cellular resistance 
 to anticancer agents constitutes a major obstacle 
 in the successful chemotherapeutic treatment of cancers with multidrug 
 resistance ( mdr ) being responsible for failure in > 90% of metastatic 
 cancer patients . over the last few decades 
 the notions of tumor heterogeneity and cancer stem cells have transformed 
 our understanding of cancer and chemoresistance . previously , tumors 
 were depicted using the clonal evolution model as a collection of 
 homogeneous cancer cells , with little variation , that had equal potential 
 to initiate and propagate tumorgenesis . however , current research 
 depicts tumors as hierarchically organized with intratumor heterogeneity 
 giving rise to a subclass of cells with increased ability to initiate 
 tumor formation . this subclass of cells , 
 referred to as tumor - initiating cells or cancer stem cells ( cscs ) , 
 was initially reported by bonnet and dick in 1997 and since then has been isolated in various types of cancers 
 including breast cancer , ovarian cancer , acute myeloid leukemia ( aml ) , 
 glioblastoma , and pancreatic cancer , among others . furthermore , it has been shown that cscs play a major role 
 in resistance through a variety of acquired and intrinsic mechanisms 
 that include overexpression of atp - binding cassette ( abc ) efflux transporters 
 and detoxification enzymes , increased ability to repair dna , down - regulation 
 of apoptotic pathways , and changes in the cell cycle kinetics and 
 microenvironment . one of many reported contributing factors to csc resistance and 
 mdr is the overexpression of a class of efflux pumps belonging to 
 the abc superfamily of proteins . resistance is conferred from abc 
 proteins by their ability to specifically efflux chemotherapeutic 
 agents out of cells and may contribute to resistance in cscs . initially , 
 abc transporters , particularly p - glycoprotein ( p - gp , abcb1 ) , emerged as a promising strategy for directly 
 addressing the mechanism of mdr and has been actively pursued for 
 the past 30 years . while several reasons 
 for the clinical failure of this approach have been put forward , an 
 overarching problem of targeting p - gp p - gp is expressed in many tissue types ( e.g. , intestine , kidney , liver , 
 placenta , blood  brain barrier ) and plays an important role 
 in xenobiotic transport . owing to its critical , protective role , a 
 safer strategy would focus on targeting abc transporters upregulated 
 in cscs and mdr cancers but whose inhibition would not prove toxic 
 to the organism . blockade or inhibition 
 of these abc transporters may prove to be novel targets to overcome 
 chemoresistance . the taxanes are a class of chemotherapeutic 
 agents affected by 
 cscs and mdr that are widely used in the treatment of breast cancer . several abc transporters , including p - gp and 
 abcc10 , are known to efflux anticancer agents such as taxanes out 
 of cancer cells . moreover , in vitro studies 
 and a recent in vivo study have shown unambiguously that abcc10 overexpression 
 confers resistance to taxanes . importantly , in recently published 
 work , it was shown that abcc10 is expressed in 100% of her2-positive , 
 85% of her2-negative , and 64% of triple - negative breast cancer tumor 
 samples . furthermore , it was shown that abcc10-null mammary tumors 
 are sensitized to taxanes and that there is a significant increase 
 in survival in abcc10 mice compared to 
 wild - type counterparts following docetaxel treatment . intriguingly , it was also shown that abcc10 affects multiple 
 parameters of breast tumor biology relevant to disease progression , 
 including metastasis , proliferation , and migration . these data and 
 another recently published report support the idea that abc transporters 
 impact tumor biology , which drives other mechanisms of resistance 
 outside of drug efflux , including their 
 roles in cancer initiation and propagation , thus supporting their validity as drug targets . selective , potent 
 inhibitors of abcc10 that possess a lower affinity for p - gp would 
 enable the resensitization of tumors to chemotherapeutic ( e.g. , taxanes ) . currently , there are only a few inhibitors of abcc10 , the most potent 
 inhibitor to date being cepharanthine . moreover , none of these inhibitors 
 are specific for abcc10 over other efflux pumps , such as p - gp ( abcb1 ) . over 75% of chemotherapeutic agents employed kam and co - workers isolated novel 
 pentacyclic strychnos alkaloids alstolucines 
 a  f , from alstonia spatulata ( figure 1 ) . in 
 addition , they were evaluated for their ability to resensitize vincristine - resistant 
 kb cells , which overexpress p - gp . structurally , the alstolucines are 
 closely related to the classical strychnos alkaloid akuammicine ( 1 ) , which we have prepared by 
 total synthesis in both racemic and asymmetric 
 form . we reasoned that these complex 
 pentacyclic strychnos alkaloids , while 
 structurally related , would inhibit abcb1 ( p - gp ) and abcc10 to varying 
 extents , allowing us to obtain valuable structure  activity 
 relationship data and accordingly develop potent and selective inhibitors 
 for the latter over the former . structures of ( )-akuammicine ( 1 ) and novel 
 pentacyclic strychnos alkaloids ( )-alstolucine 
 b ( 2 ) , ( )-alstolucine f ( 3 ) , ( )-n - demethylalstogucine ( 4 ) , and ( )-alstolucine 
 a ( 5 ) . to determine the capacity 
 of these novel alkaloids to reverse abcc10 
 mdr and their affinity for p - gp , we prepared 25 by asymmetric total synthesis from ( )-1 and evaluated their biological activity . specifically , we measured 
 the inhibitory effect of 25 on ( 1 ) 
 abcc10 atpase activity , ( 2 ) p - gp atpase activity , and ( 3 ) the ability 
 of 15 to resensitize abcc10-transfected 
 cell lines to paclitaxel . the 
 asymmetric total syntheses of alkaloids 15 are outlined in scheme 1 . utilizing 
 the methodology of yus and co - workers , n - tosyl indole-3-carboxaldehyde 
 ( 6 ) was condensed with ( r)-n - tert - butanesulfinylamide in the presence of ti(oet)4 and in(0 ) . the addition of allyl 
 bromide and in situ formation of an allylindium species enabled a 
 stereoselective allylation of the preformed n - sulfinimine , 
 which afforded homoallylic sulfinamide 7 in 87% yield 
 ( dr = 10:1 ) . the sequential removal of the auxiliary and n - tosyl group was achieved by treating 7 with 4 m hcl 
 in dioxane followed by mg(0 ) in meoh to give 8 in 75% 
 overall yield . the primary amine was condensed with ethyl glyoxylate 
 and reduced to the primary alcohol using lithium aluminum hydride . the resulting secondary amine was treated with boc anhydride in the 
 presence of hnig s base to furnish 9 in 
 57% yield over two steps . reagents and conditions : ( a ) 
 ( r)-n - tert - butanesulfinamide , 
 in(0 ) , ti(oet)4 and then allyl bromide , thf , 87% ( dr = 
 10:1 ) ; ( b ) 4 m hcl in dioxane then mg(0 ) , meoh , 75% over two steps ; ( c ) ethyl glyoxaldehyde , 4  molecular sieves in thf and then 
 lithium aluminum hydride , thf ; ( d ) ( boc)2o , i - pr2net , 57% over two steps ; ( e ) methyl acrylate , 10 mol 
 % hoveyda  grubbs second - generation catalyst [ 1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(o - isopropoxyphenylmethylene)ruthenium ] , ch2cl2 , 80% ; ( f ) pph3 , dead , rt then dbu , toluene , 80 
 c , 12 h , 56% ; ( g ) tfa , ch2cl2 , quant . ; 
 ( h ) ( z)-2-iodobutenyl bromide , k2co3 , mecn , 71% ; ( i ) pd(oac)2 , pph3 , et3n , 87% ; ( j ) nmo , oso4 , t - buoh / meoh / thf , 
 86% ; ( k ) ncs , dms , et3n , ch2cl2 , 
 66% ; ( l ) ac2o , et3n , dmap , ch2cl2 , 95% ; ( m ) smi2 , thf / meoh , 71% ; ( n ) nabh4 , cecl37h2o , meoh , 83% ; ( o ) etco2cl , et3n , ch2cl2 , 73% ; ( p ) 
 nabh4 , meoh , 85% . cross - metathesis 
 of 9 and methyl acrylate in the presence 
 of 10 mol % hoveyda  grubbs second - generation catalyst gave 
 enoate 10 in 80% yield . inspired 
 by ellman s elegant work on ( )-aurantioclavine , we employed a novel bis - cyclization protocol 
 wherein the pentacyclic e - ring was closed using mitsunobu conditions , 
 followed by treatment with dbu to cyclize the c - ring and access tetracycle 11 in 56% yield . removal of the n - boc group with tfa and subsequent alkylation with ( z)-2-iodobutenyl bromide and potassium 
 carbonate afforded 12 in 71% yield . heck cyclization 
 of 12 using pd(oac)2 , pph3 , and 
 et3n , which was inspired by rawal s elegant approach 
 to the strychnos alkaloids , accessed the d - ring to deliver ( )-akuammicine 
 ( 1 ) in 87% yield ( 10% overall yield in nine steps ) . with 1 in hand , we were tempted 
 to employ an anti - markovnikov 
 hydroboration / carbinol oxidation sequence to access alstolucine b 
 ( 2 ) in two steps from 1 . however , levy and 
 co - workers had shown that the hydroboration of 1 proceeds 
 in a markovnikov manner , affording the tertiary carbinol product . in the light of this , we reasoned that a viable 
 alternative route to 25 would feature 
 ( 1 ) dihydroxylation of the c20c19 double bond , ( 2 ) oxidation 
 of the diol to install the requisite ketone functionality at c19 , 
 and ( 3 ) a deoxygenation of the acyloin intermediate to deliver the 
 alstolucines . toward this end , acetylation of the tertiary c20 
 alcohol using standard conditions furnished acetate 15 in 95% yield . reductive removal of the o - acetate 
 moiety in 15 was realized using molander s method 
 of smi2 in thf at 78 c to afford alstolucine 
 b ( 2 , 44% ) and alstolucine f ( 3 , 27% ) in 
 a 1.6:1 ratio , which were separable by flash column silica gel chromatography . stereoselective luche reduction of alstolucine 
 f ( 3 ) afforded n - demethylalstogucine 
 ( 4 ) in 87% yield . acylation 
 of the newly formed alcohol using clco2et and et3n furnished alstolucine a ( 5 ) in 73% yield . alternatively , 
 the stereoselective reduction of alstolucine b ( 2 ) yielded 
 echitamidine ( 16 ) in 85% . to determine if compounds 25 and 
 intermediate compounds inhibited 
 abcc10 atpase activities , we performed atpase assays using end point 
 inorganic phosphate as previously described . as depicted in figure 2 , we have shown that 
 alstolucines b ( 2 ) and f ( 3 ) inhibited abcc10 
 4050% at 12.5 and 50 m , respectively . all other compounds 
 tested showed modest to no inhibition of abcc10 . the effect of pentacyclic strychnos alkaloids on abcc10 atpase activity . the 
 ability of alstolucines 
 and demethylalstogucine to inhibit abcc10 atpase activity was tested 
 using a range of concentrations of alstolucine a ( 5 ) , 
 alstolucine b ( 2 ) , alstolucine f ( 3 ) , and n - demethylalstogucine ( 4 ) . alstolucine b ( 2 ) and alstolucine f ( 3 ) showed the best inhibition 
 ( 4050% ) at 12.5 and 50 m , respectively . atpase activity assays were run in a similar matter 
 to test inhibition of p - gp as depicted in figure 3 . it was determined that alstolucines a ( 5 ) , 
 b ( 2 ) , and f ( 3 ) do not inhibit p - gp , while n - demethylalstogucine ( 4 ) inhibits p - gp ( 3537% ) 
 at 2.08 and 6.25 m . in comparison to cepharanthine ( i.e. , the 
 best inhibitor of abcc10 to date ) , alstolucines b ( 2 ) 
 and f ( 3 ) are more potent inhibitors in abcc10 atpase 
 assays ( 50% inhibition versus 20% inhibition ) ; however , they selectively 
 inhibit abcc10 over p - gp , unlike cepharanthine and other known inhibitors 
 of abcc10 . the ability of alstolucines 
 and demethylalstogucine to inhibit abcb1 atpase activity was tested 
 using a range of concentrations of alstolucine a ( 5 ) , 
 alstolucine b ( 2 ) , alstolucine f ( 3 ) , and n - demethylalstogucine ( 4 ) . n - demethylalstogucine ( 4 ) showed the only inhibition 
 ( 3537% ) at 2.08 and 6.25 m , respectively . we previously determined the resistance 
 profile for abcc10 using 
 two hek 293 cell lines , which overexpressed abcc10 , and a vector - only 
 transfected line . in this report , we 
 use these previously described cell lines to assess if the alstolucines 
 that inhibited abcc10 in atpase assays inhibited abcc10 in vitro . standard cellular proliferation assays were performed to determine 
 if the alstolucines were able to modulate abcc10 s in vitro 
 resistance capabilities for the taxane paclitaxel . as shown in figure 4 , both alstolucines b ( 2 ) and f ( 3 ) modulated abcc10 s resistance to taxanes . alstolucines 
 b ( 2 ) and f ( 3 ) completely reversed abcc10-mediated 
 resistance to paclitaxel by resensitizing the transfectants to levels 
 similar to the vector - only transfected line . alstolucines inhibit 
 abcc10-mediated paclitaxel resistance : ( a ) 
 alstolucine f ( 3 , red ) , ( b ) alstolucine b ( 2 , blue ) , and ( c ) akuammicine ( 1 , purple ) reverse abcc10-mediated 
 paclitaxel resistance in two abcc10 transfectants ( , ) 
 to levels of nontransfected control cell lines (  ) . ( d ) alstolucine 
 a ( 5 , orange ) does not reverse abcc10-mediated resistance . black 
 lines represent the survival of abcc10 overexpressing cell lines in 
 the absence of inhibitor . we 
 have accomplished the first asymmetric total syntheses of novel 
 pentacyclic strychnos alkaloids ( )-alstolucine 
 b ( 2 ) , f ( 3 ) , and a ( 5 ) and n - demethylalstogucine ( 4 ) in an efficient manner , 
 in addition to ( )-echitamidine ( 16 ) . we have 
 shown that abcc10 plays an important role in modulating taxane resistance 
 both in vitro and in vivo . additionally , overexpression of abcc10 
 confers 310-fold resistance to paclitaxel and 812-fold 
 resistance to docetaxel . results of in vitro and cell - based assays 
 have identified alstolucine a ( 5 ) , alstolucine b ( 2 ) , and alstolucine f ( 3 ) , at 12.5 m , 
 as potent inhibitors of abcc10 atpase activity . importantly , these 
 compounds do not inhibit the function of p - gp atpase activity , like 
 current inhibitors of abcc10 . furthermore , it was found that alstolucine 
 b ( 2 ) and alstolucine f ( 3 ) at 10 m 
 were able to resensitize the abcc10-transfected cell lines to paclitaxel 
 when compared to vector - transfected control lines to levels comparable 
 to or greater than that of cepharanthine , the most potent inhibitor 
 found to date . altogether , these data 
 justify the synthesis and evaluation of alstolucine analogs with increased 
 potency and favorable physicochemical properties . all reactions containing water- or air - sensitive 
 reagents were performed in oven - dried glassware under nitrogen or 
 argon . toluene was passed through one column 
 of neutral alumina and one column of q5 reactant . triethylamine was 
 distilled from calcium hydride prior to use , and 4  molecular 
 sieves were activated by flame - drying under vacuum . ( z)-2-iodobutenyl bromide was prepared according 
 to the procedure of cook . for cross - metathesis 
 reactions flash column chromatography 
 was performed with icn silitech 32 - 63 d 60  silica gel with 
 the indicated solvents . detection was performed 
 using uv light , kmno4 stain , pma stain , and subsequent 
 heating . h and c nmr spectra were recorded 
 at the indicated field strength in cdcl3 at room temperature 
 ( rt ) . the purity of each compound tested was determined on an agilent 
 1200 lc / ms instrument using a kinetex 2.6u c18 column ( 30  2.1 
 mm , with a flow rate of 1 ml / min and detection at 254 nm ) employing 
 a 5100% acetonitrile / water/0.1% formic acid gradient . n - methylmorpholine n - oxide ( 607 
 mg , 5.18 mmol ) was added to 1 ( 334 mg , 1.04 mmol ) . the 
 compounds were dissolved in a mixture of t - buoh / thf / h2o ( 3:2:1 ) . a catalytic amount of oso4 ( 4 wt % solution 
 in h2o , 0.15 ml ) was added dropwise to the solution . the 
 reaction was stirred at rt for 18 h. the reaction was quenched by 
 adding 40% na2so3 solution ( 25 ml ) . the residue was purified by flash 
 column chromatography eluting with meoh / ch2cl2/net3 ( 9:1:0.05 ) to give 316 mg of 13 ( 86% ) 
 as a white foam : [ ]d20 100.5 ( c 0.9 , chcl3 ) ; ir ( neat ) 3355 , 3053 , 2948 , 1669 , 1540 , 1474 , 790 cm ; h nmr ( 400 mhz , cdcl3 )  
 9.10 ( s , 1h ) , 7.18 ( d , j = 7.4 hz , 1h ) , 7.13 ( td , j = 7.7 , 1.2 hz , 1h ) , 6.91 ( td , j = 7.5 , 
 0.9 hz , 1h ) , 6.81 ( d , j = 7.8 hz , 1h ) , 3.86 ( s , 1h ) , 
 3.77 ( s , 3h ) , 3.59 ( q , j = 6.2 hz , 1h ) , 3.153.00 
 ( m , 2h ) , 3.012.80 ( m , 3h ) , 2.722.60 ( m , 1h ) , 2.48 
 ( d , j = 12.7 hz , 1h ) , 1.87 ( dd , j = 13.2 , 6.6 hz , 1h ) , 1.39 ( d , j = 6.2 hz , 3h ) , 
 1.201.13 ( m , 1h ) ; c nmr ( 126 mhz , cdcl3 )  172.17 , 168.21 , 144.04 , 135.51 , 127.79 , 121.38 , 119.90 , 
 109.75 , 100.57 , 73.95 , 72.74 , 60.17 , 56.57 , 54.11 , 52.59 , 51.05 , 42.77 , 
 33.89 , 25.91 , 17.40 ; hrms ( fab ) calcd for c20h24n2o4 + h = 357.1982 , found 357.1808 . to a solution of n - chlorosuccinimide 
 ( 80.4 mg , 0.401 
 mmol ) in ch2cl2 ( 4 ml ) at 20 c 
 was added dimethyl sulfide ( 47 mg , 0.602 mmol ) . the reaction was stirred 
 for 20 min at 20 c , a solution of the diol 13 ( 143 mg , 0.401 mmol ) in dry ch2cl2 ( 4 ml ) 
 was added to the reaction , and the mixture stirred for 1.5 h. at this 
 time , triethylamine ( 81 mg , 0.802 mmol ) was added . the reaction was 
 allowed to warm to rt , while being stirred for 1 h. the reaction was 
 quenched with satd aq nahco3 ( 3 ml ) and diluted with ch2cl2 ( 25 ml ) . the organic layer was washed with 
 satd aq nahco3 ( 10 ml ) and brine ( 10 ml ) . the residue was purified by flash column chromatography eluting 
 with meoh / ch2cl2 ( 0.2:9.8 ) to afford 94 mg of 14 ( 66% ) as a white foam : [ ]d20 610.7 ( c 0.09 , 
 chcl3 ) ; ir ( neat ) 3355 , 3052 , 2948 , 2877 , 1705 , 1675 , 1238 , 
 962 , 748 cm ; h nmr ( 400 mhz , cdcl3 )  8.96 ( s , 1h ) , 7.18 ( d , j = 7.3 
 hz , 1h ) , 7.14 ( td , j = 7.7 , 1.1 hz , 1h ) , 6.91 ( td , j = 7.5 , 0.7 hz , 1h ) , 6.81 ( d , j = 7.7 
 hz , 1h ) , 3.94 ( s , 1h ) , 3.743.69 ( q , 1h ) , 3.68 ( s , 3h ) , 3.13 
 ( d , j = 13.2 hz , 1h ) , 3.07 ( s , 3h ) , 2.81 ( dd , j = 14.8 , 9.0 hz , 2h ) , 2.64 ( d , j = 13.3 
 hz , 1h ) , 2.36 ( s , 3h ) , 1.961.88 ( m , 1h ) , 1.23 ( t , j = 7.0 hz , 1h ) ; c nmr ( 101 mhz , cdcl3 )  204.65 , 170.90 , 167.43 , 144.03 , 142.47 , 127.97 , 121.57 , 
 119.92 , 109.99 , 96.87 , 77.48 , 77.16 , 76.84 , 59.96 , 53.93 , 51.17 , 45.87 , 
 43.30 , 34.17 , 29.75 , 25.70 , 24.48 , 21.66 ; hrms ( fab ) calcd c20h22n2o4 + h = 355.4128 , found 355.1580 . acetic 
 anhydride ( 45 mg , 0.444 mmol ) and triethylamine ( 49 mg , 0.484 
 mmol ) were added to a solution of hydroxyl ketone ( 143 mg , 0.403 mmol ) 
 in dry ch2cl2 ( 3 ml ) . a catalytic amount of 
 dmap ( 4.9 mg , 0.040 mmol ) was added and the reaction was stirred at 
 rt for 4 h. the reaction was quenched with a saturated nahco3 solution ( 5 ml ) and extracted with ch2cl2 ( 3 
  10 ml ) . the combined organic layers were washed with brine 
 ( 10 ml ) , dried over na2so4 , and concentrated 
 under reduced pressure . the residue was purified by flash column chromatography 
 eluting with meoh / ch2cl2 ( 0.1:9.9 ) to afford 
 152 mg of 15 ( 95% ) as a white foam : [ ]d20 487.8 
 ( c 0.1 , chcl3 ) ; ir ( neat ) 3352 , 3053 , 
 2949 , 2879 , 1731 , 1590 , 1474 , 1462 , 1435 , 1240 , 866 , 734 cm ; h nmr ( 500 mhz ) :  9.0 ( s , 1h ) , 7.16 ( d , j = 5.0 hz , 1h ) , 7.147.11 ( m , 1h ) , 6.926.89 
 ( m , 1h ) , 6.80 ( d , j = 10.0 hz , 1h ) , 3.85 ( s , 1h ) , 
 3.68 ( s , 1h ) , 3.30 ( dd , j = 15.5 hz , 1.5 hz , 1h ) , 
 3.22 ( s , 1h ) , 3.092.98 ( m , 3h ) , 2.84 ( m , 1h ) , 2.68 ( td , j = 16.5 hz , 3.0 hz , 1h ) , 2.22 ( s , 3h ) , 2.17 ( s , 3h ) , 1.861.85 
 ( m , 1h ) , 1.281.25 ( m , 1h ) ; c nmr ( 101 mhz , cdcl3 )  204.2 , 172.6 , 170.5 , 167.3 , 144.0 , 135.4 , 127.7 , 
 121.3 , 119.7 , 109.7 , 96.9 , 85.1 , 59.8 , 57.0 , 53.9 , 50.8 , 45.9 , 43.2 , 
 34.2 , 25.7 , 24.3 , 21.4 ; hrms ( fab ) calcd c22h24n2o5 + h = 397.1234 , found 397.1749 . to a round - bottomed flask containing 15 ( 87 mg , 0.219 mmol ) were added deaerated thf ( 7 ml ) and 
 meoh ( 3 ml ) under inert atmosphere . c , and a solution of smi2 ( 10.9 ml , 0.1 m , 1.10 mmol ) , 
 prepared using molander s method , was added slowly . the reaction mixture was stirred at 78 
 c for 30 min , warmed to rt , and stirred overnight . the reaction 
 was quenched with a satd aq solution of na2co3 ( 3 ml ) and extracted with etoac ( 3  10 ml ) . the combined organic 
 phases were washed with brine ( 10 ml ) , concentrated under reduced 
 pressure , and purified by flash column chromatography eluting with 
 meoh / dcm ( 0:10  0.2:9.8 ) to give 33 mg of 2 ( 44% ) 
 as a white foam and 20 mg of 3 ( 27% ) as a yellow oil . h and c nmr spectra were identical with reported 
 literature values ; however , the optical rotation for synthetic 2 was [ ]d20 464 ( c 0.35 , chcl3 ) , 
 whereas the reported value was [ ]d20 515 ( c 1.28 , chcl3 ) . the optical rotation for synthetic 3 was [ ]d20 307 ( c 0.29 , chcl3 ) , 
 whereas the reported value was [ ]d20 371 ( c 0.15 , 
 chcl3 ) . to a solution 
 of 2 ( 20 mg , 0.059 mmol ) in meoh ( 1.5 ml ) at 0 c 
 was added nabh4 ( 3.9 mg , 0.103 mmol ) . the reaction was 
 warmed to rt and stirred for 1 h. the reaction was quenched with a 
 satd aq solution of nahco3 ( 3 ml ) and extracted with ch2cl2 ( 3  10 ml ) . the solution was concentrated 
 under reduced pressure , and the residue was purified by flash column 
 chromatography eluting with meoh / ch2cl2 ( 0.7:9.3 ) . h and c nmr spectra were identical with reported 
 literature values ; however , the optical rotation for synthetic ( )-echitamidine 
 ( 16 ) was [ ]d20 509 ( c 0.10 , chcl3 ) , whereas the reported literature value was [ ]d16 515 ( c 5.0 , etoh ) . to a stirred 
 solution of 3 ( 8 mg , 0.023 mmol ) 
 in meoh ( 1 ml ) was added cerium(iii ) chloride heptahydrate ( 9.6 mg , 
 0.026 mmol ) at rt . after stirring for 10 min , the solution was cooled 
 to 0 c , nabh4 ( 2.1 mg , 0.057 mmol ) was added , and 
 the solution was stirred for 1 h at 0 c . the reaction was quenched 
 with a half - saturated solution of nahco3 ( 1 ml ) and extracted 
 with ethyl acetate ( 3  10 ml ) . the combined organic extractions 
 were washed with brine ( 10 ml ) and dried over na2so4 , and the solvent was concentrated under reduced pressure . the residue was purified by flash column chromatography eluting with 
 meoh / ch2cl2 ( 0.4:9.6  0.6:9.4 ) to give 
 6.7 mg of 4 ( 87% ) . h and c nmr 
 spectra were identical with reported literature values ; however , the 
 optical rotation for synthetic material was [ ]d20 382 ( c 0.10 , chcl3 ) , whereas the literature value 
 was [ ]d20 399 ( c 0.33 , chcl3 ) . triethylamine 
 ( 0.028 ml , 0.295 mmol ) and ethyl chloroformate ( 0.04 ml , 0.295 mmol ) 
 were added to a stirred solution of 4 ( 20 mg , 0.059 mmol ) 
 in ch2cl2 ( 5 ml ) at 0 c . the reaction 
 was warmed to room temperature and stirred for 1 h. the reaction was 
 quenched with saturated nh4cl ( 3 ml ) . the reaction mixture 
 was extracted with ch2cl2 ( 3  10 ml ) and 
 dried over na2so4 . the solvent was concentrated 
 under reduced pressure , and the residue was purified by flash column 
 chromatography eluting with meoh / ch2cl2 ( 0.4:9.6 ) 
 to give 18 mg of 5 ( 73% ) . h and c nmr spectra were identical with reported literature values ; however , 
 the optical rotation of synthetic material was [ ]d20 413 ( c 0.40 , chcl3 ) , whereas the literature value 
 was [ ]d20 438 ( c 0.12 , chcl3 ) . drug sensitivity 
 was analyzed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium inner salt / phenazine methosulfate microtiter 
 plate assay ( cell - titer 96 cell proliferation assay ; promega , madison , 
 wi ) . hek293-pcdna3 and mrp7-transfected cell lines hek293-mrp7-c17 
 and hek293-mrp7-c18 were seeded in triplicate at 5000 cells / well in 
 96-well dishes in dmem containing 10% fetal bovine serum . the following 
 day , drugs were added at various concentrations to the growth medium . growth assays were performed after 72 h of incubation in the presence 
 of drug . paclitaxel was purchased from sigma chemical co. ( st . louis , 
 mo ) . the amount of inorganic phosphate released over 20 min at 
 37 c was measured ; 2 atpase assay buffer ( 100 mm tris - hcl 
 ph 7.5 , 1 m kcl , 0.25 m sodium azide , 0.125 m egta , 1 mm ouabain , 
 1 m dtt ) was combined with 2 m mgcl2 , 510 g 
 of membrane protein , and various drugs or substrates for a 5 min preincubation 
 at 37 c . the reaction was initiated by 5 mm atp addition and 
 quenched with 100 l of 5% sds .
the selective modulation of atp - binding 
 cassette ( abc ) efflux pumps 
 overexpressed in multidrug resistant cancers ( mdr ) and attendant resensitization 
 to chemotherapeutic agents represent a promising strategy for treating 
 cancer . 
 we have synthesized four novel pentacyclic strychnos alkaloids alstolucines b ( 2 ) , f ( 3 ) , and a ( 5 ) and n - demethylalstogucine ( 4 ) , in addition to known strychnos alkaloid echitamidine ( 16 ) , 
 and we evaluated compounds 15 in 
 biochemical assays with abcc10 and p - glycoprotein ( p - gp ) . 
 alstolucines 
 b ( 2 ) and f ( 3 ) inhibited abcc10 atpase 
 activity at 12.5 m without affecting p - gp function ; moreover , 
 they resensitized abcc10-transfected cell lines to paclitaxel at 10 
 m . altogether , 
 the alstolucines represent promising lead candidates 
 in the development of modulators of abcc10 for mdr cancers overexpressing 
 this pump .