Elongation of the side chain at C9 was achieved by an epoxide-opening reaction with a variety of nucleophiles, and the functional group manipulations completed the syntheses of these natural products. Stereochemistries of desertine could be established by the transformations.Total syntheses of the labdane diterpene lactones marrubiin, marrulibacetal, desertine, marrulibacetal A, marrubasch F, cyllenine C, marrulanic acid, and marrulactone are described. The  Marrubium (Lamiaceae) are distributed in the Mediterranean and temperate regions of the Eurasian zone, and most of the plants are used in folk medicine 2-catalyzed PKRs of substituted 5-(pent-4-ynyl)cyclohexa-1,3-dienes are precedented to afford cis-decalin derivatives stereoselectively 2-catalyzed reaction  in 65% yield. However, Grignard reagent 32 is prone to isomerization due to the high acidity of the furan 2-position, resulting in the formation of isomer 33 in 9% yield D20 9% yield . This prperature % yield pyran is spirolinked to a trans-decalin ring system, by an electrochemical oxidation of hispanolone , in 36% yield along with isomer 41 (38% yield) and other diastereomers . It is noteworthy that the dehydration of commercially available benzene with 3 Å molecular sieves prior to use affected the transacetalization, leading to the decomposition of substrates, although the reason is unclear at present.An inspection of the structures of marrulibacetal  D25−17), , with m-CPBA being the optimal oxidizing agent, whereas only desilylation occurred to give marrubiin (1) with either performic acid or magnesium monoperoxyphthalate (MMPP).Kuwajima and Urabe reported that 2-(trimethylsilyl)furans could be oxidized regioselectively with peracetic acid , and the-workers . On the 3)  were inconsistent with those observed for natural cyllenine C (12), marrulactone (14), and marrulanic acid (10) -3-methyl-2-(1-methylcyclohex-2-en-1-yl)-2-(trimethylsilyl)oxyhex-5-ynoate dicobalt hexacarbonyl complex (25). Co2(CO)8  was added to an ice-cooled (0 °C) solution of enyne 20  in CH2Cl2 (16 mL). After 1.5 h of stirring at room temperature, the reaction mixture was concentrated in vacuo, and the reddish brown residue was chromatographed twice  to give dicobalt complex 25  as a dark red oil. fR 0.51 (20:1 n-hexane/AcOEt); IR (neat) 2951, 2091, 2048, 2016, 1748, 1250, 1175, 1138, 841 cm−1; 1H-NMR  δ 0.19 3), 0.87 , 1.00 , 1.42 , 1.63 , 1.71 , 1.89−2.03 , 2.28 , 2.60 , 3.49 , 3.71 , 5.71 , 5.82 , 6.01 ; 13C-NMR  δ 2.8 (CH3), 16.1 (CH3), 20.0 (CH2), 24.4 (CH2), 25.1 (CH3), 31.5 (CH2), 36.9 (CH2), 41.3 (CH), 42.4 (C), 51.5 (CH3), 74.2 (C), 88.5 (C), 95.8 (CH), 126.8 (CH), 132.8 (CH), 175.1 (C), 200.0 (C).4,12]dodec-3-ene-7-carboxylateMethyl -6,8-dimethyl-2-oxo-7-(trimethylsilyl)oxytricyclo+ calcd for C19H30O4SiNa 373.1806; found 373.1823.One-pot reaction. Co2(CO)8  was added to an ice-cooled (0 °C) solution of enyne 20  in 1,2-dichloroethane (100 mL), and the mixture was stirred at room temperature for 3 h. The resulting dark red suspension was diluted with 1,2-dichloroethane (900 mL), and cyclohexylamine  was added. After 5 h of heating at reflux, the reaction mixture was cooled to room temperature, and the volatile elements were removed in vacuo. The residue was suspended in 4:1 n-hexane/AcOEt and filtered through a Celite pad. Evaporation of the filtrate in vacuo furnished the crude product (5.75 g), which was purified by column chromatography  to give enone trans-24  as a white solid.4,12]dodec-3-ene-7-carboxylateMethyl -1,6,8-trimethyl-2-oxo-7-(trimethylsilyl)oxytricyclo+ calcd for C20H32O4SiNa 387.1962; found 387.1950.-5-(Methoxycarbonyl)-1,4a,6-trimethyl-8-oxo-5-(trimethylsilyl)oxydecahydronaphthalene-1-carboxylic acid (27). KMnO4  was added to a solution of NaIO4  in H2O (7.0 mL), and the mixture was stirred for 30 min. To the mixture was added K2CO3 , followed by a solution of enone 26  in t-BuOH (1.4 mL plus 2 × 1 mL rinse). After 24 h of stirring, the reaction was quenched with NaHSO3 , and the resulting mixture was extracted with AcOEt (3 × 20 mL). The combined organic extracts were washed with brine (2 × 15 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the crude product (70.9 mg), which was used without further purification.28 (70.9 mg) in AcOEt (14 mL). After 63 h of stirring at room temperature, the resulting suspension was filtered through a Celite pad, and the filtrate was evaporated in vacuo. Purification of the crude product (74.7 mg) by column chromatography  gave γ-ketocarboxylic acid 27  as an amorphous solid. fR 0.60 (1:1 n-hexane/AcOEt); + calcd for C19H32O6SiNa 407.1860; found 407.1847.Activated carbon (708 mg) was added to an ice-cooled (0 °C) solution of the crude α-ketocarboxylic acid 4,12]dodecane-7-carboxylateMethyl -1,6,8-trimethyl-2-oxo-7-(trimethylsilyl)oxy-3-oxatricyclo+ calcd for C19H32O5SiNa 391.1911; found 391.1917.Ethyl chloroformate  was added to an ice-cooled (0 °C) mixture of crude γ-hydroxycarboxylic acid (42.7 mg) and Et4,12]dodecane-7-carboxylateMethyl -7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatricyclo+ calcd for C16H24O5Na 319.1516; found 319.1519; Anal. Calcd for C16H24O5: C, 64.84; H, 8.16. Found: C, 64.82; H, 8.06.-2a,5a,7-Trimethyloctahydrospirofuran-6,2′-oxiran]-2(2aH)-one (22). Sodium bis(2-methoxyethoxy)aluminum hydride in toluene  was diluted with CH2Cl2 (5.6 mL), and the solution was cooled to −78 °C. A solution of α-hydroxyester 29  in CH2Cl2 (2 mL plus 2 × 0.5 mL rinse) was added, and the mixture was stirred at −23 °C for 22 h. The reaction was quenched by addition of MeOH (3 mL) followed by 10% aqueous potassium sodium tartrate (12 mL). After 3 h of stirring, the resulting mixture was extracted with AcOEt (2 × 40 mL), and the combined organic extracts were washed with brine (2 × 20 mL) and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the crude product (235 mg), which was used without further purification.2Cl2 (4 mL) with the reaction time of 16 h at −23 °C. The crude product (216 mg) was used without further purification.This sequence was repeated employing bis(2-methoxyethoxy)aluminum hydride in toluene  and CH31 (216 mg) in THF (2 mL plus 1 mL and 2 × 0.5 mL rinse) was added dropwise to an ice-cooled (0 °C) suspension of NaH  in THF (4.6 mL). After 30 min of stirring at 0 °C, p-toluenesulfonyl imidazole  was added, and the mixture was stirred at 0 °C for 11 h. The reaction was quenched with saturated aqueous NH4Cl (10 mL), and the resulting mixture was extracted with AcOEt (2 × 40 mL). The combined organic extracts were washed with brine (2 × 20 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the crude product (563 mg), which was purified by column chromatography  to give epoxide 22  as a white solid. fR 0.53 (2:1 n-hexane/AcOEt); mp 83−84 °C (colorless needles from 8:1 n-hexane/Et2O); + calcd for C15H22O3Na 273.1461; found 273.1469.A solution of crude diol Marrubiin (1). To a cooled (−10 °C) suspension of CuBr·SMe2  in Et2O (1.5 mL) was added a 0.14 M solution of (3-furylmethyl)magnesium bromide (32) in Et2O  [prepared from 3-(bromomethyl)furan  and magnesium  in Et2O (4 mL) at 0 °C], followed by addition of a solution of epoxide 22  in Et2O (0.5 mL plus 2 × 0.5 mL rinse). After 2 h, the reaction was quenched with saturated aqueous NH4Cl (5 mL), and the resulting mixture was extracted with AcOEt (3 × 10 mL). The combined organic extracts were washed with brine (20 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the crude product (200 mg), which was chromatographed twice  to give marrubiin  and isomer 33  as white solids. fR 0.59 (1:1 n-hexane/AcOEt); mp 160−161 °C (colorless needles from 4:1 n-hexane/AcOEt)  ; IR (KBr) 3466, 2940, 2870, 1740, 1468, 1395, 1356, 1304, 1269, 1200, 1153, 1101, 1024, 984 cm−1; 1H-NMR  δ 0.97 , 1.07 , 1.26 , 1.29 , 1.32 , 1.43−1.55 , 1.66−1.79 , 1.90 , 2.09−2.18 , 2.23 , 2.48−2.58 , 4.74 , 6.27 , 7.24 , 7.37 ; 13C-NMR  δ 16.6 (CH3), 18.2 (CH2), 21.0 (CH2), 22.3 (CH3), 22.9 (CH3), 28.3 (CH2), 28.6 (CH2), 31.5 (CH2), 32.4 (CH), 35.1 (CH2), 39.7 (C), 43.8 (C), 44.8 (CH), 75.8 (C), 76.2 (CH), 110.7 (CH), 125.0 (C), 138.6 (CH), 143.1 (CH), 183.8 (C); HRMS (ESI) m/z [M + Na]+ calcd for C20H28O4Na 355.1880; found 355.1867.t)  D20 S,4R,6R,7R,8S,12R)-7-[(3-methylfuran-2-yl)methyl]-7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatricyclododecane (33): fR 0.27 (3:1 n-hexane/AcOEt); mp 173−174 °C (colorless needles from 3:1 n-hexane/AcOEt); + calcd for C20H28O4Na 355.1880; found 355.1871.Data for -7-[2-[2-(Trimethylsilyl)furan-3-yl]ethyl]]-7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatricyclo[6.3.1.0 (35). Phosphorus tribromide  was added to an ice-cooled (0 °C) solution of [2-(trimethylsilyl)furan-3-yl]methanol  in Et2O (30 mL). After 30 min of stirring, the reaction was quenched with brine (30 mL), and the resulting mixture was extracted with Et2O (30 mL). The organic extract was dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the pale brown oil, which was purified by distillation to give 3-(bromomethyl)-2-(trimethylsilyl)furan  as a pale brown oil.2O (1 mL) was added to a cooled (−10 °C) suspension of magnesium tuning  in Et2O (1 mL), and the reaction mixture was stirred for 1.5 h. The 0.23 M solution of [2-(trimethylsilyl)furan-3-yl]methylmagnesium bromide (34) in Et2O  thus obtained was added to a cooled (−10 °C) suspension of CuBr·SMe2  in Et2O (0.6 mL), followed by addition of a solution of epoxide 22  in Et2O (1.0 mL). After 4 h of stirring, the reaction was quenched with saturated aqueous NH4Cl (3 mL), and the resulting mixture was extracted with AcOEt (3 × 10 mL). The combined organic extracts were washed with brine (10 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the pale brown oil (206 mg), which was purified by flash column chromatography  to give silylated marrubiin 35  as a colorless solid. fR 0.32 (3:1 n-hexane/AcOEt); + calcd for C23H36O4SiNa 427.2275; found 427.2259.A solution of 3-(bromomethyl)-2-(trimethylsilyl)furan  in EtMarrubiin (1). Bu4NF in THF  was added to a solution of silylated marrubiin 35  in THF (0.2 mL). After 4 h of stirring, the mixture was partitioned between AcOEt (8 mL) and H2O (8 mL), and the aqueous layer was extracted with AcOEt (8 mL). The combined organic extracts were washed with brine (8 mL) and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the colorless oil (0.5 mg), which was purified by column chromatography  to give marrubiin  as a white solid.4,12]dodecane-7-[2-ethyl]-7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatricyclo+ calcd for C24H38O6Na 445.2561; found 445.2550.4,12]dodecane-7-[2-ethyl]-7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatricyclododecaneand -7-[2-ethyl]-7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatri cyclo+ calcd for C24H40O8Na 479.2615; found 479.2631.Marrulibacetal (13). TsOH  was added to a mixture of triols 38 and 39  in benzene (1 mL), and the mixture was stirred for 1.5 h. The reaction was quenched with saturated aqueous NaHCO3 (5 mL), and the resulting mixture was extracted with AcOEt (2 × 15 mL). The combined organic extracts were washed with brine (15 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the crude product (19.2 mg), which was purified by flash column chromatography  to give a mixture of marrulibacetal (13) and its diastereomers. The mixture was flash chromatographed  to provide a mixture of marrulibacetal (13) and C13,14,15,16-epimer 41, along with a 1:1 mixture of C15-epimer and C13,14,16-epimer . Separation of marrulibacetal (13) and 41 by flash column chromatography  yielded marrulibacetal  and C13,14,15,16-epimer 41  as white solids. fR 0.44 (4:1 CH2Cl2/acetone); mp 177−179 °C (colorless needles from n-hexane/benzene); ; IR (neat) 3435, 2961, 2928, 1773, 1740, 1458, 1389, 1244, 1200, 1111, 1053, 935 cm−1; 1H-NMR  δ 1.03 , 1.11 , 1.19 , 1.22 , 1.28 , 1.42 , 1.49 , 1.71−1.78 , 1.80−1.90 , 1.98 , 2.05−2.20 , 2.36 , 2.60 , 2.61 , 3.51 , 3.82 , 3.95 , 4.77 , 5.04 , 5.46 ; 13C-NMR  δ 15.0 (CH3), 17.9 (CH2), 19.5 (CH3), 21.1 (CH2), 22.1 (CH3), 23.1 (CH3), 27.8 (CH2), 28.2 (CH2), 29.6 (CH2), 32.3 (CH2), 33.6 (CH), 40.9 (C), 43.9 (C), 44.6 (CH), 63.9 (CH2), 75.6 (C), 76.5 (CH), 78.5 (CH), 80.4 (C), 105.3 (CH), 108.7 (CH), 184.0 (C); HRMS (EI) m/z [M+] calcd for C22H34O7 410.2305; found 410.2300.S,2′aS,3S,3aR,5′aS,6R,7′R,7aR,8′aR,8′bR)-2-ethoxy-3,3a-dihydroxy-2′a,5′a,7′-trimethyltetradecahydrospiropyran-6,6′-[6H]naphthofuran]-2′(2′aH)-one (41). fR 0.56 (4:1 CH2Cl2/acetone);  calcd for C22H34O7 410.2305; found 410.2297.Data for ,8S,12R]-7-[2-ethyl]-7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatricyclo+ calcd for C22H36O8Na 451.2302; found 451.2308.A 0.157 M solution of OsOMarrulibacetal A (17). TsOH  was added to a mixture of desertine (18) and its diastereomer 40  in benzene (1.4 mL). After 5 h of stirring, an additional portion of TsOH  was added, and the reaction mixture was stirred for 3.5 h. The reaction was quenched with saturated aqueous NaHCO3 (2 mL), and the resulting mixture was extracted with AcOEt (3 × 5 mL). The combined organic extracts were washed with brine (5 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the brown oil (68.1 mg), which was purified by flash column chromatography  to give marrulibacetal A  and C13,14,15,16-isomer 42  as colorless amorphous solids. fR 0.73 (AcOEt); + calcd for C21H32O7Na 419.2045; found 419.2037.S,2′aS,3S,3aR,5′aS,6R,7′R,7aR,8′aR,8′bR)-3,3a-dihydroxy-2-methoxy-2′a,5′a,7′-trimethyl tetradecahydrospiropyran-6,6′-[6H]naphthofuran]-2′(2′aH)-one (42): fR 0.78 (AcOEt); + calcd for C21H32O7Na 419.2045; found 419.2059.Data for (2Marrubasch F (19). m-CPBA  was azeotropically dried with benzene, and dissolved in CH2Cl2 (0.2 mL). The m-CPBA solution was added to an ice-cooled (0 °C) solution of silylated marrubiin 35  in CH2Cl2 (0.8 mL). After 1.5 h of stirring, the reaction was quenched with a mixture of 1 M aqueous Na2S2O3 (2 mL) and saturated aqueous NaHCO3 (2 mL), and the resulting mixture was extracted with AcOEt (10 mL). The organic extract was washed with brine (4 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the colorless oil (28.4 mg), which was purified by preparative thin layer chromatography (200 mm × 100 mm × 0.25 mm preparative silica gel plate and elution with 1:1 n-hexane/AcOEt) to give marrubasch F  as a white solid. fR 0.32 (1:1 n-hexane/AcOEt); mp 195−196 °C   [lit. [D + 41.5 ]; IR (neat) 3536, 2926, 2873, 1747, 1456, 1199, 1139, 1095, 1076, 1041, 983 cm−1; 1H-NMR  δ 0.96 , 1.04 , 1.25 , 1.30 , 1.45 , 1.53 , 1.69−1.84 , 2.05−2.17 , 2.28 , 2.41−2.52 , 4.75 , 4.80 , 7.14 ; 13C-NMR  δ 16.7 (CH3), 18.1 (CH2), 21.2 (CH2), 22.3 (CH3), 22.9 (CH3), 28.3 (CH2), 28.6 (CH2), 31.6 (CH2), 32.4 (CH2), 32.6 (CH2), 39.9 (C), 43.8 (C), 44.9 (CH), 70.4 (CH2), 75.2 (C), 76.2 (CH), 134.7 (C), 144.3 (CH), 174.9 (C), 183.9 (C); HRMS (ESI) m/z [M + Na]+ calcd for C20H28O5Na 371.1830; found 371.1834. 8.1 mg, .0 μmol i 8.1 mg, .0 μmol i4,12]dodecane-7-Hydroxy-1,6,8-trimethyl-2-oxo-7-propargyl-3-oxatricyclo+ calcd for C17H24O3Na 299.1618; found 299.1623.Cyllenine C (12). To an ice-cooled (0 °C) mixture of alcohol 43  and m-CPBA  in CH2Cl2 (0.3 mL) was added a 0.123 M solution of methanesulfonic acid in CH2Cl2  followed by Ph3PAuNTf2 . After 1 h of stirring at room temperature, the reaction was quenched with saturated aqueous NaHCO3 (1 mL), and the resulting mixture was extracted with AcOEt (2 × 5 mL). The combined organic extracts were washed with brine (5 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the colorless oil (13.7 mg), which was purified by column chromatography  to give cyllenine C  as a white solid. fR 0.32 (1:2 n-hexane/AcOEt); mp 164−165 °C (colorless needles from n-hexane/Et2O); ; IR (neat) 3019, 2934, 2870, 1763, 1474, 1458, 1420, 1391, 1273, 1120, 1067, 1042, 993 cm−1; 1H-NMR  δ 0.93 , 1.09 , 1.30 , 1.31 , 1.46 , 1.49 , 1.52 , 1.72 , 1.78 , 1.95 , 2.12 , 2.18 , 2.19 , 2.22 , 2.28 , 2.54 , 2.62 , 4.75 ; 13C-NMR  δ 15.4 (CH3), 17.7 (CH2), 22.2 (CH3), 22.4 (CH3), 24.5 (CH2), 27.7 (CH2), 28.2 (CH2), 29.3 (CH2), 31.2 (CH2), 32.3 (CH), 38.6 (C), 44.0 (C), 45.3 (CH), 75.6 (CH), 91.2 (C), 176.9 (C), 183.3 (C); HRMS (ESI) m/z [M + Na]+ calcd for C17H24O4Na 315.1567; found 315.1566.2) D20 4,12]dodecane-7-[4-(tert-Butyldimethylsilyl)oxybutyl]-7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatricyclo[6.3.1.0 (45). To an ice-cooled (0 °C) solution of 3-(tert-butyldimethylsilyl)oxypropylmagnesium bromide (44) [prepared from (3-bromopropoxy)(tert-butyl)dimethylsilane  and magnesium tuning ] in THF (14 mL) was added CuBr·SMe2 , followed by addition of a solution of epoxide 22  in THF (0.20 mL plus 2 × 0.20 mL rinse). After 5 h of stirring at room temperature, the reaction was quenched with saturated aqueous NH4Cl (15 mL), and the resulting mixture was extracted with AcOEt (3 × 20 mL). The combined organic extracts were washed with brine (20 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the pale yellow oil (869 mg), which was purified by column chromatography  to give TBS ether 45  as a pale yellow amorphous. fR 0.46 (3:1 n-hexane/AcOEt); + calcd for C24H44O4SiNa 447.2907; found 447.2891.4,12]dodecane-7-Hydroxy-7-(4-hydroxybutyl)-1,6,8-trimethyl-2-oxo-3-oxatricyclo+ calcd for C18H30O4Na 333.2042; found 333.2052.Marrulactone (14). TEMPO  was added to an ice-cooled (0 °C) mixture of diol 46  and PhI(OAc)2  in CH2Cl2 (0.3 mL). After 4 h of stirring at room temperature, the reaction was quenched with 1 M aqueous Na2S2O3 (1 mL), and the resulting mixture was extracted with AcOEt (3 × 1 mL). The combined organic extracts were washed with brine (3 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the colorless oil (64.0 mg), which was purified by column chromatography  to give marrulactone  as a colorless amorphous. fR 0.60 (1:2 n-hexane/AcOEt); ; IR (neat) 3017, 2961, 2928, 2872, 1769, 1719, 1458, 1391, 1329, 1261, 1244, 1215, 1188, 1117, 1070, 1030, 1003 cm−1; 1H-NMR  δ 0.98 , 1.11 , 1.31 , 1.34−1.42 , 1.48 , 1.55 , 1.74−1.80 , 1.80−1.94 , 2.09−2.20 , 2.25 , 2.32 , 2.57 , 4.78 ; 13C-NMR  δ 16.5 (CH3), 17.8 (CH2), 18.9 (CH2), 22.0 (CH3), 22.8 (CH3), 26.1 (CH2), 28.1 (CH2), 28.6 (CH2), 30.4 (CH2), 30.9 (CH2), 34.3 (CH), 40.8 (C), 44.0 (C), 44.3 (CH), 75.8 (CH), 88.3 (C), 172.2 (C), 183.5 (C); HRMS (ESI) m/z [M + Na]+ calcd for C18H26O4Na 329.1723; found 329.1711.Marrulanic acid (10). LiOH  was added to a solution of marrulactone  in THF/H2O . After 31 h of stirring, the mixture was acidified with 1 M aqueous HCl (3 mL), and the resulting mixture was extracted with AcOEt (3 × 3 mL). The combined organic extracts were washed with brine (5 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the colorless oil (11.7 mg), which was purified by column chromatography  to give marrulanic acid  as a colorless amorphous. fR 0.19 (1:2 n-hexane/AcOEt); ; IR (neat) 3536, 3196, 2949, 2928, 2868, 1761, 1732, 1464, 1412, 1391, 1287, 1260, 1196, 1179, 1159, 1126, 1094, 1078, 1042, 1018 cm−1; 1H-NMR  δ 0.92 , 1.04 , 1.26 , 1.29 , 1.44−1.80 , 2.04−2.17 , 2.24 , 2.38 , 4.74 ; 13C-NMR  δ 16.5 (CH3), 18.2 (CH2), 20.3 (CH2), 22.3 (CH3), 22.9 (CH3), 28.3 (CH2), 28.6 (CH2), 31.5 (CH2), 32.2 (CH), 34.29 (CH2), 34.31 (CH2), 39.7 (C), 43.8 (C), 44.8 (CH), 75.6 (C), 76.2 (CH), 178.5 (C), 183.9 (C); HRMS (ESI) m/z [M + Na]+ calcd for C18H28O5Na 347.1834; found 347.1813.3) D25 4,12]dodecane-7-[2-ethyl]-7-hydroxy-1,6,8-trimethyl-2-oxo-3-oxatricyclo+ calcd for C22H36O8Na 451.2302; found 451.2308.Desertine (18). TsOH  was added to a solution of bisacetal 42  in MeOH (0.5 mL), and the mixture was heated at reflux for 3.5 h. After cooling to 0 °C, the reaction was quenched with saturated aqueous NaHCO3 (2 mL), and the resulting mixture was extracted with AcOEt (2 × 5 mL). The combined organic extracts were washed with brine (10 mL), and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the pale yellow oil (3.6 mg), which was purified by flash column chromatography  to give desertine  as a colorless oil. fR 0.30 (1:1 n-hexane/AcOEt); + calcd for C22H36O8Na 451.2302; found 451.2313.e) , marrulanic acid (10), cyllenine C (12), marrulibacetal (13), marrulactone (14), marrulibacetal A (17), desertine (18), and marrubasch F (19), have been accomplished. Since chiral building block 20 could be prepared from -6-[(tert-butyldimethylsilyl)oxymethyl]-4-methyltetrahydro-2H-pyran-2-one [37 and transacetalization of the diastereomer of marrulibacetal A with MeOH. The syntheses illustrate the synthetic utility of chiral building block 20 for the synthesis of this class of diterpenoids. The synthetic strategy also features elongation of the C9 side chain through an epoxide-opening reaction as well as high convergency and flexibility. The syntheses of other natural products and non-natural analogues for biological and pharmaceutical investigations are in progress in our laboratory and will be reported in due course.Total syntheses of eight labdane diterpene lactones, marrubiin (an-2-one  in 37% y


The linkage type of fructooligosaccharides (FOS) in Fig. 2 and in the text  was incorrectly written as β-1,2 and should be β-2,1.Volume 11, no. 1, e02179-19, 2020,


Myristica fragrans Houtt) (MF) is a well-known traditional Chinese medicine; its major toxic components, safrole and myristicin, are rich in essential oils. Essential oils of MF (MFO) were extracted by hydrodistillation; the residue was extracted using 50% methanol (MFE-M). The minimum inhibitory concentration (MIC) of MFE-M against Cutibacterium acnes and Staphylococcus aureus was 0.64 mg. Four compounds were obtained from MFE-M: myristicin (1), (+)-erythro-Δ8′-7S,8R- dihydroxy-3,3,5′-trimethoxy-8-O-4′-neolignan (2), (+)-erythro-Δ8’-7-hydroxy-3,4,3’,5’-tetramethoxy 8-O-4-neolignan (3), and erythro-Δ8′-7-acetoxy-3,4,3′,5′-tetramethoxy-8-O-4′-neolignan (4). Compound 2 exerted the strongest antimicrobial activity, with MICs of 6.25 and 3.12 μg/mL against C. acnes and S. aureus, respectively. Moreover, 2 inhibited NO, PGE2, iNOS, and COX-2 levels in RAW 264.7 cells induced by LPS or heat-killed C. acnes; NO production at 50% inhibitory concentrations (IC50) was 11.07 and 11.53 μg/mL, respectively. Myristicin and safrole content was higher in MFO than in MFE-M. MFO and MFE-M caused no skin irritation after a single topical application in Wistar rats. MFE-M, with low safrole and myristicin content, did not cause skin irritation and exhibited an anti-acne effect; moreover, 2 was identified as the active substance. Therefore, MFE-M could be employed to develop anti-acne compounds for use in cosmetics.Acne is a common skin condition observed in adolescents. Nutmeg ( Cutibacterium acnes (previously Propionibacterium acnes) and Staphylococcus aureus), and (4) skin inflammation . 1H NMR  δ: 6.39 , 6.35 , 5.92 , 5.90 , 5.10-5.05 , 3.89 , 3.29 . 13C NMR  δ: 148.8 (C-2), 143.5 (C-1), 137.3 (C-3), 134.6 (C-2′), 133.5 (C-5), 115.8 (C-3′), 107.7 (C-4), 102.7 (C-6), 101.2 (O-CH2-O), 56.5 (OCH3), 40.2 (C-1′) −. 1H NMR  δ: 6.93 , 6.72 , 6.68 , 6.52 , 5.97 , 5.12 , 5.05 , 4.74 , 4.27 , 3.81 , 3.34 , 1.09 . 13C NMR  δ: 154.7 (C-3′ and C-5′), 148.7 (C-4), 146.7 (C-3), 138.8 (C-8′), 137.7 (C-1′), 134.8 (C-4′), 133.5 (C-1), 120.1 (C-6), 116.1 (C-9′), 115.8 (C-5), 111.1 (C-2), 106.9 (C-2′ and C-6′), 83.8 (C-8), 75.5 (C-7), 56.6 (3′-OMe and 5′-OMe), 56.4 (3-OMe), 41.4 (C-7′), 13.8 (C-9) +. 1H NMR  δ: 6.95 , 6.84 , 6.77 , 6.46 , 5.96 , 5.12 , 5.09 , 4.80 , 4.33 , 3.86 , 3.85 , 3.83 , 3.35 , 1.10 . 13C NMR  δ: 153.5 (C-3′ and C-5′), 148.8 (C-4), 147.9 (C-3), 137.1 (C-8′), 136.1 (C-4′), 133.0 (C-1′), 132.7 (C-1), 118.1 (C-6), 116.2 (C-9′), 110.8 (C-2), 109.3 (C-5), 105.5 (C-2′ and C-6′), 82.3 (C-8), 72.8 (C-7), 56.1 (3′-OMe and 5′-OMe), 55.9 (3-OMe and 4-OMe), 40.6 (C-7′), 12.8 (C-9) +. 1H NMR  δ: 6.88 , 6.80 , 6.84 , 6.39 , 5.96 , 5.86 , 5.11 , 5.08 , 4.43 , 3.86 , 3.85 , 3.77 , 3.33 , 2.17 , 1.28 . ). 13C NMR  δ: 170.2 (7-OAc),153.4 (C-3′ and C-5′), 148.7 (C-4),148.5 (C-3), 137.2 (C-8′), 135.7 (C-4′), 133.8 (C-1′), 130.6 (C-1), 119.3 (C-6), 116.0 (C-9′), 110.8 (C-2), 110.3 (C-5), 105.6 (C-2′ and C-6′), 80.1 (C-8), 76.4 (C-7), 56.0 (3′-OMe and 5′-OMe), 55.9 (3-OMe and 4-OMe), 40.5 (C-7′), 21.2 (7-OAc), 14.5 (C-9) [C. acnes  and S. aureus  were obtained from the Bioresource Collection and Research Center . C. acnes were cultured in BAP, BactoTM TSB, and BactoTM TSA in an anaerobic condition using MGC AnaeroPack-Anaero and MGC AnaeroPack-Jar. S. aureus was cultured in BactoTM TSB and BactoTM TSA.The strains of 1, 2, 3, and 4 were tested against C. acnes by determining the MIC values through the broth-dilution method [8 CFU/mL). Triton X-100 was used as a positive control. Samples were diluted in sterile broth and then mixed with broth inoculated with C. acnes. Dilutions were prepared at 10-fold the desired final concentration. The test concentrations of MFE-M, MFE-E, and MFE-H were 0.02.10.24 mg/mL. The test concentrations of compounds 1, 2, 3 and 4 were 3.12–100 µg/mL. Test samples were incubated under anaerobic conditions at 37 °C for 24 h until visible growth of the test microorganisms was observed in the control. The O.D. value was measured using an ELISA reader at 600 nm. The inhibition ratio was compared to that of Triton X-100. The MIC experiment was repeated in triplicate. Moreover, S. aureus (0.5 × 108 CFU/mL) was prepared. The experimental protocol was the same as that previously described, with some modifications. The antimicrobial activity was expressed as the O.D. value of the inhibition rate against the test microorganisms: Ratio (%) = (O.D. of sample/O.D. Triton X-100) × 100.The experimental protocol followed a previous report with modifications . MFO, MFn method . A fresh2 atmosphere. RAW 264.7 cells (4.0 × 105 cells/mL) were seeded in 96-well plates and co-treated with LPS (500 ng/mL) and the test samples. After 24 h, NO production was determined with Griess reagent, and the absorption was detected at 530 nm. The inhibitory percentage of NO production was presented as an anti-inflammatory activity. Prostaglandin E2 (PGE2) concentrations in the cell culture media were determined using a PGE2 enzyme-linked immunosorbent assay (ELISA) kit . The survival rate of RAW 264.7 cells was detected by an MTT assay.The anti-inflammatory activities of the compounds were evaluated in RAW 264.7 cells cultured in DMEM with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin, and maintained at 37 °C in a 5% COC. acnes (4 × 108 cfu/mL) was heated to 80 °C for 30 min to kill the bacteria. HKC was freeze-dried as a powder and maintained at 4 °C until use. RAW 264.7 cells were cultured in DMEM with 10% FBS, 1% penicillin-streptomycin, and 1% l-glutamine at 37 °C in a 5% CO2 atmosphere. RAW 264.7 cells (4 × 105 cells/mL) were seeded in six-well plates and co-treated with HKC (300 μg/mL) and test samples. NO and PGE2 production were determined according to previously described methods [ methods . Whole-ccontroller, an LC-10ATvp liquid chromatographic pump, an SPD-M10A diode array detector, an SIL-10Avp auto-injector, a CTO-10A column oven, FCV-10Avp flow-channel selection valves , and an ERC-3415 degasser . The stationary phase comprised a Purospher® STAR RP-18e reverse-phase column , and the mobile phase system included 0.05% trifluoroacetic acid : CH3CN isocratic at a ratio of 40:60. The flow rate was 1 mL/min, and the column oven temperature was maintained at 40 °C. The ultraviolet wavelength was set to 220 nm to detect myristicin and safrole in MFO and MFE-M. The concentration range of myristicin and safrole was 0.78 to 1000 μg/mL.The HPLC apparatus included an SCL-10Avp system ad libitum and maintained on a 12-h light/dark cycle. At 24 h before the test, the backs of the rats were shaved free of hair (2.5 × 2.5 cm at each site) and checked for any abnormalities . An appropriate concentration of MFO and MFE-E (50 μL per sample) was smeared onto the shaved back of a Wistar rat. The patches were secured for a 4-h exposure period and removed with distilled water. At 24 and 48 h after the application of MFO and MFE-E, skin irritation was observed, and the extent of an evident skin allergic reaction was evaluated for each test animal. An acute primary irritation test was performed following the classic Draize test.The skin toxicity of MFO and MFE-E was evaluated by a single topical application in Wistar rats . The folt-test. Differences were considered significant at p < 0.05.The data are presented as the mean and standard deviation (S.D.). Significance was calculated using Student’s


In Havelange (Belgium), two farms are experiencing an ecological transition. We aimed to evaluate the impact of their agricultural activities on insect pollinator communities. This article depicts the situation at the very early stage of the farm transition. This study supports the fact that the maintenance of farm-level natural habitats provides environmental benefits, such as the conservation of two important pollinator communities: wild bees and hoverflies.Andrenaschencki Morawitz 1866, Bombuscampestris (Panzer 1801), Euceralongicornis (L.) and Halictusmaculatus Smith 1848 or for data deficient species, such as A.semilaevis Pérez 1903, A.fulvata (Müller 1766), A.trimmerana (Kirby 1802) and Hylaeusbrevicornis Nylander 1852.Over two years (2018-2019), by using nets and coloured pan-traps, we collected 6301 bee and hoverfly specimens amongst contrasting habitats within two farmsteads undergoing ecological transition in Havelange (Belgium). We reported 101 bee species and morphospecies from 15 genera within six families and 31 hoverfly species and morphospecies from 18 genera. This list reinforces the national pollinator database by providing new distribution data for extinction-threatened species, such as  Nowadays, the greatest challenge faced by agriculture is to provide food for everyone, without altering the agro-biodiversity and the related ecosystem services . Indeed,Agroecological farming systems grow crops on small areas, alongside heterogeneous habitats and complex arrangements  that provide shelters and abundant food resources to beneficial insects . DiversiHymenoptera: Anthophila) is amongst the most efficient pollinator groups in temperate agriculture landscapes. In Belgium, the latest inventory recorded 403 bee species, which represents almost one quarter of the European bee diversity (The bee community (iversity . Their miversity . The riciversity . Since tiversity .Diptera order represents one of the largest and most diverse groups in the pollinator community (Syrphidae) play a pivotal role in the pollen transmission of over 70% of wildflowers . The general impacts of farm-scale landscape diversification on bee and hoverfly fauna are discussed. In future research, such inventory will allow an assessment of the impacts of regenerating agricultural landscapes on the pollinator community structure. Moreover, this study feeds in new records and new locations for the national repository of the wild bee and hoverfly communities, owned by the Laboratory of Functional and Evolutionary Entomology (Prof. Frédéric Francis), Gembloux Agro-Bio Tech and the Laboratory of Zoology (Prof. Pierre Rasmont), University of Mons.The study was conducted in two neighbouring agricultural sites, located in the Municipality of Havelange Fig. A: the FrThe Froidefontaine farmstead50°23'6''N, 5°8'34.799''E) covers an area of 55 hectares, with a mosaic of varied habitats. One of the management objectives is diversifying the land use by conserving natural areas  and hosting different farming projects in a collaborative way on farming areas. Thus, the farm aims at creating a rich and welcoming landscape for diversity, including biodiversity.The Froidefontaine farm  covers an area of just over 40 ha. In 2016, the farm managers and a committee of various partners converted conventionally-managed fields to agroecological farming methods. To allow a complexification of the ecological network and creating an agricultural landscape enriched with biodiversity, the first actions were: laying hedges and grass strips; planting rustic apple trees; breeding Angus cattle (Bostaurustaurus L.) in an orchard; alternating temporary and permanent meadows; arranging of flowered grass strips; using no pesticides and amendments.The Emeville farm (Festucaarundinacea Schreb 1771 and Dactylisglomerata L. 1753 sown at 20 kg/ha. The feeder flower patch was composed of a mix of 40% of clover (Trifoliumpratense L. 1753) and 60% of alfalfa (Medicagosativa L. 1753) sown at 25 kg/ha. In order to match Agri-Environmental and Climate Measures (AECM) specifications, the pollinator flower patch was sown at 30 kg/ha and was composed of a mix including 85% of grasses , 2% of leguminous species , 3% of annual flower  and 10% of other flower species .The sampling zone covered 15 ha and was divided into seven parcels Fig. B was comTo assess wild bee and hoverfly diversity, we conducted standardised sampling methods by combining coloured pantraps and netting transects . SamplinWe followed the protocol of In 2019, we decided to let the yellow pantraps to be continuously activated from mid-May to the end of July with sampling every 10 days to maximise the capture of syrphids and considering that hoverflies have a predilection for the yellow colour .Halictidae, Andrenidae and Apidae, Colletidae, Megachilidae and Melittidae). All Halicitidae and Andrenidae specimens were confirmed by Alain Pauly  and Thomas James Wood (University of Mons), respectively. Other bee specimens were confirmed by the reference collections of Gembloux Agro-Bio Tech. Hoverfly specimens were identified at the species level using the identification key of Bee specimens were identified at the species level following identification keys of Thanks to Data Fauna-Flora v.5.1 software , we querggplot2 and ggpubr packages 26086597-4FB4-571F-9533-CDBA94D99EB6PolylecticNear ThreatenedTable 2Smith 1847773B4C42-F312-55F1-A798-F2BB489EB06ESalicaceaeOligolectic on Least ConcernTable 2Fabricius 1775AD109654-7ACA-53D3-BDCF-F9CDDEC27AC1PolylecticLeast ConcernTable 2Pérez 190287EB1626-D85F-55A6-B7AA-7F30B5637E80PolylecticLeast ConcernTable 2(Kirby 1802)6E930963-BB1F-514D-9246-C4CD3A7DC3E5PolylecticLeast ConcernTable 2(Linnaeus 1758)BB080AAB-19F3-50ED-B714-78A2D50B0F24PolylecticLeast ConcernTable 2(Kirby 1802)81C96F69-0E63-5D14-8FCE-516BA09F275ESalicaceaeOligolectic on Least ConcernTable S(Kirby 1802)ED29E1DB-B7AC-5ADC-9E94-9CDE6778C581PolylecticLeast ConcernTable 2Panzer 1799C4A9DB13-2481-53A0-9C3D-7BB49EB4A3D7PolylecticLeast ConcernTable 2(Müller 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184155CC1179-8DCE-5C1D-A06E-7A4FD614D928Cuckoo beeLeast ConcernTable (Linnaeus 1758)D8D544B6-87EC-5E22-8623-0FED8C8162FFCuckoo beeLeast ConcernTable 2Fabricius 179306000374-DEBA-546D-844E-71E384D5E8FECuckoo beeLeast ConcernTable Jurine 18076BF255D1-42E2-562A-A60F-A1DC7B0CBA32Cuckoo beeLeast ConcernTable 2Panzer 1798D6C94E98-8489-5156-B17C-77F5CD03AA46Cuckoo beeLeast ConcernTable 2Panzer 179860709FC6-A497-5FE3-A8C0-E50BC33FF16BCuckoo beeLeast ConcernTable 2(Schrank 1781)948C4CAF-20AC-5B9F-8FFD-0E90A5FBEA2APolylecticLeast ConcernTable 2(Linnaeus 1758)491182D3-2281-5F9A-8B14-5CB8E9B4720APolylecticLeast ConcernTable 2(Latreille 1805)560596E3-1F94-50A9-B1E4-A9054049D5D8PolylecticLeast ConcernTable 2(Kirby 1802)CABA8176-0B1E-55F3-9B7B-72D3C19042B4AsteraceaeOligolectic on Least ConcernTable 2(Kirby 1802)23711571-D04D-5663-AB09-638C6B483ADBPolylecticLeast ConcernTable 2Dufour & Perris 1840D33C3091-21CA-5E49-B160-C814AC38702DPolylecticLeast ConcernTable 2(Linnaeus 1758)64AB9D91-37AB-5194-83D8-5A4311AA2ADFPolylecticLeast ConcernTable 2(Linnaeus 1767)7F29730F-2965-586C-BFC1-D8CADDB49A3ECuckoo beeLeast ConcernTable 2Hagens 18825CD3E34E-6546-5806-BBD7-4EDD0A124711Cuckoo beeLeast ConcernTable 2(Linnaeus 1758)8FC2E3A1-A810-5BC0-BACA-D5C7D2D0E5BDCuckoo beeLeast ConcernTable 2(Kirby 1802)76A571DE-6A83-59DA-8058-E74BC4176C49Cuckoo beeLeast ConcernTable 2Thomson 1870E9CF53B2-A974-51ED-99DD-168D066F0762Cuckoo beeLeast ConcernTable 25C8E9C69-5C10-5A06-9F9F-F0600257FC2BCuckoo beeTable 2Meigen 182290050123-9179-5E3F-8E07-2EDAA3F9F3AFPolylecticNot ApplicableTable 2(De Geer 1776)53CF093A-375A-5B54-AD1B-B8F688A04037PolylecticNot ApplicableTable 2(Linnaeus 1758)F5FEA349-0334-5849-8ADC-53CC3A9818C9PolylecticNot ApplicableTable 2(Linnaeus 1758)BA3E4C59-DBF8-5529-8093-97F86233CFD4PolylecticNot ApplicableTable 2(Scopoli 1763)BB83C0FB-CBEE-5FD3-9F69-410762CC14A9PolylecticNot ApplicableTable 2(Linnaeus 1758)93FF6A50-A0D4-5117-B746-91EE0DD66B8CPolylecticNot ApplicableTable 2F89CB51F-CF0A-5D74-9E32-19E1A7A2B421PolylecticNot ApplicableTable 2(Linnaeus 1758)38B88F15-9565-5863-A262-EAA792C67AC3PolylecticNot ApplicableTable 2(Meigen 1822)C838CF26-339D-59ED-8A20-93B7FB48C12CPolylecticNot ApplicableTable 2(Scopoli 1763)7D8EA026-B075-5EB9-90B5-FE9A80268201PolylecticNot ApplicableTable 2(Fabricius 1805)EDEACC39-7F2D-53BB-AFA9-4C3ABC0D3CE0PolylecticNot ApplicableTable 2(Linnaeus 1758)481349F2-DD00-5900-ABDE-5D750AA7A354PolylecticNot ApplicableTable 2(Fabricius 1794)BF63ABD8-86F0-5ABB-B60D-62979A7E15CBPolylecticNot ApplicableTable 2(Macquart 1829)61023682-D938-50BD-8BB7-E2E6FC8A5E51PolylecticNot ApplicableTable 2(Linnaeus 1758)B251790A-E0FA-5E46-935D-612EB37AB9F5PolylecticNot ApplicableTable 2(Fabricius 1781)290814F9-DC8A-5E26-BA25-ADDCB4733474PolylecticNot ApplicableTable 2(Meigen 1822)7F670446-E654-5BA3-9D0D-1EBA9F50B8EBPolylecticNot ApplicableTable 2(Zetterstedt 1849)7B152F87-B0F6-562C-A25E-35A4A4C4E6FCCyperaceaeOligolectic on Not ApplicableTable 2(Meigen 1822)B7F1B06F-01ED-52C0-970D-4F85A42CF4B1PolylecticNot ApplicableTable 2(Staeger 1843)4F178E3D-398F-5E6F-8C92-D1282CA7E6E5PolylecticNot ApplicableTable 2Meigen 1822B0EFBFC0-2AC5-515D-B792-F22104462E47PolylecticNot ApplicableTable 2(Linnaeus 1758)DF7175F0-5E96-5F27-BEF1-7E2AD03539C2PolylecticNot ApplicableTable 2(Linnaeus 1758)2B6BC5BE-26CA-50ED-A3CB-0A6837186BBFPolylecticNot ApplicableTable 2(Linnaeus 1758)CD7067BB-457C-5FB1-BDFA-12FC949916D4PolylecticNot ApplicableTable 2(Linnaeus 1758)50F55B3F-2078-5B9D-877E-0576C6AA7543PolylecticNot ApplicableTable 2Meigen 1822EEBD48C0-87E0-5828-99B5-DB19EE78F53DPolylecticNot ApplicableTable 2(Linnaeus 1758)A3F2FF42-4B36-58EA-84A0-9CA20C7B2EB1PolylecticNot ApplicableTable 2(Linnaeus 1758)3296E90F-34DA-550D-8AC4-22300A23FE44PolylecticNot ApplicableTable 2(Harris 1776)226D97AC-74A4-570F-B298-AFCF80C8226FPolylecticNot ApplicableTable 2(Linnaeus 1758)F9132A36-B891-5F8F-B53F-492985E4EC15PolylecticNot ApplicableTable 2(Linnaeus 1758)9D5FAD45-007C-5431-B5CD-E6176EFA5110PolylecticNot ApplicableTable 2Platycheirusimmarginatus (Table Over 2 years 2018-2019) of sampling, we collected 4,303 bees and 1,998 syrphids, representing 92 species and morphospecies from 15 genera and six families for the bees and 31 species and morphospecies from 18 genera for the hoverflies , Apismellifera (12.94%), A.haemorrhoa (10.71%), A.flavipes (7.25%) and Lasioglossumpauxillum (6.83%).In our study, we identified 101 different bee species, corresponding to almost one quarter of the Belgian bee fauna . DepictiTaraxacum spp. (Asteraceae), Salix spp. , Craetegus spp. (Rosaceae) and fruit trees could explain the dominance of A.cineraria, A.haemorrhoa and A.flavipes populations. Moreover, they usually nest in south-exposed sites, in bare soils or in areas with sparse and short vegetation , Centaurea spp. (Asteraceae) or Daucuscarota (Apiaceae) , orchards, umbellifers or Cirsium spp. (Asteraceae) were flowering. Two specimens of Colletescunicularius were sampled from both farms. This species is specialised on Salix spp.  or Prunuscerasus L., (Rosaceae) , Taraxacum spp. (Asteraceae) and D.carota - this species is considered as "near threatened" according to the IUCN Red List Criteria in Belgium  , which wosaceae) . While L Belgium .Andrenaschencki . This species nests in calcareous soils and forages principally on Asteraceae flowers, such as Taraxacum spp.   .Melittidae family, Melittaleporina    , which weraceae) .Chelostomarapunculi, Euceralongicornis, Hylaeussignatus, Megachileericertorum and Osmialeaiana, probably because their preferred flowers were partially present:Trifolium sp., Medicago sp., Cirsium sp., Rubus sp., Centaurea sp. and Stachyssylvatica L. 1753.In Froidefontaine habitats, we also sampled a few specimens of subgenusPsithyrus Lepeletier), Bombuscampestris and B.vestalis, in Froidefontaine wetland and in Froidefontaine orchard , we identified 15 species representing 4.6% of the collected material. They especially parasitise Andrena spp. and their relative abundance is dependent on the proportion of their host bee species  and A.apicata, respectively. On the contrary, N.goodeniana and N.ruficornis were largely present due to the strong dominance of A.cineraria and A.haemorrhoa    . The spesticides . On the .) Table  (Speight.) Table . We did Xylotasylvarum and X.segnis, whose larvae are saproxylic and live close to roots and dead wood   . The culctively)  and the ctively) D showed ctively) D. The flra Table . Moreovera Table C, couplera Table . Indeed,ra Table .A.schencki, one specimen of B.campestris, one specimen of E.longicornis and nine specimens of H.maculatus. We also mitigated the data deficiency in Belgium for a few records of bee species, such as A.semilaevis, A.trimmerana and Hylaeusbrevicornis  reflect current taxonomic impediments for 9.4% of the Belgian bee richness (According to the Belgian Red List of bees , we havenis Fig. E. Taxonorichness .Pollinator composition of each farmstead harboured both common and rare species, which indicates that on-farm diversification and organic practices may be an important refuge for rare, Red-Listed or oligolectic pollinator species . RestoriFA2F290F-667A-5394-B421-364EC9D03A7810.3897/BDJ.9.e60665.suppl1Supplementary material 1Havelange bee historical dataData typeoccurencesFile: oo_472961.xlsmhttps://binary.pensoft.net/file/472961Grégoire Noël


O-alkylated eugenol derivatives, bearing a propyl chain with terminals like hydrogen, hydroxyl, ester, chlorine, and carboxylic acid, were synthesized in the present work. These compounds were later subjected to epoxidation conditions to give the corresponding oxiranes. All derivatives were evaluated against their effect upon the viability of insect cell line Sf9 (Spodoptera frugiperda), demonstrating that structural changes elicit marked effects in terms of potency. In addition, the most promising molecules were evaluated for their impact in cell morphology, caspase-like activity, and potential toxicity towards human cells. Some molecules stood out in terms of toxicity towards insect cells, with morphological assessment of treated cells showing chromatin condensation and fragmentation, which are compatible with the occurrence of programmed cell death, later confirmed by evaluation of caspase-like activity. These findings point out the potential use of eugenol derivatives as semisynthetic insecticides from plant natural products.Eugenol, the generic name of 4-allyl-2-methoxyphenol, is the major component of clove essential oil, and has demonstrated relevant biological potential with well-known antimicrobial and antioxidant actions. New  Due to the exponential increase in population, it is necessary to ensure that agricultural production follows the resulting food needs. The need for the prevention and control of plant diseases, as well as insect pests, is a crucial issue facing crop protection. To date, the most common strategy for controlling these issues has depended on the use of conventional pesticides, most of which are synthetic pesticides, including insecticides ,2,3.The intensive use of synthetic pesticides has resulted in damage to the environment, health hazards, and loss of biodiversity ,5, so itPlants offer an extraordinary diversity of secondary metabolites, with proven efficacy against mosquito species of medical and veterinary importance, as well as against other noxious arthropod pests and vectors ,14. In rIn fact, EOs extracted by steam distillation of many aromatic plants have recently received a lot of attention due to their broad spectrum of action. These natural ingredients, composed of complex mixtures of monoterpenes, biogenetically related phenols, and sesquiterpenes ,18,19, dSyzygium aromaticum (clove) oil, has been used as a starting material and building block molecule for the manufacturing of bioactive compounds, on account of its particular structure and ready availability, in addition to numerous applications found in pharmaceutical, food, agricultural, and cosmetics industries + = 207.1380; found = 207.1381.Starting from compound 1  and using 1-bromo-3-chloro-propane , compound 2b was obtained as a light yellow oil . Rf = 0.67 (ethyl acetate/light petroleum = 1:10). 1H NMR : δH = 2.28 , 3.35 , 3.78 , 3.86 , 4.15 , 5.06–5.13 , 5.93–6.03 , 6.72–6.74 , 6.86  ppm. 13C NMR : δC = 32.31 (OCH2CH2CH2Cl), 39.72 (CH2Ph), 41.64 (OCH2CH2CH2Cl), 55.85 (OCH3), 65.88 (OCH2CH2CH2Cl), 112.45 (C-6), 113.91 (C-3), 115.60 (CH=CH2), 120.48 (C-5), 133.37 (C-4), 137.55 (CH=CH2), 146.48 (C-1), 149.52 (C-2) ppm. HRMS: m/z (ESI) calculated for C13H1735ClNaO2 + = 265.0783; found = 265.0780.Starting from compound 1  and using 3-bromopropan-1-ol , compound 2c was obtained as a light yellow oil . Rf = 0.23 (ethyl acetate/light petroleum 1:3). 1H NMR : δH = 2.07 , 3.34 , 3.85 , 3.88 , 4.18 , 5.05–5.12 , 5.91–6.01 , 6.71–6.73 , 6.84  ppm. 13C NMR : δC = 31.75 (OCH2CH2CH2OH), 39.82 (CH2Ph), 55.75 (OCH3), 61.56 (OCH2CH2CH2OH), 68.69 (OCH2CH2CH2OH), 112.06 (C-3), 113.48 (C-6), 115.64 (CH = CH2), 120.44 (C-5), 133.41 (C-4), 137.57 (CH=CH2), 146.46 (C-1), 149.37 (C-2) ppm. HRMS: m/z (ESI) calculated for C13H19O3 [M+1]+ = 223.1329; found = 223.1329.Starting from compound 1  and using ethyl 4-bromobutyrate , compound 2e was obtained as a colorless oil . Rf = 0.44 (ethyl acetate/light petroleum 1:10). 1H NMR : δH = 1.24 , 2.14 , 2.53 , 3.33 , 3.85 , 4.04 , 4.14 , 5.04–5.11 , 5.91–6.01  6.69–6.72 , 6.82  ppm. 13C NMR : δC = 14.14 (OCH2CH2CH2CO2CH2CH3), 24.57 (OCH2CH2CH2CO2CH2CH3), 30.73 (OCH2CH2CH2CO2CH2CH3), 39.74 (CH2Ph), 55.85 (OCH3), 60.31 (OCH2CH2CH2CO2CH2CH3), 68.09 (COCH2CH2CH2CO2CH2CH3), 112.41 (C-3) 113.60 (C-6), 115.54 (CH = CH2), 120.44 (C-5), 133.06 (C-4), 137.60 (CH = CH2), 146.58 (C-1), 149.46 (C-2), 173.20 (CO2CH2CH3) ppm. HRMS: m/z (ESI) calculated for C16H23O4 [M+1]+ = 279.1591; found = 279.1592.Starting from compound 2c  and using acetic anhydride , compound 2d was obtained as a light yellow oil . Rf = 0.29 (ethyl acetate/light petroleum 1:10). 1H NMR : δH = 2.02 , 2.11 , 3.30 , 3.81 , 4.05 , 4.25 , 5.01–5.08 , 5.88–5.98 , 6.67–6.69 , 6.80  ppm. 13C NMR : δC = 20.66 (CO2CH3), 28.43 (OCH2CH2CH2CO2CH3), 39.57 (CH2Ph), 55.64 (OCH3), 61.18 (OCH2CH2CH2CO2CH3), 65.60 (OCH2CH2CH2CO2CH3), 112.30 (C-3), 113.53 (C-6), 115.38 (CH=CH2), 120.26 (C-5), 133.03 (C-4), 137.40 (CH=CH2), 146.38 (C-1), 149.35 (C-2), 170.76 (CO2CH3) ppm. HRMS: m/z (ESI) calculated for C15H20NaO4 [M+Na]+ = 287.1254; found = 287.1253.Starting from compound 2e  in 1,4-dioxane (3.0 mL), 1 M aqueous sodium hydroxide  was added, and compound 2f was obtained as a white solid . Rf = 0.78 (ethyl acetate/light petroleum 1:10). 1H NMR : δH = 2.15 , 2.62 , 3.34 , 3.85 , 4.06 , 5.05 – 5.12 , 5.92–6.02 , 6.70–6.83 , 6.83  ppm.13C NMR : δC = 24.31 (OCH2CH2CH2CO2H), 30.55 (OCH2CH2CH2CO2H), 39.77 (CH2Ph), 55.85 (OCH3), 67.99 (OCH2CH2CH2CO2H), 112.45 (C-3), 113.73 (C-6), 115.60 (CH=CH2), 120.48 (C-5), 133.26 (C-4), 137.60 (CH=CH2), 146.49 (C-1), 149.50 (C-2), 179.15 (CO2H) ppm. HRMS: m/z (ESI) calculated for C14H19O4 [M+1]+ = 251.1278; found = 251.1274.To a suspension of compound 2a  and using m-chloroperbenzoic acid , compound 3a was obtained as a yellow oil . Rf = 0.30 (ethyl acetate/light petroleum 1:10). 1H NMR : δH = 1.04 , 1.82–1.91 , 2.55 , 2.76–2.87 , 3.12–3.17 , 3.86 , 3.97 , 6.76–6.84 , 6.83  ppm. 13C NMR : δC = 10.39 (OCH2CH2CH3), 22.47 (OCH2CH2CH3), 38.27 (CH2Ph), 46.78 (CH2 oxirane), 52.57 (CH oxirane) 55.99 (OCH3), 70.59 (OCH2CH2CH3), 112.79 (C-2), 113.13 (C-5), 120.94 (C-6), 129.72 (C-1), 147.36 (C-4), 149.36 (C-3) ppm. HRMS: m/z (ESI) calculated for C13H18NaO3 [M+Na]+ = 0245.1148; found = 245.1148.Starting compound 2b  and using m-chloroperbenzoic acid , compound 3b was obtained as a yellow oil . Rf = 0.52 (ethyl acetate/light petroleum 1:3). 1H NMR : δH = 2.28 , 2.55 , 2.79–2.83 , 3.12–3.16 , 3.77 , 3.86 , 4.15 , 6.77–6.79 , 6.86  ppm. 13C NMR : δC = 32.27 (OCH2CH2CH2Cl), 38.26 (CH2Ph), 41.63 (OCH2CH2CH2Cl), 46.76 (CH2 oxirane), 52.53 (CH oxirane), 55.93 (OCH3), 65.82 (OCH2CH2CH2Cl), 112.88 (C-6), 113.85 (C-5), 121.01 (C-2), 130.47 (C-1), 146.96 (C-4), 149.54 (C-3) ppm. HRMS: m/z (ESI) calculated for C13H1735ClO3 [M+Na]+ = 279.0758; found = 279.0754; calculated for C13H1737ClO3 [M+Na]+ = 281.0733; found = 281.0722.Starting from compound 2c  and using m-chloroperbenzoic acid , compound 3c was obtained as a yellow oil . Rf = 0.58 (ethyl acetate). 1H NMR : δH = 2.08 , 2.55 , 2.80–2.85 , 3.13–3.17 , 3.86 , 3.89 , 4.19 , 6.71–6.73 , 6.84  ppm. 13C NMR : δC = 31.75 (OCH2CH2CH2OH), 38.34 (CH2Ph), 46.78 (CH2 oxirane), 52.56 (CH oxirane), 55.83 (OCH3), 61.57 (OCH2CH2CH2OH), 68.64 (OCH2CH2CH2OH), 112.50 (C-3), 113.45 (C-6), 120.96 (C-5), 130.52 (C-4), 146.95 (C-1), 149.41 (C-2) ppm. HRMS: m/z (ESI) calculated for C13H18NaO4 [M+Na]+ = 261.1097; found = 261.1098.Starting compound 2d  and using m-chloroperbenzoic acid , compound 3c was obtained as light yellow oil . Rf = 0.32 (ethyl acetate/light petroleum 1:3). 1H NMR : δH = 2.05 , 2.15 , 2.55 , 2.79–2.82 , 3.12–3.16 , 3.86 , 4.09 , 4.28 , 6.77–6.79 , 6.84  ppm. 13C NMR : δC = 20.90 (CO2CH3), 23.79 (OCH2CH2CH2CO2CH3), 38.26 (CH2Ph), 46.77 (CH2 oxirane), 52.55 (CH oxirane), 55.93 (OCH3), 61.37 (OCH2CH2CH2CO2CH3), 65.73 (OCH2CH2CH2CO2CH3), 112.88 (C-3), 113.59 (C-6), 120.96 (C-5), 130.33 (C-4), 147.02 (C-1), 149.51 (C-2), 170.07 (CO2CH3) ppm. HRMS: m/z (ESI) calculated for C15H20NaO5 [M+Na]+ = 303.1203; found = 303.1202.Starting from compound 2e  and m-chloroperbenzoic acid , compound 3e was obtained as a yellow oil . Rf = 0.31 (ethyl acetate/light petroleum 1:3). 1H NMR : δH = 1.26 , 2.14 , 2.51–2.56 , 2.76–2.86 , 3.12–3.16 , 3.86 , 4.05 , 4.14 , 6.75–6.79 , 6.84  ppm. 13C NMR : δC = 14.19 (CO2CH2CH3), 24.57 (OCH2CH2CH2CO2CH2CH3), 30.74 (OCH2CH2CH2CO2CH2CH3), 38.29 (CH2Ph), 46.78 (CH2 oxirane), 52.55 (CH oxirane), 55.95 (OCH3), 60.36 (CO2CH2CH3), 68.07 (OCH2CH2CH2CO2CH2CH3), 112.87 (C-3), 113.58 (C-6), 120.98 (C-5), 130.18 (C-4), 147.09 (C-1), 149.50 (C-2), 173.20 (CO2CH2CH3) ppm. HRMS: m/z (ESI) alculated for C15H22NaO5 [M+Na]+ = 317.1359; found = 317.1359.Starting from compound Sf9 cells, which derive from the ovary cells of Spodoptera frugiperda (a common pest) were used. All molecules were assayed at the same concentration (100 µg/mL) in order to allow direct comparison of their potency. The starting material, eugenol 1, was nearly devoid of any toxicity, causing a marginal decrease of viability (2a–f) displayed higher toxicity than the starting eugenol 1, with cells showing around 55–65% of viability  cultures of iability . All eugiability . When anrpyrifos . For thi3b and 3e had in the morphology of the insect cells under study. For this reason, treated cells were imaged to evaluate chromatin status and overall cell morphology. We used both molecules at the same concentration that had been assessed for viability studies (100 µg/mL); however, the effect of 3e was so pronounced that no cells remained. For this reason, in the specific case of this molecule, the concentration of 50 µg/mL was used.We were interested in assessing the impact that Incubation with either molecules resulted in reduced cell density when compared with control cells, which is in line with the results reported for their impact in cell viability.3b- and 3e-treated cells exhibited chromatin changes, karyorrhexis , and pyknosis . We have quantified this effect  channel , it was s effect , results3b resulted in an about three-fold increase in caspase-like activity, reaching over six-fold in the case of 3e. For benchmarking purposes, the commercial insecticide chlorpyrifos was also evaluated, and its effect was not statistically different from control cells.Considering the lack of commercial options for the assessment of insect counterparts of mammalian caspases, it was hypothesized that the degree of homology described between insect and mammalian caspases  should b3b and 3e in human cells has been evaluated. Considering the usual routes of poisoning, specifically skin, human keratinocytes, were chosen. As it can be seen in 3b caused around 40% loss of cell viability , which is still less pronounced than the effect elicited in insect cells. In the case of 3e, Bearing in mind the importance of developing new and more potent insecticides that have a safe toxicological profile towards mammalian organisms, the impact of 1, obtained by hydrodistillation of clove, were carried out. Alkylation of the hydroxyl group of 4-allyl-2-methoxyphenol 1 with 1-bromopropane,1-bromo-3-chloropropane, 3-bromopropan-1-ol, and ethyl 4-bromobutanoate, using cesium carbonate as a base and by heating at 65 °C in acetonitrile, gave 4-allyl-2-methoxy-1-propoxybenzene 2a, 4-allyl-1-(3-chloropropoxy)-2-methoxybenzene 2b, 3-propan-1-ol 2c, and ethyl 4-butanoate 2e, respectively.Structural modifications in the hydroxyl group and double bond of 4-allyl-2-methoxyphenol, eugenol 2c was further reacted with acetic anhydride by heating at 65 °C to obtain 3-propyl acetate 2d. In addition, compound 2e was subjected to hydrolysis with aqueous 1M NaOH in 1,4-dioxane at room temperature to give 4-butanoic acid 2f. Compounds 2a–e were obtained as oils or a solid material (2f) in 53% to 88% yields, and were fully characterized by 1H and 13C NMR spectroscopy, as well as HRMS. The 1H NMR spectra of compounds 2a–f showed the different characteristic signals for the aliphatic protons of methylene and methyl groups (δ = 1.03–4.30 ppm), as well as the expected protons for the eugenol’s double bond as multiplets, CH2 (δ = 5.01–5.14 ppm) and CH (δ = 5.90–6.04 ppm). 13C NMR spectra of all compounds showed signals of the aliphatic carbons from the methylene and methyl groups (δ = 10.23–70.44 ppm), and for compounds 2d, 2e, and 2f was visible the presence of signals for the carbonyl groups (δ = 170.76–179.15 ppm).Compound 2a–e reacted with m-chloroperbenzoic acid in dichloromethane at room temperature, and the respective derivatives, namely 2-(3-methoxy-4-propoxybenzyl)oxirane 3a, 2-(4-(3-chloropropoxy)-3-methoxybenzyl)oxirane 3b, 3-(2-methoxy-4-(oxiran-2-ylmethyl)phenoxy)propan-1-ol 3c, 3-(2-methoxy-4-(oxiran-2-ylmethyl)phenoxy)propyl acetate 3d, and ethyl 4-(2-methoxy-4-(oxiran-2-ylmethyl)phenoxy)butanoate 3e were obtained. These compounds 3a–e were isolated as yellow oils in yields up to 67%, and were fully characterized by the usual analytical techniques. It stands out that epoxidation of compounds 2a–e was verified by the presence of the protons signals related the oxirane ring (δ = 2.52–3.17 ppm) and the absence of the signals of protons for the double bond of eugenol skeleton. The presence of carbon signals relative to oxirane ring, CH2  also confirmed the structure of expected eugenol derivatives 3a–e.To perform epoxidation of the double bond of eugenol derivatives, compounds 2a–f) displayed higher toxicity than the starting molecule 1 . The 1-Bromopropane, 3-bromopropan-1-ol and ethyl 4-bromobutanoate were from Sigma-Aldrich . The anhydrous magnesium sulphate and acetic anhydride were PanReac Applichem  products. Chloroform-d was produced by Eurisotop . Thin-layer chromatography (TLC) analyses were carried out on 0.25 mm-thick, precoated silica plates , and spots were visualized under UV light. Chromatography on silica gel was carried out on Merck Kieselgel (230–240 mesh).Dichloromethane, acetonitrile, ethyl acetate, light petroleum, 1,4-dioxane, 1-bromo-3-chloropropane, cesium carbonate, and 1H NMR and 100.6 MHz for 13C NMR, using the solvent peak as internal reference at 25 °C. All chemical shifts are given in ppm using δ Me4Si = 0 ppm as reference, and J values are given in hertz. Assignments were made by a comparison of chemical shifts, peak multiplicities, and J values, and were supported by spin decoupling–double resonance and bidimensional heteronuclear correlation techniques. High-resolution mass spectrometry analyses were performed at the CACTUS, Unidade de Masas e Proteómica, at the University of Santiago de Compostela, Spain.The NMR spectra were obtained on a Bruker Avance III at an operating frequency of 400.0 MHz for 1, was made from Syzygium aromaticum (cloves) in a round-bottom flask containing distilled water (200 mL) and the cloves (21.415 g). Hydrodistillation assembly was performed, and the mixture was refluxed during 2 h. The distillate was extracted with dichloromethane (3 × 150 mL), the organic phase was dried over anhydrous magnesium sulphate, and solvent evaporation under vacuum yielded 4-allyl-2-methoxyphenol, eugenol (1).The extraction of 4-allyl-2-methoxyphenol, eugenol 1 (1 equiv) in acetonitrile (5 mL), the corresponding alkyl halide (1.1 equiv) and cesium carbonate (5 equiv) were added, and the resulting mixture was heated at 60 °C for 2 h 30 min. The progress of the reaction was monitored by TLC (ethyl acetate/light petroleum = 1:10). The excess of base was filtered, the solvent was evaporated, and the crude mixture was purified by column chromatography on silica gel using ethyl acetate/light petroleum (1:10) as the eluent.To a solution of 4-allyl-2-methoxyphenol 2c acetic anhydride was added, and the resulting mixture was stirred at 65 °C for 12 h. The reaction was monitored by TLC (ethyl acetate/light petroleum = 1:10). After completion, the mixture was diluted with ethyl acetate and washed with sodium bicarbonate (2 × 5 mL); the organic phase was dried with anhydrous magnesium sulfate, and the solvent was evaporated.To compound 2e in 1,4-dioxane, 1 M aqueous sodium hydroxide was added. The solution was stirred at room temperature for 8 h, and acidified to pH 2–3 with 1 M aqueous potassium hydrogen sulfate. The reaction mixture was evaporated and dichloromethane was added, giving a precipitate that was filtered. Then the solvent was evaporated.To a suspension of compound 2a–e (4 mL) dissolved in dichloromethane was added dropwise to a solution of m-chloroperbenzoic acid (1 equiv) in dichloromethane (6 mL) at 0 °C. After stirring for 1 h, m-chloroperbenzoic acid was again added (1 equiv), and the reaction mixture was stirred for more 12 h. A 10% aqueous solution of sodium sulfate (10 mL) was added, and the resulting solution was washed with 5% aqueous solution of sodium hydrogen carbonate (2 × 10 mL). The organic phase was dried with anhydrous magnesium sulfate, and the solvent was evaporated.The respective precursor (1 equiv) Sf9 (Spodoptera frugiperda) cells were maintained as a suspension culture and cultivated in Grace’s medium with 10% FBS and 1% penicillin/streptomycin, at 28 °C. Cells were used in experiments while in the exponential phase of growth. On the other hand, HaCaT (human keratinocyte) cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C, in a humidified atmosphere of 5% CO2.Sf9 and HaCaT cells were plated at a density of 3.0 × 104 and 1.5 × 104 cells/well, respectively, incubated for 24 h, and then exposed to the molecules under study for 24 h. After this period, a commercial solution of resazurin was added (1:10), and the kinetic reaction of fluorescence increase monitored at 560/590 nm. For HaCaT and Sf9 cells, 30 and 60 min of incubation were used, respectively.For the assessment of viability, a resazurin-based method was used. Sf9 cells were cultured in 96-well plates at the same density used for viability experiments, in the presence of the molecules under study. After incubation, cells were washed with Hanks’ balanced salt solution (HBSS) and fixed in 10% formalin solution for 30 min, at room temperature. CF543 (5 U/mL) and DAPI (0.25 µg/mL) were added, and cells were stained for 25 min at room temperature and washed with HBSS.For morphological studies, Images were acquired in an inverted Eclipse Ts2R-FL (Nikon) equipped with a Retiga R1 camera and an S Plan Fluor ELWD 20x DIC N1 objective. Images were analyzed with Fiji . For quaSf9 cells were plated at the same density described for viability studies and exposed to the molecules under study for the designated time. Generally, the same method described by the authors previously for mammalian cells [an cells  was usedp < 0.05.For biological assays, the Shapiro–Wilks normality test was performed in the data to ensure that it followed a normal distribution. Comparison between the means of controls and each experimental condition was performed using ANOVA. Outliers were identified by the Grubbs’ test. Data was expressed as the mean ± standard deviation (SD) of at least three independent experiments. GraphPad Prism 7.0 software was used, and values were considered statistically significant when 1, it was possible to obtain several molecules with enhanced activity, some of them highly potent at concentrations as low as 100 μg/mL. The most potent molecules were shown to elicit morphological changes compatible with some processes of programmed cell death, being also capable of increasing the activity of serine proteases pivotal to some of these death pathways, notably caspases.Overall, with the present work it was demonstrated that medicinal chemistry approaches are valid strategies for obtaining new semisynthetic derivatives of a natural essential oil, which can act as promising alternatives to the available synthetic insecticides. Having started with a natural molecule devoid of activity, eugenol 3e exhibits a more favorable safety profile towards human cells than that of the commercial insecticide chlorpyrifos, thus paving the way for the design of new alternatives to insecticides currently in use.Finally, it was showed that ethyl 4-(2-methoxy-4-(oxiran-2-ylmethyl)phenoxy)butanoate


H)-one derivatives and their inhibitory activity for 11β-HSD1 and 11β-HSD2. The derivative containing the spiro system of thiazole and cyclohexane rings shows the highest degree of 11β-HSD1 inhibition (54.53% at 10 µM) and is the most selective inhibitor of this enzyme among the tested compounds. In turn, derivatives containing ethyl and n-propyl group at C-5 of thiazole ring inhibit the activity of 11β-HSD2 to a high degree (47.08 and 54.59% at 10 µM respectively) and are completely selective. Inhibition of the activity of these enzymes may have a significant impact on the process of formation and course of tumors. Therefore, these compounds can be considered as potential pharmaceuticals supporting anti-cancer therapy.Glucocorticoid metabolism at the tissue level is regulated by two isoenzymes 11β-hydroxysteroid dehydrogenase (11β-HSD), which mutually convert biologically active cortisol and inactive cortisone. Recent research is focused on the role of 11β-HSD1 and 11β-HSD2 as autocrine factors of tumor cell proliferation and differentiation. Herein, we report the synthesis of novel 2-(isopropylamino)thiazol-4(5 Cancer is a major public health problem and a leading cause of death worldwide. Increased investment in basic and clinical research to find the metabolic processes underlying carcinogenesis would help to advance treatment options for patients . In the The most important factor affecting its secretion and access to target cells is a microsomal enzyme named 11β-hydroxysteroid dehydrogenase (11β-HSD). This enzyme exists in two isoforms 11β-HSD1 and 11β-HSD2. The first one catalyzes the reaction that converts biochemically inactive cortisone into its active counterpart cortisol. On the other hand, 11β-HSD2 is used catalytically in a reverse reaction, in a way of inactivation of cortisol, transforming it into cortisone  11,12]..12]. ChrIn turn, 11β-HSD2 is involved in carcinogenesis, tumor progression and metastasis along with its role in inflammation and hypertension regulation ,19. TherO-hemisuccinate) is the known inhibitor of 11β-HSD1. It is the hemisuccinate ester derivative of glycyrrhetinic acid, a natural product found in liquorice root. Not only does carbenoxolone inhibit 11β-HSD1, but it is also—To a lesser degree, though an inhibitor of 11β-hydroxysteroid dehydrogenase type 2. Likewise, the glycyrrhetinic acid  itself is potent non-selective inhibitor of both 11β-HSD1 and 11β-HSD2, although it inhibits the type 2 enzyme to a higher degree at different concentrations. +) in positive ESI mode. The results of the measurements were processed using the MassLynx 4.1 software .High-resolution mass spectrometry (HRMS) measurements were performed using Synapt G2-Si mass spectrometer (Waters) equipped with an ESI source and quadrupole-Time-of-flight mass analyzer. To ensure accurate mass measurements, data were collected in centroid mode and mass was corrected during acquisition using leucine enkephalin solution as an external reference (Lock-SprayTM), which generated reference ion at All chemicals and solvents were purchased commercially and used without further purification.Solvents: ethyl alcohol, methyl alcohol, chloroform, diethyl ether, ethyl acetate, dimethylsulfoxide .Reagents for Synthesis: N-isopropylthiourea 97% , ethyl 2-bromopropionate 99%, ethyl 2-bromobutyrate 98%, ethyl 2-bromovalerate 99%, ethyl 2-bromo-3-methylbutyrate 95%, ethyl 2-bromoisobutyrate 98%, ethyl 2-bromophenyl acetate 97%, ethyl 2-bromo(4-bromophenyl) acetate 97%, methyl 1-bromocyclohexane carboxylate 97%, ethyl bromocyclobutane carboxylate 95% purchased from Alfa Aesar.Auxiliary Reagents: N-ethyldiisopropylamine 99% (Alfa Aesar), sodium, magnesium sulfate, hydrochloric acid, sodium hydroxide TLC and Column Chromatography: 5 × 10 cm TLC plates coated with silica gel with F-254 Merck, silica gel MN kieselgel 60M with 0.04–0.063 mm grain diameter .11β-HSD1 Assays: 18-beta-glycyrrhetinic acid , phosphate buffer powder, cortisone, NADPH tetrasodium salt , carbenoxolone , Pooled human liver microsomes, mixed gender, 1 mL, 20 mg/mL Lot No.1410013, XenoTech, Cortisol Elisa Ref DkO001 Lot No. 4715A , ELISA Kit for 11-Beta-Hydroxysteroid Dehydrogenase Type 1 Lot No.L160706125 , PBS Lot No. H161008 .11β-HSD2 Assays: 18-beta-glycyrrhetinic acid (Acros Organic), phosphate buffer powder, cortisone, NAD cofactor (Sigma-Aldrich), carbenoxolone , Human Kidney Microsomes, mixed gender, 0.5 mL, 10 mg/mL Lot No. 1,710,160 XenoTech, Cortisol Elisa Ref DkO001 Lot No. 4715A (DiaMetra), Enzyme-Linked Immunosorbent Assay (ELISA) Kit for 11-Beta-Hydroxysteroid Dehydrogenase Type 2 Lot No. L191113457 (Cloud-Clone Corp.), PBS Lot No. H161008 .N-isopropylthiourea (1) and 0.0055 mol of the corresponding 2-bromoester (2a–2e) were added. It was heated for 7–15 h depending on the type of ester (TLC control-ethyl acetate). After the heating stopped, the solvent was evaporated on a rotary evaporator and the residue was dissolved in 10 mL of water and neutralized with 2 M HCl to pH = 7–8. The product was extracted with chloroform (4 × 20 mL). The organic layer was dried with MgSO4, then filtered and the solvent was evaporated. The oily liquid was crystallized from diethyl ether. The resulting precipitate was filtered under reduced pressure. The NMR and MS spectra of all obtained compounds (3a–3i) are attached in 10 mL of methanol were measured into the flask and 0.23 g (0.01 mol) of sodium was added. After completion of the reaction, 0.59 g (0.005 mol) of H)-one (3a)—Yield: 34%. M.p. 115–117 °C. 1H-NMR : 10.77 , 4.07 , 3.61 2, 7.0), 1.62 , 1.40 2, 7.0 9.1). 13C-NMR : 188.54 (C-4), 179.79 (C-2), 48.71 (C-5), 48.61 (CH(CH3)2), 22.00 (CH3CH), 21.96 (CH3CH), 18.70 (C5-CH3). HR-MS m/z 173.0747 [M+ + 1] . Rf : 0.31.2-(Isopropylamino)-5-methylthiazol-4(5H)-one (3b)—Yield: 28%. M.p. 130–131 °C. 1H-NMR : 10.35 , 4.11 , 3.67 2, 6.4), 2.11–2.21 , 1.85–1.96 , 1.43, 2, 5.6 6.4), 1.04 . 13C-NMR : 187.98 (C-4), 180.55 (C-2), 56.86 (C-5), 49.04 (CH(CH3)2), 26.21 (C5-CH2CH3), 22.39 2CH), 11.47 (C5-CH2CH3). HR-MS m/z 187.0905 [M+ + 1] . Rf : 0.38.5-Ethyl-2-(isopropylamino)thiazol-4(5H)-one (3c)—Yield: 47%. M.p. 110–112 °C. 1H-NMR : 10.37 , 4.11 , 3.66 2, 6.4), 2.12–2.20 , 1.74–1.84 , 1.43 2, 5.6 6.4), 1.45–1.55 , 0.98 . 13C-NMR : 190.37 (C-4), 180.55 (C-2), 55.37 (C-5), 49.02 (CH(CH3)2), 35.33 (C-5-CH2CH2CH3), 22.34 2CH), 21.13 (C5-CH2CH2CH3), 13.58 (C5-CH2CH2CH3). HR-MS m/z 201.1060 [M+ + 1] . Rf : 0.45.2-(Isopropylamino)-5-propylthiazol-4(5H)-one (3d)—Yield: 25%. M.p. 153–155 °C. 1H-NMR : 10.54 , 4.20 , 3.71 2, 6.8), 2.52–2.63 , 1.43 2, 6.0 6.4), 1.07 , 0.91 . 13C-NMR : 187.70 (C-4), 180.83 (C-2), 62.95 (C-5), 49.04 (CH(CH3)2), 30.53 (C5-CH), 22.33 2CH), 16.30 2). HR-MS m/z 201.1062 [M+ + 1] . Rf : 0.43.5-Isopropyl-2-(isopropylamino)thiazol-4(5H)-one (3e)—Yield: 10%. M.p. 193–194 °C. 1H-NMR : 10.43 , 3.59 2, 6.8), 1.64 2H), 1.42 2, 6.8).m 13C-NMR : 193.09 (C-4), 178.76 (C-2), 59.84 (C-5), 49.04 (CH(CH3)2), 27.92 2) 22.46 2CH). HR-MS m/z 187.0907 [M+ + 1] . Rf : 0.44.2-(Isopropylamino)-5,5-dimethylthiazol-4(5N-isopropylthiourea (1) and 0.011 mol of the corresponding 2-bromoester  were added to 50 mL of chloroform. The mixture was stirred at room temperature for 168–240 h (TLC control-ethyl acetate). The resulting crude product was filtered off under reduced pressure and crystallized from ethyl acetate  . Rf : 0.60.2-(Isopropylamino)-5-phenylthiazol-4(5H)-one (3g)—Yield: 85%. M.p. 236–237 °C. 1H-NMR : 9.72 , 7.54–7.59 , 7.24–7.32 , 5.49 , 3.75 2, 1.84), 1.22 2, 6.6 9.0). 13C-NMR : 185.47 (C-4), 176.68 (C-2), 137.04 , 132.05 , 131.10 , 121.60 , 57.03 (C-5), 48.25 (CH(CH3)2), 22.30 2CH). 22.27 2CH). HR-MS m/z 313.0011 [M+ + 1] . Rf : 0.60.5-(4-Bromophenyl)-2-(isopropylamino)thiazol-4 N-isopropylthiourea (1) and 0.01 mol of the corresponding 2-bromoester  were added to 3.5 mL of ethyl alcohol. It was heated to reflux for 168–240 h (TLC control-ethyl acetate). After the end of heating, the solvent was evaporated and the residue was dissolved in 10 mL of water, the pH of the solution was about 7. The product was extracted with chloroform (4 × 20 mL). The organic layer was dried with anhydrous MgSO4. Then it was filtered off and the solvent was evaporated. The reaction mixture was purified by column chromatography  . Rf : 0.56.2-(Isopropylamino)-1-thia-3-azaspiro dec-2-en-4-one (3i)—Yield: 15%. M.p. 151–152 °C. 1H-NMR : 10.15 , 3.58 2, 6.4), 2.73–2.87 , 2.46–2.59 , 2.26–2.39 , 1.97–2.10 , 1.44 2, 6.4). 13C-NMR : 190.84 (C-4), 178.76 (C-2), 60.48 (C-5), 49.08 (CH(CH3)2), 34.17 , 22.40 2CH), 16.89 . HR-MS m/z 199.0910 [M+ + 1] . Rf : 0.48.2-(Isopropylamino)-1-thia-3-azaspiro oct-2-en-4-one , 10 μL of microsomes (1.13 μg/mL 11β-HSD1) solution in PBS , 60 μL of phosphate buffer (pH 7.4) and 10 μL of inhibitor solution  were placed in the well. Mixtures were incubated for 150 min at 37 °C. The reaction was stopped by the addition of 10 μL solution of 100 μM 18β-glycyrrhetinic acid in PBS. The amount of cortisol obtained was measured using a cortisol ELISA kit  [+ , 10 μL of microsomes (0.127 μg/mL 11β-HSD2) solution in PBS , 60 μL of phosphate buffer (pH 7.4) and 10 μL of inhibitor solution  were placed in the well. Mixtures were incubated for 150 min at 37 °C. The reaction was stopped by the addition of 10 μL solution containing 100 μM carbenoxolone in PBS. The amount of unreacted cortisol was measured using a cortisol ELISA kit [Transformation of cortisol to cortisone was conducted on 96-well microtiter plates in the presence of human kidney microsomes as 11β-HSD2 sources in a total volume of 100 μL. 20 μL of substrate mixture cortisol/NAD50 values were determined by carrying out analogous determinations as those described in point 3.6 (for 11β-HSD1 inhibitors) or in point 3.7 (for 11β-HSD2 inhibitors) using inhibitors at the following concentrations: 10, 5, 2.5, 1.25, 0.625 µM. Then, graphs of the dependence of the concentration of cortisol (in the case of 11β-HSD1 inhibitors) and cortisone (in the case of 11β-HSD2 inhibitors) obtained in reaction to the concentration of the inhibitor were prepared. The inhibitor concentration at which there was a 50% reduction of cortisol (or cortisone) concentration compared to the blank (reaction without inhibitor) was read from the graph.ICH)-one derivatives and examined their inhibitory activity against isoforms 1 and 2 of 11β-HSD. The derivative 3h, containing the spiro system of thiazole and cyclohexane rings in its structure, is characterized by the highest degree of 11β-HSD1 inhibition (54.53% at the concentration 10 µM) and is the most selective inhibitor of this enzyme among the tested compounds. In turn, derivatives 3b and 3c inhibit the activity of 11β-HSD2 to a high degree (47.08 and 54.59% respectively at the concentration 10 µM) and are completely selective. Therefore, they can potentially contribute to a reduction in PGE2 production, thereby preventing the proliferation of tumor cells, tumor growth, and metastasis in, for example, colorectal cancer. The obtained results may constitute a new therapeutic approach targeting the action of 11β-HSD1 and 11β-HSD2 in order to restore the correct response to the action of glucocorticoids, which may be important in the inhibition of the proliferation of neoplastic cells.In conclusion, we described the synthesis of new 2-(isopropylamino)thiazol-4(5


The structures of these compounds were determined based on various spectroscopic and chemical analyses. All of the isolated compounds were evaluated on bone formation parameters, such as ALP  and mineralization stimulatory activities of MC3T3-E1 cell lines. The results showed that the new compound, 1,3-butanediol 3-pyroglutamate (4), 2-deoxy-d-ribono-1,4-lactone (6), methyl pyroglutamate (7), ampelopsisionoside (10), icariside B1 (11), and benzyl α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside (12) stimulated both ALP and mineralization activities.In our continuing research for bioactive constituents from natural resources, a new methyl threonolactone glucopyranoside ( Osteoporosis is an age-related chronic disease characterized by a decrease of bone mineral density and an increased risk of bone fracture. As the population in the world ages, osteoporosis is becoming an important social problem. In the world, more than 200 million people are suffering from osteoporosis. Approximately 50% of women over 50 years old will have an osteoporosis-related fracture in their lifetime. An imbalance of bone remodeling causes osteoporosis through bone resorption by osteoclasts and bone formation by osteoblasts. Insufficient bone formation is an essential cause of osteoporosis. Mesenchymal stem cells differentiate to osteoblasts with activation of alkaline phosphatase (ALP) and bone mineralization, which are regulated by a variety of molecules such as Runt-related transcription factor 2 (Runx2), bone morphogenetic proteins (BMPs), and estrogen .Spilanthes is a genus comprising over 60 species that are widely distributed in tropical and subtropical regions of the world, such as Africa, America, Borneo, India, Sri Lanka, and Asia + . The IR spectrum implied the presence of carbonyl (1777 cm−1), hydroxy groups (3392 cm−1), and C-O linkage (1210 and 1078 cm−1). The 1H-NMR spectrum ; four oxygenated methylene protons at δH 3.60 , 3.91 , 4.10 , and 4.52 ; five oxygenated methine protons at δH 3.238 , 3.237 , 3.34 , 3.36 , and 4.43 ; and an anomeric proton at δH 4.37  that indicated the presence of a glycosyl moiety. A lactone moiety was also indicated from three degrees of unsaturation, the relatively higher wavenumber of carbonyl (1777 cm−1), the esterified low field-shifted chemical shifts, and non-equivalency of H-5α  and H-5β .Compound spectrum  displaye13C-NMR spectrum , two oxygenated methylene carbons (δC 63.2 and 69.6), five oxygenated methine carbons , a quaternary carbon (δC 75.7), an anomeric carbon (δC 104.0) and a carbonyl carbon at δC 179.4. The NMR spectroscopic data of 1 closely resembled that of 2-C-methyl-d-threono-1,4-lactone (5) + . The 1H and 13C-NMR spectra . The 13C-NMR also showed two secondary carbons at δC 60.6 and 62.9; three tertiary carbons at δC 79.0, 82.6, and 84.7; and a quaternary carbon at δC 109.2, indicating a fructofuranose moiety. Zhang et al. (2009) described that α and β orientations of D-fructofuranose were distinguishable by the J value of H-3 and the chemical shift of C-2. The characteristic values of α orientation are 3JH3,H4 = 3–4 Hz and C-2 (δC 107–109), while the β orientation is 3JH3,H4 = 7–9 Hz and C-2 (δC 103–106) + ion at an m/z of 224.0890  corresponding to a molecular formula of C9H15O4N. The IR spectrum showed a strong absorption band for the hydroxy (3331 cm−1) and carbonyl (1735 and 1684 cm−1). The 1H and 13C-NMR spectra of 3 showed signals assignable to two methylenes ) and δH 2.37  / δC 30.4 (C-3), and δH 2.16  and 2.48 /δC 26.0 (C-4)), a methine /δC 57.3 (C-5)), and two carbonyls (δC 181.2 (C-2) and 174.2 (C-6)) /δC 23.9 (Me-10)), a methylene  and 1.79 /δC 38.8 (C-2′)), an oxygenated methine +  in HR-ESI-MS indicated that 4 was an isomer of 3. The shielding of H-1′  and δH 3.68 /C-1′ (δC 60.5)) and deshielding of H-3′ /C-3′ (δC 65.5)) suggested that the ester linkage of 1,3-butanediol must be changed from primary alcohol (C-1′) to the secondary alcohol (C-3′) of 1,3-butanediol in 4 . Silica gel open column chromatography (CC) and reversed-phase (ODS) CC were performed on silica gel 60 , and Cosmosil 75C18-OPN , respectively. HPLC was performed on an ODS column , and the eluate was monitored with a JASCO RI-930 intelligent detector and a JASCO PU-1580 intelligent pump unless otherwise specified.Spilanthes acmella (L.) L. were collected in late June 2007 in Kebun Raya Purwodadi, Malang, Indonesia (07°46′09″–07°47′23″ South latitude and 112°16′23″–112°17′17″ East Longitude), and voucher specimens were deposited at the Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Airlangga University as SA30062007 , yielding 12 fractions (Fr. Sab1–Sab12). The fraction Sab11 (2.75 g) was subjected to ODS CC in 10% aq. MeOH (400 mL)–100% MeOH (400 mL), linear-gradient, led 10 fractions (Fr. Sab11-1–Sab11-10). The fraction Sab11-3 (442 mg) and Sab11-4 (123 mg) were purified by HPLC with 35% aq. MeOH to give 1 (10.1 mg) and 8 , respectively. The fraction Sab10 (1.81 g) was subjected to ODS CC in 10% aq. MeOH (400 mL)–100% MeOH (400 mL) and led seven fractions (Fr. Sab10-1–Sab10-7). The fraction Sab10-1 (770 mg) was purified by HPLC with 100% H2O, YMC Triart C18 column. Three peaks appeared at 5, 18, and 35 minutes and were collected to give 2 (7.62 mg), 5 , and 14 . The fraction Sab10-2 (193 mg) was purified by HPLC (20% aq. MeOH) to give 11  and 13 . The fraction Sab10-3 (142 mg) was purified by HPLC (35% aq. MeOH) to give 9 . The fraction Sab5 (710 mg) was subjected to ODS CC in 10% aq. MeOH (400 mL)–100% MeOH (400 mL) and led 10 fractions (Fr. Sab5-1–Sab5-10). The fraction Sab5-1 (483 mg) and Sab5-2 (68.3 mg) were purified by HPLC  to give 3 (7.80 mg), 4 (4.21 mg), and 7 , respectively. The mixture of fraction Sab6, Sab7, Sab8, and Sab9 (2.06 g) was subjected to ODS CC in 10% aq. MeOH (400 mL)–100% MeOH (400 mL) and led 10 fractions (Fr. Sab6-9-1–Sab6-9-10). The fraction Sab6-9-1 (340 mg) was purified by HPLC  to give 6 . The fraction, Sab6-9-4 (114 mg), was purified by HPLC (40% aq. MeOH) to give 10 . The fraction Sab12 (5.36 g) was subjected to ODS CC in 10% aq. MeOH (400 mL)–100% MeOH (400 mL) and led 10 fractions (Fr. Sab12-1–Sab12-10). The fraction Sab12-3 (129 mg) was purified by HPLC (35% aq. MeOH) to give 12 (benzyl α-l-arabinopyranosyl (1→6)-β-d-glucopyranoside, 4.51 mg).The 1-butanol layer (40.0 g) was subjected on silica gel (300 g) CC with increasing amounts of MeOH in CHClD26.7 -28.6 ; IR (film) ⱱmax cm−1: 3392, 2924, 1777, 1713, 1650, 1557, 1456, 1391, 1210, 1078, 899, 647; 1H-NMR and 13C-NMR, see m/z): 317.0845 [M + Na]+.Colorless amorphous powder; [α]D26.9 -10.4 ; IR (film) ⱱmax cm−1: 3386, 2938, 1774, 1732, 1651, 1540, 1456, 1339, 1206, 1073, 870, 669; 1H-NMR and 13C-NMR, see m/z): 317.0844 [M + Na]+.Colorless amorphous powder; [α]D27.7 −0.86 ; IR (film) ⱱmax cm−1: 3331, 2966, 2926, 1735, 1684, 1557, 1457, 1338, 1207, 1052, 670; 1H-NMR and 13C-NMR, see m/z): 224.0890 [M + Na]+.Colorless amorphous powder; [α]D27.1 +1.40 ; IR (film) ⱱmax cm−1: 3314, 2969, 2931, 1735, 1683, 1557, 1457, 1338, 1229, 1054, 669; 1H-NMR and 13C-NMR, see m/z): 224.0893 [M + Na]+ .Colorless amorphous powder; [α]1 or 2 (1 mg) in 1 N aq. HCl (0.1 mL) was heated at 80 °C for 2 h. The mixture was neutralized by the addition of amberlite IRA400 (OH- form), and the resin was removed by filtration. Then, the filtrates were extracted with EtOAc. The aqueous layers were subjected to HPLC analysis ; flow rate: 1.0 mL/min) to identify D-glucose and D-fructose, which were identified by the comparison of their retention times with those of authentic samples; tR: 5.11  and tR: 6.10 . They also yielded the aglycon of 2-C-methyl-d-threono-1,4-lactone (1a and 2a) D27.5 −15.0 ; IRⱱmax (film) cm−1: 3286, 2923, 1729, 1649, 1558, 1456, 1339, 1205, 1092, 667; 1H-NMR : 1.35 , 3.96 , 4.17 , 4.49 ; positive HR-ESI-MS m/z 155.0312 [M + Na]+ and tR = 10.20 . (2a): Colorless amorphous powder; [α]D27.9 −15.7 ; IRⱱmax (film) cm−1: 3373, 2927, 1717, 1649, 1558, 1455, 1338, 1207, 1098, 668; 1H-NMR : 1.35 , 3.96 , 4.17 , 4.49 ; positive HR-ESI-MS m/z 155.0319 [M + Na]+  and tR = 10.20  together with authentic sample 5.A solution of  and 2a) . (1a): C2 incubator at 37 °C and sub-cultured every 3 days by trypsin (0.25%) treatment. The 5 × 104 cells were seeded in 24-well plates and incubated for the following ALP and mineralization assays.An osteoblast-like cell of MC3T3-E1 was purchased from Riken Cell Bank, Tsukuba, Japan. The cells were cultured in α-MEM medium containing 10% FBS in a CO1–14). On harvesting, the medium was removed, and the cell monolayer was gently washed twice with phosphate-buffered saline. The cells were lysed with 0.2% Triton X-100, and centrifuged at 14,000 × g for 5 min. The clear supernatant was used to measure ALP activity using p-nitrophenylphosphate [The cells were treated at 90% confluence with the culture medium containing 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid to initiate in vitro proliferation. The medium was changed every 2–3 d. After 6 days, the cells were cultured individually for 3 days with medium containing 0.3% bovine serum and isolated compounds . On harvesting, the cells were fixed with 70% ethanol for 1 hour and then stained with 40 mM Alizarin Red S for 10 min with gentle shaking. To quantify the bound dye, the stain was solubilized with 10% cetylpyridinium chloride by shaking for 15 min. The absorbance of the solubilized stain was measured at 561 nm [The cells were treated, at 90% confluence, with culture medium containing 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid, to initiate in vitro mineralization. After 12 days, the cells were cultured individually for 2 days with medium containing 0.3% bovine serum and isolated compounds (t 561 nm .Spilanthes acmella (L.) L. gave 14 compounds (1–14), including 2 new methyl threonolactone glycosides, 2-C-methyl-d-threono-1,4-lactone-3-O-β-d-glucopyranoside (1) and 2-C-methyl-d-threono-1,4-lactone-2-O-α-d-fructofuranoside (2); 2 new pyroglutamates, 1,3-butanediol 1-pyroglutamate (3) and 1,3-butanediol 3-pyroglutamate (4); and 10 known compounds, 2-C-methyl-d-threono-1,4-lactone (5), 2-deoxy-d-ribono-1,4-lactone (6), methyl pyroglutamate (7), dendranthemoside A (8), dendranthemoside B (9), ampelopsisionoside (10), icariside B1 (11), benzyl α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside (12), chicoriin (13), and uridine (14). All the isolated compounds were evaluated for ALP and calcium deposition as markers for osteogenic differentiation and mineralization stimulatory activities. Our study demonstrated that compounds 4, 6, 7, 10, 11, and 12 stimulated ALP activity and calcium deposition in osteoblastic MC3T3-E1 cell in vitro. Compounds 6, 10, and 12 stimulated ALP and mineralization up to 112% at 25 µM, comparable to that of the positive control, 17β-estradiol, at 0.02 and 0.01 μM. Of these active compounds, compounds 4, 6, 7, 10, and 11 are the first time to be reported their osteoblastic activity. While compound 12 has already isolated as an osteoblastic active compound from the fruit of Prunus mume [12) from the different plants through independent study strengthened and supported the reliability of our results. Overall, the compounds 4, 6, 7, 10, 11, and 12 are the active principle of S. acmella on the stimulation of osteoblastic bone formation and may play an important role in bone remodeling as drug seeds in osteoporosis therapy.The chemical investigation of the 1-butanol layer of the methanol extract of nus mume . The iso