacin, ofloxacin
  • Quinolone antibiotics include fluoroquinolones (e.g., having a fluorine substituent at the C-6 position), and non-fluoroquinolones. Also included within the scope of quinolone antibiotics are derivatives in which a quinolone is conjugated with, e.g., covalently bound to, another core structure.
  • U.S. Pub. No. 20040215017 discloses compounds in which an oxazolidinone, isoxazolinone, or isoxazoline is covalently bonded to a quinolone.
  • quinolone antibiotics that can be utilized in accordance with the present invention are compounds that have a core structure related to the 4-oxo-l,4-dihydroquinoline and 4-oxo-l,4 dihydronapthyridine systems, e.g., 2-pyridones, 2-naphthyridinones, and benzo[b]napthyridones.
  • R ai group in Formula (AI) has the structure of Formula (I): (D
  • X is oxygen, sulfur, or N(R);
  • R 1 is hydrogen, or an optionally substituted group selected from a Ci -6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
  • each R 2 is independently halogen, R 3 , OR 3 , SR 3 , S(O)R 3 , N(R 3 ) 2 , C(O)R 3 ,
  • each R 3 is independently hydrogen or an optionally substituted group selected from a Ci -6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0—5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
  • Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched Ci_ 6 hydrocarbon chain, wherein 0-2 methylene units of Q are independently replaced by -0-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO 2 -, -NRSO 2 -, -SO 2 NR-, -NRC(O)-, -C(O)NR-, -OC(O)NR-, NRC(O)NR or -NRC(O)O-; each R is independently hydrogen or an optionally substituted aliphatic group; and Ring A is an optionally substituted 3-8 membered bivalent, saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring
  • Formula (I) (and formulae herein) are those: wherein X is O; wherein A bl is selected from quinolones, ⁇ -lactams (e.g., penicillins or cephalosporins), carbapenems, aminoglycosides, macrolides, ketolides, tetracyclines, glycycyclines, lincomycins, oxazolidinones, amphenicols, ansamycins, polymyxins, aminomethlycyclines, lincosamides, streptogramins, 2,4-diaminopyrimidines, nitrofurans, sulfonamides, sulfones, rifabutins, dapsones, peptides, glycopeptides, or combinations thereof; wherein A bl is a quinolone antibiotic; wherein L comprises between 2-15 atoms selected from carbon, nitrogen or sulfur; wherein L is divalent al
  • the R 1 group of formula I is hydrogen, or an optionally substituted group selected from a Ci -6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • the R 1 group of formula I is a bicyclic 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 1 group of formula I is selected from any of the following groups:
  • each wavy line depicts the point of attachment to Q and the depicted bond can be attached at any atom of the depicted structure (i.e., attached at any atom in place of a depicted hydrogen atom) or
  • R 1 group of formula I is selected from any of the following groups:
  • each wavy line depicts the point of attachment to Q and the depicted bond can be attached at any atom of the depicted structure (i.e., attached at any atom in place of a depicted hydrogen atom) or
  • the R 1 group of formula I is a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 1 is phenyl substituted with 1 to 3 groups independently selected from halogen, -(CH 2 ) O ⁇ 4 R 0 , and -(CH 2 )o_ 4 ⁇ R°.
  • groups include chloro, fluoro, bromo, OH, OMe, methyl, ethyl, propyl, cyclopropyl, isopropyl, and the like.
  • OH or OMe groups are present.
  • R 1 group of formula I is selected from:
  • the Q group of formula I is a valence bond or a bivalent, saturated or unsaturated, straight or branched Ci_ 6 hydrocarbon chain, wherein 0-2 methylene units of Q are independently replaced by -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO 2 -, -NRSO 2 -, -SO 2 NR-, -NRC(O)-, -C(O)NR-, -0C(0)NR-,-NRC(0)0- or -NRC(O)NR-.
  • Q is a valence bond such that R 1 is directly attached to Ring A.
  • Q is a bivalent, saturated, and straight Ci -3 hydrocarbon chain, wherein 0-1 methylene units of Q is replaced by -0-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO 2 -, -NRSO 2 -, -SO 2 NR-, -NRC(O)-, -C(O)NR-, -OC(O)NR-, or -NRC(O)O-.
  • Q is -0-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO 2 -, -NRSO 2 -, -SO 2 NR-, -NRC(O)-, -C(O)NR-, -OC(O)NR-, -NRC(O)O- or -NRC(O)NR.
  • Q is -O-.
  • Ring A group of formula I is an optionally substituted 3-8 membered bivalent, saturated, partially unsaturated, or aryl monocyclic ring having 0—4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Ring A is an optionally substituted 3-8 membered bivalent, saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Ring A is an optionally substituted 5-6 membered bivalent aryl ring having 0-2 nitrogen atoms.
  • Ring A is phenylene optionally substituted with 1-2 groups independently selected from halogen, and -(CH 2 )o_ 4 ⁇ R°.
  • groups include chloro, fluoro, bromo, OH, OMe, methyl, ethyl, propyl, cyclopropyl, isopropyl, and the like.
  • OH and/or OMe group(s) are present.
  • each R 2 group of formula I is independently halogen, R 3 , OR 3 , SR 3 , S(O)R 3 , N(R 3 ) 2 , C(O)R 3 , C(O)OR 3 , NR 3 C(O)R 3 , C(O)NR 3 , SO 2 R 3 , NR 3 SO 2 R 3 , SO 2 N(R 3 ) 2 , NR 3 C(O)NR 3 wherein each R 3 is independently hydrogen or an optionally substituted group selected from a Ci -6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • each R 2 group is independently halogen, R 3 , OR 3 , SR 3 , S(O)R 3 , S(O) 2 R 3 or N(R 3 ) 2 , wherein each R is as defined above.
  • at least one R group is OH.
  • one R 2 group is R 3 wherein R 3 is an optionally substituted bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • one R 2 group is R 3 wherein R 3 is a bicyclic 10 membered partially unsaturated ring having one oxygen atom optionally substituted with 1 to 3 substituents independently selected from halogen, -(CH 2 ) 0 _ 4 R°, -(CH 2 )o- 4 ⁇ R°, -(CH 2 ) O- ⁇ SR 0 , -(CH 2 )( M Ph, optionally substituted with R° or OR 0 , -(CH 2 ) O ⁇ O(CH 2 ) O- .
  • R° optionally substituted with R° or 0R°
  • -CH CHPh, optionally substituted with R° or 0R°, -(CH 2 )o- 4 N(R°) 2
  • each R° may be substituted as defined herein and is independently hydrogen, Ci_ 6 aliphatic, -CH 2 Ph, -O(CH 2 ) 0 -iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 3 is substituted with 1 to 3 groups independently selected from OH and OMe and optionally substituted phenyl.
  • the R 2 group formula I is OH, OMe, or is selected from any of the following groups:
  • each wavy line depicts the point of attachment to Q and the depicted bond can be attached at any atom of the depicted structure (i.e., attached at any atom in place of a depicted hydrogen atom).
  • the R 2 group formula I is OH, OMe, or is selected from any of the following groups:
  • each wavy line depicts the point of attachment to Q and the depicted bond can be attached at any atom of the depicted structure (i.e., attached at any atom in place of a depicted hydrogen atom).
  • the instant invention provides for the compounds of Formula (Al) delineated below:
  • the compounds from the following examples can be prepared by linking the RecA inhibitor compound (with or without suitable protecting group manipulation) to the antibacterial compound via, but not limited to, ester, amide, ether,oxime, imine or amine linkage. It will be apparent to one skilled in the art that many such combinations of RecA inhibitors and antibiotics are possible. The following examples are meant to be illustrative only and not limiting.
  • compositions comprising an effective amount of a compound of any of the formulae herein (e.g., including Formula I, Al, etc.), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph or prodrug, if applicable, of said compound; and an acceptable carrier.
  • a composition of this invention is formulated for pharmaceutical use ("a pharmaceutical composition"), wherein the carrier is a pharmaceutically acceptable carrier.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in amounts typically used in such compositions.
  • compositions comprising an effective amount of a compound of Formula Al and a pharmaceutically acceptable carrier
  • a pharmaceutical composition which comprises the product prepared by combining (e.g., mixing) an effective amount of a compound of Formula Al and a pharmaceutically acceptable carrier
  • the pharmaceutical composition(s) above further comprising an effective amount of an additional therapeutic agent, for example, anti-infective agents, antibiotic agents, ⁇ -lactamase inhibitor agents, antiviral agents, antimicrobial agents, and the like.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphate
  • compositions of the invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the compound of the formulae herein is administered transdermally (e.g., using a transdermal patch techniques).
  • Other formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA (17th ed. 1985).
  • Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients.
  • ingredients such as the carrier that constitutes one or more accessory ingredients.
  • the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.
  • the compound is administered orally.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc.
  • Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, 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 optionally may be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • compositions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.
  • compositions suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • Such injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 ,3- butanediol.
  • the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
  • compositions of this invention may be administered in the form of suppositories for rectal administration.
  • These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • compositions of this invention may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches and ionophoretic administration are also included in this invention.
  • the compounds of this invention may be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents, or catheters.
  • an implantable medical device such as prostheses, artificial valves, vascular grafts, stents, or catheters.
  • Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in US Patents 6,099,562; 5,886,026; and 5,304, 121.
  • the coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof.
  • the coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition.
  • Coatings for invasive devices are to be included within the definition of pharmaceutically acceptable carrier, adjuvant or vehicle, as those terms are used herein.
  • the compounds of the present invention are preferably administered using the formulations of the invention.
  • the compounds of the present invention are administered by IV infusion over a period of from about 10 minutes to about 24 hours, e.g., about 30 minutes to about 4 hours, about 45 minutes to 90 minutes, or about 1 hour.
  • One of ordinary skill in the art would readily know how to determine doses based on both the height and or weight of the patient.
  • the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by the compounds (or compositions thereof) herein comprising the step of administering to said subject an effective amount of a compound or a composition of this invention.
  • the present invention thus provides methods for the treatment or prevention of infection and related disease, disorders, or symptoms thereof (including those bacteria delineated herein) comprising the step of administering to a subject in need thereof at least one compound of any of the formulae herein (e.g., Formula Al) in an amount effective therefor.
  • Other therapeutic agents such as those described below may be employed with the inventive compounds in the present methods.
  • such other therapeutic agent(s) may be administered prior to, simultaneously with or following the administration of the compound(s) of the present invention.
  • Other aspects of the methods include those further comprising wherein a subject sample is assessed; and also further with one or more of: assessing bacterial levels of the subject; assessing that is performed prior to administration of the compound; assessing that is performed after administration of the compound; or assessing that is performed both prior to and after administration of the compound.
  • Methods delineated herein include those wherein the subject is identified as in need of a particular stated treatment. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). For example, such methods include clinical diagnosis based at least in part on symptoms (e.g., diagnosed or subject self-reported), imaging studies, immunodiagnostic assays, nucleic acid based diagnostics, and/or isolation and culture of potentially causative microorganisms from samples, such as blood, urine, sputum, synovial fluid, cerebrospinal fluid, pus, or any sample of body fluid or tissue.
  • symptoms e.g., diagnosed or subject self-reported
  • imaging studies e.g., immunodiagnostic assays
  • nucleic acid based diagnostics e.g., and/or isolation and culture of potentially causative microorganisms from samples, such as blood,
  • Inventive compounds herein are useful to inhibit growth of a wide variety of microbial types including, for example, gram negative bacteria, gram positive bacteria and/or acid fast bacteria.
  • bacteria whose growth or proliferation can be inhibited include, but are not limited to, members of the following genuses: Actinomyces, Staphylococcus, Streptococcus, Enterococcus, Erysipelothrix, Neisseria, Branhamella, Listeria, Bacillus, Corynbacterium, Erysipelothrix, Gardnerella, Mycobacterium, Nocardia, Enterobacteriaceae, Escherichia, Salmonella, Shigella, Yersinia, Enterobacter, Klebsiella, Citrobacter, Serratia, Providencia, Proteus, Morganella, Edwardsiella, Erwinia, Vibrio, Aeromonas, Helicobacter, Campylobacter, Eikenella, Pasteurella, Pseudomon
  • the bacteria are species that are causative agents of disease in humans and/or animals.
  • examples include, but are not limited to, Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Enterobacter cloacae, Campylobacter jejuni, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella oxytoca, Legionella pneumophila, Pasteurella multocida, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Helicobacter pylori, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhimu ⁇ um, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and combinations thereof.
  • the bacterial species or strain is one that is sensitive to a particular antibiotic agent or class of antibiotic agents; in some embodiments, the bacterial species or strain is sensitive to one or more bacterial type II topoisomerase inhibitors such as a quinolone antibiotic.
  • Infections and infection-related conditions that can be treated using an inventive compound herein (and optionally another antibiotic agent) include, but are not limited to, pneumonia, meningitis, sepsis, septic shock, sinusitis, otitis media, mastoiditis, conjunctivitis, keratitis, external otitis (e.g., necrotizing otitis externa and perichondritis), laryngeal infections (e.g., acute epiglottitis, croup and tuberculous laryngitis), endocarditis, infections of prosthetic valves, abscesses, peritonitis, infectious diarrheal diseases, bacterial food poisoning, sexually transmitted diseases and related conditions, urinary tract infections, pyelonephritis, infectious arthritis, osteomyelitis, infections of prosthetic joints, skin and soft tissue infections, oral infections, dental infections, nocardiosis and actinomycosis, mastitis, brucellosis, Q
  • the bacterial species or strain is one that is resistant to a particular antibiotic agent or class of antibiotic agents; in some embodiments, the bacterial species or strain is resistant to one or more bacterial type II topoisomerase inhibitors such as a quinolone antibiotic.
  • Inventive compounds and compositions containing them can be used to inhibit microbial growth and/or survival in a variety of contexts. For example, they may be employed to inhibit growth and/or survival of organisms maintained in cell culture or inhabiting locations in the environment, e.g., inert surfaces, clothing, towels, bedding, utensils, etc.
  • locations in the environment e.g., inert surfaces, clothing, towels, bedding, utensils, etc.
  • fomites i.e., inanimate objects that may be contaminated with disease-causing microorganisms and may serve to transmit disease to a human or animal.
  • Such locations or objects can be contacted with a solution containing an inventive compound, and optionally including one or more other antibiotic agents.
  • Inventive compounds herein, alone or together with one or more other antibiotic agents can be added to food or water, particularly for the prevention of microbial disease in animals.
  • the methods herein include those further comprising monitoring subject response to the treatment administrations. Such monitoring may include periodic sampling of subject tissue, fluids, specimens, cells, proteins, chemical markers, genetic materials, etc. as markers or indicators of the treatment regimen.
  • the subject is prescreened or identified as in need of such treatment by assessment for a relevant marker or indicator of suitability for such treatment.
  • the invention provides a method of monitoring treatment progress.
  • the method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target or cell type delineated herein modulated by a compound herein, bacterial type or count) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof delineated herein, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof.
  • the level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status.
  • a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy.
  • a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.
  • a level of Marker or Marker activity in a subject is determined at least once. Comparison of Marker levels, e.g., to another measurement of Marker level obtained previously or subsequently from the same patient, another patient, or a normal subject, may be useful in determining whether therapy according to the invention is having the desired effect, and thereby permitting adjustment of dosage levels as appropriate. Determination of Marker levels may be performed using any suitable sampling/expression assay method known in the art or described herein. Preferably, a tissue or fluid sample is first removed from a subject. Examples of suitable samples include blood, urine, tissue, mouth or cheek cells, and hair samples containing roots. Other suitable samples would be known to the person skilled in the art.
  • Determination of bacterial levels, protein levels and/or mRNA levels (e.g., Marker levels) in the sample can be performed using any suitable technique known in the art, including, but not limited to, enzyme immunoassay, ELISA, radiolabelling/assay techniques, blotting/chemiluminescence methods, realtime PCR, and the like.
  • co-administered means that the second therapeutic agent may be administered together with a compound of this invention as part of a single dosage form (such as a composition of this invention comprising a compound of the invention and an second therapeutic agent as described above) or as separate, multiple dosage forms.
  • the additional agent may be administered prior to, consecutively with, or following the administration of a compound of this invention.
  • both the compounds of this invention and the second therapeutic agent(s) are administered by conventional methods.
  • composition of this invention comprising both a compound of the invention and a second therapeutic agent to a subject does not preclude the separate administration of that same therapeutic agent, any other second therapeutic agent or any compound of this invention to said subject at another time during a course of treatment.
  • the invention provides the use of a compound of the formulae herein (e.g., formula AI, formula I) alone or together with one or more of the above-described second therapeutic agents in the manufacture of a medicament, either as a single composition or as separate dosage forms, for treatment or prevention in a subject of a disease, disorder or symptom set forth above.
  • a compound of the formulae herein for use in the treatment or prevention in a subject of a disease, disorder or symptom thereof delineated herein. Recitation of a numerical range includes each individual number in that range as well as is inclusive of the delineated beginning and ending numbers of the range.
  • n is independently 0-4"
  • n can be 0, 1 , 2, 3 or 4 at any designated instance.
  • the synthesis of compounds of the formulae herein can be readily effected by synthetic chemists of ordinary skill. A convenient method for producing compounds of the formulae herein is provided in the schemes herein. Other approaches to synthesizing compounds of the formulae herein can readily be adapted from references cited herein. Variations of these procedures and their optimization are within the skill of the ordinary practitioner.
  • the compounds delineated herein can be made from reaction (nucleophilic displacement, nucleophilic substitution, esterification, etherif ⁇ cation, alkylation,oxime formation, imine formaton, etc.) of appropriate recA or antibiotic compounds (or appropriate derivatives thereof) and appropriate linker moiety precursors.
  • reaction nucleophilic displacement, nucleophilic substitution, esterification, etherif ⁇ cation, alkylation,oxime formation, imine formaton, etc.
  • antibiotic compounds or appropriate derivatives thereof
  • LG leaving group, e.g., halo, sulfonate, alkoxy
  • Additional methods of synthesizing compounds of the formulae herein and their synthetic precursors, including those within routes not explicitly shown in schemes herein, are within the means of chemists of ordinary skill in the art. Methods for optimizing reaction conditions, if necessary minimizing competing by-products, are known in the art. In addition, various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the applicable compounds are known in the art and include, for example, those described in R.
  • the synthetic methods described herein may also additionally include steps, either before or after any of the steps described in the preceding schemes, to add or remove suitable protecting groups in order to ultimately allow synthesis of the compound of the formulae described herein.
  • the methods delineated herein contemplate converting compounds of one formula to compounds of another formula.
  • the process of converting refers to one or more chemical transformations, which can be performed in situ, or with isolation of intermediate compounds.
  • the transformations can include reacting the starting compounds or intermediates with additional reagents using techniques and protocols known in the art, including those in the references cited herein. Intermediates can be used with or without purification (e.g., filtration, distillation, crystallization, trituration, chromatography, etc.).
  • the compounds of the invention can be assessed for antibacterial activity using any of a variety of standard protocols (e.g., in vitro, in vivo, cellular, animal) known in the art.
  • growth or survival of cells can be assessed using cells growing in liquid media or on solid or semi-solid media. Any method known in the art can be used to determine whether an agent inhibits growth, proliferation, and/or survival. Examples include measuring optical density in liquid culture, measuring colony formation, or measuring bacterial viability. Bacterial viability can be assessed based on metabolic characteristics such as oxidation/reduction state, ability to metabolize particular substrate(s) or produce particular metabolite(s), or based on membrane integrity, which can be detected by evaluating ability of a bacterial cell to exclude a particular substance such as a detectable molecule (e.g., a fluorescent or luminescent molecule) from the cell interior.
  • a detectable molecule e.g., a fluorescent or luminescent molecule
  • LIVE/DEAD BacLight Bacterial Viability assay (Molecular Probes, now owned by Invitrogen, Carlsbad, CA).
  • This assay utilizes mixtures of SYTO® 9 green fluorescent nucleic acid stain and the red fluorescent nucleic acid stain, propidium iodide. These stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells.
  • SYTO 9 stain labels bacteria with both intact and damaged membranes. Propidium iodide, however, penetrates only bacteria with damaged membranes, competing with the SYTO 9 stain for nucleic acid binding sites when both dyes are present.
  • SYTO 9 stain and propidium iodide When mixed in recommended proportions, SYTO 9 stain and propidium iodide produce green fluorescent staining of bacteria with intact cell membranes and red fluorescent staining of bacteria with damaged membranes. The background remains virtually nonfluorescent. The ratio of green to red fluorescence intensities therefore provides a quantitative index of bacterial viability.
  • a fiuorometer can be used to detect the fluorescence intensities.
  • Another suitable assay for determining the number of viable bacterial cells in culture is based on quantitation of the ATP present. ATP is an indicator of metabolically active cells.
  • the BacTiter- GloTM Assay (Promega, Madison, WI) is a commercially available assay based on this principle that involves adding a single reagent (BacTiter-GloTM Reagent) directly to bacterial cells in medium and measuring luminescence.
  • BocTiter-GloTM Reagent a single reagent directly to bacterial cells in medium and measuring luminescence.
  • Many additional assays suitable for assessing bacterial viability are described in "Handbook of Fluorescent Probes and Research Products” (Molecular Probes, 9th edition, 2002) and “The Handbook — A Guide to Fluorescent Probes and Labeling Technologies” (Invitrogen, 10th edition, available at the Invitrogen web site). Examples
  • RecA Activity Assay Luciferase ATP ase Assay RecA is a DNA-dependent ATPase (i.e., it catalyzes the reaction adenosine triphosphate [ATP] ⁇ adenosine diphosphate [ADP]).
  • An assay for RecA ATPase activity based on detection of the amount of ATP remaining in a reaction mixture following incubation of RecA protein, DNA, and ATP is utilized for test compounds.
  • E. coli RecA protein is incubated in reaction buffer with DNA, ATP, and any test compound or compounds for a measured about of time. The quantity of ATP remaining after the RecA reaction is quantitated using a subsequent luciferase assay.
  • the amount of ATP remaining can be compared to controls containing either a known inhibitor (full inhibition control) or no test compound (full activity control).
  • Example 2. Cell-based Assay for RecA Inhibitor Compounds
  • the recA promoter is induced when RecA becomes activated as a result of exposure to DNA damaging agents.
  • a low copy reporter plasmid in which the recA promoter controls transcription of the mRNA encoding green flourescent protein (GFP) is used.
  • the plasmid, referred to herein as prec::GFP has been previously described (Ronen, 2002). See also Kuang, 2004.
  • prec::GFP contains 2821707-2821893 of the E. coli MGl 655 genome (numbering based on the sequenced E.
  • the prec::GFP plasmid is introduced into MG1655 and MG1655 recA null strains and the fluorescence emitted by GFP in the presence or absence of quinolone as compared with background fluorescence detected from cultures of MG 1655 lacking the plasmid is measured.
  • Cells of these three strains are grown to early log phase in LB at 37 0 C with shaking.
  • Various amounts of Nor (norfloxacin) are added to lOOul cultures of each strain to achieve final concentrations ranging from 0 ⁇ g/ml to 31.25 ⁇ g/ml. After 60 min, fluorescence due to GFP expression from the reporter plasmid is measured using a spectrophotometer.
  • RecA is a DNA-dependent ATPase.
  • One assay for RecA activity is based on detection of the amount of ATP remaining in a reaction mixture following incubation of RecA protein, DNA, and ATP. 40-50 ⁇ g/ml of RecA (New England Biolabs) is combined with 20 ⁇ M ATP in a final volume of 50 ul PNK buffer Ix either with or without and 250 ng/ul of Ml 3 DNA, and the reaction mixtures are incubated at 25°C for 30 minutes.
  • InM ATP-Gamma a non-hydrolyzable form of ATP that inhibits RecA, is added to some of the reactions 10 minutes after the start of the incubation.
  • the assay measures the amount of ATPase (adenosine triphosphate (ATP) - adenosine diphosphate (ADP)) activity catalyzed by E. CoIi RecA protein.
  • ATPase adenosine triphosphate (ATP) - adenosine diphosphate (ADP)
  • recA is incubated in reaction buffer with DNA, ATP and compounds of interest for a measured amount of time.
  • the quantity of ATP remaining after the RecA reaction is quantitated using a subsequent luciferase assay.
  • the amount of ATP remaining can be compared to inhibited and fully active control samples. Representative details of the assay are provided below:
  • RecA activity in the presence of DNA can be readily detected based on the reduced luminescence detected from reactions in which ATP has been consumed by active
  • RecA activity is DNA dependent in this assay.
  • Test compounds are housed in wells in microwell plates in which 100 ⁇ g of each compound is resuspended in 50 ul of DMSO (2ug/ul). The screen is performed using 384- well plates. A reaction mixture containing 2mg/ml of RecA (New England Biolabs), 250 ng/ul of Ml 3 DNA, and ImM ATP in a final volume of 50 ul PNL buffer Ix is dispensed into each well. 5ul of a different compound solution (10 ⁇ g of compound) is added to each well, one compound per well.
  • the plate includes 4 negative control wells in which no test compound is added to the reaction mixture, and 4 positive control wells, in which the reaction mixture also contains 1 nM ATP- gamma to maximally inhibit RecA and does not contain a test compound.
  • the plates are incubated for 1 hour, following which 50 ul of the Promega Kinase-GloTM Reagent (Promega) is added to each well.
  • Luminescence is measured. Test compounds in wells in which the luminescence is lower than the average luminescence detected in the negative control wells are identified as RecA inhibitors. The extent of RecA inhibition by any particular test compound is assessed by comparing the luminescence with that detected in the positive control well, which represent 100% inhibition of RecA.
  • test compound For each test compound, the difference between the OD600 for the well containing the test compound and Nor and the OD600 for the well containing the test compound but no Nor is calculated. If the OD600 for the well containing a particular test compound and Nor is significantly lower than the OD600 for the well containing the same test compound and not containing Nor, then the test compound is identified as a quinolone potentiating compound.
  • Example 5 Identification of Active Agents Using a Cell-based RecA Reporter Assay Test compounds are housed in wells in microwell plates in which 100 ⁇ g of each compound is resuspended in 50 ul of DMSO (2ug/ul). The screen is performed using 384-well plates. 50 ⁇ l of an early log phase culture of MG 1655 containing the prec:GFP plasmid are dispensed into each well, and Nor is added to a final concentration of 10 ⁇ g/ml. A different test compound is added to each well to a final concentration of 50 ⁇ g/ml. Each plate contains 4 control wells to which no test compound is added and 4 control wells to which 50 nM ATP-gamma is added to inhibit RecA.
  • RecA inhibitors Compounds in wells that display decreased fluorescence relative to the control wells to which no test compound is added are identified as RecA inhibitors. The extent of RecA inhibition is assessed by comparison with fluorescence in the wells that contain ATP-gamma, which represents maximal inhibition of RecA (and thus approximately background levels of fluorescence).

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Abstract

The present invention relates to novel compounds derived from compounds having antibiotic activities. The invention further provides compositions comprising a compound of this invention and the use of such compositions in methods of treating diseases and conditions beneficially treated by anti-infective agents, particularly those relating to the management of bacterial infection.

Description

Novel Fusion Compounds
RELATED SUBJECT MATTER
This application claims the benefit of U.S. provisional patent application Ser. No. 60/897,981 filed January 26, 2007. The disclosure of the aforementioned patent application is incorporated herein in its entirety by this reference.
TECHNICAL FIELD OF THE INVENTION
The present invention relates to novel compounds derived from compounds having antibiotic activities. The invention further provides compositions comprising a compound of this invention and the use of such compositions in methods of treating diseases and conditions beneficially treated by anti-infective agents, particularly those relating to the management of bacterial infection.
BACKGROUND OF THE INVENTION
The emergence of resistance to antibacterial agents is a growing problem for human and animal health. In many instances, new drugs to treat infections, even as early as during their clinical development, are found to due to encounter microorganisms that display resistance to the new drug and/or currently used antibiotics. Efforts to overcome the growing problem of resistance have included modification of known antibiotics, classical screening of new compound libraries and natural product libraries, and genomic efforts to identify novel targets to which no cross resistance with existing antibiotics would be anticipated. Even with this significant antibiotic discovery effort, only a few agents that represent new chemical classes of antibiotic agents have been approved by regulatory agencies in recent years. Furthermore, a number of potent antibiotic agents are too toxic for clinical use or have significant side effects that limit their therapeutic utility. As such, novel approaches and new antibiotic agents to address microorganisms with increasing drug resistance profiles are urgently needed.
SUMMARY OF THE INVENTION
The present invention provides compounds, compositions comprising the compounds, and methods of treating and preventing disease by administration of the compounds and compositions thereof. The invention also relates to methods of making the compounds/compositions of the invention as well.
DETAILED DESCRIPTION OF THE INVENTION In one aspect, the invention provides a compound that comprises a recA inhibitor compound bonded to a second therapeutic agent, particularly a compound having antibiotic activity. The second therapeutic agent having antibiotic activity can be, for example, any compound with antibiotic activity, including those delineated herein (e.g., any antibiotic, a second recA inhibitor). In other aspects, the compound is that wherein the RecA inhibitor and the second compound are bonded via a linker moiety; wherein the linker moiety comprises between 2-15 atoms selected from carbon, nitrogen or sulfur; wherein the second compound is a quinolone antibiotic; or wherein the second compound is a macrocyclic antibiotic.
In one aspect, the invention relates to a compound of Formula Al : Ra'-L-Abl (Al) wherein,
Raι is a RecA inhibitor compound; L is a linker;
Abl is an antibiotic compound; or acid addition salt, solvate, hydrate or polymorph thereof.
The terms "ameliorate" and "treat" are used interchangeably and both mean decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or disorder (e.g., disease or disorders, or symptom thereof, such as those delineated herein). The term "antibiotic agent" refers to an agent that inhibitis and/or stops growth an/or proliferation of one or more species of microorganism (e.g., bacteria or fungus). An antibiotic agent may display activity in vitro (e.g., when contacted with cells in culture), in vivo (e.g., when administered to a subject at risk of or suffering from an infection), or both. The term "antibiotic agent" may be used to refer to bactericidal agents (i.e., agents that kill bacteria) and/or bacteriostatic agents (i.e., agents that inhibit or stop bacterial growth or proliferation but does not kill the cells). The invention contemplates bactericidal antibiotics and bacteriostatic antibiotics. Bactericidal antibiotics kill bacteria; bacteriostatic antibiotics slow the growth or reproduction of bacteria. The term "aryl", used alone or as part of a larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term "aryl" may be used interchangeably with the term "aryl ring". The aryl may be optionally substituted as delineated herein.
By "disease" or "disorder" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ; including those delineated herein. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
In general, an "effective amount" of a biologically and/or pharmacologically active agent is an amount sufficient to achieve a desired biological and/or pharmacological effect when delivered to a cell or organism according to a selected administration form, route, and/or schedule. As will be appreciated by those of ordinary skill in this art, the absolute amount of a particular agent that is effective may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the target tissue, etc. Those of ordinary skill in the art will further understand that an "effective amount" may be administered in a single dose, or may be achieved by administration of multiple doses. For example, an effective amount of an antibiotic agent may be an amount sufficient to achieve one or more of the following: (i) inhibit microbial growth in culture or in vivo; (ii) reduce the severity of or prevent one or more symptoms or signs of an infection; (iii) significantly reduce the risk of recurrence of an infection; (iv) significantly reduce the risk of a clinically significant infection in a subject who has been exposed to an infectious agent, etc.
Comparably, an effective amount of a potentiating agent may be an amount sufficient to achieve the same level of antibiotic activity with a particular antibiotic agent as is achieved when that antibiotic agent is administered at its conventional dose, in circumstances where the antibiotic agent is administered at a reduced dose as compared with its conventional dose.
The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon. This includes any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen, or; a substitutable nitrogen of a heterocyclic ring including =N- as in 3,4-dihydro-2//-pyrrolyl, -NH- as in pyrrolidinyl, or =N(R*)- as in N-substituted pyrrolidinyl.
The term "infection", as used herein, refers to the invasion of a host, whether the host is a vertebrate, invertebrate, fish, plant, bird, or mammal, by pathogenic microbes, e.g., bacteria. The term encompasses excessive growth of microbes that are normally present in or on the body of a mammal or other organism. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host organism. Thus, an organism is "suffering" from a microbial infection when excessive numbers of a microbial population are present in or on the organism's body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of an organism. The agents and compositions of certain embodiments of the invention are also useful in treating microbial growth or contamination of cell cultures or other media, or inanimate surfaces or objects, and nothing herein should limit the invention to treatment of higher organisms, except when explicitly so specified in the claims.
As used herein, agent or entity is "isolated" if it is separated from at least some materials or components with which it is associated in nature or when initially generated. In general, such separation involves activity of the hand of man. In aspects, the chemical compounds delineated herein are isolated. The term "linker" or "L" refers to a divalent chemical moiety containing atoms selected from carbon, nitrogen or sulfur. In one aspect the linker contains between 2- 15 atoms, or in another aspect 2-10 atoms, each independently selected from carbon, nitrogen or sulfur atoms; in another aspect the linker is a divalent alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, wherein any carbon atom is replaced with a heteroatom selected from N, O, S, or P, and any atom is optionally substituted with H, alkyl, oxo, hydroxyl, alkoxy, thioalkoxy, NH2, NRR, S(O), S(O)2, C(O)OR, OC(O)OR, OC(O)NR, RNC(O)NR or C(O)NRR, wherein R is H, optionally substituted alkyl (e.g., alkyl substituted with R*, R0, etc.), or any "R" group defined herein (e.g., R3, R*, R0, etc.); in another aspect the linker is an ester, amide, cycloalkyl, heterocycloalkyl, hydroxyl, sulfonate, sulfoxide, phosphate, phosphonate, primary or secondary amine, hydroxylamine, imine, oxime -C(O)O- , -OC(O)O, - OC(O)NR, RNC(O)CNR or -C(O)N(R)- group. Linkers utilized in the invention include cleavable and non-cleavable linker. Cleavable linkers may be cleaved by enzymes in a subject. Typical cleavable linkers include, but are not limited to, esters, amides, phosphonates, sulfonates, carbon esters, phosphorus esters, and sulfur esters.
The term "minimal inhibitory concentration" (MIC) are used herein consistently with its use in the art, i.e., to indicate the concentration of an agent that will inhibit bacterial proliferation (growth) (MIC). MIC values may be for example, the concentration of agent that inhibits visible growth or may be expressed as MIC5O, MIC90 or MIC99 values i.e., the concentration of an agent that reduces bacterial proliferation to 50% or less, 10% or less, or 1% or less, respectively, of the control value that would occur in the absence of the agent. As is well known in the art, MIC can be measured by a variety of methods, including automated and non-automated methods. Suitable methods are described in publications of the Clinical Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), as set forth in NCCLS: Performance Standards documents.
The term "potentiate", as used herein, means to enhance or increase at least one biological effect or activity of a biologically and/or pharmacologically active agent so that either (i) a given concentration or amount of the agent results in a greater biological effect or activity when the agent is potentiated than the biological effect or activity that would result from the same concentration or amount of the agent when not potentiated; or (ii) a lower concentration or amount of the agent is required to achieve a particular biological effect or activity when the agent is potentiated than when the agent is not potentiated; or (iii) both (i) and (ii). The biological effect or activity may be, for example, the ability to catalyze or inhibit one or more chemical reactions, the ability to activate or inhibit a biological or biochemical pathway, the ability to reduce or inhibit microbial proliferation, the ability to kill a microorganism, etc. An agent whose presence potentiates another agent may be referred to as a "potentiating agent".
The term "proliferation" refers to an increase in microbial number. Since bacterial proliferation, rather than mere increase in cell mass without cell division, is usually of primary concern, and since under most circumstances of interest herein proliferation is accompanied by an increase in microbial biomass, the term "growth" is generally understood to mean "proliferation", and the two terms are used interchangeably herein although it is recognized that different assays may measure either or both of these parameters. For example, optical density reflects biomass and does not specifically reflect cell number, whereas an assay based on detecting colonies formed from individual cells reflects cell number rather than biomass.
As used herein, the term "purified" refers to agents or entities that have been separated from most of the components with which they are associated in nature or when originally generated. In general, such purification involves action of the hand of man. Purified agents or entities may be partially purified, substantially purified, or pure. Such agents or entities may be, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% pure; or X% pure wherein X is any number between 0-100%, inclusive.
The term "quinolone antibiotic" means an agent containing a quinolone or a naphthyridine nucleus with any of a variety of different side chains and substituents as known and understood in the art and that displays inhibitory activity towards one or more microbial species, e.g., various quinolone carboxylic acids. The term "quinolone antibiotic" encompasses isolated enantiomers, salts, hydrates, solvates, or the free base form of any quinolone antibiotic.
According to the present invention, an agent is a RecA inhibitor if one or more RecA activities is reduced in the agent's presence as compared with its absence, or if the level or amount of RecA protein or gene product is reduced in the agent's presence as compared with its absence. In certain embodiments, RecA inhibitors act directly on RecA in that they physically interact with RecA. In other embodiments, inhibitors act indirectly on RecA. In general, agents that inhibit one or more activities of RecA, and/or that inhibit RecA expression levels, may be useful in accordance with the present invention. Exemplary RecA activities that may be inhibited include, but are not limited to, DNA binding, monomer interaction, helicase activity, filament formation, ATP binding and/or hydrolysis, co-protease activity (e.g., toward LexA and/or UmuD), recombinase activity, and replication function. In some embodiments, inventive RecA inhibitors inhibit one or more such activities with an IC50 below about 100 μg /ml, 50 μg /ml, 15 μg /ml; 10 μg /ml; 5 μg /ml, 3 μg /ml, or 1 μg /ml, or below X μg /ml, wherein X is a number between 0 and 100, inclusive. According to the present invention, desirable RecA ATPase inhibitors may even have an ICs0 well below 1 μg/ml, or even below 500 ng/ml, 100 ng/ml, 50 ng/ml, 30 ng/ml,. 25 ng/ml, 20 ng/ml, 15 ng/ml, 10 ng/ml, 5 ng/ml, 1 ng/ml, or less.
In some embodiments of the present invention, RecA inhibitors are broad spectrum in that they inhibit RecA (or the relevant RecA homolog) from more than one different microbial source. In some embodiments, however, the RecA inhibitors (which may be broad spectrum with regard to microbes) do not inhibit RecA (or the relevant RecA homolog) from one or more higher organisms (e.g., mammals, humans). For example, in some embodiments, inventive RecA inhibitors do not inhibit RAD51. In some embodiments, the present invention provides RecA inhibitors that inhibit the RecA ATPase activity. For example, the present invention demonstrates that a variety of compounds inhibit RecA ATPase activity in an in vitro luciferase assay (see e.g., Examples herein). The present invention provides the compounds (including portions thereof) depicted herein as RecA inhibitors with the indicated IC5oS in the in vitro luciferase assay.
In some embodiments, inventive RecA inhibitors that bind directly to RecA bind to a site comprised of amino acid residues including R85, F270, Y271 , K310, and/or R324. According to the present invention, this site can be found on the outer surface of RecA, as positioned in a filament. According to the present invention, hinokiflavone may bind to this RecA site. Further according to the present invention, compounds that compete with hinokiflavone for binding to RecA may be desirable RecA inhibitors., inventive RecA inhibitors do not bind to the RecA ATP binding site. In certain embodiments, inventive RecA inhibitors bind two or more different sites on the RecA protein. In some embodiments of the present invention, RecA inhibitors are small molecule agents, typically having some cyclic character (e.g., including one or more aryl rings). Certain RecA inhibitors according to the present invention are flavones or isoflavones; certain RecA inhibitors according to the present invention are bisflavones. In some embodiments of the invention, the RecA inhibitor is or includes hinokiflavone.
In general, a small molecule is understood in the art to be an organic molecule that is less than about kilodaltons (Kd) in size. In some embodiments, the small molecule is less than about 3 Kd, 2 Kd, or 1 Kd. In some embodiments, the small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, small molecules are non-polymeric. In some embodiments, small molecules are not amino acids. In some embodiments, small molecules are not nucleotides. In some embodiments, small molecules are not saccharides. A "subject", as used herein, is an individual to whom an agent is to be delivered, e.g., for experimental, diagnostic, and/or therapeutic purposes. Subjects of interest herein include animals, particularly agriculturally significant animals or companion animals (e.g., ruminants, cows, steer, sheep, goats, horses, swine, dogs, cats, rabbits, birds, fish, etc.), zoo animals (giraffe, bears, zebra, lions, tigers, etc.), reptiles, laboratory animals (e.g., mice, rats), mammals, primates, or humans.
A "sublethal" concentration of an antibiotic refers to a concentration that is less than the MIC of the antibiotic. In certain embodiments of the invention a sublethal concentration is not sufficient to significantly reduce the growth rate (proliferation) of a microbial cell, e.g., the growth rate is reduced by less than 20%, preferably less than 10%. Such a concentration is referred to herein as a "non- inhibiting concentration". A "lethal" concentration of an antibiotic is one that is equal to or greater than the MIC and would ultimately result in microbial death and complete or essentially complete sterilization of a culture medium containing the microbe if continued indefinitely assuming that no resistant strains arise during the incubation period.
As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen; -(CH2)o_4R°; -(CH2)<MORO; -0-(CH2)O-4C(O)OR0; -(CH2)o-4CH(OR°)2; -(CH2)(MSR°; -(CH2)(MPh, which may be substituted with R°; -(CH2)o_4θ(CH2)o_iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -NO2; -CN; -N3; -(CH2)(MN(R°)2; -(CH2)o_4N(R°)C(0)R0; -N(R°)C(S)R°; -(CH2)(MN(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2)o-4N(R°)C(0)OR°; -N(R°)N(R°)C(0)R°; -N(R°)N(R°)C(O)NR°2; -N(RO)N(R°)C(O)OR°; -(CH2)(MC(0)R°; -C(S)R0; -(CH2)0-4C(O)OR°; -(CH2)O_ 4C(O)SR°; -(CH2)o-4C(0)OSiR°3; -(CH2)0^OC(O)R°; -OC(O)(CH2)0-4SR-, SC(S)SR0; -(CH2)(MSC(O)R0; -(CH2)o-4C(0)NR°2; -C(S)NR°2; -C(S)SR0; -
SC(S)SR0, -(CH2)(MOC(O)NR°2; -C(O)N(ORO)R°; -C(O)C(O)R0; -C(O)CH2C(O)R0; -C(NOR°)R°; -(CH2)o-4SSR°; -(CH2)(MS(O)2R°; -(CH2)(MS(O)2ORO; -(CH2)O_ 4OS(O)2R°; -S(O)2NR°2; -(CH2)(MS(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -P(O)2R0; -P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; SiR°3; - (Ci-4 straight or branched alkylene)O-N(R°)2; or -(Ci-4 straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, Ci_6 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-ό-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bi cyclic ring having 0^- heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.
Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o_2R*, -(haloR*), -(CH2)o-2OH, -(CH2)o-2OR#, -(CH2)0- 2CH(OR*)2; -O(haloR#), -CN, -N3, -(CH2)o-2C(0)R#, -(CH2)0-2C(O)OH, -(CH2)o_ 2C(O)OR#, -(CH2)O-2SR', -(CH2)O-2SH, -(CH2)o_2NH2, -(CH2)0-2NHR#, -(CH2)0_ 2NR# 2, -NO2, -SiR*3, -OSiR*3, -C(O)SR', -(Ci-4 straight or branched alkylene)C(O)OR#, or -SSR* wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from C i-4 aliphatic, -CH2Ph, -0(CH2)o_iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 (inclusive) heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.
Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =O, =S, =NNR* 2, =NNHC(O)R*,
=NNHC(O)OR*, =NNHS(O)2R*, =NR*, =NOR*, -O(C(R* 2))2_3O- or -S(C(R* 2))2_3S- , wherein each independent occurrence of R* is selected from hydrogen, Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0_4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted" group include: - O(CR* 2)2_3O-, wherein each independent occurrence of R* is selected from hydrogen, Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on the aliphatic group of R include halogen, -R', -(haloR'), -OH, -OR', -O(haloR'), -CN, -C(O)OH, -C(O)OR', -NH2, -NHR', -NR'2, or -NO2, wherein each R' is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C^ aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R+, -NR+ 2, -C(O)R+, -C(O)OR+, -C(O)C(O)R+, -C(O)CH2C(O)R+, -S(O)2R+, -S(O)2NR+ 2, -C(S)NR+ 2, -C(NH)NR+ 2, or -N(R+)S(O)2R+; wherein each R+ is independently hydrogen, Ci_6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R+, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on the aliphatic group of R+ are independently halogen, -R', -(haloR'), -OH, -OR', -O(haloR'), -CN, -C(O)OH, -C(O)OR', -NH2, -NHR', -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_4 aliphatic, - CH2Ph, -0(CH2)o_iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. As used herein, the term "treatment" refers to the provision of any type of medical or surgical management to a subject. Treating can include, but is not limited to, administering a pharmaceutical composition to a subject. Treating is typically undertaken in an effort to alter the course of a disease, disorder, or undesirable condition in a manner beneficial to the subject. The effect of treating can generally include reversing, alleviating, reducing, delaying the onset of, inhibiting the progression of, and/or reducing the likelihood of occurrence of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder or condition. A composition of this invention can be administered to a subject who has developed an infection or is at increased risk of developing an infection relative to a member of the general population. A composition of this invention can be administered prophylactically, i.e., before development of any symptom or manifestation of a condition. Typically in this case the subject will be at risk of developing the condition. The composition can be administered prior to exposure of the subject to an infectious agent or prior to the occurrence of a pathogenic event.
The term "stable compounds", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., formulation into therapeutic products, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds, treating a disease or condition responsive to therapeutic agents).
A salt of a compound of this invention is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. According to another preferred embodiment, the compound is a pharmaceutically acceptable acid addition salt. Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic, lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenyl acetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2- sulfonate, mandelate and the like salts. Preferred pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.
As used herein and unless otherwise indicated, the term "prodrug" means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of this invention. Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds of any one of the formulae disclosed herein that comprise moieties such as amides, esters, carbamates, carbonates, ureides, and phosphate analogues. Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs".
The term "pharmaceutically acceptable," as used herein, refers to a component that is suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. As used herein, the term "hydrate" means a compound which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
As used herein, the term "solvate" means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, ethanol, methanol, dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The RecA protein is a key sensor and activator in response to DNA damage and plays a major role in inducing the SOS response pathway following such damage. It is known that RecA is also involved in other cellular processes in addition to recombination and DNA damage repair.
One fundamental event in both homologous recombination and SOS response induction is the formation of a RecA-ssDNA-ATP nucleoprotein filament. In this conformation, RecA acts as both a recombinase and co-protease. In the latter function, it activates the SOS response by cleaving the LexA repressor protein, which results in the induction of genes that are repressed by LexA under normal conditions. Over 30 SOS genes, and UmuD, a sub-unit of polymerase IV, also involved in the SOS response are induced (Courcelle & Hanawalt, Annu Rev Genet. 37:61 1-646, 2003; Sutton et al., Annu Rev Genet. 34:479, 2000). Another fundamental role of RecA is to maintain the integrity of the genetic material. The binding of RecA to single-stranded DNA regions that block replication forks serves as the sensor that replication is blocked and maintains the integrity of the replication fork itself until replication can resume (Courcelle & Hanawalt, Annu Rev Genet. 37:611-646, 2003). The RecA protein is highly conserved and is cross species functional. For example, recA homologs from Yersinia pestis, Bacillus anthracis and M. tuberculosis have been shown to complement the E. coli recA- mutation (Suchkov & Mishan'kin, MoI Gen Mikrobiol Virusol. 5:34, 1989; Ko et al., J Bacteriol. 184:3917, 2002; Nair & Steyn, J Gen Microbiol. 137:2409, 1991 ). Thus inhibitors of RecA identified using the RecA protein from one species would be expected to show activity in a wide variety of bacteria.
The RecA protein has many functional features that present points of intervention to inhibit its activity in accordance with the present invention. Multiple alignment of the sequence reveals a canonical structure of RecA-like proteins consisting of distinct segments or motifs. These segments or modules are highly conserved and have been assigned functional roles based on genetic, biochemical and structural studies. Such modules are involved in DNA damage recognition and binding, monomer interaction, filament formation, helicase motifs, ATP binding and hydrolysis, recombination, replication and co-protease activity. Mutational studies have identified residues that are critical to these processes.
For example, the Glyl57 change generates a constitutive co-protease form of RecA and results in a lower survival in response to UV treatment, a phenotype itself associated to a low recombination competent form of RecA. The present invention makes use of this information to precisely map regions of RecA to be targeted for compound discovery, e.g., using computational approaches (see below).
For example, RecX (also called OraA) is an inhibitor of RecA for both recombinase and co-protease activities (Stohl et al., J Biol Chem. 278:2278, 2003). RecX appears to inhibit the ATP ase activity of RecA. Genetic and biochemical evidence identifies sites of interaction between RecA and LexA, suggesting that amino acids at positions 67, 154-157, 229 and 243 are responsible at least in part for the binding to LexA (VanLook et al., J MoI Biol. 333:345, 2003). Also, amino acid changes at positions 122-123 and 150-161 dramatically decrease the ability of mutant cells ability to survive in response to UV radiation treatment. A domain in RecA that likely forms part of the co-protease substrate binding site has also been identified (Nastri et al., MoI Microbiol. 25:967, 1997).
Exemplary structural classes of antibiotics for use in combination with RecA inhibitors that provide compounds of according to the present invention include, but are not limited to, quinolones (e.g., cinoxacin, nalidixic acid, pipemidic acid, cipro floxiacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and sparfloxacin), fluoroquinolones, β-lactams (penicillins, e.g., ampicillin, ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate, lanate, piperacillin, piperacillin/tazoba, ctam, pencillin G, pencillin V, azlocillin, carbenicillin, mezlocillin, ticarcillin, ticarcillin/clavulanate, or cephalosporins, e.g., cefazolin, cephalexin, cephalothin, cephradine, cefaclor, cefamandole, cefuroxime, loracarbef, cefepime, cefpirome, cefoselis, cefixime, cefatazime, cefpodoxime, ceftazidime, ceftizoxime, cefoperazone, cefoperazone/sulbactam, and ceftriaxone), carbapenems (e.g., ertapenem, faropenem, imipenem, and meropenem), aminoglycosides (e.g., amikacin, arbekacin, gentamicin, kanamycin, netilmicin, neomycin, tobramycin, and strptomysin), macrolides (e.g., azithromycin, clarithromycin, erythromycin, midecamycin, roxithromycin, spiramycin, and telithromycin), ketolides, tetracyclines (e.g., chlortetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline), glycycyclines, lincomycins, oxazolidinones (e.g., linezolid), amphenicols (e.g., chloramphenicol and thiamphenicol), ansamycins (e.g., rifabutin, rifampin, and rifaximin), polymyxins (e.g., colistin and polymyxin B), aminomethlycyclines, lincosamides (e.g., clindamycin and lincomycin), streptogramins (e.g. quinupristin/dalfopristin and pristinamycin), 2,4-diaminopyrimidines, nitrofurans (e.g., metronidazole and tinidazole), sulfonamides (para-aminobenzoic acid, pyrimethamine, sulfadiazine, sulfamethoxazole, sulfapyridine, sulfisoxazole, and trimethoprim), sulfones, rifabutins, dapsones, peptides, glycopeptides (e.g., teicoplanin, vancomycin), lipopeptides (e.g., daptomycin), cephamycins (e.g., cefotetan and cefoxitin), clofazimine monobactams (e.g., aztreonam), amidonopenicillins (e.g., mecillinam), spectinomycins, cyclic polypeptides (e.g., bacitracin), fosfomycin, fusidic acid, mupirocin, nitroimidazoles, cycloserine, ethambutol, ethionamide, isoniazid, para- aminosalicyclic acid, pyranzinamde, and combinations thereof.
Exemplary mechanistic classes of antibiotics for use in combination with RecA inhibitors according to the present invention include, but are not limited to, those that inhibit protein synthesis, cell wall synthesis, DNA replication, transcription, and/or cell division. It will be appreciated that biological and biochemical pathways are not mutually exclusive and that some biological or biochemical pathways may be considered to be subsets or sub-pathways of other biological or biochemical pathways. Mechanisms of action more specifically include, but are not limited to, inhibiting protein synthesis (e.g., by binding ribosomal RNA or proteins, blocking tRNA binding to ribosome-mRNA complex, inhibiting peptidyl transferase), inhibiting or interfering with synthesis of a cell wall component (e.g., inhibition of peptidoglycan synthesis, disruption of peptidoglycan cross-linkage, disruption of movement of peptidoglycan precursors, disruption of mycolic acid or arabinoglycan synthesis), cell membrane disruption, inhibiting or interfering with nucleic acid synthesis or processing, acting as "antimetabolites" and either inhibiting an essential bacterial enzyme or competing with a substrate of an essential bacterial enzyme, inhibiting or interfering with cell division.
Quinolone antibiotics are compounds that contain a quinolone or a naphthyridine nucleus with any of a variety of different side chains and substituents. Numerous modifications of the originally identified core structures have been made resulting in a large number of compounds with activity against differing groups of bacteria. Quinolone antibiotics are described, e.g., in Ronald, A.R. and Low, D. E., (eds.), "Fluoroquinolone Antibiotics", Birkhauser Verlag, Basel, 2003; DaSilva, et al., Curr Med Chem 10(l):21, 2003; Van Bambeke et al., Clin Microbiol. Infect., 1 1 :256, 2005; U.S. Pat. Nos. 3,669,965; 4,563,459; 4,620,007; 4,382,892; 4,985,557 5,053,407; and 5,142,046). Exemplary quinolone antibiotics include, but are not limited to, any of the antibacterial agents disclosed in the foregoing references including, but not limited to, ciprofloxacin, oxolinic acid, cinoxacin, flumequine, miloxacin, rosoxacin, pipemidic acid, norfloxacin, enoxacin, moxifloxacin, gatifloxacin, ofloxacin, lomefloxacin, temafloxacin, fleroxacin, pefloxacin, amifloxacin, sparfloxacin, levofloxacin, clinafloxacin, nalidixic acid, enoxacin, grepafloxacin, levofloxacin, lomefloxacin norfloxacin, ofloxacin, trovafloxacin, olamufloxacin, cadrofloxacin, alatrofloxacin, gatifloxacin, rufloxacin, irloxacin, prulifloxacin, pazufloxacin, gemifloxacin, sitafloxacin, tosulfloxacin, amifloxacin, nitrosoxacin-A, DX-619, and ABT-492. Quinolone antibiotics include fluoroquinolones (e.g., having a fluorine substituent at the C-6 position), and non-fluoroquinolones. Also included within the scope of quinolone antibiotics are derivatives in which a quinolone is conjugated with, e.g., covalently bound to, another core structure. For example, U.S. Pub. No. 20040215017 discloses compounds in which an oxazolidinone, isoxazolinone, or isoxazoline is covalently bonded to a quinolone. Included within the scope of quinolone antibiotics that can be utilized in accordance with the present invention are compounds that have a core structure related to the 4-oxo-l,4-dihydroquinoline and 4-oxo-l,4 dihydronapthyridine systems, e.g., 2-pyridones, 2-naphthyridinones, and benzo[b]napthyridones.
In one aspect, the Rai group in Formula (AI) has the structure of Formula (I): (D
Figure imgf000018_0001
or a pharmaceutically acceptable salt, hydrate, solvate, or derivative thereof, wherein: X is oxygen, sulfur, or N(R); Each n is independently 0, 1 , 2, 3 or 4; R1 is hydrogen, or an optionally substituted group selected from a Ci-6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R2 is independently halogen, R3, OR3, SR3, S(O)R3, N(R3)2, C(O)R3,
C(O)OR3, NR3C(O)R3, C(O)NR3, OC(O)NHR3, NHR3C(O)O, NHR3C(O)NHR3, P(O)(OR3)2, SO2R3, NR3SO2R3, SO2N(R3)2; each R3 is independently hydrogen or an optionally substituted group selected from a Ci-6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0—5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched Ci_6 hydrocarbon chain, wherein 0-2 methylene units of Q are independently replaced by -0-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRC(O)-, -C(O)NR-, -OC(O)NR-, NRC(O)NR or -NRC(O)O-; each R is independently hydrogen or an optionally substituted aliphatic group; and Ring A is an optionally substituted 3-8 membered bivalent, saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; wherein the structure of Formula (I) can be attached to L at any atom in Formula (I) (e.g., as depicted generally by the wavy line bond in Formula (I)).
Other aspects of Formula (I) (and formulae herein) are those: wherein X is O; wherein Abl is selected from quinolones, β-lactams (e.g., penicillins or cephalosporins), carbapenems, aminoglycosides, macrolides, ketolides, tetracyclines, glycycyclines, lincomycins, oxazolidinones, amphenicols, ansamycins, polymyxins, aminomethlycyclines, lincosamides, streptogramins, 2,4-diaminopyrimidines, nitrofurans, sulfonamides, sulfones, rifabutins, dapsones, peptides, glycopeptides, or combinations thereof; wherein Abl is a quinolone antibiotic; wherein L comprises between 2-15 atoms selected from carbon, nitrogen or sulfur; wherein L is divalent alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, wherein any carbon atom is replaced with a heteroatom selected from N, O, S, or P, and any atom is optionally substituted with H, alkyl, oxo, hydroxyl, alkoxy, thioalkoxy, NH2, NRR, C(O)OR, S(O), S(0)2, OC(O)OR, OC(O)NR, RNC(O)NR or C(O)NRR; wherein L comprises a moiety that is an ester, thioester, amide, cycloalkyl, heterocycloalkyl, hydroxyl, alkoxyl, sulfoxide, sulfonate, phosphate, phosphonate, carbamate, carbonate, urea, thiourea, primary or secondary amine, hydroxylamine , oxime or imine; wherein L is attached to Rai and Abl , respectively, by carbon atoms on L; wherein L is attached to Raι and Abl , respectively, by one carbon atom and one heteroatom on L; or wherein L is attached to Rai and A ' , respectively, by heteroatoms atoms on L;
As defined generally above, the R1 group of formula I is hydrogen, or an optionally substituted group selected from a Ci-6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, the R1 group of formula I is a bicyclic 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In still other embodiments, R1 is a bicyclic 10 membered partially unsaturated ring having one oxygen atom optionally substituted with 1 to 3 substituents independently selected from halogen, -(CH2)O-4R0, -(CH2)CMORO, -(CH2)CMSRO, -(CH2)O-4Ph, optionally substituted with R° or OR0, -(CH2)0-40(CH2)o-iPh optionally substituted with R° or OR0, -CH=CHPh, optionally substituted with R° or OR°, -(CH2)o-4N(R°)2, wherein each R° may be substituted as defined herein and is independently hydrogen, Ci_6 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. According to one embodiments, R1 is substituted with 1 to 3 groups independently selected from OH and OMe.
According to one embodiment of the invention, the R1 group of formula I is selected from any of the following groups:
Figure imgf000020_0001
wherein each wavy line depicts the point of attachment to Q and the depicted bond can be attached at any atom of the depicted structure (i.e., attached at any atom in place of a depicted hydrogen atom) or
Figure imgf000020_0002
wherein each wavy line depicts the point of attachment to Q.
According to another embodiment of the invention, the R1 group of formula I is selected from any of the following groups:
Figure imgf000020_0003
wherein each wavy line depicts the point of attachment to Q and the depicted bond can be attached at any atom of the depicted structure (i.e., attached at any atom in place of a depicted hydrogen atom) or
Figure imgf000021_0001
wherein each wavy line depicts the point of attachment to Q.
In certain embodiments, the R1 group of formula I is a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In other embodiments, the R1 group of formula I is a monocyclic 5-6 membered aryl ring having 0-2 nitrogen atoms, wherein R1 is optionally substituted with 1 to 3 substituents independently selected from halogen, -(CH2)o-4R0, -(CH2)<M0R°, -(CH2)(MSR°, -(CH2)o^Ph, optionally substituted with R° or OR°,
Figure imgf000021_0002
optionally substituted with R° or OR°, -CH=CHPh, optionally substituted with R° or OR°, -(CH2)0-4N(R°)2, wherein each R° may be substituted as defined herein and is independently hydrogen, C|_6 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0—4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. According to one embodiment of the present invention, R1 is phenyl substituted with 1 to 3 groups independently selected from halogen, -(CH2)O^4R0, and -(CH2)o_4θR°. Such groups include chloro, fluoro, bromo, OH, OMe, methyl, ethyl, propyl, cyclopropyl, isopropyl, and the like. In some embodiments, OH or OMe groups are present.
According to another embodiment, the R1 group of formula I is selected from:
Figure imgf000021_0003
wherein each wavy line indicates the point of attachment to Q. As defined generally above, the Q group of formula I is a valence bond or a bivalent, saturated or unsaturated, straight or branched Ci_6 hydrocarbon chain, wherein 0-2 methylene units of Q are independently replaced by -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRC(O)-, -C(O)NR-, -0C(0)NR-,-NRC(0)0- or -NRC(O)NR-. In certain embodiments, Q is a valence bond such that R1 is directly attached to Ring A. In other embodiments, Q is a bivalent, saturated, and straight Ci-3 hydrocarbon chain, wherein 0-1 methylene units of Q is replaced by -0-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRC(O)-, -C(O)NR-, -OC(O)NR-, or -NRC(O)O-. In still other embodiments, Q is -0-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRC(O)-, -C(O)NR-, -OC(O)NR-, -NRC(O)O- or -NRC(O)NR. According to another embodiment, Q is -O-.
As defined generally above, the Ring A group of formula I is an optionally substituted 3-8 membered bivalent, saturated, partially unsaturated, or aryl monocyclic ring having 0—4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, Ring A is an optionally substituted 3-8 membered bivalent, saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In other embodiments, Ring A is an optionally substituted 5-6 membered bivalent aryl ring having 0-2 nitrogen atoms. In still other embodiments, Ring A is phenylene optionally substituted with 1 to 4 groups independently selected from halogen, -(CH2)<MR°, -(CH2)O^OR°, -(CH2)(^SR0, -(CH2)(MPh, optionally substituted with R° or 0R°, -(CH2)o-4θ(CH2)0-iPh optionally substituted with R° or 0R°, -CH=CHPh, optionally substituted with R° or OR°, -(CH2)0-4N(R°)2, wherein each R° may be substituted as defined herein and is independently hydrogen, Ci_6 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0—4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. According to another embodiment, Ring A is phenylene optionally substituted with 1-2 groups independently selected from halogen,
Figure imgf000022_0001
and -(CH2)o_4θR°. Such groups include chloro, fluoro, bromo, OH, OMe, methyl, ethyl, propyl, cyclopropyl, isopropyl, and the like. In some embodiments, OH and/or OMe group(s) are present.
As defined generally above, each R2 group of formula I is independently halogen, R3, OR3, SR3, S(O)R3, N(R3)2, C(O)R3, C(O)OR3, NR3C(O)R3, C(O)NR3, SO2R3, NR3SO2R3, SO2N(R3)2, NR3C(O)NR3 wherein each R3 is independently hydrogen or an optionally substituted group selected from a Ci-6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, each R2 group is independently halogen, R3, OR3, SR3, S(O)R3, S(O)2R3 or N(R3)2, wherein each R is as defined above. According to one embodiment, at least one R group is OH. According to another embodiment, one R2 group is R3 wherein R3 is an optionally substituted bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In still other embodiments, one R2 group is R3 wherein R3 is a bicyclic 10 membered partially unsaturated ring having one oxygen atom optionally substituted with 1 to 3 substituents independently selected from halogen, -(CH2)0_4R°, -(CH2)o-4θR°, -(CH2)O-^SR0, -(CH2)(MPh, optionally substituted with R° or OR0, -(CH2)O^O(CH2)O-. iPh optionally substituted with R° or 0R°, -CH=CHPh, optionally substituted with R° or 0R°, -(CH2)o-4N(R°)2, wherein each R° may be substituted as defined herein and is independently hydrogen, Ci_6 aliphatic, -CH2Ph, -O(CH2)0-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. According to one embodiments, R3 is substituted with 1 to 3 groups independently selected from OH and OMe and optionally substituted phenyl.
According to some embodiments of the invention, the R2 group formula I is OH, OMe, or is selected from any of the following groups:
Figure imgf000023_0001
Figure imgf000024_0001
wherein each wavy line depicts the point of attachment to Q and the depicted bond can be attached at any atom of the depicted structure (i.e., attached at any atom in place of a depicted hydrogen atom).
According to some embodiments of the invention, the R2 group formula I is OH, OMe, or is selected from any of the following groups:
Figure imgf000024_0002
wherein each wavy line depicts the point of attachment to Q and the depicted bond can be attached at any atom of the depicted structure (i.e., attached at any atom in place of a depicted hydrogen atom).
In certain embodiments, the instant invention provides for the compounds of Formula (Al) delineated below:
Figure imgf000025_0001
The compounds from the following examples can be prepared by linking the RecA inhibitor compound (with or without suitable protecting group manipulation) to the antibacterial compound via, but not limited to, ester, amide, ether,oxime, imine or amine linkage. It will be apparent to one skilled in the art that many such combinations of RecA inhibitors and antibiotics are possible. The following examples are meant to be illustrative only and not limiting.
Figure imgf000025_0002
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
The invention also provides compositions comprising an effective amount of a compound of any of the formulae herein (e.g., including Formula I, Al, etc.), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph or prodrug, if applicable, of said compound; and an acceptable carrier. Preferably, a composition of this invention is formulated for pharmaceutical use ("a pharmaceutical composition"), wherein the carrier is a pharmaceutically acceptable carrier. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in amounts typically used in such compositions.
Other embodiments of the present invention include the following: a pharmaceutical composition comprising an effective amount of a compound of Formula Al and a pharmaceutically acceptable carrier; a pharmaceutical composition which comprises the product prepared by combining (e.g., mixing) an effective amount of a compound of Formula Al and a pharmaceutically acceptable carrier; the pharmaceutical composition(s) above, further comprising an effective amount of an additional therapeutic agent, for example, anti-infective agents, antibiotic agents, β-lactamase inhibitor agents, antiviral agents, antimicrobial agents, and the like. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. The pharmaceutical compositions of the invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. In certain embodiments, the compound of the formulae herein is administered transdermally (e.g., using a transdermal patch techniques). Other formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA (17th ed. 1985).
Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product. In certain preferred embodiments, the compound is administered orally.
Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, 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 optionally may be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. Methods of formulating such slow or controlled release compositions of pharmaceutically active ingredients, such as those herein and other compounds known in the art, are known in the art and described in several issued US Patents, some of which include, but are not limited to, US Patent Nos. 4,369, 172; and 4,842,866, and references cited therein. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
Compositions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.
Compositions suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
Such injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 ,3- butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
The pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches and ionophoretic administration are also included in this invention.
Thus, according to yet another embodiment, the compounds of this invention may be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents, or catheters. Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in US Patents 6,099,562; 5,886,026; and 5,304, 121. The coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition. Coatings for invasive devices are to be included within the definition of pharmaceutically acceptable carrier, adjuvant or vehicle, as those terms are used herein.
When administered intravenously, the compounds of the present invention, are preferably administered using the formulations of the invention. Generally, the compounds of the present invention are administered by IV infusion over a period of from about 10 minutes to about 24 hours, e.g., about 30 minutes to about 4 hours, about 45 minutes to 90 minutes, or about 1 hour. One of ordinary skill in the art would readily know how to determine doses based on both the height and or weight of the patient.
According to another embodiment, the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by the compounds (or compositions thereof) herein comprising the step of administering to said subject an effective amount of a compound or a composition of this invention. The present invention thus provides methods for the treatment or prevention of infection and related disease, disorders, or symptoms thereof (including those bacteria delineated herein) comprising the step of administering to a subject in need thereof at least one compound of any of the formulae herein (e.g., Formula Al) in an amount effective therefor. Other therapeutic agents such as those described below may be employed with the inventive compounds in the present methods. In the methods of the present invention, such other therapeutic agent(s) may be administered prior to, simultaneously with or following the administration of the compound(s) of the present invention. Other aspects of the methods include those further comprising wherein a subject sample is assessed; and also further with one or more of: assessing bacterial levels of the subject; assessing that is performed prior to administration of the compound; assessing that is performed after administration of the compound; or assessing that is performed both prior to and after administration of the compound.
Methods delineated herein include those wherein the subject is identified as in need of a particular stated treatment. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). For example, such methods include clinical diagnosis based at least in part on symptoms (e.g., diagnosed or subject self-reported), imaging studies, immunodiagnostic assays, nucleic acid based diagnostics, and/or isolation and culture of potentially causative microorganisms from samples, such as blood, urine, sputum, synovial fluid, cerebrospinal fluid, pus, or any sample of body fluid or tissue.
Inventive compounds herein are useful to inhibit growth of a wide variety of microbial types including, for example, gram negative bacteria, gram positive bacteria and/or acid fast bacteria. Particular examples of bacteria whose growth or proliferation can be inhibited include, but are not limited to, members of the following genuses: Actinomyces, Staphylococcus, Streptococcus, Enterococcus, Erysipelothrix, Neisseria, Branhamella, Listeria, Bacillus, Corynbacterium, Erysipelothrix, Gardnerella, Mycobacterium, Nocardia, Enterobacteriaceae, Escherichia, Salmonella, Shigella, Yersinia, Enterobacter, Klebsiella, Citrobacter, Serratia, Providencia, Proteus, Morganella, Edwardsiella, Erwinia, Vibrio, Aeromonas, Helicobacter, Campylobacter, Eikenella, Pasteurella, Pseudomonas, Burkholderia, Stenotrophomonas, Acinetobacter, Ralstonia, Alcaligenes, Moraxella, Mycoplasma, Legionella, Francisella, Brucella, Haemophilus, Bordetella, Clostridium, Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Borrelia, Chlamydia, Rickettsia, Ehrlichia, Bartonella, Trichomonas, Treponema, and combinations thereof (i.e., infections established by more than one bacterial strain).
In particular embodiments of the invention the bacteria are species that are causative agents of disease in humans and/or animals. Examples include, but are not limited to, Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Enterobacter cloacae, Campylobacter jejuni, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella oxytoca, Legionella pneumophila, Pasteurella multocida, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Helicobacter pylori, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhimuήum, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and combinations thereof.
In certain embodiments of the invention, the bacterial species or strain is one that is sensitive to a particular antibiotic agent or class of antibiotic agents; in some embodiments, the bacterial species or strain is sensitive to one or more bacterial type II topoisomerase inhibitors such as a quinolone antibiotic.
Infections and infection-related conditions that can be treated using an inventive compound herein (and optionally another antibiotic agent) include, but are not limited to, pneumonia, meningitis, sepsis, septic shock, sinusitis, otitis media, mastoiditis, conjunctivitis, keratitis, external otitis (e.g., necrotizing otitis externa and perichondritis), laryngeal infections (e.g., acute epiglottitis, croup and tuberculous laryngitis), endocarditis, infections of prosthetic valves, abscesses, peritonitis, infectious diarrheal diseases, bacterial food poisoning, sexually transmitted diseases and related conditions, urinary tract infections, pyelonephritis, infectious arthritis, osteomyelitis, infections of prosthetic joints, skin and soft tissue infections, oral infections, dental infections, nocardiosis and actinomycosis, mastitis, brucellosis, Q fever, anthrax, wound infections, etc.
In certain embodiments of the invention, the bacterial species or strain is one that is resistant to a particular antibiotic agent or class of antibiotic agents; in some embodiments, the bacterial species or strain is resistant to one or more bacterial type II topoisomerase inhibitors such as a quinolone antibiotic.
Inventive compounds and compositions containing them can be used to inhibit microbial growth and/or survival in a variety of contexts. For example, they may be employed to inhibit growth and/or survival of organisms maintained in cell culture or inhabiting locations in the environment, e.g., inert surfaces, clothing, towels, bedding, utensils, etc. Of particular interest are fomites, i.e., inanimate objects that may be contaminated with disease-causing microorganisms and may serve to transmit disease to a human or animal. Such locations or objects can be contacted with a solution containing an inventive compound, and optionally including one or more other antibiotic agents. Inventive compounds herein, alone or together with one or more other antibiotic agents, can be added to food or water, particularly for the prevention of microbial disease in animals. In other aspects, the methods herein include those further comprising monitoring subject response to the treatment administrations. Such monitoring may include periodic sampling of subject tissue, fluids, specimens, cells, proteins, chemical markers, genetic materials, etc. as markers or indicators of the treatment regimen. In other methods, the subject is prescreened or identified as in need of such treatment by assessment for a relevant marker or indicator of suitability for such treatment.
In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target or cell type delineated herein modulated by a compound herein, bacterial type or count) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof delineated herein, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.
In certain method embodiments, a level of Marker or Marker activity in a subject is determined at least once. Comparison of Marker levels, e.g., to another measurement of Marker level obtained previously or subsequently from the same patient, another patient, or a normal subject, may be useful in determining whether therapy according to the invention is having the desired effect, and thereby permitting adjustment of dosage levels as appropriate. Determination of Marker levels may be performed using any suitable sampling/expression assay method known in the art or described herein. Preferably, a tissue or fluid sample is first removed from a subject. Examples of suitable samples include blood, urine, tissue, mouth or cheek cells, and hair samples containing roots. Other suitable samples would be known to the person skilled in the art. Determination of bacterial levels, protein levels and/or mRNA levels (e.g., Marker levels) in the sample can be performed using any suitable technique known in the art, including, but not limited to, enzyme immunoassay, ELISA, radiolabelling/assay techniques, blotting/chemiluminescence methods, realtime PCR, and the like.
The term "co-administered" as used herein means that the second therapeutic agent may be administered together with a compound of this invention as part of a single dosage form (such as a composition of this invention comprising a compound of the invention and an second therapeutic agent as described above) or as separate, multiple dosage forms. Alternatively, the additional agent may be administered prior to, consecutively with, or following the administration of a compound of this invention. In such combination therapy treatment, both the compounds of this invention and the second therapeutic agent(s) are administered by conventional methods. The administration of a composition of this invention comprising both a compound of the invention and a second therapeutic agent to a subject does not preclude the separate administration of that same therapeutic agent, any other second therapeutic agent or any compound of this invention to said subject at another time during a course of treatment.
In yet another aspect, the invention provides the use of a compound of the formulae herein (e.g., formula AI, formula I) alone or together with one or more of the above-described second therapeutic agents in the manufacture of a medicament, either as a single composition or as separate dosage forms, for treatment or prevention in a subject of a disease, disorder or symptom set forth above. Another aspect of the invention is a compound of the formulae herein for use in the treatment or prevention in a subject of a disease, disorder or symptom thereof delineated herein. Recitation of a numerical range includes each individual number in that range as well as is inclusive of the delineated beginning and ending numbers of the range. For example, a recitation of "n is independently 0-4" means n can be 0, 1 , 2, 3 or 4 at any designated instance. The synthesis of compounds of the formulae herein can be readily effected by synthetic chemists of ordinary skill. A convenient method for producing compounds of the formulae herein is provided in the schemes herein. Other approaches to synthesizing compounds of the formulae herein can readily be adapted from references cited herein. Variations of these procedures and their optimization are within the skill of the ordinary practitioner.
For example, the compounds delineated herein can be made from reaction (nucleophilic displacement, nucleophilic substitution, esterification, etherifϊcation, alkylation,oxime formation, imine formaton, etc.) of appropriate recA or antibiotic compounds (or appropriate derivatives thereof) and appropriate linker moiety precursors. Such chemical reactions are known in the art and are also described in the references herein.
The chemical structures in the schemes herein depict variables that are hereby defined commensurately with chemical groups (moieties, atoms, etc.) of the corresponding position in the compound formulae herein, whether identified by the same variable name (e.g., any R group (Ri, R2, etc.), A, L, X, Q, etc.) or not. The suitability of a chemical group in a compound structure for use in synthesis of another compound structure is within the knowledge of one of ordinary skill in the art. Scheme 1. Synthesis
Raι-L-Abl
Figure imgf000038_0001
P = protecting group
LG = leaving group, e.g., halo, sulfonate, alkoxy The specific approaches and compounds shown above are not intended to be limiting. Additional methods of synthesizing compounds of the formulae herein and their synthetic precursors, including those within routes not explicitly shown in schemes herein, are within the means of chemists of ordinary skill in the art. Methods for optimizing reaction conditions, if necessary minimizing competing by-products, are known in the art. In addition, various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the applicable compounds are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser 's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.
The synthetic methods described herein may also additionally include steps, either before or after any of the steps described in the preceding schemes, to add or remove suitable protecting groups in order to ultimately allow synthesis of the compound of the formulae described herein. The methods delineated herein contemplate converting compounds of one formula to compounds of another formula. The process of converting refers to one or more chemical transformations, which can be performed in situ, or with isolation of intermediate compounds. The transformations can include reacting the starting compounds or intermediates with additional reagents using techniques and protocols known in the art, including those in the references cited herein. Intermediates can be used with or without purification (e.g., filtration, distillation, crystallization, trituration, chromatography, etc.).
The compounds of the invention can be assessed for antibacterial activity using any of a variety of standard protocols (e.g., in vitro, in vivo, cellular, animal) known in the art.
Generally, growth or survival of cells can be assessed using cells growing in liquid media or on solid or semi-solid media. Any method known in the art can be used to determine whether an agent inhibits growth, proliferation, and/or survival. Examples include measuring optical density in liquid culture, measuring colony formation, or measuring bacterial viability. Bacterial viability can be assessed based on metabolic characteristics such as oxidation/reduction state, ability to metabolize particular substrate(s) or produce particular metabolite(s), or based on membrane integrity, which can be detected by evaluating ability of a bacterial cell to exclude a particular substance such as a detectable molecule (e.g., a fluorescent or luminescent molecule) from the cell interior. One example of a commercially available assay suitable for use in assaying compounds is the LIVE/DEAD BacLight Bacterial Viability assay (Molecular Probes, now owned by Invitrogen, Carlsbad, CA). This assay utilizes mixtures of SYTO® 9 green fluorescent nucleic acid stain and the red fluorescent nucleic acid stain, propidium iodide. These stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, the SYTO 9 stain labels bacteria with both intact and damaged membranes. Propidium iodide, however, penetrates only bacteria with damaged membranes, competing with the SYTO 9 stain for nucleic acid binding sites when both dyes are present. When mixed in recommended proportions, SYTO 9 stain and propidium iodide produce green fluorescent staining of bacteria with intact cell membranes and red fluorescent staining of bacteria with damaged membranes. The background remains virtually nonfluorescent. The ratio of green to red fluorescence intensities therefore provides a quantitative index of bacterial viability. A fiuorometer can be used to detect the fluorescence intensities. Another suitable assay for determining the number of viable bacterial cells in culture is based on quantitation of the ATP present. ATP is an indicator of metabolically active cells. The BacTiter- Glo™ Assay (Promega, Madison, WI) is a commercially available assay based on this principle that involves adding a single reagent (BacTiter-Glo™ Reagent) directly to bacterial cells in medium and measuring luminescence. Many additional assays suitable for assessing bacterial viability are described in "Handbook of Fluorescent Probes and Research Products" (Molecular Probes, 9th edition, 2002) and "The Handbook — A Guide to Fluorescent Probes and Labeling Technologies" (Invitrogen, 10th edition, available at the Invitrogen web site). Examples
Example 1: RecA Activity Assay: Luciferase ATP ase Assay RecA is a DNA-dependent ATPase (i.e., it catalyzes the reaction adenosine triphosphate [ATP] → adenosine diphosphate [ADP]). An assay for RecA ATPase activity based on detection of the amount of ATP remaining in a reaction mixture following incubation of RecA protein, DNA, and ATP is utilized for test compounds. In the assay, E. coli RecA protein is incubated in reaction buffer with DNA, ATP, and any test compound or compounds for a measured about of time. The quantity of ATP remaining after the RecA reaction is quantitated using a subsequent luciferase assay. The amount of ATP remaining can be compared to controls containing either a known inhibitor (full inhibition control) or no test compound (full activity control). Example 2. Cell-based Assay for RecA Inhibitor Compounds The recA promoter is induced when RecA becomes activated as a result of exposure to DNA damaging agents. A low copy reporter plasmid in which the recA promoter controls transcription of the mRNA encoding green flourescent protein (GFP) is used. The plasmid, referred to herein as prec::GFP, has been previously described (Ronen, 2002). See also Kuang, 2004. prec::GFP contains 2821707-2821893 of the E. coli MGl 655 genome (numbering based on the sequenced E. coli genome as reported in Blattner, F.R., et al., Science, 277, pp. 1453-1474, 1997) cloned upstream of a promoterless GFPmuύ gene in a low copy pSClOl origin plasmid as described (Kalir, S., et al., Science, 292, pp. 2080-2083, 2001).
The prec::GFP plasmid is introduced into MG1655 and MG1655 recA null strains and the fluorescence emitted by GFP in the presence or absence of quinolone as compared with background fluorescence detected from cultures of MG 1655 lacking the plasmid is measured. Cells of these three strains are grown to early log phase in LB at 370C with shaking. Various amounts of Nor (norfloxacin) are added to lOOul cultures of each strain to achieve final concentrations ranging from 0 μg/ml to 31.25 μg/ml. After 60 min, fluorescence due to GFP expression from the reporter plasmid is measured using a spectrophotometer.
It is expected that a compound that inhibits expression of RecA or inhibits any of a variety of functions of RecA will result in a decrease in GFP fluorescence in cells containing the reporter plasmid.
Example 3. In-vitro Assay for RecA Inhibitor Compounds
RecA is a DNA-dependent ATPase. One assay for RecA activity is based on detection of the amount of ATP remaining in a reaction mixture following incubation of RecA protein, DNA, and ATP. 40-50 μg/ml of RecA (New England Biolabs) is combined with 20 μM ATP in a final volume of 50 ul PNK buffer Ix either with or without and 250 ng/ul of Ml 3 DNA, and the reaction mixtures are incubated at 25°C for 30 minutes. InM ATP-Gamma, a non-hydrolyzable form of ATP that inhibits RecA, is added to some of the reactions 10 minutes after the start of the incubation. The assay measures the amount of ATPase (adenosine triphosphate (ATP) - adenosine diphosphate (ADP)) activity catalyzed by E. CoIi RecA protein. In the assay, recA is incubated in reaction buffer with DNA, ATP and compounds of interest for a measured amount of time. The quantity of ATP remaining after the RecA reaction is quantitated using a subsequent luciferase assay. The amount of ATP remaining can be compared to inhibited and fully active control samples. Representative details of the assay are provided below:
Materials-
Fisher PVR3771 Kingase glo plus
New England
Biolabs N4040S M13mpl 8 Single-stranded DNA
New England
Biolabs MO249L RecA Protein
Fisher S75022 ADENOSINE TRIPHOSPHATE(ATP)
Fisher AA448800006666M001 Adenosine 5'-O-(3-thiotriphosphate) New England Biolabs B0201 S T4 Polynucleotide Kinase Reaction Buffer = PNK Fisher 07-200-648 384 well plates, white
PNK:
70 mM Tris-CL pH 7.5 10 mM MgC12
5 mM DTT
Assay Procedure-
Complete Reaction example 0.5ul M 13 single stranded DNA (250ug/ml) l .Oul 1Ox PNK buffer
0.25 ul RecA (2mg/ml)
0.25ul lmM ATP
0.50ul compound in DMSO 12ul H2O
Potential inhibitors are left to incubate 5 min at room temp in the presence of RecA protein & DNA. Therefore, 2 mixes are made as below. A no DNA and DNA/DMSO control sample can be included.
mix 1 ATP mix M13
1Ox PNK buffer recA
ImM ATP
ImM ATP-G or compound
H20 to each
Figure imgf000043_0001
Add 7.75 ul of mix 1 to the well of a 384 well plate.
Add 0.5 ul of the compound of interest. Incubate at room temperature for 5min.
Add 2.5 ul of the ATP mix. Incubate at room temperature for 30min.
Add 12.5 ul of Promega Kinase glo plus.
Incubate at room temp for 10 min or more.
Measure luminescence of the reaction.
RecA activity in the presence of DNA can be readily detected based on the reduced luminescence detected from reactions in which ATP has been consumed by active
RecA. RecA activity is DNA dependent in this assay.
Example 4. Identification of Quinolone Potentiating Agents
Test compounds are housed in wells in microwell plates in which 100 μg of each compound is resuspended in 50 ul of DMSO (2ug/ul). The screen is performed using 384- well plates. A reaction mixture containing 2mg/ml of RecA (New England Biolabs), 250 ng/ul of Ml 3 DNA, and ImM ATP in a final volume of 50 ul PNL buffer Ix is dispensed into each well. 5ul of a different compound solution (10 μg of compound) is added to each well, one compound per well. The plate includes 4 negative control wells in which no test compound is added to the reaction mixture, and 4 positive control wells, in which the reaction mixture also contains 1 nM ATP- gamma to maximally inhibit RecA and does not contain a test compound. The plates are incubated for 1 hour, following which 50 ul of the Promega Kinase-Glo™ Reagent (Promega) is added to each well. Luminescence is measured. Test compounds in wells in which the luminescence is lower than the average luminescence detected in the negative control wells are identified as RecA inhibitors. The extent of RecA inhibition by any particular test compound is assessed by comparing the luminescence with that detected in the positive control well, which represent 100% inhibition of RecA. For each test compound, the difference between the OD600 for the well containing the test compound and Nor and the OD600 for the well containing the test compound but no Nor is calculated. If the OD600 for the well containing a particular test compound and Nor is significantly lower than the OD600 for the well containing the same test compound and not containing Nor, then the test compound is identified as a quinolone potentiating compound.
Example 5. Identification of Active Agents Using a Cell-based RecA Reporter Assay Test compounds are housed in wells in microwell plates in which 100 μg of each compound is resuspended in 50 ul of DMSO (2ug/ul). The screen is performed using 384-well plates. 50 μl of an early log phase culture of MG 1655 containing the prec:GFP plasmid are dispensed into each well, and Nor is added to a final concentration of 10 μg/ml. A different test compound is added to each well to a final concentration of 50 μg/ml. Each plate contains 4 control wells to which no test compound is added and 4 control wells to which 50 nM ATP-gamma is added to inhibit RecA. The wells are incubated at 37°C for 60 minutes, following which GFP fluorescence is detected. Compounds in wells that display decreased fluorescence relative to the control wells to which no test compound is added are identified as RecA inhibitors. The extent of RecA inhibition is assessed by comparison with fluorescence in the wells that contain ATP-gamma, which represents maximal inhibition of RecA (and thus approximately background levels of fluorescence).
All references cited herein, whether in print, electronic, computer readable storage media or other form, are expressly incorporated by reference in their entirety, including but not limited to, abstracts, articles, journals, publications, texts, treatises, technical data sheets, internet web sites, databases, patents, patent applications, and patent publications.

Claims

CLAIMS What is claimed is:
L A compound comprising a RecA inhibitor compound bonded to a second compound having a therapeutic activity, or acid addition salt, solvate, hydrate or polymorph thereof.
2. The compound of claim 1 , wherein the second compound has antibiotic activity.
3. The compound of claim 1 , wherein the RecA inhibitor and the second compound are bonded via a linker moiety.
4. The compound of claim 1 , wherein the linker moiety comprises between 2-15 atoms selected from carbon, nitrogen, oxygen or sulfur.
5. The compound of claim 1, wherein the second compound is a quinolone antibiotic.
6. The compound of claim 1, wherein the second compound is a macrocyclic antibiotic.
7. A compound of Formula Al :
Rai-L-Abi (Al) wherein,
Raι is a RecA inhibitor compound; L is a linker;
Abl is an antibiotic compound; or acid addition salt, solvate, hydrate or polymorph thereof.
8. The compound of claim 7, wherein Rai is of the formula (I), or a pharmaceutically acceptable derivative thereof, wherein:
Figure imgf000045_0001
wherein, X is oxygen, sulfur, or N(R);
Each n is independently 0, 1 , 2, 3 or 4; R1 is hydrogen, or an optionally substituted group selected from a Ci-6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R2 is independently halogen, R3, OR3, SR3, S(O)R3, S(O)2R3 N(R3)2, C(O)R3, C(O)OR3, NR3C(O)R3, C(O)NR3, SO2R3, NR3SO2R3, SO2N(R3)2; each R3 is independently hydrogen or an optionally substituted group selected from a Ci-6 aliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched Ci_6 hydrocarbon chain, wherein 0-2 methylene units of Q are independently replaced by -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -C(O)-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRC(O)-, -C(O)NR-, -OC(O)NR-, or -NRC(O)O-; each R is independently hydrogen or an optionally substituted aliphatic group; and Ring A is an optionally substituted 3-8 membered bivalent, saturated, partially unsaturated, or aryl monocyclic ring having 0-^4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
9. The compound of claim 8, wherein X is O.
10. The compound of claim 7, wherein Abl is selected from quinolones, β- lactams (e.g., penicillins or cephalosporins), carbapenems, aminoglycosides, macrolides, ketolides, tetracyclines, glycycyclines, lincomycins, oxazolidinones, amphenicols, ansamycins, polymyxins, aminomethlycyclines, lincosamides, streptogramins, 2,4-diaminopyrimidines, nitrofurans, sulfonamides, sulfones, rifabutins, dapsones, peptides, glycopeptides, or combinations thereof.
11. The compound of claim 7,wherein Abl is a quinolone antibiotic.
12. The compound of claim 7, wherein L comprises between 2-15 atoms selected from carbon, nitrogen or sulfur.
13. The compound of claim 7, wherein L is divalent alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, wherein any carbon atom is replaced with a heteroatom selected from N, O, S, or P, and any atom is optionally substituted with H, alkyl, oxo, hydroxyl, alkoxy, thioalkoxy, NH2, NRR, C(O)OR, or C(O)NRR.
14. The compound of claim 7, wherein L comprises a moiety that is an ester, amide, cycloalkyl, heterocycloalkyl, hydroxyl, sulfonate, phosphate, primary or secondary amine, or imine.
15. The compound of claim 7, wherein L is attached to Rai and Abl , respectively, by carbon atoms on L.
16. The compound of claim 7, wherein L is attached to Ra' and Abl , respectively, by one carbon atom and one heteroatom on L.
17. The compound of claim 7, wherein L is attached to Raι and Abl , respectively, by heteroatoms atoms on L.
18. A method of treating a subject suffering from infection comprising administering to the subject an effective amount of a compound according to claim 1 or 7.
19. A method of preventing infection in a subject comprising administering to the subject an effective amount of a compound according to claim 1 or 7.
20. The method of claim 18, wherein the infection is pneumonia, meningitis, sepsis, septic shock, sinusitis, otitis media, mastoiditis, conjunctivitis, keratitis, external otitis (e.g., necrotizing otitis externa and perichondritis), laryngeal infections (e.g., acute epiglottitis, croup and tuberculous laryngitis), endocarditis, infections of prosthetic valves, abscesses, peritonitis, infectious diarrheal diseases, bacterial food poisoning, sexually transmitted diseases and related conditions, urinary tract infections, pyelonephritis, infectious arthritis, osteomyelitis, infections of prosthetic joints, skin and soft tissue infections, oral infections, dental infections, nocardiosis and actinomycosis, mastitis, brucellosis, Q fever, anthrax, or wound infections.
21. The method of claim 18, wherein the infection is related to Actinomyces, Staphylococcus, Streptococcus, Enterococcus, Erysipelothrix, Neisseria, Branhamella, Listeria, Bacillus, Corynbacterium, Erysipelothrix, Gardnerella, Mycobacterium, Nocardia, Enter obacteriaceae, Escherichia, Salmonella, Shigella, Yersinia, Enterobacter, Klebsiella, Citrobacter, Serratia, Providencia, Proteus, Morganella, Edwardsiella, Erwinia, Vibrio, Aeromonas, Helicobacter, Campylobacter, Eikenella, Pasteurella, Pseudomonas, Burkholderia,
Stenotrophomonas, Acinetobacter, Ralstonia, Alcaligenes, Moraxella, Mycoplasma, Legionella, Francisella, Brucella, Haemophilus, Bordetella, Clostridium, Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Borrelia, Chlamydia, Rickettsia, Ehrlichia, Bartonella, Trichomonas, Treponema, or combinations thereof (i.e., infections established by more than one bacterial strain).
22. The method of claim 18, further comprising administering an additional therapeutic agent.
23. The method of claim 18, further comprising administering an additional antibacterial agent.
24. The method of claim 23, wherein the additional antibacterial agent is selected from quinolones, β-lactams (e.g., penicillins or cephalosporins), carbapenems, aminoglycosides, macrolides, ketolides, tetracyclines, glycycyclines, lincomycins, oxazolidinones, amphenicols, ansamycins, polymyxins, aminomethlycyclines, lincosamides, streptogramins, 2,4-diaminopyrimidines, nitrofurans, sulfonamides, sulfones, rifabutins, dapsones, peptides, glycopeptides, or combinations thereof.
25. The method of claim 18, further comprising assessing bacterial levels of the subject.
26. The method of claim 25, wherein the assessing is performed prior to administration of the compound.
27. The method of claim 25, wherein the assessing is performed after administration of the compound.
28. The method of claim 25, wherein the assessing is performed both prior to and after administration of the compound.
29. The method of claim 25, wherein a subject sample is assessed.
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