2.2.3. *In Vitro* Cytotoxicity Screening at the NCI

With the discovery that **1**/**3** and **2**/**4** exhibit contrasting mechanisms of cell death towards Jurkat cells, the set of **1**–**6** were further screened for *in vitro* antitumor activity at the National Cancer Institute [10]. In one dose (10 μM) testing, dioxothiazine regioisomers **3** and **4** exhibited more potent panel average cell kill than the natural products **1** and **2**, with precursor quinones **5** and **6** being found to be the least active (see Supplementary Materials). Selectivity towards melanoma cell lines, in particular MDA-MB-435 (all compounds) and MALME-3M (**3** and **4**), was observed. More comprehensive 5-dose testing of **1**–**4** identified **3**, the regioisomer of thiaplidiaquinone A, to be the more active compound, showing good activity towards MDA-MB-435 (LC50 0.66 μM, TGI 0.18 μM, GI50 0.052 μM) and MALME-3M (LC50 3.4 μM, TGI 0.60 μM, GI50 0.10 μM) cell lines (see Supplementary Materials). This compound has been selected for further *in vivo* evaluation. Our findings that non-natural dioxothiazine regioisomers can exhibit more potent cytotoxicity to tumour cells than the corresponding natural product is in agreement with results observed by Aiello *et al*., as part of their SAR studies of the dioxothiazine-ring containing ascidian natural product aplidinone A [11]. Their study investigated the effect of variation in lipophilic sidechain structure and dioxothiazine ring regiochemistry on a range of biological activities. They identified one structurally simplified analogue of the natural product containing the non-natural dioxothiazine ring regiochemistry that exhibited enhanced cytotoxicity towards a number of tumour cell lines as well as enhanced inhibition of TNFα-induced Nf-κB activation.
