cystic fibrosis ( cf ) is one of the most 
 common genetic diseases 
 among caucasians .
the illness is best known for affecting the respiratory 
 system , where the lungs become obstructed with thick sticky mucus , 
 resulting in difficulty breathing and increased susceptibility to 
 bacterial infections .
the disease also affects the pancreas , liver , 
 intestines , and male reproductive system .
cf is caused by mutations 
 in the gene for the cystic fibrosis transmembrane 
 conductance regulator protein ( cftr ) . in healthy individuals ,
cftr 
 acts as a phosphorylation and nucleotide regulated channel which mediates 
 the flux of chloride across the apical membrane of epithelial cells.(1 ) in cf patients , mutant cftr fails to correctly 
 mediate chloride flux , and as a consequence , transepithelial chloride , 
 salt , and water transport is impaired .
this transport defect leads 
 to dehydration of the airway surface fluid , mucus desiccation , and 
 obstruction with recurrent episodes of inflammation and infection . 

currently , cf is treated through careful control of diet , physiotherapy , 
 antibiotics and , when lung function is severely degraded , lung transplantation.(2 ) the average life expectancy for a cf patient 
 is 47 years.(3 ) there are currently no pharmacological 
 agents approved for use in the clinic that address the basic molecular 
 defect underlying the disease .
the most common mutation in cftr , 
 occurring on at least one allele 
 in 70% of cf patients , is the deletion of a phenylalanine residue 
 at position 508 in the amino acid sequence ( f508del - cftr).(4 ) this defect results in protein misfolding , retention 
 in the endoplasmic reticulum , and its failure to reach the cell membrane.(5 ) in cell culture expression systems , this trafficking 
 defect can be partially overcome by incubating cells at 27 c,(6 ) however the protein s ability to function 
 as a chloride channel at the cell surface remains impaired relative 
 to that of the wild - type cftr.(7 ) another 
 less common mutation : g551d - cftr undergoes normal biogenesis and trafficking 
 to the cell surface(5 ) yet it exhibits defective 
 channel activation at that location.(8 ) thus , 
 two classes of small molecules have been identified , which may prove 
 to be useful in the pharmacological treatment of cf .  correctors  are those molecules which , through either 
 direct interactions with mutant cftr or perhaps any of the various 
 chaperone proteins , rescue the cell s ability to correctly 
 traffic the protein to the cell membrane . 
potentiators  
 are those molecules which result in a restoration of normal channel 
 activity in the mutant protein once it appears in the cell membrane . in 2006 ,
a group from vertex pharmaceuticals reported that 4-methyl-2-(5-phenyl-1h - pyrazol-3-yl)phenol ( vrt-532 , 1 ) was identified 
 in a high - throughput screen for the ability to potentiate channel 
 activity in mouse nih 3t3 cells expressing wild - type cftr or f508del - cftr 
 after low - temperature correction of the trafficking defect.(11 ) subsequent reports have shown that 1 also partially promotes proper protein trafficking of f508del - cftr 
 from the endoplasmic reticulum ( er ) to the cell membrane.(12 ) thus , 1 is both a potentiator and 
 a corrector .
it has been suggested on the basis of limited proteolysis 
 studies that this molecule may modify the conformation of f508del - cftr 
 such that it more closely resembles that of the wild - type protein , 
 and that this interaction is sufficient to partially rescue both protein 
 biogenesis and channel activity.(13 ) understanding 
 the molecular basis for this interaction will provide insight into 
 the mechanism of action of 1 as a modulator of cftr and 
 provide a template for development of therapeutically efficacious 
 compounds . to help elucidate the nature of the interaction between 1 and mutant cftr proteins , we have designed and prepared 
 derivatives 
 of 1 , modified with functional groups useful in biochemical 
 studies .
we have also developed a protocol for hydrogen isotope exchange 
 ( deuteration by d2o , as a model for tritiation ) .
we show 
 in functional assays of the channel activity of the mutant cftr proteins 
 that these derivatives retain the potentiator activity of the parent 
 compound and , hence , have the potential to define the molecular basis 
 for this activity .
a photoaffinity label is a molecule that contains a recognition 
 element for specific interactions with a site on a target protein 
 as well as a photoactivatible functional group that reacts to form 
 a covalent bond to the protein upon photoirradiation .
the labeled 
 site can then be identified using any of a number of biochemical techniques , 
 for instance limited proteolysis followed by gel electrophoresis and/or 
 mass spectrometry to suggest the location of the binding site for 
 the unlabeled ligand.(14 ) our synthetic 
 efforts to prepare derivatives of 1 including the photoaffinity 
 label , aryl azide 11 are illustrated in scheme 1 .
reaction of 2-hydroxy-5-methylacetophenone 2 with 4-iodobenzoyl chloride 3 afforded the 
 expected ester 6 in 86% yield .
venkataraman rearrangement by heating with t - buok in thf , intending to isolate diketone 8.(15 ) the crude product of this reaction 
 was a yellow solid which by h nmr spectroscopy suggested 
 the presence of numerous species .
enol 
 tautomers , which ought to be competent in the subsequent pyrazole 
 formation , it was decided to carry the crude yellow solid through 
 the next step .
treatment with hydrazine in acetic acid afforded the 
 desired pyrazole 9 in a serviceable 68% yield ( from ester 6 ) .
exchange of the iodide for azide using a cu / proline catalyzed 
 methodology reported by ma(16 ) was hampered 
 by the fact that the desired azide 11 and starting material 
 iodide 9 have identical chromatographic properties on 
 silica gel .
it was therefore impossible to gauge reaction progress 
 by tlc and impossible to isolate 11 from the reaction 
 mixture by column chromatography .
in fact , it was only by h nmr analysis of what appeared by tlc analysis to be clean recovered 9 that we were able to observe that the material was actually 
 a nearly 1:1 mixture of 9 and another compound with the 
 same nmr splitting pattern .
after this discovery , tlc analysis of 
 this material using a variety of common eluents failed to resolve 
 the two compounds .
treatment of a portion of the mixture with triphenylphosphine 
 in wet thf ( staudinger reaction conditions)(17 ) led to the formation of the amine compound 12 , confirming 
 that the second component of the 1:1 mixture was in fact the azide 11 , formed in only about 50% conversion in the iodide - for - azide 
 exchange reaction . given this poor conversion , as well as the inability 
 to separate 11 from 9 by any practical means
diazotization of 12 by treatment with t - butyl nitrite , followed by treatment with azidotrimethylsilane,(18 ) provided the desired azide 11 in 
 excellent 94% yield .
reagents and conditions : 
 ( i ) 
 pyridine , 0 c , 1 h ; ( ii ) kot - bu , thf , 50 c , 
 30 min ; ( iii ) h2n 
nh2xh2o , acoh , 65 c , 16 h ; ( iv ) nan3 , cui , l - proline , naoh , dmso , 100 c , 16 h ; ( v ) pph3 , h2o , thf ; ( vi ) sncl22h2o , 
 etoh , 78 c , 16 h ; ( vii ) t - buono , tms - n3 , mecn , 0 c to rt , 1 h. subsequent 
 to these experiments , we discovered that the entire 
 synthesis of the pyrazole core from acetophenone 2 and 
 benzoyl chloride 5 could be accomplished in one pot , 
 using pyridine as a solvent , at 50 c .
with monitoring by tlc , 
 additional 5 was added until 2 was completely 
 converted . at this point
, t - buok was added , again 
 using tlc to gauge complete consumption of the intermediate ester . 

thus , 
 in less than one day s reaction time , in one pot , and with 
 only a single , final chromatographic purification step , 1 was isolated in 65% yield from 2 .
using the same one - pot 
 sequence , 10 has also been prepared from 2 and 4 in 24% yield . under uv irradiation ,
aryl 
 azides extrude a molecule of n2 , transiently forming a 
 reactive nitrene intermediate , which rearranges 
 to an electrophilic cyclic ketenimine.(14 ) in photoaffinity labeling experiments , this occurs within a protein 
 binding site , and the ketenimine is quenched by a nucleophilic functional 
 group from the protein , thus forming a covalent linkage between the 
 label and the protein ( scheme 2a ) . to demonstrate 
 that azide 11 is capable of undergoing this chemistry , 
 a benzene solution of 11 , containing an excess of diethylamine , 
 was irradiated with an unfiltered ( full spectrum ) mercury lamp ( scheme 2b ) .
after workup and chromatographic purification , 
 diethylamine adduct 13 was isolated in 40% yield .
, the azide decomposes 
 to the reactive nitrene , which rearranges to the cyclic ketenimine , 
 which is ultimately quenched by a nucleophilic functional group from 
 the protein target .
the amino group on compound 12 was envisaged to be a 
 handle for further functionalization , for instance by reaction with 
 fluorescent electrophiles like 4-chloro-7-nitrobenzofurazan ( nbd chloride)(19 ) or 5-(dimethylamino)naphthalene-1-sulfonyl chloride 
 ( dansyl chloride),(20 ) however no reaction 
 was observed on treatment of 12 with either of these 
 reagents under their prescribed reaction conditions . rather than attempt 
 to optimize reaction with these costly reagents , we reasoned that 
 incorporation of a glycine amide might provide a more reactive aliphatic 
 amino group for further derivatization .
unfortunately , attempts to 
 acylate 12 with boc - glycine using a variety of standard 
 peptide coupling agents also failed .
ultimately , we decided to abandon 
 this approach , instead choosing to focus on palladium - catalyzed cross - coupling 
 chemistry to install a fluorophore .
the n - propargyl 
 dansyl amide 14 was prepared(21 ) and found to react cleanly with iodide 9 under sonogashira 
 reaction(22 ) conditions to afford 15 in 47% yield ( scheme 3 ) . given the success 
 of this reaction , boc - propargylamine 16 was also prepared(23 ) and reacted with 9 , affording 17 in 69% yield .
upon boc - deprotection , this compound will 
 afford a nucleophilic primary aliphatic amine , which can be readily 
 derivatized with electrophiles , should the need arise to prepare new 
 derivatives in the future . reagents and conditions : 
 ( i ) 
 pdcl2dppfch2cl2 , cui , 
 net3 , thf , 45 c , 15 h. dansyl 
 sulfonamides typically display fluorescence that is sensitive 
 to their local environments .
figure 1 shows 
 the emission spectrum of 15 , recorded in solvents of 
 different polarity .
when dissolved in chloroform , fluorescence emission 
 is maximal and centered at about 504 nm . in the most polar of the 
 media tested , an aqueous buffer solution , the emission maximum was 
 red - shifted by 37 nm to 541 nm , with an intensity 32% that of the 
 emission in chloroform . in the organic solvents dimethyl sulfoxide 
 and methanol , which are increasingly more polar than chloroform , but 
 less so than water
, fluorescent emissions maxima were intermediate 
 ( 529 nm in both ) and intensity decreased with increasing polarity . 

probe 15 contains the recognition element of 1 , conjugated to a fluorescent dansyl group through a propargylamine 
 linker and , as such , is expected to be a useful probe for the binding 
 interaction between 1 and cftr .
the direct binding of 
 probe 15 to cftr , and/or the conformational changes which 
 mediate protein folding and function , may be detected in fluorescence 
 anisotropy measurements.(24 ) the probe s 
 fluorescence intensity or emission maximum may change upon binding 
 to a hydrophobic pocket of cftr , which would be monitored by fluorescence 
 experiments . alternatively , as the emission for the tryptophan residues 
 in cftr overlaps with the excitation of this dansyl derivative , there 
 may be measurable resonance energy transfer upon binding to the protein.(25 ) lastly , we anticipate that competition experiments 
 with unlabeled drugs will identify other small molecules that bind 
 directly to cftr and permit quantification of their relative binding 
 constants .
fluorescence emission spectra of 20 m solution of 15 in chcl3 ( black solid line ) , dmso ( black dotted 
 line ) , meoh ( gray solid line ) , and aqueous buffer containing 20 mm 
 mops , 75 mm ki , 1 mm n - dodecyl--d - maltoside ( gray dotted line )
. a radiolabeled version of 1 would also be useful for quantitative studies of its binding 
 properties to cftr .
radioligands have shown to be invaluable tools 
 for the understanding of the mechanism of action of membrane proteins , 
 receptors , transporters , and channels.(26 ) to date , binding of cftr modulators has been detected using functional 
 assays which report the consequences of binding rather than binding 
 per se .
hence , the 
 development of radiolabeled 1 would enable direct assay 
 of the relative affinities for binding to cftr and mutant cftr proteins , 
 thereby providing insight into potential genotype - specific structural 
 differences in the binding site .
further , this tool would enable comparison 
 of the binding sites for various cftr modulators as they are identified . while it would be possible to incorporate a radioactive electrophile 
 onto the propargylamino group accessible from 17
, a method 
 that would be much less disturbing to the original structure of 1 would be to replace a 
non - exchangeable  hydrogen 
 atom for a tritium atom . as a model for this ,
a study was conducted 
 on the incorporation of less expensive and more safely handled nonradioactive 
 deuterium .
a small sample of 1 was dissolved in 1 m naoh 
 in d2o and heated at 90 c . under these conditions , 
 in addition to the classically exchangeable phenolic o  h and 
 pyrazole n 
h , the pyrazole c4h also gradually exchanged 
 for deuterium ( figure 2a ) .
the singlet 
 at 6.7 ppm , arising from the pyrazole c4h , was observed to 
 gradually decrease in integration , reaching 50% deuterium 
 incorporation over 24 h , decreasing to 20% ( i.e. , 80% 
 deuterium incorporation ) after 48 h. parts b and c of figure 2 show the aromatic region of the h nmr 
 spectrum of 1 at the beginning ( t = 
 0 ) and end ( t = 48 h ) of the experiment , respectively . 

after workup ( including aqueous acid to re - exchange the phenolic o  h 
 and the pyrazole n 
h for protons ) and tlc purification , 1 was reisolated in 88% yield and determined to have 80% deuterium 
 incorporation at pyrazole c4 .
if these conditions were to be repeated 
 using commercially available tritiated water ( radioactivity level 
 185 gbq / g),(29 ) they would be expected to 
 provide [ h]-1 at a level of 1.33  10 bq / mol,(30 ) without the need for 
 multiple synthetic steps or purification of radioactive materials .
( b ) 
 aromatic region of the h nmr spectrum of 1 in 1 m naoh / d2o , prior to any heating .
( c ) the same 
 region of the spectrum after heating at 90 c for 48 h , wherein 
 80% of the pyrazole c4 protons have been exchanged for deuterium . for these molecular probes to be 
 of use in biochemical experiments , it must be demonstrated that the 
 substitutions , in every case at the 4-position of the phenyl ring 
 of the parent structure 1 , do not interfere with the 
 compound s ability to interact with the protein . to determine 
 this ,
the effects of 912 and 15 on cftr channel function were assessed using patch clamp 
 electrophysiology . in our version of this assay ,
baby hamster kidney 
 ( bhk ) cells expressing f508del - cftr at the cell membrane ( after biosynthetic 
 rescue by low temperature culture conditions)(6 ) are loaded with nai , stimulated using forskolin ( 10 m ) , and 
 then treated acutely with the compounds at a final concentration of 
 10 m at approximately the 500 s mark .
the potentiation of channel 
 function is observed as an increased rate of iodide efflux from the 
 cells , measured using an iodide - sensitive electrode positioned in 
 the cell bath . after sufficient data is collected ( around 800 s ) , 
 cells are lysed , releasing all remaining iodide .
figure 3a depicts the measured level of iodide in the cell bath over 
 time , after acute treatment with either 1 or 11 at a final concentration of 10 m or dmso control .
the experiment 
 was also repeated with addition of 9 , 10 , 12 , and 15 ( traces not shown ) .
the channel 
 potentiating effects of the compounds ( measured as the maximum slope 
 of the line prior to addition subtracted from the maximum slope after 
 addition of the compound ) are depicted in figure 3b .
the dmso control showed negligible change in halide efflux 
 post forskolin stimulation ( 1.4  0.4 nm / s , n = 4 ) whereas acute addition of 1 displayed a marked 
 increase in efflux post forskolin stimulation ( 9.9  2.3 nm / s , n = 7 ) .
analogues 912 were 
 found to be biologically active , with efflux rates ranging from 5.1 
 to 7.5 nm / s . in this assay ,
fluorescent analogue 15 proved 
 to be inactive ( 1.7  1.2 nm / s , n = 6 ) .
the 
 effects of 1 and 912 were all determined to be significantly different from dmso control , 
 as assessed using analysis of variance ( anova ) statistics .
importantly , 
 the rates of efflux promoted by 912 were not significantly different than the rate observed for the 
 parent compound 1 , thus suggesting that substitution 
 of a small functional group at the 4-position of the phenyl ring is 
 not detrimental to the function of the molecule as a potentiator of 
 cftr channel function .
( a ) typical iodide efflux traces for 1 ( black 
 line ) , 11 ( dark - gray ) , and dmso control ( light - gray ) . 

cells were 
 activated with forskolin at 120 s ( arrow i ) , treated with compound 
 at 500 s ( arrow ii ) , and completely lysed at 800 s ( arrow iii ) .
( b ) 
 potentiating effects of all compounds tested ( measured as the maximum 
 slope of the iodide efflux trace prior to addition of the compound , 
 subtracted from the maximum slope post addition ) .
the reduced activity in the iodide efflux assay 
 of dansyl derivative 15 may be attributable to the compound s 
 reduced ability 
 to permeate the cell membrane rather than some hindered interaction 
 with cftr . to test this possibility , we examined the effect of this 
 molecule in a less complex reconstitution system , which allows the 
 direct interactions of small molecules with isolated cftr to be studied . 

g551d - cftr , a clinically relevant mutant which exhibits more severely 
 defective channel activity than f508del - cftr , was reconstituted into 
 liposomes of egg phosphatidylcholine . in this reconstituted system
, 
 a population of protein molecules are oriented inside out within the 
 liposome ( i.e. , domains that are normally intracellular in live cells 
 face out of the liposome , and vice versa ) .
therefore a compound does 
 not need to cross the membrane in order to interact with the intracellular 
 domains of the protein . if 15 retains the ability to 
 interact with cftr , it should potentiate channel activity in this 
 population of protein molecules .
the liposomes were loaded with 
 potassium iodide and suspended in 
 a 10 m solution of the compound to be tested in buffer lacking 
 iodide .
the concentration of iodide outside the liposomes is continuously 
 measured using an iodide sensitive probe , and cftr activity is observed 
 after addition of protein kinase a and atp to activate cftr , and valinomycin 
 to prevent charge build - up across the membrane due to cftr - mediated 
 iodide efflux which would slow and eventually stop further efflux . 

figure 4a shows the traces of iodide concentration 
 vs time for compound 1 ( black line ) and 15 ( dark - gray ) as well as a dmso control ( light - gray ) .
figure 4b shows the effect of each compound , measured as 
 the maximum slope of the iodide efflux trace following valinomycin 
 activation . under these conditions ,
compound 15 proved 
 to be an effective potentiator of channel activity , with an iodide 
 efflux rate 11.18  2.08 nm / s , n = 3 , although 
 with slightly diminished activity compared to 1 ( 16.85 
  1.26 nm / s , n = 5 ) .
these data suggest that 15 maintains the ability of the parent compound 1 to interact with cftr and modulate its activity but has a diminished 
 ability to cross the cell membrane .
therefore 15 should 
 be a useful fluorescent probe in systems of purified and reconstituted 
 protein and other in vitro experiments not involving whole live cells .
( a ) iodide 
 efflux assay on purified g551d - cftr , reconstituted in 
 proteoliposomes for compound 1 ( black line ) , fluorescent 
 derivative 15 ( dark - gray ) and dmso control ( light - gray ) . 

( b ) the effect of 1 and 15 on iodide efflux 
 in this system , measured as the maximum slope of the iodide efflux 
 trace after valinomycin activation .
the interaction between 1 and 
 mutants of cftr imbues 
 the protein with properties more like the wild - type , including improved 
 protein maturation and halide channel function ( the two key deficiencies 
 which lead to the symptoms of cystic fibrosis ) .
however , little is 
 known about the exact nature of this interaction . as tools to help 
 elucidate these details , useful molecular probes based on the structure 
 of 1
compound 11 bears an azide group substituted at the 4-position of the 
 parent compound s phenyl ring .
the compound ( and other derivatives 
 bearing small substitutions at the same position ) maintains the ability 
 to potentiate channel function in live cells expressing the major 
 mutant f508del - cftr and also undergoes a photoreaction generating 
 a strong electrophile .
sonogashira 
 coupling between iodide 9 and propargylamine derivatives 
 provided further useful compounds , including the fluorescent dansyl 
 sulfonamide 15 .
this compound is active as a potentiator 
 in a purified proteoliposome assay , however exhibits almost no activity 
 in live cells , suggesting that 15 maintains the ability 
 to interact with cftr mutants but that the larger substituent abrogates 
 the ability to cross the cell membrane . the environment - dependent 
 fluorescence properties of 15 will make it a useful probe 
 molecule for experiments in purified protein and/or cell lysates . 

finally , it has been demonstrated that the pyrazole c4h in 1 undergoes gradual exchange in hot alkaline water , and a 
 protocol to exploit this property to incorporate radioactive tritium 
 has been developed .
these molecular probes enable an array of biochemical 
 experiments to determine how and where 1 interacts with 
 mutant cftr , which will in turn enable the design of new , more potent 
 and specific compounds as potential treatments for cystic fibrosis . 

these experiments are currently underway , and their results will be 
 reported in due course .
all reagents and solvents were 
 used as received from commercial suppliers , except for tetrahydrofuran , 
 which was dispensed under nitrogen from an mbraun solvent purification 
 system immediately prior to use as a reaction solvent .
propargylamine 
 derivatives 14(21 ) and 16(23 ) were prepared according to 
 literature methods .
tlc was carried out on silica gel 60 f254 aluminum 
 backed plates , supplied by emd chemicals , eluting with the solvent 
 system indicated , and visualizing with uv light .
column chromatography 
 was carried out on silicycle siliaflash p60 , 4063 m 
 silica gel , eluting with the solvent system indicated below for each 
 compound . nuclear magnetic resonance ( h nmr ,
400 mhz , 
 and c nmr , 100 mhz ) spectroscopy was carried out on a 
 bruker avance 400 instrument in the deuterated solvents indicated 
 below for each compound , and spectra were referenced to the solvent 
 residual proton and c peaks .
high - resolution mass spectrometry 
 was performed using esi - tof or dart - tof as indicated .
uv / visible spectroscopy 
 was performed using a perkin - elmer lambda 20 instrument .
the purity of all 
 compounds subjected to biological assays was determined to be 95% 
 by hplc analysis , using an agilent zorbax rx - c8 4.6 mm  150 
 mm column , eluting at 0.6 ml / min with a binary gradient of 100% h2o to 100% mecn over the course of 1 h , except for compound 15 , for which the gradient was 100% h2o to 50% 
 mecn over the course of 1 h. solid 2-hydroxy-5-methylacetophenone 
 ( 2 , 3.948 g , 26.3 mmol ) was added to an ice - bath cooled 
 suspension of 4-iodobenzoyl chloride ( 3 , 7.008 g , 26.3 
 mmol ) in pyridine ( 140 ml ) .
the resultant clear yellow solution was 
 stirred over an ice - bath for one hour , after which time it was quenched 
 by pouring into an ice / hcl mixture ( 2 m , 600 ml ) .
this aqueous phase 
 was extracted three times with ch2cl2 ( 200 ml 
 each time ) .
the combined organic extracts were washed aqueous hcl 
 ( 1 m , 200 ml ) , then nahco3 ( saturated , 200 ml ) , and finally 
 nacl ( saturated , 200 ml ) , before drying over na2so4 and evaporating to afford the title ester 6 as 
 a yellow solid , which was used without further purification .
h nmr ( 400 mhz , ( cd3)2so ) :  7.997.95 ( m , 2h ) , 7.847.81 ( m , 2h ) , 
 7.76 ( d , j = 1.5 hz , 1h ) , 7.45 ( ddd , j = 8.5 hz , j = 2.0 hz , j = 0.5 
 hz , 1h ) , 7.22 ( d , j = 8.5 hz , 1h ) , 2.46 ( s , 3h ) , 
 2.38 ( s , 3h ) .
c nmr ( 100 mhz , ( cd3)2so )  196.87 , 163.89 , 145.74 , 137.41 , 135.39 , 133.53 , 130.92 , 
 130.27 , 129.58 , 127.97 , 123.19 , 102.14 , 28.83 , 19.81 .
prepared as described above for 6 , but using 2 ( 2.508 g , 16.7 mmol ) and 4-nitrobenzoyl 
 chloride ( 4 , 4.324 g , 23.3 mmol ) . as above for 6 , this compound was used after extractive workup without 
 further purification .
h nmr ( 400 mhz , cdcl3 ) :  8.388.36 
 ( m , 4h ) , 7.68 ( d , j = 2 hz , 1h ) , 7.42 ( ddd , j = 12 hz , j = 2 hz , j = 1 hz , 1h ) , 7.13 ( d , j = 8 hz , 1h ) , 2.54 ( s , 3h ) , 
 2.45 ( s , 3h ) .
c nmr ( 100 mhz , ( cd3)2co )  196.79 , 163.47 , 150.91 , 146.75 , 136.45 , 135.19 , 134.02 , 
 131.32 , 131.03 , 130.22 , 123.66 , 123.55 , 28.65 , 19.97 .
potassium tert - butoxide 
 ( 613 mg , 5.46 mmol ) was dissolved in thf ( 25 ml ) .
the solution was 
 warmed to 50 c , and ester 6 ( 2.000 g , 5.26 mmol ) 
 was added , upon which a yellow precipitate was observed to form .
the 
 mixture was stirred at 50 c for 30 min , then cooled in an ice - bath 
 and quenched by addition of aqueous acetic acid ( 10% v / v , 20 ml ) . 

the aqueous mixture was extracted three times with ch2cl2 ( 50 ml each time ) .
the combined organic extracts were dried 
 over na2so4 and evaporated to afford a yellow 
 solid .
this solid was taken up in glacial acetic acid ( 30 ml ) and 
 cooled in an ice - bath .
hydrazine hydrate (  5060%  , 
 5.7 ml , 100 mmol ) was added dropwise to the mixture .
the ice 
 bath was removed , and the mixture was warmed to 65 c for 16 
 h. the mixture was then cooled to room temperature and quenched with 
 water ( 30 ml ) and then extracted three times with ch2cl2 ( 50 ml each time ) .
the crude product 
 was purified by silica gel column chromatography , eluting with a gradient 
 of 1:10 to 1:1 ethyl acetate / hexanes , to afford the title compound 9 as a yellow solid .
h nmr ( 400 mhz , ( cd3)2co ) 
  13.03 ( bs , 1h ) , 10.64 ( bs , 1h ) , 7.86 ( d , j = 8 hz , 2h ) , 7.68 ( d , j = 8 hz , 2h ) , 7.58 ( s , 1h ) , 
 7.28 ( s , 1h ) , 7.01 ( d , j = 8 hz , 1h ) , 6.84 ( d , j = 8 hz , 1 h ) , 2.29 ( s , 3h ) .
c nmr ( 100 mhz , 
 ( cd3)2co )  177.09 , 138.16 , 135.28 , 135.11 , 
 129.74 , 127.91 , 127.44 , 127.03 , 124.44 , 123.56 , 118.14 , 116.49 , 116.23 , 
 19.77 .
prepared as described above for 9 but beginning with ester 7 ( 3.500 g , 11.7 mmol ) and 
 driving the rearrangement step with 17.6 mmol of sodium tert - butoxide .
the 
 crude product was purified by silica gel column chromatography , eluting 
 with a 3:17 ethyl acetate / hexanes , to afford the title compound 10 as an orange solid .
h nmr ( 400 mhz , ( cd3)2co ) 
  12.94 ( bs , 1h ) , 10.81 ( bs , 1h ) , 8.35 ( d , j = 1.5 hz , 2h ) , 8.17 ( d , j = 9 hz .
2h ) , 7.62 ( d , j = 1.5 hz , 1h ) , 7.46 ( s , 1h ) , 7.04 ( dd , j = 8.5 hz , j = 1.5 hz , 1h ) , 6.89 ( d , j = 6 hz , 1h ) , 2.30 ( s , 3h ) .
c nmr ( 100 mhz , ( cd3)2co )  161.24 , 148.25 , 136.27 , 130.83 , 129.35 , 
 128.17 , 127.12 , 125.08 , 124.46 , 119.15 , 117.37 , 116.78 , 102.01 , 20.55 . 

stannous chloride dihydrate ( 3.850 g , 17 
 mmol ) was added to a solution of 10 ( 1 g , 3.4 mmol ) in 
 95% ethanol ( 30 ml ) .
the mixture was heated to reflux for 16 h. the 
 mixture was cooled to room temperature and poured into aqueous naoh 
 ( 1 m , 150 ml ) .
the mixture was extracted twice with ethyl acetate 
 ( 75 ml each time ) .
the combined organic extracts were washed with 
 aqueous nacl ( saturated , 75 ml ) , then dried over na2so4 and evaporated .
the crude product was purified by silica 
 gel column chromatography , eluting with a 1:4 ethyl acetate / hexanes , 
 to afford the title compound to afford the title compound 12 as a pale - yellow solid .
h nmr ( 400 mhz , ( cd3)2co )  13.2 
 ( s , 1h ) , 10.9 ( s , 1h ) , 7.54 ( s , 1h ) , 7.48 ( d , j = 
 8.5 hz , 2h ) , 7.03 ( s , 1h ) , 6.97 ( d , j = 8 hz , 1h ) , 
 6.79 ( d , j = 8 hz , 1h ) , 6.63 ( d , j = 8 hz , 2h ) , 5.42 ( s , 2h ) , 2.27 ( s , 3h ) .
c nmr ( 100 
 mhz , ( cd3)2co )  154.17 , 149.38 , 129.54 , 
 129.34 , 127.66 , 126.77 , 126.69 , 126.32 , 120.08 , 116.72 , 116.35 , 114.36 , 
 97.06 , 19.70 .
esi - tof ms : m / z = 
 266.1288 ( m + h ) calculated for c16h16n3o : 266.1287 .
conversion of the amine to the azide was 
 conducted using the method reported by moses.(18 ) amine 12 ( 265 mg , 1 mmol ) was dissolved in acetonitrile 
 ( 10 ml ) and cooled in an ice - bath . to this was added tert - butyl nitrite ( 178 l , 154 mg , 1.5 mmol ) , followed by azidotrimethylsilane 
 ( 158 l , 138 mg , 1.2 mmol ) .
the mixture was warmed to room temperature 
 and stirred for one hour , after which the solvent was evaporated and 
 the crude product was purified by silica gel column chromatography , 
 eluting with a gradient of 1:10 to 1:1 ethyl acetate / hexanes , to afford 
 the title compound 11 as a yellow solid .
h nmr ( 400 mhz , ( cd3)2co )  12.82 ( bs , 1h ) , 10.70 ( bs , 1h ) , 7.93 
 ( d , j = 8.5 hz , 2h ) , 7.59 ( d , j = 
 2 hz , 1 h ) , 7.287.22 ( m , 3h ) , 7.02 ( dd , j = 8.5 hz , j = 2 hz , 1h ) , 6.83 ( d , j = 8 hz , 1h ) , 2.30 ( s , 3h ) .
c nmr ( 100 mhz , ( cd3)2co )  153.89 , 143.72 , 140.18 , 129.68 , 127.99 , 
 127.14 , 126.97 , 126.38 , 125.50 , 119.63 , 116.49 , 116.37 , 99.14 , 19.76 . 

benzoyl chloride ( 5 , 510 l , 617 mg , 4.4 mmol ) was added to a solution 
 of 2 ( 601 mg , 4 mmol ) in pyridine ( 10 ml ) , and the mixture 
 was warmed to 50 c . at 15 min intervals , more 5 ( 50 l ,
61 mg , 0.4 mmol ) was added , until tlc analysis ( 1:3 , 
 ethyl acetate / hexanes ) indicated complete conversion of 2 ( rf = 0.8 ) into the ester ( rf = 0.7 ) ( three aliquots were added over 45 min , for a 
 total of 660 l , 800 mg , 5.7 mmol benzoyl chloride in the reaction ) . 
 while stirring at 50 c , solid potassium tert - butoxide ( 987 mg , 8.8 mmol ) was gradually added to the reaction 
 mixture .
after 15 min , tlc analysis ( 1:3 , ethyl acetate / hexanes ) indicated 
 substantial but incomplete conversion of the ester ( rf = 0.7 ) to the rearranged  dione  ( under 
 these conditions , the tlc of this material was a streak extending 
 from the baseline to a major spot with rf = 0.6 ) .
more potassium tert - butoxide ( 258 mg , 2.3 
 mmol ) was added , and after an additional 30 min of stirring , tlc analysis 
 indicated complete consumption of the ester .
hydrazine hydrate (  5060%  , 
 2.5 ml , 44 mmol ) was added over the course of one minute , 
 followed by glacial acetic acid ( 2.0 ml , 2.1 g , 35 mmol ) .
after 1.5 
 h , tlc analysis indicated completed conversion to the desired product 
 ( 1:3 , ethyl acetate / hexanes , rf = 0.5 ) . 

the reaction mixture was poured into an ice / hcl mixture ( 1 m , 250 ml ) , 
 which was then extracted three times with ethyl acetate ( 100 ml each 
 time ) .
the combined organic extracts were washed with nahco3 ( saturated , 100 ml ) and nacl ( saturated , 100 ml ) and then dried 
 over mgso4 and evaporated .
the crude product was purified 
 by silica gel column chromatography , eluting with a 1:9 ethyl acetate / hexanes , 
 to afford the title compound 1 as a yellow solid .
yield 
 65% ( 648 mg ) ; mp 160161 c ( lit.(33 ) 156157 c ) .
h nmr ( 400 mhz , ( cd3)2co )  12.78 ( bs , 1h ) , 10.65 ( bs , 1h ) , 7.9087.876 
 ( m , 2h ) , 7.606 ( d , j = 2 hz , 1h ) , 7.5427.488 
 ( m , 2h ) , 7.4547.397 ( m , 1h ) , 7.274 ( s , 1h ) , 7.022 ( ddd , j = 8.5 hz , j = 2 hz , j = 0.5 , 1h ) , 6.838 ( d , j = 7.5 hz , 1h ) , 2.303 ( s , 
 3h ) .
c nmr ( 100 mhz , ( cd3)2co ) 
  154.82 , 152.95 , 145.16 , 130.50 , 130.28 , 129.92 , 129.57 , 128.79 , 
 127.79 , 126.48 , 117.33 , 117.28 , 100.04 , 20.59 .
dart - tof ms : m / z = 251.1175 ( m + h ) calculated for c16h15n2o : 251.1184 .
the synthesis of 1 has been previously reported , however no characterization data aside from melting point have been 
 published . prepared by a method analogous 
 to 1 above , beginning with 1 g ( 6.7 mmol ) of 2 and employing a total of 1.781 g ( 9.6 mmol ) of 4-nitrobenzoyl chloride 4 .
a solution of 11 ( 40 mg , 0.14 mmol ) and diethylamine ( 0.4 ml ) in c6d6 ( 1.5 ml ) in a quartz nmr tube was irradiated with a 
 140 w hanovia utility ultraviolet quartz lamp .
the tube was held 10 
 cm from the light bulb , and a stream of air was blown over the tube 
 to maintain the contents at room temperature .
after 5 h of exposure , 
 the mixture was evaporated under reduced pressure , and the crude product 
 was purified by silica gel column chromatography , eluting with 3:7 
 ethyl acetate / hexanes to afford the diethylamine adduct 13 .
h nmr ( 400 mhz , ( cd3)2co )  10.75 ( bs , 1h ) , 7.54 ( s , 1h ) , 7.21 ( d , j = 8.0 hz , 1h ) , 6.98 ( dd , j = 8.5 hz , j = 1.5 hz , 1h ) , 6.92 ( s , 1h ) , 6.80 ( d , j = 8.0 hz , 1h ) , 6.04 ( d , j = 8.0 hz , 1h ) , 5.73 ( t , j = 7.5 hz , 1h ) , 3.46 ( q , j = 7.0 hz , 4h ) , 
 2.28 ( s , 3h ) , 1.29 ( bs , 2h ) , 1.261.01(m , 6h ) .
c nmr ( 100 mhz , ( cd3)2co )  154.05 , 152.09 , 
 146.01 , 144.21 , 142.88 , 129.44 , 129.40 , 127.74 , 126.79 , 116.56 , 116.35 , 
 109.33 , 106.77 , 99.40 , 42.95 , 30.97 , 19.66 , 13.46 .
a 10 ml schlenck tube was fitted with a 
 magnetic stirring bar and a glass stopper and flushed with argon gas . 

the tube was then charged with 9 ( 203 mg , 0.54 mmol ) , 14(21 ) ( 312 mg , 1.08 mmol ) , and pdcl2dppfch2cl2 ( 22 mg , 0.027 
 mmol ) .
the tube was evacuated and backfilled with argon . under a stream 
 of argon ,
thf ( 4 ml ) , triethylamine ( 0.12 ml , 0.8 mmol ) , and finally 
 cui ( 4 mg , 0.02 mmol ) were added . the tube was sealed , and the mixture 
 was heated to 45 c for 15 h and then cooled back to room temperature . 

the mixture was diluted with ethyl acetate ( 25 ml ) and extracted with 
 0.1 m hcl ( 10 ml ) , then brine ( 10 ml ) , then dried over anhydrous sodium 
 sulfate and evaporated under reduced pressure .
the crude material 
 was purified by silica gel column chromatography , eluting with 1:4 
 ethyl acetate / hexanes , to afford the title compound 15 as a yellow solid .
h nmr ( 400 mhz , ( cd3)2co )  12.82 
 ( bs , 1h ) , 10.62 ( bs , 1h ) , 8.51 ( d , j = 8.5 hz , 1h ) , 
 8.42 ( d , j = 8.5 , 1h ) , 8.33 ( dd , j = 7.5 hz , j = 1.0 hz , 1h ) , 7.72 ( d , j = 8 hz , 2h ) , 
 7.66  7.57 ( m , 3h ) , 7.29  7.23 ( m , 3h ) , 7.02 ( dd , j = 8 hz , j = 2 hz , 1h ) , 6.94 ( d , j = 8 hz , 2h ) , 6.84 ( d , j = 8 hz , 1 h ) , 
 4.12 ( d , j = 5 hz , 2h ) , 2.81 ( s , 6h ) , 2.30 ( s , 3h ) .
c nmr ( 100 mhz , ( cd3)2co )  152.85 , 
 137.24 , 137.21 , 132.72 , 130.98 , 130.90 , 130.75 , 130.59 , 130.29 , 130.27 , 
 128.93 , 128.73 , 127.86 , 126.08 , 124.27 , 123.27 , 120.40 , 117.36 , 117.28 , 
 117.12 , 115.97 , 100.47 , 86.47 , 83.50 , 45.59 , 33.77 , 20.57 ( one c signal was not observed after 15000 scans ) .
dart - tof ms : m / z = 537.1958 ( m + h ) calculated for c31h29n4o3s : 537.1960 .
hplc 
 rt = 25.2 min . prepared by a method analogous to 15 above , but using 9 ( 610 mg , 1.62 mmol ) , 16(23 ) ( 500 mg , 3.24 mmol ) , pdcl2dppfch2cl2 ( 66 mg , 0.084 
 mmol ) , thf ( 12 ml ) , triethylamine ( 0.35 ml , 2.5 mmol ) , and cui ( 11 
 mg , 0.06 mmol ) .
h nmr ( 400 mhz , ( cd3)2co )  12.86 
 ( bs , 1h ) , 10.66 ( bs , 1h ) , 7.87 ( d , j = 8 hz , 2h ) , 
 7.60 ( d. j = 1.5 hz , 1h ) , 7.54 ( d , j = 7 hz , 2h ) , 7.31 ( s , 1h ) , 7.03 ( dd , j = 8.5 hz , j = 2 hz , 1h ) , 6.876.82 ( m , 1h ) , 6.45 ( bs , 1h ) , 
 4.13 ( d , j = 6 hz , 2h ) , 2.30 ( s , 3h ) , 1.45 ( s , 9h ) .
c nmr ( 100 mhz , ( cd3)2co )  156.32 , 
 154.70 , 136.16 , 135.98 , 133.02 , 130.59 , 128.90 , 127.87 , 126.46 , 124.12 , 
 119.04 , 117.36 , 117.16 , 100.53 , 89.87 , 82.18 , 79.38 , 31.30 , 28.59 , 
 20.58 .
dart - tof ms : m / z = 404.1966 
 ( m + h ) calculated for c24h26n3o3 : 404.1974 .
to a standard 
 nmr tube containing 1 ml of 1 m naoh in d2o was added 1 ( 25 mg , 0.1 mmol ) .
reaction progress was monitored by periodically 
 collecting a h nmr spectrum and observing the relative 
 integration of the pyrazole c4h signal at  6.7 ppm . 

after 48 h , the reaction was cooled to room temperature and diluted 
 with 10 ml of h2o .
the mixture was cooled in an ice bath 
 and acidified to ph 1 by dropwise addition of 1 m hcl .
the 
 mixture was extracted with 3  10 ml et2o , concentrated 
 under reduced pressure , and purified by preparative tlc eluting with 
 1:3 etoac / hexanes to afford 22 mg ( 88% ) of 1c4-d with 80% deuteration as determined by h nmr 
 integration .
h nmr ( 400 mhz , ( cd3)2co )  12.78 ( bs , 1h ) , 10.65 ( bs , 1h ) , 7.917.88 ( m , 
 2h ) , 7.61 ( d , j = 2 hz , 1h ) , 7.537.49 ( m , 
 2h ) , 7.457.41 ( m , 1h ) , 7.27 ( s , 0.2h ) , 7.02 ( dd , j = 8.5 hz , j = 1.5 hz , 1h ) , 6.84 ( d , j = 8.5 hz , 1h ) , 2.30 ( s , 3h ) .
c nmr ( 100 mhz , ( cd3)2co )  155.94 , 154.82 , 142.80 , 130.49 , 129.97 , 
 129.56 , 128.80 , 127.82 , 126.48 , 117.33 , 117.30 , 100.06 , 20.58 ( one c signal not observed after 13686 scans ) .
a stock solution of 15 ( 5 mm in dmso ) was 
 diluted to 20 m in 100% chloroform , 100% methanol , 100% dmso 
 or buffer containing 20 mm mops , 75 mm ki , and 1 mm n - dodecyl--d - maltoside ( ddm ) .
emission spectra were 
 obtained with an excitation wavelength of 360 nm , and 4 nm excitation 
 and emission bandpass , at 22 c in a 0.5 cm micro quartz cuvette 
 on a pti ( photon technology international , london , on , canada ) quantamaster 
 qm/80 steady state spectrofluorimeter .
curves are an average of two 
 measurements at 0.5 nm increments with a 1 s integration time and 
 were corrected using a solvent blank measured under identical conditions .
using bhk cells stably expressing f508del - cftr , continuous recording 
 cell - based iodide efflux assays were performed as previously described.(27 ) in brief , cells were grown to approximately 
 90100% confluency .
cells were loaded with nai loading buffer 
 ( 3 mm kno3 , 2 mm ca(no3)2 , 11 mm 
 glucose , 20 mm hepes , and 136 mm nai ) at 37 c for 1 h. nai loading 
 buffer was aspirated , and cells were washed four times in iodide - free 
 efflux buffer ( 3 mm kno3 , 2 mm ca(no3)2 , 11 mm glucose , 20 mm hepes , and 136 mm nano3 ) .
cells 
 were scraped in 1 ml of iodide - free efflux buffer and collected by 
 centrifugation ( 350 g for 5 min at 25 c ) .
iodide - free efflux 
 buffer was removed , and the cell pellet was resuspended in 400 l 
 of iodide - free efflux buffer .
iodide efflux was measured at room temperature 
 using an iodide - sensitive electrode ( lazar research laboratories , 
 los angeles , ca ) .
f508del - cftr at the cell surface was stimulated 
 with 10 m forskolin , followed by addition of 10 m of 
 compound ( 1 , 9 , 10 , 11 , 12 , or 15 ) . five minutes after addition 
 of each compound , triton x-100 ( sigma ) was used to lyse the cells 
 to ensure iodide was properly loaded .
the maximal iodide efflux rate 
 was quantified over a 1 min interval associated with the largest positive 
 slope during the 4- to 5-min time period after the addition of the 
 activation cocktail .
traces were recorded using the digidata 1320a 
 data acquisition system with clampex 8 software ( molecular devices , 
 sunnyvale , ca ) .
mutant g551d - cftr 
 was purified from sf9 cells expressing the channel with a c - terminal 
 his10 tag from 0.5 l of culture .
cells were homogenized 
 in the presence of protease inhibitors using an emulsiflex c3 high - pressure 
 homogenizer ( avenstin , ottawa , on .
canada ) , and plasma membranes were 
 isolated on a 35% sucrose cushion by ultracentrifugation .
one - fifth 
 of the membrane pellet was solubilized by the presence of 2% fos - choline 14 ( anatrace , maumee , oh ) for 1 
 h with resuspension by 30 g needle , and insoluble material was removed 
 by ultracentrifugation .
mississauga , on , canada ) , and the beads were washed with 1050 
 mm imidazole buffer containing 1 mm dodecylmaltoside ( ddm ) .
g551d - cftr 
 was eluted with 600 mm imidazole buffer containing 1 mm ddm , and lower 
 molecular weight contaminants and imidazole were removed by centrifugation 
 with an amicon ultra centrifugal filter device ( milipore corp .
billerica , 
 ma ) , yielding up to 0.25 mg of pure g551d - cftr protein per 
 liter of culture used , in 20 mm mops , 75 mm ki , and 1 mm ddm , ph 7.4 . 

g551d - cftr was reconstituted into 5 mg of egg phosphatidylcholine 
 ( avanti polar lipids , alabaster , ab ) at a protein : lipid ratio of approximately 
 1:300 ( w / w ) by incubation in the presence of 20 mm mops , 75 mm ki , 
 1 mm ddm , and lipid for 30 min , followed by passage through an extracti - gel 
 d detergent - binding column ( pierce corp .
the external iodide in the reconstituted protein sample was exchanged 
 for 75 mm k - glutamate by sephadex g50 gel filtration resin equilibrated 
 with 20 mm mops , 75 mm k - glutamate , yielding g551d - cftr liposomes 
 containing 75 mm ki on the vesicle interior and 75 mm k - glutamate 
 in the bulk solution .
the increase in external iodide concentrations 
 was monitored continuously using an iodide - selective electrode ( lazar 
 research laboratories , los angeles , ca ) interfaced to the digidata 
 1320a data acquisition system and controlled by clampex 8 software 
 ( axon instruments , sunnyvale , ca ) , as described above for the cell - based 
 assay .
madison , 
 wi ) and mg - atp ( 200 nm and 1 mm , respectively ) were added to the external 
 solution to initiate iodide flux , and 20 nm valinomycin was added 
 to prevent charge - buildup across the membrane that would limit iodide 
 flux .
control liposomes were prepared and treated identically but 
 in the absence of g551d - cftr .
verification that sufficient iodide 
 was trapped in the proteoliposomes was done by lysing the liposomes 
 with 0.5% triton x-100 at late time points .
a working solution 
 of 20 m was prepared from the stock in 20 mm mops and 75 mm 
 k - glutamate buffer and added to the vesicle solution at a ratio of 
 1:1 to yield a final concentration of 10 m , 5 min before initiation 
 of flux by valinomycin .
 cystic fibrosis is a genetic disease caused by mutations 
 in the 
 gene for the cystic fibrosis transmembrane conductance regulator ( cftr ) 
 protein . in vitro experiments 
 have demonstrated that 4-methyl-2-(5-phenyl-1h - pyrazol-3-yl)phenol ( vrt-532 , 1 ) is able 
 to partially restore the function of mutant cftr proteins . to help 
 elucidate the nature of the interactions between 1 and 
 mutant cftr , molecular probes based on the structure of 1 have been prepared . 
 these include a photoreactive aryl azide derivative 11 and a fluorescent dansyl sulfonamide 15 . 
 additionally , 
 a method for hydrogen isotope exchange on 1 has been 
 developed , which could be used for the incorporation of radioactive 
 tritium . using iodide efflux assays , 
 the probe molecules have been 
 demonstrated to modulate the activity of mutant cftr in the same manner 
 as 1 . 
 these probe molecules enable a number of biochemical 
 experiments aimed at understanding how 1 rescues the 
 function of mutant cftr . 
 this understanding can in turn aid in the 
 design and development of more efficacious compounds which may serve 
 as therapeutic agents in the treatment of cystic fibrosis . 
Introduction
Results and Discussion
Conclusion
Experimental Section