Protein arginine methyltransferase 5 (PRMT5) is an attractive molecular target in anticancer drug discovery due to its extensive involvement in transcriptional control, RNA processing, and other cellular pathways that are causally related to tumor initiation and progression. In recent years, various compounds have been screened or designed to target either the substrate- or cofactor-binding site of PRMT5. To expand the diversity of chemotypes for inhibitory binding to PRMT5 and other AdoMet-dependent methyltransferases, in this work, we designed a series of triazole-containing adenosine analogs aimed at targeting the cofactor-binding site of PRMT5. Triazole rings have commonly been utilized in drug discovery due to their ease of synthesis and functionalization as bioisosteres of amide bonds. Herein, we utilized the electronic properties of the triazole ring as a novel way to specifically target the cofactor-binding site of PRMT5. A total of about 30 compounds were synthesized using the modular alkyne-azide cycloaddition reaction. Biochemical tests showed that these compounds exhibited inhibitory activity of PRMT5 at varying degrees and several showed single micromolar potency, with clear selectivity for PRMT5 over PRMT1. Docking-based structural analysis showed that the triazole ring plays a key role in binding to the characteristic residue Phe327 in the active pocket of PRMT5, explaining the compounds’ selectivity for this type-II enzyme. Overall, this work provides new structure–activity relationship information on the design of AdoMet analogs for selective inhibition of PRMT5. Further structural optimization work will further improve the potency of the top leads. S-adenosyl-l-methionine (SAM) onto the terminal nitrogens of the guanidino group of the arginine side chain, producing S-adenosyl-l-homocysteine (SAH) as a byproduct. The nine members of the human PRMT family are classified into three types based on their end-methylation products. All members can catalyze the first methylation step, which forms NG-monomethylarginine (MMA), and additionally type I and type II PRMTs can further catalyze a second methylation reaction. Type I PRMTs  add a second methyl group onto the same nitrogen as the first to form NG, NG-dimethylarginine (ADMA). Type II PRMTs  add a second methyl group onto the other omega nitrogen to form NG, N’G-dimethylarginine (SDMA). PRMT7 is a type III enzyme and can only catalyze the formation of MMA  was used as the peptide substrate, at a final concentration of 0.5 μM. 3H-SAM was used as the methyl donor at 0.5 μM. SAH was used as a control inhibitor. Compounds that showed strong inhibition of PRMT5 in the single point well-plate assay and subsequently resynthesized at a milligram scale had their IC50 determined in a concentration-dependent activity assay under the same experimental condition. Analysis of the biochemical activity results against PRMT5 disclosed a few interesting structure–activity relationship (SAR) trends. Firstly, substitution of the 4-position of the triazole ring with a trimethylsilyl (TMS) group (7) gave a low micromolar potency of 8.3 ± 2.2 μM. Removal of the TMS group (2) resulted in a reduction in the potency (IC50 27.0 ± 4.5 μM). The SPA test also revealed that compound 8 (R = tBu) had a similar potency to 7, which was to be expected. Substitution with a polar functional group  resulted in an increased potency, with IC50 of 4.4 ± 0.3 μM. Lengthening the substituent generally resulted in a decrease in the potency. Increasing the TMS group of 7 to –CH2TMS in 9 resulted in a big loss of activity (IC50 > 200 μM). Addition of a methylene group to 3  or methylation of 3 to give 4 also reduced the potency. If the hydroxyl group of 3 was changed to an amine (6), the potency was improved; however, the selectivity was abolished. Generally, compounds that contained positive charges  near the triazole ring had further improved potencies. Compounds with negative charges varied in their potencies, with compounds that had the negative charge near the ring (11 and 19) having dramatically better potencies than those compounds where the charge was further from the ring (12 and 13). Surprisingly, bisubstrate-like compounds 29 and 30 had only modest potencies towards PRMT5, with IC50 of 53.7 and 45.6 μM, respectively, with only 29 showing selectivity towards PRMT5.The methyltransferase activities of PRMT5 and PRMT1 in the absence and presence of the synthetic inhibitors were measured by using the scintillation proximity assay (SPA) protocol  Table 1Table 1. 3, 4, 7, and 9, all of which had poor activities, with the exception of 3. The observed changes in potencies can largely be attributed to how well the substituent fit into the arginine-binding pocket. More polar groups tended to do better than their less polar counterparts . With the hydrogen-bonding donor capability of 3, it was able to form a stronger hydrogen bond with E444 at the hydroxyl group rather than at H5. When comparing compounds 3, 4, and 5, the polar chain of 3 was able to form hydrogen bonds with E435 and E444. Increasing the length of the chain to give 5 reduced its ability to form these bonds and was only able to form bonds with E435, resulting in its loss of activity. Methylation of 3 to give 4 removed its hydrogen bond donor ability, and increased its hydrophobicity, so 4 was only able to form an H-bond to E435 (7 vs. 9). The aromatic group of 14 was more preferential as it also joined the triazole ring in forming a pi–pi interaction with Phe327. The loss of activity in 9 was found to be due to the bulky TMS group forming steric clashes further into the binding pocket, primarily with W579 (To better understand the observed structure–activity relationship (SAR) of these molecules, the previous docking analysis was further examined using Discovery Studios  Figure . We were to E435 . Bulky hith W579 .12 and 13, this held true. It is also possible that the long aliphatic chains on these molecules also provided little to no interactions in the arginine-binding pocket. In the case of 11, however, we were surprised to see that it had a potency comparable to 6: its positive charge analog. This result is believed to be due to the ring rotating back to avoid negative interactions associated with placing the substituent in the arginine pocket such that this group is allowed to point into the methionine-binding pocket, so the acid group is able to lie in the pocket that would normally be occupied by the carboxylic acid of SAM. Lengthening the chain of 11 to give either 12 or 13 would make this rotation sterically disfavored, so the ring is forced to assume its usual conformation and have the substituent go through the arginine pocket. Of the basic substituents, the simplest substituent 6 had an activity comparable to 7; however, it had a loss of selectivity K20biotin was synthesized using solid-phase peptide synthesis method.5′-azido-5′-deoxy-2′,3′-O-isopropylideneadenosine (32). In total, 1.49 g of triphenylphosphine (5.7 mmol) was dissolved in 5 mL of THF. The solution was placed on an ice bath and stirred for 10 min. In total, 3 mL of DEAD (40% in toluene) was added dropwise to the triphenylphosphine solution, and the resulting yellow solution was stirred for another 10 min. Then, 1 mL of DPPA (5.7 mmol) was added dropwise over 1 min to the solution, and the solution was removed from ice and allowed to stir at room temperature for 10 min. In total, 500 mg of 5′-deoxy-2′,3′-O-isopropylideneadenosine (1.63 mmol) was added portion-wise to the solution. The flask was then stirred overnight at room temperature. After completion, the solution was diluted with 2 mL of ethyl acetate and concentrated to afford a yellow oil. The oil was then purified through column chromatography (50–100% ethyl acetate in hexanes) to give a clear oil. Yield: 500 mg, 93%. ESI-HRMS calc for C13H16N8O3 [M+H]+: 333.1419 found 333.1398 1H NMR  δ 8.35 , 8.19 , 7.38 , 6.22 , 5.53 , 5.01 , 4.31 , 3.67–3.50 , 1.55 , 1.34 . 13C NMR  δ 156.64, 153.27, 149.23, 140.49, 119.62, 114.00, 89.54, 84.98, 83.35, 81.99, 51.96, 27.43, 25.63.5′-azido-5′-deoxyadenosine (1). In total, 500 mg of 32 (1.71 mmol) was dissolved in 9 mL of a 1:8 mixture of water and formic acid. This solution was stirred at room temperature overnight. The reaction progress was monitored via analytical HPLC . The compound was purified via column chromatography (1–10% methanol in ethyl acetate). Yield: 281 mg, 64%. ESI-HRMS calc for C10H12N8O3 [M+H]+: 293.1106, found 293.1101 1H NMR  δ 8.37 , 8.19 , 7.32 , 5.95 , 5.61 , 5.41 , 4.77 , 4.23 , 4.08–4.00 , 3.74–3.52 . 13C NMR  δ 156.55, 153.14, 149.86, 140.41, 119.67, 88.25, 83.42, 73.20, 71.39, 52.19.4·5H2O (0.05 mmol) were dissolved in 1 mL of water. In total, 566 µL of a 53 mg/mL solution of 1  was added to the solution, followed by 2 equivalents of the appropriate alkyne. The reaction mixture was stirred at room temperature for 6 h. After reaction completion, the compounds were purified using reverse-phase HPLC (5–50% gradient CH3CN in H2O over 45 min) and lyophilized to give the final products as solids. For the hydrophobic alkynes used to synthesize 18, 21, and 24, a solvent system of 2:1 DMSO:H2O was used. For the acidic alkynes used to synthesize 11–13, the associated sodium salt was used.In total, 42 mg of sodium ascorbate (0.21 mmol) and 12 mg of CuSO-2-methyl)-5-(6-amino-9H-purin-9-yl)tetrahydrofuran-3,4-diol (2). In total, 22.5 mg (0.06 mmol) of 7 was dissolved in 1 mL of THF. In total, 115 µL of a 1M solution of TBAF in THF (0.12 mmol) was added to the solution, and it was stirred at room temperature overnight. The following day, the solution was evaporated, and the resulting residue was purified by reverse-phase HPLC and lyophilized to give a white powder. Yield: 15 mg, 82% ESI-HRMS calc for C12H15N8O3 [M+H]+: 319.1262 found 319.1252 1H NMR  δ 8.46 , 8.36 , 8.02 , 7.68 , 5.95 , 4.87–4.73 , 4.63 , 4.34–4.21 .-2-(6-amino-9H-purin-9-yl)-5-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydrofuran-3,4-diol (3). Synthesized using the general procedure detailed above. Yield: 17 mg, 48% ESI-HRMS calc for C13H17N8O4 [M+H]+: 349.1368 found 349.1362 1H NMR  δ 8.53 , 8.39 , 7.85 , 5.96 , 4.74 , 4.60 , 4.46 .-2-(6-amino-9H-purin-9-yl)-5-((4-(methoxymethyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydrofuran-3,4-diol (4). Synthesized using the general procedure detailed above. Yield 21 mg, 56% ESI-HRMS calc for C14H19N8O4 [M+H]+: 363.1524 found 363.1514 1H NMR  δ 8.37 , 7.94 , 6.05 , 4.62 , 4.51 , 4.42 , 3.38 .-2-(6-amino-9H-purin-9-yl)-5-((4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydrofuran-3,4-diol (5). Synthesized using the general procedure detailed above. Yield: 16 mg, 43% ESI-HRMS calc for C14H19N8O4 [M+H]+: 363.1524 found 363.1519 1H NMR  δ 8.47 , 8.39 , 7.77 , 7.70 , 5.90 , 3.53 , 2.98 , 2.65 .-2-(6-amino-9H-purin-9-yl)-5-((4-(aminomethyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydrofuran-3,4-diol (6). Synthesized using the general procedure detailed above. Yield: 21 mg, 59% ESI-HRMS calc for C13H19N9O3 [M+H]+: 348.1528 found 348.1524 1H NMR  δ 8.54 , 8.38 , 8.06 , 6.01 , 4.90 , 4.77 .-2-(6-amino-9H-purin-9-yl)-5-((4-(trimethylsilyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydrofuran-3,4-diol (7). Synthesized using the general procedure detailed above. Yield: 25 mg, 62% ESI-HRMS calc for C15H23N8O3Si [M+H]+: 391.1657 found 391.6566 1H NMR  δ 8.06 , 5.94 , 4.77 , 4.52 , 0.21 .-2-(6-amino-9H-purin-9-yl)-5-((4-(tert-butyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydrofuran-3,4-diol (8). Synthesized using the general procedure detailed above. Yield: 23 mg. 60% ESI-HRMS calc for C16H23N8O3 [M+H]+: 375.1888 found 375.1876 1H NMR  δ 8.60 , 5.98 , 4.70 , 4.20 , 4.08 , 3.74–3.52 .-2-(6-amino-9H-purin-9-yl)-5-((4-((trimethylsilyl)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydrofuran-3,4-diol (9). Synthesized using the general procedure detailed above. Yield: 26 mg, 63% ESI-HRMS calc for C16H25N8O3Si [M+H]+: 405.1814 found 405.1806 1H NMR  δ 8.47 , 7.75 , 6.08 , 4.82 , 4.69 , 4.45 , 2.07 , 0.00 .1--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazole-4-carboxylic acid (11). Synthesized using the general procedure detailed above. Yield: 15 mg, 40% ESI-HRMS calc for C13H15N8O5 [M+H]+: 363.1160 found 363.1161 1H NMR  δ 8.59 , 8.38 , 5.97 , 4.70 , 4.19 , 4.08 , 3.69–3.57 . 13C NMR  δ 154.44, 150.44, 141.39, 130.70, 88.31, 83.56, 73.40, 71.32, 52.16.4--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)butanoic acid (12). Synthesized using the general procedure detailed above. Yield: 20 mg, 48% ESI-HRMS calc for C16H21N8O5 [M+H]+: 405.1630 found 405.1629 1H NMR  δ 6.07 , 4.65 , 4.44 , 2.68 , 2.34 , 1.91 .5--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)pentanoic acid (13). Synthesized using the general procedure detailed above. Yield: 25 mg, 58% ESI-HRMS calc for C17H23N8O5 [M+H]+: 419.1786 found 419.1788 1H NMR  δ 6.03 , 4.61 , 4.42 , 2.33 , 2.19 , 1.74–1.45 .-2-(6-amino-9H-purin-9-yl)-5-methyl)tetrahydrofuran-3,4-diol (14). Synthesized using the general procedure detailed above. Yield: 24 mg, 59% ESI-HRMS calc for C18H19N8O3 [M+H]+: 395.1575 found 395.1575 1H NMR  δ 8.03 , 7.62 , 7.49–7.32 , 6.05 , 4.64 , 4.46 .13C NMR  δ 146.77, 131.09, 129.34, 128.32, 125.59, 122.60, 88.42, 82.80, 73.25, 71.34, 51.96.N--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (15). Synthesized using the general procedure detailed above. Yield: 27 mg, 58% ESI-HRMS calc for C20H22N9O4 [M+H]+: 452.1790 found 452.1769 1H NMR  δ 8.99 , 8.54 , 8.39 , 7.89 , 7.85–7.80 , 7.58–7.48 , 7.45 , 5.95 , 4.74 , 4.62 , 4.46 , 4.30 , 4.24 .(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)propanoic acid (18). Synthesized using the general procedure detailed above. Yield: 20 mg, 31% MALDI-MS calc for C30H30N9O7 [M+H]+: 628.23 found 628.5 1H NMR  δ 7.88 , 7.75 , 7.66 , 7.41 , 7.30 , 5.94 , 4.72 , 4.59 , 4.23 .(S)-2-amino-3--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)propanoic acid (19). In total, 15 mg (0.024 mmol) of 18 was dissolved in 2 mL of a 20% solution of piperidine in THF. The solution was stirred at room temperature for 90 min at which point the solution was concentrated and the resulting residue was purified by reverse-phase HPLC and lyophilized to give a white powder. Yield: 7 mg, 72% ESI-HRMS calc for C15H20N9O5 [M+H]+: 406.1582 found 406.1568 1H NMR  δ 8.36 , 8.23 , 7.82 , 5.93 , 4.82–4.74 , 4.68 , 4.26 .-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-valinate (Boc-20). Synthesized using the general procedure detailed above. Yield: 25 mg, 44% ESI-HRMS calc for C23H34N9O7 [M+H]+: 548.2576 found 548.2587 1H NMR  δ 6.03 , 4.59 , 4.40 , 4.03 , 2.02 , 1.50–1.22 , 0.83 .-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl L-valinate (20). In total, 12 mg of Boc-protected 20 was dissolved in 2 mL of a 9:1 TFA:MeOH solution and was stirred at room temperature for 4 h. The TFA was removed using rotary evaporation and the resulting residue was purified using reverse-phase HPLC and lyophilized to give a white powder. Yield: 8 mg, 82% ESI-HRMS calc for C18H26N9O5 [M+H]+: 448.2052 found 448.2045 1H NMR  δ 8.46 , 8.33 , 8.08 , 5.94 , 5.31–5.21 , 4.81 , 4.66 , 4.28 , 0.87 .13C NMR  δ 141.32, 126.29, 88.34, 82.91, 73.08, 71.45, 58.87, 52.03, 29.89, 18.45, 17.87.2--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-N-(m-tolyl)acetamide (21). Synthesized using the general procedure detailed above. Yield: 16 mg, 31% ESI-HRMS calc for C22H27N10O4 [M+H]+: 495.2212 found 495.2205 1H NMR  δ 10.36 , 8.36 , 8.21 , 8.06 , 7.36 , 7.26–7.16 , 6.94 , 5.94 , 4.90–4.84 , 4.75 , 4.29 , 3.94 .Methyl 4--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)benzoate (22). Synthesized using the general procedure detailed above. Yield: 15 mg, 30% ESI-HRMS calc for C22H26N9O5 [M+H]+: 496.2052 found 496.2054 1H NMR  δ 8.45–8.36 , 8.23 , 7.98 , 5.93 , 4.96–4.80 , 4.74 , 4.28 , 4.07 .2--5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-N-(2-fluorophenyl)acetamide (24). Synthesized using the general procedure detailed above. Yield: 19 mg, 37% ESI-HRMS calc for C21H24FN10O4 [M+H]+: 499.1961 found 499.1962 1H NMR  δ 10.33 , 8.46 , 8.29 , 8.07 , 7.99–7.84 , 7.34–7.18 , 5.95 , 4.86 , 4.72 , 4.34–4.23 , 4.03 .29 and 30 was conducted using the FOCUS XC instrument from AAPPTec. Prior to synthesis, Fmoc-amino acids were dissolved in N-methyl-2-pyrrolidone to create 0.2 M stocks. Rink amide resin, Fmoc-amino acids, and other reagents were added to the appropriate reaction vials. Resin was swollen in 15 mL of DMF and mixed using nitrogen gas for 10 min. After draining of the solvent, deprotection was conducted using 8 mL of 20% piperidine in DMF. The deprotection reaction was mixed for 30 min, drained, and repeated with another 8 mL of 20% piperidine in DMF. The resin was drained and washed with 15 mL of DMF. Prior to coupling, 5 mL of 0.2 M HCTU in DMF, 5 mL of 22% N-methyl morpholine in DMF, and 5 mL of the appropriate amino acid were mixed and added to the reaction vessel. Coupling was conducted using nitrogen gas mixing for 1 h after which the solution was drained, and resin was washed with 15 mL of DMF. For 30, this coupling step was repeated for each amino acid addition. Following coupling, the cycles of deprotection and coupling were conducted until the sequence was complete and the final N-terminus was deprotected. Acetyl capping was conducted by the addition of 4 mL of DMF and 1 mL of acetic anhydride. The resin was mixed mechanically for 5 min; then, 1.5 mL of DIPEA was added and the resin continued to be mixed for an additional 30 min. The resin was then washed using DMF and dried using DCM.Synthesis of 1 in 1 mL of DMSO in the second vial, and 9.91 mg of sodium ascorbate in 0.45 mL of water in the third vial. After swelling, the resin was drained and the solutions of CuBr and 1 were added to the resin followed by 58.2 μL of 2,6-lutidine, 86.5 μL of DIPEA, and lastly the solution of sodium ascorbate. The reaction was allowed to mix overnight. The following morning, the resin was drained; washed with DMSO, water, and DCM; and dried. Cleavage was conducted using a cocktail containing 200 mg of phenol, 0.2 mL of water, 0.2 mL of thioanisole, 0.1 mL of 1,2-ethanedithiol, 40 μL of triisopropylsilane, and 3.46 mL of trifluoroacetic acid and mixing for 4 h. After cleavage, the peptide was precipitated in 8 mL of diethyl ether and centrifuged at 5000 rpm at 4 °C for 10 min. The supernatant was removed and pellet dried. The peptide was then purified using reverse-phase HPLC and lyophilized to obtain the product. MALDI-MS calc for compound 29: C41H70N26O9 [M]+: 1070.6, found 1071.7. MALDI-MS calc for compound 30: C30H45N15O10 [M]+: 775.3, found 776.2.On-resin click reactions were conducted first by swelling the resin in 7 mL of DMSO for 10 min. Stock solutions of click reagents were made in separate vials by dissolving 7.17 mg of CuBr was in 1.5 mL of DMSO in the first vial, 14.6 mg of E. coli to obtain the recombinant bacmids, and the bacmids were extracted using the PureLink HiPure Plasmid Miniprep Kit . The purity and concentration of the plasmid were measured by a Nanodrop. Sf9 cells were cultured in Sf-900 II SFM medium until the cell density reached 1.5 × 106 cells/mL and cell viability was more than 95%. Cells were plated in a 6-well tissue culture plate at a density of 4 × 105 cells/mL per well. In total, 2 µg of PRMT5 and MEP50 recombinant bacmid were transfected into each well of cells. After 5 h of transfection, the transfection medium was replaced by 2 mL of Sf-900 II SFM + 100 U P/S per well. The medium was collected and centrifuged at 1000 rpm for 5 min to obtain the supernatant P1 viral stock after 7 days of inoculation at 27.5 °C. To obtain a higher titer of viral stock, P1 viral stock was amplified in a 6-well tissue culture plate containing 2 × 106 cells per well. In total, 0.1 MOI of P1 virus stock was added into each well. After 7 days of incubation at 27.5 °C, the cell medium was centrifuged at 1000 rpm for 5 min to obtain the P2 viral stock in the supernatant. The Baculovirus plaque assay was performed to determine the viral titer concentration; the target concentration range was 1 × 107–1 × 108 IFU/mL.Human recombinant His-tag PRMT5 was co-expressed with His-tag MEP50 using the Bac-to-Bac baculovirus expression system  as previously described . Briefly6 cells/mL. After incubation for 72 h at 27.5 °C, the cells were harvested and centrifuged. PRMT5 and MEP50 protein were collected in the cell pellet for further purification. Cell pellets were disrupted twice at 100 psi in 25 mL of cell lysis buffer . Before the Ni-NTA purification, 10 mL of Ni-NTA resin  was loaded into a 50 mL column and equilibrated with equilibration buffer . After 60 min of cell lysate centrifugation at 4 °C at 18,000 rpm, the cell lysate supernatant was incubated with the equilibrated nickel resin. The resin was washed with equilibration buffer and protein was eluted with elution buffer . For the dialysis of protein, elution samples were loaded into a 10,000 MWCO SnakeSkin  dialysis bag in the storage buffer . Afterwards, proteins were concentrated using 10,000 MWCO Vivaspin 20 ultrafiltration devices  by centrifugation at 6000 rcf, 4 °C. Bradford assay was carried out to determine the concentration of PRMT5/MEP50 protein. Purified protein was verified by running an SDS-PAGE.For the expression of PRMT5 and MEP50, Sf9 insect cells were cultured in suspension at 27.5 °C at a speed of 130 rpm in Expression Systems ESF 921 medium with 100 U P/S. In total, 200 mL of Sf9 cell culture was inoculated with PRMT5 and MEP50 P2 viral stocks when the cell density reached 1.5 × 102O) to create a series of working concentrations centered around an anticipated IC50. [H]3-SAM and substrate were diluted together in buffer to working concentrations of 2.5 and 5 μM, respectively. To each well in a 96-well plate, 6 μL of the inhibitor solution was added, with negative and positive controls receiving buffer. In total, 6 μL of the [H]3-SAM/substrate solution was then added to each well. The enzyme was then diluted in buffer to a working concentration of 0.83 μM, and 18 μL was added to each well, with negative controls receiving 18 μL of buffer. The plate was covered, and the reaction was allowed to run for 30 min and was then quenched with the addition of 30 μL of 100% iPrOH followed by 60 μL of 50% iPrOH in H2O. In total, 10 μL of 20 mg/mL streptavidin-coated scintillant beads were added and the plate was read using a MicroBeta  plate reader. The CPM counts in the absence of an inhibitor for each data set were considered as 100% activity. In the absence of the enzyme and inhibitor, the CPM counts in each data set were defined as background (0%).Inhibitor was dissolved in water to create a 10 mM stock concentration. This was then diluted in buffer . Ligands were prepared using the Prepare Ligands module and ionization states were generated at pH 7.0 ± 1.5. Protein structure was prepared with Prepare Protein using the module within Discovery Studios and energy minimized using the CHARMM force field. Binding sites were determined using PDB records and analysis of the enzyme cavities forming a sphere centered at the active site with a radius of 11.6 Å. A series of conformations of the ligands were generated by using molecular dynamics with various random seeds. Each of the conformations was translated through the active site using flexible ligand docking. The binding affinities were predicted using the resulting CDOCKER score. Each ligand was screened for the formation of crucial interactions on the nucleoside portion of the ligand and optimum poses were saved.CDOCKER from Discovery Studio (version 4.0) was used to predict the binding poses of the synthesized compounds In this study, we developed a modular, easily diversifiable set of chemical probes for selective targeting of PRMT5. We demonstrated that triazole-containing adenosine analogs have the capability of selectively targeting PRMT5 over the type-I enzyme PRMT1. Through the use of CuAAC click chemistry, our scaffold can be linked to a subset of molecules to create a wide array of potential inhibitors containing the targeting moiety. Our SAR analysis showed that the triazole ring is positioned well to form a pi–pi interaction with the conserved residue in PRMT5: Phe327, and allows for substituents at the C4 position to easily access the arginine-binding pocket. With these results, it is reasonable to conclude that this targeting scaffold can be potentially used to selectively target PRMT5 and open the door to potentially a new class of bisubstrate inhibitors for PRMT5. Future directions for this scaffold could involve a thorough investigation into the selective mechanisms behind the triazole ring and further diversification of the C4 and C5 positions to provide targeted bisubstrate inhibitors of PRMT5.


N-aryl-2-cyano-3-mercapto-3-(phenylamino)acrylamide derivatives with N-(4-acetylphenyl)-2-chloroacetamide in alcoholic sodium ethoxide. IR, 1H NMR, and mass spectroscopic analyses were used to characterize the synthesized derivatives. In addition, molecular, electronic properties of the synthesized products were studied by the density functional theory (DFT) where they exhibited close HOMO–LUMO energy gap (ΔEH-L) in which the amino derivatives 7a-c have the highest while the methyl derivatives 5a-c were the lowest. Using the ABTS method, the antioxidant properties of the produced compounds were evaluated, where amino thiophene-2-carboxamide 7a exhibit significant inhibition activity 62.0% compared to ascorbic acid The antibacterial activity against two pathogenic Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and two of pathogenic Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) revealed that 7b records the highest activity index compared to ampicillin 83.3, 82.6, 64.0, 86.9%, respectively. Furthermore, the thiophene-2-carboxamide derivatives were docked with five different proteins with the use molecular docking tools and the results explained interactions between amino acid residue of enzyme and compounds. Compounds 3b and 3c showed the highest binding score with 2AS1 protein. Synthetic strategy for the synthesis of thiophene 2-carboxamide derivatives substituted with hydroxyl, methyl and amino groups at position-3 was proposed. The strategy includes the cyclization of the precursor ethyl 2-arylazo-3-mercapto-3-(phenylamino)acrylate derivatives, 2-acetyl-2-arylazo-thioacetanilide derivatives and The online version contains supplementary material available at 10.1186/s13065-023-00917-2. E. coli, S. aureus, P. aeuroginosa) [4 was used as a narrow spectrum antibacterial lead compound [N-(thiophen-2-ylmethyl)thiophene-2-carboxamide displays close experimental and theoretical structure parameters with (ΔEH-L) 5.031 eV [2, OH groups aiming at increasing anti-oxidant and antibacterial inhibition power.The antibacterial resistance threatens the human health around the world. It appeared due to the human abuse of antibiotics and it reached to danger limit. In 2019, 1.27 out of 4.95 million deaths attributed to antimicrobial resistance, with three leading pathogens for deaths (oginosa) , 4. Manyoginosa) , industroginosa) , antibacoginosa) –19, antioginosa) , inflammoginosa)  and antioginosa) –26. Someoginosa)  and Rivaoginosa) . Moreoveoginosa) . Additiooginosa) , 32; forcompound  and thiocompound . Previou5.031 eV . While t5.031 eV . As a re3, 5 and 7 was aimed as one-step method by condensation of N-(4-acetylphenyl)-2-chloroacetamide (1) and various functionalized thiocarbamoyl compounds 2, 4, and 6, respectively. The highly versatile chloroacetamide compound, N-(4-acetylphenyl)-2-chloroacetamide (1) has been prepared by the reported chloroacetylation reaction of 4-aminoacetopheone [1 was reacted with ethyl 2-arylazo-3-mercapto-3-(phenylamino)acrylate derivatives (2) [A   in ethancetamide  and vari5a-c was performed. Thus, reaction of N-(4-acetylphenyl)-2-chloroacetamide (1) with 2-acetyl-2-arylazo-thioacetanilide derivatives (4) [5a–c   in boili1 with N-aryl-2-cyano-3-mercapto-3-(phenylamino)acrylamide derivatives 6 [7a-c. The reaction proceeded via the formation of alkylated sulphide intermediate C, which was then subjected to intramolecular methylene group addition on the nitrile function to yield the corresponding 3-aminothiophene derivatives 7a-c . Furthermore, 3-amino thiophene-2-carboxamide derivatives 7a-c promoted the highest antioxidant activity with percent inhibition 62.0–46.9%. While, 3-hydroxy thiophene-2-carboxamide derivatives 3a-c shows moderate inhibition percentage 54.9–28.4% and 3-methyl thiophene-2-carboxamide derivatives 5a-c the lowest 22.9–12.0%.In this study, the antioxidant activity of the synthesized thiophene-2-carboxamide derivatives nt assay . The decin Table . The res3, 5 and 7 were evaluated for their antibacterial activity against a panel of two pathogenic Gram-positive bacteria (S. aureus and B. subtilis) and two of pathogenic Gram-negative bacteria (E. coli and P. aeruginosa), as shown in  than their corresponding 3-hydroxy thiophene-2-carboxamide compounds 3a-c (from 20.0 to 78.3%) and the lowest inhibition values displayed by 3-methyl thiophene-2-carboxamide compounds 5a-c (from no activity to 47.8%). The thiophene-2-carboxamide derivatives 3, 5, 7b (substituted with methoxy group at aryl Th4) showed the best inhibition activity against Gram-positive bacteria (S. aureus and B. subtilis) and against Gram-negative bacterium (P. aeruginosa) rather than their corresponding derivatives substituted with methyl group or chlorine atom.The newly synthesized compounds in Table . The ant7b containing methoxy group showed excellent activity against P. aeruginosa (86.9%), S. aureus (83.3%) and B. subtilis (82.6%) with inhibition zones 20, 20 and 19 mm, respectively. Hydroxy thiophene-2-carboxamide compound 3b having methoxy group showed very good effect against B. subtilis and P. aeruginosa 78.3% (inhibition zone 18 mm) and inhibition activity against S. aureus 70.8% (inhibition zone 17 mm).Amino thiophene-2-carboxamide compound 3a-c, 5a-c, and 7a-c can be derived from antioxidant and antibacterial testing results: (i) Antioxidant and antibacterial activity of the amino thiophene-2-carboxamide derivatives 7a-c are more potent than hydroxyl or methyl thiophene-2-carboxamide 3a-c; which may be attributed to the absence of azo moiety and presence of amino group. Increasing the antioxidant power in 7a-c is caused by presence of electron donating amino group which increases the resonating electron on the thiophene ring, and faceplate the electron trapping for peroxide radical [3a-c showing higher activity than 5a-c, perhaps it increase the solubility of this class of compound [7a possess potent activity among them; which may be due to absence of substituents on benzene ring Th4. While, compounds 3b and 5b is more potent than their derivatives which may be attributable to the presence of (-OMe group Th4). (iii) Antibacterial activity of amino thiophene-2-carboxamide derivatives 7b was boosted due to presence of (4-Me on carboxamide Th2) against E. coli, P. aeruginosa, S. aureus and B. subtilis. The structural variations such as (4-OMe on carboxamide Th4) and replacement of carboxamide with azo moieties 3b and 5b favors the activity in positive manner [3b, 5b, 7b may be attributed to the increase in the hydrophilicity power of the antibacterial drug agent caused by methoxy group [The following structure–activity relationship of newly synthesized thiophene 2-carboxamide derivatives  radical , 45. Morcompound , 47.  are shown in  is involved in intramolecular hydrogen bond with N1 of azo group(Th4), in 3a-c and 5a-c, while with oxygen of the carboxamide(Th4) in 7a-c. For 3a-c and 7a-c, the H-atoms of thiophene-OH and thiophene-NH2 formed hydrogen bonds with the O-carboxamide (Th2) and not the azo group’s nitrogen atom, -Th4-Th5-S and Th4-Nazo(1)-Nazo(2)-CPh(Azo), we can see that the thienyl is flat and coplanar with the phenylazo moiety in compounds 3a-c and 5a-c. Despite, the NH group was in the same plane as the thienyl ring (NH-Th5-S-Th2 = 176.56–177.90°), the phenyl ring was angled on the aminothienyl plane between 21 and 24° in compounds 3a-c and 5a-c and at an angle of 29° in compounds 7a-c. Furthermore, the 2-carboxamide group was skewed for compounds 7a-c = 152° and lied in plane with the thienyl ring as S-Th2-Th3-OH(th) in range 179–176° for compounds 3a-c and 7a-c.Using the average values of the dihedral angles 3a-c and 5a-c derivatives HOMO plots are composed primarily of the π-orbitals of the thienyl, phenyl, and azo groups, as well as S, N, and O, non-bonding lone pairs except OH(Th3), which has a slight corporation. LUMO orbitals consist of π∗-orbitals of thienyl, phenyl, and azo groups with minimal heteroatom contributions. For 7a-c derivatives, the HOMO orbitals were distributed over the π-orbitals of the thienyl, phenyl, and amide groups, while their LUMO showed completely different composition, where it is localized only on the 2-carboxamide phenyl thiophene substituent. As shown in  ranged between 3.11–3.83 eV. It reveals that 7a-c derivatives have the maximum value. Also, the hydroxyl derivatives 3a-c were higher than corresponding methyl derivatives 5a-c. Finally, the amino derivatives, 7a-c showed higher ΔEH-L than other derivatives.Electronic properties and the chemical reactivity of molecules are chiefly determined by Frontier molecular orbitals (FMOs) , 56. TheHOMO and ELUMO we managed to determine some chemical descriptors such as electronegativity (χ), global hardness (η) and softness (δ) and electrophilicity (ω), respectively  results, the HOMO–LUMO electron flow in the 5c derivative resulted in a greater decrease in energy compared to the 3c and 7c derivatives.The results showed that Nazo(2), NHCO and NHTh5, each of which has positive charges, showing that its lone pair is involved in the resonance of phenyl ring. Much more, in derivatives 3a-c, the carbon atom of thienyl (Th3) has close negative charges, but in derivatives 5a-c, they have close positive charges. This could be due to the OH group electron donating effect. On parallel, the thienyl carbon atoms Th3 in derivatives 7a-c have close negative charges and lower than 3a-c and the reason behind this decrease is replacing azo group with amide group in position 4. Moreover, determination of the Fukui indices (q k (N), q k (N + 1) and q k (N − 1) are the systems atomic charges with N, N + 1 and N-1 electrons, respectively [Describing the electronegativity and charge transfer processes was offered as an output of quantum chemical calculations. This calculation process is described as the Mulliken’s atomic charges  , Nazo2 (7a-c) and S (7a-c),Nazo1(7a-c), Oacetyl. The data showed Th3 has higher It is clear for all derivatives mentclass2pt{minim3a displayed one intermolecular H-bond between N-aniline (Th5) with Pro 145 (2.99 ˚A) offering a score for binding energy, S = −8.1675 kcal/mol. Derivative 3b showed π- H interaction between aniline (Th5) with Ser 8 (3.84 ˚A) and π- π stick interactions between aryl azo (Th4) and Ph (Th2) with Trp 51 (3.83 ˚A) and His 175 (3.84 ˚A), respectively. An eminent binding energy score, S = −9.3283 kcal/mol was offered for derivative 3b  with Arg 84 (3.02 ˚A). The second binding type was π-H and π- π stick between Ph (Th2), aryl azo (Th4) and aniline (Th5) with Asp 146, Trp 51 and His 175 , respectively.To get a promising approach of synthesized ligands interactions with antioxidant and bacterial protein receptors, docking studies were performed. A molecular docking investigation was conducted on the newly synthesized thiophene-2-carboxamide derivatives in order to investigate their interaction with the crystallographic coordinates available in the RCSB Protein Data Bank. Downloaded from the Protein Data Bank  and deli5a exhibited notable binding score . The π-H and π- π stick forces between aniline (Th5) and aryl azo(Th4) with Arg 48 (4.14 ˚A) and Trp 51 (3.70 ˚A) was the reason for binding. A weak score of binding  showed by derivative 5b. This is due to two H-bonds exhibited between O-carboxamide (Th2) and O-acetyl with Arg 72 (3.04 ˚A) Glu 135 (3.12 ˚A) as well π- π stick forces between aniline (Th5) and His 96 (3.65˚A). Moreover, derivative 5c demonstrated a respectable binding score,  due to H-bond between N1-azo group with Ser 81(3.65˚A) and π-H interaction between thiophene ring with Asp 146 (4.37 ˚A). However, 3-amino thiophene carboxamide derivative 7a revealed two intermolecular H-bond between Trp 51 and His 52 with the O-carboxamide (Th2) (3.59 ˚A), (3.23 ˚A) respectively. Derivative 7a exhibited a highly regarded binding score with the amino acids of 2AS . Derivative 7b exhibited three intermolecular hydrogen bonds between N-carboxamide (Th4) with Asp 148 (3.49˚A), and N-amino group (Th3) with Asp 146 (2.90˚A) besides, O-acetyl group with Arg 48 (3.10 ˚A). It also showed π-H interaction between thiophene ring with Ala 147 (4.32˚A), and π-cation interaction between Ph (Th4) with Lys 149 (3.44˚A) recording a weak score . Moreover, derivative 7c presented two intermolecular H-bond between Ser 81 and Leu 177 with the S-atom and Cl atom (3.91 ˚A), (3.27 ˚A) through very good binding score . The ascorbic acid was subjected to amino acid 2AS1 and shows low score  resulted from two H-bonds between O(Lac3) and O(Et1) with His 181 and Leu 177 (3.16 ˚A), (2.88 ˚A) respectively.Meanwhile, 3-methyl thiophene carboxamide derivative P. aeruginosa and sortase A (PDB ID 2MLM) from S. aureus [E. coli [B. subtilis [P. aeruginosa  as a result of π-H interaction between anilide (Th2) with Trp 28 (4.17 ˚A). Moreover, derivative 3b demonstrated intermolecular H-bonds between N-aniline with Leu 39 (2.89 ˚A) and one π- H interaction between aniline (Th5) with Glu 150 (4.30 ˚A) through binding score . Additionally, derivative 3c showed a weak binding score . It has two H-bond interaction between O-carboxamide (Th2) with Tyr 45 (3.08 ˚A) and Lys 71 (2.84˚A) plus π- H interaction between aniline (Th4) with Tyr 97 (3.83 ˚A) and aryl azo (Th5) with Tyr 97(3.85 ˚A).For antibacterial target, IMP-1 metallo beta-lactamase (PDB ID 1DD6), from . aureus  and rhom[E. coli  and Bacisubtilis  was selesa Table . The 3-h5a through two interaction modes  outcomes from one H-bond interaction between O-carboxamide with Asn 167 (3.32 ˚A), and two π-H interaction between aniline with Val 25 (4.11 ˚A) and aryl azo with Ser 80 (4.24 ˚A). Furthermore, derivative 5c shows weak score  as a consequence of one H-bond interaction between O-acetyl group with Lys 215 (3.02 ˚A), and π-H interaction between aniline with Tyr 163 (4.51 ˚A).Meanwhile, a good score of binding  offered by 3-methyl thiophene carboxamide derivative 7a revealed two intermolecular H-bond between N-aniline (Th5) and O-carboxamide (Th4) with Leu 39 (3.07 ˚A) and Asn 41 (3.05 ˚A) besides, π-H interaction between anilide with Ala 42 (4.65 ˚A). Derivative 7a was displayed a highly regarded binding score . Furthermore, derivative 7b exhibited three intermolecular hydrogen bonds between O-carboxamide (Th2) with Lys 127 (3.42˚A), (2.88˚A), respectively. In addition to, O-carboxamide (Th4) with Tyr 97 (2.92 ˚A). It also showed two π-H interaction between anilide(Th4) with Tyr 97 (4.37˚A), and π-H interaction between aniline (Th5) with Try 124 (4.43˚A) offering a weak score . Moreover, derivative 7c presented one H-bond between Asp 170 with N-amino (Th3) (3.29 ˚A). It also displayed π-H and π-π interaction between thiophene ring with Ser 80 (4.95 ˚A) and His 79 (3.96 ˚A) showing a good score . Ampicillin was also docked with 1DD6 protein and showed good binding score  resulted from intermolecular H-bond between O-cyclic amide and O-carboxyl group with His 179 (3.06 ˚A) and Lys 161 (2.88 ˚A).However, 3-amino thiophene carboxamide derivative Staphyllococcus aureus (Table . The 3-hydroxy thiophene carboxamide derivative 3a exhibited two intermolecular H-bonds between S-thiophene ring with Asn 56 (3.69 ˚A) and O-carboxamide (Th2) with Lys 117 (3.03 ˚A) through a binding score . Also, derivative 3b demonstrated two intermolecular H-bonds between S-thiophene ring with Asn 56 (3.08 ˚A) and N-aniline (Th5) with Thr 122 (3.17 ˚A) through binding score . Moreover, derivative 3c demonstrated two types of interactions. Two intermolecular H-bonds between S-thiophene ring with Gln 114 (3.67 ˚A) and O-carboxamide (Th2) with Lys 117 (3.06 ˚A) and one π- H interaction between thiophene ring with Gln 120 (3.95 ˚A).For anti-bacterial protein sortase A (PDB ID 2MLM), the ligands inhibited the target by developing various associations with amino acid residues of active site of us Table . The 3-h5a exhibited a binding score . The binding interactions are three H-bonds between N-aniline (Th5) with Glu 47 (3.02 ˚A) (3.12 ˚A) and S-thiophene ring with Asn 56 (3.63 ˚A). A weak binding score was assigned to derivative 5b . Derivative 5b displayed four H-bond between S-thiophene ring and N-aniline (Th5) with Val 108 in addition to O-carboxamide (Th2) and (Th5) with Arg 139 (4.38 ˚A), (3.17 ˚A), (3.20 ˚A), (3.14 ˚A), respectively. Derivative 5c demonstrated a proper binding score, . The interactions in derivative 5c involves H-bond between O-acetyl group with Gln 120 (3.14˚A) and π-H interaction between arylazo (Th4) with Ala 46 (4.06 ˚A).Meanwhile, 3-methyl thiophene carboxamide derivative 7a displayed a highly regarded score of binding with the amino acids of 2MLM  rose from two H-bond between O-acetyl group with Gln 120 (3.00 ˚A) and S-thiophene ring with Glu 113 (3.68 ˚A). Also, a π-cation interaction between aniline (Th5) with Arg 139 (3.93 ˚A) was noticed  along with O-carboxamide (Th2) with Lys 117 (2.97 ˚A) through abundant binding score . Additionally, derivative 7c presented H-bond between Gln 120 with N-amide (Th2) (3.16 ˚A). It also exhibited two π-H interactions between aniline (Th5) and (Th4) with Ser 99 and Thr 122 (3.68 ˚A), (4.12 ˚A) respectively through good binding score . Ampicillin showed with 2MLM protein good binding score  resulted from six H-bonds between C-benzyl and S-thiazole with Glu 47 (3.35 ˚A), (3.63 ˚A), respectively. As well, N-amino group with Asn 56 (3.23 ˚A) and O-carboxylic ketone with Gln 55 (2.93 ˚A) was also appeared. Lys 117 was engaged in two H-bonds with O-cyclic ketone and O-β-lactam (3.00 ˚A and 3.07 ˚A).However, 3-amino thiophene carboxamide derivative lu 113 3.8 ˚A. AlsE. coli , the ligands inhibited the target by developing various associations with amino acid residues of active site of li Table .Table 7T3a displayed two H-bonds between O-carboxamide (Th2) with Ser 201 (2.96 ˚A), O-acetyl group with Met 208 (3.40 ˚A) and π- H interaction between thiophene ring with His 150 (4.12 ˚A). These interactions have a strong binding score . Derivative 3b demonstrated binding score  presented in a H-bond between O-carboxamide (Th2) with Arg 92 (2.92˚A). Moreover, derivative 3c shows two mode of interactions via a binding score . Two H-bonds between N-aniline (Th5) (3.17 ˚A) and N1-azo group with Ser 201 (3.35˚A) and π- π interaction between thiophene ring with His 150 (3.58 ˚A).The 3-hydroxy thiophene carboxamide derivative 5a exhibited binding score  resulted from H-bonds between S-thiophene ring with Leu 152 (4.14 ˚A). But, derivative 5b showed a rising binding score . Binding modes are, one H-bond displayed between N2-azo group with Ser 201 (3.00 ˚A) as well as π- π stick forces between Ph(Th2) with Phe 146 (3.58 ˚A) and thiophene ring with His 150 (3.88 ˚A). Derivative 5c have a relatively high binding score, . It also forms a π- π stick forces between thiophene ring with His 150 (3.78 ˚A) and Ph (Th2) with Phe 146 (3.95 ˚A).Meanwhile, 3-methyl thiophene carboxamide derivative 7a established two interaction modes with a binding score . Derivative 7a shows two H-bond between O-carboxamide (Th4) with Ser 201 and His 254 (3.27˚A), (3.00˚A), respectively. A π-H type of interaction between thiophene ring with Gly 198 (4.78 ˚A) and π- π stick forces between Ph (Th2) Phe 146 (3.68 ˚A). Derivative 7b demonstrated one H-bond between O-carboxamide (Th2) with Ala 250 (2.96 ˚A) through a binding score . Derivative 7c presented one π-π stick interaction between His 150 and Ph (Th4) (3.95 ˚A) via a unique binding score  , the ligands inhibited the target by developing various associations with amino acid residues of active site of 3a exhibited two types of interactions offering an eminent binding score . A H-bond between N-aniline (Th5) with Trp 329 (3.17 ˚A) and π- H interaction between Ph (Th2) with Gly 68 (3.98 ˚A)  through two modes of interactions. Two H-bonds between N-carboxamide (Th2) with Met 221 (4.23˚A) and O-acetyl group with Lys 360 (2.92˚A). Besides π- H interaction and π- π stick forces between aniline (Th5) and Ph (Th2) with Arg 65 (4.11 ˚A) and His 128 (3.90 ˚A). Derivative 3c showed two modes of interactions and a score of binding . A H-bond between N2-azo group with Lys 211 (3.14 ˚A) and π- H interaction between Ph (Th2) with Glu 194 (3.78 ˚A). Furthermore, 3-methyl thiophene carboxamide derivative 5a revealed binding score  via π-H interaction between Ph (Th2) with Arg 65 (4.19 ˚A). Derivative 5b have a fallen binding score  through π-H interaction between thiophene ring with Pro 196 (3.78 ˚A). Derivative 5c demonstrated a relatively high binding score,  through three H-bonds between S-thiophene ring with Tyr 357 (3.37 ˚A) and O-carboxamide (Th2) with Arg 65 (2.89 ˚A) and O-acetyl group with Asn 64 (3.08 ˚A). Along with π- H interaction between Ph (Th2) with Arg 65 (4.83 ˚A).The 3-hydroxy thiophene carboxamide derivative 7a presented two interactions through a binding score . It shows H-π interaction between H-acetyl group with Trp 238 (3.87 ˚A) and π-H interaction between Ph (Th5) with Trp 329 (4.45 ˚A). Derivative 7b established excellent binding score  caused by two H-bonds between N-amino (Th3) and N-aniline (Th5) with Cys 66 (3.68 ˚A) and Met 221 (3.70 ˚A) besides two π-H interaction between Ph (Th4) and thiophene ring with Arg 65 (3.77 ˚A) and Phe 235 (4.65 ˚A). Furthermore, derivative 7c presented π-H interaction between Lys 259 with Ph (Th4) (4.34 ˚A) in addition to four H-bonds through binding score . Asp 256 established H-bonds with N-carboxamide (Th4) (3.29 ˚A) and (Th2) (2.96 ˚A). Lys 257 and Lys 260 showed H- bonds with O-acetyl group (2.93 ˚A) (2.98 ˚A). Ampicillin showed with 4D3V protein binding score  resulted from H-bond between Glu 243 with O-carboxyl group (2.86 ˚A).However, 3-amino thiophene carboxamide derivative 2) were synthesized. In addition to intramolecular H-bonds, the DFT modelling data suggested a comparable Mulliken's charge and HOMO–LUMO contribution. The newly synthesized 3-substituted thiophene 2-carboxamide 3a-c, 5a-c, 7a-c antioxidant activity was examined through ABTS method, while the antibacterial activity was assessed against 4 types of bacteria. Derivatives 7a-c inhibited better than derivatives 3a-c and 4a-c. SAR-study of the obtained compounds shows the enhancement of OMe substituent on the activity and absence of substituent was also a positive impact. In addition, the synthesized compounds were subjected to a molecular docking analysis using (PDB ID 2AS1) (PDB ID 1DD6) (PDB ID 2MLM) (PDB ID 3ZMI) (PDB ID 4D3V). Thiophene derivatives 3b, 5a, 7a, 7c and 3a had better score of binding  toward the selected proteins.A series of 3-substituted thiophene 2-carboxamide with functional groups  in sodium ethoxide solution (which was prepared from 0.05 g Na and 20 mL absolute ethanol) was stirred for 10 min and then N-(4-acetylphenyl)-2-chloroacetamide (1)  was added. The mixture was refluxed for 2 h then was permitted to be cool, and poured onto ice-water. The obtained product was dried out and crystallized from ethanol to give the targeted hydroxythiophenes 3a-c.A suspension of each 2-(arylhydrazono)-2-ethoxycarbonyl-thioacetanilide derivative −1): 3363 (N–H stretch), 1654 (C = O stretch). 1H NMR (δ/ppm): 2.34 , 2.52 , 7.29 , 7.44–7.52 , 7.75 , 7.93 , 9.86 , 12.38 . 13C NMR (δ/ppm): 20.80, 26.46, 118.55 (3C), 118.92 (2C), 121.36 (4C), 125.93, 129.64 (4C), 129.72 (5C), 129.93 (3C), 160.83, 196.51. MS: m/z (%) = 470 , 430.94 (52.51), 405.13 (78.21), 400.79 (100.00), 284.79 (51.30), 284.28 (46.29), 254.68 (44.82), 244.49 (51.91), 237.61 (50.33), 236.67 (45.29), 175.37 (51.74), 172.90 (44.75), 90.64 (45.29), 40.61 (52.01). Analysis calculated for C26H22N4O3S (470.14): C, 66.37; H, 4.71; N, 11.91%. Found: C, 66.07; H, 4.63; N, 12.07%.Red solid, yield = 56%, m.p. = 270–271 °C. IR (ν/cm−1): 3366 (N–H stretch), 1654 (C = O stretch). 1H NMR (δ/ppm): 2.52 , 3.84 , 7.07 , 7.26 , 7.46–7.52 , 7.74 , 7.93 , 9.73 , 12.40 . 13C NMR (δ/ppm): 26.48, 55.58, 114.62 (2C), 114.76, 118.55 (2C), 118.99, 121.13 (3C), 122.13 (2C), 125.63, 129.67 (4C), 129.79 (4C), 131.51, 153.91, 159.95, 196.50. Analysis calculated for C26H22N4O4S (486.14): C, 64.18; H, 4.56; N, 11.52%. Found: C, 63.96; H, 4.48; N, 11.68%.Dark red solid, yield = 48%, m.p. = 234–235 °C. IR (ν/cm−1): 3366 (N–H stretch), 1653 (C = O stretch). 1H NMR (δ/ppm): 2.52 , 7.29 , 7.42 , 7.49–7.58 , 7.74 , 7.87 , 7.93 , 9.88 , 12.47 . Analysis calculated for C25H19ClN4O3S (490.09): C, 61.16; H, 3.90; N, 11.41%. Found: C, 61.32; H, 3.85; N, 11.50%.Red crystals, yield = 52%, m.p. = 252–253 °C. IR  in 20 mL dioxane, sodium methoxide  and N-(4-acetylphenyl)-2-chloroacetamide (1)  were added. The mixture was refluxed for an hour and then sodium methoxide  was added again. Refluxing was continued for additional 2 h and then the reaction mixture was permitted to cool. The formed solid product was collected to give the targeted methylthiophenes 5a-c.To a solution of each 2-acetyl-2-arylazo-thioacetanilide derivative −1): 3447, 3277 (N–H stretch), 1675, 1640 (C = O). 1H NMR (δ/ppm): 2.32 , 2.49 , 2.51 , 7.25 , 7.30 , 7.38 , 7.48–7.55 , 7.75 , 7.90 . 13C NMR (δ/ppm): 12.94, 20.65, 26.47, 115.09, 116.81 (2C), 119.86 (2C), 120.86 (3C), 121.05 (2C), 125.23, 125.82, 129.26 (2C), 129.36 (2C), 129.66 (3C), 129.98, 130.07 (2C), 170.10, 196.56. Analysis calculated for C27H24N4O2S (468.16): C, 69.21; H, 5.16; N, 11.96%. Found: C, 69.43; H, 5.07; N, 12.11%.Pale red crystals, yield = 70%, m.p. = 290–291 °C. IR (ν/cm−1): 3447, 3289 (N–H stretch), 1676, 1639 (C = O). 1H NMR (δ/ppm): 2.52 , 2.57 , 3.80 , 7.05 , 7.24 , 7.42–7.51 , 7.70 , 7.79 , 7.91 , 8.49 , 10.28 . 13C NMR (δ/ppm): 13.32, 26.50, 55.51, 114.78 (2C), 119.66 (2C), 120.14 (2C), 120.51 (3C), 125.33, 129.29 (3C), 129.76 (3C), 134.35, 141.41, 143.91, 158.76, 161.61, 166.67, 175.08, 196.59. MS: m/z (%) = 484 , 475.20 (41.81), 408.21 (33.89), 380.32( 31.14), 370.21 (76.02), 352.11 (30.03), 328.12 (34.06), 327.54 (59.69), 313.37 (55.63), 298.16 (48.14), 297.22 (52.83), 253.13 (44.02), 233.13 (30.90), 206.22 (30.80), 165.38 (85.57), 86.55 (63.81), 75.22 (100.00), 66.44 (83.76). Analysis calculated for C27H24N4O3S (484.16): C, 66.92; H, 4.99; N, 11.56%. Found: C, 66.75; H, 5.06; N, 11.67%.Red crystals, yield = 63%, m.p. = 319–320 °C. IR (ν/cm−1): 3448, 3277 (N–H stretch), 1674, 1638 (C = O). 1H NMR (δ/ppm): 2.45 , 2.52 , 7.28 , 7.39 , 7.47–7.52 , 7.63 , 7.78 , 7.92 , 8.50 , 10.27 . Analysis calculated for C26H21ClN4O2S (488.11): C, 63.86; H, 4.33; N, 11.46%. Found: C, 63.64; H, 4.28; N, 11.61%.Orange crystals, yield = 81%, m.p. = 270–271 °C. IR (ν/cmN-phenyl-3-(phenylamino)acrylamide derivative 6a, 6b or 6c (0.002 mol), N-(4-acetylphenyl)-2-chloroacetamide  and sodium methoxide  was refluxed in 20 mL dioxane for 2 h. The mixture was poured into ice-water. The formed solid was collected and recrystallized from ethanol to produce the corresponding 3-aminothiophene compounds 7a-c.A mixture of each 2-cyano-3-mercapto-−1): 3346, 3276, 3192 (NH2 & NH), 1654 . 1H NMR (δ/ppm): 2.51 , 5.12 , 7.03–7.08 , 7.29–7.38 , 7.57 , 7.68 , 7.94 , 8.80 , 9.31 , 10.71 , 11.85 . 13C NMR (δ/ppm): 26.50, 118.54 (2C), 119.44 (2C), 119.94, 120.29, 121.21 (2C), 123.21, 124.50, 128.49, 129.01 (2C), 129.28 (2C), 129.63 (2C), 132.20, 138.58, 142.88, 149.95, 163.38, 165.85, 167.48, 196.57. MS: m/z (%) = 470 , 453.06 (58.69), 449 (63.16), 44.27 (52.26), 429.94 (52.39), 354.88 (52.44), 174.09 (62.15), 147.41 (75.00), 117.02 (57.18), 104.56 (100), 43.95 (72.07). Analysis calculated for C26H22N4O3S (470.14): C, 66.37; H, 4.71; N, 11.91%. Found: C, 66.48; H, 4.65; N, 11.82%.White crystals, yield = 47%, m.p. = 190–191 °C. IR (ν/cm−1): 3390, 3252 (NH2 & NH), 1670, 1649 (C = O stretch). 1H NMR (δ/ppm): 2.24 , 2.51 , 5.10 , 7.06–7.14 , 7.36–7.52 , 7.67 , 7.94 , 8.76 , 9.33 , 10.71 , 11.75 . Analysis calculated for C27H24N4O3S (484.16): C, 66.92; H, 4.99; N, 11.56%. Found: C, 67.08; H, 5.06; N, 11.45%.Yellow solid, yield = 54%, m.p. = 230–231 °C. IR (ν/cm−1): 3348, 3234 (NH2 & NH), 1646 . 1H NMR (δ/ppm): 2.52 , 3.77 , 5.12 , 6.94 , 7.38–7.43 , 7.68 , 7.74 , 7.98 , 8.78 , 9.25 , 10.70 , 11.81 . Analysis calculated for C27H24N4O4S (500.15): C, 64.79; H, 4.83; N, 11.19%. Found: C, 64.84; H, 4.89; N, 11.31%.Orange solid, yield = 62%, m.p. = 244–245 °C. IR  software.The three-dimensional structure (3D) of the selected proteins (PDB ID 2AS1), (PDB ID 1DD6), (PDB ID 2MLM), (PDB ID 3ZMI), (PDB ID 4D3V) were downloaded from the PDB website. The water molecules and repeated chains were removed. Protons were added and the energy of the protein was minimized. The preparation of thiophene 2-carboxamide derivatives Additional file 1: Experimental general remarks. Table S1. Bond length, bond angle, dihedral angle for compounds 3a-c. Table S2. Bond length, bond angle, dihedral angle for compounds 5a-c. Table S3. Bond length, bond angle, dihedral angle for compounds 7a-c. Table S4. The atomic Mulliken’s charges and Fukui’s indices of investigated compounds. Fig. S1. DFT optimized structures for compounds 3a-c, 5a-c, and 7a-c. Fig. S2. The binding interaction of 3-hydroxythiophene 3a with (PDB ID: 2AS1). Fig. S3. The binding interaction of 3-hydroxythiophene 5a with (PDB ID: 2AS1). Fig. S4. The binding interaction of 3-hydroxythiophene 5b with (PDB ID: 2AS1). Fig. S5. The binding interaction of 3-hydroxythiophene 5c with (PDB ID: 2AS1). Fig. S6. The binding interaction of 3-hydroxythiophene 7a with (PDB ID: 2AS1). Fig. S7. The binding interaction of 3-hydroxythiophene 7b with (PDB ID: 2AS1). Fig. S8. The binding interaction of 3-hydroxythiophene 7c with (PDB ID: 2AS1). Fig. S9. The binding interaction ascorbic acid with (PDB ID: 2AS1). Fig. S10. The binding interaction of 3-hydroxythiophene 3a with (PDB ID: 1DD6). Fig. S11. The binding interaction of 3-hydroxythiophene 3b with (PDB ID: 1DD6). Fig. S12. The binding interaction of 3-hydroxythiophene 3c with (PDB ID: 1DD6). Fig. S13. The binding interaction of 3-methylthiophene 5b with (PDB ID: 1DD6). Fig. S14. The binding interaction of 3-methylthiophene 5c with (PDB ID: 1DD6). Fig. S15. The binding interaction of 3-aminothiophene 7a with (PDB ID: 1DD6). Fig. S16. The binding interaction of 3-aminothiophene 7b with (PDB ID: 1DD6). Fig. S17. The binding interaction of 3-aminothiophene 7c with (PDB ID: 1DD6). Fig. S18. The binding interaction of with ampicillin (PDB ID: 1DD6). Fig. S19. The binding interaction of 3-hydroxythiophene 3a with (PDB ID: 2MLM). Fig. S20. The binding interaction of 3-hydroxythiophene 3b with (PDB ID: 2MLM). Fig. S21. The binding interaction of 3-hydroxythiophene 3c with (PDB ID: 2MLM). Fig. S22. The binding interaction of 3-methylthiophene 5a with (PDB ID: 2MLM). Fig. S23. The binding interaction of 3-methylthiophene 5b with (PDB ID: 2MLM). Fig. S24. The binding interaction of 3-methylthiophene 5c with (PDB ID: 2MLM). Fig. S25. The binding interaction of 3-methylthiophene 7b with (PDB ID: 2MLM). Fig. S26. The binding interaction of 3-methylthiophene 7c with (PDB ID: 2MLM). Fig. S27. The binding interaction of ampicillin with (PDB ID: 2MLM). Fig. S28. The binding interaction of 3-hydroxythiophene 3a with (PDB ID: 3ZMI). Fig. S29. The binding interaction of 3-hydroxythiophene 3b with (PDB ID: 3ZMI). Fig. S30. The binding interaction of 3-hydroxythiophene 3c with (PDB ID: 3ZMI). Fig. S31. The binding interaction of 3-methylthiophene 5a with (PDB ID: 3ZMI). Fig. S32. The binding interaction of 3-methylthiophene 5b with (PDB ID: 3ZMI). Fig. S33. The binding interaction of 3-methylthiophene 5c with (PDB ID: 3ZMI). Fig. S34. The binding interaction of 3-aminothiophene 7a with (PDB ID: 3ZMI). Fig. S35. The binding interaction of 3-aminothiophene 7b with (PDB ID: 3ZMI). Fig. S36. The binding interaction of with ampicillin (PDB ID: 3ZMI). Fig. S37. The binding interaction of 3-hydroxythiophene 3b with (PDB ID: 4d3v). Fig. S38. The binding interaction of 3-hydroxythiophene 3c with (PDB ID: 4d3v). Fig. S39. The binding interaction of 3-methylthiophene 5a with (PDB ID: 4d3v). Fig. S40. The binding interaction of 3-methylthiophene 5b with (PDB ID: 4d3v). Fig. S41. The binding interaction of 3-methylthiophene 5c with (PDB ID: 4d3v). Fig. S42. The binding interaction of 3-aminothiophene 7a with (PDB ID: 4d3v). Fig. S43. The binding interaction of 3-aminothiophene 7b with (PDB ID: 4d3v). Fig. S44. The binding interaction of 3-aminothiophene 7c with (PDB ID: 4d3v). Fig. S45. The binding interaction of with ampicillin (PDB ID: 4d3v). Fig. S46. IR spectrum of compound 3a. Fig. S47.1H NMR spectrum of compound 3a. Fig. S48.13C NMR spectrum of compound 3a. Fig. S49. IR spectrum of compound 3b. Fig. S50.1H NMR spectrum of compound 3b. Fig. S51.13C NMR spectrum of compound 3b. Fig. S52. IR spectrum of compound 3c. Fig. S53.1H NMR spectrum of compound 3c. Fig. S54. IR spectrum of compound 5a. Fig. S55.1H NMR spectrum of compound 5a. Fig. S56.13C NMR spectrum of compound 5a. Fig. S57. IR spectrum of compound 5b. Fig. S58.1H NMR spectrum of compound 5b. Fig. S59.13C NMR spectrum of compound 5b. Fig. S60. IR spectrum of compound 5c. Fig. S61.1H NMR spectrum of compound 5c. Fig. S62. IR spectrum of compound 7a. Fig. S63.1H NMR spectrum of compound 7a. Fig. S64.13C NMR spectrum of compound 7a. Fig. S65. IR spectrum of compound 7b. Fig. S66.1H NMR spectrum of compound 7b. Fig. S67. IR spectrum of compound 7c. Fig. S68.1H NMR spectrum of compound 7c.