the tetracycline class of antibacterial agents has seen widespread clinical use for over 50 years due to its broad spectrum antibacterial activity.(1 ) tetracyclines inhibit bacterial growth by preventing protein biosynthesis through binding to the 30s ribosome , thereby blocking the binding of aminoacyl - trna to the acceptor site.(2 ) this class includes a number of naturally occurring compounds , such as chlorotetracycline ( 1)(3 ) and tetracycline ( 2),(4 ) as well as several semisynthetic analogues , such as doxycycline ( 3)(5 ) and minocycline ( 4 ) ( figure 1).(6 ) in addition to their use as antibiotics for serious infections , tetracyclines have also been prescribed for use in acne and rosacea.(7 ) as with many classes of antibiotics , widespread use of tetracyclines has led to resistance and has limited their use in more complicated bacterial infections . examples of naturally occurring ( 1 and 2 ) and semisynthetic ( 36 ) tetracyclines . two major bacterial resistance mechanisms have been identified for tetracyclines : ( 1 ) active transport from bacterial cells via efflux pumps(10 ) and ( 2 ) ribosomal protection.(11 ) the efflux pumps are comprised of inner membrane tetracycline efflux proteins that were first identified in gram - negative organisms ( tet(ae ) ) and were later found in gram - positive bacteria ( tet(kl)).(10c ) the ribosomal protection mechanism ( tet(mo ) ) involves the expression of proteins that bind to the ribosome and allow protein synthesis to proceed even in the presence of tetracyclines . since these early discoveries , the number of efflux and ribosomal protection mechanisms identified has increased.(12 ) the presence of the strongly activating 10-hydroxy group readily enables chemical modification of the tetracycline d - ring at the 7- and 9-positions . this strategy led to the introduction of the 7-dimethylamino group in compounds 46 , all of which have demonstrated improved activity in bacterial strains bearing efflux pump resistance mechanisms . more recently , the 9-substituted glycylcyclines , such as tigecycline ( 5),(13 ) and the aminomethylcyclines , such as amadacycline ( 6),(14 ) have shown improved activity for resistant organisms bearing the ribosomal protection mechanism . in 2005 , myers and co - workers published a fully synthetic route to the tetracyclines , the key step of which involves the michaeldieckmann condensation of the ab precursor 8 with structurally diverse d - ring precursors . this route provides potential access to a broad range of tetracyclines that are inaccessible via traditional semisynthesis.(17 ) one example of this is the introduction of a nitrogen atom into the d - ring , resulting in  azatetracyclines . one 7-azatetracycline and one 9-azatetracycline were disclosed in myers original work , resulting in compounds with only modest antibacterial activity . herein we demonstrate this powerful approach using pyridyl d - ring precursors 7 to generate novel 8-azatetracyclines , 10 ( scheme 1 ) . to explore 7- and 9-substituted-8-azatetracyclines , we required intermediates that could be readily modified either prior to or after construction of the protected compound 9 . thus , the halopyridines 15 and 16 were prepared according to schemes 2 and 3 . 3,5-dichloroisonicotinic acid was treated with sodium benzylate to give compound 12 , which was esterified to the phenyl ester 13 by conversion to the acid chloride , followed by treatment with phenol and 4-(dimethylamino)pyridine ( dmap ) . suzuki coupling(18 ) using methylboronic acid and pdcl2cy2 catalyst(19 ) gave the parent d - ring precursor 14 in good yield . n - oxide formation with hydrogen peroxide in acetic acid followed by treatment with either phosphorousoxychloride or phosphorousoxybromide gave 15a and 15b , respectively . for 15a , the reaction gave a 4.5:1.0 ratio of the desired regioisomer , while a 25:1 ratio was observed for 15b . the regiochemistry was assigned by conversion to the dimethylamino compound 15c through a palladium - mediated amination.(20)h nmr studies indicated an noe between the methyl groups of the amine and the adjacent ring methyl group . the methoxy derivative 15d was prepared from 15b by treatment with sodium methoxide in 2-methylpyrrolidinone ( nmp ) at 120 c . this gave the acid directly , which was re - esterified to the phenyl ester . reagents and conditions : ( i ) nah ( 2 equiv ) , bnoh , nmp , 80 c ; ( ii ) ( cocl)2 , cat . dmf , ch2cl2 ; ( iii ) phoh , et3n , dmap , ch2cl2 ; ( iv ) ch3b(oh)2 , pdcl2cy2 , k3po4 , toluene , water , 100 c ; ( v ) h2o2 , acoh , 80 c ; ( vi ) pocl3 , toluene , 100 c ; ( vii ) pobr3 , toluene , 100 c ; ( viii ) dimethylamine in thf , pd2dba3 , xantphos , k3po4 , 1,4-dioxane , 100 c ( sealed ) ; reagents and conditions : ( i ) for 15a , mcpba , ch2cl2 ; for 15b , h2o2 , acoh , 80 c ; ( ii ) pobr3 , toluene , 100 c ; ( iii ) bocnh2 , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 80 c ; ( iv ) hcl , 1,4-dioxane ; ( v ) hf - pyridine , nano2 , pyridine ; ( vi ) dimethylamine in thf , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 60 c ( sealed ) . the dihalo compounds 16a and 16b were prepared by n - oxidation of 15a and 15b followed by treatment with pobr3 ( scheme 3 ) . yields were typically moderate ( 4550% ) , with reduction of the n - oxide to give the starting pyridines 15 observed as major side reactions in both cases . palladium - mediated coupling of 16b with t - butylcarbamate gave a 2.5:1 mixture of regioisomers , which were readily separated after conversion to the free amine 16c upon treatment with hcl . treatment of 16c with sodium nitrite in the presence of hf - pyridine gave the fluoro derivative 16d in good yield.(21 ) similarly , a 2.4:1.0 ratio of regioisomers was observed in the palladium - mediated reaction of 16b with dimethylamine to give 16e and 16f . interestingly , the regioselectivity could be reversed by heating 16b in the presence of dimethylamine without catalyst to give 16f as the major isomer in a 3:1 ratio . the 9-amino d - ring precursors 18a and 18b were prepared according to scheme 4 . once again , palladium - mediated couplings using t - butylcarbamate proved to be highly effective for introduction of the amino group . further protection of the mono - boc intermediates 17 with boc2o and dmap in dmf gave the di - boc precursors 18 . reagents and conditions : ( i ) bocnh2 , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 80 c ; ( ii ) boc2o , dmap , dmf . most of the d - ring precursors were lithiated with lithium diisopropylamide ( lda ) at 78 c , followed by treatment with the enone 8 . warming to 20 c yielded the fully protected tetracycline precursors 19 . for d - ring precursors 16a , 16d , and 18b , a one - pot reaction was performed in which lithium bis(trimethylsilyl)amide ( lhmds ) was added to a 78 c solution of the precursor and the enone 8 followed by warming to 20 c . yields varied from 20 to 84% depending on substrate , with larger reaction scales generally providing better results . the lhmds procedure also typically gave higher yields but was only possible on the more highly electron deficient d - ring precursors . the 7-bromo intermediate 19c was further derivatized at this stage via suzuki couplings with methylboronic acid and phenylboronic acid to give 19d and 19e , respectively . similarly , palladium - mediated amination with 19h and t - butylcarbamate provided the 7-chloro-9-amino compound 19j . deprotection of the intermediates 19 with hf followed by hydrogenation in the presence of palladium on carbon gave the final 8-azatetracyclines 20ak . reagents and conditions : ( i ) for 14 , 15bd , 16e , and 18a , lda , tmeda , 78 c , thf then 8 , 78 c to 20 c ; ( ii ) for 16a , 16d , and 18b , lhmds , 8 , thf , 78 c to 20 c ; ( iii ) ch3b(oh)2 or phb(oh)2 , pdcl2dppf2-ch2cl2 , k3po4 , toluene , 1,4-dioxane , water , 100 c ; ( iv ) bocnh2 , pd2dba3 , xantphos , k3po4 , 1,4-dioxane , 100 c ; ( v ) aqueous hf , ch3cn or 1,4-dioxane ; ( vi ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane . to compare the 8-azatetracyclines directly to tigecycline , the 9-glycylamido-8-azatetracyclines 21ac were prepared ( scheme 6 ) . treatment with bromoacetylchloride in thf followed by excess t - butylamine gave the acylated intermediates , which were deprotected under standard conditions to give the final analogues 21ac . reagents and conditions : ( i ) hcl , 1,4-dioxane ; ( ii ) bromoacetylchloride , thf then t - bunh2 , 50 c ; ( iii ) aqueous hf , ch3cn or 1,4-dioxane ; ( iv ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane . the bromopyridine intermediates proved to be useful for the introduction of alkylamines via palladium - mediated aminations and alkyl groups via suzuki couplings , ultimately providing the 9-alkylamino-8-azatetracyclines 24 and 9-alkyl-8-azatetracyclines 26 ( scheme 7 ) . thus , 19h was treated with various amines in the presence of pd2dba3 and 9,9-dimethyl-4,5-bis-(diphenylphosphino)xanthenes ( xantphos ) in 1,4-dioxane at 100 c . potassium phosphate was found to be the most effective base for this transformation , with cesium carbonate and sodium t - butoxide leading to rapid decomposition . low yields were generally observed due to decomposition of both the starting material and products upon heating . reaction times of 24 h were eventually settled on as a balance between product formation and decomposition . copper - catalyzed aminations were also explored , but rapid decomposition of the starting material was observed . hindered amines generally gave low yields as did reactions with the 7-dimethylamino compound 19 m . alternatively , the alkylamines could be introduced prior to the michaeldieckmann reaction using either palladium or copper - mediated aminations(22 ) with 16a , 16d , and 16e . protection of the resulting aminopyridines upon treatment with lhmds followed by boc2o or cbzcl gave the d - ring precursors 22 . suzuki reactions with 19h or 19 m and various boronic acids were also carried out in moderate yields , providing the 9-alkyl intermediates 25 . reagents and conditions : ( i ) r2r3nh2 , pd2dba3 , xantphos , k3po4 or cs2co3 , 1,4-dioxane , 100 c or n - propylamine , cui , 2-acetylcyclohexanone , k3po4 , dmf , 100 c ; ( ii ) lhmds , thf then boc2o or cbzcl ; ( iii ) lda , tmeda , 78 c , thf then 8 , 78 c to 20 c or lhmds , 8 , thf , 78 c to 20 c ; ( iv ) aqueous hf , ch3cn , or 1,4-dioxane ; ( v ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane ; ( vi ) r2b(oh)2 , ( ph3p)4pd , k3po4 , toluene , 1,4-dioxane , h2o , 90 c . the in vitro antibacterial activities of all analogues were determined for a panel of gram - positive ( e.g. , staphylococcus aureus and streptococcus pneumoniae ) and gram - negative ( e.g. , escherichia coli and klebsiella pneumoniae ) bacteria . the activities of the 7-substituted-8-azatetracycline as well as several control tetracyclines for a representative set of these organisms are found in table 1 . the parent 8-azatetracycline 20a showed comparable activity to tetracycline , with reasonable minimum inhibitory concentrations ( mics ) of 0.251 g / ml for the tetracycline - susceptible strains . lacking a substituent at either c-7 or c-9 , 20a demonstrated poor activity for the strains bearing tet(m ) , tet(k ) , and tet(a ) . overall , the 7-position appears to be very tolerant of substitution , allowing for bulky ( 20e ) , electron withdrawing ( 20b and 20i ) , and electron donating ( 20f and 20 g ) groups while maintaining activity against the tetracycline - susceptible strains . as with minocycline , introduction of the 7-dimethylamino group in 20f led to improved activities for most strains tested . modest ( 12 dilution ) improvements in mics were observed for the tet(m ) strains , while a more significant improvement was seen for the strain bearing the tet(k ) resistance mechanism . with the exception of the 7-chloro analogue , all of the 7-substituted-8-aza compounds exhibited improved mics for the tet(k ) strain , correlating well with literature reports on 7-substituted tetracyclines . unfortunately , all of the 8-azatetracyclines appear to be substrates for the tet(a ) efflux pump , exhibiting no antibacterial activity at the highest concentration tested ( 32 g / ml ) . in general , substitution at the 7-position appears to be inefficient at overcoming the ribosomal protection resistance mechanism of the tet(m ) strains . the only compound with two dilution improvements for both tet(m ) strains was compound 20i , the 7-fluoro analogue . obtained from marilyn roberts lab at the university of washington . to make a direct comparison to tigecycline , the 9-glycylamido-8-azatetracyclines were prepared ( table 2 ) . whereas tigecycline is a very potent antibacterial agent against all of the organisms in our panel , including the four strains bearing tetracycline resistance mechanisms , the corresponding 8-azatetracycline analogues 21ac were found to have poor overall activity . the aniline precursors 20j and 20k , however , showed significant improvements in mics for both tet(m ) strains relative to the corresponding 9-unsubstituted compounds 20b and 20i . for the 7-cl analogue 20j , antibacterial activity also improved for the tet(k ) and wild type strains . these compounds were also assessed for their ability to inhibit protein synthesis in a cell - free transcription / translation assay ( table 3 ) . in this assay , compounds 20j and 20k were about 2-fold more potent than tetracycline , in agreement with their relative mics for e. coli . compounds 21ac , on the other hand , inhibited protein synthesis with ic50s between 0.42 and 0.90 g / ml , similar to tigecycline , despite having only modest antibacterial activity in the whole - cell assays . these data suggest that the compounds are active at the target , but that compounds 21ac are not capable of getting to the target in the whole - cell assay , possibly due to poor cell permeability . atcc 25922 is a tetracycline - susceptible strain . on the basis of the results of compounds 20j and 20k , compounds bearing a small , unbranched alkylamino group at the 9-position ( 24ac ) proved to be very potent antibacterial agents against the two tetracycline - susceptible gram - positive strains , with mics between 0.016 and 0.25 g / ml . a 4-fold improvement in activity against the tet(m ) s. aureus strain was observed for all of these compounds relative to 20j , while mics for the s. pneumoniae tet(m ) strain were largely unchanged . for the tet(k ) s. aureus strain , antibacterial activity improved with increasing size of the alkyl group . among the tetracycline - susceptible gram - negative strains , however , there was a clear loss in potency as the size of the alkyl group increased , a trend that continued for the larger branched alkyl compounds 24df . this is not surprising , as it is known that gram - negative antibacterial agents tend to be smaller and more polar.(23 ) these larger side chains also adversely affected the antibacterial activity for s. pneumoniae while having less effect on the mics for the s. aureus strains . the 7-fluoro-9-n - propylamino ( 24n ) and 7-dimethylamino-9-n - propylamino ( 24o ) analogues were prepared as comparisons to 24c . while the 7-fluoro compound had mics that tracked fairly closely to the 7-chloro analogue , the 7-dimethylamino compound exhibited no antibacterial activity for any of the strains at the highest concentration tested ( 32 g / ml ) with the exception of the tetracycline - susceptible s. pneumoniae strain . all three of the compounds had similar , albeit weak , activity in the transcription / translation assay , indicating the differences in mics might be due to poor permeability of the 7-dimethylamino analogue . introduction of a more polar heteroatom into the side chain ( 24gi , k ) resulted in improved antibacterial activity against the s. pneumoniae and gram - negative strains but had minimal effect on activity toward either resistant s. aureus organisms . interestingly , the oxygen bearing side chains ( 24 g and 24h ) yielded significantly improved antibacterial activity toward tetracycline - susceptible gram - positive strains relative to the all carbon side chains ( 24e and 24f ) , while the nitrogen analogues ( 24k and 24i ) were less potent against s. aureus and showed more modest improvements against s. pneumoniae . the combination of a branched side chain with a distal amino group gave the most balanced antibacterial compounds in this series ( 24l , 24 m , and 24j ) . these compounds combine good antibacterial activity toward both gram - positive and gram - negative organisms , with modest activity against tet(a ) e. coli . both compounds 24l and 24 m are less potent than tigecycline but compare favorably to amadacycline . the antibacterial activity of the 9-alkyl and phenyl substituted compounds were generally modest ( table 5 ) . here again , a larger substituent at c-9 yielded improved activity for tet(m ) and tet(k ) strains but resulted in loss of activity for wild type s. pneumoniae and gram - negative strains . introduction of a 7-cl or 7-dimethylamino group also gave improved activity for most of the strains in the panel . when comparing the 7-cl-9-alkyl compounds ( 26b ) to the 7-cl-9-aminoalkyl compounds ( 24c ) , it is clear that the nitrogen confers significant improvements in antibacterial activity . in the 7-dimethylamino case , however , the 7-dimethylamino-9-alkyl compound 26a is a balanced gram - positive antibacterial agent while the 9-aminoalkyl compound 24o has no significant activity . were selected for further profiling . both compounds inhibited protein synthesis in the transcription / translation assay with ic50s comparable to tigecycline . susceptibility testing was performed , and mic50 and mic90 values were calculated for recent clinical isolates of s. aureus , s. pneumoniae , haemophilus influenzae , and e. coli ( table 6 ) . the panels contained both tetracycline - susceptible and resistant strains , including 12 methicillin - resistant s. aureus ( mrsa ) strains . as expected from the initial mic data , compound 20f had very potent antibacterial activity for the tetracycline - susceptible strains but performed poorly against the resistant organisms . the compound did have mic90s that were significantly lower than tetracycline . despite having higher mics in the primary screen relative to tigecycline , compound 24l performed similarly in the s. aureus , s. pneumoniae , and h. influenzae panels , exhibiting clinically relevant mic90s in each case . these organisms are particularly relevant , as they are responsible for infections in a large portion of community - acquired bacterial pneumonia cases . the pharmacokinetic profiles of the two 8-azatetracyclines were determined in spraguedawley rats ( table 7 ) . compound 20f had very similar auc , clearance , and volume of distribution profiles to both tetracycline and tigecycline , although the half - life was somewhat shorter . low oral bioavailability ( % f ) of 12.7% was observed but was comparable to tetracycline ( 12.1% ) . in humans , tetracycline is generally reported to have oral bioavailability of 5070%.(24 ) we and others have found the oral bioavailability of several tetracycline derivatives to be significantly lower in rat than reported data in humans . compound 24l had a 4-fold higher auc and 5-fold lower clearance than the other three compounds as well as a significantly lower volume of distribution . compounds were dosed at 1 mg / kg iv and 10 mg / kg po with sterile water as vehicle . the compounds were also investigated in mouse s. aureus and e. coli septicemia models ( table 8) . both 8-azatetracyclines performed well in the s. aureus model following iv administration , with pd50 values comparable to the two marketed control tetracyclines . 20f , the most potent of the four compounds , also provided the best protection , with 100% survival at the lowest dose at which it was administered ( 0.30 mg / kg ) . despite having an 8-fold higher mic , compound 24l provided equivalent protection relative to tigecycline , likely due to its higher auc and lower clearance.(25 ) the e. coli model proved to be more challenging , requiring higher doses for protection . compound 20f had a pd50 of 4.3 mg / kg compared to 2.1 mg / kg for tigecycline . compound 24l was 4-fold less potent than 20f and was comparable to tetracycline in this model . s. aureus atcc 13709 ( smith ) or e. coli atcc 25922 was mixed with 5% mucin and inoculated by intraperitoneal injection at 2.1  10 cfu / mouse ( s. aureus ) or 2.0  10 cfu / mouse ( e. coli ) . one hour postchallenge , mice received iv treatment ( vehicle = sterile water ) with the test article at concentrations ranging from 0.0510 mg / kg ( s. aureus ) or 0.3030 mg / kg ( e. coli ) . the pd50 in mg / kg was calculated as survival after 48 h. lowest dose was 0.30 mg / kg with 100% survival at all doses . a novel , fully synthetic series of 8-azatetracycline analogues was prepared and optimized . through modification of the 7- and 9-positions , compounds were found that overcome both tet(k ) efflux and tet(m)-mediated ribosomal protection . the 7-chloro-9-alkylamino-8-azatetracycline 24l exhibits clinically relevant mic90 profiles against both gram - positive and gram - negative organisms that include these resistance mechanisms . this compound also demonstrated in vivo efficacy in murine models of infection that is equivalent to currently marketed tetracycline antibacterial agents . the 7-dimethylamino-8-azatetracycline 20f was efficacious when administered intravenously and had oral bioavailability in rat equivalent to tetracycline . the current work demonstrates the ability of this chemistry platform to generate antibacterial compounds that are inaccessible through traditional semisynthesis , providing the medicinal chemist a broader array of tools through which essential antibacterial properties can be modulated . importantly , this includes the ability to overcome ever evolving resistance mechanisms and to identify structureactivity relationships to deliver compounds with desired pharmacokinetic properties . air and moisture sensitive liquids and reagents were transferred via syringe or cannula and were introduced into flame - dried glassware under a positive pressure of dry nitrogen through rubber septa . analytical thin - layer chromatography was performed on em science precoated glass - backed silica gel 60  f-254 250 m plates . visualization of the plates was effected by ultraviolet illumination and/or immersion of the plate in a basic solution of potassium permanganate in water followed by heating . column chromatography was performed on a flashmaster personal system using isolute flash si ii silica gel prepacked cartridges ( available from biotage ) . preparative reversed - phase hplc chromatography ( hplc ) was accomplished using a waters autopurification system with mass - directed fraction collection . all intermediate compounds were purified with a waters sunfire prep c18 obd column ( 5 m , 19 mm  50 mm ; flow rate = 20 ml / min ) using a mobile phase mixture of h2o ( a ) and ch3cn ( b ) containing 0.1% hco2h . the final 8-azatetracycline compounds were purified using a phenomenex polymerx 10  rp 100a column ( 10 m , 30 mm  21.2 mm ; flow rate = 20 ml / min ) using a mobile phase mixture of 0.05n hcl in h2o ( a ) and ch3cn ( b ) . all final 8-azatetracycline compounds were isolated as mono- , di- , or trihydrochloride salts following freeze - drying . h nmr spectra were recorded on a jeol ecx-400 ( 400 mhz ) spectrometer and are reported in ppm using residual solvent as the internal standard ( cdcl3 at 7.24 ppm , dmso - d6 at 2.50 ppm , or cd3od at 3.31 ppm ) . high performance liquid chromatographyelectrospray mass spectra ( lc - ms ) were obtained using a waters alliance hplc system equipped with a binary pump , a diode array detector , a waters sunfire c18 ( 5 m , 4.6 mm i.d . 50 mm ) column , and a waters 3100 series mass spectrometer with electrospray ionization . the eluent was a mixture of h2o ( a ) and ch3cn ( b ) containing 0.1% hco2h at a flow rate of 1 ml / min . purity of the final compounds was assessed by the following method : ( a ) time = 0 , 100% a ; ( b ) time = 0.5 min , 100% a ; ( c ) time = 3.5 min , 100% b ; ( d ) time = 5 min , 100% b ( e ) time = 6 min , 100% a ; ( f ) time = 7 min , 100% a. all final products were 95% purity as assessed by this method . sodium hydride ( 60% dispersion in mineral oil , 4.37 g , 109 mmol ) was added portionwise to a solution of 3,5-dichloroisonicotinic acid ( 10.2 g , 53.3 mmol ) in nmp ( 100 ml ) . after gas evolution ceased , benzyl alcohol ( 5.52 ml , 53.3 mmol ) was added dropwise . after 1 h , the reaction was complete and was allowed to cool to room temperature and stand overnight . the reaction mixture was diluted with water ( 300 ml ) and was washed with et2o ( 2  100 ml , discarded ) . the aqueous layer was brought to ph 2 with conc hcl , causing a precipitate to form . the mixture was diluted with brine ( 100 ml ) and was allowed to stand for 30 min . the solid was collected by filtration , was washed with water ( 3 ) , and was dried in a 45 c vacuum oven overnight . this gave 9.36 g ( 67% ) of the title compound as a white solid . h nmr ( 400 mhz , dmso - d6 )  14.3014.10 ( bs , 1 h ) , 8.52 ( s , 1h ) , 8.34 ( s , 1 h ) , 7.507.30 ( m , 5 h ) , 5.33 ( s , 2 h ) . oxalyl chloride ( 4.9 ml , 56 mmol ) was added to a suspension of 12 ( 3.71 g , 14.1 mmol ) in ch2cl2 ( 50 ml ) over 2 min . dmf was added dropwise in 20 l portions every 5 min until complete solution was achieved . after stirring for an additional 30 min , the resulting solid was dissolved in ch2cl2 ( 50 ml ) , and phenol ( 2.65 g , 28.1 mmol ) , dmap ( 0.172 g , 1.41 mmol ) , and et3n ( 9.76 ml , 70.4 mmol ) were added . after 1 h , the reaction mixture was diluted with ch2cl2 ( 50 ml ) and was washed with water ( 2  100 ml ) and nahco3 ( saturated , aqueous , 100 ml ) . the material was purified by column chromatography ( biotage 50 g column , 025% etoac in hexanes gradient ) , yielding 4.20 g ( 88% ) of the product as an off - white solid . h nmr ( 400 mhz , dmso - d6 )  8.70 ( s , 1h ) , 8.48 ( s , 1 h ) , 7.527.30 ( m , 8 h ) , 7.157.08 ( m , 2 h ) , 5.44 ( s , 2 h ) . compound 13 ( 2.01 g , 5.92 mmol ) , methyl boronic acid ( 1.06 g , 17.7 mmol ) , dichlorobis(tricyclohexylphosphine)palladium(ii ) ( 102 mg , 0.296 mmol ) , and k3po4 ( 3.76 g , 17.7 mmol ) were heated to 100 c in toluene ( 20 ml ) and water ( 2 ml ) . after 16 h , the reaction mixture was allowed to cool to room temperature and was diluted with etoac ( 20 ml ) . this was washed with water ( 20 ml ) and brine ( 20 ml ) , dried over na2so4 , filtered , and concentrated under reduced pressure . the material was purified by column chromatography ( biotage 50 g column , 040% etoac in hexanes gradient ) , yielding 1.66 g ( 88% ) of the product as a white solid . h nmr ( 400 mhz , dmso - d6 )  8.50 ( s , 1h ) , 8.26 ( s , 1 h ) , 7.527.30 ( m , 8 h ) , 7.207.10 ( m , 2 h ) , 5.37 ( s , 2 h ) , 2.38 ( s , 3 h ) . ms ( esi ) m / z 320.27 ( m + h ) . compound 14 ( 9.66 g , 30.3 mmol ) was heated to 80 c in acetic acid ( 50 ml ) and hydrogen peroxide ( 30% aqueous solution , 17 ml ) . after 6 h , lc / ms indicated that the starting material was consumed and the n - oxide present . most of the acetic acid and water were removed under reduced pressure . the material was then diluted with etoac ( 200 ml ) , and the mixture was washed with nahco3 ( saturated aqueous solution , 3  50 ml ) . the organics were dried over na2so4 , filtered , and concentrated under reduced pressure , yielding 9.37 g ( 92% ) of the crude n - oxide . the material was dissolved in toluene ( 60 ml ) , phosphorousoxychloride ( 7.69 g , 50.1 mmol ) was added , and the reaction mixture was heated to 100 c . after 2 h , lc / ms indicated that the reaction was complete . upon cooling to room temperature , the reaction mixture was poured into a solution of k2co3 ( 40 g ) in water ( 200 ml ) . this mixture was extracted with etoac ( 3  150 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was redissolved in etoac ( 30 ml ) with heating and was then allowed to cool to room temperature and stand overnight . another 1.92 g ( 19% ) of the white crystalline solid was obtained from the mother liquor after standing in a refrigerator overnight . h nmr ( 400 mhz , cdcl3 )  8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) . compound 14 ( 25.9 g , 81.1 mmol ) was heated to 80 c in acetic acid ( 160 ml ) and hydrogen peroxide ( 30% aqueous solution , 40 ml ) . after stirring overnight , lc / ms indicated that the starting material was consumed and the n - oxide present . the reaction mixture was concentrated under reduced pressure , was diluted with etoac , and was washed with nahco3 ( saturated aqueous solution , 3 ) . the organics were dried over na2so4 , filtered , and concentrated under reduced pressure , yielding 25.4 g ( 93% crude ) of the n - oxide as a tan solid . the material was suspended in toluene ( 200 ml ) and was heated to 80 c , at which point most of the solid had dissolved . upon cooling to room temperature , the reaction mixture was poured into a solution of k2co3 ( 83 g ) in water ( 300 ml ) . the combined extracts were dried over mgso4 , filtered through celite , and concentrated under reduced pressure . the material was recrystallized from etoac / hexanes ( 1:1 ) , yielding 20.6 g ( 64% ) of the product as an off - white solid . h nmr ( 400 mhz , cdcl3 )  8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) . compound 15b ( 1.06 g , 2.66 mmol ) , dimethylamine ( 2.0 m solution in thf , 4.0 ml , 8.0 mmol ) , k3po4 ( 1.7 g , 8.0 mmol ) , tris(dibenzylideneacetone)dipalladium ( 120 mg , 0.13 mmol ) , and xantphos ( 220 mg , 0.40 mmol ) were heated to 100 c in 1,4-dioxane ( 10 ml ) in a sealed pressure vessel . after heating overnight , the reaction mixture was cooled to room temperature , was diluted with etoac ( 100 ml ) , and was washed with water ( 3  50 ml ) and brine ( 50 ml ) . the material was purified by column chromatography ( biotage 50 g column , 015% etoac in hexanes gradient ) , yielding 388 mg ( 40% ) of the title compound as a white solid . h nmr ( 400 mhz , cdcl3 )  7.96 ( s , 1h ) , 7.457.20 ( m , 8 h ) , 7.08 ( d , j = 8.2 hz , 2 h ) , 5.16 ( s , 2 h ) , 2.76 ( s , 6 h ) , 2.37 ( s , 3 h ) . ms ( esi ) m / z 363.45 ( m + h ) . sodium methoxide ( 38 mg , 0.70 mmol ) was added to a solution of compound 15b ( 118 mg , 0.351 mmol ) in nmp ( 2 ml ) , and the reaction mixture was heated to 100 c . after heating overnight , additional sodium methoxide ( 57 mg , 1.1 mmol ) was added and heating was continued at 100 c . after an additional 1 h , the reaction mixture was cooled to room temperature , water ( 20 ml ) was added , and the ph was adjusted to 2 with 6 n aqueous hcl . this was extracted with etoac ( 3  20 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the crude acid intermediate was dissolved in ch2cl2 ( 10 ml ) , and oxalyl chloride ( 0.123 ml , 1.40 mmol ) was added . the material was dissolved in ch2cl2 ( 10 ml ) , and phenol ( 66 mg , 0.70 mmol ) , dmap ( 4 mg , 0.04 mmol ) , and et3n ( 0.245 ml , 1.76 mmol ) were added . after 1 h , the reaction mixture was diluted with etoac ( 50 ml ) and was washed with water ( 2  30 ml ) and brine ( 30 ml ) . the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 20.3 mg ( 17% ) of clean product . h nmr ( 400 mhz , cdcl3 )  7.75 ( s , 1h ) , 7.457.24 ( m , 9 h ) , 7.10 ( d , j = 7.3 hz , 2 h ) , 5.13 ( s , 2 h ) , 3.91 ( s , 3 h ) , 2.67 ( s , 3 h ) . ms ( esi ) m / z 336.1 , 338.1 ( m + h ) . compound 15a ( 4.99 g , 14.1 mmol ) was dissolved in ch2cl2 ( 30 ml ) , and 3-chloroperbenzoic acid ( 77% , 6.33 g , 28.2 mmol ) was added in one portion . after 6 h , an additional portion of 3-chloroperbenzoic acid ( 3.15 g , 14.1 mmol ) was added , and the reaction was stirred at room temperature overnight . the reaction mixture was diluted with ch2cl2 ( 200 ml ) and was washed with na2co3 ( saturated aqueous solution , 100 ml ) and brine . the material was purified by column chromatography ( biotage 70 g column , 550% etoac in hexanes gradient ) , yielding 3.90 g ( 75% ) of the pure n - oxide as an off - white solid ( rf = 0.33 in 66% etoac / hexanes ) . the n - oxide was dissolved in toluene ( 30 ml ) , phosphorousoxybromide ( 6.36 g , 22.1 mmol ) was added , and the reaction mixture was heated to 80 c . after 1 h , the reaction mixture was allowed to cool to room temperature and was poured into a solution of k2co3 ( 10 g ) in water ( 50 ml ) . this mixture was extracted with etoac ( 3  100 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was purified by column chromatography ( biotage 50 g column , 010% etoac in hexanes gradient ) , yielding 2.06 g ( 45% ) of the desired product 16a as an oil which slowly solidified on standing overnight . h nmr ( 400 mhz , cdcl3 )  8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) . compound 15b ( 8.54 g , 21.4 mmol ) was heated to 80 c in acetic acid ( 120 ml ) and hydrogen peroxide ( 30% aqueous solution , 30 ml ) . after stirring overnight , lc / ms indicated that the starting material was mostly consumed and the n - oxide present . the reaction mixture was cooled to room temperature and was diluted with brine ( 150 ml ) . the resulting precipitate was collected by filtration and was washed with water / brine ( 1:1 , 2 ) . the solid was dissolved in ch2cl2 and was dried over na2so4 , filtered , and concentrated under reduced pressure . this gave 7.83 g ( 88% crude ) of the n - oxide as a tan solid . the material was suspended in toluene ( 200 ml ) and was heated to 100 c , at which point most of the solid had dissolved . a solution of phosphorousoxybromide ( 10.8 g , 37.8 mmol ) in toluene ( 10 ml ) was added rapidly . after 45 min , the reaction mixture was allowed to cool to room temperature and was poured into a solution of k2co3 ( 40 g ) in water ( 150 ml ) . after stirring for 30 min at room temperature , the combined extracts were dried over mgso4 , filtered , and concentrated under reduced pressure . the material was purified by column chromatography ( biotage 50 g column , 08% etoac in hexanes gradient to isolate the product then 15% etoac in hexanes to recover 15b ) , yielding 5.02 g ( 49% ) of the product as an off - white solid . h nmr ( 400 mhz , dmso - d6 )  7.497.32 ( m , 8 h ) , 7.10 ( d , j = 7.3 hz , 2 h ) , 5.15 ( s , 2 h ) , 2.41 ( s , 3 h ) . ms ( esi ) m / z 476.18 , 478.16 , 480.16 ( m + h ) . compound 16b ( 2.15 g , 4.50 mmol ) , t - butylcarbamate ( 633 mg , 5.40 mmol ) , cs2co3 ( 2.93 g , 9.00 mmol ) , tris(dibenzylideneacetone)dipalladium ( 206 mg , 0.225 mmol ) , and xantphos ( 380 mg , 0.673 mmol ) were weighed into a flask . this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 15 ml ) was added . the reaction mixture was cooled to room temperature , was diluted with etoac ( 100 ml ) , and was washed with water ( 2  50 ml ) . the material was purified by column chromatography ( biotage 50 g column , 012% etoac in hexanes gradient ) , yielding 1.43 g ( 62% ) of a 4:1 mixture of the two regioisomeric compounds . h nmr ( 400 mhz , cdcl3 )  7.517.46 ( m , 2 h ) , 7.427.32 ( m , 7 h ) , 7.307.24 ( m , 1 h ) , 7.12 ( d , j = 8.2 hz , 0.5 h ) , 7.03 ( d , j = 8.2 hz , 2 h ) , 6.88 ( s , 0.25 h ) , 6.71 ( s , 1 h ) , 5.12 ( s , 2 h ) , 5.02 ( s , 0.5 h ) , 2.43 ( s , 0.75 h ) , 2.34 ( s , 3 h ) , 1.50 ( s , 9 h ) , 1.47 ( s , 2.25 h ) . ms ( esi ) m / z 535.10 , 537.10 ( m + na ) . the above regioisomeric mixture ( 1.43 g , 2.78 mmol ) was stirred in 4 m hcl in 1,4-dioxane ( 20 ml ) and 1,4-dioxane ( 5 ml ) overnight . the reaction mixture was diluted with etoac ( 100 ml ) and was washed with nahco3 ( saturated aqueous solution , 2  100 ml ) . the material was purified by column chromatography ( biotage 20 g column , 030% etoac in hexanes gradient ) , yielding 805 mg ( 70% ) of the major regioisomer 16c and 270 mg ( 24% ) of the minor regioisomer . h nmr ( 400 mhz , cdcl3 )  7.507.42 ( m , 2 h ) , 7.407.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.04 ( d , j = 8.2 hz , 2 h ) , 5.06 ( s , 2 h ) , 4.56 ( s , 2 h ) , 2.16 ( s , 3 h ) . ms ( esi ) m / z 413.11 , 415.01 ( m + h ) . nmr ( 400 mhz , cdcl3 )  7.427.34 ( m , 8 h ) , 7.137.10 ( m , 2 h ) , 5.01 ( s , 2 h ) , 4.64 ( s , 2 h ) , 2.35 ( s , 3 h ) ; ms ( esi ) m / z 413.09 , 415.09 ( m + h ) . hf - pyridine solution ( 70% hf , 2 ml ) was added to a 0 c solution of compound 16c ( 884 mg , 2.14 mmol ) in pyridine ( 1 ml ) . sodium nitrite ( 177 mg , 2.57 mmol ) was added ( bubbling observed ) , and the reaction mixture was allowed to slowly warm to room temperature . after 3 days , the reaction mixture was poured into na2co3 ( saturated aqueous solution , 30 ml ) and was extracted with etoac ( 3  30 ml ) . the combined extracts were washed with hcl ( 1 n aqueous solution , 2  50 ml ) and were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was purified by column chromatography ( biotage 20 g column , 06% etoac in hexanes gradient ) , yielding 687 mg ( 77% ) of the product as a colorless oil that slowly solidified on standing . h nmr ( 400 mhz , cdcl3 )  7.507.42 ( m , 2 h ) , 7.407.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.14 ( s , 2 h ) , 2.33 ( s , 3 h ) . ms ( esi ) m / z 416.17 , 418.15 ( m + h ) . compound 16b ( 1.51 g , 3.16 mmol ) , cs2co3 ( 3.1 g , 9.5 mmol ) , tris(dibenzylideneacetone)dipalladium ( 145 mg , 0.158 mmol ) , and xantphos ( 267 mg , 0.474 mmol ) were weighed into a vial equipped with a septum . this was evacuated and flushed with nitrogen ( 3 ) , and 1,4-dioxane ( 7 ml ) and dimethylamine ( 2.0 m solution in thf , 4.75 ml , 9.50 mmol ) were added . the reaction mixture was heated to 60 c and was stirred for 4 h. the reaction mixture was allowed to cool to room temperature , was filtered through celite , and was concentrated under reduced pressure . the material was purified by column chromatography ( biotage 50 g column , 010% etoac in hexanes gradient ) , yielding 868 mg ( 58% ) of the major regioisomer 16e and 354 mg ( 24% ) of the minor regioisomer 16f . data for 16e : h nmr ( 400 mhz , cdcl3 )  7.507.46 ( m , 2 h ) , 7.417.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.08 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.32 ( s , 3 h ) ; ms ( esi ) m / z 441.19 , 443.18 ( m + h ) ; rf = 0.29 in 10% etoac / hexanes . the regiochemistry was confirmed by an noe between the methyl protons and the n , n - dimethylamino protons ( 0.61% ) . data for 16f : h nmr ( 400 mhz , cdcl3 )  7.417.31 ( m , 7 h ) , 7.307.20 ( m , 1 h ) , 7.01 ( d , j = 7.3 hz , 2 h ) , 4.94 ( s , 2 h ) , 3.04 ( s , 6 h ) , 2.33 ( s , 3 h ) ; ms ( esi ) m / z 441.19 , 443.18 ( m + h ) ; rf = 0.40 in 10% etoac / hexanes . the regiochemistry was confirmed by noe between the benzylic protons of the o - benzyl group and the n , n - dimethylamino protons ( 0.44% ) . compound 16d ( 538 mg , 1.29 mmol ) , t - butylcarbamate ( 302 mg , 2.58 mmol ) , cs2co3 ( 840 mg , 2.58 mmol ) , tris(dibenzylideneacetone)dipalladium ( 59 mg , 0.065 mmol ) , and xantphos ( 109 mg , 0.190 mmol ) were weighed into a flask . this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 3 ml ) was added . after 4 h , the reaction mixture was cooled to room temperature , was diluted with etoac ( 50 ml ) , and was washed with water ( 2  25 ml ) . the material was purified by column chromatography ( biotage 20 g column , 012% etoac in hexanes gradient ) , yielding 451 mg ( 77% ) of the product . rf = 0.35 in 20% etoac / hexanes . h nmr ( 400 mhz , cdcl3 )  7.427.24 ( m , 8 h ) , 7.046.99 ( m , 2 h ) , 4.99 ( s , 2 h ) , 2.38 ( s , 3 h ) , 1.40 ( s , 18 h ) . compound 17a ( 451 mg , 1.00 mmol ) was treated with di - t - butyldicarbonate ( 1.09 g , 5.00 mmol ) and dmap ( 24 mg , 0.20 mmol ) in dmf ( 15 ml ) . after 30 min , the reaction mixture was diluted with etoac ( 100 ml ) and was washed with water ( 3  50 ml ) and brine ( 50 ml ) . the material was purified by column chromatography ( biotage 10 g column , 08% etoac in hexanes gradient ) , yielding 481 mg ( 87% ) of the title compound . h nmr ( 400 mhz , cdcl3 )  7.427.24 ( m , 8 h ) , 7.046.99 ( m , 2 h ) , 4.99 ( s , 2 h ) , 2.38 ( s , 3 h ) , 1.40 ( s , 18 h ) . compound 16e ( 467 mg , 1.06 mmol ) , t - butylcarbamate ( 372 mg , 3.17 mmol ) , cs2co3 ( 1.04 g , 3.17 mmol ) , tris(dibenzylideneacetone)dipalladium ( 48 mg , 0.053 mmol ) , and xantphos ( 89.3 mg , 0.159 mmol ) were weighed into a vial . this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 3 ml ) was added . after 4 h , the reaction mixture was cooled to room temperature and was filtered through celite . the filtrate was concentrated under reduced pressure , and the material was purified by column chromatography ( biotage 20 g column , 016% etoac in hexanes gradient ) , yielding 493 mg ( 98% ) of the product containing 20% of an unidentified impurity . h nmr ( 400 mhz , cdcl3 )  7.437.32 ( m , 8 h ) , 7.307.24 ( m , 1 h ) , 7.157.10 ( m , 2 h ) , 6.80 ( s , 1 h ) , 4.94 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.31 ( s , 3 h ) , 1.48 ( s , 9 h ) . ms ( esi ) m / z 478.27 ( m + h ) . compound 17b ( 490 mg , 1.03 mmol ) was treated with di - t - butyldicarbonate ( 672 mg , 3.08 mmol ) and dmap ( 12.5 mg , 0.103 mmol ) in dmf ( 5 ml ) . after stirring overnight , the reaction mixture was diluted with etoac ( 25 ml ) and was washed with water ( 3  20 ml ) and brine ( 20 ml ) . the material was purified by column chromatography ( biotage 10 g column , 010% etoac in hexanes gradient ) , yielding 424 mg ( 72% ) of the product . h nmr ( 400 mhz , cdcl3 )  7.427.24 ( m , 8 h ) , 7.067.00 ( m , 2 h ) , 4.92 ( s , 2 h ) , 2.79 ( s , 6 h ) , 2.36 ( s , 3 h ) , 1.39 ( s , 18 h ) . ms ( esi ) m / z 578.33 ( m + h ) . lda was prepared at 78 c from n - butyllithium ( 1.6 m solution in hexane , 0.495 ml , 0.792 mmol ) and diisopropylamine ( 0.112 ml , 0.792 mmol ) in thf ( 5 ml ) . tmeda ( 0.318 ml , 2.11 mmol ) was added , followed by dropwise addition of a solution of compound 14 ( 169 mg , 0.529 mmol ) in thf ( 2 ml ) . after 5 min , a solution of 8 ( 128 mg , 0.264 mmol ) in thf ( 2 ml ) was added . after complete addition , the reaction mixture was allowed to warm to 20 c over 1 h. the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution , 20 ml ) and was extracted with etoac ( 2  20 ml ) . the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 65.5 mg ( 35% ) of the title compound as a yellow solid . h nmr ( 400 mhz , cdcl3 )  15.77 ( s , 1 h ) , 8.36 ( s , 1 h ) , 8.16 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.405.25 ( m , 4 h ) , 3.93 ( d , j = 11.0 hz , 1 h ) , 3.163.04 ( m , 1 h ) , 2.982.90 ( m , 1 h ) , 2.762.64 ( m , 1 h ) , 2.602.40 ( m , 8 h ) , 2.12 ( d , j = 14 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 708.72 ( m + h ) . the following compounds were synthesized by methods similar to 19a . prepared from 15a in 38% yield , yellow solid . h nmr ( 400 mhz , cdcl3 )  15.6 ( s , 1 h ) , 8.12 ( s , 1 h ) , 7.567.49 ( m , 4 h ) , 7.427.32 ( m , 6 h ) , 5.37 ( s , 2 h ) , 5.07 ( s , 2 h ) , 3.91 ( d , j = 11.0 hz , 1 h ) , 3.133.02 ( m , 1 h ) , 2.97 ( dd , j = 15.6 4.9 hz , 1 h ) , 2.652.45 ( m , 3 h ) , 2.50 ( s , 6 h ) , 2.14 ( d , j = 15.6 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.27 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 742.56 ( m + h ) . prepared from 15b in 71% yield , yellow solid . h nmr ( 400 mhz , cdcl3 )  15.55 ( s , 1 h ) , 8.12 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.405.22 ( m , 4 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.25 ( dd , j = 16.5 hz , j = 4.88 hz , 1 h ) , 3.123.02 ( m , 1 h ) , 2.622.42 ( m , 9 h ) , 2.14 ( d , j = 14.0 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 786.63 , 788.63 ( m + h ) . prepared from 15c in 50% yield , yellow solid . h nmr ( 400 mhz , cdcl3 )  15.65 ( s , 1 h ) , 8.01 ( s , 1 h ) , 7.527.22 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 11.0 hz , 2 h ) , 4.02 ( d , j = 11.0 hz , 1 h ) , 3.05 ( dd , j = 17.2 hz , j = 11.6 hz , 1 h ) , 2.982.86 ( m , 1 h ) , 2.73 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.12 ( d , j = 13.4 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 751.79 ( m + h ) . prepared from 15d in 27% yield , yellow solid . h nmr ( 400 mhz , cdcl3 )  15.70 ( s , 1 h ) , 7.83 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 13.4 hz , 2 h ) , 3.98 ( d , j = 11.0 hz , 1 h ) , 3.72 ( s , 3 h ) , 3.18 ( d , j = 16.5 hz , 1 h ) , 3.04 2.96 ( m , 1 h ) , 2.602.30 ( m , 9 h ) , 2.15 ( d , j = 14 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 738.66 ( m + h ) . h nmr ( 400 mhz , cdcl3 )  15.59 ( s , 1 h ) , 7.567.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.92 ( q , j = 67.8 hz , j = 9.16 hz , 2 h ) , 3.91 ( d , j = 11.0 hz , 1 h ) , 3.203.02 ( m , 2 h ) , 2.622.45 ( m , 9 h ) , 2.192.12 ( m , 1 h ) , 1.37 , ( s , 18 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 941.59 ( m + h ) . prepared from 16e in 54% yield , yellow solid . h nmr ( 400 mhz , cdcl3 )  15.65 ( s , 1 h ) , 7.527.22 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 11.0 hz , 2 h ) , 4.01 ( d , j = 11.0 hz , 1 h ) , 3.05 ( dd , j = 17.2 hz , j = 11.6 hz , 1 h ) , 2.982.86 ( m , 1 h ) , 2.73 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.12 ( d , j = 13.4 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 829.49 , 831.48 ( m + h ) . compound 16a ( 793 mg , 1.84 mmol ) and compound 8 ( 885 mg , 1.84 mmol ) were dissolved in thf ( 16 ml ) , and the solution was cooled to 78 c . lhmds ( 1.0 m solution in thf , 5.5 ml , 5.5 mmol ) was added slowly via syringe . after 10 min , the reaction mixture was allowed to slowly warm to 0 c over 1 h. a phosphate buffer solution ( ph 7.0 , 20 ml ) was added , followed by the addition of ammonium chloride ( saturated , aqueous solution , 50 ml ) . the resulting mixture was extracted with ch2cl2 ( 3  50 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the resulting brown solid was washed with cold methanol ( 3  5 ml ) to afford 1.11 g ( 74% ) of the title compound as a yellowbrown powder . the organics were concentrated under reduced pressure and purified by column chromatography ( biotage 20 g column , 520% etoac in hexanes gradient ) , yielding an additional 150 mg ( 10% ) of the product . h nmr ( 400 mhz , cdcl3 )  15.45 ( br , 1 h ) , 7.547.48 ( m , 4 h ) , 7.407.30 ( m , 6 h ) , 5.36 ( s , 2 h ) , 5.03 ( abq , j = 10.4 hz , 2 h ) , 3.87 ( d , j = 11.0 hz , 1 h ) , 3.273.23 ( m , 1 h ) , 3.103.00 ( m , 1 h ) , 2.652.57 ( m , 1 h ) , 2.572.43 ( m , 8 h ) , 2.16 ( d , j = 11.0 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 820.37 , 822.37 ( m + h ) . the following compounds were prepared by methods similar to 19h . prepared from 16d in 45% yield , yellow solid . h nmr ( 400 mhz , cdcl3 )  15.55 ( s , 1 h ) , 7.567.26 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.02 ( s , 2 h ) , 3.88 ( d , j = 10.4 hz , 1 h ) , 3.183.04 ( m , 2 h ) , 2.622.58 ( m , 1 h ) , 2.582.40 ( m , 8 h ) , 2.17 ( d , j = 14.6 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 804.34 , 806.34 ( m + h ) . prepared from 18b in 53% yield , yellow solid . h nmr ( 400 mhz , cdcl3 )  15.50 ( s , 1 h ) , 7.527.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.85 ( q , j = 86.1 hz , j = 9.76 hz , 2 h ) , 4.02 ( d , j = 10.4 hz , 1 h ) , 3.083.00 ( m , 1 h ) , 3.002.82 ( m , 1 h ) , 2.77 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.202.12 ( m , 1 h ) , 1.35 , ( br s , 18 h ) , 0.79 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 966.59 ( m + h ) . a solution of compound 19c ( 52 mg , 0.067 mmol ) , methylboronic acid ( 40 mg , 0.67 mmol ) , dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(ii ) dichloromethane adduct ( 3 mg , 0.003 mmol ) , and k3po4 ( 142 mg , 0.667 mmol ) in toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.2 ml ) was heated to 70 c . after 2 h , the reaction mixture was heated to 100 c . after an additional 2 h , the reaction mixture was diluted with etoac ( 20 ml ) and was washed with water ( 15 ml ) and brine ( 15 ml ) . the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 18.3 mg ( 38% ) of the title compound as a yellow solid . h nmr ( 400 mhz , cdcl3 )  15.71 ( s , 1 h ) , 8.24 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.305.20 ( m , 2 h ) , 3.95 ( d , j = 10.4 hz , 1 h ) , 3.102.92 ( m , 2 h ) , 2.622.42 ( m , 12 h ) , 2.12 ( d , j = 14.0 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.14 ( s , 3 h ) . ms ( esi ) m / z 722.72 ( m + h ) . a solution of compound 19c ( 31 mg , 0.040 mmol ) , phenylboronic acid ( 24.5 mg , 0.201 mmol ) , dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(ii ) dichloromethane adduct ( 1.6 mg , 0.002 mmol ) , and sodium carbonate ( 21.3 mg , 0.201 mmol ) in toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.2 ml ) was heated to 100 c via microwave reactor for 10 min . the reaction mixture was diluted with etoac ( 10 ml ) and was washed with water ( 5 ml ) and brine ( 5 ml ) . the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 16.9 mg ( 54% ) of the title compound as a yellow solid . h nmr ( 400 mhz , cdcl3 )  15.66 ( s , 1 h ) , 8.48 ( s , 1 h ) , 7.527.24 ( m , 15 h ) , 5.425.30 ( m , 4 h ) , 3.97 ( d , j = 10.4 hz , 1 h ) , 3.002.86 ( m , 2 h ) , 2.782.62 ( m , 1 h ) , 2.582.30 ( m , 8 h ) , 2.00 ( d , j = 14.0 hz , 1 h ) , 0.80 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.14 ( s , 3 h ) . ms ( esi ) m / z 784.75 ( m + h ) . compound 19h ( 100 mg , 0.122 mmol ) , t - butylcarbamate ( 42.9 mg , 0.366 mmol ) , k3po4 ( 77.7 mg , 0.366 mmol ) , tris(dibenzylideneacetone)dipalladium ( 5.6 mg , 0.006 mmol ) , and xantphos ( 10.3 mg , 0.018 mmol ) were weighed into a vial equipped with a septum . the vial was evacuated and flushed with nitrogen ( 3 ) , and 1,4-dioxane ( 0.5 ml ) was added . the reaction mixture was heated to 100 c and stirred for 2 h. the reaction mixture was allowed to cool to room temperature , was filtered through celite , and was concentrated under reduced pressure . the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 41.7 mg ( 40% ) of the title compound as a yellow solid . h nmr ( 400 mhz , cdcl3 )  15.6515.45 ( br s , 1 h ) , 7.567.50 ( m , 2 h ) , 7.437.34 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 5.38 ( s , 2 h ) , 4.94 ( dd , j = 29.3 hz , j = 11.0 hz , 2 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.27 ( dd , j = 16.4 hz , j = 11.6 hz , 1 h ) , 3.123.03 ( m , 1 h ) , 2.682.60 ( m , 1 h ) , 2.602.45 ( m , 8 h ) , 2.18 ( d , j = 14.6 hz , 1 h ) , 1.47 ( s , 9 h ) , 0.85 ( s , 9 h ) , 0.29 ( s , 3 h ) , 0.15 ( s , 3 h ) . ms ( esi ) m / z 857.67 ( m + h ) . aqueous hf ( 48% , 0.4 ml ) was added to a solution of 19a ( 65.5 mg , 0.093 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water ( 20 ml ) . the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added . an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 2 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure . the material was purified by preparative hplc ( phenomenex polymerx column , 0100% b gradient ) . fractions with the desired mw were collected and freeze - dried to yield 18.4 mg ( 41% , 2 steps ) of the title compound as a yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  8.55 ( s , 1 h ) , 8.36 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.262.98 ( m , 9 h ) , 2.71 ( t , j = 14.2 hz , 1 h ) , 2.392.32 ( m , 1 h ) , 1.751.63 ( m , 1 h ) . the following compounds were synthesized by similar methods to 20a . prepared from 19b in 24% yield , 2 steps , yellow solid . h nmr ( 400 mhz , cd3od )  8.08 ( d , j = 6.0 hz , 1 h ) , 4.09 ( s , 1 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.70 1.58 ( m , 1 h ) . ms ( esi ) m / z 450.18 ( m + h ) . prepared from 19d in 34% yield , 2 steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  8.35 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.252.95 ( m , 9 h ) , 2.802.25 ( m , 5 h ) , 1.801.63 ( m , 1 h ) . ms ( esi ) m / z 430.46 ( m + h ) . prepared from 19e in 24% yield , 2 steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  8.53 ( s , 1 h ) , 7.62 ( s , 5 h ) , 4.18 ( s , 1 h ) , 3.142.82 ( m , 9 h ) , 2.812.66 ( m , 1 h ) , 2.242.14 ( m , 1 h ) , 1.661.52 ( m , 1 h ) . ms ( esi ) m / z 492.48 ( m + h ) . prepared from 19f in 96% yield , 2 steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  8.22 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.383.30 ( m , 2 h ) , 3.24 ( s , 6 h ) , 3.122.95 ( m , 7 h ) , 2.60 ( t , j = 14.2 hz , 1 h ) , 2.422.34 ( m , 1 h ) , 1.751.63 ( m , 1 h ) . ms ( esi ) m / z 459.50 ( m + h ) . prepared from 19 g in 51% yield , 2 steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  7.80 ( s , 1 h ) , 4.15 ( s , 1 h ) , 3.97 ( s , 3 h ) , 3.402.98 ( m , 9 h ) , 2.322.20 ( m , 2 h ) , 1.721.56 ( m , 1 h ) . ms ( esi ) m / z 446.39 ( m + h ) . prepared from 19i in 68% yield , 2 steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  7.81 ( s , 1 h ) , 4.17 ( s , 1 h ) , 3.262.96 ( m , 9 h ) , 2.422.25 ( m , 2 h ) , 1.701.58 ( m , 1 h ) . ms ( esi ) m / z 434.27 ( m + h ) . prepared from 19j in 68% yield , 2 steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.19 ( s , 1 h ) , 3.202.98 ( m , 8 h ) , 2.352.22 ( m , 2 h ) , 1.681.56 ( m , 1 h ) . ms ( esi ) m / z 465.32 ( m + h ) . prepared from 19k in 49% yield , 2 steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.20 ( s , 1 h ) , 3.402.90 ( m , 9 h ) , 2.422.22 ( m , 2 h ) , 1.701.54 ( m , 1 h ) . compound 19j ( 68.3 mg , 0.080 mmol ) was stirred in 4 m hcl in 1,4-dioxane for 2 h. the reaction mixture was concentrated under reduced pressure . the material was dissolved in thf ( 2 ml ) , and bromoacetylchloride ( 12.5 mg , 0.080 mmol ) was added . after stirring overnight , an additional portion of bromoacetylchloride ( 0.020 ml ) was added . t - butylamine ( 0.063 ml , 0.60 mmol ) was added to a solution of the intermediate ( 35 mg , 0.040 mmol ) in thf ( 1 ml ) , and the reaction mixture was heated to 50 c . after stirring overnight , the reaction mixture was concentrated under reduced pressure and was purified by preparative hplc ( sunfire prep c18 column , 50100% b gradient ) . this gave 16.8 mg ( 49% ) of the title compound as a yellow solid . h nmr ( 400 mhz , cdcl3 )  8.20 ( br s , 1 h ) , 7.527.46 ( m , 2 h ) , 7.437.30 ( m , 8 h ) , 5.36 ( s , 2 h ) , 4.95 ( dd , j = 40.6 hz , j = 11.0 hz , 2 h ) , 3.88 ( d , j = 11.0 hz , 1 h ) , 3.323.24 ( m , 1 h ) , 3.123.03 ( m , 1 h ) , 2.662.59 ( m , 1 h ) , 2.582.42 ( m , 8 h ) , 2.18 ( d , j = 14.6 hz , 1 h ) , 1.301.10 ( br s , 9 h ) , 0.82 ( s , 9 h ) , 0.27 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 870.60 ( m + h ) . aqueous hf ( 48% , 0.4 ml ) was added to a solution of the preceding intermediate ( 16.8 mg , 0.019 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 7.8 g ) in water ( 15 ml ) . sodium chloride ( 10 g ) was added to the aqueous layer , and this was extracted with etoac ( 2 ) . the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added . an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure . the material was purified by preparative hplc ( phenomenex polymerx 10  rp 100a column , 0100% b gradient ) . fractions with the desired mw were collected and freeze - dried to yield 1.2 mg ( 10% , 2 steps ) of the title compound as a yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.304.20 ( s , 2 h ) , 4.19 ( s , 1 h ) , 3.252.96 ( m , 9 h ) , 2.482.28 ( m , 2 h ) , 1.721.58 ( m , 1 h ) , 1.43 ( s , 9 h ) . the following compounds were prepared according to methods similar to 21a : prepared from 19k in 68% yield for the acylation / amine displacement and 58% yield for the deprotections , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.244.65 ( m , 3 h ) , 3.262.97 ( m , 9 h ) , 2.382.28 ( m , 2 h ) , 1.691.56 ( m , 1 h ) , 1.44 ( s , 9 h ) . ms ( esi ) m / z 562.37 ( m + h ) . prepared from 19l in 43% yield for the acylation / amine displacement and 55% yield for the deprotections , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.304.40 ( m , 3 h ) , 3.403.25 ( m , 8 h ) , 3.123.04 ( m , 4 h ) , 2.99 ( s , 3 h ) , 2.622.52 ( m , 1 h ) , 2.412.35 ( m , 1 h ) , 1.731.60 ( m , 1 h ) , 1.45 ( s , 9 h ) . compound 19h ( 50 mg , 0.061 mmol ) , k3po4 ( 39 mg , 0.18 mmol ) , tris(dibenzylideneacetone)dipalladium ( 2.8 mg , 0.003 mmol ) , and xantphos ( 5.1 mg , 0.009 mmol ) were added to a small vial equipped with a septum . after the flask was evacuated and flushed with nitrogen ( 3 ) , 1,4-dioxane ( 0.5 ml ) and ethylamine ( 2.0 m solution in thf , 0.091 ml , 0.18 mmol ) were added . the reaction mixture was heated to 100 c and stirred for 3 h. the reaction mixture was allowed to cool to room temperature and was filtered through celite . the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient ) . fractions with the desired mw were collected and freeze - dried to yield 11 mg ( 23% ) of the title compound as a yellow solid . h nmr ( 400 mhz , cdcl3 )  15.6 ( br , 1 h ) , 7.567.49 ( m , 4 h ) , 7.427.30 ( m , 6 h ) , 5.37 ( s , 2 h ) , 5.03 ( s , 2 h ) , 3.89 ( d , j = 11.0 hz , 1 h ) , 3.243.04 ( m , 3 h ) , 2.97 ( dd , j = 15.6 , 4.9 hz , 1 h ) , 2.642.43 ( m , 3 h ) , 2.49 ( s , 6 h ) , 2.14 ( d , j = 15.6 hz , 1 h ) , 1.25 ( t , j = 6.8 hz , 3 h ) , 0.81 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.11 ( s , 3 h ) . ms ( esi ) m / z 785.57 ( m + h ) . aqueous hf ( 48% , 0.3 ml ) was added to a solution of the preceding intermediate ( 11 mg , 0.014 mmol ) in ch3cn ( 7 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 2 g ) in water ( 10 ml ) . the material was dissolved in meoh ( 2 ml ) and 1,4-dioxane ( 2 ml ) , and palladium on carbon ( degussa , 10 wt % , 5.6 mg ) was added . an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure . the material was purified by preparative hplc ( phenomenex polymerx 10  rp 100a column , 0100% b gradient ) . fractions with the desired mw were collected and freeze - dried to yield 3.2 mg ( 46% , 2 steps ) of the title compound as a yellow solid . h nmr ( 400 mhz , cd3od )  4.08 ( s , 1 h ) , 3.43 ( q , j = 7.4 hz , 2 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.671.55 ( m , 1 h ) , 1.22 ( t , j = 7.4 hz , 3h ) . ms ( esi ) m / z 493.24 ( m + h ) . the following compounds were prepared by similar methods to 24a : prepared from 19h and methylamine ( 1.0 m solution in thf ) in 21% yield for the amination step , 19% for the 2 deprotection steps , yellow solid . h nmr ( 400 mhz , cd3od )  4.08 ( s , 1 h ) , 3.41 ( s , 3 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.671.55 ( m , 1 h ) . ms ( esi ) m / z 479.22 ( m + h ) . prepared from 19h and n - propylamine in 24% yield for the amination step , 40% for the 2 deprotection steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.16 ( s , 1h ) , 3.47 ( t , j = 7.3 hz , 2 h ) , 3.152.96 ( m , 9 h ) , 2.322.20 ( m , 2 h ) , 1.781.55 ( m , 3 h ) , 1.01 ( t , j = 7.4 hz , 3 h ) . ms ( esi ) m / z 507.29 ( m + h ) . prepared from 19h and 2-propylamine in 11% yield for the amination step , 16% for the 2 deprotection steps , yellow solid . h nmr ( 400 mhz , cd3od )  4.214.13 ( m , 1 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.312.15 ( m , 2 h ) , 1.671.56 ( m , 1 h ) , 1.23 ( dd , j = 6.4 , 1.8 hz , 6 h ) . ms ( esi ) m / z 507.24 ( m + h ) . prepared from 19h and 2-methylpropan-1-amine in 15% yield for the amination step , 57% for the 2 deprotection steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.18 ( s , 1h ) , 3.38 ( d , j = 7.3 hz , 2 h ) , 3.202.96 ( m , 9 h ) , 2.342.20 ( m , 2 h ) , 2.101.98 ( m , 1h ) , 1.681.54 ( m , 1 h ) , 1.00 ( d , j = 6.4 hz , 6 h ) . ms ( esi ) m / z 521.40 ( m + h ) . prepared from 19h and 3-methyl - butan-1-amine in 25% yield for the amination step , 66% for the 2 deprotection steps , yellow solid . h nmr ( 400 mhz , cd3od )  4.09 ( s , 1 h ) , 3.453.40 ( m , 2 h ) , 3.20 ( s , 1 h ) , 3.082.93 ( m , 9 h ) , 2.302.16 ( m , 2 h ) , 1.721.58 ( m , 2 h ) , 1.551.48 ( m , 2 h ) , 0.96 ( d , j = 6.4 hz , 6 h ) . ms ( esi ) m / z 535.30 ( m + h ) . prepared from 19h and 2-methoxyethylamine in 19% yield for the amination step , 44% for the 2 deprotection steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.13 ( s , 1h ) , 3.723.60 ( m , 4h ) , 3.40 ( s , 3 h ) , 3.122.95 ( m , 9 h ) , 2.322.21 ( m , 2 h ) , 1.691.56 ( m , 1 h ) . ms ( esi ) m / z 523.32 ( m + h ) . prepared from 19h and 2-(propan-2-yloxy)ethan-1-amine in 9% yield for the amination step , 30% for the 2 deprotection steps , yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.19 ( s , 1h ) , 3.823.69 ( m , 5h ) , 3.202.98 ( m , 9 h ) , 2.352.22 ( m , 2 h ) , 1.681.56 ( m , 1 h ) , 1.18 ( d , j = 6.4 hz , 6h ) . ms ( esi ) m / z 551.41 ( m + h ) . prepared from 19h and ( 3-aminopropyl)dimethylamine in 9% yield for the amination step , 21% for the 2 deprotection steps , yellow solid . h nmr ( 400 mhz , cd3od )  4.09 ( s , 1 h ) , 3.703.46 ( m , 4 h ) , 3.102.90 ( m , 15 h ) , 2.312.17 ( m , 2 h ) , 2.102.00 ( m , 2 h ) , 1.681.56 ( m , 1 h ) . compound 16d ( 100 mg , 0.24 mmol ) , cs2co3 ( 235 mg , 0.720 mmol ) , tris(dibenzylideneacetone)dipalladium ( 11 mg , 0.012 mmol ) , and xantphos ( 20 mg , 0.036 mmol ) were weighed into a flask . this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 0.5 ml ) and n , n - dimethylneopentanediamine ( 0.057 ml , 0.36 mmol ) were added . after 1.5 h , the reaction mixture was cooled to room temperature and was filtered through celite . the filtrate was concentrated under reduced pressure and was purified by column chromatography ( biotage 10 g column , 03% meoh in ch2cl2 gradient ) , yielding 76.9 mg ( 69% ) of the intermediate . lhmds ( 1.0 m in thf , 0.22 ml , 0.22 mmol ) was added dropwise to a 78 c solution of the above intermediate ( 93 mg , 0.20 mmol ) in thf ( 5 ml ) . after 5 min , benzyl chloroformate ( 0.084 ml , 0.60 mmol ) after 15 min , the reaction mixture was quenched by the addition of nh4cl ( saturated , aqueous solution , 15 ml ) and was extracted with etoac . the material was purified by column chromatography ( biotage 10 g column , 07% meoh in ch2cl2 gradient ) , yielding 111 mg ( 93% ) of the product . nmr ( 400 mhz , cdcl3 )  7.457.20 ( m , 13 h ) , 7.066.98 ( m , 2 h ) , 5.16 ( s , 2 h ) , 4.87 ( s , 2 h ) , 3.78 ( br s , 2 h ) , 2.38 ( s , 3 h ) , 2.18 ( br s , 6 h ) , 1.62 ( br s , 2 h ) , 0.90 ( br s , 6 h ) . ms ( esi ) m / z 600.28 ( m + h ) . a solution of compound 22a ( 54 mg , 0.090 mmol ) in thf ( 0.5 ml ) was added dropwise to a 78 c solution of lda ( 2.0 m solution in thf , 0.112 ml , 0.224 mmol ) and tmeda ( 0.081 ml , 0.54 mmol ) in thf ( 2 ml ) , resulting in an orange - colored solution . after 10 min , a solution of 8 ( 43 mg , 0.090 mmol ) in thf ( 0.5 ml ) was added dropwise over 30 s. after complete addition , the reaction mixture was allowed to warm to 10 c over 1 h. the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) , was diluted with water , and was extracted with etoac ( 2 ) . the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was purified by preparative hplc ( sunfire prep c18 column , 20100% b gradient ) , yielding 22.9 mg ( 26% ) of the desired product as a yellow solid . h nmr ( 400 mhz , cdcl3 )  15.55 ( br s , 1 h ) , 7.527.20 ( m , 15 h ) , 5.36 ( s , 2 h ) , 5.205.00 ( m , 2 h ) , 4.80 ( s , 2 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.193.05 ( m , 2 h ) , 2.622.57 ( m , 1 h ) , 2.562.12 ( m , 19 h ) , 0.940.74 ( m , 15 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 988.59 ( m + h ) . aqueous hf ( 48% , 0.4 ml ) was added to a solution of 23a ( 22.9 mg , 0.0232 mmol ) in ch3cn ( 0.8 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water . the mixture was extracted with etoac , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) , 0.5 m hcl in meoh ( 0.2 ml ) , and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 2 mg ) was added . an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 3 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure . the material was purified by preparative hplc ( phenomenex polymerx column , 0100% b gradient ) . fractions with the desired mw were collected and freeze - dried to yield 9.9 mg ( 67% , 2 steps ) of the desired product as a yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.16 ( s , 1 h ) , 3.43 ( s , 2 h ) , 3.152.90 ( m , 17 h ) , 2.302.22 ( m , 1 h ) , 2.222.12 ( m , 1 h ) , 1.651.54 ( m , 1 h ) , 1.17 , ( s , 6 h ) . the following compounds were prepared according to methods similar to 24j : prepared from 16a and n , n - dimethylethylenediamine . h nmr ( 400 mhz , cd3od )  4.13 ( s , 1 h ) , 3.843.77 ( m , 2 h ) , 3.443.38 ( m , 2 h ) , 3.102.92 ( m , 15 h ) , 2.322.20 ( m , 2 h ) , 1.681.55 ( m , 1 h ) . ms ( esi ) m / z 536.25 ( m + h ) . prepared from 16a and n , n - dimethylneopentanediamine . h nmr ( 400 mhz , cd3od )  4.10 ( s , 1 h ) , 3.513.36 ( m , 4 h ) , 3.102.92 ( m , 15 h ) , 2.332.18 ( m , 2 h ) , 1.681.57 ( m , 1 h ) , 1.174 ( s , 3 h ) , 1.169 ( s , 3 h ) . ms ( esi ) m / z 578.35 ( m + h ) . prepared from 16a and 2,2-dimethyl-3-(pyrrolidin-1-yl)propan-1-amine . h nmr ( 400 mhz , cd3od )  4.09 ( s , 1 h ) , 3.883.78 ( m , 2 h ) , 3.503.39 ( m , 2 h ) , 3.18 2.92 ( m , 9 h ) , 2.322.08 ( m , 6 h ) , 1.671.56 ( m , 1 h ) , 1.15 ( s , 6 h ) . ms ( esi ) m / z 604.29 ( m + h ) . prepared from 16d and n - propylamine . h nmr ( 400 mhz , cd3od )  4.09 ( s , 1 h ) , 3.33 ( t , j = 6.9 hz , 2 h ) 3.423.38 ( m , 2 h ) , 3.20 ( s , 1 h ) , 3.072.94 ( m , 8 h ) , 2.91 ( dd , j = 14.6 , 4.6 hz , 1 h ) , 2.232.12 ( m , 2 h ) , 1.681.55 ( m , 3 h ) , 0.96 ( t , j = 7.3 hz , 3 h ) . copper(i ) iodide ( 4.7 mg , 0.025 mmol ) and k3po4 ( 209 mg , 0.984 mmol ) were added to a vial equipped with a septum . after the vial was evacuated and flushed with nitrogen ( 3 ) , a solution of compound 16e ( 217 mg , 0.492 mmol ) in dmf ( 2 ml ) , 1-propylamine ( 0.162 ml , 1.97 mmol ) , and 2-acetylcyclohexanone ( 0.013 ml , 0.098 mmol ) were added . the reaction mixture was heated to 100 c for 3 h. the reaction mixture was allowed to cool to room temperature , was diluted with etoac ( 50 ml ) , and was washed with water ( 3  40 ml ) . the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 74.6 mg ( 36% ) of the title compound as a thick oil . h nmr ( 400 mhz , cdcl3 )  7.507.46 ( m , 2 h ) , 7.417.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.08 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.32 ( s , 3 h ) . ms ( esi ) m / z 420.34 ( m + h ) . lhmds ( 1.0 m solution in thf , 0.167 ml , 0.167 mmol ) was added to a solution of phenyl 5-benzyloxy-2-(dimethylamino)-3-methyl-6-(propylamino)isonicotinate ( 63.5 mg , 0.152 mmol ) in thf ( 2 ml ) , resulting in a bright - yellow solution . a solution of di - t - butyldicarbonate ( 99.5 mg , 0.456 mmol ) in thf ( 1 ml ) was added . after 10 min , the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) and was extracted with etoac ( 2 ) . the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 13.6 mg ( 17% ) of the product . h nmr ( 400 mhz , cdcl3 )  7.407.28 ( m , 7 h ) , 7.287.22 ( m , 1 h ) , 7.057.00 ( m , 2 h ) , 4.90 ( s , 2 h ) , 2.79 ( s , 6 h ) , 2.34 ( s , 3 h ) , 1.761.66 ( m , 2 h ) , 1.561.50 ( m , 2 h ) , 0.91 ( t , j = 7.56 hz , 3 h ) . ms ( esi ) m / z 520.38 ( m + h ) . a solution of 22b ( 24 mg , 0.047 mmol ) in thf ( 0.5 ml ) was added dropwise to a 78 c solution of lda ( 0.5 m solution in thf , 0.102 ml , 0.051 mmol ) and tmeda ( 0.041 ml , 0.27 mmol ) in thf ( 2 ml ) , resulting in a yellowishorange colored solution . after 10 min , a solution of 8 ( 16.4 mg , 0.034 mmol ) in thf ( 0.5 ml ) was added dropwise . after complete addition , the reaction mixture was allowed to warm to 20 c over 45 min . the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) and was extracted with etoac ( 2 ) . the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient ) , yielding 6.1 mg ( 20% ) of the desired product as a yellow solid . h nmr ( 400 mhz , cdcl3 )  15.56 ( s , 1 h ) , 7.527.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.994.64 ( m , 2 h ) , 4.02 ( d , j = 11.0 hz , 1 h ) , 3.102.81 ( m , 2 h ) , 2.78 ( s , 6 h ) , 2.622.40 ( m , 10 h ) , 2.202.14 ( m , 1 h ) , 1.801.45 ( m , 2 h ) , 1.441.15 ( m , 11 h ) , 1.000.70 ( m , 12 h ) , 0.25 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 908.65 ( m + h ) . aqueous hf ( 48% , 0.4 ml ) was added to a solution of 20o ( 6.1 mg , 0.007 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water ( 15 ml ) . the combined etoac extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 2 mg ) was added . an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 4 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure . . fractions with the desired mw were collected and freeze - dried to yield 2.8 mg ( 70% , 2 steps ) of the desired product as a yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.19 ( s , 1 h ) , 3.44 ( t , j = 7.6 hz , 2 h ) , 3.243.12 ( m , 7 h ) , 3.122.97 ( m , 8 h ) , 2.392.26 ( m , 2 h ) , 1.731.56 ( m , 3 h ) , 0.98 ( t , j = 8.4 hz , 3 h ) ; ms ( esi ) m / z 516.42 ( m + h ) . compound 19 m ( 105 mg , 0.126 mmol ) , tetrakis(triphenylphosphine)palladium(0 ) ( 15 mg , 0.013 mmol ) , and k3po4 ( 80 mg , 0.38 mmol ) were weighed into an 8 ml vial . this was sealed with a septum and was evacuated and backflushed with nitrogen ( 3 ) . toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.5 ml ) were added , followed by allylboronic acid pinacol ester ( 63.7 mg , 0.379 mmol ) . after 2.5 h , the reaction mixture was cooled to room temperature and was concentrated under reduced pressure . the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient ) , yielding 28.6 mg ( 29% ) of the title compound as a yellow solid . aqueous hf ( 48% , 0.4 ml ) was added to a solution of 25a ( 28.6 mg , 0.036 mmol ) in 1,4-dioxane ( 1 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water . the mixture was extracted with etoac , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) , 0.5 m hcl in meoh ( 0.5 ml ) , and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added . an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure . the material was purified by preparative hplc ( phenomenex polymerx column , 070% b gradient ) . fractions with the desired mw were collected and freeze - dried to yield 14.3 mg ( 69% ) of the title compound as a yellow solid . h nmr ( 400 mhz , cd3od with 1 drop dcl )  4.20 ( s , 1 h ) , 3.383.15 ( m , 7 h ) , 3.142.94 ( m , 8 h ) , 2.912.82 ( m , 2 h ) , 2.56 ( t , j = 13.8 hz , 1 h ) , 2.402.32 ( m , 1 h ) , 1.861.76 ( m , 2 h ) , 1.741.62 ( m , 1 h ) , 1.01 ( t , j = 7.34 hz , 3 h ) . the following compounds were prepared according to methods similar to 26a : prepared from 19h and allylboronic acid pinacol ester , yellow solid . h nmr ( 400 mhz , cd3od )  4.10 ( s , 1 h ) , 3.222.92 ( m , 9 h ) , 2.812.74 ( m , 2 h ) , 2.492.38 ( m , 1 h ) , 2.262.19 ( m , 1 h ) , 1.77 1.60 ( m , 3 h ) , 0.97 ( t , j = 7.3 hz , 3 h ) . ms ( esi ) m / z 492.27 ( m + h ) . prepared from 19h and methylboronic acid , yellow solid . h nmr ( 400 mhz , cd3od )  4.09 ( s , 1 h ) , 3.222.92 ( m , 9 h ) , 2.85 ( s , 3 h ) , 2.492.38 ( m , 1 h ) , 2.262.19 ( m , 1 h ) , 1.77 1.60 ( m , 1h ) . ms ( esi ) m / z 464.30 ( m + h ) . prepared from 19h as an over - reduced side product in the synthesis of 26c . h nmr ( 400 mhz , cd3od )  7.78 ( s , 1 h ) , 4.12 ( s , 1 h ) , 3.222.91 ( m , 9 h ) , 2.78 ( s , 3 h ) , 2.492.21 ( m , 1 h ) , 1.771.62 ( m , 1h ) . ms ( esi ) m / z 430.46 ( m + h ) . prepared from 19h and phenylboronic acid . h nmr ( 400 mhz , cd3od )  8.047.98 ( m , 2 h ) , 7.477.39 ( m , 3 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.572.47 ( m , 1 h ) , 2.282.07 ( m , 1 h ) , 1.731.65 ( m , 1h ) . ms ( esi ) m / z 526.26 ( m + h ) . prepared from 19h as an over - reduced side product in the synthesis of 26e . h nmr ( 400 mhz , cd3od )  8.047.98 ( m , 2 h ) , 7.477.39 ( m , 3 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.572.47 ( m , 1 h ) , 2.282.07 ( m , 1 h ) , 1.731.65 ( m , 1h ) . tetracycline - susceptible isolates sa100 ( s. aureus atcc 13709 , smith ) , sa101 ( s. aureus atcc 29213 ) , sp106 ( s. pneumoniae atcc 49619 ) , ef103 ( e. faecalis atcc 29212 ) , ec107 ( e. coli atcc 25922 ) , kp109 ( k. pneumoniae atcc 13883 ) , ab110 ( acinetobacter baumannii atcc 19606 ) , ec108 ( enterobacter cloacae atcc 13047 ) , and pa111 ( pseudomonas aeruginosa atcc 27853 ) were obtained from the american type culture collection ( atcc , manassas , va ) . tetracycline - resistant isolates s. aureus sa158 ( tet(k ) ) , s. pneumoniae sp160 ( tet(m ) ) , e. faecalis ef159 ( tet(m ) ) , e. coli ec155 ( tet(a ) ) , k. pneumoniae kp153 ( tet(a ) ) were obtained from marilyn roberts lab at the university of washington . s. aureus sa161 ( tet(m ) ) was obtained from micromyx ( kalamazoo , mi ) . minimal inhibitory concentration ( mic ) determinations were performed in liquid medium in 96-well microtiter plates according to the methods described by the clinical and laboratory standards institute ( clsi).(26 ) cation - adjusted mueller hinton broth was obtained from bbl ( cat . 212322 , becton dickinson , sparks , md ) , prepared fresh and kept at 4 c prior to testing . a0432 , pml microbiologicals , wilsonville , or ) was used to supplement medium , as appropriate . all test methods met acceptable standards based on recommended quality control ranges for all comparator antibiotics and the appropriate atcc quality control strains . compound stocks prepared and diluted in sterile deionized water were assayed for inhibition of coupled in vitro transcription / translation using an e. coli s30 extract system with a firefly luciferase readout from promega ( cat . briefly , compounds were diluted into water and added to reaction mix aliquoted to black - walled 96-well microtiter plates ( cat . an appropriate three - point titration was used for each compound , and reactions were run in duplicate . plates were incubated at 37 c for one hour and then placed on ice for 5 min to arrest transcription / translation . e1500 ) was added to each well and luminescence was detected on a bmg labtech lumistar - optima instrument . positive assay control values , from reactions without inhibitor , were averaged per plate to determine percent inhibition of luciferase production . results were plotted using microsoft excel and 50% inhibition values ( ic50 ) were determined .
a novel series of fully synthetic 8-azatetracyclines was prepared and evaluated for antibacterial activity . 
 compounds were identified that overcome both efflux ( tet(k ) ) and ribosomal protection ( tet(m ) ) tetracycline resistance mechanisms and are active against gram - positive and gram - negative organisms . 
 two compounds were identified that exhibit comparable efficacy to marketed tetracyclines in in vivo models of bacterial infection .