Heterocyclic rimantadine analogues with antiviral activity

2-(1-Adamantyl)-2-methyl-pyrrolidines 3 and 4, 2-(1-adamantyl)-2-methyl-azetidines 5 and 6, and 2-(1-adamantyl)-2-methyl-aziridines 7 and 8 were synthesized and tested for their antiviral activity against influenza A. Parent molecules 3, 5, and 7 contain the alpha-methyl-1-adamantan-methanamine 2 pharmacophoric moiety (rimantadine). The ring size effect on anti-influenza A activity was investigated. Pyrrolidine 3 was the most potent anti-influenza virus A compound, 9-fold more potent than rimantadine 2, 27-fold more potent than amantadine 1, and 22-fold more potent than ribavirin. Azetidines 5 and 6 were both markedly active against influenza A H2N2 virus, 10- to 20-fold more potent than amantadine. Aziridine 7 was almost devoid of any activity against H2N2 virus but exhibited borderline activity against H3N2 influenza A strain. Thus, it appears that changing the five-, to four- to a three-membered ring results in a drop of activity against influenza A virus.     

Comparisons of the influenza virus A M2 channel binding affinities, anti-influenza virus potencies and NMDA antagonistic activities of 2-alkyl-2-aminoadamantanes and analogues

The new 2-alkyl-2-aminoadamantanes and analogues 4-10 were designed and synthesized by simplification of the structure of the potent anti-influenza virus A spiranic aminoadamantane heterocycles 2 and 3. The aim of the present work was to examine the effects of bulky and extended lipophilic moieties attached to amantadine 1 on binding to the M2 channel and the resulting antiviral potency. The binding affinities of the compounds to the M2 protein of influenza virus A/chicken/Germany/27 (Weybridge strain; H7N7) were measured for the first time using an assay based on quenching of Trp-41 fluorescence by His-37 protonation, and their antiviral potencies were evaluated against the replication of influenza virus A H2N2 and H3N2 subtypes and influenza virus B in MDCK cells. Of the various 2-alkyl-2-aminoadamantanes, and analogues, spiro[piperidine-2,2'-adamantane] 3 had the strongest M2 binding and antiviral potency, which were similar those of amantadine 1. The relative binding affinities suggested that the rigid carbon framework provided by the pyrrolidine or piperidine rings results in a more favorable orientation inside the M2 channel pore as compared to large, freely rotating alkyl groups. The aminoadamantane derivatives exhibited similar NMDA antagonistic activity to amantadine 1. A striking finding was the antiviral activity of the adamantanols 4, and 6, which lack any NMDA antagonist activity.     

Novel 3-(2-adamantyl)pyrrolidines with potent activity against influenza A virus-identification of aminoadamantane derivatives bearing two pharmacophoric amine groups

The 3-(2-adamantyl)pyrrolidines 8a-g, 14 were synthesized and evaluated for activity against influenza A virus. The parent N-H compound 14 was several times more active than amantadine against H(2)N(2) and H(3)N(2) influenza A virus. The combined use of NMR spectroscopy and computational chemistry showed that the conformation around the pyrrolidine-adamantyl carbon-carbon bond is trans and the pyrrolidine heterocycle has an envelope conformation with C-2 out of the plane of the other ring atoms. N-Dialkylaminoethyl substitution of compound 14 resulted in the potent diamine analogues 8e,f,g. Interestingly, their lactam amine precursors were also active. Compounds 8e,f,g are the first adamantane derivatives, bearing two amine groups, reported to be active against influenza A virus.     

Synthesis and structure-activity relationships of novel camphecene analogues as anti-influenza agents

A chemical library was constructed based on the scaffold of camphecene (2-(E)-((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene-aminoethanol). The modifications included introduction of mono-and bicyclic heterocyclic moieties in place of the terminal hydroxyl group of camphecene. All compounds were tested for cytotoxicity and anti-viral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells. Among 15 tested compounds 11 demonstrated a selectivity index (SI) higher than 10 and IC₅₀ values in the micromolar range. The antiviral activity and toxicity were shown to strongly depend on the nature of the heterocyclic substituent. Compounds 2 and 14 demonstrated the highest virus-inhibiting activity with SIs of 106 and 183, and bearing pyrrolidine and piperidine moieties, correspondingly. Compound 14 was shown to interfere with viral reproduction at early stages of the viral life cycle (0–2 h post-infection). Taken together, our data suggest potential of camphecene derivatives in particular and camphor-based imine derivatives in general as effective anti-influenza compounds.     

Design and synthesis of 1,2-annulated adamantane piperidines with anti-influenza virus activity

1–2 Annulated adamantane piperidines 4, 6, 16, 17, 19, 23 and 25 were synthesized and evaluated for anti-influenza A virus activity. The stereoelectronic requirements for optimal antiviral potency were investigated. Piperidine 23 proved to be the most active of the compounds tested against influenza A virus, being 3.5-fold more active than amantadine, equipotent to rimantadine and 15-fold more potent than ribavirin. It is noteworthy that piperidine 23 displayed one of the highest selectivity indexes (SI > 732) among aminoadamantanes or other cage structure amines tested till now.     

Effects of Clinacanthus siamensis leaf extract on influenza virus infection

Ethanolic extracts of 20 medicinal plants were screened for influenza virus NA inhibition and in vitro antiviral activities using MDCK cells in an MTT assay. The vaccine proteins of influenza virus A/New Caledonia/20/99 (H1N1), mouse-adapted influenza virus A/Guizhou/54/89 (A/G)(H3N2) and mouse-adapted influenza virus B/Ibaraki/2/85 (B/I) were used in the NA inhibition assay, and mouse-adapted influenza viruses A/PR/8/34 (H1N1), A/G and B/I were used in the in vitro antiviral assay. The results of the in vitro antiviral assay indicated that the A/G virus was the most susceptible and an extract of the leaf of CS possessed the highest in vitro anti-A/G virus activity (41.98%). Therefore, the A/G virus and the CS extract were selected for studying in vivo anti-influenza virus activity. BALB/c mice were treated with CS extract (100 mg/kg per day, 5 times) orally from 4 hr before to 4 days after infection. CS extract elicited significant production of anti-influenza virus IgG₁ antibody in BAW and increased mouse weight compared to oseltamivir (0.1 mg/kg per day) on day 19 or water on days 17–19 of infection. Moreover, CS extract produced a higher anti-influenza virus IgA antibody level in BAW compared to oseltamivir, and a tendency towards an increase in anti-influenza virus IgA compared to water was shown. The results suggest that CS extract has a protective effect against influenza virus infection.     

Synthesis, conformational characteristics and anti-influenza virus A activity of some 2-adamantylsubstituted azacycles

The broad-spectrum antiviral activity of 2-(2-adamantyl)piperidines 11, 13a,b, and 15, 3-(2-adamantyl)pyrrolidines 27, 21a-g and 2-(2-adamantylmethyl)piperidines 30, 32a-c, and 35a-d was examined. Several compounds in the new series were potent against influenza A H(3)N(2) virus. When 1-aminoethyl pharmacophore group of 2-rimantadine 4 (2-isomer of rimantadine) is included into a saturated nitrogen heterocycle, see compound 11, potency was retained. The diamine derivatives 21e-g and particularly 35a-c possessing three pharmocophoric groups, that is, the adamantyl and the two amine groups, exhibited high potency. The new compounds did not afford specific activity at non-toxic concentrations against any of the other viruses tested. According to NMR spectroscopy and molecular mechanics calculations it is striking that the parent structures 11 and 27 adopt a fixed trans conformation around C2-C2' bond. In the parent amines, which proved to be active compounds, the distance between nitrogen and adamantyl pharmacophoric groups was different; N-C2' distance is 3.7, 3.8 A for 27, 30 and 2.5 A for 11 suggesting that M2 receptor site can accommodate different in size and orientation lipophilic cages.     

In Vivo Antiviral Properties of Biologically Active Compounds: II. Studies with Influenza and Vaccinia Viruses

The in vivo anti-influenza virus and antivaccinia virus activity of 156 biologically active compounds was determined. One of two criteria was used for evaluating activity against the influenza virus. The criteria were increase in survivor number and mean survival time, and reduction in virus-induced lung consolidation in treated, infected Swiss mice. Increase in survivor number and mean survival time were the criteria for evaluation of antivaccinia virus activity. Several drug doses were tested against two virus concentrations to demonstrate antiviral activity more clearly. Two compounds were considered significantly active against the influenza virus: DL-noformicin (NSC 72942) and amantadine hydrochloride (NSC 83653). Eleven compounds had reproducible activity against vaccinia virus: isatin-beta-thiosemicarbazone (NSC 721), 6-azauracil (NSC 3425), 9-alpha-fluoro-2alpha-methylhydrocortisone 21-acetate (NSC 12601), 5-[bis(2-chloroethyl)amino]uracil (NSC 34462), 5-iodo-2'-deoxyuridine (NSC 39661), streptonigrin (NSC 45383), N-methylisatin beta-thiosemicarbazone (NSC 69811), cytovirin (NSC 91770), 9-beta-D-arabinofuranosyladenine (NSC 404241), and 5-(mercaptomethyl)uracil (NSC 529351).     

Synthesis, antimicrobial, and anti-HIV-1 activity of certain 5-(1-adamantyl)-2-substituted thio-1,3,4-oxadiazoles and 5-(1-adamantyl)-3-substituted aminomethyl-1,3,4-oxadiazoline-2-thiones

The reaction of 5-(1-adamantyl)-1,3,4-oxadiazoline-2-thione 2 with iodoethane, 2-dimethylaminoethyl chloride hydrochloride or 2-piperidinoethyl chloride hydrochloride in ethanolic potassium hydroxide yielded the corresponding 5-(1-adamantyl)-2-ethyl or substituted ethylthio-1,3,4-oxadiazoles 3a-c. Interaction of 2 with formaldehyde solution and primary aromatic amines or 1-substituted piperazines, in ethanol at room temperature yielded the corresponding 5-(1-adamantyl)-3-arylaminomethyl-1,3,4-oxadiazoline-2-thiones 4a-m or 5-(1-adamantyl)-3-(4-substituted-1-piperazinylmethyl)-1,3,4-oxadiazoline-2-thiones 5a-h, respectively. All the synthesized compounds were tested for in vitro activities against certain strains of Gram-positive and Gram-negative bacteria and the yeast-like pathogenic fungus Candida albicans. Compounds 2, 5a, and 5e were found as the most active derivatives, particularly against the Gram-positive bacteria. In addition, the antiviral activity of compounds 2, 4a-m, and 5a-h against HIV-1 using the XTT assay was carried out. Compound 2 produced 100%, 43%, and 37% reduction of viral replication at 50, 10, and 2microg/mL concentrations, respectively.     

Abnormal humoral immune response to influenza vaccination in pediatric type-1 human immunodeficiency virus infected patients receiving highly active antiretroviral therapy

Given that highly active antiretroviral therapy (HAART) has been demonstrated useful to restore immune competence in type-1 human immunodeficiency virus (HIV-1)-infected subjects, we evaluated the specific antibody response to influenza vaccine in a cohort of HIV-1-infected children on HAART so as to analyze the quality of this immune response in patients under antiretroviral therapy. Sixteen HIV-1-infected children and 10 HIV-1 seronegative controls were immunized with a commercially available trivalent inactivated influenza vaccine containing the strains A/H1N1, A/H3N2, and B. Serum hemagglutinin inhibition (HI) antibody titers were determined for the three viral strains at the time of vaccination and 1 month later. Immunization induced a significantly increased humoral response against the three influenza virus strains in controls, and only against A/H3N2 in HIV-1-infected children. The comparison of post-vaccination HI titers between HIV-1+ patients and HIV-1 negative controls showed significantly higher HI titers against the three strains in controls. In addition, post vaccination protective HI titers (defined as equal to or higher than 1:40) against the strains A/H3N2 and B were observed in a lower proportion of HIV-1+ children than in controls, while a similar proportion of individuals from each group achieved protective HI titers against the A/H1N1 strain. The CD4+ T cell count, CD4/CD8 T cells ratio, and serum viral load were not affected by influenza virus vaccination when pre- vs post-vaccination values were compared. These findings suggest that despite the fact that HAART is efficient in controlling HIV-1 replication and in increasing CD4+ T cell count in HIV-1-infected children, restoration of immune competence and response to cognate antigens remain incomplete, indicating that additional therapeutic strategies are required to achieve a full reconstitution of immune functions.     

Synthesis and evaluation of diterpenic Mannich bases as antiviral agents against influenza A and SARS-CoV-2

A chemical library was constructed based on the resin acids (abietic, dehydroabietic, and 12-formylabietic) and its diene adducts (maleopimaric and quinopimaric acid derivatives). The one-pot three-component CuCl-catalyzed aminomethylation of the abietane diterpenoid propargyl derivatives was carried out by formaldehyde and secondary amines (diethylamine, pyrrolidine, morpholine, and homopiperazine). All compounds were tested for cytotoxicity and antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells and SARS-CoV-2 pseudovirus in BHK-21-hACE2 cells. Among 21 tested compounds, six derivatives demonstrated a selectivity index (SI) higher than 10, and their IC₅₀ values ranged from 0.19 to 5.0 μM. Moreover, two derivatives exhibited potent anti-SARS-CoV-2 infection activity. The antiviral activity and toxicity strongly depended on the nature of the diterpene core and heterocyclic substituent. Compounds 12 and 21 bearing pyrrolidine moieties demonstrated the highest virus-inhibiting activity with SIs of 128.6 and 146.8, respectively, and appeared to be most effective when added at the time points 0–10 and 1–10 h of the viral life cycle. Molecular docking and dynamics modeling were adopted to investigate the binding mode of compound 12 into the binding pocket of influenza A virus M2 protein. Compound 9 with a pyrrolidine group at C20 of 17-formylabietic acid was a promising anti-SARS-CoV-2 agent with an EC₅₀ of 10.97 µM and a good SI value > 18.2. Collectively, our data suggested the potency of diterpenic Mannich bases as effective anti-influenza and anti-COVID-19 compounds.     

Andrographolide inhibits influenza A virus-induced inflammation in a murine model through NF-κB and JAK-STAT signaling pathway

Influenza viruses, the main cause of respiratory tract diseases, cause high morbidity and mortality in humans. Excessive inflammation in the lungs is proposed to be a hallmark for the severe influenza virus infection, especially influenza A virus infection. Strategies against inflammation induced by influenza A virus infection could be a potential anti-influenza therapy. Here, lethal dose of mouse-adapted H1N1 strain PR8A/PR/8/34 was inoculated C57BL/6 mice to detect the anti-influenza activity of andrographolide, the active component of traditional Chinese medicinal herb Andrographis paniculata, with or without influenza virus entry inhibitor CL-385319. Treatment was initiated on 4 days after infection. The survival rate, body weight, lung pathology, viral loads, cytokine expression were monitored in 14 days post inoculation. The combination group had the highest survival rate. Andrographolide treatment could increase the survival rate, diminish lung pathology, decrease the virus loads and the inflammatory cytokines expression induced by infection. Mechanism studies showed the NF-κB and JAK-STAT signaling pathway were involved in the activity of andrographolide. In conclusion, combination of virus entry inhibitor with immunomodulator might be a promising therapeutic approach for influenza.     

Synthesis of 2-(2-adamantyl)piperidines and structure anti-influenza virus A activity relationship study using a combination of NMR spectroscopy and molecular modeling

The 2-(2-adamantyl)piperidines 13 and 15a-c were synthesized and evaluated for anti-influenza virus A and B activity. The parent N-H compound 13 was 3–4 times more active than amantadine and rimantadine against H₂N₂ influenza A. N-alkylation of 13 resulted in derivatives 15a-c that were devoid of biological activity. This dramatic reduction in biological activity may be attributed to the different conformational properties between N-H and N-alkyl piperidines, as deduced from the combination of computational chemistry and NMR spectroscopy.     

Synthesis and biological evaluation of novel 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of novel S-DABO analogues of 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were compounds 6c1,6c6, and 6b1 (EC(50)=0.24 ± 0.05, 0.38 ± 0.13, 0.39 ± 0.05 μM, respectively), which possess improved or similar HIV-1 inhibitory activity compared with nevirapine (NVP) (EC(50)=0.21 μM) and delavirdine (DLV) (EC(50)=0.32 μM). None of these compounds were active against HIV-2 replication. Furthermore, enzyme inhibitory assays were performed with selected derivatives against HIV-1 wtRT, confirming that the main target of these compounds is the HIV-1 RT and these new S-DABOs are acting as NNRTIs. The preliminary structure-activity relationship (SAR) of these new congeners is discussed briefly and rationalized by docking studies.     

Influenza virus partially counteracts restriction imposed by tetherin/BST-2

Influenza virus infections lead to a burst of type I interferon (IFN) in the human respiratory tract, which most probably accounts for a rapid control of the virus. Although in mice, IFN-induced Mx1 factor mediates a major part of this response, the situation is less clear in humans. Interestingly, a recently identified IFN-induced cellular protein, tetherin (also known as CD317, BST-2, or HM1.24), exerts potent antiviral activity against a broad range of retroviruses, as well as several other enveloped viruses, by impeding the release of newly generated viral particles from the cell surface. Here we show that influenza virus belongs to the targets of this potent antiviral factor. Ectopic expression of tetherin strongly inhibited fully replicative influenza virus. In addition, depleting endogenous tetherin increased viral production of influenza virions, both in cells constitutively expressing tetherin and upon its induction by IFN. We further demonstrate, by biochemical and morphological means, that tetherin exerts its antiviral action by tethering newly budded viral particles, a mechanism similar to the one that operates against HIV-1. In addition, we determined that the magnitude of tetherin antiviral activity is comparable with or higher than the one of several previously identified anti-influenza cellular factors, such as MxA, ADAR1, ISG15, and viperin. Finally, we demonstrate that influenza virus reduces the impact of tetherin-mediated restriction on its replication by several mechanisms. First, the influenza virus NS1 protein impedes IFN-mediated tetherin induction. Second, influenza infection leads to a decrease of tetherin steady state levels, and the neuraminidase surface protein partly counteracts its activity. Overall, our study helps to delineate the intricate molecular battle taking place between influenza virus and its host cells.     

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model

Background

Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.

Principal Findings

We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection.

Conclusion

A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.     

Dihydromyricetin is a new inhibitor of influenza polymerase PB2 subunit and influenza-induced inflammation

Development of new and effective anti-influenza drugs is critical for the treatment of influenza virus infection. The polymerase basic 2 (PB2) subunit as a core subunit of influenza A virus RNA polymerase complex is considered to be an attractive drug target for anti-influenza drug discovery. Dihydromyricetin, as a natural flavonoid, has a wide range of biological activities, but its anti-influenza A virus activity is ambiguous. Here, we found dihydromyricetin could inhibit the replication of a variety of influenza A virus strains. Mechanism studies demonstrated that dihydromyricetin reduced viral polymerase activity via selective inhibition of viral PB2 subunit, and decreased relative amounts of viral mRNA and genomic RNA during influenza A virus infection. The binding affinity and molecular docking analyses revealed that dihydromyricetin interacted with the PB2 cap-binding pocket, functioned as a cap-binding competitor. Interestingly, dihydromyricetin also reduced cellular immune injury by inhibiting TLR3 signaling pathway. Additionally, combination treatment of dihydromyricetin with zanamivir exerted a synergistic anti-influenza effect. Altogether, our experiments reveal the antiviral and anti-inflammatory activities of dihydromyricetin in vitro against influenza virus infection, which provides a new insight into the development of novel anti-influenza drugs.     

Spiro[pyrrolidine-2,2'-adamantanes]: synthesis,anti-influenza virus activity and conformational properties

Synthetic spiro[pyrrolidine-2,2'-adamantanes] 2, 3, 11, 15, 12, 16, 18, 20 were evaluated in vitro and found to be active anti-influenza virus A compounds; the effect of the position of C-Me pyrrolidine ring substituent on antiviral activity was examined. Pyrrolidine 5-Me substitution appears to be optimal for H(2)N(2) strain activity. From the four different possible protonated conformers, experimental observation using NMR spectroscopy and molecular mechanics calculations demonstrated only a pair of conformers A(+)H (N-Me (ps-ax), C-Me (ps-eq)) and B(+)H ((N-Me ps-ax, C-Me ps-ax)) which can contribute to the biological activity of C-Me, N-Me protonated derivatives 15(+)H, 16(+)H and 20(+)H. The relative populations were calculated from NMR spectra. For compounds 15(+)H and 20(+)H conformer A(+)H (cis dimethyl orientation) is the major one whereas a similar population of conformers A(+)H and B(+)H (trans dimethyl orientation) was observed for compound 16(+)H. Since this new series is characterized by a lipophilic part, that is the pyrrolidine ring, in addition to adamantane, that can interact with influenza A M2 protein, an ultimate future goal would be the in vitro mapping of M2 lipophilic pocket.     

Anti-influenza diarylheptanoids from the bark of Alnus japonica

This study to investigate anti-influenza components from the bark of Alnus japonica resulted in the isolation of two rare acylated diarylheptanoids, named oregonoyl A (5) and oregonoyl B (6), along with nine known compounds (1–4 and 7–11). Their structures were elucidated on the basis of extensive spectroscopic and chemical methods. Antiviral testing of compounds 1–11 against KBNP-0028 (H9N2) avian influenza virus showed that platyphyllone (10) was strongly active, and platyphyllonol-5-xylopyranoside (9) was moderately active against KBNP-0028 as compared with the positive control, zanamivir, respectively.