Patent Application: US-201113641713-A

Abstract:
this invention relates generally to compounds that are glucuronidase inhibitors . glucuronidase inhibitors described include phenoxy thiophene sulfonamides . other compounds , for instance pyridine sulfonyls , benzene sulfonyls , thiophene sulfonyls , thiazole sulfonyls , thiophene carbonyls , and thiazole carbonyls , are also contemplated . also contemplated are compositions including one or more of such compounds for use inhibiting glucuronidase and methods of using one or more of such compounds as a co - drug to be used in combination with the anticancer drug cpt - 11 .

Description:
for purposes of the following detailed description , it is to be understood that the invention may assume various alternative variations and step sequences , except where expressly specified to the contrary . moreover , other than in any operating examples , or where otherwise indicated , all numbers expressing , for example , quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term “ about ”. accordingly , unless indicated to the contrary , the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention . at the very least , and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims , each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques . notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations , the numerical values set forth in the specific examples are reported as precisely as possible . any numerical value , however , inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements . also , it should be understood that any numerical range recited herein is intended to include all sub - ranges subsumed therein . for example , a range of “ 1 to 10 ” is intended to include all sub - ranges between ( and including ) the recited minimum value of 1 and the recited maximum value of 10 , that is , having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10 . in this application , the use of the singular includes the plural and plural encompasses singular , unless specifically stated otherwise . in addition , in this application , the use of “ or ” means “ and / or ” unless specifically stated otherwise , even though “ and / or ” may be explicitly used in certain instances . in the present invention , 76 phenoxythiophene sulfonamides from a 35 , 000 compound diversity set library were tested for their ability to inhibit the bacterial enzyme β - glucuronidase . the structures and inhibitory activity of the compounds are shown in table 1 . in the present invention , 18 analogs of brite - 355252 were synthesized and tested to initially explore the structural relationship these compounds display towards inhibition of β - glucuronidase . the structures and inhibitory activity of the 18 analogs of brite - 355252 are shown in table 2 . wherein r 1 , r 2 , r 3 , r 4 and r 5 are as defined above can be prepared by a process comprising the steps of ( a ) reacting a halo thiophene - sulfonyl halo and r 1 — n — h 2 , to form a resultant n - monoprotected thiophene sulfonamide having a first n - protecting group comprising r 1 , ( b ) reacting the resultant n - monoprotected amide with r 2 — n - halo and a catalyst in a base , to form a resultant n , n - diprotected thiophene sulfonamide having also a second n - protecting group comprising r 2 , ( c ) reacting the resultant n , n - diprotected thiophene sulfonamide with cs 2 co 3 and phenol group substituted by r ; wherein r is selected from piperazin , 4 -( c 1 - c 4 alkyl ) piperazin , 4 - methylpiperazin , 4 - ethyl - piperazin , and 4 - propylpiperazin , in a solvent , and then removing the solvent , to obtain a resultant n , n - diprotected phenoxy thiophene sulfonamide , and ( d ) reacting the resultant n , n - diprotected phenoxy thiophene sulfonamide with a deprotecting agent that is selective for deprotecting the second n - protecting group , thereby removing the second n - protecting group , and forming a n - monoprotected phenoxy thiophene sulfonamide . the halogen atom of the halo thiophene - sulfonyl halo compound is selected from bromine , chlorine , fluorine and iodine . any base that will in combination with the n - monoprotected amide with r 2 — n - halo and a catalyst result in a n , n - diprotected thiophene sulfonamide can be used . non - limiting examples of bases that can be used are et 3 n , na 2 co 3 , k 2 co 3 and nah and any base described in the examples . in an embodiment of the invention the halo thiophene - sulfonyl halo is dichlorothiophene - sulfonyl chloride and r1 - n — h 2 is naphthylmethylamine . these groups are mixed and cooled to form a n - monoprotected thiophene sulfonamide , having a first n - protecting group that comprises naphthylmethyl . in an embodiment of the invention the resultant n - monoprotected thiophene sulfonamide , is mixed with methoxybenzyl bromide and a catalyst that can be used in a finkelstein reaction in sodium hydride , and cooled thereby forming a n , n - diprotected thiophene sulfonamide having also a second n - protecting group that comprises methoxybenzyl , and the resultant n , n - monoprotected thiophene sulfonamide , and cs 2 co 3 and tea - butyl ( hydroxyphenyl ) piperazine - carboxylate in a solvent , are mixed and heated . the solvent is then removed to obtain a resultant n , n - diprotected phenoxy thiophene sulfonamide . in an embodiment of the invention the resultant n , n - diprotected phenoxy thiophene sulfonamide is mixed with a deprotecting agent that is selective for deprotecting the second n - protecting group , thereby removing the methoxy benzyl that is the second n - protecting group , and thereby forming a n - alkyl or n - aryl phenoxy thiophene sulfonamide . examples of non - limiting embodiments of the invention are where : the dichlorothiophene - sulfonyl chloride is 4 , 5 - dichlorothiophene - 2 - sulfonyl chloride ; the naphthylmethylamine is 1 - naphthylmethylamine ; the methoxybenzyl bromide is 4 - methoxybenzyl bromide ; the catalyst is tetrabutyl - ammonium iodide ; the butyl ( hydroxyphenyl ) piperazine - carboxylate is tert - butyl - 4 -( 3 - hydroxyphenyl ) piperazine - 1 - carboxylate ; the solvent is dimethyl formamide and / or the selective deprotecting agent comprises dichloromethane and trifluoroacetic acid ; or a combination thereof . in addition to dimethyl formamide , non - limiting examples of solvents that can be used are dmso and dioxane and the solvents described in the examples . the following reaction scheme 1 illustrates the preparation of compounds within the scope of the present invention : scheme 1 refers to the preparation of compounds of formula i . referring to scheme 1 , compounds of the formula i are prepared by reacting commercially available 4 , 5 - dichlorothiophene - 2 - sulfonyl chloride 1 with an amine to generate dichlorothiophene sulfonamide 2 . pmb ( p - methoxybenzyl ) protected 4 , 5 - dichlorothiophene sulfonamide 3 is generated by reacting compound 2 with nah in dmf , pmethoxybenzyl bromide and a catalytic amount of tbai . nucleophilic displacement of the c - 5 chlorine with a phenol in the presence of cs 2 co 3 produce n , n - diprotected 5 -( 3 - phenoxy )- thiophene - 2 - sulfonamide 3 . in the final step , the protecting group is removed using tfa in dcm ( 1 : 1 ) to give the desired compound . the term “ pharmaceutically acceptable salts ” refers to the non - toxic , inorganic and organic acid addition salts and base addition salts of compounds of the present invention . such conventional non - toxic salts include those derived from inorganic acids such as hydrochloric , hydrobromic , sulfuric , sulfamic , phosphoric , and nitric acid ; and the salts prepared from organic acids such as acetic , propionic , succinic , glycolic , stearic , lactic , malic , tartaric , citric , ascorbic , palmoic , maleic , hydroxy - maleic , phenylacetic , glutamic , benzoic , salicylic , sulfanilic , 2 - acetoxybenzoic , fumaric , toluenesulfonic , methanesulfonic , ethane disulfonic , oxalic , and isethionic acid . pharmaceutically acceptable salts from amino acids may also be used . such as salts of arginine and lysine . pharmaceutically acceptable salts may be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods . generally , such salts may be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent , or in a mixture of the two . as used herein , the terms “ treatment ” and “ therapy ” and the like refer to alleviate , slow the progression , prophylaxis , or attenuation of existing disease . as used herein , the terms “ inhibit ,” “ inhibiting ,” and the like means that the activity of glucuronidase is reduced . as used herein , the term “ subject ” means an animal or human . the pharmaceutical compositions of this invention comprise one or more compounds that inhibit glucuronidase and one or more pharmaceutically acceptable carriers , diluents , and excipients . pharmaceutical compositions of the present invention may be in a form suitable for use in this invention for examples compositions may be formulated for i ) oral use , for example , aqueous or oily suspensions , dispersible powders or granules , elixirs , emulsions , hard or soft capsules , lozenges , syrups , tablets or trouches ; ii ) parenteral administration , for example , sterile aqueous or oily solution for intravenous , subcutaneous , intraperitoneal , or intramuscular ; iii ) delivered intracerebrally or iv ) topical administration , for example , a suppository or ointment . as used herein the term “ pharmaceutically acceptable ” is meant that the carrier , diluent , excipients , and / or salt must be compatible with the other ingredients of the formulation including the active ingredient ( s ), and not deleterious to the recipient thereof . “ pharmaceutically acceptable ” also means that the compositions , or dosage forms are within the scope of sound medical judgment , suitable for use for an animal or human without excessive toxicity , irritation , allergic response , or other problem or complication , commensurate with a reasonable benefit / risk ratio . a compound can also be used in the manufacture of a medicament . this medicament can be used for the purposes described herein . the compositions or medicaments normally contain about 1 to 99 %, for example , about 5 to 70 %, or from about 5 to about 30 % by weight of the compound or its pharmaceutically acceptable salt . the amount of the compound or its pharmaceutically acceptable salt in the composition will depend on the type of dosage form and the pharmaceutically acceptable excipients used to prepare it . the dose of the compounds of this invention , which is to be administered , can cover a wide range . the dose to be administered daily is to be selected to suit the desired effect . actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient , which is effective to achieve the desired therapeutic response for a particular patient , composition , and mode of administration without causing undue side effects or being toxic to the patient . the selected dosage level will depend upon a variety of factors , including the activity of the particular compound of the present invention employed , the route of administration , the time of administration , the rate of excretion of the particular compound being employed , the duration of the treatment , other drugs , compounds and / or materials used in combination with the particular compounds employed , the age , sex , weight , condition , general health and prior medical history of the patient being treated , and like factors well known in the medical arts . as used herein , “ effective amount ” and the like means the amount of the compound or composition necessary to achieve a therapeutic effect . an effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the age , sex , and weight of the subject . dosage regimens can be adjusted to provide the optimum therapeutic response . for example , several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation . compounds of the invention can be formulated into compositions that can be administered to a subject in need of a glucuronidase inhibitor . the compounds or compositions thereof are used for inhibition of glucuronidase . the compounds or compositions thereof are used in methods for treating a subject in need of a glucuronidase inhibitor . the compounds or compositions are administered in an amount that is effective to inhibit the glucuronidase . in some embodiments of the invention it is ∃ glucuronidase or bacterial ∃ glucuronidase that is inhibited . the compounds or compositions can be administered prior to , concurrently with or after administration of a camptothecin - derived anticancer agent such as cpt - 11 . administration of the compounds or compositions may result in certain benefits such as decreasing the dose of the anticancer drug , increasing the tolerance of the anticancer drug and alleviating side effects from the use of the anticancer drug . side effects include gastrointestinal side effects . the invention is further understood by reference to the following examples , which are intended to be purely exemplary of the invention . the present invention is not limited in scope by the exemplified embodiments , which are intended as illustrations of single aspects of the invention only . any methods that are functionally equivalent to those described in the examples are within the scope of the invention . various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description . such modifications fall within the scope of the appended claims . the full - length e . coli β - glucuronidase gene was obtained from bacterial genomic dna and was cloned into the pet - 28a expression plasmid ( novagen ) with an n - terminal 6 ×- histidine tag . bl21 - de3 competent cells were transformed with the expression plasmid and grown in the presence of kanamycin ( 25 ug / ml ) in lb medium with vigorous shaking at 37 ° c . until an od 600 of 0 . 6 was attained . the expression was induced with the addition of 0 . 3 mm isopropyl - 1 - thio - d - galactopyranoside ( iptg ) and further incubated at 37 ° c . for 4 hours . cells were collected by centrifugation at 4500 × g for 20 min at 4 ° c . cell pellets were resuspended in buffer a ( 20 mm potassium phosphate , ph 7 . 4 , 25 mm imidazole , 500 mm nacl ), along with pmsf ( 2 μl / ml from 100 mm stock ) and 0 . 05 μl / ml of protease inhibitors containing 1 mg / ml of aprotinin and leupeptin . resuspended cells were sonicated and centrifuged at 14 , 500 × g for 30 min to clarify the lysate . the cell lysate was flowed over a pre - formed ni - nta his - trap gravity column and washed with buffer a . the ni - bound protein was eluted with buffer b ( 20 mm potassium phosphate , ph 7 . 4 , 500 mm imidazole , 500 mm nacl ). collected fractions were then tested for initial purity by sds - page . relatively pure (˜ 85 %) fractions were combined and loaded into the äktaxpress fplc system and passed over a hiload ™ 16 / 60 superdex ™ 200 gel filtration column . the protein was eluted into 20 mm hepes , ph 7 . 4 , and 50 mm nacl for crystallization and activity assays . two milliliter fractions were collected based on highest ultraviolet absorbance at 280 nm . fractions were analyzed by sds - page ( which indicated & gt ; 95 % purity ), combined , and concentrated to 10 mg / ml for long - term storage at − 80 ° c . the β - glucuronidase assay was performed by the addition of 0 . 5 μl of compound ( or dmso ) to the well of a black 384 - well plate followed by the addition of 30 μl of diluted β - glucuronidase enzyme . the enzyme was diluted in assay buffer ( 50 mm hepes , ph 7 . 4 ) plus 0 . 0166 % triton x - 100 for a final enzyme concentration of 50 pm and final detergent concentration of 0 . 01 %. after a 15 minute incubation at room temperature ( 23 ° c . ), 20 ul of substrate , 4 - methylumbelliferyl β - d - glucuronide hydrate ( 4mug ) diluted into assay buffer , was added to the reaction for a final concentration of 125 um . β - glucuronidase hydrolyzes the non - fluorescent 4mug resulting in a fluorescent product , 4 - methylumbelliferyl . after a 30 minute incubation at room temperature , the reaction was stopped by the addition of 20 ul 1 m na 2 co 3 . the fluorescence ( in relative fluorescence units , rfu ) was measured using a 355 nm excitation filter and 460 nm emission filter using a victor v ( perkin elmer ) plate reader . minimum ( min ) controls were performed using reactions with no enzyme . maximum ( max ) controls were performed using no compound . 1 % dmso was maintained in all reactions . percent inhibition was calculated using rfu data by the following formula : [ 1 −( assay readout - average of min )/( average of max - average of min )]× 100 . the known β - glucuronidase inhibitor , d - glucaric acid - 1 , 4 - lactone monohydrate , was used to validate the assay and serve as a positive control . ic 50 value was defined as the concentration of inhibitor calculated to inhibit 50 % of the assay signal based on a serial dilution of compound . values were calculated using either a four or three - parameter dose response ( variable slope ) equation in graphpad prism ™ or activitybase ™. for the ic 50 determinations , serial dilutions of compounds were performed in 100 % dmso with a two - fold dilution scheme resulting in 10 concentrations of compound . these results are shown in tables 1 and 2 . all solvents and reagents were obtained from commercial sources and used without further purification unless otherwise stated . all reactions were performed in oven - dried glassware ( either in rb flasks or 20 ml vials equipped with septa ) under an atmosphere of nitrogen and the progress of reactions was monitored by thin - layer chromatography and lc - ms . analytical thin - layer chromatography was performed on precoated 250 μm layer thickness silica gel 60 f 254 plates ( emd chemicals inc .). visualization was performed by ultraviolet light and / or by staining with phosphomolybdic acid ( pma ) or p - anisaldehyde . all the silica gel chromatography purifications were carried out by using combiflash ® rf ( teledyne isco ) and combiflash ® companion ® ( teledyne isco ) either with etoac / hexane or meoh / ch 2 cl 2 mixtures as the eluants . melting points were measured on a mel - temp ® capillary melting point apparatus and are uncorrected . proton nuclear magnetic resonance ( 1 h nmr ) spectra and carbon nuclear magnetic resonance ( 13 c nmr ) spectra were recorded on a varian vnmrs - 500 ( 500 mhz ) spectrometer . chemical shifts ( δ ) for proton are reported in parts per million ( ppm ) downfield from tetramethylsilane and are referenced to it ( tms 0 . 0 ppm ). coupling constants ( j ) are reported in hertz . multiplicities are reported using the following abbreviations : br = broad ; s = singlet ; d = doublet ; t = triplet ; q = quartet ; m = multiplet . chemical shifts ( δ ) for carbon are reported in parts per million ( ppm ) downfield from tetramethylsilane and are referenced to residual solvent peaks : carbon ( cdcl 3 77 . 0 ppm ). mass spectra were recorded on an agilent 1200 series lc / ms instrument equipped with a xterra ® ms ( c - 18 , 3 . 5 μm ) 3 . 0 × 100 mm column . to a solution of 4 , 5 - dichlorothiophene - 2 - sulfonyl chloride ( 1 . 000 g , 4 . 002 mmol ) in anhydrous ch 2 cl 2 ( 20 ml ) was added 1 - naphthylmethylamine ( 0 . 630 g , 4 . 007 mmol ) followed by et 3 n ( 0 . 84 ml , 6 . 027 mmol ) and stirred at room temperature for 2 h . the reaction mixture was diluted with water ( 20 ml ) and extracted with ch 2 cl 2 ( 100 ml ), washed with brine , dried ( na 2 so 4 ) and concentrated under vacuo . the residue was purified by recrystallization from ch 2 cl 2 - hexane to afford the pure 4 , 5 - dichloro - n -( naphthalen - 1 - ylmethyl ) thiophene - 2 - sulfonamide ( 1 . 350 g , 91 %) as a white crystalline product . sodium hydride ( 0 . 081 g , 3 . 375 mmol ) was slowly added in portions to a solution of 4 , 5 - dichloro - n -( naphthalen - 1 - ylmethyl ) thiophene - 2 - sulfonamide ( 1 . 250 g , 3 . 358 mmol ) in anhydrous dmf ( 10 ml ) at 0 ° c . and stirred for 15 min . then , 4 - methoxybenzyl bromide ( pmbbr ) ( 0 . 675 g , 3 . 357 mmol ), and a catalytic amount of tbai ( 0 . 030 g , 0 . 081 mmol ) were added at 0 ° c ., and allowed to stir at room temperature for 2 h . after completion of the reaction , it was quenched by slow addition of water ( 5 ml ) and extracted with etoac ( 100 ml ), washed with water and brine , dried ( na 2 so 4 ), concentrated under vacuo and the residue purified by flash silica gel column chromatography ( combiflash ® rf ) using etoac - hexane ( 1 : 9 ) as eluant to afford 4 , 5 - dichloro - n -( 4 - methoxybenzyl )- n -( naphthalen - 1 - ylmethyl ) thiophene - 2 - sulfonamide ( 1 . 500 g , 91 %) as a white solid . representative procedure for the coupling of phenols with pmb protected 4 , 5 - dichloro - n -( aryl / alkyl ) thiophene - 2 - sulfonamides a mixture of 4 , 5 - dichloro - n -( 4 - methoxybenzyl )- n -( naphthalen - 1 - ylmethyl ) thiophene - 2 - sulfonamide ( 0 . 100 g , 0 . 203 mmol ), tert - butyl 4 -( 3 - hydroxyphenyl ) piperazine - 1 - carboxylate ( 0 . 068 g , 0 . 244 mmol ) and cs 2 co 3 ( 0 . 099 g , 0 . 304 mmol ) in anhydrous dmf ( 2 ml ) was heated at 80 ° c . for 2 . 5 h . the solvent was removed under vacuo and the residue was purified by combiflash ® rf ( isco ) using etoac - hexanes ( 1 : 9 ) to obtain a white solid ( 0 . 140 g , 94 %). to a solution of tert - butyl 4 -( 3 -( 3 - chloro - 5 -( n -( 4 - methoxybenzyl )- n -( naphthalen - 1 - ylmethyl ) sulfamoyl ) thio - phen - 2 - yloxy ) phenyl ) piperazine - 1 - carboxylate ( 0 . 085 g , 0 . 116 mmol ) in anhydrous ch 2 cl 2 ( 2 ml ) was added tfa ( 2 ml ) and stirred at room temperature for 3 h . the solvent mixture was removed under vacuo and the residue was re - dissolved in ch 2 cl 2 ( 20 ml ), washed with aqueous sat . nahco 3 followed by brine , dried ( na 2 so 4 ), and concentrated under vacuo . the crude product was purified by flash silica gel column chromatography using meoh — ch 2 cl 2 ( 1 : 9 ) to afford a light orange solid ( 0 . 055 g , 92 %). 1 h nmr ( 500 mhz , dmso - d 6 ): δ ( ppm ): 2 . 81 ( t , 4h , j = 5 . 0 hz ), 3 . 09 ( t , 4h , j = 5 . 0 hz ), 4 . 56 ( s , 2h ), 6 . 43 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 75 ( t , 1h , j = 2 . 5 hz ), 6 . 83 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 7 . 26 ( t , 1h , j = 8 . 0 hz ), 7 . 43 - 7 . 48 ( m , 3h ), 7 . 54 - 7 . 58 ( m , 2h ), 7 . 87 ( dd , 1h , j = 1 . 5 , 7 . 5 hz ), 7 . 93 - 7 . 96 ( m , 1h ), 8 . 06 - 8 . 09 ( m , 1h ). apci / esi ms : m / z 513 . 9 [ m + h ] + the product was prepared in 89 %% yield ; white solid , mp : 144 - 146 ° c . ; 1 h nmr ( 500 mhz , dmso - d 6 ): δ ( ppm ): 2 . 55 ( s , 3h ), 2 . 86 ( t , 4h , j = 4 . 5 hz ), 3 . 14 ( t , 4h , j = 4 . 5 hz ), 4 . 63 ( s , 2h ), 6 . 62 - 6 . 66 ( m , 1h ), 6 . 84 - 6 . 88 ( m , 2h ), 7 . 30 ( t , 1h , j = 8 . 0 hz ), 7 . 48 - 7 . 62 ( m , 4h ), 7 . 91 ( s , 1h ), 7 . 94 ( d , 1h , j = 8 . 0 hz ), 7 . 98 ( d , 1h , j = 9 . 0 hz ), 8 . 29 ( d , 1h , j = 8 . 0 hz ). apci / esi ms m / z 527 . 9 [ m + h ] + the product was prepared in 89 %% yield ; white solid , mp : 156 - 158 ° c . ; 1 h nmr ( 500 mhz , dmso - d 6 ): δ ( ppm ): 2 . 21 ( s , 3h ), 2 . 43 ( t , 4h , j = 5 . 0 hz ), 3 . 17 ( t , 4h , j = 5 . 0 hz ), 4 . 55 ( d , 2h , j = 4 . 5 hz ), 6 . 44 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 78 ( t , 1h , j = 2 . 0 hz ), 6 . 84 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 7 . 27 ( t , 1h , j = 8 . 0 hz ), 7 . 43 - 7 . 48 ( m , 3h ), 7 . 53 - 7 . 58 ( m , 2h ), 7 . 88 ( dd , 1h , j = 1 . 5 , 7 . 5 hz ), 7 . 93 - 7 . 97 ( m , 1h ), 8 . 05 - 8 . 09 ( m , 1h ), 8 . 52 ( t , 1h , j = 4 . 5 hz , nh ). apci / esi ms im / z 527 . 9 [ m + h ] + the product was prepared in 75 % yield ; white solid , mp : 86 - 88 ° c . ( decomposed ); 1 h nmr ( 500 mhz , dmso - d 6 ): δ ( ppm ): 3 . 06 ( t , 4h , j = 5 . 0 hz ), 3 . 20 ( t , 4h , j = 5 . 0 hz ), 4 . 23 ( s , 2h ), 6 . 54 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 6 . 67 ( t , 1h , j = 2 . 5 hz ), 6 . 77 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 6 . 95 - 7 . 02 ( m , 2h ), 7 . 04 ( d , 1h , j = 7 . 0 hz ), 7 . 23 - 7 . 32 ( m , 2h ), 7 . 33 ( s , 1h ). the product was prepared in 71 % yield ; white solid , mp : 58 - 60 ° c . ( decomposed ): 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 3 . 03 ( t , 4h , j = 5 . 0 hz ), 3 . 17 ( t , 4h , j = 5 . 0 hz ), 4 . 29 ( s , 2h ), 6 . 53 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 6 . 66 ( t , 1h , j = 2 . 5 hz ), 6 . 76 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 7 . 22 - 7 . 25 ( m , 1h ), 7 . 31 ( s , 1h ), 7 . 43 - 7 . 50 ( m , 3h ), 7 . 56 ( d , 1h , j = 7 . 0 hz ). the product was prepared in 71 % yield ; white solid , mp : 122 - 124 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 2 . 33 ( s , 3h ), 3 . 03 ( t , 4h , j = 5 . 0 hz ), 3 . 17 ( t , 4h , j = 5 . 0 hz ), 4 . 20 ( s , 2h ), 6 . 52 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 6 . 66 ( t , 1h , j = 2 . 5 hz ), 6 . 76 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 7 . 02 ( d , 1h , j = 8 . 0 hz ), 7 . 04 ( s , 1h ), 7 . 11 ( d , 1h , j = 7 . 5 hz ), 7 . 20 ( d , 1h , j = 8 . 0 hz ), 7 . 23 ( d , 1h , j = 8 . 0 hz ), 7 . 32 ( s , 1h ). apci esi - ms m / z 477 . 9 [ m + h ] + the product was prepared in 89 % yield ; yellowish syrup : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 3 . 06 ( t , 4h , j = 5 . 0 hz ), 3 . 20 ( t , 4h , j = 5 . 0 hz ), 4 . 19 ( s , 2h ), 6 . 56 ( dd , 1h , j = 1 . 5 , 8 . 0 hz ), 6 . 68 ( s , 1h ), 6 . 77 ( dd , 1h , j = 1 . 5 , 8 . 0 hz ), 7 . 14 ( d , 2h , j = 0 . 5 hz ), 7 . 24 - 7 . 29 ( m , 2h ), 7 . 31 ( s , 1h ). apci / esi ms m / z 531 . 8 [ m + h ] + the product was prepared in 89 % yield ; white solid , mp : 118 - 120 ° c . : 1 h nmr ( 500 mhz , cdcl 3 + cd 3 od ): δ ( ppm ): 3 . 02 ( t , 4h , j = 5 . 0 hz ), 3 . 16 ( t , 4h , j = 5 . 0 hz ), 3 . 79 ( s , 3h ), 6 . 48 ( dd , 1h , j = 1 . 5 , 8 . 0 hz ), 6 . 60 ( t , 1h , j = 1 . 5 hz ), 6 . 74 ( dd , 1h , j = 2 . 0 , 8 . 5 hz ), 6 . 81 - 6 . 85 ( m , 2h ), 7 . 05 - 7 . 09 ( m , 2h ), 7 . 18 ( s , 1h ), 7 . 22 ( t , 1h , j = 8 . 0 hz ). apci / esi ms m / z 479 . 9 [ m + h ] + the product was prepared in 97 % yield ; light orange solid , mp : 156 - 158 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 3 . 04 - 3 . 07 ( m , 4h ), 3 . 13 - 3 . 16 ( m , 4h ), 4 . 65 ( s , 2h ), 6 . 89 - 6 . 92 ( m , 2h ), 7 . 02 - 7 . 05 ( m , 2h ), 7 . 32 ( s , 1h ), 7 . 37 - 7 . 42 ( m , 2h ), 7 . 52 - 7 . 55 ( m , 2h ), 7 . 83 ( dd , 1h , j = 2 . 0 , 7 . 0 hz ), 7 . 86 - 7 . 89 ( m , 1h ), 7 . 92 - 7 . 94 ( m , 1h ). the product was prepared in 83 % yield ; light orange solid , mp : 99 - 101 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 3 . 02 - 3 . 04 ( m , 4h ), 3 . 15 - 3 . 18 ( m , 4h ), 4 . 35 ( s , 2h ), 6 . 49 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 64 ( t , 1h , j = 2 . 0 hz ), 6 . 75 ( dd , 1h , j = 2 . 5 , 8 . 5 hz ), 7 . 20 ( s , 1h ), 7 . 22 ( d , 1h , j = 0 . 5 hz ), 7 . 24 ( s , 1h ), 7 . 30 ( s , 1h ), 7 . 32 - 7 . 35 ( m , 2h ). the product was prepared in 85 % yield ; white solid , mp : 123 - 125 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 3 . 01 - 3 . 04 ( m , 4h ), 3 . 15 - 3 . 18 ( m , 4h ), 4 . 21 ( s , 2h ), 6 . 54 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 6 . 67 ( t , 1h , j = 2 . 5 hz ), 6 . 77 ( dd , 1h , j = 2 . 5 , 8 . 0 hz ), 7 . 13 - 7 . 16 ( m , 1h ), 7 . 21 - 7 . 30 ( m , 4h ), 7 . 32 ( s , 1h ). apci / esi ms m / z 497 . 9 [ m + h ] + the product was prepared in 78 % yield ; light orange solid , mp : 118 - 120 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 3 . 01 - 3 . 04 ( m , 4h ), 3 . 15 - 3 . 18 ( m , 4h ), 4 . 20 ( s , 2h ), 6 . 53 ( ddd , 1h , j = 0 . 5 , 2 . 0 , 8 . 0 hz ), 6 . 67 ( t , 1h , j = 2 . 0 hz ), 6 . 77 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 7 . 18 - 7 . 21 ( m , 2h ), 7 . 22 ( s , 1h ), 7 . 29 - 7 . 32 ( m , 2h ), 7 . 34 ( s , 1h ). the product was prepared in 48 % yield ; white solid , mp : 106 - 108 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 2 . 97 ( t , 4h , j = 5 . 0 hz ), 3 . 12 ( t , 4h , j = 5 . 0 hz ), 3 . 77 ( s , 3h ), 4 . 14 ( s , 2h ), 6 . 51 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 64 ( t , 1h , j = 2 . 0 hz ), 6 . 73 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 82 ( d , 2h , j = 8 . 5 hz ), 7 . 14 ( d , 2h , j = 8 . 5 hz ), 7 . 23 ( t , 1h , j = 8 . 5 hz ), 7 . 28 ( s , 1h ). apci / esi ms m / z 494 . 0 [ m + h ] + the product was prepared in 31 % yield ; white solid , mp : 60 - 62 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 2 . 97 ( t , 4h , j = 5 . 0 hz ), 3 . 12 ( t , 4h , j = 5 . 0 hz ), 3 . 76 ( s , 3h ), 4 . 18 ( s , 2h ), 6 . 52 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 64 ( t , 1h , j = 2 . 0 hz ), 6 . 73 ( dd , 1h , j = 2 . 0 , 8 . 5 hz ), 6 . 76 ( s , 1h ), 6 . 78 - 6 . 83 ( m , 2h ), 7 . 22 ( abq , 2h , j = 8 . 5 hz ), 7 . 28 ( s , 1h ). apci / esi ms m / z 494 . 1 [ m + h ] + the product was prepared in 78 % yield ; white solid , mp : 154 - 156 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 3 . 17 ( t , 4h , j = 5 . 0 hz ), 3 . 28 ( t , 4h , j = 5 . 0 hz ), 6 . 49 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 74 ( s , 1h ), 6 . 78 ( d , 1h , j = 7 . 5 hz ), 6 . 83 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 86 ( dd , 1h , j = 1 . 0 , 7 . 5 hz ), 6 . 96 ( s , 1h ), 7 . 10 ( t , 1h , j = 8 . 0 hz ), 7 . 23 ( s , 1h ), 7 . 26 ( t , 1h , j = 8 . 0 hz ), 8 . 34 ( br s , 1h , nh ). apci / esims m / z 484 . 0 [ m + h ] + the product was prepared in 78 % yield ; white solid , mp : 180 - 182 ° c . : 1 h nmr ( 500 mhz , cdcl 3 ): δ ( ppm ): 3 . 03 ( t , 4h , j = 5 . 0 hz ), 3 . 14 ( t , 4h , j = 5 . 0 hz ), 3 . 78 ( s , 3h ), 6 . 47 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 59 ( t , 1h , j = 2 . 0 hz ), 6 . 65 - 6 . 68 ( m , 1h ), 6 . 71 - 6 . 76 ( m , 3h ), 7 . 18 - 7 . 23 ( m , 2h ), 7 . 27 ( s , 1h ). apci / esi ms m / z 480 . 0 [ m + h ] + to a solution of tert - butyl 4 -( 3 -( 5 -( n , n - bis ( 4 - methoxybenzyl ) sulfamoyl )- 3 - chlorothiophen - 2 - yloxy ) phenyl ) piperazine - 1 - carboxylate ( 0 . 430 g , 0 . 602 mmol ) in anhydrous ch 2 cl 2 ( 0 . 5 ml ) was added tfa ( 4 . 5 ml ) and stirred at room temperature for 4 h . the solvent mixture was removed under vacuo and the residue was re - dissolved in ch 2 cl 2 ( 30 ml ), washed with aqueous sat . nahco 3 followed by brine , dried ( na 2 so 4 ), and concentrated under vacuo . the residue was purified by combiflash ® rf ( isco ) using meoh — ch 2 cl 2 ( 1 : 5 ) to give a white solid ( 0 . 180 g , 80 %). 1 h nmr ( 500 mhz , cd 3 od ): δ ( ppm ): 3 . 02 - 3 . 05 ( m , 4h ), 3 . 19 - 3 . 22 ( m , 4h ), 6 . 56 ( dd , 1h , j = 2 . 0 , 8 . 0 hz ), 6 . 73 ( t , 1h , j = 2 . 0 hz ), 6 . 84 ( dd , 1h , j = 2 . 0 , 8 . 5 hz ), 7 . 27 ( t , 1h , j = 8 . 5 hz ), 7 . 40 ( s , 1h ). the product was prepared in 87 % yield ; 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