apratoxins are potent 
 cytotoxins derived from marine cyanobacteria . because of their biological activity and intriguing 
 structures ,
we recently established that 
 apratoxins prevent cotranslational translocation and thereby downregulate 
 various receptors , including receptor tyrosine kinases ( rtks ) , and 
 inhibit trafficking of other secretory molecules , including growth 
 factors that act on rtks .
rtks such as epidermal 
 growth factor receptors and corresponding ligands such as vascular 
 endothelial growth factor a ( vegf - a ) individually are validated drug 
 targets , which resulted in the approval of small molecules and antibodies 
 against these proteins for colorectal cancer and other cancers . the combined indirect inhibition of both classes 
 of molecules by apratoxins has proven very powerful and an alternative 
 to the specific targeting of selected secretory proteins in cancers 
 that rely on autocrine loops such as 
 colorectal cancer .
apratoxin a was rigorously 
 profiled and shown to possess broad - spectrum yet differential in vitro 
 activity ; however , it also showed irreversible 
 toxicity in vivo and was not well tolerated .
our initial investigation of the structure  activity relationships 
 indicated that the irreversible toxicity may be an off - target effect 
 rather than mechanism - based and may be related to the presence of 
 the michael acceptor in the molecule that might be prone to nonspecific 
 addition of cellular nucleophiles .
indeed , 
 in vitro experiments showed that michael addition at c29 of apratoxin 
 a can occur with thiol - containing compounds such as glutathione , cysteine , 
 and n - acetylcysteine ( supporting 
 information , scheme s1 ) .
unfortunately , 
 the only natural apratoxin without that ,-unsaturated 
 carbonyl system has been apratoxin e , which , however , exerts much 
 reduced activity , presumably due to the dehydration of the tertiary 
 alcohol at c35 . therefore , we designed 
 and synthesized apratoxin s4 ( 1a ) , which represents a 
 hybrid of the natural products apratoxins a and e , containing the 
 hydroxy group as in apratoxin a that is important for potent activity 
 and lacking the michael acceptor as in apratoxin e ( figure 1 ) .
compound 1a showed greater potency and efficacy in a colorectal tumor xenograft 
 model than the parent compound ( apratoxin a ) and was much better tolerated 
 in vivo , providing evidence that the apratoxin scaffold can serve 
 as a new modality to treat cancer but also validating the mechanism 
 of action as a new therapeutic strategy .
natural 
 apratoxins a and e and synthetic apratoxin s4 . in our previous work ,
some fragments 
 of 1a were prepared with modified methods that paralleled 
 those by other groups . to enable the large - scale synthesis of 1a for rigorous preclinical assessment ,
several steps needed to be 
 improved or replaced due to their low efficiency or harsh operation . 

an improved synthesis would also provide easier access to additional 
 apratoxin analogues , which we designed based on earlier observations 
 with the goal of improving metabolic stability and reducing complexity 
 of the molecule . in apratoxin
a , the chiral center at c34 bears the 
 risk of epimerization ; however , the c34 epimer has the same potency 
 as apratoxin a and the same holds true for 1a and 34-epi-1a ( apratoxin 
 s6 ) .
it is also known that the hydroxy 
 group at c35 in apratoxin a is sensitive toward acid - induced dehydration , 
 leading to the double bond between c34 and c35 , and the product has greatly decreased activity , thus presenting a potential major deactivation pathway for 1a as well .
on the basis of the above , it was necessary to 
 explore and investigate analogues at c34 : ( 1 ) nonmethyl - c34 , to remove 
 the chiral center , thereby simplifying the molecule ( apratoxin s7 , 1b ) , and ( 2 ) gem - dimethyl - c34 , to remove 
 the chiral center to simplify the molecule and reduce 
 the possibility to dehydrate to form a conjugated system with the 
 thiazoline ( apratoxin s8 , 1c ) .
both analogues were expected 
 to retain potent activity based on our previous results with 34-epi-1a . to continue 
 our systematic sar investigation in the nonpeptide portions ,
we also 
 aimed to explore the synthesis and activity of 30-epi-1a ( apratoxin s9 , 1d ) . here
we show 
 the details of an improved synthesis of 1a and the synthesis 
 of analogues with different c34 methylation status 
 ( 1b , c ) and configuration of c30 ( 1d ) . during the development of this improved synthesis , several new 
 reactions were applied ,
key reactions were modified to meet different 
 substrate requirements , and many steps were optimized , resulting in 
 an efficient synthesis that enables the generation of large - scale 
 quantities of apratoxins for preclinical evaluation .
we studied the 
 sar by subjecting all new synthetic apratoxins to a multidimensional 
 assay platform , measuring cell growth , receptor tyrosine kinase receptor 
 ( rtk ) levels , and growth factor secretion .
we also investigated the 
 in vitro stability of these compounds and tested the compound with 
 the least propensity to be deactivated via dehydration ( 1c ) for in vivo antitumor efficacy .
the main strategies and procedures in the 
 synthesis of all analogues we designed paralleled those of reported 
 procedures but with notable modifications .
on the basis of the retrosynthetic 
 analysis depicted in scheme 1 , 1a and its analogues 1b  d can be synthesized 
 from two parts : tripeptide 6 and thiazole - containing 
 long chain aliphatic acid 2 .
the key step for the synthesis 
 of each analogue is the formation of the thiazoline ring in 2 from the open - chain precursor 3 .
experiments 
 below showed that different degrees of methylation at c34 or different 
 configuration of c30 affected the ring formation to different extents . 

compound 3 can be disconnected into modified cysteine 5 and proline ester based carboxylic acid 4 , 
 which in turn can be synthesized from aldehyde 7 through 
 a series of known reactions .
scheme 2 details the steps which were improved 
 from published procedures to meet practical large - scale production .
-hydroxy ketone 8 was prepared via d - proline catalyzed aldol reaction 
 of pivalaldehyde with acetone .
after protection with tbs to 9 , reduction with nabh4 to 10 , and 
 elimination through mesylate , 8 was transformed into 
 allyl tbs ether 11 .
the cleavage of the tbs from 11 afforded allyl alcohol 12 , which is the most 
 critical step in the preparation of 12 due to its volatile 
 property .
the solvent ( et2o 
 and thf ) in 12-containing fractions can be removed by 
 distillation using a vigreux fractionation column for small - scale 
 reactions under normal pressure ; however , for large - scale reactions , 
 this was not feasible and we used the combination of cooling - concentration 
 method under ambient reduced pressure ( see supporting 
 information , figure s1 ) and vigreux fraction concentration . 

even though our reaction sequence consisted of more steps to prepare 12 than brown s method using ipc2bome , the latter caused purification problems which 
 we did not encounter utilizing our optimized method .
we found that this is indeed a practical route to the large - scale 
 production of 12 , which we could easily achieve on 10 
 g scale . according to published procedures with 
 minor modifications ,
aldehyde 7 was prepared starting 
 from allyl alcohol 12 through formation of acryloyl ester 13 , grubbs catalyst - effected rcm reaction ( 14 ) , methylation with me2(cucn)li2 ( 15 ) , weinreb amide formation ( 16 ) , protection of hydroxy 
 group with pmb ( 17 ) , and reduction with dibal - h ( 7 ) .
the yield was low ( 1025% ) when we used p - methoxybenzyl bromide ( pmbbr ) to protect the hydroxy group 
 of 16 in the presence of nah / tetra - n - butyl - ammonium iodide ( tbai ) in thf or nah in dmf .
fortunately , 
 we found that the pmboc(nh)ccl3/tfoh method was effective in smoothly converting 16 into pmb ether in moderate yield ( 56% ) .
furthermore , unreacted starting 
 material 16 was recovered quantitatively and could be 
 used in the next cycle .
it is noteworthy that , to shorten the 
 synthetic route , we also 
 tried to apply a grignard reaction to the syn - cyclic 
 sulfate derivative of 8 to afford aldehyde 7 ( supporting information , scheme s2 ) . using this strategy , 8 should be 
 stereoselectively reduced to the syn - diol .
when 
 we performed small - scale reactions ( < 2 g ) , we obtained adequate 
 results ; however , for large - scale reactions ( > 4 g ) , both methods 
 provided 
 unsatisfactory results : low yields , low diastereoselectivity , and 
 most of the product was obtained as an inseparable mixture .
we speculate 
 that an extremely slow rate of addition of reducing reagents is crucial 
 for the success of this reaction .
with aldehyde 7 in hand , we prepared proline ester - based 
 aliphatic acid 4a  c using modified 
 published procedures ( scheme 3 ) .
c34-methyl 4a and c34-nonmethyl 4b were synthesized utilizing 
 a similar strategy ( scheme 3a ) . in our previous 
 work
, we constructed the c34(me)c35(oh ) chiral unit of 19a through roush s crotylation with roush s 
 ( e)-crotylborate at 78 c in toluene following published procedures .
roush s ( e)-crotylborate is not 
 commercially available , it is laborious to purify crude roush s 
 ( e)-crotylborate prepared in the lab by distillation 
 and , if crude product was used without purification , the impurity 
 reduced the yield to as low as 55% .
here we now applied leighton s 
 silanes 18a , b instead of roush s 
 crotylborate to construct the c34c35 units in 19a , b .
silanes 18a and 18b are commercially available and inexpensive ; they are solid 
 and easy to handle .
compound 19a and 19b were obtained smoothly in high yields when 
 aldehyde 7 was treated with 18a and 18b along with sc(otf)3 in ch2cl2 at 0 c , respectively .
compound 19a was 
 converted into troc ester 20a with 2,2,2-trichloroethoxycarbonyl 
 chloride ( troccl ) in the presence of pyridine and dmap .
the troc ester 
 of 19b was not stable and partly dehydrated to form a 
 conjugate double bond , which led to low yield and caused problems 
 with purification .
therefore , we protected hydroxy group 19b with tbs and smoothly obtained the stable tbs ether 20b .
the removal of the pmb group 
 and subsequent esterification with 
 fmoc - pro - oh by yamaguchi s method provided prolyl ester 21a and 21b .
the combination of oxidants , oso4/oxone and 
 naio4 , was effective to oxidize 21a to carboxylic 
 acid 4a ; however , when 21b was exposed to 
 the same conditions , the tbs group was cleaved simultaneously and 
 gave carboxylic acid 4b with a free hydroxy group .
the 
 cleavage of the tbs group probably resulted from the acidic property 
 of oxone which is a triple salt 2khso5khso4k2so4 . both 4a and
the enantioselective chiral borane - mediated aldol reaction developed 
 by kiyooka was used to construct the 
 -hydroxy-,-dimethyl acid part of c34-gem - dimethyl acid 4c ( scheme 3b ) .
treatment of aldehyde 7 with methyl trimethylsilyl 
 dimethylketene acetal at 78 c in the presence of chiral 
 oxazaborolidinone 22 ( derived from d - val ) provided 
 ( s)--hydroxy ester 23 . with 23 in hand , we first tried to protect the hydroxy with the 
 troc group and then hydrolyze the methyl ester with lioh .
however , 
 we found that troco-23 was not compatible to the lioh 
 aq solution during hydrolysis in which a complex mixture including 
 troc - cleaved acid was generated .
we planned to couple 24 with modified cysteine ( mocys ) unit 5a first and then 
 protect the hydroxy group with troc and finally remove pmb to form 
 proline ester .
however , we found the esterification from this reaction 
 sequence provided a very low yield and caused difficulty in the purification . 
 therefore , we adjusted the reaction sequence to esterify with fmoc - pro - oh 
 first and then install the mocys unit .
hydroxy acid 24 was converted into allyl ester 25 with allylbr in the 
 presence of k2co3 in 93% yield .
similarly to 
 the preparation of 21a , b , hydroxy ester 25 was protected with troc and the pmb group was removed followed 
 by esterification with fmoc - pro - oh to provide prolyl ester 27 , which was transformed into the carboxylic acid 4c by 
 treatment with pd catalyst and n - methylaniline . 

the synthesis 
 of modified cysteine unit 5a and its 
 enantiomer 5b was achieved by following published procedures with minor modifications as depicted in scheme 4 . the weinreb amide 28 from s- ( or r- ) n - boc - cys(s - trt)-oh was reduced to amino aldehyde 29 , 
 which was selectively converted into chain - extended modified cysteine 
 ( e)-30 by wittig olefination . during 
 this conversion
, the choice of reductant was of importance because 
 -amino aldehydes are easily racemized .
the method using lithium aluminum hydride ( lialh4 ) in 
 thf at 0 c decreased the racemization rate compared with that 
 using dibal - h in toluene at 78 c in this preparation .
subsequent reduction of the conjugated double bond in 30a and 30b using nabh4 in 95% ethanol afforded 
 saturated compounds 31a and 31b , respectively , 
 but only in 35% yield for both of them . to increase this yield , we also attempted hydrogenation with rhcl(pph3)3/h2 in toluene at 50 c .
this reaction , however , gave a similar yield 
 but also posed problems during the chromatographic purification of 
 the product , which was considered a greater problem .
fortunately , 
 our apratoxin synthesis is convergent and this low yield is still 
 not detrimental because it is not part of the longest linear sequence . 

finally , ethyl esters 31 were transformed into allyl 
 ester 5 through hydrolysis and subsequent alkylation .
using modified reported procedures,3a  d and 2a  d were synthesized 
 as depicted in scheme 5 .
the n - boc groups in 4a  c were selectively 
 removed with tmsotf in the presence of 2,6-lutidine and subsequent 
 coupling of deprotected 4a  c with 5a or 5b gave 3a  d .
first , we screened several coupling reagents for the preparation 
 of 3a ( table 1 , entry 1 ) .
even though 
 hatu did not provide a good yield in the preparation of 3a , the yield was as high as 82% when it was used in the preparation 
 of 3b .
acknowledging 
 that it may be difficult to prepare 3c due to the steric 
 hindrance of -gem - dimethyl 
 in acid 4c , we also screened coupling reagents for its 
 preparation ( table 1 , entry 2 ) : pyaop gave 
 the highest yield of 89% and surprisingly the bep coupling product 
 gave a complex proton nmr as well as low yield while hatu gave an 
 even lower yield of 20% . considering the similarity of 3d to 3a and the problem with bep to obtain 3c ,
we used bop as coupling reagent but only obtained product in moderate 
 yield ( 62% ) ; however , pyaop provided a high yield of 91% ( table 1 , entry 3 ) . with 3a  d in hand
, we synthesized 
 thiazoline - based acids 2a  d ( scheme 5 ) .
the formation of thiazoline ring is the key step 
 in the preparation 2a  d . replicating 
 published procedures using kelly s method ,
ph3p = o / tf2o induced thiazoline 
 ring formation smoothly for 3a , b to give 33a , b in ch2cl2 at 0 c 
 within 30 min . however , for 3d , it took 3 h to consume 
 all starting material and to finally form cyclized compound 33d . for gem - dimethyl 3c
, it 
 was surprising to us that ph3p = o / tf2o 
 was unable to induce thiazoline formation effectively ( table 2 ) , despite extended reaction time or increased reaction 
 temperature .
solely the reaction at 60 c for 24 h gave 29% of 33c ( table 2 , entry 5 ) .
under other 
 conditions , we only detected trace amounts of product ( table 2 , entries 14 ) . then we turned to kelly s 
 ticl4 method using conditions 
 as shown in table 2
. for ticl4 mediated 
 thiazoline formation , at 2540 c in 540 h , only 
 3034% yield was obtained ( entries 67 ) ; however , the 
 yield improved to 72% when the reaction temperature was increased 
 to 60 c ( table 2 , entry 8) .
thiazoline - containing 
 intermediates 33a  d were immediately 
 treated with zn - nh4oac to remove the troc group to avoid 
 elimination of the o - troc group , providing 34a  d in 5590% yield . in the preparation 
 of 34a  b , the percentage of their 
 dehydration products was : dehyd-34b = 15.5% 
  dehyd-34d trace >
the 
 removal of allyl esters of 34a  d using 
 pd(pph3)4/n - methylaniline provided 
 acids 2a  d .
reactions were 
 carried out using 
 ph3p = o ( 8 equiv ) and tf2o ( 4 equiv ) for 
 entries 15 .
ticl4 ( 5 equiv ) for entries 68 
 ( 10 mg scale reactions ) or 2.53.0 equiv ticl4 used 
 for > 30 mg scale reactions ) .
this reaction first was carried 
 out at 25 c for 24 h , when large amounts of starting material 
 were still found by tlc and ms , and then this reaction was heated 
 under reflux for another 16 h. reactions were monitored by ms , 
 the bands of starting material and product on tlc were very close .
products were isolated using 
 preparative 
 tlc plates . with 2a  d in hand , we synthesized 
 the final targets 1a  d ( scheme 6 )
. fmoc - protected tripeptide 6(2a2c ) was treated with et2nh in mecn to liberate the corresponding 
 amine and then coupled with acids 2a  d to provide 36a  d in yields of 7195% . 

pyaop was chosen as the coupling reagent in the preparation of 36a , c , d with acceptable results .
however , for c34-nonmethyl - acid 2b ( table 3 ) , when pyaop or hatu were used as coupling reagents , 
 product 36b was obtained in only low yield ( 1035% ) 
 along with dedydration product ( dehyd-36b ) ( 520% isolated yield ) .
one of the reasons for the low yield 
 of 36b was that starting material 2b was 
 consumed by intramolecular dehydration ( via elimination to form a 
 double bond at c34c35 or intramolecular cyclization to form 
 lactone between cooh and c35-oh of 2b ) .
when we used 
 depbt , a specific reagent for amine coupling , the desired product 36b was obtained in 72% yield and 
 13% of dehyd-36b was isolated , while only 
 trace amount of intramolecular dehydration compound of 2b was detected .
the cleavage of o - allyl ester by pd(pph3)4/n - methylaniline and the removal of 
 fmoc by et2nh / mecn from linear precursors 36a  d gave unmasked reactive precursors , which were 
 then macrocyclized with pyaop or depbt in diluted solution .
subsequent 
 purification by semipreparative hplc afforded final targets 1a  d in yields of 60% , 25% , 70% , and 45% , 
 respectively .
along with 1b and 1d , there 
 were 10% and 5% dehydrated cyclized compounds isolated , respectively . 

the yields of cyclization and final total yields for the longest linear 
 sequence from pivalaldehyde are summarized in table 4 . the most optimized yield ( 5.0% )
the lower yields of 1b and 1d resulted 
 mainly from their dehydration propensity at c35 during the formation 
 of the thiazoline ring and macrocyclization steps .
however , the lower 
 total yield of 1c is due to the lower efficiency of the 
 thiazoline ring formation , presumably as a result of steric hindrance 
 of gem - dimethyl at c34 .
nevertheless , the prevention 
 of dehydration of gem - dimethyl in the formation of 
 the thiazoline ring ( 34c ) and the best yield ( 70% ) during 
 macrocyclization ( 1c ) supported the utility and synthetic 
 accessibility of the gem - dimethyl analogue at c34 . based 
 on the reaction sequence : 
 pivalaldehyde  7  4  3  2  1 .
compounds 1b  d retained potent activity compared 
 with 1a in all biological assays . as hypothesized at 
 the onset of this study and consistent with our previous data that 
 the cytotoxic activity is independent of the configuration of methyl - c34 , 
 we found that the nonmethylated compound ( 1b ) as well 
 as the gem - dimethyl analogue ( 1c ) have 
 similar antiproliferative activity between 1 and 2 nm ( table 5 ) .
this indicates that the complexity of apratoxins 
 by eliminating one chiral center ( c34 ) can be somewhat reduced .
we 
 have previously shown that apratoxins inhibit cotranslational translocation 
 of secretory molecules , including receptors and growth factors , and therefore we measured representative key members of these protein 
 classes .
the low - nanomolar antiproliferative activity is paralleled 
 by a similar potency in reducing levels of met proto - oncogene receptor 
 ( met ) ( figure 2 ) , representative for receptor 
 tyrosine kinases ( rtks ) overexpressed in cancers .
we also measured 
 secretion of the angiogenic vegf - a , which is potently inhibited even 
 at subnanomolar concentrations by 1b and 1c with ic50 values of 300 and 470
we had been unable to predict the biological 
 consequences of inverting the c30 configuration of the thiazoline 
 ring .
interestingly , the 30-epi-1a , 
 compound 1d , showed superior , picomolar ( subnanomolar ) 
 potency in all three assays , outperforming the other apratoxins by 
 23-fold ( table 5 , figure 2 ) . determined after 
 48 h ( n = 4 ) .
sar for synthetic apratoxins by immunoblot analysis 
 for rtk ( met ) 
 levels .
compounds 1a  d were remarkably stable 
 ( t1/2 > 24 h ) under aqueous conditions 
 at physiological ( ph 7.4 ) and lysosomal 
 ph ( 4.88 ) ( figures 3a , b ) and possessed excellent 
 plasma and cellular stability ( figures 3c , d ) . 
 under these conditions ,
the c34c35 dehydrated and hydrated 
 compounds were probably also not interconverting because we also did 
 not observe any hydration at c28c29 of the conjugated system 
 in apratoxin a. we had been concerned about the tendency of apratoxins 
 a , 1a , 1b , and 1d to dehydrate 
 during synthesis as well as upon prolonged exposure to acidic organic 
 solvents ( e.g. , chloroform ) , which could represent 
 a major deactivation pathway for the apratoxins because the dehydrated 
 compounds possess much reduced activity . while an issue during synthesis 
 ( see above ) and in organic solvents , however , this appeared to not 
 be a major concern in our biological in vitro systems .
nevertheless , 1a and 1b were generally somewhat less stable 
 than 1c , as expected ( figure 3 ) , especially under acidic conditions ( figure 3b ) .
the stability of 1d was also slightly enhanced compared 
 with that of 1a , suggesting that the configuration at 
 c30 may affect the tendency to dehydrate ( possibly by changing the 
 macrocycle conformation ) , assuming that dehydration is indeed the 
 mode of primary apratoxin modification . in the synthesis , however , 
 the dehydration propensity at c35 during the formation of the thiazoline 
 ring and macrocyclization steps was slightly greater for 1d compared with 1a .
microsomal metabolism of all apratoxins 
 was strongly accelerated by nadph and stability was found to be low 
 ( t1/2 < 5 min , table 6 ) , which may suggest that certain apratoxin biotransformation 
 products could also retain activity , considering that 1a was extremely potent and active in vivo as well . in vitro stability of apratoxins under various conditions .
apratoxins 
 were incubated as indicated and extracted with ethyl acetate , subjected 
 to lc - ms and monitored by using compound - specific mrm mode with harmine 
 as internal standard .
( d ) cellular stability upon exposure to hct116 protein lysate 
 ( 0.7 mg / ml ) .
mean values are shown  
 sd . even though 1c had slightly lower 
 activity than our other synthetic apratoxins , 
 it was still very potent in vitro and had the potential advantage 
 that it can not dehydrate to form a conjugated system . therefore , we 
 tested 1c for efficacy in the same hct116 xenograft mouse 
 model as previously performed for 1a(2a ) and administered 1c at the previous efficacious 
 dose for 1a ( 0.25
mg / kg , 5 g / mouse ) and 
 a 2.5-fold lower dose ( 0.1 mg / kg , 2 g / mouse ) daily 
 via intraperitoneal ( ip ) injection for 16 days .
there was a clear 
 dose - dependent tumor growth inhibition ( figure 4a ) .
the higher dose strongly retarded the tumor growth , paralleling 
 the effects of 1a .
the lower 
 dose showed a weaker yet pronounced effect ( figure 4a ) ; however , the tumor growth could only be inhibited by about 
 50% using 0.1 mg / kg . it is noteworthy that we did not observe signs 
 of toxicity based on lack of both weight loss and abnormal behavior . 

although vegfr2 expression in hct116 xenografts was found to be low 
 ( consistent with literature data ) , at 
 the higher dose of 1c , there was a detectable reduction 
 in vegfr2 expression levels , indicating target engagement in vivo 
 ( figure 4b ) .
dose - dependent in vivo activity of 1c using a hct116 
 xenograft mouse model .
( a ) subcutaneous tumor - bearing mice were injected 
 daily ip with 1c ( n = 7 ) or vehicle 
 ( n = 6 ) , and
error bars indicate sem ( b ) at the end of the 
 efficacy study , tumors were analyzed by immunoblot analysis for levels 
 of an rtk .
apratoxin 
 s4 ( 1a ) along with its hitherto unknown 
 c30 epimer ( 1d ) and two analogues with achiral c34 ( 1b , c ) were synthesized using improved procedures . 

the innovative points in the synthesis are : ( 1 ) preparation 
 of homoallylic alcohol 12 from its tbs ether directly 
 working up by cooling - concentration , which is critical to the scale 
 up of aldehyde 7 , ( 2 ) leighton s silanes were 
 introduced to efficiently prepare -hydroxy acid 4 , one key segment of composing apratoxins , ( 3 ) lialh4 was 
 used instead of dibal - h to prepare amino aldehyde in the preparation 
 of mocys part to greatly decrease the racemization , ( 4 ) kelly s 
 thiazoline formation methods ( both ph3p = o / tf2o and ticl4 ) were developed to meet different substrate 
 requirements , and ( 5 ) the optimized steps are efficient for the synthesis 
 of 1a and viable to generate 1c and 1d .
as hypothesized , sar studies demonstrated that 1b and 1c retain activity compared with 1a .
unexpectedly , we identified 1d as the most potent 
 apratoxin to date , exhibiting 23-fold improved in vitro activity , 
 even though 1d still has the potential to be deactivated 
 by dehydration .
however , compound 1d was found to be 
 stable during repurifications and stability tests .
the propensity 
 of apratoxins to dehydrate to form a c34c35 double bond is 
 decreased with each methylation at c34 ; this is possibly a reason 
 why the methylene - c34 has not been isolated from cyanobacteria but 
 only the dehydrated form , apratoxin e ( figure 1 ) , which could have been formed enzymatically 
 or nonenzymatically .
while dehydration appears to not be a problem 
 during in vitro incubations and stability studies , it may lead to 
 apratoxin deactivation in vivo so that the corresponding gem - dimethyl compound 1c was further characterized and 
 shown to possess in vivo antitumor efficacy .
the low microsomal stability 
 combined with the potent in vivo activity may suggest that apratoxin 
 biotransformation products could play a role in the overall activity , 
 and future experiments are aimed at characterizing the in vivo biotransformation 
 products .
overall , our study further corroborates that the development 
 of this class of anticancer agents is warranted .
tetrahydrofuran ( thf ) 
 and diethyl ether ( et2o ) were distilled from sodium chips 
 in the presence of a small amount of benzophenone ; ch2cl2 and toluene were distilled from cah2 ; mecn , n , n - dimethylformamide ( dmf ) were dried 
 with 4  molecular sieves ( ms ) and meoh dried with 3  ms ; 
 4 m hydrochloric acid ( hcl ) solution in ethyl acetate was prepared 
 by dissolving hcl gas ( yielding by dropping aqueous hydrochloric acid 
 ( 34% ) to concentrated sulfuric acid ( 98% ) ) to ethyl acetate .
all reactions 
 were performed in heat - gun dried flasks ( 400 c under reduced 
 pressure ) under an inert atmosphere of anhydrous ar unless otherwise 
 noted .
thin layer chromatography was performed on emd silica gel 60  
 f254 glass plates and preparative thin layer chromatography 
 was performed on whatman silica gel 60  f254 glass 
 plates ( layer thick 1000 m ) .
nuclear magnetic 
 resonance ( nmr ) spectra were recorded on a varian mercury 400 mhz , 
 bruker avance ii 600 , bruker avance iii 600 mhz , or agilent vnmr 600 
 mhz spectrometer as indicated in the data list .
chemical shifts for 
 proton nuclear magnetic resonance ( h nmr ) spectra are 
 reported in parts per million relative to the signal residual cdcl3 at 7.26 ppm .
chemicals shifts for carbon nuclear magnetic 
 resonance ( c nmr ) spectra are reported in parts per million 
 relative to the center line of the cdcl3 triplet at 77.16 
 ppm .
the abbreviations s , d , dd , ddd , dddd , t , q , br , and m stand 
 for the resonance multiplicity singlet , doublet , doublet of doublets , 
 doublet of doublet of doublets , doublet of doublet of doublet of doublets , 
 triplet , quartet , broad and multiplet , respectively .
optical rotation 
 was measured on a perkin - elmer 341 polarimeter ( na d line ) using a 
 microcell of 1 dm path length .
high resolution mass spectra ( hrms ) 
 data were obtained using an agilent - lc - tof mass spectrometer with 
 an apci / esi multimode ion source detector .
lr - ms data was obtained 
 using a 3200 qtrap triple quadrupole mass spectrometer and detection 
 by electrospray ionization - ms in the positive ion mode .
the purity 
 ( > 95% ) of all tested compounds was determined by hplc analysis 
 ( shimadzu ) 
 equipped with a phenomenex ultracarb ods column ( 250 mm  10 
 mm , 5 m ) and a shimadzu spd - m20a detector at 200/220 nm wavelength . 

the solution of tetra - n - butylammonium floride trihydrate ( tbaf ) ( 82.07 g , 260.116 
 mmol ) in thf ( 220 ml ) was added to the mixture of compound 11 ( 21.0 g , 86.705 mmol ) and 4  molecular sieves ( 22 g , predried 
 at 450 c under reduced pressure 1 h ) in anhydrous thf ( 300 ml ) 
 at 0 c .
then the reaction mixture was stirred at room temperature 
 overnight and filtered through a small pad of celite ( washed with 
 diethyl ether ) .
the filtrate was quenched with 200 ml of water , extracted 
 with diethyl ether ( 300 ml  3 ) , washed with brine ( 200 ml  
 2 ) , dried over anhydrous mgso4 , concentrated with cooling / condensing 
 fraction distillation system under moderate vacuum , and further concentrated 
 by vigreux fraction distillation column .
the concentrated mixture 
 was purified by column chromatography eluted by 35% diethyl 
 ether in pentane .
the eluted product fractions were also concentrated 
 by cooling / condensing fraction distillation system under moderate 
 vacuum to give product 12 and further distilled by vigreux 
 fraction distillation column to provide product 12 ( 10.5 
 g , 95% ) .
h nmr ( 400 mhz , cdcl3 ) :  5.86 
 ( dddd , j = 14.4 , 10.4 , 8.8 , 6.0 hz , 1h ) , 5.14 ( m , 
 2h ) , 3.25 ( dd , j = 10.4 , 2.0 hz , 1h ) , 2.392.33 
 ( m , 1h ) , 1.98 ( ddd , j = 13.6 , 9.6 , 9.6 hz , 1h ) , 0.91 
 ( s , 9h ) ppm .
c nmr ( 100 mhz , cdcl3 ) :  
 136.7 , 117.9 , 78.2 , 36.7 , 34.7 , 25.9 ppm .
4-methoxybenzyl-2,2,2-trichloroacetimidate ( 3.7 ml , 17.8 
 mmol ) and trifluoromethane sulfonic acid ( tfoh ) ( 7.9 l , 0.089 
 mmol ) was added sequentially to the solution of 16 ( 2.05 
 g , 8.9 mmol ) in thf ( 20 ml ) at 0 c .
the resulting mixture was 
 stirred at room temperature overnight and was then diluted with ethyl 
 acetate ( 20 ml ) , quenched with saturated nahco3 ( 20 ml ) , 
 extracted with ethyl acetate ( 20 ml  3 ) , dried with anhydrous 
 mgso4 , and evaporated in vacuo .
hexane ( 100 ml ) was added 
 to the residue , which resulted in the precipitation of a white solid 
 ( 2,2,2-trichloroacetimidate ) .
the solid was filtered off , and the 
 filtrate was concentrated and purified by column chromatography ( eluted 
 by 2050% ethyl acetate in hexane ) to give product 17 ( 1.74 g , 56% , 100% brsm ) ( recovered starting material 1.08 g and 
 used in next cycle ) . to the solution of 7 ( 234.1 mg , 0.801 mmol ) in ch2cl2 ( 8 
 ml )
was added ( s , s)-trans ez - crotylmix ( mixture of 18a and sc(otf)3 ) ( 943 mg , 1.602 mmol ) at room temperature .
this mixture was 
 stirred vigorously at the same temperature for 2.5 h , and then it 
 was treated with 12 ml of et2o and 12 ml of 1n aq hcl . 
 after this quenched mixture
was stirred at room temperature for 30 
 min , it was filtered off to remove precipitated solid , extracted with 
 et2o ( 15 ml  3 ) , washed with saturated nahco3 ( 20 ml  2 ) and brine ( 20 ml ) , dried with anhydrous 
 mgso4 , evaporated in vacuo , and purified by chromatography 
 column ( eluted by 3.54.5% ethyl acetate in hexane ) to give 
 product 19a ( 245.7 mg , 88% ) as a colorless oil . to the solution of 7 ( 203.3 
 mg , 0.696 mmol ) 
 in ch2cl2 ( 7 ml ) was added ( s , s)-18b ( 772.1 mg , 1.392 mmol ) and 
 sc(otf)3 ( 28.5 mg , 0.058 mmol ) at 0 c .
this mixture 
 was stirred vigorously at the same temperature for 2.0 h , and then 
 it was treated with 12 ml of et2o and 12 ml of 1n aq hcl . 

afterwards , the quenched mixture was stirred at room temperature for 
 30 min , filtered to remove the precipitate , extracted with et2o ( 15 ml  3 ) , washed with saturated nahco3 ( 20 ml  2 ) and brine ( 20 ml ) , dried with anhydrous mgso4 , evaporated in vacuo , and purified by column chromatography 
 ( eluted by 4.2% ethyl acetate in hexane ) to give product 19b ( 197.6 mg , 85% ) as a colorless oil ; [ ]d 57.8 ( c 0.32 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 ) :  7.30 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.4 hz , 2h ) , 
 5.895.79 ( m , 1h ) , 5.175.13 ( m , 2h),4.63 ( d , j = 10.4 hz , 1h ) , 4.52 ( d , j = 10.4 , 1h ) , 
 3.79 ( s , 3h ) , 3.793.74 ( m , 1h ) , 3.11 ( dd , j = 9.2 , 2.4 hz , 1h ) , 2.302.24 ( m , 1h ) , 2.212.14 ( m , 
 1h ) , 2.001.90 ( br m , 1h ) , 1.65 ( ddd , j = 
 13.6 , 10.6 , 2.8 hz , 1h ) , 1.46 ( ddd , j = 14.0 , 8.8 , 
 4.0 hz , 1h ) , 1.35 ( ddd , j = 14.0 , 9.2 , 2.4 hz , 1h ) , 
 1.09 ( ddd , j = 13.6 , 10.4 , 2.0 hz , 1h ) , 0.97 ( d , j = 6.4 hz , 3h ) , 0.94 ( s , 9h ) ppm .
c nmr ( 100 
 mhz , cdcl3 ) :  159.0 , 135.0 , 131.7 , 129.2 , 118.1 , 
 113.7 , 85.3 , 74.3 , 68.4 , 55.3 , 43.5 , 43.3 , 39.8 , 36.2 , 26.6 , 21.1 
 ppm . hrms ( esi ) m / z calcd for c21h34o3na ( m + na ) 357.2400 , 
 found 357.2409 . to the solution of 19b ( 178.6 
 mg , 0.534 mmol ) in ch2cl2 ( 8 ml ) were added 
 2,6-lutidine ( 310 l , 2.672 mmol ) and tert - butyldimethylsilyl 
 trifluoromethanesulfonate ( tbsotf ) ( 368.2 l , 1.603 mmol ) at 
 0 c under ar . after being stirred at the same temperature for 
 1.5 h ,
the reaction was quenched with meoh ( 5 ml ) and saturated aq 
 nh4cl ( 7 ml ) , extracted with ethyl acetate ( 10 ml  
 4 ) , washed with brine ( 10 ml  2 ) , dried with anhydrous mgso4 , evaporated in vacuo , and purified by column chromatography 
 ( eluted by 3.6% ethyl acetate in hexane ) to give product 20b ( 224.4 mg , 94% ) as a colorless oil ; [ ]d 49.0 ( c 0.31 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 ) :  7.28 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.8 hz , 2h ) , 
 5.865.76 ( m , 1h ) , 5.075.03 ( m , 2h ) , 4.65 ( d , j = 10.8 hz , 1h ) , 4.45 ( d , j = 10.8 hz , 
 1h ) , 3.863.80 ( m , 1h ) , 3.80 ( s , 3h ) , 3.08 ( dd , j = 7.2 , 3.6 hz , 1h ) , 2.312.20 ( m , 2h ) , 1.87 ( br m , 1h ) , 1.66 
 ( ddd , j = 13.2 , 9.2 , 2.8 hz , 1h),1.461.34 
 ( m , 2h ) , 1.06 ( d , j = 6.8 hz , 3h ) , 0.91 ( s , 9h ) , 
 0.86 ( s , 9h ) ppm .
c nmr ( 100 mhz , cdcl3 ) : 
  159.0 , 135.2 , 131.9 , 128.9 , 117.0 , 113.7 , 85.6 , 73.9 , 69.8 , 
 55.4 , 43.9 , 43.4 , 40.5 , 36.2 , 26.8 , 26.6 , 26.1 , 21.0 , 18.2 , 4.1 , 
 4.3 ppm . hrms ( esi ) m / z calcd 
 for c27h48o3sina ( m + na ) 471.3265 , found 471.3264 . to a solution 
 of 20 ( 0.806 mmol ) in the mixture of ch2cl2 ( 5.0 ml ) and h2o ( 0.5 ml )
was added 2,3-dichloro-5,6-dibenzoquinone 
 ( ddq ) ( 220 mg , 0.968 mmol ) at 0 c .
the reaction mixture was 
 stirred at the same temperature for 1 h , quenched with saturated aqueous 
 nahco3 ( 10 ml ) , and filtered in vacuo .
the organic layer 
 was separated and the water layer was extracted with ch2cl2 ( 30 ml  3 ) .
the organic phase was combined and 
 washed with brine ( 20 ml  2 ) , dried with anhydrous mgso4 , filtered , and concentrated in vacuo .
to the suspended 
 solution of fmoc - pro - oh ( 549.5 mg , 1.629 mmol ) in toluene ( 5.0 ml ) 
 were added n , n - diisopropylethylamine 
 ( diea ) ( 435 l , 2.5 mmol ) and 2,4,6-trichlorobenzoyl chloride 
 ( 390 l , 2.5 mmol ) at room temperature under argon , and the 
 mixture was stirred at the same temperature for 30 min
. then the crude 
 alcohol in toluene ( 3.0 ml ) and dmap ( 350 mg , 2.862 mmol ) were added 
 to the above mixture at 10 c . after being stirred at room temperature 
 for 3 h ,
the reaction mixture was quenched and extracted with diethyl 
 ether ( 10 ml  3 ) .
the combined organic layer was washed with 
 saturated nh4cl ( 15 ml  2 ) , saturated nahco3 ( 15 ml  2 ) , and brine ( 15 ml ) , dried over mgso4 , and concentrated in vacuo .
the residue was purified by silica gel 
 column chromatography ( eluted by 10% ethyl acetate in hexane ) to give 
 ester 21 .
h nmr ( 400 mhz , cdcl3 , mixture 
 of rotamers ) :  7.787.75 ( m,2 h ) , 7.677.55 ( m , 
 2h ) , 7.427.38 ( m , 2h ) , 7.337.30 ( m , 2h ) , 5.855.71 
 ( m , 1h ) , 5.054.99 ( m , 2h ) , 4.80 ( dd , j = 
 8.4 , 2.0 hz , 1h ) , 4.534.40 ( m , 2h ) , 4.354.17 ( m , 2h ) , 
 3.813.71 ( m , 1h ) , 3.703.62 ( m , 1h ) , 3.603.50 
 ( m , 1h ) , 2.322.15 ( m , 3h ) , 2.141.93 ( m , 3h ) , 1.621.49 
 ( m , 3h ) , 1.391.30 ( m , 1h ) , 1.201.03 ( m , 1h ) , 0.95 
 ( d , j = 6.4 hz , 2h ) , 0.87 ( s , 9 h ) , 0.87 ( s , 9 h ) , 
 0.82 ( d , j = 6.4 hz , 1h ) , 0.050.02 ( m , 6h ) 
 ppm .
c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 
  172.2 , 172.1 , 154.7 , 154.5 , 144.3 , 144.2 , 144.1 , 143.8 , 141.4 , 
 141.4 , 141.3 , 135.6 , 135.3 , 127.8 , 127.8 , 127.2 , 127.1 , 127.1 , 125.5 , 
 125.3 , 125.2 , 120.0 , 116.9 , 116.8 , 80.6 , 80.4 , 77.5 , 77.2 , 76.8 , 70.2 , 
 70.0 , 67.8 , 67.5 , 59.8 , 59.6 , 47.4 , 47.0 , 46.4 , 44.2 , 44.0 , 42.6 , 
 42.5 , 39.3 , 39.1 , 35.2 , 35.1 , 31.3 , 30.1 , 27.1 , 27.0 , 26.1 , 26.0 , 
 26.0 , 24.4 , 23.4 , 20.9 , 20.6 , 18.2 ppm . hrms ( esi ) m / z calcd for c39h57no5sina ( m + na ) 670.3898 , found 670.3920 . to the solution 
 of 21 ( 0.646 mmol ) in dmf ( 6.0 ml ) were added oxone ( 1.588 
 g , 2.583 mmol ) , nahco3 ( 217.0 mg , 2.583 mmol ) , and oso4 ( 2.5% solution in tert - buoh ) ( 81 l , 
 6.5 mol ) at room temperature . after being stirred at the same 
 temperature for 15 h
, the reaction mixture was diluted with water 
 ( 4 ml ) and tert - buoh ( 7.5 ml ) , and then naio4 ( 276.3 mg , 1.292 mmol ) was added .
the reaction mixture was 
 stirred at room temperature for an additional 5 h and poured into 
 aqueous hcl ( 1 m for 4a , 0.5 m for 4b to 
 ph 1 ; 25 ml ) and ch2cl2 ( 25 ml ) .
the water layer 
 was extracted with ch2cl2 ( 50 ml  3 ) . 

the combined ch2cl2 layer was washed with 10 
 wt % na2s2o3 ( 50 ml  3 ) and 
 brine ( 50 ml  1 ) , dried over mgso4 , and concentrated 
 in vacuo .
the residue was purified by silica gel column chromatography 
 to give product 4 as a white solid ( 4a eluted 
 by 1725% ethyl acetate in hexane ; 4b eluted by 
 2.510% meoh in ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 , mixture of rotamers , major ) :  
 7.75 ( d , j = 7.6 hz , 2h ) , 7.627.55 ( m , 2h ) , 
 7.39 ( dd , j = 7.6 , 7.4 hz , 2h ) , 7.337.29 
 ( m , 2h ) , 5.40 ( br , 1h ) , 4.91 ( d , j = 10.8 hz , 1h ) , 
 4.424.38 ( m , 1h ) , 4.344.29 ( m , 1h ) , 4.124.06 
 ( m , 1h ) , 3.683.62 ( m , 1h ) , 3.523.47 ( m , 1h ) , 2.442.43 
 ( m , 2h ) , 2.332.23 ( m , 1h ) , 2.081.91 ( m , 3h ) , 1.861.80 
 ( m , 1h ) , 1.75 ( dd , j = 13.6 , 2.4 hz , 1h ) , 1.66 ( dd , j = 14.4 , 2.0 hz , 1h ) , 1.331.26 ( m , 1h ) , 1.040.98 
 ( m , 1h ) , 0.98 ( d , j = 6.4 hz , 3h ) , 0.89 ( s , 9h ) ppm .
c nmr ( 100 mhz , cdcl3 , mixture of rotamers , major ) :  
 174.6 , 172.4,155.4 , 144.0 , 143.7 , 127.9 , 127.8 , 127.3 , 127.2 , 125.3 , 
 125.2 , 120.1 , 120.1 , 78.5 , 68.0 , 66.0 , 59.6 , 47.2 , 46.7 , 42.7 , 41.7 , 
 37.2 , 34.8 , 30.0 , 26.1 , 25.0 , 24.6 , 20.6 ppm . hrms
( esi ) m / z calcd for c32h41no7na ( m + na ) 574.2775 , found 574.2970 . to the solution of n - tosyl - d - val - oh ( 145.6 mg , 0.537 mmol ) in ch2cl2 ( 7 ml ) at 0 c under argon
was added the solution of bh3 in thf ( 1 m , 537 l , 0.537 mmol ) dropwise .
the resulting 
 mixture was stirred at room temperature for 20 min and then cooled 
 to 78 c .
aldehyde 7 ( 157.0 mg , 0.537 mmol ) 
 and methyl trimethylsilyl dimethylketene acetal ( 120.0 l , 0.591 
 mmol ) were added successively to the above mixture at 78 c . 
 after being stirred at 78 c for 3 h ,
the reaction mixture 
 was quenched with buffer ( ph 7 ; 8 ml ) and then it was warmed to room 
 temperature and another 5 ml of buffer ( ph 7 ) was added .
the quenched 
 reaction mixture was extracted with et2o ( 25 ml  
 3 ) , washed with saturated nahco3 ( 15 ml  2 ) and brine 
 ( 15 ml  2 ) , dried with anhydrous mgso4 , and purified 
 by column chromatography ( eluted by 8.5% ethyl acetate in hexane ) 
 to give product 23 as a colorless oil ( 171.5 mg , 81% ) ; 
 [ ]d 55.8 ( c 0.12 , ch2cl2 ) .
h nmr ( 400 mhz , 
 cdcl3 ) :  7.29 ( d , j = 8.4 hz , 2h ) , 
 6.85 ( d , j = 8.4 hz , 2h ) , 4.63 ( d , j = 10.4 hz , 1h ) , 4.50 ( d , j = 10.4 hz , 1h ) , 3.77 
 ( s , 3h ) , 3.74 ( br m , 1h ) , 3.69 ( s , 3h ) , 3.09 ( dd , j = 9.2 , 2.4 hz , 1h ) , 2.47 ( br , 1h ) , 2.031.95 ( br m , 1h ) , 
 1.581.45 ( m , 2h ) , 1.371.30 ( m , 1h ) , 1.18 ( s , 6h ) , 
 1.031.00 ( m , 1h ) , 0.96 ( d , j = 6.4 hz , 3h ) , 
 0.93 ( s , 9h ) ppm .
c nmr ( 100 mhz , cdcl3 ) : 
  178.3 , 159.0 , 131.6 , 129.2 , 113.7 , 85.2 , 74.4 , 74.2 , 55.3 , 
 51.9 , 47.3 , 39.7 , 38.2 , 36.2 , 26.8 , 26.6 , 22.1 , 20.9 , 20.6 ppm .
hrms 
 ( esi ) m / z calcd for c23h38o5na ( m + na ) 417.2611 , found 
 417.2628 . to the solution of 23 ( 223.8 mg , 0.568 mmol ) in the mixture of thf 
meoh  h2o ( 7 ml3.5 ml0.7 ml ) was added aq lioh ( 119.1 
 mg in 2.8 ml h2o , 2.838 mmol ) . after being stirred at room 
 temperature for 5 h
, the reaction mixture was diluted with 10 ml of 
 water , acidified by addition of aq hcl ( 2 m ) to ph 2 , extracted with 
 ethyl acetate ( 10 ml  4 ) , washed with brine ( 10 ml  2 ) , 
 dried with anhydrous mgso4 , and purified by column chromatography 
 ( eluted by 8.517% acetone in hexane ) to give 24 ( 225 mg , 100% ) as a white solid ; [ ]d 52.3 ( c 0.26 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 ) :  7.30 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.4 hz , 2h ) , 
 4.63 ( d , j = 10.4 hz , 1h ) , 4.53 ( d , j = 10.4 hz , 1h ) , 3.80 ( d , j = 13.0 , 1h ) , 3.78 ( s , 
 3h),3.13 ( dd , j = 8.0 , 2.0 hz , 1h ) , 1.80 ( br m , 1h ) , 
 1.621.49 ( m , 2h ) , 1.401.34 ( m , 1h ) , 1.21 ( s , 3h ) , 
 1.19 ( s , 3h ) , 1.09 ( dd , j = 12.4 , 12.4 ) , 0.98 ( d , j = 6.4 hz , 3h ) , 0.95 ( s , 9h ) ppm .
c nmr ( 100 
 mhz , cdcl3 ) :  182.9 , 159.0 , 131.4 , 129.3 , 113.7 , 
 85.2 , 74.3 , 74.3 , 55.3 , 47.1 , 39.4 , 37.9 , 36.2 , 26.8 , 26.6 , 22.3 , 
 20.8 , 20.2 ppm .
hrms ( esi ) m / z calcd 
 for c22h36o5na ( m + na ) 403.2455 , found 403.2472 . to the solution of compound 24
( 222.4 mg , 0.585 mmol ) in dimethyl sulfoxide ( dmso ) ( 5.0 ml ) were 
 added k2co3 ( 242.5 mg , 1.754 mmol ) and allyl 
 bromide ( allylbr ) ( 99 l , 1.170 mmol ) at room temperature .
after 
 being stirred at the same temperature overnight , the reaction was 
 quenched with water ( 10 ml ) and extracted with ethyl acetate ( 20 ml 
  4 ) .
the combined organic layer was washed with brine ( 10 ml 
  5 ) , filtered , concentrated in vacuo , and purified by silica 
 gel column chromatography ( eluted by 6.5% ethyl acetate in hexane ) 
 to give product 25 ( 228.6 mg , 93% ) as a colorless oil ; 
 [ ]d 56.0 ( c 0.175 , ch2cl2 ) .
h nmr ( 400 mhz , 
 cdcl3 ) :  7.30 ( d , j = 8.4 hz , 2h ) , 
 6.85 ( d , j = 8.4 hz , 2h ) , 5.965.86 ( m , 1h ) , 
 5.355.22 ( m , 2h ) , 4.64 ( d , j = 10.4 hz , 1h ) , 
 4.60 ( d , j = 5.6 hz , 2h ) , 4.50 ( d , j = 10.4 hz , 1h ) , 3.78 ( br m , 1h ) , 3.77 ( s , 3h ) , 3.10 ( dd , j = 8.0 , 2.0 hz , 1h ) , 2.54 ( br , 1h ) , 2.0 ( br m , 1h ) , 1.611.55 
 ( m , h ) , 1.49 ( ddd , j = 17.6 , 9.6 , 4.0 hz , 1h ) , 1.371.31 
 ( m , 1h ) , 1.21 ( s , 6h ) , 1.050.98 ( m , 1h ) , 0.97 ( d , j = 6.8 hz , 3h ) , 0.94 ( s , 9h ) ppm .
c nmr ( 100 
 mhz , cdcl3 ) :  177.3 , 158.9 , 132.0 , 131.5 , 129.1 , 
 118.2 , 113.6 , 85.0 , 74.3 , 74.2 , 65.2 , 55.2 , 47.3 , 39.7 , 38.1 , 36.1 , 
 26.6 , 26.5 , 21.9 , 20.9 , 20.7 ppm . hrms ( esi ) m / z calcd for c25h40o5na 
 ( m + na ) 443.2768 , found 443.2769 . to the solution of 25 ( 220.0 

mg , 0.523 mmol ) in ch2cl2 ( 5.0 ml ) at 0 c 
 under ar were added 4-dimethylaminopyridine ( dmap ) ( 1.3 mg , 10.5 mol ) , 
 pyridine ( 423 l , 5.234 mmol ) , and 2,2,2-trichloroethoxylcarbonyl 
 chloride ( troc - cl ) ( 554.5 mg/360.0 l , 2.617 mmol ) . after being 
 stirred at 0 c for 10 min and room temperature for 1 h
, the 
 reaction was quenched with water ( 10 ml ) and extracted with ethyl 
 acetate ( 15 ml  4 ) .
the combined organic layer was washed with 
 saturated nahco3 and brine , dried with mgso4 , concentrated in vacuo , and purified by silica gel column chromatography 
 ( 2.51015% ethyl acetate in hexane ) to give 26 ( 295.1 mg , 95.0% ) ; [ ]d 7.6 
 ( c 0.37 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 ) :  7.27 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.4 hz , 2h ) , 5.965.86 
 ( m , 1h ) , 5.375.23 ( m , 3h ) , 4.78 ( d , j = 12.0 
 hz , 1h ) , 4.59 ( d , j = 5.6 hz , 2h ) , 4.55 ( d , j = 10.8 hz , 1h ) , 4.49 ( d , j = 10.8 hz , 
 1h ) , 4.30 ( d , j = 12.0 hz , 1h ) , 3.78 ( s , 3h ) , 3.05 
 ( dd , j = 9.6 , 2.0 hz , 1h),1.991.93 ( m , 1h ) , 
 1.75 ( br m , 1h ) , 1.50 ( ddd , j = 17.2 , 9.2 , 3.0 hz , 
 1h ) , 1.351.29 ( m , 1h ) , 1.27 ( s , 3h ) , 1.25 ( s , 3h ) , 1.081.01 
 ( m , 4h ) , 0.92 ( s , 9h ) ppm .
c nmr ( 100 mhz , cdcl3 ) :  174.8 , 159.0 , 154.4 , 131.9 , 131.5 , 128.7 , 118.6 , 113.7 , 
 94.6 , 85.0 , 80.9,76.6 , 74.4 , 65.6 , 55.3 , 47.0 , 39.8 , 36.8 , 36.1 , 26.5 , 
 26.4 , 22.4 , 20.8 , 19.7 ppm .
hrms ( esi ) m / z calcd for c28h41cl3o7na ( m + na ) 617.1810 , found 617.1832 . to a solution of 26 ( 291.5 mg , 0.491 mmol ) in a mixture 
 of ch2cl2 ( 5.0 ml ) and h2o ( 0.5 ml ) 
 was added 2,3-dichloro-5,6-dibenzoquinone ( ddq ) ( 133.6 mg , 0.589 mmol ) 
 at 0 c .
the reaction mixture was stirred at the same temperature 
 for 1 h , quenched with saturated aqueous nahco3 ( 6 ml ) , 
 and filtered in vacuo .
the organic layer was separated , and the water 
 layer was extracted with ch2cl2 ( 15 ml  
 4 ) . the organic phase was combined and washed with brine ( 3  
 15 ml ) , dried with anhydrous mgso4 , filtered , and concentrated 
 in vacuo .
this residue was used for the next reaction without further 
 purification . to the suspended solution of
fmoc - pro - oh ( 334.3 
 mg , 0.991 mmol ) in toluene ( 3.0 ml ) were added n , n - diisopropylethylamine ( diea ) ( 0.26 ml , 1.49 mmol ) and 
 2,4,6-trichlorobenzoyl chloride ( 233 l , 1.491 mmol ) at room 
 temperature under argon , which was stirred at the same temperature 
 for 10 min .
then the crude alcohol in toluene ( 1.5 ml ) and dmap ( 212.8 
 mg , 1.742 mmol ) were added to the above mixture . after being stirred 
 at room temperature overnight
, the reaction mixture was quenched with 
 water and extracted with diethyl ether ( 20 ml  4 ) .
the combined 
 organic layer was washed with saturated nh4cl ( 20 ml  
 2 ) , saturated nahco3 ( 20 ml  2 ) , and brine ( 20 ml ) , 
 dried over mgso4 , and concentrated in vacuo .
the residue 
 was purified by silica gel column chromatography ( eluted by 10% ethyl 
 acetate in hexane ) to give ester 27 ( 360.8 mg , 90.4% ) 
 as a colorless oil ; [ ]d 58.9 
 ( c 0.124 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) :  7.777.75 
 ( m , 2h ) , 7.677.62 ( m , 2h ) , 7.40 ( dd , j = 
 7.4 , 7.2 hz , 2h ) , 7.347.29 ( m , 2h ) , 6.005.79 ( m , 1h ) , 
 5.345.12 ( m , 3h ) , 4.97 ( d , j = 12.0 hz , 0.6h ) , 
 4.87 ( d , j = 12.0 hz , 0.4h ) , 4.78 ( d , j = 10.0 hz , 1h ) , 4.69 ( d , j = 12.0 hz , 0.4h ) , 4.64 
 ( d , j = 12.0 hz , 0.6h ) , 4.594.16 ( m , 6h ) , 
 3.673.61 ( m , 1h ) , 3.593.48 ( m , 1h ) , 2.382.28 
 ( m , 0.6h ) , 2.242.16 ( m , 0.4h ) , 2.152.06 ( m , 1h ) , 2.041.93 
 ( m , 2h ) , 1.921.80 ( m , 1h ) , 1.551.48 ( m , 1h ) , 1.461.42 
 ( m , 1h ) , 1.381.29 ( m , 1h ) , 1.251.19 ( m , 6h ) , 1.151.07 
 ( m , 0.6h ) , 1.000.96 ( m , 1.6h ) , 0.89 ( s , 5.4h ) , 0.86 ( s , 3.6h ) , 
 0.67(d , j = 6.4 hz , 1.8h ) ppm .
c nmr 
 ( 100 mhz , cdcl3 , mixture of rotamers ) :  175.0,175.0 , 
 172.9 , 172.4 , 154.7 , 154.5 , 154.3 , 154.2 , 144.4 , 144.3 , 144.0 , 143.9 , 
 141.4 , 141.4 , 141.3 , 141.3 , 132.0 , 131.9 , 127.8 , 127.2 , 127.1 , 125.5 , 
 125.4 , 125.3 , 125.2 , 120.0 , 120.0 , 118.6 , 94.9 , 94.8 , 80.8 , 80.6 , 
 79.8 , 79.5 , 77.1 , 76.9 , 68.0 , 67.5 , 65.7 , 65.6 , 59.9 , 59.4 , 47.3 , 
 47.3 , 47.2 , 47.1 , 46.5 , 38.2 , 38.0 , 37.3 , 36.8 , 34.9 , 34.8 , 34.7 , 
 31.7 , 31.4 , 30.1 , 26.9 , 26.7 , 25.9 , 25.9 , 25.4 , 23.5 , 22.8 , 22.0 , 
 22.0 , 20.5 , 20.4 , 19.8 , 19.7 , 14.2 ppm . hrms ( esi ) m / z calcd for c40h50cl3no9na ( m + na ) 816.2443 , found 816.2420 . to a solution of 27 ( 349.3 mg , 0.440 mmol ) in thf 
 ( 8 ml )
were added pd(pph3)4 ( 76.3 mg , 0.066 
 mmol ) and n - methyl aniline ( 144.3 l , 1.321 
 mmol ) at room temperature under argon .
this reaction was protected 
 with aluminum foil . after being stirred at room temperature for 1 
 h ,
the reaction mixture was concentrated in vacuo and purified by 
 column chromatography ( eluted by acetone / hexane 1:3 ) to give acid 4c ( 354.1 mg , 95% ) as a pale - yellow solid ; [ ]d 51.1 ( c 0.131 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 , mixture 
 of rotamers ) :  9.90 ( br , 1h ) , 7.777.74 ( m , 2h ) , 7.687.63 
 ( m , 2h ) , 7.427.38 ( m , 2h ) , 7.347.29 ( m , 2h ) , 5.23 
 ( d , j = 10.4 hz , 0.4h ) , 5.13 ( d , j = 10.4 hz , 0.6h ) , 
 5.01 ( d , j = 12.0 hz , 0.6h ) , 4.90 ( d , j = 12.0 hz , 0.4h ) , 4.854.79 ( m , 1h ) , 4.70 ( d , j = 12.0 hz , 0.4h ) , 4.63 ( d , j = 12.0 hz , 0.6h ) , 
 4.524.17 ( m , 4h ) , 3.673.48 ( m , 1h ) , 2.372.27 
 ( m , 0.6h ) , 2.242.19 ( m , 0.4h ) , 2.162.10 ( m , 1h ) , 2.011.83 
 ( m , 3h ) , 1.781.69 ( m , 0.4h ) , 1.621.49 ( m , 1.6h ) , 1.431.35 
 ( m , 1h ) , 1.311.20 ( m , 6h ) , 1.091.00 ( m , 1h ) , 1.01 
 ( d , j = 6.4 hz , 1.2 h ) , 0.94 ( s , 5.4h ) , 0.87 ( s , 
 3.6h ) , 0.69 ( d , j = 6.4 hz , 1.8h ) ppm .
c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) :  181.1 , 
 180.7 , 172.8 , 172.3 , 154.8 , 154.4 , 154.2 , 144.3 , 144.1 , 143.9 , 143.7 , 
 141.3 , 141.3 , 141.2 , 141.2 , 135.1 , 135.0 , 135.0 , 130.6 , 128.1 , 128.1 , 
 128.0 , 127.7 , 127.1 , 127.1 , 127.0 , 125.5 , 125.3 , 125.2 , 125.1 , 120.0 , 
 119.9 , 94.9 , 94.8 , 80.5 , 80.3 , 79.8 , 79.4 , 77.0 , 76.8 , 68.0 , 67.6 , 
 59.8 , 59.3 , 47.2 , 47.1 , 46.9 , 46.4 , 38.1 , 37.5 , 37.1 , 36.7 , 34.9 , 
 34.7 , 34.6 , 34.5 , 31.6 , 31.3 , 30.0 , 29.1 , 29.1 , 26.7 , 26.5 , 25.9 , 
 25.3 , 24.3 , 23.5 , 22.7 , 22.2 , 21.9 , 20.8 , 20.4 , 19.9 , 19.6 , 19.4 , 
 18.8 , 14.2 , 11.5 ppm .
hrms ( esi ) m / z calcd for c37h46cl3no9na ( m + na ) 776.2130 , found 776.2149 . to the solution of 
 weinreb amide 28 ( 2.505 g , 4.945 
 mmol ) in thf ( 50 ml ) was added lialh4 ( 234.6 mg , 6.182 
 mmol ) in one portion at 0 c . after being stirred at 0 c 
 for 30 min
, the reaction mixture was quenched with 0.2n aq khso4 ( 30 ml ) and extracted with et2o ( 50 ml  
 3 ) .
the combined organic layer was washed with 1n aq hcl ( 30 ml  
 3 ) and brine ( 30 ml  3 ) , dried with anhydrous mgso4 , and evaporated in vacuo to give the crude aldehyde 29 , which was used in the next step without further purification . to 
 the crude aldehyde 28 in toluene ( 30 ml )
was added ph3p = chco2et ( 3.101 g , 8.901 mmol ) at 0 c under 
 ar . after being stirred at room temperature for 3 h
, the reaction 
 mixture was concentrated in vacuo and purified by column chromatography 
 ( eluted by 1217% ethyl acetate in hexane ) to give product(s ) 30 as a colorless oil .
yield 88% , 2 steps ; [ ]d + 6.0 ( c 0.20 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 ) :  
 7.447.42 ( m , 6h ) , 7.317.27 ( m , 6h ) , 7.247.20 
 ( m , 3h ) , 6.72 ( dd , j = 15.6 , 4.4 hz , 1h ) , 5.84 ( d , j = 15.6 hz , 1h ) , 4.80 ( d , j = 8.4 hz , 
 1h ) , 4.29 ( br , 1h ) , 4.17 ( q , j = 7.2 hz , 2h ) , 2.532.41 
 ( m , 2h ) , 1.45 ( s , 9h ) , 1.27 ( t , j = 7.2 hz , 3h ) ppm .
c nmr ( 100 mhz , cdcl3 ) :  169.9 , 154.7 , 
 146.6 , 144.3 , 129.5 , 128.0 , 126.8 , 121.5 , 79.7 , 67.0 , 60.4 , 50.5 , 
 36.2 , 28.5 , 14.2 ppm .
hrms ( esi ) m / z calcd for c31h35no4sna ( ( m + na ) 540.2210 , found 540.2223 . to the 
 solution of compound 30 ( 3.379 
 g , 6.532 mmol ) in 95% etoh ( 35 ml ) was added nabh4 ( 247.1 
 mg , 6.532 mmol ) at room temperature .
the resulting mixture was stirred 
 at room temperature for 30 h and then quenched with water ( 50 ml ) 
 and extracted with et2o ( 50 ml  4 ) .
the combined 
 organic layer was dried with anhydrous mgso4 , evaporated 
 in vacuo , and purified by column chromatography ( eluted with the mixture 
 of ch2cl2/hexane / acetone from 50:148:2  
 50:98:2  50:48:2 ) to give product 31 ( 1.187 g , 
 35% ) as a thick colorless oil .
h nmr ( 400 mhz , cdcl3 ) :  7.427.40 ( m , 6h ) , 
 7.307.27 ( m , 6h ) , 7.237.19 ( m , 3h ) , 4.49 ( d , j = 9.2 hz , 1h ) , 4.10 ( q , j = 7.2 hz , 2h ) , 
 3.64 ( br , 1h ) , 2.34 ( br m , 2h ) , 2.21 ( t , j = 7.6 
 hz , 2h ) , 1.781.59 ( m , 2h ) , 1.43 ( s , 9h ) , 1.23 ( t , j = 7.2 hz , 3h ) ppm .
c nmr ( 100 mhz , cdcl3 ) :  173.3 , 155.3 , 144.7 , 129.7 , 128.0 , 126.8 , 79.4 , 
 66.7 , 60.5 , 49.6 , 37.2 , 31.1 , 29.7 , 28.5 , 14.3 ppm . hrms ( esi ) m / z calcd for c31h37no4sna ( m + na ) 542.2336 , found 542.2331 . to the solution of 31 ( 1.045 g , 2.013 
 mmol ) in 95% ethanol
( 7.5 ml ) was added aq naoh ( 1 m , 4.0 ml ) at room 
 temperature . after being stirred at the same temperature for 2 h
, 
 the reaction mixture was diluted with water ( 10 ml ) , acidified with 
 10.5 m aq hcl to ph 45 , and extracted with diethyl 
 ether ( 20 ml  3 ) .
the combined organic layer was dried over 
 mgso4 , filtered , and concentrated in vacuo to give crude 
 acid 32 , which was used in the next step without further 
 purification . to the above crude acid 32 solution in 
 dmso
( 15 ml ) was added k2co3 ( 556.5 mg , 4.027 
 mmol ) and allyl bromide ( 255.5 l , 3.020 mmol ) at room temperature . 
 after being stirred at room temperature 5 h ,
the reaction was quenched 
 with water ( 30 ml ) and extracted with ethyl acetate ( 50 ml  
 3 ) .
the combined organic layer was washed with brine , filtered , and 
 concentrated in vacuo , and the residue was purified by column chromatography 
 on silica gel ( eluted by 10% ethyl acetate in hexane ) to give product 5 as a colorless oil . yield 0.75 g , 70% : [ ]d + 10.0 ( c 0.16 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 ) :  
 7.427.41 ( m , 6h ) , 7.317.27 ( m , 6h ) , 7.237.20 
 ( m , 3h ) , 5.955.85 ( m , 1h ) , 5.325.21(m , 2h ) , 4.55 ( d , j = 5.6 hz , 2h ) , 4.49 ( d , j = 8.8 hz , 1h ) , 
 3.65 ( br m , 1h ) , 2.34 ( br m , 2h ) , 2.26 ( t , j = 7.6 
 hz , 2h ) , 1.801.62 ( m , 2h ) , 1.44 ( s , 9h ) ppm .
c nmr ( 100 mhz , cdcl3 ) :  172.9 , 155.3 , 144.7 , 132.2 , 
 129.6 , 128.1 , 126.8 , 118.3 , 79.4 , 66.7,65.3 , 49.5 , 37.2 , 31.0 , 29.7 , 
 28.5 ppm . hrms ( esi ) m / z calcd for 
 c32h37no4sna ( ( m + na ) 554.2336 , found 554.2341 . to the solution of 5 ( 169.6 mg , 0.319 mmol ) 
 in ch2cl2 ( 4 
 ml ) were added 2,6-lutidine ( 556 l , 4.790 mmol ) and trimethylsilyl 
 trifluoro - methanesulfonate ( tmsotf ) ( 694 l , 3.832 mmol ) dropwise 
 at room temperature under argon . after being stirred at the same temperature 
 for 1.5 h ,
the reaction mixture was quenched with meoh ( 6 ml ) and 
 water ( 10 ml ) at 0 c and extracted with ch2cl2 ( 15 ml  4 ) .
the combined organic layer was washed with 
 brine , dried over anhydrous mgso4 , and evaporated in vacuo 
 to give the crude free amine of 5 , which was used in 
 next step without further purification . to the crude free amine 
 of 5 ( 1.3 equiv ) in ch2cl2 ( 4 ml ) 

were added corresponding acid 4 ( based in scheme 5 ) ( 0.245 mmol , 1 equiv ) , coupling reagent ( cr , 0.368 
 mmol , 1.5 equiv ) , and diea ( 140 l , 0.735 mmol , 3.0 equiv ) at 
 room temperature or 0 c . after being stirred at room temperature 
 for 2.524 h
, the resulting reaction mixture was evaporated 
 in vacuo and purified by column chromatography , eluting with ethyl 
 acetate in hexane to give product 3a , c , d or 34 as a colorless oil . from 4a , 5a ; cr bep added at 0 c ; reaction time 2.5 h ; yield 245.6 mg , 
 87% ) . from 4b , 5a ; cr hatu added at rt , reaction time 3.0 
 h ; yield 193.8 mg , 82% ; [ ]d 31.2 
 ( c 0.24 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) :  7.74 
 ( d , j = 7.6 hz , 2h ) , 7.637.54 ( m , 2h ) , 7.417.36 
 ( m , 8h ) , 7.307.15 ( m , 11h ) , 6.69 ( d , j = 
 8.8 hz , 0.84h ) , 6.06 ( d , j = 8.4 hz , 0.16h ) , 5.925.77 
 ( m , 1h ) , 5.305.15 ( m , 2h ) , 4.89 ( d , j = 11.2 
 hz , 0.84h ) , 4.79 ( d , j = 10.4 hz , 0.16h ) , 4.534.44 
 ( m , 2h ) , 4.394.29 ( m , 2h ) , 4.234.16 ( m , 1h ) , 4.01(br 
 m , 1h ) , 3.953.85 ( m , 1h ) , 3.79 ( br , 1h ) , 3.663.61 
 ( m , 1h ) , 3.583.44 ( m , 1h ) , 2.402.15 ( m , 6h ) , 2.112.01 
 ( m , 3h ) , 1.981.88 ( m , 2h ) , 1.851.75 ( m , 2h ) , 1.711.60 
 ( m , 3h ) , 1.551.47 ( m , 1h ) , 1.401.30 ( m , 1h ) , 1.040.97 
 ( m , 1h ) , 0.96 ( d , j = 6.8 hz , 2.52h ) , 0.88 ( s , 9h ) , 
 0.77 ( d , j = 6.8 hz , 0.48h ) ppm .
c nmr 
 ( 100 mhz , cdcl3 , mixture of rotamers ) :  172.7 , 172.3 , 
 172.0 , 155.2 , 144.8 , 144.7 , 144.0,143.9 , 141.4 , 132.3 , 129.7 , 128.1 , 
 128.0 , 127.8 , 127.3 , 127.2 , 126.9 , 126.8 , 125.2 , 125.2 , 120.1 , 120.1 , 
 118.3 , 78.7 , 67.7 , 66.6 , 66.3 , 65.2 , 59.6 , 47.9 , 47.3 , 46.6 , 44.3 , 
 42.9 , 42.2 , 41.4 , 38.0 , 37.1 , 36.8 , 34.9 , 34.6 , 31.7 , 31.3 , 31.0 , 
 30.0 , 29.8 , 29.3 , 26.1 , 25.2 , 24.5 , 23.5 , 22.8 , 20.7 , 14.3 ppm .
hrms 
 ( esi ) m / z calcd for c59h68n2o8sna ( m + na ) 987.4589 , 
 found 987.4628 . from 4c , 5a ; cr pyaop added at rt , reaction 
 time 24 h ; yield 254.3 mg , 89% ; [ ]d 48.5 ( c 0.20 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 
  7.787.75 ( m , 2h ) , 7.67 ( d , j = 7.6 
 hz , 0.6h ) , 7.62 ( d , j = 7.2 hz , 1.4h ) , 7.417.38 
 ( m , 8h ) , 7.347.27 ( m , 8h ) , 7.227.20 ( m , 3h ) , 6.02 
 ( d , j = 8.0 hz , 0.4h ) , 5.96 ( d , j = 8.4 hz , 0.6 h ) , 5.925.80 ( m , 1h ) , 5.305.18 ( m , 
 2h ) , 5.13 ( d , j = 10.0 hz , 0.4h ) , 5.04 ( d , j = 10.0 hz , 0.6h ) , 4.95 ( d , j = 12.0 hz , 
 0.6h ) , 4.83 ( d , j = 12.0 hz , 0.4h ) , 4.794.75 
 ( m , 1h ) , 4.70 ( d , j = 12.0 hz , 0.4h ) , 4.66 ( d , j = 12.0 hz , 0.6h ) , 4.524.34 ( m , 4.4h ) , 4.284.23 
 ( m , 1h ) , 4.204.16 ( m , 0.6h ) , 3.993.87 ( m , 1h ) , 3.673.45 
 ( m , 2h ) , 2.362.27 ( m , 2.6h ) , 2.242.16 ( m , 2.4h ) , 2.12 
 ( br m , 1h ) , 2.011.90 ( m , 2h ) , 1.851.74 ( m , 3h ) , 1.531.44 
 ( m , 2h ) , 1.371.27 ( m , 1h ) , 1.191.16 ( m , 6h ) , 1.061.00 
 ( m , 1h ) , 0.97 ( d , j = 6.4 hz , 1.8h ) , 0.88 ( s , 5.4h ) , 
 0.85 ( s , 3.6h ) , 0.69 ( d , j = 6.4 hz , 1.2h ) ppm .
c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) :  
 174.2 , 172.7 , 172.6 , 172.2 , 154.6 , 154.3 , 154.2 , 154.1 , 144.5 , 144.5 , 
 144.3 , 144.1 , 143.9 , 143.7 , 141.3 , 141.2 , 132.1 , 132.0 , 129.5 , 128.0 , 
 127.7 , 127.1 , 127.0 , 126.8 , 125.4 , 125.3 , 125.1 , 125.1 , 120.0 , 119.9 , 
 118.3 , 94.8 , 94.8 , 81.6 , 81.4 , 79.6 , 79.4 , 79.3 , 76.7 , 67.8 , 67.4 , 
 66.5 , 65.2 , 59.8 , 59.3 , 48.3 , 47.2 , 47.0 , 46.8 , 46.3 , 38.1 , 37.8 , 
 36.8 , 36.3 , 36.3 , 34.8 , 34.7 , 34.5 , 31.6 , 31.3 , 30.8 , 30.0 , 26.8 , 
 26.5 , 25.8 , 25.8 , 25.3 , 24.4 , 23.4 , 23.1 , 22.7 , 20.4 , 20.3 , 20.1 , 
 14.2 ppm . hrms ( esi ) m / z calcd for 
 c64h73cl3n2o10sna ( m + na ) 1189.3944 , found 1189.3964 . from 4a , 5b ; cr pyaop added at rt , reaction 
 time 24 h ; yield 257.0 mg , 91% ; [ ]d 40.0 ( c 0.05 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 
  7.877.78 ( m , 2h ) , 7.717.67 ( dd , j = 8.4 , 8.0 hz , 2h),7.517.46 ( m , 8h ) , 7.407.28 ( m , 
 11h ) , 6.17 ( d , j = 8.4 hz , 0.6h ) , 6.025.92 
 ( m , 1h ) , 5.51 ( d , j = 8.4 hz , 0.4h ) , 5.405.30 
 ( m , 2h ) , 5.10 ( ddd , j = 7.6 , 7.4 , 7.2 hz , 1h ) , 4.974.80 
 ( m , 2.4h ) , 4.75 ( d , j = 12.0 hz , 0.6h ) , 4.624.30 
 ( m , 6h ) , 4.00 ( br m , 1h ) , 3.743.55 ( m , 2h ) , 2.602.57 
 ( m , 0.4h ) , 2.422.23 ( m , 6.6 h ) , 2.06 ( br m , 2h ) , 1.981.86 
 ( m , 1h ) , 1.821.70 ( m , 3h ) , 1.661.58 ( m , 2h ) , 1.551.30 
 ( m , 1h ) , 1.24 ( d , j = 6.8 hz , 1.8h ) , 1.20 ( d , j = 7.2 hz , 1.2 h ) , 1.07 ( d , j = 6.0 hz , 
 1.8h ) , 0.98 ( s , 3.6h ) , 0.96 ( s , 5.4h ) , 0.88 ( d , j = 6.4 hz , 1.2h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) :  172.8 , 172.8 , 172.6 , 172.5 , 172.4 , 
 172.3 , 154.9 , 154.5 , 153.7 , 153.7 , 144.6 , 144.5 , 144.2 , 144.0 , 143.8 , 
 141.4 , 141.2 , 132.2 , 129.8 , 129.6 , 128.1 , 128.0 , 127.8 , 127.3 , 127.2 , 
 127.1 , 126.9 , 126.9 , 125.7 , 125.5 , 125.2 , 120.0 , 118.6 , 118.5 , 79.5 , 
 79.2 , 79.0 , 78.7 , 76.7 , 68.0 , 67.5 , 66.8 , 66.7 , 65.4 , 65.4 , 59.8 , 
 59.5 , 48.1 , 47.3 , 47.3 , 47.1 , 46.4 , 45.7 , 45.4 , 38.3 , 37.6 , 37.1 , 
 36.6 , 36.5 , 35.1 , 34.8 , 34.8 , 31.7 , 31.4 , 29.9 , 30.1 , 29.8 , 29.2 , 
 29.2 , 26.2 , 26.1 , 26.0 , 25.9 , 25.4 , 24.4 , 23.5 , 22.8 , 20.6 , 20.2 , 
 14.3 , 14.0 , 13.8 ppm . hrms ( esi ) m / z calcd for c63h71cl3n2o10sna ( m + na ) 1175.3787 , found 1175.3805 .
to the solution of compound 35 ( 166.3 mg , 0.172 mmol ) 
 and pyridine ( 140 l , 1.742 mmol ) in ch2cl2 ( 5.0 ml ) were added 2,2,2-trichloroethoxylcarbonyl chloride ( troc - cl ) 
 ( 182.6 mg/118.7 l , 0.862 mmol ) and 4-dimethylaminopyridine 
 ( dmap)(0.36 mg , 2.93 mol ) at 0 c . after being stirred 
 at the same temperature for 1 h
the water layer was extracted with ethyl acetate ( 20 ml  
 4 ) .
the combined organic layer was washed with 5% nahco3 ( 20 ml  2 ) and brine ( 20 ml ) , dried with mgso4 , 
 and concentrated in vacuo .
the residue was purified by chromatography 
 column on silica gel ( 2030% ethyl acetate in hexane ) to give 3b ( 156.5 mg , 80% ) ; [ ]d 45.0 
 ( c 0.111 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) :  7.777.74 
 ( m , 2h ) , 7.67 ( d , j = 7.6 hz , 0.4h),7.60 ( d , j = 7.6 hz , 1.6h ) , 7.417.37 ( m , 8h ) , 7.337.17 
 ( m , 11h ) , 6.13 ( d , j = 8.4 hz , 0.6h ) , 5.925.80 
 ( m , 1h ) , 5.62 ( d , j = 8.4 hz , 0.4h ) , 5.305.18 
 ( m , 2.4h ) , 4.854.80 ( m , 1.2h ) , 4.77 ( d , j = 12.0 hz , 0.4h ) , 4.61 ( d , j = 12.0 hz , 0.4h ) , 
 4.54 ( d , j = 12.0 hz , 0.6h ) , 4.534.45 ( m , 
 3h ) , 4.404.30 ( m , 1.4h ) , 4.264.18 ( m , 1.6h ) , 3.933.82 
 ( m , 1h ) , 3.663.60 ( m , 1h ) , 3.583.45 ( m , 1h ) , 2.542.56 
 ( m , 4h ) , 2.222.10 ( m , 3h ) , 2.001.85 ( m , 3h ) , 1.751.60 
 ( m , 2h ) , 1.551.24 ( m , 3h ) , 0.99 ( d , j = 6.4 
 hz , 1.8h ) , 0.88 ( s , 3.6h ) , 0.87 ( s , 5.4h ) , 0.80 ( d , j = 6.4 hz , 1.2 h ) ppm .
c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) :  172.8 , 172.7 , 172.7 , 172.3 , 168.7 , 
 168.3 , 154.9 , 154.4 , 153.6 , 153.5 , 144.6 , 144.6 , 144.2 , 144.1 , 143.9 , 
 143.8 , 141.3 , 141.3 , 132.2 , 132.1 , 129.6 , 128.0 , 128.0 , 127.8 , 127.8 , 
 127.1 , 126.9 , 126.8 , 125.5 , 125.4 , 125.2 , 120.0 , 120.0 , 118.4 , 118.3 , 
 94.8 , 94.8 , 79.5 , 79.1 , 76.7 , 76.6 , 75.3 , 74.8 , 67.8 , 67.5 , 65.3 , 
 65.2 , 59.8 , 59.5 , 48.4 , 47.3 , 47.0 , 46.5 , 41.6 , 41.0 , 39.8 , 39.4 , 
 38.1 , 37.7 , 36.4 , 36.3 , 35.0 , 34.7 , 31.7 , 31.3 , 29.8 , 29.1 , 25.9 , 
 25.9 , 25.4 , 24.4 , 23.4 , 22.7 , 20.8 , 20.7 , 14.2 ppm . hrms ( esi ) m / z calcd for c62h69cl3n2o10sna ( m + na ) 1161.3631 , found 1161.3634 . to the 
 solution of triphenylphosphine oxide 
 ( 223.3 mg , 0.802 mmol ) in ch2cl2 ( 1 ml )
was 
 added dropwise trifluoromethanesulfonic anhydride ( tf2o ) 
 ( 68 l , 0.401 mmol ) at 0 c under argon . after being stirred 
 at the same temperature for 10 min , compound 3 ( 0.100 
 mmol ) in ch2cl2 ( 0.5 ml )
the reaction mixture was stirred at the same temperature for 30 min 
 for 3a/3b and 3 h for 3d .
this 
 reaction was monitored by mass spectrometry and was quenched with 
 saturated nahco3 ( 6 ml ) at 0 c when starting material 
 disappeared .
the aqueous layer was extracted with ethyl acetate ( 10 
 ml  4 ) , washed with brine ( 10 ml ) , dried with mgso4 , filtered , and concentrated in vacuo to give troc protected thiazoline 
 intermediate 33a , b , or d. 
 the residue was used in the next step immediately without further 
 purification .
the above residue 33 was dissolved 
 in thf ( 4 ml ) , and then aqueous nh4oac ( 1 m , 1.0 ml ) and 
 zinc powder ( freshly activated with 1 m aqueous hcl ) ( 100 mg ) were 
 added at room temperature . after being stirred at the same temperature 
 for 30 min , ethyl acetate ( 5 ml ) and brine ( 5 ml ) and
the aqueous layer was extracted with ethyl acetate 
 ( 5 ml  4 ) .
the combined organic layer was dried with mgso4 , filtered , concentrated in vacuo , and purified by column 
 chromatography on silica gel ( eluted by ethyl acetate / hexane 1:3 , 
 v / v ) to give thiazoline ring product 34a , b , or d as a colorless oil .
yield 
 53.2 mg , 76% ; [ ]d 78.0 ( c 0.10 , ch2cl2 ) .
h nmr 
 ( 400 mhz , cdcl3 , mixture of rotamers ) :  7.74 ( d , j = 7.6 hz , 2h ) , 7.63 ( dd , j = 5.6 , 6.0 
 hz , 1.6 h ) , 7.56 ( d , j = 7.6 hz , 0.4h ) , 7.37 ( dd , j = 7.2 , 7.2 hz , 2h ) , 7.327.27 ( m , 2h ) , 5.935.83 
 ( m , 1h ) , 5.305.19 ( m , 2h ) , 4.90 ( d , j = 10.8 
 hz , 0.8h ) , 4.83 ( d , j = 9.6 hz , 0.2h ) , 4.584.51 
 ( m , 2h ) , 4.464.34 ( m , 3.8h ) , 4.284.18 ( m , 1.2h ) , 3.97 
 ( br m , 1h ) , 3.683.61 ( m , 2h ) , 3.573.47 ( m , 1h ) , 3.33 
 ( dd , j = 10.4 , 8.8 hz , 0.2h ) , 3.24 ( dd , j = 10.4 , 8.8 hz , 0.8h ) , 2.88 ( dd , j = 10.0 , 8.4 
 hz , 0.2h ) , 2.81 ( dd , j = 10.4 , 8.4 hz , 0.8h ) , 2.552.48 
 ( m , 4h ) , 2.292.19 ( m , 1h ) , 2.081.80 ( m , 5h ) , 1.751.58 
 ( m , 2h ) , 1.481.25 ( m , 2h ) , 1.040.97 ( m , 1h ) , 0.96 
 ( d , j = 6.4 hz , 2.4h ) , 0.87 ( s , 9h ) , 0.79 ( d , j = 6.4 hz , 0.6h ) ppm .
c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) :  172.9 , 172.9 , 172.8 , 172.3 , 
 169.2 , 169.1 , 154.9 , 154.3 , 144.3 , 144.1 , 144.0 , 143.8 , 141.3 , 141.3 , 
 141.2 , 132.2 , 132.2 , 127.6 , 127.6 , 127.1 , 127.0 , 127.0 , 127.0 , 125.4 , 
 125.3 , 125.3 , 125.2 , 79.5 , 78.5 , 76.0 , 75.8 , 67.8 , 67.7 , 67.2 , 66.7 , 
 65.2 , 65.1 , 59.5 , 47.2 , 47.1 , 47.0 , 46.5 , 42.9 , 42.2 , 42.0 , 41.9 , 
 38.0 , 37.9 , 37.4 , 34.8 , 34.7 , 34.6 , 31.6 , 31.5 , 31.4 , 31.2 , 30.4 , 
 29.9 , 26.0 , 25.9 , 25.7 , 25.3 , 25.1 , 24.5 , 23.3 , 22.7 , 20.6 , 20.5 , 
 14.2 ppm .
hrms ( esi ) m / z calcd for 
 c40h52n2o7sna ( m + na ) 727.3387 , found 727.3399 .
yield 
 57.0 mg , 79% ; [ ]d 22.5 ( c 0.12 , ch2cl2 ) .
h nmr 
 ( 400 mhz , cdcl3 , mixture of rotamers ) :  7.76 ( d , j = 7.2 hz , 2h ) , 7.63 ( dd , j = 6.6 , 6.4 
 hz , 1.7 h ) , 7.57 ( d , j = 7.2 hz , 0.3h ) , 7.39 ( dd , j = 7.2 , 7.2 hz , 2h ) , 7.30 ( dd , j = 7.4 , 
 7.2 hz , 2h ) , 5.945.85 ( m , 1h ) , 5.315.20 ( m , 2h ) , 4.88 
 ( d , j = 10.0 hz , 0.7h ) , 4.81 ( d , j = 8.8 hz , 0.3h ) , 4.594.51 ( m , 2.3h ) , 4.454.33 ( m , 
 3h ) , 4.314.18 ( m , 1.7h ) , 3.823.72 ( m , 0.7h ) , 3.683.62 
 ( m , 1.3h ) , 3.583.49 ( m , 1.3h ) , 3.353.24 ( m , 1h ) , 2.902.85 
 ( m , 0.3h ) , 2.81 ( dd , j = 10.8 , 7.6 hz , 0.7h ) , 2.722.65 
 ( m , 0.7h ) , 2.612.44 ( m , 2.3h ) , 2.342.18 ( m , 1.7h ) , 
 2.101.89 ( m , 4.4h ) , 1.82 ( m , 0.7h ) , 1.701.57 ( m , 2h ) , 
 1.371.29 ( m , 1h ) , 1.19 ( d , j = 7.0 hz , 0.9h ) , 
 1.17 ( d , j = 7.0 hz , 2.1h ) , 1.061.00 ( m , 
 1h ) , 0.96 ( d , j = 6.4 hz , 2.1h ) , 0.87 ( s , 9h ) , 0.79 
 ( d , j = 6.4 hz , 0.9h ) ppm .
c nmr ( 100 
 mhz , cdcl3 , mixture of rotamers ) :  174.2 , 173.1 , 
 173.0 , 172.8 , 172.5 , 155.0 , 154.5 , 144.4 , 144.3 , 144.1 , 143.9 , 141.4 , 
 141.4 , 141.3 , 132.3 , 132.3 , 128.0 , 127.7 , 127.4 , 127.2 , 127.1 , 125.6 , 
 125.4 , 125.3 , 125.3 , 121.0 , 120.0 , 118.4 , 118.3 , 79.6 , 78.6 , 77.4 , 
 76.2 , 75.7 , 71.5 , 71.5 , 70.7 , 67.9 , 67.8 , 65.3 , 65.3 , 59.7 , 59.7 , 
 47.3 , 47.2 , 47.1 , 46.6 , 46.0 , 45.3 , 40.4 , 39.3 , 38.2 , 37.8 , 37.4 , 
 37.3 , 34.8 , 34.7 , 31.7 , 31.5 , 31.3 , 30.3 , 30.2 , 30.1 , 29.8 , 26.1 , 
 26.1 , 25.9 , 25.5 , 25.3 , 24.6 , 23.4 , 20.7 , 20.6 , 16.3 , 15.8 ppm .
hrms 
 ( esi ) m / z calcd for c41h54n2o7sna ( m + na ) 741.3544 , 
 found 741.3567 . to 
 the solution of 3c ( 97.8 mg , 0.084 mmol ) in 1,2-dichloroethane 
 ( 5 ml ) was added ticl4 ( 1 m in ch2cl2 , 0.294 ml , 0.293 mmol , 3.5 equiv ) at room temperature .
the resulting 
 solution was heated to 60 c and stirred at this temperature 
 for 2.5 h. this reaction was monitored by mass spectrometry .
when 
 the starting material was consumed completely , the reaction was cooled 
 to 0 c and quenched with saturated aq nahco3 ( 7 ml ) , 
 stirred at room temperature for another 10 min , extracted with ethyl 
 acetate ( 10 ml  4 ) , dried over anhydrous mgso4 , filtered , 
 and evaporated in vacuo to give crude intermediate 33c , which was used in the next step without further purification .
the above crude 33c was dissolved in thf ( 4 ml ) , and 
 then aqueous nh4oac ( 1 m , 1.0 ml ) and zinc powder ( freshly 
 activated with 1 m aqueous hcl ) ( 80 mg ) were added at room temperature . 
 after being stirred at the same temperature for 30 min , to the reaction 

the aqueous layer 
 was extracted with ethyl acetate ( 7 ml  4 ) .
the combined organic 
 layer was dried with mgso4 , filtered , concentrated in vacuo , 
 and purified by column chromatography on silica gel ( eluted by ethyl 
 acetate / hexane 1:3 , v / v ) to give thiazoline ring product 34c ( 101.3 mg , 55% ) as a colorless oil ; [ ]d 80.8 ( c 0.12 , ch2cl2 ) .
h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 
  7.767.74 ( m , 2h ) , 7.667.61 ( m , 1.7h ) , 7.57 
 ( d , j = 7.2 hz , 0.3h ) , 7.39 ( dd , j = 7.4 , 7.2 hz , 2h ) , 7.317.28 ( m , 2h ) , 5.945.84 ( m , 
 1h ) , 5.315.19 ( m , 2h ) , 4.88 ( dd , j = 11.6 , 
 1.6 hz , 0.7h ) , 4.81 ( dd , j = 11.6 , 1.6 hz , 0.3h ) , 
 4.584.54 ( m , 2h ) , 4.524.38 ( m , 3h ) , 4.354.19 
 ( m , 2h ) , 3.663.59 ( m , 2h ) , 3.583.48 ( m , 2h ) , 3.303.23 
 ( m , 1h ) , 2.862.81 ( m , 1h ) , 2.592.46 ( m , 2h ) , 2.362.19 
 ( m , 1h ) , 2.121.91 ( m , 5h ) , 1.78 ( br m , 1h ) , 1.671.56 
 ( m , 2h ) , 1.371.27 ( m , 2h ) , 1.22 ( s , 0.9h ) , 1.191.18 
 ( m , 5.1h ) , 0.96 ( d , j = 6.4 hz , 2.1h ) , 0.87 ( s , 9h ) , 
 0.79 ( d , j = 6.8 hz , 0.9h ) ppm .
c nmr 
 ( 100 mhz , cdcl3 , mixture of rotamers ) :  178.8 , 178.3 , 
 173.1 , 173.0 , 172.9 , 172.5 , 154.9 , 154.4 , 144.4 , 144.3 , 144.0 , 143.9 , 
 141.4 , 141.3 , 141.2 , 132.3 , 127.7 , 127.7 , 127.1 , 127.1 , 125.5 , 125.4 , 
 125.3 , 125.2 , 120.0 , 118.3 , 118.3 , 79.4 , 78.6 , 76.0 , 75.8 , 75.0 , 74.9 , 
 67.9 , 67.7 , 65.2 , 65.2 , 59.7 , 59.7 , 47.3 , 47.2 , 47.0 , 46.5 , 45.9 , 
 45.6 , 38.0 , 37.8 , 37.0 , 37.0 , 36.9 , 36.6 , 34.8 , 34.6 , 31.6 , 31.4 , 
 31.3 , 30.3 , 30.2 , 30.0 , 29.8 , 26.1 , 25.8 , 25.3 , 24.5 , 24.3 , 23.7 , 
 23.4 , 23.1 , 20.6 , 20.5 ppm . hrms ( esi ) m / z calcd for c42h56n2o7sna ( m + na ) 755.3700 , found 755.3718 . to a solution of 34 ( 0.054 mmol ) in 
 thf
( 1.5 ml ) were added pd(pph3)4 ( 7.1 mg , 0.005 
 mmol ) and n - methylaniline ( 0.015 ml , 0.136 mmol ) 
 at room temperature under argon .
this reaction was protected with 
 aluminum foil . after being stirred at the same temperature for 1 h
, 
 the reaction mixture was concentrated in vacuo and purified by preparative 
 tlc ( 20 cm  20 cm plate ) to give acid 2 .
yield 
 31.8 mg , 89% ; [ ]d 90.0 ( c 0.04 , ch2cl2 ) .
h nmr 
 ( 400 mhz , cdcl3 , mixture of rotamers ) :  7.74 ( d , j = 7.6 hz , 2h ) , 7.63(d , j = 7.2 hz , 1.8h ) , 
 7.57 ( d , j = 7.6 hz , 0.2h ) , 7.38 ( dd , j = 7.6 , 7.6 hz , 2h ) , 7.327.28 ( m , 2h ) , 6.22 ( br , 1h ) , 4.90 
 ( d , j = 10.0 hz , 0.8h ) , 4.84 ( d , j = 9.2 hz , 0.2h ) , 4.534.34 ( m , 3.8h ) , 4.294.17 ( m , 
 1.2h ) , 4.003.92 ( m , 1h ) , 3.683.61 ( m , 1h ) , 3.573.45 
 ( m , 1h ) , 3.35 ( dd , j = 10.8 , 8.8 hz , 0.2h ) , 3.23 
 ( dd , j = 10.8 , 8.4 hz , 0.8h ) , 2.91 ( dd , j = 10.8 , 8.4 hz , 0.2h ) , 2.80 ( dd , j = 10.8 , 8.4 
 hz , 0.8h ) , 2.602.43 ( m , 4h ) , 2.352.17 ( m , 1h ) , 2.081.82 
 ( m , 5h ) , 1.741.59 ( m , 2h ) , 1.481.25 ( m , 2h ) , 1.030.96 
 ( m , 1h ) , 0.96 ( d , j = 6.4 hz , 2.4h ) , 0.87 ( s , 9h ) , 
 0.77 ( d , j = 6.4 hz , 0.6h ) ppm .
c nmr 
 ( 100 mhz , cdcl3 , mixture of rotamers ) :  176.7 , 176.5 , 
 173.1 , 172.5 , 171.8 , 170.5 , 155.1 , 154.5 , 144.3 , 144.1 , 144.0 , 143.8 , 
 141.4 , 141.4 , 141.3 , 141.3 , 127.7 , 127.7 , 127.2 , 127.1 , 127.1 , 125.5 , 
 125.4 , 125.3 , 120.0 , 79.8 , 75.7 , 75.3 , 67.9 , 67.8 , 67.4 , 67.0 , 59.6 , 
 47.2 , 47.2 , 47.0 , 46.6 , 42.7 , 42.3 , 42.1 , 41.9 , 38.2 , 38.1 , 38.0 , 
 37.4 , 36.8 , 34.8 , 34.7 , 32.0 , 31.7 , 31.3 , 30.1 , 30.0 , 30.0 , 26.1 , 
 26.0 , 25.7 , 25.1 , 24.6 , 23.4 , 20.7 , 20.5 ppm .
hrms ( esi ) m / z calcd for c37h48n2o7sna ( m + na ) 687.3074 , found 687.3070 . yield 
 31.2 mg , 84% ; [ ]d 57.7 ( c 0.052 , ch2cl2 ) .
h nmr 
 ( 400 mhz , cdcl3 , mixture of rotamers ) :  7.76 ( d , j = 7.6 hz , 2h ) , 7.657.58 ( m , 2h ) , 7.39 ( dd , j = 7.4 , 7.2 hz , 2h ) , 7.327.28 ( m , 2h ) , 4.88 ( d , j = 12.0 , 0.7h ) , 4.80 ( d , j = 12.0 hz , 
 0.3h ) , 4.51 ( dd , j = 8.8 , 2.8 hz , 0.3h ) , 4.46 ( d , j = 7.2 hz , 0.3h ) , 4.43 ( d , j = 7.2 hz , 
 0.7h ) , 4.414.32 ( m , 2h ) , 4.304.25 ( m , 1.7h ) , 3.653.56 
 ( m , 2h ) , 3.533.45 ( m , 1h ) , 3.30 ( dd , j = 
 11.0 , 8.4 hz , 0.3h ) , 3.19 ( dd , j = 11.0 , 8.4 hz , 
 0.7h ) , 2.88 ( dd , j = 10.8 , 10.8 hz , 1h ) , 2.702.53 
 ( m , 2h ) , 2.372.14 ( m , 1h ) , 2.081.90 ( m , 5h ) , 1.79 
 ( br m , 1h ) , 1.681.60 ( m , 2h ) , 1.351.26 ( m , 2h ) , 1.26 
 ( s , 0.9h ) , 1.19 ( s , 2.1h ) , 1.18 ( s , 3h ) , 0.95 ( d , j = 6.6 hz , 2.1h ) , 0.87 ( s , 9h ) , 0.74 ( d , j = 6.6 
 hz , 0.9h ) ppm .
c nmr ( 100 mhz , cdcl3 , mixture 
 of rotamers ) :  182.4 , 180.5 , 175.7 , 175.5 , 173.2 , 172.7 , 155.1 , 
 154.7 , 144.5 , 144.2 , 144.1 , 143.8 , 141.5 , 141.4 , 141.3 , 127.8 , 127.8 , 
 127.2 , 127.1 , 127.1 , 125.5 , 125.4 , 125.4 , 125.3 , 120.0 , 79.7 , 78.6 , 
 76.0 , 75.8 , 75.4 , 75.1 , 68.1 , 67.9 , 59.7 , 59.6 , 47.3 , 47.2 , 47.1 , 
 46.6 , 46.5 , 45.8 , 38.0 , 37.8 , 37.5 , 37.4 , 37.1 , 36.3 , 34.8 , 34.6 , 
 33.4 , 32.7 , 32.7 , 31.7 , 31.3 , 30.2 , 30.0 , 29.8 , 29.7 26.1 , 25.6 , 25.1 , 
 24.9 , 24.7 , 23.8 , 23.7 , 23.5 , 22.8 , 20.7 , 20.4 , 14.3 ppm . hrms ( esi ) m / z calcd for c39h52n2o7sna ( m + na ) 715.3387 , found 
 715.3397 . yield 
 33.0 mg , 90% ; [ ]d 60.0 ( c 0.04 , ch2cl2 ) .
h nmr 
 ( 400 mhz , cdcl3 , mixture of rotamers ) :  7.75 ( d , j = 7.6 hz , 2h ) , 7.677.57 ( m , 2h ) , 7.39 ( dd , j = 7.4 , 7.2 hz , 2h ) , 7.30 ( dd , j = 7.6 , 
 7.4 hz , 2h ) , 5.90 ( br , 1h ) , 4.91(dd , j = 12.8 , 12.4 
 hz , 0.7h ) , 4.864.82 ( m , 0.3h ) , 4.544.50 ( m , 0.3h ) , 
 4.454.40 ( m , 1h ) , 4.384.33 ( m , 1.7h ) , 4.304.27 
 ( m , 1.3h ) , 4.254.16 ( m , 0.7h ) , 3.823.80 ( m , 0.7h ) , 
 3.753.70 ( m , 0.3h ) , 3.64 ( br m , 1h ) , 3.513.49 ( m , 
 1h ) , 3.363.30 ( m , 0.3h ) , 3.263.15 ( m , 0.7h ) , 2.912.85 
 ( m , 0.3h ) , 2.842.72 ( m , 0.7h ) , 2.682.50 ( m , 3h ) , 2.352.21 
 ( m , 1h ) , 2.212.15 ( m , 0.3h ) , 2.051.89 ( m , 5h ) , 1.841.79 
 ( m , 0.7h ) , 1.751.70 ( m , 1h ) , 1.661.60 ( m , 1h ) , 1.541.45 
 ( m , 0.7h ) , 1.451.40 ( m , 0.3h ) , 1.191.16 ( m , 3h ) , 1.031.00 
 ( m , 1h ) , 0.96 ( d , j = 6.0 hz , 2.1h ) , 0.88 ( s , 9h ) , 
 0.74 ( d , j = 6.0 hz , 0.9h ) ppm .
c nmr 
 ( 100 mhz , cdcl3 , mixture of rotamers ) :  177.8 , 177.6 , 
 175.9 , 173.4 , 173.4 , 173.3 , 172.7 , 155.1 , 155.1 , 154.6 , 144.4 , 144.4 , 
 144.2 , 144.2 , 144.2 , 144.0 , 143.9 , 141.4 , 141.4 , 141.4 , 141.3 , 141.3 , 
 128.0 , 128.0 , 127.8 , 127.8 , 127.5 , 127.2 , 127.1 , 125.5 , 125.4 , 125.4 , 
 125.3 , 121.0 , 121.0 , 120.0 , 120.0 , 80.0 , 80.0 , 79.2 , 78.7 , 77.4 , 76.2 , 
 76.0 , 75.6 , 75.5 , 71.8 , 70.8 , 70.4 , 68.0 , 67.9 , 67.9 , 67.7 , 67.5 , 
 59.7 , 59.6 , 47.3 , 47.3 , 47.1 , 46.6 , 46.0 , 45.9 , 45.4 , 40.7 , 39.7 , 
 39.4 , 39.2 , 38.1 , 38.0 , 37.8 , 37.6 , 37.0 , 34.8 , 34.7 , 32.9 , 32.9 , 
 32.3 , 31.7 , 31.3 , 31.1 , 30.2 , 30.0 , 30.0 , 29.8 , 29.4 , 29.2 , 28.7 , 
 26.1 , 26.0 , 25.8 , 25.4 , 25.2 , 25.1 , 24.6 , 23.4 , 22.8 , 20.8 , 20.8 , 
 20.6 , 20.5 , 16.6 , 16.0 , 14.3 , 14.2 ppm .
hrms ( esi ) m / z calcd for c38h50n2o7sna ( m + na ) 701.3231 , found 701.3253 . to a solution of fmoc protected tripeptide 6(2a2c ) ( 28.1 mg , 0.042 mmol ) in mecn ( 1.2 ml ) was added 
 diethylamine ( 0.6 ml ) at room temperature . after being stirred at 
 the same temperature for 30 min ,
the reaction mixture was evaporated 
 in vacuo , then azeotroped with toluene and ch2cl2 two times , respectively , and dried under reduced pressure for 1 
 h to give the free amine tripeptide , which was used in the next coupling 
 reaction without further purification .
the above crude free 
 amine tripeptide was dissolved in ch2cl2 ( thf 
 for 2b ) ( 2 ml ) . to this solution
were added acid 2 ( 0.028 mmol ) , corresponding coupling reagent ( 0.056 mmol ) 
 ( pyaop for 2a , c , d and depbt 
 for 2b ) , and diea ( 0.014 ml , 0.083 mmol ) at room temperature . 
 after being stirred at the same temperature for 1524 h ,
the 
 reaction mixture was concentrated in vacuo and purified by preparative 
 tlc plate ( developed by acetone / hexane ( 2:3 , v / v ) ) to give the precursor 36a  d as a colorless oil .
yield 22.0 mg , 72% ; 
 [ ]d 99.3 ( c 0.14 , ch2cl2 ) .
h nmr ( 400 mhz , 
 cdcl3 , mixture of rotamers ) :  7.777.74 ( m , 
 2h ) , 7.647.62 ( m , 1.7h ) , 7.56 ( d , j = 7.6 
 hz , 0.3h ) , 7.39 ( dd , j = 7.6 , 7.4 hz , 2h ) , 7.327.26 
 ( m , 2h ) , 7.09 ( d , j = 8.0 hz , 2h ) , 6.76 ( d , j = 8.0 hz , 2h ) , 6.63 ( d , j = 8.0 hz , 0.3h ) , 
 6.49 ( br m , 0.3h ) , 6.346.27 ( m , 1h ) , 6.206.12 ( m , 
 0.3h ) , 5.925.83 ( m , 0.7h ) , 5.545.47 ( m , 0.3h ) , 5.425.35 
 ( m , 0.7h ) , 5.315.17 ( m , 2.7h ) , 5.115.06 ( m , 0.3h ) , 
 4.944.85 ( m , 1.7h ) , 4.624.50 ( m , 2h ) , 4.474.35 
 ( m , 2.3h ) , 4.27 ( t , j = 7.2 hz , 1h ) , 4.204.16 
 ( m , 1.4h ) , 3.74 ( s , 3h ) , 3.723.64 ( m , 1.3h ) , 3.603.54 
 ( m , 1h ) , 3.263.07 ( m , 1h ) , 3.022.90 ( m , 4h ) , 2.832.77 
 ( m , 2h ) , 2.772.60 ( m , 4h ) , 2.502.46 ( m , 0.7h ) , 2.392.19 
 ( m , 4h ) , 2.181.90 ( m , 6.3h ) , 1.871.78 ( m , 1h ) , 1.73 
 ( br m , 1h ) , 1.61 ( br m , 0.7h ) , 1.551.49 ( m , 1h ) , 1.421.38 
 ( m , 1h ) , 1.271.21 ( m , 3h ) , 1.000.84 ( m , 19.1h ) , 0.68 
 ( d , j = 6.4 hz , 0.9h ) ppm .
c nmr ( 100 
 mhz , cdcl3 , mixture of rotamers ) :  172.7 , 172.1 , 
 172.0 , 171.9 , 171.6 , 171.6 , 170.8 , 158.7 , 154.8 , 154.4 , 144.3 , 144.2 , 
 144.1 , 143.9 , 141.4 , 141.3 , 131.8 , 131.4 , 130.5 , 130.4 , 128.2 , 128.2 , 
 127.8 , 127.8 , 127.2 , 127.1 , 127.1 , 126.4 , 125.5 , 125.4 , 125.3 , 120.1 , 
 118.8 , 114.0 , 79.6 , 79.1 , 77.4 , 75.9 , 75.7 , 67.9 , 67.7 , 66.1 , 65.5 , 
 60.6 , 59.6 , 59.4 , 55.3 , 50.4 , 49.8 , 49.2 , 47.3 , 47.1 , 46.6 , 38.7 , 
 38.2 , 37.9 , 37.2 , 37.1 , 36.8 , 34.8 , 34.5 , 33.7 , 33.4 , 32.1 , 31.7 , 
 31.5 , 31.1 , 31.0 , 30.7 , 30.2 , 29.8 , 29.6 , 29.5 , 28.8 , 26.1 , 25.1 , 
 24.6 , 23.5 , 22.8 , 21.1 , 20.7 , 16.2 , 15.9 , 14.5 , 14.4 , 14.3 , 11.8 , 
 10.7 ppm .
hrms ( esi ) m / z calcd for 
 c61h83n5o11sna ( m + na ) 1116.5702 , found 1116.5742 . yield 28.3 mg , 90% ; 
 [ ]d 130.0 ( c 0.10 , ch2cl2 ) .
h nmr ( 400 mhz , 
 cdcl3 , mixture of rotamers ) :  7.767.73 ( m , 
 2h ) , 7.657.59 ( m , 2h ) , 7.407.36 ( m , 2h ) , 7.317.26 
 ( m , 2h ) , 7.107.08 ( m , 2h ) , 6.85 ( d , j = 8.4 
 hz , 0.3h ) , 6.776.74 ( m , 2h ) , 6.65 ( d , j = 
 8.0 hz , 0.7h ) , 5.925.82 ( m , 1h ) , 5.37 ( dddd , j = 6.8 , 6.8 , 6.8 , 6.8 hz , 1h ) , 5.315.20 ( m , 2h ) , 5.205.14 
 ( m , 1h ) , 4.934.87 ( m , 1.7h ) , 4.81 ( d , j = 
 10.8 hz , 0.3h ) , 4.624.51 ( m , 3h ) , 4.464.41 ( m , 2h ) , 
 4.334.24 ( m , 3h ) , 3.74 ( s , 3h ) , 3.683.62 ( m , 1.3h ) , 
 3.603.48 ( m , 1.7h ) , 3.18 ( ddd , j = 18.8 , 
 10.8 , 8.8 hz , 1h ) , 3.032.95 ( m , 4h ) , 2.822.73 ( m , 
 2h ) , 2.68 ( s , 2.1h ) , 2.65 ( s , 0.9h ) , 2.432.17 ( m , 4h ) , 2.151.86 
 ( m , 5h ) , 1.851.75 ( m , 2h ) , 1.701.55 ( m , 2.3h ) , 1.501.45 
 ( m , 0.7h ) , 1.351.17 ( m , 13h ) , 0.970.91 ( m , 6h ) , 0.910.82 
 ( m , 14.1h ) , 0.76 ( d , j = 6.4 hz , 0.9h ) ppm .
c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) :  
 178.8 , 178.3 , 173.0 , 172.7 , 172.3 , 172.2 , 171.9 , 171.6 , 171.6 , 171.4 , 
 170.8 , 169.7 , 158.6 , 154.9 , 154.6 , 144.6 , 144.3 , 144.0 , 143.9 , 141.4 , 
 141.4 , 141.3 , 131.8 , 130.5 , 130.5 , 130.4 , 128.4 , 128.3 , 127.8 , 127.7 , 
 127.1 , 125.5 , 125.4 , 125.4 , 125.3 , 120.0 , 120.0 , 118.8 , 113.9 , 79.4 , 
 78.7 , 77.4 , 76.1 , 75.7 , 75.2 , 75.0 , 68.0 , 67.7 , 66.1 , 65.5 , 64.6 , 
 60.5 , 59.8 , 59.6 , 55.3 , 53.6 , 50.4 , 49.8 , 49.3 , 47.3 , 47.2 , 47.1 , 
 46.6 , 45.9 , 45.4 , 38.1 , 37.9 , 37.0 , 36.6 , 34.8 , 34.7 , 34.5 , 33.7 , 
 33.5 , 33.3 , 32.0 , 31.7 , 31.3 , 31.0 , 31.0 , 30.9 , 30.6 , 30.1 , 29.8 , 
 26.1 , 25.6 , 25.4 , 25.1 , 24.6 , 24.0 , 23.7 , 23.5 , 22.8 , 20.8 , 20.7 , 
 20.6 , 16.2 , 15.9 , 15.0 , 14.9 , 14.4 , 14.3 , 11.7 , 10.7 ppm .
hrms ( esi ) m / z calcd for c63h87n5o11sna ( m + na ) 1144.6015 , found 
 1144.6041 .
yield 22.0 mg , 71% ; 
 [ ]d 66.0 ( c 0.05 , ch2cl2 ) .
h nmr ( 400 mhz , 
 cdcl3 , mixture of rotamers ) :  7.777.74 ( m , 
 2h ) , 7.657.60 ( m , 2h ) , 7.39 ( dd , j = 7.6 , 
 7.2 hz , 2h ) , 7.30 ( dd , j = 7.2 , 6.8 hz , 2h ) , 7.107.07 
 ( m , 2h ) , 6.786.75 ( m , 2h ) , 5.925.81 ( m , 1h ) , 5.395.35 
 ( m , 1h ) , 5.315.21 ( m , 2h ) , 5.185.11 ( m , 1h ) , 4.934.81 
 ( m , 2h ) , 4.624.53 ( m , 2h ) , 4.464.21 ( m , 5h ) , 3.813.69 
 ( m , 4h ) , 3.693.60 ( m , 1h ) , 3.583.49 ( m , 1h ) , 3.223.18 
 ( m , 1h ) , 3.032.88 ( m , 4h ) , 2.822.76 ( m , 3h ) , 2.712.57 
 ( m , 4h ) , 2.342.21 ( m , 4h ) , 2.111.79 ( m , 8h ) , 1.71 
 ( br m , 1h ) , 1.641.58 ( m , 1h ) , 1.341.18 ( m , 5h ) , 0.970.78 
 ( m , 21h ) ppm .
c nmr ( 100 mhz , cdcl3 , mixture 
 of rotamers ) :  175.1 , 174.6 , 173.2 , 172.9 , 172.7 , 172.6 , 172.3 , 
 172.2 , 172.0 , 172.0 , 171.9 , 171.9 , 171.8 , 171.6 , 171.5 , 171.4 , 170.8 , 
 169.7 , 158.7 , 158.6 , 155.1 , 155.0 , 154.5 , 144.5 , 144.4 , 144.3 , 144.2 , 
 144.1 , 144.0 , 143.9 , 141.4 , 141.4 , 141.3 , 131.8 , 130.5 , 128.5 , 128.3 , 
 128.0 , 127.8 , 127.1 , 125.5 , 125.4 , 125.4 , 125.3 , 120.0 , 118.8 , 114.0 , 
 113.9 , 79.7 , 78.8 , 77.4 , 76.2 , 75.9 , 75.6 , 71.8 , 70.9 , 70.3 , 67.9 , 
 67.8 , 67.6 , 66.1 , 65.5 , 60.5 , 59.8 , 59.7 , 59.7 , 55.3 , 50.4 , 49.9 , 
 47.3 , 47.3 , 47.2 , 47.1 , 46.6 , 45.8 , 45.2 , 45.0 , 40.8 , 39.8 , 39.6 , 
 38.1 , 37.8 , 37.7 , 37.3 , 37.3 , 34.8 , 34.7 , 33.5 , 33.4 , 32.1 , 31.7 , 
 31.4 , 31.0 , 31.0 , 30.7 , 30.6 , 30.1 , 30.1 , 29.8 , 26.1 , 25.7 , 25.4 , 
 25.2 , 25.1 , 24.6 , 23.4 , 22.8 , 20.7 , 20.6 , 16.8 , 16.4 , 15.9 , 14.4 , 
 14.4 , 14.3 , 13.9 , 11.8 , 11.6 , 10.7 ppm .
hrms ( esi ) m / z calcd for c62h85n5o11sna ( m + na ) 1130.5859 , found 1130.5905 . to a solution of cyclic precursor 36 ( 11.6 
 mol ) in thf ( 1.0 ml ) were added pd(pph3)4 ( 2.7 mg , 2.32 mol ) and n - methylaniline 
 ( 6.3 l , 58.0 mol ) at room temperature under argon .
this 
 reaction was protected with aluminum foil . after being stirred at 
 the same temperature for 1 h
, the reaction mixture was concentrated 
 in vacuo and purified by preparative tlc plate ( developed with meoh / ch2cl2 1:9 , v / v ) to give the free acid cyclic precursor .
to the solution of free acid cyclic precursor in mecn ( 1.5 ml ) 
 was added n , n - diethylamine ( 0.75 
 ml ) . after being stirred at room temperature for 30 min ,
the reaction 
 mixture was evaporated in vacuo , azeotroped with toluene ( three times ) 
 and ch2cl2 ( two times ) , and then dried under 
 reduced pressure for 1 h to give the unmasked precursor as a foam 
 solid . then the unmasked precursor was dissolved in ch2cl2 ( dmf for 36b ) ( 20 ml ) . to this solution 

were added diea ( 20.0 l , 0.116 mmol ) and corresponding coupling 
 reagent ( 34.8 mol ) at 0 c ( pyaop for 36a , c , d and depbt for 36b ) .
after 
 being stirred at 0 c for 30 min , the reaction was allowed to 
 warm up to room temperature and stirred for additional 15 h. then 
 the reaction was concentrated in vacuo and purified by semipreparative 
 reversed - phase hplc ( phenomenex ultracarb , ods 250 mm  10 mm , 
 5 m , 3.0 ml / min , uv detection at 200/220 nm ) using an isocratic 
 system of 80% aqueous mecn for 30 min , 80100% mecn for 3040 
 min , and 100% mecn for 4060 min to afford 1a  d .
yield 
 2.3 mg , 25% in 
 3 steps ; [ ]d 106.2 ( c 0.024 , ch2cl2 ) ; tr = 17.5 min .
h nmr ( 600 mhz , cdcl3 , mixture of rotamers , major and minor ( 6/4 ) ) :  7.14 ( d , j = 8.4 hz , 1.2h ) , 7.10 ( d , j = 8.4 hz , 
 0.8h ) , 6.80 ( d , j = 8.4 hz , 0.8h ) , 6.79 ( d , j = 8.4 hz , 1.2h ) , 6.17 ( d , j = 9.0 hz , 
 0.4h ) , 5.78 ( d , j = 10.2 hz , 0.6h ) , 5.26 ( d , j = 11.4 hz , 0.6h ) , 5.205.16 ( m , 0.4h ) , 5.15 ( ddd , j = 15.0 , 10.2 , 4.8 hz , 0.6h ) , 4.96 ( dd , j = 12.6 , 2.4 hz , 0.6h ) , 4.89 ( d , j = 11.4 hz , 0.4h ) , 
 4.87 ( dd , j = 12.6 , 2.4 hz , 0.4h ) , 4.64 ( q , j = 6.6 hz , 0.4h ) , 4.60 ( d , j = 10.8 hz , 
 0.6h ) , 4.384.31 ( m , 1.2h ) , 4.244.19 ( m , 1h ) , 4.114.08 
 ( m , 0.4h ) , 4.05 ( br m , 0.4h ) , 4.003.95 ( m , 0.6h ) , 3.90 ( br 
 m , 0.4h ) , 3.77 ( s , 1.8h ) , 3.77 ( s , 1.2h ) , 3.703.66 ( m , 0.6h ) , 
 3.653.61 ( m , 0.4h ) , 3.39 ( dd , j = 10.8 , 8.4 
 hz , 0.6h ) , 3.32 ( dd , j = 10.8 , 8.4 hz , 0.4h ) , 3.29 ( q , j = 6.6 hz , 0.6h ) , 3.09 ( dd , j = 12.0 , 11.4 hz , 1h ) , 
 3.053.01 ( m , 1h ) , 2.94 ( dd , j = 12.6 , 4.2 
 hz , 0.4h ) , 2.88 ( s , 1.2h ) , 2.80 ( s , 1.8h ) , 2.77 ( dd , j = 12.6 , 4.8 hz , 0.6h ) , 2.74 ( s , 1.8h ) , 2.642.58 ( m , 1.8h ) , 
 2.52 ( dd , j = 13.5 , 11.4 hz , 0.6h ) , 2.452.32 
 ( m , 1.6h ) , 2.312.23 ( m , 1.6h ) , 2.151.96 ( m , 2.4h ) , 
 1.961.83 ( m , 3.6h ) , 1.811.69 ( m , 4h ) , 1.62 ( ddd , j 
 = 13.8 , 9.6 , 3.6 hz , 1h ) , 1.561.51 ( m , 1h ) , 1.311.25 
 ( m , 1h ) , 1.23 ( d , j = 7.2 hz , 1.8h ) , 1.211.17 
 ( m , 0.4h ) , 1.09 ( d , j = 6.6 hz , 1.2h ) , 1.03 ( t , j = 6.6 hz , 1.8h ) , 1.03 ( d , j = 6.6 hz , 
 1.8h ) , 1.00 ( d , j = 6.6 hz , 1.2h ) , 0.98 ( d , j = 6.6 hz , 1.2h ) , 0.980.92 ( m , 0.6h ) , 0.87 ( s , 
 9h ) , 0.84 ( t , j = 7.2 hz , 1.2h ) , 0.57 ( d , j = 6.6 hz , 1.2h ) ppm .
c nmr ( 150 mhz , cdcl3 , mixture of rotamers , major and minor ) :  172.7 , 172.1 , 
 171.9 , 171.2 , 170.7 , 170.5 , 170.3 , 170.2 , 170.0 , 168.6 , 158.8 , 158.7 , 
 130.7 , 130.6 , 128.7 , 128.5 , 114.2 , 114.0 , 78.1 , 77.6 , 76.2 , 75.8 , 
 68.6 , 67.3 , 60.7 , 59.9 , 59.5 , 58.1 , 57.9 , 57.4 , 55.5 , 55.4 , 54.1 , 
 50.9 , 49.8 , 47.8 , 45.0 , 43.9 , 42.9 , 41.0 , 39.8 , 38.8 , 37.6 , 37.5 , 
 37.4 , 37.0 , 36.9 , 34.1 , 34.1 , 33.2 , 33.0 , 31.5 , 30.6 , 30.6 , 29.3 , 
 29.3 , 29.0 , 26.2 , 26.2 , 25.7 , 25.6 , 25.3 , 25.2 , 24.8 , 20.9 , 19.9 , 
 15.1 , 14.3 , 14.1 , 14.1 , 10.0 , 10.0 ppm .
hrms ( esi ) m / z calcd for c43h67n5o8sna ( m + na ) 836.4603 , found 836.4607 . yield 6.8 mg , 70% in 
 3 steps ; [ ]d 59.5 ( c 0.037 , ch2cl2 ) ; tr = 26.2 min .
h nmr ( 600 mhz , cdcl3 , mixture of rotamers , major and minor ( 7/3 ) ) :  7.14 ( d , j = 8.4 hz , 0.6h ) , 7.12 ( d , j = 8.4 hz , 
 1.4h ) , 6.80 ( d , j = 8.4 hz , 1.4h ) , 6.78 ( d , j = 8.4 hz , 0.6h ) , 6.18 ( d , j = 9.0 hz , 
 0.7h ) , 5.73 ( d , j = 10.2 hz , 0.3h ) , 5.27 ( d , j = 11.4 hz , 0.3h ) , 5.185.11 ( m , 1h ) , 4.96 ( d , j = 12.0 hz , 0.3h ) , 4.89 ( d , j = 10.8 hz , 
 1h ) , 4.60 ( q , j = 6.0 hz , 0.7h ) , 4.454.37 
 ( m , 1h ) , 4.354.31 ( m , 0.7h ) , 4.15 ( t , j = 
 8.4 hz , 0.3h ) , 4.104.06 ( m , 0.7h ) , 3.87 ( d , j = 10.8 hz , 0.7h ) , 3.76 ( s , 3h ) , 3.70 ( t , j = 10.8 hz , 0.7h ) , 3.633.59 
 ( m , 1h ) , 3.34 ( dd , j = 10.8 , 8.4 hz , 0.3h ) , 3.31 
 ( q , j = 6.6 hz , 0.3h ) , 3.24 ( dd , j = 10.8 , 8.4 hz , 0.7h ) , 3.12 ( dd , j = 12.6 , 11.4 
 hz , 0.3h ) , 3.093.04 ( m , 1.4h ) , 2.95 ( dd , j = 12.6 , 3.6 hz , 0.7h ) , 2.88 ( s , 2.1h ) , 2.87 ( s , 0.9h ) , 2.76 ( s , 
 0.9h ) , 2.752.73 ( m , 0.3h ) , 2.58 ( s , 2.1h ) , 2.41 ( ddd , j = 13.2 , 13.2 , 4.8 hz , 0.7h ) , 2.362.26 ( m , 2.3h ) , 
 2.232.18 ( m , 1h ) , 2.142.10 ( br m , 0.7h ) , 2.082.02 
 ( m , 1.3h ) , 1.981.91 ( m , 3.3h ) , 1.891.82 ( m , 2.3h ) , 
 1.781.73 ( m , 3.3h ) , 1.601.56 ( m , 0.7h ) , 1.51 ( m , 1h ) , 
 1.291.23 ( m , 2.8h ) , 1.17 ( s , 0.9h ) , 1.16 ( s , 2.1h ) , 1.071.06 
 ( m , 5.1h ) , 1.03 ( t , j = 7.2 hz , 0.9h ) , 0.98 ( d , j = 6.6 hz , 3h),0.87 ( s , 9h ) , 0.83 ( t , j = 7.2 hz , 2.1h ) , 0.49 ( d , j = 6.6 hz , 2.1h ) ppm .
c nmr ( 150 mhz , cdcl3 , mixture of rotamers , major 
 and minor ) :  179.6 , 178.4 , 172.2 , 172.0 , 172.0 , 171.8 , 170.9 , 
 170.4 , 170.3 , 170.2 , 170.1 , 169.8 , 158.9 , 158.6 , 130.7 , 130.6 , 128.7 , 
 128.5 , 114.2 , 113.9 , 77.7 , 75.8 , 75.3 , 74.8 , 73.9 , 60.8 , 60.0 , 59.3 , 
 57.8 , 57.4 , 55.5 , 55.4 , 54.2 , 50.9 , 49.8 , 47.8 , 46.7 , 46.5 , 40.0 , 
 38.2 , 38.0 , 37.6 , 37.3 , 37.0 , 36.0 , 35.2 , 35.1 , 34.9 , 34.6 , 34.1 , 
 33.7 , 31.7 , 31.4 , 30.8 , 30.8 , 30.7 , 29.8 , 29.5 , 29.3 , 28.9 , 26.3 , 
 26.3 , 25.8 , 25.6 , 25.6 , 25.5 , 25.3 , 25.0 , 24.2 , 22.8 , 20.7 , 20.3 , 
 18.7 , 18.3 , 14.9 , 14.6 , 14.3 , 14.1 , 14.1 , 10.7 , 10.0 ppm . hrms ( esi ) m / z calcd for c45h71n5o8sna ( m + na ) 864.4916 , found 
 864.4918 . yield 
 4.3 mg , 45% in 
 3 steps ; [ ]d 82.6 ( c 0.023 , ch2cl2 ) ; tr = 24.8 min .
h nmr ( 600 mhz , cdcl3 , mixture of rotamers , major and minor ( 6/4 ) ) :  7.13 ( d , j = 9.0 hz , 1.2h ) , 7.13 ( d , j = 9.0 hz , 
 0.8h ) , 6.82 ( d , j = 9.0 hz , 0.8h ) , 6.79 ( d , j = 9.0 hz , 1.2h ) , 6.23 ( d , j = 8.4 hz , 
 0.4h ) , 5.86 ( d , j = 9.6 hz , 0.6h ) , 5.23 ( d , j = 11.4 hz , 0.6h ) , 5.19 ( ddd , j = 10.5 , 
 10.5 , 4.8 hz , 0.6h ) , 5.05 ( ddd , j = 10.8 , 8.4 , 4.2 
 hz , 0.4h ) , 4.95 ( dd , j = 13.2 , 3.0 hz , 0.6h ) , 4.88 
 ( dd , j = 12.6 , 3.6 hz , 0.4h ) , 4.82 ( d , j = 11.4 hz , 0.4h ) , 4.73 ( d , j = 11.4 hz , 0.6h ) , 
 4.59 ( q , j = 6.6 hz , 0.4h ) , 4.23 ( qd , j = 8.4 , 4.8 hz , 0.4h ) , 4.364.34 ( m , 0.4h ) , 4.314.23 
 ( m , 1h ) , 4.23 ( t , j = 7.8 hz , 0.6h ) , 4.19 ( d , j = 10.2 hz , 0.4h ) , 4.134.09 ( m , 0.4h ) , 3.77 ( s , 
 1.8h ) , 3.77 ( s , 1.2h ) , 3.693.64 ( m , 0.6h ) , 3.643.60 
 ( m , 0.4h ) , 3.553.51 ( m , 0.4h ) , 3.50 ( dd , j = 10.8 , 7.8 hz , 0.4h ) , 3.46 ( qd , j = 11.1 , 3.6 hz , 0.6h ) , 3.38 ( dd , j = 10.8 , 7.8 hz , 0.6h ) , 3.26 ( q , j = 6.6 
 hz , 0.6h ) , 3.092.98 ( m , 1.4h ) , 2.94 ( s , 1.2h ) , 2.802.76 
 ( m , 1.6h ) , 2.74 ( s , 1.8h ) , 2.74 ( s , 1.8h ) , 2.70 ( dd , j = 11.4 , 11.4 hz , 0.6h ) , 2.632.57 ( m , 2.2h ) , 2.44 ( ddd , j = 16.8 , 5.4 , 3.0 hz , 0.6h ) , 2.412.34 ( m , 1h ) , 
 2.302.22 ( m , 1.2h ) , 2.202.17 ( m , 0.4h ) , 2.162.10 
 ( m , 1h ) , 2.092.03 ( m , 1.4h ) , 1.981.84 ( m , 2.4h ) , 1.79 
 ( td , j = 13.2 , 3.6 hz , 0.6h ) , 1.751.61 ( m , 
 4.4h ) , 1.581.52 ( m , 0.6h ) , 1.49 ( td , j = 
 11.4 , 3.6 hz , 0.6h ) , 1.33 ( ddd , j = 13.8 , 11.1 , 3.0 
 hz , 0.4h ) , 1.291.26 ( m , 1.4h ) , 1.23 ( d , j = 7.2 hz , 1.8h ) , 1.121.09 ( m , 2.2h ) , 1.07 ( d , j = 6.6 h , 1.8h ) , 1.05 ( d , j = 7.2 hz , 1.8h ) , 1.01 
 ( t , j = 7.2 hz , 1.8h ) , 0.970.95 ( m , 3.6h ) , 
 0.92 ( m , 0.4h ) , 0.87 ( s , 5.4h ) , 0.87 ( s , 3.6h ) , 0.85 ( t , j = 7.2 hz , 1.2h ) , 0.43 ( d , j = 6.6 hz , 1.2h ) ppm . c nmr ( 150 mhz , cdcl3 , mixture of rotamers , major 
 and minor ) :  175.3 , 174.9 , 172.6 , 172.2 , 171.9 , 171.4 , 171.1 , 
 170.7 , 170.5 , 170.5 , 170.3 , 170.0 , 158.9 , 158.7 , 130.7 , 130.5 , 128.7 , 
 128.4 , 114.3 , 114.0 , 78.0 , 77.6 , 75.5 , 74.9 , 72.5 , 72.1 , 60.7 , 59.8 , 
 59.3 , 58.3 , 56.9 , 55.5 , 55.4 , 53.8 , 51.6 , 49.6 , 49.0 , 47.8 , 47.5 , 
 40.0 , 39.4 , 38.3 , 38.0 , 37.8 , 37.6 , 37.5 , 37.1 , 36.8 , 35.2 , 35.2 , 
 34.2 , 34.0 , 33.2 , 32.5 , 31.6 , 31.1 , 30.8 , 30.5 , 29.3 , 29.2 , 28.8 , 
 26.2 , 25.8 , 25.7 , 25.3 , 25.2 , 24.8 , 24.7 , 20.6 , 20.1 , 16.5 , 16.4 , 
 14.6 , 14.3 , 14.0 , 14.0 , 10.1 , 9.9 ppm . hrms ( esi ) m / z calcd for c44h69n5o8sna ( m + na ) 850.4759 , found 850.4773 . human 
 colon adenocarcinoma hct116 cells 
 were purchased from atcc ( manassas , va ) and cultured in dulbecco s 
 modified eagle s medium ( invitrogen , carlsbad , ca ) supplemented 
 with 10% fetal bovine serum ( hyclone , logan , ut ) at 37 c humidified 
 air and 5% co2 .
hct116 
 cells were seeded 
 at a density of 1  10 cells per well in 96-well 
 plates . then 24 h later , the cells were treated with various concentrations 
 of apratoxins or solvent control ( 1% etoh ) .
after 48 h of incubation , 
 cell viability was detected using mtt according to the manufacturer s 
 instructions ( promega , madison , wi ) .
hct116 cells ( 1  
 10 cells per well ) were seeded in 96-well plates and one 
 day later treated with various concentrations of apratoxins or solvent 
 control .
after 12 h of incubation , culture supernatants were collected 
 for detection of vegf - a by using an alphalisa kit ( perkinelmer , waltham , 
 ma ) following the manufacturer s instructions .
briefly , acceptor 
 bead and anti - vegf antibody were incubated first with the supernatants 
 for 60 min , donor beads were added later and incubated for another 
 30 min , and then vegf - a levels were detected using envision ( perkinelmer ) .
hct116 cells were seeded in 6-well 
 plates at a density of 4  10 cells and the next 
 day treated with various concentrations of apratoxins or solvent control 
 ( 1% etoh ) . then 24 h later , whole cell lysates were collected using 
 phosphosafe buffer ( emd chemicals , inc . , gibbstown , nj ) .
the protein 
 concentration was measured with the bca protein assay kit ( thermo 
 fisher scientific , rockford , il ) .
lysates containing equal amounts 
 of protein were separated by sds polyacrylamide gel electrophoresis 
 ( 412% ) , transferred to polyvinylidene difluoride membranes , 
 probed with primary and secondary antibodies , and detected with the 
 supersignal west femto maximum sensitivity substrate ( thermo fisher 
 scientific ) .
anti - met , anti - vegfr2 , and secondary antimouse antibodies 
 were from cell signaling technology , inc .
three  five week old 
 female nude mice ( nu / nu ) were obtained 
 from charles river laboratory ( wilmington , ma ) .
one 10 hct116 cells in a volume of 100 l of sterile saline were 
 injected subcutaneously on the left rear flank of a nude mouse to 
 establish tumors .
tumor dimensions were measured using calipers every 
 day , and tumor volumes were calculated using the formula w  l  0.5 , where width ( w )  length ( l ) .
tumors with a starting 
 volume bigger than 100 mm were excluded from the analysis . 

mice were injected intraperitoneally with the doses of 2 g / mouse 
 ( 0.1 mg / kg ) , 5 g / mouse ( 0.25 mg / kg ) of 1c or solvent 
 ( dmso ) control every day ( 25 l ) until the tumor size in one 
 dimension reached 15 mm and tumor tissue was harvested on the following 
 day .
finally , 50 mg of tumor tissue was sonicated in phosphosafe lysis 
 buffer ( emd chemicals , inc . ) and used for immunoblot analysis described 
 as the above .
all studies were carried out under the protocol approved 
 by the institutional animal care and use committee at the university 
 of florida .
hplc - ms was done on 
 a 3200 qtrap ( applied biosystems ) equipped with a shimadzu ( kyoto , 
 japan ) uflc system .
pooled cd1 mouse liver ( female ) microsomes were purchased from xenotech , 
 lcc ( lenexa , ks ) with protein concentrations of 0.5 mg / ml .
cell lysates were prepared with phosphosafe lysis buffer ( novagen , 
 san diego , ca ) .
stock solutions 
 of 1a  d were prepared by dissolving 
 the compounds 
 in ethanol to give a 1 mg / ml solution .
aliquots of this stock solution 
 were then obtained to afford a 40 g / ml solution in acetonitrile . 

serial dilution of the 40 g / ml solution in acetonitrile gave 
 standard solutions with concentrations of 25 , 12.5 , 2.5 , 1.25 , 0.25 , 
 0.125 , 0.025 , and 0.0125 g / ml . a 1 mg / ml stock solution of 
 the internal standard harmine was prepared in ethanol , which subsequently 
 was used to prepare a 100 g / ml solution with ethanol .
an aliquot 
 of the 100 g / ml harmine solution was diluted to 35 ng / ml with 
 ethyl acetate to serve as the working internal standard solution . in vitro plasma stability of 1a
first , 10 l 
 of apratoxins ( 25 g / ml ) were added to 100 l of mouse 
 serum , and the solution was then vortex - mixed for 15 s and incubated 
 for 0.25 min to 24 h. at the end of each incubation period , 400 l 
 of ethyl acetate was added to each tube , followed by 200 l 
 of harmine to quench the reaction and extract remaining apratoxin . 

samples were further incubated in a thermomixer at 27 c ( 750 
 rpm , 5 min ) and later centrifuged for 5 min at 1643 g .
a volume of 10 l of the reconstituted solution was injected 
 into the hplc - ms system .
the stability 
 of 1a  d in the presence of mouse 
 microsomes was determined 
 by using an adaptation of a published procedure . in brief , microsomes were added to prewarmed phosphate 
 buffer ( 100 mm , ph 7.4 ) at 37 c .
apratoxins ( 3 l ) were 
 added to the microsomal preparation followed by nadph cofactor solution 
 ( 1.3 mm nadp , 3.3 mm glucose 6-phosphate , 0.4 u / ml glucose-6-phosphate 
 dehydrogenase , 3.3 mm mgcl2 ) .
the reaction was allowed 
 to proceed for 3 min to 2 h at 37 c ( thermomixer , 1050 rpm ) . 

the reaction was quenched by addition of ethyl acetate and subsequently 
 spiked with harmine .
the zero time point was defined by denaturing 
 the microsomes with ethyl acetate before the addition of apratoxins . 

incubation of apratoxins with microsomes alone was also performed 
 following the same procedure to determine nadph - independent metabolism . 

the final concentration of the incubation mixture contained 0.5 mg / ml 
 protein concentration and 1 m apratoxins .
aliquots of hct116 cell lysates 
 were diluted with 25 mm tris - hcl buffer , ph 8.0 , to give a final reaction 
 volume of 100 l and protein concentration of 0.7 mg / ml . cell 
 lysate solutions were incubated with 10 l of apratoxins ( 25 
 g / ml ) for 0.25 min to 24 h. remaining apratoxins were extracted 
 from the reaction solution at the end of the incubation periods with 
 ethyl acetate using the same procedure as described for the plasma 
 stability assay .
the stability 
 of 1a  d in aqueous solution was determined 
 in 100 mm phosphate buffer , 
 ph 4.88 , 100 mm phosphate buffer , ph 7.4 .
portions of each solution 
 ( 100 l ) were spiked with 10 l of apratoxin solution 
 ( 25 g / ml ) and allowed to incubate for 0.25 min to 24 h. the 
 reaction was quenched at the end of each time point , and the remaining 
 apratoxins were extracted with ethyl acetate , as in the plasma stability 
 study .
analysis of 1a  d was done by using hplc - ms [ column , onyx monolithic c18 ( 3.0 
 mm  100 mm ) , phenomenex ( torrance , ca ) ; solvent , water ( solvent 
 a ) acetonitrile ( solvent b ) ; flow rate , 0.5 ml / min ; detection by electrospray 
 ionization  ms in positive ion mode ( mrm scan ) ] .
a stepwise 
 gradient elution was used starting at 60% b and 40% a , then increasing 
 to 80% b at 5 min and maintained at this condition for 5 min .
parameters 
 were optimized before analysis by using direct syringe infusion . the 
 retention times ( tr , min ; mrm ion pair ) 
 of the analytes and internal standard
are as follows : harmine ( 2.2 ; 
 213.1  169.9 ) , 1a ( 4.1 ; 828.5  432.2 ) , 1b ( 4.05 ; 814.5  418.2 ) , 1c ( 5.2 ; 842.5 
  446.2 ) , 1d ( 4.9 ; 828.5  432.2 ) .
compound - dependent 
 parameters used were as follows : apratoxins , declustering potential 
 ( dp ) 51 , entrance potential ( ep ) 12 , collision energy ( ce ) 45 , collision 
 cell exit potential ( cxp ) 6 , collision cell entrance potential ( cep ) 
 32 ; and harmine , dp 56.0 , ep 4.5 , ce 44.0 , cxp 5 , cep 16.0 .
source 
 gas parameters used were as follows : curtain gas , 15.0 ; collision 
 gas low , ionspray voltage 5500 ; temperature , 600.0 ; ion source gas 
 1 50.0 ; ion source gas 2 60.0 .
calibration curves for 1a  d in the 
 presence of mouse serum , hct116 cell 
 lysates , and aqueous solutions were generated by least - squares linear 
 regression analysis of the analyte peak area and internal standard 
 peak area ratio against the nominal concentration of the standard 
 solutions .
a linear regression analysis was performed , and the concentration 
 of remaining apratoxins at each time point was determined through 
 interpolation for plasma , cellular , and aqueous stability experiments . 
 the amount of remaining apratoxins with microsome incubation was determined 
 from the peak area ratio of apratoxins at tx ( 3 min to 2 h ) and t0 .
all calculations 
 were done by using analyst 1.4.2 ( applied biosystems ) quantitate mode .
human 
 colon adenocarcinoma hct116 cells 
 were purchased from atcc ( manassas , va ) and cultured in dulbecco s 
 modified eagle s medium ( invitrogen , carlsbad , ca ) supplemented 
 with 10% fetal bovine serum ( hyclone , logan , ut ) at 37 c humidified 
 air and 5% co2 .
hct116 
 cells were seeded 
 at a density of 1  10 cells per well in 96-well 
 plates . then 24 h later , the cells were treated with various concentrations 
 of apratoxins or solvent control ( 1% etoh ) .
after 48 h of incubation , 
 cell viability was detected using mtt according to the manufacturer s 
 instructions ( promega , madison , wi ) .
hct116 cells ( 1  
 10 cells per well ) were seeded in 96-well plates and one 
 day later treated with various concentrations of apratoxins or solvent 
 control .
after 12 h of incubation , culture supernatants were collected 
 for detection of vegf - a by using an alphalisa kit ( perkinelmer , waltham , 
 ma ) following the manufacturer s instructions .
briefly , acceptor 
 bead and anti - vegf antibody were incubated first with the supernatants 
 for 60 min , donor beads were added later and incubated for another 
 30 min , and then vegf - a levels were detected using envision ( perkinelmer ) .
hct116 cells were seeded in 6-well 
 plates at a density of 4  10 cells and the next 
 day treated with various concentrations of apratoxins or solvent control 
 ( 1% etoh ) . then 24 h later , whole cell lysates were collected using 
 phosphosafe buffer ( emd chemicals , inc . , gibbstown , nj ) .
the protein 
 concentration was measured with the bca protein assay kit ( thermo 
 fisher scientific , rockford , il ) .
lysates containing equal amounts 
 of protein were separated by sds polyacrylamide gel electrophoresis 
 ( 412% ) , transferred to polyvinylidene difluoride membranes , 
 probed with primary and secondary antibodies , and detected with the 
 supersignal west femto maximum sensitivity substrate ( thermo fisher 
 scientific ) .
anti - met , anti - vegfr2 , and secondary antimouse antibodies 
 were from cell signaling technology , inc .
three  five week old 
 female nude mice ( nu / nu ) were obtained 
 from charles river laboratory ( wilmington , ma ) .
one 10 hct116 cells in a volume of 100 l of sterile saline were 
 injected subcutaneously on the left rear flank of a nude mouse to 
 establish tumors .
tumor dimensions were measured using calipers every 
 day , and tumor volumes were calculated using the formula w  l  0.5 , where width ( w )  length ( l ) .
tumors with a starting 
 volume bigger than 100 mm were excluded from the analysis . 

mice were injected intraperitoneally with the doses of 2 g / mouse 
 ( 0.1 mg / kg ) , 5 g / mouse ( 0.25 mg / kg ) of 1c or solvent 
 ( dmso ) control every day ( 25 l ) until the tumor size in one 
 dimension reached 15 mm and tumor tissue was harvested on the following 
 day .
finally , 50 mg of tumor tissue was sonicated in phosphosafe lysis 
 buffer ( emd chemicals , inc . ) and used for immunoblot analysis described 
 as the above .
all studies were carried out under the protocol approved 
 by the institutional animal care and use committee at the university 
 of florida .
hplc - ms was done on 
 a 3200 qtrap ( applied biosystems ) equipped with a shimadzu ( kyoto , 
 japan ) uflc system . mouse serum and harmine
pooled cd1 mouse liver ( female ) microsomes were purchased from xenotech , 
 lcc ( lenexa , ks ) with protein concentrations of 0.5 mg / ml . hct116 
 cell lysates were prepared with phosphosafe lysis buffer ( novagen , 
 san diego , ca ) .
stock solutions 
 of 1a  d were prepared by dissolving 
 the compounds 
 in ethanol to give a 1 mg / ml solution .
aliquots of this stock solution 
 were then obtained to afford a 40 g / ml solution in acetonitrile . 

serial dilution of the 40 g / ml solution in acetonitrile gave 
 standard solutions with concentrations of 25 , 12.5 , 2.5 , 1.25 , 0.25 , 
 0.125 , 0.025 , and 0.0125 g / ml . a 1 mg / ml stock solution of 
 the internal standard harmine was prepared in ethanol , which subsequently 
 was used to prepare a 100 g / ml solution with ethanol .
an aliquot 
 of the 100 g / ml harmine solution was diluted to 35 ng / ml with 
 ethyl acetate to serve as the working internal standard solution . in vitro plasma stability of 1a
first , 10 l 
 of apratoxins ( 25 g / ml ) were added to 100 l of mouse 
 serum , and the solution was then vortex - mixed for 15 s and incubated 
 for 0.25 min to 24 h. at the end of each incubation period , 400 l 
 of ethyl acetate was added to each tube , followed by 200 l 
 of harmine to quench the reaction and extract remaining apratoxin . 

samples were further incubated in a thermomixer at 27 c ( 750 
 rpm , 5 min ) and later centrifuged for 5 min at 1643 g .
a volume of 10 l of the reconstituted solution was injected 
 into the hplc - ms system .
the stability 
 of 1a  d in the presence of mouse 
 microsomes was determined 
 by using an adaptation of a published procedure . in brief
, microsomes were added to prewarmed phosphate 
 buffer ( 100 mm , ph 7.4 ) at 37 c .
apratoxins ( 3 l ) were 
 added to the microsomal preparation followed by nadph cofactor solution 
 ( 1.3 mm nadp , 3.3 mm glucose 6-phosphate , 0.4 u / ml glucose-6-phosphate 
 dehydrogenase , 3.3 mm mgcl2 ) .
the reaction was allowed 
 to proceed for 3 min to 2 h at 37 c ( thermomixer , 1050 rpm ) . 

the reaction was quenched by addition of ethyl acetate and subsequently 
 spiked with harmine .
the zero time point was defined by denaturing 
 the microsomes with ethyl acetate before the addition of apratoxins . 

incubation of apratoxins with microsomes alone was also performed 
 following the same procedure to determine nadph - independent metabolism . 

the final concentration of the incubation mixture contained 0.5 mg / ml 
 protein concentration and 1 m apratoxins .
aliquots of hct116 cell lysates 
 were diluted with 25 mm tris - hcl buffer , ph 8.0 , to give a final reaction 
 volume of 100 l and protein concentration of 0.7 mg / ml . cell 
 lysate solutions were incubated with 10 l of apratoxins ( 25 
 g / ml ) for 0.25 min to 24 h. remaining apratoxins were extracted 
 from the reaction solution at the end of the incubation periods with 
 ethyl acetate using the same procedure as described for the plasma 
 stability assay .
the stability 
 of 1a  d in aqueous solution was determined 
 in 100 mm phosphate buffer , 
 ph 4.88 , 100 mm phosphate buffer , ph 7.4 .
portions of each solution 
 ( 100 l ) were spiked with 10 l of apratoxin solution 
 ( 25 g / ml ) and allowed to incubate for 0.25 min to 24 h. the 
 reaction was quenched at the end of each time point , and the remaining 
 apratoxins were extracted with ethyl acetate , as in the plasma stability 
 study .
analysis of 1a  d was done by using hplc - ms [ column , onyx monolithic c18 ( 3.0 
 mm  100 mm ) , phenomenex ( torrance , ca ) ; solvent , water ( solvent 
 a ) acetonitrile ( solvent b ) ; flow rate , 0.5 ml / min ; detection by electrospray 
 ionization  ms in positive ion mode ( mrm scan ) ] .
a stepwise 
 gradient elution was used starting at 60% b and 40% a , then increasing 
 to 80% b at 5 min and maintained at this condition for 5 min .
the 
 retention times ( tr , min ; mrm ion pair ) 
 of the analytes and internal standard are as follows : harmine ( 2.2 ; 
 213.1  169.9 ) , 1a ( 4.1 ; 828.5  432.2 ) , 1b ( 4.05 ; 814.5  418.2 ) , 1c ( 5.2 ; 842.5 
  446.2 ) , 1d ( 4.9 ; 828.5  432.2 ) .
compound - dependent 
 parameters used were as follows : apratoxins , declustering potential 
 ( dp ) 51 , entrance potential ( ep ) 12 , collision energy ( ce ) 45 , collision 
 cell exit potential ( cxp ) 6 , collision cell entrance potential ( cep ) 
 32 ; and harmine , dp 56.0 , ep 4.5 , ce 44.0 , cxp 5 , cep 16.0 .
source 
 gas parameters used were as follows : curtain gas , 15.0 ; collision 
 gas low , ionspray voltage 5500 ; temperature , 600.0 ; ion source gas 
 1 50.0 ; ion source gas 2 60.0 .
calibration curves for 1a  d in the 
 presence of mouse serum , hct116 cell 
 lysates , and aqueous solutions were generated by least - squares linear 
 regression analysis of the analyte peak area and internal standard 
 peak area ratio against the nominal concentration of the standard 
 solutions .
a linear regression analysis was performed , and the concentration 
 of remaining apratoxins at each time point was determined through 
 interpolation for plasma , cellular , and aqueous stability experiments . 
 the amount of remaining apratoxins with microsome incubation was determined 
 from the peak area ratio of apratoxins at tx ( 3 min to 2 h ) and t0 .
all calculations 
 were done by using analyst 1.4.2 ( applied biosystems ) quantitate mode .
 apratoxins 
 are cytotoxic natural products originally isolated from 
 marine cyanobacteria that act by preventing cotranslational translocation 
 early in the secretory pathway to downregulate receptor levels and 
 inhibit growth factor secretion , leading to potent antiproliferative 
 activity . through rational design and total synthesis of an apratoxin 
 a / e hybrid , apratoxin s4 ( 1a ) , 
 we have previously improved 
 the antitumor activity and tolerability in vivo . 
 compound 1a and newly designed analogues apratoxins s7s9 ( 1b  d ) , with various degrees of methylation at c34 
 ( 1b , c ) or epimeric configuration at c30 
 ( 1d ) , were efficiently synthesized utilizing improved 
 procedures . 
 optimizations have been applied to the synthesis of key 
 intermediate aldehyde 7 and further include the application 
 of leighton s silanes and modifications of kelly s methods 
 to induce thiazoline ring formation in other crucial steps of the 
 apratoxin synthesis . 
 apratoxin s9 ( 1d ) exhibited increased 
 activity with subnanomolar potency . 
 apratoxin s8 ( 1c ) 
 lacks the propensity to be deactivated by dehydration and showed efficacy 
 in a human hct116 xenograft mouse model . 
Introduction
Results and Discussion
Conclusion
Experimental
Section
Biological Material and Methods
Metabolite Analyses