dengue is the most common arthropod - borne viral infection in the world and is estimated to transmit 390 million new infections every year . the genome of denv is comprised of a 10.7  kb , single , positive - stranded rna with at least four different circulating serotypes ( denv-1 to denv-4 ) . reportedly , a fifth serotype now complicates vaccine development . with increased levels of population growth , urbanization , and international travel , most of the current research on dengue infections is focused on the treatment of symptoms , which often can be a tedious and intensive process . as there is neither a drug nor any vaccine available to combat denv infections till date , it is highly important to explore and uncover small molecules that have potent anti - dengue activity . in the past half - decade these include an adenosine analog 1 
 , a n - sulfonylanthranilic acid derivate 2 
 , the chlorophenyl - thiophene derivate 3 
 , and most recently some 2,4-diaminoquinazoline derivatives 4 
  ( see fig .  1 ) . hence , further development of new chemical entities endowed with dengue inhibitory properties is warranted . our previous efforts herein , focused on tritylated and alkylated nucleoside analogs , resulting in compounds endowed with strong anti - flavivirus inhibitory properties . the five - membered imidazole ring is a structural unit found in many biologically active compounds . the strong therapeutic properties of imidazole containing drugs have encouraged medicinal chemists to synthesize a large number of novel chemotherapeutic agents comprising this entity . amongst others , imidazole core structures are found in different carboxypeptidase , hemeoxygenase and lactamase inhibitors , as well as among anti - inflammatory , anticancer , antibacterial , antifungal , antitubercular , antidiabetic and antiviral products , further highlighted with some examples . synthesized a series of novel 5-(nitro / bromo)-styryl-2-benzimidazole derivatives ( 5 ) and tested them for their antibacterial activity against staphylococcus aureus , escherichia coli and likewise evaluated the antifungal activity against candida albicans and aspergillus fumigates 
 . et  al . synthesized a series of 2-methylaminobenzimidazole derivatives in which compound 6 showed analgesic and anti - inflammatory activity comparable with the standard drug nimesulide . synthesized a series of 1 , 4-diarylimidazole-2(3h)-one derivatives 7 and their 2-thione analogs and found antitumor activity . synthesized imidazole derivatives for antiviral screening and different [ 2-(substituted phenyl)-imidazol-1-yl]-benzamides like 8 and 9 were selected as the most promising antiviral agents   ( see fig .  2 ) . imidazole-4,5-dicarboxylic acid ( i45dc ) and its derivatives such as those bearing primary or secondary amides have previously been reported to be active against hiv-1 protease . likewise , some derivatives of this scaffold were uncovered as potential kinase inhibitors with antiproliferative activity against hl-60 cells . in addition , inhibition of protein  protein interaction between hepatitis c glycoprotein e2 and cd81 has been reported previously . the i45dcs are known as well to be a structural component of some antibiotics   and also to affect memory . substitution on the imidazole moiety herein has been reported , both in solution phase as well as in a combinatorial fashion on solid support . high throughput screening of a compound library led to the identification of n-(4-fluorophenyl)-n-(4-methyl-2-(3-methyl-1h - pyrazol-5-yl)phenyl)-1h - imidazole-4,5-dicarboxamide 15a ( see fig . 3 ) with an ec50 of 2.50  m and 3.47  m against denv and yfv , respectively . in search for new compounds with potential for clinical use as antiviral agents , a series of compounds based on this i45dc scaffold were synthesized with regiospecific attachment of the substituents and these analogs were investigated for their inhibitory properties against denv and yfv . in addition , the imidazole core was substituted for a pyrazine ring leading to a series of pyrazine-2,3-dicarboxylic acids ( p23dc ) . the intended substitution of the central imidazole ring would result in slightly different orientation of the attached amide groups , and in a change of the hydrogen bonding pattern exchanging the combination of a hydrogen donating and a hydrogen accepting nitrogen into two hydrogen accepting nitrogen atoms . in this communication , we report on the synthesis and biological activity of both new series of dissymmetric i45dcs and p23dcs , which we studied for their inhibitory properties against yfv and denv . the general procedure employed for the synthesis of dissymmetrically disubstituted i45dcs is shown in scheme 1 . imidazole-4,5-dicarboxylic acid 10 was allowed to reflux with socl2 in toluene with catalytic dmf to afford pyrazinedione diacid dichloride 11 in 92% yield . the literature route for amide formation suggests hydrolysis of the acid chlorides to carboxylic acids by the addition of water . amines are then added to open both acyl imidazole bonds affording two identical imidazole analogs . baures et  al . , replaced the water with phenol , thereby modulating the reactivity of the acid chloride in comparison with an acyl imidazole bond for the subsequent addition of two different amines . the difficulty in preparing such analogs is that their synthesis is highly dependent upon the substituents to be introduced . primary benzyl amines , for example , are too reactive to provide selectivity in the reaction , whereas anilines can be added in a proper stoichiometric ratio in order to provide a pyrazine dione intermediate 12 which can often be purified by crystallization . in the case of aniline derivatives , the resulting pyrazine intermediates are often insoluble in the reaction solvent and can be isolated simply by vacuum filtration . in parallel , different bromoanilines or benzylamines were subsequently coupled with commercially available heterocyclic boronic acids through palladium catalyzed suzuki reaction to give the desired second aniline in 6075% yield . addition of this second aniline to the pyrazine intermediates 12a  c resulted in opening of the acyl imidazole bond to obtain the expected product . based on this approach , a series of novel molecules was prepared within this i45dc family containing various substituted phenyl rings . for some of the final products 15 , the reaction sequence was changed with prior attachment of the aniline carrying a heterocycle . pyrazines are important pharmacophores present in a number of biologically active compounds such as antimycobacterial , antibacterial , antidiabetic , and hypnotic agents . to further explore previous structure  activity relationship , we planned to substitute the five - membered imidazole ring with a pyrazine ring . hereto , regioselective functionalization of the pyrazine started from commercial pyrazine-2,3-dicarboxylic acid ( p23da ) and a four - step synthesis of the target compounds has been described in scheme 2 . p23da was converted to its corresponding anhydride 17 by treatment with acetic anhydride at room temperature . reaction with p - fluoroaniline or p - toluidine resulted in opening of the five - membered ring with concomitant formation of the first amide bond ( 18a , b ) . reaction with trifluoroacetic anhydride at 0  c afforded the anhydric activated product 19a or 19b allowing introduction of the second amide . hereto , the intermediate was reacted with preformed anilines or benzyl amines 14a  m to give the target compounds 20a  n in 6080% overall yields . all final products were purified by silica gel chromatography and characterized by h nmr , c nmr and hrms before evaluation of their inhibitory properties against denv and yfv . as shown in fig .  3 and schemes 1 and 2 , four main structural domains were chosen for optimization of hit 15a : the aromatic moiety ( ring a ) , the central pyrazine / imidazole core ( ring b ) , the substituted aromatic moiety ( ring c ) and the heterocyclic group ( ring d ) . these efforts have provided a few compounds that exhibited an improved biological profile with respect to the initial lead compound 15a . overall , improvement or reduction of the inhibitory activities for either denv or yfv is mostly running in parallel . the overall structure of 15a comprises an imidazole core carrying two aniline moieties coupled via an amide bond , with one of the aniline rings substituted with an additional heterocyclic ring ( ring d ) . therefore , our efforts to understand the structure  activity relationship of the initial lead started with modifying the heterocyclic moiety as shown in table  1 . using p - fluoroaniline ( ring a ) and the i45dc core as a fixed fragment , substitution of the five - membered pyrazole moiety ( ring d ) with a thienyl or furanyl ring mostly led to considerable loss of activity ( i.e. , compare 15h , 15i , 15j versus 15a ) . however , activity remarkably was restored upon substitution of a methyl group for the para - fluorine in ring a ( 15k , 15l ) . the benzylamine substitution for ring a likewise annihilated the inhibitory properties ( 15 m ) , while in contrast a p - fluoro - phenylethylamine again displayed nice activity ( 15n ) . exchanging aniline ring c for a benzylamine preserved the activity when the supplementary heterocycle d was attached at the ortho position ( 15c , 15d ) , while para substitution led to considerable loss of activity ( 15e  g ) within this context . a single attempt with an aminopyridine ring at site c proved inactive ( 15o ) . overall , the compounds 15b ( ec50  =  1.85  m , yfv ) , 15c ( ec50  =  1.93  m , denv ) , 15d ( ec50  =  2.61  m , yfv ) and 15k ( ec50  =  2.02  m , denv ) are endowed with the best inhibitory properties within this imidazole series . with these results in mind , we hoped to further improve the potency via replacement of the central five - membered imidazole ( ring b ) with a pyrazine scaffold , both rings containing two nitrogen atoms but providing a slightly different orientation for the aniline substituents . focusing on compound 15c as most potent representative for inhibition of denv ( ec50  =  1.92  m ) , we synthesized a series of pyrazine analogs with a pyridine substituent as the heterocyclic moiety d , attached at different positions of ring c. the inhibitory potency of these compounds was likewise examined against denv and yfv and is summarized in table  2 ( compounds 20a20n ) . unfortunately , the compound 20f corresponding to 15c , exhibited 20-fold decrease in activity against denv ( ec50  =  40.70  m ) and 6-fold against yfv ( ec50  =  21.29  m ) . concentrating on anti - denv activity , we noticed within the imidazole series that the para attachment of the heterocycle to ring c proved deleterious for the biological activity ( see 15e  g ) . in contrast with these results , the para - pyrimidin-4-yl substituent on ring c proved advantageous within the pyrazine series and led to compound 20a displaying a 2-fold increase in anti - denv potency with ec50 of 0.93  m . a distinction however can be made having either a benzylamine ( n  =  1 ) or an aniline ( n  =  0 ) aromatic ring c. within the benzylamine series , the heterocycle is preferred at the para position with preference for concomitant presence of fluoroaniline at the a - site ( 20a , 20c ) over toluidine ( 20h , 20j ) . however , ortho attachment of the heterocycle on the benzylamine ring seriously reduced the activity ( 20f , 20 m ) . having an aniline ring at site c the results were less clear with mostly intermediate inhibitory properties ( 525  m ) , except for the strongly inhibiting compound 20b and the non - active analog 20e . within this pyrazine series ( 20a  n ) the yfv inhibitory properties in general were slightly weaker compared to denv inhibition by these compounds . a series of tetracyclic inhibitors were prepared using i45cd and p23dc scaffolds and which further were part of a structureactivity investigation . the sar results of both series , i45dcs and p23dcs derivatives , as potent anti - dengue and anti - yellow fever virus agents are described above . our investigation led to the discovery of 15b , which is the most potent inhibitor of the series ( ec50  =  1.85  m ) against yfv , but followed closely in potency by the quite deviating structure 15d . the structural analog 15c on the other hand proved most inhibitory for dengue virus ( see fig . in addition , within the pyrazine series both compounds 20a and 20b were found to have potent denv inhibitory properties ( ec50  =  0.93  m ) uncovered to date . key features of these inhibitors include the central bis - amide arrangement which is found to be a metal - chelating component in many drugs . in summary , determination of the antiviral activities shows that i45dc and p23dc are attractive candidate scaffolds for further studies and the interesting activities observed so far , warrant an in depth study of their mechanism of action . all other chemicals were provided by aldrich or acros and were of the highest quality . h and c nmr spectra were determined with a 300  mhz varian gemini apparatus with tetramethylsilane as internal standard for the h nmr spectra ( s  =  singlet , d  =  doublet , dd  =  double doublet , t  =  triplet , br . d  =  broad doublet , m  =  multiplet ) and the solvent signal  dmso - d6 (   =  39.6  ppm ) or cdcl3 (   =  76.9  ppm )  for the c nmr spectra . exact mass measurements were performed with a quadrupole / orthogonal acceleration time - of - flight tandem mass spectrometer ( qtof2 , micromass , manchester , uk ) fitted with a standard electrospray ionization ( esi ) interface . all solvents were carefully dried or bought as such . to a dry round - bottom flask was added 1.0  g of imidazole-4,5-dicarboxylic acid ( 10 , 1  mmol ) and 10  ml of toluene . to this stirred suspension were added 3.0  ml of thionyl chloride ( 6.5  mmol ) and 0.250  ml of dmf ( catalytic ) . the resulting mixture was refluxed for 16  h. after the mixture was cooled in ice bath , the solid product was collected by vacuum filtration , washed with two 20  ml portions of toluene , and dried under vacuum to yield 0.95  g of crude 11 ( yield : 95% ) as a yellow solid . this product was used further without characterization . to a suspension of 11 ( 1.0  mmol ) in 10  ml of dichloromethane was added n , n - diethylaniline ( 2  mmol ) , and corresponding substituted aniline ( 1.7  mmol ) at  10  c . the reaction mixture was stirred at room temperature for 35  h before collecting highly insoluble colored solid 12a the product was washed with portions of 10  ml dcm , cold water , and acetone , respectively . note : addition of excess of aniline results in opening of acyl imidazole bonds affording unwanted products . yield : 85% ; orange solid ; h nmr ( 300  mhz , cdcl3 ) :  10.74 ( s , 2h ) , 9.09 ( s , 2h ) , 7.80 ( d , 4h , j  =  7.8  hz ) , 7.41 ( t , 4h , j  =  8.1  hz ) , 7.17 ( t , 2h , j  =  7.5  hz ) . c nmr ( 75  mhz , cdcl3 ) :  162.3 , 158.1 , 149.2 , 145.2 , 138.7 , 138.2 , 129.0 , 124.5 , 120.1 . yield : 87% ; yellow solid ; h nmr ( 300  mhz , cdcl3 ) :  10.76 ( s , 2h ) , 9.09 ( s , 2h ) , 7.857.81 ( m , 4h ) , 7.25 ( m , 4h , j  =  9.0  hz ) . c nmr ( 75  mhz , cdcl3 ) :  160.3 , 158.1 , 157.2 , 149.0 , 145.0 , 138.6 , 134.8 , 134.8 , 122.1 , 122.0 , 120.7 , 115.5 . hrms calcd for c22h15n6o4 [ m+h ] : 427.1149 ; found : 427.1151 . yield : 89% ; red solid ; h nmr ( 600  mhz , dmso ) :  10.62 ( s , 2h ) , 9.07 ( s , 2h ) , 7.67 ( d , 4h , j  =  8.4  hz ) , 7.20 ( d , 4h , j  =  7.8  hz ) , 2.30 ( s , 6h ) . c nmr ( 125  mhz , dmso ) : 157.8 , 149.2 , 145.2 , 138.6 , 135.9 , 133.5 , 129.4 , 120.4 , 120.0 , 20.6 . yield : 74% ; red solid ; h nmr ( 300  mhz , dmso ) : 10.01 ( s , 2h ) , 8.97 ( s , 2h ) , 7.797.63 ( m , 6h ) , 7.357.23 ( m , 6h ) , 2.37 ( s , 6h , 2ch3 ) . hrms calcd for c32h23n6o4s2 [ m+h ] : 619.1237 ; found : 619.1238 . in view of insufficient solubility c nmr yield : 72% ; yellow solid ; h nmr ( 300  mhz , dmso ) : 10.46 ( s , 2h ) , 9.07 ( s , 2h ) , 7.817.61 ( m , 6h ) , 7.256.63 ( m , 6h ) , 2.38 ( s , 6h , 2ch3 ) . hrms calcd for c32h23n6o6 [ m+h ] : 587.1673 ; found : 587.1672 . in view of insufficient solubility c nmr to a solution of corresponding aniline / benzyl amine ( 1.0  mmol ) in 10  ml dioxane : water ( 1:1 ) , was added anhydrous k2co3 ( 1.5  mmol ) , aryl boronic acids ( 1.2  mmol ) and pd(tpp)2cl2 ( 0.025  mmol ) in a seal tube . the mixture was purged with argon for 30  min at rt and heated at 100  c for 1  h in microwave . after completion , the solvent was evaporated under reduced pressure and the residue was purified by column chromatography on a silica gel to give the desired product . yield : 52% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  7.50 ( d , 1h , j  =  7.2  hz ) , 7.13 ( t , 1h , j  =  7.5  hz ) , 6.796.75 ( m , 2h ) , 6.39 ( s , 1h ) , 2.37 ( s , 3h , ch3 ) . yield : 62% ; yellow oil ; h nmr ( 300  mhz , cdcl3 ) :  8.70 ( s , 1h ) , 8.618.59 ( m , 1h ) , 7.837.80 ( m , 1h),7.407.35 ( m , 1h ) , 7.057.01 ( m , 1h ) , 6.94 ( m , 1h ) , 6.736.71 ( d , 1h , j  =  8.1  hz ) , 3.60 ( bs , 2h , nh2 ) , 2.30 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  149.2 , 147.4 , 140.3 , 135.7 , 130.0 , 129.0 , 127.3 , 122.6 , 115.2 , 19.5 . yield : 59% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  8.698.67 ( m , 2h ) , 7.447.42 ( m , 2h ) , 7.066.96 ( m , 2h ) , 6.72 ( d , 1h , j  =  8.1  hz ) , 3.69 ( bs , 2h , nh ) , 2.30 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  150.4 , 130.6 , 130.5 , 124.0 , 116.4 , 20.5 . yield : 72% ; yellow solid ; h nmr ( 300  mhz , cdcl3 ) :  8.73 ( s , 1h ) , 8.628.60 ( m , 1h ) , 7.847.80 ( m , 1h ) , 7.417.36 ( m , 1h ) , 7.247.11 ( m , 2h ) , 6.896.79 ( m , 2h ) , 3.75 ( bs , 2h , nh2 ) . c nmr ( 75  mhz , cdcl3 ) :  150.2 , 148.6 , 143.9 , 136.6 , 135.4 , 130.7 , 129.5 , 123.6 , 119.0 , 116.0 . yield : 69% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  8.698.67 ( m , 2h ) , 7.447.28 ( m , 2h ) , 7.257.13 ( m , 2h ) , 6.896.78 ( m , 2h ) , 3.82 ( bs , 2h , nh2 ) . c nmr ( 75  mhz , cdcl3 ) :  149.4 , 129.2 , 128.8 , 123.0 , 118.0 , 115.2 . yield : 55% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  8.86 ( m , 2h ) , 8.618.59 ( m , 1h ) , 7.917.87 ( m , 1h ) , 7.607.57 ( m , 2h ) , 7.477.40 ( m , 2h ) , 7.387.36 ( m , 1h ) , 3.96 ( s , 2h , ch2 ) , 1.62 ( bs , 2h , nh2 ) . c nmr ( 75  mhz , cdcl3 ) :  149.9 , 127.5 , 126.8 , 121.1 , 45.7 . yield : 59% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  8.648.62 ( m , 2h ) , 7.617.41 ( m , 6h ) , 3.92 ( s , 2h , ch2 ) , 1.77 ( bs , 2h , nh2 ) . c nmr ( 75  mhz , cdcl3 ) :  149.2 , 126.9 , 126.2 , 120.5 , 45.1 . yield : 65% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  8.768.74 ( m , 1h ) , 7.567.10 ( m , 8h ) , 3.92 ( s , 2h , ch2 ) . c nmr ( 75  mhz , cdcl3 ) :  149.1 , 132.0 , 131.3 , 131.1 , 131.1 , 128.2 , 127.7 , 127.6 , 127.5 , 126.9 , 122.7 , 120.5 , 46.0 . yield : 62% ; off - white solid ; h nmr ( 300  mhz , cdcl3 ) :  7.477.40 ( m , 2h ) , 7.337.31 ( m , 1h ) , 7.09 ( s , 1h ) , 7.037.00 ( m , 1h ) , 6.756.72 ( d , 1h , j  =  8.1  hz ) , 3.73 ( bs , 2h , nh2 ) , 2.33 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  141.5 , 140.1 , 130.7 , 129.1 , 128.5 , 127.8 , 126.0 , 122.6 , 122.5 , 116.0 , 20.5 . hrms calcd for c11h12n1s1 [ m+h ] : 190.0685 ; found : 190.0686 . yield : 58% ; off - white solid ; h nmr ( 300  mhz , cdcl3 ) :  7.617.58 ( m , 2h ) , 7.477.28 ( m , 6h ) , 3.92 ( s , 2h , ch2 ) . c nmr ( 75  mhz , cdcl3 ) :  127.7 , 126.8 , 126.5 , 126.3 , 120.2 , 46.3 . yield : 52% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  7.587.12 ( m , 6h ) , 3.91 ( s , 2h , ch2 ) . c nmr ( 150  mhz , cdcl3 ) :  162.1 , 160.8 , 160.5 , 142.0 , 141.1 , 135.8 , 132.7 , 130.1 , 129.8 , 129.3 , 129.0 , 128.8 , 128.6 , 128.4 , 128.2 , 127.7 , 127.5 , 127.4 , 127.1 , 127.0 , 126.8 , 125.5 , 125.0 , 123.4 , 122.7 , 46.8 , 44.2 . yield : 63% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  7.52 ( s , 1h ) , 7.32 ( s , 1h ) , 6.976.94 ( m , 1h ) , 6.716.52 ( m , 3h ) , 2.30 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  152.5 , 141.2 , 140.8 , 129.5 , 127.8 , 127.6 , 116.9 , 116.2 , 111.3 , 106.3 , 20.4 . hrms calcd for c11h12n1o1 [ m+h ] : 174.0913 ; found : 174.0910 . yield : 57% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  7.84 ( s , 1h ) , 7.28 ( s , 1h ) , 6.99 ( d , j  =  3.3  hz , 1h ) , 6.736.72 ( m , 1h ) , 4.96 ( bs , 2h , nh2 ) , 2.49 ( s , 3h , ch3 ) , 2.18 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  153.6 , 146.5 , 139.8 , 138.5 , 137.1 , 125.6 , 125.5 , 122.7 , 114.5 , 17.0 , 14.9 . to a stirred solution of 12a e ( 1.0  mmol ) in 10  ml dcm , was added dipea ( 3.0  mmol ) , and 14a  m ( 3.0  mmol ) under argon atmosphere at room temperature . the mixture was allowed to reflux for 1648  h. two alternative workup procedures were followed depending on whether the product precipitated out of solution or not . workup 1 ( for soluble products ) : the dcm was evaporated using a rotavapor . the product was purified by flash chromatography on silica gel . workup 2 ( for insoluble products ) : the precipitate was collected by filtration using a buchner funnel and washed on the frit with dcm ( 2025  ml ) until the yellow impurities had disappeared . yield : 86% ; h nmr ( 500  mhz , dmso ) :  13.69 ( bs , 1h , nh ) , 13.30 ( bs , 1h , nh ) , 13.05 ( bs , 1h , nh ) , 12.78 ( bs , 1h , nh ) , 8.65 ( d , 1h , j  =  8.0  hz ) , 7.96 ( s , 1h ) , 7.787.23 ( m , 9h ) , 6.54 ( s , 1h ) , 2.31 ( s , 3h , ch3 ) . c nmr ( 125  mhz , dmso ) :  163.3 , 159.6 , 158.0 , 156.4 , 150.3 , 140.2 , 137.3 , 135.2 , 135.0 , 133.9 , 130.0 , 128.7 , 124.8 , 123.2 , 121.9 , 121.6 , 116.4 , 116.2 , 103.1 , 10.8 . yield : 83% ; h nmr ( 600  mhz , dmso ) :  8.76 ( d , 0.5h , j  =  6.6  hz ) , 7.967.17 ( m , 10h ) , 4.57 ( d , 2h , j  =  6.0  hz , ch2 ) . c nmr ( 150  mhz , dmso ) :  159.5 , 157.9 , 150.0 , 137.4 , 127.1 , 134.8 , 132.7 , 132.4 , 131.8 , 131.7 , 129.3 , 129.1 , 129.0 , 128.7 , 128.1 , 122.4 , 122.0 , 116.0 , 115.8 , 43.1 . yield : 86% ; h nmr ( 600  mhz , dmso ) :  13.53 ( bs , 0.5h , nh ) , 13.45 ( bs , 0.5h , nh ) , 9.40 ( bs , 0.5h , nh ) , 9.23 ( bs , 0.5h , nh ) , 7.997.18 ( m , 12h ) , 4.58 ( d , 2h , j  =  6.6  hz , ch2 ) . c nmr ( 150  mhz , dmso ) :  164.3 , 156.1 , 140.5 , 137.3 , 137.0 , 132.7 , 132.5 , 129.9 , 129.0 , 128.0 , 127.6 , 127.1 , 126.3 , 123.7 , 121.1 , 115.9 , 115.7 , 42.9 . hrms calcd for c22h16f1n4o2s1 [ mh ] : 419.0973 ; found : 419.0967 . yield : 76% ; h nmr ( 600  mhz , dmso ) :  13.55 ( s , 1h , nh ) , 13.49 ( bs , 1h , nh ) , 10.49 ( s , 1h ) , 10.06 ( s , 1h ) , 9.47 ( s , 1h ) , 8.857.20 ( m , 9h ) , 4.59 ( d , 2h , j  =  6.6  hz , ch2 ) . c nmr ( 150  mhz , dmso ) :  164.2 , 156.4 , 148.6 , 147.7 , 139.3 , 137.2 , 136.0 , 135.6 , 135.1 , 134.4 , 133.0 , 128.9 , 128.4 , 128.3 , 127.3 , 127.2 , 124.2 , 121.4 , 121.4 , 116.1 , 116.9 , 42.3 . hrms calcd for c23h17f1n5o2 [ mh ] : 414.1372 ; found : 414.1372 . yield : 78% ; h nmr ( 300  mhz , dmso ) :  9.84 ( bs , 1h , nh ) , 7.97 ( s , 1h ) , 7.877.19 ( m , 15h ) , 4.57 ( d , 2h , j  =  6.3  hz , ch2 ) . c nmr ( 75  mhz , dmso ) :  160.1 , 157.0 , 141.4 , 141.4 , 138.0 , 137.0 , 134.9 , 134.0 , 128.1 , 128.0 , 127.6 , 127.1 , 126.3 , 126.0 , 121.7 , 121.6 , 120.9 , 120.7 , 115.9 , 115.6 , 42.2 . yield : 81% ; h nmr ( 600  mhz , dmso )  13.56 ( s , 1h , nh ) , 13.47 ( bs , 1h , nh ) , 9.50 ( s , 1h , nh ) , 8.617.20 ( m , 13h ) , 4.59 ( d , 2h , j  =  6.0  hz , ch2 ) . c nmr ( 150  mhz , dmso ) :  164.2 , 156.2 , 150.5 , 150.3 , 146.9 , 140.4 , 137.1 , 135.9 , 135.0 , 133.0 , 128.8 , 128.3 , 128.2 , 127.1 , 127.0 , 121.3 , 121.2 , 116.0 , 115.9 , 42.2 . hrms calcd for c23h17f1n5o2 [ mh ] : 414.1372 ; found : 414.1369 . yield : 89% ; h nmr ( 500  mhz , dmso ) :  13.64 ( s , 0.7h , nh ) , 13.56 ( s , 0.3h , nh ) , 13.22 ( s , 0.7h , nh ) , 12.44 ( s , 0.3h , nh ) , 10.46 ( s , 0.3h , nh ) , 10.12 ( s , 0.7h , nh ) , 7.997.15 ( m , 13h ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 125  mhz , dmso ) :  162.5 , 156.0 , 138.3 , 137.1 , 136.7 , 135.2 , 134.9 , 132.9 , 131.5 , 130.7 , 130.6 , 130.0 , 129.2 , 128.6 , 128.6 , 128.4 , 128.4 , 127.0 , 126.2 , 124.9 , 124.2 , 124.0 , 122.9 , 199.9 , 121.2 , 121.2 , 116.0 , 115.8 , 115.2 , 115.1 , 20.6 . yield : 77% ; h nmr ( 300  mhz , dmso ) :  13.69 ( bs , 1h , nh ) , 13.20 ( bs , 1h , nh ) , 10.60 ( s , 1h , nh ) , 8.057.55 ( m , 6h ) , 7.276.56 ( m , 5h ) , 2.38 ( s , 3h , ch3 ) . yield : 81% ; h nmr ( 600  mhz , dmso ) :  13.63 ( s , 0.7h , nh ) , 13.56 ( s , 0.3h , nh ) , 13.18 ( s , 0.7h , nh ) , 12.47 ( s , 0.3h , nh ) , 10.34 ( s , 0.3h , nh ) , 10.12 ( s , 0.7h , nh ) , 7.967.12 ( m , 12h ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 125  mhz , dmso ) :  162.4 , 156.0 , 138.5 , 138.3 , 137.1 , 135.2 , 132.8 , 131.5 , 130.6 , 129.9 , 129.3 , 128.6 , 128.4 , 127.0 , 124.2 , 124.0 , 120.9 , 119.4 , 20.6 . yield : 79% ; h nmr ( 600  mhz , dmso ) :  13.59 ( s , 0.7h , nh ) , 13.54 ( s , 0.3h , nh ) , 13.09 ( s , 0.3h , nh ) , 12.52 ( s , 0.7h , nh ) , 10.26 ( s , 0.3h , nh ) , 10.10 ( s , 0.7h , nh ) , 7.987.18 ( m , 10h ) , 2.36 ( s , 3h , ch3 ) , 2.28 ( s , 3h , ch3 ) . c nmr ( 125  mhz , dmso ) :  162.4 , 155.8 , 138.3 , 137.0 , 136.0 , 135.1 , 133.1 , 132.7 , 131.5 , 130.6 , 129.9 , 129.6 , 129.4 , 129.0 , 128.6 , 128.5 , 128.4 , 128.4 , 127.0 , 124.2 , 124.0 , 120.9 , 119.4 , 20.6 . hrms calcd for c23h21n4o2s1 [ m+h ] : 417.1380 ; found : 417.1377 . yield : 76% ; 13.58 ( s , 0.7h , nh ) , 13.52 ( s , 0.3h , nh ) , 13.07 ( s , 0.3h , nh ) , 12.50 ( s , 0.7h , nh ) , 10.23 ( s , 0.3h , nh ) , 10.08 ( s , 0.7h , nh ) , 7.977.93 ( m , 2h ) , 7.717.56 ( m , 4h ) , 7.337.11 ( m , 5h ) , 2.35 ( s , 3h , ch3 ) , 2.28 ( s , 3h , ch3 ) . c nmr ( 125  mhz , dmso ) :  162.4 , 155.8 , 138.3 , 136.9 , 136.0 , 135.1 , 133.1 , 132.7 , 131.5 , 130.6 , 129.8 , 129.6 , 129.4 , 129.0 , 128.6 , 128.5 , 128.4 , 128.4 , 127.0 , 124.2 , 124.0 , 120.9 , 119.4 , 20.6 , 20.6 . hrms calcd for c23h21n4o3 [ m+h ] : 401.1608 ; found : 401.1602 . yield : 83% ; h nmr ( 300  mhz , cdcl3 ) :  11.84 ( bs , 1h , nh ) , 11.57 ( bs , 1h , nh ) , 10.32 ( bs , 1h , nh ) , 8.16 ( d , 1h , j  =  8.4  hz ) , 7.617.18 ( m , 10h ) , 6.74 ( d , 1h , j  =  3.3  hz ) , 6.576.55 ( m , 1h ) , 4.70 ( d , 2h , j  =  6.0  hz , ch2 ) , 2.40 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  161.7 , 159.0 , 142.4 , 134.9 , 134.8 , 130.5 , 129.1 , 128.6 , 127.9 , 127.4 , 127.2 , 123.0 , 111.7 , 108.5 , 43.5 , 21.0 . yield : 75% ; h nmr ( 300  mhz , cdcl3 ) :  11.13 ( bs , 1h , nh ) , 10.32 ( bs , 1h , nh ) , 8.18 ( d , 1h , j  =  8.7  hz ) , 7.676.55 ( m , 10h ) , 3.73 ( m , 2h , ch2 ) , 3.00 ( t , 2h , j  =  7.5  hz ch2 ) , 2.42 ( s , 3h , ch3 ) . c nmr ( 150  mhz , dmso ) :  161.5 , 157.9 , 157.1 , 150.6 , 150.3 , 143.1 , 142.8 , 142.3 , 142.2 , 138.4 , 137.4 , 136.8 , 136.5 , 135.7 , 135.0 , 133.3 , 132.6 , 130.4 , 130.2 , 129.0 , 128.7 , 128.3 , 127.6 , 127.3 , 127.1 , 125.5 , 125.0 , 124.5 , 123.8 , 118.0 , 115.7 , 115.4 , 113.1 , 112.8 , 112.1 , 109.1 , 108.9 , 77.1 , 60.9 , 34.81 . hrms calcd for c24h22fn4o3 [ m+h ] : 433.1670 ; found : 433.1668 . yield : 76% ; h nmr ( 600  mhz , cdcl3 ) :  13.63 ( bs , 1h , nh ) , 13.08 ( bs , 0.7h , nh ) , 12.92 ( bs , 0.3h , nh ) , 10.70 ( bs , 0.7h , nh ) , 10.54 ( bs , 0.3h , nh ) , 8.317.08 ( m , 8h ) , 6.796.76 ( m , 1h ) , 2.39 ( s , 3h , ch3 ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 150  mhz , cdcl3 ) :  184.6 , 149.5 , 147.4 , 144.6 , 143.7 , 141.2 , 138.3 , 138.2 , 137.4 , 137.3 , 135.6 , 132.9 , 129.3 , 129.2 , 129.1 , 128.5 , 127.1 , 126.8 , 126.3 , 128.5 , 17.5 , 15.1 . hrms calcd for c22h19n5o2s1 [ mh ] : 416.1187 ; found : 416.1187 . sodium dodecyl sulfate ( sds , 6  mmol ) was added to a stirred heterogeneous suspension of amine ( 5  mmol ) in water ( 20  ml ) until a homogeneous solution was formed , ( in case of turbidity , the mixture was warmed to obtain a clear solution ) . anhydride 17 ( 5  mmol ) dissolved in acetonitrile ( 5  ml ) was added to this solution in one lot . after stirring for 1  h at room temperature , the acetonitrile was evaporated and the product precipitated from the aqueous layer . to the aqueous solution containing precipitate , solid sodium hydrogen carbonate was added pinch - wise until the effervescence ceased and the ph was 6.0 . the remaining precipitated product was filtered , washed with water ( 20  ml ) , dried in a vacuum desiccator . in cases where the product did not precipitate , the reaction mixture was extracted with ethyl acetate ( 2    25  ml ) . the combined organic extracts were dried with anhydrous na2so4 and the solvent was removed in a rotary evaporator under reduced pressure to yield the pure product . yield : 72% ; white solid ; h nmr ( 300  mhz , dmso ) :  10.86 ( bs , 1h ) , 8.90 ( s , 2h ) , 7.837.78 ( m , 2h ) , 7.22 ( t , 2h , j  =  9.0  hz ) . yield : 65% ; white solid ; h nmr ( 300  mhz , dmso ) :  10.68 ( bs , 1h ) , 8.88 ( s , 2h ) , 7.67 ( d , j  =  8.4  hz , 2h ) , 7.17 ( t , j  =  8.4  hz , 2h ) , 2.29 ( s , 3h , ch3 ) . c nmr ( 75  mhz , dmso ) :  166.5 , 162.3 , 146.5 , 145.8 , 145.6 , 144.6 , 136.0 , 133.4 , 129.3 , 120.2 , 20.7 . trifluoroacetic anhydride ( 1.5  equiv ) was added dropwise to a stirring solution of acid 18a  b ( 1  equiv ) and et3n ( tea , 3  equiv ) in 1,4-dioxane ( 20 ml ) that was kept at 0  c with an ice bath . after 15  min the yellow solution was allowed to warm to room temperature and was stirred for 30  min , then it was poured in cold h2o ( 100  ml ) . a precipitate formed , which was collected by filtration using a buchner funnel and washed with h2o . yield : 70% ; off - white solid ; h nmr ( 300  mhz , cdcl3 ) :  9.06 ( d , 2h , j  =  5.1  hz ) , 7.737.69 ( m , 2h ) , 7.15 ( t , j  =  8.4  hz , 2h ) . c nmr ( 75  mhz , cdcl3 ) :  150.9 , 149.5 , 128.7 , 128.6 , 116.2 , 116.0 . yield : 66% ; white solid ; h nmr ( 300  mhz , cdcl3 ) :  9.079.03 ( m , 2h ) , 7.697.28 ( m , 4h ) , 2.43 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  150.5 , 149.0 , 129.5 , 126.4 , 21.0 . phthalisoimide 19a  b ( 1  equiv ) was added to stirring solution of the aniline / benzylamine 14a  m ( 1.2  equiv ) in thf . the mixture was diluted with etoac ( triple the volume of thf ) and washed three times with 1  m hcl . yield : 76% ; h nmr ( 300  mhz , dmso ) :  10.65 ( bs , 1h , nh ) , 9.45 ( t , j  =  6.6  hz , 1h , nh ) , 8.88 ( q , 2h , j  =  2.4  hz ) , 8.648.61 ( m , 2h ) , 7.787.69 ( m , 6h ) , 7.50 ( d , j  =  8.4  hz , 2h ) , 7.21 ( t , j  =  9.0  hz , 2h ) , 2.39 ( d , j  =  6.3  hz , 2h , ch2 ) . c nmr ( 75  mhz , dmso ) :  164.0 , 163.7 , 160.1 , 156.9 , 150.3 , 148.3 , 146.9 , 145.4 , 145.3 , 144.5 , 140.5 , 135.8 , 135.5 , 128.3 , 126.9 , 121.7 , 121.6 , 121.2 , 115.7 , 115.4 , 42.2 . yield : 74% ; h nmr ( 300  mhz , cdcl3 ) :  9.02 ( bs , 1h , nh ) , 8.698.54 ( m , 5h ) , 8.27 ( d , j  =  8.4  hz , 1h ) , 7.84 ( d , j  =  8.1  hz , 1h ) , 7.707.66 ( m , 2h ) , 7.367.26 ( m , 2h ) , 7.117.03 ( m , 3h ) , 2.39 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  161.8 , 161.5 , 150.1 , 149.1 , 144.3 , 137.3 , 135.7 , 134.2 , 131.8 , 131.0 , 130.1 , 123.7 , 123.2 , 122.2 , 122.1 , 116.1 , 115.8 , 21.1 . hrms calcd for c24h19f1n5o2 [ m+h ] : 428.1517 ; found : 428.1514 . yield : 72% ; h nmr ( 300  mhz , cdcl3 ) :  9.33 ( bs , 1h , nh ) , 8.76 ( bs , 1h , nh ) , 8.63 ( s , 2h ) , 8.548.52 ( d , j  =  4.8  hz , 1h ) , 7.867.27 ( m , 9h ) , 7.05 ( m , j  =  8.7  hz , 2h ) , 4.76 ( d , j  =  6.0  hz , 2h , ch2 ) . c nmr ( 75  mhz , cdcl3 ) : 164.9 , 161.6 , 161.5 , 148.6 , 148.3 , 147.6 , 146.1 , 144.6 , 144.0 , 137.8 , 137.3 , 136.3 , 134.4 , 133.6 , 128.8 , 127.6 , 123.7 , 122.1 , 122.0 , 116.0 , 115.7 , 43.7 . hrms calcd for c24h19f1n5o2 [ m+h ] : 428.1517 ; found : 428.1517 . yield : 66% ; h nmr ( 300  mhz , cdcl3 ) :  10.72 ( bs , 1h , nh ) , 10.27 ( bs , 1h , nh ) , 8.908.86 ( m , 2h ) , 8.588.56 ( m , 2h ) , 7.857.76 ( m , 3h ) , 7.557.37 ( m , 5h ) , 7.22 ( t , j  =  9.0  hz , 2h ) . c nmr ( 75  mhz , cdcl3 ) :  163.2 , 163.1 , 149.8 , 147.0 , 146.2 , 145.1 , 144.9 , 135.2 , 134.4 , 133.6 , 130.3 , 129.3 , 126.6 , 126.2 , 124.0 , 121.9 , 121.8 , 115.7 , 115.4 , 79.3 . hrms calcd for c23h17f1n5o2 [ m+h ] : 414.1361 ; found : 414.1363 . yield : 76% ; h nmr ( 300  mhz , cdcl3 ) :  9.04 ( bs , 1h , nh ) , 8.78 ( bs , 1h , nh ) , 8.708.56 ( m , 4h ) , 8.45 ( d , j  =  8.1  hz , 1h ) , 7.87 ( d , j  =  7.8  hz , 1h ) , 7.727.69 ( m , 2h ) , 7.527.7.28 ( m , 4h ) , 7.07 ( t , j  =  8.7  hz , 2h ) . c nmr ( 75  mhz , cdcl3 ) :  161.7 , 161.5 , 150.1 , 149.2 , 144.3 , 137.3 , 134.4 , 134.0 , 133.5 , 130.5 , 129.9 , 129.5 , 125.8 , 123.7 , 123.1 , 122.2 , 122.1 , 116.1 , 115.8 . hrms calcd for c23h17f1n5o2 [ m+h ] : 414.1361 ; found : 414.1365 . yield : 68% ; h nmr ( 600  mhz , dmso ) :  10.67 ( bs , 1h , nh ) , 9.41 ( t , j  =  6.0  hz , 1h ) , 8.918.89 ( m , 2h ) , 7.757.19 ( m , 8h ) , 4.51 ( d , j  =  6.0  hz , 2h ) . c nmr ( 125  mhz , dmso ) :  164.3 , 163.6 , 159.2 , 157.6 , 150.6 , 149.0 , 148.1 , 145.3 , 144.5 , 137.4 , 135.3 , 132.4 , 129.0 , 128.5 , 127.8 , 122.1 , 121.5 , 115.5 , 115.4 , 42.9 . yield : 76% ; h nmr ( 300  mhz , cdcl3 ) :  8.95 ( bs , 1h , nh ) , 8.668.58 ( m , 5h ) , 8.32 ( d , j  =  8.4  hz , 1h ) , 7.727.67 ( m , 2h ) , 7.43 ( d , j  =  8.4  hz , 2h ) , 7.30 ( d , j  =  8.4  hz , 2h ) , 7.137.7.04 ( m , 3h ) , 2.40 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  150.0 , 145.9 , 144.0 , 143.8 , 130.9 , 130.0 , 129.9 , 124.0 , 122.6 , 121.7 , 121.6 , 115.6 , 115.3 , 20.6 . yield : 78% ; h nmr ( 300  mhz , cdcl3 ) :  9.19 ( bs , 1h , nh ) , 8.668.62 ( m , 4h ) , 7.647.47 ( m , 8h ) , 7.17 ( d , j  =  8.1  hz , 3h ) , 4.78 ( d , j  =  6.0  hz , 2h , ch2 ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  165.1 , 161.2 , 150.4 , 148.1 , 148.0 , 145.8 , 144.8 , 143.8 , 139.0 , 137.6 , 134.9 , 134.8 , 129.7 , 128.8 , 127.5 , 121.7 , 120.3 , 43.7 , 21.1 . hrms calcd for c25h22n5o2 [ m+h ] : 424.1768 ; found : 424.1765 . yield : 76% ; h nmr ( 300  mhz , cdcl3 ) :  8.95 ( bs , 1h , nh ) , 8.728.56 ( m , 5h ) , 8.33 ( d , j  =  8.1  hz , 1h ) , 7.89 ( d , j  =  7.5  hz , 1h ) , 7.62 ( d , j  =  8.1  hz , 2h ) , 7.387.14 ( m , 4h ) , 2.42 ( s , 3h , ch3 ) , 2.37 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  150.2 , 149.2 , 144.5 , 144.1 , 137.3 , 135.5 , 134.8 , 134.2 , 131.9 , 131.0 , 130.1 , 123.6 , 123.3 , 120.3 , 21.1 . hrms calcd for c25h22n5o2 [ m+h ] : 424.1768 ; found : 424.1767 . yield : 71% ; h nmr ( 300  mhz , cdcl3 ) :  9.24 ( bs , 1h , nh ) , 8.788.54 ( m , 4h ) , 7.887.84 ( m , 1h ) , 7.63 ( d , j  =  8.4  hz , 2h ) , 7.567.17 ( m , 8h ) , 4.79 ( d , j  =  6.0  hz , 2h , ch2 ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  165.2 , 161.2 , 148.6 , 148.3 , 148.1 , 145.7 , 144.6 , 143.8 , 137.9 , 137.2 , 136.4 , 134.9 , 134.7 , 134.4 , 129.7 , 128.9 , 127.6 , 123.7 , 120.3 , 43.7 , 21.1 . yield : 72% ; h nmr ( 300  mhz , cdcl3 ) :  8.95 ( bs , 1h , nh ) , 8.688.51 ( m , 6h ) , 7.63 ( d , j  =  8.4  hz , 2h ) , 7.507.19 ( m , 7h ) , 2.37 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  150.5 , 146.2 , 144.6 , 144.1 , 134.9 , 134.1 , 129.9 , 129.8 , 125.7 , 124.5 , 123.0 , 120.3 , 21.1 . hrms calcd for c24h20n5o2 [ m+h ] : 410.1611 ; found : 410.1608 . yield : 68% ; h nmr ( 300  mhz , cdcl3 ) :  8.70 ( bs , 1h , nh ) , 8.708.47 ( m , 6h ) , 7.90 ( d , j  =  7.8  hz , 2h ) , 7.637.18 ( m , 8h ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  162.0 , 160.1 , 149.6 , 148.5 , 144.0 , 143.7 , 137.0 , 123.2 , 134.0 , 133.6 , 120.0 , 129.2 , 129.1 , 125.2 , 123.2 , 122.8 , 119.8 , 20.6 . h nmr ( 300  mhz , cdcl3 ) :  9.13 ( bs , 1h , nh ) , 8.778.66 ( m , 3h ) , 7.717.16 ( m , 11h ) , 4.82 ( s , 2h , ch2 ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  161.3 , 150.8 , 144.6 , 143.9 , 136.9 , 132.9 , 130.5 , 129.7 , 129.4 , 128.7 , 128.6 , 128.0 , 121.6 , 120.3 , 44.3 , 21.1 . hrms calcd for c25h22n5o2 [ m+h ] : 424.1768 ; found : 424.1768 . yield : 73% ; h nmr ( 300  mhz , cdcl3 ) :  8.94 ( bs , 1h , nh ) , 8.688.62 ( m , 4h ) , 8.49 ( bs , 1h , nh ) , 8.37 ( d , j  =  8.4  hz , 1h ) , 7.63 ( d , j  =  8.4  hz , 2h ) , 7.487.14 ( m , 6h ) , 2.42 ( s , 3h , ch3 ) , 2.37 ( s , 3h , ch3 ) . c nmr ( 75  mhz , cdcl3 ) :  161.3 , 150.5 , 146.4 , 144.5 , 144.1 , 134.8 , 131.5 , 130.4 , 129.8 , 124.5 , 123.2 , 120.3 , 21.1 . hrms calcd for c25h22n5o2 [ m+h ] : 424.1768 ; found : 424.1770 . green monkey kidney cells [ vero - b cells as obtained from the european collection of cell cultures ( ecacc ) ] were grown in minimum essential medium ( mem ; gibco , merelbeke , belgium ) supplemented with 10% fetal calf serum ( fcs ) , 1% l - glutamine and 1% sodium bicarbonate . vero - b cells were seeded at a density 7    10  cells / well in 100  l assay medium and allowed to adhere overnight . antiviral assays were performed in medium supplemented with 2% fcs , 1% l - glutamine and 1% sodium bicarbonate . after washing cells twice with 2% fcs medium , serial compound dilutions ( 1:2 ) were added to each well ( starting concentration 100  g / ml ) , following by adding 100  l of 2% of phosphate buffered saline ( pbs ) culture medium containing 100  l 50% cell culture infectious doses ( i.e. , ccid50 ) of virus . after 7 days of incubation , 2% fcs culture medium was discarded and cells were fixed with ethanol and stained with 1% methylene blue , and ec50 and cc50 were determinate visually . the 50% effective concentration ( ec50 ) , which is defined as the compound concentration that is required to inhibit the virus - induced cytopathogenic effect ( cpe ) by 50% , and 50% cytotoxic concentration ( cc50 ) , which is defined as the compound concentration that is required to inhibit the cell growth by 50% .
the results of a high - throughput screening assay using the dengue virus-2 replicon showed that the imidazole 4,5-dicarboxamide ( i45dc ) derivative ( 15a ) has a high dengue virus inhibitory activity . based on 15a as a lead compound , 
 a novel class of both disubstituted i45dcs and the resembling pyrazine 2,3-dicarboxamides ( p23dcs ) were synthesized . here 
 , we report on their in  vitro inhibitory activity against dengue virus ( denv ) and yellow fever virus ( yfv ) . 
 some of these first generation compounds have shown activity against both viruses in the micromolar range . within this series , compound 15b was observed to display the highest antiviral potency against yfv with an ec50  =  1.85  m . 
 in addition , compounds 20a and 20b both potently inhibited replication of denv ( ec50  =  0.93  m ) in vero cells .