RS)-2-(6-chloro-9H-carbazol-2-yl)propanonic acid (carprofen) treatment with methanol, yielding methyl (RS)-2-(6-chloro-9H-carbazol-2-yl)propanoate; (ii) the resulted methylic ester was converted to (RS)-2-(6-chloro-9H-carbazol-2-yl)propane hydrazide (carprofen hydrazide) by treatment with hydrazine hydrate; (iii) reaction of the hydrazide derivative with acyl chlorides led to N-[(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoil]-N′-R-substituted-benzoylhydrazine formation, which; (iv) in reaction with phosphorus oxychloride gave the (RS)-1-(6-chloro-9H-carbazol-2-yl)-1-ethane derivatives. In the second synthesis pathway, new 1,3,4-oxadiazole ring compounds were obtained starting from carprofen which was reacted with isoniazid, in the presence of phosphorus oxychloride to form (RS)-1-(6-chloro-9H-carbazol-2-yl)-1-ethane. The synthesized compounds were characterized by IR, 1H-NMR and 13C-NMR, screened for their drug-like properties and evaluated for in vitro cytotoxicity and antimicrobial activity. The obtained compounds exhibited a good antimicrobial activity, some of the compounds being particularly active on E. coli, while others on C. albicans. The most significant result is represented by their exceptional anti-biofilm activity, particularly against the P. aeruginosa biofilm. The cytotoxicity assay revealed that at concentrations lower than 100 μg/mL, the tested compounds do not induce cytotoxicity and do not alter the mammalian cell cycle. The new synthesized compounds show good drug-like properties. The ADME-Tox profiles indicate a good oral absorption and average permeability through the blood brain barrier. However, further research is needed to reduce the predicted mutagenic potential and the hepatotoxicity.In this paper, we aimed to exploit and combine in the same molecule the carbazole and the 1,3,4-oxadiazole pharmacophores, to obtain novel carprofen derivatives, by using two synthesis pathways. For the first route, the following steps have been followed: (i) ( Carbazole and its derivatives represent an important group of aromatic heterocyclic compounds containing a nitrogen atom. Compounds containing this scaffold have important electronic and charge-transport properties, as well as a conjugated pi-electron system, which facilitate the easy introduction of various functional groups into the structurally rigid carbazole ring .These properties result in the extensive applications of carbazole derivatives in the medical field, such as antitumor, antimicrobial, antihistaminic, antioxidant, anti-inflammatory, and psychotropic agents .Murraya euchrestifolia Hayata has shown a strong fungicidal effect on Cladosporium cucumerinum -N-[(substituted phenyl)(piperazin-1-yl)methyl]-1,3,4-oxadiazol-2-amines derivatives proved an antimicrobial effect and anti-proliferative activity, against the breast cancer cell line MCF7 ..21].The most commonly used synthetic route for the synthesis of 1,3,4-oxadiazoles includes the reactions of acid hydrazides with acid chlorides or carboxylic acids and cyclization of the formed acylhydrazines using a dehydrating agent, such as phosphorous pentaoxide, thionyl chloride, or phosphorous oxychloride .1H-NMR and 13C-NMR, screened for their drug-like properties and evaluated for in vitro cytotoxicity and antimicrobial activity.In the present study, we aimed to combine in the same molecular framework the carbazole and the 1,3,4-oxadiazole pharmacophores, to obtain novel carprofen derivatives. The new synthesized compounds were characterized by IR, The synthesis routes approached to obtain the target compounds are outlined in RS)-2-(6-chloro-9H-carbazol-2-yl)propanoic acid (carprofen) (1) with methanol, in the presence of concentrated sulfuric acid leading to methyl (2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoate (carprofen methyl ester) (2). The reaction of compound 2 with hydrazine hydrate in refluxing ethanol gave (2RS)-2-(6-chloro-9H-carbazol-2-yl)propane hydrazide (carprofen hydrazide) (3).The reaction of  were obtained by stirring the carprofen hydrazide (3) and the acyl chloride at room temperature in anhydrous pyridine.The 4a–c showed characteristic absorption band at 3201–3215 cm−1 due to N–H group and also to carbonyl absorption bands at 1706–1702 and 1649–1651 cm−1. Its 1H-NMR spectrum revealed singlets at δ 10.15–10.19 and 10.34–10.66 ppm due to two NH protons of the hydrazide moiety, in addition to singlet at δ 11.38–11.41 ppm assignable for -NH- proton of the carbazole core. The 13C-NMR spectral data for compounds 4a–c revealed signals in the aromatic region and additional two singlets around δ 172.74–172.90 and in the region 164–165.93 ppm for the two carbonyl groups, which are in agreement with the assigned structures.The IR spectrum of compounds 5a–c) the corresponding acylhydrazide was heated on the water bath with phosphorus oxychloride.For the synthesis of 2,5-disubstituted 1,3,4-oxadiazoles (RS)-1-(6-chloro-9H-carbazol-2-yl)-1-ethane (5d) by reaction with isoniazid, in the presence of phosphorus oxychloride.Starting from carprofen we also obtained  values ranged in the same interval as the MIC ones, being equal or one time higher than that corresponding MIC values, suggesting a microbicidal mechanism of action .P. aeruginosa biofilm.All tested compounds exhibited excellent anti-biofilm properties against the bacterial and fungal strains, with Minimal Biofilm Eradication Concentration (MBEC) values generally ranging from 0.009 to 2.5 mg/mL . The MBEFor the in vitro cytotoxicity assay, the results show that the tested compounds become toxic at concentrations higher than 100 μg/mL, with the HeLa cells and HaCaT cells viability gradually decreasing after 48 h of incubation, in a dose-dependent manner . All tesAfter 48 h, at 100 μg/mL, the compounds do not alter cell cycle phases of HeLa cells, but a fluctuation of the synthesis (S) phase is observed for HaCaT cells .The tested compounds comply with Lipinski rule and are predicted to have a good bioavailability according to the Veber rule .4a–c and 5a–d with other already known chemicals compounds, we noticed that compounds 1a, c recorded a higher similarity with compound 2-(6-chloro-9H-carbazol-2-yl)-N-phenylpropanamide, with biological activity, i.e., inhibition of Faah—fatty acid amide hydrolase active site etil]-1,3,4-oxadiazol-2-il]-1~{H}-indol-4-il]-~{N},~{N}-dimetiletanamine, compound 5c is similar with 2-(3~{H}-benzimidazol-5-yl)-5-[2-[4-(trifluoromethyl)phenyl]ethyl]-1,3,4-oxadiazole, a cyclotransferase inhibitor.Considering that the structural similarities may induce similarities in biological activities, we identified the possible structural similarities of the analyzed compounds with other ligands . Interes5d we founded a poor similarity with other know compounds  and the carprofen methyl ester were confirmed on the basis of spectral studies.We synthesized novel derivatives based on 6-chloro-94a showing the best activity against E. coli. In contrast, 5c is a potentially antifungal agent. It is to be highlighted that the obtained compounds acted preferentially on biofilm embedded cells, as revealed by the very low MBEC values obtained for some compound-microbial strain pairs, in comparison with the MIC and MBC values. Also, it is to be noticed the very good anti-biofilm activity of all tested compounds against the P. aeruginosa biofilm, well known for its high resistance to all currently available antimicrobial agents. The in vitro cytotoxicity of the tested compounds performed on two cellular lines  reveal a dose- and time-related effect, the cellular viability decreasing with the increase of concentration (>100 μg/mL) and incubation time (48 h). However, at concentrations lower than 100 μg/mL, the tested compounds do not induce cytotoxicity and do not alter the mammalian cell cycle.The biological activity data have shown that the synthesized compounds have a good antimicrobial activity, especially on Gram-negative strains, The new synthesized compounds show good drug-like properties. All studied compounds comply with Lipinski rule and also, have a good predicted bioavailability according to the Veber rule.4a–c and 5a–d, we evaluated by bioinformatics tools the molecular similarity of our compounds and other compounds, already included into most popular databases. Our results have shown that compounds 4a–c exhibit a molecular similarity with Faah inhibitors, while the compounds 5a–c, appeared to have nociceptive and QPCT—glutaminyl-peptide cyclotransferase inhibitory activities.To predict a possible biological activity of compounds The organic cation transporter OCT2 is involved in the renal excretion of cationic drugs and raises the possibility of drug–drug interactions between an inhibitor and a substrate drug, which will have a decreased renal clearance .5a, 5b, and 5c show an average permeability through the blood brain barrier. Unfortunately, the compounds may also have mutagenic potential and increased hepatotoxicity.The ADMET predictions show that the compounds may have good oral absorption. Plus, Research may continue in this direction, by synthesizing similar compounds with lower cytotoxic, mutagenic and hepatotoxic potential.Melting points were determined in open glass capillary tube on an Electrothermal 9100  capillary melting point apparatus and are uncorrected.The FT-IR ATR  spectra were taken in solid state on a Bruker Vertex 70 spectrophotometer .1H-NMR and 13C-NMR spectra were recorded in dimethylsulfoxide-d6 (DMSO-d6) or deuterated chloroform on a Bruker Fourier 300 MHz or Bruker AvanceIII 500 MHz instrument , using tetramethylsilane as the internal standard, the chemical shifts are expressed in δ ppm.The 1H-NMR data are reported in the following order: chemical shifts, multiplicity, the coupling constants, number of protons and signal/atom attribution. The apparent resonance multiplicity is described as s (singlet), d (doublet), q (quartet), m (multiplet), dd (double doublet), and br (broad) signal.The 13C-NMR data the order is the following: chemical shifts and signal/atom attribution.For the 5d was recorded on a Maxis Bruker 4G spectrometer  with an electrospray ionization source (ESI). The samples were dissolved in DMSO 1 mg/1 mL and then the solution was diluted with methanol to a final concentration of 1 μg/1 mL. The scanning range of molecular ions (m/z) was 50–1250.The mass spectrum of compound Methyl (2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoat (carprofen methyl ester) (2)RS)-2-(6-Chloro-9H-carbazol-2-yl)propanoic acid (carprofen)  was dissolved with stirring in 400 mL methanol absolute. Then 1.2 mL concentrated sulfuric acid (98%) was dropwise added as a catalyst and stirring was continued at room temperature for 8 h. The reaction mixture was left overnight at room temperature, and then the methanol was evaporated at low pressure until a precipitate appeared. Over the reaction mixture, 200 mL of water was added and the precipitate was filtered off at low pressure and washed well with water on the filter to remove the acid traces. The white or slightly yellow precipitate was air dried for 24 h.(We obtained 10 g (yield 95%) of carprofen methyl ester (m.p. 107–110 °C).−1): 3450w; 3405vs; 2983w; 2944w; 2875w; 1729vs; 1697m; 1624m; 1448m; 1429m; 1325w; 1269w; 1236w; 1193m; 1172m; 1093w; 1059m; 873w; 806m; 731w; 695w.FT-IR : 8.26 ; 7.96 ; 7.92 ; 7.33 ; 7.31 ; 7.25 ; 7.17 ; 3.90 ; 3.71 ; 1.60 ;13C-NMR : 175.49 (C-12); 140.29 (C-8a); 139.01 (C-1a); 138.00 (C-2); 125.70 (C-7); 124.74 (C-5a); 124.11 (C-4a); 121.53 (C-6); 120.54 (C-4); 119.84 (C-5); 119.47 (C-3); 111.54 (C-8); 109.49 (C-1); 52.17 (C-13); 45.74 (C-10); 18.86 (C-11).(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanohydrazide (carprofen hydrazide) (3)Carprofen methyl ester  in ethanol 96% or absolute (40 mL) is treated under magnetic stirring, with hydrazine hydrate 100% (7 mL) and refluxed for 8 h with continuous stirring. Then the mixture is cooled and the carprofen hydrazide is filtered off at low pressure and washed with cold alcohol on the filter; 4.2 g of white carprofen hydrazide are obtained with a melting point of 241–243 °C (yield 70%).−1): 3347s; 3260m; 2979w;2873w; 1632vs; 1517m; 1462s; 1428m; 1379w; 1338m; 1269m; 1238s; 1120w; 1061m; 988m; 926w; 885m; 828w; 800m; 733w; 690w.FT-IR : 11.36 ; 9.24 ; 8.15 ; 8.05 ; 7.48 ; 7.47 ; 7.35 ; 7.16 ; 4.21 ; 3.70 ; 1.42 ;13C-NMR : 173.05 (C-12); 140.67 (C-8a); 140.56 (C-1a); 138.36 (C-2); 123.71 (C-5a); 123.56 (C-4a); 122.85 (C-6); 125.02 (C-7); 120.34 (C-4); 119.64 (C-5); 118.90 (C-3); 112.34 (C-8); 109.71 (C-1); 43.81 (C-10); 18.79 (C-11).N-[(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoyl]-N’-benzoylhydrazine (4a)In a round bottomed flask equipped with stirrer, carprofen hydrazide  and benzoyl chloride  and anhydrous pyridine (20 mL) are introduced. The mixture was stirred at room temperature for 4 h. After the reaction time has expired, the mixture was poured into a dilute, cold solution of hydrochloric acid (40 mL 10% HCl solution). A precipitate was obtained, isolated by filtration, washed on the filter with water and used in the steps in the raw state.N,N’-disubstituted hydrazine were obtained, with a melting point (m.p.) of 262–263 °C at a yield of 87% toward hydrazide. The compound is soluble at room temperature in pyridine, dimethylsulfoxide, dimethylformamide, after heating in methanol, ethanol, isobutanol, and hardly soluble after heating in isopropanol, xylene, insoluble in ethyl acetate, hexane and water.After drying, 1.02 g of −1): 3356s; 3215m; 3084w; 3024w; 2974w; 2934w; 1706vs; 1651vs; 1576m; 1466s; 1367w; 1273m; 1241m; 1180w; 1144w; 1066w; 947w; 890w; 804w; 739w; 705m; 647w; 601m.FT-IR : 11.38 ; 10.34 ; 10.15 ; 8.17 ; 8.09 ; 7.86 ; 7.58- 7.44 ; 7.53 ; 7.35 ; 7.23 ; 3.92 ; 1.50 ;13C-NMR : 172.74 (C-12); 165.93 (C-13); 140.28 (C-8a); 138.91 (C-1a); 138.11 (C-2); 132.32 (C-14); 131.70 (C-17); 128.35 ; 127.30 ; 124.93 (C-7); 123.53 (C-5a); 122.72 (C-4a); 120.30 (C-4); 120.26 (C-6); 119.56 (C-5); 118.89 (C-3); 112.20 (C-8); 109.72 (C-1); 43.56 (C-10); 18.85 (C-11).N-[(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoyl]-N’-(4-chlorobenzoyl)hydrazine (4b)4a), from 4-chlorobenzoyl chloride .The compound was prepared by the method described for (There were obtained 1.04 g of compound (81% yield toward hydrazide), with m.p. 268–270 °C.The compound is soluble at room temperature in pyridine, dimethylsulfoxide, dimethylformamide, after heating in isobutanol, hardly soluble after heating in methanol, ethanol, isopropanol, ethyl acetate, and insoluble in xylene, hexane and water.−1): 3414w; 3201m; 1599vs; 1567m; 1454m; 1273w; 1221w; 1152w; 1093w; 848w; 802w; 754w; 730w; 659w.FT-IR : 11.41 ; 10.46 ; 10.21 ; 8.17 ; 8.09 ; 7.87 ; 7.56 ; 7.52 ; 7.48 ; 7.36 ; 7.22 ; 3.93 ; 1.49 ;13C-NMR : 172.90 (C-12); 164.49 (C-13); 140.55 (C-8a); 139.98 (C-1a); 138.38 (C-2); 136.69 (C-17); 131.16 (C-14); 129.35 ; 128.63 ; 125.06 (C-7); 123.62 (C-4a); 122.83 (C-5a); 120.43 (C-4); 120.37 (C-6); 119.67 (C-5); 118.97 (C-3); 112.31 (C-8); 109.81 (C-1); 43.57 (C-10); 18.83 (C-11).N-[(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoyl]-N’-(3-trifluoromethylbenzoyl)hydrazine (4c)4a), using of 3-trifluoromethylbenzoyl chloride , there were obtained 1.06 g of the compound, resulted after recrystallization from isopropanol:water 1:3, yield 77% toward hydrazide, m.p. 216–219 °C.Following the synthesis procedure described for : 3405m; 3357vs; 3209m; 3079w; 3023w; 2967w; 2932w; 1708vs; 1649vs; 1550w; 1474m; 1449m; 1331s; 1273m; 1225m; 1174s; 1125s; 1071m; 923w; 880w; 818w; 742w; 693m; 603m;FT-IR : 11.38 ; 10.66 ; 10.29 ; 8.20 ; 8.17 ; 8.16 ; 8.09 ; 7.95 ; 7.75 ; 7.53 ; 7.48 ; 7.36 ; 7.22 ; 3.94 ; 1.49 ;13C-NMR : 172.82 (C-12); 164.00 (C-13); 140.53 (C-8a); 139.94 (C-1a); 138.36 (C-2); 133.29 (C-14); 131.53 (C-19); 130.06 (C-18); 129.52 =31.7 Hz, C-16); 128.43 =3.8 Hz, C-17); 125.02 (C-7); 124.05 =3.8 Hz, C-15); 123.88 (C-4a); 123.70 =267.8 Hz, -CF3); 123.63 (C-5a); 120.40 (C-6); 120.45 (C-4); 119.65 (C-5); 118.91 (C-3); 112.33 (C-8); 109.83 (C-1); 43.56 (C-10); 18.78 (C-11).(RS)-1-(6-chloro-9H-carbazol-2-yl)-1-ethane (5a)4a) and 8 mL of phosphorus oxychloride were introduced in a round bottom flask equipped with a reflux condenser to which a calcium chloride tube was attached. The mixture was heated on the water bath for 9 h, then cooled and poured, under stirring, over ice water. The resulting precipitate was filtered off, washed with water and recrystallized from isopropanol:water 1:2.5.An amount of 0.784 g (0.002 mol) of corresponding acylhydrazide :3238s; 2979w; 2940w; 2780w; 1609m; 1551s; 1454vs; 1375w; 1330w; 1268s; 1244m; 1210m; 1062m; 1017w; 925w; 887w; 816m; 775w; 752m; 685s; 649w; 621m.FT-IR : 8.70 ; 8.01- 7.94 ; 7.52 - 7.39 ; 7.33 ; 7.26 ; 4.58 ; 1.90 ;13C-NMR : 169.17 (C-12); 165.10 (C-13); 140.51 (C-8a); 138.77 (C-2); 138.24 (C-1a); 131.71 (C-17); 129.00 ; 126.87 ; 125.96 (C-7); 124.90 (C-14); 124.10 (C-5a); 123.82 (C-4a); 121.91 (C-6); 120.89 (C-4); 119.98 (C-5); 119.18 (C-3); 111.75 (C-8); 109.58 (C-1); 38.01 (C-10); 20.04 (C-11).(RS)-1-(6-chloro-9H-carbazol-2-yl)-1-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)ethane (5b)5a, using corresponding acyl hydrazide (4b) . Purification was done from isopropanol.The synthesis followed the procedure described for the preparation of compound An amount of 0.56 g of the compound was obtained, yield 69% toward acyl hydrazide with m.p. 200–204 °C, soluble at room temperature in pyridine, ethyl acetate, dimethylsulfoxide, dimethylformamide, after heating in ethanol, isopropanol, isobutanol, xylene, hardly soluble after heating in methanol and insoluble in hexane and water.−1): 3268m; 3088w; 2978w; 1606s; 1563m; 1481vs; 1462s; 1432m; 1408w; 1338w; 1274s; 1246m; 1095m; 1063m; 1013m; 873w; 834m; 817s; 792m; 627w;FT-IR : 11.41 ; 8.18 ; 8.13 ; 7.93 ; 7.61 ; 7.48 ; 7.46 ; 7.36 ; 7.18 ; 4.73 ; 1.79 ;13C-NMR : 169.18 (C-12); 163.39 (C-13); 140.57 (C-8a); 138.98 (C-1a); 138.42 (C-2); 136.61 (C-14); 129.56 ; 128.22 ; 125.33 (C-7); 123.46 (C-4a); 122.99 (C-5a); 122.21 (C-17); 121.14 (C-4); 120.82 (C-6); 119.77 (C-5); 118.38 (C-3); 112.44 (C-8); 109.83 (C-1); 36.95 (C-10); 19.91 (C-11).(RS)-1-(6-chloro-9H-carbazol-2-yl)-1-(5-(3-trifluoromethylphenyl)-1,3,4-oxadiazol-2-yl)ethane (5c)5a, of the corresponding acyl hydrazide (4c) (0.919 g 0.002 mol) were used. Purification was done from isopropanol.Following the synthesis procedure described for the preparation of compound An amount of 0.55 g of compound was obtained, 62% toward acyl hydrazide, m.p. 169–172 °C, soluble at room temperature in pyridine, ethyl acetate, dimethylsulfoxide, dimethylformamide, after heating in methanol, ethanol, isopropanol, isobutanol, xylene and insoluble in hexane and water.−1): 3416w; 3239w; 3083w; 2984w; 1616w; 1559m; 1469m; 1428m; 1378w; 1328s; 1272m; 1244m; 1170m; 1131vs; 1068m; 1014w; 971w; 927w; 869w; 811m; 752w; 732w; 697m.FT-IR : 11.40 ; 8.22 ; 8.18 ; 8.17 ; 8.13 ; 7.96 ; 7.79 ; 7.48 ; 7.48 ; 7.36 ; 7.19 ; 4.76 ; 1.80 ;13C-NMR : 169.54 (C-12); 163.08 (C-13); 140.43 (C-8a); 138.94 (C-1a); 138.29 (C-2); 130.85 (C-18); 130.44 (C-19); 130.08 =32.5 Hz, C-16); 128.37 =3.8 Hz, C-17); 125.35 (C-7); 124.45 (C-14); 123.60 =212.5 Hz, -CF3); 123.47 (C-4a); 123.43 (C-5a); 122.73 =3.8 Hz, C-15); 121.14 (C-4); 120.84 (C-6); 119.78 (C-5); 118.42 (C-3); 112.41 (C-8); 109.82 (C-1); 36.98(C-10); 19.99 (C-11).(RS)-1-(6-chloro-9H-carbazol-2-yl)-1-ethane (5d)3 (2 mL), carprofen , and isoniazid (0.001 mol). The mixture was stirred for a few minutes and then heated under reflux for 2.5 h. Benzene or toluene (10 mL) was added and the mixture evaporated under reduced pressure on a rotary evaporator. The residue was treated with ice-water (10 g) and the red brick precipitate was filtered off under vacuum and washed several times with cold water (ice water). The orange product was crystallized from a mixture water/ethanol (2:1). To a round bottom flask equipped with a stirrer bar and a condenser was introduced in sequence, POCl21H15ON4Cl) was obtained, with yield 91% toward carprofen, m.p. 173–175 °C, soluble at room temperature in acetone, dimethylsulfoxide, dimethylformamide, and 1:1 dichloromethane: methanol, by heating in methanol, benzene, hexane, dichloromethane, dimethylsulphoxide, hardly soluble by heating in ethanol, chloroform, toluene, petroleum ether, and insoluble in xylene, ethyl ether, ethyl acetate and water.An amount of 0.34 g compound (C−1): 3241m; 3104w; 2979w; 2934w; 1691m; 1610w; 1551m; 1473vs; 1461vs; 1432m; 1358w; 1337w; 1273vs; 1217m; 1173m; 1065s; 928w; 871w; 817m; 753w; 699w; 584w.FT-IR : 11.41 ; 8.80 ; 8.19 ; 8.13- 8.19 ; 7.90 ; 7.49 ; 7.48 ; 7.36 ; 7.18 ; 4.80 ; 1.80 ;13C-NMR : 170.03 (C-12); 162.65 (C-13); 150.74 ; 140.56 (C-8a); 138.79 (C-1a); 130.65 (C-2); 125.86 (C-7); 125.36 (C-14); 123.45 (C-4a); 123.0 (C-5a); 122.99 (C-4); 120.86 (C-6); 120.28 , 120.20 (C-5); 118.41 (C-3); 112.47 (C-8); 109.90 (C-1); 36.98 (C-10); 19.50 (C-11).5d was also analyzed by mass spectroscopy, revealing the appearance of a peak corresponding to the molecular ion 375.1 [M + H], which confirms the molecular weight of this compound.In order to confirm the structure, the compound Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) and Gram-positive  bacteria, as well as the fungal strain Candida albicans ATCC 90029.The antimicrobial activity of the synthesized compounds was determined using three methods, against a panel of Gram-negative  assay: The minimum amount of the tested compounds that inhibited the microbial growth was evaluated using the microdilution method in liquid Mueller Hinton medium. The tested binary concentrations in the range of 5–0.009 mg/mL were achieved starting from a stock solution prepared in DMSO of 10 mg/mL, in a 100 µL culture medium final volume, seeded with a 20 µL microbial suspension of 0.5 MacFarland density. In each test, a microbial culture positive control and a sterile medium negative control were performed. The plates were incubated for 24 h at 37 °C. The results are shown in Minimal bactericidal concentration (MBC) assay: after reading the MIC value, a volume of 10 µL from the well corresponding to the MIC value and from all the previous wells was seeded on solid PCA (plate count agar) medium to determine the MBC value, corresponding to the concentration at which the total inhibition of microbial growth on the solid medium was obtained  was determined to be the lowest concentration of the tested compounds at which the decrease in absorbance value, measured at 490 nm, was observed in comparison to the positive control ( control  28]..Anti-bio® CCL-2™) and the HaCaT immortalized keratinocyte line. They were maintained in DMEM: F12 medium supplemented with 10% fetal bovine serum.The cell cultures used in this study were HeLa -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. Toxicity assessment was performed at 2 h, by reading the absorbance at 490 nm, using the Tristar Berthold Technologies spectrophotometer .The cells were seeded at a density of 7.5 × 105/well in 24-well plates. The medium contained 1 mg/mL compound. After 24 h, cell viability was assessed by staining with FDA (10 μg/mL) and PI (20 μg/mL). Cells were observed on the Zeiss fluorescence microscope .The cells were seeded at a density of 7.5 × 105/well in 24-well plates and maintained in culture medium containing the tested compounds, at a concentration of 100 μg/mL, for 48 h. After incubation, the cells were trypsinized, washed in phosphate buffer saline, fixed in 70% cold ethanol and stained with 50 μg/mL propidium iodide solution. The evaluation was performed using the Beckman Coulter XLM flow cytometer  and Flow Jo7 software .The cells were seeded at a density of 7.5 × 10The series of molecules were modeled in the Discovery Studio software , using carprofen as template compound. The analysis consisted of modeling 2D structures, adding specific substituents and completing valences with hydrogen atoms and passing these structures into three-dimensional space, in order to obtain 3D spatial structures.Minimization of these molecules was done using the Hamiltonian method: Forcefield MMFF94x at a gradient of 0.05. After calculating the minimum energy, the Gasteiger partial loads (PEOE) were applied.For establishing the pharmacological character of the newly synthesized compounds, the compliance with at least two Lipinski rules (drug-like nature) and with the Veber rule (bioavailability) was checked ,30.By accessing the Expasy/medicinal chemistry database we identified the possible structural similarities of the analyzed compounds with other chemical compounds. For the ADME prediction, a pkCSM platform was used , and forWe have selected the following properties: 1. Intestinal absorption, 2. blood–brain barrier permeability (BBB), 3. central nervous system permeability (CNS), 4. AMES toxicity, 5. maximum tolerated human dose, ability to inhibit human ether-a-gogo gene (hERG I and hERG II), 6. Lethal Dose 50 (on rat), 7. hepatotoxicity, 8. the ability of the substance to produce carcinogenic effects.


Given tMoreover, the early work of Lemal and McGregor on the generation of dienes via the stereospecific deamination of 3-pyrrolines with Angeli’s salt  proffere3 from 2,3-diethyl-1-(phenylsulfonyl)indole (8) (8 from 2-iodoaniline via a Larock indolization indole 10 with benzylamine, and then debenzylated using Yang’s method  shift in 18% overall yield from 9.We initially focused on the generation of 2,3-diethylidene-1-(phenylsulfonyl)indoline dole (8) . Howeverlization  with 3-h fashion . Treatme5% yield . The strears ago . Indole s method  to give 13 as opposed to the isomeric 2-ethyl-1-(phenylsulfonyl)-3-vinylindole (14) was established by both 2D NMR and an independent synthesis of 14 -3-vinylindole (14) in modest yield. The NMR data  clearly distinguished 3-vinylindole 14 from 2-vinylindole 13. This observation of a  shift (3 to 13) during the formation of an indole-2,3-quinodiethane has been reported by Pindur [b]indol-3-one, and by Bergman  shift process, we turned our attention to the possible generation of indole-2,3-quinodimethane inivasan ,35 to gizylation  with α-c17 with p-benzoquinone and dimethyl acetylenedicarboxylate have been unsuccessful. For identification with the anticipated adduct of p-benzoquinone with 17, we prepared adduct 25 following Cava’s conditions indole (10)  in anhydrous methylene chloride (30 mL), with stirring at −10 °C. The reaction mixture turned orange-brown. After 35 min the solvent was removed via vacuum pump. Methanol (20 mL) was added to the orange-brown residue. The reaction mixture was refluxed for 55 min, after which it was allowed to cool to room temperature. The solvent was evaporated in vacuo yielding 11 as a brown oil with a white solid. This crude product was taken on to the next step without any further purification: MS m/z 363 (M), 220 (100%), 141, 114, 77.A solution of α-chloroethyl chloroformate  in anhydrous methylene chloride (20 mL) was added dropwise via addition funnel to the reaction mixture of crude 2-benzyl-1,3-dimethyl-4-(phenylsulfonyl)-1,2,3,8-tetrahydropyrroloindole (21)  in anhydrous methylene chloride (10 mL) with stirring at −10 °C. The peach-colored solution was stirred for 1 h. The solvent was removed via vacuum pump. Methanol (10 mL) was added to the peach-colored, solid residue. The white suspension was refluxed for 1.75 h. The reaction mixture was allowed to cool to room temperature. The solvent was evaporated in vacuo, yielding 22 as a gray-white solid. The solid was recrystallized with a mixture of hexanes:methylene chloride (1:1). A white solid was obtained : mp 219–220°; 1H-NMR (CDCl3) δ 7.98 , 7.91 , 7.59 , 7.52 , 7.41–7.27 , 4.99 , 4.69 ; Anal. Calcd for C16H15ClN2O2S: C, 57.40; H, 4.52; Cl, 10.59; N, 8.37; S, 9.58. Found: C, 55.58; H, 4.50; Cl, 10.58; N, 7.79; S, 8.96.A clear, colorless solution of α-chloroethyl chloroformate  in anhydrous methylene chloride (10 mL) was added dropwise via addition funnel to a pale orange solution of 2-benzyl-4-(phenylsulfonyl)-1,2,3,4-tetrahydropyrroloíndole hydrogen chloride (22)  and Angeli’s salt  were stirred in a 1:1 mixture of methanol (15 mL) and distilled water (15 mL). Hydrochloric acid  was added via syringe. The reaction mixture was stirred at room temperature for 27 min, after which it was refluxed for 2 h. It was allowed to cool to room temperature and was subsequently was cooled to −78 °C. It was allowed to gradually warm up to room temperature. The organic layer was extracted with methylene chloride (3 × 25 mL), washed with saturated aqueous sodium bicarbonate (2 × 25 mL), and dried with anhydrous magnesium sulfate. The solvent was evaporated in vacuo, yielding a red-brown oil, which was purified via column chromatography (2:1 hexanes: ethyl acetate). Dimer 24 was obtained as a tan solid : mp 134.5–135 °C; 1H-NMR (CD2Cl2) δ 7.97 , 7.91 , 7.84 , 7.63–7.53 , 7.51–7.43 , 7.37 , 7.31 , 7.24 , 5.83–4.78 , 4.27 , 4.04 , 3.92 ; 13C-NMR (CDCl3) δ 134.7 (2C), 134.6 (2C), 129.9 (4C), 127.0 (4C), 125.5 (2C), 125.4 (2C), 124.5 (2C), 123.9 (2C), 119.9 (2C), 119.7 (2C), 114.6 (2C), 114.5 (2C), 52.3, 51.0, 49.2, 47.6; MS m/z 566 (M), 532, 457, 387, 315, 284, 245, 220, 173, 142, 91 (100%); HRMS Calcd for C32H26N2O4S2: 566.1334; Found: 566.1328.4-(Phenylsulfonyl)-1,2,3,4-tetrahydropyrrolo[3,4-20) , sodium iodide , and p-benzoquinone  were stirred in DMF (10.0 mL). The dark red-brown reaction mixture was heated to 78 °C and later cooled to 73.5 °C. It was heated for 18 h and was subsequently allowed to cool to room temperature. The dark-brown mixture was filtered through a Celite pad with methylene chloride. The filtrate was poured onto aqueous sodium bisulfite. The organic layer was extracted with methylene chloride (3 × 20 mL), washed with brine (2 × 20 mL), and dried first with sodium sulfate, then with anhydrous magnesium sulfate. The solvent was evaporated in vacuo giving a brown oil, which was purified via column chromatography. Quinone 25 was obtained as a bright yellow solid : mp 214 °C dec.; 1H-NMR (CDCl3) δ 9.00 , 8.66 , 8.40 , 8.08 , 7.93 , 7.67 , 7.55–7.46 , 7.40 , 7.05 ; 13C-NMR (CDCl3) δ 184.9, 184.7, 139.5, 139.1, 134.7 (2C), 130.0 (2C), 129.7 (2C), 128.3, 126.9 (2C), 125.2, 125.1 (2C), 121.7 (2C), 119.4, 115.3, 113.4 (2C); UV λmax (95% EtOH) 195, 216, 284 nm; MS m/z 387 (M), 246 (100%), 218, 190, 164, 141, 115, 77; HRMS Calcd for C22H13NO4S: 387.0565; Found: 387.0556.2,3-Dibromomethyl-1-(phenylsulfonyl)indole  was added via syringe at room temperature to the yellow filtrate. The filtrate was cooled in an ice bath. A cream-colored solid  was collected via vacuum filtration and immediately used for the deamination reaction step.The procedure of Addison was used . A 21% s


Clavularia flava, which potentiates proteasome inhibition. Chemical structure elucidation revealed they are dolabellane- and secosteroid-based compounds including a new dolabellane, clavinflol C (1), three known dolabellanes, stolonidiol (2), stolonidiol-17-acetate (3), and clavinflol B (4) as well as two new secosteroids, 3β,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9,23-dione (5) and 3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9,23-dione (6). All six compounds show less cytotoxicity than those of known proteasome inhibitors, bortezomib and MG132. In summary, the high-content measurements of control inhibitors, bortezomib and MG132, manifest the highest ratio >2 in high-content measurement. Of the isolated compounds, 2 and 5 showed higher activity, followed by 3 and 6, and then 1 and 4 exhibited moderate inhibition.We performed a high-content screening (HCS) assay aiming to discover bioactive molecules with proteasome inhibitory activity. By structural elucidation, we identified six compounds purified from soft coral  The three known dolabellane diterpenes, 2–4 were identified by comparison of their spectral data with those of reported literatures . A,8. A1), , 2.12 (p < 0.0010), 1.74 (p = 0.0020), 1.33 (p < 0.0010), 2.03 (p < 0.0010), and 1.72 (p < 0.0010), respectively. The highest ratios of average intensity per cell presented for compounds 1, 2, 3, 4, 5, and 6 were 1.32 (p = 0.0371), 1.75 (p = 0.0021), 1.40 (p < 0.0010), 1.19 (p = 0.0117), 1.85 (p < 0.0010), and 1.34 (p = 0.0089), respectively , 0.91 (p = 0.0359), and 0.88 (p < 0.0010), respectively (50 for compound 2 was greater than 25 μg/mL for HEK293T cell in both assays (3 were 0.91 (p < 0.0010), 0.96 (p = 0.1484), and 0.86 (p < 0.0010), respectively. The protein ratios for compound 4 were 0.92 (p = 0.0045), 0.95 (p = 0.0420), and 0.86 (p = 0.0014), respectively. However, HEK293T cells treated with compound 3 and 4 at 25 μg/mL exhibited a lighter toxicity with ED50 values of 19.56 and 21.33 μg/mL, respectively. Compound 5 exhibited significant reduction at 5 and 25 μg/mL; the ratios were 0.78 (p < 0.0010) and 0.15 (p < 0.0010), respectively, which is consistent with cytotoxicity ED50 values , 1.73 (p < 0.0010), 1.76 (p < 0.0010), 1.48 (p < 0.0010), 1.29 (p = 0.0236), and 1.32 (p = 0.0062), respectively; for vesicle aggresomes, 1.43 (p = 0.0028), 1.88 (p < 0.0010), 1.84 (p < 0.0010), 1.54 (p < 0.0010), 1.53 (p < 0.0010), and 1.37 (p = 0.0071), respectively. Similar profiles were observed for the ratios of integrated intensity per cell , 1.48 (p < 0.0010), 1.31 (p = 0.0055), 1.33 (p < 0.0010), 1.41 (p < 0.0010), and 1.21 (p = 0.0097), respectively; for vesicle aggresomes, 1.16 (p = 0.0220), 1.73 (p < 0.0010), 1.76 (p < 0.0010), 1.05 (p = 0.5352), 1.29 (p = 0.0236), and 1.32 (p =0.0063), respectively. The ratios of average intensity per cell were the same for pit and vesicle aggresomes , 1.42 (p < 0.0010), 1.25 (p = 0.0034), 1.22 (p = 0.0628), 1.42 (p < 0.0010), and 1.19 (p = 0.0135), respectively.After the results, we investigated the phenotypic size of aggresomes and analyzed the high-content data from 1), stolonidiol (2), and stolonidiol-17-acetate (3) and clavinflol B (4) with proteasome inhibition activities. Several groups demonstrated that stolonidiol (2) and stolonidiol-17-acetate (3) enhance the activities of choline acetyl transferase (ChAT) [2) 5 μg/mL treatment, the EGFP-UL76 displayed the highest increase ratios of 2.12 and 1.75 of integrated and average intensity, respectively (2) potentiated the highest ratios of aggresome pit and vesicle integrated and average intensities  , which le (ChAT) . ChAT cae (ChAT) . Therefoe (ChAT) ,10,11. Aectively . Consistensities . For the6 showed the highest inhibitory measurements of EGFP-UL76 integrated and average intensities and at 1 μg/mL , which was collected at Green Island, Taiwan. Briefly, corals were minced and exhaustively extracted with acetone. The organic extract was partitioned between ethyl acetate and water, and the ethyl acetate layer was separated further over a normal phase silica gel by column chromatography eluted with n-hexane, ethyl acetate, and methanol to yield 15 fractions. Fraction 14 (116.5 mg) eluted with n-hexane–EtOAc (1:5) was subjected to a RP-18 gravity column  to separate 6 subfractions. Subsequently, a subfraction 14-3 (23 mg) was purified by RP-18 HPLC (50% acetonitrile in H2O) to obtain 1 (10.2 mg) and 2 (3.2 mg). Fraction 13 (96.7 mg) eluted with n-hexane–EtOAc (1:3) was subjected to column chromatography on silica gel using n-hexane–EtOAc gradient (10:1 to 1:10) for elution to give 11 subfractions. Subsequently, a subfraction 13-6 (22 mg) was purified by RP-18 HPLC (60% MeOH in H2O) to obtain 3 (5.2 mg). Fraction 15 (86.5 mg) eluted with n-hexane–EtOAc (1:10) was subjected to a RP-18 gravity column  to separate 7 subfractions. Subsequently, a subfraction 15-4 (20 mg) was purified by RP-18 HPLC (50% ACN in H2O) to obtain 4 (2.4 mg), 5 (3.2 mg), and 6 (3.0 mg).In the present study, compounds 1–6  were isoWe followed the HCS assay routinely conducted in the lab. In brief, the complete assay includes a series of experiments . Experim


Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of the plant pathogens that produce it in various biochemical processes. The kinetic and molecular modeling studies were performed and for selected compounds, the docking results were discussed. Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24–674 µM with the predicted and desirable competitive type of inhibition. The structure–activity relationship (SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger 3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site of laccase and, interestingly, acted as non-competitive (Ki = 32.0 µM) or uncompetitive (Ki = 17.9 µM) inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to environment and non-specific, was over 6 times more active than the above compounds.A series of hydrazide-hydrazones  Sclerotniaceae family, for instance, gray mold (Botrytis cinerea) which attacks flowers, green tissues of crop plants methyl}benzenesulfonate (1d). For this compound, the spectra were measured in fully deuterated CH3OH. In the 1H-NMR spectra measured in DMSO-d6, the amide proton (NHCO), phenolic hydroxy group of the 4-HBAH fragment (4–OH) and conjugate imine (CH=N) resonances were observed at ca.: 11.24–12.18, 9.83–10.80 and 8.32–8.93 ppm, respectively. In the 13C-NMR spectra, the resonances of the conjugated carbonyl and imine C=O and C=N carbond, and tertiary C-4 phenolic carbon atoms, and ArC-2 were observed at ca.: 161.93–162.80 and 142.19–150.36, 160.43–161.04 and 154.72–160.77 ppm, respectively. For the sulfonic acid sodium salt derivative 1d, similar chemical shifts were observed in the 1H-NMR resonance. The hydrazide-hydrazones which had phenyl or alkyl group near the salicylic hydroxy group caused a weak bathochromic effect. The spectroscopic data were consistent with the data published in the literature ..tert-but6d) and 3-isopropyl-6-methylsalicylaldehyde (7e) were prepared by the Reimer-Tiemann formylation of 4-bromophenol (10d) + or [M + Na]+ molecular species are reported. The literature procedure was adapted for the preparation of benzoic acid hydrazide 4b–c 4 and CaH4 and CaH 4 and CaH 4 and CaH 4 and CaH 4 and CaH 4 and CaH 4 and CaH 4 and CaH 4 and CaH 4 and CaH4 and CaHr 1c 4 and CaH benzohydrazide (1a). The general procedure starting from benzaldehyde , 4-hydroxybenzohydrazide , CH3OH (5.0 mL), and AcOH (100 µL) was employed with a 2 h reaction time, and solvent was slowly distilled to a volume of cas 2.5 mL before crystallization to obtain hydrazide-hydrazone 1a. Colorless crystals; 462 mg, 1.92 mmol, 96% yield; m.p. 242–244 °C (from CH3OH) , 3056 (C-H), 3039 (C-H), 3027 (C-H), 1614 (C=O), 1588 (C=C), 1548 (CH=N), 1509, 1356, 1280 (C-O), 1212, 1172, 1110, 1056, 960, 844, 763, 661, 630, 614, 510, 492; 1H-NMR : δ 11.64 , 10.13 , 8.43 , 7.81 , 7.71 , 7.38–7.48 , 6.87  ppm; 13C-NMR : δ 162.77 (C=O), 160.66 (C-4), 146.82 (CH=N), 130.47 (ArC-1), 129.79 (ArC-4), 129.65 , 128.75 , 126.90 , 123.81 (C-1), 114.98  ppm; HRMS (Time-of-Flight (TOF), Mass spectrometry (MS), Electrospray ionization (ESI)) calculated for C14H12N2O2 [M + Na]+m/z 263.0786, found 263.0806.4-Hydroxy-N’-[(E)-3-phenylpropylidene]benzohydrazide (1b) +m/z 269.1279, found 269.1288.ide (1b) . The gen4-Hydroxy-N’-[(E)-(4-methylphenyl)methylidene]benzohydrazide monomethanolate (1c). The general procedure starting from 4-methylbenzaldehyde , 4-hydroxybenzohydrazide , in CH3OH (15 mL) was employed with a 2 h reaction time to obtain hydrazide-hydrazone 1c in 92% yield, which was recrystallized slowly from hot CH3OH (30 mL/g) to obtain hydrazide-hydrazone 1c crystallized with one molecule of CH3OH. Colorless crystals; 456 mg, 1.59 mmol, 80% yield; m.p. 246–247 °C , 1622 (C=C), 1605 (C=O), 1538 (CH=N), 1500, 1351, 1280 (C-O), 1265, 1208, 1173, 1110, 1012, 956, 907, 854, 843, 814, 767, 742, 591 (br), 513, 478; 1H-NMR : δ 11.59 , 10.13 , 8.39 , 7.81 , 7.60 , 7.25 , 6.86 , 4.12 , 3.17 , 2.33  ppm; 13C-NMR : δ 162.67 (C=O), 160.61 (C-4), 146.85 (CH=N), 139.60 (ArC-4), 131.76 (ArC-1), 129.60 , 129.38 , 126.89 , 123.88 (C-1), 114.96 , 48.56 (CH3OH), 20.97 (Me) ppm.Sodium 2-{(E)-[2-(4-hydroxybenzoyl)hydrazinylidene]methyl}benzenesulfonate (1d). The general procedure starting from ≥95% sodium 2-formylbenzenesulfonate , 4-hydroxybenzohydrazide , CH3OH (5.0 mL), in the absence of AcOH, was employed with a 2 h reaction time. The reaction mixture was concentrated in vacuum to a small volume of ca. 2 mL, then the product was isolated by slow crystallization in an open vessel to obtain hydrazide-hydrazone 1d which crystallized with 2/3 a molecule of CH3OH. Yellow powder; 720 mg, 1.94 mmol, 97% yield, m.p. 251 °C (from CH3OH) with decomposition; selected FT-IR (ATR) νmax/cm−1: 3635 (CH3OH), 3377 (O-H), 3307 (N-H), 1608 (C=O), 1578 (C=C), 1556 (CH=N), 1510, 1272 (C-O), 1178 (SO3Na), 1134, 1017, 850, 760, 615, 563, 526, 482; 1H-NMR : δ 9.24 , 8.32 , 7.98 , 7.87 , 7.51 , 7.45 , 6.88 , 3.35  ppm; 13C-NMR : δ 167.07 (C=O), 162.80 (C-4), 148.93 (CH=N), 145.22 (ArC-2), 132.84 (ArC-1), 131.51 (ArC-5), 131.02 , 130.60 (ArC-4), 128.29 , 124.62 (C-1), 116.32 , 49.90 (CH3OH) ppm; HRMS  calculated for C14H11N2O5S [M + Na]+m/z 365.0173, found 365.0185.4-Hydroxy-N’-[(E)-(2-hydroxyphenyl)methylidene]benzohydrazide (1e). The general procedure starting from salicylic aldehyde , 4-hydroxybenzohydrazide , CH3OH (40 mL) and AcOH (200 µL) was employed, with a 2 h reaction time. The reaction mixture was slowly concentrated to a small volume before crystallization to obtain compound 1e. Colorless powder; 440 mg, 1.72 mmol, 86% yield; m.p. 265.0–266.5 °C (from CH3OH) (257–258 °C +m/z 279.0735, found 279.0745.7–258 °C ); select4-Hydroxy-N’-[(E)-(3-hydroxyphenyl)methylidene]benzohydrazide monohydrate (1f). The general procedure starting from 3-hydroxybenzaldehyde , 4-hydroxybenzohydrazide , CH3OH (2.5 mL), and AcOH (100 μL) was employed, with a 2 h reaction time. To the reaction mixture, cooled to RT, H2O (2.5 mL) was added before crystallization, to obtain 1f which crystallized with one molecule of H2O. Pale crystals; 255 mg, 0.93 mmol, 93% yield; m.p. 254 °C  with decomposition (>250 °C +m/z 279.0735, found 279.0747. 1H-NMR and 13C-NMR data and elementary analysis calculated for C14H12N2O3 × 1H2O are consistent with literature values benzohydrazide (1g). The general procedure starting from 4-hydroxybenzaldehyde , 4-hydroxybenzohydrazide , CH3OH (2.5 mL) and AcOH (200 μL) was employed, with a 2 h reaction time to obtain of hydrazide-hydrazone 1g which crystallizes with 1/3 a molecule of CH3OH. Pale crystals; 512 mg, 1.92 mmol, 96% yield; m.p. 261 °C (from CH3OH) with decomposition (>265 °C +m/z 279.0735, found 279.0747.(>265 °C ); select4-Hydroxy-N’-[(E)-(2-methoxyphenyl)methylidene]benzohydrazide (1h). The general procedure starting from 2-methoxybenzaldehyde , 4-hydroxybenzohydrazide , CH3OH (10 mL), and AcOH (150 μL) was employed, with a 4 h reaction time. The reaction mixture was cooled to RT and H2O (2.5 mL) was added before crystallization to obtain hydrazide-hydrazone 1h. Pale powder; 383 mg, 1.42 mmol, 71% yield; m.p. 236–237 °C  , 2971 (C-H), 2942 (C-H), 2840 (C-H), 1607 (C=O), 1558 (C=N), 1508, 1468, 1360, 1315, 1286 (C-O), 1253 (C-O), 1235, 1172, 1055 (C-O), 1046, 835, 755, 618, 489; 1H-NMR : δ 11.66 , 10.11 , 8.79 , 7.86 , 7.83 , 7.40 , 7.09 , 7.01 , 6.86 , 3.86  ppm; 13C-NMR : δ 162.52 (C=O), 160.57 (C-4), 157.57 (ArC-2), 142.19 (CH=N), 131.24 (ArC-4), 129.59 , 125.36 (ArC-6), 123.79 (C-1), 122.46 (ArC-1), 120.66 (ArC-5), 114.92 , 111.73 (ArC-3), 55.58 (OMe) ppm; HRMS  calculated for C15H14N2O3 [M + Na]+m/z 293.0891, found 293.0889.nd AcOH 1 μL was e4-Hydroxy-N’-[(E)-(3-methoxyphenyl)methylidene]benzohydrazide (1i). The general procedure starting from 3-methoxybenzaldehyde , 4-hydroxybenzohydrazide , CH3OH (5.0 mL), and AcOH (100 µL) was employed, with a 5 h reaction time. The reaction mixture was concentrated to a volume of ca. 2.2 mL, and H2O (300 µL) was added before crystallization to obtain hydrazide-hydrazone 1i. Colorless powder; 213 mg, 0.79 mmol, 79% yield; m.p. 210–212 °C  (205–206 °C +m/z 293.0891, found 293.0895.5–206 °C ); select4-Hydroxy-N’-[(E)-(4-methoxyphenyl)methylidene]benzohydrazide (1j). The general procedure starting from 4-methoxybenzaldehyde , 4-hydroxybenzohydrazide , and CH3OH (10 mL) in the absence of AcOH, with a 5 h reaction time to obtain hydrazide-hydrazone 1j. White needless; 443 mg, 1.64 mmol, 82% yield; m.p. 223–224 °C (from CH3OH) , 3043 (C-H), 3006 (C-H), 2839 (C-H), 1595 (C=O), 1576 (C=C), 1553 (CH=N), 1505, 1439, 1361, 1312, 1277, 1249 (C-O), 1230 (C-O), 1167, 1060 (C-O), 1020, 962, 842, 827, 756, 682, 619, 546, 529; 1H-NMR : δ 11.50 , 10.09 , 8.37 , 7.79 , 7.65 , 7.01 , 6.85 , 3.80  ppm; 13C-NMR : δ 162.58 (C=O), 160.60 (ArC-4), 160.52 (C-4), 146.73 (CH=N), 129.52 , 128.47 , 127.01 (ArC-1), 123.96 (C-1), 114.93 , 114.23 , 55.20 (OMe) ppm; 1H-NMR : δ 11.52 , 10.11 , 8.37 , 7.80 , 7.65 , 7.01 , 6.86 , 3.80  ppm; 13C-NMR ; δ 162.56 (C=O), 160.61 (ArC-4), 160.52 (C-4), 146.71 (CH=N), 129.53 , 128.47 , 127.02 (ArC-1), 123.97 (C-1), 114.93 , 114.25 , 55.21 (OMe) ppm; HRMS  calculated for C15H14N2O3 [M + H]+m/z 271.1072, found 271.1098.0–221 °C ); select4-Hydroxy-N’-[(E)-(2-hydroxy-3-phenyl-phenyl)methylidene]benzohydrazide monomethanolate (2a). The general procedure starting from 3-phenylsalicylic aldehyde  +m/z 333.1228, found: 333.1227.50 mmol) , 4-hydro4-Hydroxy-N’-[(E)-(3-tert-butyl-2-hydroxyphenyl)methylidene]benzohydrazide (2b). The general procedure starting from 3-tert-butyl-salicylic aldehyde  +m/z 313.1547, found: 313.1540..0 mmol) , 4-hydro4-Hydroxy-N’-[(E)-methylidene]benzohydrazide (2c) +m/z 273.0870, found 273.0872.ide (2c) . The gen4-Hydroxy-N’-[(E)-(5-bromo-2-hydroxyphenyl)methylidene]benzohydrazide (2d). The general procedure starting from 5-bromosalicylic aldehyde  +m/z 356.9845, found 356.9857..0 mmol) , 4-hydro9–280 °C ); select4-Hydroxy-N’-[(E)-(6-methoxy-2-hydroxyphenyl)methylidene]benzohydrazide (2e). The general procedure starting from 6-methoxysalicylic aldehyde  +m/z 287.10263, found 287.1035..0 mmol) , 4-hydro4-Hydroxy-N’-[(E)-methylidene]benzohydrazide (2f). The general procedure starting from 2,6-dimethoxybenzaldehyde  +m/z 323.1002, found 323.1002..0 mmol) , 4-hydro4-Hydroxy-N’-[(E)-(4-hydroxy-3-methoxyphenyl)methylidene]benzohydrazide (2g) +m/z 309.0851, found 309.0845.ide (2g) . The gen(226–227 ); select4-Methoxy-N’-[(E)-2-hydroxy-3-phenyl-benzylidene]benzohydrazide (2h). The general procedure starting from 3-phenylsalicylic aldehyde  +m/z 369.1210, found 369.1198..0 mmol) , 4-metho.0 mmol) , CH3OH (4-Hydroxy-N’-[(E)-2-hydroxy-3-hydroxymethyl-5-methyl-benzylidene]benzohydrazide (3a). The general procedure starting from 3-hydroxymethyl-5-methylsalicylic aldehyde  +m/z 323.1002, found 323.1008..0 mmol) , 4-hydro4-Hydroxy-N’-[(E)-(5-hydroxymethyl-3-methyl-2-hydroxyphenyl)methylidene]benzohydrazide (3b). The general procedure starting from 5-hydroxymethyl-3-methylsalicylic aldehyde  +m/z 323.1002, found 323.1002.50 mmol) , 4-hydro4-Hydroxy-N’-[(E)-methylidene]benzohydrazide (3c). The general procedure starting from 3,5-di-tert-butyl-salicylic aldehyde , 4-hydroxybenzohydrazide , CH3OH (10 mL), and AcOH (100 µL) was employed, with a 6 h reaction time to obtain of compound 3c. Colorless needles; 1.22 g, 3.31 mmol, 83% yield; m.p. 274.5–275.5 °C (from CH3OH); the second fraction of 3c was obtain from concentrated filtrates: 156 mg, 0.42 mmol, 10.5% yield; m.p. 274–275 °C; selected FT-IR (ATR) νmax/cm−1: 3161 , 2955 (C-H), 2907 (C-H), 2867 (C-H), 1642 (C=C), 1607 (C=O), 1583 (CH=N), 1558, 1511, 1435, 1355, 1280, 1248, 1233 (C-O), 1172 (C-O), 891, 844, 769, 665, 618, 494; 1H-NMR : δ 12.35 , 11.99 , 10.21 , 8.54 , 7.84 , 7.30 , 7.20 , 6.89 , 1.41 , 1.28  ppm; 13C-NMR : δ 162.37 (C=O), 160.95 (C-4), 154.60 (ArC-2), 150.26 (CH=N), 140.26 (ArC-5), 135.53 (ArC-3), 129.70 , 125.52 (ArC-6), 125.26 (ArC-4), 123.00 (C-1), 117.06 (ArC-1), 115.13 , 34.59 (C-3-tBu), 33.81 (C-5-tBu), 31.25 (3 × CH3 – 5-tBu), 29.24 (3 × CH3 – 3-tBu) ppm; HRMS  calculated for C22H28N2O3 [M + H]+m/z 369.2173, found 369.2177.4-Hydroxy-N’-[(E)-3-tert-butyl-2-hydroxy-5-methylbenzylidene]benzohydrazide (3d). The general procedure starting from 3-tert-butyl-5-methylsalicylic aldehyde  +m/z 349.1523, found 349.1517..0 mmol) , 4-hydro4-Hydroxy-N’-[(E)-(2-hydroxy-3-isopropyl-6-methylphenyl)methylidene]benzohydrazide (3e). The general procedure starting from 3-isopropyl-6-methylsalicylic aldehyde  +m/z 313.1547, found: 313.1548..0 mmol) , 4-hydro4-Hydroxy-N’-[(E)-methylidene]benzohydrazide (3f). The general procedure starting from 4,6-dimethoxysalicylic aldehyde  +m/z 317.1132, found 317.1145..0 mmol)  and 4-hy3-Hydroxy-N’-[(E)-(3-tert-butyl-2-hydroxy-5-methylphenyl)methylidene]benzohydrazide (3g). The general procedure starting from 3-tert-butyl-5-methylsalicylic aldehyde  +m/z 349.1523, found 349.1524..0 mmol) , 3-hydrov/v) at 25 °C was investigated (see 0 = 65000 M−1·cm−1 , k3 [min–1] – kinetic constants, Vmax – maximal velocity [µM·min–1], CS – substrate concentration [µM], CE – enzyme concentration [µM].The activity of laccase was determined using the procedure described by Leonowicz and Grzywnowicz . Basicalated see . The samM−1·cm−1 . Kinetiction (1) :V = Vmax2); the sum of squared errors (SSE) and root mean squared error (RMSE). The 25 hydrazide-hydrazones, 4-hydroxybenzoic acid , 4-hydroxybenzohydrazide (4a – 4-HBAH), and sodium azide (control) were tested against laccase in the standard reaction mixture. Prior to the substrate addition, the laccase and tested compound were pre-incubated for 10 min at 25 °C. At least three inhibitor concentrations were used in each experiment. All measurements were performed in triplicate. To determine the type of inhibition Lineweaver–Burk transformation was used applying the following equations for competitive (2), uncompetitive (3) and non-competitive model (4) [Km [µM]—Michaelis-Menten constant, Vmax—maximal velocity [µM·min–1], Ki—inhibition constant [µM], CS—substrate concentration [µM], Ci—inhibitor concentration [µM].The parameters of the function were estimated using the nonlinear least square method in Matlab . The method was used simultaneously for datasets obtain for different enzyme concertation. The common minimum of error function was found for all considered datasets resulting in one common estimate of each model parameter. The goodness of fit was assessed using the coefficient of determination (Rodel (4) . The parBind energy for each inhibitor were selected as the final results.A crystal structure of an enzyme was obtained from the RCSB Protein Data Bank (PDB). The 1GYC  was preppara-hydroxybenzoic acid unit that served as a decoy, reminiscent of typical phenolic laccase substrates. In addition, salicylic aldehydes with bulky groups in the adjacent 3rd position provided strong interaction with the hydrophobic part of the substrate-binding centre. In this regard, the reversible competitive action on laccase of the tested hydrazide-hydrazones is beneficial for further design of inhibitors for bio-application.In this article, we identified a novel class of low-molecular-weight hydrazide-hydrazones, which contained naturally derived units of aldehydes having an aromatic motif and/or hydroxybenzoic acids. We presented the characteristics of 25 molecules, including 14 new ones, and their inhibition potency toward laccase of fungal origin. The results of the syringaldazine oxidation in the presence of the compounds indicated that they essentially acted as reversible competitive inhibitors, with some exceptions. The computational docking simulations showed that both aromatic fragments connected by a hydrazide linker in the molecules were crucial for their activity. The hydrazide fragment contained a


E)-2-methyl-but-2-enyl nucleosides phosphonate prodrugs bearing 4-substituted-1,2,3-triazoles were prepared in a straight approach through an olefin acyclic cross metathesis as the key synthetic step. All novel compounds were evaluated for their antiviral activities against HBV, HIV and SARS-CoV-2. Among these molecules, only compound 15j, a hexadecyloxypropyl (HDP)/(isopropyloxycarbonyl-oxymethyl)-ester (POC) prodrug, showed activity against HBV in Huh7 cell cultures with 62% inhibition at 10 μM, without significant cytotoxicity  at 10 μM.A series of hitherto unknown -( R)-PMPA [9-[9(R)-2-(phosphonomethoxy)propyl]adenine, 1] and PMEA [9-[2-(phosphonomethoxy)ethyl] adenine, 2] discovered by A. Holý and E. De Clercq in 1986, led to a new family of nucleotide analogs which has attracted considerable attention, [bis(POC)-PMPA (3) or bis(POM)-PMEA (4)]; prodrug moiety cytosine) while maintaining an electronic contribution similar to that brought by the oxygen atom + calcd for C15H28O10P: 399.14146, found: 399.14151.To a solution of E)-4-Azido-3-methyl-but-2-enyl-bis(POC)phosphonate (10)(E)-4-hydroxy-3-methyl-but-2-enyl-bis(POC)phosphonate 9  in anhydrous CH2Cl2 (5 mL) were added dropwise methanesulfonyl chloride  and anhydrous triethylamine  at 0 °C under the argon. After 30 min stirring at room temperature, the mixture was diluted with CH2Cl2 and washed with brine solution (3 × 10 mL). The organic layers were dried over MgSO4, filtrated and concentrated in vacuo. The residue was directly used for the next step without further purification. Thus, it was dissolved in anhydrous DMF (8 mL) and sodium azide  was added under nitrogen atmosphere. After 5 h stirring at room temperature, the mixture was diluted with EtOAc, washed with water and then extracted with EtOAc. The combined organic layers were washed with brine solution (5 × 10 mL), dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography  to give 10  as a colorless oil. 1H NMR  δ 5.71–5.61 , 5.48–5.42 , 4.92 2), 3.71 , 2.75 , 1.73 , 1.32 2). 13C NMR  δ 153.2 (C=O), 136.2, 136.1 (C=CH), 116.4, 116.3 (CH=C), 84.2, 84.1 (O-CH2-O), 73.3 (CH(CH3)2), 58.6, 58.6 (CH2-N3), 27.6 (CH2-P), 26.2 (CH2-P), 21.6 (CH(CH3)2), 14.9, 14.9 (CH3). 31P NMR  δ 27.85. IR νmax 2985.61, 2937.37, 2100.12, 1755.01, 1255.06, 1031.52, 984.34, 949.97, 904.26, 832.67, 788.47 cm−1. HRMS (ESI): m/z [M+Na]+ calcd for C15H26N3NaO9P: 446.12988, found: 446.12990.To a solution of -4-Azido-3-methyl-but-2-enyl-bis(POC)phosphonate 10 (1.0 equiv.) and alkyne (1.3 equiv.) in tBuOH/H2O (2:1) were added sodium ascorbate (0.6 equiv.) and CuSO4.5H2O (0.1 equiv.). The resulting suspension was stirred at 40 °C until completion (followed by TLC), then the crude mixture was co-evaporated five times with methanol (10 mL). The residue was purified by silica gel column chromatography using elution gradient of petroleum ether/ethyl acetate to give the desired bis(POC) phosphonate derivatives.To a solution of (E)-4-[4-(4-Propylphenyl)triazol-1-yl]-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11a)+ calcd for C26H39N3O9P: 568.24184, found: 568.24217.The title compound was prepared from E)-4-[4-(4-Butylphenyl)triazol-1-yl]-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11b)+ calcd for C27H40N3NaO9P: 604.23943, found: 604.23935.The title compound was prepared from E)-4-[4-(4-Pentylphenyl)triazol-1-yl]-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11c)+ calcd for C28H42N3NaO9P: 618.25508, found: 618.25473.The title compound was prepared from E)-4-[4-(4-Hexylphenyl)triazol-1-yl]-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11d)+ calcd for C29H44N3NaO9P: 632.27073, found: 632.27074.The title compound was prepared from E)-4-[4-(4-Heptylphenyl)triazol-1-yl]-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11e)+ calcd for C30H47N3O9P: 624.30444, found: 624.30373.The title compound was prepared from E)-4-(4-Cyclopropyltriazol-1-yl)-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11f)+ calcd for C20H33N3O9P: 490.19489, found: 490.19492.The title compound was prepared from E)-4-(4-tert-Butyltriazol-1-yl)-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11g)+ calcd for C21H37N3O9P: 506.22619, found: 506.22601.The title compound was prepared from E)-4-[4-(Hydroxymethyl)triazol-1-yl]-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11h)+ calcd for C18H30N3NaO10P: 502.156102, found: 502.155885.The title compound was prepared from E)-4-(4-Formyltriazol-1-yl)-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11i)+ calcd for C18H29N3O10P: 478.158507, found: 478.158468.The title compound was prepared from E)-4-(4-Carbamoyltriazol-1-yl)-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11j)+ calcd for C18H30N4O10P: 493.169406, found: 493.169337.The title compound was prepared from E)-4-(4-Octanoylaminomethyltriazol-1-yl)-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11k)+ calcd for C26H45N4NaO10P: 627.276551, found: 627.275980.The title compound was prepared from E)-4-(4-Nitrophenyltriazol-1-yl)-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11l)+ calcd for C23H32N4O11P: 571.179971, found: 571.179797.The title compound was prepared from E)-4-[Bis(isopropoxycarbonyloxymethoxy)phosphoryl]-2-methyl-but-2-enyl]triazole-4-carboxylic acid (11m)1-+ calcd for C18H29N3O11P: 494.153422, found: 494.153404.To a solution of propiolic acid  and (E)-4-[bis(isopropoxycarbonyloxymethoxy)phosphoryl]-2-methyl-but-2-enyl]triazole-4-carboxylate (11n)Methyl-1-+ calcd for C19H30N3NaO11P: 530.151016, found: 530.151002.The title compound was prepared from E)-4-[bis(isopropoxycarbonyloxymethoxy)phosphoryl]-2-methyl-but-2-enyl]triazole-4-carboxylate (11o)Ethyl-1-+ calcd for C20H32N3NaO11P: 544.166666, found: 544.166837.The title compound was prepared from E)-4-(4-Methylcarbamoyltriazol-1-yl)-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11p)+ calcd for C19H31N4NaO10P: 529.167001, found: 529.166551.The title compound was prepared from E)-4-[4-(Ethylcarbamoyl)triazol-1-yl]-3-methyl-but-2-enyl]-(isopropoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate (11q)+ calcd for C20H33N4NaO10P: 543.182651, found: 543.182603.The title compound was prepared from E)-4-Hydroxy-3-methyl-but-2-enyl-(HDP/POC) phosphonate (13)(12)  and 2-methyl-2-propen-1-ol (8)  in dry CH2Cl2 (10 mL), Hoveyda–Grubbs catalyst  was added. The catalyst addition was performed in five equal portions of 3 mol%  at t = 0, 3, 6, 9 and 21 h over the course of the reaction. The solution was sonicated at 55 °C under nitrogen atmosphere for 24 h. Volatiles were evaporated and the residue was purified by silica gel column chromatography  to give the desired phosphonate derivative 13  as brown oil. 1H NMR  δ 5.67–5.59 , 5.47–5.41 , 4.92 2), 4.21–4.08 , 4.02 , 3.47 , 3.38 14CH2-O), 2.67 , 1.90 , 1.69 , 1.54 13CH2CH2-O), 1.32–1.24 13CH2CH2-O, CH(CH3)2), 0.87 15O). 13C NMR  δ 153.3 (C=O), 140.8, 140.7 (CH=C), 112.6, 112.5 (CH=C), 84.4, 84.3 (O-CH2-O), 73.1 (CH(CH3)2), 71.2 (CH3(CH2)14CH2-O), 68.1, 68.0 (CH2-OH), 66.5, 66.4 (Cc), 63.3, 63.2 (Ca), 31.9, 30.7, 30.6 (Cb), 29.7, 29.5, 29.4, 27.1 (CH2-P), 26.2, 25.7 (CH2-P), 22.7, 21.6 (CH(CH3)2), 14.1 (CH3(CH2)15O), 13.9, 13.8 (CH3). 31P NMR  δ 26.71. HRMS (ESI): m/z [M+H]+ calcd for C29H58O8P: 565.386382, found: 565.385868.To a solution of (HDP/POC) allylphosphonate (E)-4-Azido-3-methyl-but-2-enyl-(HDP/POC) phosphonate (14)(E)-4-hydroxy-3-methyl-but-2-enyl-(HDP/POC)phosphonate 13  in anhydrous CH2Cl2 (5 mL) were added dropwise methansulfonyl chloride  and anhydrous triethylamine  at 0 °C under the argon. After 40 min stirring at room temperature, the mixture was diluted with CH2Cl2 and washed with water (3 × 10 mL) and brine. The organic layer was dried over MgSO4, filtrated and concentrated in vacuo. The residue was directly used for the next step without further purification. To a solution of the above product in anhydrous DMF (8 mL) was added sodium azide  under nitrogen atmosphere. After 5 h stirring at room temperature, the mixture was diluted with EtOAc and water and then extracted with EtOAc. The combined organic layer was washed with water (5 × 10 mL), brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography  to give (E)-4-Azido-3-methyl-but-2-enyl-(HDP/POC) phosphonate 14  as a colorless oil. 1H NMR  δ 5.69–5.58 , 5.51–5.41 , 4.91 2), 4.22–4.07 , 3.70 , 3.46 , 3.37 14CH2-O), 2.68 , 1.90 , 1.73 , 1.53 13CH2CH2-O), 1.32–1.22 13CH2CH2-O, CH(CH3)2), 0.86 15O). 13C NMR  δ 153.3 (C=O), 135.4, 135.3 (CH=C), 117.3, 117.2 (CH=C), 84.5, 84.4 (O-CH2-O), 73.1 (CH(CH3)2), 71.2 (CH3(CH2)14CH2-O), 66.5 (Cc), 63.5, 63.4 (Ca), 58.7, 58.6 (CH2-N3), 32.0, 30.8, 30.7 (Cb), 29.6 (CH2-P), 26.1, 26.0 (CH2-P), 22.7, 21.6 (CH(CH3)2), 14.8, 14.7 (CH=CCH3), 14.1 (CH3(CH2)15O). 31P NMR  δ 27.80. IR νmax : 2922.60, 2851.80, 2094.55, 1755.94, 1460.42, 1257.26, 1100.26, 1047.93, 992.52, 949.43 cm−1. HRMS (ESI): m/z [M+H]+ calcd for C29H57N3O7P: 590.392864, found: 590.393411.To a solution of -4-Azido-3-methyl-but-2-enyl-(HDP/POC) phosphonate 14 (1.0 equiv.) in tBuOH/H2O (2:1) were added sodium ascorbate (0.6 equiv.) and CuSO4.5H2O (0.1 equiv.). The resulting suspension was stirred at 40 °C until completion (followed by TLC), then the crude mixture was co-evaporated five times with methanol. The residue was purified by silica gel column chromatography using an elution gradient of petroleum ether/ethyl acetate to give the desired HDP/POC phosphonate derivatives.To a solution of terminal alkyne (1.3 equiv.) and (E)-3-methyl-4-[4-(4-propylphenyl]triazol-1-yl]but-2-enyl]phosphoryl]oxymethyl isopropyl carbonate (15a)+ calcd for C40H69N3NaO7P: 734.486765, found: 734.486600.The title compound was prepared from E)-3-methyl-4-[4-(4-pentylphenyl)triazol-1-yl]but-2-enyl]phosphoryl]oxymethyl isopropyl carbonate (15c)+ calcd for C42H72N3NaO7P: 784.500009, found: 784.499917.The title compound was prepared from E)-3-methyl-4-[4-(heptylphenyl)triazol-1-yl]but-2-enyl]phosphoryl]oxymethyl isopropyl carbonate (15e)+ calcd for C44H77N3O7P: 790.549365, found: 790.549322.The title compound was prepared from E)-4-(4-Carbamoyltriazol-1-yl)-3-methyl-but-2-enyl]-(3-hexadecyloxypropoxy)phosphoryl]oxymethyl isopropyl carbonate (15j)+ calcd for C32H60N4O8P: 659.414328, found: 659.414346.The title compound was prepared from E)-3-methyl-4-[4-(nitrophenyl)triazol-1-yl]but-2-enyl]phosphoryl]oxymethyl isopropyl carbonate (15l)+ calcd for C37H61N4NaO9P: 759.406837, found: 759.406817.The title compound was prepared from E)-2′-methyl-but-2′-enyl aliphatic side-chain with 1,4-substituted-1,2,3-triazoles as nucleobase. The convergent synthesis was based on olefin acyclic cross-metathesis and CuAAC cross-coupling reaction as the key steps. All those compounds were evaluated against HBV, HIV and SARS-CoV-2 viruses for their antiviral properties. Among them, compound 15j, a HDP/POC prodrug with R = C(O)NH2, a ribavirin analog, showed 62% inhibition (at 10 μM) without significant cytotoxicity (IC50 66.4 μM in HepG2 cells and IC50 = 43.1 μM in HepG2 cells). Further structural optimization of both the (E)-2′-methyl-but-2′-enyl aliphatic side-chain and the heterocycle is underway, alongside more detailed biological testing of the most active compound, with the aim of improving further its antiviral potency.In summary, we have efficiently synthesized various hitherto unknown ANP prodrugs bearing the (


Mode of action studies revealed that compound 21 is an allosteric dual-site compound inhibiting both HIV-1 RT functions, blocking the polymerase function also in presence of mutations carried by circulating variants resistant to non-nucleoside inhibitors, and the RNase H function interacting with conserved regions within the RNase H domain. Proving compound 21 as a promising lead for the design of new allosteric RNase H inhibitors active against viral replication with not significant cytotoxic effects.The persistence of the AIDS epidemic, and the life-long treatment required, indicate the constant need of novel HIV-1 inhibitors. In this scenario the HIV-1 Reverse Transcriptase (RT)-associated ribonuclease H (RNase H) function is a promising drug target. Here we report a series of compounds, developed on the 2-amino-6-(trifluoromethyl)nicotinic acid scaffold, studied as promising RNase H dual inhibitors. Among the 44 tested compounds, 34 inhibited HIV-1 RT-associated RNase H function in the low micromolar range, and seven of them showed also to inhibit viral replication in cell-based assays with a selectivity index up to 10. The most promising compound,  The acquired immunodeficiency syndrome (AIDS) pandemic is still one of the major world health concerns. Presently approved treatments do not allow immunization of people or the eradication of the infection once acquired, and among the estimated 37.9 million people living with HIV worldwide in 2019, only 24.5 million people were accessing the current antiretroviral therapy ,7. These2+ ions, and by a tertiary structure [2+ ions coordinated in the enzyme site by the DDE motif; (ii) RNase H selective allosteric inhibitors; (iii) allosteric inhibitors that bind at the p66/p51 interface; (iv) dual allosteric inhibitors of both the RDDP and RNase H functions. None of them has reached the clinical trial so far, enlightening the level of difficulty represented by the target [HIV-1 RT-associated RNase H is an enzymatic function present in all the members of the Ortervirales order  and it btructure  conservetructure ,13,14,15tructure  and; thetructure . Two dectructure ,18,19:  was first treated with the 2-methyl-5-chloroaniline (2) in MeCN solution to give the non-isolable 3-amino-3-(2-methyl-5-chlorophenyl)aminopropenenitrile (3) and then with 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-one (4). The temperature was gradually allowed to reach 60 °C. After 3 h, pyridinecarbonitrile 5 was obtained in good yields. Sodium hydroxide catalyzed hydrolysis of nitrile 5 afforded pyridine-3-carboxamide 6. High yield of 6-trifluoromethyl-3-pyridinecarboxylic acid 10 was achieved by hydrolysis of derivative 5 in 50% aqueous sulfuric acid. Chemistry similar to that described in the synthesis of compound 5 was used for the preparation of ethyl ester 9 . As reported in 7) was sequentially treated with the amine 2, then with enol ether 4 in MeCN solution to give the ester 9. Treatment of acid 10 with the appropriate phenol in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and hydroxybenzotriazole (HOBt) in MeCN solution gave esters 11–24 (25–52 (10 with the appropriate amine by EDCI method [The target derivatives of 2-(2-methyl-5-chloro)-6-trifluoromethyl-3-pyridinecarboxylic acid 9, 11–52  were synrs 11–24  in 52$%–4 (25–52  were obtI method . All the9 showed promising inhibitory activity (IC50 24 μM) of the RNase H function. The replacement of ethoxy group with 4-substituted aryl rings, produced changes in potency, with the 4-chlorine (13), 4-methoxy (18) and 4-thiomethyl (18) aryl esters being the most potent of the ester series. Interestingly the shift of the 4-chlorine atom into 2- or 3-position led to drop in activity (esters 11 and 12). The shift of the 4-hydroxy group of ester 18 into 2-position to give the analog 16 produced a small increase in inhibition potency, while the 3-hydroxyaryl ester 17 was less potent as compared to compounds 16 and 18. The methylation of the hydroxy group of compound 18, as well as its replacement with a thiomethyl group, , produced about two-fold enhancement of potency. On the contrary, the introduction of further methoxy groups, as in compounds 20–22, slightly reduced the potency of inhibition. The introduction of a 2-chlorine atom on compound 13 to give the ester 14 abolished the activity, while the 2,4,6-trichlorophenyl ester 15 showed about four-fold reduction in potency as compared to ester 13. The 3,5-dimethoxyphenyl ester 21 resulted about 1.5-fold more potent on the RNase H function as compared to the 3,4-dimethoxyphenyl analog 20.To establish the potential biologic effect of the synthesized compounds we tested them on the HIV-1 RT-associated RNase H activity, using as a control the RNase H inhibitors RDS1759  and beta6 was completely inactive, while the oxime 25 showed more potent anti-RNase H activity, being the best inhibitor with an IC50 value of 0.7 μM. The monosubstituted aryl amides 28–30 and 37 inhibited RNase H activity with IC50 in the 5.6-20 μM range. The replacement of the 3-methyl group of amide 29 with a 3-methoxy group to give compound 33 abolished the activity, while the presence of two or three methoxy groups as in compounds 34–36 repristinated the anti-RNase H efficacy. The benzylamides 38–47 showed RNase H inhibitory activity (IC50 in the 9–24 μM concentration range). The arylpiperazino amides 49 and 51 displayed some RNase H inhibitory potency . The introduction of a second chlorine atom on amide 49, to give the 3,4-dichlorophenyl piperazine analog 52, produced an increase in potency (IC50 7 μM).The amides showed a RNase H inhibitory activity rather different from esters. The nicotinamide 50 value below 30 μM were tested for the ability to inhibit HIV-1 replication in TZMbl cells  of 3.6. The 3,4-dimethoxyphenyl ester 21 resulted as the best ester inhibitor of the HIV replication activity with an EC50 value of 5 μM and a SI greater than 10. Amides 34 and 36 showed to be able to inhibit HIV-1 replication  although residual cytotoxic effect activity caused a poor selectivity , and also the amides 49 and 52 displayed HIV inhibitory activity . Noteworthy, the most active RNase H inhibitor in biochemical assays, compound 25, was inactive in the HIV replication assay.The 28 compounds that were able to inhibit the HIV-1 RNase H activity with an ICbl cells . The 4-cRDS1759. Results , very similar to the ones obtained on the RNase H function, indicating a dual-function allosteric inhibitory profile. In order to further dissect the mode of action the most promising compound, we selected ester function , showing21 and 49 were tested against both RT-associated functions of a panel of enzymes carrying single amino acid substitutions in the polymerase domain: mutations K103N, Y181C that are related to resistance to the NNRTIs [To clarify their binding mode, and also ascertain the possibility that these compounds could be active against circulating drug-resistant variants, compounds e NNRTIs , and mute NNRTIs . Besidese NNRTIs , that hae NNRTIs ,37,40.21 and 49  on the V108A RT RDDP function. Of note, all the inhibitors were able to inhibit the RT-associated RDDP activity of A502F and Q475A RTs, except for 49 that showed to increase its RDDP IC50 value on Q475A RT of 2.5-fold.We tested the inhibition of the HIV-1 RDDP activity of mutated HIV-1 RTs by compounds 1 and 49 , using a21 and 49 was analyzed using as control the RNase H active site inhibitor BTP  and more toxic (CC50 = 44 μM). The designed molecules showed a consistent inhibition of the HIV-1 RT-associated RNase H function, with the amide derivatives being overall slightly more potent than the ester counterparts. Cell-based assays revealed that the scaffold is more promising than the previous reported attempts of RNase H/RDDP dual inhibitors. Among the 44 2-(arylamino)-6-(trifluoromethyl)nicotinic acid derivatives, 7 compounds inhibited viral replication, while no active compound has been identified so far among the isatin-derivatives ,42. Furt21 was able to block HIV-1 replication with a SI > 10. The mode of action studies revealed that the compound acts differently form NNRTI inhibitors, with an activity profile against RDDP activity different from EFV on K103N and Y181C RTs, since these amino acid substitutions had a lower impact on its efficacy as compared to previously reported compounds [21 binds independently to two different allosteric sites previously described: S first pocket (pocket 1), contiguous with the NNRTI binding pocket, was described for hydrazones [21 on that sole function, proving also that these two conserved residues [21.The 3,5-dimethoxyphenyl ester ompounds ,42,64. Sdrazones , isatinedrazones ,42 and pdrazones . A secondrazones ,37, and drazones ,42 and pdrazones . For yearesidues  play a kresidues ,42, sinc21 as a promising good hit to be developed.These results provide useful insights for the design of novel and more potent HIV-1 RT RNase H/RDDP allosteric dual inhibitors, active against selected drug-resistant variants. With this aim, a major focus should be pointed on pocket 1 as the most suitable to be exploited for drug design, and on compound 6). Infrared spectra were recorded on a Vector 22 spectrometer  in Nujol mulls. The main bands are given in cm−1. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-focusing MAT 95 instrument  with BE geometry. Melting points (mp) were determined on a SMP1 Melting Point apparatus  and were uncorrected. All reported products showed NMR spectra in agreement with the assigned structures. The purity of the tested compounds was determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the Department of Chemical and Pharmaceutical Sciences of the University of Ferrara with a MT-5 CHN recorder elemental analyzer , and the values found were within 0.4% of theoretical values. Compounds 6, 9 [11–15, 19, 21, 23, 27, 29–37 and 49–51 were prepared as previously described [All commercially available solvents and reagents were used without further purification. NMR spectra were recorded on an Inova 500 spectrometer . The chemical shifts (δ) are reported in parts per million downfield from tetramethylsilane (TMS), which was used as internal standard, and the spectra were recorded in hexadeuteriodimethylsulphoxide , EDCI , and HOBt  in dry MeCN (10 mL) was stirred at room temperature for 30 min and then treated with the appropriate phenol (1 mmol). The mixture was stirred at room temperature for an additional 24 h. Then the solution was evaporated to dryness in vacuo. The residue was dissolved in ethyl acetate (20 mL) and washed with brine (2 × 5 mL), 5% aqueous sodium hydroxide (2 × 5 mL), and water (2 × 5 mL). The organic layer was dried over anhydrous sodium sulfate. Concentration of the dried extract yielded a residue which was triturated with di-isopropyl ether. The formed precipitate was filtered off and purified by crystallization from the adequate solvent to give the ester derivatives 11–24.A mixture of acid 16).2-Hydroxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate ( Yield 86%. Mp 179–180 °C (MeCN). 1H NMR (DMSO-d6): δ 2.38 , 6.98–7.41 , 7.52 d, 1H, J = 8.1 Hz, pyridyl), 7.46 , 8.26 , 8.88 , 9.98 , 10.06 . IR (Nujol) 3330, 1712, 1615, 1590 cm−1. m/z 423 (M + H)+. Anal. Calcd for C20H14ClF3N2O3 (422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.87, %H 3.32, %N 6.61.17).3-Hydroxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate ( Yield 87%. Mp 188-190 °C (MeCN). 1H NMR (DMSO-d6): δ 2.27 , 7.30-7.66 , 8.11 , 8.79 , 9.88 , 10.05 . IR (Nujol) 3294, 1712, 1620, 1590 cm−1. m/z 423 (M + H)+. Anal. Calcd for C20H14ClF3N2O3 (422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.78, %H 3.33, %N 6.60.18).4-Hydroxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate ( Yield 63%. Mp 184-186 °C (EtOH). 1H NMR (DMSO-d6): δ 2.27 , 6.83 , 7.13 , 7.37 , 8.14 , 8.73 , 9.55 , 9.86 . IR (Nujol) 3474, 3345, 1687, 1617, 1589 cm−1. m/z 423 (M + H)+. Anal. Calcd for C20H14ClF3N2O3 (422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.79, %H 3.36, %N 6.66.20).3,4-Dimethoxyphenyl-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate ( Yield 52%. Mp 127-129 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.30 , 3.79 , 3.86 , 6.85–7.98 , 8.24 , 8.76 , 10.51 . IR (Nujol) 3345, 3278, 1748, 1699, 1622, 1608, 1591 cm−1. m/z 467 (M + H)+. Anal. Calcd for C22H18ClF3N2O4 (466.84) %C 56.60, %H 3.89, %N 6.00. Found %C 56.65, %H 3.90, %N 6.02.22).3,4,5-Trimethoxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate ( Yield 98%. Mp 138-140 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.26 , 3.67 , 3.76 , 6.73 , 7.28 , 7.35–7.40 , 8.08 , 8.72 , 9.89 . IR (Nujol) 3331, 3293, 1697, 1618, 1590 cm−1. m/z 497 (M + H)+. Anal. Calcd for C23H20ClF3N2O4 (496.86) %C 55.60, %H 4.06, %N 5.64. Found %C 55.54, %H 4.07, %N 5.66.24).4-(Methylthio)phenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate ( Yield 63%. Mp 125–127 °C (2-propanol). 1H NMR (DMSO-d6): δ , 7.41–7.52 , 8.24 , 8.87 , 10.03 . IR (Nujol) 3294, 1712, 1620, 1590 cm−1. m/z 453 (M + H)+. Anal. Calcd for C21H16ClF3N2O2S (452.88) %C 55.69, %H 3.56, %N 6.19. Found %C 55.74, %H 3.58, %N 6.17.10 , EDCI , and HOBt  in dry MeCN (10 mL) was stirred at room temperature for 30 min and then treated with the appropriate amine (1 mmol). The mixture was stirred at room temperature for an additional 24 h. Then the solution was evaporated to dryness in vacuo. The residue was dissolved in ethyl acetate (20 mL) and washed sequentially with brine (2 × 5 mL), 10% aqueous sodium carbonate (2 × 5 mL), 10% aqueous citric acid (2 × 5 mL), and water (2 × 5 mL). The organic layer was dried over anhydrous magnesium sulfate. Concentration of the dried extracts yielded a solid residue which was washed with ethyl ether, filtered off, and dried to give the amide derivatives in analytically pure form without additional purification by crystallization if not elsewhere indicated.A mixture of acid 25).2-((5-Chloro-2-methylphenyl)amino)-N-hydroxy-6-(trifluoromethyl)nicotinamide ( Yield 61%. Mp 154–155 °C. 1H NMR (DMSO-d6): δ 2.37 , 7.36–7.68 , 8.34 , 8.86 , 9.87 , 10.59  12.01 . IR (Nujol) 3350, 1686, 1618, 1589 cm−1. m/z 346 (M + H)+. Anal. Calcd for C14H11ClF3N3O2 (345.70) %C 48.64, %H 3.21, %N 12.15. Found %C 48.69, %H 3.20, %N 12.18.26).Ethyl 2-(2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamido)acetate ( Yield 96%. Mp 128-130 °C. 1H NMR (DMSO-d6): δ 1.22 , 2.39 , 4.27 , 4.15 , 7.25–7.39 , 8.20 , 8.56 , 9.59 , 10.69 . IR (Nujol) 3429, 1737, 1722, 1655 cm−1. m/z 416 (M + H)+. Anal. Calcd for C18H17ClF3N3O3 (415.79) %C 52.00, %H 4.12, %N 10.11. Found %C 52.05, %H 4.10, %N 10.08.28).2-((5-Chloro-2-methylphenyl)amino)-N-(4-(methylthio)phenyl)-6-(trifluoromethyl)nicotinamide ( Yield 64%. Mp 152-154 °C. 1H NMR (DMSO-d6): δ 2.27 , 2.48 , 7.23 , 7.28 , 7.36 , 7.65 , 8.15 , 8.46 , 10.35 , 10.64 . IR (Nujol) 3380, 3300, 1639, 1588 cm−1. m/z 452 (M + H)+. Anal. Calcd for C21H17ClF3N3OS  %C 55.82, %H 3.79, %N 9.30.38).N-(2-chlorobenzyl)-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamide ( Yield 93%. Mp 169-170 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.39 , 4.72 , 7.36–7.61 , 8.35 , 8.56 , 9.68 , 10.90 . IR (Nujol) 3300, 1636, 1613, 1597 cm−1. m/z 450 (M + H)+. Anal. Calcd for C21H16ClF3N3O (449.85) %C 58.74, %H 4.26, %N 9.34. Found %C 58.69, %H 4.27, %N 9.30.39).2-((5-Chloro-2-methylphenyl)amino)-N-(2-methoxybenzyl)-6-(trifluoromethyl)nicotinamide ( Yield 53%. Mp 148-150 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.40 , 3.96 , 4.63 , 7.02–7.48 , 8.37 , 8.55 , 9.52 , 11.00 . IR (Nujol) 3315, 1639, 1617, 1599 cm−1. m/z 450 (M + H)+. Anal. Calcd for C21H16ClF3N3O (449.85) %C 58.74, %H 4.26, %N 9.34. Found %C 58.79, %H 4.24, %N 9.37.40).N-(4-chlorobenzyl)-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamide ( Yield 46%. Mp 130–131 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.28 , 4.52 , 7.24–7.40 , 8.23 , 8.40 , 9.60 , 10.96 . IR (Nujol) 3283, 1634, 1612, 1599 cm−1. m/z 452 (M + H)+. Anal. Calcd for C21H16ClF3N3O (451.89) %C 55.52, %H 3.55, % N 9.25. Found %C 55.58, %H 3.54, % N 9.22.41).2-((5-Chloro-2-methylphenyl)amino)-N-(4-fluorobenzyl)-6-(trifluoromethyl)nicotinamide ( Yield 43%. Mp 184–185 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.40 , 4.63 , 7.25–7.54 , 8.35 , 8.51 , 9.69 , 10.97 . IR (Nujol) 3276, 1635, 1613 cm−1. m/z 438 (M + H)+. Anal. Calcd for C21H16ClF4N3O (437.82) %C 55.52, %H 3.55, %N 9.25. Found %C 55.57, %H 3.54, %N 9.23.42).2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)-N-(4-(trifluoromethyl)benzyl)nicotinamide ( Yield 62%. Mp 170–171 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.38 , 4.74 , 7.37 , 7.43 , 7.84 , 7.70 , 7.82 , 8.34 , 8.54 , 9.78 , 10.92 . IR (Nujol) 3325, 1635, 1613, 1597 cm−1. m/z 488 (M + H)+. Anal. Calcd for C21H16ClF4N3O (487.83) %C 54.17, %H 3.31, %N 8.61.43).2-((5-Chloro-2-methylphenyl)amino)-N-(4-methoxybenzyl)-6-(trifluoromethyl)nicotinamide ( Yield 39%. Mp 126-128 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.40 , 3.84 , 4.58 , 7.02 , 7.43 , 7.34–7.45 , 8.35 , 8.48 , 9.63 , 11.00 . IR (Nujol) 3292, 1634, 1612, 1598 cm−1. m/z 450 (M + H)+. Anal. Calcd for C22H19ClF3N3O2 (449.85) %C 58.74, %H 4.86, %N 9.34. Found %C 58.68, %H 4.88, %N 9.37.44).2-((5-Chloro-2-methylphenyl)amino)-N-(4-methylbenzyl)-6-(trifluoromethyl)nicotinamide ( Yield 95%. Mp 189–190 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.39 , 2.43 , 4.60 , 7.25–7.47 , 9.66 , 10.99 . IR (Nujol) 3277, 1634, 1612, 1598 cm−1. m/z 434 (M + H)+. Anal. Calcd for C22H19ClF3N3O (433.85) %C 60.90, %H 4.41, %N 9.69. Found %C 60.97, %H 4.43, %N 9.66.45).2-((5-Chloro-2-methylphenyl)amino)-N--6-(trifluoromethyl)nicotinamide ( Yield 65%. Mp 153–155 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.38 , 4.69 , 7.36–7.57 , 7.76, , 8.34 , 8.54 , 9.69 , 10.88 . IR (Nujol) 3304, 1635, 1614, 1598 cm−1. m/z 489 (M + H)+. Anal. Calcd for C21H15ClF3N3O (488.72) %C 51.91, %H 3.09, %N 8.60. Found %C 51.96, %H 3.09, %N 8.64.46).2-((5-Chloro-2-methylphenyl)amino)-N--6-(trifluoromethyl)nicotinamide ( Yield 79%. Mp 179–180 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.39 , 4.65 , 7.27–7.82 , 8.34 , 8.53 , 9.80 , 10.92 . IR (Nujol) 3283, 1644, 1615, 1597 cm−1. m/z 489 (M + H)+. Anal. Calcd for C21H15ClF3N3O (488.72) %C 51.91, %H 3.09, %N 8.60. Found %C 51.85, %H 3.10, %N 8.63.47).2-((5-Chloro-2-methylphenyl)amino)-N-(4-hydroxy-3-methoxybenzyl)-6-(trifluoromethyl)nicotinamide ( Yield 95%. Mp 142–143 °C (2-propanol). 1H NMR (DMSO-d6): δ 2.33 , 3.85 , 4.53 , 6.83–7.50 , 8.34 , 8.54 , 9.04 , 9.60 , 11.01 . IR (Nujol) 3305, 1645, 1616 cm−1. m/z 466 (M + H)+. Anal. Calcd for C22H19Cl3F3N3O (465.85) %C 56.72, %H 4.11, %N 9.02. Found %C 56.67, %H 4.12, %N 9.06.48).(2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)pyridin-3-yl)(4-phenylpiperazin-1-yl)methanone ( Yield 81%. Mp 118–120 °C (n-hexane). 1H NMR (DMSO-d6): δ 2.25 , 3.17–3.21 , 3.36–3.41 , 6.80–7.32 , 7.55 , 7.84 , 8.55 . IR (Nujol) 3365, 1630, 1614, 1599 cm−1. m/z 475 (M + H)+. Anal. Calcd for C24H22ClF3N4O (474.91) %C 60.70, %H 4.67, %N 11.80. Found %C 60.64, %H 4.69, %N 11.84.52).(2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)pyridin-3-yl)piperazin-1-yl)methanone ( Yield 87%. Mp 165–167°C (MeCN). 1H NMR (DMSO-d6): δ 2.27 , 3.41–3.45 , 3.73–3.75 , 7.05–7.55 , 7.65 , 7.96 , 8.65 . IR (Nujol) 3323, 1638, 1609 cm−1. m/z 544 (M + H)+. Anal. Calcd for C24H20Cl3F3N4O (543.80) %C 53.01, %H 3.71, %N 10.30. Found %C 53.07, %H 3.70, %N 10.27.Escherichia coli strain M15 containing the p6HRT-prot vector were grown up to an OD600 of 0.7 and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) 1.7 mM for 4 h. Cell pellets were resuspended in Lysis buffer , incubated on ice for 20 min, added 0.3 M final NaCl, sonicated and centrifuged at 30,000× g for 1 h. The supernatant was loaded into a Ni2+-sepharose column pre-equilibrated with loading buffer  and washed thoroughly with wash buffer . RT was gradient-eluted with elute buffer (wash buffer with 0.5 M imidazole). Fractions were collected, protein purity was checked by SDS-PAGE and found to be higher than 90%. RT containing fractions were pooled and diluted 1:1 with dilute buffer  then loaded into a Hi-trap Heparine HP GE  pre-equilibrated with 10-column volume of loading buffer 2 . Column was then washed with loading buffer 2 and RT was gradient-eluted with elute buffer 2 . Fractions were collected, protein was dialyzed and stored in buffer containing 50 mM Tris HCl pH 7.0, 25 mM NaCl, 1 mM EDTA, 50% glycerol. Catalytic activities and protein concentration were determined. Enzyme-containing fractions were pooled and aliquots were stored at −80 °C.p6HRT-prot vector was kindly provided by Dr. Stuart Le Grice Laboratory. Heterodimeric RT was expressed essentially as described . Briefly2 in a water solution, using a Nano Drop ONE reader (Thermo Scientific). Results are plotted as absorbance vs wavelength using Prisme 6, version 6.01.The inhibitors were diluted in water to a final concentration of 100 μM. UV spectra recorded in the absence and after addition of 6 mM MgClAmino acid substitutions were introduced into the p66 HIV-1 RT subunit coded in a p6HRT-prot plasmid using the QuikChange protocol .2, 1 mM dithiothreitol (DTT), 80 mM KCl, hybrid RNA/DNA , and different amounts of enzymes according to a linear range of dose-response curve—20 ng of wt RT; 50 ng K103N RT; 100 ng V108A RT; 4 ng Y181C RT; 100 ng Q475A RT; 100 ng A502F RT. The reaction mixture was incubated for 1 h at 37 °C, the reaction was stopped by addition of EDTA and products were measured with a Victor 3 (Perkin) at 490/528 nm.The RT-associated RNase H activity of wt and mutated HIV RTs was measured as described , in 100 2, 60 mM KCl, 13 mM DTT, poly(A)-oligo(dT), 100 µM dTTP, and 6 ng wt RT . After enzyme addition, the reaction mixture was incubated for 30 min at 37 °C and enzymatic reaction was stopped by addition of EDTA. Reaction products were detected by PicoGreen addition and measured with a Victor 3 (Perkin) at 502/523 nm.The HIV-1 RT-associated RDDP activity was measured as described . Briefly4 TZMbl cells were infected with HIV-1 IIIB strain at a multiplicity of infection of 0.1. Serial dilutions of drugs were added immediately after infection. Luciferase activity was measured as reported [50) was defined as the concentration that caused a 50% reduction of luciferase activity (relative light units) compared to virus control wells. Cell viability in CEM cells was quantified 5 days after infection with the MTT-dye reduction method.Drug-mediated inhibition of virus-induced cytotoxicity was assayed in TZM-BL cells. Triplicate wells of 96-well plates containing 1 × 10reported . Two day21, active against viral replication in low micromolar range with no significant cytotoxic effects, showed to be a dual inhibitor active also against the RNA dependent DNA polymerase activity of HIV-1 RT, being active against RTs carrying NNRTI-resistant mutations. Mode-of-action and single-site mutagenesis studies indicated that compound 21 potency of inhibition is strongly impaired by the substitutions of amino acidic residues in two different pockets, located in two different domains of RT enzyme, hence supporting the hypothesis of a dual-site binding mode. Overall compound 21 is a promising lead for the design of new allosteric RNase H inhibitors more potent against HIV-1 replication.We identified a new promising 2-amino-6-(trifluoromethyl)nicotinic acid scaffold for compounds that can inhibit both HIV-1 RT activities and are also active against viral replication. Compound