Abstract 1260 Site-Specific Isopropylation of Parent Molecule As a Way to Develop Artemisinin Long-Acting Antimalarial and AntiCytomegalovirus Formulation

Objective: A sesquiterpene endoperoxide lactone artemisinin (AM) isolated from the plant Artemisia annua, has become first-line natural antimalarial drug highly efficient in areas of multidrug resistance (https://doi.org/10.1002/anie.201601967). AM was also shown inhibiting replication of human cytomegalovirus (CMV) in vitro and in vivo (https://doi.org/10.1016/j.antiviral.2005.10.003). AM is characterized by a short half-life. To prevent development of the resistant malaria, a AM-based combination therapy is widely utilized, which however is attributed to much lower efficacy of AM. A unique 1,2,4-trioxane ring is essential for activity of the parent substrate, as all known AM metabolites were inactive due to the endoperoxide bridge has been reduced to an epoxide. It was reaffirmed, both in vivo and in human liver microsomes that AM to be principally metabolized by CYP2B6 monooxygenase isozyme (https://doi.org/10.1046/j.1365-2125.1999.00044.x). Previously, under similar circumstances of highly potent parent drugs being demethylated by CYP2B6 into non-active products, we had developed site-directed modification of a parent molecule by substituting its methyl groups with isopropyl groups (https://doi.org/10.1007/BF03189961). The modification creates steric hindrance at CYP2B6 active site thus affecting ligand binding and lowering catalysis. Such strategy was applied in this study. Materials and Methods: While AM is biosynthesized in Artemisia annual plant extracts from mevalonic acid via dimethylallyl and isopentenyl pyrophosphates, here C-2 dimethylallyl groups at pyrophosphate were substituted with isopropylallyl groups. Isopropylartemisidin (IPAM) molecule appears to retain an intact 1,2,4-trioxane ring incorporating an endoperoxide bridge determined by a specific LC-MS/MS assay. The IPAM was incubated with human liver microsomes from eight different healthy donors, and concentration-time data were assessed by a first-order depletion model. Also, the hydroxylated and glucuronyl-conjugated derivatives of covalent heme-AM and heme-IPAM adducts were determined in urine of Plasmodium infected mice by means of LC-MS. The human monocytic line THP-1 (ATCC), passages 20-40, was used for infections with pp28-luciferase Towne CMV or the GCV-resistant strain (moi = 1). Thereafter, media containing virus was removed and replaced by DMEM with 4% FBS containing anti-viral compounds. In treated with AM or IPAN THP-1 cells (as well as in THP-1-derived macrophages) the CMV and GCV strain virus production and viral DNA replication were measured by a plague assay and dot blot hybridization. Results: With correlation to the metabolic rate constants for CYP2B6 probe substrates, efavirenz and bupropion, our data revealed that IPAM was metabolized by human CYP2B6 at least 12-13 times slower than AM. Also, the amounts of covalent heme-drug adducts were identically high in infected mice treated with 40 nM AM or 40 nM IPAM, compared to mice treated with 40 nM AM metabolites lacking trioxane ring endoperoxide bridge. We measured the inhibition of GCV-resistant strain by AM and IPAM using the PRNT50 value, and found that IPAM completely inhibited plague formation at 0.5 mM while the parent AM and GCV both showed ∼ 40% plague reduction even at 5.0 mM. Conclusion: Isopropylartemisinin appears to represent a new potent antimalarial and antiCMV drug that can inhibit, respectively, multiple stages of the malaria parasite's life cycle, and production of the resistant strain of human herpesvirus.