voltage - gated sodium 
 ( nav ) channels are the fundamental generators of electrical 
 impulses ( action potentials ) in the membranes of excitable cells . 

these drugs are primarily used in the treatment 
 of central nervous system ( cns ) disorders , such as epilepsy and neuropathic 
 pain , but are increasingly being scrutinized as potential treatments 
 for neurodegenerative conditions occurring acutely , such as in ischemic 
 stroke , or chronically , as in multiple sclerosis ( ms ) .
it exerts a voltage - dependent 
 and frequency - dependent block of na currents .
this selectivity 
 is suggested to be the reason for its lack of significant cognitive 
 side effects .
other first - generation antiepileptics 
 that act on nav channels include lamotrigine 2 , and carbamazepine 3(4 ) ( figure 1 ) .
recently , 
 lacosamide 4 was approved for use in partial - onset seizures 
 and neuropathic pain .
lacosamide is proposed to act via an unique 
 mechanism of state - dependent blockade of nav channels : 
 stabilizing channels in a slow inactivated state without effects on 
 fast inactivation . compounds with sodium 
 channel modulatory activity .
nine nav channel isoforms have been identified , 
 of which , 
 nav1.1 , 1.2 , 1.3 , and 1.6 are predominately found within 
 the cns , whereas nav1.7 , 1.8 , and 1.9 are located in the 
 peripheral nervous system .
nav1.4 is found within skeletal 
 muscle , and nav1.5 is found in the heart .
recently , the first x - ray crystal structure of 
 a voltage - gated sodium channel was solved : initially in the closed 
 state and subsequently in two presumed inactivated states . under normal physiological conditions ,
the channels interconvert 
 between different functional states : the resting state in which the 
 channel pore is closed , an open state in which the channel pore is 
 open and permeable to na , and one or more inactivation 
 states where the pore is open but blocked .
recent studies have highlighted the importance of nav1.3 , 1.7 , 1.8 , and 1.9 in various pain syndromes .
notably , genetically 
 inherited pain disorders have been associated with mutation of these 
 nav encoding genes .
clinically , 
 carbamazepine , lamotrigine , and phenytoin are all used to treat neuropathic 
 pain .
the merck , pfizer , astrazeneca , 
 and glaxosmithkline pharmaceutical groups have all independently reported 
 their own series of 1.3- , 1.7- , and 1.9-selective sodium channel modulators 
 for use in neuropathic pain , exemplified 
 here by the benzazepinone compounds from merck pharmaceuticals , such 
 as 5 ( figure 1 ) .
influx of sodium from the extracellular 
 space during reperfusion plays a major role in excitotoxic neuronal 
 death after cerebral ischemia .
block 
 of nav channels can attenuate the primary events that occur 
 in an ischemic attack , most likely by blocking na permeability , 
 resulting in reduced atp depletion and reduced ischemic - induced release 
 of glutamate .
a number of drugs , including 
 lamotrigine , bw-1003c87 , the local anesthetic lidocaine , and sipatrigine , 
 have all shown neuroprotective effects in animal models of stroke . however , as of yet , these results have failed to translate into 
 beneficial effects in clinical trials .
thus , there 
 is a need for drugs with a better clinical efficacy and therapeutic 
 index .
treatment of stroke is limited by the fact that administration 
 of treatment in humans only occurs sometime after the onset of the 
 neurodegenerative insult .
in contrast , for slowly evolving neurodegenerative 
 conditions , it is possible to treat during or even before individual 
 neurodegenerative episodes occur .
carbamazepine ( 3 ) is 
 commonly used to alleviate the symptoms of ms , such as spasms , nerve 
 pain , and trigeminal neuralgia , and lamotrigine has been trialled 
 in neuroprotection .
research into the 
 potential neuroprotective effects of these compounds is gaining increasing 
 momentum because of a better understanding of the role of nav channels in nerve damage . to 
 understand the role of individual nav channel isoforms 
 in neuroprotection
although 
 most of the sodium channel variants are expressed at different levels 
 in the cns , nav1.6 expression is widespread but is more abundant in nerves of the retina , 
 cerebellum , and cortex tissue . in a mature , 
 myelinated axon , nav1.6 tends to cluster and is the predominant 
 isoform at the nodes of ranvier .
a significant 
 increase in nav1.6 density can be seen subsequent to nerve 
 fiber injury in traumatic brain injury and multiple sclerosis .
mechanical , ischemic , and inflammatory insult can , 
 over time , contribute to this degeneration .
shear and stretch forces , 
 despite not destroying the cells or severing the axons can , nonetheless , 
 produce secondary degeneration in brain tissues .
rapid tetrodotoxin - sensitive calcium overload occurs in 
 stretch - traumatized axons , which suggests that in neuropathologic 
 conditions structural degradation of the axolemmal bilayer can foster 
 a chronically left - shifted nav channel operation .
this 
 points to leaky nav channels playing an important role 
 in determining neuronal cell survival .
other nav channel isoforms , namely , nav1.1 and nav1.3 , have also been implicated 
 in traumatic brain injury models .
overall , 
 there is no clear understanding of the specific contributions of each 
 isoform to the neuroprotective effect ; we anticipate that investigations 
 of the action of well - characterized compounds will aid in this regard . 

previous work by this group designed a class of voltage - dependent 
 sodium channel modulators based on the indazolyloxadiazolyl scaffold .
further development of analogues around this 
 scaffold has now identified a series of imidazolylethylindazoles with 
 good neuroprotective effects .
following assessment of sodium channel 
 isoform selectivity and functional activity , a candidate molecule , 
 cfm6104 , 6 ( figure 1 ) , was tested 
 in an in vivo optic neuritis ( inflammation of the optic nerve ) mouse 
 model of ms and found to prevent the consequent loss of retinal nerve 
 cells .
in our previous investigations , 
 we demonstrated 
 that aminomethyloxadiazolylindazoles had good neuroprotective activity . 

if the amino group was protected as a boc group , then the activity 
 was generally lost . however , exploration of alternative groups to 
 the indazole n1/n2 benzyl revealed a boc - protected 
 intermediate ( vide infra ) with surprising neuroprotective activity . 

this molecule exhibited a different sar profile to the previous series 
 with lower cytochrome p450 inhibition .
we therefore carried out a 
 detailed investigation of the neuroprotective activities of this series . 

key objectives were to maintain the neuroprotective activity and to 
 improve the metabolic stability to allow evaluation in vivo .
the synthesis of 3-[3-tert - butoxycarbonylaminomethyl-1,2,4-oxadiazol-5-yl]indazole 7 ( scheme 1 ) has been previously described .
this compound was utilized as a key synthetic 
 intermediate for the production of a further series of compounds . 

the stereoselective synthesis of alkyl n1- and n2-heteroaromatic - substituted compounds ( scheme 1 ) was carried out using a cesium- or potassium carbonate - mediated 
 alkylation of an alkyl halide - functionalized heteroaromatic compound 
 ( n1 ) , 8 , or a mitsunobu reaction with an alcohol - functionalized 
 heteroaromatic compound ( n2 ) , 9 .
boc removal 
 was performed using trifluoroacetic acid , triisopropylsilane , and 
 water to yield the free amines 10 and 11 .
the benzamides were formed via a coupling reaction between the free 
 amine and r - substituted benzoic acid in the presence of dmf , hatu , 
 and dipea at room temperature .
this reaction occurred at various efficiencies 
 ( 975% ) across the range of substituted oxadiazolylindazoles 
 and substituted benzoic acids .
the sulfonamide analogues were formed 
 by reaction of the free amine with r - substituted benzene sulfonyl 
 chloride in the presence of sodium carbonate at room temperature with 
 reaction efficiencies of 2439% .
activity against sodium channels 
 was assessed using a radioligand binding assay using [ h]sipatrigine as the ligand and by patch - clamp 
 electrophysiology on cloned human channels .
the in vitro neuroprotection 
 assay was an adaptation of an original study by fowler and li . in this system ,
rat hippocampal slices are subjected 
 to oxygen - glucose deprivation , and the subsequent progression to cell 
 death is monitored by determining their atp levels .
nav channel isoform activity was determined 
 using an automated patch - clamp system against hnav1.1hnav1.8/3 expressing cells .
t - cell immunosuppressive activity 
 was assessed using a simple contact hypersensitivity model that detects 
 t - cell proliferation to oxazolone in lymphnodes . in vivo neuroprotection 

was evaluated in an optic neuritis model using double - transgenic mice expressing both a thy1-promoter - driven 
 cyan fluorescent protein restricted to retinal ganglion cells and 
 a myelin oligodendrocyte glycoprotein t - cell receptor ( tcr ) . following injection of bordetella 
 pertussis toxin ,
the mice develop subneurological 
 experimental autoimmune encephalomyelitis ( eae ) and optic neuritis 
 in the optic nerve , which then leads to retinal ganglion cell loss 
 secondary to axon damage .
initial testing with n1- and n2-functionalized tert - butyl carbamate - protected analogues showed encouraging 
 results in the [ h]sipatrigine binding and neuroprotection 
 assays ( table 1 ) .
benzyl - functionalized 
 molecules 14 and 15 , reported in previous 
 work , are shown for comparison .
a combination 
 of reasonable potency ( 20 m ) and > 30% neuroprotection identifies 
 active compounds .
results are 
 normalized to the effect of ttx and are based on the mean of at least 
 three experiments .
replacement 
 of the benzyl group with substituted and nonsubstituted 
 heteroaromatic groups showed a general decrease in the ability of 
 the compounds to displace the radioactive ligand , with the notable 
 exceptions of the ethylimidazole analogues 16 and 17 ( table 1 ) .
other side chains with 
 weakly basic pyridyl moieties , 18 and 19 , demonstrated moderate binding and neuroprotective activity , whereas 
 nonbasic heterocycles 20 and 21 or bulkier 
 groups 22 and 23 displayed weak binding 
 or low neuroprotection . in the previous series , a free amine 
 group led to a general increase 
 in the neuroprotection by the benzylindazole analogues ( e.g. , compounds 24 and 25 ; table 2 ) . that 
 trend was not observed here .
the modest neuroprotection observed for 
 the imidazole and pyridine analogues was removed with free amines 
 such as 26 and 27 .
the neutral side chains ethylthiophene 30 , methylisoxazole 31 , and substituted benzyls 32 and 33 all showed good binding and modest neuroprotection .
results are 
 normalized to the effect of ttx and are based on the mean of at least 
 three experiments .
these results 
 supported the idea that the activity observed for 
 the indazoles substituted by basic heterocycles at the n1 position was not following a similar sar pattern to the original 
 series and required a lipophilic group at the oxadiazolylmethylamine . 
 when the free amine analogues were functionalized with a benzamide 
 group at the amine moiety , the neuroprotection and the radioligand - displacement 
 ability of some of the analogues was improved , with moderate to good 
 levels of binding observed for n1 and n2 imidazole 
 analogues 6 and 34 ( table 3 ) .
thus , it appeared that a benzamide group could mimic the 
 action of the tert - butyl carbamate .
methylpyridyl 
 analogue 35 showed modest binding , whereas ethylthiophene 36 and methyloxazole 37 were essentially inactive .
results are 
 normalized to the effect of ttx and are based on the mean of at least 
 four experiments
on 
 the basis of these encouraging effects , a new set of n1 and n2 imidazole analogues was designed and synthesized 
 to see if improved binding and neuroprotective activity was achievable . 

the new set of n1 and n2 ethyl-2-imidazoles 
 ( compounds 3864 , table 4 ) showed a range of activity , with most showing 
 good ability to displace the radioligand [ h]sipatrigine , 
 and a range of neuroprotective activity , many with moderate to good 
 neuroprotection . in the n1-substituted series , all but 
 one of the 11 new analogues had a binding ic50 < 13 
 m , with some compounds having values as low as 1 m . 

similarly , in the n2-substituted series , all but one of 
 the 16 new analogues had a binding ic50 < 17 m , 
 with some as low as 0.4 m .
neuroprotection efficacy also showed 
 a general increase , with compound 50 reaching 88% neuroprotection . 
 in the n1 series ,
the most profound effect was the 2,3-dimethoxy 
 substitution of the benzamide ring , 41 , which led to 
 a marked increase in displacement ability ( ic50 13 to 7.1 
 m ) and neuroprotection ( 46 to 71% ) .
interestingly , the analogous 
 trisubstitution at the 2,3,4-positions , 38 , resulted 
 in a decrease in neuroprotection .
these aside , in the n1 series , functionalization the benzamide changed little in terms 
 of activity from the parent , 6 ( ic50 range 
 of 113 m and neuroprotection range of 3752% ) , 
 with the potential to add larger groups , such as a indole 39 or t - butyl 40 , without significant 
 loss of binding or neuroprotection . compared to the n1 series , 
 the n2 benzamide series had a much greater range of activities , 
 both in binding ( 0.459 m ) and neuroprotection ( 289% ) . 

the 2- , 3- , and 4-methoxybenzamides all showed good binding ( ic50 < 8 m ) and neuroprotection ( 5463% ) , with 
 the 2,3-dimethoxy - substituted benzamide once again showing the best 
 activity with good binding ( ic50 = 5 m ) and excellent 
 neuroprotection ( 89% , ec50 = 12.3  5.8 ) .
the benzamide 
 substituted at the 3 position with ocf3 showed comparable 
 activity with binding ( ic50 = 0.8 m ) and neuroprotection 
 ( 80% ) . the only other marked effect was an almost total ablation of 
 neuroprotective effect by fluorination at the 2 and 3 positions , despite 
 showing an ic50 of 4 m in the radioligand - displacement 
 assay .
of all of the sulfonamide compounds in the n1 series , 
 only one improved on the neuroprotection of parent benzamide 6 , although many showed better binding .
the 2- and 3-substituted 
 sulfonamide showed good binding and modest neuroprotection , but substitution 
 at the 4 position seemed to provide compounds with poor neuroprotective 
 ability , if not binding , such as 57 , 59 , 
 and 62 . in the n2 series ,
the sulfonamide 
 analogue of 34 , 63 , showed comparable binding 
 and neuroprotection , but once again , a 4-substituted sulfonamide , 64 , had poorer binding and reduced neuroprotection .
in fact , 
 4-substituted sulfonamide and benzamide analogues across the group 
 had reduced capacity for neuroprotection in the hippocampal slice 
 assay when compared to the 2- or 3-substituted or unsubstituted compounds , 
 despite no significant difference in binding .
the presence of unfavorable 
 interactions at the 4 positions is indicated , and this may also account 
 for the observation that small substituents such as cl have a lesser 
 detrimental effect on activity than larger groups . in
the limited 
 number of halide - functionalized benzamides and sulfonamides tested , 
 none showed improved neuroprotection .
results are 
 normalized to the effect of ttx and are based on the mean of at least 
 three experiments .
tested at 
 3 m ( values in 
 bold are ic50 s in micromolar ) .
the n1 and n2 ethyl-2-imidazole - substituted 
 series was examined 
 for metabolic stability in rat liver microsomes .
table 5 shows a select subset of the compounds screened for rate 
 of turnover . when examined , 14 of 21 compounds tested in this series 
 had turnovers of greater than 90% after 40 min .
boc - protected and 
 free amine n2 analogues 17 and 27 showed better metabolic resistance , with turnovers of 70 and 57 
 min , respectively .
however the n1 analogue , 6 , showed better stability with more 
 than half of the original compound left after 40 min . despite this 
 promising result ,
none of the further analogues managed to maintain 
 or improve on this stability , with any substitution making the compounds 
 significantly more labile to metabolic degradation ( > 90% ) .
five 
 benzamide 
 and sulphonamide analogues that were fluorine- or chlorine - substituted 
 at the 4 , 5 , and 6 positions on the indazole ring were also tested 
 to see if this modification could arrest the rate of metabolism .
the only 
 compound 
 with moderately increased stability was the co-3-ocf3ph - substituted 
 benzamide , 42 .
similarly , substitution of the benzamide 
 or changing to a sulfonamide did not provide much improvement in the 
 metabolic stability of the n2 analogues of benzamide 34 .
interestingly , however , substitution at the 4 position 
 with the amide group , nhcoch3 , in both the sulfonamide 64 and benzamide 34 analogues gave significant 
 improvement in stability , with turnovers of 54 and 45% , respectively , 
 after 40 min .
unfortunately , these 4 position - substituted nhcoch3 compounds were also some of the most inactive compounds in 
 the neuroprotective assay .
compounds 6 and 51 were examined by patch - clamp 
 electrophysiology 
 to determine their effects on sodium channel isoforms ( table 6 and figure 2 ) .
compound 6 showed preferential activity for the inactivated state ( figure 2a ) but little discrimination between subtypes .
the 
 block of nav1.1 , 1.3 , and 1.6 is implicated in the pharmacological 
 basis of neuroprotection ( vide supra ) , and activity was noted against 
 these isoforms consistent with the compounds ability to displace 
 radiolabeled sipatrigine ( table 6 ) .
compound 51 showed good activity in both the radioligand - displacement 
 and neuroprotection assays , and this is reflected in its potent block 
 across the range of isoforms .
it blocks many of the isoforms at an 
 order of magnitude lower concentration than 6 , with its 
 most potent block against the nav1.5 sodium channel , an 
 isoform found primarily in cardiac muscle .
there also appears to be 
 less functional selectivity compared to 6 ( figure 2b ) .
the potency of 51 against nav1.1 , 1.3 , and 1.6 when compared to 6 may account 
 for the observed increase in neuroprotection seen with this compound 
 ( table 6 ) .
detail of activity of 6 ( a ) 
 and 51 ( b ) 
 against the nav1.6 isoform .
blue triangles are block of 
 the inactivated state , red circles are block of 10 hz current , and 
 black squares are block of tonic current .
the ratio of ic50 a / c 
 and b / c illustrates the degree of functional selectivity for the inactivated 
 state .
compound 6 was selected for detailed 
 in vivo evaluation 
 on the basis of its mix of properties : in vitro sodium channel activity , 
 activity against the neuronal nav isoforms , functional 
 selectivity , and stability in rat liver microsomes .
the influence 
 of 6 was assessed in vivo in wild - type and myelin oligodendrocyte 
 glycoprotein - specific t - cell receptor transgenic mice ( figure 4 ; 5 ) .
first , 6 was examined for t - cell immunosuppressive activity , as the 
 eae model of ms is well - known to be t - cell - dependent .
rather than use transgenic mice where all t - cells have 
 the same specificity , a contact - hypersensitivity model was used because 
 it is known to be predictive in identifying in vivo immunosuppressive 
 doses for eae studies .
doses of 6 ranging from 550 
 mg / kg were administered intraperitoneally ( i.p . ) from day 1 
 to day 2 , and the in vivo induced proliferative response was assessed 
 in the draining lymph nodes 3 days ( the peak time of t - cell proliferation ) 
 after the sensitization to the contact - sensitizer oxazolone on day 
 0 .
there was no evidence suggestive of any inhibitory effect of sensitization 
 based on cell numbers within the lymph nodes or based on a per cell 
 proliferative response ( figure 3 ) .
importantly , 
 this study demonstrated that the drug was well - tolerated and there 
 was no apparent toxicity .
we next sought to assess whether 6 could exert a neuroprotective effect and limit the amount of axonal 
 and nerve loss that results as a consequence of t - cell autoimmunity 
 within the cns . following the development of optic neuritis in our 
 model mice , demyelination was augmented following injection of 250 
 g of z12 rat igg2b demyelinating antibody on day 14 .
retinal ganglion cell survival following treatment 
 of animals with 5 mg / kg i.p .
6 was assessed on day 21 
 postinduction in retinal flatmounts ( figures 3a , b and 4 ) .
it 
 was found that 6 increased ganglion cell survival ( p < 0.02 ) and reduced the mean retinal loss from 914 
  117 to 1298  77 cells / mm compared to 1653 
  89 cells / mm in a group of age - matched control animals 
 without optic neuritis ( figure 4 ) .
this study 
 suggests that this class of compounds may have some utility in controlling 
 the accumulation of nerve loss that occurs in diseases such as ms .
mice 
 received epicutaneous application of 25 l of 2.5% oxazolone 
 ( ox ) in acetone olive oil ( aoo ) 4:1 on day 0 on the dorsum of the 
 ear .
injections of either 
 vehicle ( dmso / cremophor / phosphate buffered saline , 1:1:18 ) or 6 from day 1 to day 2 . on day 3
, the draining auricular 
 lymph nodes were removed from three mice and pooled , and a single - cell 
 suspension was made .
lymph node cells were counted , and 5  10 cells per well , in triplicate , were cultured overnight at 
 37 c .
t - cell proliferation was assessed using the celltiter 
 96 aqueous cell proliferation assay , which uses a mts tetrazolium 
 compound that is bioreduced by live cells .
retinal ganglion cells in the retina of mice ( a ) before or ( b ) 
 after the development of optic neuritis .
differences between vehicle and drug - treated controls were assessed 
 using student s t test .
during ms , nav channels are redistributed 
 along demyelinated 
 nerves , which can help to restore function to some extent but leaves 
 them vulnerable to excessive sodium entry .
our strategy was to develop compounds with good activity toward 
 sodium channel inhibition , in particular against the neuronal isoforms 
 nav1.1 , 1.3 , and 1.6 , but , most importantly , which could 
 also display neuroprotective capabilities in an in vitro hippocampal 
 slice assay system .
the selection of a candidate molecule for in vivo 
 studies was based on a balanced assessment of properties , principally 
 sodium channel activity , neuroprotection , and in vitro stability . 

the use of the hippocampal slice model , measuring atp levels and tuned 
 for sodium channel blockade , was key in ensuring an appropriate functional 
 effect . in general , the series was found to exhibit poor stability 
 in rat liver microsomes in vitro , with 6 being a notable 
 exception that allowed us to test this paradigm in vivo .
there are 
 reports on the metabolism of 1,2,4-oxadiazoles , and this may be the site of the metabolic fragility .
studies 
 detailing the pharmacokinetics and localization of 6 to 
 brain and spinal cord lesions in other eae models are published elsewhere . sodium channel blockade with 6 in vivo
was found to 
 be neuroprotective , preventing retinal ganglion cell loss in optic 
 neuritis in this refined eae model . in 
 this case
, the mechanism of neuroprotection is unlikely to be due 
 to peripheral immunosuppression that prevents disease from developing .
there was no evidence of t - cell immunosuppression seen here in a 
 paradigm that can detect immunosuppressive doses of drugs even following 
 a single dose .
furthermore , in our hands , overt immunosuppression 
 does not affect the development of disease in conventional eae using 
 a similar delivery of sodium channel - blocking compounds including 
 phenytoin , sipatrigine , carbamazepine , and oxcarbazepine , which is in contrast to some studies where immunosuppression 
 of sodium channel blockers delivered in the drinking water was noted .
neuroprotection with 6 is most 
 likely to occur at the level of the axon or nerve and perhaps through 
 inhibition of microglial activity , which are probably central to the 
 pathogenesis of progressive ms .
therefore , blockade of sodium channels 
 could result in neuroprotective effects on immune and nonimmune components 
 that may drive progressive nerve loss in ms .
neurodegeneration accumulates slowly over time and therefore allows 
 a larger time window for treatment onset compared to acute conditions 
 such as stroke .
lesions in ms can develop throughout the neuroaxis 
 but are common within the optic nerve , where optic neuritis is often 
 an initial sign of disease .
the visual pathway is the most accessible 
 part of the human nervous system , and optic neuritis in ms is considered 
 to be an ideal tissue to study the effect of neuroprotective treatments 
 in ms and is being targeted in a number of studies .
amiloride and phenytoin are currently in phase ii clinical trials 
 for the treatment of optic neuritis in ms ( nct01802489 , nct01451593 ) .
we chose to utilize an optic neuritis model 
 for in vivo evaluation of neuroprotection in this study . recently
, 
 lamotrigine was examined in progressive ms , but it did 
 not appear to inhibit the development of brain atrophy , which was 
 the primary outcome measure , although significant effects of slowing 
 of walking deterioration were noted .
there 
 is a clear need to evaluate better tolerated and more effective neuroprotective 
 agents .
the chemical series described here may provide a platform 
 for the development of pharmaceutical agents for the treatment of 
 ms and other neurological diseases .
all compounds were at least 95% pure as assayed by 
 lcms ( electrospray 
 positive ) .
the preparations of analogues 6 and 51 are shown below , and the preparation of the remainder of 
 the analogues is provided in the supporting information . to a solution of r - substituted benzoic acid ( 0.10 g , 0.82 mmol ) 
 in dmf ( 5 ml ) were added hatu ( 0.34 g , 0.9 mmol , 1.1 equiv ) and diisopropylethylamine 
 ( 0.23 g , 0.31 ml , 1.8 mmol , 2.2 equiv ) , and the solution allowed to 
 stir for half of an hour at room temperature under nitrogen .
the amine 
 ( 0.9 mmol , 1.1 equiv ) was then added , and the brown solution was allowed 
 to stir overnight .
the dmf was removed in vacuo , and the resulting 
 crude oil was dissolved in ethyl acetate and washed with water , 1 
 n hcl , 1 n nahco3 , and brine .
the organic phase was then 
 dried over magnesium sulfate , and the solvent was removed in vacuo . 

the crude oil was purified using flash chromatography , eluting with 
 10% methanol in dichloromethane .
to a solution 
 of the appropriate amine ( 0.22 
 g , 0.71 mmol , 1 equiv ) in water ( 10 ml ) was added r - substituted benzene 
 sulfonyl chloride ( 0.78 mmol , 1.1 equiv ) .
the solution was allowed 
 to stir , and saturated sodium carbonate(aq ) was added gradually to 
 adjust the acidic ph to 8 .
the white precipitate was filtered off , washed 
 with diethyl ether , and dried in vacuo .
h nmr ( 300 mhz , dmso )  9.27 ( t , j = 
 5.7 hz , 1h , nh ) , 8.13 ( d , j = 8.1 hz , 1h , arh ) , 7.92 
 ( d , j = 6.7 hz , 2h , arh ) , 7.64 ( d , j = 8.5 hz , 1h , arh ) , 7.617.41 ( m , 4h , arh ) , 7.417.27 
 ( m , 2h , arh ) , 7.02 ( s , 1h , arh ) , 6.71 ( s , 1h , arh ) , 4.96 ( t , j = 5.8 hz , 2h , ch2 ) , 4.73 ( d , j = 5.7 hz , 2h , ch2 ) , 4.55 ( t , j = 5.8 
 hz , 2h , ch2 ) .
c nmr ( 75 mhz , cdcl3 )  170.1 , 168.9 , 166.5 , 140.8 , 137.2 , 133.7 , 131.5 , 129.3 , 
 128.4 , 127.7 , 127.4 , 127.3 , 123.7 , 121.5 , 120.3 , 119.6 , 110.5 , 49.7 , 
 45.7 , 35.3 . hrms ( tof ms es ) calcd for c22h20n7o2 ( mh ) , 414.1678 ; found , 
 414.1668 .
h nmr ( 300 mhz , cdcl3 )  8.18 ( s , 1h , nh ) , 8.09 ( d , j = 8.4 hz , 
 1h , arh ) , 7.99 ( d , j = 7.7 hz , 1h , arh ) , 7.95 ( s , 
 1h , arh ) , 7.81 ( d , j = 8.6 hz , 1h , arh ) , 7.547.30 
 ( m , 5h , arh ) , 6.69 ( s , 1h , arh ) , 6.30 ( s , 1h , arh ) , 5.37 ( t , j = 5.5 hz , 2h , ch2 ) , 4.82 ( d , j = 5.1 hz , 2h , ch2 ) , 4.55 ( t , j = 5.5 
 hz , 2h , ch2).c nmr ( 75 mhz , dmso )  
 237.95 , 168.13 , 167.26 , 164.96 , 151.20 , 148.34 , 147.35 , 137.33 , 135.81 , 
 130.68 , 128.25 , 127.08 , 126.41 , 125.70 , 124.15 , 122.28 , 119.96 , 119.79 , 
 119.23 , 118.23 , 53.29 , 45.60 , 35.20 .
hrms ( tof ms es ) 
 calcd for c25h30n10o5 ( mh ) , 498.1501 ; found , 498.1500 .
six to eight week old male and female wild - type 
 and double - transgenic c57bl/6-cg - tg(thy1-cfp)23jrs.tg ( tcra2d2,tcrb2d2)1kuch / j 
 mice were used from stock bred at queen 
 mary university of london .
founder mice were originally obtained from 
 jackson laboratories , bar habor , me , usa ) .
these transgenic mice express 
 both a h-2a - restricted t - cell receptor ( v3 , v11 ) 
 specific for myelin oligodendrocyte glycoprotein residues 3555 
 and a neuronally restricted thy1 ( cd90)-promoter - driven expression 
 of cyan fluorescent protein , which is only present in the retinal 
 ganglion cells within the retina .
mice 
 were maintained on a 12 h light / dark cycle with controlled humidity 
 and temperature and housed as described previously .
the reporting of most aspects of the experimental design , 
 which conform with the arrive guidelines , has been described previously .
all animal studies were approved by the queen mary ethical review 
 panel and the united kingdom government home office inspectorate . 

these studies conformed to the united kingdom animals ( scientific 
 procedures ) act 1986 for the use of animals in research .
mice ( n = 34/group ) 
 received epicutaneous application of 25 l 
 of acetone / olive oil ( 4:1 , aoo ) or 25 l of 2.5% oxazolone ( ox , 
 sigma - aldrich , poole , uk ) on day 0 on the dorsum of the ear . on day 
 3
, the draining auricular lymph nodes were removed , and a single - cell 
 suspension was made .
lymph node cells were counted , and 5  10 cells / well , in triplicate , were cultured overnight at 37 
 c/5% co2 in 200 l of rpmi-1640 medium supplemented 
 with 10% heat - inactivated fetal calf serum , l - glutamine , 
 sodium - pyruvate , and antibiotics as described previously .
t - cell proliferation was assessed using the 
 celltiter 96 aqueous non - radioactive cell proliferation 
 assay according to the manufacturer s instructions ( promega , 
 southhampton , uk ) .
optic 
 neuritis was induced in c57bl/6-cg - tg(thy1-cfp)23jrs.tg(tcra2d2 , tcrb2d2)1kuch / j 
 transgenic mice .
subclinical eae in the absence of paralytic disease 
 was induced following administration of 150 ng of bordetella 
 pertussis toxin ( sigma - aldrich ) in phosphate - buffered 
 saline .
optic neuritis within the optic 
 nerve was augmented following the injection of 250 g of z12 
 rat igg2b mouse myelin oligodendrocyte - specific demyelinating antibody 
 on day 14 .
animals were injected daily 
 from day 021 of the experiment with 5 mg / kg i.p .
animals were 
 killed on day 21 postinduction , and eyes were enucleated and immersed 
 in 4% paraformaldehyde ( sigma - aldrich ) in pbs ( ph 7.4 ) overnight . 

the retinae were dissected , and the cornea , sclera , lens , hyaloid 
 vasculature , and connective tissue were removed in 2 normal - strength 
 pbs .
four radial incisions were cut around the retina , and flatmounts 
 were mounted onto slides and coverslipped with antifade glycerol ( citifluor 
 ltd . ,
the retinal flatmounts were imaged by fluorescent 
 microscopy , retinal ganglion cell density was calculated by counting 
 cyan fluorescent protein - expressing retinal ganglion cells using stereology 
 software with a fractionator probe , and the number cells in the four 
 quadrants around the optic nerve head was counted .
the parametric data was assessed using student s t test , incorporating tests for equality of variance using 
 sigmastat software ( systat software , inc . , san jose , ca , usa ) .
 a series of imidazol-1-ylethylindazole 
 sodium channel ligands were 
 developed and optimized for sodium channel inhibition and in vitro 
 neuroprotective activity . 
 the molecules exhibited displacement of 
 a radiolabeled sodium channel ligand and selectivity for blockade 
 of the inactivated state of cloned neuronal nav channels . 
 
 metabolically stable analogue 6 was able to protect retinal 
 ganglion cells during optic neuritis in a mouse model of multiple 
 sclerosis . 
Introduction
Results
Discussion
Experimental
Section