# Patent

## Title
Molecules and compositions that inhibit gram negative bacteria and their uses

## Abstract

Antivirulence strategies to combat Pseudomonas aeruginosa, are described. One strategy encompasses synthesis of a series of compounds that inhibit the production of pyocyanin, a redox-active virulence factor produced by this pathogen. A related strategy encompasses synthesis of compounds that inhibit the two P. aeruginosa quorum-sensing receptors, LasR and RhlR, inhibit production of pyocyanin, and inhibit biofilm formation.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/880,610, filed Sep. 20, 2013 and U.S. Provisional Application No. 61/881,204, filed Sep. 23, 2013, all of which are herein incorporated by reference in their entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under Grant No. GM065859 awarded by the National Institutes of Health, Grant No. MCB-0343821 awarded by the National Science Foundation, and Grant No. FA9550-12-1-0368 awarded by the Air Force Office of Scientific Research. The government has certain rights in the invention.


BACKGROUND
In a process referred to as quorum sensing, bacteria communicate using chemical signal molecules called autoinducers. By monitoring increases and decreases in autoinducer concentration, quorum-sensing bacteria track changes in cell-population density and synchronously switch into and out of group behaviors. Quorum sensing allows bacteria to collectively carry out tasks that would be unsuccessful if carried out by an individual bacterium acting alone.
Both Gram-positive and Gram-negative infectious bacteria, which include human, animal, plant, and marine pathogens, use quorum sensing strategies to control virulence. Typically, bacterial infections are treated with bactericidal or bacteriostatic molecules that impede four major processes: DNA replication, transcription, translation or tetrahydrofolic acid synthesis. Existing methods for treating bacterial infection unfortunately exacerbate the growing antibiotic resistance problem because they inherently select for growth of bacteria that in turn can resist the drug. What is needed are new treatments that avoid selecting for drug resistant bacteria.
Quorum sensing also controls biofilm formation. Biofilms are communities of bacterial cells adhered to surfaces and are highly problematic, for example in industrial processes (e.g., clogging of cooling towers in manufacturing plants) and in hospital or other clinical settings (e.g., catheter and implant infections). Initial studies with Staphylococcus aureus and Staphylococcus epidermidis indicated that manipulation of a form of quorum sensing that is peptide-mediated would not have successful results. Most notably, disruption of the peptide quorum-sensing circuit in S. epidermidis by deleting necessary quorum sensing genes led unexpectedly to increased biofilm formation on implanted medical devices. Therefore what is needed are new treatments for bacterial infection that can more subtly manipulate bacterial behaviors that promote health problems.
The bacterium Pseudomonas aeruginosa is the major pathogen associated with cystic fibrosis lung infection, keratitis eye infection, and third-degree burn-associated skin infections. P. aeruginosa has a complex signaling pathway that governs quorum sensing and virulence (FIG. 1B). The signaling pathway includes LasI, a synthase enzyme that makes the native acyl-homoserine lactone (AHL) signal, 3OC12-HSL. The native signal is detected by the transcriptional regulator LasR, forming a LasR:3OC12-HSL complex. The LasR:3OC12-HSL complex affects gene transcription, turning on virulence factors, the Rhl system and additional quorum-sensing circuits.
Another synthase, RhlI, produces another AHL (C4-HSL), which is detected by the transcriptional regulator RhlR. The RhlR:C4-HSL complex also regulates virulence genes and other components of the signaling pathway. Virulence production is impacted by multiple other factors, including the transcription factor QscR and the PQS system that produces and detects quinolone signals.
This tandem regulatory arrangement allows LasI/R to control the first wave of quorum-sensing-controlled gene expression and RhlI/R to control the second. Because LasR activates expression of rhlR, deletion of lasR reduces expression of both LasR- and RhlR-regulated target genes.
Additionally one key factor in pathogenicity of a bacterial infection is the production of virulence factor produced at high cell density, such as pyocyanin. This small molecule is redox active and is important for maintaining the redox balance in P. aeruginosa, particularly under low oxygen or anaerobic conditions. RhlR is a key transcriptional regulator controlling the up-regulation of the pyocyanin biosynthetic pathway, which in turn is induced by the LasR:3OC12-HSL complex (FIG. 1B). Thus, new compounds and methods of treating bacterial infection and/or contamination are needed.
SUMMARY OF THE INVENTION
The inventive concept is for anti-infective and prophylactic therapies to protect humans against gram negative bacteria, such as, for example, P. aeruginosa. This includes methods to block gram negative bacteria virulence and biofilm formation.
In one aspect, the invention is a compound having the formula:




wherein

    Py is a pyridine ring attached in the 2, 3, or 4 position and substituted with one or more additional substituents selected from the group consisting of alkyl, trifluoromethyl, methoxy, F, Cl and Br;
    Aryl is a benzene ring with one or more additional substituents selected from the group consisting of methyl, trifluoromethyl, cyano, nitro, F, Cl, Br and methoxy;
    X is O, NH, S or —CH2—; and
    n is 0 to 4 —CH2— units.


In one embodiment of this aspect, the compound has the formula:




wherein

    R is a substituent selected from the group consisting of alkyl, trifluoromethyl, methoxy, and Cl; and
    n is 4 to 5 —CH2— units.


In yet another embodiment, the compound has the formula:




wherein

R is a substituent selected from the group consisting of I, F, and Cl.
In another aspect, the invention is a compound having the formula:




where n=1, 2, 4 or 5

and a compound having the formula:





where n=1 or 3.

In the aspect of the invention which is inventive compounds, the compound is not meta-bromothiolactone (mBTL); is not chlorolactone (CL); and is not chlorothiolactone (CTL).

Another embodiment is a composition comprising the inventive compound.
Another aspect of the invention is the use of the compounds or the composition of the invention to inhibit gram negative bacteria. In the present invention, preferred examples of gram negative bacteria that can be inhibited by molecules of the invention, include, but are not limited to Burkholderia cepaci, C. violaceum, V harveyi, Pseudomonas, including, but not limited to Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, Bordetell pertussis, Haemophilus influenzae, Legionella pneuinophila, Brucella, Francisella, Xanthomonas, Agrobacterium, enteric bacteria, such as Escherichia coli and its relatives, the members of the family Enterobacteriaceae, such as Salmonella and Shigella, Proteus, and Yersinia pestis. 
It is contemplated that the use of the compounds or the composition of the invention inhibits quorum sensing and production of a biofilm or virulence factor, preferably pyocyanin. It is also contemplated that the use of the compounds of the invention inhibit LasR/RhlR receptor signaling.
Another aspect of the invention is the use wherein an effective amount of the compounds or composition is administered to a subject. Preferably the subject is a cow, pig, horse, chicken, cat, or dog, and even more preferably, a human. It is contemplated that the subject may have an infection, which may be, for example, opportunistic, antibiotic resistant, or have respiratory illness, dental plaque, gingivitis, chronic sinusitis, endocarditis, burn, wound, or may be immunosuppressed, immunocompromised, or may have bacterial invasion of a device in contact with the subject such as coating of contact lenses, medical device or other implanted device. The medical device may be a catheter, stent, joint prosthesis, prosthetic cardiac valve, ventilator or intrauterine device. The infection may be of the pulmonary tract and may be pneumonia. The respiratory illness may be cystic fibrosis and it may be in conjunction with pneumonia.
It is contemplated that the administration is therapeutic or prophylactic. Some of the preferable prophylactic uses are when the subject is undergoing surgery, a dental procedure or implantation of a medical device. It is also contemplated that the administration may be a co-administration with one or more drugs, preferably antibiotics. It is contemplated that administration may be topical, intravenous or intranasal.
Another aspect the invention is use of the compound or composition on or within a medical instrument or device, a filtration device, a tubing, a pipe, a pipeline, a sewage system, water tower cooling system, or a work surface. Preferably the medical device is a joint prosthesis, a prosthetic cardiac valve, a ventilator, a stent, or an intrauterine device.
The use of the compounds is also contemplated as a method comprising contacting the bacteria with the compound or composition of the invention. Preferably, the method comprises administering the inventive compound or composition to a subject. Alternatively the compound or composition is applied to surfaces, tubes, pipes or devices in a fluid, aerosol, gel or cream formulation.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Small molecule control of pyocyanin production in P. aeruginosa PA14. A. Structures of autoinducers and inhibitors. B. Simplified schematic of the major components of the P. aeruginosa quorum-sensing circuit. C. Pyocyanin production measured at OD600=695 nm in cell-free culture fluids prepared from WT P. aeruginosa PA14, lasR and rhlR single and double mutants, and in WT treated with 100 μM CL, CTL, mCTL, and mBTL. Error bars represent S.D. for two replicates. D. Pyocyanin inhibition titrations performed with WT P. aeruginosa PA14 in triplicate with CL (inverted triangles), CTL (squares), mCTL (diamonds) and mBTL, (asterisks). Error bars represent S.D. of three replicates.
FIG. 2. mBTL binds and inhibits LasR and RhlR. A. LasR activation of expression of rsaL-gfp in E. coli. B. RhlR activation of expression of rhlA-gfp in E. coli. In each panel, gfp expression in the presence of the cognate autoinducer (100 nM 3OC12-HSL or 20 μM C4-HSL) is set to 100%. mBTL was tested for inhibition at 1 mM. Agonism was examined at 100 nM for LasR and 20 μM for RhlR. Error bars represent S.D. of three replicates. C. SDS-PAGE analysis of whole cell (WC) and soluble (S) extracts from E. coli cultures expressing LasR in the presence of DMSO, 100 μM 3OC12-HSL, or 100 μM mBTL. An uninduced control is shown for comparison. D. Same as panel c with RhlR in the presence of DMSO, 100 μM C4-HSL, or 100 μM mBTL. E. Hierarchical clustering, heat maps and the root mean square (RMS) of the fold-change (login) of mBTL treated (+) or DMSO treated (−) WT P. aeruginosa, lasR, rhlR, and rhlI mutants. Dendrogram to the left of the map indicates average Euclidean linkage distances between the gene expression profiles. Data are the average of three independent biological experiments, one in which the Cy3 and Cy5 dyes were swapped.
FIG. 3. mBTL inhibits P. aeruginosa PA14 virulence toward C. elegans and human A549 lung cells. A. C. elegans were applied to lawns of E. coli HB101 (circles), WT P. aeruginosa PA14 (squares), lasR mutant (triangles), rhlR mutant (inverted triangles), and lasR, rhlR double mutant (diamonds) strains. The % live worms was calculated every hr for the first 5 hr and again at 24 hr. Error bars represent S.E.M. of three replicates. B. Same as panel a. E. coli HB101 (circles), WT P. aeruginosa PA14 (squares), and WT P. aeruginosa in the presence of 50 μM mBTL (asterisks). C. The % cell death was calculated using propidium iodide uptake into A549 lung cells after 8 hr and normalized to cells lysed with detergent. Error bars represent S.E.M. of three replicates.
FIG. 4. mBTL inhibits quorum-sensing-regulated clogging of microfludic chambers and biofilm formation in static cultures. A. Time to clogging was measured for the designated P. aeruginosa PA14 strains and for the WT in the presence of 100 μM mBTL. Error bars represent S.D. of six replicates. B. Biofilms were grown in static cultures at the base of a glass-bottom microtiter plate in the presence or absence of 100 μM mBTL. Biofilm thickness was measured using confocal microscopy. Error bars indicate S.D. of 5-8 biological replicates.
FIG. 5. Treatment of cell-free culture fluids with mBTL or mCTL does not alter pyocyanin levels. Cell-free culture fluids from WT P. aeruginosa PA14 following 17 hr growth were left untreated or supplied with 100 mM mCTL, mBTL, or an equivalent amount of DMSO. Subsequently, the fluids were incubated shaking at 37° C. for 17 hr. Pyocyanin levels were assessed by absorption. Error bars indicate S.D. of three replicates.
FIG. 6. Inhibition of pyocyanin production in P. aeruginosa PA14 by previously reported LasR inhibitors. a. Structures of four previously published LasR inhibitors. b. Pyocyanin inhibition assays were performed with 100 mM of each of the four molecules in panel a. on WT P. aeruginosa PA14. c. Pyocyanin inhibition titration assays were performed with itc-13 (circles) and V-06-018 (squares) at the concentrations shown using WT P. aeruginosa PA14. Error bars represent S.D. of three replicates.
FIG. 7. Structure-function analysis of mBTL and closely related derivatives. a. Structures of mBTL and derivatives tested for inhibition of pyocyanin production. b. Pyocyanin inhibition titrations were performed with the (R) (squares) and (S) (circles) enantiomers of mBTL and the racemic mixture (asterisks). c. mBTL derivatives containing 2-6 carbon chains were tested at 100 mM. d. Lactone variants were examined at 100 mM. In all panels, error bars indicate S.D. of three replicates.
FIG. 8. mBTL rescues C. elegans from killing by the P. aeruginosa PA14 lasR strain. The % living worms was scored on plates containing the lasR P. aeruginosa mutant in the presence of 100 mM mBTL and compared to plates containing DMSO after 5 hr. HB101 denotes the E. coli control strain. Error bars represent S.E.M. of three replicates.
FIG. 9. mBTL is not cytotoxic to A549 human lung cells. A549 human lung cells were treated with 100 mM mBTL or an equivalent amount of DMSO in the absence of bacteria. Fluorescence from propidium iodide uptake was measured after 8 hr. Error bars represent S.E.M. of six replicates. Lung cells treated with WT P. aeruginosa PA14 show the maximum level of propidium iodide uptake.
FIG. 10. CL inhibits LasR- and RhlR-controlled transcription in E. coli. A. LasR activation of expression of rsaL-gfp in E. coli. B. RhlR activation of expression of rhlA-gfp in E. coli. In each panel, gfp expression in the presence of the cognate autoinducer (100 nM 3OC12-HSL or 20 μM C4-HSL) is set to 100%. CL was tested for inhibition at 1 mM and agonism at 100 nM for LasR and 20 μM for RhlR. Error bars represent S.D. of three replicates.
FIG. 11. mBTL functions as a prophylactic. Pelleted P. aeruginosa PA14 cells that had been grown for 17 hr in the presence of 100 mM mBTL or an equivalent amount of DMSO were washed, resuspended in PBS, and added to human lung A549 cells. An additional 100 μM mBTL or an equivalent amount of DMSO was added at the start of infection. Cell death was measured using fluorescence from propidium iodide uptake. Error bars indicate S.E.M. of six replicates.
FIG. 12. Key interactions of the homoserine lactone with LasR and modeled interactions of 3 with LasR.
FIG. 13. Library design and representative examples.
FIG. 14. Synthesis of head group library and hybrid library.
FIG. 15. Activities of selected hits and previously reported compounds.
FIG. 16. Inhibitors do not affect the redox state of pyocyanin. Incubation of cell-free supernatant with DMSO or inhibitor leads to no change in pyocyanin absorbance.
FIG. 17. Pyocyanin production of WT P. aeruginosa and ΔlasRrhlR mutant after treatment with DMSO or 16. P<0.05 of DMSO versus 16-treated (Student's t test).
FIG. 18. LasR and RhlR assays in E. coli with gfp reporter. Compounds tested at 100 μM. For antagonism assays, the native AHL was also added at its EC95. a) LasR agonism b) LasR antagonism c) RhlR agonism d) RhlR antagonism.



DETAILED DESCRIPTION OF THE INVENTION
One aspect of the invention is directed to a class of molecules that have the ability to inhibit gram negative bacteria, whether by inhibiting quorum sensing, pathogenicity, virulence factor, and/or pyrocyanin production, and/or biofilm production,
For example, disabling quorum-sensing circuits with small molecules is one strategy to prevent bacterial pathogenicity. Synthetic molecules were prepared and assayed for inhibition of the two P. aeruginosa quorum-sensing receptors, LasR and RhlR. The most effective compound, the small molecule meta-bromothiolactone (mBTL) inhibits both the production of the virulence factor pyocyanin and biofilm formation in our assays. In tissue culture and in an animal model, mBTL protects cells from P. aeruginosa. mBTL partially inhibited both the LasR and RhlR receptors in vivo and in vitro. In the tested assays, more potent antagonists did not exhibit superior function in impeding virulence which may be because mBTL displays a more appropriate tuning of the two receptors. In the present invention, one strategy described herein for blocking pathogenesis in vivo comprises developing inhibitors that appropriately tune the two P. aeruginosa receptors, as well as the corresponding receptors found in other gram negative bacteria. These findings are the basis for the inventive concept of anti-infective and prophylactic therapies to protect humans against gram negative bacteria, such as, for example, Burkholderia cepaci, C. violaceum, harveyi, Pseudomonas, including, but not limited to Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, Bordetella pertussis, Haemophilus influenzae, Legionella pneumophila, Brucella, Francisella, Xanthomonas, Agrobacterium, enteric bacteria, such as Escherichia coli and its relatives, the members of the family Enterobacteriaceae, such as Salmonella and Shigella, Proteus, and Yersinia pestis. 
Moreover, the gram negative bacteria, such as the pathogen P. aeroginosa owes its virulence to virulence factors, such as, for example, pyocyanin that it produces under the control of a quorum-sensing system. Compounds of the invention have been designed that attenuate the virulence of gram negative bacteria (for example, P. aeroginosa, working by means of a regulator of the quorum-sensing system.
The native QS autoinducers in P. aeroginosa are 3OC12-HSL and C4-HSL which form complexes with the LuxR-type proteins LasR and RhlR, respectively. With the lactone moiety as a starting point for the design of substitute compounds that bind these proteins, a library of candidates was synthesized and inhibitory compounds were identified.
In the present tested assays, the most potent of these compounds as an inhibitor of pyocyanin production is meta-bromo thiolactone (mBTL) (FIG. 1a). mBTL was used as a lead compound to design other inhibitors, either retaining the thiolactone moiety or substituting a lactone moiety. Of the thiolactone derivatives of mBTL, C5-mBTL was the most effective inhibitor of pyocyanin production in the assays tested. Of the lactone derivatives of mBTL, C4-mBL was the most effective inhibitor of pyocyanin production in the assays tested. The S enantiomer of mBTL is active as an inhibitor of pyocyanin production (IC50=4 μM) and the R enantiomer has residual activity (IC50=100 μM).
mBTL was found to be an effective as an attenuator of virulence in P. aeroginosa in the assays tested. mBTL or derivatives of mBTL find use as compounds as described herein. They can act through the quorum sensing system by binding to a QS LuxR-type protein.
In a broader context, what is contemplated is the use of the compounds of the invention to attenuate bacterial virulence. In one embodiment, the compound or compounds are a component of a composition and have efficacy to inhibit the bacterial virulence, preferably of gram negative bacteria, such as, for example, P. aeroginosa. Preferably these compositions comprise mBTL or derivatives of mBTL. In another embodiment procedures are contemplated comprising administering mBTL, derivatives of mBTL, or the compositions to an individual who is free of bacterial disease. Preferably, administration is in advance of an anticipated health-related procedure known to increase susceptibility to a gram negative bacteria, and preferably, P. aeroginosa pathogenicity, for example, in advance of a surgical procedure, including dental procedures, especially procedures involving implants, or insertion of catheters or other devices. In yet another embodiment, it is contemplated to contact surfaces of work areas, medical instruments, medical devices and the like with the compositions of the invention in order to attenuate the virulence of a gram negative bacteria, such as P. aeroginosa, that might come into contact with these surfaces.
In another aspect of the invention, what is contemplated is deploying the compounds of the invention to prevent the failure of devices that are prone to fouling by biofilms. These compounds are useful in industrial settings and in contexts requiring medical implants. The compounds of the invention may be administered in the liquid phase, may be embedded in materials used for production of such devices, or may coat such devices resulting in products that are innately resistant to biofilms. These compounds also may be used to inhibit biofilms from forming in situations where liquids are flowing, as, for example, through pipes, pipelines, tubing, water cooling systems, stents or filtration devices.
Indications
Gram negative bacteria are typically free-living organisms often found in soil and water, and play an important role in decomposition, biodegradation, and the C and N cycles. However, many gram negative bacteria are pathogenic. Examples of gram negative bacteria that can be inhibited by compounds of the invention, include, but are not limited to Burkholderia cepaci, C. violaceum, harveyi, Pseudomonas, including, but not limited to Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, Bordetella pertussis, Haemophilus influenzae, Legionella pneurnophila, Brucella, Francsella, Xanthomonas, Agrobacterium, enteric bacteria, such as Escherichia coli and its relatives, the members of the family Enterobacteriaceae, such as Salmonella and Shigella, Proteus, and Yersinia pestis. 
For example, gram negative bacteria often cause opportunistic infections in immunocompromised or immunosuppressed individuals. One example of such a bacteria is P. aeruginosa. These infections are spread by heath care workers or patients to surfaces, machinery or instruments in health care facilities. P. aeruginosa typically infects the pulmonary tract, urinary tract, burns, and wounds. P. aeruginosa also causes catheter-associated infections, blood infections, middle ear infections, formation of dental plaque, gingivitis, chronic sinusitis, endocarditis, coating of contact lenses, and infections associated with implanted devices, for example, catheters, joint prostheses, prosthetic cardiac valves and intrauterine devices. P. aeruginosa causes infections of the central nervous system, gastrointestinal tract, bones, joints, ears and eyes. The compounds or compound compositions of the invention can be used to treat these infections and conditions.
Specifically, the compound or compound compositions of the invention can be administered to treat, inhibit, and/or ameliorate infections including opportunistic infections and/or antibiotic resistant bacterial infections caused by gram negative bacteria. Examples of such opportunistic infections, include, but are not limited to P. aeruginosa. or poly-microbial infections of P. aeruginosa with, for example, Staphylococcus aureus or Burkholderia cepacia. Examples of patients who may acquire such opportunistic and/or resistant infections include, but are not limited to patients who are immunocompromised or immunosuppressed, who have cystic fibrosis or HIV. who have implanted medical devices, subcutaneous devices or who are on ventilators, patients who have been intubated or who have catheters, nosocomial infections, patients who are undergoing bone marrow transplant or other types of surgery, including, but not limited to dental surgery and patients who are TV drug users, especially with regard to heart valve infection.
Burns and/or other traumatic wounds as well as common or uncommon infections can also be prophylactically treated and/or ameliorated by administration of the compound or compound compositions. Examples of such wounds and infection disorders include, but are not limited to puncture wounds, radial keratotomy, ecthyma gangrenosum, osteomyelitis, external otitis or dermatitis.
In one embodiment, the compound or compound compositions of the invention can be administered to treat, prevent, and/or ameliorate pulmonary infections. More preferably, the compound or compound compositions of the invention can be administered to treat, prevent, diagnose, and/or ameliorate pneumonia. More preferably, the compound or compound compositions of the invention can be administered to treat, prevent, and/or ameliorate lung infections, such as pneumonia, in cystic fibrosis patients. More preferably, the compound or compound compositions of the invention can be administered to treat, prevent, and/or ameliorate gram negative infections such as by P. aeruginosa in cystic fibrosis patients. Pneumonia can be caused by colonization of medical devices, such. as ventilator-associated pneumonia, and other nosocomial pneumonia, and the compound or compound compositions of the invention can be administered to treat and/or prevent these types of pneumonia or bacterial infections as well.
Additionally, the compound or compound compositions of the invention can be administered to treat and prevent septic shock. More preferably, the compound or compound compositions of the invention can be administered to treat, prevent, and/or ameliorate septic shock in neutropenic, immunocompromised, and/or immunosuppressed patients or patients infected with antibiotic resistant bacteria, such as, for example, antibiotic resistant P. aeruginosa.
Additionally, the compound or compound compositions of the invention can be administered to treat, prevent, and/or ameliorate urinary tract or pelvic infections. In another preferred embodiment, the compound or compound compositions of the invention can be administered to treat, prevent, and/or ameliorate gastrointestinal infections, such as necrotizing enterocolitis, often seen in premature infants and/or neutropenic cancer patients.
Additionally, the compound or compound compositions of the invention can be administered to treat, prevent, and/or ameliorate urinary dysenteriae (for example, dysenteria caused by bacillary dysentery), food poisoning and/or gastroenteritis (for example, caused by Salmonella enterica), typhoid fever (for example, caused by Salmonella typhi), whooping cough (or pertussis) as is caused by Bordetella pertussis, Legionnaires' pneumonia, caused by Legionella pneumophila, sexually transmitted diseases, such as gonorrhea, caused by Neisseria gonorrhoeae, or meningitis, caused by, for example, Neisseria meningitidis or Haemophilus influenzae, brucellosis which is caused by brucellae, and more specifically, Brucella abortus. 
Formulations and Methods of Administration
The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of the compound or a pharmaceutical composition of the invention. In a preferred aspect, the compound is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to mammals, amphibians, birds, and fish. The subject is more preferably either a mammal, including but not limited to animals such as cows, pigs, horses, cats, dogs, etc., or an avian species, including but not limited to chickens, ducks and other domestic poultry. The subject is most preferably a human.
Formulations and methods of administration that can be employed with the compound or compound compositions, additional appropriate formulations and routes of administration can be selected from among those described herein below. The compound or compound compositions of the invention may be administered therapeutically, such as, for example, in the case of infection of a susceptible patient with burn or other traumatic wound injury or lung infection, such as in a cystic fibrosis patient infected with P. aeruginosa or Burkholderia cepacia separately or in combination. Alternatively, the compound or compound compositions may be administered prophylactically, such as, for example, to prevent opportunistic gram negative bacterial infection, such as by P. aeruginosa, prior to surgery, dental work, or implantation of a medical device such as a catheter or ventilator tube continuously, such as, for example in the case of an immunosuppressed or immunocompromised patient.
Various delivery systems are known and can be used to administer compound, e.g., encapsulation in liposomes, microp articles, microcapsules. Methods of introduction include, but are not limited to, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, inhalation, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In one embodiment the compound of the invention is formulated in 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, 0.01% polysorbate 80, pH 6.5 (±0.3). In another embodiment, the compound of the invention is formulated in 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, 0.01% polysorbate 80, pH 6.5 (±0.3) for intravenous administration.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
In another embodiment, the composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
In yet another embodiment, the composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit, Ref Biomed. Eng. 14:20 1 (1987); Buchwald et al, Surgery 88:507 (1980); Saudek et al, N. Engl. J. Med. 321:574 (1989)), In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, Macromol. Sci. Rev. Macromol. Chem. 2.3:61 (1983); see also Levy et al, Science 2.28:190 (1985); During et al, Ann. Neurol. 25:35 1 (1989); Howard et al, J. Neurosurg. 7 1:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of compound and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a. suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc, Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, topical or pulmonary administration to human beings. Typically, compositions for such administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocanme to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compositions of the invention can be formulated as neutral or salt forms, Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the composition of the invention that will be effective in the treatment, inhibition and prevention of a disease or disorder can be determined by standard clinical techniques. Additionally, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For example, the dosage administered to a patient should typically be 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. In preferred embodiments, a dose of 1, 4, 10, or 20 mg/kg is administered intravenously to a patient. Further, the dosage and frequency of administration, of therapeutic or pharmaceutical compositions of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the skin and/or lungs) of by modifications such as, for example, lipidation.
The compound or compound compositions of the invention may be administered alone or in combination with other compounds, such as adjuvants. In one embodiment the compounds may be administered in combination with one or more antibiotics, for example, gentamicin, tobramycin, colistin, and fluoroquinolins. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
In the treatment of burns or other traumatic wound injuries that are susceptible to bacterial infection such as, for example, P. aeruginosa infection, the presently described compound can be prepared in a medicament and the preparation applied generously (e.g., topically) to the entire burn area as quickly as possible. Repeated applications are made, if necessary and as needed to relieve pain, increase healing and decrease infection. If necessary, resuscitation is started by the introduction of the conventional intravenous fluids. Pain killers, toxin neutralizers, vitamins and antibiotics may be employed as indicated. Moreover, intravenous treatment of the compound or compound composition may also be needed to treat the burn or other traumatic wound injury.
The wound to which the compound or compound compositions have been applied may be covered with gauze and sheet wadding and thereafter dressed daily. At the time of dressing, all devitalized tissue and crusts Which can be removed readily should be removed. Tissue which is attached firmly is permitted to separate normally,
In the use of the compound or compound compositions for the treatment of lung infections, preferably, for example, in patients suffering from cystic fibrosis, pneumonia (regardless of the etiology), and/or antibiotic resistant bacterial pulmonary infection, the compound will generally be administered for symptomatic treatment in the form of a conventional pharmaceutical composition, for example, as generally described in U.S. Pat. No. 4,910,190, and preferably as an aerosol. A formulation providing a solution containing a concentration, for example, of 10 mg/mL, 20 mg/ml, or 30 mg/ml of the compound and suitable for use with a. nebulizer (preferably) or as an injectable solution. A suitable nebulizer for use is, for example, a RETECTM nebulizer, in which the solution is nebulized with compressed air.
In general, the compound or compound compositions will be administered to humans at a daily dose in the range of, for example, 5 to 100 mg of the compound by aerosol or 50 to 1000 mg intravenously, or a combination of the two. However, it readily will be understood that it may be necessary to vary the dose of therapeutic product administered in accordance with well-known medical practice to take account of the nature and severity of the lung disease (for example, cystic fibrosis) under treatment, concurrent therapy, and the age, weight and sex of the patient receiving treatment. it similarly will be understood that generally equivalent amounts of a pharmaceutically acceptable salt of the compound also may be used.
Industrial Uses
Compounds of the invention can be used in industrial settings to inhibit biofilm production and/or to remove antibiotic resistant bacteria, such as in a hospital or other public setting. For example, the compounds of the invention can be used to remove biofilms that have grown in moist and warm environments, such as showers, water and sewage pipes, cooling or heating water systems, (e.g., cooling towers), marine engineering systems, such as, for example, pipelines of the offshore oil and gas industry. The compounds of the invention can also be used, for example, to remove and/or prevent bacterial adhesion to boat hulls, since once a biofilm of bacteria forms, it is easier for other marine organisms such as barnacles to attach. The compounds of the invention can be used to reduce, for example, the time a boat is in dry dock for refitting and repainting, thereby increasing productivity of shipping assets, and useful life of the ships. The compounds of the invention can also be used to remove biofilm production intentionally used to eliminate petroleum oil from contaminated oceans or marine systems, once the contamination is removed.
Additionally, the compound of the invention can be used to wash, rinse or swab floors and counters, such as in food preparation areas or medical facilities, as well as medical devices, including but not limited to, stents, catheters, intubation tubes, or ventilator equipment. Still further the compounds can be used as a handwash to help eliminate spread of virulent bacteria by health workers, patients and others.
Particular species of bacteria may be especially problematic. For example, Pseudomonas aeruginosa is a pathogen that can survive in a wide range of environments. The bacterium is a public health threat because it causes a variety of secondary infections in humans, where those with burn wounds, cystic fibrosis, and implanted medical devices are particularly at risk. With an outer membrane of low permeability, a multitude of efflux pumps, and various degradative enzymes to disable antibiotics, P. aeruginosa is difficult to treat. As with other common pathogenic bacteria, antibiotic-resistant strains are an increasing problem.
Blocking virulence is one of the strategies contemplated to combat these bacteria. This approach provides less selective pressure for the spread of resistant mutants and leads to drug therapies that are effective over a greater time span compared to traditional antibiotics. Rather than preventing growth or killing the bacteria, an antivirulence approach prevents the expression of virulence traits. The bacteria that have been treated and are thus benign should then be more easily cleared by the host immune system.
In yet another embodiment, another series of potent molecules were designed that inhibit pyocyanin production. The invention encompasses the design, synthesis, and evaluation of these inhibitors. In a preferred embodiment, the structures of these molecules were evolved from the native signal, 3OC12-HSL, and a thiolactone inhibitor mBTL (FIG. 15, 1). The structures of the inhibitors substitute the lactone moiety of the native signal with a chemically stable moiety that was chosen to maintain native binding contacts with LasR, according to modeling based on the LasR crystal structure.
In considering the natural QS components (FIG. 1A), the homoserine lactone (HSL) head group is the common feature of a series of AHL quorum sensing signals and this head group takes part in key hydrogen-bonding interactions with the receptor binding pocket (2, FIG. 12), while the tails generally have only van der Waals interactions in a hydrophobic pocket of the protein. A replacement for the HSL should maintain the key hydrogen-bond interactions with the receptor and perhaps find additional opportunities for enhanced binding. Using the LasR crystal structure as a template for HSL binding, a set of head group candidates was identified in a virtual screen using AutoDock. Heterocycles, including aminopyridine (3, FIG. 12) were identified as good candidates that could maintain the native binding contacts and could have potential for additional hydrogen-bonding interactions. In addition to its chemical stability, the aminopyridine moiety was attractive as the platform for the synthesis of a focused library to explore structure-activity relationships.
Despite having been designed to bind the LasR and/or RhlR receptors of P. aeruginosa, subsequent studies suggest that the inhibitor compounds reduce pyocyanin levels by a pathway different from the LasR or RHLR receptors.
The compounds of the present invention can be described according to the following formula:




where X═O, S, or NH;

    
    
        Y═O; H, H; or H, OH,
        n=2-8;
        Ar=a phenyl group or a substituted phenyl group, wherein the substituted phenyl group is represented by:
    
    






where R1-R5 can be Cl, Br, F, NO2, CN, alkyl, or phenyl.

The following examples set forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention.
EXAMPLE 1
Strains and Media
E. coli strains were grown at 37° C. in Luria broth (LB) (Fisher). Plasmid pET23b (Novagen) was used to express lasR and rhlR in E. coli strain BL21-Gold (DE3) (Stratagene). Plasmids were maintained with 100 μg/mL ampicillin. Plasmid pEVS141 was used for rhlA-gfp or rsaL-gfp expression and maintained with 50 mg/mL of kanamycin. P. aeruginosa strains were grown with shaking at 37° C. in LB. C. elegans wild-type strain N2 was propagated on NGM with an E. coli HB101 lawn as the food source at 20° C. A549 human lung carcinoma cells (ATCC #CCL-185) were grown in DMEM medium (Gibco) plus 20% fetal bovine serum and 1× PenStep (Sigma) at 37° C.
The P. aeruginosa rhlR strain (rhlR::MAR2xT7) and the rhlI strain (rhlI::MAR2xT7) come from the P. aeruginosa PA14 ordered transposon library. The P. aeruginosa lasR and lasR, rhlR double mutant strains were constructed using red recombination. The region spanning approximately 600 bp upstream of lasR and including the start codon (called lasR′) and the sequence encoding the C-terminal 6 amino acids of LasR and approximately 600 bp downstream (called 'lasR) were amplified by PCR. The FRT-aacCl-FRT region in pAS03 was amplified using primers that span sequences in lasR′ or ‘lasR. The lasR’, FRT-aacCl-FRT and lasR PCR products were combined through overlap extension PCR and amplified. The resulting lasR′-FRT-aacCl-FRT-′lasR product was transformed into P. aeruginosa PA14 harboring pUCP18-RedS. Gentamicin resistance was selected to yield lasR::aacCl in the chromosome. Following excision of the gentamicin resistance gene, the lasR, rhlR double mutant strain was constructed by inserting rhlR::MAR2xT7 into the lasR strain background followed by selection for gentamicin resistance. This strategy yielded the lasR::FRT, rhlR::MAR2xT7 strain.
EXAMPLE 2
Chemistry, Materials and Methods
Unless otherwise stated, reactions were performed in flame-dried glassware fitted with rubber septa under a nitrogen atmosphere and were stirred with Teflon-coated magnetic stirring bars. Liquid reagents and solvents were transferred via syringe using standard Schlenk techniques. Reaction solvents were dried by passage over a column of activated alumina. All other solvents and reagents were used as received unless otherwise noted. Reaction temperatures above 23° C. refer to the oil bath temperature, which was controlled by an OptiCHEM temperature modulator. Thin layer chromatography was performed using SiliCycle silica gel 60 F-254 precoated plates (0.25 mm) and visualized by UV irradiation and anisaldehyde, ceric ammonium molybdate, or potassium permanganate stain. Sorbent standard silica gel (particle size 40-63 μm) was used for flash chromatography. 1H and 13C NMR spectra were recorded on Bruker Avance II (500 MHz for 1H; 125 MHz for 13C) spectrometer fitted with either a 1H-optimized TCI (H/C/N) cryoprobe or a 13C-optimized dual C/H cryoprobe. Chemical shifts (δ) are reported in ppm relative to the residual solvent signal (δ=7.26 for 1H NMR and δ=77.0 for 13C NMR). Data for 1H NMR spectra are reported as follows: chemical shift (multiplicity, coupling constants, number of hydrogens). Abbreviations are as follows: s (singlet), d (doublet), t (triplet), m (multiples). High-resolution mass spectral analysis was performed using an Agilent 1200-series electrospray ionization—time-of-flight (ESI-TOF) mass spectrometer in the positive ESI mode. The following compounds were synthesized as previously described: CL, CTL, mBTL, mCTL, itc-13, PD-12. Methods used to prepare and evaluate molecules described above are described in example 14 and in Swem et al. (2009) (Reference 4, below).
EXAMPLE 3
Pyocyanin Analyses
Methods. The oxidized form of pyocyanin imparts a green color to P. aeruginosa cultures, making the production of pyocyanin convenient to monitor by UV/Vis absorbance. Overnight P. aeruginosa cultures were subcultured into 5 mL fresh medium at 1:1000 dilution. Synthetic compounds were assayed at 100 μM for end point assays and at concentrations ranging from 200 nM to 200 μM for titrations following 17 hr of aerobic growth with shaking at 37° C. Cells were separated from culture fluids via centrifugation at 13,000 rpm for 15 min. Culture fluids were passed through 0.22 μm syringe driven filters (Millipore). Cell-free culture fluids were analyzed for pyocyanin using wavelength scans on a Beckman Coulter DU-800 spectrophotometer from 200 nm to 800 nm 695 nm was chosen for graphical representation. Titration data were fit with a variable-slope sigmoidal dose-response curve using GraphPad Prism to determine the IC50 values.
Results. The molecule CL (FIG. 1a) is an inhibitor of the LasR/RhlR homolog CviR from Chromobacterium violaceum. Based on the CL structure, design and synthesis of a focused library of approximately 30 molecules was completed. Analyses for CL and three additional molecules are provided as examples (CTL, mCTL, and mBTL; FIG. 1a). The first experiment was of the molecules for inhibition of quorum sensing in wild-type P. aeruginosa PA14 using the quorum-sensing-regulated production of the virulence factor pyocyanin as the readout (FIG. 1c). Following activation by LasR:3OC12-HSL, the RhlR:C4-HSL complex activates expression of the pyocyanin operon (FIG. 1b). Therefore, P. aeruginosa PA14 produces pyocyanin at high cell density, and limited pyocyanin production occurs in a lasR or a rhlR null mutant and in the lasR, rhlR double mutant (FIG. 1c).
Neither CL nor CTL inhibited pyocyanin production in vivo (FIG. 1c). By contrast, mCTL and mBTL showed potent inhibition of pyocyanin (FIG. 1c) while not affecting P. aeruginosa PA14 growth. Calculated IC50 values were 8 μM (±2) for mBTL and 9 μM (±2) for mCTL (FIG. 1d). Inhibition was not due to interaction of these molecules with pyocyanin, as incubation of pyocyanin-containing cell-free culture fluids from untreated P. aeruginosa PA14 with 100 μM mBTL or mCTL did not alter pyocyanin levels (FIG. 5). The next experiment involved synthesis of four previously reported LasR inhibitors for comparison in the pyocyanin assay (FIG. 6a). Two compounds, itc-13 and V-06-018, inhibited pyocyanin production in P. aeruginosa PA14 with higher IC50 values (56 μM (±10) and 18 μM (±2), respectively) than mBTL and mCTL (FIG. 6b,c). The other molecules (PD-12 and B7) showed limited or no inhibition in vivo. (FIG. 6b). Thus, mBTL is more potent than mCTL and these previously identified inhibitors.
EXAMPLE 4
Chiral Resolution of mBTL
Methods. mBTL enantiomers were resolved using a Berger Multigram II SFC system equipped with two Varian SD-1 pumps, a Knauer K-2501 multi-wavelength detector set at 220 nm, a Knauer K-1900 pump, a Vatran SGP-50-100 condenser, and using a Chiralpak IC (2×15 cm) column. An isocratic method using a mixture of 30% MeOH/CO2 (100 bar) at 60 mL/min was employed. The two peaks eluted at 1.66 min and 2.13 min. The identity of the enantiomers was determined through comparison of the HPLC trace with that of authentic (S)-mBTL synthesized from L-homocysteine thiolactone hydrochloride. Based on this analysis, peak 1 (>99:1 er) is (S)-mBTL and peak 2 (>99:1 er) is (R)-mBTL.
Results. To determine the enantiomer of mBTL responsible for inhibition, a chiral separation was performed. The S enantiomer is active (IC50=4 μM) while the R enantiomer displays residual activity (IC50=100 μM) (FIG. 7a,b). Because the racemic mixture has an IC50 value only 2-fold higher than the isolated S enantiomer, we conclude that the R enantiomer does not influence inhibition. For ease of synthesis, racemic mixtures of mBTL were used for the other examples.
EXAMPLE 5
LasR and RhlR GFP Assays
Methods. The LasR-GFP assays were performed in E. coli strain BL21 DE3 Gold (Agilent) carrying pET23b (Novagen) containing lasR (maintained with 100 μg/mL ampicillin) and carrying plasmid pEVS141 (31) containing the rsaL promoter driving expression of gfp (maintained with 50 mg/mL of kanamycin.) The RhlR-GFP assays were performed in E. coli strain BL21 DE3 Gold (Agilent) carrying pET23b (Novagen) containing rhlR (maintained with 100 μg/mL ampicillin) and carrying plasmid pEVS141 containing the rhlA promoter driving expression of gfp (maintained with 50 μg/mL of kanamycin.) These E. coli strains were grown overnight and subcultured into fresh medium with appropriate antibiotics at a 1:100 dilution and grown shaking for 8 hr at 37° C. for the LasR-GFP strain and 12 hr for the RhlR-GFP construct. 50 nM 3OC12-HSL or 20 μM C4-HSL was added to the LasR-GFP and RhlR-GFP preparations, respectively. Compounds were tested at 1 mM for antagonism and at 100 nM or 20 μM for agonism. These concentrations were chosen for agonism studies to match the concentrations of autoinducers used in the experiments. For antagonism studies, the EC95 concentration was used for each receptor. GFP was measured on an Envision plate reader.
Results. Investigation of whether mBTL interacts with LasR, RhlR, or both receptors proceeded using recombinant E. coli strains producing the receptor proteins and containing target gfp reporter fusions (rsaL-gfp for LasR and rhIA-gfp for RhlR). In the absence of ligand, neither receptor activates expression of the target-gfp fusion (FIG. 2a,b). When autoinducer is supplied at the EC95, LasR and RhlR activate gene expression. mBTL partially antagonizes this effect (22%±4 and 43%±10 inhibition, respectively, FIG. 2a,b). In the absence of autoinducer, mBTL, when provided at the EC95 of the native ligand for each receptor, acts as a partial agonist of both LasR and RhlR (72%±3 and 80%±25, respectively). Molecules such as mBTL, that resemble autoinducers, induce conformational changes in LuxR-type receptors that impair their ability to interact with RNA polymerase which lowers their transcriptional activation potential. In the presence of autoinducer, this reduction in transcription potential manifests as antagonism. In the absence of autoinducer, some transcription activation can still occur. Thus, without autoinducer present, molecules such as mBTL appear as partial agonists.
mBTL, the most potent in vivo inhibitor, is a partial agonist/partial antagonist of RhlR and LasR in the recombinant E. coli assay (FIG. 2a,b) and according to P. aeruginosa microarrays (Table 7). This feature of mBTL may be critical for its ability to function in vivo. Because LasR and RhlR act reciprocally to control key virulence genes (FIG. 1b), molecules that function as pure antagonists of both receptors might not be superior anti-virulence therapies. Consistent with this notion, CL is a more potent inhibitor of both LasR- and RhlR-directed transcription in E. coli than is mBTL, and CL does not agonize either receptor (FIG. 10). Nevertheless, CL exhibits no in vivo inhibition of pyocyanin production (FIG. 1b,c) and it does not attenuate killing of A549 lung cells by P. aeruginosa. This might be termed that a “sweet spot” exists in terms of antagonist potency for inhibitors of LasR and/or RhlR, and that mBTL has the appropriate level of potency to be effective in vivo. Consider a case in which LasR activates and RhlR represses a particular virulence factor (see FIG. 1b).
EXAMPLE 6
Binding of LuxR-Type Proteins LasR and RhlR to mBTL and to Cognate Autoinducers
Methods. Overnight cultures of E. coli BL21-Gold (DE3) carrying the LasR and RhlR overexpression constructs were diluted 1:100 into fresh LB supplemented with antibiotics and grown shaking at 37° C. to an OD600 of 0.4. Autoinducer or antagonist molecules were added at 100 μM and incubated an additional 30 min at 20° C., after which protein production was induced by the addition of 1 mM IPTG for 6 hr at 20° C. Cells were harvested by centrifugation and resuspended in 1 mL of 20 mM Tris (pH 7.5), 0.5 mM EDTA, 300 mM NaCl, 1 mM DTT, and 5% glycerol and 100 μM of the appropriate ligand. Resuspended pellets were sonicated twice for 15 seconds to produce lysates containing all of the cell contents. This preparation is referred to as the whole cell (WC) fraction. The WC fraction was subjected to centrifugation at 4° C. at 13,300 RPM for 15 min to remove insoluble material and the membrane fraction. The supernatant from this pellet is referred to as the soluble (S) fraction. SDS-PAGE gels (4% stacking and 12% resolving) were used followed by Coomassie blue (BioRad) staining to visualize protein. Contrast was uniformly adjusted for both gels.
Results. LuxR-type proteins require cognate autoinducers to fold. Consistent with this, LasR and RhlR are insoluble in the absence of autoinducer, and are present in the whole cell (WC) fraction but not the soluble (S) fraction following SDS-PAGE (FIG. 2c,d). Addition of 100 μM 3OC12-HSL or mBTL solubilizes LasR (FIG. 2c). mBTL also solubilizes RhlR; surprisingly, C4-HSL, its native autoinducer, does not (FIG. 2d). It appears that autoinducer-bound RhlR is not particularly soluble when over-produced in E. coli and the protein likely aggregates. mBTL appears to protect the protein from aggregation. Similar protection occurs with CviR bound to CL.
EXAMPLE 7
RNA Extraction and Microarray Analysis
Methods. Overnight P. aeruginosa PA14 cultures were back-diluted 1:1000 into 5 mL of fresh LB. 100 μM mBTL, or an equivalent amount of DMSO, was added to cultures which were grown aerobically with shaking at 37° C. for 17 hr. 9 ODs of cells were harvested for each treatment. Lysozyme (1 mg/mL in TE buffer) (Sigma) was added for 10 min at room temperature. Total RNA was prepared using the RNeasy Midi Kit (Qiagen). RNA was treated with RNase-Free DNaseI (Ambion) for 1 hr at 37° C., inactivated using DNaseI Inactivation Reagent Resin (Ambion), and re-purified using the RNeasy Mini Kit. A cDNA library containing Cy3- or Cy5-labeled dUTP (Enzo Life Sciences) was synthesized from the purified RNA using SuperScript III Reverse Transcriptase (Invitrogen). Sodium hydroxide was added to degrade RNA, and the reaction was subsequently neutralized by addition of hydrochloric acid. The library was purified using the PCR Purification kit (Qiagen) and assessed for Cy3 and Cy5 incorporation using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies). Libraries were normalized for cDNA concentration and hybridized using the Gene Expression Hybridization Kit (Agilent) to a custom microarray (Agilent design number 43307), which was designed using the Agilent eArray tool with 2 probes for most genes. Samples were hybridized for 22 hr at 65° C. with continuous rotation at 10 rpm. Microarrays were scanned using an Agilent G2505C scanner and analyzed using Agilent Feature Extract software version 9.5. Resulting microarray intensity data were submitted to the PUMA Database (http://puma.princeton.edu) for archiving and analyzed using Matlab R2013a.
Results. To verify that mBTL functions by inhibiting quorum sensing in vivo, microarrays were used to examine the consequences of administering mBTL to wild-type P. aeruginosa and to the lasR and rhlR mutants. Treatment of wild-type P. aeruginosa PA14 caused alterations in expression of many of the known quorum-sensing targets (Table 7). For example, LasR-regulon genes including rpoS and nor were down-regulated (Tables S1 and S2). RhlR-controlled virulence genes, for example those encoding rhamnolipids (rhlA and rhlB), and phenazine (phzA2, phzB1, and phzB2, were also repressed (Table 7 and S3). Indeed, the profile of wild-type cells treated with mBTL matches well with the combined profiles of the lasR and rhlR mutants (Table 7). However, the fold-activation and fold-repression is not as dramatic in the mBTL-treated wild-type as in the mutants, confirming that mBTL does not fully inhibit either regulator (Tables S1, S2 and S3). Thirteen genes are activated 2-fold or more in wild-type P. aeruginosa PA14 when treated with mBTL (Table 10). By contrast, two hundred and thirteen genes are down-regulated 2-fold or more when wild-type is treated with mBTL (Table 7). These data indicate that the major role of mBTL in wild-type P. aeruginosa PA14 is as an antagonist that exerts control over virulence through partial-inhibition of LasR and RhlR not via up-regulation of other genes. Both the LasR and RhlR quorum-sensing receptors are partially inhibited by mBTL, however, as shown in the following experiment, in the contexts that we have examined, RhlR, not LasR, is the relevant in vivo target.
EXAMPLE 8
Analysis of lasR and rhlR Mutants Gene Expression
Results. The most important comparisons for defining the target of mBTL are the mBTL-treated and untreated lasR and rhlR mutants (Tables 11 and 12 and FIG. 2e). In the absence of LasR, mBTL treatment still alters expression of some genes. By contrast, there is essentially no difference between the untreated and mBTL-treated rhlR mutant profiles. Thus, while some of the mBTL effects occur through LasR, all of the mBTL effects depend on RhlR (Tables 11 and 12 and FIG. 2e). Thus, mBTL functions in vivo by inhibiting quorum sensing via RhlR.
EXAMPLE 9
mBTL Agonism of RhlR In Vivo
Given that mBTL acts as a partial agonist of RhlR in recombinant E. coli when the cognate autoinducer C4-HSL is not present (FIG. 2b), further examination was made of the extent of mBTL agonism of RhlR in vivo. To do this, a P. aeruginosa rh11 null strain was treated with mBTL and microarrays were performed. mBTL activated 2-fold or higher expression of forty-four genes. For comparison, RhlR bound to C4-HSL activated nearly 300 genes (Tables 13 and 9). In every case in which a gene could be activated by C4-HSL or by mBTL, C4-HSL was a much more potent activator. Importantly, only one gene that was activated by mBTL in the rh11 mutant was activated by mBTL in wild-type P. aeruginosa (Tables 13 and 10). These data indicate that mBTL can act as a weak RhlR agonist in vivo, but only when the native autoinducer is absent; a situation that is not likely to occur in wild-type bacteria.
EXAMPLE 10
mBTL Limits Virulence in an Animal Model
Methods. C. elegans fast killing assays were conducted with 90 wild-type N2 worms for each condition (30 worms/replicate, 3 replicates performed). C. elegans were propagated on NGM plates prior to eggs being harvested from gravid adults using a standard bleaching protocol (30 mL 5% bleach, 15 mL 5 M KOH, 55 mL dH2O). Harvested eggs were placed on lawns of fresh E. coli HB101 and allowed to grow for 48 hr (to reach the L4 stage) at 20° C. prior to being moved to lawns of P. aeruginosa and placed at 25° C. on sorbitol, glucose, cholesterol plates. Nematodes were scored for survival every hr for 5 hr and again at 24 hr. The % living worms was calculated in triplicate for each time point. 50 μM mBTL or an equivalent volume of DMSO was added to plates and to the bacterial cultures during growth.
Results. To determine if mBTL can limit virulence, a C. elegans fast-kill infection assay was used. Wild-type P. aeruginosa PA14 and the lasR mutant rapidly kill C. elegans: 77% and 90% of worms die after 24 hr, respectively (FIG. 3a), indicating that LasR is not required for pathogenesis in this assay. Much less killing occurs when the nematodes are exposed to the rhlR or the lasR, rhlR double null strain (31% and 17%, respectively, died in 24 hr, FIG. 3a) showing that RhlR is required for virulence in this assay. Treatment of wild-type and the lasR mutant P. aeruginosa PA14 strains with 50 μM mBTL protects C. elegans from killing (23% and 50% death, respectively, FIG. 3b and FIG. 8). Together, these results confirm that the relevant in vivo target of mBTL is RhlR and, importantly, that inhibiting RhlR could form the basis of an anti-bacterial therapeutic strategy.
The double lasR, rhlR null mutant is not hyper-virulent in the nematode assay. The quorum-sensing-controlled virulence factors required for pathogenicity in mammalian cells are not precisely identical to those that are essential for virulence in nematodes which presumably accounts for this discrepancy.
EXAMPLE 11
A549 Human Lung Cell Infections
Methods. Human A549 cells were grown in CellStar tissue culture flasks. Prior to infection, the A549 cells were treated with trypsin-EDTA (CellGro), split, counted, and aliquotted into 96 well plates at 20,000 mammalian cells/well (cell counts were estimated using Trypan Blue (CellGro) exclusion). Cells were grown for 20 hr at 37° C. in DMEM (Invitrogen). Cells were washed 3× with warm PBS (Gibco). 100 μL of “master mix” was added to each well for each condition. Master mix contained 1 mL pre-warmed-PBS, 5 μL of 2 mg/mL propidium iodide (Bioprobes), 1 μL of a 100 mM inhibitor stock or DMSO, and 10 μL of OD600=2 P. aeruginosa PA14 grown in the presence of mBTL or DMSO. Infections were monitored using an EnVision plate reader every 2 hr with the RFP filter supplied by GE.
Results. To test whether mBTL could improve the outcome for mammalian cells during infection the human lung carcinoma cell line A549 was used. mBTL at 100 μM is not toxic to A549 cells (FIG. 9). Wild-type, lasR and rhlR single mutants, and the lasR, rhlR double mutant P. aeruginosa are all capable of killing A549 cells (FIG. 3c). Treatment with mBTL reduces killing by the wild-type and the lasR mutant strain but not by the rhlR or the lasR, rhlR double mutant strains (FIG. 3c). These results validate the conclusion reached on the basis of the microarray data presented above. Specifically, the relevant target of mBTL is present in the wild-type and the lasR mutant but is not in the rhlR single and lasR, rhlR double mutant strains. Thus, RhlR appears to be the major in vivo target.
In the nematode and lung cell experiments shown here, the bacteria were pregrown with mBTL and supplied a dose of inhibitor at the start of infection. Virulence was not reduced when P. aeruginosa PA14 was pre-grown in the absence of inhibitor (FIG. 11), indicating that mBTL may also function prophylactically.
It is noteworthy that, in the lung cell assay, the lasR, rhlR double mutant causes more cell death than does the wild-type (FIG. 3c). This result may stem from mis-regulation of virulence factors that are controlled in opposing directions by RhlR and LasR (FIG. 1b). The phenotype of the double mutant in the lung cell assay supports the proposition that potent inhibition of both LasR and RhlR could result in increased pathogenicity. Complete inhibition of LasR and RhlR would yield the same phenotype as deletion of both receptors. Apparently, that phenotype is high virulence.
EXAMPLE 12
mBTL Inhibits P. aeruginosa Biofilms
Microfluidic Flow Cells. Methods. Overnight P. aeruginosa PA14 cultures were back-diluted 1:1000 into 800 mL of tryptone broth (1% tryptone in H2O) with or without 100 μM mBTL and grown to mid-logarithmic phase (OD600=0.5). These cultures were used to fill 100 mL reservoirs that fed into microfluidic flow channels via Tygon tubing with an inner diameter of 2.4 mm. Similar tubing connected the outlet of the microfluidic channel to a collection dish on an analytical balance controlled via LabVIEW. The elevation of the culture reservoir above the collection dish on the balance set the constant pressure difference that drove the flow through the microfluidic channel. The microfluidic channel is 200 μm wide, 90 μm high, and contains a sequence of 37 bends that mimic corners in porous materials. The weight of the effluent culture was measured as a function of time t with measurement intervals of 4 s, and the data were converted into a flow rate Q(t) via the equation




  
    
      Q
      ⁡
      
        (
        t
        )
      
    
    =
    
      
        
          
            w
            ⁡
            
              (
              
                t
                +
                
                  Δ
                  ⁢
                  
                    
                  
                  ⁢
                  t
                
              
              )
            
          
          -
          
            w
            ⁡
            
              (
              
                t
                -
                
                  Δ
                  ⁢
                  
                    
                  
                  ⁢
                  t
                
              
              )
            
          
        
        
          1
          ⁢
          
            
          
          ⁢
          min
        
      
      ⁢
      
        1
        density
      
    
  
  ,




where Δt=30 s and the density is assumed to be that of water, 1 kg/L. To the resulting flow rate time series Q(t), the function





  
    Q
    0
  
  /
  
    [
    
      1
      +
      
        exp
        ⁡
        
          (
          
            
              t
              +
              T
            
            
              τ
              /
              2
            
          
          )
        
      
    
    ]
  




was fitted which yields the measurement of the time until clogging (corresponding to the time at which the flow rate declined to 50% of its baseline value Q0).

Results. Beyond being a clinically-relevant pathogen, P. aeruginosa is an industrial and medical nuisance because it causes blockages in filtration devices and stents. P. aeruginosa PA14 also clogs microfluidic chambers that model such devices. Clogging is due to biofilms that produce exopolysaccharide-containing streamers that act as sieves to catch passing cells. Compared to wild-type, lasR and rhlR single and double null mutants exhibit dramatically delayed clogging, demonstrating that quorum sensing is required to form blockages (FIG. 4a). In the presence of 100 μm mBTL, wild-type P. aeruginosa PA14 exhibits a phenotype indistinguishable from the mutants (FIG. 4a).
The results showing that mBTL prevents biofilm formation and clogging in microfluidic devices (FIG. 4) suggest that, in addition to the implications of mBTL as an anti-infective, deploying anti-quorum-sensing molecules could prevent the failure of devices that are prone to fouling by biofilms.
Static culture. Methods. Overnight P. aeruginosa cultures were back-diluted 1:1000 into tryptone broth with 100 μM mBTL, or an equivalent concentration of DMSO, and grown to mid-logarithmic phase (OD600=0.5). A 96-well plate with glass bottom (Thermo Fisher), which was filled with 200 μL of tryptone broth containing 100 μM mBTL or DMSO, was then inoculated with 2 μL of the mid-logarithmic culture. The 96-well plates were incubated for 24 h, prior to adding 5 μM SYTO 9 nucleic acid stain (Invitrogen). Biofilm thickness was measured using confocal microscopy (Nikon).
Results. The ability of mBTL to influence static biofilm formation in P. aeruginosa PA14 was examined. Wild-type P. aeruginosa PA14 forms biofilms with an average height of 27.5+/−11.5 μm (FIG. 4b). Treatment of wild-type with 100 μM mBTL results in a decrease in the average height to 10+/−4 μm (FIG. 4b). Treatment of the lasR null strain with mBTL also decreases the average height of the biofilm (15.7+/−4.9 μm to 6.6+/−2.3 μm). However, treatment of the rhlR null strain (25+/−10.6 μm untreated vs. 22.1+/−7.5 μm treated) or the lasR, rhlR double null strain (11.9+/−5.5 μm untreated vs. 16.2+/−5.9 μm treated) did not influence the height of the biofilm, indicating that the action of mBTL relies on the presence of RhlR (FIG. 4b). Taken together these findings show that mBTL inhibits quorum-sensing-controlled biofilm formation in P. aeruginosa PA14.
EXAMPLE 13
Synthetic Compounds Evolved from mBTL
Methods. Synthesis of the compounds is described in Example 14. The pyocyanin production assay is described in Example 3.
Results. mBTL contains a four-carbon linker (FIG. 1a). Synthesis and testing was performed of mBTL derivatives with two- to six-carbon linkers, and lactone versions of mBTL harboring four- or six-carbon linkers, containing or lacking a 3-oxo-moiety. Of the thiolactone derivatives of mBTL, C5-mBTL was the most effective inhibitor of pyocyanin production. Of the lactone derivatives of mBTL, C4-mBL was the most effective inhibitor of pyocyanin production. None of these compounds exhibited increased potency over mBTL (FIG. 7a, c, d). Thus, mBTL remains the most potent in vivo inhibitor in our collection.
EXAMPLE 14
Synthetic Chemistry General Procedures




General Procedure A. Synthesis of acids: To a flame-dried flask was added the 3-bromophenol (1.0 equiv), the appropriate bromo-ester (1.0 equiv), potassium carbonate (1.2 equiv), and DMF (0.50 M). The reaction was stirred for 3 d or until complete by TLC. After completion, H2O was added, and the aqueous layer was extracted 3× with Et2O. The combined organic layer was washed 3× with H2O and 1× with 1 M NaOH. The solution was dried over Na2SO4, filtered, and concentrated. The product was purified by column chromatography to remove excess 3-bromophenol if necessary. The resulting ester (1.0 equiv) was added to a solution of sodium hydroxide (5.0 equiv) in 3:1 THF/H2O (0.30 M). The reaction was heated to 65° C. for 12 hr, or until complete by TLC. The reaction was cooled and acidified with 1 M HCl. The aqueous layer was extracted 3× with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The product was carried forward crude.




General Procedure B. Synthesis of amides: To a flame-dried flask were added the acid (1.0 equiv), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.1 equiv), 1-hydroxybenzotriazole (0.25 equiv), triethylamine (2.2 equiv), the appropriate (thio)lactone (1.0 equiv), and CH2Cl2 (0.10 M). After the mixture was stirred at room temperature for 24 hr, H2O was added, and the aqueous layer was extracted 3× with EtOAc. The combined organic layer was washed sequentially with 1 M NaHSO4, saturated aqueous NaHCO3, and brine. The solution was dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography with a hexanes/EtOAc gradient.




General Procedure C. Synthesis of β-keto amides: The acid (1.0 equiv) was combined with CH2Cl2 (0.5 M) and cooled to 0° C. N,N-dicyclohexylcarbodiimide (1.0 equiv) was added, and the reaction was stirred at 0° C. for 30 min. Meldrum's acid (1.0 equiv) and 4-(dimethylamino)pyridine (1.0 equiv) were added, and the reaction mixture was stirred at room temperature overnight. The solution was filtered through a Celite plug and concentrated. The residue was dissolved in CH3CN (0.10 M). After L-homoserine lactone hydrobromide (1.0 equiv) and trifluoroacetic acid (1.0 equiv) were added, the reaction was heated to 65° C. for 4 hr. The reaction mixture was cooled, diluted with EtOAc, and washed sequentially with 1 M NaHSO4, saturated aqueous NaHCO3, and brine. The solution was dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography with a hexanes/EtOAc gradient.




C4 acid (S1): Prepared from ethyl 4-bromobutyrate using general procedure A to give S1 in 90% yield over two steps. 1H NMR (500 MHz, CDCl3) δ 7.16-7.01 (m, 3H), 6.84-6.79 (m, 1H), 4.00 (t, J=6.0 Hz, 2H), 2.59 (t, J=7.2 Hz, 2H), 2.16-2.08 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 179.1, 159.4, 130.5, 123.9, 122.8, 117.6, 113.4, 66.6, 30.4, 24.2; HRMS (ESI-TOF) calculated for C10H12BrO3 [M+H]+: m/z 258.9971, found 258.9967.




C5 acid (S2): Prepared from ethyl 5-bromovalerate using general procedure A to give S2 in 93% yield over two steps. 1H NMR (500 MHz, CDCl3) δ 7.18-7.01 (m, 3H), 6.85-6.77 (m, 1H), 3.96 (t, J=5.6 Hz, 2H), 2.50-2.41 (m, 2H), 1.89-1.80 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 178.6, 159.6, 130.5, 123.7, 122.8, 117.6, 113.5, 67.5, 33.4, 28.4, 21.3; HRMS (ESI-TOF) calculated for C11H14BrO3 [M+H]+: m/z 273.0127. found 273.0135.




C6 acid (S3): Prepared from methyl 6-bromohexanoate using general procedure A to give S3 in 80% yield over two steps. 1H NMR (500 MHz, CDCl3) δ 7.16-7.01 (m, 3H), 6.85-6.78 (m, 1H), 3.94 (t, J=6.4 Hz, 2H), 2.40 (t, J=7.4 Hz, 2H), 1.84-1.76 (m, 2H), 1.76-1.67 (m, 2H), 1.60-1.49 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 179.0, 159.7, 130.5, 123.6, 122.8, 117.6, 113.5, 67.8, 33.7, 28.8, 25.5, 24.3; HRMS (ESI-TOF) calculated for C12H16BrO3 [M+H]+: m/z 287.0283. found 287.0277.




V-06-018: Ethyl benzylacetate (0.10 mL, 0.58 mmol, 1.0 equiv) was combined with ethanol (5.8 mL, 10 M). Nonylamine (0.11 mL, 0.58 mmol, 1.0 equiv) was added dropwise, and the mixture was heated to reflux for 6 hr. The reaction mixture was concentrated, and the residue was dissolved in EtOAc. The solution was washed sequentially 2× with 1 M HCl, 1× with brine, then dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (hexanes/EtOAc gradient) to afford 8.3 mg of V-06-018 in a 5.0% yield. 1H NMR (500 MHz, CDCl3) δ 8.00 (d, J=7.5 Hz, 2H), 7.62 (t, J=7.4 Hz, 1H), 7.50 (t, J=7.8 Hz, 2H), 7.15 (s, 1H), 3.95 (s, 2H), 3.31-3.27 (m, 2H), 1.55-1.50 (m, 2H), 1.38-1.17 (m, 12H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 196.4, 165.5, 136.1, 134.1, 128.9, 128.6, 45.2, 39.7, 31.8, 29.5, 29.4, 29.2, 29.2, 26.9, 22.7, 14.1; HRMS (ESI-TOF) calculated for C18H28NO2 [M+H]+: m/z 290.2120. found 290.2120.




B7: Prepared with L-homoserine lactone and 3-(4-bromophenyl)propionic acid using general procedure B to give B7 in a 52% yield. The spectral data agreed with that reported for B7 (17). 1H NMR (500 MHz, CDCl3) δ 7.41 (d, J=8.3 Hz, 2H), 7.07 (t, J=9.1 Hz, 2H), 5.86 (s, 1H), 4.57-4.41 (m, 2H), 4.30-4.25 (m, 1H), 2.93 (t, J=7.5 Hz, 2H), 2.89-2.78 (m, 1H), 2.61-2.43 (m, 2H), 2.09-2.00 (m, 1H).; 13C NMR (125 MHz, CDCl3) δ 175.2, 172.2, 139.3, 131.6, 130.1, 120.2, 66.1, 49.3, 37.5, 30.6, 30.6; HRMS (ESI-TOF) calculated for C13H15BrNO3 [M+H]+: m/z 312.0236. found 312.0239.




2C-mBTL: Prepared with homocysteine thiolactone hydrochloride and (3-bromophenoxy)acetic acid using general procedure B to give 2C-mBTL in a 50% yield. 1H NMR (500 MHz, CDCl3) δ 7.22-7.09 (m, 3H), 6.95 (s, 1H), 6.89-6.84 (m, 1H), 4.68-4.58 (m, 1H), 4.57-4.46 (m, 2H), 3.44-3.35 (m, 1H), 3.34-3.26 (m, 1H), 3.00-2.91 (m, 1H), 2.08-1.95 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 204.6, 168.1, 157.5, 130.9, 125.5, 123.0, 118.3, 113.4, 67.2, 58.9, 31.6, 27.5; HRMS (ESI-TOF) calculated for C12H13BrNO3S [M+H]+: m/z 329.9800. found 329.9830.




3C-mBTL: Prepared with homocysteine thiolactone hydrochloride and 3-(3-bromo-phenoxy)-propionic acid using general procedure B to give 3C-mBTL in a 42% yield. 1H NMR (500 MHz, CDCl3) δ 7.21-7.07 (m, 3H), 6.90-6.84 (m, 1H), 6.32 (s, 1H), 4.57-4.49 (m, 1H), 4.30-4.21 (m, 2H), 3.43-3.33 (m, 1H), 3.32-3.24 (m, 1H), 3.04-2.96 (m, 1H), 2.80-2.67 (m, 2H), 2.01-1.87 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 205.3, 170.7, 158.9, 130.6, 124.4, 122.8, 117.9, 113.6, 64.0, 59.6, 36.2, 32.0, 27.7; HRMS (ESI-TOF) calculated for C13H15BrNO3S [M+H]+: m/z 343.9956. found 343.9984.




5C-mBTL: Prepared with homocysteine thiolactone hydrochloride and S2 using general procedure B to give 5C-mBTL in a 68% yield. 1H NMR (500 MHz, CDCl3) δ 7.16-6.99 (m, 3H), 6.85-6.77 (m, 1H), 5.91 (s, 1H), 4.56-4.46 (m, 1H), 3.95 (t, J=5.5 Hz, 2H), 3.42-3.31 (m, 1H), 3.30-3.22 (m, 1H), 3.01-2.90 (m, 1H), 2.37-2.29 (m, 2H), 1.97-1.78 (m, 5H); 13C NMR (125 MHz, CDCl3) δ 205.6, 173.0, 159.6, 130.5, 123.7, 122.8, 117.6, 113.4, 67.6, 59.5, 35.8, 32.1, 28.5, 27.6, 22.1; HRMS (ESI-TOF) calculated for C15H19BrNO3S [M+H]+: m/z 372.0269. found 372.0300.




6C-mBTL: Prepared with homocysteine thiolactone hydrochloride and S3 using general procedure B to give 6C-mBTL in a 74% yield. 1H NMR (500 MHz, CDCl3) δ 7.16-7.00 (m, 3H), 6.85-6.76 (m, 1H), 5.89 (s, 1H), 4.57-4.45 (m, 1H), 3.93 (t, J=6.4 Hz, 2H), 3.41-3.31 (m, 1H), 3.30-3.20 (m, 1H), 3.03-2.91 (m, 1H), 2.35-2.21 (m, 2H), 1.98-1.83 (m, 1H), 1.83-1.66 (m, 4H), 1.54-1.44 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 205.7, 173.3, 159.7, 130.5, 123.6, 122.7, 117.6, 113.4, 67.8, 59.5, 36.2, 32.1, 28.8, 27.6, 25.6, 25.1; HRMS (ESI-TOF) calculated for C16H21BrNO3S [M+H]+: m/z 386.0426. found 386.0427.




mBL: Prepared with L-homoserine lactone hydrobromide and S1 using general procedure B to give mBL in a 62% yield. 1H NMR (500 MHz, CDCl3) δ 7.14-6.98 (m, 3H), 6.83-6.76 (m, 1H), 5.98 (s, 1H), 4.56-4.47 (m, 1H), 4.44 (t, J=9.0 Hz, 1H), 4.30-4.21 (m, 1H), 3.97 (t, J=5.9 Hz, 2H), 2.87-2.78 (m, 1H), 2.44 (t, J=6.8 Hz, 2H), 2.17-2.02 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 175.3, 172.7, 159.5, 130.6, 123.9, 122.8, 117.7, 113.4, 66.9, 66.1, 49.3, 32.2, 30.6, 24.7; HRMS (ESI-TOF) calculated for C14H17BrNO4 [M+H]+: m/z 342.0341. found 342.0345.




C6-mBL: Prepared with L-homoserine lactone hydrobromide and S3 using general procedure B to give C6-mBL in a 61% yield. 1H NMR (500 MHz, CDCl3) δ 7.17-7.01 (m, 3H), 6.85-6.77 (m, 1H), 5.95 (s, 1H), 4.59-4.50 (m, 1H), 4.48 (t, J=9.0 Hz, 1H), 4.34-4.25 (m, 1H), 3.93 (t, J=6.3 Hz, 2H), 2.93-2.82 (m, 1H), 2.35-2.23 (m, 2H), 2.19-2.06 (m, 1H), 1.86-1.68 (m, 4H), 1.55-1.46 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 175.4, 173.3, 159.7, 130.5, 123.6, 122.8, 117.6, 113.4, 67.7, 66.1, 49.3, 36.0, 30.7, 28.8, 25.6, 25.0; HRMS (ESI-TOF) calculated for C16H21BrNO4 [M+H]+: m/z 370.0654. found 370.0666.




3O-mBL: Prepared with L-homoserine lactone hydrobromide and (3-bromophenoxy)acetic acid using general procedure C to give 3O-mBL in a 46% yield. 1H NMR (500 MHz, CDCl3) δ 7.20-7.03 (m, 3H), 6.87-6.80 (m, 1H), 4.68 (s, 2H), 4.64-4.56 (m, 1H), 4.48 (t, J=8.9 Hz, 1H), 4.35-4.24 (m, 1H), 3.66 (s, 2H), 2.93-2.88 (m, 1H), 2.81-2.75 (m, 1H), 2.29-2.15 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 201.7, 174.8, 165.6, 157.9, 130.9, 125.3, 123.0, 118.0, 113.3, 72.6, 66.0, 49.3, 45.5, 29.9; HRMS (ESI-TOF) calculated for C14H15BrNO5 [M+H]+: m/z 356.0134. found 356.0127.




3O—C6-mBL: Prepared with L-homoserine lactone hydrobromide and S1 using general procedure C to give 3O-C6-mBL in a 34% yield. 1H NMR (500 MHz, CDCl3) δ 7.59 (s, 1H), 7.18-6.99 (m, 3H), 6.85-6.76 (m, 1H), 4.63-4.55 (m, 1H), 4.48 (t, J=8.6 Hz, 1H), 4.32-4.23 (m, 1H), 3.96 (t, J=5.9 Hz, 2H), 3.51 (s, 2H), 2.83-2.70 (m, 3H), 2.23-2.15 (m, 1H), 2.14-2.02 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 205.6, 174.7, 166.1, 159.3, 130.6, 124.0, 122.8, 117.6, 113.4, 66.6, 65.9, 49.1, 48.2, 40.1, 29.9, 22.9; HRMS (ESI-TOF) calculated for C16H19BrNO5 [M+H]+: m/z 384.0447. found 384.0455.
Chiral Resolution of mBTL: mBTL enantiomers were resolved using a Berger Multigram II SFC system equipped with two Varian SD-1 pumps, a Knauer K-2501 multi-wavelength detector set at 220 nm, a Knauer K-1900 pump, a Vatran SGP-50-100 condenser, and using a Chiralpak IC (2×15 cm) column. An isocratic method using a mixture of 30% MeOH/CO2 (100 bar) at 60 mL/min was employed. The two peaks eluted at 1.66 min and 2.13 min. The identity of the enantiomers was determined through comparison of the HPLC trace with that of authentic (S)-mBTL synthesized from L-homocysteine thiolactone hydrochloride. Based on this analysis, peak 1 (>99:1 er) is (S)-mBTL and peak 2 (>99:1 er) is (R)-mBTL.
REFERENCES
1. Müh U, Schuster, M., Heim, R., Singh, A., Olson, E. R., Greenberg, E. P. (2006) Novel Pseudomonas aeruginosa Quorum-Sensing Inhibitors Identified in an Ultra-High-Throughput Screen. Antimicrobial Agents and Chemotherapy 50(11):3674-3679.
2. Geske G D, O'Neill, Jennifer C., Miller, David M., Mattmann, Margrith E., Blackwell, Helen E. (2007) Modulation of Bacterial Quorum Sensing with Synthetic Ligands: Systematic Evaluation of N-Acylated Homoserine Lactones in Multiple Species and New Insights into Their Mechanisms of Action. Journal of the American Chemical Society 129(44):13613-13625.
3. Amara N, et. al. (2009) Covalent Inhibition of Bacterial Quorum Sensing. Journal of the American Chemical Society 131(30):10610-10619.
4. Swem L R, Swem, D. L., O'Loughlin, C. T., Gatmaitan, R., Zhao, B., Ulrich, S. M., et. al. (2009) A Quorum-Sensing Antagonist Targets Both Membrane-Bound and Cytoplasmic Receptors and Controls Bacterial Pathogenicity. Molecular Cell 35(2):143-153.
5. Morkunas B, Galloway, Warren R. J. D., Wright, Megan, Ibbeson, Brett M., Hodgkinson, James T., O'Connell, Kieron M. G., et. al. (2012) Inhibition of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells by quorum sensing autoinducer-mimics Organic & Biomolecular Chemistry 10(42):8452-8464.
EXAMPLE 15
Synthesis of Surrogate Head Groups with 3OC12 Tail
The initial investigation focused on the binding of surrogate head groups using the native 3OC12 tail of the LasR signal or a simplified C12 tail (e.g., 4-5, FIG. 13) to evaluate the effectiveness of the head group as a replacement for the HSL. In principle, any head group that binds LasR is of interest, even if the compound were an agonist.
Based on previous work, the working hypothesis was that the addition of the appropriately functionalized tail could transform an agonist into an antagonist. Nonetheless, a new head group structure with inherent antagonistic activity is particularly interesting. Hits from the initial head group study are then combined with a functionalized tail to form hybrid analogs (e.g., 6, FIG. 13) that are designed to have higher potency as antagonists and better stability in vivo.
For the first library, acylation of an amino-heterocycle furnished the C12 tail analogs (8, FIG. 14). To install the β-ketoamide of the 3OC12 tail, the Meldrum's acid adduct was first made and then an amino-heterocycle was added to furnish 10. Hybrids (13) were generally synthesized via an SN2 displacement of an alkyl halide with a phenol to incorporate the tail functionality and an amide formation to append the head group.
EXAMPLE 16
Bioassay of Compounds of the First Library
The compounds of the first library were assayed in wild-type (WT) P. aeruginosa PA14. Pyocyanin was used as a read-out for quorum sensing activity based on its absorbance at 695 nm. The efficacy of the compounds at reducing pyocyanin levels was calculated with respect to WT levels of pyocyanin, where WT levels of pyocyanin would lead to a 0% efficacy, and an absorbance equal to the background medium would lead to a 100% efficacy. Agonists that increase pyocyanin production have negative efficacy values. The growth of P. aeruginosa was also monitored by absorbance at 600 nm in the presence of the compounds to ensure that the potential inhibitors did not impact growth.
Since the modeled interactions of compound 3 were promising (FIG. 12), it was followed by a systematic examination of a series of pyridine, pyrimidine and pyrazine head groups with a 3OC12 or C12 tail (entries 1-6, 7-12, respectively; Table 1). The 4-aminopyridine (entry 3) and 4-aminopyrimidine (entry 4) were the most active compounds. The 2-aminopyridine (entry 1) was a less effective inhibitor, suggesting that the nitrogen in the heterocycle para to the amine is key to activity and a second nitrogen in the ortho-position is also tolerated.







TABLE 1







Pyridine, pyrimidine, and pyrazine head groups.











Entry
Substrate
Efficacy (%)















1




44 ± 13



 



2




10 ± 9 



 



3




63 ± 2 



 



4




 64 ± 0.8



 



5




21 ± 13



 



6




−0.51 ± 4    



 



7




55 ± 3 



 



8




34 ± 3 



 



9




28 ± 11



 



10




55 ± 8 



 



11




−38 ± 3   



 



12




23 ± 3 










EXAMPLE 17
Bioassay of Aminopyridine and other Head Group Compounds
The 4-aminopyridine scaffold was further explored, incorporating a variety of substituents about the pyridine ring (e.g., entries 1-10, Table 2). The exploration included related pyridines (entries 11-14), as well as indole and benzofuran motifs (entries 15-23). Most analogs were less effective than the parent 4-aminopyridine. However, the incorporation of a fluoride at the 2-position along with the removal of the ketone in the tail led to a compound that decreased pyocyanin levels by 70% (entry 7).







TABLE 2







4-Aminopyridine and other head groups









Entry
Substrate
Efficacy (%)












1




−19 ± 3 


 


2




−24 ± 2  


 


3




−17 ± 2 


 


4




−5.1 ± 1  


 


5




24 ± 1


 


6




34 ± 5


 


7




70 ± 2


 


8




−8.5 ± 3  


 


9




−12 ± 3 


 


10




27 ± 3


 


11




13 ± 3


 


12




7.7 ± 5 


 


13




−15 ± 4 


 


14




−42 ± 2 


 


15




−11 ± 3 


 


16




1.5 ± 8 


 


17




12 ± 3


 


18




−1.0 ± 3  


 


19




9.3 ± 4 


 


20




−9.2 ± 4  


 


21




4.4 ± 3 


 


22




0.60 ± 4  


 


23




6.1 ± 4 









EXAMPLE 18
Bioassay of Head Group/Tail Group Hybrids
The next library of compounds (Table 3) encompassed an initial study of the combination of 4-aminopyridine head group analogs with the 3-bromophenol tail group from previously identified inhibitor 1, examining two linker lengths (entries 1-5, 7, Table 3). For this initial set of hybrid compounds, a methoxy group in the 2-position of the head group along with the longer linker was the most effective, reducing pyocyanin levels by 73% (entry 7). A subsequent set of compounds allowed more extensive study of the importance of the linker (entries 5-10). The four-methylene linker is the most active (81%, entry 6). Increasing to a 5- or 6-methylene linker drops the activity to 73-75% (entries 7 and 9), and an additional methylene group leads to further deterioration of activity (60%, entry 10). The incorporation of a ketone at C3 in the linker also decreases activity to 40% (entry 7 vs. entry 8).
In the initial head group studies, the 4-aminopyrimidine was as efficacious as the 4-aminopyridine (Table 1, entries 3-4). This motif was revisited in the hybrid studies (entries 11-14, Table 3). The addition of a methoxy group to the 6-position increased activity (entry 11 vs. entry 12), but a methoxy group in the 2-position or a second methoxy group was not tolerated (entries 13-14). The best analog of this series had an efficacy of only 41% (entry 12), so further investigation returned to the 4-aminopyridine scaffold.
Other inductively withdrawing groups at the 2-position in the head group were more effective, with a chloride substituent (entry 15) affording 89% efficacy and a trifluoromethyl group (entry 18) effectively shutting down pyocyanin production completely (99% efficacy). Exploration of the importance of chain length on this potent analog (entries 16-19) revealed that the five-methylene linker was the most active (entry 18), followed by nearly equal activity with the four-methylene linker (95% efficacy, entry 17). The pyridine nitrogen is important for activity, as its removal led to a compound with only 62% efficacy (entry 20), and an additional trifluoromethyl group fails to rescue activity (44%, entry 21). While the electronics of the 2-position substituents are important, an analog with a simple methyl group is still fairly active (80%, entry 22). Moving the trifluoromethyl group to the 3-position was not tolerated, leading to a compound with only 15% efficacy (entry 23).







TABLE 3







Head group study of hybrids.









Entry
Substrate
Efficacy (%)












1




21 ± 5


 


2




53 ± 1


 


3




32 ± 4


 


4




31 ± 6


 


5




20 ± 1


 


6




81 ± 6


 


7




73 ± 2


 


8




40 ± 6


 


9




75 ± 3


 


10




60 ± 2


 


11




27 ± 7


 


12




41 ± 9


 


13




16 ± 5


 


14




15 ± 7


 


15




89 ± 5


 


16




43 ± 8


 


17




95 ± 5


 


18




  99 ± 0.3


 


19




23 ± 7


 


20




62 ± 6


 


21




44 ± 8


 


22




80 ± 8


 


23




15 ± 7









EXAMPLE 19
Optimizing Tail Group for Trifluoromethylpyridine Head Group
Working with the 4-amino-2-trifluoromethylpyridine head group, optimization of the tail group was studied. Moving the bromide to the 2-position of the aryl tail group (94%, entry 1, Table 4) was better than a move to the 4-position (88%, entry 2), but both modifications led to a decrease in activity in comparison to the parent compound (99%, Table 3, entry 18). Evaluating other halides in the 3-position also gave potent analogs: an iodide was slightly less active (95%, entry 3, Table 4), while the chloride (101%, entry 4) and fluoride derivatives (103%, entry 5) were very effective at inhibiting pyocyanin production. Moving the fluoride around the ring led to similar trends as in the bromide case (entries 6-7 vs. entries 1-2), where substitution at the 4-position was the least active (54%, entry 7), and the 3-position was superior overall (entry 5). The incorporation of additional fluorides only decreased activity (entries 8-10).
Substrates with other inductively withdrawing groups at the 3-position were less effective inhibitors (entries 11-13). A methyl group is still effective (81%, entry 14), but less so than the halides at the 3-position. A hydroxyl group at this position also had moderate activity (70%, entry 15), but was more potent at the 2-position (95%, entry 16).
The importance of the ether linkage between the aryl tail and the rest of the substrate was examined. Substituting the oxygen with a sulfur or carbon lead to a small decrease in activity (94%, entry 17 and 96%, entry 18) and suggested that the electronic character of this linker atom was not significant to the activity. A nitrogen displayed similar efficacy as the parent oxygen (100%, entry 19). An alkyne was also tolerated in the linker (97%, entry 20).







TABLE 4







Hybrid tail group optimization









Entry
Substrate
Efficacy (%)












1




94 ± 4 


 


2




88 ± 4 


 


3




95 ± 3 


 


4




101 ± 3 


 


5




103 ± 3 


 


6




78 ± 3 


 


7




54 ± 4 


 


8




49 ± 3 


 


9




66 ± 4 


 


10




21 ± 14


 


11




39 ± 36


 


12




0.48 ± 7  


 


13




59 ± 3 


 


14




81 ± 4 


 


15




70 ± 7 


 


16




95 ± 4 


 


17




94 ± 4 


 


18




96 ± 4 


 


19




100 ± 4 


 


20




97 ± 4 









EXAMPLE 20
Inhibitory Activity of the Synthetic Compounds
The IC50 values for top hits from each of the libraries was determined (FIG. 15). Most of the inhibitors had low micromolar activities, while 14 was an order of magnitude more active (IC50=0.16 μM). Strikingly, the hybrid compounds (16-17) were highly effective at blocking pyocyanin production and are superior to inhibitor 1 or compound 18, a very effective pyocyanin inhibitor in P. aeruginosa PAO1. To ensure that the observed activity in the compounds was not due to the alteration of the oxidation state of pyocyanin, cell-free supernatant containing pyocyanin was incubated with the inhibitors. No change in absorbance occurred over 17 hours (FIG. 16).
EXAMPLE 21
Biological Targets of the Synthetic Compounds
The compounds were designed to be AHL analogs that bind and antagonize LasR and/or RhlR. If either receptor were the target, a ΔlasRrhlR strain of P. aeruginosa would not be expected to have a further reduction of pyocyanin when treated with an inhibitor. Instead, in a mutant P. aeruginosa strain that lacks LasR and RhlR, pyocyanin levels are further reduced upon treatment with hybrid 16 (FIG. 17).
EXAMPLE 22
LasR and RhlR GFP Assays of Synthetic Compounds
The LasR-GFP assays were performed in E. coli strain BL21 DE3 Gold (Agilent) carrying pET23b (Novagen) containing lasR (maintained with 100 μg/mL ampicillin) and carrying plasmid pEVS141 (31) containing the rsaL promoter driving expression of gfp (maintained with 50 μg/mL of kanamycin.) The RhlR-GFP assays were performed in E. coli strain BL21 DE3 Gold (Agilent) carrying pET23b (Novagen) containing rhlR (maintained with 100 μg/mL ampicillin) and carrying plasmid pEVS141 (31) containing the rhlA promoter driving expression of gfp (maintained with 50 μg/mL of kanamycin.) These E. coli strains were grown overnight and subcultured into fresh medium with appropriate antibiotics at a 1:100 dilution and grown shaking for 8 hr at 37° C. for the LasR-GFP strain and 12 hr for the RhlR-GFP construct. 50 nM 3OC12-HSL or 20 μM C4-HSL was added to the LasR-GFP and RhlR-GFP preparations, respectively. Compounds were tested at 1 mM for antagonism and at 100 nM or 20 μM for agonism. These concentrations were chosen for agonism studies to match the concentrations of autoinducers used in our experiments. For antagonism studies, we used the EC95 concentration for each receptor. GFP was measured on an Envision plate reader.
The activity of the pyocyanin inhibitors was investigated in a heterologous E. coli system, where the relevant transcriptional regulator and target-gfp fusions are present on plasmids. Upon addition of an agonist such as the native AHL, GFP is produced. None of the compounds acted as agonists for LasR or RhlR at 100 μM (FIG. 18). In the presence of the native AHL, an inhibitor should decrease the production of GFP. None of the compounds acted as antagonists for LasR or RhlR at 100 μM. Despite having been designed to hit the LasR and/or RhlR receptors of P. aeruginosa, clearly the inhibitors are reducing pyocyanin levels by influencing a different pathway, in a more effective manner than the LasR/RhlR inhibitors 1 and 18.
Due to the importance of pyocyanin to maintain the redox balance of P. aeruginosa, it is reasonable that an environmental response regulator could also control pyocyanin production.
EXAMPLE 23
RNA Extraction and Microarray Analysis
Overnight P. aeruginosa PA14 cultures were back-diluted 1:1000 into 5 mL of fresh LB. 100 μM inhibitor compound, or an equivalent amount of DMSO, was added to cultures which were grown aerobically with shaking at 37° C. for 17 hr. 9 ODs of cells were harvested for each treatment. Lysozyme (1 mg/mL in TE buffer) (Sigma) was added for 10 min at room temperature. Total RNA was prepared using the RNeasy Midi Kit (Qiagen). RNA was treated with RNase-Free DNaseI (Ambion) for 1 hr at 37° C., inactivated using DNaseI Inactivation Reagent Resin (Ambion), and re-purified using the RNeasy Mini Kit. A cDNA library containing Cy3- or Cy5-labeled dUTP (Enzo Life Sciences) was synthesized from the purified RNA using SuperScript III Reverse Transcriptase (Invitrogen). Sodium hydroxide was added to degrade RNA, and the reaction was subsequently neutralized by addition of hydrochloric acid. The library was purified using the PCR Purification kit (Qiagen) and assessed for Cy3 and Cy5 incorporation using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies). Libraries were normalized for cDNA concentration and hybridized using the Gene Expression Hybridization Kit (Agilent) to a custom microarray (Agilent design number 43307), which was designed using the Agilent eArray tool with 2 probes for most genes. Samples were hybridized for 22 hr at 65° C. with continuous rotation at 10 rpm. Microarrays were scanned using an Agilent G2505C scanner and analyzed using Agilent Feature Extract software version 9.5. Resulting microarray intensity data were submitted to the PUMA Database (http://puma.princeton.edu) for archiving and analyzed using Matlab R2013a.
Microarray analysis allows observation of the effects of the inhibitor in vivo. WT P. aeruginosa PA14 was treated with hybrid 16 and compared with the untreated bacterium. Pyocyanin acts as a terminal signal in P. aerguinosa, upregulating the production of putative monooxygenase PA14_35160 as well as transporters through SoxR. 24If there were no pyocyanin produced, decreased expression of the genes controlled by SoxR would be expected. Treatment with inhibitor 16 does lead to the expected down-regulation of the SoxR-regulated genes (Table 5).







TABLE 5







Changes in the SoxR regulon after treatment with 16.











Gene

Fold Down-


Gene Locus
Name
Description
Regulated













PA14_35160

hypothetical protein
4.93


PA14_16310

MFS permease
3.27


PA14_09530
mexH
RND efflux membrane fusion
2.93




protein


PA14_09520
mexI
RND efflux transporter
2.84


PA14_09540
mexG
hypothetical protein
2.68


PA14_09500
opmD
outer membrane protein
2.50









Examining the rest of the microarray data, none of the pyocyanin biosynthetic genes are impacted by hybrid 16, suggesting a post-transcriptional regulation of the virulence factor. Indeed, few genes of the quorum-sensing regulon as a whole were affected. Instead, the majority of genes with the greatest down-regulation are associated with the oxidative stress response (Table 6).







TABLE 6







Oxidative stress genes impacted by 16.











Gene

Fold Down-


Gene Locus
Name
Description
regulated













PA14_21530

ankyrin domain-containing protein
44.39


PA14_22320

hypothetical protein
35.99


PA14_01720
ahpF
alkyl hydroperoxide reductase
33.03


PA14_53290
trxB2
thioredoxin reductase 2
27.79


PA14_09150
katA
catalase
20.80


PA14_03090

hypothetical protein
14.35


PA14_58040

hypothetical protein
5.67


PA14_51830

DNA-binding stress protein
5.60


PA14_61040
katB
catalase
4.15


PA14_58030
fumC
fumarate hydratase
2.71









EXAMPLE 24
Chemistry Materials and Methods
Unless otherwise stated, reactions were performed in flame-dried glassware fitted with rubber septa under a nitrogen atmosphere and were stirred with Teflon-coated magnetic stirring bars. Liquid reagents and solvents were transferred via syringe using standard Schlenk techniques. Reaction solvents were dried by passage over a column of activated alumina. All other solvents and reagents were used as received unless otherwise noted. Reaction temperatures above 23° C. refer to oil bath temperature, which was controlled by an OptiCHEM temperature modulator. Thin layer chromatography was performed using SiliCycle silica gel 60 F-254 precoated plates (0.25 mm) and visualized by UV irradiation and anisaldehyde or potassium permanganate stain. Sorbent standard silica gel (particle size 40-63 μm) was used for flash chromatography. 1H and 13C NMR spectra were recorded on Bruker Avance III (500 MHz for 1H; 125 MHz for 13C) spectrometer fitted with either a 1H-optimized TCI (H/C/N) cryoprobe or a 13C-optimized dual C/H cryoprobe or a Bruker NanoBay (300 MHz). Chemical shifts (δ) are reported in ppm relative to the residual solvent signal (δ=7.26 for 1H NMR and 6=77.0 for 13C NMR for CDCl3, δ=3.31 for 1H NMR and δ=49.0 for 13C NMR for CD3OD, δ=2.05 for 1H NMR and δ=29.8 for 13C NMR for acetone-d6). Data for 1H NMR spectra are reported as follows: chemical shift (multiplicity, coupling constants, number of hydrogens). Abbreviations are as follows: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), p (pentet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dt (doublet of triplets), td (triplet of doublets), m (multiplet). High-resolution mass spectral analysis was performed using an Agilent 1200-series electrospray ionization—time-of-flight (ESI-TOF) mass spectrometer in the positive ESI mode.
General Procedures:
Synthesis of β-keto Amide Compounds




General procedure A: To a flame-dried flask was added Meldrum's acid (1 equiv) and CH2Cl2 (0.34 M). The reaction mixture was cooled to 0° C., and pyridine (2 equiv) was added over 20 min. Decanoyl chloride (1 equiv) was then added dropwise. The reaction mixture was stirred at 0° C. for 2 h and was allowed to return to room temperature over 1 h. The reaction was diluted with CH2Cl2 and a 2 M HCl/ice mixture. After stirring for 10 min., the phases were separated. The organic phase was washed sequentially with 2 M HCl and brine, dried over Na2SO4 and concentrated. The residue was dissolved in CH3CN (0.1 M) and the amino-heterocycle (1 equiv) was added. The reaction was heated to 65° C. for 4 h. The reaction mixture was then concentrated and the crude product was purified by column chromatography.
Synthesis of Amide Compounds:




General procedure B: The amino-heterocycle (1 equiv), CH2Cl2 (0.15 M), and Et3N (2 equiv) were combined in a flame-dried flask. The reaction mixture was cooled to 0° C., and dodecanoyl chloride (1 equiv) was added dropwise. The reaction mixture was allowed to warm to room temperature over 3 h. The reaction was then quenched with saturated aqueous NaHCO3 solution. The layers were separated, and the aqueous layer was extracted 3× with CH2Cl2. The combined organic layer was washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by column chromatography.




General procedure C: To a flame-dried flask were added the carboxylic acid (1.0 equiv), dicyclohexylcarbodiimide (1.1 equiv), dimethylaminopyridine (1.1 equiv), dodecylamine (1.0 equiv), and CH2Cl2 (0.40 M). After stirring at room temperature for 24 h, the reaction mixture was filtered through a Celite plug and concentrated. The crude product was purified by column chromatography.
Synthesis of 4-amino-2-trifluoromethylpyridine analogs:




General procedure D: S63 (1 equiv), anhydrous potassium iodide (12 equiv), anhydrous potassium carbonate (7.5 equiv), and the corresponding aryl nucleophile (3.8 equiv) were dissolved in isopropanol (0.68 M) in a vial. The vial was sealed, and the reaction mixture was heated to 100° C. behind a blast shield for at least 60 hours, or until done. After cooling, the reaction was quenched with water and extracted with CH2Cl2. The combined organic layer was washed sequentially with saturated aqueous NaHCO3 (2×), 1 M HCl, and brine. The solution was dried over Na2SO4 and concentrated. The crude product was purified by column chromatography.




3OC12-4-aminopyridine (14): Prepared from 4-aminopyridine using general procedure A to furnish 14 in a 51% yield. HRMS (ESI-TOF) calculated for C17H27N2O2 [M+H]+: m/z 291.2073. found 291.2077; 1H NMR (500 MHz, CDCl3) δ 9.61 (s, 1H), 8.51 (d, J=5.7 Hz, 2H), 7.58-7.48 (m, 2H), 3.59 (s, 2H), 2.58 (t, J=7.3 Hz, 2H), 1.62 (p, J=6.7 Hz, 2H), 1.44-1.15 (m, 12H), 0.87 (t, J=6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 208.0, 164.2, 150.8, 144.3, 113.9, 48.3, 44.3, 31.8, 29.3, 29.3, 29.2, 28.9, 23.3, 22.6, 14.1.




C12-4-amino-2-fluoropyridine (15): Prepared from 4-amino-2-fluoropyridine using general procedure B to furnish 15 in a 45% yield. HRMS (ESI-TOF) calculated for C17H28FN2O [M+H]+: m/z 295.2186. found 295.2188; 1H NMR (500 MHz, CDCl3) δ 8.09 (d, J=5.6 Hz, 1H), 7.40-7.31 (m, 2H), 7.17 (dt, J=5.7, 1.5 Hz, 1H), 2.40 (t, J=7.6 Hz, 2H), 1.72 (p, J=7.5 Hz, 2H), 1.26 (d, J=6.6 Hz, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.9, 164.8 (d, J=236 Hz), 148.8 (d, J=12 Hz), 148.1 (d, J=17 Hz), 111.2 (d, J=4 Hz), 98.6 (d, J=43 Hz), 37.8, 31.9, 29.6, 29.6, 29.4, 29.3, 29.3, 29.1, 25.1, 22.7, 14.1.




4-amino-2-trifluoromethylpyridine-C6-3-bromophenoxyhybrid (16): Prepared from 4-amino-2-trifluoromethylpyridine and S381 using general procedure C to furnish 16 in a 42% yield. HRMS (ESI-TOF) calculated for C18H19BrF3N2O2 [M+H]: m/z 431.0582. found 431.0571; 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J=5.5 Hz, 1H), 7.89 (d, J=2.1 Hz, 1H), 7.67 (dd, J=5.5, 2.1 Hz, 1H), 7.47 (s, 1H), 7.13 (t, J=8.1 Hz, 1H), 7.10-6.99 (m, 2H), 6.81 (ddd, J=8.2, 2.5, 1.1 Hz, 1H), 3.95 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.89-1.74 (m, 4H), 1.63-1.50 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.8, 159.7, 151.0, 149.3 (q, J=35 Hz), 146.1, 130.6, 124.7-117.9 (m), 123.7, 122.8, 117.6, 115.3, 113.4, 110.3 (q, J=3 Hz), 67.7, 37.5, 28.8, 25.6, 24.7.




4-amino-2-trifluoromethylpyridine-C6-3-fluorophenoxyhybrid (17): Prepared from 3-fluorophenol and S63 using general procedure D to furnish 17 in a 40% yield. HRMS (ESI-TOF) calculated for C18H19F4N2O2 [M+H]: m/z 371.1383. found is 371.1367; 1H NMR (500 MHz, CDCl3) δ 8.59 (s, 1H), 7.92 (s, 1H), 7.77 (s, 1H), 7.73-7.65 (m, 1H), 7.24-7.16 (m, 1H), 6.68-6.61 (m, 2H), 6.58 (dt, J=11.0, 2.4 Hz, 1H), 3.94 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.86-1.76 (m, 4H), 1.60-1.51 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.9, 163.6 (d, J=245 Hz), 160.3 (d, J=11 Hz), 150.9, 149.3 (q, J=35 Hz), 146.3, 130.2 (d, J=10 Hz), 121.3 (q, J=274 Hz), 115.4, 110.5-110.1 (m, 2C), 107.4 (d, J=21 Hz), 102.1 (d, J=25 Hz), 67.7, 37.5, 28.8, 25.7, 24.7.




3OC12-2-aminopyridine (S1): Prepared from 2-aminopyridine using general procedure A to furnish S1 in a 60% yield. HRMS (ESI-TOF) calculated for C17H27N2O2 [M+H]+: m/z 291.2073. found 291.2099; 1H NMR (500 MHz, CDCl3) δ 9.55 (s, 1H), 8.30 (d, J=4.9 Hz, 1H), 8.15 (d, J=8.4 Hz, 1H), 7.69 (t, J=7.9 Hz, 1H), 7.10-7.00 (m, 1H), 3.57 (s, 2H), 2.58 (t, J=7.3 Hz, 2H), 1.60 (p, J=7.0 Hz, 2H), 1.46-1.14 (m, 12H), 0.87 (t, J=6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 205.9, 164.2, 151.0, 147.9, 138.3, 120.0, 114.3, 49.9, 44.0, 31.8, 29.4, 29.3, 29.2, 29.0, 23.3, 22.6, 14.1.




3OC12-3-aminopyridine (S2): Prepared from 3-aminopyridine using general procedure A to furnish S2 in a 65% yield. HRMS (ESI-TOF) calculated for C17H27N2O2 [M+H]+: m/z 291.2073. found 291.2100; 1H NMR (500 MHz, CDCl3) δ 9.48 (s, 1H), 8.66 (d, J=2.6 Hz, 1H), 8.37 (d, J=3.2 Hz, 1H), 8.13 (d, J=8.9 Hz, 1H), 7.28 (d, J=6.1 Hz, 1H), 3.60 (s, 2H), 2.59 (t, J=7.4 Hz, 2H), 1.71-1.52 (m, 2H), 1.39-1.17 (m, 12H), 0.88 (t, J=6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 208.1, 164.1, 145.5, 141.5, 134.3, 127.3, 123.7, 48.2, 44.3, 31.8, 29.3, 29.3, 29.2, 28.9, 23.3, 22.6, 14.1.




3OC12-4-aminopyrimidine (S3): Prepared from 4-aminopyrimidine using general procedure A to furnish S3 in a 58% yield. HRMS (ESI-TOF) calculated for C16H26N3O2 [M+H]+: m/z 292.2025. found 292.2020; 1H NMR (500 MHz, CDCl3) δ 9.67 (s, 1H), 8.92-8.89 (m, 1H), 8.63 (d, J=5.7 Hz, 1H), 8.11 (d, J=5.7 Hz, 1H), 3.61 (s, 2H), 2.58 (t, J=7.4 Hz, 2H), 1.77-1.52 (m, 2H), 1.41-1.14 (m, 12H), 0.87 (t, J=6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 206.0, 165.0, 158.5, 158.5, 156.6, 110.5, 49.1, 44.3, 31.8, 29.3, 29.3, 29.2, 28.9, 23.3, 22.6, 14.1.




3OC12-2-aminopyrazine (S4): Prepared from 2-aminopyrazine using general procedure A to furnish S4 in a 50% yield. HRMS (ESI-TOF) calculated for C16H26N3O2 [M+H]+: m/z 292.2025. found 292.2016; 1H NMR (500 MHz, CDCl3) δ 9.63 (s, 1H), 9.48 (s, 1H), 8.38-8.33 (m, 1H), 8.31-8.25 (m, 1H), 3.62 (s, 2H), 2.59 (t, J=7.4 Hz, 2H), 1.67-1.58 (m, 2H), 1.38-1.11 (m, 12H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 206.5, 164.0, 147.7, 142.3, 140.5, 137.2, 48.8, 44.3, 31.8, 29.4, 29.3, 29.2, 28.9, 23.3, 22.6, 14.1.




3OC12-2-aminopyrimidine (S5): Prepared from 2-aminopyrimidine using general procedure A to furnish S5 in a 15% yield. HRMS (ESI-TOF) calculated for C16H26N3O2 [M+H]+: m/z 292.2025. found 292.2024; 1H NMR (500 MHz, CDCl3) δ 9.21 (s, 1H) 8.77-8.54 (m, 2H), 7.12-6.96 (m, 1H), 3.94 (s, 2H), 2.58 (t, J=7.4 Hz, 2H), 1.61 (p, J=7.3 Hz, 2H), 1.45-1.15 (m, 12H), 0.88 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 182.4, 171.5, 158.2, 157.1, 116.5, 51.4, 43.3, 31.8, 29.4, 29.4, 29.2, 29.1, 23.4, 22.6, 14.1.




C12-2-aminopyridine (S6): Prepared from 2-aminopyridine using general procedure B to furnish S6 in a 4% yield. HRMS (ESI-TOF) calculated for C17H29N2O [M+H]+: m/z 277.2281. found 277.2284; 1H NMR (500 MHz, CDCl3) δ 8.25 (ddd, J=4.9, 2.0, 0.9 Hz, 1H), 8.22 (d, J=8.3 Hz, 1H), 7.90 (s, 1H), 7.70 (ddd, J=8.7, 7.3, 1.9 Hz, 1H), 7.03 (ddd, J=7.4, 4.9, 1.1 Hz, 1H), 2.39 (t, J=7.6 Hz, 2H), 1.72 (p, J=7.5 Hz, 2H), 1.43-1.17 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.9, 151.4, 147.6, 138.5, 119.6, 114.0, 37.8, 31.9, 29.6, 29.6, 29.4, 29.3, 29.3, 29.2, 25.4, 22.7, 14.1.




C12-3-aminopyridine (S7): Prepared from 3-aminopyridine using general procedure B to furnish S7 in a 57% yield. HRMS (ESI-TOF) calculated for C17H29N2O [M+H]+: m/z 277.2281. found 277.2277; 1H NMR (500 MHz, CDCl3) δ 8.53 (d, J=2.6 Hz, 1H), 8.34 (dd, J=4.8, 1.5 Hz, 1H), 8.20 (dt, J=8.4, 2.0 Hz, 1H), 7.35 (s, 1H), 7.31-7.26 (m, 1H), 2.39 (t, J=7.6 Hz, 2H), 1.73 (p, J=7.5 Hz, 2H), 1.44-1.17 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.9, 145.2, 140.9, 134.7, 127.1, 123.7, 37.6, 31.9, 29.6, 29.6, 29.4, 29.3, 29.3, 29.2, 25.5, 22.7, 14.1.




C12-4-aminopyridine (S8): Prepared from 4-aminopyridine using general procedure B to furnish S8 in a 69% yield. HRMS (ESI-TOF) calculated for C17H29N2O [M+H]+: m/z 277.2281. found 277.2278; 1H NMR (500 MHz, CDCl3) δ 8.57-8.44 (m, 2H), 7.55-7.46 (m, 2H), 7.44 (s, 1H), 2.39 (t, J=7.6 Hz, 2H), 1.72 (p, J=7.5 Hz, 2H), 1.41-1.17 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 172.1, 150.7, 144.9, 113.3, 37.9, 31.9, 29.6, 29.6, 29.4, 29.3, 29.3, 29.2, 25.3, 22.7, 14.1.




C12-4-aminopyrimidine (S9): Prepared from 4-aminopyrimidine using general procedure B to furnish S9 in a 69% yield. HRMS (ESI-TOF) calculated for C16H28N3O [M+H]+: m/z 278.2232. found 278.2225; 1H NMR (500 MHz, CDCl3) δ 8.85 (d, J=1.4 Hz, 1H), 8.62 (d, J=5.8 Hz, 1H), 8.18 (dd, J=5.8, 1.4 Hz, 1H), 7.91 (s, 1H), 2.42 (t, J=7.6 Hz, 2H), 1.72 (p, J=7.5 Hz, 2H), 1.44-1.17 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 172.5, 158.4, 158.3, 156.8, 110.1, 37.8, 31.9, 29.6, 29.6, 29.4, 29.3, 29.3, 29.1, 25.0, 22.7, 14.1.




C12-2-aminopyrazine (S10): Prepared from 2-aminopyrazine using general procedure B to furnish S10 in a 15% yield. HRMS (ESI-TOF) calculated for C16H28N30 [M+H]+: m/z 278.2232. found 278.2232; 1H NMR (500 MHz, CDCl3) δ 9.56 (s, 1H), 8.34 (d, J=2.5 Hz, 1H), 8.22 (dd, J=2.7, 1.6 Hz, 1H), 7.92 (s, 1H), 2.44 (t, J=7.6 Hz, 2H), 1.74 (p, J=7.5 Hz, 2H), 1.26 (d, J=11.6 Hz, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.6, 148.0, 141.8, 140.1, 137.0, 37.5, 31.9, 29.6, 29.6, 29.4, 29.3, 29.3, 29.1, 25.2, 22.7, 14.1.




C12-2-aminopyrimidine (S11): Prepared from 2-aminopyrimidine using general procedure B to furnish S11 in a 5% yield. HRMS (ESI-TOF) calculated for C16H28N3O [M+H]+: m/z 278.2232. found 278.2247. Spectra were consistent with those reported by Ref 2.




C12-4-amino-2-methylpyridine (S12): Prepared from 4-amino-2-methylpyridine using general procedure B to furnish S12 in a 90% yield. HRMS (ESI-TOF) calculated for C18H31N2O [M+H]+: m/z 291.2437. found 291.2431; 1H NMR (500 MHz, CDCl3) δ 8.37 (d, J=5.6 Hz, 1H), 7.42 (s, 1H), 7.27 (s, 1H), 7.22 (dd, J=5.7, 2.1 Hz, 1H), 2.52 (s, 3H), 2.37 (t, J=7.6 Hz, 2H), 1.71 (p, J=7.5 Hz, 2H), 1.27 (d, J=19.0 Hz, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 172.0, 159.7, 150.0, 145.1, 112.6, 110.7, 37.9, 31.9, 29.6, 29.6, 29.4, 29.3, 29.3, 29.2, 25.3, 24.6, 22.7, 14.1.




C12-4-amino-2-methylquinoline (S13): Prepared from 4-amino-2-methylquinoline using general procedure B to furnish S13 in a 25% yield. HRMS (ESI-TOF) calculated for C22H33N2O [M+H]+: m/z 341.2593. found 341.2593; 1H NMR (500 MHz, CDCl3) 8.24 (s, 1H), 8.04 (d, J=8.4 Hz, 1H), 7.77 (s, 1H), 7.75-7.65 (m, 2H), 7.53 (t, J=7.6 Hz, 1H), 2.73 (s, 3H), 2.54 (t, J=7.6 Hz, 2H), 1.81 (p, J=7.4 Hz, 2H), 1.48-1.39 (m, 2H), 1.39-1.19 (m, 14H), 0.87 (t, J=6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 172.0, 160.2, 148.4, 140.1, 129.8, 129.4, 125.6, 118.5, 118.2, 111.4, 38.1, 31.9, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2, 25.7, 25.5, 22.7, 14.1.




C12-4-amino-3-trifluoromethylpyridine (S14): Prepared from 4-amino-3-trifluoromethylpyridine using general procedure B to furnish S14 in a 56% yield. HRMS (ESI-TOF) calculated for C18H28F3N2O [M+H]+: m/z 345.2154. found 345.2146; 1H NMR (500 MHz, CDCl3) δ 8.77 (s, 1H), 8.69 (d, J=5.9 Hz, 1H), 8.50 (d, J=5.8 Hz, 1H), 7.58 (s, 1H), 2.44 (t, J=7.5 Hz, 2H), 1.72 (p, J=7.4 Hz, 2H), 1.39-1.17 (m, 16H), 0.87 (t, J=6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.8, 164.3-161.7 (m), 154.4, 147.3 (q, J=6 Hz), 142.8, 128.2-119.4 (m), 115.0, 38.2, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2, 29.0, 25.1, 22.7, 14.1.




C12-4-amino-2-methoxypyridine (S15): Prepared from 4-amino-2-methoxypyridine using general procedure B to furnish S15 in a 93% yield. HRMS (ESI-TOF) calculated for C18H31N2O2 [M+H]+: m/z 307.2386. found 307.2414; 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J=5.6 Hz, 1H), 7.16 (s, 1H), 7.04-6.96 (m, 2H), 3.92 (s, 3H), 2.36 (t, J=7.6 Hz, 2H), 1.71 (p, J=7.5 Hz, 2H), 1.26 (d, J=12.8 Hz, 16H), 0.87 (t, J=6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.9, 165.5, 147.7, 146.9, 108.0, 99.3, 53.6, 38.0, 32.0, 29.6, 29.6, 29.5, 29.4, 29.4, 29.2, 25.3, 22.7, 14.2.




3OC12-4-amino-2-methoxypyridine (S16): Prepared from 4-amino-2-methoxypyridine using general procedure A to furnish S16 in a 43% yield. HRMS (ESI-TOF) calculated for C18H29N2O3 [M+H]: m/z 321.2178. found 321.2177; 1H NMR (500 MHz, CDCl3) δ 9.53 (s, 1H), 8.05 (d, J=5.7 Hz, 1H), 7.08 (d, J=1.9 Hz, 1H), 7.00 (dd, J=5.7, 1.9 Hz, 1H), 3.91 (s, 3H), 3.56 (s, 2H), 2.57 (t, J=7.3 Hz, 2H), 1.71-1.49 (m, 2H), 1.38-1.06 (m, 12H), 0.86 (t, J=6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 207.9, 165.4, 164.0, 147.6, 146.4, 108.4, 99.9, 53.6, 48.4, 44.3, 31.8, 29.3, 29.3, 29.2, 28.9, 23.3, 22.6, 14.1.




C12-4-amino-2-bromopyridine (S17): Prepared from 4-amino-2-bromopyridine using general procedure B to furnish S17 in a 52% yield. HRMS (ESI-TOF) calculated for C17H28BrN2O [M+H]: m/z 355.1385. found 355.1398; 1H NMR (500 MHz, CDCl3) δ 8.23 (d, J=5.5 Hz, 1H), 7.78 (d, J=1.9 Hz, 1H), 7.39 (dd, J=5.6, 1.9 Hz, 1H), 7.26 (s, 1H), 2.43-2.36 (m, 2H), 1.71 (p, J=7.5 Hz, 2H), 1.35-1.16 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.9, 150.6, 146.5, 142.9, 116.9, 112.6, 37.8, 31.9, 29.6, 29.6, 29.4, 29.3, 29.3, 29.1, 25.1, 22.7, 14.1.




3OC12-4-amino-2-fluoropyridine (S18): Prepared from 4-amino-2-fluoropyridine using general procedure A to furnish S18 in a 42% yield. HRMS (ESI-TOF) calculated for Ci7H26FN202 [M+H]+: m/z 309.1978. found 309.1978; 1H NMR (500 MHz, CDCl3) δ 9.82 (s, 1H), 8.11 (d, J=5.6 Hz, 1H), 7.39-7.36 (m, 1H), 7.21 (d, J=5.6 Hz, 1H), 3.60 (s, 2H), 2.58 (t, J=7.3 Hz, 2H), 1.70-1.52 (m, 2H), 1.39-1.16 (m, 12H), 0.88 (t, J=6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 208.1, 164.7 (d, J=235 Hz), 164.2, 148.4 (d, J=11 Hz), 148.1 (d, J=17 Hz), 111.9 (d, J=4 Hz), 99.3 (d, J=43 Hz), 48.0, 44.4, 31.8, 29.3, 29.3, 29.2, 28.9, 23.3, 22.6, 14.1.




C12-4-amino-2,3,5,6-tetrafluoropyridine (S19): Prepared from 4-amino-2,3,5,6-tetrafluoropyridine using general procedure B to furnish S19 in a 10% yield. HRMS (ESI-TOF) calculated for C17H25F4N2O [M+H]+: m/z 349.1903. found 349.1879; 1H NMR (500 MHz, CDCl3) 2.38 (t, J=7.5 Hz, 2H), 1.63-1.49 (m, 2H), 1.38-1.15 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 211.9, 42.8, 31.9, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 23.9, 22.7, 14.1. The aryl signals were obscured by the C—F splitting.




C12-4-amino-2-trifluoromethylpyridine (S20): Prepared from 4-amino-2-trifluoromethylpyridine using general procedure B to furnish S20 in a 92% yield. HRMS (ESI-TOF) calculated for C18H28F3N2O [M+H]+: m/z 345.2154. found 345.2137; 1H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H), 8.52 (d, J=5.6 Hz, 1H), 7.97 (s, 1H), 7.76-7.66 (m, 1H), 2.40 (t, J=7.6 Hz, 2H), 1.69 (p, J=7.4 Hz, 2H), 1.52-0.99 (m, 16H), 0.85 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 173.3, 150.6, 148.9 (q, J=34 Hz), 147.0, 121.4 (q, J=274 Hz), 115.7, 110.7 (q, J=3 Hz), 37.6, 34.2, 31.9, 29.6, 29.5, 29.4, 29.2, 25.2, 24.9, 22.7, 14.1.




C12-isonicotinic amide (S21): Prepared from isonicotinic acid and dodecylamine using general procedure C. The crude product was purified by recrystallization from CH2Cl2/hexanes to provide S21 in a 40% yield. HRMS (ESI-TOF) calculated for C18H31N2O [M+H]+: m/z 291.2437. found 291.2425; 1H NMR (500 MHz, CDCl3) δ 8.80-8.68 (m, 2H), 7.68-7.52 (m, 2H), 6.16 (s, 1H), 3.55-3.37 (m, 2H), 1.68-1.48 (m, 2H), 1.42-1.20 (m, 14H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 165.5, 150.6, 141.8, 120.8, 40.3, 31.9, 29.6, 29.6, 29.6, 29.5, 29.5, 29.3, 29.3, 26.9, 22.7, 14.1.




C12-4-(aminomethyl)pyridine (S22): Prepared from 4-(aminomethyl)pyridine using general procedure B to furnish S22 in a 10% yield. HRMS (ESI-TOF) calculated for C18H31N2O [M+H]+: m/z 291.2437. found 291.2434; 1H NMR (500 MHz, CDCl3) δ 8.55 (d, J=5.0 Hz, 2H), 7.23-7.10 (m, 2H), 5.84 (s, 1H), 4.47 (d, J=6.1 Hz, 2H), 2.26 (t, J=8.0 Hz, 2H), 1.81-1.59 (m, 2H), 1.45-1.06 (m, 16H), 0.87 (t, J=6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 173.3, 150.0, 147.5, 122.3, 120.8, 42.2, 36.7, 31.9, 29.6, 29.6, 29.5, 29.3, 29.3, 25.7, 22.7, 14.1.




C12-4-(hydroxymethyl)pyridine (S23): Prepared from 4-(hydroxymethyl)pyridine using general procedure B to furnish S23 in a 66% yield. HRMS (ESI-TOF) calculated for C18H30NO2 [M+H]+: m/z 292.2277. found 292.2280; 1H NMR (500 MHz, CDCl3) δ 8.61 (d, J=4.9 Hz, 2H), 7.25-7.23 (m, 2H), 5.12 (s, 2H), 2.40 (t, J=7.6 Hz, 2H), 1.66 (p, J=7.4 Hz, 2H), 1.38-1.15 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 173.4, 149.9, 145.1, 121.9, 64.0, 34.1, 31.9, 29.6, 29.6, 29.4, 29.3, 29.2, 29.1, 24.9, 22.7, 14.1.




C12-4-(piperazine)pyridine (S24): Prepared from 4-(piperazine)pyridine using general procedure B to furnish S24 in a 24% yield. HRMS (ESI-TOF) calculated for C21H36N3O [M+H]+: m/z 346.2858. found 346.2851; 1H NMR (500 MHz, CDCl3) δ 8.38-8.24 (m, 2H), 6.72-6.51 (m, 2H), 3.77 (t, J=5.4 Hz, 2H), 3.63 (t, J=5.3 Hz, 2H), 3.37 (t, J=5.3 Hz, 2H), 3.34 (t, J=5.4 Hz, 2H), 2.42-2.30 (m, 2H), 1.65 (p, J=7.5 Hz, 2H), 1.25 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.9, 154.5, 150.5, 108.5, 46.0, 44.8, 40.7, 33.3, 31.9, 29.6, 29.5, 29.5, 29.4, 29.3, 25.3, 22.7, 14.1.




C12-5-aminoindole (S25): Prepared from 5-aminoindole using general procedure B to furnish S25 in an 89% yield. HRMS (ESI-TOF) calculated for C20H31N2O [M+H]+: m/z 315.2437. found 315.2415; 1H NMR (500 MHz, CDCl3) δ 8.12 (s, 1H), 7.84 (s, 1H), 7.33 (d, J=8.7 Hz, 1H), 7.25-7.19 (m, 2H), 7.12 (s, 1H), 6.52 (s, 1H), 2.37 (t, J=7.5 Hz, 2H), 1.75 (p, J=7.6 Hz, 2H), 1.46-1.13 (m, 16H), 0.88 (t, J=6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.3, 133.1, 130.5, 128.0, 125.1, 116.3, 112.6, 111.1, 102.8, 37.8, 31.9, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 25.8, 22.7, 14.1.




Methoxyarylester (S26): 2-Methoxyphenylacetic acid (0.10 g, 0.60 mmol, 1.0 equiv), n-hexanol (90 μL, 0.72 mmol, 1.2 equiv), diisopropylcarbodiimide (0.19 mL, 1.2 mmol, 1.7 equiv), dimethylaminopyridine (7.4 mg, 0.060 mmol, 0.01 equiv), and CH2Cl2 (8 mL) were combined. The reaction was stirred for 17 h, then poured onto water (5 mL) and extracted with CH2Cl2 (3×8 mL). The combined organic layer was washed sequentially with saturated NaHCO3 (5 mL), water (5 mL), and brine (5 mL). The solution was then dried over Na2SO4 and concentrated. The crude product was purified by column chromatography to provide 0.12 g of S26 (78% yield); 1H NMR (500 MHz, CDCl3) δ 7.30-7.22 (m, 1H), 7.21-7.15 (m, 1H), 6.92 (t, J=7.4 Hz, 1H), 6.87 (d, J=8.2 Hz, 1H), 4.09 (t, J=6.7 Hz, 2H), 3.81 (s, 3H), 3.62 (s, 2H), 1.60 (p, J=7.4 Hz, 2H), 1.36-1.20 (m, 6H), 0.88 (t, J=6.6 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 172.0, 157.5, 130.8, 128.5, 123.2, 120.4, 110.3, 64.8, 55.3, 36.1, 31.4, 28.5, 25.5, 22.5, 14.0.




Methoxyaryl diazoester (S27): S26 (0.12 g, 0.46 mmol, 1.0 equiv) was combined with 4-acetamidobenzenesulfonyl azide (0.27 g, 1.1 mmol, 2.4 equiv) in 2.4 mL CH3CN. The reaction was cooled to 0° C. and 1,8-diazabiocyclo[5.4.0]undec-7-ene (0.28 mL, 1.9 mmol, 4.0 equiv). After stirring at room temperature for 24 h, the reaction was quenched with saturated aqueous NH4Cl (3 mL) and extracted with diethyl ether (3×5 mL). The organic layer was dried over Na2SO4 and concentrated. Using a 20:1 petroleum ether/diethyl ether eluent, the crude product was purified by column chromatography to furnish S27 in a quantitative yield. 1H NMR (500 MHz, CDCl3) δ 7.59-7.50 (m, 1H), 7.32-7.22 (m, 1H), 7.06-6.98 (m, 1H), 6.94-6.86 (m, 1H), 4.23 (t, J=6.7 Hz, 2H), 3.86 (s, 3H), 1.68 (p, J=7.0 Hz, 2H), 1.44-1.25 (m, 6H), 0.92-0.88 (m, 3H).




C6-dihydrobenzofuran ester (S28): Based on Ref. 3. To a flask containing Rh2(S-DOSP)4 (9.4 mg, 5.0×10−3 mmol, 0.010 equiv) in 2.1 mL hexanes was added S27 (0.13 g, 0.48 mmol, 1.0 equiv) in mL hexanes over 3 h by syringe pump. After stirring for 72 h, the reaction mixture was concentrated and was purified by column chromatography to furnish a quantitative yield of S28. HRMS (ESI-TOF) calculated for C15H21O3 [M+H]+: m/z 249.1491. found 249.1489; 1H NMR (500 MHz, CDCl3) δ 7.37 (d, J=7.6 Hz, 1H), 7.23-7.13 (m, 1H), 6.89 (td, J=7.5, 1.0 Hz, 1H), 6.82 (d, J=8.1 Hz, 1H), 4.94 (dd, J=9.2, 6.6 Hz, 1H), 4.67 (t, J=9.6 Hz, 1H), 4.33 (dd, J=9.8, 6.6 Hz, 1H), 4.16 (td, J=6.7, 2.7 Hz, 2H), 1.66 (p, J=6.7 Hz, 2H), 1.42-1.24 (m, 6H), 0.89 (t, J=6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.2, 159.7, 129.4, 125.3, 124.3, 120.6, 109.9, 72.4, 65.7, 47.2, 31.3, 28.5, 25.5, 22.5, 14.0.




C12-dihydrobenzofuran acylhydroxylamine (S29): 3-coumarone (50 mg, 0.37 mmol, 1.0 equiv), HONH2.HCl (69 mg, 0.99 mmol, 2.7 equiv), and 0.75 mL pyridine were combined in a flame-dried flask and stirred for 3 h. The reaction was diluted with 3 mL EtOAc and washed sequentially with a 10% CuSO4 solution (3×1 mL) and with brine (1×1 mL). The solution was dried over Na2SO4 and concentrated. The crude oxime was combined with 3.75 mL CH3CN and Et3N (0.16 mL, 1.1 mmol, 3.0 equiv). The reaction was cooled to 0° C., and dodecanoyl chloride (0.09 mL, 0.37 mmol, 1.0 equiv) was added dropwise. The reaction was allowed to return to room temperature over 4 h. The reaction was quenched with 2 mL saturated aqueous NaHCO3 and extracted with CH2Cl2 (3×4 mL). The organic layer was washed with brine (5 mL), dried over Na2SO4, and concentrated. The crude product was purified by column chromatography to provide S29 in a 43% yield. HRMS (ESI-TOF) calculated for C20H30NO3 [M+H]+: m/z 332.2226. found 332.2231; 1H NMR (500 MHz, CDCl3) δ 7.82 (d, J=7.8 Hz, 1H), 7.46 (t, J=7.9 Hz, 1H), 7.05 (t, J=7.6 Hz, 1H), 7.01 (d, J=8.5 Hz, 1H), 5.19 (d, J=2.3 Hz, 2H), 2.47 (t, J=7.6 Hz, 2H), 1.73 (p, J=7.0 Hz, 2H), 1.45-1.17 (m, 16H), 0.88 t, J=6.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 170.8, 166.7, 164.4, 134.7, 123.7, 122.0, 117.9, 111.8, 70.9, 32.8, 31.9, 29.6, 29.6, 29.4, 29.3, 29.2, 29.1, 24.9, 22.7, 14.1.




C12-3-amino-1-ethylcarbazole (S30): Prepared from 3-amino-1-ethylcarbazole using general procedure B to furnish S30 in a 53% yield. HRMS (ESI-TOF) calculated for C-26H37N2O [M+H]+: m/z 393.2906. found 393.2895; 1H NMR (500 MHz, CDCl3) δ 8.30 (s, 1H), 8.03 (d, J=7.8 Hz, 1H), 7.60-7.51 (m, 1H), 7.48 (d, J=9.3 Hz, 1H), 7.44 (d, J=7.5 Hz, 1H), 7.37 (d, J=8.2 Hz, 1H), 7.31-7.24 (m, 1H), 7.18 (t, J=7.4 Hz, 1H), 4.34-4.24 (m, 2H), 2.39 (t, J=7.6 Hz, 2H), 1.77 (p, J=7.6 Hz, 2H), 1.56-1.04 (m, 19H), 0.89 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.7, 140.4, 137.1, 129.8, 125.8, 123.0, 122.8, 120.7, 119.6, 118.7, 112.9, 108.5, 108.4, 37.8, 37.6, 32.0, 29.7, 29.7, 29.6, 29.5, 29.4, 29.4, 25.9, 22.8, 14.2, 13.9




3OC12-3-amino-1-ethylcarbazole (S31): Prepared from 3-amino-1-ethylcarbazole using general procedure A to furnish S31 in a 17% yield. HRMS (ESI-TOF) calculated for C26H35N2O2 [M+H]+: m/z 407.2699. found 407.2734; 1H NMR (500 MHz, CDCl3) δ 9.22 (s, 1H), 8.33 (s, 1H), 8.08 (d, J=7.8 Hz, 1H), 7.55 (d, J=8.6 Hz, 1H), 7.46 (t, J=7.7 Hz, 1H), 7.39 (d, J=8.3 Hz, 1H), 7.35 (d, J=8.7 Hz, 1H), 7.21 (t, J=7.4 Hz, 1H), 4.35 (q, J=7.2 Hz, 2H), 3.62 (s, 2H), 2.62 (t, J=7.3 Hz, 2H), 1.70-1.60 (m, 2H), 1.42 (t, J=7.2 Hz, 3H), 1.38-1.06 (m, 12H), 0.88 (t, J=6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 208.4, 163.4, 140.4, 137.3, 129.3, 125.9, 123.0, 122.8, 120.8, 119.5, 118.8, 112.9, 108.6, 108.5, 48.8, 44.3, 37.6, 31.9, 29.4, 29.4, 29.3, 29.0, 23.4, 22.7, 14.2, 13.9.




C12-2-amido-1-methylindole (S32): Prepared from 1-methylindole-2-carboxylic acid and dodecylamine using general procedure C to furnish S32 in a 29% yield. HRMS (ESI-TOF) calculated for C22H35N2O [M+H]+: m/z 343.2749. found 343.2745; 1H NMR (500 MHz, CDCl3) δ 7.65 (d, J=8.2 Hz, 1H), 7.42-7.29 (m, 2H), 7.22 (t, J=7.4 Hz, 1H), 6.83-6.75 (m, 1H), 4.05 (s, 3H), 3.54-3.47 (m, 2H), 1.71-1.62 (m, 2H), 1.50-1.08 (m, 18H), 0.87 (t, J=6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 160.7, 136.8, 125.2, 124.0, 121.7, 121.0, 120.5, 119.4, 110.3, 103.3, 77.3, 39.8, 32.0, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 27.0, 22.7, 14.2.




C12-3-amidoindole (S33): Prepared from indole-3-carboxylic acid and dodecylamine using general procedure C to furnish S33 in a 47% yield. HRMS (ESI-TOF) calculated for C21H33N2O [M+H]+: m/z 329.2593. found 329.2586; 1H NMR (500 MHz, CDCl3) δ 9.32 (s, 1H), 7.98-7.85 (m, 1H), 7.74-7.69 (m, 1H), 7.47-7.41 (m, 1H), 7.26-7.22 (m, 2H), 6.08-6.00 (m, 1H), 3.55-3.47 (m, 2H), 1.65 (p, J=7.5 Hz, 2H), 1.49-1.13 (m, 18H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 165.6, 136.5, 128.2, 124.5, 122.8, 121.5, 119.6, 112.5, 112.2, 39.7, 32.0, 30.0, 29.7, 29.7, 29.7, 29.6, 29.4, 29.4, 27.1, 22.7, 14.2.




C12-3-(methylamido)indole (S34): Prepared from 2-(1H-indol-3-yl)acetic acid and dodecylamine using general procedure C to furnish S34 in a 40% yield. HRMS (ESI-TOF) calculated for C22H35N2O [M+H]+: m/z 343.2749. found 343.2740; 1H NMR (500 MHz, CDCl3) δ 8.27 (s, 1H), 7.56 (d, J=7.9 Hz, 1H), 7.42 (d, J=8.2 Hz, 1H), 7.26-7.21 (m, 1H), 7.19-7.11 (m, 2H), 5.68 (s, 1H), 3.74 (s, 2H), 3.20-3.11 (m, 2H), 1.38-1.06 (m, 20H), 0.88 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.3, 136.4, 127.0, 123.7, 122.7, 120.2, 118.8, 111.4, 109.2, 39.6, 33.5, 32.0, 29.7, 29.7, 29.6, 29.5, 29.5, 29.4, 29.3, 26.8, 22.8, 14.2.




C12-2-amino-tetrahydrodibenzofuran (S35): Prepared from 6,7,8,9-tetrahydrodibenzo[b,d]furan-2-amine using general procedure B to furnish S35 in a 68% yield. HRMS (ESI-TOF) calculated for C24H36NO2 [M+H]+: m/z 370.2746. found 370.2745; 1H NMR (500 MHz, CDCl3) δ 7.75 (s, 1H), 7.40 (s, 1H), 7.30-7.22 (m, 1H), 7.09 (d, J=8.7 Hz, 1H), 2.76-2.67 (m, 2H), 2.63-2.50 (m, 2H), 2.35 (t, J=7.6 Hz, 2H), 1.96-1.86 (m, 2H), 1.84-1.78 (m, 2H), 1.73 (p, J=7.5 Hz, 2H), 1.50-1.08 (m, 16H), 0.87 (t, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.6, 155.1, 151.2, 132.6, 129.3, 115.9, 113.2, 110.6, 37.8, 32.0, 29.7, 29.7, 29.6, 29.5, 29.4, 29.4, 29.1, 25.8, 23.5, 22.9, 22.7, 22.6, 20.4, 14.2




4-aminopyridine-C4-3-bromophenoxyhybrid (S37): Prepared from 4-aminopyridine and S361 using general procedure C to furnish S37 in a 75% yield. HRMS (ESI-TOF) calculated for C15H16BrN2O2 [M+H]+: m/z 335.0395. found 335.0388; 1H NMR (500 MHz, CDCl3) δ 8.59-8.39 (m, 2H), 8.23 (s, 1H), 7.49 (d, J=5.4 Hz, 2H), 7.12 (t, J=8.0 Hz, 1H), 7.07 (d, J=8.2 Hz, 1H), 7.03-6.99 (m, 1H), 6.78 (dd, J=8.2, 2.4 Hz, 1H), 4.02 (t, J=5.8 Hz, 2H), 2.61 (t, J=7.1 Hz, 2H), 2.27-2.14 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.4, 159.3, 150.5, 145.1, 130.6, 124.0, 122.8, 117.7, 113.5, 113.2, 66.8, 33.9, 24.5.




4-aminopyridine-C6-3-bromophenoxyhybrid (S39): Prepared from 4-aminopyridine and S381 using general procedure C to furnish S39 in a 70% yield. HRMS (ESI-TOF) calculated for C17H20BrN2O2 [M+H]+: m/z 363.0708. found 363.0710; 1H NMR (500 MHz, CDCl3) δ 8.50 (s, 2H), 7.48 (d, J=5.3 Hz, 2H), 7.30 (s, 1H), 7.13 (t, J=8.1 Hz, 1H), 7.09-7.00 (m, 2H), 6.83-6.78 (m, 1H), 3.95 (t, J=6.3 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 1.88-1.76 (m, 4H), 1.60-1.49 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.1, 159.7, 150.0, 145.6, 130.5, 123.7, 122.7, 117.6, 113.4, 113.4, 67.7, 37.5, 28.8, 25.6, 24.8.




4-amino-2-fluoropyridine-C4-3-bromophenoxyhybrid (S40): Prepared from 4-amino-2-fluoropyridine and S361 using general procedure C to furnish S40 in a 48% yield. HRMS (ESI-TOF) calculated for C15H15BrFN2O2 [M+H]+: m/z 353.0301. found 353.0290; 1H NMR (500 MHz, CDCl3) δ 8.09 (d, J=5.7 Hz, 1H), 7.47 (s, 1H), 7.41-7.30 (m, 1H), 7.19-7.07 (m, 3H), 7.07-6.99 (m, 1H), 6.88-6.75 (m, 1H), 4.05 (t, J=5.7 Hz, 2H), 2.63 (t, J=7.0 Hz, 2H), 2.22 (p, J=6.8 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 171.1, 164.8 (d, J=235 Hz), 159.3, 148.2 (d, J=17 Hz), 148.1, 130.7, 124.1, 122.9, 117.7, 113.3, 111.3, 98.7 (d, J=43 Hz), 66.7, 34.0, 24.4.




4-amino-2-fluoropyridine-C6-3-bromophenoxyhybrid (S41): Prepared from 4-amino-2-fluoropyridine and S381 using general procedure C to furnish S41 in a 14% yield. HRMS (ESI-TOF) calculated for C17H19BrFN2O2 [M+H]+: m/z 381.0614. found 381.0595; 1H NMR (500 MHz, CDCl3) δ 8.09 (d, J=5.7 Hz, 1H), 7.53 (s, 1H), 7.35 (d, J=1.8 Hz, 1H), 7.18 (dt, J=5.7, 1.6 Hz, 1H), 7.13 (t, J=8.1 Hz, 1H), 7.06 (ddd, J=7.9, 1.8, 1.0 Hz, 1H), 7.03 (t, J=2.1 Hz, 1H), 6.80 (ddd, J=8.2, 2.5, 1.1 Hz, 1H), 3.94 (1, J=6.2 Hz, 2H), 2.44 (t, J=7.4 Hz, 2H), 1.87-1.73 (m, 4H), 1.61-1.49 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.7, 164.8 (d, J=235 Hz), 159.7, 148.9 (d, J=12 Hz), 148.1 (d, J=17 Hz), 130.5, 123.7, 122.8, 117.6, 113.4, 111.3 (d, J=4 Hz), 98.7 (d, J=43 Hz), 67.7, 37.6, 28.8, 25.6, 24.7.




4-amino-2-methoxypyridine-C4-3-bromophenoxyhybrid (S42): Prepared from 4-amino-2-methoxypyridine and S36′ using general procedure C to furnish S42 in a 72% yield. HRMS (ESI-TOF) calculated for C16H18BrN2O3 [M+H]+: m/z 365.0501. found 365.0486; 1H NMR (500 MHz, CDCl3) δ 8.04 (d, J=5.8 Hz, 1H), 7.13 (t, J=8.0 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 7.05-6.99 (m, 2H), 6.97 (dd, J=5.8, 1.9 Hz, 1H), 6.81 (ddd, J=8.2, 2.5, 1.1 Hz, 1H), 4.03 (t, J=5.8 Hz, 2H), 3.91 (s, 3H), 2.59 (t, J=7.1 Hz, 2H), 2.29-2.14 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.0, 165.4, 159.3, 147.6, 146.7, 130.6, 124.0, 122.8, 117.7, 113.3, 107.9, 99.3, 66.8, 53.6, 34.0, 24.5.




4-amino-2-methoxypyridine-C5-3-bromophenoxyhybrid (S44): Prepared from 4-amino-2-methoxypyridine and S431 using general procedure C to furnish S44 in a 58% yield. HRMS (ESI-TOF) calculated for C17H20BrN2O3 [M+1-1]′: m/z 379.06573. found 379.0633; 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J=5.7 Hz, 1H), 7.13 (t, J=8.0 Hz, 1H), 7.10-7.05 (m, 1H), 7.05-6.96 (m, 3H), 6.81 (ddd, J=8.2, 2.5, 1.1 Hz, 1H), 3.98 (t, J=5.7 Hz, 2H), 3.92 (s, 3H), 2.46 (t, J=7.0 Hz, 2H), 2.02-1.81 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 171.2, 165.4, 159.5, 147.6, 146.8, 130.6, 123.8, 122.8, 117.6, 113.4, 107.9, 99.3, 67.7, 53.6, 37.2, 28.3, 22.0.




4-amino-2-methoxypyridine-C6-3-bromophenoxyhybrid (S45): Prepared from 4-amino-2-methoxypyridine and S381 using general procedure C to furnish S45 in a 13% yield. HRMS (ESI-TOF) calculated for C18H22BrN2O3 [M+H]+: m/z 393.0814. found 393.0805; 1H NMR (500 MHz, CDCl3) δ 8.11-7.98 (m, 1H), 7.18-7.09 (m, 2H), 7.07 (t, J=1.5 Hz, 1H), 7.06-6.97 (m, 3H), 6.81 (ddd, J=8.0, 2.4, 1.3 Hz, 1H), 3.95 (t, J=6.4 Hz, 2H), 3.92 (s, 3H), 2.41 (t, J=7.4 Hz, 2H), 1.88-1.72 (m, 4H), 1.63-1.48 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 171.4, 165.4, 159.7, 147.6, 146.8, 130.5, 123.7, 122.8, 117.6, 113.4, 107.9, 99.3, 67.7, 53.6, 37.7, 28.8, 25.6, 24.9.




4-amino-2-methoxypyridine-3OC6-3-bromophenoxyhybrid (S46): S361 (0.83 g, 3.2 mmol, 1.0 equiv) was dissolved in 6.4 mL CH2Cl2. The reaction mixture was cooled to 0° C. and oxalylchloride (0.27 mL, 3.2 mmol, 1.0 equiv) was added dropwise. After 5 min, 1 drop of DMF was added to the reaction mixture. The reaction mixture was stirred at 0° C. for 30 min and then at room temperature for 90 min. The acid chloride was then used in general procedure A with 4-amino-2-methoxypyridine to furnish S46 in a 59% yield. HRMS (ESI-TOF) calculated for C18H20BrN2O4 [M+H]+: m/z 407.0606. found 407.0583; 1H NMR (500 MHz, CDCl3) δ 9.58 (s, 1H), 8.05 (d, J=5.7 Hz, 1H), 7.16 (s, 1H), 7.11 (t, J=8.0 Hz, 1H), 7.09-6.99 (m, 3H), 6.78 (d, J=8.1 Hz, 1H), 3.98 (t, J=5.9 Hz, 2H), 3.94 (s, 3H), 3.65 (s, 2H), 2.80 (t, J=7.0 Hz, 2H), 2.12 (p, J=6.0 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 206.4, 165.0, 164.2, 159.3, 147.2, 146.7, 130.6, 124.1, 122.8, 117.6, 113.4, 108.5, 99.6, 66.6, 54.0, 49.1, 40.6, 23.0.




C7 acid (S47): Sodium hydride (60%, 22 mg, 0.49 mmol, 1.3 equiv) was added to 3-bromophenol (0.10 mL, 0.37 mmol, 1.0 equiv) in 0.50 mL DMF. The reaction was heated to 100° C. for 1 h. After cooling, methyl-7-bromoheptanoate (0.10 g, 0.45 mmol, 1.2 equiv), potassium iodide (7.4 mg, 0.045 mmol, 0.12 equiv), and potassium carbonate (0.18 g, 1.1 mmol, 3.0 equiv were added). After stirring at room temperature for 1 week, the reaction was diluted with 5 mL diethyl ether and washed sequentially with water (3×5 mL) and 2 M potassium hydroxide (3×5 mL). The organic layer was dried over Na2SO4 and concentrated. Column chromatography furnished the C7 aryl ester in a 55% yield. The ester (80 mg, 0.25 mmol, 1.0 equiv) was combined with lithium hydroxide monohydrate (57 mg, 1.3 mmol, 5 equiv) in 4:1 THF/H2O (2.5 mL). The reaction was heated to 65° C. for 17 h or until complete. After cooling, the reaction was acidified with 1 M HCl. The aqueous layer was extracted with EtOAc (3×4 mL). The combined organic was washed with brine (5 mL), dried over Na2SO4, and concentrated to provide S47 in an 80% yield. The material was used without further purification. 1H NMR (500 MHz, CDCl3) δ 10.99 (bs, 1H), 7.13 (t, J=8.0 Hz, 1H), 7.09-7.01 (m, 2H), 6.86-6.78 (m, 1H), 3.92 (t, J=6.4 Hz, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.83-1.73 (m, 2H), 1.67 (p, J=7.5 Hz, 2H), 1.55-1.37 (m, 4H).




4-amino-2-methoxypyridine-C7-3-bromophenoxyhybrid (S48): Prepared from 4-amino-2-methoxypyridine and S47 using general procedure C to furnish S48 in a 21% yield. HRMS (ESI-TOF) calculated for C19H24BrN2O3 [M+H]+: m/z 407.0970. found 407.0944; 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J=5.7, 1H), 7.18 (s, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.08-6.95 (m, 4H), 6.81 (ddd, J=8.2, 2.5, 1.0 Hz, 1H), 3.95-3.89 (m, 5H), 2.38 (t, J=7.5 Hz, 2H), 1.87-1.69 (m, 4H), 1.54-1.37 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 171.6, 165.4, 159.8, 147.6, 146.8, 130.5, 123.6, 122.8, 117.6, 113.5, 107.9, 99.2, 67.9, 53.6, 37.7, 28.9, 28.8, 25.8, 25.1.




C8 acid (S49): The acid was prepared using the same procedure as for S47, but starting with methyl-8-bromooctanoate to provide S49 in a quantitative yield. HRMS (ESI-TOF) calculated for C14H20BrO3 [M+H]+: m/z 315.0596. found 315.0607; 1H NMR (500 MHz, CDCl3) δ 9.90 (bs, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.08-7.02 (m, 2H), 6.82 (ddd, J=8.2, 2.4, 1.1 Hz, 1H), 3.92 (t, J=6.5 Hz, 2H), 2.37 (t, J=7.5 Hz, 2H), 1.81-1.73 (m, 2H), 1.70-1.62 (m, 2H), 1.50-1.42 (m, 2H), 1.42-0.34 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 177.9, 159.8, 130.5, 123.5, 122.7, 117.6, 113.5, 68.1, 33.6, 29.0, 28.9, 28.9, 25.8, 24.6.




4-amino-2-methoxypyridine-C8-3-bromophenoxyhybrid (S50): Prepared from 4-amino-2-methoxypyridine and S49 using general procedure C to furnish S50 in a 36% yield. HRMS (ESI-TOF) calculated for C20H26BrN2O3 [M+H]+: m/z 421.1127. found 421.1121; 1H NMR (500 MHz, CDCl3) δ 8.08 (d, J=5.6 Hz, 1H), 7.21-7.13 (m, 2H), 7.13-6.98 (m, 4H), 6.85 (ddd, J=8.2, 2.5, 1.1 Hz, 1H), 4.03-3.91 (m, 5H), 2.41 (t, J=7.5 Hz, 2H), 1.86-1.71 (m, 4H), 1.54-1.37 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 171.7, 165.4, 159.8, 147.6, 146.8, 130.5, 123.6, 122.7, 117.6, 113.5, 107.9, 99.2, 68.0, 53.6, 37.8, 29.0, 29.0, 29.0, 25.8, 25.1.




4-aminopyrimidine-C6-3-bromophenoxyhybrid (S51): Prepared from 4-aminopyrimidine and S381 using general procedure C to furnish S51 in a 48% yield. HRMS (ESI-TOF) calculated for C16H19BrN3O2 [M+H]+: m/z 364.0661. found 364.0648; 1H NMR (500 MHz, CDCl3) δ 8.85 (s, 1H), 8.63 (d, J=5.7 Hz, 1H), 8.27-8.09 (m, 2H), 7.12 (t, J=8.0 Hz, 1H), 7.09-7.01 (m, 2H), 6.84-6.78 (m, 1H), 3.95 (t, J=6.3 Hz, 2H), 2.47 (t, J=7.4 Hz, 2H), 1.88-1.77 (m, 4H), 1.61-1.49 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.2, 159.7, 158.5, 158.2, 156.9, 130.5, 123.7, 122.8, 117.6, 113.4, 110.2, 67.7, 37.6, 28.8, 25.6, 24.6




4-amino-6-methoxypyrimidine-C6-3-bromophenoxyhybrid (S52): Prepared from 4-amino-6-methoxypyrimidine and S38′ using general procedure C to furnish S52 in a 23% yield. HRMS (ESI-TOF) calculated for C17H21BrN3O3 [M+H]: m/z 394.0766. found 394.0752; 1H NMR (500 MHz, CDCl3) δ 8.45 (d, J=1.1 Hz, 1H), 7.82 (s, 1H), 7.56 (d, J=1.1 Hz, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.09-7.01 (m, 2H), 6.81 (ddd, J=8.2, 2.5, 1.1 Hz, 1H), 3.97 (s, 3H), 3.94 (t, J=6.3 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 1.87-1.73 (m, 4H), 1.59-1.49 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.9, 171.3, 159.7, 157.5, 157.4, 130.5, 123.7, 122.8, 117.6, 113.4, 95.0, 67.7, 54.2, 37.6, 28.8, 25.6, 24.7.




4-amino-2-methoxypyrimidine-C6-3-bromophenoxyhybrid (S53): Prepared from 4-amino-2-methoxypyrimidine and S381 using general procedure C to furnish S53 in a 14% yield. HRMS (ESI-TOF) calculated for C17H21BrN3O3 [M+H]: m/z 394.0766. found 394.0763; 1H NMR (500 MHz, CDCl3) δ 8.41 (d, J=5.6 Hz, 1H), 7.86 (s, 1H), 7.79 (d, J=5.6 Hz, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.09-7.00 (m, 2H), 6.81 (dd, J=8.2, 2.4 Hz, 1H), 4.01-3.89 (m, 5H), 2.45 (t, J=7.5 Hz, 2H), 1.87-1.75 (m, 4H), 1.57-1.50 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 13C NMR (126 MHz, CDCl3) 172.1, 165.0, 160.5, 159.7, 158.9, 130.5, 123.7, 122.8, 117.7, 113.4, 103.9, 67.7, 54.8, 37.5, 28.8, 25.6, 24.6.




4-amino-2,6-dimethoxypyrimidine-C6-3-bromophenoxyhybrid (S54): Prepared from 4-amino-2,6-dimethoxypyrimidine and S381 using general procedure C to furnish S54 in a 13% yield. HRMS (ESI-TOF) calculated for C18H23BrN3O4 [M+H]+: m/z 424.0872. found 424.0863; 1H NMR (500 MHz, CDCl3) δ 7.69 (s, 1H), 7.20 (s, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.09-7.01 (m, 2H), 6.81 (ddd, J=8.2, 2.5, 1.0 Hz, 1H), 3.97-3.92 (m, 8H), 2.42 (t, J=7.5 Hz, 2H), 1.88-1.70 (m, 4H), 1.63-1.47 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 173.3, 171.7, 164.5, 159.7, 158.5, 130.5, 123.6, 122.8, 117.6, 113.4, 88.4, 67.7, 54.7, 54.2, 37.6, 28.8, 25.6, 24.7.




4-amino-2-chloropyridine-C6-3-bromophenoxyhybrid (S55): Prepared from 4-amino-2-chloropyridine and S381 using general procedure C to furnish S55 in a 6.4% yield. HRMS (ESI-TOF) calculated for C17H19BrClN2O2 [M+H]+: m/z 397.0318. found 397.0299; 1H NMR (500 MHz, CDCl3) δ 8.26 (d, J=5.6 Hz, 1H), 7.64 (d, J=2.0 Hz, 1H), 7.41-7.35 (m, 1H), 7.34 (dd, J=5.7, 1.9 Hz, 1H), 7.13 (t, J=8.0 Hz, 1H), 7.10-7.05 (m, 1H), 7.03 (t, J=2.1 Hz, 1H), 6.81 (ddd, J=8.3, 2.5, 1.1 Hz, 1H), 3.95 (t, J=6.2 Hz, 2H), 2.43 (t, J=7.4 Hz, 2H), 1.89-1.74 (m, 4H), 1.65-1.48 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.6, 159.7, 152.5, 150.2, 146.8, 130.5, 123.7, 122.9, 117.6, 113.4, 113.3, 112.2, 67.7, 37.6, 28.8, 25.6, 24.7.




4-amino-2-trifluoromethylpyridine-C4-3-bromophenoxyhybrid (S56): Prepared from 4-amino-2-trifluoromethylpyridine and S361 using general procedure C to furnish S56 in a 4.0% yield. HRMS (ESI-TOF) calculated for C16H15BrF3N2O2 [M+H]+: m/z 403.0269. found 403.0259; 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J=5.5 Hz, 1H), 7.87 (d, J=2.0 Hz, 1H), 7.70-7.57 (m, 2H), 7.14 (t, J=8.0 Hz, 1H), 7.09 (dt, J=8.0, 1.4 Hz, 1H), 7.03 (t, J=2.1 Hz, 1H), 6.80 (ddd, J=8.1, 2.5, 1.0 Hz, 1H), 4.05 (t, J=5.8 Hz, 2H), 2.65 (t, J=7.0 Hz, 2H), 2.27-2.18 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.3, 159.2, 151.1, 149.9-148.7 (m), 146.0, 130.7, 124.5-118.0 (m), 124.2, 122.9, 117.7, 115.3, 113.3, 110.4-110.2 (m), 66.7, 34.0, 24.4.




4-amino-2-trifluoromethylpyridine-C5-3-bromophenoxyhybrid (S57): Prepared from 4-amino-2-trifluoromethylpyridine and S431 using general procedure C to furnish S57 in a 6.1% yield. HRMS (ESI-TOF) calculated for C17H17BrF3N2O2 [M+H]+: m/z 417.0425. found 417.0431; 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J=5.6 Hz, 1H), 7.87 (d, J=2.1 Hz, 1H), 7.66 (dd, J=5.5, 2.1 Hz, 1H), 7.46 (d, J=2.7 Hz, 1H), 7.13 (t, J=8.0 Hz, 1H), 7.08 (dt, J=7.9, 1.3 Hz, 1H), 7.04 (t, J=2.1 Hz, 1H), 6.81 (ddd, J=8.2, 2.5, 1.1 Hz, 1H), 4.00 (t, J=5.7 Hz, 2H), 2.52 (t, J=7.0 Hz, 2H), 2.04-1.82 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 171.6, 159.4, 151.0, 149.3 (q, J=35 Hz), 146.0, 130.6, 123.9, 122.8, 117.6, 121.3 (q, J=274 Hz), 115.3, 113.4, 110.3-110.2 (m), 67.7, 37.1, 28.3, 21.9.




4-amino-2-trifluoromethylpyridine-C7-3-bromophenoxyhybrid (S58): Prepared from 4-amino-2-trifluoromethylpyridine and S471 using general procedure C to furnish S58 in a 17% yield. HRMS (ESI-TOF) calculated for C19H21BrF3N2O2 [M+H]: m/z 445.0738. found 445.0719; 1H NMR (500 MHz, CDCl3) δ 8.61 (d, J=5.5 Hz, 1H), 7.93 (d, J=2.0 Hz, 1H), 7.70 (dd, J=5.6, 2.1 Hz, 1H), 7.57 (s, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.09-7.00 (m, 2H), 6.81 (ddd, J=8.2, 2.4, 1.1 Hz, 1H), 3.92 (t, J=6.4 Hz, 2H), 2.38 (t, J=7.4 Hz, 2H), 1.84-1.72 (m, 2H), 1.67 (p, J=7.5 Hz, 2H), 1.55-1.37 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 178.9, 177.4, 159.8, 151.0, 149.5-148.9 (m), 130.5, 124.8-116.7 (m), 123.6, 122.7, 117.6, 115.6, 113.5, 110.7-110.5 (m), 68.0, 33.7, 28.9, 28.7, 25.7, 24.5.




1-amino-3-trifluoromethyl-C6-3-bromophenoxyhybrid (S59): Prepared from 3-(trifluoromethyl)aniline and S381 using general procedure C to furnish S59 in a 70% yield. HRMS (ESI-TOF) calculated for C19H20BrF3NO2 [M+H]+: m/z 430.0630. found 430.0595; 1H NMR (500 MHz, CDCl3) δ 7.83 (s, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.44 (t, J=8.1 Hz, 1H), 7.39-7.32 (m, 1H), 7.28 (s, 1H), 7.12 (t, J=8.3 Hz, 1H), 7.09-7.00 (m, 2H), 6.81 (d, J=8.3 Hz, 1H), 3.95 (t, J=6.4 Hz, 2H), 2.42 (t, J=7.5 Hz, 2H), 1.93-1.76 (m, 4H), 1.64-1.52 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.2, 159.7, 138.3, 131.3 (q, J=32 Hz), 130.5, 129.5, 127.1-120.3 (m), 123.7, 122.8, 122.7, 120.8-120.7 (m), 117.6, 116.4-116.3 (m), 113.4, 67.7, 37.5, 28.9, 25.7, 25.0.




1-amino-3,5-bis(trifluoromethyl)-C6-3-bromophenoxyhybrid (S60): Prepared from 3,5-bis(trifluoromethyl)aniline and S381 using general procedure C to furnish S60 in a 22% yield. HRMS (ESI-TOF) calculated for C20H19BrF6NO2 [M+H]+: m/z 498.0503. found 498.0488; 1H NMR (500 MHz, CDCl3) δ 8.04 (s, 2H), 7.60 (s, 1H), 7.36 (s, 1H), 7.13 (t, J=8.1 Hz, 1H), 7.10-6.99 (m, 2H), 6.81 (ddd, J=8.2, 2.5, 1.1 Hz, 1H), 3.95 (t, J=6.3 Hz, 2H), 2.45 (t, J=7.4 Hz, 2H), 1.89-1.74 (m, 4H), 1.64-1.50 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.3, 159.7, 139.1, 132.3 (q, J=33 Hz), 130.5, 123.7, 122.8 (t, J=137 Hz), 122.8, 119.4-119.1 (m), 117.6, 117.6-117.4 (m), 113.4, 67.7, 37.4, 28.8, 25.7, 24.9.




4-amino-2-methylpyridine-C6-3-bromophenoxyhybrid (S61):Prepared from 4-amino-2-methylpyridine and S381 using general procedure C to furnish S61 in a 52% yield. HRMS (ESI-TOF) calculated for C18H22BrN2O2 [M+H]+: m/z 377.0865. found 377.0871; 1H NMR (500 MHz, CDCl3) δ 8.31 (d, J=5.8 Hz, 1H), 8.20 (s, 1H), 7.62 (s, 1H), 7.46-7.37 (m, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.06 (dt, J=7.9, 1.2 Hz, 1H), 7.02 (t, J=2.1 Hz, 1H), 6.85-6.76 (m, 1H), 3.94 (t, J=6.3 Hz, 2H), 2.56 (s, 3H), 2.48 (t, J=7.4 Hz, 2H), 1.87-1.72 (m, 4H), 1.62-1.49 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.2, 160.6, 159.7, 158.4, 147.8, 130.5, 123.7, 122.8, 117.6, 113.4, 113.1, 111.2, 67.7, 37.6, 28.8, 25.6, 24.8, 23.6.




4-amino-3-trifluoromethylpyridine-C6-3-bromophenoxyhybrid (S62): Prepared from 4-amino-3-trifluoromethylpyridine and S381 using general procedure C to furnish S62 in a 44% yield. HRMS (ESI-TOF) calculated for C18H19BrF3N2O2 [M+H]+: m/z 431.0582. found 431.0573. 1H NMR (500 MHz, CDCl3) δ 8.90-8.64 (m, 2H), 8.50 (d, J=5.3 Hz, 1H), 7.57 (s, 1H), 7.13 (t, J=8.0 Hz, 1H), 7.09-6.99 (m, 2H), 6.81 (ddd, J=8.3, 2.5, 1.0 Hz, 1H), 3.95 (t, J=6.3 Hz, 2H), 2.49 (t, J=7.4 Hz, 2H), 1.89-1.76 (m, 4H), 1.64-1.49 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 171.4, 159.7, 154.3, 147.4-147.0 (m), 142.7, 130.5, 127.0-120.1 (m, 2C), 123.7, 122.7, 117.6, 115.4-115.1 (m), 113.4, 67.6, 38.0, 28.8, 25.5, 24.7.




6-chloro-N-(2-(trifluoromethyl)pyridin-4-yl)hexanamide (S63): Pyridine (0.30 mL, 3.7 mmol, 1.0 equiv) was added to a mixture of 4-amino-2-trifluoromethylpyridine (0.60 g, 3.7 mmol, 1.0 equiv) in CH2Cl2 (25 mL). After cooling the reaction to 0° C., 6-chlorohexanoyl chloride4 (0.45 mL, 3.7 mmol, 1.0 equiv) was added dropwise. The reaction mixture was allowed to warm to room temperature overnight. The reaction was quenched with saturated aqueous NH4Cl (10 mL) and was extracted with CH2Cl2 (3×25 mL). The combined organic layers were washed sequentially with 1 M HCl (3×25 mL) and brine (1×25 mL), dried over Na2SO4, and concentrated to provide S63 in an 80% yield. S63 was used without further purification. HRMS (ESI-TOF) calculated for C12H15C1F3N2O [M+H]+: m/z 295.0825. found 295.0823; 1H NMR: (500 MHz, CDCl3) δ 8.57 (d, J=5.6 Hz, 1H), 7.98 (s, 1H), 7.93 (d, J=2.1 Hz, 1H), 7.69 (dd, J=5.5, 2.0 Hz, 1H), 3.54 (t, J=6.5 Hz, 2H), 2.44 (t, J=7.4 Hz, 2H), 1.96-1.66 (m, 4H), 1.59-1.46 (m, 2H); 13C NMR: (125 MHz, CDCl3) δ 172.1, 150.8, 149.2 (q, J=35 Hz), 146.5, 121.4 (q, J=274 Hz), 115.5, 110.5 (q, J=3 Hz), 44.8, 37.4, 32.2, 26.3, 24.3.




4-amino-2-trifluoromethylpyridine-C6-4-bromophenoxyhybrid (S64): Prepared from 4-bromophenol and S63 using general procedure D to furnish S64 in a 24% yield. HRMS (ESI-TOF) calculated for C18H19BrF3N2O2 [M+H]+: m/z 431.0582. found 431.0580; 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J=5.4 Hz, 1H), 7.90 (s, 1H), 7.73-7.66 (m, 1H), 7.54 (s, 1H), 7.42-7.31 (m, 2H), 6.85-6.65 (m, 2H), 3.94 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.93-1.72 (m, 4H), 1.66-1.47 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.0, 158.0, 149.3 (q, J=34 Hz), 148.8, 146.3, 132.2, 121.3 (q, J=274 Hz), 116.2, 115.4, 112.7, 110.4 (q, J=3 Hz), 67.7, 37.5, 28.8, 25.6, 24.7.




4-amino-2-trifluoromethylpyridine-C6-2-bromophenoxyhybrid (S65): Prepared from 2-bromophenol and S63 using general procedure D to furnish S65 in a 38% yield. HRMS (ESI-TOF) calculated for C18H19BrF3N2O2 [M+H]+: m/z 431.0582. found 431.0562; 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J=5.5 Hz, 1H), 7.90 (s, 1H), 7.72 (s, 1H), 7.68 (dd, J=5.6, 2.1 Hz, 1H), 7.52 (dd, J=7.9, 1.6 Hz, 1H), 7.26-7.22 (m, 1H), 6.87 (dd, J=8.3, 1.4 Hz, 1H), 6.83 (td, J=7.6, 1.4 Hz, 1H), 4.04 (t, J=6.0 Hz, 2H), 2.48 (t, J=7.5 Hz, 2H), 1.93-1.80 (m, 4H), 1.68-1.58 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.0, 155.1, 150.9, 149.2 (q, J=35 Hz), 146.2, 133.3, 128.5, 121.8, 121.2 (q, J=274 Hz), 115.4, 113.1, 112.0, 110.4-110.3 (m), 68.7, 37.6, 28.6, 25.8, 24.7.




4-amino-2-trifluoromethylpyridine-C6-3-iodophenoxyhybrid (S66): Prepared from 3-iodophenol and S63 using general procedure D to furnish S66 in a 34% yield. HRMS (ESI-TOF) calculated for C18H19F3IN2O2 [M+H]+: m/z 479.0443. found 479.04262; 1H NMR (500 MHz, CDCl3) δ 8.59 (d, J=5.2 Hz, 1H), 7.92 (s, 1H), 7.77-7.58 (m, 2H), 7.29-7.25 (m, 1H), 7.24-7.20 (m, 1H), 6.98 (t, J=8.0 Hz, 1H), 6.84 (dd, J=8.4, 2.4 Hz, 1H), 3.93 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.87-1.76 (m, 4H), 1.60-1.50 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.0, 159.5, 150.9, 149.2 (q, J=34 Hz), 146.3, 130.8, 129.8, 123.6, 121.3 (q, J=274 Hz), 115.4, 114.1, 110.4 (q, J=3 Hz), 94.3, 67.6, 37.5, 28.8, 25.6, 24.7.




4-amino-2-trifluoromethylpyridine-C6-3-chlorophenoxyhybrid (S67): Prepared from 3-chlorophenol and S63 using general procedure D to furnish S67 in a 46% yield. HRMS (ESI-TOF) calculated for C18H19C1F3N2O2 [M+H]+: m/z 387.1087. found 387.1079; 1H NMR (500 MHz, CDCl3) δ 8.59 (d, J=5.5 Hz, 1H), 7.91 (s, 1H), 7.75-7.60 (m, 2H), 7.18 (t, J=8.1 Hz, 1H), 6.95-6.89 (m, 1H), 6.89-6.85 (m, 1H), 6.76 (dd, J=8.2, 2.4 Hz, 1H), 3.95 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.89-1.75 (m, 4H), 1.62-1.50 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.9, 159.6, 150.9, 149.3 (q, J=35 Hz), 146.2, 134.8, 130.2, 120.8, 121.3 (q, J=274 Hz), 115.4, 114.7, 112.9, 110.5-110.3 (m), 67.7, 37.5, 28.8, 25.6, 24.7.




4-amino-2-trifluoromethylpyridine-C6-4-fluorophenoxyhybrid (S68): Prepared from 4-fluorophenol and S63 using general procedure D to furnish S68 in a 17% yield. HRMS (ESI-TOF) calculated for C181-119F4N2O2 [M+H]+: m/z 371.1383. found 371.1364; 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J=5.5 Hz, 1H), 7.90-7.86 (m, 1H), 7.79-7.61 (m, 1H), 7.48 (s, 1H), 7.00-6.92 (m, 2H), 6.84-6.79 (m, 2H), 3.93 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.87-1.76 (m, 4H), 1.59-1.52 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.8, 158.1, 155.6 (d, J=152 Hz), 151.0, 149.3 (q, J=35 Hz), 146.0, 121.3 (q, J=274 Hz), 115.8 (d, J=23 Hz), 115.4-115.2 (m, 2C), 110.3 (q, J=3 Hz), 68.1, 37.6, 29.0, 25.7, 24.7.




4-amino-2-trifluoromethylpyridine-C6-2-fluorophenoxyhybrid (S69): Prepared from 2-fluorophenol and S63 using general procedure D to furnish S69 in a 37% yield. HRMS (ESI-TOF) calculated for C18H19F4N2O2 [M+H]+: m/z 371.1383. found 371.1377; 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J=5.5 Hz, 1H), 7.93-7.84 (m, 1H), 7.78-7.62 (m, 2H), 7.12-7.01 (m, 2H), 6.99-6.92 (m, 1H), 6.92-6.85 (m, 1H), 4.05 (t, J=6.1 Hz, 2H), 2.46 (t, J=7.5 Hz, 2H), 1.93-1.77 (m, 4H), 1.64-1.55 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.1, 152.6 (d, J=244 Hz), 151.0, 149.2 (q, J=35 Hz), 146.9 (d, J=11 Hz), 146.2, 124.4 (d, J=4 Hz), 121.3 (q, J=275 Hz), 121.0 (d, J=7 Hz), 116.1 (d, J=18 Hz), 115.4, 114.6 (d, J=2 Hz), 110.4 (q, J=3 Hz), 68.9, 37.5, 28.6, 25.7, 24.8.




4-amino-2-trifluoromethylpyridine-C6-2,4-bisfluorophenoxyhybrid (S70): Prepared from 2,4-bisfluorophenol and S63 using general procedure D to furnish S70 in a 49% yield. HRMS (ESI-TOF) calculated for C18H18F5N2O2 [M-ql]f: m/z 389.1288. found 389.1288; 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J=5.5 Hz, 1H), 7.91 (d, J=2.1 Hz, 1H), 7.81 (s, 1H), 7.68 (dd, J=5.6, 2.1 Hz, 1H), 6.93-6.81 (m, 2H), 6.80-6.74 (m, 1H), 4.00 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.89-1.77 (m, 4H), 1.62-1.53 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.0, 156.3 (dd, J=242, 10 Hz), 152.4 (dd, J=248, 12 Hz), 150.93, 149.2 (q, J=34 Hz), 146.2, 143.4 (dd, J=11, 4 Hz), 121.3 (q, J=274 Hz), 115.5-115.2 (m, 2C), 110.6-110.2 (m, 2C), 104.8 (dd, J=27, 22 Hz), 69.7, 37.5, 28.8, 25.6, 24.7.




4-amino-2-trifluoromethylpyridine-C6-2,6-bisfluorophenoxyhybrid (S71): Prepared from 2,6-bisfluorophenol and S63 using general procedure D to furnish S71 in a 47% yield. HRMS (ESI-TOF) calculated for C18H18F5N2O2 m/z 389.1288. found 389.1290; 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J=5.5 Hz, 1H), 7.89 (d, J=2.0 Hz, 1H), 7.74 (s, 1H), 7.69 (dd, J=5.6, 2.0 Hz, 1H), 7.00-6.92 (m, 1H), 6.91-6.82 (m, 2H), 4.13 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.92-1.76 (m, 4H), 1.64-1.56 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.1, 156.2 (dd, J=248, 6 Hz), 151.0, 149.2 (q, J=34 Hz), 146.2, 136.4-134.5 (m), 122.8 (t, J=9 Hz), 121.3 (q, J=274 Hz), 115.4, 112.5-111.9 (m), 110.3, 74.5, 37.6, 29.5, 25.3, 24.7.




4-amino-2-trifluoromethylpyridine-C6-perfluorophenoxyhybrid (S72): Prepared from pentafluorophenol and S63 using general procedure D to furnish S72 in a 57% yield. HRMS (ESI-TOF) calculated for C18H15F8N2O2 [M+H]: m/z 443.1006. found 443.1007; 1H NMR (500 MHz, CDCl3) δ 8.59 (d, J=5.5 Hz, 1H), 7.91 (d, J=2.1 Hz, 1H), 7.74 (s, 1H), 7.69 (dd, J=5.6, 2.1 Hz, 1H), 4.15 (t, J=6.2 Hz, 2H), 2.47 (t, J=7.4 Hz, 2H), 1.93-1.75 (m, 4H), 1.62-1.53 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.8, 151.0, 149.3 (q, J=35 Hz), 146.2, 121.3 (d, J=274 Hz), 115.3, 110.3 (q, J=3 Hz), 75.3, 37.4, 29.5, 25.2, 24.5. The aryl signals of the perfluoronated aryl phenol were obscured by the C—F splitting.




4-amino-2-trifluoromethylpyridine-C6-3-nitrophenoxyhybrid (S73): Prepared from 3-nitrophenol and S63 using general procedure D to furnish S73 in a 25% yield. HRMS (ESI-TOF) calculated for C18H19F3N3O4 [M+H]+: m/z 398.1328. found 398.1300; 1H NMR (500 MHz, CDCl3) δ 8.59 (d, J=5.5 Hz, 1H), 7.93 (s, 1H), 7.84-7.73 (m, 2H), 7.72-7.67 (m, 2H), 7.41 (t, J=8.2 Hz, 1H), 7.20 (dd, J=8.4, 2.5 Hz, 1H), 4.04 (t, J=6.2 Hz, 2H), 2.48 (t, J=7.4 Hz, 2H), 1.97-1.70 (m, 4H), 1.67-1.51 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.8, 159.4, 151.0, 149.2 (q, J=40.0 Hz), 149.1, 146.1, 130.0, 121.6, 121.3 (q, J=274 Hz), 115.7, 115.3, 110.3 (q, J=3 Hz), 108.6, 68.2, 37.5, 28.7, 25.6, 24.6.




4-amino-2-trifluoromethylpyridine-C6-3-cyanophenoxyhybrid (S74): Prepared from 3-cyanophenol and S63 using general procedure D to furnish S74 in a 22% yield. HRMS (ESI-TOF) calculated for C19H19F3N3O2 [M+H]+: m/z 378.1429. found 378.1413; 1H NMR (300 MHz, CD3OD) δ 8.52 (d, J=5.6 Hz, 1H), 8.11 (d, J=2.0 Hz, 1H), 7.77 (dd, J=5.6, 2.1 Hz, 1H), 7.49-7.37 (m, 1H), 7.31-7.14 (m, 3H), 4.04 (t, J=6.3 Hz, 2H), 2.48 (t, J=7.4 Hz, 2H), 1.92-1.72 (m, 4H), 1.65-1.49 (m, 2H); 13C NMR (125 MHz, acetone-d6) δ 173.4, 160.3, 151.9, 149.3 (q, J=34 Hz), 148.4, 131.6, 125.1, 132.2-118.7 (m), 120.9, 119.3, 118.0, 116.2, 114.2, 110.8-110.5 (m), 69.0, 37.7, 29.6, 26.3, 25.5.




4-amino-2-trifluoromethylpyridine-C6-3-trifluoromethylphenoxyhybrid (S75): Prepared from 3-trifluoromethylphenol and S63 using general procedure D to furnish S75 in a 16% yield. HRMS (ESI-TOF) calculated for C19H19F6N2O2 [M+H]+: m/z 421.1351. found 421.1353. 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J=5.5 Hz, 1H), 7.90 (d, J=2.1 Hz, 1H), 7.68 (dd, J=5.6, 2.1 Hz, 1H), 7.52 (s, 1H), 7.37 (t, J=8.0 Hz, 1H), 7.19 (d, J=7.7 Hz, 1H), 7.12-7.08 (m, 1H), 7.06-7.02 (m, 1H), 4.00 (t, J=6.2 Hz, 2H), 2.47 (t, J=7.4 Hz, 2H), 1.97-1.75 (m, 4H), 1.65-1.53 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 171.7, 159.0, 151.0, 149.3 (q, J=34 Hz), 146.1, 131.7 (q, J=32 Hz), 130.00, 125.7 (q, J=183 Hz), 121.3 (q, J=274 Hz), 117.9-117.8 (m), 117.3 (q, J=4 Hz), 115.3, 111.1 (q, J=4 Hz), 110.3 (d, J=3 Hz), 67.7, 37.5, 28.8, 25.6, 24.7.




4-amino-2-trifluoromethylpyridine-C6-3-methylphenoxyhybrid (S76): Prepared from 3-methylphenol and S63 using general procedure D to furnish S76 in a 30% yield. HRMS (ESI-TOF) calculated for C19H22F3N2O2 [M+1-1]′: m/z 367.1633. found 367.1618; 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J=5.4 Hz, 1H), 7.89 (d, J=2.0 Hz, 1H), 7.73-7.62 (m, 2H), 7.16 (t, J=7.8 Hz, 1H), 6.76 (d, J=7.6 Hz, 1H), 6.72-6.64 (m, 2H), 3.96 (t, J=6.2 Hz, 2H), 2.45 (t, J=7.4 Hz, 2H), 2.32 (s, 3H), 1.88-1.76 (m, 4H), 1.62-1.50 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.0, 158.8, 151.0, 149.3 (q, J=34 Hz), 146.1, 139.5, 129.2, 121.5, 121.3 (q, J=274 Hz), 115.9, 115.3, 111.2, 110.3 (q, J=3.0 Hz), 67.3, 37.6, 28.9, 25.7, 24.8, 21.5.




4-amino-2-trifluoromethylpyridine-C6-3-hydroxyphenoxyhybrid (S77): Prepared from 1,3-dihydroxybenzene and S63 using general procedure D to furnish S77 in a 4% yield. HRMS (ESI-TOF) calculated for C18H20F3N2O3 [M+H]+: m/z 369.1426. found 369.1398; 1H NMR (500 MHz, CD3OD) δ 8.52 (d, J=5.6 Hz, 1H), 8.11 (d, J=2.0 Hz, 1H), 7.78 (dd, J=5.6, 2.0 Hz, 1H), 7.02 (t, J=8.0 Hz, 1H), 6.42-6.28 (m, 3H), 3.94 (t, J=6.3 Hz, 2H), 2.48 (t, J=7.4 Hz, 2H), 1.87-1.71 (m, 4H), 1.64-1.48 (m, 2H); 13C NMR (125 MHz, CD3OD) δ 175.6, 161.9, 159.8, 152.0, 151.4-150.6 (m), 149.4, 131.0, 125.8-118.1 (m), 117.0, 111.6 (m), 108.8, 106.8, 102.9, 68.6, 38.1, 30.3, 27.0, 26.2.




4-amino-2-trifluoromethylpyridine-C6-2-hydroxyphenoxyhybrid (S78): Prepared from 1,2-dihydroxybenzene and S63 using general procedure D to furnish S78 in an 18% yield. HRMS (ESI-TOF) calculated for C18H20F3N2O3 [M+H]+: m/z 369.1426. found 369.1415; 1H NMR (500 MHz, CDCl3) δ 8.59 (d, J=5.5 Hz, 1H), 7.89 (d, J=2.0 Hz, 1H), 7.69 (dd, J=5.6, 2.1 Hz, 1H), 7.64 (s, 1H), 6.96-6.91 (m, 1H), 6.90-6.80 (m, 3H), 5.73 (s, 1H), 4.06 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.97-1.73 (m, 4H), 1.62-1.51 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.8, 151.0, 149.3 (q, J=34 Hz), 146.1, 145.7, 145.6, 125.0-117.3 (m) 121.4, 120.2, 115.4, 114.6, 111.6, 110.3 (q, J=3 Hz), 68.3, 37.4, 28.8, 25.6, 24.6.




4-amino-2-trifluoromethylpyridine-C6-3-fluoroarylsulfide hybrid (S79): Prepared from 3-fluorothiophenol and S63 using general procedure D to furnish S79 in a 55% yield. HRMS (ESI-TOF) calculated for C18H19F4N2OS [M+H]+: m/z 387.1154. found 387.1139; 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J=5.5 Hz, 1H), 7.90 (d, J=2.0 Hz, 1H), 7.76 (s, 1H), 7.67 (dd, J=5.6, 2.1 Hz, 1H), 7.25-7.19 (m, 1H), 7.07-7.01 (m, 1H), 6.97 (dt, J=9.6, 2.2 Hz, 1H), 6.84 (td, J=8.4, 2.5 Hz, 1H), 2.93 (t, J=7.2 Hz, 2H), 2.42 (t, J=7.4 Hz, 2H), 1.80-1.65 (m, 4H), 1.58-1.42 (m, 2H); NMR (125 MHz, CDCl3) δ 171.8, 162.8 (d, J=247 Hz), 150.9, 149.2 (q, J=34 Hz), 146.2, 139.1 (d, J=8 Hz), 130.1 (d, J=9 Hz), 123.8 (d, J=3 Hz), 121.3 (q, J=274 Hz), 115.4, 114.9 (d, J=23 Hz), 112.4 (d, J=24 Hz), 110.3 (q, J=3 Hz). 37.4, 32.8, 28.5, 28.1, 24.4.




4-amino-2-trifluoromethylpyridine-C6-3-fluoroaniline hybrid (S80): Prepared from 3-fluoroaniline and S63 using general procedure D to furnish S80 in a 23% yield. HRMS (ESI-TOF) calculated for C18H20F4N3O [M+H]+: m/z 370.1543. found 370.1528; 1H NMR (500 MHz, CDCl3) δ 8.59 (d, J=5.5 Hz, 1H), 7.90 (d, J=2.1 Hz, 1H), 7.67 (dd, J=5.5, 2.1 Hz, 1H), 7.58 (s, 1H), 7.12-7.04 (m, 1H), 6.41-6.32 (m, 2H), 6.27 (dt, J=11.7, 2.3 Hz, 1H), 3.79 (s, 1H), 3.11 (t, J=7.0 Hz, 2H), 2.44 (t, J=7.3 Hz, 2H), 1.79 (p, J=7.5 Hz, 2H), 1.71-1.61 (m, 2H), 1.53-1.43 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.8, 164.1 (d, J=243 Hz), 151.0, 150.0 (d, J=11 Hz), 149.3 (q, J=34 Hz), 146.1, 130.3 (d, J=10 Hz), 121.3 (q, J=274 Hz), 115.3, 110.3 (q, J=3 Hz), 108.6 (d, J=2 Hz), 103.6 (d, J=22 Hz), 99.1 (d, J=25 Hz), 43.4, 37.5, 29.0, 26.5, 24.7.




N-(2-(trifluoromethyl)pyridin-4-yl)hept-6-ynamide (S81): 6-Heptynoic acid (0.10 mL, 0.79 mmol, 1.0 equiv) was dissolved in CH2Cl2 (1.6 mL). Then oxalyl chloride (0.40 mL, 4.8 mmol, 6.1 equiv) was added dropwise. The reaction mixture was heated at reflux for 1 hour, or until complete. The reaction mixture was concentrated, and then dissolved in CH2Cl2 (5 mL). 4-Amino-2-(trifluoromethyl)pyridine (0.12 g, 0.79 mmol, 1.0 equiv) and pyridine (60 μL, 0.79 mmol, 1.0 equiv) were added to the solution. The reaction mixture was stirred for 12 h, and was then quenched with NH4Cl (10 mL). The aqueous layer was extracted with CH2Cl2 (3×10 mL). The combined organic layer was washed sequentially with 1 M HCl (2×10 mL) and brine (20 mL), dried over Na2SO4 and concentrated, providing S81 in a 75% yield over two steps. S81 was taken onward without further purification. HRMS (ESI-TOF) calculated for C13H14F3N2O [M+H]+: m/z 271.1058. found 271.1048; 1H NMR: (500 MHz, CDCl3) δ 8.54 (d, J=5.5 Hz, 1H), 8.51-8.32 (m, 1H), 7.97-7.93 (m, 1H), 7.71-7.67 (m, 1H), 2.45 (t, J=7.5 Hz, 2H), 2.27-2.18 (m, 2H), 1.99-1.93 (m, 1H), 1.84 (p, J=7.5 Hz, 2H), 1.58 (p, J=7.1 Hz, 2H); 13C NMR: (125 MHz, CDCl3) 172.3, 150.7, 149.0 (q, J=36 Hz), 146.6, 121.3 (q, J=274 Hz), 115.6, 110.6-110.4 (m), 83.7, 68.9, 36.9, 27.6, 24.0, 18.1.




7-(3-fluorophenyl)-N-(2-(trifluoromethyl)pyridin-4-yl)hept-6-ynamide (S82): S81 (40 mg, 0.15 mmol, 1.5 equiv), 1-bromo-3-fluorobenzene (12 pt, 0.10 mmol, 1.0 equiv), bis(triphenylphosphine)palladium(II) dichloride (4.0 mg, 5 mol %), triphenylphosphine (1.0 mg, 2.5 mol %), and triethylamine (20 μL, 0.15 mmol, 1.5 equiv) were dissolved in THF (1 mL). The reaction mixture was stirred for 20 minutes at room temperature, and then copper(I) iodide (1.0 mg, 2 mol %) was added. After 16 hours, the reaction mixture was filtered through Celite and concentrated. The crude product was purified using flash chromatography to furnish S82 in a 22% yield. HRMS (ESI-TOF) calculated for C19H17F4N2O [M+H]+: m/z 365.1277. found 365.1251; 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J=5.5 Hz, 1H), 7.87 (d, J=2.0 Hz, 1H), 7.74-7.61 (m, 2H), 7.26-7.20 (m, 1H), 7.18-7.13 (m, 1H), 7.10-7.04 (m, 1H), 7.02-6.95 (m, 1H), 2.53-2.44 (m, 4H), 1.92 (p, J=7.6 Hz, 2H), 1.80-1.59 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 171.7, 162.3 (d, J=246 Hz), 151.0, 149.3 (q, J=34 Hz), 146.1, 129.8 (d, J=9 Hz), 127.4, 125.4 (d, J=10 Hz), 121.3 (q, J=274 Hz), 118.3 (d, J=23 Hz), 115.3, 115.1 (d, J=21 Hz), 110.3 (q, J=3 Hz), 90.5, 80.1, 37.0, 27.7, 24.2, 19.1




7-(3-fluorophenyl)-N-(2-(trifluoromethyl)pyridin-4-yl)heptanamide (S83): S82 (12 mg, 0.033 mmol) was dissolved in methanol (1 mL). Palladium on carbon (2.0 mg, 10 wt %) was added to the solution, and a hydrogen balloon was added. After 12 h, the reaction mixture was filtered through Celite, concentrated, and purified using flash chromatography to give an 89% yield of S83. HRMS (ESI-TOF) calculated for C19H21F4N2O [M+H]+: m/z C19H21F4N2O. found 369.1578; 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J=5.5 Hz, 1H), 7.90 (d, J=2.1 Hz, 1H), 7.75-7.53 (m, 2H), 7.25-7.18 (m, 1H), 6.93 (d, J=7.4 Hz, 1H), 6.90-6.82 (m, 2H), 2.59 (t, J=7.7 Hz, 2H), 2.40 (t, J=7.5 Hz, 2H), 1.72 (p, J=7.5 Hz, 2H), 1.62 (p, J=7.7 Hz, 2H), 1.49-1.30 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 172.1, 162.8 (d, J=245 Hz), 151.0, 149.3 (q, J=35 Hz), 146.2, 145.1 (d, J=7 Hz), 129.6 (d, J=8 Hz), 124.0 (d, J=3 Hz), 121.3 (q, J=274 Hz), 115.2, 115.1 (d, J=21 Hz), 112.5 (d, J=21 Hz), 110.3 (q, J=3 Hz), 37.6, 35.5, 30.9, 28.9, 28.8, 24.9.







TABLE 7







Genes down-regulated by mBTL in the wild-type strain determined through microarray analysis.


The ratio of gene expression in untreated cells to that of mBTL treated cells (fold


down-regulation) is displayed for genes that exhibited changes greater than 2-fold.


Additional columns indicate the genes that are also downregulated in the lasR and rhlR


null strains. Ratios are the average of three independent experiments.













Gene


LasR-
RhlR-


Gene locus
name
Description
Ratio
regulated
regulated















PA14_20020
hasAp
heme acquisition protein HasAp
13.6
*
*


PA14_21530

ankyrin domain-containing protein
9.2
*
*


PA14_54840

tRNA-Gly
7.6
*
*


PA14_53300

alkyl hydroperoxide reductase
7.5
*
*


PA14_30720

tRNA-Cys
7.0
*
*


PA14_39420

hypothetical protein
6.6
*
*


PA14_07470

tRNA-Met
6.5
*
*


PA14_03090

hypothetical protein
6.5
*
*


PA14_39960
phzB2
phenazine biosynthesis protein
6.2

*


PA14_26990

hypothetical protein
5.9
*
*


PA14_14570

tRNA-Leu
5.7
*
*


PA14_58710

tRNA-Thr
5.7
*
*


PA14_27190

tRNA-Ser
5.6
*
*


PA14_39970
phzA2
phenazine biosynthesis protein
5.6

*


PA14_30420

tRNA-Ser
5.5
*
*


PA14_62790

tRNA-Met
5.5
*
*


PA14_51830

DNA-binding stress protein
5.5
*
*


PA14_52320

tRNA-Met
5.5
*
*


PA14_24870

tRNA-Pro
5.4
*
*


PA14_51230

tRNA-Ser
5.4
*
*


PA14_09150
katA
catalase
5.3
*
*


PA14_68150

tRNA-Thr
5.0
*
*


PA14_06810
norC
nitric-oxide reductase subunit C
4.9
*
*


PA14_61830

tRNA-Met
4.9
*
*


PA14_60160

tRNA-Pro
4.8
*
*


PA14_22320

hypothetical protein
4.8
*
*


PA14_18590

hypothetical protein
4.7
*
*


PA14_51420
pqsB
PqsB
4.6


PA14_41330

tRNA-His
4.5
*
*


PA14_27330

phospho-2-dehydro-3-deoxyheptonate aldolase
4.3
*
*


PA14_28190

tRNA-Val
4.1
*
*


PA14_05860

hypothetical protein
4.0
*
*


PA14_18800

hypothetical protein
4.0
*
*


PA14_06830
norB
nitric-oxide reductase subunit B
3.9
*
*


PA14_23570

tRNA-Ala
3.8
*
*


PA14_06875

rsmYregulatoryRNA
3.7
*
*


PA14_01490

hemolysin
3.7

*


PA14_48060
aprA
alkaline metalloproteinase
3.6
*
*


PA14_28660
infC
translation initiation factor IF-3
3.5


PA14_01710
ahpC
alkyl hydroperoxide reductase
3.4
*
*


PA14_36330
hcnA
hydrogen cyanide synthase HcnA
3.4

*


PA14_20500

tRNA-Arg
3.4
*
*


PA14_51410
pqsC
PqsC
3.3


PA14_28740

tRNA-Pro
3.3

*


PA14_13950

hypothetical protein
3.2


PA14_50290
fliC
flagellin type B
3.2


PA14_08900
rplV
50S ribosomal protein L22
3.2


PA14_08220

hypothetical protein
3.2


PA14_58040

hypothetical protein
3.1
*
*


PA14_15990
trmD
tRNA (guanine-N(1)-)-methyltransferase
3.1


PA14_01720
ahpF
alkyl hydroperoxide reductase
3.1
*
*


PA14_51610

hypothetical protein
3.1


PA14_53290
trxB2
thioredoxin reductase 2
3.1
*
*


PA14_09470
phzB1
phenazine biosynthesis protein
3.1

*


PA14_26980

hypothetical protein
3.0
*
*


PA14_63460

tRNA-Sec
3.0

*


PA14_41340

tRNA-Arg
3.0
*
*


PA14_28720
ihfA
integration host factor subunit alpha
3.0


PA14_08930
rpmC
50S ribosomal protein L29
3.0


PA14_59230

colicin immunity protein
3.0


PA14_39470

hypothetical protein
3.0


PA14_69200
trxA
thioredoxin
3.0
*
*


PA14_61040
katB
catalase
2.9
*
*


PA14_60480

hypothetical protein
2.9


PA14_08880
rplB
50S ribosomal protein L2
2.9


PA14_14560

hypothetical protein
2.9


PA14_08660

tRNA-Gly
2.9
*
*


PA14_36320
hcnB
hydrogen cyanide synthase HcnB
2.8

*


PA14_03410

tRNA-Arg
2.8
*
*


PA14_28670
rpmI
50S ribosomal protein L35
2.8


PA14_53610

hypothetical protein
2.8


PA14_09130
rplQ
50S ribosomal protein L17
2.7


PA14_07680

hypothetical protein
2.7


PA14_41320

tRNA-Leu
2.7
*


PA14_68030

tRNA-Phe
2.7
*
*


PA14_62680

hypothetical protein
2.7


PA14_38180

hypothetical protein
2.7


PA14_10230
adh
2,3-butanediol dehydrogenase
2.6


PA14_58010

hypothetical protein
2.6


PA14_20290

DNA binding-protein
2.6


PA14_09080
rpsM
30S ribosomal protein S13
2.6


PA14_49520
pyoS3A
pyocin killing protein
2.6


PA14_55940

hypothetical protein
2.6
*
*


PA14_70190
rpmB
50S ribosomal protein L28
2.6


PA14_09540
mexG
hypothetical protein
2.6

*


PA14_58000
sodM
superoxide dismutase
2.5


PA14_51380
pqsE
quinolone signal response protein
2.5


PA14_35840

hypothetical protein
2.5


PA14_18690

peroxidase
2.5


PA14_49030

hypothetical protein
2.5


PA14_28680
rplT
50S ribosomal protein L20
2.5


PA14_48140

hypothetical protein
2.5
*
*


PA14_23410
orfJ
glycosyl transferase family protein
2.5


PA14_58030
fumC
fumarate hydratase
2.5


PA14_18070

periplasrnic metal-binding protein
2.5
*
*


PA14_51390
pqsD
3-oxoacyl-ACP synthase
2.5


PA14_23330
rpsA
30S ribosomal protein S1
2.5


PA14_24650
rmf
ribosome modulation factor
2.5


PA14_14300

zinc-binding oxidoreductase
2.5


PA14_49470
nrdB
ribonucleotide-diphosphate reductase subunit beta
2.5


PA14_57460

cell division protein MraZ
2.5


PA14_08820
fusA1
elongation factor G
2.4


PA14_17480
rpoS
RNA polymerase sigma factor RpoS
2.4
*


PA14_09090
rpsK
30S ribosomal protein S11
2.4


PA14_35160

hypothetical protein
2.4

*


PA14_62690

hypothetical protein
2.4
*


PA14_19590

molybdopterin-binding protein
2.4

*


PA14_57950

hypothetical protein
2.4


PA14_23360
wzz
O-antigen chain length regulator
2.4


PA14_20050

outer membrane protein
2.4
*


PA14_41230
clpX
ATP-dependent protease ATP-binding subunit ClpX
2.4


PA14_59430

hypothetical protein
2.4


PA14_50880

hypothetical protein
2.4


PA14_06750
nirS
nitrite reductase
2.4
*
*


PA14_49510
pyoS3I
immunity protein S3I structureal gene
2.4


PA14_64500

transcriptional regulator
2.4
*
*


PA14_28780

hypothetical protein
2.3


PA14_30210
clpS
ATP-dependent Clp protease adaptor protein ClpS
2.3


PA14_69370
algP
alginate regulatory protein AlgP
2.3


PA14_41570
oprF
major porin and structural outer membrane porin OprF
2.3




precursor


PA14_09270
pchE
dihydroaeruginoic acid synthetase
2.3

*


PA14_08790
rpsL
30S ribosomal protein S12
2.3


PA14_35760

hypothetical protein
2.3


PA14_53590

hypothetical protein
2.3


PA14_19110
rhlB
rhamnosyltransferase chain B
2.3

*


PA14_13940

S-type pyocin protein
2.3


PA14_35850

hypothetical protein
2.3


PA14_08810
rpsG
30S ribosomal protein S7
2.3


PA14_09340
fptA
Fe(III)-pyochelin outer membrane receptor
2.3


PA14_65180
rpsF
30S ribosomal protein S6
2.2


PA14_48040
aprI
alkaline proteinase inhibitor AprI
2.2
*
*


PA14_68940

hypothetical protein
2.2

*


PA14_51430
pqsA
coenzyme A ligase
2.2


PA14_29570

hypothetical protein
2.2


PA14_08720
rplK
50S ribosomal protein L11
2.2


PA14_08910
rpsC
30S ribosomal protein S3
2.2


PA14_59400

hypothetical protein
2.2


PA14_09210
pchA
salicylate biosynthesis isochorismate synthase
2.2


PA14_59610

hypothetical protein
2.2


PA14_59190

hypothetical protein
2.2


PA14_14750

iron-binding protein IscA
2.2

*


PA14_56070
mvaT
transcriptional regulator MvaT, P16 subunit
2.2


PA14_08690

tRNA-Trp
2.2

*


PA14_19100
rhlA
rhamnosyltransferase chain A
2.2

*


PA14_33750

outer membrane protein
2.2
*
*


PA14_08840
rpsJ
30S ribosomal protein S10
2.2


PA14_45960
lasR
transcriptional regulator LasR
2.2
*


PA14_60960

hypothetical protein
2.2


PA14_68340
arcB
ornithine carbamoyltransferase
2.2


PA14_19490

antioxidant protein
2.2

*


PA14_61760

tRNA-Gln
2.2
*
*


PA14_25630
rpmF
50S ribosomal protein L32
2.2


PA14_23400

hypothetical protein
2.2


PA14_70180
rpmG
50S ribosomal protein L33
2.2


PA14_51810

hypothetical protein
2.2


PA14_08730
rplA
50S ribosomal protein L1
2.2


PA14_23390
orfE
polysaccharide biosynthesis protein
2.2


PA14_30190
icd
isocitrate dehydrogenase
2.2


PA14_15850

hypothetical protein
2.2


PA14_10560

hypothetical protein
2.2

*


PA14_23340
ihfB
integration host factor subunit beta
2.2


PA14_53070
hpd
4-hydroxyphenylpyruvate dioxygenase
2.2


PA14_20010
hasR
heme uptake outer membrane receptor HasR
2.2


PA14_41250
tig
trigger factor
2.2


PA14_08850
rplC
50S ribosomal protein L3
2.2


PA14_08960
rplX
50S ribosomal protein L24
2.2


PA14_51350
phnB
anthranilate synthase component II
2.2


PA14_09115
rpoA
DNA-directed RNA polymerase subunit alpha
2.2


PA14_59390

hypothetical protein
2.1

*


PA14_67190

hypothetical protein
2.1


PA14_37520

hypothetical protein
2.1


PA14_61380

hypothetical protein
2.1
*


PA14_17440
truD
tRNA pseudouridine synthase D
2.1


PA14_20530

hypothetical protein
2.1


PA14_08890
rpsS
30S ribosomal protein S19
2.1


PA14_14730
iscS
cysteine desulfurase
2.1


PA14_73300
atpE
F0F1 ATP synthase subunit C
2.1


PA14_54490

hypothetical protein
2.1
*


PA14_09480
phzA1
phenazine biosynthesis protein
2.1


PA14_12260

hypothetical protein
2.1


PA14_59620

hypothetical protein
2.1


PA14_28710
pheT
phenylalanyl-tRNA synthetase subunit beta
2.1


PA14_20610
lecB
fucose-binding lectin PA-IIL
2.1

*


PA14_08970
rplE
50S ribosomal protein L5
2.1


PA14_56780
sodB
superoxide dismutase
2.1


PA14_31350

hypothetical protein
2.1

*


PA14_16830

hypothetical protein
2.1


PA14_52990
phhA
phenylalanine 4-monooxygenase
2.1


PA14_49200
oprH
PhoP/Q and low Mg2+ inducible outer membrane prote
2.1


PA14_53340

hypothetical protein
2.1


PA14_49860

hypothetical protein
2.1


PA14_08750
rplL
50S ribosomal protein L7/L12
2.1


PA14_13460

RNA polymerase sigma factor
2.1
*
*


PA14_07480

reverse transcriptase
2.0


PA14_08870
rplW
50S ribosomal protein L23
2.0


PA14_09220
pchB
isochorismate-pyruvate lyase
2.0


PA14_59590

hypothetical protein
2.0


PA14_62720
rpsO
30S ribosomal protein S15
2.0
*
*


PA14_66875
phaF
polyhydroxyalkanoate synthesis protein PhaF
2.0


PA14_04300

hypothetical protein
2.0


PA14_37420

transmembrane sensor protein
2.0


PA14_14740

scaffold protein
2.0

*


PA14_55810

two-component response regulator
2.0


PA14_41210
hupB
DNA-binding protein HU
2.0

*


PA14_10490

hypothetical protein
2.0

*


PA14_16910

hypothetical protein
2.0


PA14_57010
groEL
chaperonin GroEL
2.0


PA14_02260

two-component response regulator
2.0
*


PA14_50280

hypothetical protein
2.0


PA14_09000
rplF
50S ribosomal protein L6
2.0


PA14_66620
pilQ
type 4 fimbrial biogenesis outer membrane protein PilQ
2.0




precursor


PA14_59380

hypothetical protein
2.0


PA14_09020
rpsE
30S ribosomal protein S5
2.0
















TABLE 8







Genes down-regulated by disruption of the lasR gene determined through


microarray analysis. The ratio of gene expression of the wild-type strain to


that of the lasR null strain (fold down-regulation) is displayed for genes


that exhibited changes greater than 2-fold. Ratios are the average


of three independent experiments.











Gene




Gene locus
name
Description
Ratio













PA14_45960
lasR
transcriptional regulator LasR
81.7


PA14_06810
norC
nitric-oxide reductase subunit C
74.5


PA14_22320

hypothetical protein
52.0


PA14_20020
hasAp
heme acquisition protein HasAp
34.9


PA14_21530

ankyrin domain-containing protein
33.1


PA14_33290

hypothetical protein
28.1


PA14_45950
rsaL
regulatory protein RsaL
23.4


PA14_01720
ahpF
alkyl hydroperoxide reductase
23.3


PA14_26990

hypothetical protein
21.5


PA14_53290
trxB2
thioredoxin reductase 2
20.9


PA14_09150
katA
catalase
16.7


PA14_03090

hypothetical protein
15.8


PA14_53300

alkyl hydroperoxide reductase
15.3


PA14_06830
norB
nitric-oxide reductase subunit B
15.3


PA14_40260

hypothetical protein
12.8


PA14_01710
ahpC
alkyl hydroperoxide reductase
12.8


PA14_52320

tRNA-Met
12.4


PA14_62790

tRNA-Met
12.3


PA14_10380

hypothetical protein
11.8


PA14_58710

tRNA-Thr
11.2


PA14_39420

hypothetical protein
11.1


PA14_06875

rsmYregulatoryRNA
11.1


PA14_68150

tRNA-Thr
11.0


PA14_18590

hypothetical protein
9.7


PA14_51230

tRNA-Ser
8.7


PA14_30420

tRNA-Ser
8.7


PA14_05860

hypothetical protein
8.6


PA14_72370

hypothetical protein
7.9


PA14_27330

phospho-2-dehydro-3-deoxyheptonate aldolase
7.5


PA14_55940

hypothetical protein
7.3


PA14_20500

tRNA-Arg
7.3


PA14_07470

tRNA-Met
7.2


PA14_39780

hypothetical protein
6.8


PA14_48060
aprA
alkaline metalloproteinase
6.4


PA14_54840

tRNA-Gly
6.3


PA14_28190

tRNA-Val
6.2


PA14_41340

tRNA-Arg
5.9


PA14_24870

tRNA-Pro
5.7


PA14_51830

DNA-binding stress protein
5.7


PA14_18800

hypothetical protein
5.6


PA14_61040
katB
catalase
5.4


PA14_14570

tRNA-Leu
5.2


PA14_09900
prpL
Pvds-regulated endoprotease, lysyl class
5.1


PA14_26980

hypothetical protein
5.0


PA14_03410

tRNA-Arg
4.8


PA14_52560

tRNA-Ser
4.7


PA14_60160

tRNA-Pro
4.4


PA14_23570

tRNA-Ala
4.1


PA14_64500

transcriptional regulator
4.0


PA14_36820

hypothetical protein
4.0


PA14_08670

tRNA-Thr
4.0


PA14_41330

tRNA-His
4.0


PA14_40240

ABC transporter ATP-binding protein/permease
4.0


PA14_36560

hypothetical protein
3.9


PA14_01290
coxB
cytochrome c oxidase subunit II
3.9


PA14_27190

tRNA-Ser
3.8


PA14_48040
aprI
alkaline proteinase inhibitor AprI
3.8


PA14_40230

secretion protein
3.8


PA14_16720

hypothetical protein
3.7


PA14_35340

2-ketogluconate kinase
3.7


PA14_61380

hypothetical protein
3.7


PA14_28600

hypothetical protein
3.6


PA14_48610

sparagine synthase
3.6


PA14_72360

hypothetical protein
3.6


PA14_61760

tRNA-Gln
3.5


PA14_13390

hypothetical protein
3.5


PA14_17980
glpF
glycerol uptake facilitator protein
3.5


PA14_22100

hypothetical protein
3.5


PA14_01780

nucleoside 2-deoxyribosyltransferase
3.5


PA14_49130
dctA
C4-dicarboxylate transporter DctA
3.4


PA14_72060

hypothetical protein
3.3


PA14_24860
snr1
cytochrome c Snr1
3.3


PA14_46750

hypothetical protein
3.3


PA14_62720
rpsO
30S ribosomal protein S15
3.2


PA14_01300
coxA
cytochrome c oxidase subunit I
3.2


PA14_28620

hypothetical protein
3.2


PA14_56910

hypothetical protein
3.1


PA14_66880

hypothetical protein
3.1


PA14_55790

hypothetical protein
3.0


PA14_06750
nirS
nitrite reductase
3.0


PA14_61830

tRNA-Met
3.0


PA14_01320
coIII
cytochrome c oxidase subunit III
2.9


PA14_01350

hypothetical protein
2.9


PA14_00300
plcB
phospholipase C, PlcB
2.8


PA14_62810
secG
preprotein translocase subunit SecG
2.7


PA14_22880

Fe—S protein
2.7


PA14_13350

hypothetical protein
2.7


PA14_13460

RNA polymerase sigma factor
2.7


PA14_62870
rrmJ
cell division protein FtsJ
2.7


PA14_68030

tRNA-Phe
2.7


PA14_69200
trxA
thioredoxin
2.7


PA14_53250
cpbD
chitin-binding protein CbpD
2.7


PA14_58040

hypothetical protein
2.6


PA14_57030
fxsA
FxsA protein
2.6


PA14_08660

tRNA-Gly
2.6


PA14_53630

hypothetical protein
2.6


PA14_17480
rpoS
RNA polymerase sigma factor RpoS
2.6


PA14_33050

hypothetical protein
2.6


PA14_54490

hypothetical protein
2.5


PA14_02500
exbB1
transport protein ExbB
2.5


PA14_20050

outer membrane protein
2.5


PA14_62860
ftsH
cell division protein FtsH
2.5


PA14_06770
nirQ
regulatory protein NirQ
2.5


PA14_53840

hypothetical protein
2.5


PA14_38550
maiA
maleylacetoacetate isomerase
2.5


PA14_00790
prlC
oligopeptidase A
2.5


PA14_18070

periplasmic metal-binding protein
2.5


PA14_07890

ABC transporter permease
2.4


PA14_38825
pqqA
coenzyme PQQ synthesis protein PqqA
2.4


PA14_55920

type II secretion system protein
2.4


PA14_13380

hypothetical protein
2.4


PA14_20770

hypothetical protein
2.4


PA14_22310

hypothetical protein
2.4


PA14_63170

transcriptional regulator
2.4


PA14_47190
cyoB
cytochrome o ubiquinol oxidase subunit I
2.3


PA14_02510
exbD1
transport protein ExbD
2.3


PA14_67770
pgm
phosphoglyceromutase
2.3


PA14_62690

hypothetical protein
2.3


PA14_26020

aminopeptidase
2.3


PA14_61060

oxidoreductase
2.3


PA14_30720

tRNA-Cys
2.3


PA14_48140

hypothetical protein
2.3


PA14_36920

hypothetical protein
2.3


PA14_25180
psrA
transcriptional regulator PsrA
2.3


PA14_58070

hypothetical protein
2.3


PA14_53160

hypothetical protein
2.3


PA14_42200

hypothetical protein
2.3


PA14_35980

acyl-CoA dehydrogenase
2.3


PA14_49050

hypothetical protein
2.3


PA14_12130
lis
lipoyl synthase
2.3


PA14_40250

outer membrane protein
2.3


PA14_60750
pra
protein activator
2.3


PA14_05020

hypothetical protein
2.2


PA14_13370

hypothetical protein
2.2


PA14_23680
ibpA
heat-shock protein IbpA
2.2


PA14_20480

hypothetical protein
2.2


PA14_11510
ribA
GTP cyclohydrolase II
2.2


PA14_53210

hypothetical protein
2.2


PA14_41320

tRNA-Leu
2.2


PA14_35360

hypothetical protein
2.1


PA14_36980

hypothetical protein
2.1


PA14_72870

aminotransferase
2.1


PA14_20780

two-component response regulator
2.1


PA14_63480

amino acid permease
2.1


PA14_54430
algU
RNA polymerase sigma factor AlgU
2.1


PA14_33060

hypothetical protein
2.1


PA14_28140

hypothetical protein
2.1


PA14_72170

citrate transporter
2.1


PA14_53040

lysozyme inhibitor
2.1


PA14_11610

ABC transporter permease
2.1


PA14_02260

two-component response regulator
2.1


PA14_61020

hypothetical protein
2.1


PA14_19870
ldh
leucine dehydrogenase
2.1


PA14_72970
tonB
TonB protein
2.1


PA14_33750

outer membrane protein
2.1


PA14_34330

hypothetical protein
2.1


PA14_00480

hypothetical protein
2.1


PA14_60500
fklB
peptidyl-prolyl cis-trans isomerase FklB
2.1


PA14_09700

monooxygenase
2.1


PA14_16250
lasB
elastase LasB
2.0


PA14_49460
nrdA
ribonucleotide-diphosphate reductase subunit alpha
2.0


PA14_72140

hypothetical protein
2.0


PA14_35370
ptxS
transcriptional regulator PtxS
2.0


PA14_66550
hemE
uroporphyrinogen decarboxylase
2.0


PA14_12030

hypothetical protein
2.0


PA14_70490

lipoprotein
2.0


PA14_38060

hypothetical protein
2.0


PA14_73230
atpC
F0F1 ATP synthase subunit epsilon
2.0


PA14_72770

hypothetical protein
2.0


PA14_47530

hypothetical protein
2.0


PA14_66400

potassium efflux protein KefA
2.0


PA14_37410

hypothetical protein
2.0


PA14_44700
alkB2
alkane-1 monooxygenase
2.0
















TABLE 9







Genes down-regulated by disruption of the rhlR gene determined


through microarray analysis. The ratio of gene expression of the


wild-type strain to that of the rhlR null strain (fold down-regulation)


is displayed for genes that exhibited changes greater than 2-fold.


Ratios are the average of three independent experiments.











Gene




Gene locus
name
Description
Ratio













PA14_10360

hypothetical protein
54.5


PA14_22320

hypothetical protein
47.2


PA14_34870
chiC
chitinase
44.6


PA14_15350

integrase
43.8


PA14_18800

hypothetical protein
42.7


PA14_15600

hypothetical protein
35.7


PA14_01720
ahpF
alkyl hydroperoxide reductase
34.7


PA14_53250
cpbD
chitin-binding protein CbpD
33.7


PA14_21530

ankyrin domain-containing protein
32.7


PA14_20610
lecB
fucose-binding lectin PA-IIL
29.6


PA14_53290
trxB2
thioredoxin reductase 2
26.8


PA14_19100
rhlA
rhamnosyltransferase chain A
24.4


PA14_06810
norC
nitric-oxide reductase subunit C
23.6


PA14_01490

hemolysin
23.2


PA14_15480
merR
transcriptional regulator MerR
23.1


PA14_15610

hypothetical protein
22.7


PA14_26990

hypothetical protein
22.6


PA14_53300

alkyl hydroperoxide reductase
19.6


PA14_11140

nonribosomal peptide synthetase
18.4


PA14_09540
mexG
hypothetical protein
16.3


PA14_01710
ahpC
alkyl hydroperoxide reductase
16.0


PA14_40310

acyl carrier protein
15.9


PA14_03090

hypothetical protein
15.6


PA14_28360

hypothetical protein
15.2


PA14_38270

hypothetical protein
15.1


PA14_37745

carbamoyl transferase
14.3


PA14_16250
lasB
elastase LasB
14.2


PA14_06830
norB
nitric-oxide reductase subunit B
13.9


PA14_09150
katA
catalase
13.6


PA14_15560

hypothetical protein
11.1


PA14_51230

tRNA-Ser
10.5


PA14_09530
mexH
RND efflux membrane fusion protein
10.3


PA14_05860

hypothetical protein
9.9


PA14_54840

tRNA-Gly
9.8


PA14_03510

hypothetical protein
9.7


PA14_68940

hypothetical protein
9.7


PA14_39420

hypothetical protein
9.2


PA14_31290
pa1L
PA-I galactophilic lectin
9.0


PA14_19590

molybdopterin-binding protein
8.7


PA14_16100

hypothetical protein
8.4


PA14_68150

tRNA-Thr
8.2


PA14_52320

tRNA-Met
8.1


PA14_62790

tRNA-Met
7.7


PA14_15520
trbJ
conjugal transfer protein TrbJ
7.3


PA14_40290
lasA
LasA protease
7.2


PA14_30720

tRNA-Cys
7.1


PA14_58710

tRNA-Thr
6.6


PA14_03520

hypothetical protein
6.4


PA14_31360

hypothetical protein
6.4


PA14_24870

tRNA-Pro
6.2


PA14_15580

Type II restriction enzyme, methylase subunit
6.2


PA14_32950

hypothetical protein
6.1


PA14_51830

DNA-binding stress protein
6.1


PA14_53840

hypothetical protein
6.0


PA14_18590

hypothetical protein
5.8


PA14_34880

GntR family transcriptional regulator
5.6


PA14_28190

tRNA-Val
5.5


PA14_49760
rhlC
rhamnosyltransferase 2
5.5


PA14_10490

hypothetical protein
5.4


PA14_68210
rmlC
dTDP-4-dehydrorhamnose 3,5-epimerase
5.4


PA14_59390

hypothetical protein
5.3


PA14_30420

tRNA-Ser
5.3


PA14_21030

ATP-dependent Clp protease proteolytic subunit
5.3


PA14_09520
mexI
RND efflux transporter
5.2


PA14_41330

tRNA-His
5.2


PA14_55940

hypothetical protein
5.1


PA14_63210

two-component response regulator
5.0


PA14_20500

tRNA-Arg
4.9


PA14_27330

phospho-2-dehydro-3-deoxyheptonate aldolase
4.9


PA14_16720

hypothetical protein
4.9


PA14_34750

taurine catabolism dioxygenase
4.9


PA14_15400

replication protein, RepC
4.9


PA14_23570

tRNA-Ala
4.9


PA14_49130
dctA
C4-dicarboxylate transporter DctA
4.8


PA14_20020
hasAp
heme acquisition protein HasAp
4.7


PA14_15360

hypothetical protein
4.7


PA14_56590

hypothetical protein
4.6


PA14_15460
merA
mercuric reductase
4.6


PA14_48040
aprI
alkaline proteinase inhibitor AprI
4.5


PA14_27190

tRNA-Ser
4.5


PA14_61040
katB
catalase
4.4


PA14_68190
rmlD
dTDP-4-dehydrorhamnose reductase
4.3


PA14_11130

short chain dehydrogenase
4.3


PA14_36330
hcnA
hydrogen cyanide synthase HcnA
4.3


PA14_31350

hypothetical protein
4.2


PA14_41340

tRNA-Arg
4.2


PA14_36320
hcnB
hydrogen cyanide synthase HcnB
4.2


PA14_52560

tRNA-Ser
4.1


PA14_09500
opmD
outer membrane protein
4.0


PA14_19110
rhlB
rhamnosyltransferase chain B
4.0


PA14_61830

tRNA-Met
4.0


PA14_26980

hypothetical protein
3.9


PA14_34780

ABC transporter ATP-binding protein
3.9


PA14_09470
phzB1
phenazine biosynthesis protein
3.9


PA14_60160

tRNA-Pro
3.9


PA14_06875

rsmYregulatoryRNA
3.9


PA14_18680

hypothetical protein
3.9


PA14_03410

tRNA-Arg
3.8


PA14_15590

hypothetical protein
3.8


PA14_13460

RNA polymerase sigma factor
3.8


PA14_39960
phzB2
phenazine biosynthesis protein
3.8


PA14_43040

hypothetical protein
3.8


PA14_02500
exbB1
transport protein ExbB
3.8


PA14_61060

oxidoreductase
3.6


PA14_62390

hypothetical protein
3.6


PA14_15510
traJ
conjugal transfer relaxosome component TraJ
3.6


PA14_17980
glpF
glycerol uptake facilitator protein
3.6


PA14_15500

oriT-binding protein, Trak
3.6


PA14_50740

hypothetical protein
3.6


PA14_45950
rsaL
regulatory protein RsaL
3.5


PA14_15450
merD
transcriptional regulator MerD
3.5


PA14_19540

hypothetical protein
3.5


PA14_48140

hypothetical protein
3.5


PA14_40860

hypothetical protein
3.4


PA14_03490

hypothetical protein
3.4


PA14_33290

hypothetical protein
3.3


PA14_15490

hypothetical protein
3.3


PA14_68170
rmlB
dTDP-D-glucose 4,6-dehydratase
3.3


PA14_00640
phzH
potential phenazine-modifying enzyme
3.3


PA14_62860
ftsH
cell division protein FtsH
3.3


PA14_55580
nemO
heme oxygenase
3.3


PA14_69200
trxA
thioredoxin
3.3


PA14_35160

hypothetical protein
3.3


PA14_31170

hypothetical protein
3.3


PA14_72060

hypothetical protein
3.3


PA14_66460

hypothetical protein
3.2


PA14_14570

tRNA-Leu
3.2


PA14_07470

tRNA-Met
3.1


PA14_30620

AraC family transcriptional regulator
3.1


PA14_38825
pqqA
coenzyme PQQ synthesis protein PqqA
3.1


PA14_64930

hypothetical protein
3.1


PA14_61760

tRNA-Gln
3.1


PA14_10350

secretion protein
3.1


PA14_53040

lysozyme inhibitor
3.1


PA14_02510
exbD1
transport protein ExbD
3.0


PA14_19530

NAD(P)H-dependent FMN reductase
3.0


PA14_28050

chemotaxis transducer
3.0


PA14_72970
tonB
TonB protein
3.0


PA14_62810
secG
preprotein translocase subunit SecG
2.9


PA14_25180
psrA
transcriptional regulator PsrA
2.9


PA14_35170

redox-sensing activator of soxS
2.9


PA14_08670

tRNA-Thr
2.9


PA14_66550
hemE
uroporphyrinogen decarboxylase
2.9


PA14_58040

hypothetical protein
2.9


PA14_06750
nirS
nitrite reductase
2.9


PA14_41210
hupB
DNA-binding protein HU
2.9


PA14_00790
prlC
oligopeptidase A
2.8


PA14_68930

permease
2.8


PA14_62720
rpsO
30S ribosomal protein S15
2.8


PA14_34730

XRE family transcriptional regulator
2.8


PA14_57030
fxsA
FxsA protein
2.8


PA14_64500

transcriptional regulator
2.8


PA14_09490
phzM
phenazine-specific methyltransferase
2.8


PA14_53160

hypothetical protein
2.8


PA14_32520

hypothetical protein
2.8


PA14_14710

Rrf2 family protein
2.8


PA14_06770
nirQ
regulatory protein NirQ
2.7


PA14_12970
tauD
taurine dioxygenase
2.7


PA14_23980
xcpP
secretion protein XcpP
2.7


PA14_24860
snr1
cytochrome c Snr1
2.7


PA14_43050

hypothetical protein
2.7


PA14_38550
maiA
maleylacetoacetate isomerase
2.7


PA14_34770

ABC transporter substrate-binding protein
2.7


PA14_30410

hypothetical protein
2.7


PA14_19490

antioxidant protein
2.6


PA14_26780

hypothetical protein
2.6


PA14_39800

ECF subfamily RNA polymerase sigma-70 factor
2.6


PA14_30670
pgsA
CDP-diacylglycerol--glycerol-3-phosphate 3-
2.6




phosphatidyltransferase


PA14_10330

outer membrane protein
2.6


PA14_06180

RNA polymerase sigma factor
2.6


PA14_12920

taurine ABC transporter periplasmic protein
2.6


PA14_28620

hypothetical protein
2.6


PA14_60030

hypothetical protein
2.6


PA14_40260

hypothetical protein
2.6


PA14_39780

hypothetical protein
2.6


PA14_36820

hypothetical protein
2.6


PA14_71900

hypothetical protein
2.5


PA14_34460

hypothetical protein
2.5


PA14_19520

ABC transporter ATP-binding protein
2.5


PA14_13210

hypothetical protein
2.5


PA14_09270
pchE
dihydroaeruginoic acid synthetase
2.5


PA14_08660

tRNA-Gly
2.5


PA14_06130

hypothetical protein
2.5


PA14_05020

hypothetical protein
2.5


PA14_62800

tRNA-Leu
2.5


PA14_31370

hypothetical protein
2.5


PA14_15370

hypothetical protein
2.5


PA14_60750
pra
protein activator
2.5


PA14_37470

flavin-dependent oxidoreductase
2.5


PA14_03080

acetyltransferase
2.4


PA14_06580

hypothetical protein
2.4


PA14_37990

RNA polymerase sigma factor
2.4


PA14_68200
rmlA
glucose-1-phosphate thymidylyltransferase
2.4


PA14_30550

periplasmic aliphatic sulfonate-binding protein
2.4


PA14_12130
lis
lipoyl synthase
2.4


PA14_58580

hydroxylase
2.4


PA14_15110

hypothetical protein
2.4


PA14_11010

hypothetical protein
2.4


PA14_00800

hypothetical protein
2.4


PA14_18070

periplasmic metal-binding protein
2.3


PA14_41930

hypothetical protein
2.3


PA14_28250

secreted acid phosphatase
2.3


PA14_09370

hypothetical protein
2.3


PA14_10560

hypothetical protein
2.3


PA14_10500

cbb3-type cytochrome c oxidase subunit I
2.3


PA14_31700

CDP-alcohol phosphatidyltransferase
2.3


PA14_72870

aminotransferase
2.3


PA14_21260

hypothetical protein
2.3


PA14_22880

Fe—S protein
2.3


PA14_26190

hypothetical protein
2.3


PA14_23090

keto-hydroxyglutarate-aldolase/keto-deoxy-
2.3




phosphogluconate aldolase


PA14_37440

MFS transporter
2.3


PA14_52250

two-component response regulator
2.3


PA14_08690

tRNA-Trp
2.2


PA14_13140

hypothetical protein
2.2


PA14_16110

hypothetical protein
2.2


PA14_63460

tRNA-Sec
2.2


PA14_20770

hypothetical protein
2.2


PA14_28740

tRNA-Pro
2.2


PA14_40510
ccoN-2
cbb3-type cytochrome c oxidase subunit I
2.2


PA14_60540

hypothetical protein
2.2


PA14_38060

hypothetical protein
2.2


PA14_35340

2-ketogluconate kinase
2.2


PA14_39970
phzA2
phenazine biosynthesis protein
2.2


PA14_72550

adhesin
2.2


PA14_42200

hypothetical protein
2.2


PA14_68030

tRNA-Phe
2.2


PA14_33750

outer membrane protein
2.2


PA14_56910

hypothetical protein
2.2


PA14_41450

hypothetical protein
2.2


PA14_52130

hypothetical protein
2.2


PA14_62870
rrmJ
cell division protein FtsJ
2.2


PA14_52690
aruG
arginine/ornithine succinyltransferase All subunit
2.2


PA14_36310
hcnC
hydrogen cyanide synthase HcnC
2.2


PA14_49800

oxidoreductase
2.2


PA14_34820

regulatory protein
2.1


PA14_43520

hypothetical protein
2.1


PA14_38220

hypothetical protein
2.1


PA14_15570

hypothetical protein
2.1


PA14_40240

ABC transporter ATP-binding protein/permease
2.1


PA14_31050

hypothetical protein
2.1


PA14_47190
cyoB
cytochrome o ubiquinol oxidase subunit I
2.1


PA14_34830

regulatory protein
2.1


PA14_14750

iron-binding protein IscA
2.1


PA14_58690

hypothetical protein
2.1


PA14_12940

taurine ABC transporter ATP-binding protein
2.1


PA14_68070

periplasmic binding protein
2.1


PA14_19120
rhlR
transcriptional regulator RhlR
2.1


PA14_40630

hypothetical protein
2.1


PA14_37770

hydrolase
2.1


PA14_02530

hypothetical protein
2.1


PA14_64940

hypothetical protein
2.1


PA14_09290
pchG
pyochelin biosynthetic protein PchG
2.1


PA14_06170

transmembrane sensor
2.0


PA14_48060
aprA
alkaline metalloproteinase
2.0


PA14_15540

mating pair formation protein TrbL
2.0


PA14_34510

hypothetical protein
2.0


PA14_65320
miaA
tRNA delta(2)-isopentenylpyrophosphate transferase
2.0


PA14_52700
aruF
arginine/ornithine succinyltransferase Al subunit
2.0


PA14_13000

transcriptional regulator
2.0


PA14_19370

asparagine synthetase
2.0


PA14_14740

scaffold protein
2.0


PA14_30630
pqsH
FAD-dependent monooxygenase
2.0
















TABLE 10







Genes up-regulated by mBTL in the wild-type strain determined


through microarray analysis. The ratio of gene expression in mBTL


treated cells to that of untreated cells (fold up-regulation) is


displayed for genes that exhibited changes greater than 2-fold.


Ratios are the average of three independent experiments.











Gene




Gene locus
name
Description
Ratio





PA14_10380

hypothetical protein
2.6


PA14_31290
pa1L
PA-I galactophilic lectin
2.4


PA14_10370

hypothetical protein
2.3


PA14_61200

hypothetical protein
2.3


PA14_49300

lipoxygenase
2.3


PA14_10350

secretion protein
2.2


PA14_31840

hypothetical protein
2.2


PA14_52000

hypothetical protein
2.2


PA14_45310
ccmF
cytochrome C-type biogenesis protein
2.1




CcmF


PA14_37070

hypothetical protein
2.1


PA14_31280

integrase
2.1


PA14_31890

RND efflux transporter
2.0


PA14_20640

hypothetical protein
2.0
















TABLE 11







Genes down-regulated by mBTL in the lasR mutant determined


through microarray analysis. The ratio of gene expression in


untreated cells to that of mBTL treated cells (fold down-regulation)


is displayed for genes that exhibited changes greater than 2-fold.


Ratios are the average of three independent experiments.











Gene




Gene locus
name
Description
Ratio













PA14_33810
pvdA
L-ornithine N5-oxygenase
18.4


PA14_09490
phzM
phenazine-specific methyltransferase
12.8


PA14_33510

hypothetical protein
12.6


PA14_33820
pvdQ
penicillin acylase-related protein
11.0


PA14_33610

peptide synthase
10.9


PA14_51380
pqsE
quinolone signal response protein
7.8


PA14_09480
phzA1
phenazine biosynthesis protein
7.7


PA14_51420
pqsB
PqsB
6.7


PA14_09470
phzB1
phenazine biosynthesis protein
6.6


PA14_49720

hypothetical protein
5.7


PA14_33730

dipeptidase
5.4


PA14_51410
pqsC
PqsC
5.4


PA14_33280
pvdL
peptide synthase
5.1


PA14_33650
pvdD
pyoverdine synthetase D
4.6


PA14_51430
pqsA
coenzyme A ligase
4.5


PA14_36320
hcnB
hydrogen cyanide synthase HcnB
4.4


PA14_28470

hypothetical protein
4.4


PA14_51390
pqsD
3-oxoacyl-ACP synthase
4.2


PA14_36330
hcnA
hydrogen cyanide synthase HcnA
4.1


PA14_51350
phnB
anthranilate synthase component II
4.0


PA14_39970
phzA2
phenazine biosynthesis protein
3.9


PA14_39960
phzB2
phenazine biosynthesis protein
3.9


PA14_33690
pvdE
pyoverdine biosynthesis protein PvdE
3.8


PA14_09400
phzS
hypothetical protein
3.7


PA14_10960

ferredoxin oxidoreductase subunit
3.7


PA14_54910

thioesterase
3.6


PA14_03370

hypothetical protein
3.5


PA14_25640
plsX
glycerol-3-phosphate acyltransferase PlsX
3.5


PA14_21020

non-ribosomal peptide synthetase
3.5


PA14_34490

hypothetical protein
3.5


PA14_29560

hypothetical protein
3.4


PA14_33760

ABC transporter ATP-binding protein/permease
3.3


PA14_52800
acsA
acetyl-CoA synthetase
3.3


PA14_19710

alpha/beta hydrolase
3.3


PA14_53230

oxidoreductase
3.3


PA14_67190

hypothetical protein
3.3


PA14_02970

hypothetical protein
3.2


PA14_33630
pvdJ
protein PvdJ
3.1


PA14_30720

tRNA-Cys
3.1


PA14_08220

hypothetical protein
3.1


PA14_54870

hypothetical protein
3.0


PA14_55110

hypothetical protein
3.0


PA14_14560

hypothetical protein
3.0


PA14_59410

hypothetical protein
3.0


PA14_29250

hypothetical protein
2.9


PA14_55550

ECF subfamily RNA polymerase sigma-70 factor
2.9


PA14_68040

short-chain dehydrogenase
2.9


PA14_41800

transcriptional regulator
2.9


PA14_51610

hypothetical protein
2.9


PA14_55360
exbB2
transport protein ExbB2
2.9


PA14_51920

acylphosphatase
2.8


PA14_33270
pvdG
protein PvdG
2.8


PA14_34420

maltose/mannitol ABC transporter substrate-binding prote
2.8


PA14_69550

hypothetical protein
2.8


PA14_50050

MFS family transporter
2.8


PA14_63070

GntR family transcriptional regulator
2.7


PA14_48950

hypothetical protein
2.7


PA14_18350

bifunctional UDP-glucuronic acid decarboxylase/UDP-4-al
2.7


PA14_32790

hypothetical protein
2.7


PA14_20330
phpE
phosphonate ABC transporter permease
2.7


PA14_35880

gamma-aminobutyraldehyde dehydrogenase
2.7


PA14_33700
pvdF
pyoverdine synthetase F
2.7


PA14_57460

cell division protein MraZ
2.7


PA14_18150

acetyl-coa synthetase
2.7


PA14_23080
pgl
6-phosphogluconolactonase
2.7


PA14_33250

hypothetical protein
2.7


PA14_68380
nudE
ADP-ribose diphosphatase NudE
2.6


PA14_01600

aldehyde dehydrogenase
2.6


PA14_41740

hypothetical protein
2.6


PA14_49010

hypothetical protein
2.6


PA14_38170

hypothetical protein
2.6


PA14_09660

acyl-CoA synthetase
2.6


PA14_19960

hypothetical protein
2.6


PA14_35950

dehydrogenase
2.6


PA14_50310

sugar nucleotidyltransferase
2.5


PA14_19750

hypothetical protein
2.5


PA14_23670

hypothetical protein
2.5


PA14_63330

glycerolphosphodiesterase
2.5


PA14_42910

hypothetical protein
2.5


PA14_51850

hypothetical protein
2.5


PA14_69430

hypothetical protein
2.5


PA14_59590

hypothetical protein
2.5


PA14_35390
pvcD
pyoverdine biosynthesis protein PvcD
2.5


PA14_38420

hypothetical protein
2.5


PA14_58900

large exoprotein
2.5


PA14_57770
hisC1
histidinol-phosphate aminotransferase
2.4


PA14_33560

adhesion protein
2.4


PA14_22550

LysR family transcriptional regulator
2.4


PA14_55160
toxR
transcriptional regulator ToxR
2.4


PA14_67350
hutU
urocanate hydratase
2.4


PA14_21680

hypothetical protein
2.4


PA14_47650
cobS
cobalamin synthase
2.4


PA14_33740
pvdP
protein PvdP
2.4


PA14_51050

aldehyde dehydrogenase
2.4


PA14_52720
argD
bifunctional N-succinyldiaminopimelate-aminotransferase/
2.4


PA14_01250

sulfate transporter
2.4


PA14_72980

G3E family GTPase
2.4


PA14_10130

short chain dehydrogenase
2.4


PA14_29390

hypothetical protein
2.4


PA14_61620

MerR family transcriptional regulator
2.4


PA14_20950
fabH2
3-oxoacyl-ACP synthase
2.4


PA14_05890

stomatin-like protein
2.3


PA14_54740

hypothetical protein
2.3


PA14_72960

MFS dicarboxylate transporter
2.3


PA14_36310
hcnC
hydrogen cyanide synthase HunC
2.3


PA14_04030

hypothetical protein
2.3


PA14_61170

transmembrane protein
2.3


PA14_33550

ABC transporter ATP-binding protein
2.3


PA14_46170

LysR family transcriptional regulator
2.3


PA14_46000

hypothetical protein
2.3


PA14_63605
fdnG
formate dehydrogenase-O, major subunit
2.3


PA14_72230

hypothetical protein
2.3


PA14_35940

acyl-CoA synthetase
2.3


PA14_28630

hydrolase
2.3


PA14_05430

chemotaxis protein
2.3


PA14_22600

glycosyl transferase family protein
2.3


PA14_34320

DszC family monooxygenase
2.3


PA14_51020

hypothetical protein
2.3


PA14_29500

type II secretion system protein
2.3


PA14_18880
nth
endonuclease III
2.3


PA14_29050

molybdopterin oxidoreductase
2.3


PA14_54110

transporter
2.3


PA14_52900

acyl-CoA dehydrogenase
2.3


PA14_42100

hypothetical protein
2.3


PA14_33720
pvdN
protein PvdN
2.3


PA14_20130

LysR family transcriptional regulator
2.2


PA14_03265

hypothetical protein
2.2


PA14_33240

hypothetical protein
2.2


PA14_73000

hypothetical protein
2.2


PA14_27410

outer membrane protein
2.2


PA14_41020

Orn/Arg/Lys decarboxylase
2.2


PA14_58110

Maf-like protein
2.2


PA14_32650

glutathione S-transferase
2.2


PA14_03250

hypothetical protein
2.2


PA14_27700

transcriptional regulator
2.2


PA14_26810

two-component sensor
2.2


PA14_67540

hypothetical protein
2.2


PA14_31740

hypothetical protein
2.2


PA14_33770

hypothetical protein
2.2


PA14_33570

hypothetical protein
2.2


PA14_03320

hypothetical protein
2.2


PA14_22060

hypothetical protein
2.2


PA14_17260
dnaE
DNA polymerase III subunit alpha
2.2


PA14_03360

hypothetical protein
2.2


PA14_14630
secD
preprotein translocase subunit SecD
2.2


PA14_34250

glycerophosphoryl diester phosphodiesterase
2.2


PA14_66700

nuclease
2.2


PA14_53740

hypothetical protein
2.2


PA14_20480

hypothetical protein
2.1


PA14_55390

hypothetical protein
2.1


PA14_34460

hypothetical protein
2.1


PA14_69890

multidrug efflux protein NorA
2.1


PA14_43510

hypothetical protein
2.1


PA14_61340

hypothetical protein
2.1


PA14_00560
exoT
exoenzyme T
2.1


PA14_33710
pvdO
protein PvdO
2.1


PA14_13330

hypothetical protein
2.1


PA14_27450

hypothetical protein
2.1


PA14_43100

Rhs family protein
2.1


PA14_72750

hypothetical protein
2.1


PA14_54890

hypothetical protein
2.1


PA14_40900

short-chain dehydrogenase
2.1


PA14_41780

hypothetical protein
2.1


PA14_31770

oxidoreductase
2.1


PA14_50570

hypothetical protein
2.1


PA14_33580

hypothetical protein
2.1


PA14_45830

hypothetical protein
2.1


PA14_34510

hypothetical protein
2.1


PA14_10600

MFS transporter
2.1


PA14_08630

pantothenate kinase
2.1


PA14_26890
pyrF
orotidine 5′-phosphate decarboxylase
2.1


PA14_33540

ABC transporter permease
2.1


PA14_37380

flavin-binding monooxygenase
2.1


PA14_06650
nirN
c-type cytochrome
2.1


PA14_28020

hypothetical protein
2.1


PA14_17650

hypothetical protein
2.1


PA14_23640

hypothetical protein
2.1


PA14_46290

TetR family transcriptional regulator
2.1


PA14_65390

hypothetical protein
2.1


PA14_68740
argA
N-acetylglutamate synthase
2.1


PA14_55000

ABC transporter periplasmic protein
2.1


PA14_28660
infC
translation initiation factor IF-3
2.1


PA14_33530

hypothetical protein
2.1


PA14_51540

transposase
2.1


PA14_29000

hypothetical protein
2.1


PA14_58980

hypothetical protein
2.1


PA14_55040

ferric enterobactin transporter ATP-binding protein
2.1


PA14_33500
pvdH
diaminobutyrate--2-oxoglutarate aminotransferase
2.1


PA14_59940

hypothetical protein
2.1


PA14_26610

hypothetical protein
2.1


PA14_59540

hypothetical protein
2.1


PA14_08620
birA
biotin--protein ligase
2.1


PA14_38020

ntibiotic biosynthesis monooxygenase
2.1


PA14_06030

acyltransferase
2.0


PA14_40790

transcriptional regulator
2.0


PA14_28170

formate/nitrate transporter
2.0


PA14_36090

porin
2.0


PA14_36400

hypothetical protein
2.0


PA14_22350
actP
acetate permease
2.0


PA14_63380

hypothetical protein
2.0


PA14_46010

ABC transporter ATP-binding protein
2.0


PA14_44480

hypothetical protein
2.0


PA14_52080

hypothetical protein
2.0


PA14_51360
phnA
anthranilate synthase component I
2.0


PA14_73100

hypothetical protein
2.0


PA14_26390

hypothetical protein
2.0


PA14_64580

two-component sensor
2.0


PA14_41563
cobA
uroporphyrin-III C-methyltransferase
2.0


PA14_20940

acyl carrier protein
2.0


PA14_26070

hypothetical protein
2.0


PA14_67260

histidine/phenylalanine ammonia-lyase
2.0
















TABLE 12







Genes down-regulated by mBTL in the rhlR mutant determined


through microarray analysis. The ratio of gene expression in


untreated cells to that of mBTL treated cells (fold down-regulation)


is displayed for genes that exhibited changes greater than 2-fold.


Ratios are the average of three independent experiments.













Gene





Gene locus
name
Description
Ratio















PA14_58970
hypothetical protein
3.0



PA14_33360
hypothetical protein
2.2



PA14_52320
tRNA-Met
2.0



PA14_62790
tRNA-Met
2.0

















TABLE 13







Genes up-regulated by mBTL in a rhlI null strain determined through microarray


analysis. The ratio of gene expression in mBTL treated cells to that of untreated


cells (fold up-regulation) is displayed for genes that exhibited changes greater than


2-fold. Additional columns indicate the genes that are down-regulated in the lasR


and rhlR null strains. Ratios are the average of three independent experiments.













Gene


LasR-
RhlR


Gene locus
name
Description
Ratio
regulated
regulated















PA14_10360

hypothetical protein
39.5

*


PA14_53250
cpbD
chitin-binding protein CbpD
10.1
*
*


PA14_01710
ahpC
alkyl hydroperoxide reductase
94
*
*


PA14_06810
norC
nitric-oxide reductase subunit C
5.8
*
*


PA14_10380

hypothetical protein
4.9
*


PA14_11140

nonribosomal peptide synthetase
4.7

*


PA14_28360

hypothetical protein
4.2

*


PA14_66720
priA
primosome assembly protein PriA
3.9


PA14_03520

hypothetical protein
3.8

*


PA14_58040

hypothetical protein
3.7
*
*


PA14_14710

Rrf2 family protein
3.5

*


PA14_17690

hypothetical protein
3.4


PA14_46450
aceK
bifunctional isocitrate dehydrogenase
3.3




kinase/phosphatase


PA14_35730

hypothetical protein
3.1


PA14_22320

hypothetical protein
3.0
*
*


PA14_38270

hypothetical protein
3.0

*


PA14_16250
lasB
elastase LasB
2.9
*
*


PA14_18800

hypothetical protein
2.9
*
*


PA14_53290
trxB2
thioredoxin reductase 2
2.9
*
*


PA14_19640

short chain dehydrogenase
2.8


PA14_52380

cytochrome b561
2.7


PA14_33360

hypothetical protein
2.5


PA14_26990

hypothetical protein
2.5
*
*


PA14_40290
lasA
LasA protease
2.5

*


PA14_03490

hypothetical protein
2.5

*


PA14_03510

hypothetical protein
2.4

*


PA14_03770
speB1
agmatinase
2.4


PA14_06830
norB
nitric-oxide reductase subunit B
2.3
*
*


PA14_44140

hypothetical protein
2.3


PA14_56110

hypothetical protein
2.3


PA14_05860

hypothetical protein
2.2
*
*


PA14_06990

hypothetical protein
2.2


PA14_17510

hypothetical protein
2.2


PA14_64390
ureC
urease subunit alpha
2.2


PA14_01720
ahpF
alkyl hydroperoxide reductase
2.1
*
*


PA14_51830

DNA-binding stress protein
2.1
*
*


PA14_71840

hypothetical protein
2.1


PA14_05660

transcriptional regulator
2.1


PA14_60480

hypothetical protein
2.1


PA14_18860

hypothetical protein
2.0


PA14_19210

hypothetical protein
2.0


PA14_46420

short chain dehydrogenase
2.0


PA14_24440

lipoprotein
2.0


PA14_14740

scaffold protein
2.0

*

## Claims

1. A compound having the formula:



wherein
Py is a pyridine ring attached in the 2, 3, or 4 position and substituted with one or more additional substituents selected from the group consisting of alkyl, trifluoromethyl, methoxy, F, Cl and Br;
Aryl is a benzene ring with one or more additional substituents selected from the group consisting of methyl, trifluoromethyl, cyano, nitro, F, Cl, Br and methoxy;
X is O, NH, S or CH2; and
n is 0 to 4 —CH2— units.

2. A compound having the formula:



where n=1 or 3.

3. A composition comprising the compound of claim 1 or 2.

4. A method for inhibiting gram negative bacteria biofilm production, virulence factor production, pyocyanin production, or quorum sensing, the method comprising the steps of:
providing a composition comprising a compound of claim 1 or 2; and
contacting at least one gram negative bacteria with the composition.

5. The method of claim 4, wherein the gram negative bacteria is selected from Pseudomonas, Burkholderia cepaci C. violaceum, V. harveyi, Neisseria gonorrhoeae, Neisseria meningitidis, Bordetella pertussis, Haemophilus influenzae, Legionella pneumophila, Brucella, Francisella, Xanthomonas, Agrobacterium, Escherichia coli, Salmonella, Shigella, Proteus, or Yersinia pestisi.

6. The method of claim 5, wherein the Pseudomonas is P. aeruginosa.

7. The method of claim 4, wherein the composition inhibits the f LasR/RhlR receptor signaling of the at least one gram negative bacteria.

8. The method of claim 4, wherein the subject is selected from a cow, a pig, a horse, a chicken, a cat, a dog, or a human.

9. The method of claim 4, wherein the subject has an infection selected from a respiratory illness, pulmonary tract infection, a urinary tract infection, a catheter-associated infection, a blood infection, a middle ear infection, dental plaque, gingivitis, chronic sinusitis, endocarditis, eye infections, eye infections resulting from contact lens use, implanted device infection, a medical device infection, a central nervous infection, a gastrointestinal tract infection, a bone infection, a joint infection, an ear infection, an eye infection, a burn, a wound, an antibiotic resistant infection, is immunocompromised, is immunosuppressed, or has an opportunistic infection.

10. The method of claim 9, wherein the medical device infection occurs in a patient having a catheter, a stent, a joint prosthesis, a prosthetic cardiac valve, on a ventilator or an intrauterine device.

11. The method of claim 9, wherein the pulmonary tract infection is pneumonia.

12. The method of claim 9, wherein the respiratory illness is Cystic Fibrosis.

13. The method of claim 12, wherein the patient has both Cystic Fibrosis and pneumonia.

14. The method of claim 4, wherein the composition is administered therapeutically.

15. The method of claim 4, wherein the composition is administered prophylactically.

16. The method of claim 15, wherein the subject is undergoing surgery, implantation of medical devices, or dental procedures.

17. The method of claim 4, wherein the composition is co-administered with one or more additional drugs or other therapeutic agents.

18. The method according to claim 17, wherein said one or more additional drugs or other therapeutic agents comprise one or more antibiotics.

19. The method of claim 4, wherein the composition is administered topically.

20. The method of claim 4, wherein the composition is administered intravenously.

21. The method of claim 4, wherein the composition is administered intranasally.

22. The method of claim 4, wherein the composition is used in an industrial setting.

23. The method of claim 22, wherein the industrial setting is a work area, a medical instrument, a medical device, a chemical unit operation, a pipe, a sewage system, a pipeline, a tubing, or a filtration device.

24. The method of claim 23, wherein the medical device is a catheter, a joint prosthesis, a prosthetic cardiac valve, a ventilator, a stent, or an intrauterine device.

25. The method of claim 23, wherein the chemical unit operation comprises a microfluidic platform, a microelectromechanical system, a 3D printer, or a system for coating nanoparticle surfaces.