Fluorine-containing substituents: metabolism of the α,α-difluoroethyl thioether motif

We report the metabolism of the recently introduced α,α-difluoroethyl thioether motif to explore further its potential as a substituent for bioactives discovery chemistry. Incubation of two aryl–SCF2CH3 ethers with the model yeast organism Cunninghamella elegans, indicates that the sulfur of the thioether is rapidly converted to the corresponding sulfoxide, and then significantly more slowly to the sulfone. When the substrate was (p-OMe)PhSCF2CH3, then the resultant (demethylated) phenol sulfoxide had an enantiomeric excess of 60%, and when the substrate was the β-substituted-SCF2CH3 naphthalene, then the enantiomeric excess of the resultant sulfoxide was 54%. There was no evidence of defluorination, unlike the corresponding oxygen ether (p-OMe)PhOCF2CH3, which was converted to the (demethylated) phenol acetate ester during C. elegans incubation. We conclude that the aryl–S–CF2CH3 motif is metabolised in a similar manner to aryl–SCF3, a motif that is being widely explored in discovery chemistry. It is however, significantly less lipophilic than aryl-SCF3 which may offer a practical advantage in tuning overall pharmacokinetic profiles of molecules in development.

All the thioethers, sulfones and sulfoxides used were synthesised in the laboratory of Prof. David O'Hagan, either as published before (Tomita et al., 2018) or as detailed in the following sections. The rest of the chemicals, media and materials were bought from Sigma-Aldrich or Alfa Aesar. Fungus Cunninghamella elegans was originally donated by Dr. Cormac Murphy (University College Dublin, Dublin, Ireland), and stored as an agar gel at 4 °C, from which our current agar gels were prepared.
All the glassware, materials and media used for microbiological purposes were sterilised by autoclaving prior to their use. The aseptic conditions were maintained during the preparation, growth and incubation of the fungal cultures.
All the glassware used for chemical synthesis was oven-dried, cooled and used under nitrogen atmosphere, unless otherwise stated.
The progress of reactions was followed by thin-layer chromatography (TLC), using aluminium plates coated with silica gel (60F245 Merck). The TLC plates were examined under UV light at 254 and 266 nm, before being visualised with alkaline potassium permanganate.
Crude extracts were analysed by 1 H and 19 F NMR. Proton and proton decoupled ( 19 F{ 1 H}, 13 C{ 1 H}) nuclear magnetic resonance spectra were recorded on Bruker Avance III 500 or Bruker Avance III 500 HD spectrometers (500 MHz 1 H, 476 MHz 19 F, 126 MHz 13 C). Bidimensional correlation spectra were also analysed for the correct assignment of signals. Chemical shifts (δ) are expressed in ppm, and quoted relative to the residual solvent signal. Proton coupling constants (J) are given in Hz, and quoted to the nearest 0.1 Hz. Identical coupling constants are averaged.
High-resolution mass spectrometry was acquired using electrospray ionisation (ESI), on a ThermoFisher Excalibur Orbitrap Spectrometer, operating in positive and negative mode, from solutions of the analyate in methanol or acetonitrile. Mass analysis was done at the University of St Andrews Mass Spectrometry facility by Mrs. Caroline Horsburgh. Mass units are reported in Daltons (Da).
X-ray crystal structures were obtained on a Rigaku XtaLAB P200 diffractometer, using multi-layer mirror monochromed Mo-Kα radiation, by Prof. Alexandra Slawin (University of St Andrews). The data was analysed using CrystalMaker.

Preparation of cultures and inoculation of xenobiotics
Sterile Saboraud Dextrose Medium (SBD, 50 mL) was inoculated with a piece of fungal agar plate (1 cm x 1 cm) at room temperature. The cultures were left to grow for 72 h at 28 o C and rotary agitation (180 rpm). After 72 h, the corresponding thioethers were added dissolved in DMF solution (5-10 mg in 50 µL) to the grown cultures, and left to incubate for further 72 h at 28 °C and 180 rpm.

Extraction and purification of the metabolites
After incubation, the fungal biomass was separated from the liquid culture, and the supernatant was extracted with ethyl ether (3 × 50 mL) and DCM (3 × 50 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The extracts were analysed by 1 H and 19 F NMR before further purification.
Purification of the metabolites was carried out by reversed-phase HPLC, using an eluent system of 60:40 AcCN/water (both supplemented with 0.05% TFA), at a flow rate of 1 mL/min. Structural analysis of the resulting metabolites and remaining starting materials was carried out by full NMR characterisation ( 1 H, 19 F, 13 C, COSY, HSQC and HMBC) and accurate mass spectrometry. X-ray structures were obtained when possible.

Thio-and oxyether metabolism
3.1 (1,1-Difluoroethyl)(4-methoxyphenyl)sulfane (4) (1,1-Difluoroethyl)(4-methoxyphenyl)sulfane (4) was dissolved in DMF (50 μL) and added to a mature culture of C. elegans. The compound was left to incubate with the fungus for 72 h. at 28 ºC and 180 rpm. After 72 h, the fungus was centrifuged down, and the supernatant was extracted with DCM (3 x 50 mL) and Et2O (3 × 50 mL). The combined organic phases were dried over Na2SO4, filtered and evaporated under reduced pressure. Purification of the metabolite and residual starting material was achieved by reversed-phase HPLC, in a Phenomenex Luna, and eluting with a mixture of 60:40 AcCN:water, both supplemented with 0.05% of TFA, at a flow rate of 1 mL/min. This afforded 6 at a tR = 24 min, 7 at a tR = 16 min and 8 at a tR = 21 min.   3.2 (1,1-Difluoroethyl)(naphthalene-2-yl)sulfane (5) (1,1-Difluoroethyl)(naphthalene-2-yl)sulfane (5) was dissolved in DMF (50 μL) and added to a mature culture of C. elegans. The compound was left to incubate with the fungus for 72 h. at 28 ºC and 180 rpm. After 72 h, the fungus was centrifuged down, and the supernatant was extracted with DCM (3 × 50 mL) and Et2O (3 × 50 mL). The combined organic phases were dried over Na2SO4, filtered and evaporated under reduced pressure. Purification of the metabolite and residual starting material was achieved by reversed-phase HPLC, in a Phenomenex Luna, and eluting with a mixture of 60:40 AcCN:water, both supplemented with 0.05% of TFA, at a flow rate of 1 mL/min. This afforded 11 at a tR = 37 min, 12 at a tR = 13 min and 13 at a tR = 17 min.