AbstractOrthoptera conservation, greater efforts must be gathered to implement specific monitoring schemes. Despite recent surveys, little is known about Portuguese Orthoptera populations. This study was performed in 2014 and 2015 mainly in Castro Verde Special Protection Area (SPA), southern Portugal, and is the first Orthoptera inventory conducted in the area. A total of 35 Orthoptera species was recorded, with two new species reported for Portugal. We provide species’ habitat occurrences within the protected area and use information on the conservation status and the Iberian distribution of each documented species to discuss the importance of Castro Verde SPA for Orthoptera conservation. The data presented here sheds new light on Castro Verde SPA biodiversity and emphasizes the inclusion of this area in the conservation of Orthoptera diversity, particularly in the protection of threatened endemic species.With the increasing awareness of the need for  The results of the recent Red List assessment of Europe’s grasshoppers, crickets and bush-crickets indicate that the highest species diversity and the greatest concentration of threatened species are found in the Mediterranean region. Moreover, the highest number of Data Deficient species are found in the Mediterranean region and many are also found in the Iberian Peninsula along with endemic species . The cliOrthoptera fauna has been more comprehensively studied in Spain, while there is still a lack of knowledge regarding the Portuguese Orthoptera  consists mainly of extensive cereal-steppes, however in recent years the afforested area has increased. The area houses the most significant diversity and abundance of steppe birds in Portugal and, therefore, was designated for steppe bird conservation under the European Union Birds Directive (79/409/EEC). Several bird species listed in Annex I of the Birds Directive are regularly found there, including the lesser kestrel , the great bustard (Otistarda) and the little bustard (Tetraxtetrax). While the biological and conservation importance of birds in Castro Verde SPA has long been recognized, little is known about its value for other groups, namely for arthropods. Many orthopterans, especially grasshoppers, are highly dependent on grassland habitats and are major primary herbivores , oaks (Quercusrotundifolia and Q.suber) and umbrella pines (Pinuspinea). Pine and oak plantations often have a grassy understorey grazed by livestock. The area occupied by forest plantations increased since the 1990’s, mostly in the periphery of the Castro Verde SPA, due to Common Agricultural Policy (CAP) subsidies for farmland afforestation  and females (F) recorded. Species are coded according to their occurrence in the studied habitats, as follows: GCI) and the standardised Grasshopper Conservation Index (GCIn) proposed by To evaluate the conservation value of the different habitat types, we calculated the Grasshopper Conservation Index  and Pyrgomorphaconica . Two species are recorded for the first time for Portugal: Dociostaurushispanicus Bolivar, 1898 and Euryparyphesterrulentus . Furthermore, Platystolusmartinezii , first recorded around 100 years ago in Portugal  was found for eucalyptus plantations (0.44±0.12) and pine plantations (0.44±0.12), while the highest was found for fallows (0.52±0.18) and mixed pine-oak plantations (0.49±0.21). An intermediate value of GCIn was found for edges (0.48±0.18) and oak plantations (0.47±0.14). The species with highest possible GCI value (1) was Euryparyphesterrulentus.The number of species was broadly similar in fallows (29), edges (24) and tree plantations (27), despite the highest number of transects placed in fallows. Among the forest types, the most speciose were oak plantations (21), whereas only 13 species were found in eucalyptus plantations. The lowest value of Grasshopper Conservation Index , Platycleisaffinis Fieber, 1853, Platystolusmartinezii and the Red-Listed species Dociostaurushispanicus and Platycleisfalx  were only recorded in fallows. The species Truxalisnasuta , was only recorded in edges. Three species, Platycleisintermedia , Pyrgomorphaconica and Tettigoniaviridissima  were only recorded in tree plantations  apicalis , Calliptamuswattenwylianus Pantel, 1896 and Tessellanatessellata  were collected in the majority of the sampling sites (>80%)  occurred most frequently, possibly due to shade, patches of bare ground and sparse vegetation dominated by grasses characteristic of these plantations (Sphingonotus (Sphingonotus) lluciapomaresi  was the species occurring most frequently. This is an Iberian endemic that occurs in areas with bare and rocky ground (Llucià-Pomares & Fernández-Ortín 2009), which characterize some of the pine plantations surveyed. In fallows, the species most frequently encountered was Dociostaurusmaroccanus   Figure . Overallntations . In pine) Figure . This sp) Figure , which and Spain , and elsnd Spain . Althougnditions .http://ipt.gbif.pt/ipt/archive.do?r=orthopt_castroverde .The data underpinning the analysis reported in this paper are deposited at GBIF, the Global Biodiversity Information Facility, Decticusalbifrons  IUCN category: Least ConcernMaterial examined. A29: 15/05/2015, OAK (1 M). A79: 26/05/2015, FAL (1 F). P20: 24/05/2014, MIX (1 M).Iberian distribution. Widespread and very common all over the Peninsula.Platycleisaffinis Fieber, 1853 IUCN category: Least ConcernMaterial examined. A68: 20/06/2015, FAL (1 F). B5: 16/06/2015, FAL (1 F). P1: 25/06/2014, FAL (1 F). P46: 16/06/2015, FAL (1 F).Iberian distribution. Present all over the Peninsula, except in the Pyrenees.Platycleisfalx  IUCN category: Vulnerable B2abMaterial examined. B8-1: 21/06/2015, FAL (1 F).Iberian distribution. Occurs mostly in the south-eastern part of the Iberian Peninsula and, specifically, it is known to occur in the central region of Portugal  IUCN category: Least ConcernMaterial examined. A85: 21/06/2015, OAK (1 F). P52: 31/05/2014, PIN (1 F).Iberian distribution. This species occurs throughout continental Spain, except in the Northern region with a more Atlantic climate influence and most of the Pyrenees. Prior to this research, the species was only accounted for in the central region of Portugal. However, our study suggests that this species may have a larger distribution range in southern Portugal.PageBreakPlatycleissabulosa Azam, 1901 IUCN category: Least ConcernMaterial examined. P15: 18/06/2014, EDG (1 F). P22: 25/06/2014, PIN (1 F). P23: 18/06/2015, FAL (1 F). P48: 24/06/2014, OAK (1 F). P51: 24/06/2014, PIN (1 F). P52: 25/06/2014, FAL (1 F).Iberian distribution. In Spain, it is widespread in the south and centre, but is apparently scarce in the northern regions. In Portugal, the species occurs from the centre to the south.Tessellanatessellata  IUCN category: Least ConcernMaterial examined. A29: 22/06/2015, FAL (2 F), OAK (1F). A33: 20/06/2015, FAL , PIN (1 F). A57: 20/06/2015, EDG (1 F), FAL (1F). A64: 22/06/2015, FAL . A68: 20/06/2015 FAL (3 F), OAK (2 M). A76: 27/05/2015, FAL (3 M); 19/06/2015, EDG (1 M), FAL . A78: 21/06/2015, FAL (1 F). A79: 23/06/2015, FAL (2 M), PIN (1 F). A85: 21/06/2015, FAL (2 F), EDG (1 F), OAK (1 F). A90: 24/06/2014, FAL (4 M), EDG (1 M). A98: 19/06/2015, FAL (2 F), EDG (1 M), PIN (1 M). A99: 19/06/2015, EDG (1 F), FAL (2 F). A152: 15/06/2015, EDG (1 M), FAL , OAK . B1: 16/06/2015, FAL (1 F). B5: 16/06/2015, FAL . B6: 18/06/2015, FAL . B7: 18/06/2015, FAL . B8-1: 21/06/2015, FAL . B13-2: 22/06/2015, FAL , EDG (1 M), MIX (1 M). B14: 01/06/2015, FAL ; 23/06/2015, FAL . P1: 03/06/2014, FAL , EDG (2 M); 25/06/2014, FAL , EDG (1 M). P3: 18/06/2015, FAL (3 F), EDG (1 F), OAK . P5: 18/06/2014, FAL . P6: 15/06/2014, FAL , EDG (1 M), PIN . P8: 16/06/2014, FAL , EDG (3 F). P9: 20/06/2014, FAL , EUC (1 F). P11: 20/06/2014, FAL , EDG , OAK . P12: 20/06/2014, FAL , EDG (1 M). P13: 16/06/2014, FAL , EDG (2 M). P15: 18/06/2014, FAL , EDG , PIN (2 F). P17: 15/06/2014, FAL , EDG (1 M). P18: 14/06/2014, FAL , EDG , OAK . P20: 18/06/2014, FAL (3 F), EDG (1 F), MIX . P22: 04/06/2014, FAL , EDG (1 F), PIN ; 25/06/2014, FAL , PIN (2 F). P23: 18/06/2015, FAL (1 M). P24: 14/06/2014, FAL (2 M). P27: 16/06/2015, FAL . P28: 17/06/2014, FAL , EDG (1 M), OAK . P29: 19/06/2014, FAL (2 F). P30: 15/06/2015, FAL , EDG (1 F), MIX (1 F). P31: 19/06/2014, FAL (1 M), MIX . P32: 19/06/2014, FAL , OAK . P36: 24/06/2014, FAL (2 F). P39: 15/06/2015, FAL (2 F), EDG (1 F), OAK (1 M). P46: 16/06/2015, FAL , EDG (1 M). P47: 16/06/2014 , EDG (1 M), OAK (3 M). P48: 24/06/2014, FAL , EDG, (1 M). P49: 01/07/2014, FAL , EDG , MIX . P51: 24/06/2014, FAL , EDG (1 F). P52: 25/06/2014, FAL (4 F), EDG , PIN (1 F).Iberian distribution. Widespread and very common all over the Peninsula.PageBreakTettigoniaviridissima  IUCN category: Least ConcernMaterial examined. A85: 12/05/2015, OAK (1 M).Iberian distribution. Widespread and very common all over the Peninsula.Odontura (Odontura) glabricauda  IUCN category: Least ConcernMaterial examined. A63: 26/05/2015, FAL (1 M); B7: 22/04/2015, FAL (1 F); P18: 17/04/2014, OAK (1 F); P20: 26/04/2014, MIX (1 M); P39: 16/04/2015, EDG (1 M).Iberian distribution. This is an Iberian endemic species and is restricted to the southwestern quadrant of the Peninsula, where it is widespread and common.Tylopsislilifolia  IUCN category: Least ConcernMaterial examined. A29: 22/06/2015, EDG (1 M), FAL (3 M), OAK (1 M); A33: 20/06/2015, EDG (2 M), FAL (3 F), PIN (1 M); B7: 18/06/2015, FAL ; B8-1: 21/06/2015, EDG (1 M); P5: 18/06/2014, FAL ; P6: 15/06/2014, PIN (2 M).Iberian distribution. In Spain it occurs mostly in the southern Mediterranean regions; in Portugal it is mainly known from the central region. It was Platystolusmartinezii  IUCN category: Least ConcernMaterial examined. A29: 31/05/2015, FAL .Iberian distribution. This is an Iberian endemic species mainly distributed in the centre of the Peninsula  IUCN category: Least ConcernMaterial examined. B1: 16/06/2015, FAL (1 F); P3: 18/06/2015, OAK (1 F); P17: 15/06/2014, FAL (1 F); P29: 19/06/2014, EDG ; P30: 15/06/2015, PageBreakFAL (1 F); P31: 19/06/2014, FAL (1 F); P51: 24/06/2014, EDG (1 F); P52: 25/06/2014, FAL (1 F).Iberian distribution. Widespread and very common all over the Peninsula.Paratettixmeridionalis  IUCN category: Least ConcernMaterial examined. B14: 01/06/2015, EUC ; 23/06/2015, EUC ; P31: 19/06/2014, MIX ; P52: 25/06/2014, FAL (1 F).Iberian distribution. Widespread and very common all over the Peninsula.Euryparyphesterrulentus  IUCN category: Least ConcernMaterial examined. P3: 25/05/2015, EDG (1 M); P13: 22/05/2014, FAL ; P15: 02/05/2014, FAL (1 M); P20: 24/05/2014, MIX (1 F).Iberian distribution. This species is new to Portugal. E.terrulentus was previously considered being endemic to Spain, where it is common in the south. This is the first record of E.terrulentus and the first confirmed record of Euryparyphes genus for Portugal, expanding its endemic Iberian distribution to southern Portugal.Pyrgomorphaconica  IUCN category: Least ConcernMaterial examined. A76: 05/05/2015, PIN (1 F).Iberian distribution. Present in most of the Peninsula, but seems to be absent in a few regions of high altitude in the North.Truxalisnasuta  IUCN category: Least ConcernMaterial examined. A33: 17/05/2015, EDG (1 M), 28/05/2015 (1 M); P36: 30/05/2014, EDG (1 F).Iberian distribution. Widely distributed throughout the whole of Portugal, except for a few localities in the North. In Spain it has a meridional distribution, occurring in PageBreakthe north-eastern part of the country and in the southern half, although it seems to be absent in the centre.Calliptamusbarbarus  IUCN category: Least ConcernMaterial examined. A29: 22/06/2015, OAK (3 F); A33: 20/06/2015, EDG (1 F), FAL ; A64: 22/06/2015, FAL (1 M); A76: 19/06/2015, EDG (1 F), FAL (1 F), PIN (1 M); A78: 21/06/2015, FAL (1 F); A85: 21/06/2015, FAL , OAK (1 M); A90: 24/06/2014, FAL (1 F); A98: 19/06/2015, PIN ; A99: 19/06/2015, FAL (1 F); A152: 15/06/2015, FAL ; B7: 18/06/2015, EDG (2 M); B8-1: 21/06/2015, FAL , EUC (1 M); B13-2: 22/06/2015, EDG (1 F), MIX (1 F); B14: 23/06/2015, FAL (1 F); P5: 18/06/2014, FAL (1 F); P6: 15/06/2014, PIN (3 M); P11: 20/06/2014, OAK ; P13: 16/06/2014, EDG (1 F); P15: 18/06/2014, PIN (2 M); P18: 14/06/2014, EDG (1 M), OAK (1 M); P20: 18/06/2014, EDG (1 M), MIX ; P22: 04/06/2014, EDG (1 M), 25/06/2014, FAL (2 M); P23: 18/06/2015, FAL (1 M); P27: 16/06/2015, FAL (1 F); P28: 17/06/2014, EDG (1 M), FAL (1 F); P29: 19/06/2014, FAL (1 M); P30: 15/06/2015, MIX (1 F); P31: 19/06/2014, FAL (3 M); P32: 19/06/2014, OAK (2 M); P39: 15/06/2015, OAK (1 F); P46: 16/06/2015, PIN (1 M); P48: 24/06/2014, EDG , FAL (1 M), OAK ; P49: 01/07/2014, EDG (1 F), MIX ; P52: 25/06/2014, FAL .Iberian distribution. Widespread and very common all over the Peninsula.Calliptamuswattenwylianus Pantel, 1896 IUCN category: Least ConcernMaterial examined. A29: 31/05/2015, EDG (1 M), FAL , 22/06/2015, EDG (1 F), FAL , OAK (2 F); A33: 28/05/2015, FAL , 20/06/2015, FAL (4 M); A57: 20/06/2015, EDG (2 M), FAL ; A64: 01/06/2015, FAL , 22/06/2015, FAL , OAK (1 F); A68: 20/06/2015, FAL (2 M), OAK (1 M); A76: 27/05/2015, FAL , EDG (1 M), PIN (2 M), 19/06/2015, FAL (2 M), EDG (1 M), PIN (1 M); A78: 30/05/2015, FAL , PIN (1 F), 21/06/2015, FAL , EDG (1 M), PIN (1 F); A79: 25/05/2015, EDG (1 M), 26/05/2015, FAL , 23/06/2015, FAL ; A85: 30/05/2015, FAL , EDG (1 F), OAK , 21/06/2015, FAL , EDG (1 M), OAK (1 M); A90: 24/06/2014, FAL (1 F), EDG (1 F); A98: 27/05/2015, FAL , EDG , PIN (3 M), 19/06/2015, FAL (2 M), EDG (1 M), PIN (1 M); A99: 25/05/2015, FAL (2 M), 19/06/2015, FAL (3 M), EDG (1 M); A152: 15/06/2015, FAL , OAK, ; B1: 16/06/2015, FAL , EDG (1 M); B5: 16/06/2015, FAL (4 M); B6: 22/05/2015, FAL , 18/06/2015, FAL , EDG (1 M), EUC (1 F); B7: 18/06/2015, EUC (2 M); B8-1: 16/05/2015, FAL (1 F), PageBreak30/05/2015, FAL , 21/06/2015, FAL ; B13-2: 31/05/2015, FAL (5 M), EDG (2 M), 22/06/2015, FAL , EDG (1 M), MIX (1 M); B14: 01/06/2015, FAL , 23/06/2015, FAL , EDG (1 M), EUC (1 M); P1: 03/06/2014, FAL (3 F), 25/06/2014, FAL ; P3: 25/05/2015, FAL , OAK , 18/06/2015, FAL , EDG (1 M), OAK ; P5: 18/06/2014, FAL ; P6: 15/06/2014, FAL ; P8: 16/06/2014, FAL , EDG (2 M), OAK (1 M); P9: 20/06/2014, FAL ; P11: 29/05/2014, FAL (4 F), EDG (1 M), OAK, , 20/06/2014, FAL (2 M), EDG (1 M), OAK ; P12: 29/05/2014, FAL (1 F), 20/06/2014, FAL , OAK (1 F); P13: 21/05/2014, PIN , 22/05/2014, PIN , 16/06/2014, FAL , EDG ; P15: 25/05/2014, PIN (2 M), 26/05/2014, FAL (3 M), 18/06/2014, FAL , EDG (1 M), PIN (1 F); P17: 15/06/2014, FAL , EDG (1 M); P18: 14/06/2014, FAL , OAK ; P19: 31/05/2014, FAL , EDG , OAK ; P20: 24/05/2014, MIX (1 F), 18/06/2014, FAL , EDG , MIX ; P22: 04/06/2014, FAL , EDG (1 F), 25/06/2014, FAL , EDG (1 M), PIN (2 M); P23: 22/05/2015, FAL , 18/06/2015, FAL , EDG (1 M); P24: 14/06/2014, FAL (1 M); P27: 16/06/2015, FAL ; P28: 17/06/2014, FAL , EDG (1 M), OAK ; P29: 19/06/2014, FAL ; P30: 15/06/2015, FAL ; P31: 19/06/2014, FAL , EDG (1 M), MIX (1 F); P32: 19/06/2014, FAL , EDG (2 F), OAK ; P36: 30/05/2014, FAL (1 F), 24/06/2014, FAL ; P39: 15/06/2015, FAL (2 F), EDG (1 M), OAK (1 M); P46: 16/06/2015, FAL ; P47: 16/06/2014, FAL , OAK ; P48: 24/06/2014, FAL ; P49: 01/07/2014, FAL ; P50: 01/06/2014, FAL (1 F); P51: 24/06/2014, FAL ; P52: 31/05/2014, FAL , 25/06/2014, FAL , EDG , PIN (4 M).Iberian distribution. Very abundant in the meridional half of the Peninsula but more scattered in the northern half, in the areas with more Atlantic influence.Pezotettixgiornae  IUCN category: Least ConcernMaterial examined. A29: 22/06/2015, OAK (2 F); A33: 20/06/2015, FAL (3 F), EDG (1 F); A57: 20/06/2015, FAL (1 F); A64: 22/06/2015, FAL (1 F), EDG (1 F), OAK (1 F); A68: 20/06/2015, FAL (3 F), EDG (1 F), OAK (2 F); A76: 19/06/2015, PIN (1 F); A78: 30/05/2015, FAL (1 F), EDG (1 F), PIN (1 F); A79: 23/06/2015, PIN (1 F); A85: 30/05/2015, OAK (1 F), 21/06/2015, FAL (2 F), OAK (1 F); A90: 24/06/2014, FAL ; A98: 19/06/2015, PIN (1 F); A99: 19/06/2015, PIN (1 F); A152: 15/06/2015, FAL , OAK (4 F); B1: 16/06/2015, FAL ; B5: 16/06/2015, FAL (2 F), EUC (1 M); B6: PageBreak18/06/2015, EDG (1 F); B7: 18/06/2015, EDG (1 F); B8-1: 30/05/2015, FAL , 21/06/2015, FAL (1 F), EDG (1 F); B13-2: 22/06/2015, FAL (3 F), MIX (1 F); B14: 23/06/2015, FAL (1 F), EDG (1 M); P1: 25/06/2014, EDG (1 F); P3: 18/06/2015, FAL (1 M), OAK ; P5: 18/06/2014, FAL , EUC ; P6: 15/06/2014, FAL (1 F), EDG (2 F), PIN (1 M); P8: 16/06/2014, FAL ; P11: 20/06/2014, FAL (1 F), EDG (1 F), OAK ; P12: 20/06/2014, FAL , OAK ; P15: 18/06/2014, PIN (4 F); P17: 15/06/2014, FAL ; P18: 14/06/2014, FAL , OAK (1 M); P20: 18/06/2014, EDG , MIX (1 F); P22: 04/06/2014, FAL (1 F), PIN , 25/06/2014, PIN (1 F); P27: 16/06/2015, EDG (1 F), EUC (1 F); P28: 17/06/2014, FAL (4 F), OAK (1 F); P29: 19/06/2014, FAL (1 F); P30: 15/06/2015, FAL (3 F), MIX (1 F); P31: 19/06/2014, FAL (2 F), MIX ; P32: 19/06/2014, FAL (2 F), EDG (1 F), OAK ; P36: 24/06/2014, FAL (1 F), EUC (1 M); P39: 15/06/2015, FAL (3 F), EDG (1 F), OAK (2 F); P46: 16/06/2015, FAL (2 F); P47: 22/04/2014, FAL (1 F), 16/06/2014, FAL (3 F), EDG (2 F); P48: 24/06/2014, EDG ; P49: 01/07/2014, FAL (2 F), MIX (1 F); P51: 24/06/2014, FAL (1 F), EDG (1 F).Iberian distribution. This species is common and widely distributed throughout all of the Peninsula, particularly in the south.Anacridiumaegyptium  IUCN category: Least ConcernMaterial examined. B1: 22/04/2015, EDG (1 F); B5: 20/04/2015, EUC (1 M).Iberian distribution. Widespread and very common all over the Peninsula.Chorthippus (Glyptobothrus) apicalis  IUCN category: Least ConcernMaterial examined. A29: 15/05/2015, FAL , EDG (5 M), OAK , 31/05/2015, FAL , EDG (2 M), OAK ; A33: 17/05/2015, FAL , EDG (1 F), PIN (3 M), 28/05/2015, FAL (2 M), EDG (1 M), PIN (1 M); A57: 05/05/2015, FAL , 26/05/2015, FAL (2 M); A63: 14/05/2015, FAL , EDG (1 M), 26/05/2015, FAL (1 F); A64: 14/05/2015, FAL (2 M), OAK (2 M); A68: 15/05/2015, EDG (2 M), OAK , 28/05/2015, OAK (1 M); A76: 05/05/2015, FAL , EDG , PIN ; A78: 13/05/2015, FAL , EDG , PIN ; A79: 02/05/2015, FAL , EDG , PIN ; A85: 12/05/2015, FAL , EDG , OAK ; A90: 04/06/2014, FAL (1 M); A98: 13/05/2015, FAL , EDG (2 M), PIN ; A99: 02/05/2015, FAL , EDG (1 M); PageBreakA152: 18/05/2015, FAL (2 M), EDG (1 M), OAK (2 M); B1: 24/05/2015, EDG (3 F); B4: 21/04/2015, FAL (1 M), EDG (1 M); B5: 20/04/2015, FAL , 21/05/2015, FAL , EDG (1 M); B6: 01/05/2015, FAL , EDG (1 F), 22/05/2015, FAL (1 M); B7: 01/05/2015, FAL , EDG (1 M); B8-1: 16/05/2015, FAL ; B13-2: 31/05/2015, EDG (1 F); B14: 01/06/2015, EDG (1 M); P3: 03/05/2015, FAL , EDG , OAK , 25/05/2015, FAL , OAK ; P5: 24/04/2014, FAL ; P6: 23/04/2014, FAL (1 M), PIN (1 F), 20/05/2014, PIN (1 F); P8: 22/04/2014, FAL , 19/05/2014, FAL , EDG , OAK ; P9: 30/04/2014, FAL , 23/05/2014, FAL ; P10: 30/04/2014, FAL , EDG (1 F), OAK , 17/05/2014, FAL , EDG (2 M), OAK ; P11: 29/04/2014, FAL , EDG , OAK , 29/05/2014, FAL (2 F), EDG (1 F); P12: 29/04/2014, FAL , 29/05/2014, FAL ; P13: 25/04/2014, FAL , EDG , PIN , 22/05/2014, FAL (2 F); P15: 02/05/2014, FAL (2 M), EDG (3 M), PIN , 25/05/2014, PIN ; P16: 18/04/2014, FAL (1 M); P17: 19/04/2014, FAL (1 M), EDG (1 F), 16/05/2014, FAL ; P18: 17/04/2014, FAL , EDG , OAK , 16/05/2014, FAL (3 M), EDG , OAK ; P19: 09/05/2014, FAL (10 M), EDG , OAK (9 M), 31/05/2014, OAK (1 M); P20: 25/04/2014, FAL , EDG , MIX (1 F), 26/04/2014, MIX (4 M), 24/05/2014, EDG (1 M); P22: 13/05/2014, FAL , PIN ; P23: 18/04/2015, FAL , EDG (1 M), 22/05/2015, FAL (2 F), EDG ; P24: 15/05/2014, FAL ; P25: 16/04/2015, FAL (1 F), OAK (1 M); P28: 22/05/2014, FAL , EDG (1 M), OAK (1 M); P29: 27/05/2014, FAL (1 M); P30: 18/04/2015, MIX (1 F), EDG (1 M), 19/04/2015, FAL , 21/05/2015, EDG (1 M); P31: 28/04/2014, MIX (1 M), 26/05/2014, EDG (2 M); P32: 01/05/2014, FAL , EDG (1 M), OAK , 27/05/2014, OAK , 19/06/2014, OAK (1 M); P33: 26/04/2014, FAL (1 M), 17/05/2014, FAL (2 M), OAK (1 M); P39: 16/04/2015, FAL (2 M), OAK (2 M), 18/05/2015, FAL , OAK ; P42: 14/05/2014, FAL (2 M); P46: 19/04/2015, FAL (2 M), EDG (1 F), PIN (1 M), 21/05/2015, FAL ; P47: 21/04/2014, OAK , 19/05/2014, FAL , OAK ; P48: 07/05/2014, FAL (1 M), EDG , 08/05/2014, OAK ; P49: 08/05/2014, FAL , EDG , MIX , 02/06/2014, EDG (1 F), MIX ; P50: 14/05/2014, EDG (1 M), OAK ; P51: 27/04/2014, FAL , EDG (1 F); P52: 09/05/2014, FAL , EDG , PIN (4 M), 31/05/2014, FAL .Iberian distribution. Distributed in nearly all regions of the Peninsula, with the exception of the extreme north-west.Chorthippusvagans  IUCN category: Least ConcernMaterial examined. A57: 20/06/2015, EDG (2 M); A63: 14/05/2015, EUC (1 M), 26/05/2015, EUC (1 F); A68: 20/06/2015, OAK (1 F); A74: 03/06/2014, PageBreakEUC ; A90: 24/06/2014, EUC (2 M); B1: 24/05/2015, EDG (1 M), 16/06/2015, EUC ; B3: 17/05/2015, PIN (1 M); B5: 21/05/2015, EUC (2 F), 16/06/2015, EUC (2 M); B7: 18/06/2015, EUC (2 F); P5: 23/05/2014, EUC ; P6: 15/06/2014, PIN ; P9: 22/05/2014, EUC (1 M), 20/06/2014 ; P27: 24/05/2015, EDG , EUC (1 M), 16/06/2015, EDG , EUC ; P29: 26/05/2014, EUC , 19/06/2014, EDG (1 M), EUC ; P30: 15/06/2015, MIX (3 F); P31: 19/06/2014, MIX (1 F); P36: 30/05/2014, EUC , 24/06/2014, EDG , EUC ; P39: 15/06/2015, EUC ; P42: 14/05/2014, EUC (1 F); P51: 30/05/2014, PIN (1 F).Iberian distribution. Widespread and very common all over the Peninsula.Dociostaurusgenei  IUCN category: Least ConcernMaterial examined. A78: 21/06/2015, FAL ; A79: 23/06/2015, EDG , FAL (3 M); A98: 19/06/2015, EDG , FAL ; A99: 19/06/2015, FAL ; A152: 15/06/2015, FAL (1 M); B8-1: 21/06/2015, FAL (1 M); B14: 23/06/2015, FAL (1 F); P13: 16/06/2014, PIN (2 F); P15: 18/06/2014, FAL ; P17: 15/06/2014, PIN ; P23: 18/06/2015, FAL (1 F), PIN ; P28: 17/06/2014, EDG (1 M).Iberian distribution. Present throughout most of the Peninsula with exception of the extreme north-west.Dociostaurushispanicus Bolivar, 1898 IUCN category: Near ThreatenedMaterial examined. P28: 17/06/2014, FAL (1 F).Iberian distribution. First record for Portugal. This is an Iberian endemic species, has in Spain has a more western distribution and can be found from north to south , EDG (1 M), OAK (1 F); A33: 20/06/2015, FAL ; A64: 22/06/2015, FAL , EDG (1 M); A68: 20/06/2015, FAL , EDG (1 M); A76: 19/06/2015, FAL , EDG (2 F), PIN (2 F); A78: 21/06/2015, FAL , EDG , PIN ; A79: 23/06/2015, FAL , EDG , PIN ; A85: 21/06/2015, FAL , EDG (1 M); A98: 19/06/2015, FAL , EDG (1 M), PIN ; A99: 19/06/2015, FAL , EDG ; A152: 15/06/2015, FAL (3 M), OAK (1 M); B6: 18/06/2015, FAL , EUC (1 F); B7: 18/06/2015, FAL (1 F), EUC (1 F); B8-1: 21/06/2015, FAL , EUC (1 M); B13-2: 22/06/2015, FAL , MIX (1 M); B14: 23/06/2015, FAL ; P1: 25/06/2014, FAL ; P3: 18/06/2015, FAL (3 M), EDG (1 M), OAK (1 F); P5: 18/06/2014, FAL (3 M), EDG , EUC (1 M); P6: 15/06/2014, PIN ; PageBreakP9: 20/06/2014, FAL (1 M); P11: 20/06/2014, FAL , EDG , OAK ; P12: 20/06/2014, FAL , OAK ; P13: 16/06/2014, FAL , EDG (1 M), PIN (1 M); P15: 18/06/2014, FAL (4 F), EDG , PIN (2 F); P18: 14/06/2014, OAK ; P20: 18/06/2014, FAL (2 M), EDG (1 M), MIX ; P22: 25/06/2014, FAL , PIN (2 M); P23: 18/06/2015, FAL , EDG , PIN (2 M); P27: 16/06/2015, EUC (3 M); P28: 17/06/2014, FAL , OAK (1 F); P31: 19/06/2014, FAL (1 F), MIX ; P32: 19/06/2014, OAK (1 M); P36: 24/06/2014, FAL , EDG (1 M); P39: 15/06/2015, FAL , EDG (4 M), OAK ; P46: 16/06/2015, FAL (1 M), PIN (1 F); P48: 24/06/2014, FAL , OAK ; P49: 01/07/2014, FAL (2 M), EDG (1 M), MIX ; P51: 24/06/2014, FAL ; P52: 25/06/2014, FAL (1 M).Iberian distribution. The area of occurrence extends throughout the Peninsula, with the exception of the northern strip faced to the Atlantic Ocean.Dociostaurusmaroccanus  IUCN category: Least ConcernMaterial examined. A33: 17/05/2015, FAL (1 M), 20/06/2015, FAL (1 M); A76: 27/05/2015, FAL , PIN (1M), 19/06/2015, FAL (1 M), EDG (1 F); A78: 13/05/2015, FAL , EDG (1 M), 30/05/2015, FAL ; A79: 26/05/2015, FAL ; A85: 30/05/2015, FAL ; A98: 13/05/2015, FAL , 27/05/2015, FAL (3 F), EDG (1 F), PIN (1 F); A99: 25/05/2015, FAL (3 M), 19/06/2015, FAL , A152: 15/06/2015, FAL (1 F); B5: 21/05/2015, FAL , 16/06/2015, FAL ; B6: 22/05/2015, FAL ; B13-2: 31/05/2015, FAL ; B14: 01/06/2015, FAL ; P3: 25/05/2015, FAL , 18/06/2015, FAL (2 M); P15: 26/05/2014, FAL ; P17: 16/05/2014, FAL (1 M), 15/06/2014, FAL (1 F); P19: 31/05/2014, FAL ; P22: 13/05/2014, FAL (3 F), 04/06/2014, FAL ; P23: 22/05/2015, FAL , 18/06/2015, FAL ; P24: 14/06/2014, FAL (1 M); P27: 16/06/2015, FAL (1 M); P28: 22/05/2014, FAL (1 F), 17/06/2014, FAL (1 M); P29: 26/05/2014, FAL , 19/06/2014, FAL (2 M); P30: 21/05/2015, FAL (1 F), 15/06/2015, FAL (1 M); P39: 18/05/2015, FAL (1 F), 15/06/2015, FAL (2 M); P47: 19/05/2014, FAL (1 M), 16/06/2014, FAL ; P49: 02/06/2014, FAL (1 F), 01/07/2014, FAL (1 F); P50: 01/06/2014, FAL ; P52: 31/05/2014, FAL (1 F).Iberian distribution. Widely distributed in the Mediterranean area of the Peninsula up to north-eastern Portugal. Seems to be absent in the northern strip faced to the Atlantic Ocean of Spain.Euchorthippuselegantulus Zeuner, 1940 IUCN category: Least ConcernMaterial examined. A33: 28/05/2015, FAL , EDG (1 F), 20/06/2015, FAL , EDG (1 F), PIN (2 M); A57: 20/06/2015, FAL (2 M); A64: 01/06/2015, FAL (1 M), 22/06/2015, FAL , EDG , OAKPageBreak(1 M); A68: 20/06/2015, FAL , EDG (1 M), OAK (2 M); A85: 21/06/2015, EDG (1 M); A90: 04/06/2014, FAL (2 M), 24/06/2014, FAL ; A99: 25/05/2015, FAL (1 M), EDG (1 F), 19/06/2015, FAL ; A152: 15/06/2015, FAL , EDG , OAK ; B5: 16/06/2015, FAL ; B6: 18/06/2015, FAL (1 F); B8-1: 30/05/2015, FAL (1 F); B13-2: 31/05/2015, FAL , 22/06/2015, FAL , EDG ; B14: 01/06/2015, EDG (1 M), 23/06/2015, FAL (1 M), EDG (2 M); P3: 18/06/2015, FAL (1 M); P6: 15/06/2014, EDG (1 M); P9: 20/06/2014, FAL ; P11: 29/05/2014, FAL (1 M), OAK (1 M); P12: 29/05/2014, FAL (1 M), 20/06/2014, FAL ; P18: 14/06/2014, EDG (1 M), OAK ; P20: 18/06/2014, EDG , MIX (1 M); P23: 18/06/2015, FAL (3 M); P27: 16/06/2015, FAL ; P28: 17/06/2014, EDG ; P29: 19/06/2014, FAL , EDG (1 M); P30: 15/06/2015, FAL , EDG (1 F), MIX (1 F); P31: 19/06/2014, FAL (1 M), EDG (1 M), MIX ; P32: 19/06/2014, FAL ; P36: 30/05/2014, FAL (1 M), 24/06/2014, FAL (1 M); P39: 18/05/2015, EDG (1 M), 15/06/2015, EDG (2 F), OAK ; P46: 21/05/2015, PIN (1 M), 16/06/2015, FAL , EDG , PIN (3 M); P47: 16/06/2014, OAK ; P48: 24/06/2014, OAK (1 F); P51: 24/06/2014, FAL ; P52: 25/06/2014, FAL , EDG .Iberian distribution. Present in almost in all of the Peninsula, being absent in the northern third.Omocestuspanteli  IUCN category: Least ConcernMaterial examined. A33: 17/05/2015, FAL (2 F), PIN (1 F), 28/05/2015, EDG (1 F); A64: 14/05/2015, OAK , 01/06/2015, OAK (3 M), 22/06/2015, OAK (1 M); A90: 04/06/2014, FAL (1 M); A99: 02/05/2015, EDG (2 M), PIN (1 M); A152: 18/05/2015, EDG (1 M); B13-2: 31/05/2015, FAL (2 M); B14: 01/06/2015, EDG , EUC (1 M); P25: 16/04/2015, EDG (1 M); P30: 18/04/2015, MIX (1 F), 19/05/2015, FAL ; P31: 25/05/2014, MIX (1 M), 19/06/2014, MIX ; P39: 16/04/2015, OAK (2 F), 18/05/2015, OAK ; P46: 19/04/2015, PIN (1 F), 21/05/2015, FAL (1 M); P50: 01/06/2014, OAK (1 M); P52: 09/05/2014, FAL (1 M), 31/05/2014, FAL .Iberian distribution. This is an Iberian endemic species and occurs in most of the Iberian territory, with the exception of a few localities in the North, particularly in the Pyrenees.Acrotyluspatruelis  IUCN category: Least ConcernMaterial examined. A29: 31/05/2015, EDG , 22/06/2015, EDG ; A33: 28/05/2015,EDG , PIN (1 M); A79: 25/05/2015, PIN (1 F), PageBreak26/05/2015, FAL (1 F); A90: 04/06/2014, EDG (1 F); A99: 25/05/2015, FAL (1 F); A152: 15/06/2015, EDG (1 F); B1: 24/05/2015, EDG (1 F), 16/06/2015, EDG (1 M); B7: 22/05/2015, EDG (2 F); B8-1: 16/05/2015, FAL (1 M); B13-2: 31/05/2015, FAL (1 M), 22/06/2015, FAL (1 F); B14: 01/06/2015, EUC (1 F); P1: 03/06/2014, FAL (2 F); P3: 25/05/2015, FAL (1 F); P13: 22/05/2014, PIN (1 F); P19: 31/05/2014, OAK (1 F), P22: 04/06/2014, PIN (1 F); P23: 22/05/2015, EDG (1 F); P29: 19/06/2014, FAL (1 F); P30: 21/05/2015, MIX (1 F); P31: 19/06/2014, MIX ; P39: 18/05/2015, EDG (1 F), OAK , 15/06/2015, OAK (1 F); P42: 14/05/2014, EDG (1 M); P50: 01/06/2014, EDG .Iberian distribution. Distributed mostly in the meridional half and centre of Spain. In Portugal, it is apparently absent in the northern half of the country and in some localities of the littoral west of the South.Aiolopuspuissanti Defaut, 2005 IUCN category: Least ConcernMaterial examined. A29: 31/05/2015, EDG ; A33: 17/05/2015, FAL (2 F), EDG (2 M), 28/05/2015, EDG (1 M); A57: 20/06/2015, FAL ; A64: 01/06/2015, EDG (1 F); A68: 28/05/2015, FAL ; A74: 03/06/2014, FAL , EDG (1 M); B13-2: 31/05/2015, FAL , 22/06/2015, FAL (1 F); B14: 23/06/2015, EUC (1 F); P1: 03/06/2014, EDG ; P10: 17/05/2014, OAK (1 M); P13: 22/05/2014, FAL (1 M); P23: 22/05/2015, FAL , EDG (1 M); P29: 26/05/2014, EDG ; P30: 19/05/2015, MIX (1 F), 21/05/2015, FAL (1 M), 15/06/2015, EDG (1 F); P31: 25/05/2014, MIX (1 M), 19/06/2014, MIX ; P32: 27/05/2014, EDG ; P36: 30/05/2014, EDG (1 M); P39: 16/04/2015, EDG (1 F), 18/05/2015, OAK (4 F); P46: 21/05/2015, FAL , 16/06/2015, FAL (1 F); P50: 01/06/2014, EDG (1 F), OAK ; P52: 31/05/2014, FAL , 25/06/2014, FAL (1 F).Iberian distribution. Present in almost all of the Peninsula with exception of the northern third.Aiolopusstrepens  IUCN category: Least ConcernMaterial examined. A74: 03/06/2014, FAL (1 F); B13-2: 17/07/2015, FAL (1 F).Iberian distribution. Widespread and very common all over the Peninsula.Locustamigratoria  IUCN category: Least ConcernMaterial examined. A33: 28/05/2015, EDG (1 M); P3: 25/05/2015, FAL (1 F), 18/06/2015, FAL (1 M); P46: 16/06/2015, FAL (1 F).Iberian distribution. Widespread and very common all over the Peninsula.Oedaleusdecorus  IUCN category: Least ConcernMaterial examined. B13-2: 22/06/2015, FAL (1 M); P31: 19/06/2014, FAL (1 M).Iberian distribution. Widespread and very common all over the Peninsula.PageBreakOedipodacaerulescens  IUCN category: Least ConcernMaterial examined. A29: 31/05/2015, OAK (1 F), 22/06/2015, OAK (1 M); A33: 28/05/2015, FAL , EDG (2 F), PIN (1 M), 20/06/2015, FAL (3 F), EDG (1 F), PIN (1 M); A57: 20/06/2015, EUC (1 M); A63: 26/05/2015, EDG (3 M); A68: 20/06/2015, OAK (1 F); A76: 27/05/2015, PIN (1 M), 19/06/2015, PIN (1 F); A78: 13/05/2015, PIN (1 M), 30/05/2015, FAL (1 F), PIN (1 F), 21/06/2015, PIN ; A79: 25/05/2015, PIN (1 M), 26/05/2015, FAL (1 M), 23/06/2015, PIN (1 M); A85: 30/05/2015, OAK (2 F); A90: 24/06/2014, EDG (1 M); A98: 27/05/2015, PIN (1 M); A99: 25/05/2015, EDG (1 F), PIN (1 F), 19/06/2015, EDG (1 M), FAL (2 F); A152: 15/06/2015, FAL (1 F); B1: 16/06/2015, EDG (1 F), EUC ; B5: 16/06/2015, EUC (3 M); B6: 18/06/2015, EDG (1 M), EUC ; B13-2: 22/06/2015, MIX (1 M); B14: 01/06/2015, EUC (1 M), 23/06/2015, EUC (1 M); P3: 18/06/2015, EDG (1 M); P5: 18/06/2014, EDG (2 M); P6: 15/06/2014, PIN (1 F); P9: 20/06/2014, EUC (2 M); P13: 16/06/2014, PIN (1 F); P17: 15/06/2014, FAL (1 F), PIN (1 M); P18: 14/06/2014, OAK ; P20: 18/06/2014, MIX (1 F); P22: 04/06/2014, PIN , 25/06/2014 ; P24: 13/06/2014, EDG (2 F); P27: 16/06/2015, EDG (1 F), EUC ; P29: 19/06/2014, EDG (1 F), EUC (1 M); P30: 15/06/2015, MIX ; P32: 19/06/2014, OAK ; P39: 18/05/2015, EDG (1 M), 15/06/2015, FAL , OAK ; P47: 16/06/2014, FAL (1 F); P48: 02/06/2014, OAK , 24/06/2014, OAK ; P49: 01/07/2014, MIX .Iberian distribution. Widespread and very common all over the Peninsula.Oedipodacharpentieri Fieber, 1853 IUCN category: Least ConcernMaterial examined. A76: 27/05/2015, FAL (1 M), 19/06/2015, FAL ; A78: 30/05/2015, FAL (6 F); A79: 26/05/2015, FAL (4 M); A85: 21/06/2015, OAK (1 F); A90: 04/06/2014, EDG (1 M); A98: 27/05/2015, FAL , 19/06/2015, FAL ; A99: 19/06/2015, FAL (3 F); B6: 18/06/2015, FAL ; B14: 01/06/2015, FAL (1 M); P1: 25/06/2014, FAL (1 F); P6: 15/06/2014, FAL ; P11: 29/05/2014, FAL (1 F), OAK ; P15: 18/06/2014, FAL (1 M); P17: 15/06/2014, FAL (1 F); P19: 31/05/2014, FAL (2 F); P20: 18/06/2014, EDG (1 F); P22: 25/06/2014, FAL (1 M); P23: 22/05/2015, FAL (1 F), 18/06/2015, FAL (2 M); P32: 19/06/2014, FAL (1 M); P47: 16/06/2014, FAL (1 F); P48: 02/06/2014, EDG (1 M), 24/06/2014, FAL (1 M).Iberian distribution. This species is widespread throughout the Mediterranean region of the Peninsula but is apparently absent in most of the extreme north-west, in the northern Atlantic littoral and in the Pyrenees.Sphingonotus (Sphingonotus) lluciapomaresi  IUCN category: Least ConcernMaterial examined. A76: 19/06/2015, PIN (1 M); A78: 21/06/2015, EDG (1 M), PIN (2 F); A79: 23/06/2015, PIN (1 M); A98: 19/06/2015, FAL (1 M), PINPageBreak(1 M); A99: 19/06/2015, FAL (1 M); P12: 20/06/2014, OAK (1 M); P13: 16/06/2014, PIN (1 M); P22: 25/06/2014, PIN ; P23: 18/06/2015, EDG , PIN (1 F).Iberian distribution. This species is endemic to the Iberian Peninsula and has a wide and central distribution in the Peninsula, occurring also in the south of Spain. Our record is the south-westernmost, extending the species known distribution range.Orthoptera species known for Portugal, and the species richness recorded augment the relevance of Castro Verde SPA in terms of biodiversity. The 35 species recorded are distributed among six of the 11 families known to occur in Portugal. From the species recorded, two are Red-Listed as threatened or near-threatened at the European level, and five are Iberian endemics. The diversity of species observed was probably driven by landscape heterogeneity, as we found species exclusive of both fallow land and forest plantations. Overall, our results point out the importance of Castro Verde SPA for the conservation of Orthoptera, and help identify some threats that may affect this value in the near future.The results of the present study expand the list of SPA in terms of Orthoptera diversity, it is likely that other species are present in the area. A more comprehensive species list can be obtained by using other methods, given the distinct phenologic and ecologic characteristics of certain groups and their ability for crypsis. For instance, beating should be used to collect tree and shrub-dwelling species, which are often difficult to detect. The use of bioacoustic exploration can also be advantageous to obtain information on less conspicuous orthopterans such as crickets and bush-crickets . In fact, despite the occurrence of Gryllotalpa spp. in Castro Verde SPA, which is referred in the work of Thyreonotusbidens , whose adults typically occur in autumn (September-November). This species is likely to occur in Castro Verde SPA, as it is usually found in forests with undergrowth  and five are Iberian endemics: Dociostaurushispanicus, Euryparyphesterrulentus, Omocestuspanteli, Platystolusmartinezii and Sphingonotuslluciapomaresi. The presence of these elements underpins the natural value of Castro Verde SPA, as endemic and threatened species are often used in marking biodiversity hotspots and prioritizing areas for conservation (Myers 2000). Some of our records complement the current knowledge on species geographic distribution in southern Europe. Five species occurring in southern Spain expanded their known distribution to southern Portugal, namely: Euryparyphesterrulentus, Platycleisfalx, Platycleisintermedia, Sphingonotuslluciapomaresi and Tylopsislilifolia. The two endemic species, Platystolusmartinezii and Dociostaurushispanicus, previously recorded mostly in the centre of the Iberian Peninsula, extend their known distribution to the south-westernmost location in Europe. These new data referring to species diversity and distribution reflect the scarcity of studies on the orthopterofauna in Portugal, and indicate that some species could have a wider distribution in the Iberian Peninsula than is currently known.Out of the 35 species recorded, two are considered threatened or near-threatened under the IUCN criteria  and the maximum number of species (29) were found in fallows, whereas the maximum GCIn (0.49) in plantations was found in mixed pine-oak forests. The importance of fallows is also highlighted by the presence of uncommon species, which includes the two species of conservation concern recorded in our study, Dociostaurushispanicus and Platycleisfalx. In contrast to fallows, mixed pine-oak plantations had a relatively low species richness (16), with the high GCIn observed therein being a consequence of the high number of endemic, rare and flightless species. For instance, within forest plantations the species with the highest GCIn, Euryparyphesterrulentus, was only found in mixed pine-oak. The lowest value of both GCIn (0.44) and species richness (13) was observed in eucalyptus plantations. This was probably because eucalyptus plantations were composed by old trees, with a more closed canopy than other forest types. Moreover, the understorey often had overgrown and dense bushes and few grassy areas, and these conditions are largely unfavourable for open-area species that dominated the Orthoptera assemblages in our study area. These results are in line with other studies showing low abundance of macro-arthropods in Eucalyptus plantations in the Mediterranean region  changes in the rotational farming system, with a shift from the production of dry cereals and extensive sheep grazing on fallows and pastures, to the specialized production of sheep and cattle; and (ii) the expansion of permanent crops, particularly olive groves, though these occur mostly in the periphery of Castro Verde SPA due to legal restrictions within the area . As shown in the Castro Verde SPA, a large number of endemic and threatened species may be associated to low-intensity farmland systems (SPA.Overall, our study provided new information on the diversity and ecology of reatened , there m schemes . Third, lsewhere . This islsewhere . Finally systems , and thu


Both turned out to be tight binding inhibitors, with IC50 values of 7 nM (4a) and 5 nM (5) and an apparent Ki value of 0.4 nM for both. Compounds 4a and 5 reduced cellular CK2 activity, indicating cell permeability. Cell viability was substantially impaired in LNCaP cells, as well as apoptosis was induced, which was not appearing in non-neoplastic ARPE-19 cells. Co-crystallization of 4a and 5 revealed an unexpected π-halogen bond of the chloro substituent at C9 with the gatekeeper amino acid Phe113, leading to an inverted binding mode in comparison to parent compound 4b, with the Cl at C6 instead, which was co-crystallized as a control. This indicates that the position of the chloro substituent on ring A of the dibenzofuran scaffold is responsible for an inversion of the binding mode that enhances potency.Human protein kinase CK2 is an emerging target for neoplastic diseases. Potent lead structures for human CK2 inhibitors are derived from dibenzofuranones. Two new derivatives, 7,9-dichloro-1,2-dihydro-8-hydroxy-4-[(4-methoxyphenylamino)-methylene]dibenzo[ Human CK2 is an ubiquitous protein kinase, which catalyzes the phosphorylation of serine/threonine residues within a consensus sequence which is present in a remarkable number of human proteins . Meanwhi2β2 stoichiometry. Despite its denomination, the “regulatory” β-subunit is not directly involved in general regulating CK2 enzymatic activity, but appears to be rather responsible for substrate specificity and stabilization of the holoenzyme furan-3 (2H)-one , to similar extent as CK2, and turned out to be cell-permeable and capable of inducing apoptosis in the prostate cancer cell LNCaP. Most recently it was shown, that—depending on the salt concentration—4-carboxy-6,8-dibromo-flavonol, also a nanomolar inhibitor of CK2, forms an unusual π-halogen bond with an aromatic side chain of the catalytic α-subunit dibenzofuran-3(2H)-one) and 5 ((E)-1,3-dichloro-6-[(4-methoxyphenylimino)methyl]dibenzofuran-2,7-diol)  , both co2, Supplementary Information Scheme S1) to afford tetrahydrocarbazoles cyclohexanone derivatives to yield the dibenzofurans as described here furan-3(2H)-one (4a), 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylene]dibenzofuran-3(2H)-one (4b), and (E)-1,3-dichloro-6-[(4-methoxyphenylimino)methyl]dibenzofuran-2,7-diol (5) has been described before dibenzocyclohexanone  were heated under reflux in dichloromethane (50 mL) for 60 min to yield a green coloured solution. After cooling to room temperature 2,6-dichloro-1,4-benzoquinone  dissolved in glacial acetic acid (10 mL), followed by refluxing again for 30 min. Filtration of the precipitate formed after standing overnight yielded 1.2 g (14.9%) of a pure orange product (mp 204 °C). IR (KBr) 1665 cm−1. 1H-NMR (pyridine-d5) δ 2.20 , 2.73 , 7.17 , 8.00 , 8.73 , 11.93 . C20H15Cl2NO3 (388.25): C, 61.87; H, 3.89; Cl, 18.26; N, 3.61. Found C, 61.72; H, 3.88; Cl, 18.39; N, 3.50. Purity was >95%.2,6-Dichloro-1,4-benzoquinone  was mixed with 2-[(4-methylphenylamino)methylene]cyclohexanone  in dichloromethane (50 mL) and heated under reflux for 60 min to yield a green coloured solution. After cooling to room temperature, 2,6-dichloro-1,4-benzoquinone  dissolved in glacial acetic acid (10 mL) was added followed by refluxing again for 30 min. After standing overnight, filtration of the precipitate yielded 1.55 g (20%) of pure product (mp 263 °C). Yellow needles (Ethanol). IR (KBr) 1650 cm−1. C20H13Cl2NO4 (402.2). 1H-NMR (DMSO-d6) δ 3.83 , 6.95 , 7.0–7.6 , 7.73 , 8.14 , 9.16 . C, 59.72; H, 3.26; Cl, 17.63; N, 3.48. Found C, 59.49; H, 3.10; Cl, 18.00; N, 3.64. Purity was determined to be >95%.2-[(4-Methoxyphenylamino)methylene]cyclohexanone  and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone  were suspended in dichloromethane (50 mL), glacial acetic acid (30 mL) was added and the mixture was refluxed for 10 min. After cooling the red precipitate was filtered and recrystallized from toluene to yield 0.56 g (70%). Mp 210 °C. IR (KBr) 1625 cmb,d]furan derivatives were subjected to the CE-based CK2 assay furan derivatives 4b, 4a and 5 were tested on selective inhibition toward one of the two isoform CK2 holoenzymes (CK2α2β2 and CK2α’2β2). For this purpose, their IC50 were determined following the protocol of the recently described whole-cell CK2 selectivity assay  for 1 min followed by vitrification in liquid nitrogen.The crystals obtained in this way were cryo-protected by incubation in a vitrification solution [32%  in order to unambiguously validate the positions of the inhibitors’ Cl-substituents via their contribution to anomalous diffraction. PDB codes: 4a: 5N9N; 4b: 5N9L; 5: 5N9K.The subsequent X-ray diffractometry experiments were performed at beamline ID23-2 of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, and at beamline X06DA (PXIII) of the Swiss Light Source (SLS) in Villigen, Switzerland. We collected high resolution data from each of the crystals ; in the All X-ray diffraction data were integrated with XDS  followedhsCK2α1–335 complexes with 4b and 4a were generated with the corresponding map calculation routine of PHENIX [The structures were solved by molecular replacement with PHASER  using thf PHENIX .


The obtained 3-bromo-4-phenylcoumarin was transformed into 4-phenylcoumarin derivatives bearing C–H, C–S, C–N, and C–C bonds at 3-position.Various 4-aryl-3-bromocoumarins were smoothly obtained in moderate yields in one pot by treating 3-aryl-2-propynoic acids with diaryliodonium triflates and K Coumarin is a benzo-α-pyrone and one of the typical heterocyclic compounds. The importance of coumarins arises from the fact that the coumarin skeleton is present in many natural products extracted from plants –3 and so3-catalyzed microwave irradiation of phenols and propynoic acids [2-catalyzed oxidative cyclocarbonylation of 2-vinylphenols at 110 °C [3-catalyzed areneselenyl-cyclization of aryl 2-alkynoates with ArSeSeAr at rt [2 at 120 °C [2H/AgNO3(cat.)/K2S2O8 at 60 °C [2/1-trifluoromethyl-3,3-dimethyl-1,2-benziodoxole (Togni reagent) at 60 °C [2P(=O)H/Ag2CO3(cat.)/Mg(NO3)2 at 100 °C [2CO2Et/fac-Ir(ppy)3(cat.) under irradiation at rt [n-Bu)4NBr /K2S2O8 at 90 °C [2H/Eosin Y(cat.)/tert-butyl hydrogen peroxide (TBHP) at rt [2NHNH2/n-Bu4NI(cat.)/TBHP at 80 °C [Comprehensive synthetic studies of coumarins and their derivatives have been carried out –16. Typiic acids , the Pd at rt , and witat 80 °C .2S2O8 at 90 °C [tert-butyl peroxybenzoate (TBPB)/acetonitrile at 130 °C [N-iodosuccinimide (NIS) at rt [N-bromosuccinimide (NBS) at rt [In addition, the formation of coumarins via the bromine-radical-mediated reaction of aryl 2-alkynoates with TBAB/Kat 90 °C , the cyat 130 °C , the sunS) at rt , and theS) at rt , where tt-BuOK under toluene refluxing conditions provides the corresponding aryl carboxylates in good yields [On the other hand, diaryliodonium salts are very useful for the C-arylation of active CH groups, the O-arylation of OH groups, and the N-arylation of NH groups under metal-free conditions –39. For d yields –41. Howed yields . Therefo2S2O8 in a mixture of 1,2-dichloroethane and water [Here, as part of our ongoing investigation of the synthetic use of diaryliodonium salts for the preparation of heterocyclic compounds –46, we wnd water .1a, 0.5 mmol) with diphenyliodonium triflate  in the presence of CuCl (5 mol %) and K2CO3 (1.0 equiv) in dichloromethane (3.0 mL) at 40 °C based on a previous report [2Aa) in 46% yield, as shown in 2Aa was increased to 74%  with N-iodosuccinimide /BF3·Et2O (2.0 or 1.1 equiv) was studied based on the previous reports [3Aa’) was obtained in low to moderate yields /BF3·Et2O (1.1 equiv), with 1,3-diiodo-5,5-dimethylhydantoin /BF3·Et2O (1.1 equiv), and with 1,3-dibromo-5,5-dimethylhydantoin /BF3·Et2O (1.1 equiv) was carried out to form 3-bromo-4-phenylcoumarin (3Aa), 3-iodo-4-phenylcoumarin (3Aa’), and 3-bromo-4-phenylcoumarin (3Aa) in 28, 49 and 46% yields, respectively (2Aa with molecular iodine (2.0 equiv)/K2CO3 (2.0 equiv) did not generate 3-iodo-4-phenylcoumarin (3Aa’) at all (2Aa) does not proceed efficiently. Then, the bromo-radical-based cyclization of phenyl 3-phenyl-2-propynoate (2Aa) with tetrabutylammonium bromide /Na2S2O8 (1.5 equiv) [3Aa) in 68% yield /Na2S2O8 (1.5 equiv) was carried out, the yield of iodocyclization product 3Aa’ was decreased to 45% (2Aa with TBAB (2.0 equiv)/Na2S2O8 (1.0 equiv) in a mixture of DCE and water at 90 °C was carried out, 3-bromo-4-phenylcoumarin (3Aa) was obtained in 79% yield  were decreased to 51 and 69%, respectively 2S2O8, and Oxone® (2KHSO5·KHSO4·K2SO4), the yields of 3-bromo-4-phenylcoumarin (3Aa) were decreased to 71, 69 and 37%, respectively (2Aa with TBAB (2.0 equiv)/Na2S2O8 (1.0 equiv) in a mixture of DCE and water at 90 °C for 19 h was the most efficient, giving 3-bromo-4-phenylcoumarin (3Aa) in good yield  at all , entry 65 equiv)  in a mix8% yield , entry 7d to 45% , entry 89% yield , entry 9od yield , entry 93 from 3-aryl-2-propynoic acids 1 was carried out. 3-Aryl-2-propynoic acids 1, such as 3-phenyl-2-propynoic acid (1a), 3-(o-methylphenyl)-2-propynoic acid (1b), 3-(m-methylphenyl)-2-propynoic acid (1c), 3-(p-methylphenyl)-2-propynoic acid (1d), 3-(p-methoxyphenyl)-2-propynoic acid (1e), 3-(p-fluorophenyl)-2-propynoic acid (1f), 3-(p-chlorophenyl)-2-propynoic acid (1g), 3-(o-chlorophenyl)-2-propynoic acid (1h), 3-(m-chlorophenyl)-2-propynoic acid (1i), 3-(p-bromophenyl)-2-propynoic acid (1j), 3-(p-biphenyl)-2-propynoic acid (1k), 3--2-propynoic acid (1l), and 3--2-propynoic acid (1m), were treated with diphenyliodonium triflate  in the presence of CuCl and K2CO3 in CH2Cl2 for 3 h under refluxing conditions. After removal of the solvent, the second-step treatment of the reaction mixture with TBAB (2.0 equiv) and Na2S2O8 (2.0 equiv) in a mixture of DCE and water at 90 °C for 19 h gave 4-aryl-3-bromocoumarins 3Aa–3Am in moderate yields, respectively, as shown in 1a, 8 mmol) with diphenyliodonium triflate A in the presence of CuCl and K2CO3 in CH2Cl2 for 3 h, followed by removal of the solvent and the reaction with TBAB and Na2S2O8 in a mixture of DCE and water at 90 °C for 19 h gave 3-bromo-4-phenylcoumarin (3Aa) in 52% yield. For 3-aryl-2-propynoic acids bearing heteroaromatic groups, treatment of 3-(benzothiophen-2’-yl)-2-propynoic acid (1n) and 3-(benzofuran-2’-yl)-2-propynoic acid (1o) under the same procedure and conditions gave the corresponding coumarins 3An and 3Ao in moderate yields, respectively. Under the present procedure and conditions, use of 2-hexynoic acid (1p), a 3-alkyl-2-propynoic acid, provided 3-bromo-4-propylcoumarin (3Ap) in 42% yield, as shown in Finally, based on the results in A). Treatment of 3-phenyl-2-propynoic acid (1a) with diaryliodonium triflates (1.0 equiv), such as di(p-methylphenyl)iodonium triflate (B), di(tert-butylphenyl)iodonium triflate (C), di(p-chlorophenyl)iodonium triflate (D), and di(p-bromophenyl)iodonium triflate (E), in the presence of CuCl and K2CO3 in CH2Cl2 for 3 h under refluxing conditions, followed by removal of the solvent and the reaction with TBAB (2.0 equiv) and Na2S2O8 (2.0 equiv) in a mixture of DCE and water at 90 °C for 19 h gave 3-bromo-4-phenylcoumarin derivatives 3Ba–3Ea bearing methyl, tert-butyl, chloro, and bromo groups at 7-position in good to moderate yields, respectively, as shown in Then, other diaryliodonium triflates were used instead of diphenyliodonium triflate (3Aa) with Zn in ethanol under refluxing conditions gave 4-phenylcoumarin (4Aa) in 81% yield. Treatment of 3-bromo-4-phenylcoumarin (3Aa) with p-toluenethiol/N,N’-dimethylethylenediamine (DMEDA)/K2CO3 in the presence of CuI in toluene at refluxing temperature and with p-methoxybenzamide/DMEDA/K2CO3 in the presence of CuI in toluene at refluxing temperature generated 3-(4-methylbenzenesulfenyl)-4-phenylcoumarin (5Aa) and 3-(4-methoxylbenzoylamino)-4-phenylcoumarin (6Aa) in 62 and 51% yields, respectively. The Pd-catalyzed coupling reactions of 3-bromo-4-phenylcoumarin (3Aa) with 4-methylstyrene/K2CO3/PdCl2(Ph3P)2, with phenylacetylene/PdCl2(Ph3P)2/Et3N and with PhB(OH)2/K2CO3/PdCl2(Ph3P)2 provided the corresponding C–C bonded coumarin derivatives 7Aa, 8Aa, and 9Aa in 79, 60 and 76% yields, respectively  from 3-phenyl-2-propynoic acid (1a) was carried out in the presence of 2,2,6,6-tetramethylpiperidine 1-oxyl radical  or 2,6-di at the second step under the same procedure and conditions, but 3-bromo-4-phenylcoumarin (3Aa) was not obtained at all in both reactions. Thus, the present bromocyclization of the formed phenyl 3-phenyl-2-propynoate (2Aa) with TBAB and Na2S2O8 in a mixture of DCE and water is a radical-mediated bromocyclization reaction. X-ray crystallographic analysis of 3-bromo-7-chloro-4-phenylcoumarin (3Da), which was formed by the subsequent treatment of 3-phenyl-2-propynoic acid (1a) with di(p-chlorophenyl)iodonium triflate (D) and then with TBAB and Na2S2O8, was carried out, as shown in To support the present bromocyclization reaction to form 4-aryl-3-bromocoumarins with TBAB and Na1 with diaryliodonium triflate in the presence of K2CO3 and CuCl occurs to form aryl 3-aryl-2-propynoate 2 (1st step). The bromocyclization of aryl 3-aryl-2-propynoate 2 with TBAB and Na2S2O8 proceeds via a bromoradical addition to the triple bond to form very reactive vinyl radical I [Ipso-cyclization of the vinyl radical I occurs to form spiro radical intermediate II. Then, β-cleavage of the spiro radical intermediate II proceeds to form carboxyl radical III. 6-Exo-trig cyclization of the carboxyl radical III onto the aromatic ring takes place to form adduct radical IV, which would be rapidly oxidized by Na2S2O8 to form cation intermediate V. Smooth deprotonation of cation intermediate V occurs to generate 4-aryl-3-bromocoumarin 3 (2nd step). The radical ipso-cyclization of the formed vinyl radical and its 1,2-carboxyl group migration agree with previously reported results [The O-arylation of 3-aryl-2-propynoic acid adical I . Ipso-cy results –34.2CO3 and CuCl, and then with tetrabutylammonium bromide (TBAB) and Na2S2O8 gave 4-aryl-3-bromocoumarins bearing hydrogen, methyl, tert-butyl, chloro, and bromo groups at 7-position in moderate yields, respectively. In one of the obtained 4-aryl-3-bromocoumarins, the C–Br bond of 3-bromo-4-phenylcoumarin was smoothly converted into 4-phenylcoumarins bearing C–H, C–S, C–N, and C–C bonds at 3-position. We believe the present method will be useful for the preparation of various 4-arylcoumarin derivatives due to its simple one-pot synthesis.The successive treatment of 3-aryl-2-propynoic acids with diaryliodonium triflates in the presence of KFile 13Aa–3Ap, 3Ba–3Ea, and 4Aa–9Aa, and X-ray analytical data of 3Da.NMR charts of all coumarin derivatives


E)-2-(2-aryl)ethenyl]-2,3-dihydroquinazolin-4-ones – products of three-component reactions between benzylamine, isatoic anhydride and furyl- or thienyl-acrolein in the presence of catalytic quantity of p-TsOH – were studied by X-ray diffraction.The molecular and crystal structures of two 3-benzyl-2-, whereas the amido N atom is flat [sum of the bond angles is 359.3° for both (I) and (II)]. The furyl- and thienylethenyl substituents in (I) and (II) are planar and the conformation about the bridging C=C bond is E. These bulky fragments occupy the axial position at the quaternary C atom of the tetrahydropyrimidine ring, apparently, due to steric reasons. In the crystals, molecules of (I) and (II) form hydrogen-bonded helicoidal chains propagating along [010] by strong intermolecular N—H⋯O hydrogen bonds.The chiral title compounds, C Remarkably, these bulky fragments occupy the axial position at the quaternary C2 carbon atom of the tetrahydropyrimidine ring, apparently, due to the steric interaction with the benzyl substituent.The tetrahydropyrimidine ring in (I)The molecules of (I)In the crystals of (I)E)-2-(2-aryl)ethenyl]-2,3-dihydroquinazolin-4-ones (I)et al., 20173-Benzyl-2- or EtOAc–EtOH [for (II)] to afford the analytically pure samples of the target products.3-Benzyl-2-[(E)-2-(furan-2-yl)ethenyl]-2,3-dihydroquinazolin-4(1H)-one (I). Colourless prisms. Yield is 2.31 g (76%). M.p. = 427.1 K (hexane–EtOAc). IR (KBr), ν (cm−1): 3376, 1645, 1611. 1H NMR : δ = 3.86 , 4.61 , 4.98 , 5.59 , 6.24 , 6.25 , 6.34 , 6.59 , 6.83 , 7.24–7.34 , 7.96 . 13C NMR : δ = 46.7 (CH2N), 69.8 (C2), 109.9, 111.5, 114.8, 119.1, 121.1, 123.6, 127.5, 127.9, 128.7, 128.7, 133.6, 115.7, 136.9, 145.4, 151.1, 142.7 , 162.9 (NCO). MS : m/z = 330 [M]+ (93), 239 (100), 197 (71), 170 (20), 160 (19), 120 (40), 106 (55), 91 (81), 76 (58), 65 (45), 51 (37), 43 (20).3-Benzyl-2-[(E)-2-(thiophen-2-yl)ethenyl]-2,3-dihydroquinazolin-4(1H)-one (II). Yellow prisms. Yield is 2.39 g (75%). M.p. = 434.1–435.1 K (EtOAc–EtOH). IR (KBr), ν (cm−1): 3306, 1625, 1506. 1H NMR : δ = 4.05 , 5.15-5.17 , 6.00 , 6.69–6.76 , 6.96 , 7.07 , 7.07 , 7.23–7.32 , 7.38 , 7.66 . 13C NMR : δ = 47.0 (CH2N), 69.6 (C2), 115.1, 115.2, 118.0, 125.7 (2C), 126.4, 127.7, 127.9, 128.1, 128.3, 128.4, 129.0, 134.0, 138.3, 140.8, 147.1 , 162.4 (NCO). MS : m/z = 346 [M]+ (76), 255 (100), 237 (93), 213 (37), 106 (14), 91 (99), 65 (13).SCALA program  = 1.2Ueq(N)]. The other hydrogen atoms were placed in calculated positions with C—H = 0.95–1.00 Å and refined in the riding model with fixed isotropic displacement parameters [Uĩso(H) = 1.2Ueq(C)].The hydrogen atoms of the amino groups were localized in difference-Fourier maps and refined isotropically with fixed displacement parameters [I/σ statistics for high-angle reflections we selected a larger exposure time, which resulted in some intensity overloads in the low-angle part of the area. These corrupted intensities were excluded from final steps of the refinement. (2) In the current setup of the instrument, the low-temperature device eclipses a small region of the detector near its high-angle limit. This resulted in zero intensity of some reflections. (3) In the case of (II)A relatively large number of reflections (a few dozen) were omitted due to the following reasons: (1) In order to achieve better 10.1107/S2056989017017479/ld2142sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989017017479/ld2142Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017017479/ld2142IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017017479/ld2142Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017017479/ld2142IIsup5.cmlSupporting information file. DOI: 1589395, 1589394CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their structures were defined by analyzing spectra data, especially 2D NMR and electronic circular dichroism (ECD). The structure of the known compound lobocrasol (7) was revised. Cytotoxicity potential of the isolated compounds was reported, too.Five new isoprenoids, 3,4,8,16-tetra- Sarcophyton are the predominant species in many coral reefs. They are endowed with diverse secondary metabolites, including cembranoids + ion peak obtained by (+)-HRESIMS. The 13C NMR data showed 20 carbon signals, including evidence of an α,β-unsaturated enone , an additional double bond (δC 133.3 and 128.4), and four oxygenated carbons  , 1.69 , 1.35 , 1.03 ], an olefinic methine [δH 5.15 ], and two oxygenated methines  (7)  . The abo2-5/H2-6/H-7, H2-9/H2-10/H-11, and H2-13/H2-14  data, analysis of 3JH-H values, and comparison of carbon chemical shifts. As depicted in 3-18/H-7 supported that they oriented on the same face, and casually assigned these protons to be α-oriented. The downfield-shifted proton H-5a (δH 3.04), deshielded by the C-2 carbonyl group, showed an NOE correlation with H-9b (δH 1.25), while H3-19 showed correlations with both H-7 and H-9a (δH 1.50), suggesting that H-7 and C-9 were located in anti-orientation, thus suggesting a β-orientation for H3-19. The strong NOE cross-peaks of H3-18/H-16 suggested the α-orientation of H-16. A weak NOE correlation was observed between H-16 and H-3, while H-3 also showed a correlation with H3-18, and suggested H-3 should be located anti to H-16. This was confirmed by comparing the small 3JH-H values with those of cyclopent-2-enone analogues, in which adjacent protons with a trans configuration showed small coupling constants D of 4 was repeatedly measured and the positive sign was always obtained. The reason for this discrepancy with literature might need to be studied further.pic data  were suphine (6) b, but ar5) showed a sodiated adduct ion peak at m/z 501.3552, implying a molecular formula of C29H50O5. Its 1H NMR spectrum disclosed three methyl singlets (one assignable to be olefinic methyl), four methyl doublets, four oxygenated-methine protons , as well as a complex array of aliphatic methine and methylene protons (13C NMR spectrum showed a tetra-substituted double bond at δC 148.2 (C-17) and 134.2 (C-20) and five oxygenated carbons at δC 68.2 (C-3), 77.4 (C-5), 76.6 (C-6), 69.6 (C-11), and 73.0 (C-16). The above spectroscopic data were very similar to those of a Δ17(20) sterol, halicrasterol B, with the acetyl signals disappeared in 5, suggesting that 5 is a deacetyl derivative of halicrasterol B. This was secured by analyzing the 1H–1H COSY correlations and the HMBC experiments , MDA-MB-231 (human breast adenocarcinoma), and A-549  cancer cells were assayed. Furthermore, compounds 6, 8, 9, and 12 were also assayed for cytotoxicity against MOLT-4 (human acute lymphoblastic leukemia), SUP-T1 (human T-cell lymphoblastic lymphoma), and U-937 (human histiocytic lymphoma) cell lines. The results showed that compound 12 exhibited cytotoxicity effect against MDA-MB-231, MOLT-4, SUP-T, and U-937 cell lines with IC50 values of 13.8, 6.7, 10.5, and 17.7 μg/mL, respectively, while compound 13 was found to possess cytotoxicity against HepG2, MDA-MB-231, and A-549 cell lines with IC50 values of 9.7, 14.0, and 15.8 μg/mL. The remaining compounds were found to be not cytotoxic against the above cancer cell lines, with IC50 values higher than 20 μg/mL.The cytotoxicity of the isolates 1H NMR and 13C NMR spectra with the chemical shifts shown as ppm referenced to the solvent residue of CDCl3 (δH 7.26 ppm and δC 77.0 ppm), DMSO-d6 (δH 2.50 ppm and δC 39.5 ppm), and CD3OD (δH 3.31 ppm and δC 49.0 ppm). A Bruker APEX II mass spectrometer equipped with an ESI ionization source  was used for acquiring high-resolution mass data. The HPLC system was composed of a Shimadzu LC-10ATVP series pump, a UV detector , and an ODS column .The optical rotation values and IR spectra were recorded on a JASCO P-1020 digital polarimeter  and JASCO J-815 spectrophotometer (JASCO Corporation), respectively. A Varian 400 NMR instrument was used to record the S. glaucum was performed off the coast of Jihui Fishing Port, Taitung county, Taiwan, in March 2013. A −20 °C freezer was used for specimen storage until extraction. Prof. Chang-Feng Dai performed the species identification, and a voucher specimen (JiH-201304) of this soft coral has been deposited in National Sun Yat-sen University.The collection of S. glaucum (195 g) were chopped and soaked (4 × 24 h) with EtOAc (4 × 2 L). After the solvent was evaporated, a residue (17.35 g) was obtained. The residue of the EtOAc layer was chromatographed by a silica gel column, using a stepwise gradient system composed of EtOAc/n-hexane  and acetone/EtOAc , and eventually washed by MeOH to afford 25 fractions according to TLC analysis. Fraction 19 was subjected to repeated column chromatography (CC) by Sephadex LH-20 , C18 gel , and silica gel  to obtain subfractions (SFr.) 19-1 and 19-2. In turn, SFr.19-1 was subjected to semipreparative HPLC  to give compound 6 (23.4 mg), while compound 2 (1.3 mg) was obtained from SFr.19-2 using a CH3CN-H2O (53%) solvent system. Fraction 20 was subjected to an open column chromatography using Sephadex LH-20  and subsequently purified by C18 column chromatography  to yield two subfractions (SFr. 20-1 and 20-2). Compounds 11 (2.7 mg) and 10 (2.0 mg) were purified from SFr. 20-1 using HPLC . SFr.20-2 was fractionated into two subfractions (SFr. 20-2-1 and SFr. 20-2-2) using C18 CC . Compounds 8 (1.7 mg) and 9 (6.9 mg) were yielded from SFr. 20-2-1 using HPLC , and compounds 3 (2.7 mg) and 15 (2.1 mg) were obtained from SFr. 20-2-2 using eluent of CH3CN-H2O (33%). Four subfractions (SFr. 22-1–SFr.22-4) were obtained from fraction 22 by C18 CC . Compounds 4 (4.9 mg) and 7 (4.7 mg) were purified from SFr.22-1 using HPLC . Compounds 1 (2.2 mg), 12 (33.6 mg), and 13 (4.8 mg) were isolated from SFr. 22-2, SFr. 22-3, and SFr.22-4, respectively, using HPLC . Fraction 23 was subjected to CC using a C18 column  and subsequently by HPLC  to give compounds 14 (3.6 mg) and 5 (1.4 mg).The lyophilized samples of epi-lobocrasol (1): colorless oil; c 1.00, CHCl3); ECD (MeOH) λmax (Δε) 211 (−0.62), 233 (+1.79), 324 (−0.62); IR (KBr) νmax 3419, 2925, 2855, 1698, 1651, 1455, 1380, 1332, 1246, 1080, and 1049 cm−1; 13C and 1H NMR data, see m/z 357 [M + Na]+; HRESIMS m/z 357.2034 [M + Na]+  for C20H30O4Na, 357.2036).3,4,8,16-Tetra-2): colorless oil; c 0.63, CHCl3); IR (KBr) νmax 2926, 2855, 1653, 1514, 1455, 1381, 1239, 1164, and 1039 cm−1; 13C and 1H NMR data, see m/z 341 [M + Na]+; HRESIMS m/z 341.2085 [M + Na]+ .1,15β-Epoxy-deoxysarcophine (2-sarcophine (3): colorless oil; c 1.28, CHCl3); IR (KBr) νmax 3444, 2965, 2929, 2857, 1747, 1668, 1638, 1455, 1373, 1317, 1260, 1127, 1062, and 1018 cm−1; 13C and 1H NMR data, see m/z 373 [M + Na]+; HRESIMS m/z 373.1986 [M + Na]+ .3,4-Dihydro-4α,7β,8α-trihydroxy-Δent-Sarcophyolide E (4): colorless oil; c 1.70, CHCl3); ECD (MeOH) λmax (Δε) 230 (+1.41), 254 (−0.40); 13C and 1H NMR data, see m/z 373 [M + Na]+; HRESIMS m/z 373.1984 [M + Na]+ .5): white powder; c 0.78, CHCl3); IR (KBr) νmax 3330, 2922, 2854,1652, 1598, 1455, 1374, and 1023 cm−1; 13C and 1H NMR data, see m/z 501 [M + Na]+; HRESIMS m/z 501.3552 [M + Na]+ .16-Deacetyl-halicrasterol B (6): colorless oil; c 4.98, CHCl3); lit. c 0.01, CHCl3); ECD (MeOH) λmax (Δε) 229 (+1.22), 248 (−1.58); MS, 1H and 13C NMR data were found to be in full agreement with literature data [Sarcophine (ure data .7): colorless oil; c 0.05, CHCl3); lit. c 0.10, CHCl3); ECD (MeOH) λmax (Δε) 215 (+0.57), 240 (−1.66), 325 (+0.81); MS, 1H and 13C NMR data were found to be in full agreement with literature data [Lobocrasol (2-sarcophine (8): colorless oil; c 0.50, CHCl3); lit. c 0.10, CHCl3); MS, 1H and 13C NMR data were found to be in full agreement with literature data [3,4-Dihydro-4α-hydroxy-Δure data ,13.2-sarcophine (9): colorless oil; c 1.53, CHCl3); lit. c 0.10, CHCl3); MS, 1H and 13C NMR data were found to be in full agreement with literature data [3,4-Dihydro-4β-hydroxy-Δure data ,13.10): colorless oil; c 0.49, CHCl3); lit. c 0.10, CHCl3); MS, 1H and 13C NMR data were found to be in full agreement with literature data [Srassumol A (ure data .11): colorless oil; c 1.28, CHCl3); lit. c 0.60, CHCl3); MS, 1H and 13C NMR data were found to be in full agreement with literature data [Klyflaccicembranol F (ure data .12): amorphous solid; c 0.67, CHCl3); lit. c 0.08, CHCl3); MS, 1H and 13C NMR data were found to be in full agreement with literature data [Sarcomilasterol (ure data .13): amorphous solid; c 1.50, MeOH); lit. c 0.23, MeOH); MS, 1H and 13C NMR data were found to be in full agreement with literature data [Sarcoaldesterol B (ure data .14): amorphous solid; c 0.45, MeOH); lit. c 0.11, MeOH); MS, 1H and 13C NMR data were found to be in full agreement with literature data [Sarglaucsterol (ure data .15): colorless oil; c 0.73, CHCl3); lit. c 1.10, CHCl3); MS, 1H and 13C NMR data were found to be in full agreement with literature data [Loliolide . The MTT assays were performed as previous reported . After a1–4 and a new steroid 5 as well as ten known compounds 6–15. The 3,4,8,16-tetra-epi-lobocrasol (1) represent the second example of lobocrasol-type diterpenoid, while the Δ11 double in lobocrasol (7) was revised as E geometry in this study. It is interesting that, similar to sarcophine and ent-sarcophine [ent-sarcophyolide E (4) were also isolated from the same genus.Our present study discovered four new diterpenoids rcophine , sarcoph


AbstractCheiracanthium C. L. Koch, 1839 are reported from Jinggang Mountains, Jiangxi Province, China. Two of them are described as new to science: C.auriculatumsp. n. (♀♂) and C.echinulatumsp. n. (♂). Cheiracanthiumtaiwanicum Chen, Huang, Chen & Wang, 2006 is recorded from Mainland China for the first time. Cheiracanthiumzhejiangense Hu & Song, 1982, the most similar species to C.auriculatumsp. n., is a newly recorded species of Jiangxi Province. Detailed descriptions, diagnoses, and photographs of the two new species are given. Cheiracanthiumtaiwanicum and C.zhejiangense are also illustrated.Four species of spider genus  Cheiracanthium C. L. Koch, 1839 contains 210 catalogued species and is mainly distributed in the Old World . The type specimens of the new species are deposited in College of Chemistry and Life Sciences, Guizhou Education University, Guiyang, Guizhou, China.Abbreviations used are:A atrium;AER anterior eye row;AL abdomen lengthALE anterior lateral eyes;AME anterior median eyes;AME–AME distance between AMEs;AME–ALE distance between AME and ALE;AW abdomen width;C conductor;CD copulatory duct;CF cymbial fold;CI carapace index;CL carapace length;CLL clypeal length;CO copulatory opening;CS cymbial spur;CW carapace width;DTA dorsal tibial apophysis;E embolus;EB embolic base;FD fertilisation duct;LL total length of leg I;LL:CL leg I / carapace length;MOQ median ocular quadrangle;MOQAMOQ anterior width;MOQPMOQ posterior width;OAL ocular area length;OAW ocular area width;PER posterior eye row;PLE posterior lateral eyes;PME posterior median eyes;PME–PME distance between PMEs;PME–PLE distance between PME and PLE;PTA prolateral tibial apophysis;RTA retrolateral tibial apophysis;R receptacle;STL sternum length;STW sternum width;TA tegular apophysis;TL total body length.Leg setae: v, ventral; p, prolateral; r, retrolateral. Most of the terminologies used in text and figure legends followed Taxon classificationAnimaliaAraneaeEutichuridaehttp://zoobank.org/45045AEA-20E9-4C6E-8BBD-F104775A8E6B26°35'30.23"N, 114°15'59.93"E), 26 April 2011, Hao Yu and Zhenyu Jin leg. Paratypes: 1♂ and 3 ♀, same data as holotype.Holotype ♂ (SYSU-JX-11-177): China, Jiangxi Province, Jinggang Mountains Nature Reserve, Xiangzhou village , referring to the tegular apophysis which is like the contour of an ear in ventral view.The specific epithet is an adjective and is derived from a Latin word “Cheiracanthiumauriculatum sp. n. is distinguished from all other Cheiracanthium species, except C.zhejiangense Hu & Song, 1982  , the more or less lengthwise receptacles    Figs ; 4A–C, tse) Figs ; 4D–E, ase) Figs ; 4E. In  n. Fig. , but witnse Fig. .Male. Total length 8.58–9.15. Holotype  1.55; AL 4.05, AW 2.42. Carapace , II 12.09 , III 8.61 , IV 12.47 ; LL:CL 5.03. Leg spines: I 0-0-1p, 2v-1v-1p, 2v1p-1p1v-1v; II 0-0-1p, 1v-2v-1p, 2v1p-1v1p-1v; III 0-0-1p1r, 0-1p1r-0, 2v1p-1p1r-1v2p2r; IV 0-0-1p1r, 1v-1v1p-0, 2v1p-1v1p1r-1v2p2r. Abdomen  about 20% of tibia length, with a more or less bifurcate apex and hiding behind tegulum; prolateral tibial apophysis (PTA) small and round; cymbial spur (CS) is approximately equal in length to tibia, tapering off into a filiform; cymbial fold (CF) poorly developed, for approximately 4/5 the length of cymbium; tip of cymbium short, about 1/4 of cymbium length. Tegulum oblong, 1.3 times longer than wide; tegular apophysis (TA) long and sinuate, more than 4/5 of tegulum length, filamentous and like an ear’s contour in ventral view; embolus (E) arising at approximately 10 o’clock position, terminating at approximately 11 o’clock position, it’s tip covered by conductor (C); conductor large, falciform.alp Fig. . Tibia eFemale. Total length 8.66–9.30. Slightly larger in size and lighter in colour. Allotype  = 1.36; AL = 4.95, AW = 2.92. Eye diameters and inter-distances: OAL 0.37, OAW 1.23; AME 0.14, ALE 0.19, PME 0.13, PLE 0.14; AME–AME 0.23, AME–ALE 0.11, PME–PME 0.31, PME–PLE 0.23; MOQA 0.46, MOQP 0.58, CLL 0.24. PMT: RMT = 6:6.STL 1.47, STW 1.23. Leg measurements: I 12.70 , II 8.51 , III 6.42 , IV 9.67 ; LL:CL 4.19. Leg spines: I 0-1p-1p, 2v-2v-0, 2v-1p1r-1v; II 0-0-1p, 1v-2v-1p, 2v1p-1p1r-1v; III 0-1p-1p1r, 1v-1p1r-0, 2v1p1r-1p1r-1v2p2r; IV 0-0-1p1r, 1v-1v1p1r-0, 2v1p1r-1v1p1r-1v2p2r.ype Fig.  measuredEpigyne  located at lateral borders of atrium; the transparent copulatory ducts (CD) running spirally (length of spira about 1.4 times longer than receptacles), forming 7 entwined loops (including 4 ascending coils and 3 descending coils); receptacle sickle-shaped, separated by three diameters.yne Fig. . Atrium Presently known only from the type locality, Jinggang Mountains, Jiangxi, China Fig. .Taxon classificationAnimaliaAraneaeEutichuridaehttp://zoobank.org/A1935AC9-A0A9-45F2-8BFA-22E0F317240126°35'30.23"N, 114°15'59.93"E), 26 April 2011, Hao Yu and Zhenyu Jin leg. Paratypes: 1♂, same data as holotype.Holotype ♂ (SYSU-JX-11-182): China, Jiangxi Province, Jinggang Mountains Nature Reserve, Xiangzhou village  in having the similar beak-shaped cymbial spur, and stalk-like dorsal tibial apophysis, but can be distinguished by: (1) the embolus originated at 3 o’clock position ; (2) tegular apophysis smaller, straight and acicular ; (3) RTA straight and digitiform .This new species resembles ion Fig. , vs. orilar Fig. , insteadorm Fig. , but witMale. Total length 9.06–9.12. Holotype  1.53; AL 4.99, AW 2.27. Carapace , II 14.92 , III 11.10 , IV 16.42 ; LL:CL 6.62. Leg spines: I 0-1p1r-1p1r, 3v-3v-1v1p, 2v-0-1v; II 0-1p1r-1p1r, 3v-2v-1v1p, 2v1p-2v1p-1v; III 0-1p1r-1p1r, 2v1p1r-1p1r-0, 2v1p1r-2v1p1r-2v1p2r; IV 0-1p1r-1p1r, 1v1p1r-1v2r-1v1r, 2v1p1r-2v1p1r-1v1p3r. Abdomen  about 50% of tibia length, heavily sclerotised and with a fingerlike apex; prolateral tibial apophysis (PTA) distinctly elevated and relatively short, about 30% of tibia length, coniform in prolateral view and digitiform in ventral view; dorsal tibial apophysis (DTA) thin and stalk-shaped, about as long as RTA; cymbial spur (CS) beak-shaped, twice shorter than tibia; cymbial fold (CF) strongly developed and well visible in ventral and retrolateral view, for approximately 2/3 the length of cymbium; tip of cymbium long, about 1/3 of cymbium length. Tegulum 1.3 longer than wide, membranous and semitransparent except its margin in ventral view; tegular apophysis (TA) short and thin, spiculate; embolus (E) starts on the retrolateral flank (approximately 3 o’clock of tegulum), surrounds the base and ends at conductor (C) apex, its tip filiform and curved behind conductor; conductor large and membranous.alp Fig. . Tibia tFemale. Unknown.Cheiracanthium species are known from females only in China: C.approximatum O. P.-Cambridge, 1885, C.escaladae Barrion et al., 2013, C.fujianense Gong, 1983, C.hypocyrtum Zhang & Zhu, 1993, C.liuyangense Xie et al., 1996, C.olliforme Zhang & Zhu, 1993, C.potanini Schenkel, 1963, C.rupicola , C.solidum Zhang et al., 1993, C.sphaericum Zhang et al., 1993, C.longtailen Xu, 1993. Among them, C.escaladae is supposedly a Clubiona species based on epigyne morphology, while C.potanini is supposededly doubtful because of the poor original illustrations and description. The other nine can be considered tentatively as valid Cheiracanthium species. However, none of them could be matched with C.echinulatum sp. n. due to their different habitus  and different number of cheliceral teeth .According to the Presently known only from the type locality, Jinggang Mountains, Jiangxi, China Fig. .Taxon classificationAnimaliaAraneaeEutichuridaeChen, Huang, Chen & Wang, 2006Cheiracanthiumtaiwanicum Chen et al., 2006: 10, fig. 1A–E; Chen and Huang 2012: 25, fig. 7A–G, pl. 2C–D, 3A–B.26°31'51.54"N, 114°8'46.02"E), 30 April 2011, Hao Yu leg.1♂ and 1♀, China, Jiangxi Province, Jinggang Mountains Nature Reserve, Hexiliong village , 26 April 2011, Hao Yu leg.1♂ and 2♀, China, Jiangxi Province, Jinggang Mountains Nature Reserve, Xiangzhou village  and Korea.


Absidia cylindrospora AM336, Mortierella isabellina AM212 and Mortierella vinaceae AM149. As a result of regio- and enantioselective hydroxylation four new oxygenated derivatives were obtained. Regardless of the biocatalyst applied, the δ-iodo- and δ-bromo-γ-lactones were hydroxylated in an inactivated position C-5 of cyclohexane ring. The analogous transformation of chlorolactone was observed in Mortierella isabellina AM212 culture but in the case of two other biocatalysts the hydroxy group was introduced at C-3 position. All obtained hydroxylactones were enantiomerically pure (ee = 100%) or enriched (ee = 50%). The highest enantioselectivity of hydroxylation was observed for M. isabellina AM212. The cytotoxic activity of halolactones was also examined by WST-1 assay wherein tested compounds did not exhibit significant effect on the viability of tumor HeLa and normal CHO-K1 cells.Three β-cyclocitral-derived halolactones, which exhibit antifeedant activity towards storage product pests, were subjected to microbial transformation processes. Among the thirty tested strains of filamentous fungi and yeast, the most effective biocatalysts were  Absidia and Mortierella genus were found to be efficient catalysts. The different Mortierella species are investigated as a potent producer of arachidonic acid but only few examples of their use for biohydroxylation are described in literature nonan-8-one (2) and 1-chloromethyl-2,2,6-trimethyl-9-oxabicyclo[4.3.0]nonan-8-one (3) were synthesized from β-cyclocitral in four steps chemical synthesis, according to the procedure described earlier nonan-8-one 1-Iodomethyl-2,2,6-trimethyl-9-oxabicyclo [4.3.0]nonan-8-one δ: 1.10 and 1.17 2C<), 1.34 , 1.38–1.75 , 2.21 and 3.26 , 3.52 and 3.84 ; 13C NMR  δ: 6.09 (C-10), 17.26 (C-4), 21.74 (CH3-6), 25.53 and 27.63 ((CH3)2C<), 38.18 (C-2), 38.31 (C-3), 39.86 (C-5), 42.39 (C-6), 47.84 (C-7), 88.02 (C-1), 175.62 (C-8); IR : 2928 (s), 1767 (s), 1245 (m), 633 (w);mp = 104–107°C; 2)1-Bromomethyl-2,2,6-trimethyl-9-oxabicyclo [4.3.0]nonan-8-one  δ: 1.07, 1.15 2C<), 1.37 , 1.38 , 1.42–1.48 , 1.53 , 1.62–1.71 , 2.17 and 3.25 , 3.73 and 3.97 ; 13C NMR  δ: 17.42 (C-4), 21.90 (CH3-6), 25.34 and 27.43 ((CH3)2C<), 35.15 (C-10), 38.07 (C-2), 38.57 (C-3), 40.08 (C-5), 42.07 (C-6), 47.85 (C-7), 88.67 (C-1), 175.93 (C-8); IR : 2976 (s), 1767 (s), 1243 (s), 656 (m);mp = 156–161°C; 3)1-Chloromethyl-2,2,6-trimethyl-9-oxabicyclo [4.3.0]nonan-8-one  δ: 1.05, 1.14 2C<), 1.34 , 1.36 , 1.42–1.48 , 1.53 , 1.61–1.70 , 2.16 and 3.14 , 3.88 and 4.05 ; 13C NMR  δ: 17.50 (C-4), 21.93 (CH3-6), 25.08 and 27.38 (CH3)2C<), 37.55 (C-2), 38.61 (C-3), 40.10 (C-5), 41.74 (C-6), 46.93 (C-10), 47.82 (C-7), 89.34 (C-1), 176.08 (C-8); IR : 2934 (s), 1773 (s), 1254 (s), 731 (m);mp = 97–101°C; Absidia coerulea AM93, Absidia glauca AM254, Absidia cylindrospora AM336, Armillaria mellea AM296, Beauveria bassiana AM278, Chaetomium sp. AM432, Fusarium avenaceum AM12, Fusarium culmorum AM10, Fusarium culmorum AM196, Fusarium scirpi AM199A, Fusarium solani AM203, Fusarium tricinctum AM16, Laetiporus sulphurens AM524, Mortierella isabellina AM212, Mortierella vinaceae AM149, Mucor circinelloides AM385, Nigrospora oryzae AM8, Penicillium camembertii AM83, Penicillium chermesinum AM113, Penicillium frequentans AM351, Penicillium lilacinum AM111, Penicillium vermiculatum AM81, Pholiota aurivella AM522, Rhodotorula marina AM77, Rhodotorula rubra AM82, Saccharomyces cerevisiae AM464, Spicaria fusispora AM136, Syncephalastrum racemosum AM105, Trametes versicolor AM536) and from the collection of the Department of Phytopatology, Kraków Agricultural University (Cenangium ferruginosum AR56). Cultivation of microorganisms was carried out on Sabouraud agar slants of following composition: aminobac (5.0 g), peptone K (5.0 g), glucose (40.0 g) in distilled water (1 L). The microorganisms were cultivated at 28°C and stored in refrigerator at 4°C.Thirty strains of filamentous fungi and yeasts used in this work came from the collection of the Institute of Biology and Botany, Wrocław Medical University (1) dissolved in 1 mL of acetone were added to the shaken cultures (150 rpm). The incubation was continued for 15 days. After 1, 2, 3, 6, 9, 12 and 15 days of incubation, the products were extracted with methylene chloride and analyzed by TLC and GC. Three strains of fungi  were able to transform the iodolactone (1). For selected strains the screening experiments with bromo- (2) and chlorolactone (3) as substrates were carried out as described above.The strains were cultivated at 25°C in 300 mL Erlenmeyer flasks containing 50 mL of medium . After 4 days of growth 10 mg of iodolactone (1–3) . For isolation and identification of products, the biotransformation were performed in multiplied scale. Selected strains were cultivated in 10 Erlenmeyer flasks with 50 mL of medium to which 100 mg of substrate dissolved in acetone was totally added (reaction conditions the same as described in screening procedure). After the optimum time for each biotransformation process the products were extracted two times with methylene chloride (50 mL for each flask). The organic layers were pooled, dried over anhydrous MgSO4 and the solvent was evaporated in vacuo. The transformation products were separated and purified by column chromatography (hexane:acetone from 7:1 to 4:1). As a result of biotransformation of (1) in A. cylindrospora AM336 culture, 27 mg (26% yield) of hydroxyiodolactone (4) was obtained, for M. isabellina AM212 and M. vinaceae AM149 21 mg (20% yield) and 19 mg (18% yield), were isolated respectively. Products of bromolactone (2) transformations were isolated in 14% yields (15 mg) using A. cylindrospora AM336, in 24% yield (20 mg) using M. isabellina AM212 and in 16% yield (17 mg) using M. vinaceae AM149. Hydroxylated metabolites of chlorolactone (3) were obtained using A. cylindrospora AM336 culture in 30% yield (32 mg), using M. vinaceae AM149 in 31% yield (33 mg) and using M. isabellina AM212 in 49% yield (52 mg). The physical and spectral data of biotransformation products are given below.The screening experiments allowed as to choose three strains of fungi capable to transform iodo-, bromo- and chlorolactone (4)(+)-5-Hydroxy-1-iodomethyl-2,2,6-trimethyl-9-oxabicyclo [4.3.0]nonan-8-one ; 1H NMR  δ: 1.09 and 1.17 2C<), 1.26 , 1.36 , 1.61–1.65 , 1.71 , 2.72 and 3.16 , 3.39 , 3.55 and 3.80 ; 13C NMR  δ: 5.87 (C-10), 13.47 (CH3-6), 25.28 and 27.52 ((CH3)2C<), 26.44 (C-4), 36.72 (C-3), 38.15 (C-2), 44.64 (C-7), 48.27 (C-6), 75.11 (C-5), 88.91 (C-1), 175.54 (C-8); IR : 3549 (s), 3479 (s), 2981 (s), 1758 (s), 1259 (s), 622 (m). HRMS (ESI-TOF) m/z [M+H]+ calcd for C12H19IO3 339.0457; found 339.0449.White crystals, mp = 168–172°C; 5)(+)-5-Hydroxy-1-bromomethyl-2,2,6-trimethyl-9-oxabicyclo [4.3.0]nonan-8-one ; 1H NMR  δ: 1.07 and 1.14 2C<), 1.29 , 1.39 , 1.61–1.66 , 1.71 , 2.67 and 3.18 , 3.41 , 3.76 and 3.94 ; 13C NMR  δ: 13.54 (CH3-6), 25.10 and 27.20 ((CH3)2C<), 26.54 (C-4), 35.26 (C-10), 36.94 (C-3), 38.13 (C-2), 44.77 (C-7), 47.88 (C-6), 75.23 (C-5), 89.67 (C-1), 175.88 (C-8); IR : 3485 (s), 2988 (s), 1762 (s), 1259 (s), 661 (m). HRMS (ESI-TOF) m/z [M+H]+ calcd for C12H19BrO3 291.0596; found 291.0599.White crystals, mp = 165–169°C; 6)(+)-3-Hydroxy-1-chloromethyl-2,2,6-trimethyl-9-oxabicyclo [4.3.0]nonan-8-one ; 1H NMR  δ: 0.99 and 1.23 2C<), 1.37 , 1.54–1.65 , 1.72 , 2.13 and 3.18 , 3.63 , 3.93 and 4.08 ; 13C NMR δ; 16.34 and 22.40 ((CH3)2C<), 21.82 (CH3-6), 26.22 (C-4), 37.86 (C-5), 41.84 (C-6), 42.93 (C-2), 46.78 (C-10), 47.92 (C-7), 73.48 (C-3), 90.70 (C-1), 175.70 (C-8);); IR: 3508 (s), 2978 (s), 1752 (s), 1269 (s) 731 (m). HRMS (ESI-TOF) m/z [M+H]+ calcd for C12H19ClO3 247.1101; found 247.1114.mp = 152–157°C, 7)(+)-5-Hydroxy-1-chloromethyl-2,2,6-trimethyl-9-oxabicyclo [4.3.0]nonan-8-one ; 1H NMR  δ: 1.05 and 1.12 2C<), 1.29 , 1.37 ; 1.59–1.77 , 2.66 and 3.06 , 3.41 , 3.91 and 4.01 ; 13C NMR  δ: 13.64 (CH3-6), 24.87 and 27.15 ((CH3)2C<), 26.54 (C-4), 36.97 (C-3), 37.58 (C-2), 44.68 (C-7), 47.22 (C-10), 47.51 (C-6), 75.17 (C-5), 90.41 (C-1), 176.10 (C-8); IR : 3491 (s), 2981 (s), 1756 (s), 1261 (s), 733 (m). HRMS (ESI-TOF) m/z [M+H]+ calcd for C12H19ClO3 247.1101; found 247.1113.White crystals, mp = 165–170°C, 1, 2, 3) on proliferation of HeLa  and CHO-K1 (ATCC CCL-61) cells was examined by WST-1 assay. Cells were cultured in DME medium supplemented with 10% (v/v) FBS in the incubator with 5% CO2 and humidified atmosphere at 37°C. HeLa and CHO-K1 cells from exponential growth phase were seeded in 96-well plates at initial concentration of 5 × 10−4 cells per well in 100 μL of media. After 24 h of inoculation, culture media was replaced by fresh one containing 100 and 200 μM of tested lactones. Stock solutions of lactones  were prepared in DMSO, and filtered through 0.22 filters. After 72 h of treatment, 10 μL of WST-1 reagent was added to each well and cells were incubated for further 4 h. Absorbance was measured at 450 nm on the microplate reader . The experiments were performed in triplicate and data were expressed as means ± S.D. of percentages of treated cells versus control (untreated cells).The effect of three halolactones (1). Thirty strains of filamentous fungi and yeast, listed in the experimental part, were checked to select efficient biocatalyst. Among them, three strains: Absidia cylindrospora AM336, Mortierella isabellina AM212 and Mortierella vinaceae AM149 were able to modify the iodolactone structure . In the next step these selected microorganisms were tested for their ability are to transform also the bromo- and chlorolactone (2 and 3). The results of screening experiments as the composition of product mixtures during the biotransformation processes are presented in Table 1. The Mortierella vinaceae AM149 was found to transform all substrates at the highest rate. On the contrary, the longest time of transformation was observed in the case of found for processes catalyzed by Absidia cylindrospora AM336 and even after 15 days substrates were not consumed completely.The metabolism of three β-cyclocitral-derived halolactones which exhibit antifeedant activity towards storage product pests  was inveAbsidia cylindrospora AM336 was twelve days, for Mortierella isabellina AM212 and Mortierella vinaceae AM149 nine and three days, respectively.Based on the data obtained in the screening experiments, the biotransformation processes in multiplied scales were carried out in order to isolate and determine the structures of obtained products. Because of intensification of metabolites production in the last days of processes, we decided to shorten the transformation time to simplify the isolation procedure. The optimal biotransformation time for 1) in the culture of all selected fungal strains resulted in the formation of the same (+)-enantiomer of product, but its enantiomeric excess depended on the biocatalyst (Table 2). Pure enantiomer of product 4 was produced by M. isabellina AM212. The process catalyzed by A. cylindrospora AM336 was the least enantioselective (ee of product—50%). The isolated yields of the product were rather moderate and did not exceed 26% as obtained in A. cylindrospora AM336—catalyzed transformation. The structure of product was established on the basis of its spectroscopic data  characteristic doublets from CH2-10 groups can be observed. The presence of those signals excluded the dehalogenation process. Incorporation of hydroxy group into the molecule was additionally confirmed by the deshielding effect of oxygen from OH function which resulted in the shift of characteristic doublet of doublets from H-5 proton to 3.39 ppm. Coupling constant value between one of the CH2-4 protons and H-5 proton (J = 9.4 Hz) suggested the axial orientation of the latter and consequently equatorial location of hydroxy group. The crucial was HMBC correlation, with allowed to confirm the exact location of hydroxy group. The coupling between carbon C-7, CH3-6 and proton H-5, undoubtedly confirms the introduction of the OH group at C-5.Biotransformation of iodolactone  . In those processes also the enantiomerically enriched (+)-isomer of hydroxybromolactone (5) was obtained as the only product. The highest enantiomeric excess (ee = 95%) was determined for the product obtained in M. isabellina AM212 culture as in the case of transformation of iodolactone (1). Analogically, the lowest ee value of hydroxybromolactone (5) (ee = 76%) was observed when Absidia cylindrospora AM336 was used as biocatalyst (Table 2).Strong similarities can be noticed for biotransformation of bromolactone (2) and product of biotransformation (5) (4). The presence of absorption bands at 1762 cm-1 and 3549 cm-1 on IR spectrum proved the retention of the γ-lactone moiety and incorporation of hydroxy function. On 1H NMR spectrum at 3.41 ppm the characteristic doublet of doublets from proton H-5 is present. The coupling constant J = 9.1 Hz found in this signal confirmed axial orientation of proton H-5 and as a consequence the equatorial position of hydroxy group. Further, the coupling between the proton H-5 and carbon atoms C-7 and CH3-6 on HMBC spectrum, confirmed the incorporation of OH group into C-5 position.The spectroscopic data of the substrate (tion (5) –S6 Figs 3  exhibited lower enantiomeric excess (52–67%) compare to iodo- and bromoderivatives (Table 2). In all experiments (+)-isomer of product 6 or 7 was the major one. The hydroxychlorolactone 7 obtained in Mortierella isabellina AM212 culture was the product of biohydroxylation of substrate at C-5. The spectral data  of proton H-5. Taking into consideration numerous similarities in the biotransformation of δ-iodo-γ-lactone 1 and δ-bromo-γ-lactone 2, the transformation of δ-chloro-γ-lactone 3 in Absidia cylindrospora AM336 and Mortierella vinaceae AM149 cultures resulted in the formation of unexpected product 6 . The analysis of its spectral data revealed that this time the substrate 3 was hydroxylated at C-3 position of cyclohexane ring  for product of C-3 hydroxylation comparing with 1.12 ppm of hydroxylactone 7. The axial orientation of proton H-3 and consequently equatorial position of hydroxy group is confirmed by constant coupling values (J = 12.0 Hz) found for H-3 signal. The product of biotransformation of chlorolactone 3 were obtained in high isolated yields within the range from 30% in the case of Absidia cylindrospora AM336 to even almost 50% in the case of Mortierella isabellina AM212 culture.The products of biohydroxylations of chlorolactone ral data –S9 Figs ane ring –S12 FigsAbsidia is one of the most potent biological systems involving different metabolic pathways in transformations processes. The ability of Absidia cylindrospora for catalyzing hydroxylation reactions, various dehalogenation reactions or epoxydation of double bond is well documented. Regardless of the biocatalyst the hydroxy group is commonly incorporated at a tertiary carbon atom [The most popular biocatalytic process used to obtain the optically active alcohols is asymmetric reduction of prochiral ketones –27 and lbon atom , 21, 34 bon atom . For unsbon atom . Alternabon atom  or unsatbon atom , 37. In 1, 2, 3), we evaluated their in vitro cytotoxicity towards HeLa and CHO-K1 cell lines by WST-1 assay. Obtained results expressed as a percentage of cell viability are presented in Table 3. Tested compounds did not show the significant decrease of cell viability in both cell lines even at 200 μM concentration. Nevertheless, the obtained cytotoxicity results are valuable as a guide how to direct the synthesis of similar analogues with improved biological properties.To confirm possible antiproliferative effect of halolactones . Absidia cylindrospora AM336 and Mortierella vinaceae AM149 exhibited the tendency of C-3 hydroxylation and Mortierella isabellina AM212 strain hydroxylated C-5 carbon atom regardless of the halogen type in substrate structure. Presented biohydroxylations is efficient method to obtained enantiomerically pure or enrich hydroxyhalolactones—the multifunctional compounds that potentially can be used as the chiral building blocks.Among thirty strains of filamentous fungi and yeast three of them: S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.


Euphorbia denticulata Lam were collected from Sanandaj city in Kurdistan province at the West of Iran. It was extracted by maceration using acetone as solvent. The isolation of compounds were carried out with repeated column chromatography using silica gel and normal preparative HPLC using YMC-Pack-Sil column and hexane: ethyl acetate as mobile phase. The structures of isolated compounds were elucidated by extensive 1 and 2D-NMR as well as HRESI-MS spectra and the cytotoxicity was done against DU-145 human prostate cancer cell line using standard MTT assay. It afforded a new 12-taraxastane derivative and two known cycloartane triterpenes including: taraxast-12-ene-3β,20,21(α)-triol (1), cycloartane-3β,25-diol (2), and cycloartane-3β,24,25-triol (3). They exhibited cytotoxic effects, with IC50 values of 12.2 ± 2.9, 27.5 ± 4.9, and 18.3 ± 1.4 µM, respectively. In this research, aerial parts of  Euphorbia is one of the largest groups in angiosperm genera with about 2000 species. According to Rechinger, there are ninety six species in Flora Iranica classified into five sections, from which Tithymalus is the largest one in Iran. E. denticulata is a perennial herb, belonging to section Tithymalus, with expanded leaves without stipules, cyathia arranged in cymose rays around a cyathium and with large and denticulata glands in outer margins  and normal preparative HPLC using a YMC-Pack-Sil column . The structures of the compounds were elucidated by Plant materialE. denticulata was collected from Sanandaj city in Kurdistan province in the West of Iran. It was identified by Professor Hojjatollah Saeedi in the Department of Biology, Faculty of Science, University of Isfahan and a voucher specimen number 19001 was deposited there in the herbarium of the University of Isfahan (Iran).Extraction and isolation1H-NMR, Fr.2, Fr.3, and Fr.4 contained characteristic signals of steroids and triterpenoids which were previously reported from this plant by the same authors  was macerated for three days with acetone (6 L×3). Filtration and in vacuum evaporation resulted in a green gum 85 g), which was applied on VLC over RP-18 (40-63 µm) using MeOH:H2O (7:3) as eluent. Then, defatted extract (12 g) was concentrated and column chromatographed on flash silica gel  using hexane/EtOAc, with increasing polarity (5→50 %) to afford eight fractions. Inspecting by 5 g, whic authors .Compound 1: Amorphous white powder, MW (g/mol): 458; yield: 0.001%; IR (KBr) υmax: 3430, 2945, 2870, 1640, 1465, 1145, 1106 cm-1.1H-NMR : see 13C-NMR data : see m/z 459.3865 .Compound 2: Amorphous white powder, MW(g/mol): 444 yield: 0.0015%; 1H-NMR : δH 3.32 , 1.25 , 1.19 , 1.00 , 1.00 , 0.92 , 0.91 , 0.58 , 0.36 . 13C-NMR data : 78.8 (C-3), 73.3 (C-25), 52.3 (C-17), 48.8 (C-14), 48.0 (C-8), 47.1 (C-5), 45.3 (C-13), 40.5 (C-4), 36.4 (C-24), 35.9 (C-20), 35.6 (C-12), 33.5 (C-22), 32.9 (C-15), 32.0 (C-1), 30.4 (C-2), 29.9 (C-19), 29.7 (C-7), 26.6 (C-27), 26.5 (C-16), 26.0 (C-10), 26.0 (C-11), 25.4 (C-30), 23.2 (C-26), 22.7 (C-23), 21.1 (C-6), 20.0 (C-9), 19.3 (C-28), 18.4 (C-21), 18.1 (C-18), 14.0 (C-29). Positive HR-ESIMS m/z 445.4054 .Compound 3: Amorphous white powder. MW(g/mol): 460; yield: 0.0015%; 1H-NMR : δH 3.37 , 3.31 , 1.24 , 1.19 , 0.99 , 0.99 , 0.92 , 0.90 , 0.57 , 0.36 . 13C-NMR data : 78.8 (C-24), 78.7 (C-3), 73.2 (C-25), 52.4 (C-17), 48.8 (C-14), 48.0 (C-8), 47.1 (C-5), 45.3 (C-13), 40.4 (C-4), 35.9 (C-20), 35.5 (C-12), 33.1 (C-22), 32.9 (C-15), 31.9 (C-1), 30.3 (C-2), 29.9 (C-19), 28.4 (C-23), 28.2 (C-7), 26.6 (C-27), 26.4 (C-16), 26.0 (C-10), 26.0 (C-11), 25.4 (C-30), 23.2 (C-26), 21.1 (C-6), 19.9 (C-9), 19.3 (C-28), 18.1 (C-21), 18.0 (C-18), 14.0 (C-29). Positive HR-ESIMS m/z 461.4012 .Cytotoxicity MTT assay2. The cells were seeded at 5000 cells per well in 5% CO2 at 37 °C in RPMI with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin, in 96-well plates. After 24 h incubation, the cells had been treated with concentrations  of compounds 1-3 for 2 days. MTT was added to the wells and incubated for another 4 hours. The experiment was done in triplicate and the absorbance was read by the microplate reader  at 570 nm. Cell viability percentages calculated by the formula: (mean OD of treated cells /mean OD of control cells) ×100 and were expressed as percent of control cells which were not treated . 1D and 2D-NMR spectra showed signals for eight methyls, eight methylenes, seven methines, and seven quaternary carbons, including two oxy-methine groups, one oxy-carbon, and an olefin bond. A total of 30 carbons were observed -characteristic of a triterpene skeleton. Application of 1H-1H correlation spectroscopy (COSY) allowed the multiplets of the triterpene structure to be sequenced into six spin systems: H-1 to H-3, H-5 to H-7, H-9 to H-12, H-15 to H-16, H-21 to H-22, and H-19 to Me-29  at C-20 ,25-triol as known cycloartane type triterpenes. To the best of our knowledge, it is the first time that NMR data of cycloartane-3β, 25-diol are reported. It had been identified previously by HR-GCMS analysis alone from matured Wheat straw by Gaspar in 1992  25-triol, is isolated and reported for the first time from natural origin. It is semi-synthesized and reported before by Della Greca and coworkers after treating of 24, 25-epoxide-3 (β)-cycloartanol with methylmagnesium N-cyclohexylisopropylamide  of (1):12.2 ± 2, (P < 0.05). But, their cytotoxic effects were not significant in lower concentration of 1 µM (P > 0.05). In comparison between compounds 2 and 3 with similar cyloartane structure at concentration of 10 µM, compound 2 showed weaker cytotoxicity effect than compound 3 (P > 0.05).To assess the effects of the isolated compounds on the viability of cancer cell lines, 12-taraxast-3β, 19, 21 (α)-triol (E. denticulata. Taraxastan triterpens are very rare in nature and it is the first report of their occurrence in endemic spurges of Iran. Compounds 1, and 2 both showed potent cytotoxic activity against DU-145 human prostate cancer cell line, in-vitro. However, MTT assay could be used only as an indicative of mitochondrial function and only help in determination of toxicity mechanisms. But, other confirmation tests including intrinsic and extrinsic pathways are required.In this study a new 12-taraxastane derivative with three hydroxyl group located at C-3, C-19, and C-21 as well as two known cycloartane triterpenes were extracted from