interferons ( ifn ) are 
 a linchpin of inflammatory signaling , assisting 
 in host defense against pathogens , antigen presentation , and immunomodulation
. 
 there are two main classes of interferons : type i ( ifn-/ ) 
 and type ii ( ifn- ) .
interferons 
 bind to their respective transmembrane receptors , inducing dimerization 
 and regulation of inflammatory gene expression through the jak / stat 
 signaling pathway .
janus kinases ( jaks ) 
 are tyrosine kinases that interact with interferon receptors , resulting 
 in recruitment and phosphorylation of signal transduction and activator 
 of transcription ( stat ) proteins .
the 
 jak / stat association in turn promotes transcription of pro - inflammatory 
 genes including inducible nitric oxide synthase ( inos ) .
interferons coordinate the inflammation 
 response in concert with 
 other innate immune pathways , particularly toll - like receptor ( tlr ) 
 signaling .
tlrs are pattern recognition receptors that respond to 
 infectious markers and induce a pro - inflammatory response .
these two pathways synergistically interact in macrophages to elicit 
 an immune response toward infective threats .
macrophage priming with 
 ifn- improves the inflammatory response to tlr ligands , such 
 as lipopolysaccharide ( lps ) for tlr4 . in turn , tlrs upregulate type 
 i interferons , and nf-b assists in transcription of multiple 
 interferon - inducible genes .
of particular 
 interest , the inos promoter has binding sites for both stat1 and nf-b .
transcription of inos may be activated by multiple 
 inflammatory factors , including lps , type i , and type ii interferons .
stat1 activation by ifn- has an autocrine / paracrine 
 mechanism preceding inos activation and serves as a necessary transcription 
 factor for synthesis .
it has been demonstrated 
 that knockdown of the interferon-/ receptor 1 ( ifnar1 ) 
 mitigates inos expression , even in the presence of lps .
thus , activation of the tlr and interferon interrelated 
 pathways can be regulated at their convergence in jak / stat signaling . 

nonetheless , it has been a significant challenge to regulate individual 
 pathways with high specificity and selectivity .
generally , inflammatory 
 signaling is beneficial and may protect 
 the host against infection .
however , in autoimmune pathologies such 
 as systemic lupus erythematosus and multiple sclerosis , an overabundance 
 of interferon signaling can have deleterious effects .
indeed , it has been noted that among centenarian women , polymorphisms 
 that result in decreased ifn- may contribute to longevity .
additionally , a small molecule modulator of 
 ifn signaling may provide a useful tool in the study and treatment 
 of autoimmune diseases . here , we report a small molecule that is able 
 to inhibit the interferon - induced jak / stat1 signaling pathway without 
 compromising the effectiveness of other components of the innate immune 
 system such as tlrs .
we started our search 
 for anti - inflammatory agents by looking for inhibitors of lps - induced 
 tlr4 activation .
the c2d2 pilot 
 library consists of 2200 drug - like compounds that represent a variety 
 of diverse and commercially available scaffolds .
the initial screen 
 was performed using lps - activated raw 264.7 cells in a 96-well plate 
 format to monitor nitric oxide ( no ) production .
this assay uses a 
 sandmeyer reaction to convert 2,3-diaminonapthalene to fluorescent 
 napthalenetriazole in the presence of no .
as no is produced in the 
 tlr inflammatory response , this readout provides information on the 
 extent of tlr signaling .
initial screening yielded 31 hits , representing 
 5 scaffolds ( figure s1 , supporting information ) .
we selected the scaffold with an isothiazolone 1,1-dioxide core 
 for further development as it produced the most numerous and potent 
 hits .
compounds 1 and 2 ( figure 1 ) represent two of the more potent hits . generic scaffold 
 of hit molecules selected from the screening of 
 the colorado center for drug discovery ( c2d2 ) pilot library . both 
 variable functionalities ( ar = substituted benzene ring , r= various 
 amide moieties ) shown in these representative structures have been 
 subsequently optimized .
we have developed an efficient synthetic route for the generic 
 scaffold in figure 1 .
as an example , the synthesis 
 of the most potent hit molecule , 1 ( scheme 1 ) , began with the oxidative dimerization of commercially available 3 to give dithiodipropionic acid 4 .
conversion to the dithiodipropionyl chloride 
 was completed with thionyl chloride , and subsequent treatment with 
 anhydrous ammonia gave dithiodipropionamide 5 as a mixture 
 of diastereomers .
as previously described 
 by lewis and co - workers , oxidative cyclization was performed with 
 sulfuryl chloride to give an inseparable mixture of 6 and 7 .
this mixture of 
 isothiazolinones was then deprotonated with sodium hydride , and alkylated 
 with benzyl chloride 9 .
the alkylation yields two synthetically 
 useful intermediates 12 and 13 , which are 
 easily separable by column chromatography .
chloro - intermediate 12 was oxidized with meta - chloroperoxybenzoic 
 acid ( m - cpba ) to give the isothiazolone 1,1-dioxide 
 core , 14 .
( i ) i2 , ki , naoh , 
 h2o , ( 4 = 99% ) ; ( ii ) ( 1 ) socl2 , 
 ( 2 ) nh3 , ch2cl2 ( 5 = 
 66% ) ; ( iii ) so2cl2 , etoac ( 6 and 7 = 51% ) ; ( iv ) piperidine or furfurylamine , dipea , ch2cl2 ( 9 = 95% , 10 = 99% ) ; 
 ( v ) boc2o , dmap , thf ( 11 = 81% ) ; ( vi ) ( 1 ) 
 nah , dmf , ( 2 ) 9 ( 12 = 70% , 13 = 67% ) ; ( vii ) m - cpba , ch2cl2 ( 14 = 49% ) ; ( viii ) arb(oh)2 , pd(dppf)cl2ch2cl2 , k2co3 , 1,4-dioxane ( 1 = 47% , 15 = 32% ) ; ( ix ) 
 4-bromopyridinehcl , pd(oac)2 , koac , dma ( 16 = 54% ) ; ( x ) m - cpba , ch2cl2 ( 17 = 84% ) .
chloro - intermediate 14 was coupled to 3,4-dimethylphenylboronic 
 acid , using previously reported conditions to give the parent library 
 hit , 1 .
these conditions failed , however , when 4-pyridinylboronic 
 acid was used . making use of intermediate 13 with heck 
 conditions and
the coupling product 16 was fully oxidized at both 
 sulfur and the pyridyl nitrogen to give 17 .
the synthesis 
 of compounds 15 and 17 provide analogues 
 that exhibit both higher and lower calculated log p values , 4.70 and 1.29 respectively , as compared to 1 ( 4.46 ) .
however , 15 only showed a small improvement 
 in potency , and 17 showed greatly reduced potency as 
 compared to 1 ( table 1 ) .
ic50 values were obtained using raw 264.7 cells treated with 
 20 ng / ml lps and varying concentrations of compound .
a cell viability 
 assay was used to determine cytotoxicity at each tested concentration . 

the purity of tested compounds was evaluated 
 via h nmr ( > 95% sample purity ) .
the 2-benzyl-4-methyl-5-phenylisothiazol-3-one 
 1,1-dioxide core 
 of 1 , 15 , and 17 was sensitive 
 to a variety of mild reaction conditions .
the few successfully 
 synthesized analogues exhibited only modest activity , so we thought 
 it best to make more drastic structural changes . in an effort to simplify 
 synthesis and increase stability , we took inspiration from saccharin 18 ( scheme 2 ) .
( xi ) ( 1 ) nah , dmf , ( 2 ) 9 ( 19 = 65% ) or 11 ( 103 = 62% ) ; 
 ( xii ) tfa , ch2cl2 ( 20 = 87% ) .
saccharin inspired analogues , 19 and cu - cpd103 ( 103 ) , were easily synthesized from commercially 
 available 
 saccharin and previously synthesized benzyl chlorides 9 and 11 .
a significant improvement in activity 
 was observed with intermediate 103 , so we sought related 
 analogues 2643 ( scheme 3 ; detailed syntheses can be found in the supporting information ) .
compounds 19 , 20 , 103 , and 2643 contain the same piperidine or furfuryl amide moieties 
 ( figure 1 ) that were present in our initial 
 library screen so we could have a consistent basis for comparison . 

the lithium aluminum hydride reduction of saccharin 18 , previously described by porter and co - workers , provided 2,3-dihydro-1,1-dioxo-1,2-benzisothiazole 21 .
alkylation of this sultam with 9 and 11 provided analogues 26 and 27 . 
 commercially available 1-isoindolinone 22 , phthalimide 
 potassium salt 23 , 1,2-benzisothiazol-3(2h)-one 24 , and 3-hydroxybenzisoxazole 25 ,
were alkylated with 9 and 11 to give 
 analogues 29 , 30 , 32 , 33 , 35 , 36 , 38 , and 39 ( scheme 3 ) .
treatment of analogues 35 and 36 with m - cpba at 0 c 
 gave analogues 38 and 39 , respectively , 
 as racemic mixtures .
all boc protected analogues were treated with 
 trifluoroacetic acid in methylene chloride to give deprotected analogues 28 , 31 , 34 , 37 , 40 , and 43 .
( xiv ) ( 1 ) nah , dmf , ( 2 ) 9 ( 26 = 84% ) 
 or 11 ( 27 = 81% ) ; ( xv ) tfa , ch2cl2 ( 28 = 
 92% ) ; ( xvi ) ( 1 ) nah , dmf , ( 2 ) 9 ( 29 = 57% ) 
 or 11 ( 30 = 10% ) ; ( xvii ) tfa , ch2cl2 ( 31 = 71% ) ; ( xviii ) 18-crown-6 , dmf , 9 ( 32 = 84% ) or 11 ( 33 = 81% ) ; ( xix ) tfa , ch2cl2 ( 34 = 81% ) ; ( xx ) ( 1 ) nah , dmf , ( 2 ) 9 ( 35 = 
 48% ) or 11 ( 36 = 27% ) ; ( xxi ) tfa , ch2cl2 ( 37 = 97% ) ; ( xxii ) m - cpba , ch2cl2 , 35 ( 38 = 85% ) or 36 ( 39 = 79% ) ; ( xxiii ) tfa , 
 ch2cl2 ( 40 = 92% ) ; ( xxiv ) ( 1 ) nah , 
 dmf , ( 2 ) 9 ( 41 = 55% ) or 11 ( 42 = 14% ) ; ( xxv ) tfa , ch2cl2 ( 43 = 87% ) .
the removal of 
 the saccharin series 3-position carbonyl gave us 
 the sultam series 26 , 27 , and 28 ( table 2 ) .
as we had seen with the parent 
 saccharin series , the boc protected amide 27 was the 
 most potent of the series .
for this reason , we believe 
 the 3-position carbonyl to be somewhat important to either binding 
 or cell permeability .
replacement of the saccharin series 1-position 
 so2 moiety with a methylene unit gave us the isoindolinone 
 series 29 , 30 , and 31 .
unlike 
 others , the analogue bearing a boc protected amide 30 shows the lowest potency .
. this led us to believe that the 1-position so2 moiety makes a significant interaction , perhaps as a hydrogen 
 bond acceptor .
as previously discussed , the 3-position carbonyl of 103 appears less significant , so the carbonyl of the isoindolinone 30 may be making a weaker hydrogen bond interaction as a poor 
 bioisostere of the so2 moiety .
ic50 values were obtained using raw 264.7 cells treated with 20 ng / ml 
 lps and varying concentrations of compound .
the purity of tested compounds was evaluated via h nmr ( > 95% sample purity ) . to further investigate the role of the so2 moiety ,
the 
 3-position carbonyl of 103 was maintained and the so2 moiety replaced with an additional carbonyl , as shown with 
 the phthalimide analogues 32 , 33 , and 34 .
this suggests that 
 a carbonyl is a suitable bioisostere to the so2 moiety 
 so long as the 3-position carbonyl is intact , unlike 30 .
furthermore , a loss of one , or both , oxygen atoms from the 
 so2 moiety of 103 results in a complete loss 
 of 
 activity , as seen with 36 and 39 . as seen 
 before
as expected , the piperidine amide analogue , 38 shows a further decrease in potency .
however , 35 inexplicably shows comparable potency as compared to 103 and is a great improvement over the related analogue 26 . when the unoxidized sulfur of 36 is replaced with 
 a smaller oxygen atom 42 , the activity is once again 
 comparable to that of 103 .
again , the deprotected analogue 43 shows significantly lower activity , although 41 shows some modest activity .
we further investigated the role 
 of the so2 moiety by 
 synthesizing the fully reduced and partially reduced variants , 3540 .
if the so2 moiety does 
 play the role of hydrogen bond acceptor , then these reduced analogues 
 would be less efficacious . as expected
, we observed either a loss 
 in activity or no change in activity from analogues 3640 . the fully reduced analogue 35 , however , inexplicably shows activity comparable to that of 103 .
this modification resulted with a drop in activity for 41 as compared to 35 and a great improvement 
 with 42 as compared to 36 .
in fact , the 
 potency of 42 is comparable to that of 103 , 27 , and 33 .
the steric constrains 
 of this position seem flexible , given two of the more active analogues 
 in this series , 103 and 42 , are the largest 
 and smallest of the series , respectively .
the less active of this 
 series are of varying size , but are consistently less basic than the 
 more active analogues . from this series of molecules
, there 
 are five analogues that have 
 ic50 values of less than or roughly equal to 10 m . 

four of these five analogues have the same boc - protected furfurylamide 
 ( 103 , 27 , 33 , and 42 ) .
this implies that there is an important interaction(s ) being made 
 with the boc group , and/or that an amide n 
analogues 50 , 52 , 54 , 55 , 56 , 57 , and 58 were synthesized 
 from the corresponding carboxylic acid 45 ( scheme 4 ) .
( xxvi ) ( 1 ) nah , dmf , ( 2 ) 4-bromomethylbenzoic 
 acid tert - butyl ester ( 44 = 62% ) .
( xxiv ) procedure a , 46 in 
 ch2cl2 , then ammonia in thf ( 49 = 92% ) , 
 or methylamine in thf ( 51 = 79% ) , or 2-furan-2-yl - ethylamine 
 and dipea ( 53 = 85% ) , or n - methylfurfurylamine 
 and dipea ( 55 = 86% ) ; procedure b , ( 1 ) tert - butylcarbamate , lihmds , thf , ( 2 ) 46 in ch2cl2 ; procedure c , ( 1 ) nah in dmf , then 47 or 48 , ( 2 ) 46 in ch2cl2 ( 57 = 41% , 58 = 17% ) .
interestingly , 
 intermediate ester 44 showed modest 
 activity ( table 3 ) while intermediate carboxylic 
 acid 45 shows no activity .
this may imply that the previously 
 successful analogues bearing boc groups might be benefiting from hydrophobic 
 interactions with the tertiary butyl moiety .
boc protected amides 52 and 56 , however , have shown very poor activity . 

initial efforts to synthesize 56 produced the double - boc 
 protected amide 50 , which also has poor activity , suggesting 
 that the furan substituent was necessary .
we also investigated a very 
 minor change by synthesizing the extended linker analogue 54 .
ic50 values were obtained using raw 264.7 cells treated with 20 ng / ml 
 lps and varying concentrations of compound .
the purity of tested compounds was evaluated via h nmr ( > 95% sample purity ) . to test
if the presence of an acidic amide n  h 
 could be 
 causing a negative effect on activity , we synthesized 55 . however , we observed significantly lower potency , perhaps suggesting 
 that the rotational constraints of a tertiary amide are not conducive 
 to activity . given this observation , we speculated that perhaps a 
 carbonyl component was still required for activity .
however , the n - methylcarbamate and n - acyl analogues , 57 and 58 , were significantly less potent than 
 even 20 .
we concluded that the furfurylamide component 
 was essential , and that boc protection was the optimal substituent . 

on the basis of these results , 103 was selected as our 
 lead compound .
our initial screen identified compounds that 
 could inhibit the lps - induced 
 inflammatory response . to determine if 103 specifically 
 targets tlrs ,
lps ( tlr4 ) , poly i : c ( tlr3 ) , and pam2csk4 ( tlr2/6 ) 
 were chosen to encompass the most variety in signaling , including 
 differences in tlr localization and adaptor proteins ( figure s3 , supporting information ) . as figure 2 shows
this suggests that 103 does not 
 bind specifically to an individual tlr but rather inhibits a common 
 downstream factor of these tlrs . the dose  response curves for 
 all three ligands show comparable inhibition , with minor deviations 
 due to tlr expression levels and the effectiveness of the ligand to 
 induce inflammation .
primary macrophage cells demonstrated the same 
 behavior as raw 264.7 cells , with 103 inhibiting lps 
 signaling with an ic50 value of 9.61  1.45 m 
 ( figure s2 , supporting information ) .
it 
 is important to note that 103 shows no cytotoxicity at 
 concentrations up to 100 m ( figure s4 , 
 supporting information ) .
raw 264.7 cells were treated with 
 lps ( tlr4 ligand ) , poly i : c ( tlr3 ligand ) , or pam2csk4
the ic50 values are 2.61  0.40 , 10.9  0.74 , 
 and 1.69  0.43 m , respectively .
treatment with 103 decreased no production with all tlr ligands in a dose - dependent 
 fashion .
these results demonstrate that 103 does not 
 specifically inhibit a particular tlr but rather , a common downstream 
 effector .
data was normalized [ ( raw data - untreated cells)/(tlr agonist 
 + solvent control - untreated cells ) ] such that tlr agonist + solvent 
 is 100% activation , and untreated cells are 0% activation .
data points 
 shown are the average of nine replicates , with error bars represented 
 as the standard deviation . to further confirm that inos was being down regulated by 
 treatment 
 with 103 , quantitative real - time polymerase chain reaction 
 ( rt - pcr ) and western blot experiments were performed .
rt - pcr data 
 was obtained using raw 264.7 cells treated with lps and varying concentrations 
 of 103 .
figure 3 demonstrates 
 that treatment with 103 decreases inos mrna in a dose - dependent 
 fashion .
western blots were performed with a pan nos antibody and 
 again inos is seen to decrease in a dose - dependent fashion ( figure 4 ) , indicating that 103 suppresses inos 
 at mrna , protein , and cell signaling levels . while lps was used as 
 the inflammation - inducing ligand in the no production assay to maintain 
 consistency , its effects are indirect .
therefore , a secondary assay 
 that monitors ifn- induced mrna changes was carried out to 
 confirm the validity of the no assay as the primary readout for understanding 
 sar and compound optimization ( figure s7 , supporting 
 information ) .
these results demonstrated the same trend of 
 inhibition for 103 , 36 , and 49 ( 49 is commercially available from enamine ltd . ) .
compound 103 is able to inhibit greater than 70% of ifn- induced 
 mrna , whereas 36 and 49 show a weaker potency , 
 with maximum inhibition of 50% .
importantly , these results corroborate 
 the ic50 values determined in the previously described 
 no production assay . 103 treatment decreases inos mrna in a dose - dependent 
 fashion .
raw 264.7 cells were incubated with 20 ng / ml lps and varying 
 concentrations of 103 for 20 h. data is shown with ligand - induced 
 cells normalized to a fold change of 1 .
data shown is the average quantification 
 of three biological replicates , each in technical duplicate , with 
 error bars represented as the standard deviation . *
( a ) 103 treatment reduces inos protein expression 
 in a dose - dependent fashion .
the inos protein is induced by lps treatment 
 and decreases with compound treatment , suggesting that compound reduces 
 the inflammation that results in inos expression .
the image shown 
 is a representative image , with brightness and contrast adjusted for 
 clarity .
data shown is the average quantification 
 of three biological replicates , with error bars represented as the 
 standard deviation . * * * p  0.001 .
regardless of their ligand or localization , all 
 tlrs activate nf-b 
 ( figure s3 , supporting information ) .
to 
 test the inhibitory effects of 103 on nf-b activity , 
 a secreted embryonic alkaline phosphatase ( seap ) assay was performed 
 in hek 293 t cells .
when tested at concentrations up to 100 m , 
 the compound did not down - regulate nf-b activation through 
 tlr3 or tlr4 ( figure s5 , supporting information ) . to determine if any modulation occurs through other nf-b 
 pathways , tnf- was used to activate nf-b signaling . 
 as seen in the figure s4 in the supporting information ,
this data suggests that 103 does 
 not directly modulate the tlr signaling pathway at any point , as nf-b 
 is essential to all tlr signaling .
we next sought to confirm this 
 result through observation of nf-b - induced cytokines , particularly 
 tnf-. a commercially available enzyme - linked immunosorbent 
 assay ( elisa ) was used to measure tnf- in raw 264.7 cells .
figure s6 in the supporting information shows 
 that there was no change in tnf- cytokine levels with compound 
 treatment .
these results confirm in two cell types that there is no 
 modulation of nf-b by 103 , regardless of ligand 
 or signaling pathway .
however , previous results demonstrated that 
 tlr - induced no activation is inhibited by 103 .
the inos 
 promotor has binding sites for both nf-b and stat1 . because 
 nf-b activation is not being affected with 103 treatment , the inhibition of inos was therefore likely to occur 
 within the jak / stat1 pathway .
as no direct antagonism was observed 
 through tlrs and nf-b , 
 additional tests were carried out to identify the potential anti - inflammatory 
 mechanism of 103 .
( ifn-/ ) 
 and interferon ii ( ifn- ) pathways cause upregulation of inos , 
 which results in production of no . as such , we speculated that observed 
 no inhibition might occur through inhibition of the jak / stat signaling 
 pathway . to test this hypothesis ,
inhibition of no occurred in 
 a dose - dependent fashion with treatment of 103 .
the ic50 value with ifn- is 7.88  1.25 m , which 
 corroborates the ic50 value of lps ( figure 5 ) .
this indicated that the jak / stat1 pathway is involved in 
 the inhibitory function of 103 . additionally , as tlr 
 activation results in production of type i interferons , synonymous 
 inhibition with ifn- proposes a shared target between these 
 two pathways .
thus , it is likely that the molecular target of 103 lies in the interferon - induced stat1 pathway .
raw 264.7 cells were treated with 5 ng / ml ifn- to 
 activate jak / stat1 signaling .
data was normalized 
 [ ( raw data - untreated cells)/(tlr agonist + solvent control - untreated 
 cells ) ] such that tlr ligand + solvent is 100% activation , and untreated 
 cells are 0% activation .
data points shown are the average of nine 
 replicates , with error bars represented as the standard deviation .
additionally , a commercial jak / stat 
 rt - pcr array was used to determine 
 if jak / stat pathway signaling is by and large modulated with 103 treatment .
the modified 
 genes , including nos2 , cebpb , and gtp1 , suggest that 103 modulates stat1 signaling . additional validation is required to target the specific site of 
 action for 103 .
taken together , our results provide consistent 
 evidence that 103 functions through the interferon - induced 
 jak / stat1 signaling pathway , suppressing inos .
in summary , we report the identification of a group of novel ifn 
 inhibitors based on a saccharine core .
extensive sar studies have 
 shown a narrow tolerance for change at the lone amido position .
a 
 boc protected furfurylamide has proven to be the most consistently 
 successful substituent .
our results demonstrate that 103 is a potent inhibitor of inos on both an mrna and protein expression 
 level .
however , this occurs without nf-b modulation , indicating 
 a tlr - independent mechanism .
further biochemical studies imply potential 
 inhibition in the stat1 pathway , as this is shared between type i 
 and ii interferons , and associated genes were observed to change via 
 pcr array .
103 may provide therapeutic insight for inflammatory 
 diseases such as systemic lupus erythematosus and multiple sclerosis .
 a series of novel , saccharin - based 
 antagonists have been identified 
 for the interferon signaling pathway . through in vitro high - throughput 
 screening with the colorado center for drug discovery ( c2d2 ) pilot 
 library , we identified hit compound 1 , which was the 
 basis for extensive structure  activity relationship studies . 
 
 our efforts produced a lead anti - inflammatory compound , tert - butyl n-(furan-2-ylmethyl)-n-{4-[(1,1,3-trioxo-2,3-dihydro-16,2-benzothiazol-2-yl)methyl]benzoyl}carbamate cu - cpd103 ( 103 ) , as a potent inhibitor using an established nitric oxide 
 ( no ) signaling assay . 
 with further studies of its inhibitory mechanisms , 
 we demonstrated that 103 carries out this inhibition 
 through the jak / stat1 pathway , providing a drug - like small molecule 
 inflammation suppressant for possible therapeutic uses . 
Introduction
Results and Discussion
Conclusions