Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogues has revolved around the synthesis of novel adamantane derivatives, we have recently found that other polycyclic scaffolds effectively block the M2 proton channel, including amantadine-resistant mutant channels. In this work, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogues of amantadine. Inhibition of the wild-type M2 channel and the A/M2-S31N, A/M2-V27A, and A/M2-L26F mutant forms of the channel were measured in Xenopus oocytes using two-electrode voltage clamp assays. Most of the novel compounds inhibited the wild-type ion channel in the low micromolar range. Of note, two of the compounds inhibited the amantadine-resistant A/M2-V27A and A/M2-L26F mutant ion channels with submicromolar and low micromolar IC50, respectively. None of the compounds was found to inhibit the S31N mutant ion channel.

We have synthesized and characterized a series of compounds containing the 3-azatetracyclo[5.2.1.15,8.01,5]undecane scaffold designed as analogs of amantadine, an inhibitor of the M2 proton channel of influenza A virus. Inhibition of the wild-type (wt) M2 channel and the amantadine-resistant A/M2-S31N and A/M2-V27A mutant ion channels were measured in Xenopus oocytes using two-electrode voltage clamp (TEV) assays. Most of the novel compounds inhibited the wt ion channel in the low micromolar range. Of note, several compounds inhibited the A/M2 V27A mutant ion channel, one of them with submicromolar IC50. None of the compounds was found to inhibit the S31N mutant ion channel. The antiviral activity of three novel dual wt and A/M2-V27A channels inhibitors was confirmed by influenza virus yield assays.

SummaryThe synthesis of phenylene-ethynylene rods and their use as rigid spacers is described. Alternation of a Sonogashira reaction and silyl group cleavage was used to obtain rigid spacers with even and odd numbers of phenylene units. Preliminary applications of these rods in divalent systems are shown. Inhibition studies with Pseudomonas Aeruginosa lectin LecA showed that the rigid spacer proved greatly beneficial for the inhibitory potency.

The synthesis of several [4,4,3], [4,3,3], and [3,3,3]azapropellanes is reported. Several of the novel amines displayed low-micromolar activities against an amantadine-resistant H1N1 strain, but they did not show activity against an amantadine-sensitive H3N2 strain. None of the tested compounds inhibit the influenza A/M2 proton channel function. Most of the compounds did not show cytotoxicity for MDCK cells. KEYWORDS: Amantadine, influenza, M2 channel, hemagglutinin, propellane H uman influenza A virus, a member of the Orthomyxoviridae, causes significant morbidity and mortality, particularly in infants, elderly people, and those suffering from previous pathology or immunodeficiency. 1 Its capacity to cause widespread epidemics is related to its fast droplet transmission and ability to escape from existing immunity.The life cycle of influenza viruses is well documented and most viral proteins are regarded as potential therapeutic targets. 2−6 However, presently available drugs for the treatment of influenza virus infections only comprise the M2 ion channel blockers amantadine (Amt) and rimantadine (Chart 1), 7,8 and the neuraminidase inhibitors oseltamivir, zanamivir, peramivir, and laninamivir. 5 Taking into account that most of the currently circulating influenza strains are resistant to the M2 ion channel blockers and as resistance to the neuraminidase inhibitors (in particular oseltamivir) is also rising, 9,10 novel antiinfluenza virus drugs, preferably with a novel mechanism of action, are urgently needed.The influenza virus enters its target cells by receptormediated endocytosis, which is followed by acid-induced fusion of the viral and endosomal membranes. This fusion event is mediated by a conformational change of the influenza hemagglutinin (HA) protein, triggered by the low pH in the endosomal lumen. 11 The endosomal acidic pH also activates the membrane-spanning A/M2 protein, which acts as a proton channel to conduct protons into the virion interior. The decrease in intravirion pH results in uncoating of the viral ribonucleoprotein, which is transferred, through the fusion pore, to the cytoplasm. 12 Since this endosomal escape of the virus critically depends on the activity of the M2 proton channel and the HA protein, dually acting agents which combine blockade of the M2 channel with an inhibitory effect on HA refolding, appear highly attractive. This dual approach could also, at least in theory, increase the barrier for selecting Amt resistance. 13 Optimized Amt analogues might be able to exert this dual pharmacological effect, provided that both the M2 and HA inhibition occur at similar and clinically relevant compound

Two new polycyclic scaffolds were synthesized and evaluated as anti-influenza A compounds. The 5-azapentacyclo[6.4.0.0(2,10).0(3,7).0(9,11)]dodecane derivatives were only active against the wild-type M2 channel in the low-micromolar range. However, some of the 14-azaheptacyclo[8.6.1.0(2,5).0(3,11).0(4,9).0(6,17).0(12,16)]heptadecane derivatives were dual inhibitors of the wild-type and the V27A mutant M2 channels. The antiviral activity of these molecules was confirmed by cell culture assays. Their binding mode was analysed through molecular dynamics simulations, which showed the existence of distinct binding modes in the wild type M2 channel and its V27A variant.