Glycyrrhizic acid conjugates with amino acid methyl esters target the main protease, exhibiting antiviral activity against wild-type and nirmatrelvir-resistant SARS-CoV-2 variants.

COVID-19 pandemic is predominantly caused by SARS-CoV-2, with its main protease, Mpro, playing a pivotal role in viral replication and serving as a potential target for inhibiting different variants. In this study, potent Mpro inhibitors were identified from glycyrrhizic acid (GL) derivatives with amino acid methyl/ethyl esters. Out of the 17 derivatives semisynthesized, Compounds 2, 6, 9, and 15, with methionine methyl esters, D-tyrosine methyl esters, glutamic acid methyl esters, and methionines in the carbohydrate moiety, respectively, significantly inhibited wild-type SARS-CoV-2 Mpro-mediated proteolysis, with IC50 values ranging from 0.06 µM to 0.84 µM. They also demonstrated efficacy in inhibiting trans-cleavage by mutant Mpro variants (Mpro_P132H, Mpro_E166V, Mpro_P168A, Mpro_Q189I), with IC50 values ranging from 0.05 to 0.92 µM, surpassing nirmatrelvir (IC50: 1.17-152.9 µM). Molecular modeling revealed stronger interactions with Valine166 in the structural complex of Mpro_E166V with the compounds compared to nirmatrelvir. Moreover, these compounds efficiently inhibited the post-entry viral processes of wild-type SARS-CoV-2 single-round infectious particles (SRIPs), mitigating viral cytopathic effects and reducing replicon-driven GFP reporter signals, as well as in vitro infectivity of wild-type, Mpro_E166V, and Mpro_Q189I SRIPs, with EC50 values ranging from 0.02 to 0.53 µM. However, nirmatrelvir showed a significant decrease in inhibiting the replication of mutant SARS-CoV-2 SRIPs carrying Mpro_E166V (EC50: >20 µM) and Mpro_Q189I (EC50: 13.2 µM) compared to wild-type SRIPs (EC50: 0.06 µM). Overall, this study identifies four GL derivatives as promising lead compounds for developing treatments against various SARS-CoV-2 strains, including Omicron, and nirmatrelvir-resistant variants.

Synthesis, modification and biological activity of 2,3-indoles of Glycyrrhetinic acid.

The synthesis of 2,3-indoles of Glycyrrhetinic acid (GLA) and its methyl ester was carried out by the Fischer reaction. Reductive transformations of GLA methyl ester 2,3-indole 3a were carried out to obtain 11-deoxo- and 9,12-diene analogs. N-methylation of 2,3-indole 3a gave N-methyl-indole-11-oxo-18ß-olean-12-en-30-oic acid. The antiulcer and anti-inflammatory activity of 2,3-indole 3a was studied in rats and mice. It was found, compound 3a exhibied a pronounced antiulcer activity in the indomethacin model of ulcers in rats and anti-inflammatory activity in the carrageenan model of acute edoema in mice, at a dose of 50 mg/kg. This is the first report of anti-ulcer and anti-inflammatory activities of 2,3-indolo-GLA derivatives.

Glycyrrhizic Acid Derivatives Bearing Amino Acid Residues in the Carbohydrate Part as Dengue Virus E Protein Inhibitors: Synthesis and Antiviral Activity.

Dengue virus (DENV) is one of the most geographically distributed mosquito-borne flaviviruses, like Japanese encephalitis virus (JEV), and Zika virus (ZIKV). In this study, a library of the known and novel Glycyrrhizic acid (GL) derivatives bearing amino acid residues or their methyl/ethyl esters in the carbohydrate part were synthesized and studied as DENV inhibitors in vitro using the cytopathic effect (CPE), viral infectivity and virus yield assays with DENV1 and DENV-2 in Vero E6 and A549 cells. Among the GL conjugates tested, compound hits GL-D-ValOMe 3, GL-TyrOMe 6, GL-PheOEt 11, and GL-LysOMe 21 were discovered to have better antiviral activity than GL, with IC50 values ranging from <0.1 to 5.98 µM on the in vitro infectivity of DENV1 and DENV2 in Vero E6 and A549 cells. Compound hits 3, 6, 11, and 21 had a concentration-dependent inhibition on the virus yield in Vero E6, in which GL-D-ValOMe 3 and GL-PheOEt 11 were the most active inhibitors of DENV2 yield. Meanwhile, the time-of-addition assay indicated that conjugates GL-D-ValOMe 3 and GL-PheOEt 11 exhibited a substantial decrease in the DENV2 attachment stage. Subsequently, chimeric single-round infectious particles (SRIPs) of DENV2 C-prM-E protein/JEV replicon and DENV2 prM-E/ZIKV replicon were utilized for the DENV envelope I protein-mediated attachment assay. GL conjugates 3 and 11 significantly reduced the attachment of chimeric DENV2 C-prM-E/JEV and DENV2 prM-E/ZIKV SRIPs onto Vero E6 cells in a concentration-dependent manner but did not impede the attachment of wild-type JEV CprME/JEV and ZIKV prM-E/ZIKV SRIPs, indicating the inhibition of Compounds 3 and 11 on DENV2 E-mediated attachment. Molecular docking data revealed that Compounds 3 and 11 have hydrophobic interactions within a hydrophobic pocket among the interfaces of Domains I, II, and the stem region of the DENV2 envelope (E) protein. These results displayed that Compounds 3 and 11 were the lead compounds targeting the DENV E protein. Altogether, our findings provide new insights into the structure−activity relationship of GL derivatives conjugated with amino acid residues and can be the new fundamental basis for the search and development of novel flavivirus inhibitors based on natural compounds.

Glycyrrhetinic acid derivatives as Zika virus inhibitors: Synthesis and antiviral activity in vitro.

Zika virus (ZIKV) is an arbovirus of the Flaviviridae family (Flavivirus genus), causing serious neurological complications, such as Guillain-Barre Syndrome (GBS) in adults and fetal microcephaly. Licensed vaccines or specific antiviral agents against ZIKV do not currently exist. Therefore, the search and development of anti-ZIKV agents are particularly relevant and necessary. Glycyrrhetinic (3ß-hydroxy-11-oxo-18ßH-Olean-12-en-30-oic acid) (GA) 1 is one of the well-known pentacyclic triterpenoids isolated from licorice root (Glycyrrhiza glabra L., Gl. uralensis Fisher) (Leguminosae) possessing many biological features, including antiviral activity. This paper is devoted to the synthesis and studies of a number of nitrogen and sulfur-containing GA derivatives as ZIKV inhibitors. Sixteen GA and related triterpenoids (3ß-hydroxy-18ßH-Olean-12-en-30-oic acid and 3ß-hydroxy-11-oxo-18ßH-Olean-12(13),18(19)-dien-30-oic acid) derivatives were synthesized (amides, semi- and thiosemicarbazones, and 1,2,3-thiadiazoles) and antiviral activity against ZIKV was studied in vitro, including the inhibitory assays on cytopathic effect (CPE), viral protein synthesis, and replication stages. Four active compounds were found among GA derivatives tested, 13 (3-O-acetyl-30-aminopyridine GA), 16 (3-semicarbazone-30-butyl GA), 18 (1,2,3-thiadiazole-30-methyl GA), and 19 (1,2,3-thiadiazole-30-butyl GA) with IC50 < 1 µM against ZIKV replication. These compounds had a stronger inhibitory activity on ZIKV-induced CPE and viral protein translation in infected cells as compared to derivatives of 11-desoxo-GA. The most active compound was amide 13 (IC50 0.13 µM, TI ˃ 384). Time-of-addition assays indicated that 1,2,3-thiadiazole ring is important for inhibiting viral entry stage (compounds 18 and 19), while the 30-butyl ester group influenced on post-entry stage (compound 19). The molecular docking analysis demonstrated that lead compounds 13 and 19 forms a hydrogen-bond interaction with the catalytic triad (His51-Asp75-Ser135) of ZIKV NS2B-NS3 protease. Therefore, the active GA derivatives are promising for developing new antiviral agents against ZIKV infection.

Antiviral activity of glycyrrhizic acid conjugates with amino acid esters against Zika virus.

Zika virus (ZIKV) is a new pathogenic flavivirus transmitted by mosquitoes Aedes spp. ZIKV infection is accompanied by serious neurological complications and is especially dangerous for pregnant women, in which it can lead to congenital malformations of the fetus and microcephaly in neonates. Currently, there are no licensed vaccines or specific post-infectious therapies for ZIKV infection. This report is devoted to the study of glycyrrhizic acid (GL) derivatives as ZIKV inhibitors. The inhibitory assays on the cytopathic effect (CPE) and viral infectivity of ZIKV in three different human cell lines revealed that the conjugation of GL with amino acids and their esters (methyl, ethyl) is influenced by the antiviral activity of the compounds. GL conjugates with Glu(OMe)-OMe 11, Glu(OH)-OMe 12, Asp(OMe)-OMe 13, TyrOMe 14, LeuOEt 15, and PheOEt 16 with free COOH groups in the triterpene moiety were active against ZIKV. The most active compounds 13 and 14 have IC50 values of 0.23 µM and 0.09 µM against low doses (MOI = 0.05) of ZIKV strain PRVABC59, 1.20 µM and 0.74 µM against high doses (MOI = 10) of ZIKV strain Natal RGN single-round infectious particles, respectively. The lead compound was 14 with a high selectivity index (SI < 500). Compound 13 showed a higher inhibitory effect on the early stage (entry) of ZIKV replication than compound 14, and was less potent than compound 14 at the post-entry stage, consistent with the docking models. Compounds 13 and 14 also had a strong interaction with the active site pocket of NS5 MTase. Compounds 13 and 14 are recommended for expanded antiviral studies against ZIKV infection.

Paeoniflorin benzoates: synthesis and influence on learning and memory of aged rats in the passive avoidance task.

Paeoniflorin per-O-benzoates with the preserved pinane structure 2 and rearranged aglycone 3, containing C4 = O function, were obtained and their influence on learning and memory of aged rats was studied in the passive avoidance task. It was found that the chemical modification of paeoniflorin affected the cognitive functions of aged rats. The introduction of C4 = O function into the pinane part of benzoate 3 led to the improvement in learning process and preservation of the memory trace in aged rats as compared to the natural glycoside. This compound can be considered as the promising for further studies on in vivo models of disorders characteristic for Alzheimer's disease.

Glycyrrhizic acid derivatives as Dengue virus inhibitors.

Dengue virus (DENV) is one of the most geographically distributed pathogenic flaviviruses transmitted by mosquitoes Aedes sps. In this study, the structure-antiviral activity relationships of Glycyrrhizic acid (GL) derivatives was evaluated by the inhibitory assays on the cytopathic effect (CPE) and viral infectivity of DENV type 2 (DENV2) in Vero E6 cells. GL (96% purity) had a low cytotoxicity to Vero E6 cells, inhibited DENV2-induced CPE, and reduced the DENV-2 infectivity with the IC50 of 8.1 µM. Conjugation of GL with amino acids or their methyl esters and the introduction of aromatic acylhydrazide residues into the carbohydrate part strongly influenced on the antiviral activity. Among compounds tested GL conjugates with isoleucine 13 and 11-aminoundecanoic acid 17 were found as potent anti-DENV2 inhibitors (IC50 1.2-1.3 µM). Therefore, modification of GL is a perspective way in the search of new antivirals against DENV2 infection.

Synthesis and Antiviral Activity of Quercetin Brominated Derivatives.

Reaction of quercetin (QR) (1) with bromine under various conditions was studied. Interaction of QR with 2-3 equiv. of bromine in glacial acetic acid at 35-40°C for 2-4 h and 20-22°C for 24 h led to the formation of QR 6,8-dibromide (2) (52-54% yields, 96-98% purity by HPLC). Interaction of QR with 2-5 equiv. bromine in absolute ethanol at 0-5°C and 20-22°C for 24 h led to the formation of 3-O-ethyl-QR-2,3,6,8,5'-pentabromide (3) (95-97% purity by HPLC) the output of which depends on the quantity of bromine. It was shown in MDCK cell culture that compound 2 exhibits a moderate inhibitory activity against pandemic influenza virus A/H1N1/pdm09 (EC50 6.0 µg/mL, CTD50 97.7 µg/mL, SI 16). Compound 3 was inactive.

Glycyrrhizic acid derivatives as influenza A/H1N1 virus inhibitors.

This Letter describes the synthesis and antiviral activity study of some glycyrrhizic acid (GL) derivatives against influenza A/H1N1/pdm09 virus in MDCK cells. Conjugation of GL with l-amino acids or their methyl esters, and amino sugar (d-galactose amine) dramatically changed its activity. The most active compounds were GL conjugates with aromatic amino acids methyl esters (phenylalanine and tyrosine) (SI=61 and 38), and S-benzyl-cysteine (SI=71). Thus modification of GL is a perspective route in the search of new antivirals, and some of GL derivatives are potent as anti-influenza A/H1N1 agents.

Inhibitory effects of some derivatives of glycyrrhizic acid against Epstein-Barr virus infection: structure-activity relationships.

Glycyrrhizic acid (18beta-GL or GL) is a herbal drug with a broad spectrum of antiviral activities and pharmacological effects and multiple sites of action. Previously we showed that GL inhibits Epstein-Barr virus (EBV) infection in vitro by interfering with an early step of the EBV replication cycle (possibly attachment/penetration). Here we tested the effects of 15 GL derivatives against EBV infection by scoring the numbers of cell expressing viral antigens and quantifying EBV DNA copy numbers in superinfected Raji cells. The derivatives were made either by transformation of GL on carboxyl and hydroxyl groups or by conjugation of amino acid residues into the carbohydrate part. We identified seven compounds active against EBV and all showed dose-dependent inhibition as determined by both assays. Among these active compounds, the introduction of amino acid residues into the GL carbohydrate part enhanced the antiviral activity in three of the seven active compounds. However, when Glu(OH)-OMe was substituted by Glu(OMe)-OMe, its antiviral activity was completely abolished. Introduction of potassium or ammonium salt to GL reduced the antiviral activity with no significant effect on cytotoxicity. The alpha-isomer (18alpha-GL) of 18beta-GL was as potent as the beta-form, but its sodium salt lost antiviral activity. The metabolic product of GL, 18beta-glycyrrhetinic acid (18beta-GA or GA), was 7.5-fold more active against EBV than its parental compound GL but, concomitantly, exhibited increased cytotoxicity resulting in a decreased therapeutic index.

Antiviral activity of glycyrrhizic acid derivatives against SARS-coronavirus.

Glycyrrhizin (GL) was shown to inhibit SARS-coronavirus (SARS-CoV) replication in vitro. Here the anti-SARS-CoV activity of 15 GL derivatives was tested. The introduction of 2-acetamido-beta-d-glucopyranosylamine into the glycoside chain of GL resulted in 10-fold increased anti-SARS-CoV activity compared to GL. Amides of GL and conjugates of GL with two amino acid residues and a free 30-COOH function presented up to 70-fold increased activity against SARS-CoV but also increased cytotoxicity resulting in decreased selectivity index.