General experimental procedures
Melting points were performed on a Büchi SMP-20 melting point apparatus. The NMR spectra in chloroform-d and methanol-d4 were obtained using Brucker BioSpin GmbH instruments, operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR. Chemical shifts are given in δ (ppm) using tetramethylsilane (TMS) as internal standard. EI-MS was obtained with JEOL JMS-600H mass spectrometer. The extraction of the plant material was done using a digital ultrasonic cleaner LMUC series of mark LABMAN (serial number L1328). Flash chromatography was performed on SCHOTT Buchner (Duran, West Germany) with a porosity of 4. Analytical thin-layer chromatography (TLC) was performed on precoated silica gel plates (Merck 60F254; 20x20, 0.25 mm). Column chromatography was performed on silica gel (70-230 mesh; Merck). Chromatograms were visualized by spraying with a solution of 10 % H2SO4, iodine vapor or under ultraviolet light of wavelengths 254 and 366 nm.
Molecular docking studies
Preparation of Ligand
4000 compounds were selected from the CANAPL database and distributed in 39 classes namely Alkane, Alcohol, Alkaloid, Amide, Anthranol, Enthrone, Aromatic compounds, Benzophenone, Carotenoids, Ceramide, Cerebroside, Chromone, Coumarin, Cyanogenic glycoside, Cyclitol (polyol), Depsidone, Endiandric acid, Fatty acid, Flavonoid, Ionone, Iridoid, Labdane, Lactone, Lignane, Limonoid, Monoterpene, Peptide cyclic, Pterocarpans, Pyrone, Quinone, Sesquiterpene lactone, Saponins, Sesquiterpenoid, Sphingolipid, Steroid, Stilbenes, Sugar, Tannin, Terpenoids and Xanthone. The 3D structures of the phytoconstituents were retrieved from our chemical database and saved in .pdb format using Chem3D 15.0. The ligands were imported to the workspace and preparation was done for docking studies. The docking results of different constituents were compared against the reference drugs (heptelidic acid, iodoacetate and pentalenolactone) (Cane and Sohng 1989; Karja et al. 2009) obtained from the drug bank in chemdraw (*cdx) format.
Preparation of Enzyme
The target for docking studies selected was Glyceraldehyde 3-phosphate dehydrogenase. Docking analysis was done by initially selecting the target for the disease and followed by obtaining the 3D structure of Glyceraldehyde 3-phosphate dehydrogenase (1ywg) (Satchell et al. 2005) from protein data bank (http:\\www.rcsb.org) in .pdb format.
The AutoDockTools (Morris et al. 2009) (ADT) was used to prepare the ligand and receptor structures, add appropriate Gasteiger and Kollman charges, identify and modify ligand rotatable bonds (Sanner 1999). The potential binding sites of target were calculated using the Lamarckian GA (4.2) algorithm implemented in Autodock4 (Morris et al. 1998). The population size, maximum number of evaluation (medium) and maximum number of generations were set at 150, 27 000 and 2 500 000 respectively (Bahadoria et al. 2016). The search space of the simulation exploited in the docking studies was studied as a subset region of 0.375Å around the active site (Robien et al. 2006). The water molecules were removed from the enzyme to decrease interactions between functional group of ligands and water molecules.
Parameters for docking functions
At this time, more than 30 molecular docking programs are available (Missier et al. 2010). The commonly used are AutoDock (Dhanick et al. 2012), GOLD, FlexX, DOCK and ICM. According to Sousa et al., (Sousa et al. 2006) we performed in-silico docking analysis by using Autodock4.0 (Lindstrom et al. 2008). This software uses two programs:
AutoGrid program calculates the interactions cards of grid in order to maximize evaluation step of different configurations of ligands. A grid surrounds the receptor protein and the ligand is placed at each intersection. The energy of interaction of this molecule with the protein is calculated and assigned to the location of the probe atom on the grid. An affinity grid is calculated for each type of ligand atom. The energy calculation time using the grids is proportional to the number of atoms of the ligand but it’s independent of the number of atoms in the enzyme.
AutoDock program performs the research and evaluation of the different ligand configurations. It is possible to use several techniques to obtain the configurations (by simulated annealing, genetic algorithm or by Lamarckian genetic algorithm).
A grid-based method was used to enhance the quick evaluation of the binding energy of conformations of the complexes formed. The grid boxes were centered using coordinates of a virtual center of mass atom for the enzymes (Forlemu et al. 2017). A grid box had dimensions of 44 Å × 44 Å × 58 Å respectively in x, y and z dimension according to amino acids which formed active site (Cys153 - His180). The affinity of the docked complexes was described using dissociation constants (Ki) (Equation (1)), binding energy based on the semi empirical force field expression as described in Equation (2) (Huey et al. 2006) and H-Bond interactions.
Synthesis
Procedure for the oxidation of some triterpenoids with selenium oxide SeO2
To a dry DCM or Dioxane solution of the olefin in a 100 mL conical flask, SeO2 was added (1.5 eq) at room temperature. The reaction mixture was refluxed while stirring for 1-3 h. After partial evaporation of the solvent to reduce the volume of the mixture, the residue was fixed to silica gel and submitted to column chromatography using Hex/AcOEt in various proportions.
Betulonic acid acrylaldehyde 14: white solid (CH2Cl2); (Hex/AcOEt 18%); mp 182°C; 1H NMR (CDCl3, 500 MHz): δ = 9.50 (1H, s, H-30), δ = 6.30 and 5.94 (each 1H, br s, H-29), 1.27 and 1.09 (each 3H, 2XCH3), 1.01-0.99 (6H, s), 0.95 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz): δ = 218.1 (C, C-3), δ = 198.2 (C, C-30), 180.9 (C, C-28), 147.3 (C, C-20), 105.8 (CH2, C-29).
Betuline acrylaldehyde 15: white solid (CH2Cl2/MeOH); (Hex/AcOEt 27%); mp 258 °C; 1H NMR (CDCl3, 500 MHz): δ = 9.51 (1H, s, H-30), δ = 6.31 and 5.93 (each 1H, br s, H-29), 1.27 and 1.09 (each 3H, 2XCH3), 1.01-0.99 (6H, s), 0.95 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz): δ = 198.4 (C, C-30), 180.9 (C, C-28), 148.3 (C, C-20), 105.8 (CH2, C-29), δ = 79.01 (C, C-3).
3β-(trans-p-Coumaroyl)oxylup-20(29)-en-28-oïc acid acrylaldehyde 16: white solid (CH2Cl2/MeOH); (Hex/AcOEt 28.5%); mp 300 °C; 1H NMR (CDCl3, 500 MHz): δ = 9.53 (1H, s, H-30), δ = 7.48 (1H, d, J = 15.0 Hz, H-3’), 7.31 (2H, d, J = 5.0 Hz, H-3”, H5”), 6.72 (2H, d, J = 5.0 Hz, H-2”, H-6”), 6.15 (1H, d, J = 15.0 Hz, H-2'), 6.47 and 6.10 (each 1H, br s, H-29), 4.43 (1H, m, H-3α), 0.98, 0.94, 0.90, 0.88 and 0.87 (each 3H, s, H-23, H-24, H-25, H-26 and H-27 respectively).
30-hydroxy betulinic acid 17: white solid (CH2Cl2/MeOH); (Hex/AcOEt 21%); mp 250 °C; 1H NMR (CDCl3, 500 MHz): δ = 4.14 (1H, dd, H-3), 3.20-2.80 (2H, dd, H-30), 0.88 (3H, d, J = 10Hz, H-29), 0.87, 0.86 and 0.85 (each 3H, s, 3xCH3), 0.73 (3H, s, CH3), 0.65 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz): δ = 179.1 (C, C-28), 78.8 (CH, C-3), 60.4 (CH2, C-30).
Betulinic acid acrylaldehyde 18: white solid (CH2Cl2/MeOH); (Hex/AcOEt 23%); mp 284 °C; 1H NMR (CDCl3, 500 MHz): δ = 9.55 (1H, s, H-30), δ = 6.31 and 5.94 (each 1H, br s, H-29), 3.20 (1H, m, H-3), 1.27 and 1.09 (each 3H, 2XCH3), 1.01-0.99 (6H, s), 0.95 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz): δ = 194.9 (C, C-30), 180.9 (C, C-28), 147.3 (C, C-20), 105.8 (CH2, C-29), δ = 78.9 (C, C-3).
(3β-trans cinnamoyloxylup-20(29)-ene) acrylaldehyde 19: white solid (CH2Cl2/MeOH); (Hex/AcOEt 6%); mp °C; 1H NMR (CDCl3, 500 MHz): δ = 9.54 (1H, s, H-30), δ = 7.68 (1H, d, J = 15.0 Hz, H-3’), 7.54-7.40 (5H, m, H-2”,H-3”, H-4”, H5”, H-6”), 6.46 (1H, d, J = 15.0 Hz, H-2'), 6.32-5.95 (each 1H, br s, H-29), 4.63 (1H, m, H-3α), 0.96, 1.05, 0.91, 0.89, 0.85, 0.94 (each 3H, s, H-23, H-24, H-25, H-26, H-27 and H-28 respectively).
Biological activity
Plasmodium falciparum culture and growth inhibition assay
Plasmodium falciparum 3D7 (chloroquine-sensitive) strain was obtained from the Biodefense and Emerging Infections (BEI) Research Resources (Manassas, VA) and maintained using a modified Trager and Jensen method. Briefly, parasites were cultured in fresh O+ human red blood cells at 3% (v/v) hematocrit in RPMI 1640 culture media containing glutamax and NaHCO3 (Gibco, UK), supplemented with 25 mM HEPES (Gibco, UK), 1X hypoxanthine (Gibco, USA), 20 µg/mL gentamicin (Gibco, China), and 0.5% Albumax II (Gibco, USA). When needed, parasites were synchronized at the ring stage by sorbitol treatment and cultured through one cycle before treatment.
Stock compound solutions were diluted in incomplete RPMI 1640 and mixed with parasite cultures (1% parasitemia and 1.5% hematocrit) in 96-well plates to a final drug concentration of 10 μM for hit identification studies, or 10 – 0.078 μM for activity confirmation assays. The final dimethyl sulfoxide (DMSO) concentration per 100 μL culture per well was 0.1%. Artemisinin at 1 µM was used as negative growth control, while the solvent treated culture (0.1% DMSO) was used as positive growth control. Following 72 h incubation at 37 °C, parasite growth was assessed by a SYBR green I-based DNA quantification assay. In brief, a 3X concentrated SYBR Green lysis buffer was added to each plate well containing parasitized erythrocytes and kept in the dark for about 30 minutes. Fluorescence was measured using a Fluoroskan Ascent multi-well plate reader with excitation and emission wavelengths at 485 and 538 nm, respectively. Mean half-maximal inhibitory concentrations (IC50 values) were derived by plotting percent growth against log drug concentration and fitting the response data to a variable slope sigmoidal curve function using GraphPad Prism v8.0.