poly adp - ribose polymerase 
 1 ( parp-1 ) is a critical enzyme in the 
 repair of dna strand breaks . this 116 kda nuclear protein detects 
 dna single strand breaks and utilizes nad as a substrate 
 to poly(adp - ribosyl)ate nuclear proteins , resulting in relaxation 
 of chromatin and recruitment of other repair proteins to the damaged 
 site . parp-1 is an attractive antitumor target because of this vital 
 role in dna repair . the current clinical approaches to the development 
 of parp-1 inhibitors include either ( 1 ) the utilization as a single 
 agent in brca1 or brca2-deficient cancers where inhibition of parp 
 results in synthetic lethality or ( 2 ) the utilization in combination 
 with dna damaging therapeutics ( radiation or chemotherapy ) to increase 
 maximum therapeutic benefit of these agents by blocking the repair 
 process . there are multiple ongoing clinical trials evaluating the 
 efficacy of parp-1 inhibitors as chemopotentiators in several cancers , 
 including non - small - cell lung cancer ( nsclc ) . however , early phase 
 clinical studies of parp-1 inhibitor , compound 1 , in 
 combination with topotecan , dacarbazine , or cisplatin plus gemcitabine showed dose - limiting toxicity that was more pronounced 
 than that seen with the chemotherapeutic agents alone . therefore , 
 targeted delivery of parp inhibitors selectively to cancer cells could 
 be a solution to overcome these systemic toxicity problems . diazeniumdiolate - based nitric oxide ( no ) releasing prodrugs developed 
 in our laboratory have proven to be effective as anticancer agents 
 in a number of in vitro and in vivo models . the lead compound , o-(2,4-dinitrophenyl ) 
 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate 
 ( js - k , 2 ) , exhibits a multifaceted mechanism of action 
 initiated by depletion of intracellular gsh and induction of oxidative 
 stress , followed by the activation of stress signaling , abrogated 
 mitochondria function , dna damage , and apoptosis . dna single strand 
 break damage ( measured by comet assay ) was observed in the h1703 nsclc 
 cell line after a 1 h treatment with 2 , and massive dna 
 damage was seen after 24 h. in multiple myeloma cells , 2 caused dna double strand break damage . we hypothesized that inhibition of parp may enhance the potency 
 of 2 and other no - releasing prodrugs of the arylated 
 diazeniumdiolate class . in this work , we have designed and synthesized 
 no - releasing parp-1-inhibitor 
 prodrugs that are activated by reaction with glutathione ( gsh ) , a 
 reaction that is accelerated by glutathione s - transferase ( gst ) , a 
 family of phase ii detoxification enzymes . we show that these hybrid 
 prodrugs are effective against cancer cells in vitro and in vivo . activation of the compounds by gstp1 , an isoform of gst that is frequently 
 overexpressed in cancers , should result in the preferential metabolism 
 and thus release of cytotoxins in cancer cells , potentially diminishing 
 systemic toxicity associated with parp inhibition . structural features to be 
 merged in designing no - releasing parp inhibitors for the present study 
 are color coded : green for the arylating fragment that is transferred 
 to gsh in the activation step ; red for the parp inhibitor moiety ; 
 and blue for the two molecules of cytolytic nitric oxide released 
 on activation . our specific strategy 
 here is to combine the structural features of the established parp-1 
 inhibitor 1 and arylated diazeniumdiolate 2 , structures shown in scheme 1 . specifically , 
 we wanted to produce molecules that link the parp - inhibitory skeleton 
 of 1 ( the red portion of structure 1 in 
 scheme 1 ; the black cyclopropane carboxamide 
 group is not necessary for parp-1 inhibition ) with the electrophilic 
 aryl ring shown in green and the caged no molecules shown in blue 
 in scheme 1 . accordingly , we reacted known 
 compounds 3a and 3b(10 ) with 4(7 ) to obtain the first 
 two drug candidates , 5a and 5b , as shown 
 in scheme 2 . our hypothesis was that attack 
 by gsh on these molecules would lead to consumption of a reducing 
 equivalent through irreversible arylation of the gsh , with simultaneous 
 generation of two nos plus known parp inhibitors 6a and 6b , as illustrated in scheme 3 . the first step in 
 the activation of these prodrugs is the arylation of gsh , liberating 
 a diazeniumdiolate ion that spontaneously decomposes to no , freeing 
 the parp - inhibitory part of the molecule . accordingly , 5a and 5b reacted smoothly with gsh to generate the expected 
 products , shown in scheme 3 . the rates of reaction 
 of 5a and 5b with 4 mm gsh in aqueous ph 
 7.4 phosphate buffer ( 0.1 m ) containing 50 m diethylenetriaminepentaacetic 
 acid at 37 c were determined . the t1/2 for 5a was 11.2 min , and the t1/2 for 5b was 11.6 min . liquid chromatography / mass 
 spectrometry ( lc / ms ) analysis of the reaction mixture after 2 half - lives 
 confirmed that 25% of the parent compound was still present while 
 75% consisted of deacylated compound 1 ( 6b ) and the aryl - glutathione conjugate ( 7 ) ( scheme 3 , figure s1 ) . the ability 
 of the new compounds 
 to inhibit parp-1 enzymatic activity was tested using an assay that 
 measures incorporation of biotinylated poly adp - ribose onto histone 
 proteins . 5a and 5b were compared to 1 and authentic compounds 6a and 6b that are products of 5a and 5b activation 
 ( scheme 3 ) . parp-1 enzyme ic50 values estimated for 6a , 6b , and 1 were 30.5 , 6.9 , and 
 15.5 nm , respectively ( figure 1 ) . both prodrugs 5a and 5b , without activation by gsh , were less 
 potent , with ic50 of 123 nm for 5a and 58 
 nm for 5b . inhibition of parp-1 enzyme activity by 5a , 5b , 6a , and 6b , 
 compared with 1 . compounds 5a and 5b inhibited 
 proliferation of an extensive 
 panel of nsclc cell lines with ic50 concentrations ranging 
 from 3 to 20 m ( table 1 ) . ic50 values for no - releasing parp-1 inhibitor prodrugs were significantly 
 lower than those of 1 for the most sensitive cell lines . the ic50 values correlated with endogenous ros levels 
 and with levels of antioxidant enzyme peroxiredoxin 1 and dna repair 
 enzyme 8-oxoguanine dna glycosylase ( ogg1 , figure 
 s2 ) . depleting the cell s gsh during prodrug activation 
 combined with no release led to induction of oxidative / nitrosative 
 stress ( figure s3 ) and cytotoxicity through 
 cellular stress overload , especially in cells with high endogenous 
 ros levels . since the proliferation screen and measurements of parp-1 
 inhibitory activities of both prodrugs showed fluoro - substituted analogue 5b to be more potent than 5a , we decided to focus 
 on 5b for further development . cellular levels of atp fall below 
 a critical level , and cells with substantial dna damage can not enter 
 apoptosis , rendering them resistant to this form of cell death or 
 susceptible to necrosis . therefore , inhibition of parp-1 in combination 
 with substantial dna damage would conserve cellular energy and could 
 allow tumor cells to undergo apoptosis in response to dna - damaging 
 no , eliminating toxic effects or immune responses associated with 
 necrosis . twenty - four - hour treatment with no - releasing parp-1 
 inhibitor prodrug 5b resulted in significant dna strand 
 break damage as observed by comet assay . treatment with 5b at 5 m resulted in a stronger comet signal , compared with 
 20 m 1 ( figure 2a ) . a strong 
 apoptotic signal ( as evidenced by cleaved caspase 7 ) was seen in cells 
 treated with 5b , while the same concentration of 1 did not trigger apoptosis ( figure 2b ) . treatment with no - releasing parp-1 inhibitor prodrug 5b resulted in dna damage ( a ) , as shown by the comet assay in h1703 
 nsclc cells . the dna strand break damage caused by 5b was more extensive than that resulting from 1 treatment . actin served as a loading control . to validate 
 the release of the parp-1 inhibitor in cells , we evaluated cellular 
 uptake and metabolism of 5b in a549 nsclc cells . cells 
 were treated with 5b for varying time periods , and cell 
 lysates were analyzed for the presence of the parent compound and 
 its metabolites by lc / ms . the parent compound was not detected at 
 any measured time point including a brief treatment of 5 min ( asterisk 
 (  ) in figure 3 ) . at all time points 
 measured up to 1 h , compound 6b and arylated gsh metabolite 7 were detected in equally abundant amounts , suggesting very 
 rapid , presumably catalyzed , cellular metabolism ( figure 3 ) . in vitro metabolism of compound 5b . the human 
 a549 
 ncslc cell line was treated with 10 m 5b . then 
 the products were extracted at various time points and analyzed by 
 lc / ms / ms . shown are extracted ion chromatograms ( eics ) , which were 
 extracted at various m / z ratios 
 including the [ m + h ] of 6b ( retention time 
 ( tr ) of 11.9 min ) , arylated gsh ( 7 , tr = 13.8 min ) , and compound 5b ( tr = 18.2 min ) ( scheme 3 ) . the asterisk (  ) indicates where compound 5b should be , based on the elution profile of a lysate spiked 
 with authentic compound 5b ( bottom panel ) . lung cancer cells frequently express high levels 
 of gstp1 , the 
 gst isoform that is often overexpressed in cancer cells . we have screened 
 a panel of 20 nsclc cell lines for the level of expression of gstp1 
 protein using western blot . this experiment confirmed that the enzyme 
 is highly expressed in a majority of nsclc cell lines , with little 
 to none of the liver - specific a1 isoform ( gsta1 ) detected ( figure s4 ) . therefore , we studied the rate of 
 reaction of 5b with gsh in the presence of recombinant 
 gstp1 and found that the reaction was enhanced over 10-fold when the 
 enzyme was present ( table 2 , row 3 ) . this strongly 
 suggests that the prodrug , despite its reactivity with gsh alone , 
 will be preferentially activated when gstp1 is present . importantly , 
 the presence of gstp1 did not alter product distribution ( figure s5 ) . next , 5b was independently 
 reacted with glutathione in the presence of gsta1 ; the a1 isoform 
 was found to be a better catalyst for glutathione attack than gstp1 
 ( table 2 , row 3 ) . further synthetic approaches 
 were taken to diminish gsta1 catalysis . only two measurements 
 made in the 
 first half - life for the gsta1 trials . in an effort to modify the structure of 5b to make 
 it less susceptible to gsta1-induced metabolism and to improve its 
 suitability as a substrate for gstp1 , we employed a strategy developed 
 previously to do so with compound 2 . in that case , molecular modeling suggested that adding 
 and reducing steric bulk at different specific locations in the structure 
 of 2 would accomplish this goal . as predicted , compound 8 ( paba / no , structure in scheme 4a ) 
 was prepared and shown to be metabolized at similar rates by gstp1 
 and gsta1 , in contrast to 2 , which was an order of magnitude 
 more efficiently metabolized by gsta1 ( table 2 , rows 1 and 2 ) . we chose a similar strategy in designing the 
 bis - diazeniumdiolated parp-1 inhibitor hybrid 13 shown 
 in scheme 4b as a synthetic target . thus , boc - protected 
 piperazine diazeniumdiolate 9 was reacted with aryl fluoride 
 substituted diazeniumdiolate 10 to give adduct 11 , which upon removal of the protecting group gave 12 . this compound contains nucleophilic nitrogen in the piperazine 
 ring that was reacted with 3b to produce the bis - diazeniumdiolated 
 parp-1 inhibitor 13 . compound 13 was 
 subjected to a similar reactivity 
 and metabolism analysis as 5b . not only was compound 13 more stable than 5b in reactions with gsh 
 alone , but the rates of catalysis suggest that compound 13 is better accommodated in the active site of gstp1 than that of 
 gsta1 ( table 2 , row 4 ) . molecular modeling 
 experiments suggested that compound 13 fits nicely into 
 the gstp1 active site while a steric clash may occur in the gsta1 
 active site ( figure 4 ) . compound 13 parp-1 enzyme inhibitory activity in the 
 presence or absence of gsh and gstp1 was also analyzed . top panels : transition state modeling 
 of the 13glutathione 
 adduct in the gsta1 and gstp1 active sites . bottom panel : inhibition 
 of parp-1 enzyme by compound 13 in the presence or absence 
 of gsh / gstp1 . mass spectrometric analysis 
 of the cellular metabolism of compound 13 in a549 lung 
 adenocarcinoma cells and u937 human leukemia 
 cells revealed that the compound is metabolized as designed to release 
 no upon gsh / gst attack . the dearylation of the first diazeniumdiolate 
 moiety results in spontaneous release of no and frees the parp-1-inhibitory 
 component . the resulting glutathione conjugate reacts further with 
 gsh , leading to attachment of two glutathionyl substituents to the 
 dinitrophenyl ring ( scheme 4c ) . alternatively , 
 the single glutathione conjugates could react with protein thiols 
 instead of gsh , resulting in a protein adduct , in which gsh and the 
 protein s thiol group are covalently cross - linked through the 
 dinitrophenyl ring . this thiol modification ( cross - linking glutathionylation ) appears to be irreversible and likely plays an important role in mediating 
 the cytotoxic activity of bis - diazeniumdiolates . it is also predictable that this irreversible modification 
 will affect proper protein folding , which , if unresolved , leads to 
 the unfolded protein response ( upr ) and endoplasmic reticulum ( er ) 
 stress . we exploited this hypothesis and found that indeed 
 treatment with 13 results in cellular protein glutathionylation 
 and er stress ( figure 5a ) . treated cells were 
 lysed and processed for immunoblotting under nonreducing conditions , 
 and protein / gsh cross - links were detected with specific antibodies 
 recognizing glutathione conjugated to proteins . as shown in figure 5a , glutathione attached to protein was detected 
 within 1 h of treatment , and the signal became gradually stronger 
 with time . the decrease in protein / gsh conjugate signal observed at 
 8 h was only seen upon the onset of apoptosis , as indicated by cleaved 
 parp ( figure 5a ) . this essentially irreversible 
 modification consumes gsh and results in a shift in the cellular redox 
 balance toward a more oxidizing environment , generating oxidative / nitrosative 
 stress . er stress induction was evidenced by phosphorylation of eif2 
 and up - regulation of chop , bip , and atf4 proteins ( figure 5b ) . activation of stress kinase p38 was seen within 
 15 min after treatment with 13 was initiated ( figure 5c ) and could be a result of ros / rns stress . we have 
 observed rapid and persistent phosphorylation of p38 in u937 leukemia 
 cells upon treatment with compound 2 . to determine whether the induction of ros / rns and 
 stress signaling were associated with cell death , cells were treated 
 with compound 13 and then analyzed by western blot for 
 markers of apoptosis . apoptosis was activated through the extrinsic 
 pathway , as evidenced by caspase 8 activation within 6 h of treatment 
 with 1 m 13 ( figure 5e ) . cleaved effector caspases 3 and 7 and cleaved parp signals were also 
 observed ( figure 5e ) . ( a ) compound 13 causes cross - linking of gsh to protein 
 thiols ( protein glutathionylation ) that is irreversible and induces 
 endoplasmic reticulum ( er ) stress in a549 lung adenocarcinoma cells 
 ( b ) . ( c ) activation of the p38 stress signaling pathway in a549 cells 
 after treatment with 13 occurred rapidly , within 15 min 
 after treatment was initiated , and phosphorylation persisted for 8 
 h. ( d ) tumor suppressor p53 and its effector , puma , are involved in 
 cells bearing wild - type p53 ( a549 nsclc cell line ) . ( e ) treatment with 13 induced the extrinsic apoptosis pathway in a549 cells . cleavage of caspase 8 and the effector caspases 3 and 7 , as well as 
 parp , was observed : f.l . , full length parp ; cl , cleaved . human lung adenocarcinoma cell 
 line a549 , 
 which expresses moderate levels of gstp1 , was chosen for assessment 
 of activity of the prodrugs in vivo against xenografted cells in athymic 
 mice . both compounds were administered at 92 mol / kg 
 intravenously two times a week for a 4-week period to assess anticancer 
 activity . figure 6 shows significant reduction 
 of the tumor growth in animals treated with compound 13 ( p < 0.02 ) , compared to 1 or saline 
 controls . importantly , treatment with either controls or diazeniumdiolate - based 
 drugs did not affect body weights ( see caption to figure 6 ) . tumors from animals in each group were resected 
 and extracted for analysis of metabolites . both the parent drugs and 
 the liberated parp-1 inhibitor were detected in some tumors from animals 
 treated with compounds 5b and 13 , indicating 
 unambiguous delivery of compound to tumors remote from the site of 
 injection . compounds were administered intravenously at 92 mol / kg , 
 2 times a week for 4 weeks , and tumors were measured with a caliper . values are medians , and the relevant 95% confidence interval bars 
 are shown ( mann  whitney test ) . stars indicate the significance 
 of the differences between 13-treated and 1-treated and control mice at each time point . the average body weight for all mice was 22.9 
  0.31 g ( mean  se ) at the beginning of the experiment . 
 at the termination , the average weights of the control groups were 
 25.6  0.76 g ( n = 12 ) , 25.0  0.78 g ( n = 10 ) , and 25.0  0.77 g ( n = 11 ) 
 for 1 , vehicle , and saline control , respectively . the 
 weights of animals treated with 13 were 25.3  0.49 
 g ( n = 10 ) , and those treated with 5b were 27.7  0.82 g ( n = 11 ) . bortezomib ( ps-341 , 14 ) is a boronic acid dipeptide derivative 
 that selectively and potently inhibits the 26s proteasome . it has clinically validated activity against 
 multiple myeloma and has undergone extensive evaluation in nsclc . preliminary in vitro studies established that 14 alone 
 induces growth inhibition in several nsclc cell lines . it has been shown that , combined with cytotoxic agents in vitro , 14 enhanced the antitumor effect in nsclc and other solid 
 tumors , and recently proteasome inhibition 
 as a treatment for nsclc advanced to clinical trials . it has previously been shown that the proteasome 
 inhibitor 14 has synergistic activity with compound 2.(6)14 induced ros 
 generation in h460 nsclc cells . having 
 shown that cytotoxicity of diazeniumdiolate - based no - releasing prodrugs 
 correlated with intracellular ros levels , we hypothesized that co - treatment 
 of lung adenocarcinoma cells with 14 and our prodrugs 
 would result in enhanced cytotoxicity through ros / rns stress . also , 
 proteasome inhibitors are known to induce the unfolded protein response 
 and er stress . therefore , a combination of 14 with compound 13 , which causes irreversible , aryl - cross - linking glutathionylation 
 of cellular proteins and induces er stress , might result in enhanced 
 cytotoxicity . indeed , combination treatment of 14 and 13 exhibited enhanced toxicity as evaluated by colony forming 
 assay ( figure 7 ) . this promising result suggests 
 that further in vitro and in vivo trials of compound 13 combined with 14 are indicated . colony forming assay 
 ( a549 cells ) indicated enhanced cytotoxicity 
 of the combination of compound 13 with proteasome inhibitor 14 . we 
 have designed prodrugs combining an arylated diazeniumdiolate 
 with a parp-1 inhibitor to achieve simultaneous delivery of a dna 
 damaging agent and a dna repair inhibitor to the cell . previous work 
 has shown that arylated diazeniumdiolates cause significant dna strand 
 break damage . concurrent treatment with a dna strand break repair 
 inhibitor dramatically exacerbates dna damage and cytotoxicity . second - generation 
 molecules have been designed to be preferentially metabolized by cells 
 containing high levels of gstp1 , e.g. , cancer cells . we conclude that 
 compound 13 , a bis - diazeniumdiolate , is capable of irreversibly 
 cross - linking gsh to protein thiols , causing the accumulation of misfolded 
 proteins , leading to er stress ( figure 8) . this additional cytotoxic stress adds to the multifaceted proapoptotic 
 mechanisms of arylated diazeniumdiolate action . starting materials were purchased from aldrich 
 chemical co. ( milwaukee , wi ) unless otherwise indicated . chemical shifts 
 (  ) are reported in parts per million ( ppm ) downfield from tetramethylsilane . ultraviolet ( uv ) spectra were recorded on an agilent model 8453 or 
 a hewlett - packard model 8451a diode array spectrophotometer . prepacked 
 silica gel flash chromatography columns were purchased from silicycle 
 ( quebec city , canada ) or from yanazen science inc . in addition to nmr , uv , and hplc with hrms , chn combustion 
 analysis was performed to determine purity of compounds . 3-[(4-oxo-3h - phthalazin-1-yl)methyl]benzoic acid , 3a , was 
 prepared as described by menear et al . to a slurry of 557 mg ( 1.6 mmol ) of the hydrochloride salt of 4 , 448 mg ( 1.6 mmol ) of 3a , and 608 mg ( 1.6 mmol ) of hatu in 5 ml of dimethylacetamide the resulting 
 solution was stirred at room temperature for 1 h , followed by addition 
 of 40 ml of water . the resulting precipitate was collected by filtration 
 and recrystallized from ethanol , giving 827 mg of an off - white solid : 
 mp 114117 c ; uv ( acetonitrile ) max 294 nm (  = 18.9 mm cm ) ; h nmr ( dmso - d6 )  
 3.633.68 ( m , 8h ) , 4.38 ( s , 2h ) , 7.287.30 ( m , 1h ) , 
 7.397.46 ( m , 3h ) , 7.817.98 ( m , 4h ) , 8.26 ( d , 1h ) j = 7.4 hz , 8.568.59 ( dd , 1h ) j = 2.7 , 7.4 hz , 8.88 ( d , 1h ) j = 2.7 hz , 12.06 ( s , 
 1h ) ; c nmr ( dmso - d6 )  
 37.7 , 50.2 , 116.5 , 122.5 , 125/6 , 125.9 , 126.5 , 127.8 , 128.3 , 129.2 , 
 129.6 , 130.2 , 130.7 , 132.0 , 133.9 , 135.6 , 137.3 , 138.93 , 142.6 , 145.3 , 
 153.2 , 159.8 , 169.4 ; hrms ( esi ) m / z calculated for c26h23n8o8 [ m + h ] = 575.1639 , found 575.1548 ,   ppm 
 = 1.5 calcd for c26h22n8o8h2o : c , 52.70 ; h , 4.08 ; n , 18.91 . found : 
 c , 52.66 ; h , 3.73 , n , 18.50 . a solution 
 of 685 mg ( 2.3 
 mmol ) of 3b(10 ) in 30 ml of n , n - dimethylformamide was cooled to 4 c . hatu ( 950 mg , 2.5 mmol ) was added , and the resulting slurry was stirred 
 under nitrogen . a solution of 765 mg ( 2.19 mmol ) of 4(5 ) and 753 l ( 5 mmol ) of diisopropylethylamine 
 in 60 ml of dimethylformamide was added , and the resulting solution 
 was stirred at room temperature overnight . the solution was cooled 
 to 4 c and ice  water ( 200 ml ) was added , giving a solid 
 precipitate . the product was collected by filtration , washed with 
 water , and allowed to dry to give 1.3 g of crude material . recrystallization 
 from ethanol afforded 853 mg of pure 5b : mp 150153 
 c ; uv ( acetonitrile ) max 295 nm (  = 
 20.5 mm cm ) ; h 
 nmr ( dmso - d6 )  3.41 ( b , 1h ) , 3.58 
 ( b , 1h ) , 3.72 ( b , 1h ) , 3.84 ( b , 1h ) , 4.33 ( s , 2h ) , 7.25 ( t , 1h ) j = 9.0 hz , 7.377.46 ( m , 2h ) , 7.82 ( t , 2h ) j = 9.0 hz , 7.877.97 ( m , 3h ) , 8.24 ( d , 1h ) j = 7.8 hz , 8.548.57 ( dd , 1h ) j = 2.4 , 7.8 hz , 8.86 ( d , 1h ) j = 2.4 hz , 12.58 ( s , 
 1h ) ; c nmr ( dmso - d6 )  
 36.9 , 45.2 , 50.5 , 116.9 , 118.52 , 119.5 , 122.6 , 124.0 , 125.8 , 126.5 , 
 127.9 , 129.3 , 130.2 , 131.9 , 132.6 , 133.9 , 135.2 , 137.3 , 142.6 , 145.2 , 
 154.1 ( d ) jc  f = 246 hz , 159.8 , 
 164.4 ; hrms ( esi ) m / z calculated 
 for c26h22fn8o8 [ m + h ] = 593.1545 , found 593.1538 ,   ppm = 1.1 calcd for c26h21fn8o8h2o : c , 51.15 ; h , 3.80 ; n , 18.35 ; f , 3.11 . found : c , 51.45 ; 
 h , 3.70 , n , 18.41 ; f , 3.42 . a partial solution 
 of 2.52 g ( 0.0087 mol ) of o-(2,4-dinitro-5-fluorophenyl ) 
 1-(n , n - dimethylamino)diazen-1-ium-1,2-diolate ( 10)(5 ) in 120 ml of tert - butanol was stirred at room temperature . a solution of 2.34 g ( 0.0087 
 mol ) of sodium 1-[4-(tert - butoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate 
 ( 9 ) in 120 ml of 5% aqueous 
 sodium bicarbonate was added gradually through a pressure equalizing 
 addition funnel , and the resulting mixture was stirred at room temperature 
 overnight . the reaction mixture was treated with 150 ml of water and 
 the resulting yellow solid was collected by filtration , washed with 
 water , and dried to give 1.33 g of product . the material was placed 
 on a silica gel column and eluted with hexane / ethyl acetate to give 
 3.6 g ( 80% ) of 11 as a yellow powder : mp 123125 
 c ; uv ( acetonitrile ) max 292 nm (  = 
 23 mm cm ) ; h nmr 
 ( cdcl3 )  1.49 ( s , 9h ) , 3.28 ( s , 6h ) , 3.603.61 
 ( m , 4h ) , 3.653.67 ( m , 4h ) , 7.57 ( s , 1h ) , 8.86 ( s , 1h ) ; c nmr ( cdcl3 )  28.3 , 41.8 , 50.5 , 81.0 , 
 105.3 , 125.5 , 154.2 , 154.3 . 24.46 . found : 
 c , 39.44 ; h , 4.79 ; n , 24.09 . to a solution of 3.6 g ( 0.007 mol ) 
 of 11 in 300 ml 
 of ethyl acetate was added 70 ml of 2 m hcl in ether . the product was collected 
 by filtration , washed with ethyl acetate , and allowed to dry , giving 
 3.1 g ( 98% ) of 12 : mp 135137 c ; uv ( ethanol ) 
 max 288 nm (  = 28 mm cm ) ; h nmr ( dmso - d6 )  3.26 ( s , 6h ) , 3.333.35 ( b , 9h ; 4h for piperazine , 
 5h for water ) , 3.893.91 ( m , 4h ) , 7.82 ( s , 1h ) , 8.90 ( s , 1h ) , 
 8.41 ( b , 2h ) ; c nmr ( dmso - d6 )  41.6 , 47.2 , 105.6 , 125.7 , 131.5 , 131.9 , 153.6 , 154.1 . this 
 salt was used for the next step without further purification . to a solution 
 of 2.05 g ( 0.006  87 mol ) of 3b(10 ) and 2.85 g ( 0.0075 mol ) of hatu in 150 ml of n , n - dimethylformamide was added 2.6 ml ( 0.015 mol ) 
 of dipea . to the solution was gradually added 3.1 g ( 0.006  87 
 mol ) of 12 in 150 ml of n , n - dimethylformamide , and the resulting solution was stirred at room 
 temperature overnight . the solution was treated with 250 ml of cold 
 aqueous ammonium chloride solution , and the resulting precipitate 
 was collected by filtration and washed with water . the solid containing 
 water and dimethylformamide was taken up in dichloromethane , dried 
 over sodium sulfate , filtered through a layer of anhydrous magnesium 
 sulfate , and evaporated in vacuo . the yellow moist solid was triturated 
 with ether to give , after filtration , 2.16 g of 13 . purification was carried 
 out with preparative hplc : mp 122125 c ; uv ( 0.2% dmso / ethanol ) 
 max 287 nm (  = 32 mm cm ) ; h nmr ( acetone - d6 )  3.30 ( s , 6h ) , 3.583.97 ( m , 8h ) , 7.147.22 
 ( m , 1h ) , 7.447.54 ( m , 2h ) , 7.79 ( s , 1h ) , 7.807.88 
 ( m , 1h ) , 7.947.96 ( m , 2h ) , 8.328.34 ( m , 1h ) , 8.86 
 ( s , 1h ) , 11.75 ( s , 1h ) ; c nmr ( acetone - d6 )  37.9 , 41.7 , 51.0 , 51.2 , 105.5 , 110.75 , 116.5 , 
 116.9 , 124.8 , 125.9 , 126.4 , 127.2 , 129.5 , 132.1 , 134.1 , 136.0 , 145.6 , 
 153.4 , 155.1 , 155.6 ( d ) jc  f = 
 201.0 hz , 160.3 , 165.1 . for further decomposition and biological screening , 
 a portion of the compound was purified on a phenomenex luna c18 column , 
 3 m , 150 mm  2.0 mm , with a gradient consisting of water 
 and acetonitrile containing 0.1% formic acid . hrms ( esi ) m / z calculated for c28h27fn11o10 [ m + h ] = 696.1921 , found 696.1928 , 
   ppm = 0.96 . calcd for c28h26n11fo10h2o : c , 47.13 ; h , 3.95 ; 
 f , 2.66 ; n , 21.59 . found : c , 47.14 ; h , 4.08 ; f , 2.73 ; n , 21.45 . intracellular levels of reactive oxygen / nitrogen 
 species were quantified by oxidation of the ros / rns - sensitive fluorophore 
 5-(and -6)-chloromethyl-2,7-dichlorodihydrofluorescein 
 diacetate ( dcf - da , invitrogen , carlsbad , ca ) . cells growing on six - well 
 plates ( 6  10/well ) were loaded with 5 m 
 dcf - da in hanks balanced salt solution ( hbss ) at 37 c 
 and 5% co2 . after 30 min of incubation , hbss containing 
 the probe was removed , cells were rinsed with hbss , and 3 ml of fresh 
 hbss was added to each well followed by addition of compounds ( 10 
 m ) or dmso as a control . after 60 min the cells were collected 
 by scraping in hbss , and dcf fluorescence was measured by using a 
 perkinelmer life and analytical sciences ( waltham , ma ) ls50b luminescence 
 spectrometer with the excitation source at 488 nm and emission at 
 530 nm . the intracellular level of nitric oxide and its oxidation 
 products after treatment with compounds was estimated by using the 
 fluorophore 4-amino-5-methylamino-2,7-difluorofluorescein ( daf - fm ) 
 diacetate ( invitrogen ) . cells growing in six - well plates were loaded 
 with 2.5 m daf - fm diacetate in hbss at 37 c and 5% co2 . after 30 min of incubation the cells were rinsed with hbss 
 to remove excess probe . test compounds in fresh hbss were added to 
 the cells at 10 m final concentration . after 30 min of incubation , 
 the fluorescence of the benzotriazole derivative formed on daf - fm s 
 reaction with aerobic no was analyzed by using a perkinelmer life 
 and analytical sciences ls50b luminescence spectrometer with the excitation 
 source at 495 nm and emission at 515 nm . all experiments were performed 
 at least three times , each time at least in triplicate . parp enzyme inhibition was measured 
 using an ht universal colorimetric 96-well parp assay kit ( trevigen , 
 gaithersburg , md ) , according to the manufacturer s protocol 
 with the following small modifications : when inhibitory activities 
 of activated prodrugs were studied , gsh ( 4 mm ) was added in the presence 
 and absence of gstp1 in pbs , ph 7.4 . reactions were initiated by the 
 addition of prodrug after a 10 min incubation at 37 c and carried 
 out for another 10 min . cell lines were 
 obtained from the american type culture collection ( manassas , va ) 
 and cultured according to the supplier s protocol . for proliferation 
 assays , cells were seeded at 1  10 per well ( h1693 , 
 h322 m , h1703 , h1944 , h1355 , h2122 , h441 , h1568 ) or 5  10 per well ( h460 , h1792 , a549 , h2023 , h2030 , h23 ) in 96-well 
 plates and allowed to adhere for 24 h. compounds were prepared as 
 10 mm stock solutions in dmso . increasing drug concentrations in 10 
 l of pbs were added to 100 l of the culture medium and 
 incubated for 72 h. the mtt assay ( promega , madison , wi ) was performed 
 according to the manufacturer s protocol . each concentration 
 was represented in six repeats , and the screening was performed as 
 at least two independent experiments . ic50 values were 
 calculated by using sigma plot software ( systat software , inc . gsh ( 4 mm ) was 
 added in the presence and absence of gstp1 or gsta1 in 0.1 m phosphate 
 buffer solution , ph 7.4 , containing 50 m diethylenetriaminepentaacetic 
 acid ( dtpa ) . reactions were initiated by the addition of substrate 
 after the gsh - containing buffer and enzyme reached thermal equilibrium . typical substrate concentrations were 10 m with a gst concentration 
 of 40 nm . in each experiment the data were analyzed at 302 nm and 
 the rate was derived by fitting the data to an exponential curve typical 
 for first order processes . the metabolism of each compound was 
 studied in the human 
 a549 nsclc cell line and the human u937 leukemia cell line . in each 
 case cells the a549 cell line was treated with 10 m of 
 each compound and incubated for varying time points . at each time point the cells were lysed via scraping in 400 l of 10 mm hcl 
 and 400 l of hplc grade acetonitrile . the u937 cell line was 
 treated with 5 m of each compound and by two cycles of freeze 
 ( 80 c ) and thaw ( 37 c ) in 400 l of 10 mm 
 hcl and 400 l of hplc grade acetonitrile . to each lysate the precipitate 
 was removed by centrifugation at 12000 g for 15 min , 
 followed by syringe filtration of the supernatant and analysis by 
 lc / ms . the system used for analysis is an agilent 1200 hplc 
 instrument coupled with an agilent 6520 accurate - mass quadrupole time - of - flight 
 ( q - tof ) lc / ms / ms instrument . separations were performed 
 on a phenomenex luna column , c18 5 m , 2.1 mm  150 mm , 
 at a flow rate of 0.2 ml / min under h2o / acetonitrile/0.1% 
 formic acid gradient conditions . the 
 alkaline comet assay was performed 
 as described . transition state modeling of the 13glutathione 
 adduct in the gsta1 and gstp1 active 
 sites was carried out as described . briefly , 
 the initial models of the meisenheimer complex of compound 13 ( gs13 ) bound to gstp1 or gsta1 was 
 built on the basis of the gstcd in the gstp1gstcd structure ( pdb entry 1aqx ) or in the 
 gsta1gstcd model complex . the initial gsta1gstcd model complex 
 was built on the basis of the crystal structures of the gstcd found in the active sites of gstm1 ( pdb entry 4gst ) and gstp1 ( pdb 
 entry 1aqx ) , 
 and then it was docked into the active site of gsta1 in complex with 
 the gsh adduct of ethacrynic acid ( pdb entry 1gse ) . the gstp1gs13 and gsta1gs13 complexes built in dimeric forms because gsh interacts 
 with the side chains from both subunits of gst . the dimeric model 
 complexes were subject to geometry optimization using the macromodel 
 module of schrdinger , llc ( macromodel , version 9.6 ) . most of the primary antibodies 
 were from cell signaling technology ( danvers , ma ) , with the exception 
 of atf3 which was from santa cruz biotechnology ( santa cruz , ca ) . to analyze for glutathionylation of cellular proteins , lysates were 
 separated by gel electrophoresis ( 412% nupage gel ) under nonreducing 
 conditions , transferred to pvdf membranes , and probed with anti - glutathione 
 conjugated to protein monoclonal antibody ( virogen ) . human lung adenocarcinoma 
 cell line a549 was obtained from the american 
 type culture collection and cultured in rpmi 1640 medium with 10% 
 fetal bovine serum , according to the cell supplier s protocol , 
 for a maximum of four passages before use . cells were harvested at 
 7080% confluence , washed with phosphate buffered saline , suspended 
 in phosphate buffered saline , and implanted subcutaneously at 5  
 10 cells/0.2 ml into ncr nu / nu athymic mice , obtained 
 from charles river . frederick national laboratory for cancer research 
 is accredited by aaalac international and follows the public health 
 service policy for the care and use of laboratory animals . animal 
 care was provided in accordance with the procedures outlined in the 
  guide for care and use of laboratory animals ( national 
 research council , 1996 ; national academy press , washington , dc ) . when 
 the tumors reached approximately 3 mm  3 mm , the mice were distributed 
 randomly into groups of 12 for treatment . compounds were injected 
 into the tail veins of the mice , at 92 mol / kg body weight ( in 
 20 l of dmso solution ) , 2 times per week for 4 weeks . control 
 groups were treated with saline or dmso . tumor volumes in mm were 
 estimated by the formula ( /2  length 
  width ) . blood was collected under isoflurane anesthesia 
 into eppendorf tubes containing 50% acetonitrile/10 mm hcl for drug 
 metabolite analysis . all experiments ( with the exception 
 of the proliferation assay and in vivo study ) were performed at least 
 three times , each time at least in triplicate . statistical tests were carried out by using 
 instat , version 3.00 ( graphpad software inc . pairwise 
 comparisons included the t test , with the welch correction 
 or application of the mann significance 
 of correlations was assessed by the pearson linear correlation or 
 the spearman test as appropriate .
we 
 report the antitumor effects of nitric oxide ( no ) releasing 
 derivatives of the parp-1 inhibitor olaparib ( 1 ) . 
 compound 5b was prepared by coupling the carboxyl group of 3b and the free amino group of arylated diazeniumdiolated piperazine 4 . 
 analogue 5a has the same structure except 
 that the f is replaced by h. compound 13 is the same 
 as 5b except that a me2n 
 n(o)=no 
 group was added para and ortho to the nitro groups of the dinitrophenyl 
 ring . 
 the resulting prodrugs are activated by glutathione in a reaction 
 accelerated by glutathione s - transferase p1 ( gstp1 ) , an enzyme frequently 
 overexpressed in cancers . 
 this metabolism generates no plus a parp-1 
 inhibitor simultaneously , consuming reducing equivalents , leading 
 to dna damage concomitant with inhibition of dna repair , and in the 
 case of 13 inducing cross - linking glutathionylation of 
 proteins . 
 compounds 5b and 13 reduced the 
 growth rates of a549 human lung adenocarcinoma xenografts with no 
 evidence of systemic toxicity .