epigenetics are widely 
 implicated in tumor initiation and progression 
 by different modifications of dna and histones . there is a growing 
 body of evidence demonstrating the importance of histone modification 
 in silencing tumor suppressor genes . among 
 the various histone modifiers , histone acetyltransferases ( hats ) and 
 histone deacetylases ( hdacs ) are two reversible enzymes regulating 
 histone acetylation status , attaching or removing acetyl groups from 
 the lysine residues in histone tails . hats 
 prevent the positive lysine residues from interacting more closely 
 with the dna phosphate backbone , resulting in an  open  
 chromatin state , whereas the hdacs remove acetyl groups , resulting 
 in a  closed  configuration , which blocks the access 
 of the transcription machinery to dna , suppressing gene expression . to date , 18 mammalian hdac isoforms have 
 been identified in humans ; 
 11 of them ( class i , ii , iv ) are zn - dependent hdac enzymes 
 ( classical hdacs ) . class i hdac , comprising 
 hdac 1 , 2 , 3 , and 8 , are homologous to yeast rpd3 ( reduced potassium dependency-3 ) protein ; class ii members include 
 hdac 4 , 5 , 6 , 7 , 9 , and 10 and are structurally related to yeast hda 
 1 ( histone deacetylases 1 ) . class iii hdacs , called sirtuins ( sirt17 ) , require nad for their activity . hdac1 
 mainly localizes in the nucleus due to lack of the nucleus 
 export signal . the knockout of hdac1 in mice caused proliferation 
 defects and development retardation because of an arrest of cell growth 
 that is associated with upregulation of the p21 and p27 ( cyclin - dependent 
 kinase inhibitors ) . a recent study showed 
 that hdac1 largely accumulated in mature areas of differentiated tumor 
 on the mice with teratoma , indicating that hdac1 is a possible biomarker 
 of benign teratoma . hdac3 is one of the 
 most frequently upregulated genes in human cancers and is involved in each of the three major targets of cancer 
 therapy : cell cycle control , differentiation , and apoptosis . there are many direct links between hdac3 and 
 various tumor types . in colon cancer cells hct116 and caco-2 , silencing 
 of hdac3 expression resulted in growth inhibition , a decrease in cell 
 survival , and increased apoptosis through stimulating p21 promoter 
 activity and expression . in hela cells , the majority of cellular hdac3 is found to associate with smrt and 
 n - cor complexes , and knockdown of hdac3 resulted in hyperacetylation 
 of histone h3 and apoptosis . in metastatic breast 
 cancer cell mda - mb-231 , hdac3 efficiently inhibited creb3-enhanced 
 nf-b activation . moreover , high hdac3 expression is also associated 
 with gastric cancer , glioma , renal cancer , liver 
 cancer , and chronic lymphocytic leukemia . on the basis of the above evidence , developing 
 hdacis more specifically against hdac1/3 may prove to be a worthwhile 
 goal . in the past 10 years , over 490 clinical trials of more 
 than 20 
 hdacis candidates have been initiated , culminating in the approval 
 of two antitumor drugs vorinostat ( saha ) and romidepsin ( fk228 ) . recently , 
 development of class or isoform selective hdacis has drawn increased 
 attention . although selective hdacis were hypothesized to have fewer 
 side effects than other pan - hdacis , their therapeutic advantages have 
 yet to be confirmed clinically . hdacis are classified into different 
 classes depending on their chemical structures , namely , hydroxamates , 
 benzamides , aliphatic acids , cyclic tetra peptides , electrophillic 
 ketones , and miscellaneous groups , among which hydroxamates are considered 
 as the most common hdacis . although some class i selective , hdac6 ( class iib ) selective , and class 
 iia selective hydroxamates inhibitors 
 have been reported ( figure 1 ) , hydroxamates 
 are generally thought to have limitation in selectivity against desired 
 hdac isoforms due to their very strong chelating ability with zinc 
 ion . to the best of our knowledge , most 
 of the subclass i selective inhibitors in research are benzamides 
 hdacis , such as ms-275 ( 30 ) , mgcd0103 ( 31 ) , ci994 ( 32 ) , and so on ( figure 1 ) . additionally , recent study demonstrated 
 aryl substituents in the o - phenylenediamine of benzamide 
 hdacis led to dramatic improvements in potency and selectivity for 
 hdac1 and 2 versus hdac3 ( figure 1 ) . recently , we have embarked on development of n - hydroxycinnamamide - based hdacis ; in our previous study , compound 
 ( s , e)-tert - butyl 
 ( 1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-2-methoxyphenoxy)-3-(1h - indol-3-yl)propan-2-yl)carbamate ( 14a ) exhibited 
 modest hdac inhibitory . here in this 
 article , we report the design , synthesis , and biological evaluation 
 of a new series of selective hydroxymates hdacis describing their 
 structure  activity relationship ( sar ) studies , isotype - selectivity , 
 and anticancer activities . several 
 compounds show an excellent effect in proliferation inhibition against 
 u937 , hel , kg1 , hl60 , k562 , pc-3 , a549 , mda - mb-231 , hct116 , and mcf-7 
 cell lines . in vivo antitumor assay in u937 xenograft model showed 
 compound 11r is a potent , orally active hdaci . we designed and synthesized 
 a library of 
 cinnamamide derivatives based on the lead compound 41 ( 14a ) ( figure 2 ) . the library 
 was designed to fully explore the influence of variation in the methoxy 
 group of ferulic acid and the cap group . synthesis of 3 was started with l - try by methyl ester protection 
 and boc - protection . reduction of 3 by lialh4 led to 4 , which was further subjected to mitsunobu 
 reaction with 6 and gave 7 . intermediate 8 , the n - deprotected 
 product of 7 , reacted with different carboxylic acids 
 or boc - protected amino acids by tbtu - mediated amide formation to afford 9b9z . compounds 9b9z were converted to hydroxamic acid compounds 11a11z by nh2ok in dry methanol . reagents and conditions : ( a ) 
 ch3cocl , ch3oh , 95% ; ( b ) ( boc)2o , 
 et3n , ch2cl2 , 90% ; ( c ) lialh4 , anhydrous thf , 86% ; ( d ) pph3 , dead , anhydrous 
 thf , 55% ; ( e ) acoet / hcl , 80% ; ( f ) carboxylic acids or boc - protected 
 amino acids , tbtu , et3n , anhydrous ch2cl2 ( 6065% ) ; ( g ) nh2oh , koh , anhydrous ch3oh , ( 3040% ) . compound 4 was treated with ch3i and phase 
 transfer catalyst tbabr in h2o / thf containing koh to get 
 the methyl protected 12 ( scheme 2 ) . subsequent reaction of 12 with 6 through 
 mitsunobu reaction in the presence of pph3 and dead afforded 
 compound 13 . finally , the hydroxamic acid group was achieved 
 in usual conditions to get compound 14 . reagents and conditions : 
 ( a ) 
 ch3i , koh , tbabr , h2o , thf , 50% ; ( b ) pph3 , dead , anhydrous thf , 55% ; ( c ) nh2ok , anhydrous 
 ch3oh , 30% . scheme 3 shows the preparation of secondary 
 amine 21a21c . intermediate 8 was mixed with benzaldehyde , 4-chlorobenzaldehyde , or 4-fluorobenzaldehyde 
 in anhydrous ch3oh , respectively , then nabh4 was added to give 18a18c . reagents and conditions : ( a ) 
 benzaldehyde ( 4-chlorobenzaldehyde or 4-fluorobenzal dehyde ) , et3n , nabh4 , anhydrous ch3oh , 85% ; ( b ) 
 ( boc)2o , et3n , ch3oh , 65% ; ( c ) nh2ok , anhydrous ch3oh , 3040% ; ( d ) acoet / hcl , 
 70% . unlike scheme 1 , scheme 4 used 3-hydroxycinnamic acid instead 
 of 4-hydroxycinnamic 
 acid to give another new series of compounds 27a27c . the reagents and conditions of scheme 4 are the same as those in scheme 1 . reagents and conditions : ( a ) 
 ch3cocl , ch3oh , 95% ; ( b ) pph3 , dead , 
 anhydrous thf , 55% ; ( c ) acoet / hcl , 80% ; ( d ) carboxylic acids , tbtu , 
 et3n , anhydrous ch2cl2 ( 6065% ) ; 
 ( e ) nh2oh , koh , anhydrous ch3oh , ( 3040% ) . in our previous study we found the lead compound 41 displayed 
 modest inhibition toward hdacs . at the 
 beginning of our present research , we made an attempt to find a new 
 lead compound by modifying the r1 and r2 groups 
 of 41 ( table 1 ) . we changed the 
 r2 group of 41 by replacing the methoxy group 
 with h to get compound 10a ( scheme 1 ) and changed the r1 group of 10a with a 
 methyl group to get compound 14 ( scheme 2 ) . we used hela cell extract as the enzyme source to efficiently 
 screen our target compounds . the result in table 1 showed that compound 10a exhibited slightly 
 increased hdac inhibitory activity compared with 41 and 14 . this indicated that the methoxy group in r2 and methyl group in r1 were not necessary , so we selected 10a as the lead compound for further optimization and modification . assays were performed 
 in replicate 
 ( n  2 ) ; data are shown as mean  sd . in the following work , we designed 
 an analogue to probe the effect 
 of the n - substituent by replacing the boc group of 10a with other functional groups ( scheme 1 ) . first , we synthesized compounds 11a11o with different substituents including boc - protected amino 
 acids , carboxylic acids , sulfo acids , and several anti - inflammatory 
 acids ( table 2 ) . it was obvious that the compounds 
 with the structure of acid amide substituents were more potent than 
 those with sulfamide substituents ( table 2 ) . for example , the ic50 value of 11 g in hela 
 extract was 1703.4 nm , much lower than compound 11h ( > 5000 
 nm ) . the same result was also shown in compounds 11e and 11f . additionally , compound 11i was more potent 
 than 11j , which indicated that a short side alkyl chain 
 might advance hdac inhibitory activity . among compounds 11a11o , the most potent compounds , 11e and 11k , with ic50 values of 15.4 and 20.8 
 nm , respectively , were far more potent than saha ( 120.8 nm ) . the common 
 element they all share is a simple benzamide as their n - substituent , so further modification focused on compound 11e . assays were performed 
 in replicate 
 ( n  2 ) ; data are shown as mean  sd . next , we synthesized 11p and 11q , which 
 had different alkyl chain lengths relative to 11e . activity 
 data showed 11e with the shortest side chain had the 
 best activity , which also agreed with the conclusion shown in 11i and 11j , that a short side alkyl chain may 
 promote their activity . then we synthesized compounds 11r11y ( scheme 1 ) to probe 
 the effect of substituent in benzene ring of benzamide ( table 3 ) . surprisingly , the para - substituted compounds 11r , 11w , 11x , and 11y showed a marked increase in the hdac inhibition potency compared 
 with 11e , the ic50 values of 11r , 11w , 11x , and 11y were 5.6 , 
 6.7 , 20.4 , and 4.8 nm , respectively , however , the ortho - substituted , 
 metha - substituted , and disubstituted compounds 11s , 11 t , 11u , and 11v exhibited inferior 
 activity relative to 11e . the result showed para - substituents 
 on a benzene ring have an obvious promoting effect on hdac inhibition . assays were performed 
 in replicate 
 ( n  2 ) ; data are shown as mean  sd . scheme 4 was designed to evaluate the activity 
 of 3-hydroxycinnamide - based series . we modified some of the most potent 
 compounds , 11e , 11r , and 11w , to achieve compounds 27a27c . 
 the hdac inhibition result in table 4 showed 
 compounds 27a27c exhibited poor 
 activity compared with 11e , 11r , and 11w , which revealed that 4-hydroxycinnamide - based series were 
 superior to the 3-hydroxycinnamide - based series . then 21a21c were designed to improve water solubility and chemical stability 
 by replacing the amide of 11e , 11r , and 11w with a secondary amine . compounds 21a21c showed mild inhibition , and the activity was 15 times lower than 11e , 11r , and 11w . the possible 
 explanation for why compounds 11r , 21c , 
 and 27c with similar structure exhibited quite different 
 activity was described in the molecule docking section . assays were performed in replicate 
 ( n  2 ) ; values are shown as mean  sd . in order to explore the hdac isoform selectivity 
 profile , we chose 
 the most potent compounds 11e , 11p , 11r , 11w , and 11y described in the 
 above discussion , as well as secondary amine analogue 21c and 3-hydroxycinnamide - based analogue 27c to conduct 
 enzyme inhibitory assays against hdac1 , hdac2 , hdac3 , and hdac6 . results 
 presented in table 5 reveal that compounds 11e , 11r , 11w , and 11y exhibited dual selectivity against hdac1/3 , among which compound 11r exhibited the most obvious dual selectivity , particularly 
 against hdac3 . ic50 value of 11r against hdac3 
 was 3.9 nm , which was 80-fold and 130-fold lower than 
 that of hdac6 and hdac2 , respectively . the positive control , saha , 
 had almost no selectivity toward these hdac isoforms . to ascertain 
 the selectivities of our compounds across the broader family of hdac 
 isoforms , we next profiled representative 11r against 
 hdac8 ( class i ) , hdac4 ( class iia ) , and hdac11 ( class iv ) . compound 11r also displayed low micromolar activity against hdac8 , 
 hdac4 , and hdac11 compared to the low nanomolar activity against hdac1 
 and hdac3 ( table 6 ) . assays were performed in replicate 
 ( n  2 ) ; the sd values are < 20% of the 
 mean . assays were performed in replicate 
 ( n  2 ) ; the sd values are < 20% of the 
 mean . compounds 11e , 11p , 11r , 11w , and 11y with the most 
 potent hdacs inhibitory activity were selected 
 to test their antiproliferative activity against 10 types of solid 
 or hematological tumor cell lines , which were most frequently used 
 in evaluating hdacis ( table 7 ) . data showed 
 cell lines hl60 , mcf-7 , and a549 had lower sensitivity with our tested 
 compounds than the other cell lines . all the tested compounds exhibited 
 much more superior antiproliferative potency than saha against u937 , 
 k562 , hel , kg1 , mda - mb-231 , pc-3 , and hct116 cells . compounds 11e , 11r , 11w , and 11y , which displayed the highest hdac enzyme inhibitory potency , also 
 exhibited the best antiproliferative activity . among these analogues , 
 compound 11r exhibited the highest potency , and human 
 leukemic monocyte lymphoma u937 seemed to be the most sensitive cell 
 line to our hdacis . furthermore , cytotoxicity of 11r against human umbilical 
 vein endothelial cells ( huvec ) was tested in our lab , and the ic50 value was 61.48  6.17 m , which revealed our 
 compounds selectivity over nontransformed cells when compared 
 with tumor cells . assays were performed in replicate 
 ( n  2 ) ; the sd values are < 20% of the 
 mean . although 
 the hdac inhibitory activity of our hdacis was confirmed in cell - free 
 assays , the results were not as clear in cell based assays . in order 
 to investigate the hdac inhibitory activity of our hdacis in cells 
 further , we chose compounds that were used in evaluating hdac isoform - selectivity 
 to conduct western blot assay in u937 cell . intracellular levels of 
 acetylated histoneh3 , acetylated histoneh4 ( known substrates for hdac1 , 
 2 , and 3 ) , and acetylated -tubulin ( a known substrate for hdac6 ) were compared with saha and lbh589 ( one of the 
 most potent pan - hdacis ) as reference compounds ( figure 3 ) . results show compounds 11e , 11p , 11r , 11w , and 11y can markedly 
 increase the level of acetylated histoneh3 and acetylated histoneh4 , 
 which were much superior to saha , while comparable to lbh589 . however , 
 the levels of acetylated -tubulin of these compounds , especially 11r , were much lower than lbh589 . these data demonstrated 
 that some of our compounds showed lower potency against hdac6 than 
 class i hdacs , which is in agreement with the isoform selectivity 
 data observed in cell - free assays . western blot analysis of acetylated tubulin , 
 acetylated histone 
 h3 , acetylated histone h4 , and pro - caspase3 in u937 cell lines after 
 24 h treatment with compounds at 1 m using saha ( sa ) and lbh589 
 ( lbh ) as positive control . as our compounds displayed hdac1/3 
 dual selectivity and some studies 
 reported that knockdown of hdac1 and hdac3 could increase the percent 
 of apoptotic cells in carcinoma cells , we also evaluated the level of procaspase3 ( inactive form of apoptotic 
 effector caspase3 ) in u937 cells ( figure 4 ) . cleavage of pro - caspases3 results in the production of an active effector , 
 caspase3 , which can cleave essential structural proteins such as cytokeratins , 
 nuclear lamins , and also an inhibitor of caspase - activated dnase ( icad ) , 
 which liberates the dnase ( cad ) to digest chromosomal dna and cause 
 cell death . we can see from the result 
 that our compounds 11e , 11k , 11r , 11w , and 11y dramatically reduced the 
 level of procaspase3 , which was most likely due to the fact that procaspase3 
 was cleaved to the active form ( caspase3 ) , which could promote apoptosis 
 of u937 cells . at the same time , we performed flow cytometry analysis 
 to confirm the effect of these compounds in inducing apoptosis . in 
 this assay , we chose compounds 11r and 11w as representative compounds and saha as the positive control . results 
 demonstrated that 11r , 11w , and saha can 
 significantly induce apoptosis in a time - dependent and dose - dependent 
 manner . at the same concentration ( 1 m ) , 11r and 11w induced 61.76% and 64.25% cell apoptosis , respectively , 
 which were much higher than that of saha ( 26.83% ) . detailed activity 
 evaluation and apoptosis mechanism studies are currently underway 
 in our laboratory . induction of apoptosis at 12 and 24 h by compounds 11r , 11w , and saha in different concentrations 
 in u937 
 cells . having demonstrated that 11r had ideal in vitro activity , 
 we next set out to assess its tumor growth inhibitory effects in subcutaneous 
 cell lines and primary tumor derived xenograft models . before we conducted 
 the in vivo study , we developed hplc methods to briefly study the 
 stability of 11r in artificial gastric juice , artificial 
 intestinal juice , rat liver homogenate , and human plasma . compound 11r was incubated under each condition at 37 c for 24 
 h , extracted , and analyzed by hplc . we observed that 11r was stable in artificial gastric juice , artificial intestinal juice , 
 rat liver homogenate , and human plasma ( figure 5 ) . stability of compound 11r in artificial gastric juice , 
 artificial intestinal juice , rat liver homogenate , and human plasma . points were achieved after 0 , 1 , 2 , 4 , 6 , 8 , and 24 h , respectively , 
 and values are shown as mean  sd . because of the great 
 stability of 11r , it was feasible to evaluate its in 
 vivo activity orally . u937 cells ( 5  10 ) were subcutaneously implanted in the right flanks of female 
 nude mice ( balb / c - nu ) . when tumor size reached about 100 mm , mice were randomized to five per group and were treated with compound 11r or saha ( 100 mg / kg / day ) by oral gavage for 16 days . tumor 
 growth inhibition ( tgi ) and relative increment ratio ( t / c ) were calculated 
 at the end of treatment to reveal the antitumor effects in tumor weight 
 and tumor volume , respectively . compound 11r demonstrated 
 potent in vivo oral antitumor activity with higher tgi value ( 55.1% ) 
 and lower t / c value ( 37% ) than saha ( tgi = 32.1% ; t / c = 47% ) . in the 
 mice group treated by 11r , no significant body weight 
 loss and no evident toxic signs in liver and spleen were detected 
 ( figures 6 and 7 ) . antitumor 
 activity comparison of 11r and saha against 
 u937 human tumor xenografts implanted in mice , expressed as mean tumor 
 volume . figure 8a revealed 
 the docked conformation of compounds 11r , 21c , and 27c . the docked conformations of 11r and 21c were very similar in the linker and zbg , while 
 they were quite different in the aromatic ring and indole ring of 
 cap group . moreover , conformation of 27c was different 
 from 11r and 21c , leading to diverse binding 
 modes shown in figure 8c  e . figure 8c , d showed 11r and 21c had similar docking mode and hydrogen bonds ; the greater potency 
 of 11r might be amenable to the stiffening of the scaffold 
 introduced by the amide bond , which forces the lipophilic p - chlorophenyl moiety to yield closer van der waals interactions 
 with the protein rather than projecting itself toward solvent as in 21c . furthermore , in cap group , hydrogen bonds formed between 21c and residues asp92 and asp93 had a longer bond length 
 ( 2.19 and 1.97  ) than 11r ( 1.95 and 1.59  ) , 
 which also might be factors that made 11r more effective 
 against hdac3 than 21c . compared with 11r ( figure 8c ) , compound 27c ( figure 8e ) could form two diverse hydrogen bonds with amide 
 n  h of phe200 and imidazole n  h of his172 through carbonyl 
 oxygen atom and etheric oxygen atom , respectively , but at the same 
 time , 27c lost two hydrogen bonds with asp92 in the cap 
 group and tyr298 around zinc ion . in addition , the carbonyl oxygen 
 of 27c had the same distance with 11r to 
 zinc ion ( 2.1  ) , but the hydroxyl oxygen of 27c had a longer distance to the zinc ion ( 4.4  ) than 11r ( 1.9  ) , which indicated that 27c could only form 
 a weak monodentate interaction with zinc , while 11r could 
 form a strong bidentate interaction with zinc . this might be the reason 
 why 27c was much less potent than 11r ( figure 8b ) . ( a ) docked conformational alignment modeling of compounds 11r , 21c , and 27c . for clarity , hdac3 ( derived by modification of pdb code 4a69 using tripos sybyl 8.0 ) used for docking is not shown . ( b ) proposed 
 docked mode of 27c and 11r for binding zinc . ( c  e ) proposed binding preferences comparison of compounds 11r ( c ) , 21c ( d ) , and 27c ( e ) at 
 hdac3 . the zinc ion is shown as a red sphere , and the dashed lines 
 stand for the hydrogen bonds ( atom types : h , white ; n , blue ; o , red ) . for 
 the first time , n - hydroxycinnamamide - based 
 derivatives have been shown to be potent dual hdac1/3 selective hdacis . the representative compound 11r showed low nanomolar 
 ic50 values against hdac1 ( 11.8 nm ) and hdac3 ( 3.9 nm ) , 
 with micromolar or submicromolar ic50 values against hdac2 , 
 hdac8 , hdac4 , hdac6 , and hdac11 . in addition , a few of the examples 
 of the new series in antiproliferative study exhibited high potency 
 against several solid or hematological cells , even though antiproliferative 
 activity of the compounds seemed inferior to their hdac inhibition 
 activity . for example , antiproliferative activity of 11r was about 10 times higher than saha , while its hdac inhibition activity 
 was about 100 times higher than saha . we supposed 11r had poor transcellular permeability ; this speculation was confirmed 
 by parallel artificial membrane permeation assay ( pampa ) conducted on 11r and saha ( data 
 not shown ) . compound 11e , 11r , 11w , 
 and 11y increased histone h3 and h4 acetylation in the 
 same level of lbh589 ( one of the most potent hydroximates hdacis in 
 clinical ) in u937 cell line . at the same time , some of the compounds 
 decreased the level of pro - caspase3 , which was consistent with the 
 result of flow cytometry analysis in inducing apoptosis . compound 11r was evaluated for its in vivo antitumor study in a u937 
 xenograft mice model and showed higher efficacy compared to the approved 
 drug saha . more detailed studies of the antitumor profiles of these 
 promising compounds are underway in our lab . using 11r as lead , continued studies are underway to search for more promising 
 hdacis with improved transcellular permeability and isoform selectivity . h nmr spectra were 
 obtained on a bruker drx spectrometer at 300 mhz with tms as an internal 
 standard ,  in parts per million , and j in 
 hertz . high - resolution mass spectrometry was performed by shandong 
 analysis and test center in jinan , china . all reactions were monitored 
 by tlc using 0.25 mm silica gel plates ( 60gf-254 ) . flash chromatography was accomplished 
 using the automated combiflash rf system from teledyne isco and was 
 done using silica gel of 200300 mesh . all tested 
 compounds are > 95% pure by hplc analysis , performed on an agilent 
 1100 hplc instrument using an ods hypersil column ( 5 m , 4.6 
 mm  250 mm ) according to one of the following methods . method 
 a : compounds 10a , 11a11x , and 27a27c were eluted with 35% 
 acetonitrile/65% water ( containing 0.4% formic acid)65% acetonitrile/35% 
 water ( containing 0.4% formic acid ) over 20 min , with detection at 
 290 nm and a flow rate of 1 ml / min . m potassium dihydrogen phosphate / phosphoric acid ( ph = 3.0 ) over 20 
 min , with detection at 290 nm and a flow rate of 1 ml / min . the temperature 
 of the column was 25 c , and quantity of injection was 20 m . ( s)-methyl 2-amino-3-(1h - indol-3-yl)propanoate 
 hydrochloride ( 2 ) , ( s)-methyl 2-((tert - butoxycarbonyl)amino)-3-(1h - indol-3-yl)propanoate(3 ) , and ( s)-tert - butyl(1-hydroxy-3-(1h - indol-3-yl)propan-2-yl)carbamate ( 4 ) were 
 synthesized as described previously . p - coumaric acid ( 16.4 g , 100mmol ) 
 was dissolved in 200 ml of meoh , then acetyl chloride ( 24 g , 300mmol ) 
 was added dropwise at 0 c ; the mixed solution was refluxed at 
 75 c for 5 h. the solvent was evaporated under vacuum ; the product 
 was dissolved with etoac , washed by saturated nahco3 solution 
 ( 2  100 ml ) , 1 m aqueous citric acid ( 2  100 ml ) , and 
 brine ( 2  100 ml ) , dried over mgso4 overnight , and 
 evaporated under vacuum to obtained product compound 6 , a white solid ( 15.6 g , 87.5% ) . h nmr ( 300 mhz , dmso - d6 )  
 3.69 ( s , 3h ) , 6.43 ( d , j = 16.2 hz , 1h ) , 6.81 ( d , j = 8.7 hz , 2h ) , 7.547.59 ( m , 3h ) , 10.01 ( s , 1h ) . h nmr ( 300 
 mhz , dmso - d6 )  3.72 ( s , 1h ) , 6.54 
 ( d , j = 15.9 hz , 1h ) , 6.836.86 ( m , 1h ) , 7.047.05 
 ( m , 1h ) , 7.15 ( d , j = 7.5 hz , 1h ) , 7.207.25 
 ( m , 1h ) , 7.60 ( d , j = 15.9 hz , 1h ) , 7.62 ( s , 1h ) . 
 esi - ms m / z : 179.2 [ m + h ] . compound 4 ( 2.9 g , 10mmol ) 
 was taken up in thf ( 50 ml ) , 
 then ( ch3ch2ch2ch2)4n(br ) ( tbabr , 0.5 g ) was added followed by ch3i 
 ( 3.5 g , 25mmol ) . the reaction was stirred overnight , thf was evaporated 
 under vacuum , and the residue was extracted with etoac ( 3  50 
 ml ) . the organic layer was washed with saturated nahco3 solution ( 2  100 ml ) , 1 m aqueous citric acid ( 2  100 
 ml ) , and brine ( 2  100 ml ) and dried over mgso4 overnight . h nmr ( 300 mhz , dmso - d6 )  
 1.35 ( s , 9h ) , 2.602.73 ( m , 1h ) , 2.842.91 ( m , 1h ) , 
 3.223.40 ( m , 2h ) , 3.583.67 ( m , 1h ) , 3.72 ( s , 3h ) , 
 4.64 ( t , j = 5.4 hz , 1h ) , 6.53 ( d , j = 8.1 hz , 1h ) , 7.697.07 ( m , 2h ) , 7.15 ( t , j = 7.8 hz , 1h ) , 7.37 ( d , j = 8.1 hz , 1h ) , 7.60 ( d , j = 7.8 hz , 1h ) . compound 4 ( 5.8 g , 20mmol ) , 6 ( 4.2 g , 24mmol ) , and pph3 ( 7.9 g , 30mmol ) were 
 dissolved in anhydrous thf , and then dead ( 5.2 g , 30mmol ) was added 
 dropwise at 0 c ; the mixture was stirred for 4 h , and then thf 
 was evaporated . the crude material was purified via flash chromatography 
 to afford the compound 7 , a white solid ( 5.4 g , 60% ) . h nmr ( 300 mhz , dmso - d6 )  1.36 ( s , 9h ) , 2.853.00 ( m , 
 2h ) , 3.70 ( s , 3h ) , 3.954.07 ( m , 3h ) , 6.51 ( d , j = 15.9 hz , 1h ) , 6.936.99 ( m , 4h ) , 7.08 ( t , j = 7.4 hz , 1h ) , 7.13 ( d , j = 2.1 hz , 1h ) , 7.34 ( d , j = 7.8 hz , 1h ) , 7.56 ( d , j = 7.8 hz , 1h ) , 
 7.63 ( d , j = 15.9 hz , 1h ) , 7.66 ( d , j = 8.4 hz , 2h ) , 10.82 ( d , j = 1.5 hz , 1h ) . compound 7 was 
 dissolved in a solution 
 of etoac ( 8 ml ) saturated by dry hcl gas . the filtered precipitate was washed 
 by diethyl ether to give the compound 8 , a white solid 
 powder ( 3.8 g , 83% ) . h nmr 
 ( 300 mhz , dmso - d6 )  3.103.24 
 ( m , 2h ) , 3.71 ( s , 3h ) , 3.713.75 ( m , 1h ) , 4.07 ( dd , j = 5.7 hz , j = 10.5 hz , 1h ) , 4.21 ( dd , j = 5.7 hz , j = 10.5 hz , 1h ) , 6.53 ( d , j = 15.9 hz , 1h ) , 6.977.01 ( m , 3h ) , 7.11 ( t , j = 6.9 hz , 1h ) , 7.27 ( d , j = 2.4 hz , 1h ) , 
 7.38 ( d , j = 8.1 hz , 1h ) , 7.597.64 ( m , 2h ) , 
 7.70 ( d , j = 8.7 hz , 2h ) , 8.40 ( s , 2h ) , 11.05 ( s , 
 1h ) . esi - ms m / z : 351.5 [ m + h ] . pale yellow oil . esi - ms m / z : 351.5 [ m + h ] . to a solution of p - toluene 
 sulfonic acid ( 0.34 g , 2mmol ) in anhydrous ch2cl2 was added 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium 
 tetrafluoroborate ( tbtu , 0.71 g , 2.2mmol ) , followed by et3n ( 0.3 g , 3mmol ) . after 30 min , compound 8 ( 0.85 g , 2.2mmol ) 
 was added followed by tea ( 0.3 g , 3mmol ) . four hours later , the solution 
 of ch2cl2 was washed with 1 n hcl ( 2  
 30 ml ) , saturated na2co3 ( 2  30 ml ) , and 
 brine ( 2  30 ml ) , dried over mgso4 overnight , and 
 the solvent was evaporated under vacuum . the crude product was purified 
 by recrystallization with etoac and petroleum ether to achieve a white 
 pure solid ( 0.51 g , 50% yield ) . h nmr ( 300 mhz , dmso - d6 )  2.30 
 ( s , 3h ) , 2.85 ( dd , j = 6.0 hz , j = 14.4 hz , 1h ) , 3.01 ( dd , j = 8.1 hz , j = 14.4 hz , 1h ) , 3.603.65 ( m , 1h ) , 3.70 ( s , 3h ) , 3.86 ( d , j = 4.8 hz , 2h ) , 6.50 ( d , j = 16.2 hz , 
 1h ) , 6.76 ( d , j = 8.7 hz , 2h ) , 6.91 ( t , j = 7.4 hz , 1h ) , 7.007.06 ( m , 2h ) , 7.20 ( d , j = 8.1 hz , 2h ) , 7.247.30 ( m , 2h ) , 7.567.62 ( m , 5h ) , 
 7.99 ( d , j = 7.2 hz,1h ) , 10.77 ( d , j = 1.5 hz , 1h ) . h nmr ( 300 mhz , dmso - d6 )  0.730.80 ( m , 6h ) , 0.961.00 ( m , 2h ) , 1.041.11 
 ( m , 4h ) , 1.301.42 ( m , 2h ) , 2.102.17 ( m , 1h ) , 2.863.03 
 ( m , 2h ) , 3.70 ( s , 3h ) , 3.984.07 ( m , 2h ) , 4.344.42 
 ( m , 1h ) , 6.51 ( d , j = 15.9 hz , 1h ) , 6.926.95 
 ( m , 3h ) , 7.07 ( t , j = 7.5 hz , 1h ) , 7.14 ( d , j = 2.4 hz , 1h ) , 7.33 ( d , j = 7.8 hz , 1h ) , 
 7.58 ( d , j = 9.0 hz , 1h ) , 7.64 ( d , j = 17.0 hz , 1h ) , 7.67 ( d , j = 9.0 hz , 2h ) , 7.94 
 ( d , j = 7.8 hz , 1h ) , 10.82 ( d , j = 1.5 hz , 1h ) . h nmr 
 ( 300 mhz , dmso - d6 )  2.08 
 ( s , 3h ) , 3.27 ( s , 3h ) , 3.083.16 ( m , 2h ) , 3.70 ( s , 3h ) , 4.114.22 
 ( m , 2h ) , 4.554.64 ( m , 1h ) , 6.49 ( d , j = 15.9 
 hz , 1h ) , 6.74 ( t , j = 7.8 hz , 1h ) , 6.83 ( d , j = 7.8 hz , 1h ) , 6.916.99 ( m , 4h ) , 7.037.09 
 ( m , 3h ) , 7.207.26 ( m , 2h ) , 7.34 ( d , j = 8.1 
 hz , 1h ) , 7.577.64 ( m , 5h ) , 8.57 ( d , j = 7.8 
 hz , 1h ) , 9.39 ( s , 1h ) , 10.84 ( d , j = 1.5 hz , 1h ) . h nmr ( 300 mhz , dmso - d6 )  2.903.06 ( m , 2h ) , 3.43 ( s , 2h ) , 3.71 ( s , 3h ) , 4.01 
 ( d , j = 5.4 hz , 2h ) , 4.274.35 ( m , 1h ) , 6.52 
 ( d , j = 15.9 hz , 1h ) , 6.916.96 ( m , 3h ) , 7.08 
 ( t , j = 7.5 hz , 1h ) , 7.10 ( d , j = 
 2.1 hz , 1h ) , 7.177.27 ( m , 5h ) , 7.34 ( d , j = 8.1 hz , 1h ) , 7.57 ( d , j = 7.8 hz , 1h ) , 7.64 ( d , j = 16.2 hz , 1h ) , 7.67 ( d , j = 8.7 hz , 
 2h ) , 8.30 ( d , j = 7.8 hz , 1h ) , 10.83 ( d , j = 1.5 hz , 1h ) . the solution of compound 8 ( 0.76 g , 2mmol ) in ch3oh was added et3n ( 0.22 g , 
 2.2mmol ) for neutralization , 
 followed by the addition of methanol ( 0.21 g , 2mmol ) ; the resulting 
 solution was stirred for 2 h and the solvent evaporated . the residue 
 was dissolved in anhydrous ch3oh , nabh4 ( 0.15 g , 
 4mmol ) was added slowly at 0 c , the mixture was stirred at room 
 temperature overnight , and after the reaction finished , the reaction 
 was quenched by adding ice water slowly . ch3oh was evaporated , 
 and the residue was extracted by etoac ( 3  30 ml ) . the organic 
 layer was washed with brine ( 2  30 ml ) and saturated na2co3 ( 2  30 ml ) and dried over mgso4 overnight . the solvent was evaporated under vacuum to get crude 
 product 11a , a colorless oil ( 0.79 g , 90% ) . h nmr ( 300 mhz , dmso - d6 )  2.95 
 ( d , j = 6.3 hz , 2h ) , 3.143.18 ( m , 1h ) , 3.70 
 ( s , 3h ) , 3.87 ( s , 2h ) , 3.94 ( d , j = 5.1 hz , 2h ) , 
 6.50 ( d , j = 15.9 hz , 1h ) , 6.92 ( t , j = 7.5 hz , 1h ) , 6.95 ( d , j = 8.7 hz , 2h ) , 7.06 ( t , j = 7.4 hz , 1h ) , 7.14 ( d , j = 2.4 hz , 1h ) , 
 7.167.22 ( m , 1h ) , 7.267.33 ( m , 5h ) , 7.43 ( d , j = 7.8 hz , 1h ) , 7.63 ( d , j = 15.9 hz , 
 1h ) , 7.65 ( d , j = 8.7 hz , 2h ) , 8.99 ( s , 1h ) , 10.83 
 ( s , 1h ) . et3n ( 0.61 g , 6mmol ) followed 
 by ( boc)2o ( 0.78 g , 3.6mmol ) were added to the solution 
 of compound 11a ( 0.79 g , 1.8mmol ) in ch2cl2 . the resulting solution was washed by saturated nahco3 solution 
 ( 2  30 ml ) , 1 m aqueous citric acid ( 2  30 ml ) , and brine 
 ( 2  30 ml ) and dried over mgso4 overnight . the solvent 
 was evaporated under vacuum to get crude product 12a , 
 a colorless oil ( 0.92 g , 95% ) . h nmr ( 300 mhz , dmso - d6 )  1.431.47 ( m , 9h ) , 2.903.16 
 ( m , 2h ) , 3.70 ( s , 3h ) , 4.004.07 ( m , 2h ) , 4.314.37 
 ( m , 2h ) , 4.56 ( m , 1h ) , 6.49 ( d , j = 16.2 hz , 1h ) , 
 6.776.80 ( m , 2h ) , 6.98 ( t , j = 7.8 hz , 1h ) , 
 7.09 ( m , 2h ) , 7.127.17 ( m , 1h ) , 7.207.24 ( m , 4h ) , 
 7.35 ( d , j = 7.8 hz , 1h ) , 7.417.47 ( m , 1h ) , 
 7.567.64 ( m , 3h ) , 10.84 ( s , 1h ) . koh ( 28 g , 509mmol ) and nh2ohhcl ( 23.35 g , 343mmol ) 
 were dissolved , respectively , in 70 and 120 ml of meoh to get solution 
 a and solution b. next , solution a was added dropwise to solution 
 b. after filtering the precipitate ( kcl ) , a nh2ok solution 
 was obtained . compound 7 ( 0.45 g , 1 mmol ) was dissolved 
 in the nh2ok solution and stirred overnight . the residue 
 was acidified with 1 n hcl to a ph 34 and then extracted with 
 etoac ( 3  20 ml ) . the organic layer was washed with brine ( 2 
  30 ml ) and dried over na2so4 overnight . the crude material was purified via flash chromatography to afford 
 the compound 10a ( 0.15 g , 30% yield ) . h nmr ( 300 mhz , dmso - d6 )  1.37 ( s , 9h ) , 2.793.00 ( m , 2h ) , 3.904.04 
 ( m , 3h ) , 6.33 ( d , j = 15.6 hz , 1h ) , 6.927.00 
 ( m , 4h ) , 7.08 ( td , j = 0.9 hz , j = 7.6 hz , 1h ) , 7.13 ( d , j = 1.8 hz , 1h ) , 7.34 ( d , j = 7.8 hz , 1h ) , 7.42 ( d , j = 15.6 hz , 
 1h ) , 7.48 ( d , j = 8.7 hz , 2h ) , 7.56 ( d , j = 7.8 hz , 1h ) , 8.97 ( s , 1h ) , 10.66 ( s , 1h ) , 10.83 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c25h29n3o5 [ m + h ] 452.2180 , found 452.2182 . retention time : 18.9 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  1.39 ( s , 9h ) , 2.853.10 ( m , 2h ) , 3.483.57 
 ( m , 2h ) , 3.96 ( m , 2h ) , 4.314.35 ( m , 1h ) , 6.33 ( d , j = 15.9 , 1h ) , 6.996.90 ( m , 4h ) , 7.08 ( t , j = 6.9 hz , 1h ) , 7.14 ( d , j = 2.1 hz , 1h ) , 
 7.33 ( d , j = 8.1 hz , 1h ) , 7.42 ( d , j = 15.9 hz , 1h ) , 7.49 ( d , j = 8.4 hz , 2h ) , 7.59 
 ( d , j = 7.8 hz , 1h ) , 7.98 ( d , j = 
 7.8 hz , 1h ) , 8.98 ( s , 1h ) , 10.66 ( s , 1h ) , 10.85 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h32n4o6 [ m + h ] 509.2395 , found 509.2392 . retention time : 7.4 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  1.37 ( s , 9h ) , 1.681.82 ( m , 2h ) , 2.42 ( t , j = 7.8 hz , 2h ) , 2.973.04 ( m , 2h ) , 3.36 ( s , 3h ) , 
 3.904.05 ( m , 3h ) , 4.254.42 ( m , 1h ) , 6.32 ( d , j = 15.6 hz , 1h ) , 6.906.97 ( m , 4h ) , 7.08 ( t , j = 6.9 hz , 1h ) , 7.20 ( d , j = 2.1 hz , 1h ) , 
 7.34 ( d , j = 7.8 hz , 1h ) , 7.41 ( d , 15.9 hz , 1h ) , 
 7.48 ( d , 8.4 hz , 2h ) , 7.60 ( d , j = 7.8 hz , 1h ) , 7.99 
 ( d , j = 7.5 hz , 1h ) , 8.97 ( s , 1h ) , 10.66 ( s , 1h ) , 
 10.85 ( s , 1h ) . hrms ( ap - esi ) m / z calcd for c30h38n4o6s [ m + h ] 583.2585 , found 583.2486 . retention time : 10.55 
 min , eluted with 47% acetonitrile/53% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  1.36 ( s , 9h ) , 2.913.05 ( m , 2h ) , 3.513.54 
 ( m , 2h ) , 3.934.02 ( m , 3h ) , 4.314.38 ( m , 1h ) , 4.84 
 ( t , j = 6.3 hz , 1h ) , 6.33 ( d , j = 
 15.6 hz , 1h ) , 6.65 ( d , 8.1 hz , 1h ) , 6.926.97 ( m , 3h ) , 7.08 
 ( t , j = 6.9 hz , 1h ) , 7.16 ( d , j = 
 2.1 hz , 1h ) , 7.34 ( d , j = 8.4 hz , 1h ) , 7.42 ( d , j = 15.9 hz , 1h ) , 7.49 ( d , j = 8.4 hz , 
 2h ) , 7.59 ( d , j = 7.8 hz , 1h ) , 7.96 ( d , j = 8.1 hz , 1h ) , 8.98 ( s , 1h ) , 10.66 ( s , 1h ) , 10.85 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c28h34n4o7 [ m + h ] 539.2500 , found 539.2496 . retention time : 5.3 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  1.01 ( d , j = 5.7 hz , 3h ) , 1.37 ( s , 9h ) , 
 2.923.05 ( m , 2h ) , 3.863.94 ( m , 4h ) , 4.344.36 
 ( m , 1h ) , 4.78 ( d , j = 5.4 hz , 1h ) , 6.276.35 
 ( m , 2h ) , 6.916.97 ( m , 3h ) , 7.08 ( t , j = 7.2 
 hz , 1h ) , 7.16 ( d , j = 1.8 hz , 1h ) , 7.317.48 
 ( m , 4h ) , 7.60 ( d , j = 7.8 hz , 1h ) , 7.96 ( d , j = 7.8 hz , 1h ) , 8.96 ( s , 1h ) , 10.65 ( s , 1h ) , 10.83 ( s , 
 1h ) . hrms ( ap - esi ) m / z calcd for 
 c29h36n4o7 [ m + h ] 553.2657 , found 553.2658 . retention time : 6.5 min , eluted 
 with 40% acetonitrile/60% water ( containing 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.12 ( d , j = 6.6 hz , 2h ) , 4.044.21 ( m , 2h ) , 
 4.544.65 ( m , 1h ) , 6.33 ( d , j = 15.9 hz , 1h ) , 
 6.936.99 ( m , 3h ) , 7.08 ( td , j = 0.9 hz , j = 7.4 hz , 1h ) , 7.19 ( d , j = 2.1 hz , 1h ) , 
 7.34 ( d , j = 8.1 hz , 1h ) , 7.42 ( d , j = 15.9 hz , 1h ) , 7.437.55 ( m , 5h ) , 7.64 ( d , j = 7.8 hz , 1h ) , 7.85 ( d , j = k7.5 hz , 2h ) , 8.53 
 ( d , j = 7.8 hz , 1h ) , 8.98 ( s , 1h ) , 10.66 ( s , 1h ) , 
 10.83 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h25n3o4 [ m + h ] 456.1918 , found 456.1916 . retention time : 12.6 min , eluted with 40% acetonitrile/60% water ( containing 
 0.4% formic acid ) . h nmr ( 300 
 mhz , dmso - d6 )  2.31 ( s , 3h ) , 2.85 
 ( dd , j = 6.0 hz , j = 14.4 hz , 1h ) , 
 3.01 ( dd , j = 8.1 hz , j = 14.4 hz , 
 1h ) , 3.603.62 ( m , 1h ) , 3.84 ( d , j = 4.8 hz , 
 2h ) , 6.32 ( d , j = 15.9 hz , 1h ) , 6.76 ( d , j = 8.7 hz , 2h ) , 6.92 ( td , j = 0.9 hz , j = 7.4 hz , 1h ) , 7.007.06 ( m , 2h ) , 7.21 ( d , j = 8.1 hz , 2h ) , 7.27 ( d , j = 10.2 hz , 
 1h ) , 7.30 ( d , j = 8.1 hz , 1h ) , 7.41 ( d , j = 15.9 hz , 1h ) , 7.44 ( d , j = 8.7 hz , 2h ) , 7.59 
 ( d , j = 8.4 hz , 2h ) , 8.00 ( d , j = 
 6.9 hz , 1h ) , 8.99 ( s , 1h ) , 10.66 ( s , 1h ) , 10.78 ( d , j = 1,8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h27n3o5s [ m + h ] 506.1715 , found 506.1744 . retention time : 22.9 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  3.14 ( d , j = 6.9 hz , 2h ) , 3.994.12 
 ( m , 1h ) , 4.184.28 ( m , 1h ) , 4.564.67 ( m , 1h ) , 6.33 
 ( d , j = 15.9 hz , 1h ) , 6.947.00 ( m , 3h ) , 7.09 
 ( t , j = 7.2 hz , 1h ) , 7.20 ( d , j = 
 2.1 hz , 1h ) , 7.35 ( d , j = 8.1 hz , 1h ) , 7.377.43 
 ( m , 2h ) , 7.477.52 ( m , 4h ) , 7.66 ( d , j = 7.8 
 hz , 1h ) , 7.727.78 ( m , 4h ) , 7.96 ( d , j = 8.4 
 hz , 2h ) , 8.60 ( d , j = 6.9 hz , 1h ) , 10.67 ( s , 1h ) , 
 10.84 ( s , 1h ) . hrms ( ap - esi ) m / z calcd for c33h29n3o4 [ m + h ] 532.2192 , found 532.2231 . retention time : 5.0 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  2.852.91 ( m , 1h ) , 2.983.05 ( m , 1h ) , 3.643.75 
 ( m , 1h ) , 3.823.91 ( m , 1h ) , 6.29 ( d , j = 15.9 
 hz , 1h ) , 6.75 ( d , j = 8.7 hz , 2h ) , 6.91 ( t , j = 7.8 hz , 1h ) , 7.01 ( t , j = 7.8 hz , 1h ) , 
 7.10 ( d , j = 2.1 hz , 1h ) , 7.24 ( d , j = 8.1 hz , 1h ) , 7.307.45 ( m , 5h ) , 7.487.53 ( m , 2h ) , 
 7.647.68 ( m , 4h ) , 7.77 ( d , j = 8.4 hz , 2h ) , 
 8.17 ( d , j = 7.2 hz , 1h ) , 8.99 ( s , 1h ) , 10.65 ( s , 
 1h ) , 10.80 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c32h29n3o5s [ m + h ] 568.1861 , found 568.1901 . retention time : 5.1 min , eluted with 65% acetonitrile/35% water ( containing 
 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 0.69 ( t , j = 7.2 hz , 3h ) , 0.79 ( t , j = 7.5 hz , 3h ) , 1.231.47 ( m , 4h ) , 1.912.02 ( m , 1h ) , 
 2.903.04 ( m , 2h ) , 3.99 ( d , j = 5.4 hz , 2h ) , 
 4.364.44 ( m , 1h ) , 6.33 ( d , j = 15.9 hz , 1h ) , 
 6.95 ( d , 8.4 hz , 2h ) , 6.98 ( td , j = 0.9 hz , j = 7.1 hz , 1h ) , 7.08 ( td , j = 0.9 hz , j = 7.6 hz , 1h ) , 7.14 ( d , j = 2.1 hz , 1h ) , 
 7.33 ( d , j = 8.1 hz , 1h ) , 7.42 ( d , j = 15.6 hz , 1h ) , 7.49 ( d , j = 8.4 hz , 2h ) , 7.59 
 ( d , j = 7.8 hz , 1h ) , 7.97 ( d , j = 
 8.1 hz , 1h ) , 8.97 ( s , 1h ) , 10.66 ( s , 1h ) , 10.82 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c26h31n3o4 [ m + h ] 450.2387 , found 450.2389 . retention time : 10.9 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  
 0.730.80 ( m , 6h ) , 1.971.20 ( m , 6h ) , 1.301.46 
 ( m , 2h ) , 2.122.18 ( m , 1h ) , 2.863.03 ( m , 2h ) , 3.98 
 ( d , j = 5.1 hz , 2h ) , 4.344.42 ( m , 1h ) , 6.33 
 ( d , j = 15.9 hz , 1h ) , 6.95 ( d , j = 8.7 hz , 2h ) , 6.98 ( td , j = 0.9 hz , j = 6.8 hz , 1h ) , 7.08 ( td , j = 0.9 hz , j = 7.2 hz , 1h ) , 7.14 ( d , j = 2.1 hz , 1h ) , 7.33 ( d , j = 7.8 hz , 1h ) , 7.42 ( d , j = 15.6 hz , 
 1h ) , 7.49 ( d , j = 8.7 hz , 2h ) , 7.58 ( d , j = 7.8 hz , 1h ) , 7.63 ( d , j = 9.0 hz , 1h ) , 7.94 ( d , j = 8.1 hz , 1h ) , 10.66 ( s , 1h ) , 10.83 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c28h35n3o4 [ m + h ] 478.2699 , found 478.2699 . retention time : 24.5 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.14 ( d , j = 6.9 , 2h ) , 4.084.22 ( m , 2h ) , 
 4.594.70 ( m , 1h ) , 6.33 ( d , j = 15.9 hz , 1h ) , 
 6.866.91 ( m , 2h ) , 6.967.00 ( m , 3h ) , 7.09 ( t , j = 7.4 hz , 1h ) , 7.18 ( d , j = 2.1 hz , 1h ) , 
 7.35 ( d , j = 8.1 hz , 1h ) , 7.377.42 ( m , 2h ) , 
 7.49 ( d , j = 8.4 hz , 2h ) , 7.64 ( d , j = 7.8 hz , 1h ) , 7.92 ( dd , j = 1.5 hz , j = 8.1 hz , 1h ) , 8.87 ( d , j = 8.1 hz , 1h ) , 8.98 ( s , 
 1h ) , 10.66 ( s , 1h ) , 10.85 ( d , j = 1.8 hz , 1h ) , 12.37 
 ( s , 1h ) . hrms ( ap - esi ) m / z calcd 
 for c27h25n3o5 [ m + h ] 472.1867 , found 472.1868 . retention time : 17.2 min , eluted 
 with 40% acetonitrile/60% water ( containing 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 2.08 ( s , 3h ) , 2.27 ( s , 3h ) , 3.053.12 ( m , 2h ) , 4.084.20 
 ( m , 2h ) , 4.554.67 ( m , 1h ) , 6.32 ( d , j = 15.9 
 hz , 1h ) , 6.74 ( t , j = 7.5h , 1h ) , 6.83 ( d , j = 8.4 hz , 1h ) , 6.916.98 ( m , 4h ) , 7.037.09 
 ( m , 3h ) , 7.237.26 ( m , 2h ) , 7.34 ( d , j = 8.1 
 hz , 1h ) , 7.41 ( d , j = 15.9 hz , 1h ) , 7.47 ( d , j = 8.1 hz , 2h ) , 7.64 ( t , j = 7.2 hz , 2h ) , 
 8.58 ( d , j = 8.1 hz , 1h ) , 8.97 ( s , 1h ) , 9.40 ( s , 
 1h ) , 10.65 ( s , 1h ) , 10.84 ( d , j = 1.5 hz , 1h ) . hrms 
 ( ap - esi ) m / z calcd for c35h34n4o4 [ m + h ] 534.1023 , 
 found 575.2653 . retention time : 10.2 min , eluted with 40% acetonitrile/60% 
 water ( containing 0.4% formic acid ) . h nmr ( 300 
 mhz , dmso - d6 )  0.810.86 
 ( m , 6h ) , 1.241.31 ( m , 3h ) , 1.711.85 ( m , 1h ) , 2.352.41 
 ( m , 2h ) , 2.843.05 ( m , 2h ) , 3.593.62 ( m , 1h ) , 3.92 
 ( m , 1h ) , 3.963.98 ( m , 1h ) , 4.264.36 ( m , 1h ) , 6.33 
 ( dd , j = 4.5 hz , j = 15.9 hz , 1h ) , 
 6.847.08 ( m , 6h ) , 7.137.19 ( m , 3h ) , 7.427.61 
 ( m , 5h ) , 8.14 ( t , j = 6.4 hz , 1h ) , 8.97 ( s , 1h ) , 
 10.66 ( s , 1h ) , 10.83 ( s , 1h ) . hrms ( ap - esi ) m / z calcd for c33h37n3o4 [ m + h ] 540.2857 , found 540.2856 . retention time : 
 7.7 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 
 mhz , dmso - d6 )  1.351.42 
 ( m , 3h ) , 2.893.08 ( m , 2h ) , 3.743.80 ( m , 1h ) , 3.853.86 
 ( m , 3h ) , 3.924.00 ( m , 2h ) , 4.294.39 ( m , 1h ) , 6.31 
 ( d , j = 15.9 hz , 1h ) , 6.84 ( d , j = 8.7 hz , 2h ) , 6.947.09 ( m , 3h ) , 7.117.51 ( m , 8h ) , 
 7.62 ( d , j = 7.8 hz , 1h ) , 7.677.75 ( m , 3h ) , 
 8.23 ( d , j = 7.8 hz , 1h ) , 8.98 ( s , 1h ) , 10.66 ( s , 
 1h ) , 10.85 ( s , 1h ) . hrms ( ap - esi ) m / z calcd for c36h33n3o5 [ m + h ] 564.2493 , found 564.2495 . h nmr ( 300 mhz , 
 dmso - d6 )  2.903.06 ( m , 
 2h ) , 3.43 ( s , 2h ) , 3.99 ( d , j = 5.1 hz , 2h ) , 4.274.38 
 ( m , 1h ) , 6.34 ( d , j = 15.9 hz , 1h ) , 6.916.97 
 ( m , 3h ) , 7.08 ( td , j = 0.9 , j = 
 7.4 hz , 1h ) , 7.11 ( d , j = 2.1 hz , 1h ) , 7.177.28 
 ( m , 5h ) , 7.35 ( d , j = 8.1 hz , 1h ) , 7.42 ( d , j = 15.9 hz , 1h ) , 7.50 ( d , j = 8.7 hz , 
 2h ) , 7.58 ( d , j = 7.8 hz , 1h ) , 8.31 ( d , j = 7.8 hz , 1h ) , 8.99 ( s , 1h ) , 10.66 ( s , 1h ) , 10.84 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c28h27n3o4 [ m + h ] 470.2074 , found 470.2074 . retention time : 12.8 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , 
 dmso - d6 )  2.41 ( t , j = 8.1 hz , 2h ) , 2.81 ( t , j = 7.8 hz , 2h ) , 2.853.02 
 ( m , 2h ) , 3.94 ( d , j = 5.1 hz , 2h ) , 4.284.38 
 ( m , 1h ) , 6.33 ( d , j = 15.9 hz , 1h ) , 6.96 ( d , j = 8.7 hz , 2h ) , 6.07 ( t , j = 6.9 hz , 1h ) , 
 7.037.09 ( m , 2h ) , 7.137.35 ( m , 5h ) , 7.34 ( d , j = 7.8 hz , 1h ) , 7.42 ( d , j = 15.9 hz , 
 1h ) , 7.50 ( d , j = 8.7 hz , 2h ) , 7.58 ( d , j = 7.8 hz , 1h ) , 8.06 ( d , j = 8.1 hz , 1h ) , 8.99 ( s , 
 1h ) , 10.67 ( s , 1h ) , 10.83 ( d , j = 1.8 hz , 1h ) . hrms 
 ( ap - esi ) m / z calcd for c29h29n3o4 [ m + h ] 484.2236 , 
 found 484.2231 . retention time : 17.4 min , eluted with 40% acetonitrile/60% 
 water ( containing 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.11 ( d , j = 6.6 hz , 2h ) , 4.064.11 ( m , 1h ) , 
 4.144.20 ( m , 1h ) , 4.544.61 ( m , 1h ) , 6.32 ( d , j = 15.6 hz , 1h ) , 6.936.98 ( m , 3h ) , 7.08 ( t , j = 6.9 hz , 1h ) , 7.18 ( d , j = 2.1 hz , 1h ) , 
 7.34 ( d , j = 7.8 hz , 1h ) , 7.42 ( d , j = 15.9 hz , 1h ) , 7.49 ( d , j = 8.4 hz , 2h ) , 7.55 
 ( d , j = 8.7 hz , 2h ) , 7.63 ( d , j = 
 7.8 hz , 1h ) , 7.88 ( d , j = 8.7 hz , 2h ) , 8.63 ( d , j = 7.8 hz , 1h ) , 8.98 ( s , 1h ) , 10.66 ( s , 1h ) , 10.83 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h24cln3o4 [ m + h ] 490.1526 , found 490.1528 . retention time : 
 6.0 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.003.13 ( m , 2h ) , 4.10 ( d , j = 5.4 hz , 2h ) , 
 4.494.57 ( m , 1h ) , 6.33 ( d , j = 15.9 hz , 1h ) , 
 6.957.00 ( m , 3h ) , 7.10 ( t , j = 7.4 hz , 1h ) , 
 7.22 ( d , j = 2.1 hz , 1h ) , 7.29 ( dd , j = 1.8 hz , j = 7.5 hz , 1h ) , 7.327.40 ( m , 
 3h ) , 7.427.50 ( m , 4h ) , 7.63 ( d , j = 7.8 hz , 
 1h ) , 8.66 ( d , j = 8.1 hz , 1h ) , 8.98 ( s , 1h ) , 10.67 
 ( s , 1h ) , 10.87 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h24cln3o4 [ m + h ] 490.1528 , found 
 490.1528 . retention time : 10.8 min , eluted with 40% acetonitrile/60% 
 water ( containing 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.13 ( d , j = 6.9 hz , 2h ) , 4.064.11 ( m , 1h ) , 
 4.144.21 ( m , 1h ) , 4.544.65 ( m , 1h ) , 6.48 ( d , j = 15.9 hz , 1h ) , 6.946.99 ( m , 2h ) , 7.037.08 
 ( m , 1h ) , 7.127.19 ( m , 2h ) , 7.277.32 ( m , 2h ) , 7.347.55 
 ( m , 4h ) , 7.65 ( d , j = 7.8 hz , 1h ) , 7.837.85 
 ( m , 2h ) , 8.53 ( d , j = 8.1 hz , 1h ) , 9.06 ( s , 1h ) , 
 10.72 ( s , 1h ) , 10.83 ( s , 1h ) . hrms ( ap - esi ) m / z calcd for c27h24cln3o4 [ m + h ] 490.1528 , found 490.1528 . retention time : 
 18.8 min , eluted with 40% acetonitrile/60% water ( containing 0.4% 
 formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.033.16 ( m , 2h ) , 4.104.19 ( m , 2h ) , 4.514.60 
 ( m , 1h ) , 6.33 ( d , j = 15.6 hz , 1h ) , 6.936.99 
 ( m , 3h ) , 7.08 ( t , j = 6.9 hz , 1h ) , 7.18 ( d , j = 2.1 hz , 1h ) , 7.34 ( d , j = 7.8 hz , 1h ) , 
 7.42 ( d , j = 15.9 hz , 1h ) , 7.49 ( d , j = 8.7 hz , 2h ) , 7.63 ( d , j = 7.8 hz , 1h ) , 7.81 ( d , j = 1.8 hz , 2h ) , 7.83 ( d , j = 1.8 hz , 1h ) , 
 8.77 ( d , j = 7.8 hz , 1h ) , 8.97 ( s , 1h ) , 10.66 ( s , 
 1h ) , 10.85 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h23cl2n3o4 [ m + h ] 534.1023 , 
 found 524.1138 . retention time : 10.1 min , eluted with 51% acetonitrile/49% 
 water ( containing 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.003.13 ( m , 2h ) , 4.084.10 ( m , 2h ) , 4.474.58 
 ( m , 1h ) , 6.33 ( d , j = 15.9 hz , 1h ) , 6.956.99 
 ( m , 3h ) , 7.10 ( t , j = 7.2 hz , 1h ) , 7.21 ( d , j = 2.1 hz , 1h ) , 7.31 ( d , j = 8.1 hz , 1h ) , 
 7.36 ( d , j = 8.1 hz , 1h ) , 7.42 ( d , j = 15.9 hz , 1h ) , 7.48 ( dd , j = 2.1 hz , j = 8.1 hz , 1h ) , 7.50 ( d , j = 8.2 hz , 2h ) , 7.63 ( d , j = 7.8 hz , 1h ) , 7.67 ( d , j = 2.1 hz , 1h ) , 
 8.72 ( d , j = 8.1 hz , 1h ) , 8.99 ( s , 1h ) , 10.66 ( s , 
 1h ) , 10.86 ( s , 1h ) . hrms ( ap - esi ) m / z calcd for c27h23cl2n3o4 [ m + h ] 524.1138 , found 524.1138 . retention 
 time : 14.2 min , eluted with 43% acetonitrile/57% water ( containing 
 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.11 ( d , j = 6.9 hz , 2h ) , 4.054.20 ( m , 2h ) , 
 4.524.60 ( m , 1h ) , 6.32 ( d , j = 15.9 hz , 1h ) , 
 6.936.99 ( m , 3h ) , 7.08 ( t , j = 6.9 hz , 1h ) , 
 7.18 ( d , j = 2.1 hz , 1h ) , 7.267.33 ( m , 3h ) , 
 7.41 ( d , j = 15.9 hz , 1h ) , 7.49 ( d , j = 8.7 hz , 2h ) , 7.63 ( d , j = 7.8 hz , 1h ) , 7.897.94 
 ( m , 2h ) , 8.56 ( d , j = 7.8 hz , 1h ) , 8.98 ( s , 1h ) , 
 10.66 ( s , 1h ) , 10.83 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h24fn3o4 [ m + h ] 474.1824 , found 474.1824 . retention time : 12.0 min , eluted with 40% acetonitrile/60% water ( containing 
 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  
 3.11 ( d , j = 6.9 hz , 2h ) , 4.064.20 ( m , 2h ) , 
 4.514.61 ( m , 1h ) , 6.32 ( d , j = 15.6 hz , 1h ) , 
 6.936.98 ( m , 3h ) , 7.08 ( td , j = 0.9 , j = 7.8 hz , 1h ) , 7.17 ( d , j = 2.1 hz , 1h ) , 
 7.36 ( d , j = 8.1 hz , 1h ) , 7.41 ( d , j = 15.6 hz , 1h ) , 7.49 ( d , j = 8.4 hz , 2h ) , 7.66 
 ( d , j = 9.3 hz , 1h ) , 7.69 ( d , j = 
 1.8 hz , 2h ) , 7.81 ( d , j = 1.8 hz , 2h ) , 8.63 ( d , j = 7.8 hz , 1h ) , 8.98 ( s , 1h ) , 10.66 ( s , 1h ) , 10.83 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h24brn3o4 [ m + h ] 534.1023 , found 534.1021 . h nmr ( 300 mhz , dmso - d6 )  3.10 ( d , j = 6.9 
 hz , 2h ) , 3.80 ( s , 3h ) , 4.024.20 ( m , 2h ) , 4.524.63 
 ( m , 1h ) , 6.33 ( d , j = 15.9 hz , 1h ) , 6.937.00 
 ( m , 5h ) , 7.08 ( td , j = 0.9 hz , j = 7.4 hz , 1h ) , 7.17 ( d , j = 2.1 hz , 1h ) , 7.34 ( d , j = 8.1 hz , 1h ) , 7.42 ( d , j = 15.9 hz , 
 1h ) , 7.49 ( d , j = 8.4 hz , 2h ) , 7.64 ( d , j = 7.8 hz , 1h ) , 7.85 ( d , j = 8.7 hz , 2h ) , 8.38 ( d , j = 8.1 hz , 1h ) , 8.98 ( s , 1h ) , 10.66 ( s , 1h ) , 10.82 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c28h27n3o5 [ m + h ] 486.2023 , found 486.2023 . retention time : 
 10.1 min , eluted with 47% acetonitrile/53% water ( containing 0.4% 
 formic acid ) . h nmr ( 300 mhz , dmso - d6 )  1.36 
 ( s , 9h ) , 2.832.99 ( m , 2h ) , 3.72 ( s , 3h ) , 3.934.04 
 ( m , 3h ) , 6.33 ( dd , j = 4.5 hz , j = 15.9 hz , 1h ) , 6.95 ( d , j = 8.4 hz , 2h ) , 6.977.02 
 ( m , 2h ) , 7.107.15 ( m , 2h ) , 7.367.42 ( m , 2h ) , 7.49 
 ( d , j = 8.4 hz , 2h ) , 7.58 ( d , j = 
 7.8 hz , 1h ) , 8.97 ( s , 1h ) , 10.66 ( s , 1h ) . hrms ( ap - esi ) m / z calcd for c26h31n3o5 [ m + h ] 466.2336 , found 466.2332 . retention 
 time : 13.8 min , eluted with 47% acetonitrile/53% water ( containing 
 0.4% formic acid ) . h nmr ( 300 mhz , dmso - d6 )  1.151.25 ( m , 9h ) , 2.903.06 
 ( m , 2h ) , 3.974.06 ( m , 2h ) , 4.314.40 ( m , 2h ) , 4.56 
 ( s , 1h ) , 6.31 ( d , j = 15.6 hz , 1h ) , 6.766.79 
 ( m , 2h ) , 6.98 ( t , j = 7.2 hz , 1h ) , 7.017.07 
 ( m , 2h ) , 7.097.25 ( m , 5h ) , 7.327.43 ( m , 5h ) , 8.96 
 ( s , 1h ) , 10.64 ( s , 1h ) , 10.84 ( s , 1h ) . esi - ms m / z : 541.4 [ m + h ] . a pale yellow oil , 80% yield . a pale yellow oil , 85% yield . a pale yellow powder , 30% yield . h nmr ( 300 mhz , dmso - d6 )  
 3.15 ( d , j = 8.4 hz , 2h ) , 4.064.11 ( m , 1h ) , 
 4.164.22 ( m , 1h ) , 4.544.64 ( m , 1h ) , 6.48 ( d , j = 15.6 hz , 1h ) , 6.946.99 ( m , 2h ) , 7.08 ( t , j = 7.6 hz , 1h ) , 7.127.14 ( m , 2h ) , 7.19 ( d , j = 2.4 hz , 1h ) , 7.30 ( d , j = 8.1 hz , 1h ) , 
 7.34 ( d , j = 7.8 hz , 1h ) , 7.38 ( d , j = 15.9 hz , 1h ) , 7.427.54 ( m , 3h ) , 7.65 ( d , j = 7.8 hz , 1h ) , 7.827.85 ( m , 2h ) , 8.52 ( d , j = 8.1 hz , 1h ) , 9.05 ( s , 1h ) , 10.71 ( s , 1h ) , 10.82 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h25n3o4 [ m + h ] 456.1918 , found 456.1919 . retention time : 11.5 
 min , eluted with 46% acetonitrile/54% water ( containing 0.4% formic 
 acid ) . a pale yellow powder , 28% yield . h nmr ( 300 mhz , dmso - d6 )  
 3.053.19 ( m , 2h ) , 4.054.11 ( m , 1h ) , 4.134.20 
 ( m , 1h ) , 4.524.61 ( m , 1h ) , 6.48 ( d , j = 15.6 
 hz , 1h ) , 6.946.98 ( m , 2h ) , 7.08 ( t , j = 7.2 
 hz , 1h ) , 7.117.18 ( m , 3h ) , 7.267.34 ( m , 4h ) , 7.42 
 ( d , j = 15.9 hz , 1h ) , 7.64 ( d , j = 7.5 hz , 1h ) , 7.897.94 ( m , 2h ) , 8.54 ( d , j = 7.8 hz , 1h ) , 9.04 ( s , 1h ) , 10.70 ( s , 1h ) , 10.80 ( s , 1h ) . hrms 
 ( ap - esi ) m / z calcd for c27h24fn3o4 [ m + h ] 474.1824 , 
 found 474.1825 . a pale yellow powder , 33% yield . h nmr ( 300 mhz , dmso - d6 )  
 3.093.12 ( m , 2h ) , 4.064.20 ( m , 2h ) , 4.544.61 
 ( m , 1h ) , 6.48 ( d , j = 15.9 hz , 1h ) , 6.946.99 
 ( m , 2h ) , 7.08 ( t , j = 7.4 hz , 1h ) , 7.127.14 
 ( m , 2h ) , 7.18 ( d , j = 2.1 hz , 1h ) , 7.277.34 
 ( m , 2h ) , 7.42 ( d , j = 15.6 hz , 1h ) , 7.55 ( d , j = 8.7 hz , 2h ) , 7.64 ( d , j = 7.5 hz , 1h ) , 
 7.88 ( d , j = 8.7 hz , 2h ) , 8.62 ( d , j = 7.8 hz , 1h ) , 9.05 ( s , 1h ) , 10.71 ( s , 1h ) , 10.82 ( d , j = 1.5 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h24cln3o4 [ m + h ] 456.1918 , found 456.1919 . retention time : 17.2 
 min , eluted with 40% acetonitrile/60% water ( containing 0.4% formic 
 acid ) . this method was the same as the general procedure for the preparation 
 of 8 and 25 . a pale yellow oil , 65% 
 yield . h nmr ( 300 mhz , dmso - d6 )  3.173.27 ( m , 1h ) , 3.413.47 
 ( m , 1h ) , 3.67 ( s , 1h ) , 4.124.17 ( m , 1h ) , 4.254.27 
 ( m , 1h ) , 4.36 ( s , 2h ) , 6.38 ( d , j = 15.6 hz , 1h ) , 
 6.916.99 ( m , 3h ) , 7.10 ( t , j = 7.2 hz , 1h ) , 
 7.22 ( d , j = 2.4 hz , 1h ) , 7.37 ( d , j = 7.8 hz , 2h ) , 7.427.50 ( m , 6h ) , 7.647.68 ( m , 2h ) , 
 9.72 ( s , 1h ) , 9.92 ( s , 1h ) , 10.31 ( s , 1h ) , 11.05 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h27n3o3 [ m + h ] 442.2125 , found 442.2125 . h nmr ( 300 mhz , dmso - d6 )  
 3.173.26 ( m , 1h ) , 3.413.48 ( m , 1h ) , 3.70 ( s , 1h ) , 
 4.124.17 ( m , 1h ) , 4.254.28 ( m , 1h ) , 4.36 ( s , 2h ) , 
 6.39 ( d , j = 15.9 hz , 1h ) , 6.926.99 ( m , 3h ) , 
 7.10 ( t , j = 6.9 hz , 1h ) , 7.23 ( d , j = 2.1 hz , 1h ) , 7.29 ( d , j = 8.7 hz , 2h ) , 7.357.42 
 ( m , 2h ) , 7.487.53 ( m , 3h ) , 7.697.74 ( m , 2h ) , 9.74 
 ( s , 1h ) , 9.94 ( s , 1h ) , 11.05 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h26fn3o3 [ m + h ] 460.2031 , found 460.2033 . h nmr ( 300 mhz , dmso - d6 )  
 3.173.26 ( m , 1h ) , 3.413.47 ( m , 2h ) , 4.114.16 
 ( m , 1h ) , 4.244.27 ( m , 1h ) , 4.37 ( s , 2h ) , 6.38 ( d , j = 15.9 hz , 1h ) , 6.926.99 ( m , 3h ) , 7.11 ( t , j = 7.2 hz , 1h ) , 7.23 ( d , j = 2.1 hz , 1h ) , 
 7.37 ( d , j = 8.1 hz , 1h ) , 7.42 ( d , j = 15.6 hz , 1h ) , 7.487.53 ( m , 5h ) , 7.70 ( d , j = 8.4 hz , 2h ) , 9.75 ( s , 1h ) , 9.69 ( s , 1h ) , 10.30 ( s , 1h ) , 11.05 
 ( d , j = 1.8 hz , 1h ) . hrms ( ap - esi ) m / z calcd for c27h26cln3o3 [ m + h ] 476.1735 , found 476.1735 . were conducted as previously described . in brief , 10 l of enzyme solution ( hela 
 nuclear extract , hdac1 , hdac2 , hdac3 . or hdac6 ) was mixed with various 
 concentrations of tested compound ( 50 l ) . the mixture was incubated 
 at 37 c for 5 min , then luorogenic substrate boc - lys(acetyl)-amc 
 ( 40 l ) was added . after incubation at 37 c for 30 min , 
 the mixture was stopped by the addition of 100 l of developer 
 containing trypsin and tsa . twenty minutes later , fluorescence intensity 
 was measured using a microplate reader at excitation and emission 
 wavelengths of 390 and 460 nm , respectively . the inhibition ratios 
 were calculated from the fluorescence intensity readings of tested 
 wells relative to those of control wells , and the ic50 values 
 were calculated using a regression analysis of the concentration / inhibition 
 data . all cell lines were 
 maintained in rpmi1640 medium containing 10% fbs at 37 c in 
 a 5% co2 humidified incubator . briefly , cells were passaged the 
 day before dosing into a 96-well cell plate , allowed to grow for 12 
 h and then treated with different concentrations of compound sample 
 for 48 h. a 0.5% mtt solution was added to each well . after incubation 
 for another 4 h , formazan formed from mtt was extracted by adding 
 150 l of dmso rocking for 15 min . briefly , u937 cells with different 
 treatment were collected and lysed with lysis buffer ( 20 mm tris - hcl 
 [ ph 7.5 ] , 1% np-40 , 1 mm sodium vanadate , 1 mm edta , 1 mm egta , 50 
 mm naf , and 1 mm phenylmethyl sulfonylfluoride [ pmsf ] ) for 30 min , 
 then centrifuged for 15 min at 14  000 rpm at 4 c , and 
 the supernatant was the whole - cell extracts . total protein extracts 
 ( 30 g per lane ) were separated by 12% sds polyacrylamide gel 
 electrophoresis and transferred onto pvdf membranes ( cat . membrane was blocked with 5% s36 milk in tbs - t ( 10 mm 
 tris [ ph 7.4 ] , 150 mm nacl , and 0.1% tween 20 ) for 1 h at room temperature , 
 then incubated with a 1:1000 or 1:2000 dilution of primary antibody 
 overnight at 4 c . then the membrane was washed three times and 
 incubated at 1:2000 dilution of antimouse or antirabbit goat - hrp - conjugated 
 secondary antibodies for 2 h at room temperature . finally the membrane 
 was washed another three times and developed by enhanced chemiluminescence 
 ( ecl , cat . u937 cells ( 1.5  10 ) were treated with different 
 concentrations of 11r , 11w , or saha for 
 12 or 24 h. cells were harvested following 
 incubation , washed twice in cold pbs , centrifuged , and resuspended 
 in 1 annexin - binding buffer . cells were diluted in 1 annexin - binding 
 buffer to 1  10 cells / ml , preparing a sufficient 
 volume to have 100 l per assay . five microliters of alexa fluor 
 488 annexin v and 1 l of 100 g / ml pi were added to each 
 100 l of cell suspension . , 150 l of 1 annexin - binding 
 buffer was added , mixed gently , and kept on ice . the stained cells 
 were analyzed by flow cytometry , measuring the fluorescence emission 
 at 530 and 575 nm ( or equivalent ) using 488 nm excitation . alexa fluor 
 488 annexin v / dead cell apoptosis kit with alexa fluor 488 annexin 
 v and pi for flow cytometry was used for this assay ( invitrogen ) . preparation of artificial gastric juice : 160 ml of water and 2 
 g of pepsin were added to 3.28 ml of diluted hydrochloric acid . preparation 
 of artificial intestinal juice : 1.36 g of sodium dihydrogen phosphate 
 was dissolved in 100 ml of water , and the ph was adjusted to 6.8 using 
 0.4% naoh . then 2 g of pancreatin was added , and the mixture was diluted 
 to 200 ml with water . preparation of rat liver homogenate : wistar 
 rats ( experimental animal center , shandong university , female , 200  
 20 g ) were sacrificed after fasting 12 h. liver was homogenized in 
 quadruple phosphate butter ( ph 7.4 ) and centrifuged ( 4000 rpm ) , 10 
 min . artificial gastric juice , artificial intestinal juice , rat 
 liver homogenate , and human plasma were preincubated at 37 c 
 prior to the addition of 50 l of stock solution of mutual prodrugs 
 ( 1 mg / ml in ch3oh ) . sample aliquots were taken for processing 
 and analysis after 24 h incubation at 37 c . all of the samples 
 underwent extraction using 600 l acetonitrile and were filtered 
 ( 0.22 m ) after shocking 30 s and centrifugation at 12  000 
 rpm , 10 min . chromatographic analysis was achieved by hplc method 
 on an agilent 1100 hplc instrument using a ods hypersil column ( 5 
 m , 4.6 mm  100 mm ) according the following method : compound 
 was eluted with 42% acetonitrile/58% water ( containing 0.4% formic 
 acid ) over 20 min , with detection at 290 nm and a flow rate of 1 ml / min . the temperature of the column was 25 c , and the quantity of 
 injection was 20 m . for 
 in vivo antitumor efficacy research , 1  10 human 
 histiocytic lymphoma cells ( u937 ) were inoculated subcutaneously in 
 the right flank of male athymic nude mice ( 56 weeks old , slac 
 laboratory animal , shanghai ) . ten days after injection , tumors were 
 palpable , and mice were randomized into treatment and control groups 
 ( 5 mice per group ) . the treatment groups received compound 11r ( 100 mg / kg / d ) or saha ( 100 mg / kg / d ) by oral administration , and 
 the blank control group received equal volume of pbs solution containing 
 40% dmso . during treatment , subcutaneous tumors were measured with 
 a vernier caliper every three days , and body weight was monitored 
 regularly . after treatment , mice were sacrificed and dissected to 
 weigh the tumor tissues and to examine the internal organ injury . 
 tumor growth inhibition ( tgi ) and relative increment ratio ( t / c ) were 
 used as the evaluation indicators to reveal the antitumor effects 
 in tumor weight and tumor volume , respectively . tumor 
 volumes ( v ) 
 were estimated using the equation ( v = ab/2 , where a and b stand 
 for the longest and shortest diameter , respectively ) . t / c was calculated 
 according to the following formula : where rtv is the relative tumor volume = vt / v0 ( vt , the tumor volume measured at the end of treatment ; v0 , the tumor volume measured at the beginning 
 of treatment ) . compounds 
 were docked into 
 the active site of hdac3 ( pdb code 4a69 ) using tripos sybyl 8.0 . before the docking 
 process , the protein structure was treated by deleting water molecules , 
 adding hydrogen atoms , fixing atom types , and assigning amber7 ff99 
 charges . the molecular structures were generated 
 with the sybyl / sketch module and optimized using powell s method 
 with the tripos force field with convergence criterion set at 0.05 
 kcal/( mol ) and assigned charges with the gasteiger
in our previous study , we designed 
 and synthesized a novel series 
 of n - hydroxycinnamamide - based hdac inhibitors ( hdacis ) , 
 among which the representative compound 14a exhibited 
 promising hdacs inhibition and antitumor activity . in this current 
 study , we report the development of a more potent class of n - hydroxycinnamamide - based hdacis , using 14a as lead , among which , compound 11r gave ic50 values of 11.8 , 498.1 , 3.9 , 2000.8 , 5700.4 , 308.2 , and 900.4 nm 
 for the inhibition of hdac1 , hdac2 , hdac3 , hdac8 , hdac4 , hdac6 , and 
 hdac11 , exhibiting dual hdac1/3 selectivity . 
 compounds 11e , 11r , 11w , and 11y showed 
 excellent growth inhibition in multiple tumor cell lines . 
 in vivo 
 antitumor assay in u937 xenograft model identified compound 11r as a potent , orally active hdaci . to the best of our knowledge 
 , 
 this work constitutes the first report of oral active n - hydroxycinnamamide - based hdacis with dual hdac1/3 selectivity .