Design and synthesis of biphenyl and biphenyl ether inhibitors of sulfatases

Two series of inhibitors of sulfatase 2, ARSA and ARSB were designed based on biphenyl and biphenyl ether scaffolds substituted with e.g. sulfamate and carboxylate groups.


Sulf-2 assay protocol
Compounds were screened using 4-MUS as a substrate for Sulf-2 according to a protocol described by Morimoto-Tomita et al. 1 Briefly 293T cells were transiently transfected with pcDNA3.1/Myc-His(-)-HSulf-2 DNA (Addgene) and TransIT-LT1 Transfection Reagent (Mirus) using a transfection mixture at the ratio 1:3 (µg DNA: µL transfection reagent) in Opti-MEM I reduced serum medium (Gibco). Conditioned medium containing Sulf-2 was collected after 3 days and bound to HIS-Select Nickel affinity gel (Sigma) overnight at 4 °C. Beads were washed three times with washing buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 0.05% Tween 20, followed by washing once with washing buffer containing no tween. Beads were suspended in 50 mM Hepes (pH 7.5) and used in inhibition assays. 20 μL of bead slurry was incubated with 1 mM compound (in DMSO) plus 10X reaction buffer (500 mM HEPES pH 7.5, 100 mM CaCl 2 ) for 1 h at 37 °C. The reaction was started by the addition of 20 μL of 20 mM 4-MUS (final concentration of 8 mM) and incubated at 37 °C for 1 h. The reaction was stopped with 100 μL 1 M Tris buffer (pH 10.4) and read at 460 nm following excitation at 355 nm in FLUOstar Omega plate reader (BMG Labtech) using Omega data analysis software.

ARSA and ARSB assay protocols
Compounds were screened in a 96-well black plate (Sterilin) using 4-MUS as a substrate, using 50 μL reaction mixture containing 40 ng of the commercially available enzymes (ARSA or ARSB from R & D Systems), 50 mM HEPES (pH = 4.5), 10 mM CaCl 2 , 1 mM test compound (dissolved in DMSO; final concentration of DMSO in reaction = 2%), and H 2 O (45 μL). The assay mixture was incubated for 1 h at 37 °C, followed by addition of 5 μL of 4-MUS (Km = 1.6 mM for ARSA and 612 μM for ARSB), and incubation for a further 1 h at 37 °C. The reaction was stopped with 100 μL of 1 M Tris (pH = 10.5) and read at 460 nm following excitation at 355 nm in FLUOstar Omega plate reader (BMG Labtech) using Omega data analysis software.

3-Summary of Generic Analytical and Chromatographic Conditions
All commercial reagents were purchased from Sigma-Aldrich Chemical Company, Alfa Aesar, Apollo Scientific or Tokyo Chemical Industry UK Ltd. The chemicals were of the highest available purity. Unless otherwise stated, chemicals were used as supplied without further purification. Anhydrous solvents were obtained from AcroSeal TM or Aldrich SureSeal™ bottles and were stored under nitrogen. Petrol refers to the fraction with a boiling point between 40 and 60 °C.
Thin layer chromatography utilised to monitor reaction progress was conducted on plates pre-coated with silica gel Merck 60F 254 or Merck NH 2 F 254S . The eluent was as stated (where this consisted of more than one solvent, the ratio is stated as volume:volume) and visualisation was either by short wave (254 nm) ultraviolet light, or by treatment with the visualisation reagent stated followed by heating. 'Flash' medium pressure liquid chromatography (MPLC) was carried out either on a Biotage SP4 automated purification system or a Varian 971-FP automated purification system, using pre-packed Varian or Grace silica or amino-bonded silica cartridges.
All reactions carried out in a microwave were performed in a Biotage Initiator with Sixty robot.
Melting points were determined using a VWR Stuart SMP40 apparatus and are uncorrected. 1 H, 13 C and 19 F nuclear magnetic resonance (NMR) spectra were obtained as either