key: cord-0988821-y04vuiq2 authors: Fayrushina, A. I.; Baltina, L. A.; Baltina, L. A.; Konovalova, N. I.; Petrova, P. A.; Eropkin, M. Yu. title: Synthesis and antiviral activity of novel glycyrrhizic acid conjugates with D-amino acid esters date: 2017-08-02 journal: Russ J Bioorgan Chem DOI: 10.1134/s1068162017040045 sha: e043a23f7e10c7e80f7e63ba50bd12893e272be4 doc_id: 988821 cord_uid: y04vuiq2 Glycyrrhizic acid (GA) conjugates with methyl and ethyl esters of D-amino acids (D-Trp, D-Phe, D-Tyr, D-Val, D-Leu) have been synthesized by the activated esters method using mixtures of N-hydroxybenzotriazole or N-hydroxysuccinimide with N,N′-dicyclohexylcarbodiimide. GA conjugate with D-Trp ethyl ester exhibited antiviral activity against influenza viruses A/H3N2, A/H1N1/pdm09, A/H5N1, B (SI > 10–29), and HRSV (SI > 25). GA conjugate with D-Trp methyl ester inhibited influenza virus A/H1N1/pdm09 (SI > 30). Glycyrrhizic acid (GA) (I) is the major triterpene glycoside in roots of common licorice Glycyrrhiza glabra L. and Ural licorice Gl. uralensis Fisher and a promising natural compound in the synthesis of new antiviral agents [1, 2] . GA is used to treat chronic hepatitis B and C infections in China and Japan (GMP F0115, Beijing Qinwutian Pharmaceutical Technology Co., Ltd). GA and its derivatives possess antiviral activity against the herpesviruses (the herpes simplex and varicella-zoster viruses) [3] , Epstein-Barr virus [4] , SARS-associated coronaviruses causing atypical pneumonia [5] , human immunodeficiency virus [6] , and influenza virus A/H1N1 [7] . GA is a natural stimulator of gamma-interferon; it inhibits viruses at early stages of the replicative cycle thus inducing no resistance. Highly purified GA is a lightly toxic compound (LD 50 5000 mg/kg); it is characterized by high bioavailability and is metabolized in the gastrointestinal tract and liver similar to corticosteroid hormones [1] . Chemical modification of GA and functional design of the derivatives are a promising approach to the preparation of new antiviral agents and immunomodulators [8] . As part of our ongoing work on the synthesis of GA derivatives with antiviral properties of potential interest for medicine, we were the first to synthesize GA conjugates with methyl and ethyl esters of D-amino acids (Scheme 1). GA (I) carboxyl groups were activated with hydroxybenzotriazol (HOBt) or N-hydrosuccinimide (HONSu) and N,N'-dicyclohexylcarbodiimide (DCC) [9, 10] . Treatment of GA (I) with HOBt/DCC at GA-HOBt-DCC molar ratio 1 : 3.5 : 3.2 or HONSu/DCC at GA-HOSu-DCC molar ratio 1 : 5.2 : 3.2 in tetrahydrofuran/dioxane at 20-22°C resulted in activated tris(oxybenzotriazol) (II) and tris(oxysuccinimide) (III) esters and N,N'-dicyclohexylurea precipitate. Activated esters (II) and (III), after separation of the N,N'-dicyclohexylurea precipitate, were introduced into the reaction with D-amino acid ester hydrochlorides dissolved in the mixture of tetrahydrofuran-dioxane-DMF in the presence of N-ethylmorpholine (NEM) excess at room temperature (20-22°C) during 24 h. Individual (as evidenced by TLC) carboxyprotected conjugates (IV-X) were isolated by column chromatography on silica gel (SG) with 55-60% yields in case of HOBt and 45-50% in case of HONSu. Compound (X) was synthesized upon GA activation with HONSu/DCC at 0-5°C at GA-HONSu-DCC molar ratio 1 : 4.3 : 5.2. Structures of the conjugates were confirmed by spectral techniques (IR, 1 H, and 13 C NMR). For example, in 13 C NMR spectra signals of C=O group carbons in amino acid esters were revealed by the additional signals in the weak field (174.9-170.0 ppm), and signals of OCH 3 groups, in the range 52.0-50.9 ppm. In 1 H NMR spectra of conjugates (V-X) signals of methoxycarbonyl groups were observed in the range 3.30-3.72 ppm. Aromatic amino acid residues (Trp, Phe, Tyr) produced a set of carbon atom signals in the range 138-111 ppm. In 13 C NMR spectrum of conjugate (X) carbon atom signal of free COOH group had a chemical shift value of 180.4 ppm, as in the GA spectrum [11] . Antiviral activity of a number of synthesized GA conjugates was assessed in the culture of MDCK cells using various influenza viruses type A and B: influenza virus A/Pert/16//09(H3N2), pandemic influenza virus A/California/07/09(H1N1)pdm09, avian influenza virus A/NIBRG/14/H5N1, and influenza virus B/Brisben/60/08 (see Table 1 ). For compounds under study, the toxic dose considerably exceeded 200 μg/mL, thus the median toxic dose, or dose causing 50% cell death (CTD 50 ), was evaluated as >200 μg/mL. Quantitative evaluation of antiviral activity was performed according to the selectivity index (SI), or therapeutic index, which is the product of CTD 50 divided by effective concentration (EC 50 ) (a dose causing 50% decrease in the virus infectious titer): SI = CTD 50 /EC 50 . In a screening study using the A/Pert/16/09(H3N2) strain, GA conjugates with methyl and ethyl esters of D-tryptophan (IV) and (V) were the most active: the decrease of infectious activity (virus titer expressed as a decimal logarithm of virus dose causing 50% cell death in a monolayer, logTCID 50 ) under concentration safe for the cells in vitro (50 and 100 μg/mL) was 3.75log for compound (IV) and 4.3log for compound (V), which are very good values since compounds decreasing the virus infectious titer in vitro by -2log and beyond are considered promising candidates. Compounds (VI-X) did not exhibit significant antiviral activity. Antiviral activity of GA conjugates (IV) and (V) was studied against other influenza virus subtypes ( Virus-inhibiting activity of compounds (IV) and (V) was studied in the MA-104 cell culture with respect to the standard human respiratory syncytial virus (HRSV). The results for compound (V) are presented in Table 2 . Conjugate (V) was found to be active against the HRSV virus as well, efficiently inhibiting replication of the virus (EC 50 18.8 μg/mL, SI > 25). Compound (IV), even at a concentration of 6.3 μg/mL, caused degradation of cell monolayer in the given model. Therefore, antiviral activity of this group of GA conjugates with respect to studied influenza virus and HRSV subtypes depended on the structure of amino acid residue and the ester group. Even for structurally related compounds, such as conjugates (IV) and (V) differing only by a single methylene group (methyl vs. ethyl ester), activity differed considerably. EXPERIMENTAL 1 H and 13 C NMR spectra were registered on an Avance-III (Brüker, Germany) pulse spectrometer at a working frequency of 500.13 ( 1 H) and 125.47 ( 13 C) MHz and an AVX-300 (Brüker) spectrometer at working frequencies of 300 and 75.5 MHz (δ, ppm; spinspin coupling constant, Hz); internal standard, tetramethylsilane. NMR spectral signals were referenced under common mode using the ACD LABS software package and literature data for GA and its amino acid conjugates [6, [9] [10] [11] . IR spectra were recorded on a Prestige-21 (Shimadzu, Japan) IR spectrophotometer in a paste with paraffin oil. Optical activity was measured on a Perkin-Elmer 341 (Germany) polarimeter in a 1-dm tube at 20-22°C (λ Na 546 nm). Melting points were determined on a Boetius apparatus. Elemental analysis data matched the calculated values. TLC was performed on Sorbfil (Sorbopolimer, Russia) plates. Substances were detected with 5% H 2 SO 4 in ethanol with further heating at 110-120°C for 2-3 min. Column chromatography was performed on the KSK (fraction 50-150, dry classification) silica gel by Sorbopolimer. Solvents were purified according to techniques described in [12] . Solvents were vacuum dried at 50-60°C. GA isolated from roots of Glycyrrhiza uralensis Fisher of Siberian populations according to [13] was used in the work. HOBt, HONSu, DCC, and D-amino acids were from Sigma-Aldrich. Acid Methyl Ester Hydrochlorides To a suspension of 5 g of D-amino acid in 100 mL dry methanol at 0-5°С, 8-10 mL thionyl chloride were added dropwise, the mixture was stirred for 2 h and kept at 20-22°С for 24 h. The solvent was evaporated under vacuum at 30-40°С, the residue was mixed with dry ether, filtered off, dried, and recrystallized from methanol-ether mixture. To a solution of 0.82 g (1 mmol) GA in 20 mL dioxane, 0.47 g (3.5 mmol) N-hydroxybenzotriazol and 0.70 g (3.2 mmol) DCC were added at 0-5°С; the mixture was stirred at the same temperature for 1 h and at 22-24°С, for 5 h. The dicyclohexylurea precipitate was filtered off; 4.0 mmol amino acid ester hydrochloride, 5 mL DMF, and 0.7 mL (6 mmol) N-ethylmorpholine were added to the filtrate. The mixture was kept under intermittent stirring for 24 h at 20-22°С, diluted with cold water, and acidified with citric acid to pH of 3-4; the precipitate was filtered off, washed with water, and dried. The product was isolated on a silica gel column with To a solution of 0.82 g (1 mmol) GA in 20 mL tetrahydrofuran, 0.60 g (5.2 mmol) N-HONSu and 0.70 g (3.2 mmol) DCC were added at 0-5°С; the mixture was stirred at the same temperature for 1 h and at 20-22°С, for 6 h. The N,N'-dicyclohexylurea precipitate was filtered off; 4.0 mmol amino acid ester hydrochloride, 5 mL DMF, and 0.7 mL (6 mmol) N-ethylmorpholine were added to the filtrate. The mixture was kept under intermittent stirring for 24 h at 20-22°С, diluted with cold water, and acidified with citric acid to pH of 3-4; the precipitate was filtered off, washed with water, and dried. The product was isolated on a silica gel column with CHCl 3 -MeOH-H 2 O mixtures 300 : 10 : 1, 200 : 10 : 1, 100 : 10 : 1, and 50 : 10 : 1 (v/v, step gradient) under TLC control. Individual (according to TLC) fractions were joined and vacuum-dried. 13 13 Study of Toxicity and Antiviral Activity Viruses and cells. Studies were conducted on a primary culture of dog kidney cells MDCK for all influenza viruses and MA-104 cells, for human respiratory syncytial virus (HRSV). For screening of antiviral activity of the conjugates, reference virus A/Pert/16/09(H3N2) was used. Antiviral effect of the most active compounds was studied on international standard influenza virus strains of various subtypes: A/California/07/09(H1N1)pdm09, avian influenza virus A/NIBRG/14/H5N1, influenza virus B/Brisben/60/08, as well as the HRSV standard. To study toxicity, as well as antiviral activity, the conjugates were dissolved in DMSO at a concentration of 20 mg/mL thus forming a standard solution. Immediately before the experiment, the standard solution was diluted with cell incubation medium 100-fold, so that the final DMSO concentration did not exceed 1% (maximum concentration affording unaffected cell viability and metabolism). Therefore, the maximum working concentration of the compounds was 200 μg/mL. Toxicity of GA conjugates (IV) and (V) was studied at higher concentrations (>500 μg/mL). Further on, a series of six two-fold dilutions was prepared (to produce solutions of 100, 50, 25, 12.5, 6.25, and 3.13 μg/mL, respectively) with MDCK cell growth medium (α-МЕМ, Biolot, Russia), which were used to evaluate the conjugate toxicity. The compounds were introduced into a one-day MDCK culture grown in 96-well plates, then the plates were incubated for 20 [ ] D α 72 h at 37°С in the presence of 5% CO 2 . After visual evaluation of cytotoxicity by the level of monolayer integrity, toxicity was determined quantitatively by measuring the residual ability of cell in culture to reduce a tetrazole dye MTT (thiazolyl blue) by mitochondrial and partially cytoplasmic dehydrogenases of cells. The intensity of the reducing dye color restoration correlates with cell viability rate. The test is often used in virology for evaluation of cytopathogenic effect of viruses on cells [15] . Its results can be interpreted as the level of cell stability towards the effect of viruses. The microtetrazolium test is also widely used to evaluate the effects of the toxicants, pharmacological preparations, and unfavorable environmental factors on cells [16] . The MTT reduction was assayed on a Varioscan (ThermoFischer) microplate reader at 550 nm. Median toxic concentration (CTD 50 ) was determined by photometry analysis of the results by using linear regression in Excel 2010 software. Antiviral activity of each compound was screened at a single maximum concentration (in two repeats), at which 100% monolayer cells survived. A one-day MDCK culture grown in a 96-well plate was inspected visually in an inverted microscope to verify the monolayer integrity. Further, 10-fold virus dilutions in supporting growth medium supplemented with trypsin were performed: dilution logarithm (logc) was in the range from -1 to -7. Plates with cell monolayers were washed with serum-free medium twice and then the agents were introduced into relevant wells of the plate in the volume of 50 μL. Control wells were filled with equal volume of growth medium. The plates were incubated for 60 min at 37°С in the presence of CO 2 . Then virus was introduced into cells at relevant dilution. Each concentration of a conjugate was analyzed thrice for each of the virus dilutions. Control wells were filled with the same volume of growth medium. Some wells were used to verify the absence of toxicity by evaluation of the monolayer cell integrity compared to cell control and the rate of cytopathic effect of the virus on cells. Virus reproduction was evaluated in the blood agglutination reaction (BAR) and expressed as titer of hemagglutination (-logTCID 50 /50 μL) compared to control (intact virus 10-times more active compared to the control (intact virus at decimal dilutions). Cytopathic effect of the virus was evaluated quantitatively using the above-mentioned technique (MTT) and expressed as ΔlogTCID 50 /50 μL compared to control virus. Table 1 provides the mean value obtained in two tests. A compound or conjugate typically is considered a promising antiviral agent candidate if in vitro ΔlogTCID 50 ≥ 2.0 [17] . The decrease in the infectious titer of a virus evidenced antiviral activity of the compounds. Virus titer is the decimal logarithm of the virus suspension dilutions causing 50% monolayer cell (-logTCID 50 ) death. Using the data obtained we calculated the 50-% efficiency concentration (EC 50 ) which caused decrease in the virus titer twice (by 0.3 logTCID 50 ) and the selectivity index (SI) was calculated as the CTD 50 /EC 50 ratio. 2 (С3), 82.0 (С2'), 76.3 (С5'') -glucopyranosyluronoyl)-D-leucine methyl ester}-(3β,20β)-11-oxo-30-(N-carbonyl-D-leucine methyl ester)-30-norolean-12-ene (IX). Yield 0.60 g (50%) (method B) 25 (m, СН, СН 2 ), 3.30 (3Н, s, ОСН 3 ), 3.40-3.56 (m, СН On the next day, the N,N'-dicyclohexylurea sediment was filtered off, 0.42 g (2.5 mmol) amino acid ester hydrochloride and 0.6 mL (5.0 mmol) N-ethylmorpholine 20-22°С for 20 h; then cold water was added and the solution was acidified with citric acid to рН 3-4. The precipitate was filtered off, washed with water, dried, and separated on a silica gel column with the СНСl 3 -MeOH-H 2 O mixtures 1532 (CONH). 1 Н NMR (300 MHz Solodka: bioraznoobrazie, khimiya, primenenie v meditsine (Licorice: Biodiversity, Chemistry, and Application in Medicine) A Handbook of Practical Data, Techniques, and References Rukovodstvo po provedeniyu doklinicheskikh issledovanii lekarstvennykh sredstv. Chast' pervaya (Guidelines for Preclinical Drug Research The work was supported by the Russian Science Foundation (project no. 14-03-01307).