O-β-d-glucuronopyranosyl-30-norolean-12,20(29)-dien-23-oxo-28-oic acid, namely bigelovii D (11), was isolated from the hydroalcoholic extract of herbs of Salicornia bigelovii along with 10 known saponins (1–10). Their chemical structures were identified on the basis of spectroscopic analyses including two-dimensional NMR and a comparison with literature data. Some of these compounds showed potent antifungal activities in vitro. Compounds 3, 4, 5, 6, 7, 10 and 11 demonstrated potent inhibitory activities against Colletotrichum gloeosporioides and compound 11 displayed broad-spectrum inhibitory activity against Alternaria alternata, A. solani, Botrytis cinerea, C. gloeosporioides, Fusarium graminearum, F. verticilloides, Thanatephorus cucumeris and Sclerotinia sclerotiorum, with EC50 values ranging from 13.6 to 36.3 μg/mL.A new nortriterpene saponin, 3- Salicornia bigelovii is a salt-tolerant land plant that grows on salt marshes and muddy seashores and is widespread in tropical and subtropical North America, Asia and Europe. It belongs to Salicornia (Chenopodiaceae), which is a genus of annual, apparently leafless, halophytic herbs that have articulated and succulent stems − at m/z 629.3261 . The 1H-NMR and 13C-NMR ], one methine bearing an oxygen function [δH 4.17 ], three olefinic protons , an aldehyde group [δH 9.73 ]. These data were correlated with information from 13C-NMR spectrum. Two signals at δH 122.7 and 144.2 ascribable to C-12 and C-13 suggested a ∆12 oleanene skeleton. Four methyl resonances , four sp2-hybridized carbon signals  and an aldehyde group (δC 206.8), and two carboxy carbon  were shown in its 13C-NMR spectrum. The 13C-NMR spectrum of 11 displayed 35 carbon resonances of which 29 carbon signals were assigned to a noroleanic-acid-type triterpene moiety and six to a monosaccharide portion. The proton and carbon signals of 11 in the 1H- and 13C-NMR spectra resembled those of glucopyranosiduronic acid (6′-COOH). This assumption was supported by a set of 2D NMR experiments that provided decisive information concerning the structure of compound 11. The chemical shifts of all the individual protons and carbons of the sugar unit were assigned on the basis of 1H-1H COSY spectra analysis. The β-glucopyranosyluronic group, which was deduced from its 3JH-1, H-2 coupling constant of 7.8 Hz, was placed at C-3 of the aglycon on the basis of the HMBC correlation between the anomeric proton at δH 4.84  and the C-3 carbon resonance at δC 82.0. In the heteronuclear multiple bond correlations spectroscopy (HMBC) experiment -dien-23-oxo-28-oic acid and named bigelovii D.Compound , oleanolic acid 28-O-β-d-glucopyranoside (2), bigelovii A (3), 3-O-[(6’-ethyl-ester)-d-glucuronopyranosyl]-oleanolicacid-28-O-β-d-glucopyranoside (4), 3-O-[(6’-methyl-ester)-β-d-glucuronopyranosyl]-30-norolean-12,20(29)-dien-28-oic-28-O-β-d-glucuronopyranosyl ester (5), chikusetsusaponin IVa methyl ester (6), bigelovii B (7), boussingoside A2 (8), 3-O-β-d-glucuronopyranosyl-oleanolic acid 28-O-β-d-glucopyranoside (9) and 3-O-β-d-glucuronopyranosyl-oleanolic acid (10), respectively, by comparing their physical data  with those reported in the literature  to give five subfractions (Fr.2-1-Fr.2-5), and Fr.2-3 (1.5 g) was further subjected to RP-C18 CC  with MeOH:H2O (90:10–0:100 v/v) and RP-HPLC [MeOH:H2O ] to provide compound 1 (13.67 mg) and 2 (11.49 mg). Fr.3 (17.29 g) was separated by silica-gel CC  with a gradient elution of EtOAc–MeOH  to obtain seven subfractions (Fr.3-1-Fr.3-8). Fr.3-1 and Fr.3-2 was purified using a Sephadex LH-20 CC with a CHCl3:MeOH eluent  to give compound 3 (12.43 mg) and compound 4 (17.39 mg), respectively. Fr.4 (37.53 g) was separated by RP-C18  and eluted with MeOH–H2O  to produce nine subfractions (Fr.4-1-Fr.4-9). Fr.4-3 (153.93 mg) was purified by RP-HPLC [MeOH:H2O ] to give compound 5 (17.34 mg) and 6 (14.43 mg). Fr.4-4 (1037.61 mg) was purified by RP-HPLC [MeOH:H2O ] to give compound 7 (619.37 mg). Fr.4-5 (1025.47 mg) was purified by RP-HPLC using [MeOH:H2O ] to give compound 8 (521.92 mg) and 9 (16.33 mg). Fr.5 (17.61 g) was separated through Sephadex LH-20 CC  to give three subfractions (Fr.5-1→Fr.5-3). Fr.5-2 (1.7 g) was further separated via RP-C18 CC  with MeOH–H2O  and purified by RP-HPLC [MeOH:H2O ] to provide compound 10 (16.57 mg) and 11 (14.47 mg).The air-dried powder of the herbs (5 kg) of 11: white amorphous powder, m.p. 205–206 °C, c 0.479, MeOH), IR vmax cm−1: 3412, 1705 and 1645, HREI-MS (negative ion mode) m/z: 629.3261 [M − H]− . 1H-NMR, 13C-NMR, DEPT, 1H-1H COSY and HMBC data see Compound The pathogens, Alternaria alternata, A. solani, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium graminearum, F. verticilloides, Thanatephorus cucumeris, Sclerotinia sclerotiorum, used in the bioassays were identified by Pingping Song, at Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, China. Positive controls Amphotericin B (AMB) was purchased from Sigma-Aldrich .50 and 95% confidence intervals.Inhibitory activity against the hyphal growth of pathogenic fungus was determined using a growth rate method . A test 11) was isolated from the herbs of S. bigelovii, and it has a significant inhibitory effect on plant pathogenic fungus. Comparison of the structures of compound 3, 4, 5, 6, 7, 10 and 11 with those of inactive compounds, suggested that the aldehyde group at C-23, the carboxyl group at C-28 and the ester group at C-6’ of glucuronopyranosyl moiety might have contributed to the inhibition of the growth of C. gloeosporioides synergistically.In the present study, a new nortriterpenoid saponin (


Lactobacillus delbrueckii and cyclodextrin glucanotransferase (CGTase) as biocatalysts was investigated. The cells of L. delbrueckii glucosylated glycitein and daidzein to give their corresponding 4'- and 7-O-β-glucosides. The β-glucosides of glycitein and daidzein were converted into the corresponding β-maltooligosides by CGTase. The 7-O-β-glucosides of glycitein and daidzein and 7-O-β-maltoside of glycitein showed inhibitory effects on IgE antibody production. On the other hand, β-glucosides of glycitein and daidzein exerted 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity and supeoxide-radical scavenging activity.The production of β-maltooligosaccharides of glycitein and daidzein using  Glycitein and daidzein are important and bioactive isoflavones isolated from soybeans whose pharmacological properties such as anticancer, anti-inflammatory, neuroprotective, anticarcinogenic effects, and protective effects against bone loss, hormone-dependent and -independent cancers, cardiovascular diseases, and autoimmune diseases and have been widely studied ,9,10,11.et al. reported that glucosyl conjugation was far more effective than cyclodextrin complexation at enhancing the water solubility of hydrophobic compounds such as curcumin + peaks at 631.1640 for 4 and 631.1641 for 6 , 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 their chemical shifts of the carbon signals. The 1H-NMR spectra showed two proton signals at δ 5.05  and 5.11  for 4, and δ 5.07  and 5.19  for 6, indicating that the glucoside linkage in these compounds had each of α- and β-orientations. The HMBC spectra of 4 and 6 included correlations 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 5.11 (H-1'') and the carbon signal at δ 157.3 (C-4') for 4, and between the proton signal at δ 5.07 (H-1''') and the carbon signal at δ 79.9 (C-4'') and between the proton signal at δ 5.19 (H-1'') and the carbon signal at δ 152.5 (C-7) for 6. These data indicated that 4 and 6 were β-maltosyl analogues of 1, the sugar moiety of which was attached at their 4'- (4) and 7-positions (6). Thus, products 4 and 6 were identified as glycitein 4'-O-β-maltoside (4) and glycitein 7-O-β-maltoside (6), respectively.The HRFABMS spectra of 5 and 7 with pseudomolecular ion [M+Na]+ peak [m/z 793.2170 (5) and 793.2177 (7)] established a molecular formula of C34H42O20 , indicating that each product consisted of one substrate and three hexoses. The sugar components in these products were identified as glucose on the basis of the chemical shifts of the carbon signals. The 1H-NMR spectra showed three proton signals at δ 5.05 , 5.07 , and 5.12  for 5, and δ 5.07 , 5.09 , and 5.19  for 7, indicating that the glucoside linkage in these compounds had two α-orientations and one β-orientation. Correlations were observed in the HMBC spectra of 5 and 7 between the proton signal at δ 5.05 (H-1'''') and the carbon signal at δ 78.9 (C-4'''), between the proton signal at δ 5.07 (H-1''') and the carbon signal at δ 78.8 (C-4''), and between the proton signal at δ 5.12 (H-1'') and the carbon signal at δ 157.6 (C-4') for 5, and between the proton signal at δ 5.07 (H-1'''') and the carbon signal at δ 79.9 (C-4'''), between the proton signal at δ 5.09 (H-1''') and the carbon signal at δ 79.7 (C-4''), and between the proton signal at δ 5.19 (H-1'') and the carbon signal at δ 152.5 (C-7) for 7. These data indicated that 5 and 7 were β-maltotriosyl analogues of 1, the sugar moiety of which was attached at their 4'- (5) and 7-positions (7). Thus, the structures of products 5 and 7 were determined to be glycitein 4'-O-β-maltotrioside (5) and glycitein 7-O-β-maltotrioside (6), respectively.The HRFABMS spectrum of 8) with L. delbrueckii cells in the presence of glucose for 5 days gave daidzein 4'-O-β-D-glucoside  . Glycitein and daidzein were purchased from Sigma-Aldrich Co. CGTase was purchased from Amano Pharmaceutical Co. Ltd. The NMR spectra were recorded in DMSO-L. delbrueckii subsp. bulgaricus (Okayama University of Science) had the following composition (in grams per liter): 20 g of lactose, 5 g of yeast nitrogen base, 20 g of bacto casitone, 1 g of sorbitan monooleate, 2 g of ammonium citrate, 5 g of sodium acetate, 2 g of K2HPO4, 0.05 g of MnSO4, 0.1 g of MgSO4. The cells were grown in the culture medium with continuous shaking on a rotary shaker (120 rpm) at 30 °C.Culture medium used for growth of L. delbrueckii 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. The β-glucosides of glycitein and daidzein were prepared as follows: substrate  was added to ten 300 mL conical flasks containing L. delbrueckii cells (5 g) and glucose (1 g) in freshly prepared culture medium (100 mL). The mixture was incubated with 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 to remove the cells and the supernatant was extracted with n-butanol. The n-butanol fraction was purified by preparative HPLC on YMC-Pack R&D ODS column to give the β-glucoside products.The cultures of To a solution containing β-glucosides of glycitein or daidzein (0.1 mmol) and starch (5 g) in sodium phosphate buffer  was added CGTase (100 U). 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 the β-maltooligoside products. Spectral data of new compounds are as follows:Glycitein 4'-O-β-maltoside (4): FABMS m/z: 631.1640 [M+Na]+ ; 1H -NMR (DMSO-d6): δ 3.05-3.80 , 3.92 , 5.05 , 5.11 , 7.00 , 7.46 , 7.51 , 7.75 , 8.41 ; 13C-NMR (DMSO-d6): δ 56.1 (OCH3), 60.7 (C-6'''), 60.8 (C-6''), 69.5 (C-4'''), 72.0 (C-2'''), 72.8 (C-5'''), 73.3 (C-2''), 73.5 (C-3'''), 75.3 (C-5''), 75.9 (C-3''), 80.5 (C-4''), 98.0 (C-1''), 100.5 (C-1'''), 101.1 (C-8), 105.0 (C-5), 115.1 , 117.7 (C-10), 122.5 (C-1'), 124.6 (C-3), 130.3 , 148.8 (C-6), 151.0 (C-9), 152.2 (C-7), 153.8 (C-2), 157.3 (C-4'), 178.1 (C-4).Glycitein 4'-O-β-maltotrioside (5): FABMS m/z: 793.2170 [M+Na]+ ; 1H-NMR (DMSO-d6): δ 3.09-3.85 , 3.92 , 5.05 , 5.07 , 5.12 , 7.01 , 7.46 , 7.50 , 7.76 , 8.41 ; 13C-NMR (DMSO-d6): δ 56.1 (OCH3), 60.5 , 60.7 (C-6''), 69.5 (C-4''''), 71.5 (C-5'''), 72.0 (C-2'''), 72.4 , 73.0 (C-3''''), 73.3 (C-3'''), 73.8 (C-2''), 75.1 (C-5''), 75.9 (C-3''), 78.8 (C-4''), 78.9 (C-4'''), 98.1 (C-1''), 101.5 , 101.9 (C-8), 105.1 (C-5), 115.0 , 117.6 (C-10), 122.5 (C-1'), 124.5 (C-3), 130.3 , 148.7 (C-6), 151.0 (C-9), 152.2 (C-7), 153.8 (C-2), 157.6 (C-4'), 178.0 (C-4).Glycitein 7-O-β-maltoside (6): FABMS m/z: 631.1641 [M+Na]+ ; 1H-NMR (DMSO-d6): δ 3.09-3.77 , 3.90 , 5.07 , 5.19 , 7.01 , 7.46 , 7.52 , 7.76 , 8.41 ; 13C-NMR (DMSO-d6): δ 56.1 (OCH3), 60.2 (C-6'''), 60.9 (C-6''), 69.7 (C-4'''), 72.0 (C-2'''), 72.7 (C-5'''), 73.3 (C-2''), 73.5 (C-3'''), 75.1 (C-5''), 75.9 (C-3''), 79.9 (C-4''), 98.8 (C-1''), 100.6 (C-1'''), 101.0 (C-8), 105.1 (C-5), 115.2 , 117.7 (C-10), 122.5 (C-1'), 124.5 (C-3), 130.3 , 148.8 (C-6), 151.0 (C-9), 152.5 (C-7), 153.8 (C-2), 157.0 (C-4'), 178.0 (C-4).Glycitein 7-O-β-maltotrioside (7): FABMS m/z: 793.2177 [M+Na]+ ; 1H-NMR (DMSO-d6): δ 3.05-3.88 , 3.92 , 5.07 , 5.09 , 5.19 , 7.00 , 7.45 , 7.51 , 7.76 , 8.41 ; 13C-NMR (DMSO-d6): δ 56.1 (OCH3), 60.5 (C-6''''), 60.7 , 69.7 (C-4''''), 71.5 (C-5'''), 72.1 (C-2'''), 72.5 , 73.0 (C-3''''), 73.5 (C-3'''), 73.8 (C-2''), 75.1 (C-5''), 75.7 (C-3''), 79.7 (C-4''), 79.9 (C-4'''), 98.1 (C-1''), 100.5, 100.6 , 101.9 (C-8), 105.1 (C-5), 115.0 , 117.6 (C-10), 122.5 (C-1'), 124.5 (C-3), 130.2 , 148.8 (C-6), 151.0 (C-9), 152.5 (C-7), 153.8 (C-2), 157.0 (C-4'), 178.1 (C-4).3 and pertussis vaccine were used as the adjuvants . Sensitization was made by injection of 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 d starting on the day of grouping. Hydrocortisone was used as the positive control. The amount of IgE was measured by the passive cutaneous anaphylaxis method on the 15th day  × 100, where At and Ac are the respective absorbance at 517 nm of sample solutions with and without the test compounds. Antioxidant activity was expressed as the 50% inhibitory concentration (IC50).DPPH free-radical scavenging activities of β-glycosides of glycitein or daidzein were determined as follows: DPPH was dissolved in ethanol (500 μM) . The sam50 value is shown as the sample concentration at which 50% of superoxide-radical was scavenged.Superoxide was generated by the xanthine-xanthine oxidase system . ReactioL. delbrueckii and CGTase. The 7-O-β-glucoside of glycitein and daidzein and 7-O-β-maltoside of glycitein inhibited IgE antibody formation. On the other hand, β-glucosides of glycitein and daidzein exerted DPPH free-radical scavenging activity and supeoxide-radical scaventing activity.The β-maltooligosaccharides of glycitein and daidzein were successfully produced through two-step biocatalytic glycosylation by


Their structures were determined on the basis of spectroscopic techniques, including IR, NMR, and HR-ESI-MS.A new dicoumarinyl ether, 3-hydroxy-6-methoxy-7,7'-dicoumarinyl ether ( Stellera chamaejasme L. , has been used in China as an important traditional medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer, and tuberculosis −. The IR spectrum of 1 displayed absorptions of hydroxyl , α,β-unsaturated lactone , and phenyl  functionalities. In the 1H-NMR spectrum, vicinal pairs of signals typical of the H-3' and H-4' protons of an AX system at δH 6.31  and 7.96 , were observed, along with an ABX coupling pattern , 7.10  and 7.06 ), suggesting the presence of an 7-oxygenated coumarin moiety, which was further confirmed by the NOESY correlation of H-4' and H-5' (Compound and H-5' . δH 7.80) and H-5 (δH 7.24) showed a correlation in the NOESY experiment. Accordingly, cross-peaks between H-4 and C-5 (δC 109.9) and between H-5 and C-4 (δC 131.5) were evident in the HMBC experiment (δH 3.92) and H-5 in the NOESY spectrum indicated that the methoxy group was attached to C-6 (δC 146.6), which was further supported by the long-range correlation between the methoxy protons and C-6 in the HMBC spectrum. Compared with the known compound daphnoretin that was isolated from the same plant − 351.0505 .Umbelliferone (2) was obtained as colorless crystals, mp 222–224 °C, from MeOH; 1H-NMR (CD3OD) δ: 7.83 , 7.44 , 6.79 , 6.70 , 6.17 ; 13C-NMR (CD3OD) δ: 162.3, 161.8, 155.8, 144.6, 129.2, 113.1, 111.6, 102.0.1) was isolated from the EtOH extract of the roots of Stellera chamaejasme L together with the known compound umbelliferone (2).A new dicoumarinyl ether, 3-hydroxy-6-methoxy-7,7'-dicoumarinyl ether (


Cholestane-3β,5α,6β-triol  is formed from cholestan-5,6-epoxide  in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7β-hydroxycholesterol (7β-HC) can also be formed by free radical oxidation of cholesterol. Here we investigate how 3β,5α,6β-triol, 7-OC and 7β-HC are metabolised to bile acids. We show, by monitoring oxysterol metabolites in plasma samples rich in 3β,5α,6β-triol, 7-OC and 7β-HC, that these three oxysterols fall into novel branches of the acidic pathway of bile acid biosynthesis becoming (25R)26-hydroxylated then carboxylated, 24-hydroxylated and side-chain shortened to give the final products 3β,5α,6β-trihydroxycholanoic, 3β-hydroxy-7-oxochol-5-enoic and 3β,7β-dihydroxychol-5-enoic acids, respectively. The intermediates in these pathways may be causative of some phenotypical features of, and/or have diagnostic value for, the lysosomal storage diseases, Niemann Pick types C and B and lysosomal acid lipase deficiency. Free radical derived oxysterols are metabolised in human to unusual bile acids via novel branches of the acidic pathway, intermediates in these pathways are observed in plasma.  •Free radical derived oxysterols metabolised to unusual bile acids.•Metabolism proceeds via novel branches of the acidic pathway.•Bile acids with:- 3β,5α,6β-triol; 3β,7β-diol-5-ene or 3β-ol-5-en-7-one in A/B-ring.•Unusual bile acids found in plasma from patients with NPC, NPB and LALD.•Intermediates in the biosynthesis of unusual bile acids found in plasma. ACOTacyl-CoA thioesterasesACOX2acyl-coenzyme A oxidase 2AMACRalpha-methylacyl-CoA-racemaseBACSbile acyl CoA-synthetaseChEHcholesterol epoxide hydrolaseChOxcholesterol oxidaseCYPcytochrome P450DBPD bifunctional proteinDDAdendrogenin AHhHedgehogHSDhydroxysteroid dehydrogenaseGlcNAcN-acetylglucosamineGPGirard P reagentLALDlysosomal acid lipase deficiencyLC-MSliquid chromatography mass spectrometryMRMmultiple reaction monitoringnMSmass spectrometry with multistage fragmentationNPANiemann-Pick disease type ANPBNiemann-Pick disease type BNPCNiemann-Pick disease type CRICsreconstructed ion chromatogramsSLOSSmith-Lemli-Opitz syndromeSmoSmoothenedSPCxsterol carrier protein x3β-HCA3β-hydroxycholest-5-en-(25R)26-oic acid3βH,7O-CA3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid5-BA3βH,7O-Δ3β-hydroxy-7-oxochol-5-enoic acid3β,5α-diHC-6O3β,5α-dihydroxycholestan-6-one3β,5α,6β-triHBA3β,5α,6β-trihydroxycholanoic acid3β,5α,6β-triHCa3β,5α,6β-trihydroxycholestan-(25R)26-oic acid3β,5α,6β-triolcholestane-3β,5α,6β-triol3β,5α,6β,24-tetraHCa3β,5α,6β,24-tetrahydroxycholestan-26-oic acid3β,5α,6β,26-tetrolcholestane-3β,5α,6β,(25R)26-tetrol3β,7α-diHCA3β,7α-dihydroxycholest-5-en-(25R)26-oic acid3β,7β-diHCA3β,7β-dihydroxycholest-5-en-(25R)26-oic acid5-BA3β,7β-diH-Δ3β,7β-dihydroxychol-5-enoic acid3β,7β,24-triHCA3β,7β,24-trihydroxycholest-5-en-26-oic acid3β,24-diH,7O-CA3β,24-dihydroxy-7-oxocholest-5-en-26-oic acid5α,6-EC5α,6-epoxycholesterol5β,6-EC5β,6-epoxycholesterol7-DHC7-dehydrocholesterol7-OC7-oxocholesterol7α-HCO7α-hydroxycholest-4-en-3-one7α-HC7α-hydroxycholesterol7αH,3O-CA7α-hydroxy-3-oxocholest-4-en-(25R)26-oic acid7β-HC7β-hydroxycholesterol7β,26-diHC7β,26-dihydroxycholesterol24S-HC24S-hydroxycholesterol25-HC25-hydroxycholesterol25H,7O-C25-hydroxy-7-oxocholesterol26-HC(25R)26-hydroxycholesterol26H,7O-C26-hydroxy-7-oxocholesterol.1ex vivo from cholesterol during sample handling -labelled Girard P (GP) reagent  with parallel multistage fragmentation (MSn). Sterols with a natural oxo group were derivatised with [2H0]GP reagent in the absence of cholesterol oxidase and analysed together with the [2H5]GP-derivatised sterols in a single LC-MS(MSn) run. Quantification was by the isotope dilution method.The LC-MS methods have been described in detail elsewhere ,31. In b reagent , then an33.1Streptomyces sp converts sterols with a 3β-hydroxy-5-ene function to analogues with a 3-oxo-4-ene structure, which are substrates for derivatisation with [2H5]GP  in the absence of authentic standards. This is also true for 7-oxo- and 3β,7β-dihydroxysterols [Cholesterol oxidase from  [2H5]GP . The dered triol . 7-Oxostf enzyme . GP-deriysterols .3.2in vivo by ChEH to 3β,5α,6β-triol [ex vivo oxidation of cholesterol producing 5,6-EC artefacts, cholesterol was removed from samples by reversed-phase solid phase extraction as a first step in sample preparation, consequently 5,6-EC and 3β,5α,6β-triol, the latter of which may be formed by ex vivo acid catalysed hydrolysis of 5,6-EC, are minimised in plasma samples from healthy donors, including disease carriers , see also Both 5α,6-EC and 5β,6-EC are converted 6β-triol ,35 . Further, metabolism catalysed by CYP27A1 should lead to 3β,5α,6β-trihydroxycholestan-(25R)26-oic acid . Evidence for the presence of 3β,5α,6β-triHCa in plasma samples from patients with NPC (6–16 ng/mL), NPB (31.5–47.5 ng/mL) and LALD (2–21 ng/mL) was again provided by LC retention time, exact mass and MS3 spectra . 3β,5α,6β-triHCa was present at low levels in control and carrier plasma  27A1, should be cholestane-3β,5α,6β,(25R)26-tetrol , NPB (2.5–4 ng/mL) and LALD  after dehydrogenation by DBP (HSD17B4). As indicated above, free bile acids are preferentially observed with the current method and 3β,5α,6β-triHBA was detected in NPC (1–3.5 ng/mL), NPB (1–2 ng/mL) and one of the two LALD (0–1.5 ng/mL) patient samples, but not controls or carriers (≤0.1 ng/mL) , its epimerisation at C-25 by alpha-methylacyl-CoA-racemase (AMACR), dehydrogenation of the side-chain between C-24 and C-25 by acyl-coenzyme A oxidase 2 (ACOX2), then hydration of the resultant double bond by peroxisomal multifunctional enzyme type 2, also known as D bifunctional protein (DBP), to give 3β,5α,6β,24-tetrahydroxycholestan-26-oic acid  as the CoA thioester (in  in Ref. ). There 1 ng/mL) B. A pero3.45-bile acids in SLOS patients where the 7-DHC concentrations are high in plasma and tissue [The metabolism of 7-OC has been of interest to many investigators. Lyons and Brown reported that it could be metabolised to 26H,7O-C by CYP27A1 in HepG2 cells, while Heo et al. showed that 26H,7O-C and the down-stream CYP27A1 metabolite 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid  could be formed by retinal pigment epithelial cells ,41. An ad tissue . We next5-BA) and 3β-hydroxy-7-oxochol-5-enoic  acid as metabolic products , but present in two of the three NPC (0–1.5 ng/mL), all three of the NPB (2–4.5 ng/mL) and both of the LALD (0.5–2.5 ng/mL) plasma samples (5-BA was present in control and carrier plasma (0.5–2 ng/mL), but at higher levels in NPC (19–50 ng/mL), NPB (14–40 ng/mL) and in LALD (4.5–14 ng/mL) 26-oic acid  and 3β,7β-diH-Δ-epimers . 7β,26-d samples E. The ul4 ng/mL) B.3.4.23 spectra it was presumptively identified in two of the three NPC (0–0.5 ng/mL), all three NPB (0.5–1.5 ng/mL) and both LALD (1–3 ng/mL) plasma samples (5-BA. It was presumptively identified in control and carrier (≤0.5 ng/mL), NPC (1.5–6.5 ng/mL), NPB (2–6.5 ng/mL) and LALD (1–5.5 ng/mL) plasma  and is found to be elevated in NPC (1–2.5 ng/mL), NPB (1–2 ng/mL) but not LALD (<0.5 ng/mL) plasma B. The 7-3.4.35-BA, are more abundant than their 3-oxo analogues 3βH,7O-CA and 3βH,7O-Δ5-BA in the ratio of about 3.5 and 8 for the C27  and C24  acids, respectively. Both ratios are also greater than 1 for the LALD patients  and carriers  and controls. In contrast to the situation for 3β,5α,6β-triHCa, where concentrations are higher in NPB than NPC plasma (see Unlike the situation for 7-OC and 7β-HC which are in a concentration ratio of about 2.5 for both NPC and NPB patents, the 7β-hydroxy acids, 3β,7β-diHCA and 3β,7β-diH-Δasma see  the conc4in vivo. The absence of high levels of 7-DHC in NPC and NPB also points to the formation of 7-OC via in vivo free radical reactions.The observation here and elsewhere of elevated levels of 3β,5α,6β-triol and 7-OC in the lysosomal storage diseases NPC and NPB points to their endogenous formation . While 74.1Until recently little was known about the metabolism of 5,6-EC. It is established that 5,6-EC can be enzymatically converted to the 3β,5α,6β-triol by ChEH , but onlThere are two major pathways of bile acid biosynthesis, the neutral and acidic pathways, and two more minor pathways, the sterol 24-hydroxylase and 25-hydroxylase pathways . The neu4.25-BA. The data reported here confirms this hypothesis as judged by the preponderance of relevant pathway intermediates identified in plasma of NPC and NPB patients. 7-OC can alternatively be converted to 7β-HC [5-BA. These 7-OC and 7β-HC branches of the acidic may be interconvertible through HSD11B1 which can convert 7-oxo to 7β-hydroxy groups and HSD11B2 which can catalyse the reverse reactions (5-BA and 3β,7β-diH-Δ5-BA as their sulphate and glycine conjugates in urine of an NPC patient [The metabolism of 7-OC has been the subject of greater interest than that of 3β,5α,6β-triol ,41,46, ato 7β-HC  and thiseactions . In fact patient , althoug patient . The HSD patient ,48,49. DHSD11B2 also has activity towards 3β,5α,6β-triol, oxidising it to the oncometabolite 3β,5α-diHC-6O, while HSD11B1 catalyses the reverse reaction [27 acids 3β,5α,6β-triHCa, 3β,7β-diHCA (In light of the low number of patient samples analysed the diagnostic value of bile acid precursors can only be speculated on. However, the high abundance of the C7β-diHCA D and 3βH7β-diHCA C in NPC 5In man, most of the primary bile acids are synthesised in the liver through the neutral pathway of bile acid biosynthesis. The acidic pathway can proceed extrahepatically , while q


Propargylic amines are important multifunctional building blocks that are frequently exploited in the synthesis of privileged heterocyclic entities. Herein we report on a novel flow process that achieves the safe and effective on-demand synthesis of propargylic amines in a telescoped manner. This process minimizes exposure to hazardous azide intermediates and renders a streamlined route into these building blocks. The value of this approach is demonstrated by the rapid generation of a small selection of drug-like thiazolines that result from a high-yielding reaction cascade between propargylic amines with different aryl isothiocyanates. As modern drug development programs continue to rely on suitably functionalized molecular building blocks, efforts towards their effective preparation remain a challenge at the forefront of modern synthetic chemistry. Oftentimes however, such key building blocks may not be readily available or cannot be stored for long periods of time due to unavoidable degradation, making on-demand synthesis the only option for synthetic chemists. Propargylic amines  represen1, the classical metal-catalyzed three-component reaction between amines, alkynes and aldehydes stands out as the most widely utilized protocol [2 typically requires a stepwise and therefore more cumbersome approach that cannot be performed in a single-pot fashion. Therefore, the stepwise conversion of propargylic alcohols 4 via the activation and subsequent displacement of the hydroxy group with a suitable nitrogen nucleophile is typically required. Commonly, azide is the preferred nucleophile that subsequently must be converted into the desired amine functionality by suitable reduction processes  giving gram quantities of the desired entities  as a readily available and bench stable azide donor that we [Having established a rapid entry into the desired substrates  that we  and othe that we ,26 had e that we  sequence8, THF, 2 M, 2 equiv., stream A) via a T-piece with a second stream containing the substrate  and DBU (1.3 equiv.) as a base that we had found to efficiently promote this transformation. The resulting mixture was then directed into a heated flow coil reactor  and collected after passing a back-pressure regulator (75 psi).As depicted in 1H-NMR analysis of the resulting reaction mixture revealed that elevated temperature (60 °C) in combination with residence times of 30 min were optimal to reach full conversion of 4 to the desired azide products 5 that are isolable entities. It was furthermore found that using an excess of DPPA (2 equiv.) was required to reliably achieve this transformation in a short period of time. Using these conditions allowed to generate the desired azide products (5a–c) in good isolated yields also permitting their full spectroscopic characterization . Furthermore, the hydrolysis of the iminophosphorane turned out to be a facile process yielding the desired propargylic amines upon addition of water (10% by volume) to this reaction mixture. Subsequent batch test reactions for the desired Staudinger reduction of intermediates 5 in the initial reaction stream and mitigate any dispersion-related problems a slight excess of the triphenylphosphine reagent (1.5 equiv.) was used [2a–e were isolated after extractive acid-base work-up in good yields and excellent purity as listed in To translate these preliminary results into a telescoped flow process, we expanded on the original set-up by mixing the azide stream with a solution of triphenylphosphine  to larger scale (5 to >10 mmol) operations. To this end an additional HPLC pump was used to deliver a stream of aqueous acid  that was mixed via a T-piece with the crude mixture from the azidation/Staudinger reduction sequence.After collection of the resulting biphasic mixture in a separating funnel, the desired propargylic amine products were again obtained in pure form upon neutralization and back-extraction into either DCM or EtOAc. Although the mixing in a conventional T-piece aided in emulsifying and consequently protonating the propargylic amine species, it was found that THF as reaction solvent led to slow phase separation and thus potential loss of product (around 10–15%). A quick reevaluation of suitable solvent alternatives revealed that 2-methyltetrahydrofuran (2-MeTHF) could effectively replace THF as it resulted in faster phase separation. Additionally, this solvent swap would be attractive as 2-MeTHF is reportedly more stable towards acids, whilst being both bioderived and biodegradable ,31,32. T11. Such a process was expected to yield valuable heterocyclic derivatives via intermediate thiourea adducts 12 that would cyclize in an acid-catalyzed reaction cascade. This sequence is interesting as it allows direct access to either thiazoline or thiazole derivatives 13 or 14 as reported in the literature [To exploit our effective route into different aryl-propargyl amines and demonstrate the value of the flow process developed to generate these materials on-demand, we studied their reaction with different isothiocyanates terature ,35,36,372  with stoichiometric amounts of suitable aryl isothiocyanates (11). The expected thiourea intermediate 12 formed within 30 min and upon addition of acid  smoothly converted into the heterocyclic target structures within a short time . 13C-NMR spectra were recorded on the same instruments (100 MHz) and are reported relative to CHCl3 (δ 77.16 ppm). Data for 1H-NMR are reported as follows: chemical shift (δ/ppm) ). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, br. s = broad singlet, app = apparent. Data for 13C-NMR are reported in terms of chemical shift (δ/ppm) and multiplicity . DEPT-135, COSY, HSQC, HMBC and NOESY experiments were used in the structural assignment. IR spectra were obtained by use of a Platinum spectrometer  with the intensities of the characteristic signals and are reported as weak , medium  or strong . High-resolution mass spectrometry was performed using the indicated techniques on a Micromass LCT orthogonal time-of-flight mass spectrometer  as an internal lock mass. Continuous flow experiments were performed on a Vapourtec E-series system  in conjunction with Omnifit glass columns.2(PPh3)2  and CuI . The resulting solution changed colour from yellow to orange and finally brown in two minutes. The reaction mixture was heated at 50 °C and stirred until complete consumption of the aryl iodide was observed . After filtering the crude material over a pad of silica (5 g) and evaporation of the solvent the crude material (1H-NMR) was purified by silica column chromatography . After removal of the volatiles, the desired hydroxyl product was typically isolated as yellow oil with a purity > 98% (by 1H-NMR) and was used directly in the next step. Copies of NMR spectra are provided in the electronic To a solution of aryl iodide  in THF  was added 2-propyn-1-ol , diisopropylamine , PdCl4a–e, 1 M, THF, 1 equiv.) and DBU (1.3 equiv.). Each stream was pumped at a flow rate of 0.15 mL/min and blended in a T-piece. The resulting mixture was passed into a tubular flow coil  to affect the azidation reaction. The exiting stream was directed through an Omnifit glass column  containing a mixture of washed scavenger resins  to remove acidic and basic by-products. After passing a back-pressure regulator (75 psi) the product was collected in a flask. Purification was achieved by silica column chromatography (2–5% EtOAc/hexanes) giving the desired products typically as oils.A flow set-up was constructed in which a first stream containing DPPA  was mixed with a stream containing the substrate  was combined in a T-piece with a stream of triphenylphosphine (2 equiv.) in aqueous THF  at equal flow rates of 0.3 mL/min. The resulting mixture then entered two consecutive flow coils  before passing a back-pressure regulator (75 psi) and collection in a flask. After evaporation of the volatiles, the crude mixture was partitioned between DCM (2 × 25 mL) and aqueous HCl (1 M). After discarding the organic phase, the pH of the aqueous layer was adjusted to 8–9 and the mixture was extracted with DCM (2 × 25 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered and evaporated in vacuo giving the desired products either as oils or amorphous solids.2a–e (1 equiv.) in MeCN (0.5 M) was added a stoichiometric amount of aryl isothiocyanate. The resulting mixture was stirred at 50 °C for 30 min before addition of acid . Once complete consumption of substrates to a new product was observed by tlc (0.5-1 h), the pH was adjusted to 7 by addition of Na2CO3 (sat. aqueous). After evaporation of volatiles under reduced pressure, the crude material was triturated from mixtures of MeCN and water (4:1). Alternatively, silica column chromatography .To a solution of the amine 3-(p-Tolyl)prop-2-yn-1-ol (4a): Yield: 85% . Appearance: yellow oil. 1H-NMR  δ 7.33 , 7.12 , 4.48 , 2.35 , 1.72 . 13C-NMR  δ 138.7 (C), 131.6 (2CH), 129.1 (2CH), 119.4 (C), 86.5 (C), 85.7 (C), 51.7 (CH2), 21.5 (CH3). IR  3296 (m), 2919 (m), 2863 (m), 2237 (s), 1651 (w), 1607 (w), 1562 (w), 1509 (s), 1407 (m), 1379 (m), 1260 (m), 1026 (s), 816 (s). Rf = 0.41 (10% EtOAc in pentanes). HR-MS (TOF-ES+) calculated for C10H10O 146.0732, found 146.0716 (Δ = −4.1 ppm).3-(4-(Trifluoromethyl)phenyl)prop-2-yn-1-ol (4b): Yield: 82% . Appearance: yellow oil. 1H-NMR  δ 7.55 , 7.51 , 4.51 , 2.16 . 13C-NMR  δ 131.9 (2CH), 130.1 , 126.3 , 125.5 , 123.8 , 89.6 (C), 84.3 (C), 51.4 (CH2). 19F-NMR  δ −62.9. Rf = 0.44 (10% EtOAc in pentanes). IR  3256 (m), 2920 (w), 2863 (w), 2242 (w), 1929 (w),1803 (w), 1685 (w), 1614 (m), 1567 (w), 1481 (w),1403 (w), 1316 (s), 1170 (s), 1119 (s), 1104 (s), 1015 (s), 952 (m), 841 (s), 710 (m), 598 (m). HR-MS (TOF-ES+) calculated for C10H7F3O 200.0449, found 200.0445 (Δ = −2.0 ppm).3-(3-(Trifluoromethyl)phenyl)prop-2-yn-1-ol (4c): Yield: 87% . Appearance: yellow oil. 1H-NMR  δ 7.68 , 7.57 , 7.41 , 4.50 , 2.25 .13C-NMR  δ 134.7 , 130.9 , 128.8 (CH), 128.4 , 125.0 , 123.6  123.5 (C), 88.8 (C), 84.1 (C), 51.4 (CH2). 19F-NMR  δ −63.1. Rf = 0.43 (10% EtOAc in pentanes). IR  3297 (m), 2917 (s), 2868 (s), 2112 (s), 1611 (w), 1588 (w), 1486 (m), 1433 (m), 1331 (s), 1237 (s), 1125 (s), 1029 (m), 973 (m), 902 (m), 802 (s), 696 (s). HR-MS (TOF-ES+) calculated for C10H7F3O 200.0449, found 200.0453 (Δ = 2.0 ppm).3-(2-Bromophenyl)prop-2-yn-1-ol (4d): Yield: 80% . Appearance: yellow oil. 1H-NMR  δ 7.57 , 7.47 , 7.26 , 7.17 , 4.55 , 1.78 . 13C-NMR  δ 133.5 (CH), 132.4 (CH), 129.7 (CH), 127.0 (CH), 125.4 (C), 124.7 (C), 91.8 (C), 84.2 (C), 51.7 (CH2). IR  3303 (broad), 2913 (w), 1587 (w), 1468 (s), 1433 (m), 1052 (m), 1023 (s), 953 (m), 749 (s), 653 (m), 574 (m).3-prop-2-yn-1-ol (4e): Yield: 91% . Appearance: yellow oil. 1H-NMR  δ 8.36 , 7.87–7.80 , 7.71 – 7.67 , 7.58 , 7.55–7.49 , 7.43–7.38 , 4.67 , 2.45 . 13C-NMR  δ 133.3 (C), 133.1 (C), 130.7 (CH), 129.0 (CH), 128.3 (CH), 126.8 (CH), 126.4 (CH), 126.1 (CH), 125.2 (CH), 120.2 (C), 92.2 (C), 83.8 (C), 51.8 (CH2). IR  3040 (w), 2933 (w), 2170 (w), 2121 (m), 1643 (s), 1589 (s), 1487 (s), 1249 (s), 1205 (s), 1161 (m), 1090 (s), 961 (m), 896 (s), 771 (s), 680 (s), 528 (s). HR-MS (TOF-ES+) calculated for C13H9 (M-CH2OH) 165.0704, found 165.0706 (Δ = 1.1 ppm).1-(3-Azidoprop-1-yn-1-yl)-4-methylbenzene (5a): Yield: 75% . Appearance: yellow oil. 1H-NMR  δ 7.36 , 7.13 , 4.13 , 2.36 . 13C-NMR  δ 139.0 (C), 131.8 (2CH), 129.1 (2CH), 118.9 (C), 87.6 (C), 80.3 (C), 40.6 (CH2), 21.5 (CH3). Rf = 0.80 (5% EtOAc in pentanes). IR  3029 (w), 2920 (w), 2860 (w), 2217 (w), 2116 (s), 1607 (w), 1509 (s), 1442 (m), 1338 (m), 1267 (m), 1238 (s), 865 (m), 867 (s), 732 (m). HR-MS (TOF-ES+) calculated for C10H9N (M-N2) 143.0735, found 143.0739 (Δ = 2.8 ppm).1-(3-Azidoprop-1-yn-1-yl)-3-(trifluoromethyl)benzene (5b): Yield: 83% . Appearance: yellow oil. 1H-NMR  δ 7.73 , 7.63 , 7.47 , 4.17 . 13C-NMR  δ 135.0 (CH), 131.0 , 128.9 (CH), 128.6 , 125.4 , 123.6 ,122.9 (C), 85.4 (C), 82.8 (C), 40.4 (CH2). 19F-NMR  δ −63.0. Rf = 0.78 (5% EtOAc in pentanes). IR  3080 (w), 2940 (w), 2105 (s), 1635 (s), 1590 (s), 1486 (m), 1434 (m), 1331 (s), 1237 (m), 1167 (s), 1127 (s), 1095 (m), 1747 (m), 1005 (m), 903 (m), 803 (s), 695 (s), 656 (m). HR-MS (TOF-ES+) calculated for C10H6F3N (M-N2)197.0452, found 197.0450 (Δ = −1.0 ppm).1-(3-Azidoprop-1-yn-1-yl)-4-(trifluoromethyl)benzene (5c): Yield: 77% . Appearance: yellow oil. 1H-NMR  δ 7.55-7.60 , 4.16 . 13C-NMR  δ 132.1 (2CH), 130.6 , 125.7  125.3 , 125.8 , 85.9 (C), 83.6 (C), 40.4 (CH2). 19F-NMR  δ −63.0. Rf = 0.78 (5% EtOAc in pentanes). IR  2916 (w), 2115 (s), 1922 (s), 1570 (m), 1406 (m), 1321 (s), 1269 (m), 1241 (m), 1166 (m), 1126 (s), 1106 (m), 1068 (s), 1018 (m), 842 (s), 688 (s), 554 (m). HR-MS (TOF-ES+) calculated for C10H6F3N (M-N2) 197.0452, found 197.0458 (Δ = 3.0 ppm).1-(3-Azidoprop-1-yn-1-yl)-2-bromobenzene (5d): Yield: 82% . Appearance: yellow oil. 1H-NMR  δ 7.59 , 7.50 , 7.30–7.25 , 7.20 , 4.19 . 13C-NMR  δ 133.8 (CH), 132.5 (CH), 130.0 (CH), 127.0 (CH), 125.4 (C), 124.2 (C), 85.8 (C), 85.6 (C), 40.6 (CH2). IR  2910 (w), 2119 (s), 2100 (s), 1469 (s), 1434 (m), 1335 (m), 1246 (m), 1233 (m), 1052 (m), 1026 (m), 988 (m), 865 (m), 750 (s), 688 (m), 650 (m), 552 (m), 512 (m), 445 (m). HR-MS (TOF-ES+) calculated for C9H6Br (M-N3) 192.9653, found 192.9657 (Δ = 2.1 ppm).1-(3-Azidoprop-1-yn-1-yl)naphthalene (5e): Yield: 79% . Appearance: pale yellow oil. 1H-NMR  δ 8.33 , 7.89–7.85 , 7.73 , 7.60 , 7.56–7.51 , 7.44 , 4.31 . 13C-NMR  δ 133.1 (C), 133.1 (C), 131.1 (CH), 129.4 (CH), 128.3 (CH), 127.0 (CH), 126.5 (CH), 125.9 (CH), 125.1 (CH), 119.6 (C), 85.8 (C), 85.4 (C), 40.9 (CH2). IR  3058 (w), 2119 (s), 2094 (s), 1586 (w), 1507 (w), 1395 (m), 1335 (m), 1247 (s), 865 (m), 797 (s), 770 (s).3-(p-Tolyl)prop-2-yn-1-amine (2a): Yield: 79% . Appearance: yellow solid. 1H-NMR  δ 7.29 , 7.10 , 3.63 , 2.33 . 13C-NMR  δ 138.1 (C), 131.4 (2CH), 129.0 (2CH), 120.1 (C), 89.5 (C), 82.5 (C), 32.2 (CH2), 21.4 (CH3). IR  3347 (w), 3265 (m), 3027 (w), 2916 (w), 2247 (w), 2131 (w), 1906 (s), 1607 (s), 1555 (m), 1433 (s), 1330 (s), 1306 (s), 969 (m), 813 (s), 544 (m), 495 (m). HR-MS (TOF-ES+) calculated for C10H12N (M + H) 146.0970, found 146.0969 (Δ = −0.5 ppm).3-(3-(Trifluoromethyl)phenyl)prop-2-yn-1-amine (2b): Yield: 86% . Appearance: yellow oil. 1H-NMR  δ 7.67 , 7.55 , 7.41 , 3.66 . 13C-NMR  δ 134.6 , 130.9 , 128.8 (CH), 128.4 , 124.6 , 124.2 (C), 123.7 , 91.8 (C), 81.0 (C), 32.1 (CH2). 19F-NMR  δ −63.0. IR  2919 (w), 2118 (w), 1587 (w), 1486 (m), 1328 (s), 1236 (m), 1163 (m), 1123 (s), 1073 (m), 1001 (w), 902 (m), 696 (m), 658 (s). HR-MS (TOF-ES+) calculated for C10H9NF3 (M + H) 200.0687, found 200.0683 (Δ = −2.0 ppm).3-(4-(Trifluoromethyl)phenyl)prop-2-yn-1-amine (2c): Yield: 74% . Appearance: yellow oil. 1H-NMR  δ 7.53 , 7.48 , 3.66 , 1.47 . 13C-NMR  δ 131.8 (2CH), 129.7 , 127.0 , 125.2 , 123.9 , 92.9 (C), 81.1 (C), 32.0 (CH2). 19F-NMR  δ −62.9. IR  3285 (w), 2921 (w), 2851 (w), 2239 (w), 1614 (m), 1437 (s), 1321 (s), 1261 (w), 1164 (s), 1122 (s), 1005 (m), 1168 (s), 1017 (m), 967 (s), 598 (m), 577 (w). HR-MS (TOF-ES+) calculated for C10H9NF3 (M + H) 200.0687, found 200.0697 (Δ = 5.0 ppm).3-(2-Bromophenyl)prop-2-yn-1-amine (2d): Yield: 80% . Appearance: yellow oil. 1H-NMR  δ 7.56 , 7.43 , 7.23 , 7.13 , 3.70 , 1.51 . 13C-NMR  δ 133.3 (CH), 132.3 (CH), 129.2 (CH), 127.0 (CH), 125.4 (C), 125.3 (C), 81.1 (C), 32.3 (CH2); 1 C not observed. IR  3367 (w), 2914 (w), 1586 (w), 1468 (s), 1433 (m), 1330 (m), 1067 (m), 1048 (s), 1025 (m), 967 (m), 860 (m), 749 (s), 652 (m), 524 (m), 445 (m). HR-MS (TOF-ES+) calculated for C9H9NBr (M + H) 209.9918, found 209.9924 (Δ = 2.7 ppm).3-prop-2-yn-1-amine (2e): Yield: 81% . Appearance: colourless oil. 1H-NMR  δ 8.33 , 7.84 , 7.81 , 7.65 , 7.56 , 7.51 , 7.44–7.39 , 3.81 , 1.56 . 13C-NMR  δ 133.3 (C), 133.1 (C), 130.2 (CH), 128.5 (CH), 128.2 (CH), 126.6 (CH), 126.3 (CH), 126.1 (CH), 125.2 (CH), 120.8 (C), 95.2 (C), 80.5 (C), 32.5 (CH2). IR  3368 (w), 3056 (w), 2913 (w), 1584 (m), 1506 (w), 1395 (m), 1330 (m), 948 (m), 862 (m), 797 (s), 770 (s), 566 (m). HR-MS (TOF-ES+) calculated for C13H12N (M + H) 182.0970, found 182.0969 (Δ = −0.4 ppm).(Z)-N-(4-Fluorophenyl)-5-(4-methylbenzylidene)-4,5-dihydrothiazol-2-amine (13a): Yield: 70% . Appearance: yellow solid. 1H-NMR  δ 7.44–7.47 , 7.26–7.32 , 7.24 , 7.19 , 6.94 , 4.95 , 2.34 . 13C-NMR  δ 162.4 , 138.4 (C), 132.1 (C), 129.2 (2CH), 127.4 (2CH), 127.0 , 124.2 (C), 124.1 (CH), 116.8 , 56.0 (CH2), 19.9 (CH3), 2 carbons not observed. 19F-NMR  δ −113.7. IR  3117 (w), 3048 (m), 2855 (m), 2675 (s), 1605 (s), 1555 (m), 1507 (s), 1460 (m), 1352 (s), 845 (s), 790 (m), 650 (m), 514 (m). HR-MS (TOF-ES+) calculated for C17H15FN2S (M + H) 299.1018, found 299.1008 (Δ = −3.4 ppm).(Z)-N-(4-Fluorophenyl)-5-(3-(trifluoromethyl)benzylidene)-4,5-dihydrothiazol-2-amine (13b): Yield: 67% . Appearance: colourless solid. 1H-NMR  δ 7.58–7.67 , 7.46–7.49 , 7.29 , 7.06 , 5.02 . 13C-NMR  δ 162.5 , 136.0 (C), 131.5 (C), 130.9 , 130.6 (CH), 129.6 (CH), 128.3 (C), 127.0 , 124.4 , 124.2 , 123.9 , 122.4 (CH), 116.8 , 56.2 (CH2); 1 C not observed. 19F-NMR  δ −64.4 (3F), −113.6 (1F). IR  3390 (w), 3190 (m), 2827 (m), 2650 (m), 1629 (s), 1510 (s), 1487 (s), 1170 (s), 909 (m), 878 (s), 797 (m), 758 (m), 645 (m), 520 (w). HR-MS (TOF-ES+) calculated for C17H13N2F4S (M + H) 357.0736, found 353.0737 (Δ = 0.4 ppm).(Z)-N-(4-Fluorophenyl)-5-(4-(trifluoromethyl)benzylidene)-4,5-dihydrothiazol-2-amine (13c): Yield: 74% . Appearance: colourless solid. 1H-NMR  δ 7.74 , 7.52 , 7.47–7.50 , 7.30 , 7.07 , 5.04 . 13C-NMR  δ 161.8 , 138.7 (C), 131.5 (C), 129.5 , 128.9 (C), 128.0 (2CH), 127.0 (2CH), 125.5 , 124.0 , 122.4 (CH), 116.8 , 56.3 (CH2); 1 C not observed. 19F-NMR  δ −64.2 (3F), δ −113.6 (1F). IR  2926 (w), 2842 (m), 2704 (m), 1654 (m), 1614 (m), 1577 (m), 1506 (s), 1319 (s), 1110 (m), 832 (s), 688 (m), 646 (m), 507 (s). HR-MS (TOF-ES+) calculated for C17H12F4N2S (M + H) 357.0736, found 353.0742 (Δ = 1.8 ppm).(Z)-N-(4-Chloro-2-(trifluoromethyl)phenyl)-5-(4-methylbenzylidene)-4,5-dihydrothiazol-2-amine (13d): Yield: 78% . Appearance: yellow solid. 1H-NMR  δ 7.59 , 7.41–7.44 , 7.10–7.17 , 6.54 , 4.57 , 2.33 . 13C-NMR  δ 137.3 (C), 132.6 (C), 132.8 (CH), 129.3 (2CH), 128.5 (C), 127.7 (2CH), 126.6 , 125.1 (CH), 120.7 (CH), 53.8 (CH2), 21.2 (CH3), not all C observed. 19F-NMR  δ −61.9. IR  3083 (w), 3045 (w), 2124 (w), 1902 (w), 1764 (w), 1764 (s), 1625 (m), 1597 (m), 1481 (m), 1468 (m), 1371 (w), 1306 (s), 1268 (m), 1250 (m), 1209 (m), 1195 (m), 1119 (s), 1049 (s), 855 (m), 836 (s), 796 (s), 617 (s), 521 (s), 441 (m). HR-MS (TOF-ES+) calculated for C18H14ClF3N2S (M + H) 383.0585, found 383.0597 (Δ = −1.2 ppm).(Z)-5-(2-Bromobenzylidene)-N-(4-chloro-2-(trifluoromethyl)phenyl)-4,5-dihydrothiazol-2-amine (13e): Yield: 75% . Appearance: yellow solid. 1H-NMR  δ 7.58 , 7.55 , 7.40 , 7.32 , 7.30–7.22 , 7.11–7.04 , 6.73 , 4.59 . 13C-NMR  δ 161.0 (C), 146.6 (C), 135.7 (C), 134.3 (C), 132.9 (CH), 132.7 (CH), 128.9 (CH), 128.7 (C), 128.3 (CH), 127.5 (CH), 126.6 , 125.5 (CH), 124.2 , 123.8 (C), 123.2 , 119.9 (CH), 53.5 (CH2). 19F-NMR  δ −61.8. IR  3049 (m), 2898 (m), 1653 (s), 1622 (s), 1483 (s), 1433 (m), 1309 (s), 1193 (m), 1161 (m), 1124 (s), 1101 (m), 1050 (s), 891 (m), 839 (m), 671 (m). HR-MS (TOF-ES+) calculated for C17H12ClBrF3N2S (M + H) 446.9545, found 446.9559 (Δ = 3.1 ppm).(Z)-N-(4-Chloro-2-(trifluoromethyl)phenyl)-5--4,5-dihydro-thiazol-2-amine (13f): Yield: 80% . Appearance: yellow solid. 1H-NMR  δ 7.97–7.92 , 7.87–7.82 , 7.76 , 7.55 , 7.54–7.47 , 7.44–7.40 , 7.36 , 7.16–7.12 , 7.05 , 6.61 , 4.69 . 13C-NMR  δ 133.5 (C), 133.1 (C), 132.6 (CH), 131.1 (C), 128.6 (CH), 128.4 (C), 128.3 (CH), 126.5 , 126.3 (CH), 126.1 (CH), 125.4 (CH), 125.3 (CH), 125.1 (CH), 123.8 (CH), 123.8 (C), 118.2 (CH), 53.2 (CH2); several C not observed. 19F-NMR  δ −61.9. IR  2982 (w), 1627 (m), 1590 (m), 1539 (m), 1484 (m), 1419 (m), 1309 (s), 1290 (m), 1174 (m), 1119 (s), 1050 (s), 795 (m), 686 (m), 641 (m). HR-MS (TOF-ES+) calculated for C21H15ClF3N2S (M + H) 419.0597, found 419.0610 (Δ = 3.2 ppm).In conclusion, we have developed a straightforward access to valuable propargylic amines that exploits a telescoped continuous flow approach. This sequence features the safe use of DPPA as the azide source for converting propargylic alcohols into the intermediate azide counterparts. A Staudinger reduction process was used as part of the same sequence to yield the desired propargylic amine species in good yield and purity. Final optimization of this telescoped flow sequence furthermore led to using the more sustainable 2-MeTHF as solvent as opposed to THF and included an in-line extractive work-up. The value of generating these amine products on demand was demonstrated in their effective transformation into drug-like thiazolines, whose correct structural assignment was accomplished by means of a set of single crystal X-ray structures. We believe that this effective entry into propargylic amines will facilitate their further exploitation in future synthesis programs yielding valuable chemical entities.


The structures of compounds 1–4 were elucidated by comprehensive spectroscopic techniques  and MS analysis, as well as by comparison with the literature. Compounds 1, 2, and 5 showed moderate cytotoxicity against human tumor K562, BEL-7402, and SGC-7901 cell lines with IC50 values of 5.76 to 20.1 µM.Three new 2-(2-phenylethyl)chromone derivatives ( Aquilaria or Gyrinops genus that belong to the family of Thymelaeaceae −). The IR spectrum revealed the presence of hydroxyl (3434 cm−1) and α,β-unsaturated carbonyl (1632 cm−1) functionalities. The 1H-NMR  and 9.70 , two para-position aromatic protons at δH 7.22  and 6.84 , five characteristic protons on the mono-substituted aromatic ring at δH 7.26  and δH 7.19 , one olefinic proton at δH 5.99 , as well as four methylene protons at δH 2.97  and δH 2.90  which connected to each other according to the 1H-1H COSY correlation from H2-7′ to H2-8′ , 144.3 (C-6), 152.0 (C-7), 102.8 (C-8), 115.8 (C-10) and 151.0 (C-9), as well as an α,β-unsaturated carbonyl moiety by signals at δC 167.2 (C-2), 108.6 (C-3) and 176.0 (C-4). Additional signals included a mono-substituted aromatic ring (C-1′ (δC 140.2), C-2′/6′ (δC 128.4), C-3′/5′ (δC 128.3), C-4′ (δC 126.2)) and two methylene carbons at δC 32.1 (C-7′) and 34.7 (C-8′). These data were quite similar to those of compound 5 chromone.Compound chromone , except C 157.8) . Thus, t3 was obtained as a yellow powder. Its molecular formula was deduced to be C18H16O6 on the basis of HR-ESI-MS (m/z 327.0873 [M − H]−). The 1H-NMR spectrum showed the presence of a methoxy group at δH 3.71 (4′-OCH3), three hydroxyl groups at δH 9.67 (8-OH), 11.85 (5-OH) and 8.85 (3′-OH), one set of signals with two ortho-coupled doublets at δH 6.61  and δH 7.17 , and another set of ABX coupling aromatic system signals at δH 6.61 , δH 6.67  and δH 6.80 . The comparison of NMR data between 3 and 5,8-dihydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone chromone.Compound C 115.7)  further 4 was obtained as a yellow powder, and its molecular formula was deduced to be C18H16O4 on the basis of HR-ESI-MS (m/z 293.1787 [M − H]−). The IR spectrum indicated the presence of a hydroxyl (3435 cm−1) and an α,β-unsaturated carbonyl (1631 cm−1) group. Its 1H-NMR spectroscopic data showed two trans-olefinic protons at δH 7.59  and δH 6.63 , a set of AA′BB′ coupling aromatic systems at δH 7.54  and δH 6.95 , three mutual coupled aromatic protons at δH 6.79 , δH 7.51  and δH 6.96  corresponding to a 1,2,3-substituted phenyl moiety in the chromone nucleus, and a methoxy group at δH 3.86 . The 1H- and 13C-NMR spectroscopic data of 4 showed a high degree of similarity with those of 5-hydroxy-2-(2-phenyletheyl)chromone chromone by a comprehensive analysis of its 2D-NMR data ethyl]chromone (5) − .6-Hydroxy-7-methoxy-2-[2-(4-methoxyphenyl)ethyl]chromone (2): colorless oil; UV (MeOH) λmax (log ε): 230 (4.78), 280 (4.41), 298 (4.24), 322 (4.39) nm; IR (KBr) vmax 3404, 1639, 1385, 1017 cm−1; 1H- (500 MHz) and 13C- (125 MHz) NMR spectral data see m/z 327.1224 [M + H]+ .5,8-Dihydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone (3): yellow powder; UV (MeOH) λmax (log ε): 204 (5.12), 224 (4.68), 256 (4.55), 296 (4.65), 320 (4.05) nm; IR (KBr) vmax 3467, 1634, 1015, 670 cm−1; 1H- (500 MHz) and 13C- (125 MHz) NMR spectral data see m/z 327.0873 [M − H]– .5-Hydroxy-2-[2-(4-methoxyphenyl)ethenyl]chromone (4): yellow powder; UV (MeOH) λmax (log ε): 204 (5.04), 224 (4.56), 256 (4.15), 296 (3.92), 306 (3.73) nm; IR (KBr) vmax 3435, 1631, 1384, 772 cm−1; 1H- (500 MHz) and 13C- (125 MHz) NMR spectral data see m/z 293.1787 [M − H]– .2 with 90% humidity. The logarithmic phase cells (90 µL) were seeded onto 96-well plates at the concentration of 5 × 104 cell/mL. The following specific experimental procedures were the same as those described previously [Human cancer cell lines, gastric carcinoma (SGC-7901), myeloid leukemia (K562), and hepatocellular carcinoma (BEL-7402), were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology. MTT assay ,21 was ueviously . The res1–3, 5 and 6 isolated from the agarwood of Gyrinops salicifolia, are very common in nature, and have been reported in agarwoods from Aquilaria species [Aquilaria sinensis. Interestingly, compound 4, discovered during this study, was the second 2-(2-phenylethenyl)chromone derivative from agarwood. Furthermore, eight known 2-(2-phenylethyl)chromone derivatives and a series of sesquiterpenes have been isolated from the agarwood of Gyrinops salicifolia in our previous study [Aquilaria sp. [Gyrinops salicifolia are similar to the agarwood resins from Aquilaria sp., so it can be considered that Gyrinops salicifolia is one of the commercial agarwood-producing species.To the best of our knowledge, 2-(2-phenylethyl)chromone derivatives, the compounds with a chromone skeleton, such as  species . For 2-( species , was fouus study ,24, and aria sp. . As we c


The structures of new compounds were determined to be 19(R)-n-butanoxy-5β,19-epoxycucurbita-6,23-diene-3β,25-diol 3-O-β-glucopyranoside (1), 23-O-β-allopyranosyle-cucurbita-5,24-dien-7α,3β,22(R),23(S)-tetraol 3-O-β-allopyranoside. (2), 23(R),24(S),25-trihydroxycucurbit-5-ene 3-O-{[β-glucopyranosyl(1→6)]-O-β-glucopyranosyl}-25-O-β-glucopyranoside (3), and 24(R)-stigmastan-3β,5α,6β-triol-25-ene 3-O-β-glucopyranoside (4), respectively. Their structures were elucidated by the combination of mass spectrometry (MS), one and two-dimensional NMR experiments and chemical reactions.Three new cucurbitane triterpenoids  Momordica charantia L (cucurbitaceae) is cultivated in Asian countries. Its fruit, called kugua in Chinese and bitter melon in English, is a popular vegetable in Asian countries and is becoming a popular food supplement to lower blood glucose worldwide , kar, karM. c1 was obtained as a white powder. The negative-ion quasimolecular ion peak was observed at m/z 689 [M-H]−, and the molecular formula C40H66O9 was determined by negative-ion HRESIMS measurement . The 1H-NMR spectrum of 1 , a secondary methyl at δ 0.97 , a third methyl at 0.80 , and four olefinic protons at δ 6.15 , 5.63 , 5.94 , and 5.94 , as well as one anomeric proton at δ 4.96 . Acid hydrolysis of 1 furnished glucose, which was identified by TLC comparison with an authentic sample. In the 13C-NMR of 1 -n-butanoxy-5β,19-epoxycucurbita-6,23-diene-3β,25-diol 3-O-β-glucopyranoside.Compound rum of 1  showed sNMR of 1 , 30 aglycoside-a , except ESY data  confirme2 was obtained as a white powder. HRESIMS afforded C42H69O14 as a possible molecular formula of 2 . The 1H-NMR spectrum of 2 , a secondary methyl at δ 1.14 , two allylic methyls at 1.60, 1.80 , and two olefinic protons at δ 6.08 , 5.51 , as well as two anomeric protons at δ 5.32  and 5.61 . Compound rum of 2  showed s13C-NMR of 2 ,23(S)-tetraol 3-O-β-allopyranoside.The NMR of 2  showed 3loside V , with theduction . The steeduction , which w48H82O19 of compound 3 was deduced by HRESIMS . 1D-NMR data  and C-3′ (δ 87.6), H-1′′ (δ 5.21) and C-6′ (δ 70.3), H-1′′′ (δ 5.30) and C-25 (δ 81.5), indicating that two glucoses were attached to C-3 and C-25 as well as the remain glucose was attached to C-6′ of the glucose linked to C-3 of aglycone. Based on the above results, compound 3 was identified as 23(R),24(S),25-trihydroxycucurbit-5-ene 3-O-{[β-glucopyranosyl(1→6)]-O-β-glucopyranosyl}-25-O-β-glucopyranoside.In the HMBC spectrum , long-ra4 was obtained as a white powder. The molecular formula C35H60O8 was established by negative-ion HRESIMS measurement at m/z 643.3951 [M+Cl]− . The 1H-NMR spectrum of 4 , a secondary methyl at δ 0.96 , one allylic methyl group at 1.59 , one third methyl at δ 0.83  and two anomeric olefinic protons at δ 4.85 , 4.78  as well as one anomeric proton at δ 4.95 . The 13C-NMR data indicated that the presence of a steroidal glycoside bearing five methyls and a sugar unit. Acid hydrolysis of 4 afforded a glucose which was confirmed by TLC comparison with an authentic sample. All above data suggested that it was almost same as 24(R)-stigmastan-3β,5α,6β-triol 3-O-β-glucopuranoside −; HRESIMS: [M-H]− m/z 689.4630 ; IR (KBr) νmax: 3413, 2930, 1633, 1453, 1375, 1073 cm−1; 1H-NMR  and 13C-NMR : see 23-O-β-Allopyranosyle-cucurbita-5,24-dien-7α,3β,22(R),23(S)-tetraol-3-O-β-allopyranoside (2). C42H69O14, white powder; m/z 797 [M−H]−; HRESIMS: [M−H]− m/z 797.4690 ; IR (KBr) νmax: 3404, 2930, 1642, 1453, 1372, 1071, 970 cm−1; 1H-NMR  and 13C-NMR : see 23(R),24(S),25-Trihydroxycucurbita-5-ene-3-O-{[β-glucopyranosyl(1→6)]-O-β-glucopyranosyl}-25-O-β-glucopyranoside (3). C48H82O19, white powder; m/z 961 [M−H]−; HRESIMS: [M-H]− m/z 961.5366 ; IR (KBr) νmax: 3400, 2926, 1648, 1455, 1377, 1072, 985 cm−1; 1H-NMR  and 13C-NMR : see 24(R)-Stigmastan-3β,5α,6β-triol-25-ene 3-O-β-glucopyranoside (4). C35H60O8, white powder; m/z 607 [M−H]−; HRESIMS: [M+Cl]− m/z 643.3951 ; IR (KBr) νmax: 3400, 2920, 1640, 1450, 1370, 1070, 980 cm−1; 1H-NMR  and 13C-NMR : see M. charantia, together with ten known compounds karaviloside II, karaviloside III, momordicoside K, kuguaglycoside B, momordicoside L, momordicoside M, momordicoside N, momordicoside B, momordicoside S, and momordicoside A. Although we researched on the same specie from different localities [M. charantia as a food supplement or a plant extract to lower blood glucose, we had better pay attention to their chemical constitutions in different localities. We have isolated three new triterpenes and one new steroidal glycoside from the fruits of calities ,7, the c


Their structures were elucidated by chemical evidence and spectral analyses, including HR-ESI-MS, and 1D- and 2D-NMR techniques.A new 3,3′′-biflavanone, sikokianin D ( Wikstroemia indica (Linn.) C. A. Mey., a shrub of the Thymelaeaceae family, is wildely distributed in the southeast of China. Known as Liaogewang, it has long been used as a folk medicine in southern China for treating arthritis, tuberculosis, syphilis and pertussis : + 231). The HR-ESI-MS of 1 exhibited a quasi-molecular-ion peak ([M+H]+) at m/z 557.1442 , corresponding to the molecular formula C31H24O10. Moreover, this compound showed positive reaction with HCl-Mg reagent, indicating that it is a flavonoid. The 1H-NMR spectrum of 1 , two H-atoms corresponding to H-2  and H-2" , and two H-atoms corresponding to H-3  and H-3"  at the rings C and C' of the biflavanone. In the 1H- and 13C-NMR established by 1H-1H COSY and HMQC experiments , and two sets of para-oxygenated B rings . From the 13C-NMR data  were also observed. These structural fragments were connected to form the given carbon framework of 1 as a dimer of flavanonol derivatives. The partial (-CH-CH-CH-CH-) structure inferred from the 1H-1H COSY spectrum  with C-3" (δC 51.0). The B ring could be located at C-2, based on the observation of the clear cross-peaks of H-2' and H-6' (δH 7.22) with C-2 (δC 81.2). In the same way, linkage of the B' ring to C-2" of the C' ring was deduced by the correlations of H-2"'and H-6"'(δH 6.93) with C-2" (δC 83.3). The HMBC cross-peak between the methoxyl group and C-4' on the B ring indicated that the methoxyl group was connected to C-4'.Compound rum of 1  displayeeriments , the speNMR data , two car1 was determined as cis-trans by comparison of the J values (JH-2 = 5.0 Hz and JH-2" = 9.5 Hz) with those of the known 3,3"-biflavanones. The key NOESY correlations between H-2" (δH 5.19) with H-2'(H-6') (δH 7.22) further confirmed the conclusion above. The relative stereochemistry of compound 1 was confirmed as shown in The stereochemistry at the C-2/C-3 and C-2"/C-3" positions in 2 was first reported as sikokianin B of which the location of MeO group was unsettled +, calcd for C31H25O10, 557.1448.Sikokianin B (2). Yellow amorphous powder. 1H-NMR: δH 3.23 , 3.33 , 3.76 , 5.17 , 5.53 , 5.75 , 5.84 , 5.86 , 5.97 , 6.74~7.16 . HR-ESI-MS: m/z 557.1446 [M+H]+. Spectral data were in accordance with those reported in the literature +. Spectral data were in accordance with those reported in the literature -4,4'-dione (1), together with two known compounds, sikokianin B (2) and sikokianin A (3) was isolated from the EtOH extract of the roots of


It is based on the sequential concatenation of two catalytic cycles, both catalyzed by the same metal species : the first cycle corresponds to the oxidative monoaminocarbonylation of the triple bond of propargylic amines to give the corresponding 2-ynamide intermediates, while the second one involves the cyclocarbonylation of the latter to yield 2-(2-oxooxazolidin-5-ylidene)-acetamides. Reactions are carried out using a simple catalytic system consisting of PdI2 in conjunction with an excess of KI, and the catalyst/solvent system could be recycled several times without appreciable loss of activity after extraction of the organic product with Et2O.A convenient carbonylative approach to 2-oxazolidinone derivatives carried out using an ionic liquid (1-ethyl-3-methylimidazolium ethyl sulfate, EmimEtSO In this work, we report on the direct synthesis of 2-oxazolidinone derivatives through an auto-tandem catalysis process consisting of the concatenation of two carbonylative catalytic cycles, both catalyzed by the same catalytic system acetamides nylation ,17,18,19 oxidant . The prople bond  of 1 wit4. Considering the importance of the class of products obtained, which are known to possess important pharmacological activities ,23,24,25N-benzyl-2-methylbut-3-yn-2-amine (1a) as the substrate. This compound was allowed to react with CO, morpholine (2a), O2, and water in the presence of the catalytic system PdI2/KI under the following conditions: PdI2/KI/1a/2a/H2O molar ratio = 1:10:50:250:250, T = 100 °C, P(CO) = 16 atm, P(air) = 4 atm, EmimEtSO4 as the solvent (0.5 mmol of 1a per mL of solvent) . The yield turned out to be lower using only 2 equiv of 2a instead of 5 . These results confirmed the possibility to carry out the auto-tandem catalysis process leading to oxazolidinones 3 in an ionic liquid (IL) as unconventional solvent. We accordingly verified the possibility to recycle the catalyst/solvent system. Thus, the residue obtained after product extraction with diethyl ether , after drying under vacuum , was used again by adding to it fresh propargylamine 1a and morpholine (2a) (1:5 ratio). After stirring at 100 °C for 24 h, 3aa was obtained again as a 3.2:1 Z/E mixture in 71% total isolated yield  and a cyclic chain such as –(CH2)4, (1c)), different groups on nitrogen (such as benzyl (1a–c) and butyl ), and different nucleophilic secondary amines, both cyclic (as in the case of morpholine (2a), piperidine (2b), and pyrrolidine (2c)) and acyclic (as in the case of diethylamine (2d)). In all cases, good yields of the corresponding 2-(2-oxooxazolidin-5-ylidene)acetamides 3 were obtained (69%–76%), and the PdI2/KI/EmimEtSO4 system could be conveniently recycled up to six times without loss of activity.In order to assess the generality of the method, the reaction was then performed using different combinations of propargylic amines 1H-NMR and 13C-NMR spectra were recorded at 25 °C in CDCl3 solutions with a Bruker DPX Avance 300 spectrometer  operating at 300 MHz and 75 MHz, respectively, with Me4Si as internal standard. IR spectra were taken with a JASCO FT-IR 4200 spectrometer . Mass spectra were obtained using a Shimadzu QP-2010 GC-MS apparatus  at 70 eV ionization voltage. Microanalyses were carried out with a Thermo-Fischer Elemental Analyzer Flash 2000 . All reactions were analyzed by TLC  on silica gel 60 F254  or on neutral alumina  and by GLC using a Shimadzu GC-2010 gas chromatograph  and capillary columns with polymethylsilicone + 5% polyphenylsilicone as the stationary phase (HP-5). Column chromatography was performed on neutral alumina 90 . Evaporation refers to the removal of solvent under reduced pressure.Chemicals were purchased from Sigma-Aldrich Italia  and were used as such without further purification. Melting points were taken on a Reichert Thermovar apparatus and are uncorrected. 1a–e were prepared and characterized as already described , 299 (5), 286 (2), 255 (1), 244 (2), 223 (13), 216 (2), 181 (2), 166 (4), 146 (2), 140 (8), 132 (2), 112 (2), 98 (9), 91 (100); anal. calcd for C18H22N2O3 (314.38): C, 68.77; H, 7.05; N, 8.91; found C, 68.75; H, 7.04; N, 8.89.(Z)-3-Benzyl-4,4-dimethyl-5-(2-oxo-2-pyrrolidin-1-ylethylidene)-oxazolidin-2-one (3ac-Z). Yellow solid, m.p. = 105–106 °C. IR (KBr): ν = 1780 (s), 1685 (s), 1616 (m), 1438 (m), 1401 (m), 1225 (w), 1036 (m), 754 (w) cm−1; 1H-NMR (CDCl3): δ = 7.48–7.22 , 5.21 , 4.48 , 3.59–3.43 , 2.03–1.84 , 1.35 ; 13C-NMR (CDCl3): δ = 162.7, 160.5, 153.8, 137.1, 128.8, 128.0, 127.8, 94.6, 62.4, 47.1, 45.7, 44.2, 27.5, 25.9, 24.4; GC-MS m/z = 314 (8) [M+], 299 (4), 286 (1), 255 (1), 243 (3), 223 (11), 216 (1), 181 (2), 166 (2), 147 (4), 140 (6), 132 (1), 112 (2), 98 (7), 91 (100); anal. calcd for C18H22N2O3 (314.38): C, 68.77; H, 7.05; N, 8.91; found C, 68.78; H, 7.03; N, 8.92.(E)-2-(3-Butyl-4-ethyl-4-methyl-2-oxooxazolidin-5-ylidene)-N,N-diethylacetamide (3dd-E). Yellow oil. IR (film): ν = 1786 (s), 1687 (m), 1618 (m), 1401 (w), 1052 (m), 1082 (m) cm−1; 1H-NMR (CDCl3): δ = 5.92 , 3.46–3.30 , 3.29–3.16 , 3.06–2.93 , 2.76–2.60 , 1.70–1.53 , 1.42–1.30 , 1.66 , 1.19 , 1.14 , 0.95 , 0.79 ; 13C-NMR (CDCl3): δ = 164.1, 163.5, 153.8, 96.7, 68.0, 43.0, 40.5, 40.0, 31.1, 28.5, 23.7, 20.3, 14.5, 13.7, 13.1, 8.2; GC-MS m/z = 296 (25) [M+], 281 (15), 267 (100), 224 (8), 180 (15), 168 (98), 140 (15), 124 (19), 112 (9), 100 (54), 72 (71); anal. calcd for C16H28N2O3 (296.41): C, 64.83; H, 9.52; N, 9.45; found C, 64.80; H, 9.54; N, 9.42.(Z)-2-(3-Butyl-4-ethyl-4-methyl-2-oxooxazolidin-5-ylidene)-N,N-diethylacetamide (3dd-Z). Yellow oil, purity: ca. 60% (by GLC). GC-MS m/z =296 (26) [M+], 281 (11), 267 (100), 224 (13), 197 (8), 180 (26), 168 (74), 138 (13), 124 (33), 112 (14), 100 (37), 72 (89).2/KI-catalyzed auto-tandem catalysis oxidative carbonylative process leading to oxazolidone derivatives 3 in an ionic liquid, such as EmimEtSO4, as unconventional solvent, and that the catalyst/solvent system can be easily recycled several times without loss of activity.In conclusion, we have shown that it is possible to successfully perform the PdI


R)-pluthiophenol (1), (3′′R)-pluthiophenol-4′′-acetate (2), 3′′-ethoxy-(3′′S)-pluthiophenol (3), 3′′-ethoxy-(3′′S)-pluthiophenol-4′′-acetate (4), together with twenty-five known compounds were obtained from the 70% ethanol-water extract of the aerial parts of Pluchea indica Less. Their structures were elucidated by spectroscopic methods. Among the known isolates, compounds 7, 8, 11, 14, 15, 18, 20, 23, 25–27 were isolated from Asteraceae family firstly, while compounds 6, 9, 10, 12, 13, 16, 19, 21, 28 were isolated from Pluchea genus for the first time. Meanwhile, compounds 1, 2, 10, 13, 18, 23 displayed significant inhibitory activities on LPS-induced NO production at 40 µM from RAW 264.7 macrophages, while compounds 3, 4, 26–29 possessed moderate inhibitory effects.Four new thiophenes, (3′′ Pluchea indica Less., belongs to Pluchea genus, Asteraceae family, is a 1 to 2 meters high shrub. It mainly distributes in the tropical and subtropical regions of Africa, Asia, America, Australia, and China’s southern provinces. As an amphibious woody semi-mangrove plant, it plays an important role in maintaining the ecological balance in the coastal areas of Southeast Asia in China , one hydroxymethyl , one oxygenated methine [δ 4.55 ], and a couple of olefinic protons . The four carbon signals  in the low field area of 13C-NMR  spectrum, combining with the special coupling constant  and MS data confirmed the existence of the thiophene ring. The 1H-1H COSY spectrum of 1 indicated the presence of two partial structures written in bold lines as shown in H 7.15 (H-3) to δC 66.8 (C-1′), 124.9 (C-2), 125.9 (C-5); δH 7.08 (H-4) to δC 78.1 (C-1′′), 124.9 (C-2), 125.9 (C-5); δH 2.02 (H3-5′) to δC 64.6 (C-3′), 80.1 (C-2′), 84.5 (C-4′); δH 4.55 (H-3′′) to δC 78.1 (C-1′′), 94.5 (C-2′′), 125.9 (C-5); δH 3.64, 3.68 (H2-4′′) to δC 64.6 (C-3′′), 94.5 (C-2′′). Consequently, the planar structure of 1 was determined. Finally, through the comparison of the optical rotation {1 with those of (R)- and (S)-(3E)-2-hydroxy-2-methyl-4-but-3-enyl acetate, {R: S: R.(3′′500 MHz)  spectrumctively} , its absR)-Pluthiophenol-4′′-acetate (2) was obtained as yellow oil with positive optical rotation 15H12O3S of 2 was determined from ESI-Q-Orbitrap MS  analysis, which was 42 Da more than that of 1, suggesting that there was one more acetyl group in 2. Meanwhile, the 1H-, 13C- (3OD) and 2D-  NMR spectra verified the existence of the acetyl group . The acetyl group was elucidated to substitute in C-4′′ by the long-range correlations observed from H-4′′ to C-1′′′ in the HMBC experiment. Similarly, according to the optical rotation, the absolute configuration of 2 was determined to be 3′′R  in 3. The ethoxy was clarified to link to C-3′′ position by the long-range correlation observed from δH 3.55, 3.83 (H2-5′′) to δC 15.5 (C-6′′), 72.7 (C-3′′). At last, its absolute configuration was elucidated to be 3′′S by the optical rotation {3′′-Ethoxy--pluthiophenol-4′′-acetate (4) was isolated as yellow oil. The ESI-Q-Orbitrap MS {m/z 301.08969 [M + H]+ } and 1H-, 13C- (3OD), 2D-  NMR experiments suggested that there was one more acetyl group  at C-4′′ of aglycone than 3. Finally, comparing the optical rotation {4 was revealed to be 3′′S. Thus, its structure was determined as 3′′-ethoxy-(3′′S)-pluthiophenol-4′′-acetate.3′′-Ethoxy- and 95% EtOH eluent (PIE) and compounds 1–29 obtained from the aerial parts of P. indica on LPS-stimulated NO production were accomplished by pretreating RAW 264.7 macrophages cells with them for 1 h before stimulating with LPS (500 ng/mL) for 24 h, respectively. Griess reagent  was used to measure NO concentrations in the culture medium. Comparing to unstimulated normal (negative control), NO production in LPS-stimulated RAW 264.7 macrophages was markedly induced . Positive and negative -ion HRESI-TOF/Orbitrap-MS were determined on Thermo UHPLC-ESI-Q-Orbitrap MS spectrometer  and Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS spectrometer . Optical rotations, UV and IR spectra were run on a Rudolph Autopol® IV automatic polarimeter (l = 50 mm) , Varian Cary 50 UV-Vis  and Varian 640-IR FT-IR spectrophotometer , respectively.NMR spectra were tested on a Bruker 500 MR NMR spectrometer  at 500 MHz for 18-MS-II (4.6 mm × 250 mm) and ; Wacopak Navi C30-5 (4.6 mm × 250 mm) and  were used to separate the constituents.CC were performed on macroporous resin D101 , Silica gel , ODS , and Sephadex LH-20 . Preparative high-performance liquid chromatography (Prep-HPLC) column: Cosmosil 5Cof Pluchea indica Less. were collected from Hepu city, Guangxi province, China and identified by Dr. Wei Songji . The voucher specimen was deposited at the Academy of traditional Chinese Medicine of Tianjin University of TCM.The aerial parts P. indica (10.0 kg) were refluxed three times with 70% EtOH. A 70% EtOH extract (1851.0 g) was provided by evaporating the solvent under pressure. Dissolved the residue in H2O, and the residue was then subjected to D101 CC (H2O → 95% EtOH), H2O (1110.2 g) and 95% EtOH (224.7 g) eluent were afforded, respectively.The dried aerial parts of 3-MeOH  → CHCl3-MeOH-H2O  → MeOH] to yield nine fractions (Fraction 1–Fraction 9).The 95% EtOH eluent (160.7 g) was subjected to silica gel CC [CHClv/v) → EtOAc], and eight fractions (Fraction 2-1–Fraction 2-8) were obtained. Fraction 2-4 (89.2 mg) was purified by pHPLC  to yield 3′′-ethoxy-(3′′S)-pluthiophenol-4′′-acetate  and fraxinellone .Fraction 2 (0.6 g) was separated by silica gel CC [Hexane → Hexane-EtOAc  → EtOAc], eleven fractions (Frction 3-1–Fraction 3-11) were obtained. Fraction 3-4 (219.6 mg) was separated by pHPLC  to afford stigmasterol . Fraction 3-5 (253.9 mg) was isolated by pHPLC  to yield (3′′R)-pluthiophenol-4′′-acetate  and dibutylphthalate . Fraction 3-6 (139.7 mg) was purified by pHPLC  to obtain vanillin . Fraction 3-7 (195.8 mg) was separated by pHPLC  to afford triethyl citrate . Fraction 3-8 (342.9 mg) was isolated by pHPLC  to obtain trans-coniferyl aldehyde . Fraction 3-10 (133.8 mg) was purified by pHPLC  to yield syringicaldehyde . Fraction 3 (4.2 g) was subjected to SiO2O ] to afford thirteen fractions (Fraction 4-1–Fraction 4-13). Fraction 4-5 (244.9 mg) was subjected to pHPLC , eight fractions (Fraction 4-5-1–Fraction 4-5-8) were obtained. Fraction 4-5-4 (40.6 mg) was further separated by pHPLC  to afford seven fractions (Fraction 4-5-4-1–Fraction 4-5-4-7). Among them, Fraction 4-5-4-5 (11.9 mg) was identified as (–)--4,4′-dihydroxy-3,3′,5,5′-pentamethoxy-7,9′:7′,9-diepoxylignane . Fraction 4-5-4-2 (6.1 mg) was purified by [CHCl3-MeOH  → MeOH] to yield threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-ethoxypropan-1-ol  and erythro-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-ethoxypropan-1-ol . Fraction 4-6 (405.9 mg) was isolated by pHPLC  to obtain ethyl caffeate . Fraction 4-10 (536.4 mg) was purified by pHPLC  to yield (3′′R)-pluthiophenol  and caryolane-1,9β-diol . Fraction 4-11 (414.0 mg) was subjected to pHPLC , six fractions were obtained (Fraction 4-11-1–Fraction 4-11-6). Among them, fractions 4-11-3 and 4-11-4 were elucidated as -isocaryolane-8,9-diol  and (+)-9′-isovaleryllariciresinol , respectively. Fraction 4-11-2 (14.7 mg) was purified by pHPLC  to afford clovane-2α,9β-diol . Fraction 4-11-5 (66.3 mg) was further isolated by pHPLC , and 3′′-ethoxy-(3′′S)-pluthiophenol  was yield. Fraction 4-12 (314.8 mg) was separated by pHPLC  to obtain valenc-1(10)-ene-8,11-diol .Fraction 4 (5.3 g) was isolated by ODS CC [MeOH-H3-MeOH ] to afford four fractions (Fraction 5-1–Fraction 5-4). Fraction 5-2 (3.3 g) was isolated by ODS CC [MeOH-H2O ], and ten fractions (Fraction 5-2-1–Fraction 5-2-10) were yielded. Fraction 5-2-1 (394.5 mg) was isolated by pHPLC  to afford 3,4-dihydroxy benzaldehyde , 3,4-dihydroxy-5-methoxybenzaldehyde , 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one , and esculetin . Fraction 5-2-8 (207.7 mg) was subjected to pHPLC , methyl 9-hydroxynonanoate  was obtained. Fraction 5-2-9 (143.3 mg) was further purified by pHPLC  to yield (3′′R)-pluthiophenol , 9,12,13-trihydroxyoctadeca-10(E),15(Z)-dienoic acid  and pinellic acid .Fraction 5 (8.0 g) was separated by Sephadex LH-20 CC [CHCl3-MeOH ] to obtained three fractions (Fraction 7-1–Fraction 7-3). Fraction 7-2 (15.5 g) was further separated by ODS CC [MeOH-H2O ], and ten fractions (Fraction 7-2-1–Fraction 7-2-10) were given. Fraction 7-2-3 (1500.0 mg) was subjected to pHPLC  to obtain eleven fractions (Fraction 7-2-3-1–Fraction 7-2-3-11). Fraction 7-2-3-1 (156.2 mg) was purified by pHPLC  to yield adenosine . Fraction 7-2-3-7 (178.3 mg) was further isolated by pHPLC  to obtain (+)-isolariciresinol .Fraction 7 (46.1 g) was isolated by Sephadex LH-20 CC + .(3′′R)-Pluthiophenol-4′′-acetate (2): Yellow oil; λmax (MeOH) nm (log ε): 208 (4.54), 235 (4.01), 246 (4.16), 319 (4.58), 340 (4.56); IR νmax (KBr) cm−1: 3099, 2977, 2233, 1745, 1520, 1448, 1381, 1326, 1229, 1106, 1046, 807; 1H- and 13C-NMR data, see m/z 273.05781 [M + H]+ .3′′-Ethoxy-(3′′S)-pluthiophenol (3): Yellow oil; λmax (MeOH) nm (log ε): 208 (4.47), 235 (3.99), 246 (4.11), 319 (4.50), 340 (4.47); IR νmax (KBr) cm–1: 3439, 3097, 2975, 2931, 2876, 2231, 1447, 1376, 1327, 1118, 807; 1H- and 13C-NMR data, see m/z 259.07875 [M + H]+ .3′′-Ethoxy-(3′′S)-pluthiophenol-4′′-acetate (4): Yellow oil; λmax (MeOH) nm (log ε): 208 (4.72), 235 (4.18), 246 (4.34), 319 (4.76), 340 (4.73); IR νmax (KBr) cm−1: 3098, 2976, 2228, 1745, 1445, 1377, 1330, 1231, 1105, 1048, 808; 1H- and 13C-NMR data, see m/z 301.08969 [M + H]+ .Lipopolysaccharides (LPS) and dexamethasone (Dex) were purchased from Sigma Chemical ; penicillin and streptomycin were purchased from Thermo Fisher Scientific ; dulbecco’s modified eagle medium (DMEM) medium was purchased from HyClone ; fetal bovine serum (FBS) were purchased from Biological Industries ; nitric oxide fluorometric assay kit was purchased from Beyotime Biotechnology .2 at 37 °C.RAW 264.7 macrophages  were maintained in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere containing 5% COP. indica were used to pretreat the cells for 1 h before stimulating with LPS (500 ng/mL) for 24 h. After incubation, each culture medium (50 µL) was mixed with an equal volume of Griess reagent. An ELISA plate reader was used to determine the nitrite levels at 540 nm, and the concentrations were calculated by referring to a NaNO2 standard calibration curve [Nitrite, as a major stable product of NO, the level of it measured by Griess reagent was considered to reflect the concentration of NO in culture supernatants. Extract, eluent and compounds obtained from the aerial parts of on curve .p < 0.05 was considered to indicate statistical significance. One-way analysis of variance (ANOVA) and Tukey’s Studentized range test were used for the evaluation of the significant differences between means and post hoc, respectively.Values are expressed as mean ± S.D. SPSS 11.0 was used to conduct the statistics of all the grouped data. P. indica, twenty-nine compounds, including four new ones, (3′′R)-pluthiophenol (1), (3′′R)-pluthiophenol-4′′-acetate (2), 3′′-ethoxy-(3′′S)-pluthiophenol (3), 3′′-ethoxy-(3′′S)-pluthiophenol-4′′-acetate (4), along with twenty-five known ones (5–29) were obtained. The structures of them were determined by means of spectroscopic methods.In summary, during the investigation of the chemical compositions from the aerial parts of 1–29 on LPS-stimulated NO production were examined. As a result, compounds 1, 2, 10, 13, 18, 23 displayed significant inhibitory activities on LPS-induced NO production at 40 μM, while 3, 4, 26–29 possessed moderate inhibitory effects. These results suggested that compounds 1, 2, 10, 13, 18, 23 may have potent anti-inflammatory activity.Meanwhile, the potential anti-inflammatory effects of compounds


All the synthesized compounds were screened for their anti-cancer potentiality over human breast carcinoma (MCF-7) and human lung carcinoma (A-549). Most of the tested compounds showed remarkable anti-breast cancer activity. However, compound 4 showed the most anti-lung cancer activity. Then, compounds with cytotoxic activity ≥ 80% over breast and lung cells were subjected to investigate their specificity on human normal skin cell line (BJ-1). Compounds 9b and 9g were chosen owing to their high breast anti-cancer efficacy and their safety, in order to study the possible anti-cancer mode of action. Otherwise, drug delivery provides a means to overcome the low solubility, un-targeted release, and limited bioavailability of the prepared 1,3,4-thiadiazole drug-like substances. Compounds 9b and 9g were chosen to be encapsulated in Na-alginate microspheres. The release profile and mechanism of both compounds were investigated, and the results revealed that the release profiles of both microspheres showed a sustained release, and the release mechanism was controlled by Fickian diffusion. Accordingly, these compounds are promising for their use in chemotherapy for cancer treatment, and their hydrophilicity was improved by polymer encapsulation to become more effective in their pharmaceutical application.The 1,3,4-thiadiazole derivatives ( Cancer is one of the major causes of death today . UsuallyInvestigation of novel structures, designing and synthesis of some new, potent, and less toxic anti-cancer agents remains a major challenge for medical chemists.The 1,3,4-Thiadiazoles- and 1,2,3-Triazole-based heterocycles have been well exploited for the production of many new medicinal scaffolds. While, the 1,3,4-Thiadiazole ring system is one of the most well-known important heterocyclic nuclei, as a core structural component, common and integral feature of a variety of biomedical agents and natural products. They attract considerable interest owing to their wide spectrum of biological and pharmaceutical applications. They have anti-tuberculosis, antibacterial, antihepatitis B viral, antifungal, antileishmanial, anti-inflammatory, CNS depressant, analgesic, anticancer, antioxidant, diuretic, molluscicidal, antihypertensive, analgesic, antimicrobial, antidiabetic, antitubercular, and anticonvulsant activities ,6,7,8,9.β3-selective adrenergic receptor agonists : 511 , 496 (22), 450 (18), 378 (3), 273 (21), 250 (36), 177 (100), 165 (18), 77 (90), 55 (43). Analysis: calcd. For C21H18BrN7S2 (511): C, 49.22; H, 3.54; N, 19.13%. Found: C, 49.35; H, 3.45; N, 19.05%.Grey Crystals, Yield: 92%, m.p.: 156–158 °C; FT-IR  and the appropriate hydrazonoyl halides 5a–i (5 m mol) with the addition of 2–3 drops of DIPEA, were mixed and ground with a pestle in an open mortar at room temperature for 3–5 min until the mixture turned into a melt. The initial syrup grinding proceeded for 5–10 min, and the reaction was monitored by TLC (Thin Layer Chromatography). The solid was washed with water, then ethanol, and recrystallized from acetic acid to give 9a–i, respectively.Methyl hydrazinecarbodithioate derivative Ethyl 5-((-(3-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-phenyl-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (9a) Yellow Crystals, Yield: 89%, m.p.: 196–198 °C; FT-IR : v 2987 (C–H), 1725 (C=O), 1630 (C=N), 1600 (C=C); 1H NMR : δ 1.32 , 2.51 , 4.35 , 7.38–8.01 , 9.02 ; 13C NMR : δ 10.34, 14.43, 63.20, 118.26, 119.17, 122.84, 123.37, 127.63, 129.54, 133.14, 135.40, 138.45, 139.02, 144.34, 149.85, 158.48; MS: m/z [%]: 656 , 654 (50), 639 (12), 624 (9), 611 (18), 578 (5), 530(6), 482 (21), 430 (12), 377 (13), 278 (2), 265 (3), 178 (80), 160 (10), 66 (90), 50 (100). Analysis: calcd. For C30H24BrN9O2S (654): C, 55.05; H, 3.70; N, 19.26%. Found: C, 55.12; H, 3.62; N, 19.17%.Ethyl 5-((-(3-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-(p-tolyl)-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (9b). Yellow Crystals, Yield: 93%, m.p.: 205–207 °C; FT-IR : v 2991 (C–H), 1727 (C=O), 1620 (C=N), 1610 (C=C); 1H NMR : δ 1.37 , 2.58 , 2.64 , 4.22 , 7.5–8.13 , 9.12 ; MS: m/z [%]: 667 , 653 (15), 637 (1), 622 (9), 608 (22), 560 (15), 527(16), 482 (21), 430 (12), 270 (21), 267 (13), 176 (11), 166 (19), 70 (100). Analysis: calcd. For C31H26BrN9O2S (667): C, 55.69; H, 3.92; N, 18.86%. Found: C, 55.58; H, 3.81; N, 18.85%.Ethyl 5-((-(3-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-(4-chlorophenyl)-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (9c). Yellow Crystals, Yield: 85%, m.p.: 220–222 °C; FT-IR : v 2982 (C–H), 1730 (C=O), 1622 (C=N), 1612 (C=C); 1H NMR : δ 1.33 , 2.59 , 4.35 , 7.15–8.95 , 8.97 , 9.04 ; MS: m/z [%]: 687 , 642 (17), 612 (21), 610 (2), 572 (19), 532(6), 491 (11), 430 (12), 270 (21), 272 (43), 180 (18), 167 (19), 55 (100). Analysis: calcd. For C30H23BrClN9O2S (687): C, 52.30; H, 3.36; N, 18.30%. Found: C, 52.25; H, 3.31; N, 18.19%.Ethyl 5-(((3-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-(4-nitrophenyl)-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (9d). Yellow Crystals, Yield: 83%, m.p.: 255–257 °C; FT-IR : v 2977 (C–H), 1727 (C=O), 1628 (C=N), 1600 (C=C); 1H NMR : δ 1.32 , 2.56 , 4.30 , 7.11–8.85 , 8.98 ; MS: m/z [%]: 699 , 672 (8), 651 (15), 640 (25), 602 (7), 600 (21), 565 (14), 537 (16), 480 (21), 412 (12), 278 (24), 266 (33), 186 (8), 177 (100), 65 (50). Analysis: calcd. For C30H23BrN10O4S (699): C, 51.51; H, 3.31; N, 20.02%. Found: C, 51.58; H, 3.27; N, 19.95%.1-(5-((-(3-(1-(4-Bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-phenyl-4,5-dihydro-1,3,4-thiadiazol-2-yl)ethan-1-one (9e). Yellow Crystals, Yield: 91%, m.p.: 199–201 °C; FT-IR : v 1685 (C=O), 1630 (C=N), 1597 (C=C); 1H NMR : 2.51 , 2.57 , 7.33–8.15 , 9.02 , 9.05 ; MS: m/z [%]: 624 , 601 (18), 591 (5), 570 (21), 523 (17), 500 (14), 485 (4), 465 (6), 432 (2), 381 (9), 280 (3), 256 (33), 180 (18), 176 (100), 66 (30). Analysis: calcd. For C29H22BrN9OS (624): C, 55.77; H, 3.55; N, 20.19%. Found: C, 55.65; H, 3.42; N, 20.13%.1-(5-((-(3-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-(p-tolyl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)ethan-1-one (9f). Yellow Crystals, Yield: 91%, m.p.: 285–287 °C; FT-IR : v 1680 (C=O), 1635 (C=N), 1611 (C=C); 1H NMR : 2.37 , 2.51 , 2.63 , 7.37–8.05 , 9.02 , 9.08 ; 13C NMR : δ 10.32, 21.12, 25.45, 118.31, 119.25, 123.10, 127.61, 130.01, 133.17, 135.41, 136.60, 138.41, 139.32, 144.34, 165.5; MS: m/z [%]: 624 , 601 (18), 591 (5), 570 (21), 523 (17), 500 (14), 485 (4), 465 (6), 432 (2), 381 (9), 280 (3), 256 (33), 180 (18), 176 (100), 66 (30). Analysis: calcd. For C29H24BrN9OS (637): C, 56.43; H, 3.79; N, 19.74%. Found: C, 56.32; H, 3.73; N, 19.65%.1-(5-((-(3-(1-(4-Bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-(4-chlorophenyl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)ethan-1-one (9g). Yellow Crystals, Yield: 89%, m.p.: 212–214 °C; FT-IR : v 1686 (C=O), 1620 (C=N), 1601 (C=C); 1H NMR : 2.53 , 2.61 , 7.41–8.36 , 9.08 ; MS: m/z [%]: 660 , 658 , 632 (3), 605 (1), 590(15), 578 (31), 543 (19), 509 (25), 486 (14), 463 (16), 427 (12), 391 (91), 287 (32), 257 (30), 190 (8), 177 (100), 60 (52). Analysis: calcd. For C29H21BrClN9OS (658): C, 52.86; H, 3.21; N, 19.13%. Found: C, 52.93; H, 3.16; N, 19.02%.1-(5-((-(3-(1-(4-Bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-(4-nitrophenyl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)ethan-1-one (9h). Yellow Crystals, Yield: 95%, m.p.: 218–220 °C; FT-IR : v 1680 (C=O), 1625 (C=N), 1600 (C=C); 1H NMR : 2.57 , 2.63 , 7.31–8.39 , 8.96 ; MS: m/z [%]: 669 , 652 (30), 639 (21), 623 (9), 607 (6), 593 (2), 590 (35), 580 (14), 534 (29), 518 (2), 480 (41), 465 (61), 430 (12), 397 (1), 282 (23), 259 (32), 197 (18), 178 (90), 50 (100). Analysis: calcd. For C29H21BrN10O3S (669): C, 52.03; H, 3.16; N, 20.92%. Found: C, 52.12; H, 3.01; N, 20.87%.(-5-((-(3-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-phenyl-4,5-dihydro-1,3,4-thiadiazol-2-yl)(phenyl)methanone (9i). Brownish Yellow Crystals, Yield: 86%, m.p.: 230–232 °C; FT-IR : v 1687 (C=O), 1633 (C=N), 1609 (C=C); 1H NMR : 2.53 , 7.17–8.98 , 9.08 , 9.14 ; MS: m/z [%]: 686 , 671 (3), 592 (27), 525 (19), 459 (16), 393 (32), 281 (13), 266 (37), 195 (100), 177 (20), 55 (60). Analysis: calcd. For C34H24BrN9OS (686): C, 59.48; H, 3.16; 3.52 N, 18.36%. Found: C, 59.35; H, 3.13; N, 18.31%.Human breast carcinoma (MCF-7 cell line), human lung carcinoma (A-549 cell line) and skin normal human cell line (BJ-1); “a telomerase immortalized normal foreskin fibroblast cell line” were obtained from Karolinska Center, Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden.The procedure was carried out in a sterile area using a laminar air flow cabinet biosafety class II level. The culture was maintained in RPMI 1640 medium with 1% antibiotic-antimycotic mixture , 1% L-glutamine, and supplemented with 10% heat inactivated fetal bovine serum. Culturing and sub culturing were carried out according to Thabrew et al. [3cells per well in case of MCF-7, 20 × 103 cells/well in case of A-549 cell lines and 35–45 × 103 cells/well in case of BJ-1 using 96-well plates at 37 °C. After 48 h of incubation, the medium was aspirated and 40 μL MTT salt (2.5 mg/mL) were added and further incubated for 4 h. Then, 200 μL 10% sodium dodecyl sulphate (SDS) was added. The absorbance was measured at 595 nm.This was done according to Selim et al. , as desc50 values were calculated, using a probit analysis, and by utilizing the SPSS computer program .ICv/v) Triton X-100, and 5 mivi Tris (pH 8.0) for 15 min on ice. The cells were then centrifuged for 20 min, at 27,000× g, to separate intact chromatin (pellet) from DNA fragments (supernatant). The amount of DNA in both the pellet and the supernatant was measured by using a diphenylamine reagent. The optical density is a colorimetric, measured at 600 nm. The percentage of DNA fragmentation was calculated as the ratio of DNA in supernatant and DNA in pellet.DNA fragmentation of the cells was assayed, as described by Cohen and Duke & Burton ,44. BrieThe micro ELISA plate provided in this kit pre-coated with CASP7 specific antibody. A biotinylated CASP7 antibody and Avidin-Horseradish Peroxidase (HRP) conjugate was added. The excess components were aspired. The substrate solution was then added. The wells, which contained CASP7, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The color turned yellow following the addition of sulfuric acid solution. The optical density (OD) was measured at a wavelength of 450 nm ± 2 nm [BCL-2 in the samples and standards were estimated according to Barbareschi et al. . A biotiBax protein levels were evaluated according to Onur et al. . Monoclo9b and 9g, were prepared by w/o emulsion and internal gelation method [w/v) sodium alginate  solution was prepared by dissolution of the polymer in distilled water. The solutions of both compounds 9b and 9g were prepared by dissolving them separately in DMSO solvent with a concentration of 10 mg/mL. After the complete dissolution of polymers, the compounds 9b and 9g were added to the polymer solution individually with volume ratio 1:4 (the microsphere samples were coded as M9b and M9g). Thereafter, 10% of Span® 85 and paraffin oil  were added to the above solutions and stirred at 1000 rpm at 70 °C for 15 min, and then CaCl2 of 2 M concentration was added to the mixtures with volume ratio 1:5 and stirred for an additional 15 min at the same stirring speed and temperature. The solutions were suddenly cooled at 4 °C. The obtained microspheres were washed with isopropanol several times, to remove the paraffin oil, centrifuged and dried. For comparison, the microspheres without chemical compounds were prepared following the above protocol (coded as B). The morphology and thermal behavior of microspheres were determined by SEM , and TGA (TGA Q500), respectively. On the other hand, the amounts of encapsulated 9b and 9g in alginate microspheres were determined by dissolving 10 mg of microspheres in 5 mL DMSO and the concentrations of 9b and 9g were determined by UV-VIS spectroscopy.Microspheres, encapsulated by compounds n method . For micM9b and M9g) was investigated by immersing 50 mg of microspheres in 4.5 mL of phosphate buffer saline (PBS) with pH 7.4. The drug release was evaluated at 37 °C up to 6 days. At each predetermined time , 1.5 mL of immersed solution was collected and replaced by 1.5 mL fresh one. The collected solutions were kept at −20 °C up until the measurement. The concentrations of released drug were determined by measuring the absorbance by UV/VIS spectroscopy at wavelength 355 nm and plotting it on the standard curve. This wavelength was determined from the maxima in UV/VIS absorption curve using Jasco.The release behavior of the synthetic chemical compounds encapsulated in Na-alginate microspheres  is set at < 0.05.All experimental data stated in this work were expressed as the average ± standard deviation (SD) for n = 3 and were analyzed using standard analysis of Student’s t-test. The level of significance -1-phenyl-1H-pyrazol-4-yl)methylene)hydrazine-1-carbodithioate (4) with selective derivatives of hydrazonoyl halides via eco-friendly grinding clean method under solvent free conditions. Compounds 9b and 9g have anti-cancer effects on breast cancer with safety effects on normal cells. Compound 9b and 9g induce apoptosis inside the cells by disturbing the balance between pro-apoptotic and anti-apoptotic protein, and increase the percentage DNA fragmentation, which triggers signals to activate Caspase 7. In turn this plays an executionary role in apoptosis. Furthermore, 9b and 9g were chosen to be encapsulated in Na-alginate microspheres, the encapsulation of selected compounds in alginate microspheres controlled their release, so that the release sustained, and the release mechanism was controlled by Fickian diffusion.Briefly, our target compounds were prepared through reaction of methyl 2-((3-(1-(4-bromophenyl)-5-methyl-1Accordingly, this work introduced new promising anti-cancer agents for chemotherapy, and solved the defects associated with their water poor solubility, and hence, poor bioavailability, by encapsulating in hydrophilic Na-alginate microspheres to increase the hydrophilicity. This formulation controlled the release of chemical compounds into the solution to a sustained release.