(NRH):quinone oxidoreductase 2 (NQO2) is associated with various processes involved in cancer initiation and progression probably via the production of ROS during quinone metabolism. Thus, there is a need to develop inhibitors of NQO2 that are active in vitro and in vivo. As part of a strategy to achieve this we have used the 4-aminoquinoline backbone as a starting point and synthesized 21 novel analogues. The syntheses utilised p-anisidine with Meldrum's acid and trimethyl orthoacetate or trimethyl orthobenzoate to give the 4-hydrazin-quinoline scaffold, which was derivatised with aldehydes or acid chlorides to give hydrazone or hydrazide analogues, respectively. The hydrazones were the most potent inhibitors of NQO2 in cell free systems, some with low nano-molar IC50 values. Structure-activity analysis highlighted the importance of a small substituent at the 2-position of the 4-aminoquinoline ring, to reduce steric hindrance and improve engagement of the scaffold within the NQO2 active site. Cytotoxicity and NQO2-inhibitory activity in vitro was evaluated using ovarian cancer SKOV-3 and TOV-112 cells (expressing high and low levels of NQO2, respectively). Generally, the hydrazones were more toxic than hydrazide analogues and further, toxicity is unrelated to cellular NQO2 activity. Pharmacological inhibition of NQO2 in cells was measured using the toxicity of CB1954 as a surrogate end-point. Both the hydrazone and hydrazide derivatives are functionally active as inhibitors of NQO2 in the cells, but at different inhibitory potency levels. In particular, 4-((2-(6-methoxy-2-methylquinolin-4-yl)hydrazono)methyl)phenol has the greatest potency of any compound yet evaluated (53 nM), which is 50-fold lower than its toxicity IC50. This compound and some of its analogues could serve as useful pharmacological probes to determine the functional role of NQO2 in cancer development and response to therapy.

NRH:quinone oxidoreductase 2 enzyme (NQO2) is a potential therapeutic target in cancer and neurodegenerative diseases, with roles in either chemoprevention or chemotherapy. Here we report the design, synthesis and evaluation of non-symmetrical furan-amidines and their analogues as novel selective NQO2 inhibitors with reduced adverse off-target effects, such as binding to DNA. A pathway for the synthesis of the non-symmetrical furan-amidines was established from the corresponding 1,4-diketones. The synthesized non-symmetrical furan-amidines and their analogues showed potent NQO2 inhibition activity with nano-molar IC50 values. The most active compounds were non-symmetrical furan-amidines with meta- and para-nitro substitution on the aromatic ring, with IC50 values of 15 nM. In contrast to the symmetric furan-amidines, which showed potent intercalation in the minor grooves of DNA, the synthesized non-symmetrical furan-amidines showed no affinity towards DNA, as demonstrated by DNA melting temperature experiments. In addition, Plasmodium parasites, which possess their own quinone oxidoreductase PfNDH2, were inhibited by the non-symmetrical furan-amidines, the most active possessing a para-fluoro substituent (IC50 9.6 nM). The high NQO2 inhibition activity and nanomolar antimalarial effect of some of these analogues suggest the lead compounds are worthy of further development and optimization as potential drugs for novel anti-cancer and antimalarial strategies.