Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability, apoptosis, cell proliferation and survival, angiogenesis, or ER-stress response. As CK2 activity is found perturbed in many pathological states, including cancers, it becomes an attractive target for the pharma. A large number of low-mass ATP-competitive inhibitors have already been developed, the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic ligands derived from the commercially available 4,5-dihalo-benzene-1,2-diamines. These ligand series were selected to enable the separation of the scaffold effect from the hydrophobic interactions attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied to test the capability of each ligand for binding at the ATP-binding site of CK2. HPLC-derived ligand hydrophobicity data are compared with the binding affinity assessed by low-volume differential scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory activity against CK2α and toxicity against four reference cell lines have been determined for eight compounds identified as the most promising in nanoDSF assay. Protein kinase CK2 is considered as one of the essential human proteins, which is involved in numerous cellular processes. This is a highly pleiotropic kinase, which can phosphorylate hundreds of protein substrates including transcriptional factors, proteins affecting the structure of DNA/ RNA, and/or involved in RNA synthesis and translation, signaling proteins and also pathogenic virus replication machinery proteins . The numSince kinase CK2 was found to be overexpressed in many forms of human cancers , its rolH-benzotriazole (TBBt) and 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were identified as strong and selective kinase CK2 inhibitors  imidazole (K44), containing an additional imidazolidine ring in its structure, was also reported to strongly inhibit CK2 imidazol-2(3H)-ones  were synthesized from 1a–e and N,N′-carbonyldiimidazole (CDI) mixed in DMF imidazol-2(3H)-one (5a) is identical. A similar trend is also observed in the case of quinoxaline-2,3-diol (7a) and benzo-1,2-diamine (1a), however, for the latter, the effect of halogenation is shifted downwards by 0.1 log(τ) units. The largest changes in hydrophobicity experience quinoxaline (6a) derivatives, which should be directly attributed to the lack of the effect of electronegative halogen atom on the proximal proton-donating groups. Such effect, partially compensating halogen hydrophobicity, was evidenced in protein-ligand structures, in which hydrogen bonds proximal to a halogen atom were statistically shortened (so must be regarded as stronger) when a nitrogen atom close to the halogen atom  was a hydrogen bond donor imidazol-2(3H)-one derivatives (5a–d) moderately interact with CK2 -one inhibits CK2 stronger (IC50 = 0.39 μM and Ki = 0.20 μM) imidazol-2(3H)-one counterpart (7 vs. 13 μM). This again points out the subtle interplay between the scaffold and the substitution with halogen atoms, indicating that the interaction of 2-trifluoromethyl-1H- benzimidazole with Lys68 is slightly stronger than for its isostructural analog-carrying carbonyl group.It is worth noting that for these two 5,6-halogenated scaffolds, the observed tendency is opposite to that observed for their tetrahalogenated analogs. Thus, 2-trifluoromethyl- 17d,e) might lead to novel potent CK2 inhibitors, as both inhibit hCK2α comparable to 2-trifluoromethyl-1H-benzimidazole derivatives , stabilizing the complex for approx. 4 °C , but have not been reported as kinase CK2 inhibitors yet.In this context, quinoxaline-2,3-diol derivatives . The reaction progress was monitored by the thin-layer chromatography analysis using silica gel plates . Column chromatography was performed on Silica Gel 60 .All starting materials and solvents for reactions were purchased from Sigma Aldrich , Fluorochem , ABCR , or Chempur . Detailed experimental data, as well as spectral data for all synthesized compounds, are available in the 2a–e)Synthesis of 2,1,3-benzothiadiazole derivatives  was dissolved in dry DCM (15 mL). Triethylamine (3 mL) was added, then thionyl chloride  dissolved in 3 mL of dry DCM was added dropwise at 0 °C. The reaction mixture was allowed to reach room temperature, then it was heated at 40 °C overnight. Afterward, the reaction mixture was filtered. The filtrate was evaporated, the crude product was purified by column chromatography on silica gel using DCM as eluent.2a) Yield = 66%. 1H-NMR  δ 7.62–7.72 , 7.99–8.80 ; 13C-NMR  δ 121.24, 129.83, 154.19; HRMS calcd. for C6H5N2S [M + H]+: 137.01680, found: 137.01673.2,1,3-benzothiadiazole (2b) Yield = 66%. 1H-NMR  δ 8.18–8.27 ; 13C-NMR  δ 106.37, 106.54, 150.46, 1651.77, 151.94, 153.81, 153.97; HRMS calcd. for C6H3F2N2S [M + H]+: 172.99795, found: 172.99803.5,6-difluoro-2,1,3-benzothiadiazole (2c) Yield = 75%. 1H-NMR  δ 8.53 ; 13C-NMR  δ 121.75, 133.54, 152.61; HRMS calcd. for C6H3Cl2N2S [M + H]+: 204.93885, found: 204.93872.5,6-dichloro-2,1,3-benzothiadiazole (2d) Yield = 78%. 1H-NMR  δ 8.67 ; 13C-NMR  δ 124.95, 126.26, 153.32; HRMS calcd. for C6H3Br2N2S [M + H]+: 292.83782, found: 292.83741.5,6-dibromo-2,1,3-benzothiadiazole (2e) Yield = 47%. 1H-NMR  δ 8.79 ; 13C-NMR  δ 111.39, 130.38, 154.22; HRMS calcd. for C6H3I2N2S [M + H]+: 388.81008, found: 388.30973.5,6-diiodo-2,1,3-benzothiadiazole (3a–e)Synthesis of 2-trifluoromethylo-1H-benzimidazole derivatives . Combined organic layers were dried by MgSO4, then the solvent was evaporated. The crude product was purified by column chromatography on silica gel using 85:15 (hexane:AcOEt) as eluent (compounds 3d and 3e) or by recrystallization from hexane .Benzene-1,2-diamine derivative (2 mmol) was dissolved in trifluoroacetic acid (2 mL) and a catalytic amount of concentrated HCl was added. The reaction mixture was heated at reflux overnight. The reaction was quenched by the addition of 50 mL of concentrated NaHCOH-benzimidazole (3a) Yield = 73%. 1H-NMR  δ 7.34–7.42, 7.68-7.77; 13C-NMR  δ 115.88, 118.02, 120.17, 122.32, 124.09, 139.90, 140.22; HRMS calcd. for C8H6F3N2 [M + H]+: 187.04776, found: 187.04773.2-trifluoromethylo-1H-benzimidazole (3b) Yield = 67%. 1H-NMR  δ 7.71–7.85 ; HRMS calcd. for C8H4F5N2 [M + H]+: 223.02892, found: 223.02882.5,6-difluoro-2-trifluoromethylo-1H-benzimidazole (3c) Yield = 85%. 1H-NMR  δ 8.00 ; HRMS calcd. for C8H4F3Cl2N2 [M + H]+: 254.96981, found: 254.96976.5,6-dichlorlo-2-trifluoromethylo-1H-benzimidazole (3d) Yield = 40%. 1H-NMR  δ 8.16 ; 13C-NMR  δ 115.88; HRMS calcd. for C8H4F3Br2N2 [M + H]+: 342.86878, found: 342.86896.5,6-dibromo-2-trifluoromethylo-1H-benzimidazole (3e) Yield = 33%. 1H-NMR  δ 8.32 ; HRMS calcd. for C8H4F3I2N2 [M + H]+: 438.84105, found: 438.84123.5,6-diiodo-2-trifluoromethylo-14a,c–e)Synthesis of 2-hydroxymethylo-1H-benzimidazole derivatives  and hydroxyacetic acid  were dissolved in water (1.5 mL) and concentrated HCl (0.5 mL) was added. The reaction mixture was heated at reflux overnight. The reaction was cooled to RT and then 20% NaOH solution was added until pH = 13. The formed participate was filtered and washed several times with water. Compound H-benzimidazole (4a) Yield = 85%. 1H-NMR  δ 4.70 , 5.69 , 7.11-7.15 , 7.49 , 12.30 ; 13C-NMR  δ 57.74, 121.25, 155.02; HRMS calcd. for C8H9N2O [M + H]+: 149.07094, found: 149.07089.2-hydroxymethylo-1H-benzimidazole (4c) Yield = 84%. 1H-NMR  δ 4.70 , 5.81 , 7.73 , 12.04 ; 13C-NMR  δ 57.57, 123.71, 158.10; HRMS calcd. for C8H7Cl2N2O [M + H]+: 216.99299, found: 216.99308.5,6-dichloro-2-hydroxymethylo-1H-benzimidazole (4d) Yield = 81%. 1H-NMR  δ 4.70 , 5.83 , 7.88 ; 13C-NMR  δ 57.50, 115.47, 119.18, 157.91; HRMS calcd. for C8H7Br2N2O [M + H]+: 304.89196, found: 304.89222.5,6-dibromo-2-hydroxymethylo-1H-benzimidazole (4e) Yield = 66%. 1H-NMR  δ 4.68 , 5.80 , 8.07 , 12.52 ; 13C-NMR  δ 57.48, 98.13, 157.17; HRMS calcd. for C8H7I2N2O [M + H]+: 400.86423, found: 400.86446.5,6-diiodo-2-hydroxymethylo-15a,c–e)Synthesis of 1H-benzo[d]imidazol-2(3H)-one derivatives   were mixed in DMF (4 mL) overnight at RT. The reaction was quenched by the addition of water (20 mL). The formed participate was filtered and washed several times with water. Compound 5d was additionally purified by recrystallization from ethyl acetate, then MeOH.Benzene-1,2-diamine derivative (3 mmol) and H-benzo[d]imidazol-2(3H)-one (5a) Yield = 45%. 1H-NMR  δ 6.91 , 10.56 ; 13C-NMR  δ 108.46, 120.40, 129.65, 155.25; HRMS calcd. for C7H7N2O [M + H]+: 135.05529, found: 135.05526.1H-benzo[d]imidazol-2(3H)-one (5c) Yield = 67%. 1H-NMR  δ 7.08 , 10.91 ; 13C-NMR  δ 109.73, 122.35, 129.83, 155.18; HRMS calcd. for C7H5Cl2N2O [M + H]+: 202.97734, found: 202.97743.5,6-dichloro-1H-benzo[d]imidazol-2(3H)-one (5d) Yield = 67%. 1H-NMR  δ 7.20 , 10.90 ; 13C-NMR  δ 112.66, 113.88, 130.63, 154.93; HRMS calcd. for C7H5Br2N2O [M + H]+: 290.87631, found: 290.87626.5,6-dibromo-1H-benzo[d]imidazol-2(3H)-one (5e) Yield = 67%. 1H-NMR  δ 7.41 , 10.80 ; 13C-NMR  δ 96.40, 118.00, 131.46, 154.58; HRMS calcd. for C7H5I2N2O [M + H]+: 386.84858, found: 386.84861.5,6-diiodo-16a,c–e)Synthesis of quinoxaline derivatives (6a was extracted with AcOEt (3 × 30 mL). Compounds 6c and 6d were purified by recrystallization from MeOH. Compounds 6a and 6e were purified by column chromatography on silica gel, using 8:2 (hexane:AcOEt) as eluent.Benzene-1,2-diamine derivative (3 mmol) and 40% glyoxal solution in water  were heated at reflux in EtOH (6 mL) overnight. The reaction was quenched by the addition of water (50 mL). The formed participate was filtered and washed several times with water (compound 6a) Yield = 70%. 1H-NMR  δ 7.84–7.92 , 8.08–8.16 , 8.96 ; 13C-NMR  δ 129.14, 130.21, 142.23, 145.72; HRMS calcd. for C8H7N2 [M + H]+: 131.06037, found: 131.06042.quinoxaline (6c) Yield = 99%. 1H-NMR  δ 8.46 , 9.02 ; 13C-NMR  δ 130.13, 133.01, 141.15, 147.13; HRMS calcd. for C8H5Cl2N2 [M + H]+: 198.98234, found: 198.98252.6,7-dichloroquinoxaline (6d) Yield = 89%. 1H-NMR  δ 8.56 , 9.01 ; 13C-NMR  δ 125.77, 133.31, 141.56, 147.14; HRMS calcd. for C8H5Br2N2 [M + H]+: 286.88140, found: 286.88165.6,7-dibromoquinoxaline (6e) Yield = 25%. 1H-NMR  δ 8.68 , 8.96 ; 13C-NMR  δ 111.06, 138.74, 141.92, 1467.83; HRMS calcd. for C8H5I2N2 [M + H]+: 382.85366, found: 382.85426.6,7-diiodoquinoxaline (7a–e)Synthesis of quinoxaline-2,3-diol derivatives (6a was extracted with AcOEt (3 × 10 mL)). Crude products were purified by recrystallization from EtOH.Benzene-1,2-diamine derivative (3 mmol) and oxalic acid  were heated at 120 °C in DMF (1 mL) overnight. The reaction was quenched by the addition of water (20 mL). The formed participate was filtered and washed several times with water (compound 7a) Yield = 57%. 1H-NMR  δ 7.02–7.10 , 7.10–7.16 , 11.90 ; 13C-NMR  δ 115.12, 123.00, 125.60, 155.17; HRMS calcd. for C8H7N2O2 [M + H]+: 163.05020, found: 163.05011.quinoxaline-1,3-diol (7b) Yield = 68%. 1H-NMR  δ 7.00–7.11 , 11.92 ; 13C-NMR  δ 103.63, 103.74, 103.81, 122.26, 122.32, 143.91, 144.04, 145.83, 145.96, 154.70; HRMS calcd. for C8H5F2N2O2 [M + H]+: 199.03136, found: 199.03138.6,7-dichloroquinoxaline-1,3-diol (7c) Yield = 86%. 1H-NMR  δ 7.21 , 12.00 ; 13C-NMR  δ 115.99, 124.29, 126.03, 154.71; HRMS calcd. for C8H5Cl2N2O2 [M + H]+: 230.97226, found: 230.97218.6,7-difluoroquinoxaline-1,3-diol (7d) Yield = 91%. 1H-NMR  δ 7.38 , 11.99 ; 13C-NMR  δ 116.15, 118.96, 126.64, 154.73; HRMS calcd. for C8H5Br2N2O2 [M + H]+: 318.87123, found: 318.87105.6,7-dibromooquinoxaline-1,3-diol (7e) Yield = 63%. 1H-NMR  δ 7.51 , 11.77 ; 13C-NMR  δ 99.11, 124.80, 128.06, 155.49; HRMS calcd. for C8H5I2N2O2 [M + H]+: 414.84349, found: 414.84366.6,7-diiodooquinoxaline-1,3-diol (v/v) DMSO. Protein was kept in constant 2.5 μM concentration.hCK2α was expressed and purified according to the method described previously . Thermal50 values were further estimated globally using the sigmoidal dose-response equation implemented in Origin 9.4 package .The inhibitory effect of 8 selected compounds was monitored based on the luminescence measured with the SpectraMax iD3 Multi-Mode Microplate Reader , using the previously described method with the aid of ADP-Glo kinase assay  . Inhibit® CRL-1555), (ii) colorectal carcinoma HCT116 (ATCC® CCL-247), (iii) colorectal carcinoma HCT116p53−/−, and (iv) normal fibroblasts BJ (ATCC® CRL-2522). The HCT116p53−/− cell line was a kind gift from professor Bert Vogelstein . The remaining cell lines were purchased from ATCC  or Sigma Aldrich by Merck .The following human cell lines were used in this study: (i) epidermoid carcinoma A-431 (ATCC2) in a humidified incubator with atmospheric oxygen.Cells were cultured in Dulbecco’s modified Eagle’s or Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% or 15% fetal bovine serum  under standard culture conditions -2,5-diphenyltetrazolium bromide) to the formazan . Cells were seeded in a 96-well plate at a density of 1500–2500 cells per well, 24 h before treatment. Treatment with tested compounds and cisplatin (0.25–160 µM) was performed for 72 h. MTT stock solution (Sigma-Aldrich) was added at the final concentration 0.5 mg/mL. After 2 h of incubation at 37 °C, water-insoluble formazan was dissolved in a lysis buffer containing 20% SDS, 50% DMF, 2.5% hydrochloric acid, and 2.5% acetic acid. Optical densities were measured at 570 nm using a scanning multi-well spectrophotometer .® C18 4 μm, 4.6 × 250 mm. Eluent: 2:1 methanol: 20 mM aqueous ammonium formate (pH = 8) (v/v). Flow: 0.7 mL/min. Detector wavelength: 280 nm. The individual retention times (Tret) were converted to a hydrophobicity scale according to the formula τ = (Tret − T0)/T0. where the retention time of unretained solvent (T0) was estimated to 300 s [Chromatographic hydrophobicity index (log(τ)) was determined with the reverse phase HPLC (RP-HPLC). Column: Nova-pakto 300 s .www.yasara.com, version 20.12.24) using amber14 force-field with 8 Å cutoff for long-range electrostatic interactions. We used 8 just-resolved structures of complexes of hCK2α with variously brominated benzotriazoles  as the reference protein structures. This, including alternative protein conformations, stated 18 structures to be tested as the ‘receptor’. Such an extended set of template protein structures was chosen to allow the sampling of a larger conformational space that allows multiple ligand orientations at the binding site (see Docking of all tested ligands at the binding site of the catalytic subunit of human protein kinase CK2 was performed with VINA algorithm implemented in Yasara Structure package (site see . In sili


Pappea capensis for the treatment of malaria. The present study aimed to investigate the phytochemical and antiplasmodial properties of the plant leaves. The bioactive compounds were isolated using chromatographic techniques. The structures were established using NMR, HRMS, and UV spectroscopy. Antiplasmodial activity of P. capensis leaf extract and isolated compounds against chloroquine-sensitive 3D7 P. falciparum was evaluated using the parasite lactate dehydrogenase assay. Cytotoxicity against HeLa (human cervix adenocarcinoma) cells was determined using the resazurin assay. The extract inhibited the viability of Plasmodium falciparum by more than 80% at 50 µg/mL, but it was also cytotoxic against HeLa cells at the same concentration. Chromatographic purification of the extract led to the isolation of four flavonoid glycosides and epicatechin. The compounds displayed a similar activity pattern with the extract against P. falciparum and HeLa cells. The results from this study suggest that the widespread use of P. capensis in traditional medicine for the treatment of malaria might have some merits. However, more selectivity studies are needed to determine whether the leaf extract is cytotoxic against noncancerous cells.Ethnobotanical surveys indicate that the Masai and Kikuyu in Kenya, the Venda in South Africa, and the Gumuz people of Ethiopia use  Malaria continues to cause serious morbidity and mortality, especially in Africa, which accounted for over 90% of the approximately 409,000 global malaria deaths in 2019. The problem is complicated by the fact that 67% of the deaths occur in children under the age of five, as well as the high prevalence among pregnant women D29 -42.12 , 1H NMR : δH 6.98 , 6.80 , 6.76 , 5.95 , 5.93 , 4.82 , 4.18 , 2.86 , 2.74 . 13C NMR : δC 157.9 (C-5), 157.6 (C-7), 157.3 (C-9), 145.9 (C-3′), 145.7 (C-4′), 132.2 (C-1′), 119.4 (C-6′), 115.9 (C-5′), 115.3 (C-2′), 100.1 (C-10), 96.4 (C-6), 95.9 (C-8), 79.8 (C-2), 67.4 (C-3), 29.2 (C-4). HR-ESI-(-)-MS: m/z 289.0705 [M-H]− .Epicatechin (O-arabinopyranoside (guaijaverin) (2): yellow solid, [α]D29 -54.07 , UV (MeOH/ACN): λmax 255, 355 nm; 1H NMR  δH 7.74 , 7.57 , 6.88 , 6.39 , 6.20 , 5.16 , 3.90 , 3.81-3.84 , 3.65 , 3.44 . 13C NMR  δC 179.5 (C-4), 166.1 (C-7), 163.0 (C-5), 158.7 (C-2), 158.5 (C-9), 150.0 (C-4′), 146.0 (C-3′), 135.6 (C-3), 123.1 (C-1′), 122.9 (C-6′), 117.5 (C-2′), 116.2 (C-5′), 105.6 (C-10), 104.7 (C-1″), 100.0 (C-6), 94.8 (C-8), 74.1 (C-3″), 72.9 (C-2″), 69.1 (C-4″), 66.9 (C-5″). HPLC Rt: 25.334 min; HR-ESI-(-)-MS: m/z 433.0775 [M-H]− .Quercetin 3-O-α-L-rhamnoside (quercitrin) (3): yellow solid, [α]D29 -136.93 , UV (MeOH/ACN): λmax 255, 350 nm; 1H NMR : δH 7.34 , 7.30 , 6.87 , 6.36 , 6.19 , 5.35 , 4.22 , 3.75 , 3.45 , 3.34 , 0.94 . 13C NMR : δC 179.6 (C-4), 165.9 (C-7), 163.2 (C-5), 159.3 (C-2), 158.5 (C-9), 149.8 (C-3′), 146.4 (C-4′), 136.2 (C-3), 123.0 (C-1′), 122.9 (C-6′), 117.0 (C-2′), 116.4 (C-5′), 105.9 (C-10), 103.5 (C-1″), 99.8 (C6-), 94.8 (C-8), 73.3 (C-4″), 72.1 (C-3″), 72.0 (C-2″), 71.9 (C-5″), 17.7 (C-6″). HPLC Rt: 29.504 min; HR-ESI-(-)-MS: m/z 447.0915 [M-H]− .Quercetin 3-O-arabinopyranoside (juglanin) (4): yellow solid, UV (MeOH/ACN): λmax 265, 347 nm; 1H NMR : δH 8.06 , 6.89 , 6.41 , 6.21 , 5.13 , 3.89 , 3.79-3.76 , 3.63 , 3.40 . 13C NMR : δC 179.6 (C-4), 166.2 (C-7), 163.1 (C-5), 161.6 (C-4′), 158.9 (C-2), 158.5 (C-9), 135.6 (C-3), 132.3 , 122.7 (C-1′), 116.3 , 105.6 (C-10), 104.4 (C-1″), 100.0 (C-6), 94.8 (C-8), 74.0 (C-3″), 72.7 (C-2″), 68.9 (C-4″), 66.7 (C-5″). HPLC Rt: 34.737 min; HR-ESI-(-)-MS: m/z 417.0829 [M-H]− .Kaempferol 3-O-β-D-glucoside (isoquercitrin) (5): yellow solid, [α]D29 -32.70 , UV (MeOH/ACN): λmax 255, 355 nm; 1H NMR : δH 7.70 , 7.59 , 6.87 , 6.4 , 6.21 , 5.24 , 3.71-3.42 (undefined sugar protons). 13C NMR : δC 179.6 (C-4), 166.4 (C-7), 161.6 (C-5), 158.6 (C-2), 158.2 (C-9), 150.4 (C-4′), 145.7 (C-3′), 135.2 (C-3), 122.6 (C-6′), 121.9 (C-1′), 117.4 (C-2′), 116.0 (C-5′), 105.8 (C-10), 105.2 (C-1″), 100.1 (C-6), 94.4 (C-8), 77.7 (C-5″), 75.5 (C-3″), 71.0 (C-2″), 70.0 (C-4″), 62.0 (C-6″). HPLC Rt: 20.216 min; HR-ESI-(-)-MS: m/z 463.0881 [M-H]− .Quercetin 3-Plasmodium falciparum strain 3D7) were maintained in RPMI 1640 medium containing 2 mM L-glutamine and 25 mM Hepes (Lonza). The medium was further supplemented with 0.5% w/v Albumax II, 20 mM glucose, 0.65 mM hypoxanthine, 60 µg/mL gentamycin and 2–4% haematocrit human red blood cells. The parasites were cultured at 37 °C under an atmosphere of 5% CO2, 5% O2, 90% N2 in a sealed T75 culture flask [Malaria parasites , as described by Makler et al. [P. falciparum (strain 3D7) in 96-well plates . After 48 h of incubation, 20 µL of culture was removed from each well and combined with 125 µL of a mixture of Malstat  and NBT/PES  solutions in a fresh 96-well plate. The purple product formed, which indicates the presence of pLDH, was quantified in a Spectramax M3 microplate reader (Abs620). The Abs620 reading in each well is an indication of the pLDH activity, and hence the number of parasites present. The % parasite viability, as indicated by the pLDH activity in treated wells relative to untreated controls, was calculated for each compound/extract. The parasitized RBCs in the absence of test compounds were taken as untreated control with 100% viability. For each compound, percentage viability was plotted against Log of compound concentration and the IC50 value (50% inhibitory concentration) was obtained from the resulting dose–response curve by non-linear regression. Chloroquine was used as a positive control drug .The extracts and pure compounds were initially screened in duplicate at a single concentration of 50 µg/mL, followed by 10 µg/mL. Compounds showing less than 50% viability without cytotoxicity at 10 µg/mL were screened for an ICr et al. . Serial 4 cells per well in medium consisting of DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin/amphotericin B antibiotics. After an overnight incubation at 37 °C in a humidified 5% CO2 incubator, test samples were added to a final concentration of 50 µg/mL or 10 µg/mL to the cells in triplicate wells, bringing the total medium volume per well to 200 µL, and incubation continued for 24 h. Twenty µL resazurin stock solution  was added to each well, and, after a 4 h incubation, fluorescence (Exc560/Emm590) was measured in a Spectramax M3 plate reader. After subtracting background readings obtained from empty wells, the fluorescence values were used to calculate percentage cell viability in treated wells relative to wells containing untreated control cells.HeLa (human cervix adenocarcinoma) cells  were plated in 96-well plates at a density of 2 × 10P. capensis displayed antiplasmodial activity against P. falciparum at a concentration at which it was also cytotoxic to cancerous HeLa cells. Investigation of the extract afforded five compounds, of which three flavonoid glycosides were identified for the first time in the plant leaves. The glycosides showed similar activity pattern as the extract. However, the reported use of P. capensis leaves in traditional medicine suggests that there might be some other compounds present in the extract that act to mitigate the cytotoxicity.The leaf extract of


All the synthesized hybrid molecules were assessed for their in vitro antiproliferative activity against more than one cancer cell lines. Compound 3--ylamino)-3-phenylacrylic acid (5a) with IC50 = 1.89 μM against HCT-116 was proved to the most potent compound in this study, as compared to standard drug staurosporin. DNA flow cytometry assay of compound 5a revealed G2/M phase arrest and pre-G1 apoptosis. Annexin V-FITC showed that the percentage of early and late apoptosis was increased. The results of topoisomerase enzyme inhibition activity showed that the hybrid molecule 5a displays potent inhibitory activity compared with control.A new series of hybrid molecules containing cinnamic acid and 2-quinolinone derivatives were designed and synthesized. Their structures were confirmed by  Molecular hybridization is a commonly molecular modification approach to obtain multiple compounds with therapeutic advantages over the two different drugs ,5,6,7,8.2-quinolones (1-azacoumarins) play an important role in anticancer drug design since their derivatives have shown excellent anticancer activity through different mechanisms such as growth inhibition via apoptosis or inhibition of angiogenesis ,12,13,14Cinnamic acid and its natural analogues are known for the treatment of cancer for over centuries ,19. The Based on the potent anticancer activity displayed by known quinolone derivatives, a new series of hybrid molecules were designed and synthesized. The hybrid structure contains cinnamic acids bearing 7,8-dihydroxy-4-methyl-1-amino-2-quinolinone as shown in 3) and 3-(2-oxoquinolin-1(2H)-ylamino)-3-arylacrylic acid derivatives 4 and 5 is outlined in The synthetic pathway leading to the 7,8-dihydroxy-4-methyl-1-amino-quinoline-2-one (2) was obtained via the condensation of pyrogallol with ethyl acetoacetate in the presence of acid catalyst according to literature procedure [2) with hydrazine hydrate in pyridine led to the formation of 1-amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (3). FT-IR spectrum of compound 3 showed the absence absorption band of carbonyl function for the coumarin ring, in addition to new absorption band at 1688 cm−1 due to carbonyl function of the amide group, in addition two new bands at 3225, and 3178 cm−1 related to NH2 group. 1H-NMR spectrum of compound 3 showed characteristic two singlet signals at δ 2.35 and 6.12 ppm due to the protons of methyl group (CH3) and H-3 of quinolinone ring. Protons of the aromatic ring were appeared at δ 6.82 and 7.10 as doublet signal of H-5 and H-6 for the quinolinone ring. The 13C-NMR spectrum of compound 3 showed four carbon signals at δ 160.73, 154.44, 150.01 and 143.75 ppm assigned to carbonyl function (C=O), two carbons (C-O) and one carbon (C-N) groups.7,8-dihydroxy-4-methylcoumarin (rocedure . ReactioH)-one (3) with substituted cinnamic acids in reflexing ethanol in the presence of fused sodium acetate provided the corresponding 3--ylamino)-3-arylacrylic acid 4a–c.Treatment of 1-amino-7,8-dihydroxy-4-methylquinolin-2-ylamino)-3-arylacrylic acid 5a,b. In the 1H-NMR spectrum of compound 5a Protons of the aromatic and H-6 of quinolinone ring were observed as multiplet signals within the expected chemical shift region at δ 7.35–7.69 ppm. The 13C-NMR spectrum of 5a showed four carbons appeared as four signals at δ 152.09, 147.53, 144.41 and 141.54 ppm due to two carbons of C-O and C-N groups. In addition, carbons of the aromatic and quinolinone rings were observed a characteristic carbon signals in the region δ 138.68–107.12 ppm.The halogenation of compounds 3, 4a,b and 5a,b on the viability of three cancer cell lines were studied using MTT assay . Five dose concentrations were used and IC50 concentration was determined. Podophyllotoxin (podo) was employed as the standard. It is observed that, compound 5a revealed strong inhibitory activity against topo II with IC50 value of 75.82 ng/mL, compared to the reference compound podo (IC50 = 31.24 ng/mL) (Compound 4 ng/mL) .3, 4a,b, and 5a,b) against breast cancer protein (PDB: 3HB5). The docking results showed a potential structure-activity relationship between the target compounds  against 3HB5 protein as shown in  receptor which is compatible with cytotoxicity and biological results.Molecular docking is a vital tool in drug design ,32,33,34shown in . Compounshown in . Compoun1H and 13C-NMR spectra were measured with a Bruker Avance 400 MHz spectrometer  using the DMSO-6d as solvent. The IR data were obtained with a shimadzu 470 spectrometer . The molecular weight of the synthesized compounds was determined by electron ionization (EI) mass spectrometer performed using a probe Agilent MSD 5975 spectrometer  operating at 70 eV. The elemental analysis was performed on a Perkin-Elmer 2400 series CHN elemental analyzer  chemical and reagents were purchased from either Aldrich or Sigma and all reagents were analytical grade.The melting point was measured on electro thermal 200 digital melting point device and was uncorrected. The A mixture of pyrogallol (0.01 mol) and ethyl acetoacetate (0.01 mol) in the presence of concentrated sulfuric acid (2 mL) as acid catalyzed was heated under reflux on a water bath for 2 h. The reaction mixture was cooled and poured into water, the solid formed was filtered off, washed with water and dried. Finally, the product was crystallized from hot water.2, known). IR (KBr) νmax: 3458 (br. OH), 1725 (C=O), 1605, 1583 (C=C), 1170, 1065, 1035 (C-O) cm−1. 1H-NMR (DMSO-d6) δ: 2.34 , 6.21 , 6.91 , 7.21 , 9.68 , 9.14  ppm. MS  = 192 . Anal. Calcd. for C8H8O4 (192): C, 62.50; H, 4.17. Found: C, 62.33; H, 4.03.7,8-Dihydroxy-4-methyl coumarin  in pyridine (30 mL) was added hydrazine hydrate (0.02 mol), then heating under reflux for 6 h. The reaction mixture was cooled, poured into ice-water and neutralized with dilute hydrochloric acid (1N). The solid obtained was filtered off, washed with water, dried and purified by crystallization from absolute ethanol.3) as yellow crystals, yield 71%, m.p. 265–267 °C. IR (K Br) νmax: 3418 (br OH), 3225, 3178 (NH2), 1648 (C=O), 1619, 1587 (C=C), 1061, 1006 (C-O) cm−1. 1H-NMR (DMSO-6d) δ: 2.35 , 4.12 , 6.12 , 6.82 , 7.09  ppm. 13C-NMR (DMSO-6d) δ: 160.73 (C-2), 154.44 (C-7), 150.01 (C-8), 143.75 (C-9), 132.69 (C-4), 115.92 (C-10), 113.17 (C-5), 112.60 (C-6), 110.59 (C-3), 18.73 (C-11) ppm. MS  = 206 . Anal. Calcd. for C10H10N2O2 (206): C, 58.25; H, 4.85; N, 13.59. Found: C, 58.09; H, 4.66; N, 13.39.1-Amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (H)-one  in ethanol (30 mL), in the presence of fused sodium acetate (0.03 mol) was refluxed for 4 h. The reaction mixture was cooled down, poured into water, and the solid formed was filtered. The product was crystallized from a suitable solvent to give compound 4a–c.A suspension of a suitable cinnamic acid derivative (0.01 mol) and 1-amino-7,8-dihydroxy-4-methylquinolin-2(14a) Pale yellow crystals, yield 76%, m.p. 185–187 °C. Crystallized from ethanol/H2O (3:1). IR (KBr) νmax: 3550–3350 (br. OH), 3232 (NH), 1705–1675 (br. C=O), 1629, 1584 (C=C), 1229, 1061, 1006 (C-O) cm−1. 1H-NMR (DMSO-6d) δ: 2.36 , 6.13 , 6.56 , 6.38 , 7.10 , 7.41–7.69  ppm. 13C-NMR (DMSO-6d) δ: 168.31 (C-21), 160.74 (C-7),150.00 (C-8), 144.11 (C-9), 143.82 (C-13), 134.77 (C-17), 132.72 (C-4), 130.62 (C-14), 129.37 , 128.63 , 120.09 (C-20), 115.92 (C-10), 113.21 (C-5), 112.67 (C-6), 110.62 (C-3), 18.73 (C-H) ppm. MS  = 352 , Anal. Calcd. for C19H16N3O5 (352), C, 64.77; H, 4.54; N, 7.95. Found: C, 64.58; H, 4.33; N, 7.77.3--ylamino)-3-phenylacrylic acid (4b) Pale yellow crystals, yield 69%, m.p. 181–183 °C. Crystallized from ethanol/H2O (3:1). IR (KBr) νmax: 3560–3100 (br. OH), 3232 (NH), 1701–1660 (C=O), 1626, 1593 (C=C), 1170, 1062, 1007 (C-O) cm−1. 1H-NMR (DMSO-6d) δ: 2.35 , 6.12 , 6.28–7.53  ppm. 13C-NMR (DMSO-6d) δ: 168.45 (C-21), 160.70 (C-2), 160.70 (C-17), 154.39 (C-7), 149.87 (C-8), 144.65 (C-9), 143.79 (C-13), 132.63 (C-4), 130.56 , 125.74 (C-14), 116.22 , 115.95 (C-10), 115.81 (C-20), 113.24 (C-5), 112.60 (C-6), 110.66 (C-3), 18.71 (C-11) ppm. MS  = 368 . Anal. Calcd. for C19H16N2O6 (368): C, 61.96; H, 3.35; N, 7.61. Found: C, 61.73; H, 3.11; N, 7.51.3--ylamino)-3-(4-hydroxyphenyl)acrylic acid (4c). Pale yellow crystals, yield 67%, m.p. 175–177 °C. Crystallized from DMF/H2O (1:1). IR (KBr) νmax: 3550–2984 (br. OH), 3232 (NH), 1700–1652 (C=O), 1620, 1595 (C=C), 1172, 1141, 1062 (C-O) cm−1. 1H-NMR (DMSO-6d) δ: 2.35 , 3.82 , 6.12 , 6.36–7.52 , 9.35 , 10.02  ppm. 13C-NMR (DMSO-6d) δ: 168.51 (C-21), 160.70(C-2), 154.41 (C-7), 149.87 (C-8), 149.53 (C-17), 148.37 (C-18), 144.99 (C-9), 143.78 (C-13), 132.26 (C-4), 126.23 (C-15), 123.21 (C-19), 116.10 (C-10), 115.95 , 113.23 (C-5), 112.58 (C-6), 111.56 (C-20), 110.65 (C-3), 56.13 (C-22), 18.72 (C-11) ppm. MS  = 398 . Anal. Calcd. for C20H18N2O7 (398): C, 60.30; H, 4.52; N, 7.03. Found: C, 60.11; H, 4.29; N, 6.89.3--ylamino)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid (4 (0.01 mol) was dissolved. Then, 10 mL of bromine (0.02 mol) in glacial acetic acid was added dropwise to compound 4 solution with stirring at 60 °C. After 5–10 min. the bromine color was discharged and a yellow solution remained. At this point, an additional 0.5–1 mL of the bromine-AcOH solution was added with stirring at room temperature for 30–45 min. The reaction mixture was poured into water and the resulting product was filtered off, washed with water and dried. Finally, The product was crystallized from DMF/H2O (1:1) to give 5a,b.In 15 mL of glacial acetic acid, compound 5a) Yellow crystals, yield 71%, m.p. 225–227 °C. IR(KBr) νmax: 3452 (br. OH), 3254 (NH),1710–1705 (C=O), 1627, 1604, 1558 (C=C), 1223, 1072, 1026 (C-O) cm−1. 1H-NMR (DMSO-6d) δ: 2.55 , 5.32  5.54 , 6.54 , 7.38–7.69  10.35 , 10.53  ppm. 13C-NMR (DMSO-6d) δ: 169.56, 168.05 (C=O of acid for two isomer), 156.46 (C-2), 152.09, 147.5, (C-O), 144.41, 141.54 (C-N), 138.68, 134.67, 133.30,130.68, 129.54, 129.35, 129.15, 128.65, 128.65, 119.66, 119.38, 113.62, 109.24, 107.12 (C-aromatic acid Quinolinone ring), 52.38, 47.69 (C-olefinic), 20.01 (CH3) ppm. MS  = 508. Anal. Calcd. for C19H14N2Br2O5 (508): C, 44,88, H, 2.75; N, 5.51. Found: C, 44.64; H, 2.52; N, 5.33.3--ylamino)-3-phenylacrylic acid (5b). Pale yellow crystals, yield 69%, m.p. 233–235 °C. IR (KBr) νmax: 3580–2955 (br. OH and NH), 1717–1705 (br. C=O), 1607, 1558 (C=C), 1239, 1160, 1101, 1028 (C-O) cm−1. 1H-NMR (DMSO-d6) δ: 2.53 , 7.01–8.16  ppm. 13C-NMR (DMSO-6d) δ: 166.21, 156.46 (C=O), 152.08, 151.84, 151.04 (C-O), 147.52, 141.53 (C-N), 134.49, 134.31, 133.31, 132.87, 132.46, 130.87, 130.77, 130.23, 119.36, 116.83, 113.61, 112.63, 112.34, 111.94, 109.24, 108.50, 107.78, 106.22 , 20.16, 20.01 . MS:  = 524. Anal. Calcd. for C19H14N2Br2O6 (524): C, 43.51; H, 2.67; N, 5.34. Found: C, 43.34; H, 2.42; N, 5.51. 1H and 13C NMR of compound 3, 4a–c and 5a–b are available in 3--ylamino)-3-(4-hydroxyphenyl)acrylic acid (5 cells/well) for 24 h before treatment with the compounds. Test compounds were dissolved in dimethyl sulfoxide (DMSO) which did not exceed 1% final concentration. Different concentrations of the compound under test  were added to the cell’s monolayer. Triplicate wells were prepared for each individual concentration. Monolayer cells were incubated with the compound(s) for 48 h at 37 °C and in atmosphere of 5% CO2. After 48 h, cells were fixed, washed with phosphate buffer saline  and stained with 40 μL of MTT solution (5 mg/mL of MTT in 0.9% NaCl) in each well was added and incubated for an additional 4 h. MTT crystals were solubilized by adding 180 μL of acidified isopropanol/well and the plate was shaken at room temperature, followed by photometric determination of the absorbance at 570 nm using ELISA reader. The molar concentration required to inhibit 50% of cell viability (IC50) was calculated and compared with the reference drug STU. The surviving fractions were expressed as means ± SD.The cytotoxic activity was measured in vitro using the MTT assay. Cells were plated in 96-multiwall plate (105 cells/well) and incubated at 37 °C for 12 h. The target cells were then treated with the compound 5a at its IC50 concentration dose value for 48 h. After treatment, cells were collected and fixed with 75% ethanol at 20 °C overnight, then, cells were washed with PBS followed by centrifugation and incubated with (10 mg/mL) Rnase  and (5 mg/mL) propidium iodide  before flow cytometry analysis .HCT-116 cells, (3.0 × 105/well) were treated with compound 5a at its IC50 concentration value for 48 h. After treatment, cells were harvested and washed twice  with PBS. Each cell well was resuspended in 100 μL of binding buffer, and 5 µL Annexin V-FITC were added. After an incubation time of 10 min at room temperature, additional 400 µL of binding buffer were added for a final volume of 500 µL. Cells were stained with PI immediately before measurement. Cells were the analyzed by using FACS Calibur Flow cytometer . Data thus obtained were analyzed with Cell-Quest software .The HCT-116 cells  ELISA kit according to manufacturer’s instructions. Prepare all reagents, working standards, and samples. Add 100 μL of standard and sample per well and incubate for 2 h at 37 °C. Remove the liquid of each well, do not wash. Add 100 μL of biotin-antibody to each well and incubate for 1 h at 37 °C. Aspirate each well and wash three times. Add 100 μL of horseradish peroxidase (HRP-avidin) to each well and incubate for 1 h at 37 °C. Repeat the aspiration/wash process for five times. Add 90 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to each well and incubate for 15–30 min at 37 °C, protect from light. Add 50 μL of stop solution to each well and determine the optical density of each well within 5 min, using a microplate reader set to 450 nm. The values of % activity versus a series of compound concentrations  were then plotted using non-linear regression analysis of sigmoidal dose-response curve. The IC50 values for compound 5a against topoisomerase II was determined by the concentration causing a half-maximal percent activity and the data were compared with Dox as standard.Compound 5 were applied.Docking calculations were estimated using a Docking Server . Docking4a–c) were synthesized via the reaction of cinnamic acid derivatives with 1-amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (3), which was obtainable in the reaction of 7,8-hydroxy-4-methylcoumarin (2) with hydrazine hydrate in pyridine. Brominated derivatives 5a,b were prepared via the halogenation of compounds 4a,b with bromine in glacial acetic acid. Cinnamic acids bearing 2-quinolone 4a,b and their brominated derivatives 5a,b were assessed for their in vitro antiproliferative activity against three cancer cell lines and the brominated derivatives were found to be more active. DNA flow cytometry assay of compound 5a revealed G2/M phase arrest and pre-G1 apoptosis. Annexin V-FITC showed the percentage of early and late apoptosis was increased. The results of topoisomerase enzyme inhibition activity showed that the hybrid molecule 5a display potent inhibitory activity compared with control. In conclusion, the preliminary study of the anticancer activity of the prepared compounds represents a novel strategy for the discovery of anticancer compounds which needs further investigation.In conclusion, hybrid molecules containing cinnamic acid and 2-quinolinone derivatives (


Dolichandrone spathacea iridoids are promising anti-diabetic inhibitors towards α-glucosidase protein (PDB-3W37) and oligo-1,6-glucosidase protein (PDB-3AJ7). Five catalpol iridoids  were isolated from mangrove plant D. spathacea, and their derivatives  were obtained from reduction, acetylation, O-alkylation, acetonisation, or hydrolysation starting from naturally isolated compounds. They were identified by spectral methods such as IR, MS, and 1D and 2D NMR. Their glucosidase-related (3W37 and 3AJ7) inhibitability and physiological compatibility were predicted by molecular docking simulation and prescreened based on Lipinski's rule of five. Experimental α-glucosidase inhibition of 1–15 was evaluated using enzyme assays. Compounds 3, 4, 5, 6, and 9 are new iridoid derivatives, introduced to the literature for the first time, while all fifteen compounds 1–15 are studied for molecular docking for the first time. Regarding protein 3W37, the five strongest predicted inhibitors assemble in the order 2 > 10 > 1 > 9 > 14. In respect to 3AJ7, the corresponding order is 14 > 2 > 10 > 5 > 1 = 9. Lipinski's criteria suggest 10 as the candidate with the most potential for oral administration. The in vitro bioassay revealed that compound 10 is the most effective inhibitor with a respective IC50 value of 0.05 μM, in the order 10 > 2 > 14 > 13 > 1. The computational and experimental results show good consistency. The study opens an alternative approach for diabetes treatment based on inhibitability of natural and semi-synthesised catalpol iridoid derivatives towards carbohydrate-hydrolases. Dolichandrone spathacea catalpol derivatives as glucosidase inhibitors: in silico and in vitro research. According to the International Diabetes Federation, it was responsible for about 4.2 million deaths in 20195 especially from sugar beet seeds.6 This type of glucosidase was demonstrated only catalysing hydrolysis of α-(1→4) and α-(1→6) bonds to yield monosaccharides.7 Protein structural data of α-glucosidase can be referenced at Worldwide Protein Data Bank under entry PDB-3W37 (DOI: 10.2210/pdb3W37/pdb). Second, oligo-1,6-glucosidase, often called isomaltase, is a debranching endoenzyme, hydrolysing only the α-1,6 linkage in starch and glycogen to produce sugars with an α-configuration.8 Unlike α-glucosidase, they are not responsible for α-1,4 linkage hydrolysis. Oligo-1,6-glucosidase is mainly found in animals although some bacterial species, such as Bacillus cereus, can also synthesise it.9 In humans, it is located on the small intestine brush border.10 Information for isomaltase crystal structure can be downloaded directly from Worldwide Protein Data Bank database under entry PDB-3AJ7 (DOI: 10.2210/pdb3AJ7/pdb). Therefore, 3W37 and 3AJ7 are considered as highly promising drug targets for effective treatment of type-2 diabetes, especially medication through oral administration. In principle, multiple inhibition of these enzymes (3W37 and 3AJ7) is considered as an efficacious strategy to suppress the hyperglycemia and improve insulin sensitisation, simultaneously. Crystal structure of the proteins are shown in There are many kinds of medicines for the treatment of type-2 diabetes, based on insulin sensitizers, sodium glucose transporter-2 (SGLT-2) inhibitors, glucagon like peptidase-1 (GLP-1) analogues and dipeptidyl peptidase-4 (DPP-IV) inhibitor mechanisms. However, these therapies have significant side effects such as weight gain, cardiovascular risks, and atherosclerosis as well as limited tolerability. Therefore, the approach for type 2 DM changes into the inhibition of glucosidase enzymes, which slows down of glucose absorption through intestinal wall. There are two main types of glucosidase responsible for carbohydrate break-down processes in the body, regarding their sources: α-glucosidase and oligo-1,6-glucosidase. First, α-glucosidase is an exoenzyme found in animal, plant, bacterial, or fungal organisms,In silico technique, based on computational simulation and computing calculation, is currently seeing a gain of their popularity for medical science as prescreening research. It is for reducing cost and time of wet laboratory experiments by predicting the compounds with undesirable properties and the most promising candidates. The former substances are deemed to be eliminated from the next analysis or further-developed research, while the latter justifies the selection.11 Regarding ligand–protein interaction, molecular docking simulation is an effective method to investigate the potency of a ligand as an inhibitor towards its targeted protein. The method can estimate ligand-target binding energy and intermolecular interaction, thus predicting static stability of the inhibitory systems.12 Effectively inhibited by external ligands, the enzyme is likely to undergo conformational changes, thus loss of enzymatic functionality inevitably ensuing. This might help mitigating the amount of glucose catalytically synthesised and excreted to bloodstream. According to the methodology of molecular docking simulation applied for ligand–protein inhibitory systems in general and to the evaluation from.−1 indicates good binding capacity.13–15 In principle, the figure is yielded by the free-energy sum of all individual intermolecular interactions, of which affinity stems from hydrophilic bonding, i.e. various hydrogen-bond types, and hydrophobic binding, i.e. van der Waals forces. Besides, a root-mean-square deviation (RMSD) value over 3 Å reveals that the inhibition expects failure; meanwhile, the threshold of docking success is widely acceptable if ≤2 Å.16 Also, visual illustration for inhibitory morphology and interaction description is often provided. Specification to drug design for diabetes, molecular docking technique demonstrated its contributive significance regarding a variety of ligand families, e.g. thiazolidinedione,17 pyrazole–rhodanine,18 or wedelolactone.19Molecular Operating Environment (MOE)-based algorithms in particular, an associated docking score (DS) is considered as the main indicator for inhibitory effects, of which value lower than −3.2 kcal mol20 Recently, there is an increasing numbers of papers reported about the anti-diabetic efficacy of catalpol derivatives in the management of diabetes and its complications.20,21 It has been showed that catalpol-type iridoids are able to restore diabetic liver function by improving glucose and lipid metabolism, ameliorating oxidative stress, and restoring mitochondrial function. They improve glucose uptake and lipolysis by upregulating the expression of glucose transporter as well as downregulating the expressions of peroxisome proliferators in mice.20 Therefore, catalpol derivatives have become a very promising scaffold for the development of anti-diabetic drug candidates. Our previous study on chemical constituents of the mangrove plant Dolichandrone spathacea led to the isolation of four catalpol iridoids.22 This seems reasonable because according to folk experiences, the plant is currently reported as a natural symptom reliever for diabetes, especially in Mekong Delta, Vietnam. Nevertheless, its chemical properties and medical potentiality still remain almost untouched possibly because of their narrow distribution.Catalpol and catalpol analogues, containing epoxy ring at C-7 and C-8, are widely found in many plants belonging to some families such as Plantaginaceae, Lamiaceae, Bignonicaceae.3W37) and oligo-1,6-glucosidase protein (PDB-3AJ7). Three commercial medicines acarbose (D1), miglitol (D2), and voglibose (D3) had been reported able to lengthen the duration time of carbohydrate absorption and flatten the blood-glucose concentrations, thus widely used in the treatment for patients with type 2 diabetes.23 Therefore, they were specified as references in this study. Their structural formula is presented in In this paper, ten derivatives from the isolated compounds were synthesized, together with natural compounds they were studied in molecular docking experiment on sugar beet α-glucosidase protein  and 2D  NMR spectra were recorded on a Bruker Avance 500 MHz using tetramethylsilane (TMS) as the internal standard for 1H and solvent signal for 13C NMR. Thin-layer chromatography (TLC) was carried out on a pre-coated silica gel 60 F254 (Merck & Co.), visualised under ultraviolet (UV) radiation at λmax 254 nm, and stained by a solution of 1% (w/w) vanillin in sulfuric acid. Column chromatography (CC) was performed on silica gel 300–400 mesh (Merck & Co.). Enzyme α-glucosidase was purchased from Sigma-Aldrich under the name code G0660, detected by a Biotek Epoch 2 microplate readers (USA).All analytically graded reagents were purchased from either Sigma-Aldrich or Merck & Co. and used without further purification. Solvents for column chromatography were distilled before use. Melting points (in °C) were determined on a Thermo Mel-temp 3.0 (USA). Infrared (IR) spectra were recorded on an IMPACT 410 Nicolet Spectrometer. ESI-MS spectra were obtained from an 1100 Agilent LC/MS ion Trap. 1D ,24,25 6-O-catalpol (2),26,27 minecoside (13),28 specioside (14).29The procedures for the isolation of 1, 2, 13 and 14 were described in ref. 2.1.2.2Dolichandrone spathacea collected in Phu Loc district, Thua Thien-Hue province, Vietnam in December 2018. The plant was identified by Dr Do Xuan Cam, University of Agriculture and Forestry, Hue University. The dried powder of the leaves of Dolichandrone spathacea (250 g) was extracted with EtOH 90% at 50 °C (3 × 3 hours × 1 L). The EtOH extracts were combined and evaporated the solvent under reduced pressure. The obtained residue (37 g) was suspended in 350 mL MeOH/H2O (1 : 2) and partitioned with CH2Cl2. The organic layer was separated, then the remaining aqueous layer was dried to obtain the residue (25 g). The residue was subjected to a silica gel column with a gradient of CH2Cl2/MeOH 100 : 0 to 70 : 30 to give four fractions Fr.1–Fr.4. Fraction Fr.1 (2.0 g) was further purified by RP-18 column chromatography, eluting with MeOH/H2O (1 : 1) to afford compound 10 (5.1 mg).Compound 10 was isolated from the leaves of 2.1.32.1.3.12·6H2O (0.5 mmol). The reaction mixture was stirred for 10 minutes at room temperature and cooled to 0 °C. NaBH4  was slowly added to the mixture, which was then allowed to stir at 0 °C for 30 minutes and one additional hour at room temperature. The reaction mixture was then removed the solvent to obtain a white solid. The solid was dissolved in CH2Cl2 and the solution was washed with water (2 × 30 mL), dried over anhydrous Na2SO4, and evaporated the solvent. The crude product was purified over a silica gel column (CH2Cl2/MeOH 9 : 1) to obtain 3  or 4 .To a stirred solution of 1 or 2 (0.1 mmol) dissolved in MeOH (5 mL) was added NiCl2.1.3.22Cl2 (20 mL) and HCl (1 N) to pH = 6, following by washing with a solution of NaHCO3 (5%) (2 × 10 mL) and water (2 × 10 mL). The organic layer was evaporated to obtain the crude product which was purified by silica gel column chromatography (n-hexane/ethyl acetate 2 : 1) to afford 5 .To a solution of 4  in pyridine (2 mL) was added anhydride acetic acid (2 mL). After stirring for 12 hours at room temperature, the reaction mixture was added CH2.1.3.33 (3 mL) and pyridine (0.5 mL) was added triphenylmethyl chloride . The reaction mixture was allowed to stir for 12 hours at room temperature. The solvent was then removed under reduced pressure to give white solid which was dissolved in CHCl3 (30 mL) and washed with water (2 × 30 mL). The organic layer was dried over anhydrous Na2SO4, evaporated the solvent to afford a yellow solid, which was purified by silica gel column chromatography (CH2Cl2/MeOH/pyridine 98 : 2 : 0.5) to obtain 6.To a solution of 2  in dried CHCl2.1.3.42SO4, evaporated the solvent to give yellow solid. The crude product was subjected to a Sephadex LH-20 column (100% MeOH) to obtain the corresponding acetylated catalpol iridoids.To a solution of 1 or 2 or 13 or 14 (0.1 mmol) in pyridine (1 mL) was added anhydride acetic acid (1 mL). The solution was stirred for 24 h at room temperature then ice cold water (10 mL) was added and extracted with EtOAc (3 × 30 mL). The EtOAc extract was combined, dried with anhydrous Na2.1.3.52SO4 and evaporated the solvent to give a white solid, this was then purified by a silica gel column chromatography (DCM/MeOH 95 : 5) to obtain 9 .To a solution of 1  and TsOH (4 mg) in dried DMF was added dropwise 2,2-dimethoxypropane . The reaction mixture was heated to 50 °C for three hours, neutralized by triethylamine, following by the addition of EtOAc (100 mL) and water (50 mL). The organic layer was separated, washed with water (2 × 50 mL), dried over anhydrous Na2.1.3.62O/THF/EtOH 1 : 1 : 1) and TEA (1 mL). Hydroxyl amine hydrochloride  was then added to the solution. The reaction mixture was heated to 100–110 °C for 6 hours. The solvent was evaporated to give a residue which was then suspended in water and partitioned with EtOAc (3 × 20 mL). The organic layers were combined, dried over anhydrous Na2SO4 and evaporated the solvent to afford a white solid, this was subjected to a silica gel column chromatography (CH2Cl2/MeOH 9 : 1) to obtain 15 .Compound 2  was dissolved in a mixture of (H2.1.4−1. Each concentration of the test sample (10 μL) was incubated at 37 °C with phosphate buffer 100 mM , α-glucosidase 0.4 U mL−1 (25 μL), p-nitrophenyl α-d-glucopyranoside 2.5 mM (25 μL). After 30 minutes, the reaction was terminated by adding 100 μL of 0.1 M Na2CO3 solution. The enzyme activities were determined by measuring the absorbance at λmax 410 nm. The control was prepared using the same procedure replacing the test sample by distilled water, while activity of the reference was tested by replacing the test sample with acarbose30 Percentage of inhibition was calculated as followed:31Each assay was performed on 96 well-plates (200 μL per well). The test samples were dissolved in DMSO 10% and further diluted to the concentrations: 1024, 256, 64, 16, 4, 1 μg.mL2.2.2.2.13W37 and 3AJ7) and docked ligands (iridoid compounds and referenced drugs) were required as the precursors. As conducted, this served as inputs to simulate intermolecular interaction between the agents, thereby yielding information on the respective ligand-protein inhibitory complex. The output included inhibitory configurations, docking score (DS) energy, root-mean-square deviation (RMSD), interaction types, and distances between ligands and proteins. In a typical procedure, a molecular docking simulation follows three steps.13–15,32The simulation was carried out on MOE 2015.10 software. The structural information of targeted proteins (2.2.1.13W37 (DOI: 10.2210/pdb3W37/pdb) and 3AJ7 (DOI: 10.2210/pdb3AJ7/pdb) were downloaded from Worldwide Protein Data Bank. The proteins and their 3D protonation were prepared by MOE QuickPrep functionality. Determination of protein active zones based upon a radius set at 4.5 Å from their amino acids and the inhibitory ligands. The preparatory protein structures obtained were saved in format *.pdb, ready for docking simulation. Independently, the ligands, including iridoid compounds and referenced drugs, were structurally optimised by Conj Grad for energy minimisation. The optimisation configuration was set: termination for energy change = 0.0001 kcal mol−1; max interactions = 1000; charge modification by Gasteiger–Huckel method.Structural data of carbohydrate-hydrolase proteins 2.2.1.2Simulation on intermolecular interaction between the investigated agents was performed on MOE 2015.10 and the obtained inhibitory structures were saved in format *.sdf. Docking simulation parameters were configured as: poses retaining for further inhibition analysis = 10; maximum solutions per iteration = 1000; maximum solutions per fragmentation = 200.2.2.1.3e.g. electron-transferring (H-acceptor/donor), cation–arene (H–π), arene–arene (π–π), and ionic; while, van der Waals forces constitute the latter. The results provide in-bonding amino acids, bonding lengths, and their Gibbs free energy. Besides, binding stability of the complex static conformation was evaluated by the respective values of root-mean-square deviation (RMSD) calculated. This is based on the fact that RMSD represent the average between neighbouring atoms; therefore, a smaller value means a more tightly bound conformation is formed. In addition, ligand conformation and orientation in its inhibited-protein active site was visualised on 2D and 3D planes.The inhibitability of a certain duo-system was primarily predicted by docking score (DS) energy, which represents binding affinity of ligands and their targeted proteins in the site–site distance. Intermolecular interactions formed between the ligands and in-pose amino acids of the proteins were also probed. These include hydrophilic binding and hydrophobic interaction. The former is a variety of hydrogen-bond types, 2.2.2average , molecular mass (Da), polarizability (Å3) and volume or size (Å), and dispersion coefficients (log P and log S) were achieved by Gasteiger–Marsili method using QSARIS system.33 They were then prescreened in an attempt to evaluate their orally pharmacological compatibility. This was based on Lipinski's rule of five, a well-known set of indicators to predict drug-likeness.34 According to Lipinski's criteria, a well membrane-permeable molecule should satisfy the requirements: (1) molecular mass < 500 Da; (2) no more than 5 groups for hydrogen bonds; (3) no more than 10 groups receiving hydrogen bonds; (4) the value of log P is less than +5 (log P < 5).35,36The docking parameters including DS3.3.1.3.1.125H30O12, ESI-MS (m/z): 523.1 [M + H]+, 521.0 [M − H]−; IR : 2936 , 1716  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O ester), 1633 , 1602 (CC–Ar), 1063 (C–O ether); 1H-NMR , δH (ppm), J (Hz): 7.69 , 7.55 , 6.95 , 6.41 , 6.36 , 5.15 , 5.05 , 5.00 , 4.81 , 4.16 , 3.94 , 3.87 , 3.84 , 3.73 , 3.68 , 3.46–3.42 , 3.35–3.34 , 3.33–3.32 , 3.30–3.28 , 2.67–2.64 , 2.63–2.61 ; 13C-NMR , δC (ppm): 168.61 (C-9′′), 162.98 (C-4′′), 146.69 (C-7′′), 142.17 (C-3), 130.98 (C-6′′), 130.90 (C-2′′), 127.92 (C-1′′), 115.31 (C-8′′), 115.26 , 102.74 (C-4), 99.47 (C-1′), 94.88 (C-1), 81.09 (C-6), 78.25 (C-5′), 77.38 (C-3′), 74.50 (C-2′), 71.39 (C-4′), 66.67 (C-8), 62.63 (C-6′), 61.12 (C-10), 60.05 (C-7), 55.81 (4′′-OCH3), 42.89 (C-9), 36.46 (C-5).White amorphous powder, C3.1.226H32O13, (+)-ESI-MS (m/z): 588.2 [M + Cl]−; IR : 2903 , 1717 (CO ester), 1635 , 1594 (CC–Ar), 1086 (C–O ether); 1H-NMR , δH (ppm), J (Hz): 7.68 , 7.14 , 7.09 , 6.90 , 6.38 , 6.34 , 5.05–5.00 , 4.84 , 4.03 , 3.95 , 3.92 , 3.88 , 3.78 , 3.74 , 3.52–3.48 , 3.39–3.36 , 2.71–2.66 , 2.65–2.64 ; 13C-NMR , δC (ppm): 167.32 (C-9′′), 151.30 (C-4′′), 149.10 (C-3′′), 145.90 (C-7′′), 141.05 (C-3), 127.01 (C-1′′), 122.86 (C-6′′), 114.64 (C-8′′), 111.00 (C-2′′), 109.72 (C-5′′), 102.06 (C-4), 98.58 (C-1′), 94.26 (C-1), 79.65 (C-6), 76.41 (C5′), 75.97 (C-3′), 73.02 (C-2′), 69.29 (C-4′), 65.42 (C-8), 60.91 (C-6′), 60.53 (C-10), 59.12 (C-7), 55.81 (3′′-OCH3), 55.76 (4′′-OCH3), 41.76 (C-9), 35.17 (C-5).White amorphous powder, C3.1.325H32O12, HR-ESI-MS (m/z): 547.1779 [M + Na]+ , ESI-MS (m/z): 559.0 [M + Cl]−; 1H-NMR , δH (ppm), J (Hz): 7.15 , 6.85 , 6.34 , 5.12 , 4.92 , 4.79 , 4.15 , 3.94 , 3.82 , 3.77 , 3.65 , 3.61 , 3.41 , 3.35–3.26 , 2.91 , 2.69 , 2.60–2.57 , 2.48–2.44 ; 13C-NMR , δC (ppm): 174.52 (C-9′′), 159.70 (C-4′′), 142.25 (C-3), 133.69 (C-1′′), 130.34 , 114.93 , 102.85 (C-4), 99.70 (C-1′), 95.04 (C-1), 81.32 (C-6), 78.62 (C-5′), 77.70 (C-3′), 74.83 (C-2′), 71.77 (C-4′), 66.77 (C-8), 62.92 (C-10), 61.24 (C-6′), 60.07 (C-7), 55.68 (4′′-OCH3), 43.13 (C-9), 37.04 (C-8′′), 36.60 (C-5), 31.15 (C-7′′).White amorphous powder, C3.1.426H34O13, HR-ESI-MS (m/z): 577.1867 [M + Na]+ , ESI-MS (m/z): 589.1 [M + Cl]−; 1H-NMR , δH (ppm), J (Hz): 6.88 , 6.80 , 6.34 , 5.12 , 4.92 , 4.79 , 4.15 , 3.94 , 3.84–3.82 , 3.83 , 3.81 , 3.65 , 3.61 , 3.41 , 3.33–3.25 , 2.93 , 2.72 , 2.60–2.57 , 2.48–2.43 ; 13C-NMR , δC (ppm): 174.55 (C-9′′), 150.47 (C-4′′), 149.07 (C-3′′), 142.28 (C-3), 134.74 (C-1′′), 121.70 (C-6′′), 113.59 (C-2′′), 113.33 (C-5′′), 102.82 (C-4), 99.70 (C-1′), 95.04 (C-1), 81.36 (C-6), 78.64 (C-5′), 77.71 (C-3′), 74.84 (C-2′), 71.78 (C-4′), 66.78 (C-8), 62.93 (C-10), 61.24 (C-6′), 60.07 (C-7), 56.61 (4′′-OCH3), 56.49 (3′′-OCH3), 43.14 (C-9), 36.96 (C-8′′), 36.62 (C-5), 31.60 (C-7′′).White amorphous powder, C3.1.536H44O18, HR-ESI-MS (m/z): 787.2389 [M + Na]+ , ESI-MS (m/z): 765.0 [M + H]+; 1H-NMR , δH (ppm), J (Hz): 6.88 , 6.78 , 6.34 , 5.30 , 5.11–5.03 , 4.93–4.87 , 4.36 , 4.23 , 3.90 , 3.84 , 3.81 , 3.80 , 3.63 , 2.93 , 2.72 , 2.56–2.52 , 2.45–2.41 , 10,2′,3′,4′,6′-OCOCH3: 2.13, 2.10, 2.04, 2.03, 2.00. 13C-NMR , δC (ppm): 174.42 (C-9′′), 172.64, 172.45, 171.60, 171.14, 170.92 (5×-OCOCH3), 150.49 (C-4′′), 149.10 (C-3′′), 142.29 (C-3), 134.68 (C-1′′), 121.70 (C-6′′), 113.61 (C-2′′), 113.35 (C-5′′), 102.96 (C-4), 98.09 (C-1′), 95.92 (C-1), 81.01 (C-6), 74.04 (C-5′), 73.33 (C-3′), 72.22 (C-2′), 69.61 (C-4′), 64.68 (C-10), 63.65 (C-8), 62.28 (C-6′), 59.93 (C-7), 56.61 (4′′-OCH3), 56.49 (3′′-OCH3), 42.72 (C-9), 36.88 (C-8′′), 36.44 (C-5), 31.56 (C-7′′), 20.75, 20.71, 20.57, 20.54, 20.51 (5×-OCOC̲H3).White amorphous powder, C3.1.664H60O13, HR-ESI-MS (m/z): 817.2788 (100) [C45H46O13Na]+ [M − C19H15 + Na + H]+ ; 1H-NMR , δH (ppm), J (Hz): 7.69 , 7.39–7.15 3), 7.13 , 7.07 , 6.88 , 6.38 , 6.31 , 5.01 , 4.96 , 4.74 , 4.59 , 3.91 , 3.86 , 3.75 , 3.45 , 3.36 , 3.27 , 3.11 , 2.76 , 2.68 ; 13C-NMR , δC (ppm): 166.98 (C-9′′), 151.43 (C-4′′), 149.34 (C-3′′), 146.92 (2×-OC̲(C6H5)3), 128.80–127.25 (2×-OC(C̲6H5)3), 87.48 and 86.60 (C-10 and C-6′), 145.68 (C-7′′), 141.09 (C-3), 127.09 (C-1′′), 122.83 (C-6′′), 115.15 (C-8′′), 111.18 (C-2′′), 109.82 (C-5′′), 102.44 (C-4), 97.27 (C-1′), 93.72 (C-1), 79.65 (C-6), 76.80 (C-5′), 76.30 (C-3′), 73.57 (C-2′), 72.54 (C-4′), 65.09 (C-8), 64.48 (C-6′), 61.22 (C-10), 59.04 (C-7), 55.93 , 42.80 (C-9), 35.53 (C-5).White amorphous powder, C3.1.737H42O19, HR-ESI-MS (m/z): 813.2209 [M + Na]+ , 791.2336 [M + H]+ ; 1H-NMR , δH (ppm), J (Hz): 7.63 , 7.08–7.07 , 7.00 , 6.38 , 6.27 , 5.18–5.15 , 5.09–5.05 , 4.96–4.90 , 4.82–4.79 , 4.26 , 4.13 , 3.92 , 3.80 , 3.69 , 3.66 , 2.61–2.58 , 2.57–2.54 , 2.25 , 2.06 , 1.98 , 1.97 , 1.95  (5×-OCOC̲H3); 13C-NMR , δC (ppm): 170.96, 170.87, 170.37, 169.51, 169.32, 168.97 (5×-OC̲OCH3), 166.72 (C-9′′), 151.32 (C-4′′), 245.26 (C-7′′), 141.57 (C-3′′), 141.03 (C-3), 132.97 (C-1′′), 123.18 (C-6′′), 121.32 (C-8′′), 117.20 (C-2′′), 111.30 (C-5′′), 101.89 (C-4), 96.50 (C-1′), 94.16 (C-1), 79.50 (C-6), 72.50 (C-5′), 72.12 (C-3′), 68.12 (C-4′), 62.58 (C-8), 62.32 (C-6′), 58.77 (C-7), 55.80 (4′′-OCH3), 41.47 (C-9), 34.96 (C-5), 20.51 (–OCOC̲H3), 20.46 (–OCOC̲H3), 20.43 (3×-OCOC̲H3).White amorphous powder, C3.1.836H42O18, (+)-ESI-MS (m/z): 763.9 [M + H]+; 1H-NMR , δH (ppm), J (Hz): 7.68 , 7.12 , 7.07 , 6.87 , 6.36 , 6.32 , 5.25–5.21 , 5.16–5.12 , 5.03–4.96 , 4.89–4.85 , 4.31 , 4.21 , 3.99 , 3.91 , 3.73 , 3.71 , 2.70–2.65 , 2.64–2.62 ; 13C-NMR , δC (ppm): 170.70, 170.54, 170.22, 169.28, 169.07 (5×OC̲OCH3), 167.05 (C-9′′), 151.39 (C-4′′), 149.21 (C-3′′), 145.92 (C-7′′), 141.06 (C-3), 127.07 (C-1′′), 122.91 (C-6′′), 114.72 (C-8′′), 111.01 (C-2′′), 109.59 (C-5′′), 102.09 (C-4), 96.57 (C-1′), 94.23 (C-1), 79.28 (C-6), 72.51 (C-5′), 72.26 (C-3′), 68.15 (C-4′), 62.57 (C-8), 62.35 (C-6′), 61.16 (C-10), 58.85 (C-7), 55.95 (3′′-OCH3), 55.85 (4′′-OCH3), 41.53 (C-9), 35.01 (C-5), 20.70 (–OCOC̲H3), 20.65 (–OCOC̲H3), 20.61 (–OCOC̲H3), 20.57 (2×-OCOC̲H3).White amorphous powder, C3.1.928H34O12, HR-ESI-MS (m/z): 585.1956 [M + Na]+ ; 1H-NMR , δH (ppm), J (Hz): 7.68 , 7.39 , 6.91 , 6.37 , 6.35 , 5.04 , 5.02 , 4.93 , 4.89 , 4.03 , 3.97 , 3.76 , 3.53 , 3.38 , 2.73 , 2.66 , 1.53 and 1.45 ; 13C-NMR , δC (ppm): 167.21 (C-9′′), 161.69 (C-4′′), 145.66 (C-7′′), 141.06 (C-3), 129.95 , 125.96 (C-1′′), 114.67 (C-8′′), 114.44 , 102.51 (C-4), 99.97 (C-7′), 99.43 (C-1′), 94.84 (C-1), 79.46 (C-6), 74.25 (C-5′), 73.54 (C-3′), 72.92 (C-2′), 67.89 (C-4′), 65.20 (C-8), 61.92 (C-6′), 61.42 (C-10), 59.19 (C-7), 55.40 (4′′-OCH3), 42.10 (C-9), 36.50 (C-5), 29.00 and 20.70 (7′-CH3).White amorphous powder, C3.1.1025H36O12; (+)-ESI-MS (m/z): 551.1 [M + Na]+, (−)-ESI-MS (m/z): 563.0 [M + Cl]−; 1H-NMR , δH (ppm), J (Hz): 6.74 , 6.26 , 5.30 , 5.06 , 4.85 , 4.70 , 4.05 , 3.99 , 3.83 , 3.72 , 3.57 , 3.54 , 3.30 , 3.21 , 3.16–3.14 , 2.52–2.46 , 2.26 , 2.08 , 1.77 , 1.60 ; 13C-NMR , δC (ppm): 169.34 (C-1′′), 144.15 (C-3′′), 142.38 (C-3), 138.37 (C-6′′), 128.60 (C-2′′), 125.74 (C-7′′), 102.91 (C-4), 99.72 (C-1′), 95.08 (C-1), 81.62 (C-6), 78.65 (C-3′), 77.72 (C-2′), 74.85 (C-5′), 71.78 (C-4′), 66.79 (C-8), 62.93 (C-10), 61.27 (C-6′), 60.17 (C-7), 59.37 (C-8′′), 43.17 (C-9), 39.06 (C-5′′), 36.67 (C-5), 28.06 (C-4′′), 16.18 (C-10′′), 12.47 (C-9′′).White amorphous powder, C3.1.1135H40O17, ESI-MS (m/z): 755.0 [M + Na + H2O]+, 791.1 [M + Na + 2H2O]+; 1H-NMR , δH (ppm), J (Hz): 7.70 , 7.49 , 6.91 , 6.35 , 6.31 , 5.25–5.23 , 5.16–5.12 , 5.02–4.97 , 4.98–4.96 , 4.89–4.86 , 4.30 , 4.21 , 3.99 , 3.83 , 3.73 , 3.71 , 2.69–2.66 , 2.64–2.62 , 2.13 , 2.12 , 2.05 , 2.05 , 2.04 , 2.01  (6×-OCOC̲H3); 13C-NMR , δC (ppm): 170.62 (2×-OC̲OCH3), 170.46 (–OC̲OCH3), 170.16 (–OC̲OCH3), 169.23 (–OC̲OCH3), 169.02 (–OC̲OCH3), 167.12 (C-9′′), 161.64 (C-4′′), 145.63 (C-7′′), 141.01 (C-3), 129.88 , 126.85 (C-1′′), 114.54 (C-8′′), 114.38 , 102.13 (C-4), 96.59 (C-1′), 94.26 (C-1), 79.26 (C-6), 72.53 (C-5′), 72.28 (C-3′), 70.57 (C-2′), 68.20 (C-4′), 62.53 (C-8), 62.32 (C-6′), 61.19 (C-10), 58.83 (C-7), 55.34 (4′′-OCH3), 41.55 (C-9), 34.99 (C-5), 20.66 (–OCOC̲H3), 20.61 (–OCOC̲H3), 20.58 (–OCOC̲H3), 20.54 (3×-OCOC̲H3).White amorphous powder, C3.1.1236H40O18; (+)-ESI-MS (m/z): 783.2 [M + Na]+; 1H-NMR , δH (ppm), J (Hz): 7.77 , 7.70 , 7.19 , 6.60 , 6.40 , 5.31 , 5.13–5.12 , 5.09–5.02 , 5.00–4.98 , 4.94 , 4.38 , 4.23 , 3.93 , 3.81 , 3.76 , 2.61–2.60 , 2.30 , 2.14 , 2.12 , 2.04 , 2.00  (6×-OCOC̲H3); 13C-NMR , δC (ppm): 172.68, 172.49, 171.62, 171.16, 170.95, 170.77 (6×OC̲OCH3), 168.13 (C-9′′), 154.03 (C-4′′), 146.03 (C-7′′), 142.43 (C-3), 133.31 (C-1′′), 130.56 , 123.45 , 118.39 (C-8′′), 103.02 (C-4), 98.13 (C-1′), 95.98 (C-1), 81.26 (C-6), 74.05 (C-5′), 73.36 (C-3′), 72.23 (C-2′), 69.63 (C-4′), 64.76 (C-8), 63.73 (C-6′), 60.06 (C-7), 42.79 (C-9), 36.59 (C-5), 20.90, 20.77, 20.73, 20.59, 20.54, 20.52 (6× OCOC̲H3).White amorphous powder, C3.1.1325H30O13; 1H-NMR , δH (ppm), J (Hz): 7.68 , 7.21 , 7.11 , 6.82 , 6.40–6.38 , 6.39 , 5.18 , 5.05 , 5.00 , 4.78 , 4.18 , 3.95 , 3.92 , 3.88 , 3.72 , 3.67 , 3.45–3.41 , 3.35–3.34 , 3.31–3.27 , 2.66–2.62 , 2.61–2.60 ; 13C-NMR , δC (ppm): 167.54 (C-9′′), 151.25 (C-4′′), 147.85 (C-3′′), 145.74 (C-7′′), 142.39 (C-3), 127.98 (C-1′′), 122.59 (C-6′′), 114.62 (C-8′′), 112.06 (C-2′′), 110.85 (C-5′′), 102.93 (C-4), 99.69 (C-1′), 95.07 (C-1), 81.29 (C-6), 78.66 (C-5′), 77.70 (C-3′), 74.85 (C-2′), 71.78 (C-4′), 66.82 (C-8), 62.93 (C-6′), 61.30 (C-10), 60.27 (C7), 56.41 (4′′-OCH3), 43.17 (C-9), 36.76 (C-5).White amorphous powder, C3.1.1424H28O12; (+)-ESI-MS (m/z): 509.1 [M − H]+; 1H-NMR , δH (ppm), J (Hz): 7.70 , 7.49 , 6.83 , 6.40 , 6.39–6.38 , 5.18 , 5.04 , 5.00 , 4.81 , 4.20 , 3.95 , 3.87 , 3.72 , 3.67 , 3.45–3.42 , 3.37–3.35 , 3.34–3.27 , 2.66–2.62 , 2.61–2.59 ; 13C-NMR , δC (ppm): 168.61 (C-9′′), 161.39 (C-4′′), 147.20 (C-7′′), 142.36 (C-3), 131.30 , 127.08 (C-1′′), 116.86 (C-3′′C-5′′), 114.61 (C-8′′), 102.94 (C-4), 99.71 (C-1′), 95.09 (C-1), 81.30 (C-6), 78.61 (C-5′), 77.69 (C-3′), 74.84 (C-2′), 71.75 (C-4′), 66.82 (C-8), 62.92 (C-6′), 61.29 (C-10), 60.26 (C-7), 43.19 (C-9), 36.74 (C-5).White amorphous powder, C3.1.1515H22O10; HR-ESI-MS (m/z): 385.1111 [M + Na]+ ; 1H-NMR , δH (ppm), J (Hz): 6.36 , 5.09 , 5.06 , 4.80 , 4.13 , 3.93 , 3.91 , 3.81 , 3.65 , 3.47 , 3.43 , 3.33 , 3.29 , 3.26 , 2.55 , 2.29 ; 13C-NMR , δC (ppm): 141.73 (C-3), 104.01 (C-4), 99.70 (C-1′), 95.30 (C-1), 79.51 (C-6), 78.53 (C-5′), 77.65 (C-3′), 74.79 (C-2′), 71.70 (C-4′), 66.24 (C-8), 62.84 (C-6′), 62.57 (C-7), 61.51 (C-10), 43.57 (C-9), 39.05 (C-5).White amorphous powder, C3.2.Dolichandrone spathacea, five iridoid compounds  have been isolated and structural elucidated. Four of them , with the exception of 10, due to small amount, were converted into their derivatives by acetylation, reduction, O-alkylation, acetonisation and hydrolysation to obtain 3, 4, 5, 6, 7, 8, 9, 11, 12 and 15. Initially, the reduction of the olefin group in cinnamoyl and iridoid moiety in the structure of 1 and 2 was designed. The reduction was carried out by different kind of reductive reagents such as NaBH4/MeOH, LiAlH4/MeOH. However, many unwanted products were afforded under these condition. This might due to the strong base condition formed during the working up, causing a damage of iridoid ring. The trial of reduction with hydrogen catalyzed by Pd/C did not work, even though the reaction was heated to high temperature. We therefore used an additional catalyst Lewis acid NiCl2·6H2O in combination with NaBH4 reagent. The reaction was carried out under mild condition at 0 °C for 30 minutes. In this case, the products 3, 4 were obtain in good yield . Interestingly, this reductive condition gave regioselective reduction only at olefinic bond of cinnamoyl group, but not at the Δ-3,4-double bond of iridoid ring. The dihydro derivative 4 was continuously acetylated with acetic anhydride in pyridine, giving the corresponding pentaacetate 5 in 80% yield. In order to investigate the effect of free hydroxyl groups on hyperglycemic activity in catalpol iridoids, compound 2 was O-alkylated with triphenylmethyl chloride and compound 1 was reacted with 2,2-dimethoxypropane/TsOH·H2O to provide product 6 and 9, respectively. The trityl groups were attached only to the primary oxymethylene groups at C-10 in catalpol ring and at C-6′ in glucose unit as well. In case of 9, cyclic ketal was formed only at C-4′ and C-6′ of the glucose unit, due to the steric hindrance of the iridoid and cinnamoyl moiety. Iridoids 1, 2, 13 and 14 were reacted with acetic anhydride in pyridine to give the corresponding acetyl products 7, 8, 11 and 12. To find out the role of cinnamoyl moiety, this moiety was tried to remove under different basic condition such as NaOH, LiOH, or KOH. However, these conditions gave many unwanted products. Therefore, an another milder basic condition using NH2OH. HCl in TEA was applied to obtain the hydrolysation derivative 15  from compound 2.The synthetic derivations are schematically summarised in 3.3.Elucidations of the structures for compounds 1, 2 and 13, 14 using spectral methods as FT-IR, MS, HR-MS, NMR (1D and 2D) were reported in ref. Structural elucidation of compound10: The 1H and 13C NMR spectra of 10 exhibit typical signals for a catalpol moiety quite similar to that of compounds 1, 2, 13 and 14. The side chain seems to be a monoterpenoid with ten carbon signals in the 13C NMR spectrum, among them 2,3-substituted double bonds at δC (ppm): 144.15 (C-3′′), 138.37 (C-6′′), 128.60 (C-2′′), 125.74 (C-7′′), one ester carbonyl , one oxymethylene  and two vinylic methyls , the linkage between the side chain and the catalpol moiety is determined as C-6 to C-1 by the correlation of signals at δC 169.34 (C-1′′) to δH 4.85 (H-6) in the HMBC spectrum. The structure of 10 is elucidated using its 1D and 2D NMR spectra. Its ESI-MS spectrum strongly supports this evidence by the pseudomolecular ion peaks at m/z 551.1 [M + Na]+ and m/z 563.0 [M + 2H2O–H]− in positive and negative mode. In comparison with the reported data,37 compound 10 is determined as nemoroside, C25H36O12.δH/δC 2.91 /31.15 (C-7′′) and 2.69 /37.04 (C-8′′) for 3 and 2.93 /31.60 (C-7′′) and 2.72 /36.96 (C-8′′) for 4. The signals for the double bond in the iridoid ring (C-3 and C-4) are still remained in the NMR spectra of 3 and 4. Compound 5 was the acetylated product of 4. Five acetyl groups are formed in the structure of 5, deducing by the appearance of five acetyl methyl singlet at δH/δC 2.00, 2.03, 2.04, 2.10, 2.13 /20.51, 20.54, 20.57, 20.71, 20.75 as well as the observation of five carbonyl groups at δC 172.64, 172.45, 171.60, 171.14, 170.92. Similarly, the totally acetylation of hydroxyl groups in 11 and 8 is confirmed by the signals of five acetyl groups in the NMR spectra of these compounds. NMR spectra of 7 and 12 indicate six acetyl groups corresponding to the acetylation of six hydroxyl groups in their structures. The cleavage of the cinnamoyl moiety is determined by the disappearance of the signal of cinnamoyl group in the NMR spectra of 15. The highfield chemical shift of H-6/C-6 at δH/δC 3.93/79.51, compared to those of 2 (H-6/C-6 at δH/δC 5.05–5.00/79.65), is due to the free hydroxyl group forming after hydrolysation.The hydrogenation of the main iridoid components 1 and 2 led to the formation of the dihydro product 3 and 4. The reduction occurs only at the double bond of cinnamoyl moiety of 1 and 2, evidenced by the appearance of two methylene at All the elucidated structural formulae are presented in 3.4.Static interaction between the studied inhibitory agents (iridoid compounds and referenced drugs) and the targeted proteins (3W37 and 3AJ7) was simulated by molecular docking technique. List of descriptive symbols are given in ESIOverall, preliminary screening groups all the iridoid compounds into two inhibitability-based categories; in detail, 1, 2, 3, 4, 5, 9, 10, 14, and 15 are considered performing elevated inhibitory effects in comparison to those of 6, 7, 8, 11, 12, and 13. This is regardless of the targeted proteins, either 3W37 or 3AJ7. The justification is due to referencing of corresponding simulations on selected commercial drugs D1, D2, and D3.3.4.1−1, the lowest value calculated. This means the complex is at the lowest free-energy level, thereby most stable. In detail, compound 2 seems to exhibit great affinity with a specific amino acid in 3W37 structure, aka. arginine, by creating seven ionic and four H-acceptor bonds with four different arginine molecule, i.e. Arg 814, Arg 670, Arg 676, and Arg 699, and one ionic bond with Asp 666. They altogether compose −39.2 kcal mol−1 in respect of hydrophilic attraction, of which bonding lengths are in 2.78–3.95 Å. This stability is followed by those of 10-3W37 and 1-3W37 with the DS values −14.7 and −14.1 kcal mol−1, respectively, both composed by seven hydrogen bonds to a variety of amino acid types. However, their hydrophilic energy distribution sees different patterns. While each 10-3W37 ligand–amino acid hydrogen bond contributes rather evenly to the total hydrophilic free energy of −11.9 kcal mol−1, C-Agr 670 H-acceptor bonding holds −8.7 kcal mol−1 of free energy accounting for over a haft of 1-3W37 total figure, −17.2 kcal mol−1. The former contains the binding performances in distance 2.71–3.30 Å, while the corresponding figures for the latter are slightly elevated, i.e. 2.75–3.80 Å. This also justifies the slight outweighing of 10-3W37 stability, previously evaluated by DS values. On the other hand, hydrophobic binding contributes fourteen van der Waals interactions to each 2-3W37, 10-3W37 or 1-3W37 inhibitory structure. Otherwise, 3-3W37, 4-3W37, 5-3W37, 9-3W37, 14-3W37, and 15-3W37 seem less stable given their inferior DS values, i.e. −12.9, −12.3, −11.6, −13.3, −13.0, and −12.3 kcal mol−1. Therefore, the inhibitability of the regarded compounds accords with an order: 2 > 10 > 1 > 9 > 14 > 3 > 4 = 15 > 5. In addition, all RMSD values registering under 2 Å indicates that the associated complexes are tolerable as biological rigid bodies. In particular, although exhibiting a moderate DS value, the average distance between neighbouring atoms in 15-3W37 structure is significantly short given its RMSD value of 0.67 Å. This suggests a ligand-bound conformation if the inhibitory structure is formed. The reason might relate to ligand-pose spatial complementarity. Also, illustrative description for the intermolecular interactions detected in each active site is projected on a 2D diagram for each complex. It is also clear that all iridoid ligands are in high exposure to their targeted protein shown by densely blurred-violet zones, while long and continuous proximity contour bounding large in-pose areas likely indicate unintermittent interactability between the docking agents. Finally, visual observations reinforce the inhibitability since the docked sites are still spacious after the inhibition of 1, 2, 3, 4, 5, 9, 10, 14, or 15.Regarding 1, 2, 3, 4, 5, 9, 10, 14, and 15, the docking results related to their inhibitory complexes with protein 3W37 are summarised in −1, thus deemed as less competent inhibitors towards the protein. The respective calculated RMSD varies between 1.41 and 1.84 Å, acceptable values for a firm conformation. Furthermore, in-depth results of the inhibitory complexes are also summarised in In respect of 7, 8, 11, 12, and 13, DS values associated with their ligand-3W37 inhibitory complexes register over −10.5 kcal mol3.4.2−1, responsible for the stability of 14-3AJ7 by representing the lowest free-energy level. However, there are only five hydrophilic interactions detected as the complex forms, including four ionic and one H-donor bonds. The targeted amino acids are aspartic acid  and glutamic acid (Glu 277), which create hydrogen-bond binding with 14 carbon atoms in distance 2.93–3.82 Å. The total hydrophilic potential calculated is −9.9 kcal mol−1, insignificantly. Hence, 14-3AJ7 stability seems accumulated from van der Waals forces arisen between its two components as they include twenty different 3AJ7 amino acids in involvement. This is followed by 2-3AJ7 of which stability is expressed via −13.5 kcal mol−1 of DS value. The value is likely cultivated from both hydrophilic and hydrophobic contribution. There are nine hydrogen bonds formed between molecule 2 and 3AJ7 amino acids in distance 2.58–4.0 Å, accounting for −28.8 kcal mol−1 of the former free energy; while, the latter is from van der Waals attraction constituted by fifteen different in-pose amino acids. In contrast, 10-3AJ7 binding affinity is likely based more on hydrophobic interactions than on hydrophilic counterparts as the former includes twenty-one amino acids while the latter results in a total energy just −7.5 kcal mol−1. Its DS value registers −12.4 kcal mol−1, the third highest figure. Otherwise, DS values of 1-3AJ7, 3-3AJ7, 4-3AJ7, 5-3AJ7, 9-3AJ7, and 15-3AJ7 are −11.4, −10.6, −10.2, −11.7, −11.5, and −11.0 kcal mol−1, respectively. Therefore, the ligand inhibitability towards protein 3AJ7 follows an order: 14 > 2 > 10 > 5 > 1 = 9 > 15 > 3 > 4. In addition, by possessing RMSD value under 2 Å, all the inhibitory complexes are accepted performing biological rigidity. Moreover, visual illustration of interacting activity between the ligand and in-pose amino acids clarify the predominance of hydrophobic interactions in the establishment of ligand-3AJ7 inhibitory structures. This is justified by a myriad of amino acids presenting around the docked compounds. Resembling to observations on ligand-3W37, spaciousness inside the active sites are highly conducive to motion of 1, 2, 3, 4, 5, 9, 10, 14, or 15 when inhibition processes are on, thus their inhibitability towards protein 3AJ7 affirmed.Regarding 1, 2, 3, 4, 5, 9, 10, 14, and 15, their inhibitory complexes with protein 3AJ7 are virtually presented in −1, clearly expressing inferiority of overall stability. Their RMSD values are all under 1.99 Å. Also, their corresponding 3D morphology and 2D interaction map are given in ESI3W37, failure was also recorded in an attempt to establish a 6-3AJ7 inhibitory structure. No possible intermolecular interaction is given, thus no inhibitability of molecule 6 towards protein 3AJ7 ensuing. The reason for both could be referred to spatial restraint due to 6 structural bulkiness and branching, which possibly prohibit its entry into the host active site. However, more in-depth investigations, either theoretical or experimental, are needed to reach a more accurate conclusion.In respect of 7, 8, 11, 12, and 13, DS values associated with their ligand-3AJ7 inhibitory complexes are also included in 3.4.3−1, respectively. These were followed by the corresponding figures for D3-protein structures, i.e. −12.7 and −13.6 kcal mol−1, respectively. Calculation on D2-protein inhibition results in relatively inferior DS values, i.e. 11.1 kcal mol−1 for D2-3W37 and −10.7 kcal mol−1 for D2-3AJ7. Nevertheless, these complexes are considered possessing ligand-bound conformations given their short average distance between in-active atoms represented by RMSD of 0.68 and 1.03 Å. Other RMSD values register under 2 Å and most binding length are predicted under 3 Å. The inhibitability of the medicines towards carbohydrate-hydrolase proteins (3W37 and 3AJ7) can be briefed as: acarbose (D1) > voglibose (D3) > miglitol (D2). In referencing, there are no significant differentials of the inhibitory indicators between these commercial medicines and the selective iridoid compounds. This implies promising applicability of these substances as carbohydrate-hydrolase inhibitors in particular and in diabetes treatment in general. 1, 2, 10 regarding 3W37 inhibition and 2, 10, 14 regarding 3AJ7 inhibition are even highly competent.The inhibitability of three commercial medicines acarbose (D1), miglitol (D2), and voglibose (D3) towards the carbohydrate-hydrolase proteins (3W37 and 3AJ7) were investigated for referencing purposes. The inhibitions are virtually rendered in 3.5.ca. 500 amu and possess negative dispersion coefficients (log P and log S). Only 10 creates less than both ten hydrogen bond acceptors and five hydrogen bond donors regardless of the targeted protein, either 3W37 or 3AJ7. This preliminarily suggests 10 as the most potential compound for oral administration in attempt to tackle diabetes. However, other promising candidates should be in great consideration. First, 507.1 amu 14 might still be suitable since the commercial product Acarbose weigh 645.6 amu, over Lipinski's first criterion. The highly efficacy-demonstrated medicine also forms seven hydrogen bond donor with protein 3AJ7. This justifies the applicability of 1 and 2. In addition, it is noticeable that their polarisability is over 10 Å3, indicating high polarisation. The property is of significance because it is highly conducive to protein inhibition as the polypeptide molecule is made of polarised amino acids. Therefore, the selective iridoid compounds, especially 10, are likely compatible with pharmaceutical applications in physiological medium. However, in vitro and in vivo experiments are required in order to confirm the computational predictions.Several properties of studied ligands are summarised in In summary, the results obtained altogether are likely to justify the applicability of the novel-synthesised iridoid compounds in cytological in general, and for inhibition of carbohydrate-hydrolase proteins (3W37 and 3AJ7) in particular.3.6.50 is determined registering 285.00 μM in the resembling assay conditions. The obtained results are shown in 50 values register lower than 25 μM. Compound 10 shows its marked inhibition towards the enzyme with an IC50 value of 0.05 μM, higher than that of acarbose by 5700 times. This was followed by 2 and 14, both exhibiting significant α-glucosidase inhibition with IC50 value 7.24 and 8.65 μM, respectively. The corresponding figure for 13 is 20.84 μM and for 1 is 24.89 μM. The predominance of 10 inhibitability seems signifying the role of -8-hydroxy-2,6-dimethyl-2,6-octadienoate moiety as it is the only compound containing this functional group. Its flexibility and polarity acquired from an octadienoate configuration might be advantageous to structural transformation, which in turn conducive to either protein-site entrance, polar–polar interaction, or in-pose topographical fitting. However, in-depth investigations are still required in order to reach more accurate view. Although predicted exhibiting insignificant inhibitability towards α-glucosidase molecule (3W37) by molecular docking simulation, experimental results reflect otherwise. On the other hand, 9 also sees noticeable simulation-experiment inconsistency, in which the compound is expected to perform pronounced α-glucosidase inhibition.The enzyme inhibitory activity of fourteen compounds (excluding 15) were assayed towards α-glucosidase. Acarbose was used as a reference whose IC50 167.76 μM) and 12 (IC50 71.56 μM) as α-glucosidase inhibitors. The corresponding computing also predicts this unfavourableness. The lessening of polarity, caused by total acetyl-hydroxyl substitution, might reason this, thus justifying the important role of catalpol hydroxyl groups to the inhibition. In addition, computationally expected possessing no inhibitability towards 3W37, 6 performs assaying inhibition towards α-glucosidase with a notably high IC50 value of 224.78 μM. This reinforces the implication that two bulky triphenyl methyl groups at C-10 and C-6′ seem to limit the host-molecule entrance into the protein sites.Compounds 3, 4, and 5, without double bond at C-7′′ and C-8′′, are inactive towards α-glucosidase observed from enzyme assays. This is relatively consistent with the computational prediction as these compounds are not expected to be effective 3W37 inhibitors by the simulation. Therefore, double bond at C-7′′ and C-8′′ could be subjected for further investigations on α-glucosidase inhibition. Regarding acetylation, the corresponding derivatives are either inactive including 8 and 11, or unfavoured such as 7  instead of cinnamoyl moiety significantly rises the α-glucosidase inhibitory activity. Nemoroside (10) is the most active compound with an IC4.Dolichandrone spathaceae as α-glucosidase (protein 3W37) and oligo-1,6-glucosidase (protein 3AJ7) inhibitors. Five isolated natural iridoids and ten synthetic derivatives were successfully identified by structural elucidation. These include five new iridoid-derived structures not found having reported before in the literature, i.e.3, 4, 5, 6, and 9. Molecular docking simulation indicates that 1, 2, 3, 4, 5, 9, 10, 14, and 15 could perform elevated inhibitability towards both carbohydrate-hydrolase proteins. Regarding protein 3W37, five strongest predicted inhibitors accords with an order assemble 2  > 10  > 1  > 9  > 14 . In respect to 3AJ7, the corresponding order is 14  > 2  > 10  > 5  > 1 = 9 . Exceptionally, compound 6 is predicted unable to establish an inhibitory structure with any given proteins. Lipinski's rule of five suggests 10 as the most potential candidate for oral administration; while, 1, 2, and 14 are highly promising for pharmaceutical applications in physiological medium. All compounds possess high polarisability, which is suitable for inhibition of polarised protein molecules. Enzyme assays on α-glucosidase revealed that compound 10 is the most effective inhibitor with an IC50 value of 0.05 μM, higher than that of acarbose by 5700 times. It is followed by 1, 2, 13, and 14 in the order 10 (IC50 0.05 μM) > 2 (IC50 7.24 μM) > 14 (IC50 8.65 μM) > 13 (IC50 20.84 μM) > 1 (IC50 24.89 μM), showing good consistency with the docking-based predictions and significant predominant in comparison to acarbose (IC50 285.00 μM). Compound 6 performs unfavoured inhibitability in the in vitro bioassay with IC50 value of 224.78 μM. The results are considered highly advantageous to preparation of therapeutic medicines based on iridoid chemotype in an attempt to develop an alternative for diabetes treatment.This study suggests a promising approach to the use of iridoid chemotype from There are no potential conflicts of interest by the authors.RA-011-D1RA00441G-s001


PLoS Biology, volume 4, issue 3: doi: 10.1371/journal.pbio.0040072In 1. In the supplementary materials, the intended simulation models for the population growth scenarios in the Yoruba were incorrectly specified. The corrected command lines, matching the models that were the intended simulations, are as follows:A to N0, occurring tonset generations in the past.Exponential growth from initial size NA = 11156, N0 = 11156, tonset = 1000: ms 120 1 -s S -r ρ L -G 0 -eG 0.02241 0.0NA = 10659, N0 = 22600, tonset = 1000: ms 120 1 -s S -r ρ L -G 67.94 -eG 0.011062 0.0NA = 7834, N0 = 57900, tonset = 4000: ms 120 1 -s S -r ρ L -G 115.81 -eG 0.017271 0.0NA = 10424, N0 = 938000, tonset = 500: ms 120 1 -s S -r ρ L -G 33765.3 -eG 0.00013326 0.0NA = 9944, N0 = 44600, tonset = 1500: ms 120 1 -s S -r ρ L -G 178.49 -eG 0.008408 0.0NA = 11020, N00 = 23300, tonset = 250: ms 120 1 -s S -r ρ L -G 279.13 -eG 0.002682 0.0NA = 9912, N0 = 199000, tonset = 1000: ms 120 1 -s S -r ρ L -G 2387.65 -eG 0.0012563 0.0NA = 10018, N0 = 1910000, tonset = 750: ms 120 1 -s S -r ρ L -G 53484.84 -eG 9.8167E-5 0.0N2. As a result of the above, Figure 4 as presented in the manuscript is incorrect, and the revision is presented below. The fundamental result and interpretation of the figure as they pertain to the manuscript are unchanged: The observed iHS scores for the Yoruban Hapmap sample are more extreme than under the (correctly) simulated growth models presented above.We regret any confusion this may have caused readers.


In a pilot study six patients received 4 days' treatment with interleukin 2 (IL-2) [cumulative dose (CD) 264 +/- 26 x 10(6) IU m-2] and C1 esterase inhibitor (C1-INH) . Toxicity was compared with that in patients given 4 days' treatment with standard (CD 66 +/- 12 x 10(6) IU m-2) or escalating-dose (CD 99 +/- 8 x 10(6) IU m-2) IL-2. IL-2-induced hypotension was equivalent and complement activation was less after IL-2 + C1-INH (C3a = 10.5 +/- 3.2 nmol l-1) than following standard (14.1 +/- 8.4 nmol l-1) or escalating-dose (18.3 +/- 2.9 nmol l-1) IL-2. This study demonstrates that C1-INH administration during IL-2 treatment is safe and warrants further study to evaluate its ability to ameliorate IL-2-induced toxicity.


Several 1,2-dithiol-3-thione and dithioester compounds were assayed for radioprotective capabilities in EMT6 cells in vitro. The 1,2-dithiol-3-thiones were generally more cytotoxic than the dithioesters and in some instances were more cytotoxic toward hypoxic cells than toward normally oxygenated cells. When the drugs were present at a concentration of 500 microM for 1 h prior to and during radiation delivery, the 5-(2-thienyl)-1,2-dithiol-3-thione produced a radiation protection factor (RPF) of 2.7 at 1 log of cell kill. The 4-methyl analogue of this same compound was, however, much less effective, producing a RPF of only 1.2. The 4-ethoxycarbonyl analogue was moderately active, producing a RPF of 1.7. The 4-methyl-5-(2-pyrazinyl)-1,2-dithiol-3-thione (Oltipraz) was least effective, yielding a RPF of only 1.1. Of the dithioesters tested, methyl 3-pyrrolidino-2-phenylpropene dithiocarboxylate produced a RPF of 2.6, methyl 3-piperidino-2-phenylpropenedithiocarboxylate a RPF of 2.7, and the corresponding 3-morpholino and 3-thiomorpholino derivatives RPF values of 2.7 and 2.9, respectively. The iodide salt of 4-ethoxycarbonyl-5-(2-thienyl)-1,2-dithiol-3-thione produced a RPF of 2.6 Methyl 3-cyclohexylamino-2-phenylpropenedithiocarboxylate was equally effective (RPF = 2.6). Finally, methyl 3-morpholino-3-thienyl-2-methylpropenedithiocarboxylate and methyl 3-morpholino-3-(2-pyrazinyl)-2-methylpropenedithiocarboxylate were less effective, producing RPF values of 2.0 and 1.6, respectively. These results demonstrate that several of these compounds are highly effective radioprotectors. In vitro and in vivo studies testing their efficacy are underway.


We examined the ability of dipyridamole (DPM) to act synergistically with vinblastine (VBL) in HT1080 fibrosarcoma cells and a drug-resistant variant, HT1080/DR4, which lacks mdr1 expression, in order to determine whether DPM requires P-glycoprotein to modulate drug sensitivity. Median effect analysis of clonogenic assay was used to produce the combination index . DPM was mildly synergistic with VBL producing a CI50 of 0.83 +/- 0.13 for HT1080 cells and 0.80 +/- 0.16 for HT1080/DR4 cells. HT1080 and HT1080/DR4 cells accumulated 6.7 +/- 0.7 and 5.6 +/- 0.9 pmol 3H-VBL mg cells-1 at steady state (Css) and 20 microM DPM elevated the Css by 1.8 and 2.9-fold, respectively. In comparison, the CI50 was 1.1 +/- 0.2 in parental KB-3-1 cells and 0.1 +/- 0.1 in mdr1-expressing KB-GRC1 cells. The KB-3-1 and KB-GRC1 cells had a Css of 3.8 +/- 0.8 and 0.7 +/- 0.2 pmol 3H-VBL mg cells-1, respectively, and DPM elevated the Css by 9.2-fold in KB-GRC1 cells. These studies demonstrate that DPM can produce synergy independently of mdr1 expression but that much greater levels of synergy are achievable in mdr1-expressing tumour cells.


The structures of the new compounds were determined by extensive analysis of spectroscopic data.A novel cytotoxic diterpenoid, chabrolin A ( Nephthea and Paralemnalia have proven to be a rich source of bioactive terpenoids  , 0.36 m, 0.49 m, 0.78 dd ; δC 8.6 CH2, 15.6 CH, 23.7 CH] . The presence of the oxygen bearing sp3 methine (δC 74.1 CH) was shown in the 13C NMR spectrum. By interpretation of 1H-1H COSY correlations  H2-19 to C-8, C-9, C-10, C-13, and C-14, (c) H-8 to C-10, (d) CH3-20 to C-12, C-13, C-14, and C-19 connected four partial structures concluding the planar structure of 1, as shown in 1) possesses a novel diterpene tricyclic skeleton. The relative configuration of all chiral centers in 1 was deduced from a NOESY experiment  Cperiment . Assuminmolecule .2) exhibited a pseudomolecular ion peak at m/z 343.1887 [M + Na]+, consistent with the molecular formula C19H28O4, and six degrees of unsaturation. The IR spectrum of 2 revealed the presence of ester carbonyl group (νmax 1743 cm−1). The 13C NMR spectrum of 2 , δC 20.8 (CH3), and 170.1 (CO); δH 2.06 (s), δC 21.2 (CH3), and 170.3 (CO)], two trisubstituted double bonds , and two oxygen-bearing methines . Thus, the bicyclic structure of 2 was revealed. From the 1H-1H COSY spectrum  and H3-13 showed NOESY correlations with H3-15 , H-7β , and H-2 (δ 5.29 brs), H3-15 and H-2 should also be positioned on the β-face. NOESY correlations observed from H-12 (δ 1.71 m) to H-4, H-6α  to H-5 , and H-5 to H-4  reflected the α-orientation of H-12, H-4, and H-5. Therefore, 2 was found to be the C-5 epimer of paralemnolin E +; (+)-HRESIMS m/z 311.23465 .Parathyrsoidin E (2): Colorless amorphous solid; c 0.3, CHCl3); IR (neat) νmax 2964, 2927, 1743, 1455, 1369, 1247, and 871 cm−1; 1H NMR data, see 13C NMR data, see m/z 343 [M + Na]+; (+)-HRESIMS m/z 343.1887 .Parathyrsoidin F (3): Colorless amorphous solid; c 0.3, CHCl3); IR (neat) νmax 3421, 2926, 1736, 1714, 1376, 1245, 1111, and 859 cm−1; 1H NMR data, see 13C NMR data, see m/z 321 [M + Na]+; (+)-HRESIMS m/z 321.1676 .Parathyrsoidin G (4): Colorless amorphous solid; c 0.1, CHCl3); IR (neat) νmax 3421, 2964, 1739, 1714, 1362, 1236, and 869 cm−1; 1H NMR data, see 13C NMR data, see m/z 303 [M + Na]+; (+)-HRESIMS m/z 303.1573 .Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549  tumor cells using a modification of the MTT colorimetric method according to a previously described procedure ,21. The 50 (50% inhibitory concentration), or a compound concentration required to reduce virus-induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [To determine the effects of natural products upon the HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products with three replications. Ganciclovir was used as a positive control. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of a 24-well dish. Antiviral activity was expressed as ICemployed .N. chablroli and P. thyrsoides led to the isolation of a novel diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton) as well as three new sesquiterpenoids. Compounds 1–4 displayed cytotoxicity against P-388, with ED50 values of 3.18, 2.59, 3.31, and 2.49 μg/mL, respectively. However, Compounds 1–4 were not cytotoxic to A549 and HT-29 cell lines and did not show anti-HCMV activity. A plausible biosynthetic pathway of 1 is proposed in Cytotoxicity-guided fractionation of the ethyl acetate extract of the soft corals