Compounds A3 and A4 exhibited broad and potent antiviral activity with the 50% effective concentration (EC50) values in the range of 1.23–1.76 μM. Moreover, the selectivity indices of A3 and A4 were significantly higher than those of the reference compound, pirodavir. The western blotting experiment indicated that the viral VP1 was significantly decreased at both the protein and RNA level in a dose-dependent manner following treatment with compound A3. Moreover, compound A3 inhibited the viral replication by acting on the virus entry stage. In summary, this study led to the discovery of 2-aryl-isoindolin-1-ones as a promising scaffold with potent anti-EV-A71 activities, which deserves further in-depth studies.Enterovirus 71 (EV-A71) is the main causative pathogen of childhood hand, foot and mouth disease. Effective medicine is currently unavailable for the treatment of this viral disease. Using the fragment-hopping strategy, a series of 2-aryl-isoindolin-1-one compounds were designed, synthesized and investigated for their in vitro antiviral activity towards multiple EV-A71 clinical isolates  in Vero cell culture in this study. The structure–activity relationship (SAR) studies identified 2-phenyl-isoindolin-1-ones as a new potent chemotype with potent antiviral activity against EV-A71. Ten out of the 24 tested compounds showed significant antiviral activity (EC Hand, foot and mouth disease (HFMD) is a seasonal epidemic infectious disease that mainly affects children under five years old ,2. Its cN-aryl benzamide was identified as a new broad antiviral chemotype in our previous research. The representative compound 3-amino-N-(4-bromophenyl)-4-methoxybenzamide  to yield 2-aryl isoindolin-1-ones A1–A10 by a modified Ullmann coupling reaction. Amides A11–A13 were prepared by condensing A10 with amines in the presence of 1-Hydroxybenzotriazole (HOBt) and N,N′-diisopropylcarbodiimide (DIC) in N,N-dimethylformide (DMF)  in 9 was subsequently obtained through bromination and a microwave-aided cyclization. Eventually, a phenyl, phenylamino or methylpiperazin group was introduced at the C6-position of isoindolinon 9 by a Buchwarld or Suzuki coupling reaction to afford the final compounds D1–D3 with a yield of less than 40%.As shown in de (DMF) . As show50) of each compound was calculated according to its inhibitory rate values with the Reed–Muench method. The 50% cytotoxic concentration (CC50) was defined as the concentration that inhibited 50% cellular growth in comparison with the controls. The selectivity index (SI) was calculated as CC50/EC50. As shown in 50 values in the submicromolar range (EC50 = 0.38~0.76 µM) as well as high SI values (between 88 and 44) in this test model. Among the 24 tested compounds, 10 compounds  showed significant antiviral activity (EC50 < 10 µM) and acceptable SI values towards four EV-A71 strains. Moreover, all EV-A71 strains showed similar sensitivity to the tested compounds. Importantly, compounds A3 and A4 possessed superior antiviral profiles (SI = 98–199) derived from their significantly lower cytotoxicity, although their EC50 values were about 2–4 fold higher (EC50 = 1.23~1.76 µM) than those of the reference compound, pirodavir. Generally, the EV-A71 H strain was less sensitive to most of the active compounds as compared to the other virus strains.An in-vitro antiviral assay was established using the Africa monkey kidney cell line Vero which was infected with different EV-A71 clinical isolates . A total of 24 synthetic compounds were initially screened in such an assay to determine their inhibitory effect on virus replication at 3-fold diluted concentrations serial (400~0.18 μM). The 50% effective concentration (ECA1) with 4-halophenyl (A2–A4) increased the antiviral activity, whereas 4-methoxyphenyl substitution (A5) did not influence much of the antiviral activity against the four tested virus strains. Furthermore, 4-bromophenyl substitution (A4) provided comparable activity to 4-chlorophenyl substitution (A3), both of which were more potent than 4-fluorophenyl A2. Therefore, it can be concluded that lipophilicity is more important for potency than the electronic effect. Meanwhile, a halogen bond may exist within the interaction between compound A3 or A4 and their target. Although 3,4-dichlorophenyl substitution (A5) showed a similar potency as 4-chlorophenyl (A3), the former has a lower SI due to its increased cytotoxicity. The unfavorable meta-position substitution was further evidenced by the increased cytotoxicity of A8 with multiple substitution as compared to A7, which has a single substituent at the para-position. The importance of lipophilicity at the C4-position of the 2-phenyl group for antiviral activity was further confirmed by the comparison between the halogen group (A2–A5) and isoquinoline (A9) or the carboxyl group (A10). The same trend was also observed for 5 and 6. The potency was decreased when the carboxylic acid group of A10 was further transformed into corresponding amide derivatives (A11–A13). Moreover, the potency was significantly compromised and even lost when the 2-phenyl group was replaced with a bulkier group such as biphenyl (A6). These results suggested that a steric limitation exists on the substituents at the pare-position of the 2-phenyl ring.Most of the active compounds were 2-substitutedphenyl isoindolin-1-ones, whereas only a minority of the active compounds contained an amino group on the isoindolin-1-one moiety. Specifically, the replacement of phenyl  slightly increased antiviral activity. On the contrary, the same substitution at the C5-position (5) decreased the activity as compared to A7. The presence of alkylamino group at the C6-postion (B1) led to a dramatically increased potency as compared to B3 with the same substitution at the C5-position. The same trend was observed for the dialkylamino substituted compounds B2 and B4. Of note, compounds with dialkylamino substitution on the isoindolin-1-one fragment consistently displayed weaker or even no activity compared to those with alkylamino group substitution. In addition, the higher potency of B1 over A7 illustrated the role of NH at the C6-position of isoindolin-1-one in antiviral activity. However, the acylation of C6-amino (C1) induced a several-fold decrease in potency in comparison with amino alkylation (B1). Among compounds C1–C3, only the C6-amide compound C1 showed moderate activity, while the C5-amide compounds (C2 and C3) completely lost their antiviral potency. These results reiterated the importance of the substitution position on isoindolin-1-one fragments. Encouraged by the good tolerance of 6-alkylamino substitution for anti-EV-A71 activity, we decided to further explore modification at that position to facilitate the SAR study. Unfortunately, the introduction of 6-phenyl (D1) resulted in a substantial loss in potency relative to that of A3. Similarly, 6-phenylamino substitution (D2) resulted in a 10-fold loss in activity against four in vitro models. Interestingly, 6-(4-methylpiperazin)-2-(4-chlorophenyl)-isoindolin-1-one (D3) showed comparable antiviral activity to that of A3. This is in contradiction to the former speculation about the importance of having the NH group at the C6-postion as a hydrogen-bond donor. A possible explanation for this disparity is that steric limitation at the C6-position plays a more crucial role in maintaining antiviral potency than the hydrogen bond.Compounds of the B, C and D series as well as compounds Altogether, it appears that 2-substitutedphenyl-isoindolin-1-one is an important pharmacophore for anti-EV-A71 activity. A lipophilic group with size restriction was shown to be more favorable at the C4-position of the phenyl ring. A small amino group at the C6-position of the isoindolin-1-one moiety was also well tolerated.A3 of various concentrations for another 24 h. As shown in A3 treatment decreased the levels of viral VP1 protein in a dose-dependent manner in vitro. Moreover, compound A3 significantly reduced VP1 RNA expression level in a dose-dependent way in the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay  assay B. Meanwhnfection C. Those A3 on the EV-A71 viral life cycle, EV-A71 viral protein VP1 was measured via Western blot when Vero cells were treated with compound A3 prior to, during, or after EV-A71 viral incubation . The chemical shift was reported in parts per million, relative to tetramethylsilane as the internal standard. High Resolution Mass Spectrum (HRMS) was recorded on a Triple TOF 5600+LC/MS/MS system (CADM-YQ-086) with an Electrospray Ionization (ESI) mass selective detector. The reaction was monitored through TLC Silica gel 60F254 Aluminium plate . Flash chromatography was performed on a CombiflashRf 200  with gel silica column.All reagents and solvents used are commercially available and were used without further purification. 2CO3 , CuI  and N1,N2-dimethylethane-1,2-diamine  were added to the solution. The resulting mixture was stirred at room temperature for 10 min, after which iodobenzene (2.25 mmol) was added. Then the mixture was heated to 120 °C. When TLC showed that isoindolin-1-one had been fully converted, the reaction was stopped. The mixture was extracted with ethyl acetate (20 mL) and H2O (10 mL). The water phase was re-extracted with ethyl acetate (20 mL). The organic layer was combined and washed with brine (10 mL). Then the solution was dried over anhydrous MgSO4, filtered and concentrated, and the crude residue was purified by flash chromatography over silica gel using CH2Cl2/CH3OH as the gradient elution to afford the title compounds.General Procedure A for the Synthesis of Compounds A1~A12. Isoindolin-1-one  was dissolved in super-dry DMSO (4 mL), and Cs22-phenylisoindolin-1-one (A1). Compound A1 was synthesized from isoindolin-1-one and iodobenzene using general procedure A as a white solid. The yield was 50%. 1H-NMR  δ 7.98 , 7.92 , 7.64 , 7.56 , 7.49 , 7.22 , 4.91 . HRMS(ESI+) m/z calcd for C14H12NO [M + H]+ 210.0919, found 210.0921.2-(4-fluorophenyl)isoindolin-1-one (A2). Compound A2 was synthesized from isoindolin-1-one and 1-fluoro-4-iodobenzene using general procedure A as a yellow solid. The yield was 32%. 1H-NMR  δ 7.98 , 7.84 , 7.73 , 7.61 , 7.35 , 5.08 . HRMS(ESI+) m/z calcd for C14H11FNO [M + H]+ 228.0825, found 228.0827.2-(4-chlorophenyl)isoindolin-1-one (A3). Compound A3 was synthesized from isolindolin-1-one and 1-chloro-4-iodobenzene using general procedure A as a white solid. The yield was 49%. 1H-NMR  δ 7.97 , 7.89 , 7.66 , 7.57 , 7.44 , 4.89 . HRMS(ESI+) m/z calcd for C14H11NOCl [M + H]+ 244.0529, found 244.0522.2-(4-bromophenyl)isoindolin-1-one (A4). Compound A4 was synthesized from isoindolin-1-one and 1-bromo-4-iodobenzene using general procedure A as a brown solid. The yield was 44%. 1H-NMR  δ 7.93 , 7.83 , 7.75–7.62 , 7.58 , 5.06 . HRMS(ESI+) m/z calcd for C14H11BrNO [M + H]+ 288.0024, found 288.0026.2-isoindolin-1-one (A5). Compound A5 was synthesized from isolindolin-1-one and 2,4-dichloro-1-iodobenzene using general procedure A as a white solid. The yield was 48%. 1H-NMR  δ 8.01 , 7.68 , 7.58 , 7.43 , 4.85 . HRMS(ESI+) m/z calcd for C14H10NOCl2 [M + H]+ 278.0139, found 278.0132.2-isoindolin-1-one (A6). Compound A6 was synthesized from isoindolin-1-one and 4-iodo-1,1’-biphenyl using general procedure A as a yellow solid. The yield was 26%. 1H-NMR  δ 8.05 , 7.84 , 7.80 , 7.73 , 7.60 , 7.51 , 7.39 , 5.12 . HRMS(ESI+) m/z calcd for C20H16NO [M + H]+ 286.1232, found 286.1220.2-(4-methoxyphenyl)isoindolin-1-one (A7). Compound A7 was synthesized from isolindolin-1-one and 1-iodo-4-methoxybenzene using general procedure A as a white solid. The yield was 53%. 1H-NMR  δ 7.96 , 7.78 , 7.62 , 7.54 , 7.01 , 4.85 , 3.87 . HRMS(ESI+) m/z calcd for C15H14NO2 [M + H]+ 240.1025, found 240.1020.2-isoindolin-1-one (A8). Compound A8 was synthesized from isoindolin-1-one and 3,4,5-trimethoxy-1-iodobenzene using general procedure A as a yellow solid. The yield was 37%. 1H-NMR  δ 7.77 , 7.69 , 7.60–7.51 , 7.28 , 5.05 , 3.83 , 3.67 . HRMS(ESI+) m/z calcd for C17H18NO4 [M + H]+ 300.1236, found 300.1222.2-(isoquinolin-7-yl)isoindolin-1-one (A9). Compound A9 was synthesized from isoindolin-1-one and 7-iodoisoquinoline using general procedure A as a yellow solid. The yield was 48%. 1H-NMR  δ 9.34 , 8.56 , 8.32 , 8.01 , 7.94 , 7.69 , 7.66–7.50 , 5.05 . HRMS(ESI+) m/z calcd for C17H13N2O [M + H]+ 261.1028, found 261.1034.4-(1-oxoisoindolin-2-yl)benzoic acid (A10). Compound A10 was synthesized from isoindolin-1-one and 4-iodobenzoic acid using general procedure A as a white solid. The yield was 31%. 1H-NMR  δ 12.87 , 8.11–8.05 , 8.02 , 7.83 , 7.75–7.67 , 7.59–7.54 , 5.09 . HRMS(ESI+) m/z calcd for C15H12NO3 [M + H]+ 254.0817, found 254.0805A11–A13. Compound A10  was dissolved in DMF (3 mL). HOBt  and DIC  were added to the solution and the resulting solution was stirred for 20 min at room temperature. Amine was added into the resulting mixture and stirred at room temperature overnight. When TLC showed that the reaction was complete, the mixture was extracted with EA and H2O. The water phase was re-extracted with EA. The organic layer was combined and washed with brine (10 mL). Then the solution was dried over anhydrous MgSO4, filtered and concentrated, and the crude residue was purified by flash chromatography over silica gel using CH2Cl2/CH3OH as the gradient elution to afford the title compounds.General Procedure for the Synthesis of Compounds 2-(4-(4-methylpiperazine-1-carbonyl)phenyl)isoindolin-1-one (A11). Compound A11 was prepared from A10 and 1-methypiperazine using the above procedure as a white solid. The yield was 61%. 1H-NMR  δ 8.03–7.98 , 7.82 , 7.74–7.67 , 7.59–7.54 , 7.52–7.47 , 5.07 , 3.45 , 2.35 , 2.20 . HRMS(ESI+) m/z calcd for C20H22N3O2 [M + H]+ 336.1712, found 336.1697.2-(4-(morpholin-4-carbonyl)phenyl)isoindolin-1-one (A12). Compound A12 was prepared from A10 and morpholine using the above procedure as a white solid. The yield was 65%. 1H-NMR  δ 8.01 , 7.82 , 7.76–7.65 , 7.61–7.47 , 5.08 , 3.62 . HRMS(ESI+) m/z calcd for C19H19N2O3 [M + H]+ 323.1396, found 323.1383.N-(3-(dimethylamino)propyl)-N-methyl-4-(1-oxoisoindolin-2-yl)benzamide (A13). Compound A13 was prepared from A10 and 3-dimethylamino-propylamine using the above procedure as a shallow yellow solid. The yield was 40%. 1H-NMR  δ 8.54 , 8.02 , 7.93 , 7.81 , 7.76–7.65 , 7.61–7.52 , 5.08 , 3.30 , 2.41 , 2.25 , 1.75–1.63 . HRMS(ESI+) m/z calcd for C20H24N3O2 [M + H]+ 338.1869, found 338.1852.6-amino-2-(4-methoxyphenyl)isoindolin-1-one (4). Compound 4 was synthesized from 6-aminoisoindolin-1-one 2 and 1-iodo-4-methoxybenzene using general procedure A as a white solid. The yield was 56.97%. 1H-NMR  δ 7.80–7.73 , 7.26 , 7.03–6.95 , 6.93–6.82 , 5.38 , 4.77 , 3.77 , 2.55 . HRMS(ESI+) m/z calcd for C15H15N2O2 [M + H]+ 255.1134, found 255.1140.5-amino-2-(4-methoxyphenyl)isoindolin-1-one (5). Compound 5 was synthesized from 5-aminoisolindolin-1-one 3 and 1-iodo-4-methoxybenzene using general procedure A as a yellow solid. The yield was 51.54%. 1H-NMR  δ 7.77 , 7.42 , 6.99 , 6.87–6.50 , 5.92 , 4.78 , 3.78 . HRMS(ESI+) m/z calcd for C15H15N2O2 [M + H]+ 255.1134, found 255.1124.5-amino-2-(p-tolyl)isoindolin-1-one (6). Compound 6 was synthesized from 5-aminoisoindolin-1-one 3 and 4-iodotoluene using general procedure A as a yellow solid. The yield was 45.69%. 1H-NMR  δ 7.74 , 7.39 , 7.26–7.12 , 6.65 , 5.93 , 4.77 , 2.29 . HRMS(ESI+) m/z calcd for C15H15N2O [M + H]+ 239.1184, found 239.1185.B1–B4. 6-amino-2-(4-methoxyphenyl)isoindolin-1-one 4  was dissolved in superdry CH3CN (5 mL), and K2CO3  was added to the solution. The resulting mixture was stirred at room temperature, after which 1-iodopropane (2.25 mmol) was added. When TLC showed that 6-amino-2-(4-methoxyphenyl)isoindolin-1-one was fully converted, the reaction was stopped. The mixture was filtered and the filtrate was evaporated in vacuo. The residue was dissolved in CH2Cl2 and then washed with H2O. The water phase was re-extracted with CH2Cl2. The organic layer was combined and washed with brine (10 mL). Then the solution was dried over anhydrous MgSO4, filtered and concentrated, and the crude residue was purified by flash chromatography over silica gel using CH2Cl2/CH3OH as the gradient elution to afford 2-(4-methoxyphenyl)-6-(propylamino)isoindolin-1-one (B1) and 6-(dipropylamino)-2-(4-methoxyphenyl)isoindolin-1-one (B2) as a yellow solid with yields of 32% and 26% for B1 and B2, respectively.General Procedure B for the Synthesis of Compounds B11H-NMR  δ 7.75 , 7.41 , 7.22 , 7.00 , 4.77 , 3.88 , 3.25 , 1.81 , 1.07 . HRMS(ESI+) m/z calcd for C18H21N2O2 [M + H]+ 297.1603, found 297.1611.Compound B21H-NMR  δ 7.77 , 7.16 , 7.01 , 6.90 , 4.87–4.66 , 3.87 , 3.35 , 1.68 , 0.98 . HRMS(ESI+) m/z calcd for C21H27N2O2 [M + H]+ 339.2073, found 339.2055.Compound B3) and 5-(dipropylamino)-2-(4-methoxyphenyl)isoindolin-1-one (B4) were synthesized from 5-amino-2-(4-methoxyphenyl)isoindolin-1-one (5) and 1-iodopropane using general procedure B, which were both yellow solids. The yields were 34% and 28% for B3 and B4, respectively.2-(4-methoxyphenyl)-5-(propylamino)isoindolin-1-one  δ 7.74 , 6.99 , 6.71 , 4.75 , 3.87 , 3.21 , 1.75 , 1.07 .Compound B41H-NMR  δ 7.75 , 6.99 , 6.75 , 6.66 , 4.76 , 3.87 , 3.37 , 1.71 , 1.01 . HRMS(ESI+) m/z calcd for C21H27N2O2 [M + H]+ 339.2073, found 339.2076.Compound C1–C3. 5-amino-2-(p-tolyl)isoindolin-1-one 6  was dissolved in CH2Cl2 (3 mL), and DIPEA  was added to the solution. Propionyl chloride was dissolved in CH2Cl2 (1 mL) and transferred to a drip funnel, and dropped into the flask, at a rate of about 1–2 drop(s) per second. After completion of the addition, the mixture was stirred at room temperature. When TLC showed that 6-amino-2-(p-tolyl)isoindolin-1-one had been fully converted, the reaction was stopped. The mixture was extracted with H2O (3 mL). The water phase was re-extracted with CH2Cl2 (10 mL). The organic layer was combined and washed with brine (10 mL). Then the solution was dried over anhydrous MgSO4, filtered and concentrated, and the crude residue was purified by flash chromatography over silica gel using CH2Cl2/CH3OH as the gradient elution to afford compound N-(1-oxo-2-(p-tolyl)isoindolin-5-yl)propionamide (C3) as a yellow solid. The yield was 46%. 1H-NMR  δ 10.27 , 8.07 , 7.80 , 7.71 , 7.63 , 7.26 , 4.97 , 2.41 , 2.33 , 1.14 . HRMS(ESI+) m/z calcd for C18H19N2O2 [M + H]+ 295.1447, found 295.1433.General Procedure C for the Synthesis of Compounds N-(2-(4-methoxyphenyl)-3-oxoisoindolin-5-yl)propionamide (C1). Compound C1 was synthesized from 6-amino-2-(4-methoxyphenyl)isoindolin-1-one (4) and propionyl chloride using general procedure C as a yellow solid. The yield was 49%. 1H-NMR  δ 8.22 , 7.85 , 7.75 , 7.63 , 7.00 , 4.83 , 3.87 , 2.77–2.30 , 1.31 . HRMS(ESI+) calcd for C18H19N2O3 [M + H]+ 311.1396, found 311.1386.N-(2-(4-methoxyphenyl)-1-oxoisoindolin-5-yl)propionamide (C2). Compound C2 was synthesized from 5-amino-2-(4-methoxyphenyl)isoindolin-1-one (5) and propionyl chloride using general procedure C as a yellow solid. The yield was 50%. 1H-NMR  δ 10.24 , 8.04 , 7.82–7.75 , 7.68 , 7.61 , 7.04–6.97 , 4.94 , 3.77 , 2.39 , 1.11 . HRMS(ESI+) calcd for C18H19N2O3 [M + H]+ 311.1396, found 311.1382.D1–D3. Methyl 5-bromo-2-methylbenzoate  was added to a flask containing 20 mL CCl4, and then N-bromosuccinimide  and TsOH  were added. The mixture was refluxed until methyl 5-bromo-2-methylbenzoate was fully converted. The solution was washed with saturated NaHCO3 solution, water and brine successively. The organic phase was evaporated under vacuum to afford the crude product methyl 5-bromo-2-(bromomethyl)benzoate (9).Procedure D for the synthesis of compounds 10  as a white solid. 1H-NMR  δ 7.96 , 7.90 , 7.68 , 7.54 , 5.03 .A 10 mL microwave vial was charged with 4-chloroaniline , methyl 5-bromo-2-(bromomethyl)benzoate , and DMF (4 mL). The vial was sealed and microwaved at 110 °C for 25 min. The reaction mixture was diluted with MeOH (50 mL) and an off-white precipitate was formed, which was filtered and dried under vacuum to give 10 , phenylboronic acid , NaOBu-t , DMSO (2 mL), and H2O (0.3 mL). The solution was degassed with nitrogen for 10 min, and Pd(dppf)Cl2  was added. The vial was sealed and microwaved at 130 °C for 2 h. The reaction mixture was poured into EtOAc/H2O and the organic layer was separated and subsequently passed through a plug of florisil. The obtained organic phase was concentrated into a residue, which was triturated with MeOH, filtered, and dried under vacuum to afford D1  as a white solid. 1H-NMR  δ 8.05–7.98 , 7.80 , 7.58–7.50 , 7.45 , 5.11 .A microwave vial was charged with D2 was synthesized using 10 and aniline using the same method of synthesis as that of D1. D2 was purified by flash chromatography to afford a yellow solid. The yield was 20%. 1H-NMR  δ 8.46 , 8.00–7.89 , 7.51 , 7.45–7.24 , 7.14 , 6.91 , 4.93 .Compound D3 was synthesized using 10 and 4-methylpiperazine using the same method of synthesis as that of D1. D3 was purified by flash chromatography to afford a pale yellow solid. The yield was 22%. 1H-NMR  δ 7.96 , 7.90 , 7.68 , 7.54 , 5.03 .Compound 2 incubator.African green monkey kidney (Vero) cells were purchased from the American Type Culture Collection (ATCC) and were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C in a 5% COEV-A71 strain ShenZhen98 (SZ98) was isolated from the throat swab sample of an HFMD case which occurred in 1998 in China. This was kindly provided by Dr. Qi Jin, Institute of Pathogen Biology, Chinese Academy of Medical Science and Peking Union Medical School, Beijing, China. EV-A71 strains BrCr (VR-1775) and H (VR-1432) were purchased from the ATCC. The EV-A71 strain JS52 was a kind gift from Dr. Xiangzhong Ye, Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. EV-A71 were all passaged in Vero cells.4 cells/well) were seeded into 96-well culture plates and incubated overnight. Then, the medium was removed and different concentrations of compounds were applied in triplicate. After 3 days of incubation, the CC50 was defined as the concentration that inhibited 50% cellular growth in comparison with the untreated controls, as calculated by the Reed–Muench method.The cytotoxic effects of compounds on Vero cells were detected by the cytopathic effect (CPE) assay. Briefly, cells (3 × 104 cells/well) were plated into 96-well culture plates for incubation for 24 h. The medium was then removed and cells were infected with EV-A71 with 100TCID50 (50% tissue culture infective doses) in serum-free medium for 1 h at 37 °C. Then, the unbound viruses were removed and various concentrations of compounds were supplemented and incubated for another 48 h. The EC50 was determined by the Reed–Muench method. The selectivity index was calculated as the ratio of CC50/EC50 [The anti-EV-A71 activity of compounds was determined by the CPE inhibition method. Briefly, cells (3 × 10C50/EC50 .5 cells/well) were inoculated with EV-A71 (MOI = 1.0) at 37 °C for 1 h. The media with or without treatment were added during the periods of −24~0 h, −1~0 h, 0~10 h, 1~10 h, 2~10 h, 4~10 h, 6~10 h and 8~10 h. After each incubation period, the collected cells were washed with PBS, and then the viral protein was determined by Western blot.The antiviral activity of the tested compound was examined at different time periods prior to, during or after viral infection. Briefly, Vero cells (9 × 105 cells/well) were plated into 6-well culture plates for incubation for 16 h. The medium was removed and cells were infected with EV-A71 . After 1 h, various concentrations of A3 were supplemented for incubation for another 24 h. The cells were lysed in the M-PER mammalian protein extraction reagent  containing halt protease inhibitor single-use cocktail (Thermo). The protein concentration was determined by the BCA reagents (Thermo). About 15 μg of proteins was denatured and applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis products were transferred to a polyvinyl idenefluoride (PVDF) film and PVDF membranes were then incubated at room temperature with a specific primary antibody. After a standard washing, membranes were incubated with horse radish peroxidase (HRP)-labeled secondary antibody. The assay was developed using a chemiluminescent substrate. The primary antibodies used in this study included antibodies against β-actin , and EV-A71-VP1 (Abnova). Goat anti-mouse HRP-labeled antibodies were obtained from Cell Signaling Technology [Vero cells (9 × 105cells/well) were plated into 6-well culture plates for incubation for 16 h. The medium was removed and cells were infected with EV-A71 . After 1 h, various concentrations of A3 were supplemented and incubated for another 24 h. The total RNA of the infected cells was extracted using the RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. The one-step qRT-PCR was performed with SuperScript III Platinum SYBR Green One-step RT PCR Kit (Invitrogen) using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems). The RNA expression of EV-A71 VP1 was detected with the sense primer 5′-GATATCCCACATTCGGTGA-3′ and the antisense primer 5′-TAGGACACGCTCCATACTCAAG -3′ targeting a conserved region of the VP1 gene. β-Actin mRNA was detected using the sense primers 5′-TGACGGGGT CACCCACA CTGTGCCCATCTA-3′ and the antisense primer 5′-CTAGAAGCATTTG CGGTGGACG ATG-3′. The PCR assay was carried out in a 25 μL volume and the target fragment amplification was carried out as follows: reverse transcription at 50 °C for 3 min; initial activation of HotStar Taq DNA Polymerase at 95 °C for 10 min; 40 cycles in two steps: 95 °C for 15 s, 60 °C for 30 s. The relative amounts of EV-A71 VP1 mRNA were calculated by the comparative Ct method after normalizing the quantity of β-actin [Vero cells . A threshold of A3 and A4) exerted more favorable antiviral profiles (SI = 98–199), which is attributed to their significantly lower cytotoxicity as compared to the reference compound pirodavir in four virus strain tests. Preliminary SAR studies suggested that 2-substitutedphenyl-isoindolin-1-one is an important pharmacophore for anti-EV-A71 activity. The small amino group on the isoindolin-1-one moiety was shown to be acceptable for antiviral potency. Lipophilic substitution with size restriction at the para-position of the phenyl group might be beneficial for maintaining antiviral activity. Although the EV-A71 H strain presented a weaker sensitivity to this class of compounds than the other strains, such as BrCr, Shenzhen98 and Jiangsu52, in this study, A3 treatment significantly decreased the viral VP1 expression at both the protein and RNA levels in a dose-dependent manner. Although the mechanism of action of this class of compounds is still undetermined, the time-of-addition experiment indicated that compound A3 played a role in the early stage of EV-A71 viral cycle, and presumably acts on the virus entry stage. In summary, 2-aryl-isoindolin-1-one is a promising scaffold with broad anti-EV-A71 activity for further study. On-going studies include detailed SAR, the mechanism of action and in vivo evaluation of compound A3 in an animal model.In this study, twenty-four 2-aryl-isoindolin-1-one analogs were designed and synthesized using the fragment-hopping strategy. Anti-EV-A71 activity was evaluated in Vero cell cultures infected by different EV-A71 clinical isolates. Ten of the 2-aryl-isoindolin-1-ones showed broad and significant antiviral activity at the submicromolar concentration level. 2--isoindolin-1-ones (


O-β-d-xylopyranoside (1), 28-hydroxy-foetidinol-3-O-β-d-xylopyranoside (2) and foetidinol-3-O-β-d-xylopyranosyl-(1”→3’)-β-d-xylopyranoside (3) together with the known compound foetidinol-3-O-β-d-xylopyranoside (4) were isolated from the n-BuOH fraction of the roots of Cimicifuga foetida. Their structures were elucidated on the basis of spectroscopic and chemical reaction data.Three new trinor-cycloartane glycosides, 15α-hydroxy-16-dehydroxy-16(24)-en-foetidinol-3- Cimicifuga species  as the crude drug source  suggested the presence of an α,β-unsaturated ketone group. The 13C-NMR , 71.3 d (C-4’), 75.6 d (C-2’), 78.7 d (C-3’), 107.6 d (C-1’)]. The trinor-triterpene structure included characteristic signals of five methyls , three oxygenated methines , an isolated olefinic bond  and an enone system . The 1H-NMR spectrum of 1 , 1.12 , 1.14 , 1.40  and 1.47 , cyclopropane methylene protons at δH 1.01  and 1.97 , three oxygenated methine protons at δH 3.59 , δH 4.63 , δH 5.36  and an olefinic hydrogen at δH 6.17 . The chemical shifts assignable to rings A, B and C were similar to those of foetidinol-3-O-β-d-xyloside - peak in the negative HRFABMS inferred a molecular formula of C32H48O10 . It has the same molecular formula as 15α-hydroxy-foetidinol-3-O-β-d-xyloside - peak in the negative HRESIMS corresponds to a molecular formula of C37H56O13 . The NMR data  and 5.27  in 1H-NMR spectrum indicated the existence of two pentoses in 3. The 13C-NMR data of the pentoses revealed the presence of xylose, and in the ROESY spectrum, H-1’ showed associations with H-3’ and H-5’ (δH 3.70 m) while H-1” showed associations with H-3” and H-5”. This was confirmed by acid hydrolysis and TLC comparison with an authentic sample. A linkage of the diglycoside of xylosyl-(1”→3’)-xylosyl to C-3 of the aglycone was proved by the HMBC correlations of H-3 with C-1’, C-4, C-29 and C-30; of H-1’ with C-3 and C-5’, of H-1” with C-3’, C-3” and C-5”; of H-2’, H-4’, 2H-5’ and H-1” with C-3’ and of H-2”, H-4” and 2H-5” with C-3” (3 was established as foetidinol-3-O-β-d-xylopyranosyl-(1”→3’)-β-d-xylopyranoside.Compound NMR data  of the aith C-3” . In sum,1H-, 13C-NMR and 2D-NMR spectra: Bruker AM-400 MHz or DRX-500 spectrometers with TMS as internal standard. MS: VG Autospec-300, Finnigan MAT-90 and API Qstar-Plusar-1 spectrometers. The pentose authentic samples were purchased from Acros Organics.Optical rotations: Horiba SEPA-300 polarimeter. UV spectra: Shimadzu UV-2401A spectrophotometer. IR spectra: Bio-Rad FTS-135 infrared spectrophotometer. C. foetida were collected in Daju Village of Lijiang County, Yunnan Province in July 2004 and identified by Prof. Pei Shengji . The voucher specimen (KIB 04072601) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.The roots of C. foetida (10 kg) were extracted three times with MeOH (25 L) under reflux. After removal of the solvent by evaporation, the residue (950 g) was suspended in H2O and partitioned sequentially with CHCl3 and n-BuOH. The n-BuOH fraction (250 g) was subjected to silica gel chromatography and eluted with CHCl3-MeOH  to give four fractions (Fr. A - D). Fr. B (20 g) was chromatographed repeatedly over RP-18 (45-70% MeOH-H2O) to successively yield compounds 2 (8 mg), 4 (200 mg), 1 (20 mg) and 3 (25 mg).Air-dried and powdered roots of d-xylopyranoside15 α-Hydroxy-16-dehydroxy-16(24)-en-foetidinol-3-O-β- (1): colorless needles; [α32H45O9, 573.3063). IR (KBr) cm-1: 3426 , 1671 (C=O); UV (MeOH) λmax (logε) nm: 282 (3.4), 321 (3.4); 1H- and 13C-NMR data, see d-xylopyranoside28-Hydroxyfoetidinol-3-O-β- (2): white powder, [α32H48O10, 573.3169). IR (KBr) cm-1: 3460 , 1724 (C=O), 1636 (C=C); 1H- and 13C-NMR data, see d-xylopyranosyl-(1”→3’)-β-d-xylopyranosideFoetidinol-3-O-β- (3): white powder, [α5H5N); HRESIMS . IR (KBr) cm-1: 3410 , 1724 (C=O), 1633 (C=C); 1H- and 13C-NMR data, see 1-3 (2 mg of each) were refluxed with 6% HCl-MeOH-n-BuOH  for 1 h at 90 °C, then neutralized with 12 M NaOH. The concentrated methanol soluble part showed a TLC spot  matching that of an authentic sample of d-xylose.Compounds C. foetida between 1994 and 1996, no such type of structure have been reported in past ten years. The new compounds found were trace constituents compared with foetidinol-3-O-β-d-xylopyranoside, and presumably are formed as side products or intermediates due to elimination, oxygenation or glycosylation reactions during the biosynthesis of this compound [C. foetida growing in this area a new resource of novel trinor-cycloartane structures.Since three trinor-cycloartanes were found for the first time in compound . They su


At the same time 4,5-dihydropyrazole-1-carbothioamide and 4,5-dihydropyrazole-1-isonicotinoyl derivatives were prepared from the reaction of chalcones with either thiosemicarbazide or isonicotinic acid hydrazide, respectively. The synthesized compounds were structurally characterized on the basis of IR, 1H-NMR, 13C-NMR spectral data and microanalyses. All of the newly isolated compounds were tested for their antimicrobial activities. The antimicrobial screening using the agar well-diffusion method revealed that the chloro derivatives are the most active ones. Moreover, the antioxidant and anti-inflammatory activity of these chloro derivatives are also studied using the DPPH radical scavenging and NO radical scavenging methods, respectively.The present work deals with the synthesis of acetoxysulfonamide pyrazole derivatives, substituted 4,5-dihydropyrazole-1-carbothioamide and 4,5-dihydropyrazole-1-isonicotinoyl derivatives starting from substituted vanillin chalcones. Acetoxysulfonamide pyrazole derivatives were prepared from the reaction of chalcones with  Pyrazole and its derivatives represent one of the most active classes of heterocyclic compounds possessing a wide spectrum of biological activities. In particular, they are used as antitumor , antibac3a–e and dihydropyrazoles 4a–e and 5a–e starting from chalcones 1a–e. which exhibit efficient antimicrobial, antioxidant and anti-inflammatory activities.Similarly, there has been a considerable interest in the chemistry of the 2-pyrazoline ring system which is a core structure in various synthetic pharmaceuticals with a broad spectrum of biological activities. Their pharmaceutical importance lies in the fact that they can be effectively utilized as antibacterial, potential antimicrobial , anti-in1a–e was accomplished by a one-pot Claisen-Schmidt condensation [trans double bond was obtained (on the basis of the corresponding NMR coupling constant). All synthesized compounds were characterized by spectral data  that was consistent with the proposed structures. The IR spectra of 1a–e revealed the characteristic bands for C=O at 1661–1684, vinyl CH=CH that appeared at 1593–1618 and OH at 3424–3441 cm−1. The 1H-NMR spectra showed the presence of a broad singlet at δ = 11.22–11.72 ppm for the phenolic OH proton, multiplets at δ = 7.30–7.97 characteristic of the aromatic protons, a doublet (J = 15 Hz) at δ = 7.22–7.29 ppm characteristic of the olefinic COCH=CH, another doublet (J = 15 Hz) at δ = 6.61–6.89 ppm characteristic of the olefinic COCH=CH. The methyl protons appeared as a singlet in the δ = 3.21–3.36 ppm range.The synthesis of chalcones ensation ,45 in 602a–e were obtained by treatment of 1a–e with p-sulphamylphenyl- hydrazine in glacial acetic acid. The IR spectra of 2a–e showed the characteristic bands for a vinyl CH=CH group at 1603–1619 cm−1, the phenolic OH in the 3430–3441 cm−1 range and a primary or secondary amine band at 3379–3391 and 3310–3350 cm−1, respectively. The 1H-NMR spectra showed the presence of a singlet at δ = 11.22–11.39 ppm for the OH proton, a singlet equivalent to one proton in the δ = 8.21–8.73 ppm range characteristic of a hydrazone NH proton, while the primary amine NH2 protons appeared at δ = 9.23–9.40 ppm. A multiplet at δ = 7.26–7.82 ppm is characteristic of the aromatic protons, while a doublet at δ = 7.53–7.61 ppm for N=C–CH=CH (J = 13 Hz), another doublet at δ = 6.69–6.85 ppm (J = 13 Hz) for N=C–CH=CH and a singlet equivalent to three protons at δ = 3.19–3.37 ppm are characteristic of the CH3 protons, respectively.The hydrazone derivatives 2a–e with acetic anhydride produced the pyrazole acetate derivatives 3a–e in good yields. The structures of 3a–e was confirmed by their IR, 1H-NMR and 13C-NMR spectra. The IR spectra of 3a–e showed the characteristic bands for C=N at 1632–1656 cm−1, ester carbonyl band at 1746–1752 cm−1and the NH2 band appeared at 3379–3393 and 3250–3330 cm−1. Also, the 1H-NMR spectra of 3a–e revealed the following signals: a singlet equivalent to two protons at δ = 9.33–10.37 ppm characteristic of NH2 protons, a multiplet at δ = 7.13–8.29 ppm characteristic for the aromatic protons, a singlet for the pyrazole C4-H at δ = 6.72–6.93 ppm, beside the presence of two singlets at δ = 2.22–2.39 ppm and δ = 3.21–3.33 ppm corresponding to the methyl protons of OCOCH3 and OCH3, respectively. Moreover, the 13C-NMR spectrum of 3d, as an example of this series, showed different characteristic signals at δ 20.7 (CH3), 56.0 (OCH3), 168.8 (C=N) and 189.3 (C=O).Reaction of 1a–e with either thiosemicarbazide or isonicotinic acid hydrazide in ethanol containing a few drops of acetic acid afforded the 4,5-dihydropyrazole derivatives 4a–e and 5a–e, respectively. The key reactions involved the intermediate formation of the hydrazones and subsequent addition of N-H on the olefinic bond of the propenone moiety that forms the ring-closed final products. The spectral data  of 4a–e and 5a–e were in full agreement with the proposed structures. The absence of the carbonyl (CO) and olefinic (C=C) bands in the IR spectra of the 4,5-dihydropyrazole derivatives 4a–e and 5a–e proved the ring closure of the final products. The IR spectra of 4a–e showed thiocarbonyl C=S stretching bands at 1232–1248 cm−1 and NH2 absorption bands at 3387–3394 cm−1, whereas the IR spectra of 5a–e showed an amide carbonyl stretching band at 1628–1634 cm−1. Also, the 1H-NMR spectra of either 4a–e or 5a–e, revealed the presence of a pair of doublets of doublets corresponding to the ring protons (HA and HB) of 4,5-dihydropyrazole. The CH protons (HX) appeared as doublets of doublets due to vicinal coupling with the two magnetically non-equivalent protons of the methylene group HA (up field shift of CH2) and HB (downfield shift of CH2) at position 4 of the dihydropyrazole ring . Moreover, the NH2 protons of the thiocarbamoyl group of the dihydropyrazoles 4a–e appeared at δ = 10.71–10.93 ppm, generally as broad bands. At the same time the 13C-NMR spectrum of 4b showed different signals at δ: 48.2, 70.6 (C3 and C4 dihydropyrazole), 56.2, 56.5 (2 OCH3), 169.4 (C=N), 177.8 (C=S) and that for 5c revealed the signals at δ: 40.3, 66.8 (C3 and C4 dihydropyrazole), 56.3 (OCH3), 164.3 (C=N), 188.4 (C=O). All the results are shown in Condensation of chalcones 1a–e, hydrazones 2a–e, pyrazoles 3a–e, and dihydropyrazoles 4a–e and 5a–e were tested for their antimicrobial activity against four test organisms, namely Staphylococcus aureus ATCC6538P, Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC9027, and Candida albicans ATCC2091 using rifampicin (5 μg/disc) and ampicillin (10 μg/disc) as standard drugs. The agar well-diffusion method [3a–e showed no significant effect, whereas all other compounds showed potent activity only against Staphylococcus aureus and Candida albicans in the following order: 2a–e > 4a–e > 1a–e ≥ 5a–e. Minimum inhibitory concentration (MIC) values for the individual compounds that showed inhibition zones > 10 mm were determined by means of the agar well-diffusion method in DMSO. The trend of activity was observed as follows: X > H > OMe > NO2 where X = Cl, Br. It is obvious that the presence of pharmacophores such as chloro and bromo substituents with lipophilic properties increases the antimicrobial activity. The activity results of our synthesized compounds against S. aureus, C. albicans are shown in All of our synthesized compounds, chalcones n method  was used1c, 2c, 4c and 5c which exhibited high activities. These results were listed in Also, minimum bactericidal concentrations (MBC) were determined for all the chloro derivatives 1c, 2c, 3c, 4c, 5c. These activities vary according to their structures and functional groups.Two pharmacological activities, namely antioxidant and anti-inflammatory activities, were tested for the chloro derivatives The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) has been widely used to evaluate the free radical scavenging capacity of different antioxidants ,48,49,50Resulting from a color change from purple to yellow, the absorbance decreased when the DPPH is scavenged by an antioxidant, through donation of hydrogen to form a stable DPPH molecule, in the radical form this molecule had an absorbance at 517 nm, which disappeared after acceptance of an electron or hydrogen radical from an antioxidant compound to form the reduced DPPH-R .2c was found to be the most efficacious antioxidant among all the listed compounds. The antioxidant activity of 2c is directly proportional to the concentrations used. Antioxidant results of the synthesized compounds 1c, 2c, 3c, 4c and 5c are reported in 4c > 5c > 3c > 1c, while converted to pro-oxidant compounds at higher concentrations.Moreover antioxidants are known to interrupt the free-radical chain of oxidation and to donate hydrogen from phenolic hydroxy groups, thereby, forming stable free radicals, which do not initiate further oxidation . Further1c, 2c, 3c, 4c, 5c were tested for in vitro anti-inflammatory activity compared to the standard vitamin C, showing acceptable anti-inflammatory activity. All tested compounds act as anti-inflammatory in a concentration dependent matter. Among all the tested compounds, 4c was the most potent compound, followed by: 5c > 3c > 1c > 2c. The in vitro anti-inflammatory activity of tested compounds is summarized in Nitric oxide (NO) is a potent inhibitor of physiological processes such as smooth muscle relaxation, neuronal signaling, and inhibition of platelet aggregation and regulation of cell mediated toxicity . In addimax) cm−1. 1H-NMR and 13C-NMR spectra were measured in deuterated chloroform (CDCl3) or deuterated dimethyl sulphoxide (DMSO-d6) on an EAC 500 MHz FT-NMR spectrophotometer . Chemical Shifts were recorded in δ as parts per million (ppm) downfield from tetramethylsilane (TMS) used as internal standard. Reaction progress and compound purity were monitored by Thin Layer Chromatography (TLC) using Alugram Sil G/UV254 silica gel plates  and chloroform or chloroform-ethanol (9:1) or (19:1) as eluent systems. The spots were visualized using an ultraviolet lamp  at λ = 254 and 266 nm. The antimicrobial activities were determined at the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University. Antioxidant and anti-inflammatory activity tests were performed at the Biochemistry Lab, Faculty of Science, Alexandria University.Melting points were determined in open capillary tubes using Electrothermal apparatus 9100  and are uncorrected. Microanalyses were operated at Faculty of Science, Cairo University, Cairo, Egypt, using an Elementary Vario El III C, H, N, S Analyzer . IR spectra were recorded using the potassium bromide method on a Tensor 37 FT-IR spectrometer : and expressed in wave number (υp-substituted acetophenone (10 mmol) was added to a cold stirred solution of sodium hydroxide (3 g in 50 mL of 60%aqueous ethanol) followed by addition of vanillin (10 mmol) dropwise with continuous stirring for five hours. The resulting crude solid was filtered, washed successively with water, dried and crystallized from ethanol (95%) [The appropriate ol (95%) ,59,60,613-(4-Hydroxy-3-methoxyphenyl)-1-(phenyl)prop-2-en-1-one (1a). The chalcone was obtained in 73% yield; m.p. 93 °C; IR : 3426 (OH), 1666 (C=O), CH=CH (1593); 1H-NMR (DMSO-d6): δ 3.35 , 6.88  , 7.26 , 7.34–7.86 , 11.32 ; Anal. Calcd. for C16H14O3: C, 75.57; H, 5.55. Found: C, 75.42; H, 5.97.3-(4-Hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl) prop-2-en-1-one (1b). The chalcone was obtained in 68% yield; m.p. 117 °C; IR : 3441 (OH), 1661 (C=O), CH=CH (1502); 1H-NMR (DMSO-d6): δ 3.21 , 3.33 , 6.72 , 7.25 , 7.32–7.85 , 11.44 ; Anal. Calcd for C17H16O4: C, 71.82; H, 5.67. Found: C, 72.00; H, 5.61.1-(4-Chlorophenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (1c). The chalcone was obtained in 77% yield; m.p.105 °C; IR : 3433 (OH), 1664 (C=O), CH=CH (1614); 1H-NMR (DMSO-d6): δ 3.31 , 6.61 , 7.28 , 7.33–7.87 , 11.44 ; Anal. Calcd for C16H13ClO3: C, 66.56; H, 4.54. Found: C, 66.41; H, 4.57.1-(4-Bromophenyl)-3-(4-hydroxy-3-methoxyphenyl) prop-2-en-1-one (1d). The chalcone was obtained in 82% yield; m.p. 97 °C; IR : 3429 (OH), 1672 (C=O), CH=CH (1606); 1H-NMR (DMSO-d6): δ 3.36 , 6.89 , 7.22 , 7.30–7.74 , 11.22 ; Anal. Calcd for C16H13BrO3: C, 57.68; H, 3.93. Found: C, 57.72; H, 3.82.3-(4-Hydroxy-3-methoxyphenyl)-1-(4-nitrophenyl) prop-2-en-1-one (1e). The chalcone was obtained in 85% yield; m.p. 95 °C; IR : 3424 (OH), 1684 (C=O), CH=CH (1618); 1H-NMR (DMSO-d6): δ 3.30 , 6.89 , 7.29 , 7.36–7.97 , 11.72 ; Anal. Calcd. for C16H13NO5: C, 64.21; H, 4.38; N, 4.68. Found: C, 64.19; H, 4.31; N, 4.69.1a–e (10 mmol) in ethanol  was refluxed with the appropriate amount of p-sulphamylphenylhydrazine (10 mmol) in glacial acetic acid (2 mL) for six hours. The reaction mixture was poured into crushed ice and kept overnight at room temperature. The separated crude solid was filtered off, washed successively with water, dried and crystallized from ethanol (95%) to give 2a–e as needles.A solution of chalcone 4-(2-(3-(4-Hydroxy-3-methoxyphenyl)-1-phenylallylidene)hydrazinyl)benzenesulfonamide (2a). The hydrazone was obtained in 67% yield; m.p. 147 °C; IR : 3430 (OH), 3391, 3322 , 1632 (C=N), 1603 (CH=CH); 1H-NMR (DMSO-d6): δ 3.33 , 6.85 , 7.54 , 7.33–7.82 , 8.21 , 9.23 , 11.22 . Anal. Calcd. for C22H21N3O4S: C, 62.40; H, 5.00; N, 9.92. Found: C, 62.47; H, 4.89; N, 9.88.4-(2-(3-(4-Hydroxy-3-methoxyphenyl)-1-hydrazinyl)benzenesulfonamide (2b). The hydrazone was obtained in 90% yield; m.p. 160 °C; IR : 3432 (OH), 3389, 3320 , 1629 (C=N), 1609 (CH=CH); 1H-NMR (DMSO-d6): δ 3.22 , 3.26 , 6.83 , 7.53 , 7.31–7.81 , 8.46 , 9.29 , 11.31 . Anal. Calcd. for C23H23N3O5S: C, 60.91; H, 5.11; N, 9.27. Found: C, 60.87; H, 5.08; N, 9.23.4-(2-(1-(4-Chlorophenyl)-3-hydrazinyl)benzenesulfonamide (2c). The hydrazone was obtained in 87% yield; m.p. 103 °C; IR : 3439 (OH), 3382, 3313 , 1626 (C=N), 1612 (CH=CH); 1H-NMR (DMSO-d6): δ 3.22 , 6.69 , 7.56 , 7.36–7.61 , 8.54 , 9.31 , 11.33 . Anal. Calcd. for C22H20ClN3O4S: C, 57.70; H, 4.40; N, 9.18. Found: C, 57.76; H, 4.35; N, 9.22.4-(2-(1-(4-Bromophenyl)-3-hydrazinyl)benzenesulfonamide (2d). The hydrazone was obtained in 93% yield; m.p. 110 °C; IR : 3435 (OH), 3383, 3314 , 1633 (C=N), 1604 (CH=CH); 1H-NMR (DMSO-d6): δ 3.19 , 6.81 , 7.54 , 7.41–7.71 , 8.61 , 9.39 , 11.39 . Anal. Calcd. for C22H20BrN3O4S: C, 52.60; H, 4.01; N, 8.36. Found: C, 52.61; H, 3.89; N, 8.42.4-(2-(3-(4-Hydroxy-3-methoxyphenyl)-1-hydrazinyl)benzenesulfonamide (2e). The hydrazone was obtained in 79% yield; m.p. 135 °C; IR : 3441 (OH), 3379, 3310 , 1627 (C=N), 1619 (CH=CH); 1H-NMR (DMSO-d6): δ 3.37 , 6.79 , 7.61 , 7.26–7.76 , 8.73 , 9.40 , 11.38 . Anal. Calcd. for C22H20N4O6S: C, 56.40; H, 4.30; N, 11.96. Found: C, 56.39; H, 4.31; N, 11.95.2a–e (10 mmol) and acetic anhydride (15 mL) was heated under reflux for three hours. After the reaction mixture attained room temperature, it was poured into crushed ice and the oily product deposited was decanted from water and extracted with ether. The ether layer was washed three times with water, dried over anhydrous sodium sulphate and evaporated. The precipitate obtained was crystallized from ethanol (95%) to afford the corresponding pyrazoles 3a–e as needles.A mixture of the appropriate hydrazone 2-Methoxy-4-(3-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazol-5-yl)phenyl acetate (3a). The pyrazole was obtained in 69% yield; m.p. 187 °C; IR : 3386 (NH2), 1750 (C=O), 1632 (C=N); 1H-NMR (DMSO-d6): δ 2.39 , 3.33 , 6.72 , 7.24–8.29 , 9.33 . Anal. Calcd. for C24H21N3O5S: C, 62.19; H, 4.57; N, 9.07. Found: C, 62.19; H, 4.40; N, 9.09.2-Methoxy-4-(3-(4-methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-5-yl)phenyl acetate (3b). The pyrazole was obtained in 91% yield; m.p. 193 °C; IR : 3384 (NH2), 1751 (C=O), 1641 (C=N); 1H-NMR (DMSO-d6): δ 2.28 , 3.21 , 3.34 , 6.87 , 7.35–7.94 , 9.76 . Anal. Calcd. for C25H23N3O6S: C, 60.84; H, 4.70; N, 8.51. Found: C, 60.75; H, 4. 68; N, 8.59.4-(3-(4-Chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-5-yl)-2-methoxyphenyl acetate (3c).The pyrazole was obtained in 76% yield; m.p. 172 °C; IR : 3379 (NH2), 1748 (C=O), 1644 (C=N); 1H-NMR (DMSO-d6): δ 2.22 , 3.27 , 6.83 , 7.29–7.86 , 9.92 . Anal. Calcd. for C24H20ClN3O5S: C, 57.89; H, 4.05; N, 8.44. Found: C, 57.80; H, 4.10; N, 8.40.4-(3-(4-Bromophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-5-yl)-2-methoxyphenyl acetate (3d). The pyrazole was obtained in 78% yield; m.p. 177 °C; IR : 3391 (NH2), 1746 (C=O), 1654 (C=N); 1H-NMR (CDCl3): δ 2.30 , 3.31 , 6.85 , 7.13–7.64 , 9.71 ; 13C-NMR (CDCl3): δ 20.7 (CH3), 56.0 (OCH3), 111.9, 121.5, 121.8, 123.4, 128.0, 130.1, 132.0, 133.7, 136.9, 140.4, 141.8, 142.2, 144.8, 151.5 , 168.8 (C=N) and 189.3 (C=O). Anal. Calcd. for C24H20BrN3O5S: C, 53.14; H, 3.72; N, 7.75. Found: C, 53.10; H, 3.66; N, 7.80.2-Methoxy-4-(3-(4-nitrophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-5-yl)phenyl acetate (3e). The pyrazole was obtained in 69% yield; m.p.197 °C; IR : 3393 (NH2), 1752 (C=O), 1656 (C=N); 1H-NMR (DMSO-d6): δ 2.26 , 3.33 , 6.93 , 7.29–8.10 , 10.37 . Anal. Calcd. for C24H20N4O7S: C, 56.69; H, 3.96; N, 11.02. Found: C, 56.72; H, 3.95; N, 11.10.1a–e (10 mmol) in ethanol (30 mL) was heated under reflux with thiosemicarbazide (12 mmol) in glacial acetic acid (2 mL) for 7 h, then the reaction mixture was poured into crushed ice and kept overnight at room temperature. The separated crude solid was filtered off, washed successively with water, dried and crystallized from ethanol/chloroform to give 4a–e as needles.A mixture of the appropriate chalcone 5-(4-Hydroxy-3-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4a). The 4,5-dihydropyrazole was obtained in 81% yield; m.p. 149 °C; IR : 3430 (OH), 3394 (NH2), 1647 (C=N), 1246 (C=S); 1H-NMR (DMSO-d6): δ 3.11 , 3.31 , 3.72 , 5.39 , 7.26–7.82 , 10.73 , 11.21 . Anal. Calcd. for C17H17N3O2S: C, 62.36; H, 5.23; N, 12.83. Found: C, 62.33; H, 5.20; N, 12.88.5-(4-Hydroxy-3-methoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4b). The 4,5-dihydropyrazole was obtained in 83% yield; m.p. 156 °C; IR : 3432 (OH), 3390 (NH2), 1650 (C=N), 1245 (C=S); 1H-NMR (DMSO-d6): δ 3.20 , 3.32 , 3.39 , 3.75 , 5.39 , 7.27–7.77 , 10.73 , 11.30 ; 13C-NMR (DMSO-d6): δ 48.2, 70.6 (C3 and C4 dihydropyrazole), 56.2, 56.5 (2 OCH3), 109.7, 113.2, 115.7, 119.4, 122.9, 126.0, 134.3, 143.4, 148.6, 149.2 (C6H3 and C6H4 C), 169.4 (C=N) and 177.8 (C=S). Anal. Calcd. for C18H19N3O3S: C, 60.49; H, 5.36; N, 11.76. Found: C, 60.50; H, 5.34; N, 11.81.3-(4-Chlorophenyl)-5-(4-hydroxy-3-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4c). The 4,5-dihydropyrazole was obtained in 87% yield; m.p. 172 °C; IR : 3439 (OH), 3390 (NH2), 1652 (C=N), 1248 (C=S); 1H-NMR (DMSO-d6): δ 3.08 , 3.27 , 3.79 , 5.44 , 7.29–8.01 , 10.79 , 11.33 . Anal. Calcd. for C17H16ClN3O2S: C, 56.43; H, 4.46; N, 11.61. Found: C, 56.33; H, 4.42; N, 11.71.3-(4-Bromophenyl)-5-(4-hydroxy-3-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4d). The 4,5-dihydropyrazole was obtained in 79% yield; m.p. 146 °C; IR : 3440 (OH), 3393 (NH2), 1657 (C=N), 1247 (C=S); 1H-NMR (DMSO-d6): δ 3.12 , 3.29 , 3.76 , 5.46 , 7.23–7.61 , 10.93 , 11.27 . Anal. Calcd. for C17H16BrN3O2S: C, 50.25; H, 3.97; N, 10.34. Found: C, 50.23; H, 3.89; N, 10.37.5-(4-Hydroxy-3-methoxyphenyl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4e). The 4,5-dihydropyrazole was obtained in 93% yield; m.p.: 166 °C; IR : 3451 (OH), 3387 (NH2), 1646 (C=N), 1232 (C=S); 1H-NMR (DMSO-d6): δ 3.07 , 3.32 , 3.74 , 3.39 , 7.27–7.99 , 10.71 , 11.24 . Anal. Calcd. for C17H16N4O4S: C, 54.83; H, 4.33; N, 15.04. Found: C, 54.87; H, 4.33; N, 15.08.1a–e (10 mmol) in ethanol (95%) (30 mL) was heated under reflux with isonicotinic acid hydrazide (10 mmol) in glacial acetic acid (2 mL) for five hours. The reaction mixture was treated as mentioned for the preparation of 4a–e to give the corresponding 4,5-dihydropyrazoles 5a–e.A mixture of the appropriate chalcone (5-(4-Hydroxy-3-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (5a). The corresponding 4,5-dihydropyrazole was obtained in 81% yield; m.p. 159 °C; IR : 3472 (OH), 1645 (C=N), 1630 (C=O); 1H-NMR (DMSO-d6): δ 3.14 , 3.32 , 3.84 , 5.40 , 6.81–7.48 , 11.27 . Anal. Calcd. for C22H19N3O3: C, 70.76; H, 5.13; N, 11.25. Found: C, 70.77; H, 5.12; N, 11.27.(5-(4-Hydroxy-3-methoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (5b). The corresponding 4,5-dihydropyrazole was obtained in 69% yield; m.p. 166 °C; IR : 3492 (OH), 1649 (C=N), 1628 (C=O); 1H-NMR (DMSO-d6): δ 3.16 , 3.25 , 3.31 , 3.75 , 5.37 , 6.79–7.49 , 11.31 . Anal. Calcd. for C23H21N3O4: C, 68.47; H, 5.25; N, 10.42. Found: C, 68.51; H, 5.26; N, 10.48.(3-(4-Chlorophenyl)-5-(4-hydroxy-3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (5c). The corresponding 4,5-dihydropyrazole was obtained in 89% yield; m.p. 142 °C; IR : 3481 (OH), 1655 (C=N), 1631 (C=O); 1H-NMR (DMSO-d6): δ 3.18 , 3.27 , 3.80 , 5.46 , 6.83–7.51 , 11.22 ; 13C-NMR (DMSO-d6): δ 40.3, 66.8 (C3 and C4 dihydropyrazole), 56.3 (OCH3), 110.6, 116.1, 118.7, 123.5, 124.8, 129.1, 130.8, 137.1 138.3, 146.0, 146.5, 148.5, 150.2 , 164.3 (C=N) and 188.4 (C=O) . Anal. Calcd. for C22H18ClN3O3: C, 64.79; H, 4.45; N, 10.30. Found: C, 64.69; H, 4.45; N, 10.29.(3-(4-Bromophenyl)-5-(4-hydroxy-3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (5d). The 4,5-dihydropyrazole was obtained in 86% yield; m.p. 168 °C; IR : 3466 (OH), 1654 (C=N), 1633 (C=O); 1H-NMR (DMSO-d6): δ 3.20 , 3.22 , 3.77 , 5.47 , 6.82–7.52 , 11.20 . Anal. Calcd. for C22H18BrN3O3: C, 58.42; H, 4.01; N, 9.29. Found: C, 58.31; H, 3.89; N, 9.28.(5-(4-Hydroxy-3-methoxyphenyl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl) methanone (5e). The 4,5-dihydropyrazole was obtained in 97% yield; m.p. 171 °C; IR : 3471 (OH), 1649 (C=N), 1634 (C=O); 1H-NMR (DMSO-d6): δ 3.17 , 3.39 , 3.69 , 5.42 , 6.84–7.71 , 11.89 . Anal. Calcd. for C22H18N4O5: C, 63.15; H, 4.34; N, 13.39. Found: C, 63.14; H, 4.28; N, 13.47.Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans. The agar well-diffusion method was applied for the determination of inhibition zone and minimum inhibitory concentration (MIC). Briefly, 0.75 mL of broth culture containing ca. 106 colony-forming units (CFU) per mL of the test strain was added to 75 mL of nutrient agar medium at 45 °C, mixed well, and then poured into a 15 cm sterile metallic Petri plate. The medium was allowed to solidify, and 8 mm wells were dug with a sterile metallic borer. Then, a DMSO solution of the test sample (1 mL) at 1 mg/mL was added to the respective wells. DMSO served as negative control, and the standard antimicrobial drugs Rifampicin (5 μg/disc) and Ampicillin (10 μg/disc) were used as positive controls. Triplicate plates for each microorganism strain were prepared and were incubated aerobically at 37 °C for 24 h. The activity was determined by measuring the diameter of zone showing complete inhibition (mm), thereby, the zones were precisely measured with the aid of a Vernier caliper (precision 0.1 mm). The growth inhibition was calculated with reference to the positive control. For the individual compounds that showed inhibition zones >10 mm, MIC values were determined by means of the agar well-diffusion method for concentrations of 1.0, 0.50, 0.25, 0.125, 0.063 and 0.031 mg/mL in DMSO. The tests were performed in triplicate, and the results were averaged. Also minimum bactericidal concentrations (MBC) were determined for all chloro derivatives which exhibited high activities  for concentrations of 1.0, 0.50, 0.25, 0.125, 0.063 and 0.031 mg/mL in DMSO. All our results are listed in All compounds were tested against four different microorganisms Since DPPH is a stable free radical containing an odd electron in its structure, it is usually utilized for detection of the radical scavenging activity. Aliquots of different concentrations (20–100 µg/mL) of the test sample is added to 100 µL solution DPPH (4 mg/100 mL methanol). Absorbance at 517 nm is determined after 30 min. Each experiment was done in triplicate and average is taken. Vitamin C was used as a positive control and percentage of free radical scavenging was expressed as inhibition from the given formula:Efficacy was calculated for 0.25 mg/mL of each compound by using the following equation:Calculated antioxidant data of all the tested samples were summarized in Nitric oxide was generated from sodium nitroprusside and measured by Griess’ reaction ,63. ReagEfficacy was calculated for 0.25 mg/mL of each compound by using the following equations:Calculated anti-inflammatory data of all the tested samples were summarized in .1H-NMR and 13C-NMR) and the structures were consistent with the data. All the synthesized compounds were tested for their antimicrobial activity against four test organisms. The results showed that the compounds that having pharmacophores with lipophilic properties such as chloro and bromo substituents exhibited the greatest antimicrobial activities. Also two pharmacological activities namely antioxidant and anti-inflammatory activity, were tested for the chloro derivatives 1c, 2c, 3c, 4c, 5c. These activities vary according to their structures and functional groups.This work demonstrates a rapid, efficient method for the synthesis of new pyrazole and dihydropyrazole derivatives. All synthesized compounds were characterized by spectral data (IR,


In the alternative procedure, the di-oxidized product 6, obtained in the oxidation of methyl hyodeoxycholate 5, was converted almost quantitatively into the target monoketone 7 by stereoselective reduction with NaBH4, increasing the overall yield of this synthetic route to 96.8%. The complete 1H- and 13C-NMR assignments for all compounds synthesized in this work have been made by 1D and 2D heteronuclear correlation gs-HSQC and gs-HMBC techniques. Thus, it was possible to update the spectroscopic information of 1H-NMR and to accomplish a complete assignment of all 13C-NMR signals for analogues 5–16, which were previously reported only in partial form.An improved synthesis route for obtaining known brassinosteroid analogues, i.e., methyl 2α,3α-dihydroxy-6-oxo-5α-cholan-24-oate ( It has steroids , whereassteroids .4), and maintaining the native side chain. Additionally, the full NMR spectroscopic characterization of these derivatives is performed.In this work, we report an improved synthesis route to obtain known brassinosteroid analogues, starting from hyodeoxycholic acid (4) with the H2SO4-MeOH system gave the methyl hyodeoxycholate (5) in almost quantitative yield (98%). Subsequently, selective oxidation of the C-6 hydroxyl group with PCC/CH2Cl2 afforded the di- and mono-oxidized compounds 6 and 7 with 30% and 68% yield, respectively. These yields are similar to those reported when PDC/CH2Cl2 was the oxidizing agent [7 was 67%–70% when K2CrO4 was used as an oxidizing agent [6 was conveniently converted to the desired monoketone 7 by selective reduction with NaBH4/MeOH at a low temperature (0–5 °C) with 96.8% yield  of compounds 6 and 7 were consistent with those previously reported [13C-NMR spectroscopic data for compound 7 has been informed but not assigned [Fischer esterification of hyodeoxycholic acid  under acid conditions (2.5% HCl/MeOH) to give the derivative 8 possessing 5α-cholestan-6-one skeleton [8, an additional esterification reaction was carried out with 94% yield. The physical properties, IR, and 1H-NMR spectroscopic data of compound 8 have been previously reported, but the signals of 13C-NMR were not assigned [cis for trans fusion) implies a very important structural modification, and therefore a detailed analysis of NMR spectroscopic data for compounds 7 and 8 must be performed. The main differences found in the 1H- and 13C-NMR data are shown in H = 4.16 and δH = 2.70 ppm, respectively) in compound 8, are displaced to downfield as compared to compound 7 . On the other hand, the signal of CH3-19 in the 13C-NMR of compound 8 appears at a higher field (δC = 12.29 ppm) than in the spectrum of compound 7 (δC = 23.16 ppm).Compound skeleton ,32. Thisskeleton . Thus, t8 with methanesulfonyl chloride (mesyl chloride) in CH2Cl2/DMAP produces the mesylated derivative 9 with 61% yield [1H-NMR spectroscopic evidence for formation of this derivative is the presence of signals at δH = 5.02 and 2.97 ppm assigned to hydrogen H-3β (b.s. 1H) and CH3-SO2- , respectively, whereas in the 13C-NMR spectrum, a signal corresponding to CH3-SO2 was observed at δC = 38.38 ppm. Subsequent treatment of compound 9 with the Li2CO3/LiBr system at 80 °C in dimethylformamide (DMF) produces the alquene 10 with 74.9% yield [1H-NMR spectroscopic data of alquene 10 was consistent with that previously reported [13C-NMR spectrum the signals at δC = 124.49 (C-2) and 124.95 (C-3) ppm are observed. Both signals were assigned from the 2D HMBC spectrum, where the H-3  showed 3JH-C correlation with C-1 (δC = 39.34 ppm) and 2JH-C with C-4 (δC = 27.88 ppm), while H-2  showed 3JH-C correlation with C-4 (δC = 27.88 ppm) and 2JH-C with C-1 (δC = 39.34 ppm).The reaction of compound 1% yield . The maj9% yield   in a mixture of (CH3)2CO-H2O (3:1) and 2.0 mL of pyridine, and in the presence of N-methylmorpholine N-oxide [1H- and 13C-NMR spectroscopic data of diol 11 were consistent with those reported [J = 11.1 Hz, 1H) showed a long-range spatial correlation with CH3-19 , while the observed signals at δC = 68.29 and 68.39 ppm in the 13C-NMR spectrum were assigned to C-2 and C-3, respectively.Compound  N-oxide . The 1H-reported ,32. In a8 was acetylated under standard conditions (Ac2O/DMAP/CH2Cl2), and commercial compound 12 was obtained with 95% yield. The synthesis of this derivative has been previously reported, but no NMR spectroscopic data was included [1H-NMR spectrum of the acetylated derivative 12 shows a singlet at δH = 2.04 ppm  and a multiplet at δH = 5.12 ppm corresponding to H-3β . The latter is shifted to downfield as compared with H-3β  in compound 8. Additionally, in the 13C-NMR spectrum the signals appearing at δC = 170.31 (C=O) and 21.39 (CH3CO) ppm  ppm  confirme13 by saponification of 8 with NaOH/dioxane under reflux conditions has been previously informed [8 under mild conditions  produces the carboxylic acid 13 with similar yields (82.2% yield)  (2O/DMAP/CH2Cl2) of 13 produces compound 14 with 72.8% yield. Compound 14 showed signals at 3373–2495 (OH), 1736 (C=O) and 1708 (C=O) cm−1 in the IR spectrum, while in 1H-NMR the signals observed at δH = 5.12 ppm  and 2.03 ppm  were assigned to the H-3β and CH3CO, respectively. In the 13C-NMR spectrum the observed signals at δC = 170.33 and 21.41 ppm (C = 179.20 ppm (14.The synthesis of compound informed . Saponif% yield) . The phyreported . The 13Cpm (C=O) , confirm1.41 ppm  were ass9.20 ppm  was assi8 with m-CPBA/CH2Cl2 gave a mixture of known 7-oxalactone 15 and 6-oxalactone 16 with 10.8% and 14.9% yields, respectively. The physical and IR, 1H-NMR spectroscopic data of 15 and 16 were consistent with those reported for these compounds [1H-NMR spectra were recorded with a 60 MHz spectrometer, and no 13C-NMR data was reported, we believe that it is worth updating the analysis of NMR data for these compounds.The Baeyer-Villiger oxidation of 3α-hydroxy-6-oxo-5α-cholanate ompounds . Conside15 and in particular the 7-oxalactone position was mainly assigned by 1H, 13C, 2D HSQC and 2D HMBC NMR spectroscopy. In the 1H-NMR spectrum of compound 15 appears a signal at δH = 4.08–4.06 ppm , which was assigned to the two hydrogens H-7 and correlated by 2D 1H-13C HSQC with the signal δC = 70.44 ppm (C-7) with pair multiplicity . Additionally, from the 2D 1H-13C HMBC spectrum important heteronuclear correlations were observed, i.e., 2JHC correlations between H-5α  with the signals at δC = 176.76 (C=O of lactone function), δC = 36.29 and δC = 30.81 ppm which were assigned to the carbons C-6, quaternary C-10 and C-4, respectively. Additionally, H-5α showed 3JHC correlation with signals at δC = 30.81, δC = 14.52, δC = 32.88 and δC = 58.35 ppm, which were assigned to the carbons C-4, CH3-19, C-1 and C-9, respectively  was observed, correlated by 2D 1H-13C HSQC with the signal at δC = 79.65 ppm (C-7) with impair multiplicity  and assigned to H-5α. Additionally, H-5 α showed 2JHC correlation with signals at δC = 35.63 ppm and 3JHC correlation with signals at δC = 11.53, δC = 57.97 and δC = 175.35 ppm, which were assigned to the carbons C-4, CH3-19, C-9 and C-6 , respectively , operating at 400.1 MHz for 1H and 100.6 MHz for 13C. Chemical shifts are reported in δ ppm and coupling constants (J) are given in Hz. IR spectra were recorded as KBr disks in a FT-IR Nicolet 6700 spectrometer  and frequencies are reported in cm−1. For analytical TLC, silica gel 60 (Merck) in 0.25 mm layer was used and TLC spots were detected by heating after spraying with 25% H2SO4 in H2O. Chromatographic separations were carried out by conventional column on silica gel 60  using EtOAc-hexane gradients of increasing polarity. All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure, below 40 °C.All reagents were purchased from commercial suppliers , and used without further purification. Melting points were measured on a Stuart-Scientific SMP3 apparatus  and are uncorrected. Methyl 3α, 6α-dihydroxy-5β-cholan-24-oate (5). A solution of 4  in MeOH (150 mL) and 1.0 mL of H2SO4 was refluxed for 2.0 h. The end of reaction was verified by TLC. Then the solvent was removed (until a 40 mL approximate volume) and diluted with EtOAc (90 mL). The organic layer was washed with saturated solution of NaHCO3 (40 mL) and water (2 × 30 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (15 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 7.6:2.4). Compound 5 (19.50 g 98% yield) was a colorless solid ; IR (cm−1): 3385 (O-H); 2938 (C-CH3); 2858 (C-CH2-C); 1743 (C=O); 1452 (CH2); 1376 (CH3); 1169 (C-O). 1H-NMR: 4.05 ; 3.66 ; 3.62 ; 2.35 ; 2.21 ; 0.91 ; 0.90 ; 0.63 . 13C-NMR: See Methyl 3, 6-dioxo-5β-cholan-24-oate (6) and Methyl 3α-hydroxy-6-oxo-5β-cholan-24-oate (7). To a solution of 5  in DCM (100 mL), 8.75 g, (40.58 mmol) of PCC in 60 mL of DCM, were added by slow dripping. The reaction mixture was slowly stirred for 48 h at room temperature and the end of reaction was verified by TLC, filtered on silica  with DCM 30 mL. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 5.8:4.2). Two fractions were obtained: Fraction I, 4.95 g (30.0% yield) of compound 6; Fraction II, 11.2 g (68% yield) of compound 7. Compound 6 was a colorless solid . IR (cm−1): 2947 (C-CH3); 2874 (C-CH3); 1743 (C=O); 1717 (C=O); 1689 (C=O); 1469 (CH2); 1383 (CH3); 1164 (C-O). 1H-NMR: 3.64 ; 2.63 ; 0.93 ; 0.91 ; 0.67 . 13C-NMR: See 7 was a colorless solid . IR (cm−1): 3301 (O-H); 2943 (C-CH3); 2867 (C-CH2-C); 2849 (C-CH2-C); 2828 (CH3O); 1740 (C=O); 1709 (C=O); 1436 (CH2); 1326 (CH3); 1171 (C-O). 1H-NMR: 3.66 ; 3.62 ; 2.33 ; 0.92 ; 0.83 ; 0.64 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Methyl 3α-hydroxy-6-oxo-5β-cholan-24-oate (7). A solution of compound 6  was prepared in 100 mL of MeOH. This solution was placed in a bath of ice-water between 0–5 °C. Subsequently 0.47 g (12.3 mmol) of NaBH4 were added in four portions (approximately 0.118 g each) maintaining the temperature and with slow stirring. The end of reaction was verified by TLC, so 20 mL of acetone and 5 mL of HCl 2.5% were added, maintaining the reaction temperature. The reaction mixture was concentrated by evaporation under reduced pressure to a volume of about 15 mL, and then AcOEt (50 mL) was added. The organic layer was washed with saturated solution of NaHCO3 (20 mL) and water (2 × 30 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 5.8:4.2) Two fractions were obtained: Fraction I, 2.88 g of unreacted compound 6; Fraction II, 2.04 g (41.2%) of compound 7. Later reduction with NaHB4/CH3OH at 0–5 °C of recovered compound 6 (2.88 g), produced 2.75 g of 7 with 95.5% yield. The total yield in both reductions for compound 7 was 96.8%. The physical and spectroscopic properties of compound 7 were identical to those reported above for oxidation of compound 5. 1H-NMR and 13C-NMR are shown in Methyl 3α-hydroxy-6-oxo-5α-cholan-24-oate (8). Compound 7  was dissolved in 100 mL of 2.5% v/v HCl-MeOH, at room temperature and constant agitation for 24 h. The end of reaction was verified by TLC. The solvent was evaporated under reduced pressure and the crude was re-dissolved in 40 mL of AcOEt. The organic layer was washed with saturated solution of NaHCO3 (30 mL) and water (2 × 30 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 6.0:4.0). Compound 8  was a colorless solid  IR (cm−1): 3302 (O-H); 2943 (C-CH3); 2867 (C-CH2-C); 2849 (C-CH2-C); 2828 (CH3O); 1740 (C=O); 1709 (C=O); 1436 (CH2); 1375 (CH3); 1257 (C-O); 1172 (C-O). 1H-NMR: 4.16 ; 3.66 ; 2.70 ; 2.33 ; 0.91 ; 0.72 ; 0.65 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Methyl 3α-methanesulfonyl-6-oxo-5α-cholan-24-oate (9). Compound 8  was dissolved in 20 mL of DCM, 5 mg of DMAP and 1 mL of pyridine were added, then the mixture was cooled to 0–5 °C. Later 1.0 mL (12.9 mmol) of CH3SO2Cl slowly was added with gentle agitation. The mixture was kept at 0 °C for 30 min and subsequently left at room temperature. The end of reaction was verified by TLC. The solvent was evaporated under reduced pressure and the crude was re-dissolved in 20 mL of AcOEt. The organic layer was washed with saturated solution of NaHCO3 (40 mL) and water (2 × 30 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 5.8:4.2). Two fractions were obtained: Fraction I, 726 mg (61.0% yield) of compound 9, and Fraction II, 230 mg of unreacted compound 8. Compound 9 was a colorless solid  IR (cm−1): 2945 (C-CH3); 2867 (C-CH2-C); 1736 (C=O); 1709 (C=O); 1435 (CH2); 1351 (CH3); 1255 (C-O); 1171 (C-O). 1H-NMR: 5.02 ; 3.64 ; 2.97 ; 2.60 ; 0.901 ; 0.713 ; 0.639 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Methyl 2-en-6-oxo-5α-cholan-24-oate (10). To a solution of compound 9  in 40 mL of DMF, 200 mg Li2CO3 (2.71 mmol) and LiBr  were added. Then the reaction mixture was stirred and refluxed at 80 °C for 1.5 h. The end of reaction was verified by TLC, the mixture was then filtered and the mother liquor concentrated to a volume of approximately 15 mL under reduced pressure. Then AcOEt (30 mL) were added. The organic layer was washed with water (2 × 20 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 2.4:7.6). Compound 10  was a colorless solid  IR (cm−1): 3020 (CH=); 2968 (CH3-); 2939 (CH3-); 2899 (C-CH2-C); 2867 (C-CH2-C); 1737 (C=O); 1703 (C=O); 1635 (C=C); 1435 (CH2); 1383 (CH3); 1250, (C-O); 1168 (C-O). 1H-NMR: 5.69–5.66 ; 5.57–5.55 ; 3.66 ; 0.924 ; 0.701 ; 0.665 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Methyl 2α,3α-dihydroxy-6-oxo-5α-cholan-24-oate (11). A solution of compound 10  in 30 mL of acetone and 10 mL water was prepared. Later 10 mg of NMMNO, 2 mL 4.0%  OsO4 solution and 2 mL of pyridine were added. The mixture was kept under constant stirring at room temperature for 72 h. The end of reaction was verified by TLC, and then 25 mL of Na2S2O3 saturated solution were added and allowed to stir for 30 min. The reaction was concentrated to a volume of approximately 15 mL under reduced pressure. Then AcOEt (40 mL) were added. The organic layer was washed with water (2 × 25 mL), dried over anhydrous Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 8.2:1.8). Two fractions were obtained: Fraction I, 218 mg of unreacted compound 10 and Fraction II, 277 mg (51.0% yield) of compound 11. Compound 11 was a colorless solid . IR (cm−1): 3392 (OH); 2944 (CH3-); 2867 (C-CH2-C); 1738 (C=O); 1709 (C=O); 1435 (CH2); 1376 (CH3); 1255, C-O); 1170 (C-O). 1H-NMR: 4.05 ; 3.77 ; 3.66 ; 2.67 ; 0.926 ; 0.752 ; 0.659 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Methyl 3α-acetoxy-6-oxo-5α-cholan-24-oate (12). To a solution of compound 8  in 20 mL of DCM, 5 mg of DMAP, 1 mL of pyridine and 0.5 mL (5.3 mmol) of Ac2O were added. The reaction mixture was kept under constant stirring and room temperature for 30 min. The end of reaction was verified by TLC, the mixture was then concentrated to a volume approximately 5 mL under reduced pressure. Then AcOEt (30 mL) were added. The organic layer was washed with water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 4.7:5.3). Compound 12  was a colorless solid  IR (cm−1): 2943 (CH3); 2869 (CH2); 1737 (C=O); 1708 (C=O); 1435 (CH2); 1376 (CH3); 1262 (C-O); 1221 (C-O); 1172 (C-O). 1H-NMR: 5.12 ; 3.66 ; 2.56 ; 2.38 ; 2.04 ; 0.94 ; 0.74 ; 0.67 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Acid 3α-hydroxy-6-oxo-5α-cholan-24-oic (13). A solution of compound 8  in 60 mL of MeOH was prepared. Later 20 mL of 15% P/V K2CO3 solution were added, and the reaction mixture was refluxed for 1 h. The end of reaction was verified by TLC, the mixture was then concentrated to a volume approximately 15 mL under reduced pressure. This was acidified with 20 mL of 10% HCl solution. Then AcOEt (40 mL) were added. The organic layer was washed with water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and a colorless solid  was obtained. Compound 13  IR (cm−1): 3403–2500 (O-H); 2945 (CH3); 2869 (CH2); 1712 (C=O); 1689 (C=O); 1442 (CH2); 1380 (CH3). 1H-NMR (CD3OD): 4.04 ; 2.75 ; 2.31 ; 0.96 ; 0.730 ; 0.71 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Acid 3α-acetoxy-6-oxo-5α-cholan-24-oic (14). To a solution of compound 13  in 60 mL of DCM, 10 mg of DMAP, 1 mL of pyridine and 1.2 mL (12.8 mmol) of Ac2O were added. The reaction mixture was kept under constant stirring and room temperature for 1 h. The end of reaction was verified by TLC, the mixture was then concentrated to a volume approximately 10 mL under reduced pressure. Then AcOEt (40 mL) were added. The organic layer was washed with 5% HCl (1 × 10 mL), water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 8.8:11.2). Compound 14  was a colorless solid  IR (cm−1): 3373-2495 (O-H); 2947 (CH3); 2869 (CH2); 1736 (C=O); 1708 (C=O); 1444 (CH2); 1376 (CH3). 1H-NMR: 5.12 ; 2.56 ; 2.38 ; 2.03 ; 0.94 ; 0.74 ; 0,67 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Methyl 3α-hydroxy-6-oxo-7-oxa-5α-cholan-24-oate (15) and methyl 3α-hydroxy-6-oxa-7-oxo-5α-cholan-24-oate (16). To a solution of 8  in 60 mL of DCM, 55.3 mg (0.320 mmol) of m-CPBA (77%) and 269 mg (0.320 mmol) of NaHCO3 were added and the mixture was stirred at room temperature by 24 h. The end of reaction was verified by TLC, the mixture was filtered then concentrated to a volume approximately 5 mL under reduced pressure. Then AcOEt (40 mL) were added. The organic layer was washed with saturated NaHCO3 solution (2 × 20 mL), water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 6.0:4.0) Three fractions were obtained. Fraction I: 37 mg of unreacted compound 8. Fraction II: 11.2 mg (10.8% yield) of compound 15 and Fraction III: 15.5 mg (14.9% yield) of compound 16. Compound 15 was a colorless solid  IR (cm−1): 3446 (O-H); 2947 (C-CH3); 2871 (C-CH2-C); 2849 (C-CH2-C); 1732 (C=O); 1436 (CH2); 1373 (CH3); 1251 (C-O); 1168 (C-O). 1H-NMR: 4.15 ; 4.08-4.06 ; 3.66 ; 3.17 ; 0.907 ; 0.878 ; 0.700 . 13C-NMR: See 16 was a colorless solid (m.p. = 155–157 °C). IR (cm−1): 3446 (O-H); 2946 (C-CH3); 1732 (C=O); 1444 (CH2); 1377 (CH3); 1250 (C-O); 1166 (C-O). 1H-NMR: 4.60 ; 4.20 ; 3.65 ; 0.897 ; 0.878 ; 0.678 . 13C-NMR: See 1H-NMR and 13C-NMR are shown in Supplementary Materials15, 6-oxalactone 16 and the 2α,3α-diol 11 from hyodeoxycholic acid has been described (4). These compounds and intermediates were obtained through modification of hyodeoxycholic acid as described previously. In the alternative procedure described here, the co-product 6 obtained in the oxidation of methyl hyodeoxycholate 5 was converted into the target monoketone 7 by stereoselective reduction with NaBH4, increasing the overall yield of this synthetic route to 96.8%.An improved synthesis of known brassinosteroid analogues 7-oxalactone 1H-NMR spectroscopic information for derivatives of hyodeoxycholic acid 5–16, and a complete assignment of all 13C-NMR signals has also been made.Additionally, using mono- and bi-dimensional NMR techniques, we have updated the 8, 13 and 14 will be used in the synthesis of new brassinosteroid analogues, while derivatives 8, 11, 15 and 16 will be evaluated as plant-growth regulators in the Rice Lamina Inclination Assay and for antifungal activity in plants. These results will be reported later.Derivatives


Three of the synthesized compounds showed potent activity against fungal strains with IC50 values in the range of 0.055–0.092 µM. The results revealed that these compounds showed better IC50 values while compared with positive controls.Recently, a new generation of highly promising inhibitors bearing β-keto-enol functionality has emerged. Reported herein is the first synthesis and use of novel designed drugs based on the β-keto-enol group embedded with heterocyclic moieties such as pyrazole, pyridine, and furan, prepared in a one-step procedure by mixed Claisen condensation. All the newly synthesized compounds were characterized by FT-IR,  The reaction, as a mixed Claisen condensation, was carried out under mild conditions , using toluene as a solvent and sodium metal as the base. The reaction was slow and gave better results (with respect to the purity of the compounds).A).The synthesis began with the formation of a ketone enolate nucleophile in cool conditions (0 °C). After adding the appropriate heterocyclic carboxylate, the resulting mixture was stirred at room temperature for two days. The formed enolate initially underwent nucleophilic attack at the ester carbonyl to produce tetrahedral intermediate (B). The expelled base (EtO−Na+) then removed an acidic alpha proton from the β-diketone to generate a stabilized enolate ion product (C) as a precipitated salt. This formed precipitate was filtered, washed with toluene, dissolved in water, and neutralized with acetic acid to pH = 5 to afford the title products in acceptable yield after being filtered through silica using CH2Cl2/MeOH as an eluant. The mechanism for the formation of these target products is given in The expulsion of the ethoxide ion from the unstable tetrahedral intermediate of the initial Claisen adduct yielded a β-diketone (5 and 6) were obtained in previous work by others, in the “4-hydroxy-alk-3-en-2-one” tautomeric form under different conditions  [It is emphasized that our products exist exclusively in “3-hydroxy-alk-2-en-1-one” form as confirmed by the spectral data; these tautomeric forms were also confirmed by XRD and the results will appear in due course . However reflux) .1H-NMR integration of signals from the enol =C-H and the ketone CH2. Indeed, according to the NMR spectra, the parent β-diketones exist almost exclusively in the enol form and only a trace of the keto form is seen around 4 ppm. In DEPT-135, a very small negative signal from CH2 was also observed. Finally, crystals of most of the β-keto-enols were isolated from methanol by slow evaporation. Compound 6 was recently reported in a different way [The β-keto-enol form is greatly favored over the β-diketone form because of the conjugation of the enol with the carbonyl group, and the stability gained, by a strong six-centered intramolecular hydrogen bond. The β-keto-enol interconversion rate (>90%) was determined using the rent way .Fusarium oxysporum f.sp albedinis FAO) using the agar diffusion technique (ADT) [Echerichia coli, Bacillus subtilis, and Micrococcus luteus), but no significant effect was observed against these organisms.All synthesized β-keto-enol heterocycles were evaluated for their activity against breast cancer (MDA-MB241) human cell lines using normoxic conditions , and agaue (ADT) . It is o6 and 10. The concentration required to induce the activity (IC50) was more pronounced for compounds 3, 6 and 10 with IC50 values of 21.95, 17.62, and 18.79 μg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity.However, the results of the anticancer and antifungal effects were very significant and are given in 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 μM, respectively. This result was better than all the described products. We noted that the substitution of methyl in the R position was essential for this biological activity. The aryl groups strongly decreased the activity. This suggests that the marked bioactivity of the heterocyclic compounds was sensitive to modifications and could be further exploited to determine the structure activity relationship around this novel class of fungal inhibitors. Other structural modifications to these active compounds as antifungal and anti-HIV candidates are currently in progress.These structures have also led to unexpected antifungal activity. Indeed, compounds 1H- and 13C-NMR spectra were performed on a Bruker AC 300 spectrometer  (300 MHz for 1H and 75.47 MHz for 13C spectra). JEOL JMS DX-300 mass spectrometer  was used for the determination of molecular weights. Infrared (IR) spectra were recorded on a Shimadzu infrared spectrophotometer  using the KBr disc technique. The formazan obtained at the end of the experiment in MTT assays was measured by means of a Perkin Elmer Victor X4 Microplate reader .All commercial reagents were analytical grade . Melting points were measured using a BUCHÏ 510 m.p. apparatus . 2Cl2, the organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The obtained residue was filtered through silica using CH2Cl2/MeOH as eluant to give the desired products 1–10 as a white solid in 35%–48% yield. β-keto-enol forms were recrystallized from methanol (95%) to obtain target compounds 1–10 which were confirmed by FT-IR, 1H-NMR, 13C-NMR, elemental analysis, and mass spectroscopy.To a suspension of sodium (15.21 mmol) in 20 mL of toluene, the appropriate heterocyclic carboxylate (12.01 mmol) in 25 mL of toluene was slowly added; then acetone or aryl methyl ketones (12.01 mmol) in 10 mL of toluene was added at 0 °C. The resulting mixture was stirred at room temperature for two days. The precipitate formed was filtered, washed with toluene, dissolved in water, and neutralized with acetic acid to pH 5. After extraction with CH(Z)-1-(1.5-Dimethyl-1H-pyrazol-3-yl)-3-hydroxybut-2-en-1-one (1). Yellow crystal; yield: 35%; m.p. 83–84 °C; fR = 0.66 (diethyl ether)/silica. IR : ν (OH) = 3422; ν (C=O) = 1619; ν (enolic C=C) = 1509; 1H-NMR (CDCl3): δ 2.11 ; 2.27 ; 3.83 ; 6.29 . 6.54 ; 13C-NMR (CDCl3): δ 11.2 ; 24.6 ; 36.7 ; 96.7 ; 106.1  140.5 ; 146.9 ; 180.4 ; 190.0 (C-OH); MS: m/z, 181.00 (M + H)+. Anal. Cald. for C9H12N2O2: C, 59.99; H, 6.71; N, 15.55. Found: C, 60.11; H, 6.83; N, 15.43.(Z)-1--3-hydroxy-3-phenylprop-2-en-1-one (2). Yellow crystal; yield: 32%; m.p. 108–110 °C; fR: 0.88 (CH2Cl2/MeOH 9/1/silica), IR : ν (OH) = 3416; ν (C=O) = 1639; ν (enolic C=C) = 1518; 1H-NMR (DMSO-d6): δ 2.29 ; 3.83 ; 6.60 ; 7.02 ; 7.58 ; 7.95 . 13C-NMR (DMSO-d6): δ 11.9 ; 37.3 ; 93.3 ; 106.1 ; 127.3 ; 128.8 ; 129.3 ; 148.8 ; 181.7 ; 183.5 ; MS: m/z, 243.10 (M + H)+.(Z)-1--3-hydroxy-3-p-tolyprop-2-en-1-one (3). Yellow crystal; yield: 22%; m.p. 132–134 °C; fR: 0.29 (diethyl ether)/silica. IR : ν (OH) = 3412; ν (C=O) = 1642; ν (enolic C=C) = 1536; 1H-NMR (DMSO-d6): δ 2.26 ; 2.36 ; 3.82 ; 6.67 ; 6.98 ; 7.32 ; 7.85 . 13C-NMR (DMSO-d6): δ 11.2 ; 21.6 ; 37.3 ; 92.8 ; 106.0 ; 128.1 ; 129.9 ; 182.2 ; 182.9 ; MS: m/z, 257.11 (M + H)+.(Z)-1--3-hydroxy-3-(4-methoxyphenyl)prop-2-en-1-one (4). Yellow crystal; yield: 31%; m.p. 122–124 °C; fR: 0.61 (CH2Cl2/MeOH 8/2)/silica. IR : ν (OH) = 3432; ν (C=O) = 1678; ν (enolic C=C) = 1528; 1H-NMR (DMSO-d6): δ 2.26 ; 3.82 ; 3.83 : 6.65 ; 6.94 ; 7.04 ; 7.94 ; 13C-NMR (DMSO-d6): δ 11.1 ; 37.3 ; 56.0 ; 92.2 ; 106.1 ; 114.6 ; 129.6 ; 181.7 ; 182.9 ; MS: m/z, 273.06 (M + H)+.(Z)-3-Hydroxy-1-(pyridin-2-yl)but-2-en-1-one (5). Brown powder; yield: 48%; m.p. = 59–61 °C; fR: 0.27 (CH2Cl2/MeOH 9/1)/silica. IR : ν (OH) = 3448; ν (C=O) = 1611; ν (enolic C=C) = 1565; 1H-NMR (CDCl3): δ 2.23 ; 6.94 ; 7.42 ; 7.85 ; 8.08 ; 8.66 ; 13C-NMR (CDCl3): δ 26.2 ; 97.8 ; 123.0 ; 126.4 ; 138.0 ; 148.5 ; 151.4 ; 179.3 , 195.6 ; MS: m/z, 164.07 (M + H)+. Anal. Calcd. for C9H9NO2: C, 66.25, H, 5.56; N, 8.58. Found: C, 66.31; H, 5.62, N, 5.40.(Z)-3-Hydroxy-3-phenyl-1-(pyridin-2-yl)prop-2-en-1-one (6). Red powder; yield: 32%; m.p. = 78–80 °C; fR: 0.54 (CH2Cl2/MeOH 9/1)/silica. IR : ν (OH) = 3438; ν (C=O) = 1600; ν (enolic C=C) = 1549; 1H-NMR (DMSO-d6): δ 7.51 ; 7.61 ; 7.66 ; 8.00 ; 8.04 ; 8.11 ; 8.77 . 13C-NMR (DMSO-d6): δ 93.6 ; 122.5 ; 127.7 ; 128.8 ; 129.4 ; 133.7 ; 138.3 ; 150.1 ; 184.4 ; 186.1 ; MS: m/z, 226.13 (M + H)+. Anal. Calcd. for C14H11NO2: C, 74.65; H, 4.92; N, 6.22. Found: C, 74.74, H, 4.89; N, 6.25.(Z)-3-Hydroxy-1-(pyridin-2-yl)-3-p-tolylprop-2-en-1-one (7). Brown powder, yield 27%, m.p.: 174–176 °C; fR: 0.75 (CH2Cl2/MeOH 6/4)/silica. IR : ν (OH) = 3444; ν (C=O) = 1602; ν (enolic C=C) = 1542; 1H-NMR (DMSO-d6): δ 2.49 ; 6.78 ; 7.31 ; 7.46 ; 7.91 ; 8.12 ; 8.89 . 13C-NMR (DMSO-d6): δ 21.3 ; 93.8 ; 121.7 ; 126.1 ; 128.1 ; 128.9 ; 136.1 ; 149.6 ; 178.1 ; 186.1 ; MS: m/z, 240.13 (M + H)+. Anal. Calcd. for C15H13NO2: C, 75.30; H, 5.48, N, 5.85. Found: C, 75.49; H, 5.44; N, 5.80.(Z)-3-Hydroxy-3-(4-methoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one (8). Brown clear crystal, yield 33%, m.p.: 112–114 °C; fR: 0.33 (CH2Cl2/MeOH 9/1)/silica. IR : ν (OH) = 3444; ν (C=O) = 1599; ν (enolic C=C) = 1549; 1H-NMR (DMSO-d6): δ 3.83 ; 7.08 ; 7.50 ; 7.62 ; 7.97 8.04 ; 8.08 ; 8.76 . 13C-NMR (DMSO-d6): δ 56.1 ; 92.8 ; 114.5 ; 122.2 ; 127.5 ; 138.2 ; 150.1 ; 182.1 ; 186.6 ; MS: m/z, 256.08 (M + H)+. Anal. Calcd. for C15H13NO3: C, 70.58; H, 5.13; N, 5.49. Found: C, 70.76; H, 5.20, N, 5.59.(Z)-1-(Furan-2-yl)-3-hydroxybut-2-en-1-one (9). Red hygroscopic; yield: 39%; fR: 0.91 (CH2Cl2/MeOH 8/2)/silica. IR : ν (OH) = 3434 cm−1; ν (C=O) = 1620 cm−1; ν (enolic C=C) = 1468; 1H-NMR (CDCl3): δ 2.13 ; 6.06 ; 6.52 ; 7.14 ; 7.55 ; 13C-NMR (CDCl3): δ 24.4 ; 96.0 ; 112.4 ; 115.5 ; 145.9 ; 150.0 ; 176.1 , 19.5 ; MS: m/z, 153.10 (M + H)+. Anal. Calcd. for C8H8O3: C, 63.15; H, 5.30. Found: C, 63.25, H, 5.38.(Z)-1-(Furan-2-yl)-3-hydroxy-3-phenylprop-2-en-one (10). Red powder; yield 42%; m.p. 64–66 °C; fR: 0.61 (CH2Cl2/silica). IR : ν (OH) =3431; ν (C=O) = 1621; ν (enolic C=C) = 1531; 1H-NMR (DMSO-d6): δ 6.72 ; 7.01 ; 7.52 ; 7.58 ; 7.64 ; 8.00 ; 8.04 ; 13C-NMR (DMSO-d6): δ 93.3 ; 113.5 ; 120.2 ; 127.3 ; 129.2 ; 148.7 ; 178.5 ; 180.8 ; MS: m/z, 215.12 (M + H)+.Prepared compounds were screened against breast cancer (MDA-MB241) human cell lines using normoxic conditions . Tests win vitro antifungal activities were tested by the agar diffusion technique [Fusarium oxysporum f.sp albedinis FAO).The echnique  using fuin vitro antibacterial activities were tested using bacterial strains .The results were compared with positive controls (benomyl and thiophanate-methyl).The in vitro anticancer and antifungal activities. Most of the compounds showed modest antiproliferative activity against breast cancer (MDA-MB241) human cell lines. Among the synthesized products, compounds 1, 5, and 9 successfully showed the most potent antifungal activity with IC50 values in the range of 0.055–0.092 μM as compared with positive controls.In summary, we have described the first synthesis of novel β-keto-enols embedded with heterocyclic moieties and the evaluation of their