in the 1920s , the german 
 biochemist otto warburg described metabolic 
 differences between cancerous and normal cells , where he noted that 
 tumor cells rely upon a high rate of aerobic glycolysis rather than 
 oxidative phosphorylation to produce energy for maintenance of cellular 
 functions . indeed , cancer cells have up to a 60-fold 
 enhanced rate of glycolysis relative to normal cells , even with sufficient 
 oxygen . this dependence upon glycolysis , 
 and its consequences , is termed  the warburg effect  . highly glycolytic cells produce excessive 
 amounts of lactate , the 
 end product of glycolysis , which is actively transported out of the 
 cell to normalize intracellular ph levels . lactate homeostasis is 
 maintained via a family of 12-membrane pass cell surface proteins 
 coined monocarboxylate transporters ( mcts ; also known as the slc16a 
 transporter family ) . fourteen mcts are known , but only mct1 , mct2 , 
 mct3 , and mct4 transport small monocarboxylates such as lactate , pyruvate , 
 and ketone bodies ( acetoacetate and -hydroxybutyrate ) across 
 plasma membranes in a proton - linked exchange . expression profiling studies have established that most aggressive 
 tumor types express markedly elevated levels of mct1 , mct4 , or both . notably , the expression of mct1 and mct4 is regulated by two major 
 oncogenic transcription factors , myc and hypoxia 
 inducible factor-1 ( hif-1 ) , respectively , that direct marked increases in the production of key proteins that 
 support aerobic glycolysis , including amino acid transporters and 
 enzymes involved in the catabolism of glutamine and glucose . malignancies having myc involvement 
 and hypoxic tumors are generally resistant to current frontline therapies , 
 with high rates of treatment failure , relapse , and high patient mortality . importantly , inhibition of mct1 or mct4 can kill tumor cells ex 
 vivo and provoke tumor regression in vivo , and 
 their potency is augmented by agents such as metformin that force 
 a glycolytic phenotype upon the cancer cell . many weak mct1 inhibitors ( i.e. , those effective at high micromolar 
 levels ) have been described , including -cyano-4-hydroxycinnamate , stilbene disulfonates , phloretin , and related flavonoids . the most potent known mct1 inhibitors 
 are the pyrrolopyridazinones and the thienopyrimidine diones ( e.g. , 
 compounds 12 , figure 1 ) . indeed , compound 2 has advanced into phase i clinical 
 trials for treating some human malignancies . these compounds , and to our knowledge all mct1 inhibitors yet described , 
 are dual mct1/mct2 inhibitors . mct2 has very high sequence homology 
 with mct1 , yet it likely has a lesser role than mct1 and mct4 for 
 monocarboxylate transport in human cancers based upon expression studies . 
 however , mct2 inhibition may play a role in potential off - target effects 
 of current agents that could arise from blocking lactate transport 
 in normal cells . improved mct1 inhibitors could be accessed by performing 
 additional 
 structure activity relationship ( sar ) studies around scaffold 1 . indeed , toward this goal we have made some refinements 
 in astra - zeneca s original synthetic strategy for 1 . however , a more desirable approach 
 is to seek alternative scaffolds for mct1 inhibition that are readily 
 synthesized and that may form similar transporter contacts as do compounds 12 . we considered   heterocyclic 
 ring systems as alternatives to the   ring systems present in 
 compounds 12 , expecting divergence 
 of sar and possibly favoring side chains and substituents that would 
 positively alter the physical properties of the resulting mct1 inhibitors . a core structure of particular interest was the pteridine scaffold 5 , a heterocyclic core that is present in many natural products and that has been widely used in drug discovery efforts . accordingly , we targeted appropriately substituted pteridine trione / dione 
 scaffolds 6 and 7 ( figure 2 ) . routes to 6,7-disubsituted pteridines have been reported , but to our knowledge the synthetic chemistry of substituted pteridinone 
 scaffolds 67 has not been explored . 
 here we report the synthesis of these substituted pteridinone scaffolds 
 and their activity as mct1-specific lactate transport inhibitors that 
 selectively block the growth of mct1-expressing human lymphoma cells . from an efficiency standpoint , 
 it is desirable to introduce structural 
 diversity ( e.g. , r and/or r in scaffolds 67 ) late in a synthetic sequence . our 
 synthesis of these scaffolds began with the commercially available 
 chloride 8 ( scheme 1 ) . alkylation 
 of 8 using isobutyl iodide gave an inseparable 5:1 mixture 
 of n- and o - alkylated products in 87% yield . after nitration , the n - alkyl regioisomer 9 was isolated in 75% yield after column chromatography . treatment 
 of 9 with protected naphthylalanine 10(40 ) under basic conditions gave 11 in 
 67% yield . nitro group reduction gave an intermediate lactam that 
 readily aromatized upon treatment with ddq , giving pteridine 2,4,6-trione 12 in 66% yield . alternatively , 
 alkylation of 12 with -bromoalcohols to give r - substituted compounds in scaffold 6 , afforded 
 the expected n5-alkylated pteridine 2,4,6-trione derivatives 14a  d , the first members of the targeted 
 core scaffold 6 available for biological evaluation . reagents and conditions : ( a ) 
 ( ch3)2chch2i , k2co3 , dmso , 60 c , 24 h , 87% ; ( b ) hno3 , h2so4 , 0 c , 2 h , 75% ; ( c ) na2co3 , dmf , 65 c , 1 h , 67% ; ( d ) zn , acoh , 80 c , 1 h , 
 100% ; ( e ) ddq , ch3cn , room temperature , 1 h , 66% ; ( f ) tf2o , tea , ch2cl2 , 0 c , 30 min , 98% ; 
 ( g ) k2co3 , butanone , reflux . we recognized that the choice of the naphthylalanine ester 10 and isobutyl iodide in the syntheses of 13 and 14a  d ( scheme 1 ) limited our early sar studies to lipophilic analogues in 
 scaffolds 6 and 7 , with r = 
 1-naphthylmethyl and r = isobutyl ( see figure 2 ) . strategically , once the c5 group 
 ( r in scaffold 6 ) or the c6 group ( r in scaffold 7 ) was optimized , we would then 
 use more polar amino acid esters in place of reagent 10 to give analogues having less lipophilic r groups . r could then also be varied by choosing different alkylating 
 agents in the first step of scheme 1 . access to triflate 13 allowed for the rapid syntheses 
 of c6-substituted compounds ( varying r ) in the core scaffold 7 . treatment of 13 with -mercaptoalcohols 
 gave thioethers 15a  c via snar substitution ( scheme 2 ) . these thioethers 
 were partially or fully oxidized , selectively , to form the racemic 
 sulfoxide 16 and the sulfone 17 . pd - catalyzed 
 cyanation of 13 and nitrile 
 hydrolysis gave acid 18 , which was then coupled with 
 tbs - protected amino alcohols to give , after deprotection , amides 1920 . reagents 
 and conditions : ( a ) 
 et3n , meoh , rt ; ( b ) m - cpba , dce , 0 c 
 or dcm , rt ; ( c ) zn(cn)2 , pd(pph3)4 , zn , nmp , 105 c ; ( d ) aq h2so4 1,4-dioxane ; 
 ( e ) hobt , edc , dmf dcm , rt ; ( f ) ( n - bu)4nf , thf , rt . introduction of a c6 
 side chain via sonogashira coupling of 13 with alkyne alcohols 21a  c afforded alkynes 22a  c ( scheme 3 ) , which were 
 reduced to compounds 23a  c by hydrogenation . compound 22b ( q = 3 ) was also converted 
 to triazole 24 by huisgen triazole 24 was further transformed to compounds 25 and 26 ( as a separable mixture of regioisomers ) via n - methylation . triflate 13 was readily converted to the 
 methyl ether 28 by a two - step sequence . reagents and conditions : ( a ) 
 pd(pph3)4 , cui , et3n , dmf , microwave , 
 175 c , 20 min ; ( b ) pd / c , h2 , meoh , rt ; ( c ) nan3 , cu cat . dmf , 120 c ; ( d ) mei , k2co3 , acetone , reflux , 5 h ; ( e ) lindlar s catalyst , h2 , thf ; ( f ) hcc(ch2)3ome , pd(pph3)4 , cui , et3n , dmf , microwave , 175 c , 
 20 min ; pd / c , h2 , meoh , thf . the 
 above methods gave a substantial test set of compounds with 
 differing r substituents in scaffolds 67 . in parallel , we took cues from the clinical mct1/2 inhibitor 2 and installed a dimethylpyrazole - containing side chain as 
 the r group in scaffold 7 . the same methods 
 used in scheme 2 were followed ( scheme 4 ) to convert the pyrazole - containing amino ester 29(44,45 ) to the triflate 30 and then to the test compound 31 . reagents and conditions : ( a ) 
 compound 21b , pd(pph3)4 , cui , dmf , 
 microwave , 175 c , 20 min ; ( b ) pd / c , h2 , meoh ; ( c ) 
 lioh , meoh . in our previous report that focused 
 on biological studies of mct1/mct2 
 inhibitors 12 , we showed that these compounds rapidly blocked the growth 
 of mct1-expressing tumor cells . in particular , they halted growth 
 of burkitt b cell lymphomas , which express high levels of myc , by virtue of immunoglobulin / myc gene translocations , and thus a high level of mct1 , 
 which is a direct transcription target induced by myc . raji burkitt lymphoma cells express high level of mct1 
 mrna and protein but not mrna and protein of other mcts , including 
 mct4 . thus , the proliferation of these 
 cells , as measured by an mtt assay , is a specific marker for mct1 
 inhibition . we used a 96 h mtt assay to test the ability of pteridinones 
 to block the growth of human raji lymphoma cells . to test for potential 
 off - target toxicity in the mtt assay , we also assessed the ability 
 of compounds to impede transport of c - lactate in human 
 estrogen receptor - positive mcf7 breast cancer cells using established 
 methods . as expected , in the mtt assay 
 inhibitors 1 and 2 dramatically impeded 
 tumor cell growth , with ec50 = 0.5 and 18 nm , respectively , 
 in general agreement with published data . in the lactate transport inhibition assay , curiously , all mct1 inhibitors 
 that we have tested are substantially more potent in the inhibition 
 of raji cell proliferation assay than in blocking lactate transport 
 in mcf7 cells . while this might suggest shared off - target effects 
 of all classes of mct1 inhibitors , it more likely reflects the very 
 different conditions , time frames , end points , and cell types used 
 in these assays . to confirm that 
 the antiproliferative effects seen in the raji 
 lymphoma - based mtt assays were indeed due to selective effects on 
 mct , we also assessed their activity in mcf7 breast cancer cells engineered 
 to overexpress mouse mct1 ( termed mcf7_mct1 cells , which were the 
 same cells used in lactate transport assays ) or human mct4 ( termed 
 mcf7_mct4 cells ) . we then measured the 
 effect of compound 1 on c lactate uptake 
 using each of these cell lines . mcf7_mct1 cells showed dramatically 
 increased c lactate uptake compared to parental mcf7 
 cells , and this was abolished by treatment with compound 1 . in contrast , mcf7_mct4 cells also showed increased c lactate uptake that was not inhibited by compound 1 . finally , in mtt assays , mcf7_mct1 cells were highly sensitive 
 to compound 1 ( growth inhibition ec50 = 21 
 nm ) , whereas mcf7_mct4 cells were resistant to compound 1 ( growth inhibition ec50 > 10 m , figure 3 ) . thus , studies using mcf7 breast 
 cancer cells and raji lymphoma 
 cells indicate that ( 1 ) the vast majority of c lactate 
 uptake is mediated by transport through mct1 in mcf7_mct1 cells , ( 2 ) 
 inhibition of lactate uptake is indicative of mct1 inhibition , ( 3 ) 
 the lack of an effect of compounds on the growth of mcf7_mct4 cells 
 in mtt assays is a marker for non - mct mediated cytotoxicity , and ( 4 ) 
 the robust mtt assay using the raji lymphoma cell line is well - suited 
 to served as a front - line screen for assessing the activity of test 
 mct1 inhibitors . actives from this screen are then profiled for c lactate uptake in mcf7_mct1 cells and are finally cross - checked 
 for cytoxicity in an mtt assay using mcf7_mct4 cells . the antiproliferative 
 effects of our compounds in raji lymphoma 
 cells are shown in the left column of table 1 . compound 12 , which lacks an r group , was 
 inactive , whereas the c5-alkylated pteridine triones 14a  c blocked lymphoma cell proliferation , although 14d was also inactive . the potency of the compounds varies 
 with the r chain length , where a four - methylene spacer 
 ( m = 4 ) is optimal ( see 14b , ec50 = 150  16 nm ) . for thioethers 15a  c , the compound with a five - atom tether , r = a 
 sulfur atom and four methylene groups ( n = 4 ) was 
 most potent ( 15c , ec50 = 37  10 nm ) . the racemic sulfoxide 16 and the sulfone 17 , possible thioether oxidative metabolites of thioether 15b , were less potent . the amide - linked 
 compounds 1920 were inactive in 
 blocking raji lymphoma cell proliferation . ec50 values ( cell - based ) 
 were determined using an mtt assay of human raji lymphoma cell proliferation . ic50 values were 
 determined 
 by a labeled lactate transport assay using mcf7 breast cancer cells 
 engineered to overexpress mct1 . value from one cell growth experiment , 
 run in triplicate with standard 
 error of mean , from 
 at least three independent raji mtt experiments , each run in triplicate . compound 23b , which like compound 15c also has r with 
 a five - atom tether to the primary hydroxyl group ( q = 3 ) , showed strong growth inhibition in the mtt assay ( ec50 = 58  10 nm ) . compound 23a , with one less methylene 
 group , was less active ( ec50 = 285  41 nm ) , while 
 compound 23c , with one more methylene group , had even 
 lower activity ( ec50  1.7 m ) . c , where proper positioning 
 of the side chain hydroxyl group in r is critical for 
 maximizing activity . incorporation of an nh or nme triazole moiety 
 at the 6-position led to a loss in potency ( compounds 2426 ) . the cis olefin 27 ( ec50 = 70  12 nm ) was roughly equipotent to its 
 saturated analogue 23b . the methyl ether analogue 28 was largely inactive ; thus , the h - bond donating hydroxyl 
 group is an essential component of the mct1 inhibitor pharmacophore . compound 31 was designed based upon clinical compound 2 , so quite to our surprise it was inactive in the raji lymphoma 
 cell proliferation assay . thus , different binding preferences exist 
 for the pteridine dione scaffold 7 versus the thienopyrimidine 
 diones ( e.g. , compound 2 ) . another scaffold difference is that the sulfur atom in compound 1 can not be replaced with a methylene group and retain potency 
 ( not shown ) , whereas compound 23b , with an all - carbon 
 link to the hydroxyl group , has similar potency to its thioether analogue 15c . our most potent new compounds in the mtt assay are compounds 15c , 23b , and 27 , which displayed 
 a ca . 24-fold higher ec50 than did the clinical 
 compound 2 , which , although less potent than compound 1 , is presumably preferable due to dmpk and drug - likeness criteria . compounds showing activity in 
 the mtt assay were then tested for 
 their ability to inhibit the transport of c - lactate in 
 mcf7 breast cancer cells engineered to overexpress mct1 ( right column , table 1 ) . the antiproliferative effects of test compounds , including potent 
 inhibitors 14b , 15b , 15c , 23b , 27 , and 1 , on mct1-expressing 
 tumor cells generally correlates with their ability to inhibit lactate 
 transport , supporting the proposed mode of action for compounds in 
 these distinct chemical series . our most 
 potent compound in the lactate transport assay is 27 , 
 which is roughly equal in ic50 to compound 1 , the compound in table 1 that is most potent 
 in both of the assays shown . when our new compounds tested in both 
 of these assays are ranked by potency in the lactate transport assay , 
 the order is 27 > when the same compounds are instead ranked 
 by potency in the mtt assay , the order is 15c  23b  27 > 14b  15b > 23a > 14a . thus , rank 
 orders 
 generally correlate , with the exception being the high potency of 
 compound 15c in the mtt assay . while rank ordering for 
 compound 27 differs , it is quite effective in both assays . the results of mtt assays in mcf7_mct1 and mcf7_mct4 cells using 
 a set of top - performing compounds are shown in figure 3 . compound 1 was more effective , although the potency ( ec50 value ) was reduced in mcf7_mct1 cells versus raji lymphoma , 
 with values that were more similar to ic50 values obtained 
 in the mcf7_mct1 lactate transport assay . as shown in panel b , mcf7_mct4 
 cells were unaffected by all test compounds , except perhaps compound 27 at the highest dose tested ( 10 m ) . thus , these compounds 
 selectively inhibit mct1 but not mct4 and off - target effects do not 
 contribute to the performance of compound 15c in the 
 raji mtt assay . our sar 
 studies have established that several newly designed pteridinones 
 impair raji lymphoma cell proliferation at submicromolar doses and 
 that their potency generally correlates with their ability to inhibit 
 lactate transport . notably , our sar analyses indicate that ( i ) side 
 chains at either c5 or c6 ( r in scaffolds 67 ) bearing an appropriately positioned hydroxyl 
 group are essential for potent mct1 inhibition in these scaffolds , 
 and ( ii ) compounds having the hydroxyl group present in alkyl and 
 thioether - containing tethers are also active . in contrast , related 
 sulfoxides , sulfones , amides , and triazoles are weakly active or inactive . the new compounds reported 
 here ( see table 1 ) with a -fused ring 
 system are novel . these small molecules , like other compounds described 
 as mct1 inhibitors , are likely dual mct1/mct2 inhibitors , although 
 we have not specifically measured activity at mct2 . further improvements 
 in the series are needed to augment their potency and to enhance their 
 physical properties . the sar trends in the - and -systems 
 diverge , and these altered substituent effects may provide new opportunities 
 in mct inhibitor design . importantly , the synthetic methods shown 
 are also versatile and will allow additional sar development : the 
 r , r , r , and r groups 
 in scaffolds 67 can be selectively 
 and systematically altered . efforts to revise the synthesis 
 strategy shown herein are underway 
 and will permit more extensive investigation of sar . analyses of additional 
 highly biologically active compounds in these scaffolds , including 
 new synthetic methodologies , dmpk studies , animal efficacy studies , and computational efforts to define the 
 mode of interaction of these inhibitors with mct1 , as well as the 
 design of mct4 inhibitors , are a focus of our current investigations . reactions were performed in flame - dried 
 glassware under positive pressure of n2 or ar . anhydrous 
 thf , et2o , dmf , ch3cn , and dcm were purchased 
 from aldrich and used as received . analytical hplc was performed on agilent technologies 
 1200 series instruments with ch3cn / water gradient mixtures 
 as eluent and 0.1% tfa as modifier . the targeted products were detected 
 by uv in the detection range of 215310 nm . nmr spectra were 
 recorded at on brker instruments at 400 mhz ( h ) 
 or 100 mhz ( c ) in cdcl3 , unless otherwise 
 stated . preparative and analytical hplc and lcms analysis and microwave - promoted 
 reactions were performed using standard techniques and commercial 
 instruments , listed in the supporting information , which provides complete experimental details . methods for synthesis 
 of specific key compounds include : a mixture of 9 ( 1.44 g , 5.51 
 mmol ) , 10 ( 1.54 g , 5.78 mmol ) , and na2co3 ( 1.46 g , 13.8 mmol ) in dmf ( 11.0 ml ) was heated to 65 c 
 for 2 h , cooled to room temperature , quenched with saturated nh4cl , and extracted with ethyl acetate ( ea ) . the solvent was removed and the residue was purified by 
 flash column chromatography ( hexanes : ea = 3:2 ) to afford 1.69 g ( 67% ) 
 of 11 as a yellow oil . lc - ms ( esi ) : m / z 455 [ m + 1 ] . h nmr  ( ppm ) 
 0.36 ( d , j = 6.6 hz , 3h ) , 0.44 ( d , j = 6.6 hz , 3h ) , 1.55 ( sep , j = 6.6 hz , 1h ) , 2.852.91 
 ( m , 1h ) , 3.24 ( s , 3h ) , 3.333.37 ( m , 2h ) , 3.86 ( s , 3h ) , 3.984.03 
 ( m , 1h ) , 4.584.66 ( m , 1h ) , 7.34 ( d , j = 6.8 
 hz , 1h ) , 7.427.46 ( m , 1h ) , 7.537.61 ( m , 2h ) , 7.83 
 ( d , j = 8.0 hz , 1h ) , 7.90 ( d , j = 
 8.0 hz , 1h ) , 8.02 ( d , j = 8.0 hz , 1h ) . c nmr  ( ppm ) 19.0 , 19.1 , 26.8 , 28.4 , 36.1 , 53.4 , 53.6 , 59.8 , 
 116.5 , 122.5 , 125.5 , 126.2 , 126.9 , 128.4 , 128.7 , 129.2 , 130.8 , 131.4 , 
 133.9 , 149.0 , 154.9 , 155.2 , 170.6 . a suspension 
 of 12 ( 2.3 g , 5.9 mmol ) in dcm ( 60 ml ) was cooled to 
 0 c under n2 . tf2o ( 3.0 g , 10.6 mmol ) 
 was added , followed by et3n ( 1.8 g , 17.6 mmol ) . the mixture 
 was stirred 1h at 0 c , quenched with saturated nh4cl , extracted with dcm , washed with brine , and dried over na2so4 . the solvent was removed , and the residue was 
 purified by flash column chromatography ( hexanes : ea = 4:1 to 2:1 ) 
 to afford 3.01 g ( 98% ) of 13 as a yellow solid . lc - ms ( esi ) : m / z 523 [ m + 1 ] . h nmr  ( ppm ) 0.50 ( d , j = 6.8 hz , 6h ) , 1.89 
 ( sep , j = 6.8 hz , 1h ) , 3.47 ( s , 3h ) , 3.65 ( d , j = 7.6 hz , 2h ) , 4.80 ( s , 2h ) , 7.477.52 ( m , 4h ) , 
 7.857.93 ( m , 3h ) . c nmr  ( ppm ) 19.4 , 26.6 , 29.0 , 36.3 , 49.9 , 117.0 , 
 120.2 , 122.3 , 123.8 , 125.5 , 125.9 , 126.5 , 128.5 , 129.0 , 131.0 , 132.0 , 
 134.0 , 146.1 , 147.1 , 150.2 , 153.5 , 158.2 . step 1 : a mixture of 13 ( 314 mg , 0.60 mmol ) , zn(cn)2 ( 70 mg , 0.60 mmol ) , and 
 zn ( 8.0 mg , 0.12 mmol ) in nmp ( 6.0 ml ) was degassed . pd(pph3)4 ( 69 mg , 0.06 mmol ) was added , and the mixture was stirred 
 at 100 c for 24 h , cooled to rt , quenched with nh4cl , and extracted with ea . the combined extracts were washed with 
 brine , dried over na2so4 , concentrated , and 
 purified by flash column chromatography ( hexanes : ea = 3:1 ) to give 
 151 mg ( 63% ) of 1-isobutyl-3-methyl-7-(naphthalene-1-ylmethyl)-2,4-dioxo-1,2,3,4-tetrahydropteridine-6-carbonitrile as a yellow solid . lc - ms ( esi ) : m / z 400 [ m + 1 ] . h nmr  ( ppm ) 0.55 ( d , j = 6.8 hz , 6h ) , 1.66 ( sep , j = 6.8 hz , 
 1h ) , 3.50 ( s , 3h ) , 3.72 ( d , j = 7.6 hz , 2h ) , 4.94 
 ( s , 2h ) , 7.477.56 ( m , 4h ) , 7.858.01 ( m , 3h ) . step 
 2 : a solution of 1-isobutyl-3-methyl-7-(naphthalene-1-ylmethyl)-2,4-dioxo-1,2,3,4-tetrahydropteridine-6-carbonitrile ( 145 mg , 0.363 mmol ) in 1,4-dioxane ( 18 ml ) was treated with h2so4 ( 11 ml , 60% ) . the resultant mixture was stirred 
 at 110 c for 60 h , cooled to room temperature , diluted with 
 h2o , and extracted with ea . the solution was concentrated to afford 137 mg ( 90% ) of 18 as a brown solid . lc - ms ( esi ) : m / z 419 [ m + 1 ] . h nmr ( dmso - d6 )  ( ppm ) 0.34 ( d , j = 6.8 hz , 
 6h ) , 1.45 ( sep , j = 6.8 hz , 1h ) , 3.26 ( s , 3h ) , 3.41 
 ( d , j = 7.6 hz , 2h ) , 3.41 ( t , j = 
 7.6 hz , 2h ) , 5.05 ( s , 2h ) , 7.417.52 ( m , 4h ) , 7.867,98 
 ( m , 3h ) . a mixture of 13 ( 261 mg , 0.50 mmol ) , 21b ( 50 mg , 0.60 mmol ) , and cui ( 9.5 mg , 0.05 mmol ) in dmf ( 7.5 ml ) 
 was degassed . pd(pph3)4 ( 29 mg , 0.025 mmol ) 
 was added , followed by et3n ( 506 mg , 5.0 mmol ) . the mixture 
 was again degassed and heated by microwave to 175 c for 20 min . the reaction mixture was diluted with ea , washed with brine , dried 
 over na2so4 , concentrated , and purified by flash 
 column chromatography ( hexanes : ea = 2:3 ) to give 105 mg ( 46% ) of 22b as a yellow solid . lc - ms ( esi ) : m / z 457 [ m + 1 ] . h nmr  ( ppm ) 0.39 ( d , j = 6.8 hz , 6h ) , 1.52 ( sep , j = 6.8 hz , 
 1h ) , 1.811.87 ( m , 2h ) , 2.62 ( t , j = 7.0 hz , 
 2h ) , 3.38 ( s , 3h ) , 3.57 ( d , j = 7.6 hz , 2h ) , 3.703.74 
 ( m , 2h ) , 4.77 ( s , 2h ) , 7.317.40 ( m , 4h ) , 7.717.84 
 ( m , 3h ) . c nmr  ( ppm ) 16.3 , 19.4 , 26.6 , 29.0 , 
 30.8 , 39.0 , 49.1 , 61.3 , 77.3 , 98.4 , 124.1 , 124.5 , 125.4 , 125.7 , 126.2 , 
 127.9 , 128.0 , 128.8 , 132.1 , 133.2 , 133.9 , 134.6 , 145.4 , 150.4 , 159.6 , 
 161.8 . a solution of 22b ( 20 mg , 0.044 mmol ) in thf ( 2.0 ml ) 
 was treated with 10% pd / c ( 5.0 mg ) . the mixture was stirred 5 h under 
 h2 , filtered , concentrated , purified by flash column chromatography 
 ( hexanes : ea = 2:3 ) , and then by preparative hplc to afford 10 mg of 23b as a white solid . lc - ms ( esi ) : m / z 461 [ m + 
 1 ] . h nmr  ( ppm ) 0.37 ( d , j = 6.8 hz , 6h ) , 1.431.57 ( m , 4h ) , 1.781.81 ( m , 3h ) , 
 3.01 ( t , j = 7.4 hz , 2h ) , 3.39 ( s , 3h ) , 3.52 ( d , j = 7.6 hz , 2h ) , 3.603.70 ( m , 2h ) , 4.63 ( s , 2h ) , 
 7.14 ( d , j = 6.8 hz , 1h ) , 7.337.43 ( m , 3h ) , 
 7.697.83 ( m , 3h ) . human raji burkitt 
 lymphoma cells were seeded in 96-well plates at 20000 cells / well and 
 were cultured  compounds generated herein , or with vehicle , 
 in 5% co2 incubator for 4 days . mtt reagents ( millipore 
 ct0-a ) were added at 10 l per well , and cells were incubated 
 for 5 h. then 100 l of 2-propanol/0.04 n hcl was added to each 
 well , pipetted to mix , and plates were read using biotek synergy ii 
 plate reader at 570 and 630 nm . experiments were performed in triplicate , 
 and assays of the most active compounds were repeated three times . methods 
 used were as previously described . briefly , 
 mcf7 breast cancer cells engineered to overexpress mct1 were cultured 
 at 35000 cell / well in 24-well plates for 2 days . assays were performed on ice by removing medium and washing 
 once with cold buffer ( 150 mm nacl , 10 mm hepes , 5 mm kcl , 1 mm mgcl2 , 1 mm cacl2 , 0.2% bsa at ph 7.4 ) containing compound 
 ( 1 nm to 1 m ) or dmso for 5 min . cells were incubated in 200 
 l of cold buffer containing 0.5 ci / well l-14c(u)-lactic 
 acid , sodium salt ( perkinelmer ) and compounds for 10 min on ice . cells 
 were washed three times in cold buffer containing 0.1 mm pholoretin 
 ( sigma ) and lysed with 200 l of 0.1 m naoh for 30 min at room 
 temperature . samples were done in triplicate , and the log of compound concentration 
 versus cpm incorporated was plotted using graphpad prism and used 
 to calculate the ic50 for each compound . mcf7_mct1 and all methods for culture and cell - handling followed the procedure 
 that was described above for the raji lymphoma - based mtt assays .
novel 
 substituted pteridine - derived inhibitors of monocarboxylate 
 transporter 1 ( mct1 ) , an emerging target for cancer therapy , are reported . 
 the activity of these compounds as inhibitors of lactate transport 
 was confirmed using a 14c - lactate transport assay , and 
 their potency against mct1-expressing human tumor cells was established 
 using mtt assays . 
 the four most potent compounds showed substantial 
 anticancer activity ( ec50 37150 nm ) vs mct1-expressing 
 human raji lymphoma cells .