Interrogation of the Structure–Activity Relationship of a Lipophilic Nitroaromatic Prodrug Series Designed for Cancer Gene Therapy Applications

Abstract

PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned >2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.

Design and Biological Evaluation of Piperazine-Bearing Nitrobenzamide Hypoxia/GDEPT Prodrugs: The Discovery of CP-506

“…In contrast, under anoxic conditions (95% N 2 , 5% CO 2 ), a significant increase in cell death was observed across the analogue series, with LCK values ranging from 2.3 to 3.7. Unsurprisingly, LCK values were highest for the most lipophilic compounds ( 16 , 17 , and 20 ), consistent with our previous observations that increasing LogD 7.4 correlated with increasing LCK at tissuelike cell densities . Overall, these data indicated that all compounds in the dibromo and bromo/mesylate mustard analogue series were hypoxia-selective.…”

“…We recently reported a novel analogue of PR-104A, termed SN29176 ( 3 ) (Figure ), that was designed to be resistant to off-target activation by human AKR1C3 while retaining hypoxia selectivity in vitro and in vivo when administered as the pre-prodrug SN35141 ( 4 ) . In addition, we explored a series of analogues of SN29176 of varying lipophilicity and identified prodrug 5 (and its pre-prodrug 6 ) (Figure ) as a preferred hypoxia/GDEPT prodrug with an optimized bystander effect (the ability of cytotoxic metabolites to diffuse into and sterilize metabolically naïve neighboring cells) . While elimination of AKR1C3 activation is a significant advance, prodrugs 3 and 5 still contain an alcohol side chain and only achieve sufficient aqueous solubility for in vivo administration through use of a phosphate pre-prodrug strategy, rendering them susceptible to rapid clearance through the glucuronidation pathway.…”

“…We previously compared and contrasted two alternate routes in the preparation of prodrug 5 and analogues . Similar methods were employed in the synthesis of the current compounds.…”

“…Chlorine to bromine halogen exchange using lithium bromide in 3-methyl-2-butanone at reflux then gave the dibromo compounds 17 and 18 , respectively. An improved route to the remaining compounds involved conversion of bismesylates 7 and 9 to their acid chlorides using oxalyl chloride in the presence of MgO to avoid undesired displacement of the mesylate groups with chloride ions. Coupling of the freshly prepared acid chlorides with various alkylpiperazines in situ then gave the bismesylate carboxamides ( 10 , 13 – 15 ).…”

“…We then determined the hypoxia-selective cytotoxicity of the analogue series in HCT116 three-dimensional MCLs, where the bystander effect of compounds may play a more significant role (Figure ). Following exposure to 100 μM drug, cell death was minimal under hyperoxic conditions (95% O 2 , 5% CO 2 ), with LCK values ranging from 0.016 to 0.38.…”