optical rotations ( chcl3 ) were measured at 589 nm on a perkin - elmer 192 polarimeter equipped with a sodium lamp (  = 589 nm ) and a 10 cm microcell . h ( 500 mhz ) and c ( 125 mhz ) nmr spectra were measured on a varian inova spectrometer . chemical shifts were referenced to the residual solvent signal ( cdcl3 : h 7.26 , c 77.0 ) . two- and three - bond hc connectivities were determined by hmbc experiments optimized for a j of 7 hz . through - space h connectivities were evidenced using a roesy experiment with a mixing time of 500 ms . two- and three - bond hc connectivities were determined by gradient 2d hmbc experiments optimized for a j of 9 hz . low- and high - resolution eims spectra ( 70 ev ) were performed on a vg prospec ( fisons ) mass spectrometer . reactions were monitored by tlc on merck 60 f254 ( 0.25 mm ) plates , which were visualized by uv inspection and/or staining with 5% h2so4 in ethanol and heating . oenanthe fistulosa was collected near san basilio ( ca , sardinia , italy ) on september 14 , 2007 . o. crocata was collected near bonacardo ( abbasanta , nu , italy ) in may 8 , 2007 , and a voucher specimen ( ocr080506bo ) is held at the university of cagliari . ( a ) powdered , dried underground parts of o. fistulosa ( 608 g ) were extracted with acetone ( 2  2 l ) at room temperature , affording 7.94 g of a dark oil , which was fractionated by gravity column chromatography on silica gel ( n - hexaneetoac , 9:1 to 6:4 , as solvent gradient system ) . fractions eluted with n - hexaneetoac ( 9:1 ) were further purified by hplc ( n - hexaneetoac , 85:15 ) to yield falcarindiol ( 3 , 344 mg ) . fractions eluted with n - hexaneetoac ( 6:4 ) were further purified by hplc ( n - hexaneetoac , 65:35 ) to yield oenathotoxin ( 1a , 68 mg ) and dihydrooenanthotoxin ( 1b , 67 mg ) . ( b ) powdered , dried seeds of o. fistulosa ( 379 g ) were extracted with acetone ( 2  2 l ) at rt , affording 21.3 g of a yellowish oil , which was fractionated by gravity column chromatography on silica gel ( n - hexaneetoac , 9:1 to 6:4 , as solvent gradient system ) . fractions eluted with n - hexaneetoac ( 7:3 ) were further purified by hplc ( n - hexaneetoac , 55:45 ) to yield epoxyfalcarindiol ( 2 , 432 mg ) . ( c ) dried underground parts of o. crocata ( 95 g ) were extracted with acetone ( 2  1 l ) to afford 3.1 g of a brownish oil , which was purified with the biotage sp1 system with a silica gel ( 10 g ) flash 12+m column and a n - hexaneetoac gradient of increasing polarities ( flow rate 9 ml / min , uv detector 316 nm ; fractionation mode : volume 9 ml / fraction ) . fractions 2931 and 3436 ( n - hexaneetoac , 6:4 ) afforded about 88 mg of oenanthotoxin ( 1a ) and 89 mg of dihydrooenathotoxin ( 1b ) , respectively . these compounds were obtained as colorless foams and could not be stored in this form . however , solutions in hydrocarbon solvents ( benzene , toluene ) or dmso could be stored for at least 6 months at 4 c without any apparent ( h nmr analysis ) degradation or development of a reddish color . amorphous foam ; [ ]d + 34 ( c 0.5 , chcl3 ) ; h nmr ( 500 mhz , cdcl3 )  6.71 ( 1h , dd , j = 15.5 , 11.0 hz , h-9 ) , 6.39 ( 1h , dt , j = 15.0 , 5.0 hz , h-2 ) ; 6.12 ( 1h , dd , j = 15.0 , 11.0 hz , h-10 ) , 5.87 ( 1h , overlapped h-3 ) , 5.86 ( 1h , overlapped , h-11 ) ; 5.57 ( 1h , d , j = 15.5 hz , h-8 ) , 4.25 ( 2h , d , j = 5.0 hz h2 - 1 ) , 3.60 ( 1h , m , h-14 ) , 2.30 ( 1h , m , h-12a ) , 2.20 ( 1h , m , h-12b ) , 1.78 ( 1h , m , h-13a ) , 1.50 ( 1h , overlapped , h-13b ) , 1.49 ( 1h , overlapped , h-15a ) , 1.40 ( 1h , m , h-15b ) , 1.30 ( 2h , m , h2 - 16 ) , 0.92 ( 3h , d , j = 7.3 , hz h3 - 17 ) ; esims ( positive ion ) m / z 281 [ m + na ] . amorphous foam ; h nmr ( 500 mhz , cdcl3 )  6.65 ( 1h , dd , j = 15.5 , 10.6 hz , h-9 ) , 6.12 ( 1h , dd , j = 15.0 , 11.0 hz , h-10 ) , 5.84 ( 1h , dt , j = 15.0 , 5.0 hz , h-11 ) , 5.50 ( 1h , d , j = 15.5 hz , h-8 ) , 3.77 ( 2h , t , j = 5.0 hz , h2 - 1 ) , 3.60 ( 1h , m , h-14 ) , 2.45 ( 2h , t , j = 5.0 hz , h2 - 3 ) , 2.30 ( 1h , m , h-12a ) , 2.21 ( 1h , m , h-12b ) , 1.79 ( 2h , t , j = 5.0 hz , h2 - 2 ) , 1.75 ( 1h , m , h-13a ) , 1.50 ( 1h , overlapped , h-13b ) , 1.49 ( 1h , overlapped , h-15a ) , 1.40 ( 1h , m , h-13b ) , 1.30 ( 2h , m , h2 - 16 ) , 0.93 ( 3h , d , j = 7.3 hz , h3 - 17 ) ; esims ( positive ion ) : m / z 283 [ m + na ] . colorless oil , [ ]d + 107 ( c 0.9 , meoh ) ; ir ( liquid film ) max 3400 , 3120 , 2080 , 1451 , 1293 , 1238 , 1164 , 983 cm ; h nmr ( 500 mhz , c6d6 )  5.78 ( 1h , ddd , j = 17.0 , 10.2 , 5.45 hz , h-2 ) , 5.37 ( 1h , brd , j = 17.0 hz , h-1a ) , 5.03 ( 1h , brd , j = 10.3 hz , h-1b ) , 4.67 ( 1h , d , j = 5.4 hz , h-3 ) , 4.20 ( 1h , d , j = 7.1 hz , h-8 ) , 3.06 ( 1h , dd , j = 7.1 , 4.0 hz , h-9 ) , 2.80 ( 1h , dt , j = 5.9 , 4.0 hz , h-10 ) , 1.53 ( 2h , brm , h-11a , b ) , 1.451.30 ( 10h , m , h-12a , b + h-13a , b + h-14a , b + h-15a , b + h-16a , b ) , 1.01 ( 3h , t , j = 7.1 hz , h-17 ) ; c nmr ( 125 mhz , cdcl3 )  136.1 ( d , c-2 ) , 116.2 ( t , c-1 ) , 79.3 ( s , c-7 ) , 79.1 ( s , c-4 ) , 70.3 ( s , c-5 ) , 70.1 ( s , c-6 ) , 63.2 ( d , c-3 ) , 61.0 ( d , c-8 ) , 57.9 ( d , c-9 ) , 57.4 ( d , c-10 ) , 31.9 ( t , c-15 ) , 27.6 ( t , c-11 ) , 29.4 , 29.3 ( t , c-13 and c-14 ) 26.6 ( t , c-12 ) , 22.8 ( t , c-16 ) , 14.1 ( q , c-17 ) . hreims m / z 276.1737 [ m ] ( calcd for c17h24o3 , 276.1725 ) . oenanthotoxin ( 1a , 2.5 mg ) was dissolved in 0.5 ml of dry pyridine and treated with ( )-r - mtpa chloride ( 20 l ) and n , n - dimethylaminopyridine ( dmap , a spatula tip ) , then maintained at room temperature under stirring overnight . after removal of the solvent , the reaction mixture was purified by hplc on a si60 column ( eluent n - hexaneetoac , 9:1 ) , affording the ( s)-mtpa diester 1c in a pure state ( 3.5 mg , 53% yield ) . using ( + ) -s - mtpa chloride , the same procedure afforded the ( r)-mtpa diester 1d in the same yield . this procedure was repeated for dihydrooenanthotoxin ( 1b , 2.0 mg ) to obtain the ( s)-mtpa diester 1e and ( r)-mtpa diester 1f in the same yield . amorphous solid ; h nmr ( 500 mhz , cdcl3 )  7.45 and 7.35 ( mtpa phenyl protons ) , 6.66 ( h-9 , dd , j = 15.5 , 10.6 hz ) , 6.28 ( h-2 , dt , j = 15.0 , 5.0 hz ) , 6.09 ( h-10 , dd , j = 15.0 , 10.6 hz ) , 5.84 ( h-3 , overlapped ) , 5.82 ( h-11 , overlapped ) , 5.57 ( h-8 , d , j = 15.5 hz ) , 5.10 ( h-14 , m ) , 4.86 ( h2 - 1 , d , j = 5.0 hz ) , 3.55 ( mtpa och3 , s ) , 2.16 ( h-12a , overlapped ) , 2.12 ( h-12b , overlapped ) , 1.81 ( h-13a , m ) , 1.73 ( h-15a , m ) , 1.65 ( h-13b , m ) , 1.58 ( h-15b , m ) , 1.29 ( h2 - 16 , m ) , 0.88 ( h3 - 17 , d , j = 7.3 hz ) ; fabms ( glycerol matrix , positive ions ) m / z 691 [ m + h ] . amorphous solid ; h nmr ( 500 mhz , cdcl3 )  7.55 and 7.53 ( mtpa phenyl protons ) , 6.69 ( h-9 , dd , j = 15.5 , 10.6 hz ) , 6.26 ( h-2 , dt , j = 15.0 , 5.0 hz ) , 6.16 ( h-10 , dd , j = 15.0 , 10.6 hz ) , 5.91 ( h-11 , overlapped ) , 5.86 ( h-3 , overlapped ) , 5.57 ( h-8 , d , j = 15.5 hz ) , 5.10 ( h-14 , m ) , 4.86 ( h2 - 1 , d , j = 5.0 hz ) , 3.58 ( mtpa och3 , s ) , 2.35 ( h-12a , m ) , 2.23 ( h-12b , m ) , 1.98 ( h-13a , m ) , 1.68 ( h-13b , m ) , 1.61 ( h-15a , m ) , 1.57 ( h-15b , m ) , 1.28 ( h2 - 16 , m ) , 0.88 ( h3 - 17 , d , j = 7.3 hz ) ; fabms ( glycerol matrix , positive ions ) m / z 691 [ m + h ] . amorphous solid ; h nmr ( 500 mhz , cdcl3 )  7.45 and 7.35 ( mtpa phenyl protons ) , 6.64 ( h-9 , dd , j = 15.5 , 10.6 hz ) , 6.09 ( h-10 , dd , j = 15.0 , 10.6 hz ) , 5.83 ( h-11 , dt , j = 15.0 , 5.0 hz ) , 5.45 ( h-8 , d , j = 15.5 hz ) , 5.20 ( h-14 , m ) , 4.39 ( h2 - 1 , m ) , 3.55 ( mtpa och3 , s ) , 2.36 ( h2 - 3 , t , j = 6.5 hz ) , 2.25 ( h-12a , overlapped ) , 2.21 ( h-12b , overlapped ) , 1.91 ( h2 - 2 , m ) , 1.75 ( h-15a , m ) , 1.71 ( h-13a , m ) , 1.60 ( h-15b , m ) , 1.56 ( h-13b , m ) , 1.30 ( h2 - 16 , m ) , 0.90 ( h3 - 17 , d , j = 7.3 hz ) ; fabms ( glycerol matrix , positive ions ) m / z 693 [ m + h ] . amorphous solid ; h nmr ( 500 mhz , cdcl3 )  7.55 and 7.53 ( mtpa phenyl protons ) , 6.70 ( h-9 , dd , j = 15.5 , 10.6 hz ) , 6.11 ( h-10 , dd , j = 15.0 , 10.6 hz ) , 5.85 ( h-11 , dt , j = 15.0 , 5.0 hz ) , 5.48 ( h-8 , d , j = 15.5 hz ) , 5.20 ( h-14 , m ) , 4.39 ( h2 - 1 , m ) , 3.58 ( mtpa och3 , s ) , 2.36 ( h2 - 3 , overlapped ) , 2.35 ( h-12a , overlapped ) , 2.28 ( h-12b , overlapped ) , 1.91 ( h2 - 2 , m ) , 1.90 ( h-13a , m ) , 1.68 ( h-15a , overlapped ) , 1.67 ( h-13b , overlapped ) , 1.54 ( h-15b , m ) , 1.27 ( h2 - 16 , m ) , 0.90 ( h3 - 17 , d , j = 7.3 hz ) ; fabms ( glycerol matrix , positive ions ) m / z 693 [ m + h ] . to a solution of falcarindiol ( 85 mg , 0.36 mmol ) in dry ch2cl2 ( 3 ml ) was added 85% m - chloroperbenzoic acid ( mcpba , 73 mg , 0.36 mmol , 1 molar equiv ) . the course of the reaction was followed by h nmr analysis of a cdcl3 solution containing an equimolecular mixture of falcarindiol and mcpba . after stirring 2 h at rt , the reaction was worked up by dilution with ch2cl2 and the addition of silica gel ( 1 g ) impregnated with a 5% water solution of na2s2o3 . after stirring a few minutes , the slurry was filtered and the filtrate was dried ( na2so4 ) and evaporated , to afford 60 mg ( 66% ) of 9-epoxyfalcarindiol ( 2 ) , spectroscopically ( h and c nmr ) identical to the natural product and having [ ]d + 101 ( c 1.1 , meoh ) . neuronal cell culture was prepared as previously described in detail by andjus et al.(19 ) briefly , p1p3-day - old wistar rats were decapitated . hippocampi were removed , manually sliced , treated with trypsin , mechanically dissociated , centrifuged twice at 40 g , plated in petri dishes , and cultured . currents were recorded in the whole - cell configuration of the patch - clamp technique using the axopatch 200b amplifier ( molecular instruments , sunnyvale , ca ) at a holding potential ( vh ) of 40 mv . the intrapipet solution contained ( in mm ) cscl 137 , cacl2 1 , mgcl2 2 , 1,2-bis(2-aminophenoxy)ethane - n , n , n-tetraacetic acid ( bapta ) 11 , atp 2 , and hepes 10 ( ph 7.2 with csoh ) . the composition of the standard external solution was ( in mm ) nacl 137 , kcl 5 , cacl2 2 , mgcl2 1 , glucose 20 , and hepes 10 ( ph 7.2 with naoh ) . for the whole - cell recordings , patch pipets had a resistance of 2.54.0 m when filled with internal solution . the whole - cell recordings were included in the statistics when the access resistance was below 10 m. the cells in which series resistance showed a clear tendency to increase during recordings were not considered in the analysis . recordings in the whole - cell mode were started at least 3 min after establishing the whole - cell mode . the agonist was applied using the rsc-200 multibarrel rapid perfusion system ( bio - logic , grenoble , france ) . with this system , in the whole - cell configuration , the solution exchange occurred within 30100 ms . before each control recording , cells were washed with normal external solution for at least 3 min . in studies aiming at assessment of the impact of considered test compounds [ oneantotoxin ( 1a ) , dihydrooneantotoxin ( 1b ) ] on gaba - elicited responses , these compounds were present both in the washing solution ( for at least 3 min before agonist application ) and in the agonist - containing saline . for acquisition and data analysis , pclamp 9.2 software was used ( molecular devices corporation ) . to avoid the data scatter due to cell - to - cell variability , the effect of studied drugs was assessed by calculating the relative amplitude values with respect to the controls recorded from the same cell . for the analysis of currents , recorded in the whole - cell configuration , the current signals were low - pass filtered at 3 khz with a butterworth filter and sampled at 10 khz using the analog - to - digital converter digidata 1322a ( molecular device corporation ) and stored on a computer hard disk . a paired student t test was used to assess the significance of differences between considered data sets .
an investigation of oenanthe fistulosa from sardinia afforded oenanthotoxin ( 1a ) and dihydrooenanthotoxin ( 1b ) from the roots and the diacetylenic epoxydiol 2 from the seeds . 
 the absolute configuration of 1a and 1b was established as r by the modified mosher s method , and the structure of 2 by chemical correlation with ( + ) -(3r,8s)-falcarindiol . oenanthotoxin ( 1a ) and dihydrooenanthotoxin ( 1b ) were found to potently block gabaergic responses , providing a molecular rationale for the symptoms of poisoning from water - dropwort ( oenanthe crocata ) and related plants . 
 these observations bear relevance for a series of historical and ethnopharmacological observations on the identification of the sardonic herb and the molecular details of the facial muscular contraction caused by its ingestion ( risus sardonicus ) .