Drx 400 spectrometer

Optical rotations were measured using a JASCO DIP-360 (Tokyo, Japan automatic digital polarimeter). IR and UV spectra were recorded using the JASCOFT-IR 620 spectrophotometer and UV-2600 instrument, respectively. 1D and 2D NMR spectra were recorded on Bruker DRX-400 spectrometer (400 MHz for ¹H-NMR, Karlsruhe, Germany) with TMS used as internal standard. The mass spectra were obtained on Agilent Series1100 SL mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA), and Bruker Daltonics mass spectrometer (Bruker Daltonics Inc. Billerica, MA, USA) with an electrospray ionization source. Circular Dichroism (CD) was obtained using the Chirascan, Applied Photophysics Ltd. (Surrey, UK). HPLC was performed using a system comprised of a CCPM pump (Tosoh, Tokyo, Japan), a CCP PX-8010 controller (Tosoh), an RI-8010 detector (Tosoh) or a Shodex OR-2 detector (Showa-Denko, Tokyo, Japan), and a Rheodyne injection port. A Capcell Pak C18 UG120 column (10 mm i.d. × 250 mm, 5 μm, Shiseido, Tokyo, Japan) was employed for preparative HPLC. Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, SE, USA) was used for column chromatography (CC), and a silica gel GF₂₅₄ (10–40 mm, Haiyang Co., Qingdao, China) was used for preparative TLC as precoated plates. TLC spots were visualized under UV light through dipping into 5% H₂SO₄ in alcohol. All chemicals used were analytical grade.

All reagents and solvents (analytical grade) were purchased from commercial sources and were used without further purification. The NMR spectra were collected on a Bruker DRX-400 spectrometer (400 MHz for ¹H and 101 MHz for ¹³C) with the residual solvent signal as chemical shift reference. Mass spectra were recorded on a Micromass LCT (ESI-TOF) mass spectrometer. Compound 1, 4-Phenylbutyric acid sodium salt (PBA, CAS#: 1716-12-7), was purchased from Tocris Bioscience (Bristol, UK), Pyridin-3-ylmethyl (4-((2-aminophenyl)carbamoyl)benzyl)-carbamate (5, MS-275, CAS#: 209783-80-2) and N¹-hydroxy-N⁸-phenyloctanediamide (17, Vorinostat, CAS#: 149647-78-9) was purchased from LC laboratories (Woburn, MA, USA), N-(4-Methoxybenzyl)-1,2-benzenediamine (12, CAS#: 5729-16-8) from Fluorochem Ltd (Hadfield, UK), 2-(benzyloxy)aniline (13, CAS#: 20012-63-9) from Acros Organics (Geel, Belgium), and Trichostatin A (18, CAS#: 58880-19-6) from Sigma-Aldrich Sweden AB (Stockholm, Sweden).

Optical rotations were measured on a Krüss polarimeter (A. KRÜSS Optronic GmbH, Hamburg, Germany) equipped with a 0.5 dm cell. UV spectra were recorded on a Lambda 40 UV/Vis spectrophotometer (Perkin Elmer Ltd., Beaconsfield, UK). IR spectra were obtained on an Alpha II FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany). Low-resolution EI mass spectra were measured on a Thermo Electron Corporation DSQ mass spectrometer (Thermo Electron Corporation, Austin, TX, USA) using a Direct-Exposure Probe (Thermo Electron Corporation, Austin, TX, USA). NMR spectra were recorded on a DRX 400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequences. Column chromatography separations were performed with Kieselgel 60 (Merck, Darmstadt, Germany). HPLC separations were conducted on a Pharmacia LKB 2248 liquid chromatography pump (Pharmacia LKB Biotechnology, Uppsala, Sweden) equipped with a RI-102 Shodex refractive index detector (ECOM spol. s r.o., Prague, Czech Republic) using an Econosphere Silica 10 μm (250 × 10 mm i.d.; Grace, Columbia, MD, USA) column. TLC were performed with Kieselgel 60 F₂₅₄ aluminum plates (Merck, Darmstadt, Germany) and spots were detected after spraying with 20% H₂SO₄ in MeOH reagent and heating at 100 °C for 1 min.

Melting points were measured in the open capillaries with a Stuart SMP3 melting-point apparatus (Bibby Scientific Limited, Staffordshire, UK). Two FT-IR spectrometer (Perkin-Elmer, Waltham, MA, USA) using the frustrated total internal reflection accessory with a diamond crystal. The ¹H and ¹⁹F NMR spectra were registered on a Bruker DRX-400 spectrometer (400 or 376 MHz, respectively) or a Bruker AvanceIII 500 spectrometer (500 or 470 MHz, respectively) (Bruker, Karlsruhe, Germany). The ¹³C NMR spectra were recorded on a Bruker AvanceIII 500 spectrometer (125 MHz). The internal standard was SiMe4 (for ¹H and ¹³C NMR spectra) and C₆F₆). The ¹³C chemical shifts were calibrated using the solvent signal DMSO-d₆ (δ_C 39.5 ppm). For compounds 4a-d, 8a-d, 5, 5’, 11a, 11b signals in ¹H and ¹³C spectra were assigned based on 2D ¹H-¹³C HSQC and HMBC experiments. The high-resolution mass spectra (HRMS) were recorded on a Bruker maXis impact mass spectrometer (ESI) (Bruker, Karlsruhe, Germany). The column chromatography was performed on silica gel 60 (0.062–0.2 mm) (Macherey-Nagel GmbH & Co KG, Duren, Germany). The initial ethyl-2-hydroxy-4-methyl-4-oxo-2-(trifluoromethyl)butanoate (aldol 6a) [35 (link),36 (link)] and diethyl 2,6-dihydroxy-4-oxo-2,6-bis(trifluoromethyl)heptanedioate (bis aldol 7a) [35 (link)] were synthesized by referring previously published methods.

The isolated constituents obtained by the HSCCC separation were analyzed by ¹H and ¹³C-NMR and recorded on a Brüker DRX 400 spectrometer. The chemical shifts were determined in CDCl3, using TMS as the internal standard. The signals of the NMR analyses were compared to the literature data [28 (link),29 (link)].