Synthesis and biological activity of methylated derivatives of the Pseudomonas metabolites HHQ, HQNO and PQS

Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine–metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively.


Assay on the formation of static biofilms
To evaluate the influence of AQ derivatives on the formation of static biofilms, the method of O'Toole was slightly modified [1]. In brief, cultures of S. aureus Newman were adjusted to an OD 600 nm of 0.1 in LB and 100 µL were used to inoculate 96-well microtiter plates. After incubation for 24 hours at 37 °C without shaking, the biofilm was stained with 0.1 % (w/v) crystal violet and the absorbance at 560 nm (A560 nm) was measured. Prior to staining, OD 600 nm was measured, indicating the total biomass. By normalizing the biofilm to the total biomass, effects of AQs on biofilm formation rather than growth are indicated. Analyses included six biological replicates (n = 6), each measured in quadruplicate. Figure S1: Effect of AQs on biofilm formation by S. aureus. Cell suspensions were incubated for 24 h with 100 µM of the indicated AQ (1-12), PQS (P) or an equal amount of DMSO as the control (C). The total biofilm mass, as measured by crystal violet staining, was normalized to the cell density measured prior to biofilm staining. Box-plot shows the medians (center lines), the means (squares), interquartile ranges (box), and 1.5 × interquartile ranges (whiskers) of 6 biological replicates (n = 6) with 4 technical repeats per replicate. The Mann-Whitney test was used for statistical analysis, but no significant differences (P < 0.05) were found. Figure S2: Competition of AQ derivatives with PQS for the binding to PqsR, the transcriptional regulator of the AQ biosynthesis gene cluster. The bioreporter strain Pseudomonas putida KT2440 mini-Tn7T::pqsR-P pqsA -lux harbors the gene pqsR (constitutively expressed) for the transcriptional regulator PqsR, which upon binding of PQS (or HHQ) upregulates the expression of the AQ biosynthesis gene cluster, and the luciferase genes luxCDABE under control of the PqsR-responsive pqsA promoter [2]. The bioreporter was used to verify that the newly synthesized AQ derivatives have direct impact on the PqsR-dependent transcription and to identify agonistic or antagonistic effects. To this end, cultures of the bioreporter strain (OD 600 nm of 0.2 in LB) were supplemented with 0.5 µM of PQS, and additionally with 10 µM of the indicated AQ (1-12), PQS (P), or an equal amount of DMSO (C). After 4 h of incubation at 20 °C, Lux-based bioluminescence was measured and normalized to the OD 600 nm of the culture (relative luminescence). Bars and errors represent the means and standard errors of biological triplicates.

General information
Manipulations involving air-or moisture-sensitive reagents were carried out under argon and performed by using standard Schlenk-techniques. Dry and oxygen-free solvents were employed for such reactions. Solvents for extraction and flash chromatography were distilled before use. Commercial compounds were purchased from standard suppliers (ABCR, Acros, Alfa Aesar, Sigma-Aldrich or TCI) and used without further purification. Flash chromatography (FC) was carried out on Acros silica gel (0.035-0.070 mm; 60 Å) under slight overpressure. IR spectra were recorded with a Digilab FTS 4000 with an attenuated total reflectance (ATR) unit. NMR spectroscopic data were recorded on a Bruker DPX-300 (300 MHz), Bruker AV400 (400 MHz) or Agilent DD2 600 (600 MHz). Chemical shifts (δ) are reported in ppm relative to SiMe 4 ( 1 H, 13 C) and referenced to either the residual solvent signal for 1 H NMR or the solvent signal for 13 C NMR (δ = 7.26/77.2 ppm for CDCl 3 ; δ = 2.50/39.5 ppm for DMSO-d 6 ). NMR multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, sept = septet, m = multiplet, br = broad signal. Mass spectra with electrospray ionization (MS-ESI-EM, m/z) were recorded on a Bruker MicroTof or a Thermo Scientific Orbitrap LTQ XL (Nanospray).

Methyl 3-oxodecanoate [3,4]
Meldrum's acid (28.8 g, 200 mmol, 1.0 equiv) was dissolved in dry CH 2 Cl 2 (200 mL) and pyridine (32 mL, 400 mmol, 2.0 equiv) and DMAP (488 mg, 4 mmol, 0.02 equiv) was added. The solution was cooled to 0 °C and octanoyl chloride (37.6 mL, 220 mmol, 1.1 equiv) was added dropwise at this temperature. The solution was allowed to warm to rt and stirred for 2 h. Aqueous HCl was added (2 M, 200 mL) and the phases were separated. The organic phase was washed with aqueous HCl (2 M, 2 × 200 mL) and water (2 × 200 mL), dried over sodium sulfate and the solvent was removed in vacuo. The crude product was dissolved in MeOH (160 mL) and heated to 65 °C for 16 h. The solvent was removed in vacuo and the residue was dissolved in EtOAc (200 mL). The solution was washed with aqueous K 2 CO 3 (10% in H 2 O, 2*80 mL) and water (80 mL), dried over sodium sulfate and the solvent was removed in vacuo. The residue was distilled under vacuum using a Vigreux-column to give the product (bp 75 °C at 0.01 Torr, 20.3 g, 101 mmol, 51%) as a colourless oil.

2-Heptyl-4(1H)-quinolone (HHQ, 1) [3,4]
Methyl 3-oxodecanoate (20.3 g, 101 mmol, 1.00 equiv) was dissolved in toluene (100 mL). Aniline (9.7 mL, 106 mmol, 1.05 equiv) and p-toluenesulfonic acid monohydrate (386 mg, 2 mmol, 0.02 equiv) were added and the solution was heated to 110 °C for 3 h (until no water was formed anymore) using a Dean-Stark apparatus. After cooling to rt the solvent was removed in vacuo (removal of traces of toluene under high vacuum is recommended to facilitate the next step) to provide a yellowish oil. In a two-neck round-bottom flask equipped with a condenser and a dropping funnel diphenyl ether (25 mL) was heated to boiling temperature (258 °C). The yellowish oil from the first step was dissolved in diphenyl ether (10 mL) and added dropwise to the refluxing diphenyl ether via the dropping funnel over a period of 30 min. The resulting solution was heated to 258 °C for another 30 min. The solution was allowed to cool to rt and heptane (70 mL) was added when the solution had cooled to about 50 °C. The solution was allowed to stand at rt for 14 h. The white precipitate was filtered off, washed with heptane (35 mL) and suspended in heptane (70 mL). The suspension was heated to 98 °C for 1 h, filtered and the white solid was collected and dried under vacuum to provide the product (10.7 g, 44 mmol, 44%

N-Hydroxy-2-heptyl-4(1H)-quinolone (HQNO) [5]
HHQ (1944 mg, 8.0 mmol, 1.0 equiv) was dissolved in THF (40 mL) and KOt-Bu (1120 mg, 10.0 mmol, 1.25 equiv) was added at rt. The solution was stirred at rt for 30 min, Boc 2 O (1918 mg, 8.8 mmol, 1.1 equiv) was added and the solution was stirred at rt for 14 h. The reaction mixture was diluted with EtOAc (120 mL), washed with water (100 mL), dried over MgSO 4 and the solvent was removed in vacuo. The crude product was dissolved in CH 2 Cl 2 (40 mL), cooled to 0 °C and mCPBA (wt.75%, 2024 mg, 8.8 mmol, 1.1 equiv) was added at this temperature. The solution was stirred for 4 h at 0 °C. CH 2 Cl 2 (40 mL) was added, the solution was washed with aqueous, saturated Na 2 CO 3 solution (2 × 60 mL) and water (60 mL), dried over MgSO 4 and the solvent was removed in vacuo. The crude product was dissolved in EtOH (40 mL) and aqueous KOH-solution (5 M, 20 mL) was added at rt. The solution was stirred at rt for 1 h. Water (120 mL) was added and the pH of the solution was adjusted to about pH 2 using concentrated aqueous HCl. The resulting white precipitate was filtered off and crystallized from EtOH/water (4:1) to give the product (1432 mg, 5.5 mmol, 69%) as a crystalline white solid.