*2.2. Biology and Structure-Activity Relationships*

All compounds (**10**–**17**) were tested for *in vitro* antiplasmodial activity against chloroquine-sensitive (3D7) and multidrug-resistant (Dd2) *P. falciparum* parasite lines, and for mammalian toxicity using human neonatal foreskin fibroblast (NFF) cells. The simple indolequinones, **10** and **17**, were essentially inactive (Table 1), highlighting the importance of the 1,1-dioxo-thiazine subunit for antiplasmodial activity. The original thiaplakortone report [1] showed that unsaturation of the thiazine ring conferred enhanced antiplasmodial activity. This trend was also observed in the side-chain truncated compounds, with the unsaturated compounds, **15** and **16** displaying greater potency than their saturated counterparts, **13** and **14**, respectively. Specifically, exchanging the saturated system of **13** and **14** with the unsaturated motif present in **15** and **16** increased 3D7 activity by 23.9- and 27.3-fold, respectively; a similar SAR trend was also observed for this series towards the Dd2 line.


**Table 1.** Biological Data for Compounds **10**–**17**.

*<sup>a</sup>* 3D7 = *P. falciparum* chloroquine-sensitive line; *<sup>b</sup>* Dd2 = *P. falciparum* multidrug-resistant line; *<sup>c</sup>* NFF = neonatal foreskin fibroblast cells; *<sup>d</sup>* SI = selectivity index = NFF cell-line IC50/*P. falciparum* IC50; *<sup>e</sup>* CQ = chloroquine (positive control).

The regiochemistry of the thiazine moiety in the original thiaplakortone report was shown to have minimal influence on the overall antiplasmodial activity and selectivity [1]. Reversal of the thiazine orientation in thiaplakortones C (**3**) and D (**4**) only showed an antiplasmodial activity increase of 1.1-fold towards both the 3D7 and Dd2 lines. In a similar manner to the earlier report, the current studies showed minimal differences in parasite potency between the side-chain truncated regioisomeric pairs, **11** and **12**, **13** and **14**, and **15** and **16**. However, when comparing NFF toxicity of the regioisomers (**11** *vs.* **12**; **13** *vs.* **14**; **15** *vs.* **16**) a clear trend was observed, with the thiazine regiochemistry present in **12**, **14** and **16** conveying reduced cytotoxicity ranging from 1.6- to 13.6-fold. Furthermore, the reduction in toxicity improved the selectivity indices for analogues **12**, **14** and **16**.

Biological data for compounds **11** and **12** identified that *N*-tosylation of the pyrrole moiety is well tolerated and improves antiparasitic activity, even in the absence of thiazine unsaturation. It is interesting to note that, consistent with the thiaplakortone natural products [1], the most active side-chain truncated analogues, **15** and **16**, are more potent against the drug-resistant line (Dd2) than the chloroquine-sensitive line (3D7). While it is clear that the ethylamine side-chain present in thiaplakortones A and B translates to more potent and selective antiplasmodial agents, the current study shows that the tricyclic core motif present in **11**–**16** represents a minimum antiplasmodial pharmacophore for the thiaplakortone chemotype.

In order to assess the drug-like properties of compounds **10**–**17**, *in silico* physicochemical parameters (Table 2) were calculated using ChemAxon MarvinSketch software (with calculator plugins) (http://www.chemaxon.com) and the data compared to Lipinski's drug-like "Rule of Five" [10]. All compounds complied with Lipinski's rules (LogP < 5, HBA < 10, HBD < 5, MW < 500). In addition, all compounds had relatively low LogD7.4 values (except compound **10**), and had appropriate polar surface area (PSA) values for membrane penetration.


**Table 2.** *In silico* physicochemical parameters for compounds **<sup>10</sup>**−**<sup>17</sup>** *<sup>a</sup>*.

*<sup>a</sup> In silico* calculations performed using ChemAxon MarvinSketch software (with calculator plugins). MW = molecular weight (Da); HBA = H-bond acceptors; HBD = H-bond donors; PSA = polar surface area.
