( s)-perillyl alcohol ( poh , also known as p - metha-1,8-diene-7-ol 
 or 4-isopropenyl cyclohexene carbinol ) 
 is a monoterpene produced by a number of plants , including cherries , 
 lavendin , mints , and celery seeds via oxidative modification of d - limonene . interest in poh as a potential 
 anticancer agent stems from its ability to cause g1 cancer cell cycle 
 arrest via the putative inhibition of post - translational modification 
 of signal transduction proteins involved in the ras / mapk pathway . while the fundamental mechanism(s ) and/or target(s ) 
 of poh remain unclear , poh has been implicated in a range of functions 
 including inhibition of small g protein isoprenylation and induction 
 of proto - oncogenes , inhibition of na / k - atpase , disruption of htert  mtor raptor 
 protein complex , suppression of 3-hydroxy-3-methylglutaryl 
 coenzyme a ( hmg - coa ) reductase synthesis in mammalian cells , as well as inhibition of 4e - bp1(ser65 ) phosphorylation and cap - dependent 
 translation . poh was initially evaluated 
 in phase i and phase ii clinical trials for the treatment of a range 
 of cancers ( breast , colon , ovarian , and prostrate ) . while these studies revealed poh to be well tolerated at high doses , 
 tumor responses were low . however , more recent clinical studies focused 
 upon intranasal delivery of poh to treat recurrent malignant glioblastoma 
 led to notable tumor regression with high doses of poh . this promising precedent suggests that poh analogues with improved 
 potency and/or drug properties may serve to reinvigorate the clinical 
 utility of this unique plant metabolite and also potentially offer 
 new probes for further mechanistic interrogation . the glycosylation 
 of small - molecule - based or natural product - based 
 leads / drugs can often dramatically influence pharmacological properties 
 and admet . in the context of poh , a small 
 set of naturally occurring poh glycosides ( figure 1 ; 1a , 2a , 9a , 10a ) isolated from perilla frutescens leaves a few additional poh glycosides were generated via conventional 
 or enzymatic synthesis , ( figure 1 ; 3a8a , 11a ) and , cumulatively , these studies revealed certain 
 poh glucosides ( figure 1 ; 1a , 3a ) as equipotent to poh against select cancer cell lines 
 in vitro . yet , studies designed to systematically assess and/or exploit 
 the impact of poh glycosylation are lacking . neoglycorandomization , 
 a divergent chemoselective glycosylation method , is advantageous in 
 this regard as it offers a rapid strategy for differential glycosylation 
 of a selected target scaffold . herein 
 we report the synthesis and in vitro anticancer activity of a set 
 of 34 distinct poh neoglycosides , several of which displayed improved 
 in vitro anticancer activity over the parent natural product . the 
 best among these ( 4-azido - d - glucoside pg9 ) displayed a striking enhancement in potency ( > 85-fold against 
 a549 
 nonsmall cell lung ; 15-fold against pc3 prostate ) over poh and represents 
 the most active perillyl glycoside reported to date . in contrast to 
 previous reports , the subsequent study 
 of the influence of poh and representative improved poh glycosides 
 upon 4e - bp1 phosphorylation in a549 cells suggests the antiproliferative 
 effects of the improved poh glycosides may be associated with the 
 inhibition of cap - dependent translation by targeted inhibition of 
 phosphorylation of s6 ribosomal protein rather than 4e - bp1 . this study 
 also highlights , for the first time , the compatibility of neoglycosides 
 with cu(i)-catalyzed huisgen 1,3-dipolar cycloaddition . perillyl 
 neoaglycon 3 was synthesized in three simple 
 steps ( scheme 1 ) . specifically , poh was oxidized 
 to ( s)-perillaldehyde ( 1 ) using o - iodoxybenzoic acid ( ibx ) and acetic acid . subsequent reductive amination proceeded via formation 
 of oxime 2 with methoxyamine hydrochloride in the presence 
 of triethylamine , followed by reduction with nacnbh3 in 
 the presence of acetic acid to provide the requisite neoaglycon 3 . optimization of 3 neoglycosylation with d - glucose using a range of general 
 neoglycosylation conditions revealed meoh : hoac ( 5:1 ) as most 
 effective , providing the corresponding d - glucoside pg1 in 69% isolated yield and , consistent with prior studies , the -anomer exclusively . the scope of 
 sugars selected for this study included representative pentoses ( d / l - arabinose , pg3/pg28 ; l - ribose , pg17 ; d - xylose , pg22 ) , 
 hexoses ( d / l - glucose , pg1/pg6 ; 
 3-o - methyl - d - glucose , pg4 ; l - rhamnose , pg32 ) , azidosugars ( 6-deoxy-6-azido - d - glucose , pg2 ; 3-deoxy-3-azido - d - glucose , pg8 ; 4-deoxy-4-azido - d - glucose , pg9 ; 
 2-deoxy-2-azido - d - glucose , pg13 ; 4,6-deoxy-4,6-diazido - d - glucose , pg20 , 4-deoxy-4-azido - l - glucose , pg24 ; 4-deoxy-4-azido - d - xylose , pg34 ) , fluorosugars ( 3-deoxy-3-fluoro - d - glucose , pg18 ; 4-deoxy-4-fluoro - d - glucose , pg21 ; 2-deoxy-2-fluoro - d - glucose , pg29 ; 2-deoxy-2-fluoro - d - mannose , pg30 ) n - acyl sugars ( n - acetyl - d - galactosamine , pg5 ; n - acetylmuramic 
 acid , pg12 ; streptozocin , pg16 ; 3-n - decanoyl - d - glucosamine , pg23 ; 6-n - decanoyl - d - glucos amine , pg26 ; 3-n - allyloxylcarbonyl - d - glucosamine , pg27 ; 2-n - allyloxylcarbonyl - d - glucosamine , pg31 ; 6-n - allyloxylcarbonyl - d - glucosamine , pg33 ) , an acid - bearing sugar ( d - glucuronic acid , pg25 ) , and a dissacharide ( d - cellobiose , pg14 ) with a bias toward glucosides and acyl glucosides based upon the 
 previously reported active glycosides ( 1a and 3a ) . the inclusion of azidosugars served as a starting point for subsequent 
 divergence via chemoselective modification [ via cu(i)-catalyzed huisgen 
 1,3-dipolar cycloaddition ] or selective reduction 
 to afford the corresponding aminosugar conjugates ( scheme 2 ) . a library of 34 distinct neoglycosides ( pg1pg34 ) were synthesized ( figure s1 and table s1 in supporting information ) in good 
 to excellent yields ( 2382% ) , with the -anomer as predominate 
 product in most cases , consistent with previous studies . for aminosugar conjugates , azidosugar glycosides ( pg2 , pg8 , pg9 , pg13 ) were readily 
 reduced to their corresponding aminosugar counterparts ( pg7 , pg10 , pg11 , pg19 ) in the 
 presence of pme3 ( 1.0 m in thf ) , with yields ranging from 
 29 to 46% . to assess the compatibility of neoglycosides with cu(i)-catalyzed 
 huisgen 1,3-dipolar cycloaddition and the potential impact of sugar 
 triazole substitution upon poh bioactivity , the 6-azido perillyl 
 glucoside was also converted to the corresponding 6-triazole 
 perillyl glucoside pg15 via cu(i)-catalyzed huisgen 1,3-dipolar 
 cycloaddition in 82% yield ( scheme 2 ) . reagents 
 and conditions : ( a ) 
 ibx , hoac , acetonitrile ( 88% ) ; ( b ) meonh3cl , et3n , etoh ( 84% ) ; ( c ) nacnbh3 , hoac , meoh ( 50% ) ; ( d ) reducing 
 sugar , meoh : hoac ( 5:1 ) , 40 c ( 23%77% ) . reagent and conditions : ( a ) pme3 in thf , overnight , ( 46% ) ; ( b ) 4-pentyn-1-ol , cui , et3n , acn , overnight , ( 82% ) . the anticancer 
 properties of poh , 1 , 2 , 3 , 
 and pg1pg34 against 
 two human cancer cell lines ( nonsmall - cell lung a549 and prostate 
 pc3 ) was first assessed via percent inhibition of cell viability at 
 a single dose of 250 m ( figure 3 ) . compounds 
 which displayed > 40% inhibition in one or both cell lines ( oxime 2 , pg2 , pg9 , pg13 , pg16 , pg20 , pg22 , pg23 , pg24 , pg26 , pg31 , pg33 , pg34 ) from the single dose analysis were subsequently subjected 
 to a full dose study for ic50 determination along with 
 the parental poh ( figure 2 , 4 ) . first , substitutions of key sugar hydroxyls 
 with an azide present the greatest improvements . for example , while d - glucoside pg1 was relatively inactive ( > 500 
 m 
 in both cell lines ) , c6 ( pg2 : 163 m , a549 ; 
 182 m , pc3 ) , c4 ( pg9 : 4 m , a549 ; 
 22 m , pc3 ) , or c2 ( pg13 : 221 m , 
 a549 ; 176 m , pc3 ) azido substitution afforded moderate to dramatic 
 improvements in potency over the parent natural product ( poh : 350 
 m , a549 ; 380 m , pc3 ) . from this series , the c4-azidosugar 
 variant pg9 stands out as the best analogue , with > 15-fold 
 improved potency against pc3 and > 85-fold increased potency against 
 a549 in vitro , highlighting regiospecificity as an important contributor . second , the benefits from sugar azido substitution are not additive , 
 as evidenced by a comparison of pg2 ( c6-azido ) 
 and pg9 ( c4-azido ) to the c4,c6-diazido 
 analogue pg20 . third , while the facile synthesis of the 
 c6-triazole - substituted analogue pg15 highlights 
 the compatibility of neoglycosides with cu(i)-catalyzed huisgen 1,3-dipolar 
 cycloaddition chemistry , c6-triazole substitution is detrimental 
 to activity based upon the comparative activities of pg2 ( c6-azido ) and pg15 . fourth , sugar n - acylation also generally improved activity as both the 
 3-n - decanoyl and 6-n - decanoyl aminoglucosides ( pg23 and pg26 , respectively ) displayed relatively similar improvements in potency 
 over poh ( 1220-fold , pc3 ; 2025-fold , a549 ) with shorter 
 c2 , c3 and c6 n - acyl substitutions 
 ( e.g. , pg16 , pg27 , pg31 , pg33 ) generally being less favorable . fifth , while the d - glucoside pg1 displayed activity similar to the 
 parent poh ( in a manner reminiscent to the prior work with 1a ) , removal of the c6-ch2oh ( pg22 ) 
 in this context led to slight improvements and , consistent with pg9 , the addition of a 4-azido - substitution ( pg34 : 76 m , a549 ; 170 m , pc3 ) led to further 
 improvements in potency . finally , on the basis of the comparative 
 activities of pg9 and pg24 , the d - enantiomer offered the greatest enhancement . structures of the most 
 antiproliferative poh neoglycosides against 
 a549 and pc3 cell lines . ( a ) single dose ( 250 m ) comparisons of poh neoglycosides 
 and poh against a549 and pc3 cell lines . reciprocal ic50 values for poh ( ic50 350 
 m , a549 ; 380 m , pc3 ) and the most active poh neoglycosides 
 ( the most active of which is pg9 , with an ic50 4 m , a549 ; 22 m , pc3 ) . ic50 values and 
  sd are summarized in supporting information , 
 table s2 and represent a triplicate of dose response 
 experiments conducted over nine concentrations at 2-fold dilution . while a number of putative anticancer 
 mechanisms for poh have been 
 put forth , one reported effect centers around cap - dependent translation 
 in tumor cell lines . specifically , peffley and co - workers reported 
 poh at 400 m to suppress 4e - bp1 ( ser65/thr37 ) phosphorylation 
 and to disrupt interactions between critical components of the capped 
 mrna - binding complex ( eif4e and eif4 g ) . thus , the effect of poh and representative glycosides pg20 and pg23 on 4e - bp1 phosphorylation was assessed using 
 the cell line in which the most dramatic antiproliferative improvements 
 were observed ( a549 ) . as illustrated in figure 5 , in a549 cells poh had 
 no effect upon the phosphorylation of 4e - bp1 at thr37/46 , ser65 , or 
 thr70 , but 4e - bp1 phosphorylation was induced by pg20 and pg23 . unlike poh , pg20 and pg23 also profoundly inhibited phosphorylation of s6 ribosomal protein , 
 another important regulator of cap - dependent translation . furthermore , pg20 and pg23 substantially increased levels of cleaved parp , an indicator of 
 apoptosis , consistent with their increased antiproliferative activities . 
 cumulatively , these data suggest that the cytotoxicity of pg20 and pg23 toward a549 cells may be associated with the 
 inhibition of phosphorylation of s6 ribosomal protein rather than 
 4e - bp1 . western blot representing the effects of 250 and 500 m perillyl 
 alcohol ( poh ) , pg20 , and pg23 treatments 
 of a549 on phosphorylation of 4e - bp1 at 6 h. this study highlights a facile four - step process 
 for poh glycodiversification 
 and the discovery of a series of new poh glycosides that display improvements 
 in potency approaching 2 orders of magnitude . for comparison , a survey 
 of the previously reported activities of poh analogues ( including 
 glycosides , prodrug conjugates , and additional functionalized variations ) revealed c7-n , n - disubstituted amino derivatives as among the 
 most potent to date , the best of which displayed an ic50 > 50 m against a549 . thus , the activity of the poh 4-azido - d - glucoside ( pg9 ) reported herein stands out among 
 most , if not all , poh derivatives reported to date . cumulatively , 
 the sar presented suggests lipophilicity ( azido or n - acyl ) to be of greatest benefit . while the fundamental antiproliferative mechanism of poh and poh congeners 
 remains unclear , the current study also supports the contention that 
 the newly reported poh glycosides may derive , at least in part , from 
 the inhibition of phosphorylation of s6 ribosomal protein rather than 
 4e - bp1 as previously put forth . additional 
 studies to specifically identify the target and assess the in vivo 
 efficacy of these newly discovered poh glycosides are underway . sugar donors 2-deoxy-2-azido - d - glucose and 6-deoxy-6-azido - d - glucose were purchased 
 from carbosynth ( berkshire , uk ) . solvents were of acs grade and purchased 
 from pharmco - aaper ( brookfield , ct ) . tlc silica gel plates ( 60 f254 ) 
 were purchased from emd chemicals inc . nmr spectra 
 were obtained on either a varian unity inova 400 or 500 mhz instrument 
 ( palo alto , ca ) at ambient temperature using 99.8% cdcl3 and 99.8% cd3od ( cambridge isotope laboratories , ma , 
 usa ) . h and c chemical shifts were referenced 
 to internal solvent resonances and reported in parts per million ( ppm ) , 
 with coupling constants j given in hz . multiplicities 
 are indicated by s ( singlet ) , d ( doublet ) , t ( triplet ) , dt ( doublet 
 of triplet ) , m ( multiplet ) , and br ( broad ) . hr - esi - ms spectra were 
 recorded on ab sciex triple tof 5600 system ( ab sciex , framingham , 
 ma , usa ) . flash column chromatography was performed with redisep rf gold columns from teledyne isco ( lincoln , ne ) on a biotage 
 isolera 4 ( biotage , charlotte , nc , usa ) . the purity of starting material , ( neoaglycon 3 ) and all corresponding neoglycosides was determined to be 
 95% unless specified otherwise ( supporting 
 information , table s1 ) . a suspension of poh ( 1 g , 6.6 mmol ) , ibx 
 ( 45 wt % , 3.7 g , 2.0 mmol ) , and acetic acid ( 0.75 ml , 2.0 mmol ) in 
 acetonitrile ( 5 ml ) was stirred vigorously at room temperature overnight . after completion of the reaction based upon tlc , sodium bicarbonate 
 ( 200 mg ) was added . the resulting mixture was filtered and the solvent 
 was removed in vacuo to afford the crude product , which was purified 
 by normal - phase flash chromatography using n - hexane / ethyl 
 acetate as eluent ( 0.9 g , 88% yield ) . h nmr ( 500 mhz , 
 cdcl3 )  9.41 ( s , 1h ) , 6.816.79 ( m , 1h ) , 
 4.73 ( d , 2h ) , 2.492.39 ( m , 2h ) , 2.252.17 ( m , 2h ) , 
 2.132.06 ( m , 1h ) , 1.901.86 ( m , 1h ) , 1.74 ( s , 3h ) , 
 1.451.39 ( m , 1h ) ; c nmr ( 100 mhz , cdcl3 )  194.0 , 150.7 , 148.4 , 141.4 , 109.6 , 40.8 , 31.8 , 26.5 , 21.7 , 
 20.8 . hrms - esi ( m / z ) : calcd for 
 c11h18no ( mh ) 180.1383 , found 180.1385 . perillyl aldehyde 1 ( 2 g , 13.3 mmol ) was dissolved in absolute etoh ( 40 ml ) . et3n ( 2.4 ml , 17.3 mmol ) and methoxylamine hydrochloride 
 ( 1.4 g , 17.3 mmol ) were added , and the reaction was stirred overnight 
 at room temperature . the solvent was removed in vacuo , and the crude 
 product was purified by normal - phase flash chromatography using n - hexane / ethyl acetate as eluent ( 2.0 g , 84% yield ) . h nmr ( 500 mhz , cdcl3 )  7.59 ( s , 1h ) , 5.93 
 ( t , 1h ) , 4.70 ( d , 2h ) , 3.80 ( s , 3h ) , 2.43 ( dd , 1h ) , 2.292.11 
 ( m , 3h ) , 2.092.01 ( m , 1h ) , 1.861.80 ( m , 1h ) , 1.70 
 ( s , 3h ) , 1.481.38 ( m , 1h ) . c nmr ( 100 mhz , cdcl3 )  151.5 , 149.1 , 134.8 , 132.7 , 109.1 , 61.6 , 41.0 , 31.4 , 
 26.9 , 24.0 , 20.8 . hrms - esi ( m / z ) : 
 calcd for c11h18no ( mh ) 180.1383 , 
 found 180.1385 . oxime 2 ( 2.0 
 g , 11.2 mmol ) was dissolved in meoh ( 50 ml ) under argon followed by 
 the addition of nacnbh3 ( 7.0 g , 111.6 mmol ) and hoac ( 3.2 
 ml , 55.8 mmol ) . the reaction was stirred at room temperature for 48 
 h , subsequently quenched with saturated aqueous nahco3 ( 45 
 ml ) , and meoh was removed in vacuo . the recovered organics were 
 washed with water and brine , dried over na2so4 , and subsequently filtered and concentrated in vacuo to provide 
 crude product which was purified by normal - phase flash chromatography 
 using n - hexane / ethyl acetate as eluent ( 1.0 g , 50% 
 yield ) . h nmr ( 500 mhz , cdcl3 )  5.61 
 ( s , 1h ) , 5.52 ( s , 1h ) , 4.67 ( s , 2h ) , 3.48 ( s , 3h ) , 3.37 ( s , 2h ) , 2.192.02 
 ( m , 4h ) , 1.981.85 ( m , 1h ) , 1.841.74 ( m , 1h ) , 1.69 
 ( s , 3h ) , 1.481.38 ( m , 1h ) . c nmr ( 100 mhz , cdcl3 )  149.9 , 134.0 , 124.4 , 108.7 , 61.8 , 58.1 , 41.1 , 30.7 , 
 27.7 , 27.7 , 20.9 . hrms - esi ( m / z ) : 
 calcd for c11h20no ( mh ) 182.1539 , 
 found 180.1542 . neoaglycon 
 ( 3 ) was dissolved in meoh / acoh ( 5:1 ) in 1 dram vials 
 along with stir fleas at a final concentration of 300350 mm . reducing sugar ( 2 equiv ) was added and the vial capped , and the reaction 
 was stirred at 40 c for 2448 h. reaction progress was 
 monitored by tlc . upon completion , solvent was removed in vacuo to 
 afford the crude product , which was purified by normal - phase flash 
 chromatography using chcl3/meoh as eluent . the perillyl 
 neoglycosides were obtained with an isolated yield ranging from 23% 
 to 77% from perillyl neoaglycon 3 ( supporting information , table s1 ) . anomeric ratios were obtained 
 by comparison of anomeric proton integration ( supporting information , table s1 ) . to a 
 100 mm solution of azidosugar glycoside in thf was added pme3 in thf ( 1.2 equiv ) , and the reaction was stirred at 50 c for 
 1 h. after removal of the solvent in vacuo , the crude residue was 
 purified by normal - phase column chromatography using chcl3/meoh as eluent to afford the desired aminosugar glycoside . the aminosugar - appended 
 perillyl neoglycosides were obtained with an isolated yield yield 
 ranging from 29% to 46% ( supporting information , 
 table s1 ) . human cancer cell 
 line ( lung adenocarcinoma a549 and prostate cancer pc3 ) viability 
 was determined using the alamar blue ( or resazurine reduction ) assay 
 as previously described . briefly , cells were seeded in f-12k medium ( kaighn s modification 
 of ham s f-12 medium , invitrogen ) , supplemented with 10% fbs , 
 2 mm l - glutamine , 100 g / ml penicillin , and 100 g / ml 
 streptomycin onto flat - bottomed 96-well tissue culture plates ( corning , 
 ny , usa ) at a density of 3  103 cells / well and allowed to adhere 
 overnight . after removing the medium , 100 l of fresh medium 
 containing nine 2- or 3-fold dilutions ( 500 to 75 nm ) of each compound 
 were added . all assays were conducted in the presence of both negative 
 ( 0.5% dmso vehicle control ) and positive ( 1.5 mm h2o2 ) controls . the plates were incubated for 48 h under standard 
 conditions , 5% co2 and 37 c , in a humidity control 
 incubator . at the end of incubation period , 10 l of resazurin 
 ( 0.25 mg / ml in water ) plates were incubated 
 for an additional 3 h at standard culture conditions and shaken for 
 510 s , and fluorescence of resarufin ( ex = 600 nm , em = 590 nm ) was recorded as a basis 
 for ic50 determination using a nonlinear interpolation 
 of dose - dependent curves using prizm . data presented ( figure 4 and supporting information , 
 table s2 ) represent mean values ( sd ) of triplicate determinations 
 from three independent experiments . a549 cells were treated with 250 
 and 500 m of perillyl alcohol ( poh ) , pg20 , and pg23 for 6 h. cells were harvested , lysed , and the corresponding 
 extracts subjected to sds - page , and analyzed by western analysis as 
 previously described . commercial primary antibodies 
 for p-4e - bp1(t37/46 ) , p-4e - bp1(s65 ) , p-4e - bp1(t70 ) , p - s6 ( s235/236 ) , 
 eif4e , and cleaved parp ( cell signaling technology , danvers , ma ) were 
 employed , and secondary antibodies were detected using chemiluminescence 
 ( ge healthcare bio - sciences , pittsburgh , pa ) .
a facile 
 route to perillyl alcohol ( poh ) differential glycosylation 
 and the corresponding synthesis of a set of 34 poh glycosides is reported . 
 subsequent in vitro studies revealed a sugar dependent antiproliferative 
 activity and the inhibition of s6 ribosomal protein phosphorylation 
 as a putative mechanism of representative poh glycosides . 
 the most 
 active glycoside from this cumulative study ( 4-azido - d - glucoside , pg9 ) represents one of the most cytotoxic 
 poh analogues reported to date .