Unity plus 500 spectrometer

Reagents were purchased from Aldrich or Merck, of the higher grade available and used without further purification. Solvents were used as received from commercial suppliers, and no further attempts were made to purify or dry them. Melting points were determined with Electrothermal 9001 Digital Melting Point apparatus (Electrothermal, Essex, UK). Microwave oven Plazmatronika 1,000 W equipped with a single-mode cavity suitable for the microscale synthesis and microwave choked outlet connected to external condenser set to 30 % power was used (http://www.plazmatronika.com.pl). High-resolution mass spectra were recorded on Quattro LCT (TOF). ¹H NMR, ¹³C NMR, HSQC, and HMBC spectra in solution were recorded at 25 °C with a Varian Unity plus-500 spectrometer and standard Varian software was employed (Varian, Inc., Palo Alto, CA, USA). The calculated shielding constants were used as an aid in an assignment of resonances of ¹³C atoms. The CPHF-GIAO approach for the NMR shielding constants calculations using Gaussian 09 program was employed [26 (link)]. Chemical shifts δ/ppm were referenced to TMS. TLC was carried out using Kieselgel 60 F₂₅₄ sheets (Merck, Darmstadt, Germany); spots were visualized by UV (254 and 365 nm). Kieselgel 60 was used for column chromatography.

¹H NMR spectra were recorded at 25 °C with a Varian UNITY PLUS-500 spectrometer at 499.9 and 125.7 MHz respectively, using standard pulse programs from the Varian library. Two-dimensional experiments (COSY, TOCSY, HSQC, and NOESY) were performed using 1K data points, 256 increments. The mixing times for NOESY were 200–400 ms. Spectra were referred to the solvent signal.

Optical rotations were considered using JASCO DIP-370 digital polarimeter. IR spectra were obtained with JASCO FT/IR-410 spectrophotometers. ¹H and ¹³C NMR, HSQC, ¹H-¹H COSY, HMBC, and NOESY spectra were obtained using Unity plus 500 spectrometer (Varian Inc., Palo Alto, CA, USA) operating at 125 MHz for ¹³C and 500 MHz for ¹H. Chemical shifts of ¹H-NMR and ¹³C NMR are expressed in δ values referring to the solvent peak δ_H 7.19, 7.55 and 8.71, δ_C 123.5, 135.5 and 149.9 for pyridine-d₅, and coupling constants are expressed in Hz. TLC was carried out on aluminum-backed plates (Merck, Kieselgel 60 F₂₅₄, 0.25 mm) and RP-18 F_254s plates (Merck). Si-gel F254 with a particle size of 0.0045–0.075 mm mesh (Wako Pure Chemical Industries Ltd., Osaka, Japan), Cosmosil 5C18-140 PREP (Nacalai tesque, No.379-34), and Sephadex LH-20 (Sigma-Aldrich, Darmstadt, Germany) were used as stationary phases for column chromatography. High resolution FABMS were obtained using JMS DX-303 spectrometer (JEOL Ltd., Tokyo, Japan). Preparative HPLC was utilized using a Develosil C-30-UG-5 (250 × 4.6 mm i.d Nomura Chemical Co., Aichi, Japan) adjusting the rate of flow at 1.5 mL/min, and a TOSOH RI-8020 detector.

Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F₂₅₄, 0.25 mm) and precoated RP-18 F_254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High resolution FAB-MS and ESI-MS were recorded on JEOL JMS700N and JMS-100TD, respectively. ¹H- and ¹³C-NMR, ¹H-¹H COSY, NOESY, HSQC, and HMBC spectra were recorded with a Unity plus 500 spectrometer (Varian Inc., U.S.A.) operating at 500 MHz for ¹H and 125 MHz for ¹³C, respectively. ¹H-NMR chemicals shifts are expressed in δ values referring to the solvent peak δ_H 2.49 for DMSO and coupling constants are expressed in Hz. ¹³C-NMR chemical shifts are expressed in δ values referring to the solvent peak δ_C 39.5 for DMSO. Piperonylic acid was purchased from commercial sources (TCI) and used without further purification.