QUINOLINONE OR QUINAZOLINONE COMPRISING ANTIBIOFILM COMPOSITIONS, COMPOUNDS AND METHODS AND USES RELATING THERETO

Abstract

An antibiofilm composition comprising a compound of formula (A1), (A2) or (A3):

##STR00001##

wherein each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is independently selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy, hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, alkyl carboxy and amido; and X may be O or S, wherein the composition does not comprise a compound of formula (B1) or (B2):

##STR00002##

Claims

1. A composition comprising a compound of formula (A1), (A2) or (A3): ##STR00011## wherein each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is independently selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy, hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, alkyl carboxy and amido; and X may be O or S, wherein the composition does not comprise a compound of formula (B1) or (B2): ##STR00012##

2. The composition according to claim 1 wherein R.sup.1 is hydrogen or methyl; R.sup.2 is hydrogen, C.sub.1 to C.sub.8 alkyl, halo or alkoxy; R.sup.3 is hydrogen, alkyl or alkoxy; R.sup.4 is hydrogen or halo; R.sup.5 is hydrogen or alkyl; R.sup.6 is alkyl; R.sup.7 is hydrogen or amino and X is O.

3. The composition according to claim 1 comprising a compound selected from: (i) a compound of formula (A1) in which R.sup.1 is hydrogen or methyl; R.sup.2 is hydrogen, C.sub.1 to C.sub.6 alkyl, chloro or methoxy; R.sup.3 is hydrogen; R.sup.4 is hydrogen or chloro; R.sup.5 is hydrogen; R.sup.6 is C.sub.7 to C.sub.9 alkyl; and R.sup.7 is hydrogen; (ii) a compound of formula (A2) wherein R.sup.1 is hydrogen; R.sup.2 is hydrogen; R.sup.3 is hydrogen; R.sup.4 is hydrogen; R.sup.6 is C.sub.7 to C.sub.9 alkyl; and R.sup.7 is hydrogen or amino; and (iii) a compound of formula (A3) wherein R.sup.1 is hydrogen; R.sup.2 is hydrogen; R.sup.4 is hydrogen; R.sup.5 is C.sub.7 to C.sub.9 alkyl; R.sup.7 is hydrogen and X is O.

4. The composition according to claim 1 which is effective against Candida albicans and/or Aspergillus fumigatus.

5. The composition according to claim 1 which is effective against Candida albicans and includes a compound selected from: (i) a compound of formula (A1) in which R.sup.1 is hydrogen; R.sup.2 is hydrogen, methyl or chloro; R.sup.3 is hydrogen; R.sup.4 is hydrogen or chloro; R.sup.5 is hydrogen; R.sup.6 is C.sub.7 alkyl; and R.sup.7 is hydrogen; (ii) a compound of formula (A2) wherein R.sup.1 is hydrogen; R.sup.2 is hydrogen; R.sup.3 is hydrogen; R.sup.4 is hydrogen; R.sup.6 is C.sub.7 alkyl and R.sup.7 is hydrogen or amino; and (iii) a compound of formula (A3) wherein R.sup.1 is hydrogen; R.sup.2 is hydrogen; R.sup.3 is hydrogen; R.sup.4 is hydrogen; R.sup.5 is C.sub.7 alkyl; R.sup.7 is hydrogen and X is O.

6. The composition according to claim 1 which is effective against Aspergillus fumigatus and includes a compound selected from: (i) a compound of formula (A1) in which R.sup.1 is hydrogen; R.sup.2 is hydrogen, n-hexyl or methoxy; R.sup.3 is hydrogen or methoxy; R.sup.4 is hydrogen or chloro; R.sup.5 is hydrogen; R.sup.6 is C.sub.7 to C.sub.9 alkyl; and R.sup.7 hydrogen; and (ii) a compound of formula (A2) wherein R.sup.1 is hydrogen; R.sup.2 is hydrogen; R.sup.3 is hydrogen; R.sup.4 is hydrogen; R.sup.6 is C.sub.7 alkyl and R.sup.7 is hydrogen or amino.

7. A composition according to claim 1 which is a pharmaceutical preparation that further comprises one or more additional antifungal agents.

8. A method of combatting a biofilm at a locus, the method comprising contacting the locus with the composition of claim 1.

9. A method of treating a fungal biofilm infection comprising administering the compound of claim 1.

10. A medical device having a coating on at least a portion thereof; wherein the coating comprises the compound of claim 1.

11. A compound of formula (A3): ##STR00013## wherein each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.7 is independently selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy, hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, alkyl carboxy and amido; and X may be O or S.

12. The compound of claim 11, for use in therapy.

Description

EXAMPLE 1

[0121] The following compounds were prepared using the methods described in Org. Biomol. Chem., 2015, 13, 5537-5541 and analogous processes:

##STR00008## ##STR00009##

[0122] The properties of these compounds were tested and compared to the properties of:

##STR00010##

[0123] The following general conditions were used in the biological testing of some of the above compounds to assess their effectiveness against Candida albicans:

C. albicans Stock Maintenance and Culturing Conditions.

[0124] C. albicans strain SC5314 was sub-cultured from 15% (v/v) glycerol stocks at 80 C. onto Yeast Peptone Dextrose (YPD) medium [1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) dextrose] and incubated at 30 C. overnight.

P. aeruginosa Stock Maintenance and Culturing Conditions.

[0125] P. aeruginosa strains, PAO1 and pqsA mutant, containing the chromosomally inserted pqsA-lacZ promoter fusion on plasmid pUC18-mini-Tn7, were sub-cultured from glycerol stocks onto LB agar plates, supplemented with Carbenicillin (200 g/ml) and X-gal (40 g/ml), and incubated at 37 C. overnight. Single colonies were inoculated into LB broth (20 ml), supplemented with Carbenicillin (200 g/ml), and incubated at 37 C., shaking at 180 rpm overnight. For subsequent experiments, the OD.sub.600nm was recorded and a starting OD.sub.600 nm of 0.02 was inoculated into fresh LB broth, supplemented with Carbenicillin (200 g/ml) and incubated at 37 C., shaking at 180 rpm.

Test Compounds

[0126] The test compounds in desiccated form were re-suspended in Methanol to create a 10 mM stock. A working concentration of 100 M was used in all experiments.

TLC Analysis.

[0127] Silica TLC plates, activated by soaking in 5% (w/v) K.sub.2HPO.sub.4 for 30 min were placed in an oven at 100 C. for 1 hr. Test compounds (5 l, 10 mM) were spotted approximately 1 cm from the bottom. The spots were dried and the plate placed in a mobile phase comprising 95:5 dichloromethane:methanol. The plate was viewed under UV light when the mobile phase had run 5 cm below the top of the plate.

EXAMPLE 2

[0128] The ability of some of the compounds detailed in example 1 to disrupt Candida albicans was tested using Confocal Scanning Laser Microscopy and an XTT Metabolic Assay. The XTT assay is a commonly used quantitative method assessing Candida biofilm mass and growth often in response to novel drug therapies.

[0129] C. albicans biofilm formation was carried out in 96 well plates by a method of the prior art. Briefly, C. albicans was inoculated into Yeast Nitrogen Base [10% (w/v)] and glucose/maltose [10% (w/v)] and incubated overnight at 30 C. Seeding densities for all subsequent experiments (N=3) were OD.sub.600=0.05. Cells were grown in Yeast Nitrogen Base supplemented with 1 M Phosphate buffer and acetyl-D-glucosamine (YNB-NP) along with appropriate volumes of test compound to provide the desired concentration. Cells were cultured in 96 well plates for 1 hr at 37 C. to facilitate yeast attachment. After this incubation, the supernatants were aspirated, the wells washed twice with YNB-NP media, and fresh media with the same concentration of test compound added to the appropriate wells. The plate was incubated for 24 hr at 37 C. The next day, the cultures were aspirated and the wells washed once with YNB-NP media.

[0130] C. albicans biofilm quantification was carried out in 96 well plates using a semi-quantitative Tetrazolium salt, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H tetrazolium-5-carboxanilide inner salt (XTT) reduction assay (Hawser, Tunney et al, Antimicrob Agents Chemother. 2004 May; 48(5):1879-81). XTT (0.01 g) was dissolved in sterile water (20 ml) and filter sterilized. 10 l of Menadione dissolved in Acetone was added to the XTT solution, just before use. The XTT-Menadione Solution (100 l) was added to each well. The plate was incubated in the dark at 37 C. for up to 2 hr to allow for colour development. The OD.sub.492 nm was recorded for each well. Experiments were repeated at least three times, with at least eight technical replicates.

[0131] For the microscopy experiments C. albicans biofilms were grown on glass coverslips in 6 well plates, using the biofilm formation protocol above. Briefly, an overnight culture of C. albicans was added to YNB-NP media to give an OD.sub.600=0.05. Test compounds were added at appropriate concentrations. The 6 well plates were incubated at 37 C. for 1 hr to facilitate attachment after which they were washed once in YNB-NP and then fresh test compound in YNB-NP added. Plates were incubated overnight at 37 C. Next day, glass coverslips were washed once in YNB-NP and stained. Calcofluor (1 mg/ml) and 10% (w/v) potassium hydroxide were added drop-wise to coverslips, washed in PBS and viewed. Concavalin A and FUN-1 were added at 50 g/ml in 1 ml PBS and incubated at 37 C. for 30 mins. Coverslips were washed in PBS and viewed. All imaging was carried out on a Zeiss LSM5 confocal microscope. Confocal images were recorded under a bright field lens using 20 objective magnification. Filter cubes facilitating fluorescent imaging were used to record images for Calcofluor at 405 nm, Con A at 488 nm and FUN-1 at 543 nm. All images were captured using the Zeiss HBO-100 microscope illuminating system, processed using the Zen AIM application imaging program and converted to JPGs using Axiovision 40 Ver. 4.6.3.0. A minimum of three independent biological repetitions were carried out.

[0132] Experiments were first carried out using the test compound at concentration of 100 M.

[0133] FIG. 1 shows graphically the XTT results for compounds B1, B2, methanol (i.e. the solvent alone) and untreated biofilms.

[0134] FIG. 2 shows the microscopy results for compounds C1, C2, C3, C4, C5, C6, C8, C10, C11 and C12. The results of the XTT tests for these compounds are detailed in table 1:

TABLE-US-00001 TABLE 1 Absorbance at 492 nm Compound Mean Standard deviation Untreated 0.851 0.079 MeOH (control) 0.647 0.007 B1 0.200 0.027 B2 0.999 0.120 C1 0.162 0.021 C2 0.178 0.003 C3 0.307 0.062 C4 0.302 0.054 C5 0.242 0.029 C6 0.336 0.067 C8 0.457 0.014 C10 0.419 0.001 C11 0.242 0.041 C12 0.298 0.029

[0135] The above experiments were repeated, this time using concentrations of test compound of 100 M, 50 M and 10 M to show that the compounds can still be effective at lower concentrations. The results are shown in table 2.

TABLE-US-00002 TABLE 2 Compound Mean Standard Deviation MeOH (control) 1 0 B1 0.45 0.047 B2 1.2 0.005 C4 10 M 0.93 0.011 C2 10 M 0.68 0.081 C3 10 M 0.71 0.024 C4 10 M 0.86 0.018 C10 10 M 0.58 0.006 C1 50 M 0.72 0.011 C2 50 M 0.38 0.061 C3 50 M 0.57 0.001 C4 50 M 0.67 0.051 C10 50 M 0.50 0.010 C1 100 M 0.58 0.001 C2 100 M 0.25 0.055 C3 100 M 0.51 0.010 C4 100 M 0.58 0.040 C10 100 M 0.53 0.114

EXAMPLE 3

[0136] The cytotoxicity of compounds B1, B2, C2, C3, C4, C5, C6 and C12 was tested according to the following method:

[0137] Lactate dehydrogenase (LDH) release from IB3 lung epithelial cells was assayed as a measure of cytotoxicity using an LDH colorimetric kit (Roche) according to manufacturers' instructions. Briefly, IB3-1 cells were seeded onto 96 well plates and treated with methanol (control) and test compounds (100 M). Following 16 hr incubation at 37 C. and 5% CO.sub.2, supernatants were removed and added to catalyst reaction mixture in a fresh plate and further incubated at 37 C. and 5% CO.sub.2 for 30 mins to allow for colour development. After this period, the plate was analysed on an ELISA plate reader at OD.sub.490 nm. Cytotoxicity was expressed as a percentage of cells treated with 0.1% (v/v) Triton (100% cytotoxicity). The results are detailed in table 3:

TABLE-US-00003 TABLE 3 % Cytotoxicity Compound Mean Standard deviation MeOH 3.844 1.40 B1 38.93 10.89 B2 13.84 4.18 C2 28.94 6.76 C3 23.77 3.46 C4 15.46 4.35 C5 31.04 7.74 C6 19.70 10.20 C12 16.68 3.97

[0138] The above experiments were repeated, testing some compounds against further cell lines (A549, DU145 and HeLa). The results are detailed in table 4:

TABLE-US-00004 TABLE 4 Cytotoxicity Standard Cell Line Compound Mean Deviation A549 C1 8.739 1.046 A549 C12 32.37 6.63 DU145 C1 6.26 4.52 DU145 C3 32.42 14.27 DU145 C4 24.42 6.49 DU145 C12 8.28 1.43 DU145 C10 31.7 5.43 HeLa C1 15.96 11.59 HeLa C12 34.57 19.08

EXAMPLE 4

[0139] The virulence of compounds B1, B2, C2, C3, C4, C5, C6, C8 and C11 towards P. aeruginosa was assessed using the following method:

RNA Isolation and qRT-PCR Transcriptional Analysis.

[0140] Overnight Candida albicans cultures were diluted to 0.05 at OD.sub.600 in either YNB or YNB-NP (Difco). YNB cultures were supplemented with methanol whereas YNB-NP cultures were supplemented with either 10 mM HHQ or the methanol volume equivalent. Cultures were grown at 30 C. with agitation (180 rpm) for 6 hours after which they were centrifuged at 4000 rpm, supernatants discarded and pellets frozen at 20 C. until processing. RNA was isolated using the MasterPure Yeast RNA purification kit (Cambio Ltd, Cambridge UK) according to manufacturer's specifications, and was quantified using a ND-1000 Spectrophotometer (NanoDrop Technologies, USA). Genomic DNA was enzymatically removed using Turbo DNA-free DNase (Ambion), and samples were confirmed DNA free by PCR. RNA was converted to cDNA using random primers and AMV reverse transcriptase (Promega) according to manufacturer's instructions. qRT-PCR was carried out using the Universal ProbeLibrary (UPL) system (Roche) according to manufacturer's specifications, and samples were normalised to C. albicans actin transcript expression (ACT1).

Phenazine Extraction.

[0141] P. aeruginosa strains were cultured as described above for 24 hr, with the addition of test compounds (10 M). Cultures were centrifuged at 4000 rpm for 10 minutes and the cell free supernatant (5 ml) removed. Chloroform (3 ml) was added, and mixed by vortex. After centrifugation at 4000 rpm for 5 mins, the lower aqueous phase was transferred to 0.2 M HCl (2 ml). Samples were mixed by vortex and centrifuged at 4000 rpm for 5 mins to separate the phases. An aliquot of the top phase (1 ml) was removed and spectrophotmetrically analysed at OD.sub.570 nm. Phenazine production was calculated using the following formula: OD.sub.570 nm217.072 and the units expressed in g/ml.

Promoter Fusion Based Expression Analysis.

[0142] Promoter fusion analyses were performed in a 96-well format. Briefly, overnight cultures of wild-type PAO1 pqsA-lacZ (pLP0996) and mutant strain PAO1 pqsA.sup. pqsA-LacZ were diluted to OD.sub.600 nm=0.02 in LB. Test compounds at 100 M final concentration were added, mixed, aliquoted into 96 well plates and incubated overnight at 37 C. with shaking. The next day, OD.sub.600 nm values were recorded in a plate reader. Aliquots of cells (0.02 ml) were permeabilised [100 mM dibasic sodium phosphate (Na.sub.2HPO.sub.4), 20 mM KCl, 2 mM MgSO.sub.4, 0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide), 0.4 mg/mL sodium deoxycholate, 5.4 L/mL beta-mercaptoethanol] and added to substrate solution [60 mM Na.sub.2HPO.sub.4, 40 mM NaH.sub.2PO.sub.4, 1 mg/mL o-nitrophenyl--D-Galactoside (ONPG), 2.7 L/mL -mercaptoethanol]. The kinetics of colour development was monitored and the reactions were stopped using 1M NaCO.sub.3. OD.sub.420 nm were recorded as above. Miller units were calculated using the following equation; 1000[OD.sub.420 nm/(OD.sub.600 nm)0.02 mlreaction time (mins)].

[0143] The phenazine production levels and Miller units in the PAO1 pqsA-pqsA-lacZ strain are detailed in table 5.

TABLE-US-00005 TABLE 5 Phenazine (g/ml) Miller Units Standard Standard Compound Mean deviation Mean deviation Untreated 0.224 0.525 11711 4153 Methanol control 0.080 0.131 12287 4283 Water control 3.088 0.799 B1 2.697 0.537 65076 27746 B2 2.117 0.727 61429 28389 C2 0.541 0.497 16027 4749 C3 0.774 0.523 18350 4331 C4 0 0 35489 12432 C5 0 0 11940 691 C6 0.011 0.019 16678 9700 C8 0.205 0.354 11753 1695 C11 0.432 0.603 12254 1913

[0144] The virulence of compounds B2, C13 and C15 towards P. aeruginosa was assessed using an analogous method. The results are shown in table 6:

TABLE-US-00006 TABLE 6 Normalised Normalised Phenazine (g/ml) Miller Units Standard Standard Compound Mean Deviation Mean Deviation Methanol control 1 1 DMSO control 1.32174 0.522353 1.44997 1.08499 B2 4.196486 1.802723 3.078914 0.850754 C13 0.747026 0.160539 0.371541 0.173011 C15 0.498371 0.319781 0.467063 0.154079

[0145] Data (a minimum of n=2 for each datapoint) is presented normalized to the methanol control.

EXAMPLE 5

[0146] The ability of some of the compounds detailed in example 1 to disrupt Aspergillus fumigatus was tested.

Aspergillus fumigatus Stock Maintenance and Culturing Conditions.

[0147] A. fumigatus Af293 was routinely grown on Sabouraud dextrose agar (SDA) in 100 ml cell culture flasks at 37 C. for 3-4 days until a lawn of fungal growth was observed.

Spore Capture and Biofilm Assay

[0148] A. fumigatus Af293 was grown on SDA in 100 ml cell culture flasks at 37 C. for 3-4 days. Conidia were harvested by flooding the surface of the agar plates with 5 ml PBS (Oxoid) containing 0.025% (v/v) Tween 20 and gently moving the liquid over the surface of the fungal lawn. The conidial suspension was transferred into a 25 ml sterile container and conidia were counted using a Neubauer haemocytometer and light microscope. Conidia were adjusted to the required concentration in RPMI 1640 (Sigma) buffered to pH 7.0 with 0.165 M MOPS immediately prior to biofilm formation analysis.

[0149] To assess biofilm formation, counted A. fumigatus spores (110.sup.5) in MOPS buffered RPMI 1640 were inoculated into 24- and 96-well plates and grown overnight in the presence of 100 M HHQ, PQS and the test compounds. After 24 hrs, the media was removed and the biofilm washed twice with distilled water after which 0.1% crystal violet (CV) was added to each well and allowed to stand for 1 hr at room temperature. The crystal violet was removed and all wells washed in a water bath by inversion. Ethanol was added to each well to solubilise biofilms after which samples were read on a spectrophotometer at Abs.sub.595 nm.

Staining and Microscopy

[0150] A. fumigatus spores (110.sup.5) were inoculated onto glass coverslips in 24 well plates and grown in the presence of analogues overnight at 37 C. in RPMI buffered with MOPS pH 7.0. After 24 hrs, glass coverslips were washed once in PBS and stained. Staining and microscopy was performed as described in Example 2.

[0151] FIG. 3 shows the microscopy results for compounds B1, B2, methanol and untreated biofilms.

[0152] FIG. 4 shows the microscopy results for compounds C1, C2, C7, C8, C9 and C10. The results of the XTT tests are detailed in table 7:

TABLE-US-00007 TABLE 7 Relative absorbance at 595 nm compared to methanol control Compound Mean Standard deviation Methanol 1 0 C1 0.39 0.18 C2 0.37 0.10 C7 0.27 0.14 C8 0.47 0.16 C9 0.18 0.05 C10 0.24 0.12 C13 0.38 0.09 C14 0.42 0.21 C15 0.25 0.10

[0153] The above experiments were carried out using biofilms grown from laboratory sources of A. fumigatus. The experiments were repeated to test some compounds against biofilms grown from three different clinical sources of A. fumigatus (CFBRC1, CFBRC2 and CFBRC3. The results are shown in table 8:

TABLE-US-00008 TABLE 8 Compound Mean Standard Deviation CFBRC1 C1 0.432 0.091 CFBRC1 C2 0.310 0.190 CFBRC1 C5 0.297 0.171 CFBRC1 C9 0.593 0.126 CFBRC1 C10 0.772 0.267 CFBRC1 C13 0.507 0.181 CFBRC1 C14 0.550 0.068 CFBRC1 C15 0.518 0.287 CFBRC1 B1 0.412 0.160 CFBRC1 B2 0.561 0.325 CFBRC1 MeOH 1 0 CFBRC2 C1 0.497 0.147 CFBRC2 C2 0.304 0.147 CFBRC2 C5 0.359 0.170 CFBRC2 C9 0.484 0.040 CFBRC2 C10 0.567 0.157 CFBRC2 C13 0.620 0.088 CFBRC2 C14 0.823 0.246 CFBRC2 C15 0.443 0.280 CFBRC2 B1 0.509 0.204 CFBRC2 B2 0.673 0.274 CFBRC2 MeOH 1 0 CFBRC3 C1 0.471 0.182 CFBRC3 C2 0.292 0.161 CFBRC3 C5 0.483 0.247 CFBRC3 C9 0.439 0.129 CFBRC3 C10 0.670 0.018 CFBRC3 C13 0.609 0.108 CFBRC3 C14 0.501 0.073 CFBRC3 C15 0.413 0.217 CFBRC3 B1 0.484 0.187 CFBRC3 B2 0.546 0.338 CFBRC3 MeOH 1 0

EXAMPLE 6

[0154] The anti biofilm properties of some of the claimed compounds was tested by carrying out a viable biofilm assay and measuring the growth of the microbes.

Viable Colony Biofilm Assay

[0155] C. albicans biofilms, supplemented with test and comparative compounds, were grown in 6-well plates and incubated overnight at 37 C. Briefly, C. albicans Yeast Nitrogen Base (YNB) cultures were measured at OD600 nm, diluted to 0.05 in YNB-NP supplemented with analogues, plated onto 6-well plates and incubated for 1 hr at 37 C. Media was removed, wells were washed twice with sterile PBS and supplemented with fresh YNB-NP with analogues. Plates were incubated overnight at 37 C. after which media was removed and wells washed with sterile PBS. For serial dilutions, biofilms were cell-scraped into 1 ml PBS, vortexed, and serially diluted into sterile PBS. Serial 147 dilutions were plated (100 l) onto YPD agar and incubating overnight at 37 C. Colonies were counted and recorded the next day.

C. albicans Growth Curves

[0156] Overnight C. albicans cultures grown in YNB were diluted to 0.05 in YNB supplemented with analogues. Cultures (200 l) were added to each well of a 100 well plate and grown for a 24 hr period on a Bioscreen C spectrophotometer (Growth Curves USA).

[0157] The results, shown in FIGS. 5A and 5B demonstrate how the compounds interrupt the biofilm rather than simply inhibiting growth.