Trypanosoma brucei, which causes human African trypanosomiasis. It is one of only a handful of chemically validated targets for T. brucei in vivo. To identify novel inhibitors of TbTryS we screened our in-house diverse compound library that contains 62 000 compounds. This resulted in the identification of six novel hit series of TbTryS inhibitors. Herein we describe the SAR exploration of these hit series, which gave rise to one common series with potency against the enzyme target. Cellular studies on these inhibitors confirmed on-target activity, and the compounds have proven to be very useful tools for further study of the trypanothione pathway in kinetoplastids.Trypanothione synthetase (TryS) is essential for the survival of the protozoan parasite  Trypanosoma brucei gambiense and T. b. rhodesiense, which are transmitted to the human host by the bite of the tsetse fly. If left untreated the disease progresses to the central nervous system and is ultimately fatal. There is a clinical need for more effective drug therapies. Current therapies are toxic and have inappropriate treatment regimens for a rural African setting. There are also problems with treatment failures.3Human African trypanosomiasis (HAT), or sleeping sickness, is endemic in sub-Saharan Africa, claiming the lives of about 30 000 people every year and putting approximately 60 million people at risk of infection.T. brucei to oxidative stress.N1,N8-bis(glutathionyl)spermidine) as their principal thiol, in contrast to most other organisms  that use glutathione .T. brucei trypanothione is synthesised from GSH and spermidine (Spd) by an ATP-dependent C–N ligase, trypanothione synthetase , with N1- and N8-glutathionylspermidine as intermediates.11Differences in metabolic pathways have been discovered between parasite and host, which may be exploited for drug discovery programmes. An example of such a difference is found in thiol metabolism and the response of TbTryS has also been genetically validated as a drug target, with RNAi and gene knockout studies confirming that TbTryS is essential for T. brucei growth in both bloodstream and procyclic forms, and that there is no alternative bypass mechanism available to the parasite.15Selective inhibition of the trypanothione pathway with chemical agents  or classical gene knockout studies have shown a clear trypanocidal effect.TbTryS was assessed for its suitability as a drug target using the traffic light scoring system that we have developed in house.TbTryS is an attractive target for drug development, especially as it is unlikely to have resistance or toxicity issues, as there is no obvious bypass metabolism or equivalent enzyme in humans.TbTryS is large enough to accommodate trypanothione and precursors, this may be an issue if the active site is a large featureless pocket, as is observed in T. brucei trypanothione reductase (TbTryR).Leishmania major suggests this is not the case,TbTryS is a bifunctional enzyme, which catalyzes the biosynthesis and hydrolysis of the GSH–Spd adduct trypanothione. The two catalytic domains are separate in Leishmania. The N-terminal domain is a cysteine-containing amidohydrolase/peptidase amidase site, with the C-terminal ATP grasp domain responsible for the synthetase activity of the enzyme.18Before commencing a drug discovery programme, TbTryS, compound 1.Figure TbTryS, which gave rise to some potent compounds. We recently reported the biological experiments to chemically validate TbTryS using five of these compounds: 9, 20, 71, 84, and 89.20Herein we describe a medicinal chemistry programme to develop inhibitors of TbTryS was developed and validated.TbTryS was then screened against a 62 000 compound diversity set at single-point concentration (30 μm). This gave rise to >720 hits (compounds with TbTryS percentage inhibition >33 % at 30 μm). These hits were clustered and filtered down to 174 compounds that underwent potency testing . This gave rise to the six putative hit series, plus a number of singletons. Appropriate hits were then re-purchased to confirm identity and activity. A round of purchasing (where available) and synthesis of analogues was initiated to validate the series and to investigate the SAR around these potential hit series to assess optimisation towards lead series.As previously described, a high-throughput screening assay for Group 1 series were identified as having a common pharmacophore with two hydrogen bond acceptors (HBA) in 1,5 relationship, with one of the HBAs from a heterocyclic ring. The pharmacophore also includes regions of hydrophobicity, indicating a potential lipophilic pocket.1 a compounds against TbTryS are listed in Table The synthetic route to prepare this series involved the condensation of a thioamide with an appropriate α-bromoketone , based o1 a (Table 4) and fluorine (compound 5), is tolerated without loss of potency. An electron-donating substituent at the 3-position, as in compound 6, gave a 10-fold loss in potency relative to unsubstituted compound 3; however, all potency was regained if the 3-position group was changed to an electron-withdrawing group, such as trifluoromethyl (compound 7). In addition, a slight improvement in potency was observed for the 3-F (9) and 3-Cl (8) derivatives (IC50: 0.4 μm). These analogues showed sub-micromolar potency is achievable within this hit series. Substitution at the 4-position was not tolerated with either an electron-withdrawing or -donating group. Both methyl (10) and trifluoromethyl (11) were found to be inactive. However, 3,4-disubstitution with an electron-withdrawing substituent at the 3-position restored potency (compounds 12 and 13), although ligand efficiency was decreased.Exploration around the scaffold of  a Table  shows th23 was synthesised via an amide coupling of the corresponding acid, which in turn was obtained from saponification of the ethyl ester.In this series, the thiazole subunit is replaced by a tetrazole, and the sulfone by a carbonyl group. The tetrazoles were alkylated with α-bromoketones Table . Amide a1 a were observed in series 1 b , suggesting a favourable specific interaction. It was possible to modify the ketone moiety to an amide without loss of potency (compare amide 23 (IC50=0.6 μm), with ketone 20 (IC50=0.3 μm)). In going from methyl (21) to ethyl (22) to tert-butyl ketone (20), an improvement in potency was observed , indicating lipophilic bulk in this area is required. In contrast, the phenyl ketone 24 shows a marked loss in potency, with an IC50 value of 45 μm.Subtle trends in SAR similar to series ries 1 b . In part1 c was made by condensation of ethyl hydrazinoacetate with the commercially available dione. The ester was then hydrolysed, allowing amide couplings using standard methodology  were tolerated, albeit at weaker potency, but bulky rigid substitution with the phenyl 32 and cyclohexyl 33 moieties was not tolerated, with complete loss in potency observed. Tertiary amide 34 with two bulky benzyl groups also lost all potency, but the less bulky diethyl amide 35 retained some inhibitory activity.Small changes to optimise the amide group in this series generated a “flat” SAR plateau , had an IC50 value of 150 nm in the TbTryS enzyme assay  and was the most potent compound to date. Para substituents led to loss in activity . This is possible evidence for the presence of a tight binding pocket into which the aromatic subunit sits. A variety of heterocycles were tolerated, although some gave a 10-fold loss in activity, such as the 2-furanyl compound 66 and 3-pyridyl compound 67. The 3-fluorophenyl motif  was more potent than 3-chlorophenyl , and was used as the standard template for exploration of substituent SAR around the R1 position  was found to be equipotent to the ketone 60  had IC50 values similar to that of the ketone 71, with compound 88 having a ligand efficiency of 0.44. Potency was retained when fusing the dialkyl amide into an N-piperidine amide (89TbTryS IC50=135 nm), but activity was completely abolished with a larger alkyl aromatic substituent (e.g. 86 IC50>100 μm).Encouragingly, the ketone subunit could be replaced by a simple amide without loss of potency or ligand efficiency . Thus thTbTryS endogenous substrate Spd binding domain interactions and improve ligand potency, was investigated. This basic group could also improve the aqueous solubility of our compounds and lower the lipophilicity (logP). Although the piperazine-containing compounds 90 and 80 lost potency (ninefold relative to 71), and were less efficient binders (ligand efficiencies of 0.29 and 0.28 respectively), they were still sub-micromolar inhibitors of the TbTryS enzyme, with TbTryS IC50 values of 0.86 and 0.83 μm, respectively. If the second basic amine centre was removed and the compounds were truncated to make the C2- and C3-linked dimethyl amine compounds 83 and 84, a significant improvement in potency  over the analogous piperazines was observed. These compounds were also significantly more efficient binders than compounds 80 and 90, with respective ligand efficiencies of 0.38 and 0.39. The C3-linked dimethylamine compound 84 was observed to be the most potent compound to date, with a TbTryS IC50 value of 45 nm. Compound 84 shows improved physicochemical properties over compound 60, especially in decreased lipophilicity, with a clogP value of 2.8 (1.3 units lower than that of 60), and Mr=354 Da and PSA=50 Å2.Addition of an appended basic amine, to potentially pick up the 84 shows similar potency to an analogue not containing an appended amine  it is unlikely that compound 84 has picked up the Spd binding domain. This conclusion is supported by competition binding studies which revealed that the compounds displayed mixed inhibition with respect to Spd, and did not show classical competitive binding kinetics.84 with a methyl group (compound 85) resulted in a fourfold decrease in potency, and the C3-linked imidazole 81 was eightfold less potent than the dimethylamine.As compound 8, 26, and 71 are reasonably metabolically stable, suggesting nothing fundamentally problematic with these scaffolds in terms of cytochrome P450-driven metabolism.Following the discovery of promising novel TryS inhibitors, compounds were progressed into a trypanosomal cell proliferation assay and a human cell counter-screen. Selected compounds were also screened in an in vitro metabolic clearance assay , to asseTbTryS inhibitors discovered. Although these compounds were not toxic to the MRC5 mammalian cell line, there was up to a 100-fold decrease in going from enzyme to trypanosomal cell efficacy, even with the lead compounds 71 and 84 . While these cell potencies are equivalent to the drugs eflornithine (22 μm) and nifurtimox (2 μm) currently in clinical use for late-stage human African trypanosomiasis, they are much less potent than the alternative arsenic-containing drug melarsoprol (8 nm), although arsenic-based compounds do show significant toxicity in the clinic.26Table 50 values and parasite EC50 values was unexpected, further experiments were carried out to confirm whether hit compounds were entering the cell and acting on-target. As described fully elsewhere, exposing T. brucei parasites to the model TbTryS inhibitors 89 and 84 (2×EC50 for 72 h) resulted in trypanothione levels dropping to <10 % of wild-type levels.TbTryS substrate GSH, providing strong evidence that these compounds were acting on-target.As this large potency shift between the enzyme ICTbTryS single knockout (SKO) and TbTryS overexpressing (OE) cell lines. Western blot analysis and densitometry demonstrated that TbTryS protein levels were decreased in the SKO cells and elevated in the OE cell line, relative to wild-type cells, and these cell lines showed the expected changes in potency to 89  and 84 , confirming that TbTryS is the specific target of these compounds.20As previously reported, the on-target effects of these hit compounds were further confirmed by generating 71 and 84, which displayed in vitro enzyme potencies >10-fold improved over the best HTS hits. Attempts so far to co-crystallise our inhibitors with the TbTryS enzyme have failed to produce robust data.In this work we successfully took HTS hits, clustered them into putative hit series, and rationalised their activities based on common pharmacophores. Initial investigation of SAR around the hit series confirmed an overlapping pharmacophore, and the optimisation potential of group 1 hit series in particular. Following the SAR on group 1 series, a hybridisation strategy and scaffold-hopping approach led us to discover the indazole lead series. Optimisation of this series for potency and improved DMPK properties led to compounds TbTryS with IC50 values of <100 nm, they failed to show sub-micromolar potency in a trypanosome proliferation assay. This can be rationalised by the observation that parasites can survive with low levels of trypanothione beyond the timeframe of the standard whole-parasite proliferation assay. The extension of the time-course in screening assay format is prohibited by the need for repeated dilutions of samples to remain in log-phase growth, leading to unacceptable variability. The lead compounds do, however, show a robust biochemical effect in T. brucei, and are proven to act on-target, inhibiting TbTryS in cells.T. brucei procyclics are significantly more susceptible to trypanocides.TbTryS reported to date, and are extremely useful leads to further explore the trypanothione pathway in kinetoplastids.Although these indazoles inhibit 1H NMR spectra were recorded on either Bruker Avance DPX 500 or Bruker Avance 300 spectrometers. Chemical shifts (δ) are expressed in ppm. Signal splitting patterns are described as singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q), multiplet (m) or combination thereof. LC–MS analyses were performed with either an Agilent HPLC 1100 series instrument connected to a Bruker Daltonics MicrOTOF, or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC–MS; both instruments were connected to an Agilent diode-array detector. LC–MS chromatographic separations were conducted with a Phenomenex Gemini C18 column, 50×3.0 mm, 5 μm particle size; mobile phase, H2O/CH3CN +0.1 % HCOOH 80:20→5:95 over 3.5 min, and then held for 1.5 min; flow rate: 0.5 mL min−1. High-resolution electrospray MS measurements were performed on a Bruker Daltonics MicrOTOF mass spectrometer. Thin-layer chromatography (TLC) was carried out on Merck silica gel 60 F254 plates using UV light and/or KMnO4 for visualisation. TLC data are given as the Rf value with the corresponding eluent system specified in brackets. Column chromatography was performed using RediSep® 4 or 12 g silica pre-packed columns. LCMS chromatographic separations were conducted with a Waters Xbridge C18 column, 50 mm × 2.1 mm, 3.5 μm particle size; Method A: mobile phase, H2O/CH3CN + 0.1 % NH3; linear gradient 80:20→5:95 over 3.5 min, and then held for 1.5 min; flow rate 0.5 mL min−1. All reactions were carried out under dry and inert conditions, unless otherwise stated.tert-Butylsulfonylmethyl)-4-(3-fluorophenyl)thiazole (9)2-(: Synthesis previously described.2O (30 mL) and then dried (MgSO4). The solvent was then removed in vacuo to give intermediate 2-bromo-1-(3-fluorophenyl)ethanone  as a pale-yellow oil; [M+H]+=217/219. To a stirred solution of 2-bromo-1-(3-fluorophenyl)ethanone  in EtOH (20 mL) was added 2-(tert-butylsulfonyl)ethanethioamide , and the reaction was heated at reflux and stirred for 2 h. The solvent was then removed in vacuo to give a crude residue which was purified by column chromatography (CH2Cl2→10 % MeOH/CH2Cl2 eluent) to give the title compound 9  as a white solid: 1H NMR : δ=8.40 , 8.31 , 7.70 , 5.15 , 1.4 ; [M+H]+=314; HRMS C14H16FNO2S2 calcd: 314.0679, obsd: 314.0690.3–8 were prepared according to the same procedures as described above for the preparation of compound 9, using the corresponding acetophenone.Compounds tert-Butylsulfonyl)methyl)-4-(phenyl)thiazole (3)2-: δ=8.23 , 7.98 , 7.46 , 7.37 , 5.07 , 1.39 ppm ; [M+H]+=296; HRMS C14H17NO2S2 calcd: 296.0767, obsd: 296.0773.tert-Butylsulfonyl)methyl)-4-(2-methylphenyl)thiazole (4)2-: δ=7.88 , 7.59 , 7.31 , 5.06 , 2.42 , 1.38 ppm ; [M+H]+=310; HRMS C15H19NO2S2 calcd: 310.0930, obsd: 310.0943.tert-Butylsulfonyl)methyl)-4-(2-fluorophenyl)thiazole (5)2-: δ=8.10 , 7.44 , 7.35 , 5.10 , 1.39 ppm ; [M+H]+=314; HRMS C14H16FNO2S2 calcd: 314.0679, obsd: 314.0690.tert-Butylsulfonyl)methyl)-4-(3-methoxyphenyl)thiazole (6)2-: δ=8.26 , 7.55 , 7.52 , 7.38 , 6.95 , 5.08 , 3.82 , 1.39 ppm ; [M+H]+=326; HRMS C15H19NO3S2 calcd: 326.0879, obsd: 326.0888.tert-Butylsulfonylmethyl)-4-(3-trifluoromethylphenyl)thiazole (7)2-: δ=8.49 , 8.30 , 7.74 , 5.12 , 1.4 ppm ; [M+H]+=364; HRMS C15H16F3NO2S2 calcd: 364.0647, obsd: 364.0639.tert-Butylsulfonyl)methyl)-4-(3-chlorophenyl)thiazole (8)2-: δ=8.38 , 8.03 , 7.95 , 7.51 , 7.44 , 5.09 , 1.39 ; 13C NMR : δ=23.8, 52.3, 61.6, 116.9, 124.4, 126.6, 128.4, 130.1, 134.9, 135.6, 154.0, 156.9 ppm; [M+H]+=330/332; HRMS C14H16ClNO2S2 calcd: 330.0384, obsd: 330.0384.9–13 were obtained from Maybridge.Compounds H-tetrazol-2-yl)-3,3-dimethylbutan-2-one (20)1--2: Synthesis previously described.3CN (5 mL) was heated at 75 °C for 3 days. The mixture was cooled, diluted with EtOAc (15 mL), and filtered through a plug of silica. The filtrate was concentrated and purified by chromatography on silica (eluent: EtOAc/hexane 0→50 %) to give the title compound 20  as a white solid: 1H NMR : δ=8.08 , 7.48 , 5.71 , 1.36 ppm ; 13C NMR : δ=26.2, 43.7, 56.6, 125.3, 130.0, 130.3, 135.7, 163.4, 204.2 ppm; [M+H]+=313; HRMS C13H14Cl2N4O calcd: 313.0586, obsd: 313.0601.an-2-one : SynthesH-tetrazol-2-yl)-N2--2,N-dimethylacetamide (23): A mixture of 5-tetrazole , NaI  and K2CO3  in DMF (5 mL) was heated at 90 °C until effervescence was nearly complete. 2-Chloro-N,N-dimethylacetamide  was added, and the mixture was heated at 90 °C overnight. The mixture was diluted with H2O (15 mL) and allowed to stand for several hours. The precipitate was collected by filtration and washed with H2O and Et2O to give the title compound 23  as a white solid: 1H NMR : δ=8.09 , 7.47 , 5.58 , 3.17 , 3.07 ppm ; [M+H]+=300; HRMS C11H11Cl2N5O calcd: 300.0413, obsd: 300.0411.23, using the corresponding aryl tetrazole and α-haloketone.Compounds below were prepared according to the same procedures as described above for the preparation of compound H-tetrazol-2-yl)butan-2-one (14)3,3-Dimethyl-1-: δ=8.17 , 7.51 , 5.70 , 1.35 ppm ; [M+H]+=245; HRMS C13H16N4O calcd: 245.1397, obsd: 245.1387.H-tetrazol-2-yl)-3,3-dimethylbutan-2-one (17)1-(5-(3-Fluorophenyl)-2: 1H NMR : δ=7.98 , 7.88 , 7.48 , 7.19 , 5.71 , 1.32 ppm ; [M+H]+=263; HRMS C13H15FN4O calcd: 263.1303, obsd: 263.1291.H-tetrazol-2-yl)-3,3-dimethylbutan-2-one (19)1--2: 1H NMR : δ=7.72 , 6.94 , 5.71 , 1.36 ppm ; [M+H]+=281; HRMS C13H14F2N4O calcd: 281.1208, obsd: 281.1210.H-tetrazol-2-yl)propan-2-one -2): 1H NMR : δ=8.08 , 7.48 , 5.51 , 2.30 ppm ; [M+H]+=271/273; HRMS C10H8Cl2N4O calcd: 271.0148, obsd: 271.0141.H-tetrazol-2-yl)butan-2-one (22)1--2: 1H NMR : δ=8.09 , 7.50 , 5.52 , 2.57 , 1.19 ppm ; [M+H]+=285/287; HRMS C11H10Cl2N4O calcd: 285.0304, obsd: 285.0292.H-tetrazol-2-yl)-1-phenylethanone (24)2--2: 1H NMR : δ=8.02 , 7.72 , 7.58 , 6.93 , 6.16 ppm ; [M+H]+=301; HRMS C11H6F2N4O calcd: 301.0895, obsd: 301.0901.15, 16 and 18 were obtained from Maybridge.Compounds NH-indazol-1-yl)acetamide (26)-(4-Chlorobenzyl)-2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-1: To a stirred suspension of 2-(trifluoroacetyl)cyclohexanone  and ethylhydrazinoacetate hydrochloride  in EtOH (30 mL) was added Et3N  and the reaction heated at reflux for 24 h. The reaction mixture was cooled to room temperature and the solvent removed in vacuo. The resultant crude residue was taken up in CH2Cl2, washed , dried (MgSO4) and concentrated in vacuo. The resultant crude residue was purified by column chromatography, eluting with 0–50 % EtOAc/hexane, to give ethyl 2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)acetate  as a white solid; [M+H]+=277.H-indazol-1-yl)acetate  in 1:1 THF/H2O (30 mL) was added 2 m (aq) NaOH , and the reaction stirred for 3 h. The solvent was then removed in vacuo to give a crude residue which was suspended in H2O, acidified (1 m aq. HCl), stirred for 5 min, filtered and the precipitate dried in vacuo to give 2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)acetic acid  as a white solid; [M+H]+=249.To a stirred solution of ethyl 2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)acetic acid , 4-chlorobenzylamine  and 1-hydroxybenzotriazole hydrate  in N,N-dimethylformamide  at 50 °C was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride  and N,N′-diisopropylethylamine  and the reaction stirred for 16 h. The reaction mixture was cooled to room temperature, taken up in EtOAc, washed , dried (MgSO4) and concentrated in vacuo. The resultant crude residue was purified by reversed-phase HPLC (method A), to give title compound 26  as a white solid: 1H NMR : δ=8.78 , 7.41 , 7.30 , 4.84 , 4.30 , 2.56 , 2.51 , 1.74 , 1.67 ppm ; 13C NMR : δ=19.9, 21.2, 21.9, 22.2, 42.8, 52.3, 116.1, 120.6, 122.7, 128.8, 128.9, 133.5, 136.0, 140.1, 166.3 ppm; [M+H]+=372; HRMS C17H17ClF3N3O calcd: 372.1085, obsd: 372.1103.To a stirred solution of 2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-1N,NH-indazol-1-yl)acetamide (35)-Diethyl-2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-1: By proceeding in a similar manner to compound 26, using 2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)acetic acid and diethylamine the title compound 35  was obtained as a white solid. 1H NMR : δ=5.10 , 3.37 , 3.27 , 1.71 , 1.17 , 1.03 ppm ; [M+H]+=304; HRMS C14H20F3N3O calcd: 304.1631, obsd: 304.1628.25, 27, 28, 31, 32, 33 and 34 were obtained from Asinex. Compounds 29 and 30 were obtained from ChemDiv.Compounds N-methyl)propane-2-sulfonamide (42)2-Methyl-: tert-Butylsulfinyl chloride  was added to a solution of methanamine  and Et3N  in CH2Cl2 (10 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h and then quenched with aqueous NaHCO3. The phases were separated and the aqueous phase was extracted with CH2Cl2. The combined organic phases were dried (Na2SO4) and concentrated. Chromatography on silica (50–100 % EtOAc/PE 40:60) gave 2-methyl-N-methyl)propane-2-sulfinamide as an orange solid : 1H NMR : δ=8.01 , 7.47 , 7.33 , 4.50 , 4.36 , 3.60 , 2.42 , 1.27 ppm ; [M+H]+=293.N-methyl)propane-2-sulfinamide  in CH2Cl2 (7.5 mL) was treated with mCPBA  and stirred at room temperature for 45 min. The reaction was quenched with 2 m aqueous NaHSO3 (10 mL) and saturated aqueous NaHCO3 (10 mL). The organic phase was applied to a plug of silica and eluted with CH2Cl2/EtOAc (0→100 %) to give title compound 42 as a white solid : 1H NMR : δ=8.01 , 7.49 , 7.34 , 4.51 , 4.43 , 2.44 , 1.47 ppm ; [M+H]+=309; HRMS C14H20N4O2S calcd: 309.1307, obsd: 309.1312.A solution of 2-methyl-36–41 were obtained from Maybridge. Compounds 43, 44, 45, 53, and 54 were obtained from ChemDiv. Compound 46 was obtained from ChemBridge. Compounds 47–52 were obtained from Asinex. Compounds 55–57 were obtained from Life Chemicals. Compound 58 was obtained from Enamine.Compounds H-indazol-1-yl)-3,3-dimethylbutan-2-one (71)1-(3-(3-Fluorophenyl)-1: The synthesis was previously described elsewhere.20H-indazole was prepared as reported by Edwards et al;M+H]+=245.3-Iodo-1H-indazole , 3-fluorophenylboronic acid , 2 m (aq) Na2CO3  and tetrakis-(triphenylphosphine)palladium(0) in 7:3:2 DME/H2O/EtOH (3 mL) was degassed, flooded with argon and irradiated (Biotage Initiator) at 150 °C for 20 min. 3-Fluorophenylboronic acid  and 2 m (aq) Na2CO3  were added, the system degassed, flooded with argon and irradiated (Biotage Initiator) at 150 °C for 20 min. The reaction mixture was taken up in EtOAc, washed  NaOH, brine), dried (MgSO4) and concentrated in vacuo. The resultant crude residue was purified by column chromatography, eluting with 0–50 % EtOAc/hexane, to give 3-(3-fluorophenyl)-1H-indazole  as a white solid.A capped process vial containing 3-iodo-1H-indazole  and the reaction stirred for 10 min. To the reaction mixture 1-bromopinacolone  was added, the reaction heated at 75 °C and stirring continued for 66 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed , dried (MgSO4) and concentrated in vacuo. The crude residue was purified by column chromatography, eluting with 0–50 % EtOAc/hexane to give title compound 71  was obtained as a white solid: 1H NMR : δ=8.12 , 7.84 , 7.71 , 7.59 , 7.55 , 7.45 , 7.27 , 5.75 , 1.38 ppm ; [M+H]+=311; HRMS C19H19FN2O calcd: 311.1554, obsd: 311.1555.To a stirred suspension of 60 % NaH  in DMF (4 mL) was added 3-(3-fluorophenyl)-171, using 3-(3-fluorophenyl)-1H-indazole (or 3-phenyl-1H-indazole) and the corresponding alkylating agent, the following compounds were obtained.By proceeding in a similar manner to compound H-indazol-1-yl)-3,3-dimethylbutan-2-one (60)1-: δ=8.09 , 7.97 , 7.54 , 7.43 , 7.25 , 5.73 , 1.28 ppm ; 13C NMR : δ=26.4, 43.5, 53.4, 109.0, 121.2, 121.6, 122.1, 126.7, 127.6, 128.0, 128.8, 133.5, 141.8, 144.9, 207.9 ppm; [M+H]+=293; HRMS C19H20N2O calcd: 293.1648, obsd: 293.1658.H-indazole (75)3-(3-Fluorophenyl)-1-(pyridin-2-ylmethyl)-1: 1H NMR : δ=8.63 , 8.06 , 7.83 , 7.75 , 7.58 , 7.47 , 7.42 , 7.27 , 7.21 , 7.12 , 6.95 , 5.83 ppm ; [M+H]+=304; HRMS C19H14FN3 calcd: 304.1245, obsd: 304.1258.H-indazole (76)3-(3-Fluorophenyl)-1-(pyridin-3-ylmethyl)-1: 1H NMR : δ=8.63 , 8.49 , 8.13 , 7.87 , 7.72 , 7.69 , 7.58 , 7.50 , 7.35 , 7.28 , 5.81 ppm ; [M+H]+=304; HRMS C19H14FN3 calcd: 304.1245, obsd: 304.1235.H-indazol-1-yl)methyl)-2,5-dimethyloxazole (77)4-((3-(3-Fluorophenyl)-1: 1H NMR : δ=8.10 , 7.83 , 7.77 , 7.71 , 7.58 , 7.48 , 7.27 , 5.52 , 3.35 , 2.36 ppm ; [M+H]+=322; HRMS C19H16FN3O calcd: 322.1350, obsd: 322.1345.H-indazol-1-yl)methyl)-5-phenylisoxazole (78)3-((3-(3-Fluorophenyl)-1: 1H NMR : δ=8.15 , 7.86 , 7.77 , 7.59 , 7.51 , 7.30 , 6.95 ppm ; [M+H]+=370; HRMS C23H16FN3O calcd: 370.1350, obsd: 370.1345.H-indazol-1-yl)methyl)-5-methylisoxazole (79)3-((3-(3-Fluorophenyl)-1: 1H NMR : δ=8.13 , 7.86 , 7.80 , 7.74 , 7.59 , 7.51 , 7.28 , 6.08 , 5.80 , 2.32 ppm ; [M+H]+=308; HRMS C18H14FN3O calcd: 308.1194, obsd: 308.1179.71, using the corresponding boronic acid and 1-(3-iodo-1H-indazol-1-yl)-3,3-dimethylbutan-2-one, the following compounds were obtained:By proceeding in a similar manner to compound H-indazol-1-yl)-3,3-dimethylbutan-2-one (61)1-(3-(4-Chlorophenyl)-1: 1H NMR : δ=8.09 , 8.00 , 7.59 , 7.55 , 7.45 , 7.27 , 5.74 , 1.28 ppm ; [M+H]+=327/329; HRMS C19H19ClN2O calcd: 327.1259, obsd: 327.1265.H-Indol-4-yl)-1H-indazol-1-yl)-3,3-dimethylbutan-2-one (62)1-: δ=11.21 , 8.00 , 7.55 , 7.44 , 7.24 , 6.88 , 5.75 , 1.30 ppm ; [M+H]+=332; HRMS C21H21N3O calcd: 332.1757, obsd: 332.1754.H-indol-4-yl)-1H-indazol-1-yl)butan-2-one (63)3,3-Dimethyl-1-: δ=8.01 , 7.57 , 7.43 , 7.39 , 7.33 , 7.23 , 6.87 , 5.75 , 3.86 , 1.30 ppm ; [M+H]+=346; HRMS C22H23N3O calcd: 346.1914, obsd: 346.1913.H-indazol-1-yl)butan-2-one (64)3,3-Dimethyl-1--1: 1H NMR : δ=8.56 , 8.30 , 8.14 , 8.06 , 7.97 , 7.57 , 7.47 , 7.31 , 5.78 , 1.29 ppm ; [M+H]+=343; HRMS C23H22N2O calcd: 343.1805, obsd: 343.1806.H-indazol-1-yl)butan-2-one (65)3,3-Dimethyl-1-(3-(pyridin-4-yl)-1: 1H NMR : δ=8.69 , 8.22 , 7.97 , 7.59 , 7.48 , 7.32 , 5.82 , 1.28 ppm ; [M+H]+=294; HRMS C18H19N3O calcd: 294.1601, obsd: 294.1614.H-indazol-1-yl)-3,3-dimethylbutan-2-one (66)1-(3-(Furan-2-yl)-1: 1H NMR : δ=8.10 , 7.87 , 7.52 , 7.56 , 7.44 , 7.26 , 5.72 , 1.27 ppm ; [M+H]+=283; HRMS C17H18N2O2 calcd: 283.1441, obsd: 283.1452.H-indazol-1-yl)-3,3-dimethylbutan-2-one (67)1-(3-(3-Pyridyl)-1: 1H NMR : δ=9.17 , 8.63 , 8.35 , 8.12 , 7.56 , 7.47 , 7.29 , 5.77 , 1.28 ppm ; [M+H]+=294; HRMS C18H19N3O calcd: 294.1601, obsd: 294.1616.H-indazol-1-yl)butan-2-one (68)3,3-Dimethyl-1-(3-(thiophen-3-yl)-1: 1H NMR : δ=8.13 , 7.69 , 7.49 , 7.42 , 7.25 , 5.70 , 1.27 ppm ; [M+H]+=299; HRMS C17H18N2OS calcd: 299.1213, obsd: 299.1219.H-pyrazol-4-yl)-1H-indazol-1-yl)-3,3-dimethylbutan-2-one (69)1-: δ=8.39 , 8.04 , 7.99 , 7.45 , 7.39 , 7.19 , 5.66 , 4.01 , 2.20 , 1.26 , 0.88 ppm ; [M+H]+=339; HRMS C20H26N4O calcd: 339.2179, obsd: 339.2183.H-indazol-1-yl)-3,3-dimethylbutan-2-one (70)1-(3-(3-Chlorophenyl)-1: 1H NMR : δ=8.11 , 7.97 , 7.95 , 7.56 , 7.47 , 7.28 , 5.77 , 1.30 ppm ; [M+H]+=327/329; HRMS C19H19ClN2O calcd: 327.1259, obsd: 327.1262.59 was obtained from ChemDiv.Compound tert-Butylsulfonylmethyl)-1-(3-fluorophenyl)-1H-indazole (73)3- in MeOH (125 mL) was added concd H2SO4 (0.5 mL) and the reaction heated at reflux for 16 h. The reaction mixture was cooled to room temperature and the solvent removed in vacuo. The resultant crude residue was taken up in CH2Cl2, washed (saturated NaHCO3 solution), dried (MgSO4) and concentrated in vacuo to give methyl 1H-indazole-3-carboxylate  as a white solid; [M+H]+=177.H-indazole-3-carboxylate  in CH2Cl2 (125 mL), was added 3-fluorophenylboronic acid , pyridine , copper(II) acetate  and molecular sieves  and the reaction stirred (open to air) for 46 h. The reaction mixture was then filtered through a pad of Celite and concentrated in vacuo to give a crude residue which was purified by column chromatography, eluting with 0–50 % Et2O/hexane, to give methyl 1-(3-fluorophenyl)-1H-indazole-3-carboxylate  as a white solid; [M+H]+=271.To a stirred solution of methyl 1H-indazole-3-carboxylate  in THF (10 mL) at 0 °C, under an inert atmosphere, was added LiAlH4 , the reaction warmed to room temperature and stirred for 16 h. The reaction mixture was quenched (Na2SO4⋅10 H2O), filtered through a pad of Celite and concentrated in vacuo. The resultant crude residue was taken up in 3:1 THF/CH2Cl2 (12 mL), PPh3  and N-chlorosuccinimide  added and the reaction stirred for 24 h. Extra PPh3  and NCS  were then added and stirring continued for 24 h. The reaction mixture was then concentrated in vacuo to give a crude residue which was taken up in EtOAc, washed , dried (MgSO4) and concentrated in vacuo. The resultant crude residue was purified by column chromatography, eluting with 0–50 % Et2O/hexane, to give 3-(chloromethyl)-1-(3-fluorophenyl)-1H-indazole  as a straw-coloured oil; [M+H]+=261/263.To a stirred solution of methyl 1-(3-fluorophenyl)-1H-indazole  in CH3CN (3 mL) was added Cs2CO3  and tert-butylthiol  and the reaction stirred for 16 h. Extra Cs2CO3  and tert-butylthiol  were then added and stirring continued for 6 h. The reaction mixture was taken up in CH2Cl2, washed , dried (MgSO4) and concentrated in vacuo. The resultant crude residue was purified by column chromatography, eluting with 0–50 % Et2O/hexane, to give 3-(tert-butylthiomethyl)-1-(3-fluorophenyl)-1H-indazole  as a colourless oil; [M+H]+=315.To a stirred solution of 3-(chloromethyl)-1-(3-fluorophenyl)-1tert-butylthiomethyl)-1-(3-fluorophenyl)-1H-indazole  in CH2Cl2 (5 mL) was added 70 % mCPBA  and the reaction stirred for 2 h. NaHSO3 solution (2 m aq) was added, the layers separated, the organic layer washed (saturated NaHCO3 solution) and filtered through a pad of silica, eluting with CH2Cl2 and EtOAc. The filtrate was concentrated in vacuo to give a crude residue which was purified by reversed-phase HPLC (method A), to give title compound 73  as a white solid: 1H NMR : δ=7.97 , 7.95 , 7.67 , 7.57 , 7.36 , 7.30 , 5.00 , 1.44 ppm ; [M+H]+=347; HRMS C18H19FN2O2S calcd: 347.1224, obsd: 347.1236.To a stirred solution of 3-(tert-Butylsulfonylmethyl)-1-phenyl-1H-indazole (72)3-(: By proceeding in a similar manner to 3-(tert-butylsulfonylmethyl)-1-(3-fluorophenyl)-1H-indazole (see compound 73), except using phenylboronic acid, title compound 72  was obtained as a white solid. 1H NMR : δ=7.96 , 7.87 , 7.79 , 7.63 , 7.54 , 7.45 , 7.33 , 5.00 , 1.44 ppm ; 13C NMR : δ=23.9, 48.0, 61.1, 110.5, 121.9, 122.3, 123.0, 124.8, 127.1, 127.7, 129.5, 135.8, 139.7, 140.1 ppm; [M+H]+=329; HRMS C18H20N2O2S calcd: 329.1318, obsd: 329.1302.tert-Butylsulfonylmethyl)-3-(3-fluorophenyl)-1H-indazole (74)1-(: 3-(3-Fluorophenyl)-1H-indazole (see compound 71)  was alkylated with tert-butyl(chloromethyl)sulfane , as in accordance with reported procedurestert-butylthiomethyl)-3-(3-fluorophenyl)-1H-indazole . 1H NMR  δ=8.03 , 7.79 , 7.71 , 7.65 , 7.51-7.47 , 7.29 , 7.12 , 5.68 , 1.34 ppm ; [M+H]+=315.2Cl2 (2 mL) was treated with mCPBA  and stirred at room temperature for 90 min. The reaction was quenched with 2 m aqueous NaHSO3 (10 mL) and saturated aqueous NaHCO3 (10 mL). The organic phase was applied to a plug of silica and eluted with CH2Cl2/EtOAc (0→100 %) to give the title compound 74 as a white solid : 1H NMR : δ=8.01 , 7.78 , 7.76 , 7.67 , 7.55  7.50 , 7.34 , 7.15 , 5.76 , 1.43 ppm ; 13C NMR : δ=23.4, 61.2, 64.6, 110.0, 114.4, 115.5, 121.1, 122.3, 123.2, 127.9, 130.5, 134.8, 142.2, 145.1, 161.2, 164.1 ppm; [M+H]+=347; HRMS C18H19FN2O2S calcd: 347.1224, obsd: 347.1214.A solution of this sulfide in CHH-indazol-1-yl)-N-(3-(4-methylpiperazin-1-yl)propyl)acetamide (80)2-(3-(3-Fluorophenyl)-1: To a stirred suspension of 60 % NaH  in DMF (20 mL) under an inert atmosphere, was added 3-(3-fluorophenyl)-1H-indazole  in DMF (10 mL) and the reaction was stirred for 15 min. To the reaction mixture was added ethyl bromoacetate , the reaction was warmed to 75 °C and stirring continued for 66 h. The reaction mixture was cooled to room temperature, taken up in EtOAc, washed , dried (MgSO4) and concentrated in vacuo. The crude residue was taken up in 1:1 THF/H2O (30 mL) and 2 m (aq) NaOH , the reaction was heated at 50 °C and stirred for 3 h. The reaction was cooled to room temperature, diluted with H2O, extracted (EtOAc), the aqueous layer acidified (1 m (aq) HCl, pH 2), extracted (EtOAc). The organic layer was dried (MgSO4) and concentrated in vacuo to give 2-(3-(3-fluorophenyl)-1H-indazol-1-yl)acetic acid  as a white foam; [M+H]+=471.26, except using 2-(3-(3-fluorophenyl)-1H-indazol-1-yl)acetic acid and 1-(2-aminopropyl)-4-methylpiperazine, the title compound 80  was obtained as a white solid: 1H NMR : δ=8.25 , 8.11 , 7.85 , 7.72 , 7.68 , 7.58 , 7.47 , 7.27 , 5.16 , 3.12 , 2.51 , 2.27 , 2.12 , 1.58 ppm ; [M+H]+=410; HRMS C23H28FN5O calcd: 410.2351, obsd: 410.2363.By proceeding in a similar manner to 26, except using 2-(3-(3-fluorophenyl)-1H-indazol-1-yl)acetic acid and the appropriate amine, the following compounds were prepared:By proceeding in a similar manner to NH-Imidazol-1-yl)propyl)-2-(3-(3-fluorophenyl)-1H-indazol-1-yl)acetamide (81)-: δ=8.41 , 8.25 , 8.12 , 7.84 , 7.72 , 7.58 , 7.48 , 7.44 , 7.27 , 7.23 , 5.20 , 4.10 , 3.09 , 1.93 ppm ; [M+H]+=378; HRMS C21H20FN5O calcd: 378.1725, obsd: 378.1739.tertH-indazol-1-yl)acetamido)ethyl)piperazine-1-carboxylate (82)-Butyl-4-(2-(2-(3-(3-fluorophenyl)-1: 1H NMR  δ mixture of rotamers δ=8.12 , 7.86 , 7.72 , 7.59 , 7.48 , 7.28 , 5.22 , 3.20 , 2.51 , 2.37 , 1.40, 1.39 ppm ; [M+H]+=482; HRMS C26H32FN5O3 calcd: 482.2562, obsd: 482.2570.NH-indazol-1-yl)acetamide (83)-(2-(Dimethylamino)ethyl)-2-(3-(3-fluorophenyl)-1: NMR 1H NMR : δ=8.22 , 8.12 , 7.85 , 7.72 , 7.69 , 7.59 , 7.47 , 7.26 , 5.19 , 3.20 , 2.31 , 2.15 ppm ; [M+H]+=341; HRMS C19H21FN4O calcd: 341.1772, obsd: 341.1767.NH-indazol-1-yl)acetamide (84)-(3-(Dimethylamino)propyl)-2-(3-(3-fluorophenyl)-1: 1H NMR : δ=8.26 , 8.13 , 7.85 , 7.71 , 7.68 , 7.59 , 7.48 , 7.26 , 5.16 , 3.13 , 2.20 , 2.01 , 1.54 ppm ; 13C NMR : δ=24.4, 40.7, 44.5, 52.6, 59.4, 109.5, 114.2, 115.1, 121.2, 122.1, 123.0, 127.3, 130.5, 135.3, 141.7, 144.0, 162.2, 164.2, 167.3 ppm; [M+H]+=355; HRMS C20H23FN4O calcd: 355.1856, obsd: 355.1862.NH-indazol-1-yl)acetamide (85)-Methyl-(3-(dimethylamino)propyl)-2-(3-(3-fluorophenyl)-1: 1H NMR  mixture of rotamers; δ=8.12 , 7.85 , 7.71 , 7.60, 7.44 , 7.25 , 5.60 , 5.50 , 3.48 , 3.30 , 3.14 , 2.84 , 2.27 , 2.20 , 2.16 , 2.09 , 1.77 , 1.60 ppm ; [M+H]+=369; HRMS C21H25FN4O calcd: 369.2085, obsd: 369.2097.NH-indazol-1-yl)acetamide (86)-(4-Chlorobenzyl)-2-(3-(3-fluorophenyl)-1: 1H NMR : δ=8.78 , 8.12 , 7.85 , 7.71 , 7.59 , 7.48 , 7.38 , 7.32 , 7.28 , 5.26 , 4.32 ppm ; [M+H]+=394; HRMS C22H17ClFN3O calcd: 394.1117, obsd: 394.1111.H-indazol-1-yl)-N-(2-(4-methylpiperazin-1-yl)ethyl)acetamide (90)2-(3-(3-Fluorophenyl)-1: 1H NMR : δ=8.12 , 8.02 , 7.86 , 7.72 , 7.71 , 7.59 , 7.47 , 7.27 , 5.19 , 3.20 , 2.51 , 2.34 , 2.10 ppm ; [M+H]+=396; HRMS C22H26FN5O calcd: 396.2194, obsd: 396.2212.N,NH-indazol-1-yl)acetamide (87)-Diethyl-2-(3-(3-fluorophenyl)-1: 3-(3-Fluorophenyl)-1H-indazole , prepared as in 71, was alkylated with 2-chloro-N,N-diethylacetamide  to give the title compound 87  as a white solid: 1H NMR : δ=8.02 , 7.78 , 7.70 , 7.53 , 7.50-7.45 , 7.27 , 7.11 , 5.30 , 3.54 , 3.43 , 1.17 ppm ; [M+H]+=326; HRMS C19H20FN3O calcd: 326.1663, obsd: 326.1663.H-indazol-1-yl)-N2-(3-(3-Fluorophenyl)-1,N-dimethylacetamide (88): 3-(3-Fluorophenyl)-1H-indazole , prepared as in 71, was alkylated with 2-chloro-N,N-dimethylacetamide  to give the title compound 88  as a white solid. 1H NMR : δ=1 H, d, J=8.2 Hz), 7.78 , 7.70 , 7.52-7.45 , 7.27 , 7.10 , 5.32 , 3.18 , 3.01 ppm ; [M+H]+=298; HRMS C17H16FN3O calcd: 298.1350, obsd: 298.1348.H-indazol-1-yl)-1-(piperidin-1-yl)ethanone (89)2-(3-(3-Fluorophenyl)-1: Synthesis previously described.H-indazole , prepared as in 71, was alkylated with 2-chloro-1-piperidin-1-ylethanone  to give the title compound  as a white solid: 1H NMR : δ=8.03 , 7.77 , 7.69 , 7.55 , 7.50-7.45 , 7.27 , 7.11 , 5.32 , 3.60 , 1.64 , 1.54 , 1.48 ppm ; [M+H]+=338; HRMS C20H20FN3O calcd: 338.1663, obsd: 338.1660.Trypanothione synthetase assay: The TryS enzyme assays were conducted as reported.20Cell-based assays: Proliferation assays using bloodstream form T. brucei and human MRC5 fibroblasts were conducted as reported.17Glutathione (GSH); human African trypanosomiasis (HAT); spermidine (Spd); single knockout (SKO); Trypanothione synthetase (TryS).


The first economical, one-step synthesis of protoapigenone from apigenin is described on up to gram scale. 13 new 1′-O-alkylflavone analogs were also synthesized, either from apigenin or β-naphthoflavone. The in vitro cytotoxic activity of each compound was tested on six human cancer cell lines . In the case of 1′-O-alkyl-protoapigenone derivatives, structure-activity relationships were found depending on the side-chain, and protoapigenone 1′-O-butyl ether was found to exert significantly stronger activity against three of the cell lines  than its non-substituted analog, protoapigenone itself. In contrast to this, all β-naphthoflavone derivatives bearing the same pharmacophore on their B-ring showed decreased cytotoxic activities when substituted with an O-alkyl side-chain at position 1′, comparing to that of the non-substituted compound.Protoapigenone, a natural flavonoid possessing an unusual  Flavonoids are among the most widespread secondary plant metabolites and play a significant role in the prevention of several chronic diseases, including cancer 1) was first isolated by our group from the Formosan fern Thelypteris torresiana2), a common 4′-hydroxy-flavone . The compound was also found to induce apoptosis in human prostate cancer cells in vitro and in vivo in nude mice while causing no major side effects in the latter case 1 was shown to exert dose-dependent DNA damage, apoptosis, and G2/M arrest at micromolar concentrations in a lung cancer cell line (H1299) 1 was chosen by the National Science Council of Taiwan as the lead compound for the development of a new class of anticancer drugs. Presently, in vivo toxicology studies are being performed as part of the preclinical phase, which have created a need for an economical way to drastically increase the production of 1.1 was reported by our group 1 from 2, we used methoxymethyl-diprotected trihydroxyacetophenones and 4-benzyloxybenzaldehyde as starting materials to prevent the A-ring of 2 from oxidation, and we applied a hypervalent iodine reaction to the 7-protected apigenin skeleton in the second-to-last step to obtain the p-quinol moiety on the B-ring. Finally, 1 was successfully obtained after the removal of the protecting group. To carry out the key oxidation step that formed the pharmacophore on the B-ring, we applied 2 equivalent (eq.) of [bis(trifluoroacetoxy)iodo]benzene (commonly referred to as PIFA) in the presence of 0.2 eq. of TEMPO, a commercially available free radical catalyst. Although in some related oxidative dearomatization reactions phenyliodine diacetate (PIDA) was found to give higher yield of product as compared to when PIFA was used 2, and, after deprotection, 1 was successfully obtained. In 2007, a six-step total synthesis of 1. Because this means of production was unable to satisfy the high material needs for the extensive preclinical pharmacological investigation, the improvement of the synthetic route has become an urgent need and a primary objective of our research program.The procedure resulted in the synthesis of protoapigenone as well as several of its derivatives which allowed SAR data to be gathered. However, due to the long reaction times and the slow chromatographic purifications, the procedure took approximately one to two months to complete, and resulted in only a 3.3% overall yield of 1 could be directly synthesized from 2 without the need for protecting groups. Optimization of the direct semisynthesis provided the following insights:Our attempt to improve the yield of the original method began with the optimization of the oxidation of 7-MOM-apigenin but led to the surprising observation that with appropriate reaction conditions, By decreasing the concentration of the starting material to 1 mg/mL, the yield could be improved significantly; further decreases of concentration did not provide further increases in the yield.1 may be prevented completely by increasing the amount of TEMPO used. Thus, it should be omitted from the reaction.TEMPO significantly decreases the yield, and the formation of 1, while 1,1,1,3,3,3-hexafluoro-isopropanol∶water  resulted in about half the yield of that obtained using the ACN∶water mixture.The reaction is highly dependent on the solvent used. For success, the majority of the solvent should be a non-nucleophilic polar solvent, preferably acetonitrile. When using acetone∶water  and THF∶water , the reaction failed. EtOAc saturated with water gave traces of Low energy microwave heating  can increase the yield by more than two-fold, compared to when the reaction is performed at room temperature.1 does not suffer quick decomposition, but it is slightly sensitive to acidic environments, so it is recommended that it be purified as soon as possible. On the other hand, neutralizing the TFA is not favorable, as the remaining ionizable phenolic hydroxyl groups may lead to a less complete separation at the purification process.The TFA (trifluoroacetic acid) derived from the PIFA in the reaction significantly decreases the pH. Compound 2  with 2 eq. of PIFA, which resulted in a 31% isolated yield for 100 mg of starting material. This was a great improvement over the previous method 1′ and 1″. Based on its NMR spectra and chromatographic properties in comparison with available testcompound and literature data, 1′ was identified as luteolin, a common flavone and an expected side-product of the performed reaction 1″ was found to have a molecular mass of 554 and a constitution of C30H18O11 corresponding to an apigenin-protoapigenone dimer. The 1H NMR spectrum suggested, that a ring corresponding to the B-ring of apigenin is present . Interestingly, four separate hydrogens were shown as doublet-doublets with the larger coupling constant of 10.0 Hz. This coupling would be characteristic to the two doublets, two hydrogens each, of the B-ring of protoapigenone. Based on the 1H-1H COSY spectrum, these four hydrogens were also on the same ring, suggesting a cyclohexadienone ring, as in protoapigenone, also to be present. Differences between the pairs of corresponding protons on this ring are suggested to arise from the effect of the close aromatic B-ring of the apigenin part, which was also shown by NOESY correlations between hydrogens of these two rings. The number of non-OH hydrogens and the presence of only two meta-coupling doublets accompanied by two singlets in the 1H spectrum suggested a C-C bond between parts of the dimer. Further assignments were made based on the HMQC and HMBC spectra, and an 8-3″ junction was suggested. Structure, numbering and key HMBC and NOESY correlations of 1″ are shown in As mentioned above, we performed the reaction via a quick, microwave-assisted oxidation of Hypervalent iodine reagents are widely used for the oxidative dearomatization of 4-substituted phenols in the presence of an appropriate nucleophile to obtain 4,4-disubstituted cyclohexadienones 1″ as a side-product also provides a possible explanation for the failure of the previous conditions, which most likely failed due to the much higher concentration of starting material and thus the more favorable dimerization or even oligomer formation via C-C coupling side-reactions. Considering the large variety of other side-reactions also available in this system, the 31% yield of 1 shows an unexpectedly good selectivity of PIFA for the 4′-OH group. This selectivity may arise from two phenomena: (1) the greater acidity of the 7-OH group compared to that of the 4′-OH group  pKa = 6.9 and 8.6, respectively 2 to mostly exist in the 7-mono-anionic form at the neutral pH of the reaction's start, and (2) the intramolecular hydrogen-bond to the 5-OH group makes it more stable than the 4′-OH.The presence of 1, we scaled-up the procedure by oxidizing 800.0 mg, 2.0 g and 5.0 g of 2, which provided 251.3 mg (29.6%), 546.2 mg (25.8%) and 1.18 g (22.3%) of 1, respectively , which served as the basis for the most active synthetic derivative to date. This reaction yielded a remarkable 59% of the corresponding analog (11), compared to the 16% yield reported previously The optimized reaction was also performed on 50.0 mg of β-naphthoflavone , Ca9-22 , A549 (lung), as well as MCF-7 and MDA-MB-231 (breast) cancer cells, with doxorubicin as a positive control. ICO-methyl substitution decreases the activity of 1O-alkyl side-chain on the protoapigenone skeletone typically increases the activity comparing to that of the methyl-substituted derivative (compound 3). As a result of this tendency, several analogs were found to exert practically the same, or, as in case of the butyl-substituted compound 7 against the Hep3B, MCF-7 and MDA-MB-231 cell lines, even higher in vitro cytotoxic activities comparing to that of the non-substituted compound 1. On the other hand, a 2″ to 3″ unsaturated side-chain  decreased the activity on the A549 cell line comparing to that of compound 5 bearing a saturated propyl side-chain . In addition to this, presence of a branching side-chain  strongly decreased the activity on each cell line as compared to that of 5 , resulting in low activities comparable to those of the methyl-substituted compound 3. 7 was found to exert significantly stronger activity than that of the lead compound 1.To elaborate on the findings of the previous investigation, which showed that 1′-12–18) does not seem to follow the same trend, except for the effect of the side-chain branching  on most of the cell lines with the exception of A-549, and all of these derivatives showed a marked decrease in the activity as compared to that of 11.Interestingly, the activity of the β-naphthoflavone derivatives  is the most promising one. The in vivo study of this compound is planned in the near future, and the synthesis of further derivatives is currently in progress.Based on these findings, new candidates for further studies were discovered, among which protoapigenone 1′-O-butyl ether (1″ (89.1%), 3 (94.8%) and 6 (94.8%). Compound purities were checked using HPLC-UV with one of the solvent systems described below at 245 nm using a gradient system consisting of two Jasco PU2080 LC pump and a Jasco UV2075 ultraviolet detector connected to a Hercule 2000 chromatographic interface. Columns used: Agilent Zorbax XDB-C8, 5 µm, 150×4.6 mm (RP-HPLC), and Agilent Zorbax-sil 5 µm, 250×4.6 mm (NP-HPLC). Solvent systems: RP-HPLC1: 55% aqueous MeOH increasing to 80% in 10 minutes; RP-HPLC2: 35% v/v aqueous MeOH increasing to 80% in 10 minutes; NP-HPLC: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 minutes and flow rate was 1 mL/min in each case. Chromatograms are shown in 1H NMR spectra were taken using a Varian Gemini-2000 200 MHz FT-NMR, and 13C and 2D NMR spectra were taken using a Varian Mercury-plus 400 MHz FT-NMR spectrometer. Shifts are given in ppm downfield of TMS and were referenced to internal residual protosolvent. 1H NMR spectra of all new compounds are shown in 1″ which could not be crystallized. Hence, http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi . The melting points were measured on VEB Wagetechnik Rapido PHNK melt microscope (upper limit: 360°C). The structures of 1, 1′, 11 and 12 were determined based on literature data 1 of natural origin: 180–181°C 1′ of natural origin: 328–330°C 1, 11 and 12 are as follows. 1. RP-HPLC1: 7.057 min . Apigenin was synthesized from naringenine (Indofine Ltd.) by Chuang DW in previous experiments (not discussed here). All compounds tested possessed a purity of over 95%, except for .057 min , purity:.733 min , purity:.580 min , purity:2 and 95% air. Cell viability was measured by the MTT colorimetric method 50 was recorded as the concentration of the agent that reduced cell viability by 50% under the experimental conditions, and it was determined by applying variable slope nonlinear regression with automatic outlier elimination at Q = 1.0% by using GraphPad Prism 5.0 (GraphPad Software Inc.). Statistical evaluation of the individual IC50 values was done by one way ANOVA with Dunnet's Multiple Comparison Test by choosing compounds 1 and 11 as references to their corresponding 1′-O-alkylated analogs. To reveal structure-activity relationships, the activites of each compound were compared by Bonferroni tests, and, in case of comparing the activities of 1 and 7, unpaired T-tests were also performed.Human breast (MCF-7 and MDA-MB-231), liver (HepG2 and Hep3B), and lung (A549) cancer cell lines were obtained from the American Type Culture Collection. The human oral squamous cell carcinoma Ca9-22 was a kind gift from Dr. Jeff Yi-Fu Chen, Kaohsiung Medical University, Kaohsiung, Taiwan. All cell lines were propagated in RPMI-1640 medium supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2 (100.0 mg) was dissolved in ACN∶water . A quantity of PIFA  was added to the container, and microwave heating was applied  with continuous stirring. The resulting mixture was evaporated, adsorbed onto 2.0 g of octadecyl silica, layered on the top of 2.0 g of octadecyl silica, washed with 10 mL of 20% aqueous MeOH and eluted with 20 mL of 60% aq. MeOH. The latter fraction was evaporated, redissolved in MeOH, and purified on a Sephadex LH-20 column . Fractions of 10 mL were collected, and fractions 11 through 14 gave protoapigenone . The side-products 1′ and 1″ were isolated from fractions 15 through 18 and 19 through 21, respectively, with repeated purification on the same Sephadex LH-20 column.Compound 2  were dissolved in ACN∶water  in staunch containers that were then pre-heated to 70°C. After releasing the pressure, PIFA  was added to each solution in a careful manner to prevent sudden explosive boiling, and after re-sealing the containers, microwave heating was applied  with continuous stirring. The samples obtained were cooled under a stream of water and subsequently with an ice bath. Each sample was immediately evaporated in a 10 L roundbottom flask using a Büchi Rotavapor R-220 at 50°C. After redissolving them, the samples were adsorbed onto octadecyl silica  that was layered on top of the same amount of octadecyl silica. SPE was performed by eluting with 15% aqueous MeOH  followed by 50% aqueous MeOH  to give 251.3 mg (29.6%), 546.2 mg (25.8%) and 1.18 g (22.3%) of protoapigenone, respectively.Different quantities of 2 or 10 was dissolved in 50 mL of a 9∶1 v/v mixture of acetonitrile and the corresponding alcohol  or water in the case of 11 and reacted with PIFA  under the same conditions used to obtain 1. To purify the products, flash chromatography (Biotage Flash+) on silica columns  was performed using a dry-loading technique and n-hexane∶EtOAc∶acetone solvent systems ) at flow rates of 24 mL/min. Finally, 3 through 9 and 12 were crystallized from hexane∶acetone , while 13 through 18 were crystallized from hexane∶CH2Cl2 .A total of 50.0 mg of 2 into 1; RP-HPLC2: 6.843 min (1H NMR (acetone-d6) δ 12.87 , 12.65 , 7.38 , 6.81 , 6.78 , 6.76 , 6.57 , 6.50 , 6.30 , 6.29 , 6.08 , 5.85  (13C NMR (acetone-d6) δ 185.2 (C-4′), 184.1 (C-4), 182.5 (C-4″), 168.8 (C-2), 165.8 (C-7″), 165.4 (C-2″), 164.0 (C-8a), 163.9 , 163.5 (C-5), 161.4 (C-4″′), 159.5 (C-8a″), 148.1 (C-2′ or C-6′), 148.0 (C-6′ or C-2′), 131.6 , 130.6 (C-3′ or C-5′), 130.5 (C-5′ or C-3′), 125.3 (C-1″′), 116.7 , 111.3 (C-3″), 108.0 (C-3), 106.1 (C-4a), 105.3 (C-4a″), 101.3 (C-8), 100.5 (C-6), 100.3 (C-6″), 95.4 (C-8″), 71.3 (C-1′); ESI-MS : 555 , 577 ; HRMS: C30H18O11Na, calculated 577.0747; found 577.0750.Yield: 4% (8.2 mg) as a side-product of the reaction of .843 min , purity:or H-3′)  and S22;1: 3.860 min (1H NMR (acetone-d6) δ 12.59 , 6.99 , 6.55 , 6.50 , 6.29 , 6.24 , 3.42  (13C NMR (acetone-d6) δ 183.4 (C-4′), 181.6 (C-4), 164.7 (C-2), 164.0 (C-7), 162.0 (C-5), 157.5 (C-8a), 144.8 (C-2′ and C-6′), 132.4 (C-3′ and C-5′), 106.9 (C-3), 104.3 (C-4a), 98.8 (C-6), 93.5 (C-8), 74.2 (C-1′), 51.6 (C-1″); ESI-MS : 323 , 301; HRMS: C16H12O6Na, calculated 323.0532; found 323.0530.Yield: 42% (23.3 mg); RP-HPLC.860 min , purity:OCH3-1′)  and S24;1: 5.157 min (1H NMR (acetone-d6) δ 12.59 , 7.01 , 6.56 , 6.51 , 6.28 , 6.24 , 3.63 , 1.27  (13C NMR (acetone-d6) δ 183.6 (C-4′), 181.7 (C-4), 164.9 (C-2), 163.9 (C-7), 162.0 (C-5), 157.5 (C-8a), 145.3 (C-2′ and C-6′), 131.9 (C-3′ and C-5′), 106.9 (C-3), 104.3 (C-4a), 98.7 (C-6), 93.4 (C-8), 74.0 (C-1′), 60.0 (C-1″), 14.6 (C-2″); ESI-MS : 337 , 315 ; HRMS C17H14O6Na, calculated 337.0688; found 337.0686.Yield: 33% (19.2 mg); RP-HPLC.157 min , purity:s, H-2″)  and S27;1: 8.240 min (1H NMR (acetone-d6) δ 12.59 , 7.01 , 6.56 , 6.52 , 6.28 , 6.24 , 3.54 , 1.68 , 0.98  (13C NMR (acetone-d6) δ 183.6 (C-4′), 181.7 (C-4), 164.8 (C-2), 163.9 (C-7), 162.0 (C-5), 157.5 (C-8a), 145.4 (C-2′ and C-6′), 131.9 (C-3′ and C-5′), 106.9 (C-3), 104.3 (C-4a), 98.7 (C-6), 93.5 (C-8), 74.0 (C-1′), 65.9 (C-1″), 22.7 (C-2″), 9.6 (C-3″); ESI-MS : 329 , 351 ; HRMS C18H16O6Na, calculated 351.0845; found 351.0846.Yield: 36% (21.9 mg); RP-HPLC.240 min , purity:z, H-3″)  and S30;1: 7.637 min (1H NMR (acetone-d6) δ 12.59 , 7.07 , 6.56 , 6.50 , 6.28 , 6.24 , 3.94 , 1.24  (13C NMR (acetone-d6) δ 162.0 (C-5), 145.7 (C-2′ and C-6′), 131.3 (C-3′ and C-5′), 107.0 (C-3), 104.2 (C-4a), 98.7 (C-6), 93.4 (C-8), 68.0 (C-1″), 23.5 (C-2″ and C-3″); ESI-MS : 329 , 351 , 270 ; HRMS C18H16O6Na, calculated 351.0845; found 351.0844.Yield: 26% (15.8 mg); RP-HPLC.637 min , purity:nd H-3″)  and S34;1: 9.437 min (1H NMR (acetone-d6) δ 12.60 , 7.01 , 6.55 , 6.52 , 6.28 , 6.25 , 3.58 , 1.63 , 1.44 , 0.93  (13C NMR (acetone-d6) δ 183.6 (C-4′), 181.7 (C-4), 164.9 (C-2), 163.9 (C-7), 162.0 (C-5), 157.5 (C-8a), 145.4 (C-2′ and C-6′), 132.0 (C-3′ and C-5′), 106.9 (C-3), 104.3 (C-4a), 98.7 (C-6), 93.4 (C-8), 74.0 (C-1′), 64.1 (C-1″), 31.5 (C-2″), 18.6 (C-3″), 12.8 (C-4″); HRMS C19H18O6Na, calculated 365.1001; found 365.1000.Yield: 44% (27.9 mg); RP-HPLC.437 min , purity:z, H-4″)  and S37;1: 8.283 min (1H NMR (acetone-d6) δ 12.59 , 7.04 , 6.57 , 6.53 , 6.29 , 6.24 , 6.00 , 5.37 , 5.20 , 4.14  (13C NMR (acetone-d6) δ 164.9 (C-2), 163.9 (C-7), 162.0 (C-5), 157.5 (C-8a), 145.4 (C-2′ and C-6′), 132.0 (C-3′ and C-5′), 106.9 (C-3), 98.7 (C-6), 93.4 (C-8), 72.9 (C-1′), 65.9 (C-1″); ESI-MS : 349 , 327 ; HRMS C18H14O6Na, calculated 349.0688; found 349.0690; X-ray data are shown in Yield: 29% (17.5 mg); RP-HPLC.283 min , purity:z, H-1″)  and S39;1: 4.180 min (1H NMR (acetone-d6) δ 12.55 , 7.08 , 6.55 , 6.53 , 6.30 , 6.24 , 4.34 , 3.11   δ 185.4 (C-4′), 183.5 (C-4), 166.1 (C-2), 166.0 (C-7), 163.9 (C-5), 159.4 (C-8a), 145.9 (C-2′ and C-6′), 134.3 (C-3′ and C-5′), 109.0 (C-3), 106.2 (C-4a), 100.8 (C-6), 95.4 (C-8), 81.3 (C-1′), 78.0 (C-3″), 76.4 (C-2″), 54.8 (C-1″); HRMS C18H12O6Na, calculated 347.0532; found 347.0535.Yield: 36% (21.6 mg); RP-HPLC.180 min , purity:1H NMR (CDCl3) δ 9.97 , 8.04 , 7.87 , 7.74 , 7.60 , 7.36 , 6.95 , 6.89 , 6.55 , 3.61 , 1.307  (13C NMR (CDCl3) δ 184.2 (C-4′), 179.5 (C-4), 160.9 (C-2), 156.9 (C-10a), 145.8 (C-2′ and C-6′), 132.2 (C-3′ and C-5′), 135.3, 130.2, 129.9, 129.0, 127.8, 126.6, 126.4 , 116.9 (C-10), 116.9 (C-4a), 112.1 (C-3), 73.9 (C-1′), 60.5 (C-1″), 15,3 (C-2″); ESI-MS : 355 , 333 ; HRMS C21H16O4Na, calculated 355.0946; found 355.0945.Yield: 65% (37.5 mg); NP-HPLC: 7.507 min , purity:z, H-2″)  and S45;1H NMR (CDCl3) δ 9.96 , 8.01 , 7.85 , 7.73 , 7.58 , 7.33 , 6.92 , 6.87 , 6.55 , 3.50 , 1.68 , 0.99  (13C NMR (CDCl3) δ 145.8 (C-2′ and C-6′), 132.2 (C-3′ and C-5′), 135.3, 129.0, 127.8, 126.6, 126.4 , 116.9 (C-10), 116.9 (C-4a), 112.1 (C-3), 73.9 (C-1′), 66.5 (C-1″), 23.0 (C-2″), 10.2 (C-3″); ESI-MS : 369 , 347 ; HRMS C22H18O4Na, calculated 369.1103; found 369.1105; X-ray data are shown in Yield: 64% (38.4 mg); NP-HPLC: 5.977 min , purity:z, H-3″)  and S48;1H NMR (CDCl3) δ 9.97 , 8.03 , 7.94 , 7.74 , 7.60 , 7.35 , 6.94 , 6.91 , 6.53 , 3.89 , 1.24  (13C NMR (CDCl3) δ 184.5 (C-4′), 179.6 (C-4), 161.2 (C-2), 156.9 (C-10a), 146.2 (C-2′ and C-6′), 131.5 , 135.3, 130.2, 129.9, 129.0, 127.7, 126.6, 126.4 , 116.9 (C-10), 116.9 (C-4a), 112.2 (C-3), 74.1 (C-1′), 68.5 (C-1″), 24.3 (C-2″ and C-3″); ESI-MS : 369 , 347 ; HRMS C22H18O4Na, calculated 369.1103; found 369.1104.Yield: 52% (31.2 mg); NP-HPLC: 6.307 min , purity:nd H-3″)  and S51;1H NMR (CDCl3) δ 9.97 , 8.03 , 7.86 , 7.74 , 7.59 , 7.34 , 6.92 , 6.87 , 6.55 , 3.54 , 1.65 , 1.45 , 0.95  (13C NMR (CDCl3) δ 184.3 (C-4′), 179.5 (C-4), 160.1 (C-2), 156.9 (C-10a), 145.9 (C-2′ and C-6′), 132.2 (C-3′ and C-5′), 135.3, 130.2, 129.9, 129.0, 127.8, 126.6, 126.4 , 116.9 (C-10), 116.9 (C-4a), 112.1 (C-3), 73.8 (C-1′), 64.6 (C-1″), 31.7 (C-2″), 18.8 (C-3″), 13.4 (C-4″); ESI-MS : 383 , 361 ; HRMS C23H20O4Na, calculated 383.1259; found 383.1258.Yield: 54% (33.8 mg); NP-HPLC: 5.230 min , purity:z, H-4″)  and S54;1H NMR (CDCl3) δ 9.96 , 8.03 , 7.86 , 7.74 , 7.60 , 7.35 , 6.94 , 6.91 , 6.57 , 5.95 , 5.38 , 5.25 , 4.09  (13C NMR (CDCl3) δ 184.1 (C-4′), 179.4 (C-4), 160.6 (C-2), 156.9 (C-10a), 145.3 (C-2′ and C-6′), 132.4 (C-3′ and C-5′), 135.3, 130.2, 129.9, 129.0, 127.8, 126.6, 126.4 , 116.9 (C-10), 116.9 (C-4a), 112.2 (C-3), 74.1 (C-1′), 65.7(C-1″), 133.2 (C-2″), 117.2 (C-3″); ESI-MS : 367 , 345 ; HRMS C22H16O4Na, calculated 367.0946; found 367.0949; X-ray data are shown in Yield: 51% (30.5 mg); NP-HPLC: 6.650 min , purity:z, H-1″)  and S57;1H NMR (CDCl3) δ 9.95 , 8.04 , 7.87 , 7.74 , 7.60 , 7.37 , 6.97 , 6.91 , 6.59 , 4.26 , 2.55  (13C NMR (CDCl3) δ 183.9 (C-4′), 179.3 (C-4), 159.9 (C-2), 156.9 (C-10a), 144.2 (C-2′ and C-6′), 132.6 (C-3′ and C-5′), 135.4, 130.2, 129.8, 129.1, 127.8, 126.6, 126.5 , 116.8 (C-10), 116.8 (C-4a), 112.2 (C-3), 74.6 (C-1′), 53.2 (C-1″), 75.7 (C-3″),79.0 (C-2″); ESI-MS : 365 , 343 ; HRMS C22H14O4Na, calculated 365.0790; found 365.0788.Yield: 45% (26.7 mg), NP-HPLC: 9.527 min , purity:z, H-3″)  and S60;Figure S1X-ray diffraction data for compound 8. CCDC 762721; formula: C18H14O6; unit cell parameters: a 6.9461(20) b 30.2804(66) c 7.6340(13) beta 93.752(19) space group P21/c.(TIF)Click here for additional data file.Figure S2X-ray diffraction data for compound 14. CCDC 762722; formula: C22H18O4; unit cell parameters: a 7.8452(8) b 25.285(5) c 18.144(2) beta 96.096(9) space group P21/c.(TIF)Click here for additional data file.Figure S3X-ray diffraction data for compound 17. CCDC 762723; formula: C22H16O4; unit cell parameters: a 7.9122(8) b 24.817(5) c 17.8259(16) beta 96.380(8) space group P21/c.(TIF)Click here for additional data file.Figure S4RP-HPLC chromatogram of compound 1. Solvent: 35% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 35%; flow rate: 1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S5RP-HPLC chromatogram of compound 1″. Solvent: 35% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 35%; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S6RP-HPLC chromatogram of compound 3. Solvent: 55% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 55%; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S7RP-HPLC chromatogram of compound 4. Solvent: 55% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 55%; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S8RP-HPLC chromatogram of compound 5. Solvent: 55% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 55%; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S9RP-HPLC chromatogram of compound 6. Solvent: 55% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 55%; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S10RP-HPLC chromatogram of compound 7. Solvent: 55% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 55%; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S11RP-HPLC chromatogram of compound 8. Solvent: 55% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 55%; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S12RP-HPLC chromatogram of compound 9. Solvent: 55% v/v aqueous MeOH increasing to 80% in 10 min and subsequently changing back to 55%; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S13NP-HPLC chromatogram of compound 11. Solvent: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 min; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S14NP-HPLC chromatogram of compound 12. Solvent: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 min; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S15NP-HPLC chromatogram of compound 13. Solvent: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 min; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S16NP-HPLC chromatogram of compound 14. Solvent: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 min; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S17NP-HPLC chromatogram of compound 15. Solvent: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 min; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S18NP-HPLC chromatogram of compound 16. Solvent: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 min; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S19NP-HPLC chromatogram of compound 17. Solvent: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 min; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S20NP-HPLC chromatogram of compound 18. Solvent: 5% v/v isopropanol in dichloromethane increasing to 17% in 12 min; flow rate:1 mL/min, λ = 245 nm(TIF)Click here for additional data file.Figure S211H NMR spectrum of compound 1″.400 MHz (TIF)Click here for additional data file.Figure S221H NMR spectrum of compound 1″.Zoom of 400 MHz (TIF)Click here for additional data file.Figure S231H NMR spectrum of compound 3 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S241H NMR spectrum of compound 3 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S251H NMR spectrum of compound 4 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S261H NMR spectrum of compound 4 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S271H NMR spectrum of compound 4 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S281H NMR spectrum of compound 5 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S291H NMR spectrum of compound 5 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S301H NMR spectrum of compound 5 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S311H NMR spectrum of compound 6 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S321H NMR spectrum of compound 6 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S331H NMR spectrum of compound 6 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S341H NMR spectrum of compound 6 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S351H NMR spectrum of compound 7 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S361H NMR spectrum of compound 7 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S371H NMR spectrum of compound 7 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S381H NMR spectrum of compound 8 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S391H NMR spectrum of compound 8 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S401H NMR spectrum of compound 9 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S411H NMR spectrum of compound 9 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S421H NMR spectrum of compound 9 before crystallization.400 MHz (TIF)Click here for additional data file.Figure S431H NMR spectrum of compound 13 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S441H NMR spectrum of compound 13 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S451H NMR spectrum of compound 13 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S461H NMR spectrum of compound 14 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S471H NMR spectrum of compound 14 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S481H NMR spectrum of compound 14 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S491H NMR spectrum of compound 15 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S501H NMR spectrum of compound 15 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S511H NMR spectrum of compound 15 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S521H NMR spectrum of compound 16 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S531H NMR spectrum of compound 16 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S541H NMR spectrum of compound 16 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S551H NMR spectrum of compound 17 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S561H NMR spectrum of compound 17 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S571H NMR spectrum of compound 17 before crystallization.Zoom 200 MHz (TIF)Click here for additional data file.Figure S581H NMR spectrum of compound 18 before crystallization.200 MHz (TIF)Click here for additional data file.Figure S591H NMR spectrum of compound 18 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.Figure S601H NMR spectrum of compound 18 before crystallization.Zoom of 200 MHz (TIF)Click here for additional data file.


The structures of compounds 1–4 were elucidated based on spectroscopic analysis, especially 2D NMR  and HRMS. Compounds 1 and 4 showed weak anti-inflammatory activity as tested by superoxide anion generation and elastase release by human neutrophil in response to fMLP/CB. Compound 3 showed selective inhibition on elastase release in vitro.Four new 8-hydroxybriarane diterpenoids, frajunolides L–O ( Junceella grown in the tropical and subtropical waters of Indo-West Pacific regions are well known as a source of highly oxidized briarane-type diterpenoids with a γ-lactone moiety + at m/z 589 and the isotope fragment [M + Na + 2]+ at m/z 591 in ESIMS, with the typical ratio of relative intensity (3:1) in the mass spectrum. In the infrared spectrum, strong absorption bands were found at 3436, 1735 and 1780 cm−1 characteristic for hydroxyl, ester carbonyl (acetyl) and five-membered γ-lactone ring, suggesting a briarane-type diterpenoid similar to compounds 1 and 2. It was found that the 1H- and 13C NMR spectra of 3 in CDCl3 showed mostly broad peaks and in some cases, certain peaks were not observed. In order to mark more optimum signals of the NMR spectra, compound 3 was dissolved in pyridine-d5. The 1H- and 13C NMR data , an exocyclic double bond  and a γ-lactone carbonyl carbon . Judging from the molecular formula and NMR data of 3, six degrees of unsaturation were counted for, indicating that compound 3 contained a tetracyclic system including an exocyclic epoxide . The HMBC correlations , H-4 , H-9 , and H-14  with one of ester carbonyl carbons, respectively, revealed that four acetyl groups were connected to the C-2, C-4, C-9, and C-14 positions. By interpretation of the NMR spectroscopic data, the planar structure of compound 3 was elucidated. The relative configuration of 3 was determined by NOESY (δC 58.2) and C-20 (δC 52.6), and the NOESY correlations between H2-20 and Me-15 agreed with β-face of H2-20, 11R-configuration regarding the exocyclic epoxide, and chair conformation of the cyclohexane ring. Furthermore, NOESY correlations of H-10/H-2, H-4/H-2 and H-10/H-9 suggested that H-2, H-4 and H-9 were located on the same face and could be assigned as α.The HRESI mass spectrum of NMR data  of 3 revelations  between by NOESY  and deta4 showed a pair of quasi-molecular ion peaks at m/z 607 and 609 [M + H]+ with a ratio of 3:1 in the ESIMS, indicating the presence of a chlorine atom. Moreover, a molecular formula C28H37ClO11 was established by HRESIMS and confirmed by 1H- and 13C NMR spectroscopic analysis /C-4 (δC 33.4), C-5 (δC 144.9), and C-6 (δC 56.2), and H2-20 /C-10 (δC 44.6), C-11 (δC 147.0), and C-12 (δC 38.6), respectively. In addition, the oxygenated methine proton H-2 , H-9 , H-13 , and H-14  showed HMBC correlations with the acetate carbonyl carbons . Furthermore, detailed analysis of 2D NMR spectroscopic data (1H-1H COSY and HMBC) established the planar structure of 4. The configuration of compound 4 was determined on the basis of NOESY correlations /H-14 and H-13/H-14 implied that H-13 and H-14 are on the β-face while correlations of H-2/H-10, H-10/H-9, H-9/Me-19, H-17/H-7 and H-6/H-7 confirmed that the configuration of these protons are identical to those of compound 3.Compound analysis . The IR periment , which selations . The NOE1–4 were evaluated by measuring superoxide anion generation and elastase release by human neutrophils in response to fMet-Leu-Phe (fMLP)/Cytochalasin B (CB) 24 D +6.0 ; IR νmax 3439, 2922, 2749, 1768, 1735, 1370, 1248, 1221, 1040 cm−1; 1H NMR data , see 13C NMR data , see m/z 573 [M + Na]+; HRESIMS m/z 573.2313 [M + Na]+ .Frajunolide L (2): colorless amorphous powder; [α]24 D +8.0 ; IR νmax 3447, 2923, 2853, 1773, 1735, 1645, 1375, 1240, 1223, 1041 cm−1; 1H NMR data , see 13C NMR data , see m/z 573 [M + Na]+; HRESIMS m/z 573.2315 [M + Na]+ .Frajunolide M (3): colorless amorphous powder; [α]24 D +18.0 ; IR νmax 3436, 2933, 1780, 1735, 1376, 1255, 1235, 1212, 1044, 1017 cm−1; 1H NMR data , see 13C NMR data , see m/z 589 [M + Na]+, 591 [M + Na + 2]+; HRESIMS m/z 589.2266 [M + Na]+ .Frajunolide N (4): colorless amorphous gum; [α]24 D +6.7 ; IR νmax 3467, 2927, 1780, 1739, 1368, 1250, 1223, 1041 cm−1; 1H NMR data , see 13C NMR data , see m/z 607 [M]+; HRESIMS m/z 607.1925 [M + Na]+ , 609.1892 [M + Na + 2]+.Frajunolide O (2 •− generation was based on the SOD-inhibitable reduction of ferricytochrome c. Degranulation of azurophilic granules was determined by elastase release as described previously [p-nitroanilide as the elastase substrate. The fMet-Leu-Phe (fMLP), activated by Cytochalasin B (CB), was used as a stimulant. Genistein was used as a standard compound.Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. The assay of Oeviously . The elaJunceella fragilis has resulted in the isolation of four new briarane diterpenoids, frajunolides L–O (1–4). Among them, compounds 1, 3 and 4 exhibited mild or selective anti-inflammatory activity.Chemical investigation of the Taiwanese gorgonian


Recently, the crystal structure of rat DNPH1, a potential target for anti-cancer therapies, suggested that various analogs of AMP may inhibit this enzyme. From this result, we asked whether N6-substituted AMPs, and among them, cytotoxic cytokinin riboside 5’-monophosphates, may inhibit DNPH1. Here, we characterized the structural and thermodynamic aspects of the interactions of these various analogs with DNPH1. Our results indicate that DNPH1 is inhibited by cytotoxic cytokinins at concentrations that inhibit cell growth. The gene  Nucleoside analogs are widely used in chemotherapy to fight against cancer and viral infections. Most of the anticancer nucleoside analogs exert their effect after incorporation into DNA. They interfere with one or more enzymes that are critical for DNA synthesis or DNA repair and cause DNA damage then apoptosis induction . For exaIn fact, multiple steps of the nucleotide biosynthesis may also be targeted upstream from DNA synthesis and repair and multi-target strategy may help in preventing drug resistance. N6-isopentenyladenosine, N6-furfuryladenosine (kinetin riboside) and N6-benzyladenosine have cytotoxic effects for different human cell lines derived from solid tumours + 430.1492, found 430.1482.White powder (42 mg); Rt = 6.02 min (10-50% of CH3CN in 10 mM TEAA buffer); 1H NMR (DMSO-d6) δ 1.63-1.70 , 2.12 , 2.25 , 3.85 , 4.03-4.05 , 4.21-4.23 , 4.64 , 4.70 , 5.92 , 7.95 , 8.19 , 8.45 ; 13C NMR (DMSO-d6) d 15.0 (C-3 cBu), 30.7 , 52.7 (C-1 cBu), 64.7 , 71.6 (C-3'), 74.4 (C-2'), 84.5 , 87.1 (C-1'), 120.0 (C-5), 139.6 (C-8), 146.9 (C-4), 153.0 (C-2), 155.0 (C-6); 31P NMR (DMSO-d6) δ -1.14; HRMS (ESI-TOF) m/z calcd for [C14H20N5O7P+H]+ 402.1179, found 402.1177. White powder (6 mg); Rt = 11.50 min (0-20% of CH3CN in 10 mM TEAA buffer); 1H NMR (DMSO-d6) δ 0.60-0.63 , 0.70-0.75 , 3.09 , 3.84 , 4.03-4.05 , 4.21-4.23 , 4.64 , 5.93 , 7.86 , 8.24 , 8.44 ; 13C NMR (DMSO-d6) δ 6.9 , 24.8 (C-1 cPr), 64.8 , 71.5 (C-3'), 74.5 (C-2'), 84.6 , 87.2 (C-1'), 120.2 (C-5), 139.6 (C-8), 149.9 (C-4), 153.0 (C-2), 155.3 (C-6); 31P NMR (DMSO-d6) δ -1.14; HRMS (ESI-TOF) m/z calcd for [C13H18N5O7P+H]+ 388.1022, found 388.1026.White powder (26 mg); Rt = 8.85 min (10-50% of CH3CN in 10 mM TEAA buffer); 1H NMR (DMSO-d6) δ 3.86-3.92 , 4.09 , 4.26 , 4.70 , 6.00 , 7.02-7.06 , 7.31-7.35 , 7.93-7.96 , 8.41 , 8.64 , 9.86 ; 13C NMR (DMSO-d6) δ 64.8 , 71.5 (C-3'), 74.5 (C-2'), 84.7 , 87.4 (C-1'), 120.3 (C-5), 121.2 , 123.0 (C-4 Arom.), 128.8 , 140.1 (C-1 Arom.), 140.8 (C-8), 150.4 (C-4), 152.5 (C-2), 153.9 (C-6); 31P NMR (DMSO-d6) δ 1.20; HRMS (ESI-TOF) m/z calcd for [C16H18N5O7P+H]+ 424.1022, found 424.1006.White powder (10 mg); Rt = 10.38 min (10-50% of CH3CN in 10 mM TEAA buffer); 1H NMR (DMSO-d6) δ 2.95 , 3.72 , 3.84-3.89 , 4.03-4.07 , 4.20-4.22 , 4.65 , 5.93 , 7.16-7.21 , 7.25-7.31 , 7.78 , 8.24 , 8.44 ; 13C NMR (DMSO-d6) δ 35.6 (CH2-Ph), 41.8 (CH2-NH), 64.8 , 71.5 (C-3'), 74.4 (C-2'), 84.6 , 87.3 (C-1'), 119.8 (C-5), 126.5 (C-4 Arom.), 128.7 , 129.1 , 139.7 (C-8), 140.0 (C-1 Arom.), 149.6 (C-4), 153.1 (C-2), 155.7 (C-6); 31P NMR (DMSO-d6) δ 1.08; HRMS (ESI-TOF) m/z calcd for [C18H22N5O7P+H]+ 452.1335, found 452.1329.White powder (38 mg); Rt = 10.70 min (0-10% of CH3CN in 10 mM aq TEAA buffer); 1H NMR (MeOD-d4) δ 2.63 , 3.90 , 4.00-4.20 , 4.27-4.29 , 4.46 , 4.70 , 6.14 , 8.28 , 8.51 ; 13C NMR (MeOD-d4) δ 37.6 (CH2-COO), 39.6 (NH-CH2), 65.8 , 72.3 (C-3'), 76.4 (C-2'), 85.9 , 88.9 (C-1'), 120.5 (C-5), 140.5 (C-8), 149.5 (C-4), 153.9 (C-2), 156.0 (C-6), 179.1 (COO); 31P NMR (MeOD-d4) δ 2.36; HRMS (ESI-TOF) m/z calcd for [C13H18N5O9P+H]+ 420.0920, found 420.0918.White powder (30 mg); Rt = 8.98 min (0-20% of CH3CN in 10 mM TEAA buffer); 1H NMR (D2O) δ 1.80-2.11 , 2.38 , 3.63 , 4.10-4.23 , 4.37-4.47 , 4.55 , 4.81 , 6.17 , 8.27 , 8.48 ; 13C NMR (D2O) δ 28.1 (CH2-CH2-COO), 37.2 (CH2-COO), 43.2 (NH-CH2), 66.9 , 73.1 (C-3'), 77.0 (C-2'), 86.6 , 89.6 (C-1'), 121.5 (C-5), 141.8 (C-8), 150.6 (C-4), 155.3 (C-2), 157.1 (C-6), 185.0 (COO); 31P NMR (D2O) δ 2.18; HRMS (ESI-TOF) m/z calcd for [C14H20N5O9P+H]+ 434.1077, found 434.1062.White powder (32 mg); dnph1 gene was amplified from the mammalian gene ATCC® Number: MGC-19540 using oligonucleotides olhumrclnde: 5’-ATGAATTCCATATGGCTGCTGCCATGGTGCCG-3’ and olhumrclHind 5’- CCCAAGCTTTCAAGTGGTTGGGTCAGGGGAG -3’ in a standard PCR reaction. The PCR product was digested with NdeI and HindIII, purified and ligated to pET28a digested with the same restriction enzymes. After transformation into E.coli DH5α strain, the plasmid DNA of several colonies was extracted and purified and sequenced. The pET28a rat dnph1  phosphine (TCEP). In the case of compound 14 (Ki = 11.2 µM), a concentration of 1 mM was used and [DNPH1] = 20 µM. The heat of dilution obtained from injecting the ligand into buffer was substracted before fitting. Heat effects were integrated with ITC Origin 7.0 software and a best fit was found using a single binding-site model.ITC was performed in a MicroCal VP-ITC calorimeter at 25°C. Protein samples were prepared as indicated above except that they were further purified by gel filtration on a Superdex 200 column. Following thermal equilibration, titrant additions were made at 600-s intervals to the 1.41 mL protein samples by adding 5 µL aliquots of 300 µM compounds to protein samples ([DNPH1] = 15 µM) in 25 mM Na4)2SO4 from Qiagen. Starting from selected conditions, having the best crystal-diffraction quality, further refinement was done with 1.1 to 1.3 M (NH4)2SO4 and 1.2 to 1.4 M Li2SO4, in mild basic buffer (100 mM Tris pH 7.4 to 8.0) at the protein concentration of 12 mg/mL in 50 mM citrate buffer pH 7.5, which gave rise to diffracting crystals from 2.0 and up to 1.6 Å resolution of rat DNPH1 in complex with the compounds 1, 5, 7 and 12. The other compounds, 8, 9, 10, 11 and 13 failed to co-crystallize and our attempts to soak those ligands failed to displace the original one. The crystal structures were solved by molecular replacement using Molrep [1, 5 and 7 were performed using Refmac5 [Crystal growth conditions have been previously described  and are g Molrep  and as ag Molrep ). The fi Refmac5  with ite Refmac5 . The lig Refmac5  and wereSince the majority of data concerning DNPH1 were performed with human cells, tissues and tumors, we decided to purify and characterize the human DNPH1.Km of 57 µM for dGMP, and a Vmax of 0.005 units.mg-1 corresponding to a kcat of 0.002 s-1. As reported previously for the rat DNPH1 enzyme [Km of human DNPH1 for dCMP (2.5 mM), dUMP (7.8 mM) and dTMP (23 mM) are high compared to dGMP (57 µM), dAMP (97 µM) and dIMP (104 µM). Whereas rat and human DNPH1s have different kcat, they have similar Km values. These differences of kcat should be interpreted with caution since both enzymes were produced in Escherichia coli. However, we cannot exclude that post-translational modifications enhance or regulate the enzymatic activity in the cellular context. Initial velocity experiments were carried out at variable concentrations of the six 2'-deoxynucleoside 5’-monophosphates, dAMP, dCMP, dGMP, dIMP, dTMP and dUMP . The kin1 enzyme , purine These data showed, however, that both enzymes possess rather similar functional features in agreement with their highly similar sequences . This obWe have previously shown that purine ribonucleotides inhibit the rat DNPH1 enzyme and that small substituents on the purine ring are well tolerated . StructuKi = 40 µM) [Ki ranging from 1.2 to 15.5 µM depending on the substitution).Different cycloalkyls, saturated and unsaturated alkyl chains with aromatic or functional groups were considered at the N6 position. As summarized in = 40 µM) , with in5, benzyl 6, and phenylethyl 12), as confirmed by the similar Ki values (from 1.8 to 2.3 µM). Surprisingly, AMP bearing a phenyl ring (compound 11) revealed a moderate Ki (10.6 µM) illustrating the benefit of a methylene group between N6 and the cycloalkyl or aromatic group.The size of the cycloalkyl group (from 3- to 6- membered ring) appears to have little influence on the inhibition potency, as well as the nature of aromatic rings (methylfuryl All these substitutions significantly improved the inhibition potency towards rat DNPH1 as compared to AMP (up to 40 fold higher). This could be explained by a contribution of the hydrophobic substituents. 1 is a good inhibitor , the presence of a hydroxyl group on the C4 chain  resulted in a significant decrease in activity (up to 5-fold for 2 compared to 1).By contrast, the presence of polar functional groups  significantly decreased the inhibition of DNPH1s. Indeed, whereas compound These results were quite surprising regarding the largely solvent-accessible active site where the nucleobase is sandwiched between two hydrophobic isoleucine residues I18 and I65) while its N1/N6 edge faces the solvent [ and I65 N6-substituted AMP derivatives to rat and human DNPH1s were determined using isothermal titration calorimetry (ITC) , with the exception of 14 (Kd of 9.4 µM). Thus, the free energies are lower as compared to GMP  [5 and 6). By contrast, no gain in entropy was observed. Only the cyclopentyl group (compound 7) showed a favorable entropic contribution, although the ∆S term remains negative. To further characterize the ligand-DNPH1 interactions, the thermodynamic binding parameters of some ry (ITC) . Represel mol-1)  suggestil mol-1) . The benThese binding parameters are very similar to those obtained with human DNPH1 . Kd. In order to diminish the experimental constraints on ITC measurements, the thermodynamic data were transformed into ∆∆-plots according to Olsson . A stricIn an attempt to rationalize these biochemical and thermodynamic data, a structural study was performed.In a previous study, we solved the crystal structure of rat DNPH1 bound to three distinct nucleotide analogs . These cP1211) that differs from the orthorhombic form previously described despite the similar conditions used to grow the crystals. These results suggest that we can survey the impact of the different crystal packing on the protein conformation. Having one complex - rat DNPH1-7 - in common in the two available space groups, we could conclude that the absence of electron density for the long loop (residues 47-59) was due to its intrinsic flexibility rather than packing issue. This is in agreement with published NMR data [N6-substituent on the inhibitory activity of the various cytokinins against DNPH1.High-resolution data were obtained for three complexes while the other ligands failed to provide us with decent crystals. The new structures were all obtained in the same monoclinic space group . In some monomers, the N6-substitution points toward a hydrophobic subpocket formed by the side chain of isoleucine I18 and part of the side chain of arginine R19 , one may conclude that alternating conformations can be predicted for all the N6-substituted AMP bound to mammalian DNPH1. None of the tested substition can fit a buried sub-pocket to maximize additional interactions. This suggests that the few favorable interactions observed in one configuration or another (inward for 7 or 1 and outward for 5) are not sufficient to dramatically alter the binding affinity. This is in agreement with our thermodynamic data  in one orientation.Indeed, the three novel crystal structures showed little structural variation for protein structure. The two dimers in the asymmetric unit from these three crystal structures can be superimposed  pathway and inhibits the Akt activation [Nevertheless, the actual molecular targets are still unknown and the precise mechanism of action of these cytokinin ribosides remains to be determined. By similarity with triciribine, Akt might be one possible target. Indeed, tivation . Ki values for DNPH1 may be explained by the phosphorylation capacity of ADK [N6-substituted purine and 2-aminopurine nucleosides 5’-monophosphates [dnph1 expression.The kinetic, thermodynamic and structural data reported here indicate that DNPH1 may be a potential cytokinin target. Some cancer cell lines are killed by cytokinins at low micromolar concentrations ,38, a coy of ADK , the exiosphates  and the N6-substituted AMP derivatives might have to inhibit both DNPH1 and LDH-A to efficiently inhibit cell growth. It is likely that cytokinins may also bind other targets such as the lactate dehydrogenase LDH-A, which is the form of LDH expressed in many cancer cells . It is iortho-topolin riboside, the most active cytokinin reported so far, (See Pubchem CID 13506406 Compound BioActivity Data). Another argument in favor of other targets than DNPH1 for the inhibition of cell growth is, for example, provided by the identification of human tyrosyl-DNA phosphodiesterase 1 (TDP1), SMAD3 or PAX8 with micromolar affinity for These data suggest that an intricate network of activation and inhibition may play a role in the anti-cancer activity of triciribine or other nucleoside analogs such as cytokinins.Stringent affinity chromatography or drug affinity responsive target stability (DARTS) may help to identify cytokinin protein targets . N6-substituted AMP suggests further modifications. Derivatizations at the N6 position are currently underway and are expected to compensate the observed alternation in binding modes. These chemical tools will contribute to elucidate the role of DNPH1 in cellular proliferation.Our current study shows that cytokinin ribosides 5’-monophosphates inhibit mammalian DNPH1. Thermodynamic and structural characterization of DNPH1 in complex with


Lobophytum durum resulted in the isolation of five new cembranolides, durumolides M–Q (1–5). The structures of compounds 1–5 were characterized by the interpretation of extensive spectroscopic analysis. Compound 4 exhibited cytotoxicity against P-388 (mouse lymphocytic leukemia) cell line with an ED50 of 3.8 μg/mL. Moreover, compound 5 showed significant antiviral activity against human cytomegalovirus with an IC50 of 5.2 μg/mL.Chemical investigations of the Dongsha Atoll soft coral  Comparison of the NMR data  correlated to the oxymethine carbon at δC 70.0 (C-17), and oxymethylene protons at δH 3.81  and 3.73  correlated to the methine carbons at δC 40.8 (C-1), and 44.5 (C-15), as well as the lactone carbonyl carbon at δC 176.9 (C-16) in the HMBC spectrum  and the NOESY correlations between H-7/H-9b and H-11/H-13 , H-3/H-5b (δ 1.11), H-14/H-2a (δ 1.75), H-7/H-9b, and H-14/H3-20. Moreover, the large coupling constant (J = 9.2 Hz) between H-1 and H-15 suggested that the vicinal protons were either in an anticoplanar or eclipse relationship. The latter relationship should be correct because the signal at δH 2.86  showed a strong NOESY correlation with δH 2.28 , implying the R configuration at C-15. On the basis of the above-mentioned observations, the structure of durumolide M (1) was characterized as -13,18-dihydroxy-17-methoxy-3,4-epoxycembra-7,11-dien-16,14-olide.The geometries of the two trisubstituted olefins were proposed as both -11/H-13 . Compoun1 and 2 had the same molecular formula (C21H32O6). Analysis of their 1H- and 13C-NMR data  was unambiguously established and elucidated as -13,18-dihydroxy-17-methoxy-3,4-epoxycembra-7,11-dien-16,14-olide.Compounds NMR data  and 2 re2-17/H-1 . Therefo3) was analyzed for a molecular formula of C23H34O7 by its HR-ESI-MS coupled with the DEPT and 13C NMR spectroscopic data -13-hydroxy-18-acetoxy-17-methoxy-3,4-epoxycembra-7,11-dien-16,14-olide.Durumolide O  exhibited a pseudomolecular ion peak at m/z 387.2148 [M + Na]+, consistent with a molecular formula of C21H32O5, which is 16 mass units smaller than that of 1. Comparison of the NMR data  and 2.45 ; δC 43.7 (CH2)] in 4. The observed COSY correlation between H-13 and H-14 and the key HMBC correlations from H3-20 to C-13 confirmed the location of the methylene group. The relative stereochemistry of 4 was in agreement with that of 2 due to the similar NOESY correlations. Judging from the undisputable evidence, the structure of 4 was unambiguously determined and deduced as -18-hydroxy-17-methoxy-3,4-epoxycembra-7,11-dien-16,14-olide.The positive HR-ESI-MS of durumolide P -MTPA ester 5a were less shielded by the phenyl ring of MTPA products. Therefore, the absolute stereochemistry of 5 was unambiguously established as -13-hydroxy-17-methoxy-3,4-epoxycembra-7,11-dien-16,14-olide.The molecular formula of CNMR data , indicatNMR data  and 3 ofmolide J , with thalcohols . AnalysiR values  accordin3J1,14) is dubious 25 D −45 ; IR (KBr) νmax 3394, 2924, 1766, 1449, 1027, 765 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 403 [M + Na]+ HR-ESI-MS m/z 403.2099 .Durumolide M (2): colorless oil; [α]25 D −98 ; IR (KBr) νmax 3436, 2926, 1766, 1386, 1178, 1033 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 403 [M + Na]+; HR-ESI-MS m/z 403.2094 .Durumolide N : colorle3): colorless oil; [α]25 D −94 ; IR (KBr) νmax 3425, 2924, 1773, 1460, 1181, 1035 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 445 [M + Na]+; HR-ESI-MS m/z 445.2199 .Durumolide O (4): colorless oil; [α]25 D −121 ; IR (KBr) νmax 3460, 2926, 1769, 1445, 1181, 1031 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 387 [M + Na]+; HR-ESI-MS m/z 387.2148 .Durumolide P (5): colorless oil; [α]25 D −99 ; IR (KBr) νmax 3446, 2924, 1773, 1383, 1177, 1031 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 387 [M + Na]+; HR-ESI-MS m/z 387.2146 .Durumolide Q  samples of 5 were dissolved in 0.6 mL of pyridine-d5 and allowed to react for 3 hr at room temperature with (R)- and (S)-MTPA chloride (one drop) to yield (S)-MTPA ester 5a and (R)-MTPA ester 5b, respectively. Selected 1H-NMR  of 5a: δH 7.41–7.61 , 5.93 , 5.74 , 5.00 , 4.44 , 4.01 , 3.26 , 2.88 , 2.82 , 2.47 , 2.20 , 1.95 , 1.78 , 1.54 , 1.21 ; Selected 1H-NMR  of 5b: 7.42–7.63 , 5.83 , 5.60 , 4.94 , 4.50 , 4.02 , 3.27 , 2.93 , 2.83 , 2.38 , 2.19 , 1.97 , 1.54 , 1.49 , 1.16 .Preparation of Mosher’s Esters of 3.4.Cytotoxicity was determined against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549  tumor cells using a modification of the MTT colorimetric method. The provision of the P-388 cell line was supported by J. M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection. The experimental details of this assay were carried out according to a previously described procedure ,26,27.3.5.50 (50% inhibitory concentration), or the compound concentration required to reduce the 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 will be incubated for 1 h in the presence or absence of various concentrations of tested natural product. Then, cells will be infected with HCMV at an input of 1000 plaque forming units (pfu) per well of a 24-well dish. Antiviral activity is expressed as ICemployed .4.1–5) with those of cembranolides previously isolated by our group from Lobophtum michaelae [Comparison of cytotoxicity of durumolides M–Q (ichaelae , showed