Patent Application: US-83891104-A

Abstract:
a novel tagged triazine library is provided in which three building blocks are prepared separately and assembled orthogonally to yield 1536 highly pure compounds . each library compound contains a variety of a triethyleneglycol linker at one of the diversity sites of the triazine scaffold .

Description:
incorporating linkers in a library of compounds prior to biological screening provides for a straightforward method for isolating a target protein without compromising the lead compound &# 39 ; s activity or performing further sar experiments . the libraries produced by the present invention are useful in screening compounds in a biological assay in search for novel phenotypes and elucidating protein functions . the linkers for use in the present invention comprise triethyleneglycol aryl or alkyl linkers . in order to provide detection capability , or to add labels for assays , the end group of the linker carries moieties such as azide , amino , nhboc , biotin , acetylene , or fluorescent moieties . for producing building block i , a primary amine or amino alcohol is used . the primary amine or amino alcohol may be a substituted or unsubstituted alkyl , alkenyl , alkynyl , or aryl primary amine or amino alcohol . the linker compound shown in fig1 a and the affinity matrix shown in fig1 c comprise a trisubstituted triazine substituted with r1 , r2 and r3 , which may be the same or different . r1 , r2 , and r3 can be substituted or unsubstituted aryl , alkyl , alkenyl , alkynyl , or cyclic or heterocyclic group . as used herein , alkyl , alkenyl and alkynyl carbon chains , if not specified , contain from 1 to 20 carbon atoms , preferably from 1 to 16 carbon atoms , and are straight or branched . alkenyl carbon chains of from 1 to 20 carbon atoms preferably contain 1 to 8 double bonds ; the alkenyl carbon chains of 1 to 16 carbon atoms preferably contain from 1 to 5 double bonds . alkynyl carbon chains of from 1 to 20 carbon atoms preferably contain 1 to 8 triple bonds , and the alkynyl carbon chains of 1 to 16 carbon atoms preferably contain 1 to 5 triple bonds . the alkyl , alkenyl , and alkynyl groups may be optionally substituted , with one or more groups , preferably alkyl group substituents that may be the same or different . as used herein , lower alkyl , lower alkenyl , and lower alkynyl refer to carbon chains having fewer than or equal to about 6 carbon atoms . as used herein an alkyl group substituent includes halos , haloalkyl , preferably halo lower alkyl , aryl , hydroxy , alkoxy , aryloxy , alkoxy , alkylthio , arylthio , aralkyloxy , aralkylthio , carboxy , alkoxycarbonyl , oxo , and cycloalkyl . for the present invention , “ cyclic ” refers to cyclic groups preferably containing from 3 to 19 carbon atoms , preferably 3 to 10 members , more preferably 5 to 7 members . cyclic groups include hetero atoms , and may include bridged rings , fused rings , either heterocyclic , cyclic , or aryl rings . the term “ aryl ” herein refers to aromatic cyclic compounds having up to 10 atoms , including carbon atoms , oxygen atoms , sulfur atoms , selenium atoms , etc . aryl groups include , but are not limited to , groups such as phenyl , substituted phenyl , naphthyl , substituted naphthyl , in which the substituent is preferably lower alkyl , halogen , or lower alkyl . “ aryl ” may also refer to fused rings systems having aromatic unsaturation . the fused ring systems can contain up to about 7 rings . an “ aryl group substituent ” as used herein includes alkyl , cycloalkyl , cycloaryl , aryl , heteroaryl , optionally substituted with 1 or more , preferably 1 to 3 , substituents selected from halo , haloalkyl , and alkyl , arylalkyl , heteroarylalkyl , alkenyl containing 1 to 2 double bonds , alkynyl containing 1 to 2 triple bonds , halo , hydroxy , polyhaloalkyl , preferably trifluoromethyl , formyl , alkylcarbonyl , arylcarbonyl , optionally substituted with 1 or more , preferably 1 to 3 , substituents selected from halo , haloalkyl , alkyl , heteroarylcarbonyl , carboxyl , alkoxycarbonyl , aryloxycarbonyl , aminocarbonyl , alkylaminocarbonyl , dialkylaminocarbonyl , arylalkylaminocarbonyl , alkoxy , aryloxy , perfluoroalkoxy , alkenyloxy , alkynyloxy , arylalkoxy , aminoalkyl , alkylaminoalkyl , dialkylaminoalkyl , arylaminoalkyl , amino , alkylamino , dialkylamino , arylamino , alkylarylamino , alkylcarbonylamino , arylcarbonylamino , amido , nitro , mercapto , alkylthio , arylthio , perfluoroalkylthio , thiocyano , isothiocyano , alkylsufinyl , alkylsulfonyl , arylsulfinyl , arylsulfonyl , aminosulfonyl , alkylaminosulfinyl , dialkylaminosulfonyl , and arylaminosulfonyl . the term “ arylalkyl ” as used herein refers to an alkyl group which is substituted with one or more aryl groups . examples of arylalkyl groups include benzyl , 9 - fluorenylmethyl , naphthylmethyl , diphenylmethyl , and triphenylmethyl . “ cycloalkyl ” as used herein refers to a saturated mono - or multicyclic ring system , preferably of 3 to 10 carbon atoms , more preferably from 3 to 6 carbon atoms . cycloalkenyl and cycloalkynyl refer to mono - or multicyclic ring systems that respectively include at least one double bond and at least one triple bond . cycloalkenyl and cycloalkynyl groups may preferably contain 3 to 10 carbon atoms , with cycloalkenyl groups more preferably containing 4 to 7 carbon atoms and cycloalkynyl groups more preferably containing 8 to 10 carbon atoms . the ring systems of the cycloalkyl , cycloalkenyl and cycloalkynyl groups may be composed of one ring or two or more rings which may be joined together in a fused , bridged , or spiro - connected fashion , and may be optionally substituted with one or more alkyl group substituents . the term “ heteroaryl ” for purposes of the present application refers to a monocyclic or multicyclic ring system , preferably about 5 to about 15 members , in which at least one atom , preferably 1 to 3 atoms , is a heteroatom , that is , an element other than carbon , including nitrogen , oxygen , or sulfur atoms . the heteroaryl may be optionally substituted with one or more , preferably 1 to 3 , aryl group substituents . exemplary heteroaryl groups include , for example , furanyl , thienyl , pyridyl , pyrrolyl , n - methylpyrrolyl , quinolyinyl and isoquinolinyl . the term “ heterocyclic ” refers to a monocyclic or multicyclic ring system , preferably of 3 to 10 members , more preferably 4 to 7 members , where one or more , preferably 1 to 3 , of the atoms in the ring system is a heteroatom , i . e ., an atom that is other than carbon , such as nitrogen , oxygen , or sulfur . the heterocycle may be optionally substituted with one or more , preferably 1 to 3 , aryl group substituents . preferred substituents of the heterocyclic group include hydroxy , alkoxy , halo lower alkyl . the term heterocyclic may include heteroaryl . exemplary heterocyclics include , for example , pyrrolidinyl , piperidinyl , alkylpiperidinyl , morpholinyl , oxadiazolyl , or triazolyl . the nomenclature alkyl , alkoxy , carbonyl , etc , is used as is generally understood by those of skilled this art . as used herein , aryl refers to saturated carbon chains that contain one or more carbon atoms ; the chains may be straight or branched or include cyclic portions or may be cyclic . the term “ halogen ” or “ halide ” includes f , cl , br , and i . this can include pseudohalides , which are anions that behave substantially similarly to halides . these compounds can be used in the same manner and treated in the same manner as halides . pseudohalides include , but are not limited to , cyanide , cyanate , thiocyanate , selenocyanate , trifluoromethyl , and azide . the term “ haloalkyl ” refers to a lower alkyl radical in which one or more of the hydrogen atoms are replaced by halogen , including but not limited to , chloromethyl , trifluoromethyl , 1 - chloro - 2 - fluoroethyl , and the like . “ haloalkoxy ” refers to ro — in which r is a haloalkyl group . “ alkylene ” refers to a straight , branched , or cyclic , preferably straight or branched , bivalent aliphatic hydrocarbon group , preferably having from 1 to about 20 carbon atoms . the alkylene group is optionally substituted with one or more alkyl group substituents . there may be optionally inserted along the alkylene group one or more oxygen , sulfur , or substituted or unsubstituted nitrogen atoms , wherein the nitrogen substituent is alkyl . exemplary alkylene groups include methylene , ethylene , propylene , cyclohexylene , methylenedioxy , and ethylenedioxy . the term “ lower alkylene ” refers to alkylene groups having from 1 to 6 carbon atoms . preferred alkylene groups are lower alkylene , with alkylene of 1 to 3 atoms being particularly preferred . the term “ alkenylene ” as used herein refers to a straight , branched or cyclic , preferably straight or branched , bivalent aliphatic hydrocarbon group , preferably having from about 1 to 20 carbon atoms and at least one double bond . the alkenylene group is optionally substituted with one or more alkyl group substituents . there may be optionally inserted along the alkenylene group one or more oxygen , sulfur , or substituted or unsubstituted nitrogen atoms , where the nitrogen substituent is alkyl as previously described . as used herein , “ alkynylene ” refers to a straight , branched or cyclic bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms and at least one triple bond . the alkynylene group is optionally substituted with one or more alkyl group substituents . there may be optionally inserted along the alkynylene group one or more oxygen , sulfur , or substituted or unsubstituted nitrogen atoms , where the nitrogen substituent is alkyl . the term “ lower alkynylene ” refers to alkynylene groups having from 2 to 6 carbon atoms . the term “ arylene ” as used herein refers to a monocyclic or polycyclic bivalent aromatic group preferably having from 1 to 20 carbon atoms and at least one aromatic ring . the arylene group is optionally substituted with one or more alkyl group substituents . there may be optionally inserted around the arylene group one or more oxygen , sulfur , or substituted or unsubstituted nitrogen atoms , where the nitrogen substituent is alkyl . “ heteroarylene ” refers to a bivalent monocyclic or multicyclic ring system , preferably of about 5 to about 15 members , wherein one or more of the atoms in the ring system is a heteroatom . the heteroarylene may be optionally substituted with one or more aryl group substituents . as used herein , “ alkylidene ” refers to a bivalent group , such as ═ cr ′ r ″, which is attached to one atom of another group , forming a double bond . “ arylalkylidene ” refers to an alkylidene group in which either r ′ or r ″ is an aryl group . as used herein , when any particular group , such as phenyl or pyridyl , is specified , this means that the group is substituted or unsubstituted . preferred substituents , where not specified , are halo , halo lower alkyl , and lower alkyl . general procedure for building block i synthesis ( fig2 ) the desired primary amine or amino alcohol is added to a suspension of a pal aldehyde resin in anhydrous tetrahydrofuran ( thf ) containing 2 % acetic acid at room temperature . the reaction mixture is shaken for one hour at room temperature , followed by addition of sodium triacetoxyborohydride . the reaction mixture is stirred for twelve hours and filtered . the resin is washed with n , n - dimethylformamide five times , alternatively with dichloromethane and methanol five times , and finally five times with dichloromethane . the resin was dried in vacuum . 2 , 2 ′-( ethylenedioxy ) bis ( ethylamine ) was dissolved in dichloromethane and the solution cooled to 0 ° c . di - tert - butyl - dicarbonate ( boc anhydride ) was dissolved in dichloromethane and added to the solution of 2 , 2 ′-( ethylenedioxy ) bis ( ethylamine ) dropwise over a period of three hours . the reaction mixture was allowed to stir for ten hours followed by extraction with saturated nacl solution . the organic layers were combined and dried over mgso 4 . the solvent was removed in vacuo . the appropriate amino benzoic acid was dissolved in dmf ( except for fmoc - aminomethylbenzoic acid , which was dissolved in thf ) at room temperature . to the reaction mixture , 1 - hydroxybenzo - triazole ( hobt ) was added and allowed to stir for several minutes , followed by addition of 1 , 3 - diisopropylcarboniimide ( dic ) and allowed to stir for several minutes . then , n - boc - 2 , 2 ′-( ethylenedioxy ) bis ( ethylamine ) dissolved in either dmf , or thf in the case of n - boc - 2 , 2 ′-( ethylenedioxy ) bis ( ethylamine ), was slowly added drop - wise . the reaction mixture was allowed to stir overnight at room temperature . a solid dic urea byproduct precipitated out of the reaction mixture , was filtered , and washed with ethyl acetate ( ea ). the filtrate was extracted three times with saturated aqueous sodium bicarbonate . the aqueous layer was back - extracted with ethyl acetate and saturated aqueous nacl . the organic layers were combined and dried over mgso 4 . the solvent was removed in vacuo . general procedure for coupling cyanuric trichloride to the dic intermediate ( fig3 b ) cyanuric chloride was dissolved in thf with diea at 0 ° c . intermediate dic coupled linker in thf was added dropwise . the reaction mixture was stirred and monitored by tlc . reaction time was about 45 minutes to 1 hour . a solid precipitate slowly formed . upon completion of the reaction , the reaction mixture was quickly filtered through a plug of flash silica and washed with ethyl acetate . the filtrate was evaporated in vacuo . the general dic coupling procedure was followed and the compound 1 was purified using flash column chromatography ( particle size 32 - 63 μm ) using a gradient of 100 % ea — 10 : 1 ea : meoh . tlc in ea ( r f : 0 . 22 ). the compound was identified by esims : ( m + h ) + calcd , 367 . 2 ; found , 368 . 2 . h 1 nmr ( 200 mhz , cdcl 3 ) δ 7 . 52 ( d , 2h , j = 7 . 6 ), 6 . 67 ( s , 1h ) 6 . 54 ( d , 2h , j = 8 . 5 ), 5 . 12 ( s , 1h ) 4 . 14 ( s , 2h ), 3 . 48 ( m ), 1 . 35 ( s , 9h ) product was a viscous orange oil ( 87 % yield ). the general cyanuric chloride coupling procedure was followed for the intermediate linker 1 . tlc conditions ( 100 % ea r f = 0 . 36 ). the product was crystallized from a small amount of thf and ea with larger amounts of hexane . the product , pp , was filtered , washed with hexane , and dried under vacuum . pp was a white solid ( 37 % yield ). esims : ( m + h ) + calcd , 514 . 1 ; found , 515 . 1 . h 1 nmr ( 200 mhz , cdcl 3 ) δ11 . 35 ( s , 1h ), 8 . 51 ( s , 1h ), 7 . 9 ( d , 2h , j = 8 . 5 hz ), 7 . 7 ( d , 2h , j = 8 . 7 hz ), 6 . 78 ( s , 1h ) 3 . 4 ( m ), 3 . 0 ( s , 2h ), 1 . 37 ( s , 9h ). the general dic coupling procedure was followed and the compound , 2 was purified using flash column chromatography ( particle size 32 - 63 μm ) using a gradient of dichloromethane : meoh 35 : 1 — dichloromethane : meoh 20 : 1 ea . tlc in dichloromethane : meoh 30 : 1 ( r f : 0 . 12 ) ( 68 % yield ). the compound was identified by esims : ( m + h ) + calcd , 367 . 2 ; found , 368 . 2 and h 1 nmr ( 200 mhz , cdcl 3 ) δ7 . 0 ( m , 3h ) 6 . 8 ( m , 1h ), 5 . 22 ( s , 1h ), 3 . 4 ( m ), 3 . 2 ( m , 2h ) 1 . 36 ( s , 9h ). the general cyanuric chloride coupling procedure was followed for the intermediate linker 2 . tlc conditions ( 100 % ea r f = 0 . 43 ). the product was crystallized by evaporating the concentrated filtrate with thf and hexane . the re - concentrated reaction mixture was crystallized from 1 : 10 thf : ethyl ether . the product , mp , was filtered , washed with ether , and dried under vacuum . mp was a white solid ( 31 % yield ). esims : ( m + h ) + calcd , 514 . 1 ; found , 515 . 1 . h 1 nmr ( 200 mhz , cdcl 3 ) δ 11 . 23 ( s , 1h ), 8 . 54 ( s , 1h ), 8 . 02 ( s , 1h ), 7 . 75 ( m , 1h ), 7 . 6 ( m , 1h ), 7 . 4 ( m , 1h ), 6 . 75 ( s , 1h ), 3 . 52 - 3 . 35 ( m ), 3 . 03 ( m , 2h ) 1 . 35 ( s , 9h ) the general dic coupling procedure was followed and the compound , 3 , was purified using flash column chromatography ( particle size 32 - 63 μm ) using a gradient of dichloromethane : meoh 35 : 1 — dichloromethane : meoh 25 : 1 ea . tlc in dichloromethane : meoh 30 : 1 ( r f : 0 . 12 ) ( 77 % yield ). the compound was identified by esims : ( m + h ) + calcd , 381 . 2 ; found , 382 . 1 and h 1 nmr ( 200 mhz , cdcl 3 ) δ7 . 03 ( d , 2h , j = 6 mhz , 6 . 8 ( d , 2h , 8 . 36 mhz , 6 . 0 ( s , 1h ) 5 . 15 ( s , 1h ) 3 . 75 ( s , 2h ) 3 . 4 ( m ) 1 . 86 ( s , 2h ) 1 . 45 ( s , 9h ). the general cyanuric chloride coupling procedure was followed for the intermediate linker 3 . tlc conditions ( 100 % ea r f = 0 . 25 ). the product , ap , was isolated using flash chromatography ( particle size 32 - 63 μm ) with a 100 % ea — 1 : 10 ea : meoh gradient . ap is an off white solid ( 59 % yield ). esims : ( m + h ) + calcd , 528 . 1 ; found , 529 . 1 . h 1 nmr ( 200 mhz , cdcl 3 ) δ 11 . 1 ( s , 1h ), 8 . 14 ( s , 1 . h ) 7 . 5 ( d , 2h , j = 8 . 48 ), 7 . 26 ( d , 2h , j = 8 . 42 ) 3 . 4 ( m ), 3 . 2 ( m , 2h ) 3 . 1 ( m , 2h ) 1 . 36 ( 9h ) ( 4 - aminomethyl )- benzoic acid ( 4 . 9 g , 1 . 1 equiv .) was dissolved in 5 % nahco 3 150 ml in water and stirred . n -( 9 - fluorenylmethoxycarbonyl )- l - proline ( fmoc , 1 equiv ., 10 g ) was dissolved in an equivalent amount of dioxane and added . the reaction was allowed to stir at room temperature . after two hours , 10 % citric acid in water 75 ml was added . a white solid precipitated upon addition . the solid was washed filtered and washed with hexane . the solid was dissolved in thf and allowed to dry overnight over mgso 4 . the following day , the solution was filtered , crystallized from thf and hexane , and placed under a drying vacuum ( 90 % yield ). esims : ( m + h ) + calcd , 373 . 1 ; found , 374 . 1 . the general dic coupling procedure was followed in thf , the compound , 4 was isolated using flash chromatography ( particle size 32 - 63 μm ) with 100 % ea tlc ( 100 % ea r f : 0 . 23 ) ( 35 % yield ); esims : ( m + h ) + calcd , 603 . 3 ; found , 604 . 3 . h 1 nmr ( 200 mhz , cdcl 3 ) 7 . 74 - 7 . 26 ( m ), 6 . 7 ( s , 1h ), 5 . 3 ( s , 1h ) 3 . 7 - 4 . 0 ( m ), 3 . 2 ( m , 3h ) 1 . 4 ( s , 9h ), 1 . 0 ( s , 7h ) fmoc cleavage was performed in 10 % piperidine and dichloromethane with stirring at room temperature , the reaction progress was monitored by tlc . the reaction time was 2 . 5 hrs . the solvent was removed in vacuo . the compound , 4a was isolated using flash chromatography ( particle size 32 - 63 μm ) with a 100 % ea — 1 : 20 ea : meoh ( with 1 % triethylamine ) gradient ( 80 % yield ). esims : ( m + h ) + calcd , 381 . 2 ; found , 382 . 2 . the general cyanuric chloride coupling procedure was followed for the intermediate linker 4a . ( 100 % dichloromethane : acetone r f 5 : 1 = 0 . 21 ) the product , am was isolated using flash chromatography ( particle size 32 - 63 μm ) with a dichloromethane : acetone 5 : 1 — dichloromethane : acetone 3 : 1 gradient . the product was an orange oil ( 52 % yield ). tlc ( dichloromethane : acetone 5 : 1 , r f = 0 . 21 ) esims : ( m + h ) + calcd , 528 . 1 ; found , 529 . 1 . h 1 nmr ( 200 mhz , cdcl 3 ) δ 7 . 6 - 7 . 8 ( d , 2h , j = 4 hz ) 7 . 35 - 7 . 37 ( d , 2h , 4 hz ), 6 . 7 - 6 . 8 ( m , 2h ) 4 . 9 ( s , 1h ), 4 . 72 ( d , 2h , j = 4 hz ,) 3 . 5 - 3 . 7 ( m ) 3 . 2 ( m , 2h ) 1 . 8 ( s , 2h ) 1 . 4 ( s , 9h ). building block ii ( 0 . 44 mmole ) was added to building block i ( 0 . 11 mmole ) in diea ( 1 ml ) and anhydrous thf ( 10 ml ) at room temperature . the reaction mixture was heated to 60 ° c . for 3 hr and filtered . the resin was washed with dmf ( 5 times ), alternatively with dichloromethane and methanol ( 5 times ), and finally dichloromethane ( 5 times ). the resin was dried in vacuum . general procedure for the final amination on the resin and product cleavage desired amines ( 4 equiv .) were added to the resin ( 10 mg ), coupled with building block i and building block ii , in diea ( 8 μl ) and 1 ml of nmp : n - buoh ( 1 : 1 ). the reaction mixture was heated to 120 ° c . for 3 hr . the resin was washed with dmf ( 5 times ), alternatively with dichloromethane and methanol ( 5 times ), and finally dichloromethane ( 5 times ). the resin was dried in vacuum . the product cleavage reaction was performed using 10 % trifluoroacetic acid ( tfa ) in dichloromethane ( 1 ml ) for 30 min at room temperature and washed with dichloromethane ( 0 . 5 ml ). free hydroxyl containing compounds were further treated with a piperazine resin in 0 . 5 ml thf at room temperature for 5 hrs to cleave the trifluoroacetic ester that was formed upon treatment with tfa . the resin was filtered out and washed with 0 . 1 ml thf . the purity and identity of all the products were monitored by lc - ms at 250 nm ( agilent 1100 model ); more than 90 % of compounds demonstrated & gt ; 90 % purity . unless otherwise noted , materials and solvents were obtained from commercial suppliers ( acros and aldrich ) and were used without further purification . pal - aldehyde ( 4 - formyl - 3 , 5 - dimethoxyphenoxymethyl ) resin from midwest bio - tech ( cat #: 20840 , lot #: sy03470 , loading level 1 . 10 mmole / g ) was used for the generation of library compounds . building block ii compounds , made by solution phase chemistry , were purified by flash column chromatography on merck silca gel 60 - pf254 . all library products were identified by an lc - ms at 250 nm ( agilent technology , hp1100 ) using a c18 column ( 20 × 4 . 0 mm ) with a gradient of 5 - 95 % ch 3 cn — h 2 o ( containing 0 . 1 % acetic acid ) as an eluant over 4 min . thermal libraries were synthesized using a standard heat block from vwr scientific products using 4 ml glass vials with paper - lined caps purchased from fisher scientific . resin filtration procedures were carried out using a 70μ pe frit cartridges from applied separations ( cat . # 2449 ). affi - gel - 10 was purchased from biorad . edta , egta , glycine , naf , phenylmethylsulfonylfluoride ( pmsf ) and trizma base were from sigma chemicals . silver stain kit and pre - casted tris - glycine gel were obtained from invitrogen . protein inhibitor cocktail was purchased from roche . nonidet p - 40 was from fluka . the desired primary amine or amino alcohol ( 2 . 8 mmole , 5 equiv .) was added to a suspension of the pal aldehyde resin ( 1 . 3 g , 1 . 43 mmole ) in anhydrous tetrahydrofuran ( thf ) ( 50 ml containing 2 % of acetic acid ) at room temperature . the reaction mixture was shaken for 1 hr at room temperature followed by addition of sodium triacetoxyborohydride ( 2 . 1 g , 9 . 9 mmole , 7 equiv .). the reaction mixture was stirred for 12 hr and filtered . the resin was washed with n , n - dimethylformamide ( dmf ) ( 5 times ), alternatively with dichloromethane and methanol ( meoh ) ( 5 times ), and finally with dichloromethane ( 5 times ). the resin was dried in vacuum . 2 , 2 ′-( ethylenedioxy ) bis ( ethylamine ) ( 10 equiv .) was dissolved in dichloromethane and solution was cooled down to 0 ° c . di - tert - butyl dicarbonate ( boc anhydride ) ( 1 equiv .) was dissolved in dichloromethane and added to the solution of 2 , 2 ′-( ethylenedioxy ) bis ( ethylamine ) dropwise over a period of 3 hours . the reaction mixture was allowed to stir for 10 hours followed by extraction with saturated nacl solution . the organic layers were combined and dried over mgso 4 . the solvent was removed in vacuo ( 64 % yield ). the appropriate amino benzoic acid ( 3 equiv .) was dissolved in dmf ( except for fmocaminomethylbenzoic acid which was dissolved in thf ) at room temperature . to the reaction mixture , 1 equiv . of 1 - hydroxybenzo - triazole ( hobt ) was added and allowed to stir for several minutes followed by 1 , 3 - diisopropylcarboniimide ( dic , 6 equiv .) and allowed to stir for several minutes . then , n - boc - 2 , 2 ′( ethylenedioxy ) bis ( ethylamine ) dissolved in either dmf , or thf in the case of fmoc - aminomethylbenzoic acid , was slowly added drop - wise . the reaction mixture was allowed to stir overnight at room temperature . a solid dic urea byproduct precipitated out of the reaction mixture was filtered and washed with ethyl acetate ( ea ). the filtrate was extracted ( 3 × 10 times the volume of saturated sodium bicarbonate ( nahco 3 ) in water ). the aqueous layer was back - extracted with ea and sat . nacl in water . the organic layers were combined and dried over mgso 4 . the solvent was removed in vacuo . general procedure for coupling of cyanuric trichloride to the dic intermediate cyanuric chloride ( 1 equiv .) was dissolved in thf with diea ( 10 equiv .) at 0 ° c . intermediate dic coupled linker ( 1 . 2 ) equiv . in thf was added dropwise . the reaction mixture was stirred and monitored by tlc . reaction time was 45 min . to 1 hr . a solid precipitate slowly formed . upon completion of the reaction , the reaction mixture was quickly filtered through a plug of flash silica and washed with ea . the filtrate was evaporated in vacuo . the general dic coupling procedure was followed and the compound 1 was purified using flash column chromatography ( particle size 32 - 63 μm ) using a gradient of 100 % ea — 10 : 1 ea : meoh . tlc in ea ( rf : 0 . 22 ). the compound was identified by esims : ( m + h )+ calcd , 367 . 2 ; found , 368 . 2 . h 1 nmr ( 200 mhz , cdcl 3 ) δ 7 . 52 ( d , 2h , j = 7 . 6 ), 6 . 67 ( s , 1h ) 6 . 54 ( d , 2h , j = 8 . 5 ), 5 . 12 ( s , 1h ) 4 . 14 ( s , 2h ), 3 . 48 ( m ), 1 . 35 ( s , 9h ) product was a viscous orange oil ( 87 % yield ). the general cyanuric chloride coupling procedure was followed for the intermediate linker 1 . tlc conditions ( 100 % ea r f = 0 . 36 ). the product was crystallized from a small amount of thf and ea with larger amounts of hexane . the product , pp , was filtered , washed with hexane , and dried under vacuum . pp was a white solid ( 37 % yield ). esims : ( m + h )+ calcd , 514 . 1 ; found , 515 . 1 . h 1 nmr ( 200 mhz , cdcl 3 ) δ11 . 35 ( s , 1h ), 8 . 51 ( s , 1h ), 7 . 9 ( d , 2h , j = 8 . 5 hz ), 7 . 7 ( d , 2h , j = 8 . 7 hz ), 6 . 78 ( s , 1h ) 3 . 4 ( m ), 3 . 0 ( s , 2h ), 1 . 37 ( s , 9h ). the general dic coupling procedure was followed and the compound , 2 was purified using flash column chromatography ( particle size 32 - 63 μm ) using a gradient of dichloromethane : meoh 35 : 1 — dichloromethane : meoh 20 : 1 ea . tlc in dichloromethane : meoh 30 : 1 ( r f : 0 . 12 ) ( 68 % yield ). the compound was identified by esims : ( m + h )+ calcd , 367 . 2 ; found , 368 . 2 and h 1 nmr ( 200 mhz , cdcl 3 ) δ 7 . 0 ( m , 3h ) 6 . 8 ( m , 1h ), 5 . 22 ( s , 1h ), 3 . 4 ( m ), 3 . 2 ( m , 2h ) 1 . 36 ( s , 9h ). the general cyanuric chloride coupling procedure was followed for the intermediate linker 2 . tlc conditions ( 100 % ea r f = 0 . 43 ) the product was crystallized by evaporating the concentrated filtrate with thf and hexane . the re - concentrated reaction mixture was crystallized from 1 : 10 thf : ethyl ether . the product , mp , was filtered , washed with ether , and dried under vacuum . mp was a white solid ( 31 % yield ). esims : ( m + h )+ calcd , 514 . 1 ; found , 515 . 1 . h 1 nmr ( 200 mhz , cdcl 3 ) δ 11 . 23 ( s , 1h ), 8 . 54 ( s , 1h ), 8 . 02 ( s , 1h ), 7 . 75 ( m , 1h ), 7 . 6 ( m , 1h ), 7 . 4 ( m , 1h ), 6 . 75 ( s , 1h ), 3 . 52 - 3 . 35 ( m ), 3 . 03 ( m , 2h ) 1 . 35 ( s , 9h ). the general dic coupling procedure was followed and the compound , 3 , was purified using flash column chromatography ( particle size 32 - 63 μm ) using a gradient of dichloromethane : meoh 35 : 1 — dichloromethane : meoh 25 : 1 ea . tlc in dichloromethane : meoh 30 : 1 ( r f : 0 . 12 ) ( 77 % yield ). the compound was identified by esims : ( m + h )+ calcd , 381 . 2 ; found , 382 . 1 and h 1 nmr ( 200 mhz , cdcl 3 ) δ7 . 03 ( d , 2h , j = 6 mhz , 6 . 8 ( d , 2h , 8 . 36 mhz , 6 . 0 ( s , 1h ) 5 . 15 ( s , 1h ) 3 . 75 ( s , 2h ) 3 . 4 ( m ) 1 . 86 ( s , 2h ) 1 . 45 ( s , 9h ). the general cyanuric chloride coupling procedure was followed for the intermediate linker 3 . tlc conditions ( 100 % ea r f = 0 . 25 ). the product , ap , was isolated using flash chromatography ( particle size 32 - 63 μm ) with a 100 % ea — 1 : 10 ea : meoh gradient . ap is an off white solid ( 59 % yield ). esims : ( m + h )+ calcd , 528 . 1 ; found , 529 . 1 h 1 nmr ( 200 mhz , cdcl 3 ) δ 11 . 1 ( s , 1h ), 8 . 14 ( s , 1 . h ) 7 . 5 ( d , 2h , j = 8 . 48 ), 7 . 26 ( d , 2h , j = 8 . 42 ) 3 . 4 ( m ), 3 . 2 ( m , 2h ) 3 . 1 ( m , 2h ) 1 . 36 ( 9h ). ( 4 - aminomethyl )- benzoic acid ( 4 . 9 g , 1 . 1 equiv .) was dissolved in 5 % nahco 3 150 ml in water and stirred . n -( 9 - fluorenylmethoxycarbonyl )- l - proline ( fmoc , 1 equiv ., 10 g ) was dissolved in an equivalent amount of dioxane and added . the reaction was allowed to stir at room temperature . after two hours , 10 % citric acid in water 75 ml was added . a white solid precipitated upon addition . the solid was washed filtered and washed with hexane . the solid was dissolved in thf and allowed to dry overnight over mgso 4 . the following day , the solution was filtered , crystallized from thf and hexane , and placed under a drying vacuum ( 90 % yield ). esims : ( m + h )+ calcd , 373 . 1 ; found , 374 . 1 . the general dic coupling procedure was followed in thf , the compound , 4 was isolated using flash chromatography ( particle size 32 - 63 μm ) with 100 % ea tlc ( 100 % ea r f : 0 . 23 ) ( 35 % yield ); esims : ( m + h )+ calcd , 603 . 3 ; found , 604 . 3 . h 1 nmr ( 200 mhz , cdcl 3 ) 7 . 74 - 7 . 26 ( m ), 6 . 7 ( s , 1h ), 5 . 3 ( s , 1h ) 3 . 7 - 4 . 0 ( m ), 3 . 2 ( m , 3h ) 1 . 4 ( s , 9h ), 1 . 0 ( s , 7h ). fmoc cleavage was performed in 10 % piperidine and dichloromethane with stirring at room temperature , the reaction progress was monitored by tlc . the reaction time was 2 . 5 hrs . the solvent was removed in vacuo . the compound , 4a was isolated using flash chromatography ( particle size 32 - 63 μm ) with a 100 % ea — 1 : 20 ea : meoh ( with 1 % triethylamine ) gradient ( 80 % yield ). esims :( m + h )+ calcd , 381 . 2 ; found , 382 . 2 . the general cyanuric chloride coupling procedure was followed for the intermediate linker 4a . ( 100 % dichloromethane : acetone r f 5 : 1 = 0 . 21 ). the product , am was isolated using flash chromatography ( particle size 32 - 63 μm ) with a dichloromethane : acetone 5 : 1 — dichloromethane : acetone 3 : 1 gradient . the product was an orange oil ( 52 % yield ). tlc ( dichloromethane : acetone 5 : 1 , r f = 0 . 21 ) esims : ( m + h )+ calcd , 528 . 1 ; found , 529 . 1 . h 1 nmr ( 200 mhz , cdcl 3 ) δ 7 . 6 - 7 . 8 ( d , 2h , j = 4 hz ) 7 . 35 - 7 . 37 ( d , 2h , 4 hz ), 6 . 7 - 6 . 8 ( m , 2h ) 4 . 9 ( s , 1h ), 4 . 72 ( d , 2h , j = 4 hz ,) 3 . 5 - 3 . 7 ( m ) 3 . 2 ( m , 2h ) 1 . 8 ( s , 2h ) 1 . 4 ( s , 9h ). building block ii ( 0 . 44 mmole ) was added to building block i ( 0 . 11 mmole ) in diea ( 1 ml ) and anhydrous thf ( 10 ml ) at room temperature . the reaction mixture was heated to 60 ° c . for 3 hr and filtered . the resin was washed with dmf ( 5 times ), alternatively with dichloromethane and methanol ( 5 times ), and finally dichloromethane ( 5 times ). the resin was dried in vacuum . general procedure for the final amination on the resin and product cleavage desired amines ( 4 equiv .) were added to the resin ( 10 mg ), coupled with building block i and building block ii , in diea ( 8 μl ) and 1 ml of nmp : n - buoh ( 1 : 1 ). the reaction mixture was heated to 120 ° c . for 3 hr . the resin was washed with dmf ( 5 times ), alternatively with dichloromethane and methanol ( 5 times ), and finally dichloromethane ( 5 times ). the resin was dried in vacuum . the product cleavage reaction was performed using 10 % trifluoroacetic acid ( tfa ) in dichloromethane ( 1 ml ) for 30 min at room temperature and washed with dichloromethane ( 0 . 5 ml ). free hydroxyl containing compounds were further treated with a piperazine resin in 0 . 5 ml thf at room temperature for 5 hrs to cleave the trifluoroacetic ester that was formed upon treatment with tfa . the resin was filtered out and washed with 0 . 1 ml thf . the purity and identity of all the products were monitored by lc - ms at 250 nm ( agilent 1100 model ); more than 90 % of compounds demonstrated & gt ; 90 % purity . the foregoing description of the specific embodiments will so fully reveal the general nature of the invention that other can , by applying current knowledge , readily modify and / or adapt for various application such specific embodiments without undue experimentation and without departing from the generic concept . therefore , such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments . it is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation . the means and materials for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention . thus , the expressions “ means to . . . ” and “ means for . . . ” as may be found in the specification above and / or in the claims below , followed by a functional statement , are intended to define and cover whatever structural , physical , chemical , or electrical element or structures which may now or in the future exist for carrying out the recited function , whether or nor precisely equivalent to the embodiment or embodiments disclosed in the specification above . it is intended that such expressions be given their broadest interpretation . 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