Reagents and instrumentsChemicals, reagents, solvents were purchased from Sigma Aldrich, and they were used as received. The two compounds A and B were synthesized according to a previous reported procedures69. Melting points of the different derivatives were measured using Gallen Kamp melting point equipment (Sanyo Gallen Kamp, UK). Digital Ultrasonic Cleaner CD-4830 (35 kHz, 310 W) was used to carry out the ultrasound-aided reactions. The IR spectra (KBr pellets, cm−1) were captured using a Pye-Unicam SP-3-300 FT-IR spectrophotometer. Bruker Avance III NMR spectrometer was used for the 1H- and 13C-NMR spectra in DMSO-d6. Mass spectra were performed on a Shimadzu GCMS-QP-1000EX mass spectrometer. Microanalytical analyses (CHN) were executed on a Perkin-Elmer analyzer (CHN-2400). Thin-layer chromatography (TLC) sheets covered with Merck 60 F254 plates’ UV fluorescent silica gel were used to monitor the reactions, and they were visualized using a UV lamp and various solvents as mobile phases. All reagents and solvents were purified and dried by standard techniques.SynthesisMethod aA mixture of cyclohexanone (0.01 mol), thioglycolic acid (0.01 mol), and some selected aromatic amines (0.01 mol) in 20 mL dry toluene was heated to reflux for 8 h. The reaction mixture was cooled, and the formed precipitate was filtered off, dried, and then recrystallized from DMF to give the desired compound (A–L).Method bA mixture of cyclohexanone (0.01 mol), thioglycolic acid (0.01 mol), the selected aromatic amines (0.01 mol) and a catalytic amount of iron oxide nanoparticles in 20 mL dry toluene was placed in Erlenmyer flask (50 mL). The reaction mixture was then subjected to ultrasound waves at room temperature for 60 min. The formed precipitate was filtered, dried, and recrystallized from DMF to afford the desired compound (A–L).4-(3,4-dimethylphenyl)-1-thia-4-azaspiro[4.5]decan-3-one (C)White crystals; m.p.185–186 °C; IR (KBr, cm−1): 3014 (CH aromatic), 2920 (CH aliphatic), 1668 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.49–1.82 (m, 10H, Cyclohexane), 2.20 (s, 3H, CH3), 2.22 (s, 3H, CH3), 3.74 (s, 2H, CH2 Thiazolone), 6.86 (d, 1H, Ar–H), 7.04 (d, 1H, Ar–H), 7.23 (s, 1H, Ar–H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 171.3 (C=O), 139.2 (Ar–C–NThiazolone), 136.6 (Ar–C), 133.9 (Ar–C), 131.3 (Ar–C), 129.3 (Ar–C), 125.7 (Ar–C), 66.3 (Spiro-C), 35.29 (2 CH2 Cyclohexane), 31.0 (CH2 Thiazolone), 26.9 (CH2 Cyclohexane), 24.5 (2 CH2 Cyclohexane), 18.1 (CH3), 17.3 (CH3); MS (m/z) (%): 275 (M+, 17), 130 (100); Anal. Calcd for: C16H21NOS (275.41): C, 69.78; H, 7.69; N, 5.09; Found: C, 69.67; H, 7.55; N, 4.96%.4-(3-nitrophenyl)-1-thia-4-azaspiro[4.5]decan-3-one (D)Off white crystals; m.p.111–113 °C; IR (KBr, cm−1): 3010 (CH aromatic), 1684 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.26–1.95 (m, 10H, Cyclohexane), 3.89 (s, 2H, CH2 Thiazolone), 7.49–7.83 (m, 3H, Ar–H), 8.03 (s, 1H, Ar–H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.1 (C=O), 149.9 (Ar–C-NO2), 141.8 (Ar–C–NThiazolone), 133.9 (Ar–C), 130.4 (Ar–C), 124.7 (Ar–C), 119.8 (Ar–C), 66.8 (Spiro-C), 34.4 (2 CH2 Cyclohexane), 31.2 ( CH2 Thiazolone), 26.9 (CH2 Cyclohexane), 23.1 (2 CH2 Cyclohexane); Anal. Calcd for: C14H16N2O3S (292.35): C, 57.52; H, 5.52; N, 9.58; Found: C, 57.45; H, 5.39; N, 9.51%.4-(2-fluorophenyl)-1-thia-4-azaspiro[4.5]decan-3-one (E)White crystals; m.p.125–127 °C; IR (KBr, cm−1): 3073 (CH aromatic), 1667 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.35–1.90 (m, 10H, Cyclohexane), 3.79 (s, 2H, CH2 Thiazolone), 7.12–7.91 (m, 4H, Ar–H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 174.4 (C=O), 162.2 (Ar–C–F), 131.3 (Ar–C–NThiazolone), 128.9 (Ar–C), 125.7 (Ar–C), 123.5 (Ar–C), 119.8 (Ar–C), 66.3 (Spiro-C), 37.6 (2 CH2 Cyclohexane), 31.9 (CH2 Thiazolone), 26.3 (CH2 Cyclohexane), 24.5 (2 CH2 Cyclohexane); MS (m/z) (%): 265 (M+, 36), 91 (100); Anal. Calcd for: C14H16FNOS (265.35): C, 63.37; H, 6.08; N, 5.28; Found: C, 63.21; H, 5.92; N, 5.09%.4-(2-chlorophenyl)-1-thia-4-azaspiro[4.5]decan-3-one (F)White crystals; m.p.105–107 °C; IR (KBr, cm−1): 3062 (CH aromatic), 1670 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.33–2.08 (m, 10H, Cyclohexane), 3.79 (s, 2H, CH2 Thiazolone), 7.13–7.92 (m, 4H, Ar–H); Anal. Calcd for: C14H16ClNOS (281.06): C, 59.67; H, 5.72; N, 4.97; Found: C, 59.49; H, 5.55; N, 4.78%.4-(2,5-dichlorophenyl)-1-thia-4-azaspiro[4.5]decan-3-one (G)White crystals; m.p.105–107°C; IR (KBr, cm−1): 3061 (CH aromatic), 1680 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.46–2.26 (m, 10H, Cyclohexane), 3.83 (s, 2H, CH2 Thiazolone), 7.22 (d, 1H, Ar–H), 7.49 (d, 1H, Ar–H), 7.93 (s, 1H, Ar–H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.9 (C=O), 142.4 (Ar–C), 138.7 (Ar–C), 133.1 (Ar–C), 129.9 (Ar–C), 124.8 (Ar–C), 119.9 (Ar–C), 65.38 (Spiro-C), 35.3 (2 CH2 Cyclohexane), 31.6 (CH2 Thiazolone), 26.0 (CH2 Cyclohexane), 23.4 (2 CH2 Cyclohexane); Anal. Calcd for: C14H15Cl2NOS (316.24): C, 53.17; H, 4.78; N, 4.43; Found: C, 53.04; H, 4.68; N, 4.36%.4-(4,6-dimethylpyrimidin-2-yl)-1-thia-4-azaspiro[4.5]decan-3-one (H)White crystals; m.p.182–183°C; IR (KBr, cm−1): 3012 (CH aromatic), 1921 (CH aliphatic), 1693 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.22–1.87 (m, 10H, Cyclohexane), 2.12 (s, 6H, 2CH3), 3.62 (s, 2H, CH2 Thiazolone), 6.29 (s, 1H, Ar–H); MS (m/z) (%): 277 (M+, 38), 111 (100); Anal. Calcd for: C14H19N3OS (277.39): C, 60.62; H, 6.90; N, 15.15; Found: C, 60.55; H, 6.82; N, 15.07%.4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1-thia-4-azaspiro[4.5]decan-3-one (I)White crystals; m.p.166–167 °C; IR (KBr, cm−1): 3022 (CH aromatic), 1916 (CH aliphatic),1713 (C=O), 1665 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.14–2.17 (m, 10H, Cyclohexane), 2.27 (s, 3H, CH3), 3.14 (s, 3H, CH3), 3.64 (s, 2H, CH2 Thiazolone), 7.11–7.49 (m, 5H, Ar–H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 168.1 (C=O), 161.9 (C=O), 134.2 (C Pyrazolone), 133.9 (Ar–C), 130.7 (2) (Ar–C), 126.3 (2) (Ar–C), 103.6 (C Pyrazolone), 64.7 (Spiro-C), 37.6 (2 CH2 Cyclohexane), 33.4 (N–CH3), 30.1 (CH2 Thiazolone), 26.7 (CH2 Cyclohexane), 22.8 (2 CH2 Cyclohexane), 14.6 (CH3); Anal. Calcd for: C19H23N3O2S (357.14): C, 63.84; H, 6.49; N, 11.76; Found: C, 63.77; H, 6.41; N, 11.68%.4-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-1-thia-4-azaspiro[4.5]decan-3-one (J)White crystals; m.p.156–158°C; IR (KBr, cm−1): 3082 (CH aromatic), 1673 (C=O), 1706 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.01–2.22 (m, 10H, Cyclohexane), 3.15 (s, 2H, CH2), 3.65 (s, 2H, CH2 Thiazolone), 7.32–7.49 (m, 5H, Ar–H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 174.5 (C=O), 173.0 (C=O), 156.4 (N–C=N Pyrazolone), 139.7 (Ar–C–N), 130.1 (2) (Ar–C), 127.8 (Ar–C), 124.0 (2) (Ar–C), 65.7 (CH2 Pyrazolone), 64.3 (Spiro-C), 35.6 (2 CH2 Cyclohexane), 31.0 ( CH2 Thiazolone), 25.7 (CH2 Cyclohexane), 23.4 (2 CH2 Cyclohexane); Anal. Calcd for: C17H19N3O2S (329.42): C, 61.98; H, 5.81; N, 12.76; Found: C, 61.88; H, 5.73; N, 12.69%.4-(thiazol-2-yl)-1-thia-4-azaspiro[4.5]decan-3-one (K)White crystals; m.p.172–173 °C; IR (KBr, cm−1): 3084 (CH aromatic), 1684 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.45–1.82 (m, 10H, Cyclohexane), 3.88 (s, 2H, CH2 Thiazolone), 7.40 (d, 2H, Ar–H), 7.59 (d, 2H, Ar–H); MS (m/z) (%): 254 (M+, 11), 150 (100); Anal. Calcd for: C11H14N2OS2 (254.37): C, 51.94; H, 5.55; N, 11.01; Found: C, 51.82; H, 5.42; N, 10.91%.4-(benzo[d]thiazol-2-yl)-1-thia-4-azaspiro[4.5]decan-3-one (L)White crystals; m.p.143–144°C; IR (KBr, cm−1): 3015 (CH aromatic), 1673 (C=O); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.52–1.89 (m, 10H, Cyclohexane), 3.93 (s, 2H, CH2 Thiazolone), 7.34 (dd, 1H, Ar–H), 7.44 (dd, 1H, Ar–H), 7.83 (d, 2H, Ar–H), 7.99 (d, 2H, Ar–H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 174.4 (C=O), 163.4 (N–C=N Pyrazolone), 149.7 (Ar–C), 130.1 (Ar–C), 127.2 (Ar–C), 124.4 (Ar–C), 121.4 (Ar–C), 119.9 (Ar–C), 67.8 (Spiro-C), 35.0 (2 CH2 Cyclohexane), 31.1 (CH2 Thiazolone), 26.7 (CH2 Cyclohexane), 24.8 (2 CH2 Cyclohexane); Anal. Calcd for: C15H16N2OS2 (304.43): C, 59.18; H, 5.30; N, 9.20; Found: C, 59.03; H, 5.24; N, 9.13%.X-ray structure analysis of A and BThe X-ray data for colorless block crystals of A and B with size of 0.17 × 0.03 × 0.03 and 0.20 × 0.14 × 0.02 mm were collected at 100(2) K with Rigaku XtaLAB Synergy-S (Rigaku-Oxford Diffraction) four circle diffractometer with a mirror monochromator and a microfocus CuKα radiation source (λ = 1.54184 Å). The obtained data set was processed with CrysAlisPro software70. The structures were solved by direct methods and refined with full-matrix least-squares method on F2 with the use of SHELX2018 program packages5. Details of the data processing and refinements were given before71,72. All non-hydrogen atoms were refined anisotropically, while hydrogen atoms were refined isotropically, and were positioned geometrically and constrained to ride on their parent atoms with CH = 0.95–0.99 Å. A summary of the crystal data and refinement details are given in Table 2. The structural data have been deposited at the Cambridge Crystallographic Data Center (CCDC No. 2295434 and 2,295,435 for A and B, respectively).Hirshfeld surface studyHirshfeld surface analyses were performed to assess the inter- and intra-molecular interactions of the molecular crystal of A and B using the Crystal Explorer 21.5 software73. Calculations were done by using the crystallographic information (cif) files of both A and B compounds.Stereochemistry and reactivity descriptors calculationsThe energetically optimized structures, energies and the global reactivity parameters for some selected compounds were performed using Gaussian 09w software55. The DFT (B3LYP) method with the basis set 6-31G (d,p) and double zeta plus polarization for hydrogen, carbon, nitrogen, oxygen, sulfur and chloride was adopted.Antimicrobial analysisThe antimicrobial activities of the synthesized compounds were screened against two Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two fungi (Candida albicans and Aspergillus flavus). Details of the experimental procedures were previously reported59,74. Ampicillin was used as an antibiotic standard and Clotrimazole was performed as standard for the antifungal estimations. The percentage activity index for the compounds was calculated using the formula:$${\text{Activity}}\;{\text{index}} = \frac{{{\text{Diameter}}\;{\text{of}}\;{\text{zone}}\;{\text{inhibition}}\;{\text{by}}\;{\text{tested}}\;{\text{compound}}\;({\text{mm}})}}{{{\text{Diameter}}\;{\text{of}}\;{\text{zone}}\;{\text{inhibition}}\;{\text{by}}\;{\text{standard}}\;({\text{mm}})}} \times 100$$Molecular docking studiesThe molecular docking studies were performed by Molecular Operating Environment (MOE) software package version 2014.0901. The X-ray crystal structure of a B-DNA with the PDB code 1BNA was downloaded from Research Collaboratory for Structural Bioinformatics (RCSB) database55.
