Synthesis and Characterization of Arylquin Inhibitors

Nutlin-3a, an inhibitor of MDM2 that is reported to bind directly to MDM2, release, stabilize and activate p53 ^{10 (link)}, was acquired from Cayman Chemical Company. Brefeldin A, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone(zVAD-fmk) and other chemicals were purchased from Sigma Aldrich or Fisher Scientific or were synthesized according to literature procedures. The synthesis of Arylquin 1, which utilized 4-(N,N-dimethylamino)-2-aminobenzaldehyde in a Friedländer condensation with 2-fluorophenylacetontrile ¹⁵ , and other heterocyclic families is described in Supplementary Note. The condensation of 2-amino-4-(N,N-dimethylamino)benzaldehyde with 2-(2-fluorophenyl)acetyl chloride secured 7-(dimethylamino)-3-(2-fluorophenyl)quinolin-2(1H)-one, and treatment with Lawesson's reagent ¹⁶ provided 7-(dimethylamino)-3-(2-fluorophenyl)quinoline-2(1H)-thione. S-alkylation of this intermediate with (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine led to biotinylated Arylquin 9 (Supplementary Note). Solvents were used from commercial vendors without further purification unless otherwise noted. Nuclear magnetic resonance spectra were determined on a Varian instrument (¹H, 400MHz; ¹³C, 100Mz). High resolution electrospray ionization (ESI) mass spectra were recorded on a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The FT resolution was set at 100,000 (at 400 m/z). Samples were introduced through direct infusion using a syringe pump with a flow rate of 5 µL/min. MALDI mass spectra were obtained on a Bruker Utraflexstreme time-of-flight mass spectrometer (Billerica, MA), using DHB (2,5-dihydroxybenzoic acid) matrix. Purity of compounds was established by combustion analyses by Atlantic Microlabs, Inc., Norcross, GA. Compounds were chromatographed on preparative layer Merck silica gel F254 unless otherwise indicated.