conventional cancer 
 chemotherapy relies heavily on drugs that trigger 
 programmed cell death via activation of apoptosis pathways . however , many cancers harbor mutations in tumor suppressor genes 
 that function in apoptosis signaling . combined with increased 
 capacity for drug efflux and dna repair , these mutations attenuate 
 apoptosis and contribute to the chemoresistance frequently encountered 
 in recurrent tumors . glioblastoma multiforme 
 ( gbm ) is the most common primary brain tumor in adults , and it is 
 a notorious example of cancer that is refractory to most forms of 
 chemotherapy . treatment for gbm typically includes surgical excision , 
 followed by radiotherapy and administration of the dna alkylating 
 agent temozolomide . this standard - of - care 
 therapy marginally increases patient survival time , but no curative 
 treatment exists . identification of molecules that 
 can kill cancer cells via nonapoptotic cell death mechanisms may provide 
 inroads to circumvent the insensitivity of gbm and other cancers to 
 apoptosis . a number of nonapoptotic cell death pathways have been 
 defined , and associated small molecules capable of 
 inducing these pathways have been reported . our group has focused on the development of compounds that can induce 
 a novel form of cell death termed methuosis . first observed as a caspase - independent form of cell death triggered 
 by overexpression of activated ras in gbm cells , methuosis is characterized by the accumulation of large cytoplasmic 
 vacuoles derived from macropinosomes and nonclathrin - coated endosomes . the vacuolated cells eventually undergo metabolic failure and lose 
 membrane integrity . in pursuit of small molecules that might be used 
 to induce methuosis in a therapeutic context , we initially identified 
 indolyl - pyridinyl - propenones ( also referred to as indole - based chalcones ) 
 that can trigger this form of cell death in gbm cells , including those 
 resistant to temozolomide . preliminary 
 structure  activity relationship ( sar ) studies defined key requirements 
 for methuosis - inducing activity , resulting in a lead compound , 3-(5-methoxy-2-methyl-1h - indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one , abbreviated 
 momipp ( 1a , figure 1 ) , with activity 
 at low micromolar concentrations in cell - based assays . the following features contributed to optimal 
 activity of the pharmacophore : ( i ) an indole - propenone - pyridine scaffold 
 where the pyridine s attachment is in the para ( 4 ) 
 position ; ( ii ) a methoxy group at the 5-indolyl position ; and , ( iii ) 
 small alkyl substitutions at the 2-indolyl position , i.e. , r = me 
 ( 1a ) and et ( 1b ) ( figure 1 ) . more recent studies of longer - chain 
 aliphatic substitutions on the 2-indolyl position identified compounds 
 that retained the ability to induce cellular vacuolization but lost 
 the ability to kill gbm cells , as exemplified by compound 1c(17 ) ( figure 1 ) . these surprising findings suggest that induction of methuotic cell 
 death by specific indolyl - pyridinyl - propenones may involve pleiotropic 
 effects on cellular pathways beyond those responsible for perturbation 
 of macropinosome trafficking and vacuolization . herein we evaluated 
 a new series of compounds with additional modifications at the 2- 
 and 5-indolyl positions . the results show that these compounds can 
 either maintain good methuosis - inducing activity or uncouple vacuolization 
 from cell death . an unexpected finding was that certain substitutions 
 at the 2-indolyl position conferred a substantially altered biological 
 profile , with disruption of microtubule polymerization becoming a 
 prominent feature . these compounds appear to represent a new class of potent 
 microtubule - active anticancer agents . the propyl 
 derivative , 1c , induces cytoplasmic vacuolization , but 
 is not cytotoxic . the 2- and 5-substituted indolyl - pyridinyl - propenones 
 ( table 1 , 2a2q ) were prepared by one of the following methodologies : ( 1 ) regioselective 
 acylation from 4-substituted n - boc - protected 2-methylanilines 
 ( 6a6d , 18 ) and weinreb 
 amides ( 7 , 19 ) followed by acid - catalyzed cyclization and deprotections ( schemes 1 and 4 ) to afford 1h - indole 
 rings ( 8a  d , 20 ) ; or ( 2 ) synthetic manipulation at specific positions 
 on the formed indole ring to obtain 5-substituted amides ( 11 , 12 , 16 in schemes 2 and 3 ) , alkyl 2-indolylcarboxylates ( 23a  d in scheme 5 ) or functionalized hydroxyl - based 2-indole intermediates ( 25 , 30 , 31 in schemes 6 and 7 ) . synthesized indoles were then 
 formylated under vilsmeier haack conditions and subsequently 
 condensed with 4-acetylpyridine producing final targets ( 2a  f , 2h , 2j n , 2p , 2q ) in a manner similar to 
 our previous reports . targets 2 g , 2i , and 2o were prepared from their corresponding 
 indolyl - pyridinyl - propenone scaffolds . reagents and conditions : ( a ) 
 diethylsulfate in the case of 4a , or alkyl bromide , k2co3 , 2-butanone , reflux ; ( b ) h2 , pd / c , 
 etoac ; ( c ) di - tert - butyl dicarbonate , thf , reflux ; 
 ( d ) 1 . pocl3 , dmf ; 2 . 1 n naoh ; ( f ) 4-acetylpyridine , piperidine , meoh , 
 reflux . ( * ) > 10 : growth inhibition relative 
 to control did not reach 50% at the highest concentration tested ( 10 
 m ) . ( * * ) ni : no significant growth inhibition was detected 
 relative to vehicle control . schemes 13 depict 
 the specific preparations of several 5-substituted analogues 
 designed to probe the effects of increasing the hydrocarbon chain 
 length in the ether linkage or replacing methoxy with nitrogen - containing 
 functionalities . the 5-alkyloxyindole analogues ( scheme 1 ) were synthesized from commercially available 3-methyl-4-nitrophenol 
 ( 3 ) by reaction with various alkyl - bromides , as in the 
 case of 4b  d , or diethylsulfate for 4a . hydrogenation reduced the nitro - group to amines 5a5d , which were then protected with 
 boc ( 6a6d ) . regioselective acylation 
 at the 2-methyl position was achieved using sec - butyllithium 
 and weinreb amide 7 . the resulting ketone intermediate 
 was cyclized and deprotected in a one - pot fashion utilizing excess 
 tfa to yield the 5-alkyloxy-2-methylindoles 8a8d . reaction of these indoles with pocl3 and dmf , 
 followed by treatment with 1 n naoh , provided aldehydes 9a9d which conveniently precipitated from the 
 reaction media . the aldehydes were condensed with 4-acetylpyridine 
 to provide the 5-substituted target compounds 2a2d . analogues of 1a containing either amide 
 or amine substitution 
 at the 5-indolyl position were prepared as shown in schemes 2 and 3 . 5-amino-2-methylindole 
 ( 10 , scheme 2 ) was prepared by 
 previously described methods and used 
 to synthesize amide intermediates 11 and 12 by reaction with acetic anydride and di - tert - butyl 
 dicarbonate , respectively . interestingly , compound 11 was isolated as the diacetylated amide under conditions of reflux 
 and excess reagent . the literature provides precedent for diacylation 
 to occur when aniline is subjected to similar conditions . conveniently , this readily isolable material 
 could be deployed directly in the next step because a single acetyl - group 
 was hydrolyzed during the naoh - water workup leading to 13 . aldehydes 13 and 14 were condensed with 
 4-acetylpyridine to provide target compounds 2e and 2f . tfa deprotection of 2f in meoh at rt yielded 
 the free amine probe 2 g . the des - methyl 
 derivatives 2h and 2i ( scheme 3 ) were prepared from commercially available 15 in a fashion analogous to that for 2f and 2 g but contain a methylene substitution between the amine and the indole 
 ring . reagents and conditions : ( a ) 
 acetic anhydride , reflux ; ( b ) di - tert - butyldicarbonate , 
 ch3cn , reflux ; ( c ) 1 . pocl3 , dmf ; 2 . 1 n naoh ; 
 ( d ) 4-acetylpyridine , meoh , reflux ; ( e ) 2f , tfa / meoh . reagents 
 and conditions : ( a ) 
 di - tert - butyl dicarbonate , ch3cn , rt ; 
 ( b ) 1 . pocl3 , dmf ; 2 . 1 n naoh ; ( c ) 4-acetylpyridine , piperidine , 
 meoh , reflux ; ( d ) tfa , meoh , reflux . expanding 
 previous sar studies of 2-indolyl alkyl substitutions , additional functionalized probes were prepared 
 as depicted in schemes 47 . trifluoromethyl derivative 2j ( scheme 4 ) , an isostere of 1a , was synthesized by acylating the 2-methyl substituent 
 on the n - boc - protected aniline with trifluoromethyl 
 amide 19 . excellent regioselectivity was 
 achieved by the dilithio species derived from 18 and sec - butyllithium in thf at 40 c . cyclization and deprotection occurred in a one - pot 
 fashion with excess tfa affording 5-methoxy-2-trifluoromethylindole 
 ( 20 ) . synthesis of aldehyde 21 and condensation 
 to the final trifluoromethyl target 2j were accomplished 
 by our standard methods . alkyl carboxylates ( 23a  d ) were synthesized from commercially available acid 22 by fisher esterification conditions using their corresponding 
 alcohols ( scheme 5 ) . formylation reactions to yield aldehydes 24a24d , followed by claisen - schmidt condensation to provide 2k2n , were conducted as described previously . interestingly , the isopropyl ester 2n appeared to be 
 the most sensitive to hydrolysis during workup and subsequent manipulation 
 or storage , likely owing to its better leaving group among this series . 
 nevertheless , after saponification in aqueous base and meoh , free 
 acid 2o was also readily obtained from 2k which was available in greater supply . thf , sec - butyllithium , 40 c to 50 
 c ; 2 . weinreb amide 19 , 50 c to 10 
 c ; 3 . pocl3 , dmf ; 2 . 1 n naoh ; ( c ) 
 4-acetylpyridine , piperidine , meoh . reagents and conditions : ( a ) 
 hcl ( g ) , r - oh , reflux ; ( b ) 1 . pocl3 , dmf ; 2 . 1 n naoh ; 
 ( c ) 4-acetylpyridine , piperidine , r - oh , reflux ; ( d ) 2k , meoh , 1 n naoh . derivatives substituted 
 with hydroxyl functionality were synthesized 
 based upon the results obtained in our previous and current sar studies 
 for 2-substituted indole . subtle chemical modifications at this position 
 can alter activity and appear to be sensitive to both steric and electronic 
 properties . previously 
 prepared intermediate ethyl 5-methoxyindole-2-carboxylate ( 23b ) was reduced by lah to 25 , which was then protected 
 by acetylating the primary hydroxyl group ( 26 ) . conveniently , the acetyl group was then hydrolyzed 
 during the alkaline conditions used to quench the acidic reagents 
 generated during the vilsmeier s step that produce aldehyde 27 . this final target compound proved to be unstable when 
 stored at rt in dmso for 48 h ( see associated note in the experimental section ) , presumably due to the presence 
 of a benzylic alcohol conjugated to an ,-unsaturated 
 ketone . to circumvent the inherent instability of 2p , 
 we also synthesized an analogue containing a hydroxypropyl functionality 
 using the methodology in scheme 7 . alcohol 25 was oxidized to 5-methoxyindole-2-carboxaldehyde 
 ( 28 ) by mno2 in etoac heated to reflux . the 
 aldehyde was subjected to wittig conditions using phosphonium ylide 
 ( ph3pchco2c2h5 ) to produce 
 the ,-unsaturated ester 29 , which was reduced 
 to 2-hydroxypropylindole 30 using excess lah . the primary 
 hydroxyl group was protected as the o - acetate ( 31 ) under 
 conditions of excess acetic anhydride , tea , and ch3cn . reagents and conditions : ( a ) 
 lialh4 , thf , rt ; ( b ) acetic anhydride , tea , ch3cn , rt ; ( c ) 1 . pocl3 , dmf ; 2 . 1 n naoh ; ( d ) 4-acetylpyridine , 
 piperidine , meoh , reflux . reagents and conditions : ( a ) 
 mno2 , etoac , reflux ; ( b ) ph3pchco2c2h5 , thf , rt ; ( c ) 1 . h2 , pd / c , etoac / meoh ; ( d ) 30 , acetic anhydride , tea , ch3cn , rt ; ( e ) 1 . pocl3 , dmf ; 2 . 1 n naoh ; ( f ) 4-acetylpyridine , piperidine , meoh , reflux . cells experiencing methuosis initially undergo extreme cytoplasmic 
 vacuolization which can be readily assessed by phase contrast microscopy . 
 as viability is compromised ( usually between 24 and 48 h ) , previous 
 studies have established that the sulforhodamine b ( srb ) colorimetric 
 assay , which measures protein associated with adherent cells , is useful 
 for evaluating the loss of viable cells and for ranking the relative 
 potency of methuosis - inducing compounds . although methuosis can occur in a broad spectrum of human tumor 
 cell lines , our previous 
 sar studies were carried out with u251 glioblastoma cells . growth 
 inhibitory ( gi ) activities for all target compounds are recorded in 
 table 1 as the dose able to achieve 50% inhibition 
 ( gi50 ) compared to growth within control cultures treated 
 with only vehicle . when 1a s methoxy - group 
 is replaced by ethoxy ( 2a ) , isopropoxy ( 2c ) , or butoxy ( 2d ) gi activity was markedly reduced . in contrast , the gi50 value for the 5-propoxy compound 
 ( 2b ) remained similar to that of 1a . as 
 shown in figure 2 , the morphology of cells 
 treated with 2.5 m 1a was consistent with methuosis . robust cytoplasmic vacuolization was observed within 4 h. the vacuoles 
 persisted for the duration of the 48 h time - course with no increase 
 in cell density . by 48 h cytotoxicity was more evident at 10 m , with 
 very few viable cells remaining on the dish . the effects of 2b at 2.5 m were essentially the same as those observed 
 with 1a . in accord with its complete lack of gi activity , 
 the morphological effect of the butoxy derivative 2d contrasted 
 sharply with that of the propoxy 2b in that the former 
 produced much fewer and smaller vacuoles and had little effect on 
 cell density . interestingly , the ethoxy ( 2a ) and isopropoxy 
 ( 2c ) derivatives exhibited intermediate activity , with 
 some induction of cellular vacuolization , but little or no inhibition 
 of cell growth . this apparent dissociation between vacuolization and 
 inhibition of cell growth / viability was similar to what we observed 
 previously with some of the 2-indolyl - substituted pyridinyl - propenones 
 upon lengthening the alkyl chain . evaluation 
 of the impact of increasing the indolyl 5-alkyloxy chain 
 length on the morphological effects of 2-methylindolyl - pyridinyl - propenones 
 in u251 glioblastoma cells . phase contrast images of live cells were 
 obtained as described in the experimental section . the control cells shown in the top panel received an equivalent 
 volume of the dmso vehicle . the 5-amino derivative 2 g had no gi activity 
 in the 
 concentration range tested ( table 1 ) . it also 
 failed to induce vacuolization at 2.5 m , although it did cause 
 vacuolization after 48 h at 10 m ( figure 3 ) . likewise , neither the acetamide ( 2e ) , nor n - boc ( 2f ) analogues showed substantial growth 
 inhibition below 10 m ( table 1 ) , although 
 both compounds induced vacuolization when applied at 2.5 m 
 and 10 m ( figure 3 ) . thus , the activities 
 of 2e , 2f , and 2 g were similar 
 to 2a and 2c , insofar as all of these compounds 
 triggered the accumulation of vacuoles without causing growth arrest 
 or cytotoxicity . the 2-position des - methyl versions 
 of the preceding n - boc and free amine compounds , 
 with a methylene spacer between the nitrogen and the indole ring at 
 the 5-position ( 2h and 2i ) , gave mixed results . primary amine 2i was completely inactive in both growth 
 inhibition and vacuolization assays while the behavior of the n - boc derivative 2h was somewhat anomalous . it did not induce vacuolization or cell death ( figure 3 ) but caused a modest reduction in the growth rate of the 
 cells at concentrations above 2.5 m ( table 1 ) . evaluation of the impact of amine , acetamide , and n - boc substitutions 
 at the 5-position of the indole ring on the morphological effects 
 of 2-methylindolyl - pyridinyl - propenones . phase contrast images of 
 live cells were obtained as described in the experimental 
 section . compounds were added at the concentrations listed 
 at the top of each panel . to further explore the influence of the 2-position on the biological 
 activity of the pharmacophore , we substituted the methyl group of 
 the prototype 1a with a lipophilic cf3 group . surprisingly , the gi activity of this probe ( 2j ) was 
 increased by nearly an order of magnitude compared with 1a ( table 1 ) . as shown in figure 4 , the majority of the cells treated with 2j at 
 2.5 m rounded up and detached by 2448 h , and most of 
 the floating cells were determined to be nonviable by trypan blue 
 dye exclusion . similar morphological effects were observed at concentrations 
 as low as 0.6 m . in cultures examined at 4 h , before the cells 
 began to detach from the surface , we observed the formation of small 
 cytoplasmic vacuoles and extensive blebbing of the plasma membrane 
 ( figure 4 ) . this phenotype is quite distinct 
 from the extensive accumulation of large macropinocytotic vacuoles 
 that we have come to associate with the onset of methuosis ( e.g. , 
 figure 2 , 1a and 2b ) . it suggests that the increased cytotoxicity of the 2-trifluoromethyl 
 derivative may be related to a different biological activity . evaluation 
 of the impact of different 2-indolyl substitutions on 
 the morphological effects of 5-methoyxyindolyl - pyridinyl - propenones . phase contrast images of live cells were obtained as described in 
 the experimental section . compounds were added 
 at the concentrations listed at the top of each panel . in light of the preceding findings , we expanded 
 our compound library 
 to include other lipophilic - linked arrangements such as derivatives 
 containing alkyl 2-indolecarboxylates . as noted in table 1 , the methyl ( 2k ) , ethyl ( 2l ) , and n - propyl ( 2 m ) esters stood out 
 as being much more potent inhibitors of cell growth and viability 
 than any of the previously tested indolyl - pyridinyl - propenones , with 
 the gi50 of 2 m approaching 10 nm ( table 1 ) . morphologically , the cells subjected to these 
 probes resembled those exposed to the trifluoromethyl derivative , 2j , with early membrane blebbing and cell rounding , followed 
 by extensive loss of adherent cells by 24 h ( figure 4 ) . in the cases of the most potent ethyl and propyl esters 
 ( 2l and 2 m ) , cytotoxic effects were observed 
 as low as 0.1 m , where the less potent methyl ester ( 2k ) and cf3 ( 2j ) analogues were no 
 longer effective at this concentration . interestingly , the gi activity 
 and morphological effects of the isopropyl ester ( 2n ) 
 and the carboxylic acid ( 2o ) were either markedly attenuated 
 or completely eliminated ( table 1 , figure 4 ) . the striking influence of the nature of the aliphatic 
 chain on the biological activity of this series of alkyl carboxylates 
 underscores the previously reported sensitivity of the indolyl - pyridinyl - propenones 
 to synthetic manipulations at the 2-position of the indole ring , although this time within the context of lipophilic 
 substituents that also appear to prompt a different mechanism . a third type of 2-indolyl substitution is displayed by the hydroxymethyl 
 ( 2p ) and hydroxypropyl ( 2q ) probes . remarkably , 
 reduction of the ester reverted the biological activity of these compounds 
 to the methuosis phenotype , with gi values in the low micromolar range 
 typical of the other methuosis - inducing compounds ( e.g. , 1a and 2b , table 1 ) . likewise , 
 cell morphology was characterized by extensive accumulation of large 
 cytoplasmic vacuoles , with loss of viable adherent cells at concentrations 
  2.5 m . the striking increase in cytotoxicity 
 and the distinct morphological effects of the alkyl esters 2k2 m suggested that these compounds might be affecting 
 cell growth and viability via a mechanism distinct from the methuosis - inducing 
 compounds like 1a . cell contraction , rounding , and detachment 
 from the substratum are typically observed in cultured cells treated 
 with mitotic inhibitors such as colchicine and vinblastine . therefore , 
 we utilized flow cytometry to generate dna histograms from cells treated 
 for 24 h with the two most potent agents , 2l and 2 m . at a concentration of 3 m , both compounds caused 
 a substantial accumulation of cells in the g2/m phase of the cell 
 cycle ( figure 5a ) . the apparent mitotic arrest 
 was accompanied by a significant increase in the percentage of cells 
 in the sub - g1/g0 compartment ( figure 5b ) , indicative 
 of cell death by mitotic catastrophe . similar cell cycle effects of 2l and 2 m were observed at 0.1 m ( figure 5c ) , which represents the lower end of the concentration 
 range at which substantial morphological perturbations were observed 
 in figure 4 . interestingly , the percentage 
 of cells in the g2/m phase actually was slightly higher in cultures 
 treated with 2 m at 0.1 m compared with 3 m 
 ( figure 5b , c ) . this could be related to a higher 
 percentage of dead cells derived from the g2/m - arrested population 
 in the cultures treated with the higher concentration of 2 m . effects 
 of selected indolyl - pyridinyl - propenones on cell cycle 
 distribution in u251 cells . ( a ) dna histograms of cells treated with 
 the indicated compounds at 3 m for 24 h were generated by flow 
 cytometry as described in the experimental section . ( b ) cells were treated with each compound for 24 h and the percentage 
 of cells registering as having less than the g1/g0 dna content ( an 
 indication of non - viable cells ) is depicted in the left panel . in 
 the same study the percentage of viable cells in each phase of the 
 cell cycle was determined after gating out the sub - g1/g0 counts ( right 
 panel ) . ( c ) the assessment of cell death and cell cycle distribution 
 was repeated in u251 cells treated with compounds at 0.1 m 
 instead of 3 m . values in b and c are means (  s.d . ) derived 
 from three separate cultures . the g2/m - phase and sub - g0/g1 cell populations 
 in the cultures treated with 2l and 2 m were 
 significantly increased compared to the dmso control at all concentrations 
 ( p  0.05 ) . in contrast to the results with 2l and 2 m , the cell cycle distribution of cells treated with methuosis - inducing 
 compounds such as 1a and 2b was similar 
 to the dmso - treated control ( figure 5 ) . the 
 latter finding is consistent with our previous studies of cells undergoing 
 methuosis in response to overexpression of h - ras ( g12v ) , wherein we observed that the cell cycle distribution 
 of the vacuolated cells did not change significantly prior to the 
 loss of viability . it should also be noted that the presence of normal 
 g1/g0 and s - phase populations in the cultures treated with 1a or 2b is not unexpected , since methuotic cell death 
 triggered by 1a typically takes longer than 24 h at drug 
 concentrations below 10 m . in figure 6 , several additional compounds were tested for cell cycle effects 
 at a single concentration ( 3 m ) . arrest of u251 cells in the 
 g2/m phase , with a corresponding accumulation of sub - g1/g0 cells , 
 was clearly evident in cultures treated with the 2-trifluoromethyl 
 ( 2j ) and methyl ester ( 2k ) derivatives ( figure 6 ) . the effects were very similar to those observed 
 with colcemid , a known microtubule disruptor ( figure 6 ) . in contrast , free acid 2o had no detectable 
 effect on cell cycle distribution , reflecting its lack of activity 
 in the srb and morphology assays . effects of additional indolyl - pyridinyl - propenones 
 on cell cycle 
 distribution in u251 cells . dna histograms of cells treated with the 
 indicated compounds at 3 m for 24 h were generated by flow 
 cytometry as described in the experimental section . the results were analyzed to determine ( a ) the percentage of nonviable 
 cells with sub - g1/g0 dna content and ( b ) the percentage of viable 
 cells in each phase of the cell cycle . the g2/m - phase and sub - g0/g1 
 cell populations in the cultures treated with 2j , 2k , and colcemid were significantly increased compared to 
 the dmso control ( p  0.05 ) . the apparent 
 mitotic arrest of cells treated with 2j2 m prompted us to examine the effects of this class of compounds 
 on microtubule polymerization in intact cells . we began by assessing 
 the integrity of the microtubule network by immunofluorescence microscopy , 
 using an antibody against -tubulin . as shown in figure 7 , the dmso - treated control cells displayed a well - developed 
 array of microtubules radiating from the juxtanuclear microtubule 
 organizing center . m 2 m had a tubulin staining pattern that was diffuse and disorganized . 
 at a lower concentration ( 0.1 m ) , 2 m had a similar 
 but somewhat less severe effect on microtubule organization . cells 
 treated with the methuosis - inducer 1a appeared to contain 
 intact cable - like microtubules , although their organization was distorted 
 by the accumulation of large vacuoles throughout the cytoplasm . immunofluorescence 
 imaging of tubulin ( red fluorescence ) in cells 
 treated for 24 h with the methuosis - inducing compound 1a and the 2-indolyl propyl ester 2 m . the nuclei are visualized 
 with dapi ( blue fluorescence ) . to obtain a more direct indication of the effects of the 
 compounds 
 on tubulin polymerization , we utilized an established biochemical 
 approach in which the drug - treated cells are lysed under conditions 
 designed to preserve the native polymerization state of the microtubules . the polymerized tubulin is then separated from soluble tubulin by 
 high - speed centrifugation and quantified by western blot analysis . the results , depicted in figure 8 , demonstrate 
 that the three most potent compounds , which caused mitotic arrest 
 in figures 5 and 6 ( 2j , 2l , and 2 m ) , produced substantial 
 declines in the relative amount of tubulin detected in the polymerized 
 fraction . lowering the concentration from 3 m to 0.1 m 
 diminished but did not eliminate the effect on tubulin polymerization , 
 consistent with the immunofluorescence observations in figure 7 . the results were very similar to those obtained 
 with the positive control , colchicine , applied at the same concentrations . the proportion of polymerized tubulin in cells treated with the methuosis - inducing 
 compounds , 1a and 2b , and the inactive derivative meta - momipp , was similar to 
 that detected in the dmso control . effects of selected indolyl - pyridinyl - propenones 
 on tubulin polymerization 
 in cultured u251 cells . cells were treated for 4 h with the indicated 
 compounds at a final concentration of 3 m and then fractionated 
 under conditions designed to preserve the native polymerization state 
 of the microtubules . the percentage of polymerized versus soluble 
 tubulin was determined by immunoblot analysis as described in the experimental section . for each compound the fraction 
 of tubulin in the polymerized state was expressed as a percentage 
 of the polymerized tubulin determined in a parallel control culture 
 treated with dmso alone . the results represent the mean ( s.d . ) 
 of determinations from three separate experiments . the decreases in 
 the percentage of polymerized tubulin in cells treated with 2j , 2l , 2 m and colchicine ( at 3 
 m ) were significant at p  0.05 . the 
 indolyl - pyridinyl - propenones described in this report are related 
 to naturally occurring flavonoid precursors termed chalcones , which 
 consist of an ,-unsaturated ketone linking two nonheterocyclic 
 aromatic rings . over the past two decades a large number of naturally 
 occurring and synthetic chalcones and chalcone derivatives have been 
 characterized as having anticancer activities . our group has 
 focused on a unique class of such molecules wherein the aromatic ring 
 systems consist of indolyl- and pyridinyl - moieties . in particular , 
 we have established that certain members of this family , namely , 1a and its des - methoxy analogue ( mipp ) , can 
 induce methuosis and kill temozolomide - resistant glioma cells and 
 doxorubicin - resistant breast cancer cells at low micromolar concentrations . the cytostatic and cytotoxic effects of 1a are moderately 
 selective for cancer cells compared with normal fibroblasts ( supporting information 
 figure s1 ) . recently , others have described unrelated small 
 molecules ( vacquinols ) that also induce methuosis in cultured glioma 
 cells and suppress the growth of tumor xenografts in mouse models . these studies have stimulated interest in evaluating 
 the therapeutic potential of methuosis - inducing compounds . previous studies suggested that induction of methuosis is 
 due to drug interaction with specific proteins , rather than general 
 covalent protein modification by michael addition . in particular , 
 the reversibility of methuosis upon drug wash - out during the early 
 stages and the strict dependency of the biological effects on the para - configuration of the pyridinyl nitrogen support this 
 concept . while reassuring in terms of highlighting 
 specificity , the tendency of these compounds to be inactivated by 
 many modifications on the pyridinyl or indolyl moieties has made the 
 use of conventional affinity - based approaches for drug target identification 
 quite challenging . a second key question concerns the mechanism(s ) 
 leading to cell 
 death , particularly the relationship between the formation of cytoplasmic 
 vacuoles and the ultimate loss of cell viability . our initial sar 
 studies with a directed library of compounds revealed that analogues 
 that failed to induce vacuolization generally failed to cause cell 
 death . thus , we inferred that vacuolization was 
 an important contributing factor in the cell death program . however , 
 our recent studies with certain aliphatic 2-indolyl substitutions , coupled with the present observations with some 
 of the substituents at the indolyl 5-position ( e.g. , 2a , 2c , 2e , 2f ) , indicate that 
 vacuolization does not necessarily lead to growth arrest and cell 
 death in every case . thus , our current working hypothesis is that 
 the cytotoxicity of certain indolyl - pyridinyl - propenones is due to 
 pleiotropic effects , combining perturbations of macropinosome / endosome 
 trafficking with alterations of cellular signaling or metabolic pathways 
 that remain to be defined . in the present study we have identified 
 several new indolyl - pyridinyl - propenones 
 that exhibit robust biological activities . for the purpose of discussion , we consider the active compounds as falling into three discernible 
 classes based on the distinct cellular phenotypes that they induce 
 in cultured cells ( figure 9 ) . the compounds 
 in class 1 cause striking cellular vacuolization , but the cells continue 
 to proliferate and remain viable . examples of class 1 compounds are 
 drawn from this report ( 2a , 2c , 2e , 2f ) and our previous publication . we envision that the class 1 compounds target proteins 
 involved in the formation of macropinosomes , the recycling of these 
 structures , or the fusion of macropinosomes with lysosomes . the complexity 
 of the phenotype suggests that there may be multiple class 1 targets , 
 but we can not exclude the possibility that perturbation of a single 
 enzyme , structural protein , or signaling molecule could be responsible . we also identified several new compounds with substantial methuosis - inducing 
 activity ( 2b , 2p , 2q ) , comparable to that of our previously 
 characterized lead compound 1a . in the context of these 
 studies , we equate methuosis with the class 2 phenotype . it is similar 
 to the class 1 phenotype in terms of massive cellular vacuolization , 
 but in this case the cells detach and undergo metabolic failure , culminating 
 with rupture of the cell membrane ( i.e. , methuosis ) . we speculate 
 that in addition to interacting with class 1 targets , the class 2 
 compounds have affinity for a separate set of protein targets involved 
 in metabolic and/or signaling pathways essential for cell viability . 
 through these combined effects , the class 2 compounds trigger methuotic 
 cell death . the most striking observation from the present study is 
 that certain modifications at the 2-position of the indole ring increased 
 the cytotoxic activity of the compounds by 1 or 2 orders of magnitude 
 ( e.g. , 2j2 m ) . the increase in potency 
 was accompanied by notable changes in the phenotype observed in glioma 
 cells treated with these compounds , which we term class 3 . in contrast 
 to the typical vacuolated morphology seen in cells undergoing methuosis , 
 the cells treated with the 2-trifluoromethylindole and the alkyl 2-indolylcarboxylate 
 esters underwent a rapid transition from early formation of small 
 vacuoles to very active membrane blebbing and cell contraction . this 
 was followed by microtubule depolymerization , mitotic arrest , and 
 massive detachment of cells from the substratum . these changes lead 
 us to conclude that compounds 2j2 m acquired the capacity to interact with one or more protein targets 
 involved in microtubule assembly or maintenance ( class 3 targets ) . the early hint of vacuolization in cells treated with these compounds 
 suggests that macropinocytosis may also have been affected , possibly 
 via class 1 or class 2 targets . however , with the superimposition 
 of potent microtubule - destabilizing activity , disruption of the cytoskeleton 
 and consequent detachment of the cells occur before the class 1 or 
 class 2 phenotypes can develop . classification of biologically active 
 idolyl - pyridinyl - propenones 
 based on distinct cellular phenotypes elicited by modifications at 
 r1 and r2 . in the hypothetical model , class 
 1 compounds are envisioned as interacting with one or more class 1 
 protein targets to induce perturbations in macropinosome trafficking , 
 resulting in cellular vacuolization without impairment of cell proliferation 
 or viability . class 2 compounds interact with the same class 1 targets , 
 but acquire the ability to bind additional class 2 protein targets 
 that function in metabolic or pro - survival signaling pathways . the 
 combination of effects on class 1 and class 2 proteins results in 
 a distinct phenotype characterized by extreme vacuolization and cell 
 death via methuosis . the novel compounds in class 3 gain the ability 
 to inhibit proteins involved in microtubule assembly in a concentration 
 range where class 1 or 2 compounds have no effect on tubulin polymerization . hence , mitotic arrest , cell rounding , and death ( presumably by mitotic 
 catastrophe ) predominate as the main features of the class 3 phenotype . it is unclear whether the class 3 compounds retain the capacity to 
 interact with class 1 and 2 targets , although the early detection 
 of vacuoles prior to microtubule disruption and cell rounding suggests 
 that there may be some overlap . in terms of summarizing specific sar features that might 
 be gleaned 
 from the assembled compounds , first , it appears that the distinctive microtubule - related actions 
 described above for the trifluoromethyl and ester analogues 2j2 m are prompted by the presence of 
 an electron withdrawing substituent placed at the indolyl 2-position . 
 in 1a the 2-methyl group is modestly electron donating 
 and the biological profile is that of the class 2 phenotype . substitution 
 with an electron withdrawing trifluoromethyl group ( 2j ) redirects the profile to that of class 3 and substitution with 
 electron withdrawing carbonyl systems such as those present in esters 2l and 2 m prompts class 3 with even stronger 
 potency . alternatively , when the ester substituent is branched ( 2n ) or is removed entirely so as to expose a carboxylic acid 
 moiety ( 2o ) , activity is significantly reduced or lost 
 completely . this is similar to our previous findings where exploration 
 of this vicinity with various alkyl substituents suggested that there 
 may be a pocket in 1a s methuosis - related protein 
 targets that prefers to accommodate a methyl or ethyl group . for that series , larger groups at the indolyl 
 2-position still caused vacuolization , but the cells did not proceed 
 to methuotic death . indeed , it was our suspicion about this pocket 
 that led to the design of the more lipophilic 2j in order 
 to further probe the importance of this feature for interaction with 
 class 2 targets . however , in addition to providing the desired increase 
 in lipophilicity relative to 1a , the cf3 substitution 
 also confers a significant electron withdrawing effect . consistent 
 with the results from the esters 2l and 2 m , the latter appears to induce a shift in the biological activity 
 of 2j from a class 2 to class 3 phenotype . contributing 
 to construction of a topological map near the indolyl 
 2-position , relative to interactions with methuosis - related protein 
 targets , are the unanticipated results for the two alcohols 2p and 2q . they are not complicated by having 
 additional electron withdrawing effects and , as expected , do not evoke 
 mitotic arrest and cell death by interaction with microtubules ( class 
 3 ) . instead , these substituents endow strong class 2 effects even 
 though they are less lipophilic than their corresponding alkyl systems . furthermore , the high potency exhibited by the n - propyl 
 alcohol 2q does not correspond to the previously limited 
 size for simple alkyl arrangements wherein methyl or ethyl were preferred 
 for methuosis activity . indeed , the preference 
 for a propyl in this case resembles the sar for the class 3 family 
 of microtubule - interacting esters wherein the best alcohol adduct 
 was also n - propyl . this could suggest that there 
 are some commonalities among the protein targets engaged by the class 
 2 and class 3 indolyl - pyridinyl - propenones . at this point , for the 
 specific methuosis - related proteins , we speculate that the topology 
 of the putative binding pocket for the indolyl s 2-position 
 may approximate a narrow groove that can accommodate simple ( nonbranched ) 
 alkyl groups that are lipophilic at the proximal end and capable of 
 hydrogen bonding at the distal end , provided that ionizable moieties 
 are not present anywhere along the alkyl chain . preferences 
 for the topology around the indolyl 5-position appear 
 to be more stringent , but the overall sar is equally intriguing . similar 
 to our former study where the indolyl 
 2-position was probed with various alkyl groups , many of the substituents 
 placed at the 5-position were able to retain class 1 activity , but 
 only a few demonstrated class 2 methuosis activity . as shown previously , a phenolic substitution is not active , while 
 a methoxy , as is present in 1a , prompts significant methuosis . 
 such activity is greatly reduced by ethoxy , is returned by n - propoxy , and then is greatly reduced again by branched 
 alkyl or n - butoxy ( 2a , 2b , 2c , and 2d , respectively ) . assuming that 
 a model similar to that postulated for the indolyl 2-position also 
 pertains to the indolyl 5-position , one might ask why a binding pocket 
 that accommodates methoxy would optimally accept an n - propoxy substituent , but not an ethoxy or butoxy group . in this 
 regard , it is worth noting that similar anomalies have been reported 
 for well - known systems that have undergone extensive sar studies . 
 in one example , the  goldilocks  nature of an ethyl group inserted between a bulky aryl - system 
 and a simple methyl ester carbonyl moiety ( thus constituting a 3-carbon 
 system ) was exploited as being  just right  to allow 
 ready access for attack of -adrenergic antagonists by esterases . while the 3-carbon system endowed an ultrashort duration of action , 
 similar effects were not afforded by methyl ( 2-carbon system ) or n - propyl ( 4-carbon system ) spacers . in this case the author speculated that the additional conformational 
 freedom afforded by the n - propyl group could cause 
 the ester to become tucked back toward other bulky features present 
 within the overall molecular framework . a second example of preferred 
 conformational arrangements comes from the literature on dopamine 
 receptor agonists . in this case , dopamine loses significant activity 
 when its primary amine becomes substituted with a broad range of alkyl 
 functionalities except for a distinct disubstitution situation wherein 
 at least one of the substituents is an n - propyl group . in this heavily studied research arena , this remarkable effect is 
 referred to as the n - propyl phenomenon  , 
 and it has prompted speculation that the dopamine receptor s 
 ligand - binding pocket maintains a  unique geometry to accommodate 
 [ just ] an n - propyl group  . our prior computational 
 studies on substituted indolyl - pyridinyl - propenones suggest that their 
 side - chain substitutions remain quite flexible , with only subtle differences 
 in energy across several conformational possibilities for each increment 
 of added carbon atoms to the 2-position . thus , in our case , various arrangements being uniquely adopted by 
 only certain homologues during interaction with a protein surface 
 can not be ruled out . further insight regarding the possible 
 topography around the indolyl 
 5-position comes from our finding that an ionizable free amine ( 2 g , 2i ) , a neutral amide ( 2e ) , and 
 a pair of carboxamides having hydrogen bonding capabilities ( 2f , 2h ) are all unable to induce methuosis . although 
 weak gi activity was observed in the specific case where a boc group 
 was synthesized on an amine spaced one methylene unit away from the 
 indolyl moiety ( 2h ) , this activity did not match the 
 class 2 or 3 cytotoxic phenotypes . thus , similar to the model for 
 the indolyl 2-position , we speculate that the protein region interacting 
 with the 5-position also may have a restrictive groove that can accommodate 
 a methoxy group or the special case of a n - propoxy 
 group , with the provisos that the latter must remain lipophilic throughout , 
 and that additional substituents capable of hydrogen bonding or ionization 
 are excluded anywhere along the alkyl chain . in conclusion for 
 our sar , we find it quite remarkable that such 
 profound changes in biological profiles can be unmasked around the 
 indolyl - pyridinyl - propenone structural motif with subtle manipulations 
 at either of just two positions . while we offer proposals for what 
 the topography may look like in the relevant binding domains of the 
 unknown class 2 protein targets , the assumptions are based upon the 
 present state of sar with a limited spectrum of structural probes . nevertheless , until specific proteins are identified , these early 
 models can serve as useful conceptual tools for the design of new 
 probes to map the structural determinants responsible for the distinct 
 biological activities of the indolyl - pyridinyl - propenones . numerous 
 chalcones and chalcone - derivatives displaying antimitotic 
 activity have been characterized previously . while many of these compounds were identified 
 by screening collections of synthetic chalcones in cell - based proliferation 
 assays , others emerged from molecular modeling and 
 design studies aimed at creating new structures that might compete 
 for tubulin sites known to bind established microtubule inhibitors 
 ( e.g. , colchicine , combretastatins ) . most recently , efforts to develop curcumin analogues that 
 might kill cancer cells by targeting the nfb activation pathway 
 serendipitously yielded a series of potent ortho - aryl 
 substituted chalcones with the ability to bind to the colchicine site 
 on tubulin and induce mitotic arrest and apoptotic cell death . because of their distinct structural profiles 
 and potencies , the trifluoromethyl and ester analogues described herein 
 appear to represent a new class of potent microtubule - active anticancer 
 compounds . among the various traditional chalcones investigated 
 as potential 
 anticancer agents , indole - based chalcones have received comparatively 
 little attention until recently . in addition to the methuosis - inducing 
 indolyl - pyridinyl - propenones described by our group , the other class of antiproliferative / cytotoxic indolyl - chalcones 
 comprises the 1-(n - methylindolyl)-3-phenylpropenones . sar studies of analogues with different substituents on the phenyl 
 ring revealed that 3-(1-methyl-1h - indol-3-yl)-1-(2,4,6-trimethoxyphenyl)-2-propen-1-one , 33 ( jai-51 ) produced mitotic arrest at low micromolar concentrations . this was attributed to direct inhibition of tubulin polymerization 
 based on the results of cell - free tubulin assembly assays . although it is possible that our 2-trifluoromethylindole 
 and ester derivatives might operate in a similar way to disrupt microtubules , 
 we have not yet obtained definitive evidence to discriminate between 
 direct tubulin binding and other indirect mechanisms , such as interference 
 with microtubule - associated proteins . the possibility that the mechanism 
 might differ from that described for 33 is suggested 
 by the much higher potency and unique structural features of the indolyl - pyridinyl - propenones . in particular , our class 3 microtubule - disrupting compounds lack the n - methyl group on the indole ring found in 33 and other antimitotic 1-(n - methylindolyl)-3-phenylpropenones . another key difference between our current set of compounds and previously 
 reported antimitotic chalcones relates to the pyridinyl moiety which 
 is uniquely present in our scaffold and requisite for optimal activity . specifically , cytotoxicity ( via methuosis ) was eliminated by switching 
 the position of the pyridinyl nitrogen from para- 
 to meta- . in a preliminary 
 study , we have determined that altering the position of the pyridinyl 
 nitrogen from para- to meta- in 
 the context of 2l and 2 m , has a similar 
 negative influence on the antiproliferative activity of these microtubule - disrupting 
 compounds , raising their gi50 s by approximately 
 2 orders of magnitude ( supporting information 
 figure s2 ) . drugs that affect microtubule dynamics are 
 among the most widely 
 employed anticancer agents . distinct classes of 
 antimitotic compounds include the vinca alkaloids 
 ( e.g. , vincristine , vinblastine ) and colchicine analogues ( e.g , combretastatins ) 
 both of which are considered microtubule destabilizers , and the taxanes 
 ( e.g. , paclitaxel ) which function as microtubule overstabilizing agents . although they are effective against many types of cancer , these 
 compounds are not without drawbacks in the clinic . side effects , such 
 as peripheral neuropathy and neutropenia , may limit the doses and 
 treatment regiments that can be tolerated . development of drug resistance 
 is also a factor that limits efficacy in many cases . the latter may 
 entail both induction of drug efflux pumps and alterations in the 
 intrinsic properties of the microtubules ( e.g. , tubulin isotypes or 
 posttranslational modifications ) . finally , the vinca alkaloids and taxanes exhibit low permeability through the blood  brain 
 barrier , minimizing efficacy for treating primary or metastatic tumors 
 in the central nervous system . for these reasons , the identification 
 of new microtubule - directed agents with unique profiles continues 
 to be of interest . indolyl - pyridinyl - propenones are particularly intriguing 
 candidates for further study because published findings with other 
 indole - based chalcones suggest that they are poor substrates for drug 
 efflux pumps and are able to cross the blood  brain barrier . indeed , several functionalized chalcones have 
 been shown to actively impair drug transporters like p - glycoprotein 
 and breast cancer resistance protein . it remains to be determined if this property extends 
 to the compounds described in this report . our novel trifluoromethyl 
 and ester analogues could be particularly attractive prototypes for 
 future study , as they may operate through a combination of mechanisms 
 to induce features of both methuosis ( disruption of macropinosome 
 trafficking ) and microtubule destabilization . in this regard , it will 
 be important to discern if these compounds trigger cell death through 
 pathways similar to those promoted by conventional antitubulin compounds 
 ( i.e. , apoptosis , mitotic catastrophe ) or instead 
 operate through a novel cell death program . ultimately , the potential 
 therapeutic utility of this class of compounds will depend on further 
 evaluation of cytotoxicity and selectivity in a broad panel of transformed 
 and nontransformed cell lines and determination of their pharmacokinetic 
 properties in vivo . all reactions were performed in 
 oven - dried 2-neck round - bottom flasks under an atmosphere of either 
 ar or n2 and stirred with teflon - coated magnetic bars . tlc ( silica gel f254 plates , baker - flex ) was used to monitor 
 progress of all reactions with visualization performed under 254 nm 
 uv light . reagent grade and anhydrous solvents were purchased from 
 sigma - aldrich and used without further purification unless otherwise 
 noted . samples to be purified by column chromatography 
 were dissolved in a minimal volume of solvent and then adsorbed onto 
 silica gel ( 510 the amount of sample by weight ) by evaporation 
 under reduced pressure until the solid composite was free - flowing . the dry loaded sample was then applied to the top of the prepacked 
 column bed allowing the solvent line of the column to be above the 
 added adsorbent . unless indicated otherwise , chromatography was conducted 
 by flash column methods as described previously utilizing a gradient of increasingly polar eluent specifically 
 indicated for each compound . gradients were performed in a stepwise 
 fashion in 200 ml increments . for etoac / hexanes systems , the column 
 was charged with the defined starting eluent percentage . after the 
 sample was applied , 200 ml of the initial eluent was used , followed 
 by 200 ml at 10% increments until the product eluted into the final 
 gradient . for meoh / dcm systems , after the sample was applied , 200 ml of 
 the initial eluent was used , followed by 200 ml at 2.5% increments 
 until the product eluted into the final gradient . appropriate fractions 
 were combined , and solvents were evaporated in vacuo ( rotary evaporator 
 under water aspirator vacuum ) and then further dried by a vacuum pump 
 ( 0.5 mm hg ) for 24 h unless described otherwise . samples that were 
 heated in a vacuum desiccator were equipped to a vacuum pump ( 0.5 
 mm hg ) and dried for a specified time and temperature denoted for 
 the individual procedure . solvent solutions dried with na2so4 were stored in a sealed flask and allowed to sit for 
 at least 12 h. upon completion , the drying agent was removed by vacuum 
 filtration , and the solvents were evaporated in vacuo and then further 
 dried by a vacuum pump ( 0.5 mm hg ) for 24 h. samples reduced by hydrogenation 
 utilized a parr hydrogenator . melting points were performed 
 in triplicate on an electrothermal digital melting point apparatus 
 and are uncorrected . proton ( h ) and carbon ( c ) nmr experiments were recorded on either a 600 mhz bruker avance , 
 inova 600 mhz , or an inova 400 mhz instrument . samples were referenced 
 to tms when present , or the solvent residual peak for h and c , respectively : ( cdcl3 ; 7.27 , 77.13 ; 
 dmso - d6 ; 2.50 , 39.51 ; meoh - d4 ; 3.31 , 49.15 ) . h nmr chemical shifts were 
 given in ppm , and coupling constants ( j values ) were 
 expressed in hertz ( hz ) using the following designations : s ( singlet ) , 
 d ( doublet ) , t ( triplet ) , q ( quartet ) , quin ( quintet ) , sex ( sextet ) , 
 sep ( septet ) , dd ( doublet of doublets ) , m ( multiplet ) . the c chemical shifts are reported for each compound in the experimental section and in all cases confirm structure . 
 in a few cases fluorine ( f ) nmr was recorded on an inova 
 400 mhz instrument at 376 mhz . purity for tested compounds ( 2a2f , 2h , 2j2q ) was determined by combustion analysis ( atlantic microlabs , norcross 
 ga ) , or in the cases of 2 g and 2i by hplc . intermediate compounds 
 were determined to possess 95% purity by combustion analysis 
 except 4a and 6a . for these intermediates , 
 the mp and spectral data matched literature values . observed values 
 for combustion analysis were considered acceptable within 0.4% 
 of calculated values . synthetic derivatives reported as solvates are 
 denoted in the text and were calculated by incorporating the minimal 
 amount of appropriate solvent that was subjected to the compound during 
 the purification process in order to be within the acceptable range 
 ( 0.4% ) . hplc was performed on an alliance instrument ( # 2659 ) 
 equipped with a quaternary pump , an inline membrane degasser , autosampler , 
 and photodiode array ( pda ) detector ( # 2996 ) from waters corporation 
 ( milford , ma ) . the column was a nova - pakc18 column , 4 m particle 
 size ( 150 mm  3.9 mm ) . details for hplc analysis are described 
 in the individual procedures for 2 g and 2i . high resolution mass spectrometry ( hrms ) was performed by the university 
 of michigan s department of chemistry as a technical service . compounds 3 , 15 , and 22 were 
 purchased commercially from sigma - aldrich . 5-ethoxy-2-methylindole-3-carboxaldehyde 9a ( 188 mg , 
 0.93 mmol ) was dissolved in anhydrous methanol ( 12 ml ) . 4-acetylpyridine 
 ( 168 mg , 1.38 mmol ) and piperidine ( 118 mg , 1.38 mmol ) were added , 
 and the solution was heated at reflux for 24 h. a precipitate slowly 
 formed which was collected , washed with ice - cold meoh ( 30 ml ) and 
 dried at 40 c in a vacuum desiccator for 24 h to yield a bright 
 yellow powder ( 244 mg , 86% ) : mp 255257 c . h nmr ( 600 mhz , dmso - d6 )  
 11.88 ( s,1h ) , 8.80 ( m , 2h ) , 8.098.06 ( d , 1h , j = 15.3 hz ) , 7.937.92 ( m , 2h ) , 7.41 ( d,1h , j = 2.22 hz ) , 7.347.32 ( d , 1h , j = 15.3 hz ) , 
 7.307.29 ( d , 1h , j = 8.64 hz ) , 6.846.82 
 ( dd , 1h , j1 = 8.7 hz , j2 = 2.28 hz ) , 4.144.11 ( q , 2h , j = 6.96 hz ) , 2.57 ( s , 3h ) , 1.371.36 ( t , 3h , j = 6.96 hz ) . c nmr ( 150 mhz , dmso - d6 )  188.1 , 154.4 , 150.6 , 145.8 , 145.1 , 139.6 , 131.0 , 
 126.6 , 121.4 , 112.82 , 112.24 , 111.3 , 109.3 , 104.4 , 63.6 , 14.9 , 12.2 . elemental analysis calculated for c19h18n2o2 : c , 74.49 ; h , 5.92 ; n , 9.14 . found : c , 74.30 ; 
 h , 6.02 ; n , 9.04 . 2-methyl-5-propoxyindole-3-carboxaldehyde 9b ( 200 mg , 
 0.920 mmol ) was reacted in a similar manner to that for 2a to yield a bright yellow powder ( 204 mg , 69% ) : mp 228229 
 c . h nmr ( 600 mhz , dmso - d6 )  11.88 ( s,1h ) , 8.818.80 ( m , 2h ) , 8.088.06 
 ( d , 1h , j = 15.3 hz ) , 7.937.92 ( m , 2h ) , 7.41 
 ( d , 1h , j = 2.22 hz ) , 7.347.31 ( d , 1h , j = 15.24 hz ) , 7.307.29 ( d , 1h , j = 8.64 hz ) , 6.856.83 ( dd , 1h , j1 = 8.64 hz , j2 = 2.28 hz ) , 4.044.02 
 ( t , 2h , 6.54 hz ) , 2.56 ( s , 3h ) , 1.801.74 ( sex , 2h , j = 6.66 hz ) , 1.031.01 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , dmso - d6 )  188.2 , 154.6 , 150.6 , 145.75 , 145.18 , 139.6 , 
 131.0 , 126.6 , 121.4 , 112.90 , 112.25 , 111.4 , 109.3 , 104.4 , 69.6 , 22.3 , 
 12.2 , 10.6 . elemental analysis calculated for c20h20n2o2 : c , 74.98 ; h , 6.29 ; n , 8.74 . found : 
 c , 75.00 ; h , 6.23 ; n , 8.69 . 5-isopropyloxy-2-methylindole-3-carboxaldehyde 9c ( 225 
 mg , 1.04 mmol ) was reacted in a similar manner to that for 2a to yield an orange powder ( 294 mg , 88% ) : mp 245247 c . h nmr ( 600 mhz , dmso - d6 )  
 11.88 ( s,1h ) , 8.818.80 ( m , 2h ) , 8.088.05 ( d , 1h , j = 15.24 hz ) , 7.927.91 ( m , 2h ) , 7.42 ( d,1h , j = 2.22 hz ) , 7.337.30 ( d , 1h , j = 15.3 hz ) , 7.307.28 ( d , 1h , j = 8.76 hz ) , 
 6.846.82 ( dd , 1h , j1 = 8.7 hz , j2 = 2.28 hz ) , 4.704.66 ( quin , 1h , 6 
 hz ) , 2.56 ( s , 3h ) , 1.301.29 ( d , 6h , j = 6 
 hz ) . c nmr ( 150 mhz , dmso - d6 )  188.2 , 153.1 , 150.6 , 145.88 , 145.17 , 139.6 , 131.1 , 126.7 , 
 121.4 , 112.82 , 112.74 , 112.23 , 109.2 , 106.8 , 70.1 , 22.0 , 12.1 . elemental 
 analysis calculated for c20h20n2o2 : c , 74.98 ; h , 6.29 ; n , 8.74 . found : c , 74.86 ; h , 6.22 ; n , 
 8.62 . 5-butoxy-2-methylindole-3-carboxaldehyde 9d ( 250 mg , 
 1.08 mmol ) was reacted in a similar manner to that for 2a to yield a bright yellow powder ( 206 mg , 57% ) : mp 227228 
 c . h nmr ( 600 mhz , dmso - d6 )  11.39 ( s , 1h ) , 8.818.80 ( m , 2h ) , 8.088.06 
 ( d , 1h , j = 15.3 hz ) , 7.937.92 ( m , 2h ) , 7.41 
 ( d , 1h , j = 12.3 hz ) , 7.347.31 ( d , 1h , j = 15.24 hz ) , 7.307.28 ( d , 1h , j = 8.64 hz ) , 6.846.82 ( dd , 1h , j1 = 8.64 hz , j2 = 2.34 hz ) , 4.084.06 
 ( t , 2h , j = 6.48 hz ) , 2.57 ( s , 3h ) , 1.761.71 
 ( m , 2h ) , 1.521.45 ( m , 2h ) , 0.970.94 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , dmso - d6 )  188.2 , 154.6 , 150.6 , 145.74 , 145.19 , 
 139.6 , 131.0 , 126.6 , 121.4 , 112.90 , 112.25 , 111.44 , 109.3 , 104.3 , 
 64.8 , 31.0 , 18.9 , 13.8 , 12.2 . elemental analysis calculated for c21h22n2o2 : c , 75.42 ; h , 6.63 ; 
 n , 8.38 . found : c , 75.39 ; h , 6.55 ; n , 8.37 5-acetamido-2-methylindol-3-caboxaldehyde 13 ( 150 mg , 
 0.69 mmol ) was dissolved in meoh ( 6 ml ) and stirred vigorously until 
 dissolution was complete . piperidine ( 91 mg , 1.07 mmol ) and 4-acetylpyridine 
 ( 208 mg , 1.72 mmol ) were added , and the mixture was heated to reflux 
 while stirring vigorously for 20 h. the resulting precipitate was 
 filtered , washed with ice - cold meoh ( 10 ml ) , and dried at 40 c 
 in a vacuum desiccator for 24 h to yield an orange solid ( 193 mg , 
 87% ) : mp > 300 c . h nmr ( 600 mhz , dmso - d6 ) 11.94 ( s , 1h ) , 9.89 ( s , 1h ) , 8.848.83 
 ( m , 2h ) , 8.26 ( d , 1h , j = 1.9 hz ) , 8.078.05 
 ( d , 1h , j = 15 hz ) , 7.877.86 ( m , 1h ) , 7.487.46 
 ( dd , 1h , j1 = 8.6 hz , j2 = 1.9 hz ) , 7.337.30 ( m , 2h ) , 2.57 ( s , 3h ) , 2.08 
 ( s , 3h ) . c nmr ( 150 mhz , dmso - d6 )  188.0 , 167.8 , 150.6 , 145.82 , 145.17 , 139.6 , 134.0 , 
 132.4 , 125.7 , 121.1 , 114.9 , 112.8 , 111.5 , 110.03 , 109.26 , 24.0 , 11.9 . 
 elemental analysis calculated for c19h17n3o20.25 meoh hrms esi calculated m / z for c19h17n3o2 ( m + h ) 320.1394 . found ( m + h ) 320.1392 . 5-(n - boc - amino)-2-methylindol-3-carboxaldehyde 14 ( 250 mg , 0.91 mmol ) was reacted in a similar manner to 
 that for 2e to yield an orange solid ( 332 mg , 96% ) : mp 
 > 300 c ( 263 c color change ) . h 
 nmr ( 600 mhz , dmso - d6 ) 11.92 ( s , 
 1h ) , 9.28 ( s , 1h ) , 8.858.84 ( m , 2h ) , 8.088.05 ( m , 
 2h ) , 7.927.91 ( m , 2h ) , 7.50 ( s , 1h ) , 7.397.37 ( d , 
 1h , j = 15 hz ) , 7.307.29 ( d , 1h , j = 9 hz ) , 2.57 ( s , 3h ) , 1.52 ( s , 9h ) ; c nmr 
 ( 150 mhz , dmso - d6 )  187.6 , 153.0 , 
 150.6 , 145.8 , 139.6 , 134.1 , 132.0 , 125.9 , 121.2 , 114.0 , 112.5 , 111.4 , 
 109.2 , 78.8 . elemental analysis calculated for c22h23n3o3 : c , 70.01 ; h , 6.14 ; n , 11.13 . 
 found : c , 69.91 ; h , 6.10 ; n , 11.04 . compound 2f ( 35 mg , 0.0927 mmol ) was suspended 
 in meoh 
 ( 5 ml ) and tfa ( 1 ml ) and heated to reflux for 24 h. the volatiles 
 were evaporated in vacuo , and the sample was redissolved in h2o ( 5 ml ) and neutralized with 1 n naoh ( determined by ph paper ) . purification by column chromatography ( 2.5% to 10% meoh / dcm ) and subsequent 
 drying at 40 c in a vacuum desiccator for 24 h yielded an orange 
 solid ( 15 mg , 58% ) : mp 263264 c . h 
 nmr ( 600 mhz , d4-meoh )  8.75 ( m , 
 2h ) , 8.268.23 ( d , 1h , j = 15.06 hz ) , 7.96 
 ( m , 2h ) , 7.40 ( d , 1h , j = 1.8 hz ) , 7.377.35 
 ( d , 1h , j = 15 hz ) , 7.167.14 ( d , 1h , j = 8.4 hz ) , 6.746.72 ( dd , 1h , j1 = 8.4 hz , j2 = 1.8 hz ) , 
 2.56 ( s , 3h ) . c nmr ( 150 mhz , meoh - d4 )  190.8 , 151.2 , 148.4 , 147.6 , 143.66 , 143.21 , 
 132.6 , 128.7 , 123.4 , 114.4 , 113.2 , 111.6 , 108.1 , 12.1 . hrms esi calculated m / z for c17h16n3o ( m + h ) 278.1288 . 
 found ( m + h ) 278.1286 . hplc analysis : retention time 
 = 2.642 min ; peak area , 99.48% ; eluent a , 10 mm tea solution with 0.1% 
 formic acid ( ph 3 ) ; eluent b , ch3cn ; isocratic ( 15% eluent 
 b ) over 10 min with a flow rate of 1 ml min and 
 detection at 280 nm ; injection of 10 l of 59 m 2 g . 5-n - boc-(aminomethyl)-indole-3-carboxaldehyde 17 ( 152 mg , 0.554 mmol ) was reacted in a similar manner to 
 that for 2e to yield a yellow solid ( 150 mg , 72% ) : mp 
 227 c . h nmr ( 600 mhz , dmso - d6 )  11.97 ( s , 1h ) , 8.838.82 ( m , 2h ) , 8.158.14 
 ( d , 1h , j = 2.6 hz ) , 8.088.06 ( d , 1h , j = 15.6 hz ) , 7.927.91 ( m , 3h ) , 7.527.50 
 ( d , 1h , j = 15.6 hz ) , 7.447.42 ( m , 2h ) , 7.177.16 
 ( d,1h , j = 8.4 hz ) , 4.304.29 ( d , 2h , j = 6 hz ) , 1.38 ( s , 9h ) . c nmr ( 150 mhz , dmso - d6 )  188.6 , 155.8 , 150.5 , 144.8 , 141.0 , 
 136.5 , 134.5 , 133.4 , 125.0 , 122.5 , 121.3 , 118.6 , 114.7 , 112.55 , 112.22 , 
 77.6 , 43.7 , 28.2 . elemental analysis calculated for c22h23n3o3 : c , 70.01 ; h , 6.14 ; n , 11.13 . 
 found : c , 70.20 ; h , 5.98 ; n , 11.16 . compound 2h ( 100 mg , 0.265 
 mmol ) was reacted in a similar manner to that for 2 g except 
 the reaction time was extended to 48 h , and the isocratic chromatography 
 eluent was dcm / meoh / tea ( 90:9:1 ) . an orange / yellow solid was obtained 
 ( 69 mg , 94% ) : mp 170172 c ( darkened 155158 c ) . h nmr ( 600 mhz , meoh - d4 )  8.788.77 ( m , 2h ) , 8.198.17 ( d , 1h , j = 15.6 hz ) , 8.11 ( d , j = 0.6 hz , 1h ) , 
 7.997.98 ( m , 2h ) , 7.96 ( s , 1h ) , 7.607.57 ( d , 1h , j = 15.6 hz ) , 7.537.51 ( d , 1h , j = 8.4 hz ) , 7.347.32 ( dd , 1h , j1 = 8.4 hz , j2 = 1.8 hz ) , 4.19 ( s , 2h ) . c nmr ( 150 mhz , meoh - d4 )  
 191.4 , 151.3 , 147.7 , 143.3 , 139.5 , 135.7 , 130.9 , 127.3 , 125.1 , 123.5 , 
 121.8 , 116.7 , 115.1 , 114.1 , 45.8 . hrms esi calculated m / z for c17h16n3o ( m + h ) 278.1288 . found ( m+h ) 278.1291 . 
 hplc analysis : retention time = 2.869 min ; peak area , 99.14% ; eluent 
 a , 10 mm tea solution with 0.1% formic acid ( ph 3 ) ; eluent b , ch3cn ; isocratic ( 15% eluent b ) over 10 min with a flow rate 
 of 1 ml min and detection at 280 nm ; injection 
 of 10 l of 44 m 2i . 5-methoxy-2-trifluoromethylindole-3-carboxyaldehyde 21 ( 156 mg , 0.641 mmol ) was dissolved in anhydrous meoh ( 10 
 ml ) . 4-acetylpyridine ( 157 mg , 1.30 mmol ) and piperdine ( 111 mg , 1.30 
 mmol ) were added , and the mixture was heated to reflux for 20 h. upon 
 completion , volatiles were evaporated in vacuo , and the sample was 
 purified by column chromatography ( 3070% etoac / hexanes ) . the 
 resulting solid was dried at 40 c in a vacuum desiccator for 
 36 h to yield a bright yellow solid ( 60 mg , 27% ) : mp 230232 
 c . h nmr ( 600 mhz , dmso - d6 )  13.03 ( s , 1h ) , 8.868.85 ( m 2h ) , 8.078.04 
 ( dd , 1h , j1 = 15.7 hz , j2 = 1.2 hz ) , 7.997.98 ( m , 2h ) , 7.757.72 
 ( d , 1h , j = 15.6 hz ) , 7.60 ( d , 1h , j = 2.28 hz ) , 7.527.51 ( d , 1h , j = 8.94 hz ) , 
 7.127.10 ( dd,1h , j1 = 8.94 hz , j2 = 2.34 hz ) , 3.92 ( s , 3h ) . c nmr 
 ( 150 mhz , dmso - d6 )  188.9 , 155.8 , 
 150.6 , 143.8 , 135.2 , 131.0 , 127.2 ( q , jcf = 36.5 hz ) 125.0 , 121.48 , 121.23 ( q , jcf = 268.8 hz ) , 119.9 , 115.8 , 114.2 , 111.4 , 
 103.3 , 55.6 ; f nmr ( 376 mhz , dmso - d6 )  51.4 ( s , 3f ) . elemental analysis calculated 
 for c18h13f3n2o2 : c , 62.43 ; h , 3.78 ; n , 8.09 . found : c , 62.59 ; h , 3.91 ; n , 8.04 . methyl 5-methoxyindole-3-carboxyaldehyde-2-carboxylate 24a ( 100 mg , 0.404 mmol ) , 4-acetylpyridine ( 73 mg , 0.607 mmol ) , 
 and piperidine ( 52 mg , 0.607 mmol ) were dissolved in anhydrous meoh 
 ( 10 ml ) and heated to reflux for 20 h. the resulting yellow precipitate 
 was filtered , washed with ice - cold meoh ( 20 ml ) , and then dried at 
 40 c in a vacuum desiccator for 36 h ( 110 mg , 81% ) : mp 272273 
 c . h nmr ( 600 mhz , dmso - d6 )  12.58 ( s , 1h ) , 8.858.84 ( d , 2h , j = 4.44 hz ) , 8.748.71 ( d , 2h , j = 16.02 hz ) , 7.957.94 ( d , 2h , j = 4.5 hz ) , 
 7.717.68 ( d , 1h , j = 16.0 hz ) , 7.54 ( s , 1h ) , 
 7.497.48 ( d , 1h , j = 8.88 hz ) , 7.107.09 
 ( d , 1h , j = 8.94 hz ) , 3.94 ( s , 3h ) , 3.90 ( s , 3h ) . c nmr ( 150 mhz , dmso - d6 )  
 189.6 , 161.0 , 155.7 , 150.6 , 144.2 , 139.1 , 131.8 , 128.4 , 125.3 , 121.5 , 
 120.2 , 116.6 , 115.6 , 114.4 , 103.0 , 55.5 , 52.2 . elemental analysis 
 calculated for c19h16n2o4 : c , 67.85 ; h , 4.80 ; n , 8.33 . found : c , 67.71 ; h , 4.76 ; n , 8.37 . ethyl 5-methoxyindole-3-carboxyaldehyde-2-carboxylate 24b ( 250 mg , 1.01 mmol ) was reacted in a manner similar to 
 that for 2k except 2 equiv of 4-acetylpyridine and piperidine 
 were added and cold etoh ( 10 ml ) was used to wash the precipitate 
 to yield a bright yellow solid ( 303 mg , 86% ) : mp 245247 c . h nmr ( 600 mhz , dmso - d6 )  
 12.51 ( s , 1h ) , 8.848.83 ( m 2h ) , 8.758.72 ( d , 1h , j = 16.02 hz ) , 7.947.93 ( m , 2h ) , 7.687.66 
 ( d , 1h , j = 15.96 hz ) , 7.54 ( d , 1h , j = 2.22 hz ) , 7.507.49 ( d , 1h , j = 8.94 hz ) , 
 7.107.08 ( dd,1h , j1 = 8.94 hz , j2 = 2.34 hz ) , 4.414.38 ( q , 2h , j = 7.14 hz ) , 3.90 ( s , 3h ) , 1.361.33 ( t , 3h , j = 7.14 hz ) . c nmr ( 150 mhz , dmso - d6 )  189.8 , 160.6 , 155.7 , 150.6 , 144.3 , 
 139.4 , 131.7 , 128.6 , 125.3 , 121.5 , 120.2 , 116.6 , 115.4 , 114.3 , 103.0 , 
 61.1 , 55.4 , 14.0 . elemental analysis calculated for c20h18n2o4 : c , 68.56 ; h , 5.18 ; n , 8.00 . 
 found : c , 68.40 ; h , 5.07 ; n , 7.95 . propyl 5-methoxyindole-3-carboxyaldehyde-2-carboxylate 24c ( 223 mg , 0.853 mmol ) was dissolved in n - proh ( 20 ml , dried over 3  molecular sieves ) . 4-acetylpyridine 
 ( 155 mg , 1.28 mmol ) and piperidine ( 109 mg , 1.28 mmol ) were added , 
 and the was mixture heated to reflux for 24 h. an orange precipitate 
 slowly began to form . upon completion , the orange precipitate was 
 collected , washed with ice - cold n - proh ( 20 ml ) , and 
 dried at 40 c in a vacuum desiccator for 36 h to yield an orange 
 solid ( 245 mg , 79% ) : mp 221 c . h nmr ( 600 mhz , dmso - d6 )  12.49 ( s , 1h ) , 8.848.83 ( m 
 2h ) , 8.748.71 ( d , 1h , j = 16.02 hz ) , 7.947.92 
 ( m , 2h ) , 7.687.65 ( d , 1h , j = 15.96 hz ) , 
 7.54 ( d , 1h , j = 2.28 hz ) , 7.517.49 ( d , 1h , j = 8.94 hz ) , 7.107.08 ( dd , 1h , j1 = 8.94 hz , j2 = 2.34 hz ) , 
 4.324.30 ( t , 2h , j = 6.48 hz ) , 3.90 ( s , 3h ) , 
 1.761.71 ( sex , 2h , j = 7.32 hz ) , 1.000.98 
 ( t , 3h , j = 7.44 hz ) . c nmr ( 150 mhz , 
 dmso - d6 )  189.9 , 160.8 , 155.8 , 
 150.7 , 144.4 , 139.4 , 131.9 , 128.8 , 125.4 , 121.66 , 120.41 , 116.70 , 
 115.50 , 114.47 , 103.1 , 66.7 , 55.6 , 21.6 , 10.4 ; elemental analysis 
 calculated for c21h20n2o4 : c , 69.22 ; h , 5.53 ; n , 7.69 . found : c , 68.93 ; h , 5.66 ; n , 7.75 . isopropyl 5-methoxyindole-3-carboxyaldehyde-2-carboxylate 24d ( 210 mg , 0.804 mmol ) was reacted in a manner similar to 
 that for 2 m except i - proh was used instead 
 of n - proh to yield a bright yellow solid ( 238 mg , 
 81% ) : mp 233236 c . h nmr ( 600 
 mhz , dmso - d6 )  12.44 ( s , 1h ) , 8.848.83 
 ( m 2h ) , 8.748.71 ( d , 1h , j = 16.02 hz ) , 7.92 
 ( m , 2h ) , 7.657.62 ( d , 1h , j = 16.02 hz ) , 
 7.53 ( d , 1h , j = 2.28 hz ) , 7.507.49 ( d , 1h , j = 9 hz ) , 7.107.08 ( dd,1h , j1 = 8.94 hz , j2 = 2.34 hz ) , 5.245.18 
 ( sep , 1h , j = 6.24 hz ) , 3.90 ( s , 3h ) , 1.341.33 
 ( d , 6h , j = 6.24 hz ) . c nmr ( 150 mhz , dmso - d6 )  190.1 , 160.3 , 155.8 , 150.7 , 144.5 , 
 139.7 , 131.8 , 129.2 , 125.4 , 121.7 , 120.4 , 116.61 , 115.37 , 114.47 , 
 103.1 , 69.0 , 55.6 , 21.7 . elemental analysis calculated for c21h20n2o40.25 c3h8o : c , 68.85 ; h , 5.84 ; n , 7.38 . compound 2k ( 52 mg , 0.18 mmol ) 
 was 
 dissolved in methanol ( 3 ml ) and naoh ( 1 n , 2 ml ) . the reaction mixture 
 was heated to reflux for 30 h. upon completion , the final volume was 
 reduced by approximately one - half of the original volume in vacuo , 
 and the sample was stored at 4 c overnight resulting in the 
 formation of an orange precipitate . the orange precipitate was collected , 
 washed dropwise with a 50% mixture of meoh / h2o ( 10 ml , 
 chilled at 4 c for 1 h ) , and dried at 40 c in a vacuum 
 desiccator for 36 h to yield an orange salt ( 16 mg , 28% ) : mp > 300 
 c . h nmr ( 600 mhz , dmso - d6 )  11.72 ( s , 1h ) , 9.349.32 ( d , 
 1h , j = 16.02 hz ) , 8.798.78 ( m , 2h ) , 7.887.87 
 ( m , 2h ) , 7.43 ( d , 1h , j = 1.8 hz ) , 7.407.37 
 ( m , 2h ) , 6.886.87 ( dd , 1h , j1 = 
 8.76 hz , j2 = 2.16 hz ) , 3.86 ( s , 3h ) ; c nmr ( 150 mhz , dmso - d6 )  
 189.2 , 163.5 , 155.0 , 150.5 , 145.6 , 144.8 , 130.2 , 126.5 , 121.6 , 114.8 , 
 113.6 , 112.4 , 110.8 , 103.8 , 55.5 . elemental analysis calculated for 
 c18h13n2nao42 h2o : c , 56.84 ; h , 4.51 ; n , 7.37 . found : c , 56.64 ; h , 
 4.60 ; n , 7.27 . hrms esi calculated m / z for c18h13n2o4 ( m  h ) 321.0881 . found ( m 
  h ) 321.0884 . 5-methoxy-2-hydroxymethylindole-3-carboxaldehyde 27 ( 75 mg , 0.37 mmol ) , 4-acetylpyridine ( 118 mg , 0.37 mmol ) , 
 and piperidine ( 82.6 mg , 0.37 mmol ) were dissolved in methanol ( 1 
 ml ) and heated to reflux . the reaction mixture was stirred for 45 
 min during which the solution became bright orange . the flask was 
 then removed from heat and allowed to stir for 1 h. a precipitate 
 formed which was collected , washed with ice - cold meoh ( 5 ml ) , and 
 dried in a vacuum desiccator for 24 h to yield an orange solid ( 28 
 mg , 24% ) . tlc rf 0.65 ( 6% meoh / dcm ) , 0.21 ( 80% etoac / hexanes ) . h nmr ( 600 mhz , dmso - d6 )  
 11.96 ( s , 1h ) , 8.828.81 ( m , 2h ) , 8.178.14 ( d , 1h , j = 15.36 hz ) , 7.947.93 ( m , 2h ) , 7.457.44 
 ( d , 1h , j = 2.28 hz ) , 7.387.34 ( m , 2h ) , 6.886.86 
 ( dd , 1h , j1 = 8.7 hz , j2 = 2.34 hz ) , 5.705.68 ( t , 1h , j = 5.46 hz ) , 4.854.84 ( d , 2h , j = 5.46 hz ) , 
 3.86 ( s , 3h ) . c nmr ( 150 mhz , dmso - d6 )  188.2 , 155.2 , 150.6 , 148.0 , 145.0 , 139.3 , 131.0 , 
 126.7 , 121.4 , 113.8 , 113.0 , 111.6 , 108.3 , 103.4 , 55.57 , 55.32 . elemental 
 analysis calculated for c18h16n2o30.033 meoh : c , 70.00 ; h , 5.26 ; n , 9.05 . found : c , 69.61 ; 
 h , 5.09 ; n , 8.90 . this compound showed significant degradation when 
 stored in dmso - d6 for 48 h as determined 
 by h nmr . after 48 h , 50% of 2p was 
 degraded , and the spectrum showed a number of unidentifiable compounds . all other derivatives ( 1a  c , 2a  o , 2q ) were stable in 
 prolonged exposure ( up to 12 months ) to dmso . when stored in its isolated 
 precipitate form , this compound is stable . 2-hydroxypropyl-5-methoxyindole-3-carboxaldehyde - o - acetate 32 ( 87 mg , 0.31 mmol ) in meoh ( 6 ml ) , piperidine ( 40 mg , 0.47 
 mmol ) , and 4-acetylpyridine ( 57 mg , 0.47 mmol ) were heated to reflux 
 and stirred for 20 h. the solvent was reduced by approximately one - half 
 of the original volume in vacuo , and the flask was placed in a 20 
 c freezer for 30 min . the resulting bright orange - red precipitate 
 was collected , washed with ice - cold meoh ( 5 ml ) and dried in a vacuum 
 desiccator for 24 h ( 16 mg , 15% ) : mp 221223 c . h nmr ( 600 mhz , dmso - d6 )  
 11.87 ( s , 1h ) , 8.818.80 ( m , 2h ) , 8.128.10 ( d , 1h , j = 15.24 hz ) , 7.947.93 ( m , 2h ) , 7.44 ( d , 1h , j = 2.28 hz ) , 7.407.37 ( d , 1h , j = 15.24 hz ) , 7.337.31 ( d , 1h , j = 8.64 
 hz ) , 6.876.85 ( dd , 1h , j1 = 8.7 
 hz , j2 = 2.4 hz ) , 4.684.66 ( t , 
 1h , j = 4.62 hz ) , 3.86 ( s , 3h ) , 3.483.45 
 ( q , 2h , j = 6.0 hz ) , 2.992.96 ( t , 2h , j = 7.62 hz ) , 1.861.81 ( quin , 2h , j = 6.24 hz ) . c nmr ( 150 mhz , dmso - d6 )  188.1 , 155.2 , 150.62 , 149.80 , 145.1 , 139.5 , 
 131.2 , 126.4 , 121.4 , 113.01 , 112.42 , 111.1 , 108.9 , 103.7 , 59.9 , 55.6 , 
 32.7 , 22.7 . elemental analysis for c20h20n2o3 : c , 71.41 ; h , 5.99 ; n , 8.33 . found : c , 71.12 ; 
 h , 6.05 ; n , 8.34 . 3-methyl-4-nitrophenol 3 ( 500 mg , 3.0 mmol ) 
 was dissolved 
 in 2-butanone ( 10 ml ) . potassium carbonate ( 903 mg , 6.5 mmol ) was 
 added to the solution followed by diethylsulfate ( 0.45 ml , 3.2 mmol ) . the reaction mixture was heated at 70 c for 2 h. upon completion , 
 the mixture was cooled to rt , and aqueous ammonia ( 30% , 1 ml ) was 
 added and stirred for 18 h at rt . the filtrate was collected and 
 evaporated in vacuo to yield a white solid ( 580 mg , 97% ) . h nmr ( 600 mhz , cdcl3 )  8.118.09 ( d , 1h , j = 8.88 hz ) , 6.826.78 ( m , 2h ) , 4.144.10 
 ( q , 2h , j = 6.96 hz ) , 2.65 ( s , 3h ) , 1.481.46 
 ( t , 3h , j = 6.95 hz ) . c nmr ( 150 mhz , 
 cdcl3 )  162.5 , 142.0 , 137.1 , 127.6 , 117.9 , 112.2 , 
 64.2 , 21.8 , 14.6 . compound 3 ( 1.5 g , 9.8 mmol ) was 
 dissolved in 2-butanone 
 ( 40 ml ) . potassium carbonate ( 3.4 g , 24.5 mmol ) was added , and the 
 solution was heated at reflux . after 5 min , 1-bromopropane ( 18.5 mmol , 
 2 ml ) was added dropwise , and the reaction mixture continued to stir 
 for 18 h. the solvent was evaporated in vacuo , and the crude residue 
 was partitioned between etoac ( 150 ml ) and saturated nahco3 ( 150 ml ) . the organic layer was separated , washed with brine ( 100 
 ml ) , and dried over na2so4 . the resulting oil 
 was purified by column chromatography ( 0% to 20% etoac / hexanes ) to 
 yield a yellow oil ( 1.89 g , 99% ) : tlc rf 0.70 ( 20% etoac / hexanes ) . h nmr ( 600 
 mhz , cdcl3 )  8.098.07 ( d , 1h , j = 8.76 hz ) , 6.796.77 ( m , 2h ) , 3.993.97 ( t , 2h , j = 6.54 hz ) , 2.63 ( s , 3h ) , 1.851.82 ( sex , 2h , j = 7.44 hz ) , 1.061.04 ( t , 3h , j = 7.44 hz ) . c nmr ( 150 mhz , cdcl3 )  
 162.9 , 142.2 , 137.3 , 127.8 , 118.1 , 112.4 , 70.3 , 22.60 , 21.98 , 10.6 . elemental analysis calculated for c10h13no30.03 hexanes : c , 61.81 ; h , 6.84 ; n , 7.08 . found : c , 
 62.20 ; h , 6.78 ; n , 7.23 . this compound was prepared in a similar 
 manner to that for 4b except that 2-bromopropane was 
 deployed for the alkyl ether 
 adduct and chromatography utilized a 015% gradient for elution 
 to yield a yellow oil ( 1.87 g , 98% ) : tlc rf 0.68 ( 20% etoac / hexanes ) . h nmr ( 600 
 mhz , cdcl3 )  8.088.07 ( d , 1h , j = 8.82 hz ) , 6.776.75 ( m , 2h ) , 4.664.62 ( sep , 1h , j = 6.06 hz ) , 2.62 ( s , 3h ) , 1.381.37 ( d , 6h , j = 6.12 hz ) . c nmr ( 150 mhz , cdcl3 )  162.6 , 141.7 , 137.1 , 127.6 , 118.9 , 112.8 , 70.6 , 21.84 , 
 21.78 . elemental analysis calculated for c10h13no30.06 hexanes : c , 62.10 ; h , 6.96 ; n , 6.99 . found : 
 c , 62.45 ; h , 6.69 ; n , 7.09 . this compound was prepared in a similar 
 manner to that for 4b except 1-bromobutane was deployed 
 for the alkyl ether adduct 
 and the partition step during workup utilized dcm ( 150 ml ) and water 
 ( 150 ml ) to provide a yellow oil ( 2.0 g , 99% ) : tlc rf 0.70 ( 20% etoac / hexanes ) . h nmr ( 600 mhz , cdcl3 )  8.098.07 ( d , 1h , j = 8.88 hz ) , 6.796.76 ( m , 2h ) , 4.034.01 
 ( t , 2h , j = 6.48 hz ) , 2.63 ( s , 3h ) , 1.801.78 
 ( quin , 2h , j = 7.44 hz ) , 1.531.47 ( sex , 2h , j = 7.44 hz ) , 1.00.97 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , cdcl3 )  
 162.7 , 141.9 , 137.1 , 127.6 , 117.9 , 112.2 , 68.3 , 31.0 , 21.8 , 19.1 , 
 13.8 . elemental analysis calculated for c11h15no3 : c , 63.14 ; h , 7.23 ; n , 6.69 . compound 4a ( 250 mg , 1.38 mmol ) was dissolved 
 in etoac ( 10 ml ) and 
 meoh ( 10 ml ) and transferred to a 250 ml hydrogenation flask . 10% 
 pd / c ( 25 mg , 10% w / w ) was added , and the sample was hydrogenated for 
 4 h at 35 psi h2 . upon completion , the mixture was filtered 
 over a bed of celite and then concentrated in vacuo . the residue was 
 dissolved in etoac ( 50 ml ) and washed with nahco3 ( 50 ml 
  3 ) . the organic layer was dried over na2so4 to yield a dark - red oil ( 192 mg , 92% ) : tlc rf 0.30 ( 20% etoac / hexanes ) . nmr 
 ( 600 mhz , cdcl3 )  6.67 ( s , 1h ) , 6.62 ( m , 2h ) , 3.973.93 
 ( q , 2h , j = 6.96 hz ) , 3.42 ( s , 2h ) , 2.16 ( s , 3h ) , 
 1.381.35 ( t , 3h , j = 7.02 hz ) . c nmr ( 150 mhz , cdcl3 )  152.3 , 138.1 , 124.4 , 117.45 , 
 116.34 , 113.1 , 64.2 , 17.9 , 15.2 . elemental analysis calculated for 
 c9h13no : c , 71.49 ; h , 8.67 ; n , 9.26 . found : 
 c , 71.42 ; h , 8.76 ; n , 9.04 . compound 4b ( 3.78 g , 19.4 
 mmol ) was dissolved in etoac ( 20 ml ) and 
 meoh ( 20 ml ) and transferred to a 250 ml hydrogenation flask . 10% 
 pd / c ( 378 mg , 10% w / w ) was added , and the sample was hydrogenated 
 for 4 h at 35 psi h2 . upon completion , the mixture was 
 filtered over a bed of celite and then concentrated in vacuo . the 
 residue was dissolved in etoac ( 100 ml ) and washed with nahco3 ( 100 ml  3 ) . the organic layer was separated and then 
 dried over na2so4 to provide a crude brown oil 
 which was further purified by chromatography ( 20% to 50% etoac / hexanes ) 
 to yield an amber oil ( 2.72 g , 85% ) : tlc rf 0.27 ( 20% etoac / hexanes ) . nmr ( 600 mhz , 
 cdcl3 )  6.67 ( s , 1h ) , 6.62 ( m , 2h ) , 3.853.38 
 ( t , 2h , j = 6.66 hz ) , 2.16 ( s , 3h ) , 1.791.73 
 ( sex , 2h , j = 7.44 hz ) , 1.021.00 ( t , 3h , j = 7.44 hz ) . c nmr ( 150 mhz , cdcl3 )  152.3 , 137.8 , 124.1 , 117.2 , 116.1 , 112.9 , 70.1 , 22.7 , 17.7 , 
 10.5 . elemental analysis calculated for c10h15no : c , 72.69 ; h , 9.15 ; n , 8.48 . found : c , 72.42 ; h , 9.14 ; n , 8.46 . this 
 compound was prepared from 4c ( 3.64 g , 18.7 mmol ) in 
 a manner similar to that for 5b to yield an amber oil 
 ( 2.5 g , 81% ) : tlc rf 0.33 
 ( 20% etoac / hexanes ) . nmr ( 600 mhz , cdcl3 )  
 6.67 ( s , 1h ) , 6.62 ( m , 2h ) , 4.394.35 ( sep , 1h , j = 6.06 hz ) , 2.15 ( s , 3h ) , 1.291.28 ( d , 6h , j = 6.06 hz ) . c nmr ( 150 mhz , cdcl3 )  
 150.8 , 138.0 , 124.1 , 119.4 , 116.10 , 115.09 , 71.0 , 22.2 , 17.7 . elemental 
 analysis calculated for c10h15no : c , 72.69 ; 
 h , 9.15 ; n , 8.48 . found : c , 72.75 ; h , 9.11 ; n , 8.33 . this compound 
 was prepared from 4d ( 1.92 g , 9.2 mmol ) in a manner similar 
 to that for 5b yielding an amber oil ( 1.48 g , 90% ) : tlc rf 0.28 ( 20% etoac / hexanes ) . nmr ( 600 mhz , cdcl3 )  6.67 ( s , 1h ) , 6.62 
 ( m , 2h ) , 3.893.87 ( t , 2h , j = 6.54 hz ) , 2.16 
 ( s , 3h ) , 1.731.70 ( quin , 2h , j = 6.6 hz ) , 
 1.491.45 ( sex , 2h , j = 7.5 hz ) , 0.970.95 
 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , 
 cdcl3 )  152.4 , 137.8 , 124.2 , 117.24 , 116.13 , 112.9 , 
 68.3 , 31.5 , 19.3 , 17.7 , 13.9 . elemental analysis calculated for c11h17no : c , 73.70 ; h , 9.56 ; n , 7.81 . found : c , 73.55 ; 
 h , 9.51 ; n , 7.96 . compound 5a ( 210 mg , 1.39 
 mmol ) and di - tert - butyl - dicarbonate ( 334 mg , 1.53 
 mmol ) in thf ( 10 ml ) were heated to reflux for 20 h. the reaction 
 mixture was concentrated in vacuo and redissolved in dcm ( 30 ml ) . this mixture was washed with saturated nahco3 ( 30 ml ) and 
 brine ( 30 ml ) . the organic layer was separated and dried over na2so4 to produce a dark - red oil which was purified 
 by chromatography ( 0% to 20% etoac / hexanes ) to yield an orange solid 
 ( 293 mg , 84% ) : mp 6668 c ( lit . h nmr ( 600 mhz , cdcl3 )  7.51 ( s , 1h ) , 6.72 ( m , 2h ) , 6.06 ( s , 1h ) , 4.013.97 
 ( q , 2h , j = 6.96 hz ) , 2.22 ( s , 3h ) , 1.53 ( s , 9h ) , 
 1.401.37 ( t , 3h , j = 6.9 hz ) . c nmr ( 150 mhz , cdcl3 )  155.8 , 153.7 , 146.7 , 129.0 , 
 124.0 , 116.6 , 112.2 , 80.1 , 63.6 , 28.4 , 18.1 , 14.9 . compound 5b ( 2.58 g , 15.6 
 mmol ) and di - tert - butyl dicarbonate ( 5.11 g , 23.4 
 mmol ) in thf ( 75 ml ) were heated to reflux for 24 h. volatiles were 
 evaporated in vacuo , and the residue was purified by chromatography 
 ( 0% to 20% etoac / hexanes ) to yield a light - purple solid ( 4.06 g , 98% ) : 
 mp 7476 c . h nmr ( 600 mhz , cdcl3 )  7.50 ( s , 1h ) , 6.726.70 ( m , 2h ) , 6.06 ( s , 
 1h ) , 3.893.86 ( t , 2h , j = 6.6 hz ) , 2.22 ( s , 
 3h ) , 1.811.75 ( sex , 2h , j = 7.38 hz ) , 1.50 
 ( s , 9h ) , 1.031.00 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , cdcl3 )  156.1 , 153.8 , 131.0 , 
 129.0 , 124.0 , 116.6 , 112.2 , 80.1 , 69.7 , 28.4 , 22.6 , 18.1 , 10.5 . elemental 
 analysis calculated for c15h23no3 : c , 67.90 ; h , 8.74 ; n , 5.28 . found : c , 67.78 ; h , 8.68 ; n , 5.24 . this compound was prepared from 5c in a similar manner to that for 6b except that chromatography 
 used a gradient of 0% to 15% . a white solid was obtained ( 3.78 g , 
 99% ) : mp 5759 c . h nmr ( 600 mhz , cdcl3 ) 
  7.50 ( s , 1h ) , 6.726.70 ( m , 2h ) , 6.06 ( s , 1h ) , 4.504.45 
 ( sep , 1h , j = 6.06 hz ) , 2.21 ( s , 3h ) , 1.59 ( s , 9h ) , 
 1.311.30 ( d , 6h , j = 6.06 hz ) . c nmr ( 150 mhz , cdcl3 )  154.63 , 153.72 , 131.2 , 
 129.0 , 123.9 , 118.2 , 113.7 , 80.1 , 70.1 , 28.4 , 22.1 , 18.0 . elemental 
 analysis calculated for c15h23no3 : c , 67.90 ; h , 8.74 ; n , 5.28 . found : c , 67.97 ; h , 8.76 ; n , 5.24 . compound 5d ( 1.18 g , 6.6 mmol ) 
 and di - tert - butyl dicarbonate ( 1.58 g , 7.24 mmol ) 
 in thf ( 50 ml ) were heated to reflux for 24 h. the solvent was evaporated 
 in vacuo , and the residue was dissolved in etoac ( 50 ml ) and washed 
 with brine ( 50 ml  3 ) . the residue was purified 
 by chromatography ( 020% etoac / hexanes ) to provide a light - orange 
 solid ( 4.06 g , 98% ) : mp 5355 c . h nmr ( 600 mhz , cdcl3 )  7.50 ( s , 1h ) , 6.726.70 
 ( m , 2h ) , 6.06 ( s , 1h ) , 3.933.91 ( t , 2h , j = 6.54 hz ) , 2.22 ( s , 3h ) , 1.761.71 ( quin , 2h , j = 6.6 hz ) , 1.50 ( s , 9h ) , 1.491.44 ( m , 2h ) , 0.980.95 
 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , 
 cdcl3 )  156.0 , 153.7 , 131.1 , 128.9 , 124.0 , 116.5 , 
 112.2 , 80.1 , 67.8 , 31.3 , 28.4 , 19.2 , 18.0 , 13.8 . elemental analysis 
 calculated for c16h25no3 : c , 68.79 ; 
 h , 9.02 ; n , 5.01 . found : c , 68.87 ; h , 8.94 ; n , 5.02 . compound 6a ( 900 mg , 3.58 
 mmol ) was dissolved in thf ( 15 ml ) under 
 an atmosphere of argon . c 
 over 10 min and sec - butyllithium ( 1.4 m , 5.37 ml ) 
 was added slowly as to maintain an internal temperature of < 25 
 c . after reaching 1 equiv of sec - butyllithium 
 ( 2.7 ml ) , the reaction mixture turned a bright yellow signifying deprotonation 
 of the amide nitrogen . the reaction mixture c , and a solution of n - methoxy - n - methylacetylamide 7 ( 442.8 mg , 4.78 mmol ) in thf ( 3 
 ml ) was added over 5 min . the mixture was partitioned between et2o ( 75 ml ) and 0.5 n hcl ( 75 ml ) . the aqueous layer was separated 
 and extracted an additional two times with et2o ( 50 ml ) . the et2o phases were combined and washed with brine ( 75 
 ml ) and then dried over na2so4 to yield a dark - brown 
 oil . tfa ( 3 ml ) 
 was added to the mixture which was then stirred at rt for 48 h. upon 
 completion , the reaction mixture was added to a separatory funnel 
 and washed with nahco3 ( 50 ml ) followed by brine ( 50 ml ) . the organic layer was separated and then dried over na2so4 to provide a crude black oil ( 1 g ) which was purified 
 by chromatography ( 020% etoac / hexanes ) to yield a brown solid 
 ( 335 mg , 53% ) : mp 8890 c . h 
 nmr ( 600 mhz , cdcl3 )  7.72 ( s , 1h ) , 7.167.14 
 ( d , 1h , j = 8.64 hz ) , 6.99 ( d , 1h , j = 2.4 hz ) , 6.776.75 ( dd , 1h , j1 = 8.7 hz , j2 = 2.4 hz ) , 6.13 ( s , 1h ) , 
 4.074.04 ( q , 2h , j = 6.96 hz ) , 2.41 ( s , 3h ) , 
 1.431.40 ( t , 3h , j = 6.96 hz ) . c nmr ( 150 mhz , cdcl3 )  153.3 , 135.8 , 131.2 , 129.5 , 
 111.33 , 110.75 , 103.1 , 100.3 , 64.2 , 15.1 , 13.8 . elemental analysis 
 calculated for c11h13no : c , 75.40 ; h , 7.48 ; 
 n , 7.99 . found : this compound 
 was prepared from 6b ( 1.00 g , 3.77 mmol ) in a manner 
 similar to that for 8a except : after the addition of 
 tfa the mixture was stirred for 24 h at rt then heated at reflux for 
 an additional 2 h to ensure complete boc indole deprotection , and 
 the gradient for chromatography was 015% . a yellow solid was 
 obtained ( 0.28 g , 39% ) : mp 6667 c . h nmr ( 600 
 mhz , cdcl3 )  7.72 ( s , 1h ) , 7.167.14 ( d , 
 1h , j = 8.7 hz ) , 6.99 ( d , 1h , j = 
 2.34 hz ) , 6.776.76 ( dd , 1h , j1 = 8.7 hz , j2 = 2.4 hz ) , 6.13 ( s , 1h ) , 
 3.963.93 ( t , 2h , j = 6.66 hz ) , 2.41 ( s , 3h ) , 
 1.841.78 ( sex , 2h , j = 7.32 hz ) , 1.061.03 
 ( t , 3h , j = 7.44 hz ) . c nmr ( 150 mhz , 
 cdcl3 )  153.5 , 135.8 , 131.1 , 129.5 , 111.3 , 110.7 , 
 103.1 , 100.3 , 70.4 , 22.8 , 13.8 , 10.6 . elemental analysis calculated 
 for c12h15no : c , 76.16 ; h , 7.99 ; n , 7.40 . found : 
 c , 76.13 ; h , 7.90 ; n , 7.36 . this 
 compound was prepared from 6c ( 1.00 g , 3.77 mmol ) in 
 a manner similar to that for 8b to yield a yellow solid 
 ( 0.43 g , 60% ) : mp 6465 c . h 
 nmr ( 600 mhz , cdcl3 )  7.69 ( s , 1h ) , 7.147.13 
 ( d , 1h , j = 8.7 hz ) , 7.01 ( d , 1h , j = 2.28 hz ) , 6.756.74 ( dd , 1h , j1 = 8.64 hz , j2 = 2.34 hz ) , 6.12 ( s , 1h ) , 
 4.504.46 ( sep , 1h , j = 6.06 hz ) , 2.40 ( s , 
 3h ) , 1.331.32 ( d , 6h , j = 6.06 hz ) . c nmr ( 150 mhz , cdcl3 )  152.2 , 135.8 , 131.6 , 
 129.7 , 113.1 , 110.6 , 106.2 , 100.3 , 71.5 , 22.3 , 13.8 . elemental analysis 
 calculated for c12h15no : c , 76.16 ; h , 7.99 ; 
 n , 7.40 . found : c , 76.12 ; h , 7.86 ; n , 7.29 . this compound 
 was prepared from 6d in a manner similar to that for 8a to yield a brown solid ( 0.356 g , 49% ) : mp 5860 
 c . h nmr ( 600 mhz , cdcl3 ) 
  7.72 ( s , 1h ) , 7.167.14 ( d , 1h , j = 
 8.7 hz ) , 6.99 ( d , 1h , j = 2.4 hz ) , 6.776.75 
 ( dd , 1h , j1 = 8.7 hz , j2 = 2.4 hz ) , 6.13 ( s , 1h ) , 4.003.98 ( t , 2h , j = 6.6 hz ) , 2.41 ( s , 3h ) , 1.801.75 ( quin , 2h , j = 6.6 hz ) , 1.541.49 ( sex , 2h , j = 7.5 hz ) , 0.990.96 ( t , 3h , j = 7.44 hz ) . c nmr ( 150 mhz , cdcl3 )  153.6 , 135.8 , 131.1 , 
 129.5 , 111.3 , 110.7 , 103.0 , 100.3 , 68.5 , 31.6 , 19.3 , 13.94 , 13.81 . elemental analysis calculated for c13h17no : 
 c , 76.81 ; h , 8.43 ; n , 6.89 . pocl3 ( 0.5 ml ) was 
 added and the reaction mixture was stirred for 10 min . a solution 
 of compound 8a ( 250 mg , 1.43 mmol ) in dmf ( 3 ml ) was 
 added dropwise over 10 min and then stirred for an additional 40 min 
 while warming to rt . the reaction mixture was added to ice - cold 1 
 n naoh ( 35 ml ) , and the solution was stirred for 30 min in an ice - bath . the resulting precipitate was collected , washed with ice - cold h2o ( 20 ml ) , and dried at 40 c for 24 h in a vacuum desiccator 
 to yield a light - brown solid ( 238 mg , 82% ) : mp 199200 c . h nmr ( 600 mhz , dmso - d6 )  
 11.85 ( s , h ) 10.00 ( s , 1h ) , 7.55 ( d , 1h , j = 2.34 
 hz ) , 7.277.25 ( d , 1h , j = 8.7 hz ) , 6.786.76 
 ( dd , 1h , j1 = 8.7 hz j2 = 2.4 hz ) , 4.033.99 ( q , 2h , j = 6.96 hz ) , 2.64 ( s , 3h ) , 1.351.33 ( t , 3h , j = 6.96 hz ) . c nmr ( 150 mhz , dmso - d6 )  184.5 , 155.2 , 149.0 , 130.5 , 126.8 , 114.1 , 112.71 , 
 112.52 , 103.6 , 63.7 , 15.3 , 12.0 . elemental analysis calculated for 
 c12h13no2 : c , 70.92 ; h , 6.45 ; n , 
 6.89 . found : c , 70.66 ; h , 6.57 ; n , 6.97 . this compound was prepared from 8b ( 263 mg , 1.39 mmol ) 
 in a manner similar to that for 9a except for after the 
 dropwise addition of substituted indole , the solution was stirred 
 for 1 h. a cream - colored powder was obtained ( 228 mg , 75% ) : mp 156158 
 c . h nmr ( 600 mhz , dmso - d6 )  11.85 ( s , 1h ) , 10.00 ( s , 1h ) , 7.55 ( d , 1h , j = 2.46 hz ) , 7.277.25 ( d , 1h , j = 8.7 hz ) , 6.796.77 ( dd , 1h , j1 = 8.7 hz , j2 = 2.52 hz ) , 3.923.90 
 ( t , 2h , j = 6.54 hz ) , 2.64 ( s , 3h ) , 1.771.71 
 ( sex , 2h , j = 7.38 hz ) , 1.000.98 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , dmso - d6 )  184.0 , 154.9 , 148.5 , 130.0 , 126.4 , 
 113.6 , 112.26 , 112.04 , 103.2 , 69.3 , 22.2 , 11.5 , 10.5 . elemental analysis 
 calculated for c13h15no2 : c , 71.87 ; 
 h , 6.96 ; n , 6.45 . found : c , 71.74 ; h , 6.81 ; n , 6.42 . this compound was prepared from 8c ( 347 mg , 
 1.83 mmol ) in a manner similar to that for 9a except 
 for after the dropwise addition of substituted indole , the solution 
 was stirred for 90 min . a cream - colored powder was obtained ( 313 mg , 
 78% ) : mp 179181 c . h nmr ( 600 
 mhz , dmso - d6 )  11.84 ( s , 1h ) , 10.00 
 ( s , 1h ) , 7.56 ( d , 1h , j = 2.4 hz ) , 7.267.25 
 ( d , 1h , j = 8.64 hz ) , 6.776.76 ( dd , 1h , j1 = 8.7 hz , j2 = 
 2.46 hz ) , 4.564.50 ( sep , 1h , j = 6 hz ) , 2.64 
 ( s , 3h ) , 1.271.26 ( d , 6h , j = 6 hz ) . c nmr ( 150 mhz , dmso - d6 )  
 184.0 , 153.4 , 148.6 , 130.1 , 126.4 , 113.58 , 113.56 , 112.0 , 105.5 , 69.9 , 
 22.0 , 11.5 . elemental analysis calculated for c13h15no2 : c , 71.87 ; h , 6.96 ; n , 6.45 . found : c , 71.70 ; 
 h , 6.83 ; n , 6.60 . this compound was prepared from 8d ( 300 mg , 1.48 mmol ) 
 in a manner similar to that for 9b to yield a cream - colored 
 powder ( 320 mg , 94% ) : mp 160161 c . h nmr ( 600 mhz , dmso - d6 )  11.85 
 ( s , 1h ) , 10.00 ( s , 1h ) , 7.55 ( d , 1h , j = 2.46 hz ) , 
 7.267.25 ( d , 1h , j = 8.7 hz ) , 6.796.77 
 ( dd , 1h , j1 = 8.7 hz , j2 = 2.46 hz ) , 3.963.94 ( t , 2h , j = 6.48 hz ) , 2.64 ( s , 3h ) , 1.731.68 ( quin , 2h , j = 6.48 hz ) , 1.491.42 ( sex , 2h , j = 7.38 
 hz ) , 0.950.93 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , dmso - d6 )  
 185.0 , 154.9 , 148.5 , 130.0 , 126.4 , 113.6 , 112.26 , 112.04 , 103.2 , 67.4 , 
 31.0 , 18.8 , 13.8 , 11.5 . elemental analysis calculated for c14h17no2 : c , 72.70 ; h , 7.41 ; n , 6.06 . found : 
 c , 72.87 ; h , 7.47 ; n , 6.13 . 5-amino-2-methylindole 10 ( 200 mg , 
 1.37 mmol ) was dissolved in acetic anhydride ( 25 ml , 2.65 mmol ) , and 
 the solution was heated at 60 c for 20 h. upon completion , the 
 volatiles were evaporated and the oil partitioned between dcm ( 15 
 ml ) and water ( 10 ml ) . the organic layer was separated , dried over 
 na2so4 , and purified by chromatography ( isocratic 
 50% etoac / hexanes ) to yield an orange - brown solid ( 200 mg , 64% ) : mp 
 130134 c . h nmr ( 400 mhz , cdcl3 )  8.12 ( s , 1h ) , 7.26 ( s , 1h ) , 7.257.24 ( d , 
 1h , j = 0.8 hz ) , 6.826.80 ( dd , 1h , j1 = 8.4 hz , j2 = 
 2 hz ) , 6.21 ( s , 1h ) , 2.42 ( s , 3h ) , 2.31 ( s , 6h ) ; c nmr 
 ( 150 mhz , cdcl3 )  174.2 , 137.3 , 135.8 , 130.9 , 129.6 , 
 120.3 , 119.0 , 111.6 , 100.2 , 27.1 , 13.5 . elemental analysis calculated 
 for c13h14n2o2 : c , 67.81 ; 
 h , 6.13 ; n , 12.17 . found : c , 67.73 ; h , 6.16 ; n 11.99 . 5-amino-2-methylindole 10 ( 400 mg , 1.62 mmol ) 
 and di - tert - butyl - dicarbonate ( 712 mg , 3.26 mmol ) 
 were dissolved in ch3cn ( 12 ml ) and stirred at rt for 20 
 h. the volatiles were evaporated in vacuo , the residue was purified 
 by chromatography ( isocratic 50% etoac / hexanes ) , and the product was 
 dried in a vacuum desiccator for 20 h to yield a light - brown solid 
 ( 615 mg , 91% ) : mp 134137 c . h 
 nmr ( 600 mhz , cdcl3 )  7.85 ( s , 1h ) , 7.54 ( s , 1h ) , 
 7.147.13 ( d , 1h , j = 8.4 hz ) , 7.016.99 
 ( d , 1h , j = 7.8 hz ) , 6.44 ( s , 1h ) , 6.13 ( s , 1h ) , 
 2.39 ( s , 3h ) , 1.53 ( s , 9h ) ; c nmr ( 150 mhz , cdcl3 )  153.9 , 136.4 , 133.2 , 130.5 , 129.3 , 114.5 , 110.6 , 
 100.2 , 80.0 , 28.6 , 13.7 . elemental analysis calculated for c14h18n2o2 : c , 68.27 ; h , 7.37 ; n , 11.37 . 
 found : c , 68.30 ; h , 7.39 ; n 11.21 . this compound was prepared from 11 ( 320 mg , 
 1.39 mmol ) in a manner similar to that for 9a except 
 for after dropwise addition of substituted indole the solution was 
 stirred for 2 h to yield an orange precipitate . the sample was further 
 purified by chromatography ( 0% to 10% meoh / dcm ) to yield a white solid 
 ( 222 mg , 74% ) : mp 263 c ( darkening ) . h nmr ( 600 
 mhz , cd3od )  9.99 ( s , 1h ) , 8.18 ( d , 1h , j = 1.8 hz ) , 7.477.45 ( dd , 1h , j1 = 8.4 hz j2 = 1.8 hz ) , 7.307.29 
 ( d , 1h , j = 9 hz ) , 2.70 ( s , 3h ) , 2.14 ( s , 3h ) . c nmr ( 150 mhz , cd3od )  186.2 , 171.6 , 151.4 , 
 135.03 , 134.29 , 127.4 , 118.6 , 115.6 , 113.8 , 112.3 , 23.7 , 11.8 . elemental 
 analysis calculated for c12h12n2o20.15 h2o : c , 65.83 ; h , 5.66 ; n , 12.80 . found : 
 c , 65.75 ; h , 5.59 ; n , 12.42 . this compound was prepared from 12 ( 615 mg , 2.24 mmol ) in a manner similar to that for 9a except for after the dropwise addition of substituted indole the 
 solution was stirred for 4 h to provide an orange precipitate . the 
 precipitate was further purified by chromatography ( isocratic 5% meoh / dcm ) 
 to yield a white solid ( 250 mg , 36% ) : mp 201203 c . h nmr ( 600 mhz , dmso - d6 )  
 9.70 ( s , 1h ) , 8.08 ( s , 1h ) , 7.267.24 ( m , 2h ) , 2.69 ( s , 3h ) , 
 1.52 ( s , 9h ) . c nmr ( 150 mhz , dmso - d6 )  184.4 , 153.4 , 149.4 , 134.5 , 131.6 , 125.9 , 115.6 , 
 113.9 , 111.4 , 110.3 , 79.0 , 28.5 , 11.7 . elemental analysis calculated 
 for c15h18n2o30.125 
 h2o : c , 65.14 ; h , 6.65 ; n , 10.13 . found : c , 64.82 ; h , 6.81 ; 
 n , 10.14 . hrms esi calculated for c15h18n2o3 ( m + h ) 275.1390 ; 
 ( m + na ) 297.1210 . found ( m + h ) 275.1393 ; 
 ( m + na ) 297.1210 . compound 15 ( 200 mg , 1.37 
 mmol ) and di - tert - butyl dicarbonate ( 358 mg , 1.64 
 mmol ) in ch3cn ( 10 ml ) were stirred at rt for 48 h. upon 
 completion , solvents 
 were evaporated in vacuo . the residue was purified by chromatography 
 ( 1050% etoac / hexanes ) and dried at 40 c in a vacuum 
 desiccator for 24 h to yield a colorless oil ( 297 mg , 88% ) : tlc rf 0.69 ( 50% etoac / hexanes ) . h nmr ( 600 mhz , cdcl3 )  8.51 ( s , 1h ) , 7.53 
 ( s , 1h ) , 7.327.31 ( d , 1h , j = 8.4 hz ) , 7.18 
 ( s , 1h ) , 7.127.11 ( d , 1h , j = 7.8 hz ) , 6.50 
 ( s , 1h ) , 4.89 ( s , 1h ) , 4.414.40 ( d , 2h , j = 4.2 hz ) , 1.48 ( s , 9h ) . c nmr ( 150 mhz , cdcl3 )  156.2 , 135.4 , 130.2 , 128.2 , 125.0 , 122.2 , 120.0 , 111.5 , 
 102.6 , 79.5 , 45.5 , 28.7 . elemental analysis calculated for c14h18n2o2 : c , 68.27 ; h , 7.37 ; n , 11.37 . 
 found : c , 68.11 ; h , 7.24 ; n , 11.17 . although we obtained a colorless 
 oil , a previous report notes a light yellow solid with a mp 8689 
 c . this compound was prepared from 16 ( 297 mg , 1.21 mmol ) in a manner similar to that for 9a to yield a cream - colored solid ( 243 mg , 73% ) : mp 126128 
 c . h nmr ( 600 mhz , dmso - d6 )  12.10 ( s , 1h ) , 9.91 ( s , 1h ) , 8.27 ( m , 1h ) , 
 8.00 ( s , 1h ) , 7.457.43 ( m , 2h ) , 7.177.15 ( d , 1h , j = 8.4 hz ) , 4.224.20 ( d , 2h , j = 6 hz ) , 1.40 ( s , 9h ) . c nmr ( 150 mhz , cdcl3 )  184.7 , 155.7 , 138.6 , 136.0 , 134.0 , 124.0 , 123.1 , 119.2 , 
 118.0 , 112.0 , 77.6 , 43.7 , 28.2 . elemental analysis calculated for 
 c15h18n2o3 : c , 65.68 ; 
 h , 6.61 ; n , 10.21 . o , n - dimethylhydroxylamine 
 hydrochloride ( 0.77 g , 7.8 mmol ) and trifluoroacetic anhydride ( 1.5 
 g , 1.0 ml , 7.1 mmol ) in anhydrous dcm ( 25 ml ) were stirred for 15 
 min at 0 c . pyridine ( 1.27 ml , 15.7 mmol ) was added dropwise 
 over 15 min . the reaction was then removed from the ice bath and stirred 
 for 3.5 h at rt . the reaction mixture was washed with 0.5 n hcl ( 50 
 ml  2 ) , saturated nahco3 ( 50 ml ) and brine ( 50 ml ) . bulb - to - bulb distillation was performed on the sample under 
 water aspirator vacuum to obtain a colorless oil ( 578 mg , 52% ) : h nmr ( 600 mhz , cdcl3 )  3.78 ( s , 3h ) , 3.30 
 ( s , 3h ) . c nmr ( 150 mhz , cdcl3 )  156.9 
 ( q , jfc = 39 hz ) , 116.1(q , jfc = 284 hz ) , 62.2 , 32.9 . f nmr ( 376 mhz , cdcl3 )  72.2 ( s , 
 3f ) . elemental analysis calculated for c4h6f3no2 : c , 30.58 ; h , 3.85 ; n , 8.92 . found : c , 30.65 ; 
 h , 4.04 ; n , 8.83 . this compound was prepared from 18 ( 1.0 g , 4.2 mmol ) 
 in a manner similar to that for 8a except weinreb amide 19 ( 726 mg , 4.6 mmol ) was utilized as the acylating agent 
 to yield a yellow powder ( 0.390 g , 43% ) : mp 55 c . h nmr ( 600 mhz , cdcl3 )  8.30 ( s , 1h ) , 7.327.31 
 ( d , 1h , j = 8.9 hz ) , 7.10 ( d , 1h , j = 2.4 hz ) , 7.006.98 ( dd , 1h , j1 = 8.9 hz , j2 = 2.4 hz ) , 6.85 ( s , 1h ) , 
 3.85 ( s , 3h ) . c nmr ( 150 mhz , cdcl3 )  
 154.9 , 131.2 , 127.09 , 126.15 ( q , jfc = 38 hz ) , 121.2 ( q , jfc = 266 hz ) , 115.8 , 112.6 , 104.0 ( q , jfc = 3 hz ) , 102.7 , 55.8 . f nmr ( 376 mhz , 
 cdcl3 )  60.9 ( s , 3f ) . elemental analysis 
 calculated for c10h8f3no 0.04 
 hexanes : c , 56.26 ; h , 3.95 ; n , 6.41 . found : c , 56.63 ; h , 3.95 ; n , 
 6.37 . dmf ( 1 ml ) was cooled to 0 c and 
 pocl3 ( 0.25 ml ) was added and stirred for 10 min . compound 20 ( 336 mg , 1.69 mmol ) in dmf ( 2 ml ) was added dropwise over 
 10 min . the solution was stirred for an additional 1 h warming to rt and then 
 heated to 85 c and allowed to react for an additional 4 h. the 
 solution was poured into ice - cold 1 n naoh ( 40 ml ) and stirred for 
 30 min in an ice - bath resulting in the formation of a precipitate . the precipitate was collected , washed with cold h2o , and 
 dried overnight at 40 c in a vacuum desiccator to yield a cream - colored 
 solid . the sample was further purified by chromatography ( 720% 
 etoac / hexanes ) to obtain a white solid ( 178 mg , 43% ) : mp 236239 
 c . h nmr ( 600 mhz , meoh - d4 )  10.22 ( s , 1h ) , 7.79 ( d , 1h , j = 2.46 hz ) , 7.457.43 ( d , 1h , j = 9 hz ) , 
 7.067.04 ( dd , 1h , j1 = 8.94 hz , j2 = 2.52 hz ) , 3.86 ( s , 3h ) . c nmr 
 ( 150 mhz , meoh - d4 )  186.2 , 159.0 , 
 132.0 , 127.1 , 123.4 , 121.6 , 118.2 , 117.2 , 114.8 , 104.1 , 56.2 . f nmr ( 376 mhz , cdcl3 )  58.7 ( s , 
 3f ) . elemental analysis calculated for c11h8f3no2 : c , 54.33 ; h , 3.32 ; n , 5.76 . found : c , 
 54.53 ; h , 3.32 ; n , 5.76 . methanolic hcl was prepared by bubbling 
 anhydrous hcl ( g ) into anhydrous 
 methanol ( 50 ml ) for 5 min . 5-methoxyindole-2-carboxylic acid 22 ( 2.0 g , 10.5 mmol ) was added to the solution , and the reaction 
 was heated to reflux for 24 h. the mixture was concentrated in vacuo 
 and partitioned between 1 n naoh ( 15 ml ) /saturated nahco3 solution ( 85 ml ) and etoac ( 100 ml ) . the organic layer was separated , 
 washed with brine ( 50 ml ) , and then dried over na2so4 . the resulting crude brown solid was purified by chromatography 
 ( 20% to 75% etoac / hexane ) to produce a light - brown solid ( 1.69 g , 
 78% ) . h nmr ( 600 mhz , meoh - d4 )  7.337.31 ( d , 1h , j = 9 hz ) , 7.08 ( m , 2h ) , 6.936.91 ( dd , 1 h , j1 = 9 hz , j2 = 2.5 
 hz ) , 3.90 ( s , 3h ) , 3.80 ( s , 3 h ) . c nmr ( 150 mhz , meoh - d4 )  164.1 , 156.1 , 134.6 , 129.07 , 128.85 , 
 117.9 , 114.2 , 109.1 , 103.2 , 56.1 , 52.3 . elemental analysis calculated 
 for c11h11no3 : c , 64.38 ; h , 5.40 ; 
 n , 6.83 . found : c , 64.55 ; h , 5.45 ; n ; 6.95 . this compound 
 was prepared in a manner similar to that for 23a except 
 ethanolic hcl was deployed as the reactive solvent 
 to produce a light - brown solid ( 1.74 g , 76% ) : mp 158160 c 
 ( lit . h nmr ( 600 mhz , cdcl3 )  8.88 ( s , 1h ) , 7.327.31 
 ( d , 1h , j = 8.9 hz ) , 7.14 ( m , 1h ) , 7.08 ( d , 1h , j = 2.3 hz ) , 7.016.99 ( dd , 1 h , j1 = 8.9 hz , j2 = 2.5 hz ) , 
 4.424.39 ( q , 2h , j = 7.1 hz ) , 3.85 ( s , 3 
 h ) , 1.421.40 ( t , 3h , j = 7.1 hz ) . c nmr ( 150 mhz , cdcl3 )  161.9 , 154.7 , 132.1 , 127.87 , 
 127.84 , 117.0 , 112.8 , 108.2 , 102.5 , 61.0 , 55.7 , 14.4 . elemental analysis 
 calculated for c12h13no3 : c , 65.74 ; 
 h , 5.98 ; n , 6.39 . found : c , 66.02 ; h , 6.09 ; n ; 6.45 . to a suspension 
 of 22 ( 670 mg , 3.50 mmol ) in anhydrous n - proh ( 20 ml ) was added hcl ( 4 n in dioxane , 2.5 ml ) . the 
 reaction mixture was heated at reflux for 36 h. upon completion , volatiles 
 were evaporated in vacuo , and the resulting oil was purified by chromatography 
 ( 0% to 20% etoac / hexanes ) to produce a white powder ( 721 mg , 88% ) : 
 mp 104107 c . h nmr ( 600 mhz , cdcl3 ) 
  8.82 ( s , 1h ) , 7.327.31 ( d , 1h , j = 
 8.94 hz ) , 7.15 ( m , 1h ) , 7.08 ( d , 1h , j = 2.4 hz ) , 
 7.016.99 ( dd , 1 h , j1 = 8.94 hz , j2 = 2.46 hz ) , 4.324.29 ( t , 2h , j = 6.72 hz ) , 3.85 ( s , 3h ) , 1.841.78 ( sex , 2h , j = 7.38 hz ) , 1.051.03 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , cdcl3 )  
 162.0 , 154.7 , 132.1 , 127.88 , 127.85 , 117.0 , 112.7 , 108.2 , 102.5 , 66.5 , 
 55.7 , 22.2 , 10.5 . elemental analysis calculated for c13h15no3 : c , 66.94 ; h , 6.48 ; n , 6.00 . found : 
 c , 66.87 ; h , 6.44 ; n ; 6.00 . this compound was prepared in a manner 
 similar to that for 23c except anhydrous i - proh was deployed 
 as the reactive solvent , and the mixture was reacted under conditions 
 of reflux for 4 d to produce a white powder ( 415 mg , 53% ) : mp 140144 
 c . h nmr ( 600 mhz , cdcl3 ) 
  8.78 ( s , 1h ) , 7.327.30 ( d , 1h , j = 
 8.94 hz ) , 7.13 ( m , 1h ) , 7.07 ( d , 1h , j = 2.34 hz ) , 
 7.006.98 ( dd , 1 h , j1= 8.94 hz , j2= 2.4 hz ) , 5.305.25 ( sep , 1h , j = 6.3 hz ) , 3.85 ( s , 3 h ) , 1.391.38 ( d , 6h , j = 6.3 hz ) . c nmr ( 150 mhz , cdcl3 )  161.4 , 154.7 , 132.0 , 128.31 , 127.86 , 116.8 , 112.7 , 108.0 , 
 102.5 , 68.5 , 55.7 , 22.0 . elemental analysis calculated for c13h15no3 : c , 66.94 ; h , 6.48 ; n , 6.00 . found : 
 c , 66.80 ; h , 6.42 ; n ; 6.16 . pocl3 ( 0.75 ml , 8 mmol ) and 
 dmf ( 2 
 ml ) were stirred at 0 c for 10 min . to this solution was added 23a ( 516 mg , 2.5 mmol ) in dmf ( 2 ml ) dropwise over 10 min . the reaction was stirred for an additional 30 min , removed from the 
 ice bath , and warmed to rt . the reaction mixture was heated to 90 
 c for 1.5 h and then poured into ice - cold 1 n naoh ( 40 ml ) . the crude precipitate was filtered , washed with ice - cold water ( 25 
 ml ) , and dried at 40 c in a vacuum desiccator for 24 h. the 
 sample was purified by chromatography ( 4070% etoac / hexanes ) 
 to provide a yellow solid ( 265 mg , 45% ) : mp 243246 c . h nmr ( 600 mhz , dmso - d6 )  
 12.82 ( s , 1h ) , 10.58 ( s , 1h ) , 7.697.68 ( d , 1h , j = 2.4 hz ) , 7.477.45 ( d , 1h , j = 9 hz ) , 
 7.057.03 ( dd , 1h , j1 = 9.6 hz , j2 = 2.5 hz , 1h ) , 3.98 ( s , 3h ) , 3.80 ( s , 3h ) . c nmr ( 150 mhz , dmso - d6 )  
 187.6 , 160.6 , 156.6 , 132.1 , 130.9 , 125.6 , 118.18 , 117.28 , 114.2 , 102.3 , 
 55.3 , 52.7 . elemental analysis calculated for c12h11no4 : c , 61.80 ; h , 4.75 , n , 6.01 . found : c , 61.83 ; 
 h , 4.85 ; n , 6.18 . this compound was prepared from 23b ( 553 mg , 2.52 mmol ) in a manner similar to that for 24a except no chromatographic purification was required for 
 the precipitate 
 formed upon quenching with 1 n naoh . a cream - colored solid was obtained 
 ( 580 mg , 93% ) : mp 233237 c ( lit . h nmr ( 600 mhz , dmso - d6 )  12.77 ( s , 1h ) , 10.60 ( s , 1h ) , 7.70 
 ( d , 1h , j = 2.46 hz ) , 7.487.47 ( d , 1h , j = 8.94 hz ) , 7.057.04 ( dd , 1h , j1 = 8.94 hz , j2 = 2.52 hz ) , 
 4.464.43 ( q , 2h , j = 7.08 hz ) , 3.81 ( s , 3h ) , 
 1.411.38 ( t , 3h , j = 7.08 hz ) . c nmr ( 150 mhz , dmso - d6 )  187.4 , 
 160.0 , 156.5 , 132.3 , 130.7 , 125.5 , 118.0 , 117.1 , 114.1 , 102.2 , 61.6 , 
 55.2 , 14.0 . elemental analysis calculated for c13h13no4 : c , 63.15 ; h , 5.30 , n , 5.67 . found : c , 62.93 ; 
 h , 5.40 ; n , 5.76 . this compound was prepared from 23c ( 310 mg , 1.33 mmol ) in a manner similar to that for 24b except after warming to rt the solution was stirred for 
 3 h to yield 
 a cream - colored solid ( 322 mg , 92% ) : mp 198201 c . h nmr ( 600 mhz , dmso - d6 )  
 12.74 ( s , 1h ) , 10.59 ( s , 1h ) , 7.70 ( d , 1h , j = 2.46 
 hz ) , 7.497.47 ( d , 1h , j = 8.94 hz ) , 7.057.03 
 ( dd , 1h , j1 = 8.94 hz , j2 = 2.52 hz ) , 4.374.35 ( t , 2h , j = 6.6 hz ) , 3.80 ( s , 3h ) , 1.821.76 ( sex , 2h , j = 7.38 hz ) , 1.020.99 ( t , 3h , j = 7.38 hz ) . c nmr ( 150 mhz , dmso - d6 )  
 187.9 , 160.7 , 157.1 , 132.80 , 131.33 , 126.1 , 118.59 , 117.72 , 114.7 , 
 102.8 , 67.6 , 55.7 , 22.0 , 10.9 . elemental analysis calculated for c14h15no4 : c , 64.36 ; h , 5.79 , n , 5.36 . 
 found : c , 64.24 ; h , 5.84 ; n , 5.40 . this compound was prepared from 23d ( 248 mg , 1.06 mmol ) in a manner similar to that for 24c to yield a light yellow powder ( 261 mg , 93% ) : mp 201203 
 c . h nmr ( 600 mhz , dmso - d6 )  12.70 ( s , 1h ) , 10.59 ( s , 1h ) , 7.69 ( d , 1h , j = 2.46 hz ) , 7.487.47 ( d , 1h , j = 8.94 hz ) , 7.057.03 ( dd , 1h , j1 = 8.94 hz , j2 = 2.52 hz ) , 5.285.24 
 ( sep , 1h , j = 6.3 hz ) , 3.80 ( s , 3h ) , 1.401.39 
 ( d , 6h , j = 6.3 hz ) . c nmr ( 150 mhz , 
 dmso - d6 )  187.5 , 159.7 , 156.6 , 
 132.7 , 130.8 , 125.6 , 118.04 , 117.20 , 114.2 , 102.3 , 69.7 , 55.3 , 21.6 . elemental analysis calculated for c14h15no40.2 h2o : c , 63.48 ; h , 5.86 , n , 5.29 . found : 
 c , 63.24 ; h , 5.73 ; n , 5.22 . compound 23b ( 2.12 g , 9.67 mmol ) in thf ( 40 
 ml ) was stirred under ar at 0 c for 15 min . 2 m lah in thf ( 3 
 eq , 15 ml ) was added to the solution dropwise . the reaction mixture 
 was stirred at rt for 1 h before the solvent was evaporated in vacuo . the residue was carefully quenched with ice - cold 0.5 n hcl ( 200 ml ) 
 and then extracted with etoac ( 200 ml ) . the resulting crude residue 
 was purified by chromatography ( 30% to 70% etoac / hexanes ) to yield 
 a yellow oil . drying at 40 c in a vacuum desiccator for 24 h 
 produced a yellow solid ( 1.35 g , 79% ) : mp 8588 c ( lit . h nmr ( 600 
 mhz , dmso - d6 )  10.81 ( s , 1h ) , 7.207.18 
 ( d , 1h , j = 8.7 hz ) , 6.95 ( d , 1h , j = 2.4 hz ) , 6.676.65 ( dd , 1h , j1 = 8.7 hz , j2 = 2.46 hz ) , 6.18 ( t , 1h , j = 1.2 hz ) , 5.205.18 ( t , 1h , j = 5.64 hz ) , 4.56- 4.55 ( d , 2h , j = 5.58 hz ) , 3.72 
 ( s , 3h ) . c nmr ( 150 mhz , dmso - d6 )  153.0 , 140.6 , 131.1 , 128.1 , 111.5 , 110.3 , 101.4 , 
 98.3 , 56.8 , 55.1 ; elemental analysis calculated for c10h11no2 : c , 67.78 ; h , 6.26 ; n , 7.90 . found : 
 c , 67.56 ; h , 6.23 ; n , 7.89 . compound 25 ( 3.05 mmol , 540 
 mg ) , triethylamine ( 3.36 
 mmol , 0.47 ml ) , and acetic anhydride ( 3.66 mmol , 0.35 ml ) in ch3cn ( 20 ml ) was stirred at rt for 3 h. the solvent was evaporated 
 in vacuo and partitioned between etoac ( 50 ml ) and saturated nahco3 ( 75 ml ) . the organic layer was separated , washed with brine 
 ( 75 ml ) , and dried over na2so4 . the residue 
 was purified by chromatography ( 1040% etoac / hexanes ) to provide 
 a light - yellow solid ( 600 mg , 90% ) : mp 8788 c . h nmr ( 600 mhz , cdcl3 )  8.47 ( s , 1h ) , 7.247.23 
 ( d , 1h , j = 8.8 hz ) , 7.057.04 ( d , 1h , j = 2.4 hz ) , 6.886.86 ( dd , 1h , j1 = 8.82 hz , j2 = 2.5 hz ) , 
 6.466.45 ( d , 1h , j = 1.6 hz ) , 5.20 ( s , 2h ) , 
 3.84 ( s , 3h ) , 2.10 ( s , 3h ) . c nmr ( 150 mhz , cdcl3 )  172.3 , 154.3 , 133.6 , 131.7 , 127.9 , 113.3 , 111.9 , 
 103.7 , 102.3 , 59.8 , 55.8 , 21.0 . elemental analysis calculated for 
 c12h13no3 : c , 65.74 ; h , 5.98 ; n , 
 6.39 . found : c , 65.91 ; h , 5.86 ; n , 6.37 . pocl3 ( 0.6 ml , 2.28 mmol ) in 
 dmf ( 2 ml ) was 
 stirred for 10 min at 0 c . compound 26 ( 470 mg , 
 2.14 mmol ) in dmf ( 2 ml ) was added dropwise over 10 min . the solution 
 continued to stir at 0 c for an additional 10 min and then warmed 
 to rt , after which it was stirred for an additional 45 min . the mixture 
 was poured into an ice - cold solution of 1 n naoh ( 60 ml ) . the aqueous 
 mixture was extracted with etoac ( 75 ml  2 ) and the latter dried 
 over na2so4 to provide a crude brown solid which 
 was purified by chromatography ( 05% meoh / dcm ) to yield an 
 off - white solid ( 348 mg , 66% ) : mp 195197 c . h nmr ( 600 
 mhz , dmso - d6 )  11.98 ( s , 1h ) , 10.07 
 ( s , 1h ) , 7.57 ( d , 1h , j = 2.64 hz ) , 7.357.33 
 ( d , 1h , j = 8.7 hz ) , 6.836.81 ( dd , 1h , j1 = 8.76 hz , j2 = 
 2.52 hz ) , 5.735.71 ( t , 1h , j = 5.58 hz ) , 
 4.964.95 ( d , 2h , j = 5.52 hz ) , 3.77 ( s , 3h ) . c nmr ( 150 mhz , dmso - d6 )  
 184.1 , 155.6 , 151.2 , 130.2 , 126.4 , 112.80 , 112.52 , 112.34 , 102.3 , 
 55.25 , 55.16 . elemental analysis calculated for c11h11no3 : c , 64.38 ; h , 5.40 ; n , 6.83 . found : c , 64.18 ; 
 h , 5.25 ; n , 6.73 . compound 25 ( 100 mg , 0.56 mmol ) and mno2 ( 490 
 mg , 5.6 mmol ) in etoac ( 5 ml ) were heated to reflux for 24 h. the 
 mixture was filtered over celite , and the filtrate was concentrated 
 in vacuo and purified by chromatography ( 0% to 10% etoac / hexanes ) 
 to yield a pale - yellow powder ( 75 mg , 76% ) : mp 143144 c 
 ( lit . h nmr ( 600 mhz , cdcl3 )  9.81 ( s , 1h ) , 9.05 
 ( s , 1h ) , 7.367.34 ( d , 1h , j = 12 hz ) , 7.207.19 
 ( m , 1h ) , 7.127.11 ( d , 1h , j = 6 hz ) , 7.097.07 
 ( dd , 1h , j1 = 9 hz , j2 = 2.5 hz ) , 3.86 ( s , 3h ) . c nmr ( 150 mhz , 
 cdcl3 )  181.8 , 155.0 , 136.3 , 133.4 , 127.7 , 119.4 , 
 114.15 , 113.36 , 102.8 , 55.7 . elemental analysis calculated for c10h9no2 : c , 68.56 ; h , 5.18 ; n , 8.00 . 
 found : c , 68.42 ; h , 5.34 ; n , 7.85 . to compound 28 ( 603 mg , 3.44 
 mmol ) in thf ( 25 ml ) was added ( carbethoxymethylene)triphenylphosphorane 
 ( 1.8 g , 5.16 mmol ) . the solution was stirred at rt for 20 h. the solvent 
 was evaporated in vacuo , and the residue was dissolved in dcm ( 50 
 ml ) , washed with brine ( 50 ml ) and then dried over na2so4 . the resulting material was purified by chromatography ( 10% 
 to 40% etoac / hexanes ) to provide a pale - yellow powder ( 715 mg , 85% ) : 
 mp 138142 c ( lit . h nmr ( 600 mhz , cdcl3 ) 
  8.35 ( s , 1h ) , 7.677.64 ( d , 1h , j = 
 16.02 hz ) , 7.24 ( s , 1h ) , 7.047.03 ( d , 1h , j = 2.34 hz ) , 6.946.92 ( dd , 1h , j1 = 8.82 hz , j2 = 2.4 hz ) , 6.74 ( d , 1h , j = 1.62 hz ) , 6.226.20 ( d , 1h , j = 16.02 hz ) , 4.304.26 ( q , 2h , j = 7.14 
 hz ) , 3.84 ( s , 3h ) , 1.361.33 ( t , 3h , j = 7.08 
 hz ) . c nmr ( 150 mhz , cdcl3 )  167.3 , 
 154.9 , 134.69 , 134.23 , 133.33 , 129.2 , 115.97 , 115.53 , 112.3 , 108.8 , 
 102.5 , 60.9 , 56.0 , 14.7 . elemental analysis calculated for c14h15no30.15 ch2cl2 : c , 65.87 ; h , 5.98 ; n , 5.43 . found : c , 66.07 ; h , 6.04 ; n , 5.45 . compound 29 ( 631 mg , 2.57 mmol ) was dissolved in thf ( 20 ml ) and stirred 
 at 0 c under argon . 2 m lah in thf ( 3 eq , 15 ml ) was added dropwise . the resulting mixture was warmed to rt for 1 h , and volatiles were 
 evaporated in vacuo . the residue was carefully quenched with ice - cold 
 0.5 n hcl ( 200 ml ) and then extracted with etoac ( 200 ml ) . the organic 
 layer was separated and dried over na2so4 . the 
 resulting crude oil was purified by chromatography ( 30% to 70% etoac / hexanes ) 
 to provide a crude yellow solid , which demonstrated 85% purity 
 based on h nmr . the yellow solid was further reacted 
 by dissolving in etoac ( 10 ml ) and meoh ( 10 ml ) in a glass hydrogenation 
 flask . 10% pd / c ( 58 mg , 10% w / w ) was added and the sample was hydrogenated 
 for 4 h at 35 psi of h2 to ensure complete reduction . the 
 mixture was filtered over celite and purified by chromatography ( 20% 
 to 70% etoac / hexanes ) to yield a light - yellow solid ( 216 mg , 41% ) : 
 mp 8588 c ( lit . h nmr ( 600 mhz , cdcl3 ) 
  8.07 ( s , 1h ) , 7.187.17 ( d , 1h , j = 
 8.7 hz ) , 7.01 ( d , 1h , j = 2.4 hz ) , 6.786.77 
 ( dd , 1h , j1 = 8.7 hz , j2 = 2.46 hz ) , 6.196.18 ( d , 1h , j = 1.98 hz ) , 3.84 ( s , 3h ) , 3.753.73 ( t , 2h , j = 6.06 hz ) , 2.872.85 ( t , 2h , j = 7.2 hz ) , 
 1.971.94 ( quin , 2h , j = 6.12 hz ) . c nmr ( 150 mhz , cdcl3 )  154.2 , 140.0 , 131.2 , 129.3 , 
 111.16 , 111.04 , 102.1 , 99.7 , 62.3 , 56.0 , 31.9 , 24.9 . elemental analysis 
 for c12h15no2 : c , 70.22 ; h , 7.37 ; 
 n , 6.82 . found : c , 70.10 ; h , 7.23 ; n , 6.74 . to compound 30 ( 300 mg , 1.46 
 mmol ) in ch3cn ( 10 ml ) was added tea ( 0.22 ml , 1.61 mmol ) 
 and acetic anhydride 
 ( 0.18 ml , 1.75 mmol ) . the solution was stirred at rt for 20 h. volatiles 
 were evaporated in vacuo , and the residue was dissolved in etoac ( 50 
 ml ) , washed with saturated nahco3 ( 75 ml ) and brine ( 50 
 ml ) . the organic layer was separated , dried over na2so4 , and purified by chromatography ( 0% to 50% etoac / hexanes 
 to yield a pale - yellow solid ( 190 mg , 53% ) : tlc rf 0.18 ( 17% etoac / hexanes ) . h nmr ( 600 mhz , cdcl3 )  7.98 
 ( s , 1h ) , 7.207.19 ( d , 1h , j = 8.7 hz ) , 7.01 
 ( d , 1h , j = 2.46 hz ) , 6.796.77 ( dd , 1h , j1 = 8.7 hz , j2 = 
 2.4 hz ) , 6.19 ( d , 1h , j = 2.1 hz ) , 4.184.16 
 ( t , 2h , j = 6.36 hz ) , 3.84 ( s , 3h ) , 2.822.80 
 ( t , 2h , j = 7.38 hz ) , 2.07 ( s , 3h ) , 2.062.02 
 ( quin , 2h , j = 6.36 hz ) . c nmr ( 150 
 mhz , cdcl3 )  171.6 , 154.4 , 139.4 , 131.2 , 129.4 , 
 111.29 , 111.25 , 102.2 , 100.0 , 63.8 , 56.1 , 28.7 , 24.9 , 21.2 . elemental 
 analysis calculated for c14h17no3 : c , 68.00 ; h , 6.93 ; n , 5.66 . found : c , 67.95 ; h , 6.91 ; n , 5.65 . pocl3 ( 0.17 ml , 1.82 mmol ) and dmf 
 ( 1.5 ml ) stirred at 0 c for 10 min . compound 31 ( 150 mg , 0.61 mmol ) in dmf ( 2 ml ) was added dropwise at 0 c 
 for an additional 10 min . the sample was removed from the ice - bath 
 and reacted for 2 h at rt . the reaction mixture was poured into ice - cold 
 h2o ( 15 ml ) and 1 n naoh was added dropwise until a ph 
 of 10 was determined by ph paper ( 10 ml ) . the flask was refrigerated 
 for 20 min to facilitate the formation of a precipitate . the precipitate 
 was collected , washed with ice - cold h2o ( 20 ml ) and dried 
 at 40 c in a vacuum desiccator for 24 h to provide a cream - colored 
 solid ( 108 mg , 64% ) : mp 170174 c . h nmr ( 600 
 mhz , dmso - d6 )  11.91 ( s , 1h ) , 10.03 
 ( s , 1h ) , 7.58 ( d , j = 2.52 hz , 1h ) , 7.307.29 
 ( d , j = 8.76 hz , 1h ) , 6.826.80 ( dd , j1 = 8.7 hz , j2 = 
 2.52 hz , 1h ) , 4.044.02 ( t , j = 6.42 hz , 2h ) , 
 3.77 ( s , 3h ) , 3.133.10 ( t , j = 7.5 hz , 2h ) , 
 2.062.01 ( quin , j = 6.42 hz , 2h ) , 1.97 ( s , 
 3h ) . c nmr ( 150 mhz , dmso - d6 )  184.0 , 170.4 , 155.5 , 151.3 , 130.2 , 126.3 , 113.5 , 112.26 , 
 112.15 , 102.3 , 63.0 , 55.3 , 28.5 , 22.2 , 20.7 . elemental analysis calculated 
 for c15h17no40.19h2o : c , 64.64 ; h , 6.28 ; n , 5.03 . found : c , 64.25 ; h , 6.49 ; n , 5.04 . the human u251 glioblastoma cell 
 line was obtained from the dct tumor repository ( national cancer institute , 
 fredrick , md ) . cells were maintained in dulbecco s modified 
 eagle medium supplemented with 10% fetal bovine serum , as described 
 previously . the effects of compounds 
 on cell growth were assessed using the sulphorhodamine b ( srb ) colorimetric 
 assay , as described previously . cells were seeded at an initial density of 2000 
 cells per well in 96-well plates , with four replicate wells for each 
 drug concentration . all test compounds were dissolved in dmso and 
 serially diluted in dmso so that the desired final drug concentrations 
 could be achieved by making a 1/1000 dilution into the culture medium . one day after plating , four wells were assayed to establish a predrug 
 ( time-0 ) baseline . at the same time srb assays were performed at a 48 h end point . to minimize 
 the impact of potential variations in stability among the compounds , 
 medium with test compound was replenished after the first 24 h. the 
 concentration of each compound producing 50% growth inhibition ( gi50 ) relative to the control without drug was calculated as 
 described in the nci-60 human cell line screening protocol ( http://dtp.nci.nih.gov/branches/btb/ivclsp.html ) . for acquisition of phase - contrast images 
 of live cells , u251 cells were seeded on 35 mm diameter plastic culture 
 dishes at 10 cells per dish . images were obtained at the indicated 
 time intervals after addition of drug , using an olympus ix70 inverted 
 microscope equipped with a dp-80 digital camera and cellsens 1.9 imaging 
 software ( olympus america , center valley , pa ) . guava cell cycle reagent was purchased 
 from emd millipore corporation , hayward , ca . u251 cells were seeded 
 at 200  000 cells per 35 mm dish 1 day prior to addition of 
 test compounds . cell cycle analyses were performed 24 h after addition 
 of test compounds , using a guava personal cytometer ( emd millipore ) 
 according to the manufacturer s protocol for the guava cell 
 cycle reagent . cytosoft 2.1.4 software was used to analyze the resulting 
 dna histograms and calculate the percentage of cells in each phase 
 of the cell cycle , based on a total of 5000 events per sample . statistical 
 significance of differences between cells treated with specific compounds 
 and control ( dmso - treated ) cells was assessed using student s 
 two tailed t test . u251 cells were seeded 
 on glass coverslips in 60 mm dishes at 350  000 cells per dish . one day after plating , fresh medium was added with compounds at the 
 indicated concentration . cells were fixed with ice - cold methanol , 
 and -tubulin was detected by immunofluorescence microscopy , 
 using a primary mouse monoclonal antibody ( sigma chemical co. , st . louis , mo ) followed by alexa fluor 568-labeled goat antimouse igg 
 ( life technologies , grand island , ny ) . after washing away excess antibodies , 
 nuclear dna was stained for 5 min with 300 nm 4,6-diamidino-2-phenyl - indole 
 ( dapi ) ( sigma chemical co. ) . one day after seeding u251 
 cells at 1.4  10 cells per 10 cm dish , cells were 
 treated for 4 h with the indicated concentration of drugs . at the 
 end of the incubation , the medium was aspirated , and the cells were 
 quickly scraped into 700 l of lysis buffer consisting of 100 
 mm pipes , ph 6.9 , 5 mm mgcl2 , 1 mm egta , 30% glycerol , 
 0.1% np-40 , 0.1% triton x-100 , 0.1% tween-20 , 0.1% 2-mercaptoethanol , 
 2 mm gtp , and protease inhibitor cocktail ( cystoskeleton inc . the lysis buffer was briefly prewarmed to 37 c prior to 
 addition to the cells . 600 l of the lysate was transferred 
 to a prewarmed tube and centrifuged at 100000 g for 
 1 h at 37 c . the resulting supernatant solution contained the 
 nonpolymerized tubulin and the pellet contained the polymerized tubulin . 0.24% of each fraction was separated on a 10% sds - polyacrylamide gel , 
 and tubulin was detected by immunoblot analysis using a mouse monoclonal 
 antibody against -tubulin ( sigma ) using established methods . chemiluminescent signals from the tubulin immunoblots 
 were quantified using an alpha innotech fluorchem hd2 imaging system 
 ( san leandro , ca ) . statistical significance of differences between 
 cells treated with specific compounds and control ( dmso - treated ) cells 
 was assessed using student s two tailed t test .
methuosis is a form of nonapoptotic 
 cell death characterized by 
 an accumulation of macropinosome - derived vacuoles with eventual loss 
 of membrane integrity . 
 small molecules inducing methuosis could offer 
 significant advantages compared to more traditional anticancer drug 
 therapies that typically rely on apoptosis . 
 herein we further define 
 the effects of chemical substitutions at the 2- and 5-indolyl positions 
 on our lead compound 3-(5-methoxy-2-methyl-1h - indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one 
 ( momipp ) . 
 we have identified a number of compounds that induce methuosis 
 at similar potencies , including an interesting analogue having a hydroxypropyl 
 substituent at the 2-position . 
 in addition , we have discovered that 
 certain substitutions on the 2-indolyl position redirect the mode 
 of cytotoxicity from methuosis to microtubule disruption . 
 this switch 
 in activity is associated with an increase in potency as large as 
 2 orders of magnitude . 
 these compounds appear to represent a new class 
 of potent microtubule - active anticancer agents .