prior and ongoing work in our groups indicates 
 that the p7c3 class 
 of compounds exert their neuroprotective effects by stabilizing mitrochondria 
 against the depolarization events that are known to trigger apoptosis . nonetheless , our most robust assay involves monitoring 
 neuroprotection of newborn hippocampal neurons in mice because this 
 assay gives a simultaneous readout of efficacy , acute toxicity , and 
 cell permeability . for this reason , all analogues of 1 were evaluated initially by infusing them directly into the left 
 lateral ventricle of environmentally deprived live mice using a subcutaneously implanted osmotic minipump . compounds 
 were delivered intracerebroventricularly ( icv ) over a 7 day period 
 at a concentration of 10 m ( 12 l / day ) . mice were additionally dosed intraperitoneally ( ip ) daily 
 with brdu ( 50 mg / kg ) to mark newly born hippocampal neural precursor 
 cells . at the end of the 1 week dosing , animals were sacrificed and 
 transcardially perfused . brain slices were stained with antibodies 
 to brdu , and the number of surviving neural precursor cells was quantified 
 by counting the number of brdu+ cells in the hippocampal dentate gyrus . 
 to avoid complications arising from the surgical implantation of the 
 pump , we quantified neurogenesis in the hemisphere opposite to compound 
 infusion . finally , to obtain consistent data across many animals , 
 the number of brdu+ cells was normalized to the volume of the dentate 
 gyrus . our initial objective was to replace the aniline ring 
 with a heterocyclic 
 ring . previous studies had shown that the n - phenyl 
 ring of 1 could be replaced with a 2-pyridyl group ( giving 3 ) , but not a 3- or 4-pyridyl group , while maintaining activity 
 in the in vivo hippocampal neuroprotection assay . moreover , we had discovered that fluorination of the central 
 methylene and substitution of the n - aryl ring improved 
 the activity , but neither change alone had substantial impact . for 
 example , 4 ( i.e. , methoxy-1 ) and 5 ( i.e. , fluoro-1 ) showed similar activity as 1 . however , fluorinating the anisidine congener 4 to 
 provide 2 substantially improved the activity in the 
 hippocampal neuroprotection assay . in this context , we targeted 15 , a fluorinated compound containing a methoxy - substituted 
 2-aminopyridine . following the approach we described for the large - scale 
 synthesis of 2 , terminal 
 epoxide 7 was opened with 4-nitrobenzenesulfonyl ( 4-nosyl , 
 4-ns)-protected 2-methoxy-6-aminopyridine ( scheme 1 ) . surprisingly , when the resultant amino alcohol 8 was subjected to fluorination conditions with morphodast , hplc / ms 
 and h nmr analyses indicated the formal loss of methanol . we speculated that the pyridine nitrogen displaced an activated alcohol 
 and the cyclized pyridinium salt ( not shown ) was demethylated to yield 
 aminopyridone 9 . the resulting secondary alcohol 10 was fluorinated 
 to provide azido fluoride 11 , and the azide was reduced 
 with triphenylphosphine to yield fluoroamine 12 . reaction 
 of this amine with iodopyridine 13(12 ) in the presence of cui and diketone ligand 14 provided the desired analogue 15 in high yield on a 
 multigram scale . alternative conditions 
 featuring pd catalysts or lacking the diketone ligand provided less 
 than 20% yield of the intended product . in a similar manner , amino 
 alcohol 16 was arylated to provide 17 , the 
 hydroxy congener of 15 . amino alcohol 16 proved to be much less reactive than fluoroamine 12 , resulting in incomplete conversion . moreover , in this case adding 
 the diketone ligand improved the yield by only around 10% , suggesting 
 that the amino alcohol itself can serve as a ligand for cu . as described above , compounds were administered 
 to mice icv over 
 a 1 week period concurrent with dosing of brdu ip . figure 1 shows that aminopyridine 15 approximately 
 doubled the number of surviving newborn hippocampal neurons over this 
 time period and displayed an activity superior to that of 1 and comparable to that of 2 . several of the synthetic 
 intermediates also displayed neuroprotective activity in this assay . while azide 11 was inactive under the conditions of this 
 experiment , the primary amines 12 and 16 as well as the n - pyridyl amino alcohol 17 showed an increase in the number of brdu+ cells compared with vehicle . however , these intermediates were less potent than 15 and even slightly less active than our initial hit 1 . we originally adopted intracerebroventricular delivery to avoid 
 complications related to brain penetration . nonetheless , icv is clearly 
 not an ideal method of dosing . fortunately , the optimized lead 15 showed a smooth dose  response curve upon intraperitoneal 
 ( ip ) administration . it was active at a dose of 1 mg kg day and reached its ceiling of efficacy at 10 
 mg kg day , results which parallel 
 those found in the mptp model of parkinson s disease ( see below ) . control experiments demonstrated that the increase in brdu+ neurons 
 within the hippocampus was due to neuroprotection rather than increased 
 neural stem cell proliferation ( figure s1 in the supporting information ) . compounds 
 were administered using the indicated dose and method for 7 days , 
 during which time mice were dosed ip daily with brdu ( 50 mg kg day ) to label newborn hippocampal 
 neurons . data are shown as mean  standard error of the mean 
 ( sem ) . previous work had demonstrated 
 that the preponderance of neuroprotective 
 activity resided in a single enantiomer of the p7c3 class of compounds . the enantiomers of 18 , 19 , and 20 that are shown in scheme 2 were the more 
 active of each pair , suggesting a specific protein - binding partner . we also determined that tertiary alcohol 21 was more 
 active than the initial hit 1 , suggesting the presence 
 of a hydrophobic binding site into which the methyl group could project . these observations prompted the question of which , 
 if either , enantiomer of fluorinated analogues such as 2 and 15 would display higher potency . in particular , 
 we sought to determine whether fluorine would act as a polar group 
 and occupy the binding site favored by the hydroxyl group of 18 , 19 , and 20 or act as a hydrophobic 
 group and bind similarly to 21 . unfortunately , previous 
 attempts to fluorinate an optically active precursor to 2 provided material with 020% ee , so developing an enantioselective 
 synthesis remained a high priority . condensation of 3,6-dibromocarbazole ( 6 ) 
 with the 
 3-ns - protected glycidol derivative 22 provided the epoxide 
 ( )-7 in 97% yield with 96% ee . epoxide ring opening with azide was quantitative and provided 
 a substrate for fluorination , ( + ) -10 . comparison with 
 the material formed from a mitsunobu reaction with ( s)-glycidol demonstrated that , as described by sharpless and co - workers , sulfonate 22 reacted by direct 
 displacement of the leaving group rather than by epoxide opening / epoxide 
 closure . consistent with previous results , we found that reaction 
 with morphodast was not stereospecific , as azidofluoride 11 was recovered with only 63% ee . we speculate that the common invertive 
 mechanistic pathway was compromised by an intramolecular displacement 
 with the carbazole nitrogen , yielding an aziridinium ion intermediate 
 ( not shown ) . in contrast , we discovered that the reagent 
 combination perfluorobutanesulfonyl fluoride and tetrabutylammonium 
 difluorotriphenylsilicate ( tbat ) yielded the desired fluoride with 
 clean inversion . the pure enantiomers of 15 were first evaluated in 
 the mouse model of hippocampal neuroprotection after they were dosed 
 icv . as shown in figure 2 , ( )-15 is highly active , whereas its enantiomer shows no neuroprotective 
 activity in this assay . the more active enantiomer shows roughly the 
 same activity as the racemic mixture at identical doses . we interpret 
 this observation to mean that this in vivo assay can not resolve the 
 2-fold difference in concentration between , for example , a single 
 enantiomer at 10 m and a racemic mixture at 10 m ( 5 
 m each enantiomer ) . x - ray crystallographic analysis of a single 
 crystal of ( )-15 revealed it to be the s enantiomer ( figure s2 in the supporting 
 information ) . interestingly , the more active fluoride is enantiomeric 
 to the more active enantiomer in the hydroxyl series ( 1820 ) . this observation implies that the binding 
 partner for the p7c3 class of neuroprotective chemicals contains both 
 a hydrophobic pocket , which can be occupied by the fluoride or methyl 
 group , and a polar pocket , which preferentially accommodates a hydroxyl 
 group . compound was administered icv at the indicated concentration 
 ( 0.5 l / h ) for 7 days , during which time mice were dosed ip 
 daily with brdu ( 50 mg kg day ) to label newborn hippocampal neurons . the graph shows the mean 
  sem for each group ( n = 4 or 5 ) . the mouse model of neurogenesis involves protecting 
 newborn hippocampal 
 neurons from premature apoptotic cell death . we also sought to determine 
 whether 15 could protect mature neurons 
 from cell death . in this context , we had previously demonstrated that 1 and 2 could protect mature dopaminergic neurons 
 within the snc from toxicity associated with mptp . this mouse model of pd entails treating mice for 5 days 
 with a toxic dose of mptp ( 30 mg kg day ) to initiate cell death within the snc . in the brain , mptp is oxidized 
 to 1-methyl-4-phenylpyridinium cation ( mpp ) , which is 
 selectively transported into dopaminergic neurons within the snc . within these cells , mpp acts as 
 a mitochondrial poison , generating a pathology that resembles that 
 of parkinsonian patients . under our assay 
 conditions , treatment with drug this dosing regime ensured that any effect of 
 the p7c3 class of compounds could be attributed to neuroprotective 
 activity rather than to simply blocking the uptake or metabolism of 
 mptp . mice were administered drug twice 
 daily for 21 days , after which time they were sacrificed by transcardial 
 perfusion . every fourth section was stained with antibodies specific 
 to the enzyme tyrosine hydroxylase ( th ) . th catalyzes the oxidation 
 of l - tyrosine to l-3,4-dihydroxyphenylalanine ( l - dopa ) , which is the first step in the biosynthesis of dopamine . accordingly , the number of th+ cells provides a quantification of 
 the neuroprotective efficacy of test compounds following mptp exposure . as shown in figure 3 , mptp effects an approximately 
 50% reduction in the number of th+ neurons under the chronic dosing 
 protocol described above . consistent with our earlier reports , 2 exhibits marked neuroprotective activity , showing a significant 
 neuroprotective effect when administered at 5 mg kg day and protection resulting in around 75% rescue 
 at a dose of 10 mg kg day . encouragingly , 15 demonstrated even 
 higher neuroprotective activity than 2 , with the former 
 showing significant activity at a dose of 1 mg kg day and rescuing over 85% of the th+ neurons 
 at the highest dose tested ( 10 mg kg day ) . moreover , the enantiomeric specificity observed in the mptp model 
 of pd mirrored that observed in the mouse model of neurogenesis . specifically , 
 ( )-(s)-15 was strongly protective , 
 while the ( + ) -r enantiomer was indistinguishable 
 from vehicle treatment under the conditions of the assay . in this 
 side - by - side experiment , 15 appeared to be at least as 
 effective as 2 , a compound that has proven efficacy in 
 several animal models of neurodegenerative disease . mice were administered mptp ( 30 mg kg day ) for 5 days . on the sixth day , treatment 
 with drug was initiated at the indicated dose ( ip , bid , 21 days ) . bars indicate numbers of tyrosine hydroxylase - positive ( th+ ) cells 
 detected by immunohistochemical staining of the substantia nigra ( mean 
  sem ) . asterisks indicate p < 0.01 ( * ) , p < 0.001 ( * * ) , or p < 0.0001 ( * * * ) 
 relative to the vehicle ( veh ) + mptp group . representative immunohistochemical 
 pictures of th staining in the snc are shown for 5 mg kg day treatment groups . the pharmacokinetic and toxicity 
 profiles of 15 were 
 evaluated using both in vivo and in vitro methods . we observed nearly 
 complete metabolic stability in the presence of murine hepatocytes 
 ( t1/2 > 240 min ) and good stability 
 in 
 the presence of liver microsomes ( t1/2 values : mouse , 49 min ; rat , > 240 min ; dog , 45 min ; monkey , 81 
 min ; 
 human , 117 min ) . we then monitored plasma and brain levels of the 
 compound following intravenous , oral , or intraperitoneal dosing . as 
 shown in table 1 , 15 exhibited 
 a dose - proportional increase in brain levels when dosed ip . plasma 
 bioavailability was higher when the compound was dosed ip as opposed 
 to per os ( po ) by oral gavage , but partitioning into the brain was 
 superior when the compound was administered by oral gavage . we noted 
 the same effect with 2 , and thus , comparable brain levels 
 are achieved either ip or by oral gavage . by all routes of administration , 15 was metabolically stable . while full pharmacokinetic profiling 
 was performed with ( )-15 , we additionally observed 
 indistinguishable plasma and brain concentrations for racemic and 
 ( )-15 at a single time point following intraperitoneal 
 injection of 1 , 3 , 10 , or 30 mg / kg doses ( figure s3 in the supporting information ) . these data are consistent 
 with similar adme properties for the racemic and optically active 
 forms of 15 . ( )-15 did not show significant 
 toxicity toward 
 the three human cancer cell lines tested ( ic50 > 10 
 m 
 vs h2122 , h2009 , and hcc44 ) . likewise , we observed < 15% inhibition 
 of the herg channel at 50 m using a patch clamp assay . several 
 cytochrome p450s ( cyp3a4 , -2c9 , -3d6 , -2c8 , -2b6 ) were insensitive 
 to ( )-15 , while cyp1a2 and cyp2c19 showed modest 
 ic50 values of 20 and 1.9 m , respectively . on the 
 basis of the high protein binding of 15 ( > 99% ) , free concentrations of the drug are unlikely 
 to reach those concentrations . finally , ( )-15 was screened through the nih s psychoactive drug screening 
 program , and no compelling binding partners were identified . of 47 
 neuronal receptors and channels tested , only the -opioid receptor 
 ( ic50 = 8.2 m ) and the peripheral benzodiazepine 
 receptor [ also known as translocator protein ( tspo ) ] ( ic50 = 0.35 m ) showed any ligand displacement . the binding affinity 
 of the former is clearly too weak to be causative for the biological 
 activity we observed , provided that the in vitro data are predictive 
 of binding in vivo . while the total brain concentration remains in the low micromolar 
 range for several hours after dosing , the free concentration is much 
 lower . moreover , while we observed enantiomeric specificity in the 
 neurogenesis ( figure 2 ) and mptp assays ( figure 3 ) , both enantiomers of 15 bind tspo 
 with similarly weak affinities ( 0.35 and 0.68 m ) . finally , 2 , which shows similar in vivo activity as ( )-15 , binds tspo more weakly ( ic50 = 2.0 m ) . 
 a wide structural range of chemicals have been found to bind tspo , 
 from the eponymous benzodiazapines to cholesterol to porphyrins , suggesting that it might represent a nonspecific 
 binding partner for small molecules . moreover , a tight - binding inhibitor 
 of this protein showed no toxicity in phase i and ii clinical trials 
 for als and is currently in phase ii trials for spinal muscular atrophy . overall , the clean receptor binding results , 
 which are shown in full in table s1 in the supporting 
 information , are consistent with our observation that long - term 
 treatment with 2 ( up to 40 mg kg day for 21 days ) or 15 ( up to 10 mg kg day for 21 days ) has no negative 
 impact on the weight , behavior , or appearance of the treated animals . we have discovered a novel class of potent neuroprotective agents 
 known as the p7c3 series of aminopropyl carbazoles . we have now synthesized 
 and characterized an optimized member of this series that lacks an 
 aniline moiety . moreover , it exhibits improved polarity and is readily 
 available as a single enantiomer . we have demonstrated that ( )-15 potently blocks 
 neuron cell death under two conditions : apoptosis of newborn hippocampal 
 neurons and mptp - mediated killing of dopaminergic neurons in the substantia 
 nigra . the latter effect is notable for almost complete protection 
 in this model of parkinson s disease . future reports will describe 
 the effect of this neuroprotective chemical in a rat model of pd and 
 mouse models of tbi . likewise , we are actively seeking to validate 
 the molecular target of the p7c3 class of compounds and will report 
 the results of those studies in due course . taken together , the data 
 suggest that ( )-15 is a promising candidate for 
 treating some of the most devastating neurological disorders disorders 
 that are likely to increase in prevalence and currently lack effective 
 therapies . unless otherwise stated , commercially 
 available materials were used without further purification , and reactions 
 were performed under a nitrogen atmosphere using freshly purified 
 solvents . solvents were purified using solvent purification columns 
 purchased from glass contour ( laguna beach , ca ) . all of the reactions 
 were monitored by thin - layer chromatography ( tlc ) using e. merck silica 
 gel 60 f254 precoated plates ( 0.25 mm ) . flash chromatography was performed 
 with the indicated solvents using silica gel ( particle size 0.0320.063 
 m ) purchased from sorbent technologies . h and c nmr spectra were recorded on a varian inova 400 or 500 
 spectrometer . electrospray ionization mass spectrometry ( esi - ms ) was performed on 
 an agilent 2010 lc optical rotations were measured 
 at 20 c on a rudolph research analytical autopol iv polarimeter . all 
 of the synthetic compounds were confirmed to be 95% pure by 
 reversed - phase hplc and h nmr analyses . a solution 
 of 6-methoxypyridin-2-ylamine ( 206.8 
 mg , 1.66 mmol ) and pyridine ( 0.2 ml ) in methylene chloride 
 ( 8.0 ml ) was stirred for 30 min before being cooled in an ice bath . 4-nitrobenzenesulfonyl chloride ( 383.9 mg , 1.73 mmol ) was added slowly 
 to the cold stirring solution , and the ice bath was removed 10 min 
 later . the reaction mixture was stirred overnight and then condensed , 
 and the dark - colored mixture was purified on an automated flash chromatography 
 system in 010% meoh / dichloromethane ( dcm ) to provide the nosylate 
 as a yellow liquid in 55% yield . h nmr ( cdcl3 , 400 mhz ) :  8.34 ( d , j = 8.9 hz , 2h ) , 8.14 
 ( d , j = 8.9 hz , 2h ) , 7.53 ( t , j = 
 8.0 hz , 1h ) , 7.357.29 ( m , 1h ) , 6.81 ( d , j = 7.7 hz , 1h ) , 6.46 ( d , j = 8.2 hz , 1h ) , 3.74 ( s , 
 3h ) . c nmr ( cdcl3 , 100 mhz ) :  53.9 , 
 103.7 , 106.7 , 124.5 , 141.1 , 145.4 , 147.6 , 163.7 . esi - ms ( m / z ) : 309.9 [ m + 1 ] ; c12h11n3o5s requires 309.04 . in a modification of a published method , a stirred solution of 3,6-dibromocarbazole ( 6.27 g , 19.3 
 mmol ) and powdered potassium hydroxide ( 1.30 g , 23.2 mmol ) in dimethylformamide 
 ( dmf ) ( 100 ml ) was cooled in ice before the dropwise addition of ( r)-glycidyl-3-nitrobenzenesulfonate ( 22 ) ( 5.01 g , 19.3 mmol ) . the ice bath was removed , 
 and the reaction mixture was stirred overnight at ambient temperature , 
 diluted with water , and washed several times with water and then brine . the organic layer was dried over na2so4 , filtered , 
 and concentrated to give epoxide ( )-7 in quantitative 
 yield , which was used without additional purification . h nmr ( cdcl3 , 400 mhz ) :  8.13 ( d , j = 2.0 hz , 2h ) , 7.57 ( dd , j = 8.7 , 1.8 hz , 2h ) , 
 7.397.31 ( m , 2h ) , 4.65 ( dd , j = 16.0 , 2.5 
 hz , 1h ) , 4.28 ( dd , j = 16.1 , 5.0 hz , 1h ) , 3.32 ( ddd , j = 5.2 , 2.6 , 1.3 hz , 1h ) , 2.82 ( t , j = 
 4.3 hz , 1h ) , 2.50 ( dd , j = 4.7 , 2.6 hz , 1h ) . c nmr ( cdcl3 , 100 mhz ) :  45.0 , 45.1 , 50.6 , 
 110.7 , 112.8 , 123.4 , 123.8 , 129.5 , 139.8 . hplc ( chiralpak ad - h , 1.0 ml / min 1% 
 iproh / hexanes ) : tr(major ) = 24.334.0 
 min ; tr(minor ) = 48.458.6 min ; 
 ee = 95.9% . [ ]d20 = 5.74 ( c = 0.244 , thf ) . n - buli ( 0.65 ml of a 2.5 m solution 
 in hexanes ) was added dropwise to a solution of n-(6-methoxypyridin-2-yl)-4-nitrobenzenesulfonamide ( 278.7 mg , 0.90 
 mmol ) in dmf ( 3.0 ml ) at 0 c . the solution was stirred for 15 
 min before the addition of 3,6-dibromo-9-(oxiran-2-ylmethyl)-9h - carbazole ( 7 ) ( 265.1 mg , 0.70 mmol ) . the 
 reaction mixture was heated to 80 c for 4 h , after which more 
 epoxide 7 ( 113.0 mg , 0.3 mmol ) was added . the reaction 
 mixture was stirred overnight at 80 c , cooled , diluted with 
 etoac , and washed twice with water , twice with 1 n naoh , twice more 
 with water , and finally brine . the crude mixture was purified on an 
 automated flash chromatography system in 010% meoh / dcm to 
 provide a light - orange solid in 12.5% yield . h nmr ( cdcl3 , 400 mhz ) :  8.17 ( d , j = 1.9 hz , 
 2h ) , 8.04 ( d , j = 9.2 hz , 2h ) , 7.56 ( dd , j = 8.7 , 2.0 hz , 2h ) , 7.44 ( t , j = 7.9 
 hz , 1h ) , 7.38 ( d , j = 8.7 hz , 2h ) , 7.056.77 
 ( m , 2h ) , 6.48 ( d , j = 7.7 hz , 1h ) , 6.40 ( d , j = 8.1 hz , 1h ) , 4.43 ( ddd , j = 15.5 , 13.8 , 
 7.5 hz , 3h ) , 4.224.03 ( m , 1h ) , 3.92 ( dd , j = 15.3 , 8.9 hz , 1h ) , 3.49 ( s , 3h ) . c nmr ( cdcl3 , 100 mhz ) :  163.9 , 155.5 , 140.9 , 140.0 , 129.4 , 126.1 , 
 125.7 , 123.7 , 123.4 , 120.2 , 114.6 , 112.8 , 111.2 , 107.4 , 105.0 , 69.1 , 
 68.1 , 53.7 , 25.8 . esi - ms ( m / z ) : 
 732.6 [ m + hcoo ] ; c27h22br2n4o6s requires 688.0 . morphodast ( 100 l , 0.82 mmol ) was added to a solution of 
 alcohol 8 ( 60.2 mg , 0.087 mmol ) in methylene chloride 
 ( 2.0 ml ) . the reaction mixture was stirred overnight , and the reaction 
 was quenched by the addition by an equivalent volume of saturated 
 aqueous sodium bicarbonate . the combined organic layers were dried over 
 na2so4 , filtered , concentrated , and purified 
 by preparative tlc in 5% meoh / dcm to provide the pyridone as a yellow 
 solid in 21% yield . h nmr ( cdcl3 , 400 mhz ) : 
  8.268.13 ( m , 2h ) , 8.06 ( d , j = 1.8 
 hz , 2h ) , 7.48 ( dd , j = 8.7 , 1.9 hz , 2h ) , 7.39 ( d , j = 8.7 hz , 2h ) , 7.35 ( dd , j = 9.1 , 7.5 
 hz , 1h ) , 7.016.80 ( m , 2h ) , 6.22 ( dd , j = 
 9.1 , 0.8 hz , 1h ) , 5.87 ( dd , j = 7.5 , 0.8 hz , 1h ) , 
 5.31 ( s , 1h ) , 5.28 ( dd , j = 7.4 , 3.2 hz , 1h ) , 4.88 
 ( dd , j = 15.2 , 3.1 hz , 1h ) , 4.70 ( dd , j = 15.1 , 7.5 hz , 1h ) , 4.15 ( dd , j = 10.2 , 8.3 hz , 
 1h ) , 3.87 ( dd , j = 10.2 , 1.3 hz , 1h ) . c nmr ( cdcl3 , 100 mhz ) :  161.3 , 146.3 , 144.9 , 142.4 , 
 139.4 , 129.8 , 125.7 , 123.9 , 123.6 , 117.8 , 113.2 , 111.4 , 110.3 , 95.2 , 
 86.3 , 53.7 , 52.0 . esi - ms ( m / z ) : 
 700.6 [ m + hcoo ] ; c26h18br2n4o5s requires 655.9 .   a 20% aqueous 
 solution of sodium azide ( 75 ml ) was added 
 to a mixture of ( s)-()-7 ( 7.79 
 g , 20.4 mmol ) and tetrahydrofuran ( thf ) ( 65 ml ) , and the mixture was 
 heated to 75 c . a further 20 ml of a 20% aqueous solution of 
 sodium azide was added after 24 h to ensure complete conversion of 
 the epoxide . the biphasic mixture was condensed under reduced pressure , 
 diluted with etoac , and washed twice with water and then brine . the 
 organic layer was dried over na2so4 , filtered , 
 and concentrated to give the azido alcohol in 91% yield , which was 
 used without further purification . h nmr ( cdcl3 , 400 mhz ) :  8.06 ( d , j = 1.9 hz , 2h ) , 7.53 
 ( dd , j = 8.7 , 2.0 hz , 2h ) , 7.28 ( d , j = 8.4 hz , 2h ) , 4.26 ( dd , j = 6.1 , 1.6 hz , 2h ) , 
 4.234.14 ( m , 1h ) , 3.44 ( dd , j = 12.6 , 4.3 
 hz , 1h ) , 3.30 ( dd , j = 12.6 , 5.6 hz , 1h ) , 2.48 ( s , 
 1h ) . c nmr ( cdcl3 , 100 mhz ) :  46.4 , 
 54.1 , 69.5 , 110.6 , 110.7 , 112.7 , 123.3 , 123.3 , 123.6 , 129.4 , 139.5 . 
 esi - ms ( m / z ) : 466.6 [ m + hcoo ] ; c15h12br2n4o requires 421.9 . following a published procedure , diisopropylethylamine 
 ( 7.75 ml , 44.5 mmol ) and toluene ( 140 ml ) were added alternatingly 
 ( ca . 20 portions each ) to a mixture of tetrabutylammonium difluorotriphenylsilicate 
 ( tbat ) ( 9.50 g , 17.6 mmol ) and ( s)-1-azido-3-(3,6-dibromo-9h - carbazol-9-yl)propan-2-ol ( 7.41 g , 17.5 mmol ) at room 
 temperature . perfluoro-1-butanesulfonyl 
 fluoride ( pbsf ) ( 6.91 ml , 38.5 mmol ) was added dropwise , and the reaction 
 mixture was stirred overnight at ambient temperature . the mixture 
 was concentrated and purified by automated chromatography ( sio2 , 50% dcm / hexanes ) to provide the fluoro azide in 70% yield 
 as a white solid . h nmr ( cdcl3 , 400 mhz ) :  
 8.14 ( t , j = 2.3 hz , 2h ) , 7.647.52 ( m , 2h ) , 
 7.32 ( dd , j = 8.7 , 1.8 hz , 2h ) , 5.124.85 
 ( m , 1h ) , 4.54 ( ddt , j = 17.9 , 5.4 , 2.4 hz , 2h ) , 3.58 
 ( ddd , j = 17.7 , 13.5 , 4.5 hz , 1h ) , 3.40 ( ddd , j = 24.1 , 13.5 , 4.7 hz , 1h ) . c nmr ( cdcl3 , 100 mhz ) :  44.2 ( d , jc f = 23.9 hz ) , 51.6 ( d , jc  f = 23.9 
 hz ) , 90.0 ( d , jc f = 177.6 hz ) , 
 110.5 ( d , jc  f = 1.3 hz ) , 113.1 , 
 123.5 ( d , jc esi - ms ( m / z ) : 468.7 
 [ m + hcoo ] ; c15h11br2fn4 requires 423.9 . hplc ( chiralpak ad - h , 1.0 ml / min 
 0.5% iproh / hexanes ) : tr(minor ) = 25.029.1 
 min ; tr(major ) = 29.440.7 min ; 
 ee = 96.2% . triphenylphosphine 
 ( 3.24 , 12.3 mmol ) was added to a solution of azide ( )-(r)-11 ( 5.10 g , 12.0 mmol ) in thf ( 80 ml ) . water was added 
 to the cooled reaction mixture , and the resulting mixture was stirred 
 for 6 h before volatile solvents were removed under reduced pressure . the viscous mixture was diluted with etoac and washed twice with water 
 and then brine . the organic layer was dried over na2so4 , filtered , and concentrated to give a yellow waxy residue . 100 ml ) , and then at 0 c 1 m hcl was added dropwise 
 to precipitate the hydrochloride salt . the slurry was stirred for 
 about 20 min before it was filtered and then washed with cold dcm 
 several times and finally with hexanes . the powdery solid ( 5.29 g , 
 quantitative ) was dried on a fritted funnel and contained less than 
 1% pph3o byproduct . the salt was freebased by adding a 
 saturated solution of sodium bicarbonate ( 90.0 ml ) to a milky mixture 
 of the amine hcl salt ( 5.29 g ) in methylene chloride ( 90.0 ml ) with 
 et3n ( 3.0 ml ) . the organic layer was separated , and 
 the aqueous phase was extracted with methylene chloride . the combined 
 organic layers were dried over na2so4 , filtered , 
 and concentrated to give the free amine as an off - white solid . h nmr ( cdcl3 , 400 mhz ) :  8.15 ( d , j = 2.0 hz , 2h ) , 7.57 ( dd , j = 8.8 , 1.9 
 hz , 2h ) , 7.36 ( d , j = 8.7 hz , 2h ) , 4.85 ( dtt , j = 47.9 , 6.1 , 4.4 hz , 1h ) , 4.674.39 ( m , 2h ) , 3.222.78 
 ( m , 2h ) . c nmr ( cd3od , 100 mhz ) :  141.1 , 
 130.5 , 125.1 , 124.3 , 113.8 , 112.4 , 112.4 , 91.2 ( d , jc f = 172.4 hz ) , 45.5 ( d , jc  f = 20.4 hz ) , 42.1 ( d , jc  f = 20.4 hz ) . esi - ms ( m / z ) : 398.7 [ m + 1 ] ; c15h13br2fn2 requires 397.94 . 1-amino-3-(3,6-dibromo-9h - carbazol-9-yl)propan-2-ol ( 16 ) ( 60.1 mg , 0.15 mmol ) , 
 2-iodo-6-methoxypyridine ( 13 ) ( 35.9 mg , 0.15 mmol ) , copper(i ) 
 iodide ( 2.3 mg , 0.012 mmol ) , and cesium carbonate ( 100.9 mg , 0.31 
 mmol ) were combined in an oven - dried vial under nitrogen . dimethyl 
 sulfoxide ( dmso ) ( 0.3 ml ) and 2,2,6,6-tetramethyl-3,5-heptanedione 
 ( 14 ) ( 3.5 l , 0.017 mmol ) were then added . the 
 reaction mixture was stirred overnight at ambient temperature , diluted 
 with etoac , and washed several times with water and then brine . the well - dried solid was dissolved in 80% dcm / hexanes , 
 passed through a short silica plug , and washed with dcm to remove 
 unreacted amine . h nmr ( cdcl3 , 500 mhz ) :  8.15 
 ( d , j = 2.0 hz , 2h ) , 7.57 ( dd , j = 8.6 , 2.0 hz , 2h ) , 7.36 ( m , 3h ) , 6.09 ( d , j = 
 7.9 hz , 1h ) , 6.02 ( d , j = 7.8 hz , 1h ) , 4.55 ( bs , 
 1h ) , 4.454.22 ( m , 3h ) , 3.76 ( s , 3h ) , 3.50 ( d , j = 14.7 hz , 1h ) , 3.26 ( dt , j = 14.1 , 5.6 hz , 1h ) . c nmr ( cdcl3 , 100 mhz ) :  163.3 , 157.4 , 
 140.2 , 139.2 , 128.8 , 123.2 , 122.9 , 112.0 , 110.5 , 99.9 , 97.4 , 70.2 , 
 53.3 , 46.5 , 46.3 . esi - ms ( m / z ) : 
 503.7 [ m + h ] ; c21h19br2n3o2 requires 503.0 . first , ( s)-12 ( 72.2 mg , 0.18 mmol ) , 2-iodo-6-methoxypyridine ( 45.4 mg , 0.19 mmol ) , copper(i ) iodide ( 2.9 
 mg , 0.015 mmol ) , and cesium carbonate ( 119.4 mg , 0.36 mmol ) were added 
 to an oven - dried vial , which was then purged with nitrogen for 10 
 min . then dmso ( 0.36 ml ) and 14 ( 4 l , 0.019 mmol ) 
 were added . the reaction mixture was heated at 40 c for 3 h. 
 the cooled mixture was diluted with etoac and washed three times with 
 a 9:1 saturated nh4cl / nh4oh solution , three 
 times with water , and then brine . the well - dried 
 solid was dissolved in methylene chloride and passed through a short 
 silica plug , which removed the small amount of unreacted amine . the 
 filtrate was condensed to give a white solid , which was purified on 
 an automated flash chromatography system in 5080% dcm / hexanes 
 to provide ( )-(s)-15 as a white 
 solid in 61% yield with 96% ee , which was stored under nitrogen . recrystallization 
 to enhance the enantiomeric excess was carried out by heating a solution 
 of 15.8 mg of the purified product in 1.0 ml of isopropanol . single 
 crystals suitable for x - ray diffraction were grown by diffusion of 
 hexanes into a concentrated solution of ( )-15 in dichloroethane ( see figure s2 in the supporting 
 information ) . h nmr ( cdcl3 , 400 mhz ) : 
  8.16 ( d , j = 1.9 hz , 2h ) , 7.56 ( d , j = 1.9 hz , 2h ) , 7.35 ( t , j = 7.8 hz , 1h ) , 
 7.30 ( d , j = 8.7 hz , 1h ) , 6.04 ( dd , j = 32.7 , 8.0 hz , 2h ) , 5.295.02 ( dm , 1h ) , 4.654.46 
 ( m , 3h ) , 3.873.74 ( m , 1h ) , 3.70 ( s , 3h ) , 3.663.49 
 ( m , 1h ) . c nmr ( cdcl3 , 100 mhz ) :  163.7 , 
 156.9 , 140.3 , 139.7 , 129.5 , 123.9 , 123.4 , 112.8 , 110.7 ( d , jc f = 1.8 hz ) , 99.6 , 98.2 , 91.4 ( d , jc f = 23.9 hz ) , 43.6 ( d , jc  f = 22.0 hz ) . esi - ms ( m / z ) : 505.8 [ m + 1 ] ; c22h19br2fn2o requires 505.0 . hplc ( chiralpak ia , 1.0 ml / min 
 100% meoh ) : tr(major ) = 8.3710.02 
 min ; tr(minor ) = 10.2611.26 min . all of the animal studies were performed 
 in accordance with the ethical guidelines established by ut southwestern 
 and the university of iowa . compounds were 
 infused icv into the left lateral ventricle of four adult ( 12 week 
 old ) wild - type c57bl / j6 mice by means of surgically implanted alzet 
 osmotic minipumps that delivered solution into the animals at a constant 
 rate of 0.5 l / h for 7 days . bromodeoxyuridine ( brdu ) was injected ip at 50 mg 
 kg day for 6 days during pump 
 infusion . twenty - four hours after the final brdu administration , mice 
 were sacrificed by transcardial perfusion with 4% paraformaldehyde 
 at ph 7.4 , and their brains were processed for immunohistochemical 
 detection of incorporated brdu in the subgranular zone ( sgz ) . dissected 
 brains were immersed in 4% paraformaldehyde overnight at 4 c 
 and then cryoprotected in sucrose before being sectioned into 40 m 
 thick free - floating sections . unmasking of brdu antigen was achieved 
 by incubation of tissue sections for 2 h in 50% formamide/2 
 saline sodium citrate ( ssc ) at 65 c followed by a 5 min wash 
 in 2 ssc and subsequent incubation for 30 min in 2 m hcl at 
 37 c . sections were processed for immunohistochemical staining 
 with mouse monoclonal anti - brdu ( 1:100 ) . the number of brdu+ cells 
 in the entire dentate gyrus sgz in the contralateral hemisphere ( opposite 
 side of the surgically implanted pump ) was quantified by counting 
 brdu+ cells within the sgz and dentate gyrus in every fifth section 
 throughout the entire hippocampus and then normalizing for dentate 
 gyrus volume . as previously reported , adult male c57bl/6 mice were individually housed 
 for 1 
 week and then injected ip daily for 5 days with 30 mg kg day free - base mptp ( sigma ) . on day 6 , 24 h after 
 the mice received the fifth and final dose of mptp , daily treatment 
 with compound was initiated . mice received twice - daily doses of each 
 compound ( or vehicle ) for the following 21 days , after which mice 
 were sacrificed by transcardial perfusion with 4% paraformaldehyde . the brains were dissected , fixed overnight in 4% paraformaldehyde , 
 and cryoprotected in sucrose for freezing by standard procedures . frozen brains were sectioned through the striatum and snc at 30 m 
 intervals , and every fourth section ( spaced 120 m apart ) was 
 stained with antibodies directed against tyrosine hydroxylase ( th ) 
 ( abcam , rabbit anti - th , 1:2500 ) . diaminobenzidine was used as a chromagen , 
 and tissue was counter - stained with hematoxylin to aid in visualization 
 of the neuroanatomy . images were analyzed with a nikon eclipse 90i 
 motorized research microscope with plan apo lenses coupled with metamorph 
 image acquisition software ( nikon ) . th+ neurons were counted with 
 imagej software ( nih ) in every section by two blinded investigators , 
 and the results were averaged and multiplied by the sectioning interval 
 to determine the total number of th+ neurons per snc . adult mice were dosed iv , ip or via oral gavage 
 ( po ) in a total volume of 0.2 ml . whole 
 blood was collected with a syringe and needle coated with anticoagulant 
 citrate dextrose ( acd ) solution ( sodium citrate ) . the blood was subsequently 
 centrifuged at 9300 g for 10 min to isolate plasma . brains were 
 isolated from mice immediately after sacrifice , rinsed extensively 
 with phosphate - buffered saline ( pbs ) , blotted dry , weighed , and snap - frozen 
 in liquid nitrogen . lysates were prepared by homogenizing the brain 
 tissue in a 3-fold volume of pbs ( weight of brain in g  3 = 
 volume in ml of added pbs ) . the total lysate volume was estimated 
 as the volume of added pbs plus the volume of brain . either plasma 
 or brain ( 100 l ) was processed by addition of 200 l 
 of acetonitrile containing 0.15% formic acid to precipitate plasma 
 or tissue protein and release bound drug . this mixture was vortexed 
 for 15 s and then centrifuged at 13120 g for 5 min , 
 and the supernatant was analyzed directly by hplc / ms / ms . standard 
 curves were prepared by addition of compound to blank plasma or blank 
 brain lysate . a value 3-fold above the signal obtained from blank 
 plasma or brain lysate was designated as the limit of detection ( lod ) . the limit of quantitation ( loq ) was defined as the lowest concentration 
 at which back - calculation yielded a concentration within 20% of the 
 theoretical value . pharmacokinetic parameters were calculated using 
 the noncompartmental analysis tool of winnonlin ( pharsight ) . bioavailability 
 was calculated as ( aucoral / auciv )  ( doseiv / doseoral )  100% , where auc denotes the 
 area under the concentration the brain : blood ratio 
 was calculated using auc values . protein binding of compound 15 was determined using 
 a recently described ultracentrifugation 
 method ; 100% compound recovery was achieved 
 during the assay , indicating no loss due to absorption to plastic .
( )-p7c3-s243 
 is a neuroprotective aminopropyl carbazole 
 with improved druglike properties compared with previously reported 
 compounds in the p7c3 class . 
 it protects developing neurons in a mouse 
 model of hippocampal neurogenesis and protects mature neurons within 
 the substantia nigra in a mouse model of parkinson s disease . 
 a short 
 , enantioselective synthesis provides the neuroprotective agent 
 in optically pure form . 
 it is nontoxic , orally bioavailable , metabolically 
 stable , and able to cross the blood  brain barrier . as such 
 , 
 it represents a valuable lead compound for the development of drugs 
 to treat neurodegenerative diseases and traumatic brain injury .