Plant material
The plant material, P. grandidentatus Gürke was cultivated in Parque Botânico da Tapada da Ajuda (Instituto Superior Agrário, Lisbon, Portugal) from cuttings obtained from the Kirstenbosch National Botanical Garden (Cape Town, South Africa). Voucher specimens (572/2008) were deposited in Herbarium João de Carvalho e Vasconcellos (ISA). The plant name has been checked with http://www.theplantlist.org.46
Extraction And Isolation
Acetone ultrasonic-assisted extraction was adapted from Bernardes C.E.S. et al,27 P. grandidentatus (2.334 Kg) were extracted with acetone (15 x 3.4 L), at room temperature, in presence of ultrasounds. The ultrasonic bath (Sonorex Super RK 510 H; Bandelin, Berlin, Germany) operated for 30 minutes, for 3 times, at 35 Hz with maximum input power of 320 W. Filtration and evaporation of the solvent (under vacuum, 40°C) yielded a residue of 52.74 g (2.3% w/w).
Supercritical fluid extraction was carried out in an apparatus, equipped with a 500 cm3 sample cell, which had been previously described by Pereira et al.47 a sample of 130.11 g of powder plant was extracted with supercritical CO2 for 4 h at 40°C and 230 bar, using a fixed CO2 flow rate of 0.3 kg⋅h− 1. The supercritical fluid extract was recovered by washing the collection vessel and tubing the expansion line with acetone. The solvent was subsequently removed in a rotary evaporator and yielded a residue of 4.67 g (3.6% w/w).
The isolation process was adapted from Bernardes et al.27 The crude extract was subjected to sequential liquid and dry flash chromatographic separations. The liquid flash chromatographic column used silica gel (Merck 9385) as stationary phase and CH2Cl2 as eluent. The dry flash chromatography used silica gel (Merck 9385) as stationary phase and mixtures of Hex:AcOEt and AcOEt:MeOH as eluents. The fractions obtained were compared with one sample of Roy by TLC (Eluent Hex: AcOEt 90:10, 80:20, or 70:30). Roy was obtained from recrystallization from Hex.
Quantification Of Roy By Hplc-dad
The quantification of Roy in P. grandidentatus extracts was adapted from Matias et al. 2019.34 The The quantification was performed using HPLC-DAD and complementary spectroscopic methodologies. The quantification of the identified compound was carried out in a Dionex Ultimate 3000 UHPLC system with diode array detector (DAD; Thermo Fisher Scientific Inc. MA, USA), equipped with a Nucleodur 100-5 C18ec, (250 x 4.0 mm i.d., 5 µm) column, from Macherey-Nagel and Thermo Scientific™ Chromeleon™ 7.3 Chromatography Data System software (Thermo Fisher Scientific Inc. MA, USA). Each sample was analysed (after 20 µL injection) and a gradient elution mixture composed of solution A (methanol), solution B (acetonitrile), and solution C (0.3% trifluoroacetic acid in water) was used as follows: 0 min, 15% A, 5% B, and 80% C; 10 min, 70% A, 30% B, and 0% C; 25 min, 70% A, 30% B, and 0% C; 28 min, 15% A, 5% B, and 80% C; and 31 min, 15% A, 5% B, and 80% C. The flow rate was set at 1 mL⋅min− 1. Compound identification was based on retention time. The time of analysis was 31 min, including the stabilization of the RP-18 column. For quantification and identification purposes, Roy was detected using chromatograms corresponding to 270 nm, and its content in the plant extracts was estimated from the peak areas based on a calibration curve obtained with an authentic standard of Roy. All analyses were performed in triplicate.
Synthesis General Procedure
For the general procedure, Roy (around 20 µmol) was dissolved in of dichloromethane (DCM), 2 mL) or pyridine (0.5-1 mL) with stirring (400 rpm) in a 5 mL round bottom flask, at room temperature, under heating or in an ice bath. When DCM is used as solvent, excess of pyridine was added (2.5–18 eq). Then, benzoyl chloride (or acetic anhydride) (1-100 eq) was added to the reaction flask. Reactions were followed by TLC (eluent CH2Cl2:Acetone, 98:2) until total consumption of Roy, then concentrated under reduced pressure. Products purification was performed by preparative chromatography, using as the eluent a mixture of CHCl3:Acetone (99:1) to purify RoyBz and CH2Cl2:Acetone (99:1) for Roy-12-Bz, Roy-12-Ac and, RoyAc.
Compounds Characterization
Roy-12-Ac (7α-acetoxy-6β-hydroxy-12- O -acetylroyleanone): Yellow amorphous solid, 87%. IR λmax: 3525, 2969, 2932, 2872, 1778, 1742, 1728, 1668, 1461, 1370, 1276, 1229, 1184, 1157, 139, 1109, 1022, 966, 900, 819, 736, 644 cm− 1. 1H NMR (300 MHz, Chloroform-d, ppm): δ 5.65 (d, J = 1.6 Hz, 1H, H-7β), 4.32 (s, 1H, H-6α), 3.11 (sept, J = 7.1 Hz, 1H, H-15), 2.51 (d, J = 13.3 Hz, 1H, H-1β), 2.34 (s, 3H, 12- OAc), 2.05 (s, 3H, 7α-OAc), 1.85–1.76 (m, 1H, H-2β), 1.63 (s, 3H, Me-20), 1.58–1.50* (m, 1H, H-2β), 1.46 (d, J = 14.2 Hz, 1H, H-3β), 1.34 (s, 1H, H-5α), 1.22* (s, 3H, Me-19), 1.21* (s, 3H, Me-17), 1.20* (s, 1H, H-3α+), 1.19* (s, 3H, Me-16), 1.18* (s, 1H, H-1α+), 0.94 (s, 3H, Me-18). 13C NMR (75 MHz, Chloroform-d, ppm): δ 185.96 (C-14), 179.92 (C-11), 169.78 (7α-COCH3), 168.42 (12-COCH3), 153.06 (C-9), 149.54 (C-12), 139.46 (C-13), 135.84 (C-8), 68.36 (C-7), 68.03 (C-6), 50.74 (C-5), 39.06 (C-10), 33.87 (C-4), 33.84 (C-18), 26.11 (C-15), 24.59 (12-COO), 21.86 (C-19), 21.23 (C-20), 20.65 (7α-COCH3), 19.58 (C-16), 19.55 (C-17).
RoyAc (7α,6β-diacetoxy-12- O -acetylroyleanone): Yellow amorphous solid, 48%. 1H NMR (300 MHz, Chloroform-d, ppm): δ 5.70 (d, J = 1.9 Hz, 1H, H-7β), 5.50 (s, 1H, H-6α), 3.19–3.02 (m, 1H, H-15), 2.55 (d, J = 13.0 Hz, 1H, H-1β), 2.35 (s, 3H, 12-OAc), 2.05 (s, 3H, 6β-OAc), 2.04 (s, 3H, 7α-OAc), 1.84–1.77 (m, 1H, H-2β), 1.61 (s, 3H, Me-20), 1.53–1.43* (m, 3H, H-2α, H-3β, H-5α), 1.28* (s, 2H, H-1α, H-3α), 1.19 (dd, J = 7.0 Hz, 6H, Me-16, Me-17), 1.00 (s, 3H, Me-19), 0.99 (s, 3H, Me-18).
Roy (7α-acetoxy-6β-hydroxyroyleanone): Yellow crystals. 1H-NMR (300 MHz, Chloroform-d, ppm): δ 7.22 (s, 1H, 12-OH), 5.66 (dd, J = 2.2, 0.7 Hz, 1H, H-7β), 4.31 (s, 1H, H-6α), 3.16 (sept, J = 7.1 Hz, 1H, H-15), 2.63 (d, J = 12.8 Hz, 1H, H-1β), 2.04 (s, 3H, Me-7α-OAc), 1.89– 1.78 (m, 1H, H-2β), 1.61 (s, 3H, Me-20), 1.55–1.46* (m, 2H, H-2α and H-3β), 1.33 (s, 1H, H-5α), 1.23* (s, 3H, Me-19), 1.22* (s, 3H, Me-17), 1.21* (s, 1H, H-3α+), 1.20* (s, 3H Me-16), 1.18* (s,1H H-1α+), 0.94 (s, 3H, Me-18) ppm. 13C-NMR (75 MHz, Chloroform-d, ppm): δ 185.91 (C11), 183.40 (C14), 169.83 (7α-COCH3), 151.04 (C12), 150.04 (C9), 137.19 (C8), 124.76 (C13), 68.86 (C7), 67.06 (C6), 49.86 (C5), 42.39 (C3), 38.75 (C10), 38.55 (C1), 33.80 (C18), 24.28 (C15), 23.94 (C19), 21.60 (C20), 21.08 (7α-COCH3), 19.97 (C16), 19.84 (C17), 19.10 (C2) ppm.
Roy-12-Bz (7α-acetoxy-6β-hydroxy-12- O -benzoylroyleanone): Yellow amorphous solid, 69%. 1H-NMR (300 MHz, Chloroform-d, ppm): δ 8.15 (d, J = 7.1 Hz, 2H, H-23), 7.72–7.61 (m, 1H, H-25), 7.52 (t, J = 7.6 Hz, 2H, H-24), 5.70 (d, J = 1.7 Hz, 1H, H-7β), 4.34 (s, 1H, H-6α), 3.19 (sept, J = 6.9 Hz, 1H, H-15), 2.52 (d, J = 9.4 Hz, 1H, H-1β), 2.07 (s, 3H, Me-7α-OAc), 1.88–1.71 (m, 1H, H-2β), 1.64 (s, 3H, Me-20), 1.60–1.52 (m, 1H, H-2α), 1.51–1.42 (m, 1H, H-3β), 1.38 (s, 1H, H-5α), 1.28–1.19* (m, 11H, Me-19, Me-17, H-3α, H-1α, Me-16), 0.95 (s, 3H, Me-18).
RoyBz (7α-acetoxy-6β-benzoyloxy-12- O -benzoylroyleanone): Yellow crystals, 79%. 1H-NMR (300 MHz, Chloroform-d, ppm): δ 8.16 (d, J = 7.3 Hz, 2H, H-23), 8.00 (d, J = 7.4 Hz, 2H, H-28), 7.69 (t, J = 7.4 Hz, 1H, H-30), 7.61–7.57 (m, 1H, H-25), 7.54 (t, J = 7.4 Hz, 2H, H-29), 7.43 (t, J = 7.3 Hz, 2H, H-24), 5.90 (d, J = 1.9 Hz, 1H, H-7β), 6.37 (s, J = 1.9 Hz, 1H, H-6α), 3.19 (sept, J = 7.0 Hz, 1H, H-15), 2.67–2.53 (m, 1H, H-1β), 2.11 (s, 3H, Me-7α-OAc), 1.84–1.82 (m, 1H, H-2β), 1.78 (s, 3H, Me-20), 1.64–1.55* (m, 2H, H-2α and H-5α), 1.51–1.46 (m, 1H, H-3β), 1.42–1.36 (m, 1H, H-3α), 1.33–1.22* (m, 7H, H-1α, Me-16 and Me-17), 1.07 (s, 3H, Me-18), 1.00 (s, 3H, Me-19).
DiRoy (7α,6β-dihydroxyroyleanone): Yellow needles, 92%. 1H NMR (300 MHz, Chloroform-d, ppm): δ 7.29 (s, 1H, 12-OH), 4.51 (s, 1H,H-7β), 4.46 (s, 1H, H-6α), 3.17 (hept, J = 7.1 Hz, 1H, H-15), 2.95 (s, 1H, H-1β), 2.60 (dt, J = 13.7, 3.3 Hz, 2H, H-1β), 1.92–1.76 (m, 1H, H-2β), 1.61 (s, 3H, Me-20), 1.58–1.31* (m, 3H, H-2α, H-3β, H-5α), 1.26 (s, 3H, Me-19), 1.24–1.20 (m, 8H, H-1α, H-3α, Me 16, Me-17), 1.05 (s, 3H, Me-18).
*overlapped signal, +Interchangeable signals.
Molecular Docking
Molecular docking experiments were conducted in a similar way as our previous successful predictions for PKC-δ,5 with AutoDock v4.2.6.43 The 1PTR PKC-δ isoform structure was obtained from the PDB and the needed mutations (M239G, W252Y, V255I and K256H) performed in MOE.48 The aminoacid protonation states were assigned using the Protonate 3D module within MOE and exported as PDB file. All tested molecules where built and energy minimized in MOE and their energy minimized using default parameters. The PBD files were converted to the respective PDBQT ligand or receptor files using python scripts available in MGLTools. Docking poses and interactions were visually inspected within MOE.
Yeast Pkc Screening Assay
The royleanones Roy, RoyBz and RoyPr were tested on the yeast assay as reported in5. Briefly, Saccharomyces cerevisiae cells individually expressing mammalian PKC-α, -βI, -δ, -ε, or -ζ, and control yeast (transformed with empty vector) obtained in previous works were used. Cells were grown in galactose selective medium in the presence of 0.1–30 µM of compounds (or 0.1% DMSO only) for approximately 42 h. Yeast growth was analysed by counting the number of colony-forming units, after 2 days incubation at 30 ºC. The growth of yeast transformed with the empty vector (control) was considered as 100%. From the dose-response curves obtained, EC50 (half maximal effective concentration) values were determined for the tested compounds.