fusion 
 of human immunodeficiency virus type 1 ( hiv-1 ) with host 
 cells is mediated by viral envelope glycoprotein 41 ( gp41 ) through 
 a series of conformational rearrangements culminating in the formation 
 of a six - helix bundle ( 6-hb ) between n - heptad repeat ( nhr ) and c - heptad 
 repeat ( chr ) regions of the extracellular domain of gp41 trimer . any chemical entity that disrupts 6-hb formation has the potential 
 to inhibit the fusion process , thereby blocking hiv-1 entry into the 
 target cells .
c - peptide inhibitors derived 
 from the chr have been shown to be potent inhibitors of hiv fusion , and time of addition experiments have revealed 
 that they typically remain active for 3090 min after initiation 
 of viral infection .
thus , it appears that a fairly 
 long - lived intermediate conformation of gp41 exists in which the nhr 
 coiled coil in the gp41 trimer is exposed and susceptible to inhibition . 

peptides directed against different regions of gp41 have been developed 
 into drugs or drug candidates .
they are useful in salvage therapy 
 against hiv strains resistant to haart therapy or can be used as vaccine 
 antigens or microbicides , but suffer from the usual limitations of peptide 
 drugs , including shelf life , stability , bioavailability , and cost .
multiple studies have shown that d - peptides and small 
 molecules targeting a conserved hydrophobic pocket on the coiled coil have fusion inhibitory properties .
antiviral activity at low nanomolar concentrations was described 
 for some of the small molecules , although they had ic50 values of several micromolar against cell  cell fusion .
low molecular weight compounds able to potently inhibit hiv-1 fusion 
 remain elusive , notwithstanding the important role they could play 
 in countering multidrug resistance , viral latency , and the cell - to - cell 
 route of transmission thought to be responsible for rapid spread and 
 resurgence of the virus .
we recently 
 described the development of a series of indole compounds as hydrophobic 
 pocket binding fusion inhibitors .
two 
 benzyl - substituted bisindole compounds containing four aromatic ring 
 systems demonstrated 0.9 m activity against cell 
cell 
 and virus  cell fusion ( figure 1 ) . in 
 this work , we have performed lead optimization based on this scaffold , 
 where we describe sar studies examining isomeric forms , alternative 
 benzyl ring and other substituents , and compounds containing benzimidazoles . 
 the work has resulted in several 66 linked bisindole 
 compounds with submicromolar activity against cell  cell and 
 virus  cell fusion , including 6j with ec50 = 200 nm , and has provided an assessment of molecular properties 
 associated with potency .
the two most active compounds 6j and 6k were tested and found to be effective against 
 multiple strains of hiv , including a strain that is resistant to t20 .
the most active previously synthesized 
 compounds 1a , 1b(22 ) with four aromatic 
 systems labeled a  d ( figure 1 ) had virtually 
 identical binding affinity and in vitro potency .
binding affinity ( ki ) to the 
 hydrophobic pocket was on the order of 12 m and the 
 compounds exhibited comparable activity against cell  cell fusion 
 ( ec50 = 0.51.0 m ) and viral 
 infectivity ( ec50 = 0.9 m ) . the following 
 aromatic system substitutions were made : indole b was replaced with 
 benzimidazole ( 10a , 10c , 20a , 20b ) ; the benzoic acid substituent was moved to the 3-position ( 14b , 14c , 16 , 18a , 18b ) ; indole c was replaced with benzimidazole ( 10b , 10d ) , benzene ( 21a , 21b ) , 
 or quinoline ( 22a , 22b ) ; ring d was moved 
 to the 3-position ( 14a ) ; ring d was replaced with a proton 
 ( 18a , 18b ) or methyl group ( 19 ) or substituted with a variety of polar and nonpolar groups ( 6a  m ) .
the connectivity between b and 
 c was also varied ( 6o  q ) . these substitutions 
 allowed us to explore variation of shape , charge , hydrophobic contact 
 surface area , and polar interactions .
these studies were intended 
 to verify that the hydrophobic and electrostatic interactions of the 
 inhibitors are specific , rather than being due to amorphous long - range 
 electrostatic or nonspecific hydrophobic effects , and to test the potential of the compounds to form additional 
 hydrogen bonds .
many of the isomers were also expected to have improved 
 druglike properties including a lower lipophilicity .
ccf : cell  cell fusion , 
 env 
 subtype hxb2 , performed in optimem ( 0% serum ) , in m .
vcf : virus  cell fusion ( measured 
 as viral replication ) , env subtypes ba - l and iiib , 2% serum ; in m .
the linkage between indoles b and c 
 was examined as a means of changing the overall shape of the molecule . 

compounds with 56 ( 6o ) , 65 
 ( 6p ) , and 55 ( 6q ) linkages 
 were synthesized and evaluated .
the activity data indicated that all 
 three compounds had reduced activity compared to 1a , 
 not only in hydrophobic pocket binding affinity ( 34 times 
 less active ) but also in cell  cell fusion and viral replication 
 assays ( 420 times less active ) ( table 1 ) .
conformational space sampled by the isomeric forms 1a , 6o  q was examined by generating 
 20 low energy conformers of each compound using omega2 , version 2.5.1.4 
 ( openeye , inc . ) .
a sample of three conformers 
 of each compound is shown in figure s2 ( supporting 
 information ) to illustrate accessible shapes of the molecules . 

the 66 linked bisindole scaffold was able to adopt 
 the most compact shape overall .
the 55 isomer adopted 
 the most extended solution conformations and was also the worst performing 
 of the four isomers . continued synthetic exploration
was restricted 
 to 66 linked indoles , which apparently better conform 
 to the shape of the hydrophobic pocket . since the hydrophobic pocket 
 is located on the long groove formed along the nhr , we next tested 
 the effect of increasing van der waals contacts by introducing additional 
 hydrophobic substituents to ring d. compounds 6e , 6f , and 6 g all contain an additional phenyl group 
 attached to ring d , and compound 6h has ring d replaced 
 with a benzotriazole ( table 1 ) .
activity did 
 not improve , leading us to believe that optimization should focus 
 on specific interactions in the hydrophobic pocket rather than on 
 extending the contact surface area .
we also could not rule out whether 
 the compounds might exhibit lower potency due to lower solubility 
 under the conditions of in vitro assays .
we then tested the 
 effect of reduced compound size by eliminating ring d , which automatically 
 reduces total surface area .
compounds 18a , b , 19 , 20a , b , 23 ( scheme 4 ) and four previously studied compounds 21a , 21b , 22a , 22b(22 ) all contain three aromatic ring systems ( table 2 ) .
compound 23 , the precursor of 6a  m , demonstrated 
 that ring d is essential for antiviral potency ; it had an ec50 of 60 m against hiv-1 ba - l , while our best four - aromatic 
 system compound 6j had 0.2 m potency .
two of the 
 compounds had submicromolar binding affinity : 18b with 
 a 3-substituted indole b and two free indole nh protons as well as 19 containing n - me ( scheme 4 ) , but 
 this did not translate into submicromolar antiviral activity .
the 
 discrepancy between binding and fusion of compounds 18b and 19 suggests that factors other than hp binding 
 may play a role in inhibition in the biological milieu , either by 
 a change in pocket shape or accessibility or by contribution of a 
 secondary interaction .
ccf : cell  cell fusion , 
 env 
 subtype hxb2 , performed in optimem ( 0% serum ) , in m .
vcf : viral replication , env subtypes 
 ba - l and iiib , 2% serum , in m . in the context of three aromatic system compounds , 
 the character 
 of rings b and c
were important , and binding affinity decreased in 
 the order indole  quinoline > phenyl > benzimidazole . 

further 
 examination was confined to compounds with four aromatic systems , 
 given the inability of smaller compounds to yield submicromolar inhibition 
 against viral fusion .
benzimidazole 
 replacement 
 of either indole b or c was introduced ( scheme 2 ) as a way to increase polarity in the core of the scaffold without 
 altering the shape of compounds with four aromatic systems .
in addition , 
 coupling of ring a and/or ring d to the 3 position of the 
 indoles was tested ( scheme 3 ) , which leaves 
 a free indole nh proton that could potentially make hydrogen bonds 
 to residues of the protein .
compounds 14a  c and 16 with at least one free indole nh proton 
 had low micromolar to submicromolar ki values and moderate in vitro ec50 values .
compound 16 , having shape equivalent to 6j but with two 
 free indole nh protons , forms a compact structure and is the most 
 active of these variants . while binding affinity is similar , bioactivity 
 is lower than for 6j , and while it appears that 3-substitution 
 does not significantly affect ki , it is 
 detrimental in the local environment of in situ gp41 . by virtue of 
 the 3-substituted indoles , compounds 14a ,
14b , and 14c have extended shapes similar to 6p , 6o , and 6q , respectively , and have similar 
 activity .
replacement of indole groups with benzimidazole increased ki and practically abrogated antifusion activity 
 entirely .
it is clear from table 1 that addition of ring d to compound 23 ( ki = 3 m ) had a significant 
 effect on compound potency , leading to ki values as low as 0.6 m or as high as 12 m and over 
 2 orders of magnitude variation in fusion inhibitory activity .
we observed that m - methoxy 
 ( 6b ) or p - cf3 ( 6d ) substitution had no effect on binding affinity or potency , while 
 a 3,5-dimethoxy substitution on ring d ( 6c ) caused a 
 barely perceptible decrease in biological activity but no change in 
 binding affinity .
bulky additions to ring d , 
 discussed earlier , compromised binding and antiviral potency compared 
 to 1 ( 6e  h ) .
we observed 
 an overall agreement between cell  cell fusion and viral infectivity 
 ec50 values and , with the exception of compound 6e , between ki and ec50 .
this suggests that a hydrophobic pocket binding mechanism 
 is central to the potency of the compounds . in all cases , a carboxylate , 

an amide , or an ester as a meta substituent on ring d improved binding 
 affinity to mostly submicromolar levels .
however , carboxylic acid 
 and amide groups in 6i and 6m(30 ) invariably resulted in significantly reduced 
 activity against fusion .
conversely , retaining the methyl or ethyl 
 ester resulted in enhanced fusion inhibitory activity that exceeded 
 the ki in potency by a factor of 23 . 

this result suggests that the characteristics required for hydrophobic 
 pocket binding in solution begin to deviate from those required for 
 fusion inhibition below a 1 m threshold of potency .
it is possible 
 that the proximity of the membrane to the hydrophobic pocket of gp41 
 in situ modifies the requirements for a compound to bind effectively 
 and inhibit fusion . in this scenario ,
a charge on ring d would have 
 a deleterious effect on the ability of the compound to reach the site 
 of action , while uncharged groups such as esters can still approach 
 and bind tightly . we have observed that the indole inhibitors can 
 partition into popc / cholesterol liposomes , cells , and virus particles , 
 with a reduced interaction for 6i compared to 6b ( unpublished ) . furthermore , the relationship between membrane partitioning 
 and efficacy has been established for gp41 c - peptide inhibitors . the best performing 
 compounds 6j and 6k inhibited cell 
cell 
 fusion with ec50 = 200700 nm and 
 inhibited viral replication with ec50 = 200300 
 nm .
we measured the antiviral activity of the compounds against various 
 primary strains of hiv-1 in pbmcs , finding that they retained high 
 potency even against a t20 resistant strain ( table 3 ) .
the methyl ester containing compound 6j gave 
 ec50 values in the range 250460 nm , tested against 
 six different strains of hiv-1 , including a strain with t20 ec50 > 2000 nm .
submicromolar ec90 values 
 were typically obtained . for two viral strains with partial insensitivity 
 to t20 ,
retention of activity against 
 t20 resistant strains is an expected property of compounds targeting 
 the hydrophobic pocket , since they do not overlap the binding surface 
 of t20 .
it suggests that the hydrophobic pocket binders could be useful 
 in combination therapy with t20 .
the mechanism of inhibition of hiv 
 entry was further confirmed by studies showing that 6j and 6k had minimal effect in a magi attachment assay 
 ( supporting information table s1 and figure 
 s3 ) , which is performed with a washout of compound and unbound virus 
 following a short - term incubation that coincides with the virus attachment 
 and fusion steps of virus infection . coupled with the results observed 
 in the other assays , loss of compound activity in this assay indicates 
 the virus is able to attach to cells in the presence of the compounds 
 and continue with fusion and subsequent steps of virus infection and 
 replication after the compounds are washed out of the assay .
ti ( therapeutic index ) = tc50/ec50 ; all values in nm . the diversity 
 of the compound set has allowed us to obtain an informative sar data 
 set and to evaluate potential binding modes of the compounds in the 
 pocket .
the low binding and fusion inhibitory properties of 20a and 20b , mirrored also in 10a and 10c , in which ring b is benzimidazole , suggests 
 that an indole group is desirable for ring b , which is likely to be 
 buried in a hydrophobic part of the pocket .
a lone - pair - containing 
 nitrogen atom at the 3 position can not be accommodated , in contrast 
 to an indole nh that was tolerated ( 14b , 14c , 16 ) .
similar functionality was required for ring c , 
 suggesting that the bisindole core might anchor the compounds in the 
 pocket .
variation of a factor of 20 in ki and a factor of 250 in ec50 was observed 
 for different ring d substituents , and methyl and ethyl esters were 
 clearly favored .
ring d was required for submicromolar in vitro 
 potency against fusion , although it was not essential for submicromolar 
 hydrophobic pocket binding . on the basis of previous reports 
 of the importance of salt - bridge interactions between peptide and 
 small molecule inhibitors and pocket lys574 , we evaluated the binding affinity of select compounds to a mutant 
 receptor in which lys574 was replaced with norleucine ( nle )
. nle contains 
 the hydrophobic side chain of a lys but is missing the -amino 
 group .
binding affinity was slightly impaired by a factor of 23 
 ( supporting information table s2 ) , which 
 is within the range of experimental error of measurement of ki , but was consistently observed for almost 
 all the compounds measured .
it appears likely that a specific salt 
 bridge between lys574 and the ligand carboxylate , if it exists , does 
 not contribute substantially to potency .
evaluation of binding 
 modes is complicated by the variability of the gp41 hydrophobic pocket 
 in the available protein data bank ( pdb ) structures .
pockets containing 
 small molecules , peptides or antibodies show significant side chain 
 variations , and it is likely that binding 
 of the indole compounds would result in induced fits that are difficult 
 to predict a priori .
interestingly , most gp41 structures in the pdb 
 have a pocket structure that is not large enough to accommodate compounds 
 with four aromatic systems fully within the groove .
the exception 
 to this is the structure 2xra , which has an antibody hk20 bound to it .
we investigated the poses and calculated affinities 
 of the compounds listed in tables 1 and 2 using fred 2.2.5 oedocking software ( openeye , inc . ) , 
 which includes options to analyze poses using a variety of scoring 
 functions .
we used scoring functions oechemscore , shapegauss , piecewise 
 linear potential , zapbind , screenscore , and chemgauss3 to evaluate 
 the lowest energy pose and 20 alternative conformations of each ligand . 

these energy functions are described in detail in fred documentation 
 and are briefly explained in the supporting information .
because of the much lower dynamic range of ki and ec50 data compared to ec50 , we used the latter as a more discriminating 
 data set to distinguish between different poses and structures .
extensive 
 hk20 side chain interactions in and surrounding the hydrophobic pocket 
 create a large core cavity for accommodating a ligand in 2xra .
in this structure 
 we obtained a highly consistent lowest energy consensus pose for almost 
 all 66 four - ring compounds , shown in figure 2a .
oechemscore is a modification 
 of chemscore , using simple contact terms 
 to define lipophilic interactions , an explicit term for hydrogen bonds , 
 and a penalty for clashes between protein and ligand .
this scoring 
 function sums over the pairwise interactions between ligand and protein 
 and is likely to reflect the total contact area between them .
figure 3 shows the calculated oechemscore values for the 
 lowest energy conformer , plotted against pec50 .
compounds were separated into three categories based on the observed 
 results : ( 1 ) 66 four - ring systems not containing benzimidazole , 
 and all three - ring systems ; ( 2 ) benzimidazole - containing compounds 
 and alternatively linked four - ring compounds ; and ( 3 ) two outliers 6f and 6i not included in the previous two categories . 

a remarkable correlation coefficient r = 0.844 was obtained for category 1 , which included most of the 
 compounds in our data set ( 23 compounds ) .
category 2 is a miscellaneous 
 combination of 11 compounds with different shapes and charge distribution , 
 as a result of which they gave a variety of different bound poses 
 and there was insufficient sampling within each subgroup to reach 
 detailed conclusions .
it is clear , however , that these compounds are 
 predicted to be more active than what was found experimentally , which 
 could be a result of bioavailability considerations or a flexible 
 receptor structure . in this context ,
four - ring system compounds with 66 
 bisindole and 
 1,1-benzyl linkages , docked using fred 2.2.5 into pdb structure 2xra ( five - helix , antibody 
 hk20 binding site ) .
( a ) shown overlaid are 1a , 6a  d , 6f , 6h  k , 10a  b .
scores of the lowest energy conformer selected by fred docking 
 are included for three categories described in the text : ( 1 ) 66 
 linked bisindole compounds , 3-substituted bisindoles with complementary 
 shape , and all three - ring systems ( black ) ; ( 2 ) benzimidazole containing 
 compounds and alternatively linked bisindole compounds ( red ) ; ( 3 ) 
 outliers not included in the calculation of correlation coefficient 
 ( green ) . the data for this graph are provided in the supporting information . observed ki and antiviral activity 
 of 6i and 6 m showed the greatest discrepancy 
 of all compounds , and this was mirrored in the calculation for 6i , which adopts the same pose as 6j ( figures 2 and 3 ) .
interestingly , a 
 similar observation of low antiviral activity for compounds containing 
 two carboxylates , and much improved activity for the methyl ester , 
 was obtained in a recent study of elongated pyrrole - thiazolidinyl 
 containing compounds .
carbomethoxy substituents 
 on a terminal benzyl ring were at least 10 times more active ( ec50 = 1.8 m ) than the corresponding free 
 carboxylic acid ( ec50 = 2168 m ) . 

it is possible that the loss of potency is due to the local lipophilic 
 environment of in situ gp41 , since 6i binds strongly 
 to gp41 in solution and is not an outlier in correlations with pki .
compound 6 m formed an internal 
 h - bond in the calculations and could not assume the extended pose 
 shown in figure 2a .
the compact pose made far 
 fewer contacts with the protein surface , consequently resulting in 
 poor calculated activity , which correlated with its poor vcf activity . 

the remaining outlier is 6f , a five - ring compound in 
 which the extensive contact area with the protein is not matched by 
 experimental efficacy . with such compounds , solubility could play 
 a role in the diminished potency .
a few additional outliers were found 
 in the pki correlation ( supporting information table s3 and figure s4 ) , but this is 
 not considered as reliable because of the available low dynamic range . in the bound pose in 2xra in figure 2b , compounds 6j and 6k , were the top ranked compounds and 
 they are clearly also the most potent compounds experimentally .
they 
 ranked at positions 1 and 3 in the data set of 36 compounds .
in fact , 
 several scoring functions ranked these compounds at or near to top 
 of the list .
table s3 shows the calculated 
 correlation coefficients for all scoring functions and the rank of 
 the top compounds .
five of the six scoring functions yielded correlation 
 coefficients r > 0.53 with vcf data , 
 and 6j was ranked as the top scoring compound using oechemscore , 
 shapegauss , piecewise linear potential , and chemgauss3 ; 6k ranked third , second , ninth , and sixth , respectively , out of 36 
 compounds .
this encourages us to believe that the pose found in 2xra 
 ( figure 2b ) is independent of scoring function 
 and may well represent an accurate pose .
in this study , we designed , synthesized , and tested a series of 
 indole derivatives as entry inhibitors against gp41-mediated hiv-1 
 fusion .
sar studies revealed a defined shape , charge , and hydrophobic 
 contact surface required to fit into the hydrophobic pocket .
cell 
 fusion inhibition , with several compounds having submicromolar activity 
 and the most active compound 6j exhibiting binding affinity 
 with ki of 0.6 m and ec50 of 0.2 m in cell  cell fusion inhibition and viral 
 replication inhibition .
we found that submicromolar inhibition constants 
 for binding to 
 the hydrophobic pocket on the gp41 coiled coil were a necessary but 
 not sufficient condition for observing submicromolar inhibition of 
 cell  cell and virus  cell fusion .
below ki = 1 m , the correlation between ki and ec50 was diminished , indicating a factor 
 other than hydrophobic pocket binding required for fusion inhibition . 

polar and nonpolar groups at opposite ends of the elongated molecules 
 were a dominant feature of submicromolar fusion inhibitors , but compounds 
 containing polar groups at both ends were poor fusion inhibitors while 
 equally potent hp binders .
we surmise that proximity of the target 
 to lipid membranes may influence accessibility of the pocket and bioavailability 
 of the inhibitors , given the lipophilic character of the best inhibitors . 

future work will focus on improving the therapeutic index and understanding 
 the mechanism that yields submicromolar activity in cell culture .
starting 
 materials and solvents were purchased 
 from commercial sources and used as is .
hplc was performed on a waters 
 breeze hplc with multiwavelength fluorescence detector and dual wavelength 
 uv detector , using a water / acetonitrile or water / methanol gradient 
 with 0.05% tfa .
mass spectra were obtained on lcms instrument ( finnigan 
 lcq duo , h2o / methanol gradient with 0.1% formic acid ) by 
 esi or by high - resolution mass spectrometry ( hrms ) on a thermofisher 
 orbitrap xl ( uc davis ) .
h nmr and c nmr spectra 
 were collected on a bruker spectrometer at 400 and 100 mhz , respectively , 
 in dmso - d6 unless otherwise stated and 
 calibrated using the residual solvent signal .
multiplicities are abbreviated 
 as follows : s = singlet , d = doublet , t = triplet , q = quartet , m 
 = multiplet , and br = broad . where consistent coupling constants have 
 been observed in the nmr spectrum , the apparent multiplicity of the 
 proton signal concerned is reported .
flash column chromatography 
 was carried out with silicycle siliaflash p60 4063 m 
 silica gel .
reactions and chromatography fractions were monitored 
 with sorbent technologies silica gel 60 f254 tlc plates 
 and visualized using uv light .
reactions were carried out under inert 
 gas atmosphere in oven - dried glassware , and reaction solutions were 
 magnetically stirred .
all other reagents and solvents were used without 
 further purification from commercial sources unless otherwise noted . 

all target compounds were purified by preparative hplc or analyzed 
 by hplc to ensure 95% purity .
the organic synthesis 
 was carried out according to schemes 14 and scheme s1 in 
 the supporting information .
representative 
 synthetic procedures for the preparation of compounds 4 , 5 , 6b , 6i , 6j , 6k , 6 m , 11a , 12a , 19 , and 23 are described below .
characterization 
 data for other target compounds and synthetic intermediates are provided 
 in the supporting information .
to a solution of methyl 3-(bromomethyl)benzoate 3a ( 458 mg , 2.00 mmol ) and 6-bromoindole 2a ( 382 
 mg , 1.95 mmol ) in dry dmf ( 5.0 ml ) was added k2co3 ( 1.38 g , 10.0 mmol ) .
tlc indicated no starting material remained ; the reaction 
 was quenched by adding 25 ml of water .
the organic solvent was combined 
 and evaporated , and the product was purified by silica gel chromatography 
 using etoac / hexane ( 15:85 ) as eluent .
655.2 mg of target compound 
 was achieved as a pale yellow oil , 95% .
h nmr  
 7.86 ( d , j = 7.6 hz , 1h ) , 7 .
79 ( s , 1h ) , 7.41 ( d , j = 8.4 hz , 1h ) , 7.31 ( s , 1h ) , 7.27 ( t , j = 8.0 hz , 1h ) , 7.147.06 ( m , 2h ) , 7.00 ( d , j = 3.2 hz , 1h ) , 6.45 ( d , j = 3.2 hz , 1h ) , 5.20 ( s , 
 2h ) , 3.81 ( s , 3h ) ; c nmr  166.99 , 137.74 , 137.32 , 
 131.41 , 131.08 , 129.40 , 129.11 , 128.20 , 127.91 , 123.32 , 122.61 , 115.81 , 
 114.25 , 112.81 , 102.66 , 52.58 , 50.09 .
to a solution of indole-6-boronic acid ( 274 
 mg , 1.7 mmol ) and compound 4 ( 516 mg , 1.5 mmol ) in thf 
 ( 15 ml ) was added pd(pph3)4 ( 87.2 mg , 0.075 
 mmol ) , followed by k2co3 ( 2.0 m , 3.0 ml ) . the 
 mixture was stirred and heated to reflux under argon for about 4 h. 
 after the reaction was completed , the mixture was cooled to room temperature , 
 water ( 20.0 ml ) was added , and the product was extracted with ethyl 
 acetate ( 20 ml  3 ) .
the organic solvent was combined , dried 
 over anhydrous na2so4 , filtered , and evaporated . 

the crude product was purified by silica gel chromatography ( 20% etoac / hexanes ) . 

ms 
 calculated for c25h20n2o2 , 380.2 ; found , 381.1 ( m + h ) ; h nmr  
 8.19 ( br , 1h ) , 7.97 ( s , 1h ) , 7.93 ( d , j = 7.8 hz , 
 1h ) , 7.68 ( dd , j = 12.8 hz , j = 
 8.2 hz , 2h ) , 7.58 ( s , 1h ) , 7.49 ( s , 1h ) , 7.44 ( d , j = 8.2 hz , 1h ) , 7.39 ( d , j = 8.0 hz , 1h ) , 7.34 ( t , j = 7.6 hz , 1h ) , 7.25 ( br , 1h ) , 7.20 ( t , j = 2.4 hz , 1h ) , 7.14 ( d , j = 3.0 hz , 1h ) , 6.59 ( d , j = 3.0 hz , 1h ) , 6.56 ( br , 1h ) , 5.40 ( s , 2h ) , 3.89 ( s , 3h ) ; c nmr  167.14 , 138.32 , 137.16 , 137.01 , 136.94 , 136.79 , 
 131.64 , 130.97 , 129.36 , 129.22 , 128.82 , 128.39 , 127.98 , 127.69 , 127.12 , 
 123.32 , 122.61 , 115.81 , 114.25 , 110.16 , 108.56 , 102.68 , 102.27 , 52.56 , 
 50.07 . to a stirred solution of compound 5
( 19 mg , 0.05 mmol ) in anhydrous dmf ( 3.0 ml ) at 0 c 
 was added sodium hydride in oil ( 60% , 15 mg ) , and the reaction mixture 
 was allowed to warm to rt over 60 min .
3-methoxybenzyl bromide ( 10.5 
 mg , 0.052 mmol ) was added , and the mixture was stirred at room temperature 
 overnight .
the organic 
 solvent was combined and evaporated ; the crude product ( 22.5 mg , 90% ) 
 was directly used for saponification by adding 5.0 ml of 4.0 m naoh 
 and stirring for 1 h , and neutralized by adding 2.0 m hcl to ph 3.0 . 

the solution was extracted with ethyl acetate ( 15 ml  3 ) , and 
 the organic solvent was combined and concentrated .
data for 6b : ms calculated for c32h26n2o3 , 486.2 ; found , 485.1 ( m  h ) ; h nmr  7.82 ( d , j = 8.5 hz , 
 2h ) , 7.68 ( d , j = 7.8 hz , 2h ) , 7.60 ( t , j = 7.8 hz , 2h ) , 7.52 ( d , j = 2.9 hz , 2h ) , 7.46 ( m , 
 2h ) , 7.35 ( m , 2h ) , 7.20 ( t , j = 7.3 hz , 1h ) , 6.78 
 ( m , 3h ) , 6.51 ( d , j = 2.8 hz , 1h ) , 6.48 ( d , j = 2.8 hz , 1h ) , 5.58 ( s , 2h ) , 5.45 ( s , 2h ) , 3.67 ( s , 3h ) . to a solution of compound 23 

( 19 mg , 0.05 mmol ) in anhydrous dmf ( 3.0 ml ) at 0 c was 
 added sodium hydride in oil ( 60% , 15 mg ) , and the reaction mixture 
 was allowed to warm to rt over 60 min .
methyl 3-(bromomethyl)benzoate 
 ( 11.9 mg , 0.052 mmol ) was added , then the mixture was stirred at room 
 temperature overnight .
saponification was achieved by adding 5.0 ml 
 of 4.0 m naoh and stirring for 1 h. the reaction was neutralized by 
 adding 2.0 m hcl to ph 3.0 and then extracted with ethyl acetate ( 15 
 ml  3 ) .
the organic solvent was combined and evaporated ; the 
 crude product ( 23.0 mg , 92% ) was purified by hplc .
data for 6i : ms ( hrms ) calculated for c32h24n2o4 ( m  h ) , 499.1652 ; found , 499.1648 ( m  
 h ) ; h nmr  12.85 ( s , 2h ) , 7.82 
 ( br , 4h ) , 7.68 ( s , 2h ) , 7.60 ( d , j = 8.5 hz , 2h ) , 
 7.52 ( s , 2h ) , 7.45 ( m , 4h ) , 7.36 ( d , j = 8.0 hz , 
 2h ) , 6.51 ( s , 2h ) , 5.57 ( s , 4h ) ; c nmr  167.0 , 
 138.8 , 136.4 , 135.0 , 131.3 , 131.2 , 129.5 , 128.8 , 128.2 , 127.8 , 127.2 , 
 120.7 , 119.0 , 108.0 , 101.0 , 48.6 . to a stirred solution of 23 ( 232 mg , 0.632 mmol ) in dmf ( 7.0 ml ) at 0 c was added nah 
 ( 60% in mineral oil , 61.5 mg , 1.54 mmol ) , and the reaction mixture 
 was allowed to warm to rt over 30 min .
methyl 3-(bromomethyl)benzoate 
 ( 145 mg , 0.633 mmol ) was added , and the mixture was stirred at room 
 temperature overnight .
the reaction mixture was quenched by adding 
 2.0 m hcl ( 20 ml ) , extracted with dcm ( 30 ml  3 ) , and the combined 
 organic fractions were concentrated .
etoac / hexanes ) afforded 6j ( 249 mg , 76% ) as a light - brown oil .
data for 6j : rf = 0.13 ( 40% etoac / hexanes ) ; ms ( hrms ) calculated 
 for c33h25n2o4 ( m  
 h ) , 513.1809 ; found , 513.1803 ; h nmr 
 ( cdcl3 )  10.68 ( br , 1h ) , 7.99 ( m , 3h ) , 7.91 ( d , 
 1h , j = 7.7 hz ) , 7.69 ( d , 2h , j = 
 8.3 hz ) , 7.46 ( s , 2h ) , 7.41 ( d , 2h , j = 8.3 hz ) , 
 7.347.18 ( m , 4h ) , 7.11 ( d , 1h , j = 3.0 hz ) , 
 7.09 ( d , 1h , j = 3.0 hz ) , 6.57 ( m , 2h ) , 5.35 ( s , 
 2h ) , 5.33 ( s , 2h ) , 3.86 ( s , 3h ) ; c nmr ( cdcl3 )  ( 5 pairs of signals are overlapping ) 171.7 , 167.0 , 138.4 , 
 138.2 , 137.0 , 136.9 , 136.8 , 132.3 , 131.5 , 130.7 , 130.0 , 129.6 , 129.3 , 
 129.1 , 129.0 , 128.8 , 128.7 , 128.3 , 127.9 , 121.3 , 120.31 , 120.29 , 108.4 , 
 102.2 , 102.1 , 52.4 , 49.8 , 49.7 . to a stirred solution of 23 ( 107 mg , 0.292 mmol ) in dmf ( 3.0 ml ) at 0 c was added nah 
 ( 60% in mineral oil , 30.2 mg , 0.755 mmol ) , and the reaction mixture 
 was allowed to warm to rt over 30 min .
ethyl 3-(bromomethyl)benzoate 
 ( 73 mg , 0.30 mmol ) was added , and the mixture was stirred at room 
 temperature overnight .
the reaction mixture was quenched by adding 
 2.0 m hcl ( 10 ml ) , extracted with dcm ( 30 ml  3 ) , and the combined 
 organic fractions were concentrated .
etoac / hexanes ) afforded 6k ( 128 mg , 
 83% ) as a light - brown oil .
data for 6k : rf = 0.48 ( 50% etoac / hexanes ) ; ms ( esi ) calculated 
 for c34h26n2o4 ( m  
 h ) , 527.1965 ; found , 527.1963 ; h nmr 
 ( cdcl3 )  8.04 ( m , 3h ) , 7.97 ( d , 1h , j = 7.8 hz ) , 7.73 ( m , 2h ) , 7.50 ( s , 2h ) , 7.44 ( d , 2h , j = 8.0 hz ) , 7.417.30 ( m , 3h ) , 7.23 ( d , 1h , j = 7.9 hz ) , 7.17 ( d , 1h , j = 3.1 hz ) , 7.15 ( d , 1h , j = 3.1 hz ) , 6.61 ( m , 2h ) 5.43 ( s , 2h ) , 5.41 ( s , 2h ) , 4.38 
 ( q , 2h , j = 7.2 hz ) , 1.39 ( t , 3h , j = 7.2 hz ) ; c nmr ( cdcl3 )  ( 2 pairs 
 of signals are overlapping ) 171.6 , 166.6 , 138.4 , 138.1 , 137.01 , 136.99 , 
 136.93 , 136.8 , 132.3 , 131.4 , 131.1 , 130.0 , 129.7 , 129.3 , 129.1 , 129.0 , 
 128.8 , 128.70 , 128.67 , 128.9 , 127.9 , 121.35 , 121.33 , 120.36 , 120.31 , 
 108.5 , 102.2 , 102.1 , 61.4 , 49.88 , 49.85 , 14.5 . to a stirred solution of compound 6l
( 4.8 mg , 0.01 mmol ) in dmso ( 1.0 ml ) cooled in an ice 
water 
 bath were added h2o2 ( 30% , 20.0 ul ) and k2co3 ( 10.0 mg ) .
then distilled water was added , extracted with 
 dcm ( 10 ml  3 ) .
after lyophilization , 2.4 
 mg of compound was achieved as an off - white powder .
h nmr  
 7.817.78 ( br , 3h ) , 7.67 ( s , 2h ) , 7.567.51 ( m , 3h ) , 
 7.41 ( br , 3h ) , 7.337.26 ( m , 5h ) , 6.49 ( s , 1h ) , 6.45 ( s , 1h ) , 
 5.55 ( s , 2h ) , 5.51 ( s , 2h ) . to a vigorously stirred solution of 6-bromoindole 
 ( 2b ) ( 390 mg , 1.99 mmol ) in toluene ( 8.0 ml ) was added 
 tetrabutylammonium iodide ( 367 mg , 0.994 mmol ) , zn(otf)2 ( 450 mg , 1.24 mmol ) , followed by dipea ( 0.36 ml , 2.1 mmol ) .
after 
 the suspension was stirred for 20 min , methyl 3-bromomethylbenzoate 
 ( 231 mg , 1.01 mmol ) was added and the reaction mixture was stirred 
 overnight .
reaction mixture was diluted with etoac ( 50 ml ) , water 
 ( 10 ml ) , and saturated aqueous nh4cl ( 30 ml ) .
the organic 
 phase was separated and washed with water ( 30 ml ) and brine ( 10 ml ) 
 and then concentrated .
purification by silica gel chromatography ( 15% 
  25% etoac / hexanes ) afforded 11a ( 338 mg , 97% ) 
 as a pink oil .
data for 11a : rf = 0.07 ( 15% etoac / hexanes ) ; ms calculated for c17h13brno2 ( m  h ) , 342.01 ; found , 342.01 ; h nmr ( cdcl3 )  
 8.39 ( br s , 1h ) , 8.04 ( s , 1h ) , 7.95 ( d , 1h , j = 7.6 
 hz ) , 7.47 ( m , 2h ) , 7.38 ( d , 1h , j = 7.6 hz ) , 7.35 
 ( d , 1h , j = 8.1 hz ) , 7.20 ( dd , 1h , j1 = 8.4 hz , j2 = 1.7 hz ) , 
 6.86 ( d , 1h , j = 2.2 hz ) , 4.12 ( s , 2h ) , 3.93 ( s , 
 3h ) ; c nmr ( cdcl3 )  167.6 , 141.5 , 137.3 , 
 133.4 , 130.2 , 129.8 , 128.6 , 127.4 , 126.2 , 123.4 , 122.6 , 120.2 , 115.6 , 
 115.0 , 114.3 , 52.2 , 31.3 . to a 
 stirred solution of 11a ( 338 mg , 0.982 mmol ) in thf ( 9.0 
 ml ) were added boc2o ( 0.24 ml , 1.04 mmol ) and dmap ( 10.0 
 mg ) , and the mixture was stirred overnight .
concentration and purification 
 by silica gel chromatography ( 10% etoac / hexanes ) afforded 12a ( 429 mg , 98% ) as a colorless oil .
data for 12a : rf = 0.32 ( 10% etoac / hexanes ) ; ms ( esi ) calculated 
 for c22h23brno4 ( m + 
 h ) , 444.08 ; found , 444.08 ; h nmr ( cdcl3 )  8.34 ( br s , 1h ) , 7.96 ( s , 1h ) , 7.89 ( d , 1h , j = 7.8 hz ) , 7.43 ( d , 1h , j = 7.6 hz ) , 
 7.34 ( m , 2h ) , 7.26 ( dd , 1h , j1 = 8.4 hz , j2 = 1.9 hz ) , 7.18 ( d , 1h , j = 8.4 hz ) , 4.03 ( s , 2h ) , 3.89 ( s , 3h ) , 1.67 ( s , 9h ) ; c nmr ( cdcl3 )  167.1 , 149.4 , 139.9 , 136.5 , 133.2 , 
 130.5 , 129.8 , 129.1 , 128.7 , 127.8 , 125.8 , 124.2 , 120.5 , 119.4 , 118.6 , 
 118.3 , 84.2 , 52.2 , 31.2 , 28.2 . to compound 5 ( 19 mg , 
 0.05 mmol ) 
 in anhydrous dmf ( 2.0 ml ) were added iodomethane ( 35 mg , 0.25 mmol ) 
 and naoh ( 20.0 mg ) .
the mixture was stirred overnight , quenched by 
 h2o ( 10.0 ml ) , then adjusted to ph 3.0 using hcl ( 2.0 m ) . 

part of the crude product ( 16.2 mg , 84% ) was purified by hplc ; 2.4 
 mg of target compound was obtained as a yellow powder .
h nmr  
 7.85 ( s , 1h ) , 7.81 ( d , j = 7.6 hz , 1h ) , 7.77 ( s , 
 1h ) , 7.647.61 ( m,2h ) , 7.57 ( d , j = 8.0 hz , 
 1h ) , 7.52 ( d , j = 3.2 hz , 1h ) , 7.467.41 ( m,3h ) , 
 7.35 ( dd , j = 8.0 hz , j = 1.6 hz , 
 1h ) , 7.30 ( d , j = 2.8 hz , 1h ) , 6.52 ( d , j = 2.8 hz , 1h ) , 6.41 ( d , j = 2.8 hz , 1h ) , 5.59 ( s , 
 2h ) , 3.84 ( s , 3h ) . to a vigorously stirred solution of 
 compound 5 ( 444 mg , 1.17 mmol ) in thf / meoh ( 4:1 , 20.0 
 ml ) was added aq naoh ( 25% , 5.0 ml ) .
the mixture was stirred at room 
 temperature for 3 h , then quenched with 3.0 m hcl ( 24 ml ) , extracted 
 with dcm ( 50 ml x 3 ) , dried , filtered , and concentrated .
purification 
 by silica gel chromatography ( 10:1 dcm / meoh ) afforded 23 ( 400 mg , 93% ) as an off - white solid .
data for 23 : rf = 0.13 ( 40% etoac / hexanes ) ; ms ( esi ) calculated 
 for c24h17n2o2 ( m  
 h ) , 365.13 ; found , 365.13 ; h nmr ( dmso - d6 )  12.87 ( s , 1h ) , 11.02 ( s , 1h ) , 7.75 
 ( m , 2h ) , 7.66 ( s , 1h ) , 7.55 ( m , 3h ) , 7.47 ( d , j = 
 4.0 hz , 1h ) , 7.40 ( m , 2h ) , 7.28 ( m , 3h ) , 6.46 ( d , j = 3.1 hz , 1h ) , 6.36 ( s , 1h ) , 5.54 ( s , 2h ) ; c nmr ( dmso - d6 )  167.1 , 139.1 , 136.6 , 136.5 , 135.5 , 
 134.7 , 131.4 , 131.1 , 129.5 , 128.9 , 128.2 , 127.7 , 127.2 , 126.6 , 125.8 , 
 120.8 , 120.2 , 119.0 , 118.8 , 109.4 , 108.1 , 101.1 , 100.9 , 48.6 .
inhibition constants ki for binding in the hydrophobic pocket were 
 determined using a fluorescence intensity assay as previously described .
briefly , fe(env2.0)3 was used to mimic the 
 hydrophobic pocket in the gp41 nhr coiled coil .
it is n - terminally capped by 2,2-bipyridine-5-carboxylate , 
 a bidentate ferrous iron chelator that ensures the trimeric structure 
 of the nhr upon metal binding .
a fluorescein ( fl ) labeled pocket - binding 
 c - peptide c18fl was used to probe inhibitor binding .
ki was determined by measuring 
 the dose dependent fluorescence recovery in the presence of a competitive 
 inhibitor .
a modified metallopeptide construct with k574 replaced with norleucine was employed to measure the 
 effect of the lysine side chain amino group on inhibitor binding affinity .
cell fusion 
 was measured as previously published and 
 using cell lines obtained through the nih aids research and reference 
 reagent program , division of aids , niaid , nih .
target cells were tzm - bl 
 cells ( no . 8129 , contributed by j.c .
kappes , x. wu , and tranzyme inc . ) 
 expressing cd4 , ccr5 , and cxcr4 , and 
 containing an integrated reporter gene for firefly luciferase under 
 control of hiv-1 ltr .
1294 , contributed by b. k. felber and g. n. pavlakis ) 
 which produce hxb2 env , tat , and rev .
serially diluted inhibitors were added to 96-well plates containing 
 25  000 tzm - bl cells per well cultured overnight .
50  000 
 hl2/3 cells were added per well , and fusion was allowed to proceed 
 for 6 h in reduced serum medium ( gibco ) with a final concentration 
 of 1% dmso .
luciferase expression was measured using luciferase assay 
 reagent ( promega ) according to the manufacturer s instructions . 

controls containing 1 l of dmso with and without hl2/3 cells 
 were measured for each compound , and experiments were performed in 
 triplicate . inhibition 
 of hiv-1 replication was determined in ccr5- and cxcr4-tropic magi 
 antiviral assays , and inhibition of hiv-1 attachment / entry was determined 
 in ccr5-tropic magi attachment assays as previously described .
hiv-1 isolates and cells were obtained from the nih aids research 
 and reference reagent program , division of aids , niaid , nih , as follows : 
 hiv-1 ba - l from suzanne gartner , mikulas popovic , and robert gallo .
for magi antiviral 
 assays , magi - ccr5 cells were grown overnight in 96-well plates in 
 dmem supplemented with 10% fbs , using 10  000 cells per well . 

the following day the medium was removed and compounds diluted in 
 medium were added ( six dilutions in triplicate at each dilution ) , 
 followed by the addition of either hiv-1 ba - l ( ccr5-tropic assay ) 
 or hiv-1 iiib ( cxcr4-tropic assay ) at approximately 10 50% tissue 
 culture infective doses per well ( 10 tcid50/well ) . 

assay plates were incubated for 48 h , after which medium was removed 
 and hiv-1 tat - induced -gal enzyme expression was determined 
 by chemiluminescence using tropix gal - screen ( applied biosystems ) 
 according to the manufacturer s instructions .
magi attachment 
 assays were performed similarly but with a washout of unbound virus 
 and compounds 3 h postinfection .
, colmar , pa ) by ficoll gradient purification and stimulated 
 using 4 g / ml phytohemagglutinin and subsequently maintained 
 in media containing 20 u / ml of il-2 .
cells from two donors were mixed , 
 adjusted to 1  10 cells / ml , and added to 96-well 
 round - bottom plates at 50 l / well ( 5  10 cells / well ) . 

test compound dilutions ( nine total serial dilutions ) were prepared 
 in media at 2 concentrations and added to triplicate wells at 
 100 l / well followed by the addition of a predetermined dilution 
 of virus stock at 50 l / well ( final multiplicity of infection 
 [ moi ] of 0.1 ) .
antiviral efficacy was determined 6 days later 
 based on supernatant reverse transcriptase ( rt ) activity using a microtiter 
 plate - based rt reaction .
the following 
 virus isolates were obtained from the nih aids research and reference 
 reagent program , division of aids , niaid , nih : hiv-1 isolates 92br014 
 ( catalog no .
1647 ) from the unaids 
 network for hiv isolation and characterization ; hiv-1 isolates cam0005bby ( catalog no .
the cytotoxic 
 effect of the compounds 
 was determined using the cell culture procedure identical to that 
 described above for viral replication or cell  cell fusion but 
 measuring cell viability using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium ( mts , celltiter 96 reagent ; promega ; used 
 for magi and pbmc assays ) or using a resazurin cell viability reagent 
 ( alamar blue or presto blue , life technologies ; used for cell  cell 
 fusion assays ) following the manufacturers protocols .
the openeye 
 software suite ( openeye scientific software , santa fe , nm , http://www.eyesopen.com ) was used for ligand analysis , conformer predictions , compound overlays , 
 and computational docking .
charges and 3d coordinates of each ligand 
 were generated from smiles strings using omega2 , version 2.5.1.4 .
multiconformer representations of each ligand were docked into the 
 receptor 2xra ( protein data bank ) using the oedocking utility and fred , version 
 2.2.5 .
 we 
 previously described indole - containing compounds with the potential 
 to inhibit hiv-1 fusion by targeting the hydrophobic pocket of transmembrane 
 glycoprotein gp41 . here 
 we report optimization and structure  activity 
 relationship studies on the basic scaffold , defining the role of shape , 
 contact surface area , and molecular properties . 
 thirty new compounds 
 were evaluated in binding , cell  cell fusion , and viral replication 
 assays . below a 1 m threshold , 
 correlation between binding 
 and biological activity was diminished , indicating an amphipathic 
 requirement for activity in cells . 
 the most active inhibitor 6j exhibited 0.6 m binding affinity and 0.2 m 
 ec50 against cell  cell fusion and live virus replication 
 and was active against t20 resistant strains . 
 twenty - two compounds 
 with the same connectivity displayed a consensus pose in docking calculations , 
 with rank order matching the biological activity . 
 the work provides 
 insight into requirements for small molecule inhibition of hiv-1 fusion 
 and demonstrates a potent low molecular weight fusion inhibitor . 
Introduction
Results and Discussion
Conclusions
Experimental Section