The discovery of the G protein-coupled estrogen receptor GPER  and the resulting development of selective chemical probes have revealed new aspects of estrogen receptor biology. The potential clinical relevance of this receptor has been suggested from numerous studies that have identified GPER expression in breast, endometrial, ovarian and other cancers. Thus GPER can be considered a candidate biomarker and target for non-invasive imaging and therapy. We have designed and synthesized a series of organometallic tricarbonyl-rhenium complexes conjugated to a GPER-selective small molecule derived from tetrahydro-3H-cyclopenta[c]quinoline. The activity and selectivity of these chelates in GPER-mediated signaling pathways were evaluated. These results demonstrate that GPER targeting characteristics depend strongly on the structure of the chelate and linkage. Ethanone conjugates functioned as agonists, a 1,2,3-triazole spacer yielded an antagonist, and derivatives with increased steric volume exhibited decreased activities. Promising GPER selectivity was observed, as none of the complexes interacted with the nuclear estrogen receptors. Radiolabeling with technetium-99m in aqueous media was efficient and gave radioligands with high radiochemical yields and purity. These chelates have favorable physicochemical properties, show excellent stability in biologically relevant media, exhibit receptor specificity and are promising candidates for continuing development as diagnostic imaging agents targeting GPER expression in cancer. Estrogens are involved in a diverse array of physiological responses. The genomic roles of the nuclear estrogen receptors ERα and ERβ have been characterized in greatest detail. ERα, and to some extent ERβ, are important drug targets because of their roles in development, reproduction, skeletal physiology and the nervous, cardiovascular, and immune systems. Estrogen also rapidly triggers a variety of secondary messenger (non-genomic) signaling events that contribute to complex physiological, morphological and behavioral effects. Estrogen-responsiveness constitutes a major determinant of therapy selection and prognosis in breast cancer with both genomic and non-genomic pathways regulating tumor biology G-1, a tetrahydro-3H-cyclopenta[c]quinoline, and the structurally related antagonists G15 and G36, all of which exhibit high affinity for GPER and high selectivity for GPER over ERα/β  chelates to determine the effect of ionic charge on GPER targeting in vitro. The neutral DOTA conjugate 4 was capable of activating rapid GPER-mediated signaling pathways while charged complexes were inactive. Rapid initiation of GPER-mediated signaling by neutral, but not charged, synthetic estrogen probes has also been demonstrated 4 exhibited receptor-mediated uptake in the uterus, mammary glands, adrenal glands and tumor with the tumor being clearly visualized, although high uptake in the intestines, hepatobiliary excretion and the formation of polar metabolites limit the usefulness of this agent. These studies have identified the need for alternative radiolabeling modalities to improve intracellular targeting properties and obtain enhanced in vivo performance.We recently described the generation of the first radiolabeled agents based on the GPER-targeting tetrahydro-3H-cyclopenta[c]quinoline ( = “G”) scaffold of nd 111In  [14], + provides a promising new class of GPER-targeted SPECT imaging agents.In this study, we describe the synthesis of a promising new class of GPER-targeted tricarbonyl-Re(I) chelates 5–10 , and pro5–10) using the tetrahydro-3H-cyclopenta[c]quinoline scaffold shared by G-1 and G15. We have previously described an efficient, diastereoselective synthetic route for G-1 5,7–9) 6) 10) We designed a series of six new GPER-targeted organorhenium probes triflate-mediated removal of the silyl group  reaction ; however, attempts to improve this using the scandium catalyst Sc(OTf)3 were unsuccessful due to competing chelation by the hydrazine-2-yl pyridine. Removal of the tbutyl-protecting groups using TFA, gave the ethane-linked pyridin-2-yl-hydrazinylethanoic acid ligand 18.Facile palladium-catalyzed hydrodebromination of the bromo-benzodioxole group complicated direct attempts to prepare the ethane-bridged chelate ligand reaction . The paltBoc-protected 5-bromopyridin-2-yl-hydrazinylethanoate derivative 11 was converted to the boronic ester using PdCl2(dppf) and bis-pinacolatodiboron 3 and gave 22 in high yield 22 with the protected 5-ethynyl derivative 12 in aqueous tert-butanol gave the triazole-linked derivative in moderate yield. Deprotection with TFA, followed by silica gel chromatography yielded the triazole-linked pyridin-2-yl-hydrazinyl ligand 23. The “click” to chelate approach was used for the coupling of 22 with the protected N-propargyl-glycine derivative 24tbutyl-groups with TFA gave the 1,2,3-triazol-4-yl-methylaminoethanoic acid ligand 25.The synthetic approaches used for the preparation of triazole ligands diate 22 . The aza3(H2O)3 in aqueous ethanol as shown in 3 at 90°C showed no observable decomposition after 1 h. The different solubilities of the ligands were considered when selecting the EtOH/H2O solvent mixture. The picoline-based chelate 6-Re was more hydrophobic than the pyridylhydrazine complex 5-Re, and while this may be expected from the isosteric replacement of “NH” with “CH2” groups, this substitution resulted in large differences in chromatographic mobility using reverse phase HPLC where the retention times were 20.63 min and 16.32 min respectively. The complexation of triazol-4-yl-methylamine ligand 25 at room temperature was relatively slow. Increasing the reaction temperature to 65°C for 1 h gave complex 10-Re in high yield (92%).All of the aminocarboxylate conjugates evaluated were effective for complexation of Re(CO)5-Re : 10-Re was evaluated using established functional assays for GPER and profiled for potential cross-reactivity via the nuclear estrogen receptor ERα  complexes ptor ERα . The cap5-Re and 6-Re were again found to be agonists of GPER-mediated PI3K activation, confirming their activity profile. Switching the assay protocol to detect antagonism of PI3K activation by 17β-estradiol, the complex 9-Re was similarly identified as an antagonist. None of the complexes were able to activate PI3K through ERα, demonstrating that the conjugation of linked chelate groups did not alter the selectivity profile to allow interaction with the nuclear receptor, consistent with the observation that there was no significant binding to ERα or ERβ at concentrations up to 10 µM, where 17β-estradiol yields a Ki of 0.3–0.5 nM (data not shown).The stimulation of GPER as well as ERα and ERβ by the native hormone 17β-estradiol activates PI3K and results in the nuclear accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), as shown by the nuclear translocation of an Akt PH domain fluorescent protein fusion protein reporter (PH-RFP). To determine whether these complexes activated the PI3K pathway, we employed COS7 cells transiently transfected with either GPER-GFP or ERα-GFP together with PH-RFP that serves as a reporter of PIP3 production. The complexes 5-Re and 6-Re possess ethanone linkages that are analogous to the methyl ketone group of G-1, and were similarly found to be potent agonists of GPER signaling in both the calcium and PI3K assays. In contrast, the triazole-linked complex 9-Re antagonized GPER-mediated signaling in both of these functional assays. The 1,2,3-triazole linkage is capable of functioning as a hydrogen-bond acceptor; however, the increased steric constraints and rigid planar ring structure in 9-Re may prevent the required conformational alignment in the receptor-bound complex. The ethane-linked complex 7-Re was inactive towards initiating or blocking GPER-mediated signaling. The conformational mobility of the flexible ethane linkage would produce a relatively large rotational steric volume and unfavorable entropic contribution that may impede the process of ligand-binding, accompanied by reduced affinity due to the hydrophobicity and absence of a H-bond accepting group in this linkage. The direct connection of heterocyclic chelates to the quinoline scaffold in compounds 8-Re and 10-Re contributes a relatively large steric volume in this region which precludes interactions with GPER. The extension of the linkage through a planar triazole group yielded antagonist complex 9, and emphasizes the importance of the linkage structure on receptor targeting properties.The activity profile in receptor-mediated signaling obtained from functional assays reveals the importance of structural effects associated with the linkage to the heterocyclic aminocarboxylate ligand at the C8 position of the tetrahydroquinoline scaffold. The pyridylhydrazine and picolinamine complexes 99mTc-radiolabeled complexes 5–8. The [99mTc(CO)3(H2O)3]+ intermediate was successfully prepared with a radiochemical purity of >95% (n>50). The resultant [99mTc(CO)3(H2O)3]+ intermediate was mixed with the corresponding ligand and stirred at room temperature for 2 hours. These conditions resulted in over 95% incorporation of the [99mTc(CO)3(H2O)3]+ into the pyridin-2-yl hydrazine 5-Tc and picolylamine 6-Tc complexes. A reduction to 85% incorporation was observed for the directly linked pyridin-2-yl hydrazine 8-Tc complex. The radiolabeled complexes were conveniently purified by reverse-phase solid phase extraction, efficiently removing excess ligand and inorganics. In order to assess a more rapid radiosynthetic method for improved specific activity, the [99mTc(CO)3(H2O)3]+ intermediate was mixed with the ligands and heated at 80°C for 30 min. Under these complexation conditions, degradation products were evident by HPLC analysis and the radiochemical purity was less than 70%. All 99mTc-labeled complexes demonstrated good stability (>95%) in mouse plasma and PBS buffer after incubation at 37°C for 24 h. The complexes were stable in the presence of biologically relevant chelating ligands, exhibiting less than 10% transchelation upon incubation with 1 mM cysteine solution or 1 mM histidine solution at 37°C for 24 h. The log P(o/w) values  of the ethanone-linked pyridin-2-yl hydrazine complex 5-Tc (4.6±0.3) was lower than that for the picolinamine complex 6-Tc (5.0±0.1), revealing the contribution of the hydrazine group to the hydrophilicity of the complex. Increased log P(o/w) values were also observed for the ethane-linked 7-Tc (4.9±0.1) and directly connected pyridin-2-yl hydrazine complex 8-Tc (5.5±0.1). Excessive lipophilicity was previously identified as a major limitation resulting in non-target tissue uptake for the urea- and hydrazone-linked I-125 radiolabeled analogs 2 and 3 with log P(o/w) values of 7.00 and 6.35 respectively 5-Tc therefore represents a significant improvement in log P(o/w) values compared to previous generations of GPER-targeted imaging agents.The tricarbonyl approach was used to prepare the 3(H2O)3]+ in aqueous ethanol. These conditions were successfully adapted for efficient 99mTc-radiolabeling. The ethanone-linked complexes 5-Re and 6-Re yielded selective activation of GPER-mediated signaling pathways comparable to the parent ligand G-1, while the triazole-linked complex 9-Re was an antagonist capable of blocking GPER signaling. These results demonstrate that structural features of the linkage and chelate were critical determinants of receptor-mediated signaling through GPER. We anticipate that continuing in vivo SPECT imaging studies of GPER expression with the promising new probe complex 5-Tc using will reveal important new insights on the role of GPER in normal and disease states.We have identified a new class of organometallic probes that selectively targets GPER in cellular assays. The tridentate heterocyclic aminocarboxylate ligands formed stable complexes from the cationic precursor  + for C28H25BrN4O5 577.20, found 577.19.Following general procedure A, the residue was purified by silica gel column chromatography using MeOH/CH2Cl2 (20∶ 80) to isolate the product 16  as a colorless solid. 1H NMR  δ 8.34, 7.53, 7.47, 7.28, 7.04, 6.98, 6.59, 5.96, 5.94, 5.87–5.84, 5.63–5.57, 4.89, 4.38–4.01, 3.51, 3.17–3.07, 2.45–2.34, 1.77–1.69; IR (KBr): 3437, 1684(CO), 1597(C = C) cm−1; HPLC-MS: Elution with 33% CH3CN in H2O containing 0.01% formic acid, exhibited single peak at Rt = 17.28 min. ESI-MS m/z (ES+) calcd [M+H]+ for C29H26BrN3O5 576.11, found 576.40.Following general procedure A, the residue was purified by silica gel column chromatography using MeOH/CH2Cl2 (10∶ 90) to isolate the product 18  as a colorless solid consisting of a mixture of syn: anti (3.3∶ 1) determined by NMR; 1H NMR  δ 7.66, 7.47, 7.14, 7.01, 6.93, 6.71, 6.68, 6.59, 5.97, 5.95, 5.76–5.71, 5.66–5.55, 4.73, 3.94, 3.61, 3.15–3.05, 2.84–2.71, 2.54- 2.46, 1.71–1.63; 13C NMR  δ 154.2(CO), 148.9(Py), 148.7(Ar), 146.3(Py), 145.5(Py), 136.0(Py), 135.4(C-1), 133.9(Py), 130.7(Ar), 130.1(C-2), 128.6(Ar), 127.4(Ar), 127.0(Ar), 123.9(Ar), 120.0(Ar), 117.4(Py), 116.1(Ar), 113.6(Ar), 113.5(Ar), 112.3(Ar), 112.2(Ar), 109.3(Ar), 103.2(OCH2O), 57.9(C-4), 52.8(NCH2CO), 47.3(C-9b), 43.5(C-3), 36.8(CH2), 34.4(CH2), 32.3(C-3a); IR (KBr): 3453(NH and OH), 1682(CO) cm−1. HPLC-MS: Elution with 3–93% CH3CN (gradient 4.5% min-1) in H2O containing 0.01% formic acid, exhibited two peak at Rt = 7.55 and 7.15 min. ESI-MS m/z (ES-) calcd [M−H] - for C28H27BrN4O4 561.12, found 561.33.Following general procedure A, the residue was purified by silica gel column chromatography using MeOH/CH2Cl2(15∶ 85) to isolate the product 20  as a colorless solid consisting of a mixture of syn: anti (3∶ 1) determined by NMR; 1H NMR  δ 8.13, 7.98, 7.25, 7.18–7.14, 7.08–7.04, 6.80, 6.00, 5.98, 5.95–5.92, 5.65–5.62, 4.83–4.82, 4.08, 3.62, 3.21–3.13, 2.57–2.48, 1.75–1.67; IR (KBr): 3435(NH and OH), 1678(CO)cm−1; HPLC-MS: Elution with 3–93% CH3CN (gradient 4.5% min−1) in H2O containing 0.01% formic acid, exhibited a single peak Rt = 6.22 min. ESI-MS m/z (ES+) calcd for C26H23BrN4O4 (M+H)+ 535.09; found 535.07.Following general procedure A, the residue was purified by silica gel column chromatography using MeOH/CH2Cl2(15∶ 85) to isolate the product 23  as a colorless solid. 1H NMR  δ 8.36, 8.31–8.21, 7.40, 7.29, 7.18, 7.10, 7.0, 6.73, 5.97, 5.96, 5.90–5.86, 5.70–5.65, 4.89, 4.12, 3.52, 3.24–3.08, 2.57–2.50, 1.83–1.76; IR (neat): 1696 (CO) cm−1HPLC-MS: Eluting with 10–90% CH3CN (gradient 2.66% min−1) in H2O exhibited single peak at Rt = 23.17 min. ESI-MS m/z (ES-) calcd [M−H]− for C28H24BrN7O4 600.11, found 600.09.Following general procedure A, the residue was purified by silica gel column chromatography using MeOH/CH2Cl2 (20∶ 70) to isolate the product 25  as a white solid. 1H NMR  δ 8.41, 7.37, 7.29, 7.14, 7.01, 6.83, 5.98, 5.97, 5.90–5.85, 5.67–5.63, 4.86, 4.43, 4.08, 3.62, 3.23–3.12, 2.57–2.49, 1.79–1.70; IR (KBr): 3430(OH), 1683(CO) cm−1; HPLC-MS: Eluting with 10–90% CH3CN (gradient 3% min−1) in H2O, exhibited single peak at Rt = 18.85 min. ESI-MS m/z (ES+) calcd [M+H]+ for C24H22BrN5O4 524.09, found 524.32.Following general procedure A, the residue was purified by addition of water, producing a solid that was filtered and purified by silica gel column chromatography using MeOH/CH3(H2O)3(1.1 eq) and NaHCO3(1.0 eq) in water was added to the ligand in EtOH and allowed to stir at rt for 3–6 h. Volatiles were removed, water was added, the resulting precipitate was filtered and washed with water. The residue was purified as described to isolate the rhenium complex.A mixture of ReBr(CO)2O (1.3∶ 1), crude residue was purified by silica gel column chromatography using MeOH/CH2Cl2 (05∶ 95) to isolate the complex 5  as a white solid. 1H NMR  δ 10.01, 9.50 , 8.14, 7.74, 7.62, 7.25, 7.11, 6.84–6.76, 6.54, 6.10, 6.07, 6.02–5.97, 5.64–5.58, 4.80, 4.33–4.16, 4.06, 3.75–3.61, 3.07–2.98, 2.45–2.35, 1.71–1.63; 13C NMR  δ 197.8(CO), 197.2(CO), 196.5(CO), 194.5(CO), 176.6(CO), 158.0(Py), 151.1(Ar), 148.5(Py), 147.2(Py), 147.1(Ar), 142.5(Ar), 134.5(C-1), 133.3(Ar), 130.1(Ar), 130.0(Ar), 129.6(C-2), 127.1(Ar), 125.9(Ar), 123.6(Ar), 123.1(Ar), 115.1(Ar), 112.2(Ar), 112.2(Ar), 108.4 (Ar), 107.0 (Ar), 101.9(OCH2O), 58.6(C-4), 55.2(N-CH2-CO),44.7(CO-CH2-Py), 41.7(C-3), 31.2 (C-3a); IR (KBr): 3429(NH), 2027(CO), 1918(CO), 1893(CO), 1630(CO) cm−1; HPLC-MS: Eluting with 63–93% CH3CN (gradient 1% min−1) in H2O containing 0.01% formic acid, exhibited single peak at Rt = 16.32 min. ESI-MS m/z (ES+) calcd [M+H] + for C31H24BrN4O8Re 847.04, found 847.30. HRMS: calcd [M+H]+ for C31H24BrN4O8Re 847.0413, found 847.0391.Following the general procedure B using EtOH/H2O (2∶ 1), the crude residue was purified by silica gel column chromatography using MeOH/CH2Cl2 (8∶ 92) to isolate the product 6  as a white solid 1H NMR  δ 8.71–8.69, 7.97–7.94, 7.76, 7.65, 7.25, 7.22–7.18, 7.12, 6.79, 6.58, 6.10, 6.08, 6.04–5.98, 5.65–5.59, 4.82, 4.60–4.38, 4.08, 3.62–3.53, 3.25–3.20, 3.09–2.99, 2.44–2.35 , 1.71–1.63; 13C NMR  δ 197.6(CO), 197.4(CO), 197.4(CO), 193.8(CO), 179.4(CO), 157.8(Py), 152.5(Py), 151.2(Ar), 147.2(Py), 147.1(Py), 141.7(Py), 134.5(C-1), 133.7(Ar), 133.2(Ar), 130.1(Ar), 129.7(C-2), 127.2(Ar), 125.8(Ar), 123.7(Ar), 122.8(Ar),115.7(Ar),112.4(Ar),112.2(Ar),108.4(Ar),104.4(Ar),102.0(OCH2O), 61.7(C-4), 55.2(NCH2CO), 53.9(PyCH2N), 44.7(COCH2Py), 41.7(C-3), 31.2 (C-3a); IR (KBr): 3429(NH), 2023(CO), 1920(CO), 1889(CO), 1635(CO) cm−1; HPLC-MS: Eluting with 63–93% CH3CN in H2O (gradient 1% min−1) containing 0.01% formic acid, exhibited single peak at Rt = 20.63 min. ESI-MS m/z (ES+) calcd [M+H]+ for C32H25BrN3O8Re 846.04, found 846.31; HRMS: Calcd [M+H]+ for C32H25BrN3O8Re 846.0461, found 846.0428.Following the general procedure B using EtOH/H2O (3∶1) the crude residue was purified by silica gel column chromatography using MeOH/CH2Cl2 (94∶ 06) to isolate the complex 7  as a white solid consisting of a mixture of syn: anti (4.5∶ 1) determined by NMR; 1H NMR  δ 9.93, 9.46, 7.96, 7.65, 7.22, 7.13, 7.13, 6.87–6.72, 6.63–6.59, 6.08, 6.06, 5.84–5.81, 5.59–5.56, 5.40, 4.63, 3.97–3.94, 3.67–3.65, 3.01–2.95, 2.77–2.65, 1.67–1.58; IR (KBr): 3428(NH), 2025(CO), 1916(CO), 1887(CO), 1631(CO) cm−1; HPLC-MS: Eluting with 3–93% CH3CN (gradient 4.5% min−1) in H2O containing 0.01% formic acid, exhibited single peak at Rt = 17.68 min. ESI-MS m/z (ES+) calcd [M+H] + for C31H26BrN4O7Re 833.06 found 833.19. HRMS: Calcd [M+H]+ for C31H26BrN4O7Re 833.0621, found 833.0602.Following the general procedure B using EtOH/H2O (2∶ 1). The crude complex was purified by column chromatography using MeOH/CH2Cl2 (8∶ 92) to isolate the product 8  as a white solid. 1H NMR  δ 10.11, 9.57, 8.25, 7.98, 7.27–7.15, 6.91, 6.81, 6.10, 6.08, 5.98–5.94, 5.86, 5.63–5.58, 4.70, 4.09, 3.69, 3.06–3.03, 2.48–2.42, 1.72–1.63; IR (KBr): 3252(NH), 2026(CO), 1916(CO), 1898(CO), 1667(CO) cm−1; HPLC-MS: Eluting with 3–93% CH3CN (gradient 4.5% min−1) in H2O containing 0.01% formic acid, exhibited single peak at Rt = 15.37 min. ESI-MS m/z (ES+) calcd [M−H]+ for C29H22BrN4O7Re 803.02 found 803.07; HRMS: Calcd [M+H] + for C29H22BrN4O7Re 805.0308, found 833.0309.Following the general procedure B using EtOH/H2O (2∶ 1) the crude residue was purified by silica gel (SiO2) flash column chromatography using MeOH/CH2Cl2 (05∶ 95) to isolate the complex 9  as a yellow solid 1H NMR  δ 8.79, 8.69, 8.17, 7.48, 7.38, 7.15, 7.03, 6.92, 6.84, 5.99, 5.98, 5.96–5.92, 5.70–5.65, 4.88, 4.12, 3.89, 3.21–3.14, 2.58–2.50, 1.80–1.71; HPLC-MS: Eluting with 40–90% CH3CN (gradient 1.5% min−1) in H2O exhibited single peak at Rt = 8.22 min. ESI-MS m/z (ES+) calcd [M+H]+ for C31H23BrN7O7Re 872.04, found 872.05.Following the general procedure B using EtOH/H3(H2O)3  and NaHCO3  in water (5 mL) was added to the triazole ligand 25  in EtOH (10 mL) heated at 65°C for 1 h. The reaction mixture was cooled and solvents were evaporated in vacuo, water (10 mL) was added, and the solids were separated by filtration and washed with water (30 mL) to isolate the complex 10 . 1H NMR  δ 8.85, 7.56, 7.45–7.38, 7.26, 7.15, 6.89, 6.25, 6.10, 6.08, 6.00–5.97, 5.63–5.62, 4.76, 4.35–4.25, 4.12, 3.63–3.57, 3.37–3.35, 3.10–3.01, 2.44–2.35, 1.72–1.66; 13C NMR  δ 197.4(CO), 196.9(CO), 196.6(CO), 179.1(CO), 148.9(Tri), 147.8(Tri), 147.19(Ar), 147.14(Ar), 134.2(Ar), 133.5(Ar), 130.0(Ar), 126.3(Ar), 125.7(Ar), 121.5(Ar), 121.1(Ar), 119.1(Ar), 116.4(Ar), 112.4(Ar), 112.2(C-1), 108.4(C-2), 101.9(OCH2O), 55.6(C-4), 55.0(N-CH2-CO), 51.8(TriCH2N), 45.2(C-9b), 41.6(C-3), 31.2(C-3a). IR (KBr): 2025(CO), 1990(CO), 1675(CO) cm−1; HPLC-MS: Eluting with 10–90% CH3CN (gradient 3% min−1) in H2O, exhibited single peak at Rt = 25.15 min. ESI-MS m/z (ES+) calcd [M+H]+ for C27H21BrN5O7Re 794.04, found 794.26; HRMS: Calcd [M+H]+ for C27H21BrN5O7Re 794.0255, found 794.0258.A mixture of ReBr(CO)2 and 95% air. Lipofectamine 2000 was used according to manufacturer's directions for all transfections except PH-RFP was transfected at ¼ the recommended amount to achieve reasonable expression levels.ER α/β-negative and GPER-expressing human breast carcinoma SKBr3 cells and ER α/β and GPER-negative monkey kidney COS7 cells were cultured in DMEM tissue media (COS7) or RPMI-1640 tissue media (SKBr3), with fetal bovine serum (10%), 2 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin. Cells were grown as a monolayer at 37°C, in a humidified atmosphere of 5% CO7/mL) were incubated in HBSS containing 3 µM Indo1-AM (Invitrogen) and 0.05% pluronic acid F-127 for 1 h at RT. Cells were then washed twice with HBSS, incubated at RT for 20 min, washed again with HBSS, resuspended in HBSS at a density of 108cells/mL and kept on ice until assay, performed at a density of 2×106cells/mL. Ca++ mobilization was determined ratiometrically using λex 340 nm and λem 400/490 nm at 37°C in a spectrofluorometer  equipped with a magnetic stirrer.SKBr3 cells (1×10The PIP3 binding domain of Akt fused to mRFP1 (PH-RFP) was used to localize cellular PIP3. COS7 cells co-transfected with GPER-GFP or ERα-GFP and PH-RFP were plated on coverslips and serum starved for 24 h followed by stimulation with ligands as indicated. The cells were fixed with 2% PFA in PBS, washed, mounted in Vectashield containing DAPI (Vector Labs) and analyzed by confocal microscopy using a Zeiss LSM510 confocal fluorescent microscope.4) were incubated with Re-labeled derivatives for 10 min in a final volume of 10 µL prior to addition of 10 µL of 20 nM E2-Alexa633 in saponin-based permeabilization buffer. Following 5 min at RT, cells were washed once with 1 mL PBS/2%BSA, resuspended in 200 µL and analyzed on a FACS Calibur flow cytometer (BD Biosciences).Binding assays for ERα and ERβ were performed as previously described 99mTc(CO)3(H2O)3]+ was prepared by adding 3.7 GBq of freshly eluated Na-99mTcO4 to the Isolink® kit  as previously described 99mTc(CO)3(H2O)3]+ mixture was then neutralized to pH 7 with acetic acid. The ligands were dissolved in ethanol and 10 µg of each of the derivatives was added to the prepared [99mTc(CO)3(H2O)3]+ mixture. The reaction mixture was stirred for 2 h at room temperature. The inorganic impurities from Isolink® kit, aqua ions of 99mTc (if any) and excess ligand were separated using solid phase extraction (SPE) technique. SPE was performed using C-18 SepPak Plus cartridges . The impurities and excess of ligand were eluted with 4 fractions of 0.5 mL of the weak solvent (50% ethanol in water). Elution of the final 99mTc-labeled complexes was performed with 4 fractions of 0.5 mL strong solvent (100% ethanol). HPLC was performed to assess radiochemical purity and specific activity. Ten µL of the final product was added to 200 µL of HPLC grade ethanol . To asses radiochemical purity and specific activity, 10 µL of the diluted quality control sample was injected on a reverse-phase C-18 column  using HPLC grade ethanol and HPLC grade water as previously described The carbonyl complex [Text S1Additional experimental details for synthesis of ligands 13, 16, 18, 20, 23, 25.(DOC)Click here for additional data file.Figure S1Synthetic scheme for preparation of 5-ethynyl derivative 12.(TIF)Click here for additional data file.Figure S2Synthetic scheme for preparation of G-CC-5-pyridyl-2-N(tBoc)N(t2CO2Boc)CHtBu.(TIF)Click here for additional data file.Figure S3Synthetic scheme for preparation of picoline amine derivative 15.(TIF)Click here for additional data file.Figure S4Synthetic scheme for preparation of tert-butyl-2-(tert-butoxycarbonyl((5-ethynylpyridine-2-yl)methyl)amino)ethanoate.(TIF)Click here for additional data file.Figure S52-N(Synthetic scheme for preparation of G-CC-5-pyridyl-2-CHt2CO2Boc)CHtBu.(TIF)Click here for additional data file.Figure S6Synthetic scheme for preparation of aniline derivative 17.(TIF)Click here for additional data file.Figure S72CH2-5-pyridyl-2-N(Synthetic scheme for preparation of G-CHtBoc)-N(t2CO2Boc)CHtBu.(TIF)Click here for additional data file.Figure S8Synthetic scheme for preparation of aniline derivative 19.(TIF)Click here for additional data file.Figure S9Synthetic scheme for preparation of G-5-pyridyl-2-N(tBoc) N(t2CO2Boc)CHtBu.(TIF)Click here for additional data file.Figure S10Synthetic scheme for preparation of azide product 22.(TIF)Click here for additional data file.Figure S11Synthetic scheme for preparation of G-triazole-5-pyridyl-2-N(tBoc) N(t2CO2Boc)CHtBu.(TIF)Click here for additional data file.Figure S122N(tBoc)CH2CO2Synthetic scheme for preparation of G-triazole-CHtBu.(TIF)Click here for additional data file.


This is the first time that these compounds (3-6) are reported as natural sterols. The structures of the new compounds were determined on the basis of the extensive spectroscopic analysis, including two-dimensional (2D) NMR and HR-ESI-MS data. Compounds 1-4, 7 and 10-13 were evaluated for their cytotoxicity against HL-60 human myeloid leukemia cell line, and all of the evaluated compounds exhibited moderate cytotoxicity to HL-60 cells with a range of IC50 values from 14.73 to 22.11 µg/mL except for compounds 12 and 13.Six new sterols ( Bugula neritina are a well known example + in the positive ion mode EI-MS, enabled the determination of the molecular formula C28H46O2, with the help of NMR data -double bond (δC 140.9 and 121.8), which was consistent with the literature +) and HR-ESI-MS ), with the help of 1D NMR spectral data. Fragment at m/z 382 [M −OCH3 −H]+ due to loss of a methoxy group, as well as the two ion fragments at m/z 271 [M −C8H15 −OCH3 −H]+ and 253 [M −C8H15 −OCH3 −H −H2O]+, suggested the presence of the mono-hydroxylated and mono-methoxylated steroid with a mono-unsaturated side chain. The presence of a strong peak in the EI-MS at m/z 111 confirmed the presence of the C8H15 side chain. The partial structure of a Δ22 mono-unsaturated side chain was established from the HMBC spectrum of 2  due to a strong broad singlet at δH3.35  correlated to C-7 in the HMBC spectrum, and the partial structure of the steroidal nucleus was confirmed by the observation of the HMBC cross peaks from H2-4 to C-3, C-5 and C-6; H-6 to C-4, C-8 and C-10; H3-18 to C-13, C-14 and C-17, and H3-19 to C-1 and C-10  between H-22 and H-23. The β-orientation of the hydroxyl group on C-3 was deduced also from the NOESY correlations of H-3/H-4α and H-4α/H-6, and the β-orientation of the methoxy group on C-7 was deduced by observation of correlations of H-7α/H-6 and H-7α/H-14 in the NOESY experiment (2 can be defined as (22E)-7β-methoxy-cholesta-5,22-dien-3β-ol.Compound rum of 2 , which sand C-10 . The conperiment . Accordi3 was isolated as a white amorphous powder and was also positive to the Libermann-Burchard test. Its molecular formula was established as C28H48O2 by observation of the molecular ion peak at m/z 439.3550 [M + Na]+  in the HR-ESI-MS spectrumand ion peak at m/z 416 [M]+ in the EI-MS spectrum. Comparison of the 1H NMR and 13C NMR spectra of 3 with those of 2 revealed that they shared the same 3β-hydroxy, 7β-methoxy Δ5-steroid nucleus but differed in the side chain. Compound 3 was finally assigned as 7β-methoxy-cholest-5-en-3β-ol due to the missing trans-double bonds between C-22 and C-23 in the 13C NMR spectrum by comparison with 2. Compound 3 has been reported as a synthetic sterol with effective inhibition of cholesterol acyltransferase (ACAT) [Compound 4 was obtained as a white amorphous powder and was positive to Liebermann-Burchard test. The HR-ESI-MS spectrum showed the molecular ion peak at m/z 407.2928 [M + Na]+  and EI-MS spectrum showed the molecular ion peak at m/z 384 [M]+ corresponding to the molecular formula C26H40O2, with the help of NMR data. Compound 4 was assumed to have the same typical nucleus of 3β-hydroxy Δ5-steroid by comparing the 1D NMR data with those of 1, but differed in the side chain. The protons of trans-olefinic bonds in the side chain appeared at δH6.07  and 6.78 . The HSQC spectral data indicated that the proton H-24 (δH6.07) was connected to the carbon at δC132.8 (C-24) and H-23 (δH6.78) was connected to the carbon at δC147.6 (C-23), while the protons H2-22  were connected to the carbon at δC39.5 (C-22). The correlation of H-23 with H2-22 and H-24 in the 1H-1H COSY spectrum indicated that the double bonds were in C-23 and C-24. This was also supported by the key cross-peaks H-24 with C-22 and H-23 with C-22 in the HMBC experiment. Similarly, the downfield singlet methyl protons at δH 2.25 (H3-26) exhibited HMBC correlations with C-25 (δC198.7) and C-24, and upfield methyl proton signals at δH0.95 (H3-21) with C-17 (δC55.9), C-20 (δC36.0) and C-22, suggesting the remnant connectivities of the side chain in compound 4. Accordingly, 4 was finally assigned as (23E)-3β-hydroxy-27-norcholesta-5,23-dien-25-one, which had been obtained by synthesis +) and HR-ESI-MS data. The 1H NMR and 13C NMR data of 5 and 6 agreed with those of cholesta-5,25-diene-3β,24ξ-diol from red alga Galaxaura marginata + (100), 382 [M − OCH3 − H]+ (26), 367 (18), 349 (11), 301 [M − C6H10OCH3]+ (25), 300 (30), 283 [M − C6H10OCH3 − H2O]+ (37), 271 (46), 241 (12), 227 (10), 215 (24), 183 (19), 159 (27), 133 (31), 113 [C6H10OCH3]+ (28), 99 (47), 85 (61), 55 (42); HR-ESI-MS (positive) m/z: 437.3398 [M + Na]+ .White amorphous powder; [α]22D − 40.7° ; 1H NMR and 13C NMR data, see + (38), 396 [M − H2O]+ (42), 382 [M − OCH3 − H]+ (100), 367 (8), 349 (12), 303 [M − C8H15]+ (10), 298 (15), 271 [M − C8H15 − OCH3 − H]+ (22), 253 (21), 211 (13), 197 (9), 175 (14), 159 (17), 145 (19), 135 (17), 119 (16), 111 [C8H15]+ (22), 69 (30), 55 (37); HR-ESI-MS (positive) m/z: 437.3394 [M + Na]+ .White amorphous powder; [α]22D − 67.5° ; 1H NMR  δ: 0.66 , 0.86 , 0.87 , 0.91 , 0.98 , 2.34 , 2.29 , 3.29 , 3.36 , 3.62 , 5.73 ; 13C NMR data, see + (26), 398 [M − H2O]+ (37), 384 [M − OCH3 − H]+ (100), 369 (11), 351 (12), 271 [M − C8H17 − OCH3 − H]+ (7), 253 (5), 213 (6), 211 (7), 185 (5), 175 (9),159 (12), 145 (12), 119 (12), 95 (15), 81 (14), 69 (11), 55 (15); HR-ESI-MS (positive) m/z: 439.3550 [M + Na]+ .White amorphous powder; [α]22D − 72.4° ; 1H NMR  δ: 0.70 , 0.95 , 1.01 , 2.25 , 3.52 , 5.35 , 6.07 , 6.78 ; 13C NMR data, see + (100), 366 [M − H2O]+ (66), 351 [M − CH3 − H2O]+ (40), 324 (9), 299 (29), 283 (32), 273 [M − Side Chain]+ (41), 255 [M − H2O − Side Chain]+ (23), 213 (45), 199 (19), 189 (29), 173 (24), 159 (48), 145 (54), 133 (44), 119 (46), 107 (62), 95 (52), 81 (54), 67 (38); HR-ESI-MS (positive) m/z: 407.2928 [M + Na]+ White amorphous powder; [α]22D − 46.7° ; 1H NMR  δ: 0.68 , 0.93 , 1.00 , 1.72 , 3.52 , 4.00 , 4.83 , 4.92 , 5.35 ; 13C NMR data, see + (41), 382 [M − H2O]+ (56), 367 [M − CH3 − H2O]+ (33), 349 (25), 340 (10), 328 (13), 315 (22), 300 [M − C6H10 − H2O]+ (27), 271 [M − C8H15 − H2O]+ (100), 255 (36), 243 (17), 229 (31), 213 (46), 199 (23), 187 (26), 173 (30), 161 (49), 145 (52), 133 (46), 119 (46), 107 (58), 95 (56), 81 (60), 71 (65), 55 (63); HR-ESI-MS (positive) m/z: 423.3241 [M + Na]+ .White amorphous powder; [α]22D − 19.4° ; 1H NMR  δ: 0.68 , 0.93 , 1.00 , 1.72 , 3.52 , 4.00 , 4.83 , 4.93 , 5.35 ; 13C NMR data, see + (41), other ion fragments identical with 5; HR-ESI-MS (positive) m/z: 423.3237 [M + Na]+ .White amorphous powder, [α]22D − 27.9°  assay [5 cells/mL. 2 mL of this cell suspension was placed into 96-well microculture plates and allowed to adhere in 5% CO2/air for 24 h at 37 °C before drug addition. 20 µL of DMSO solution containing the sample was added to give the various concentrations in triplicate for 72 h, with adriamycin (Sigma) as positive control. After the incubation, 20 µL of MTT solution (5 mg/mL) was added to each well, and the incubation continued for 4 h at 37 °C. Then, 150 µL of DMSO solution was added to each well, and the formazan crystals in each well were dissolved by stirring with a pipette. The optical density (OD) was read on a plate reader on an ELISA reader  at a wavelength of 570 nm. Each assay was done in triplicate, and inhibition was expressed as IC50 value, which stands for inhibition of cell growth by 50%.The cytotoxicity of compounds T) assay . The cel


Stevia rebaudiana Bertoni, under conditions that simulated extreme pH and temperature conditions has been studied. Thus, rebaudioside M was treated with 0.1 M phosphoric acid solution (pH 2.0) and 80 °C temperature for 24 h. Experimental results indicated that rebaudioside M under low pH and higher temperature yielded three minor degradation compounds, whose structural characterization was performed on the basis of 1D  & 2D  NMR, HRMS, MS/MS spectral data as well as enzymatic and acid hydrolysis studies.Degradation of rebaudioside M, a minor sweet component of  The 1H-NMR spectrum of 1 showed the presence of three methyl singlets at δ 1.33, 1.35 and 1.89, eight methylene and two methine protons between δ 0.77–2.69, and a trisubstituted olefinic proton at δ 5.03, suggesting the presence of an ent-13-hydroxykaur-15-en-19-oic acid skeleton in its structure + ion. Acid hydrolysis of 2 afforded d-glucose which was identified by preparing the corresponding thiocarbamoyl-thiazolidine carboxylate derivatives and in comparison of its retention times with the standard sugars as described in the literature  ent-kaur-15-en-19-oic acid 2-O-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl ester, which was further confirmed from the key COSY and HMBC correlations as shown in A close comparison of the 3 was determined to be C56H92O34 by the HRMS data that showed (M + H)+ and (M + Na)+ ions at m/z 1309.5588 and 1331.5414, respectively. The 1H-NMR spectrum of 3 showed the presence of three methyl singlets at δ 1.28, 1.31, 1.32, nine methylene and two methine protons between δ 0.78–2.67. In the absence of any unsaturated protons or carbons together with the appearance of a methyl group at δ 1.32 corresponds to a methyl group connected to a tertiary hydroxyl group suggested the structure of 3 should be similar to ent-13,16β-dihydroxykauran-19-oic acid + ion and this was supported by the six anomeric protons observed at δ 5.22, 5.53, 5.63, 5.76, 5.79 and 6.35 in its 1H-NMR spectral data. Acid hydrolysis of 3 afforded D-glucose which was identified in comparison of its retention times with the standard sugars as described in 2 -16β-hydroxy-ent-kauran-19-oic acid [O-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]-16β-hydroxy ent-kauran-19-oic acid 2-O-β-d-glucopyranosyl- 3-O-β-d-glucopyranosyl-β-d-glucopyranosyl ester.The molecular formula of compound oic acid . The prebed in 2 . The 1H-elations . A closeelations . The larelations  of the d4 was deduced as C38H60O18 from the [M + H]+ and [M + Na]+ ions at m/z 805.3890 and 827.3707, respectively in the positive ESI mass spectrum. This was also supported by the 13C-NMR spectral data. The 1H-NMR spectrum of 4 showed the presence of three methyl singlets at δ 0.81, 0.98 and 1.42; nine methylene and two methine protons. Enzymatic hydrolysis of 3 furnished an aglycon that was found to be identical to isosteviol on the basis of its NMR spectral data reported in the literature  ent-kaur-15-en-19-oic acid (2-O-β-d-glucopyranosyl-3-O-β-d-gluopyranosyl-β-d-glucopyranosyl) ester (2): White powder; 1H-NMR  and 13C-NMR  spectroscopic data see +m/z 1291.5439 ; 1313.5254 .13-[(2-O-β-d-glucopyranosyl-3-O-β-d-gluopyranosyl-β-d-glucopyranosyl)oxy]-17-hydroxy-ent-kaur-16β-hydroxy-19-acid (2-O-β-d-glucopyranosyl-3-O-β-d-gluopyranosyl-β-d-glucopyranosyl) (3): White powder; 1H-NMR  and 13C-NMR  spectroscopic data see +m/z 1309.5588 ; 1331.5404 .13- [Each compound 3.5.4.4 (250 μg) was dissolved in 2.5 mL of 0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillus niger  was added. The mixture was stirred at 50 °C for 48 h. The product precipitated out during the reaction and was filtered and then crystallized. The resulting product obtained from the hydrolysis was identified as isosteviol, characterized by comparison of its co-TLC with standard compound and 1H-NMR spectral data [Compound ral data –21.4.1) under acidic conditions has been studied at high temperature. The complete 1H- and 13C-NMR spectral data for the degradation compounds 2–4 are reported herewith for the first time based on COSY, HSQC, and HMBC spectroscopic data as well as enzymatic and acid hydrolysis studies.The degradation of rebaudioside M (


Xanthomonas campestris and cyclodextrin glucanotransferase (CGTase). X. campestris glucosylated hesperetin to its 3'-, 5-, and 7-O-glucosides, and CGTase converted hesperetin glucosides into the corresponding maltosides. The resulting 7-O-glucoside and 7-O-maltoside of hesperetin showed inhibitory effects on IgE antibody production and on O2- generation from rat neutrophils.The production of hesperetin glycosides was investigated using glycosylation with  Citrus species, tomatoes, and apples, is a kind of vitamin P and has been well documented for its medicinal properties as an important Chinese traditional medicine + peaks at 649.1751 for 5, 649.1755 for 6, and 649.1750 for 7 , indicating that each product consisted of one substrate and two hexoses. The sugar components in these products were determined to be glucose on the basis of the chemical shifts of their carbon signals. The 1H NMR spectra showed two proton signals at δ 5.01  and 5.06  for 5, δ 5.03  and 5.40  for 6, and δ 5.05  and 5.17  for 7, indicating that the glucoside linkage in these three compounds had α-orientations. The HMBC spectra of 5-7 included correlations between the proton signal at δ 5.01 (H-1''') and the carbon signal at δ 80.7 (C-4'') and between the proton signal at δ 5.06 (H-1'') and the carbon signal at δ 146.2 (C-3') for 5, between the proton signal at δ 5.03 (H-1''') and the carbon signal at δ 80.5 (C-4'') and between the proton signal at δ 5.40 (H-1'') and the carbon signal at δ 165.1 (C-5) for 6, and between the proton signal at δ 5.05 (H-1''') and the carbon signal at δ 80.5 (C-4'') and between the proton signal at δ 5.17 (H-1'') and the carbon signal at δ 166.9 (C-7) for 7. These data indicated that 5-7 were α-maltosyl analogues of 1, the sugar moiety of which attached at their 3'-position (5), 5-position (6), and 7-position (7). Thus, products 5-7 were identified as hesperetin 3'-O-α-maltoside (5), hesperetin 5-O-α-maltoside (6), and hesperetin 7-O-α-maltoside (7), respectively. The substrate hesperetin used for this experiment was a chiral compound, the stereochemistry of which was 2S x100, where Pt and Pc are the photon count of sample solutions with and without the test compound. As a result, inhibitory effects of hesperetin 7-O-α-glycosides 4 and 7 and an authentic anti-allergic drug on fMLP-induced O2- generation from rat neutrophils are as follows: 4, 59%; 7, 46%; mequitazine, 62% (O-α-glucoside (4) exhibited significant inhibitory activity for O2- generation, to the same level as mequitazine. This is the first time that hesperetin α-glycosides have been shown to have a strong inhibitory effect on O2- generation. Hesperetin 3'-O-α-glycosides 2 and 5 and hesperetin 5-O-α-glycosides 3 and 6 showed no inhibitory action on O2- generation. Previously, we reported that 7-O-β-glycosides of polyphenols, i.e., genistein and quercetin, showed anti-allergic activities, whereas polyphenol glycosides, where the is attached at other phenolic hydroxyl groups, exhibited no anti-allergic actions . Substrate, hesperetin, was purchased from Sigma-Aldrich Co. CGTase was purchased from Amano Pharmaceutical Co. Ltd. The NMR spectra were recorded in CDX. campestris was a gift from the Department of Chemistry of Okayama University of Science. Culture medium used for growth of X. campestris had the following composition (in grams per liter): 5 g of maltose, 3 g of peptone, 0.4 g of yeast extract, 0.2 g of MgSO4. The cells were grown in the culture medium with continuous shaking on a rotary shaker (120 rpm) at 30°C.X. campestris were grown in 500 mL conical flasks containing 200 mL of culture medium at 30 °C. Prior to use for the experiments, the cells were harvested by centrifugation at 8,000 g for 15 min and were lyophilized. Hesperetin α-glucosides were prepared as follows. A total of 2 mmol of hesperetin was added to ten 300-ml conical flasks (0.2 mmol/flask), each of which contained 5 g of lyophilized X. campestris cells and 1 mmol of maltose in 100 ml of 10 mM HEPES-NaOH buffer (pH 7.5). The mixture was incubated continuous shaking on a rotary shaker (120 rpm) for 5 days at 30 °C. The reaction mixture was centrifuged at 8,000 g for 15 min and the supernatant was extracted with n-butanol. The butanol fraction was purified by preparative HPLC on YMC-Pack R&D ODS column to give hesperetin α-glucosides.The cultures of To a solution containing 0.1 mmol of hesperetin α-glucoside and 5 g of starch in 25 mM of sodium phosphate buffer (pH 7.0) was added 100 U of CGTase. The reaction mixture was stirred at 40 °C for 24 h, and then the mixture was centrifuged at 3,000 g for 10 min. The supernatant was subjected on to a Sephadex G-25 column equilibrated with water to remove CGTase. The fractions containing glycosides were purified by preparative HPLC on YMC-Pack R&D ODS column to give hesperetin α-maltosides.Spectral data of selected new compounds are as follows.O-α-maltoside (5): FABMS m/z: 649.1751 [M+Na]+; 1H NMR : δ 2.77 , 3.08-3.88 , 3.33 , 3.78 , 5.01 , 5.06 , 5.50 , 5.89 , 5.90 , 7.03 , 7.08 , 7.25 ; 13C NMR : δ 43.3 (C-3), 55.5 (OCH3), 60.5 (C-6'''), 60.7 (C-6''), 69.5 (C-4'''), 72.2 (C-5''), 72.4 (C-2''), 73.5 , 73.9 , 78.0 (C-2), 80.7 (C-4''), 94.7 (C-8), 95.7 (C-6), 98.0 (C-1''), 100.5 (C-1'''), 101.4 (C-10), 111.5 (C-5'), 113.6 (C-2'), 120.5 (C-6'), 130.2 (C-1'), 146.2 (C-3'), 149.0 (C-4'), 162.4 (C-9), 163.2 (C-5), 166.3 (C-7), 196.9 (C-4).Hesperetin 3'-O-α-maltoside (6): FABMS m/z: 649.1755 [M+Na]+; 1H NMR (DMSO-d6): δ 2.76 , 3.05-3.89 , 3.33 , 3.78 , 5.03 , 5.40 , 5.51 , 6.08 , 6.12 , 6.90 , 7.00 ; 13C NMR (DMSO-d6): δ 43.0 (C-3), 55.5 (OCH3), 60.3 (C-6'''), 60.6 (C-6''), 69.5 (C-4'''), 72.2 (C-5''), 72.5 (C-2''), 73.5 , 74.1 , 78.0 (C-2), 80.5 (C-4''), 93.7 (C-8), 94.8 (C-6), 99.7 (C-1''), 100.6 (C-1'''), 103.5 (C-10), 111.7 (C-5'), 113.8 (C-2'), 117.7 (C-6'), 130.8 (C-1'), 146.0 (C-3'), 147.7 (C-4'), 162.5 (C-9), 165.1 (C-5), 165.0 (C-7), 198.9 (C-4).Hesperetin 5-O-α-maltoside (7): FABMS m/z: 649.1750 [M+Na]+; 1H NMR (DMSO-d6): δ 2.76 , 3.07-3.83 , 3.32 , 3.78 , 5.05 , 5.17 , 5.49 , 6.16 , 6.22 , 6.88 , 6.92 ; 13C NMR (DMSO-d6): δ 42.2 (C-3), 55.5 (OCH3), 60.4 (C-6'''), 60.6 (C-6''), 69.5 (C-4'''), 72.2 (C-5''), 72.5 (C-2''), 73.8 , 74.1 , 78.2 (C-2), 80.5 (C-4''), 95.4 (C-6), 95.9 (C-8), 100.1 (C-1''), 100.7 (C-1'''), 103.2 (C-10), 111.5 (C-5'), 113.9 (C-2'), 117.6 (C-6'), 130.7 (C-1'), 146.0 (C-3'), 147.8 (C-4'), 162.2 (C-9), 162.7 (C-5), 166.9 (C-7), 196.7 (C-4).Hesperetin 7-3 and pertussis vaccine were used as the adjuvants . Sensitization was made by injecting a mixture (0.6 mL) of the antigen and the adjuvant into the paws of each rat . Paw edema was measured 24 h after injection and the treated rats were divided in groups with an equal average swelling volume. Each sample was dissolved in physiological saline containing 10% Nikkol and the solution was injected daily into the rat for 11 days starting on the day of grouping. The amount of IgE was measured by the passive cutaneous anaphylaxis method on the 15th day [The inhibitory action of hesperetin α-glycosides on IgE antibody formation was examined as follows. Ovalbumin was used as the antigen (1 mg/rat), and Al(OH)15th day . The res2- generation from rat neutrophils were examined as follows. Male Wistar rats, each weighing 250 to 300 g, were used. Under ether anesthesia, whole blood was collected from the carotid artery and diluted twice with Hanks' balanced salt solution (HBSS) (pH 7.4). Neutrophils were purified by Percoll density gradient centrifugation. O2- generation from rat neutrophils was measured by the cypridina luciferin analog-dependent chemiluminescence method [6 cells/mL) were incubated for 3 min in HBSS containing 0.4 mM of cypridina luciferin analog and 50 μM of hesperetin α-glycoside or authentic antiallergic agent at 37 °C in the dark. Five seconds later, N-formyl-Met-Leu-Phe (fMLP) (2.5 μM) was added into the assay mixture. Cypridina luciferin analog-dependent chemiluminescence was monitored with a Lumicounter ATP-237 (Advantec Co. Ltd.). The results are expressed in terms of the percentage reduction of the O2- generation from rat neutrophils at 5 min after the administration of fMLP by test compounds.Effects of hesperetin α-glycosides on Oe method . An authi.e., α-glucosides and α-maltosides, were successfully synthesized through sequential biocatalytic glycosylation by X. campestris and CGTase for the first time. It should be emphasized that new vitamin P glycosides, i.e., hesperetin α-maltosides, which are glycosylated at each phenolic hydroxyl group of hesperetin, can be prepared by the present two-step glycosylation system. Hesperetin 7-O-α-glucoside and hesperetin 7-O-α-maltoside showed suppressive action on IgE antibody formation and exhibited inhibitory effects on O2- generation from rat neutrophils. These new vitamin P compounds would be useful food ingredients with potent anti-allergic activities. Further studies on the physiological activity of hesperetin α-glycosides, such as cytotoxicity and anticancer activity, are now in progress.Hesperetin α-glycosides,


Their structures were elucidated through spectralanalysis using MS and NMR spectrometers.Two new monocyclic monoterpenoid glycosides, Mentha species are used for their flavoring and medicinal properties widely throughoutthe world + at m/z 373.1521([C16H30O8Na]+), and the EI-MS gave fragment-ion peaksat m/z 171 [M + 1–162(glucosyl)-H2O]+and 153 [M + 1–162(glucosyl)-2H2O]+corresponding to a molecular formula C16H30O8, with thepresence of 16 carbon signals in the 13C-NMR spectrum.Compound 1 was obtained as a pale amorphous powder. Its HR-ESI-MS displayed quasi-molecular-ionpeak , a tertiary methyl δ 1.21 ], two methylenes δ 1.37 , 1.40  and 1.57 ], four methines (two of them was oxygenated) δ 3.82 , 3.33 , 2.31 , and 1.69 ],and an oxygentated quaternary carbon, suggesting that compound 1 was a menthane-type monoterpenewith three OH-groups  was evident from 1H- and 13C-NMR of 1. The J value(8.2 Hz) of the anomeric proton concluded the β-configuration of the glucosemoiety, suggesting that 1 was a p-menthane-1,2,3-triol glycoside. This was furtherconfirmed by the HMBC experiment, in which correlations of the glucosyl H-1′ (δ4.33) with the C(3) (δ 81.9) were observed. Furthermore, other HMBC correlationsconfirmed the structure of compound 1. Thus, these 2D-NMR methods deduced compound 1 asp-menthane-1,2,3-triol 2-Oβ-D- glucopyranoside. The couplingconstants of 10.8 Hz for H-C(3)/H-C(2), 9.2 Hz for H-C(3)/H-C(4) for 1 showed thatH-C(2), H-C(3) and H-C(4) were axial protons. The relative configuration at C(1) was determined fromROESY correlation of δ 1.21 (Me(7)) with H-2 (δ 3.33). It was in goodagreement with those of rel-p-menthane-1,2,3,6-tetrol [rel--configuration.The 2 was obtained as a white amorphous powder. Its molecular formula was assigned asC16H28O8 on the basis of the 13C-NMR data and negativeHR-ESI-MS (m/z 347.1711 [M-H]-), which was 2 amu less than that of 1.Compound 1H- and 13C-NMR spectra of compounds 1 and 2 were very similar and gavesame signals assignable to two secondary methyl, a tertiary methyl, two methylenes, two oxygenatedmethines, an oxygenated quaternary carbon and a β-D-glucopyranosyl unit. Comparison ofthe NMR data of 2 and 1 indicated that the only difference was the presence of two olefinic carbonsin 2 instead of the two methines in 1. It suggested that a double bond situated at C(4) and C(8)positions in 2. This was supported by the IR spectrum showing a strong band at1649 cm-1, probably due to a tetra-substituted double bond and the correlations ofthe H-C(5) with the olefinic carbons C-4 and C-8 observed in the HMBC spectrum. The1H-1H COSY interactions of H-2 (δ 3.37)/H-3 (δ5.02), and H-5 /H-6  provided C-2 and C-3positions. This was sustained by the HMBC experiment showing correlations of H-3 (δ5.02) with the C(1), C(2), C(4), C(5) and C(8) observed, respectively. It reveals aterpinolene-1,2,3-triol fragment in 2 [1H-1H COSY and HMBC experiments. The HMBC correlations of glucosyl H-1′(δ 4.09) in 2 with the C(3) at δ 76.1 confirmed the location of glucosylat C(2). The coupling patterns [ and ] demonstrated that H-C(2) and H-C(3)were axial-equatorial or equatorial-equatorial couplings. In the ROESY experiments that H-2 and Me-7ROESY correlation was missing, while H-3 and Me-7 was present. It was illustrated that H-2 and H-3trans located on the alpha and beta face, respectively. Therefore, the structure of 2 wasdetermined to be rel-  terpinolene-1,2,3-triol3-Oβ-D- glucopyranoside c = 0.187, MeOH), IR(KBr): 3363, 2919, 1372, 1259, 1162, 1034. 1H-NMR  and13C-NMR : Table m/z 171 [M + 1 − 162(glucosyl)-H2O]+ and 153 [M + 1 − 162(glucosyl)-2H2O]+, 135, 127, 112, 97, 84, 73, 55. HR-ESI-MS: m/z 373.1521([C16H30O8Na]+), calcd forC16H30O8Na, 373.1990. terpinolene −1,2,3-triol3-O-β-D- glucopyranoside (2): pale amorphous powder,[α]c = 0.323, MeOH), IR(KBr): 3308, 2938, 1649, 1449, 1341, 1259, 1076. 1H-NMR and 13C-NMR : Table m/z 347.2 [M-H]-, 185.1[M-H-162(glucosyl)]-. HR-ESI-MS: m/z 347.1711[M-H]-, calcd for C16H27O8, 347.1710.S,2R,3R tThe authors declare that they have no competing interests.GS carried out the chemical analysis-structure elucidation and drafted the Manuscript; CX carriedout the chemical and biological studies; BL conceived of the study and its design and coordinationof the scientific teams. All authors have read and approved the final manuscript.


A three-dimensional supramolecular network is formed by multiple O—H⋯O hydrogen-bonding interactions between the coordinated water donors and the uncoordinated carboxyl acceptors.In the title compound, [Fe(C N-benzoyldithiocarbazate to 5-phenyl-1,3,4-oxadiazole-2-thiol (pot) in the presence of ethylenediamine, and its first-row transition-metal complexes, see: Tripathi et al. -1,3,4-oxadiazole-2-thiol, see: Du et al. (2006         For the synthesis of 5-(4-pyridyl)-1,3,4-oxadiazole-2-thione, see: Young & Wood 1955. For met al. 2007. For ZnI al. 2006.       9H6N3O3S)2(H2O)4] = 0.036                           wR(F                           2) = 0.078                           S = 1.13                           2285 reflections185 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.33 e Å−3                        Δρmin = −0.25 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97  I. DOI: 10.1107/S1600536811038918/si2366Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Molecular docking study gave a clear insight into the structural activity relationship of the compounds in comparison with monastrol.Biginelli dihydropyrimidinone derivatives as structural analogs of monastrol, a known human kinesin Eg5 inhibitor, were synthesized. IC Their involvement as bases in nucleic acids has a great significance in drug design. Recent progress in the DHPM class of the anticancer agent monastrol, an inhibitor of human kinesin Eg5 , has ledConsiderable work has also been devoted to gain insights into the structure-activity relationship in the monastrol derivative series . Recentl3/DMSO-d6/CD3OD. Chemical shifts are reported in parts per million (ppm) from tetramethylsilane with tetramethylsilane as the internal standard. Data reported are as follows: chemical shift, multiplicity as singlet (s), doublet (d), triplet (t), quartet (q), broad singlet (br s) and multiplet (m), and coupling constants (Hz). Mass spectra were obtained on a Shimadzu GCMS-QP5050A. Combustion analyses were performed on a Perkin-Elmer 2400-II analyzer. The progress of the reactions was monitored by thin layer chromatography using F254 silica gel pre-coated sheets . Column chromatography was performed on silica gel (100 to 200 mesh). Solvents for extraction and chromatography were AR/HPLC grade  spectra were recorded on Bruker 400/500 MHz spectrometer in CDClA solution of arylaldehyde (0.28 mmol), ethyl acetoacetate (0.28 mmol), urea (0.42 mmol) and boric acid (0.056 mmol) in glacial acetic acid (15 mL) was heated at 100°C, stirring for 9 h. Progress of the reaction was monitored by thin layer chromatography (TLC) using hexane:ethyl acetate (4:6) as mobile phase. Then, the reaction mixture was cooled to room temperature, poured into ice-cold water (150 to 200 mL) and stirred for 15 min. The solid precipitate obtained was filtered, washed with ice-cold water and recrystallized with absolute alcohol to afford white solid crystals with a quantitative yield +.C. (Lit.  Mp. 212.v/v HCl and then extracted with ethyl acetate (3 × 15 mL). The ethyl acetate layer was separated, pooled over anhydrous sodium sulfate and evaporated under vacuum to give the crude acid. The crude acid was purified by column chromatography using chloroform:methanol (95:5) as mobile phase and silica gel (100 to 200 mesh) as stationary phase to afford the pure product +.A mixture of compounds 2a to 2d was added to tetramethyluronium tetraflouroborate (TBTU) (1.5 eq.) and diisopropylethylamine (2 eq.) in 2 mL dimethylformamide under nitrogen atmosphere. The mixture was stirred for 2 min at 25°C, and then, pyrrolidine/piperidine/morpholine  was added to it. Another 2 equivalents of diisopropylethylamine (DIPEA) was again added to it. The reaction mixture was stirred for 10 min. The reaction was monitored by TLC using chloroform:methanol (9:1). The reaction mixture was poured into 50 mL ice-cold water and was extracted with dichloromethane 3 × 20 mL). The extract was purified by column chromatography using chloroform:methanol (97:3) and silica gel (100 to 200 mesh) as stationary phase to afford the desired product ,20. mL. The 6-Methyl-4-phenyl-5-(pyrrolidine-1-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3a): White solid, Yield 81%. Mp. 258°C to 260°C. Rf: 0.46 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,224, 3,145 (N-H str.), 1,696 (C = O str.), 2,881 (C-H str.). 13 C NMR : δ 166.34 (C = O), 161.83 (C = O), 140.42 to 124.32 , 151.74 (=C-NH), 25.32 to 23.96 , 15.56 (CH3). 1 H NMR : 7.26 to 7.24 , 6.38 , 5.41 , 5.11 , 3.26 to 2.10 , 1.73 . MS (EI): 285 [M]+, 270 [M-CH3]+, 214 [M-C5H10N]+, 187 [M-C6H10NO]+, 172 [M-CH3-C6H10NO]+, 132 [M-C9H14N2O]+.6-Methyl-4-phenyl-5-(piperidine-1-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3b): White solid, Yield 83%. Mp. 266°C to 268°C. Rf 0.49 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,223, 3,095 (N-H str.), 1,681 (C = O str.), 2,858 (C-H str.). 13 C NMR : 165.88 (C = O), 162.28 (C = O), 141.56 to 125.56 (6 C aromatic), 152.26 (=C-NH), 104.48, 56.24 (CH), 26.32 to 23.80 , 15.78 (CH3). 1 H NMR : δ 8.44 , 7.35 , 7.33 , 7.31 , δ 7.26 , 7.19 , 7.17 , 2.95 , 1.34 , 1.10 . MS (EI): 299 [M]+, 284 [M-CH3]+, 214 [M-C5H10N]+, 187 [M-C6H10NO]+, 172 [M-CH3-C6H10NO]+, 133 [M-C9H14N2O]+.4-Methyl-5-(morpholine-4-carbonyl)-6-phenyltetrahydropyrimidin-2(1 H)-one (3c): White solid, Yield 83%. Mp. 233°C to 234°C. Rf 0.52 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,240 (N-H str.), 1,695 (C = O str.), 2,858 (C-H str.). 13 C NMR : 166.68 (C = O), 162.28 (C = O), 142.28 to 126.23 , 152.38 (=C-NH), 103.76, 56.22 (CH), 30.42 to 26.68 , 15.32 (CH3). 1 H NMR : δ 7.35 to 7.31 7.21  5.49 , 5.30 , 3.22 to 3.05  1.27 to 1.29 . MS (EI): 301 [M]+, 286 [M-CH3]+, 214 [M-C4H8NO]+, 186 [M-C5H8NO2]+, 172[M-CH3-C5H8NO2]+, 132 [M-C8H12N2O2]+.4-(4-Fluorophenyl)-6-methyl-5-(pyrrolidine-1-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3d): White solid, Yield 80%. Mp. 242°C to 244°C. Rf 0.48 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,242, 3,142 (N-H str.), 1,689, 1,660 (C = O str.), 2,954 (C-H str.). 13 C NMR : δ 166.57 (C = O), 160.15 (C = O), 162.15 to 114(6 C aromatic), 152.58 (=C-NH), 105.79, 55.6 (CH), 25.11 to 23.80 , 15.96 (CH3). 1 H NMR : 7.26 to 6.29 , 6.87 , 5.41 , 5.23 , 3.26 to 2.59 , 1.67 , 1.66 to 1.18 . MS (EI): 303 [M]+, 288 [M-CH3]+, 233 [M-C4H8NO]+, 205 [M-C5H8NO2]+, 190 [M-CH3-C5H8NO2]+, 150 [M-C8H12N2O]+.4-(4-Fluorophenyl)-6-methyl-5-(piperidine-1-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3e): White solid, Yield 82%. Mp. 264°C to 266°C. Rf: 0.53 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,240 (N-H str), 1,691 (C = O str), 1,658 (amide C = O str). 13 C NMR : δ 165.76 (C = O), 160.52 (C = O), 163.18 to 113.32 , 153.26 (=C-NH), 106.72, 55.4 (CH), 26.52 to 23.20 , 15.40 (CH3). 1 H NMR : δ 7.55 , 7.35 to 7.30 , 7.05 to 7.01 , 5.45 , 5.43 , 3.86 to 3.04 , 1.76 , 1.60 to 1.55 , 1.46 to 1.41 , 1.29 to 1.24. MS (EI): 317 [M]+, 302 [M-CH3]+, 233 [M-C5H10N]+, 222 [M-C6H4F]+, 205 [M-C6H10NO]+, 193 [M-CH3-C6H10NO]+, 150 [M-C9H14N2O]+.4-(4-Fluorophenyl)-6-methyl-5-(morpholine-4-carbonyl)-3, 4-dihydropyrimidin-2(1 H)-one (3f): White solid, Yield 82%. Mp. 230°C to 232°C. Rf 0.51 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,248, 3,150 (N-H str.), 1,681, 1,608 (C = O str.), 2,875 (C-H str.). 13 C NMR : δ 166.22 (C = O), 161.36 (C = O), 162.78 to 113.76 , 152.64 (=C-NH), 105.63, 55.28 (CH), 29.22 to 24.46 , 15.63 (CH3). 1 H NMR : 7.27 to 6.96 , 6.41 , 5.42 , 5.10 , 3.25 to 3.027 , 1.72 , 1.48 to 1.18 . 319 [M]+, 303 [M-CH3]+, 232 [M-C4H8NO]+, 205 [M-C5H8NO2]+, 190 [M-CH3-C5H8NO2]+, 150 [M-C8H12N2O2]+.4-(4-Chlorophenyl)-6-methyl-5-(pyrrolidine-1-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3 g): White solid, Yield 84%. Mp. 260°C to 262°C. Rf 0.42 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,227, 3,151(N-H str.), 1,676, 1,608 (C = O str.), 2,862 (C-H str.). 13 C NMR : δ 165.82 (C = O), 162.28 (C = O), 139.70 to 115.92 , 153.72 (+C-NH), 103.02, 56.75 (CH), 24.78 to 23.20 , 15.34 (CH3). 1 H NMR : δ 7.55 , 7.32 to 7.29 , 7.28 to 7.26 , 5.44 , 5.46 , 3.33 , 3.02 , 2.68 , 1.80 , 1.72 to 1.61 . ESI-MS: 320 [M]+, 249 [M-C5H8N]+, 303 [M-CH4]+, 207 [M-C6H5Cl]+.4-(4-Chlorophenyl)-6-methyl-5-(piperidine-1-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3 h): White solid, Yield 86%. Mp. 253°C to 255°C. Rf 0.48 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,227, 3,151(N-H str.), 1,676, 1,608 (C = O str.), 2,862 (C-H str.). 13 C NMR : δ 166.25 (C = O), 161.48 (C = O), 138.78 to 116.26 , 152.58 (=C-NH), 103.38, 56.53 (CH), 25.20 to 23.64 , 15.62 (CH3). 1 H NMR : δ 7.70 , 7.32 to 7.24 , 3.48 to 3.02 , 1.79 , 1.66 to 1.25 . MS (EI): 333[M-1]+, 318 [M-CH3]+, 249[M-C4H8NO]+, 221 [M-C5H8NO2]+, 206[M-CH3-C5H8NO2]+, 166 [M-C9H14N20]+.4-(4-Chlorophenyl)-6-methyl-5-(morpholine-4-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3i): White solid, Yield 84%. Mp. 228°C to 230°C. Rf 0.44 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,264, 3,102 (N-H str.), 1,681, 1,624(C = O str.), 2,858 (C-H str.). 13 C NMR : 165.62 (C = O), 162.36 (C = O), 139.28 to 116.44 , 152.76 (=C-NH), 104.28, 56.24 (CH), 29.76 to 24.38 , 15.78 (CH3). 1 H NMR : 7.28 to 7.20 , 7.04 , 5.41 , 5.27 , 3.25 to 3.03 , 2.22 to 2.10 , 1.72 . MS (EI): 335[M]+, 320 [M-CH3]+, 249 [M-C4H8NO]+, 221[M-C5H8NO2]+, 207 [M-CH3-C5H8NO2]+, 166[M-C8H12N2O2]+.4-(2-Chlorophenyl)-6-methyl-5-(pyrrolidine-1-carbonyl)-3, 4-dihydropyrimidin-2(1 H)-one (3j): White solid, Yield 83%. Mp. 223°C to 224°C. Rf 0.53 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,221, 3,095 (N-H str.), 1,686 (C = O str.), 2,877 (C-H str.). 13 C NMR : 166.24 (C = O), 162.36 (C = O), 150.25 (=C-NH), 143.66 to 121.54 , 105.65, 53.44 (CH), 26.28 to 23.93 , 15.65 (CH3). 1 H NMR : δ 7.52 to 7.50 , 7.34 to 7.32 , 7.30 to 7.23 , 7.22 to 7.20, 6.91 , 5.76 , 5.36 , 3.37 to 3.20 , 1.89 , 1.79 . MS (EI): 319 [M]+, 304 [M-CH3]+, 284 [M-Cl]+, 249 [M-C4H8N]+, 221 [M-C5H8NO], 206 [M-CH3-C5H8NO]+, 166 [M-C8H12N2O]+.4-(2-Chlorophenyl)-6-methyl-5-(piperidine-1-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3 k): White solid, Yield 80%. Mp. 240°C to 242°C. Rf 0.52 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,222, 3,109 (N-H str.), 1,685 (C = O str.), 2,949 (C-H str.). 13 C NMR : 166. 38 (C = O), 161.72 (C = O), 141.56 to 122.38 , 151.36 (=C-NH), 104.72, 54.46 (CH), 25.92 to 23.80 , 15.55 (CH3). 1 H NMR : 7.49 to 7.48 , 7.43 , 7.35 to 7.33 , 7.28 , 7.24 to 7.22 , 5.74 , 5.40 , 3.35 to 3.21 , 1.84 , 1.49 to 1.33 . MS (EI): 333 [M-1]+, 318 [M-CH3]+, 298 [M-Cl]+, 249 [M-C5H10N]+, 221 [M-C6H10NO]+, 207 [M-CH3-C6H10NO]+.4-(2-Chlorophenyl)-6-methyl-5-(morpholine-4-carbonyl)-3,4-dihydropyrimidin-2(1 H)-one (3 l): White solid, Yield 80%. Mp. 240°C to 242°C. Rf 0.52 (Chloroform:methanol (9:1)). IR (KBr) cm−1: 3,240, 3,099 (N-H str.), 1,685(C = O str.), 2,860 (C-H str.). 13 C NMR : 165.42 (C = O), 161.56 (C = O), 140.49 to 121.25 , 153.25 (=C-NH), 104.72, 54.72 (CH), 28.56 to 24.78 , 15.45 (CH3). 1 H NMR : 7.47 , 7.40 to 7.38 , 7.31 to 7.18 , 7.24 to 7.22 , 7.20 to 7.18 , 3.22 to 2.77 , 1.77 . 335[M]+, 320 [M-CH3]+, 249 [M-C4H8NO]+, 221[M-C5H8NO2]+, 207 [M-CH3-C5H8NO2]+, 166[M-C8H12N2O2]+.in vitro cytotoxicity potential of these monastrol mimics, 3--2,5-diphenyltetrazolium bromide (MTT) assay on HepG2 and HeLa cell lines was performed. Exponentially growing cell lines were harvested from 25 cm2. Tissue culture flasks and a stock cell suspension (1 × 105 cell/mL) were prepared. A 96-well flat-bottom tissue culture plate was seeded with 1 × 104 cells in 0.1 ml of MEM and DMEM mediums supplemented with 10% FBS and allowed to attach for 24 h. Test compounds were prepared just prior to the experiment in 0.5% DMSO and serially diluted with medium to get the working stock of 500, 250, 125 and 62.5 μg/mL solution. After 24 h of incubation, cells were treated with 20 μL of test solutions from the respective top stocks, 80 μL of fresh medium was added, and the cells were incubated for 48 h. The cells in the control group received only the medium containing 0.5% DMSO. Each treatment was performed in triplicates. After the treatment, the drug-containing media was removed and washed with 200 μL of phosphate buffered saline (PBS). To each well of the 96-well plate, 100 μL of MTT reagent  was added and incubated for 4 h at 37°C. After 4 h of incubation, the plate was inverted on tissue paper to remove the MTT reagent. To solubilize formazan crystals in the wells, 100 μL of 100% DMSO was added to each well. The optical density was measured by an enzyme-linked immunosorbent assay plate reader at 540 nm [In order to assess the t 540 nm .et al.[Molecular modeling studies were performed using a flexible docking method with the Glide version 2010 as described by Yan et al. and Garcet al.. The X-rThe dihydropyrimidinones in this study were prepared by the Biginelli reaction  of the sSince mild hydrolyzing agent could prevent the extent of decarboxylation, lithium hydroxide with different solvent combinations (methanol/THF/dioxane)  was usedEarlier literature reports of DHPM esters hydrolysis in ethyl acetate and sodium hydroxide   also faiAcid hydrolysis using concentrated HCl and TFA (25°C/reflux) also failed to yield any acid. Refluxing with KOH and ethanol led to decarboxylation due to higher refluxing temperature (around 80°C). These observations propelled us to use a solvent with a lower boiling point.Studies also reported that refluxing in methanol in the presence of aqueous NaOH (10%) led to hydrolysis and consequent decarboxylation ,17. HencThe synthesis of compounds 3a to 3 l with a stable amide linker between DHPMs and cyclic amines was achieved via TBTU/DIPEA-promoted coupling reaction, which was quick and high yielding. The physical data of all the synthesized compounds are summarized in Table in vitro cytotoxicity assay suggest that compounds 3 g and 3 h were found to be the most potent against the HepG2 cell lines with IC50 124.46 and 120.62 μg/mL, respectively. However, most of the compounds exhibited weak activity (IC50 200 μg/mL) against HeLa cell lines. Hence, it was anticipated that substitution of electron-withdrawing substituents, such as chlorine, at the para position may be essential for the ligand-receptor interaction. Structure-activity relationship (SAR) analysis revealed that compounds with weakly basic pyrrolidine and piperidine substitution in the side chain attenuate the anticancer activity. In contrast, morpholine was not tolerated at the side chain of DHPMs and hence resulted in decreased IC50. Molecular modeling studies showed the absence of hydrogen bonding interactions with Glu-118, which could be attributed to the missing hydrogen bond donor on the phenyl ring. Hydrogen bonding interactions of water molecules with the oxygen atom of morpholine in compounds 3c, 3f, 3i and 3 l proved to be fatal for anticancer activity. In vitro data for these compounds are summarized in Table The data obtained from 50 of 180.92, 166.96 and 207.45 μg/mL and 216, 189 and 240 μg/mL against HePG2 and HeLa cell lines, respectively, served as the template for cytotoxicity potential comparison. Replacement of the protons in compounds 3a, 3b and 3c with fluorine at the para position of the phenyl ring did not improve the cytotoxicity against both cell lines. However, compound 3d showed a better IC50 (178 μg/mL) than its analog 3a against HepG2 cell lines. Encouraged by this result, fluorine was replaced by chlorine at the same position on the phenyl ring in compounds 3d, 3e and 3f to afford compounds 3 g, 3 h and 3i. Surprisingly, compound 3 g and 3 h significantly inhibited the proliferations of HepG2 cell lines with IC50 of 124.46 and 120.62 μg/mL. The anticancer activities of these two compounds against HeLa cell lines were equally frustrating like the previous results. The possibility to change the position of chlorine from para to ortho in the phenyl ring was then explored. Interestingly, this modification proved detrimental, and compounds 3j, 3 k and 3 l displayed IC50 of 190 μg/mL.To determine the SAR, functionality modification was carried out on the phenyl ring, while different cyclic amines were introduced at the side chain of DHPMs. Unsubstituted compounds  which displayed ICIn order to further investigate the relationships between the virtual receptor-ligand binding interaction and physicochemical properties of the new compounds with their anticancer activity, molecular modeling studies were performed. Docking analysis revealed that hydrogen bond formation and hydrophobic interactions were the key factors affecting inhibitory action of the compounds. The Glu-116, Gly-117, Glu-118, Ar-119, Trp-127, Pro-137, Tyr-211 and Leu-214 of Eg5 protein were found to be directly interacting with the synthesized DHPMs. Most of the synthesized compounds showed hydrogen bonding interaction with Glu-116 as monastrol. However, the unsubstituted compounds, except 3c  with the residues Glu-116 and Glu-118 Figure .The most active compounds, 3 h against HepG2 Figure  and compin vivo studies. This shows the potential of the compounds to bind with the enzyme effectively and inhibit cell proliferation with minimized toxic effects. Optimal pharmacokinetic properties such as lipophilicity and absorption were established by the QLogP values and polar surface areas, which correlated with the number of hydrogen bond donors/acceptors -2,5-diphenyltetrazolium bromide; HCl: hydrochloric acid; TBTU- O: (Benzotriazol-1-yl)-N,N,N',N'- tetramethyluronium tetraflouroborate; DIPEA: diisopropylethylamine; ICThe authors declare that they have no competing interests. Proton NMR spectrum of compound 1d.Click here for file Mass spectrum of compound 1d.Click here for file Carbon NMR spectrum of compound 3f.Click here for file Mass spectrum of compound 3f.Click here for file Proton NMR spectrum of compound 3 g.Click here for file Mass spectrum of compound 3 g.Click here for file Proton NMR spectrum of compound 3j.Click here for file Mass spectrum of compound 3j.Click here for file


H-1λ3,2-benziodaoxol-3-one (3), a hypervalent-iodine-based electrophilic trifluoromethylating reagent, is described. Whereas considerations based on cyclic voltammetry and X-ray structural properties would predict an inferior reactivity when compared to the non-nitrated derivative 2, 19F NMR kinetic studies showed that this new derivative is almost one order of magnitude more reactive. Furthermore, differential scanning calorimetry measurements indicated that, in addition, it is also safer to handle.The synthesis of 5-nitro-1-(trifluoromethyl)-3 Re. Re4) ha13C NMR chemical shift of the quaternary phenyl carbon attached to iodine (CAr-I) in 5 and comparison to literature data available for 2-iodobenzoic acid (4) indeed confirmed the installation of an electron-poor center + calcd for C7H3ClINO4, 326.8795; found, 326.8775; Anal. calcd for C7H3ClINO4: C, 25.68; H, 0.92; N, 4.28; found: C, 25.42; H, 1.02; N, 4.28.5-Nitro-1-(trifluoromethyl)-3H-1λ3,2-benziodaoxol-3-one (3): Spray-dried KF  was flame-dried under high vacuum, then suspended with 1-chloro-5-nitro-3H-1λ3,2-benziodaoxol-3-one  in dry MeCN (125 mL) under argon and stirred at 75 °C for 3.5 h, leading to the precipitation of a white solid. The suspension was cooled to 0 °C, TMSCF3  was added and stirring continued for 2 h. The reaction mixture was filtered through a Celite pad. Subsequently, the organic layer was washed with brine (50% sat), dried over Na2SO4, and concentrated under reduced pressure to turbidity. Filtration and washing with MeCN afforded the pure product  as a pale yellow solid. If impure product is obtained, recrystallization form MeCN may be applied. X-ray quality crystals were obtained by recrystallization from boiling bromobenzene. Mp 163 °C (dec); 1H NMR  δ 8.68 , 8.64 , 8.08 ; 13C NMR  δ 164.2 (CO2), 149.9 (CNO2), 134.8 (CCO2), 130.0 , 129.3 (CH para to CCO2), 125.7 (CH ortho to CCO2), 121.8 (CI), 106.6 ; 19F NMR  δ −32.6; HRMS–EI (m/z): [M + H]+ calcd for C8H3F3INNaO4, 383.8951; found, 383.8961; Anal calcd for C8H3F3INO4: C, 26.62; H, 0.84; N, 3.88; found: C, 26.67; H, 0.93; N, 4.08.File 119F NMR monitoring experiments and data analysis.Experimental details, crystallographic data (selected intermolecular bond lengths) as well as a description of the


S,3S,4-trihydroxybutyl)pyrazine (1), along with 12 known compounds, 2-(furan-2-yl)-5-pyrazine (2), (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (3), (S)-4-isopropyl-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (4), (4S)-4-(2-methylbutyl)-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (5), (S)-4-benzyl-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (6), flazin (7), perlolyrine (8), 1-hydroxy-β-carboline (9), lumichrome (10), 1H-indole-3-carboxaldehyde (11), 2-hydroxy-1-(1H-indol-3-yl)ethanone (12), and 5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (13), were isolated and identified from the fermentation broth of an endophytic actinomycetes, Jishengella endophytica 161111. The new structure 1 and the absolute configurations of 2–6 were determined by spectroscopic methods, J-based configuration analysis (JBCA) method, lactone sector rule, and electronic circular dichroism (ECD) calculations. Compounds 8–11 were active against the influenza A virus subtype H1N1 with 50IC and selectivity index (SI) values of 38.3(±1.2)/25.0(±3.6)/39.7(±5.6)/45.9(±2.1) μg/mL and 3.0/16.1/3.1/11.4, respectively. The 50IC and SI values of positive control, ribavirin, were 23.1(±1.7) μg/mL and 32.2, respectively. The results showed that compound 9 could be a promising new hit for anti-H1N1 drugs. The absolute configurations of 2–5, 13C nuclear magnetic resonance (NMR) data and the specific rotations of 3–6 were also reported here for the first time.A new alkaloid, 2-(furan-2-yl)-6-(2 Jishengella endophytica 161111, had been isolated from the root of the mangrove plant, Xylocarpus granatum (Meliaceae) oxazine-6-carbaldehyde (3) oxazine-6-carbaldehyde (4) oxazine-6-carbaldehyde (5) oxazine-6-carbaldehyde (6) + . The similarity of 1D- and 2D-NMR data (1 and the known crotonine (2) oxazine-6-carbaldehyde.Though absolute structure terature , no refetor rule  could be nm of 6 . The dedf (3R)-6 . Thus, t3–5 were found to have the molecular formulas of C12H15NO3, C11H13NO3, and C12H15NO3 by HRESI-MS peaks at m/z 222.1131 [M + H]+, 208.0974 [M + H]+, and 222.1129 [M + H]+, respectively. The similarity of ultraviolet (UV) and NMR spectra between 3–5 and 6 indicates compounds 3–5 contain the same3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde nucleus. The remaining moieties of C4H9, C3H7 and C4H9 for 3–5 could be deduced as isobutyl, isopropyl, and sec-butyl, respectively, from their 1D NMR data  to H-7 (δH 6.31) combined with the key HMBC correlations from H-11  to C-2 (δC 167.5), C-3 (δC 56.4), C-13 (δC 21.6), and C-14 (δC 23.3), from H-9  to C-2, C-7 (δC 107.2), and C-8 (δC 132.5), from H-3 (δH 5.65) to C-2, C-8, C-12 (δC 24.7), of H-7 (δH 6.31) to C-5 (δC 130.7), C-6 (δC 125.2), of H-6 to C-7, C-8, and C-10 (δC 180.1). The same lactone sector rule and ECD quantum chemical calculation used in 6 were also applied to elucidate the absolute configurations of 3–5. Thus, the same (3S)-configurations of 3–5 could be deduced from the same negative sign of the specific rotations and the positive CD Cotton effects at the long wave length along with concordance of CD with the calculated ECD of (3S)-isomer and opposition to ECD of (3R)-isomer -isomer .1–13 were tested for antivirus effects on H1N1 by the cytopathic effect (CPE) inhibition assay + .S,3S,4-trihydroxybutyl)pyrazine (2)2-(Furan-2-yl)-5-; UV (MeOH) λmax (log ε) 240 (3.2), 273 (3.4), and 328 (3.1) nm; CD  λext (Δε) 316 (−0.54), 267 (+0.35), 235 (−1.0) nm; 1H  and 13C NMR , see m/z 273.0849 [M + Na]+ .S)-4-Isobutyl-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (3); UV (MeOH) λmax (log ε) 254 (3.3), and 291 (3.9) nm; CD  λmax (Δε) 292 (+2.9), 248 (−1.0), 207 (+2.5) nm; 1H  and 13C NMR , see m/z 222.1131 [M + H]+ .S)-4-Isopropyl-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (4); UV (MeOH) λmax (log ε) 254 (3.4) and 291 (4.03) nm; CD  λmax (Δε) 298 (+16.1), 290 (−1.9), 280 (+6.6), 253 (−8.9), 208 (+8.8) nm; 1H  and 13C NMR , see m/z 208.0974 [M + H]+ .S)-4-(2-Methylbutyl)-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (5); UV (MeOH) λmax (log ε) 254 (3.6) and 291 (4.2) nm; CD  λmax (Δε) 293 (+5.8), 252 (−2.7), 208 (+3.1) nm; 1H  and 13C NMR , see m/z 222.1129 [M + H]+ .S)-4-Benzyl-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (6); UV (MeOH) λmax (log ε) 257 (3.4) and 295 (3.8) nm; CD  λmax (Δε) 293 (+13.1), 249 (−4.7), 211 (+6.8) nm; 1H  and 13C NMR  see m/z 256.0976 [M + H]+ .50IC was calculated as the compound concentration required inhibiting influenza virus yield at 48 h post-infection by 50%. Ribavirin was used as the positive control with an 50IC value of 23.1 ± 1.7 μg/mL.The antiviral activity against H1N1 virus was evaluated by the CPE inhibition assay ,46. Conf2 and 95% air at 37 °C, cell suspensions with a density of 4.6 × 104 cells/mL (198 μL) was plated in 96-well microtiter plates and incubated for 24 h. Then, the test solutions in MeOH (2 μL) were added to each well and further incubated for 36 h. The MTT solution  was then added to each well and incubated for 4 h. Old medium containing MTT (150 μL) was then gently replaced by dimethylsulfoxide (DMSO) and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 570 nm. Ribavirin was used as positive control .Cytotoxicity was assayed by the MTT . In the S,3S,4-trihydroxybutyl)pyrazine (1), was isolated and identified from the fermentation products of Jishengella endophytica 161111 endophytic with mangrove plant, Xylocarpus granatum (Meliaceae). The absolute configurations and the key reference data of [α]D, CD and 13C NMR of 2-(furan-2-yl)-5-pyrazine (2), (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (3), (S)-4-isopropyl-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (4), and (4S)-4-(2-methylbutyl)-3-oxo-3,4-dihydro-1H-pyrrolooxazine-6-carbaldehyde (5) were reported here for the first time. In addition, β-carboline alkaloid 9 exhibited moderate anti-H1N1 virus activity and weak cytotoxicity.A new pyrazine derivative, 2-(furan-2-yl)-6-(2


Vibrionaceae family. The purpose of the present study was to investigate the phylogeny and bioactivity of five Vibrionaceae strains with pronounced antibacterial activity. These were identified as Vibrio coralliilyticus (two strains), V. neptunius (two strains), and Photobacterium halotolerans (one strain) on the basis of housekeeping gene sequences. The two related V. coralliilyticus and V. neptunius strains were isolated from distant oceanic regions. Chemotyping by LC-UV/MS underlined genetic relationships by showing highly similar metabolite profiles for each of the two V. coralliilyticus and V. neptunius strains, respectively, but a unique profile for P. halotolerans. Bioassay-guided fractionation identified two known antibiotics as being responsible for the antibacterial activity; andrimid  and holomycin . Despite the isolation of already known antibiotics, our findings show that marine Vibrionaceae are a resource of antibacterial compounds and may have potential for future natural product discovery.On a global research expedition, over 500 bacterial strains inhibitory towards pathogenic bacteria were isolated. Three hundred of the antibacterial strains were assigned to the  Bioactive secondary metabolites are believed to play a key role in microbial interactions by mediating antagonistic activity and intercellular communication . In addiPseudoalteromonas spp. D20 −62.9° ; 1H NMR δH ppm: 0.75 , 0.80 , 1.07 , 1.78 , 2.30 , 2.65 , 2.77 , 2.91 , 3.92 , 4.62 , 5.28 , 5.90 , 6.01 , 6.18 , 6.26 , 6.53 , 7.00 , 7.20 , 7.29-7.31 , 8.11 , 8.42 , 11.36 ; 13C NMR δC ppm: 14.5 (C-6), 17.2 (C-4′), 18.3 (C-8‴), 19.4 (C-5′), 28.1 (C-3′), 39.0 (C-4), 41.9 (C-2″), 57.8 (C-3), 63.1 (C-2′), 124.2 (C-2‴), 126.4 (C-5″), 126.9 (C-7″), 128.1 (C-4‴), 128.2 (C-6″), 131.5 (C-6‴), 133.4 (C-7‴), 139.0 (C-5‴), 139.4 (C-3‴), 142.9 (C-4″), 164.3 (C-1‴), 169.9 (C-1″), 173.8 (C-2), 180.0 (C-5), 203.9 (C-1′); HRESIMS m/z 479.2435 .orange-yellow amorphous solid; UV (MeCN/H2O) λmax 200 (100%), 280 (40%) nm; 1H NMR δH ppm: 2.01 , 7.04 , 9.86 , 10.69 ; 13C NMR δC ppm: 22.4 (C9), 110.6 (C-1), 115.4 (C-5), 133.7 (C-2), 133.9 (C-6), 167.9 (C-4), 168.8 (C-8); HRESIMS m/z 213.9860 .orange-yellow prisms; UV (MeCN/HVibrionaceae bioactivity and physiology by showing a worldwide occurrence of marine strains producing antibacterial compounds. In addition, we underlined that chemotyping can support gene-based species identification and help resolving phylogenetic relationships within a genetically homogenous family such as the Vibrionaceae. The discovery of known antibiotics that are also produced by evolutionary distant microbes suggests an involvement of horizontal gene transfer, and indicates that these compounds are fundamental to compete and communicate in the natural habitat. The cosmopolitanism of identical antibiotics has major implications for natural product discovery strategies and stresses the need for careful dereplication in the initial stages of screening. An alternative approach could be the screening for largely untested bioactivities, for instance, interference with quorum sensing or modulation of gene expression.The present study adds to the knowledge of


The synthetic protocol for the reduction of alcohols to hydrocarbons by using hydriodic acid, first described by Kiliani more than 140 years ago, was improved to be more applicable to organic synthesis. Instead of a strongly acidic, aqueous solution, a biphasic toluene–water reaction medium was used, which allowed the conversion of primary, secondary and tertiary benzylic alcohols, in good yields and short reaction times, into the corresponding hydrocarbons. Red phosphorous was used as the stoichiometric reducing agent. Keto, ester, amide or ether groups are tolerated, and catalytic amounts of hydriodic acid (0.2 equiv) in the presence of 0.6 equiv phosphorous are sufficient to achieve conversion. The most commonly applied method is the Barton–McCombie reaction +, 161.1 (81) [M − C3H7]+, 186.1 (5) [M − H2O]+, 204.2 [M]+∙; HRMS (m/z): [M]+ calcd for C14H20O, 204.1514; found, 204.1511.-en-3-ol , entry 1(E)-1-Phenylhex-4-en-3-ol (3 (5 mL), H2O (2 × 5 mL) and dried with MgSO4. The solvent was removed with a rotary evaporator. (E)-1-Phenylhex-4-en-3-ol was obtained in 96% yield  in analytical purity. Analytical data were identical with the literature +, 91.1 (67) [C7H7]+, 105.1 (19) [M − C4H7O]+, 176.1 (50) [M]+.-en-3-ol , entry 2terature . 1H NMR 1-(4-Methoxyphenyl)-2-phenylpropan-1-ol (3 (3 mL), H2O (2 × 2.5 mL) and dried with MgSO4. The solvent was removed with a rotary evaporator. The crude product was purified by flash chromatography . 1-(4-Methoxyphenyl)-2-phenylpropan-1-ol was isolated as a yellow oil in 57% yield . Analytical data are identical with literature +, 224.1 (2) [M − H2O]+, 242.1 (1) [M]+∙.pan-1-ol , entry 5terature . 1H NMR 6,6-Dimethyl-2-phenylhept-4-yn-3-ol  was added dropwise through a septum by syringe. The reaction mixture was allowed to warm to rt before the solution of 2-propionaldehyde  in dry THF (5 mL) was added dropwise through a septum by syringe. This solution was stirred for 4.5 h. The reaction was stopped by the addition of H2O (10 mL). The aqueous phase was extracted with diethyl ether (3 × 15 mL), and the combined organic layers were dried with MgSO4. The solvent was removed with a rotary evaporator. The crude product was purified by flash chromatography . 6,6-dimethyl-2-phenylhept-4-yn-3-ol was isolated as a colorless oil in 46% yield . 1H NMR  δ 7.40–7.19 , 4.44 , 3.03 , 1.67 , 1.64 , 1.39 , 1.17 ; 13C NMR  δ 141.9, 128.8, 128.2, 127.0, 95.5, 78.1, 67.8, 67.5, 55.0, 46.1, 31.0, 30.0, 16.3; EIMS m/z (%): 57.1 (36) [C4H9]+, 99.1 (100), 105.1 (20) [C8H10]+, 216.2 (7) [M]+∙.-yn-3-ol , entry 4File 1Spectroscopic data for the synthesis of some alcohols. Quantitative mass efficiency analysis of four alternative alcohol reduction reactions.


Our results indicated that compound 7b exhibited the best potency against the tested cell lines. These results also suggest that pyrroloisoquinoline moiety constitutes a suitable scaffold to design new anti-proliferative agents. Two set of 2-aryl-5, 6-dihydropyrrolo  isoquinolines were designed and synthesized to evaluate their biological activities as topoisomerase inhibitors. Cytotoxic activity of the synthesized compounds 4a-e and 7a-d was assessed against several human cancer cell lines, including MCF-7 (breast cancer cell), HepG2 (liver hepatocellular cells), A549 , T47D  and Hela (Human cervix cancer). According to our results, HepG2 seems to be the most sensitive cell line for these compounds with mean IC Camptotheca acuminates   . DockingGeneral All chemicals, reagents and solvents were purchased from Merck and Aldrich Chemical Company. Melting points were determined with a Thomas–Hoover capillary apparatus. J) values are calculated in hertz (Hz) and spin multiplicities are given as s (singlet), d (double), t (triplet) and m (multi). The mass spectral measurements were performed on a 6410 Agilent triple quadrupole mass spectrometer (LCMS) with an electro spray ionization (ESI) interface. Infrared spectra were acquired using a Perkin Elmer Model 1420 spectrometer. A Brucker FT-500 MHz instrument  was used to acquire 1HNMR spectra with TMS as internal standard. Chloroform-D and DMSO-D6 were used as solvents. Coupling constants  were synthesized as depicted in sheme1. Dihydroisoquinoline derivatives were prepared starting from β-arylethylamine in three steps. Treatment of β-arylethylamine with the acetyl chloride at 0 °C in anhydrous CHCl3 and 3,4-dimethoxy phenyl acetic acid under free solvent condition at 200 °C, provided desired amides 2 and 5 respectively. The amides underwent a Bischler-Napieralski cyclization in the presence of POCl3, resulting in the formation of 3, 4-dihydroisoquinolines 3 and 6 . The subsequent reaction of these dihydroisoquinolines with various phenacyl bromide derivatives in anhydrous ACN gave the residue was subjected to a plate chromatography to obtain the purified compounds 4a-e and 7a-d. All compounds were stable and kept in dry place at 25 °C. The structures of the synthesized compounds were confirmed by IR, 1HNMR and ESI-MS.3, 4-Dimethoxyphenethyl acetamide m/z = 224 (M+1), 246 (M+23).Yield: 40%; Yellow solid; Mp= 85 °C; IR (KBr): υ (cm-1) NH (3256), 1633 (C=O); LC-MS (ESI) 6, 7-Dimethoxy-1-methyl-3, 4-dihydroisoquinolinem/z = 206 .Yield: 82%; Yellow solid; Mp= 166 °C; IR (KBr): υ (cm-1) 1603, 1517 (aromatic); LC-MS (ESI) 8, 9-Dimethoxy-2-phenyl-5, 6-dihydropyrrolo isoquinoline (4a)Yield: 55%; White solid; Mp= 134 °C; IR (KBr): υ (cm-1) 1606, 1556, 1502 (aromatic);m/z = 306 , 1HNMR (CDCl3/500MHZ): 3.05 , 3.93 , 3.98 , 4.10 , 6.68 , 6.75 , 7.11 , 7.19 , 7.36 , 7.58 .LC-MS (ESI) 8, 9-Dimethoxy-2-(4-methoxyphenyl)-5, 6-dihydropyrrolo isoquinoline (4b)m/z = 336 ; 1HNMR (CDCl3/500MHZ): 3.04 , 3.71 , 3.93 , 4.03 , 4.12 , 6.52 , 6.70 , 6.92 , 7.08 , 7.29 , 7.49 .Yield: 60%; White solid; Mp= 206 °C; IR (KBr): υ (cm-1) 1517, 1474 (aromatic); LC-MS (ESI) 8, 9-Dimethoxy-2-p-tolyl-5, 6-dihydropyrrolo isoquinoline (4c)J = 6.6 Hz), 3.93 , 3.98 , 4.09 , 6.68 , 6.74 , 7.11 , 7.18 , 7.29 , 7.47 . Yield: 52%; White solid; Mp= 152 °C, IR (KBr): υ (cm-1) 1602, 1507 (aromatic); LC-MS (ESI) m/z = 320 ; 1HNMR (CDCl3/500MHZ): 2.38 , 3.05 -8, 9-dimethoxy-5, 6-dihydropyrrolo isoquinoline (4d) J= 6.6 Hz), 3.93 , 3.98 , 4.10 , 6.69 , 6.75 , 6.96 , 7.09 , 7.32 , 7.49 . Yield: 48%; White solid; Mp= 185 °C; IR (KBr): υ (cm-1) 1554, 1496 (aromatic); LC-MS (ESI) m/z = 340 ; 1HNMR (CDCl3/500MHZ): 3.05 - 8,9-dimethoxy-5,6- dihydropyrroloisoquinoline (4e) m/z = 324 ; 1HNMR (DMSO-d6/300MHZ): 2.99 , 3.87 , 3.92 , 4.04 , 6.58 , 6.69 , 6.98  and 4-fluorophenyl H3 and H5),7.44 .Yield: 55%; White solid; Mp= 186 °C; IR (KBr): υ (cm-1) 1598, 1513(aromatic); LC-MS (ESI) 3,4-Dimethoxyphenethyl-2-acetamide (5)Yield: 54%; White solid; Mp= 120 °C, IR (KBr): υ (cm-1) NH (3320), 1631 (C=O); LC-MS (ESI) m/z = 359.9 (M+1), 381.8 (M+23).3,4-Dimethoxybenzyl-6,7-dimethoxy-3,4-dihydroisoquinoline (6)Yield: 60%, Yellow solid, Mp= 130 °C, IR (KBr): υ (cm-1)1597, 1559, 1506, LC-MS (ESI) m/z = 342 1--8, 9-dimethoxy-2-phenyl-5, 6-dihydropyrrolo isoquinoline (7a)Yield: 30%; White solid; Mp= 207 °C; IR (KBr): υ (cm-1) 1470, 1510 (aromatic); LC-MS (ESI) m/z = 442 ; 1HNMR (CDCl3/500MHZ): 3.09 , 3.42 , 3.73 , 3.89 , 3.92 , 4.14 , 6.69 , 6.72 , 6.89-6.91 , 7.18-7.22 , 7.29 .1--8, 9-dimethoxy-2-(4-methoxyphenyl)-5, 6-dihydropyrrolo  isoquinoline (7b)J= 6.5 HZ), 3.42 , 3.74 , 3.79 , 3.89 , 3.92 , 4.11 , 6.69 , 6.72 , 6.77 , 6.90-6.95 , 7.12 , 7.29 .Yield: 48%; White solid; Mp= 188 °C; IR (KBr): υ (cm-1) 1550, 1503 (aromatic); LC-MS (ESI) m/z = 471 ; 1HNMR (CDCl3/500MHZ): 3.07 -8, 9-dimethoxy-2-p-tolyl-5, 6-dihydropyrrolo isoquinoline (7c)m/z = 456 ; 1HNMR (CDCl3/500MHZ): 2.32 , 3.10 , 3.41 , 3.75 , 3.89 , 3.92 , 4.13 , 6.68 , 6.72 , 6.90-6.91 , 7.03 , 7.10 , 7.29 .Yield: 47%; White solid; Mp= 192 °C, IR (KBr): υ (cm-1) 1511, 1490 (aromatic); LC-MS (ESI) 2-(4-Chlorophenyl)-1--8, 9-dimethoxy--5, 6-dihydropyrrolo  isoquinoline (7d)J= 6.4 HZ), 3.41 , 3.76 , 3.89 , 3.93 , 4.12 , 6.67 , 6.72 , 6.88 , 6.92 , 7.11 , 7.17.Yield: 53%; White solid; Mp= 210 °C, IR (KBr): υ (cm-1) 1507, 1485 (aromatic); LC-MS (ESI) m/z = 476 ; 1HNMR (CDCl3/500MHZ): 3.08 , Hep-G2 (human liver carcinoma), A549 (human lung cancer), Hela (human cervix cancer) and T47D (human breast cancer) cell lines were purchased from Pasteur Institute, Tehran, Iran. Five human tumor cell lines were used to determine the cytotoxicity of the lamellarin derivativesThe cells were cultured in RPMI1640 medium at 37 °C under 5% CO2 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Cell viability was assayed by using a MTT method which is based on the reduction of 3--2, 5-diphenyltetrazolium bromide (MTT) dye to purple formazan crystals by mitochondrial succinate dehydrogenase enzyme in living cells. The cells were seeded into 96-well plates at a concentration of 104 cells/well and allowed to incubate for 24 h. The cells were incubated with increasing concentrations of test compounds for 48h. At the end of each treatment period, 10 μL of MTT (5 mg/mL in PBS) was added to each well and the microplate was incubated at 37 °C for 4 h. The medium with MTT was removed and 100 μL DMSO was added to each well to dissolve the insoluble formazan crystals. Plates were incubated for 20 min at 37 °C and the optical densities were read at 570 nm with a reference wavelength of 630 nm as background using a spectrophotometer plate reader . Doxorubicin was used as positive control and DMSO as the solvent of the test compounds. Data are presented as the mean of triplicate number of living cells and their capacity to reduce samples. IC50 was calculated by calibration curve using Prism software.Molecular modeling (docking) studiesDocking studies were performed using Autodock Vina software for all the synthesized compounds to study their interactions with the topotecan binding site of topoisomerase were simulated. The X-ray crystal structure of the topoisomerase structure in complex with topotecan (entry code 1K4T) was obtained from the RCSB Protein Data Bank. All the compounds were built using hyperchem version 8 and subsequently minimized. The protein structure was prepared for docking using AUTODOCK Tool. Polar hydrogens were added and non-polar hydrogens were merged and finally Kollman united atom charge and atom type parameters were added to 1K4T. Grid map dimensions (28×28×28) were set surrounding active site. The energy minimized ligands were docked in binding site of topoisomerase. The quality of the docked structures was assessed by measuring the intermolecular energy of the ligand-enzyme assembly. Two set of 2-aryl-5, 6-dihydropyrrolo isoquinoline were synthesized via multi-step reactions to prepare compounds 4a-e and 7a-d. All the compounds were confirmed using IR, ESI–MS and 1HNMR spectroscopies. Yields of the resulting compounds 4a-e and 7a-d ranged from 30% to 60%. Cytotoxic activity of the synthesized compounds 4a-e and 7a-d was assessed against several human cancer cell lines, including MCF-7 (breast cancer cells), HepG2 (liver hepatocellular cells), A549 , HeLa (human cervix cancer cells) and T47D . The results are shown in para position of phenyl ring may improve the cytotoxicity activity. In addition, compounds containing an additional phenyl ring at position 2 of pyrrole ring (compound 7a-7d) did not show higher anti-proliferative activity than compound 4a-e which does not have this group. Based on these results, the presence of phenyl ring at position 2 of pyrrole ring is not essential for cytotoxicity activity. Also, our results indicated that compound 7b exhibited the best potency against the tested cell lines and the highest activity was observed against HepG2. Accordingly docking studies were performed for compound 7b in the active site of topoisomerase and compared with topotecan. As shown in According to our results HepG2 seems to be the most sensitive while T47D was the most resistant cell line to these compounds. All the compounds, possessed satisfactory activity against HepG2 with mean IC50 values raging from 4.25 to 75.02 μM. Our results indicated that all compounds exhibited weak to moderate cytotoxicity activity for HeLa  and A549 . However, compounds 7c and 7d did not show cytotoxicity activity for A549 cell line at 100 μM concentration. These results also indicated that the synthesized compounds did not have considerable anti-proliferative activity against MCF-7 and T47D cell lines except compound 7b which had good activity for MCF-7 . These results revealed that the compounds containing electron donating groups at


HVTN 078 is a randomized, double blind phase 1b clinical trial to evaluate the safety and immunogenicity of heterologous prime/boost vaccine regimens (NYVAC-B/rAd5 vs. rAd5/NYVAC-B) in healthy, HIV-1 uninfected, Ad5 seronegative adult participants.The rAd5 vaccine expressed clade B Gag-Pol and the gp140 of HIV-1 92RW020 (clade A), HxB2/Bal-V3/ΔV1V2 (clade B) and 97ZA012 (clade C). The NYVAC-B vaccine expressed clade B Gag-Pol-Nef and the gp120 of Bx08 (clade B). 80 healthy, HIV-1 uninfected, Ad5 seronegative volunteers, aged 18 to 45 years, were randomized to the placebo arm (n=5) or one of 4 treatment (T) arms: T1 (n=30), 2xNYVAC-B/1xrAd5 (10E10); T2 (n=15), 1xrAd5 (10E8)/2xNYVAC-B; T3 (n=15), 1xrAd5 (10E9)/2xNYVAC-B; T4 (n=15), 1xrAd5 (10E10)/2xNYVAC-B.Intracellular cytokine staining responses  were assessed two weeks after the final vaccination.For CD4+ T cells, the overall response rates for IFN-γ and/or IL-2 among the vaccinees were 42.9%, 93.3%, 92.3%, and 85.7% for T1-T4, respectively; and the median response magnitudes for positive responders were 0.26%, 0.76%, 0.40%, and 0.76% for T1- T4, respectively. Both response rates (p<0.01) and magnitudes (p<0.03) of CD4+ T-cell responses were significantly lower in T1 compared to the other three treatment groups. For CD8+ T cells, the overall response rates were 65.5%, 73.3%, 76.9% and 85.7% for T1-T4, respectively; and median response magnitudes for positive responders were 0.32%, 0.99%, 1.86%, and 1.65%, respectively. Response rates were not significantly different between groups; however, response magnitudes were significantly lower in T1 compared to the other three arms (p<0.04).Priming with rAd5 followed by NYVAC-B boost is superior to priming with NYVAC-B followed by rAd5 boost for both response rates and the magnitude of CD4+ and CD8+ T-cell responses.