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  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
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  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/20—Oxygen atoms
  • C07D215/22—Oxygen atoms attached in position 2 or 4
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
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  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/10—Nitrogen as only ring hetero atom
  • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/045—Culture media therefor

Definitions

• quorum sensing enables a bacterial species to sense its own number and regulate gene expression according to population density.
• quorum sensing is cell density-dependent regulation of genes that involves a freely diffusible molecule synthesized by the cell called an autoinducer (Fuqua, W. C. et al (1996) Annu. Rev. Microbiol. 50:727-751; Salmond, G. P. C. et al. (1995) Mol Microbiol. 16:615-624; Sitnikov, D. M. et al. (1995) Mol. Microbiol. 17:801-812).
• Autoinducers are described, e.g., in U.S. Pat. Nos. 5,591,872 and 5,593,827.
• Autoinducer molecules and methods for the use of autoinducer molecules are described, for example, in U.S. Pat. Nos. 5,591,872 and 6,057,288, and in published PCT international patent application Nos. WO 98/57618, WO 98/58075, WO 99/65889, and WO 00/06177.
• Bacteria at a low cell density produce a basal level of autoinducer, and, as a population grows, autoinducer concentration increases concomitantly with cell density. On reaching a threshold concentration, autoinducer binds to and thereby activates an R protein, which then induces or ceases to repress specific target genes. In this manner, intercellular signals enable a bacterial population to control the expression of specific genes in response to cell density.
• V fischeri exists at low cell densities in sea water and also at very high cell densities within the light organs of various marine organisms, such as the squid Euprymna scolopes (Pesci, E. C. et al.(1997) Trends in Microbiol. 5(4):132-135; Pesci, E. C. et al. (1997) J. Bacteriol. 179:3127-3132; Ruby, E. G. (1996) Ann. Rev. Microbiol. 50:591-624). At high cell densities, the V.
• fischeri genes encoding the enzymes required for light production are expressed. These genes are part of the lux ICDABEG operon and are regulated by the gene products of luxI and luxR (Baldwin, T. O. et al. (1989) J. of Biolum. and Chemilum. 4:326-341; Eberhard, A., et al. (1991) Arch. of Microbiol. 155:294-297; Gray, K. M. et al. (1992) J. Bacteriol. 174:4384-4390).
• LuxI is an autoinducer synthase that catalyzes the formation of the V. fischeri autoinducer (VAI), N-(3oxohexanoyl) homoserine lactone (Eberhard, A., et al. (1991) Arch. of Microbiol. 155:294-297; Seed, P. C. et al. (1995) J. Bacteriol. 177:654-659).
• VAI V. fischeri autoinducer
• the autoinducer freely diffuses across the cell membrane and at high cell densities, reaches a critical concentration (Kaplan, H. B. et al. (1985) J. Bacteriol. 163:1210-1214).
• VAI interacts with LuxR, a DNA-binding transcriptional regulator.
• the LuxR-VAI complex then binds to an upstream sequence of the lux operon called the “lux box”, and activates transcription (Devine, J. H. et al. (1989) PNAS 86: 5688-5692; Hanzelka, B. A. et al. (1995) J. Bacteriol. 177:815-817; Stevens, A. M. et al. (1994) PNAS 91:12619-12623). Since one of the genes of the operon is luxI, an autoregulatory loop is formed.
• the systems typically have acylated homoserine lactone (“HSL”) ring autoinducers, in which the homoserine lactone ring is conserved.
• HSL homoserine lactone
• the acyl side chain can vary in length and degree of substitution.
• Pseudomonas aeruginosa has two quorum sensing systems, las and rhl (Brint et al. 1995, Hanzelka et al. 1996, Baldwin et al., 1989, Passador et al. 1993, Pearson et al. 1997, Pesci et al. 1997).
• the two systems have distinct autoinducer synthases (lasI and rhlI), transcriptional regulators (lasR and rhlR), and autoinducers (N-(3-oxododecanoyl) homoserine lactone (HSL) and N-butyryl HSL) (Sitnikov et al., 1995, Stevens et al. 1994).
• N-(3-oxododecanoyl) homoserine lactone is synthesized by LasI along with a small amount of N-(3-oxooctanoyl) HSL and N-(3-oxohexanoyl) HSL, while RhlI makes primarily N-butyryl HSL and a small amount of N-hexanoyl (Pearson et al. 1994, Winson et al. 1995).
• the rhl and las systems are involved in regulating the expression of a number of secreted virulence factors, biofilm development, and the stationary phase sigma factor (RpoS) (Brint et al. 1995, Davies et al.
• Biofilms are defined as an association of microorganisms, single or multiple species, that grow attached to a surface and produce a slime layer that provides a protective environment (Costerton, J. W. (1995) J Ind Microbiol. 15(3):137-40, Costerton, J. W. et al. (1995) Annu Rev Microbiol. 49:711-45).
• biofilms produce large amounts of extracellular polysaccharides, responsible for the slimy appearance, and are characterized by an increased resistance to antibiotics (1000- to 1500-fold less susceptible).
• Several mechanisms are proposed to explain this biofilm resistance to antimicrobial agents (Costerton, J. W. et al. (1999) Science. 284(5418):1318-22).
• the extracellular matrix in which the bacterial cells are embedded provides a barrier toward penetration by the biocides.
• a further possibility is that a majority of the cells in a biofilm are in a slow-growing, nutrient-starved state, and therefore not as susceptible to the effects of antimicrobial agents.
• a third mechanism of resistance could be that the cells in a biofilm adopt a distinct and protected biofilm phenotype, e.g., by elevated expression of drug-efflux pumps.
• Biofilms of P. aeruginosa have been isolated from medical implants, such as indwelling urethral, venous or peritoneal catheters (Stickler, D. J. et al. (1998) Appl Environ Microbiol. 64(9):3486-90).
• biofilms In industrial settings, the formation of biofilms is often referred to as ‘biofouling’, or biological fouling. Biological fouling of surfaces is common and leads to material degradation, product contamination, mechanical blockage, and impedance of heat transfer in water-processing systems. Biofilms are also the primary cause of biological contamination of drinking water distribution systems, due to growth on filtration devices.
• gram-negative bacteria have been shown to possess one or more quorum sensing systems that regulate a variety of physiological processes, including the activation of virulence genes and biofilm formation.
• One such gram negative bacterium is Pseudomonas aeruginosa.
• P. aeruginosa is a soil and water bacterium that can infect animal hosts. Normally, the host defense system is adequate to prevent infection. However, in immunocompromised individuals (such as burn patients, patients with cystic fibrosis, or patients undergoing immunosuppressive therapy), P. aeruginosa is an opportunistic pathogen, and infection with P. aeruginosa can be fatal (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74; Van Delden, C. et al. (1998) Emerg Infect Dis. 4(4):551-60).
• Cystic fibrosis the most common inherited lethal disorder in Caucasian populations ( ⁇ 1 out of 2,500 life births), is characterized by bacterial colonization and chronic infections of the lungs.
• the most prominent bacterium in these infections is P. aeruginosa —by their mid-twenties, over 80% of people with CF have P. aeruginosa in their lungs (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74).
• these infections can be controlled for many years by antibiotics, ultimately they “progress to mucoidy,” meaning that the P. aeruginosa forms a biofilm that is resistant to antibiotic treatment. At this point the prognosis is poor.
• P. aeruginosa is also one of several opportunistic pathogens that infect people with AIDS, and is the main cause of bacteremia (bacterial infection of the blood) and pneumonitis in these patients (Rolston, K. V. et al. (1990) Cancer Detect Prev. 14(3):377-81; Witt, D. J. et al. (1987) Am J Med. 82(5):900-6).
• a recent study of 1635 AIDS patients admitted to a French hospital between 1991-1995 documented 41 cases of severe P. aeruginosa infection (Meynard, J. L. et al. (1999) J Infect. 38(3):176-81).
• nosocomial infections infections are often acquired in hospitals (“nosocomial infections”) when susceptible patients come into contact with other patients, hospital staff, or equipment.
• nosocomial infections In 1995 there were approximately 2 million incidents of nosocomial infections in the U.S., resulting in 88,000 deaths and an estimated cost of $4.5 billion (Weinstein, R. A. (1998) Emerg Infect Dis. 4(3):416-20).
• AIDS patients Of the AIDS patients mentioned above who died of P. aeruginosa bacteremia, more than half acquired these infections in hospitals (Meynard, J. L. et al. (1999) J Infect. 38(3):176-81).
• Nosocomial infections are especially common in patients of intensive care units as these people often have weakened immune systems and are frequently on ventilators and/or catheters.
• Catheter-associated urinary tract infections are the most common nosocomial infection (Richards, M. J. et al. (1999) Crit Care Med. 27(5):887-92) (31% of the total), and P. aeruginosa is highly associated with biofilm growth and catheter obstruction. While the catheter is in place, these infections are difficult to eliminate (Stickler, D. J. et al. (1998) Appl Environ Microbiol. 64(9):3486-90).
• the second most frequent nosocomial infection is pneumonia, with P.
• NIS National Nosocomial Infections Surveillance
• P. aeruginosa is also of great industrial concern (Bitton, G. (1994) Wastewater Microbiology. Wiley-Liss, New York, N.Y.; Steelhammer, J. C. et al. (1995) Indust. Water Treatm.: 49-55).
• the organism grows in an aggregated state, i.e., the biofilm, which causes problems in many water processing plants.
• problems include corroded pipes, loss of efficiency in heat exchangers and cooling towers, plugged water injection jets leading to increased hydraulic pressure, and biological contamination of drinking water distribution systems (Bitton, G. (1994) Wastewater Microbiology.
• Biocides in contrast to antibiotics, are antimicrobials that do not possess high specificity for bacteria, so they are often toxic to humans as well. Biocide sales in the US run at about $1 billion per year (Peaff, G. (1994) Chem. Eng. News: 15-23).
• a particularly ironic connection between industrial water contamination and public health issues is an outbreak of P. aeruginosa peritonitis that was traced back to contaminated poloxamer-iodine solution, a disinfectant used to treat the peritoneal catheters.
• P. aeruginosa is commonly found to contaminate distribution pipes and water filters used in plants that manufacture iodine solutions. Once the organism has matured into a biofilm, it becomes protected against the biocidal activity of the iodophor solution.
• a common soil organism that is harmless to the healthy population, but causes mechanical problems in industrial settings, ultimately contaminated antibacterial solutions that were used to treat the very people most susceptible to infection.
• the present invention is based, at least in part, on the discovery of a novel autoinducer molecule, 2-heptyl-3-hydroxy-4-quinolone, which functions as an intercellular signal molecule in the cell-to-cell communication system of Pseudomonas aeruginosa.
• 2-heptyl-3-hydroxy-4-quinolone can function as an intercellular signal sheds light on the role of secondary metabolites and shows that P. aeruginosa cell-to-cell signaling is not restricted to acyl-homoserine lactones.
• Pseudomonas quinolone signal are mediated by the P. aeruginosa las and rhl quorum sensing systems, respectively. Accordingly, the invention is directed to bacterial quinolone signal molecules and more particularly to Pseudomonas quinolone signal (“PQS”) molecules, e.g., 2-heptyl-3-hydroxy-4-quinolone, and analogs and derivatives thereof.
• PQS Pseudomonas quinolone signal
• the invention is a compound of formula I:
• R 1 -R 4 are independently H, alkyl, alkenyl, alkynyl, OH, NH 2 , SH, O—R 6 , N—R 7 R 8 , or a halogen;
• R 5 is H, SH, OH, O—R 6 , or N—R 7 R 8 ;
• R6 is H or C 1 -C 4 alkyl
• R 7 and R 8 are independently H, C 1 -C 4 alkyl, O, or S;
• X and Y are independently S, O, or N—R 9 ;
• R 9 is H, O, S, or C 1 -C 4 alkyl
• Q is a tail group
• Q has the formula IA
• R 10 -R 13 are independently H, C 1 -C 4 alkyl, OH, NH 2 , SH, O—R 25 , N—R 26 R 27 , or a halogen, or R 10 and R 11 taken together form a carbonyl, a sulfonyl or an imino moiety, or R 12 and R 13 taken together form a carbonyl, a sulfonyl or an imino moiety;
• R 14 -R 24 are independently H, C 1 -C 4 alkyl, OH, NH 2 , SH, O—R 25 , N—R 26 R 27 , or a halogen;
• R 25 is H or C 1 -C 4 alkyl
• R 26 and R 27 are independently H, C 1 -C 4 alkyl, O, or S.
• the invention is directed to compounds of formula I that are different than 2-heptyl-3-hydroxy-4-quinolone.
• examples of such embodiments include, but are not limited to, compounds of formula I in which: when Q is heptyl, X is O, Y is NH and R 5 is OH, R 1 -R 4 are not all hydrogen; when Q is heptyl, X is O, Y is NH, and R 1 -R 4 are all hydrogen, R 5 is not OH; when Q is heptyl, Y is NH, R 1 -R 4 are all hydrogen, and R 5 is OH, X is not O; when Q is heptyl, X is O, R 1 -R 4 are all hydrogen, and R 5 is OH, Y is not NH; and when X is O, Y is NH and R 5 is OH, and R 1 -R4 are all hydrogen, Q is not heptyl.
• the invention when Q is heptyl, X
• R 16 , R 17 , and R 18 are H.
• R 2 is halogen; or R 3 is halogen; or R4 is halogen; or X is S or N—R 9 ; or Y is O, S, or N—R 9 and R 9 is C 1 -C 4 -alkyl; or R 5 is H, SH, O—R6, or N—R 7 R 8 , and R6 is C 1 -C 4 alkyl; or R 5 is SH, O—R 6 , or N—R 7 R 8 ; X is 0; or R 5 is OH and Y is N—R 9 .
• Q is an alkylene chain having a skeleton of three to twenty carbon atoms.
• the alkylene chain contains one or more double bonds or triple bonds between the carbon atoms forming the skeleton alkylene side chain.
• one or more carbon atoms forming the skeleton of the alkylene side chain are replaced with sulfur or sulfur-substituted moieties.
• the compounds of the invention contain a chiral center, for example, the Q substituent of formula IA.
• the compounds are optically active isomers.
• the invention is a compound of formula II:
• the invention is directed to autoinducer molecules comprising the compounds hereinabove described.
• the autoinducer molecules regulate gene expression.
• the autoinducer molecule regulates gene expression in bacteria.
• the bacteria is Pseudomonas aeruginosa.
• the Pseudomonas aeruginosa gene expresses a virulence factor.
• the virulence factor is elastase.
• the autoinducer molecules of the invention regulate the activity of the LasR protein of Pseudomonas aeruginosa. In other embodiments, the autoinducer molecules of the invention regulate the activity of the RhlR protein of Pseudomonas aeruginosa. In certain embodiments, the autoinducer molecules of the invention are isolated from culture media in which Pseudomonas aeruginosa is grown.
• the invention is directed to compounds hereinabove defined that are capable of modulating the autoinducer activity of 2-heptyl-3-hydroxy-4-quinolone.
• modulation comprises inhibition of the autoinducer activity of 2-heptyl-3-hydroxy-4-quinolone.
• modulation comprises synergistic enhancement of the autoinducer activity of 2-heptyl-3-hydroxy-4-quinolone.
• the invention is directed to compounds hereinabove described that are capable of modulating the activity of the LasR and/or the RhlR proteins of Pseudomonas aeruginosa.
• the compound is an agonist of the LasR and/or the RhlR proteins of Pseudomonas aeruginosa.
• the compound is an antagonist of the LasR and/or the RhlR proteins of Pseudomonas aeruginosa.
• the invention is a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I and a pharmaceutically acceptable carrier therefor, wherein the compound inhibits the activity of one or more proteins in a microorganism that regulate expression of virulence factors.
• the compound is present in an amount effective to affect the ability of the microorganism to initially infect or further infect an organism.
• the microorganism is Pseudomonas aeruginosa.
• the compound of the pharmaceutical composition inhibits the activity of the LasR and/or the RhlR proteins of Pseudomonas aeruginosa. In certain other embodiments, the compound of the pharmaceutical composition inhibits the autoinducer activity of 2-heptyl-3-hydroxy-4-quinolone.
• the pharmaceutical composition may further comprise an antimicrobial, antibacterial or antifungal agent.
• the invention is a method of inhibiting the infectivity of Pseudomonas aeruginosa comprising administering to a subject a therapeutically effective amount of a compound of formula I, wherein the compound inhibits the activity of the LasR and/or the RhlR proteins of Pseudomonas aeruginosa.
• the compound inhibits the autoinducer activity of 2-heptyl-3-hydroxy-4-quinolone.
• Another aspect of the invention is a method of treating an immunocompromised subject infected with Pseudomonas aeruginosa comprising administering to a subject a therapeutically effective amount of a compound of formula (I), wherein the compound inhibits the activity of the LasR and/or the RhlR proteins of Pseudomonas aeruginosa.
• the compound inhibits the autoinducer activity of 2-heptyl-3-hydroxy-4-quinolone.
• the subject is afflicted with cystic fibrosis.
• the invention is a culture medium for microorganisms comprising, as an added compound, an autoinducer molecule of the invention, at a concentration effective to stimulate or promote the metabolism, growth and/or recovery of the microorganism.
• the microorganism is Pseudomonas aeruginosa.
• the autoinducer is 2-heptyl-3-hydroxy-4-quinolone.
• Yet another aspect of the invention is a method for identifying a compound that modulates an autoinducer molecule in bacteria, said method comprising:
• the test compound inhibits the autoinducer molecule.
• the compound synergizes activity of the autoinducer molecule.
• the bacteria is Pseudomonas aeruginosa.
• the autoinducer is 2-heptyl-3-hydroxy-4-quinolone.
• the compound inhibits binding of the autoinducer molecule to LasR and/or RhlR.
• Still another aspect of the invention is a method of regulating the expression of a gene in bacteria comprising:
• the method further comprises the additional steps of:
• Another aspect of the invention is an inhibitor of the autoinducer activity of 2-heptyl-3-hydroxy-4-quinolone.
• Yet another aspect of the invention is an analog of 2-heptyl-3-hydroxy-4-quinolone that inhibits the induction of virulence factors by 2-heptyl-3-hydroxy-4-quinolone, LasR or RhlR.
• the virulence factor is exotoxin A, elastase, or an alkaline protease.
• Still another aspect of the invention is an analog of 2-heptyl-3-hydroxy-4-quinolone that inhibits the induction of biofilm formation by 2-heptyl-3-hydroxy-4-quinolone, LasR or RhlR.
• the invention is a method for modulating quorum sensing signaling in bacteria, said method comprising:
• bacteria that comprise a quorum sensing controlled gene, wherein said bacteria are responsive to an autoinducer molecule; and incubating the bacteria with a compound of formula I other than 2-heptyl-3-hydroxy-4-quinolone, such that quorum sensing signaling in bacteria is modulated.
• the autoinducer is 2-heptyl-3-hydroxy-4-quinolone.
• chiral refers to molecules that have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules that are superimposable on their mirror image partner.
• halogen designates —F, —Cl, —Br or —I;
• sulfhydryl or “thiol” means —SH;
• hydroxyl means —OH.
• alkenyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described below, but which contain at least one double bond. Unless the number of carbons is otherwise specified, “lower alkenyl” refers to an alkenyl group, as defined above, but having from two to four carbon atoms in its backbone structure.
• alkyl includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
• alkyl further includes alkyl groups, which can further include heteroatoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms.
• a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone (e.g., C 1 -C 12 for straight chain, C 3 -C 12 for branched chain).
• alkyl groups contemplated by the invention include, but are not limited to, methyl, ethyl, isopropyl, isobutyl, tert-butyl, branched pentyl, branched hexyl, cyclohexyl, cyclopentyl, n-heptyl and branched heptyl groups.
• alkyl includes both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
• substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), arylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoro
• alkoxyalkyl “polyaminoalkyl” and “thioalkoxyalkyl” include alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
• alkynyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. Unless the number of carbons is otherwise specified, “lower alkynyl” refers to an alkynyl group, as defined above, but having from two to four carbon atoms in its backbone structure.
• analog includes compounds which are structurally similar but not identical to autoinducer molecules derived from bacteria, such as, for example, N-(3-oxododecanoyl)homoserine lactone and 2-heptyl-3-hydroxy-4-quinolone.
• autoinducer molecule includes molecules that diffuse across cell membranes and activate transcription of various factors that affect bacterial viability. Such compounds can affect virulence and biofilm development. Autoinducer molecules include, for example, 2-heptyl-3-hydroxy-4-quinolone. Other examples of autoinducer molecules are listed below in Table 1. In isolated form, autoinducer molecules can be obtained from naturally occurring proteins by purifying cellular extracts, synthesized chemically, or recombinantly produced.
• homoserine lactone Vibrio harveyi N- ⁇ - LuxM/LuxN- luxICDABEG, (hydroxybutyryl)- LuxO-LuxR d luminescence homoserine and poly- lactone (HAI-1) hydroxybutyrate synthesis HAI-2 Lux?/LuxPQ- luxCDABEG LuxO-LuxR d Pseudomonas N-3-(oxo- LasI/LasR lasB, lasA, aprA, aeruginosa dodecanyoyl)-L- toxA, virulence homoserine factors lactone (PAI-1) N-(butyryl)-L- RhII/RhIR rhlAB, rhamnolipid homoserine synthesis, virulence lactone (PAI-2) factors Pseudomonas (PRAI) PhzI/PhzR phz, phenazine aeure
• carotovora SCRI193 Erwinia VAI-1 CarI/CarR cap carbapenem carotovora antibiotic synthesis subsp.
• SAI-1 N-hexanoyl-L- SwrI/? swarming motility homoserine lacton
• SAI-22 Aeromonas
• AHAI AhyI/AhyR function unclear hydrophila Escherichia ?/SdiA ftsQAZ, cell coli /? g division
• antimicrobial is intended to encompass the elimination or reduction of a population of microorganisms, for example, bacteria.
• the term is also intended to include a reduction in or elimination of the pathogenic effect of bacteria, for example through inhibition of bacterial production of virulence factors and/or biofilm development.
• aryl includes aryl groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
• Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like.
• aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles”, “heteroaryls” or “heteroaromatics”.
• the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, s
• Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings that are not aromatic so as to form a polycycle (e.g., tetralin).
• aralkyl includes alkyl groups substituted with at least one aryl group and aryl groups substituted with at least one alkyl group.
• Biofilm includes biological films that develop and persist at interfaces in 25 aqueous and other environments.
• Biofilms are composed of microorganisms embedded in an organic gelatinous structure composed of one or more matrix polymers which are secreted by the resident microorganisms.
• biofilm development includes the formation growth, and modification of the bacterial colonies contained with the biofilm structures as well as the synthesis and maintenance of the exopolysaccharide matrix of the biofilm structures.
• biofilm associated states includes disorders which are characterized by the presence or potential presence of a bacterial biofilm.
• biofilm associated states include, but are not limited to, middle ear infections, cystic fibrosis, osteomyelitis, acne, dental cavities, and prostatitis.
• Biofilm associated states also include infection of the subject by one or more bacteria, e.g., Pseudomonas aeruginosa.
• chain includes moieties of atoms covalently bonded to each other linearly.
• the atoms in the chain may be substituted with any substituents that allow the compounds to perform their intended function.
• substituents examples include carbon, nitrogen, sulfur, oxygen, and phosphorous.
• the linear chain may be entirely composed of carbon atoms.
• the carbon atoms of the chain may be substituted (e.g., with carbonyl groups, halogens, hydroxy, thiol, amino, alkyl, alkenyl, alkynyl groups etc.), unsubstituted or bound to hydrogen atoms.
• the carbon atoms forming the skeleton of the chain may be replaced with one or more other atoms, e.g., sulfur.
• the chain can be saturated (e.g., contain only single bounds) or may be unsaturated (e.g., contain double or triple bonds between the atoms of the chain or their substituents.)
• the term “compound” includes molecules of any one of formulae, as described herein, or pharmaceutically acceptable salts, esters, or prodrugs thereof.
• the term “compound” includes analogs of autoinducer molecules as well as inhibitors of autoinducer molecules.
• the term “compound” includes antimicrobial agents, which inhibit or modulate the formation of bacterial virulence factors and/or bacterial biofilms.
• heteroatom includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
• an “immunocompromised subject” includes subjects, e.g., mammals, e.g., humans, which have an immune system that is incapable of reacting to pathogens.
• the subject can be immunocompromised due to a genetic disorder, disease or drugs that inhibit immune response.
• An immunocompromised subject includes an individual afflicted with cystic fibrosis or who is taking corticosteroids or immunosuppressive agents.
• the language “infected with Pseudomonas aeruginosa ” includes a subject that is found to have Pseudomonas aeruginosa, present in its body. For example, Pseudomonas aeruginosa often infects the lungs of cystic fibrosis patients. Even a small number of Pseudomonas aeruginosa found in an organism can constitute infection with Pseudomonas aeruginosa.
• inhibitor of the autoinducer molecule includes compounds of the invention and other compounds that interfere with the ability of the autoinducer molecule to stimulate, regulate, or modulate the activity of the protein which is normally responsive to it.
• inhibitors of P. aeruginosa autoinducer molecules include compounds which degrade, compete with, or bind to, for example, 2-heptyl-3-hydroxy-4-quinolone, or otherwise alter the ability of the autoinducer molecule to interact with the LasR protein and/or the RhlR protein of P. aeruginosa.
• parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
• phrases “pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
• the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
• materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
• esters refers to the relatively non-toxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent.
• Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst.
• Hydroxyls can be converted into esters via treatment with an esterifying agent such as alkanoyl halides.
• the term also includes lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. (See, for example, Berge et al, supra.)
• salts are art recognized and includes relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
• Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J Pharm. Sci. 66:1-19).
• composition includes preparations suitable for administration to mammals, e.g., humans.
• mammals e.g., humans.
• the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
• polycyclyl or “polycyclic radical” refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings”. Rings that are joined through non-adjacent atoms are termed “bridged” rings.
• Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
• the terms “effective amount” and “therapeutically effect amount” are used interchangeably and are intended to include the amount of a compound of the invention given or applied to an organism or subject that allows the compound to perform its intended therapeutic function.
• the effective amounts of the compound of the invention will vary according to factors such as the degree of infection in the subject, the age, sex, and weight of the subject, and the ability of the compound to inhibit the activity of the protein regulated by the autoinducer molecule of the bacteria upon, for example, the LasR protein of P. aeruginosa in the subject.
• Dosage regimes can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.
• subject includes organisms which are can suffer from biofilm associated states.
• subject includes mammals, e.g., horses, monkeys, bears, dogs, cats, mice, rabbits, cattle, squirrels, rats, and, preferably, humans.
• the subject may be immunocompromised.
• the language “synergist of the autoinducer molecule” is intended to include molecules that enhance the ability of the autoinducer molecule to stimulate the protein modulated by the autoinducer molecule (e.g., such as the LasR protein for P. aeruginosa ).
• systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
• tail group includes groups that allow the compound of the invention to perform its intended function.
• the tail group of the compound of the invention is hydrophobic.
• the tail group is a chain of three to twenty atoms, such as carbon, nitrogen, sulfur, oxygen, or phosphorous.
• the tail group may be substituted or unsubstituted, saturated or unsaturated.
• the tail group is unsubstituted or substituted alkyl, alkenyl, or alkynyl.
• the tail group is substituted with one or more halogens (e.g., fluorine, chlorine, bromine or iodine).
• the tail group is substituted with at least one C 1 -C 4 group (e.g., methyl, ethyl, isopropyl, n-propyl, t-butyl, iso-butyl, or n-butyl.)
• the tail group is a chain of from about 5 atoms to about 15 atoms, 5 atoms to about 14 atoms, about 5 atoms to about 13 atoms, about 5 atoms to about 12 atoms, about 5 atoms to about 11 atoms, about 5 atoms to about 10 atoms, about 5 atoms to about 9 atoms, about 6 atoms to about 9 atoms, about 7 atoms to about 8 atoms, about 7 atoms, and about seven carbons.
• the tail group is heptyl.
• modulator as in “modulator of an autoinducer molecule” is intended to encompass, in its various grammatical forms, induction and/or potentiation, as well as 25 inhibition and/or down regulation of quorum sensing controlled gene expression.
• modulator of quorum sensing signaling includes a compound or agent that is capable of modulating or regulating at least one quorum sensing controlled gene or quorum sensing controlled genetic locus, e.g., a quorum sensing controlled genetic locus in P. aeruginosa.
• a modulator may act to modulate signal generation (e.g., the synthesis of a quorum sensing signal molecule, i.e., autoinducer molecule), signal reception (e.g., the binding of a signal molecule to a receptor or target molecule), or signal transmission (e.g., signal transduction via effector molecules to generate an appropriate biological response).
• a method of the present invention encompasses the modulation of the transcription of an indicator gene in response to an autoinducer molecule.
• a method of the present invention encompasses the modulation of the transcription of an indicator gene, preferably an quorum sensing controlled indicator gene, by a test compound.
• quorum sensing signaling or “quorum sensing” is intended to include the generation of a cellular signal in response to cell density. In one embodiment, quorum sensing signaling mediates the coordinated expression of specific genes.
• a “quorum sensing controlled gene” is any gene, the expression of which is regulated in a cell density dependent fashion. In a preferred embodiment, the expression of a quorum sensing controlled gene is modulated by a quorum sensing signal molecule, e.g., an autoinducer molecule (e.g., a quinolone molecule of the invention).
• the term “quorum sensing signal molecule” is intended to include a molecule that transduces a quorum sensing signal and mediates the cellular response to cell density.
• the quorum sensing signal molecule is a freely diffusible autoinducer molecule, e.g., 2-heptyl-3-hydroxy-4-quinolone or analog thereof.
• a quorum sensing controlled gene encodes a virulence factor (e.g., exotoxin A, elastase, alkaline protease).
• a quorum sensing controlled gene encodes a protein or polypeptide that, either directly or indirectly, inhibits and/or antagonizes a bacterial host defense mechanism.
• a quorum sensing controlled gene encodes a protein or polypeptide that regulates biofilm formation.
• This novel molecule belongs to the 4-quinolone chemical family, which is best known for the antibiotic activity of many of its members.
• the signal has been shown to be 2-heptyl-3-hydroxy-4-quinolone and it has been designated as the Pseudomonas quinolone signal (PQS), shown in FIG. 1C in comparison to known autoinducers 3-oxo-C12-HSL and C4-HSL. It has also been shown that this compound induces lasB in P. aeruginosa and depends on the P. aeruginosa quorum sensing systems for its production and bioactivity.
• PQS Pseudomonas quinolone signal
• the LasB elastase (also known as elastolytic protease and elastin-hydrolyzing protease) is a significant P. aeruginosa virulence factor that is controlled by both the las and rhl quorum sensing systems (6, 8). It has been shown that the transcription of lasB is greatly reduced in either a P. aeruginosa lasI or rhlI mutant, indicating the importance of 3-oxo-C12-HSL and C4-HSL, respectively (11, 12).
• strain PAO-R1 which contains a LasR null mutation and a lasB′-lacZ fusion, does not express elastase or ⁇ -gal (6, 7).
• This phenotype results from the absence of LasR, which positively regulates genes controlled by the las quorum sensing system including rhlR, which is required for the rhl quorum sensing system to function (8, 20, 21).
• the absence of lasR also renders the lasB′-lacZ fusion in strain PAO—R1 (lasR 2) (pTS400) unresponsive to 3-oxo-C12-HSL (4).
• lasB′-lacZ in this strain is mildly activated by exogenously added C4-HSL, which is most likely attributable to the presence of low amounts of RhlR that can be produced in the absence of LasR (4, 20).
• ⁇ LasR This truncated LasR, referred to as ⁇ LasR, has a large N-terminal deletion and is encoded on plasmid pECP39.
• the ⁇ LasR protein which was based on a similar truncated construct of the Vibrio fischeri LuxR protein (a LasR homolog) (22), can activate LasR-controlled genes in the absence of 3-oxo-C12-HSL. Therefore, ⁇ LasR was used to induce the production of the novel signal in strain PAO-JP2 (lasI ⁇ , rhlI ⁇ ).
• the double autoinducer mutant, strain PAO-JP2 was transformed with plasmid pECP39, and it was found that a spent culture media extract from this strain contained the novel signal that activated lasB′-lacZ in strain PAO-R1 (lasR ⁇ ) (pTS400) (FIG. 2A, below). There were no lasB′-lacZ activating signals in spent media extracts from cultures of strain PAO-JP2 (lasI ⁇ , rhlI ⁇ ) alone or containing the control vector pUCP22 (FIG. 2A, below). This confirmed that the synthesis of the new signal depended on LasR and indicated that the signal was not produced by a known autoinducer synthase because strain PAO-JP2 did not have a functional LasI or RhlI.
• E. coli strain DH5a was transformed with plasmid pECP62.5, which contained the lasB′-lacZ reporter and the inducible tacp-rhlR gene. If the signal worked directly through RhlR, then the induction of tacp-rhlR in the presence of exogenously added signal should result in the activation of lasB′-lacZ in E. coli strain DH5a (pECP62.5). It was determined that the signal did not induce lasB′-lacZ when RhlR was expressed in E. coli strain DH5a (pECP62.5). This suggested that the signal's requirement for rhlR may be attributable to an additional P. aeruginosa component that is controlled by rhlR or, alternatively, that E. coli may be less permeable to this signal.
• P. aeruginosa produces a cell-to-cell signal that is unlike any previously reported intercellular signal molecule.
• This molecule was determined to have a 4-quinolone base structure and therefore has been designated as the Pseudomonas quinolone signal (PQS).
• PQS Pseudomonas quinolone signal
• exogenously added PQS induced a lasB′-lacZ fusion in the P. aeruginosa lasR mutant, strain PAO—R1, containing the plasmid pTS400.
• RhlR may serve to regulate the expression of a P. aeruginosa gene that is involved in the response to PQS. This suggestion is based on the fact that the PQS response was not replicated by the addition of PQS to a recombinant E. coli strain expressing RhlR in the presence of lasB′-lacZ. These initial experiments on PQS indicated that this signal depended on both P. aeruginosa quorum sensing systems and that it was involved in the regulation of the major virulence factor, LasB elastase. The addition of a third molecule to the P. aeruginosa intercellular signal repertoire increases the complexity of the quorum sensing hierarchy.
• a mutated lasR gene was constructed that encoded a truncated protein ( ⁇ LasR) capable of activating LasR-controlled genes in the absence of 3-oxo-C12-HSL.
• ⁇ LasR truncated protein
• PQS was purified by sequential reverse phase column chromatography and TLC from the double autoinducer mutant, strain PAO-JP2, that was expressing the ⁇ LasR protein. Based primarily on the spectral properties of the purified material, it was concluded that the novel signal molecule was 2-heptyl-3-hydroxy-4-quinolone.
• aeruginosa bioassays for 3-oxo-C12-HSL and C4-HSL showed that a concentration of ⁇ 1 mM was required for these signals to activate their respective assays to half of their maximum activity (4).
• 4-quinolones are secondary metabolites that can have potent antibiotic activity (28). It is now evident that at least one member of this family of molecules, 2-heptyl-3-hydroxy-4-quinolone, has a role in cell-to-cell signaling.
• the modern 4-quinolone antibiotics are halogenated at the 6 or 8 position and are commonly used to treat a variety of both Gram-positive and Gram-negative infections (29). These molecules are bactericidal and are believed to target DNA gyrase, which can be naturally mutated in P. aeruginosa to produce resistant strains (30, 31).
• PQS does not have detectable anti- Staphylococcus aureus or anti- E. coli activity. However, this molecule showed activity as an intercellular signal that induced the P. aeruginosa virulence gene lasB.
• Plasmid pECP39 which encodes a truncated form of LasR ( ⁇ LasR) that is capable of activating LasR-controlled genes in the absence of an autoinducer, was constructed as follows. First, the intermediate plasmid pKDT39 was constructed by ligating the lasR DNA that encodes LasR amino acids 160-239 in-frame to the histidine fusion site of the expression vector pTRCHisC (Invitrogen). This resulted in the trcp- ⁇ lasR fusion, which encodes for a truncated, autoinducer-independent form of LasR, the expression of which is controlled by the trc promoter.
• the trcp- ⁇ lasR-containing DNA fragment from pKDT39 then was ligated into the P. aeruginosa cloning vector pUCP22 (16) to form pECP39 (bla, lac q , trcp- ⁇ lasR, oriP, oriC).
• Plasmid pTS400 (5) contains a lasB′-lacZ translational fusion
• plasmid pECP62.5 (12) contains tacp-rhlR and a lasB′-lacZ translational fusion. Trans-formations and other molecular techniques were completed with standard procedures (17).
• the PQS analog 2-hydroxy-3- heptyl-4-quinolone (FIG. 1D) was synthesized as described (18), and 2-heptyl-4-hydroxy-quinoline-N-oxide (FIG. 1E) was purchased from Sigma.
• Extracts that were resuspended in ethyl acetate were added to bioassay tubes, and ethyl acetate was removed by drying under a stream of nitrogen. Unless otherwise specified, the amount of material assayed was extracted from 10 ml of culture fluid.
• TLC Thin Layer Chromatography
• HPLC High Performance Liquid Chromatography
• Preparative TLC plates were obtained by making 1-mm layers of a slurry of 55-g silica gel G (Machery & Nagel) in a solution of 5 g of KH2PO4 in 95 ml of water on 20- ⁇ 20-cm glass plates. Plates then were air-dried and activated at 100° C. for 1 h.
• the solvent for TLC was a 17:2:1 mixture of dichloromethane-acetonitrile-dioxane (vol/vol).
• HPLC was performed on a Beckman-Altex Ultra-sphere 10-mm ⁇ 25-cm C 18 reverse phase column.
• Extracts were resuspended in 0.25 ml of methanol and were loaded onto the column that then was eluted (2 ml/min) with an acetonitrile/water gradient (10-100% over 120 min). Fractions were collected at the indicated intervals and were dried by rotary evaporation at room temperature. Evaporated samples were stored at 220° C. before being dissolved in methanol and were assayed for PQS activity.
• PQS was purified from 1.2 liters of P. aeruginosa strain PAO-JP2 (pECP39) that was grown for 24 h at 37° C. with shaking (260 rpm; initial optical density was 0.05 at 660 nm). Cultures were centrifuged for 10 min at 10,000 ⁇ g, and spent supernatant was removed and extracted twice with ethyl acetate as described (3). The ethyl acetate extract was dried with sodium sulfate and was concentrated by rotary evaporation at room temperature. The concentrated material then was extracted and concentrated three times with progressively smaller volumes of a 1:1 mixture of ethyl acetate and acetonitrile (final volume, 0.75 ml).
• This concentrated extract was fractionated by using Short Body C18 Sep-Pak Plus cartridges (Waters). After loading the extract onto a cartridge (0.25 ml per cartridge), it was washed with 10, 30, and 40% acetonitrile in water (3 ml per cartridge). The active material then was eluted with 55% acetonitrile in water. This partially purified extract was dissolved in 0.3 ml of 90% dioxane in water and was further purified by preparative TLC. After loading the extract, the TLC plate was eluted twice with 17:2:1 dichloromethaneyacetonitrileydioxane.
• the ultraviolet spectra were recorded on a Shimadzu UV-1601 PC spectrophotometer.
• Low-resolution MS were recorded on a Hewlett- Packard 5973 mass selective detector fitted with an SIS direct insertion probe, and high-resolution electron impact spectra were recorded at the University of California-Riverside Mass Spectrometry Facility.
• Aqueous hydrogen peroxide (1.05 M, 1.49 ml, 1.56 mmol) was added to a solution of 3-formyl-2-heptylquinolone (0.41 g, 1.49 mmol) in ethanol (4.5 ml) and aqueous sodium hydroxide (1.08 M, 1.49 ml, 1.6 mmol) under argon, and the mixture was stirred at room temperature for 6 h.
• the precipitate was removed by filtration, was air dried, and was crystallized from ethyl acetate to give 2-heptyl-3-hydroxy-4-quinolone (0.29 g, 74%), as off-white needles: mp 196-198° C.

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