Synthesis of New Phosphonylated Bis-1,3,4-oxadiazoles and Study of Their Antimicrobial and Antiviral Activity

Synthesis of New Phosphonylated Bis-1,3,4-oxadiazoles and Study of Their Antimicrobial and Antiviral Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis of New Phosphonylated Bis-1,3,4-oxadiazoles and Study of Their Antimicrobial and Antiviral Activity Elizaveta Gerasimova, Dmitry Egorov, Aleksandr Krylov, Ekaterina Chernova, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7856319/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Medicinal Chemistry Research → Version 1 posted 4 You are reading this latest preprint version Abstract A series of new bis-1,3,4-oxadiazoles was synthesized by reacting the corresponding dicarboxylic acid bishydrazides with dialkyl chloroethynyl phosphonate. This reaction represents a new approach to the synthesis of 1,3,4-oxadiazoles, and the process's efficiency was demonstrated. The resulting compounds were tested against a range of test cultures: Staphylococcus aureus ATCC6538; Pseudomonas aeruginosa 0387; and Candida utilis LIA-01. All synthesized compounds exhibited moderate antistaphylococcal activity and weak fungistatic action. The cytotoxicity and antiviral activity of the obtained compounds against the influenza A (H1N1) virus was studied in vitro . Two of seven compounds under investigation (28%) have demonstrated high anti-viral activity against influenza virus A/Puerto Rico/8/34 (H1N1) with selectivity indices of 15 and 24. 1 3 4-oxadiazole phosphonate antimicrobial activity antiviral activity cytotoxicity Figures Figure 1 Figure 2 Figure 3 Introduction Heterocyclic compounds occupy a leading position in the development of new biologically active compounds [ 1 – 5 ]. 1,3,4-Oxadiazoles are one of the key classes used as pharmacophoric fragments [ 6 – 9 ]. In many ways, the great interest in this class of compounds is due to the fact that 1,3,4-oxadiazoles can be considered as bioisosteres of the cisamide group [ 10 , 11 ]. This explains their broad biological activity, which is confirmed by a large number of studies. Thus, the key areas in which research into the biological activity of 1,3,4-oxadiazoles is conducted are anticancer [ 12 – 14 ], antimicrobial [ 15 – 17 ], antifungal [ 18 – 20 ], antioxidant [ 21 – 23 ], and other [ 24 – 29 ] types of activity. It should be noted that the 1,3,4-oxadiazole fragment has proven itself well and is part of the structure of some active pharmaceutical substances – zibotentan [ 30 ], furamisole [ 31 ], thiodazosin [ 32 ], nesapidil [ 33 ], raltegravir [ 34 ], setileuton [ 35 ], phenadiazole [ 36 ] (Fig. 1 ). The biological activity of bis-1,3,4-oxadiazoles has not been extensively studied [ 37 – 41 ], despite the fact that the synthetic basis for such compounds is readily available dibasic acids. Furthermore, the ability to vary the linker may allow the structure to be tailored to the geometry required for biological objects. Results In this work, we developed a method for the synthesis of new phosphorylated bis-1,3,4-oxadiazole derivatives from the corresponding dibasic acid bishydrazides and chloroethynyl phosphonates. This synthetic approach was previously used to obtain phosphonylated 5-alkyl-1,3,4-oxadiazoles [ 42 ]. In the case of bishydrazides, the reaction proceeded under similar conditions and led to the final products in high yields 88–93%. The first step involves a nucleophilic attack by the amino group of the bishydrazide on the triple-bonded carbon atom associated with chlorine. While the proton on the nitrogen atom remains, a rearrangement typically occurs to form the corresponding ketenimine. The reaction culminates in an attack by the O-nucleophilic center on the central carbon atom of the ketenimine moiety, resulting in the formation of the final 1,3,4-oxadiazole ring. Calcium carbonate was also added to the flask to neutralize the hydrochloric acid formed during the reaction. Acetonitrile was used as a solvent. The data obtained by 1 H, 13 C, and 31 P NMR spectroscopy correspond to the structure of the obtained compounds. The characteristic signals in the 1 H NMR spectra of the obtained bis-1,3,4-oxadiazoles were as follows: a doublet signal of the methylene group at the phosphorus atom in the region of δ H 3.29–3.59 ppm with a characteristic constant of 2 J HP 21.2–21.3 Hz, doublet signals of methyl ether groups appeared in the region of δ H 3.61–3.84 ppm with characteristic constants of 3 J HP 11.2 Hz. More informative are the spectral data of NMR 13 C, in which the following characteristic signals can be distinguished: methylene groups appearing as a doublet in the region of δ C 23.38–23.84 ppm, with characteristic coupling constants 1 J CP 142.1-172.2 Hz, the signals of the carbon atoms of the oxadiazole fragment appear in the corresponding regions, for the carbon atom C 2 in the region of δ C 159.23-159.98 ppm, with characteristic coupling constants 2 J CP 9.7–10.4 Hz, for the carbon atom C 5 δ C 164.47-167.91 ppm. Antimicrobial activity of compounds. In vitro antimicrobial activity data from the synthesized bis-1,3,4-oxadiazole compounds showed that all compounds, regardless of their modifications, exhibited moderate antistaphylococcal activity. The zones of growth inhibition for the test strain Staphylococcus aureus ATCC 6538 ranged from 15 to 19 mm. No significant differences in the antistaphylococcal activity of the synthesized compounds were detected. It should also be noted that all compounds exhibited weak fungistatic activity against the Candida utilis strain LIA-01. The diameter of the zone of partial growth inhibition did not exceed 10 mm (Fig. 3 ). Antimicrobial activity against the Gram-ve bacterium Pseudomonas aeruginosa 0387 was not detected. Anti-viral activity of compounds. In order to evaluate anti-viral potential of synthesized compounds, their cytotoxicity and virus-inhibiting properties have been assessed in in vitro tests. The results obtained are summarized in Table 1 . Table 1 Cytotoxic and virus-inhibiting activity of bis-1,3,4- oxadiazole derivatives against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cell culture. Compound CС 50 , µM IC 50 , µM SI 3aa 574 ± 32 39 ± 5 15 3ab > 708 > 708 1 3ac > 685 > 685 1 3ad > 625 > 625 1 3ae > 607 26 ± 4 > 24 3bc > 607 > 607 1 3cc 69 ± 4 > 60 1 Oseltamivir carboxylate > 200 0.3 ± 0.1 > 667 As suggested from the results presented, the chemical library, in general, was of low toxicity. Indeed, the values of 6 of 7 compounds assessed have demonstrated CC 50 ’s values of hundreds of micromoles. Only compound 3cc bearing isopropyl moieties at terminal phosphates had CC 50 of 69 micromoles. Two of seven compounds appeared active against influenza virus. Indeed, compounds 3aa with bulky phenyl linker between oxadiazole moieties, as well as 3ae with long (C8) linker have demonstrated IC 50 ’s of 39 and 36 µM, correspondingly. Taken together with low toxicity, this resulted in high SI’s (15 and 24 for 3aa and 3ae , correspondingly). Other compounds were inactive against influenza virus used in the study (SI’s < 10). Conclusion An efficient method for the synthesis of bis-1,3,4-oxadiazoles using acid bishydrazides and push-pull chloroethynylphosphonate has been developed. All synthesized compounds exhibited moderate antistaphylococcal activity against the test strain Staphylococcus aureus ATCC 6538 and weak fungistatic activity against Candida utilis LIA-01. The compounds were of low cytotoxicity against MDCK cells in in vitro experiments. Two of seven compounds under investigation (28%) having demonstrated high anti-viral activity against influenza virus A/Puerto Rico/8/34 (H1N1). Taken together, these data allow to consider derivatives of bis-1,3,4-oxadiazole as prospective class of potential antimicrobial and anti-viral compounds. Experimental section Materials Syntheses were performed using reagents purchased from Sigma-Aldrich, Lenreactive and Neva reactive without additional purification. Solvents were pre-distilled and absolutized. Instruments The 1 H and 13 C NMR spectra were recorded on a Bruker Avance III HD 400 NanoBay spectrometer at frequencies of 400.17 ( 1 H), 101.62 ( 13 C). Mass spectroscopy studies were carried out on an Agilent 6530 Q-TOF LC/MS instrument using the electrospray ionization method (Dual ESI source, fragment potentials of 50 V, skimmer of 65 V, drying gas temperature—200°C). Melting points were measured on a Kofler stage (VEB Wägetechnik Rapido, PHMK 81/2969). The X-ray diffraction study of 3cc was performed at 100(2) K on Rigaku Oxford Diffraction «XtaLAB Supernova» diffractometer (HyPix-3000 type detector) using Cu Kα (λ = 1.54184 Å) radiation. Synthesis procedures General procedure for the synthesis of bis-1,3,4-oxadiazoles 3aa-3ae weighed portions of bishydrazide and potassium carbonate were added to a round-bottomed flask. Acetonitrile was added as a solvent. The required amount of dialkylchloroethynylphosphonate was then added dropwise and the mixture was stirred at room temperature for 24 hours. If necessary, the bath temperature was increased to 60–100°C to accelerate the reaction. The reaction progress was monitored using 31 P NMR spectroscopy. The resulting precipitate was separated from the main mixture using a Schott filter, and the filtrate was placed on a rotary evaporator until all the solvent had evaporated. The structure of the resulting phosphonates was confirmed using 1 H, 13 C, and 31 P NMR spectroscopy. Chemical shifts δ Р of the obtained compounds are presented in the characteristic region of ~ 20 ppm. Dimethyl ((5-(3-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)phenyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3aa). White crystals (Ethyl acetate : Pentane, 10:3). Yield 91%. Mp 82-83 o C. 1 H NMR (400.17 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 3.59 d (4H, CCH 2 , 2 J HP 21.3), 3.84 d (12H, CH 3 O, 3 J HP 11.2), 7.66 t (1H, CH, 3 J HH 7.6), 8.21 dd (2H, CH, 3 J HH 6.2, 4 J HH 1.7), 8.65 s (1H, CH). 13 C NMR (100.61 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 23.84 d (CH 2 P, 1 J CP 142.1), 53.58 d (CH 3 OP, 2 J CP 6.5), 124.66 (C C HC), 125.02 (C), 129.89 (CH C HC), 130.06 (CH C HCH), 159.83 d (C 2 , 2 J CP 9.9), 164.47 (C 5 ). 31 P NMR (162 Hz, DMSO- d 6 ): δ 20.68 ppm. Mass spectrum (HRMS-ESI), m/z: 459.0823 [M + H] + (calcd for C 16 H 21 N 4 O 8 P 2 : 459.0829). Dimethyl ((5-(3-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)propyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3ab). Viscous colorless oil. Yield 93%. 1 H NMR (400.17 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 2.11 p (2H, CH 2 , 3 J HP 7.3), 3.29 d (4H, CH 2 P, 2 J HP 21.3), 2.81 d (4H, CCH 2 , 3 J HH 7.3), d 3.61 (12H, CH 3 O, 3 J HP 11.2). 13 C NMR (100.61 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 22.73 (CH 2 C H 2 CH 2 ), 24.14 ( C H 2 CH 2 CH 2 ), 23.38 d (CH 2 P, 1 J CP 172.2), 24.14 (CH 2 CH 2 C H 2 ), 53.39 d (CH 3 OP, 2 J CP 6.5), 159.54 d (C 2 , 2 J CP 9.7), 166.51 (C 5 ). 31 P NMR (162 Hz, DMSO- d 6 ): δ 21.04 ppm. Mass spectrum (HRMS-ESI), m/z: 447.0806 [M + Na] + (calcd for C 13 H 22 N 4 NaO 8 P 2 : 447.0805). Dimethyl ((5-(4-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)butyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3ac). Viscous colorless oil. Yield 90%. 1 H NMR (400.17 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 1.89 m (4H, CH 2 ), т 2.88 d (4H, CCH 2 , 3 J HP 6.6), 3.44 d (4H, CH 2 P, 2 J HP 21.3), d 3.79 (12H, CH 3 O, 3 J HP 11.2). 13 C NMR (100.61 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 22.9 (CH 2 C H 2 C H 2 CH 2 ), 24.32 ( C H 2 CH 2 CH 2 C H 2 ), 23.61 d (CH 2 P, 1 J CP 142.3), 53.46 d (CH 3 OP, 2 J CP 6.5), 159.44 d (C 2 , 2 J CP 9.9), 167.14 (C 5 ). 31 P NMR (162 Hz, DMSO- d 6 ): δ 21.1 ppm. Mass spectrum (HRMS-ESI), m/z: 461.0964 [M + Na] + (calcd for C 14 H 24 N 4 NaO 8 P 2 : 461.0962). Diethyl ((5-(4-(5-((diethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)butyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3bc). White crystals (Ethyl acetate : Pentane, 10:3). Yield 89%. Mp 35-36 o C. 1 H NMR spectrum (400.17 MHz, DMSO- d 6 ), δ, ppm (J, Hz): 1.28 t (12H, CH 3 , 3 J HH 7.07), p 1.87 (4H, CH 2 , 3 J HH 3.76 ), d 2.84 d (4H, CCH 2 , 3 J HP 6.4), 3.39 d (4H, CH 2 P, 2 J HP 21.19), ddd 4.12 (8H, CH 2 OP, 3 J HP 8.38, 3 J HH 7.12, 3 J HH 1.56). 13 C NMR spectrum (101.62 MHz, DMSO- d 6 ), δ, ppm (J, Hz): 16.29 d (CH 3, 3 J HP 6.02), 23.86 (CH 2 C H 2 C H 2 CH 2 ), 24.56 d (CH 2 P, 1 J CP 141.49), 25.4 ( C H 2 CH 2 CH 2 C H 2 ), 63.03 d (CH 2 OP, 2 J CP 6.51), 159.66 d (C 2 , 2 J CP 10.16), 167 (C 5 ). 31 P NMR spectrum (162 MHz, DMSO- d 6 ): δ 18.34 ppm. Mass spectrum (HRMS-ESI), m/z: 517.1589 [M + Na] + (calcd for C 18 H 32 N 4 NaO 8 P 2 : 517.1588). Tetraisopropyl ((butane-1,4-diylbis(1,3,4-oxadiazole-5,2-diyl))bis(methylene))bis(phosphonate) (3cc). White crystals (Ethyl acetate : Pentane, 10:3). Yield 88%. Mp 60-61 o C. 1 H NMR spectrum (400.17 MHz, DMSO- d 6 ), δ, ppm (J, Hz): 1.33 d (12H, CH 3 , 3 J HH 6.19), 1.35 d (12H, CH 3 , 3 J HH 6.19), 1.94 p (4H, CH 2 , 3 J HH 3.55 ), 2.91 t (4H, CCH 2 , 2 J HH 6.67), 3.41 d (4H, CH 2 P, 2 J HP 21.17), 4.75 dg (4H, CHO, 3 J HP 12.4, 3 J HH 6.2). 13 C NMR spectrum (101.62 MHz, DMSO- d 6 ), δ, ppm (J, Hz): 23.85 d (4CH 3 , 3 J HP 4.87), 24.0 d (4CH 3 , 3 J HH 3.92), 24.0 d (CH 2 P, 1 J CP 141.49), 24.9 (CH 2 C H 2 CH 2 CH 2 ), 25.06 (CH 2 CH 2 C H 2 CH 2 ), 25.51 ( C H 2 CH 2 CH 2 CH 2 ), 26.47 ( C H 2 CH 2 CH 2 CH 2 ), 72.02 d (CHO, 2 J CP 6.73), 159.98 d (C 2 , 2 J CP 10.38), 166.96 (C 5 ). 31 P NMR spectrum (162 MHz, DMSO- d 6 ): δ 16.22 ppm. Mass spectrum (HRMS-ESI), m/z: 573.2206 [M + Na] + (calcd for C 22 H 40 N 4 NaO 8 P 2 : 573.2214). Dimethyl ((5-(7-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)heptyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3ad). Viscous colorless oil. Yield 92%. 1 H NMR (400.17 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 1.35 m (6H, CH 2 ), 1.75 m (4H, CH 2 ), 2.82 d (4H, CCH 2 , 3 J HH 7.5), 3.44 d (4H, CH 2 P, 2 J HP 21.3), 3.79 d (12H, CH 3 O, 3 J HP 11.2). 13 C NMR (100.61 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 23.65 d (CH 2 P, 1 J CP 142.3), 25.23 (CH 2 C H 2 CH 2 CH 2 CH 2 C H 2 CH 2 ), 26.18 ( C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 C H 2 ), 28.55 (CH 2 CH 2 C H 2 CH 2 C H 2 CH 2 CH 2 ), 28.64 (CH 2 CH 2 CH 2 C H 2 CH 2 CH 2 CH 2 ), 53.49 d (CH 3 OP, 2 J CP 6.5), 159.24 d (C 2 , 2 J CP 9.9), 167.81 (C 5 ). 31 P NMR (162 Hz, DMSO- d 6 ): δ 21.16 ppm. Mass spectrum (HRMS-ESI), m/z: 503.1427 [M + Na] + (calcd for C 17 H 30 N 4 NaO 8 P 2 : 503.1431). Dimethyl ((5-(8-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)octyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3ae). Viscous colorless oil. Yield 89%. 1 H NMR (400.17 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 1.34 m (8H, CH 2 ), 1.77 m (4H, CH 2 ), 2.83 t (4H, CCH 2 , 3 J HH 7.6), 3.46 d (4H, CH 2 P, 2 J HP 21.3), 3.81 d (12H, CH 3 O, 3 J HP 11.2). 13 C NMR (100.61 Hz, DMSO- d 6 ), δ, ppm ( J , Гц): 23.67 d (CH 2 P, 1 J CP 142.4), 25.28 (CH 2 C H 2 CH 2 CH 2 CH 2 CH 2 C H 2 CH 2 ), 26.26 ( C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 C H 2 ), 28.8 (CH 2 CH 2 C H 2 CH 2 CH 2 C H 2 CH 2 CH 2 ), 28.84 (CH 2 CH 2 CH 2 C H 2 C H 2 CH 2 CH 2 CH 2 ), 53.49 d (CH 3 OP, 2 J CP 6.5), 159.23 d (C 2 , 2 J CP 10.0), 167.91 (C 5 ). 31 P NMR (162 Hz, DMSO- d 6 ): δ 21.19 ppm. Mass spectrum (HRMS-ESI), m/z: 517.1582 [M + Na] + (calcd for C 18 H 32 N 4 NaO 8 P 2 : 517.1588). In vitro antimicrobial activity To determine the antimicrobial activity of the synthesized compounds 3aa-3ae, the following were used as test strains: Gram + ve bacterium Staphylococcus aureus АТСС, Gram-ve bacterium Pseudomonas aeruginosa 0387, yeast-like fungus Candida utilis LIA-01. The sensitivity of the strains to bis-1,3,4-oxadiazoles was determined on the Mueller-Hinton nutrient medium using the agar diffusion method (well method) [ 43 ]. The inoculum concentrations of the daily test strains grown on meat-peptone agar were 1.5 x 10 8 CFU/ml (for bacteria) and 1.5 x 10 5 CFU/ml (for yeast), which corresponds to a density of 0.5 according to the McFarland standard. Solutions of the test compounds were prepared in dimethyl sulfoxide. The procedure for applying the test strain inoculum to Mueller-Hinton agar and adding the compound solutions was presented previously. [ 43 , 44 ]. Petri dishes were incubated in a thermostat at 35°C for 24 hours. After incubation, the presence of zones of inhibition or growth retardation of the test strains was noted. Statistical processing of the obtained data was performed using STATISTICA 10.0 software. Differences in the diameter of the growth inhibition zones for each of the studied compounds were assessed using one-way ANOVA and the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05. Virus and cells . We used MDCK cells (ATCC CCL-34) from the collection of cell lines of the Saint Petersburg Pasteur Institute. Cells were cultured in 96-well culture plates in MEM medium with 10% fetal bovine serum («HyClone», USA), 40 U/ml gentamicin sulfate and 2.5 U/ml amphotericin B. Cell suspension with a concentration of 10 5 cells/ml was placed in the wells of the plates in volume of 100 µl and cultured until a complete monolayer formation for 24 h at 36°C in the presence of 5% CO 2 . The same medium without serum was used as a support medium for culturing cells with viruses. We used influenza virus A/Puerto Rico/8/34 (H1N1) from the collection of the Saint Petersburg Pasteur Institute. The infectious titer of virus was determined by end-point titration in 96-well plates with monolayers of MDCK cells. The results were evaluated visually according to the presence of the virus cytopathic action, the virus titer was calculated by the Spearman-Kerber method and represented in decimal logarithms of 50% tissue cytopathic doses in ml (lg TCID 50 /ml). Evaluation of cytotoxic properties of compounds The assessment of toxicity of compounds was carried out based on evaluation of the cell viability using the reduction reaction of the tetrazolium dye MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) by cells in culture. Its intensity shows the degree of cell viability as a result of dye reduction by mitochondrial and partially cytoplasmic dehydrogenases. The test compounds in the concentration range of 3.7–300 µg/ml dissolved in the medium for cell cultivation were added to the plate wells in a volume of 200 µl and incubated for 48 h at 36°C in atmosphere of 5% CO 2 . At the end of the incubation period, the cells were washed with MEM medium, and 100 µl of a solution (0.5 mg/ml) of MTT in the cell medium was added to the plate wells. The cells were incubated at 36°C at 5% CO 2 for 2 h and washed for 5 min with saline. The precipitate was dissolved in 100 µl of DMSO per well, and the optical density was measured using a Multiscan FC plate analyzer (Thermo Scientific) at a wavelength of 540 nm. Based on the obtained data, the 50% cytotoxic concentration (CC 50 ) was calculated, i.e. the concentration of the compound, which reduces the optical density in the wells by half compared to control cells without drugs. For this purpose, GraphPad Prism 6.01 software was used. Evaluation of antiviral activity of compounds Anti-viral properties of compounds were evaluated by cytopathogenic effect (CPE) reduction assay. Briefly, the compounds in appropriate concentrations were added to MDCK cells (0.1 ml per well). After 1 h of incubation, cells were infected with influenza virus A/Puerto Rico/8/34 (H1N1) (moi 0.01) and incubated for 48 h at 36°C and 5% CO 2 . After that, cell viability was assessed by MTT test as described above. The cytoprotective activity of compounds was considered as their ability to increase the values of OD comparing to control wells (with virus only, no drugs). Based on the results obtained, the values of IC 50 , i.e. concentration of compounds that result in 50% cells protection were calculated using GraphPad Prism 6.01 software. Values of IC 50 obtained in µg/ml were then calculated into micromoles (µM). For each compound the value of selectivity index (SI) was calculated as ratio of CC 50 to IC 50 . Compounds with SI of 10 and higher were considered active. Neuraminidase inhibitor oseltamivir carboxylate (Hoffman LaRoche, Switzerland) was used as a reference compounds. Declarations Compliance with ethical standards Conflict of interest. The authors declare no competing interests Funding The study was supported by the Russian Science Foundation grant No. 23-13-00224, https://rscf.ru/project/23-13-00224/ . Author Contribution Author contributions Synthesis, GEA; Conceptualization and methodology, EDM, ILK, ZAZ, ZVV; Spectral assays, CEN, SDV; Biological testing, ZTB, ILK, ASV, MAN, ZVV; Supervision, EDM; Writing original draft, all authors; Editing the text, all authors; All authors have read and agreed to the published version of the manuscript. 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Design, Synthesis and Anticancer Evaluation of Benzoxazole Resemble Substituted Arylamide Derivatives of Bis-1,3,4-Oxadiazole-oxazolo[4,5-b]pyridin-2-yl) as Anticancer Agents. Chem. Data Coll. 2023;43:100971. https://doi.org/10.1016/j.cdc.2022.100971 Dilanyan S.V., Buniatyan Z.M., Panosyan H.A. Synthesis and Antioxidant Activity of New Bis-1,3,4-oxadiazoles, Bis-1,3,4-thiadiazoles, and Their Derivatives. Russ. J. Org. Chem. 2024;60:1301–6. https://doi.org/10.1134/S1070428024070224 Shah S., Khan M., Ali M., Wadood A., Rehman A.U., Shah Z., et al. Bis-1,3,4-Oxadiazole Derivatives as Novel and Potential Urease Inhibitors; Synthesis, In Vitro, and In Silico Studies. Med. Chem. 2022;18(7):820–30. https://doi.org/10.2174/1573406418666220301161934 Mekky A.E.M., El-Idreesy T.T., Sanad S.M.H. Chloramine Trihydrate-Mediated Tandem Synthesis of New Pyrrole and/or Arene-Linked Mono- and Bis(1,3,4-Oxadiazole) Hybrids as Potential Bacterial Biofilm and MRSA Inhibitors. Chem. Biodivers. 2022;19(8):e202200338. https://doi.org/10.1002/cbdv.202200338 Abdelfattah A.M., Mekky A.E.M., Sanad S.M.H. Synthesis, antibacterial activity and in silico study of new bis(1,3,4-oxadiazoles). Synth. Comm. 2022;52(11–12):1421–40. https://doi.org/10.1080/00397911.2022.2095211 Gerasimova E.A., Egorov D.M. Synthesis of Phosphonylated 5-Alkyl-1,3,4-oxadiazoles Based on Dialkyl Chloroethynylphosphonates. Russ. J. Gen. Chem. 2024;94(8):1941–54. https://doi.org/10.1134/S1070363224080061 Balouiri M., Sadiki M., Ibnsouda S.K. Methods for in vitro evaluating antimicrobial activity: A review. J. Pharm. Analysis 2016;6(2):71–9. https://doi.org/10.1016/j.jpha.2015.11.005 Bespalov D.S., Egorov D.M., Krylov A.S., Chernova E.N., Kuzikova I.L., Zhakovskaya Z.A. Synthesis of 5,6-Dialkyl-4-aryl-2-aminopyridine-3-carbonitriles and In Vitro Study of Their Antimicrobial Activity. Russ. J. Gen. Chem. 2024;94:3173–80. https://doi.org/10.1134/S1070363224120065 Schemes Schemes 1 and 2 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.docx Schemes.docx Cite Share Download PDF Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Medicinal Chemistry Research → Version 1 posted Editorial decision: Revision requested 19 Oct, 2025 Editor assigned by journal 16 Oct, 2025 Submission checks completed at journal 16 Oct, 2025 First submitted to journal 14 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7856319","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":532027491,"identity":"05780233-e509-413a-9233-905a59c9da1b","order_by":0,"name":"Elizaveta Gerasimova","email":"","orcid":"","institution":"St. Petersburg Federal Research Center of the Russian Academy of Sciences (SPC RAS)","correspondingAuthor":false,"prefix":"","firstName":"Elizaveta","middleName":"","lastName":"Gerasimova","suffix":""},{"id":532027492,"identity":"4ac84698-e1da-4149-bb19-ae9ccbd9a0ec","order_by":1,"name":"Dmitry 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08:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7856319/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7856319/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00044-025-03516-z","type":"published","date":"2025-12-16T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95031501,"identity":"ad54152c-2153-4a8a-a466-c824731055c7","added_by":"auto","created_at":"2025-11-03 14:24:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44516,"visible":true,"origin":"","legend":"\u003cp\u003eCompounds with pronounced biological activity containing a 1,3,4-oxadiazole fragment\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7856319/v1/e2ad11efd46c7b681a9df91d.png"},{"id":95031503,"identity":"6455957d-5e93-4891-a3bf-fae774b81130","added_by":"auto","created_at":"2025-11-03 14:24:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":270855,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray structural analysis data of tetraisopropyl ((butane-1,4-diylbis(1,3,4-oxadiazol-5,2-diyl))bis(methylene))bis(phosphonate) \u003cstrong\u003e3сс \u003c/strong\u003e(CCDC 2493467).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7856319/v1/9e194a36b4086aafaeb3bda5.png"},{"id":95031502,"identity":"ba457a83-6ed3-47ab-b0f7-92971986a0cb","added_by":"auto","created_at":"2025-11-03 14:24:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56837,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial activity of the bis-1,3,4-oxadiazoles. Compounds concentration 5 µg/ml. \u003cstrong\u003e1 – 3aa\u003c/strong\u003e, \u003cstrong\u003e2 – 3ab\u003c/strong\u003e, \u003cstrong\u003e3 - 3ac\u003c/strong\u003e, \u003cstrong\u003e4 – 3bc\u003c/strong\u003e, \u003cstrong\u003e5 – 3cc\u003c/strong\u003e, \u003cstrong\u003e6 - 3ad\u003c/strong\u003e, \u003cstrong\u003e\u0026nbsp;7 – 3ae\u003c/strong\u003e. a - \u003cem\u003eStaphylococcus aureus \u003c/em\u003eАТСС 6538, b - \u003cem\u003eCandida utilis \u003c/em\u003eLIA-01.* - fungistatic action (partial zone of growth inhibition)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7856319/v1/b6866ba50cb95b16a3c1a78c.png"},{"id":98813819,"identity":"33da1be0-d0fa-4503-8efa-4cd756d111cb","added_by":"auto","created_at":"2025-12-22 16:00:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1533372,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7856319/v1/002ba7ea-185b-4504-9820-f1b1ce5a6a31.pdf"},{"id":95031505,"identity":"f2a689ed-78e3-48be-8883-55b2cd38537f","added_by":"auto","created_at":"2025-11-03 14:24:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5774127,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7856319/v1/a66bad36df1774a98ba0a2bb.docx"},{"id":95031504,"identity":"b5b5ab01-7875-4ae6-81fa-a06b50fbffb6","added_by":"auto","created_at":"2025-11-03 14:24:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":143975,"visible":true,"origin":"","legend":"","description":"","filename":"Schemes.docx","url":"https://assets-eu.researchsquare.com/files/rs-7856319/v1/b4c6323e1d2112e1b67537c5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSynthesis of New Phosphonylated Bis-1,3,4-oxadiazoles and Study of Their Antimicrobial and Antiviral Activity\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeterocyclic compounds occupy a leading position in the development of new biologically active compounds [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 1,3,4-Oxadiazoles are one of the key classes used as pharmacophoric fragments [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In many ways, the great interest in this class of compounds is due to the fact that 1,3,4-oxadiazoles can be considered as bioisosteres of the cisamide group [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This explains their broad biological activity, which is confirmed by a large number of studies. Thus, the key areas in which research into the biological activity of 1,3,4-oxadiazoles is conducted are anticancer [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], antimicrobial [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], antifungal [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], antioxidant [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and other [\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] types of activity. It should be noted that the 1,3,4-oxadiazole fragment has proven itself well and is part of the structure of some active pharmaceutical substances \u0026ndash; zibotentan [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], furamisole [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], thiodazosin [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], nesapidil [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], raltegravir [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], setileuton [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], phenadiazole [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe biological activity of bis-1,3,4-oxadiazoles has not been extensively studied [\u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], despite the fact that the synthetic basis for such compounds is readily available dibasic acids. Furthermore, the ability to vary the linker may allow the structure to be tailored to the geometry required for biological objects.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn this work, we developed a method for the synthesis of new phosphorylated bis-1,3,4-oxadiazole derivatives from the corresponding dibasic acid bishydrazides and chloroethynyl phosphonates. This synthetic approach was previously used to obtain phosphonylated 5-alkyl-1,3,4-oxadiazoles [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the case of bishydrazides, the reaction proceeded under similar conditions and led to the final products in high yields 88\u0026ndash;93%.\u003c/p\u003e\u003cp\u003eThe first step involves a nucleophilic attack by the amino group of the bishydrazide on the triple-bonded carbon atom associated with chlorine. While the proton on the nitrogen atom remains, a rearrangement typically occurs to form the corresponding ketenimine. The reaction culminates in an attack by the O-nucleophilic center on the central carbon atom of the ketenimine moiety, resulting in the formation of the final 1,3,4-oxadiazole ring.\u003c/p\u003e\u003cp\u003eCalcium carbonate was also added to the flask to neutralize the hydrochloric acid formed during the reaction. Acetonitrile was used as a solvent. The data obtained by \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, and \u003csup\u003e31\u003c/sup\u003eP NMR spectroscopy correspond to the structure of the obtained compounds. The characteristic signals in the \u003csup\u003e1\u003c/sup\u003eH NMR spectra of the obtained bis-1,3,4-oxadiazoles were as follows: a doublet signal of the methylene group at the phosphorus atom in the region of δ\u003csub\u003eH\u003c/sub\u003e 3.29\u0026ndash;3.59 ppm with a characteristic constant of \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 21.2\u0026ndash;21.3 Hz, doublet signals of methyl ether groups appeared in the region of δ\u003csub\u003eH\u003c/sub\u003e 3.61\u0026ndash;3.84 ppm with characteristic constants of \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 11.2 Hz. More informative are the spectral data of NMR \u003csup\u003e13\u003c/sup\u003eC, in which the following characteristic signals can be distinguished: methylene groups appearing as a doublet in the region of δ\u003csub\u003eC\u003c/sub\u003e 23.38\u0026ndash;23.84 ppm, with characteristic coupling constants \u003csup\u003e1\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 142.1-172.2 Hz, the signals of the carbon atoms of the oxadiazole fragment appear in the corresponding regions, for the carbon atom C\u003csub\u003e2\u003c/sub\u003e in the region of δ\u003csub\u003eC\u003c/sub\u003e 159.23-159.98 ppm, with characteristic coupling constants \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 9.7\u0026ndash;10.4 Hz, for the carbon atom C\u003csub\u003e5\u003c/sub\u003e δ\u003csub\u003eC\u003c/sub\u003e 164.47-167.91 ppm.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAntimicrobial activity of compounds.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e antimicrobial activity data from the synthesized bis-1,3,4-oxadiazole compounds showed that all compounds, regardless of their modifications, exhibited moderate antistaphylococcal activity. The zones of growth inhibition for the test strain \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538 ranged from 15 to 19 mm. No significant differences in the antistaphylococcal activity of the synthesized compounds were detected. It should also be noted that all compounds exhibited weak fungistatic activity against the \u003cem\u003eCandida utilis\u003c/em\u003e strain LIA-01. The diameter of the zone of partial growth inhibition did not exceed 10 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Antimicrobial activity against the \u003cem\u003eGram-ve\u003c/em\u003e bacterium \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e 0387 was not detected.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnti-viral activity of compounds.\u003c/p\u003e\u003cp\u003eIn order to evaluate anti-viral potential of synthesized compounds, their cytotoxicity and virus-inhibiting properties have been assessed in \u003cem\u003ein vitro\u003c/em\u003e tests. The results obtained are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCytotoxic and virus-inhibiting activity of bis-1,3,4- oxadiazole derivatives against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cell culture.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCС\u003csub\u003e50\u003c/sub\u003e, \u0026micro;M\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e, \u0026micro;M\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e574\u0026thinsp;\u0026plusmn;\u0026thinsp;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;708\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;708\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3ac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3ad\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3ae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;607\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;607\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;607\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3cc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e69\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOseltamivir carboxylate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;667\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAs suggested from the results presented, the chemical library, in general, was of low toxicity. Indeed, the values of 6 of 7 compounds assessed have demonstrated CC\u003csub\u003e50\u003c/sub\u003e\u0026rsquo;s values of hundreds of micromoles. Only compound \u003cb\u003e3cc\u003c/b\u003e bearing isopropyl moieties at terminal phosphates had CC\u003csub\u003e50\u003c/sub\u003e of 69 micromoles.\u003c/p\u003e\u003cp\u003eTwo of seven compounds appeared active against influenza virus. Indeed, compounds \u003cb\u003e3aa\u003c/b\u003e with bulky phenyl linker between oxadiazole moieties, as well as \u003cb\u003e3ae\u003c/b\u003e with long (C8) linker have demonstrated IC\u003csub\u003e50\u003c/sub\u003e\u0026rsquo;s of 39 and 36 \u0026micro;M, correspondingly. Taken together with low toxicity, this resulted in high SI\u0026rsquo;s (15 and 24 for \u003cb\u003e3aa\u003c/b\u003e and \u003cb\u003e3ae\u003c/b\u003e, correspondingly). Other compounds were inactive against influenza virus used in the study (SI\u0026rsquo;s\u0026thinsp;\u0026lt;\u0026thinsp;10).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAn efficient method for the synthesis of bis-1,3,4-oxadiazoles using acid bishydrazides and push-pull chloroethynylphosphonate has been developed. All synthesized compounds exhibited moderate antistaphylococcal activity against the test strain \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538 and weak fungistatic activity against \u003cem\u003eCandida utilis\u003c/em\u003e LIA-01. The compounds were of low cytotoxicity against MDCK cells in \u003cem\u003ein vitro\u003c/em\u003e experiments. Two of seven compounds under investigation (28%) having demonstrated high anti-viral activity against influenza virus A/Puerto Rico/8/34 (H1N1). Taken together, these data allow to consider derivatives of bis-1,3,4-oxadiazole as prospective class of potential antimicrobial and anti-viral compounds.\u003c/p\u003e"},{"header":"Experimental section","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eSyntheses were performed using reagents purchased from Sigma-Aldrich, Lenreactive and Neva reactive without additional purification. Solvents were pre-distilled and absolutized.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInstruments\u003c/h3\u003e\n\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded on a Bruker Avance III HD 400 NanoBay spectrometer at frequencies of 400.17 (\u003csup\u003e1\u003c/sup\u003eH), 101.62 (\u003csup\u003e13\u003c/sup\u003eC). Mass spectroscopy studies were carried out on an Agilent 6530 Q-TOF LC/MS instrument using the electrospray ionization method (Dual ESI source, fragment potentials of 50 V, skimmer of 65 V, drying gas temperature\u0026mdash;200\u0026deg;C). Melting points were measured on a Kofler stage (VEB W\u0026auml;getechnik Rapido, PHMK 81/2969). The X-ray diffraction study of \u003cb\u003e3cc\u003c/b\u003e was performed at 100(2) K on Rigaku Oxford Diffraction \u0026laquo;XtaLAB Supernova\u0026raquo; diffractometer (HyPix-3000 type detector) using Cu\u003cem\u003eKα\u003c/em\u003e (λ\u0026thinsp;=\u0026thinsp;1.54184 \u0026Aring;) radiation.\u003c/p\u003e\n\u003ch3\u003eSynthesis procedures\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral procedure for the synthesis of bis-1,3,4-oxadiazoles 3aa-3ae\u003c/strong\u003e\u003cp\u003eweighed portions of bishydrazide and potassium carbonate were added to a round-bottomed flask. Acetonitrile was added as a solvent. The required amount of dialkylchloroethynylphosphonate was then added dropwise and the mixture was stirred at room temperature for 24 hours. If necessary, the bath temperature was increased to 60\u0026ndash;100\u0026deg;C to accelerate the reaction. The reaction progress was monitored using \u003csup\u003e31\u003c/sup\u003eP NMR spectroscopy. The resulting precipitate was separated from the main mixture using a Schott filter, and the filtrate was placed on a rotary evaporator until all the solvent had evaporated. The structure of the resulting phosphonates was confirmed using \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, and \u003csup\u003e31\u003c/sup\u003eP NMR spectroscopy. Chemical shifts δ\u003csub\u003eР\u003c/sub\u003e of the obtained compounds are presented in the characteristic region of ~\u0026thinsp;20 ppm.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eDimethyl ((5-(3-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)phenyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3aa).\u003c/b\u003e White crystals (Ethyl acetate : Pentane, 10:3). Yield 91%. Mp 82-83\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR (400.17 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 3.59 d (4H, CCH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 21.3), 3.84 d (12H, CH\u003csub\u003e3\u003c/sub\u003eO, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 11.2), 7.66 t (1H, CH, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 7.6), 8.21 dd (2H, CH, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 6.2, \u003csup\u003e4\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 1.7), 8.65 s (1H, CH). \u003csup\u003e13\u003c/sup\u003eC NMR (100.61 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 23.84 d (CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e1\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 142.1), 53.58 d (CH\u003csub\u003e3\u003c/sub\u003eOP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 6.5), 124.66 (C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eHC), 125.02 (C), 129.89 (CH\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eHC), 130.06 (CH\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eHCH), 159.83 d (C\u003csup\u003e2\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 9.9), 164.47 (C\u003csup\u003e5\u003c/sup\u003e). \u003csup\u003e31\u003c/sup\u003eP NMR (162 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e): δ 20.68 ppm. Mass spectrum (HRMS-ESI), m/z: 459.0823 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e: 459.0829).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDimethyl ((5-(3-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)propyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3ab).\u003c/b\u003e Viscous colorless oil. Yield 93%.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR (400.17 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 2.11 p (2H, CH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 7.3), 3.29 d (4H, CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 21.3), 2.81 d (4H, CCH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 7.3), d 3.61 (12H, CH\u003csub\u003e3\u003c/sub\u003eO, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 11.2). \u003csup\u003e13\u003c/sup\u003eC NMR (100.61 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 22.73 (CH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 24.14 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 23.38 d (CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e1\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 172.2), 24.14 (CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 53.39 d (CH\u003csub\u003e3\u003c/sub\u003eOP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 6.5), 159.54 d (C\u003csup\u003e2\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 9.7), 166.51 (C\u003csup\u003e5\u003c/sup\u003e). \u003csup\u003e31\u003c/sup\u003eP NMR (162 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e): δ 21.04 ppm. Mass spectrum (HRMS-ESI), m/z: 447.0806 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eNaO\u003csub\u003e8\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e: 447.0805).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDimethyl ((5-(4-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)butyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3ac).\u003c/b\u003e Viscous colorless oil. Yield 90%.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR (400.17 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 1.89 m (4H, CH\u003csub\u003e2\u003c/sub\u003e), т 2.88 d (4H, CCH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 6.6), 3.44 d (4H, CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 21.3), d 3.79 (12H, CH\u003csub\u003e3\u003c/sub\u003eO, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 11.2).\u003c/p\u003e\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC NMR (100.61 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 22.9 (CH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 24.32 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 23.61 d (CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e1\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 142.3), 53.46 d (CH\u003csub\u003e3\u003c/sub\u003eOP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 6.5), 159.44 d (C\u003csup\u003e2\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 9.9), 167.14 (C\u003csup\u003e5\u003c/sup\u003e). \u003csup\u003e31\u003c/sup\u003eP NMR (162 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e): δ 21.1 ppm.\u003c/p\u003e\u003cp\u003eMass spectrum (HRMS-ESI), m/z: 461.0964 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eNaO\u003csub\u003e8\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e: 461.0962).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDiethyl ((5-(4-(5-((diethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)butyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3bc).\u003c/b\u003e White crystals (Ethyl acetate : Pentane, 10:3). Yield 89%. Mp 35-36\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectrum (400.17 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (J, Hz): 1.28 t (12H, CH\u003csub\u003e3\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 7.07), p 1.87 (4H, CH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 3.76 ), d 2.84 d (4H, CCH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 6.4), 3.39 d (4H, CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 21.19), ddd 4.12 (8H, CH\u003csub\u003e2\u003c/sub\u003eOP, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 8.38, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 7.12, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 1.56). \u003csup\u003e13\u003c/sup\u003eC NMR spectrum (101.62 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (J, Hz): 16.29 d (CH\u003csub\u003e3,\u003c/sub\u003e \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 6.02), 23.86 (CH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 24.56 d (CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e1\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 141.49), 25.4 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 63.03 d (CH\u003csub\u003e2\u003c/sub\u003eOP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 6.51), 159.66 d (C\u003csup\u003e2\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 10.16), 167 (C\u003csup\u003e5\u003c/sup\u003e). \u003csup\u003e31\u003c/sup\u003eP NMR spectrum (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e): δ 18.34 ppm. Mass spectrum (HRMS-ESI), m/z: 517.1589 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eNaO\u003csub\u003e8\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e: 517.1588).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTetraisopropyl ((butane-1,4-diylbis(1,3,4-oxadiazole-5,2-diyl))bis(methylene))bis(phosphonate) (3cc).\u003c/b\u003e White crystals (Ethyl acetate : Pentane, 10:3). Yield 88%. Mp 60-61\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectrum (400.17 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (J, Hz): 1.33 d (12H, CH\u003csub\u003e3\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 6.19), 1.35 d (12H, CH\u003csub\u003e3\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 6.19), 1.94 p (4H, CH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 3.55 ), 2.91 t (4H, CCH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 6.67), 3.41 d (4H, CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 21.17), 4.75 dg (4H, CHO, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 12.4, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 6.2). \u003csup\u003e13\u003c/sup\u003eC NMR spectrum (101.62 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (J, Hz): 23.85 d (4CH\u003csub\u003e3\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 4.87), 24.0 d (4CH\u003csub\u003e3\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 3.92), 24.0 d (CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e1\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 141.49), 24.9 (CH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 25.06 (CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 25.51 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 26.47 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 72.02 d (CHO, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 6.73), 159.98 d (C\u003csup\u003e2\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 10.38), 166.96 (C\u003csup\u003e5\u003c/sup\u003e). \u003csup\u003e31\u003c/sup\u003eP NMR spectrum (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e): δ 16.22 ppm. Mass spectrum (HRMS-ESI), m/z: 573.2206 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e40\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eNaO\u003csub\u003e8\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e: 573.2214).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eDimethyl ((5-(7-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)heptyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3ad).\u003c/b\u003e Viscous colorless oil. Yield 92%.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR (400.17 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 1.35 m (6H, CH\u003csub\u003e2\u003c/sub\u003e), 1.75 m (4H, CH\u003csub\u003e2\u003c/sub\u003e), 2.82 d (4H, CCH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 7.5), 3.44 d (4H, CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 21.3), 3.79 d (12H, CH\u003csub\u003e3\u003c/sub\u003eO, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 11.2). \u003csup\u003e13\u003c/sup\u003eC NMR (100.61 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 23.65 d (CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e1\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 142.3), 25.23 (CH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 26.18 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 28.55 (CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 28.64 (CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 53.49 d (CH\u003csub\u003e3\u003c/sub\u003eOP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 6.5), 159.24 d (C\u003csup\u003e2\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 9.9), 167.81 (C\u003csup\u003e5\u003c/sup\u003e). \u003csup\u003e31\u003c/sup\u003eP NMR (162 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e): δ 21.16 ppm. Mass spectrum (HRMS-ESI), m/z: 503.1427 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eNaO\u003csub\u003e8\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e: 503.1431).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDimethyl ((5-(8-(5-((dimethoxyphosphoryl)methyl)-1,3,4-oxadiazol-2-yl)octyl)-1,3,4-oxadiazol-2-yl)methyl)phosphonate (3ae).\u003c/b\u003e Viscous colorless oil. Yield 89%.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR (400.17 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 1.34 m (8H, CH\u003csub\u003e2\u003c/sub\u003e), 1.77 m (4H, CH\u003csub\u003e2\u003c/sub\u003e), 2.83 t (4H, CCH\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHH\u003c/sub\u003e 7.6), 3.46 d (4H, CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 21.3), 3.81 d (12H, CH\u003csub\u003e3\u003c/sub\u003eO, \u003csup\u003e3\u003c/sup\u003eJ\u003csub\u003eHP\u003c/sub\u003e 11.2). \u003csup\u003e13\u003c/sup\u003eC NMR (100.61 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e), δ, ppm (\u003cem\u003eJ\u003c/em\u003e, Гц): 23.67 d (CH\u003csub\u003e2\u003c/sub\u003eP, \u003csup\u003e1\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 142.4), 25.28 (CH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 26.26 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 28.8 (CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 28.84 (CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e), 53.49 d (CH\u003csub\u003e3\u003c/sub\u003eOP, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 6.5), 159.23 d (C\u003csup\u003e2\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eJ\u003csub\u003eCP\u003c/sub\u003e 10.0), 167.91 (C\u003csup\u003e5\u003c/sup\u003e). \u003csup\u003e31\u003c/sup\u003eP NMR (162 Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e): δ 21.19 ppm. Mass spectrum (HRMS-ESI), m/z: 517.1582 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eNaO\u003csub\u003e8\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e: 517.1588).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eantimicrobial activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the antimicrobial activity of the synthesized compounds 3aa-3ae, the following were used as test strains: \u003cem\u003eGram\u0026thinsp;+\u0026thinsp;ve\u003c/em\u003e bacterium \u003cem\u003eStaphylococcus aureus\u003c/em\u003e АТСС, \u003cem\u003eGram-ve\u003c/em\u003e bacterium \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e 0387, yeast-like fungus \u003cem\u003eCandida utilis\u003c/em\u003e LIA-01. The sensitivity of the strains to bis-1,3,4-oxadiazoles was determined on the Mueller-Hinton nutrient medium using the agar diffusion method (well method) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The inoculum concentrations of the daily test strains grown on meat-peptone agar were 1.5 x 10\u003csup\u003e8\u003c/sup\u003e CFU/ml (for bacteria) and 1.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU/ml (for yeast), which corresponds to a density of 0.5 according to the McFarland standard. Solutions of the test compounds were prepared in dimethyl sulfoxide. The procedure for applying the test strain inoculum to Mueller-Hinton agar and adding the compound solutions was presented previously. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Petri dishes were incubated in a thermostat at 35\u0026deg;C for 24 hours. After incubation, the presence of zones of inhibition or growth retardation of the test strains was noted. Statistical processing of the obtained data was performed using STATISTICA 10.0 software. Differences in the diameter of the growth inhibition zones for each of the studied compounds were assessed using one-way ANOVA and the Kruskal-Wallis test. Differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVirus and cells\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWe used MDCK cells (ATCC CCL-34) from the collection of cell lines of the Saint Petersburg Pasteur Institute. Cells were cultured in 96-well culture plates in MEM medium with 10% fetal bovine serum (\u0026laquo;HyClone\u0026raquo;, USA), 40 U/ml gentamicin sulfate and 2.5 U/ml amphotericin B. Cell suspension with a concentration of 10\u003csup\u003e5\u003c/sup\u003e cells/ml was placed in the wells of the plates in volume of 100 \u0026micro;l and cultured until a complete monolayer formation for 24 h at 36\u0026deg;C in the presence of 5% CO\u003csub\u003e2\u003c/sub\u003e. The same medium without serum was used as a support medium for culturing cells with viruses.\u003c/p\u003e\u003cp\u003eWe used influenza virus A/Puerto Rico/8/34 (H1N1) from the collection of the Saint Petersburg Pasteur Institute. The infectious titer of virus was determined by end-point titration in 96-well plates with monolayers of MDCK cells. The results were evaluated visually according to the presence of the virus cytopathic action, the virus titer was calculated by the Spearman-Kerber method and represented in decimal logarithms of 50% tissue cytopathic doses in ml (lg TCID\u003csub\u003e50\u003c/sub\u003e/ml).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of cytotoxic properties of compounds\u003c/h2\u003e\u003cp\u003eThe assessment of toxicity of compounds was carried out based on evaluation of the cell viability using the reduction reaction of the tetrazolium dye MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) by cells in culture. Its intensity shows the degree of cell viability as a result of dye reduction by mitochondrial and partially cytoplasmic dehydrogenases.\u003c/p\u003e\u003cp\u003eThe test compounds in the concentration range of 3.7\u0026ndash;300 \u0026micro;g/ml dissolved in the medium for cell cultivation were added to the plate wells in a volume of 200 \u0026micro;l and incubated for 48 h at 36\u0026deg;C in atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. At the end of the incubation period, the cells were washed with MEM medium, and 100 \u0026micro;l of a solution (0.5 mg/ml) of MTT in the cell medium was added to the plate wells. The cells were incubated at 36\u0026deg;C at 5% CO\u003csub\u003e2\u003c/sub\u003e for 2 h and washed for 5 min with saline. The precipitate was dissolved in 100 \u0026micro;l of DMSO per well, and the optical density was measured using a Multiscan FC plate analyzer (Thermo Scientific) at a wavelength of 540 nm. Based on the obtained data, the 50% cytotoxic concentration (CC\u003csub\u003e50\u003c/sub\u003e) was calculated, i.e. the concentration of the compound, which reduces the optical density in the wells by half compared to control cells without drugs. For this purpose, GraphPad Prism 6.01 software was used.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEvaluation of antiviral activity of compounds\u003c/h3\u003e\n\u003cp\u003eAnti-viral properties of compounds were evaluated by cytopathogenic effect (CPE) reduction assay. Briefly, the compounds in appropriate concentrations were added to MDCK cells (0.1 ml per well). After 1 h of incubation, cells were infected with influenza virus A/Puerto Rico/8/34 (H1N1) (moi 0.01) and incubated for 48 h at 36\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. After that, cell viability was assessed by MTT test as described above. The cytoprotective activity of compounds was considered as their ability to increase the values of OD comparing to control wells (with virus only, no drugs). Based on the results obtained, the values of IC\u003csub\u003e50\u003c/sub\u003e, i.e. concentration of compounds that result in 50% cells protection were calculated using GraphPad Prism 6.01 software. Values of IC\u003csub\u003e50\u003c/sub\u003e obtained in \u0026micro;g/ml were then calculated into micromoles (\u0026micro;M). For each compound the value of selectivity index (SI) was calculated as ratio of CC\u003csub\u003e50\u003c/sub\u003e to IC\u003csub\u003e50\u003c/sub\u003e. Compounds with SI of 10 and higher were considered active. Neuraminidase inhibitor oseltamivir carboxylate (Hoffman LaRoche, Switzerland) was used as a reference compounds.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompliance with ethical standards\u003c/h2\u003e\u003cp\u003eConflict of interest. The authors declare no competing interests\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe study was supported by the Russian Science Foundation grant No. 23-13-00224, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rscf.ru/project/23-13-00224/\u003c/span\u003e\u003cspan address=\"https://rscf.ru/project/23-13-00224/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributions Synthesis, GEA; Conceptualization and methodology, EDM, ILK, ZAZ, ZVV; Spectral assays, CEN, SDV; Biological testing, ZTB, ILK, ASV, MAN, ZVV; Supervision, EDM; Writing original draft, all authors; Editing the text, all authors; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to the Engineering Center of the St. Petersburg State Technological Institute (Technical University) and the Scientific Park of St. Petersburg State University \u0026ldquo;Research Centre for X-ray Diffraction Studies\u0026rdquo; (project no. 125021702335-5).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKumar A., Singh A.K., Singh H., Vijayan V., Kumar D., Naik J., et al. 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Chem. 2024;94:3173\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1134/S1070363224120065\u003c/span\u003e\u003cspan address=\"10.1134/S1070363224120065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medicinal-chemistry-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcre","sideBox":"Learn more about [Medicinal Chemistry Research](https://www.springer.com/journal/44)","snPcode":"44","submissionUrl":"https://submission.nature.com/new-submission/44/3","title":"Medicinal Chemistry Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"1,3,4-oxadiazole, phosphonate, antimicrobial activity, antiviral activity, cytotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-7856319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7856319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA series of new bis-1,3,4-oxadiazoles was synthesized by reacting the corresponding dicarboxylic acid bishydrazides with dialkyl chloroethynyl phosphonate. This reaction represents a new approach to the synthesis of 1,3,4-oxadiazoles, and the process's efficiency was demonstrated. The resulting compounds were tested against a range of test cultures: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC6538; \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e 0387; and \u003cem\u003eCandida utilis\u003c/em\u003e LIA-01. All synthesized compounds exhibited moderate antistaphylococcal activity and weak fungistatic action. The cytotoxicity and antiviral activity of the obtained compounds against the influenza A (H1N1) virus was studied \u003cem\u003ein vitro\u003c/em\u003e. Two of seven compounds under investigation (28%) have demonstrated high anti-viral activity against influenza virus A/Puerto Rico/8/34 (H1N1) with selectivity indices of 15 and 24.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Synthesis of New Phosphonylated Bis-1,3,4-oxadiazoles and Study of Their Antimicrobial and Antiviral Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-03 14:24:47","doi":"10.21203/rs.3.rs-7856319/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-20T01:44:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-16T14:33:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-16T14:32:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medicinal Chemistry Research","date":"2025-10-14T08:39:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medicinal-chemistry-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcre","sideBox":"Learn more about [Medicinal Chemistry Research](https://www.springer.com/journal/44)","snPcode":"44","submissionUrl":"https://submission.nature.com/new-submission/44/3","title":"Medicinal Chemistry Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a72457c8-396e-453d-9861-6d2f8f129a1b","owner":[],"postedDate":"November 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T15:58:51+00:00","versionOfRecord":{"articleIdentity":"rs-7856319","link":"https://doi.org/10.1007/s00044-025-03516-z","journal":{"identity":"medicinal-chemistry-research","isVorOnly":false,"title":"Medicinal Chemistry Research"},"publishedOn":"2025-12-16 15:56:54","publishedOnDateReadable":"December 16th, 2025"},"versionCreatedAt":"2025-11-03 14:24:47","video":"","vorDoi":"10.1007/s00044-025-03516-z","vorDoiUrl":"https://doi.org/10.1007/s00044-025-03516-z","workflowStages":[]},"version":"v1","identity":"rs-7856319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7856319","identity":"rs-7856319","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}