more than 25 years after the identification 
 of the causative agent 
 of aids , hiv / aids is still a major challenge to society . 
 the latest who / unaids report ( 2010 ) states that the number of people 
 living with hiv has risen to 33.3 million , with more than 2.6 million 
 new cases annually and almost 5000 aids - related deaths per day . with the introduction of the first hiv-1 
 protease inhibitor ( pi ) 
 ( saquinavir ) in 1995 and the development 
 of highly active antiviral therapy ( haart ) the clinical 
 outcome of hiv
/ aids changed from a lethal to a manageable , but chronic , 
 disease in the developed world .
the early pis suffered from poor 
 pharmacokinetic profiles and caused severe side effects such as hepatic 
 toxicity and lipodystrophy . for these reasons and 
 with a frequent daily dosing regimen , they were not the first - hand 
 choice in haart .
the most common combinations in early haart were 
 instead two nucleoside reverse transcriptase inhibitors ( nrtis ) together 
 with a non - nucleoside reverse transcriptase inhibitor ( nnrti ) .
the 
 development of nnrti- and/or nrti - resistant hiv strains and the introduction 
 of new pis , with a once - daily dose regime and improved effect profiles , 
 have made the combination of a pi together with two nrtis a more frequent 
 choice for first line treatment in haart .
although saquinavir has , to date , been followed 
 by eight other 
 pis ( ritonavir , indinavir , fosamprenavir , nelfinavir , lopinavir , atazanavir , 
 tipranavir , and darunavir ) , improving 
 pharmacokinetic properties and reducing adverse effects are still 
 issues that need to be addressed .
further , the rapid replication 
 and the high mutation rate of the hiv-1 virus , together with the mutation pressure induced by today s pharmacotherapies , 
 will lead to an increase in the problems associated with resistant 
 virus strains .
thus , we can not expect the good results currently seen 
 with haart to continue if new drugs are not developed and introduced 
 onto the market .
we have been engaged in 
 the development of novel hiv-1 pis since 
 1997 . in our most recent program
we developed 
 novel classes of potent hiv-1 pis incorporating a shielded tertiary 
 alcohol as part of the transition state mimic .
inspired by the structure of the potent inhibitor atazanavir ( atz ) ( figure 1 ) , we used a similar hydrazide moiety 
 in the prime side of our new tert - hydroxy - containing pis . by altering the length of the 
 central backbone , using a one- , two- , or three - carbon spacer ( figure 1 , series a , b , and c , respectively )
, we focused on optimizing the interaction with the 
 catalytically active aspartic acid residues of the enzyme .
class b , with the two - carbon spacer , yielded the best 
 results , with values of ki and ec50 as low as 1 and 3 nm , respectively .
in all three series ( a  c ) , inhibitors 
 with high membrane permeability were identified , as well as inhibitors 
 with good metabolic stability , providing pharmacokinetic properties well in the range of hiv pis 
 already on the market , e.g. , atz .
c cocrystallized with the enzyme revealed binding modes that 
 were not completely successful in establishing strong symmetric hydrogen 
 bonds ( < 3.0  ) with both the catalytic residues asp25 and 
 or asp125 , originating from each monomer of the hiv-1 protease .
therefore , we decided to further elaborate the central transition - state 
 mimic by relocating the hydroxyl group one position away from the 
 backbone .
this strategy was implemented by making use of a -hydroxy 
 -lactam moiety equipped with a secondary alcohol .
it was hypothesized 
 that the -hydroxy -lactam would provide a better hydrogen 
 bond arrangement for the catalytic asp residues and at the same time 
 reduce the flexibility , providing a more rigid inhibitor .
spacers are indicated in red : a , one - carbon 
 spacer ( ki = 5.5 nm);b , two - carbon spacer ( ki = 2.3 nm);c , three - carbon spacer ( ki = 2.8 nm);d , novel lactam - based inhibitors with two - carbon spacer ( ki = 0.8 nm ) and altered stereocenters indicated 
 by asterisks ; e , three - carbon spacer ( ki = 4.2 nm ) .
atz is included for comparison ( ki = 2.7 nm ) . modeling studies supported the hypothesis that 
 a hydroxyl group 
 in the 4-position of the -lactam might provide a new conformationally 
 constrained transition - state - mimicking scaffold for the development 
 of novel hiv-1 pis . since both the ( 3r,4s ) and the ( 3r,4r ) stereoisomers 
 provided good
docking poses , we decided to synthesize and evaluate 
 all four stereoisomers of the -lactam ( figure 1 , d ) .
in addition , two different lengths of the 
 central tether ( two or three carbons ) were investigated ( figure 1 , d and e ) .
the prime - side hydrazide moiety , inspired by atz , has been 
 successfully used in inhibitors in series a 
c and was therefore retained in the new series of lactam - based 
 inhibitors . here
we present the synthetic protocols and the 
 inhibitory potency 
 on enzyme level , as well as the activity in a cell - based assay , of 
 the new inhibitors ( d and e ) .
also included 
 are stability and permeability studies of selected compounds , together 
 with x - ray analyses of three of the inhibitors cocrystallized with 
 the hiv-1 protease .
starting from ( s)-4-hydroxydihydrofuran-2(3h)-one ( 1a ) or ( r)-4-hydroxydihydrofuran-2(3h)-one ( 1b ) , four hiv-1 pr inhibitors with 
 a two - carbon spacer and with varied stereochemistry in the lactam 
 ring were synthesized ( scheme 1 ) . encouraged 
 by
previously reported alkylations,1a and 1b were chosen as starting substrates for the two - step alkylation 
 process . upon treatment with dmpu , lda , and the first alkylating agent 
 ( allyl bromide or benzyl bromide ) at 50 c followed by 
 a second portion of lda and the addition of the second alkylating 
 agent ( benzyl bromide or allyl bromide ) at 40 c ,
the 
 dialkylated -hydroxy -lactams 2a  d were synthesized in isolated yields of 249% ( scheme 1 , paths a and b ) .
path 
 a : ( a ) dmpu , lda , allyl bromide , dry thf , added at 50 c , 
 stirred at 50 c for 1 h ; ( b ) lda , benzyl bromide added 
 at 40 c , stirred at 30 c for 1 h , giving 2a and 2c in 49% and 33% isolated yield , respectively . 

path b : ( c ) dmpu , lda , benzyl bromide , dry thf , added at 50 
 c , stirred at 40 c for 1 h ; ( d ) lda , allyl bromide , 
 added at 40 c , stirred at 30 c for 1 h , 
 giving 2b and 2d in 2% and 5% isolated yield , 
 respectively . in the first alkylation , the 
 allyl group in 2a and 2c ( or the benzyl 
 group in 2b and 2d ) was introduced trans 
 to the controlling 4-hydroxyl group as expected , 
 showing facial selectivity , as previously reported by meyers et al . and others .
in the second alkylation , the benzyl group ( or the 
 allyl group in 2b and 2d ) was introduced 
 trans to the 4-hydroxyl functionality .
consequently , the second alkylation 
 changed the stereochemistry of the first inserted group , forcing it 
 to end up cis to the 4-hydroxyl group . to be able to collect enough material of 2b and 2d , with their low - yielding synthetic pathway ,
a method was 
 developed to alter the stereochemistry at the hydroxyl group in 2a and 2c .
martin reagent to the corresponding ketones 
 was followed by reduction using nabh4 , affording 2d and 2b following paths a and b , respectively 
 ( scheme 2 ) , with ratios 2d/2a of 5.7:1 and 2b/2c of 5.9:1 . 

martin , dcm , rt , 
 1 h ; ( b ) 
 nabh4 , 1% methanol in thf , rt 2 h , 2d + 2a ( 5.9:1 ) 92% , 2b + 2c ( 5.7:1 ) 
 85% .
lactamization of the lactones 2a  d with tbs - protected indanolamine ( 3 ) was performed by 
 adopting the methodology developed by orrling et al .
lactams 4a  d were isolated in good yields using the ionic 
 liquid 1-butyl-3-methylimidazolium tetrafluoroborate ( [ bmim]bf4 ) under microwave irradiation 
 at 180 c for 35 min , followed by protection of the alcohol moiety 
 with tbsotf under basic conditions .
although the mixture was heated to 180 c , 
 these lactamization conditions are relatively mild compared to those 
 previously reported .
the use of highly 
 polar [ bmim]bf4 allowed lactamization to proceed smoothly 
 without the need of brnstedt acid .
reagents and conditions : 
 ( i ) 
 [ bmim]bf4 , 180 c , 35 min ; ( ii ) triethylamine , tbsotf , 
 dcm , 025 c , overnight , giving isolated yields of 4a 64% , 4b 53% , 4c 72% , and 4d 50% . to synthesize the prime - side 
 moiety 5a ,
hydrazone 7 was prepared in almost 
 quantitative yield starting from 
 the boc - protected hydrazine 6 , as previously reported 
 in the literature ( scheme 4 ) .
benzylation of 7 using koh and 4-bromobenzyl 
 bromide in anhydrous toluene afforded 8 in good yield . 

catalytic quantities of the phase - transfer catalyst tetrabutylammonium 
 hydrogen sulfate ( tbahs ) were used to improve solubility and increase 
 the rate of the reaction .
after the initial 
 workup of the alkylation reaction only compound 8 was 
 generated , but after flash chromatography purification , 
 compound 9 was also formed ( owing to hydrolysis of the 
 hydrazone ) .
however , purification in this step was necessary to remove 
 excess quantities of 4-bromobenzyl bromide , which was foreseen to 
 cause problems in the later steps .
the mixture of 8 and 9 was deprotected with 4 m hcl in thf to yield the pure hydrochloride 
 salt of 10 .
owing to the photosensitivity of the 
 free nitrogen in the p - bromobenzylhydrazine 10 , the coupling of 10 with 11 , 
 synthesized as previously reported , was 
 performed in a reaction vessel wrapped in 
 aluminum foil .
moreover , 10 , 11 , and hobt 
 were added under a nitrogen atmosphere at 0 c , and the mixture 
 was stirred for 30 min . subsequently ,
4-methylmorpholine ( nmm ) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide 
 hydrochloride ( edc ) were added and the reaction mixture was gradually 
 heated to 25 c and stirred under a nitrogen atmosphere for 15 
 h , giving 5a in good isolated yield ( 77% , 61% overall 
 isolated yield starting from 38 mmol of 6 ) .
reaction conditions : 
 ( a ) acetone , 
 mgso4 , acoh ( cat . ) , reflux , 1 h , 98% ; ( b ) ( i ) koh , anhydrous 
 toluene , tbahs , 50 c , 20 min ; ( ii ) 3 , 100 c , 
 2 h , 81% ; ( c ) hcl , thf , reflux , 3 h , quantitative yield ; ( d ) edci , 
 hobt , nmm , dcm , 025 c , 15 h , 77% ( 61% isolated yield 
 over four steps . ) . steps
a and c required no purification . next the allylic double bonds in lactams 4a  d were oxidatively cleaved to give the corresponding 
 aldehydes v  viii using osmium tetraoxide 
 and sodium 
 periodate in thf / water ( 3:1 ) at room temperature ( scheme 5 ) .
note 
 that the nomenclature for the absolute configuration for the lactam 
 carbon in position 3 changes when comparing the lactams 4a  d , the intermediates v  viii , and 12 and 13 because of changes 
 in the assigned priority according to the sequence rule .
reagents and conditions : 
 ( a ) 4a  d , oso4 , naio4 , thf / h2o , rt , overnight ; ( b ) 5a , 
 acetic 
 acid , na(oac)3bh , dry thf , rt , overnight , provided 12a in 35% and 12d in 54% isolated yield from 4a and 4d , respectively ; ( c ) tbaf , thf , rt , overnight , 
 provided 13a in 38% , 13b in 60% , 13c in 46% , 13d in 34% isolated yield from 4a  d , respectively .
reductive amination between the crude aldehydes and the prime side 
 ( 5a ) was performed in dry thf using acetic acid , followed 
 by treatment with na(oac)3bh , to afford the crude tbs - protected 
 products .
the tbs protecting groups were removed using tbaf , and the 
 inhibitors 13a  d , carrying a two - carbon 
 tether , were isolated in good yields ( scheme 5 ) .
the tbs - protected inhibitors 12a and 12d ( but not 12b and 12c ) were isolated , purified , 
 and fully characterized before the final deprotection . to evaluate 
 the effect of different p1 side chains , a small 
 series of p1 p - phenyl- and p - pyridyl - substituted inhibitors was produced .
the known problem of 
 rapid protodeboronation of 2-pyridylboronic acid prevented us from conducting functionalization of 12a and 12d directly via suzuki 
thus , to introduce the 2-pyridyl as a para - substituent 
 in p1 , the 2-pyridine - substituted hydrazide 5b ( scheme 6 ) was 
 synthesized starting from the 4-(2-pyridinyl)benzaldehyde , as previously 
 described .
the alcohols 14a and 14d were isolated 
 as side products in reductive amination reactions to produce 12a and 12d , respectively .
martin 
 reagent was used to oxidize 14a and 14d to 
 the corresponding aldehyde intermediates ( scheme 5 , v and viii , respectively ) , followed 
 by reductive amination with 5b using acetic acid and 
 na(oac)3bh in dry thf and subsequent tbaf - mediated deprotection 
 to give useful yields of the inhibitors 13e and 13f ( scheme 6 ) .
martin reagent , 
 dry dcm , 
 rt , 1 h ; ( b ) acetic acid , na(oac)3bh , dry thf , rt , overnight ; 
 ( c ) tbaf , thf , rt , overnight , provided isolated yields of 63% 13e and 38% 13h from 14a and 14d , respectively .
the tbs - protected 
 inhibitors 12a and 12d were decorated using 
 the corresponding phenyl- or pyridylboronic 
 acids in suzuki 
miyaura cross - coupling in which herrmann s 
 palladacycle ( 0.1 equiv ) was used as a palladium precatalyst together 
 with k2co3 ( 3.3 equiv ) and [ hp(t - bu)3]bf4 ( 0.2 equiv ) in dme / water .
the reaction 
 mixtures were heated to 140 c for 20 min under focused microwave 
 irradiation in sealed reaction vessels .
cross - coupling was followed 
 by deprotection of the hydroxyl groups using tbaf in thf at room temperature , 
 giving inhibitors 13g  j in good isolated 
 yields ( scheme 7 and table 2 ) .
reagents and conditions : 
 ( a ) 
 ( i ) 12a or 12d , herrmann s palladacycle , 
 k2co3 , 3- or 4-pyridylboronic acid , [ hp(t - bu)3]bf4 , dme , water , microwave 
 140 c , 20 min ; ( ii ) tbaf , thf , rt , overnight , providing isolated 
 yields of 63% 13 g , 59% 13h , 74% 13i , and 66% 13j . to be able to 
 incorporate the new lactam scaffold into inhibitors 
 with the three - carbon spacer , corresponding to the previously published c series ( figure 1 ) ,
the allylic compound 4a was refluxed in thf at 80 c with 9-bbn for 6 h. after 
 addition of naoh , h2o2 , and ethanol at room 
 temperature and another 2 h stirring , the primary alcohol 16 was isolated in good yield ( scheme 8) .
the alcohol 16 was oxidized to the corresponding aldehyde ( 17 ) using 50% so3py in dmso together with triethylamine 
 in dcm at 025 c .
the aldehyde 
 was thereafter used in a reductive amination reaction 
 with 5a using na(oac)3bh as reducing agent 
 at 35 c to give 18 in moderate isolated yield ( scheme 8) .
the nomenclature for the absolute configuration 
 of the lactam carbon in position 4 changes when comparing the 13a  j and 19a  e series because of changes in the assigned priority according 
 to the sequence rule in iupac s guidelines . reagents and conditions : 
 ( a ) 
 ( i ) 9-bbn , dry thf , 80 c , 6 h ; ( ii ) 2 m naoh , 30% h2o2 in h2o , ethanol , rt , 2 h , 78% ; ( b ) et3n , 50% so3py in dmso , dry dcm , 020 c , 
 3 h ; ( c ) 5a , acetic acid , na(oac)3bh , dry 
 thf , 35 c , 3 h , 35% ; ( d ) tbaf , thf , rt , overnight , 19a 61% ; ( e ) ( i ) herrmann s palladacycle , k2co3 , arylboronic acid , [ hp(t - bu)3]bf4 , 105 c , 1.5 h ; ( ii ) tbaf , thf , rt , overnight , 19b 45% , 19c 35% , and 19d 30% ; ( f ) 
 ( i ) 2-(tributylstannyl)pyridine , pd(pph3)2cl2 , cuo , dmf , 105 c , 2 h ; ( ii ) tbaf , thf , rt , overnight , 19e 16% .
deprotection of 18 using tbaf in thf gave inhibitor 19a in a good yield . 

inhibitor 18 was also used 
 as starting material in suzuki  miyaura cross - coupling with 
 phenyl- and pyridylboronic acids together with herrmann s palladacycle , 
 k2co3 , and [ hp(tbu3)]bf4 , heated by microwave irradiation to 105 c for 1.5 h. deprotection 
 of the tbs groups using tbaf in thf gave 19b  d in 3540% isolated yields . by use of 2-(tributylstannyl)pyridine
, 
 compound 18 was subjected to stille type coupling in 
 dmf under microwave irradiation ( 105 c , 2 h ) using cuo and with 
 pd(pph3)2cl2 as precatalyst .
the stille coupling was followed by tbaf - mediated deprotection giving 
 inhibitor 19e in moderate isolated yield .
since the preliminary docking studies suggested 
 that two of the 
 stereoisomers in the lactam moiety ( ( 3r,4s ) and ( 3r,4r ) ) , in the 
 two - carbon - tethered inhibitors would fit well in the enzyme , all four 
 stereoisomers were synthesized and evaluated regarding binding and 
 in a cell - based assay , giving the results summarized in table 1 .
comparisons with previous series of tertiary - alcohol - based 
 hiv-1 pis ( a  c ) could easily be conducted 
 by using the indanolamide in the p2 position and the p - bromophenyl as the p1 side chain . in accordance with 
 the initial docking studies , inhibitors
13a ( 3r,4s ) and 13d ( 3r,4r ) exhibited good activity 
 in the enzyme assay ( ki of 2.1 and 6.4 
 nm , respectively ) as well as in the cell - based evaluation ( ec50 of 0.64 and 0.35 m , respectively ) .
the stereoisomers 13b ( 3s,4s ) and 13c ( 3s,4r ) did not show any activity 
 and , as expected , neither did the tbs - protected inhibitors 12a and 12d . the metabolic stability and permeability of 
 the two active inhibitors were investigated .
because of its low solubility , inhibitor 13a was not 
 tested in the permeability assay .
compound 13b exhibited 
 slight cell toxic properties , with a cc50 of 15 m .
the lactam scaffold inhibitors 13a and 13d ( ( 3r,4s ) and ( 3r,4r ) , respectively ) yielded the most potent inhibitors 
 and were therefore selected for further optimization .
when the 
 p1 position is optimized by replacing the p - bromo substituent of the p1 phenyl group in 13a and 13d with heteroaromatic moieties , the 
 inhibitors showed improved protease inhibitor potency and , most importantly , 
 increased antiviral activity ( table 2 , 13e 
isolated 
 yields in the final reductive 
 amination ( 12a , 12d ) or reductive amination 
atz papp(caco-2 ) = 5.3  10 cm / s . for preparation of inhibitors , see 
 schemes 6 and 7 .
isolated yields of 13e  f from 14a or 14d ( scheme 6 ) or in the coupling deprotection step of 13g  j ( scheme 7 ) .
atz 
 clint = 90 l 
 min mg   [ 140 l min mg  ] .
atz papp(caco-2 ) = 5.3  
 10 cm / s . the best inhibitors , having ( 3r,4s ) configuration and 3- or 4-pyridylbenzyl as the p1 
 moiety 
 ( 13 g and 13i ) , exhibited 10 times higher 
 potency than 13a in the cell - based antiviral activity 
 assay , the best ec50 values being 40 nm ( table 2 ) .
the 2-pyridyl - substituted inhibitor ( 13e ) showed lower activity than the 3- and 4-pyridyl - substituted analogues 
 ( 13 g and 13j , respectively ) .
the improved 
 ec50 upon decorations with 2- , 3- , and 4-pyridyls has previously 
 been demonstrated showing the same trend .
the ( 3r,4r ) compounds showed 
 less improvements , but all inhibitors decorated with pyridine functionalized 
 in p1 were observed to have higher potency than the precursor 
 bromo compound 13d .
the position of the nitrogen in the 
 heteroaromatic p1 group showed the same general trend as in 
 the ( 3r,4s ) inhibitors , with the 
 meta- and para - positions providing the best potency ( table 2 ) .
heteroaromatic functionalization of p1 
 provided inhibitors 
 with increased stability compared to 13a and 13d .
compound 13h gave the best result ( clint of 120 l min mg ) . 

both 13 g and 13h were observed to possess 
 moderate permeability in the caco-2 studies , with papp of 3.8  10 and 5.1  
 10 cm / s , respectively . when the backbone 
 spacer was elongated from two to three carbons , 
 as in 19a  e , inhibitors with lower 
 potency than the 13 series were obtained ( table 3 ) .
this is in accordance with results previously 
 reported for the linear series of tertiary alcohol inhibitors , e.g. , 
 comparing the b(26 ) and c(27 ) series ( figure 1 ) .
however , with the p - phenyl or p-4-pyridyl groups in the p1 position , submicromolar 
 values of ec50 were observed in the antiviral cell based 
 assay ( 19b and 19d ) . as mentioned 
 above , permeability ( caco-2 ) and stability ( clint ) studies 
 were performed on some of the inhibitors prepared 
 ( 13a , 13d , 13 g , 13h , 19a , 19c , and 19e ) .
compound 19a showed high permeability ( > 20  10 cm / s ) , while all other inhibitors investigated showed moderate permeability 
 ( ( 320 )  10 cm / s ) .
the value of clint varied from 120 to > 300 l min mg ( tables 13 ) .
these results are in the same range as those 
 previously reported for atz ( papp = 5.3 
  10 cm / s , clint = 90 l 
 min mg   [ 140 l min mg  ] ) .
there was no major difference 
 between the 13 and the 19 series with respect 
 to clint and papp , and the 
 rigidification of the backbone seemed to be well tolerated compared 
 to the linear inhibitors .
the metabolic stability was improved when the bromo group in 13a and 19a was substituted by the heteroaromatic 
 pyridyls , although the permeability was unfortunately reduced at the 
 same time . for conditions ,
isolated yields of 19a for the deprotection step , and isolated yields of 19b  e for the coupling  deprotection step .
atz clint = 90 l 
 min mg   [ 140 l min mg  ] .
a drug - resistant strain of 
 the hiv-1 protease ( leu63pro , val82thr , ile84val ) was cocrystallized with the active pis 13i , 19b , and 19d for x - ray crystallographic 
 studies of the complexes .
data were obtained for all complexes , and 
 the structures were refined to high resolution ( for refinement statistics , 
 see supporting information ) . the resulting 
 electron density maps allowed unambiguous modeling of the inhibitors 
 within the binding site .
previously published structures 
 of hiv-1 pis 20,21 , and atz are included for comparison ( figure 3 ) .
comparison 
 of the overall x - ray conformations and binding patterns 
 of compounds 13i ( top left , pdb code 2uxz ) , 19b ( bottom left , pdb code 4a6c ) , and 19d ( bottom right , pdb code 4a6b ) in the active site 
 of hiv-1 protease .
compound 13i forms five direct hydrogen 
 bonds to the protease and five more via water molecules .
the corresponding 
 binding interactions for 19b and 19d are 
 four direct bonds and six more through water bridges . in all three 
 complexes , two of the interactions via water are due to the structural 
 water coordinating ile50 and ile150 in the protein flaps .
a complicating factor for the comparisons of the 
 inhibitor complexes 
 was the fact that compounds 20 and atz were rotated 180 
 compared to compounds 13i , 19b , 19d , and 21 .
the overall binding configurations for 13i , 19b , and 19d to the protease are , as 
 expected , in good accordance with those of previously published linear 
 inhibitors 20,21 , as well as with atz , despite the novel 
 -hydroxy -lactam moiety .
previously published pis for comparison : 
 atz ( pdb code 3el9 ) , 20 ( pdb code 2uxz ) , and 21 ( pdb code 2xye ) . on the basis of the modeling studies
, 
 it was postulated that the -hydroxyl group of the lactam moieties 
 forms hydrogen bonds with the catalytic aspartic acids ( asp25 and 
 asp125 ) .
in the two - carbon linker compound 13i this 
 -hydroxy group forms hydrogen bond interactions to the two 
 catalytic aspartic acids with 2.7 and 3.0  .
the -hydroxy 
 group in the three - carbon inhibitors 19b and 19d only form hydrogen bonds to asp25 , with 2.7 and 2.6  , respectively 
 ( figure 2 ) .
this loss of a hydrogen bond for 
 the 19 series compounds is due to the different spatial 
 conformation of the lactam ring , apparently as a result of the longer 
 central backbone ( figure 4 ) .
as the only difference 
 between the structures of 13i and 19d is 
 the length of the backbone tether , this is a likely explanation of 
 the lower antiviral potency of compound 19d compared 
 with 13i .
the position of the -hydroxy group of the lactame 
 ring , 
 involved in hydrogen binding to both asp25 and asp125 in 13i ( gold ) , is different in 19d ( purple ) and 19b ( not shown ) exhibiting the three - carbon linker .
this leads to a 
 loss of a hydrogen bond to one of the catalytic aspartates .
the position 
 of the -hydroxy group involved in hydrogen binding in 19d is 2.1  from the position observed in 13i .
none of the cocrystallized pis in these novel series 
 formed a symmetrical 
 binding pattern with the catalytic aspartic acids ( asp25 and asp125 ) 
 such as that seen in atz . together with the hydrazide carbonyl oxygen , 
 the carbonyl oxygen in the lactam ring in both 13i and 19d creates hydrogen bonds to the structural water bridging 
 the inhibitors and the ile50 and ile150 in the flap region with hydrogen 
 bond lengths of 2.73.3  ( figure 2 ) .
the position of the p2p3 indanolamide in 13i , 19b , and 19d is not markedly affected 
 by the introduction of the -hydroxy -lactam , absent 
 in 20 . while in 20 the indanolhydroxyl group 
 was close enough to form a hydrogen bond to arg108 and for the arg108 
 to make an edge - on cation interaction with the p1 phenyl 
 group , the distance to the indanol group in 13i seems 
 to prevent this bond from forming ( figure 5 ) . in accordance with the previously observed 
 results for 21 ,
the p1 
 outer phenyl group in both 13i and 19d interacts 
 through a hydrophobic interaction with pro81 ( 3.33.8  ) 
 and an edge  face  interaction to phe153 
 ( 3.73.8  ) .
the differences in length of the central 
 motif as well as in the length of the extension of 13i , 19d , 20 , 21 , and atz in 
 the p1 site are nicely accommodated through corresponding 
 shifts in the positions of phe153 and pro81 ( figure 5 ) .
these interactions are 
 likely to improve the binding constant and is the most likely explanation 
 of the better binding of compound 13i than 13d , differing only in the length of the extension in the p1 
 site . in a previously examined complex with compound 20 , the interaction with phe153 was not possible , as the corresponding 
 moiety only reached far enough for a van der waal interaction with 
 pro81 . neither is the interaction with phe153 observed in the complex 
 with atz .
since the binding modes of 19b and 19d are very similar ( figure 2 ) , only 19d was included in the analysis , as the structure of the complex could 
 be interpreted at higher resolution .
comparison of the positioning of the cocrystallized 
 inhibitors 
 in the s2s3 pocket and interaction with pro81 and phe153 in 
 the s1 pocket .
the effect on the s2s3 site is visualized 
 at residues asp29 , asp30 , arg108 , and pro181 .
( a ) superimposition 
 of 13i ( gold ) and 19d ( purple ) . as a result 
 of an additional ch2 group in 19d ,
the lactam group present in the new series of compounds as in 19d mimics the conformation of 20 , also exhibiting 
 the three - carbon linker , very well . with the lactam ring present , 
 the position of the indanol ring , and therefore also asp30/130 ,
is 
 more similar to the situation in 13i comprising the two - carbon 
 linker .
( c ) superimposition of 13i and atz ( black ) . despite 
 the differences of functional elements between 13i and 
 atz in the s2s3 site ,
the common ribbon of the compounds overlap 
 well . in the p1 site 13i and 19d
compound 19d induced side chain and main chain 
 atom displacements in phe153 and pro81 up to 2.5 and 1.7  , respectively , 
 compared to atz complex positions .
none of the new compounds 
 induced a shift in the position of arg108 , as was seen in compound 20 .
the introduction of the -hydroxy -lactams 
 as new scaffolds was intended to provide more rigid pis and to relocate 
 the hydroxyl group from the backbone to enable more symmetric binding 
 to the catalytically active asp25 and asp125 of the hiv-1 protease . 
 the outcome of the dialkylation reactions performed to obtain 2a 
d was in accordance with the results 
 described by amat et al . in 2007 , although they observed a larger 
 substrate - dependent variability . when 
 introducing the benzyl moiety in the first alkylation , as in the cases 
 of 2b and 2d , the yields were lower ( 2% 
 and 5% , respectively ) than when the allyl group was introduced before 
 the benzyl moiety ( as in 2a and 2c , with 
 yields of 49% and 33% , respectively ) .
the same trend has been reported 
 by johnson et al . with 4-substitued lactams but
was not observed in the 5-substitued examples presented by meyers 
 et al . , in which the order of addition did not affect the yields .
probable reasons for the lower yields observed 
 by johnson et al . were steric and/or electrostatic interactions between 
 the 4-hydroxy group and the bulkier 3-benzyl moiety present after 
 the first alkylation , compared to the smaller allyl group .
these findings 
 followed the reasoning presented by huang et al . , who proposed stereoelectronic factors to be the major explanation 
 in this class of stereoselective two - step alkylation reactions .
in the present work , the diastereoselectivity controlled by the stereochemistry 
 of the 4-hydroxy group was strong enough to allow highly enantiomerically 
 enriched isomers to be obtained in all cases .
there was an urgent 
 need for a robust method for the synthesis 
 of the prime side hydrazide moiety ( 5a ) .
the procedures 
 used previously were cumbersome and low yielding because of the use 
 of toxic and environmentally hazardous hydrazine hydrate and/or tedious 
 purification protocols .
previously used synthetic procedures were 
 not satisfactory , since the quantities of prime side were not sufficient 
 to support our lead optimization program throughout . the synthetic 
 route to the prime side hydrazide moiety 5a presented 
 here provided an efficient way of producing sufficient amounts and 
 constitutes an improvement in yield as well as a reduction in work 
 compared to previous methods . with this convenient 
 method
, there was no need to use hazardous hydrazine hydrate , and 
 the purification protocol resulted in a good yield .
the biological results obtained from the novel lactam - containing 
 inhibitors are summarized in tables 13 .
evaluation of the four stereoisomers ( 13a  d ) gave two active and two nonactive pis ( table 1 ) .
the ( 3r,4r ) 
 and ( 3r,4s ) stereoisomers in the 
 lactam ring showed the best results , with 13a and 13d being the most potent compounds ( ki < 10 nm and ec50 < 1 m ) .
the 
 most important structure  activity feature appears to be the 
 direction of the benzyl in the p1 position . with r - stereochemistry at the -carbon ( 13a and 13d )
, the direction of the -hydroxy substituent ( position 
 4 ) appears to be of less importance for inhibition with 13a and 13d being almost equipotent . with s - stereochemistry at the -carbon , 13b and
13c showed almost no inhibiting effect on the enzyme or in 
 the cell - based antiviral activity assay ( table 1 ) and , as expected , the tbs - protected inhibitors 12a and 12d did not show any inhibitory potency . when the p1 side chain was decorated with heteroaromatic 
 moieties ( table 2 ) ,
at best a 10-fold improvement 
 in inhibitory potency was observed ( 13 g and 13i , ec50 = 0.04 m ) .
compared to the 2-pyridyl inhibitor 13e ( ec50 = 0.190 m ) , the 3- and 4-pyridyl - substituted 
 inhibitors ( 13 g and 13i , respectively ) with 
 ( 3r,4s ) stereochemistry afforded 
 5 times higher potency , with ec50 of 40 nm . despite the 
 fact that atz contains a 2-pyridinyl in position p1 , our previous series with one- or three - carbon 
 spacers showed better potency for the 3- and 4-pyridinyl - substituted 
 inhibitors . with the linear two - carbon spacer the 2- , 3- , and 4-pyridinyls gave 
 equipotent inhibitors .
this result was 
 also obtained with the lactam - containing inhibitors with the three - carbon 
 extended pis in the 19 series .
comparing p - bromide functionalized inhibitors 13a and 19a , a 5-fold loss of potency within 
 measured ki and ec50 values 
 were observed .
however , the same trend is present in both series ( 13 and 19 , table 3 ) as 
 seen with the shorter inhibitors .
the pyridyls ( 19c  e ) showed slightly better inhibition compared to the p - bromo compound 19a .
the p - phenyl substituted 19b was among the most potent inhibitors , 
 concurring with recent reports .
we have successfully introduced -hydroxy 
 -lactams 
 providing a rigid backbone moiety and replaced the previously used tert - hydroxy group with a sec - hydroxy group . 

in addition , the length of the central spacer 
 was varied ( two or three carbons ) .
functionalization of the two most 
 potent stereoisomers ( 3r,4s ) ( 13a ) and ( 3r,4r ) ( 13d ) with heteroaromatic moieties in the p - benzyl p1 position improved the potency , rendering ki values down to 0.7 nm and ec50 values 
 down to 0.04 m .
three inhibitors were cocrystallized with the 
 hiv-1 protease enzyme providing information about the binding of the 
 hydroxy lactams to the enzyme .
the change in binding pattern between 
 the inhibitors with two- and three - carbon spacers was in good agreement 
 with the observed variation in enzyme binding activity .
 in an effort to identify a new class of druglike hiv-1 
 protease 
 inhibitors , four different stereopure -hydroxy -lactam - containing 
 inhibitors have been synthesized , biologically evaluated , and cocrystallized . 
 
 the impact of the tether length of the central spacer ( two or three 
 carbons ) was also investigated . a compound with a shorter tether and 
 ( 3r,4s ) absolute configuration exhibited 
 high activity with a ki of 2.1 nm and 
 an ec50 of 0.64 m . 
 further optimization by decoration 
 of the p1 side chain furnished an even more potent hiv-1 protease 
 inhibitor ( ki = 0.8 nm , ec50 = 0.04 m ) . 
 according to x - ray analysis , the new class of 
 inhibitors did not fully succeed in forming two symmetric hydrogen 
 bonds to the catalytic aspartates . 
 the crystal structures of the complexes 
 further explain the difference in potency between the shorter inhibitors 
 ( two - carbon spacer ) and the longer inhibitors ( three - carbon spacer ) . 