3.2.1. Cell Culture

Jurkat cells were maintained in RPMI 1640 supplemented with 10% FBS, 1% glutamax and 1% Penicillin/streptomycin. Cultures were maintained at 37 ◦C with 5% CO2, and split 2–3 times per week.

### 3.2.2. Intracellular ROS

Cells were resuspended in HBSS at 1 × 106 cells/mL and 100 <sup>μ</sup>L cells added to individual wells of a 96-well round bottom plate. DHR123 was added to the wells for a final concentration of 500 nM per well and cells incubated for 5 min to allow uptake of DHR123. Compounds were made up in DMSO to 10 mM for a stock solution. A ten-fold dilution series was made for each stock solution in HBSS from 1 mM to 10 nM and 10 μL of each added to triplicate wells, for final concentrations of 100 μM to 1 nM, along with corresponding DMSO controls. The assay was incubated at 37 ◦C for 30 min, and the cells were washed and resuspended in FACS buffer (PBS with 0.1% BSA and 0.2% sodium azide). Intracellular ROS was measured by flow cytometry analysis of DHR123 uptake.

#### 3.2.3. Jurkat Cell Cytotoxicity Assay

Cells were resuspended in cRPMI at 1 × 106 cells/mL and 100 <sup>μ</sup>L cells added to individual wells of a 96-well round bottom plate. Compounds were made up in DMSO to 10 mM for a stock solution. A ten-fold dilution series was made for each stock solution in cRPMI from 1 mM to 10 nM and 10 μL of each added to triplicate wells, for final concentrations of 100 μM to 1 nM, along with corresponding DMSO controls. The cells plus compounds were then incubated for 24 h at 37 ◦C. The cells were washed in ice-cold Annexin V binding buffer (200 mL PBS with 10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2). The cell pellets were labelled with 5 μL of Annexin V-FITC (BD Pharmingen) for 15 min at 4 ◦C. Cells were washed again with ice-cold Annexin V buffer and labelled with 10 μL of 5 μg/mL PI for 7 min at 4 ◦C. Cells were washed and resuspended in FACS buffer, and Annexin V/PI staining analysed using flow cytometry. Annexin V positive cells were undergoing apoptosis, while PI positive cells were necrotic. Double positive cells were dead, while double negative cells were live cells.

### 3.2.4. NCI Evaluation

Detailed protocols have been reported elsewhere [12].

## 3.2.5. Antimalarial and Anti-Farnesyltransferase Evaluation

Detailed protocols have been reported elsewhere [13,14].

### **4. Conclusions**

Our biological evaluation of members of a small library of compounds focused on the marine meroterpenoids thiaplidiaquinone A and B has revealed that the non-natural dioxothiazine regioisomers **3** and **4** tend to exhibit more potent biological activities against a number of cellular targets. An unexpected discovery was that the mechanism of Jurkat cell death was dominantly *via* necrosis for thiaplidiaquinone A but *via* apoptosis for the geranyl sidechain positional isomer thiaplidiaquinone B. The finding that the dioxothiazine regioisomers of the natural products exhibited more potent activities in a range of bioassays highlights the crucial role that total synthesis can play in the discovery of new bioactive scaffolds.

**Acknowledgments:** We acknowledge funding from the University of Auckland and Marcel Kaiser of Swiss Tropical and Public Health Institute for antimalarial data for compounds **5** and **6**.

**Author Contributions:** B.C., D.B., M.-L.B.-K. and J.H. conceived and designed the experiments; L.S., J.H., J.D. and A.V. performed the experiments; I.K. synthesized the compounds; J.H., M.-L.B.-K., J.D., A.V. and B.C. analyzed the data; and all authors contributed to writing the paper.

**Conflicts of Interest:** The authors declare no conflict of interest.
