O,N-heterocycles containing tetrahydro-1,4-benzoxazepine and tetrahydroquinoline moieties were prepared by a regio- and diastereoselective domino Knoevenagel–-hydride shift cyclization reaction of a 4-aryl-2-phenyl-1,4-benzoxazepine derivative obtained from flavanone. The relative configuration of products were determined by the correlation of 3JH,H coupling data with the geometry of major conformers accessed by DFT conformational analysis. Separated enantiomers of the products were characterized by HPLC-ECD data, which allowed their configurational assignment on the basis of TDDFT-ECD calculation of the solution conformers. Two compounds showed neuroprotective activities against hydrogen peroxide (H2O2) or β-amyloid25–35 (Aβ25–35)-induced cellular injuries in human neuroblastoma SH-SY5Y cells in the range of those of positive controls.Condensed  In the following step, the N-arylation of rac-10 was performed through a nucleophilic aromatic substitution by using 2-fluoro-5-nitrobenzaldehyde (11b) to give rac-5 containing a tertiary arylamine nitrogen in 71% yield. With the 2-chloro-5-nitrobenzaldehyde reagent, only 3% yield for rac-5 could be achieved. Subsequently rac-5 was reacted with 1,3-dimethylbarbituric acid (12), Meldrum’s acid (13) and malononitrile (14) all containing active methylene groups, which initiated a domino reaction  and Meldrum’s acid (13), the reaction with malonitrile (14) stopped at the stage of the Knoevenagel product rac-7c with MgSO4 in CHCl3 and further cyclization did not occur when heated in DMSO at 150 °C or refluxed in n-butanol.The Schmidt reaction of  Patonay  affordinreaction . The Knorac-6a–c , which u7a,b clearly indicated that they were single diastereomers and the determination of the relative configuration was carried out by the correlation of NMR data and DFT conformational analysis of cis- and trans-7a,b  of these methine protons , suggesting small 3J2-H,3-H couplings.Although the hydrogens 2-H and 15a-H of  protons  does notnformers . Accorditrans-7a,b  corroborate that the couplings between 2-Hax and 3-Hs derive from trans-diaxial  and gauche  orientations, which is only feasible with the trans relative configuration of 7a,b. Thus in the course of the cyclization, the attack of the carbanion to the iminium carbon of intermediate A occurred with trans-diastereoselectivity to the C-2 phenyl group. The formation of the diastereomeric cis-7a,b could not be detected.Whereas in the computed solution conformers of ans-7a,b , the oxa7a,b also confirmed that the reaction took place diastereoselectively and enantiomers of trans-7a,b were separated on a Chiralpak IA column using hexane/dichloromethane as eluent and online HPLC-ECD spectra of the separated enantiomers were recorded. Due to their similar chromophoric system, the HPLC-ECD spectra of 7a and 7b were quite similar. The first-eluting enantiomer of 7a had an intense broad positive Cotton effect (CE) at 378 nm, negative ones at 314, 305, 272 nm and positive ones at 284, 279 and 224 nm -7d  and -7b and computed ECD curves were compared with the measured HPLC-ECD ones of the separated enantiomers of trans-7a,b -7d and -7b gave good agreement with the experimental HPLC-ECD spectra of the second-eluting enantiomers . The good agreement between the experimental HPLC-ECD and computed ECD spectra of 7a,b not only allowed the configurational assignment of their separated enantiomers but also confirmed independently the trans-diastereoselectivity of the cyclization.For the configurational assignment of the separated enantiomers, TDDFT-ECD calculations were carried out on the solution conformers of , β-amyloid-25-35 (Aβ25–35) and oxygen–glucose deprivation (OGD)-induced neurotoxicity in human neuroblastoma SH-SY5Y cells -hydride shift cyclization reactions of a 4-aryl-2-phenyl-1,4-benzoxazepine derivative afforded three condensed 1H: 500 MHz; 13C: 125 MHz) and Bruker Avance II 400  spectrometers using TMS as internal standard. Chemical shifts were reported as δ in ppm and 3JH,H coupling constants in Hz. Chiral HPLC separation of 7a,b were performed on a Jasco HPLC system with Chiralpak IA column  and HPLC-ECD spectra were recorded in stopped-flow mode on a JASCO J-810 electronic circular dichroism spectropolarimeter equipped with a 10 mm HPLC flow cell. ECD ellipticity (Φ) values were not corrected for concentration. For an HPLC-ECD spectrum, three consecutive scans were recorded and averaged with 2 nm bandwidth, 1 s response, and standard sensitivity. The HPLC-ECD spectrum of the eluent recorded in the same way was used as background. The concentration of the injected sample was set so that the HT value did not exceed 500 V in the HT channel down to 230 nm. IR spectra were recorded on a JASCO FTIR-4100 spectrometer and absorption bands are presented as wavenumber in cm−1. Electrospay Quadrupole Time-of-Flight HRMS measurements were performed with a MicroTOF-Q type QqTOF MS instrument equipped with an ESI source from Bruker . Elementary analysis was carried with a Vario Micro V1.9.6 instrument.Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. The NMR spectra were recorded on Bruker-AMX 500  and TDDFT-ECD calculations were performed with Gaussian 09 -2,5-diphenyltetrazolium bromide (MTT) for 3 h under 5% CO2/95% air at 37 °C. After replacement of the medium with 100 µL DMSO, absorbance was read at 490 nm. Data were analyzed by one-way analysis of variance (ANOVA) and expressed as means ± SD with P < 0.05 as significance.2-Phenyl-3,4-dihydro-1,4-benzoxazepin-5(2H)-one (rac-9): To a stirred solution of racemic flavanone  in TFA (55 mL), sodium azide  was added in two parts and the mixture was stirred for 6 h. Cold diethyl ether (200 mL) was added to the reaction mixture and stirring was continued for 1 h. The precipitated white solid was filtered and refluxed in 180 mL water for 3 h. After cooling to room temperature, it was refrigerated for 1 h, the resultant precipitate was filtered and washed with cold water. The product rac-9 was dried and isolated as white powder  with mp 127–129 °C. 1H NMR  δ 3.54 , 3.66 , 5.46 , 7.07 , 7.19 , 7.28 , 7.36–7.44 , 7.50 , 7.85 ; 13C NMR  δ 46.3 (C-3), 85.8 (C-2), 122.4 (C-9), 123.7 (C-7), 125.9 (C-5a), 126.3 , 128.5 (C-4’), 128.7 , 130.9 (C-6), 133.3 (C-8), 139.0 (C-1’), 154.5 (C-9a), 170.9 (C-5); IR (KBr) ν: 1462, 1665, 2917, 3075, 3203, 3307 cm−1; HRMS–ESI (m/z): [M + Na]+ calcd for C15H13NO2Na, 262.0844; found, 262.0839; Anal. calcd for C15H13NO2 (239.09): C, 75.30; H, 5.48; N, 5.85; found: C, 75.28; H, 5.49; N, 5.85.2-Phenyl-2,3,4,5-tetrahydro-1,4-benzoxazepine (rac-10): To a stirred solution of rac-9  in dry THF (80 mL), 2.0 M lithium aluminium hydride solution in THF was added dropwise  and the mixture was refluxed for 1.5 h. After cooling to room temperature, ethyl acetate (5 mL), methanol (5 mL) and water (50 mL) were added and the mixture was concentrated under reduced pressure. The residue was extracted with dichloromethane (3 × 50 mL). The combined organic layers were washed with water (20 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The product rac-10 was isolated as yellow solid  with mp 73–74 °C. 1H NMR  δ 3.20 , 3.37 , 3.96 , 4.12 , 4.66 , 7.04 , 7.17 , 7.31–7.43 ; 13C NMR  δ 52.5 (C-5), 58.9 (C-3), 86.4 (C-2), 121.6 (C-9), 123.6 (C-7), 125.8 , 127.7 (C-8), 128.4 , 129.0 , 135.6 (C-5a), 140.5 (C-1’), 159.1 (C-9a); IR (KBr) ν: 1579, 2935, 3328 cm−1; HRMS–ESI (m/z): [M + H]+ calcd for C15H16NO, 226.1232; found, 226.1235; Anal. calcd for C15H15NO (225.11): C, 79.97; H, 6.71; N, 6.22; found: C, 80.03; H, 6.76; N, 6.18.5-Nitro-2--yl)benzaldehyde (rac-5): To the stirred solution of rac-10  in dry toluene (25 mL), anhydrous K2CO3 (1.85 g 13.39 mmol) and 2-fluoro-5-nitrobenzaldehyde  were added and the mixture was refluxed for 8 h. After cooling to room temperature, K2CO3 was filtered off and toluene was removed under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate/hexane 4:1) to give rac-5 as yellow solid . Mp 152–156 °C; 1H NMR  δ 3.63 , 4.04 , 4.71 , 4.94 , 5.29 , 7.03 , 7.13 , 7.26 , 7.36–7.42 , 8.17 , 8.61 , 9.97 ; 13C NMR  δ 56.1 (C-5), 64.6 (C-3), 82.7 (C-2), 117.6 (C-6”), 121.0 (C-9), 123.8 (C-7), 124.3 (C-5a), 125.9 , 127.5 (C-2’’), 128.1 (C-4”), 128.7 , 129.1 , 129.5 (C-5”), 129.8 (C-4’), 129.8 (C-6), 138.1 (C-1’), 156.8 (C-9a), 158.2 (C-1”), 188.3 (CHO); IR (KBr) ν: 1382, 1500, 1681, 1734, 2921 cm−1; HRMS–ESI (m/z): [M + Na]+ calcd for C22H18N2O4Na, 397.1164; found, 397.1158; Anal. calcd for C22H18N2O4 (374.12): C, 70.58; H, 4.85; N, 7.48; found: C, 70.59; H, 4.89; N, 7.45.trans-1,3-Dimethyl-11'-nitro-2'-phenyl-1',2'-dihydro-2H,7b'H,9'H-spirobenzoxazepine]-2,4,6-trione (rac-trans-7a): To a stirred solution of rac-5  in chloroform (5 mL), anhydrous MgSO4  and 1,3-dimethylbarbituric acid  were added and the mixture was refluxed for 4 h. After cooling to room temperature, MgSO4 was filtered off and chloroform was removed under reduced pressure. Water (10 mL) and dichloromethane (20 mL) were added and the layers were separated. The aqueous phase was extracted with dichloromethane (2 × 10 mL). The combined organic layers were washed with concentrated NaHCO3 solution, dried over MgSO4, filtered and concentrated under reduced pressure. The oily product was triturated with ether to afford rac-trans-7a as yellow solid  with mp 262–264 °C. 1H NMR  δ 2.91 , 3.12 , 3.13 , 3.87 , 4.00 , 4.16 , 4.81 , 5.13 , 6.80 , 6.85 , 7.10 , 7.15 , 7.30–7.38 , 8.08 , 8.13 ; 13C NMR  δ 28.7 (Me), 28.9 (Me), 34.7 (C-9), 47.3 (C-3) 51.9 (C-10), 71.5 (C-15a), 81.2 (C-2), 109.9 (C-19), 121.5 (C-15b), 123.8 (C-5), 124.7 (C-17), 124.8 (C-6), 125.5 (C-8a), 127.0 (C-2’ and C-6’), 128.8 (C-3’ and C-5’), 129.1 (C-8), 129.9 (C-18), 132.1 (C-16), 137.6 (C-7), 138.3 (C-1’), 147.6 (C-13), 150.1 (C-19a), 151.7 (C-4a), 167.1 (C-15), 168.9 (C-11); IR (KBr) ν: 749, 988, 1326, 1683, 2926 cm−1; HRMS–ESI (m/z): [M + Na]+ calcd for C28H24N4O6Na, 535.1594; found, 535.1588; Anal. calcd for C28H24N4O6 (512.17): C, 65.65; H, 4.72; N, 10.93; found: C, 65.64; H, 4. 70; N, 10.95. -7a: retention time (tR) 10.57 min ; HPLC-ECD data in hexane/dichloromethane 70:30 as λmax (Φ): 378 (8.97), 368 sh (8.71), 314 (−12.73), 305 sh (−12.73), 284 (2.21), 272 (−1.54), 239 (16.42), 224 sh (11.27). -7a: retention time (tR) 28.73 min ; HPLC-ECD in hexane/dichloromethane 70:30 as λmax (Φ): 381 sh (−4.83), 370 (−5.03), 314 (7.10), 305 sh (5.68), 286 (−1.24), 270 (0.78), 239 (−9.32), 221 (−11.60).trans-2,2-Dimethyl-11'-nitro-2'-phenyl-1',2'-dihydro-7b'H,9'H-spirobenzoxazepine]-4,6-dione (rac-trans-7b): To the stirred solution of rac-5  in chloroform (5 mL), anhydrous MgSO4  and Meldrum’s acid  were added and the mixture was refluxed for 6 h. After cooling to room temperature, MgSO4 was filtered off and chloroform was removed under reduced pressure. Water (10 mL) and dichloromethane (20 mL) were added and the layers were separated. The aqueous phase was extracted with dichloromethane (2 ×10 mL). The combined organic layers were washed with concentrated NaHCO3 solution, dried over MgSO4, filtered and concentrated under reduced pressure. The oily product was triturated with ether to afford rac-trans-7b as yellow solid  with mp 130–134 °C. 1H NMR  δ 1.06 , 1.62 , 3.19 , 3.90 , 3.95 , 4.16 , 5.02 , 5.22 , 6.83 , 6.87 , 7.12 , 7.22 , 7.27–7.35 , 8.09 ; 13C NMR  δ 26.9 (Me), 30.5 (Me), 35.7 (C-9), 47.2 (C-3), 50.0 (C-10), 70.7 (C-15a), 80.3 (C-2), 105.5 (C-13), 110.0 (C-19), 119.5 (C-8a), 125.1 (C-5), 125.2 (C-17), 125.6 (C-6), 126.7 (C-15b), 127.2 , 128.6 , 129.0 (C-8), 131.1 (C-4’), 131.4 (C-16), 137.3 (C-7), 138.1 (C-1’), 147.7 (C-19a), 152.2 (C-4a), 164.5 (C-15), 168.2 (C-11); IR (KBr) ν: 1197, 1322, 1507, 1737, 2925 cm−1; HRMS–ESI (m/z): [M + Na]+ calcd for C28H24N2O7Na, 523.1481; found, 523.1475; Anal. calcd for C28H24N2O7 (500.16): C, 67.19; H, 4.83; N, 5.60; found: C, 67.17; H, 4.86; N, 5.59. -7b: retention time (tR) 5.96 min ; HPLC-ECD data in hexane/dichloromethane 70:30 as λmax (Φ): 379 sh (10.62), 368 (10.90), 312 (−9.79), 303 sh (−7.89), 279 (6.49), 261 sh (−3.01), 251 (−3.30), 228 (15.44). -7b: retention time (tR) 14.09 min ; HPLC-ECD data in hexane/dichloromethane 70:30 as λmax (Φ): 383 sh (−3.29), 371 (−3.71), 311 (3.16), 302 sh (2.75), 278 (−2.26), 260 sh (1.04), 249 (1.10), 227 (−5.51).[5-Nitro-2--yl)benzylidene]propanedinitrile (7c): To the stirred solution of rac-5  in chloroform (5 mL), anhydrous MgSO4  and malononitrile  were added and the mixture was refluxed for 10 h. After cooling to room temperature, MgSO4 was filtered off and chloroform was removed under reduced pressure. Water (10 mL) and dichloromethane (20 mL) were added and the layers were separated. The aqueous phase was extracted with dichloromethane (2 × 10 mL). The combined organic layers were washed with concentrated NaHCO3 solution, dried over MgSO4, filtered and concentrated under reduced pressure. The oily product was triturated with ether to afford 7c as yellow solid  with mp 152–156 °C. 1H NMR  δ 3.78 , 4.50 , 4.58 , 5.06 , 7.15 , 7.16–7.26 , 7.32 , 7.41 , 7.75 , 8.26 , 8.85 ; 13C NMR  δ 59.4 (C-11), 63.2 (C-3), 82.9 (C-2), 84.3 (C-17), 111.8 (CN), 112.9 (CN), 119.2 (C-15), 121.5 (C-6), 121.9 (C-10), 124.5 (C-13), 125.9 , 128.4 (C-11a), 128.7 (C-7) 128.9 , 129.2 (C-4’), 129.9 (C-12), 138.2 (C-8), 141.2 (C-1’), 156.9 (C-16), 158.2 (C-15a), 158.5 (C-5) IR (KBr) ν: 759, 1222, 1335, 1488, 2227, 2923 cm−1; HRMS–ESI (m/z): [M+Na]+ calcd for C25H18N4O3Na, 445.1277; found, 445.1271; Anal. calcd for C28H18N4O3 (422.14): C, 71.08; H, 4.29; N, 13.26; found: C, 71.05; H, 4.30; N, 13.25. First eluting enantiomer of 7c: retention time (tR) 21.41 min ; HPLC-ECD data in hexane/dichloromethane 80:20 as λmax (Φ): 348 (−4.21), 279 (3.84), 252 sh (1.62), 223 (2.04). Second eluting enantiomer of 7c: retention time (tR) 21.41 min ; HPLC-ECD data in hexane/dichloromethane 80:20 as λmax (Φ): 356 (3.97), 281 (−3.48), 250 sh (−1.32).File 1Spectroscopic data and details of calculations.7b–d, 1H, 13C NMR and IR spectra are disclosed in this file.Structures and populations of the in vacuo and PCM solvent model conformers, cartesian coordinates of


Bioassay-guided purification of the active fractions of the sponge’s extract resulted in the isolation of two new glycerides, mirabolides A and B (1 and 2), together with the reported 4-methylene sterols, conicasterol (3) and swinhosterol B (4). The structures of the compounds were assigned by interpretation of their 1D , 2D  NMR spectral data and high-resolution mass determinations. Compounds 1–4 displayed marked cytotoxic activity against human breast adenocarcinoma cell line (MCF-7) with IC50 values of 16.4, 5.18, 6.23 and 3.0 μg/mL, respectively, compared to 5.4 μg/mL observed by doxorubicin as reference drug.As a part of our continuing work to find out bioactive lead molecules from marine invertebrates, the CHCl Marine sponges are multicellular animals evolved from about 600 million years ago; they belong to phylum Porifera and are considered the most primitive animals . More thTheonella is well-known producer of diverse bioactive constituents including 4-methylene sterols .,24.1 , 1.53/27.8 (H2-2′′′/C-2′′′), 1.15/37.1 (H2-13′′′/C-13′′′), 1.50/26.0 (H-14′′′/C-14′′′), 0.85/20.7 (H3-15′′′/C-15′′′), 0.85/20.7 (H3-16′′′/C-16′′′) together with the methylene cluster at 1.25/20.8–35.1 (H2-3′′′-H2-12′′′/C-3′′′-C-12′′′) supported this assignment. The two methyl doublet signals at δ 0.85 (H3-15′′′ and H3-16′′′) indicated the iso-branching of the fatty chain. This was verified by COSY correlations and HMBC cross peaks  was identified based on its GLC retention time and mass fragmentation spectrum. Two relatively intense fragment peaks at m/z 213 and 241 indicated the favored α-cleavage with the respect of the tertiary carbon atom (C-13″) carrying the methyl branch. Characteristic peaks at m/z 219 and 201 resulted from the loss of methanol and water from the fragment at m/z 241.In the negative ESIMS of ectively . The ove1 is reported here as a new natural product and was named mirabolide A.To the best of our knowledge, 2  and 2D  NMR experiments allowed the assignment of a triglyceride skeleton (H/δC 3.60/70.5 (H2-1/C-1), 5.22/71.5 (H-2/C-2) and 4.40, 4.16/63.9 (H2-3/C-3) were indicative for a trisubstituted glycerol moiety (H/δC at 175.1 (C-1′), 2.30/35.0 (H2-2′/C-2′), 1.59/26.1 (H2-3′/C-3′), 1.28/28.2–31.1 (H2-4′-H2-11′/C-4′-C-11′), 1.10/38.2 (H2-12′/C-12′), 1.31/35.7 (H-13′/C-13′), 1.28/30.8 (H2-14′/C-14′), 0.88/11.8 (H3-15′/C-15′) and 0.85/19.6 (H3-16′/C-16′). The COSY correlations and HMBC cross peaks of H-2′/C-1′, H2-3′/C-1′ supported this assignment , 2.30/35.1 (H2-2′′′/C-2′′′), 1.59/26.1 (H2-3′′′/C-3′′′), 1.28/28.2–31.1 (H2-4′′′-H2-14′′′/C-4′′′-C-14′′′), 1.19/40.2 (H2-15′′′/C-15′′′), 1.50/29.1 (H-16′′′/C-16′′′), 0.87/23.1 (H3-17′′′/C-17′′′) and 0.87/23.1 (H3-18′′′/C-18′′′). Two methyl doublets at δ 0.87 (H3-17′′′ and H3-18′′′) indicated the iso-branching of the fatty chain. The assignment was supported by COSY and HMBC cross peaks , 3.70/77.5 (H-2″/C-2″), 2.08, 2.22/29.1 (H2-3″/C-3″), 3.53, 3.67/68.6 (H2-4″/C-4″) and 3.20/52.4 (H3CO-2″) were assigned as 4-hydroxy-2-methoxybutanoyl moiety attached to position 2 of the substituted glycerol. The assignment of this moiety was deduced from HMBC cross peaks of H-2″/C-1″, H2-3″/C-1″ and H3CO-2″/C-2″ , methyl-13-methyl-pentadecanoate (m/z 270) and methyl 16-methylheptadecanoate (m/z 298), corresponding to the three fatty acyl chains attached to C-2, C-1 and C-3, respectively. Branching of methyl at position C-13′ of methyl-13-methyl-pentadecanoate was indicated by the mass fragmentation pattern as in 1.Compound 2  was obtaskeleton . The sigl moiety  23,24].,24.2 . Compound 4 was the most active compound with IC50 value of 3.0 μg/mL compared to doxorubicin (IC50 = 5.4 μg/mL). Compounds 1–3 were less active with IC50 values of 16.4, 5.18 and 6.23 μg/mL, respectively.Compounds m/z 556.2771 [M + H]+ as a reference mass. NMR spectra were obtained in CD3OD or CDCl3 on Bruker Avance DRX 600 MHz spectrometer  at 600 MHz for 1H NMR and 150 MHz for 13C NMR. NMR spectra were referenced to the residual protonated solvent signals . Silica gel  and silica gel  were used for isolation of compounds on vacuum liquid chromatography and column chromatography, respectively. The HPLC separation was performed on a RP18, 250 × 10 mm, 5 μm Cosmosil ARII column . Trace Ultra GC system  was used for GC analysis. Fatty acid methyl esters were separated on 70% (cyanopropyl-polysil phenylene siloxane) capillary column; injection temperature was set at 200 °C and detector temperature at 250 °C (MSD). The flow rate of the carrier, Helium, was 1.5 mL/min. The column temperature was 80 °C for 2 min then increased to 230 °C by the rate of 3 °C/min. For TLC, pre-coated silica gel 60 F-254 plates  were used, solvent systems were CHCl3 (100%), CHCl3/MeOH (95:5) and CHCl3/MeOH (85:15), spots were visualized by p-anisaldehyde/sulphuric acid reagent. Doxorubicin  was used as positive cytotoxic control.Optical rotations were recorded by a P800 series digital automatic high-speed polarimeter . UV spectra were recorded on a Hitachi 300 spectrometer . IR spectral analysis were carried on Nicolet™ iS™10 FT-IR spectrometer ; ESIMS spectra were obtained with a LCQ DECA mass spectrometer  coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector. Mass spectral data were obtained with a Micromass Q-tof equipped with lockspray mass spectrometer using Leucine Enkaphalin at Theonella aff. mirabilis, because it conforms closely to its original description from Micronesia. In view of the great distance from the type locality, some doubt of conspecificity is here expressed. A voucher fragment was deposited in the Naturalis Biodiversity Center at Leiden under registration number ZMA Por. 17590. Another voucher was deposited at the Department of Natural Products at King Abdulaziz University under the code #DY-60.The sponge was collected off Sharm El-Sheikh in the Red Sea at a depth of 10–14 m in July 2013. Identification of the sponge was kindly provided by Prof. Rob van Soest. The sponge consists of a group of thick-walled tubes with bluish green color in life, beige-yellow in preserved condition. The individual tubes measure up to 6 cm high and 2 cm in diameter, with a terminal oscule which measures up to 3 mm in preserved condition. The surface is optically smooth. The consistency is hard but toughly compressible. This species belongs to a group of “soft” lithistids. The skeleton is mainly a desma reticulation but in more than half the outer part of the tubes, the desmas are absent and the skeleton is there formed by bundles of strongyloxeas (strongyles with tapering endings) measuring 384–462 × 5–12 μm. The surface membrane lacks the usual phyllotriaenes but is crowded with acanthomicrorhabd microscleres measuring 9–13 × 2–3 μm, and these are densely distributed throughout the choanosome. In the inner parts of the tubes, single desmas gradually form a loose reticulation. The desmas are peculiar in being calthrops-like and having four bifurcate cladi with trilophose sharply pointed endings. The cladomes measure 240–270 μm. The primary cladi measure up to 100–120 × 20 μm, while the secondary cladi measure 30–35 × 15 μm. The specimen is assigned to the widespread Indo-Pacific species 2Cl2/MeOH (1:1) (3 × 2000 mL) at room temperature. The combined extracts were concentrated under vacuum to afford 11.0 g of dried extract. The extract was partitioned between CHCl3 and H2O and the organic layer was evaporated under vacuum to afford 6.0 g of dried residue. The residue was then chromatographed on SiO2 VLC using n-hexane/EtOAc/MeOH gradient to give five main fractions (A–E). Fraction B  was purified on silica gel column using n-hexane/CHCl3 gradient to give several fractions. The active fraction was purified on C18-reversed phase semipreparative HPLC using 70% ACN to give compound 3 (4.9 mg). Fraction C which was eluted by n-hexane/EtOAc  was purified on silica gel column using n-hexane/CHCl3 gradient and final HPLC purification of the active fraction on C18-reversed phase semipreparative HPLC using 70% ACN to yield compound 4 (14 mg). Fraction D was eluted with EtOAc/MeOH  was subjected to SiO2 column using CHCl3/MeOH gradient to give four fractions. The cytotoxic fraction was subjected to final HPLC purification on C18 semipreparative column using 60% ACN to afford compound 1 (5.8 mg). Finally, fraction E was eluted with EtOAc/MeOH mixtures  was chromatographed over silica gel column using CHCl3/MeOH gradient to give several fractions. The cytotoxic fraction was purified on HPLC using 60% ACN to give compound 2 (3.3 mg).The freeze-dried sponge (750 g) was extracted with a mixture of CH1). White precipitate; c 0.025, CH3OH); UV (MeOH) λmax 292 nm; IR: νmax 3350, 1735 cm−1; HRESIMS m/z 685.5625 ; NMR data: see Mirabolide A (2). White precipitate; c 0.025, CH3OH); UV (MeOH) λmax 292 nm; IR: νmax 3240, 1740 cm−1; HRESIMS m/z 713.5933 ; NMR data: see Mirabolide B (3). White precipitate; c 0.1, CHCl3); UV (MeOH) λmax 235 nm; IR: νmax 3450, 1712 cm−1; ESIMS: m/z 413.3 [M + H]+, C29H49O.1H NMR : δH 5.07 , 4.63 , 4.02 , 2.48 , 2.25 , 1.95 , 1.78 , 1.75 , 1.75 , 1.74 , 1.64 , 1.58 , 1.49 , 1.52 , 1.48 , 1.45 , 1.39 , 1.38 , 1.35 , 1.33 , 1.23 , 1.22 , 1.22 , 1.14 , 1.13 , 1.13 , 1.10 , 0.92 , 0.85 , 0.83 , 0.80 , 0.78 , 0.58 ; 13C NMR : δC 153.1 (C-4), 142.9 (C-14), 125.6 (C-8), 102.7 (C-29), 73.3 (C-3), 56.8 (C-17), 49.4 (C-5), 49.2 (C-9), 42.7 (C-13), 39.9 (C-10), 37.3 (C-24), 36.7 (C-12), 34.5 (C-1), 32.3 (C-20), 31.9 (C-22), 30.1 (C-25), 29.3 (C-23), 27.1 (C-2), 27.0 (C-15), 25.7 (C-6), 24.6 (C-7), 22.7 (C-16), 20.2 (C-11), 19.0 (C-27), 18.2 (C-21), 18.1 (C-26), 15.3 (C-18), 14.1 (C-28), 13.1 (C-19).Conicasterol (4). White precipitate; c 0.1, CHCl3); UV (MeOH) λmax 280 nm; IR: νmax 3460, 1735, 1710 cm−1; ESIMS: m/z 445.4 [M + H]+, C29H49O3.1H NMR : δH 5.16 , 4.67 , 4.0 , 2.41 , 2.35 , 2.29 , 2.20 , 2.12 , 2.10 , 2.04 , 1.97 , 1.97 , 1.82 , 1.77 , 1.71 , 1.63 , 1.53 , 1.51 , 1.48 , 1.47 , 1.43 , 1.41 , 1.37 , 1.29 , 1.25 , 1.23 , 1.19 , 1.08 , 0.87 , 0.82 , 0.81 , 0.81 ; 13C NMR : δC 224.9 (C-14), 211.2 (C-8), 150.9 (C-4), 104.2 (C-29), 72.9 (C-3), 62.5 (C-9), 52.5 (C-13), 48.4 (C-5), 46.6 (C-17), 44.3 (C-10), 41.6 (C-7), 38.9 (C-24), 37.9 (C-15), 37.3 (C-12), 36.5 (C-1), 34.4 (C-20), 32.4 (C-25), 32.3 (C-22), 32.2 (C-2), 30.6 (C-23), 26.0 (C-6), 23.7 (C-16), 20.2 (C-27), 18.5 (C-18), 18.2 (C-26), 18.1 (C-21), 18.0 (C-11), 15.4 (C-28), 13.0 (C-19).Swinhosterol B  were separately refluxed with 5% methanolic KOH (5 mL) for 2 h [3. The fatty acids were liberated from the aqueous layer after acidification with 1 N HCl followed by extraction with CHCl3, then were methylated using CH2N2 and subjected to GC-MS analysis.Compounds  for 2 h . The rea1–4 against breast adenocarcinoma cell line  were evaluated using sulforhodamine assay [50) was calculated from the log dose response curve. The values were the results of three determinations. Doxorubicin was used as reference drug and showed an IC50 value of 5.4 μg/mL.The cytotoxic activity of compounds ne assay . Cells wTheonella mirabilis afforded four compounds (1–4) including two sterols and two glycerides. Compounds 1 and 2 are new glycerides and are reported here for the first time. The structures of the isolated compounds were determined by careful examination of their 1D and 2D NMR, and HRESIMS spectral data. All isolated compounds demonstrated significant cytotoxic activity against human breast adenocarcinoma cell line (MCF-7) with IC50 values ranging from 3.0 to 16.4 μg/mL.Column chromatographic fractionation of the organic extract of the Red Sea sponge


Penicillium purpurogenum G59. The G59 strain originally did not produce any metabolites with antitumor activities in MTT assays using K562 cells. Tracing newly produced metabolites under guidance of MTT assay and TLC analysis by direct comparison with control G59 extract, seven new (1–7) and two known (8–9) lipopeptides were isolated together with five known polyketides 10–14 from the extract of mutant AD-2-1. Structures of the seven new compounds including their absolute configurations were determined by spectroscopic and chemical evidences and named as penicimutalides A–G (1–7). Seven known compounds were identified as fellutamide B (8), fellutamide C (9), 1′-O-methylaverantin (10), averantin (11), averufin (12), nidurufin (13), and sterigmatocystin (14). In the MTT assay, 1–14 inhibited several human cancer cell lines to varying extents. All the bioassays and HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses demonstrated that the production of 1–14 in the mutant AD-2-1 was caused by the activated production of silent metabolites in the original G59 fungal strain. Present results provided additional examples for effectiveness of the chemical mutagenesis strategy using diethyl sulphate mutagenesis to discover new compounds by activating silent metabolites in fungal isolates.AD-2-1 is an antitumor fungal mutant obtained by diethyl sulfate mutagenesis of a marine-derived  Penicillium purpurogenum G59 using dimethyl sulfoxide (DMSO) as accessorial agent +; negative ESI-MS m/z: 570 [M − H]−, 606 [M + Cl]−, 616 [M + HCOO]−, 660 [M + HC2O4]−. Positive HR-ESI-MS m/z: measured 594.3834 [M + Na]+, calcd for C28H53N5O7Na [M + Na]+ 594.3843, measured 610.3626 [M + K]+, calcd for C28H53N5O7Na [M + K]+ 610.3582. IR νmax cm−1 (KBr): 3422, 3283, 2958, 2922, 2852, 1663, 1626, 1552, 1536, 1516, 1443, 1384, 1311, 1261, 1095, 1018, 928, 879. 1H and 13C NMR data: see Penicimutalide A (2): White amorphous powder (MeOH),  −17.0 . Positive ESI-MS m/z: 594 [M + Na]+; negative ESI-MS m/z: 570 [M − H]−, 606 [M + Cl]−, 616 [M + HCOO]−, 660 [M + HC2O4]−. Positive HR-ESI-MS m/z: measured 594.3847 [M + Na]+, calcd for C28H53N5O7Na [M + Na]+ 594.3843, measured 610.3612 [M + K]+, calcd for C28H53N5O7K [M + K]+ 610.3582. IR νmax cm−1 : 3283, 3215, 2934, 2862, 1654, 1540, 1419, 1320, 1290, 1255, 1147, 1122, 1063, 883, 865. 1H and 13C NMR data: see Penicimutalide B (3): White amorphous powder (MeOH),  −23.7 . Positive ESI-MS m/z: 558 [M + H]+, 580 [M + Na]+; negative ESI-MS m/z: 592 [M + Cl]−. Positive HR-ESI-MS m/z: measured 580.3695 [M + Na]+, calcd for C27H51N5O7Na [M + Na]+ 580.3686. IR νmax cm−1 : 3268, 2934, 2862, 1650, 1536, 1418, 1325, 1282, 1256, 1196, 1145, 1114, 1062, 979, 949. 1H and 13C NMR data: see Penicimutalide C (4): White amorphous powder (MeOH),  −24.8 . Positive ESI-MS m/z: 558 [M + H]+, 580 [M + Na]+, 596 [M + K]+; negative ESI-MS m/z: 556 [M − H]−,592 [M + Cl]−. Positive HR-ESI-MS m/z: measured 580.3684 [M + Na]+, calcd for C27H51N5O7Na [M + Na]+ 580.3686, measured 596.3426 [M + K]+, calcd for C28H53N5O7K [M + K]+ 596.3426. IR νmax cm−1 : 3274, 2930, 2859, 1650, 1632, 1537, 1417, 1325, 1281, 1255, 1197, 1139, 1112, 1056, 946, 865. 1H and 13C NMR data: see Penicimutalide D (5): White amorphous powder (MeOH),  −15.0 . Positive ESI-MS m/z: 571 [M + H]+; negative ESI-MS m/z: 569 [M − H]−. Positive HR-ESI-MS m/z: measured 571.3819 [M + H]+, calcd for C27H51N6O7 [M + H]+ 571.3819; measured 593.3634 [M + Na]+, calcd for C27H50N6O7Na [M + Na]+ 593.3639. IR νmax cm−1 : 3273, 2931, 2859, 1642, 1546, 1537, 1418, 1324, 1283, 1156, 1134, 1074, 864, 783. 1H and 13C NMR data: see Penicimutalide E (6): White amorphous powder (MeOH),  −18.8 . Positive ESI-MS m/z: 571 [M + H]+; negative ESI-MS m/z: 569 [M − H]−. Positive HR-ESI-MS m/z: measured 571.3805 [M + H]+, calcd for C27H51N6O7 [M + H]+ 571.3819. IR νmax cm−1 : 3273, 2933, 2860, 1653, 1628, 1540, 1418, 1177, 1132, 1024. 1H and 13C NMR data: see Penicimutalide F (7): White amorphous powder (MeOH),  −7.5 . Positive ESI-MS m/z: 458 [M + H]+, 480 [M + Na]+; negative ESI-MS m/z: 456 [M − H]−, 492 [M + Cl]−. Positive HR-ESI-MS m/z: measured 458.2970 [M + H]+, calcd for C21H40N5O6 [M + H]+ 458.2979; measured 480.2798 [M + Na]+, calcd for C21H39N5O6Na [M + Na]+ 480.2798; measured 496.2526 [M + K]+, calcd for C27H5N5O7K [M + K]+ 496.2537. IR νmax cm−1 : 3274, 3205, 2929, 2857, 1651, 1632, 1540, 1414, 1320, 1281, 1254, 1200, 1133, 1095, 1070, 1042, 958, 866. 1H and 13C NMR data: see Penicimutalide G (8): White amorphous powder (MeOH),  −28.0 . Positive ESI-MS m/z: 556 [M + H]+; negative ESI-MSm/z: 554 [M − H]−. 1H and 13C NMR data: see Fellutamide B (9): White amorphous powder (MeOH),  −33.0 . Positive ESI-MS m/z: 558 [M + H]+, 580 [M + Na]+; negative ESI-MS m/z: 592 [M + Cl]−. Positive HR-ESI-MS m/z: measured 558.3862 [M + H]+, calcd for C27H52N5O7 [M + H]+ 558.3867; measured 580.3692 [M + Na]+, calcd for C27H51N5O7Na [M + Na]+ 580.3686; measured 596.3413 [M + K]+, calcd for C27H51N5O7K [M + K]+ 596.3426. 1H and 13C NMR data: see Fellutamide C (O-Methylaverantin (10): Crystalline orange-red powder (CH2Cl2), mp 214−216 °C,  +1.8 , ESI-MS m/z: 387 [M + H]+. 1H NMR  δ: 12.78 , 12.18 , 10.10 , 9.69  7.23 , 7.13 , 6.64 , 4.97 , 3.49 , 1.95–1.75 , 1.58–1.38 , 1.38–1.24 , 0.89 .1′-11): Crystalline orange-red powder (CH2Cl2), mp 279−280 °C,  −130.0 . ESI-MS m/z: 373 [M + H]+. 1H NMR  δ: 12.63 , 12.08 , 9.00 , 7.09 , 7.00 , 6.50 , 5.29 , 1.80–1.63 , 1.50–1.30 , 1.30–1.10 , 0.76 . 13C NMR  δ: 190.7 (C-9), 181.8 (C-10), 166.0 (C-8), 165.9 (C-1), 165.0 (C-6),161.7 (C-3), 136.4 (C-4a), 134.5 (C-10a), 122.0 (C-2), 110.3 (C-5), 110.2 (C-9a), 109.4 (C-8a), 108.9 (C-7), 108.8 (C-4), 69.9 (C-1′), 36.9 (C-2′), 32.3 (C-3′), 25.6 (C-4′), 23.2 (C-5′), 14.3 (C-6′).Averantin (12): Crystalline orange-red powder (CH2Cl2), mp 280−282 °C,  −1.7 . 1H NMR  δ: 12.51 , 12.19 , 10.09 , 7.22 , 7.09 , 6.63 , 5.29 , 2.15–1.25 , 1.55 . 13C NMR  δ: 190.7 (C-9), 181.6 (C-10), 166.0 (C-8), 165.8 (C-1), 161.5 (C-6), 159.8 (C-3), 136.3 (C-4a), 134.4 (C-10a), 117.1 (C-2), 110.2 (C-5), 109.5 (C-9a), 109.4 (C-8a), 108.9 (C-7), 108.4 (C-4), 102.2 (C-5′), 67.4 (C-1′), 36.3 (C-2′), 28.0 (C-4′), 27.9 (C-6′), 16.5 (C-3′).Averufin (13): Crystalline orange-red powder (CH2Cl2), mp 188−190 °C,  +0.3 . ESI-MS m/z: 385 [M + H]+. 1H NMR  δ: 12.58 , 12.22 , 10.17 , 7.26 , 7.14 , 6.67 , 5.17 , 3.95 , 2.30–1.30 , 1.58 .Nidurufin (14): Pale yellow needles (CH2Cl2), mp 246 °C (dec.),  −384 , ESI-MS m/z: 325 [M + H]+, 347 [M + Na]+. 1H NMR  δ: 13.23, 7.47 , 6.80 , 6.79 , 6.72 , 6.50 , 6.40 , 5.43 , 4.76 , 3.98 . 13C NMR  δ: 181.2 (C-1), 164.4 (C-10), 163.1 (C-12), 162.1 (C-3), 154.8 (C-7), 153.8 (C-8), 145.2 (C-17), 135.6 (C-5), 113.2 (C-14), 111.1 (C-4), 108.7 (C-2), 106.4 (C-6), 105.8 (C-9), 105.7 (C-13), 102.4 (C-16), 90.4 (C-11), 56.7 (C-18), 47.9 (C-15).Sterigmatocystin  to obtain an oxidized product of 8 from the n-BuOH solution. Then, the total of this oxidized product was hydrolyzed using 100 μL of aqueous 6 N HCl at 110 °C for 24 h, derivatized as mentioned above with FDAA at the same time and same conditions, and then subjected to the HPLC analysis, in the same manner as mentioned.Because we had the 18 column  at room temperature, using mixed A (H2O containing 1% HCOOH) and B (acetonitrile) solutions in a linear gradient  as mobile phase. The acquired photodiode array (PDA) data were processed by Empower PDA software and targeted derivatives were detected at 340 nm.HPLC analysis of the FDAA derivatives was performed on a Venusil MP Cd- and l-standards were separated well as given their retention times (tR) in 1–9 were analyzed by injection of the derivatives alone and co-injection with those of related l- and d-standards, respectively.In the HPLC conditions, the FDAA derivatives of 1–9 was mixed, and the mixture was suspended in 1 mL of aqueous 6 N HCl in a sealed tube and hydrolyzed at 110 °C for 1 h. The solution was then extracted three times with 1 mL of CHCl3 to obtain a CHCl3 extract. This extract was purified by silica gel PTLC  to obtain 3R-hydroxydodecanoic acid :  −16 . Positive ESI-MS m/z: 239 [M + Na]+; negative ESI-MS m/z: 215 [M − H]−. 1H NMR  δ: 4.02 , 2.57 , 2.47 , 1.40–1.58 , 1.20–1.36 , 0.87 . 13C NMR (CDCl3) δ: 176.8, 68.0, 40.8, 36.5, 31.9, 29.7, 29.5, 29.4, 29.3, 25.4, 22.7, 14.1. The  and 1H and 13C NMR data are identical with those reported for 3R-hydroxydodecanoic acid in the literature [Each 0.3 mg of terature .1–14 in MeOH at 10 mg/mL were used as reference standards in the HPLC-PDAD-UV analysis. The MeOH solutions of 1–6 at 10 mg/mL were mixed between 1/2, 3/4, and 5/6 in a ratio of 1:1, respectively, and the mixed sample solutions were used, because the two compounds in these pairs were known to be eluted as a peak with the same retention time in the given HPLC condition by pilot tests of HPLC analyses.EtOAc extracts of the control G59 strain and the mutant AD-2-1 were dissolved in MeOH to prepare sample solutions at 10 mg/mL for HPLC analysis. Crude samples of the compounds 18 column  using the Waters HPLC equipment mentioned above. The sample and standard solutions were filtered using 0.22 µm pore membrane filters, respectively, and each 5 µL of the solutions was injected into the column. Then, elution was performed using MeOH–H2O in linear gradient . The acquired photodiode array (PDA) data were processed using the Empower PDA software to obtain targeted HPLC-PDAD-UV data.The HPLC-PDAD-UV analysis was carried out on a Venusil MP C1–14 was performed on the basis of both HPLC retention times and UV absorption curves.In the HPLC-PDAD-UV analysis, examination of 1–9: 56.12 min (1/2), 54.57 min (3/4), 50.62 min (5/6), 46.52 min (7), 52.77 min (8), and 52.57 min (9). Because 1–9 showed only end UV absorptions, they were hardly identified by the HPLC-PDAD-UV analysis both in the mutant AD-2-1 and the control G59 extracts due to the lack of typical UV absorptions and the baseline drift (Retention times of ne drift .10–14: 63.90 min (10), 59.50 min (11), 63.00 min (12), 52.12 min (13), and 50.25 min (14). All of 10–14 were detected in the mutant AD-2-1 extract but not in the parent G59 extract  at the conditions the same as mentioned for the HPLC-PDAD-UV analysis. The mass detector was set to scan a range from m/z 150–1500 both in the positive and negative modes. The acquired data were processed by Analyst 1.4 software to obtain targeted HPLC-ESI-MS data.The HPLC-ESI-MS analysis was performed on an LC-MS equipment equipped with Agilent 1100 HPLC system, AB Sciex API 3000 LC-MS/MS system and AB Sciex Analyst 1.4 software. HPLC was carried out on the Venusil MP C1–14 appeared with shortened retention times than those in their HPLC-PDAD-UV analysis because of the shortened flow length from the outlet of the HPLC column to the inlet of MS. Retention times of 1–14: 52.80 min (1/2), 51.25 min (3/4), 47.48 min (5/6), 43.40 min (7), 49.59 min (8), and 49.28 min (9), 62.38 min (10), 58.87 min (11), 61.83 min (12), 52.05 min (13), and 47.74 min (14). Detection of 1–14 in the control G59 and the mutant AD-2-1 extracts was achieved by selective ion  monitoring with both extracted ion chromatograms and related MS spectra  and two known lipopeptides (8 and 9) as well as five known polyketides 10–14, were isolated by tracing newly produced metabolites in the mutant AD-2-1 extract under the guidance of MTT assay and TLC analysis using the parent G59 extract as control. Structures of the seven new compounds including their absolute configurations were determined by spectroscopic data and chemical evidences, and named penicimutalides A–G (1–7), respectively. All of 1–14 inhibited several human cancer cell lines to varying extents. Both present bioassays and HPLC-PDAD-UV and HPLC-ESI-MS analyses demonstrated that the production of 1–14 in the mutant AD-2-1 was caused by the activated production of silent metabolites in original fungal G59 strain by the DES mutagenesis.AD-2-1 is an antitumor fungal mutant obtained by the DES mutagenesis of originally inactive, parent


N-5, while compound 14 was first isolated from a natural source. The structures of the new compounds were determined by analysis of detailed spectroscopic data, and the absolute configurations were further decided by a comparison of the experimental and calculated ECD spectra. Hemiacetalmeroterpenoid A (1) possesses a unique and highly congested 6,6,6,6,5,5-hexa-cyclic skeleton. Moreover, the absolute configuration of compound 14 was also reported for the first time. Compounds 1, 5 and 10 exhibited significant antimicrobial activities against Penicillium italicum and Colletrichum gloeosporioides with MIC values ranging from 1.56 to 6.25 μg/mL.Four new compounds including three andrastin-type meroterpenoids hemiacetalmeroterpenoids A-C ( Andrastins are meroterpenoids characterized by a 6,6,6,5-tetra-carbocyclic skeleton. They are biogenetically derived from 3,5-dimethylorsellinic acid (DMOA) and farnesyl diphosphate (FPP), synthesized via a mixed polyketide-terpenoid pathway, and usually possess a keto-enol tautomerism at the cyclopentane ring ,2,3,4. TPenicillium sp. N-5, isolated from the rhizosphere soil of mangrove plant Avicennia marina, led to the isolation of four new compounds, hemiacetalmeroterpenoids A–C (1–3) and astellolide Q (15). Especially, hemiacetalmeroterpenoid A (1) was a new andrastin-type meroterpenoid containing a unique 6,6,6,6,5,5-hexa-cyclic skeleton. Meanwhile, eleven known compounds, including 3-deacetyl-citreohybridonol (4) + , indicating ten degrees of unsaturation. In the 1H NMR spectrum, the signal for one olefinic proton (δH 5.63), one methoxyl (δH 3.60), one methine (δH 1.47), four methylenes  and six methyls . The 13C NMR data exhibited 26 carbon resonances, including two olefinic carbons for one double bond , three carbonyl carbons for two ketone (δC 203.7 and 203.8), and one ester carbonyl (δC 169.3), one methine (δC 49.1), five methylenes (one oxygenated), seven methyls (one oxygenated), eight quaternary carbons (one highly oxygenated: δC 99.8) , a 6-membered ring was confirmed between C-1, C-2, C-3, C-10, and C-21. Finally, another new 5-membered ring was formed by intramolecular dehydration of hydroxyl groups at C-12 and C-16. Thus, the planar structure of 1 was established as shown in Hemiacetalmeroterpenoid A (δC 99.8) . The NMRδC 99.8)  28]. An. An1) wa1 was defined by the NOESY correlations. The correlations of H2-21 with H3-23, H3-20 with H3-19, H3-24, and H3-18 with H3-19, H3-26 were observed in the NOESY spectrum, which means H3-18, H3-19, H3-20, H2-21, H3-23, H3-24 and H3-26 were on the same side. The NOESY correlations of H1-5 with H3-22 suggested that H1-5 and H3-22 were in the opposite face  for two enantiomers 3R, 5S, 8S, 10S, 12R, 13S, 14R, 16R-1a and 3S, 5R, 8R, 10R, 12S, 13R, 14S, 16S-1b with the experimental one. Finally, the experimental ECD spectrum of 1 was nearly identical to the calculated ECD spectrum for 1a  was isolated as a white powder and had a molecular formula of C26H36O6, determined by HRESIMS data m/z 445.25772 [M+H]+  with nine degrees of unsaturation. The 1H NMR spectrum of 2 displayed the signal for one olefinic proton (δH 5.42), one methoxyl (δH 3.56), two methines (δH 1.33 and 1.89), four methylenes  and six methyls . The 13C NMR data revealed 26 carbon resonances, involving four olefinic carbons for two double bonds , two carbonyl carbons for one ketone (δC 201.4), one ester carbonyl (δC 172.6) (2 was similar to the co-isolated andrastin B (13). The obvious difference is that the acetyl group at the C-3 position of compound 2 disappears. Meanwhile, the HMBC from H2-21 to C-3 (δC 99.5) also indicated that a new 6-membered ring was formed between C-1, C-2, C-3, C-10 and C-21  . Accordiand C-21 .1-5, H1-9 and H3-22 were on the same side based on the correlations of H1-5 with H1-9 and H3-22. On the contrary, it was suggested that H3-19, H3-21, H3-23, H3-24, and H3-26 were on the other side based on the NOESY correlations of H2-21 with H3-23 and H3-24, along with H3-19 with H3-24 and H3-26  was also purified as a white powder. The molecular formula was specified as C28H38O7 (ten degrees of unsaturation) by HRESIMS (m/z 509.25015 [M+Na]+), which is 42 mass units higher than that of 2  was also acquired as a yellow powder. It molecular formula was determined as C17H24O6 according to HRESIMS analysis at m/z 347.14578 [M+Na]+ , indicating six degrees of unsaturation. The 1H NMR of 15 showed two methyls (δH 1.15 and 2.08), four methylenes , one methines (δH 1.74) one hydroxymethine (δH 4.55) and three hydroxy-methylenes . In addition, according to the HSQC data, the 13C NMR data showed the presence of 17 carbon signals, including two ester carbonyl carbons  and two olefinic carbons , one methyl, seven methylenes (three oxygenated), two methines (one oxygenated), two aliphatic quaternary carbons  at C-21 has significantly antimicrobial activity, followed by the aldehyde group (7), and hydroxymethyl (13) was the weakest. In-depth analysis showed that apart from the degree of oxidation at C-21, keto-enol tautomerism at the cyclopentane ring also had obvious influences on the antimicrobial activities of compounds. Compared to compounds 7 and 13 (enol form), compounds 8 and 9 (keto form) showed no activities against all strains (As for the study of the structure–activity relationship (SAR), it was found that the degree of oxidation at C-21 had different effects on the activities of the compounds. The compound with methyl  at room temperature. Optical rotations data were recorded on an MCP300 . UV were tested using a Shimadzu UV-2600 spectrophotometer . IR spectra were recorded on IR Affinity-1 spectrometer . HR-ESI-MS spectra were tested on a ThermoFisher LTQ-Orbitrap-LC-MS spectrometer . LC-MS/MS data was performed on a Q-TOF manufactured by Waters and a Waters Acquity UPLC BEH C18 column . Recoated silica gel plates , Column chromatography (CC) and Sephadex LH-20  were used to purify the compounds. Avicennia marina . It was identified as Penicillum sp. by the ITS region , and fungus N-5 was deposited at Sun Yat-sen University, China.Fungus N-5 was isolated from the rhizosphere soil of mangrove plant Penicillum sp. N-5 was cultured in one hundred 1000 mL Erlenmeyer flasks at 25 °C for 30 days; these contained autoclaved rice solid-substrate medium composed of 50 g rice and 50 mL 3‰ saline water. The fungus v/v) on silica gel CC and divided into six fractions (Fr.1–Fr.6). Fr. 2 (10 g) was separated to Sephadex LH-20 (methanol) to yield three sub-fractions (SFrs. 2.1–2.3). SFrs.2.3 (1.2 g) was applied to silica gel CC  and further purified by reversed-phase (RP) high performance liquid chromatography  to obtain compounds 1 (5 mg) and 3 (7 mg). Fr. 3 (16 g) was also separated to Sephadex LH-20 (methanol) to yield four sub-fractions (SFrs. 3.1–3.4). SFrs.3.1 (1.6 g) was separated to silica gel CC  and further purified by reversed-phase (RP) high performance liquid chromatography  to yield compounds 2 (11 mg) and 15 (6 mg). After incubation, the mycelia and solid rice medium were extracted four times with EtOAc, and 75 g of residue was obtained. Next, the residue was separated by a gradient of petroleum ether/EtOAc from 9:1 to 0:10 (1): white powder, m.p. 122.8–124.1 °C; c 0.02, MeOH), UV (MeOH) λmax (log ε): 206 (2.52) (max (∆ε): 240 (+5.47), 301 (−1.14), 362 (+0.61); 1H  and 13C NMR  data, see m/z 459.23709 [M+H]+ .Hemiacetalmeroterpenoid A (6 (2.52) ; ECD (Me2): white powder, m.p. 106.9–108.4 °C; c 0.02, MeOH), UV (MeOH) λmax (log ε): 205 (2.83), 238 (1.36) (max (∆ε): 206 (−11.92), 248 (+3.38), 311 (−1.19); 1H  and 13C NMR  data, see m/z 445.25772 [M+H]+ .Hemiacetalmeroterpenoid B (8 (1.36) ; ECD (Me3): white powder, m.p. 100.8–102.6 °C; c 0.02, MeOH), UV (MeOH) λmax (log ε): 205 (2.51), 260 (1.78) (max (∆ε): 206 (−18.31), 240 (+12.72), 310 (−2.18); 1H  and 13C NMR  data, see m/z 509.25015 [M+Na]+ .Hemiacetalmeroterpenoid C (0 (1.78) ; ECD (Me14): yellow powder, m.p. 202.9–204.5 °C; c 0.02, MeOH), UV (MeOH) λmax (log ε): 216 (3.15) (max (∆ε): 203 (−1.82), 225 (+3.27); 1H  and 13C NMR  data; HR-ESI-MS: m/z 305.13565 [M+Na]+ .Astellolide J (6 (3.15) ; ECD (Me15): yellow powder, m.p. 160.1–162.2 °C; c 0.02, MeOH), UV (MeOH) λmax (log ε): 218 (3.62) (max (∆ε): 232 (+5.19); 1H  and 13C NMR  data, see m/z 347.14578 [M+Na]+ .Astellolide Q (8 (3.62) ; ECD  were selected for optimization and calculation in methanol at B3LYP/6-31+G . Finally, the ECD spectra were generated by the program SpecDis 1.6  and drawn by OriginPro 8.0  from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.30 eV [Firstly, ECD calculations of compounds  0.30 eV ,30.Antimicrobial activity assay was performed as previously described in ,32.1–3), one new drimane sesquiterpenoid (15) and one sesquiterpenoid J (14) that was first isolated from a natural source, together with ten known compounds (4–13) were isolated from the cultures of the rhizosphere soil of mangrove plant Avicennia marina fungus Penicillium sp. N-5. Their structures were determined by the analysis of NMR, HR-MS and ECD spectra. All the isolated compounds were investigated for their antimicrobial activities against two phytopathogenic fungi and four bacterial strains. Among them, compounds 1, 5 and 10 exhibited significant inhibition against Penicillium italicum and Colletrichum gloeosporioides with MIC values of 6.25, 1.56, 6.25 and 6.25, 3.13, 6.25 μg/mL. Notably, compound 5 showed potential antimicrobial activity against all the strains and the MIC values were lower than 25 μg/mL. Moreover, andrastin-type meroterpenoid antimicrobial activity against phytopathogenic fungi was reported for the first time.In summary, three new andrastin-type meroterpenoids (


The mineralocorticoid receptor (MR) belongs to the steroid receptor subfamily of nuclear receptors. MR is a transcription factor key in regulating blood pressure and mineral homeostasis. In addition, it plays an important role in a broad range of biological and pathological conditions, greatly expanding its interest as a pharmacological target. Non-steroidal MR antagonists (MRAs) are of particular interest to avoid side effects and achieve tissue-specific modulation of the receptor. The 1,4-dihydropyridine  ring has been identified as an appropriate scaffold to develop non-steroidal MRAs. We report the identification of a novel series of 1,4-DHP that has been guided by structure-based drug design, focusing on the less explored DHP position 2. Interestingly, substituents at this position might interfere with MR helix H12 disposition, which is essential for the recruitment of co-regulators. Several of the newly synthesized 1,4-DHPs show interesting properties as MRAs and have a good selectivity profile. These 1,4-DHPs promote MR nuclear translocation with less efficiency than the natural agonist aldosterone, which explains, at least in part, its antagonist character. Molecular dynamic studies are suggestive of several derivatives interfering with the disposition of H12 in the agonist-associated conformation, and thus, they might stabilize an MR conformation unable to recruit co-activators. The mineralocorticoid receptor (MR) is a ligand-activated transcription factor mediating the biological effects of aldosterone, a key factor in regulating mineral homeostasis, acid-base balance, and blood pressure ,2,3. In + secretion, potentially leading to hyperkalemia  (2 groups are within a suitable distance to establish an extra hydrogen bond with the CO group of the Asn770 side chain (1 = H), which fits nicely within the orthosteric site. On the other hand, bulkier amino acids led to steric clashes. Although D-amino acids with small substituents at R1 might also be accommodated in the binding pocket, the amino acid side chains did not superimpose with the steroid ring.The analysis of the molecular docking poses showed a similar binding mode as that previously reported for other 1,4-DHPs ,30,33. AH(CH3)2] , are parde chain . The lat2 moiety at position 2 is close to the T945 residue of MR  2-OH, COCH2-COOH, and CO(CH2)2-COOH, together with amino acids Asp and Glu, were explored. The analysis of this second library showed that derivatives shown in 2 = CH2-COOH depicted in 2 and the side chain oxygen of T945 besides the Glu chiral center might allow the separation of both DHP diastereoisomers.A close inspection of the IFD pose of these 1,4-DHPs showed that the α-NHue of MR B. Theref(R1 = H) . As R2 g1 = H, CH3 and CH(CH3)2, respectively, 2 selected from the 2nd library 2 . However, only 12ab (1:1 diastereomeric mixture) could be resolved by semipreparative HPLC.The IFD studies on the 1st library also showed an interest in small hydrophobic substituent at the 2-methylene of CH(CH3)2 A. As ind11, bulkier esters were incorporated at position 3, in particular, ethyl (14) and tert-butyl (15), a 4-cyano-2-methoxi-phenyl moiety at position 4 (16), and a carboxamide at position 5 (17). As shown in 7–10.In addition to the compounds selected from the libraries, it would be of interest to explore substituents that have led to improvement in affinity in other 1,4-DHP derivatives ,29. Ther3 for 1–3 days. The need for further amounts of DHP 18 to prepare derivatives from the 2nd library motivates further studies aimed at optimizing the synthetic methodology. As the solvent CDCl3 usually has HCl traces, we envisaged that acid catalysis might improve the interconversion of DHP isomers. Consequently, the preparation of 11 (precursor of 18) using CH3CN/HCl in the last step led, after just 30 min, to this 1,4-DHP with a considerable increase in the yield, from 28% to 55%  in a cell-based assay. In 23b, were able to interact with MR, thus validating the theoretical approach used to design these new compounds.The synthesized 1,4-DHP derivatives were initially evaluated for their ability to displace [3H]-aldosterone assay to calculate inhibitory constants (Ki) for a selection of 1,4-DHP derivatives. To that end, we used a constant concentration of [3H]-aldosterone (1 nM) and increasing concentrations of each derivative. Curves obtained with this procedure are shown in We next took advantage of the methyl-4-(2-chloro-4-fluoro)phenyl-5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate (11). From methyl-4-[(benzyloxycarbonyl)amino]-3-oxo-butanoate, 2-chloro-4-fluorobenzaldehyde and 3-aminocrotonate. Method A: 1 min (40 °C), 5 min (100 °C), 1 day (CHCl3). Yield: 26%. Syrup. Eluent: Hexane:EtOAc (2:1). HPLC-MS: Gradient from 30 to 95% ACN/H2O (0.05% TFA) in 10 min, tR: 8.65, (m/z: 484.16 M+H+). HRMS (ESI pos) m/z Calculated C25H23ClFN3O4 483.13611, found 483.13436 (−3.63 ppm). 1H NMR : δ 7.37 , 7.19 , 7.08 , 6.91 , 5.79 , 5.17 , 5.14 , 4.32 , 3.97 , 2.02 , 1.07 . 13C NMR : δ 166.7 (3-CO), 161.4 , 158.5 (Cbz CO), 146.5 (2-C), 145.4 (6-C), 139.3 , 136.0 (Cbz Ph C), 133.1 , 131.5 , 128.8, 128.6 128.2 (Cbz Ph CH), 118.9 (CN), 116.8 , 114.8 , 101.8 (3-C), 86.0 (5-C), 67.7 (Cbz CH2), 60.6 (Et 1-C), 41.2 (2-CH2), 37.4 (4-C), 18.4 (6-CH3), 14.0 (Et 2-C).Ethyl-2-[(benzyloxycarbonyl)aminomethyl]-4-(2-chloro-4-fluoro)phenyl-5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate (14). From ethyl 4-[(benzyloxycarbonyl)amino]-3-oxo-butanoate, 2-chloro-4-fluorobenzaldehyde and 3-aminocrotonate. Method A: 5 min (40 °C), 15 min (100 °C), 3 days (CHCl3). Yield 29%. Method B. Yield 68%. Syrup. Eluent: Hexane:EtOAc (2:3). HPLC_MS: Gradient from 30 to 95% ACN/H2O (0.05% TFA) in 10 min, tR = 7.21, (m/z: 473.46 M+H+). HRMS (ESI pos) m/z Calculated C26H24N4O5 472.17467, found 472.17422 (−0.96 ppm). 1H NMR : δ 7.43–7.31 , 7.19–7.10 , 7.09 , 5.74 , 5.15 , 5.07 , 4.37 , 4.35 , 3.85 , 3.53 , 2.01 . 13C NMR : δ 167.2 (3-CO), 158.5 (Cbz CO), 156.7 (Ph 2-C), 147.4 (2-C), 146.2 (6-C), 138.6 (Ph C), 136.0 (Cbz Ph C), 129.7 (Ph 2-C), 128.8, 128.7, 128.3 (Cbz Ph CH), 125.1 (Ph 3-C), 119.1 (Ph CN), 119.0 (5-CN), 114.2 (Ph 3-C), 111.8 (Ph 4-C), 100.1 (3-C), 85.3 (5-C), 67.7 (Cbz CH2), 56.0 (Ph OCH3), 51.7 (COOCH3), 41.2 (2-CH2), 34.9 (4-H), 18.5 (2-CH3).Methyl 2-[(benzyloxycarbonyl)amino]methyl-5-cyano-4-(4-cyano-2-methoxy)phenyl-6-methyl-1,4-dihydropyridine-3-carboxylate (16). From methyl 4-[(benzyloxycarbonyl)amino]-3-oxo-butanoate, 2-chloro-4-fluorobenzaldehyde and 3-aminocrotonate. Method A: 1 min (40 °C), 4 min (100 °C) and 1 day , it is added at 0 °C propylene oxide  and the corresponding acid chloride (0.15 mmol). After 18 h of stirring at room temperature, the solvent was evaporated to dryness, and the resulting residue was purified in a silica gel column chromatography using the eluent system indicated for each product. Method B. To a solution of the corresponding amine (0.1 mmol) in dry THF (2 mL) it is added the corresponding acid (0.2 mmol), PyBOP , and TEA . After 18 h of stirring at room temperature, the solvent was evaporated to dryness, and the residue was dissolved in AcOEt. The organic layer was washed with 10% citric acid, 10% NaHCO3, saturated NaCl, and dried with Na2SO4. The solution was filtered and concentrated. The product was purified by column chromatography using the eluent system indicated in each product.Method A. To a solution of the corresponding amine (0.1 mmol) in CH18 and acetyl chloride. Method A: Yield 43%. Syrup. Eluent: Hexane:EtOAc (5:1). HPLC-MS: Gradient from 15 to 95% ACN/H2O (0.05% TFA) in 15 min, tR = 7.45, (m/z: 378.50 M+H+). HRMS (ESI pos) m/z Calculated C18H17ClFN3O3 377.09425 found 377.09292 (−3.52 ppm). 1H-RMN : δ 7.86  7.20 , 7.08 , 6.93 , 6.61 , 5.14 , 4.43 , 4.34 , 3.55 , 2.06 , 2.04 . 13C-RMN : δ 172.7, 167.5 (CO), 161.4 , 146.8, 145.7 , 139.3 , 133.1 , 131.6 , 119.0 (CN), 116.9 , 114.9 , 101.5 (3-C), 85.9 (5-C), 51.6 (OCH3), 39.9 (2-CH2), 37.5 (4-C), 23.1 (COCH3), 18.5 (6-CH3).Methyl 2-(acetamido)methyl-4-(2-chloro-4-fluoro)phenyl-5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate (19). From 2O (0.05% TFA) in 10 min, tR = 9.63, (m/z: 476.57 M+H+). HRMS (ESI neg) m/z Calculated C23H25ClFN3O5 447.14668, found 447.14465 (−4.24 ppm). 1H NMR : δ 7.74 , 7.22 , 7.07 , 6.93 , 5.16 , 4.56 , 4.50 , 3.57 , 3.29 , 2.06 , 1.47 . 13C NMR : δ 168.4, 168.2, 167.2 (CO), 161.4 , 146.1, 145.6 , 139.3 , 133.1 , 131.6 , 119.0 (CN), 116.9 , 114.9 , 101.6 (3-C), 86.0, 83.4 , 51.6 (OCH3), 42.3), 39.7 , 37.5 (4-C), 28.1 (tBu CH3), 18.5 (6-CH3).Methyl 2-methyl-4-(2-chloro-4-fluoro)phenyl-5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate (20). From 18 and mono-tert-butyl malonate. Method B: Yield 64%. Syrup. Eluent: Hexane:EtOAc (1:1). HPLC-MS: Gradient from 15 to 95% ACN/H22ab and 21, see For experimental details, description, and characterization of derivatives 2-terminus to the enhanced green fluorescent protein (eGFP) by subcloning in peGFP-C1  by Ouvrard-Pascaud et al. -aldosterone  for one additional hour. Cells were then washed twice with phosphate-buffered saline  and then extracted with 200 μL of an ethanol:water mix . Disintegrations were recorded using liquid scintillation in a β counter. Control conditions included non-transfected cells, transfected cells treated with unlabeled aldosterone or transfected cells, treated with 1 nM [3H]-aldosterone in the presence of 1 × 10−5 M unlabeled aldosterone. Specific binding was determined by subtracting the number of disintegrations per minute (dpm) in the presence of 1 × 10−5 M unlabeled aldosterone from the condition with 1 nM [3H]-aldosterone. Each condition was performed in triplicate. Competition of each compound for 1 nM [3H]-aldosterone binding was calculated as the percentage of the maximum binding obtained in the absence of compound. Inhibition constants (Ki) were calculated from the percentage competition of 1 × 10−9 M [3H]-aldosterone binding in the presence of eight difference compound concentrations . Non-linear regression was performed using Prism 5 (GraphPad) using a model with one binding site and assuming an aldosterone Kd of 1 × 10−9 M [Ligand-receptor binding assays were performed as described before ,61. Brie× 10−9 M , with th−5 M. After 2 h of incubation, cells were further treated with agonists  in the presence of each compound for 16 h. Assays using hMR included a control condition with 0.25 × 10−6 M eplerenone. MR maximum and minimum activity were obtained in each assay using conditions with 1 × 10−9 M aldosterone or vehicle (ethanol). Each condition was performed in three parallel wells. After treatments, firefly and Renilla luciferase activities were measured sequentially in the cell lysates using a commercially available kit . Receptor transcriptional activity was calculated as the ratio luc/ren. The activities in each condition were normalized to the activity obtained with vehicle or with agonist as indicated in each figure panel and are expressed a as percentage of the maximum value ± SD.Receptor transcriptional activity was assessed using a transactivation gene reporter assay, as described . Briefly2/chamber) in DMEM supplemented with 10% charcoal-stripped FBS. Twenty-four hours after seeding, cells were transfected with peGFP-hMR. Twenty-four hours after transfection, cells were treated with each compound at 1 × 10−5 M in the presence or absence of 1 × 10−8 M aldosterone and incubated for the indicated periods of time. Cells were then washed once with PBS and fixed 4% formaldehyde in PBS for 5 min. After fixation, cells were washed three times with PBS, the walls of the culture chambers were eliminated, and cells were mounted with a coverslip, and mounting medium  supplemented with 0.1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) to provide nuclear counterstaining. Cell preparations were then examined under a Leica SP8 confocal microscope using a 63x magnification, oil-immersion objective.COS-7 cells were seeded on glass chamber slides divided into 8 culture chambers  from an average of at least two experiments.Activity on nuclear receptors has been evaluated using a recruitment coactivator, AlphaScreen, assay following the manufacturer’s instructions. Briefly, the assays were performed in white low-volume, 384-well ProxiPlates  using a final volume of 15 μL containing the GST-NR-LBD protein and its biotinylated coactivator peptide at specific, previously optimized concentrations, for the determination of a dose-response curve for test compounds over 1 h at room temperature. In the antagonist mode, the stimulation with the compounds was done for 1 h in the presence of a fixed concentration of a reference compound at its EC80 concentration. The plate was read in EnVision instrument  after a 4 h incubation with 20 μg/mL of donor and acceptor beads. EC


Pulicaria dysenterica, in the present study, the antimicrobial potential of the extracted essential oil was investigated against a panel of eighteen microorganism strains. Additionally, anti-acetylcholinesterase and antispasmodic  activities, general acute toxicity , and immunomodulatory properties (cytotoxicity on isolated mouse macrophages) were studied. Detailed analyses of the essential oil led to the identification of 3-methoxycuminyl 2-methylbutanoate  and 3-methoxycuminyl 3-methylbutanoate . The obtained esters and intermediates in the synthesis of the starting alcohol (3-methoxycuminol) were subjected to a battery of 1D- and 2D-NMR experiments. The synthesized esters were additionally characterized by GC–MS, IR, and UV–Vis. The synthesized compounds  were biologically tested in the same way as the extracted P. dysenterica essential oil. The obtained low acute toxicity and promising antimicrobial potential suggest that the P. dysenterica essential oil might partially explain the ethnopharmacological application of P. dysenterica plant material for the treatment of gastrointestinal infections.Motivated by the ethnopharmacological use of  Artemisia annua , 192(2), 179(3), 178(23), 177(5), 176(48), 162(4), 160(8), 158(13), 151(10), 150(4), 149(25), 148(68), 147(15), 146(9), 145(8), 136(4), 135(39), 134(14), 133(39), 132(32), 131(15), 130(24), 121(8), 120(14), 119(11), 118(12), 117(19), 116(19), 115(77), 108(4), 107(36), 106(19), 105(21), 104(17), 103(45), 102(19), 101(4), 95(7), 94(11), 93(10), 92(14), 91(100), 90(8), 89(15), 87(4), 79(13), 78(21), 77(92), 76(14), 75(18), 74(14), 65(26), 64(5), 63(22), 62(8), 59(7), 53(6), 52(7), 51(31), 50(14), 43(48), 41(16), 39(20); analyzed C 62.20, H 5.73, N 7.23, O 24.84%, calculated for C10H11NO3, C 62.17, H 5.74, N 7.25, O 24.84%; 1H NMR (CDCl3) δ 1.35 , 3.47 , 7.70 , 8.07 , 8.20 , 10.04 ; 13C NMR (CDCl3) δ 23.4 , 29.1 (C-8), 125.1 (C-2), 128.7 (C-5), 132.4 (C-6), 134.9 (C-1), 148.9 (C-4), 150.1 (C-3), 189.6 (C-7).2; 12 g, 62.18 mmol) and sodium borohydride  in an anhydrous methanol/tetrahydrofuran mixture  was stirred at 0 °C for 30 min and additional 2 h at room temperature. A saturated solution of NaHCO3 (100 mL) was added, and the mixture was stirred for 10 min. The reaction mixture was extracted with diethyl ether (4 × 100 mL), followed by a usual work-up (drying with MgSO4 and solvent evaporation), and it yielded 11.88 g (60.93 mmol) of the pure 3-nitrocuminol . The yield of 3-nitrocuminol (3) was 98%. The spectral data of 3 are given below:A mixture of 3-nitrocuminaldehyde (4-Isopropyl-3-nitrophenyl)methanol (: retention index (RI) = 1685 (DB-5MS column); UV (CH3CN) λmax(log ε) 292 (3.53), 242 (3.99), 206 (4.47) nm; FTIR  3338, 2968, 2872, 1622, 1567, 1523, 1463, 1386, 1352, 1202, 1135, 1104, 1054, 887, 833, 808, 765, 675; MS (EI), m/z (%) 195(2) [M+], 194(1), 180(10), 179(5), 178(65), 160(21), 153(3), 152(5), 150(26), 149(8), 148(43), 144(4), 137(17), 136(9), 135(13), 134(28), 133(15), 132(7), 130(22), 128(9), 121(8), 120(12), 118(12), 117(35), 116(15), 115(56), 109(4), 108(6), 107(43), 106(27), 105(27), 104(4), 103(28), 102(10), 94(11), 93(14), 92(14), 91(98), 90(11), 89(25), 87(4), 79(56), 78(25), 77(100), 76(6), 75(4), 74(5), 65(20), 64(5), 63(19), 62(6), 59(4), 57(7), 55(9), 53(13), 52(9), 51(28), 50(9), 44(5), 43(52), 41(22), 39(24); analyzed C 61.55, H 6.70, N 7.20, O 24.55%, calculated for C10H13NO3, C 61.53, H 6.71, N 7.18, O 24.58%; 1H NMR (CDCl3) δ 1.29 , 3.39 , 4.33 , 4.73 , 7.46 , 7.53 , 7.70 ; 13C NMR (CDCl3) δ 23.6 , 28.4 (C-8), 63.8 (C-7), 121.9 (C-2), 127.8 (C-5), 130.7 (C-6), 139.8 (C-1), 141.6 (C-4), 149.7 (C-3).3; 11.5 g, 58.97 mmol) and 5% Pd/C (1 g) in anhydrous ethyl acetate (50 mL) was stirred under hydrogen (atmospheric pressure) at room temperature for a duration of 6 h. After the completion of the reaction (monitored by TLC and GC–MS), the mixture was filtered and concentrated under reduced pressure. Crude 3-aminocuminol (4) was purified by dry-flash chromatography on silica gel using n-hexane/Et2O mixtures of increasing polarity as the eluents. The yield of 3-aminocuminol ) was 97%. The spectral data of 4 are given below:A solution of 3-nitrocuminol (3-Amino-4-isopropylphenyl)methanol (: retention index (RI) = 1587 (DB-5MS column); UV (CH3CN) λmax(log ε) 292 (3.46), 241 (3.91), 207 (4.51) nm; FTIR  3378, 3185, 2959, 2928, 2867, 2838, 1621, 1577, 1508, 1447, 1425, 1381, 1367, 1311, 1284, 1253, 1227, 1160, 1060, 1045, 988, 955, 921, 889, 853, 798, 730; MS (EI), m/z (%) 166(4), 165(36) [M+], 151(10), 150(100), 134(3), 133(3), 132(8), 130(3), 122(3), 121(3), 120(14), 118(5), 117(5), 115(5), 106(9), 105(8), 104(3), 103(6), 94(9), 93(6), 91(8), 79(5), 78(3), 77(13), 65(5), 59(4), 51(3), 41(3), 39(4); analyzed C 72.71, H 9.12, N 8.49, O 9.68%, calculated for C10H15NO, C 72.69, H 9.15, N 8.48, O 9.68%; 1H NMR (CDCl3) δ 1.24 , 2.87 , 3.14 , 4.54 , 6.66 , 6.75 , 7.11 ; 13C NMR (CDCl3) δ 22.3 , 27.5 (C-8), 65.1 (C-7), 114.5 (C-2), 117.7 (C-6), 125.6 (C-5), 132.2 (C-4), 139.4 (C-1), 143.4 (C-3).4) were dissolved in a solution of concentrated sulfuric acid (12 mL) in 30 mL of water at 0 °C with efficient stirring. After 15 min, an aqueous solution of sodium nitrite (3.76 g (54.55 mmol) of NaNO2 dissolved in 10 mL of water) was dropwise added to this mixture . The solution was stirred for 2 h at room temperature and extracted three times with Et2O. The organic layers were combined, dried over anhydrous MgSO4, and concentrated under reduced pressure. Crude 3-hydroxycuminalcohol (5) was purified by dry-flash chromatography on silica gel using an n-hexane/Et2O mixture. The yield of 3-hydroxycuminol ) was 67%. The spectral data of 5 are given below:Nine grams (54.55 mmol) of 3-aminocuminol 5-(Hydroxymethyl)-2-isopropylphenol (: retention index (RI) = 1563 (DB-5MS column); UV (CH3CN) λmax(log ε) 282 (3.50), 276 (3.49), 218 (3.94), 202 (4.22) nm; FTIR  3271, 2960, 2870, 1616, 1585, 1504, 1424, 1382, 1362, 1288, 1236, 1193, 1152, 1112, 1087, 1060, 1002, 939, 863, 817, 755, 739, 710; MS (EI), m/z (%) 167(4), 166(37) [M+], 152(10), 151(100), 135(4), 133(10), 123(4), 121(22), 115(6), 107(7), 105(10), 103(10), 95(11), 93(4), 91(12), 79(8), 78(3), 77(17), 65(5), 53(3), 51(4), 41(3), 39(4); analyzed C 72.31, H 8.45, O 19.24%, calculated for C10H14O2, C 72.26, H 8.49, O 19.25%; 1H NMR (CDCl3) δ 1.21 , 3.22 , 4.27 , 4.54 , 6.78–6.81 , 7.14 ; 13C NMR (CDCl3) δ 22.6 , 26.8 (C-8), 65.0 (C-7), 114.2 (C-6), 119.3 (C-2), 126.5 (C-5), 134.5 (C-1), 138.9 (C-4), 153.3 (C-3).5; 5 g, 30.12 mmol) was added to a suspension of anhydrous potassium carbonate  in acetone (50 mL). Then, methyl iodide  was added, and the solution was heated for 4 h under reflux. After that, another portion of methyl iodide  was added, and the solution was stirred at room temperature for another 24 h and concentrated in vacuo. The residue was dissolved in water (50 mL) and extracted three times with Et2O. The combined organic extracts were dried with anhydrous MgSO4 and concentrated under reduced pressure. Crude 3-methoxycuminol (6) was purified by dry-flash chromatography on silica gel using n-hexane/Et2O mixtures of increasing polarity as the eluents. The yield of 3-methoxycuminol ) was 96%. The spectral data of 6 are given below:3-Hydroxycuminol (4-Isopropyl-3-methoxyphenyl)methanol (: retention index (RI) = 1492 (DB-5MS column); UV (CH3CN) λmax(log ε) 318 (3.12), 281 (3.63), 275 (3.62), 223 (4.13), 203 (4.46) nm; FTIR  3310, 2958, 2869, 1612, 1579, 1505, 1462, 1416, 1382, 1360, 1287, 1254, 1191, 1160, 1093, 1061, 1040, 921, 854, 818, 733; MS (EI), m/z (%) 181(3), 180(28) [M+], 166(11), 165(100), 149(4), 147(3), 135(9), 121(5), 117(7), 115(7), 109(4), 107(4), 105(21), 103(7), 91(17), 79(11), 78(4), 77(15), 65(5), 53(3), 51(4), 41(4), 39(4); analyzed C 73.28, H 8.96, O 17.76%, calculated for C11H16O2, C 73.30, H 8.95, O 17.75%; 1H NMR (CDCl3) δ 1.20 , 2.00 , 3.30 * , 3.83 , 4.63 * , 6.8745 * , 6.8877 * , 7.1787 * ; 13C NMR (CDCl3) δ 22.7 , 26.5 (C-8), 55.4 (C-11), 65.4 (C-7), 109.1 (C-2), 119.0 (C-6), 126.1 (C-5), 136.5 (C-1), 139.4 (C-4), 156.9 (C-3). * The values of chemical shift and coupling constants were determined by a simulation of the 1H NMR spectrum .6) with isobutanoic (7), butanoic (8), 2-methylbutanoic (9), 3-methylbutanoic (10), and pentanoic (11) acids were prepared according to the general Steglich approach /4-(dimethylamino)pyridine (DMAP)). A solution of 3-methoxycuminol , the appropriate carboxylic acid (2.3 mmol), DMAP , and DCC  in 30 mL of dry CH2Cl2 was stirred overnight at room temperature. Then, the precipitated urea was filtered off and the filtrate was concentrated in vacuo. The resulting residue was purified by dry-flash chromatography on silica gel using an n-hexane/Et2O mixture  as the eluent. The spectral data 4-Isopropyl-3-methoxybenzyl isobutanoate  = 1725 (DB-5MS column); UV (CH3CN) λmax(log ε) 281 (3.71), 275 (3.72), 224 (4.25), 204 (4.57) nm; FTIR  2961, 2872, 1732, 1613, 1581, 1507, 1463, 1418, 1386, 1362, 1342, 1289, 1256, 1188, 1148, 1110, 1094, 1062, 1041, 965, 926, 852, 817, 758, 735; MS (EI), m/z (%) 251(8), 250(54) [M+], 236(14), 235(100), 181(8), 180(76), 179(6), 165(5), 164(5), 163(37), 162(3), 149(3), 148(13), 147(16), 137(25), 135(9), 133(7), 132(3), 131(7), 121(15), 119(5), 118(3), 117(16), 116(5), 115(16), 109(12), 105(9), 103(6), 91(15), 79(4), 78(3), 77(8), 71(11), 65(3), 55(3), 43(21), 41(8), 39(3); analyzed C 71.95, H 8.85, O 19.20%, calculated for C15H22O3, C 71.97, H 8.86, O 19.17%.8; 3-methoxycuminyl butanoate)4-Isopropyl-3-methoxybenzyl butanoate  = 1776 (DB-5MS column); UV (CH3CN) λmax(log ε) 281 (3.29), 275 (3.30), 224 (3.83), 201 (4.52) nm; FTIR  2961, 2873, 1733, 1613, 1581, 1507, 1461, 1418, 1382, 1349, 1288, 1256, 1165, 1094, 1062, 1040, 972, 921, 851, 817, 734; MS (EI), m/z (%) 251(7), 250(49) [M+], 236(14), 235(100), 181(8), 180(77), 179(3), 165(6), 164(3), 163(19), 162(3), 149(3), 148(9), 147(15), 137(23), 135(5), 133(6), 132(3), 131(5), 121(10), 119(5), 118(3), 117(13), 116(4), 115(13), 109(8), 105(7), 103(5), 91(12), 79(3), 78(3), 77(7), 71(12), 65(3), 43(13), 41(6), 39(3); analyzed C 71.96, H 8.84, O 19.20%, calculated for C15H22O3, C 71.97, H 8.86, O 19.17%; 1H NMR (CDCl3) δ 0.95 , 1.20 , 1.68 , 2.34 , 3.30 , 3.83 , 5.08 , 6.83 , 6.92 , 7.19 ; 13C NMR (CDCl3) δ 13.7 (C-15), 18.5 (C-14), 22.6 , 26.6 (C-8), 36.3 (C-13), 55.4 (C-11), 66.2 (C-7), 110.3 (C-2), 120.5 (C-6), 126.1 (C-5), 134.5 (C-1), 137.1 (C-4), 156.9 (C-3), 173.6 (C-12).9; 3-methoxycuminyl 2-methylbutanoate)4-Isopropyl-3-methoxybenzyl 2-methylbutanoate  = 1808 (DB-5MS column); UV (CH3CN) λmax(log ε) 281 (3.40), 275 (3.42), 224 (3.95), 200 (4.72) nm; FTIR  2962, 2936, 2874, 1731, 1613, 1581, 1507, 1461, 1418, 1382, 1350, 1289, 1257, 1178, 1144, 1118, 1094, 1062, 1041, 1013, 957, 850, 817, 757, 735; MS (EI), m/z (%) 265(8), 264(47) [M+], 249(77), 181(11), 180(100), 179(6), 178(4), 165(5), 164(6), 163(42), 162(3), 149(3), 148(11), 147(13), 137(24), 135(7), 133(6), 131(6), 121(15), 119(4), 118(3), 117(14), 116(5), 115(14), 109(11), 105(8), 103(5), 91(13), 85(6), 79(4), 78(3), 77(7), 57(23), 55(4), 41(8), 39(3); analyzed C 71.93, H 8.88, O 19.19%, calculated for C15H22O3, C 71.97, H 8.86, O 19.17%.10; 3-methoxycuminyl 3-methylbutanoate)4-Isopropyl-3-methoxybenzyl 3-methylbutanoate  = 1817 (DB-5MS column); UV (CH3CN) λmax(log ε) 281 (3.00), 275 (3.02), 224 (3.54), 200 (4.32) nm; FTIR  2958, 2871, 1732, 1613, 1581, 1507, 1463, 1418, 1371, 1350, 1291, 1255, 1182, 1164, 1117, 1093, 1062, 1041, 986, 926, 851, 816, 736; MS (EI), m/z (%) 265(7), 264(42) [M+], 250(12), 249(79), 181(11), 180(100), 179(3), 165(6), 164(5), 163(29), 162(3), 149(3), 148(9), 147(13), 137(26), 135(5), 133(6), 131(5), 121(11), 119(4), 118(3), 117(12), 116(4), 115(12), 109(8), 105(7), 103(5), 91(11), 85(9), 79(3), 78(3), 77(6), 57(12), 55(3), 43(5), 41(7), 39(3); analyzed C 71.97, H 8.82, O 19.21%, calculated for C15H22O3, C 71.97, H 8.86, O 19.17%.11; 3-methoxycuminyl pentanoate)4-Isopropyl-3-methoxybenzyl pentanoate  = 1863 (DB-5MS column); UV (CH3CN) λmax(log ε) 281 (3.25), 275 (3.26), 224 (3.79), 200 (4.56) nm; FTIR  2958, 2872, 1736, 1613, 1581, 1507, 1463, 1418, 1380, 1349, 1288, 1259, 1166, 1094, 1062, 1020, 851, 804; MS (EI), m/z (%) 265(7), 264(40) [M+], 250(13), 249(80), 181(12), 180(100), 179(3), 165(6), 164(5), 163(26), 162(4), 149(4), 148(11), 147(14), 137(28), 135(6), 133(7), 131(6), 121(13), 119(5), 118(3), 117(16), 116(5), 115(16), 109(9), 105(9), 103(6), 91(15), 85(13), 79(4), 78(3), 77(8), 57(16), 55(6), 43(3), 41(9), 39(3); analyzed C 71.95, H 8.86, O 19.19%, calculated for C15H22O3, C 71.97, H 8.86, O 19.17%; 1H NMR (CDCl3) δ 0.91 , 1.20 , 1.35 , 1.63 , 2.36 , 3.30 , 3.83 , 5.08 , 6.83 , 6.92 , 7.19 ; 13C NMR (CDCl3) δ 13.7 (C-16), 22.3 (C-15), 22.6 , 26.6 (C-8), 27.1 (C-14), 34.1 (C-13), 55.4 (C-11), 66.3 (C-7), 110.3 (C-2), 120.5 (C-6), 126.1 (C-5), 134.5 (C-1), 137.1 (C-4), 156.9 (C-3), 173.8 (C-12).Disease-free male Wistar rats (300–350 g) were obtained from the Vivarium of the Scientific Research Center for Biomedicine, Faculty of Medicine, University of Niš, Serbia. The animals were maintained under standard husbandry conditions with a temperature of 23 ± 2 °C, relative humidity of 55 ± 10%, and 12/12 h light/dark cycle. All animals were fed with commercially available standard laboratory food pellets, and water was provided ad libitum. The experiments were performed following the declaration of Helsinki and European Community guidelines for the ethical handling of laboratory animals , and the experimental protocols were commenced after being approved by the institutional animal ethics committee (No. 323-07-06862/2016-05/2).2, 1.80 mM CaCl2, 0.42 mM NaH2PO4, 11.90 mM NaHCO3, and 5.55 mM glucose, pH 7.4. The luminal contents were flushed out using the same solution, and the distal colon strips (approximately 1.0–1.5 cm in length) were longitudinally mounted in a 20 mL tissue bath containing Tyrode’s solution bubbled with a mixture containing 5% CO2 (v/v) in oxygen and maintained at 37 °C. One edge of the distal colon was anchored with a silk suture to the bottom of the organ bath, and the other edge was connected using a cotton thread to the isometric force transducer . The data were recorded and analyzed with PC Biodata-F software .After the animals were sacrificed, their abdomens were opened and the distal colon, a few centimeters from the anus, was dissected and placed in a Petri dish filled with Tyrode’s solution of the following composition: 136.75 mM NaCl, 2.68 mM KCl, 1.05 mM MgClEO) from 0.025 µg/mL to 0.25 mg/mL. The two samples of essential oil were of very similar composition, so they were pooled and used in the biological assays. Due to the poor solubility of the essential oil in Tyrode’s solution, higher concentrations (0.25 mg/mL) were not tested. The distal colon strip was exposed to each EO concentration for 5 min, after which the tissue segments were washed with fresh Tyrode’s solution and left to stabilize for 10 min before being exposed to the corresponding EO concentration. Different EO concentrations were tested in parallel using two segments of the distal colon, and the experiments were repeated four times on distal colon segments obtained from different animals.After a stabilization period of 45 min, the distal colon tissue was exposed to increasing concentrations of the essential-oil sample (EO), the maximal and minimal amplitudes were measured during 5 min of exposure to the EO sample. The change in the amplitude of distal colon contractions, relative to the one measured in the period before the addition of the test compounds, was expressed as a percentage and used to calculate EC values. The number of contractions was counted before the addition of the EO samples or papaverine (positive control). For each of the tested concentrations of the EO, the number of contractions was counted during each minute of a 5 min exposure period, and the obtained data were used to calculate the percentage of the increase or decrease in the number of distal colon contractions.For each tested concentration of the essential-oil sample , and synthesized compounds 2–11 were measured by a quantitative colorimetric assay based on Ellman’s method [EO per mL or 0.0095–5 mmol/L of compounds 1–11 in absolute methanol; ten different concentrations) were incubated for 20 min at 37 °C. After that, Ellman’s reagent  in buffer B ) and 25 µL of 15 mM acetylthiocholine iodide were added, and the absorbance at 405 nm was recorded every 15 s over 15 min. Absolute methanol was used as the negative control . For validation, different concentrations of rivastigmine served as a positive control. Each experiment was carried out in triplicate and repeated three times.The AChE inhibitory activities of the s method . BrieflyP. dysenterica and the synthesized compounds were tested against a panel of microbial strains belonging to the American Type Culture Collection reference strains; Gram-positive bacteria (Staphylococcus aureus (ATCC 6538), S. epidermidis (ATCC 12228), Bacillus cereus (ATCC 11778), and Kocuria rhizophila (formerly Sarcina lutea under the same ATCC number of ATCC 9341)), Gram-negative bacteria (Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Salmonella enterica subsp. enterica serovar Enteritidis (ATCC 13076) and Acinetobacter baumanii (ATCC 19606)), yeast Candida albicans (ATCC 10231) and mold Aspergillus brasiliensis (ATCC 16404). The testing was also performed against eight isolates of Salmonella spp. obtained from human stool samples. Bacterial strains were maintained on Nutrient Agar (NA) at 37 °C and fungal strains were maintained on Sabouraud Dextrose Agar (SDA) at 30 °C at the Microbiology Laboratory .The essential oil of v/v) did not affect bacterial or fungal growth. These solutions were further serially diluted (the diluting factor 2) in a concentration range of 0.01–4.00 g/L. After making the dilutions of the test substances, the inoculum was added to all wells and the plates were incubated at 37 °C for 24 h in the case of bacteria or at 30 °C for 48 h in the case of fungi. Streptomycin, chloramphenicol, and nystatin served as positive controls, and one non-inoculated well, free of any antimicrobial agent, was also included to ensure medium sterility. The bacterial growth was determined by adding 20 μL of a 0.5% triphenyltetrazolium chloride (TTC) aqueous solution. MIC was defined as the lowest concentration of the test compound that inhibited visible growth (red-colored pellet on the bottom of the wells after the addition of TTC). All experiments were performed in triplicate.Antimicrobial activity was evaluated using a broth microdilution method in microtiter plates, as described earlier . BrieflyArtemia salina (brine shrimp) cyst hatching used here was previously described by Radulović et al. [EO and synthesized compounds 5, 6, 7, 9, and 10) were as follows: 3.9, 7.8, 15.6, 31.3, 62.5, and 125 µg/mL. The final concentration of DMSO was much less than 1% (v/v). The tested samples were not aerated, and the test dishes were left at room temperature under constant illumination; brine shrimps were not fed during the test. Dead nauplii were counted after 24 and 48 h. Statistical analysis determined a concentration lethal to 50% of nauplii (LC50). Sodium dodecyl sulphate (SDS) was used as a positive control. DMSO was inactive under the stated conditions, as demonstrated by a negative control. All the tests were performed in triplicate and repeated twice.The method for ć et al. . The fin6 cells/mL, and their viability was confirmed using trypan blue staining (>95% of viable cells). These cells were further cultured in 96-well cell culture plates ; each well contained 100 µL of the suspension containing the RPMI medium. Control cells were cultured with 100 µL of RPMI per well. Dexamethasone (a steroid drug with anti-inflammatory and immunosuppressant effects) was used as the positive control at a final concentration of 1 × 10−4 M in the wells. The EO sample was assayed in five different concentrations from 100 to 0.001 µg/mL. The compounds were tested in doses from 10−4 to 10−8 mol/L. The plates were incubated at 37 °C for 24 h under an atmosphere of 95% air and 5% CO2 (v/v). All experiments were performed in quadruplicate and repeated three times.Animals were sacrificed and opened under sterile conditions. To obtain a single-cell suspension, the peritoneal cavity was washed with PBS. Suspensions of the rat peritoneal macrophages obtained after centrifugation at 1200 rpm for 10 min (at 4 °C) were re-suspended in an RPMI medium, cell density was adjusted to 2.5 × 10The mitochondrial-dependent reduction of MTT to formazan crystals was used to determine cell viability in cultures. The assay was performed 24 h after the incubation of macrophages with different concentrations of the oil or appropriate control. After the removal of the cell medium, 100 µL of a fresh RPMI medium and an MTT solution (5 mg/mL) were added, and the plates were incubated for an additional 4 h. Acidified isopropanol was added to all wells, and the plates were shaken to dissolve the dark blue crystals of the formazan. A few minutes after the dissolution of crystals, the absorbance was read at 550 nm  using anp) ≤ 0.05 were considered to be statistically significant.The results are expressed as the mean ± SD. Statistically significant differences between the treatments in in vitro assays conducted on isolated rat distal colon tissue and peritoneal macrophages were determined by a One-Way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons . Probability values , 2-methylbutanoate , and 3-methylbutanoate , it did not affect spontaneous distal colon contractions. Additionally, the oil and its constituents only exerted a high cytotoxic potential when cells were exposed to the highest tested concentrations; in the subsequently tested dilutions, the toxicity almost wholly disappeared.A sum of the organic synthesis and GC–MS, UV–Vis, FTIR, and 1D and 2D NMR analyses provide unequivocal proof that tial oil ). The heP. dysenterica can be considered a natural agent that can be further explored as a crop for treating digestive problems caused by some microorganisms. However, although we have provided new data regarding the phytochemistry and bioactivity of P. dysenterica’s essential oil and oil constituents, this is just a tiny piece of the whole picture. We focused our attention on the essential oil and several volatile metabolites. To confirm P. dysenterica as medicinal taxa and potential industrial crops, we need to provide answers about the non-volatile metabolites and their bioactivity/toxicity.Based on the present results, the essential oil of


Of the new derivatives, six compounds (1–6) were found to contain a γ-hydroxybutenolide moiety capable of reversible stereoinversion at the hydroxylated carbon center. Under the influence of other adjacent functional groups, each derivative exhibited a different stereochemical behavior, which was fully deduced by ROESY experiments. All the isolated compounds were examined for their cytotoxicity by MTS assay using staurosporine as a positive control , and they were found to show weak growth inhibitory activities against HeLa and MCF-7 cells, with a minimal IC50 value of 20.0 μΜ. The compounds containing a γ-hydroxybutenolide moiety  showed cytotoxicity, with IC50 values ranging from 24.3 to 29.9 μΜ, and the most potent derivative was heteronemin (16). Although the cytotoxicities of isolated compounds were insufficient to discuss the structure–activity relationship, this research could contribute to expanding the structural diversity of scalaranes and understanding the stereochemical behavior of γ-hydroxybutenolides.Eighteen scalarane sesterterpenoids ( Cacospongia scalaris  + .12β,20β-Dihydroxy-16α-methoxy-17-scalaren-19,20-olide 2); UV λmax (MC) (log ε) 235 (3.62) nm; IR (ATR) νmax: 3297, 2925, 2858, 1724, 1466, and 1021 cm−1; 1H and 13C NMR data in CDCl3, see m/z: 433.2928 [M + H]+ .12β,20α-Dihydroxy-16β-methoxy-17-scalaren-19,20-olide 3); UV λmax (MC) (log ε) 239 (0.56) nm; IR (ATR) νmax: 3406, 3218, 2918, 2858, 1721, 1452, 1374, and 1021 cm−1; 1H and 13C NMR data in C5D5N, see m/z: 441.2604 [M + Na]+ .12β,16β,20β-Trihydroxy-17-scalaren-19,20-olide 4); UV λmax (MC) (log ε) 231 (2.32) nm; IR (ATR) νmax: 3385, 2911, 2861, 1749, 1458, 1371, and 1021 cm−1; 1H and 13C NMR data in CDCl3, see m/z: 433.2961 [M + H]+ .12β,19α(β)-Dihydroxy-16α-methoxy-17-scalaren-19,20-olide 5); UV λmax (MC) (log ε) 238 (0.70) nm; IR (ATR) νmax: 3356, 2922, 2861, 1745, 1452, 1377, 1265, and 1021 cm−1; 1H and 13C NMR data in CDCl3, see m/z: 433.2961 [M + H]+ .12β,19α(β)-Dihydroxy-16β-methoxy-17-scalaren-19,20-olide 6); UV λmax (MC) (log ε) 249 (3.48) nm; IR (ATR) νmax: 3374, 2922, 2858, 1745, 1458, 1377, and 1028 cm−1; 1H and 13C NMR data in C5D5N, see m/z: 401.2695 [M + H]+ .12β,19α-Dihydroxy-14,15-dehydrate-17-scalaren-19,20-olide epi-carboxylic-12-epi-scalaral 7); UV λmax (MC) (log ε) 238 (0.53) nm; IR (ATR) νmax: 3413, 2925, 2858, 1710, 1458, 1381, 1261, 1017, and 734 cm−1; 1H and 13C NMR data in C5D5N, see m/z: 425.2649 [M + Na]+ .12-Deacetyl-18-O-Deacetyl-12,16-di-epi-norscalaral B 8); UV λmax (MC) (log ε) 231 (2.37) nm; IR (ATR) νmax: 3470, 3127, 2925, 2855, 1717, 1458, 1377, 1303, 1265, 1027, and 735 cm−1; 1H and 13C NMR data in C5D5N, see m/z: 413.2667 [M + Na]+ .2-® AQueous One Solution Cell Proliferation Assay . For this purpose, HeLa and MCF-7 cells were plated in 384-well clear plates at densities of 3700 cells/well and 2000 cells/well, respectively. The seeded cells were incubated for 24 h at 37 °C in 5% (v/v) CO2 and then treated with the compounds at seven different final concentrations  for 48 h. DMSO was used as the vehicle control, and staurosporine was used as a positive control. The viable cell numbers were determined based on the concentration of formazan resulting from tetrazolium conversion, and the absorbance was measured at 490 nm using an EnVision Xcite Multilabel Reader .The MTS assay was performed using the CellTiter 96


In the structure of 1,2,3-I3-1,2-C2B10H9, an iodine atom attached to the boron atom (position 3) donates its lone pairs simultaneously to the σ-holes of both iodine atoms attached to the carbon atoms (positions 1 and 2) with the I⋯I distance of 3.554(2) Å and the C-I⋯I and B-I⋯I angles of 169.2(2)° and 92.2(2)°, respectively. The structure is additionally stabilized by a few B-H⋯I-shortened contacts. In the structure of 8,9,12-I3-1,2-C2B10H9, the I⋯I contacts of type II are very weak (the I⋯I distance is 4.268(4) Å, the B8-I8⋯I12 and B12-I12⋯I8 angles are 130.2(3)° and 92.2(3)°) and can only be regarded as dihalogen bonds formally. In comparison with the latter, the structure of 8,9,12-Br3-1,2-C2B10H9 demonstrates both similarities and differences. No Br⋯Br contacts of type II are observed, while there are two Br⋯Br halogen bonds of type I.The crystal structures of two isomeric triiodo derivatives of  The ability of halogens to form complexes with various electron pair donors was discovered over two hundred years ago ,2,3, andThis progress has been largely due to a better understanding of the principles on which the strength of the halogen bond depends. The performance of the halogen bond largely depends on the degree of polarization of the halogen atom; that is, the greater the positive electrostatic potential of the σ-hole, the more efficient the halogen bond donor will be ,11,12. Tsp hybridization of carbon atoms bearing a halogen is favored over 2sp followed by 3sp hybridization . Th. Th2B10Hortho-carborane containing substituents in the three most electron-withdrawing positions of the carborane cage  and the three most electron-donating positions ; in addition, a comparative analysis of the crystal packings of the 8,9,12-triiodo and 8,9,12-tribromo derivatives of ortho-carborane was performed.In this contribution, we studied intermolecular bonding in two isomers of triiodo-ortho-carborane have been synthesized, and the structures of a dozen of them have been established by single-crystal X-ray diffraction. The derivatives with a high degree of substitution such as 8,9,10,12-I4-1,2-C2B10H8 [8-1,2-C2B10H4 [10-1,2-C2B10H2 [ortho-carborane 1-I-1,2-C2B10H11 [2B10H11 [2B10H11 [2B10H11 [To date, a number of iodo derivatives of -C2B10H8 , 4,5,7,8-C2B10H4 , and 3,4-C2B10H2 , as in tC2B10H11 , 3-I-1,2[2B10H11 , 8-I-1,2[2B10H11 , and 9-I[2B10H11 .ortho-carborane, the presence of intermolecular I⋯I dihalogen bonds inside them depends on the position of the substituents. In addition to 1,12-diiodo-ortho-carborane 1,12-I2-1,2-C2B10H10, which is characterized by the presence of strong intermolecular I⋯I dihalogen bonds (3.57 Å) [2-1,2-C2B10H10 [2-1,2-C2B10H10 [2-1,2-C2B10H10 [2-1,2-C2B10H10 [ortho-carboranes containing substituents in the three most electron-withdrawing positions of the caborane cage 1,2,3-I3-1,2-C2B10H9 and the three most electron-donating positions of 8,9,12-I3-1,2-C2B10H9.As for the diiodo derivatives of (3.57 Å) , weak I⋯C2B10H10 , while tC2B10H10 , 4,7-I2-C2B10H10 , and 9,1C2B10H10  isomers 3-1,2-C2B10H9 (1) was previously reported in the iodination of ortho-carborane with molecular iodine in acetic acid in the presence of a mixture of concentrated sulfuric and nitric acids [ortho-carborane with iodine in dichloromethane in the presence of AlCl3 [3-1,2-C2B10H9 (1) have been reported [The formation of 8,9,12-Iic acids . We isolof AlCl3 . It shoureported ; however3-1,2-C2B10H9 was determined by single-crystal X-ray diffraction. A general view of 1 is presented in 3-1,2-C2B10H9 are nearly equal (B8-I8 is 2.165(7)Å, B9-I9 is 2.160(7) Å, and B12-I12 is 2.160(7) Å) and are only slightly longer than the B-I distances in 8,9,10,12-I4-1,2-C2B10H8  [The crystal structure of 8,9,12-I2.151 Å) .1 is depicted in 1. Halogen atoms participate in both types (I and II) of halogen bonding, and I⋯H-C(B) hydrogen bonds as well as B-H⋯H-B contacts are formed. It should be noted that all intermolecular contacts except for one are somewhat longer than the sum of the van-der-Waals radii. For instance, the type II halogen bond is very weak (the I⋯I distance is 4.268(4) Å, the B8-I8⋯I12 angle is 130.2(3)°, and the B12-I12⋯I8 angle is 92.2(3)°) °)  and can bc crystallographic plane are due to I⋯H-C(B) and H⋯H contacts, while in the crystallographic direction, a, molecules are linked mostly by I⋯I interactions. As a result, the crystal packing of 8,9,12-I3-1,2-C2B10H9 can be considered to be nearly isotropic.Therefore, it is impossible to choose one or two of the most important contacts that can be considered to be structure-forming. Interactions in the ortho-carborane with that of its closest analog, 8,9,12-tribromo-ortho-carborane 8,9,12-Br3-1,2-C2B10H9 (2). Despite the fact that the bromination of ortho-carborane was first described as early as the mid-1960s [ortho-carborane [ortho-carborane (up to 3:1) and the reaction time. This allowed us to isolate the desired compound 8,9,12-Br3-1,2-C2B10H9 (2) at a 17% yield . This indicates a lower acidity of the CH-carborane groups in the 8,9,12-tribromo derivative compared to the 8,9,12-triiodo derivative.It would be interesting to compare the crystal packing of 8,9,12-triiodo-id-1960s , the chearborane . Since tield see  It shoulrboranes ,60, for 3-1,2-C2B10H9 was determined by single-crystal X-ray diffraction. A general view of 2 is presented in The crystal structure of 8,9,12-Brortho-carborane was determined in 1966 [1, it is interesting to mention some other studied bromo- and iodo-derivatives of ortho-carborane. For instance, the crystal structures of 1,2-Me2-8,9,10,12-I4-1,2-C2B10H6 [2-8,9,10,12-Br4-1,2-C2B10H6 [2-1,2-C2B10H10 [2-C2B10H10 [2-1,2-C2B10H10 [2-1,2-C2B10H10 [2-1,2-C2B10H10 [It should be noted that the structure of 8,9,12-tibromo- in 1966  at room -C2B10H6  and 1,2--C2B10H6  are isosC2B10H10  and 1,12C2B10H10  do not sC2B10H10  and 9,12C2B10H10 ; howeverC2B10H10   Å, which is shorter than the sum of the van-der-Waals radii 3.79 Å)  and H⋯H. The differences in the crystal-packing properties described above result in some redistribution of the contact types  . At the ct types : the con3-1,2-C2B10H9 and 8,9,12-Br3-1,2-C2B10H9 can be clearly seen in The observed similarities and dissimilarities in the crystal packing of 8,9,12-I3-1,2-C2B10H9 (3) was prepared by the deprotonation of 3-iodo-ortho-carborane followed by a treatment of molecular iodine (see below). The crystal structure of 1,2,3-I3-1,2-C2B10H9 was determined by single-crystal X-ray diffraction. A general view of 3 is presented in 2-closo-C2B10H10 [The 1,2,3-isomer 1,2,3-IC2B10H10 .3-1,2-C2B10H9, the crystal packing of 1,2,3-I3-1,2-C2B10H9 is formed by halogen-bonded planes parallel to the bc crystallographic plane  at the PBE0/def2tzvp level followed by a topological analysis of the calculated electron density in terms of the “Atoms in Molecules” theory [In our recent study, we theoretically compared the dimer formation of 1,12- and 1,3-diiodo-rboranes . Accordi” theory . The int” theory ,67 using” theory .3 as anisotropic unlike the 8,9,12-isomer. It is interesting to note that the crystal density of the latter is somewhat higher than that of the 1,2,3-isomer. This can be explained by the increased role of the I⋯I interactions . Acros Organics silica gel (0.060–0.200 mm) was used for column chromatography. The NMR spectra at 400 MHz (1H) and 128 MHz (11B) were recorded with Varian Inova 400 spectrometer. The residual signal of the NMR solvent relative to Me4Si was taken as the internal reference for 1H spectra. 11B NMR spectra were referenced using BF3·Et2O as external standard.The reactions were carried under an inert atmosphere. 3-Iodo-terature . 1,2-Dimterature . All oth3-ortho-C2B10H9 was isolated as a by-product from the di-iodination reaction of ortho-carborane under standard conditions [3 (0.400 g) were added to a solution of ortho-carborane  in dichloromethane (30 mL) and heated under reflux for 8 h. Then, the reaction mixture was cooled and treated with a solution of Na2S2O3·5H2O (3.000 g) in water (50 mL). The organic phase was separated, and the aqueous fraction was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica using diethyl ether as eluent to yield 1.900 g (69%) of 9,12-I2-1,2-C2B10H10 and 0.102 g (3%) of 8,9,12-I3-1,2-C2B10H9 as white powders.8,9,12-Inditions . Iodine 3-1,2-C2B10H9: 1H NMR : 4.13 , 3.8−2.0 . 11B NMR : δ −6.1 , −11.5 , −13.1 , −14.7 , −16.4 , and −17.2 ).8,9,12-Iortho-carborane (1.009 mg and 7.00 mmol) in 1,2-dichloroethane (30 mL) and heated under reflux for 40 h. Then, the reaction mixture was cooled and treated with a solution of Na2S2O3·5H2O (5.000 g) in water (50 mL). The organic phase was separated, and the aqueous fraction was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica using chloroform as eluent to yield 0.450 g (17%) of 8,9,12-Br3-1,2-C2B10H9 as a white powder.Bromine  and anhydrous aluminum chloride (0.400 g) were added to a solution of 3-1,2-C2B10H9: 1H NMR : 3.87 , 3.5−1.7 . 11B NMR : 0.4 ), −5.3 ), −8.4 ), −13.9 ), −15.6 ), −17.1 ), and −20.4 ). 13C NMR : 45.5 (CHcarb).8,9,12-Brn-butyllithium  was added to a solution of 3-iodo-ortho-carborane  in 1,2-dimethoxyethane (10 mL) and stirred at room temperature for 1 h. Iodine  was added in one portion and the reaction mixture was stirred at room temperature overnight; then, it was treated with a solution of Na2S2O3·5H2O (250 mg) in water (50 mL). The organic phase was separated, and the aqueous fraction was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica using diethyl ether as eluent to yield 137 mg of mixture of 3-I-1,2-C2B10H11 and 1,2,3-I3-1,2-C2B10H9.A 2.25 M hexane solution of 1, 2, and 3  contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.Single-crystal X-ray diffraction experiments of nd 3 see  were car program . The CCD3-1,2-C2B10H9 (1): C2H9B10I3 are monoclinic; space group P21/n: a = 7.5776(6) Å, b = 24.0030(18) Å, c = 7.7535(6) Å, β = 109.487(2)°, V = 1329.46(18) Å3, and Z = 4, M = 521.89, dcryst = 2.607 g·cm−3. wR2 = 0.0794 calculated on F2hkl for all 2611 independent reflections with 2θ < 52.1° ).Crystallographic data for 8,9,12-I3-1,2-C2B10H9 (2): C2H9B10Br3 are monoclinic; space group C2/c: a = 12.1453(6) Å, b = 8.4794(5) Å, c = 23.0632(11) Å, β = 90.089(2)°, V = 2375.2(2) Å3, Z = 8, M = 380.92, dcryst = 2.131 g·cm−3. wR2 = 0.0797 calculated on F2hkl for all 2349 independent reflections with 2θ < 52.1° ).Crystallographic data for 8,9,12-Br3-1,2-C2B10H9 for (3): C2H9B10I3 are orthorhombic; space group Pnma: a = 19.1157(8) Å, b = 8.0014(3) Å, c = 8.7287(4) Å, V = 1335.08(10) Å3, Z = 4, M = 521.89, dcryst = 2.596 g·cm−3. wR2 = 0.0580 calculated on F2hkl for all 1739 independent reflections with 2θ < 56.2° ).Crystallographic data for 1,2,3-I3-1,2-C2B10H9 was carried out using the Gaussian program [Quantum chemical optimization of halogen-bonded trimeric associate of 1,2,3-I program . The ini program ,69.


Scientific Reportshttps://doi.org/10.1038/s41598-022-19983-y, published online 28 September 2022Correction to: Correction to: The original version of this Article contained an error in the legend of Figure 3.Figure 3. S. exempta future environmental suitability maps for Kenya and Tanzania for 3 different CO2 emission scenarios and two different time periods. (A) 2021-2040 SSP1-2.6, (B) 2021-2040 SSP3-7.0, (C) 2021–2040 SSP5-8.5, (D) 2061-2080 SSP1-2.6, (E) 2061-2080 SSP3-7.0, and (F) 2061-2080 SSP5-8.5.”“now reads:Figure 3. S. exempta future environmental suitability maps for Kenya and Tanzania for 3 different CO2 emission scenarios. (A) 2061-2080 SSP1-2.6, (B) 2061-2080 SSP3-7.0, and (C) 2061-2080 SSP5-8.5.”“The original Article has been corrected.