compounds 14 , 7 , 8 , and 11 were 
 synthesized following our previously reported synthetic routes .
saturated derivatives 5 and 6 , unique to this work , were synthesized via the hydrogenation 
 of diastereomers 1 and 2 , respectively . 
 palmyrolide alcohol ( 9 ) and acid ( 10 )
were 
 prepared exploiting the known acid - catalyzed ring opening of 1 , followed by treating the resultant 
 aldehyde with either nabh4 ( for 9 ) or naclo2 ( for 10 ) .
alcohol 12 could be accessed 
 in a similar manner from 14-epi - palmyrolide ( 2 ) . in order to detect an interaction of ( )-palmyrolide 
 a and 
 its analogues with voltage - gated sodium channels ( vgscs )
, we examined 
 the ability of these compounds to antagonize veratridine - stimulated 
 na influx in murine primary cerebrocortical neurons .
we 
 previously demonstrated that veratridine is a partial agonist at neurotoxin 
 site 2 on the vgsc  subunit .
murine 
 cerebrocortical neurons were loaded with the na - binding 
 benzofuran isophthalate ( sbfi ) , and the ability of palmyrolide analogues 
 to block veratridine - induced elevation of neuronal [ na]i was determined .
sodium influx was monitored with sbfi ( 340/380 ) responses to veratridine 
 in the absence and presence of ( )-palmyrolide a and its analogues . 

the compounds shown are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide a ( 5 ) ; ( c ) ( + ) -palmyrolide 
 a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide 
 a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f ) 
 [ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) .
data shown are from a representative 
 experiment performed in 2 or 3 replicates for each concentration . 
 in total 47 experiments
nonlinear regression analysis of concentration  response 
 data for active analogues of ( )-palmyrolide a. a three - parameter 
 logistic fit of the palmyrolide a analogue inhibition of the response 
 to veratridine is shown for each active compound .
the compounds shown 
 are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide 
 a ( 5 ) ; ( c ) ( + ) -palmyrolide a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f ) [ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) . data points 
 shown represent the mean  sem of 25 experiments performed 
 with 210 replicates each ( a  f ) .
values represent mean  sem 
 of 25 experiments each performed with 210 replicates . naturally occurring ( )-palmyrolide 

a is a neuroactive macrolide 
 that was previously shown to block veratridine - induced sodium influx 
 in neuro-2a cells ( ic50 = 5.2 m ) and to display significant inhibition of ca oscillation in murine cerebrocortical neurons , with an ic50 value of 3.70 m ( 2.295.98 m , 95% ci ) .
we have confirmed and extended these original 
 findings by demonstrating that synthetic ( )-palmyrolide a 
 ( 1 , figures 2a and 3a ) and its enantiomer ( 3 , figures 2c and 3c ) produced concentration - dependent 
 antagonism of veratridine - induced increase in neuronal [ na]i .
the concentration  response curves for these 
 compounds were best fit by a three - parameter logistic equation .
this 
 analysis yielded an ic50 value ( 95% confidence interval ) 
 of 2.1 m ( 1.23.1 , 95% ci ) , which was in good agreement 
 with the ic50 value reported earlier for natural ( )-palmyrolide 
 a. we found that ( )-palmyrolide 
 a ( 1 ) and macrolide analogues 5 and 3 were approximately 39-fold more potent than the 
 other analogues tested ( figures 2a 
the observation that saturated 
 congener 5 showed a comparable ic50 to synthetic 
 ( )-palmyrolide a suggests that the enamide double bond is 
 not essential for activity .
it is moreover noteworthy that acyclic 
 analogues of ( )-palmyrolide a with the natural absolute configuration 
 were inactive as blockers of veratridine - stimulated na influx .
these data indicate that the three - dimensional molecular 
 shape of ( )-palmyrolide a was crucial for the interaction 
 with vgscs . in order to support these observations and to better 

understand 
 how structure relates to biological function , an exhaustive nmr and 
 computational analysis was next conducted on 1 and three 
 diastereomers ( i.e. , 2 , 7 , and 8) .
saturated derivatives 5 and 6 exist 
 as a mixture of rotational isomers and precluded precise nmr assignments 
 from being made .
chemical shift assignments of ( )-palmyrolide 
 a ( 1 ) and synthetic diastereomers 2 , 7 , and 8 were made using dqf - cosy , hsqc , hmbc , 
 and h2bc spectra . 

the chemical shifts of these four diastereomeric compounds follow 
 the same general trends but differ slightly , indicating that the conformations 
 of the macrolide ring are different . for example , in each diastereomer 
 a different methylene group exhibited degenerate chemical shifts for 
 its two protons .
stereospecific assignments were made for most of 
 the prochiral atoms that possessed nondegenerate shifts .
the noesy 
 spectra provided 74 nonredundant distance restraints for 1 , 83 for 2 , 84 for 7 , and 77 for 8 .
h coupling in the h nmr spectra 
 and from the heteronuclear couplings measured by the hetloc experiments 
 ( see supporting information for all restraints ) . 
 applying the distance and torsion restraints during simulated annealing 
 calculations produced fairly well - defined ensembles of structures . 

table 2 summarizes the inputs 
 and results of the structure calculations . to test the specificity 
 of the restraints and their ability to discriminate between the four 
 diastereomers ,
restrained simulated annealing calculations were performed 
 for all 16 combinations of starting structure and restraint set . if 
 the restraints correctly discriminated between conformations , then 
 the correct combination should have the least restraint violations 
 and the lowest energy .
for all four structures we found the best restraint 
 set for each starting structure was the correct one , namely , the restraints 
 derived from nmr spectra of that compound ( table 3 ) .
this confirms that the restraint sets were able to correctly 
 discriminate between the diastereomers .
the four ensembles generally 
 have the same overall conformation , 
 with the major differences being due to the orientations of the substitutents . 

all four diastereomers have a cisoid configuration 
 of the n - methyl group ( n - ch3 ) and the 
 adjacent carbonyl despite the starting structures having a transoid configuration .
examining the trajectories reveals 
 that the configuration converts early in the simulated annealing as 
 the restraints take effect .
the driving force in these simulations 
 appears to be noes between h-18 and the h-2 methylene group . to quantify differences between the ensembles , we calculated root - mean - square 
 deviations ( rmsd s ) between the representative structures of 
 each ensemble ( table 4 ) .
the rmsd s 
 were calculated using the differences in the positions of the carbon , 
 nitrogen , and oxygen atoms of the macrolide ring after aligning the 
 structures to minimize these differences .
representative structures 
 were selected as the structure having the smallest rmsd among all 
 other structures in its ensemble .
the rmsd s between all four 
 representative structures were similar ( table 4 ) , with 2 having the lowest values overall , suggesting 
 that this ensemble is the most  central  in comparison 
 to the other diastereomers .
visual inspection of the ensembles 
 shows that the macrolide ring 
 is relatively flat , but the orientation of the tert - butyl and methyl groups alters the global shape ( figure 4 ) . in 1
the tert - butyl 
 and methyl groups lie close to the plane of the ring , but in 7 the tert - butyl group projects almost perpendicular 
 to the plane of the ring , and in 8 the two methyl groups 
 project in opposite directions above and below the ring . examining 
 the surface electrostatic potential of the representative structures 
 ( figure 5 ) we find that 8 is the 
 most similar to the active compound 1 . however , the c-14 
 methyl group in 1 projects to the side , whereas in 8 it is situated above the macrolide ring . taken together , 
 these differences between diastereomers may account , at least in part , 
 for the observed differences in biological activity .
in addition , 
 due to the highly flexible nature of the macrolide , as evidenced through 
 computational modeling , the minimized ligand ensembles reported here 
 may not necessarily reflect the structure these molecules adopt when 
 bound to their molecular target .
further consideration and investigations 
 into structure  activity relationships are ongoing and will 
 be reported in due course .
number of diastereotopic hydrogens 
 for which stereospecific assignments were obtained , and total number 
 of protons with nondegenerate chemical shifts .
the three numbers reported for each 
 combination of starting structure and restraint set are number of 
 selected structures , average amber energy , and average violation energy . 

the italic cells indicate the best set of restraints for each starting 
 structure , with respect to amber energy and violation energy , and 
 also correspond to the correct pairing .
the final row is the sum of the 
 three rmsd s above , a measure of the total difference from 
 the other structures .
the four structures in the top row are shown in 
 a similar orientation to figure 4 . in the lower 
 row
the structures have been rotated 180 about the horizontal 
 axis to show the rear face .
the electrostatic potential ranges from 
 red ( 2 kcal / mole ) through white ( 0 ) to blue ( + 2 kcal / mole ) .
these 
 findings suggest that both synthetic ( )-palmyrolide 
 a and its enantiomer function with equal potency as vgsc antagonists 
 to block veratridine - induced sodium influx . with regard to identification 
 of the pharmacophore of this natural product
, it appears that the 
 intact macrolide is required since acyclic versions were inactive 
 as inhibitors of vgscs .
moreover , analogues lacking the enamide double 
 bond were of high potency in this assay , and thus this functionality 
 appears unnecessary for blocking vgscs . finally ,
using detailed nmr 
 and computational approaches with the natural product and three diastereomeric 
 analogues , it was found that the c-5c-7 anti / c-7c14 syn arrangement of stereocenters 
 produces a unique combination of three - dimensional shape and electrostatic 
 potential that is responsible for the potent biological activity of 
 the natural product . in an effort to understand
the similar biological 
 activity found for the natural stereoisomer and its enantiomer , continued 
 investigations in this area will focus on uncovering the specific 
 molecular target and associated binding site , which may also assist 
 in future analogue development of novel sodium channel blocking analgesics 
 derived from palmyrolide a.
unless otherwise noted , 
 reactions were performed in flame - dried glassware under an atmosphere 
 of dry nitrogen .
reaction solvents ( ch2cl2 , 
 thf , and et2o ) were purified before use in a solvent purification 
 system under a flow of dry nitrogen .
all other solvents and reagents 
 were purchased from commercial suppliers and used as received , unless 
 otherwise specified .
thin - layer chromatography ( tlc ) was performed 
 using plates precoated with silica gel 60  f-254 ( 250 m ) 
 and visualized by uv light , kmno4 , or anisaldehyde stains , 
 followed by heating .
ir samples were prepared by evaporation 
 from chcl3 or ch2cl2 on nacl plates 
 and run on a perkinelmer spectrum one ft - ir spectrometer . for the 
 synthetic studies ,
h and c nmr spectra were 
 recorded at 300 and 75 mhz ( oxford magnet with a varian 300 console ) , 
 at 400 and 100 mhz ( oxford magnet with a varian unity 400 console ) , 
 and at 600 and 150 mhz ( magnex magnet with a bruker avance
iii 600 
 console ) , respectively , and are reported relative to residual solvent 
 peak ( h 7.26 and c 77.0 for h and c in cdcl3 ) .
high - resolution 
 mass spectra were obtained using positive electrospray ionization 
 on a bruker 12 t apex - qe fticr - ms with an apollo ii ion source at 
 the cosmic laboratory facility at old dominion university , va . to a solution of ( s , e)-6-iodo-2-methylhex-5-enoic 
 acid ( 0.096 g , 0.37 mmol ) in thf ( 3.7 ml )
was added diisopropylethylamine 
 ( 0.100 ml , 0.60 mmol ) followed by the yamaguchi reagent ( 0.071 ml , 
 0.45 mmol ) .
the mixture was allowed to stir at room temperature for 
 3 h before being concentrated in vacuo .
the resultant 
 mixed anhydride was dissolved in toluene ( 7.4 ml ) and then added , 
 via cannula , to a flask containing ( 5r,7r)-7-hydroxy-5,8,8-trimethylnonanamide ( 0.051 g , 0.23 mmol ) and dmap 
 ( 0.046 g , 0.37 mmol ) . after stirring at room temperature ( rt ) for 
 12 h
, the reaction mixture was diluted with ch2cl2 , washed with a saturated aqueous solution of nahco3 , 
 dried over mgso4 , and concentrated .
purification via flash 
 column chromatography ( 1:1 hexanes / etoac ) afforded 0.088 g ( 81% ) of 
 the coupled product as a colorless oil : [
]d22.5 + 13.3 ( c 1.98 , 
 chcl3 ) ; ir ( neat , thin film )  3429 , 3351 , 3203 , 
 2962 , 2934 , 2868 , 1725 , 1665 , 1607 , 1461 , 1380 , 1366 , 1259 , 1181 , 
 1119 , 1067 , 957 , 935 cm ; h nmr ( 300 
 mhz , cdcl3 )  6.49 ( dt , j = 7.2 , 
 14.4 hz , 1 h ) , 6.02 ( d , j = 14.4 hz , 1h ) , 5.88 ( bs , 
 1 h ) , 5.46 ( bs , 1 h ) , 4.79 ( dd , j = 4.5 , 6.3 hz , 
 1 h ) , 2.45 ( sextet , j = 6.9 hz , 1 h ) , 2.232.15 
 ( m , 2 h ) , 2.122.05 ( m , 2 h ) , 1.861.69 ( m , 2 h ) , 1.611.25 
 ( m , 6 h ) , 1.17 ( d , j = 7.2 hz , 3 h ) , 1.110.99 
 ( m , 1 h ) , 0.910.87 ( overlapping doublet / singlet , 12 h ) ; c nmr ( 75 mhz , cdcl3 )  176.5 , 175.8 , 145.6 , 
 79.0 , 75.5 , 39.3 , 37.7 , 35.7 , 34.79 , 34.77 , 33.9 , 32.3 , 29.2 , 26.1 , 
 22.8 , 21.1 , 17.5 ; hresims m / z 474.1471 
 [ m + na ]
( calcd for c19h34ino3na , 474.1476 ) . to the coupled product ( 0.088 g , 0.19 
 mmol ) in thf ( 19.0 ml )
was added cui ( 0.005 g , 0.03 mmol ) and cs2co3 ( 0.100 g , 0.30 mmol ) .
the mixture was first 
 degassed using nitrogen ( 10 min ) before n , n-dimethylethylenediamine ( 0.050 ml , 0.46 mmol ) 
 was added .
the rubber septum was quickly replaced with a glass stopper , 
 and the reaction mixture heated to 60 c for 19 h. after cooling 
 to rt , the reaction mixture was diluted with etoac , filtered through 
 a short plug of silica , and concentrated .
the crude product was purified 
 via flash column chromatography ( 8:2  7:3  1:1 hexanes / etoac ) 
 to yield the n - h enamide intermediate ( 0.022 g ) , 
 which was immediately dissolved in thf ( 0.7 ml ) and cooled to 0 c . 
 to this
was added nah ( 0.014 g , 0.34 mmol , 60% in mineral oil ) in 
 a single portion .
the reaction mixture was allowed to stir for 20 
 min before mei ( 0.100 ml ) was added dropwise , followed by removal 
 of the ice water bath . after being allowed to stir at rt for 20 min , 

the reaction mixture was again cooled to 0 c before being quenched 
 with h2o , extracted using etoac , dried over mgso4 , and concentrated .
the crude product was purified via flash column 
 chromatography ( 9:1 hexanes / etoac ) to yield 14-epi - palmyrolide a ( 0.016 g , 25% over two steps ) as a colorless oil : 
 [ ]d24.5 11.4 ( c 0.58 , chcl3 ) ; ir ( neat , 
 thin film )  2959 , 2927 , 2873 , 1728 , 1675 , 1646 , 1466 , 1413 , 
 1384 , 1366 , 1333 , 1298 , 1240 , 1205 , 1193 , 1171,1121 , 933 cm ; h nmr ( 400 mhz , cdcl3 )  6.63 ( d , j = 13.6 hz , 1 h ) , 4.92 ( ddd , j = 5.2 , 
 8.8 , 13.6 hz , 1 h ) , 4.85 ( dd , j = 2.0 , 9.6 , 1 h ) , 
 3.05 ( s , 3 h ) , 2.602.42 ( m , 3 h ) , 2.32 ( dt , j = 7.2 , 13.6 hz , 1 h ) , 2.242.17 ( m , 1 h ) , 2.031.95 
 ( m , 1 h ) ,
1.841.74 ( m , 1 h ) , 1.681.54 ( m , 2 h ) , 1.491.30 
 ( m , 4 h ) , 1.26 ( d , j = 7.2 hz , 3 h ) , 1.030.93 
 ( m , 1 h ) , 0.89 ( d , j = 6.4 hz , 3 h ) , 0.86 ( s , 9 h ) ; c nmr ( 100 mhz , cdcl3 )  175.4 , 172.8 , 129.8 , 
 110.2 , 76.5 , 37.2 , 36.6 , 35.4 , 34.3 , 33.6 , 31.0 , 29.8 , 29.5 , 27.3 , 
 25.9 , 24.9 , 20.0 , 19.2 ; hresims m / z 360.2504 [ m + na ] ( calcd for c20h35no3na , 360.2509 ) .
to a solution 
 of ( )-palmyrolidea ( 0.008 g , 0.022 mmol ) in anhydrous meoh 
 ( 1 ml ) was added 10% palladium on carbon ( 0.005 g ) .
the atmosphere 
 in the flask was replaced with hydrogen , and the reaction mixture 
 was allowed to stir at rt for 18 h before being filtered through a 
 short plug of silica .
after rinsing several times with ether , the 
 filtrate was concentrated and purified via column chromatography ( 8:2 
 hexanes / etoac ) to afford compound 5 ( 0.006 g , 77% ) as 
 a mixture of rotational isomers ( 2:1 ratio ) : [ ]d23 + 35.6 ( c 0.48 , chcl3 ) ; ir ( neat , thin film )  
 2954 , 2931 , 2871 , 1720 , 1651 , 1269 , 1250 , 1073 cm ; h nmr ( 300 mhz , cdcl3 , mixture of rotational 
 isomers with mostly overlapping peaks , making the assignment of major 
 and minor components difficult )  ( major isomer ) 4.82 ( d , j = 10.0 hz , 1 h ) , 3.30 ( t , j = 5.0 hz , 
 1 h ) , 2.98 ( s , 3 h ) , 2.532.65 ( m , 2 h ) , 2.212.33 ( m , 
 2 h ) , 1.211.73 ( m , 14 h ) , 1.18 ( d , j = 6.9 
 hz , 3 h ) , 0.94 ( d , j = 5.9 hz , 3 h ) , 0.86 ( s , 9 h ) , 
 ( minor isomer , partial ) 4.92 ( d , j = 10.8 hz , 0.6 
 h ) , 2.89 ( s , 1.5 h ) , 0.91 ( d , j = 6.1 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 , mixture of rotational isomers ) 
  175.5 , 174.8 , 173.5 , 77.8 , 77.2 , 48.9 , 46.3 , 40.4 , 40.0 , 38.3 , 
 37.2 , 35.2 , 35.0 , 33.6 , 33.2 , 32.98 , 32.96 , 31.2 , 39.7 , 27.9 , 27.6 , 
 27.0 , 26.0 , 25.9 , 25.4 , 23.5 , 23.2 , 22.2 , 20.4 , 20.1 , 18.0 , 16.5 ; 
 hresims m / z 362.2660 [ m + na ] ( calcd for c20h37no3na , 
 362.2665 ) .
the crude product was purified via column 
 chromatography ( 8:2 hexanes / etoac ) to afford compound 6 ( 0.006 g , 96% ) as a mixture of rotational isomers ( 2:1 ratio ) : 
 [ ]d23 + 42.1 ( c 0.5 , chcl3 ) ; ir ( neat , thin 
 film )  2956 , 2930 , 2872 , 1727 , 1647 , 1459 , 1192 , 1156 cm ; h nmr ( 300 mhz , cdcl3 , mixture 
 of rotational isomers with mostly overlapping peaks , making the assignment 
 of major and minor components difficult )  ( major isomer ) 4.86 
 ( d , j = 10.5 hz , 1 h ) , 3.513.42 ( m , 1 h ) , 
 3.233.14 ( m , 1 hz ) , 2.88 ( s , 3 h ) , 2.312.48 ( m , 2 
 h ) , 2.182.31 ( m , 2 h ) , 1.971.79 ( m , 2 h ) , 1.231.66 
 ( m , 15 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.92 ( d , j = 5.8 hz , 3 h ) , 0.86 ( s , 11 h ) , ( minor isomer , partial ) 
 4.83 ( d , j = 11.6 hz , 0.6 h ) , 2.91 ( s , 1 h ) , 1.15 
 ( d , j = 7.2 hz , 1.5 h ) , 0.96 ( d , j = 5.7 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 , 
 mixture of rotational isomers )  176.8 , 176.6 , 173.3 , 173.0 , 
 77.2 , 49.1 , 46.0 , 40.6 , 40.1 , 38.0 , 37.3 , 35.2 , 34.0 , 33.9 , 33.2 , 
 33.1 , 33.0 , 31.3 , 29.7 , 28.9 , 27.2 , 25.9 , 24.8 , 23.9 , 22.8 , 19.9 , 
 19.8 , 19.2 , 19.1 ; hresims m / z 362.2659 
 [ m + na ]
( calcd for c20h37no3na , 362.2665 ) . to a solution 
 of 1 ( 0.007 g , 0.021 mmol ) in chcl3 ( 2 ml ) 

was added a 6 n aqueous hcl solution dropwise until tlc analysis showed 
 complete consumption of the starting material .
the reaction mixture 
 was then quenched with a saturated aqueous solution of nahco3 , followed by extraction using etoac .
the 
 resultant crude aldehyde was dissolved in anhydrous meoh ( 1 ml ) and 
 treated with nabh4 ( 0.015 g , 0.39 mmol ) .
the reaction mixture 
 was allowed to stir at rt for 1 h before being quenched with h2o , extracted using etoac , dried over mgso4 , filtered , 
 and concentrated .
the crude alcohol was purified by column chromatography 
 ( 100% etoac ) to afford alcohol 9 ( 0.006 g , 73% over two 
 steps ) as a colorless oil : [ ]d24.3 + 26.8 ( c 0.20 , chcl3 ) ; ir ( neat , thin film )  3296 , 2956 , 1728 , 1651 , 1562 , 
 1462 , 1366 , 1164 cm ; h nmr ( 300 mhz , 
 cdcl3 )  6.10 ( bs , 1 h ) , 4.76 ( dd , j = 2.2 , 9.5 hz , 1 h ) , 3.63 ( t , j = 6.4 hz , 2 h ) , 
 2.79 ( d , j = 4.8 hz , 3 h ) , 2.392.51 ( m , 1 
 h ) , 2.002.23 ( m , 2 h ) , 1.91 ( bs , 1 h ) , 1.631.80 ( m , 
 2 h ) , 1.221.60 ( m , 10 h ) , 1.16 ( d , j = 7.0 
 hz , 3 h ) , 0.87 ( d , overlapped , 3 h ) , 0.86 ( s , 9 h ) ; c 
 nmr ( 75 mhz , cdcl3 )  176.8 , 174.0 , 78.6 , 62.5 , 40.2 , 
 37.6 , 36.2 , 34.5 , 33.3 , 32.5 , 28.8 , 26.2 , 25.9 , 25.9 , 23.5 , 23.0 , 
 20.8 , 17.4 ; hresims m / z 380.2764 
 [ m + na ] ( calcd for c20h39no4na , 380.2771 ) . approximately 0.3 
 ml of an oxidant solution prepared via the mixing of naclo2 ( 0.060 g ) and nahpo4 ( 0.040 g ) in h2o ( 1 ml ) 
 was added to a solution of palmyrolide aldehyde ( prepared as above , 
 0.006 g , 0.016 mmol ) in tert - butanol ( 0.25 ml ) and 
 2-methyl-2-butene ( 0.1 ml ) . the reaction mixture was allowed to stir 
 for 30 min before being quenched with water , extracted with etoac , 
 dried over mgso4 , filtered , and concentrated .
the crude 
 acid was purified by column chromatography ( 100% etoac ) to afford 
 acid 10 ( 0.002 g , 25% ) as a colorless oil : [ ]d23 + 10.8 ( c 0.10 , chcl3 ) ; ir ( neat , thin film )  
 3306 , 2960 , 1727 , 1633 , 1164 cm ; h nmr ( 300 mhz , cdcl3 )  6.04 ( bs , 1 h ) , 4.78 ( dd , j = 10.2 , 1.9 hz , 1 h ) , 2.82 ( d , j = 4.8 
 hz , 3 h ) , 2.48 ( q , j = 6.9 hz , 1 h ) , 2.312.41 
 ( m , 2 h ) , 2.022.23 ( m , 3 h ) , 1.601.82 ( m , 4 h ) , 1.211.59 
 ( m , 6 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.921.05 
 ( m , 1 h ) , 0.88 ( d , overlapped , 3 h ) , 0.87 ( s , 9 h ) ; c 
 nmr ( 75 mhz , cdcl3 )  176.4 , 78.5 , 40.1 , 37.6 , 36.2 , 
 34.6 , 34.4 , 33.0 , 29.7 , 28.7 , 26.3 , 26.0 , 23.0 , 22.7 , 20.7 , 17.2 ; 
 hresims m / z 394.2558 [ m + na ] ( calcd for c20h37no5na , 
 394.2563 ) .
the crude product was purified via column chromatography 
 ( 100% etoac ) to afford compound 12 ( 0.004 g , 94% ) : [ ]d25.5 + 30.8 ( c 0.24 , chcl3 ) ; ir ( neat , thin film )  
 3304 , 2950 , 1729 , 1649 , 1560 , 1462 , 1366 , 1163 , 1074 cm ; h nmr ( 300 mhz , cdcl3 )  6.25 ( bs , 
 1 h ) , 4.76 ( dd , j = 9.9 , 1.6 hz , 1 h ) , 3.553.70 
 ( m , 2 h ) , 2.77 ( d , j = 4.8 hz , 3h ) , 2.372.50 
 ( m , 1 h ) , 2.27 ( bs , 1 h ) , 2.002.20 ( m , 2 h ) , 1.631.83 
 ( m , 2 h ) , 1.211.60 ( m , 10 h ) , 1.16 ( d , j = 
 7.0 hz , 3 h ) , 0.921.04 ( m , 1 h ) , 0.86 ( d , overlapped , 3 h ) , 
 0.85 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 )  
 177.0 , 174.1 , 78.4 , 62.2 , 40.0 , 37.5 , 36.2 , 34.6 , 34.5 , 33.1 , 32.4 , 
 28.8 , 26.1 , 25.9 , 23.6 , 23.1 , 20.7 , 17.7 ; hresims m / z 380.2764 [ m + na ] ( calcd for c20h39no4na , 380.2771 ) .
primary cultures of 
 cerebrocortical neurons were obtained from embryonic day 17 swiss - webster 
 mice as described elsewhere .
briefly , 
 pregnant mice were euthanized by co2 asphyxiation , and 
 embryos were removed under sterile conditions .
cerebral cortices were 
 collected , stripped of meninges , minced by trituration with a pasteur 
 pipet , and treated with trypsin for 25 min at 37 c .
the cells 
 were then dissociated by two successive trituration and sedimentation 
 steps in soybean trypsin inhibitor and dnase - containing isolation 
 buffer , centrifuged , and resuspended in eagle s minimal essential 
 medium with earle s salt ( mem ) supplemented with 1 mm l - glutamine , 10% fetal bovine serum , 10% horse serum , 100 iu / ml penicillin , 
 and 0.10 mg / ml streptomycin ( ph 7.4 ) .
cells were plated onto poly - l - lysine - coated 96-well ( 9 mm ) , clear - bottomed , black - well culture 
 plates ( midsci ) at a density of 1.5  10 cells / well . 

cells were then incubated at 37 c in a 5% co2 and 
 95% humidity atmosphere .
the culture media was changed every other 
 day , starting from day 5 in vitro using a serum - free 
 growth medium containing neurobasal medium supplemented with b-27 , 
 100 iu / ml penicillin , 0.10 mg / ml streptomycin , and 0.2 mm l - glutamine .
all animal use protocols were 
 approved by the institutional animal care and use committee ( iacuc ) 
 of creighton university .
[ na]i measurement and full in situ calibration of sodium - binding benzofuran isophthalate fluorescence 
 ratio were performed as described previously .
cells were grown in 96-well plates and washed four times with locke s 
 buffer ( 8.6 mm hepes , 5.6 mm kcl , 154 mm nacl , 5.6 mm glucose , 1.0 
 mm mgcl2 , 2.3 mm cacl2 , 0.1 mm glycine , ph 7.4 ) 
 using an automated microplate washer ( biotek instruments ) . after measuring 
 the background fluorescence of each well , cells were incubated for 
 1 h at 37 c with dye - loading buffer ( 100 l / well ) containing 
 10 m sbfi - am ( invitrogen ) and 0.02% pluronic f-127 ( invitrogen ) . 

cells were washed five times with locke s buffer , leaving a 
 final volume of 100 l in each well .
the plate was then transferred 
 back to the incubator for 15 min to allow the cells to equilibrate 
 after washing and placed in a flexstation ii ( molecular devices ) chamber 
 to detect na - bound sbfi emission at 505 nm ( cells were 
 excited at 340 and 380 nm ) .
fluorescence readings were taken once 
 every 5 s for 60 s to establish the baseline , and then 50 l 
 of different concentrations of ( )-palmyrolide a and/or veratridine 
 was added to each well from the compound plate at a rate of 26 l / s , 
 yielding a final volume of 200 l / well . after correcting for 
 background fluorescence , sbfi fluorescence ratios ( 340/380 ) and concentration 
each palmyrolide a analogue was dissolved in 
 dmso at a stock concentration of 50 mm and stored at 20 c 
 until used . on the day of an experiment ,
this dilution in locke s 
 buffer resulted in palmyrolide a analogue solutions that were 4 times 
 the desired final concentration .
( )-palmyrolide 
 a ( 1 ) and analogues ( 2 , 7 , 
 and 8) were dissolved in cdcl3 at a concentration 
 of 2.5 mg in 50 l and transferred to a 1.7 mm nmr tube . 

the noesy mixing time was 500 ms , the hmbc delay was set to 6.25 ms 
 ( 8 hz ) , and the hetloc used a dispi-2 spinlock of 60 ms duration . 

chemical shift assignments were based on analysis of the dqf - cosy , 
 hsqc , and hmbc 2d experiments using ucsf sparky .
topspin ( bruker biospin ) was used to analyze coupling constants 
 in the h nmr and hetloc spectra .
noesy peak heights were 
 converted to upper distance restraints of 3 , 4 , 5 , or 6  using 
 in - house perl scripts . where restraints involved a group of atoms , 
 pseudoatom corrections were applied by the amber restraint generation 
 tools .
force field parameters were estimated using 
 the gaff force field and the antechamber 
 program in the amber 14 package .
molecular 
 dynamics simulations were nonperiodic , vacuum simulations with a distance - dependent 
 dielectric term .
using sander in the amber 14 package each starting 
 structure was energy minimized , then subjected to 20 ps of simulated 
 annealing with the nmr - derived restraints in which the temperature 
 was raised from 10 k to 600 k then cooled to near 0 k. the simulated 
 annealing was run 20 times for each structure with a different random 
 number seed each time .
each collection of 20 structures was sorted 
 by restraint violation energy , and outliers were discarded . to align 
 structures ,
the selected ensemble was superimposed on c17 , 
 o12 , c1318 , and n19 using cpptraj . to stereospecifically assign pairs of diastereotopic hydrogens , 
 pairs of noes that involved the diastereotopic atoms and had differential 
 intensities
were identified , then compared with the distances between 
 these atoms in the ensemble of selected structures .
if the ensemble 
 mean of one distance was more than one standard deviation from the 
 alternative , then the diastereotopic hydrogens were stereospecifically 
 assigned .
 a small library of synthetic ( )-palmyrolide 
 a diastereomers , 
 analogues , and acyclic precursors have been examined with respect 
 to their interaction with voltage - gated sodium channels ( vgscs ) . toward 
 this goal , 
 the ability of ( )-palmyrolide a and analogues to 
 antagonize veratridine - stimulated na+ influx in primary 
 cultures of mouse cerebrocortical neurons was assessed . 
 we found that 
 synthetic ( )-palmyrolide a and its enantiomer functioned as 
 vgsc antagonists to block veratridine - induced sodium influx . a detailed 
 nmr and computational analysis of four diastereomers revealed that 
 none had the same combination of shape and electrostatic potential 
 as exhibited by natural ( )-palmyrolide a. these data indicate 
 that the relative configuration about the tert - butyl 
 and methyl substituents appears to be a prerequisite for biological 
 function . additional testing revealed that the enamide double bond 
 was not necessary for blocking veratridine - induced sodium influx , 
 whereas the acyclic analogues and other macrolide diastereomers tested 
 were inactive as inhibitors of vgscs , suggesting that the intact macrolide 
 was required . 
Results and Discussion
Conclusion
Experimental Section