g - quadruplexes ( g4s ) 
 are nucleic acids secondary structures that 
 may form in single - stranded g - rich sequences under physiological conditions .
four gs bind via hoogsteen - type hydrogen bonds base - pairing to yield 
 g - quartets , which in turn stack on top of each other to form the g4 . 

g4 structures may significantly vary , both in terms of strand stoichiometry 
 ( forming both inter- and intramolecular structures ) and strand orientation / topology . 

the presence of k cations specifically supports g4 formation 
 and stability . in the human genome and in prokaryotes ,
g4 dna motifs 
 have been found in telomeres , g - rich micro- and mini - satellites , near 
 oncogene promoters , and within the rdna .
human g4 dna motifs have been reported to be associated with recombination 
 prone regions and to show mutational 
 patterns that preserve the potential to form g4 dna structures .
the identification of g4 binding proteins and their visualization in cells with antibody - based technology have also provided convincing evidence of the existence of cellular 
 g4s in vivo .
recently , the presence of g4s in viruses and their 
 involvement 
 in virus key steps has also been provided .
we and other groups have identified functionally significant 
 g4s in the nef coding region and the 
 unique long terminal repeat ( ltr ) promoter of the human 
 immunodeficiency virus ( hiv ) , the etiologic agent of the acquired 
 immune deficiency syndrome ( aids ) .
these studies have shown that g4 
 folding at the ltr promoter decreased viral transcription with an 
 effect that was augmented by a g4 ligand .
the involvement of g4 structures in several human diseases 
 propelled 
 the development of g4 ligands .
several aromatic cores with protonable 
 side chains bind the g4 conformation , such as porphyrins , acridines , such as 1 ( braco-19 , scheme 1 ) , fluoroquinolones anthraquinones , phenanthrolines , quinacridines , 
 carbazoles , bis - indole carboxamides , triazoles , and benzimidazoles ; natural compound derivatives , such as berberine , telomestatin , quindolin , several tetra - substituted perlyene tetracarboxylic and water - soluble naphthalene diimide ( ndis ) .
so far , the vast majority of molecules has been tested against cellular 
 g4s implicated in tumor pathogenesis : some compounds showed interesting 
 antiproliferative properties ; in particular , 
 quarfloxin proceeded into phase ii clinical trials , but its limited 
 bioavailability prevented further progress .
few molecules have been tested also as antiviral agents : despite the low selectivity indexes , these studies have shown the 
 possibility to develop antiviral molecules with a g - quadruplex - mediated 
 mechanism of action . the lateral expansion of the ndi core provided by our 
 investigation 
 may offer an additional opportunity to enhance both g4 binding potency 
 and selectivity , discriminating g4 structures with different accessibility 
 to the edge quartets .
examples that comprise the annulation of five- 
 and six - membered heterocyclic rings either on one side of the ndi or on both sides have been reported ( scheme 1 ) .
surprisingly , only 
 one class of these core - extended ndis ( c - exndis ) has 
 been engineered water - soluble to be investigated as g4 ligand , the others have found applications in the field 
 of material chemistry and as ph sensors . because 
 of the possibility to block hiv-1 transcription by stabilizing 
 the g4 structures present in the unique viral ltr promoter , we here 
 synthesized and tested a series of novel c - exndis for 
 both their binding to the ltr g4s and anti - hiv-1 activity . in these 
 new structures
, the ndi core has been merged to the dihydrobenzophenazine 
 polynuclear heterocycle ( highlighted in red in scheme 1 ) .
we proved that these derivatives display 
 binding and stabilization selectivity toward the viral g4s as compared 
 to a telomeric g4 and that this translates into a therapeutic window 
 where the compounds are active against the virus but not toxic for 
 the cell .
we have 
 designed and synthesized a small library of new and water - soluble c - exndis ( 211 , scheme 2 ) , where the ndi 
 core has been merged to the dihydrobenzophenazine polynuclear heterocycle 
 ( highlighted in red in scheme 1 ) .
the choice has been made based on the pka values of the n , n - dimethylpropan - amine 
 side chains : the pka values are above 
 7 ( pka1 = 8.1 , pka2 = 8.6 ) , suggesting that the 
 side chains are mainly protonated under physiological conditions . 

we discharged the shorter n , n - dimethylthyl - amine 
 side chains , as their pka values are much 
 lower ( pka1 = 5.6 , pka2 = 7.4 ) .
the c - exndis are structurally 
 characterized by a larger flat core than the ndis .
indeed , dft calculation performed at b3lyp/6 - 31+g(d , p ) level of 
 theory in gas phase indicated that the dimension of the c - exndi core supported a more extensive overlap to the quartet of the g4 
 structure ( supporting information , figure s1 ) .
enhancement of the ligand steric hindrance accessing the outside 
 g4 quartets might offer an opportunity to implement selectivity as 
 well . furthermore , the proposed structural extension on the ndi scaffold 
 should merge the useful optoelectronic properties ( absorption , emission , 
 and singlet oxygen sensitizer ) of functionalized ndis to those of 
 substituted dihydrobenzophenazines .
compounds with both h and br substituents 
 at the y position were considered to evaluate the effect of a bulky 
 substituent ( br ) at this position . to synthesize the c - exndis
, we developed the synthetic protocol highlighted in scheme 2 . according to published methods , 

the imidation reaction of the readily available 2,6-dibromo-1,4,5,8-naphthalenetetracarboxylic 
 dianhydride yielded the 2,6-dibromo - substituted ndi , under acidic 
 conditions , in almost quantitative yield .
subsequently , a two - step 
 protocol starting from a nucleophilic aromatic substitution ( snar ) in the presence of an excess ( 2.5 equiv ) of ortho - diaminophenyl derivatives in dmf as solvent at 70 c for 16 
 h afforded a 60:40 mixture of monobrominated and debrominated products 
 in an overall quantitative yield . the ring closing reaction following 
 the snar in one pot ( step a , in scheme 2 ) occurred through a tchitchibabin mechanism , 
 with the elimination of hydride favored by the formation of a six - membered 
 ring .
the second snar on the remaining aromatic bromide 
 was carried out by dissolving 3 in neat n , n - dimethylpropane-1,3-diamine . 
 to improve the reaction yield
, we optimized a microwave - assisted protocol 
 ( 150 c , 200 psi , 250 bar , 200 w , 3 min , sealed reaction vessels ) 
 to give product 9 , which crystallized from the reaction 
 mixture .
we achieved the synthesis of the permanently charged quaternary 
 ammonium salts 10 and 11 with a classical 
 exhaustive methylation , in the presence of mei at rt for 16 h. all 
 the synthesized ligands ( 211 ) were 
 purified by preparative hplc ( ch3cn : h2o and 
 0.1% cf3cooh as eluent ) .
further anion exchange yielded 29 as hydrochloride salts . on the contrary , 10 and 11
( a ) dmf , 70c , 16 h , 
 under n2 ; ( b ) n , n - dimethylpropylamine , 
 mw , 150c 3 min , closed vessel ; ( c ) mei , chcl3 , rt , 
 16 h. to detect 
 the ability of the new c - exndis to bind and stabilize 
 the g4 conformations of the hiv-1 ltr promoter , two g4-forming ltr 
 sequences were employed : ltr - iii and ltr - iv , both composed of four 
 ggg tracts ( figure 1a ) . when embedded within the full - length g - rich sequence of the ltr 
 promoter ,
ltr - iii is the most stable g4 , while ltr - iv folds when induced 
 by g4 ligands .
a fret melting assay was 
 performed to assess the degree of stabilization of the ltr - iii and 
 ltr - iv g4 by c - exndis .
the minimal intramolecular g4-forming 
 human telomeric ( htel ) sequence ( figure 1a ) was employed to evaluate the activity 
 of the c - exndis on one of the most highly represented 
 and accessible cellular g4 : in fact , the ttaggg repeat is present 
 as 20003000 double - stranded ( ds ) and 50200 single - stranded 
 sequences at the human telomeres .
a double - stranded 
 ( ds ) dna was also added to address specificity toward the g4 conformation 
 ( supporting information , table s1 ) . while 
 this kind of assay is typically performed using molar excess of the 
 tested compounds , we here initially employed equimolar amounts of 
 the target dna sequence and compounds ( i.e. , 0.25 m ) to better 
 differentiate the stabilization efficiency among the different c - exndis .
the melting temperatures of the ltr - iii , ltr - iv , 
 htel , and dsdna g4 conformations were sufficiently similar ( 66.9 , 
 61.9 , 66.9 , and 66.5 c , respectively ) to allow a meaningful 
 comparison . in the presence of the c - exndis , a marked 
 increase in the stabilization of the g4-forming oligonucleotides was 
 observed ( figure 1b 
 and supporting information , table s2 ) . 
 among the tested c - exndis , 2 , 3 ,
4 , 10 , and 11 were the most 
 efficient ligands , with a stabilization on ltr - iv g4 of 1016 
 c at equimolar amounts . at 4-fold
c - exndi : dna molar 
 ratio , the stabilization increased to above 25 c , which is the 
 maximum increase measurable in these settings ( data not shown ) .
the 
 other c - exndis showed intermediate ( 4 c  
 tm < 7 c , 5 , 6 , and 7 ) , or low ( tm < 4 c , 8 and 9 ) level 
 of stabilization on ltr - iv g4 . compounds 2 and 10 stabilized ltr - iii g4 above 10 c . among the g4 structures , 
 htel g4 was the
only compounds 10 and 11 displayed a significant stabilization on dsdna , indicating 
 that the presence of two permanent charges ( the quaternary ammonium 
 moieties ) favors an unspecific binding to the negatively charged nucleic 
 acid .
in general , the h series of all derivatives ( y = h , 2 , 4 , 6 , 10 ; scheme 2 ) was more active and more 
 selective for the viral g4s compared to the br substituted ones ( y 
 = br , 3 , 5 , 7 , 8 ; scheme 2 ) .
compounds 2 , 3 , and 10 exhibited a difference 
 in stabilization of ltr - iv g4s vs htel g4 equal or above 6 c , 
 which roughly corresponds to 40% of the overall stabilization efficiency 
 ( figure 1b and supporting 
 information , table s2 ) .
tm values of hiv-1 ltr g4-folded sequences ( ltr - iii and ltr - iv ) , 
 the g4-folded telomeric sequence ( htel ) , and a control double - stranded 
 sequence ( dsdna ) obtained upon addition of equimolar amounts of 211 ( 0.25 m ) .
taq polymerization was performed 
 in the presence / absence of k and 2 , as indicated , 
 on the htel , ltr - iii , and ltr - iv templates , which were previously 
 folded into g4 . a control template unable to fold into g4
( d ) quantification of the intensity of the stop bands obtained in 
 the taq polymerase stop assay . stop band intensity 
 of the treated samples were normalized vs the untreated sample . because oligonucleotides used 
 for fret analysis display fluorophore - modified 
 ends that may influence the measured stabilization , additional techniques 
 using label - free oligonucleotides were employed .
compound 2 , a representative of the best ligands , was assayed by circular dichroism 
 ( cd ) on ltr - iv , ltr - iii , and htel g4s . at 4:1 c - exndi : dna ratio , a stabilization higher than 27 c was obtained on 
 both ltr - iii and ltr - iv ( supporting information , figures s2a and s2b ) , while htel g4s was stabilized by only 
 19.9 c ( supporting information , figure s2c ) , confirming a preference for the viral g4s .
a taq polymerase stop assay was performed on the ltr - iii , ltr - iv , and 
 htel templates . a control template unable to fold into g4
was used 
 to exclude unspecific inhibition of the polymerase enzyme by the c - exndi .
compound 2 induced an intense stop site 
 at the most 3 g tract in each g4-forming template ( figure 1c ) , indicating effective 
 stabilization of the g4 conformation by the compound .
this effect 
 was significantly more relevant on the viral sequences vs the telomeric 
 template ( figure 1d ) , 
 confirming again a preferential stabilization of the viral g4s .
the above techniques evaluated stabilization of the g4 conformation 
 as an indicator of the efficiency of the interaction of the ligands 
 with the tested g4 structures . binding affinity was next directly 
 assessed by two different methods .
competition electrospray 
 ionization mass spectrometry ( esi - ms ) 
 experiments allowed establishment of the relative affinity of 
 the c - exndis toward the g4 sequences .
we first confirmed 
 by cd analysis that in the buffer conditions used for the esi / ms experiments 
 the topology of the g4-folded oligonucleotides did not significantly 
 modify ( supporting information , figure s3 ) equimolar amounts of ltr - iii / htel , ltr - iv / htel , and ltr - iii / ltr - iv 
 g4s were incubated with 2-fold molar excess of compounds 2 or 4 . because esi / ms transfers intact biomolecules 
 complexes in the gas phase and signal intensity is proportional to 
 the abundance of each species in solution , the ratio between m / z signals of each bound species and the 
 corresponding bound and free g4 ( figure 2a ) was calculated ( table 1 ) . while ltr - iii and ltr - iv were detected 
 mostly as all - bound species , htel was mostly unbound ( figure 2a , and supporting information , figures s4a and s4b ) .
this analysis clearly shows 
 that our c - exndis preferentially bind the two viral sequences 
 over the telomeric sequence . to corroborate the selectivity toward 
 the viral sequences , two additional cellular g4 folding sequences 

the ltr g4s were preferred over c - kit2 g4 by 
 both compounds ; in the case of c - myc g4 , compound 4 preferentially 
 bound to the ltr g4s , whereas compound 2 showed more 
 intense binding to c - myc ( table 1 ) .
therefore , we confirmed a general , even though not 
 absolute , selectivity toward the ltr g4s .
it is also possible that 
 the much higher stability of the c - myc g4 ( tm > 95 c ) vs the stability of the other tested g4s 
 ( ltr - iii 
 66.9 c , ltr - iv 61.9 c , c - kit2 72.1 c ) played a role 
 in this result .
when the two ltr g4s were competed with each other , 
 the ltr - iii sequence was preferred . at 2-fold excess of compound , 

ltr - iii bound up to two molecules of compounds , whereas only one molecule 
 was obtained for both ltr - iv and htel ( figure 2 , and supporting information , figure s5 ) . at saturating concentrations ( 10-fold 
 molar excess )
, three molecules were bound to ltr - iii g4 , whereas up 
 to two molecules could bind ltr - iv and htel g4s ( supporting information , figure s5 ) . affinity and binding site analysis .
equimolar amounts of ltr - iii and htel were 
 incubated with 2-fold excess of 2 and analyzed by esi / ms . 

( b ) spr analysis 
 of 2 binding toward the ltrs and htel g4s .
biosensor data 
 were collected at 25 c ; the compound was injected at increasing 
 concentrations ( 26 , 40 , 59 , 89 , 133 , 200 nm ) .
ltr - iv ( c ) and ltr - iii ( d ) oligonucleotides 
 were g4 folded , incubated with increasing amounts of 2 ( lanes 513 ) , alkylated with cl ( cl lanes ) , and subjected 
 to hot piperidine treatment ( pip lanes ) to induce cleavage of the 
 phosphodiester bond at the alkylated bases .
( c ) lanes 14 are 
 control lanes not treated with the c - exndi .
m is a marker lane obtained by the maxam and gilbert sequencing of 
 the oligonucleotides .
the vertical dotted bar indicates the region of the ltr - iii 
 shared with ltr - iv .
( e ) schematics of the ltr - iv and ltr - iii g4 conformations . 

the represented ltr - iv g4 topology has been solved by nmr ( pdb code 2n4y ) ; the shown ltr - iii 
 g4 conformation derives from previous low resolution structural data .
next , absolute 
 affinity of 2 was measured by surface 
 plasmon resonance ( spr ) toward the three g4-folded sequences .
this 
 analysis confirmed that ltr - iii g4 displays the highest binding affinity 
 ( kd 3.2 nm ) , followed by ltr - iv g4 ( kd 6.1 nm ) ; htel g4 exhibited the lowest affinity 
 ( kd 13.1 nm ) ( figure 2b , and supporting information , figure s6 ) .
the binding affinity of the br derivatives 
 with each of the substituents in position r against ltr - iv was next 
 assessed .
compounds 3 and 5 had kd values similar to that of 2 ( kd 6.6 and 5.9 nm , respectively ) , 10 displayed an increased binding affinity ( kd 1.7 nm ) , whereas 7 and 11 showed 
 the lowest affinity ( kd 21.6 and 12.9 
 nm , respectively ) .
these data are in line with the stabilization properties 
 observed for these derivatives by fret and cd analysis and confirm 
 the improved and reduced recognition properties of the c - exndis exhibiting permanent dicationic ( 10 ) and neutral or 
 monocationic charge states ( 7 ) , respectively , under physiological 
 conditions .
they also confirm that a bulky substituent in position 
 y of the ndi core ( 11 , scheme 2 ) highly reduces binding , whereas a no2 group in position r maintains excellent binding properties 
 ( 5 , scheme 2 ) .
we reasoned that the observed biased affinity for the ltr 
 sequences 
 could derive by specific binding of the c - exndis to structural / sequence 
 features shared by the ltr g4s . to this end , we evaluated the protection 
 exerted by compound 2 over residues of the ltr g4s against 
 clerocidin ( cl ) .
this molecule has been shown to alkylate dna single - stranded 
 g , c , and a bases and to be able to predict g4 
 structural folding .
labeled oligonucleotides 
 were folded , treated with increasing amounts of 2 , exposed 
 to cl , and further treated with piperidine to highlight cl - alkylated 
 sites . in these settings
, we expected to visualize only loop bases 
 bound by the c - exndi because base - paired gs involved 
 in g - quartet formation would not be available to cl alkylation , independently 
 of the presence of the compound .
the nmr structure of ltr - iv has been 
 solved ( pdb code 2n4y ) , and therefore we were able to assess that nucleotides affected 
 by the presence of the c - exndi were in two loops of the 
 structure : in particular , in the actg loop the a base was protected , 
 whereas c and g were exposed ; in the 1-base - loop , the c base was exposed 
 ( figure 2c and 2e ) .
the structure of ltr - iii has not been solved 
 yet , however , previous data indicated that this g4 can fold into different 
 conformations , the main one of which is likely the one depicted in figure 2e , as detected by 
 low - resolution structural data . in this 
 case ,
we observed protection of the c base in the actg loop ( figure 2d ) , which is shared 
 with the ltr - iv sequence ( see figure 1a ) ; three additional residues ( c , c , and g ) were protected 
 in the 11-base - loop ( figure 2d and 2e ) .
protection and exposure 
 of residues indicated specific interaction of 2 at the 
 affected ltr sites .
interestingly , unique ltr loops and in particular 
 the actg loop shared by both ltr - iii and ltr - iv , and absent in the 
 telomeric sequence , were involved , indicating that these are likely 
 the moieties that induce selectivity .
we can not exclude that other 
 regions are bound by the c - exndi , but probably the interaction 
 at other sites is less stable or more dynamic so that a clear protection / exposure 
 could not be observed .
we have previously shown that the hiv-1 viral 
 promoter folds into dynamic g4s and that stabilization of these g4 
 conformations by the g4 ligand 1 represses viral transcription 
 and virus production in infected cells with ic50 ( the concentration 
 required to inhibit 50% of virus production ) in the low m range . the average selectivity index ( si ) for 1 was 12 .
given 
 the c - exndi binding / stabilization selectivity observed 
 in vitro for the viral g4s vs the cellular g4 , we set out to investigate 
 the anti - hiv-1 activity of the newly synthesized c - exndi derivatives .
compound entry was preliminarily measured by phase 
 contrast and fluorescence microscopy , taking advantage of the blue - colored c - exndi dyes ( abs = 555 and 600 nm , for 
 the unsubstituted 2 ) and their red fluorescence emission 
 ( em = 610 and 655 nm , for 2 ) .
after 
 30 min of incubation , compounds concentrated into the cell ( supporting 
 information , figure s7 ) .
we next treated 
 infected cells with increasing amounts of compounds and measured virus 
 production ; concurrently , the cytotoxic concentration ( cc50 ) of the c - exndis in the same conditions but on uninfected 
 cells was measured by mtt assay .
all compounds were tested against 
 the hiv-1 nl4 - 3 strain , which is a x4 strain .
compounds 2 , 3 , 4 , and 5 displayed 
 inhibitory concentration ( ic50 ) values below 25 nm .
compound 2 displayed a promising si of 40 , the best among the tested c - exndis , whereas si values of compound 3 , 4 , and 5 were around 20 , again superior to 1 . derivatives 6 and 7 exhibited 
 low activity and low cytotoxicity , while 9 showed the 
 worst combination with low activity , high cytotoxicity , and si of 
 2.2 .
we also evaluated the range of concentration ( therapeutic window 
 or tw ) , at which we obtained antiviral activity paralleled by less 
 than 80% cytotoxicity .
as shown in figure 3a , 2 displayed the widest tw 
 ( 3.9250 nm ) ; 4 and 5 also showed 
 interesting tw values ; tw concentrations for 6 and 7 , which are likely to be mainly monoanionic under physiological 
 conditions as both the ( ch2)3nhme2 moieties exhibit a pka > 8.1 , were in the m range ; no tw was observed 
 for 8 and 9 ( table 1 ) .
finally , 10 and 11 , containing two quaternary ammonium moieties , did not display any 
 antiviral or cytotoxic activity likely due to their inability to enter 
 the cells , as already shown for ndis with permanent positive charges .
compound 2 and 3 were 
 also tested on the hiv-1 bal strain , a r5 strain with a different 
 tropism . in this case ,
ic50 values below 40 nm were obtained , 
 with si of 14 for the h derivative ( table 1 ) .
note that the h series of all derivatives 
 was more active than the br substituted one .
moreover , all compounds 
 displayed cc50 values higher than 2 m against a 
 human fibroblast cell line .
( a ) tzm - bl cells were infected with hiv-1 nl4 - 3 strain and treated 
 with increasing concentrations ( 3.91000 nm ) of the c - exndis .
antiviral activity , expressed as relative 
 luciferase units ( rlu ) , was measured on infected cells , while cell 
 viability was in parallel obtained with a mtt assay on the uninfected 
 cells .
the vertical dotted lines define the therapeutic window ( tw ) 
 where the compound is active against the virus and not toxic against 
 the cells .
this figure is representative of the antiviral / cytotoxicity / tw 
 experiments performed with the whole series of c - exndi compounds .
tzm - bl cells were treated with the compound 2 ( 50100 nm ) , azt ( 0.5 g / ml ) , or the entry 
 inhibitor ds ( 100 g / ml ) at 0 , 1 , or 2 h pi .
hela cells 
 with a stably transfected ltr - egfp reporter plasmid were treated with 
 increasing concentrations of 2 ( 62.5250 nm ) . 

cytotoxicity was estimate 
 in parallel by gating on forward scatter ( fsc ) vs side scatter ( ssc ) 
 using untreated cells as reference .
( d ) specificity of the 2-induced inhibition of ltr promoter activity on the g4 ltr conformation . 

293 t cells were transiently transfected with plasmids with the wt 
 or m4 + 5 mutant ltr promoter sequence upstream a luciferase reporter 
 gene .
luciferase signals were measured as a function of increasing 2 amounts and normalized to total protein content .
ic50 is the compound concentration 
 required to inhibit 50% of hiv-1 production ; cc50 is the 
 compound concentration at which 50% of cell toxicity is observed ; 
 si is the selectivity index ; tw is the therapeutic window concentration . 

it has been reported 
 that compounds with multiple positive charges 
 can block viral entry by interfering with the cxcr4 coreceptor . to rule out this unspecific mechanism of action 
 of the c - exndi ,
we treated tzm - bl cells at 0 , 1 , and 
 2 h post infection ( pi ) with compound 2 and tested virus 
 production at 31 h pi .
no significant difference in the activity of 
 the compound was observed at the tested times ( figure 3b ) .
zidovudine ( azt ) , a reference drug that 
 targets the reverse transcriptase step , displayed a behavior similar to that of the c - exndi , maintaining activity from 0 to 2 h pi .
in contrast , dextran sulfate 
 ( ds ) , a reference entry inhibitor , exhibited 
 antiviral effect only when added at 0 h pi .
these data indicate that the initial 
 steps are not targeted by 2 , which is thus active at / after 
 2 h pi , i.e. , after the entry process has occurred .
we next 
 tested the ability of 2 to reduce the activity of the 
 hiv-1 ltr promoter in an egfp reporter assay in hela - tat - iii cells , 
 which is a cell line stably transfected with a plasmid embedding the 
 egfp coding region under the control of the hiv-1 ltr promoter .
cells 
 were treated with increasing concentrations of the c - exndi , and the intensity of egfp fluorescence was assessed by facs analysis . 
 a steady decrease in the mean levels of egfp
was observed at noncytotoxic 
 concentrations of 2 ( up to 60% of inhibition ) ( figure 3c ) . to check
if the 
 activity of the c - exndi at the ltr promoter was specific 
 for the g4 conformation , two point mutations that abolish g4 folding 
 were introduced in the ltr sequence ( m4 + 5 ltr ) .
the wt ltr and m4 + 5 ltr luciferase reporter plasmids 
 were transiently transfected into 293 t cells either alone or in the 
 presence of increasing amounts of compound 2 , and the 
 luciferase signal was measured .
again , an important inhibition of 
 ltr activity was observed on the wt ltr ( up to 65% inhibition ) , whereas 
 no effect was detected on the non - g4-forming mutant ltr sequence ( figure 3d ) .
these data indicate 
 a g4-mediated inhibition of the viral promoter by the c - exndi .
we have here shown the synthesis , 
 g4 binding properties , and antiviral 
 activity of a new series of ndi derivatives with an extended aromatic 
 core .
first , most of the new compounds displayed improved stabilization 
 on g4 structures , with substantial increments in tm above values previously reported for ndi derivatives .
this feature is an indication that the expansion of the aromatic 
 surface allows a more effective recognition of the g4 structure .
this 
 may be due to a more extensive overlap to the quartet of the g4 structure , 
 as inferred by the dimension of the c - exndi core , evaluated 
 by dft calculation ( supporting information , figure s1 ) .
compared to 1 , the best antiviral 25 showed improved 
 ic50 values ( in the low nm vs low m range ) .
this 
 feature may derive from the selectivity observed in vitro for the 
 ltr g4s over the telomeric g4 structures .
the viral and telomeric 
 g4s are likely the two most abundant dna g4 species in the cell during 
 infection , therefore a preferential effect on the viral g4s would 
 result in higher antiviral activity and lower cytotoxicity , as observed . 

the c - exndis displayed both selective binding affinity 
 and stabilization for the viral g4s .
between the two ltr g4s , the 
 ltr - iii sequence was most efficiently bound by 2 , as 
 measured by esi / ms and spr . even though ltr - iii ( 38 nts ) and ltr - iv 
 ( 29 nts ) display an identical number of g - quartets , ltr - iii offers 
 longer loops that may accommodate additional molecules and therefore 
 may display increased binding affinity .
indeed , at saturating concentrations , 
 binding of up to three c - exndi molecules was observed 
 by esi / ms with ltr - iii compared to two molecules for ltr - iv .
in addition , 
 two loops in both ltr - iii and ltr - iv g4s were shown to be involved 
 in c - exndi interaction by footprinting analyses .
we suggest 
 that the compounds interact with the edge g - quartets through the core 
 ndi planar aromatic surface , while the extended moiety and side chains 
 interact with the side loops , affording selectivity toward the ltr 
 g4s . to our knowledge , this is the first example of this type of selectivity 
 based on chemical recognition of specific residues ; in fact , previously 
 reported selectivity rather relied on the recognition of a g4 conformation .
analysis of the binding activity of the 
 series of c - exndis led us to the following structure  activity 
 relationship ( sar ) 
 conclusions : ( i ) the nonextended side of the c - exndi core 
 [ position y ( scheme 1 ) ] , lacking substituents as the introduction of an aminoalkyl side 
 chain , highly hinders stabilization of the g4 structures ( i.e. , 9 ) ; ( ii ) br is allowed as y substituent , but it lowers binding 
 and selectivity toward the viral sequences .
the br group may both 
 hinder specific interactions ( hence lower selectivity ) and partially 
 distort the aromatic core from planarity ( hence lower g4 binding ) ; 
 ( iii ) the pyridine ring in place of the benzene in the extended core 
 is strongly detrimental ( compound 8) .
the fair antiviral 
 activity is paralleled by moderate cytotoxicity , allowing no tw , possibly 
 due to off - target activity ; ( iv ) the no2 moiety is allowed 
 ( compounds 4 and 5 ) as compound 4 is the most powerful and selective ligand toward ltr - iii ; ( v ) the 
 negatively charged carboxylate group decrease the electrostatic component 
 of the binding to the dna g4s ( compounds 6 and 7 ) ; ( vi ) on the contrary , the permanently positively charged 
 ammonium groups extensively increase binding to the target g4s by 
 adding electrostatic interaction ( compounds 10 and 11 ) .
however , compounds with permanent charges are not suitable 
 for cell entry and hence can not be exploited for antiviral purposes . 
 a scheme of the chemical properties that lead to high - affinity recognition 
 and selectivity of the c - exndi binders is reported in 
 the graphical abstract . in conclusion , we have reported a new 
 class of g4 ligands able 
 to discriminate g4 structures based on the chemical recognition of 
 structural features that are unique to the selected g4 sequences . 

on one hand this approach opens the possibility to develop anti - hiv 
 compounds with a new mechanism of action that may complement current 
 clinical aids therapies ; on the other hand , our work clearly highlights 
 the possibility to selectively recognize g4 structures with small 
 molecules , prompting the search and development of g4 ligands specific 
 for g4s implicated in several important human diseases .
reagents , oligonucleotides , 
 solvents , and chemicals were purchased from alfa aesar or sigma - aldrich 
 and were used as supplied without further purification .
tlc analysis 
 was carried out on silica gel ( merck 60f-254 ) with visualization at 
 254 and 366 nm .
hplc analysis and purifications were performed using 
 two different hplc : waters system combining a delta 600 pump , a 2489 
 uv / vis detector and fraction collector iii ( for preparative and analytical ) , 
 and an agilent system series 1260 ( for analytical ) .
the analytical 
 column was xselect csh phenyl - hexyl ( 150 mm  4.6 mm ) ( waters ) . 

the preparative column was xselect csh prep phenyl  hexyl 5 
 m ( 150 mm  30 mm ) ( waters ) . flows were 1 or 1.4 ml / min 
 for analytical and 27 ml / min for preparative .
for the analytical analysis 
 were used the following method a : ( aqueous solvent : 0.1% trifluoroacetic 
 acid in water ; organic solvent , acetonitrile ) ; method a = 1 ml / min , 
 isocratic flow over 2 min 95% of aqueous solvent ; gradient , 95% aqueous , 
 gradually to 40% aqueous over 12 min and at the end an isocratic flow 
 over 4 min . for the preparative purification
was used the following 
 method b : ( aqueous solvent : 0.1% trifluoroacetic acid in water ; organic 
 solvent , acetonitrile ) ; method b = 27 ml / min , isocratic flow over 
 4 min 95% of aqueous solvent ; gradient , 95% aqueous , gradually to 
 70% aqueous over 16 min and at the end an isocratic flow over 4 min ) .
h , c nmr spectra were recorded on a bruker advance 
 300 mhz spectrometer .
uv / vis spectra were run 
 on a varian cary 100 scan spectrophotometer with quartz cuvettes of 
 the appropriate path length ( 0.11 cm ) at 25.0  0.1 c . 

the new ligands ( 211 ) were analyzed by hplc ( see supporting information , hplc purity data ) , confirming 97.6% purity .
the 
 starting ndi ( 0.5 mmol ) was dissolved into 20 ml of dmf in a round - bottom 
 flask together with 1.0 mmol of o - phenyl - diamine 
 derivatives , the mixture was stirred at 45 c for 16 h under 
 argon .
the resulting dark - violet solution was basified with sodium 
 bicarbonate aqueous solution and extracted with three portion of chcl3 ( 50 ml ) .
the crude product was analyzed 
 and purified by hplc chromatography ( ch3cn : h2o 0.1%tfa ) according to analytical method a and preparative method 
 b. hcl 1 m solution was added to each chromatographic portion .
yield 
 = 25.2% ; dark - violet 
 solid ; mp dec > 200 c .
h nmr ( 300 mhz , cd3od ) : 7.74 ( s , 2h ) , 7.11 ( dd , j = 3.3 , 5.7 
 hz , 2h ) , 
 6.89 ( dd , j = 3.3 , 5.7 hz , 2h ) , 4.06 ( m , 4h ) ; 3.26 
 ( m , 4h ) , 2.94 ( s , 12h ) , 2.14 ( m , 4h ) .
c nmr ( 75 mhz , 
 cd3od ) : 166.4 , 164.3 , 143.2 , 128.7 , 128.0 , 127.0 , 125.9 , 
 123.0 , 118.2 , 96.0 , 57.0 , 56.0 , 43.9 , 38.5 , 38.2 , 24.9 , 24.2 .
calcd for c30h34cl2n6o4 : c , 58.73 ; h , 5.59 ; cl , 11.56 ; n , 13.70 ; o , 10.43 .
yield = 19.4% ; dark - violet 
 solid ; mp dec > 200 c .
h nmr ( 300 mhz , cd3od ) : 8.1 ( s , 1h ) , 7.15 ( dd , j = 3.4 , 6.0 
 hz , 2h ) , 
 7.03 ( dd , j = 3.4 , 6.0 hz , 2h ) , 4.17 ( m , 4h ) , 3.25 
 ( m , 4h ) , 2.94 ( s , 12h ) , 2.16 ( m , 4h ) .
c nmr ( 75 mhz , 
 cd3od ) : 166.5 , 166.1 , 163.9 , 163.6 , 152.4 , 144.3 , 132.5 , 
 130.3 , 129.0 , 128.9 , 128.3 , 128.1 , 126.6 , 123.1 , 121.1 , 120.4 , 118.4 , 
 118.2 , 97.0 , 79.8 , 57.1 , 43.9 , 38.7 , 38.5 , 24.9 , 24.7 .
calcd 
 for c30h33brcl2n6o4 : c , 52.04 ; h , 4.80 ; br , 11.54 ; cl , 10.24 ; n , 12.14 ; o , 9.24
. 
 found : c , 52.17 ; h , 4.81 ; n , 12.12 .
yield = 11.2% ; dark - violet 
 solid ; mp dec > 200 c .
h nmr ( 300 mhz , cd3od ) : 8.04 ( s , 2h ) , 7.88 ( d , j = 6.6 hz , 1h ) , 
 7.84 
 ( 1h , s ) , 7.07 ( d , j = 6.5 hz , 1h ) , 4.2 ( bs , 4h ) , 
 3.27 ( m , 4h ) , 2.94 ( s , 12h ) , 2.17 ( m , 4h ) .
c nmr ( 75 
 mhz , cd3od):166.9 , 166.7 , 164.5 , 134.6 , 132.1 , 129.6 , 127.3 , 
 127.2 , 126.9 , 126.6 , 124.3 , 124.1 , 122.8 , 117.8 , 112.9 , 99.6 , 99.4 , 
 57.0 , 43.8 , 38.4 , 24.8 . anal .
calcd for c30h33cl2n7o6 : c , 54.72 ; h , 5.05 ; cl , 
 10.77 ; n , 14.89 ; o , 14.58 .
14.92 . yield 
 = 18.9% ; dark - violet 
 solid ; mp dec > 200 c .
h nmr ( 300 mhz , cd3od ) : 8.00 ( s , 1h ) , 7.87 ( d , j = 6.6 hz , 1h ) , 
 7.79 
 ( 1h , s ) , 7.0 ( d , j = 6.5 hz , 1h ) , 3.98 ( bs , 4h ) , 
 3.14 ( m , 4h ) , 2.83 ( s , 12h ) , 2.01 ( m , 4h ) .
c nmr ( 75 
 mhz , cd3od):162.0 , 161.0 , 144.6 , 141.4 , 140.7 , 132.1 , 127.1 , 
 124.1 , 122.9 , 122.1 , 121.2 , 120.7 , 118.9 , 111.9 , 55.1 , 48.7 , 46.5 , 
 42.6 , 37.5 , 37.2 , 22.5 , 22.4 . anal . calcd for c30h32brcl2n7o6 : c , 48.86 ; h , 
 4.37 ; br , 10.84 ; cl , 9.61 ; n , 13.30 ; o , 13.02 .
yield = 18% ; dark - violet solid ; 
 mp dec > 200 c .
h nmr ( 400 mhz , cd3od ) : 
 7.85 ( s , 1h ) , 7.76 ( s , 1h ) , 7.20 ( m , 1h ) , 6.95 ( bs , 1h ) , 3.91 ( m , 
 4h ) , 2.70 ( m , 4h ) , 2.52 ( m , 12h ) , 1.93 ( m , 4h ) .
c nmr 
 ( 100 mhz , cd3od):165.8 , 162.5 , 161.5 , 146.5 , 143.8 , 141.9 , 
 133.2 , 128.7 , 125.5 , 125.1 , 123.4 , 123.1 , 121.9 , 120.5 , 97.8 , 57.9 , 
 45.2 , 45.1 , 39.9 , 39.7 , 26.0 , 25.9 .
calcd for c29h32brcl2n7o4 : c , 50.23 ; 
 h , 4.65 ; br , 11.52 ; cl , 10.22 ; n , 14.14 ; o , 9.23 . found : c , 50.17 ; 
 h , 4.71 ; n , 14.08 .
ndi 1 ( 0.5 mmol ) was dissolved into 40 ml 
 of ch3cn 
 in a round - bottom flask together with 1.0 mmol of 3,4-diaminobenzoic 
 acid , the mixture was stirred at 85 c for 72 h under argon . 

the resulting dark - violet solution was cooled at r.t . to induce precipitation 
 of the product .
the crude powder was filtered and purified by hplc 
 chromatography , ( ch3cn : h2o 0.1%tfa ) according 
 to analytical method a and preparative method b , affording the title 
 ndis 6 ( 27% ) and 7 ( 30.5% ) . yield = 27% ; 
 dark - violet solid ; 
 mp dec > 200 c .
h nmr ( 400 mhz , cd3od ) : 
 7.86 ( s , 2h ) , 7.62 ( d , j = 8.0 hz , 1h ) , 7.39 ( s , 
 1h ) , 6.89 ( d , j = 8.0 hz , 1h ) , 4.16 ( m , 4h ) , 3.26 
 ( m , 4h ) , 2.95 ( s , 12h ) , 2.16 ( m , 4h ) .
c nmr ( 100 mhz , 
 cd3od):168.2 , 166.6 , 166.5 , 164.3 , 164.2 , 143.1 , 142.8 , 
 132.6 , 129.8 , 129.2 , 128.6 , 127.0 , 126.7 , 126.6 , 126.4 , 123.6 , 123.5 , 
 119.0 , 117.7 , 98.3 , 97.7 , 57.0 , 43.8 , 40.7 , 38.4 ; 24.8 .
calcd 
 for c31h34cl2n6o6 : c , 56.63 ; h , 5.21 ; cl , 10.78 ; n , 12.78 ; o , 14.60 . found : c , 56.77 ; 
 h , 5.31 ; n , 12.72 . yield = 30.5% ; dark - violet 
 solid ; mp dec > 200 c .
h nmr ( 300 mhz , cd3od ) : 8.37 ( s , 2h ) , 7.85 ( d , j = 8.0 hz , 1h ) , 
 7.73 
 ( s , 1h ) , 7.21 ( d , j = 8.0 hz , 1h ) , 4.35 ( m , 4h ) , 
 3.41 ( m , 4h ) , 3.05 ( s , 12h ) , 2.29 ( m , 4h ) .
c nmr ( 75 
 mhz , cd3od ) : 168.3 , 166.2 , 163.5 , 161.8 , 161.7 , 160.8 , 
 152.2 , 133.2 , 132.6 , 130.1 , 129.4 , 126.5 , 123.4 , 123.3 , 121.6 , 119.12 , 
 118.21 , 117.9 , 98.3 , 97.6 , 57.04 , 43.9 , 38.3 , 38.5 , 24.8 , 24.7 .
calcd for c31h33brcl2n6o6 : c , 50.56 ; h , 4.52 ; n , 11.41 ; o , 13.03 .
found : c , 50.34 ; 
 h , 4.61 ; n , 11.29 compound 3 ( 0.5 
 mmol ) was dissolved into 4 ml of n , n - dimethyl - propan-1,3-diamine .
the mixture was stirred and heated 
 in a microwave reactor , according to a closed vessel protocol , at 
 150 c , 250 psi , 200 w , for 3 min .
the resulting green solution 
 was cooled at rt to induce precipitation of the product .
the resulting 
 deep - green powder was filtered and washed by water afforded 1 ( yield 92% ) .
further hplc preparative purification ( ch3cn : h2o and 0.1% cf3cooh as eluent ) , 
 and final trifluoroacetate - chloride anion exchange , by addition of 
 1 ml hcl 1 m , yielded 9 as trihydrochloride ( 93hcl ) ; deep - green solid ; mp dec > 200 c .
h 
 nmr ( 300 mhz , cd3od ) :  = 7.10 ( m , 1h ) , 6.96 ( m , 
 2h ) , 6.69 ( m , 2h ) , 4.11 ( m , 4h ) , 3.42 ( m , 2h ) , 3.26 ( m , 6h ) , 2.99 
 ( s , 6h ) , 2.93 ( s , 12h ) , 2.18 ( bs , 2h ) , 2.11 ( bs , 4h ) .
c nmr ( 75 mhz , cd3od ) :  = 166.7 , 166.4 , 164.5 , 
 144.2 , 139.7 , 129.8 , 129.0 , 128.9 , 127.5 , 126.6 , 125.0 , 117.6 , 117.1 , 
 115.8 , 108.2 , 104.5 , 98.1 , 97.7 , 63.4 , 57.0 , 54.0 , 53.8 , 43.8 , 41.1 , 
 38.5 , 38.1 , 25.7 , 24.9 , 24.8 , 24.1 .
calcd for c35h47cl3n8o4 : c , 56.04 ; 
 h , 6.32 ; cl , 14.18 ; n , 14.94 ; o , 8.53 . found : c , 56.17 ; h , 6.39 ; n , 
 14.90 . the c - exndis ( 2 or 3 ) purified as hydrochlorides were dissolved in a nahco3 solution and extracted 3 times with ch2cl2 .
the recovered organic layers have been dried on na2so4 and the solvent evaporated under reduced pressure . 
 the collected amine ( 2.5 mmol ) was suspended in 50 ml of chcl3 and 1.2 g ( 8.5 mmol ) of ch3i were added .
this 
 suspension was stirred under nitrogen atmosphere , at room temperature 
 for 12 h. after this period the resulting dark - violet powder was filtered 
 and dried , afforded the product as pure di - iodide salt .
yield = 99% ; dark - violet 
 crystals ; 
 mp dec > 200 c .
h nmr ( 400 mhz , cd3od ) : 
  = 7.94 ( s , 2h ) , 7.12 ( dd , j = 3.3 , 6.0 hz , 
 2h ) , 6.97 ( dd , j = 3.3 , 6.0 hz , 2h ) , 4.14 ( m , 4h ) , 
 3.49 ( m , 4h ) , 3.15 ( m , 18h ) , 2.21 ( m , 4h ) .
c nmr ( 100 
 mhz , d2o ) :  = 166.9 , 165.8 , 143.8 , 129.1 , 128.1 , 
 127.6 , 126.3 , 123.5 , 118.3 , 97.5 , 66.1 , 54.1 , 38.6 , 23.6 .
calcd 
 for c32h38i2n6o4 : c , 46.62 ; h , 4.65 ; i , 30.78 ; n , 10.19 ; o , 7.76 .
yield = 99% ; dark - violet crystals ; 
 mp dec > 200 c .
h nmr ( 400 mhz , cd3od ) : 
  = 8.04 ( s , 1h ) , 7.15 ( bs , 2h ) , 7.00 ( bs , 2h ) , 4.12 ( m , 4h ) , 
 3.51 ( m , 4h ) , 3.15 ( s , 18h ) , 2.20 ( bs , 2h ) .
c nmr ( 100 
 mhz , cd3od ) :  = 166.3 , 165.9 , 163.2 , 162.2 , 145.1 , 
 144.1 , 143.0 , 132.5 , 130.1 , 128.8 , 128.7 , 128.3 , 128.2 , 126.4 , 123.0 , 
 121.1 , 120.2 , 118.3 , 118.2 , 96.9 , 65.8 , 53.9 , 38.8 , 38.6 , 23.3 , 23.2 . 
 anal .
calcd for c32h37bri2n6o4 : c , 42.55 ; h , 4.13 ; br , 8.84 ; i , 28.09 ; n , 9.30 ; o , 
 7.08 . found : c , 42.60 ; h , 4.18 ; n , 9.39 .
all oligonucleotides 
 used in this study were from sigma - aldrich ( milan , italy ) . for fluorescence 
 melting curves ,
equimolar amounts of the compounds were added to each 
 fam ( 6-carboxyfluorescein ) 5-end- and tamra ( 6-carboxy - tetramethylrhodamine ) 
 3-end - labeled oligonucleotide ( 0.25 um ) folded in the lithium 
 cacodylate buffer supplemented with potassium ( 100 mm ) .
after stabilization 
 at 4 c , samples were processed in a lightcycler ii ( roche , milan , 
 italy ) or lightcycler 480 ( roche , milan , italy ) , and the oligonucleotide 
 melting was monitored by observing 6-carboxyfluorescein ( 6-fam ) emission 
 in the temperature range of 30 95 c with 1 c / min 
 gradient .
melting profiles were normalized as previously described.tm was defined as 
 the temperature corresponding to the 0.5 fraction of the normalized 
 fluorescence . for cd analysis , oligonucleotides were diluted 
 to a final concentration of 4 m in lithium cacodylate buffer 
 ( 10 mm , ph 7.4 ) and kcl 100 mm .
after annealing step ( 95 c for 
 5 min ) , dna samples were gradually cooled to room temperature and 
 compounds added from stock at final concentration of 16 m . 

cd spectra were recorded on a chirascan - plus ( applied photophysisics , 
 leatherhead , uk ) equipped with a peltier temperature controller using 
 a quartz cell of 5 mm optical path length and an instrument scanning 
 speed of 50 nm / min over a wavelength range of 230320 nm .
the 
 reported spectrum of each sample represents the average of 2 scans 
 is baseline - corrected for signal contributions due to the buffer . 

observed ellipticities were converted to mean residue ellipticity 
 (  ) = deg  cm  dmol ( mol ellip ) . for the determination of tm ,
spectra were recorded over a temperature range of 2095 
 c , with temperature increase of 5 c / min .
tm values were calculated according to the vant 
 hoff equation , applied for a two - state transition from a folded to 
 unfolded state , assuming that the heat capacity of the folded and 
 unfolded states are equal .
briefly , the 5-end labeled 
 primer was annealed to its template ( supporting information , table s1 ) in lithium cacodylate buffer in the 
 presence or absence of kcl 100 mm and by heating at 95 c for 
 5 min and gradually cooling to room temperature .
primer 
 extension was conducted with 2 u of amplitaq gold dna polymerase ( applied 
 biosystem , carlsbad , california , usa ) at 47 c for 30 min .
reactions 
 were stopped by ethanol precipitation , primer extension products were 
 separated on a 15% denaturing gel , and finally visualized by phosphorimaging 
 ( typhoon fla 9000 ) .
oligonucleotides 
 were heat - denatured and folded in 10 mm kcl , 150 mm trimethylammonium 
 acetate ( tmaa ) , ph 6.8 , overnight at room temperature .
the oligonucleotides 
 where diluted to final concentration of 10 m and incubated 
 with the tested compound at ratio dna : compound 1:1 overnight at 4 
 c .
samples were analyzed by direct infusion electrospray 
 ionization ( esi ) on a xevo g2-xs qtof mass spectrometer ( waters , manchester , 
 uk ) .
the injection was automatically performed by an agilent 1290 
 infinity hplc ( agilent technologies , santa clara , ca , us ) equipped 
 with an auto sampler ; the carrying buffer was tmaa 100 mm .
the absence 
 of potassium in the carrying buffer allows the dilution of the salts 
 before ms analysis decreasing the signal suppression .
the electrospray 
 capillary was at 1.8 kv , the source and desolvation temperatures were 
 45 and 65 c , respectively , and the sampling cone was at 65 v. 
 all these parameters ensured minimal fragmentation of the dnas complexes . 

the instrument was calibrated using a 2 mg / ml solution of sodium iodide 
 in 50% 2-propanol . additionally , the use of the lockspray during the 
 analysis provided a typical < 2 ppm mass accuracy .
the internal 
 standard lockspray consisted in a solution of leu - enkephalin 1 g / ml 
 in acetonitrile / water ( 50:50 , v / v ) containing 0.1% formic acid .
binding 
 affinities were calculated for each experiment using the reconstructed - ion 
 chromatogram area for each species calculated by masslynx v4.1 : this 
 analysis was made possible by the experimental design that used an 
 hplc system to inject the samples in the mass spectrometer .
the binding 
 affinity was calculated with the following formula : [ ba = ( g4b/(g4f + g4b ) )  100 ] , 
 where ba is the binding affinity , g4b is chromatogram area 
 of bound g4 dna , and g4f is the chromatogram area of free 
 g4 dna .
signals with charge 
 states 6 , 5 , and 4 were used for the free and bound htel dna and c - myc , charge states 
 6 and 5 for ltr - iii , charge 
 states 5 and 4 for c - kit2 , and 
 charge state 4 for ltr - iv .
free dna mono and dipotassium 
 adducts were included in the calculation . for stoichiometric calculation , 
 oligonucleotides were folded in 10 mm kcl , 150 mm tmaa ph 6.8 overnight 
 at rt .
the oligonucleotides , diluted to final concentration of 10 
 m , were incubated with the tested compound ( ratio 1:5 , 1:10 , 
 1:20 , and 1:50 ) at 4 c and analyzed by esi - ms .
5-biotinylated ltr - iii , ltr - iv , 
 and htel oligonucleotides were heated at 95 c for 5 min and 
 cooled down at room temperature to allow g4 folding .
immobilization 
 was performed in hepes - kcl running buffer ( 0.01 m hepes ph 7.4 , 0.2 
 m kcl , 3 mm edta ) on a steptavidin coated surface ( sa sensor chip , 
 biacore ) .
oligonucleotides were diluted in hepes - kcl running buffer 
 to a concentration of 30 nm and injected to reach the response of 
 around 500 ru .
c - exndis binding analysis was performed at a flow rate of 
 30 l / min , with contact time of 280 s and dissociation time 
 of 360 s in hepes - kcl buffer .
after each compound injection the chip 
 surface was regenerated with glycine 10 mm ph 2.0 solution ( ge healthcare ) . 

data 
 were fitted to a 1:1 binding model with rmax initial parameter set to theoretical calculated rmax using biaevaluation software ( ge healthcare ) .
all 
 experiments were performed independently at least twice , and in each 
 instance  values , which indicate the goodness of 
 fitting , were below 0.2 .
all oligonucleotides 
 were 
 gel - purified before use and prepared in desalted / lyophilized form . 

oligonucleotides were 5-end - labeled with [ - p]atp by t4 polynucleotide kinase and purified by microspin g-25 
 columns ( ge healthcare , europe ) .
they were next resuspended in lithium 
 cacodylate 10 mm , ph 7.4 , and kcl 100 mm , heat - denatured , and folded . 

reactions of the labeled g - quadruplex folded oligonucleotides ( 4 pmol / sample ) 
 with increasing amounts of compound 2 ( 4100 nm ) 
 were performed at 20 c for 24 h in resuspension buffer .
samples 
 were then reacted with cl ( 100 m ) at 37 c for 24 h. reactions 
 were stopped by ethanol precipitation , resuspended , and either kept 
 on ice or treated at 90 c for 30 min with 1 m piperidine to 
 complete strand scission according to the maxam and gilbert protocol . 

samples were then lyophilized , resuspended in formamide gel loading 
 buffer , and heated at 95 c for 3 min .
reaction products were 
 analyzed on 20% denaturing polyacrylamide gels and visualized by typhoon 
 fla 9000 phosphorimaging analysis ( ge healthcare ) .
the hiv-1 promoter activity in live 
 cells in the presence of 2 was monitored in hela - tat - iii / ltr / d1egfp 
 cells ( obtained through the nih aids reagent program , division of 
 aids , niaid , nih , from dr .
this cell line derives from hela - tat - iii 
 and is transfected with d1egfp under the control of hiv-1 ltr promoter . 

then 8 10 of hela - tat - iii / ltr / d1egfp cells were 
 seeded in a 12-well plate in 1 ml of dmem/10% fbs medium supplemented 
 with geneticin selective antibiotic ( 1 mg / ml , life technologies , monza , 
 italy ) and incubated overnight .
cells were next treated with increasing 
 concentrations of 2 ( 062.5125250 
 nm ) and incubated at 37 c .
after 48 h , cells were washed with 
 pbs 1 and resuspended in 500 l of pbs 1. to evaluate 
 the mean of egfp fluorescence , a total of 10000 events were acquired 
 for each sample with an lrs 2 instrument using facs diva software 
 ( bd bioscience ) and analyzed with flow jo ( tree star ) .
cell debris 
 and dead cells were excluded from the analysis by gating on forward 
 scatter ( fsc ) versus side scatter ( ssc ) and were quantified to estimate 
 the cytotoxicity . for the luciferase assay ,
hiv-1 ltr region 
 ( wt or m4 + 5 ) was inserted into the promoterless luciferase reporter 
 vector pgl4.10-luc2 ( promega italia , italy ) , as previously reported .
for assessment of luciferase activity , human 
 embryonic kidney 293 t ( hek293 t ) cells were seeded in a 24-well plate 
 ( 9  10 cells / well ) . then
24 h later , cells were 
 transfected with 100 ng / well of pgl4.10-ltrwt or of pgl4.10-ltr - m4 
 +
5 using transit-293 transfection reagent ( mirus bio llc , madison , 
 wi , usa ) , according to the manufacturer s protocol . after 1 
 h
luciferase activity was measured using the britelite plus reporter 
 gene assay system ( perkinelmer inc . , milan , italy ) at a victor x2 
 multilabel plate reader ( perkinelmer inc . ,
cells were lysed in ripa 
 buffer ( 50 mm tris - hcl ph 7.2 , 150 mm nacl , 1% igepal , 0.1% sds ) and 
 protein concentration was determined by bca assay ( thermo scientific 
 pierce , monza , italy ) .
luciferase signals were subsequently normalized 
 to total protein content , according to the manufacturer s protocol 
 ( http://ita.promega.com/~/pdf/resources/pubhub/cellnotes/normalizing-genetic-reporter-assays/ ) .
infectivity was measured using the tzm - bl reporter cell line ( obtained 
 through the nih aids reagent program , division of aids , niaid , nih , 
 from dr .
tzm - bl is a hela 
 cell line stably expressing large amounts of cd4 and ccr5 and containing 
 integrated copies of the luciferase and -galactosidase genes 
 under control of the hiv-1 promoter .
cells ( 1 10 cells / well ) were seeded 
 in 96-well plates and grown overnight to permit adherence prior to 
 treatment and viral infection .
cells were next infected with hiv-1 
 nl4 - 3 strain or bal strain at a moi of 0.5 , treated with serial dilutions 
 of tested compounds , and incubated at 37 c .
after 48 h , cells 
 were washed with pbs 1 and hiv-1 production was assessed following 
 the ltr - luciferase signal using the britelite plus reporter gene assay 
 system ( perkinelmer , waltman , ma , usa ) according to the manufacturer s 
 protocol .
cytotoxicity 
 of tested compounds 
 was tested in parallel to antiviral and reporter assays by mtt .
the 
 50% cytotoxic concentration ( cc50 ) was defined as the concentration 
 of test compound that was able to reduce the absorbance of the mock - infected 
 cells by 50% .
the 50% inhibitory concentration ( ic50 ) was 
 defined as the concentration of test compound that inhibit 50% of 
 ltr - luciferase signal .
the selectivity index ( si ) is the relative 
 effectiveness of the tested compound in inhibiting viral replication 
 compared to inducing cell death ( cc50 value / ic50 value ) .
the therapeutic window ( tw ) is the concentration range at 
 which the compound shows antiviral activity paralleled by less than 
 80% cytotoxicity .
tzm - bl cells ( 1 
  10 cells / well ) were seeded in 96-well plates and 
 grown overnight to 
 permit adherence prior to treatment and viral infection .
cells were 
 next infected with hiv-1 nl4 - 3 ( moi 0.5 ) and incubated at 37 c . 

compound 2 ( 50100 nm ) and the reference compounds 
 ds ( 100 g / ml , sigma - aldrich ) and azt ( 0.5 g / ml , sigma - aldrich ) 
 were added at different hours ( 0 , 1 , 2 h ) pi . after 31 h , cells were 
 washed with pbs 1 and hiv-1 production was assessed following 
 the ltr - luciferase signal using the britelite plus reporter gene assay 
 system ( perkinelmer , waltman , ma , usa ) according to the manufacturer s 
 protocol .
dft calculations 
 were performed 
 with the gaussian 09 , revision b.01 software package .
gas - phase geometry optimizations were carried out using 
 the b3lyp functional , with 6 - 31+g(d , p ) 
 basis set for all the atoms .
 we have previously reported that 
 stabilization of the g - quadruplex 
 structures in the hiv-1 long terminal repeat ( ltr ) promoter suppresses 
 viral transcription . 
 here we sought to develop new g - quadruplex ligands 
 to be exploited as antiviral compounds by enhancing binding toward 
 the viral g - quadruplex structures . 
 we synthesized naphthalene diimide 
 derivatives with a lateral expansion of the aromatic core . 
 the new 
 compounds were able to bind / stabilize the g - quadruplex to a high extent , 
 and some of them displayed clear - cut selectivity toward the viral 
 g - quadruplexes with respect to the human telomeric g - quadruplexes . 
 
 this feature translated into low nanomolar anti - hiv-1 activity toward 
 two viral strains and encouraging selectivity indexes . 
 the selectivity 
 depended on specific recognition of ltr loop residues ; the mechanism 
 of action was ascribed to inhibition of ltr promoter activity in cells . 
 
 this is the first example of g - quadruplex ligands that show increased 
 selectivity toward the viral g - quadruplexes and display remarkable 
 antiviral activity . 
Introduction
Results
Discussion and Conclusions
Experimental Section