Merck 60 f254

Phytochemistry analysis. ¹H 400 MHz and ¹³C 100 MHz NMR spectra were measured with Bruker 400 spectrometer (Bruker^®, Billerica, MA, USA). Chemical shifts were referenced to the residual solvent signal (C₃D₆O: δ_H = 2.05, δ_C = 206.7, 29.9 and CDCl₃: δ_H = 7.25, δ_C = 77.0, CD₃OD: δ_H = 3.31, δ_C = 49.00). Silica gel 60 (70−230 mesh), RP C-18 silica gel, and Celite^® 545 particle size 0.02–0.1 mm, pH 10 (100 g/L, H₂O, 20 °C), used for low-pressure chromatography and vacuum chromatography was purchased from Macherey-Nagel (Düren, Germany). Purifications were monitored by TLC on Merck 60 F254 (0.25 mm) plates, visualized by staining with 5% H₂SO₄ in EtOH and heating. Chemical reagents and solvents were from Aldrich (Darmstadt, Germany) and were used without any further purification unless stated otherwise. Flash chromatography Isolera One with DAD (Uppsala, Sweden), HPLC JASCO Hichrom, 250 × 25 mm, silica UV−vis detector-2075 plus (Oklahoma, Japan).

Non-psychoactive Cannabis sativa L. plant material, belonging to the III chemotype, was purchased from Canvasalus Srl (Monselice, Italy). A voucher specimen (Cs-CBD/03/2021) of the vegetal material is stored in Novara laboratories.
CBN (1) was synthetized starting from 100 mg of CBD (2) (Figure 1, created with the software ChemDraw 19), obtained by extraction and purification from non-woody C. sativa aerial parts, following the protocol described by [13 (link)].
The reaction led to obtaining 80 mg of residue that was further purified by HPLC (silica petroleum ether—EtOAc gradient from 95:5 to 85:15) affording 67 mg of pure CBN (1) identified by ¹H NMR data (Figure S1), according to literature [14 (link)]. ¹H 400 MHz NM spectra were measured on Bruker 400 spectrometers (Bruker^®, Billerica, MA, USA). Chemical shifts were referenced to the residual solvent signal (CDCl₃: δH = 7.26). Silica gel 60 (70–230 mesh), used for low-pressure chromatography, was purchased from Macherey-Nagel (Düren, Germany). Purifications were monitored by TLC on Merck 60 F254 (0.25 mm) plates, visualized by staining with 5% H₂SO₄ in EtOH and heating. Chemical reagents and solvents were from Aldrich (Darmstadt, Germany) and were used without any further purification unless stated otherwise. HPLC JASCO Hichrom, 250 mm × 25 mm, silica UV–vis detector-2075 plus (Oklahoma, Japan).

Mass spectral data [Electrospray ionization mass spectrometry (ESI-MS)] were measured on a Waters Synapt HDMS spectrometer. NMR Spectra were recorded with an Agillent spectrometer at 400 MHz. Chemical shifts (δ) were quoted in parts per million (ppm) from the internal standard tetramethylsilane (TMS). Deuterated dimethyl sulfoxide (DMSO-d₆), was used as solvent for the NMR experiments. Column chromatography was performed on silica gel Merck 60 F₂₅₄ [(0.2–0.5 mm) and (0.2–0.063 mm)] 70–230 and 230–400 mesh (Darmstadt, Germany). Pre-coated silica gel 60 F₂₅₄ thin layer chromatography (TLC) plates (Merck, Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm) and then sprayed with 50% sulphuric acid (H₂SO₄) followed by heating to 100 °C.

All the reagents were purchased from Merck (Darmstadt, Germany) or Alfa Aesar (Tewksbury, MA, USA) and were used as received. Melting points were obtained using a Gallenkamp (G) (Fiorano Modenese, Italy) melting point apparatus. The structures of final compounds were unambiguously assessed by ¹H NMR (nuclear magnetic resonance) and ¹³C NMR. Spectra were recorded in the indicated solvent (Chloroform- CDCl₃, Dimethyl Sulphoxide-DMSO-d₆) at 25 °C on a Bruker 300 MHz spectrometer (Bruker, Milano, Italy) or a Bruker Advance DPX400 employing TMS (tetramethyl silane)as internal standard. Chemical shifts are expressed in δ values (ppm) and coupling constants (J) in hertz (Hz). IR spectra were recorded on a PerkinElmer machine 10.4.00 (PerkinElmer, Milan, Italy). Reactions were monitored by TLC (thin layer chromatography) on silica gel plates Merck 60 F254 (Merck, Burlington, MA, USA). Final products were purified by a flash chromatography system with column chromatography, using Merck 60 silica gel, 230–400 mesh. Elemental analyses were performed on Leco Trunspec CHNS Micro elemental system (St. Joseph, MI, USA). The purity of final compounds was evaluated by C, H, N analysis, and it was confirmed to be ≥95%.

Thalli were cleaned and washed with distilled water, and dried at 60 °C. EP was extracted in acetone at room temperature (20 °C ± 2°) for 48 and 24 h successively. The extract was then purified chromatographically using a silica gel Merck 60 G (0.032–0.063 nm) column eluted with a mixture of hexane and ethyl-acetate with increasing polarity. The fractions were monitored by thin-layer chromatography (TLC) using silica gel Merck 60 F₂₅₄ plates. The blots were developed using a H₂SO₄ spray reagent and UV light (254/365 nm).

Nuclear magnetic resonance (NMR) spectra were recorded in CDCl₃ (chloroform-d) or benzene-d₆ solutions (unless indicated otherwise); chemical shifts are reported in the δ scale in ppm, with the solvent signal as the internal standard (CDCl₃, ¹H NMR 7.26 ppm; ¹³C NMR 77.00 ppm, benzene-d₆, ¹H NMR 7.16 ppm, ¹³C NMR 128.06 ppm). Column chromatography was performed on Merck silica gel 60, 230–400 mesh (Darmstadt, Germany). Thin layer chromatography (TLC) was performed on aluminum sheets, Merck 60 F₂₅₄ (Darmstadt, Germany). Anhydrous solvents were obtained by distillation over calcium chloride (CaCl₂) (DCM, Toluene). All reactions were performed under argon (Ar) in pre-dried glassware using Schlenk techniques.