New pleconaril and [(biphenyloxy)propyl]isoxazole derivatives with substitutions in the central ring exhibit antiviral activity against pleconaril-resistant coxsackievirus B3
Introduction
Coxsackievirus B3 (CVB3) belongs to the genus enterovirus of the family Picornaviridae (Racaniello, 2007). Like other enteroviruses CVB3 causes a broad spectrum of acute and chronic human diseases including respiratory infections, meningitis, encephalitis, pancreatitis and myocarditis (Melnick, 1996). Up to now, no antiviral agents that are active against enteroviruses are available for clinical use. Besides inhibition of viral RNA and protein synthesis, interference with virus attachment and uncoating is a very promising mechanism to prevent and treat enterovirus-induced diseases (Barnard, 2006, De Palma et al., 2008, Patick, 2006). According to the most recent review published by De Palma et al. (2008), the rhinovirus protease inhibitor rupintrivir reached clinical trials but was recently halted from further development and Biota’s HRV drug BTA-798 is scheduled for phase II trials in 2008. BTA-798 is structurally related to pirodavir that binds to the viral capsid (Andries et al., 1992). Compounds binding to the viral capsid, the so-called capsid function inhibitors block virus adsorption, entry and/or uncoating. The most advanced capsid function inhibitor in clinical trials is pleconaril. It exhibits a potent and highly specific in vitro activity against various serotypes of enteroviruses (Pevear et al., 1999). Pleconaril also inhibits a broad spectrum of rhinoviruses in cell culture (Kaiser et al., 2000, Ledford et al., 2005). However, oral pleconaril was not approved by the FDA for the treatment of common cold in adults, primarily based on drug interactions resulting from the activation of cytochrome P-450 3A enzymes (Ma et al., 2006) and marginal treatment effects (Hayden et al., 2002, Hayden et al., 2003). In 2007, a phase II clinical study with a pleconaril nasal spray for treatment of common cold symptoms and asthma exacerbations following rhinovirus exposure was completed (De Palma et al., 2008). The results have not yet been reported.
Moreover, it was shown that naturally resistant viruses may exist within an enterovirus serotype and that resistant mutants may emerge rapidly during pleconaril treatment in vitro. For example, the prototype strain CVB3 Nancy often used in antiviral studies as well as CVB3 Nancy variants are naturally pleconaril-resistant (Pevear et al., 1999, Schmidtke et al., 2005). Pleconaril-resistant CVB3 mutants were easily selected in vitro (Groarke and Pevear, 1999). The frequency of resistance to pleconaril in the wild type population was ∼5 × 10−5. CVB3 Nancy as well as in vitro selected resistant mutants carry the amino acid substitution Ile1092 → Leu or Ile1092 → Met in the hydrophobic pocket of viral capsid protein 1. Using recombinant viruses it could be demonstrated that a single amino acid substitution Ile1092 → Leu may induce complete pleconaril resistance (Schmidtke et al., 2005). In vitro selected pleconaril-resistant variants (Leu1092 or Met1092) showed attenuated virulence in a murine model (Groarke and Pevear, 1999). In contrast, the pleconaril-resistant CVB3 31-1-93 (Leu1092) is highly virulent in mice, infects the murine heart and causes chronic myocarditis (Schmidtke et al., 2000, Schmidtke et al., 2007). The possibility of transmission of resistant viruses cannot be excluded. From this perspective, there is a high medical need for improved antiviral agents for either the prevention or therapy of enteroviral diseases.
The correlation between amino acid 1092 in the center of the hydrophobic pocket of viral capsid protein VP1 and pleconaril susceptibility of CVB3 isolates and laboratory strains (Schmidtke et al., 2005) as well as the close vicinity of amino acid 1092 to the central ring of capsid binders (Muckelbauer et al., 1995) suggest an important role of substituents in the central phenyl of these antiviral drugs for anti-CVB3 activity. Methyl groups of capsid binders could be important for compound binding by hydrophobic interactions with the Ile1092 of pleconaril-sensitive CVB3. Alternatively, they may sterically hinder the positioning of capsid binders into the hydrophobic pocket of pleconaril-resistant CVB3 variants (Leu1092 or Met1092). Therefore, pleconaril and [(biphenyloxy)propyl]isoxazoles lacking methyl groups in the central phenyl were synthesized. Furthermore, monosubstitution with 3-methyl as well as with substituents varying in space size or lipophilicity were carried out to acquire new insights into the role of the central part and the strength of its interaction with the hydrophobic pocket of CVB3. After determining cytotoxicity, antiviral assays were performed with 3 pleconaril-resistant (Leu1092 and Met1092) as well 3 pleconaril-sensitive CVB3 variants (Ile1092) to determine the impact of individual substitutions for drug susceptibility and to discover active compounds. The antiviral spectrum of monosubstituted pleconaril analogues was determined with other CVB types and various human rhinoviruses.
Section snippets
Chemistry
The reference compound pleconaril, as well as the pleconaril derivatives and biphenyl derivatives were synthesized by Dr. Makarov (RAS Institute of Biochemistry, Moscow, Russia). The method of synthesis was the same as described for the pleconaril analogues (Fig. 1). All reagents and solvents were purchased from commercial suppliers and used without further purification. Melting points were determined on Electrothermal 9001 and are uncorrected. 1H and 13C NMR were measured at 400 MHz on Varian
Synthesis and chemical characterization of new 3-[[3-(3-methyl-5-isoxazolyl)propyl]oxy]phenyl]-1,2,4-oxadiazoles and [(biphenyloxy)propyl]isoxazoles
Several phenyl substituted analogues were synthesized for structure–activity relationship analysis with the aim to clarify the role of substituents of the central phenyl of capsid binders towards inhibition of CVB3 and to discover more active analogues that exhibit antiviral activity against pleconaril-susceptible as well as -resistant virus strains. The method of pleconaril synthesis was used as described previously (Diana et al., 1995). Some modifications were introduced for the preparation
Discussion
Capsid binders like pleconaril represent highly potential antiviral drugs for treatment of enterovirus infections. But as shown for pleconaril, a single amino acid substitution in the drug-binding pocket of entero- as well as rhinoviruses may reduce or even completely abolish the antiviral activity of this class of compounds (Groarke and Pevear, 1999, Ledford et al., 2004, Ledford et al., 2005, Schmidtke et al., 2005). This was further confirmed for the mouse-adapted CVB3 31-1-93 as well as
Acknowledgement
We thank B. Jahn for excellent technical assistance.
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