*6.2. Artemisinin and Derivatives*

Testing compounds that have anecdotal evidence suggesting that they may have antiviral activity often revolves around natural products. Various natural products have been reported to have anti-HCMV activity linked to inhibition of IE expression or function, but most remain largely unsubstantiated beyond initial observations. However, a considerable body of evidence has been generated with respect to the anti-HCMV activity of natural product artemisinin, its semi-synthetic derivative artesunate and various related compounds. Artemisinin is a natural product derived from the plant *Artemisia annua* (Sweet Wormwood), a herb used in traditional Chinese medicine [317,318]. Artemisinin and its derivatives are best known for effective antimalarial activity and treatment [317,318], which provided the premise for testing artesunate for anti-HCMV activity [319]. Artesunate, along with various related compounds, exhibit in vitro inhibitory activity against laboratory, clinical and drug-resistant strains of HCMV in a range of cell types with EC50 values generally in the low micromolar to sub-micromolar range [319–329]. Chemically linking artemisinin-related molecules into dimers and trimers significantly improves antiviral potency [320,324–326,330,331]. Examples include artemisinin-derived dimer diphenyl phosphate (838), a potent, selective HCMV inhibitor with irreversible activity [332,333] and trimeric artesunate derivative TF27 [326], which exhibits potent in vitro and in vivo activity in the MCMV model [329]. Hybridization of artemisinin-derivatives with bioactive molecules, such as quinazoline, has produced novel compounds with potent anti-HCMV activity significantly better than parental compounds and GCV [334–337].

The mechanism of action by which artesunate and the various derivatives generate their anti-HCMV activity has not been fully elucidated, but the general consensus is that artesunate primarily interferes with the NF-κB pathway [319,326,331]. The NF-κB pathway is stimulated upon HCMV infection and activates the MIEP, driving expression of IE proteins and hence subsequent steps in HCMV lytic replication and pathogenesis [179,338,339]. Indeed, artesunate, along with many derivatives, has been shown to block the IE phase of HCMV replication via a reduction in expression levels of IE2, and to a lesser extent IE1 [319,320,326,328,331,332]. Artesunate, and dimer/trimer derivatives such as TF27, have been shown to interfere with the NF-κB pathway, which is proposed to occur via a direct interaction of the compound with NF-κB subunit RelA/p65 [319,326,331]. Interaction of artesunate with a host cell factor leads to the expectation that acquisition of drug resistance would be less likely; indeed, attempts to generate artesunate drug-resistant isolates in vitro have thus far been unsuccessful [326,340]. Alternative modes of action, implicating other cell signalling pathways and modulation of cell cycle progression, have also been proposed for artesunate compounds [319,340].

Clinical use of artesunate for management of drug-resistant HCMV infections in stem cell or solid organ transplant recipients is considered feasible due the documented anti-HCMV activity discussed above, favourable results in a rodent animal model study [341] and the long and safe clinical history of artesunate treatment in malaria patients [317]. The first use of artesunate in a clinical setting was a success with artesunate being reported as an effective inhibitor of HCMV replication in the treated patient [342]. However, subsequent studies reported either mixed success or that artesunate was ineffective in controlling HCMV infection [343–345]. Further studies are required to fully determine the differences in clinical outcomes for artesunate-treated patients, and studies with more potent artesunate derivatives may hold future promise. For example, the trimeric derivative TF27 has recently been demonstrated to display antiviral efficacy in the mouse model. MCMV replication was significantly reduced and restricted to the site of infection, preventing organ dissemination without adverse effects [329].
