Design, synthesis and antiviral evaluation of novel conjugates of the 1,7,7-trimethylbicyclo[2.2.1]heptane scaffold and saturated N-heterocycles via 1,2,3-triazole linker.

A new series of heterocyclic derivatives with a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment was designed, synthesised and biologically evaluated. Synthesis of the target compounds was performed using the Cu(I) catalysed cycloaddition reaction. The key starting substances in the click reaction were an alkyne containing a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment and a series of azides with saturated nitrogen-containing heterocycles. Some of the derivatives were found to exhibit strong antiviral activity against Marburg and Ebola pseudotype viruses. Lysosomal trapping assays revealed the derivatives to possess lysosomotropic properties. The molecular modelling study demonstrated the binding affinity between the compounds investigated and the possible active site to be mainly due to hydrophobic interactions. Thus, combining a natural hydrophobic structural fragment and a lysosome-targetable heterocycle may be an effective strategy for designing antiviral agents.

Novel O-acylated amidoximes and substituted 1,2,4-oxadiazoles synthesised from (+)-ketopinic acid possessing potent virus-inhibiting activity against phylogenetically distinct influenza A viruses.

This article describes the synthesis and antiviral activity evaluation of new substituted 1,2,4-oxadiazoles containing a bicyclic substituent at position 5 of the heterocycle and O-acylated amidoximes as precursors for their synthesis. New compounds were obtained from the (+)-camphor derivative (+)-ketopinic acid. The chemical library was tested in vitro for cytotoxicity against the MDCK cell line and for antiviral activity against influenza viruses of H1N1 and H7N9 subtypes. The synthesised compounds exhibited high virus-inhibiting activity against the H1N1 influenza virus. Some synthesised compounds were also active against the influenza virus of a different antigenic subtype: H7N9. The mechanism of the virus-inhibiting activity of these compounds is based on their interference with the fusion activity of viral hemagglutinin (HA). No interference with the receptor-binding activity of HA has been demonstrated. According to molecular docking results, the selective antiviral activity of O-acylated amidoximes and 1,2,4-oxadiazoles is associated with their structural features. O-Acylated amidoximes are likely more complementary to the binding site located at the site of the fusion peptide, and 1,2,4-oxadiazoles are more complimentary to the site located at the site of proteolysis. Significant differences in the amino acid residues of the binding sites of HA's of different types allow us to explain the selective antiviral activity of the compounds under study.

Triterpenic Acid Amides as Potential Inhibitors of the SARS-CoV-2 Main Protease.

Although the incidence and mortality of SARS-CoV-2 infection has been declining during the pandemic, the problem related to designing novel antiviral drugs that could effectively resist viruses in the future remains relevant. As part of our continued search for chemical compounds that are capable of exerting an antiviral effect against the SARS-CoV-2 virus, we studied the ability of triterpenic acid amides to inhibit the SARS-CoV-2 main protease. Molecular modeling suggested that the compounds are able to bind to the active site of the main protease via non-covalent interactions. The FRET-based enzyme assay was used to reveal that compounds 1e and 1b can inhibit the SARS-CoV-2 main protease at micromolar concentrations.

Synthesis and Antiviral Properties of Camphor-Derived Iminothiazolidine-4-Ones and 2,3-Dihydrothiazoles.

A set of heterocyclic products was synthesized from natural (+)-camphor and semi-synthetic (-)-camphor. Then, 2-Imino-4-thiazolidinones and 2,3-dihydrothiazoles were obtained using a three-step procedure. For the synthesized compounds, their antiviral activity against the vaccinia virus and Marburg virus was studied. New promising agents active against both viruses were found among the tested compounds.

Synthesis and In Vitro Study of Antiviral Activity of Glycyrrhizin Nicotinate Derivatives against HIV-1 Pseudoviruses and SARS-CoV-2 Viruses.

When developing drugs against SARS-CoV-2, it is important to consider the characteristics of patients with different co-morbidities. People infected with HIV-1 are a particularly vulnerable group, as they may be at a higher risk than the general population of contracting COVID-19 with clinical complications. For such patients, drugs with a broad spectrum of antiviral activity are of paramount importance. Glycyrrhizinic acid (Glyc) and its derivatives are promising biologically active compounds for the development of such broad-spectrum antiviral agents. In this work, derivatives of Glyc obtained by acylation with nicotinic acid were investigated. The resulting preparation, Glycyvir, is a multi-component mixture containing mainly mono-, di-, tri- and tetranicotinates. The composition of Glycyvir was characterized by HPLC-MS/MS and its toxicity assessed in cell culture. Antiviral activity against three strains of SARS-CoV-2 was tested in vitro on Vero E6 cells by MTT assay. Glycyvir was shown to inhibit SARS-CoV-2 replication in vitro (IC502-8 µM) with an antiviral activity comparable to the control drug Remdesivir. In addition, Glycyvir exhibited marked inhibitory activity against HIV pseudoviruses of subtypes B, A6 and the recombinant form CRF63_02A (IC50 range 3.9-27.5 µM). The time-dependence of Glycyvir inhibitory activity on HIV pseudovirus infection of TZM-bl cells suggested that the compound interfered with virus entry into the target cell. Glycyvir is a promising candidate as an agent with low toxicity and a broad spectrum of antiviral action.

New class of hantaan virus inhibitors based on conjugation of the isoindole fragment to (+)-camphor or (-)-fenchone hydrazonesv.

This work presents the design and synthesis of camphor, fenchone, and norcamphor N-acylhydrazone derivatives as a new class of inhibitors of the Hantaan virus, which causes haemorrhagic fever with renal syndrome (HFRS). A cytopathic model was developed for testing chemotherapeutics against the Hantaan virus, strain 76-118. In addition, a study of the antiviral activity was carried out using a pseudoviral system. It was found that the hit compound possesses significant activity (IC50 = 7.6 ± 2 µM) along with low toxicity (CC50 > 1000 µM). Using molecular docking procedures, the binding with Hantavirus nucleoprotein was evaluated and the correlation between the structure of the synthesised compounds and the antiviral activity was established.

Quaternary ammonium salts based on (-)-borneol as effective inhibitors of influenza virus.

A series of compounds containing a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment were evaluated for their antiviral activity against influenza A virus strain A/Puerto Rico/8/34 (H1N1) in vitro. The most potent antiviral compound proved to be a quaternary ammonium salt based on (-)-borneol, 10a. In in vitro experiments, compound 10a inhibited influenza A viruses (H1, H1pdm09, and H3 subtypes), with an IC50 value of 2.4-16.8 µM (depending on the virus), and demonstrated low toxicity (CC50 = 1311 µM). Mechanism-of-action studies for compound 10a revealed it to be most effective when added at the early stages of the viral life cycle. In direct haemolysis inhibition tests, compound 10a was shown to decrease the membrane-disrupting activity of influenza A virus strain A/Puerto Rico/8/34. According to molecular modelling results, the lead compound 10a can bind to different sites in the stem region of the viral hemagglutinin.

(+)-Camphor and (-)-borneol derivatives as potential anti-orthopoxvirus agents.

Although the World Health Organisation had announced that smallpox was eradicated over 40 years ago, the disease and other related pathogenic poxviruses such as monkeypox remain potential bioterrorist weapons and could also re-emerge as natural infections. We have previously reported (+)-camphor and (-)-borneol derivatives with an antiviral activity against the vaccinia virus. This virus is similar to the variola virus (VARV), the causative agent of smallpox, but can be studied at BSL-2 facilities. In the present study, we evaluated the antiviral activity of the most potent compounds against VARV, cowpox virus, and ectromelia virus (ECTV). Among the compounds tested, 4-bromo-N'-((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide 18 is the most effective compound against various orthopoxviruses, including VARV, with an EC50 value of 13.9 µM and a selectivity index of 206. Also, (+)-camphor thiosemicarbazone 9 was found to be active against VARV and ECTV.

Discovery of New Ginsenol-Like Compounds with High Antiviral Activity.

A number of framework amides with a ginsenol backbone have been synthesized using the Ritter reaction. We named the acetamide as Ginsamide. A method was developed for the synthesis of the corresponding amine and thioacetamide. The new compounds revealed a high activity against H1N1 influenza, which was confirmed using an animal model. Biological experiments were performed to determine the mechanism of action of the new agents, a ginsamide-resistant strain of influenza virus was obtained, and the pathogenicity of the resistant strain and the control strain was studied. It was shown that the emergence of resistance to Ginsamide was accompanied by a reduction in the pathogenicity of the influenza virus.

Synthesis and Antiviral Activity of Camphene Derivatives against Different Types of Viruses.

To date, the 'one bug-one drug' approach to antiviral drug development cannot effectively respond to the constant threat posed by an increasing diversity of viruses causing outbreaks of viral infections that turn out to be pathogenic for humans. Evidently, there is an urgent need for new strategies to develop efficient antiviral agents with broad-spectrum activities. In this paper, we identified camphene derivatives that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses, including influenza virus A/PR/8/34 (H1N1), Ebola virus (EBOV), and the Hantaan virus. The lead-compound 2a, with pyrrolidine cycle in its structure, displayed antiviral activity against influenza virus (IC50 = 45.3 µM), Ebola pseudotype viruses (IC50 = 0.12 µM), and authentic EBOV (IC50 = 18.3 µM), as well as against pseudoviruses with Hantaan virus Gn-Gc glycoprotein (IC50 = 9.1 µM). The results of antiviral activity studies using pseudotype viruses and molecular modeling suggest that surface proteins of the viruses required for the fusion process between viral and cellular membranes are the likely target of compound 2a. The key structural fragments responsible for efficient binding are the bicyclic natural framework and the nitrogen atom. These data encourage us to conduct further investigations using bicyclic monoterpenoids as a scaffold for the rational design of membrane-fusion targeting inhibitors.

Single-stage synthesis of heterocyclic alkaloid-like compounds from (+)-camphoric acid and their antiviral activity.

An effective technique for one-stage synthesis of new polycyclic nitrogen-containing compounds has been developed. The procedure involves refluxing mixtures of camphoric acid with aliphatic or aromatic diamine without catalysts. In cases where the starting amine has a low boiling point (less than 200 °C), phenol is used as a solvent, as it is the most optimal one for obtaining products with good yields. It has been shown that the use of Lewis acids as catalysts reduces the yield of the reaction products. A set of compounds have been synthesized, which can be attributed to synthetic analogues of alkaloids. In vitro screening for activity influenza virus A was carried out for the obtained compounds. The synthesized quinazoline-like agent 14 has inhibitory activity against different strains of influenza viruses.

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.

Synthesis of Camphecene and Cytisine Conjugates Using Click Chemistry Methodology and Study of Their Antiviral Activity.

A series of camphecene and quinolizidine alkaloid (-)-cytisine conjugates has been obtained for the first time using 'click' chemistry methodology. The cytotoxicity and virus-inhibiting activity of compounds were determined against MDCK cells and influenza virus A/Puerto Rico/8/34 (H1N1), correspondingly, in in vitro tests. Based on the results obtained, values of 50 % cytotoxic dose (CC50 ), 50 % inhibition dose (IC50 ) and selectivity index (SI) were determined for each compound. It has been shown that the antiviral activity is affected by the length and nature of linkers between cytisine and camphor units. Conjugate 13 ((1R,5S)-3-(6-{4-[(2-{(E)-[(1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene]amino}ethoxy)methyl]-1H-1,2,3-triazol-1-yl}hexyl)-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyrido[1,2-a][1,5]diazocin-8-one), which contains cytisine fragment separated from triazole ring by -C6 H12 - aliphatic linker, showed the highest activity at relatively low toxicity (CC50 =168 µmol, IC50 =8 µmol, SI=20). Its selectivity index appeared higher than that of reference compound, rimantadine. According to theoretical calculations, the antiviral activity of the lead compound 13 can be explained by its influence on the functioning of neuraminidase.

Discovery of a New Class of Inhibitors of Vaccinia Virus Based on (-)-Borneol from Abies sibirica and (+)-Camphor.

A series of the bornyl ester/amide derivatives with N-containing heterocycles were designed and synthesized as vaccinia virus (VV) inhibitors. Bioassay results showed that among the designed compounds, derivatives 6, 13, 14, 34, 36 and 37 showed the best inhibitory activity against VV with the IC50 values of 12.9, 17.9, 3.4, 2.5, 12.5 and 7.5 µm, respectively, and good cytotoxicity. The primary structure-activity relationship (SAR) study suggested that the combination of a saturated N-heterocycle, such as morpholine or 4-methylpiperidine, and a 1,7,7-trimethylbicyclo[2.2.1]heptane scaffold was favorable for antiviral activity.

Synthesis of camphecene derivatives using click chemistry methodology and study of their antiviral activity.

A series of seventeen tetrazole derivatives of 1,7,7-trimethyl-[2.2.1]bicycloheptane were synthesized using click chemistry methodology and characterized by spectral data. Studies of cytotoxicity and in vitro antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells of the compounds obtained were performed. The structure-activity relationship analysis suggests that to possess virus-inhibiting activity, the compounds of this group should bear oxygen atom with a short linker (C2-C4), either as a hydroxyl group (18, 19, 29), keto-group (21) or as a part of a heterocycle (24). These compounds demonstrated low cytotoxicity along with high anti-viral activity.

(+)-fenchol and (-)-isopinocampheol derivatives targeting the entry process of filoviruses.

The increasing frequency of filovirus outbreaks in African countries has led to a pressing need for the development of effective antifilovirus agents. In continuation of our previous research on the antifilovirus activity of monoterpenoid derivatives, we synthesized a series of (+)-fenchol and (-)-isopinocampheol derivatives by varying the type of heterocycle and linker length. Derivatives with an N-alkylpiperazine cycle proved to be the most potent antiviral compounds, with half-maximal inhibitory concentration (IC50) 1.4-20 µÐœ against Lenti-EboV-GP infection and 11.3-47 µÐœ against Lenti-MarV-GP infection. Mechanism-of-action experiments revealed that the compounds may exert their action by binding to surface glycoproteins (GPs). It was demonstrated that the binding of the synthesized compounds to the Marburg virus GP is less efficient as compared to the Ebola virus GP. Furthermore, it was shown that the compounds possess lysosomotropic properties. Thus, the antiviral activity may be due to dual effects. This study offers new antiviral agents that are worthy of further exploration.

The Potential of Usnic-Acid-Based Thiazolo-Thiophenes as Inhibitors of the Main Protease of SARS-CoV-2 Viruses.

Although the COVID-19 pandemic caused by SARS-CoV-2 viruses is officially over, the search for new effective agents with activity against a wide range of coronaviruses is still an important task for medical chemists and virologists. We synthesized a series of thiazolo-thiophenes based on (+)- and (-)-usnic acid and studied their ability to inhibit the main protease of SARS-CoV-2. Substances containing unsubstituted thiophene groups or methyl- or bromo-substituted thiophene moieties showed moderate activity. Derivatives containing nitro substituents in the thiophene heterocycle-just as pure (+)- and (-)-usnic acids-showed no anti-3CLpro activity. Kinetic parameters of the most active compound, (+)-3e, were investigated, and molecular modeling of the possible interaction of the new thiazolo-thiophenes with the active site of the main protease was carried out. We evaluated the binding energies of the ligand and protein in a ligand-protein complex. Active compound (+)-3e was found to bind with minimum free energy; the binding of inactive compound (+)-3g is characterized by higher values of minimum free energy; the positioning of pure (+)-usnic acid proved to be unstable and is accompanied by the formation of intermolecular contacts with many amino acids of the catalytic binding site. Thus, the molecular dynamics results were consistent with the experimental data. In an in vitro antiviral assay against six strains (Wuhan, Delta, and four Omicron sublineages) of SARS-CoV-2, (+)-3e demonstrated pronounced antiviral activity against all the strains.

Molecular Modeling of Viral Type I Fusion Proteins: Inhibitors of Influenza Virus Hemagglutinin and the Spike Protein of Coronavirus.

The fusion of viral and cell membranes is one of the basic processes in the life cycles of viruses. A number of enveloped viruses confer fusion of the viral envelope and the cell membrane using surface viral fusion proteins. Their conformational rearrangements lead to the unification of lipid bilayers of cell membranes and viral envelopes and the formation of fusion pores through which the viral genome enters the cytoplasm of the cell. A deep understanding of all the stages of conformational transitions preceding the fusion of viral and cell membranes is necessary for the development of specific inhibitors of viral reproduction. This review systematizes knowledge about the results of molecular modeling aimed at finding and explaining the mechanisms of antiviral activity of entry inhibitors. The first section of this review describes types of viral fusion proteins and is followed by a comparison of the structural features of class I fusion proteins, namely influenza virus hemagglutinin and the S-protein of the human coronavirus.

Development of an LC-MS/MS-based method for quantification and pharmacokinetics study on SCID mice of a dehydroabietylamine-adamantylamine conjugate, a promising inhibitor of the DNA repair enzyme.

Earlier, it was found that the agent KS-389, a conjugate of dehydroabietylamine and 1-aminoadamantane, possess inhibiting activity with regard to Tdp1. It this study, LC-MS/MS-based methods of quantification of KS-389 in mice blood and several organs (brain, liver and kidney) were developed and validated. Validation of the methods was performed according to the guidelines of U.S. Food and Drug Administration and European Medicines Agency in terms of selectivity, linearity, accuracy, precision, recovery, matrix effect, stability and carry-over. Dried blood spots (DBS) method was used for blood sample preparation. HPLC separation was performed on a reversed-phase column; the total analysis time was 12 min. Mass spectral detection was performed on a 6500 QTRAP mass spectrometer in multiple reaction monitoring mode. Transitions 463.5→135.1/107.2 and 336.2→332.2/176.2 were scanned for KS-389 and 2,5-bis(4-diethylaminophenyl)-1,3,4-oxadiazole used as the internal standard, respectively. Pharmacokinetics of the compound as well as its distribution in the organs were studied on SCID mice after intraperitoneal administration of the substance at a dose of 5 mg/kg, and it was found that its maximum concentration in blood is reached in 1-1.5 h and was 80 ng/mL. The maximum concentration in all organs is reached after the same time and is approximately 1500 ng/g and 1100 ng/g in liver and kidney, respectively. This is the first report on the pharmacokinetics of Tdp1 inhibitor based on dehydroabietylamine and 1-aminoadamantane after a single administration to mice. Also, the substance was found to be able to penetrate the blood-brain barrier which is important for, and its maximum concentration was c.a. 25-30 ng/g. These results are important for glioma treatment and make it promising for this purpose.

The main protease 3CLpro of the SARS-CoV-2 virus: how to turn an enemy into a helper.

Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists.