the nitrogen mustard 
 mechlorethamine ( 1 ) , developed 
 in the 1940s , was the first cancer chemotherapeutic agent . analogues such as chlorambucil ( 2 ) , melphalan ( 3 ) , bendamustine ( 4 ) , estramustine ( 5 ) , uramustine ( 6 ) , cyclophosphamide ( 7 ) , 
 and ifosfamide ( 8) see widespread clinical use today . nitrogen mustards generate aziridinium ions that alkylate dna at 
 a variety of positions including n7-guanosine , n3-adenosine , n1-adenosine , 
 and n3-cytosine . the predominant site of dna alkylation by nitrogen mustards is the 
 n7-atom of guanine residues . nitrogen mustards cause serious side effects that 
 arise from the 
 alkylation of dna and other biomolecules in nonmalignant tissue . consequently , there have been many efforts to 
 develop  targeted  nitrogen mustards with improved potency 
 and selectivity against cancer cells . one promising approach for the design of cancer - cell - selective mustards 
 exploits selective enzymatic reduction of nitroaryl compounds in the 
 oxygen - starved ( hypoxic ) cells found in solid tumors . one - electron reductases such as nadph : cytochrome p450 reductase , 
 cytochrome b5 reductase , xanthine oxidase , 
 and aldehyde oxidase can convert nitroaryl compounds to nitroso , hydroxylamino , 
 and amino metabolites selectively under hypoxic conditions ( scheme 1 ) . in normal tissue , o2 blocks production of reduced metabolites 
 via oxidation of the radical anion intermediates involved in this 
 process ( reverse reactions , scheme 1 ) . the initial nitro - to - nitroso 
 conversion typically is a key oxygen - sensitive step in the bioreduction 
 of nitroaryl compounds , but there is also evidence that 
 the hydroxylamino - to - aniline step can be inhibited by o2 . it is well established that 
 aziridinum ion formation by n - aryl nitrogen mustards 
 is suppressed by electron - withdrawing 
 substituents and favored by electron - releasing substituents on the 
 aromatic ring . thus , hypoxia - selective conversion 
 of the electron - withdrawing nitro substituent ( hammett  = 0.78 ) 
 to the electron - donating hydroxylamino (  = 0.34 ) or 
 amino substituents (  = 0.66 ) constitutes an  electronic 
 switch  that can transform a deactivated n - aryl nitrogen mustard into an activated nitrogen mustard selectively 
 in tumor tissue ( scheme 2 ) . two anticancer drug candidates , th-302 and pr-104 , that employ this 
 design principle are currently undergoing phase i and ii clinical 
 trials . in the pursuit of clinically 
 useful hypoxia - selective dna cross - linking 
 agents , it may be important to explore the utility of oxygen - sensing 
 units other than the nitroaryl motif . in this regard , the 1,2,4-benzotriazine 
 1,4-dioxide scaffold deserves consideration . in terms of both basic 
 and clinical research , the compound 3-amino-1,2,4-benzotriazine 1,4-dioxide 
 ( tirapazamine , 9 ) may be the best - characterized hypoxia - selective 
 antitumor agent . on the basis of its potent hypoxia - selective cytotoxicity in preclinical 
 testing , this compound was examined in a wide variety of phase 
 i , ii , and iii clinical trials . in early clinical trials , the drug showed promise that was not realized in subsequent studies . the disappointing clinical performance of 9 may stem , in part , from failure to stratify patients on the basis 
 of the hypoxic character of their tumors . in addition , pharmacokinetic models suggested that 9 may be metabolized in a small zone of hypoxic tumor cells , leaving 
 a significant fraction of neighboring cancer cells untouched by the 
 drug . rational 
 use of the 1,2,4-benzotriazine 1,4-dioxides as components 
 in the design of hypoxia - selective dna - alkylating agents rests upon 
 an existing understanding of the mechanisms by which these agents 
 selectively kill hypoxic cells . intracellular one - electron reductases 
 convert 9 to an oxygen - sensitive radical intermediate 10 ( scheme 3 ) . we have presented evidence that , under hypoxic conditions , the protonated 
 drug radical 11 fragments to release the dna - damaging 
 agent , hydroxyl radical , though other mechanisms also have been considered . the deoxygenation product , 1,2,4-benzotriazine 1-oxide 12 , is the major metabolite generated by hypoxic metabolism of 9 , and other 1,2,4-benzotriazine 1,4-dioxides similarly 
 are converted to the 1-oxide products . compound 9 is not extensively metabolized to the mono - n - oxide 12 by oxygen - insensitive pathways 
 involving two - electron reductases such as dt - diaphorase . the mono - n - oxide metabolite of tirapazamine is 
 not cytotoxic on its own , although it does display 
 useful oxygen mimetic ( radiosensitizing ) properties that potentiate 
 the dna strand - cleaving properties of hydroxyl radical . we envisioned that electronic changes resulting from the 
 deoxygenation 
 of tirapazamine analogues might be exploited to activate dna - alkylating 
 functional groups selectively in hypoxic tissue . indeed , the charge 
 distribution in the mono - n - oxide metabolite 12 is quite different than that of the parent di - n - oxide 9 . we describe the 
 design , synthesis , and characterization of hypoxia - selective dna - alkylating 
 agents constructed by grafting nitrogen mustard units onto the 1,2,4-benzotriazine 
 1,4-dioxide scaffold ( scheme 4 ) . our synthetic approaches to the desired tirapazamine - mustard 
 derivatives 
 were informed by previous reports describing the synthesis of tirapazamine 
 and its analogues . the compound 6-fluoro-1,2,4-benzotriazine 
 1-oxide 13 oxidation of 13 with 
 trifluoroacetic acid/80% h2o2 gave the di - n - oxide 14 in 43% yield ( scheme 5 ) . attempts to convert 15 to the tosyl mustard 17b via treatment with tosyl chloride 
 gave a complex mixture of products , from which we isolated 19 as a major component . the structure of 19 was confirmed 
 by single - crystal x - ray crystallographic analysis ( supplementary figure s1 ) . this product may arise via the initial 
 attack of the 4-oxide unit of 15 on tosyl chloride , followed 
 by a nucleophilic deoxygenative rearrangement . as an alternate route to the target mustards , 13 was 
 treated with diethanolamine in n - methylpyrrolidone 
 to give the mono - n - oxide 16 ( 94% ) . this 
 product was converted in good yields to the ditosylate 17b by treatment with tosyl chloride or to the dimesylate 17a by treatment with mesyl chloride . oxidation to the di - n - oxides 18 was effected by oxone or m - cpba in modest yields ( 1537% ) . the chlorinated mustard 17c was synthesized in 94% yield via treatment of 17b with lithium chloride in dmf , and 18c was prepared 
 in 30% yield by oxidation with oxone . reagents and conditions : ( a ) 
 cf3cooh , 70% h2o2 , 50 c , 43% ; 
 ( b ) hn(ch2ch2oh)2 , ch3cn , 73% ; ( c ) hn(ch2ch2oh)2 , nmp , 
 100 c , 94% ; ( d ) mesyl chloride , tea , dmf , 0 c , 72% ; ( e ) 
 tosyl chloride , naoh , thf / h2o , 0 c , 74% ; ( f ) nacl , 
 dmf , 110 c , 94% ; ( g ) oxone , nahco3 , meoh / h2o , 50 c , 15 - 30% ; ( h ) m - cpba , thf , 37% . to investigate how the 3-amino substituent and the 
 location of 
 the mustard unit on the benzotriazine ring system affect reactivity 
 of an appended nitrogen mustard unit , we sought to prepare the 3-desamino 
 tirapazamine analogues 21 , 22 , 25 , and 26 . toward this end , 13 was deaminated 
 by treatment with tert - butylnitrite in dimethylformamide 
 to give 20 ( 50% yield , scheme 6 ) . nucleophilic aromatic substitution with 
 diethanolamine , followed by treatment with tosyl chloride , gave the 
 mono - n - oxide mustard 21 in 67% yield 
 for the two steps . oxidation of 21 with oxone gave the 
 di - n - oxide mustard 22 ( 10% ) . synthesis 
 of compounds with the mustard located on the 7-position of the benzotriazine 
 ring system started with preparation of 7-fluoro-1,2,4-benzotriazine 
 1-oxide 23 via condensation of 6-fluoro-2-nitroaniline 
 with cyanamide ( scheme 7 ) . treatment with tert - butylnitrite in dimethylformamide 
 gave the deaminated analogue 24 in 50% yield . subsequent 
 reaction of this compound with diethanolamine , followed by tosyl chloride , 
 gave a 57% yield of 25 . finally , oxidation with either 
 trifluoroacetic anhydride/70% h2o2 in ch2cl2 or oxone gave the target di - n - oxide 26 in low ( 1% ) yield . formation of the desired 
 compound 26 , in this case , was accompanied by extensive 
 degradation during the reaction and workup . reagents and conditions : ( a ) t - buno2 , dmf , 60 c , 50% ; ( b ) hn(ch2ch2oh)2 , ch3cn ; ( c ) tscl , 
 naoh , thf 0 c , 61% from 2 steps ; ( d ) oxone , nahco3 , meoh , h2o , 60 c , 10% . reagents and conditions : ( a ) t - buno2 , 
 dmf , 60 c , 50% ; ( b ) hn(ch2ch2oh)2 , ch3cn ; ( c ) tscl , 
 naoh , thf 0 c , 57% from 2 steps ; this work explores the idea that mustard units in the parent di - n - oxides will be relatively unreactive , while the mustard 
 units in the mono - n - oxide deoxygenation products 
 will be active alkylating agents . to learn whether the mustard units 
 in the mono - n - oxides 17 , 21 , and 25 are in fact more reactive than those in the 
 corresponding di - n - oxides ( 18 , 22 , 26 ) as illustrated in scheme 4 , we first examined the hydrolytic stability of these agents 
 in an organic / aqueous solvent mixture . hydrolysis of aromatic nitrogen 
 mustards typically proceeds via a rate - determining formation of the 
 aziridinium ion intermediate . we used h nmr to monitor the hydrolysis of these mustard derivatives ( 1 
 mm ) in 50:50 cd3cn / d2o at 50 c . this mixed 
 organic / aqueous solvent system ensured solubility of the compounds 
 at the concentrations required for the nmr experiments and also served 
 to slow aziridinium ion formation and hydrolysis to easily measurable 
 rates . the disappearance of the compounds 
 followed first - order kinetics ( figure 1 ) . the 
 mesylate mustard analogue of tirapazamine 18a disappeared 
 with a half - life of 15.4  0.6 d ( table 1 ) , while the corresponding mono - n - oxide mustard 17a disappeared approximately 5-fold faster , with a half - life 
 of 2.7  0.4 d. the tosylate mustards displayed a similar trend , 
 in which the di - n - oxide 18b was more 
 stable ( t1/2 = 15.4  0.6 d ) than 
 the mono - n - oxide 17b ( t1/2 = 5.5  0.6 d ) . the similar reactivities of the 
 tosyl ( 17b/18b ) and mesyl ( 17a/18a ) systems is consistent with previous analyses showing 
 that tosylate and mesylate are comparable leaving groups in aqueous / organic 
 solvent mixtures . the chlorinated mustard 
 analogues 17c and 18c were quite stable , 
 with 17c displaying an estimated half - life of more than 
 51 d under the conditions of our nmr experiment ( 85% of the starting 
 material remained after 10 d ) . compound 18c showed no 
 detectable reaction over the course of 10 d. due to the sluggishness 
 of these reactions we did not examine the hydrolysis of 17c and 18c further . rate of decay for compounds 17a , 17b , 18a , and 18b in 1:1 acetonitrile - d6/d2o measured by h nmr at 50 c . the products resulting from decomposition of the nitrogen 
 mustards 
 in these nmr experiments were identified as the expected diol ( 16 ) and the morpholino compound ( 27 ) , presumably 
 derived from intramolecular attack of the hydroxyethyl arm of the 
 half - mustard intermediates ( 28 ) on the adjacent mustard 
 group . the desamino analogues 21 and 22 , bearing 
 a tosyl mustard at the 6-position , disappeared with half - lives that 
 were the same within experimental error ( table 1 ) . the half - lives of these compounds ( t1/2  13 d ) were very similar to that of the deactivated dioxide 
 tosyl mustard 18b in the 3-amino series . the desamino 
 analogues 25 and 26 , bearing the tosyl mustard 
 unit on the 7-position , also disappeared at the same rate within experimental 
 error ( table 1 ) . the decomposition rates of 
 the 7-mustards 25 and 26 ( t1/2  6.5 d ) was approximately two times faster 
 than the decomposition of the desamino 6-mustards 21 and 22 ( t1/2  13 d ) . in the 
 conclusion , we consider possible reasons why there is no significant 
 difference between the reactivities of the desamino mono - n - oxide and di - n - oxide mustards . we also measured 
 decomposition of the mesyl mustards 17a and 18a ( 250 m ) at 50 c in a predominantly 
 aqueous solvent mixture composed of sodium phosphate buffer ( 25 mm , 
 ph 7 ) containing dmf ( 2.5% v / v ) . we used hplc analysis to monitor 
 the disappearance of the starting mustards ( 17a and 18a ) . as expected , the hydrolysis rates of 17a and 18a were substantially faster in this solvent as 
 compared to the 50:50 cd3cn / d2o mixture employed 
 in the nmr experiments . compounds 17a and 18a disappeared with half - lives of 12 
  0.5 h and 96  21 h , respectively , in phosphate - buffered 
 water . under these conditions , the  activated  mono - n - oxide metabolite 17a reacted approximately 
 seven times faster than the parent dioxide 18a . comparison 
 with authentic synthetic standards revealed that the major products 
 generated in the decomposition of 17a were the diol 16 and the morpholino 27 , both presumably formed 
 via the half - mustard 28 ( supplementary 
 figure s2 ) . we felt 
 it would be useful to quantitatively measure the electron - withdrawing 
 power of the triazine ring systems in 17 and 18 . electronic effects exerted by various substituents on an aromatic 
 ring typically are assessed using hammett sigma (  ) constants . hammett  values are determined by measuring 
 the affect that a substituent exerts on the acidity of benzoic acid , 
 where substituent = log(ka(substituted  benzoic  acid)/ka(benzoic  acid ) ) . therefore , we 
 prepared the carboxylic acid derivatives 29 and 30 via reaction of guanidine with 3-fluoro-4-nitrobenzoic 
 acid under basic conditions , as shown in scheme 8 . reagents and conditions : ( a ) 
 guanidine , thf , etoh 90 c ; ( b ) kotbu , thf , 90 c , 95% after 
 2 steps ; ( c ) tfa , 70% h2o2 , 50 c , 36% . we measured the acidity of 29 by 
 monitoring the changes 
 in its uv  vis spectrum as a function of ph ( supplementary figure s3 ) . from the measured pka of 2.9 , we were able to calculate a  value of 
 1.3 for the mono - n - oxide 29 . the establishes 
 the 1,2,4-triazine-1-oxide ring as a strongly electron - withdrawing 
 substituent comparable to a p - sulfonyl cyanide group 
 ( -so2cn ) . unfortunately , we 
 were not able to measure the pka for the 
 di - n - oxide 30 because the uv vis 
 spectral changes associated with protonation of the carboxylic acid 
 group were obscured by another process , perhaps involving protonation 
 of the oxygen in the 4-oxide group . uv  vis spectra of compounds 31 ( top ) and 32 ( bottom ) from ph 3.4 to 9.0 ( 31 ) and ph 2.4 
 to 9.0 ( 32 ) . electronic properties of substituents can also be measured 
 using 
 a  parameter that is obtained by determining 
 the effect of substituents on the acidity of phenol . while the  parameter is probably less 
 relevant than  to the aziridinium ion - forming reactions that 
 are the subject of this work , we felt 
 this measurement would nonetheless provide a useful evaluation of 
 the electronic properties of the triazine mono - oxide and dioxide ring 
 systems . therefore , we synthesized the phenol derivatives 31 and 32 by reaction of the corresponding fluoro compounds 
 with basic hydrogen peroxide , followed by workup with sodium thiosulfate 
 ( scheme 9 ) . we 
 then measured the acidity of these compounds by monitoring the changes 
 in their uv  vis spectra as a function of ph ( figure 2 ) . the pka of 31 was found to be 6.3 , and that of 32 was 5.3 
 ( supplementary figure s4 ) . the pka measurements allowed us to calculate  values of 1.81 for the 1,2,4-triazine 1-oxide  substituent  
 in 31 and 2.31 for the 1,2,4-triazine 1,4-dioxide  substituent  
 in 32 . these values indicate that both the triazine mono - n - oxide and the di - n - oxide rings are strongly 
 electron - withdrawing . by way of comparison , the sulfonyl perfluoropropane 
 group ( -so2(cf2)2cf3 ) 
 has a  value of 1.75 , and the diazonium 
 group ( -n2 ) has a  value of 3.43 . importantly , there is 
 a substantial difference ( 0.5 ) between the  values of the 1,2,4-triazine 1-oxide and 1,2,4-triazine 1,4-dioxide 
 substituents in 31 and 32 . this is similar 
 to the difference between  values of the 
 nitro group ( 1.27 ) and the acetyl group ( 0.84 ) . overall , the results validate the idea that transformation 
 of the 1,4-di - n - oxide ( 9 ) to the 1-n - oxide ( 12 ) is accompanied by a substantial 
 change in electron density at the 6-position of the benzo ring . reagents and conditions : ( a ) 
 naoh , 30% h2o2 , h2o / nmp 60 c , 
 50% ; ( b ) naoh , 30% h2o2 , h2o / nmp 
 50 c , 51% . nitrogen mustards alkylate dna primarily at the n7-position of 
 guanine residues , with smaller amounts of reaction also occurring 
 at the n3-position of adenine residues . the resulting lesions 
 can be converted to strand breaks by treatment of the dna with warm 
 piperidine ( maxam  gilbert workup ) . we incubated mustards 17a and 18a with 
 the 5-p - labeled dna duplex 33 in 
 hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) and dmf ( 10% v / v ) , 
 followed by piperidine workup ( figure 3 ) . the 
 resulting labeled dna fragments were resolved on a 20% denaturing 
 polyacrylamide gel and visualized by phosphorimager analysis . we found 
 that treatment of dna with the dioxide 18a , followed 
 by piperidine workup , generated little strand cleavage at the guanine 
 residues in the labeled strand of duplex 33 . the mono - n - oxide 17a generated significantly higher 
 yields of alkali - labile lesions at both guanine residues in duplex 33 . specifically , compound 17a generated a 30-fold 
 higher yield of alkylation at g2 than 18a ( lanes 3 and 
 4 , figure 3b ) . the control diol 16 did not induce strand cleavage that was significantly above background 
 ( lane 2 , figure 3b ) . we compared dna alkylation 
 by 17a to that by the clinically used nitrogen mustard 
 chlorambucil ( 2 , lanes 4 and 5 , figure 3b ) . chlorambucil generated piperidine - labile lesions predominantly 
 at the guanine residues in duplex 31 , alongside weaker 
 cleavage that may arise from alkylation at adenine residues in the 
 dna . conditions : 
 hepes buffer ( 50 
 mm , ph 7 ) , nacl ( 100 mm ) , mustard ( 1 mm unless otherwise specified ) , 
 10% dmf , 37 c for 3 d followed by piperidine workup ( 1 m , 95 
 c 30 min ) . following incubation ( a ) p - labeled duplexes 33 and 34 used to examine dna - alkylating properties 
 of mustards . ( b ) lane 1 : duplex 33 ( no mustard ) , lane 
 2 : duplex 33 with 16 , lane 3 : duplex 33 with 18a , lane 4 : duplex 33 with 17a , lane 5 : duplex 33 with 2 , lane 
 6 : g - sequencing of duplex 33 . ( c ) lane 1 : duplex 34 ( no mustard ) , lanes 24 : duplex 34 with 17a at 0.25 , 0.5 , and 1 mm concentrations , lane 5 : duplex 34 with 1 ( 0.25 mm ) , lane 6 : duplex 34 with 2 ( 0.25 mm ) , and lane 7 : g - sequencing of duplex 34 . compound 17a showed a marked selectivity for alkylation 
 at g2 of duplex 33 that is distinct from the sequence 
 specificity of 2 ( figure 3b ) . this observation inspired us to examine the alkylation of duplex 34 containing a larger number of guanine residues embedded 
 in different sequence contexts . again , compound 17a showed 
 a strong sequence preference for alkylation of a guanine residue residing 
 in a 5-gt sequence ( g1 in duplex 34 , figure 3c ) . mechlorethamine ( 1 ) displayed relatively 
 low sequence specificity compared to 17a , with 5-ggg as a favored sequence and 5-gc as a disfavored 
 sequence , consistent with literature reports ( lane 5 , figure 3c ) . possible origins 
 of the sequence specificity displayed by 17a are discussed 
 in the conclusions below . these experiments provided evidence that 
 the anticipated hypoxic metabolite 17a is a significantly 
 better dna - alkylating agent than the parent di - n - oxide 18a . successful deployment 
 of the activated 
 mustard 17a requires hypoxia - selective enzymatic reduction 
 of the dioxide 18a . thus , it was important to examine 
 whether the dialkylamine substituent at the 6-position of the tirapazamine 
 analogue perturbs bioreductive , deoxygenative metabolism of the 1,2,4-benzotriazine 
 1,4-dioxide  core  . to simplify product analysis in 
 these experiments , we examined the in vitro hypoxic metabolism of 
 an analogue , 15 , bearing the chemically stable bis(2-hydroxyethyl)amino 
 substituent . we employed recombinant human nadph : cytochrome p450 reductase 
 as the bioreductive enzyme because this , or a closely related enzyme , 
 is thought to be responsible for bioactivation of 9 and 
 related compounds in mammalian cells . hplc 
 chromatogram showing the in vitro metabolic conversion of 15 to 16 by nadph : cytochrome p450 reductase under 
 anaerobic conditions . compound 15 was incubated with nadph : cytochrome 
 p450 
 reductase ( 1 u / ml ) and nadph ( 500 m ) in sodium phosphate buffer 
 ( 25 mm , ph 7 ) under hypoxic conditions . hplc analysis of the resulting 
 metabolites revealed conversion of the di - n - oxide 15 to the corresponding mono - n - oxide 16 in approximately 16% yield ( figure 4 ) . a parallel experiment carried out under aerobic conditions produced 
 no detectable yields of 16 ( supplementary 
 figure s6 ) . a separate control experiment showed that tirapazamine 
 is a better substrate for nadph : cytochrome p450 reductase than is 15 under our reaction conditions ( 47% conversion of 9 to the mono - n - oxide versus 16% for 15 ) . likely this is because the electron - donating bis(2-hydroxyethyl)amino 
 group in 15 decreases the electron - affinity of the 1,2,4-benzotriazine 
 1,4-dioxide core . nonetheless , the results provide evidence that 15 can undergo hypoxia - selective metabolism in a manner similar 
 to that of the parent compound tirapazamine ( figure 
 s7 ) . in the work described 
 here , we explored the idea that the hypoxia - selective 
 enzymatic reduction of 1,2,4-benzotriazine 1,4-dioxides to the corresponding 
 1,2,4-benzotriazine 1-oxide metabolites can be exploited for the selective 
 generation of dna - alkylating species in the oxygen - poor cells found 
 in tumor tissue . in the case of the tirapazamine analogue 18a bearing a nitrogen mustard unit at the 6-position , it was found 
 that removal of the 4-oxide from the parent di - n - oxide 
 to generate the mono - n - oxide analogue 17a does indeed cause a substantial increase in reactivity of the mustard 
 unit , as measured by the rates of mustard hydrolysis . hammett  
 and  values measured for the 3-amino-1,2,4-triazine 
 1-oxide and 3-amino-1,2,4-triazine 1,4-dioxide  substituents in 17a and 18a , respectively , confirmed 
 that removal of the 4-oxide causes a significant decrease in the electron - withdrawing 
 effects exerted by the triazine ring system on substituents at the 
 6-position of the benzo ring . in contrast to the tirapazamine 
 analogues 17 and 18 , the reactivities of 
 the mustard units in the desamino 
 series 21 , 22 , 25 , and 26 were unaffected by the presence ( or absence ) of the 4-oxide 
 group . it is interesting to consider why the reactivities of the mustard 
 groups in the 3-desaminotirapazamine analogues ( 21 , 22 , 25 , and 26 ) are not  switched  by deoxygenation . first , it may be noteworthy 
 that the n = n(o ) group in 17 and 18 is cross - conjugated with the mustard nitrogen at the 6-position 
 and the 3-amino group . thus , in these tirapazamine analogues , the 
 3-amino group may serve to mitigate the electron - withdrawing properties 
 of the n = n(o ) group . on the other hand , in analogues 21 and 22 lacking the 3-amino group , the n = n(o ) group may exert a strong electron - withdrawing ( deactivating ) effect 
 on the mustard unit in the 6-position that altogether prevents participation 
 of the nitrogen lone pair in aziridinium ion formation . the hydrolysis 
 rates of 25 and 26 are higher than those 
 of 21 and 22 but still are unaffected by 
 the presence or absence of the 4-oxide unit . evidently , the n = n(o ) 
 unit in the meta position relative to the mustard unit is less electron - withdrawing 
 than when it resides in the para position . in this regard , the observation that the 
 reactivity of the mustard unit in the 7-position is not affected by 
 the presence or absence of the 4-oxide unit may be rationalized by 
 the fact that the nitrogen lone pair of the mustard substituent in 
 this location is not  through conjugated  with the 4-oxide 
 unit . the dna - alkylating properties of the nitrogen mustard 
 unit in the 
 mono - n - oxide 17a are  switched 
 on  compared to those of the parent di - n - oxide 18a . under our assay conditions , the activated analogue 17a generates approximately 30-fold greater yields of guanine 
 alkylation than does 18a . the mono - n - oxide 17a displays a striking and unexpected preference 
 for reaction at guanine residues located in 5-gt sequences . in contrast , typical mustards such as mechlorethamine ( 1 ) alkylate guanine residues with modest sequence selectivity ( lane 
 5 of figure 3c ) . we speculate that the sequence specificity of 17a arises 
 via formation of hoogsteen - type hydrogen bonds between the 3-amino-1,2,4-benzotriazine 
 ring system and the adenine residue in the target 5-gt/5-ac 
 sequence . such an interaction could deliver the mustard unit of 17a to the n7-atom of the guanine residue in the major groove 
 of the duplex ( figure 5 ) . a similar scenario 
 has been proposed to explain the unusual 5-gc sequence specificity 
 for uramustine 6 . finally , 
 we provided evidence that the dialkylamino substituent on the 6-position 
 of the 3-amino-1,2,4-benzotriazine 1,4-dioxide ring in analogues 18 is compatible with the hypoxia - selective enzymatic deoxygenative 
 metabolism required to deploy the activated mustards such as 17a . molecular model depicting hoogsteen base pairing by 17a with the adenine residue in a 5-gt/5-ac 
 sequence . the compounds described here result 
 from the combination of two 
 well - studied anticancer drugs . the drug tirapazamine has oxygen sensing 
 properties and generates oxidative dna damage selectively under hypoxic 
 conditions , while the nitrogen mustards are clinically used dna - alkylating agents . our work provides evidence that a judicious union 
 of these two anticancer drug motifs can yield new agents with the 
 potential to deliver both dna - alkylating and dna - oxidizing power selectively 
 to oxygen - poor tumor tissue . a potential disadvantage of tirapazamine 
 is that the agent kills only the small subset of tumor cells in which 
 bioreductive metabolism occurs . addition of a dna - alkylating unit 
 to the tirapazamine scaffold may yield agents with an ability to diffuse 
 into  and kill  neighboring cells that exist under conditions 
 of both more modest and more severe hypoxia . more generally , our results 
 provide evidence that the 1,2,4-benzotriazine 1,4-dioxide unit can 
 serve as an oxygen - sensing prodrug platform for the selective release 
 or activation of various bioactive agents in hypoxic tissue . all chemicals were used as purchased . 
 the compound 5-fluoro-2-nitroaniline was purchased from ak scientific . human cytochrome p450 reductase was purchased from sigma - aldrich . 
 for the kinetic measurements in the nmr , nmr spectra were recorded at 500 mhz for h nmr and 125 mhz for c nmr . oligonucleotides 
 were obtained from integrated dna technologies . t4 polynucleotide 
 kinase was purchased from new england biolabs . compounds 9 , 12 , 13 , 14 , and 23 were prepared according to literature methods . compound 14 ( 240 
 mg , 1.3 mmol ) was suspended in a mixture of acetonitrile ( 35 ml ) , 
 water ( 1 ml ) , and diethanolamine ( 1.4 g , 10 equiv ) . the 
 solvent was removed by rotary evaporation , the orange residue resuspended 
 in cold ethanol , and the solid collected by vacuum filtration . the 
 solid was washed with cold ethanol followed by diethyl ether and dried 
 in an oven overnight at 70 c to give 15 as an orange 
 powder ( 270 mg , 73% ) : mp 210212 c dec ; h 
 nmr ( 500 mhz , dmso - d6 )  7.94 ( d , j = 9.8 hz , 1h ) , 7.71 ( bs , 2h ) , 7.20 ( dd , j = 9.9 , 2.7 hz , 1h ) , 6.96 ( d , j = 2.7 , 1h ) , 4.93 
 ( t , j = 5.1 hz , 2h ) , 3.64 ( bs , 8h ) ; c nmr ( 125 mhz , dmso - d6 )  153.7 , 
 151.8 , 140.1 , 123.0 , 122.8 , 116.5 , 91.2 , 58.2 , 53.7 ; hrms ( esi ) m / z calculated for c11h16n5o4 ( m + h ) 282.1197 , 
 found 282.1201 . compound 13 ( 1.97 
 g , 11 mmol ) and diethanolamine ( 5.75 g , 5 equiv ) were suspended in 
 1-methyl-2-pyrrolidinone ( 18 ml ) and heated at 100 c for 18 
 h. the resulting orange suspension was cooled to room temperature , 
 triturated with cold ethanol ( 40 ml ) , and collected by vacuum filtration . the solid was washed with dichloromethane and dried in an oven overnight 
 at 70 c to give 16 as an orange powder ( 2.73 g , 
 94% ) . ( for further purification the solid was suspended in ethanol , 
 briefly heated with stirring , cooled , and collected by vacuum filtration ) : 
 mp 205210 c dec ; h nmr ( 500 mhz , dmso - d6 )  7.88 ( d , j = 9.7 
 hz , 1h ) , 6.99 ( dd , j = 9.7 , 2.4 hz , 2h ) , 6.83 ( bs , 
 2h ) , 6.41 ( d , j = 2.3 , 1h ) , 4.85 ( t , j = 5.2 hz , 2h ) , 3.603.56 ( m , 8h ) ; c nmr ( 125 
 mhz , dmso - d6 )  160.6 , 153.3 , 150.8 , 
 122.0 , 120.9 , 114.4 , 99.7 , 58.0 , 53.2 ; hrms ( esi ) m / z calculated for c11h16n5o3 ( m + h ) 266.1248 , found 266.1252 . compound 16 ( 500 
 mg , 1.9 mmol ) was dissolved in dimethylformamide ( 4.5 ml ) , followed 
 by the addition of triethylamine ( 800 l , 3 equiv ) and cooling 
 in an ice bath . methanesulfonyl chloride ( 370 l , 2.5 equiv ) 
 was added dropwise over 15 min as an ice - cold solution in dimethylformamide 
 ( 0.5 ml ) , and the resulting mixture was stirred at room temperature 
 for 2.5 h. the solution was poured into 30 ml of cold water with stirring 
 and refrigerated overnight at 4 c . the solid was washed with water 
 and diethyl ether and dried in a desiccator to give 17a ( 570 mg , 72% ) : h nmr ( 500 mhz , dmso - d6 )  7.94 ( d , j = 9.6 hz , 1h ) , 
 7.06 ( dd , j = 9.7 , 1.8 hz , 2h ) , 6.98 ( bs , 2h ) , 6.56 
 ( s , 1h ) , 4.38 ( t , j = 5.3 hz , 4h ) , 3.90 ( t , j = 5.2 hz , 4h ) , 3.17 ( s , 6h ) ; c nmr ( 125 mhz , 
 dmso - d6 )  161.0 , 152.8 , 151.1 , 
 123.1 , 121.6 , 114.4 , 101.6 , 67.4 , 49.7 , 37.1 ; hrms ( esi ) m / z calculated for c13h20n5o7s2 ( m + h ) 422.0799 , found 
 422.0800 . compound 16 ( 126 
 mg , 0.5 mmol ) was suspended in a biphasic mixture of tetrahydrofuran 
 ( 2 ml ) and naoh ( 1.5 ml of a 4 m solution in water ) and cooled in 
 an ice bath . to this orange biphasic mixture was added dropwise an 
 ice - cold solution of p - toluenesulfonyl chloride ( 200 
 mg , 2.2 equiv ) in tetrahydrofuran ( 3 ml ) . the resulting suspension 
 was stirred vigorously with cooling in an ice bath for 5 h. during 
 this time the mixture turned yellow . water slurry ( 10 ml ) and stirred for 20 min to give 
 a bright yellow powder . this solid was collected by vacuum filtration , 
 washed with water and then diethyl ether , and then dried in a desiccator 
 in to give 17b ( 202 mg , 74% ) : h nmr ( 500 
 mhz , dmso - d6 )  7.73 ( d , j = 9.7 hz , 1h ) , 7.60 ( d , j = 8.2 hz , 4h ) , 
 7.24 ( d , j = 8.1 hz , 4h ) , 6.97 ( bs , 2h ) , 6.63 ( dd , j = 9.7 , 2.5 hz , 1h ) , 6.15 ( d , j = 7.5 
 hz , 1h ) , 4.13 ( t , j = 5.1 hz , 4h ) , 3.64 ( t , j = 5.1 hz , 4h ) , 2.25 ( s , 6h ) ; c nmr ( 125 mhz , 
 dmso )  160.6 , 151.4 , 150,4 , 144.9 , 131.6 , 129.9 , 127.4 , 122.5 , 
 120.6 , 113.7 , 101.1 , 67.29 , 48.9 , 20.9 . hrms ( esi ) m / z calculated for c25h28n5o7s2 ( m + h ) 574.1425 , found 
 574.1433 . compound 17b ( 200 
 mg , 0.35 mmol ) and licl ( 148 mg , 10 equiv ) were dissolved in dimethylformamide 
 ( 1.5 ml ) and heated at 110 c for 2 h under a n2 atmosphere 
 before being cooled on ice and mixed with ice - cold water ( 20 ml ) . the resulting yellow precipitate was collected by vacuum filtration , 
 washed with water and diethyl ether , and then dried in a desiccator 
 to give 17c ( 104 mg , 94% yield ) : h nmr ( 500 
 mhz , dmso - d6 )  7.95 ( d , j = 9.6 hz , 1h ) , 7.04 ( dd , 9.7 , 2.6 hz , 1h ) , 6.98 ( bs , 2h ) , 
 6.48 ( d , j = 2.5 hz , 1h ) , 3.89 ( t , j = 6.6 hz , 4h ) , 3.80 ( t , j = 6.6 hz , 4h ) ; c nmr ( 125 mhz , dmso - d6 )  160.7 
 ( d , j = 253.90 hz ) , 152.1 , 150.8 , 122.8 , 121.4 , 113.9 , 
 101.1 , 51.8 , 40.9 ; hrms ( esi ) m / z calculated for c11h14cl2n5o ( m + h ) 302.0570 , found 302.0576 . compound 17a ( 70 
 mg , 0.17 mmol ) and nahco3 ( 86 mg , 6 equiv ) were suspended 
 in methanol ( 25 ml ) . oxone ( 245 mg , 1.2 equiv ) was added , followed 
 by water ( 10 ml ) . the mixture was stirred at 50 c under a n2 atmosphere for 24 h. thin layer chromatographic analysis 
 indicated that most of the reaction progress occurred within the first 
 5 h. the reaction was cooled to room temperature , and the white solid 
 was removed by vacuum filtration and washed with ch2cl2 ( 10 ml ) . the filtrate was extracted with ch2cl2 ( 2  125 ml ) , dried with anhydrous sodium sulfate , and 
 evaporated under reduced pressure , and the resulting residue was subjected 
 to column chromatography on silica gel eluted with a gradient of 15% 
 methanol in ch2cl2 to give 18a as 
 an orange solid ( 11 mg , 15% ) : h nmr ( 500 mhz , dmso - d6 )  8.01 ( d , j = 9.8 
 hz , 1h ) , 7.82 ( bs , 2h ) , 7.28 ( dd , j = 9.9 , 2.7 hz , 
 2h ) , 7.09 ( d , j = 2.7 , 1h ) , 4.43 ( t , j = 5.3 hz , 4h ) , 3.97 ( t , j = 5.3 hz , 4h ) , 3.19 ( s , 
 6h ) ; c nmr ( 125 mhz , dmso - d6 )  153.0 , 151.9 , 140.1 , 123.7 , 123.2 , 116.2 , 92.8 , 67.2 , 49.7 , 
 37.1 ; hrms ( esi ) m / z calculated 
 for c13h20n5o8s2 ( m + h ) 438.0748 , found 438.0746 . compound 17b ( 501 
 mg , 0.9 mmol ) was dissolved in tetrahydrofuran ( 100 ml ) and cooled 
 with stirring in an ice bath . to this mixture was added m - cpba ( 300 mg , 1.5 equiv of a 77% maximum purity material ) , 
 and the reaction was stirred overnight in an ice bath . an additional 
 portion of m - cpba was added , and the reaction was 
 stirred at room temperature for an additional 3 d. the solution was 
 concentrated under vacuum and poured into cold water , and the solid 
 was collected by vacuum filtration . the material was purified by column 
 chromatography on silica gel eluted with a gradient of 13% 
 methanol in ch2cl2 to give 18b as 
 a yellow solid ( 194 mg , 37% ) : h nmr ( 500 mhz , dmso - d6 ) 7.84 ( bs , 2h ) , 7.80 ( d , j = 9.8 hz , 1h ) , 7.60 ( d , j = 8.2 hz , 4h ) , 7.22 ( d , j = 8.1 hz , 4h ) , 6.86 ( dd , j = 9.8 , 2.7 
 hz , 1h ) , 6.65 ( d , j = 2.7 hz , 1h ) , 4.19 ( t , j = 5.0 hz , 4h ) , 3.67 ( t , j = 4.9 hz , 4h ) , 
 2.24 ( s , 6h ) ; c nmr ( 125 mhz , dmso - d6 )  151.6 , 151.5 , 144.9 , 139.3 , 131.6 , 129.9 , 127.4 , 
 123.0 , 122.1 , 115.6 , 92.2 , 67.1 , 48.9 , 20.8 . hrms ( esi ) m / z calculated for c25h28n5o8s2 ( m + h ) 590.1374 , found 
 590.1371 . compound 17c ( 50 
 mg , 0.17 mmol ) and nahco3 ( 42 mg , 3 equiv ) were suspended 
 in methanol ( 25 ml ) . oxone ( 122 mg , 1.2 equiv ) was added , followed 
 by water ( 10 ml ) . the reaction mixture was filtered while still 
 warm , and the solids were washed with methanol ( 5 ml ) . the filtrate 
 was cooled to room temperature , diluted with water ( 50 ml ) , and extracted 
 methylene chloride ( 3  100 ml ) . column chromatography on silica 
 gel eluted with a gradient of 26% methanol in ch2cl2 gave 18c as an orange solid ( 16 mg , 30% ) : h nmr ( 500 mhz , dmso - d6 )  
 8.01 ( d , j = 9.8 , hz , 1h ) , 7.81 ( bs 2h ) , 7.27 ( dd , j = 9.8 , 2.7 hz , 1h ) , 7.05 ( d , j = 2.7 
 hz , 1h ) , 3.96 ( t , j = 6.7 hz , 4h ) , 3.85 ( t j = 6.7 hz , 4h ) ; c nmr ( 125 mhz , dmso - d6 )  152.3 , 151.5 , 139.7 , 123.4 , 123.0 , 
 115.8 , 92.2 ; hrms ( esi ) m / z calculated 
 for c11h14cl2n5o2 ( m + h ) 318.0519 , found 318.0519 . compound 15 ( 52 mg , 
 0.18 mmol ) was suspended in a stirred solution of pyridine in an ice 
 bath , and tosyl chloride ( 172 mg , 5 equiv ) was added . the mixture 
 was stirred in an ice bath for 12 h , poured into ice - cold water ( 20 
 ml ) , and extracted with methylene chloride ( 5  25 ml ) . the combined 
 organic fractions were washed with brine and dried over na2so4 , and solvent was removed by rotary evaporation . column 
 chromatography on silica gel eluted with 1% methanol in methylene 
 chloride 19 as an orange solid ( 6 mg , 10% ) : h nmr ( 500 mhz , dmso - d6 )  7.85 
 ( d , j = 9.8 hz , 1h ) , 7.75 ( d , j = 
 8.2 hz , 2h ) , 7.739 ( d , j = 8.2 hz , 2h ) , 7.19 ( d , j = 9.8 hz , 1h ) , 6.94 ( bs 2h ) , 4.65 ( t , j = 5.1 hz , 2h ) , 3.473.40 ( m , 8h ) ; c nmr ( 125 
 mhz , dmso - d6 )  160.1 , 148.2 , 145.9 , 
 145.5 , 132.6 , 129.5 , 128.8 , 126.6 , 123.6 , 118.8 , 118.7 , 58.4 , 54.3 , 
 21.3 ; hrms ( esi ) m / z calculated 
 for c18h22n5o6s ( m + h ) 436.1285 , found 436.1293 . crystals suitable for x - ray crystallography 
 were prepared by vapor diffusion with ethyl acetate and hexane . the 
 crystal structure and crystallographic data for 19 are 
 shown in supplementary figure s1 and table s1 , respectively . following the general procedure of boyd et al.23 ( 930 mg , 5 mmol ) 
 was dissolved in anhydrous dimethylformamide ( 50 ml ) , and the mixture 
 was degassed by bubbling argon through the solution for 30 min . to 
 this mixture was added tert - butyl nitrite ( 3.5 ml , 
 5 equiv , 90% ) by syringe , and the mixture was heated in a 60 c 
 oil bath for 2 h under an atmosphere of argon gas . the resulting dark 
 residue was taken up in ethyl acetate ( 300 ml ) , mixed with brine ( 150 
 ml ) , and stirred vigorously . the phases were allowed to separate , 
 the organic layer was washed with brine ( 2  150 ml ) and dried 
 over anhydrous sodium sulfate , and the solvent was removed by rotary 
 evaporation . column chromatography on silica gel eluted with a gradient 
 of 010% ethyl acetate in ch2cl2 gave 24 as a pale white solid ( 438 mg , 50% ) : mp 127129 
 c ; h nmr ( 500 mhz , dmso - d6 )  9.20 ( s , 1h ) , 8.24 ( dd , j = 9.3 , 5.3 hz , 
 1h ) , 8.20 ( dd , j = 8.4 , 2.8 hz , 1h ) , 8.08 ( m , 1h ) ; c nmr ( 125 mhz , dmso - d6 )  
 162.6 ( d , j = 253.9 ) , 154.0 ( d , j = 2.6 hz ) , 144.9 , 136.0 , 132.5 ( d , j = 9.4 hz ) , 
 126.7 ( d , j = 25.9 hz ) , 105.2 ( d , j = 29.3 hz ) ; hrms ( esi ) m / z calculated 
 for c7h5fn3o ( m + h ) 
 166.0411 , found 166.0419 . compound 20 was prepared from 13 ( 980 mg ) by the same method ( 447 mg , 50% ) : mp 160162 
 c ; h nmr ( 500 mhz , dmso - d6 )  9.19 ( s , 1h ) , 8.50 ( dd , j = 9.6 , 5.5 hz , 
 1h ) , 7.99 ( dd , j = 9.1 , 2.7 hz , 1h ) , 7.82 ( m , 1h ) ; c nmr ( 125 mhz , dmso - d6 )  
 166.1 ( d , j = 256.7 ) , 155.5 , 149.3 ( d , j = 15.1 hz ) , 133.1 , 123.8 ( d , j = 11.2 hz ) , 121.8 
 ( d , j = 26.6 hz ) , 113.3 ( d , j = 
 22.7 hz ) ; hrms ( esi ) m / z calculated 
 for c7h5fn3o ( m + h ) 
 166.0411 , found 166.0417 . compound 20 ( 361 mg , 2.2 mmol ) and diethanolamine ( 500 mg , 2.5 equiv ) 
 were dissolved in acetonitrile ( 7 ml ) , and the mixture was heated 
 in an 80 c oil bath overnight . the reaction mixture was cooled , 
 and solvent was removed under reduced pressure by rotary evaporation . 
 the resulting solid was suspended in ethanol ( 10 ml ) , briefly heated 
 to 80 c with stirring , and then cooled to 0 c , and the 
 resulting precipitate was collected by vacuum filtration . the orange 
 solid was washed with cold ethanol and diethyl ether and dried in 
 an oven overnight at 70 c . the resulting solid ( 200 mg ) was 
 dissolved in a biphasic mixture of tetrahydrofuran ( 9 ml ) and naoh 
 ( 2.4 ml of a 4 m solution in water ) and cooled in an ice bath . to 
 this orange biphasic mixture was added dropwise an ice - cold solution 
 of p - toluenesulfonyl chloride ( 455 mg , 3 equiv ) in 
 tetrahydrofuran ( 1.5 ml ) . the resulting mixture was stirred vigorously 
 in an ice bath for 1.5 h. during this time the mixture turned yellow . 
 the mixture was then poured into an ice  water slurry ( 100 ml ) 
 and stirred for 30 min to give a yellow precipitate . the solid was 
 collected by vacuum filtration , then washed with water and diethyl 
 ether , and dried in a desiccator to give 21 ( 356 mg , 
 61% overall yield over two steps ) : h nmr ( 500 mhz , dmso - d6 )  8.87 ( s , 1h ) , 7.96 ( d , j = 9.2 hz , 1h ) , 7.57 ( d , j = 8.3 hz , 4h ) , 7.19 ( d , j = 8.0 , 4h ) , 7.14 ( dd , j = 9.8 , 2.6 hz , 
 1h ) , 6.64 ( d , j = 2.7 hz , 1h ) , 4.18 ( t , j = 5.1 hz , 4h ) , 3.71 ( t , j = 5.1 hz , 4h ) , 2.20 ( s , 
 6h ) ; c nmr ( 500 mhz , dmso - d6 )  154.8 , 152.1 , 149.2 , 145.2 , 131.9 , 130.2 , 127.8 , 127.4 , 
 120.7 , 120.3 , 103.7 , 67.6 , 49.3 , 21.2 . hrms ( esi ) m / z calculated for c25h27n4o7s2 ( m + h ) 559.1316 , found 
 559.1320 . compound 25 was prepared in from 24 by heating for 4 days using the same conditions ( 390 mg , 57% overall 
 yield over two steps ) : h nmr ( 500 mhz , dmso - d6 )  8.89 ( s , 1h ) , 7.73 ( d , j = 
 9.4 hz , 1h ) , 7.56 ( d , j = 8.2 hz , 4h ) , 7.44 ( dd , j = 9.5 , 2.8 , 1h ) , 7.16 ( d , j = 8.1 hz , 
 4h ) , 6.86 ( d , j = 2.8 hz , 1h ) , 4.19 ( t , j = 5.0 hz , 4h ) , 3.69 ( t , j = 5.0 hz , 4h ) , 2.17 ( s , 
 6h ) ; c nmr ( 125 mhz , dmso - d6 )  150.2 , 148.6 , 145.2 , 140.7 , 135.8 , 132.0 , 130.1 , 129.5 , 
 127.7 , 125.0 , 94.8 , 67.5 , 49.1 , 21.2 . hrms ( esi ) m / z calculated for c25h27n4o7s2 ( m + h ) 559.1316 , found 
 559.1317 . compound 21 ( 20 
 mg , 0.04 mmol ) and nahco3 ( 30 mg , 10 equiv ) were suspended 
 in methanol ( 6.25 ml ) . oxone ( 110 mg , 5 equiv ) was added , followed 
 by water ( 2.5 ml ) , and the mixture was stirred at 50 c under 
 an atmosphere of nitrogen gas for 16 h. the reaction was cooled to 
 room temperature and extracted with methylene chloride ( 5  10 
 ml ) . the organic layers were combined , washed with brine , and dried 
 over anhydrous sodium sulfate . column chromatography on silica gel 
 eluted with 1% meoh in ch2cl2 gave compound 22 as an red - orange solid ( 2 mg , 10% ) with 50% recovery of 
 starting materials : h nmr ( 500 mhz , dmso - d6 )  9.15 ( s , 1h ) , 7.96 ( d , j = 
 9.8 hz , 1h ) , 7.57 ( d , j = 8.2 hz , 4h ) , 7.257.18 
 ( m , 5h ) , 6.84 ( d , j = 2.8 hz , 1h ) , 4.21 ( t , j = 5.0 hz , 4h ) , 3.70 ( t , j = 4.9 hz , 4h ) , 
 2.25 ( s , 6h ) ; c nmr ( 125 mhz , dmso - d6 )  152.0 , 145.3 , 142.5 , 140.7 , 132.0 , 130.2 , 127.8 , 
 127.3 , 122.4 , 120.9 , 84.6 , 67.4 , 49.1 , 21.2 . hrms ( esi ) m / z calculated for c25h27n4o8s2 ( m + h ) 575.1265 , found 
 575.1263 . using a procedure adapted from 
 pchalek and hay , trifluoroacetic anhydride 
 ( 300 l ) and methylene chloride ( 1.5 ml ) were mixed with stirring 
 in an ice bath , and 70% h2o2 ( 105 l ) 
 was added dropwise . the mixture was stirred for 10 min and then allowed 
 to warm to room temperature . this solution was cooled in an ice bath 
 and slowly added to an ice - cold solution of 25 ( 110 mg , 
 0.2 mmol ) in methylene chloride ( 10 ml ) . the reaction was stirred 
 in an ice bath for 30 min before being diluted with methylene chloride 
 ( 100 ml ) and washed with cold water , cold nahco3 ( saturated ) , 
 and then brine . the organic layer was dried over anhydrous sodium 
 sulfate , and column chromatography on silica gel eluted with 0.5% 
 meoh in ch2cl2 gave 26 as a red 
 solid ( 1 mg , 1% ) : h nmr ( 500 mhz , dmso - d6 )  9.06 ( s , 1h ) , 7.96 ( d , j = 
 9.7 hz , 1h ) , 7.57 ( d , j = 8.3 hz , 4h ) , 7.41 ( dd , j = 9.7 , 2.7 , 1h ) , 7.22 ( d , j = 8.0 hz , 
 4h ) , 6.85 ( d , j = 2.6 hz , 1h ) , 4.19 ( t , j = 5.0 hz , 4h ) , 3.67 ( t , j = 4.9 hz , 4h ) , 2.25 ( s , 
 6h ) ; c nmr ( 125 mhz , dmso - d6 )  149.3 , 144.9 , 138.8 , 135.6 , 131.8 , 131.7 , 129.9 , 127.4 , 
 123.2 , 119.5 , 96.1 , 67.1 , 48.6 , 20.9 . hrms ( esi ) m / z calculated for c25h27n4o8s2 ( m + h ) 575.1265 , found 
 575.1275 . compound 13 ( 503 mg , 
 2.8 mmol ) and morpholine ( 0.72 ml , 3 equiv ) were suspended in 1-methyl-2-pyrrolidinone 
 ( 4 ml ) and heated to 100 c overnight . the resulting orange suspension was cooled to room temperature , diluted with water ( 40 ml ) , and filtered . the precipitate was washed with water and diethyl ether and then dried 
 in an oven overnight at 70 c . the orange - yellow powder was collected 
 by vacuum filtration to give 27 ( 650 mg , 94% ) : mp 245250 
 c dec ; h nmr ( 500 mhz , dmso - d6 )  7.94 ( d , j = 9.6 hz , 1h ) , 7.16 
 ( dd , j = 9.7 , 2.5 hz , 2h ) , 7.00 ( bs , 2h ) , 6.60 ( d , j = 2.5 , 1h ) , 3.73 ( t , j = 4.7 hz , 4h ) , 
 3.38 ( t , j = 4.8 hz , 4h ) ; c nmr ( 125 
 mhz , dmso - d6 )  160.7 , 155.3 , 150.8 , 
 123.3 , 120.9 , 115.2 , 103.0 , 65.8 , 46.8 ; hrms ( esi ) m / z calculated for c11h14n5o2 ( m + h ) 248.1142 , found 248.1142 using a procedure adapted from the method of 
 ligthart et al . , naoh ( 528 mg , 10 equiv ) 
 was dissolved in hot ethanol ( 30 ml ) , guanidine hydrochloride ( 1.2 
 g , 10 equiv ) was added , and the resulting mixture was stirred for 
 20 min . the reaction was cooled to room temperature and filtered to 
 remove the white precipitate . to the filtrate were added tetrahydrofuran 
 ( 20 ml ) and 3-fluoro-4-nitrobenzoic acid ( 230 mg , 1.2 mmol ) , and the 
 resulting heterogeneous mixture was refluxed for 4 h. potassium tert - butoxide ( 150 mg , 10 equiv ) was added , and the mixture 
 was stirred at reflux for an additional 2 h. after the reaction was 
 complete , the tetrahydrofuran was decanted of , f and water ( 60 ml ) 
 was added with vigorous stirring . acidification with hcl ( 1 m ) resulted 
 in precipitation of a yellow solid that was collected by vacuum filtration 
 and washed with dilute hcl , followed by minimal amounts of water and 
 diethyl ether . the solid was then dried in an oven overnight at 70 
 c to give 29 ( 244 mg , 95% yield ) : mp > 280 c ; h nmr ( 500 mhz , dmso - d6 )  
 13.63 ( bs , 1h ) , 8.20 ( d , j = 8.9 hz , 1h ) , 7.98 ( d , j = 1.5 hz , 1h ) , 7.74 ( dd , j = 2 hz , 1.6 
 hz , 1h ) , and 7.52 ( s , 2h ) ; c nmr ( 125 mhz , dmso - d6 )  166.1 , 160.6 , 148.6 , 137.1 , 131.7 , 
 127.4 , 123.6 , 120.7 ; hrms ( esi ) m / z calculated for c8h7n4o3 ( m + h ) 207.0513 , found 207.0519 . compound 29 ( 40 mg , 0.2 mmol ) 
 was suspended in trifluoroacetic acid ( 1 ml ) and h2o2 ( 70% , 0.8 ml ) , and the mixture was stirred at 50 c 
 for 48 h. after the reaction was complete ( as judged by thin layer 
 chromatography ) , the solvent was removed by rotary evaporation under 
 reduced pressure , and the resulting residue was triturated with ethanol 
 ( 5 ml ) . the resulting suspension was chilled , and the red precipitate 
 was collected by vacuum filtration . the solid was washed with cold 
 ethanol and dried under vacuum to provide compound 30 ( 16 mg , 36% ) : mp 260 c dec ; h nmr ( 500 mhz , dmso - d6 )  13.84 ( bs , 1h ) , 8.61 ( s , 1h ) , 8.28 
 ( d , j = 8.8 hz , 1h ) , 8.19 ( bs , 2h ) , and 7.94 ( d , j = 8.7 hz , 1h ) ; c nmr ( 125 mhz , dmso - d6 )  165.6 , 151.8 , 138.3 , 136.3 , 132.1 , 
 125.8 , 122.2 , 118.8 ; hrms ( esi ) m / z calculated for c8h7n4o4 ( m + h ) 223.0462 , found 223.0467 . using a procedure adapted from cantrell et al . compound 13 ( 504 mg , 2.8 mmol ) 
 was suspended in 1-methyl-2-pyrrolidinone ( 4 ml ) , and an aqueous solution 
 of naoh ( 400 mg in 20 ml , 4 equiv ) was added . to this mixture , a solution 
 of h2o2 ( 30% , 480 l , 2 equiv ) was added , 
 and the mixture was stirred for 4 h at 60 c . the reaction was 
 cooled to room temperature and filtered to remove undissolved starting 
 material . the resulting solution was acidified with 
 hcl ( 1 m ) , and the resulting orange - yellow precipitate was collected 
 by vacuum filtration . the solid was then washed with water and diethyl 
 ether and dried in an oven overnight at 70 c to give 31 ( 360 mg , 50% ) : mp 210 c dec ; h nmr ( 500 mhz , dmso - d6 )  10.94 ( bs , 1h ) , 8.00 ( d , j = 9.3 hz , 1h ) , 7.13 ( bs , 2h ) , 6.84 ( dd , j = 9.3 hz , 1.9 hz , 1h ) , and 6.68 ( d , j = 1.9 hz , 
 1h ) ; c nmr ( 125 mhz , dmso - d6 )  163.8 , 160.6 , 151.1 , 124.4 , 122.0 , 117.2 , 106.1 ; hrms ( esi ) m / z calculated for c7h7n4o2 ( m + h ) 179.0564 , found 179.0566 . compound 14 ( 160 mg , 0.83 
 mmol ) was suspended in 1-methyl-2-pyrrolidinone ( 1.2 ml ) , and naoh 
 ( 120 mg , 4 equiv , in 6 ml of water ) was added . to this mixture was 
 added h2o2 ( 30% , 144 l , 2 equiv ) , followed 
 by stirring for 1.5 h at 50 c . the reaction was cooled to room 
 temperature and acidified with hcl ( 1 m ) . the resulting precipitate 
 was collected by vacuum filtration and washed with water and diethyl 
 ether . drying under vacuum gave 32 as a dark red solid 
 ( 81 mg , 51% ) : mp 202 c dec ; h nmr ( 500 mhz , dmso - d6 )  11.51 ( bs , 1h ) , 8.08 ( d , j = 9.5 hz , 1h ) , 7.92 ( bs , 2h ) , 7.31 ( d , j = 2.5 hz , 1h ) , and 7.04 ( dd , j = 9.5 hz , 2.5 hz , 
 1h ) ; c nmr ( 125 mhz , dmso - d6 )  164.1 , 151.5 , 140.1 , 125.1 , 123.7 , 119.2 , 98.0 ; hrms ( esi ) m / z calculated for c7h7n4o3 ( m + h ) 195.0513 , found 195.0516 . solutions containing the 1,2,4-benzotriazine nitrogen 
 mustards ( 1 mm final concentration ) were prepared in cd3cn / d2o ( 1:1 ) containing disodium maleate ( 5 mm ) as an 
 internal standard . solutions were placed in a capped s500 norell nmr 
 tube and warmed in a 50 c water bath . at various time points the nmr tubes were removed from the heat bath and cooled in a room 
 temperature water bath , and h nmr spectra were obtained 
 using a 500 mhz nmr equipped with a 5 mm hcn cryo - probe . total number of scans 
 acquired was 32 with repetition delay of 4.1719923 s to ensure that 
 the integration of resonances in various compounds in the mixture 
 was quantitatively accurate . the amount of unreacted mustard at each 
 time point was assessed by measuring the integration of the ch2 resonances of the starting material in comparison to the 
 ch2 peak of the internal standard at each time point . the 
 pseudo - first - order rates for the hydrolysis reactions were obtained 
 by a least - squares fit to the equation ln a / a0 = kt . solutions of the mono- or di - n - oxide mustards ( 250 m ) were prepared in sodium phosphate 
 buffer ( 25 mm , ph 7 ) containing 2.5% dmf ( v / v ) . the samples were incubated 
 at 50 c for 24 h. at various time - points aliquots were removed 
 and frozen at 20 c for later analysis . the samples were 
 then analyzed by hplc using a varian microsorb - mv c-18 column ( 100 
  sphere size , 5 m pore size , 250 mm length , and 2.6 mm 
 i.d . eluted with a gradient mobile phase composed of solvent a ( 0.5% 
 acoh in h2o ) and solvent b ( methanol ) . for compound 17a a gradient of 2550% b over 5 min , followed by 
 50100% b over 5 min , followed by 100% b for 5 min was used . the mobile phase was returned to 25% b over 5 min and held at 25% 
 b for 5 min postrun . for compound 18a the mobile phase was held at 25% b for 5 min following injection , before increasing 
 to 50% b over 5 min and holding at 50% b for 4 min . the mobile phase 
 was then returned to 25% b over 1 min and held at 25% b for 5 min 
 postrun . the identity of the major hydrolysis products was confirmed by 
 comparison to authentic synthetic standards . vis 
 spectra were taken for compounds 29 , 30 , 31 , and 32 ( 50 m ) in solutions with ph 
 values from 0 to 9 using hcl solutions and universal buffers prepared 
 as in britton and robinson except containing 0.5% dmf ( v / v ) . absorbance values were measured at 256 and 282 nm for 29 , 260 and 270 nm for 30 , 345 and 400 nm for 31 , and 360 and 400 nm for compound 32 . changes in absorbance 
 were plotted against ph and the pka values 
 were calculated by fitting the data to the equation : pka = ph + log(dm  d)/(d  di ) , where dm is the absorbance for the 
 un - ionized species ( starting absorbance ) , di is the absorbance of the ionized species ( final absorbance ) , and d is the absorbance at any point in the ph titration . to confirm that pka values measured reflected protonation of the 6-cooh or 6-oh substituents 
 in compounds 29 , 31 , and 32 , ph - absorbance measurements were performed with 9 and 12 to show that these control compounds displayed no significant 
 changes in absorbance in the ph regions of interest . the 2-deoxyoligonucleotides 
 ( 33 and 34 , figure 3 ) were labeled on the 5-end with p and annealed 
 to their complements using standard methods . dna duplexes were mixed with the indicated compounds ( 1 mm final 
 concentrations ) and incubated at 37 c for 3 d in hepes buffer 
 ( 50 mm , ph 7 ) containing nacl ( 100 mm ) and dmf ( 10% v / v ) . the dna 
 was then ethanol precipitated , washed with 80% ethanol twice , and 
 briefly dried in a speedvac concentrator ( 5 min at room temperature ) . the dna was resuspended in piperidine ( 1 m in water ) and heated to 
 95 c for 30 min . the piperidine solution was removed under vacuum 
 in a speedvac concentrator , and the resulting residue was resuspended 
 in formamide loading buffer , warmed briefly , 
 and loaded onto a 20% denaturing polyacrylamide gel . the gel was electrophoresed 
 for 3 h at 1500 v to resolve the labeled dna fragments . the labeled 
 dna fragments in the gel were visualized using phosphorimaging . for experiments involving in vitro hypoxic metabolism , sodium phosphate 
 buffer and hplc - grade water were degassed by bubbling argon through 
 the solutions for at least 30 min inside a glovebag filled with argon 
 gas . stock solutions of compounds 15 or 9 in dmf were degassed by three cycles of freeze  pump  thaw 
 inside pyrex tubes . the tubes were then torch - sealed , scored , and 
 transferred to the argon - purged glovebag . the enzyme substrate nadph 
 was dissolved in degassed water inside the glovebag . in the glovebag , solutions containing sodium phosphate buffer ( 25 mm , ph 7 ) , the di - n - oxide 15 ( 100 m ) , nadph ( 500 m ) , 
 and nadph : cytochrome p450 reductase ( 1 unit / ml ) and dmf ( 1% v / v ) were 
 incubated in a sealed microcentrifuge tube at room temperature protected 
 from light for 24 h. a similar reaction was prepared using nondegassed 
 solutions and incubated open to air as a aerobic control . after 24 
 h , the solutions were passed through millipore ( ym-3 ) centrifuge filters 
 ( 30 min , 7500  g ) . the filtrate was analyzed 
 by hplc using a varian microsorb - mv c-18 column ( 100  sphere 
 size , 5 m pore size , 250 mm length , and 2.6 mm i.d . ) . the column 
 was eluted with a mobile phase composed of solvent a ( 0.5% acoh in 
 h2o ) and solvent b ( methanol ) . the column was eluted at 
 a flow rate of 0.9 ml / min for 2 min at 10% b , a gradient of 1050% 
 b over 8 min , 50% b for 5 min , then returned to 10% b over 5 min and 
 held at 10% b for 5 min postrun . compounds were detected by their 
 absorbance at 420 nm , and the identity of products 15 and 16 was confirmed by comparison to authentic synthetic 
 standards .
tirapazamine 
 ( 3-amino-1,2,4-benzotriazine 1,4-dioxide ) is a heterocyclic 
 di - n - oxide that undergoes enzymatic deoxygenation 
 selectively in the oxygen - poor ( hypoxic ) cells found in solid tumors 
 to generate a mono - n - oxide metabolite . 
 this work 
 explored the idea that the electronic changes resulting from the metabolic 
 deoxygenation of tirapazamine analogues might be exploited to activate 
 a dna - alkylating species selectively in hypoxic tissue . toward this 
 end , 
 tirapazamine analogues bearing nitrogen mustard units were prepared . 
 in the case of the tirapazamine analogue 18a bearing 
 a nitrogen mustard unit at the 6-position , it was found that removal 
 of the 4-oxide from the parent di - n - oxide to generate 
 the mono - n - oxide analogue 17a did indeed 
 cause a substantial increase in reactivity of the mustard unit , as 
 measured by hydrolysis rates and dna - alkylation yields . 
 hammett sigma 
 values were measured to quantitatively assess the magnitude of the 
 electronic changes induced by metabolic deoxygenation of the 3-amino-1,2,4-benzotriazine 
 1,4-dioxide heterocycle . 
 the results provide evidence that the 1,2,4-benzotiazine 
 1,4-dioxide unit can serve as an oxygen - sensing prodrug platform for 
 the selective unmasking of bioactive agents in hypoxic cells .