Bactrocera kraussi (Hardy) (Diptera: Tephritidae) is a polyphagous pest fruit fly for which the chemical profile of rectal glands is available for males but not for females. There have been no studies of the volatile emissions of either sex or of electrophysiological responses to these compounds. The present study (i) establishes the chemical profiles of rectal gland contents and volatiles emitted by both sexes of B. kraussi by gas chromatography–mass spectrometry (GC–MS) and (ii) evaluates the detection of the identified compounds by gas chromatography–electroantennogram detection (GC–EAD) and –electropalpogram detection (GC–EPD). Sixteen compounds are identified in the rectal glands of male B. kraussi and 29 compounds are identified in the rectal glands of females. Of these compounds, 5 were detected in the headspace of males and 13 were detected in the headspace of females. GC–EPD assays recorded strong signals in both sexes against -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, 2-ethyl-7-mehtyl-1,6-dioxaspiro[4.5]decane isomer 2, /-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, and -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. Male antennae responded to -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, 2-methyl-6-pentyl-3,4-dihydro-2H-pyran, 6-hexyl-2-methyl-3,4-dihydro-2H-pyran, 6-oxononan-1-ol, ethyl dodecanoate, ethyl tetradecanoate and ethyl (Z)-hexadec-9-enoate, whereas female antennae responded to -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and 2-methyl-6-pentyl-3,4-dihydro-2H-pyran only. These compounds are candidates as pheromones mediating sexual interactions in B. kraussi.Pheromones are biologically important in fruit fly mating systems, and also have potential applications as attractants or mating disrupters for pest management.  Bactrocera oleae, sex pheromones are produced by female flies undecane undecane as a major compound, its -isomer, diastereomer(s) of 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane, -2-ethyl-8-methyl-1,7-dioxaspiro-[5.5]undecane, 2,8-dimethyl-1,7-dioxaspiro[5.5]undecan-3-ol, N-(2-methylbutyl)acetamide, N-(3-methylbutyl)acetamide, diethyl butanedioate, 2-methyl-6-pentyl-3,4-dihydro-2H-pyran, 3-methylbutan-1-ol, 6-oxoundecan-2-ol and 6-oxononan-1-ol undecane, another isomer of 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane, 2-methyl-8-propyl-1,7-dioxaspiro[5.5]undecane, 2-ethyl-1-hexanol and 6-hexyl-2-methyl-3,4-dihydro-2H-pyran have been identified. Only five of the rectal gland compounds were detected in male headspace samples. The most abundant compound in both male rectal glands and headspace samples was -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (5), representing about 85.6% and 45.1% of the blends, respectively. N-(3-Methylbutyl)acetamide (4) was found as a minor compound (2.2%) in male rectal glands but appeared as a major compound (44.3%) in the headspace samples. A total of 29 compounds were identified in B. kraussi female rectal glands, including all the spiroacetals found in males, except 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane isomer 2, N-(3-methylbutyl)acetamide, and the 20 esters of saturated/unsaturated fatty acids -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (5), 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane isomer 1 (6) and -2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane (10) are in noticeably smaller amounts than in males. Of these, 13 compounds were detected in headspace samples. The predominant compound present in female gland extracts and headspace samples was ethyl dodecanoate (19), although it was found in higher proportions in the headspace samples (70.2% vs. 39.1%). The second major compound, ethyl tetradecanoate (25), had similar relative abundance in rectal gland extracts and headspace samples .The compositions of ty acids . The spiB. kraussi to the rectal gland extracts of conspecific males and females are presented in E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (5), its two diastereomers  and 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane isomer 2 (11) -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (5), the two dihydropyrans , 6-oxononan-1-ol (16), ethyl dodecanoate (19), ethyl tetradecanoate (25) and ethyl (Z)-hexadec-9-enoate (31), while female antennae produced signals corresponding to only -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (5) and 2-methyl-6-pentyl-3,4-dihydro-2H-pyran (8). -2,8-Dimethyl-1,7-dioxaspiro[5.5]undecane (2) elicited responses from both antennae and palps of both sexes.Electroantennographic (EAD) and electropalpographic detection (EPD) of male and female r 2 (11) . Male anB. kraussi rectal glands and headspace, and to describe the chemical profiles of male B. kraussi headspace. It is also the first report of electrophysiological detection to rectal gland volatiles in male and female B. kraussi. The combined analysis of rectal gland extracts and headspace (volatile) collections with GC–EAD/EPD provides a robust starting point for understanding the chemical communication of B. kraussi.This paper is the first to describe the chemical profiles of female B. kraussi males, except 3-methylbutan-1-ol and 2-hydroxyundecan-6-one undecane. The other new spiroacetals include 2,7-dimethyl-1,6-dioxaspiro[4.5]decane (2), diastereomer(s) (11) of 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane, and 2-methyl-8-propyl-1,7-dioxaspiro[5.5]undecane (14). The spiroacetal 2,7-dimethyl-1,6-dioxaspiro[4.5]decane (2) was assigned by examining an apparent molecular ion of m/z 170 and the fragment ions at m/z 101 and 98, indicative of a methyl substituted five-membered ring. The overall fragmentation patterns of 2,7-dimethyl-1,6-dioxaspiro[4.5]decane (2) are consistent with previous findings decane (11) undecane (14) was assigned by examining a molecular ion of m/z 212 and the fragment ions at m/z 143 and 140, indicative of propyl substituted, and at m/z 115, 112, indicative of methyl substituted six-membered rings. These patterns match those in the previous study decane isomer 2 (11) elicited very strong EPD signals and 6-hexyl-2-methyl-3,4-dihydro-2H-pyran (13) elicited EAD signals (N-(3-methylbutyl)acetamide (4) was a major compound in the male headspace samples but was minor in the rectal glands. Retention times and, hence, retention indexes of N-(3-methylbutyl)acetamide (4) and -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (5) are similar (see RIs in N-(3-methylbutyl)acetamide (4) and -2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (5) are 220–224 °C (Dehn 1912) and 225 °C undecane (5) originate from a fatty acid via different pathways, but 2-hydroxyundecan-6-one is the common and advanced intermediate undecane (5) undecane (5) and ethyl dodecanoate (19) are EAD active undecane isomer (9) and 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane isomer (11) that were present only in very minute amounts. These EPD results emphasize a high sensitivity of the EAD/EPD system, as well as the likely importance of the minor compounds in chemical communication. These compounds are volatile and these findings suggest that B. kraussi may utilize palps to detect some long-range chemical signals. Differences in olfactory function between antennae and maxillary palps are known in other Bactrocera species, including B. tryoni undecane (5) may be of particular biological importance as it elicited antennal and palpal responses in both sexes.Strong EPD responses to the four spiroacetals indicate that palps are more sensitive to the endogenous compounds than antennae in . tryoni ,38 and Bdepressa . EPD resdepressa ,39. The B. kraussi (G27), maintained using a carrot-based larval diet, was obtained from the Queensland Department of Agriculture and Fisheries (Cairns). Approximately 500 pupae were placed in a 47.5 × 47.5 × 47.5 cm fine mesh cage  for emergence and kept in a controlled environment room at 25 ± 0.5 °C, 65 ± 5% relative humidity (RH) and 11.5:0.5:11.5:0.5 light/dusk/dark/dawn photoperiod at Macquarie University, Sydney . Adult flies were fed with sugar and yeast hydrolysate  provided separately and tap water through a soaked sponge. Flies were reared for one generation at Macquarie University using a standard carrot diet undecane (5), 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane (6), N-(2-methylbutyl)acetamide (3), N-(3-methylbutyl)acetamide (4), 6-oxononan-1-ol (16), propyl dodecanoate (21), ethyl (Z)-hexadec-9-enoate (31) and ethyl (E)-octadec-9-enoate (35) were not available commercially, and were synthesised following literature procedures (see 2), 2-methyl-6-pentyl-3,4-dihydro-2H-pyran (8), -2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane (10), 2-ethyl-7-methyl-1,6-dioxaspiro[5.5]undecane isomer 2 (11), 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane isomers (9 and 12), 6-hexyl-2-methyl-3,4-dihydro-2H-pyran (13), 2-methyl-8-propyl-1,7-dioxaspiro[5.5]undecane (14), 2,8-dimethyl-1,7-dioxaspiro[5.5]undecan-3-ol (15) and ethyl (E)-hexadec-9-enoate (32) were tentatively identified based on the literature mass spectral fragmentation patterns . Ac. Ac43]. terature .N-(2-methylbutyl)acetamide (3) (N-(3-methylbutyl)acetamide (4) as a clear liquid . 1H NMR  δ 0.85 2), 1.33 , 1.56 , 1.92 , 3.18 , 6.21 . 13C NMR  δ 22.4, 23.1, 25.8, 38.0, 38.3, 170.4. GC–MS (EI) m/z (%) 129 , 114 , 73 (M+−CH2CH2CH(CH3)2, 100). Spectral data were consistent with the literature , Gr, Gr18], 1H NMR  δ 5.80 , 4.87–4.97 , 2.33 , 1.98 , 1.60 . 13C NMR  δ 210.9 (CO), 138.0 (CH=), 115.2 (CH2=), 41.9 (CH2), 33.1 (CH2), 22.8 (CH2). GC–MS (EI) m/z (%) 112 (14.6), 97 (30), 84 (27.8), 83 (10.6), 70 (14.1), 69 (59.5), 58 (48.7), 55 (49.6), 43 (24.5), 41 (100). Spectral data were consistent with the literature undecane. Hg(OAc)terature  under thterature .E,E)-2,8-Dimethyl-1,7-dioxaspiro[5.5]undecane (5) undecane (9). GC–MS (EI) m/z (%) 184 , 115 (CH3(C5H7O)=OH+, 100), 114 (37), 112 (CH3(C5H7O)=CH2, 39.5), 97 (73.1), 83 (11.8), 73 (27.5), 71 (12.4), 69 (59.9), 55 (41.8), 43 (38.6), 42 (24.6), 41 (38.4).(E)-Ethyl hex-4-enoate. Following the previously described method (E)-ethyl hex-4-enoate. In brief, 3-Buten-2-ol , triethylorthoacetate  and acetic acid  was heated at 140 °C for 4 h. The consumption of starting material at this time was determined by GC–MS. The reaction mixture was then cooled to room temperature and ethanol (20 mL) and water (20 mL) was added. The aqueous layer was extracted with diethyl ether (3 × 10 mL). The combined organic layers were stirred with hydrochroric acid solution  at room temperature for 30 min, then washed with brine (20 mL) and dried over magnesium sulfate. Concentration by rotary evaporation yielded (E)-ethyl hex-4-enoate as a clear yellow oil . GC–MS (EI) m/z (%) 142 (6.6), 97 (26.3), 96 (14.9), 88 (22.3), 71 (58.1), 70 (15.6), 69 (83.0), 68 (100), 67 (29.8), 60 (32.6), 55 (74.6), 43 (19.3), 42 (17.3), 41 (82.55). Experimental spectra were consistent with literature data undecane. Under this condition, a mixture of - and -isomers of 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]undecane was formed together with - and - isomers of 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane .2-Ethyl-7-methyl-1,6-dioxaspiro[4.5]undecane. Using a similar reaction and work-up conditions to 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, oxymercuration-reduction was performed to convert -2-Ethyl-7-methyl-1,6-dioxaspiro[4.5]undecane (5). GC–MS (EI) m/z (%) 184 , 169 , 155 , 140 , 126 (2.3), 115 (CH3(C5H7O)=OH+, 41.13), 114 (11.9), 113 (4.13), 112 (M+−CH2CHCH(OH)CH3, 30.7), 97 (51.82), 95 (10.2), 85 (51.7), 83 (23.4), 73 (22.9), 71 (14.5), 70 (14.7), 69 (64.3), 67 (9.7), 55 (100), 58 , 43 (89.2), 42 (44.6), 41 (79.3). Experimental spectra were consistent with literature data . A . A 48]. ure data .w/v aqueous sodium bicarbonate (10 mL) were added to the reaction mixture. The organic layer was separated and washed with 5% w/v aqueous sodium bicarbonate (3 × 10 mL) and dried over sodium sulfate  δ 5.32–5.35 , 4.11 , 2.29 , 1.57–1.66 , 1.25–1.29 , 0.93 , 0.87 . 13C NMR  δ 174.1 (C=O), 65.9 (OCH2), 34.5 (CH2), 32.0 (CH2), 29.7 (CH2), 29.6 (CH2), 29.47 (CH2), 29.40 (CH2), 29.3 (CH2), 25.1 (CH2), 22.8 (CH2), 22.1 (CH2), 14.2 (CH3), 10.5 (CH3). GC–MS (EI) m/z (%) 242 , 213 (1.4), 201 , 183 , 171 (6.6), 157 (6.8), 143 (3.3), 129 (8.7), 115 (21.8), 102 (32.5), 97 (7.7), 85 (12.2), 73 (39.3), 61 (100), 57 (30.9), 43 (80.2).A mixture of dodecanoic acid , 1-propanol (10 mL) and concentrated sulfuric acid (2 drops) was heated to reflux for 1.5 h. After cooling, diethyl ether (10 mL) and 5%  sulfate . The solZ)-hexadec-9-enoic acid , was esterified with ethanol (10 mL) in the presence of concentrated sulfuric acid (2 drops), quenched with sodium bicarbonate and diethyl ether and purified by distillation to afford ethyl (Z)-hexadec-9-enoate as a colourless oil . 1H NMR  δ 5.32–5.35 , 4.11 , 2.28 , 1.98–2.01 , 1.59–1.63 , 1.23–1.30 , 0.88 . 13C NMR  δ 174.0 (C=O), 130.1 (CH), 129.9 (CH), 60.2 (OCH2), 34.5 (CH2), 31.9 (CH2), 29.87 (CH2), 29.82 (CH2), 29.3 (CH2), 29.26 (CH2), 29.23 (CH2), 29.1 (CH2), 27.36 (CH2), 27.30 (CH2), 25.1 (CH2), 22.8 (CH2), 14.4 (CH3), 14.2 (CH3). GC–MS (EI) m/z (%) 282 , 236 , 218 (1.4), 207 (1.4), 194 , 179 (1.65), 165 (2.8), 152 (M+−(CH2)4COOCH2CH3, 14.9), 138 (6.9), 123 (11.9), 101 (31.8), 88 (50.6), 83 (44.9), 69 (64.1), 55 (100), 41 (81.8). Spectral data were not available in the literature.Using similar conditions used in the synthesis of propyl dodecanoate , (Z)-hexE)-octadec-9-enoic acid , was esterified with ethanol (10 mL) in the presence of concentrated sulfuric acid (2 drops), quenched with sodium bicarbonate and diethyl ether and purified by distillation to afford ethyl (E)-octadec-9-enoate as a colourless oil . 1H NMR  δ 5.36–5.28 , 4.11 , 2.27 , 1.95–1.96 , 1.57–1.60 , 1.23–1.28 , 0.87 . 13C NMR  δ 174.0 (C=O), 130.6 (CH), 130.4 (CH), 60.3 (OCH2), 34.5 (CH2), 32.78 (CH2), 32.73 (CH2), 32.0 (CH2), 29.8 (CH2), 29.7 (CH2), 29.6 (CH2), 29.4 (CH2), 29.36 (CH2), 29.30 (CH2), 29.1 (CH2), 25.1 (CH2), 22.8 (CH2), 14.4 (CH3), 14.2 (CH3). GC–MS (EI) m/z (%) 310 , 281 , 264 , 222 (11.3), 180 (11.2), 155 (7.0), 138 (5.6), 123 (13.5), 111 (20.6), 97 (38.6), 88 (45.6), 83 (49.0), 69 (69.0), 55 (100), 41 (76.4). Spectral data were consistent with the literature [Using similar conditions used in the synthesis of propyl dodecanoate , (E)-octterature .Coupled gas chromatography–electroantennogram detection (GC–EAD) or –electropalpogram detection (GC–EPD) was used to record electrophysiological responses. Whole fly heads were excised and secured between two silver wires with capillary electrodes filled with electrode gel . One electrode was placed at the tip of an antenna or palp and the other electrode at the bottom of the head. The antennal preparation was under charcoal filtered and humidified air flow (400 mL/min) controlled by a flow controller . The responses of antennae or maxillary palps were amplified/recorded by a data acquisition controller  and analysed using GCEAD 2014 software version 1.2.5. Separation of the individual compounds in the extracts was achieved on an Agilent 7890B gas chromatography (GC) system equipped with a split/splitless injector, fused silica capillary column SH-Rtx-5Sil MS (30 m × 0.25 mm ID × 0.25 μm film thickness) and flame ionization detector (FID). Hydrogen  was used as a carrier gas with a constant flow of 2.5 mL/min. An aliquot of 1 µL of a sample was injected at splitless mode. The temperature program was set initially at 50 °C (1 min), then increased to 260 °C (1 min) at a rate of 15 °C/min. The injector and detector temperatures were 270 °C and 290 °C, respectively. The effluent of the column was mixed with 30 mL/min make-up nitrogen gas and split with one part going to the internal FID and 1.5 passed to EAD or EPD through a heated transfer line , kept at constant temperature of 200 °C. Rectal gland extracts of both sexes were presented to antennae and palps of both sexes. The identities of FID peaks from the GC–EAD system were confirmed by comparing the retention times of GC–MS peaks that operated on the same type of column and under the same GC conditions.


Despite the fact that COVID-19 vaccines are already available on the market, there have not been any effective FDA-approved drugs to treat this disease. There are several already known drugs that through drug repositioning have shown an inhibitory activity against SARS-CoV-2 RNA-dependent RNA polymerase. These drugs are included in the family of nucleoside analogues. In our efforts, we synthesized a group of new nucleoside analogues, which are modified at the sugar moiety that is replaced by a quinazoline entity. Different nucleobase derivatives are used in order to increase the inhibition. Five new nucleoside analogues were evaluated with in vitro assays for targeting polymerase of SARS-CoV-2. The ongoing pandemic COVID-19 has already infected more than 142 million people worldwide resulting in over 3 million reported deaths, with significant social and economic repercussions − = 179.2-(6-Amino-9H-purin-9-yl) quinazolin-4-ol (6). To a two-neck round-bottom flask under inert atmosphere, a well-grounded mixture of adenine , 2-chloroquinazolin-4-ol , cesium carbonate , silica gel (3.5 g) and 20 mL of dry DMSO were added. The solution was stirred at 130 °C for 3.5 h. Cold water (200 mL) was added in the reaction mixture. Then, it was filtered and dried over-night at 50 °C. The desired product was anchored on the silica gel. Methanol (200 mL) was added, and the solution was placed in the ultra-sonic bath. After 15 min, it was filtered under vacuum and was evaporated. The mixture was purified by flash chromatography (CHCl3/MeOH) in order to obtain the desired product 2-(6-amino-9H-purin-9-yl) quinazolin-4-ol . 1H NMR  δ 9.18 , 8.17 , 7.99–7.97 , 7.57–7.52 , 7.42–7.40 , 7.22–7.18 , 13C NMR  δ 170.91, 160.57, 153.82, 153.15, 152.90, 150.67, 144.00, 131.67, 126.08, 124.71, 122.75, 121.67, 109.53. LC-MS (ESI): [M − H]− = 278.N-(9-(4-Hydroxyquinazolin-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)acetamide (7). To a two-neck round-bottom flask under inert atmosphere, a well-grounded mixture of N-acetyl guanine , 2-chloroquinazolin-4-ol , cesium carbonate , silica gel (3.5 g) and 20 mL of dry DMSO were added. The solution was stirred at 130 °C for 24 h. Cold water (200 mL) was added in the reaction mixture. Then, it was filtered and dried over-night at 50 °C. Water ultra-pure (200 mL) was added to the solid and the solution was placed in the ultra-sonic bath. After 15 min, it was filtered under vacuum and was evaporated. The mixture was purified by flash chromatography (CHCl3/MeOH) in order to obtain the desired product N-(9-(4-hydroxyquinazolin-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)acetamide . 1H NMR  δ 9.86 , 9.03 , 8.05 , 7.70 , 7.54 , 7.36 , 2.20 , 13C NMR  δ 169.27, 164.89, 161.99, 161.36, 154.84, 149.16, 146.90, 141.50, 133.79, 126.17, 125.85, 124.59, 120.88, 109.41, 24.57. LC-MS (ESI): [M − H]− = 336. Note: This compound is partially soluble in DMSO, which affects the NMR spectrum.N-(1-(4-Hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) acetamide (8). To a two-neck round-bottom flask under inert atmosphere, a well-grounded mixture of N-acetyl cytosine , 2-chloroquinazolin-4-ol , cesium carbonate , silica gel (3.5 g) and 20 mL of dry DMSO were added. The solution was stirred at 130 °C for 24 h. Cold water (200 mL) was added in the reaction mixture and was stirred for 20 min. After filtration under vacuum, a solution of HCl 1N was added and an off-white solid was precipitated. The solid was dried over-night at 50 °C in order to obtain the pure desired product N-(1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) acetamide . 1H NMR  δ 13.06 , 11.20 , 8.45–8.43 , 8.18–8.16 , 7.91–7.87 , 7.73–7.71 , 7.63–7.59 , 7.38–7.36 , 2.17 , 13C NMR  δ 171.32, 163.76, 161.45, 153.93, 147.55, 147.02, 145.80, 134.96, 127.51, 127.26, 126.17, 121.20, 96.42, 24.53. LC-MS (ESI): [M − H]− = 296.4-Amino-1-(4-hydroxyquinazolin-2-yl)pyrimidin-2(1H)-one (9). A round-bottom flask was charged with N-(1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide (8)  and 1 mL of 40% methylamine solution. The reaction was stirred at 65 °C for 24 h. The mixture was evaporated, and then it was diluted with 10 mL of MeOH and was filtered. The solid was dried over-night for 24 h at 50 °C in order to obtain the desired product 4-amino-1-(4-hydroxyquinazolin-2-yl)pyrimidin-2(1H)-one . 1H NMR  δ 13.43 , 8.39–8.37 , 8.13–8.11 , 7.90 , 7.86–7.82 , 7.65–7.63 , 7.54–7.50 , 6.05–6.03 , 13C NMR  δ 165.80, 160.95, 155.15, 147.79, 146.68, 140.93, 134.86, 126.45, 126.11, 120.55, 97.06. LC-MS (ESI): [M − H]− = 254.7-Fluoroquinazoline-2,4-dione (11). A round-bottom flask was charged with 2-amino-benzoic acid  and urea  and was placed at 150 °C for 7 h. The mixture was cooled to room temperature and 500 mL of water was added. Then, it was stirred for 2 h. The mixture was filtered under vacuum. The solid was diluted again with 100 mL of water (×3), was filtered under vacuum and dried at 50 °C over-night, in order to obtain the desired product 7-fluoroquinazoline-2,4-dione . 1H NMR  δ 11.29 , 7.96–7.92 , 7.03–6.99 , 6.90–6.87 . LC-MS (ESI): [M − H]− = 179.2,4-Dichloro-7-fluoroquinazoline (12). A round-bottom flask was charged with 7-fluoroquinazoline-2,4-dione , in POCl3 . Then, dropwise addition of N, N-dimethylaniline  was performed. The mixture was heated at 110 °C and kept at reflux for 3 h. The reaction mixture was cooled to room temperature and was poured onto an ice-CH2Cl2 mixture. Then, 30 mL of CH2Cl2 were added and washed with 15 mL of water (×3). All the organic layers were dried over Na2SO4 and evaporated to obtain a green solid. The solid was purified with flash chromatography (hexane/ethyl acetate) to afford the pure 2,4-dichloro-7-fluoroquinazoline  1H NMR  δ 8.44–8.40 , 7.95–7.91 , 7.86–7.81 . LC-MS (ESI): [M − H]− = 215.2-Chloro-7-fluoroquinazolin-4-ol (13). A round-bottom flask was charged with 2,4-dichloroquinazoline  and with solution of 2% sodium hydroxide (26.8 mL) and was stirred at 60 °C for 6 h. The reaction mixture was filtered and then was acidified with glacial acetic acid. A white solid was formed. The solid was filtered and dried for 1 h at 65 °C to afford pure 22-chloro-7-fluoroquinazolin-4-ol . 1H NMR  δ 13.38 , 8.16–8.12 , 7.44–7.38 . LC-MS (ESI): [M − H]− = 197.2-(6-Amino-9H-purin-9-yl)-7-fluoroquinazolin-4-ol (14). To a two-neck round-bottom flask under inert atmosphere, a well-grounded mixture of adenine , 2-chloro-7-fluoroquinazolin-4-ol , cesium carbonate , silica gel (0.70 g) and 2 mL of dry DMSO were added. The solution was stirred at 130 °C for 7 h. Cold water (20 mL) was added to the reaction mixture. Then, it was filtered and dried over-night at 50 °C. The desired product was anchored on the silica gel. Methanol (20 mL) was added, and the solution was placed in the ultra-sonic bath. After 15 min, it was filtered under vacuum and was evaporated. The mixture was purified by flash chromatography (CHCl3/MeOH) in order to obtain the desired product 2-(6-amino-9H-purin-9-yl)-7-fluoroquinazolin-4-ol  1H NMR  δ 9.13 , 8.28 , 8.12–8.10 , 7.28–7.26 , 7.20–7.16 , 13C NMR  δ 167.76, 166.29, 163.83, 159.72, 152.84, 152.64, 151.70, 151.57, 144.69, 129.03, 128.92, 118.04, 112.75, 112.52, 110.07, 109.86, 109.25 LC-MS (ESI): [M − H]− = 296.2-(6-Amino-9H-purin-9-yl) quinazolin-4-yl dihydrogen phosphate (15). A round-bottom flask was charged with phosphorus pentachloride , 11 mL of CH3CN and 11 mL of H2O and was placed in an ice-bath. A dropwise addition of a solution 30% H2O2 was performed within 10 min and the solution was stirred for 5 min. 2-(6-amino-9H-purin-9-yl) quinazolin-4-ol  and tetrabutylammonium bromide  were added to the flask and the reaction mixture was stirred for 5 h in the ice-bath. After completion of the reaction, the mixture was filtered and the solid was dried at 50 °C over-night. The solid was diluted to 20 mL MeOH and was stirred for 30 min. It was filtered and dried at 50 °C for 2 h to afford the desired product 2-(6-amino-9H-purin-9-yl) quinazolin-4-yl dihydrogen phosphate . 1H NMR  δ 9.01 , 8.35, , 8.17–8.15 , 7.87–7.84 , 7.66–7.64 , 7.55–7.52 , 13C NMR  δ 167.77, 160.17, 153.28, 153.62, 149.21, 148.13, 144.61, 133.22, 126.12, 125.28, 124.56, 120.90, 109.28, 31P NMR  δ −1.09 [M + H]+ = 360.In the assays, the RdRp catalytic domain of RNA-directed RNA polymerase  was used. As a template, we used the Genomic RNA from 2019 Novel Coronavirus, Strain: 2019-nCoV/USA-WA1/2020 , while primers (RNA or DNA) complementary to specific regions were obtained from Eurofins. In the reactions, we used the 10X First-Strand Buffer and RNase Inhibitor from the Amino Allyl MessageAmp™ II aRNA Amplification Kit , as well as dNTPs/rNTPs. A commercial inhibitor Cordycepin 5′-triphosphate sodium salt  was used as a positive control. The run conditions were 37 °C for 2 h, and samples were run on agarose gels.The positive sample included everything mentioned above (without the inhibitor), as well as commercial nucleotides, to verify that the synthesis is performed without any problems, while a second positive control, including the commercial inhibitor, was used to compare our analogues and their ability to terminate the reaction. Negative controls, including only primer, or without specific nucleotides, were used as well. In the reactions, where our nucleoside analogues were added, commercial normal nucleotides were also added at a ratio of 1:1. The products were run on agarose gels using an appropriate ladder. Gels were stained with MidoriGreen Dye, which stains dsDNA, ssDNA, dsRNA and ssRNA.In summary, a new class of nucleoside analogues were synthesized which contain a quinazoline moiety instead of a sugar entity. Five of these new compounds were tested in-vitro and based on the results, it was demonstrated that the addition of modified nucleoside analogues seems to inhibit the polymerization activity of SARS-CoV-2 RNA-dependent RNA polymerase.All of these results have been filed for a PCT patent.


Pinus needle tea are very popular in Eastern countries such as Japan, Russia, Korea, and China. Pine needle tea is claimed to have significant anti-aging effects, but no clear evidence has supported this until now. In the present study, five undescribed compounds (1–5) as well as seventy-two known compounds were purified and characterized from the bioactive fraction of methanol extracts of P. taiwanensis needles. Most of the isolates were examined for their anti-inflammatory bioactivity by cellular neutrophil model and six compounds  exhibited a significant inhibition on superoxide anion generation and elastase release with IC50 values ranging from 3.3 ± 0.9 to 8.3 ± 0.8 μM. These anti-inflammatory ingredients were subjected to docking computing to evaluate their binding affinity on the ghrelin receptor, which played an important role in regulating metabolism, with anti-aging effects. Compounds 49, 50, and 51 formed a stable complex with the ghrelin receptor via hydrogen bonds and different types of interactions. These results suggest the flavonoids are responsible for the potential anti-aging effects of pine needle tea. Pinus genus (Pinaceae), comprising more than 100 species and are mainly distributed in the Northern Hemisphere, are generally evergreen trees while some are shrubs 25D + 5.3 ; UV (MeOH) λmax (log ε) 284 (3.59), 253 (3.80) nm; ECD  λmax (Δε) 248 (+1.33), 224 (+1.03), 214 (+1.10) nm; IR (neat) νmax 3425, 2927, 1541, 1384, 1271 cm−1; 1H and 13C NMR; HRESIMS m/z 349.0936 .Colorless syrup; [α]25D + 30.8 ; UV (MeOH) λmax (log ε) 225 (sh) (3.83) nm; IR (neat) νmax 3450, 2937, 1648, 1252 cm−1; 1H-NMR  δ 0.63 , 1.09 , 1.21 , 1.21 , 1.42 , 1.51 , 1.59 , 1.79 , 1.91 , 1.91 , 1.98 , 2.01 , 2.08 , 2.12 , 2.14 , 2.43 , 4.03 , 4.53 , 4.91 , 5.88 ; 13C-NMR  δ 13.5 (CH3-20), 14.9 (CH3-16), 21.2 (C-2), 27.6 (C-6), 29.6 (CH3-18), 31.5 (C-11), 39.4 (C-3), 40.0 (C-7), 40.3 (C-1), 41.2 (C-10), 45.3 (C-4), 53.1 (C-9), 57.6 (C-5), 75.5 (C-12), 106.9 (C-17), 117.8 (C-14), 150.2 (C-8), 159.5 (C-13), 167.2 (C-15), 181.5 (C-19); HRESIMS m/z 349.2024 .Colorless powder; mp: 263 °C (dec.); [α]25D + 28.6 ; UV (MeOH) λmax (log ε) 237 (sh) (3.40) nm; IR (neat) νmax 3456, 2925, 1635, 1395 cm-1; 1H-NMR  0.88 , 0.98 , 1.84 , 2.02 , 2.09 , 2.31 , 2.50 , 2.70 , 6.32 ; 13C-NMR  δ 19.0 (C-9), 20.9 (C-10), 24.3 (C-4), 27.0 (C-6), 27.1 (C-8), 53.7 (C-5), 128.7 (C-2), 160.6 (C-1), 174.8 (C-7), 205.7 (C-3); HRESIMS m/z 181.0855 .Colorless tabular crystal; mp: 235 °C (dec.); [α]λmax (log ε) 308 (2.89), 237 (sh) (3.37), 222 (sh) (3.70) nm; IR (neat) νmax 3421, 2926, 1572, 1395, 1268 cm-1; 1H-NMR  1.82 , 2.61 , 3.56 , 3.83 , 6.86 , 7.31 ; 13C-NMR  δ 32.7 (C-7), 35.6 (C-8), 56.6 (OCH3-3), 62.3 (C-9), 116.3 (C-4), 120.1 (C-1), 122.8 (C-6), 131.9 (C-5), 149.2 (C-3), 150.7 (C-2), 176.2 (C-10); HRESIMS m/z 225.0767 .Colorless syrup; UV (MeOH) The study was conducted with the approval of the Institutional Review Board of Chang Gung Memorial Hospital (IRB No. 201800369A3). Blood samples were drawn from healthy human donors (20 to 30 years old), and neutrophils were isolated and purified according to the protocols described previously .c as described previously benzoic acid (1), 1--2-hydroxybenzoic acid (2), one new diterpenoid, -12-hydroxyagathic acid (3), one monoterpenoid, 5-isopropyl-3-oxocyclohex-1-ene-1-carboxylic acid (4), and one phenylpropane, styraxinolic acid (5). The chemical structures of these new compounds were constructed with the assistance of the NMR spectral elucidation and MS spectrometric analysis. Moreover, seventy-two known compounds, comprising one steroid, β-sitosterol (6); one sesquiterpenoid, (‒)-oplopan-4-one-10-α-O-β-D-glucoside (7); one coumarin, umbelliferone (8); one alkaloid, indole-3-aldehyde (9); four diterpenoids, acrostalic acid (10), 15-hydroxy-7-oxo-8,11,13-abietatrien-18-oic acid (11), 3β,13-dihydroxylabda-8(20),14-dien-19-oic acid (12), 12,15-dihydroxylabda-8(17),13-dien-19-oic acid (13); twenty-six lignans, -2,3-dihydro-3-hydroxymethyl-7-methoxy-2-(4′-hydroxy-3′-methoxyphenyl)-5-benzofuranpropanol 3α-O-α-L-rhamnopyranoside (14), -dihydrodehydrodiconiferyl alcohol-9-O-α-L-rhamnopyranoside (15), icariside E4 (16), massonianoside B (17), -dihydro-3′-hydroxy-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol (18), (±)-rel--7-O-methylcedrusin (19), cedrusinin (20), -idaeusin D (21), -4,9-dihydroxy-4′,7-epoxy-8′,9′-dinor-8,5′-neolignan-7′-oic acid (22), 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]propane-1,3-diol (23), evofolin-B (24), (S)-3-hydroxy-1,2-bis(4-hydroxy-3-methoxyphenyl)-1-propanone (25), cupressoside A (26), 1-(4′-hydroxy-3′-methoxyphenyl)-2-[2″-hydroxy-4″-(3-O-α-L-rhamnopyranosyloxypropyl)phenoxy]-1,3-propanediol (27), -3-methoxy-8,4′-oxyneoligna-3′,4,7,9,9′-pentol (28), erythro-3-methoxy-8,4′-oxyneolignan-3′,4,7,9,9′-pentol (29), pinoresinol (30), (+)-salicifoliol (31), (+)-idaeusinol A (32), schizandriside (33), (+)-isolariciresinol 2α-O-α-L-arabinoside (34), (+)-isolariciresinol (35), secoisolariciresinol (36), secoisolariciresinol-9,9′-acetonide (37), (‒)-nortrachelogenin (38), -2α-(4″-hydroxy-3″-methoxybenzyl)-3β-(4′-hydroxy-3′-methoxybenzyl)-γ-butyrolactone (39); twelve flavonoids, astragalin (40), kaempferol-3-O-β-D-galactopyranoside (41), kaempferol-3-O-α-L-furanoarabinoside (42), rhamnetin 3-O-β-D-glucopyranoside (43), apigenin (44), kaempferol-3,6-dimethyl ether (45), 5,7,8,4′-tetrahydroxy-3-methoxy-6-methylflavonol-8-O-β-D-glucopyranoside (46), 6-methylaromadendrin (47), naringenin (48), tiliroside (49), kaempferol 3-O--β-D-glucopyranoside (50), kaempferol-3-O-(5″-O-E-p-coumaroyl)-α-L-arabinofuranoside (51); seven ionones, machilusoxide A (52), (+)-(S)-dehydrovomifoliol (53), isololiolide (54), (S)-(+)-abscisic acid sodium salt (55), -3,5,6-trihydroxy-7-megastigmen-9-one (56), blumenol A (57), peltopterin B (58); nineteen benzenoids, 3,4-dihydroxybenzoic acid methyl ester (59), p-hydroxybenzoic acid (60), vanillic acid (61), 4-hydroxybenzaldehyde (62), methylparaben (63), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (64), 3-hydroxy-1-(4-hydroxyphenyl)-1-propanone (65), 2-(4-hydroxyphenyl)acetic acid (66), phenylacetic acid (67), isovanillic acid (68), benzoic acid (69), vanillin (70), p-hydroxyacetophenone (71), sodium salicylate (72), vannilic acid 4-O-α-L-rhamnoside (73), trans-ferulic acid (74), sodium p-coumarate (75), p-coumaric acid (76), trans-methyl p-coumarate (77), respectively, were identified by the examination of their physical and spectroscopic data with those previously published  and a conjugated carbonyl group (1598 cm−1). The 1H-NMR data , 6.86 , 7.24 , and 7.83 ], two oxygenated methines , and two methines . The 13C and DEPT NMR spectra  supported the relative configuration of 1 at C-7′/C-8′ as threo benzoic acid as shown in Compound NMR data  showed t spectra  of 1 discarbonyl . The 2J-rum of 1 . Moreoveas threo . The absterature ,38 and t spectra  furnishe2 was assigned as C17H18O8 on the basis of HR-ESI-MS analytical data  indicated the hydroxyl and conjugated carbonyl functionalities, respectively. Comparison of the NMR spectra of 1 and 2, it could observe that they possessed different aromatic moieties. Two sets of ABX-coupled aromatic ring and one methoxy group could be detected in the 1H-  and positive Cotton effect at 230 nm (Δε + 1.14), the absolute configuration of 2 was assigned as the threo- and -form, the same as 1 -2-hydroxybenzoic acid , and 1648 (carboxylic acid) cm−1 in its IR spectrum. It was evidenced by the 13C-NMR spectrum (C 167.2 (C-15) and 181.5 (C-19). In its 1H-NMR  and δC 75.5 (C-12) indicated the presence of a secondary alcohol group. The terminal methylene group could be established due to the proton resonances at δH 4.53  and 4.91 , and the carbon signals at δC 106.9 (C-17) and 150.2 (C-8), respectively. Two methyl groups at δH 0.63  and 1.21  were connected to the quarternary carbons (C-10 and C-4) evidenced by the HMBC correlations (3-20 (δH 0.63), suggesting its β-configuration [H 4.53) appeared in the upfield region, suggesting the 12R configuration [3-16 to C-12 and C-14; from CH3-18 to C-3, C-4 and C-19; from CH3-20 to C-1, C-5, C-9 and C-10, respectively, constructed the planar structure of 3 as previously reported for 12-hydroxyagathic acid [3 was determined as E by the NOESY analytical data -12-hydroxyagathic acid as shown  and 6.32 , two methylene groups at δH 1.84 , 2.02 , 2.50  and 2.70 , and one set of isopropyl protons at δH 0.88 , 0.98  and 2.31  appeared in the 1H-NMR spectrum of 4 (C 174.8 (C-7), and one carboxyl carbon at δC 205.7 (C-3) could be observed in its 13C- and DEPT NMR spectra  and carboxyl (1572 cm−1) functionalities could be detected. The 1H-NMR spectrum  and 7.31 , one methoxy group at δH 3.56 , and one set of propanol protons at δH 1.82 , 2.61  and 3.56 . Moreover, one carboxylic group was located at δC 176.2 (C-10) in its 13C-NMR spectrum -3-methoxybenzoic acid . Moreover, 48, 50, and 51   . Compounrophages . The aut45, 47, 48, 49, 50, and 51  and Arg283, C-5 hydroxyl and Gln120, C-7 hydroxyl and Tyr313, and C-4′ hydroxyl and Cys304, respectively. Arg199 formed a conventional hydrogen bond with a carbonyl group of p-coumaroyl. In addition, 49 was linked to the Arg283, Arg102, Asp99, Phe279, Phe312, Leu181, and Pro200 residues of the ghrelin receptor via different effects such as the π-cation, π-anion, π–π T-shaped, and π-alkyl interactions. These allowed compound 49 and protein to form a stable complex  were also observed with Asp99, Arg102, Arg283, Phe279, Phe312, Leu181, and Leu210  to link with Asp99, Arg283, Arg102, Leu181, Phe312, and Pro200 residues of ghrelin receptor could be detected (45, 47, and 48 with 49, 50, and 51, the former group possessed a flavonoid skeleton only while the latter group had sugar and coumaroyl moieties. It was reported that the coumaroyl group attached on the sugar was crucial for the binding affinity to the ghrelin receptor [49, 50, and 51 were higher than those of 45, 47, and 48, which suggested the better binding capability. The major structural characteristics were the coumaroyl groups rather than the sugar moieties and this could be further evidenced by examination of more compounds possessing coumaroyl functionalities. In this study, the active ingredients 49, 50, and 51 possessed not only anti-inflammatory bioactivity, but also the ghrelin receptor binding potential. This indicated that the claimed anti-aging effects of pine needle tea may be also based on these teaghrelin-like compounds. The lowest binding energy of each ligand was considered the best conformation. The binding affinities are listed in kcal/mol . This su complex B. 50 wasd Leu210 C. 51 alsdetected D. Comparreceptor . The gapreceptor . Moreovereceptor . AccordiP. taiwanensis needles. Their structures were characterized through spectroscopic and spectrometric analyses. Forty-three purified compounds were examined for their anti-inflammatory activity by the inhibition of superoxide anion generation and elastase release on neutrophil model. The results suggest that 45, 47, 48, 49, 50, and 51 possess significant anti-inflammatory potentials. Further molecular docking computing results supported 49, 50, and 51 exhibiting a binding affinity to the active pocket of the ghrelin receptor. Therefore, the crude extracts and purified constituents of P. taiwanensis have the potential to be developed as new anti-inflammatory lead drugs or food ingredients.A total of seventy-seven isolates comprising five undescribed compounds were purified from the methanol extracts of


Alternaria eureka 1E1BL1. Initially, oleandrin was isolated from the dried leaves of Nerium oleander L. and subjected to an acid-catalysed hydrolysis to obtain the substrate gitoxigenin . After 21 days of incubation, five new cardenolides 1, 3, 4, 6, and 8 and three previously- identified compounds 2, 5 and 7 were isolated using chromatographic methods. Structural elucidations were accomplished through 1D/2D NMR, HR-ESI-MS and FT-IR analysis. A. eureka catalyzed oxygenation, oxidation, epimerization and dimethyl acetal formation reactions on the substrate. Cytotoxicity of the metabolites were evaluated using MTT cell viability method, whereas doxorubicin and oleandrin were used as positive controls. Biotransformation products displayed less cytotoxicity than the substrate. The new metabolite 8 exhibited the highest activity with IC50 values of 8.25, 1.95 and 3.4 µM against A549, PANC-1 and MIA PaCa-2 cells, respectively, without causing toxicity on healthy cell lines (MRC-5 and HEK-293) up to concentration of 10 µM. Our results suggest that A. eureka is an effective biocatalyst for modifying cardenolide-type secondary metabolites.Microbial biotransformation is an important tool in drug discovery and for metabolism studies. To expand our bioactive natural product library via modification and to identify possible mammalian metabolites, a cytotoxic cardenolide (gitoxigenin) was biotransformed using the endophytic fungus  Microbial biotransformation is one of the most preferred and powerful procedures in natural product drug discovery and development studies enabling specific biochemical modifications of substrate molecules . An enzyNerium oleander L. is a perennial evergreen shrub that belongs to the family Apocynaceae. It is a widely cultivated ornamental herb, native to the Mediterranean region. In ethnobotanical literature, N. oleander is reported as a traditional medicine being used as abortifacient and for the treatment of a wide range of diseases such as dermatitis, eczema, skin cancer, viral infections, asthma, epilepsy, malaria, tumors, corns, leprosy, ringworm, and warts , suggested H-7 to be an oxymethine proton. The significant downfield shifts of C-6 (δC 35.9) and C-8 (δC 45.6) , and the long-distance correlations in the HMBC spectrum from C-7 (δ 69.1) to H2-6  and H-8  verified the monooxygenation at C-7 position (The molecular formula of position .H 3.84) signal showed NOESY correlation with the α-oriented H-9 (δH 1.58 m) establishing the β-orientation of the hydroxy group at C-7. Consequently, the structure of compound 1 was characterized as 7β-hydroxy-3-oxogitoxigenin.The relative orientation of the hydroxy group was determined using 2D-NOESY. The H-7  with eight indices of hydrogen deficiency (HD). Apart from the absence of hydroxymethine signal of C-3 and the downfield shift of carbon resonances belonging to A-ring, the characteristic signals of the gitoxigenin were present for 2. In the 13C-NMR spectrum, the resonance at δC 212.7 was evident for modification of C-3 secondary alcohol to a ketone, substantiating the increase in HD. Moreover, the HMBC correlations of C-3 with one of H2-2  and H2-4 (δH 2.62 m) resonances, justified the oxidation at C-3  indicating a molecular formula of C23H32O7. The disappearance of the oxymethine signal of C-3 and the presence of a carbonyl carbon signal at δC 214.5 implied a similar modification, as in 1 and 2. Examination of the HMBC spectra proved oxidation at C-3. In the spectrum, two additional low-field resonances (δH 3.91 and δH 3.36) were observed. Besides, two extra down-field carbon signals at δ 70.3 and δ 75.9 were also noted. In order to deduce the oxygenation positions, the 2D NMR spectra were studied thoroughly. Like 1, a monooxygenation at C-7 was confirmed via inspection of the COSY spectrum (H-7: δH 3.91). The second hydroxy group was located at C-12 on the basis of the HMBC spectrum, which showed a cross-peak between the C-12 (δC 75.9) signal and H-18 (δH 0.89 s). The relative orientations of hydroxy groups at C-7 and C-12 were determined based on the 2D-NOESY data. The H-12  signal showed NOE correlation with α-oriented H-17  verifying the orientation of C-12 hydroxy as β. Likewise, based on the NOE correlation observed between H-7  and α-oriented H-9 , C-7 hydroxy group was deduced as β-oriented. Finally, the structure of 3 was assigned as 7β,12β-dihdyroxy-3-oxo-gitoxigenin.The HR-ESI-MS spectrum of 4 was found to be C25H38O7 based on the major ion peak at m/z 449.2541 [M-H]− . Inspection of the 1H-NMR spectrum of 4 showed two O-methyl groups (δH 3.12 and δH 3.19) corresponding to the same carbon resonance (δC 47.7) in the HSQC spectrum suggesting a geminal dimethoxy substitution. Based on the absence of a low-field oxymethine signal due to H-3 in the 1H-NMR spectrum, and the observation of a new quaternary signal (δC 100.4) displaying long-range correlations with δH 3.12 and δH 3.19 signals in the HMBC spectrum, a geminal dimethoxy substitution at C-3 was deduced unambiguously. An additional low-field proton signal was also present at δH 3.96 indicating an oxymethine group in the structure. Examination of the COSY and HSQC spectra suggested the monooxygenation position to be C-7, as in 3. This assumption was confirmed by the HMBC experiment, which showed the long-range correlations from H2-6 (δH 1.57 and 1.92) and H-8 (δH 1.72) to the 70.9 ppm resonance (C-7). The stereochemistry of the hydroxy group at C-7 was established via 2D-NOESY spectrum. The observed cross peaks from H-7  to α-oriented protons H-9  and H-17  substantiated the orientation of C-7 hydroxy as β. Based on this evidence, the structure of 4 was determined as 3-dimethylacetal-7β-hydroxygitoxigenin.The molecular formula of 5 was identified as 3-epigitoxigenin by comparison of the spectroscopic data with those previously reported by Kawaguchi et al. [Compound i et al. .m/z 405.2280 [M-H]−  in the HR-ESI-MS spectrum of compound 6 indicated 16 amu increase over gitoxigenin and 5 (3-epi-gitoxigenin), implying a hydroxylated analogue of either of them. The 1H- and 13C-NMR spectra of 6 were found to be very similar to those of 5, except for an additional proton at δH 4.26 corresponding to a carbon signal at δC 70.5 in the HSQC spectrum /H-7 /H2-6 , together with the long-range correlations from C-7 (δC 70.5) to H2-6 and H-8 in the HMBC spectrum  and one of H-15 protons appeared at δH 2.67. In turn, the cross peak from α-oriented H-15  to H-7  confirmed the orientation of hydroxy group on C-7 as β. Additionally, the cross peak from H-3  to β-oriented H-5  in the 2D-NOESY spectrum was evident to verify the epimerization at C-3. Consequently, the structure of 6 was elucidated as 7β-hydroxy-3-epi-gitoxigenin.The major peak at spectrum . In the trum see  helped u7, the major ion peak was observed at m/z 449.2179 [M + FA-H]− , indicating the same molecular weight with that of 1. The presence of characteristic carbonyl carbon signal of C-3 ketone and appearance of a downfield proton resonance at δH 3.40  and H3-18  helped us to determine the location of hydroxy group as C-12. The relative configuration of C-12 was established via 2D-NOESY data. The strong correlation between H-12  and α- oriented H-17 , was evident to establish the orientation of C-12(OH) as β. On the basis of these evidence, 7 was identified as 3-oxo-diginatigenin, a known compound − , indicating 16 amu increase over gitoxigenin and 5 (3-epi-gitoxigenin). The 1H- and 13C-NMR spectra of 8 showed substantial resemblance with those of 5 rather than of gitoxigenin  helped us locating the hydroxy group at C-12. In the NOESY spectrum, H-12  showed correlation with α-oriented H-16 , establishing the orientation of OH-12 as β. Also, the NOESY correlation between H-3  and β-oriented H-5  verified the epimerization at C-3 indicating α-orientation of C-3(OH). In conclusion, the structure of 8 was elucidated as 3-epi-diginatigenin.The HR-ESI-MS spectrum of compound oxigenin . The 1H-1, 3, 4, 6 and 8 together with 3 previously identified compounds 2, 5 and 7. The structures and approximate yields were illustrated in The microbial biotransformation study on gitoxigenin afforded five new cardenolide derivatives 1–3 and 7 were mainly obtained. The formation of 3-oxogitoxigenin by Gibberella fujikuroi and Fusarium sp. was demonstrated in earlier biotransformation studies phenanthren-17-yl) furan-2(5H)-one) (gitoxigenin). White powder. FT-IR (CHCl3: MeOH): 3526, 3431, 2942, 2887, 1789, 1759, 1737, 1629, 1451, 1376, 1185, 1093, 1033, 888 cm−1. HR-ESI-MS (negative ion mode): m/z = 435.2387 [M + FA-H]− [Calculated for C23H34O5 [M + FA-H]− 435.2388]. [α]D18= +14.7° . 1H-NMR : see 13C-NMR : see 4--7,14,16-Trihydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one . White needles. FT-IR (CHCl3): 3405, 2942, 2873, 2156, 1739, 1712, 1631, 1617, 1449, 1341, 1284, 1110, 1051, 1032, 755 cm−1. HR-ESI-MS (negative ion mode): m/z = 403.2123 [M − H]− [Calculated for C23H32O6 [M-H]− 403.2126]. [α]D18= +20.4° . 1H-NMR : see 13C-NMR : see -14,16-Dihydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one : White crystalline powder. FT-IR (CHCl3: MeOH): 3443, 2926, 2863, 1739, 1714, 1615, 1447, 1285, 1109, 1032, 895 cm−1. HR-ESI-MS (negative ion mode): m/z = 433.2227 [M + FA-H]− [Calculated for C23H32O5 [M + FA-H]− 433.2231]. [α]D18 = +12.5° . 1H-NMR : see 13C-NMR : see -7,12,14,16-Tetrahydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one : Yellowish-white oily substance. FT-IR (CHCl3: MeOH): 3381, 2935, 2876, 1732, 1628, 1448, 1277, 1110, 1051, 1014 cm−1. HR-ESI-MS (negative ion mode): m/z = 419.2075 [M − H]− [Calculated for C23H32O7 [M − H]− 419.2075]. [α]D18= +12.7° . 1H-NMR : see 13C-NMR : see -7,14,16-Trihydroxy-3,3-dimethoxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one : White powder. FT-IR (CHCl3): 3382, 2937, 2874, 1736, 1616, 1450, 1363, 1279, 1178, 1107, 1094, 1051, 1035, 877, 755 cm−1. HR-ESI-MS (negative ion mode): m/z = 449.2541 [M + FA-H]− [Calculated for C25H38O7 [M + FA-H]− 449.2545]. [α]D18 = +20° . 1H- NMR : see 13C-NMR : see 4--3,14,16-Trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one : White needles. FT-IR (CHCl3: MeOH): 3411, 2935, 2865, 2156, 1735, 1615, 1450, 1379, 1170, 1114, 1093, 1073, 1034 cm−1. HR-ESI-MS (negative ion mode): m/z = 435.2385 [M + FA-H]− [Calculated for C23H34O5 [M + FA-H]− 435.2388]. [α]D18= + 9.2° . 1H-NMR : see 13C-NMR : see -3,7,14,16-Tetrahydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one : White needles. FT-IR (CHCl3: MeOH): 3391, 2936, 2869, 2156, 1734, 1455, 1177, 1112, 1051, 774 cm−1. HR-ESI-MS (negative ion mode): m/z = 405.2280 [M − H]− [Calculated for C23H34O6 [M − H]− 405.2283]. [α]D18 = +6.9° . 1H-NMR : see 13C-NMR : see -12,14,16-Trihydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one : White powder. FT-IR (CHCl3: MeOH): 3394, 2924, 2865, 1733, 1713, 1630, 1455, 1287, 1019, 775 cm−1. HR-ESI-MS (negative ion mode): m/z = 449.2179 [M + FA-H]− [Calculated for C23H32O6 [M + FA-H]− 449.2181]. [α]D18= +21.9° . 1H-NMR : see 13C- NMR : see -3,12,14,16-Tetrahydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one : White powder. FT-IR (CHCl3: MeOH): 3390, 2929, 2863, 1732, 1614, 1559, 1451, 1074, 1035, 1027 cm−1. HR-ESI-MS (negative ion mode): m/z = 405.2280 [M − H]- [Calculated for C23H34O6 [M − H]− [405.2355]. [α]D18 = 0.0° . 1H-NMR : see 13C-NMR : see (® CRL-1469™), MIA PaCa-2 , DU 145  and A549  and two healthy cell lines MRC-5  and HEK-293  according to manufacturer’s instructions . For this purpose, cells at 80–90% confluency were seeded to 96-well plates as 5-10x103 cells/well in Eagle Minimal Essential Medium (EMEM) or Dulbecco Modified Eagle Medium (DMEM); supplemented with 10% FBS and 1% L-glutamine depending on the cell type and incubated in a humidified incubator at 37 °C with 5% CO2 atmosphere for 24 h. After cell adhesion, gitoxigenin and the metabolites were applied to the cells in triplicate with concentrations ranging between 0.001 and 10 µM. After 48 h exposure to certain concentrations of the compounds, medium was replaced with fresh one containing 10% MTT and plates were incubated for 3 h in the dark for the conversion of tetrazolium salts to formazan crystals by mitochondrial metabolism of viable cells. After incubation, formazan crystals were dissolved in DMSO and absorbance of each well were measured at 570 nm via microplate reader . Doxorubicin  was used as positive control, whereas oleandrin  was the second positive control as a potent cardenolide.To see cytotoxic effects of the obtained metabolites, a standard MTT -2,5-diphenyltetrazolium bromide) cell viability assay was performed on human cancer cell lines PANC-1 , which is non-toxic to cells. The IC50 values of the compounds were calculated by the normalization of optical densities (OD) to negative control DMSO .All compounds were dissolved in DMSO  and the final DMSO concentration applied to cells was 0.02% (


H)-one derivatives containing different substituents at C-5 of thiazole ring and tested their activity towards inhibition of two 11β-HSD isoforms. For most of them, over 50% inhibition of 11β-HSD1 and less than 45% inhibition of 11β-HSD2 activity at the concentration of 10 µM was observed. The binding energies found during docking simulations for 11β-HSD1 correctly reproduced the experimental IC50 values for analyzed compounds. The most active compound 2-(adamantan-1-ylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one (3i) inhibits the activity of isoform 1 by 82.82%. This value is comparable to the known inhibitor-carbenoxolone. The IC50 value is twice the value determined by us for carbenoxolone, however inhibition of the enzyme isoform 2 to a lesser extent makes it an excellent material for further tests.A common mechanism in which glucocorticoids participate is suggested in the pathogenesis of such metabolic diseases as obesity, metabolic syndrome, or Cushing’s syndrome. The enzyme involved in the control of the availability of cortisol, the active form of the glucocorticoid for the glucocorticoid receptor, is 11β-HSD1. Inhibition of 11β-HSD1 activity may bring beneficial results for the alleviation of the course of metabolic diseases such as metabolic syndrome, Cushing’s syndrome or type 2 diabetes. In this work, we obtained 10 novel 2-(adamantan-1-ylamino)thiazol-4(5 According to the World Health Organization (WHO), obesity is one of the most common metabolic diseases. It is also recognized as a global epidemic of the 21st century . The pheCortisol, which is one of the main glucocorticoids (GCs) secreted by the cortex of the adrenal gland, helps maintain homeostasis. Its concentration increases during times of stress. In addition, it is involved in the regulation of carbohydrate, lipid, and protein metabolism. It causes the release of fatty acids and also increases the number of neutrophils or platelets. It also shows anti-inflammatory properties .In 1953, it was proved that the conversion of cortisol in humans is related to the activity of the 11β-HSD enzyme. Its presence was also confirmed in various tissues, including in the kidneys or placenta. Subsequent studies showed that the liver converts cortisone into cortisol, which led to the hypothesis of the existence of two isoforms of this enzyme, which were described in the late 1990s ,11. 11β-11β-HSD1 activity is elevated in subcutaneous adipose tissue in obese patients ,14,15. IThe above reports indicate that the search for selective 11β-HSD1 inhibitors may support the therapeutic process in patients with obesity and the metabolic syndrome.Heterocycles including nitrogen and sulfur have been investigated for a long time because of their synthetic diversity and therapeutic importance. Among a wide variety of heterocyclic compounds, thiazoles and their derivatives are favored candidates for the synthesis of pharmaceuticals .Compounds containing dihydrothiazoles in their structure exhibit a broad spectrum of biological activity, including antiproliferative, antibacterial, anti-inflammatory, antiparasitic, and antifungal activity ,21. ThiaOur previous research has also shown that some of the thiazolone derivatives may be selective inhibitors over one of the 11β-HSD isoforms  30,31,3,33,34.On the other hand, a group of compounds of interest from the pharmacological point of view includes the adamantane (tricyclo [3.3.1.13.7]-decane) derivatives. The introduction of the bulky and lipophilic adamantyl groups reduces the lability of the molecule and allows a better fit in the hydrophobic receptor/catalyst pocket, which may be directly related to biological activity. The adamantyl group positively modulates the therapeutic index of many experimental compounds increasing drug-like qualities of a lead compound, without increasing toxicity .Several classes of selective, non-steroidal adamantyl-based 11β-HSD1 inhibitors have been published. These include adamantine triazoles such as the Merck compound 544 , amides H)-one derivatives as potential 11β-HSD1 inhibitors.For this reason, we focused our attention on derivatives containing both the thiazolone ring and the adamantyl group by synthesizing a series of new 2-(adamantylamino)thiazol-4(5H)-one derivatives were obtained by reacting 1-(adamantan-1-yl)thiourea with 2-bromo esters  at C-5 was promising, the yield of the reaction was 60.1–75.8%. In the accordance with the same procedure, the reaction of 1-(adamantan-1-yl)thiourea with esters containing aromatic substituents allowed obtaining compounds 3g and 3h, with good (66.2%) and moderate (25.5%) yields, respectively. Unfortunately, under these conditions, the synthesis of 3e–3f derivatives with branched substituents at C-5 did not give the expected results. TLC analysis showed the formation of trace amounts of products, even within long reaction times (the progress of the reaction was monitored for 4 months). Therefore, these compounds were obtained by heating under reflux in an alkaline medium (sodium methoxide). The change of the reaction conditions allowed obtaining compounds 3e–3f with isolated yields of 15.8–18.7%. Our previous research on the synthesis of 2-aminothiazol-4(5H)-one derivatives containing the spiro thiazole and alicyclic ring system showed that compounds of that type could be obtained by prolonged heating of the reactants in ethanol in the presence of N,N-diisopropylethylamine  . Rf : 0.23.2-(adamantan-1-ylamino)thiazol-4(5H)-one (3b)—Yield: 75.8%. M.p. 265–267 °C. 1H-NMR : 11.88 , 4.30 , 2.22 , 2.16 , 1.75 , 1.72 . 13C-NMR 13C-NMR : 170.89 (C-4), 169.87 (C-2), 58.75A , 44.34 (C-5), 40.20B , 35.00C , 28.82D , 17.45 (CH3). HR-MS m/z 265.1374 [M+ + 1] . Rf : 0.38.2-(adamantan-1-ylamino)-5-methylthiazol-4(5H)-one (3c)—Yield: 66.1%. M.p. 257–259 °C. 1H-NMR : 11.98 , 4.24 , 2.23 , 2.21–2.18 , 2.17 , 2.10–2.02 , 1.72 , 1.11 . 13C-NMR : 170.39 (C-4), 170.35 (C-2), 58.66A , 51.90 (C-5), 40.15B , 34.99C , 28.82D , 24.96 (CH2), 10.40 (CH3). HR-MS m/z 279.1536 [M+ + 1] . Rf : 0.46.2-(adamantan-1-ylamino)-5-ethylthiazol-4(5H)-one (3d)—Yield: 60.1%. M.p. 247–249 °C. 1H-NMR : 11.91 , 4.26 , 2.27 , 2.21–2.18 , 2.17 , 1.98–1.90 , 1.72 , 1.56–1.46 , 1.01 . 13C-NMR : 170.52 (C-4), 170.32 (C-2), 58.66A , 50.43 (C-5), 40.16B , 35.00C , 33.53 (CH2), 28.82D , 19.94 (CH2), 12.98 (CH3). HR-MS m/z 293.1696 [M+ + 1] . Rf : 0.53.2-(adamantan-1-ylamino)-5-propylthiazol-4(5H)-one (3e)—Yield: 18.7%. M.p. 239–241 °C. 1H-NMR : 5.46 , 4.25 , 2.61–2.55  2.11 , 2.09 , 1.69 , 1.25 . 13C-NMR : 190.18 (C-4), 177.80 (C-2), 63.89A (1C. C10H15), 56.49 (C-5), 41.07B , 35.56C , 30.32 2), 29.30D , 29.10D , 28.97D , 22.15 2), 15.78 2). HR-MS m/z 293.1697 [M+ + 1] . Rf : 0.50.2-(adamantan-1-ylamino)-5-isopropylthiazol-4(5H)-one (3f)—Yield: 15.8%. M.p. 208–210 °C. 1H-NMR : 5.47 , 2.15 , 2.12 , 1.78 , 1.69 , 1.63 . 13C-NMR : 176.12 (C-4), 100.62 (C-2), 61.11A , 53.17 (C-5), 42.50B , 35.72C , 29.53D , 17.45 2). HR-MS m/z 279.1535 [M+ + 1] . Rf : 0.48.2-(adamantan-1-ylamino)-5,5-dimethylthiazol-4(5H)-one (3g)—Yield: 66.2%. M.p. 252–254 °C. 1H-NMR : 7.37–7.28 , 5.55 , 5.22 , 2.17 , 2.14 , 1.71 . 13C-NMR : 178.03 (C-4), 177.55 (C-2), 129.24 (1CPh), 128.91 (2CPh), 128.34 (1CPh), 128.23 (2CPh), 59.54A , 57.26 (C-5), 41.54B , 35.95C , 29.54D . HR-MS m/z 327.1532 [M+ + 1] . Rf : 0.53.2-(adamantan-1-ylamino)-5-phenylthiazol-4(5H)-one (3h)—Yield: 25.4%. M.p. 320 °C (dec.). 1H-NMR : 7.56–7.49 , 7.33 , 7.31–7.25 , 2.02 , 1.96 , 1.59 . HR-MS m/z 405.0639 [M+ + 1] . Rf : 0.63.2-(adamantan-1-ylamino)-5-(4-bromophenyl)thiazol-4(53i)—Yield: 14.6%. M.p. 268–270 °C. 1H-NMR : 5.43 , 2.12 , 2.05 , 1.99–1.75 , 1.70 , 1.40–1.22 . 13C-NMR : 193.39 (C-4), 177.15 (C-2), 70.66 (C-5), 56.79A , 41.58B , 36.73 , 35.97C , 29.52D , 25.50 , 24.86 . HR-MS m/z 319.1848 [M+ + 1] . Rf : 0.53.2-(adamantan-1-ylamino)-1-thia-3-azaspiro [4.5]dec-2-en-4-one (3j)—Yield: 28.3%. M.p. 265–266 °C. 1H-NMR : 5.75 , 2.86–2.76 , 2.54–2.44 , 2.33–2.23 , 2.19–2.13 , 2.10 , 2.04 , 1.70 . 13C-NMR : 193.19 (C-4), 175.71 (C-2), 61.52 (C-5), 56.37A , 41.11B , 35.56C , 33.89 , 29.11D , 16.52 . HR-MS m/z 291.1535 [M+ + 1] . Rf : 0.55.6-(adamantan-1-ylamino)-5-thia-7-azaspiro[3.4]oct-6-en-8-one , microsomes  solution in PBS , phosphate buffer , and 10 µL of compounds 3a–3j dissolved in the mixture containing 1% of DMSO and 99% of water. The resulting solution with the final volume of 100 µL was incubated for 2.5 h at 37 °C. To stop the reaction, 10 μL of a 100 μM solution of carbenoxolone in PBS was added. The level of unreacted cortisol was measured by commercially available ELISA kit.Human liver microsomes were used as a source of 11β-HSD2 enzyme to study the inhibitory effect of 50 values for compounds 3a–3j were obtained using their solutions at concentrations of 0.625, 1.25, 2.5, 5.0, and 10.0 µM and using standard procedure and conditions as described in the previous sections. As a control, analogous tests without the addition of inhibitors were used. Half the inhibitory concentration (causing 50% reduction of cortisol or cortisone) was read from the chart.Calibration curves to determine IC3 which covers the originally co-crystallized ligands present in the PDB structures as well as the closest amino-acid residues that exhibit contact with those ligands. All the default procedures and algorithms implemented in AutoDock Vina were applied during the docking procedure. In addition to the flexibility of the ligand molecules, the rotation of selected sidechains  in the proximity of the co-crystalized ligands was allowed. The predicted binding energies were averaged over all the eight protein structures. In addition, in the case of chiral ligands, the energies were averaged over two stereoisomers. The more favorable binding mode is associated with the lower binding energy value; only the lowest energy values and ligand poses associated with them were considered in the subsequent analysis.Ligand molecules see  were draThe docking methodology was initially validated by docking simulations of the ligand molecule originally included in the protein structure. The description of the validation procedure and the graphical illustration of its results can be found in . MoreoveH)-one derivatives containing different substituents at C-5 of thiazole ring and tested their activity towards inhibition of two 11β-HSD isoforms. All the obtained compounds show inhibitory activity against both isoforms to a different extent (15.3–82.8% for 11β-HSD1 and 14.4–44.7% for 11β-HSD2).In conclusion, we obtained 10 new 2-(adamantan-1-ylamino)thiazol-4(550 values for analyzed compounds. Molecular docking shows that the driving force for binding between ligands and protein related to the hydrophobic interactions of the ligand with Tyr177 and neighboring leucines, supported by the π–π interactions with Tyr183 and NADP+, whereas divergences between binding energies across the whole group of compounds can be ascribed to the interactions between the substituent attached to the thiazole ring and the region of the protein located in the vicinity of Thr124 and Thr222.The binding energies found during docking simulations for 11β-HSD1 correctly reproduces the experimental IC3i at the concentration of 10 µM inhibits the activity of isoform 1 by 82.82%. This value is comparable to the known inhibitor-carbenoxolone. The IC50 value is twice the value determined by us for carbenoxolone, however, inhibition of the enzyme isoform 2 to a lesser extent makes it an excellent material for further tests.The most active compound


Synthesized molecules were characterized according to their structure with 1H NMR, 13C NMR and FT-IR spectral foundations, which were compatible with literature informations. Antimicrobial activity and cytotoxicity studies were done by disc diffusion and NCI-60 sulphordamine B assay methods. The antimicrobial test results revealed that synthesized compounds have better activity against gram-positive species than gram-negative ones. A total analysis of the antibacterial, antifungal, and antiyeast activity revealed that newly synthesized compounds were really active againstBacillus cereus,Bacillus ehimensis, andBacillus thuringiensis species.For cytotoxicity, among three different cancer cell lines  compounds5c, 5d, 5e, 5f, 5g, 5i, 5jand5kwere seemed especially effective on HUH7 cancer cell line via moderate to good activity. More significantly, against liver carcinoma cell line (HUH7) most of the compounds of the series (5c-5gand5i-5j) have better IC50 values (IC50= 18.78 µM) than 5-Florouracil (5-FU) and also compound5dpossessed 10.1 µM value, which represents good druggable cytotoxic activity. Further, the molecules were also screened for in silico chemoinformatic and toxicity data to gather the predicted bioavailibity and safety measurements. The reaction of 3,4-dichlorophenyl-1,3,4-oxadiazole-2( Antimicrobial drugs are known as a group of pharmaceuticals that preserve various defensive effects against bacteria, fungi, virus, and parasites. It is well recognized that many antimicrobial agents are necessary to treat life-threatening infections, but improved bacterial resistance against these medications may cause worser consequences for human health. Antimicrobial resistance occurs naturally over time, when responsible strains of a microorganism exchange among microorganisms usually through genetic material. This proceeding problem of currently marketed antimicrobials have speed up the birth of new variants of mechanisms about resistance and fairly fast rise in the number of microorganisms which spread among all over the world [1–4]. To prevent the risk of increase in antimicrobial resistance, different therapy strategies should be applied according to the type of microorganisms (drug-resistant or not) and disease severity. Since 2005, World Health Organization (WHO) has developed a criteria to order antimicrobials according to their relative importance in human medicine and the study has been used by clinicians to preserve the usage quality of currently available drugs [5]. This commercially available medicines which are used for infection and cancer treatments have a common point: the emergence of resistance against multiple drugs . Other related problems are insufficient selectivity and undesirable side effects consequences of the patients . Hence, there is a massive requirement for the improvement of new antimicrobial and anticancer therapies, with higher selectivity, besides serving fewer side effects than current ones. So, the main goal is resistance prevention, good potential of action, and last but not least, diminished side effects [9–12]. As well as developing antimicrobial treatment strategies, new studies on antimicrobials have been maintained as cancer theraphy due to strong evidences about the relationship between microbial diseases and cancer. This data supports the combined usage of antimicrobial and anticancer medications in clinical area. In addition, studies on direct antiproliferative activity of certain antimicrobials and prophylactic effect of antimicrobials against post-chemotheraphy infections due to immunosupression are being discussed with their possible mechanisms about cancer treatment [13]. Today, it is reported in the literature that antiproliferative activity of clinically used antimicrobials is related to the topoisomerase enzyme inhibition , degradation of tumorigenic proteins , destabilization [18], antiangiogenic effects and apoptosis [19–24].50 values of 0.47–1.4 µM [26].1,3,4-Oxadiazole is generally used entity for pharmacophore development and has been investigated because of its good metabolic profile and hydrogen-bonding capacity within the receptor site. Presence of azole group (N=C-O) also elevates lipophilicity feature of compound, which provides advantage for its transportation through cell membrane to reach the target site and show various biological activities [25]. These co-operative properties provide great benefits to obtain desired antimicrobial and anticancer activity within various proven in vitro and in vivo models. In 2013, Du and coworkers dealed with modeling 1,3,4-oxadiazole ring to obtain both of anticancer and antimicrobial effect by targeting thymidylate synthase (TS), which is an important enzyme for DNA synthesis. Newly synthesized compounds were identified as potent inhibitors against two kinds TS proteins with ICNowadays, dual antimicrobial-anticancer activity of 1,3,4-oxadiazole core structure has been an important concern. Ahsan et al. dealed with disubstituted derivatives of 1,3,4-oxadiazole with antimicrobial-anticancer activity capacity ,and synthesized analogs showed moderate to severe potency for this binary physiological topics [27]. In another research, Savariz and coworkers studied with 3,5-disubstituted-1,3,4-oxadiazole pharmacophore group, which were derived with different functional moieties via Mannich reaction. Results showed that both anticancer and antimicrobial activity of synthesized series have intermediate to excellent effect, and especially one of the compounds, which has a heterocyclic ring from third position of core structure, improved antitumor activity, which was found to be 4.5 timefold compared to the precursor molecule [28]. Under the shadow of previous experiences, Selvaraj and coworkers synthesized fifteen 1,3,4-oxadiazole derivatives, and they were found to have moderate to severe inhibitor effect against different microbial and cancer cell lines in 2017 [29].3H)-thione derivatives carrying different piperidine side chains. The compounds were evaluated for their antimicrobial and cytotoxicity profile to investigate the effect of molecular variations on activity against different bacteria, fungi, and yeasts. Further, compounds were tested in against various cancer cell lines for their cytotoxic activity. Based on the statements above, to develop new, potent antimicrobial and anticancer agents, we aimed to synthesize a new series of 3,5-disubstituted-1,3,4-oxadiazole-2 using tetramethylsilane as the internal reference, with dimethyl Sulfoxide (DMSO-d6), as solvent, the chemical shifts were reported in parts per million (ppm), and coupling constants (J) were given in hertz (Hz). Elemental analyses were done by LECO 932 CHNS instrument  and were within ± 0.4% of the theoretical values.Melting points of compounds were checked by Mettler Toledo FP62 capillary melting point apparatus  and are uncorrected. Infrared spectral data were obtained by the use of Perkin-Elmer Spectrum One series FT-IR apparatus (Version 5.0.1) , with potassium bromide pellets and the frequencies were presented in cmSolution of aroyl hydrazine (3.13 mmol) and carbon disulfide (6.27 mmol) in absolute ethanol (15 mL) were mixed in cold media (0 °C) and after the addition of potassium hydroxide (3.13 mmol) in one portion, the mixture was refluxed for 8 h. After the reaction was over, solvent was evaporated and the residue was acidified with 2M hydrochloric acid and extracted with ethyl acetate (2 × 20 mL). The organic layers were washed with water and dried with anhydrous sodium sulphate. Filtration and concentration in vacuo gave a solid, which was recrystallized from ethanol to give the compound [30].H)-thione , an appropriate N-substituted amine (3 mmol) and 37% formaldehyde solution (1 mL) in ethanol (15 mL), was refluxed 3–5 h. The crude products were either precipitated or it was necessary to add water in case of not precipitated. The crude products were filtered, washed with water, dried, and crystallized from ethanol or ethanol/water. A mixture of 5--1,3,4-oxadiazole-2(3max : 3075-3024 (Aromatic C-H), 1611 (C=N), 1435 (C=C), 1318 (C=S), 1238 (C-O-C) cm–1; 1H-NMR  ppm: δ = 8.07 , 7.79 , 7.61 , 7.34-7.19 , 5.11 , 3.16 , 2.65 , 1.76 , 1.65 , 1.66–1.62 ; C13-NMR  δ 179.9 (C=S), 159.8 (C=N), 151.5 , 134.9 , 132.4 , 131.8 , 128.7 , 126.7 , 126.2 , 126.1 , 125.1 , 122.1 , 73.5 (N-CH2-N), 69.0 , 46.2 , 37.7 ; Anal. Calcd. for C20H19Cl2N3OS: C, 57.15; H, 4.56; N, 10.00; S, 7.63. Found: C, 57.32; H, 4.56; N, 10.14; S, 7.66.White powder, yield 87%, Mp 182.0°C; FT-IR (KBr) νmax : 3456 (O-H), 3077 (Aromatic C-H), 1435 (C=N), 1417 (C=C), 1317 (C=S), 1235 (C-O-C) cm–1; 1H-NMR  ppm: δ = 8.06 , 7.86 , 7.46 , 7.36–7.18 , 5.10 , 4.79 , 2.94 , 1.93-1.91 , 1.59 ; C13-NMR  δ 177.7 (C=S), 156.6 (C=N), 149.7 (phenyl C1’), 134.9 (phenyl C4), 132.4 (phenyl C3), 131.8 (phenyl C5), 127.7 (phenyl C3’+C5’), 127.6 (phenyl C4’), 126.1 (phenyl C6), 126.1 (phenyl C2), 124.6 (phenyl C2’+C6’), 122.8 (phenyl C1), 73.5 (piperidine C3), 69.0 (N-CH2-N), 46.2 (piperidine C1+C5), 37.7 (piperidine C2+C4); Anal. Calcd. for C20H19Cl2N3O2S: C, 55.05; H, 4.39; N, 9.63; S, 7.35. Found: C, 54.85; H, 4.27; N, 9.72; S, 7.42.White powder, yield 69.23%, Mp 182.1°C; FT-IR (KBr) νmax : 2924-2831 (Aliphatic C-H), 1698 (C=O), 1606 (C=N), 1422 (C=C), 1330 (C=S), 1244 (C-O-C) cm–1; 1H-NMR  ppm: δ = 8.01 , 7.86–7.82 , 7.38–7.31 , 5.01 , 2.92 , 2.63 , 2.43 , 1.97–1.89 , 1.84 ; C13-NMR  δ 208.6 (C=O), 177.7 (C=S), 156.8 (C=N), 141.2 (phenyl C1’), 134.8 (phenyl C4), 132.4 (phenylC3), 131.8 (phenylC5), 128.7 (phenyl C3’+C5’), 127.6 (phenylC4’), 127.0 (phenyl C6), 126.2 (phenyl C2+C6), 126.1 (phenylC2), 122.9 (phenyl C1), 70.5 (N-CH2-N), 53.5 (piperidine C3), 47.4 (piperidine C1+C5), 32.1 (piperidine C2+C4), 25.4 (COCH3); Anal. Calcd. for C22H21Cl2N3O2S: C, 57.15; H, 4.58; N, 9.09; S, 6.93. Found: C, 57.35; H, 4.40; N, 9.03; S, 6.55.White powder, yield 52.48%, Mp 149.8°C; FT-IR (KBr) νmax : 3091 (Aromatic C-H), 2931 (Aliphatic C-H), 2238 (CºN), 1607 (C=N), 1432–1415 (C=C), 1322 (C=S), 1249 (C-O-C) cm–1; 1H NMR  ppm: δ = 8.10 , 7.89 , 7.53 , 7.44 , 7.37 , 5.13 , 3.25 , 2.87 , 2.16 , 2.03 ; C13-NMR  δ 177.5 (C=S), 156.8 (C=N), 139.9 (phenyl C1’), 135.1 (phenylC4), 132.4 (phenylC3), 131.8 (phenylC5), 129.0 (phenylC3’+C5’), 128.0 (phenylC4’), 127.7 (phenylC6), 126.2 (phenylC2), 125.5 (phenyl C2’+C6’), 122.6 (phenylC1), 121.6 (C≡N), 70.1 (N-CH2-N), 47.5 (piperidine C1+C5), 41.3 (piperidine C3), 35.3 (piperidine C2+C4); Anal. Calcd. for C21H18Cl2N4OS: C, 56.63; H, 4.07; N, 12.58; S, 7.20. Found: C, 56.54; H, 4.20; N, 12.57; S, 7.24.White crystals Yield 47.68%, , Mp 168.7°C; FT-IR (KBr) νmax : 3024 (Aromatic C-H), 2914 (Aliphatic C-H), 1617 (C=N), 1436-1418 (C=C), 1318 (C=S), 1233 (C-O-C) cm–1; 1H-NMR  ppm: δ =8.04 , 7.84 , 7.25 , 7.14–7.11 , 5.03 , 3.01 , 2.45 , 2.43 , 1.54 , 1.44–1.40 , 1.17 ; C13-NMR  δ 177.8 (C=S), 156.8 (C=N), 140.1 (phenyl C1’), 134.5 (phenyl C4), 132.4 (phenyl C3), 131.7 (phenyl C5), 128.8 (phenyl C3’+C5’), 128.0 (phenyl C2’+C6’), 127.5 (phenyl C2), 126.0 (phenyl C4’), 125.7 (phenyl C6), 123.2 (phenyl C1), 71.0 (N-CH2-N), 50.0 (piperidine C1+C5), 42.1 (N-CH2-Phenyl), 36.5 (piperidine C3), 31.5 (piperidine C2+C4); Anal. Calcd. for C21H21Cl2N3OS: C, 58.07; H, 4.87; N, 9.67; S, 7.38. Found: C, 58.24; H, 4.91; N, 9.85; S, 7.55. White crystals, Yield 56.92%, Mp 94.8°C; FT-IR (KBr) νmax : 2938 (Aromatic C-H), 1610 (C=N), 1448 (C=C), 1326 (C=S), 1246 (C-O-C) cm–1; 1H-NMR  ppm: δ = 8.04 , 7.84 , 5.02 , 3.58 , 3.33 , 3.09 , 1.79 , 1.42 ; C13-NMR  δ 178.3 (C=S), 157.2 (C=N), 132.1 (phenylC4), 131.6 (phenylC3), 127.2 (phenylC5), 125.8 (phenylC6), 123.5 (phenylC2), 122.0 (phenylC1), 70.4 (N-CH2-N), 65.7 (morpholineC2+C3), 63.0 (piperidine C3), 60.8 (morpholineC1+C4), 49.0 (piperidine C1+C5), 27.1 (piperidine C2+C4); Anal. Calcd. for C18H22Cl2N4O2S: C, 50.35: H, 5.16: N, 13.05; S, 7.47. Found: C, 49.61; H, 4.81; N, 12.92; S, 7.75.White powder, yield 59.55%, Mp 156.8°C; FT-IR (KBr) νmax : 3424 (O-H), 3042 (Aromatic C-H) 2941 (Aliphatic C-H), 1725 (C=O), 1554 (C=N), 1441–1419 (C=C), 1320 (C=S), 1239 (C-O-C) cm–1; 1H-NMR  ppm: δ = 8.01 , 7.82 , 5.04 , 3.00 , 2.00 , 1.82 , 1.60–1.51 ; C13-NMR  δ 179.1 (C=O), 175.7 (C=S), 157.7 (C=N), 133.4 (phenyl C4), 132.1 (phenyl C3), 131.6 (phenyl C2+C6), 126.9 (phenyl C5), 125.5 (phenyl C1), 70.6 (N-CH2-N), 49.9 (piperidine C1+C5), 42.5 (piperidine C3), 27.6 (piperidine C2+C4); Anal. Calcd. for C15H15Cl2N3O3S: C, 46.40; H, 3.89; N, 10.82; S, 8.26. Found: C, 47.42; H, 4.49; N, 10.88; S, 7.75.White powder, yield 49.55%, Mp 181.3°C; FT-IR (KBr) νmax : 3421 (O-H), 2934 (Aromatic C-H), 1710 (C=O), 1609 (C=N), 1437–1414 (C=C), 1328 (C=S), 1235 (C-O-C) cm–1; 1H-NMR  ppm: δ= 12.25 , 8.05 , 7.87–7.85 , 5.06 , 3.34 , 3.13 , 2.91 , 2.66 , 1.78–1.63 , 1.47-1.31 ; C13-NMR  δ 177.4 (C=O), 174.6 (C=S), 156.6 (C=N), 134.9 (phenyl C4), 132.3 (phenyl C3), 131.7 (phenyl C5), 127.7 (phenyl C6), 126.2 (phenyl C2), 122.8 (phenyl C1), 70.8 (N-CH2-N), 52.1 (piperidine C1), 50.0 (piperidine C5), 41.0 (piperidine C2), 25.7 (piperidine C3), 23.9 (piperidine C4); Anal. Calcd. for C15H15Cl2N3O3S: C, 46.40; H, 3.89; N, 10.82; S, 8.26. Found: C, 46.15; H, 3.88; N, 10.86; S, 8.53.White powder, yield 70.83%, Mp 181.7°C; FT-IR (KBr) νmax : 2950 (Aromatic C-H), 1720 (C=O), 1611 (C=N), 1443–1421 (C=C), 1324 (C=S), 1241 (C-O-C) cm–1; 1H-NMR  ppm: δ = 8.06 , 7.86 , 5.04 , 4.03 , 3.02 , 2.56 , 2.24-2.21 , 1.81 , 1.57–1.52 , 1.15 ; C13-NMR  δ 177.5 (C=O), 174.0 (C=S), 156.6 (C=N), 134.9 (phenyl C4), 132.3 (phenyl C3), 131.7 (phenyl C5), 127.7 (phenyl C6), 126.2 (phenyl C2), 122.8 (phenyl C1), 70.8 (N-CH2-N), 59.7 (O-CH2CH3), 49.1 (piperidine C1+C3+C5), 28.7 (piperidine C2+C4), 13.9 (O-CH2CH3); Anal. Calcd. for C17H19Cl2N3O3S: C, 49.04; H, 4.60; N, 10.09; S, 7.70. Found: C, 48.64; H, 4.53; N, 10.00; S, 8.72.White crystals, yield 85.13%, Mp 163.8°C; IR (KBr) νmax : 2934 (Aromatic C-H), 1727 (C=O), 1607 (C=N), 1414 (C=C), 1335 (C=S), 1180 (C-O-C) cm–1; 1H-NMR  ppm: δ = 8.06 , 7.87 , 5.05 , 4.06 , 3.12 , 2.92-2.87 , 2.72 , 2.59–2.52 , 1.76–1.63 , 1.47–1.35 , 1.17 ; C13-NMR  δ 177.5 (C=O), 172.8 (C=S), 156.7 (C=N), 134.9 (phenyl C4), 132.3 (phenyl C3), 131.7 (phenyl C5), 127.6 (phenyl C6), 126.1 (phenyl C2), 122.8 (phenyl C1), 70.7 (N-CH2-N), 59.7 (O-CH2CH3), 51.9 , 49.9 , 40.9 , 25.5 , 23.7 , 13.9 (O-CH2CH3); Anal. Calcd. for C17H19Cl2N3O3S: C, 49.04; H, 4.60; N, 10.09; S, 7.70. Found: C, 48.64; H, 3.94; N, 9.99; S, 6.80.White crystals, yield 75.51%, Mp 132.9°C; FT-IR (KBr) νmax 2938 (Aromatic C-H), 1730 (C=O), 1448-1414 (C=C), 1321 (C=S), 1187 (C-O-C) cm–1; 1H-NMR  ppm: δ = 8.04 , 7.89 , 7.84 , 5.13 , 4.09–4.02 , 3.69 , 3.24–3.21 , 1.77–1.67 , 1.51-1.48 , 1.40–1.30 , 1.18 ; C13-NMR  δ 177.1 (C=O), 172.5 (C=S), 156.3 (C=N), 134.9 (phenylC4), 132.3 (phenylC3), 131.8 (phenylC5), 127.5 (phenylC6), 126.0 (phenylC2), 122.7 (phenylC1), 68.9 (N-CH2-N), 59.9 (O-CH2CH3), 48.2 (piperidine C1+ C5), 29.0 (piperidine C2), 24.8 (piperidine C4), 20.9 (piperidine C3), 13.9 (O-CH2CH3); Anal. Calcd. for C17H19Cl2N3O3S: C, 49.04; H, 4.60; N, 10.09; S, 7.70. Found: C, 49.13; H, 4.72; N, 10.20; S, 7.74.”White crystals, yield 32.05%, Mp 113.6°C; FT-IR (KBr) ν–1. The lyophilized compounds sterilized by filtration via 0.45 mm millipore filters. Disc diffusion method was performed by using 100 mL of suspension containing 108 colony forming units (CFU) mL–1 of bacteria, 106 CFU mL–1 of yeast and 104 spore mL–1 of fungi spread on nutrient agar (NA), sabour dextrose agar (SDA), and potato dextrose agar (PDA) medium, in sequence. A total of 15 mL of each synthesized compounds (300 mg/disc) at the concentration of 10 mg mL–1 were impregnated to the discs (6 mm in diameter). DMSO impregnated discs were used for negative controls. The compounds and negative controls were located in the inoculated agar. In order to determine the sensitivity of one strain/isolate standard, ofloxacin and nystatin were used as positive references for bacterial and fungus-yeast strains, respectively. The incubation at 37°C of inoculated plates took 24 h for bacterial strains, 48 h for yeast and 72 h for fungi isolates. The incubation of plant related microorganisms were held at 27°C, differently [31].Dimethylsulfoxide (DMSO) was used to dissolve and prepare the synthesized compounds with a concentration of 10 mg mL2 and 95% air. Cancer cells (range of 2000 cells/well to 5000 cells/well) were inoculated into 96-well plates in 200 μL of media and incubated in 37 °C incubators containing 5% CO2 and 95% air. After a 24 h incubation period, one plate for each cell line was fixed with 100 μL of 10% ice-cold trichloroacetic acid (TCA). This plate represents the behavior of the cells just prior to compound treatment and is accepted as the time-zero plate. The compounds to be tested were solubilized in dimethyl sulfoxide (DMSO) to a final concentration of 40 mM and stored at +4°C. While treating the cells with the compounds, the corresponding volume of the compound was applied to the cell to achieve the desired drug concentration and diluted through serial dilution . After drug treatment, the cells were incubated in 37 °C incubators containing 5% CO2 and 95% air for 72 h. Following the termination of the incubation period after drug treatment, the cells were fixed with 100 μL 10% ice-cold TCA and incubated in the dark at +4°C for 1 h. Then, the TCA was washed away with ddH2O five times and the plates were left to air dry. In the final step, the plates were stained with 100 μL of 0.4% SRB (cat.86183-5 g from Sigma) solution in 1% acetic acid solution. Following staining, the plates were incubated in dark for 10 min at room temperature. The unbound dye was washed away using 1% acetic acid and the plates were left to air dry. To measure the absorbance results, the bound stain was then solubilized using 200 μL of 10 mM Tris-Base. Camptothecin was the positive control and 5-Fluorouracil (5-FU) was standard drug for the cytotoxic effect. The OD values were obtained at 515nm [32].The human cancer cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM), with 10% fetal bovine serum (FBS) and 1% penicillin. They were incubated in 37 °C incubators containing 5% CO5a-5kseries were retrieved from Lazar software (Version 1.4.2) which is a web-based application as https://lazar.in-silico.ch [33].For determination of drug-like physicochemical, pharmacokinetics, and toxicity parameters, a combination of various online screening tools were used, which included MedChem Designer 5.5 , Chem&BioDraw 12.0 (ChemDraw version 12.0.2.10762019), SwissAdme (Swiss Institute of Bioinformatics2013_http://www.swissadme.ch/). Toxicity prediction of these newly synthesized compound3H)-thione derivatives (5a-5k) were prepared via Mannich reaction. According to chemical procedure of piperidine, derivatives were reacted with -1,3,4-oxadiazole-2(3H)-thione (4) group in alcoholic media . Compounds4,5a-5kwere characterized by FT-IR, 1H NMR, 13C NMR spectroscopy, and purity of compounds were checked with elemental analysis. All results of spectral and elemental analysis were found compatible with literature data [34–37]. Compounds4, 5a-5kwere tested for their antimicrobial and cytotoxic properties. 5--3-[(substitutedpiperidine)methyl]-1,3,4-oxadiazole-2 and thione (C=S) groups in 1,3,4-oxadiazole-2(3H)-thione structure, generated two characteristic signals approximately at 1610 and 1330 cm–1. Compounds5c, 5g-5khad an extra sharp signal around 1740–1680 cm–1, which corresponded to the carbonyl group, and compound 5d showed a spesific band at 2238 cm–1, which was claimed as nitrile group. The FT-IR spectrum of compounds displayed a strong band in range of 3080–2900 cm1H NMR spectra of compounds4, 5a-5kdemonstrated hydrogen signals of aromatic structures in the range of 8.00–7.00 ppm. Two proton integrationed and singlet coupled signal in the range of 5.13–5.01 ppm values was a strong evidence for methylene bridge protons between 1,3,4-oxadiazole and piperidine moieties which obtained viaMannichreaction procedure. Variable but compatible integrated signals between 3.60–1.30 ppm confirmed different piperidine protons for each compound. Compound5chave an acetyl group and preserved a singlet signal in 1.84 ppm for alpha protons. Signals for compounds5i-5k, which have different positioned ethyl ester groups on piperidine moiety emerged at 4.06–4.02 for methylene protons (-COOCH2-) and 1.18–1.15 ppm values for methyl protons (-COOCH2CH3). Integration and multiplicity of signals for all compounds were compatible with literature data. 13C NMR spectra of compounds4, 5a-5kpreserved two characteristic signals related to thione (C=S) and imine (C=N) groups at 179 and 158 ppm values originated from 1,3,4-oxadiazole-2(3H)-thione structure. Carbon signal of ketone carbonyl in compound5b, carboxy and ester carbonyl in compounds5g-5k were appeared at different ppm values due to different chemical environments of carbonyl functional groups. While ketone carbonyl carbon of5cshowed signal at 208 ppm, carboxy and ester carbonyl carbons of compound5g-5kindicated their carbonyl carbon signals in the range of 179–174 ppm. Due to shielded-deshielded properties of carbon atoms in magnetic field of 13C NMR, signal of ketone carbonyl in5coccured in downfield region while carboxylic acid  and ester (5i-5k) carbonyl carbons shifted through upfield part of the scale. In this direction, moderately shielded aromatic structures in compounds4, 5a-kgave their spesific signals in the range of 138–122 ppm. 13C NMR signals of remaining carbons associated with methylene and piperidine groups were observed at 70–65 and 69–32 ppm. E. coli, P. aeruginosa, P. vulgaris) and sixteen gram-positive bacterial strains. . They were also evaluated for their antifungal potential against six fungal strains  and antiyeast activity against three yeast strains . Ofloxacin and nystatin were used as positive controls. Antimicrobial data of compounds and reference drugs were given in Tables 1 and Table 2.Antimicrobial activity was tested by measuring the zone of inhibition against test organisms with disc diffusion assay method, and results were summarized in Table 1 and Table 2 with positive control ofloxacin. Eleven compounds were screened for their antibacterial activity against three gram-negative  for their antibacterial activities towards pathogenic gram-positive and gram-negative bacteria, and ofloxacin was used as positive control under the same conditions. As shown in Table 1, compound series displayed serious inhibition of growth (mm) in certain bacterial strains. Especially compounds5b, 5c, 5f-5kshowed considerable antibacterial activity against gram-positiveBacillus spp. when compared to ofloxacin, while inhibition of growth values (mm) of other compounds in the series were either equal or lower than reference molecule. In vitro antibacterial screening results also revealed that, for all bacterial strains, lower inhibition values than reference material meaned as an indicator of antibacterial inability especially for compounds5a, 5dand5e(Table 1). None of compounds didn’t show any antibacterial activity againstE.coliandP. Aeruginosa but compounds5b, 5f, 5g, 5hand5jshowed statistically significant antibacterial activity againstB. ehimensiswhen they compared with ofloxacin.In vitro disc diffusion test was carried out to evaluate newly synthesized compounds (3H)-thione compounds was important point to establish biological activity-functional group relationship. Therefore, newly synthesized molecules were designed to emphasize this correlation. As a comparable result, compounds5a, 5b, 5cand5e, which have a common phenyl group, showed different biological responses forB. ehimensisstrains. Other substituents and phenyl groups that were located on the fourth position piperidine moiety had significant functionality differences for activity. Based on above part, strong electron-donating hydroxyl containing compound5b, weak electron-withdrawing acetyl containing5c, strong electron-withdrawing nitrile containing 5d, and only phenyl substituted piperidine containing compound5a were good examples to elucidate this phenomenon. According to structure-activity relationship (SAR) studies in literature, electron-donating groups provide an elevation of inhibitor level against spesific bacterial strains [38]. The results obtained in parallel with this information was compound5b, which had high level of antimicrobial property specifically againstB. ehimensisdue to its electron-donating hydroxyl moiety. In addition of these four compounds (5a-d), morpholine containing compound 5f was also found more active than reference drug onB. ehimensisstrain. Heteroatoms in morpholine and their bonding capasities with active site of bacteria were serious indicators for biologic activity of 5f,which was an open point for further studies on heterocyclic structure substituted piperidine rings. Besides, substituent variation and their effects on biologic acitiy, structural design was modulated also to generate position effect on activity. So, on piperidine moiety, differently located carboxylic acid containing compounds5g, 5hand differently positioned ethyl ester containing compounds5i,5jand5kwere added to series. Results clearly claimed that position differences of one substituent on piperidine did not cause a significant difference for their antimicrobial response (Table 1). Chemical nature of substitution pattern related to piperidine derivatives of 5--1,3,4-oxadiazole-2, breast (MCF7), and liver (HUH7) with sulphorhodamine B (SRB) assay in triplicate aplication where 5-flourouracil (5-FU) was used as positive control. The IC50 values obtained for these compounds were shown in Table 3. All synthesized target compounds50 values ranging from 13.9–80.4 µM concentrations. It was noteworthy that the cytotoxic effects were more pronounced against liver carcinoma cell line, HUH7. Most of the compounds of the series (5c-5gand5i-5j) have better IC50 values than 5-FLU (IC50 = 18.78 µM) and also compound5dpossessed 10.1 µM value, which represents good druggable cytotoxic activity. The results of cytotoxicity studies revealed that activities of the compounds were not impressive against colon and breast cancer cells, all of the compounds showed cell viability with IC5c, 5d, 5f, 5g, 5i-5klead to not only to an increase in the antimicrobial activity on certain bacterial and fungal strains, but also to a significant level of anticancer activity against liver carcinoma cell line (HUH7). According to the preliminary antimicrobial activity, compound5bshowed the best inhibitor activity againstBacillus sppstrains, and compound5d was evaluated to have strongest cytotoxic activity against liver carcinoma cells (HUH7). A total analysis of the antibacterial, antifungal, and antiyeast activity revealed that newly synthesized compounds were really active againstBacillus cereus,Bacillus ehimensis andBacillus thuringiensis species.Our results indicate that the addition of phenyl and carbonyl group as substituents enhances the antimicrobial activity of the prepared compounds. It indicates that the structural differences is an important factor for the activity. It is notable, also, that phenyl and carbonyl substitutions in compounds5a-5kshowed acceptable consequences for Lipinski’s rule of five (RO5) analysis; molecular weight (MW) , hydrogen bond acceptor (HBA) (<10), hydrogen bond donor (HBD) (<5) and logP (<5) values [40]. In the situation of one deviation, corresponds to poor absorption of compounds. However, there are plenty of examples are available for RO5 violation amongst the existing drugs . Furthermore, the polar surface area (PSA) of a molecule is defined as the surface sum over all polar atoms, primarily oxygen and nitrogen with their attached hydrogen atoms. The PSA value of a molecule reflects the ability to permeate cells, which is used for drug’s optimization. Previous researches showed the standard value of PSA as <89 A2 [43] in which this measure is supported by our newly synthesized compound5a-5kserie. On the other hand, number of rotatable bond is a measurement for molecular flexibility and is significant in determining oral bioavailability of the compounds (rule of three-number of rotatable bonds ≤ 3), which explains oral usage of compound5a-5kmight be decrease bioavailibility (Table 4) [44].The chemo-informatic features of newly synthesized molecules were evaluated by computational tools. According to in silico data, compoundsToxicity prediction is a tool is an useful method in the drug discovery due to many of the newly synthesized potential candidates had failed in clinical trial evaluation because of some pharmacokinetics and toxicity problems. In silico predictions are improved to overcome such scenario in this detailed process. Meanwhile, toxicity predictions were clearly revelead that all our novel compounds are seemed to have no predictable central nervous system side effects, carcinogenicity, and mutagenicity (Table 5).3H)-thione structures. According to biological consequences, phenyl and carbonyl group that substituted to piperidine ring seemed to have supportive property on antimicrobial activity of the novel compounds. Some compounds like 5c, 5d, 5f, 5g, 5i-5kthat contain phenyl and carbonyl group revealed not only antimicrobial effect on certain bacterial and fungal strains but also significant level of anticancer activity against liver carcinoma cell line (HUH7). Especially compound5bshowed the best inhibitor activity againstBacillus sppwhereas compound5d represent valuable effect against liver carcinoma cells (HUH7). Besides, evaluating biological properties, compounds were predicted for the chemo-informatic and possible toxicity features within some software programmes. Synthesized molecules were calculated as to qualified the criteria to be a drug as per Lipinski Rule of Five and in silico predicted toxicology results were seemed as all of them have no mutagenic or carcinogenic profiles. In summary, we report the efficient synthesis, characterization, antimicrobial and cytotoxic activity evaluation of new compound series which contain different substituted piperidine bearing 1,3,4-oxadiazole-2(1H and 13C NMR spectra of all compounds, 5a-5k, are given as supplementary information at www.ias.ac.in/chemsci.