In an in vitro antifungal evaluation, the myristic acid derivatives were assessed against Candida albicans and Aspergillus niger strains by determining their minimal inhibitory concentrations (MIC50). The compounds 3u, 3k, 3r and 3t displayed superior antifungal capabilities against Candida albicans, and the compounds 3u, 3m and 3r displayed superior antifungal capabilities against Aspergillus niger compared to the standard drug FLZ (fluconazole). Altogether, we identified a new series of antifungal agents.In recent years, N-Myristoyltransferase (NMT) has been identified as a new target for the treatment of fungal infections. It is observed that at present, there are increased rates of morbidity and mortality due to fungal infections. Hence, a series of novel myristic acid derivatives were designed via molecular docking studies and ADMET studies by targeting NMT (N-Myristoyltransferase). The designed myristic acid derivatives were synthesized by converting myristic acid into myristoyl chloride and coupling it with aryl amines to yield corresponding myristic acid derivatives. The compounds were purified and characterized via FTIR, NMR and HRMS spectral analyses. In this study, we carried out a target NMT inhibition assay. In the NMT screening assay results, the compounds  Candida albicans and Aspergillus niger strains are responsible for the most severe pathogenic fungal infections. . Th. Th43]. Candida albicans  and Aspergillus niger (A. niger) were carried out in triplicate at concentrations ranging from 100 μg/mL to 6.25 μg/mL. The percentage cytotoxicity and MIC50 value for each compound were calculated from the observed absorbance measured at 590 nm, and the results are shown in 50) values in μg/mL.A microbroth dilution method was used to test the synthesized compounds. In vitro tests for antifungal activity against 3k-3v have good potency against Candida albicans  and Aspergillus niger (A. niger) Moreover, the compounds 3u, 3k, 3r and 3t were more active against Candida albicans  than fluconazole (MIC50 = 17.76 μg.mL−1). The compounds 3l, 3m, 3o and 3p exhibit moderate activity, with an MIC50 range of 15–22 μg.mL−1, against Candida albicans compared to fluconazole. In case of the Aspergillus niger (A. niger) strain, compound 3u (MIC50 = 14.38 μg.mL−1) shows better activity than fluconazole (MIC50 = 16.30 μg.mL−1). Compounds 3m and 3r expressed good effects, with MIC50 values of 14.68, and 15.42 μg.mL−1 compared to fluconazole, and others show poor activities. The percentage inhibition was calculated and plotted in the graph to determine the MIC50 values for the compounds 3u, 3k, 3r and 3t against Candida albicans  were analysed using spectroscopic methods . Molecular docking studies with the target N-Myristoyltransferase (NMT), ID: 1IYK, predict that the compounds 3u, 3m and 3t show good docking scores and docking poses.The present study aimed to develop novel antifungal agents based on their mechanisms of action. NMT is thought to be a molecular target for the forthcoming class of antifungal drugs and was therefore selected as a target . Conside3u, 3m and 3t showed better activity compared to other compounds and myristic acid. Some authors have reported a pseudo-peptidic NMT inhibitor with an IC50 value of 0.50 μM +: 318.2752 peak found [M+1]+: 318.3335.Colour: brown crystals, yield: 65.53%, melting point: 79–81 °C, molecular formula: C22H37NO2. IR : 3448.90 (N-H stretching), 2954.67 (Ar C-H stretching), 2919.92 (Ali C-H stretching), 1660.62 (C=O stretching), 1532.81 (Ar C=C stretching). 1H NMR: : 0.873 , 1.312 , 1.653 , 2.313 , 2.358 , 7.283 , 7.388 , 7.512 . 13C- NMR: : 14.070 (CH3), 22.672 (CH3), 24.724 (CH2), 25.836 (CH2), 29.346 (9 CH2), 31.915 (CH2), 37.693 (CH2), 121.075 (2ArCH), 129.551 (2ArCH), 133.851 (1ArC), 155.752 (1ArC), 179.507 (C=O). LC-MSMS (m/z) peak calculated for C22H37NO2 [M+1]+: 348.2858 peak found [M+1]+: 348.3464.Colour: brownish crystals: yield: 61.30%, melting point: 77–80 °C, molecular formula: C20H39NO. IR : 3447.81 (N-H stretching), 2953.98 (Ar C-H stretching), 2918.22 (Ali C-H stretching), 1701.55 (C=O stretching), 1638.86 (Ar C=C stretching). 1H NMR: : 0.878 , 1.317 , 1.663 , 2.336 , 2.374 , 7.109 , 7.284 , 7.380 . 13C- NMR: : 14.250 (CH3), 20.963 (CH3), 22.821 (CH2), 25.837 (CH2), 29.534 (9 CH2), 32.054 (CH2), 37.911 (CH2), 120.075 (2ArCH), 129.551 (2ArCH), 133.851 (1ArC), 135.593 (1ArC), 171.561 (C=O). LC-MSMS (m/z) peak calculated for C20H39NO [M+1]+: 310.3065 peak found [M+1]+: 310.3651.Colour: brown crystals: yield: 63.20%, melting point: 74–78 °C, molecular formula: C18H35NO2. IR : 3450.04 (N-H stretching), 2954.24 (Ar C-H stretching), 2917.68 (Ali C-H stretching), 1701.50 (C=O stretching), 1471.30 (Ar C=C stretching). 1H NMR: : 0.883 , 1.323 , 1.651 , 2.343 , 2.381 , 7.109 , 7.284 , 7.380 . 13C- NMR: : 14.077 (CH3), 22.678 (CH3), 24.680 (CH2), 25.837 (CH2), 29.663 (9 CH2), 31.916 (CH2), 34.104 (CH2), 120.075 (2ArCH), 129.551 (2ArCH), 133.851 (1ArC), 135.593 (1ArC), 180.288 (C=O). LC-MSMS (m/z) peak calculated for C18H35NO2 [M+1]+: 298.2701 peak found [M+1]+: 298.3297.Colour: orange crystals: yield: 65.10%, melting point: 76–78 °C, molecular Formula: C21H35NO. IR : 3679.57 (N-H stretching), 2924.51 (Ar C-H stretching), 2854.01 (Ali C-H stretching), 1665.87 (C=O stretching), 1595.60 (Ar C=C stretching). 1H NMR: : 0.882 , 1.288 , 1.644 , 2.303 , 2.341 , 7.159 , 7.283 , 7.383 . 13C- NMR: : 14.059 (CH3), 22.655 (CH3), 24.801 (CH2), 25.553 (CH2), 29.565 (9 CH2), 31.894 (CH2), 34.021 (CH2), 127.245 (2ArCH), 127.709 (2ArCH), 129.679 (1ArC), 144.185 (1ArC), 178.237 (C=O). LC-MSMS (m/z) peak calculated for C21H35NO [M+1]+: 318.2752 peak found [M+1]+: 318.3335.Colour: orange liquid, yield: 64.12%, melting point: 78–80 °C, molecular formula: C20H33NO. IR : 3447.69 (N-H stretching), 2954.20 (Ar C-H stretching), 2917.76 (Ali C-H stretching), 1701.27 (C=O stretching), 1601.47 (Ar C=C stretching). 1H NMR: : 0.884 , 1.280 , 1.651 , 2.344 , 2.381 , 7.109 , 7.259 , 7.284 . 13C- NMR: : 14.091 (CH3), 22.686 (CH3), 22.821 (CH2), 24.684 (CH2), 29.594 (9 CH2), 31.923 (CH2), 34.108 (CH2), 120.093 (2ArCH), 129.078 (2ArCH), 133.851 (1ArC), 135.593 (1ArC), 180.295 (C=O). LC-MSMS (m/z) peak calculated for C20H33NO [M+1]+: 304.2596; peak found [M+1]+: 304.3194.Colour: brown crystals: yield: 60.10%, melting point: 74–76 °C, molecular formula: C22H37NO. IR : 3446.84 (N-H stretching), 2954.25 (Ar C-H stretching), 2918.84 (Ali C-H stretching), 1701.97 (C=O stretching), 1594.84 (Ar C=C stretching). 1H NMR: : 0.896 , 1.276 , 1.648 , 2.337 , 2.375 , 7.156 , 7.284 , 7.496 . 13C- NMR: : 14.073 (CH3), 20.963 (CH3), 22.680 (CH2), 24.691 (CH2), 29.437 (9 CH2), 31.922 (CH2), 34.124 (CH2), 122.841 (2ArCH), 128.337 (2ArCH), 129.029 (1ArC), 129.612 (1ArC), 180.207 (C=O). LC-MSMS (m/z) peak calculated for C22H37NO [M+1]+: 332.2909; peak found [M+1]+: 332.3496.Colour: brown crystals: yield: 61.10%, melting point: 76–79 °C, molecular formula: C20H32FNO. IR : 3444.93 (N-H stretching), 2954.32 (Ar C-H stretching), 2919.00 (Ali C-H stretching), 1703.46 (C=O stretching), 1656.36 (Ar C=C stretching). 1H NMR: : 0.882 , 1.278 , 1.666 , 2.285 , 2.338 , 7.031 , 7.153 , 7.452 . 13C- NMR: : 14.073 (CH3), 22.672 (CH3), 24.698 (CH2), 25.651 (CH2), 29.429 (9 CH2), 31.913 (CH2), 34.066 (CH2), 115.419 (2ArCH), 115.640 (2ArCH), 121.718 (1ArC), 121.795 (1ArC), 179.846 (C=O). LC-MSMS (m/z) peak calculated for C20H32FNO [M+1]+: 322.2501; peak found [M+1]+: 322.3115.Colour: brown crystals: yield: 60.20%, melting point: 79–81 °C, molecular formula: C21H32F3NO. IR : 3445.46 (N-H stretching), 2954.92 (Ar C-H stretching), 2918.82 (Ali C-H stretching), 1672.28 (C=O stretching), 1530.34 (Ar C=C stretching). 1H NMR: : 0.884 , 1.278 , 1.655 , 2.346 , 2.414 , 7.284 , 7.573 , 7.674 . 13C- NMR: : 14.092 (CH3), 22.681 (CH3), 25.575 (CH2), 29.349 (9 CH2), 31.916 (CH2), 37.994 (CH2), 119.763 (2ArCH), 125.424 (2ArCH), 126.266 (1ArC), 141.005 (1ArC), 172.027 (C=O). LC-MSMS (m/z) peak calculated for C21H32F3NO [M_1]: 371.2436; peak found [M_1]: 370.3534.Colour: brownish crystals, yield: 61.10%, melting point: 77–79 °C, molecular formula: C20H33NO2. IR : 3448.74 (N-H stretching), 2955.53 (Ar C-H stretching), 2918.53 (Ali C-H stretching), 1654.67 (C=O stretching), 1529.53 (Ar C=C stretching). 1H NMR: : 0.882 , 1.277 , 1.667 , 2.342 , 2.379 , 7.001 , 7.031 , 7.606 . 13C- NMR: : 14.103 (CH3), 20.963 (CH3), 22.686 (CH2), 24.711 (CH2), 29.437 (9 CH2), 31.921 (CH2), 34.024 (CH2), 120.075 (2ArCH), 129.551 (2ArCH), 133.851 (1ArC), 135.593 (1ArC), 179.501 (C=O). LC-MSMS (m/z) peak calculated for C20H33NO2 [M+1]+: 320.2545; peak found [M+1]+: 320.3158.Colour: brown crystals, yield: 60.15%, melting point: 77–79 °C, molecular formula: C19H32N2O. IR : 3436.43 (N-H stretching), 2955.00 (Ar C-H stretching), 2918.11 (Ali C-H stretching), 1697.79 (C=O stretching), 1577.78 (Ar C=C stretching). 1H NMR: : 0.872 , 1.268 , 1.639 , 2.331 , 2.369 , 7.284 , 7.308 , 7.358 . 13C- NMR: : 14.082 (CH3), 20.963 (CH3), 22.675 (CH2), 24.699 (CH2), 29.392 (9 CH2), 31.913 (CH2), 34.114 (CH2), 120.075 (2ArCH), 129.551 (2ArCH), 133.851 (1ArC), 135.593 (1ArC), 180.017 (C=O). LC-MSMS (m/z) peak calculated for C19H32N2O [M+1]+: 305.2548; peak found [M+1]+: 305.3128.Colour: yellowish crystals, yield: 62.20%, melting point: 76–78 °C, molecular formula: C18H32N2OS. IR : 3315.74 (N-H stretching), 2953.12 (Ar C-H stretching), 2918.40 (Ali C-H stretching), 1656.91 (C=O stretching), 1523.82 (Ar C=C stretching). 1H NMR: : 0.856 , 1.352 , 1.665 , 2.263 , 2.446 , 7.028 , 7.272 . 13C- NMR: : 14.091 (CH3), 22.662 (CH3), 25.036 (CH2), 29.346 (9 CH2), 31.895 (CH2), 36.128 (CH2), 127.047 (2ArCH), 132.554 (1ArC), 158.745 (1ArC), 171.276 (C=O). LC-MSMS (m/z) peak calculated for C18H32N2OS [M+1]+: 325.2269; peak found [M+1]+: 325.2711.Colour: yellowish crystals, yield: 63.20%, melting point: 74–76 °C, molecular formula: CCandida albicans with the newly synthesised myristic acid derivatives using a molecular docking approach. The X-ray crystallography structure of the drug target N-Myristoyltransferase (Sogabe) from PDB (Protein databank) ID: 1IYK was retrieved from the RCSB Protein Data Bank (RCSB PDB) repository . The structures of chemical compounds were drawn and geometrically cleaned using Chem Draw Ultra (version), and the induvial structures were saved in mol format. Both the drug target and the ligands were prepared using the protein and ligand preparation wizard in BIOVIA Discovery studio 2020. The protocol of CDOCKER (Wu), a grid-based molecular dynamics docking function, was validated using a bound nonpeptidic inhibitor (MIM) to obtain the pose RMSD deviations within 2.5–3 Å. The optimized method was used to test the myristic acid derivatives and myristic acid at the same active site binding of MIM. The grid was placed at the active site along with water molecules at the 3D Cartesian space coordinates of 25.7586X, 49.4463Y and 34.8952Z. Consequently, the interpretation was based on CDOCKER’s scoring function for the interaction energy of the binding interaction. Further, ADMET, TOPKAT and Lipinski’s rule of five (RO5) were used to understand the bioavailability and toxicity profiles of the compounds using the small molecule tools in Discovery studio [In this work, the concept of a structure-based drug was implemented to probe the binding of the N-Myristoyltransferase of To further evaluate the stability and real-time behaviour of the N-Myristoyltransferase docked complex dynamics, a CHARMm simulation for 1000ps in was employed. The CHARMm heterogenous force field parameter was implemented in the BIOVIA Discovery studio suite and was applied to five complexes, such as 3u- N-Myristoyltransferase, 3m- N-Myristoyltransferase, 3t- N-Myristoyltransferase, 3k- N-Myristoyltransferase and myristic acid- N-Myristoyltransferase. The complex system of protein–ligand interactions was solvated with a TIP3 water model in an orthorhombic box and neutralized with 0.145 M of NaCl (sodium chloride). The three different complexes were processed for two stages of 500 steps of energy minimization with the steepest decent and conjugate gradient. The system was gradually heated and equilibrated and production occurred under an NPT isothermal–isobaric ensemble. A Nosé–Hoover thermostat was used as a temperature control to set the temperature at 300 k, and the pressure was fixed at 1.0 bar with a Langevin piston. The nonbond lower cutoff distance to higher cutoff distance range of 12–10 Å included with the Leapfrog Verlet Integrator was used as a dynamics integrator to perform a numerical integration of the equation of motion. The time-dependent parameter RMSD and radius of gyration were calculated to understand the stability of the complexes .v/v) Triton X-100, and a final concentration of 2.7% (v/v) dimethyl sulfoxide (DMSO). Fluorescence was measured at an excitation wavelength of 380 nm and an emission wavelength of 470 nm. These readings were obtained on an EnSight Multimode Microplate Reader, and we plotted the response vs. concentration (log nM), ranging from 0.1 nM to 10,000 nM, to obtain the IC50 values using Graph Pad Prism, according to the reported standard procedure [The NMT protein used in the study was from the supplier Ray Biotech. The assay was performed in 96-well black PP microplates with the myristic acid derivatives in concentrations ranging from 0.1 nM to 10,000 nM. The solutions were prepared in a buffer containing 20 mM of potassium phosphate (pH 7.9–8.0) and 0.5 mM of ethylenediaminetetraacetic acid (EDTA), 0.1%  and Aspergillus niger (ATCC 16888) from the American Type Culture Collection. The acquired fungal strains were used in the current experiment after being subcultured in SDB (Sabouraud dextrose broth). The 48 h young cultures were created and kept at 26 °C before the experiment.From HIMEDIA M210 Mumbai, India, test fungal strains were acquired. The conventional micro-broth dilution method was used to test the in vitro antifungal activity of the synthesized compounds against the fungi Antifungal Assay5 CFU/mL and incubated at 26 °C for 48 h. Subsequently, using a UV spectrophotometer, the absorbance at 590 nm of each culture was determined. The formula was used to compute the viability percentage [According to the CLSI’s standard M27 recommended methodology, the SDB was treated with the compounds, which were dissolved in DMSO via the serial dilution method in sterile circumstances to achieve the necessary concentration levels. All the cultures that had been exposed to the compounds were serially diluted twice in growth media, inoculated to about 1 × 10Percentage viability = (OD control−OD sample/OD control) × 10050 value for each compound  and plotted in the graph to determine the MICcompound  and  was used for statistical analysis. The MIC3u and 3m show superior NMT inhibition activity compared to myristic acid. Flexible molecular docking was used to investigate the binding mechanisms of the compounds. The results show how crucial hydrogen bonds between the aromatic ring and NMT are required for the inhibitor’s correct orientation. Thus, the results of the in silico studies predicted favourable ADMET and drug-like parameters, which can be used as a solid starting point for future research on the design and synthesis of NMT inhibitors.A new series of myristic acid derivatives were synthesized, analysed, screened in an NMT inhibition assay, and evaluated for their antifungal activity. Among the novel derivatives of myristic acid, the compounds


Streptomyces sp. CNQ-617 has led to the isolation of a new quinazolinone derivative, actinoquinazolinone (1), as well as two known compounds, 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one (2) and 7-methoxy-8-hydroxy cycloanthranilylproline (3). The interpretation of 1D, 2D NMR, and MS spectroscopic data revealed the planar structure of 1. Furthermore, compound 1 suppressed invasion ability by inhibiting epithelial–mesenchymal transition markers (EMT) in AGS cells at a concentration of 5 µM. In addition, compound 1 decreased the expression of seventeen genes related to human cell motility and slightly suppressed the signal transducer and activator of the transcription 3 (STAT3) signal pathway in AGS cells. Together, these results demonstrate that 1 is a potent inhibitor of gastric cancer cells.A HPLC-UV guided fractionation of the culture broth of  Cancer is the uncontrolled growth and division of cells in the body . GastricMarine microorganisms, including actinobacteria, have emerged as a promising source for the discovery of novel bioactive compounds with potential pharmacological properties ,9. ActinStreptomyces sp. strain CNQ-617 is a marine-derived actinobacterium isolated from sediment samples collected from La Jolla Submarine Canyon in California. This strain has been shown to produce several bioactive compounds with potential therapeutic applications + . The 1H NMR spectrum of 1 , two aromatic protons at δH 7.46  and 6.98 , one methoxy group at δH 3.88 , and one exchangeable proton at δH 10.35 . Based on spectroscopic data from 13C NMR and HSQC, six quaternary carbons were assigned at δC 159.7 (C-4), 148.2 (C-6), 153.0 (C-7), 143.9 (C-9), 113.8 (C-10), and 172.2 (C-14), two methylene carbons at δC 51.2 (C-11) and 39.8 (C-13), four methine carbons at δC 147.1 (C-2), 105.7 (C-5), 110.9 (C-8), and 65.0 (C-12), and one methoxy carbon at δC 55.7 (6-Ome).Actinoquinazolinone , 148.2 (C-6), 153.0 (C-7), 110.9 (C-8), 143.9 (C-9), and 113.8 (C-10), and two hetero-aromatic carbons at δC 147.1 (C-2) and 159.7 (C-4), along with the HMBC correlations from H-2 to C-4, C-9 and C-10; from H-5 to C-4, C-6, C-7 and C-10; and from H-8 to C-7, C-9 and C-10. In addition, HMBC correlations from 6-Ome to C-6 provided the attachment of the methoxy group at C-6. Furthermore, COSY cross peaks [H-11/H-12 and H-12/H-13] and long-range HMBC correlations of H-12 and H-13 with C-14 (δC 172.2) as well as the carbon chemical shift of C-12 (δC 65.0) allowed the construction of the 3-hydroxybutanoic acid moiety. This moiety was linked to the quinazolinone moiety through the nitrogen atom between C-2 and C-4 from the consideration of the carbon chemical shift of C-11 (δC 51.2) and from the observation of the long-range HMBC correlation from H-12 to C-2 and C-4. Additionally, the unassigned hydroxy group was determined to be located at C-7 based on the carbon chemical shift of C-7 (δC 153.0) and the molecular formula of the compound. Therefore, the final structure of actinoquinazolione (1) was determined to be 7-hydroxy-6-methoxy quinazolinone with a 3-hydroxy butanoic acid moiety, as shown in Additional 2D NMR analyses allowed the structure of 1H NMR  spectrum of 2 revealed one hetero-aromatic proton at δH 7.90 , two aromatic protons at δH 7.43  and 6.98 , one methoxyl group at δH 3.87 , and two exchangeable protons at δH 11.93  and 10.27  , 153.0 (C-7), 148.0 (C-2), 144.9 (C-6), 143.6 (C-9), 114.7 (C-10), 111.3 (C-8), 105.5 (C-5), and 55.7 (6-Ome)  . The int (6-Ome) . Compouncompound . However3) was isolated as yellowish oil and its molecular formula was determined to be C13H14N2O4 by HR-FAB-MS [M+H]+ ion at m/z 263.1029. The 1H NMR spectrum of compound 3 displayed para-coupled aromatic protons at δH 7.36  and 6.58  (13C NMR spectrum of 3 showed twelve carbons at Cδ 171.2 (C-11), 166.6 (C-5), 151.1 (C-8), 145.4 (C-7), 131.4 (C-9a), 117.9 (C-5a), 111.8 (C-6), 107.7 (C-9), 57.2 (C-11a), 47.1 (C-3), 25.8 (C-1), 23.4 (C-2), and one methoxy group at Cδ 55.4 (7-Ome)  . The 13C (7-Ome) . The che1 and 2 are quinazolinone derivatives with an N-containing heterocyclic scaffold. Quinazolinones are a class of nitrogen-containing heterocyclic compounds found in nature which are produced by plants and microorganisms +.7-methoxy-8-hydroxy cycloanthranilylproline (3): max 3214, 1693, 1607, 1519, 1437, 1274, 1201, 1179, 786 cm−1; UV (MeOH) λmax (log ε) 220 (7.76), 260 (3.79), 300 (3.31) nm, 1H , 6.58 , 4.16 (m. H-11a), 3.91 , 3.72 , 3.57 , 2.67 , 2.05 , 2.02 , 13C , 166.6 (C-5), 151.1 (C-8), 145.4 (C-7), 131.4 (C-9a), 117.9 (C-5a), 111.8 (C-6), 107.7 (C-9), 57.2 (C-11a), 55.4 (7-OCH3), 47.1 (C-3), 25.8 (C-1), 23.4 (C-2), HR-FAB-MS m/z 263.1029 [M + H]+ .2 [Cell lines A549 (lung cancer), AGS (gastric cancer), and Caco-2  were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium or Dulbecco’s Modified Eagle Medium (DMEM) , supplemented with 10% FBS and 1% penicillin–streptomycin solution C in a humid environment with 5% CO2 .3 cells/well), AGS (3 × 103 cells/well), and Caco-2 (2.5 × 103 cells/well) were seeded on 96-well plates for gown overnight, and then treated with DMSO (Sigma-Aldrich) or various concentrations of actinoquinazolione (1), 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one (2), and 7-methoxy-8-hydroxy cycloanthranilylproline (3) for 48 h. MTT was added to the cultures for 4 h after treatment. In the final step of MTT, the cells were lysed with 150 µL of DMSO and the absorbance was measured using a spectrophotometer  [A549  ,87.1 for 24 h and DMSO as a control. The lower chamber was filled with 600 µL RPMI containing 0.2% BSA and 6 µg/mL fibronectin  as a chemoattractant. Diff-Quik kit  was used for the fixation and dying of AGS cells after 24 h treatment, the cells in the upper chambers were quantified using a K1-Fluo Confocal Microscope  and i-Solution FL Auto Software , then treated with 1 and 5 µM concentrations of compound , Canada ,89.1 groups were converted to cDNA. SYBR Green  was used to evaluate relative gene expression and CFX  was applied for analysis [Total RNA of AGS cells was extracted by RNAiso Plus  according to the manufacturer’s suggestions. Using a Moloney Murine Leukemia Virus (M-MLV) kit, total RNA (1 µg) from DMSO as well as 1 and 5 μM of treatment with compound analysis ,91.1. After 24 h treatment, rinsed with PBS, then extracted using lysis buffer. The extracted protein was separated by SDS-PAGE. The membranes were incubated in blocking buffer  for one hour and antibodies against E-cadherin and N-cadherin ; Snail/Slug and Twist ; α-tubulin, STAT3, and p-STAT3  were detected by horseradish peroxidase-conjugated secondary antibodies  using the Immobilon Western Chemiluminescent HRP Substrate Kit . The Multi Gauge 3.0  software was used for analyzing the density of the bands [The AGS cells were seeded in 6-well plates for gown overnight, and then treated with DMSO as well as 1 and 5 µM concentrations of he bands . t-test was used to compare statistically significant differences between two groups. A p-value less than 0.05 was considered statistically significant. Data is represented as means ± standard deviation. All statistical analyses were carried out using the Sigma Plot software. Student’s Streptomyces sp. CNQ-617 has led to the discovery of a novel compound, actinoquinazolinone (1), along with two previously reported compounds, 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one (2) and 7-methoxy-8-hydroxy cycloanthranilylproline (3). Furthermore, compound 1 exhibited moderate antibacterial activity against the Gram-positive bacteria K. rhizophila KCTC 1915 and weak inhibitory activities against B. subtilis KCTC 1021 and S. aureus KCTC 1927. In addition, compound 1 showed higher activity than compounds 2 and 3 in decreasing the cell viability of cancer cells. Moreover, compound 1 suppressed the invasion ability of the EMT and STAT3 signal pathways, and some of the cell motility-related genes. These findings highlight the potential of marine sediment-derived Streptomyces sp. CNQ-617 as a source of novel bioactive compounds that may have therapeutic applications. Further studies are required to determine the potential therapeutic effect and the potential side effect of 1.In summary, the exploration of marine natural products from marine sediment-derived


From the reaction of β-methoxy enone with diazo compound, dioxole, and Cα-H insertion products were also obtained as side products along with 2,3-dihydrofuran derivative. When the unsaturated compound has an ester and a ketone group, only one dihydrofuran derivative was formed, which occurred by the 1,5-ring closure of keto-carbonyl ylide. Dihydrofuran derivative from the formation of ester carbonyl ylide in the reactions was not obtained.2,3-dihydrofurans were synthesized from carbonyl-ylides via 1,5-electrocyclization reactions with high yields. Dimethyl diazomalonate was reacted with several β-alkoxy and/or β-phenoxy α,β-unsaturated compounds in the presence of Cu(acac) Substituted dihydrofurans are intermediates for the synthesis of a wide variety of compounds such as furan, cyclopropyl aldehyde, γ-hydroxy aldehyde, γ-hydroxy ketone, γ-lactone, and hydroxy amino acid –2. 2,3-D4-catalyzed reactions of β-methoxy α,β-unsaturated ketone with ethyl diazoacetate gave furan compounds over 2,3-dihydrofurans via methanol elimination + 239.1072, found 239.1081.Acetophenone was used as a methyl ketone in Step 1 and toluidine was used as a phenolic compound in Step 3. The product was synthesized according to Step 1, Step 2, and Step 3, respectively. Obtained as yellow solid, m.p.: 85–88 °C; +, 5), 171 (94), 143 (51), 115(100), 97 (49), 71 (42), 53 (7); Isomer 1: tR = 8.87; 1H NMR : δ 7.48 , 4.09–3.97 , 2.19 , 1.23–1.07 ; Isomer 2: tR = 9.06; 1H NMR : δ 7.47 , 4.09-3.97 , 2.12 , 1.23–1.07 .Ethyl acetoacetate (3 mol), triethyl orthoformate (3.6 mol), and glacial acetic acid (9 g) were placed in 2 L of a three-necked round-bottom flask, which is equipped with a thermometer and distillation set-up. The system was heated and alcohol was distilled about at 125 °C. The reaction took place at about 4 h. Product was purified by vacuum distillation . The product obtained as brown oil; EI-MS (m/z): 186  and a solution of β-alkoxy/phenoxy enone  in benzene (10 mL) were heated under reflux. A solution of dimethyl diazomalonate  in benzene (1.5 mL) was added to this solution over 2.5 h under a nitrogen atmosphere. When the IR spectrum of the reaction mixture indicated the absence of a characteristic diazo band at 2130 cm−1, the mixture was filtered, concentrated, and purified by silica column chromatography.Cu(acac)1H NMR : δ 7.70 , 7.51–7.32 , 7.04 , 6.92 , 4.55 , 4.17 , 3.85 , 3.49 , 2.25 ; 13C NMR : δ 168.0 (CO2CH3), 166.4 (CO2CH3), 156.8, 145.7 (O-C(Ph) = C), 132.7, 131.6, 130.4, 129.6, 128.4, 126.8, 117.6, 102.5 (O-C(Ph)=C), 93.4 (C(CO2CH3)2), 82.9 (C-OPh), 52.7 (CO2CH3), 52.2 (CO2CH3), 20.8 (CH3); tR = 14.0; EIMS (m/z): 368 , 281 (11), 230 (84), 202 (100), 105 (51), 77 (41), 59 (8); HR-MS: calcd for C21H21O6 [M+H]+ 369.1333, found 336.1320.Isolated by silica column chromatography using hexane: ethyl acetate (4:1) as an eluent. Obtained yellow oily compound (yield 76 %); 3c was obtained with compound 4 as a mixture of pale-yellow oil . 1H NMR : δ 5.14 , 4.91 , 3.84 , 3.83 , 3.36 , 1.95 ; 13C NMR : δ 167.3 (CO2CH3), 165.5 (CO2CH3), 159.1 (O-C=C), 96.6 (O-C=C), 91.7 (C(CO2CH3)2), 87.3 (CH-OCH3), 57.7 (CH-OCH3), 53.0 (CO2CH3), 52.9 (CO2CH3), 13.7 (CH3); tR = 8.34; EI-MS (m/z): 230  198 (23), 170 (69), 155 (65), 139 (41), 109 (100), 83 (27), 69 (62), 59 (45).Isolated by silica column chromatography using hexane: ethyl acetate gradually from beginning 4:0.4 as an eluate. 1H NMR : δ 5.28 , 4.18–4.03 , 3.76 , 3.68–3.50 , 2.23 , 1.20 , 1.04 ; 13C NMR : δ 168.8 (CO2CH3), 165.2 (CO2CH3), 163.6 (CO2CH2CH3), 163.3 (O-C=C), 104.6 (C=C-O), 90.9 (C(CO2CH3)2), 83.9 (CH-OCH2CH3), 67.3 (CO2CH2CH3), 58.9 (OCH2CH3), 52.6 (CO2CH3), 52.0 (CO2CH3), 14.4 (OCH2CH3), 13.3 (CH3); tR = 11.8; EI-MS (m/z): 316 , 301 (4), 271 (51), 240 (71), 227 (100), 212 (60), 183 (81), 167 (61), 123 (21), 59 (36); HR-MS: calcd for C14H21O8 [M+H]+ 317.1231, found 317.1257.Isolated by silica preparative thin layer chromatography using hexane: ethyl acetate (4:1) as an eluate (yield 84 %); 4 was obtained with 3c as a mixture of pale-yellow oil ; 1H NMR : δ 6.25 , 5.95 , 3.85 , 3.83 , 3.81 , 1.61 ; 13C NMR : δ 169.4 (C=C-OCH3), 164.8 (CO2CH3), 133.4 (CH(OCH3)=CH), 128.9 (C-CO2CH3), 116.2 (CH(OCH3)=CH), 91.4 (C-CH3), 53.5 (OCH3), 53.3 (CO2CH3), 50.4 (OCH3), 25.7 (CH3); tR = 8.12; EI-MS (m/z): 230  215 (10), 199 (20), 171 (100), 155 (32), 139 (50), 111 (77), 109 (67), 83 (61), 59 (59).Isolated by silica column chromatography using hexane: ethyl acetate gradually from beginning 4:0.4 as an eluate. Compound 1H NMR : δ 7.40 , 3.93 , 3.73 , 4.69 , 2.27 ; 13C NMR : δ 194.3 (C=O), 168.5 (CO2CH3), 162.6 (C=CH-OCH3), 116.3(C=CH-OCH3), 62.4 (OCH3), 52.6 (CO2CH3), 46.9 (CH), 24.9 (CH3CO); tR = 10.1; EI-MS (m/z): 229 (17), 187 (74), 159 (66), 115 (100), 69 (86), 59 (82); HR-MS: calcd for C10H15O6 [M+H]+ 231.0891, found 231.0915.Isolated by silica column chromatography using hexane: ethyl acetate gradually from beginning 4:0.6 as an eluent (yield 10 %); 2 catalyst [2-catalyzed reactions of β-alkoxy/phenoxy enones and dimethyl diazomalonate were investigated in this study (E)-1-phenyl-3-(p-tolyloxy)prop-2-ene-1-one (1a) and dimethyl diazomalonate and different copper catalysts  and dimethyl diazomalonate in the presence of Cu(acac)catalyst –15. Accois study . Initialatalysts . Accordieactions .2 2  were per2-catalyzed reaction of (E)-1-phenyl-3-(p-tolyloxy)prop-2-ene-1-one (1a) and dimethyl diazomalonate, dimethyl 5-phenyl-3-(p-tolyloxy)furan-2,2(3H)-dicarboxylate (3a) was obtained as the sole product (1b (R1 = OMe) did not give any product with the diazo compound. However, a dimerization reaction of carbene occurred and only carbene dimers were obtained.In the Cu(acac) product . On the 3 = Me, 1c), yielded a 2,3-dihydrofuran derivative along with dioxole (4) and insertion (5) products. The widespread product distribution is observed in this reaction since (E)-4-methoxy-but-3-ene-2-one (1c) is less hindered than other reactants  and the reaction could not proceed chemoselectively. Carbonyl ylide intermediate of (E)-4-methoxybut-3-ene-2-one (1c) and copper-carbenoid gave a 1,5-electrocyclization reaction and formed 2,3-dihydrofuran (3c) as a major product.The reaction of the dimethyl diazomalonate with β-methoxy enone (R4) derivative as another cyclization product also occurred from the carbonyl ylide intermediate  was obtained as a mixture with 3c.Concomitantly, dioxole (rmediate . IntermeE)-4-methoxy-but-3-ene-2-one (1c) formed 5 as a minor product. Here it should be noted that no insertion product was observed in other reactions. However, 2,3-dihydrofuran (3d) was obtained as the sole product with a high yield in the copper-catalyzed reaction of ethyl 2-(ethoxymethylene)-3-oxobutanoate (1d) and dimethyl diazomalonate. Compound 1d has an ester and a ketone group in its structure, and 1d has been synthesized as two isomers. Only one of the carbonyl groups reacted with carbene to form the dihydrofuran compound (3d)  was observed from the ester-carbonyl ylide


The final derivatives were evaluated for their in vitro anti-microbial activity after thorough purification. Among all the tested compounds, the compound 12e, 12f and 12 k possess the highest growth inhibitory activity at MIC values of 4.8, 5.1 and 4.0 μg/ml respectively. The antioxidant properties of these compounds demonstrated and revealed remarkable activity compared to the standard antioxidant by using the DPPH free radical-scavenging assay. Moreover, molecular docking studies to evaluate the probable interactions with the catalytic domain of the gram-positive S. aureus topoisomerase IV enzyme may provide new insights for developing these new hybrids as potential antimicrobial agents. The binding affinities of compounds 12a-l were ranging from − 10.0 to − 11.0 kcal/mol with topoisomerase IV enzyme and with COVID-19 main protease binding affinities are ranging from − 8.2 to − 9.3 kcal/mol. These docking studies reveal that the compounds 12a-l could be the best inhibitors for the novel SARS Cov-2 virus and have more future in discovery of potent drug candidates.New series of biologically active triazole and pyrazole compounds containing 2, 4-disubstituted thiazole analogues (12a-l) were synthesized from p-hydroxy benzaldehyde and phenyl hydrazine in excellent yields and purity. All the synthesized compounds were unambiguously identified based on their spectral data analyses (IR, The online version contains supplementary material available at 10.1186/s13065-023-00965-8. The 1,2,3-triazoles are renowned scaffolds that are simple to conjugate with additional heterocyclic groups. As a result, a variety of bioactive compounds with antibacterial , 5 actio. thiamine (B1) +) ion peak, corresponding to a molecular formula C25H19N5O2. In order to construct the thiazole unit, the 1,4-disubstituted 1,2,3-triazole derivatives 8a–c were each treated with thiosemicarbazamide 9 individually to give the compounds 10a-c in excellent yield.All the compounds 12a-l were prepared in moderate to good yields via the Hantzsch reaction of compounds 10a-c treated with various substituted phenacyl bromides 11a-d in the presence of sodium bicarbonate under reflux conditions +) and corresponding to a molecular formula C34H25N8OSBr. The chemical structures of newly synthesized compounds 12b-l were confirmed by IR, 1H NMR, 13CNMR, and HRMS spectral data. The synthetic route to target compounds is given in Scheme Finally, the desired compounds nditions . The IR 12a-l) were evaluated using two strains of Gram-positive bacteria  and two strains of Gram-negative bacteria . The Minimum Inhibitory Concentration (MIC) , 4.79 , 3.30 . 13C NMR : 197.1 (CO), Ar–C , pyrazole-C , 45.5 (C), 26.9 (C), 25.9 (CH). IR: v = 3295, 1698 cm−1; ESI–MS: m/z calcd for C19H14N2O2 302; found 303 [M + 1]+.The compound  (0.1 eq) in DMF was added aryl azides (7a-c) (0.1 eq), in the presence of 10 mol % CuSO4.5H2O, sodium ascorbate and the reaction mixture was allowed to react at room temperature for 3–4 h. The reaction condition was monitored by TLC. After completion, added the ice-cold water and extracted with ethyl acetate, and the combined organic layers were removed under reduced pressure and purified by column chromatography  to get pure compounds 8 a-c with a yield of 80–85%.To the compound 1-phenyl-3-(4-(prop-2-yn-1-yloxy)-phenyl)-11H NMR  (ppm): 9.98 , 8.43 , Ar–H , 5.30 .13C NMR : 184.03 (CHO), Ar–C ,triazole-H 0.63.21 (CH2). IR: v = 3452, 2929, 1730, 1602, 1592, 1450, 1235, 1045 cm−1; Anal.Calcd. For C25H19N5O2: C, 71.25; H, 4.54; N, 16.62; Found: C, 71.24; H, 4.52; N, 16.64. ESI–MS: m/z calcd for C25H19N5O2421; found 422 [M + 1]+.White solid; yield 80%; m.p.:144–146 °C; 1H NMR  (ppm): 9.99 , 8.46 , 7.52 , Ar–H  5.30 . 13C NMR : 185.03 (CHO), Ar–C , triazole-H . 63.00 (CH2). IR: v = 3451, 2927, 1732, 1602, 1591, 1453, 1236, 1044 cm−1; Anal.Calcd. For C25H18N5O2Cl: C, 65.86; H, 3.98; N, 7.78; Found: C, 65.87; H, 3.99; N, 7.76. ESI–MS: m/z calcd for C25H18N5O2Cl 455; found 456 [M + 1]+.White solid; yield 80%; m.p.: 142–144; 1H NMR  (ppm): 10.08 , 8.55 , 7.73 , Ar–H , 5.37 , 2.38 , 2.34 . 13C NMR : 185.06 (CHO), Ar–C , triazole-C , 62.16 (CH2), 19.91 (CH3), 19.49 (CH3). IR: v = 3443, 2927, 1733, 1600, 1583, 1451, 1237, 1040 cm−1; Anal.Calcd. For C27H23N5O2: C, 76.94; H, 5.50; N, 9.97; Found: C, 76.93; H, 5.51; N, 9.96. ESI–MS: m/z calcd for C27H23N5O2449; found 450 [M + 1]+.Light white solid; yield 80%; m.p.:148–150 °C; 8a-c (1.0 mmol) and ethanol (25 mL) was added thiosemicarbazamide 9 (1.0 mmol), and the reaction mixture was refluxed for 4 h. Progress of the reaction was monitored by TLC. After completion, EtOH was removed in a vacuum. The crude product was purified by silica gel column chromatography in the ratio of ethyl acetate: hexane (25:75) to afford the pure compound 10 a-c.To a solution of substituted compound, 10 a-c (1.0 eq) and acetonitrile (25 mL) was added phenacyl bromide 11a-d (1.0 eq) in the presence of sodium bicarbonate (0.5 eq), refluxed for 1–2 h. After completion (using TLC), acetonitrile was removed in vacuo. The crude product was purified by silica gel column chromatography in the ratio of ethyl acetate: hexane (30:70) to obtain the pure compound 12 a-l in good yields.To a solution of substituted compounds 1H NMR  (ppm): 8.25 , 8.09 ,7.70 , Ar–H , 6.83 , 5.37 .13C NMR : thiazole-C , Ar–C , pyrazole-C , triazole-C ,135.55 (CN), 62.36 (CH2). IR: v = 3668, 3451, 2929, 1692, 1593, 1451, 1237, 1045 cm−1; Anal. Calcd. For C34H25N8OSBr: C, 60.63; H, 3.74; N, 16.64; Found: C, 60.61; H, 3.70; N, 16.65. ESI–MS: m/z calcd for C34H25N8OSBr 671.7; found 672.85 [M + 1]+.White solid; yield 86%; m.p.: 184–186 °C; 1H NMR  (ppm): 8.35 , 8.09 , 7.94 , Ar–H , Ar–C , triazole-C , 137.83 (CN), 62.03 (CH2). IR: v = 3668, 3453, 2929, 1692, 1602, 1593, 1451, 1237, 1045 cm−1; Anal. Calcd. For C34H25ClN8OS: C, 64.91; H, 4.01; N, 17.81; Found: C, 64.90; H, 4.00; N, 17.82. ESI–MS: m/z calcd for C34H25ClN8OS 628.8; found 629.0952 [M + 1]+.White solid; yield 82%; m.p.: 188–190 °C; 1H NMR  (ppm): 8.90 , 8.55 , 8.12 , Ar–H , 5.38 , 3.91 . 13C NMR : thiazole-C , Ar–C , triazole-C , 62.05 (CH2), 55.34 (CH3). IR: v = 3673, 3456, 2926, 1698, 1602, 1508, 1449, 1248, 1046 cm−1; Anal. Calcd. For C35H28N8O2S: C, 67.29; H, 4.52; N, 17.94; Found: C, 67.30; H, 4.50; N, 17.93. ESI–MS: m/z calcd for C35H28N8O2S 624; found 625.0 [M + 1]+.White solid; yield: 84%; m.p.: 182–184 °C; 1H NMR  (ppm): 8.23 , 8.08 , Ar–H , 5.37 .13C NMR : thiazole-C , Ar–C , triazole-C . 62.1 (CH2). IR: v = 367, 3444, 2926, 1698, 1602, 1508, 1449, 1248, 1037 cm−1; Anal.Calcd. For C34H26N8OS: C, 68.67; H, 4.41; N, 18.84; Found: C, 68.65; H, 4.40; N, 18.83. ESI–MS: m/zcalcd for C34H26N8OS 594.7; found 595.1578 [M + 1]+.White solid; yield 80%; m.p.: 176–178 °C; 1H NMR  (ppm): 8.26 , 8.08 ,7.73 , Ar–H , 6.82 , 5.36 . 13C NMR : thiazole-C , Ar–C , pyrazole-C , triazole-C , 62.0  (ppm): 8.29 , 8.08 , Ar–H , 6.82 , 5.36 . 13C NMR : thiazole-C , Ar–C , triazole-C , 62.0 (CH2). IR: v = 3664, 3452, 2932, 1690, 1602, 1599, 1451, 1246, 1033 cm−1; Anal. Calcd. For C34H24N8OSBrCl: C, 57.68; H, 3.42; N, 15.83; Found: C, 57.67; H, 3.40; N, 15.82. ESI–MS: m/z calcd for C34H24N8OSBrCl 708.1; found 709.10 [M + 1]+.White solid; yield 83%; m.p.: 192–194 °C; 1H NMR  (ppm): 8.89 , 8.54 , Ar–H , 5.39 , 3.92 .13C NMR : thiazole-C , Ar–C , triazole-C , 61.98 (CH2), 55.34 (CH3). IR: v = 3664, 3452, 2932, 1690, 1599, 1503, 1451, 1246, 1033 cm−1; Anal. Calcd. For C35H27N8O2SCl: C, 63.77; H, 4.13; N, 17.00; Found: C, 63.76; H, 4.14; N, 17.01. ESI–MS: m/z calcd for C35H27N8O2SCl 658.05; found 659.0424 [M + 1]+.White solid; yield 82%; m.p.: 186–188 °C; 1H NMR  (ppm): 8.29 , 8.08 , 7.68 , Ar–H , 6.86 , 5.38 . 13C NMR : thiazole-C , Ar–C , triazole-C , 62.04 (CH2). IR: v = 3673, 3437, 2932, 1581, 1495, 1446, 1235, 1045 cm−1; Anal. Calcd. For C34H25N8OSCl: C, 64.91; H, 4.01; N, 17.81; Found: C, 64.90; H, 4.00; N, 17.82.ESI–MS: m/z calcd for C34H25N8OSCl 628.2; found 629.1371 [M + 1]+.Yield: 85%,white solid; m.p.: 180–182 °C; 1H NMR  (ppm): 8.31, 8.02 , 7.43 , Ar–H , 6.84 , 5.37 , 3.80 , 2.33 , 2.31 . 13C NMR : thiazole-C , pyrazole-C , triazole-C , Ar–H +.White solid; yield 82%; m.p.: 170–172 °C; 1H NMR  (ppm): 8.32, 8.05 , 7.42 , Ar–H , pyrazole-C , triazole-C , Ar–H +.White solid; yield 80%; m.p.: 174–176 °C; 1H NMR  (ppm): 8.34 , 8.04 , 7.53 , Ar–H .13C NMR : thiazole-C , pyrazole-C , triazole-C , Ar–H ,61.6 (CH2), 19.9 (CH3), 19.5 (CH3). IR: v = 3535, 3435, 2928, 1578, 1448, 1236, 1048 cm−1; Anal. Calcd. For C36H29N8OSBr: C, 61.63; H, 4.17; N, 15.97; Found: C, 61.62; H, 4.18; N, 15.98.ESI–MS: m/z calcd for C36H29N8OSBr 700.0; found 701.0441 [M + 1]+.White solid; yield 84%; m.p.: 164–166 °C; 1H NMR  (ppm): 8.32 , 8.03 , Ar–H , Ar–C , pyrazole-C , triazole-C , 62.13 (CH2), 19.94 (CH3), 19.51 (CH3). IR: v = 3667, 3494, 3121, 2926, 1691, 1602, 1509, 1452, 1247, 1035 cm−1; Anal. Calcd. For C36H30N8OS: C, 69.43; H, 4.86; N, 17.99; Found: C, 69.43; H, 4.84; N, 17.98. ESI–MS: m/z calcd for C36H30N8OS 621.7; found 622.85 [M + 1]+.White solid; yield 82%; m.p.: 168–170 °C; All microbial cultures received were grown on Muller-Hinton Agar plates (20 ml/plate) at 37 °C for 24 h in sterile conditions and were treated as stock cultures. A loop full of the grown cultures was then inoculated into sterile autoclaved 10 mL Luria–Bertani Broth, Miller (LB) broth medium containing Tryptone (10 g/L), Yeast extract (5 g/L), Sodium chloride (10 g/L), pH 7.5, and kept aside in an incubator shaker for 12 h at 37 °C with continuous shaking at 220 rpm for growth. After exactly 12 h, growth was observed through the formation of turbidity in cultures and confirmed by comparison with the control which was not inoculated. Subsequently, 1% of the grown culture was re-inoculated into fresh new LB broth and incubated in the same conditions as above for another 3.5 to 4 h until it reached the log phase. The culture grew and entered into the log phase, which is very active in participating in any reactions, and was confirmed by an absorbance of OD 0.4 to 0.6 at 600 nM. Therefore, it can be recommended for testing antibacterial activity.Luria–Bertani Broth, Miller (LB broth), a potent microbiological growth medium, was prepared and sterilized using an autoclave under aseptic conditions, with 121 °C/15 Lbs pressure for 30 min, and used for antibiotic susceptibility testing. The microbial strains to be tested were grown at 37 °C for 12 h with continuous shaking at 220 rpm and maintained in the lag phase by storing them at 4 °C. The compounds synthesized for testing antimicrobial activity were dissolved in DMSO to produce a concentration of 1 mg/ml, which was treated as a stock solution. Subsequently, different concentrations of the compound ranging from minimum to maximum were tested against bacterial culture growth to determine its MIC value. The antibacterial activity of the compounds was determined by treating various concentrations of the compounds with 1ul/ml log phase cultures, followed by incubation for 12 h at 37 degrees C with continuous shaking at 220 rpm. Ampicillin, a standard drug with a 1% stock concentration, was used as a positive control for the antibacterial activity, and a blank was prepared by adding 1ul/ml culture to the broth and maintained as a negative control. Each tube contained 2 ml of fresh LB broth and 2ul of log phase bacterial culture with varying concentrations of compounds to check its MIC. All tests were performed in triplicate to validate the observed results (Mean ± SEM) by measuring the variability of data.Aspergillus niger MTCC 404 and Saccharomyces cerevisiae MTCC 1344 yeast cultures. They were obtained from the Culture Collection Division at Department of Microbiology, Osmania University and are chosen based on the nature of active most frequent contaminant of food. The fungal cultures were grown on yeast extract agar (YEP) media containing chloramphenicol (50 μg/mL) for 2–4 days at 28 °C. After 2 days, the black spores and lawn of cultures were collected and stored in an aqueous solution of 40% (v/v) glycerol at − 80 °C. Antifungal assay can be done by cylinder plate or disc method and cross streak plate method, broth dilution method. Here in the present study we used cross steak plate method to identify the minimum concentration of compound required to inhibit the fungal growth.The antifungal activities of the synthesized compounds were tested against filamentous fungi YEP agar media (commonly used to assess susceptibility of diverse mutants) was prepared by mixing the bactopeptone, sodium chloride and maintained final pH 7 at 25 °C. Sterilization of media is carried at 121 °C/15 lbs pressure for 30 min, and cooled till room temperature and poured into sterile petriplates. Aqueous culture of stock fungal strains was spreaded evenly on plates and different diluted concentration of compounds was streaked on plate. The measured zone of clear area length (mm) on YEP agar plate determined the efficacy of compound for fungal growth inhibition. Experiments were performed in triplicates to represent the data in form of Mean ± SEM.The antioxidant activity of the test compounds was checked by transferring concentrations of the compound ranging from 10 µM to 200 µM into separate sterile Eppendorf tubes. Each concentration was performed in triplicate to avoid mean differences. Transfer 100 μl of freshly prepared DPPH reagent to an Eppendorf tube and add distilled water to make up the mixture volume up to 1 mL. Gently shake and vortex the mixture and keep it in the dark at room temperature for 30 min. Further, a blank for the test is prepared by adding various concentrations of diluent compounds and a constant volume of the reagent to make the reaction up to 1 mL. The absorbance of the test compounds was measured using a UV spectrophotometer at 517 nm. Percentage antioxidant activity calculationS. aureus topoisomerase IV enzyme of catalytic domain in complex with novobiocin (PDB ID: 4URN) and the crystal structure of COVID-19 main protease (6LU7) were used as targets. Topoisomerase IV is a known efficient DNA decatenase, it plays a crucial role in separation of daughter chromosomes during DNA replication. Antibiotics like novobiocin and ampicillin acts upon bacteria by targeting this topoisomerase IV [Molecular docking studies were performed by using Vina in PyRx docking tool to predict the protein–ligand interactions at molecular level. The co-crystal structure of Gram positive erase IV , 45. Tar12a-l. The synthesized compounds 12a-l was characterized by 1H NMR, 13C NMR, ESI-Mass, HRMS spectral data. The anti-microbial activities of these compounds were evaluated against various bacterial and fungal strains. The synthesized compounds 12a, 12f, and 12 k showed good activity against the tested antimicrobial, similarly these compounds have shown potent DPPH scavenging activity and it was emerged as potential molecules for further development. Moreover, the best dock scored compounds from our current in silico study were further evaluated for minimal microbial growth-inhibitory studies. The binding affinities of compounds 12a-l with topoisomerase IV enzyme ranged from-10.0 to − 11.0 kcal/mol, while COVID-19 main protease binding affinities ranged from − 8.2 to − 9.3 kcal/mol. These docking studies reveal that the compounds 12a-l could be the best inhibitors for novel SARS Cov-2 virus and of more interested potent drug candidates.In summary, we have synthesized a new series of pyrazole, triazole, based 2,4-di substituted thiazole derivatives Additional file 1: Fig. S1.1H-NMR of 1-phenyl-3-methoxy)phenyl)-1H-pyrazole-4-carbaldehyde . Fig. S2. 13C-NMR of 1-phenyl-3-methoxy)phenyl)-1H-pyrazole-4-carbaldehyde . Fig. S3. 1H-NMR of 3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde . Fig. S4. 13C-NMR of 3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde . Fig. S5. 1H-NMR of 3--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde . Fig. S6.13C-NMR of 3--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde . Fig. S7. 1H-NMR of 4-(4-bromophenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene) hydrazinyl) thiazole . Fig. S8. 13C-NMR of 4-(4-bromophenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene) hydrazinyl) thiazole . Fig. S9. FT-IR of 4-(4-bromophenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene) hydrazinyl) thiazole . Fig. S10. Mass of 4-(4-bromophenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene) hydrazinyl) thiazole . Fig. S11. 1H-NMR of 4-(4-chlorophenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl) thiazole . Fig. S12. 13C-NMR of 4-(4-chlorophenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl) thiazole . Fig. S13. FT-IR of 4-(4-chlorophenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl) thiazole . Fig. S14. ESI-Mass of 4-(4-chlorophenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl) thiazole . Fig. S15. 1H-NMR of 4-(4-methoxyphenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl) thiazole . Fig. S16. 13C-NMR of 4-(4-methoxyphenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl) thiazole . Fig. S17. FT-IR of 4-(4-methoxyphenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl) thiazole . Fig. S18. ESI-Mass of 4-(4-methoxyphenyl)-2-meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S19. 1H-NMR of 4-phenyl-2-methoxy)phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole . Fig. S20. 13C-NMR of 4-phenyl-2-methoxy)phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole . Fig. S21. ESI-Mass of 4-phenyl-2-methoxy)phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole . Fig. S22. ESI-Mass of 4-phenyl-2-methoxy)phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole . Fig. S23. 1H-NMR of 4-(4-chlorophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S24. 13C-NMR of 4-(4-chlorophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S25. FT-IR of 4-(4-chlorophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S26. ESI-Mass of 4-(4-chlorophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S27. 1H-NMR of 4-(4-bromophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S28. 13C-NMR of 4-(4-bromophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S29. FT-IR of 4-(4-bromophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S30. ESI-Mass of 4-(4-bromophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) hydrazinyl)thiazole . Fig. S31. 1H-NMR of 2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole. Fig. S32. 13C-NMR of 2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole. Fig. S33. FT-IR of 2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole. Fig. S34. ESI-Mass of 2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole. Fig. S35. 1H-NMR of 2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-phenylthiazole. Fig. S36. 13C-NMR of 2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-phenylthiazole. Fig. S37. FT-IR of 2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-phenylthiazole. Fig. S38. ESI-Mass of 2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-phenylthiazole. Fig. S39. 1H-NMR of 2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole . Fig. S40. 13C-NMR of 2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole . Fig. S41. FT-IR of 2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole . Fig. S42. ESI-Mass of 2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole . Fig. S43. 1H-NMR of 4-(4-chlorophenyl)-2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole. Fig. S44. 13C-NMR of 4-(4-chlorophenyl)-2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole. Fig. S45. FT-IR of 4-(4-chlorophenyl)-2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole. Fig. S46. ESI-Mass of 4-(4-chlorophenyl)-2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole. Fig. S47. 1H-NMR of 4-(4-bromophenyl)-2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole. Fig. S48. 13C-NMR of 4-(4-bromophenyl)-2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole. Fig. S49. FT-IR of 4-(4-bromophenyl)-2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole. Fig. S50. ESI-Mass of 4-(4-bromophenyl)-2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole. Fig. S51. 1H-NMR of 2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)hydrazinyl)-4-phenylthiazole. Fig. S52. 13C-NMR of 2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)hydrazinyl)-4-phenylthiazole. Fig. S53. FT-IR of 2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)hydrazinyl)-4-phenylthiazole. Fig. S54. ESI-Mass of 2--1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)hydrazinyl)-4-phenylthiazole. Fig. S55. Zone of inhibition test for antimicrobial activity. Table S1. MIC of synthesized 12a-lcompounds (μg/ml) against gram-positive and gram-negative bacterial strains


Schizosaccharomyces pombeDim1 is a conserved essential component of the U4/U6.U5 tri-snRNP complex essential for pre-mRNA splicing. In a synthetic lethal screen with the temperature-sensitivedim1-35mutant, we isolated multiple alleles of non-essentialmtl16that encodes the U6 snRNA m6A methyltransferase. Further genetic analysis revealed strong and specific negative genetic interactions betweenmtl16 and a mutation in the Dim1 binding partner, Prp31, and betweendim1-35and a mutation in the Prp31 binding partner, Prp6. Our work provides additional tools to study pre-mRNA splicing inS. pombeand biological confirmation of the importance of the Prp6-Prp31-Dim1-U6 snRNA interactions for pre-mRNA splicing. S. cerevisiaeortholog is termed Dib1 (first named CDH1)S. pombe dim1-35mutant, in which amino acid 126 is changed from G to D, is defective in pre-mRNA splicinglid1-6lid1-6was isolateddim1-35strain with mousedim1cDNA expressed from thenmt1promoter inserted at the leu1locus was mutagenized with nitrosoguanidine as described dim1locus. The new alleles were not temperature-sensitive on their own. Further genetic analyses indicated that all 7 of these strains contained mutations in the same gene and they were notlid1alleles. A genomic library constructed in the pUR18 vectormtl16+geneS. cerevisiae. While the human ortholog, METTL16, is essential,S. pombeMtl16 facilitates pre-mRNA splicing of just a subset of transcripts via m6A methylation of U6 snRNA and its deletion displays only a mild pre-mRNA splicing defectmtl16gene was amplified from each of the 7 isolated strains and sequenced to determine the positions of the inactivating mutations , suggesting an impact on transcripts necessary for spindle functiondim1-35, this is consistent with the significant mitotic defects ofdim1-35cellsprp31-E1, prp6-1, andprp1-1 using the mitotic checkpoint defective mutant,mph1∆prp31-E1and particularlyprp6-1 were sensitive to TBZ, unexpectedly, we did not detect sensitivity ofmtl16∆ to TBZ. Thus, TBZ sensitivity appears to be a common phenotype associated with defective pre-mRNA splicing and perhaps related to the level of splicing deficiency. Structures of the U4/U6.U5 tri-snRNP determined by cryo-electron microscopy indicate that Dib1/Dim1 is situated adjacent to the invariant ACAGAGA sequence of U6 snRNA in the catalytic core of the spliceosomeS. pombeU6 snRNA, that is modified by Mtl16Description Dim1 is a highly conserved and essential thioredoxin-like protein that is part of the U4/U6.U5 tri-snRNP splicing complextations . Two alts tail . That aprotein . Themtt codon .mtl16:dim1-35 but viadim1-351F. Thisndprp6. Likedprp6-1. Many mYeast methods:S. pombestrains were grown in yeast extract (YE) or Edinburgh minimal medium (EMM) supplemented with appropriate amino acids with appropriate supplements and standardS. pombemating, sporulation, and tetrad dissection techniques were used for backcrossing, outcrossing, and to construct new strainsdim1from the nmt1promoter. To disrupt themtl16gene, a portion of the protein coding region in a pUC18 genomic clone was replaced withura4+ and 100 bp downstream of the stop codon (GGCTATTCAATATAAGAAGATTACCAA) (Integrated DNA technologies). The PCR product was sequenced with two additional oligonucleotides; a forward oligonucleotide at 635 bp within the mtl16 genomic DNA (CTTACTCCCTGATACCGAAGGGATG) and a reverse oligonucleotide at 780 bp within the genomic mtl16 DNA (CATTTCATGATAAACTCCAGTACAAACAC).The strains used in this study and their genotypes are listed below.Strain Genotype Source-ura4-D18 leu1-32 ade6-M210 hLab stockKGY246mtl16-R247C leu-32 ura4-D18- hThis studyKGY1193mtl16-G104D leu-32 ura4-D18- hThis studyKGY1194mtl16-W209stop leu-32 ura4-D18- hThis studyKGY1195mtl16-R378stop leu-32 ura4-D18- hThis studyKGY1196mtl16-P276stop leu-32 ura4-D18- hThis studyKGY1197mtl16-G104D leu-32 ura4-D18- hThis studyKGY1198mtl16-G215D leu-32 ura4-D18- hThis studyKGY1201mtl16-G104D dim1-35 leu1-32::nmt81-dim1This studyKGY1224ade6-M216h-+ura4-D18 leu1-32 ade6-M21X h-mtl16::ura4This studyKGY1301+ura4-D18 leu1-32 ade6-M21X h+mtl16::ura4This studyKGY1844prp31-E1 ade6-M210leu-32 ura4-D18- hKGY2457- prp1-1 ura4-D18 leu1-32 ade6-M21X hKGY1142+ura4-D18 leu1-32prp1-1 mtl16::ura4This StudyKGY8109-ade6-M21X h-prp1-4 ura4-D18 leu1-32 ade6-M210 hKGY2551+ura4-D18 leu1-32prp1-4 mtl16::ura4This StudyKGY8110-ade6-M210 h-prp7-1 ura4-D18 leu1-32 ade6-M210 hKGY7218+ura4-D18 leu1-32prp7-1 mtl16::ura4This StudyKGY8468+ade6-M21X h-prp12-1 ura4-D18 leu1-32 ade6-M210 hKGY8432+ura4-D18 leu1-32prp12-1 mtl16::ura4This StudyKGY8543-ade6-M21X h-prp6-1 ura4-D18 leu1-32 ade6-M210 hKGY1877+ura4-D18 leu1-32prp6-1 mtl16::ura4This StudyKGY8544+ade6-M21X hdim1-35 leu1-32:nmt81-dim1cDNA-ura4-D18 hLab stockKGY2671+mtl16::ura4+prp31-E1 ura4-D18 hThis studyKGY8111


H-pyrrol-1-yl)-N′-(2-(substituted)acetyl) benzohydrazides (5a–n) were prepared and new heterocycles underwent thorough characterization and evaluation for antibacterial activity; some of them underwent further testing for in vitro inhibition of enoyl ACP reductase and DHFR enzymes. The majority of the synthesized molecules exhibited appreciable action against DHFR and enoyl ACP reductase enzymes. Some of the synthesized compounds also showed strong antibacterial and antitubercular properties. In order to determine the potential mode of action of the synthesized compounds, a molecular docking investigation was conducted. The results revealed binding interactions with both the dihydrofolate reductase and enoyl ACP reductase active sites. These molecules represent excellent future therapeutic possibilities with potential uses in the biological and medical sciences due to the compounds’ pronounced docking properties and biological activity.In this study, a new series of 4-(2,5-dimethyl-1 Mycobacterium tuberculosis (Mtb) is a slow-growing, acid-fast bacterium with a highly impermeable cell wall. Mtb is an opportunistic pathogen that can remain dormant in macrophages for years before reactivating in people with weakened immune systems, such as those who are also HIV-infected. The rise of extensively drug-resistant (XDR) TB and the expansion of multidrug-resistant (MDR) TB have revived efforts to find new antitubercular drugs. As a result, there is a critical unmet medical need for TB medications that are safer and more potent and that work through various mechanisms [The WHO’s 2022 report lists tuberculosis (TB) as the eighth most common cause of death globally and the most common infectious disease, surpassing HIV/AIDS. The TB-causing bacteria chanisms .Several medications that include nitrogen heterocycles are now being tested in clinical studies to treat TB . PyrroleInhibitors with excellent specificity and the capacity to distinguish between bacterial and host DHFR have found use as antibacterial drugs since bacteria need DHFR to thrive and multiply. These remarkable characteristics make the DHFR enzyme an important target for the development of both antibacterial and antitubercular drugs.Finding new therapeutic targets relies heavily on academic research, especially with regard to understanding target biology and linkages between targets and disease states. However, research must advance from basic study to the identification and testing of a drug candidate in clinical trials, which are normally carried out by the biopharmaceutical industry, if it is to result in the development of novel pharmaceuticals. Timely attention to target evaluation factors such as target-related safety problems, druggability, and assayability, as well as the possibility for target manipulation to achieve differentiation from current medicines, might help ease this shift ,11,12.The goal of this effort was to synthesize some novel pyrrole derivatives with strong antibacterial activity in order to create new therapeutically active units. These compounds were examined for activity against typical infections. The inhibition of the enzymes DHFR and enoyl-ACP reductase was also tested for the active derivatives. Finally, the action target and binding manner were confirmed by molecular docking studies.H-pyrrol-1-yl)-N′-(2-(substituted)acetyl)benzohydrazides (5a–n) were prepared via the reaction of 4-benzohydrazide 3 and substituted phenyl acetic acids in distilled N,N-dimethyl formamide using coupling agent 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine as a catalytic reagent under cold conditions , which was equivalent to its molecular weight.In the To obtain connections and a direction to the active pocket for comparison with other recently synthesized molecules; the 2NSD ligand was re-docked5b had three hydrogen bonding interactions at the enzyme’s active site (PDB ID: 2NSD), two of which were between the oxygen atom of the CO-NH group and the hydrogen atoms of TYR158 and NAD300  and one of which was between the oxygen atom of the NHCO group and the hydrogen atom of NAD300 . As portrayed in 5e demonstrated three hydrogen bonding interactions, including two communications between the oxygen atom of the CO-NH group and the hydrogen atoms of TYR158 and NAD300  as well as a residual hydrogen bonding interface between the oxygen atom of the methoxy group and the hydrogen atom of MET98 . The docked image of the binding interaction of 1DF7_ligand with enzyme active sites shown in As shown in Another docking investigation of PDB ID: 1DF7 indicated that all of the molecules had extremely good docking scores against the combination of NADPH and methotrexate and Mycobacterium TB’ dihydrofolate reductase forms. The findings of our study were in line with previously studies, which reported the molecular docking and three-dimensional quantitative structural analysis studies of potent inhibitors of enoyl-acyl carrier protein reductase as possible antimycobacterial agents ,14.5a underwent three hydrogen bonding interactions at the enzyme’s active site (PDB ID: 1DF7), two of which were raised by the oxygen of the C=O group of benzohydrazide and involve the hydrogen atoms of ARG60  and hydrogen atom of NH group makes an interaction with oxygen atom of GLN28 . As shown in 5h formed three hydrogen bonds (PDB ID: 1DF7). These bonds were formed by the oxygen atom of the C=O group of benzohydrazide and the hydrogen atoms of ARG60 and ARG32 . As shown in 5b (cyan) and 5e (blue) with the corresponding 2NSD_ligand (green) and the superimposition of the active compounds 5a (cyan) and 5h (blue) with the corresponding 1DF7_ligand (green) are depicted in The superimposition of the active compounds All of the compounds demonstrated consensus scores in the range of 6.09 to 4.29 as shown in M. tuberculosis H37Rv strain as well as an antibacterial investigation against S. aureus (Gram +ve) and E. coli (Gram −ve) are shown in 5f, 5i, 5j, and 5n demonstrated greater action. At an MIC of 0.8 µg/mL, compound 5k exhibited the highest level of activity. Compounds exhibited antibacterial activity (expressed as MIC) in the range of 0.8 to 100 µg/mL. Previous studies have reported similar findings with novel quinolinone-based thiosemicarbazones and novel analogs of 2,4-disubstituted quinoline derivatives against M. tuberculosis [The results of the MABA method used to test all compounds for antitubercular activity against the rculosis ,18,19.50 value of 92 μM, which was in agreement with the literature data (88 μM). The majority of the studied molecules were noticeably more effective against MtDHFR (5d–5n), were stronger than those of trimethoprim.Our chemical series’ in vitro MtDHFR inhibitory activity was assessed by monitoring the fluorescence released by MtDHFR substrates when stimulated at 340 nm . As the t MtDHFR . The inhThe various in silico techniques, including molecular docking and the quantitative structure–activity relationship (QSAR), are crucial for obtaining this background information in drug discovery projects . Multidi5l and 5n did not cross BBB. The compounds showed moderate skin permeability in the range of −5.70 to −6.33. The results are shown in The ADME properties of all of the synthesized molecules were determined by using the Swiss ADME web tool, and all of the molecules complied with Lipinski’s rule of five. Swiss ADME studies showed that all of the molecules had good synthetic accessibility, moderate solubility, and GI absorption. Only compounds The toxicological summary of all of the compounds was determined by using ProTox-II, and the toxic effects are depicted in 5c, 5d, 5f, 5i, and 5k) toward the mammalian Vero cell lines and A549 (lung adenocarcinoma) cell lines up to MIC values of 62.5 µg/mL. These tested molecules showed a modest cytotoxicity compared to the standard INH /dimethylsulfoxide (DMSO-d6) as the solvent and TMS as the internal standard. The NMR spectra were split into singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartret (q), and multiplet (m). All of the compounds’ mass spectra on a Water-Q-Tof Premier-HAB213 showed data that were compatible with the projected structure. Analytical thin layer chromatography (TLC) was carried out using Silica Gel GF, and the solvent’s movement was detected using an ultraviolet lamp.The melting points of synthesized compounds were calculated using the SHITAL-Digital programmable melting point equipment (SSI -22(B)) and occasionally using a Thiles Tube and were uncorrected; IR spectra were obtained using KBr pellets on a Bruker-T spectrophotometer. Chemical shifts are expressed as δ values (ppm) for the H-pyrrol-1yl)benzoic acid hydrazide 3 (1.8 mmol) was dissolved. Then, under cold conditions (0–5 °C), substituted phenyl acetic acid (1.9 mmol) was added to it [2CO3 solution (20 mL) and 1N HCl (15 mL). After that, a 25% aq NaCl solution (10 mL) was added to the process. The combined ethyl acetate extract was concentrated with rotavapor after drying over anhydrous sodium sulphate. The resulting residue was purified using column chromatography by eluting with ethyl acetate:petroleum ether (6:4).In dry DMF (20 mL), 4-: 3245 (NH), 3025 (NH), 2926 (Ar-C=CH), 1679 (C=O), 1647 (C=O).Light brown amorphous solid. (Yield 87%). M.p106–108 °C; FTIR : 10.14 , 9.70 , 7.90-7.88 , 7.29-7.17 , 5.89 , 3.65 , 2.03 .13C NMR : 169.30 (-NHCO), 164.21 (-CONH), 133.94 (phenyl-C1), 128.61  128.39 (bridging phenyl-C4) 128.51 , 128.25 , 127.38 , 127.31 (phenyl-C4), 106.58 , 41.09(-CH2), 15.25 (2CH3).m/z): found 348.3158 (M+ + 1); Calcd. 347.42.Mass : 3232 (NH), 2922 (Ar-C=CH), 1618 (C=O), 1570 (C=C).Light brown amorphous solid. (Yield 76%). M.p109–112 °C; FTIR : 9.97 , 8.96 , 7.88-7.86 , 7.25-7.10 , 5.89 , 3.67 , 2.32 , 1.98 .13C NMR : 168.94 (-NHCO), 163.98 (-CONH), 142.66 (bridging phenyl-C1), 137.21 (phenyl-C2), 131.91 (phenyl-C1), 130.85 (phenyl-C3), 130.34 (phenyl-C4), 130.22 , 128.56 , 128.33 (bridging phenyl-C4), 128.28 (phenyl-C6), 126.63 (phenyl-C5), 128.09 , 106.59 , 39.45 (-CH2), 19.58 (CH3), 13.06 (2CH3).m/z): found 362.3349 (M+ + 1); Calcd. 361.45.Mass : 3422 (NH), 3259 (NH), 2922 (Ar-C=CH), 1625 (C=O), 1493 (C=C).Light brown amorphous solid. (Yield 65%). M.p95–98 °C; FTIR :10.03 , 9.45 , 7.90 , 7.25-7.10 , 5.89 , 3.60 , 2.30 , 1.98 .13C NMR : 168.55 (-NHCO), 163.59 (-CONH), 142.71 (bridging phenyl-C1), 137.41 (phenyl-C4), 130.31 (phenyl-C1), 129.77 , 129.23 , 128.58 , 128.42 (bridging phenyl-C4), 128.21 , 106.59 , 38.62 (-CH2), 21.10 (CH3), 13.05 (2CH3).m/z): found 362.3388 (M+ + 1); Calcd. 361.45.Mass : 3422 (NH), 3259 (NH), 29223(Ar-C=CH), 16259 (C=O), 1493 (C=C).Light brown amorphous solid. (Yield 78%). M.p118–120 °C; FTIR :10.04 . 9.47 , 7.91 , 7.25-7.17 , 6.83 , 5.89 , 3.74 , 3.58 , 1.98 .13C NMR : 169.21 (-NHCO), 163.87 (-CONH), 142.68 (bridging phenyl-C1), 159.04 (phenyl-C4), 130.41 (phenyl-C1), 130.24 , 128.54 , 128.35 , 128.29 , 114.40 , 125.3 (bridging phenyl-C4), 106.61 , 55.26 (OCH3), 40.44 (-CH2), 13.05 (2CH3).m/z): found 378.3364 (M+ + 1); Calcd. 377.44.Mass : 3180 (NH), 3026(NH), 2970 (Ar-C=CH), 1606 (C=O).Light brown amorphous solid. (Yield 85%). M.p138–142 °C; FTIR :9.78 . 9.18 , 7.88 , 7.29-6.91 , 5.89 , 3.90 , 3.67, 2.03 .13C NMR : 168.32 (-NHCO), 163.11 (-CONH), 157.04 (phenyl-C2), 142.50 (bridging phenyl-C1),131.17 (phenyl-C6),130.17 (bridging phenyl-C4), 129.23 , 128.58 , 128.33 (phenyl-C4), 128.19 , 122.27 (phenyl-C1), 122.19 (phenyl-C5), 110.83 (phenyl-C3), 106.52 , 55.62 (OCH3), 36.83 (-CH2), 13.05 (2CH3).m/z): found 378.2687 (M+ + 1); 379.2685 (M+ + 1); Calcd. 377.44Mass : 3227 (NH), 3029 (NH), 2923 (Ar-C=CH), 1695 (C=O), 1620 (C=O).Light brown amorphous solid. (Yield 67%). M.p130–132 °C; FTIR :10.23 . 10.04 , 7.92 , 7.25-7.16 , 5.89 , 3.57 , 1.97 .13C NMR : 168.32 (-NHCO), 163.99 (-CONH), 142.88 (bridging phenyl-C1), 133.53 (phenyl-C4), 132.18 (phenyl-C1),130.59 , 130.04 , 129.03 , 128.52 , 128.44 (bridging phenyl-C4), 128.27 , 106.71 , 40.39 (-CH2), 13.06 (2CH3).m/z): found 382.2198 (M+ + 1), 384.2190 (M+ + 3); Calcd. 381.86.Mass : 3222 (NH), 3031 (NH), 2923 (Ar-C=CH), 1695 (C=O), 1611 (C=O).Light brown amorphous solid. (Yield 70%). M.p145–147 °C; FTIR : 10.38 , 9.77 , 7.92 , 7.31-7.13 , 5.88 , 3.78 , 1.97 .13C NMR : 167.72 (-NHCO), 163.86 (-CONH), 142.64 (bridging phenyl-C1), 134.51 (phenyl-C1), 131.88 (phenyl-C6), 131.59 (bridging phenyl-C4), 130.17 , 129.73 (phenyl-C3), 129.12 (phenyl-C2), 128.55 , 128.36 (phenyl-C4), 128.30 , 127.27 (phenyl-C5), 106.59 , 38.96 (-CH2), 13.06 (2CH3).m/z): found 382.2158 (M+ + 1), 383.21.50 (M+ + 2); Calcd. 381.86.Mass : 3450 (NH), 3026 (NH), 2922 (Ar-C=CH), 1606 (C=O).Light brown amorphous solid. (Yield 55%). M.p104–108 °C; FTIR :10.29 , 10.20 , 7.93 , 7.30-7.12 , 5.88 , 3.59 , 2.03 .13C NMR : 167.88 (-NHCO), 163.88 (-CONH), 142.85 (bridging phenyl-C1), 134.71 (phenyl-C1), 134.61 (phenyl-C3), 130.08 (phenyl-C5), 129.37 , 128.55 , 128.45 , 128.28 (bridging phenyl-C4), 127.71 , 127.44 (phenyl-C2), 106.67 , 40.60 (-CH2), 13.06 (2CH3).m/z): found 382.2158 (M+ + 1); Calcd. 381.86.Mass : 3450 (NH), 2922 (Ar-C=CH), 1622 (C=O).Brown amorphous solid. (Yield 72%). M.p100–104 °C; FTIR : 10.06 . 9.41 , 7.95 , 7.52-7.21 , 7.14 , 5.89 , 3.82 , 1.98 .13C NMR : 168.98 (-NHCO), 164.75 (-CONH), 141.20 (bridging phenyl-C1), 135.03 (phenyl-C1), 131.76 , 131.25 , 128.38 , 128.25 , 127.87 (bridging phenyl-C4),127.43 , 119.67 (phenyl-C4), 106.38 , 38.92 (-CH2), 12.77 (2CH3).m/z): found 426.1733 (M+), 428.1699 (M+ + 2); Calcd. 426.31.Mass : 3445 (NH), 2987 (Ar-C=CH), 1611 (C=O).Light brown amorphous solid. (Yield 84%). M.p123–125 °C; FTIR :10.06 . 9.40 , 7.91 , 7.53-7.09 , 5.89 , 3.82 , 1.98 .13C NMR : 167.27 (-NHCO), 164.75 (-CONH), 142.67 (bridging phenyl-C1), 133.55 (phenyl-C1), 133.11 (phenyl-C3), 130.24 (phenyl-C4), 129.40 , 1129.26 (phenyl-C5), 128.58 (phenyl-C6), 128.38 , 128.30 (bridging phenyl-C4), 127.99 , 125.00(phenyl-C2), 106.58 , 41.62 (-CH2), 13.07 (2CH3).m/z): found 426.1691 (M+ + 1), 428.1699 (M+ + 2); Calcd. 426.31.Mass : 3448 (NH), 2937 (Ar-C=CH), 1646 (C=O).Light brown amorphous solid. (Yield 73%). M.p134–137 °C; FTIR : 10.50 . 10.25 , 9.24 , 8.01-7.15 , 5.83 , 3.57 , 1.99 .13C NMR : 168.71 (-NHCO), 163.83 (-CONH), 161.53 (phenyl-C4), 143.24 (bridging phenyl-C1), 131.81 (phenyl-C1), 131.57 (phenyl-C2), 130.19 (phenyl-C6), 129.73 , 129.32 , 128.37 , 128.32 (bridging phenyl-C4), 116.51 , 106.50 , 38.76 (-CH2), 12.96 (2CH3).m/z): found 366.2451 (M+ + 1), 367.2485 (M+ + 2); Calcd. 365.41.Mass : 3446 , 2922 (Ar-C=CH), 1643 (C=O).Light brown amorphous solid. (Yield 65%). M.p 190–193 °C; FTIR : 10.45 . 10.14 , 9.24 , 8.02 , 7.39-6.71 , 5.83 , 3.42 , 1.99 .13C NMR : 169.11 (-NHCO), 167.85 (-CONH), 163.83 (phenyl-C4), 142.85 (bridging phenyl-C1), 134.64 , 129.37 (bridging phenyl-C4), 128.50 , 128.49 , 128.28 (phenyl-C1), 127.74 , 115.64 , 105.65 , 39.63 (-CH2), 13.01 (2CH3).m/z): found 364.3542 (M+ + 1), Calcd. 363.42.Mass : 3447 (NH), 2924 (Ar-C=CH), 1641 (C=O).Light brown amorphous solid. (Yield 57%). M.p 143–149 °C; FTIR : 10.30 . 10.04 , 7.99 , 7.25-7.16 , 5.99 , 3.55 , 1.97 .13C NMR : 169.25 (-NHCO), 167.21 (-CONH), 143.63 (bridging phenyl-C1), 134.05 (phenyl-C1), 133.13 (phenyl-C4), 131.62 (phenyl-C5), 130.24 (phenyl-C2), 129.06 , 128.88 , 128.58 (bridging phenyl-C4), 128.28 (phenyl- C6), 127.79 , 125.06 (phenyl-C3), 106.10 , 40.62 (-CH2), 13.03 (2CH3)m/z): found 426.1225 (M+ + 1), Calcd. 425.07.Mass : 3252 (NH), 1703 (C=O), 1640 (C=O).Light brown amorphous solid. (Yield 78%). M.p 172–173 °C; FTIR : 10.45 . 10.14 , 8.01 , 7.39 , 7.15 , 6.72 , 5.83 , 3.47 , 1.98 .13C NMR : 169.20 (-NHCO), 164.86 (-CONH), 148.65 (phenyl-C4), 146.69 (bridging phenyl-C1), 142.63 (phenyl-C1), 130.34 , 128.24 (bridging phenyl-C4), 128.35 , 128.29 , 126.63 , 125.43 , 106.61 , 30.34 (-CH2), 13.05 (2CH3).m/z): found 493.4536 (M+ + 1), Calcd. 392.42Mass , the crystal structures of enoyl acyl carrier protein reductase InhA in association with N-(4-methylbenzoyl)-4-benzylpiperidine  and dihydrofolate reductase of Mycobacterium tuberculosis complexed with NADPH and methotrexate were obtained. The ligands and protein used in our docking technique were produced in accordance with the Sybyl-X 2.0 standard protocol [Utilizing the patented Sybyl-X 2.0 search engine, Surflex-Dock was employed for molecular docking to clarify the binding mechanism of chemicals at the active sites of the ENR enzyme and dihydrofolate reductase enzyme . This prprotocol ,31. All ProTox-II was used to predict toxicities (the results are presented in 50) of some of the compounds against A549 (lung adenocarcinoma) MV cell lines up to a concentration of 50 mg/mL using a Promega Cell Titer 96 non-radioactive cell proliferation assay with cisplatin as the positive control. The IC50 values given in Cellular conversion of MTT -2,5-diphenyl-tetrazolium bromide) into a formazan product was performed to evaluate the cytotoxic activity (ICM. tuberculosis strain H37Rv by means of a Microplate Alamar Blue assay (MABA). The results are reported in All of the newly synthesized compounds were evaluated against S. aureus (Gram +ve) and E. coli (Gram-ve) using a broth microdilution method [An antibacterial inhibition study of all molecules was conducted in comparison with ciprofloxacin as the reference drug against n method ,35. Anti5f, 5i, and 5k showed promising enzyme inhibitory activity (on both the enzymes) during the in vitro assay. Due to the size of the present novel molecules used to minimize M. tuberculosis growth, additional modification of molecular fragment changes through molecular modeling and 3D-QSAR studies might be considered as new leads for the development of druggable candidates.All of the 14 newly synthesized pyrrolyl benzohydrazide derivatives exhibited moderate to good antitubercular potency with MICs that ranged from 0.8 to 25 µg/mL. All of the derivatives were also analyzed in a molecular docking study, and these reported novel inhibitors fitted closely with the binding site of the ENR enzyme and DHFR enzyme in a manner similar to that of the 2NSD_ligand and 1DF7_ligand. Compounds


The structures of the new compounds were determined by 1D and 2D nuclear magnetic resonance (NMR) and other spectroscopic methods, as well as computational methods. Selected compounds were evaluated for their biological properties against a mouse pancreatic cancer cell line and inhibitory effects on iNOS and COX-2 in RAW264.7 cells.Six new compounds, including a tetralone  K. svenhedini  is a millipede species first described by Verhoeff in 1934 + ion peak at m/z 251.1274 . The 1H NMR data ], two methoxy signals [δH 3.96  and 3.73 ], and two methyl signals [δH 2.51  and 1.09 ]. The 13C NMR and DEPT spectra of compound 1 (2 and one of them oxygenated), one ketone, and five sp2 carbons (two of them oxygenated). Some of these signals resemble those of 8-O-methylteratosphaerone B + ion peak at m/z 317.1008 . The 1H NMR data ], a methoxy signal [δH 3.95 ], and a methyl signal [δH 2.48 ]. The 13C NMR and DEPT spectra . The 1H and 13C NMR data and the molecular formula indicated that the compound possesses the same two pentasubstituted benzene rings, suggesting an axially symmetric structure. The methoxy group is situated at C-3, as determined by the HMBC correlation (3-11/C-3 (δC 154.1). The hydroxy group and the methyl group are positioned at C-2 and C-4, respectively, as deduced by comparing the HMBC correlations of H-1/C-2 (δC 148.7), C-9a (δC 118.1), C-9 (δC 178.5), and H3-12/C-3, C-4 (δC 121.6), C-4a (δC 151.0), C-11  with those in the literature + ion peak at m/z 257.0802 . The 1H NMR data , 7.39 ], which corresponds to a 1,2,4,5-tetrasubstituted benzene substructure. Additional aromatic proton signals [δH 7.38  and δH 7.16 ] suggest the presence of a 1,2,3,5-tetrasubstituted benzene substructure. The 13C NMR and DEPT spectra of 3 . The two methyl groups are positioned at C-3 and C-5, as deduced by the HMBC correlations (3-11/C-2 (δC 153.8), C-3 (δC 137.5), C-4 (δC 120.5) and H3-12/C-5 (δC 130.2), C-6 (δC 126.1), C-10a (δC 150.1), C-8a . By comparing the HMBC correlations of H-1/C-2, C-9a (δC 120.5), C-9 (δC 179.1), and H-8/C-7 (δC 154.4), C-9 (δC 179.1) with those in the literature + ion peak at m/z 357.1680 . The 1H NMR data , 6.31 , and 6.26 ], suggesting the presence of a 1,2,4-trisubstituted benzene substructure. Additionally, two aromatic signals at δH 6.86  and δH 6.84  are observed in the 1H NMR spectrum, indicating the presence of a 1,3,4,5-tetrasubstituted benzene substructure. The 13C NMR and DEPT spectra of 4 (2 and two of them oxygenated), one oxygenated carbon, and seven sp2 carbons (four of them oxygenated). Based on this information, compound 4 was deduced to be similar to daphnegiralin C1 + ion peak at m/z 269.2116 . The 1H NMR spectrum  and 5.12 ], one oxymethylene group [δH 3.59 ], and three methyl signals . The 13C NMR and DEPT spectra of compound 7 (2), one carbonyl carbon, and two sp2 quaternary carbons. The 1H–1H COSY spectrum /H-3 /H2-4 /H2-5 /H-6 (δH 5.12), H-3 (δH 1.93)/H2-15 (δH 0.96), H2-8 /H2-9 /H-10 (δH 5.18), and H2-12 /H2-13 , which revealed three partial structures a (C-2 to C-6), b (C-8 to C-10), and c (C-12 to C-13). The partial structures a and b were connected to C-7 by the correlations of H3-16 (δH 1.61)/C-6 (δC 125.6), C-7 (δC 136.0), C-8 (δC 40.7), and H-8/C-6, C-7 in the HMBC spectrum /C-10 (δC 127.4), C-11 (δC 132.9), C-12 (δC 43.8), and H-12/C-10, C-11. The presence of a conjugated carboxylic acid was verified by the HMBC correlation of H2-2 to C-1 (δC 177.1). Concerning the geometry of 7, the ROESY correlation  deduced from its HRESIMS [M + H]+ ion peak at m/z 283.2268 . The 1H NMR data ], one oxymethylene group [δH 3.97 ], and three methyl signals . The 13C NMR and DEPT spectra (2), one carbonyl carbon, and two sp2 quaternary carbons. A comparison of the NMR data of compound 8 /H2-13 /H2-14  and the HMBC correlations (C 126.0), C-11 (δC 135.2), and H-13/C-11 revealed the structure of C-12 to C-14, which differs from 7. Owing to the overlapping signals between H2-5 and H2-8, H2-9 and H2-12, the geometry of the double bonds was determined as 6E,10E through a comparison of the 1H and 13C chemical shifts with similar compounds D25 +9.38 ; UV (MeOH) λmax, nm (log ε): 208 (2.92), 229 (2.88), 275 (2.78); ECD (MeOH) λ, nm (Δε): 210 (−1.88), 231 (+0.34), 251 (+0.02), 273 (−0.41), 327 (+0.39); HRESIMS (m/z): [M + H]+ calcd for C14H19O4, 251.1278; found, 251.1274; 1H and 13C NMR data, see Kronopoiol A (2): brown solid; UV (MeOH) λmax, nm (log ε): 202 (3.56), 242 (3.58), 280 (3.13), 324 (3.15), 367 (2.89); HRESIMS (m/z): [M + H]+ calcd for C17H17O6, 317.1020; found 317.1008 ; 1H and 13C NMR data, see Kronopoiol B (3): brown solid; UV (MeOH) λmax, nm (log ε): 201 (3.18), 241 (3.44), 260 (3.22), 324 (2.73), 378 (2.69); HRESIMS (m/z): [M + H]+ calcd for C15H13O4, 257.0808; found, 257.0802 ; 1H and 13C NMR data, see 5-O-Methyldaphnegiralin C1 (4): brown solid; [α]D25 +30.77 ; UV (MeOH) λmax, nm (log ε): 207 (3.92), 280 (3.14); ECD (MeOH) λ, nm (Δε): 208 (+15.19), 237 (−1.90), 250 (+0.23), 283 (−0.71); HRESIMS (m/z): [M + H]+ calcd for C21H25O5, 357.1697; found, 357.1680; 1H and 13C NMR data, see Kronoponoid A (7): light yellow gum; [α]D25 +7.50 ; UV (MeOH) λmax, nm (log ε): 202 (3.68); HRESIMS (m/z): [M + H]+ calcd for C16H29O3, 269.2111; found, 269.2116 ; 1H and 13C NMR data, see Kronoponoid B (8): light yellow gum; [α]D25 +2.50 ; UV (MeOH) λmax, nm (log ε): 202 (3.81); HRESIMS (m/z): [M + H]+ calcd for C17H31O3, 283.2268; found, 283.2268 ; 1H and 13C NMR data, see R,4R)-1, -1, -4, -4, -4, and -4 using the Spartan'14 software package and the Gaussian 09 program package. The ECD calculations of the predominant conformers (80%) were conducted using DFT calculations at the B3LYP/6-311G level of theory. The program SpecDis 1.62 was used to generate the CD spectra [The CONFLEX 7 searches, considering the Molecular Merck force field (MMFF94) and DFT/TDDFT calculations, were performed for model compounds  containing 10% fetal bovine serum , 100 U/mL penicillin, 10 μg/mL streptomycin, and 10 μg/mL puromycin. Cells were seeded at a density of 5000 cells/well in 96-well plates under the same incubation conditions. Following overnight culture, cells were pretreated with compounds at a 20 μM concentration or DMSO for 48 h, with gemcitabine as a positive reference drug. Subsequently, Cell Count Kit-8  was added to each well at a 10 μL concentration for 2 h. The absorbance at 450 nm was measured using a microplate reader .Panc02-h7-GP-GFP cells (derived from the transformation of mouse pancreatic cancer cell line Panc02-h7) were maintained at 37 °C in a 5% COThis assay, along with the associated experimental procedures, received approval from the Institutional Animal Care and Use Committee of Shenzhen University Health Science Center and the Animal Experimentation Ethics Committee of Shenzhen University Health Science Center (AEWC-202300026). All animal housing and handling adhered to the research ethics guidelines set forth by the Institutional Animal Care and Use Committee of Shenzhen University Health Science Center and the Animal Experimentation Ethics Committee of Shenzhen University Health Science Center.6 cells per mouse). After 14 days, tumors were excised, digested with a digestion solution, ground, and centrifuged to produce a cell suspension. Lymphocytes were isolated from the cell suspension using the Percoll method and were further enriched (via negative selection) to obtain naive CD8+ T cells. The enrichment effect and phenotype of the CD8+ cells were detected by flow cytometry . CD8+ T cells and Panc02-h7-GP-GFP cells were co-cultured with the corresponding concentrations of compounds or DMSO for 18 h. Fluorescence intensity was measured using a microplate reader .Panc02-h7-GP-GFP cells were treated with trypsin  and resuspended in PBS  following centrifugation. The cell suspension was then injected into the pancreas of mice  solution was added and incubated for 1 h. The absorbance of the solution in the 96-well plate was measured using a microplate reader , and the cell survival rate was calculated.RAW264.7 (a mouse macrophage cell line) cells were incubated in high-glucose DMEM  containing 10% fetal bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in an atmosphere of 5% COWestern blot analysis was used to detect protein levels in cells. RAW264.7 cells were pre-treated with the corresponding concentration of the compound or DMSO for 2 h and then stimulated with lipopolysaccharide  for 12 h. Subsequently, radioimmunoprecipitation assay (RIPA) buffer  containing a protease inhibitor  was used to extract total protein from cells. Protein content samples was determined by the BCA assay .Equivalent protein extracts were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA and incubated with the indicated antibodies overnight at 4 °C. Following this, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature. The ECL kit  and analysis system  were used to visualize and detect the bands. Immunoblot densitometric analysis results were processed using ImageJ software .File 1+ T cells in vitro.NMR, HRESIMS, and CD spectra for new compounds and the figures of antitumor activity assay of CD8


H)-one derivatives were synthesized, and their anticancer, antioxidant, and 11β-hydroxysteroid dehydrogenase (11β-HSD) inhibitory activities were tested. Anticancer activity has been assessed using the MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay against human colon carcinoma (Caco-2), human pancreatic carcinoma (PANC-1), glioma (U-118 MG), human breast carcinoma (MDA-MB-231), and skin melanoma (SK-MEL-30) cancer cell lines. Cell viability reductions, especially in the case of Caco-2, MDA-MB-231, and SK-MEL-30 lines, were observed for most compounds. In addition, the redox status was investigated and oxidative, but nitrosative stress was not noted at a concentration of 500 µM compounds tested. At the same time, a low level of reduced glutathione was observed in all cell lines when treated with compound 3g (5-(4-bromophenyl)-2-(cyclopentylamino)thiazol-4(5H)-one) that most inhibited tumor cell proliferation. However, the most interesting results were obtained in the study of inhibitory activity towards two 11β-HSD isoforms. Many compounds at a concentration of 10 µM showed significant inhibitory activity against 11β-HSD1 (11β-hydroxysteroid dehydrogenase type 1). The compound 3h (2-(cyclopentylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one) showed the strongest 11β-HSD1 inhibitory effect (IC50 = 0.07 µM) and was more selective than carbenoxolone. Therefore, it was selected as a candidate for further research.In this study, a series of nine new 2-(cyclopentylamino)thiazol-4(5 H)-one) is a partially hydrogenated thiazole derivative containing an amino group in the 2-position and a carbonyl group in the 4-position -one derivatives. We synthesized derivatives substituted in the amino group with allyl  . 2-(cyclopentylamino)-5-methylthiazol-4(5H)-one (3b)—Yield: 69.29%. M.p. 211.5–213.7 °C. 1H-NMR : 12.23 , 4.33 , 3.93 , 2.23–2.31 , 2.06–2.15 , C5-CHACH3), 1.91–2.01 , 1.71–1.80 , 1.14 . 13C-NMR : 171.57 (C-4), 171.29 (C-2), 58.01 , 50.67 (C-5), 34.14 , 31.37 , 24.50 , 23.81 , 20.37 (CH2), 13.29 (CH3). HR-MS m/z 213.1062 [M+ + 1] . 2-(cyclopentylamino)-5-ethylthiazol-4(5H)-one (3c)—Yield: 81.63%. M.p. 192-194 °C. 1H-NMR : 11.86 , 11.68 , 4.89 , 4.42 , 4.23 , 3.94 , 2.06–2.26 , 1.95–2.01 , 1.82-1.94 , 1.64–1.77 , 1.42–1.61 , 1.03 , 0.97 . 13C-NMR : 172.25 (C-4), 171.42 (C-2), 59.73 , 51.01 (C-5), 34.07 , 32.53 , 24.50 , 23.97 , 23,73 CH2), 20.30 (CH2), 13.29 (CH3). HR-MS m/z 227.1221 [M+ + 1] . 2-(cyclopentylamino)-5-propylthiazol-4(5N-cyclopentylthiourea (1) and 0.0022 mol of the corresponding 2-bromo ester (2d-2e) were added to the solution of sodium methoxide (prepared with 0.092 g of sodium in 8 mL of anhydrous methanol). The mixture was refluxed for 7 (3d) and 14 days (3e), respectively. After evaporation of the solvent, the resulting oily post-reaction mixture was dissolved in 20 mL of water and neutralized with HCl to pH 7–8. The reaction mixture was extracted with chloroform (4 × 20 mL), then dried over magnesium sulfate, and the solvent evaporated. The concentrated mixture was purified by column chromatography (eluent chloroform/ethanol 9:1). The pure main products were crystallized from diethyl ether.Firstly, 0.002 mol of H)-one (3d)—Yield: 81.63%. M.p. 135-138 °C. 1H-NMR : 4.21 , 3.82–3.90 , 2.55–2.63 , 2.00–2.13 , 1.83–1.98 , 1.83–1.66 , 1.08 , 0.92 . 13C-NMR : 186.43 (C-4), 181.02 (C-2), 62.82 , 57.86 (C-5), 32.63 , 30.54 2), 23.99 , 22.06 (CH(CH3A)2), 16.34 (CH(CH3B)2). HR-MS m/z 227.1222 [M+ + 1] .2-(cyclopentylamino)-5-isopropylthiazol-4(5H)-one (3e)—Yield: 9.42%. M.p. 178–181 °C. 1H-NMR : 3.69–3.78 , 1.99–2.12 , 1.81–1.97 , 1.52–1.70 , 1.64 . 13C-NMR : 190.63 (C-4), 179.38 (C-2), 59.77 , 57.86 (C-5), 32.47 , 27.86 2), 23.90 . HR-MS m/z 213.1061 [M+ + 1] . 2-(cyclopentylamino)-5,5-dimethylthiazol-4(5N-cyclopentylthiourea (1), 0.0022 mol of the corresponding 2-bromo ester (2f–2g), 0.374 mL of N,N-diisopropylethylamine, and 2 mL of anhydrous ethanol were added to the reaction vessel. The mixture was heated in a microwave reactor in two cycles . The organic fraction was dried over MgSO4, filtered, and evaporated. The main product was isolated from the reaction mixture by column chromatography (chloroform/ethanol 9:1).Firstly, 0.02 mol of H)-one (3f)—Yield: 71.06%. M.p. 183–184 °C. 1H-NMR : 7.32–7.44 , 4.40 , 3.80–3.90 , 2.03–2.13 , 1.78–2.02 , 1.53–1.77 . 13C-NMR : 181.75 (C-4), 178.63 (C-2), 134.79 (1CPh), 129.24 (1CPh), 128.82 (1CPh), 128.41 (2CPh), 58.34 , 57.24 (C-5), 32.65 , 23.91 . HR-MS m/z 261.1065 [M+ + 1] . 2-(cyclopentylamino)-5-phenylthiazol-4(5H)-one (3g)—Yield: 85.48%. M.p. 220 °C (dec.). 1H-NMR : 7.47–7.54 , 7.22–7.27 , 5.14 , 3.79–3.87 , 2.01–2.16 , 1.80–1.99 , 1.51–1.77 . 13C-NMR : 185.43 (C-4), 180.97 (C-2), 135.01 , 132.15 , 129.98 , 122.51 , 57.91 , 57.74 (C-5), 32.65 , 23.96 . HR-MS m/z 339.0170 [M+ + 1] . 2-(cyclopentylamino)-5-(4-bromophenyl)thiazol-4(53h)—Yield: 2.47%. M.p. 207.7–209.1 °C. 1H-NMR : 3.75 , 1.98–2.13 , 1.86–1.97 , 1.82–1.85 , 1.69–1.82 , 1.51–1.65 , 1.32–1.46 . 13C-NMR : 190.73 (C-4), 180.65 (C-2), 68.96 (C-5), 58.00 , 36.63 , 32.51 , 25.14 , 24.94 , 23.95 . HR-MS m/z 253.1377 [M+ + 1] . 2-(cyclopentylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one (3i)—Yield: 27.86%. M.p. 202.4–203.1 °C. 1H-NMR : 3.68–3.77 , 2.75–2.87 , 2.46–2.56 , 2.25–2.34 , 1.98–2.12 , 1.84–1.97 , 1.49–1.77 . 13C-NMR : 190.76 (C-4), 179.74 (C-2), 60.70 (C-5), 57.86 , 34.19 , 32.53 , 23.94  16.89 . HR-MS m/z 225.1062 [M+ + 1] . 6-(cyclopentylamino)-5-thia-7-azaspiro[3.4]oct-6-en-8-one , microsomes  solution in PBS , phosphate buffer , and 10 µL of compounds 3a-3j dissolved in the mixture containing 1% of DMSO and 99% of water. The resulting solution with a final volume of 100 µL was incubated for 2.5 h at 37 °C. To stop the reaction, 10 μL of a 100 μM solution of carbenoxolone in PBS was added. The level of unreacted cortisol was measured by a commercially available ELISA kit.Human liver microsomes were used as a source of 11β-HSD2 enzyme to study the inhibitory effect of 50 values for compounds 3a–3i were obtained using their solutions at concentrations of 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5.0, and 10.0 µM and using standard procedure and conditions as described in the previous sections. As a control, analogous tests without the addition of inhibitors were used. Half the inhibitory concentration (causing 50% reduction of cortisol or cortisone) was read from the chart.Calibration curves to determine ICwww.rcsb.org; accessed on 13 April 2023): PBD: 3crz, 3qqp, 4bb5, 4c7j, and 4hfr. The crystal resolutions of the particular structures are equal to 0.235, 0.272, 0.220, 0.216, and 0.273 nm, respectively. The AutoDock Vina software [3, which covers the originally co-crystallized ligands present in the PDB structures as well as the closest amino acid residues that exhibit contact with those ligands . All the default procedures and algorithms implemented in AutoDock Vina were applied during docking procedure. In addition to the flexibility of the ligand molecules, the rotation of selected sidechains  in the proximity of the co-crystalized ligands was allowed as well. The predicted binding energies were averaged over five protein structures, and each stereoconfiguration of the ligand was treated separately . The more favorable binding mode is correlated with the lower binding energy value; only the lowest energy values corresponding to the given ligand were accounted for during subsequent analysis. The visual inspection of the location and orientation of the docked ligands in order to control the uniformity of the binding pattern was performed. Ligand molecules see  were drasoftware  was applThe docking methodology was initially validated by docking simulations of several ligand molecules originally included in the protein structures . The description of the validation procedure and the graphical illustration of its results can be found in Ref. . Here, w2 at 37 °C. All cell lines were cultured in T75 cell culture flasks or cell culture dishes to reach 80–90% confluency and seeded at a standard density of 5 × 103 cells/0.32 cm2, except for BJ cells at 2 × 103 cells/0.32 cm2.The BJ (CRL-2522), Caco-2 (HTB-37), PANC-1 (CRL-1469), U-118 MG (HTB-15), SK-MEL-30 (ACC 151), and MDA-MB-231 (HTB-26) cell lines were purchased from ATCC  or DSMZ . Cells were cultured in accordance with manufacturers' recommendations in a DMEM, MEM, or RPMI-1640 , supplemented with 10% FBS, except for Caco-2 (20%)  and 1% antibiotic mix solution  in a humidified atmosphere with 5% CO3h and incubated at standard conditions, protected from light. Finally, the absorbance was read at 590 nm and 620 nm wavelengths using a Synergy H1 microplate reader . The results are presented as %, while untreated control cells were considered 100%.The MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assay was performed following previously reported method . BrieflyThe assay was performed following previously reported method . The celp-value of <0.05 was considered statistically significant between groups and is displayed as: * p < 0.05; ** p < 0.01; and ***p < 0.001.Statistical analysis of the experimental data was carried out using GraphPad Prism ver. 6.0. All results are presented as means ± standard deviation. Differences between control and test samples were assessed with one-way ANOVA of variance with Dunnett post hoc test. A H)-one derivatives, which could act as inhibitors of 11β-hydroxysteroid dehydrogenase isoenzymes, were synthesized and evaluated. All obtained compounds showed different inhibitory activity against both isoforms of 11β-HSD (10.94–90.49% for 11β-HSD1 and 36.55–46.33% for 11β-HSD2). Compound 3h containing a spiro system of cyclohexyl and thiazole rings turned out to be a stronger and more selective 11β-HSD1 inhibitor (IC50 = 0.07 µM) than the most known 11β-HSD1 inhibitor—carbenoxolone. This result makes compound 3h a strong candidate for further research. The results of molecular docking of the obtained compounds with 11β-HSD1 are consistent with the results of in vitro tests.In this study, nine new 2-(cyclopentylamino)thiazol-4(53g derivative showed the greatest anticancer activity, as it caused the greatest decrease in viability among all five cancer cell lines. The second objective of this study was to evaluate the anti-proliferative activity of the obtained compounds. All thiazolone derivatives showed antiproliferative activity, especially against three tumor cell lines: Caco-2, MDA-MB-231, and SK-MEL-30. Among the obtained compounds, the