New pleconaril and [(biphenyloxy)propyl]isoxazole derivatives with substitutions in the central ring exhibit antiviral activity against pleconaril-resistant coxsackievirus B3

doi:10.1016/j.antiviral.2008.09.002

Antiviral Research

Volume 81, Issue 1, January 2009, Pages 56-63

https://doi.org/10.1016/j.antiviral.2008.09.002 Get rights and content

Abstract

Amino acid 1092 (AA1092) in capsid protein 1 of coxsackievirus B3 (CVB3) is located in close vicinity to the central phenoxy group of capsid binders (i.e. pleconaril). Whereas isoleucine is associated with drug susceptibility, leucine and methionine confer resistance to pleconaril. In the present study, novel analogues with different substitutions in the central phenoxy group were synthesized to study their influence on anti-CVB3 activity with the aim to overcome pleconaril resistance.

Two [(biphenyloxy)propyl]isoxazoles and pleconaril were synthesized without methyl groups in the central phenoxy ring using Suzuki coupling reaction and tested for antiviral activity against the pleconaril-resistant CVB3 Nancy. Furthermore, pleconaril with 3-methyl, 3-methoxy, 3-bromine, 2,3-dimethyl in the central ring as well as the external rings in meta position were synthesized for structure–activity relationship analysis with CVB3 variants containing leucine, methionine or isoleucine at position 1092, other coxsackieviruses B (CVB) as well as several rhinoviruses.

The results demonstrate a high impact of substituents in the central ring of capsid inhibitors for anti-enteroviral activity. Pleconaril resistance of CVB3 based on Leu1092 or Met1092 was overcome by unsubstituted analogues or by monosubstitution with 3-methyl as well as 3-bromine in the central phenyl. The 3-bromine derivative inhibited a broad spectrum of CVB and rhinoviruses.

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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New pleconaril and [(biphenyloxy)propyl]isoxazole derivatives with substitutions in the central ring exhibit antiviral activity against pleconaril-resistant coxsackievirus B3

Abstract

Introduction

Section snippets

Chemistry

Synthesis and chemical characterization of new 3-[[3-(3-methyl-5-isoxazolyl)propyl]oxy]phenyl]-1,2,4-oxadiazoles and [(biphenyloxy)propyl]isoxazoles

Discussion

Acknowledgement

Antivir. Res.

Antivir. Res.

J. Mol. Biol.

Antivir. Res.

Antivir. Res.

Structure

Structure

Antivir. Res.

Virology

J. Virol. Methods

In vitro activity of pirodavir (R 77975), a substituted phenoxy-pyridazinamine with broad-spectrum antipicornaviral activity

Antimicrob. Agents Chemother.

Two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity

J. Virol.

Current status of anti-picornavirus therapies

Curr. Pharm. Des.

Mild regioselective halogenation of activated pyridines with N-bromosuccinimide

Synthesis

Selective inhibitors of picornavirus replication

Med. Res. Rev.

Antipicornavirus activity of tetrazole analogues related to disoxaril

J. Med. Chem.

Antipicornavirus compounds: use of rational drug design and molecular modelling

Antivir. Chem. Chemother.

Picornavirus inhibitors: trifluoromethyl substitution provides a global protective effect against hepatic metabolism

J. Med. Chem.