Anticonvulsant activity of prepared compounds was evaluated using MES and PTZ models. Some of the designed compounds significantly protected mice against the PTZ-induced seizure among which, compound 10 with lipophilic and flexible aromatic moiety was more potent than the reference drug phenytoin and was the most potent in this series of phthalimide derivatives. In the MES model, the prepared phthalimide did not show efficient activity. The prepared compounds are active in clonic seizure.Anticonvulsant activity of phthalimide was discovered in 2000 by molecular hybridization of thalidomide and ameltolide. In our present research we report some new 4-substituted derivatives of phthalimide with good activity against the tonic and clonic seizures. A series of novel 4-flurophthalimides designed using bioisosteric replacement were synthesized by condensation of 4-flurophthalic anhydride with appropriate arylamines. The purity of these compounds was determined by TLC and the chemical structures were confirmed by IR and  Nitro mPreparation of 2-(2-chloro-4-nitrophenyl)-5-fluoro-1H-isoindole-1, 3(2H)-dione (2)1H-NMR (CDCl3): δ 7.974 , 8.299, 8.019, 7.677, 7.573, 7.511 ; IR (KBr): ν cm- , 3103 and 3074 (CH-aromatic), 1788 and 1730 (CO), 1526, 1481 and 1375, 1340 (NO2). Anal. Cal. For (C14H6ClFN2O4): C, 52.44; H, 1.89; N, 8.74; Found: C, 52.49; H, 1.90; N, 8.72.A solution of 4-flurophthalic anhydride  and 2-chloro-4-nitroaniline  in glacial acetic acid (2 mL) was stirred and heated under reflux for 120 h. After cooling to room temperature, cold water (5 mL) was added and the resulting precipitate was collected by filtration, washed with cold water and cold ethanol, respectively, and recrystallized from ethanol to give compound 2 as pale yellow crystals . TLC tank solvent was (dichloromethane/petroleum ether 2:1), m.p. 173-175 °C. Preparation of 5-fluoro-2-(2-methyl-3-nitrophenyl)-1H-isoindole-1, 3(2H)-dione (3)1H-NMR (CDCl3): δ 7.982-8.033, 7.665, 7.484-7.546, 2.358 ppm ; IR (KBr): ν cm- , 3095 and 3076 (CH-aromatic), 2923 , 1778 and 1722 (CO), 1531, 1475 and 1263, 1240 (NO2). Anal. Cal. For (C15H9FN2O4): C, 60.01; H, 3.02; N, 9.33; Found: C, 60.09; H, 3.03; N, 9.31.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 2-methyl-3-nitroaniline provided the title compound after 96 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a brown crystals ; m.p. 152-153 °C; Preparation of 5-fluoro-2-(2-methyl-4-nitrophenyl)-1H-isoindole-1,3 (2H)-dione (4)1H-NMR (CDCl3): δ 8.266, 8.205, 8.003, 7.66, 7.566, 7.513, 7.406, 2.337 ppm . IR (KBr): ν cm- , 3103 and 3076 (CH-aromatic), 2926 , 1780 and 1720 (CO), 1520, 1485 and 1375, 1344 (NO2). Anal. Cal. For (C15H9FN2O4): C, 60.01; H, 3.02; N, 9.33; Found: C, 60.07; H, 3.03; N, 9.34.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 2-methyl-4-nitroaniline provided the title compound after 96 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a white crystals ; m.p. 170-171 °C; Preparation of 5-fluoro-2-(2-methyl-6-nitrophenyl)-1H-isoindole-1, 3(2H)-dione (5)1H-NMR (CDCl3): δ 8.053, 7.988, 7.632-7.693, 7.566, 7.501, 2.334 ppm . IR (KBr): ν cm-1, 3072(CH-aromatic), 2923, 1782 and 1728 (CO), 1531, 1481 and 1267, 1238(NO2). Anal. Cal. For (C15H9FN2O4): C, 60.01; H, 3.02; N, 9.33; Found: C, 60.08; H, 3.01; N, 9.31.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 2-methyl-6-nitroaniline provided the title compound after 144 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a pale brown crystals ; m.p. 108-110 °C; Preparation of 2--5-fluoro-1H-isoindole-1, 3(2H)-dione (6)1H-NMR (CDCl3): δ 7.975 , 7.644 , 7.478 , 7.284 , 7.19, 2.156 ppm . IR (KBr): ν cm-1, 3101, 3074 and 3035 (CH-aromatic), 2932, 1776 and 1720 (CO). Anal. Cal. For (C16H12FNO2): C, 71.37; H, 4.49; N, 5.20; Found: C, 71.31; H, 4.51; N, 5.22.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 2, 6-dimethylnitroaniline provided the title compound after 72 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a white crystals ; m.p. 146.5-148 °C; Preparation of 2-benzonitrile (7)1H-NMR (CDCl3): δ 8.019, 7.853, 7.781, 7.678, 7.61, 7.464-7.565 ppm. IR (KBr): ν cm-1, 3067 (CH-aromatic), 2230(CN), 1788 and 1732 (CO). Anal. Cal. For (C15H7FN2O2): C, 67.67; H, 2.65; N, 10.52; Found: C, 67.60; H, 2.64; N, 10.55.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 2-aminobenzonitrile provide the title compound after 24 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a pale yellow crystals ; m.p. 193.5-196 ºC: Preparation of 2-(3-chlorophenyl)-5-fluoro-1H-isoindole-1,3 (2H)-dione (8)2, 4-flurophthalic anhydride and 3-chloroaniline provided the title compound after 72 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a white crystals ; m.p. 150.5-151.5 °C: 1H-NMR (CDCl3): δ 7.981, 7.643, 7.35-7.52 ppm . IR (KBr): ν cm-1, 3074 (CH-aromatic), 1778 and 1720(CO). Anal. Cal. For (C14H7ClFNO2): C, 61.00; H, 2.56; N, 5.08; Found: C, 61.09; H, 2.57; N, 5.06.Using a procedure similar to that of Preparation of 5-fluoro-2--1H-isoindole-1,3 (2H)-dione (9)1H-NMR (CDCl3): δ 7.942-8.056, 7.697, 7.446-7.641 ppm . IR (KBr): ν cm-1, 3110, 3072 and 3040 (CH-aromatic), 1778 and 1722(CO). Anal. Cal. For (C18H10FNO2): C, 74.22; H, 3.46; N, 4.81; Found: C, 74.18; H, 3.47; N, 4.83.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 1-naphthylamine provided the title compound after 72 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a violet crystals ; m.p. 188-191 °C: Preparation of 2-(diphenylmethyl)-5-fluoro-1H-isoindole-1, 3(2H)-dione (10)1H-NMR (DMSO-d6): δ 7.842-7.898, 7.166-7.7.541, 6.290 ppm . IR (KBr): ν cm-1, 3030 and 3034 (CH-aromatic), 1692 and 1647(CO). Anal. Cal. For (C21H14FNO2): C, 76.12; H, 4.26; N, 4.23; Found: C, 76.19; H, 4.28; N, 4.20.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 1,1-diphenylmethaneamine provided the title compound after 8 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a white crystals ; m.p. 254-256 °C: Preparation of 2-(biphenyl-2-yl)-5-fluoro-1H-isoindole-1, 3(2H)-dione (11)1H-NMR (CDCl3) : δ 7.819, 7.46-7.566), 7.256-7.409 ppm. IR (KBr): ν cm-1, 3106, 3067 and 3031 (CH-aromatic), 1778 and 1724(CO). Anal. Cal. For (C20H12FNO2): C, 75.70; H, 3.81; N, 4.41; Found: C, 75.65; H, 3.82; N, 4.44.Using a procedure similar to that of 2, 4-flurophthalic anhydride and biphenyl-2-amine provided the title compound after 100 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a white crystals ; m.p. 166-168 °C: Preparation of 5-fluoro-2-(4-methoxybiphenyl-3-yl)-1H-isoindole-1,3 (2H)-dione (12)1H-NMR (CDCl3) : δ 7.964 , 7.656, 7.650, 7.559, 7.391-7.485, 7.336, 7.129, 3.845 ppm . IR (KBr): ν cm-1, 3100 and 3057 (CH-aromatic), 1776 and 1722(CO), 1105(OCH3). Anal. Cal. For (C21H14FNO3): C, 72.62; H, 4.06; N, 4.03; Found: C, 72.68; H, 4.08; N, 4.06.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 4-methoxybiphenyl-3-amine provided the title compound after 72 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a white crystals ; m.p. 201-203 °C: Preparation of 5-fluoro-2-(4-phenoxyphenyl)-1H-isoindole-1, 3(2H)-dione (13)1H-NMR (CDCl3): δ 7.968, 7.633, 7.481 , 7.387-7.434, 7.370, 7.070-7.191 ppm . IR (KBr): ν cm-1, 3105, 3059 and 3034 (CH-aromatic), 1775 and 1709(CO). Anal. Cal. For (C20H12FNO3): C, 72.07; H, 3.63; N, 4.20; Found: C, 72.02; H, 3.62; N, 4.22.Using a procedure similar to that of 2, 4-flurophthalic anhydride and 4-phenoxyaniline provided the title compound after 48 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a white crystals ; m.p. 153-154.5 °C: Preparation of 2-(4-amino-2-chlorophenyl)-5-fluoro-1H-isoindole-1, 3(2H)-dione (14)1H-NMR (CDCl3): δ 7.957, 7.62, 7.458, 7.081, 6.850, 6.675 , 3.01 ppm. IR (KBr): ν cm-1, 3417 and 3338(NH2), 3220 (CH-aromatic), 1780 and 1722 (CO). Anal. Cal. For (C14H8ClFN2O2): C, 57.85; H, 2.77; N, 9.64; Found: C, 57.89; H, 2.76; N, 9.62.A suspension of compound 2 , isopropanol (8 mL), cyclohexene (2mL) and Pd/C 10%  in a 50 mL round-bottomed flask equipped with a reflux condenser was heated and stirred vigorously under reflux for 24 h . The reaPreparation of 2-(3-amino-2-methylphenyl)-5-fluoro-1H-isoindole-1, 3(2H)-dione (15)1H-NMR ( CDCl3 ): δ 7.966, 7.633, 7.465, 7.211, 7.012, 6.788 provided the title compound after 24 h as a light yellow crystals ; m.p. 219-221 °C: 3). IR (KBr): ν cm-1, 3441 and 3352(NH2), 3110 and 3045 (CH-aromatic), 2923 , 1772 and 1706 (CO). Anal. Cal. For (C15H11FN2O2): C, 66.66; H, 4.10; N, 10.37; Found: C, 66.71; H, 4.12; N, 10.35. J = 7.25Hz, 1H, H-4-phenyl), 2.053ppm -5-fluoro-1H-isoindole-1, 3(2H)-dione (16)1H-NMR (CDCl3): δ 7.943, 7.613, 7.443, 6.959, 6.657, 6.623, 2.80, 2.092ppm . IR (KBr): ν cm-1, 3439 and 3360(NH2), 3097 and 3077 (CH-aromatic), 2966 , 1776 and 1718 (CO). Anal. Cal. For (C15H11FN2O2): C, 66.66; H, 4.10; N, 10.37; Found: C, 66.62; H, 4.08; N, 10.39.Using a procedure similar to that of 14, compound 4  provided the title compound after 24 h as a light brown crystals ; m.p. 182.5-184 °C: Preparation of 2-(2-amino-6-methylphenyl)-5-fluoro-1H-isoindole-1, 3(2H)-dione (17)1H-NMR (CDCl3): δ 8.31, 7.930, 7.439-7.70, 7.065-7.45, 2.09 ppm . IR (KBr): ν cm-1, 3430 and 3355(NH2), 3095 and 3070 (CH-aromatic), 2923, 1761 and 1722, (CO). Anal. Cal. For (C15H11FN2O2): C, 66.66; H, 4.10; N, 10.37; Found: C, 66.63; H, 4.11; N, 10.39.Using a procedure similar to that of 14, compound 5  provide the title compound after 24 h as a yellow crystals ; m.p. 153-155 °C: PharmacologyThe test compounds were evaluated for anticonvulsant activities using maximal electroshock- (MES) and pentylenetetrazole- (PTZ) induced seizure. Male NMRI mice  were used for MES test. For PTZ-induced seizure, male wistar rats (150-200 g) were used. Animals were purchased from Pasture Institute  and were kept in controlled light and temperature condition  with free access to food and tap water. The test compounds were dissolved in DMSO and injected intraperitoneally at the dose of 40 mg/kg 30 min before seizure tests. The different test compound-treated groups were compared with the control group (which received DMSO) or phenytoin-treated group (40 mg/kg) as reference drug. The volume of injected drugs/vehicle was 10 mL/kg in mice and 1 mL/kg in rats. Anticonvulsant evaluation by MES test was performed as described previously . Electrogroup.Statistical AnalysisThe results of PTZ and MES test are presented as seizured/total and protected/total, respectively and the percent protection against seizure between the groups was analyzed by Chi-square test. P values less than 0.05 were considered as statistically significant. Chemistry:1H-NMR spectroscopy.A series of novel 2, 5-disubstituted phthalimides were synthesized in good yield according to the method that was mentioned previously -26. CondExistence of absorption bands of amide, nitro, aromatic amine groups in IR spectra approved preparing of our desired compounds. In H-NMR spectroscopy, the proton on the phthalic ring in nitro and amino derivatives are shifted downfield and upfield respectively as compared with 5-unsubstituted compounds.Pharmacology2, CN and Cl, respectively) in the phenyl ring increased the potency. Comparison of compounds 9, 10, 11, 12, and 13 which contain two aromatic rings in two different position of phthalimide showed that compound 10 with more lipophilic and routable aromatic ring is more active than others.The ability of the compounds 2-17 to protect rats against PTZ- and MES- induced seizure was evaluated and the results were presented in Results of MES test (as shown in in-vivo in mice. Pharmacological results indicated that all the prepared compounds were active in clonic seizure but in the tonic model did not show any efficient activity.Sixteen analogs of 4-flurophthalimid were synthesized, purified, and characterized by thin layer chromatography, IR, elemental analysis and H-NMR. Ability of prepared compounds to protect against PTZ induced seizure and MES were evaluated


The structure of these components was elucidated by using various spectral techniques and chemical reactions. Among the known isolates, syringaldehyde (12), 3-chloro-4-hydroxybenzoic acid (13), 3-chloro-4-hydroxyl benzoic acid-4-O-β-d-glucopyranoside (14), and isotachioside (15) were isolated from the Eurycoma genus for the first time. Further, the NMR data of 14 was reported here firstly. Meanwhile, the nitric oxide (NO) inhibitory activities of all compounds were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at 40 μM. As results, piscidinol A (6), 24-epi-piscidinol A (7), bourjotinolone A (10), and scopoletin (16) were found to play important role in suppressing NO levels without cytotoxicity. Furthermore, the Western blot method was used to investigate the mechanism of compounds 6, 7, 10, and 16 by analysing the level of inflammation related proteins, such as inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in LPS-stimulated RAW264.7 cells. Consequently, compounds 6, 7, 10, and 16 were found to significantly inhibit LPS-induced protein expression of IL-6, NF-κB and iNOS in NF-κB signaling pathway. Moreover, it was found that the protein expression inhibitory effects of 6, 7, and 16 exhibited in a dose-dependent manner. The mechanism may be related to the inhibition of the iNOS expressions through suppressing the IL-6-induced NF-κB pathway.Four new phenolic components, eurylophenolosides A ( As we know, inflammation is a very common and important pathological process. Although moderate inflammation is a benefit for the body, overreaction or a lack of it can cause some adverse reactions, and even lead to other deadly chronic disease such as cancer, Alzheimer’s disease, diabetes, and atherosclerosis . Thus, tAs a kind of immune cell distributed throughout the body, macrophages play a central role in the immune surveillance system . MacrophInflammatory response is closely related to inflammatory factors and inflammatory cells. Proinflammatory cytokines regulate the secretion of inflammatory factors. Cellular signalling pathways can regulate the transcription and synthesis of pro-inflammatory cytokines, which include Janus kinase-signal transduction and transcription activator (JAK-STAT), mitogen-activated protein kinase (MAPK), and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) pathways. Among them, NF-κB is the main signaling pathway .Lipopolysaccharides (LPS) can stimulate the acute inflammatory response of RAW 264.7 cells to release typical proinflammatory cytokines, such as tumor necrosis factor (TNF-α) and interleukin 6 (IL-6) . Then, pEurycoma longifolia Jack (Simaroubaceae family) is a wild shrub. As a commonly used medicine in Southeast Asian countries, it is mainly distributed in Malaysia, Vietnam, Thailand, and India. It is also known as one of the three national treasures of Malaysia, together with bird’s nest and tin. Its roots and root bark possess multiple biological functions such as male testosterone level increasement, anti-fatigue, hypertension, fever treatment +  in the HRESI-TOF-MS. Infrared (IR) spectrum of it showed characteristic absorptions of hydroxyl (3374 cm−1), aromatic ring , and glycosyl bond (1067 cm−1). Its 1H and 13C NMR spectra (3), 3.88 ]. The long-range correlation observations from δH 6.94  to δC 134.3 (C-4), 154.4 , 155.5 (C-1); δH 3.88  to δC 154.4 ; δH 3.79 (4-OCH3) to δC 134.3 (C-4) showed in HMBC spectrum , 5.66 , 6.60 ] displayed in its 1H NMR spectrum, the existences of one hexose and two pentoses were speculated. d-glucose was obtained when compound 1 was hydrolysed with 1 M HCl, which was identified by retention time and optical rotation using chiral detection by HPLC analysis + ]. The IR spectrum of it not only presented the characteristic absorptions of hydroxyl (3357 cm−1), aromatic ring , glycosyl bond (1067 cm–1), but also the characteristic absorption of unsaturated carboxyl (1704 cm−1). Comparing its 1H and 13C NMR spectra ], two more symmetrical aromatic proton [δ 7.68 ], as well as one more carbonyl [δC 166.7 (C-7’’’’)] signals displayed in the NMR spectra of 2 than 1. The presence of them were clarified by the long-range correlations from δH 7.68  to δC 120.1 (C-1’’’’), 143.0 (C-4’’’’), 148.7 , 166.7 (C-7’’’’); δH 3.77  to δC 148.7  observed in its HMBC spectrum, and the moiety was decuced to be 3,5-dimethoxy-4-hydroxybenzoyl. Finally, the correlation from δH 4.86, 4.90 (H2-5’’’) to δC 166.7 (C-7’’’’) indicated that 3,5-dimethoxy-4-hydroxybenzoyl connected with C-5’’’ of compound 1, and eurylophenoloside B (2) was formed.Eurylophenoloside B ( spectra  with tho3) was obtained as a white powder with negative optical rotation . HRESI-TOF-MS determination result [m/z 707.2521 [M + Na]+ ] revealed its molecular formula was C32H44O16. d-glucose was analyzed from its acid hydrolysis product , one 1,3,4,5-tetrasubstituted phenyl , two β-d-glucopyranosyls , along with two methoxyls . In addition to the 26 signals represented by the above mentioned moieties and functional groups, there were six more carbon signals displaying in its 13C NMR spectrum, which suggested compound 3 was one of phenylpropane glycoside. The correlations between δH 3.97 (H-8) and δH 2.73, 2.96 (H2-7), 3.69, 3.76 (H2-9) showed in the 1H 1H COSY spectrum suggested the presence of “-CH2-CH-CH2O-” moiety. Moreover, the exitence of “-CH=CH-CH2OH” moiety was clarified by the correlations between δH 6.30 (H-8’) and δH 4.33, 4.52 (H2-9’), 6.65 (H-7’). On the other hand, the coupling constant (J = 16.0 Hz) between H-7’ and H-8’ indicated that the two protons presented trans configuration. The planar structure was clarified by the long-range correlations observed from the followling proton to carbon pairs: δH 3.69 (3-OCH3), 6.56 (H-5) to δC 148.4 (C-3); δH 6.48 (H-6), 6.57 (H-2) to δC 145.4 (C-4); δH 2.73, 2.96 (H2-7) to δC 113.8 (C-2), 122.6 (C-6), 133.2 (C-1), 139.0 (C-5’); δH 3.97 (H-8) to δC 119.4 (C-6’), 133.2 (C-1), 139.0 (C-5’), 145.2 (C-4’); δH 6.92 (H-6’), 6.95 (H-2’) to δC 145.2 (C-4’); δH 3.83 (3’-OCH3), 6.95 (H-2’) to δC 153.5 (C-3’); δH 6.65 (H-7’) to δC 109.2 (C-2’), 119.4 (C-6’), 135.2 (C-1’); δH 4.68 (H-1’’) to δC 145.2 (C-4’); δH 4.37 (H-1’’’) to δC 70.8 (C-9’)  suggested the absolute configuration of it might be 8R  appeared. The substitute position of carboxyl was clarified by the long-range correlations from δH 7.49 (H-2’) to δC 122.6 (C-6’), 148.8 (C-4’), 153.2 (C-3’), 170.4 (C-7’)  . Moreovee method ,27 as th5–16 were identified.By comparing the spectroscopic data with those reported in literature, the known compounds 1–16, the effects of compounds 1–16 on LPS-stimulated NO release were measured through the Griess reaction in RAW264.7 cells , and 19 fractions (ELG1–ELG19) were yielded. ELG2 (779.5 mg) was separated by pHPLC , and ten fractions (ELG2-1–ELG2-10) were given. ELG2-8 (42.3 mg) was isolated by pHPLC  to yield 3-chloro-4-hydroxyl benzoic acid-4-O-β-d-glucopyranoside . ELG3 (410.1 mg) was purified by pHPLC  to gain isotachioside . ELG9 (3.2 g) was subjected to pHPLC  to produce eight fractions (ELG9-1–ELG9-8). ELG9-4 (923.6 mg) was purified by pHPLC  to yield eurylophenoloside A . ELG15 (1490.0 mg) was subjected to Sephadex LH-20 CC [MeOH-H2O ], and five fractions (ELG15-1–ELG15-5). ELG15-4 (283.3 mg) was isolated by pHPLC  to yield eurylolignanoside A . ELG15-5 was purified by pHPLC  to produce eurylolignanoside B . ELG16 (841.3 mg) was separated by Sephadex LH-20 CC [MeOH-H2O ] to give two fractions (ELG16-1–ELG16-2). ELG16-2 (633.4 mg) was isolated by pHPLC  to yield six fractions (ELG16-2-1–ELG16-2-6). ELG16-2-3 (33.7 mg) was further purified by pHPLC  to gain eurylophenoloside B .The 95% EtOH eluates (25.0 g) was subjected to ODS CC [MeOH-H2Cl2-EtOAc  → MeOH → MeOH + NH3·H2O] to produce thirteen fractions (ELE-1–ELE-13). ELE-6 (2.0 g) was separated by silica gel CC [PE-EtOAc ], and twelve fractions (ELE6-1–ELE6-12). ELE6-8 (102.6 mg) was purified by pHPLC  to give bourjotinolone B . Using the same separation condition, bourjotinolone A  was obtained from ELE6-10 (179.6 mg). ELE-7 (2.2 g) was subjected to silica gel CC [CH2Cl2-EtOAc  → MeOH] to yield twenty-one fractions (ELE7-1–ELE7-21). ELE7-4 (62.1 mg) was isolated by pHPLC  to give syringaldehyde  and scopoletin . ELE7-7 (81.9 mg) was separated by pHPLC  to yield 3-methoxy-4-hydroxybenzoic acid  and 3-chloro-4-hydroxybenzoic acid . ELE7-13 (131.8 mg) was purified by pHPLC  to give piscidinol A  and 3-episapeline A . ELE7-14 (76.2 mg) was isolated by pHPLC  to gain hispidol B  and 24-epi-piscidinol A .ELE (25.0 g) was subjected to silica gel CC + .2):Eurylophenoloside B ; UV λmax (MeOH) nm (log ε): 222 , 278 (3.85); IR (KBr) υmax 3357, 2932, 2886, 2837, 1704, 1603, 1506, 1462, 1423, 1379, 1335, 1275, 1225, 1129, 1076, 1041 cm−1; 1H NMR  and 13C NMR  see m/z 813.2438 [M + H]+ .3):Eurylolignanoside A ; UV λmax (MeOH) nm (log ε): 217 , 267 (3.86); CD  mdeg (λnm): −62.6 (225 nm), −23.1 (259 nm); IR υmax (KBr): 3382, 2923, 2881, 1712, 1586, 1515, 1461, 1423, 1376, 1314, 1273, 1228, 1154, 1073, 1040 cm−1; 1H NMR  and 13C NMR  see m/z 707.2521 [M + Na]+ .4):Eurylolignanoside B ; UV λmax (MeOH) nm (log ε) 232 , 284 (3.41); CD  mdeg (λnm): −15.1 (231 nm), −4.2 (291 nm); IR (KBr) υmax 3408, 2935, 2851, 1702, 1600, 1557, 1516, 1460, 1423, 1390, 1273, 1211, 1152, 1070, 1031 cm−1; 1H NMR  and 13C NMR  see m/z 509.1664 [M − H]− .3-Chloro-4-hydroxyl benzoic acid-4-O-βd-glucopyranoside- (14): White powder; 1H NMR : δ 8.47 , 7.67 , 8.21 , 5.80 , 4.38 , 4.37 , 4.32 , 4.15 , ; 13C NMR : δ 127.1 (C-1), 132.2 (C-2), 123.0 (C-3), 157.1 (C-4), 115.9 (C-5), 130.4 (C-6), 167.8 (C-7), 101.8 (C-1’), 74.7 (C-2’), 78.6 (C-3’), 71.0 (C-4’), 79.2 (C-5’), 62.3 (C-6’) data were firstly reported here; HRESI-TOF-MS m/z 333.0383 [M − H]− .1–4: The acid hydrolysis reactions of 1–4 were conducted by using the method reported previously [d-glucose  for 1–4 was identified by a comparison of their retention time and optical rotation with that of an authentic sample.Acid hydrolysis of eviously . As a reMTT and nitrite levels were measured on a BioTek Cytation five-cell imaging multi-mode reader ; protein bands were mixed with Enhanced Chemiluminescence ; protein bands were visualized with the Amersham imager 600 luminescent image analyzer .RAW264.7 cells were obtained from the cell center at the Chinese Academy of Medical Science; LPS and Dex were purchased from Sigma Chemical ; penicillin and streptomycin were purchased from Thermo Fisher Scientific ; dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Biological Industries ; nitric oxide fluorometric assay kit was purchased from Beyotime Biotechnology ; bicinchoninic acid protein assay kit was purchased from Thermo Fisher Scientific . Rabbit anti-IL-6 was purchased from Proteintech Group, Inc . Rabbit anti-NF-κB, iNOS, and β-actin were purchased from Abcam plc. . Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) was purchased from Zhongshan Goldbridge Biotechnology .2 at 37 °C. Cells were grown to 80% confluence and then seeded at 2 × 106 cells/mL density in 24-well plates incubated before treatment.RAW 264.7 cells were maintained in high glucose DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere containing 5% COMTT colorimetric assay was used to determine cell viability. In brief, RAW 264.7 cells were seeded in 24-well plastic plates and treated without or with test samples (40 μM) for 24 h, respectively. The culture condition was the same as 3.2.2. The medium was removed, and the cells were incubated with 0.5 mg/mL of MTT solution. After 4 h incubation, the supernatant was removed and formation of formazan. The absorbance at 490 nm was measured with a microplate reader.6 cells/mL RAW264.7 cells were seeded on a 24-well plate and incubated overnight. After 24h, the media were changed, which contained LPS (0.5 μg/mL) with or without tested compounds 1–16 (40 μM), as well as the positive drug DEX (1 μg/mL), and then, the cells were incubated for 24 h. The cell supernatant was collected to detect NO levels, and cells were harvested for protein analysis.Initially, 2 × 10As described previously , the RAWFinally, protein bands were mixed with enhanced chemiluminescence . Subsequently, the protein bands were visualized with the Amersham imager 600 luminescent image analyzer. The intensities of protein bands were quantified by Image J analysis software.p < 0.05 was considered to indicate statistical significance. One-way analysis of variance (ANOVA) and Tukey’s studentized range test were used for the evaluation of the significant differences between means and post hoc, respectively.Values are expressed as mean ± S.D. SPSS 17.0 was used to conduct the statistics of all the grouped data. 1) and B (2), eurylolignanosides A (3) and B (4), along with twelve known isolates were obtained from the 70% ethanol extract of E. longifolia roots by a combination of various chromatographic methods and spectral techniques. Among the known compounds, 12–15 were isolated from the Eurycoma genus for the first time. Furthermore, the NMR data of 14 was firstly reported here.In summary, four new phenolic acids, eurylophenolosides A (6, 7, 10, and 16 showed significant inhibitory activity on NO release from RAW264.7 cells. In order to make the mechanism of their anti-inflammatory activities clear, Western blot assays were conducted. As a result, all of them were found to inhibit the LPS-induced protein expression of IL-6, NF-κB, and iNOS in the NF-κB signaling pathway. Moreover, the protein expression inhibitory effects of 6, 7, and 16 were found to be in a dose-dependent manner. The mechanism may be related to inhibiting the IL-6-induced NF-κB pathway to suppress iNOS expressions. The experiment suggested terpenes and coumarins might also be the anti-inflammatory substances in E. longifolia roots, except for alkaloids [Compounds lkaloids .16 possessed the ability to down-regulate the protein expression level of IL-6 and NF-κB [6, 7, and 10 have been reported to possess in vitro anti-inflammatory activities [16, but also complemented the blank of exisiting literature on the anti-inflammation mechanisms of compounds 6, 7, and 10. Furtherly, the strong inhibitory activities of compound 6 indicated that it could be the drug candidates for inflammation-related diseases.On the other hand, acording to references, compound nd NF-κB , as wellnd NF-κB , which wtivities ,32, thei


H. oreadica reported the presence of a wide spectrum of complex limonoids and dihydrocinnamic acids. Our interest in the Rutaceae motivated a reinvestigation of H. oreadica, H. brasiliana and H. superba searching for other secondary metabolites present in substantial amounts for taxonomic analysis. In a continuation of the investigation of the H. oreadica, three new limonoids have now been isolated 9α-hydroxyhortiolide A, 11β-hydroxyhortiolide C and 1(S*)-acetoxy-7(R*)-hydroxy-7-deoxoinchangin. All the isolated compounds from the Hortia species reinforce its position in the Rutaceae. With regard to limonoids the genus produces highly specialized compounds, whose structural variations do not occur in any other member of the Rutaceae, thus, it is evident from limonoid data that Hortia takes an isolated position within the family. In addition, H. superba afforded the unexpected coumarin 5-chloro-8-methoxy-psoralen, which may not be a genuine natural product. Solid-state cross-polarisation/magic-angle-spinning 13C nuclear magnetic resonance, X-Ray fluorescence and Field-emission gun scanning electron microscopy experiments show that the Sephadex LH-20 was modified after treatment with NaOCl, suggesting that when xanthotoxin  was extracted from cleaning of the gel column, chlorination of the aromatic system occurred.Previous investigations of  H. oreadica reported the presence of a wide spectrum of complex limonoids and dihydrocinnamic acids , Murraya paniculata , and in Triphasia trifolia  ) at m/z 251.5 and ([M+2 + H+]) 253.5 for compound 4. Simulation of isotope ratio mass spectrum for C12H7O4Cl provided the differences in naturally-occurring isotopic abundances, which showed peaks similar to those found for compound 4, the [M+2 + H+] (m/z 253) peak about one-third the intensity of the molecular ion peak ([M + H+] m/z 251), confirming the presence of chlorine. Although, the structure of 4 has been described as being a synthetic product and the 1H-NMR data for both are in close agreement with the structure proposed 5-chloro-8-methoxy-psoralen .11β-Hydroxyhortiolide C (2). Amorphous white solid;  −251.27 ; IR (film) νmax 3019 (OH), 1733 (carboxyl group), 1637  cm−1, 1H-NMR , see 13C-NMR , see 3); COSY , HMBC : H-2→C-1, C-10, H-5→C-7, C-10, C-14, C-19, H-6a→C-5, C-7, H-6b→C-7, H-11→C-9, C-10, C-13, C-14, H-12→C-11, C-13, C-14, H-15→C-9, C-13, C-14, C-16, H-17→C-13, C-14, C-20, C-21, C-22, H3-18→C-12, C-13, C-14, C-17, H3-19→C-1, C-5, C-9, C-14, H-21→ C-20, C-22, C-23, H-22→C-20, C-21, C-23, H-23→C-20, C-22, H3-28→C-3, C-5, C-29, H3-29→C-3, C-5, C-28, H3-30→C-8, C-9, C-14, OMe→C-7, HO-11→C-9, C-12, HO-15→C-14, C-16; g-NOESY : irradiated H-17→H-15, H-5, H-11→Me-30, Me-19; HREIMS, 501.2119 , 523.1950 [M + Na+].*)-Acetoxy-7(R*)-hydroxy-7-deoxoinchangin1(S (3). Amorphous white solid;  −46.80 ; IR (film) νmax 3430 (OH), 1745 (carboxyl group) cm−1; 1H-NMR , see 13C-NMR , see 3); COSY , HMBC : H-1→1-OC(O)Me, C-8, C-19, H-2a/ H-2b→C-1, C-3, C-10, H-5→C-4, C-6, C-7, C-9, C-10, C-19, Me-28, Me-29, H-6a/H-6b→C-5, C-7, C-10, H-7→C-5, C-8, Me-30, H-9→C-1, C-5, C-8, C-10, C-14, C-19, Me-30, H-11a/H-11b→C-10, C-12, H-12a/H-12b→C-13, C-14, Me-18, H-15→C-8, C-14, C-16, H-17→C-12, C-13, C-14, C-20, C-21, C-22, Me-18, H3-18→C-12, C-13, C-14, C-17, H-19a/H-19b→C-1, C-3, C-5, C-9, C-10, H-21→C-20, C-22, C-23, H-22→C-20, C-21, C-23, H-23→C-20, C-21, C-22, H3-28→C-4, C-5, Me-29, H3-29→C-4, C-5, Me-28, H3-30→C-7, C-8, C-9, C-14; g-NOESY : irradiated H-1→H-5, H-9, H-9→H-5, Me-18, Me-30→Me-19, H-7; HREIMS, 533.2250 , 555.2088 [M + Na+], 571.1839 [M + K+].H. oreadica taproots appear to be a rich source of complex limonoids. By contrast, other organs of Hortia species have long been the subject of chemical investigation, which have found many typically rutaceous compounds but the limonoids are simple and less abundant than in the H. oreadica taproots. Therefore, all Hortia roots or taproots should be re-examined for limonoids.This study shows that Clearly further chemical investigations on the interaction of Sephadex LH-20 and sodium hypochlorite will be essential to better understand the structures of the products formed. Additional experiments by enzymatic hydrolysis to obtain the main oxidized components soluble for NMR and MS analysis are in progress. However, information on the risks to recover Sephadex LH-20 with NaOCl must be clarified immediately, to avoid publication of possible chlorinated compounds as a natural occurrence.


N-(thiophen-2ylmethyl)pyrimidin-4-amine (17), the 6-fluoromethyl analogue (20), and the 6-(2-fluoroethoxy) analogue (27), were labeled in useful yields and with high molar activities by treating the 6-hydroxy analogue (26) with [11C]iodomethane, [18F]2-fluorobromoethane, and [d2-18F]fluorobromomethane, respectively. 11C]172027[ were readily purified with HPLC and formulated for intravenous injection. These methods allow these radioligands to be produced for comparative evaluation as PET radioligands for measuring COX-2 in healthy rhesus monkey and for assessing their abilities to detect inflammation.Cyclooxygenase 2 (COX-2) is an inducible enzyme responsible for the conversion of arachidonic acid into the prostaglandins, PGG2 and PGH2. Expression of this enzyme increases in inflammation. Therefore, the development of probes for imaging COX-2 with positron emission tomography (PET) has gained interest because they could be useful for the study of inflammation in vivo, and for aiding anti-inflammatory drug development targeting COX-2. Nonetheless, effective PET radioligands are still lacking. We synthesized eleven COX-2 inhibitors based on a 2(4-methylsulfonylphenyl)pyrimidine core from which we selected three as prospective PET radioligands based on desirable factors, such as high inhibitory potency for COX-2, very low inhibitory potency for COX-1, moderate lipophilicity, and amenability to labeling with a positron-emitter. These inhibitors, namely 6-methoxy-2-(4-(methylsulfonyl)phenyl- Cyclooxygenase has two isoforms, COX-1 and COX-2, that exist in endoplasmic reticulum and produce prostaglandins G2 and H2 from arachidonic acid. Prostaglandins are important mediators of symptoms of inflammation, such as fever and pain ,4, inclu(NSAID). These drugs include celecoxib (Celebrex) celecoxib (11C]1[) [18F]celecoxib (18F]1[) [11C]2[ [18F]3[ [11C]rofecoxib (11C]4[) [18F]5[) [11C]6[) [11C]7[) [11C]8[) [18F]desbromo-DuP-697 (18F]9[) [18F]pyricoxib (18F]10[) [18F]101) , [18F]ce [[11C]2  and [18F [[18F]3 , [11C]ro([11C]4) ,23,24, a[[18F]5) , a diary[[11C]6) , a diary[[11C]7) , a diary[[11C]8) , and [18([18F]9) ,29. All [18F]10)  has a hel cancer  but no ol cancer .Ka expected to be in the range 7 to 9. These groups contribute strongly to high-affinity COX-2 binding but their high degree of ionization at physiological pH (7.4) counters brain entry. Nearly all the previously tested radioligands show COX-2 inhibitory potencies with IC50’s exceeding 5 nM whereas much higher affinity (indexed by 1/IC50) may be required for successful PET imaging.Successful PET radioligands for imaging proteins in living subjects must meet many requirements, among which high affinity and selectivity for the target protein, and moderate lipophilicity are the most critical . For ima18F]pyricoxib class in being based on a substituted pyrimidinyl ring . From our set of prepared 2-(4-methylsulfonylphenyl)pyrimidines, we ultimately selected and produced three candidate PET radioligands, 11C]172027172027carbon dioxide by reduction to [11C]methane and then high temperature iodination in a commercial apparatus, as previously described [26 with [11C]iodomethane under basic conditions in DMF at RT for 5 min gave 11C]17202-fluoro-1-bromoethane was prepared from 2-bromoethyl tosylate and NCA [18F]fluoride ion [26 with [18F]2-fluoro-1-bromoethane under basic conditions at 110 °C for 15 min enabled formulated 18F]20[ to be obtained in 8.4% yield from starting [18F]fluoride ion with a molar activity of 60 GBq/µmol.For ride ion . Treatmed2-18F]27fluorobromomethane, was prepared from [d2]dibromomethane and NCA cyclotron-produced [18F]fluoride ion, as previously described [26 with [d2-18F]fluorobromomethane, Cs2CO3, and 18crown-6 at 110 °C for 15 min gave d2-18F]27[ in 27% yield after HPLC purification and formulation for sterile intravenous injection from starting [18F]fluoride ion, and with a molar activity of 66 GBq/µmol.For escribed . This laescribed . Treatme26) or derived impurities  for 11C]17[ and d2-18F]27+: calcd. for: C13H12ClFN2O3S: 331.0319, found 331.0321 (331.0321 .4-Chloro-6-(methylthio)-2-(4-(methylsulfonyl)phenyl)pyrimidine) (16). Sodium methanethiolate  and THF (6.0 mL) were loaded into a Schlenk tube (25-mL volume) under argon. A solution of 11  in THF (1.0 mL) was then added dropwise. The Schlenk tube was sealed and heated at 50 °C for 3 h. The mixture was then quenched with water (10 mL), extracted with EtOAc (10 mL × 3) and washed with aq. NaHCO3 . The product was dried (MgSO4) and purified with flash chromatography  to give 16 as a white solid ; mp: 160.2–161.9 °C; 1H NMR (CDCl3): δ = 2.69 , 3.11 , 7.18 , 8.05 , 8.64 ; 13C NMR (CDCl3): δ = 173.07, 159.75, 142.66, 143.58, 141.11, 129.44, 127.60, 116.51, 44.49, 12.95; HRMS (ESI): m/z [M + H]+: calcd. for C12H11ClN2O2S 315.0029, found 315.0025.General procedure A (non-microwave method) (GP1). This method was based on that in reference + 408.1 (100); HRMS (ESI) m/z [M + H]+: calcd. for C18H18FN3O3S2: 408.0852, found 408.0856; HPLC . From GP1 (62%).6-Thiomethyl-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (21). GP2 on 16 and thiophen-2-ylmethanamine gave 21 as an off-white solid ; mp: 159.1–160.4 °C; 1H NMR (CDCl3): δ 2.58 , 3.08 , 4.81 , 5.24  6.21 , 6.97–6.98 , 7.03–7.04 , 7.24 , 8.00 , 8.62 ; 13C NMR (CDCl3): δ 169.10, 161.23, 161.11, 143.12, 141.08, 129.12, 127.26, 126.94, 125.87, 125.31, 44.56, 40.36, 30.94, 12.71; HRMS (ESI): m/z [M + H]+ calcd. for C17H17N3O2S3: 392.0561, found 392.0559; HPLC . From GP1 (56%).N-Benzyl-6-methoxy-2-(4-(methylsulfonyl)phenyl)pyrimidin-4-amine (22). GP2 on 12 and benzylamine gave 22 as a white solid (33% yield): mp 133.4–134.6 °C; lit. mp 124.0–126.5 °C + 356.1 (100); HPLC .> 265 °C ; 1H NMR terature ; m/z (%)6-Chloro-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (25). GP2 gave 25 as a white solid ; mp 170.0–171.2 °C; lit. mp 167.0–169.1 °C + calcd. for C17H16FN3O3S2: 394.0684, found: 394.0686; HPLC .4,6-Difluoro-2-(4-(methylsulfonyl)phenyl)pyrimidine (28). Compound 11 , potassium fluoride , and 18-crown-6  in MeCN (2 mL) were added to an oven-dried screw-cap tube under argon. The mixture was stirred at RT for 24 h, quenched with water (10 mL), and extracted EtOAc (10 mL × 3). The combined organic layers were washed with brine-water (1:1 v/v: 30 mL × 2) and the solvent was evaporated off under reduced pressure. The residue was purified with flash chromatography  to give 28 as an off-white solid ; mp: 164.5–166.2 °C; 1H NMR (CDCl3): δ = 3.11 , 6.57 , 8.08 , 8.63 ; 13C NMR (CDCl3): δ = 173.69, 171.12, 163.88, 143.58, 139.45, 129.67, 127.82, 91.50 (t), 44.41; 19F NMR (CDCl3): δ = −54.91; HRMS (ESI): m/z [M + H]+ calcd. for C11H8F2N2O2S: 271.0353, found 271.0347.6-Fluoro-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (29). Microwave reaction conditions in GP2 when applied to 28 with thiophen-2-ylmethanamine gave 29 as a white solid (53%); mp: 130.4–131.4 °C; 1H NMR (CDCl3): δ = 3.09 , 4.85 , 5.65 , 5.91 , 6.98–7.01 , 7.05 , 7.25 , 8.02 , 8.58 ; 13C NMR (CDCl3): δ = 169.86, 165.53, 162.86, 162.68, 142.20, 141.73, 140.34, 129.27, 127.41, 127.05, 126.17, 125.57, 44.50, 40.73; 19F NMR (CDCl3): δ = −65.17; HRMS (ESI): m/z [M + H]+ calcd. for C16H14FN3O2S2: 364.0590, found 364.0585; HPLC . From 28  with GP1 (47%).6-Chloro-N-methyl-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (30). Compound 25 , sodium hydride,  and DMF (1.0 mL) were added to a Schlenk tube (10-mL volume) under argon and left for 1 h. Iodomethane  was then added and the tube closed. After 24 h, the tube contents were evaporated to dryness and water (10 mL) was added. The mixture was then extracted with EtOAc (10 mL × 2). The combined organic layers were washed with brine-water (1:1 v/v: 20 mL × 2) and dried (MgSO4). The residue was purified with flash chromatography ; mp: 149.9–151.9 °C; 1H NMR (CDCl3): δ = 3.08 , 3.12 , 5.08 , 6.45 , 6.95–6.97 , 7.03 , 7.24 , 8.00 , 8.63 ; 13C NMR (CDCl3): δ = 162.63, 161.91, 160.67, 142.16, 142.06, 139.20, 129.33, 127.35, 126.74, 126.42, 125.66, 100.27, 47.95, 44.51 35.44. HRMS (ESI) m/z [M + H]+ calcd. for C17H16ClN3O2S2: 394.0451; found 394.0445. HPLC .IC50’s) of test compounds for these enzymes carbon dioxide (~85 GBq) was produced with a PETtrace cyclotron  according to the 14N11C reaction [NCA [reaction  by irrad11C]Iodomethane was produced from [11C]carbon dioxide via reduction to [11C]methane followed by vapor phase iodination [11C]carbon dioxide was delivered to a PETtrace MeI Process Module  through stainless tubing  over 3 min. The [11C]carbon dioxide was reduced to [11C]methane with hydrogen over heated nickel (360 °C) and trapped on molecular sieves (13X). Finally, the [11C]methane was recirculated over iodine at 720 °C to generate [11C]iodomethane, which was trapped on Porapak Q held in the recirculation path.Iodomethane was then released into the loop and held for 5 min at RT. 11C]17[ was isolated with reversed phase HPLC on a Luna C18 column  eluted with MeCN-water (55:45 v/v) at 6 mL/min (tR = 9.9 min) (The phenol precursor 9.9 min) A. The pr11C]17[ (tR = 5.4 min) was confirmed with reversed phase HPLC on a Luna C18 column  eluted with MeCN-water (60:40 v/v) at 2 mL/min (tR = 4.85 min (m/z) [M + H]+: 376]. The radiochemical purity of 11C]17[ exceeded 99%. 11C]17fluoride ion was produced with the 18O18F reaction by irradiating 18O-enriched water  with a proton beam  from a PETtrace cyclotron.NCA [18F]fluoride ion (~7.4 GBq) in [18O]water (0.4–0.65 mL) was delivered into a glass vial containing K 2.2.2  and potassium carbonate  in MeCN-H2O . This solution was transferred to a modified version [t-butanol  was added to the dry [18F]fluoride ion-K 2.2.2-K2CO3 complex, sealed and then heated at 95 °C for 15 min. The reaction vessel was then cooled to 35 °C. Nitrogen was used to carry the volatile [18F]2-fluorobromoethane through a silica gel SepPak Plus cartridge  and into a septum-sealed vial  containing phenol precursor 26 , Cs2CO3 , and 18-crown-6  in DMF (0.5 mL). The vial was then heated at 110 °C for 15 min. 18F]20[ was separated with reversed phase HPLC on a Luna C18 column  eluted with MeCN-H2O (40:60 v/v) at 6 mL/min (tR = 49.7 min) (18F]2020[ (tR = 10.1 min) were confirmed with reversed phase HPLC on a Luna C18 column  eluted with MeCN-water (50:50 v/v) at 2 mL/min  C. The [12 mL/min D. [18F]218F]fluoride ion (~7.4 GBq) in [18O]water (0.4–0.65 mL) was delivered into a glass vial containing K 2.2.2  and potassium carbonate  in MeCN-H2O . This solution was transferred to a modified version [2Br2 (100 µL) in MeCN (1.0 mL) was added to the dry [18F]fluoride ion-K 2.2.2-K2CO3 complex, which was then heated at 95 °C for 15 min. The vessel was then cooled to 35 °C. Nitrogen was used to transfer the volatile [d2-18F]fluorobromomethane through a series of four silica gel SepPak Plus cartridges  into a septum-sealed 1-mL vial  containing phenol precursor 26 , Cs2CO3  and 18-crown-6  in DMF (0.5 mL). The vial was sealed and heated at 110 °C for 15 min. The d2-18F]27[ was separated with reversed phase HPLC on a Luna C18 column  eluted with MeCN-water (40:60 v/v) at 6 mL/min (tR = 45 min) (d2-18F]2727172027[ were measured by a method that we have described previously [n-octanol and sodium phosphate buffer  phases were determined with a γ-counter.eviously . The rad17, 20, and 27, were identified from the 2-(4-methylsulfonylphenyl)-pyrimidine class as having promising physiochemical and pharmacological properties for development as PET radioligands for imaging COX-2 in monkey and human in vivo. Methods were established for satisfactorily labeling these ligands with positron-emitters for comparative PET imaging in monkey in vivo, including a neuroinflammation model [Three COX-2 inhibitors, on model . These r


The isolation was performed using bio-assay tracking experiments. The structures of compounds 1-5 were established by spectroscopic means. The antibacterial activities of the compounds were assessed against Escherichia coli, Bacillus subtilis, Micrococcus tetragenus and Pseudomonas fluorescens. Chrysosplenol-D (4) exhibited activities against all the four spoilage microorganisms.Four phenolics, salviaplebeiaside ( Vitex negundo var. cannabifolia (Verbenaceae) is a shrub growing mainly in Yangzi River basin of China , a 1,2,4-trisubstituted benzene , and a β-D-glucopyranosyl moiety . The high field region of the 1H-NMR spectrum gave a 4H singlet (δH 2.617), which was correlated with two carbon signals at δC 45.81 and 30.20 in the HSQC spectrum. The above information was characteristic of a phenylbutanone glucoside, containing a glucopyranosyl moiety, and an aromatic ring (A-ring) with a butanone substituent  in 1 could indeed be readily deduced. The key differences between the NMR spectra of 1 and those of vitexfolin C were the chemical shifts of the 1H- and 13C-NMR signals of the 2′, 5′ and 6′ positions. In the NMR spectra of 1, the chemical shifts of H-2′, C-2′ and C-5′ were upshifted to δH 3.446, δC 74.88 and δC 75.83 ppm, respectively, while those of H-5′, H-6′a, H-6′b and C-6′ were downshifted to δH 3.641, δH 4.575, δH 4.275, and δH 64.83 ppm, respectively, suggesting that the β-D-glucopyranosyl moiety in 1 was 1′,6′-disubstituted instead of 1′,2′-disubstituted as in vitexfolin C. By analysis on the reported phenylbutanone glucosides in Vitex spp., 1 was assumed to be a 1′-(A-ring),6′-(B-ring)-disubstituted glucopyranose -3-hydroxyphenyl}-2-butanone. The structure was previously reported as salviaplebeiaside from Salvia plebeian –  in negative mode. 1H- and 13C-NMR data, see Pale yellow gum. IR (KBr) m/z 417 [M + H]+, 415 [M – H]–; 1H-NMR (CDCl3) δ: 6.372 , 2.669 , 2.136, 2.113 , 1.243 , 0.872-0.846 ; 13C-NMR (CDCl3) δ: 146.3 (C-6), 145.9 (C-8a), 125.9 (C-8), 121.7 (C-7), 118.4 (C-4a), 112.3 (C-5), 75.6 (C-2), 40.2 (C-1'), 39.5 (C-11'), 37.5 , 32.9 , 31.6 (C-3), 28.0 (C-12'), 24.9 (C-10'), 24.5 (C-6'), 24.2 (C-2a), 22.8 and 22.7 (C-12'a and C-13'), 22.4 (C-4), 21.1 (C-2'), 19.8 , 11.9 .Yellow oil; ESI-MS m/z 447 [M + H]+, 445 [M – H]–; 1H-NMR (CDCl3) δ: 2.539 , 2.032 , 2.003 , 1.228 , 0.867-0.830 ; 13C-NMR (CDCl3) δ: 187.8 (C-4), 187.3 (C-1), 144.5 (C-6), 140.6 (C-5), 140.5 (C-3), 140.3 (C-2), 72.8 (C-3'), 42.4 (C-4'), 40.3 (C-2'), 39.4 (C-14'), 37.7 (C-6'), 37.5 (C-10'), 37.4 (C-12'), 32.7 , 29.7 (C-8'), 27.9 (C-15'), 26.7 (Me-3'), 24.9 (C-13'), 24.6 (C-9'), 22.8 (Me-15'), 22.7 (Me-15'), 21.5 (C-1'), 21.4 (C-5'), 19.8 (Me-7'，11'), 12.5 (Me-2), 12.4 (Me-2), 11.9 (Me-5).Yellow oil; ESI-MS m/z 361 [M + H]+, 383 [M + Na]+, 359 [M – H]–; 1H-NMR (DMSO-d6) δ: 7.56 , 7.45 , 6.91 , 6.80 , 3.93 , 3.81 , 3.76 ; 13C-NMR (DMSO-d6) δ: 178.1 (C-4), 158.5 (C-7), 156.0 (C-9), 151.7 (C-5), 151.5 (C-2), 149.3 (C-4'), 145.4 (C-3'), 137.6 (C-3), 131.5 (C-6), 120.6 (C-1'), 120.3 (C-6'), 115.7 (C-2'), 115.3 (C-5'), 105.5 (C-10), 91.2 (C-8), 60.0, 59.6.3 , 56.4 (7-OCH3).Yellow amorphous powder; ESI-MS m/z 455 [M + Na]+, 431 [M – H]–; 1H-NMR (CD3OD) δ: 7.71 , 6.81 , 6.47 , 6.38 , 4.81 , 4.07 , 3.78 , 3.65 , 3.40-3.29 ; 13C-NMR (CD3OD) δ: 184.0 (C-4), 166.2 (C-2), 165.0 (C-7), 162.8 (C-4'), 162.0 (C-5), 158.7 (C-9), 129.4 , 123.1 (C-1'), 117.0 , 109.2 (C-6), 105.2 (C-10), 103.9 (C-3), 95.3 (C-8), 82.6 (C-5"), 80.2 (C-3"), 75.3 (C-1"), 72.6 (C-2"), 71.8 (C-4"), 62.9 (C-6").Yellow amorphous powder; ESI-MS E. coli, B. subtilis, M. tetragenus, and P. fluorescens, which were obtained from the School of Basic Medical Sciences, Anhui Medical University, Hefei, P.R. China. The EtOH extract, PE and CHCl3 fractions, as well as fractions B and C were individually dissolved and diluted with DMSO to obtain serial concentrations of 100, 50 and 25 mg·mL-1. Six mm wide holes were bored with a sterilized steel borer into the Nutrient Agar Media  in the Petri dish inoculated with the test microorganism. The solution of the compound (60 μL) at a specific concentration was added into each of the holes. DMSO was used as the negative control. The plates were then incubated at 37 °C for 24 hours. The diameters of the inhibition zones were measured and recorded. The assays were performed three times in order to guarantee reproducibility of results  were individually dissolved and diluted with DMSO to obtain serial concentrations of 1000, 500, 250, 125 and 62.5 μg·mL-1 . The anti-bacterial assays were also performed by the hole plate diffusion method as described above. The inhibition zones around the holes were measured and the MIC, which was defined as the lowest concentration being able to inhibit any visible bacterial growth, was recorded. The assays were performed three times for statistical analysis (see Compounds ysis see .P < 0.05.The data in γ-tocopherol and α-tocoquinone from the genus Vitex. Compounds 1-5 were reported from investigated species for the first time. The apoptosis-inducing and antimalarial activities, as well as vascular relaxation effects of 4 had been reported [4 on Staphylococcus aureus, Cladosporium cucumerinum and Bacillus cereus were documented [4 on four spoilage microorganisms for the first time. It is known that P. fluorescens plays an important role in rotting of meat. The results of the present assays suggest chrysosplenol-D could be used as a potential antiseptic food additive. Furthermore, this compound might also be one of the key components to account for the medicinal usage of the plant. This is the first report on the isolation of salviaplebeiaside, reported ,14,15,16cumented ,17. Our V.negundo var. cannabifolia and their antiseptic activities were analyzed [P. fluorescens. In our previous work, the compositions of the essential oil from the aerial parts of analyzed . HoweverV.negundo var. cannabifolia, even though more chemical examinations have to be done for this plant.A characteristic of phenolic-rich high-polar fraction in the fruits of the investigated species was revealed by previous phytochemical studies . Our wor


On the other hand, it was demonstrated that the Csp3–H insertion leading to β-lactams can be effectively promoted by both Pd(0) and Pd(II) catalysts, the latter being most efficient. Insight into the reaction mechanisms involved in these transformations was provided by DFT calculations.The Pd-catalyzed intramolecular carbene C–H insertion of α-diazo-α-(methoxycarbonyl)acetamides to prepare oxindoles as well as β-lactams was studied. In order to identify what factors influence the selectivity of the processes, we explored how the reactions are affected by the catalyst type, using two oxidation states of Pd and a variety of ligands. It was found that, in the synthesis of oxindoles, ((IMes)Pd(NQ)) Arsp2–H bonds . D. D56]. D2(dba)3 and ((IMes)Pd(NQ))2 as catalysts for these insertion processes. Although both Pd(0) and Pd(II) catalysts can be used to promote Csp3–H insertion, some significant differences were identified. On the whole, the Pd(II) complexes proved to be more versatile for β-lactam formation despite not always giving the highest yield. In general, the resulting β-lactams were obtained in moderate to good overall yields (53–90%), usually as mixtures of cis and trans isomers, in transformations proceeding with high site selectivity, except when using 5f. In this particular case, the use of Pd(II) catalysts to promote its decomposition resulted in the formation of trans-7f (10–14%), together with major amounts of aldehyde 10. On the other hand, when ((IMes)Pd(NQ))2 was used, a complex reaction mixture was obtained, from which cis-7f (9%), trans-7f (10%), and aldehyde 10 (15%) were isolated, while no cyclization product was obtained in the presence of Pd2(dba)3.The examples in cis β-lactam to the more stable trans isomer took place during the chromatographic purification of the reaction mixtures, which explains the observed differences in cis/trans ratios before and after purification.Notably, partial or complete isomerization of the 2(dba)3 in the presence of phosphine ligands resulted in slower reaction rates. Thus, for example, treatment of 5a with Pd2(dba)3 (10 mol%) in the presence of (o-tolyl)3P (20 mol%) under otherwise the same reaction conditions gave a mixture of 6 (12%), cis-7 (22%), and trans-7 (26%) together with some unreacted α-diazoamide (10%) (result not included in the table).The effect of adding phosphine ligands to the Pd(0) catalyst was also explored. However, similarly to the oxindole-forming reactions, the use of Pd5k -catalyzed transformations -2-oxoazetidine-3-carboxylate (cis-7f). Amorphous orange solid. 1H-NMR  δ 1.30 , 2.95 , 3.40 , 4.18 , 4.83 , 6.65 , and 7.21 .6g).Methyl 2-tert-butyl-7-chloro-3-oxo-1H-2,3-dihydrocyclohepta[c]pyrrole-3a-carboxylate  δ 1.45 , 3.64 , 4.22 , 4.47 , 5.60 , 6.27–6.30 , 6.32 , and 6.61 .6g’).Methyl 2-tert-butyl-5-chloro-3-oxo-1H-2,3-dihydrocyclohepta[c]pyrrole-3a-carboxylate  δ 1.46 , 3.64 , 4.23 , 4.42 , 5.76 , 6.18–6.21 , and 6.38–6.40 .8h).Methyl 1-(3-cyanobenzyl)-5,5-dimethyl-2-oxopyrrolidine-3-carboxylate  δ 1.16 , 1.24 , 2.19 , 2.33 , 3.62 , 3.80 , 4.33 , and 4.55 .6i).Methyl 2-tert-butyl-8-methoxy-3-oxo-1H-2,3-dihydrocyclohepta[c]pyrrole-3a-carboxylate  δ 1.47 , 3.55 , 3.69 , 4.23 , 4.48 , 5.65 , 6.29–6.33 , 6.31 , and 6.38–6.45 . 13C-NMR  δ 27.6 (3 CH3), 49.4 (CH2), 52.8 (CH3), 55.1 (C), 57.5 (CH3), 99.8 (CH), 120.6 (CH), 122.5 (CH), and 126.9 (CH).8j).Methyl 1-(2-fluorobenzyl)-5,5-dimethyl-2-oxopyrrolidine-3-carboxylate  δ 1.14 , 1.25 , 2.15 , 2.31 , 3.61 , 3.82 , 4.46 , 4.55 , 7.00 , 7.09 , 7.19–7.25 , and 7.39 .8k).Methyl 1-(2-bromobenzyl)-5,5-dimethyl-2-oxopyrrolidine-3-carboxylate  δ 1.18 , 1.23 , 2.20 , 2.35 , 3.65 , 3.83 , 4.45 , 4.64 , 7.10 , 7.24–7.30 , 7.51 . 13C-NMR  δ 26.9 , 27.8 , 38.3 (CH2), 43.1 (CH2), 47.6 (CH), 52.9 (CH3), 60.0 (C), 122.6 (C), 128.0 (CH), 128.9 (CH), 129.2 (CH), 132.7 (CH), 137.1 (C), 170.1 (C), and 171.1 (C).8l).Methyl 1-(2-iodobenzyl)-5,5-dimethyl-2-oxopyrrolidine-3-carboxylate  δ 1.20 , 1.22 , 2.20 , 2.35 , 3.65 , 3.83 , 4.35 , 4.59 , 6.93 , 7.23 , 7.30 , and 7.79 .cis-12).Methyl cis-1-tert-butyl-2-oxo-4-(pyridin-4-yl)azetidine-3-carboxylate  δ 1.32 , 3.36 , 4.28 , 4.88 , 7.32-7.35 , and 8.61–8.65 . 13C-NMR  δ 28.2 (3 CH3), 52.2 (CH3), 55.4 (CH), 55.5 (C), 59.0 (CH), 121.1 (2 CH), 146.2 (C), 150.2 (2 CH), 162.5 (C), and 165.9 (C).12).Methyl trans-1-tert-butyl-2-oxo-4-(pyridin-4-yl)azetidine-3-carboxylate  δ 1.27 , 3.67 , 3.78 , 4.84 , 7.33 , and 8.64 . 13C-NMR  δ 28.2 (3 CH3), 53.0 (CH3), 55.2 (CH), 55.7 (C), 62.1 (CH), 121.6 (2 CH), 148.5 (C), 150.7 (2 CH), 161.7 (C), and 167.0 (C).sol, by means of single point (SP) calculations at the same level with a larger basis set, def2-TZVPP [≠ values given in the text were obtained from the Gibbs energy in solution, Gsol, which was calculated by adding the thermochemistry corrections, G − E, to the refined SP energies, Esol, i.e., Gsol = Esol + G − E. See All the calculations reported in this paper were performed with the Gaussian 09 suite of programs . Electrof2-TZVPP ,72, wherIn summary, in the present paper, we report a full account of our experimental and computational studies on the Pd-catalyzed intramolecular carbene C–H insertion of α-diazo-α-(methoxycarbonyl)acetamides to prepare oxindoles and β-lactams. We have explored how the reactions are affected by the substituents on the α-diazoamide moiety and by the catalyst type, exploring the use of both Pd(0) and Pd(II) catalysts.N-aryl acetamides selectively afforded oxindoles, starting from N,N-dialkyl acetamides, the corresponding β-lactams were obtained.The chemoselectivity of the Pd-catalyzed C–H insertion reactions of α-diazo acetamides is mainly governed by the nature of the substrates. Thus, while 2 can be used as an alternative to Pd2(dba)3 to catalyze the carbene CArsp2–H insertion.The results obtained in the annulation reactions to form oxindoles show that Pd(0) catalysts were much more efficient than Pd(II) catalysts and that ((IMes)Pd(NQ))3–H insertion leading to β-lactams. However, the Pd(II) complexes proved to be more versatile for β-lactam formation since the use of Pd(0) catalysts resulted in increased amounts of Buchner products and, in some cases, of the corresponding γ-lactams.On the other hand, both Pd(0) and Pd(II) catalysts can be used to promote Csp2 as the catalyst, both CArsp2–H and Csp3–H insertions involve similar stepwise reaction mechanisms, in which a palladium-mediated hydrogen migration is followed by a reductive elimination.Our computational studies show that when using ((IMes)Pd(NQ))


Hizikia fusiformis (Harvey) Okamura is an edible marine alga that has been widely used in Korea, China, and Japan as a rich source of dietary fiber and essential minerals. In our previous study, we observed that the methanol extract of H. fusiformis and its non-polar fractions showed potent protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase inhibition. Therefore, the aim of the present study was to identify the active ingredient in the methanol extract of H. fusiformis. We isolated a new glycerol fatty acid (13) and 20 known compounds including 9 fatty acids , mixture of 24R and 24S-saringosterol (4), fucosterol (5), mixture of 24R,28R and 24S,28R-epoxy-24-ethylcholesterol (6), cedrusin (14), 1-(4-hydroxy-3-methoxyphenyl)-2-[2-hydroxy -4-(3-hydroxypropyl)phenoxy]-1,3-propanediol (15), benzyl alcohol alloside (16), madhusic acid A (17), glycyrrhizin (18), glycyrrhizin-6’-methyl ester (19), apo-9′-fucoxanthinone (20) and tyramine (21) from the non-polar fraction of H. fusiformis. New glycerol fatty acid 13 was identified as 2--icosatrienoyloxy)propoxy)-7′-oxoheptanoyl)oxymethylpropenoic acid by spectroscopic analysis using NMR, IR, and HR-ESI-MS. We investigated the effect of the 21 isolated compounds and metabolites (22 and 23) of 18 against the inhibition of PTP1B and α-glucosidase enzymes. All fatty acids showed potent PTP1B inhibition at low concentrations. In particular, new compound 13 and fucosterol epoxide (6) showed noncompetitive inhibitory activity against PTP1B. Metabolites of glycyrrhizin, 22 and 23, exhibited competitive inhibition against PTP1B. These findings suggest that H. fusiformis, a widely consumed seafood, may be effective as a dietary supplement for the management of diabetes through the inhibition of PTP1B. Diabetes mellitus (DM) is a serious chronic disease and an important public health problem. DM occurs when the pancreas does not produce enough insulin or when the body cannot effectively use insulin. In 2014, 422 million adults worldwide had DM and the prevalence of DM has been rising steadily for the past three decades . This caHizikia fusiformis (Harvey) Okamura is an edible brown seaweed in the Sargassaceae family that is widely distributed in the northwest coasts of the Pacific Ocean . As shown in δC 173.2, C-1‴) of eicosatrienoic acid and carbonyl carbon  of heptane-1,7-dioic acid by HMBC spectrum, respectively. The detailed −1. Similarly, typical absorptions for acylglycerol and FA with aliphatic chains were detected in the FT-IR data: 3705.55-3680.48-3651.07 (O-H stretching), 3005.52-3022.39 (C-H olefins), 2957.79-2923.07-2892.7-2852.69 , 1737.07 (C=O stretching), and 1055.35-1033.18-1011.96 (C-O stretching) cmc 14.2, C-20‴), 12 methylenes , six olefinic carbons , and one carbonyl carbon at δC 173.2 in the 13C-NMR spectra and a methyl signal at δH 0.88, overlapping methylene protons between δH 1.25 and 2.31, and six olefinic protons (δH 5.36) in the 1H-NMR spectra explain the presence of eicosatrienoic acid. The 1H-NMR spectrum showed two methylene groups lying between three double bonds of eicosatrienoic acid at δH 2.79 (2H), which could be assigned to H-7‴ and H-10‴. The HMBC correlations of eicosatrienoic acid were also observed from H-5‴ to C4‴, from H6‴ to C-7‴, H-8‴ to C-7‴, H-9‴ to C-10‴, H-11‴ to C-10‴, H-12‴ to C-13‴, and from H-20‴ to C-18‴ and C-19‴. The geometry of the three double bonds in this FA moiety was presumed to be cis-form based on the 13C-NMR spectrum . The signals of carbons next to a double bond usually appear at δC 27 to 28 in a cis-configuration, whereas those of a trans-configuration appear at δC 32 to 33 -1,3- propanediol (15), respectively. The ra of 14 , one aryra of 15 , one phehed data ,28 estab13C-and 1H-NMR spectra of compounds 16–21 and previously published data -1,3-propanediol (15) (5.5 mg), respectively but-3-en -2-one  (1.6 mg), respectively + . 1H-NMR (400 MHz in CDCl3): 6.42 , 5.96 , 5.36 , 5.24 , 4.36 , 4.20 , 4.16 , 4.14 , 3.64 , 2.79 , 2.31 , 2.06 , 1.59 , 1.25 , 0.88 ; 13C-NMR (100 MHz in CDCl3): 173.6 (C1′ and C7′), 173.2 (C1‴), 170.2 (C1), 136.3 (C2), 130.4, (C12‴), 130.1 , 129.8 (C9‴), 128.3 (C8‴), 128.2 (C5‴), 70.1 (C2″), 69.3 , 62.7 (C1″), 34.4 (C2‴), 34.3 , 32.1 (C18‴), 29.8 , 29.7 (C5′), 29.6 (C16‴), 29.5 (C18‴), 29.4 (C8‴), 29.3 (C17‴), 29.2 (C4′), 27.4 (C4‴), 27.3 (C13‴), 25.8 , 25.1 , 25.0 , 22.8 (C19‴), 14.2 . See Compound 15: Yellowish powder; 1H-NMR (400 MHz in CD3OD): 7.00 , 6.88 , 6.87 , 6.83 , 6.75 , 6.70 , 4.85 , 4.00 , 3.87 , 3.67 , 3.55 , 3.46 , 2.58 , 1.79 ; 13C-NMR (100 MHz in CD3OD): 149.2 (C3′), 148.4 (C4′), 145.0 (C2″), 142.9 (C1″), 136.4 (C4″), 129.6 (C1′), 126.4 (C5′), 122.4 (C5″), 121.6 (C6′), 117.6 (C3″ and C6″), 111.9 (C2′), 80.0 (C2), 77.8 (C1), 62.2 (C3 and C9″), 56.4  35.6 (C8″), 32.4 (C7″).Compound Enzyme inhibition studies were carried out spectrophotometrically in a 96-well micro-plate reader using a procedure reported by Li et al. . AcarbospNPP as a substrate  -1-naphthyl}-L-alanyl)amino] pentyl}oxy)-2-naphthoic acid (compound A) (PDB ID: 1NNY), was used to generate the grid box for catalytic inhibition mode. The 3D structures of 5, 22, and 23 were downloaded from PubChem Compound (NCBI) with compound CIDs of 5281328, 12193680 and 10114, respectively. The 3D structures of 24R,28R epoxy-24-ethylcholesterol (6a), 24S,28R-epoxy-24-ethylcholesterol (6b), and 13 were generated by Chem3D pro . The structures of ligands were adjusted to neutral (pH 7.0) using MarvinSketch  and minimized using Chem3D pro. The results were visualized and analyzed using UCSF Chimera , Discovery Studio , and Ligplot+ .For docking studies, the crystal structure of the truncated form of PTP1B protein target (amino acid 1-282) was obtained from the RCSB Protein Data Bank (PDB) with the accession code 1T49 . The co-n = 3).All experiments were carried out in triplicate and repeated on three separate days. All data are expressed as the mean ± standard deviation (SD) (


Optical measurements, X-ray diffraction analysis, and density functional theory calculations revealed relationships between the photophysical properties and molecular structures of these triads in solution and in the solid state. The compounds emitted blue to green fluorescence in CH2Cl2 solution and in the solid state. Notably, compound 2 showed fluorescence with an absolute quantum yield of 0.17 in the solid state but showed no fluorescence in CH2Cl2. Our findings confirmed that the substituent adjacent to the disilane moiety affects the conformations and emission efficiencies of compounds in solution and in the solid state.A series of disilane-linked donor‒acceptor‒donor triads (D‒Si‒Si‒A‒Si‒Si‒D) was synthesized to investigate the effects of substituents on the photophysical properties. The triads were prepared by metal-catalyzed diiodosilylation of aryl iodides using a Pd(P( Compounds in which electron-donating and electron-accepting groups are connected by π-conjugated linkers have recently attracted much attention as luminescent molecules ,2. These3 structure, and σ‒π conjugated molecules probably adopt spatially expanded structures phenyl}-2,1,3-benzothiadiazole, which had the similar structure of 2, showed decreased photoluminescence in the solid state in comparison with solution state thiadiazole, 5,7-diiodo-2,3-dimethylthienopyrazine, and 7,7′-diiodo-4,4′-dibenzo[c]thiadiazole, were prepared using literature protocols .1H-NMR spectra, and CDCl3 (δ = 77.0) was used as an internal standard for the 13C-NMR spectra. Gas chromatography-mass spectrometry (GC-MS) spectra were recorded on a GC-MS system . Low- and high-resolution FAB (fast atom bombardment) mass spectra were measured with a high-resolution mass spectrometer . Absorption spectra were measured with a spectrophotometer  using a 1 cm path length quartz cell. Fluorescence spectra were measured with a spectrofluorometer . Fluorescence quantum yields were determined by the integrating sphere method on an absolute PL (photoluminescence) quantum yield measurement system . Optical measurements were conducted using 10−5 M 1–5 in solution. Fluorescence lifetimes were measured with a Hamamatsu Quantaurus-Tau C11367-02 spectrometer . Preparative gel permeation liquid chromatography was performed using a liquid chromatography system .Melting points were obtained on a melting point apparatus  and were uncorrected. NMR spectra were measured with an NMR spectrometer . Tetramethylsilane (δ = 0.00) was used as an internal standard for the 1 using the slow evaporation method at 277 K. Structural data for 1 are summarized in λ = 0.71075 Å) at 97 K. The structures were solved by direct methods using SHELXL-97 thiadiazole  and Pd(P(t-Bu)3)2  in dry toluene (20 mL) and the mixture was stirred for 8 days at 0 °C. The reaction mixture was quenched with water and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 three times. The combined organic layer was washed with brine and dried over sodium sulfate. The solvent was evaporated under reduced pressure, partially purified by column chromatography on silica gel (eluent: hexane/ethyl acetate = 25/1), and completely purified by recycling HPLC with a GPC column (eluent: CHCl3/Et3N = 99.5/0.5) to afford 1 as a pale yellow solid in 29% yield (186 mg).Under an argon atmosphere, 4,7-Bis-1,1,2,2-tetramethyldisilaneyl)benzo[c]thiadiazole (1). Yield: 29%. Colorless powder. Mp: 90.1–92.2 °C. 1H-NMR  δ 7.48 , 7.13 , 6.39 , 6.22 , 3.76 , 3.51 , 0.44 , 0.24 . 13C-NMR  δ 164.8 (Cq), 162.1 (Cq), 158.0 (Cq), 135.5 (CH), 135.4 (Cq), 134.6 (CH), 118.3 (Cq), 104.3 (CH), 97.0 (CH), 55.1 (CH3), 54.4 (CH3), −3.2 (CH3), −3.4 (CH3). FAB–LRMS m/z 640 (M+). FAB–HRMS Calcd for C30H44O4N2SSi4: 640.2099, Found: 640.2112 (M+).4,7-Bisdisilaneyl)benzo[c]thiadiazole (2). Yield: 26%. Colorless powder. Mp: 61.4–63.1 °C. 1H-NMR  δ 7.55 , 7.51 , 7.10–7.08 , 0.52 , 0.39 . 13C-NMR  δ 158.0 (Cq), 138.5 (Cq), 135.3 (CH), 134.3 (Cq), 134.2 (CH), 130.4 (CH), 128.0 (CH), −2.0 (CH3), −3.7 (CH3). FAB–LRMS m/z 532 (M+). FAB–HRMS Calcd for C22H32N2S3Si4: 532.0805, Found: 532.0792 (M+).5,7-Bis-1,1,2,2-tetramethyldisilaneyl)-2,3-dimethylthienopyrazine (3). Yield: 8%. Colorless powder. Mp: 87.1–89.0 °C. 1H-NMR  δ 7.18 , 6.42 , 6.29 , 3.78 , 3.61 , 2.51 , 0.45 , 0.28 . 13C-NMR  δ 165.0 (Cq), 162.1 (Cq), 150.1 (Cq), 149.6 (Cq), 136.4 (Cq), 135.8 (CH), 118.5 (Cq), 104.3 (CH), 97.0 (CH), 55.1 (CH3), 54.5 (CH3), 23.4 (CH3), −2.5 (CH3), −3.2 (CH3). FAB–LRMS m/z 668 (M+). FAB–HRMS Calcd for C32H48O4N4SSi4: 668.2412, Found: 668.2393 (M+).2,3-Dimethyl-5,7-bisdisilaneyl)thienopyrazine (4). Yield: 46%. Yellow oil. 1H-NMR  δ 7.56 , 7.19–7.15 , 2.57 , 0.51 , 0.42 . 13C-NMR  δ 151.0 (Cq), 150.1 (Cq), 138.7 (Cq), 135.7 (Cq), 134.4 (CH), 130.5 (CH), 128.2 (CH), 23.5 (CH3), −1.9 (CH3), −2.7 (CH3). FAB–LRMS m/z 560 (M+). FAB–HRMS Calcd for C24H36N2S3Si4: 560.1118, Found: 560.1108 (M+).7,7′-Bis(2-(4-methoxyphenyl)-1,1,2,2-tetramethyldisilaneyl)-4,4′-bibenzo[c]thiadiazole (5). Yield: 36%. Yellow oil. 1H-NMR  δ 8.23 , 7.57 , 7.31 , 6.82 , 3.78 , 0.52 , 0.36 . 13C-NMR  δ 160.0 (Cq), 159.4 (Cq), 152.9 (Cq), 135.7 (CH), 135.3 (Cq), 135.1 (CH), 130.3 (CH), 130.0 (Cq), 129.6 (Cq), 113.5 (CH), 55.0 (CH3), −3.1 (CH3), −3.5 (CH3). FAB–LRMS m/z 714 (M+). FAB–HRMS Calcd for C34H42O2N4S2Si4: 714.1826, Found: 714.1819 (M+).1–5 were designed and synthesized by Pd-catalyzed coupling of aryl iodides with hydrosilane precursors. The compounds were characterized by NMR spectroscopy, HRMS, and UV-vis absorption and fluorescence spectroscopy. XRD analysis of 1 and computational calculations of 1–5 furthered our understanding of the optical properties of these compounds and revealed several features underlying the relationship between the structure and the photophysical properties.Disilane-linked D/A/D compounds o-position against to Si‒Si unit of the donor moiety contrastively affected the photoluminescence properties of disilane-linked D/A/D molecules in solution and in the solid state. Molecules with a substituent at the o-position may suppress intramolecular motion such as internal rotation, leading to non-radiative decay, resulting in increased emission intensity in solution. However, in the solid state, the substituent at the o-position produces a twisted conformation and reduces intramolecular interaction between the donor and acceptor moieties, leading to decreased emission intensity. Compounds with no substituent at the o-position showed low emission intensity in the solution state, whereas their emission intensity was much higher in the solid state. Accordingly, compound 2 exhibited a high solid-state fluorescence quantum yield that was nearly two orders of magnitude larger than that in solution. These results indicate that substituents increasing emission efficiency in solution are not necessarily effective even in the solid state. In addition, these results are important findings in the molecular design of disilane-linked molecules whose reported examples are still limited.The substituent at the


N-(prop-2-ynyl)-benzenamine (I) in the presence of equimolar amounts of p-substituted benzyl derivatives, yields a mixture of mono- and dibenzylated 1,2,3-triazoles. When two equivalents of the benzyl derivative are added to the multicomponent reaction, the selective preparation of the dibenzylated compounds is achieved. The reactivity of the aniline N-H bond in monobenzylated 1,2,3-triazoles was tested by treatment with one equivalent of a p-substituted benzyl chloride at 40 °C, rendering the dibenzylated derivatives quantitatively.The pseudo-four component click synthesis of dibenzylated 1,2,3-triazoles derived from aniline is reported. The cycloaddition of sodium azide to  One-pot multicomponent reactions are increasingly important in areas such as organic chemistry and materials science because they offer several advantages over classical linear syntheses ,2,3. MulWithin the most conventional multicomponent processes, those based on the peculiar reactivity of isocyanides, such as the Doemling, Ugi and Passerini reactions are the most studied ,24,25,26N-benzylated 1,2,3-triazoles under mild conditions and the functionalization of the N-H bond in the aniline moiety simultaneously through a pseudo-four component click process.Most click processes for triazole synthesis are usually based on three component reactions ,46,47,48N-(prop-2-ynyl)benzenamine (I) by deprotonation of aniline with potassium carbonate in acetone at room temperature, followed by the addition of equimolar amounts of propargyl bromide. After work up and purification through column chromatography on silica gel, alkyne I was isolated in 79% yield according to The initial synthetic step involved the preparation of I and sodium azide was carried out in a 4:1 water/ethanol mixture using Cu(OAc)2 and sodium ascorbate as the reagents to render the Cu(I) active catalyst. After addition of benzyl chloride to the reaction mixture and stirring at room temperature for 18 h we observed the complete consumption of the alkyne. At our first intention, we had targeted the preparation of the N-benzylated 1,2,3-triazole 2a, however, TLC analysis of the reaction mixture showed two main products. After separation by column chromatography using CH2Cl2 and CH2Cl2–MeOH (98:2) as gradient mixtures, the two products were isolated. The 1H-NMR spectra of the second fraction showed two single peaks corresponding to the methylene groups and a triplet signal assigned to the NH bond from the aniline moiety. All the patterns were consistent with the structure of the expected triazole 2a. On the other hand, the spectrum of the first fraction displayed three different methylene moieties and the signal for the NH group no longer appeared. Careful analysis of the 1H and 13C spectra and the M+H [355.1923] ion of 1a in HRMS (ESI-TOF) confirmed the dibenzylated triazole structure of this molecule. Thus, to our delight, the preparation of triazole 1a resulted from a one-pot pseudo-four component click process.The click reaction between p-halobenzylic derivatives yielding the mono- and di-benzylated 1,2,3-triazoles 1b‒e and 2b–e, respectively. Likewise the use of several benzylic derivatives allows for the preparation of 1,2,3-triazoles which feature several halogen substituents. As depicted in The scope of the method was extended with the use of several 1a–f and 2a–f were conveniently characterized by FT-IR, 1H and 13C-NMR spectroscopy, and also by High Resolution Mass Spectrometry. The formation of the 1,2,3-triazoles was apparent by the presence in the 1H-NMR of the characteristic singlet due to the triazolyl protons in the δ = 7.99–8.02 ppm for 1a–e and δ = 7.98–8.01 ppm region for 2a–e, respectively.Triazoles For most of the series, the click reaction provides the dibenzylated products in higher yields with the exception of the reaction with 4-iodobenzyl chloride in which the monobenzylated triazole is the major product. This may be related to a deactivating effect on the N-H bond due to the presence of the large iodo atom.p-halogenated benzyl derivatives. Thus, the original process was optimized using 2.4 equivalents of the benzylated reactant and 24 h stirring at room temperature. As denoted in 1a–e in high yields.As the dibenzylated products were an interesting example of a pseudo-four component process, we focused next on optimization of their preparation. Considering that the dibenzylated products were favored in the original process, we envisioned that we could facilitate their selective preparation by increasing the equivalents of 1a–e, we targeted the synthesis of dibenzylated compounds containing mixed benzyl moieties. With this aim, the reaction was carried out in the presence of one equivalent of p-iodobenzyl chloride and one equivalent of p-bromobenzyl chloride and stirring for 24 h showing by TLC the presence of four compounds in similar ratios. Analysis by of the 1H and 13C suggested the presence of two mono- and two benzylated compounds, however, due to the complexity of the mixture no separation was possible.Once we had selectively prepared N-benzylated 1,2,3-triazoles contained a N-H bond, we decided to test its reactivity towards benzyl derivatives in order to obtain bifunctionalized species. Monitoring the addition of equimolar amounts of p-iodobenzyl chloride to triazole 2e at room temperature in chloroform, it was observed that after 24 h, only 5% conversion to the desired benzylated aniline was reached. After several trials, we observed that carrying out the reaction at 40 °C and 12 h of stirring resulted in the full consumption of the starting material. After separation by column chromatography on silica gel with CH3Cl as eluent, compound 1e was obtained as a waxy solid in 94% yield benzenamine (I) was synthesized as reported in the literature [4Si as an internal reference; coupling constants are given in Hertz. High-resolution mass spectra (HRMS) were recorded on a JEOL JMS-SX 102a and Agilent-MSD-TOF-1069A spectrometers 41.Commercially available reagents and solvents were used as received. terature . Flash c2·H2O , 1,10-phenanthroline monohydrate , and sodium L-ascorbate . After addition of a EtOH/H2O mixture , the resulting suspension was stirred for five minutes at room temperature. Subsequently, I , sodium azide , and benzyl chloride  were added to the reaction mixture, which was stirred during 18 h at room temperature. H2O (5 mL) was added to the reaction mixture and the precipitate was filtered off, washed thoroughly with H2O, petroleum ether, and dried under vacuum. The crude mixture containing mono- and dibenzylated products was separated by column chromatography and characterized as follows:To a 20 mL round-bottomed flask equipped with a magnetic stirrer, were charged Cu(OAc)N-Benzyl-N-methyl)benzenamine (1a). Using CH2Cl2 as eluent, the first fraction affords 169 mg (42% Yield) of 1a as a white solid. mp 104–106 °C. 1H-NMR (CDCl3): δ = 4.63 , 4.64 , 5.55 , 6.59 , 6.77 , 7.07–7.11 , 7.19–7.23 , 7.24–7.28 , 7.30–7.36 , 7.99 . 13C-NMR (CDCl3): δ = 45.9 (ArCH2Ntriazole), 52.6 (ArCH2N), 53.8 (NCH2C=C), 112.5 (CHar), 116.2 (CHar), 123.1 (CHtriazole), 126.5 (CHar), 126.6 (CHar), 127.6 (CHar), 127.9 (CHar), 128.3 (CHar), 128.6 (CHar), 128.8 (CHar), 136.0 (Car), 138.9 (Car), 144.7 (Ctriazole), 147.9 (Car). FT-IR/ATR νmax cm−1: 3116, 3084, 3064, 3027, 2960, 2920, 2885, 1726, 1596, 1527, 1506, 1494, 1450. HRMS (ESI-TOF) calculated for C23H22N4+H+: 355.1923; Found: 355.1908.N-methyl)benzenamine (2a). Using CH2Cl2-MeOH (99:1) as eluent, the second fraction affords 105 mg (35% Yield) of 2a as a white solid. mp 75–77 °C. 1H-NMR (DMSO-d6): δ = 4.29 , 5.57 , 6.01 , 6.54 , 6.61 , 7.05–7.08 , 7.28–7.36 , 7.99 . 13C-NMR (DMSO-d6): δ = 39.1 (ArCH2N), 53.2 (NCH2C=C), 112.8 (CHar), 116.5 (CHar), 123.3 (CHtriazole), 128.3 (CHar), 128.5 (CHar), 129.2 (CHar), 129.3 (CHar), 136.6 (Car), 146.5 (Ctriazole), 148.8 (Car). FT-IR/ATR νmax cm−1: 3401, 3104, 3058, 2950, 2920, 2893, 2848, 1599, 1507, 1493, 1454, 1439. HRMS (ESI-TOF) calculated for C16H16N4+H+: 264.1453; Found: 264.1459.The general procedure was followed using 1.40 mmol 4-fluorobenzyl chloride. The mixture of products was purified by column chromatography and characterized as follows:N-(4-Fluorobenzyl)-N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl-)methyl)benzenamine (1b). Using CH2Cl2 as eluent, the first fraction affords 178 mg (40% yield) of 1b as a white solid. mp 93–95 °C. 1H-NMR (CDCl3): δ = 4.60 , 4.63 , 5.54 , 6.61 , 6.76 , 7.07–7.10 , 7.15–7.17 , 7.24–7.27 , 7.30–7.33 , 7.99 . 13C-NMR (CDCl3): δ = 45.8 (ArCH2Ntriazole), 51.8 (ArCH2N), 53.1 (NCH2C=C), 112.6 (CHar), 115.0 , 115.4 , 116.4 (CHar), 123.0 (CHtriazole), 128.4 , 128.8 (CHar), 130.0 , 132.2 , 134.9 , 144.6 (Ctriazole), 147.8 (Car), 160.4 , 162.3 . FT-IR/ATR νmax cm−1: 3108, 3064, 2857, 2927, 2859, 1725, 1594, 1504, 1456, 1442, 1389. HRMS (ESI-TOF) calculated for C23H20F2N4+H+: 391.1734; Found: 391.1720.N-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl-)methyl)benzenamine (2b). Using CH2Cl2:MeOH (99:1) as eluent, the second fraction affords 100 mg (31% yield) of 2b as a white solid. mp 88–90 °C. 1H-NMR (DMSO-d6): δ = 4.27 , 5.56 , 6.01 , 6.54 , 6.63 , 7.05–7.08 , 7.17–7.21 , 7.35–7.38 , 8.00 . 13C-NMR (DMSO-d6): δ = 39.1 (ArCH2N), 52.3 (NCH2C=C), 112.8 (CHar), 115.9 , 116.5 (CHar), 123.2 (CHtriazole), 129.3 (CHar), 130.6 , 132.9 , 146.6 (Ctriazole), 148.9 (Car), 162.3 . FT-IR/ATR νmax cm−1: 3397, 3105, 3058, 3048, 2952, 2920, 2893, 2846, 1719, 1599, 1506, 1459, 1439. HRMS (ESI-TOF) calculated for C16H15FN4+H+: 283.1359; Found: 283.1364.The general procedure was followed using 1.40 mmol of 4-chlorobenzyl chloride. The mixture of products was purified by column chromatography and characterized as follows:N-(4-Chlorobenzyl)-N-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl-)methyl)benzenamine (1c). Using CH2Cl2 as eluent, the first fraction affords 188 mg (39% yield) of 1c as a white solid. mp 126–128 °C. 1H-NMR (CDCl3): δ = 4.61 , 4.65 , 5.55 , 6.61 , 6.75 , 7.09–7.12 , 7.22–7.25 , 7.32 , 7.40 , 8.02 . 13C-NMR (CDCl3): δ = 45.9 (ArCH2Ntriazole), 51.8 (ArCH2N), 53.2 (NCH2C=C), 112.6 (CHar), 116.4 (CHar), 123.1 (CHtriazole), 128.2 (CHar), 128.4 (CHar), 128.6 (CHar), 128.8 (CHar), 129.6 (CHar), 131.1 (Car), 132.7 (Car), 135.0 (Car), 138.0 (Car), 144.6 (Ctriazole), 147.7 (Car). FT-IR/ATR νmax cm−1: 3129, 3085, 3064, 3041, 2925, 1758, 1597, 1575, 1505, 1491, 1458, 1435. HRMS (ESI-TOF) calculated for C23H20Cl2N4+H+: 423.1143; Found: 423.1142N-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl-)methyl)benzenamine (2c). Using CH2Cl2:MeOH (99:1) as eluent, the second fraction affords 119 mg (35% yield) of 2c as a white solid. mp 83–85 °C. 1H-NMR (DMSO-d6): δ = 4.29 , 5.57 , 6.03 , 6.54 , 6.64 , 7.05–7.08 , 7.31 , 7.42 , 8.01 . 13C-NMR (DMSO-d6): δ = 39.1 (ArCH2N), 52.4 (NCH2C=C), 112.8 (CHar), 116.5 (CHar), 123.4 (CHtriazole), 129.1 (CHar), 129.3 (CHar), 130.2 (CHar), 133.2 (Car), 135.7 (Car), 146.6 (Ctriazole), 148.8 (Car). FT-IR/ATR νmax cm−1: 3427, 3322, 3123, 3066, 3046, 2947, 2917, 2849, 1600, 1509, 1490, 1458, 1427, 1409. HRMS (ESI-TOF) calculated for C16H15ClN4+H+: 299.1019; Found: 299.1063.The general procedure was followed using (1.40 mmol) of 4-bromobenzyl chloride. The mixture of products was purified by column chromatography and characterized as follows:N-(4-Bromobenzyl)-N-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl-)methyl)benzenamine (1d). Using CH2Cl2 as eluent, the first fraction affords 245 mg (42% yield) of 1d as a white solid. mp 131–133 °C. 1H-NMR (CDCl3): δ = 4.59 , 4.65 , 5.54 , 6.61 , 6.75 , 7.09–7.12 , 7.16–7.20 , 7.46 , 7.55 , 8.02 . 13C-NMR (CDCl3): δ = 46.0 (ArCH2Ntriazole), 51.9 (ArCH2N), 53.3 (NCH2C=C), 112.7 (CHar), 116.4 (CHar), 119.5 (Car), 121.3 (Car), 123.2 (Ctriazole), 128.8 (CHar), 128.9 (CHar), 129.9 (CHar), 131.2 (CHar), 131.6 (CHar), 135.5 (Car), 138.5 (Car), 144.7 (Ctriazole), 147.7 (Car). FT-IR/ATR νmax cm−1: 3139, 3043, 3012, 2977, 2911, 2935, 2800, 1596, 1515, 1499, 1436, 1425. HRMS (ESI-TOF) calculated for C23H20Br2N4+H+: 513.0133; Found: 513.0146.N-((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl-)methyl)benzenamine (2d). Using CH2Cl2:MeOH (99:1) as eluent, the second fraction affords 113 mg (29% yield) of 2d as a white solid. mp 114–116 °C. 1H-NMR (DMSO-d6): δ = 4.29 , 5.56 , 6.01 , 6.55 , 6.62 , 7.05–7.08 , 7.24 , 7.56 , 8.00 . 13C-NMR (DMSO-d6): δ = 39.0 (ArCH2N), 52.4 (NCH2C=C), 112.8 (CHar), 116.5 (CHar), 121.8 (Car), 123.4 (CHtriazole), 129.3 (CHar), 130.6 (CHar), 132.1 (CHar), 136.1 (Car), 146.6 (Ctriazole), 148.8 (Car). FT-IR/ATR νmax cm−1: 3492, 3398, 3320, 3123, 3045, 2944, 2887, 1600, 1510, 1487, 1460, 1426. HRMS (ESI-TOF) calculated for C16H15BrN4+H+: 344.0460; Found: 344.0465.The general procedure was followed using (1.40 mmol) of 4-iodobenzyl bromide. The mixture of products was purified by column chromatography and characterized as follows:N-(4-Iodobenzyl)-N-((1-(4-iodobenzyl)-1H-1,2,3-triazol-4-yl-)methyl)benzenamine (1e). Using CH2Cl2 as eluent, the first fraction affords 172 mg (25% yield) of 1e as a white solid. mp 138–140 °C. 1H-NMR (CDCl3): δ = 4.57 , 4.64 , 5.51 , 6.61 , 6.74 , 7.02–7.04 , 7.08–7.11 , 7.63 , 7.71 , 8.01 . 13C-NMR (CDCl3): δ = 45.9 (ArCH2Ntriazole), 52.0 (ArCH2N), 53.4 (NCH2C=C), 92.1 (Car), 94.2 (Car), 112.6 (CHar), 116.4 (CHar), 123.2 (Ctriazole), 128.8 (CHar), 129.0 (CHar), 129.9 (CHar), 135.8 (Car), 137.0 (CHar), 137.4 (CHar), 138.9 (Car), 144.6 (Ctriazole), 147.7 (Car). FT-IR/ATR νmax cm−1: 3130, 3062, 3024, 2955, 2922, 2905, 2855, 1597, 1504, 1482, 1456, 1431. HRMS (ESI-TOF) calculated for C23H20I2N4+H+: 606.9855; Found: 606.9856.N-((1-(4-Iodobenzyl)-1H-1,2,3-triazol-4-yl-)methyl)benzenamine (2e). Using CH2Cl2:MeOH (99:1) as eluent, the second fraction affords 195 mg (42% yield) of 2e as a white solid. mp 128–130 °C. 1H-NMR (DMSO-d6): δ = 4.27 , 5.52 , 5.99 , 6.54 , 6.62 , 7.04–7.07 , 7.08 , 7.72 , 7.98 . 13C-NMR (DMSO-d6): δ = 38.6 (ArCH2N), 52.4 (NCH2C=C), 94.2 (Car), 112.3 (CHar), 116.0 (CHar), 122.9 (CHtriazole), 128.8 (CHar), 130.1 (CHar), 135.9 (Car), 137.4 (Car), 146.1 (Ctriazole), 148.3 (Car). FT-IR/ATR νmax cm−1: 3452, 3390, 3303, 3121, 3039, 2947, 2854, 1598, 1504, 1483, 1458, 1428. HRMS (ESI-TOF) calculated for C16H15IN4+H+: 391.0375; Found: 391.0420.2·H2O , 1,10-phenanthroline monohydrate , and sodium L-ascorbate . After addition of a mixture of EtOH/H2O , the resulting suspension was stirred for five minutes at room temperature. Subsequently, I , sodium azide , and benzyl chloride  were added to the reaction mixture, which was stirred during 24 h at room temperature. H2O (5 mL) was added to the reaction mixture and the precipitate was filtered off, washed thoroughly with H2O, petroleum ether, and dried under vacuum. The dibenzylated products 1a–e were purified by column chromatography using CH3Cl as eluent.To a 20 mL round-bottomed flask equipped with a magnetic stirrer, were charged Cu(OAc)N-(4-Fluorobenzyl)-N-((1-(4-iodobenzyl)-1H-1,2,3-Triazol-4-yl-)methyl)benzenamine (3). In a 20 mL round-bottomed flask equipped with a magnetic stirrer, p-fluorobenzyl chloride  was added to a solution of 2e  in CH2Cl2 (10 mL) and the reaction mixture was allowed to stir for 12 h at 40 °C. The mixture was dried under vacuum and the residue was purified by column chromatography using CH3Cl as eluent to give 114 mg (90% yield) of the title product as a waxy solid; mp 86–88 °C. 1H-NMR (CDCl3): δ = 4.56 , 4.68 , 5.41 , 6.76 , 6.78 , 6.95 , 6.99 , 7.19–7.21 , 7.27 , 7.71 . 13C-NMR (CDCl3): δ = 46.6 (ArCH2Ntriazole), 53.5 (ArCH2N), 54.0 (NCH2C=C), 94.5 (Car), 113.2 (CHar), 115.4 , 117.6 (CHar), 121.6 (CHtriazole), 128.4 , 129.3 (CHar), 129.6 (CHar), 134.0 , 134.3 (Car), 138.2 (CHar), 146.1 (Ctriazole), 148.3 (Car), 161.9 . FT-IR/ATR νmax cm−1: 3119, 3075, 3037, 3023, 2979, 2917, 1729, 1594, 1503, 1484, 1458, 1440. HRMS (ESI-TOF) calculated for C23H20IFN4+H+: 499.0717; Found: 499.0788.N-(4-Iodoobenzyl)-N-((1-(4-iodobenzyl)-1H-1,2,3-Triazol-4-yl-)methyl)benzenamine (1e). The above procedure was followed using 4-iodobenzyl chloride (0.281 mmol). The title product  was obtained as a waxy solid after purification by column chromatography using CH3Cl as eluent.1a–e was achieved by addition of excess of p-halogenated benzyl derivatives in a pseudo-four component click process. Particularly appealing is the reactivity of the available N-H bond in the N-benzylated triazoles 2a–e which may be tunable with a variety of functional groups. The possible applications of the synthetized 1,2,3-triazoles are the topic of current investigation in our laboratory.In conclusion, we have reported a one-pot methodology for the synthesis of a series of mono-, and dibenzylated 1,2,3-triazoles utilizing “click” chemistry. The selective preparation of dibenzylated triazoles


In a previous study, we have found a significant effect of certain derivatives on the ABCB1 transporter mediated multi-drug resistance of a transfected murine leukemia cell line. In this paper, we present a structure-activity relationship study focused on the apolar dioxolane derivatives of 20-hydroxyecdysone. Semi-synthesis and bioactivity of a total of 32 ecdysteroids, including 20 new compounds, is presented, supplemented with their complete  Ajuga, Serratula and Silene spp., containing high amounts of these compounds, are good sources of ecdysteroids of herbal origin +, 531 [M+H-H2O]+, 445, 427, 409.29α-I-butyl-29β-methyl-20,22-O-methylidene-20-hydroxyecdysone (6): white needle-like crystals; mp 198–199 °C; for 1H- and 13C-NMR data, see m/z : 563 [M+H]+, 545 [M+H-H2O]+, 445, 427, 409.29α-E-ethenylbenzyl-20,22-O-methylidene-20-hydroxyecdysone (9): white solid; mp. 161–163 °C; for 1H- and 13C-NMR data, see J = 16.0 Hz) proved the E configuration of the double bond; APCI-MS, m/z : 595 [M+H]+, 577 [M+H-H2O]+, 445, 427, 409.29α-Z-ethenylbenzyl-20,22-O-methylidene-20-hydroxyecdysone (10): white solid; mp 138–140 °C; for 1H- and 13C-NMR data, see m/z : 595 [M+H]+, 577 [M+H-H2O]+, 445, 427, 409.29α-(3-Methoxy-4-hydroxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (11): white solid; mp 163–165 °C; for 1H- and 13C-NMR data, see m/z : 615 [M+H]+, 597 [M+H-H2O]+, 445, 427, 409.29β-(3-Methoxy-4-hydroxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (12): white solid; mp 157–159 °C; for 1H- and 13C-NMR data, see m/z : 615 [M+H]+, 597 [M+H-H2O]+, 445, 427, 409.29α-(4-Benzyloxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (13): white solid; mp 144–146 °C; for 1H- and 13C-NMR data, see m/z : 675 [M+H]+, 445, 427, 409.29β-(4-Benzyloxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (14): white solid; mp 139–141 °C; for 1H- and 13C-NMR data, see m/z : 675 [M+H]+, 445, 427, 409.20-Hydroxyecdysone 2,3-acetonide (16): white solid; mp 124–126 °C; for 1H- and 13C-NMR data, see m/z : 553 [M+H+MeOH]+, 535, 503, 485, 467, 409.28α,29α-Diethyl-28β,29β-dimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (17): white solid; mp 98–100 °C; for 1H- and 13C-NMR data, see m/z : 589 [M+H]+, 571 [M+H-H2O]+, 499, 481, 409.28α,29α-Dimethyl-28β-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (18): white solid; mp 99–101 °C; for 1H- and 13C-NMR data, see m/z : 561 [M+H]+, 543 [M+H-H2O]+, 499, 481, 409.28β,29β-Dimethyl-29α-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (19): white solid; mp 79–81 °C; for 1H- and 13C-NMR data, see m/z : 561 [M+H]+, 543 [M+H-H2O]+, 471, 453, 409.29α-Ethyl-29β-methyl-20,22-O-methylidene-20-hydroxyecdysone (20): white solid; mp 140–142 °C; for 1H- and 13C-NMR data, see m/z : 535 [M+H]+, 517 [M+H-H2O]+, 445, 427, 409.28β,29α-Dibutyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (22): transparent crystals; mp 186–187 °C; for 1H- and 13C-NMR data, see m/z : 617 [M+H]+, 599 [M+H-H2O]+, 513, 495, 409.28β,29,29-Trimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (26): white solid; mp 100–102 °C; for 1H- and 13C-NMR data, see m/z : 547 [M+H]+, 517, 499, 467, 409.28α,29,29-Trimethyl-28β-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (27) white solid; mp 360–362 °C; for 1H- and 13C-NMR data, see m/z : 575 [M+H]+, 557 [M+H-H2O]+, 499, 481, 409.28,28,29β-Trimethyl-29α-i-buthyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (30): transparent solid; mp 114–115 °C; for 1H- and 13C-NMR data, see m/z : 603 [M+H]+, 585 [M+H-H2O]+, 485, 467, 409.28,28-Dimethyl-29α-phenyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (31): white solid; mp 114–117 °C; for 1H- and 13C-NMR data, see m/z : 641 [M+H+MeOH]+, 623, 517, 485, 467, 409.28,28,29β-Trimethyl-29α-phenyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (32): white solid; mp 126–128 °C; for 1H- and 13C-NMR data, see m/z : 623 [M+H]+, 605 [M+H-H2O]+, 485, 467, 409.28β-Propyl-29,29-dimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (33): transparent solid; mp 109–111 °C; for 1H- and 13C-NMR data, see m/z : 575 [M+H]+, 557 [M+H-H2O]+, 499, 481.Each compound was dissolved in 99.5% DMSO . In each protocol DMSO was always tested as solvent control and no activity was observed.2 atmosphere [Two mouse lymphoma cell lines were used in this work: a parental (PAR) cell line, L5178 mouse T-cell lymphoma cells ; and a multi-drug resistant (MDR) cell line derived from PAR by transfection with pHa MDR1/A retrovirus [mosphere . Medium,3 cells/well were incubated with serial dilutions of each compound (n = 3) in McCoy’s 5 A medium for 72 h at 37 °C, 5% CO2. Then, MTT (Sigma) [50) were calculated using non-linear regression curve fitting of log(inhibitor) vs. response and variable slope with a least squares (ordinary) fit of GraphPad Prism 5 software .Anti-proliferative activities on PAR and MDR cell lines were performed as described before . Briefly (Sigma)  was adde6 cells/mL were treated with 2 and 20 μM of each compound. After 10 min incubation, rhodamine 123 (Sigma) was added to a final concentration of 5.2 μM and the samples were incubated at 37 °C in water bath for 20 min. Samples were centrifuged  and washed twice with phosphate buffer saline . The final samples were re-suspended in 0.5 mL PBS and its fluorescence measured with a Partec CyFlow flow cytometer . Verapamil  at 20.4 μM was used as positive control.Inhibition of ABCB1 was evaluated using rhodamine 123, a fluorescent dye, which retention inside the cells was evaluated by flow cytometry (14). Briefly, 2 × 104 cells/well were incubated with doxorubicin and the compound to be tested for 48 h at 37 °C under 5% CO2. Cell viability rate was determined through MTT staining, as described above. The interaction was evaluated using the CompuSyn software  at each constant ratio of compound vs. doxorubicin (M/M), and combination index (CI) values were obtained for 50%, 75%, and 90% of growth inhibition.The combined activity of doxorubicin  and the ecdysteroids was determined using the checkerboard microplate method, as described before . BrieflyIn the present study, we have prepared 32 semi-synthetic derivatives of 20-hydroxyecdysone, following our previously observed structure-activity relationships on the strong synergistic activity of ecdysteroid dioxolanes with doxorubicin on a murine MDR cancer cell line expressing the human ABCB1 transporter. By utilizing the different reactivity of the 2,3 and 20,22 vicinal diol moieties, various bis-homo- and bis-hetero-dioxolanes were synthesized, as well as several 20,22- and two 2,3-monodioxolane derivatives. In addition to these, two epimer pairs were also obtained. Twenty compounds are reported for the first time; their chemical structures were thoroughly investigated by comprehensive 1 and 2D-NMR methods, based on which complete signal assignments are provided.The compounds showed mild to very strong synergistic effects with doxorubicin against the aforementioned MDR cancer cell line, and the diversity of the substituents allowed us to observe several new structure-activity relationships. Among these, the importance of the 2,3-dioxolane substitution and the observations concerning the role of stereochemistry at C-28 and C-29 are the most interesting results. Apparently, ecdysteroids can be engineered to become strong MDR modulators only by decreasing the polarity at the A-ring, while the polar side-chain can be kept, providing the possibility for designing such compounds with a reasonable water solubility and high drug-likeness.per os application of these compounds requires an appropriate formulation; development of such delivery systems is currently in process, investigation on their activity against MDR cancer xenografts is going to be reported in the near future.Considering the high importance of the 2,3-dioxolane group in our compounds and the fact that exactly this part is the most sensitive to an acidic environment,