15% 
 of the global population and more than 65% of the population in the 
 third and developing world , yet current drug therapies for protozoan 
 infections are woefully inadequate . as protozoan infections take their 
 toll predominantly in the developing world , market forces are insufficient 
 to promote the development of novel antiprotozoan drugs . in 2000 , 
 only ca .
0.1% of global investment in health research was spent on 
 drug discovery for tropical diseases .
one such neglected parasitic 
 disease is human african trypanosomiasis 
 ( hat ) or african sleeping sickness , which is caused by the protozoan 
 parasite trypanosoma brucei .
the world 
 health organization ( who ) estimates that hat constitutes a serious 
 health risk to 60 million people in sub - saharan africa .
the who also 
 estimates that there are < 30000 new cases per year in sub - saharan 
 africa and at present an annual death toll of 8000 . the related disease in cattle
, cattle trypanosomiasis 
 or nagana , 
 also represents a major health concern due to its devastating economic 
 ( estimated by the who to cause an annual economic loss of us$4 
 billion ) , social , and nutritional impact on african families . as such
, 
 the total burden of trypanosomiasis translates into 1598000 disability - adjusted 
 life years ( daly ) .
treatment of hat is solely dependent 
 upon a repertoire of four 
 drugs : suramin ( 1 ) , pentamidine ( 2 ) , melarsoprol 
 ( 3 ) , and eflornithine ( 4 ) ( figure 1 ) .
these therapies are often toxic , difficult to 
 administer , and increasingly have an acquired drug resistance , highlighting 
 the urgent need for new , more effective drug therapies .
developed 
 before the 1950s , suramin ( 1 ) and melarsoprol ( 3 ) are used for chemotherapy of the early stage of the disease 
 ( trypanosoma brucei rhodesiense ) , as 
 is pentamidine ( 2 ) ( trypanosoma brucei 
 gambiense ) .
the arsenical melarsoprol ( 3 ) is extremely toxic , with death in 6% of cases and treatment 
 failure rates of as high as 30% in certain areas .
treatment of the 
 second stage of the disease , where the parasites cross the blood  brain 
 barrier and invade the central nervous system , is limited to melarsoprol 
 ( 3 ) and eflornithine ( 4 ) , the ornithine 
 decarboxylase inhibitor ( difluoromethylornithine ) .
structures 
 of clinic drugs 15 for sleeping 
 sickness and clinic phase i drug fexinidazole ( 6 ) .
nifurtimox ( 5 ) ( figure 1 ) , 
 is often used to treat chagas disease , caused by trypanosoma cruzi , and has been used as a monotherapy 
 for melarsoprol - refractory hat on compassionate grounds despite its 
 low efficacy and severe toxicity .
eflornithine 
 combination therapy ( nect ) by who in the model lists of essential 
 medicines has been seen as highly advantageous in terms of cost , logistics , 
 and human resources in areas of poverty .
patients given nect , consisting 
 of oral nifurtimox over 10 days with eflornithine ( 4 ) 
 infusions for 7 days , were found to fair just as well as those given 
 the eflornithine monotherapy , with cure rates of around 97% .
the use of nect has renewed general interest 
 in the use of nitroheterocyclic 
 compounds to treat a wide range of infectious disease , including tuberculosis 
 and hepatitis c. this increased awareness of the potential of nitroheterocyclic 
 compounds has led to the reinvestigation of fexinidazole ( 6 ) ( figure 1 ) , which is presently in clinical 
 trials against both early- and late - stage african sleeping sickness .
alarmingly , it has been shown that t. brucei nifurtimox - resistant cells show cross - resistance to other nitro - containing 
 drugs , including fexinidazole , that are currently in clinical trials .
several different types of studies on nitroheterocycles have shed 
 some light on this observed drug resistance .
genome - wide rnai screens 
 of nifurtimox and benznidazole ( another trypanocidal nitroheterocyclic 
 drug ) resistant t. brucei have identified 
 that a decrease in nitroreductase activity is linked to nifurtimox 
 resistance .
this was confirmed by showing 
 that genetic deletion of one allele of the t. brucei s nitroreductase led to nifurtimox resistance .
likewise , 
 the overexpression of this nitroreductase , but not the alternative 
 proposed prostaglandin f2 synthase and cytochrome p450 reductase , 
 in t. brucei resulted in increased 
 susceptibility to nifurtimox .
collectively , 
 it is clear that this type i t. brucei nitroreductase , a nad(p)h dependent , flavin binding protein , is 
 involved in the reductive activation of nifurtimox .
the nitroreductases 
 mediate a series of two - electron reductions of the nitro group to 
 a nitroso intermediate , then to a hydroxylamine , and eventually to 
 the corresponding amine .
however , the true function of the t. brucei mitochondrial nitroreductase is unknown , 
 but its essentiality may explain why only a single allele deletion / mutation 
 is associated with nifurtimox resistance . as well as the development of nifurtimox - resistance and its 
 cross - resistance 
 to other nitro - containing drugs ,
for example , the adverse off - target side 
 effects with nifurtimox lead to treatment cessation in over 30% of 
 patients with chagas disease .
the most common side effects are anorexia , loss of weight , psychic 
 alterations , excitability or sleepiness , and digestive manifestations 
 such as nausea or vomiting and occasionally intestinal colic and diarrhea .
various human enzymes have been identified to 
 be capable of 5-nitrofuran reduction in vitro , in cells , or tissues .
recently , we have elucidated in collaboration with the patton laboratory 
 a possible role of aldehyde dehydrogenase 2 ( aldh2 ) in the toxicity 
 caused by 5-nitrofuran containing drugs such as nifurtimox .
while considerable effort has gone into 
 the synthesis of nitroaromatic 
 containing compounds over the years , there remains several unexplored areas of chemical space .
for example , 
 in the context of nifurtimox , most of the efforts have been focused 
 on changing either the furan ring to other heterocycles ( such as imidazole ) 
 or changing the substituent in the hydrazone motif , with limited increases in activity compared to nifurtimox .
recently , 
 two new types of typanocidal compounds , nitrobenzylphosphoramide mustards 
 ( for example see compound 7 , figure 2 ) and aziridinyl nitrobenzamides , 
 have been developed to target specifically t. brucei type i nitroreductase , the best having an ec50  
 1 m against both t. brucei and t. cruzi .
several 5-nitro-2-furancarboxylamide 
 containing compounds have been reported to have potent antituberculosis 
 and antimicrobial activity , however , very few 5-nitro-2-furancarboxylamides are 
 reported to have typanocidal activity , primarily low activity against t. cruzi . a nifurtimox analogue 
 ( 8) ( figure 2 ) showed trace activity 
 against t. cruzi in a mouse model .
the best 5-nitro-2-furancarboxylamides identified 
 thus far , 9 and 10 , have ec50 values of 42 and 262 nm , respectively , against t. 
 cruzi amastigotes ( figure 2 ) but have not been tested against t. brucei to the best of our knowledge .
several 
 5-nitro-2-furancarbohydrazides possess activity against t. brucei with the best , compound 11 , having an ec50 of 0.13 m but low selectivity 
 against human cells .
, we report that our recently discovered 
 nitrofuran 
 nfn1 ( 12a ) also shows promising 
 activity against t. brucei in vitro .
a lack of cross - resistance with nifurtimox 
 and significantly increased ( 23 orders of magnitude ) trypanocidal 
 activity has important implications for the future therapeutic use 
 of these nitrofuran containing compounds in the treatment of hat , 
 instead of , or in combination with , nifurtimox .
5-nitrofurans 12a  k were prepared by acylation of the corresponding 2-amino - thiophenes 13a  k with 5-nitro - furancarboxylic acid 
 chloride 14a ( scheme 1 , table 1 for substituents ) .
the required 2-amino - thiophenes 13a  h were prepared using the gewald multicomponent 
 reaction with various combinations of the 2-cyanoacetyl esters or 
 amides ( 15a  g ) with aldehydes ( 16a or 16b ) and elemental sulfur ( see supporting information table s1 for details).13h was o - silyl - protected before coupling 
 to 14a .
this reaction gave 12h directly 
 as a result of in situ removal of the silyl - protecting group .
thiophene 13i was prepared by hydrolysis of 13a and 13j prepared by catalytic amination of 2-iodothiophene using 
 cui and l - proline.13k and 13l ( table 2 ) were commercially 
 available .
reaction of 13a ( r = et , r = co2et ) with 14b gave control compound 18a .
reagents and conditions : 
 ( a ) 
 ( i ) ( cocl)2 , dcm / dmf , rt , 2 h , ( ii ) rnh2 , et3n , dcm , 52% for 15d , 49% ( 15e ) , 37% ( 15 g ) ; ( b ) s8 , 16a or 16b , et2nh , dmf , rt , o / n , 66% for 13a , 59% ( 13b ) , 76% ( 13c ) , 53% ( 13d ) , 49% ( 13e ) , 46% ( 13f ) , 48% 

( 13 g ) , 46% ( 13h ) , for synthesis of 13i  j see esi , 13k and 13l were commercially available ; ( c ) 13a  k , et3n , dcm , rt with 14a , 68% for 12a , 62% ( 12b ) , 65% ( 12c ) , 58% ( 12d ) , 57% ( 12e ) , 57% ( 12f ) , 54% 
 ( 12 g ) , 47% ( 12h ) , 25% ( 12i ) , 
 41% ( 12j ) , 51% ( 12k ) , with 14b , 56% for 18a ; ( d ) rnh2 , et3n , 
 dcm , rt with 14a , 65% for 22a , 61% ( 22b ) , 72% ( 22c ) , 67% ( 22d ) , 37% 
 ( 22e ) , 50% ( 22f ) , 45% ( 22 g ) , 
 41% ( 22h ) , 66% ( 22i ) , 39% ( 22j ) , 51% ( 22k ) , 73% ( 22l ) , 51% ( 22 m ) , 68% ( 22n ) , 67% ( 22o ) , 69% ( 22p ) , 48% ( 22q ) , with 14b 73% for 18b ( see table 2 for structure 
 of 18b ) .
( a ) 15a  c and 15f were commercially available ; ( b ) 15d , 15e , and 15 g were prepared from 17 ; ( c ) for r and r substituents , see table 1 ; ( c ) for r substituents , 
 see tables 2 and 3 . for structure ,
not applicable as no activity or 
 low activity against t. brucei was 
 observed for 18b and 25a .
analogues 20a  c and 21 ( table 2 ) based on 12 g but containing 
 alternative nitroaromatic rings were prepared by coupling the corresponding 
 acid chlorides with 13 g ( see esi for details ) .
similarly , 
 reaction of 14a with a range of amines and anilines led 
 to the synthesis of 22a  q ( scheme 1 ) .
demethylation of 22o to give 22r was followed by selective o - allylation 
 to give 22s ( scheme 2 ) , the structure 
 of which was confirmed by x - ray crystallographic analysis ( data not 
 shown ) . when a longer reaction time and an excess amount of allyl 
 bromide was used in the o - allylation of 22r , the diallylated analogue 22 t was formed ( scheme 2 ) .
methylation 
 of 22q to give 22v was achieved using mei 
 under basic conditions in moderate yield ( scheme 2 ) .
conversion of 5-nitrofuran aldehyde 23 to 
 imines 24a or 24b enabled the synthesis 
 of the corresponding amines 25a and 25b using 
 standard reductive amination conditions ( scheme 2 ) .
reagents and conditions : 
 ( a ) 
 pyridine hydrochloride , 160 c , mw , 5 min , 52% ; ( b ) 22r or 22p , allyl bromide ( 2 or 10 equiv ) , k2co3 , acetone , rt , 2 h for 22s ( 75% ) , 24 h 
 for 22 t ( 55% ) , 24 h for 22u ( 72% ) , 22q , mei , k2co3 , acetone , 3 h , for 22v ( 51% ) ; ( c ) aniline or 13 g , dcm , rt , 89% for 24a , 63% for 24b ; ( d ) nabh4 , dcm , 
 rt , 79% for 25a , 87% for 25b . in comparison 
 to nifurtimox , nfn1 was 2 orders of magnitude more potent with an 
 ec50 of 31.3  3.1 nm ( table 1 , cf .
the nitro group in nfn1 was found to be essential 
 with no activity being observed up to the solubility limit of 18a ( entry 3 ) .
removal , or worse , elongation of the alkyl 
 substituent in nfn1 led to reduced activity ( cf .
entry 2 and entries 
 4 and 5 , respectively ) , and while removal of the ester group initially 
 appeared to be tolerated in the r = h series ( cf .
entries 
 4 and 6 ) , it was found that the methyl and n - butyl 
 ester analogues ( 12c and 2d ) of nfn1 had 
 similar activity to nfn1 ( cf . entry 2 and entries 7 and 8 , respectively ) . 

conversion of the ester groups in nfn1 and 12d to the 
 corresponding amides in 12e and 12f led 
 to a significant loss in activity ( cf .
this observation , coupled with the decrease 
 in activity associated with amide 12h ( entry 11 ) , may 
 be explained by the fact that hydrolysis of the ester groups in 12a  d and 12k in the parasite 
 gives the corresponding carboxylic acid - containing bioactive metabolites , 
 something that is unlikely to occur for the amides 12e and 12f . however , acid 12i showed much 
 less activity with an ec50 of 959.0  33.9 nm ( entry 
 12 ) , which may be due to a lack of cell permeability .
the primary 
 amide 12 g was found to be the most active thiophene - containing 
 analogue with an ec50 of 17.3  2.4 nm ( entry 13 ) . 

furthermore , analogues 12a  k showed 
 no significant toxicity against the human hela cell line even at a 
 concentration above 20 m .
the highly selective toxicity of 
 these analogues against the parasite suggests these nitrofuran analogues 
 have real potential for therapeutic applications .
encouraged 
 by the potent activity of 12 g ( table 1 ) , it was decided to prepare more analogues based on this 
 structure .
fragments 19 and 13 g along with 13l were shown to be inactive against t. brucei , suggesting that both the 5-nitrofuran and thiophene fragments are 
 required ( table 2 , cf .
changing the nitrofuran motif in 12 g to either a nitrophenyl or nitropyrazole ring also led 
 to a 100-fold or more drop in activity ( cf .
interestingly , the position of the nitro group in the 
 nitrophenyl ring had an influence as 20b , an analogue 
 with the nitro group in the meta - position , was more 
 active than either 20a ( ortho - no2 ) or 20c ( para - no2 ) .
studies next focused on replacing the thiophene motif in 12a  k . by incorporating a cyclohexyl ring 
 to give 22a , a 10-fold decrease in activity was observed 
 compared to 12 g ( entry 9 ) , while the phenyl ( 22b ) and benzyl ( 22c ) analogues were essentially equipotent 
 with 12 g ( cf .
incorporation of a nitrogen atom into the 2-position of the phenyl 
 ring in 22c to give 22d led to a 10-fold 
 drop in activity ( entry 12 ) .
entries 10 and 13 ) , and the amide 
 linkage in 22b and 12 g was also shown to 
 be important , as the amine analogues 25a and 25b showed a dramatic decrease in activity ( entries 14 and 15 , respectively ) . on the basis of the potent activity of 22b ,
the effect 
 of incorporating additional substituents into the phenyl ring was 
 investigated ( table 3 ) .
substitution at the para-(r ) and meta-(r or r)-positions with either electron - withdrawing ( entries 
 3 and 57 , respectively ) or electron - donating groups ( entries 
 4 and 8 , respectively ) proved detrimental , with the exception of the 
 incorporation of the meta - cf3 group in 22e ( ec50 of 22e = 4.69  0.14 
 nm , entry 2 ) .
interestingly , incorporation of a meta - cf3 into the phenyl ring of the benzyl analogue 22c ( table 2 ) gave analogue 22l , which had an ec50 = 17.4  3.0 nm , suggesting that 
 the use of a phenyl substituent ( as in 22e ) may be preferred 
 to the benzyl substituent in 22l . combining the meta - cf3 substituent with either an additional meta - methoxy- or a second meta - cf3 substituent led to a decrease in activity ( entries 9 and 11 , respectively ) , 
 whereas the meta - cf3 group was able to 
 override the effect of a para - methoxy substituent 
 ( cf .
incorporation of a para - hydroxy 
 in both the meta - cf3 and unsubstituted 
 series led to a significant reduction in activity ( entries 13 and 
 14 , respectively ) , but extending the para - alkoxy 
 chain from methoxy in 22o to allyloxy led to the most 
 active compound prepared , analogue 22s , which had an 
 ec50 against t. brucei of 
 2.4  0.3 nm ( entry 15 ) .
the positive role of the para - allyloxy substituent was also observed in the unsubstituted series 
 ( cf .
the incorporation of an additional allyloxy 
 substituent on the amide nitrogen in 22s led to a significant 
 loss in activity for 22 t ( cf .
interestingly , the lack of significant biological 
 activity displayed 
 by the 2-trifluoromethylphenol 22r , e.g. , ec50 value of 2.2  0.1 m versus 2.4  0.3 nm for 22s or 7.8  0.3 nm for 22o , might be due 
 to the high instability of 22r in the tested conditions 
 as the result of fluorine elimination generating a quinone methide , 
 as previously reported . masking the phenol 
 as allyl or methyl ethers , e.g. , as in 22s or 22o ,
finally , the special role played 
 by the meta - cf3 substituent was further 
 highlighted by a more than 5-fold 
 drop in activity observed on its replacement by a meta - ch3 substituent ( cf .
as was the case 
 for analogues 12a  k , all the analogues 
 shown in tables 2 and 3 showed low toxicity against the human hela cell line .
the striking increase in potency 
 of the 5-nitro-2-furancarboxylamide 
 analogues compared to nifurtimox against bloodstream t. brucei suggests they have a different or possibly 
 additional mode of action to that of nifurtimox , i.e. , in addition 
 to the activation by the t. brucei nitroreductase . 
 to investigate this possibility ,
it was decided to generate drug resistant 
 cell lines to nifurtimox and the most potent of the 5-nitro-2-furancarboxylamide 
 analogues , 22s ( ec50 = 2.4  0.3 nm ) . 

previous lab - generated nifurtimox - resistant strains have shown that 
 deletion or mutation of one of the alleles of the nitroreductase confers 
 resistance . as with these previous studies ,
nifurtimox - resistant 
 parasites 
 were generated easily by culturing bloodstream t. brucei in the continuous presence of nifurtimox .
a stepwise increase in 
 the concentrations of nifurtimox , initially starting at 1.5 m , 
 increasing to 2.5 , 4 , 5 , 7.5 , 10 , 15 , 20 , 25 , 30 , 40 , 45 , and 50 m 
 on days 2 , 5 , 9 , 14 , 23 , 39 , 55 , 65 , 76 , 90 , 109 , and 120 , respectively , 
 was used ( figure 3a ) . at 143 days ,
these nifurtimox - resistant 
 parasites were cloned by serial dilution while maintaining the 50 
 m nifurtimox .
generation of nifurtimox and 22s resistance 
 cell - lines : 
 schematic representation of the generation of a ( a ) nifurtimox - resistant 
 and ( b ) 22s - resistant cell lines in t. 
 brucei .
each point represents the concentration at 
 that time point when cells were checked and divided if required .
selection studies started at 1 nm of 22s , increasing to 2 , 3 , 5 , 7.5 , 10 , 15 , 20 , 25 , 30 , 35 40 , and 50 m 
 on days 2 , 5 , 9 , 26 , 36 , 47 , 58 , 70 , 76 , 103 , 130 , and 148 , respectively 
 ( figure 3b ) . at 160 days ,
the 22s - resistant parasites were cloned by serial dilution while maintaining 
 a 50 nm concentration of 22s . both of the drug - resistant 
 cell lines were stable for 30 days in the absence of their respective 
 drugs .
the nifurtimox - resistant parasites had a doubling time of 11.5 
  0.6 h compared to wild - type 7.8  0.4 h , while 22s - resistant parasites had a doubling time of 13.3 
  1.1 h. the sensitivities of the cloned nifurtimox - resistant 
 and 22s - resistant parasites to nifurtimox , 22s , and 
 the diamidine pentamidine ( 2 ) were determined and compared 
 to wild - type cells .
the nifurtimox - resistant cells did not greatly 
 alter ( 2-fold increase ) the sensitivity to pentamidine ( 2 ) compared to wild - type cells ( table 4 ) , in accordance with previous lab generated nifurtimox - resistant 
 cell lines.22s - resistant parasites 
 also showed very little alteration in the ec50 ( 1.1  
 0.1 nm ) of pentamidine ( 2 ) compared to that of wild - type 
 cells ( table 4 ) .
ec50 literature value 
 of nifurtimox in wild - type t. brucei 2.4  0.1 m and in nifurtimox resistant cells 20.1  
 0.9 m .
ec50 literature value 
 of pentamidine ( 2 ) in wild - type t. brucei 0.95  0.02 nm and in nifurtimox resistant cells 2.6  
 0.1 nm .
the nifurtimox - resistant cells were found to be 910-fold 
 less sensitive to nifurtimox than wild - type cells , with ec50s of 20.9  1.7 and 2.1  0.2 m , respectively ( table 4 ) , in accordance with previous findings.22s - resistant cells were found to 
 be 14-fold less sensitive to 22s than wild - type 
 cells , with ec50s of 29.3  2.0 and 2.4  0.3 
 nm , respectively ( table 4 ) . even at this level 
 of resistance , 22s is still 70 times more potent 
 than nifurtimox in wild - type t. brucei . to investigate cross - resistance between nifurtimox and 22s , the ec50s of the drug - resistant cell lines 
 versus the 
 alternative nitrofuran containing compounds were determined ( table 4 ) .
the results showed that nifurtimox and 22s showed a low level of cross - resistance .
nifurtimox - resistant 
 cells showed a 3-fold increase in the ec50 of 22s , while nifurtimox also showed 3-fold increase 
 in the ec50 against 22s - resistant cells .
these 
 findings imply that these two nitrofuran compounds , nifurtimox and 22s , are trypanocidal as a result of primarily targeting different 
 biochemical process within the parasites .
however , the low level of 
 cross - resistance suggests there may be some minor overlap in the mode 
 of action .
these findings 
 have important implications for the therapeutic use of this new generation 
 of nitrofuran compounds as part of novel combination therapies or 
 more importantly potentially replacing nifurtimox in a clinical setting . as nifurtimox and
22s apparently act through primarily 
 different modes of action , this suggests that their combined action 
 against the parasite may be synergistic . as such
, a series of ec50s were determined for 22s in the presence of 
 various concentrations of nifurtimox ( figure 4 ) . at low concentrations of nifurtimox ( < 700 nm ) ,
synergy can 
 be observed as the ec50s are below the straight diagonal 
 line that represents what would be expected for an additive trypanocidal 
 effect of the two compounds . above concentrations of 700 nm nifurtimox
, 
 there is a distinct lack of synergy ; if anything , competition occurs 
 between the two inhibitors .
this , taken together with the observed 
 low level of cross - resistance ( table 4 ) , may 
 indicate that nifurtimox at high concentrations is competing with 
 the same biochemical activation process which is normally targeted 
 by 22s .
this further highlights the possible multitarget 
 nature for nifurtimox that has been suggested previously .
this type of information is relevant for the potential pharmacological 
 applications of these novel nitro - furancarboxylamides as a combinational 
 therapy would likely lead to the use of lower nifurtimox doses .
studies of 
 possible synergy between nifurtimox ( 5 ) 
 and 22s in t. brucei .
it was decided to test some of the 5-nitro-2-furancarboxylamides 
 from this study against cultured t. b. rhodesiense , one of the human - infective subspecies ( table 5 ) .
the potency of a selection of analogues against t. b. rhodesiense were similar to those observed 
 for t. b. brucei , suggesting that these 
 novel nitrofurans could be considered lead candidates for the next 
 step toward a novel treatment for hat , as well as the related animal 
 disease , nagana .
in this work , we designed and synthesized 
 a series of nitrofuran - containing 
 analogues that were tested for trypanocidal and cytotoxicity activity 
 against cultured bloodstream form t. b. brucei and human hela cells , respectively .
the analogue 22s showed an ec50 of 2.4  0.3 nm , 3 orders of magnitude 
 more potent than nifurtimox with a selectivity index > 8000 .
sar 
 studies 
 showed that all fragments of the compounds were required for activity . 

in addition , both the nitrofuran and amide functional groups and the 
 amide nh were necessary . the thiophene ring in the starting analogue 12a can be replaced by a range of other substituents .
however , 
 in the thiophene series , an ester group was required and is likely 
 converted to the corresponding carboxylic acid in the parasite .
several 
 of our analogues were also tested against t. b. rhodesiense , showing analogous activity to that in t. b. brucei .
this exciting result demonstrates that this series of analogues 
 is worthy of further study as a potential therapy for hat .
importantly , 
 the cross - resistance studies showed that the 5-nitro-2-furancarboxylamide 
 analogues in this study have low levels of resistance to nifurtimox - resistant 
 cells and vice versa while showing no resistance to pentamidine ( 2 ) .
these results indicate that the trypanocidal mode - of - action 
 of the 5-nitro-2-furancarboxylamide analogues in this study does not 
 rely upon nitroreductase activation unlike nifurtimox and other nitroheterocyclic 
 compounds that are presently in clinical trials against hat
. however , 
 the lack of expected synergy at high concentrations of nifurtimox 
 suggests it may be interacting with the novel mode of action which 
 is primarily targeted by our novel 5-nitro-2-furancarboxylamide analogues .
our data suggest that these new 5-nitro-2-furancarboxylamide analogues 
 have a potential therapeutic implication as a single reagent in regard 
 to potency and selectivity .
also , combination therapies can be considered 
 due to their low cross - resistance to other trypanocidal drugs .
this 
 idea is supported by our results that show there is a synergistic 
 relationship between nifurtimox and 22s at concentrations 
 of nifurtimox of < 700 nm ( figure 4 ) . while 
 there remain many more challenges to the development of a hat therapeutic 
 that have not been addressed here ( see supporting 
 information table s6 for a detailed discussion of drug - likeness 
 parameters associated with our analogues ) , we believe that the novel 
 mechanistic and high in vitro potency of the antitrypanosomal compounds 
 presented here renders them worthy of further study in the drug discovery 
 context .
the trypanocidal activity over 
 a 72 h period was determined using the alamar blue viability , as described 
 previously .
nifurtimox , pentamidine , 
 melarsoprol , and various novel nitrofuran analogues were tested against trypanosoma brucei brucei bloodstream - form ( strain 
 427 ) or drug - resistant strains were cultured at 37 c in hmi9 
 medium supplemented with 10% fetal calf serum and 2.5 g ml g418 as described previously.t .
brucei rhodesiense ( strain 
 z310 ) were cultured in a similar manner but in the absence of g418 . 
 for the synergistic experiments , various concentrations of nifurtimox 
 ( 0 , 200 , 400 , 600 , 800 , 1000 , 1500 , 2000 , and 2400 nm ) were used in 
 conjunction with a serial dilution of 22s .
experiments 
 were conducted in replicates of four ; the data was fitted using grafit 
 software to obtain ec50  standard deviations and 
 slope factors .
briefly , the cells were 
 cultured in dmem supplemented with 10% fetal calf serum and 2 mm l - glutamine .
cells were plated at initial cell concentration 
 of 2  10 cells / well and incubated with the compounds 
 for 65 h prior to addition of alamar blue solution for a further 
 5 h. drug - resistant t. brucei cell lines were generated by subculturing bloodstream trypanosomes 
 in the continuous presence of either nifurtimox or compound 22s .
parasites were exposed to stepwise - increased concentrations 
 of drug , starting at appropriate sublethal concentrations , until they 
 were routinely growing between 20 and 25 times their respective original 
 ec50s .
after 150160 days in culture , drug - resistant 
 parasites were cloned by limiting dilution and used for further study . 

cell doubling times were determined in replicates of 4 over a 96 h 
 period .
chemicals and reagents were obtained from either 
 aldrich or alfa - aesar , except nifurtimox ( 5 ) from bayer 
 argentina . all reactions involving moisture sensitive reagents
were 
 performed in oven - dried glassware under a positive pressure of argon . 

dichloromethane ( dcm ) was obtained dry from a solvent purification 
 system ( mbraun , sps-800 ) .
melting points were recorded in open capillaries 
 using an electrothermal 9100 melting point apparatus .
infrared spectra 
 were recorded on a perkin - elmer spectrum gx ft - ir spectrometer using 
 thin films on kbr ( for solids ) discs or nujol ( for liquids ) .
low resolution 
 ( lr ) and high resolution ( hr ) electrospray mass spectral ( es - ms ) analyses 
 were acquired by electrospray ionization ( esi ) within the school of 
 chemistry , university of st .
low and high resolution esi 
 ms were carried out on a micromass lct spectrometer or at a high performance 
 orthogonal acceleration reflecting tof mass spectrometer coupled to 
 a waters 2975 hplc .
nuclear magnetic resonance ( nmr ) spectra were 
 acquired either on a bruker avance 300 ( h , 300.1 mhz ; c , 75.5 mhz ) or on a bruker avance 400 ( h , 400 
 mhz ; c , 100.1 mhz ) spectrometer and in the deuterated 
 solvent stated .
c nmr spectra were acquired using the 
 pendant or deptq pulse sequences . for characterization of 2-aminothiophenes 13a  k , compounds 20a  c , 21 , intermediates , and other known compounds
the 
 prepared analogues were analyzed by hplc with purity above 95% ( supporting information ) .
the furoic 
 acid chlorides 14a and 14b were prepared 
 in situ : thionyl chloride ( 1.10 equiv ) was added dropwise to a mixture 
 of 5-nitrofuran-2-carboxylic acid or 2-furoic acid ( 1.10 equiv ) and 
 triethylamine ( 1.50 equiv ) in dcm ( 0.4 m ) under a n2 atmosphere . 

the reaction mixture was stirred at room temperature for 5 h. then 
 crude 14a or 14b was added to another flask 
 containing the corresponding amine or aniline ( 1.00 equiv ) and triethylamine 
 ( 2.00 equiv ) in dcm ( 0.4 m ) .
the reaction mixture was stirred at room 
 temperature for 5 h. the solvent was then removed under reduced pressure , 
 and the crude reaction mixture was purified by column chromatography .
details of the synthesis of furancarboxyl amides 12a , 12 g , 22e , 22h , and 22s are given here in the main text of the paper ; the remainder 
 ( 12b  f , 12h  k , 22a  d , 22f , 22 g , 22i 
the general procedure was followed using 
 2-amiothiophene 13a ( 3.99 g , 20.0 mmol ) .
the crude reaction 
 mixture was purified by column chromatography on silica gel ( 5:1 , 
 hexane / ethyl acetate ) to afford the product as an orange solid ( 4.60 
 g , 13.6 mmol , 68% ) ; mp 127128 c .
ir ( kbr ) max = 3120 ( s ) ( nh ) , 2958 ( m ) ( c  h ) , 1667 ( s ) ( c = o ) , 
 1569 ( s ) , 1531 ( s ) ( no2 ) , 1350 ( s ) ( no2 ) , 1280 
 ( s ) ( c  o ) , 1266 ( s ) ( c  o ) cm .
h nmr ( 400 mhz , acetone - d6 ) :  
 11.75 ( br s , 1h ) , 7.58 ( d , j = 3.9 hz , 
 1h ) , 7.42 ( d , j = 3.9 hz , 1h ) , 6.85 ( t , j = 1.1 hz , 1h ) , 4.28 ( q , j = 7.1 hz , 2h ) , 2.67 ( dq , j = 7.5 hz , j = 1.1 hz , 2h ) , 1.27 ( t , j = 7.1 hz , 3h ) , 1.17 ( t , j = 7.5 hz , 3h ) .
c nmr ( 100 mhz , acetone - d6 ) :  165.8 ( c ) , 153.6 ( c ) , 153.1 ( c ) , 
 147.4 ( c ) , 145.6 ( c ) , 139.2 ( c ) , 120.6 ( ch ) , 118.6 ( ch ) , 115.0 ( c ) , 
 113.8 ( ch ) , 61.7 ( ch2 ) , 23.2 ( ch2 ) , 15.9 ( ch3 ) , 14.6 ( ch3 ) . lrms ( es ) : m / z ( % ) 360.87 ( 100 ) [ m + na ] .
hrms ( es ) : m / z calcd for c14h14n2o6nas [ m + na ] , 
 361.0470 ; found , 361.0469 .
the general procedure was followed using 
 2-amiothiophene 13 g ( 170 mg , 1.00 mmol ) .
the crude reaction 
 mixture was purified by column chromatography on silica gel ( 1:1 , 
 hexane / ethyl acetate ) to afford the product as a yellow solid ( 167 
 mg , 0.54 mmol , 54% ) ; mp ( dec . )
ir ( kbr ) max = 3476 ( m ) , 3341 ( m ) ( nh ) , 3211 ( m ) , 3143 ( m ) , 1659 ( s ) 
 ( c = o ) , 1591 ( s ) , 1565 ( s ) , 1534 ( s ) ( no2 ) , 1349 
 ( s ) ( no2 ) , 739 ( m ) cm . h nmr ( 400 mhz , acetone - d6 ) :  
 7.56 ( d , j = 3.9 hz , 1h ) , 7.36 ( d , j = 3.9 hz , 1h ) , 7.05 ( t , j = 1.1 hz , 1h ) , 6.80 ( br s , 1h ) , 2.66 ( dq , j = 7.5 hz , j = 1.1 
 hz , 2h ) , 1.16 ( t , j = 7.5 hz , 3h ) .
c nmr ( 100 mhz , acetone - d6 ) : 
  168.3 ( c ) , 153.4 ( c ) , 152.9 ( c ) , 148.0 ( c ) , 144.2 ( c ) , 138.9 
 ( c ) , 119.4 ( ch ) , 117.9 ( ch ) , 117.0 ( c ) , 113.7 ( ch ) , 23.4 ( ch2 ) , 15.8 ( ch3 ) .
lrms ( es ) : m / z ( % ) 331.91 ( 100 ) [ m + na ] .
hrms ( es ) : m / z calcd for c12h11n3o5nas [ m + na ] , 
 332.0317 ; found , 332.0323 .
the general procedure was followed using 
 3-(trifluoromethyl)aniline ( 622 l , 5.00 mmol ) .
the crude reaction 
 mixture was purified by column chromatography on silica gel ( 2:1 , 
 hexane / ethyl acetate ) to afford the product as a yellow solid ( 550 
 mg , 1.83 mmol , 37% ) ; mp 188189 c .
ir ( kbr ) max = 3271 ( m ) ( nh ) , 1668 ( s ) ( c = o ) , 1496 ( s ) ( no2 ) , 1334 ( s ) ( no2 ) , 1266 ( s ) , 1157 ( s ) ( cf3 ) , 1119 ( s ) ( cf3 ) cm .
h nmr ( 300 mhz , acetone - d6 ) :  
 10.37 ( br s , 1h ) , 8.30 ( s , 1h ) , 8.07 ( d , j = 8.3 hz , 1h ) , 7.66 ( d , j = 3.8 hz , 
 1h ) , 7.61 ( d , j = 7.9 hz , 1h ) , 7.537.50 
 ( m , 2h ) .
c nmr ( 75 mhz , acetone - d6 ) :  156.2 ( c ) , 152.3 ( c ) , 149.2 ( c ) , 140.2 ( c ) , 132.0 
 ( c ) , 131.2 ( ch ) , 127.3 ( c ) , 125.2 ( ch ) , 122.3 ( ch ) , 118.3 ( ch ) , 118.2 
 ( ch ) , 114.1 ( ch ) . lrms ( es ) : m / z ( % ) 301.05 ( 100 ) [ m + h ] . hrms ( es ) : m / z calcd for c12h8n2o4f3 [ m + h ] , 301.0436 ; found , 301.0428
calcd for c12h7f3n2o4 : c , 48.01 ; h , 
 2.35 ; n , 9.33 . found : c , 47.57 ; h , 2.27 ; n , 9.05 .
the general procedure was followed using 
 4-(trifluoromethyl)aniline ( 622 l , 5.00 mmol ) .
the crude reaction 
 mixture was purified by column chromatography on silica gel ( 3:1 , 
 hexane / ethyl acetate ) to afford the product a yellow solid ( 615 mg , 
 2.01 mmol , 41% ) ; mp 235236 c .
ir ( kbr ) max = 3406 ( m ) ( nh ) , 1697 ( s ) ( c =
o ) , 1538 ( s ) , 1528 ( s ) ( no2 ) , 1323 ( s ) ( no2 ) , 1129 ( s ) ( cf3 ) , 1115 
 ( s ) ( cf3 ) , 1069 ( s ) , 855 ( s ) cm . h nmr ( 300 mhz , acetone - d6 ) :  
 10.29 ( br s , 1h ) , 7.94 ( d , j = 8.6 hz , 
 2h ) , 7.61 ( d , j = 8.6 hz , 2h ) , 7.54 ( d , j = 3.8 hz , 1h ) , 7.40 ( d , j = 3.8 hz , 1h ) .
c nmr ( 100 mhz , acetone - d6 ) :  155.8 ( c ) , 152.8 ( c ) , 148.8 ( c ) , 
 142.5 ( c ) , 127.0 ( 2ch ) , 126.9 ( c ) , 124.0 ( c ) , 121.3 ( 2ch ) , 117.9 ( ch ) , 
 113.7 ( ch ) .
hrms ( es ) : m / z calcd for c12h8n2o4f3 [ m + h ] , 301.0436 ; 
 found , 301.0432 . a mixture of 22r ( 64 mg ,
0.20 mmol , 1.00 equiv ) , k2co3 ( 55 
 mg , 0.40 mmol , 2.00 equiv ) , and allyl bromide ( 0.03 ml , 0.40 mmol , 
 2.00 equiv ) in acetone ( 5 ml ) was stirred at room temperature for 
 3 h. the reaction mixture was then diluted in ethyl acetate ( 20 ml ) , 
 washed with water ( 10 ml ) , dried ( na2so4 ) , and 
 concentrated under vacuum .
the crude reaction mixture was purified 
 by column chromatography ( 3:1 , hexane / ethyl acetate ) to afford the 
 product as a yellow solid ( 53 mg , 0.15 mmol , 75% ) ; mp 155156 
 c .
ir ( kbr ) max = 3350 ( s ) , 3115 ( m ) ( nh ) , 
 1681 ( s ) ( c = o ) , 1611 ( m ) , 1504 ( s ) ( no2 ) , 1331 ( s ) 
 ( no2 ) , 1250 ( s ) ( c  o ) , 1135 ( s ) ( cf3 ) , 
 1113 ( s ) ( cf3 ) cm .
h nmr 
 ( 400 mhz , acetone - d6 ) :  10.07 ( br 
 s ) , 8.03 ( d , j = 2.5 hz , 1h ) , 7.89 ( dd , j = 9.1 hz , j = 2.5 hz , 1h ) , 7.52 ( d , j = 3.9 hz , 
 1h ) , 7.35 ( d , j = 3.9 hz , 1h , ) , 7.13 
 ( d , j = 9.1 hz , 1h ) , 6.025.87 
 ( m , 1h ) , 5.35 ( d , j = 17.5 hz , 1h ) , 5.15 
 ( d , j = 10.7 hz , 1h ) , 4.644.59 
 ( m , 2h ) .
c nmr ( 100 mhz , acetone - d6 ) :  155.5 ( c ) , 153.9 ( c ) , 152.6 ( c ) , 149.1 ( c ) , 133.8 
 ( ch ) , 131.8 ( c ) , 126.7 ( ch ) , 125.9 ( c ) , 123.2 ( c ) , 120.3 ( ch ) , 117.5 
 ( ch ) , 117.4 ( ch2 ) , 115.1 ( ch ) , 113.7 ( ch ) , 70.08 ( ch2 ) . lrms ( es ) : m / z ( % ) 378.91 ( 100 ) [ m + na ] .
hrms ( es ) : m / z calcd for c15h11n2o5naf3 [ m + na ] , 379.0518 ; 
 found , 379.0526 .
 recently , the world health organization approved the 
 nifurtimox  eflornithine 
 combination therapy for the treatment of human african trypanosomiasis , 
 renewing interest in nitroheterocycle therapies for this and associated 
 diseases . in this study , we have synthesized a series of novel 5-nitro-2-furancarboxylamides 
 that show potent trypanocidal activity , 1000-fold more potent 
 than nifurtimox against in vitro trypanosoma brucei with very low cytotoxicity against human hela cells . 
 more importantly , 
 the most potent analogue showed very limited cross - resistance to nifurtimox - resistant 
 cells and vice versa . 
 this implies that our novel , relatively easy 
 to synthesize and therefore cheap , 5-nitro-2-furancarboxylamides are 
 targeting a different , but still essential , biochemical process to 
 those targeted by nifurtimox or its metabolites in the parasites . 
 
 the significant increase in potency ( smaller dose probably required ) 
 has the potential for greatly reducing unwanted side effects and also 
 reducing the likelihood of drug resistance . 
 collectively , these findings 
 have important implications for the future therapeutic treatment of 
 african sleeping sickness . 
Introduction
Results and Discussion
Cross-Resistance Studies
Conclusions
Experimental Section