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Seeking the Elusive Long-Acting Ozonide: Discovery of Artefenomel (OZ439) Ho Shin Kim, Jared T. Hammill, and R. Kiplin Guy* Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40508, United States
ABSTRACT: The majority of frontline therapies for the treatment of malaria are combination drugs containing artemisinin (or its semisynthetic analogs), known as artemisinin combination therapies (ACTs). While generally efficacious, ACTs and the first generation fully synthetic ozonide, arterolane (OZ277, 1), suffer from rapid clearance requiring 3-day dosing regimens. Extensive structure−activity studies led to the discovery of a second-generation ozonide, artefenomel (OZ439, 2), which has overcome this limitation, maintaining the rapid onset of action and potent activity of the artemisinin derivatives while exhibiting greatly improved pharmacokinetics, low projected cost of goods, prophylactic activity, and the potential for a single dose cure.
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stages of the erythrocytic life cycle including rings, trophozoites, and schizonts. However, ACTs suffer from some notable disadvantages. First, the ARTs uniformly possess short in vivo half-lives that drive the requirement for a 3-day dosing schedule.5 Second, isolation from the sweet wormwood plant is still the only practical means of accessing ART starting material, rendering both drug supply and cost variable. Finally, resistance to ACTs is emerging in Southeast Asia. Thus, there is an urgent need to discover and develop new drugs that can overcome these issues.3 Upon the basis of the MMV product definition,5 new drugs should be effective as single-dose treatments, overcome all existing resistance, be active against multiple life cycle stages, exhibit prophylactic activity, be cheap to produce, and have a high therapeutic ratio, especially with respect to children and pregnant woman. Previous efforts by the authors led to the discovery of a novel synthetic ozonide, arterolane (OZ277, 1),6,7 which is approved as a three-dose combination therapy with piperaquine for uncomplicated malaria. Compound 1 is structurally simpler than the ARTs, possesses superior antimalarial activity, has reliable manufacture methods, and has an improved biopharmaceutical profile. However, 1 has only a marginally improved half-life (t1/2 = 3 h) in humans. Compound 1 is oxidized by cytochrome P450 enzymes to form inactive adamatane-hydroxyl metabolites and also decomposed by Fe(II) in blood to form the adamantanelactone and cyclohexanone as cleavage products.7 These mechanisms directly inactivate the key pharmacophore for the antimalarial activity of compound 1. In the new work, the
n this issue of the Journal of Medicinal Chemistry, Dong et al. describe the structure−activity relationship (SAR) and structure−property relationship (SPR) studies that led to the development of artefenomel (OZ439, 2).1 These studies represent the medicinal chemistry component of an integrated interdisciplinary project carried out by a team composed of academic research groups, industry affiliates, and the Medicines for Malaria Venture (MMV) public−private partnership.2 The SAR and SPR studies that led to 2 represent an excellent example of how state-of-the-art medicinal chemistry can play off pharmacokinetic optimization, producing higher and prolonged plasma exposure, with mechanistic pharmacophore based design to translate into improved clinical outcomes. The effort also illustrates that the combination of improved plasma exposure and in vitro efficacy does not always straightforwardly predict in vivo efficacy. Malaria, a mosquito-borne infectious disease, is a leading cause of morbidity and mortality worldwide, with the World Health Organization (WHO) estimating 212 million new clinical episodes and more than 400 000 deaths caused by malaria in 2015.3 The current recommended treatment for malaria utilizes ACTs. Artemisinin (ART) and its derivatives (ARTs) contain an endo-peroxide pharmacophore that is critical to their antimalarial activity. Although the precise mechanism of action is not fully understood, evidence suggests that the endo-peroxide undergoes reductive activation by the Fe(II) in heme to generate carbon-centered free radicals, which alkylate heme and other proteins, ultimately leading to downstream effects that kill the parasites.4 Artemisinin derivatives share unique pharmacological advantages including extremely rapid action, activity against Plasmodium strains resistant to classic antimalarials, and activity against all asexual © 2017 American Chemical Society
Received: February 22, 2017 Published: March 15, 2017 2651
DOI: 10.1021/acs.jmedchem.7b00299 J. Med. Chem. 2017, 60, 2651−2653
Journal of Medicinal Chemistry
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Figure 1. Optimization of artefenomel analogs. A. General synthetic route used to prepare artefenomel analogs. B. Representative compounds from structure−activity relationship (SAR) studies of three groups of amino substituents. (ain vitro intrinsic clearance measured in human liver microsomes; values in parentheses represent intrinsic clearance in mouse, rat, and dog liver microsomes, respectively. ND = not determined.).
The SAR revealed by these studies can be broken into three trends based on whether the aromatic ring substituent contains a primary, secondary, or tertiary amine. Both primary and secondary amino substituents showed better metabolic stability than the tertiary amines. Introducing additional polar groups on the primary amine’s linker reduced in vivo efficacy. Among primary amino substituents, compounds 10 and 11 (Figure 1B) achieved the best results including potent and curative activity in vivo and prolonged exposure. Most of the secondary amino substituents exhibited good metabolic stability, high aqueous solubility, prolonged exposure, and in vivo curative efficacy. The best secondary amines (16, 17) had the highest log D7.4 value (∼3.3). In contrast to the primary amino substituents, additional polar groups did not affect the in vivo efficacy of the secondary amino substituents. Among the tertiary amines, four- and five-membered heterocylic rings showed no in vivo curative efficacy. Among the six-membered ring subseries, the methane sulfonyl piperazine, morpholine, and thiomorpholine 1-oxide showed
authors sought to reduce metabolism by driving the conformation of the cyclohexyl linker from the reactive equatorial to the more stable axial orientation. Indeed, switching from the alkyl cyclohexyl to the aryl drove the conformational equilibrium in the axial direction and increased the blood stability of their compounds without detriment to antimalarial efficacy. Final optimization of the aryl substituents afforded the highly potent and longer-acting 2 with significantly improved blood stability (t1/2 = 46−60 h).2 The compounds made during the SAR study were tested for in vitro activity against P. falciparum, aqueous solubility, and stability in human liver microsomes. All of the tested analogs proved to be potent (EC50 values of 0.8−12 nM). All compounds were also tested for in vivo efficacy using P. berghei infected mice. Selected compounds were profiled for plasma exposure following a single oral dose of 30 mg/kg. Finally, the best compounds were tested for in vivo prophylactic activity and toxicity. 2652
DOI: 10.1021/acs.jmedchem.7b00299 J. Med. Chem. 2017, 60, 2651−2653
Journal of Medicinal Chemistry
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vivo curative efficacy; (3) tertiary amino substituents were less toxic than primary or secondary amino substituents. However, the correlations between performance in the in vitro and in vivo assays were poor and not easily understood, hindering rationalization of the factors driving the performance of the best candidate. Ultimately, these efforts addressed the key halflife issue of the first generation synthetic ozonide 1 and yielded a potent second generation analog 2 with improved pharmacokinetics, low projected cost of goods, prophylactic activity, and the potential for a single-dose cure.
the best in vivo curative efficacy. Although all of the curative compounds containing tertiary amino substituents possessed poor aqueous solubility (99% on day 3, there was no simple correlation between in vitro and in vivo potency. Similarly, there were poor correlations between physiochemical properties and exposure. The authors did observe an overall trend that compounds with higher pKa and lower log D7.4 exhibited superior metabolic stability, but neither parameter directly correlated with oral exposure or efficacy. Overall, the compounds with the best in vivo efficacy (4/5 to 5/5 cures) showed 2−3 times higher plasma exposure than the compounds with low efficacy (0/5 to 3/5 cures). This result is consistent with the original design hypothesis that stabilization of peroxide bond via conformational changes should inhibit rapid clearance and increase in vivo curative efficacy. Multiple high dose exploratory toxicology studies (5 doses of 100 or 300 mg/kg) were carried out. The primary amine series (3, 4, 10, 11) showed significant signs of toxicities at higher doses (300 mg/kg). Additionally, both the primary amine 10 and secondary amine 19 displayed significant mortality (9/12, 3/12 rats respectively). In contrast to the primary and secondary amino substituents, the tertiary amines (2, 33, 38) showed only minor toxicities. Among the tested compounds, tertiary amino compound 2 exhibited the lowest toxicity. Among the analogs described, 2 exhibited the best pharmacological profile. Compound 2 is curative for P. berghei infected mice following a single oral dose (30 mg/kg) and also possesses prophylactic activity, superior to the current clinical benchmark mefloquine. Finally, multiple dose toxicology studies (up to 300 mg/kg doses) revealed only minimal adverse effects. These results suggest that 2 may be efficacious as a single-dose combination treatment, a substantial improvement over the previous generation, which required multi-dose treatments. Additionally, the synthetic ozonide derivatives were prepared using Griesbaum coozonolysis providing a simple, scalable route for synthesis that should help minimize manufacturing costs (Figure 1A). In summary, understanding the mechanism of action and degradation of synthetic ozonides led to a rational approach to stabilize the key peroxide in the consensus pharmacophore, thus improving plasma exposure. Systematic investigation of the SAR about the aryl ring substituent revealed three general trends: (1) increasing pKa and lowering log D7.4 reduced intrinsic clearance; (2) increasing plasma exposure improved in
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
R. Kiplin Guy: 0000-0002-9638-2060 Notes
The authors declare no competing financial interest.
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REFERENCES
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DOI: 10.1021/acs.jmedchem.7b00299 J. Med. Chem. 2017, 60, 2651−2653
