Plant materialThe 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.org49.Extraction and isolationAcetone ultrasonic-assisted extraction was adapted from Bernardes C.E.S. et al.24 The leaves and steams of P. grandidentatus were air dried. 2.334 kg of the dried plant was grinded to powder and with acetone (15 × 3.4 L), in an ultrasound equipment. The ultrasonic bath (Sonorex Super RK 510 H; Bandelin, Berlin, Germany) operated at room temperature, for 30 min, 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.50 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.24 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–DADThe quantification of Roy in P. grandidentatus extracts was adapted from Matias et al. 201932. 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 × 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. LOD and LOQ were assessed based on Signal-to-Noise approach51.Synthesis general procedureFor 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 DCM: 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. The purity of the compounds was assessed by HPLC, according to the methodology described above. All compounds exhibited a purity exceeding 95%.Compounds characterizationRoy (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).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).Roy-12-Bz (7α-acetoxy-6β-hydroxy-12-O-benzoylroyleanone): Yellow amorphous solid, 69%. mp: 216–218 °C. \(\left[ \upalpha \right]_{D}^{20} = \user2{ } + 33.1 ^\circ\) (c 0.151, CHCl3). IR \({\overline{\nu }}\) max: 3476.8, 2971.5, 2935.7, 2873.7, 1745.8, 1667.6, 1605.7, 1540.5, 1455.7, 1371.0, 1224.3, 1142.8, 1113.4, 1061.3, 1015.6, 771.1, 751.6, 702.7 cm−1.1H-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).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-12-Ac (7α-acetoxy-6β-hydroxy-12-O-acetylroyleanone): Yellow oil, 87%. \(\left[ \upalpha \right]_{D}^{20} = + 25.0 ^\circ\) (c 0.079, CHCl3). IR \({\overline{\nu }}\) max: 2965.0, 2929.1, 2854.2, 1762.1, 1670.9, 1615.4, 1468.7, 1371.0, 1273.2, 1234.0, 761.4 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 185.94 (C-14), 179.90 (C-11), 169.83 (7αCOCH3), 168.44 (12-COCH3), 153.01 (C-9), 149.50 (C-12), 139.43 (C-13), 135.78 (C-8), 68.98 (C-7), 67.34 (C-6), 49.88 (C-5), 39.00 (C-10), 38.43 (C-1), 33.86 (C-4), 33.65 (C-18), 25.32 (C-15), 23.98 (12- COOCH3), 21.85 (C-19), 21.04 (C-20), 20.58 (7α-COCH3), 20.41 (C-16), 20.34 (C17), 19.03 (C2). HRMS (ESI–MS): m/z calculated for C24H32O7 [M + H]+ 433.2221, found 433.22179.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 dockingMolecular docking experiments were conducted in a similar way as our previous successful predictions for PKC-δ5, with AutoDock v4.2.646. The 1PTR PKC-δ isoform structure was obtained from the PDB and the needed mutations (M239G, W252Y, V255I and K256H) performed in MOE52. 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.Human cell lines and growth conditionsRoyleanones and doxorubicin (Doxo) were tested on human breast adenocarcinoma lines. Human breast adenocarcinoma MCF‐7, MDA-MB-231 and MDA-MB-468, and non‐tumorigenic foreskin fibroblasts HFF‐1 cell lines were purchased from the ATCC (Rockville, MD, USA). Cancer cells were routinely cultured in RPMI‐1640 medium with UltraGlutamine from Biowest (VWR, Carnaxide, Portugal) supplemented with 10% FBS (fetal bovine serum) from Biowest (VWR). HFF‐1 cells were cultured in DMEM/F‐12 supplemented with 10% FBS. All cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Cells were routinely tested for mycoplasma infection using the MycoAlert™ PLUS mycoplasma detection kit (Lonza).Sulforhodamine B (SRB) assayFor the evaluation of the effect of the compounds on cell proliferation, the SRB assay was performed. Human cell lines were seeded in 96‐well plates at a density of 5.0 × 103 (MCF-7, MDA-MB-231, and HFF-1) cells and 7.5 × 103 (MDA-MB-468) cells per well and allowed to adhere for 24 h. Cells were treated for 48 h with serial dilutions of the compounds (ranging from 0.1 to 30.0 µM). The effects on cell proliferation were assessed by SRB assay, as described5, and IC50 values were determined for each cell line using the GraphPad Prism software version 7.0 (La Jolla, CA, USA).Yeast PKC screening assayThe royleanones Roy, RoyBz and RoyPr were tested on the yeast assay as reported in3. 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 analyzed 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.
